Fertiliser Control 5th Amendment Order,2025

The Gazette of India EXTRAORDINARY PART II—Section 3—Sub-section (ii) PUBLISHED BY AUTHORITY No. 2479] NEW DELHI, TUESDAY, JUNE 10, 2025/JYAISTHA 20, 1947 C.G.-D.L.-A.-11062025-263724 CG-DL-E-11062025-263724 MINISTRY OF AGRICULTURE AND FARMERS WELFARE (Department of Agriculture and Farmers Welfare) ORDER New Delhi, the 9th June, 2025 S.O. 2539(E).— In exercise of the powers conferred by section 3 of the Essential Commodities Act, 1955 (10 of 1955), the Central Government hereby makes the following Order further to amend the Fertiliser (Inorganic, Organic or Mixed) (Control) Order, 1985, namely:- 1. (1) This Order may be called the Fertiliser (Inorganic, Organic or Mixed) (Control) Fifth Amendment Order, 2025. (2) It shall come into force on the date of its publication in the Official Gazette. 2. In the Fertiliser (Inorganic, Organic or Mixed) (Control) Order, 1985, (hereinafter referred to as the said Order), in clause 20C, in sub-clause (3), for item B, the following items shall be substituted, namely:- “B. Bio-efficacy Trials: 1. Agronomic Bio-efficacy trials shall be conducted at the National Agricultural Research System, including the Indian Council of Agricultural Research and State Agricultural Universities. 2. Bio-efficacy trials to be conducted on same crop at minimum three different doses for one season at three agro-ecological locations.” 3. In the said Order, in Schedule VI,- (i) for Part D, the following Part shall be substituted, namely:- “Part-D METHODOLOGY OF TESTING 1. Estimation of pH.- As mentioned in the Schedule –IV, Part-D at serial number 1 2. Estimation of specific gravity.- As mentioned in the Schedule- II, Part-B at serial number 21 3. Estimation of bulk density.- As mentioned in the Schedule –IV, Part-D at serial number 2 4. Estimation of organic matter and organic carbon.- As mentioned in the Schedule –IV, Part-D at serial number 5 5. Estimation of water solubility- 5.1 Equipment: a. analytical balance. b. rotatory evaporator or hot air oven or hot plate. c. magnetic stirrer with temperature control. d. constant temperature water bath. e. whatman grade 934-AH RTU glass microfiber filters. 5.2 Procedure.- (i) take the test item in different quantity form lower to higher amount in fixed volume of water (eg.100 ml); (ii) visually determine the saturation of solution and record the amount required to saturate the taken water volume; (iii) weigh excess quantity of test sample (gram) (about five times quantity of test substance required to saturate the water) into each of three glass vessels fitted with glass stoppers (conical flasks); (iv) take the volume of water as taken in preliminary test (e.g 100 ml) and add to each glass vessel and tightly stopper the glass vessel and then agitate one of the vessels at 30 °C in water bath or on magnetic stirrer for twenty four hours; (v) equilibrate the other two vessels at 30 °C for another forty eight hours and seventy two hours respectively; (vi) after twenty hours, equilibrate one of the vessels for twenty four hours at 20 ± 0.5 °C with occasional shaking; (vii) after twenty four hours equilibration, filter the solution under vacuum using whatman grade 934-AH RTU glass microfiber filters or 0.45µ membrane filters; (viii) take the filtrate in round bottom flask and evaporate under vacuum up to the dryness using rotatory evaporator with the water bath set at 60oC. (Hot plate or hot air oven can also be used as alternate to rotary evaporator); (ix) carefully collect the residue after evaporation (if required dry the residue at room temperature) and record the mass of the residue using the analytical balance; (x) additionally equilibrate the other two vessels already equilibrated at 30 °C for forty eight hours and seventy two hours, respectively for twenty four hours at 20 ± 0.5 °C with occasional shaking and record the mass of the residue as per the procedure mentioned in above point; and (xi) the measured mass of residue in at least the two last vessels do not differ by more than 15%, then consider it satisfactory and the results from vessels 1, 2 and 3 show a tendency of increasing values, the whole test shall be repeated using longer equilibration times. 5.3 Calculations.- Calculate water solubility % w/w by formula as given below:- Water solubility % w/w = Mass of the residue obtained (gram) x 100 Mass of the test substance taken (gram) 6. Estimation of total dissolved solids (TDS) or total soluble solid (TSS). - 6.1 Equipment: a. rotary evaporator or hot plate. b. analytical balance. c. vortex mixture. 6.2 Procedure.- (i) take 100 ml of the liquid sample in a pre-weighted 500 ml glass beaker (W1). (ii) heat it at temperature of 60oC using either rotary evaporator or at 103oC using hot plate. (iii) after complete evaporation of the solvent till the constant weight is achieved, record the weight of the beaker (W2). 6.3 Calculation.- TDS or TSS (gram / 100 ml) = W2- W1 7. Detection and quantification of alginic acid.- 7.1 Equipment.- (a) HPLC-RID system. (b) analytical balance. (c) 0.45µ syringe filter. (d) high speed centrifuge. (e) vortex mixer. 7.2 Chemicals and Reagent preparation.- (a) Sodium alginate (CAS No. 9005-38-3). (b) Ultra pure water. (c) standard preparation- (i) prepare a series of sodium alginate standards in ultrapure water (5 ppm to 500 mg / ml); (ii) Sodium Alginate stock standard solution: weigh about 10 mg of Sodium alginate in 10 ml volumetric flask and dilute it with 10 ml ultrapure water and it gives a stock solution of 1000 mg / ml; (iii) Sodium alginate intermediate standard solution: Dilute 1.0 ml stock standard to 10 ml with ultrapure water and mix well and it gives an intermediate standard solution of 100 mg /ml; (iv) dilute 1.0 ml of intermediate standard solution to 10 ml ultrapure water and it gives an intermediate standard solution of 10 mg /ml; and (v) prepare linearity point on the basis of sample concentration as given in the table bellow:- Table +-----+----------+------------+------------+---------------+ |S. No|Linearity |Conc. of |Volume (ml) |Volume (ml) |Concentration of| | |Standard |Stock |of stock |of water to be |standard | | | |solution |solution to |used |(mg/ml) | | | |(mg/ml) |be used | | | +-----+----------+------------+------------+---------------+---------------| | (i) |Standard 1|10 |0.50 |0.50 |5 | +-----+----------+------------+------------+---------------+---------------| | (ii)|Standard 2|100 |0.25 |0.75 |25 | +-----+----------+------------+------------+---------------+---------------| | (iii)|Standard 3|100 |0.50 |0.50 |50 | +-----+----------+------------+------------+---------------+---------------| | (iv)|Standard 4|100 |1.00 |0.00 |100 | +-----+----------+------------+------------+---------------+---------------| | (v) |Standard 5|1000 |0.20 |0.80 |200 | +-----+----------+------------+------------+---------------+---------------| | (vi)|Standard 6|1000 |0.30 |0.70 |300 | +-----+----------+------------+------------+---------------+---------------| | (vii)|Standard 7|1000 |0.40 |0.60 |400 | +-----+----------+------------+------------+---------------+---------------| | (viii)|Standard 8|1000 |0.50 |0.50 |500 | +-----+----------+------------+------------+---------------+---------------| (d) Sample preparation (i) vortex the formulation to be tested before preparing the samples; (ii) for solid samples, the sample need to be vortexed with ultrapure water (1:20 w/v) and to be kept at room temperature for twelve hours followed by vigorous shaking and centrifugation; (iii) dilute the formulation (around 50 to 100 times) taking into consideration the theoretical expected values of alginate in the formulation under analysis; and (iv) the samples shall be prepared as such that it falls under the standard curve concentrations of individual standards (Alginate) and filter each sample with 0.45µ syringe filter and fill it in 2 ml HPLC glass sample vials. 7.3 Procedure.- (i) inject separately equal volumes (50 ml) of standard preparations and sample preparations in the HPLC-RID system; (ii) record the chromatograms and measure the peak response of standard preparations; (iii) calculate the quantity of the desired compound with respect to the peak response for the individual standard; and (iv) use the following Chromatographic conditions for analysis as specified in table below:- Table +-------+-----------------------------------+----------------------+ | (i) | Column | Rezex RHM Monosaccharide H + LC Column (300 | | | | mm x 7.8 mm) | +-------+-----------------------------------+----------------------+ | (ii) | Column Temperature | 65°C | +-------+-----------------------------------+----------------------+ | (iii) | Detector | Refractive Index Detector| +-------+-----------------------------------+----------------------+ | (iv) | Detector Temperature | 50°C | +-------+-----------------------------------+----------------------+ | (v) | Pump mode | Isocratic | +-------+-----------------------------------+----------------------+ | (vi) | Mobile Phase | 0.05% acetic acid | +-------+-----------------------------------+----------------------+ | (vii) | Injection Volume | 50 µl | +-------+-----------------------------------+----------------------+ | (viii)| Signal Acquisition: | Positive Polarity Mode| +-------+-----------------------------------+----------------------+ | (ix) | Flow rate | 0.5 ml/min | +-------+-----------------------------------+----------------------+ 7.4 Calculations.- Plot the standard curve with standard solutions taking concentrations along the X-axis and Area along the Y-axis and determine the concentration of alginate present in the test samples from the standard curve using Linear regression equation. y= mx +b where y is the value of second data set (peak area) x is the value of first day set (Concentration of standard) m is the slope of the line b is y-intercept of the line Slope and intercept can be calculated using the formula.- n∑xy- (∑x)(∑y) m (slope) = ------------------ n∑x² - (∑x)² (∑y) ∑x² - ∑x∑xy b (intercept)= ------------------ n ∑x² - (∑x)² Example.- +---+----+----+----+----+ | x | 2 | 4 | 6 | 8 | +---+----+----+----+----+ | y | 3 | 7 | 5 | 10 | +---+----+----+----+----+ Construct the following table- +-----+----+-----+-----+ | x | y | x² | xy | +-----+----+-----+-----+ | 2 | 3 | 4 | 6 | +-----+----+-----+-----+ | 4 | 7 | 16 | 28 | +-----+----+-----+-----+ | 6 | 5 | 36 | 30 | +-----+----+-----+-----+ | 8 | 10 | 64 | 80 | +-----+----+-----+-----+ |∑x=20|∑y=25|∑x²=120|∑xy=144| +-----+----+-----+-----+ m (slope) = 4(144) - 20 x 25/ 4(120) – 400 = 0.95 b (intercept)= 25 x 120 – 20 x 144/ 4(120)- 400 = 1.5 Equation for linear regression is y= mx+b y= 0.95 x + 1.5 7.5 Reference.- Valverde S, Hernández-Apaolaza L, Lucena JJ 2022. A Simple Analytical Method to Determine Alginic Acid, Laminarin and Mannitol and Seaweed Extracts Fertilisers, Journal of Chromatography and Separation Techniques Volume-13, Issue 1 No: 1000470. 8. Estimation of carrageenan.- 8.1 Equipment.- (a) spectrophotometer. (b) analytical balance. (c) vortex mixer. 8.2 Chemicals and Reagent preparation.-- (a) Toluidine Blue-O (CAS No.: 92-31-9). (b) κ-Carrageenan standard: (CAS No.: 11114-20-8). (c) Ultra pure water. (d) Reagent A: 0.06 M Toluidine Blue O (TBO) solution. (i) prepare a stock solution of TBO at a concentration of 2 mM in water (6 mg in 10 ml water) and vortex for three to five minutes for uniform mixing; and (ii) dilute it to the concentration of 0.06mM, by adding 0.6ml of the 2 mM TBO stock solution to 19.4 ml water. (e) Reagent B: stock solution (2 mg/ml) of Carrageenan. (vi) use -Carrageenan, a sulphated polysaccharide as standard; (vii) take 10 ml volumetric flask; (viii) weigh 20 mg of Carrageenan and transfer it to 10 ml standard flask; (ix) add 5-6 ml water to the standard flask and dissolve the carrageenan and vortex for 3-5 min for uniform mixing; and (x) Make up the final volume to 10 ml using water and mix well for homogeneity. (f) Reagent C: Working solution (0.04 mg/ml) of Carrageenan iii) take 10 ml volumetric flask; and iv) add 200 μl of reagent B in standard flask and make up the final volume to 10 ml using water and mix well for homogeneity. Note: The Toluidine blue O reagent and standard solution shall be prepared fresh before initiating each experiment. (g) preparation of standard solution: prepare a series of carrageenan standard solutions (in glass test tubes) of concentrations, 0 (blank, distilled water), 0.008, 0.016, 0.024,0.032 and 0.040 mg/ml by mixing the carrageenan working solution (Reagent C) and water as specified in the following table – Table +-----+----------+----------+---------------------+ |S. No|Reagent C |Water |Carageenan concentration| | |(ml) |(ml) |(mg/ml) | +-----+----------+----------+---------------------+ | (i) |0 |300 |0 | +-----+----------+----------+---------------------+ | (ii)|60 |240 |0.008 | +-----+----------+----------+---------------------+ | (iii)|120 |180 |0.016 | +-----+----------+----------+---------------------+ | (iv)|240 |60 |0.032 | +-----+----------+----------+---------------------+ | (v) |300 |0 |0.04 | +-----+----------+----------+---------------------+ (h) Preparation of sample- (A)For liquid and viscous (gel) product.- (i) dilute the test sample appropriately with water such that the absorbance of the sample is within the range of the standard curve; (ii) in case of coloured samples, add activated charcoal (1% v/v) to the aqueous solution of the sample and stir well for ten minutes using vortex mixer followed by the filtration; and (iii) use the filtrate for the estimation of -Carrageenan. (B) For granular/flakes product.- (i) take 10 gram of test sample granule or flake in a conical flask containing 90 ml of water; (ii) incubate at room temperature for forty five minutes and mix the components to homogeneity on a magnetic stirrer; (iii) withdraw 2 ml of sample and centrifuge at 3000 rpm for three minuets; and (iv) the supernatant thus obtained shall be used for analysis of carrageenan or sulphated sugar. Note: To estimate the concentration of carrageenan or sulphated sugar in the sample containing approximately 25 mg/ml sulphated sugar, the sample needs to be diluted by 103 fold, so that the approximate concentration of diluted sample is 0.025 mg/ml, which is within the limit of standard curve. 8.3 Procedure.- (i) take 60 μl of standard (or sample) solution as prepared above and add 240 μl of TBO solution (Reagent A); (ii) mix the solution to homogeneity; (iii) measure absorbance at 630 nm; (iv) prepare standard curve using concentration in X axis and absorbance in Y axis; and (v) calculate the concentration of carrageenan in test sample from standard curve. 8.4 Calculation.- Calculate the total carrageenan content of the test sample using the formula as below.- % Carrageenan = Concentration of carrageenan x dilution factor x 100 Weight of sample taken 8.5 Reference.- Ziolkowska D, Kaniewsk A, Lamkiewicz J, Shyichuk A. 2017, Determination of carrageenan by means of photometric titration with methylene blue and Toluidine blue dyes. Carbohydrate Polymers 165: 1-6 9. Detection and quantification of fucoidan.- 9.1 Equipment.- (a) HPLC-RID system. (b) analytical balance. (c) 0.45 µ syringe filter. (d) vortex mixer. 9.2 Chemicals and Reagent Preparation.- (a) fucoidan (CAS No. 9072-19-9). (b) ultra pure water (c) standard Preparation: (i) prepare a series of fucoidan standards in ultra pure water (5 ppm to 500 mg/ml); (ii) Fucoidan stock standard solution: weigh about 10 mg of fucoidan in 10 ml volumetric flask and dilute it with 10 ml ultrapure water and it gives a stock solution of 1000 mg/ml; (iii) Fucoidan intermediate standard solution: dilute 1.0 ml stock standard to 10 ml with ultrapure water and mix well and it gives an intermediate standard solution of 100 mg/ml; (iv) dilute 1.0 ml of intermediate standard solution to 10 ml with ultrapure water and it gives an intermediate standard solution of 10 mg/ml; and (v) prepare linearity point as specified under sub paragraph (c) (v) of paragraph 7.2 in the Part-D of schedule-VI. (d) sample preparation: same as specified under sub paragraph (d) of paragraph 7.2 in the Part-D of schedule-VI 9.3 Procedure and Calculations.- Same as specified in under paragraph 7.3 and 7.4 in the Part-D of schedule-VI. (Note:Since sodium alginate interfere with fucoidan, prior to the preparation of the sample, add 2% CaCl2 solution to precipitate out alginates and use the remaining solution for fucoidan estimation). 10. Estimation of mannitol.- 10.1 Equipment.- (a) HPLC- RID system. (b) detector: Refractive index detector. (c) online vacuum degasser. (d) quaternary pump (e) high speed centrifuge. (f) analytical balance. (g) vortex mixer. (h) sonicator. (i) 0.45µ syringe filters. 10.2 Chemicals and Reagent Preparation.- (a) D-Mannitol (CAS No.69-65-8). (b) ultra pure water. (c) acetic acid (0.05%) : Take 0.5 ml of acetic acid in 1liter of HPLC water in mobile phase container. (d) standard preparation: (i) prepare a series of mannitol standards in ultrapure water (5 ppm to 500 mg/ml); (ii) Mannitol stock standard solution that is weigh about 10 mg of mannitol in 10 ml volumetric flask and dilute it with 10 ml ultrapure water and it gives a stock solution of 1000 mg/ml; (iii) for Mannitol intermediate standard solution that is dilute 1.0 ml stock standard to 10 ml with ultrapure water and mix well and it will give an intermediate standard solution of 100 mg/ml; (iv) dilute 1.0 ml of intermediate standard solution to 10 ml with ultrapure water and it gives an intermediate standard solution of 10 mg/ml; and (v) prepare linearity point as specified under sub paragraph (c) (v) of paragraph 7.2 in the Part-D of schedule-VI. (e) Sample preparation: same as specified under sub paragraph (d) of paragraph 7.2 in the Part-D of schedule-VI. 10.3 Procedure and Calculations.- (a) Same as specified under sub paragraph 7.3 and 7.4 of the Part-D of schedule-VI. 10.4 Reference.- Valverde S, Hernández-Apaolaza L, Lucena J.J. 2022. A Simple Analytical Method to Determine Alginic Acid, Laminarin and Mannitol and Seaweed Extracts Fertilisers. Journal of Chromatography and Separation Techniques, Volume-13, Issue 1 No: 1000470. 11. Estimation of total poly-phenols.- 11.1 Equipment- (a) spectrophotometer. (b) analytical balance. (c) high speed centrifuge. (d) vortex mixer. (e) water bath. 11.2 Chemicals and Reagent Preparation.- (a) gallic acid(CAS No 149-91-7) or phloroglucinol (CAS No 108-73-6) (b) Folin- Ciocalteu Reagent (CAS No 12111-13-6). (c) 80% aqueous methanol that is to 80 ml of methanol add 20 ml of water. (d) 20% sodium carbonate that is to weigh 20 g of sodium carbonate and transfer it to 100 ml volumetric flask. Add 40 ml of water and shake vigorously to dissolve and make up the volume to 100 ml with water. (e) Sample preparation (i) Liquid and gel samples The algal or liquid biostimulant samples are to be extracted with eighty percent methanol. (1) extract 1ml of the seaweed biostimulant sample with 10 ml of eighty percent aqueous methanol (extraction ratio- 1:10 v/v) overnight at 4°C in dark; (2) vortex the mixture thoroughly and centrifuge at 12000 ×g for five minutes at 4°C; and (3) collect the supernatant and use it for further analysis. (ii) solid samples (powder, granules and flakes).- (1) extract accurately weighed homogenized algal tissue or solid test sample (0.3 ± 0.005 gram) with 15 ml of 80% aqueous methanol overnight at 4°C in dark; (2) vortex the mixture thoroughly and centrifuge at 12000 × g for five minutes at 4°C; and (3) collect the supernatant and use it for further analysis. (f) Gallic acid standard preparation.- (i) accurately weigh about 100 mg gallic acid, and transfer into a 100 ml volumetric flask and add about 75 ml water and mix well until the solids are dissolved; (ii) make up the volume to 1000 ml with water and mix well; (iii) label it as “stock standard solution.” This stock standard solution (ca 1000 mg/L gallic acid) may be used for up to 1 month when stored at 2–8°C; and (iv) prepare calibration standard solutions as shown in Table (ca 40–200 mg/l gallic acid) by pipetting the indicated amount of stock standard solution into the indicated size volume flask and diluting to volume with water and the following approximate concentrations of gallic acid in each of the calibration standard solutions are specified in table below.- Table +-----+---------------------+------------------------+----------------+---------------------+ |S. No|Calibration |volume stock |Volume of |approximate | | |Standard solution |standard |flask, ml |concentration of gallic| | | |solution, ml | |acid, mg/L | +-----+---------------------+------------------------+----------------+---------------------+ | (i) |1 |1 |25 |40 | +-----+---------------------+------------------------+----------------+---------------------+ | (ii)|2 |2 |25 |80 | +-----+---------------------+------------------------+----------------+---------------------+ | (iii)|3 |3 |25 |120 | +-----+---------------------+------------------------+----------------+---------------------+ | (iv)|4 |4 |25 |160 | +-----+---------------------+------------------------+----------------+---------------------+ | (v) |5 |5 |25 |200 | +-----+---------------------+------------------------+----------------+---------------------+ 11.3 Procedure.- (1) add an aliquot of 1 ml of supernatant to 15 ml of distilled water and 1 ml of Folin- Ciocalteu reagent in a test tube; (2) to mix the contents of the tube thoroughly and incubate for five minutes; (3) after five minutes of incubation, add 3 ml of 20% sodium carbonate followed by heating the test tubes at 50°C for thirty minutes in a water bath; (4) measure the absorbance using a spectrophotometer at 765 nm after removal from water bath; (5) to prepare a standard curve (40-200 mg/liter) with above method using Gallic acid or phloroglucinol as reference; and (6) express the concentration of phenolic compounds in gallic acid or phloroglucinol equivalents. 11.4 Calculations.- Calculate the total phenolics as mg gallic acid equivalent (GAE) / gram sample GAE (mg/g) = C x V/ M Where: C = concentration of gallic acid obtained from calibration curve in mg/ml V = Volume of extract in ml M= Mass of extract in gram 11.5 Reference Kupina S, Fields C, Roman MC 2019. Determination of total phenolic content using Folin-C-Assay: Single Laboratory validation, First Action 2017.13. Journal of AOAC International. 101: pp 1466- 1472. 12. Estimation of Humic acid and fulvic acid- . 12.1 Equipment.- (a) analytical balance. (b) drying oven. (c) high speed centrifuge. (d) rotary evaporator. (e) pH meter. (f) spectrophotometer. (g) peristaltic pump. (h) muffle furnace. (i) rotating shaking mixer. (j) desiccator 12.2 Chemicals and Reagent preparation- (a) sodium hydroxide. (b) hydrochloric acid. (c) nitrogen gas- 99.96% purity. (d) Supelco Superlite DAX-8 resin. (e) Amberlite IR 20 strong cation exchange resin. 12.3 Procedure- (a) Alkaline extraction- (i) If the material is solid or granular, prepare analytical samples by crushing them to fine powder using a pestle and mortar so that crushed sample passes through Sieve mesh size of 60–80; (ii) To determine the moisture content gravimetrically as follows- (a) weigh an aluminum weigh boat and record mass (Wt1); (b) transfer 2 ± 0.5 gram test portion of the analytical sample into the weigh boat, weigh, and record mass (W1); (c) place the sample in drying oven for 24 ± 0.2 h at 90°C; after twenty four hours, remove from drying oven and place in desiccator to cool for one hour; weigh and record mass of weigh boat and dry test portion (W2); (d) determine the moisture ratio using Equation Moisture ratio = [(W1 – W2)/(W1 – Wt1)] (iii) weigh about 2- 5 gram of second test portion from the analytical sample (W3: exact weight of sample used) and place it into a precalibrated or premarked one liter Erlenmeyer flask; (Note: The amount of test sample taken shall contain approximately 2.5 gram humic acid). (iv) add 0.1 M Sodium Hydroxide (that is, 4 gram Sodium Hydroxide per l liter distilled water) with stirring, and make to a final volume of 1 liter and determine the dry weight of the test portion using Equation: Dry test portion dry weight = (W3) (1 – moisture ratio) (v) for liquid materials, thoroughly mix the analytical sample by stirring with a glass rod for one minute, ensuring that any residue that may have fallen to the bottom of the container is thoroughly mixed; (vi) add an aliquot of a test portion of the analytical sample, noting the volume (V1), into a precalibrated or premarked 1 liter Erlenmeyer flask, and bring to a final volume of 1 liter with 0.1 M Sodium Hydroxide; Note: The test portion shall result in a concentration of 200 to 600 mg/liter humic acid and fulvic acid after dilution with 0.1 M Sodium Hydroxide, the aliquot volume will be based on the humic acid and fulvic acid product concentrations that are claimed in test sample. (vii) determine the density (gram/ml) of the liquid material by weighing 10 ml of well-mixed analytical sample in a pretared graduated cylinder (D1) and determine the weight of the liquid test portion using equation: Liquid test portion weight (gram) = (V1) (D1) (viii) add a 0.8 to 1.2 centimeter long magnetic stir bar, replace the air in the headspace with Nitrogen, and cover with parafilm, mix vigorously on a stir plate (e.g., 200–300 rpm); stir solid materials for six hours to extract humic substances and the liquid materials for one hour to ensure dissolution of all humic acid and fulvic acid; (ix) from this point on, the method is the same for both solid and liquid materials; (x) after stirring, remove the flask from the stir plate, transfer the contents to centrifuge tubes, and centrifuge the entire volume to separate any insoluble material from the dissolved humic acid and fulvic acid; (xi) centrifuge at 3900 × g for ten minutes (use 50 ml centrifuge tubes, or larger, as available), discard the insoluble precipitates and collect the alkaline supernatant containing the humic acid and fulvic acid in a clean one liter Erlenmeyer flask; (xii) while the extract solution is being mixed with a stir bar, carefully insert a pH electrode into the middle portion of the solution and to flocculate the humic acid, add concentrated hydrochloric acid (1:1) dropwise to the alkaline extract until pH is reached to 1.0 ± 0.05; (xiii) cover the flask with parafilm and mix for one hour, check pH and readjust to pH 1.0 with additional concentrated hydrochloric acid (1:1) if necessary and if the pH falls below 0.95, adjust pH back to 1.0 ± 0.05 with the 0.1 M sodium hydroxide solution and continue to let the acidified extract mix and check pH occasionally until it is stable at pH = 1 ± 0.05 for five minutes; and (xiv) once the pH is stable, remove the flask from mixer and cover with Parafilm and let the mixture sit unstirred until precipitated humic acid is fallen to the bottom of the flask. Note: The time for humic acid to precipitate from solution varies greatly among products, but a typical time range is 1 to 6 h. (b) Separation of Humic acid.- (i) once the humic acid is completely precipitated, decant the fulvic acid containing extract (fulvic fraction) into a clean one liter Erlenmeyer flask, being careful not to include any of the humic acid precipitate and with some products, the precipitated humic acid will remain in suspension as colloidal particles, if so centrifuge the entire volume to separate the flocculated humic acid from the acidified fulvic fraction; (ii) pour the remaining mixture into centrifuge tubes and centrifuge at 3900 × g for 0.5 hours to separate the humic acid precipitate; and Note: If necessary, a higher g force or longer centrifugation time may be used to obtain a clean separation of the fulvic extract supernatant from the humic acid precipitate. (iii) add the supernatant to the fulvic acid containing acidified extract. (c) Determination of Humic acid concentration.- (i) place the centrifuge tubes containing the precipitated humic acid in a drying oven set at 90°C, and dry the humic acid to constant weight (typically twenty four hours), constant weight is achieved when the tube and humic acid (or fulvic acid) weigh the same after an additional two hours drying time; and (ii) after drying, remove the tubes from the drying oven and place in a desiccator to cool to room temperature and after cooling, quantitatively transfer the residue from the tube by scraping it from the sides and bottom of the tube with a spatula, transfer to a tared weigh boat, and record the mass (W4HA) and this residue is the “Extracted humic acid”. (d) Determination of Ash Content.- (i) transfer the extracted humic acid to a pre-weighed (Wt2) ceramic dish that had been previously dried in a drying oven set at 90°C and then cooled in a desiccator to room temperature; (ii) record the combined mass of the extracted humic acid and dish (W5) and transfer the dish to a muffle oven for four hours at 500°C for combustion; (iii) while still warm, remove the dish and contents from the muffle oven and place in a desiccator to cool and once cool, weigh the dish with ash (W6) and calculate the ash ratio by the following equation Ash ratio = [(W5 – W6)/(W5 – Wt2)]; and (iv) determine the final mass of the extracted humic acid by correcting for ash content using following equation weight of extracted humic acid without ash (gram) = (W4HA)(1 – Ash Ratio). (e) Separation of Fulvic Acid.- (i) to separate fulvic acid from the other acid-soluble compounds in the fulvic fraction by using a 40 × 250 mm glass column prepared with a nonionic macroporous acrylic ester resin (that is, Supelite DAX-8); Note: Through selective adsorption, hydrophilic acid-soluble components do not bind to the resin and are removed. (ii) pass the fulvic fraction through the column using a peristaltic pump, under low pressure, via the top of the column and it is critical that the top of the resin in the column remains covered with solution until all the extract has been added to prevent drying of the resin; (iii) once the fulvic fraction has been completely loaded onto the resin, wash the resin with deionized water by pumping it through the top of the column using the peristaltic pump under low pressure and discard the effluent; (iv) wash the column until the absorbance at 350 nm of the column effluent is equal (e.g., within 0.015 absorbance units) to that of the deionized water used to wash the column, a wavelength of 350 nm gives strong absorbance by fulvic acid and allows the use of a spectrophotometer that only measures visual wavelengths; (v) desorb the fulvic acid by back elution (that is, influent introduced into the bottom of column) by pumping 0.1 M Sodium hydroxide using the peristaltic pump, most of the fulvic acid is adsorbed to the very top of the DAX-8 resin and desorption from the column bottom uses a minimal amount of 0.1 M Sodium hydroxide to fully desorb the fulvic acid; (vi) all the fulvic acid has been desorbed when the absorbance of the column effluent is equal to the absorbance of influent at 350 nm, use 0.1M Sodium hydroxide as the spectrophotometric blank and add the effluent taken to check absorbance of the desorbed fulvic acid solution; (vii) protonate and de-ash the fulvic acid by passing repeatedly (by gravity feed) through Amberlite IR120 hydrogen form ion exchange resin contained in a 5 × 50 cm column until the electrical conductivity of the effluent is <120 uS/m as measured with a conductivity meter; (viii) to ensure that all the fulvic acid is removed from the resin after the final pass, wash the column with deionized water until the absorbance of effluent at 350 nm is the same (e.g., within 0.015 absorbance units) as the deionized water used to wash the column, use deionized water as the spectrophotometric blank and add the wash and any effluent portions taken to check absorbance to the purified fulvic acid solution; (ix) to help with removal of all fulvic acid, the resin to be agitated (e.g., using a long glass or plastic rod) several times; (x) concentrate the fulvic acid to a volume of approximately 15 ± 2 ml by using a rotary evaporator at 55°C; (xi) completely transfer the 15 ml fulvic acid concentrate to a 50 ml plastic centrifuge tube and dry at 90°C to constant dryness in a drying oven (freeze drying is an alternative to oven drying); and (xii) after drying, as described for the humic acid, place the tube in a desiccator to cool and remove fulvic acid from the tube by complete scraping of the tube sides and bottom with a spatula, and weigh it on pretared weigh paper (W8) and this material is the “Extracted Fulvic acid”; (xiii) determine the residual ash content of extracted fulvic acid as described for humic acid and calculate the ash ratio; and (xiv) finally, determine the weight of the extracted Fulvic acid without ash using following equation: Weight of extracted fulvic acid without ash (gram) = (W4FA) (1-Ash Ratio). (f) Column Regeneration.- (i) regenerate the DAX-8 resin by pumping 0.1M hydrochloric acid [8.33 ml concentrated hydrochloric acid per 1000 ml final volume deionized (DI) water] through the bottom of the column until the pH of the effluent is equal to the pH of the influent, use the peristaltic pump to pump all reagents through the DAX-8 column during regeneration; (ii) next, rinse the column with deionized water by pumping it into the top of the column until the pH of the effluent equals the pH of the influent (that is of deionized water); (iii) regenerate the H+ form cation exchange resin in a batch process by pouring the resin into a large beaker (e.g., 4 L plastic beaker), pour off the water, and cover the resin with 1M hydrochloric acid [83.3 ml concentrated HCl per 1000 ml final volume deionized (DI) water]; (iv) let stand for a minimum of thirty minutes with occasional stirring(e.g., once every five minutes and remove the excess acid from the resin by pouring off the acid and covering the resin with deionized water; (v) stir vigorously with a stirring rod for fifteen seconds, then let the resin and rinse water sit for five minutes, repeat the process until the pH of the rinse water equals the pH of the deionized water; and (vi) load the regenerated resin back into the column, once loaded, rinse the resin with deionized water and check the pH of the effluent and if it is still lower than the pH of the deionized water before it is passed through the column, continue to rinse the column with deionized water until the pH of the effluent is within 0.1 pH units of the pH of the influent. 12.4 Reference.- Lamar RT, Olk DC, Mayhew L and Bloom PR 2014, A New Standardized Method for Quantification of Humic and Fulvic Acids in Humic Ores and Commercial Products. Journal of AOAC International, 97 (3): 721- 730. 13. ESTIMATION OF TOTAL CONTENT OF FREE AMINO ACIDS.- 13.1 Equipment.- (a) water bath or dry block thermostat. (b) spectrophotometer. (c) centifuge. (d) analytical balance. 13.2 Chemicals and Reagent Preparation.- (a) ninhydrin (CAS No 485-47-2). (b) hydrindantin (CAS No 5103-42-4). (c) glycine (CAS No 56-40-6). (d) acetic acid (CAS number: 64-19-7). (e) potassium acetate (CAS number: 127-08-2). (f) dimethyl sulfoxide (CAS number: 67-68-5). (g) 2-Propanol (CAS number: 67-63-0). (h) distilled water. (i) 2-Propanol/water = 1/1 (v/v) (mix 500 ml 2-propanol with 500 ml water). (j) acetate buffer- Dissolve 98.1 gram of potassium acetate and 111 ml of glacial acetic acid in water to a final volume of 500 ml. (k) ninhydrin reagent- Dissolve 550 mg ninhydrin and 22 mg hydrindantin in 11 ml DMSO and mix the solution with 11 ml of acetate buffer, use the reagent directly after preparation. (l) glycine Standard solution (10000 mg/ml)- weigh 100 mg of glycine standard and place it in 10 ml volumetric flask, dissolve it in small quantity of distilled water and make up the volume to 10 ml with distilled water. (m) sample solution- take 1gram of sample to be analysed in 100 ml of volumetric flask, dissolve it in small quantity of distilled water, make up the volume to 100 ml with distilled water, mix the contents thoroughly by shaking for half an hour and dilute the sample, if required and use the dilution factor (DF) in the calculation. 13.3 Procedure.- (i) pipette out different volumes (10 µl, 20 µl, and so on) of the glycine standard solution (10000 ppm) into a series of test tubes and make up the volume to 1 ml with distilled water and it gives working standard concentration of 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 mg/ml); (ii) take 200 µl of sample or standard in 1.5 ml reaction tubes and add 800 µl of Ninhydrin reagent to it; (iii) centrifuge the tubes for ten seconds to ensure that no drops of mixture remain in the caps of the tubes; (iv) cover the tubes with caps on top and incubate the tubes in dry block thermostat or in a water bath at 90oC for forty five minutes; (v) cool the tubes to room temperature and transfer exactly 500 µl to a 3 ml cuvette and add 2.5 ml of mixture of 2-propanol/water in 1:1 ratio (v/v) to it and mix the contents well; (vi) measure the optical density of the solutions at 570 nm against a blank; (vii) prepare a standard curve of absorbance against amino acid concentration; and (viii) determine the amount of amino acid in the unknown sample by plotting a standard curve of A570 on the Y-axis and concentration of amino acid on the X-axis. (ix) 13.4 Calculations.- Calculate the total amount of free amino acids using following formula Concentration (mg/kg) = Concentration by graph (mg/L) x Make volume x DF (if any) Weight of sample (g) For result in % = Concentration (mg/kg) 10000 13.5 Reference.- Stauß, A.C., Fuchs, C., Jansen, P., Repert, S., Alcock, K., Ludewig, S., Rozhon, W. 2024. The ninhydrin reaction revisited : optimization and application for quantification of free amino acids, Molecules, 29, 3262. 14. Estimation of free amino acid analysis (for specific amino acid).- Amino acid analysis using HPLC with a fluorescence detector and AccQ•Tag Ultra-2A reagent involves a method where amino acids from a sample are first chemically derivatized (labeled) with the AccQ•Tag Ultra 2A reagent and derivatization allows for highly sensitive detection of each amino acid by fluorescence when separated on an HPLC column, enabling accurate quantification of the individual amino acids present in the sample. 14.1 Equipment.- (a) UHPLC system with FLD (Fluorescence detector), alternative equipment may be used for this method but will require adapting the separation gradient to ensure separation of all compounds. (b) chromatography column: C18, 3.9 mm × 150 mm x 4µ (particle size). (c) adjustable micropipets (10, 20, 200, and 1000μl) and tips. (d) vortex mixer. (e) analytical balance. (f) heating block. (g) laboratory oven. 14.2 Chemicals and Reagent preparation.- (a) AccQ•Tag Ultra Derivatization kit- to prepare the reagents included in the kit following the manufacturer’s instructions: (1) AccQ•Tag Ultra Borate buffer (reagent 1), ready-to-use solution, alternative reagent: 5% (w/v) sodium tetraborate in water; and (2) AccQ•Tag Ultra reagent (vials 2A and 2B), reconstitute AccQ•Tag Ultra reagent (vial 2A) according to following the kit manufacturer’s instructions: (i) preheat a heating block to 55°C; (ii) tap vial 2A lightly before opening to ensure all AccQ•Tag Ultra reagent powder is at the bottom of the vial; (iii) rinse a clean micropipette by drawing and discarding 1 ml AccQ•Tag Ultra reagent diluent from vial 2B (ready-to-use solution), repeat two times; (iv) draw 1.0 ml from vial 2B, transfer it to the AccQ•Tag Ultra reagent powder in vial 2A and cap the vial tightly, mix on a vortex mixer for approximately ten seconds, then heat vial 2A on top of the preheated heating block at 55°C until the AccQ•Tag Ultra reagent powder is dissolved; and Note: Do not heat the reagent for longer than ten minutes. (v) once reconstituted, the AccQ•Tag Ultra reagent is approximately 10 mM. Store reconstituted AccQ•Tag Ultra reagent in a desiccator at room temperature for up to one week. Note: AccQ•Tag Ultra reagent reacts with atmospheric moisture, seal the container tightly when not in use and do not refrigerate. (b) amino acid standard solution.- Containing the following seventeen amino acids at 2.5 μmol/ml (or 2.5 mM) each (except L-cystine at 1.25 μmol/ml) L-alanine, L-arginine, L-aspartic acid, Lcystine, L glutamic acid, L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L phenylalanine, L-proline, L serine, L-threonine, L-tyrosine, and L-valine. Store 2.5 mM calibration standard stock solution at –20°C for up to six months as 250 μL aliquots. (i) 0.5 mM amino acid solution.- Add 600 μL 0.1M hydrochloric acid to 150 μL 2.5 mM amino acid solution. (ii) 0.05 mM amino acid solution.—Add 900 μL 0.1 M hydrochloric acid to 100 μL 0.5 mM amino acid solution. Note: Both 0.5 mM and 0.05 mM amino acid solutions are to be freshly prepared for each analysis. (c) chromatography solvents (mobile phases)- (i) Eluent A (Solvent A), to prepare Eluent A from AccQ•Tag Ultra Eluent A (ammonium formate eighty four per cent, formic acid six per cent, acetonitrile ten per cent, by volume) concentrate as follows: (1) Measure 850 ml water into a 1 L graduated cylinder; (2) In a separate graduated cylinder, measure 150 ml AccQ•Tag Ultra Eluent A concentrate; and (3) add the concentrate to the water and mix thoroughly. Note: Eluent A concentrate, once opened, must be stored tightly capped at around 4°C. Dilute Eluent A is stable for 1 week at room temperature. (ii) Eluent B (Solvent B), to prepare AccQ•Tag Eluent B (acetonitrile with addition two per cent of formic acid, by volume) is supplied as a working solution; no additional preparation is required Note: Eluent B, once opened, must be stored tightly capped at around 4°C for no longer than one month, for Alternative Eluent B, use HPLC grade acetonitrile supplemented with two percent (w/w) formic acid. (d) wash solvents- (i) The weak needle wash solvent is five percent (v/v) acetonitrile in water; (ii) The strong needle wash solvent is ninety five percent (v/v) acetonitrile in water; and (iii) The seal wash solvent is fifty per cent (v/v) acetonitrile in water. (e) sample preparation and neutralization.- (i) weigh about 1 gram (±10 %) of sample in 10 ml volumetric flask and make up the volume to 10 ml, sonicate for ten minutes; (ii) transfer 0.2 ml of sample into a 1.5 ml microcentrifuge tube and add 0.2 ml 6M sodium hydroxide solution and then 0.4 ml 0.1M hydrochloric acid, mix well and filter through a 0.45µ membrane filter into another 1.5ml tube. (f) Derivatisation (of samples and amino acids standards).- Derivatisation converts free amino acids into highly stable derivatives, standards and samples are derivatized following process as described below: (i) preheat a heating block to 55°C; (ii) with a micropipette, add 70 μl AccQ•Tag Ultra Borate buffer to a clean 12 × 32 mm glass screw neck total recovery vial; (iii) add 10 μl calibration standard, neutralized sample solution to the vial; (iv) mix briefly on a vortex mixer; (v) add 20 μl reconstituted AccQ•Tag Ultra reagent to the sample vial; (vi) mix the solution immediately by pipetting up and down several times; (vii) cap and mix on a vortex mixer immediately for several seconds, and tap the vial to ensure that no bubble is trapped; (viii) let stand for one minute at room temperature; and (ix) heat the vial in a heating block for ten minutes at 55 ± 1°C. (g) Ultra-High-Performance Liquid Chromatography (UHPLC) separation.- (i) prime solvent lines for five minutes; (ii) prime wash sample syringes for four cycles; (iii) allow the chromatographic system to stabilize before injecting standards and samples, make sure the system pressure and initial conditions are stable before performing injections (around 9000 psi); (iv) before starting a series of analyses, inject two blanks (water) to condition the column; (v) inject 1μl each derivatised calibration standard and then inject 1μl derivatised sample solutions; (vi) perform single injections and add a blank injection (water) at the end of each calibration series; and (vii) perform the Ultra-High-Performance Liquid Chromatography (UHPLC) under the conditions specified in the Table below and operating conditions may vary depending on the apparatus (follow the instruction manual): Table (Chromatographic Conditions) +-------+----------------------+----------------------+ | (i) | Instrument | HPLC | +-------+----------------------+----------------------+ | (ii) | Detector | FLD (Fluorescence detector)| +-------+----------------------+----------------------+ | (iii) | Column | Waters Nova-Pak C18, 150 mm x 3.9 mm, 4 µ | | | | (particle size) | +-------+----------------------+----------------------+ | (iv) | Column temperature | 30oC | +-------+----------------------+----------------------+ | (v) | Flow rate | 1 ml/minute | +-------+----------------------+----------------------+ | (vi) | Pump mode | Gradient | +-------+----------------------+----------------------+ | (vii) | Run time | 60 minutes | +-------+----------------------+----------------------+ | (viii)| Wavelength | Excitation- 250 nm and Emission- 395 nm| +-------+----------------------+----------------------+ | (ix) | Injection volume | 5 µl | +-------+----------------------+----------------------+ Table (Gradient program) +-----+----------+-----------+-----------+ |S. No|Time |Per cent A |Per cent B | +-----+----------+-----------+-----------+ | (i) |0.00 |99 |1 | +-----+----------+-----------+-----------+ | (ii)|0.50 |98 |2 | +-----+----------+-----------+-----------+ | (iii)|14 |93 |7 | +-----+----------+-----------+-----------+ | (iv)|18 |90 |10 | +-----+----------+-----------+-----------+ | (v) |21 |90 |10 | +-----+----------+-----------+-----------+ | (vi)|33 |78 |22 | +-----+----------+-----------+-----------+ | (vii)|39 |77 |23 | +-----+----------+-----------+-----------+ | (viii)|42 |67 |33 | +-----+----------+-----------+-----------+ | (ix)|49 |67 |33 | +-----+----------+-----------+-----------+ | (x) |50 |30 |70 | +-----+----------+-----------+-----------+ | (xi)|52 |30 |70 | +-----+----------+-----------+-----------+ | (xii)|53 |99 |1 | +-----+----------+-----------+-----------+ | (xiii)|60 |99 |1 | +-----+----------+-----------+-----------+ (h) Peak identification and integration.- (i) identify the amino acid peaks in the sample solution by comparison with the retention times of the corresponding peaks obtained in the calibration standards; and (ii) check that peaks are separated with a good resolution (baseline separation) and if this is not the case, adapt the chromatographic conditions (e.g., gradient, temperature, tubing length, etc.) accordingly. 14.3 Calculation.- Integrate the peak responses observed in the HPLC for sample as well as standard and calculate each Amino acid by using the formula: Amino acid in mg/gram= Sample Area x Standard concentration Standard Area x Sample weight Amino acid % (w/w) = Amino acid in mg/g x 100 1000 14.4 Reference.- Jaudzems G and J Guthrie J 2019. Total Amino Acids by UHPLC–UV in Infant Formulas and Adult Nutritionals, First Action 2018.06. Journal of AOAC International 102: 1574- 1588. 15. Estimation of total amino acids (from total nitrogen).- (1) protocol detailed as in paragraph 3 in Part B of Schedule II to be followed for estimation of total nitrogen (in nitrate free samples); (2) the total nitrogen estimated as per the above protocol, should be used for estimation of total amino acids in the formulation using the formula; Crude protein (%) = Total nitrogen (estimated by Kjeldahl method) x 6.25 Note: All the amino acids contains 16 % of nitrogen; hence the multiplying factor derived and used as 6.25 (100/16). (3) in the protein hydrolysate sample, 70% of the nitrogen content is from amino acids and after calculating crude protein, calculate the amino acid content as follows.- Amino acids (%) = Crude protein x (70/100 ) 16. Estimation of myo- inositol. - 16.1 Equipment.- (a) HPLC with Column: Amino (250 mm x 4.6mm); 5µ (Particle size). (b) evaporative light scattering detector (ELSD). (c) analytical balance. (d) 0.22µ syringe filter. 16.2 Chemicals and Reagent Preparation.- (a) myo Inositol (CAS No. 87-89-8). (b) acetonitrile(CAS No. 75-05-8). (c) ultrapure water. (d) myo Inositol standard stock preparation: weigh about 50 mg of Inositol standard in 10 ml volumetric flask and dilute it with 10 ml water and it gives a stock solution of 5000 ppm. (e) myo- Inositol intermediate standard solution: Dilute 2.0 ml stock standard to 10 ml with water and mix well. It gives an intermediate standard solution of 1000 ppm. (f) prepare following linearity point as per sample concentration specified in the table below Table +-----+----------+---------------------+------------+------------+---------------------+ |S. No|Linearity |Concentration |Volume (ml) |Volume (ml)|Concentration of | | |standard |of stock solution |of stock |of water to |standard | | | |(ppm) |solution to |be used |(mg/ml) | | | | |be used | | | +-----+----------+---------------------+------------+------------+---------------------+ | (i) |Standard 1|1000 ppm |0.10 |0.90 |100 | +-----+----------+---------------------+------------+------------+---------------------+ | (ii)|Standard 2|1000 ppm |0.25 |0.75 |250 | +-----+----------+---------------------+------------+------------+---------------------+ | (iii)|Standard 3|5000 ppm |0.10 |0.90 |500 | +-----+----------+---------------------+------------+------------+---------------------+ | (iv)|Standard 4|5000 ppm |0.16 |0.84 |800 | +-----+----------+---------------------+------------+------------+---------------------+ | (v) |Standard 5|5000 ppm |0.20 |0.80 |1000 | +-----+----------+---------------------+------------+------------+---------------------+ (g) Sample preparation.- (i) thoroughly mix or stir products prior to sampling; (ii) for liquid products, accurately weigh 0.5 to 5 g (±10%) of product into a 50 ml volumetric flask and record the weight to the nearest 0.0001 gram; (iii) for powdered products that do not require reconstitution, accurately weigh 0.25 to 2.0 g powder into a 50 ml volumetric flask and record the weight to the nearest 0.0001 gram; (iv) add approximately 10 to 15 ml methanol to 50 ml volumetric flask and swirl or stir to completely dissolve the powder and for powdered products that are not homogeneous at the sub-gram level, reconstitute following the product label instructions and accurately weigh 0.5 to 5 g reconstituted product into a 50 ml volumetric flask and record the weight to the nearest 0.0001 gram; (v) make up the volume to 50 ml with methanol; and (vi) sonicate it for thirty minutes and filter it through 0.22µ syringe filter. 16.3 Procedure.- (i) HPLC analysis.- following as specified in table below for instrument operating conditions and ELSD settings.- Table (HPLC Conditions) +-------+---------------------------------+----------------------+ | (i) | Column | Amino (250 mm x 4.6mm); 5 µ | | | | (Particle size) | +-------+---------------------------------+----------------------+ | (ii) | Detector | Evaporative light scattering Detector | | | | (ELSD) | +-------+---------------------------------+----------------------+ | (iii) | Detector temperature | 50oC | +-------+---------------------------------+----------------------+ | (iv) | ELSD gain | 6 | +-------+---------------------------------+----------------------+ | (v) | ELSD Chamber temperature | 40oC | +-------+---------------------------------+----------------------+ | (vi) | Pressure of nitrogen in nebulizer | 2.9 bar | +-------+---------------------------------+----------------------+ | (vii) | Flow rate | 2 ml/ min. | +-------+---------------------------------+----------------------+ | (viii)| Column temperature | 25oC | +-------+---------------------------------+----------------------+ | (ix) | Mobile phase | A- Acetonitrile (80%) | | | | B- Water (20%) | +-------+---------------------------------+----------------------+ | (x) | Run time | 20 minutes | +-------+---------------------------------+----------------------+ | (xi) | Injection volume | 5 µl | +-------+---------------------------------+----------------------+ 16.4 Calculation.- Myo Inositol (mg/kg)= sample area x standard concentration x dilution standard area x Sample weight 16.5 Reference.- Pazourek J. 2014. Fast separation and determination of free myo-inositol by hydrophilic liquid chromatography. Carbohydrate Research, 391: 55-60. 17. Estimation of Vitamin C (ascorbic acid)- 17.1 Equipment- (a) HPLC with Column: C18 (250 mm x 4.6mm); 5 µ (Particle size). (b) UV detector. (c) analytical balance. (d) 0.22 µ syringe filter. 17.2 Chemicals and Reagent Preparation- (a) ascorbic acid. (b) 20mM Monobasic phosphate buffer: Dissolve 2.76g of sodium dihydrogen phosphate (NaH2PO4) in approximately 800ml of distilled water in a one liter volumetric flask, adjust the pH to 8.5 using orthophosphoric acid, and finally dilute to the one liter mark with distilled water. (c) ortho phosphoric acid. (d) methanol. (e) acetonitrile. (f) methanol: acetonitrile (60: 40). (g) ultrapure water. (h) ascorbic acid stock standard solution: Weigh about 10 mg of Ascorbic acid standard in 10 ml volumetric flask and dilute it with 10 ml mobile phase, it gives a stock solution of 1000 mg/ml. (i) ascorbic acid intermediate standard solution: Dilute 1.0 ml stock standard to 10 ml with mobile phase and mix well, it gives an intermediate standard solution of 100 ppm and from this standard solution, dilute 1.0 ml of intermediate standard solution to 10 ml with mobile phase and it gives an intermediate standard solution of 10 ppm. (j) prepare linearity point as per sample concentration specified in the table below. Table +-----+----------+---------------------+---------------------+---------------------+---------------------+ |S. No|Linearity |Conc. of |Volume (ml) |Volume (ml) |Concentration of | | |standard. |stock |of stock |of water to |standard | | | |solution |solution to be |be used. |(mg/ml). | | | |(ppm). |used. | | | +-----+----------+---------------------+---------------------+---------------------+---------------------+ | (i) |Standard 1|10 ppm |0.10 |0.90 |1 | +-----+----------+---------------------+---------------------+---------------------+---------------------+ | (ii)|Standard 2|10 ppm |0.20 |0.80 |2 | +-----+----------+---------------------+---------------------+---------------------+---------------------+ | (iii)|Standard 3|100 ppm |0.05 |0.95 |5 | +-----+----------+---------------------+---------------------+---------------------+---------------------+ | (iv)|Standard 4|1000 ppm |0.10 |0.90 |10 | +-----+----------+---------------------+---------------------+---------------------+---------------------+ | (v) |Standard 5|1000 ppm |0.05 |0.95 |50 | +-----+----------+---------------------+---------------------+---------------------+---------------------+ | (vi)|Standard 6|1000 ppm |0.10 |0.90 |100 | +-----+----------+---------------------+---------------------+---------------------+---------------------+ (k) Sample preparation.- (i) thoroughly mix or stir products prior to sampling; (ii) for liquid products, accurately weigh 0.5 to 5 g (±10%) of product into a 50 ml volumetric flask and record the weight to the nearest 0.0001 g; (iii) for powdered products that do not require reconstitution, accurately weigh 1.0 gram powder into a 50 ml volumetric flask and record the weight to the nearest 0.0001 gram; (iv) add approximately 10 to 15 ml mobile phase to 50 ml volumetric flask and swirl or stir to completely dissolve the powder and for powdered products that are not homogeneous at the sub-gram level, reconstitute following the product label instructions and accurately weigh 1.0 gram reconstituted product into a 50 ml volumetric flask and record the weight to the nearest 0.0001gram; (v) Make up the volume to 50 ml with mobile phase; (vi) sonicate it for 30 minutes and filter it through 0.22µ syringe filter; and (vii) Inject 5µl to HPLC- UV for analysis. 17.3 Procedure (i) HPLC analysis: following instrument operating conditions specified in table below: Table (HPLC Conditions) +-------+----------------------+---------------------+ | (i) | Column | C18(250 mm x 4.6mm); 5 mg (Particle size)| +-------+----------------------+---------------------+ | (ii) | Detector | UV | +-------+----------------------+---------------------+ | (iii) | Detecting | 230 nm | | | wavelength | | +-------+----------------------+---------------------+ | (iv) | Flow rate | 1.0 ml/ min. | +-------+----------------------+---------------------+ | (v) | Column | 20oC | | | temperature | | +-------+----------------------+---------------------+ | (vi) | Mobile phase | A- 20 mM Monobasic phosphate buffer in water, adjust pH to | | | | 2.5 with ortho phosphoric acid | | | | B- Methanol: Acetonitrile (60:40) | +-------+----------------------+---------------------+ | (vii) | Run time | 8 minutes | +-------+----------------------+---------------------+ | (viii)| Injection | 5 µl | | | volume | | +-------+----------------------+---------------------+ Table (Gradient) +-----+----------+-----------+ |S. No|Time |% B | +-----+----------+-----------+ | (i) |0 |5 | +-----+----------+-----------+ | (ii)|2.0 |5 | +-----+----------+-----------+ | (iii)|2.1 |25 | +-----+----------+-----------+ | (iv)|13.0 |25 | +-----+----------+-----------+ | (v) |13.1 |90 | +-----+----------+-----------+ | (vi)|18.0 |90 | +-----+----------+-----------+ | (vii)|18.1 |5 | +-----+----------+-----------+ | (viii)|25.0 |5 | +-----+----------+-----------+ 17.4 Calculation.- Vitamin C (mg/kg)= Sample area x Std. concentration x dilution Std. area x Sample weight 17.5 Reference Latef SS 2011. Analysis of ascorbic acid, citric acid and benzoic acid in orange juice. Agilent Application Solution. Agilent Technologies; Published in USA, September 1, 2011. 18. Estimation of tocopherol (Vitamin E).- 18.1 Equipment.- (a) HPLC with Column: C18 (150 mm x 4.6mm); 5µ (Particle size). (b) UV detector. (c) analytical balance. (d) 0.22 µ syringe filter. 18.2 Chemicals and Reagent Preparation- (a) tocopherol(CAS No. 10191-4-0). (b) acetonitrile (CAS No. 75-05-8). (c) methanol (CAS No 67- 56-1). (d) methanol: water (98:2): mix 98 ml of methanol with 2 ml of water. (e) tocopherol (Vitamin E) stock standard solution: weigh about 50 mg of Tocopherol standard in 50 ml Volumetric flask and dilute it with 50 ml of methanol and it gives a stock solution of 1000 ppm. (f) tocopherol intermediate standard solution: dilute 1.0 ml stock standard to 10 ml with methanol and mix well and it gives an intermediate standard solution of 100 ppm and from this standard solution, dilute 1.0 ml of intermediate standard solution to 10 ml with methanol and it gives an intermediate standard solution of 10 ppm. (g) prepare linearity point as per sample concentration specified in the table below. Table +-----+----------+---------------------+---------------------+---------------------+---------------------+ |S. No|Linearity |Conc. of |Volume (ml) |Volume (ml) |Concentration of | | |standard. |stock |of stock |of water to |standard | | | |solution |solution to be |be used. |(mg/ml). | | | |(ppm). |used. | | | +-----+----------+---------------------+---------------------+---------------------+---------------------+ | (i) |Standard 1|10 ppm |0.10 |0.90 |1 | +-----+----------+---------------------+---------------------+---------------------+---------------------+ | (ii)|Standard 2|10 ppm |0.20 |0.80 |2 | +-----+----------+---------------------+---------------------+---------------------+---------------------+ | (iii)|Standard 3|100 ppm |0.05 |0.95 |5 | +-----+----------+---------------------+---------------------+---------------------+---------------------+ | (iv)|Standard 4|1000 ppm |0.10 |0.90 |10 | +-----+----------+---------------------+---------------------+---------------------+---------------------+ | (v) |Standard 5|1000 ppm |0.05 |0.95 |50 | +-----+----------+---------------------+---------------------+---------------------+---------------------+ | (vi)|Standard 6|1000 ppm |0.10 |0.90 |100 | +-----+----------+---------------------+---------------------+---------------------+---------------------+ (h) Sample preparation.- (i) thoroughly mix or stir products prior to sampling; (ii) for liquid products, accurately weigh 50 mg of product into a 50 ml volumetric flask and record the weight to the nearest 0.0001 g; (iii) for powdered products that do not require reconstitution, accurately weigh 1.0 gram powder into a 50 ml volumetric flask and record the weight to the nearest 0.0001 gram; (iv) add approximately 10 to 15 ml methanol to 50 ml volumetric flask and swirl or stir to completely dissolve the powder and for powdered products that are not homogeneous at the sub-gram level, reconstitute following the product label instructions and accurately weigh 1.0 gram reconstituted product into a 50 ml volumetric flask and record the weight to the nearest 0.0001 g; (v) make up the volume to 50 ml with methanol; and (vi) filter it through 0.22 µ syringe filter. 18.3 Procedure.- (a) HPLC analysis: following instrument operating conditions specified in table below: Table (HPLC Conditions) +-------+-----------------------+---------------------+ | (i) | Column | C18 (150 mm x 4.6mm); 5 µ (Particle size).| +-------+-----------------------+---------------------+ | (ii) | Detector | UV. | +-------+-----------------------+---------------------+ | (iii) | Detecting wavelength | 292 nm. | +-------+-----------------------+---------------------+ | (iv) | Flow rate | 1.0 ml/ minutes | +-------+-----------------------+---------------------+ | (v) | Column temperature | 25oC. | +-------+-----------------------+---------------------+ | (vi) | Mobile phase | 98% Methanol : 2% Water.| +-------+-----------------------+---------------------+ | (vii) | Run time | 20 minutes. | +-------+-----------------------+---------------------+ | (viii)| Approximate retention time| 10- 15 minutes. | +-------+-----------------------+---------------------+ | (ix) | Injection volume | 20 ml. | +-------+-----------------------+---------------------+ 18.4 Calculation.- Vitamin E (mg/kg)= Sample area x standard concentration x dilution standard area x Sample weight 18.5 Reference.- Brabcová I, Kovářová L, Šatínský D, Havlíková L and Solich P, 2013, fast HPLC method for determination of Vitamin E acetate in dietary Supplements using monolithic column. Food Anal. Methods 6: 380–385. 19. Estimation of thiamine hydrochloride (vitamin b1) - 19.1 Equipment.- (a) reverse phase LC-18 column (250 mm × 4.6 mm), 5µ particle size. (b) UV Detector. (c) analytical balance. (d) sonicator. (e) vacuum filtration assembly. (f) 0.22µ syringe filter. (g) micropipettes. 19.2 Chemicals and Reagent Preparation.- (a) pentane sulfonic acid. (b) glacial acetic acid. (c) distilled water. (d) triethylamine. (e) methanol (CAS No 67- 56-1). (f) thiamine hydrochloride. (g) mobile phase. (i) add 1.8 gram pentane sulfonic acid, 5 ml glacial acetic acid, 0.9 ml of triethyl amine and 265 ml of methanol, make up the volume to 1000 ml with distilled water; and (ii) sonicate it for forty-five minutes and vacuum filter the buffer and degas before use. (h) thiamine hydrochloride stock standard solution: weigh about 10 mg of standard in 10 ml volumetric flask and dissolve it in 1.0- 1.5 ml of acetic acid with heating and make up the volume to 10 ml with distilled water and it gives a stock solution of 1000 ppm. (i) thiamine hydrochloride intermediate standard solution: dilute 1.0 ml stock standard to 10 ml with distilled water and mix well and it gives an intermediate standard solution of 100 ppm and dilute 1.0 ml of intermediate standard solution to 10 ml with distilled water and it gives an intermediate standard solution of 10 ppm. (j) prepare the following linearity point as per sample concentration specified in the table below.- Table +-----+----------+---------------------+---------------------+---------------------+---------------------+ |S. No|Linearity |Concentration |Volume (ml) |Volume (ml) |Concentration of | | |standard. |of stock |of stock |of water to |standard | | | |solution (ppm). |solution to be |be used. |(µg/ml). | | | | |used. | | | +-----+----------+---------------------+---------------------+---------------------+---------------------+ | (i) |Standard 1|10 ppm |0.10 |0.90 |1 | +-----+----------+---------------------+---------------------+---------------------+---------------------+ | (ii)|Standard 2|10 ppm |0.20 |0.80 |2 | +-----+----------+---------------------+---------------------+---------------------+---------------------+ | (iii)|Standard 3|100 ppm |0.05 |0.95 |5 | +-----+----------+---------------------+---------------------+---------------------+---------------------+ | (iv)|Standard 4|1000 ppm |0.10 |0.90 |10 | +-----+----------+---------------------+---------------------+---------------------+---------------------+ | (v) |Standard 5|1000 ppm |0.05 |0.95 |50 | +-----+----------+---------------------+---------------------+---------------------+---------------------+ | (vi)|Standard 6|1000 ppm |0.10 |0.90 |100 | +-----+----------+---------------------+---------------------+---------------------+---------------------+ (k) Sample preparation.- (i) thoroughly mix or stir products prior to sampling; (ii) for liquid products, accurately weigh 0.5 to 5 g (±10%) of product into a 50 ml volumetric flask and record the weight to the nearest 0.0001 gram; (iii) for powdered products that do not require reconstitution, accurately weigh 1.0 gram powder into a 50 ml volumetric flask and record the weight to the nearest 0.0001 gram; (iv) add approximately 10 to 15 ml mobile phase to 50 ml volumetric flask and swirl or stir to completely dissolve the powder and for powdered products that are not homogeneous at the sub gram level, reconstitute following the product label instructions and accurately weigh 1.0 gram reconstituted product into a 50 ml volumetric flask and record the weight to the nearest 0.0001 gram; (v) make up the volume to 50 ml with mobile phase; and (vi) sonicate it for thirty minutes and filter it through 0.22µ syringe filter. 19.3 Procedure.- (i) Inject 20 ml of standard or sample to HPLC- UV detector for analysis. (ii) Chromatographic conditions.- (a) use reverse phase C-18 column (250 mm x 4.6 mm x 5 µ); (b) use mobile phase consisting of 1.8 gram pentane sulfonic acid, 5 ml glacial acetic acid, 0.9 ml of triethylamine and 265 ml of methanol and water to make up the volume to 1000 ml for isocratic elution; (c) equilibrate the column at 22 ◦C at the flow rate of 1 ml/minute, use methanol for conditioning the column; (d) filter the mobile phase through a 0.22µ membrane filter under vacuum and degas it before use; (e) use 20 µl for injection into the injector of the HPLC. (iii) HPLC analysis: following instrument operating conditions specified in table below: Table (HPLC Conditions) +-------+---------------------------------+---------------------+ | (i) | Column | C18 (250 mm x 4.6mm); 5µ (Particle size).| +-------+---------------------------------+---------------------+ | (ii) | Detector | UV. | +-------+---------------------------------+---------------------+ | (iii) | Detecting wavelength | 230 nm. | +-------+---------------------------------+---------------------+ | (iv) | Flow rate | 1.0 ml/ minute. | +-------+---------------------------------+---------------------+ | (v) | Column temperature | 40oC. | +-------+---------------------------------+---------------------+ | (vi) | Mobile phase | Isocratic elution with mobile phase consisting of | | | | 1.8 gram pentane sulfonic acid, 5 ml glacial | | | | acetic acid, 0.9 ml of triethyl amine and 265 ml | | | | of methanol, make up the volume to 1000 ml | | | | with distilled water. | +-------+---------------------------------+---------------------+ | (vii) | Run time | 8 minutes. | +-------+---------------------------------+---------------------+ | (viii)| Injection volume | 20µl | +-------+---------------------------------+---------------------+ 19.4 Calculation.- Vitamin B1 (mg/kg) = Sample area x standard concentration x dilution standard area x sample weight 19.5 Reference.- Marszałł ML, Lebiedzi´nska A, Czarnowski W and Szefer P, 2005., High-performance liquid chromatography method for the simultaneous determination of thiamine hydrochloride, pyridoxine hydrochloride and cyanocobalamin in pharmaceutical formulations using coulometric electrochemical and ultraviolet detection, Journal of Chromatography A, 1094: 91–98 20. Estimation of citric acid - The method used for estimation of ascorbic acid as detailed in paragraph 16 can be used for estimation of citric acid except for standard preparation. The standard preparation is as follows.- (i) the Citric acid stock standard solution: Weigh about 10 mg of citric acid standard in 10 ml Volumetric flask and dilute it with 10 ml mobile phase and it gives a stock solution of 1000 ppm. (ii) prepare linearity point as per sample concentration specified in the table below.- Table +-----+----------+---------------------+---------------------+---------------------+---------------------+ |S. No|Linearity |Concentration |Volume (ml) |Volume (ml) |Concentration of | | |standard. |of stock |of stock |of water to |standard | | | |solution |solution to be |be used. |(µg/ml). | | | |(ppm). |used. | | | +-----+----------+---------------------+---------------------+---------------------+---------------------+ | (i) |Standard 1|1000 ppm |1.000 |0.000 |1000 | +-----+----------+---------------------+---------------------+---------------------+---------------------+ | (ii)|Standard 2|1000 ppm |0.750 |0.250 |750 | +-----+----------+---------------------+---------------------+---------------------+---------------------+ | (iii)|Standard 3|1000 ppm |0.500 |0.500 |500 | +-----+----------+---------------------+---------------------+---------------------+---------------------+ | (iv)|Standard 4|1000 ppm |0.250 |0.750 |250 | +-----+----------+---------------------+---------------------+---------------------+---------------------+ | (v) |Standard 5|1000 ppm |0.125 |0.875 |125 | +-----+----------+---------------------+---------------------+---------------------+---------------------+ | (vi)|Standard 6|1000 ppm |0.100 |0.900 |100 | +-----+----------+---------------------+---------------------+---------------------+---------------------+ Reference.- Coppola ED and Starr MS 1986. Liquid Chromatographic Determination of Major Organic Acids in Apple Juice and Cranberry Juice Cocktail: Collaborative Study. J. Assoc. Off. Anal. Chem. 69: 594- 597. 21. Estimation of α-amylase activity.- The assay is based on the time required to obtain a standard degree of hydrolysis of a starch solution at 30 ± 0.1°C and the degree of hydrolysis is determined by comparing the iodine colour of the hydrolysate with that of a standard. 21.1 Equipment.- (a) spectophotometer. (b) analytical balance. (c) fume hood. 21.2 Chemicals and Reagent Preparation.- (a) cobaltous chloride. (b) potassium dichromate. (c) hydrochloric acid. (d) potassium dihydrogen phosphate (KH2PO4). (e) dibasic sodium phosphate (Na2HPO4). (f) iodine. (g) potassium iodide. (h) enzyme α-Amylase. (i) reference colour standard: Prepare a colour standard by dissolving 25.0 gram of cobaltous chloride (COCI26H2O) and 3.84 gram of potassium dichromate in 100 ml of 0.01 N hydrochloric acid and this standard is stable indefinitely when stored in an amber coloured stoppered bottle. (j) phosphate buffer pH 6.6.- (i) solution A: Dissolve 9.1 gram of potassium dihydrogen phosphate (KH2PO4) in sufficient water to make 1000 ml; (ii) solution B: Dissolve 9.5 gram of dibasic sodium phosphate (Na2HPO4) in sufficient water to make 1000 ml; and (iii) add 400 ml of Solution A to 600 ml of Solution B, mix, and adjust the pH to 6.6, if necessary, by the addition of Solution A or Solution B as required. (k) stock iodine solution: Dissolve 5.5 gram of iodine and 11.0 gram of potassium iodide in about 200 ml of water, dilute to 250 ml with water, and mix, store in a dark bottle, and make a fresh solution every thirty days. (l) dilute iodine solution: Dissolve 20 gram of potassium iodide in 300 ml of distilled water, and add 2.0 ml of Stock Iodine Solution, quantitatively transfer the mixture into a 500 ml volumetric flask, dilute to volume with water, and mix, prepare daily. (m) starch substrate solution: Disperse 10.0 gram (dry-weight basis) of starch in 100 ml of cold water, and slowly pour the mixture into 300 ml of boiling water, boil and stir for one to two minutes, and then cool while continuously stirring, quantitatively transfer the mixture into a 500 ml volumetric flask with the aid of water, add 10 ml of phosphate buffer pH 6.6, dilute to volume with water, and mix. (n) enzyme dilution: prepare an enzyme solution so that 10 ml of the final dilution will give an end point between 15- 35 under the conditions of the assay. 21.3 Procedure.- (a) pipet 5.0 ml of dilute iodine solution into a series of glass test tubes, and place them in a water bath maintained at 30 ± 0.1°C; (b) pipet 20.0 ml of the starch substrate solution into a 50 ml Erlenmeyer flask, stopper, and equilibrate for twenty minutes in the water bath at 30°C; (c) at zero time, rapidly pipet 5.0 ml of the sample preparation into the equilibrated substrate, mix immediately by swirling, stopper the flask, and place it back in the water bath; (d) after ten minutes, transfer 1.0 ml of the reaction mixture from the 50 ml flask into one of the test tubes containing the dilute iodine solution, shake the tube; (e) mix the solution and compare its OD against the OD of the reference colour at 660 nm against distilled water as blank and when the reaction nears the end point, an aliquot should be withdrawn at a regular interval of one minute for comparison with the reference colour standard OD and the end point is calculated by considering the OD values of the two aliquots withdrawn (2 minutes apart when the reaction nears end point); (f) wherein one aliquot has OD higher than the reference colour OD and the other has OD lower than the reference colour OD) at 660 nm. Note: The reaction time is noted at the end point and Be certain that the pipet tip does not touch the iodine solution as carry-back of iodine to the hydrolyzing mixture will interfere with enzyme action and will affect the results of the determination. 21.4 Calculations.- (i) One amylase unit (AU) is defined as that quantity of enzyme that will dextrinize starch at the rate of 1 mg/minute under the specified test conditions; (ii) Calculate the α-amylase activity of the sample, expressed as AU, by the formula AU/g= (40 x F)/ T Wherein, 40 is a factor (400/10) derived from the 400 mg of starch in (20 ml of 2% solution) and the 10 ml of aliquot of sample preparation used F is the dilution factor (total dilution volume/sample weight, in grams) and T is the dextrnising time in minutes and is calculated as Calculation at seconds (T)= (Higher test OD- Reference colour Standard OD)/60 (Higher test OD- Lower test OD)/60 21.5 Reference.- α-Amylase Activity; FAO JECFA monographs; combined compendium of food additive specifications; joint FAO/WHO expert committee on food additives; Vol 4 analytical methods, test procedures and laboratory solutions used by and referenced in the food additive specifications pp 119- 120. ISSN 1817- 7077. 22. Estimation of protease activity - This procedure is used to determine protease activity, expressed as PC units, the assay is based on a thirty minutes proteolytic hydrolysis of casein at 37°C and pH 7.0, unhydrolyzed casein is removed by filtration, and the solubilized casein is determined spectrophotometrically. 22.1 Equipment.- (a) water bath. (b) spectrophotometer. (c) pH Meter. (d) analytical Balance. (e) automatic Pipetters. (f) timer. (g) magnetic stirrer. 22.2 Chemicals and Reagent Preparation (a) tris (hydroxymethyl) aminomethane. (b) trichloroacetic acid. (c) sodium acetate trihydrate. (d) glacial acetic acid. (e) hydrochloric acid. (f) casein. (g) protease enzyme. (h) tris buffer (pH 7.0): Dissolve 12.1 gram of enzyme-grade (or equivalent) tris (hydroxymethyl) aminomethane in 800 ml of water, and titrate with 1N hydrochloric acid to pH 7.0 and transfer into a 1,000-ml volumetric flask, dilute to volume with water, and mix. (i) TCA solution: Dissolve 18 gram of trichloroacetic acid and 19 gram of sodium acetate trihydrate in 800 ml of water in a 1,000-ml volumetric flask, add 20 ml of glacial acetic acid, dilute to volume with water, and mix. (j) substrate solution: Dissolve 6.05 gram of tris (hydroxymethyl) aminomethane (enzyme grade) in 500 ml of water, add 8 ml of 1N hydrochloric acid, and mix, dissolve 7 gram of Casein in this solution, and heat for 30 minutes in a boiling water bath, stirring occasionally and cool to room temperature, and adjust to pH 7.0 with 0.2N hydrochloric acid, adding the acid slowly, with vigorous stirring, to prevent precipitation of the casein and transfer the mixture into a 1,000-ml volumetric flask, dilute to volume with water, and mix. (k) sample preparation: Using Tris Buffer, prepare a solution of the sample enzyme so that 2 ml of the final dilution will contain between 10 and 44 PC units. (l) tyrosine standard preparation: Transfer 100 mg of Tyrosine, previously dried at 105oC for one hour to 1000 ml volumetric flask and dissolve in 60 ml of hydrochloric acid and make up the volume with distilled water and the stock solution contains 100 mg of tyrosine in 1.0 ml and prepare three more dilutions to contain 75, 50 and 25µg of tyrosine per ml as specified in the table below.- +-----+-------------+----------------+----------------+---------------------+ |S. No|Tube |ml of stock |ml of distilled |Concentration | | |number. |solution. |water. |(mg /ml). | +-----+-------------+----------------+----------------+---------------------+ | (i) |S1 |2.5 |7.5 |25 | +-----+-------------+----------------+----------------+---------------------+ | (ii)|S2 |5.0 |5.0 |50 | +-----+-------------+----------------+----------------+---------------------+ | (iii)|S3 |7.5 |2.5 |75 | +-----+-------------+----------------+----------------+---------------------+ | (iv)|S4 |10.0 |0.0 |100 | +-----+-------------+----------------+----------------+---------------------+ 22.3 Procedure - (a) pipet 10.0 ml of the Substrate Solution into test tubes and keep three replicate tubes for enzyme test samples (mark as T1, T2 and T3), and one tube for blank (mark as B). (b) equilibrate the tubes for fifteen minutes in a water bath maintained at 37+ 0.1° and following equilibration to be followed, (i) rapidly add 2.0 ml of the sample preparation into the tubes with equilibrated substrate solution (T1, T2 and T3) at zero time, mix the content and place the tubes back to water bath maintained at 37+ 0.1°; and (ii) to blank tube (B), add 2 ml of Tris buffer instead of sample preparation, mix the content and place the tubes back to water bath maintained at 37+ 0.1°. (c) after exactly thirty minutes of incubation in water bath maintained at 37+ 0.1°, add 10 ml of TCA solution in all the tubes (T1, T2, T3 and B) to stop the reaction; (d) for Blank tube (B), add 2 ml of sample preparation after adding TCA, mix the contents rapidly; (e) place all the tubes back into the water bath for an additional 30 minutes at 37+ 0.1° to allow the protein to coagulate completely; (f) at the end of second heating period, shake each tube vigorously and filter through Whatman No. 42 or equivalent and discard the first 3 ml of filtrate. Collect the perfectly clear filtrate; (g) determine the absorbance of each sample filtrate instrument at zero and obtain the net absorbance by subtracting blank OD from test OD; (h) procedure is summarized in the table below: Table +-------+-------------------------------------------------+-------------+ | (i) | Test | Blank | +-------+-------------------------------------------------+-------------+ | (ii) | Substrate solution | 5.0 ml | +-------+-------------------------------------------------+-------------+ | (iii) | Equilibrate for 15 min in a water bath maintained at 37+ 0.1° | | +-------+-------------------------------------------------+-------------+ | (iv) | Then add: | | +-------+-------------------------------------------------+-------------+ | (v) | Enzyme solution | 2.0 |0.0 | +-------+-------------------------------------------------+-------------+-------------+ | (vi) | Mix by swirling and incubate in a water bath maintained at 37+ 0.1° C for 30 minutes| | +-------+-------------------------------------------------+-------------+-------------+ | (vii) | Then add: | | | +-------+-------------------------------------------------+-------------+-------------+ | (viii)| TCA reagent | 10 ml |10 ml | +-------+-------------------------------------------------+-------------+-------------+ | (ix) | Enzyme solution | 0.0 |2.0 ml | +-------+-------------------------------------------------+-------------+-------------+ | (x) | Mix by swirling and incubate at 37°C for about 30 minutes| | | +-------+-------------------------------------------------+-------------+-------------+ | (xi) | Filter through Whatman No. 42 or equivalent | | | +-------+-------------------------------------------------+-------------+-------------+ | (xii) | Measure absorbance of sample against blank at 275 nm| | | +-------+-------------------------------------------------+-------------+-------------+ 22.4 Calculations Unit Definition (PC): One Protease Unit (PCU) is defined as the quantity of enzyme that produce the equivalent of 1 µg/ml of tyrosine per minute under the conditions of the assay, calculate PC activity using formula: Activity (PC/g) = Tyrosine concentration x 22/ 30 x W Where: tyrosine concentration in the sample is obtained using the standard curve; 22 is the total volume of the reaction mixture (ml); 30 is the reaction time in minutes; and W is the weight of the original sample taken in gram. 22.5 Reference Protease Activity; FAO JECFA monographs; combined compendium of food additive specifications; joint FAO/WHO expert committee on food additives; Vol 4 analytical methods, test procedures and laboratory solutions used by and referenced in the food additive specifications p 147. ISSN 1817- 7077. 23. Estimation of lipase activity The method is based on the speed at which the enzyme hydrolyzes tributyrin at pH 7.5, the butyric acid, which is formed, is titrated with sodium hydroxide and the consumption of the latter recorded as a function of time. 23.1 Equipment- (a) pH stat titration system: Use an instrument operating in the pH Stat mode and equipped with a jacketed titration cell (Radiometer Titralab or equivalent), the instrumental assembly is composed of a thermoelectric temperature-regulating magnetic stirrer, a motor-driven burette, an automatic recorder, and a pH meter and the electrical impulses from the pH meter is transferred to the recorder input, which in turn activate the motor-driven burette through a micro switch to maintain a constant pH of the assay mixture. (b) waterbath. (c) pH electrode. (d) magnetic stirrer. 23.2 Chemicals and Reagent Preparation (a) sodium hydroxide. (b) potassium hydrogen phthalate. (c) sodium chloride. (d) monobasic potassium phosphate. (e) glycerol. (f) gum arabic. (g) tris (hydroxymethyl) aminomethane. (h) tributyrin. (i) 0.05N sodium hydroxide (NaOH): Dissolve 1.0 gram of sodium hydroxide in water and dilute to 500 ml, standardise the normality by titrating with potassium hydrogen phthalate. (j) emulsification Reagent: Dissolve 17.9 gram of sodium chloride and 0.41 gram of monobasic potassium phosphate in about 400 ml of water. Add 540 ml of glycerol, with vigorous stirring, add 6.0 gram of gum Arabic, stir until dissolved and dilute to 1000 ml. (k) 10 mMTris buffer (pH 8.0): Dissolve 12.1 gram of enzyme-grade (or equivalent) tris (hydroxymethyl) aminomethane in 800 ml of water, and titrate with 1N hydrochloric acid to pH 8.0 and transfer into a 1,000-ml volumetric flask and make up the volume to 1000 ml with distilled water. (l) substrate emulsion: Take 15.9 ml of tributyrin into a beaker, add 50 ml emulsion reagent and 235 ml of water and Sonicate with digital sonifier at 30 amplitude three times for five minutes with five minutes interval in between each sonication and equilibrate at 30oC in constant temperature bath for at least fifteen minutes before use use within four hours. (m)sample and test preparation: Dissolve an accurately weighed amount of the enzyme preparation in the tris biffer, adjust pH to 8.0, so that it contains between 5000 to 8000 lipase units/ ml and accurately serial dilute a portion of this solution with water to obtain a final solution containing 0.5 – 1.5 lipase units per ml. 23.3 Procedure.- (a) fill the titrator burette with 0.05N sodium hydroxide solution; (b) transfer 15 ml of the substrate emulsion to the 100 ml double walled reaction vessel, equipped with small stirrer bar and the reaction vessel is placed in a recording pH stat with stirring at 1000 rpm; (c) the temperature is controlled at 30°C, and the pH is adjusted to 7.0; (d) add 1-ml amount of the lipase preparation to the reaction mixture previously conditioned to 30oC; (e) after the reaction is initiated with an aliquot of lipase, there is a drop in the pH due to the release of butyric acid and the pH meter sends an electrical impulse to the recorder input, which in turn activate the motor-driven burette through a micro switch to add standard NaOH (0.05N) to tributyrin mixture to maintain a constant pH of 7.0 of the assay mixture. (f) the pH stat record volume of standardized base (0.05N NaOH) consumed versus time; and (g) stop the titration after a constant (linear) rate of addition is observed for five minutes. (A five minutes period usually allowed for the determination; volume of the 0.05N NaOH recorded from the digital readout). 23.4 Calculations.- Definition of units.- 1-TBU (lipase unit) is the amount of enzyme which releases 1 mol titratable butyric acid per minute under the given standard conditions. Calculate the activity of the enzyme preparation by the formula: LU/g= R x N x 1000/W where (i) R is the mean rate of addition of NaOH in the time interval where slope is linear, in ml/minute; (ii) N is the normality of the Sodium hydroxide solution; (iii) 1000 is to convert mM to µM; and (iv) W is the weight, in grams, of the enzyme preparation contained in 1 ml of the diluted sample preparation added to 15 ml of substrate emulsion. 23.5 Reference.- Lipase Activity: food chemicals codex, 11th Ed. committee on codex specifications, food and nutrition board, division of biological sciences, assembly of life sciences, National Research Council. national academy press, Washington, DC, 2018. 24. Estimation of 2-bromo-(1h)-indole-3-carboxaldehyde 24.1 Equipment.- (a) HPLC- UV system. (b) analytical balance. (c) 0.45µ syringe filter. 24.2 Chemicals and Reagent Preparation.- (a) 2-Bromo-(1H)-indole-3-carboxaldehyde (CAS No. 119910-45-1). (b) acetonitrile. (c) standard preparation: accurately weigh 10 mg of 2-Bromo-(1H)-indole-3-carboxaldehyde and transfer it to 10 ml volumetric flask and make up the volume with acetonitrile to give a stock solution of 1000µg/ml. (d) intermediate standard solutions: dilute 1.0 ml stock standard to 10 ml with acetonitrile and mix well to give an intermediate standard solution of 100 µg/ml, from this intermediate standard solution, dilute 1.0 ml to 10 ml with acetonitrile to give an intermediate standard solution of 10 µg/ml. (e) prepare linearity point as per sample concentration specified in the table below Table +-----+----------+---------------------+---------------------+---------------------+---------------------+ |S. No|Linearity |Concentration |Volume (ml) |Volume (ml) |Concentration of | | |standard. |of stock |of stock |of water to |standard | | | |solution (ppm). |solution to be |be used. |(µg/ml). | | | | |used. | | | +-----+----------+---------------------+---------------------+---------------------+---------------------+ | (i) |Standard 1|10 ppm |0.10 |0.90 |0.11 | +-----+----------+---------------------+---------------------+---------------------+---------------------+ | (ii)|Standard 2|10 ppm |0.25 |0.75 |2.5 | +-----+----------+---------------------+---------------------+---------------------+---------------------+ | (iii)|Standard 3|10 ppm |0.50 |0.50 |5.0 | +-----+----------+---------------------+---------------------+---------------------+---------------------+ | (iv)|Standard 4|10 ppm |0.75 |0.25 |7.5 | +-----+----------+---------------------+---------------------+---------------------+---------------------+ | (v) |Standard 5|100 ppm |0.10 |0.90 |10.0 | +-----+----------+---------------------+---------------------+---------------------+---------------------+ (f) sample preparation: accurately weigh 5.0 gram of test sample in 20 ml volumetric flask, make up the volume with acetonitrile and if required, then further dilute the sample, then filter it through 0.45µ filter. 24.3 Procedure- (a) inject 20µl of the diluted solution in the HPLC fitted with UV detector. (b) HPLC Analysis : following instrument operating conditions specified in table below: Table (HPLC Condition) +-------+------------------------+---------------------+ | (i) | Column. | C18 (250 x 4.6mm) 5µ particle size| +-------+------------------------+---------------------+ | (ii) | Detector. | UV | +-------+------------------------+---------------------+ | (iii) | Detecting wavelength. | 210 nm | +-------+------------------------+---------------------+ | (iv) | Flow rate. | 0.8ml/min | +-------+------------------------+---------------------+ | (v) | Mobile Phase. | Acetonitrile: 0.1 % Ortho Phosphoric acid in water 60 : 40 | +-------+------------------------+---------------------+ | (vi) | Run Time. | 15 Minutes | +-------+------------------------+---------------------+ | (vii) | Column Temperature. | 25 oC | +-------+------------------------+---------------------+ | (viii)| Injection Volume. | 20 µl | +-------+------------------------+---------------------+ 24.4 Calculations – Determination of 2-Bromo-(1H)-indole-3-carboxaldehyde in the test sample in mg/kg according to the below given formula X1 (mg/kg) = A x V M Where, (i) X1- the content of 2-Bromo-(1H)-indole-3-carboxaldehyde in mg/kg. (ii) A-the calculated concentration of 2-Bromo-(1H)-indole-3-carboxaldehyde in the test sample solution in gram/ml. (iii) V -the volume of the test sample in ml. (iv) M- the mass of the test sampling. The percentage (%) of 2-Bromo-(1H)-indole-3-carboxaldehyde in the test sample is calculated using below formula 2-Bromo-(1H)-indole-3-carboxaldehyde in (%)= X1 (mg/kg) 10000 25. Estimation of lipo-chitooligosaccharides 25.1 Equipment - (a) calibrated pipettes. (b) auto sampler vial with screw caps. (c) centrifuge tubes. (d) vortex mixer. (e) sonicatar. (f) analytical Balance. (g) 0.2 µ syringe filter. 25.2 Chemicals and Reagent Preparation - (a) water. (b) acetonitrile. (c) methanol. (d) dimethyl Sulfoxide. (e) lipo-chitooligosaccharides (CAS Number 168843-28-5). (f) 30:70 acetonitrile: water- (i) add 30 ml of acetonitrile to a clean glass bottle; (ii) add 70 ml of water to the bottle; and (iii) cap and mix well to ensure uniformity. (g) 45:55 acetonitrile: water- (i) add 45 ml of acetonitrile to a clean glass bottle; (ii) add 55 ml of water to the bottle; and (iii) cap and mix well to ensure uniformity. (h) 50:50 acetonitrile: water- (i) add 50 ml of acetonitrile to a clean glass bottle; (ii) add 50 ml of water to the bottle; and (iii) cap and mix well to ensure uniformity. (i) 60:40 methanol: water- (i) add 60 ml of methanol to a clean glass bottle; (ii) add 40 ml of water to the bottle; and (iii) cap and mix well to ensure uniformity. 25.3 Standards and QC Solutions- (A) Stock Standard- (i) preparation of approximately 0.09 weight % lipo-chitooligosaccharides (LCO) stock standard solutions is as follows, namely:- (a) weigh an empty 8 dram (or equivalent) vial to the nearest 0.0001 gram; (b) weigh a minimum of 0.010 gram of lipo-chitooligosaccharides to the nearest 0.0001 gram into a dram vial; and (c) calculate the amount of Dimethyl Sulfoxide needed to add to the vial to get an approximate concentration of 0.09 weight % lipo-chitooligosaccharides using the following equation- 0.09 weight % LCO = LCO % purity x gram LCO gram LCO + gram dimethyl sulfoxide Note: Input lipo-chitooligosaccharides purity (%) and gram lipo-chitooligosaccharides into the equation above, solve for gram dimethyl sulfoxide. (ii) add the calculated amount of dimethyl sulfoxide to the vial; (iii) weigh final lipo-chitooligosaccharides + Dimethyl Sulfoxide solution to the nearest 0.0001 gram; (iv) sonicate for five minutes; (v) vortex a minimum of fifteen seconds; (vi) repeat steps ‘(iv)’ and ‘(v)’ a minimum of two times, or until solution is clear; (vii) visually inspect to ensure lipo-chitooligosaccharides has gone into solution; (viii) the concentration of the Stock standard in weight % is calculated using the following equation: Stock standard weight % LCO= (grams LCO/grams total solution) x % LCO purity Example calculation: (0.0113gram LCO/12.5521 gram total solution) x 87.4% purity = 0.07868 weight % LCO (ix) Shelf life and storage conditions: the standard stock solution shall be used on the day of preparation and to be kept away from light when not in use. (B) Intermediate Standard- (i) preparation of approximately 0.001 weight % lipo-chitooligosaccharides intermediate standard solution is as follows:- (a) weigh an empty 8 dram (or equivalent) vial to the nearest 0.0001 gram; (b) add approximately 0.22 ml of stock standard to the 8 dram vial; (c) weigh the 8 dram vial + stock standards to the nearest 0.0001gram; (d) add approximately 20 ml of 30:70 acetonitrile: water to the 8 dram vial; (e) weigh final mixture to the nearest 0.0001gram; and (f) vortex a minimum of fifteen seconds (ii) The concentration of the intermediate standard in weight % is calculated using the following equation Intermediate Standard weight % LCO = Stock standard weight % LCO x (gram Stock standard / gram total solution) Example calculation of Intermediate standard- weight % for stock standard lipo chitooligosaccharides concentration of 0.07868 weight % - 0.07868 weight % LCO x (0.2444 gram Stock standard/ 19.3037 gram total solution) = 0.0009962 weight % LCO (iii) Shelf life and storage conditions: the intermediate standard solution shall be used the day of preparation and to be kept away from light when not in use. (C) Working calibration standards- (i) Working calibration standards are prepared gravimetrically to encompass the range of approximately 0.00004 weight % to 0.0008 weight % lipo-chitooligosaccharides by diluting the intermediate standard with 30 : 70 Acetonitrile : Water as described below- (a) for preparation weigh five empty eight dram (or equivalent) vials to the nearest 0.0001 gram Note: There will be one vial for each of the five working calibration levels; (b) add the following volumes of Intermediate Standard to the corresponding eight dram vial specified in the table below:- Table +-----+------------+-------------------------+ |S. No|Standard. |Intermediate standard (ml).| +-----+------------+-------------------------+ | (i) |Standard 5 |8.00 | +-----+------------+-------------------------+ | (ii)|Standard 4 |5.00 | +-----+------------+-------------------------+ | (iii)|Standard 3 |3.00 | +-----+------------+-------------------------+ | (iv)|Standard 2 |1.00 | +-----+------------+-------------------------+ | (v) |Standard 1 |0.40 | +-----+------------+-------------------------+ (c) weigh each vial + intermediate standard to the nearest 0.0001gram; (d) add the following volumes of 30:70 acetonitrile: water to the corresponding 8 dram vial as specified in the table below:- Table: +-----+----------+---------------------------+ |S. No|Standard. |30:70 acetonitrile: water (ml).| +-----+----------+---------------------------+ | (vi)|Standard 5|2.0 | +-----+----------+---------------------------+ | (vii)|Standard 4|5.0 | +-----+----------+---------------------------+ | (viii)|Standard3 |7.0 | +-----+----------+---------------------------+ | (ix)|Standard 2|9.0 | +-----+----------+---------------------------+ | (x) |Standard 1|9.6 | +-----+----------+---------------------------+ (e) weigh each full vial to the nearest 0.0001 gram; (f) vortex a minimum of fifteen seconds; (g) transfer 1ml of each standard into a HPLC vial; and (h) cap vial and analyze via HPLC (ii) The concentration of the working calibration standard in weight % is calculated using the following equation: Working calibration standard weight % LCO = Intermediate Standard weight % LCO x (gram Intermediate Standard/ gram total solution) Example calculation of Standard 1: shown using an Intermediate Standard concentration of 0.0010 weight % lipo-chitooligosaccharides 0.0010 weight % LCO x (7.5200 gram Intermediate Standard/ 9.4000 gram total solution) = 0.0008 weight % LCO (iii) shelf life and storage condition- the working calibration standards can be stored and used for up to sixty days under refrigerated temperatures (4oC) and in the dark. Note: Bring working calibration standards to room temperature and vortex a minimum of fifteen seconds before opening for future use. (D) Quality control check standard- (i) The quality control check standard is diluted directly from the stock standard solution and analyzed against the working calibration standards, this provides a check on the accuracy of the working calibration standards. (a) for preparation weigh an empty eight dram (or equivalent) vial to the nearest 0.0001gram; (b) add 0.1 ml of stock standard to the eight dram vial; (c) weigh vial + stock standard to the nearest 0.0001 gram; (d) add 25 ml of 30:70 Acetonitrile: Water to the eight dram vial; (e) weigh each full vial to the nearest 0.0001 gram; (f) vortex a minimum of fifteen seconds; (g) transfer ~1 ml of each standard into a HPLC vial; and (h) cap vial and analyze via HPLC (ii) The concentration of the quality control check standard in weight % is calculated using the following equation- Quality control check standard weight % LCO = Stock standard weight % LCO x (gram Stock standard/gram total solution) Example calculation: a Quality control check standard is shown using Stock standard concentration of 0.09 weight % lipo-chitooligosaccharides 0.09 weight % LCO x (0.1000 gram Stock standard/ 23.6438 gram total solution) = 0.000381 weight % LCO (iii) Shelf life and storage conditions-the quality control sample can be stored and used for up to sixty days under refrigerated temperatures (4°C) and in the dark. NOTE: Bring quality control sample to room temperature and vortex a minimum of fifteen seconds before opening for future use. (E) Sample preparation for HPLC-UV - Sample preparation overview This method is applicable for the lipo-chitooligosaccharides concentration: 2.5 x 10-5 weight % lipo chitooligosaccharides Overview of sample concentration step is specified in the table below: Table +-----+------------------------+--------------+------------+------------------------+ |S. No|Target lipo |Approximate |Dilution |Target lipo | | |chitooligosaccharides |volume of |factor after|chitooligosaccharides | | |concentration (weight % |sample to apply|dilution of |concentration at end of | | |lipo |to solid phase|eluted |sample preparation for | | |chitooligosaccharides) |extraction |fraction |HPLC analysis (weight | | | |cartridge (ml)|(ml) |%) | +-----+------------------------+--------------+------------+------------------------+ | (i) |2.5 x 10⁻⁵ |250 |2.5 |0.00025 | +-----+------------------------+--------------+------------+------------------------+ 25.5 Procedure- (a) To analyze the active ingredient using HPLC-UV system and standard linearity plots for quantitative determination; (b) to prepare the samples for HPLC analysis using solid phase extraction (SPE) to concentrate lipo-chitooligosaccharides and remove matrix components, which interfere with chromatography; (c) the solid phase extraction procedure- (i) add approximately 250 ml sample to a container; Note: Ensure sample is homogenous by inverting a minimum of thirty times before use; and perform duplicate preparations for each batch tested (ii) weigh sample and container to the nearest 0.01 grams; (iii) set aside for use later; (iv) condition by passing 15 ml HPLC grade acetonitrile through SPE cartridges; Tip: Take care to flow solvent through solid phase extraction cartridges at a dropwise pace and control the flow rate by adjusting the stopper on the solid phase extraction manifold. (v) the condition by passing 15 ml HPLC grade methanol through solid phase extraction cartridges; (vi) the equilibrate by passing 15 ml HPLC grade water through solid phase extraction cartridges; (vii) obtain weighted samples from step ‘(i)’ to ‘(iii)’; (viii) to load the 250 ml sample on the solid phase extraction cartridge; Tip: Control the flow rate so that the sample has enough time to interact with the solid phase extraction sorbent, the eluate shall be drop-wise. (ix) once all the sample has been added, weigh the container and sample residue to the nearest 0.01grams; (x) subtract the weights from step ‘(ix)’ and step ‘(ii)’ to calculate the grams of sample loaded on the solid phase extraction cartridges; (xi) wash by passing three 10 ml portions of HPLC Type-1 water through solid phase extraction cartridges; (xii) wash by passing three 10 ml portions of 60 : 40 methanol : water through solid phase extraction cartridges; (xiii) wash by passing three 10 ml portions of 30 : 70 acetonitrile: water through solid phase extraction cartridges; (xiv) wash by passing one 5 ml portion of 45:55 acetonitrile: water through solid phase extraction cartridges; and (xv) proceed to the elution step below for the specific lipo-chitooligosaccharides sample concentration. (d) 2.5 x 10-5 weight % lipo-chitooligosaccharides solid phase extraction elution and remaining sample preparation, namely:- (i) weigh one empty 15ml centrifuge tube for each solid phase extraction cartridge to the nearest 0.0001grams; (ii) remove eluate reservoir and replace with 15ml centrifuge tube to collect eluate; (iii) elute by passing one 10ml portion of 50:50 acetonitrile : water through solid phase extraction cartridges; (iv) collect eluted samples in 15ml centrifuge tubes and record weights to the nearest 0.0001 grams; (v) subtract steps ‘(iv)’ and ‘(i)’ to determine the final sample weight; (vi) dilute the samples by pipetting 400µl into 600µl 30:70 acetonitrile : water in an auto sampler vial; (vii) cap and vortex the diluted sample; (viii) pass approximately 1ml sample through a 0.2µm filter into a HPLC vial; and (ix) cap vial, vortex and analyze using HPLC. (e) final determination by HPLC-UV - Liquid chromatography method parameters are specified below- (i) mobile phase A: water; (ii) mobile phase B: Acetonitrile; (iii) wash solvent: Acetonitrile; (iv) column: C18, 4.6 x 250mm, 5 µm; (v) column oven setting: 50 °C; (vi) injection volume: 100 µL; (vii) flow rate: 1.0 ml/minute; (viii) wavelengths: 200 nm for analysis and quantitation; (ix) run time: sixty minutes; and (x) liquid chromatography gradient description specified in the table below:- Table +-----+----------+--------------+ |S.No.|Time |% B | | |(Minutes) |(Acetonitrile)| +-----+----------+--------------+ | (i) |0 |30 | +-----+----------+--------------+ | (ii)|5 |30 | +-----+----------+--------------+ | (iii)|50 |50 | +-----+----------+--------------+ | (iv)|52 |100 | +-----+----------+--------------+ | (v) |53 |100 | +-----+----------+--------------+ | (vi)|54 |30 | +-----+----------+--------------+ | (vii)|60 |30 | +-----+----------+--------------+ Expected retention time for lipo-chitooligosaccharides is ~45.0 minutes, exact retention time may change due to chromatographic conditions and should be confirmed with standards for each analysis. (f) Calibration procedures- (i) the calculation of results is based on peak intensity measurements (peak area) using a calibration curve; (ii) inject a set of different concentrations of standard solution samples into the HPLC-UV instrument covering the range of 0.00004 weight % to 0.0008 weight % to obtain the standard curve; (iii) obtain the calibration curves by plotting peak area against the concentration of the injected analyte solutions; (iv) establish the stability of the detector response by injecting several concentrations of standards; and (v) determine the calibration range for a batch by the acceptance criteria, namely:- (1) 80% of the standards must have back-calculated accuracy within 15% of nominal concentration; and (2) the R² of the regression analysis for the standards must be 0.985 or higher. 25.5 Calculations Samples quantified using the external standard curve for lipo-chitooligosaccharides, data from the calibration standards are fit with a linear regression to obtain a prediction equation for the concentration determination of lipo-chitooligosaccharides in the HPLC samples, using formula: x= (y-b)/m where, (i) y= peak area of lipo-chitooligosaccharides (ii) m= slope of calibration curve (iii) b= intercept of calibration curve (iv) x= measured concentration of lipo-chitooligosaccharides (weight %) in injected HPLC sample Calculations: 2.5 x 10⁵ weight % lipo-chitooligosaccharides (LCO) samples Use the following calculations to quantify LCO in samples starting with a theoretical concentration of 2.5 x 10⁻⁵ weight % LCO: (1) Determine weight of sample loaded on solid phase extraction (SPE) cartridge- Sample loaded on SPE (gram) = [container sample (gram)] - [container + sample residue (gram)] Example calculation 253.22 gram loaded on SPE =526.35 gram full- 273.13 gram empty with residue (2) Determine weight of final sample prior to HPLC analysis: Diluted sample weight (gram) = [full 15 ml tube (gram)]–[empty 15 ml tube (gram)] Example calculation: 8.75123 gram sample = 15.61920 gram full - 6.86797 gram empty (3) Calculate concentration of LCO (weight %) in each sample: weight % LCO = (HPLC weight % LCO + [final sample weight (gram)]/ sample loaded on SPE (gram)]* 2.5 In this equation, the HPLC weight % LCO is the HPLC output and 2.5 is the dilution factor. Example calculation: 2.56 x 10⁻⁵ weight % LCO = (0.000297048 weight % LCO x (8.75123gram/ 253.22gram) x 2.5 26. Estimation of vasicine 26.1 Equipment- (a) HPLC. (b) UV detector. (c) micro balance. (d) analytical Balance. (e) sonicator. 26.2 Chemicals and Reagents required- (a) Type I water. (b) HPLC grade Acetonitrile. (c) orthophosphoric acid. (d) vasicine (CAS Number: 6159-55-3). (e) mobile phase- (i) mobile phase (A): transfer 5ml of orthophosphric acid to a 1000ml of mobile phase bottle containing Type I water, mix well and sonicate for ten minutes and to be used as a Mobile Phase-A. (ii) mobile phase (B): transfer 1000 ml of HPLC grade acetonitrile in to a 1000 ml of mobile phase bottle and to be used as Mobile Phase- B. (f) preparation of diluent and blank solution: HPLC grade acetonitrile to be used as diluent and blank solution. (g) preparation of reference standard stock and working solution- (i) weigh 5.0 + 1.0 mg of vasicine standard (of known purity) and transfer it to 5 ml volumetric flasks; (ii) add 4 ml of acentonitrile to dissolve the content, make up the volume to 5 ml with acetonitrile and sonicate for ten minutes, it gives a stock solution of 1000 µg/ml; and (iii) take 0.1 ml from the above diluted stock solutions and transfer it to a 100 ml of volumetric flasks and make up the volume with acetonitrile, it gives a working solution of 1 µg/ml. (h) Preparation of test item stock and working solution- (i) accurately weigh 880± 10.0 mg of test sample and transfer it in to two different 5 ml volumetric flasks; (ii) add 4 ml of acentonitrile to dissolve the content and sonicate for ten minutes; and (iii) allow the solution to equilibrate to room temperature for ten minutes and make up the volume to 5 ml with acetonitrile. 26.3 Procedure- (a) inject 20 µl of the sample or standard into the injector of HPLC. (b) HPLC analysis: by following conditions as specified in table below: Table (HPLC Conditions) +--------+------------------------------------+---------------------+ | (i) | Instrument | High-Performance Liquid Chromatography| +--------+------------------------------------+---------------------+ | (ii) | Column | C18 (250 mm x 4.6 mm I.D x 5 um particle size)| +--------+------------------------------------+---------------------+ | (iii) | Detector | UV | +--------+------------------------------------+---------------------+ | (iv) | Mobile phase (A:B) | A: 0.5% orthophosphoric acid in water 20% (v/v)| | | | B: acetonitrile 80% (v/v) | +--------+------------------------------------+---------------------+ | (v) | Injection Volume | 20µl | +--------+------------------------------------+---------------------+ | (vi) | Flow rate | 0.5 ml/minute | +--------+------------------------------------+---------------------+ | (vii) | End time/ Stop time | 8.00 minutes | +--------+------------------------------------+---------------------+ | (viii) | Wavelength | 280 nm | +--------+------------------------------------+---------------------+ | (ix) | Column oven Temperature | 30⁰C | +--------+------------------------------------+---------------------+ | (x) | Thermostat or Auto Sampler | 25⁰C | | | Temperature | | +--------+------------------------------------+---------------------+ | (xi) | Retention time | 3.6 minutes | +--------+------------------------------------+---------------------+ 26.4 Calculation Active Ingredient = Area of sample solution x Concentration of standard x Standard purity Area of standard solution Concentration of sample Vasicine content (ppm) = Vasicine content (% w/w) x 10000 26.5 References- (i). Srivastava S, Verma RK, Gupta MM , Singh, and Kumar S 2001. HPLC determination of vasicine and vasicinone in Adhatoda vasica with photo diode array detection. Journal of Liquid Chromatography and related technologies 24: 153–159 (ii). Martolia J, Soni H and Tandel F 2021. In Vitro anti tubercular activity and physicochemical standardization of selected medicinal plant extracts. Indian Journal of Pharmaceutical Sciences 83: 230-237 27. Enumeration of Methylococcus capsulatus Methylococcus capsulatus is an obligate methanotroph, which requires methane (CH4) for its growth, this bacteria grows in Nitrate mineral salt medium when incubated in an atmosphere containing 50% air: 50% CH4, the growth temperature ranges from 37-40℃. 27.1 Equipment- (a) weighing balance. (b) pH meter. (c) water bath. (d) autoclave. (e) hot air oven. (f) biosafety cabinet or laminar airflow chamber. (g) incubators. (h) colony counters. 27.2 Growth medium and diluents- (a) growth medium: Nitrate Mineral Salt (NMS) medium and modified by American Type Culture Collection and the following table specified the composition of Nitrate mineral salt medium, namely:- (i) Composition of Nitrate mineral salt (NMS) medium is specified in table below: Table +-----+---------------------+----------+ |S.No.|Ingredient |Amount | +-----+---------------------+----------+ | (i) |MgSO₄.7H₂O |1.0 gram | +-----+---------------------+----------+ | (ii)|CaCl₂.6H₂O |0.20 gram | +-----+---------------------+----------+ | (iii)|*Chelated Iron Solution|2.0 ml | +-----+---------------------+----------+ | (iv)|KNO₃ |1.0 gram | +-----+---------------------+----------+ | (v) |**Trace Element Solution|0.5 ml | +-----+---------------------+----------+ | (vi)|Agar |15.0 gram | +-----+---------------------+----------+ | (vii)|Distilled Deionized Water|1.0 litre | +-----+---------------------+----------+ | (viii)|Adjust pH to |6.8-7.0 | +-----+---------------------+----------+ * Chelated iron solution to be prepared following as specified in table below Table +-----+---------------------------+----------+ |S. No|Ingredient |Amount | +-----+---------------------------+----------+ | (i) |Ferric (III) ammonium citrate|0.1 gram | +-----+---------------------------+----------+ | (ii)|EDTA, sodium salt |0.2 gram | +-----+---------------------------+----------+ | (iii)|HCl (concentrated) |0.3 ml | +-----+---------------------------+----------+ | (iv)|Distilled Deionized Water |100.0 ml | +-----+---------------------------+----------+ (Optional: 0.05 gram Ferric (III) chloride may be substituted) ** Trace Element Solution to be prepared following as specified in table below - Table +-----+-------------------+----------+ |S. No|Ingredient |Amount | +-----+-------------------+----------+ | (i) |EDTA |500.0 mg | +-----+-------------------+----------+ | (ii)|FeSO₄. 7H₂O |200.0 mg | +-----+-------------------+----------+ | (iii)|ZnSO₄. 7H₂O |10.0 mg | +-----+-------------------+----------+ | (iv)|MnCl₂. 4H₂O |3.0 mg | +-----+-------------------+----------+ | (v) |H₃BO₃ |30.0 mg | +-----+-------------------+----------+ | (vi)|CoCl₂. 6H₂O |20.0 mg | +-----+-------------------+----------+ | (vii)|CaCl₂. 2H₂O |1.0 mg | +-----+-------------------+----------+ | (viii)|NiCl₂. 6H₂O |2.0 mg | +-----+-------------------+----------+ | (ix)|Na₂MoO₄. 2H₂O |3.0 mg | +-----+-------------------+----------+ | (x) |Distilled water |1.0 Litre | +-----+-------------------+----------+ (ii) Autoclave the medium for 20 minutes at 121oC; and (iii) Post autoclave additions: Add 2 ml/litre *phosphate buffer (pH 6.8) to the autoclaved medium. * For phosphate buffer, pH 6.8 (gram/liter), add 3.6 gram of Na₂HPO₄ and 1.4 gram of KH₂PO₄ to 1 liter of water and autoclave, autoclaved phosphate buffer stock solution should be stored at 4-8 oC (refrigeration). (b) Diluents: Physiological saline (0.9% sodium chloride) - Add 0.9 gram of NaCl to 100 ml of water. 27.3 Procedure- (a) prepare the Nitrate Mineral Salt (NMS) medium in accordance with the composition and autoclave it at 121℃ for twenty minutes; (b) after autoclaving the medium, add 2 ml of the autoclaved phosphate buffer to 1 litre of the medium and let the medium cool to 45-50oC; (c) pour about 15-20 ml of medium into petri plates in a laminar flow bench; (d) dispense 10 gram of biostimulant formulation to 90 ml of autoclaved physiological saline in 250 ml conical flask and shake for thirty minutes on shaker at 150 rpm, this gives 10⁻¹ dilution of the sample; (e) prepare nine tubes each containing physiological saline and autoclave and cool it; (f) transfer 1 ml of aliquot from 10⁻¹ dilution to a tube with 9 ml of physiological saline, this gives 10⁻² dilution and likewise make serial dilutions up to 10⁻¹⁰ each time transferring 1 ml to 9 ml of physiological saline; (g) spread plate 0.1 ml of serially diluted sample on Nitrate Mineral Salt agar plate and incubate the plates in desiccators with an atmosphere of 50% air and 50% methane for 4-5 days and keep the desiccators in an incubator maintained at 37±2 oC. (h) observe the plates for growth after five days and count the number of colonies that shows the following colony morphology, namely:- (i) the edge of typical Methylococcus capsulatus is entire, and the colony surface appears smooth and shiny; (ii) the colony shape is circular, and the elevation is convex; and (iii) the colony color is off-white, with semi-translucent opacity. 27.4 Calculation Colony forming unit (CFU per gram) = N x Dilution factor x 10 [Example: 201 colonies appeared in 10⁻⁶ dilution, the CFU per gram will be = 201 x 10⁶ x 10=2.01 x 10⁹ ] 27.5 Reference Whittenbury, R., Davies, S. L. & Wilkinson, J. F. (1970) Enrichment, isolation and some properties of methane-utilizing bacteria. Journal of General Microbiology, 61, 205–218. 28. Enumeration of Methylobacterium symbioticum and Methylobacterium extorquens 28.1 Equipment (a) weighing balance. (b) pH meter. (c) water bath. (d) autoclave. (e) hot air oven. (f) biosafety cabinet/Laminar air flow chamber. (g) incubators. (h) colony counters. 28.2 Growth medium and diluents- (a) Growth medium- prepare Ammonium mineral salt agar medium as per the composition specified in table below, autoclave it and add 5 ml of Methanol to one litre of medium. (i) Composition of Ammonium mineral salt medium: Table +-----+------------+----------+ |S. No|Ingredient |Amount | +-----+------------+----------+ | (i) |K₂HPO₄ |0.70 gram | +-----+------------+----------+ | (ii)|KH₂PO₄ |0.54 gram | +-----+------------+----------+ | (iii)|MgSO₄. 7H₂O |1.0 gram | +-----+------------+----------+ | (iv)|CaCl₂. 2H₂O |0.2 gram | +-----+------------+----------+ | (v) |FeSO₄. 7H₂O |4 mg | +-----+------------+----------+ | (vi)|NH₄Cl |0.5 gram | +-----+------------+----------+ | (vii)|ZnSO₄.7 H₂O |0.10 mg | +-----+------------+----------+ | (viii)|MnCl₂. 4H₂O |0.03 mg | +-----+------------+----------+ | (ix)|H₃BO₃ |0.3 mg | +-----+------------+----------+ | (x) |CoCl₂. 6H₂O |0.2 mg | +-----+------------+----------+ | (xi)|CuCl₂. 2H₂O |0.1 mg | +-----+------------+----------+ | (xii)|NiCl₂. 6 H₂O|0.02 mg | +-----+------------+----------+ | (xiii)|Na₂MoO₄. 2 H₂O|0.06 mg | +-----+------------+----------+ | (xiv)|Adjust pH to|6.8 | +-----+------------+----------+ (b) Diluent: Physiological saline (0.9% sodium chloride)- Add 0.9 gram of NaCl to 100 ml of water. 28.3 Procedure (a) prepare the AMS medium in accordance with the composition and autoclave it at 121oC for twenty minutes; (b) after autoclaving the medium, add 5 ml of methanol to 1 litre of the medium, let the medium cool to 45-50oC; (c) pour about 15- 20 ml of medium into petri plates in a laminar flow bench; (d) dispense 10 gram of biostimulant formulation to 90 ml of autoclaved physiological saline in 250 ml conical flask and shake for thirty minutes on shaker at 150 rpm. And this gives 10⁻¹ dilution of the sample; (e) prepare 9 tubes each containing physiological saline and autoclave and cool it; (f) transfer 1 ml of aliquot from 10⁻¹ dilution to a tube with 9 ml of physiological saline, this gives 10⁻² diltuion and likewise, make serial dilutions up to 10⁻⁹ each time transferring 1 ml to 9 ml of physiological saline; (g) spread plate 0.1 ml of serially diluted sample on NMS agar plate and incubate the plates in an incubator maintained at 30±2 oC; and (h) observe the plates for growth after 5- 8 days, count the number of colonies that are small and pink in colour. 28.4 Calculation Colony forming unit (CFU per g) = N x Dilution factor x 10 28.5 Reference Whittenbury, R., Davies, S. L. & Wilkinson, J. F. (1970) Enrichment, isolation and some properties of methane-utilizing bacteria. Journal of General Microbiology, 61, 205–218. 29. Molecular identification of bacterial cultures 29.1 Equipment- (a) water bath. (b) refrigerated Microcentrifuge. (c) vortex mixer. (d) UV- Vis Spectrophotometer. (e) thermal cycler. (f) gel documentation system. (g) DNA sequencer. 29.2 Chemicals and Reagent Preparation- (a) tris(hydroxymethyl) aminomethane. (b) ethylenediaminetetraacetic acid (EDTA). (c) sodium Dodecyl Sulphate (SDS). (d) phenol. (e) chloroform. (f) sodium acetate. (g) ethanol. (h) tris- EDTA (TE) buffer. (i) Taq polymerase (j) 10X Taq Polymerase Buffer (k) dNTPs. (l) primer F- pA (5'-AGAGTTTGATCCTGGCTCAG-3'). (m) primer R - pH (5'AAGGAGGTGATCCAGCCGCA-3'). (n) nuclease free water. (o) 6X loading buffer (Mix 3.4 ml glycerol, 25 milligram bromophenol blue, 25 mg xylene cyanol FF and 6.7 ml dist. H2O). (p) ethidium bromide. (q) agarose. (r) horizontal gel electrophoresis system. (s) power pack. (t) sequence buffer. (u) TRR – Terminator Ready Reaction: A, G, C, T dye terminator labeled with d-Rhodamine dye, dNTPs, Taq polymerase, MgCl2 and Tris-HCl buffer (pH – 9.0) (v) Loading buffer [formamide:25 mM EDTA (4:1)]. (w) big - Dye terminator kit. (x) gel purification kit. 29.3 Procedure (A) Preparation of template DNA (a) take one loopful of bacterial from the agar plates and suspended in 0.4 ml Tris EDTA buffer in a 1.5 ml micro-centrifuge tube; (b) to this add 40 µl of 10 per cent SDS and incubate for 30 minutes at 37oC in a water bath with gentle shaking; (c) add 0.40 ml Phenol and mix well; (d) centrifuge the tube containing this suspension for five minutes at 8000 rpm; (e) collect the aqueous phase with the help of a micropippete and transfer it to a new tube; (f) add to the aqueous phase 0.20 ml of Phenol and 0.20 ml Chloroform and mix well without vortexing; (g) centrifuge the suspension for five minutes at 8000 rpm; (h) collect the aqueous phase and transfer it to another fresh tube; (i) to it add 0.4 ml chloroform and mix without vortexing and centrifuge at 8000 rpm for five minutes and collect the aqueous phase in a fresh tube; (j) to it add 0.1 volume of 3M Sodium Acetate, pH 5.2 and mix well; (k) add 2 volume of 96 per cent ethanol and keep it at -200C for one hour; (l) centrifuge the contents for ten minutes at 8000 rpm; (m) decant the supernatant and the pellet in the tube is the DNA; (n) wash the DNA pellet with cold 70 per cent ethanol and centrifuge for ten minutes at 8000 rpm; (o) discard the supernatant; (p) suspend the dried pellet in 40 µl TE buffer and store at 4oC; and (q) quantify the DNA using spectrophotometer at 260 nm (1 OD= 50.0 ng DNA per µL) (B) PCR amplification of 16S rRNA gene and gel electrophoresis (a) amplify the 16S rRNA gene by mixing the genomic DNA (50-100 nano gram) with universal primers pA (5'-AGAGTTTGATCCTGGCTCAG-3') and pH (5'AAGGAGGTGATCCAGCCGCA-3'), buffer, dNTPs and Taq polymerase; (b) add the reagents in the required volume in a 0.2 ml PCR tube as per the following specified in table below; Table +-----+--------------------------+----------------+----------------+---------------------+ |S. No|Components |Stock |Working |Volume per | | | |solution |solution |reaction | +-----+--------------------------+----------------+----------------+---------------------+ | (i) |10X Taq Polymerase |10 X |1 X |5 µl | | |Buffer | | | | +-----+--------------------------+----------------+----------------+---------------------+ | (ii)|dNTP |10 mM |0.2 mM |1 µl | +-----+--------------------------+----------------+----------------+---------------------+ | (iii)|Primer F (pA) |50 pM |1.5 pM |1.5 µl | +-----+--------------------------+----------------+----------------+---------------------+ | (iv)|Primer R (pH) |50 pM |1.5 pM |1.5 µl | +-----+--------------------------+----------------+----------------+---------------------+ | (v) |Taq polymerase |3 U/µl |1.5 U |0.5 µl | +-----+--------------------------+----------------+----------------+---------------------+ | (vi)|DNA | | |4 µl | +-----+--------------------------+----------------+----------------+---------------------+ | (vii)|Nuclease free water | | |36.5 µl | +-----+--------------------------+----------------+----------------+---------------------+ | (viii)|Total | | |51 µl | +-----+--------------------------+----------------+----------------+---------------------+ (c) carry out all the operations on ice; (d) in addition to the samples, maintain the negative control (i.e without template DNA) simultaneously to check for possible contamination. (e) cap all the PCR tubes and vortex to mix. Pulse the tubes for 2-3 seconds in a microcentrifuge to pool the contents in the bottom. (f) carry out DNA amplification in Thermal Cycler with the following temperature profile- initial incubation at 94oC for five minutes followed by 35 cycles (94oC for one minute, 52.5oC for one minute, and 72oC for two minutes) and final extension at 72oC for ten minutes using thermal cycler; (g) after completion of PCR, mix an appropriate aliquot (2-3 µl) with 0.5 µl of 5X loading buffer and load into the 1% agarose gel containing ethidium bromide (with a final concentration of 0.2–0.5 mg / ml); and (h) separate the amplified DNA fragment by running the gel at 60 Volt/cm for one and a half hours and visualize under UV light using a gel documentation system. (C) Sequencing of 16S rRNA gene- (a) cut the gel precisely with the 16S rDNA fragments and remove it from the gel; (b) using any commercially available PCR/Gel purification kit and following the manufacturer’s protocol, elute and purify the DNA. (c) use the purified PCR product for sequencing PCR with the following primers 8-27F - 5'AGAGTTTGATCCTGGCTCAG-3' 1492R - 5'-GGT TACCTTGTTACGACTT-3' (d) The protocol for sequencing PCR is specified in the following table:- Table +-----+-----------------+----------------+----------------+ |S. No|Components |Stock solution |Volume/reaction | +-----+-----------------+----------------+----------------+ | (i) |Sequence Buffer |5 X |1.5 µl | +-----+-----------------+----------------+----------------+ | (ii)|Primer |2 pM |1.5 µl | +-----+-----------------+----------------+----------------+ | (iii)|Nuclease free water| |3.0 µl | +-----+-----------------+----------------+----------------+ | (iv)|DNA | |3.0 µl | +-----+-----------------+----------------+----------------+ | (v) |TRR | |1.0 µl | +-----+-----------------+----------------+----------------+ | (vi)|Total | |10 µl | +-----+-----------------+----------------+----------------+ TRR – Terminator Ready Reaction: A, G, C, T dye terminator labeled with d-Rhodamine dye, dNTPs, Taq polymerase, MgCl₂, and Tris-HCl buffer (pH – 9.0) (e) carry out cycle sequencing in a PCR machine for 35 cycles with the following conditions: Initial denaturation at 96oC for one minute followed by 25 cycles of sequencing and each cycle consists of a denaturation step at 96oC for thirty seconds, an annealing step at 52oC for fifteen seconds and an extension step at 60oC for four minutes, Finally hold at 4˚C until ready to purify; (f) after the PCR, precipitate the products by incubating on ice for fifteen minutes using 1 µl of 3 M sodium acetate (pH 4.6) and 50 µl of ethanol; (g) recover the pellet by centrifugation at 15000 rpm for twenty minutes at 4⁰C and wash the pellet with 70% ethanol, dry it under vacuum, and dissolve in 10 µl of loading buffer [formamide:25 mM EDTA (4:1)]; and (h) determine the rDNA sequence by the dideoxy chain-termination method using the mix from the Big Dye Terminator sequencing kit in a total volume of 10 μl,.using Genetic Analyzer. (D) Molecular identification using the 16S rRNA gene sequence The full length 16S rDNA sequences of the bacteria are compared with sequences available at NCBI database by the BLAST search available in the database to identify the nearest taxa. 30. Estimation of pesticide residues contamination - 30.1 Equipment - (1) centrifuge Low Volumes. (2) GCMS/MS. (3) LCMS/MS. (4) analytical balance 0.01mg. 30.2 Chemicals and Reagent Preparation- (a) ethyl acetate (AR Grade) (b) sodium sulphate anhydrous: - Heated for four hours at 6500C. (c) methanol: - LCMS grade. (d) ammonium acetate AR grade (e) primary secondary amine (PSA) (f) standard preparation - (i) stock solution of individual pesticide, weigh individual certified reference standards around 10 mg in 10 ml volumetric flask and dissolve in Methanol for LCMS/MS and in Ethyl acetate for GCMS-MS and the concentration of pesticide in this stock solution is 1000 mg/kg; (ii) intermediate standard solutions, from stock solution (1000 mg/kg) , prepare standard solution of 100 mg/ kg by pipetting required volume in 10 ml volumetric flask and make up to the mark with appropriate solvent (Methanol for LCMS/MS and Ethyl Acetate for GCMS/MS); (iii) mixture of pesticide standards (1µg/ml), from each of individual standard stock solution (100 mg/kg), pipette out required volume of standard solution to 25ml/50ml / 100ml volumetric flask, to prepare the mixture of pesticides having concentration 1.0 µg/ml; and (iv) preparation of linear dilutions, pipette out required volume of stock solutions from step ‘(iii)’ to 5ml volumetric flasks for the different five levels of dilutions and make up to the mark with appropriate solvent. 30.3 Procedure 30.3.1. For LCMS/MS Extraction- (i) sample weight: weigh 1 gram of sample in a 15 mL centrifuge tube and add 10 ml of methanol to the sample and shake the sample vigorously for thirty seconds and vortex for thirty seconds and sonicate the sample for ten minutes. (ii) dilution-1: pipette out 1ml of extract from step ‘(i)’ in to a 10 ml standard volumetric flask and make up the volume to the mark by using methanol and water (50 : 50) (iii) clean-up: add 25 mg of Primary secondary amine in a 2 ml micro centrifuge tube and pipette out 1.0 ml of sample extract and vortex for thirty seconds. (iv) low volume centrifugation: centrifuge the sample in a refrigerated centrifuge for five minutes at 10000 rpm at -10° C; (v) filtration: take supernatant after centrifugation and filter the supernatant through 0.2µ membrane filter in to a 2 ml glass vial; and (vi) sample injection: inject 5 µL filtrate in LCMS/MS using chromatographic conditions given in Table-1. 30.3.2 For GCMS/MS Extraction - (i) weigh 1 gram of sample in 15 ml tube, add 10 mL ethyl acetate to the sample and vigorously shake the sample solution for thirty seconds, vortex for thirty seconds and sonicate for ten minutes, (ii) dilution-1: pipette out 1ml extract from step ‘(i)’ in to a 10 ml standard volumetric flask and make up the volume to the mark by ethyl acetate; (iii) clean-up: pipette out 1 ml of sample extract and add 25 mg of primary secondary amine (PSA) and 150 mg Na₂SO₄ in a 2 ml centrifuge tube and vortex for thirty seconds; (iv) centrifugation: centrifuge the sample in a refrigerated centrifuge for five minutes at 10000 rpm at -10°C; (v) filtration: take supernatant after centrifugation and filter the supernatant through 0.2µ membrane filter in to a 2 ml GC vial; and (vi) sample injection: inject 1µl filtrate in GCMS/MS using chromatographic conditions specified in the Table-2. 30.3.3 Sequence of injections (i) inject reagent blank; (ii) inject one standard of suitable concentration within calibration range; (iii) inject reagent blank; (iv) inject samples; (v) after every six samples inject standard of suitable concentration within calibration range. 30.4 Calculation Concentration of Pesticide (%) = Area of sample x Conc. of standard x DF Area of standard Wt. of sample 10⁴ where, DF = Dilution Factor =100 Table -1 (LC-MS-MS Condition) +-----------------+-----------------------------------------+ |Column |Acquity UPLC®BEH-C18, 1.7µm [100 mm] | +-----------------+-----------------------------------------+ |Mobile phase |Mobile phase A: water with10mM ammonium acetate and 0.1% formic acid: | | |methanol (98:2) | | |Mobile phase B: methanol with 0.1% formic Acid | +-----------------+-----------------------------------------+ |Flow rate |0.35 ml/minute | +-----------------+-----------------------------------------+ |Mobile phase programming| | +-----------------+-----------------------------------------+ |Time |Flow |%A |%B |Curve| |(100 mm |(ml/minute) | | | | |column) | | | | | +-----------------+-----------------------------------------+-----+----+-----| |Initial |0.35 |95.0 |5.0 |Initial| +-----------------+-----------------------------------------+-----+----+-----| |0.25 |0.35 |95.0 |5.0 |6 | +-----------------+-----------------------------------------+-----+----+-----| |5.00 |0.35 |50.0 |50.0|6 | +-----------------+-----------------------------------------+-----+----+-----| |10.00 |0.35 |37.5 |62.5|6 | +-----------------+-----------------------------------------+-----+----+-----| |10.05 |0.35 |20.0 |80.0|6 | +-----------------+-----------------------------------------+-----+----+-----| |13.00 |0.35 |20.0 |80.0|6 | +-----------------+-----------------------------------------+-----+----+-----| |16.00 |0.35 |5.00 |95.0|6 | +-----------------+-----------------------------------------+-----+----+-----| |17.50 |0.35 |95.0 |5.0 |6 | +-----------------+-----------------------------------------+-----+----+-----| |22.00 |0.35 |95.0 |5.0 |6 | +-----------------+-----------------------------------------+-----+----+-----| |MS/MS Condition | | | | | +-----------------+-----------------------------------------+-----+----+-----| |Source |Dissolution |Capillary voltage|RF lens|Dissolvation gas|Cone gas flow| |temperature |temperature |(kV) |[V] |flow |(liter/hour) | |(⁰C) |(⁰C) | | |(liter/hour) | | +-----------------+-----------------------------------------+-----+----+----------------+-------------| |120 |350 |3.0 |0.1 |1000 |50 | +-----------------+-----------------------------------------+-----+----+----------------+-------------| List of pesticides to be analyzed on LC-MS-MS +-----+---------------------+--------------------+---------------------+---------------------+ |S. No|Name of the Pesticide|Parent ion (Q1) |Quantifier ion (Q2) |Qualifier ion (Q3) | +-----+---------------------+--------------------+---------------------+---------------------+ | (1) |6-Benzyl Adenine |226.01 |90.95 |65.01 | +-----+---------------------+--------------------+---------------------+---------------------+ | (2) |Abamectin |890.34 |567.31 |305.26 | +-----+---------------------+--------------------+---------------------+---------------------+ | (3) |Acephate |184.1 |143.0 |125.1 | +-----+---------------------+--------------------+---------------------+---------------------+ | (4) |Acetamiprid |223.0 |126.0 |56.1 | +-----+---------------------+--------------------+---------------------+---------------------+ | (5) |Azoxystrobin |404.0 |372.0 |329.0 | +-----+---------------------+--------------------+---------------------+---------------------+ | (6) |Aldicarb |191.1 |116.0 |89.0 | +-----+---------------------+--------------------+---------------------+---------------------+ | (7) |Ametryn |228.0 |68.0 |95.9 | +-----+---------------------+--------------------+---------------------+---------------------+ | (8) |Bendiocarb |224.1 |167.0 |109.0 | +-----+---------------------+--------------------+---------------------+---------------------+ | (9) |Benfuracarb |411.2 |195.0 |252.0 | +-----+---------------------+--------------------+---------------------+---------------------+ | (10)|Bensulfuron-Methyl |411.0 |149.0 |182.0 | +-----+---------------------+--------------------+---------------------+---------------------+ | (11)|Bitertenol |338.1 |99.1 |70.1 | +-----+---------------------+--------------------+---------------------+---------------------+ | (12)|Buprofezin |306.1 |201.0 |57.4 | +-----+---------------------+--------------------+---------------------+---------------------+ | (13)|Carbaryl |202.0 |145.0 |117.0 | +-----+---------------------+--------------------+---------------------+---------------------+ | (14)|Carbendazim |192.1 |160.1 |132.1 | +-----+---------------------+--------------------+---------------------+---------------------+ | (15)|Carbofuran |222.1 |165.1 |123.0 | +-----+---------------------+--------------------+---------------------+---------------------+ | (16)|Carbofuran-3-Hydroxy |238.0 |163.0 |181.0 | +-----+---------------------+--------------------+---------------------+---------------------+ | (17)|Carboxin |236.0 |143.0 |87.0 | +-----+---------------------+--------------------+---------------------+---------------------+ | (18)|Carfentrazone Ethyl |414.0 |368.0 |348.0 | +-----+---------------------+--------------------+---------------------+---------------------+ | (19)|Capropamid |334.0 |139.0 |195.9 | +-----+---------------------+--------------------+---------------------+---------------------+ | (20)|Chlorantraniliprole |481.8 |111.8 |283.6 | +-----+---------------------+--------------------+---------------------+---------------------+ | (21)|Chlormequat Chloride |122.2 |62.9 |59.1 | +-----+---------------------+--------------------+---------------------+---------------------+ | (22)|Chlorpropham |214.1 |172.0 |154.0 | +-----+---------------------+--------------------+---------------------+---------------------+ | (23)|Clothianidin |250.0 |169.0 |132.0 | +-----+---------------------+--------------------+---------------------+---------------------+ | (24)|Cymoxanil |199.0 |128.0 |111.0 | +-----+---------------------+--------------------+---------------------+---------------------+ | (25)|Cyproconazole |292.0 |70.0 |124.9 | +-----+---------------------+--------------------+---------------------+---------------------+ | (26)|Diafenthiuron |385.3 |278.3 |329.2 | +-----+---------------------+--------------------+---------------------+---------------------+ | (27)|Difenconazole |406.0 |251.1 |111.1 | +-----+---------------------+--------------------+---------------------+---------------------+ | (28)|Dimethoate |230.1 |199.0 |125.0 | +-----+---------------------+--------------------+---------------------+---------------------+ | (29)|Dimethomorph |388.1 |301.0 |165.0 | +-----+---------------------+--------------------+---------------------+---------------------+ | (30)|Dinotefuran |203.0 |129.0 |113.0 | +-----+---------------------+--------------------+---------------------+---------------------+ | (31)|Emamectin Benzoate |886.6 |158.0 |126.0 | +-----+---------------------+--------------------+---------------------+---------------------+ | (32)|Ethiprole |398.93 |256.9 |352.9 | +-----+---------------------+--------------------+---------------------+---------------------+ | (33)|Ethoxy Sulfuron |398.9 |261.0 |218.0 | +-----+---------------------+--------------------+---------------------+---------------------+ | (34)|Fenamidone |312.1 |92.0 |236.1 | +-----+---------------------+--------------------+---------------------+---------------------+ | (35)|Fenazaquin |307.1 |57.0 |161.0 | +-----+---------------------+--------------------+---------------------+---------------------+ | (36)|Fenobucarb |208.0 |94.9 |152.0 | +-----+---------------------+--------------------+---------------------+---------------------+ | (37)|Fenoxaprop-Ethyl |362.1 |288.0 |121.1 | +-----+---------------------+--------------------+---------------------+---------------------+ | (38)|Flonicamide |230.0 |202.9 |173.9 | +-----+---------------------+--------------------+---------------------+---------------------+ | (39)|Fluopyram |397.1 |173.0 |208.0 | +-----+---------------------+--------------------+---------------------+---------------------+ | (40)|Fenpyroximate |422.2 |366.1 |138.1 | +-----+---------------------+--------------------+---------------------+---------------------+ | (41)|Flubendiamide |680.9 |253.9 |273.8 | +-----+---------------------+--------------------+---------------------+---------------------+ | (42)|Flufenoxuron |489.1 |158.0 |141.0 | +-----+---------------------+--------------------+---------------------+---------------------+ | (43)|Flusilazole |316.0 |247.0 |165.0 | +-----+---------------------+--------------------+---------------------+---------------------+ | (44)|Hexazinone |253.0 |171.1 |71.0 | +-----+---------------------+--------------------+---------------------+---------------------+ | (45)|Hexythiazox |353.0 |168.1 |228.1 | +-----+---------------------+--------------------+---------------------+---------------------+ | (46)|Imidacloprid |256.1 |175.1 |209.1 | +-----+---------------------+--------------------+---------------------+---------------------+ | (47)|Indoxacarb |528.0 |150.0 |203.0 | +-----+---------------------+--------------------+---------------------+---------------------+ | (48)|Iprovalicarb |321.1 |119.0 |203.1 | +-----+---------------------+--------------------+---------------------+---------------------+ | (49)|Isoproturon |207.1 |72.0 |46.0 | +-----+---------------------+--------------------+---------------------+---------------------+ | (50)|Linuron |249.1 |160.1 |181.1 | +-----+---------------------+--------------------+---------------------+---------------------+ | (51)|Lufenuron |508.9 |338.8 |174.8 | +-----+---------------------+--------------------+---------------------+---------------------+ | (52)|Malaoxon |315.2 |99.0 |127.0 | +-----+---------------------+--------------------+---------------------+---------------------+ | (53)|Malathion |331.0 |99.0 |127.0 | +-----+---------------------+--------------------+---------------------+---------------------+ | (54)|Mandipropamid |412.0 |327.9 |124.8 | +-----+---------------------+--------------------+---------------------+---------------------+ | (55)|Mepiquat Chloride |114.2 |58.0 |98.0 | +-----+---------------------+--------------------+---------------------+---------------------+ | (56)|Metalaxyl |280.1 |220.1 |192.1 | +-----+---------------------+--------------------+---------------------+---------------------+ | (57)|Methamidaphos |141.9 |94.0 |110.0 | +-----+---------------------+--------------------+---------------------+---------------------+ | (58)|Methomyl |163.0 |88.0 |64.9 | +-----+---------------------+--------------------+---------------------+---------------------+ | (59)|Metolachlor |284.1 |252.1 |176.1 | +-----+---------------------+--------------------+---------------------+---------------------+ | (60)|Metribuzin |215.0 |89.1 |131.0 | +-----+---------------------+--------------------+---------------------+---------------------+ | (61)|Monocrotophos |224.1 |127.1 |98.1 | +-----+---------------------+--------------------+---------------------+---------------------+ | (62)|Myclobutanil |289.1 |70.2 |125.0 | +-----+---------------------+--------------------+---------------------+---------------------+ | (63)|Novaluron |492.9 |157.9 |140.9 | +-----+---------------------+--------------------+---------------------+---------------------+ | (64)|Omethoate |214.1 |125.1 |183.1 | +-----+---------------------+--------------------+---------------------+---------------------+ | (65)|Oxadiazon |247.1 |169.0 |109.0 | +-----+---------------------+--------------------+---------------------+---------------------+ | (66)|Paclobutrazole |294.0 |69.9 |124.8 | +-----+---------------------+--------------------+---------------------+---------------------+ | (67)|Penconazole |284.0 |70.1 |159.0 | +-----+---------------------+--------------------+---------------------+---------------------+ | (68)|Pencycuron |329.1 |125.1 |124.9 | +-----+---------------------+--------------------+---------------------+---------------------+ | (69)|Phosmet |317.9 |160.0 |77.0 | +-----+---------------------+--------------------+---------------------+---------------------+ | (70)|Phosphomidon |300.1 |127.1 |174.1 | +-----+---------------------+--------------------+---------------------+---------------------+ | (71)|Pretillachlor |312.1 |252.1 |176.1 | +-----+---------------------+--------------------+---------------------+---------------------+ | (72)|Propanil |217.9 |161.9 |127.0 | +-----+---------------------+--------------------+---------------------+---------------------+ | (73)|Propiconazole |342.0 |69.0 |159.0 | +-----+---------------------+--------------------+---------------------+---------------------+ | (74)|Pyriproxyfen |322.1 |96.0 |227.1 | +-----+---------------------+--------------------+---------------------+---------------------+ | (75)|Simazine |202.0 |124.0 |96.0 | +-----+---------------------+--------------------+---------------------+---------------------+ | (76)|Spinetoram |748.3 |142.1 |98.0 | +-----+---------------------+--------------------+---------------------+---------------------+ | (77)|SpinosadA |732.6 |142.0 |98.0 | +-----+---------------------+--------------------+---------------------+---------------------+ | (78)|Spinosad-D |732.6 |142.0 |98.1 | +-----+---------------------+--------------------+---------------------+---------------------+ | (79)|Spirotetramat |371.1 |216.1 |302.1 | +-----+---------------------+--------------------+---------------------+---------------------+ | (80)|Tebuconazole |308.0 |70.1 |125.0 | +-----+---------------------+--------------------+---------------------+---------------------+ | (81)|Tetrachlorvinfos |366.85 |126.92 |240.78 | +-----+---------------------+--------------------+---------------------+---------------------+ | (82)|Tetraconazole |372.0 |159.0 |70.1 | +-----+---------------------+--------------------+---------------------+---------------------+ | (83)|Thiacloprid |253.0 |126.0 |90.1 | +-----+---------------------+--------------------+---------------------+---------------------+ | (84)|Thiamethaxam |292.0 |211.2 |132.0 | +-----+---------------------+--------------------+---------------------+---------------------+ | (85)|Thiobencarb |258.0 |125.0 |100.0 | +-----+---------------------+--------------------+---------------------+---------------------+ | (86)|Thiodicarb |355.0 |87.9 |107.9 | +-----+---------------------+--------------------+---------------------+---------------------+ | (87)|Thiophanate Methyl |343.0 |151.0 |93.0 | +-----+---------------------+--------------------+---------------------+---------------------+ | (88)|Tolfenpyrad |384.0 |91.0 |197.1 | +-----+---------------------+--------------------+---------------------+---------------------+ | (89)|Triadimefon |294.1 |69.3 |197.2 | +-----+---------------------+--------------------+---------------------+---------------------+ | (90)|Triadimenol |296.1 |70.2 |99.1 | +-----+---------------------+--------------------+---------------------+---------------------+ | (91)|Tridemorph |257.0 |109.0 |79.0 | +-----+---------------------+--------------------+---------------------+---------------------+ | (92)|Tricyclazole |190.0 |163.0 |136.0 | +-----+---------------------+--------------------+---------------------+---------------------+ | (93)|Trifloxystrobin |409.0 |186.0 |145.0 | +-----+---------------------+--------------------+---------------------+---------------------+ Table -2 (GC Condition) +---------------------+---------------------+---------------------+---------------------+ |Oven Temperature |Rate/ minute |Temperature |Hold time (minute) | |Programme | | | | +---------------------+---------------------+---------------------+---------------------+ | |0.0 |70⁰C |1.0 | +---------------------+---------------------+---------------------+---------------------+ | |70⁰C |180⁰C |3.0 | +---------------------+---------------------+---------------------+---------------------+ | |20⁰C |220⁰C |9.0 | +---------------------+---------------------+---------------------+---------------------+ | |8⁰C |280⁰C |9.0 | +---------------------+---------------------+---------------------+---------------------+ |Injector Temperature |Rate / minute |Temperature |Hold time (minute) | |Programme | | | | +---------------------+---------------------+---------------------+---------------------+ | |0.0 |75⁰C |0.1 | +---------------------+---------------------+---------------------+---------------------+ | |450⁰C |325⁰C |5.0 | +---------------------+---------------------+---------------------+---------------------+ | |10⁰C |250⁰C |0.0 | +---------------------+---------------------+---------------------+---------------------+ |Column |DBP-5MS, 30m, 0.25mm, 0.5µ| | | +---------------------+---------------------+---------------------+---------------------+ |Column flow |1.7 ml/ minute | | | +---------------------+---------------------+---------------------+---------------------+ |Transfer line | |280⁰C | | |Temperature | | | | +---------------------+---------------------+---------------------+---------------------+ |Mass range |50 to600 | | | +---------------------+---------------------+---------------------+---------------------+ |MS Condition: | | | | +---------------------+---------------------+---------------------+---------------------+ |Source Temperature |MS1Quad. |MS2Quad. |Collision Flow |Quench Flow | | |Temperature |Temperature | | | +---------------------+---------------------+---------------------+---------------------+---------------------+ |290⁰C |150⁰C |150⁰C |1.5 ml/minute |2.5 ml/minute | +---------------------+---------------------+---------------------+---------------------+---------------------+ List of pesticides to be analyzed on GCMS +-----+---------------------+--------------------+--------------------+---------------------+ |S. No|Name of Pesticide |Parent Ion |Quant Ion |Qualifier Ion | | | |(Q1) |(Q2) |(Q3) | +-----+---------------------+--------------------+--------------------+---------------------+ |1. |2 4DDT |234.7 |165 |199 | +-----+---------------------+--------------------+--------------------+---------------------+ |2. |44 DDD |234.8 |165 |199 | +-----+---------------------+--------------------+--------------------+---------------------+ |3. |44 DDE |245.7 |176 |211 | +-----+---------------------+--------------------+--------------------+---------------------+ |4. |44DDT |234.7 |165 |199 | +-----+---------------------+--------------------+--------------------+---------------------+ |5. |2,4,DDD |235 |199 |165 | +-----+---------------------+--------------------+--------------------+---------------------+ |6. |2,4DDE |245.8 |211 |175.6 | +-----+---------------------+--------------------+--------------------+---------------------+ |7. |Alachlor |187.8 |160 |131 | +-----+---------------------+--------------------+--------------------+---------------------+ |8. |Aldrin |262.6 |192.8 |191 | +-----+---------------------+--------------------+--------------------+---------------------+ |9. |Allethrin |122.7 |81.1 |79.1 | +-----+---------------------+--------------------+--------------------+---------------------+ |10. |Alpha HCH |180.8 |109 |111 | +-----+---------------------+--------------------+--------------------+---------------------+ |11. |Anilofos |225.5 |184 |157 | +-----+---------------------+--------------------+--------------------+---------------------+ |12. |Atrazine |214.8 |58 |200 | +-----+---------------------+--------------------+--------------------+---------------------+ |13. |Beta–HCH |180.8 |145 |109 | +-----+---------------------+--------------------+--------------------+---------------------+ |14. |Bifenazate |299.7 |214.1 |199 | +-----+---------------------+--------------------+--------------------+---------------------+ |15. |Bifenthrin |180.8 |165 |166 | +-----+---------------------+--------------------+--------------------+---------------------+ |16. |Boscalid |140 |112 |76 | +-----+---------------------+--------------------+--------------------+---------------------+ |17. |Bromuconazole |172.8 |145 |109 | +-----+---------------------+--------------------+--------------------+---------------------+ |18. |Butachlor |175.8 |147 |134 | +-----+---------------------+--------------------+--------------------+---------------------+ |19. |Captan |79 |51 |77 | +-----+---------------------+--------------------+--------------------+---------------------+ |20. |Chlorobenzilate |251.1 |139.1 |75.1 | +-----+---------------------+--------------------+--------------------+---------------------+ |21. |Chlorfenvinphos |266.7 |159 |81 | +-----+---------------------+--------------------+--------------------+---------------------+ |22. |Chlorpyrifos |213.6 |258 |286 | +-----+---------------------+--------------------+--------------------+---------------------+ |23. |Chlorpyrifosmethyl |285.7 |93 |271 | +-----+---------------------+--------------------+--------------------+---------------------+ |24. |Clomazone |124.5 |99 |88.1 | +-----+---------------------+--------------------+--------------------+---------------------+ |25. |Cyfluthrin |162.8 |127 |109 | +-----+---------------------+--------------------+--------------------+---------------------+ |26. |Cypermethrin |180.7 |152 |127 | +-----+---------------------+--------------------+--------------------+---------------------+ |27. |Cypermethrin Alpha |180.7 |152 |127 | +-----+---------------------+--------------------+--------------------+---------------------+ |28. |Delta HCH |218.8 |183 |147 | +-----+---------------------+--------------------+--------------------+---------------------+ |29. |Deltamethrin |252.8 |73 |174 | +-----+---------------------+--------------------+--------------------+---------------------+ |30. |Dicofol |138.9 |111 |75 | +-----+---------------------+--------------------+--------------------+---------------------+ |31. |Dichlorovos |184.8 |93 |109 | +-----+---------------------+--------------------+--------------------+---------------------+ |32. |Diazinon |303.8 |179 |166.7 | +-----+---------------------+--------------------+--------------------+---------------------+ |33. |Dieldrine |262.7 |193 |191 | +-----+---------------------+--------------------+--------------------+---------------------+ |34. |Diflubenzuron |156.9 |141.1 |113.1 | +-----+---------------------+--------------------+--------------------+---------------------+ |35. |Dimefluthrin |122.8 |80.9 |67 | +-----+---------------------+--------------------+--------------------+---------------------+ |36. |Dimethoate |124.9 |47 |79 | +-----+---------------------+--------------------+--------------------+---------------------+ |37. |Diflufenican |265.8 |246.1 |238 | +-----+---------------------+--------------------+--------------------+---------------------+ |38. |Edifenphos |173 |109 |65 | +-----+---------------------+--------------------+--------------------+---------------------+ |39. |Endosulfan-alpha |240.7 |206 |204 | +-----+---------------------+--------------------+--------------------+---------------------+ |40. |Endosulfan-beta |240.7 |205.8 |169.8 | +-----+---------------------+--------------------+--------------------+---------------------+ |41. |Endosulfan Sulphate |271.6 |236.8 |234.7 | +-----+---------------------+--------------------+--------------------+---------------------+ |42. |Endrin |263 |193 |191 | +-----+---------------------+--------------------+--------------------+---------------------+ |43. |Epoxyconazole |192 |138 |111 | +-----+---------------------+--------------------+--------------------+---------------------+ |44. |Ethion |230.7 |129 |175 | +-----+---------------------+--------------------+--------------------+---------------------+ |45. |Ethofenprox |163 |107.1 |135.1 | +-----+---------------------+--------------------+--------------------+---------------------+ |46. |Ethofumesate |285.9 |179 |161 | +-----+---------------------+--------------------+--------------------+---------------------+ |47. |Etoxazole |141 |113 |63.1 | +-----+---------------------+--------------------+--------------------+---------------------+ |48. |Fenitrothion |276.7 |260 |125 | +-----+---------------------+--------------------+--------------------+---------------------+ |49. |Fenpropathrin |180.8 |152 |127 | +-----+---------------------+--------------------+--------------------+---------------------+ |50. |Fenthion |124.8 |79 |47 | +-----+---------------------+--------------------+--------------------+---------------------+ |51. |Fenvalerate |166.9 |125 |89 | +-----+---------------------+--------------------+--------------------+---------------------+ |52. |Fluvalinate |249.7 |55 |208 | +-----+---------------------+--------------------+--------------------+---------------------+ |53. |Fipronilsulfone |212.7 |178 |143 | +-----+---------------------+--------------------+--------------------+---------------------+ |54. |Fipronil |366.6 |254.9 |212.9 | +-----+---------------------+--------------------+--------------------+---------------------+ |55. |Floraxy 1 Methyl Heptyl Ester|208.9 |181 |160.9 | +-----+---------------------+--------------------+--------------------+---------------------+ |56. |FluazifopPButyl |281.8 |238.1 |91 | +-----+---------------------+--------------------+--------------------+---------------------+ |57. |Fluchloralin |306 |264 |206 | +-----+---------------------+--------------------+--------------------+---------------------+ |58. |Flufenacet |150.8 |136.1 |95 | +-----+---------------------+--------------------+--------------------+---------------------+ |59. |gama HCH/ Lindane |180.8 |145 |109 | +-----+---------------------+--------------------+--------------------+---------------------+ |60. |Heptachlor |271.6 |236.8 |235 | +-----+---------------------+--------------------+--------------------+---------------------+ |61. |Hexaconazole |213.7 |159 |187 | +-----+---------------------+--------------------+--------------------+---------------------+ |62. |Isoprothiolane |289.7 |204 |118 | +-----+---------------------+--------------------+--------------------+---------------------+ |63. |Lambda Cyhalothrin |180.8 |152 |127 | +-----+---------------------+--------------------+--------------------+---------------------+ |64. |Meperfluthrin |162.8 |126.8 |90.9 | +-----+---------------------+--------------------+--------------------+---------------------+ |65. |MethylParathion |263 |109 |246 | +-----+---------------------+--------------------+--------------------+---------------------+ |66. |Parathionethyl |290.9 |109 |81 | +-----+---------------------+--------------------+--------------------+---------------------+ |67. |Pendimethalin |252.1 |162.1 |191 | +-----+---------------------+--------------------+--------------------+---------------------+ |68. |Permethrin |182.8 |168 |153 | +-----+---------------------+--------------------+--------------------+---------------------+ |69. |Phenol4-bromo-2-chloro-|207.8 |63 |98.9 | +-----+---------------------+--------------------+--------------------+---------------------+ |70. |Phenthoate |273.7 |125 |121 | +-----+---------------------+--------------------+--------------------+---------------------+ |71. |Phoratesulphone |199 |152.9 |97 | +-----+---------------------+--------------------+--------------------+---------------------+ |72. |Phoratesulfoxide |152.9 |125 |97 | +-----+---------------------+--------------------+--------------------+---------------------+ |73. |Phorate |75 |47 |41 | +-----+---------------------+--------------------+--------------------+---------------------+ |74. |Phosalone |181.8 |111 |75 | +-----+---------------------+--------------------+--------------------+---------------------+ |75. |Pirimiphosmethyl |304.7 |290.1 |276 | +-----+---------------------+--------------------+--------------------+---------------------+ |76. |Procymidone |283 |255 |96 | +-----+---------------------+--------------------+--------------------+---------------------+ |77. |Profenofos |337 |267 |309 | +-----+---------------------+--------------------+--------------------+---------------------+ |78. |Propaquizafop |299 |255 |91 | +-----+---------------------+--------------------+--------------------+---------------------+ |79. |Quinolphos |156.8 |102 |77 | +-----+---------------------+--------------------+--------------------+---------------------+ |80. |Quizalofopethyl |298.8 |255.3 |192.2 | +-----+---------------------+--------------------+--------------------+---------------------+ |81. |Tebufenpyrad |318.1 |145.1 |131.1 | +-----+---------------------+--------------------+--------------------+---------------------+ |82. |Transfluthrin |162.8 |143 |91 | +-----+---------------------+--------------------+--------------------+---------------------+ |83. |Triazophos |160.9 |134 |106 | +-----+---------------------+--------------------+--------------------+---------------------+ |84. |Trifluralin |263 |159.9 |148 | +-----+---------------------+--------------------+--------------------+---------------------+ 31. Estimation of cadmium, copper, chromium, lead and zinc - As mentioned in the Schedule –IV, Part-D at serial number 10. 32. Estimation of arsenic - As mentioned in the Schedule –IV, Part-D at serial number 12. [F.No. 7-38/2024(Fert Law/BSC] FRANKLIN L KHOBUNG, Jt. Secy. Note: The principal Order was published in the Gazette of India vide G.S.R. number 758(E), dated the 25th September, 1985 and was last amended vide S.O. 2346(E) dated 26th May, 2025. Uploaded by Dte. of Printing at Government of India Press, Ring Road, Mayapuri, New Delhi-110064 and Published by the Controller of Publications, Delhi-110054.