Full Text
REGD. No. D. L.-33004/99
The Gazette of India
CG-DL-E-06102025-266713
EXTRAORDINARY
PART II-Section 3-Sub-section (ii)
PUBLISHED BY AUTHORITY
No. 4405]
NEW DELHI, MONDAY, OCTOBER 6, 2025/ASVINA 14, 1947
MINISTRY OF AGRICULTURE AND FARMERS WELFARE
(Department of Agriculture and Farmers Welfare)
ORDER
New Delhi, the 30th September, 2025
S.O. 4533(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) Eleventh
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, in Schedule VI, in Part D “
METHODOLOGY OF TESTING”, after sub-heading 32, “Estimation of arsenic”, the following items
and the entries shall be inserted, namely.-
"33. Estimation of salicylic acid by HPLC.-
33.1 Equipment.-
(a) reverse phase C-18 column (250 mm×4.6 mm x 5μ).
(b) UV detector.
(c) analytical balance.
(d)0.22µ syringe filter.
33.2 Chemicals and Reagent Preparations.-
(a) acetonitrile (HPLC grade, CAS No. 75-05-8).
(b) orthophosphoric acid (CAS No. 7664-38-2).
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THE GAZETTE OF INDIA : EXTRAORDINARY
[PART II-SEC. 3(ii)]
(c) salicylic acid (CAS No. 69-72-7).
(d) water (HPLC grade).
(e) preparation of mobile phase: take acetonitrile and water (with 0.1% orthophosphoric acid) in the
ratio of 50: 50 v/v, sonicate it for 5 minutes and vacuum filter the solution and degas before use.
(f) preparation of stock solution of salicylic acid standard: take salicylic acid (10 mg) in 10 ml
volumetric flask, add 7 ml of mobile phase to the flask, sonicate for 10 minutes, make up the
volume to 10 ml with mobile phase, it gives a stock solution of 1000 ppm.
(g) preparation of working solution of salicylic acid: dilute 1.0 ml stock standard to 10 ml with mobile
phase and mix well, it gives working standard solution of 100 ppm, dilute 1.0 ml of working
standard solution to 10 ml with mobile phase, it gives working standard solution of 10 ppm,
prepare calibration curve as specified in table below.-
Table
S. No. | Linearity | Concentration | Volume (ml) of | Volume (ml) of | Concentration of
| standard. | of stock solution | stock solution to | water to be used. | standard (µg/ml).
| | (ppm). | be used. | |
(i) | standard 1 | 10 ppm | 0.10 | 0.90 | 1
(ii) | standard 2 | 10 ppm | 0.50 | 0.50 | 5
(iii) | standard 3 | 10 ppm | 1.0 | 0.00 | 10
(iv) | standard 4 | 100 ppm | 0.25 | 0.75 | 25
(v) | standard 5 | 100 ppm | 0.50 | 0.50 | 50
(vi) | standard 6 | 100 ppm | 1.0 | 0.00 | 100
(h) preparation of sample solution.-
(i) thoroughly mix or stir products prior to sampling;
(ii) for liquid products, accurately weigh 0.5 to 5 gram (±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, 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;
(vi) sonicate it for thirty minutes and filter it through 0.22µ syringe filter; and
(vii)
inject 10µl to HPLC- UV system for analysis.
33.3 Procedure.-
perform analysis following HPLC conditions as specified in table below.-
Table
(i) | Column | C-18 (250 mm x 4.6 mm x 5 μ)
(ii) | Detector | UV
(iii) | Detecting wavelength | 237 nm
(iv) | Flow rate | 1.0 ml/ minute
(v) | Column temperature | 40°C
(vi) | Mobile phase | Isocratic elution with mobile phase using
| | acetonitrile: water (50: 50 v/v) with 0.1%
| | orthophosphoric acid
(vii) | Run time | 50 minutes
(viii) | Injection volume | 10 μl
33.4 Calculation.-
Salicylic acid (mg/kg) = Sample area x Standard concentration x dilution
Standard area x Sample weight
33.5 Reference.-
Kowalska M, Woźniak M, Kijek M, Mitrosz P, Szakiel J, Turek P. Management of validation
of HPLC method for determination of acetylsalicylic acid impurities in a new pharmaceutical
product. Scientific Reports 2022 Jan 6;12 (1):1.
34. Estimation of cytokinin by HPLC.-
34.1 Equipment.-
(a) reverse phase C-18 column (250 mm×4.6 mm x 5 µm).
(b) UV detector.
(c) analytical balance.
(d) 0.22µ syringe filter.
34.2 Chemicals and Reagents Preparation.-
(a) acetonitrile (HPLC grade, CAS No. 75-05-8).
(b) water (HPLC grade).
(c) methanol (HPLC grade, CAS No 67-56-1).
(d) preparation of mobile phase.-
solvent A - methanol: water: glacial acetic acid (50:450:1, v/v/v); and
solvent B - methanol: glacial acetic acid (500: 1, v/v)
(e) preparation of stock solution of zeatin standard stock standard solution: take 10 mg zeatin in 10
ml volumetric flask. Add 1 ml of 0.1N NaOH and dissolve by sonication, make up the volume
to 10 ml using methanol, it gives a stock solution of 1000 ppm.
(f) preparation of working solution of zeatin: dilute 1.0 ml stock standard to 10 ml with methanol
and mix well, it gives an intermediate standard solution of 100 ppm, dilute 1.0 ml of
intermediate standard solution to 10 ml with mobile phase, it gives an intermediate standard
solution of 10 ppm, prepare calibration curve following details specified in table below.-
Table
S. | Linearity | Conc. of Stock | Volume (ml) of | Volume (ml) of | Concentration of
No. | Standard | solution (ppm) | stock solution to | methanol to be | standard (µg/ml)
| | | be used | used |
(i) | standard 1 | 10 ppm | 0.50 | 0.50 | 5
(ii) | standard 2 | 10 ppm | 1.0 | 0.00 | 10
(iii)| standard 3 | 100 ppm | 0.5 | 0.50 | 50
(iv) | Standard 4 | 100 ppm | 1.0 | 0.00 | 100
(v) | standard 5 | 1000 ppm | 0.2 | 0.80 | 200
(vi) | standard 6 | 100 ppm | 0.4 | 0.60 | 400
(g) preparation of sample solution.-
(i) thoroughly mix or stir products prior to sampling;
(ii) for liquid products, accurately weigh 0.5 to 5 gram (±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, 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) sonicate it for thirty minutes and filter it through 0.22µ syringe filter; and
(vi) inject 10µl to HPLC-UV detector for analysis.
34.3 Procedure.-
(a) Initially condition the column with a solvent mixture of 80% of Solvent A and 20% of Solvent B,
elute the compounds using solvent system given in HPLC conditions with a gradient elution of
24
THE GAZETTE OF INDIA: EXTRAORDINARY
[PART II-SEC. 3(ii)]
increasing methanol concentration as zero to twenty minutes (20% B+ 80% A) to (45 % B+ 55%
A), twenty to twenty-two minutes (45% B +55 % A), twenty-two to thirty minute (45% B + 55%
A) to (60% B + 40% A), thirty to forty minute (60 % B to 40% A); and
(b) HPLC conditions: perform HPLC analysis following HPLC operation conditions as specified in
table below:
Table
(i) | column | C18 (250 mm x 4.6mm x 5 μ)
(ii) | detector | UV
(iii) | detecting wavelength | 269 nm
(iv) | flow rate | 0.5 ml/ minute
(v) | column temperature | 30°C
(vi) | mobile phase | solvent A- Methanol: water: glacial acetic acid (50:450:
| | 1, v/v/v)
| | solvent B- Methanol: glacial acetic acid (500: 1, v/v)
(vii) | run time | 40 minutes
(viii) | injection volume | 20μ1
34.4 Calculation.-
Cytokinin (mg/kg) = sample area x standard concentration x dilution
standard area x sample weight
34.5 Reference.-
Qi, B.;Wu, C.; Liang, H.; Cui, K.; Fahad, S.; Wang, M.; Liu, B.; Nie, L.; Huang, J.; Tang, H.
Optimized High-Performance Liquid Chromatography Method for Determining Nine Cytokinins,
Indole-3-acetic Acid and Abscisic Acid. Sustainability 2021, 13, 6998.
35. Estimation of carbohydrate or saccharides.-
35.1 Equipment.-
(a) spectrophotometer.
(b) vortex mixer.
(c) heating mantle/water bath.
(d) weighing balance.
35.2 Chemicals and Reagents Preparation.-
(a) anthrone (CAS No. 90-44-8).
(b) concentrated HCl (CAS No. 7647-01-0).
(c) concentrated H2SO4(CAS No. 7664-93-9).
(d) 2.5N HCl: take 208.3 ml of concentrated HCl and make up the volume to 1000 ml with
water.
(e) anthrone reagent: dissolve 200mg anthrone in 100ml of ice cold concentrated H2SO4, prepare
fresh before use.
(f) stock standard glucose: dissolve 100mg glucose in 100ml water, it gives a stock of 1000
mg/ml glucose solution.
(g) working standard: dilute 10ml of stock solution to 100ml with distilled water, it gives a
working standard of 100 mg/ml, store refrigerated after adding a few drops of toluene.
Note: cool the contents of all the tubes on ice before adding ice-cold anthrone reagent.
35.3 Procedure.-
(a) weigh 100mg of the sample into a boiling tube;
(b) hydrolyze by keeping it in boiling water bath for 3 hours with 5ml of 2.5N HCl and cool to room
temperature;
(c) neutralize it with solid sodium carbonate until the effervescence ceases;
(d) make up the volume to 100ml and centrifuge;
(e) collect the supernatant and take 0.5ml and 1ml aliquots for analysis;
(f) prepare the standards by taking 0, 0.2, 0.4, 0.6, 0.8 and 1ml of the working standard, '0' serves as
blank;
(g) make up the volume to 1ml in all the tubes including the sample tubes by adding distilled water;
(h) add 4ml of ice cold anthrone reagent;
(i) heat for eight minutes in a boiling water bath;
(j) cool rapidly on ice and read the green to dark green color at 630nm;
(k) draw a standard graph by plotting concentration of the standard on the X-axis versus absorbance on
the Y-axis; and
(l) from the graph calculate the amount of carbohydrate present in the sample tube.
35.4 Calculation.-
Amount of carbohydrate
(in 100mg sample) = (mg of glucose ÷ volume of test sample) x 100
35.5 Reference.-
Hedge, JE and Hofreiter BT (1962) In: Carbohydrate Chemistry 17 (Eds Whistler R L and Be Miller,
J N) Academic Press New York.
36. Estimation of starch.-
36.1 Equipment.-
(a) water bath.
(b) high speed centrifuge.
(c) Spectrophotometer.
(d) analytical balance.
36.2 Chemicals and Reagents preparation.-
(a) ethanol 95%- to 95 ml of absolute ethanol add 5 ml of water.
(b) concentrated Perchloric acid.
(c) concentrated H2SO4.
36.3 Procedure.-
(a) homogenize 0.1 to 0.5gram of the sample in hot 80% ethanol to remove sugars, centrifuge and
retain the residue, wash the residue repeatedly with hot 80% ethanol till the washing do not give
color with anthrone reagent, dry the residue well over a water bath;
(b) to the residue add 5.0ml of water and 6.5ml of 52% perchloric acid;
(c) extract at 0°C for 20minutes, centrifuge and save the supernatant;
(d) repeat the extraction using fresh perchloric acid, centrifuge and pool the supernatant and make up
to 100ml;
(e) pipette out 0.1ml or 0.2ml of the supernatant and make up the volume to 1 ml with water; and
(f) proceed for analysis of reducing sugars using anthrone reagent as detailed in protocol for
estimation of carbohydrate or saccharides by anthrone method at Serial No. 35.
36.4 Calculation.-
Find out the glucose content in the sample using the standard graph, multiply the value by a factor 0.9 to
arrive at the starch content.
36.5 Reference.-
Hodge JE and Hofreiter BT 1962. In: Methods in Carbohydrate Chemistry (eds Whistler RL and Be
Miller JN) Academic Press New York.
37. Estimation of cellulase activity.-
37.1 Equipment.-
26
THE GAZETTE OF INDIA : EXTRAORDINARY
[PART II-SEC. 3(ii)]
(a) spectrophotometer.
(b) water-bath.
(c) analytical balance.
37.2 Chemicals and Reagents preparation.-
(a) carboxy methyl cellulose (CAS No. 9004-32-4).
(b) dinitrosalicyclic acid (CAS No. 609-99-4).
(c) phenol (CAS No. 108-95-2).
(d) sodium sulfite (CAS No. 7757-83-7).
(e) rochelle salt (Potassium sodium tartarate)(CAS No. 6381-59-5).
(f) citric acid monohydrate (CAS No. 5949-29-1).
(g) citrate buffer: dissolve citric acid monohydrate (210gram) in 750 ml distilled water, adjust the pH
to 4.3 using NaOH, make up the volume to 1000 ml and add NaOH to adjust the pH to 4.5 and this
gives 1M Citrate buffer (pH 4.5), now take 5 ml of 1 M Citrate buffer, add 90 ml of distilled water
and adjust the pH to 4.8 using NaOH and make up the volume to 100 ml with distilled water, this
gives a solution of 0.05M Citrate buffer (pH 4.8).
(h) dinitrosalicylic acid (DNSA) stock solution: it comprises of solution A and solution B as given
below.-
(i) solution A: dissolve 10 gram of NaOH in 1000 ml of water, mix well to dissolve and it will
give 1% NaOH solution; and
(ii) solution B: to solution A, add 200 mg of K-Na tartarate, 2 gram of crystalline phenol and
10gram of DNS, place the beaker on a heater/stirrer and warm gently, whilst stirring, to
dissolve, cool to ambient temperature and store the solution in amber coloured bottles at
4°C.
(i) DNSA working solution: to 99 ml of stock DNSA, add 1 ml of 5% sodium sulfite and mix well to
dissolve;
(j) substrate preparation: weigh 2gram of carboxymethyl cellulose (CMC) and dissolve it in 100 ml of
0.05M citrate buffer (pH 4.8), stir the mixture vigorously to promote dissolution and if necessary,
heat the solution to a moderate temperature (e.g., 60°C) to facilitate dissolution;
(k) standard stock solution of glucose: dissolve 100mg glucose in 100ml water and it gives a stock
of 1mg/ml;
(l) working standard solution of glucose: pipette out aliquots from 0.1 ml to 1 ml in a clean dry test
tube, make up the volume to 1 ml with 0.05 M citrate buffer (pH 4.8) and prepare blank by taking 1
ml 0.05 M citrate buffer (pH 4.8); and
(m) preparation of sample solution: thoroughly mix or stir products prior to sampling, for liquid
products, take 1 ml of the sample and dilute with 0.05M citrate buffer (pH 4.8) and use the dilution
factor for calculation, and for powdered products, take 1 gram of sample and dissolve it in 10ml of
0.05M citrate buffer (pH 4.8) and take 1 ml of the sample for testing.
37.3 Procedure.-
(a) take 1 ml of the test sample, blank and glucose working standards in the test tubes and incubate for
thirty minutes in water bath maintained at 50°C;
(b) add 3 ml of DNSA reagent and incubate the test tubes for fifteen minutes in a boiling water bath;
and
(c) cool the samples and read the absorbance at 575 nm in spectrophotometer.
37.4 Calculation.-
(a) prepare a standard curve of glucose and calibrate for estimation of reducing sugars in the sample;
and
(b) express the enzyme activity as IU/ml using the equation.-
CMCase activity= mg glucose released x 0.37 x DF (if any)
37.5 Reference.-
Ghose TK 1987. Measurement of cellulase activities. Pure and Applied Chemistry 59: 257-268.
38. Enumeration of Wickerhamo mycesanomolus.-
38.1 Equipments.-
(a) analytical balance.
(b) pH meter.
(c) autoclave.
(d) biosafety cabinet or laminar air flow chamber.
(e) incubators.
(f) colony counter.
38.2 Preparation of growth medium and diluents.-
(a) growth medium: prepare Rose Bengal agar medium as per the composition specified in table
below.-
Table
S. No. | Constituents | gram per liter
-------|--------------------------|---------------
(i) | dextrose | 10
(ii) | peptone | 5
(iii) | mono potassium phospahte | 1
(iv) | magnesium sulphate | 0.5
(v) | rose Bengal | 0.05
(vi) | agar | 18
(vii) | pH | 7.2±0.2
(b) autoclave prepared media at 121°C for fifteen minutes and after autoclaving add 10 ml of filter
sterilized chlorotetracycline or chloramphenicol (1% w/v).
(c) Diluent: Physiological saline (0.9% sodium chloride) - Add 0.9 gram of NaCl to 100 ml of
water.
38.3 Procedure.-
(a) prepare the Rose Bengal medium in accordance with the composition and procedure described
in Sl No. 38.2 above and let the medium cool to 45-50°C;
(b) pour about 15-20 ml of medium into petri plates in a laminar flow bench;
(c) dispense 10gram of test sample 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 10-¹dilution of the
sample;
(d) prepare nine tubes each containing physiological saline and autoclave and cool it;
(e) transfer 1 ml of aliquot from 10-¹ dilution to a tube with 9 ml of physiological saline, this gives
10-²diltuion and likewise, make serial dilutions up to 10-º each time transferring 1 ml to 9 ml of
physiological saline;
(f) spread plate 0.1 ml of serially diluted sample on rose Bengal agar plate and incubate the plates
in an incubator maintained at 28°C and for each dilution, maintain plates in triplicate; and
(g) observe the plates for growth after five days, count the number of colonies that are smooth,
glabrous (without hairs), yeast like, white to cream coloured.
38.4 Calculation.-
Colony forming unit (CFU per = number of colonies x dilution factor x 10
gram) (mean of three replicates)
38.5 Reference.-
Jarvis B 1973. Comparison of an improved rose bengal-chlortetracycline agar with other media for
the selective isolation and enumeration of moulds and yeasts in foods, Journal of Bacteriology 36:
723-727
39. Estimation of Eugenol.-
39.1 Equipment.-
(a) GC-FID.
(b) GC-MS equipped with a VA-5 MS capillary columns (30 m x 0.25 mm x 0.25 µm).
(c) weighing balance.
28
THE GAZETTE OF INDIA : EXTRAORDINARY
[PART II-SEC. 3(ii)]
39.2 Chemicals, Gas and Reagents preparation.-
(a) eugenol (CAS No. 97-53-0).
(b) hexane (CAS No. 110-54-3).
(c) nitrogen gas.
(d) air gas.
(e) hydrogen gas.
(f) standard solution: take standard eugenol (1.0mg) in 1.0 ml vial and add 1.0ml hexane and mix.
(g) sample solution: take sample (1.0mg) in 1.0 ml vial and add 1.0ml hexane and mix.
39.3 Procedure.-
GC-FID Conditions: GC-FID conditions are specified in table below.-
Table
(i) | Column | Capillary column, SH Rtx-5 (30 m × 0.25 mm × 0.25 µm)
(ii) | Carrier gas | Nitrogen
(111) | Flow-rate | 1 ml/minute (split mode, 1:20)
(iv) | Injection volume | 0.2μl
(v) | Injector temperature | 250°C
(vi) | Detector temperature | 260°C
(vii) | Oven temperature | 60°C to 250°C
(viii) | Ramp programe | 60°C and programmed at 3°C/minute to 250°C with split mode (1:20)
39.4 Calculation.-
Calculate the relative amount of eugenol based on computer calculated GC peak areas (Area under
Curve i.e., AUC) without corrections for FID response factor.
Total Eugenol % = AUC of the sample peak x concentration of standard (mg/L) x % purity of
standard
Total AUC of standard peak x concentration of sample (mg/L)
39.5 Reference.-
Ajith, M., Pankaj, Shakil, N.A., Kaushik, P., and Rana, V.S. Chemical composition and nematicidal
activity of essential oils and their major compounds against Meloidogyne graminicola (Rice Root-Knot
Nematode). Journal of Essential Oil Research 2020, 32, 526-535.
40. Determination of Folic acid (Vitamin B9).-
40.1 Equipment.-
(a) HPLC with binary gradient pump.
(b) analytical balance.
(c) centrifuge.
(d) micropipettes (100 -1000 µl, 20 -200 µl 10 -100 μl).
(e) sonicator.
(f) vortex mixer.
(g) homogenizer.
(h) 0.45µm nylon syringe filter.
40.2 Chemicals and Reagents Preparation.-
(a) ammonium hydroxide (CAS No: 1336-21-6).
(b) phosphoric acid (CAS No: 7664-38-2).
(c) monobasic potassium phosphate (CAS No: 7778-77-0).
(d) tetrabutylammonium hydroxide (CAS No: 2052-49-5).
(e) methanol (CAS No 67- 56-1).
(f) folic Acid (CAS No: 59-30-3).
(g) water (HPLC grade/ultra-pure water).
29
(h) 0.5M tetra butyl ammonium hydroxide: dissolve 129.735 gram of tetra butyl ammonium hydroxide
in 1000 ml methanol.
(i) 3N phosphoric acid: take 67.8 ml of 85% phosphoric acid in a 1000 ml flask, add water to make up
the volume to 1000 ml.
(j) 6N ammonium hydroxide: add 198 ml of concentrated ammonium hydroxide (28% NH4OH,
specific gravity 0.90) to 500 ml of purified water and dilute to 1000 ml volume.
(k) Preparation of mobile phase.-
(i) accurately weigh 2.0 gram of monobasic potassium phosphate into a 1000 ml volumetric
flask and add 650 mL of water to it;
(ii) add 15 ml of 0.5M tetra butyl ammonium hydroxide and 7.0 ml of 3-N-phosphoric acid;
(iii) add 270 ml of methanol and cool it to room temperature;
(iv) adjust pH 5.0 with 3 N-phosphoric acid or 6-N-ammonium hydroxide; and
(v) finally make the volume to 1000 ml with ultra-pure water.
(l) Preparation of sample solution.-
(i) accurately weigh 1 gram (± 0.1 gram) and add 0.1 ml of 10 % ammonium hydroxide and
then transfer into a 10 ml amber colored volumetric flask, add 5 ml mobile phase and
vortex for five minutes;
(ii) cool the sample solution at room temperature;
(iii) make up the volume to 10 ml with mobile phase and vortex for two minutes;
(iv) filter the solution through 0.45µm nylon syringe filter; and
(v) pour the filtrate into the vial, and use it for injecting into HPLC.
Note: If required, dilute the sample for desired concentration.
(m) Preparation of standard stock solution of folic acid.-
(i) accurately weigh 10 mg (± 0.1 mg) of folic acid standard in 10 ml amber coloured volumetric
flask and add 0.1 ml of 10% ammonium hydroxide solution and make up the volume to 10 ml
with mobile phase, vortex for two minutes, store the solution at -20°C in the light protected
area, it gives a standard stock solution (SSS) of 1000 mg/kg; and
(ii) prepare calibration standard solutions as per the details specified in the table below.-
Table
S. No. | calibration | Standard Stock | volume of | final make up | final
| standard | Solution (mg/kg) | Standard Stock | volume diluent | concentration
| solutions | | Solution (ml) | (ml) | (mg/kg)
(i) | Linearity | 1000 | 1.50 | 10 | 150
| Solution 6 | | | |
(ii) | Linearity | 1000 | 1.20 | 10 | 120
| Solution 5 | | | |
(iii) | Linearity | 1000 | 1.00 | 10 | 100
| Solution 4 | | | |
(iv) | Linearity | 1000 | 0.85 | 10 | 85
| Solution 3 | | | |
(v) | Linearity | 1000 | 0.50 | 10 | 50
| Solution 2 | | | |
(vi) | Linearity | 1000 | 0.20 | 10 | 20
| Solution 1 | | | |
Note: Always make fresh preparation of calibration standard solutions.
40.3 Procedure.-
Perform HPLC analysis following HPLC Conditions as specified in table below.-
Table
(i) | Instrument | HPLC
(11) | Detector | UV
(iii) | Wavelength | 280 nm
30
THE GAZETTE OF INDIA: EXTRAORDINARY
[PART II-SEC. 3(ii)]
(iv) Column | C-18 ODS Column (4.6 mm X 250 mm X 5 μm)
(v) Run time | 10 minutes
(vi) Flow rate | 1.8 ml/minute
(vii) Injection Volume | 10 μl
(viii) Column Temperature | 25°C
Note: The make, model of Instrument & Column can be changed, however instrument should be able to
achieve the desired Level of Detection (LOD) and Level of Quantification(LOQ) Value & the Column is
exactly same in terms of the composition and dimensions.
40.4 Calculations.-
Calculate the folic acid content in premix using the following equation.-
Folic Acid (Vitamin B9) (mg/kg) = Sample concentration (mg/kg) x make up volume (ml)
Sample weight (gram)
40.5 Reference.-
Method protocol: PRT/RA/PRM/2023/001, method validation report for estimation of folic acid (Vitamin
B9) in premix using HPLC. United States Pharmacopeia- Folic acid (assay).
41. Estimation of homobrassinolide.-
41.1 Equipment.-
(a) reverse phase C -18 column (150 mm×4.6 mm x 3µm).
(b) UV detector (ELSD).
(c) weighing balance.
(d) ultrasonic bath.
(e) thermostat.
(f) 0.22µ syringe filter.
41.2 Chemicals and Reagents preparation.-
(a) water (Ultra-pure/HPLC grade).
(b) acetonitrile (CAS No. 75-05-8).
(c) methanol (CAS No 67- 56-1).
(d) (22S, 23S)- 28- homobrassinolide (CAS No 82373-95-3).
(e) phenylboronic acid.
(f) preparation of mobile phase: mix Acetonitrile (80ml) and water (20ml) in a ratio of (80:20, v/v)
and sonicate for fifteen minutes.
(g) preparation of phenylboronic acid solution: weigh approximately 1500mg (to the nearest 0.01gram)
phenylboronic acid into 250ml volumetric flask, dissolve to the mark with methanol and mix
thoroughly.
(h) preparation of 28-Homobrassinolide stock standard solution:
(i) weigh accurately 10 mg of 28 Homobrassinolide in 25 ml volumetric flask and add methanol
(15 ml) to dissolve it;
(ii) add 4ml phenylboronic acid solution and keep it for thirty minutes in thermostat at 50°C;
(iii) allow the solution to cool to ambient temperature and fill to the mark with methanol;
(iv) mix thoroughly and place the flask in an ultrasonic bath for five minutes, then filter the
solution through a 0.45µm filter membrane prior to analysis;
(v) it gives a stock solution of 400 mg/liter; and
(i) prepare linearity point as specified in table below.-
Table
S. No. | Linearity Standard | concentration | volume (ml) | volume (ml) of | concentration of
| | of stock | of stock | methanol to be | standard (mg/litre)
| | solution (ppm)| solution to be| used |
| | | used | |
(i) | standard 1 | 400 | 0.20 | 0.80 | 80
(ii) | standard 2 | 400 | 0.40 | 0.60 | 160
(iii) | standard 3 | 400 | 0.60 | 0.40 | 240
(iv) standard 4 | 400 | 0.80 | 0.20 | 320
(v) standard 5 | 400 | 1.00 | 0.00 | 400
(j) Preparation of sample preparation.-
(i) thoroughly mix or stir products prior to sampling;
(ii) weigh 10 mg or sufficient sample to contain about 10mg of 28-Homobrassinolide into 25ml
volumetric flask;
(iii) add 15ml of methanol to dissolve;
(iv) add 4ml phenylboronic acid solution and keep it for thirty minutes in thermostat at 50°C;
(v) allow the solution to cool to ambient temperature and fill to the mark with methanol; and
(vi) mix thoroughly and place the flask in an ultrasonic bath for five minutes, then filter the
solution through a 0.45µm filter membrane prior to analysis.
41.3 Procedure.-
Perform HPLC analysis following conditions as specified in table below.-
Table
(i) | Column | C18 (150 mm x 4.6mm x 5 μ)
(ii) | Detector | UV
(iii) | Detecting wavelength | 220 nm
(iv) | Flow rate | 1.0 ml/ minute
(v) | Column temperature | 25°C
(vi) | Mobile phase | acetonitrile: water (80: 20)
(vii) | Run time | 20 minutes
(viii) | Injection volume | 10 μι
41.4 Calculation.-
f = mass of 28-Homobrassinolide standard (mg) x purity of 28-Homobrassinolide standard (g/kg)
------------------------------------------------------------------------------------------
peak areas of 28-Homobrassinolide in the calibration solution
Content of 28-Homobrassinolide (g/kg) = peak areas of 28-Homobrassinolide in the sample solution x f
-----------------------------------------------------------
mass of sample taken (mg)
41.5 Reference.-
CIPAC/5247/m 28-Homobrassinolide (June 2020).
42. Estimation of protein.-
(a) follow protocol detailed at Part B of Schedule II at Serial No. 3 for estimation of total nitrogen (in
nitrate free samples); and
(b) total nitrogen estimated to be used for estimation of protein using the formula.-
Protein (%) = total nitrogen x 6.25
43. Estimation of vitamin-B3 (niacin).-
43.1. Equipment.-
(a) HPLC system with fluorescence detector.
(b) column - LiChrospher® 60 RP-18 (250 mm x 4.0 mm x 5 μm).
(c) 0.22µm syringe filter
43.2 Chemicals and Reagents preparation.-
(a) sodium acetate.
(b) potassium dihydrogen phosphate, KH2PO4 .
(c) non stabilized hydrogen peroxide solution, H2O2 (30%).
32
THE GAZETTE OF INDIA : EXTRAORDINARY
[PART II-SEC. 3(ii)]
(d) copper sulfate.
(e) acetic acid.
(f) concentrated hydrochloric acid solution.
(g) sodium hydroxide.
(h) nicotinic acid (CAS No 59-76-6).
(i) nicotinamide (CAS No 98-92-0).
(j) water (HPLC grade).
(k) 0.1M hydrochloric acid solution: take a dried and cleaned 1000 ml volumetric flask and add 100 ml
of water, add 8.3 ml of concentrated hydrochloric acid, make up the volume up to the mark with
water and mix the solution thoroughly.
(l) copper sulfate solution, [Cu(II)SO4.5H2O], it should be suitable for dilution to prepare mobile
phase.
(m) potassium dihydrogen phosphate (KH2PO4) solution, it should be suitable for dilution to prepare
mobile phase.
(n) hydrogen peroxide solution (H2O2), it should be suitable for dilution to prepare mobile phase.
(o) 5M sodium hydroxide solution: dissolve 20 gram of sodium hydroxide in 80ml of water, after
cooling dilute to 100 ml.
(p) preparation of nicotinic acid stock solution (1 mg/ml): dissolve nicotinic acid standard substance
(100 mg) in water (100 ml), this solution is stable for one week at -18°1°C).
(q) nicotinic acid(NA) and nicotinamide(NAM) standard solutions (0.05 - 5 µg/ml): prepare first
solution with 1 ml of each stock solution in 100 ml of water, and from this solution prepare four
standard solutions (0.5 ml, 2.5 ml, 10 ml and 50 ml) in 100 ml of water, these solutions are stable
for one day at room temperature.
(r) sample preparation:
(i) vortex the sample (0.5-2.0 gram) with 25 ml of 0.1M hydrochloric acid;
(ii) heat the tubes for one hour in a boiling water bath;
(iii) transfer the extracts into erlenmeyer flasks (250 mL), and add 20 ml of distilled H2O and 4 ml
of 5M sodium hydroxide, autoclave the flasks at 121° 1 °C for one hour;
(iv) after cooling, adjust the pH of the extracts to 4.5 first with concentrated and then with dilute
0.1M hydrochloric acid; and
(v) dilute the extracts to achieve 100 ml with distilled H2O and filter into HPLC vials.
43.3 Procedure.-
(a) separate the niacin vitamers (NA and NAM) with a normal phase silica (HSS) T3 column (2.1 ×
150 mm, 1.8 µm).
(b) perform the chromatographic separation at 30°C using an isocratic flow of the mobile phase (0.3
ml/minute) consisting of an optimized concentration of copper sulphate (CuSO4, 5 µM) and
hydrogen peroxide (H2O2, 150 mM) in a potassium phosphate buffer (70 mM of potassium
dihydrogen phosphate; pH 4.5).
(c) expose the eluent flow from the column to a long-wavelength UV light (366 nm, 8 W) in a
knitted PTEE reaction coil (1.59 mm o.d., 0.17 mm i.d. and 5 m length).
(d) detect NA and NAM fluorometrically (322 nm excitation and 380 nm emission wavelengths).
(e) inject the sample extracts (10 µl) in duplicate.
(f) HPLC Conditions to be followed as specified in table below.-
Table
(i) | column | normal phase silica (HSS) T3 column (2.1 × 150 mm, 1.8 µm).
(ii) | mobile phase | isocratic flow of the mobile phase, consisting of an optimized
| | concentration of copper sulphate (5µM CuSO4) and hydrogen
| | peroxide (150mM H2O2) in a potassium phosphate buffer (70mM
| | potassium dihydrogen phosphate, pH 4.5).
(iii) | sample injection | 10 μl.
(iv) | retention time | 3.6 minutes (for nicotinic acid).
| | 10.6 minutes (for nicotinamide).
| | (These may vary depending on the column phase and dimensions as
| | well as mobile phase flow rate).
(v) | flow rate | 0.3 ml/minute.
(vi) temperature | 30°C.
(vii) run time | 15 minutes.
Note: The level of detection for nicotinic acid and nicotinamide is 0.02 ng and 0.01 ng respectively, the
level of quantification (3-fold the level of detection) for nicotinic acid and nicotinamide is 0.06 ng and
0.03 ng respectively.
43.4 Calculation.-
The nicotinic acid and nicotinamide concentrations to be calculated using external calibration
curves (calibration range: 0.2 - 20 ng).
43.5 Reference.-
Method for Determination of Niacin in foodstuffs, 2022 (FSSAI).
44. Method of analysis of Carrabiitol (Carrageenan oligosaccharide derivatives).-
44.1 Equipment.-
(a) HPLC CromSil LE PB + (8%) column (7.8 mm x 300 mm x 7μ) or USP L34- RPM
monosaccharide PB (+) column.
(b) refractive index detector.
(c) analytical balance.
(d) ultrasonic bath.
(e) 0.22µ syringe filter.
44.2 Chemicals and Reagents.-
(a) water (HPLC grade).
(b) carrabiitol standard.
(c) preparation of carrabiitol standard solution: weigh carrabiitol (0.01 g) in a 10ml volumetric flask
and make up the final volume with HPLC grade water and mix well.
(d) ultrasonicate solution for fifteen minutes and filter with 0.22μ syringe filter.
44.3 Procedure.-
Perform HPLC analysis following conditions as specified in table below.-
Table
(i) | Column | CromSil LE PB+ (8%) column
(ii) | Detector | RI
(iii) | Detector temperature | 35±2°C
(iv) | Flow rate | 0.6 ml/minute
(v) | Column temperature | 80+2°C
(vi) | Mobile phase | Water (HPLC grade)
(vii) | Run time | 20 minutes
(viii) | Injection volume | 10 μl
(ix) | Tri- Carrabiitol (Peak 1) | RRT: 1.75
(x) | Tetra- Carrabiitol (Peak 2) | RRT: 2.10
(xi) | Hexa- Carrabiitol (Peak 3) | RRT: 2.25
(xii) | Di- Carrabiitol (Peak 4) | Retention time: between 7- 8 minute
44.4 Calculation.-
(a) Carrabiitol Area (nRlU*s) for sample = Sum of all four peak Area (Peak 1 Area + Peak 2 Area +
Peak 3 Area + Peak 4 Area); and
(b) Carrabiitol Area % for standard = Sum of all four peak Area (Peak 1 Area + Peak 2 Area + Peak 3
Area + Peak 4 Area).
Total Carrabiitol (%) = Average of Sample peak area x Standard. weight (g) x Dilution factor x Average of standard peak area
------------------------------------------------------------------------------------------------------
Average of Standard peak area x Sample weight (gram)
34
THE GAZETTE OF INDIA : EXTRAORDINARY
[PART II-SEC. 3(ii)]
44.5 Reference.-
MPTD/HPLC/2021/E01 18 (CSIR – National Institute for Interdisciplinary Science and Technology,
Microbial Processes and Technology Division).
45. Determination of Harpin-αβ.-
45.1. Equipment.-
(a) HPLC.
(b) ultrasonic Bath.
(c) analytical balances.
(d) laboratory glassware.
(e) freezer (-18°C).
45.2 Chemicals and Reagents preparation.-
(a) harpin αβ (CAS No. 917237-54-8) standard of known purity.
(b) acetonitrile.
(c) water.
(d) trifluoroacetic acid.
(e) calibration standard stock solutions: weigh 10 mg of harpin aß and add to 50 ml volumetric flask,
make the volume to 50 ml with mobile phase A.
(f) prepare the calibration solutions from calibration standard stock solution over approximately 50-
150 µg/ml Harpin aß in mobile phase A.
(g) sample solutions: approximately 1.0 gram sample to be diluted to 100 ml with mobile phase A
(approximately 100 µg/ml Harpin αβ).
45.3 Chromatographic Conditions.-
(a) Perform HPLC analysis following conditions as specified in table below.-
Table
(i) | column | Zorbax 300SB-C8, 4.6 mm x 50 mm, 300 Ă, 5m USJK001535.
| | (including a Zorbax 300SB C8 5 pm, 4.6 mm x 12.5 mm guard
| | cartridge column).
(ii) | detection wavelength | 218 nm.
(iii) | mobile phase | Mobile Phase A: 0.1% trifluoroacetic acid + 99.9% water (v/v).
| | Mobile Phase B: 0.1% trifluoroacetic acid + 94.9% acetonitrile + 5%
| | water (v/v).
(iv) | sample injection | (20 μL).
(v) | retention time | 7.4 minutes.
(vi) | flow rate | 1.5 ml/minute.
(vii) | temperature | 50°C.
(b). mobile phase gradient as specified in table below to be followed during HPLC analysis.-
Table
S. No. | Time (Minutes) | % of Mobile Phase-B
-------|----------------|--------------------
(i) | 0.0 | 44
(ii) | 5.0 | 54
(iii) | 8.0 | 66
(iv) | 8.5 | 100
(v) | 9.5 | 100
(vi) | 9.75 | 44
(vii) | 12 | 44
45.4 Calculation.-
Harpin aẞ (mg/kilogram) = sample area x standard concentration x dilution
standard area x sample weight
46. Determination of lactose.-
46.1 Equipment.-
(a) water bath.
(b) spectrophotometer.
46.2 Chemicals and Reagents Preparation.-
(a) Zinc Acetate - Phosphotungstic acid (ZAPT): dissolve 25.0 gram of zinc acetate and 12.5 gram of
Phosphotungstic acid in water, 20 ml of glacial acetic acid is added and made up to 100 ml.
(b) Glycine-NaOH buffer: mix 150 ml of glycine solution containing 2.4768 gram of glycine and
1.9359 gram NaCl with 850 ml of 0.385 N NaOH to give pH of 12.8.
(c) Methylamine solution: dissolve 5.0 gram of methylamine HCl in distilled water and dilute to 100
ml and store in refrigerator.
(d) Sodium Sulphite solution 1% (w/v): dissolve 1.0 gram of sodium sulphite in distilled water and
dilute to 100 ml, prepare fresh.
(e) 1N NaOH solution: dissolve 40 gram of NaOH in distilled water and make the volume to one litre.
(f) Standard Lactose solution:
(i) stock solution: dissolve 2.5315 gram of lactose monohydrate (USP grade) in 200 ml of 0.1
percent (W/V) benzoic acid and store in refrigerator; and
(ii) working solution: dilute 10, 15, 20, 25 and 30 ml of stock solution to 250 ml separately to
get 0.5 to 1.5 mg of lactose per ml.
(g) Preparation of sample:
(i) to 8.0 gram of well mixed sample add 1 ml of ZAPT reagent, dilute to 10 ml, and after ten
minutes filter using Whatman No. 1 filter paper; and
(ii) to 0.5 ml of the filtrate add 0.5 ml of NaOH solution, dilute to 10 ml and filter using
Whatman No. 1 filter paper, dilute 5 ml of the filtrate to 10 ml.
46.3 Procedure.-
(a) preparation of standard curve and measurement in sample: Pipette 5 ml each of working standard
lactose and unknown solution into 25 ml test tubes;
(b) add 5 ml of glycine NaOH buffer, 0.5 ml of methylamine solution and 0.5 ml of sodium sulphite
solution in each tube, mix thoroughly;
(c) heat tubes in a thermostatically controlled water bath at 65°C for twenty five minutes and cool
immediately in an ice water bath for two minutes to stop the reaction;
(d) read absorbance against blank at 540 nm in a spectrophotometer or a suitable spectrophotometer;
and
(e) draw a standard curve by plotting absorbance against concentration of lactose and determine the
concentration of lactose from it.
46.4 Reference.-
Nickerson et al. 1976. Colorimetric estimation of lactose and its hydrolytic products. Journal Dairy Science
59, No 3, page 386".
[F.No. 3-5/2025-Biostimulants]
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. dated 4441(E) dated 29th September, 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. SARVESH KUMAR
SRIVASTAVA
Date: 2005.10.06 22:53:09+0530