ISO 6650:2023

INTERNATIONAL ISO
STANDARD 6650
First edition
2023-10
Fertilizers, soil conditioners and
beneficial substances — Simultaneous
determination of N-(n-Butyl)
thiophosphoric triamide and
dicyandiamide by high-performance
liquid chromatography
Engrais, Amendements et Substances Bénéfiques — Détermination
Simultanée du N-buthylthiophosphore Triamide (NBPT) et du
Dicyandiamide (DCD) par Chromatographie Liquide à Haute
Performance (HPLC)
Reference number
© ISO 2023
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ii
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Reagents . 2
6 Apparatus and materials .2
7 Test procedure .3
7.1 General . 3
7.2 Preparation of test sample . 3
7.3 Preparation of the test solution . 3
7.4 HPLC conditions . 3
7.5 Determination of standard working solutions and sample test solutions . 4
7.5.1 Determination of standard working solutions . 4
7.5.2 Determination of sample test solutions . 4
8 Calculation and expression of results . 4
8.1 General . 4
8.2 Precision . . . 4
8.2.1 Ring test . 4
8.2.2 Repeatability, r . 4
8.2.3 Reproducibility, R . 5
9 Test report . 5
Annex A (normative) Typical HPLC chromatogram of DCD and NBPT . 6
Annex B (informative) Interlaboratory test report of the International Laboratories Ring
Test . 7
Bibliography .20
iii
Foreword
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beneficial substances.
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iv
Introduction
The stability of urea today is receiving greater attention due to a major increase in no-tillage or minimum-
tillage crop production. N-(n-butyl) thiophosphoric triamide (NBPT) can slow urea breakdown by
controlling the activity of the urease enzyme. Dicyandiamide (DCD) temporarily inhibits nitrification
by deactivating the enzyme of ammonia monooxygenase (AMO) in ammonia-oxidizing microbes.
This document provides a method to determine the content of NBPT and DCD in nitrogen fertilizers
simultaneously, which will help governmental authorities, fertilizer producers and consumers around
the world. Also, it can save time and protect the environment by reducing experimental waste.
v
INTERNATIONAL STANDARD ISO 6650:2023(E)
Fertilizers, soil conditioners and beneficial substances —
Simultaneous determination of N-(n-Butyl) thiophosphoric
triamide and dicyandiamide by high-performance liquid
chromatography
1 Scope
This document specifies the analytical method for the simultaneous determination of N-(n-butyl)
thiophosphoric triamide (NBPT) and dicyandiamide (DCD) in fertilizers by high-performance liquid
chromatography (HPLC) method.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 3696, Water for analytical laboratory use — Specification and test methods
ISO 8157, Fertilizers, soil conditioners and beneficial substances — Vocabulary
ISO 8358, Solid fertilizers — Preparation of samples for chemical and physical analysis
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 8157 and the following apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www. iso. org/o bp
— IEC Electropedia: available at https:// www.e lectropedia. org/
3.1
N-(n-butyl) thiophosphoric triamide
NBPT
white crystalline solid major urease inhibitor of commercial and practical importance in agriculture
Note 1 to entry: CAS Registry Number® 94317-64-3.
3.2
dicyandiamide
DCD
2-cyanoguanidine
nitrification inhibitor and slow-release nitrogen source that has 4 % to 5 % solubility in water
Note 1 to entry: DCD, C H N (CAS 461-58-5).
2 4 4
1 Chemical Abstracts Service (CAS) Registry Number® is a trademark of the American Chemical Society (ACS).
This information is given for the convenience of users of this document and does not constitute an endorsement by
ISO of the product named. Equivalent products may be used if they can be shown to lead to the same results.
4 Principle
This analytical method is based on the principles of liquid chromatography, with the absorption in the
ultraviolet region is used for detection of separated compounds.
5 Reagents
During the analysis unless otherwise stated, use only reagents of recognized analytical grade (AR) and
only water in accordance with ISO 3696 with electrical resistivity ≥18,2 MΩ·cm.
5.1 Acetonitrile (ACN), HPLC grade.
5.2 N-(n-butyl) thiophosphoric triamide (NBPT), assay, of a mass fraction ≥98 %.
5.3 Dicyandiamide (DCD) assay, of a mass fraction ≥99 %.
5.4 Stock solutions.
5.4.1 NBPT stock solution (1 000 mg/l).
Weigh to the nearest 0,1 mg, approximately 250 mg of NBPT into a 250 ml volumetric flask and dissolve
to volume with water.
5.4.2 DCD stock solution (1 000 mg/l).
Weigh to the nearest 0,1 mg, approximately of 250 mg DCD into a 250 ml volumetric flask and dissolve
to volume with water.
5.5 Standard solutions.
Prepare standard solutions (including both NBPT and DCD) in volumetric flasks in concentrations
ranges listed in Table 1.
The concentration ranges for standard solutions may be adjusted according to the levels expected in
the samples.
Table 1 — Preparation of standard solutions
Standard solution NBPT DCD
mg/l mg/l
Blank 0 0
Standard 1 5,0 1,0
Standard 2 10,0 2,5
Standard 3 25,0 10,0
Standard 4 50,0 25,0
Standard 5 100,0 100,0
6 Apparatus and materials
The usual laboratory apparatus and, in particular, the following shall be used.
6.1 HPLC apparatus with photo-diode array (PDA) detector (dual or multiple wavelengths,
recommended) or UV detector.
6.2 Analytical balance, to the nearest of 0,1 mg.
6.3 Ultrasonic bath.
6.4 Membrane filter, 0,45 µm, with the usual filtration equipment.
7 Test procedure
7.1 General
Two replicate experiments shall be done for the determination.
7.2 Preparation of test sample
For solid fertilizers, prepare a test portion by reducing the fertilizer sample to 100 g in accordance
with ISO 8358. Grind the sample until it passes through a sieve of aperture size 0,5 mm and mix until
homogenous. Place in a clean and dry bottle with a lid. For liquid fertilizers, shake the fertilizer sample
up until homogenous and pour out 100 ml. Place in a clean and dry bottle with a lid.
7.3 Preparation of the test solution
Accurately weigh to the nearest 0,1 mg, an appropriate amount (0,1 g to 3 g) of the ground test portion
and mix with 100 ml ultra-pure water into a 250 ml volumetric flask and dissolve using an ultrasonic
bath for 20 min. Make up the volume to the mark with water. Filter one part of the homogenized sample
solution through the membrane filter (6.4).
7.4 HPLC conditions
7.4.1 Column: 250 mm × 4,6 mm C18 reversed-phase column.
7.4.2 Injection volume: 10 µl.
7.4.3 Detector: PDA detector (214 nm for DCD, 205 nm for NBPT, recommended) or UV-detector with
absorbance set to 214 nm only.
7.4.4 Eluent: Mixture of acetonitrile (5.1) and water, gradient shown in Table 2.
Table 2 — Gradient elution schedule
Time Acetonitrile Water
min % %
0 5 95
3 5 95
10 25 75
16 25 75
17 5 95
31 5 95
7.3.5 Flow rate: 1,0 ml/min.
7.5 Determination of standard working solutions and sample test solutions
7.5.1 Determination of standard working solutions
Use NBPT and DCD standard solutions (5.5) to prepare the standard working solutions given in Table 1.
Plot the standard curves using the concentration of NBPT and DCD, corresponding to the peak areas
obtained in the test.
7.5.2 Determination of sample test solutions
Test a blank solution and the sample test solutions under the same conditions as the standards. Use the
retention times to identify NBPT and DCD, and derive the concentrations of NBPT and DCD, in the test
solutions from the standard curves, as shown in Annex A.
It is highly recommended that the DCD shall be determined under the wavelength of 214 nm, while the
NBPT shall be determined under the wavelength of 205 nm.
Only if the HPLC equipped with a single-wavelength UV detector, consider testing the DCD and NBPT
both under the wavelength of 214 nm.
The blank solution is prepared in the same manner as the test solutions, except for adding any test
samples.
If the response value (peak area) of any compound in a test solution exceeds the linear calibration range
of a standard solution, appropriate dilutions should be prepared.
8 Calculation and expression of results
8.1 General
The mass fraction of NBPT and DCD, w, in the unit of %, is calculated as follows:
ρ××Vf
w=
m×10000
where
ρ is the mass concentration of NBPT and DCD, expressed in milligrams per litre of the test solutions;
V is the volume of the test solutions, expressed in millilitres;
f is the dilution factor of the test solutions;
m is the mass of the test portion, expressed in grams.
The determination result is the arithmetic average of two parallel determination results and shall be
rounded off to three significant figures.
8.2 Precision
8.2.1 Ring test
Details of ring test on the precision of the method are summarized in Annex B.
8.2.2 Repeatability, r
For DCD content of all levels, the repeatability limit, r, is 0,067w − 0,014, in mass fraction percentage.
For NBPT content of all levels, the repeatability limit, r, is 0,074w − 0,011, in mass fraction percentage.
8.2.3 Reproducibility, R
For DCD content of all levels, the reproducibility limit, R, is 0,160w − 0,021, in mass fraction percentage.
For NBPT content of all levels, the reproducibility limit, R, is 0,137w + 0,034, in mass fraction percentage.
9 Test report
The test report shall contain at least the following information:
a) all information necessary for the complete identification of the sample;
b) the International Standard used (including its year of publication);
c) test method used with reference to this document;
d) test results obtained (signal-to-noise ratio plots per unknown, instrument detection limits and
percent recoveries are suggested to be presented, if necessary;
e) date of sampling and sampling procedure (if known);
f) date of analysis completion;
g) whether or not the requirement of the repeatability limit is fulfilled;
h) any deviations from the procedure;
i) any unusual features observed;
j) all operating details not specified in this document, or regarded as optional, together with details
of any incidents occurred when performing the method, which can have influenced the test results.
Annex A
(normative)
Typical HPLC chromatogram of DCD and NBPT
A typical HPLC chromatogram of DCD and NBPT of urea-based fertilizer is shown in Figure A.1.
a) PDA Multi 1 214 nm, 4 nm
b) PDA Multi 2 205 nm, 4 nm
Figure A.1 — Typical HPLC chromatogram of DCD and NBPT of a urea-based fertilizer
Annex B
(informative)
Interlaboratory test report of the International Laboratories Ring
Test
B.1 Overview
The International Laboratories Ring Test for this document was done between December 2022 to
March 2023. Eighteen laboratories participated in the two parallel tests on four test samples. This
international interlaboratory test and the statistician analysis/final report was accomplished by
Shanghai Institute of Chemical Industry Testing, Co., Ltd., P. R. China and Shanghai Research Institute
of Chemical Industry, Co., Ltd., P. R. China. Eighteen participating laboratories around the world which
have successfully participated in the interlaboratory test are listed as follows:
— Center for Soil Testing and Fertilizer Analysis, Agricultural Resources and Regional Planning
Institute, Chinese Academy of Agricultural Sciences, P. R. China
— Fujian Inspection and Research Institute for Product Quality, P. R. China
— Guizhou Institute of Products Quality Inspection and Testing, P. R. China
— Hebei Research Institution for Product Quality Supervision and Inspection, P. R. China
— Hunan Provincial Institute of Product and Goods Quality Inspection, P. R. China
— Jiangsu product quality testing and inspection institute, P. R. China
— Linyi Testing and Inspection Centre, P. R. China
— Ministry of Agriculture, Food and Forestry - Dept of ICQRF - Laboratory of Catania, Italy
— National Testing and Certification International Group Jingcheng Testing Co., Ltd, P. R. China
— Office of Indiana State Chemist, USA
— Shandong Institute for Product Quality Inspection, P. R. China
— Shandong Zhongsheng Huajian Certification and Testing Co., Ltd, P. R. China
— Shanghai Institute of Chemical Industry Testing Co., Ltd., P. R. China
— Sichuan Research Institute of Chemical Quality and Safety Testing, P. R. China
— Stanley Agriculture Group Co., Ltd., P. R. China
— University of Kentucky - Division of Regulatory Services, USA
— Xinjiang Uygur Autonomous Region Product Quality Supervision and Inspection Institute, P. R.
China
— Yunnan Supervision and Inspection Institute for Product Quality, P. R. China
NOTE Participating laboratories are listed in the alphabetic order, which has no relation to the sequences
listed in Table B.1 to B.12.
The test method described in this document was adopted for this ring test to determine the DCD and
NBPT content in fertilizer samples.
Four different types of fertilizer samples were used during the ring test, each with its well-designed
mean level of DCD and NBPT content. The test samples were: sample 6650-A (urea), sample 6650-B
(urea), sample 6650-C (complex fertilizer), sample 6650-D (urea ammonium nitrate solution).
The DCD and NBPT content (mass fraction %) to be determined and involved in the statistics in the four
fertilizer samples generally lie in the range of 0,2 % to 5 %.
The precision of the test results is evaluated according to ISO 5725-2.
B.2 Statistical analysis of the test results of DCD contents
B.2.1 Original test results
Eighteen laboratories have participated in the determination of DCD contents in the four fertilizer
samples (A, B, C and D). The results are listed in Table B.1, as mass fraction %. The test results of all
four samples reported by laboratory 2 have too large a systematic error in the level of its test results.
Laboratory 2 was considered as an outlying laboratory. The data of DCD contents results from
laboratory 2 were therefore discarded.
Table B.1 — Original test results of the determination of DCD contents
Laboratory DCD at level j
mass fraction %
A B C D
1 0,885 0,915 4,593 3,870 1,802 1,843 0,198 0,201
2 — — — — — — — —
3 0,882 0,894 4,068 4,231 1,665 1,591 0,178 0,168
4 0,998 0,999 4,299 4,255 1,832 1,828 0,199 0,199
5 0,980 0,984 4,665 4,717 1,896 1,891 0,199 0,198
6 0,990 0,990 4,274 4,258 1,810 1,811 0,193 0,196
7 1,039 1,095 4,810 4,732 1,96
...