This document (prEN 18317:2026) has been prepared by Technical Committee CEN/TC 260 “Fertilizers and liming materials”, the secretariat of which is held by DIN.

This document is currently submitted to CEN Enquiry.

This document has been prepared under a standardization request addressed to CEN by the European Commission. The Standing Committee of the EFTA States subsequently approves these requests for its Member States.

1.0 Scope

This document specifies a method for the determination of the urease inhibitor
N-(n-butyl)thiophosphoric triamide (NBPT) and its oxidate form N-(n-butyl)phosphoric triamide (NBPTO) in urea based organo-mineral fertilizers, using the liquid chromatography coupled with triple quadrupole mass spectrometry (LC-MSQQQ).

This document is applicable to organo-mineral fertilizers.

NOTE It is possible to apply this method to inorganic fertilizers; in this case a validation is carried out by the laboratory for the procedure and data generated.

2.0 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.

EN 1482‑2, Fertilizers, liming materials and inhibitors — Sampling and sample preparation — Part 2: General sample preparation provisions

prEN 12944‑1, Fertilizers, liming materials and inhibitors — Vocabulary — Part 1: General terms

prEN 12944‑2, Fertilizers, liming materials and inhibitors — Vocabulary — Part 2: Terms relating to fertilizers

3.0 Terms and definitions

For the purposes of this document, the terms and definitions given in prEN 12944‑1 and prEN 12944‑2 and the following shall be applied.

ISO and IEC maintain terminology databases for use in standardization at the following addresses:

— IEC Electropedia: available at https://www.electropedia.org/

— ISO Online browsing platform: available at https://www.iso.org/obp

4.0 Principle

This analytical method is based on the principles of liquid chromatography coupled with triple quadrupole mass spectrometry (LC-MSQQQ) for determination of the separated inhibiting compounds NBPT and NBPTO.

WARNING — Persons using this document should be familiar with normal laboratory practice. This document does not purport to address all of the safety problems, if any, associated with its use. It is the responsibility of the user to establish appropriate safety and health practices and to ensure compliance with any national regulatory conditions.

IMPORTANT — It is absolutely essential that tests conducted in accordance with this document be carried out by suitably trained staff.

5.0 Reagents

5.1 General

Use only reagents of recognized analytical grade and distilled water or ultrapure water for LC-MS.

5.1.1 Reagents for liquid chromatography

5.2.1 Methanol, LC-MS grade.

5.2.2 Formic acid, LC-MS grade.

5.2.3 Ammonium formate, p.a. high purity.

5.2.4 Water, ultrapure LC-MS grade or distilled water.

5.2.5 N-(n-butyl)thiophosphoric triamide (NBPT), CAS n. 94317-64-3, minimum mass fraction of 98 %.

5.2.6 N-(n-butyl)phosphoric triamide (NBPTO), CAS n. 25316-39-6, minimum mass fraction of 80 %.

5.2.7 Urea, p.a. high purity.

5.1.2 Calibration standards

5.3.1 Stock solution, mass concentration ρ_NBPT = 0,20 mg/ml.

Weigh 50 mg NBPT (5.2.5) into a 250 ml volumetric glass flask and dissolve to volume with water (5.2.4). Store at +4 °C ± 1 °C for no more than 2 days.

5.3.2 Stock solution, ρ_NBPTO = 0,20 mg/ml.

Weigh 50 mg NBPTO (5.2.6) into a 250 ml volumetric glass flask and dissolve to volume with water (5.2.4). Store at +4 °C ± 1 °C for no more than 2 days.

5.3.3 Blank solution, water (5.2.4).

6.0 Equipment and consumables

Disposable equipment is acceptable in the same way as reusable glassware if the specifications are similar. Ordinary laboratory equipment, and particularly the following shall be used.

6.1 Analytical scale, capable of weighing to the nearest 0,000 1 g.

6.2 Graduated pipettes, for volumes 5 ml, 10 ml, 15 ml, 20 ml, 25 ml with an accuracy of 0,01 ml.

6.3 One-mark volumetric glass flasks Class A [3], capacity 100 ml and 250 ml.

6.4 Horizontal shaker agitator.

6.5 Qualitative filter paper.

6.6 LC-MSQQQ.

6.6.1 Operative conditions

— Column: a non-endcapped silica-based HSS PFP/Fluoro-Phenyl reversed phase HPLC column, 3,5 μm 4,6 mm × 150 mm (designed for low pH separations that require alternative selectivity compared to a fully endcapped, high coverage C18 phase, usable for UPLC separations)

— Mobile phase A: Water (5.2.4) with 0,1 % formic acid (5.2.2) and 5 mmol ammonium formate (5.2.3)

— Mobile phase B: methanol (5.2.1) with 0,1 % formic acid (5.2.2)

— Mobile phases working gradient: see Table 1

— Flow rate: 0,6 mL/min

— Column temperature: 30 °C

— Injection volume: 20 μL

— Run time: 15 min

— Expected Retention Time, t_R of NBPT: 8 min.

— Expected Retention Time, t_R of NBPTO: 6,9 min.

Table 1 — Mobile phases working gradient

Gradient time min	Mobile phase A %	Mobile phase B %
0	90	10
2	90	10
7	5	95
10	5	95
10,1	90	10
15	90	10

— Detector: MS triple quadrupole (MSQQQ)

— Electrospray ionization (ESI)

— Positive polarity

— Gas temperature: 135 °C

— Gas flow: 12 l/min

— VCap: 4 000 V

— NBPT and NBPTO MSQQQ setting are in Table 2 and Table 3

Table 2 — NBPT MSQQQ setting

NBPT	Molecular ion	Fragment ion	Collision energy
Qualifier	168	151	15
Quantifier	168	74	15

Table 3 — NBPTO MSQQQ setting

NBPT-O	Molecular ion	Fragment ion	Collision energy
Qualifier	152	135.3	10
Quantifier	152	74.3	15

7.0 Sampling and sample preparation

7.1 Sampling

Sampling is not part of the method specified in this document. Recommended sampling methods are given in EN 1482‑1 [11] and EN 1482‑3 [12].

It is important that the laboratory receives a sample that is representative of both the product under consideration and the given analysis. The sample should not have been damaged or changed during transport or storage.

7.1.1 Sample preparation

Sample preparation shall be carried out in accordance with EN 1482‑2.

8.0 Procedure

8.1 General

8.1.1 Preparation of the test portion

An amount of 5 g of the test sample shall be weighted to the nearest 0,000 1 g, transferred to a 250 ml volumetric glass flask (6.3), added a volume of 150 ml of water (5.2.4) and dissolved by shaking with agitator (6.4) for 30 min at a rate that keeps the sample in suspension.

It shall be made up to volume with water, homogenized thoroughly and filtered with filter paper (6.5).

An aliquot portion of the extract in water (5.2.4) shall be diluted in one or more steps so that the final concentration of the molecules to be determined falls within the calibration range (see 8.3).

All samples shall be injected in duplicate.

8.1.2 Calibration

Calibration shall be performed before the analysis.

Calibration curve shall include at least 5 calibration points chosen according to the sensitivity of the available instrument.

The solutions shall be prepared at the time of each batch of analytical determination.

The stock solutions (5.3.1 and 5.3.2) shall be used to determine the retention time of NBPT and NBPTO in the LC system.

The response factor of NBPT and NBPTO shall be calculated by analysis of the calibration standards in the LC system.

All standards shall be injected in duplicate.

8.1.3 Blank test

For each series of determinations, a blank test shall be introduced in each analytical batch using urea sample test (5.2.7) free from NBPT and NBPTO, prepared according to the procedure in 8.2.

9.0 Calculation and expression of the result

The concentration of NBPT and NBPTO in the sample solution is determined according to the principle of external standard.

The mass fraction of NBPT w_NBPT and NBPTO w_NBPTO in percent shall be calculated according to Formulae (1) and (2):

(1)

(2)

where

A	is the peak area for NBPT or NBPTO;
R	is the response factor (see Formulae (4) and (5)) (peak area/μg NBPT or NBPTO);
V	is the injection volume in μl (20 μl);
m	is the mass of the test portion weighed into the sample solution (250 ml), in g.
D	is the dilution factor calculated according to the Formula (3):

(3)

where

V_p_1,2_…n	are withdrawal volumes of the solutions for each dilution step in ml;
V_1,2_,…n	are volumes of the flasks in which dilution is carried out in ml.

The result shall be given as the average of two replicated determinations done on two different test portions from the same sample, with an accuracy of two figures, e.g. 12 %, 1,2 %, 0,12 %.

The external standard response factor R is calculated from the average of the peak areas and mass concentrations of NBPT and NBPTO of the 5 calibration standards according to Formulae (4), (5) and Formulae (6), (7):

(4)

(5)

(6)

(7)

where

R_C1,_C2…C5	is the response factor of calibration standards;
A_C1,C2…C5	is the peak area of calibration standards;
	is the mass concentration of NBPT in each calibration standard C1, C2, C3, C4, C5 in mg/ml;
	is the mass concentrations of NBPTO in each calibration standard C1, C2, C3, C4, C5 in mg/ml;
V_C1,C2…C5	is the injection volume of calibration standards in μl.

10.0 Precision

10.1 Inter-laboratory study

Details of inter-laboratory study on the precision of the method are summarized in Annex A.

Repeatability and reproducibility were calculated according to ISO 5725‑1 [4], ISO 5725‑2 [5] and ISO 5725‑3 [6].

It is possible that the values derived from this study are not applicable to concentration ranges and matrices other than those given.

10.1.1 Repeatability

The absolute difference between two independent single test results, obtained using the same method on identical test material in the same laboratory by the same operator using the same equipment within a short interval of time, will in no more than 5 % of the cases be greater than the repeatability limit r given in Table 4 and Table 5.

10.1.2 Reproducibility

The absolute difference between two independent single test results, obtained using the same method on identical test material in different laboratories with different operators using different equipment, will in no more than 5 % of the cases be greater than the reproducibility limit R given in Table 4 and Table 5.

Table 4 — NBPT: mean values, repeatability and reproducibility limits

Sample		r	R
Sample	mg/kg	mg/kg	mg/kg
FE-2–21	23,794	3,589	7,415
FE-3–24	60,089	5,828	15,133
FE-4–24	183,14	9,67	79,00

Table 5 — NBPTO: mean values, repeatability and reproducibility limits

Sample		r	R
Sample	mg/kg	mg/kg	mg/kg
FE-2–21	10,645	2,457	4,308
FE-3–24	19,331	1,746	5,089
FE-4–24	30,366	2,763	9,426

10.1.3 NBPT Stability test

Due to the scientific literature [7], [8], [9], [10] showing the low stability of NBPT turning into NBPTO and other metabolites after few days, a stability test has been performed in order to study the NBPT degradation behavior in organo-mineral fertilizers over the 14-day time period.

Details of the study and its results are reported in Annex B.

11.0 Test report

The test report shall contain at least the following information:

a) a title, e.g. “Test report”;

b) the name and address of the laboratory, and the location where the test was carried out, if different from the address of the laboratory;

c) unique identification of the test report (such as the serial number) and on each page an identification in order to ensure that the page is recognized as a part of the test report and a clear identification of the end of the test report;

d) all information necessary for the complete identification of the sample;

e) the document used (including its year of publication), i.e. prEN 18317:2026;

f) the method used (if the standard includes several);

g) date and location of sampling, sampling procedure, sampling operator if known, and details of any environmental conditions during sampling that can affect the interpretation of the test result(s);

h) date when the test was started and finished;

i) the result(s) obtained with its unit of measurement, including a reference to the clause which explains how the results were calculated;

j) any operating details not specified in this document, or regarded as optional, together with details of any incidents occurred when performing the method, which influenced the test result(s);

k) any deviations from the procedure and any unusual features observed;

l) the name(s), function(s) and signature(s) or equivalent identification of person(s) authorizing the test report;

m) where relevant, a statement to the effect that the result(s) relate only to the items tested;

n) where applicable, a statement on the estimated uncertainty of measurement; information on uncertainty is needed in the test report when it is relevant to the validity or application of the test result(s), when the uncertainty affects compliance to a specification limit/tolerance.

(informative)

Results of the inter-laboratory study
1. Inter-laboratory study

The precision of the method has been determined in the year 2025 in an inter-laboratory study with 7 laboratories participating and carried out on 3 samples (Table A.1) of organo-mineral fertilizers commercially available.

Table A.1 — Materials tested in the inter-laboratory study

Sample type	FE-2–21: NPK 12‑8‑21 with 3 % N_org and 10 % C_org, solid
	FE-3–24: NPK 20‑10‑0 with 1 % N_org, 20 % SO₃ and 7,5 % C_org, solid
	FE-4–24: NPK 30‑0‑0 with 1 % N_org, 15 % SO₃ and 7 % C_org, solid

Samples were shaken well and divided into aliquots, labelled with the appropriate anonymous code and were maintained protected from light at +4°C ± 1 °C until shipment.

The procedure for the determination of NBPT was carried out in accordance with Clause 8.

Specific instructions are sent to laboratories only for the preparation of the final samples before the test: spike of NBPT standard solution on each organo-mineral fertilizer sample. The spike event was performed two times: the first at t = 0 and the second at t = 5 days after the first; during this time range the original samples were stored at 4 °C ± 2 °C. The NBPT and NBPTO determination was done immediately after each spike event.

The statistical results are given in Table A.2 and Table A.3.

1. Statistical results for the determination of NBPT and NBPTO by LC-MSQQQ

Table A.2 —Statistical results for determination of NBPT in organo-mineral fertilizers

Sample	FE-2-21	FE-3-24	FE-4-24
L	7	7	7
*L_A*	7	7	7
N	28	28	28
*N_A*	28	28	28
O	0,0	0,0	0,0
x̿	23,794	60,089	183,14
*s_R*	2,648	5,405	28,22
*s_r*	1,282	2,081	3,46
*s_I*	2,099	2,678	6,29
*RSD_R*	11,1	9,0	15,4
*RSD_r*	5,4	3,5	1,9
R	7,415	15,133	79,00
r	3,589	5,828	9,67
*HorRat*	1,1	1,0	2,1
L L_A N N_A O x̿ s_R s_r s_I RSD_R RSD_r R r HorRat	Number of participating laboratories; Number of laboratories after elimination of outliers; Number of all analytical values; Number of analytical values after rejection of outliers; Percentage of outliers, in %; Total mean of results (without outliers), in mg/kg; Reproducibility standard deviation in mg/kg; Repeatability standard deviation in mg/kg; Intermediate standard deviation in mg/kg; Relative reproducibility standard deviation, in %; Relative repeatability standard deviation, in %; Reproducibility limit (2,77 s_R), in mg/kg; Repeatability limit (2,77 s_r), in mg/kg; HorRat index.

Table A.3 — Statistical results for determination of NBPTO in organo-mineral fertilizers

Sample	FE-2-21	FE-3-24	FE-4-24
L	7	7	7
*L_A*	6	6	6
N	28	28	28
*N_A*	24	24	24
O	14,3	14,3	14,3
x̿	10,645	19,331	30,366
*s_R*	1,539	1,817	3,366
*s_r*	0,878	0,624	0,987
*s_I*	1,515	1,493	2,806
*RSD_R*	40,5	26,3	31,0
*RSD_r*	23,1	9,0	9,1
R	4,308	5,089	9,426
r	2,457	1,746	2,763
*HorRat*	1,3	0,9	1,2
L L_A N N_A O x̿ s_R s_r s_I RSD_R RSD_r R r HorRat	Number of participating laboratories; Number of laboratories after elimination of outliers; Number of all analytical values; Number of analytical values after rejection of outliers; Percentage of outliers, in %; Total mean of results (without outliers), in mg/kg; Reproducibility standard deviation in mg/kg; Repeatability standard deviation in mg/kg; Intermediate standard deviation in mg/kg; Relative reproducibility standard deviation, in %; Relative repeatability standard deviation, in %; Reproducibility limit (2,77 s_R), in mg/kg; Repeatability limit (2,77 s_r), in mg/kg; HorRat index.

In addition, the percentage of recovery as been calculated to evaluate the accuracy of the method. Table A.4 and Table A.5 show the recovery data for NBPT and NBPTO.

Table A.4 — Recovery data for NBPT

Sample	Spike	x̿	Recovery
Sample	mg/kg	mg/kg	%
FE-2–21	25	23,794	95
FE-3–24	65	60,089	92
FE-4–24	200	183,14	92

Table A.4 — Recovery data for NBPTO

Sample	Spike	x̿	Recovery
Sample	mg/kg	mg/kg	%
FE-2–21	10	10,645	106
FE-3–24	20	19,331	97
FE-4–24	30	30,366	91

(informative)

Stability test of NBPT in organo-mineral fertilizers
1. Stability test description

Due to the scientific literature [7], [8], [9], [10] showing the low stability of NBPT turning into NBPTO and other metabolites after few days, the study of its behavior over the 14-day time period was necessary.

A stability test has been performed to evaluate the degradation of NBPT; a standard solution of only NBPT has been added on the same sample type listed in Table A.1, as a spike at 3 different concentrations as shown in Table B.1.

Table B.1 — NBPT spike concentrations

Sample	NBPT on test sample (mg/kg)
FE-2-21	25
FE-3-24	65
FE-4-24	200

During this test, the spiked samples are stored at room temperature for 14 days.

The quantitative analysis of NBPT and NBPT-O formed has been performed immediately at the spike event (t = 0 d) at the 5th day (t = 5 d) and 14th day (t = 14 d) after the spike event.

The NBPT stability test results, calculated each from 14 data provided by the laboratories participating to interlaboratory study already described in Annex A, are reported in Table B.2 and a diagram of its degradation is reported in Figure B.1 and in Figure B.2.

Table B.2 — NBPT stability test results

Sample	NBPT (mean) at t = 0 d mg/kg	NBPTO formed (mean) at t = 0 d mg/kg	NBPT (mean) after t = 5 d mg/kg	NBPTO formed (mean) at t = 5 d mg/kg	NBPT (mean) after t = 14 d mg/kg	NBPTO formed (mean) at t = 14 d mg/kg
FE-2-21	21,32	0,52	14,61	0,77	7,94	-
FE-3-24	54,69	0,76	17,14	0,70	7,06	-
FE-4-24	158,26	0,89	129,95	2,67	85,98	3,58

The results show that in the organo-mineral fertilizer samples during the 14 days of the test, the NBPT degrades into its metabolites, one of which is the NBPTO but not the only one, as published in many scientific articles [7], [8], [9], [10].

Key

X	time (days)
Y	NBPT concentration (bright bar) in mg/kg
	time in days
a	FE 2‑21 at t = 0 days
b	FE 2‑21 at t = 5 days
c	FE 2‑21 at t = 14 days
d	FE 3‑24 at t = 0 days
e	FE 3‑24 at t = 5 days
f	FE 3‑24 at t = 14 days
g	FE 4‑24 at t = 0 days
h	FE 4‑24 at t = 5 days
i	FE 4‑24 at t = 14 days

Figure B.1 — NBPT stability test results

Key

X	time (days)
Y	NBPT (bright bar) and NBPTO (dark bar) concentration in mg/kg
	time in days
a	FE 2‑21 at t = 0 days
b	FE 2‑21 at t = 5 days
c	FE 2‑21 at t = 14 days
d	FE 3‑24 at t = 0 days
e	FE 3‑24 at t = 5 days
f	FE 3‑24 at t = 14 days
g	FE 4‑24 at t = 0 days
h	FE 4‑24 at t = 5 days
i	FE 4‑24 at t = 14 days

Figure B.2 — NBPT stability showing the NBPTO formation

Bibliography

[1] Regulation (EU) 2019/1009 of the European Parliament and of the Council of 5 June 2019 laying down rules on the making available on the market of EU fertilising products and amending Regulations (EC) No 1069/2009 and (EC) No 1107/2009 and repealing Regulation (EC) No 2003/2003

[2] EN 15688:2018, Fertilizers — Determination of urease inhibitor N-(nbutyl) thiophosphoric triamide (NBPT) using high-performance liquid chromatography (HPLC)

[3] EN ISO 1042:1999, Laboratory glassware — One mark volumetric flasks (ISO 1042:1998)

[4] ISO 5725‑1:2023, Accuracy (trueness and precision) of measurement methods and results — Part 1: General principles and definitions

[5] ISO 5725‑2:2019, Accuracy (trueness and precision) of measurement methods and results — Part 2: Basic method for the determination of repeatability and reproducibility of a standard measurement method

[6] ISO 5725‑3:2023, Accuracy (trueness and precision) of measurement methods and results — Part 3: Intermediate precision and alternative designs for collaborative studies

[7] Peters N., Thiele-Bruhn S. - Major metabolites of NBPT degradation pathways contribute to urease inhibition in soil. Chemosphere. 2022, 303 p. 135163

[8] Lasisi A.A., Akinremi O.O. - Degradation of N-(n-butyl) Thiophosphoric Triamide (NBPT) with and without nitrification inhibitor in soils. Nitrogen. 2022, 3 pp. 161–169

[9] Cantarella H. et al. - Stability of urease inhibitor added to urea, 2016 International Nitrogen Initiative Conference "Solutions to improve nitrogen use efficiency for the world", 4 – 8 December 2016, Melbourne, Australia

[10] Cantarella H. et al. - Agronomic efficiency of NBPT as a urease inhibitor: A review. J. Adv. Res. 2018, 13 pp. 19–27

[11] EN 1482‑1, Fertilizers, liming materials and inhibitors — Sampling and sample preparation — Part 1: General sampling provisions

[12] EN 1482‑3, Fertilizers, liming materials and inhibitors — Sampling and sample preparation — Part 3: Sampling of static heaps

Table of Contents

1.0 Scope

2.0 Normative references

3.0 Terms and definitions

4.0 Principle

5.0 Reagents

5.1 General

5.1.1 Reagents for liquid chromatography

5.1.2 Calibration standards

6.0 Equipment and consumables

7.0 Sampling and sample preparation

7.1 Sampling

7.1.1 Sample preparation

8.0 Procedure

8.1 General

8.1.1 Preparation of the test portion

8.1.2 Calibration

8.1.3 Blank test

9.0 Calculation and expression of the result

10.0 Precision

10.1 Inter-laboratory study

10.1.1 Repeatability

10.1.2 Reproducibility

10.1.3 NBPT Stability test

11.0 Test report