A method for determining the content of nbpt in urea

Abstract

The application discloses a method for determining the content of NBPT in urea, comprising the following steps: S1, preparation of a detection reagent; S2, sample pretreatment; S3, chromatography-mass spectrometry combined detection; and S4, quantitative analysis. The method directly injects a 0.1% formic acid aqueous solution-acetonitrile mobile phase system into an LC-MS / MS system, wherein the formic acid serves as an ionization promoter to significantly improve the protonation efficiency of NBPT under ESI + source, the response intensity of [M+H] + is greater than 10 5 cps, and the derivatization step necessary for a traditional HPLC-UV method is completely eliminated; secondly, a blank matrix extraction liquid is introduced to match a standard curve, and a standard sample is diluted by a urea / fertilizer matrix extraction liquid homologous to the sample to be measured, so that the ion suppression effect of humic acid on the target object is accurately offset, the standard addition recovery rate of a complex matrix sample is stably kept at 85%-99.5%, and the quantitative deviation is less than 5%.

Description

Technical Field

[0001] This invention belongs to the field of chemical detection technology, specifically relating to a method for determining the NBPT content in urea. Background Technology

[0002] The detection of NBPT (n-butylthiophosphoric triamine) in urea and urea-containing fertilizers mainly relies on the HPLC-UV derivatization method specified in the NY / T3038-2016 standard. This method requires converting NBPT into a UV-detectable derivative through a phenyl isocyanate derivatization reaction, followed by quantification by reversed-phase chromatography. However, this procedure has significant drawbacks:

[0003] The derivatization process is cumbersome and unstable. Derivatization reagents must be stored in the dark, and the reaction must be precisely controlled by a 60°C water bath for 30 minutes. The derivatized products are easily decomposed at room temperature (≤4h shelf life), resulting in low efficiency for batch detection. The ability to resist matrix interference is weak. Components such as biuret, ammonium salts and humic acid in urea can easily encapsulate the derivatives, causing chromatographic peak tailing or false positives. The spiked recovery rate for complex matrices such as compound fertilizers / organic fertilizers is only 70%-125%, which is difficult to meet the needs of trace detection (<0.1mg / kg).

[0004] Furthermore, although some studies have attempted non-derivative detection by LC-MS / MS, the low ionization efficiency of NBPT under electrospray ionization (ESI) and the interference from the urinary salt matrix have not been effectively addressed: insufficient mobile phase optimization and the lack of introduction of ionization promoters (such as formic acid) result in NBPT exhibiting low [M+H] ionization efficiency in conventional acetonitrile-water systems. + Response strength <10 4 The sensitivity of the CPs method is lower than that of the derivatization UV method; matrix matching correction is not used, and the standard curve is prepared directly with solvent. The ion inhibition effect of high concentration of ammonium ions (>5000 mg / kg) in urea on the target analyte is ignored, resulting in a signal attenuation of 30%-70% and a quantitative deviation of >15%. Therefore, we propose a method for determining the NBPT content in urea to specifically solve the above problems. Summary of the Invention

[0005] The purpose of this invention is to provide a method for determining the NBPT content in urea, which improves the ionization efficiency of NBPT in LC-MS / MS detection without the need for derivatization, and overcomes the interference of high salt and organic components in urea and compound fertilizer matrices on the quantification of trace NBPT, thereby achieving rapid and highly accurate direct detection.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for determining the NBPT content in urea, comprising the following steps:

[0008] S1. Preparation of detection reagents: Prepare mobile phase A containing 0.1% formic acid aqueous solution and acetonitrile as mobile phase B;

[0009] Preparation of matrix-matched standard solutions: Based on the standard with NBPT purity ≥98.0%, prepare a 1000 mg / L stock solution with water-methanol at a ratio of 1:1, and then dilute it with blank matrix extract to prepare working solutions of a series of concentrations from 0.01 to 1.0 mg / L.

[0010] S2. Sample pretreatment: Weigh 1-5g of urea-based sample, add 20mL of primary water, and extract by shaking at 25±2℃ for 30min.

[0011] After centrifuging the extract at 10000 r / min for 10 min, the supernatant was passed through a 0.22 μm aqueous filter membrane.

[0012] For complex matrix samples, the supernatant was purified by a C18 solid-phase extraction column, activated with 5 mL of methanol → 5 mL of water; 5 mL of methanol was eluted, the eluent was collected and purged with nitrogen until nearly dry, redissolved in 1 mL of water and filtered.

[0013] S3, Chromatography-mass spectrometry (GC-MS) detection, using an ESI source equipped with an electrospray ionization (ESI) source. + The LC-MS / MS system used a C18 reversed-phase column, 2.1 mm × 100 mm, 1.8 μm.

[0014] Gradient elution was performed: 20% B for 0-2 min → 80% B for 5 min → 80% B for 7 min → 20% B for 7.1 min, at a flow rate of 0.3 mL / min.

[0015] The mass spectrometer uses multiple reaction monitoring (MRM) mode, with the NBPT detection channel being the parent ion m / z 182.1 → daughter ion m / z 109.0, and the collision energy being 18 eV.

[0016] S4. Quantitative analysis: Quantitative analysis using a matrix-matched standard curve; calculate the NBPT content in the sample; the standard curve R... 2 ≥0.995, limit of quantitation (LOQ) ≤0.015 mg / kg.

[0017] Preferably, in the preparation of the blank matrix extract in step S1, for the urea matrix, pure urea raw material without NBPT is selected, and after being crushed and passed through a 100-mesh sieve, it is operated according to the solid sample extraction process: 1g sample + 20mL primary water, shaken at 25℃ for 30min; the concentration gradient of the matrix matching standard working solution is 0.01, 0.05, 0.1, 0.5, 1.0mg / L.

[0018] Preferably, the purification operation of the complex matrix sample in step S2 meets the following conditions:

[0019] When the eluent from the C18 solid-phase extraction column is concentrated by purging with nitrogen, the water bath temperature is controlled to be ≤40℃. After purging to a residual volume of less than 0.5mL, the volume is adjusted to 1mL with water.

[0020] After reconstitution, a 0.22μm aqueous polyethylene filter membrane is used for filtration to avoid adsorption loss of NBPT on organic filter membranes;

[0021] The C18 column activation procedure for purifying complex matrix samples includes: sequentially passing 5 mL of methanol and 5 mL of primary water through the column at a flow rate of ≤2 mL / min, and discarding the waste liquid; loading the sample at a flow rate of 1 mL / min to prevent the target analyte from penetrating.

[0022] Preferably, in the gradient elution of step S3, the mobile phase composition during the equilibrium phase of 7.1-10 min is phase A: 0.1% formic acid aqueous solution and phase B: acetonitrile, and the proportion of phase B drops from 80% to 20% and is maintained for 10 min.

[0023] Preferred ESI for mass spectrometry detection + The source parameters must simultaneously satisfy:

[0024] The ion source temperature was 150±5℃, and the desolvation gas temperature was 350±10℃.

[0025] The desolventizing gas flow rate is 800±50L / h, and the cone voltage is set to 30±2V for NBPT.

[0026] Preferably, when the method is applied to urea compound fertilizer containing humic acid, the purification operation in step S2 is performed by C18 column solid-phase extraction, and the rinsing is done with 5 mL of 5% methanol aqueous solution to remove the inhibition of NBPT ionization by humic acid.

[0027] Preferably, the quantitative analysis in step S4 needs to meet the following requirements:

[0028] LOQ≤0.015mg / kg of NBPT in the sample;

[0029] When the NBPT concentration of the sample is ≥ LOQ, the signal-to-noise ratio (S / N) of the quantitative ion pair m / z 182.1→109.0 is ≥ 10.

[0030] Preferably, the column temperature in step S3 is set to 35±1℃, and the column pressure fluctuation range is ≤±5%. When detecting urea samples with a salt content >1%, a 0.5μm sieve plate online filter is connected in series before the injector.

[0031] Preferably, the method performs three-level validation during the spiked quality control stage:

[0032] Low concentration spiking: 0.02 mg / kg;

[0033] Medium concentration spiked at 0.1 mg / kg;

[0034] For high-concentration spikes of 0.5 mg / kg, the acceptable recovery rate is 85%-99.5%, with RSD ≤ 5%.

[0035] The technical effects and advantages of this invention are as follows:

[0036] A 0.1% formic acid aqueous solution-acetonitrile mobile phase system was directly injected into the LC-MS / MS system. Formic acid, acting as an ionization promoter, significantly improved the efficiency of NBPT in ESI. + Protonation efficiency at the source, [M+H] + Response strength > 10 5 cps completely eliminates the derivatization step required by traditional HPLC-UV methods;

[0037] Secondly, a blank matrix extract was introduced to match the standard curve. The standard was diluted with a urea / fertilizer matrix extract that was homologous with the sample to be tested. This accurately counteracted the ion inhibition effect of humic acid (high concentration of ammonium salt >5000 mg / kg) in urea on the target analyte, so that the spiked recovery rate of complex matrix samples was stabilized at 85%-99.5% and the quantitative deviation was <5%.

[0038] Meanwhile, the gradient elution coupled dual MRM ion pair monitoring was optimized to complete high-resolution analysis of NBPT within 10 minutes, with a resolution R > 1.5. The false positive rate was reduced by using dual-channel verification of the parent ion m / z 182.1 → daughter ion m / z 109.0 for quantification and m / z 140.0 for qualitative analysis. Detailed Implementation

[0039] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] This invention discloses a method for determining the NBPT content in urea, which completely overcomes the bottlenecks of existing technologies through three core innovations: derivatization-free LC-MS / MS detection using a 0.1% formic acid-acetonitrile mobile phase system, and ESI of NBPT. + The ionization response intensity is increased to >10. 5 CPS is improved compared to traditional methods;

[0041] Matrix-matched dynamic calibration uses a standard curve constructed with a homologous blank matrix extract to completely offset the inhibitory effects of biuret, humic acid, and 5000 mg / kg ammonium ions, stabilizing the recovery rate under complex fertilizer matrices at 85%-99.5%.

[0042] The dual-channel MRM validation technology (m / z 182.1→109.0 / 140.0) combined with a 10-minute gradient elution program achieves zero false detection of interfering substances (R>1.5), achieving a breakthrough in the accurate detection of trace NBPT: the detection limit is reduced to 0.005 mg / kg, and the detection time for a single sample is shortened by 70% to 30 minutes, providing key technical support for the quality monitoring of controlled-release agents in the urea industry;

[0043] The method for determining the NBPT content in urea includes the following steps:

[0044] S1. Preparation of detection reagents: Prepare mobile phase A containing 0.1% formic acid aqueous solution and acetonitrile as mobile phase B;

[0045] Preparation of matrix-matched standard solutions: Based on the standard with NBPT purity ≥98.0%, prepare a 1000 mg / L stock solution with water-methanol at a ratio of 1:1, and then dilute it with blank matrix extract to prepare working solutions of a series of concentrations from 0.01 to 1.0 mg / L.

[0046] It should be noted that when preparing the blank matrix extract, for the urea matrix, pure urea raw material without NBPT was selected, and after being crushed and passed through a 100-mesh sieve, it was operated according to the solid sample extraction procedure (1g sample + 20mL primary water, shaken at 25℃ for 30min); the concentration gradient of the matrix matching standard working solution was 0.01, 0.05, 0.1, 0.5, 1.0mg / L;

[0047] In the preparation of matrix-matched standard solutions, the blank matrix extract must be derived from the same type of fertilizer matrix (urea / compound fertilizer / organic fertilizer) as the test sample, and after extraction, it must be stored at 4°C and used for dilution within 24 hours.

[0048] S2. Sample pretreatment: Weigh 1-5g of urea-based sample, add 20mL of primary water, and extract by shaking at 25±2℃ for 30min.

[0049] After centrifuging the extract at 10000 r / min for 10 min, the supernatant was passed through a 0.22 μm aqueous filter membrane.

[0050] For complex matrix samples, the supernatant was purified by a C18 solid-phase extraction column, activated with 5 mL of methanol → 5 mL of water; 5 mL of methanol was eluted, the eluent was collected and purged with nitrogen until nearly dry, redissolved in 1 mL of water and filtered.

[0051] Furthermore, the purification process for complex matrix samples must meet the following conditions:

[0052] When the eluent from the C18 solid-phase extraction column is concentrated by purging with nitrogen, the water bath temperature is controlled to be ≤40℃. After purging to a residual volume of less than 0.5mL, the volume is adjusted to 1mL with water.

[0053] After reconstitution, filtration must be performed using a 0.22μm aqueous polyethylene filter membrane to avoid adsorption loss of NBPT on the organic filter membrane.

[0054] S3, Chromatography-mass spectrometry (GC-MS) detection, using an ESI source equipped with an electrospray ionization (ESI) source. + The LC-MS / MS system used a C18 reversed-phase column, 2.1 mm × 100 mm, 1.8 μm.

[0055] Gradient elution was performed: 20% B for 0-2 min → 80% B for 5 min → 80% B for 7 min → 20% B for 7.1 min, at a flow rate of 0.3 mL / min. In the gradient elution program, the mobile phase composition during the equilibration phase (7.1-10 min) was phase A: 0.1% formic acid aqueous solution and phase B: acetonitrile. The proportion of phase B was stepped down from 80% to 20% and maintained until 10 min. The total run time was shortened by more than 40% compared with the conventional HPLC-UV method.

[0056] Mass spectrometry employed multiple reaction monitoring (MRM) mode, with the NBPT detection channel showing the precursor ion at m / z 182.1 → daughter ion at m / z 109.0, and a collision energy of 18 eV; the ESI detection was performed using mass spectrometry. + The source parameters must simultaneously satisfy:

[0057] The ion source temperature was 150±5℃, and the desolvation gas temperature was 350±10℃.

[0058] The desolventizing gas flow rate is 800±50L / h, and the cone voltage is set to 30±2V for NBPT.

[0059] MRM monitoring channel expanded for dual ion pair verification:

[0060] Main quantitative channel: m / z 182.1→109.0 (collision energy 18eV);

[0061] Auxiliary qualitative channel: m / z 182.1→140.0 (collision energy 15eV), the allowable deviation of the peak area ratio of the two channels ≤±15%;

[0062] In addition, the column temperature was set to 35±1℃, and the column pressure fluctuation range was ≤±5%. When detecting urea samples with a salt content >1%, a 0.5μm sieve plate online filter was connected in series before the injector.

[0063] S4. Quantitative analysis: Quantitative analysis using a matrix-matched standard curve; calculate the NBPT content in the sample; the standard curve R...2 ≥0.995, limit of quantitation (LOQ) ≤0.015 mg / kg; Quantitative analysis must meet the following requirements:

[0064] LOQ≤0.015mg / kg of NBPT in the sample;

[0065] When the NBPT concentration of the sample is ≥ LOQ, the signal-to-noise ratio (S / N) of the quantitative ion pair m / z 182.1→109.0 is ≥ 10.

[0066] When the above method is applied to urea compound fertilizer containing humic acid, the purification operation in step S2 is performed by solid-phase extraction on a C18 column, and 5 mL of 5% methanol aqueous solution is used for rinsing to remove the inhibition of NBPT ionization by humic acid.

[0067] The method undergoes three-level validation during the spiked quality control phase:

[0068] Low concentration spiking: 0.02 mg / kg;

[0069] Medium concentration spiked at 0.1 mg / kg;

[0070] For high-concentration spikes of 0.5 mg / kg, the acceptable recovery rate is 85%-99.5%, with RSD ≤ 5%.

[0071] The above method provides false positive protection for dual-ion pair verification (182.1→109.0 and 182.1→140.0), with a clearly defined deviation threshold of ±15%.

[0072] The three concentration spikes and the recovery rate range (85-115%) are directly related to the accuracy parameters of the method; the coupling of the cone voltage (30±2V) and the collision energy (18eV) ensures the fragmentation efficiency of NBPT, and the column temperature control of ±1℃ reduces retention time drift; the method distinguishes between scenarios such as liquid samples without shaking, humic acid fertilizers with forced SPE purification, and high-salt samples using online filters; the method limits the use of water-based polyethylene filter membranes to avoid adsorption, and specifies the C18 column flow rate (≤2mL / min for activation, ≤1mL / min for loading) to prevent the loss of target substances.

[0073] The chromatographic conditions for the above method are shown in the table below:

[0074] Table 1 Chromatographic conditions

[0075]

[0076] Mass spectrometry conditions (ESI) + The patterns are shown in the table below:

[0077] Table 2 Mass Spectrometry Conditions

[0078]

[0079] Based on the above, the following embodiments are possible:

[0080] Example 1

[0081] Operating procedure: Weigh 10g of urea sample, add 50mL of 80% methanol aqueous solution and shake to extract; purify the extract through a 0.22μm filter membrane, and use the supernatant as the test solution; prepare a 0.1-50μg / LNBPT standard curve using blank urea matrix extract; LC-MS / MS conditions: mobile phase A 0.1% formic acid aqueous solution, B acetonitrile; gradient program: 0-2min 5%B → 6min 90%B → 8min 90%B; MRM channel: 182.1 → 109.0 (quantitative), 182.1 → 140.0 (qualitative).

[0082] Effect:

[0083] Breakthrough in ionization efficiency: NBPT's [M+H] + Response reaches 1.5×10 5 The cps (12 times higher than the system without formic acid) are high; the trace detection capability is as low as 0.005 mg / kg (5 times higher than HPLC-UV method); the resistance to matrix interference is high, with a recovery rate of 98.3±2.1% when biuret (>0.5%) coexists in urea (compared to only 85±15% by traditional derivatization method); and the timeliness is fast, with a single sample detection time of 30 minutes (compared to 3 hours by derivatization method).

[0084] Example 2

[0085] The operation procedure was as follows: the compound fertilizer (containing 35% urea and 15% humic acid) sample was extracted by ultrasonic extraction with methanol-water (1:1); the standard curve was diluted with the blank extract of humic acid and urea from the same batch; the LC conditions were improved by adding a guard column (Shim-pack Scepter HD-C18) and extending the gradient to 12 min to separate the humic acid interference peak.

[0086] Effect:

[0087] Precise separation: The resolution between NBPT and humic acid characteristic peaks was R=2.3 (>1.5 standard), with no co-eluent interference; resistant to humic acid interference: In the presence of 50 mg / L humic acid, the NBPT recovery rate was 102.5±3.8% (the recovery rate of the traditional LC-MS / MS method fluctuated to 68%); Dual-channel validation of reliability: The relative deviation of the qualitative ion ratio (109.0 / 140.0) was <0.5% (the false positive problem in the background technique was eliminated); Reproducibility: The RSD of 6 repeated detections was 2.1% (the RSD of the derivatization method was >8%).

[0088] Example 3

[0089] The operating procedure simulates an industrial controlled-release fertilizer sample (containing formaldehyde condensate + metal salt) and extracts it with 0.1% formic acid and methanol. The calibration curve uses three sets of parallel matrix matching: ordinary urea + formaldehyde condensate + zinc salt blank extract. The gradient is optimized to 0-10 min 5%-95% B to ensure that the metal complex elutes after NBPT.

[0090] Effect:

[0091] Eliminating metal ion inhibition, when 5000 mg / kg ammonium ions and 200 mg / kg zinc salt coexisted, the NBPT signal attenuation decreased from -65% to -3.5%.

[0092] Spiked recovery was consistent, with recoveries of 96.7% / 99.2% / 97.8% under the three matrix curves (direct solvent calibration deviation > 25%).

[0093] Specific enhancement: The characteristic ion (m / z 213.0) of formaldehyde condensate showed no cross-response in the NBPT channel;

[0094] In actual samples, a controlled-release fertilizer was found to contain 0.021 mg / kg of residual NBPT.

[0095] From the above three sets of examples, it can be concluded that the complexity of this method increases from basic urea to humic acid compound fertilizer to industrial-grade controlled-release fertilizer. The quantifiable effect directly addresses the pain points. The low sensitivity of LC-MS / MS is solved by improving the ionization efficiency by 12 times; the false positive caused by matrix interference is solved by the recovery rate under the coexistence of humic acid; and the high salt ion inhibition is overcome by the -3.5% signal attenuation of the metal salt matrix. The reproducibility RSD of the three sets of examples is <3% (derivatization methods are usually >8%). The detection limit of 0.005 mg / kg reaches the highest accuracy in the industry (background technology requires 0.1 mg / kg).

[0096] In summary, this invention employs a 0.1% formic acid aqueous solution-acetonitrile mobile phase system directly injected into the LC-MS / MS system. The formic acid in this system acts as an ionization promoter, significantly enhancing the performance of NBPT in ESI. + Protonation efficiency at the source, [M+H] + Response strength > 10 5 cps completely eliminates the derivatization step required by traditional HPLC-UV methods;

[0097] Secondly, a blank matrix extract was introduced to match the standard curve. The standard was diluted with a urea / fertilizer matrix extract that was homologous with the sample to be tested. This accurately counteracted the ion inhibition effect of humic acid (high concentration of ammonium salt >5000 mg / kg) in urea on the target analyte, so that the spiked recovery rate of complex matrix samples was stabilized at 85%-99.5% and the quantitative deviation was <5%.

[0098] Meanwhile, the gradient elution coupled dual MRM ion pair monitoring was optimized to complete high-resolution analysis of NBPT within 10 minutes, with a resolution R > 1.5. The false positive rate was reduced by using dual-channel verification of the parent ion m / z 182.1 → daughter ion m / z 109.0 for quantification and m / z 140.0 for qualitative analysis.

[0099] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for determining the NBPT content in urea, characterized in that, Includes the following steps: S1. Preparation of detection reagents: Prepare mobile phase A containing 0.1% formic acid aqueous solution and acetonitrile as mobile phase B; Preparation of matrix-matched standard solutions: Based on the standard with NBPT purity ≥98.0%, prepare a 1000 mg / L stock solution with water-methanol at a ratio of 1:1, and then dilute it with blank matrix extract to prepare working solutions of a series of concentrations from 0.01 to 1.0 mg / L. S2. Sample pretreatment: Weigh 1-5g of urea-based sample, add 20mL of primary water, and extract by shaking at 25±2℃ for 30min. After centrifuging the extract at 10000 r / min for 10 min, the supernatant was passed through a 0.22 μm aqueous filter membrane. For complex matrix samples, the supernatant was purified by a C18 solid-phase extraction column, activated with 5 mL of methanol → 5 mL of water; 5 mL of methanol was eluted, the eluent was collected and purged with nitrogen until nearly dry, redissolved in 1 mL of water and filtered. S3, Chromatography-mass spectrometry (GC-MS) detection, using an ESI source equipped with an electrospray ionization (ESI) source. + The LC-MS / MS system used a C18 reversed-phase column, 2.1 mm × 100 mm, 1.8 μm. Gradient elution was performed: 20% B for 0-2 min → 80% B for 5 min → 80% B for 7 min → 20% B for 7.1 min, at a flow rate of 0.3 mL / min. The mass spectrometer uses multiple reaction monitoring (MRM) mode, with the NBPT detection channel being the parent ion m / z 182.1 → daughter ion m / z 109.0, and the collision energy being 18 eV. S4. Quantitative analysis: Quantitative analysis using a matrix-matched standard curve; calculate the NBPT content in the sample; the standard curve R... 2 ≥0.995, limit of quantitation (LOQ) ≤0.015 mg / kg.

2. The method for determining the NBPT content in urea according to claim 1, characterized in that, In step S1, when preparing the blank matrix extract, pure urea raw material without NBPT is selected for the urea matrix. After being crushed and passed through a 100-mesh sieve, the solid sample extraction process is followed: 1g sample + 20mL primary water, shaken at 25℃ for 30min. The concentration gradient of the matrix matching standard working solution is 0.01, 0.05, 0.1, 0.5, 1.0mg / L.

3. The method for determining the NBPT content in urea according to claim 1, characterized in that, The purification operation of the complex matrix sample in step S2 meets the following conditions: When the eluent from the C18 solid-phase extraction column is concentrated by purging with nitrogen, the water bath temperature is controlled to be ≤40℃. After purging to a residual volume of less than 0.5mL, the volume is adjusted to 1mL with water. After reconstitution, a 0.22μm aqueous polyethylene filter membrane is used for filtration to avoid adsorption loss of NBPT on organic filter membranes; The C18 column activation procedure for purifying complex matrix samples includes: sequentially passing 5 mL of methanol and 5 mL of primary water through the column at a flow rate of ≤2 mL / min for activation, and then discarding the waste liquid; The loading flow rate is 1 mL / min to prevent the target substance from penetrating.

4. The method for determining the NBPT content in urea according to claim 1, characterized in that, In the gradient elution of step S3, the mobile phase composition during the equilibrium phase of 7.1-10 min is phase A: 0.1% formic acid aqueous solution and phase B: acetonitrile, and the proportion of phase B drops from 80% to 20% and is maintained for 10 min.

5. The method for determining the NBPT content in urea according to claim 1, characterized in that, ESI for mass spectrometry detection + The source parameters must simultaneously satisfy: The ion source temperature was 150±5℃, and the desolvation gas temperature was 350±10℃. The desolventizing gas flow rate is 800±50L / h, and the cone voltage is set to 30±2V for NBPT.

6. The method for determining the NBPT content in urea according to claim 1, characterized in that, When the method is applied to urea compound fertilizer containing humic acid, the purification operation in step S2 is performed by solid-phase extraction using a C18 column, and the rinsing is done with 5 mL of 5% methanol aqueous solution to remove the inhibition of NBPT ionization by humic acid.

7. The method for determining the NBPT content in urea according to claim 1, characterized in that, The quantitative analysis in step S4 must meet the following requirements: LOQ≤0.015mg / kg of NBPT in the sample; When the NBPT concentration of the sample is ≥ LOQ, the signal-to-noise ratio (S / N) of the quantitative ion pair m / z 182.1→109.0 is ≥ 10.

8. The method for determining the NBPT content in urea according to claim 1, characterized in that, In step S3, the column temperature is set to 35±1℃ and the column pressure fluctuation range is ≤±5%. When detecting urea samples with a salt content >1%, a 0.5μm sieve plate online filter is connected in series before the injector.

9. The method for determining the NBPT content in urea according to claim 1, characterized in that, The method undergoes three-level validation during the spiked quality control phase: Low concentration spiking: 0.02 mg / kg; Medium concentration spiked at 0.1 mg / kg; For high-concentration spikes of 0.5 mg / kg, the acceptable recovery rate is 85%-99.5%, with RSD ≤ 5%.

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