Method for detecting perfluorinated compounds in urine

Abstract

This invention discloses a method for detecting perfluorinated compounds in urine, belonging to the field of analytical detection technology. The method includes: mixing urine with a 2% formic acid-methanol solution, passing the mixture through a WAX solid-phase extraction column pre-activated with a 0.1% ammonia-methanol solution and a 1% formic acid-water solution, followed by sequential elution with a 1% formic acid-water solution, a 1% formic acid-methanol solution, and methanol; drying under pressure; eluting with a 0.1% ammonia-methanol solution; collecting the eluent; drying the eluent; redissolving the eluent with methanol; filtering to obtain the test solution; detecting the test solution using liquid chromatography-tandem mass spectrometry; and calculating the perfluorinated compound content in the urine based on a standard curve. This invention significantly improves the separation of perfluorinated compounds, exhibits low matrix effect, high sensitivity, and good reproducibility, and is suitable for trace detection of perfluorinated compounds in urine.

Description

Technical Field

[0001] This invention relates to a method for detecting perfluorinated compounds in urine, belonging to the field of analytical detection technology. Background Technology

[0002] Perfluorinated compounds (PFCs) are a class of synthetic organic compounds in which fluorine atoms partially or completely replace hydrogen atoms in the carbon chain. Due to their excellent hydrophobicity, oleophobicity, heat resistance, and chemical stability, they are widely used in textiles, leather, surfactants, fire-fighting foams, and food packaging coatings. Studies have shown that PFCs are persistent, bioaccumulative, and possess various toxicities (such as hepatotoxicity, reproductive toxicity, thyroid interference, and potential carcinogenicity), making them a new type of persistent organic pollutant of global concern. Humans are mainly exposed to PFCs through food, drinking water, inhalation, and skin contact. In the body, they primarily bind to serum proteins and are ultimately excreted in urine. Urine, as a non-invasive and readily available biological sample, is an ideal matrix for assessing human internal exposure levels of PFCs. Therefore, establishing accurate, sensitive, and reliable methods for detecting PFCs in human urine is of significant practical importance for population exposure risk assessment, environmental epidemiological studies, and health monitoring.

[0003] Currently, the detection of perfluorinated compounds in urine mainly employs liquid chromatography-tandem mass spectrometry (LC-MS / MS). This method typically includes two key steps: sample pretreatment and instrumental analysis.

[0004] In terms of sample pretreatment, existing technologies mainly fall into two categories: one is simple liquid-liquid extraction or protein precipitation methods, such as CN115754101A, which discloses a method for the simultaneous detection of perfluorinated and polyfluoroalkyl substances in urine, using acetonitrile as the extractant, repeating the extraction three times before direct injection for analysis. Although this method is simple to operate and consumes little solvent, the complex urine matrix (containing a large amount of salts, urea, proteins, metabolites, etc.) easily produces a strong matrix effect, leading to ion inhibition or enhancement, affecting quantitative accuracy and detection sensitivity. The other category is solid-phase extraction (SPE), which uses a weak anion exchange (WAX) solid-phase extraction column for purification, but the matrix effect is still quite significant, and the recovery rate and reproducibility need to be improved. In addition, although many published patents (such as CN120141972A, CN120084927A, CN116930349A, etc.) involve solid-phase extraction methods for perfluorinated compounds in matrices such as water environment, soil, and biological tissues, their rinsing and elution conditions are not designed for urine matrix. When used directly on urine, they are difficult to effectively remove endogenous interfering substances and cannot meet the requirements of trace analysis.

[0005] In instrumental analysis, the selection of liquid chromatography conditions is crucial for the resolution and sensitivity of perfluorinated compounds. In existing technologies, pure methanol or pure acetonitrile is commonly used as the organic phase in the mobile phase. For example, CN115754101A uses methanol, CN116930349A uses methanol, and CN120084927A uses acetonitrile. However, extensive experiments have revealed that when using pure methanol or pure acetonitrile as the organic phase, various perfluorinated compounds in urine (especially homologues with similar carbon chain lengths and certain sulfonic acid compounds) exhibit co-elution or insufficient resolution, leading to difficulties in accurate quantification. This technical problem has not been mentioned or resolved in existing literature and patents.

[0006] Furthermore, the sensitivity and stability of mass spectrometry for perfluorinated compounds in existing technologies are difficult to guarantee.

[0007] In summary, the existing technology lacks a detection method for urine matrix that can simultaneously achieve good separation of multiple perfluorinated compounds, with low matrix effect, high sensitivity, and high stability. Summary of the Invention

[0008] The purpose of this invention is to provide a new method for detecting perfluorinated compounds in urine.

[0009] To achieve the objective of this invention, the method for detecting perfluorinated compounds in urine includes the following steps: (1) Sample pretreatment: Mix urine with 2% formic acid methanol solution, pass it through a WAX solid phase extraction column that has been pre-activated with 0.1% ammonia methanol solution and 1% formic acid aqueous solution, then wash with 1% formic acid aqueous solution, 1% formic acid methanol solution and methanol in sequence, dry under pressure, then elute with 0.1% ammonia methanol solution, collect the eluent, dry it, redissolve it with methanol, filter it to obtain the test solution; The volume ratio of urine: 2% formic acid methanol solution: 0.1% ammonia methanol solution for activation: 1% formic acid aqueous solution for activation: 1% formic acid aqueous solution for rinsing: 1% formic acid methanol solution for rinsing: methanol for rinsing: 0.1% ammonia methanol solution for elution: methanol for reconstitution is 1 : 1 : 1.25 : 1.25 : 0.75 : 0.75 : 0.25 : 1.5 : 0.25; The percentage content in the 2% formic acid methanol solution, 1% formic acid aqueous solution, 1% formic acid methanol solution, and 0.1% ammonia methanol solution are all volume percentages. (2) The test solution was detected by liquid chromatography-tandem mass spectrometry. The liquid chromatography conditions were as follows: the column was a C18 column, the mobile phase A was 5 mM ammonium acetate aqueous solution, and the mobile phase B was a 50% methanol-50% acetonitrile mixed solution. The mobile phase was used for gradient elution. The gradient elution program was as follows: 0-10 min, A phase volume 80% to 0%, B phase volume 20% to 100%; 10-15 min, A phase volume 0%, B phase volume 100%; 15-15.01 min, A phase volume 0% to 80%, B phase volume 100% to 20%; 15.01-25 min, A phase volume 80%, B phase volume 20%; the flow rate was 0.27-0.33 mL / min, the column temperature was 30-35℃, and the injection volume was 1 μL. The mass spectrometry conditions were: electrospray negative ion mode, multiple reaction monitoring, dryer gas temperature 350℃, dryer gas flow rate 8 L / min, nebulizer pressure 35 psi, capillary voltage 4500 V, sheath gas temperature 350℃, sheath gas flow rate 10 L / min, and nozzle voltage 0 V. (3) Calculate the content of perfluorinated compounds in urine based on the standard curve.

[0010] The drying process described in step (1) can be at least one of natural evaporation, nitrogen blowing to dry, or nitrogen blowing to near dry.

[0011] In one specific embodiment, the WAX ​​solid-phase extraction column has a specification of 150 mg / 6 mL.

[0012] In one specific embodiment, the filtration uses a 0.22 μm nylon filter membrane.

[0013] In one specific embodiment, the chromatographic column is an Agilent Proshell EC-C18 column with dimensions of 2.1 mm × 100 mm and a diameter of 2.7 μm; the flow rate is 0.3 mL / min.

[0014] In one specific embodiment, the perfluorinated compound is at least one selected from perfluorobutyric acid, perfluorovaleric acid, perfluorohexanoic acid, perfluoroheptanoic acid, perfluorooctanoic acid, perfluorononanoic acid, perfluorodecanoic acid, perfluoroundecanoic acid, perfluorododecanic acid, perfluorotridecanoic acid, perfluorotetradecanoic acid, perfluorohexadecanoic acid, perfluorobutanesulfonic acid, perfluorohexanesulfonic acid, perfluorooctanesulfonic acid, and perfluorodecanesulfonic acid.

[0015] In one specific embodiment, the standard curve is a solvent standard curve, and a series of standard working solutions are prepared using methanol.

[0016] In one specific embodiment, the average spiked recovery rate of the method is 72.01% to 115.59%, the relative standard deviation is 0.09% to 3.9%, the limit of detection is 0.0075 to 0.0625 ng / mL, and the limit of quantitation is 0.025 to 0.125 ng / mL.

[0017] In one specific embodiment, under the multiple reaction monitoring mode of the tandem mass spectrometry, the parent ion, daughter ion, source fragmentation voltage, and collision energy of each perfluorinated compound are shown in Table 1:

[0018] In one specific embodiment, during the qualitative determination, the retention time of the chromatographic peak of the target analyte in the sample solution deviates from the retention time of the standard working solution by within ±2.5%, and the relative abundance of each qualitative ion compared with the standard is allowed to deviate by ±20% when the relative ion abundance is >50%, ±25% when it is >20% to 50%, ±30% when it is >10% to 20%, and ±50% when it is ≤10%.

[0019] In one specific embodiment, under chromatographic conditions of column temperature 32–37°C, flow rate 0.27–0.33 mL / min, and initial mobile phase B volume ratio of 18%–22%, the relative standard deviation of the determination results between different chromatographic conditions is less than 4.66%.

[0020] Beneficial effects

[0021] This invention solves the technical problems existing in the prior art, such as insufficient separation, strong matrix effect, non-specific purification conditions, insufficient sensitivity, and poor method robustness, as detailed below: 1. Significantly improved separation performance: This invention significantly improves the separation degree between various perfluorinated compounds, achieving baseline separation (see...). Figures 1-3 ).

[0022] 2. Low matrix effect: This invention effectively removes salts and polar interfering substances from urine, with the matrix effect controlled between -2.0% and 38.3%.

[0023] 3. High sensitivity: The detection limit of this invention is 0.0075 to 0.0625 ng / mL, and the quantitation limit is 0.025 to 0.125 ng / mL.

[0024] 4. High accuracy and precision: The average spiked recovery rate is 72.01% to 115.59%, and the relative standard deviation is 0.09% to 3.9%.

[0025] 5. The method is robust: the sample is stable within 24 hours; under chromatographic conditions of column temperature 32-37℃, flow rate 0.27-0.33 mL / min, and initial mobile phase B ratio 18%-22%, the relative standard deviation of the determination results under different conditions is less than 4.66%; the method has good transferability as verified by two external laboratories. Attached Figure Description

[0026] Figure 1 The total ion chromatogram (TIC) when mobile phase B is pure methanol shows that some perfluorinated compounds co-elute or the separation is insufficient.

[0027] Figure 2 The total ion chromatogram (TIC) when mobile phase B is pure acetonitrile shows that some perfluorinated compounds co-elute or the separation is insufficient.

[0028] Figure 3 Total ion chromatogram (TIC) of mobile phase B as a 50% methanol-50% acetonitrile mixed solution shows baseline separation of each perfluorinated compound.

[0029] Figure 4 Linearity results for perfluorobutyric acid (PFOA).

[0030] Figure 5 : Multiple reaction monitoring (MRM) extracted ion chromatogram of perfluorobutyric acid.

[0031] Figure 6 Distribution of matrix effect results for 16 perfluorinated compounds. Detailed Implementation

[0032] To achieve the objective of this invention, the method for detecting perfluorinated compounds in urine includes the following steps: (1) Sample pretreatment: Mix urine with 2% formic acid methanol solution, pass it through a WAX solid phase extraction column that has been pre-activated with 0.1% ammonia methanol solution and 1% formic acid aqueous solution, then wash with 1% formic acid aqueous solution, 1% formic acid methanol solution and methanol in sequence, dry under pressure, then elute with 0.1% ammonia methanol solution, collect the eluent, dry it, redissolve it with methanol, filter it to obtain the test solution; The volume ratio of urine: 2% formic acid methanol solution: 0.1% ammonia methanol solution for activation: 1% formic acid aqueous solution for activation: 1% formic acid aqueous solution for rinsing: 1% formic acid methanol solution for rinsing: methanol for rinsing: 0.1% ammonia methanol solution for elution: methanol for reconstitution is 1 : 1 : 1.25 : 1.25 : 0.75 : 0.75 : 0.25 : 1.5 : 0.25; The percentage content in the 2% formic acid methanol solution, 1% formic acid aqueous solution, 1% formic acid methanol solution, and 0.1% ammonia methanol solution are all volume percentages. (2) The test solution was detected by liquid chromatography-tandem mass spectrometry. The liquid chromatography conditions were as follows: the column was a C18 column, the mobile phase A was 5 mM ammonium acetate aqueous solution, and the mobile phase B was a 50% methanol-50% acetonitrile mixed solution. The mobile phase was used for gradient elution. The gradient elution program was as follows: 0-10 min, A phase volume 80% to 0%, B phase volume 20% to 100%; 10-15 min, A phase volume 0%, B phase volume 100%; 15-15.01 min, A phase volume 0% to 80%, B phase volume 100% to 20%; 15.01-25 min, A phase volume 80%, B phase volume 20%; the flow rate was 0.27-0.33 mL / min, the column temperature was 30-35℃, and the injection volume was 1 μL. The mass spectrometry conditions were: electrospray negative ion mode, multiple reaction monitoring, dryer gas temperature 350℃, dryer gas flow rate 8 L / min, nebulizer pressure 35 psi, capillary voltage 4500 V, sheath gas temperature 350℃, sheath gas flow rate 10 L / min, and nozzle voltage 0 V. (3) Calculate the content of perfluorinated compounds in urine based on the standard curve.

[0033] The drying process described in step (1) can be at least one of natural evaporation, nitrogen blowing to dry, or nitrogen blowing to near dry.

[0034] In one specific embodiment, the WAX ​​solid-phase extraction column has a specification of 150 mg / 6 mL.

[0035] In one specific embodiment, the filtration uses a 0.22 μm nylon filter membrane.

[0036] In one specific embodiment, the chromatographic column is an Agilent Proshell EC-C18 column with dimensions of 2.1 mm × 100 mm and a diameter of 2.7 μm; the flow rate is 0.3 mL / min.

[0037] In one specific embodiment, the perfluorinated compound is at least one selected from perfluorobutyric acid, perfluorovaleric acid, perfluorohexanoic acid, perfluoroheptanoic acid, perfluorooctanoic acid, perfluorononanoic acid, perfluorodecanoic acid, perfluoroundecanoic acid, perfluorododecanic acid, perfluorotridecanoic acid, perfluorotetradecanoic acid, perfluorohexadecanoic acid, perfluorobutanesulfonic acid, perfluorohexanesulfonic acid, perfluorooctanesulfonic acid, and perfluorodecanesulfonic acid.

[0038] In one specific embodiment, the standard curve is a solvent standard curve, and a series of standard working solutions are prepared using methanol.

[0039] In one specific embodiment, the average spiked recovery rate of the method is 72.01% to 115.59%, the relative standard deviation is 0.09% to 3.9%, the limit of detection is 0.0075 to 0.0625 ng / mL, and the limit of quantitation is 0.025 to 0.125 ng / mL.

[0040] In one specific embodiment, the parent ion, daughter ion, fragmentation voltage, and collision energy of each perfluorinated compound in the multi-reaction monitoring mode of the tandem mass spectrometer are shown in Table 1.

[0041] In one specific embodiment, during the qualitative determination, the retention time of the chromatographic peak of the target analyte in the sample solution deviates from the retention time of the standard working solution by within ±2.5%, and the relative abundance of each qualitative ion compared with the standard is allowed to deviate by ±20% when the relative ion abundance is >50%, ±25% when it is >20% to 50%, ±30% when it is >10% to 20%, and ±50% when it is ≤10%.

[0042] In one specific embodiment, under chromatographic conditions of column temperature 32–37°C, flow rate 0.27–0.33 mL / min, and initial mobile phase B volume ratio of 18%–22%, the relative standard deviation of the determination results between different chromatographic conditions is less than 4.66%.

[0043] In one specific embodiment, the method for detecting perfluorinated compounds in urine includes the following steps: Step 1: Sample Pretreatment Take urine and perform the following operations sequentially according to the volume ratio of urine: 2% formic acid methanol solution: 0.1% ammonia methanol solution (activated): 1% formic acid aqueous solution (activated): 1% formic acid aqueous solution (rinsing): 1% formic acid methanol solution (rinsing): methanol (rinsing): 0.1% ammonia methanol solution (eluting): methanol (redissolving) = 1:1:1.25:1.25:0.75:0.75:0.25:1.5:0.25: Urine was mixed with a 2% formic acid methanol solution and passed through a WAX solid-phase extraction column (150 mg / 6 mL) pre-activated with a 0.1% ammonia methanol solution and a 1% formic acid aqueous solution. The mixture was then eluted sequentially with a 1% formic acid aqueous solution, a 1% formic acid methanol solution, and methanol. The mixture was then dried under pressure and eluted again with a 0.1% ammonia methanol solution. The eluent was collected, dried under nitrogen at 50°C, reconstituted with methanol, and filtered through a 0.22 μm nylon membrane to obtain the test solution.

[0044] The percentages in the 2% formic acid methanol solution, 1% formic acid aqueous solution, 1% formic acid methanol solution, and 0.1% ammonia methanol solution are all volume percentages.

[0045] Step 2: Detection by liquid chromatography-tandem mass spectrometry

[0046] The test solution was detected by liquid chromatography-tandem mass spectrometry, wherein: The liquid chromatography conditions were as follows: a C18 column (preferably an Agilent Proshell EC-C18 column, 2.1 mm × 100 mm, 2.7 μm); mobile phase A was a 5 mM ammonium acetate aqueous solution; mobile phase B was a 50% methanol-50% acetonitrile mixture; gradient elution was used, with the following elution program: 0–10 min, 80% to 0% of phase A, 20% to 100% of phase B; 10–15 min, 0% of phase A, 100% of phase B; 15–15.01 min, 0% to 80% of phase A, 100% to 20% of phase B; 15.01–25 min, 80% of phase A, 20% of phase B; flow rate was 0.3 mL / min; column temperature was 35℃; and injection volume was 1 μL.

[0047] The mass spectrometry conditions were as follows: electrospray negative ion mode, multiple reaction monitoring, drying gas temperature 350℃, drying gas flow rate 8 L / min, nebulizer pressure 35 psi, capillary voltage 4500 V, sheath gas temperature 350℃, sheath gas flow rate 10 L / min, and nozzle voltage 0 V. The parent ion, daughter ion, fragmentation voltage, and collision energy of each perfluorinated compound are shown in Table 1 (Note: When the collision energies of quantitative ion pairs and qualitative ion pairs are different, they are listed separately).

[0048] Step 3: Quantitative Analysis

[0049] The content of perfluorinated compounds in urine was calculated using the solvent standard curve method. A series of standard working solutions were prepared using methanol to create the standard curve.

[0050] During qualitative determination, the retention time of the target chromatographic peak in the sample solution should deviate from that in the standard working solution by within ±2.5%. Furthermore, the relative abundance of each qualitative ion should be within ±20% of the standard when the relative ion abundance is >50%, ±25% when it is >20% to 50%, ±30% when it is >10% to 20%, and ±50% when it is ≤10%.

[0051] Example 1

[0052] 1. Experimental Materials and Instruments

[0053] 1.1 Reagents and Materials

[0054] The standard reference material for 17 perfluorinated compounds in methanol (BWY0824124), including perfluorobutyric acid (PFBA), perfluorobutane sulfonic acid (PFBS), perfluorodecanoic acid (PFDA), perfluorododecanoic acid (PFDoA), perfluorodecane sulfonic acid (PFDS), perfluoroheptanoic acid (PFHpA), perfluorohexanoic acid (PFHxA), perfluorohexadecanoic acid (PFHxDA), perfluorohexane sulfonic acid (PFHxS), perfluorononanoic acid (PFNA), perfluorooctanoic acid (PFOA), perfluorooctane sulfonic acid (PFOS), perfluorovalerate (PFPeA), perfluorotetradecanoic acid (PFTeDA), perfluorotridecanoic acid (PFTrDA), perfluoroundecanoic acid (PFUdA), and perfluorooctadecanoic acid (PFODA), all with a concentration of 10 mg / L, was purchased from Sichuan Zhongce Standard Technology Co., Ltd.

[0055] WAX solid-phase extraction column (specification: 150 mg / 6 mL); ammonium acetate (C2H7NO2; analytical grade); formic acid (CH2O2; chromatographic grade); methanol (CH3OH; chromatographic grade); acetonitrile (C2H3N; chromatographic grade); all laboratory water was Grade I water, and specific requirements are specified in GB / T6682.

[0056] 1.2 solution

[0057] 5 mmol / L ammonium acetate solution: Weigh 385.4 mg of ammonium acetate, dissolve it in 800 mL of water, transfer it to a 1 L volumetric flask, dilute with water to the mark, shake well, and filter through a 0.22 μm aqueous phase filter membrane to obtain mobile phase A.

[0058] 50% methanol-acetonitrile solution: Take 500 mL of methanol, add acetonitrile to make up to 1 L, mix well, and filter through a 0.22 μm nylon membrane to obtain mobile phase B.

[0059] Standard working solution series: Accurately transfer 0.10 mL of the perfluorinated compound standard solution into a 10 mL volumetric flask, dilute to volume with methanol, and mix well to obtain a standard working solution with a mass concentration of 100 ng / mL. Accurately transfer an appropriate amount of the mixed standard intermediate solution and dilute it stepwise with methanol to prepare a series of standard working solutions with mass concentrations of 0.10, 0.25, 0.5, 1.0, 2.5, 5.0, 10, and 25 ng / mL. Prepare fresh before use.

[0060] 1.3 Instruments

[0061] Liquid chromatography-mass spectrometry / mass spectrometer: equipped with an ESI ion source (with a dedicated pipeline for the detection of perfluorinated compounds and a perfluorinated compound trapping column in the mobile phase).

[0062] 10mL graduated pipette.

[0063] Electronic balance: accurate to 0.01 mg.

[0064] Vortex mixer.

[0065] Nitrogen blowing device.

[0066] 2 Experimental Methods

[0067] 2.1 Sample Pretreatment

[0068] Take 4.0 mL of urine and place it in a 10 mL glass test tube that has been ignited at 500 °C for 2 h. Add 4.0 mL of 2% formic acid in methanol solution, vortex to mix, and pass the solution through a WAX column (the WAX ​​column was pre-activated with 5 mL of 0.1% ammonia-methanol and 5 mL of 1% formic acid aqueous solution). Elute sequentially with 3 mL of 1% formic acid, 1% formic acid in methanol solution, and 1 mL of methanol. After elution, the column is dried under pressure and eluted with 6 mL of 0.1% ammonia-methanol. Collect the eluent in a 10 mL glass test tube that has been ignited at 500 °C for 2 h. Dry the eluent with nitrogen at 50 °C, then redissolve it with 1.0 mL of methanol. Vortex to mix, and filter through a 0.22 μm nylon membrane. Take the filtrate for analysis. Simultaneously prepare a blank sample: except for the absence of urine, follow the same pretreatment steps as the sample.

[0069] 2.2 Chromatographic Reference Conditions

[0070] a) Chromatographic column: C 18 Column, 2.1 mm × 100 mm, 2.7 μm; b) Mobile phase: A is 5mM ammonium acetate, B is 50% methanol acetonitrile solution, and the gradient elution program is shown in Table 2; c) Flow rate: 0.3 mL / min; d) Column temperature: 35 ℃; e) Injection volume: 1 μL.

[0071] Table 2 Gradient elution program

[0072] like Figure 3 As shown, the baseline separation of each perfluorinated compound is achieved.

[0073] 2.3 Mass Spectrometry Reference Conditions

[0074] a) Ion source: Electrospray ionization source (ESI source); b) Scanning mode: Negative ion mode; c) Detection method: Multiple reaction monitoring (MRM); d) Drying gas (N2) temperature: 350 ℃; e) Nebulized gas (N2) pressure: 35 psi; f) Drying airflow rate: 8 L / min; g) Capillary voltage: 4500 V; h) Sheath gas temperature: 350 ℃; i) Sheath gas flow rate: 10 L / min; j) Nozzle voltage: 0 V k) Qualitative ion pairs, quantitative ion pairs, collision gas energy, and fragmentation voltage are shown in Table 1.

[0075] Table 1 Retention time, ion pairs, fragmentation voltage and collision energy of perfluorinated compounds

[0076] 2.4 Qualitative determination

[0077] Under the same experimental conditions, if the retention time of the analyte in the sample solution is the same as that in the standard working solution (within ±2.5%), and the selected ions appear in the sample solution spectrum after background subtraction, and the relative abundance of each qualitative ion and the relative abundance ratio of the standard ions do not deviate from the range specified in Table 3, then it can be determined that the corresponding analyte exists in the sample.

[0078] Table 3 Maximum permissible deviation of relative ion abundance during qualitative confirmation.

[0079] 2.5 Quantitative determination

[0080] Under optimal instrument operating conditions, analyze samples using a series of standard working solutions. Plot a standard working curve with the peak area of ​​the target analyte on the ordinate and the concentration of the target analyte solution on the abscissa. Use the standard working curve to quantify the sample. The response value of the perfluorinated compound in the sample solution should be within the linear range measured by the instrument. If the content exceeds the range of the standard working curve, dilute with methanol to a suitable concentration before analysis.

[0081] 2.6 Results Analysis

[0082] The content of the target substance in the sample is calculated according to formula (1).

[0083] Equation (1)

[0084] In the formula: w —The content of the target substance in the sample, in nanograms per milliliter (ng / mL); ρ —The mass concentration of the target substance in the sample obtained from the standard working curve, in nanograms per milliliter (ng / mL); V —The volume of the sample is measured in milliliters (mL); V 样—Sample volume, in milliliters (mL).

[0085] The calculation results should exclude blank values, and the results should be expressed as the arithmetic mean of two independent determinations obtained under repeatability conditions, with three significant figures retained.

[0086] Example 2

[0087] Linearity and Sensitivity

[0088] A series of standard working solutions were prepared using methanol and analyzed under the instrument conditions described in Example 1. Standard working curves were plotted with peak area (y) as the ordinate and mass concentration (x, ng / mL) as the abscissa. The linear range, regression equation, correlation coefficient, limit of detection (LOD, S / N≥3), and limit of quantitation (LOQ, S / N≥10) results for the 16 perfluorinated compounds are shown in Table 4. For details, see the linearity graph for compound PFBA. Figure 4 .

[0089] Table 4. Linear range, regression equation, correlation coefficient, limit of detection, and limit of quantitation for perfluorinated compounds.

[0090] The results showed that all compounds exhibited good linearity within the defined linear range, with correlation coefficients (r) all greater than 0.997. The limits of detection and quantitation met the requirements for trace analysis.

[0091] Example 3

[0092] Twelve urine samples were collected. Three samples were pretreated using the same method as in Example 1. The other nine samples were spiked with mixed standard solutions at three concentration levels: low (1.25 ng / mL), medium (2.5 ng / mL), and high (5 ng / mL), with each concentration in triplicate. Pretreatment and detection were performed according to the method in Example 1. The recoveries and relative standard deviations (RSDs) of each compound were calculated. The results showed that the average recoveries ranged from 72.01% to 115.59%, and the RSDs ranged from 0.09% to 3.9%. All results met the requirements of GB / T27417-2017 for method recovery and precision, indicating that this method has good accuracy and precision in determining the content of perfluorinated compounds in urine. The recovery results of PFBA are shown in Table 5.

[0093] Table 5. Results of PFBA recovery rate

[0094] Example 4

[0095] To further evaluate the impact of the matrix on the detection results and ensure the accuracy of the method, this study used the relative response value method to evaluate the matrix effect: Matrix effect (ME, %) = (peak area of ​​target analyte in matrix standard solution / peak area of ​​target analyte in solvent standard solution) × 100%. Generally, matrix interference is considered acceptable when the ME is between 70% and 130%. If it exceeds this range, pretreatment optimization or matrix matching calibration is required to eliminate interference. A blank urine sample was taken and processed according to 1.3.1 to obtain 1.0 mL of blank matrix solution. 50 μL of standard working solution was added to this solution to prepare a matrix standard solution. Separately, 1.0 mL of methanol was taken and an equal volume of standard working solution was added to prepare a solvent standard solution of the same concentration. The matrix effect was calculated after HPLC-MS / MS detection of both standard solutions. The results showed that the ME values ​​of the 16 target analytes were all between 90% and 130%, which is within the acceptable range. Therefore, this experiment can directly use solvent-prepared standard solutions for quantitative analysis. Figure 6 The distribution of matrix effect results for 16 perfluorinated compounds is shown in the figure.

[0096] Example 5

[0097] stability

[0098] Standard solutions and low-spiked urine solutions (spiking concentration 1.25 ng / mL) were taken and analyzed at 0 h, 1 h, 2 h, 4 h, 6 h, 9 h, 15 h, and 24 h, respectively, following the pretreatment and detection methods of Example 1. The results showed that the RSD of the peak areas of the 16 perfluorinated compounds ranged from 1.3% to 4.5% (with the highest being 4.5% for PFDoA), as detailed in Tables 6 and 7. The results indicate that no obvious regular decrease or increase in the response results of each solution was observed within 24 hours. According to the repeatability requirements of GB / T27417-2017, the RSD of all samples met the requirements, indicating good stability of all samples within 24 hours.

[0099] Table 6 Stability of Standard Solutions

[0100] Table 7 Stability of urine spiked solutions

[0101] Example 6

[0102] Column capacity

[0103] A mixed standard solution of perfluorinated compounds was added to blank urine samples at loading amounts of 10 ng, 100 ng, and 200 ng, respectively. Pretreatment and detection were performed according to the method in Example 1. The results showed that the recoveries of each compound remained between 82.71% and 117.20% under all three loading amounts (see Table 8). The results indicate that the recoveries under all three loading amounts met the recovery requirements for their respective concentration levels, and the solid-phase extraction column capacity was sufficient to meet the sample determination requirements.

[0104] Table 8. Solid-phase extraction column capacity (n=3)

[0105] Example 7

[0106] Durability

[0107] Blank samples, 5 ppb standard solutions, samples, and spiked solutions of sample concentrations were tested at 100% limit of the spiked test sample under specified conditions, conditions A, B, C, D, E, and F, respectively. Specific chromatographic conditions are shown in Table 9, and the test results are shown in Table 10. Under varying parameters, the relative standard deviation of the test results was less than 4.66% (maximum 4.66%), indicating that the method has good robustness under the fluctuations of the stated conditions.

[0108] Table 9. Durability assessment under different chromatographic conditions

[0109] Table 10. Measured values ​​and RSD of test solution under different chromatographic conditions.

[0110] Comparative Example 1

[0111] Pure methanol as mobile phase B

[0112] The only difference from Example 1 is that mobile phase B is pure methanol (instead of 50% methanol-50% acetonitrile). All other conditions are the same. The results are as follows: Figure 1 As shown: Some perfluorinated compounds exhibit co-eluenting or insufficient separation, making baseline separation impossible.

[0113] Comparative Example 2

[0114] Pure acetonitrile as mobile phase B

[0115] The only difference from Example 1 is that mobile phase B is pure acetonitrile (instead of 50% methanol-50% acetonitrile). All other conditions are the same. The results are as follows: Figure 2 As shown, some perfluorinated compounds also suffer from insufficient separation, and the response values ​​of some compounds are lower than those in Example 1.

[0116] Comparative Example 3

[0117] For the 16 target perfluorinated compounds in Example 1, the same actual urine sample was processed using both the method of Example 1 of this invention and the sample pretreatment method disclosed in CN115754101A. Then, the samples were detected using the liquid chromatography-tandem mass spectrometry conditions of Example 1 of this invention. The specific steps of the CN115754101A method are as follows: Take 0.5 mL of urine, add a stable isotope internal standard mixed solution (5 ng of each internal standard), add 0.5 mL of acetonitrile, vortex, shake, centrifuge, repeat the extraction 3 times, combine the supernatants, blow dry with nitrogen, reconstitute with 0.5 mL of methanol, filter, and then inject the sample.

[0118] The detection results are shown in Table 11. The results show that the method of Example 1 of this invention detected PFBA (0.35 ng / mL), PFHxA (0.026 ng / mL), and PFPeA (0.071 ng / mL), while the method of CN115754101A only detected PFBA (0.33 ng / mL), and PFHxA and PFPeA were not detected (below the instrument detection limit), indicating that the method of this invention has a stronger detection capability for low concentration target substances.

[0119] Table 11 Comparison of Sample Detection Results between the Two Methods

[0120] Furthermore, at a spiking level of 10 ng / mL, the average recoveries of each target compound in the CN115754101A method were 70%–121%. The average recoveries of this invention at a spiking level of 10 ng / mL are detailed in Table 12. Table 12 Average spiked recoveries of the present invention at a spiking level of 10 ng / mL

[0121] As shown in Table 12, the average recovery rate of the present invention at a spiking level of 10 ng / mL is 72.01% to 115.59%, and the recovery rate of the method of the present invention has a narrower fluctuation range.

[0122] Furthermore, the CN115754101A method requires the use of a corresponding stable isotope internal standard for each analyte, while the method of this invention uses an external standard method for quantification, eliminating the need to purchase expensive isotope internal standards, significantly reducing detection costs, and making it more suitable for monitoring and studying the exposure levels of perfluorinated compounds in large-scale human biological samples.

[0123] Comparative Example 4

[0124] The only difference from Example 4 was that an HLB solid-phase extraction column (150 mg / 6 mL) was used instead of a WAX column, and the activation, elution, and elution conditions were performed according to the optimal HLB conditions (i.e., activation with 6 mL of methanol and 6 mL of water sequentially, elution with 3 mL of water after sample loading, pressure drying, and elution with 5 mL of acetonitrile). All other conditions remained the same. Results: The recovery rate of some target compounds using the HLB column was less than 60%, while the recovery rate after treatment with the WAX ​​column ranged from 70% to 110%, indicating that the WAX ​​column is more suitable for the enrichment and purification of perfluorinated compounds in urine matrix.

[0125] Comparative Example 5

[0126] The resolution solvent is the initial mobile phase or 50% methanol.

[0127] The only difference from Example 1 was that the resolution solvent was either the initial proportioned mobile phase (A:B=80:20) or a 50% methanol aqueous solution, instead of methanol. All other conditions remained the same. Results: When resolution was performed using the initial mobile phase, the response of some long-chain perfluorinated compounds (such as PFHxDA and PFTeDA) was significantly reduced; when resolution was performed using 50% methanol, the response improved somewhat but was still lower than when resolution was performed with methanol.

[0128] Comparative Example 6

[0129] Mobile phase A is pure water or a 0.1% formic acid aqueous solution.

[0130] The only difference from Example 1 was that mobile phase A was replaced with pure water or 0.1% formic acid aqueous solution instead of 5 mM ammonium acetate aqueous solution. All other conditions remained the same. Results: When using pure water or 0.1% formic acid, the chromatographic peak shapes of some compounds deteriorated (tailing or broadening), and the response values ​​were lower than under the 5 mM ammonium acetate condition, indicating that the addition of ammonium acetate improved peak shape and ionization efficiency.

[0131] Comparative Example 7

[0132] Column temperature is 25℃ or 45℃

[0133] The only difference from Example 1 was that the column temperatures were set to 25°C and 45°C respectively (35°C in Example 1). All other conditions were the same. Results: When the column temperature was 25°C, the retention time of some compounds was prolonged and the peak shape broadened; when the column temperature was 45°C, the resolution of compounds with poor response, such as PFDS, decreased.

[0134] Comparative Example 8

[0135] Use PFP or T3 columns

[0136] The only difference from Example 1 is that the chromatographic column used was a Phenomenon PFP column (2.1 mm × 100 mm, 1.8 μm) or an HSS T3 column (3.0 mm × 100 mm, 2.5 μm) instead of an Agilent proshell EC-C18 column. All other conditions were the same. The results are detailed in Table 13.

[0137] Table 13

[0138] From the separation and response results of the three chromatographic columns, the separation performance of the three types of columns was basically the same. Regarding retention time, the T3 column generally had a longer retention time, but it did not significantly improve separation. Example C... 18 Under the same separation conditions, the low-response compound has the best response value.

[0139] Comparative Example 9

[0140] The only difference from Example 1 was that the drying gas temperature was set to 300°C (350°C in Example 1). All other conditions were the same. Results: The response values ​​of each target compound were significantly lower than 350°C, as detailed in Table 14.

[0141] Table 14 Effect of Drying Gas Temperature on Perfluorinated Compounds

[0142] Comparative Example 10

[0143] The only difference from Example 1 was that the nozzle voltage was set to 500V (0V in Example 1). All other conditions were the same. Results: The response values ​​of each target compound were significantly reduced (see Table 15 for details).

[0144] Table 15 Effect of Nozzle Voltage on Perfluorinated Compounds

[0145] In addition, many experiments were conducted during the development of this invention. For example, the response of some compounds decreased when the drying gas flow rate deviated from 8 L / min; the nebulizer pressure was optimal at 35 psi, and the response of all compounds decreased when the pressure was increased; the capillary voltage was optimal at 4500 V, and the response decreased when it was too low; the sheath gas temperature was optimal at 350℃, and the response decreased significantly when it was too low; the sheath gas flow rate was optimal at 10 L / min, and the response of most compounds decreased when it was too low.

Claims

1. A method for detecting perfluorinated compounds in urine, characterized in that, Includes the following steps: (1) Sample pretreatment: Mix urine with 2% formic acid methanol solution, pass it through a WAX solid phase extraction column that has been pre-activated with 0.1% ammonia methanol solution and 1% formic acid aqueous solution, then wash with 1% formic acid aqueous solution, 1% formic acid methanol solution and methanol in sequence, dry under pressure, then elute with 0.1% ammonia methanol solution, collect the eluent, dry it, redissolve it with methanol, filter it to obtain the test solution; The volume ratio of urine: 2% formic acid methanol solution: 0.1% ammonia methanol solution for activation: 1% formic acid aqueous solution for activation: 1% formic acid aqueous solution for rinsing: 1% formic acid methanol solution for rinsing: methanol for rinsing: 0.1% ammonia methanol solution for elution: methanol for reconstitution is 1:1:1.25:1.25:0.75:0.75:0.25:1.5:0.25; The percentage content in the 2% formic acid methanol solution, 1% formic acid aqueous solution, 1% formic acid methanol solution, and 0.1% ammonia methanol solution are all volume percentages. (2) The test solution was detected by liquid chromatography-tandem mass spectrometry. The liquid chromatography conditions were as follows: the column was a C18 column, the mobile phase A was 5 mM ammonium acetate aqueous solution, and the mobile phase B was a 50% methanol-50% acetonitrile mixed solution. The mobile phase was used for gradient elution. The gradient elution program was as follows: 0-10 min, A phase volume 80% to 0%, B phase volume 20% to 100%; 10-15 min, A phase volume 0%, B phase volume 100%; 15-15.01 min, A phase volume 0% to 80%, B phase volume 100% to 20%; 15.01-25 min, A phase volume 80%, B phase volume 20%; the flow rate was 0.27-0.33 mL / min, the column temperature was 30-35℃, and the injection volume was 1 μL. The mass spectrometry conditions were: electrospray negative ion mode, multiple reaction monitoring, dryer gas temperature 350℃, dryer gas flow rate 8 L / min, nebulizer pressure 35 psi, capillary voltage 4500 V, sheath gas temperature 350℃, sheath gas flow rate 10 L / min, and nozzle voltage 0 V. (3) Calculate the content of perfluorinated compounds in urine based on the standard curve.

2. The method of claim 1, wherein, The drying process involved nitrogen blowing at 50°C until dry; the WAX ​​solid-phase extraction column had a specification of 150 mg / 6 mL.

3. The method of claim 1, wherein, The filtration uses a 0.22 μm nylon filter membrane.

4. The method of claim 1, wherein, The chromatographic column is an Agilent Proshell EC-C18 column with dimensions of 2.1 mm × 100 mm and 2.7 μm; the preferred liquid chromatography conditions are: flow rate 0.3 mL / min.

5. The method of claim 1, wherein, The perfluorinated compound is at least one of perfluorobutyric acid, perfluorovaleric acid, perfluorohexanoic acid, perfluoroheptanoic acid, perfluorooctanoic acid, perfluorononanoic acid, perfluorodecanoic acid, perfluoroundecanoic acid, perfluorododecanic acid, perfluorotridecanoic acid, perfluorotetradecanoic acid, perfluorohexadecanoic acid, perfluorobutanesulfonic acid, perfluorohexanesulfonic acid, perfluorooctanesulfonic acid, and perfluorodecanesulfonic acid.

6. The method of claim 1, wherein, The standard curve is a solvent standard curve, and a series of standard working solutions are prepared using methanol.

7. The method of claim 1, wherein, The average spiked recoveries of the method were 72.01%–115.59%, with relative standard deviations of 0.09%–3.9%, limits of detection of 0.0075–0.0625 ng / mL, and limits of quantitation of 0.025–0.125 ng / mL.

8. The method of claim 1, wherein, The parent ion, daughter ion, fragmentation voltage, and collision energy of each perfluorinated compound in the multiple reaction monitoring mode of the tandem mass spectrometry are shown in Table 1: Table 1 。 9. The method of claim 1, wherein, During the qualitative determination, the retention time of the chromatographic peak of the target analyte in the sample solution deviates from the retention time of the standard working solution by within ±2.5%. Furthermore, the relative abundance of each qualitative ion compared with the standard is allowed to deviate by ±20% when the relative ion abundance is >50%, ±25% when it is >20% to 50%, ±30% when it is >10% to 20%, and ±50% when it is ≤10%.

10. The method of claim 1, wherein, Under the chromatographic conditions of column temperature 32–37℃, flow rate 0.27–0.33 mL / min, and initial mobile phase B volume ratio 18%–22%, the relative standard deviation of the determination results between different chromatographic conditions is less than 4.66%.

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