An application of LC-MS / MS in determining the content of indophenol sulfate and p-cresol sulfate in a hemoperfusion device.

Abstract

This invention relates to the field of medical device testing technology, specifically to the application of LC-MS / MS in determining the content of indophenol sulfate and p-cresol sulfate in a hemoperfusion device. The method includes: mixing plasma and acetonitrile, vortexing and centrifuging, collecting the supernatant, eluting the supernatant through a C18 solid-phase extraction column, collecting the eluent, mixing the eluent with water and filtering to obtain the test solution; performing liquid chromatography and mass spectrometry on the test solution, wherein the mobile phase A of the liquid chromatography is a 3-8 mmol / L gradient concentration ammonium acetate solution containing 0.05% ammonium formate by volume, and the mobile phase B is methanol; S3: calculating the concentrations of indophenol sulfate and p-cresol sulfate in the test solution based on the labeled curve. This invention, through dual purification pretreatment and multi-ion-pair threshold matching technology, can effectively remove more than 95% of interfering protein-binding toxoids. The interference peak response value in blank plasma is less than 5% of the target analyte response value, solving the detection error problem caused by interference in traditional methods.

Description

Technical Field

[0001] This invention relates to the field of medical device testing technology, specifically to the application of LC-MS / MS in determining the content of indophenol sulfate and p-cresol sulfate in a hemoperfusion device. Background Technology

[0002] Hemoperfusion is an important adjunctive treatment for renal dysfunction in clinical practice. It removes protein-bound uremic toxins from the blood, including indophenol sulfate (IS) and p-cresol sulfate (PCS), through adsorption materials. IS and PCS have a binding rate of over 90% with human serum albumin, and the accurate detection of their adsorption performance is directly related to the clinical efficacy evaluation and quality control of hemoperfusion devices.

[0003] Currently, LC-MS / MS is widely used for the detection of IS and PCS due to its high sensitivity and specificity. However, existing technologies still have significant drawbacks: First, plasma samples contain various other protein-bound toxoids (such as indoleacetic acid and hippuric acid), which have structures similar to IS and PCS. Traditional acetonitrile precipitation protein pretreatment methods cannot effectively separate these toxoids, leading to interference with the detection results. Second, the separation effect of existing mobile phase systems is limited, and the retention times of some interfering toxoids are close to those of the target analyte, making baseline separation difficult. Third, MRM modes often use two sets of ion pairs for qualitative and quantitative analysis, and the response of a single ion pair may overlap with the interfering substance, leading to false positive results.

[0004] Among the existing technologies, CN115856119A discloses a method for simultaneously detecting three protein-bound toxoids in blood samples. However, it uses artificial plasma (bovine serum albumin solution) for simulation, which differs significantly from the actual plasma matrix. Furthermore, it lacks specified purification steps and flow rate control, resulting in insufficient anti-interference capabilities. CN113702463A can only detect a single toxoid, indophenol sulfate, and cannot simultaneously determine PCS, nor does it address interference exclusion techniques. Therefore, developing a robust and accurate LC-MS / MS method is of great significance. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides an application of LC-MS / MS in determining the content of indophenol sulfate and p-cresol sulfate in a hemoperfusion device. Through dual purification pretreatment and multi-ion pair threshold matching technology, this invention can effectively remove more than 95% of interfering protein-binding toxoids. The interference peak response value in blank plasma is less than 5% of the target analyte response value, thus solving the detection error problem caused by interference in traditional methods.

[0006] Therefore, the present invention provides the following technical solution:

[0007] In a first aspect, the present invention provides, in an optional embodiment, the application of LC-MS / MS in determining the content of indophenol sulfate and p-cresol sulfate in a hemoperfusion device, the application comprising the following steps:

[0008] S1: Mix plasma and acetonitrile, vortex and centrifuge, collect the supernatant, elute the supernatant through a C18 solid phase extraction column, collect the eluent, mix the eluent with water and filter to obtain the test solution;

[0009] S2: Perform liquid chromatography and mass spectrometry on the test solution, wherein the mobile phase A of the liquid chromatography is a 3-8 mmol / L gradient concentration ammonium acetate solution, and the ammonium acetate solution contains 0.05% ammonium formate by volume, and the mobile phase B is methanol;

[0010] S3: Calculate the concentrations of indophenol sulfate and p-cresol sulfate in the test solution based on the labeled curve.

[0011] This invention adds a C18 solid-phase extraction (SPE) column purification step to the existing acetonitrile protein precipitation method. It utilizes the difference in hydrophobic interaction between the C18 packing and protein-bound toxoids to specifically retain indophenol sulfate (IS) and p-cresol sulfate (PCS), while eluting away other interfering protein-bound toxoids (such as similar uremic toxins and other protein derivatives), thus solving the problem of incomplete purification in traditional pretreatment.

[0012] Preferably, in step S2, the mass spectrometry employs an electrospray ionization source in negative ion mode and multiple reaction monitoring mode; the number of ion pairs for both indophenol sulfate and p-cresol sulfate is 3, and a threshold of ±5% for the ion pair response intensity ratio is set. The ion pairs for the 3 groups of indophenol sulfate are 212.0→79.9, 212.0→132.0, and 212.0→93.0, respectively; the ion pairs for the 3 groups of p-cresol sulfate are 187.0→107.0, 187.0→79.9, and 187.0→125.0, respectively.

[0013] Preferably, in step S1, the volume ratio of plasma to acetonitrile is 1:9, the vortexing time is 30-60 s, the centrifugation speed is 12000 r / min, and the centrifugation time is 10 min. The activation conditions for the C18 solid-phase extraction column are: sequential rinsing with 5 mL of methanol and 5 mL of pure water at a rinsing rate of 1-2 mL / min. The rinsing uses a 5% (w / w) methanol-water solution, and the elution uses an 80% (w / w) methanol-water solution; and / or, the volume ratio of eluent to water is 1:9, and the filter membrane used for filtration has a pore size of 0.22 μm.

[0014] Preferably, in step S2, the liquid chromatography uses an InfinityLab Porashell 120 EC-C18 column with a column temperature of 40°C, a flow rate of 0.2 mL / min, and an injection volume of 10 μL. The liquid chromatography employs a gradient elution program as follows: 0-1.5 min, 95% mobile phase A; 1.5-3.0 min, 95% mobile phase A decreasing to 5% mobile phase A; 3.0-6.0 min, 5% mobile phase A; 6.0-7.0 min, 5% mobile phase A increasing to 25% mobile phase A; 7.0-9.0 min, 25% mobile phase A increasing to 65% mobile phase A; 9.0-11.0 min, 65% mobile phase A increasing to 95% mobile phase A; followed by a 5-min run. The mass spectrometer parameters include: dry gas flow rate 10 L / min, nebulizer pressure 50 psi, sheath gas flow rate 11 L / min, capillary voltage +2000V, -4000V, dry gas temperature 300℃, sheath gas temperature 350℃, and collision gas 9 psi.

[0015] Preferably, in step S3, the standard curve is prepared as follows: a series of standard solutions with IS concentrations of 0.0850-1.3080 μg / mL and PCS concentrations of 0.0905-1.3920 μg / mL are prepared; a standard curve is plotted with concentration as the abscissa and characteristic ion abundance as the ordinate, yielding the IS regression equation Y = 36045.242X + 763.841, R 2 =0.9987; PCS regression equation Y=201921.735X+32600.239, R 2 =0.9956.

[0016] Compared with the prior art, the present invention has one of the following beneficial effects:

[0017] 1. This invention, through dual purification pretreatment and multi-ion pair threshold matching technology, can effectively remove more than 95% of interfering protein-binding toxoids. The interference peak response value in blank plasma is less than 5% of the target analyte response value, thus solving the detection error problem caused by interference in traditional methods.

[0018] 2. In this invention, the single-concentration ammonium acetate solution (5 mmol / L) is changed to a gradient concentration system (3-8 mmol / L), and 0.05% ammonium formate is added as a modifier. By adjusting the ionic strength and polarity, the separation of IS, PCS and interfering toxins on the C18 chromatographic column is enhanced, so that the retention time of the target analyte is stable and completely separated from the interference peak.

[0019] 3. Based on the MRM mode, this invention adds one set of characteristic ion pairs to IS and PCS respectively (IS adds a new qualitative ion pair 212.0→93.0, PCS adds a new qualitative ion pair 187.0→125.0), and sets a threshold (±5%) for the ratio of ion pair response intensity. Only peaks that simultaneously meet the matching of 3 sets of ion pairs and whose ratios are within the threshold range are identified as target substances, thus eliminating interference caused by matching of a single ion pair. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is an experimental flowchart of Embodiment 1 of the present invention. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0023] The technical solution of the present invention will be described below with reference to the embodiments.

[0024] Example 1

[0025] See Figure 1 1. Instruments and reagents

[0026] (1) Instruments: Ultra-high performance liquid chromatography-triple quadrupole liquid chromatography-mass spectrometry system (Agilent 1290 InfinityⅡ-6475LC / TQ, Agilent Technologies); Multi-parts electronic balance (MSA225S-1CE-DU, Sartorius Scientific Instruments Co., Ltd.); High-speed refrigerated centrifuge (HRC-16KPlus, Shanghai Titan Technology Co., Ltd.); Thermostatic shaking water bath (BS-31, Jeio Tech); Vortex shaker (VG3, IKA); C18 solid phase extraction column (500mg / 6mL, Agilent Technologies); Multifunctional microplate reader (SuPerMax 3100, Shanghai Flash Spectrum Biotechnology Co., Ltd.).

[0027] (2) Reagents: IS reference standard (batch number C16097103, purity 95%, Shanghai McLean Biochemical Technology Co., Ltd.); PCS reference standard (batch number 149691, purity 97.38%, TargetMol); chromatographic grade acetonitrile, methanol, ammonium acetate, ammonium formate (Merck); bovine plasma (batch number 20241213, Guangzhou Ruite Biotechnology Co., Ltd.); ultrapure water (prepared by Millipore ultrapure water system); pH 7.4 phosphate buffer (PBS, batch number 23314817, Beijing Lanjieke Technology Co., Ltd.).

[0028] 2. Preparation of standard solutions

[0029] (1) Control stock solution: Accurately weigh 22.95 mg of IS reference standard and 23.80 mg of PCS reference standard, dissolve them in acetonitrile and make up to 10 mL to obtain a control stock solution with an IS concentration of 109 μg / mL and a PCS concentration of 116 μg / mL; take the stock solution and dilute it with acetonitrile to obtain a secondary stock solution with an IS concentration of 6.54 μg / mL and a PCS concentration of 6.96 μg / mL.

[0030] (2) Series of standard solutions: Dilute the secondary stock solution with ultrapure water to prepare a series of standard solutions with IS concentrations of 0.0850, 0.1360, 0.2040, 0.4251, 0.8502, 1.1118, and 1.3080 μg / mL and PCS concentrations of 0.0905, 0.1448, 0.2172, 0.4524, 0.9048, 1.1832, and 1.3920 μg / mL. Prepare three parallel solutions for each concentration.

[0031] 3. Sample preparation and pretreatment (improved method of this invention)

[0032] (1) Preparation of adsorbed samples: Take 35 mL of anticoagulated bovine plasma, pre-treat at 37℃ for 15 min, add 0.00286 g IS and 0.00312 g PCS to prepare bovine plasma with IS and PCS concentrations both > 80 μg / mL (simulating the severe toxin accumulation state of patients with chronic kidney disease); measure 1 mL of wet adsorbent in the perfusion device and put it into a 50 mL stoppered conical flask, add 10 mL of the above bovine plasma, shake and adsorb at (37±1)℃ and 100 rpm for 2 h, and obtain the adsorbed plasma sample after standing at room temperature. Prepare 6 parallel samples and divide them into 6 groups. One group is detected by the improved method of this invention, and the other 5 groups are detected by comparative experiment.

[0033] (2) Double purification pretreatment: ① Take 0.4 mL of the adsorbed plasma sample, add 3.6 mL of acetonitrile, vortex for 45 s, centrifuge at 12000 r / min for 10 min, and take the supernatant; ② Activate the C18 solid phase extraction column with 5 mL of methanol and 5 mL of ultrapure water in sequence, load the supernatant onto the column at a flow rate of 1.5 mL / min, rinse with 5 mL of 5% methanol aqueous solution, discard the rinsing solution, and then elute with 5 mL of 80% methanol aqueous solution, and collect the eluent; ③ Take 100 μL of eluent, add 900 μL of ultrapure water, vortex to mix, and filter through a 0.22 μm filter membrane to obtain the test solution.

[0034] 4. LC-MS / MS detection conditions of the improved method of this invention

[0035] (1) Chromatographic conditions: InfinityLab Porashell 120 EC-C18 column (100×4.6mm, 2.7μm); mobile phase A was 3-8mmol / L gradient concentration ammonium acetate solution (containing 0.05% ammonium formate), mobile phase B was methanol; column temperature 40℃; flow rate 0.2mL / min; injection volume 10μL; gradient elution program: 0-1.5min, 95% mobile phase A; 1.5-3.0min, 95% mobile phase A reduced to 5% mobile phase A; 3.0-6.0min, 5% mobile phase A; 6.0-7.0min, 5% mobile phase A increased to 25% mobile phase A; 7.0-9.0min, 25% mobile phase A increased to 65% mobile phase A; 9.0-11.0min, 65% mobile phase A increased to 95% mobile phase A; then run for 5min.

[0036] (2) Mass spectrometry conditions: Electrospray ionization (ESI) negative ion mode; dry gas flow rate 10 L / min; nebulizer pressure 50 psi; sheath gas flow rate 11 L / min; capillary voltage positive 2000 V, negative -4000 V; dry gas temperature 300 ℃; sheath gas temperature 350 ℃; collision gas 9 psi; MRM mode, IS set 3 ion pairs: 212.0→79.9, 212.0→132.0, 212.0→93.0; PCS set 3 ion pairs: 187.0→107.0, 187.0→79.9, 187.0→125.0; ion pair response intensity ratio threshold ±5%, only peaks that meet the ratio requirements are included in the detection results.

[0037] 5. Comparative Experiment Design and Operation

[0038] To verify the anti-interference effect of the improved technology of this invention, four groups of single-variable comparative experiments were set up. Except for the specified variable, all other sample pretreatment, chromatographic / mass spectrometry conditions, and operating procedures were completely consistent with the improved method of this invention described above. An additional blank matrix control group (bovine plasma only, without IS or PCS) was set up to exclude matrix interference. The experimental parameters for each group are set as follows:

[0039] Comparative Experiment 1: Liquid chromatography using a single concentration of ammonium acetate solution (5 mmol / L)

[0040] The only difference is that the mobile phase A in the chromatographic conditions is replaced with a 5 mmol / L single-concentration ammonium acetate solution (containing 0.05% ammonium formate), and the gradient concentration system is changed to a fixed concentration. All other chromatographic conditions, pretreatment steps, and mass spectrometry conditions are consistent with the improved method of this invention.

[0041] Comparative Experiment 2: No 0.05% ammonium formate was added as a modifier

[0042] The only difference in the chromatographic conditions is that the mobile phase A is replaced with a 3-8 mmol / L gradient concentration ammonium acetate solution (without ammonium formate modifier). All other chromatographic conditions, pretreatment steps, and mass spectrometry conditions are consistent with the improved method of this invention.

[0043] Comparative Experiment 3: No additional characteristic ion pairs were added for IS and PCS, and no threshold was set for the ratio of ion pair response intensities.

[0044] The mass spectrometry conditions were adjusted as follows: IS retained only 2 ion pairs (212.0→79.9, 212.0→132.0), and PCS retained only 2 ion pairs (187.0→107.0, 187.0→79.9). The requirement for the ion pair response intensity ratio threshold ±5% was removed. All peaks detected by all ion pairs were included in the results. The remaining mass spectrometry conditions, pretreatment steps, and chromatographic conditions were consistent with the improved method of this invention.

[0045] Comparative Experiment 4: The supernatant was not eluted or eluted using a C18 solid-phase extraction column.

[0046] The pretreatment steps are simplified as follows: Take 0.4 mL of the adsorbed plasma sample, add 3.6 mL of acetonitrile, vortex for 45 s, centrifuge at 12000 r / min for 10 min, take 100 μL of supernatant directly, add 900 μL of ultrapure water, vortex to mix, and filter through a 0.22 μm filter membrane to obtain the test solution. The activation, rinsing, and elution steps of the C18 solid phase extraction column are omitted. The remaining pretreatment operations and chromatographic / mass spectrometry conditions are consistent with the improved method of this invention.

[0047] 6. Detection and data processing of all experiments

[0048] (1) Standard curve plotting: The series of standard solutions were analyzed according to the pretreatment and detection conditions of the corresponding groups. Each concentration was measured twice in parallel. The standard curve was plotted with concentration (X) as the abscissa and the abundance value of characteristic ions of quantitative ion pairs (Y) as the ordinate. The regression equation and correlation coefficient R² were calculated. The improved method of this invention and all comparative experiments used the same batch of series of standard solutions to ensure the consistency of the standard curve.

[0049] (2) Sample detection: The test solutions of the improved method of this invention, the four groups of comparative experiments, and the blank matrix control group were injected into the LC-MS / MS instrument. Each group of samples was measured in parallel three times, and the quantitative ion pair response value, target retention time, number of interference peaks and response value were recorded.

[0050] (3) Adsorption performance calculation: The concentrations (Ct) of IS and PCS in the test solution after adsorption were calculated based on the standard curves of each group. Combined with the initial concentrations of IS and PCS in the plasma before adsorption (C0, which were calibrated to IS: 89.25 μg / mL and PCS: 91.68 μg / mL), the adsorption rate and adsorption amount were calculated according to the following formulas. The results were taken as the arithmetic mean of three parallel determinations:

[0051] Adsorption rate C = (C0 - Ct) / C0 × 100%

[0052] Adsorption capacity M = (C0 - Ct) × 10 (unit: μg, based on 10 mL plasma system)

[0053] (4) Evaluation of anti-interference and precision: ① Anti-interference: The interference coefficient is 100% of the response value of the interference peak / the response value of the target substance. The lower the interference coefficient, the stronger the anti-interference. ② Precision: Calculate the intraday relative standard deviation (RSD) of the adsorption rate of each group of samples. RSD = (standard deviation / average value) × 100%.

[0054] 7. Experimental Results and Analysis

[0055] 7.1 Standard Curve Results

[0056] The improved method of this invention and the series of standard solutions in the four comparative experiments all showed good linearity. The IS regression equation was Y=36045.242X+763.841 (R²≥0.9985), and the PCS regression equation was Y=201921.735X+32600.239 (R²≥0.9950). This indicates that the adjustment of a single variable did not affect the linear detection capability of the instrument, and the difference in detection results can be attributed to the difference in anti-interference and separation effect.

[0057] 7.2 Anti-interference results (interference coefficient)

[0058]

[0059] The results showed that the interference coefficients of the four comparative experiments were significantly higher than those of the improved method of this invention, and the number of interference peaks increased significantly with the adjustment of variables. Among them, comparative experiment 4 had the highest interference coefficient because C18 solid-phase extraction was cancelled, and impurities and other protein-bound toxins (such as indoleacetic acid and hippuric acid) in the plasma that were not removed directly entered the detection system. The improved method of this invention completely eliminated the interference peaks through multiple anti-interference technologies, and the interference coefficients were all below 2%, and the anti-interference ability was significantly improved.

[0060] 7.3 Precision and Adsorption Performance Test Results

[0061]

[0062] The results show that:

[0063] The adsorption rate RSD of the improved method of this invention is all <0.2%, which is much lower than that of the four comparative experiments, indicating that the improved technology effectively reduces the influence of interference on the detection results and significantly improves the detection precision.

[0064] The adsorption rate of the comparative experiment 4 was significantly lower because in the sample that did not undergo C18 solid phase extraction, the interfering substances competed with IS and PCS for the chromatographic column and mass spectrometry ion source, which led to the suppression of the target analyte response value and the distortion of the detection results.

[0065] Using a single concentration of ammonium acetate, without ammonium formate modifier, without adding new ion pairs, and without adjusting the ratio threshold will all lead to a decrease in the separation degree between the target analyte and the interfering analyte or a deviation in qualitative judgment, which in turn reduces the detection precision and causes fluctuations in the adsorption rate results.

[0066] 7.4 Retention Time Stability Results

[0067] In the improved method of this invention, the IS retention time was 9.319±0.005 min and the PCS retention time was 9.591±0.006 min, with a relative deviation of retention time <0.1%. In the four comparative experiments, the retention time fluctuation of the target analyte was >0.5%. Among them, the retention time fluctuation was the largest in comparative experiment 1 because the mobile phase ionic strength was fixed and the interaction between IS, PCS and the chromatographic column packing was unstable (IS: 9.319±0.052 min, PCS: 9.591±0.048 min). This indicates that the combination of gradient concentration ammonium acetate and ammonium formate modifiers can improve the stability of the target analyte retention time and further ensure the accuracy of the detection results.

[0068] 8. Method Validation Results (Improved Method of This Invention)

[0069] The improved method of this invention has undergone comprehensive methodological validation, and all indicators meet the high-precision requirements for medical device testing. Specific results are as follows:

[0070] (1) Specificity: Blank bovine plasma showed no interfering peaks at the IS and PCS retention times, and other protein-bound toxoids achieved baseline separation from the target, indicating good specificity;

[0071] (2) Limit of detection and limit of quantitation: The limit of detection of IS is 0.000023 μg / mL and the limit of quantitation is 0.000212 μg / mL; the limit of detection of PCS is 0.000001 μg / mL and the limit of quantitation is 0.000023 μg / mL. The sensitivity meets the requirements for trace detection.

[0072] (3) Precision: For IS at low, medium and high concentrations (55.48, 91.20, 128.59 μg / mL), the intraday RSD was 0.158%-0.164%, and the interday RSD was 0.122%-0.354%; for PCS at low, medium and high concentrations (44.79, 89.59, 103.29 μg / mL), the intraday RSD was 0.160%-0.311%, and the interday RSD was 0.066%-0.383%, showing excellent precision.

[0073] (4) Recovery rate: IS recovery rate 99.56%-109.77%, PCS recovery rate 102.54%-109.00%, and accuracy meets the requirements of the "Guiding Principles for Validation of Analytical Methods for Drug Quality Standards".

[0074] Although the principles of the present invention have been described in detail above with reference to preferred embodiments, those skilled in the art should understand that the above embodiments are merely illustrative explanations of the implementation of the present invention and are not intended to limit the scope of the present invention. The details in the embodiments do not constitute a limitation on the scope of the present invention. Any obvious changes, such as equivalent transformations or simple substitutions, based on the technical solutions of the present invention without departing from the spirit and scope of the present invention fall within the protection scope of the present invention.

Claims

1. An application of LC-MS / MS in determining the content of indophenol sulfate and p-cresol sulfate in a hemoperfusion device, characterized in that, The application includes the following steps: S1: Mix plasma and acetonitrile, vortex and centrifuge, collect the supernatant, elute the supernatant through a C18 solid phase extraction column, collect the eluent, mix the eluent with water and filter to obtain the test solution; S2: Perform liquid chromatography and mass spectrometry on the test solution, wherein the mobile phase A of the liquid chromatography is a 3-8 mmol / L gradient concentration ammonium acetate solution, and the ammonium acetate solution contains 0.05% ammonium formate by volume, and the mobile phase B is methanol; S3: Calculate the concentrations of indophenol sulfate and p-cresol sulfate in the test solution based on the labeled curve.

2. The application according to claim 1, characterized in that, In step S2, the mass spectrometry employs an electrospray ionization source in negative ion mode and multiple reaction monitoring mode; The number of ion pairs for both indophenol sulfate and p-cresol sulfate is 3, and the threshold for the ratio of ion pair response intensity is set to ±5%.

3. The application according to claim 2, characterized in that, The ion pairs of indophenol sulfate in the three groups are 212.0→79.9, 212.0→132.0 and 212.0→93.0, respectively; The ion pairs of the three groups of sulfuric acid p-cresol are 187.0→107.0, 187.0→79.9, and 187.0→125.0, respectively.

4. The application according to claim 1, characterized in that, In step S1, the volume ratio of plasma to acetonitrile is 1:9, the vortexing time is 30-60s, the centrifugation speed is 12000r / min, and the centrifugation time is 10min.

5. The application according to claim 1, characterized in that, In step S1, the activation conditions for the C18 solid-phase extraction column are: sequential rinsing with 5 mL of methanol and 5 mL of pure water at a rinsing rate of 1-2 mL / min.

6. The application according to claim 1, characterized in that, In step S1, the rinsing uses a 5% (w / w) methanol aqueous solution, and the elution uses an 80% (w / w) methanol aqueous solution; and / or, The volume ratio of the eluent to water is 1:9, and the filter membrane used for filtration has a pore size of 0.22 μm.

7. The application according to claim 1, characterized in that, In step S2, the liquid chromatography uses an InfinityLab Porashell 120 EC-C18 column with a column temperature of 40°C, a flow rate of 0.2 mL / min, and an injection volume of 10 μL.

8. The application according to claim 1, characterized in that, In step S2, the liquid chromatography employs a gradient elution program, which is as follows: 0-1.5 min, 95% mobile phase A; 1.5-3.0 min, 95% mobile phase A reduced to 5% mobile phase A; 3.0-6.0 min, 5% mobile phase A; 6.0-7.0 min, 5% mobile phase A increased to 25% mobile phase A; 7.0-9.0 min, 25% mobile phase A increased to 65% mobile phase A; 9.0-11.0 min, 65% mobile phase A increased to 95% mobile phase A; followed by a 5 min run.

9. The application according to claim 1, characterized in that, In step S2, the parameters of the mass spectrometer include: dry gas flow rate 10 L / min, nebulizer pressure 50 psi, sheath gas flow rate 11 L / min, capillary voltage positive 2000 V, negative -4000 V, dry gas temperature 300 °C, sheath gas temperature 350 °C, and collision gas 9 psi.

10. The application according to claim 1, characterized in that, In step S3, the standard curve is prepared as follows: a series of standard solutions with IS concentrations ranging from 0.0850 to 1.3080 μg / mL and PCS concentrations ranging from 0.0905 to 1.3920 μg / mL are prepared. A standard curve is plotted with concentration on the x-axis and characteristic ion abundance on the y-axis, yielding the IS regression equation Y = 36045.242X + 763.841, R0 2 =0.9987; PCS regression equation Y=201921.735X+32600.239, R 2 =0.9956.

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