High performance liquid chromatography method for determining concentration of aquacide and paraquat blood drug and urine drug

Abstract

The invention relates to a high performance liquid chromatography method for determining the blood drug concentration and the urine drug concentration of diquat and paraquat. The high performance liquid chromatography method comprises the following steps: step 1, pre-treating serum and urine samples; step 2, carrying out HILIC-UV detection; and (3) quantitative analysis: outputting an original peak area by using Emppower software developed by the Wortsph company, fitting a regression equation by using Graphpad prism 10.0, selecting a weight coefficient, and calculating a measurement result by using an external standard method. According to the method, laboratory detection is carried out by adopting the most common liquid chromatography-tandem ultraviolet detector. The detection system is low in cost and high in popularization rate in a laboratory. Compared with a Raman spectrum and a liquid chromatography-mass spectrometry technology, the method is easier to realize technical popularization, and the acute diquat or paraquat poisoning detection capability of medical institutions, especially basic medical institutions, is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of clinical toxicology detection, and particularly relates to a high performance liquid chromatography detection method for determining the concentrations of paraquat and diquat in human serum and urine. Background Art

[0002] Paraquat and diquat are dipyridyl herbicides, which are highly toxic and moderately toxic herbicides respectively. The chemical name of paraquat is N,N'-dimethyl-4,4-bipyridinium dichloride; diquat is an analogue of paraquat, and its chemical name is 1,1′-ethylidene-2,2′-bipyridinium dibromide. Paraquat has been completely banned in China since July 2016, but paraquat poisoning is still reported from time to time. The number of acute diquat poisoning incidents has been increasing year by year (Li e Xiao et al., 2021; Meng Hui, 2022). The early clinical symptoms of acute paraquat and diquat poisoning are similar, and both can cause obvious oxidative stress damage to multiple organs, especially abnormal liver and kidney functions. Existing technologies have shown that alanine aminotransferase, aspartate aminotransferase, and urea nitrogen are laboratory indicators that affect the prognosis of patients. However, the similarity of clinical symptoms and laboratory indicators has further increased the diagnostic difficulty of diquat and paraquat. Therefore, China has successively issued the Expert Consensus on the Diagnosis and Treatment of Acute Diquat Poisoning in 2020 and the Expert Consensus on the Diagnosis and Treatment of Acute Paraquat Poisoning (2022). Both of the above consensuses recommend toxicology detection of blood and urine samples to clarify the diagnosis and help judge the prognosis.

[0003] The detection of paraquat and diquat in body fluids includes qualitative, semi - quantitative and quantitative analysis, such as the qualitative or semi - quantitative / quantitative methods developed by Wang Bing, Jia Jun'e, Li Haifeng, etc. (Comparative Document 1: A quantitative / semi - quantitative detection method and application of paraquat or diquat, CN 119413740 A; Comparative Document 2: Development of a rapid identification method for diquat and paraquat and a rapid detection atlas of diquat, Journal of Toxicology, Vol. 33, No. 5, 2019; Comparative Document 3: A method for rapid detection of paraquat and diquat in urine, CN 111781199 A; Comparative Document 4: A colloidal gold immunochromatographic combined detection card for diquat and paraquat, preparation method and application, CN 116539864 A). The above - mentioned methods have the technical advantages of rapid and real - time detection, but also have the following limitations: ① Poor color development stability. In Comparative Document 1, reducing sugar was used to replace the sodium dithionite method used in Comparative Document 2 and Comparative Document 3, and the color development absorbance stability could only be maintained for about 0.5 hours; ② Poor sensitivity. Taking Comparative Document 1 as an example, in this method, the absorbance was linearly regressed with the concentration of the poison, and the linear ranges of paraquat and diquat were 1 μg / mL - 80 μg / mL and 1 μg / mL - 60 μg / mL respectively; ③ Strong subjectivity. In Comparative Document 1, Comparative Document 2 and Comparative Document 3, the standard color scale card or naked - eye observation of color change was used, which had strong subjectivity; ④ Only applicable to qualitative or semi - quantitative detection. Comparative Document 2 and Comparative Document 3 are applicable to semi - quantitative detection of urine, and Comparative Document 4 is only used for qualitative detection. However, qualitative or semi - quantitative analysis is only applicable to the initial screening of poisoning. Developing a quantitative detection technology applicable to clinical laboratories is very necessary for guiding clinical prognosis and individualized treatment.

[0004] The quantitative detection of diquat and / or paraquat mainly relies on Raman spectroscopy, liquid chromatography, and liquid chromatography-tandem mass spectrometry techniques. For example, the surface-enhanced Raman spectroscopy analysis method developed by Xie Jianwei et al. can achieve the determination of paraquat and diquat contents in whole blood, plasma, and urine matrices (Comparative Document 5: A method for rapid detection of paraquat and / or diquat, CN106153596 B). However, the enhancement of Raman spectral signals is significantly substrate-dependent (such as the preparation of core-shell gold nanoparticles); in addition, halogen ions (such as iodide ions and fluoride ions, etc.) must be added to this system to promote colloid aggregation to enhance signal response. Liquid chromatography-tandem mass spectrometry has good detection sensitivity and specificity, and can achieve the rapid detection of samples of various pesticides such as paraquat and diquat and complex biological matrices. For example, a method for detecting paraquat, diquat, glufosinate, and glyphosate in plasma disclosed in the patent with publication number CN 118348137 A (Comparative Document 6) uses a Thermo Acclaim Trinity Q1 chromatographic column and can effectively simultaneously determine the four toxic components in plasma. It should be noted that herbicide poisoning occurs frequently in rural areas of our country, and poisoning reports are particularly concentrated in agricultural provinces such as Hebei, Henan, Shandong, and Anhui (Meng Hui, 2022). The cost of liquid chromatography-mass spectrometry equipment is high (2 million - 4 million yuan) and it is highly dependent on laboratories and technical personnel, which restricts the popularization and development of this technology in primary medical institutions. High-performance liquid chromatography (HPLC) technology is an analytical technology widely used in hospital pharmacy departments and clinical laboratories. This technology is mature and stable and has lower technical requirements for operators. Relying on this technology to carry out technological innovation will be beneficial to the popularization and application of the invention technology. For this reason, Pan Jialiang, Guiyan Yuan, etc. have successively developed HPLC quantitative analysis methods based on ion-pair chromatography (Comparative Document 7: A method for determining the contents of paraquat and diquat in biological fluids, CN 108593811 B; Comparative Document 8: Simultaneous determination of paraquat and diquat in human plasma by HPLC-DAD: Its application in acute poisoning patients induced by these two herbicides, Journal of Clinical Laboratory Analysis, 2021; 35: e23669). However, the above HPLC methods have the following technical defects, which are prominently reflected in: ① The use of strong ion-pair reagents such as sodium heptanesulfonate or sodium octanesulfonate-phosphoric acid aqueous solution is extremely likely to damage the column life; ② It is impossible to take into account various types of body fluid samples or high-throughput detection: for example, the analysis time in Comparative Document 7 is as high as 26 minutes; Comparative Document 8 is only applicable to the detection of plasma samples; ③ The batch difference and non-volatility of ion-pair reagents also lead to great difficulties in method transfer and popularization.

[0005] Hydrophilic interaction chromatography (HILIC) is a normal-phase chromatography technique compatible with aqueous mobile phases and has been applied to the detection of paraquat and diquat residues in foods (Comparative Document 9, A rapid detection method for diquat and paraquat residues in foods, CN 109100450 A). For this reason, the present invention synthesizes the advantages and defects of previous studies and develops an HPLC quantitative method for simultaneously analyzing diquat and paraquat in human serum and urine specimens. To achieve the above objectives, the present invention mainly focuses on improving and innovating the biological sample pretreatment method and chromatographic analysis conditions. Summary of the Invention

[0006] The object of the present invention is to provide a high-performance liquid chromatography method for determining the blood and urine drug concentrations of diquat and paraquat.

[0007] To achieve the above object and other related objects, the technical solution provided by the present invention is: A high-performance liquid chromatography method for determining the blood and urine drug concentrations of diquat and paraquat, comprising the following steps:

[0008] Step 1: Pretreatment of serum and urine samples

[0009] Collect venous blood from patients using vacuum clot activator tubes, centrifuge to separate serum, and after collecting urine, centrifuge to obtain the supernatant to obtain the test samples; dilute the test samples with deionized water, load the diluted samples onto a solid-phase extraction column that has been pre-activated, and after the samples have naturally drained, add deionized water and methanol for rinsing respectively. After vacuum drying, add an acetonitrile-formic acid solution to the solid-phase extraction column for elution; collect the eluate in an injection vial for on-machine analysis;

[0010] Step 2: HILIC-UV detection:

[0011] Chromatographic conditions: The mobile phase consists of A: ammonium acetate with a concentration of 150 mM and B: acetonitrile, isocratic elution, flow rate of 0.6 mL / min; the chromatographic column is Agilent HILIC plus, column temperature 35 °C; the detection wavelengths for diquat and paraquat are 310 nm and 254 nm respectively, injection volume 5 μL;

[0012] (3) Quantitative analysis

[0013] Use the Empower software developed by Waters Corporation to output the original peak areas, use Graphpad prism 10.0 to fit the regression equation and select the weighting coefficient, and calculate the determination results by the external standard method.

[0014] The preferred technical solution is: The centrifugation process parameters are: 1500 - 2000 g × 3 - 8 min.

[0015] The preferred technical solution is: the volume ratio of A to B is 35:65; the pH value of A is adjusted to 4.0 with formic acid.

[0016] The preferred technical solution is: chromatographic column: 2.1 mm × 100 mm, 3.5 μm.

[0017] The preferred technical solution is: the volume ratio of acetonitrile to formic acid in the acetonitrile-formic acid solution is 9:1.

[0018] Due to the application of the above technical solution, the advantages of the present invention compared with the prior art are:

[0019] 1. The present invention uses solid-phase extraction technology for the pretreatment of serum and urine. The solid-phase extraction packing material used in the present invention is a carboxyl-modified polystyrene / divinylbenzene copolymer, which has a dual extraction effect of weak cation exchange and hydrophilic-lipophilic balance reverse phase, and is more suitable for retaining strong basic substances. Specificity investigation and recovery test confirm that the solid-phase extraction method disclosed in the present invention can not only effectively reduce endogenous and exogenous interference in serum or urine, but also efficiently enrich paraquat or diquat components in the matrix.

[0020] 2. The present invention develops a weak ion pair system (mobile phase) based on HILIC for the chromatographic separation of diquat and paraquat. The present invention selects 150 mM ammonium acetate solution as the weak ion pair reagent (formic acid is used to adjust the mobile phase to pH = 4), which can significantly promote the protonation of paraquat and diquat and improve the hydrophilic distribution of the analytes in the water-rich layer. It is found that the retention times of diquat and paraquat are about 4.9 min and 5.6 min respectively, and the two are baseline separated within 10 min.

[0021] 3. The present invention uses the most common liquid chromatography-tandem ultraviolet detector for laboratory detection. This detection system has a relatively low cost (300,000 - 400,000 yuan) and a high penetration rate in laboratories. Compared with Raman spectroscopy and liquid chromatography-mass spectrometry technology, it is easier to promote the technology and improve the detection ability of acute diquat or paraquat poisoning in medical institutions, especially primary medical institutions. Description of the Drawings

[0022] Figure 1 This is a representative chromatogram of paraquat and diquat in serum in the embodiment of the present invention, where: A is the chromatogram of diquat at the lowest quantitative limit of serum; B is the chromatogram of paraquat at the lowest quantitative limit of serum; C is the chromatogram of diquat in blank serum matrix; D is the chromatogram of paraquat in blank serum matrix; E is the chromatogram of diquat in the serum of a paraquat-poisoned patient; F is the chromatogram of paraquat in the serum of a paraquat-poisoned patient; G is the chromatogram of diquat in the serum of a diquat-poisoned patient; H is the chromatogram of paraquat in the serum of a diquat-poisoned patient;

[0023] Figure 2This is a representative chromatogram of paraquat and diquat in urine in the embodiments of the present invention, where:

[0024] A is the chromatogram of diquat at the lowest quantitation limit of urine; B is the chromatogram of paraquat at the lowest quantitation limit of urine; C is the chromatogram of diquat in blank urine matrix; D is the chromatogram of paraquat in blank urine matrix; E is the chromatogram of diquat in urine of paraquat poisoned patients; F is the chromatogram of paraquat in urine of paraquat poisoned patients; G is the chromatogram of diquat in urine of diquat poisoned patients; H is the chromatogram of paraquat in urine of diquat poisoned patients.

[0025] Figure 3 This is a representative chromatogram of paraquat and diquat in serum and urine of patients not poisoned by diquat or paraquat (taking glufosinate as an example) in the embodiments of the present invention, where: A is the chromatogram of diquat in serum of glufosinate poisoned patients; [00X]B is the chromatogram of paraquat in serum of glufosinate poisoned patients; C is the chromatogram of diquat in urine of glufosinate poisoned patients; D is the chromatogram of paraquat in urine of glufosinate poisoned patients.

[0026] Figure 4 This is a schematic diagram of the detection process of the present invention. Detailed implementation manners

[0027] The following specific embodiments illustrate the implementation manners of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this embodiment.

[0028] Please refer to Figures 1-4 . It should be noted that the structures, ratios, sizes, etc. shown in the drawings of this specification are only used to cooperate with the content disclosed in the specification for those skilled in the art to understand and read, and are not used to limit the limited conditions under which the present invention can be implemented. Therefore, they do not have any technical essence. Any modification of the structure, change of the proportional relationship, or adjustment of the size. The following embodiments are provided to better understand the present invention, rather than to limit the present invention. The experimental materials used in the following embodiments are all obtained from conventional consumables and biochemical reagent stores unless otherwise specified.

[0029] At present, the publicly available detection techniques at home and abroad can be divided into qualitative, semi-quantitative, and quantitative detections. Among them, quantitative detection can not only identify the types of poisons but also provide guidance for clinical prognosis and individualized treatment. As mentioned above, surface-enhanced Raman spectroscopy, liquid chromatography-tandem mass spectrometry, and high-performance liquid chromatography are all quantitative detection methods for paraquat and diquat poisoning. However, the above-reported publicly available studies have problems such as complex technology, high cost, or inapplicability to the detection of clinical samples. Paraquat and diquat poisoning are more common in rural areas of our country. Developing a detection method applicable to medical institutions, especially laboratories in primary medical institutions, is more conducive to the application and practice of the invention. In summary, this invention takes the HPLC method with lower equipment cost and technical difficulty as the starting point, and develops a chromatographic separation technology based on the HILIC principle to simultaneously meet the quantitative analysis of diquat and paraquat in serum and urine.

[0030] Example 1

[0031] Materials and Solutions

[0032] 1. Materials: Paraquat reference standard (Paraquat dichloride, Sigma, Lot: SZBF174XV, purity > 99.9%); Diquat reference standard (Diquat dibromide monohydrate, TMstandard, Lot: 21041176, purity > 99.9%); Methanol (chromatographic grade, Tedia, USA); Ammonium acetate (chromatographic grade, Sinopharm Chemical Reagent Co., Ltd.); Solid-phase extraction column (ProElut PWC 60mg / 3mL, Dikma).

[0033] 2. Solutions: (1) 150 mM ammonium acetate (pH = 4) solution: Weigh 5.781 g of ammonium acetate precisely, dissolve it thoroughly with deionized water, adjust the pH to 4 with formic acid, and make up the volume to 500 mL with deionized water. After ultrasonic mixing, the above solution is filtered through a water-based filter membrane (0.22 μm) and reserved for use; (2) Preparation of stock solutions: The stock solutions of paraquat and diquat are both 4000 μg / mL (the solvent is 0.05% formic acid aqueous solution); (3) Quality control and calibration curve working solutions: Precisely pipette an appropriate amount of the stock solution, dilute it to 2.4 mL with deionized water to obtain high, medium, and low-level quality control solutions with concentrations of 1500 μg / mL, 600 μg / mL, and 150 μg / mL; Precisely pipette an appropriate amount of the stock solution to 1200 mL and label it as C1 (1920 μg / mL), and then successively dilute it by a factor of two to obtain calibration curve working solutions of C2 (960 μg / mL), C3 (480 μg / mL), C4 (240 μg / mL), C5 (120 μg / mL), and C6 (60 μg / mL).

[0034] 3. Blank matrix: Serum and urine from patients who have not been exposed to diquat and paraquat (from at least 6 different individuals, from a health examination center, neurology department, and endocrinology department) were centrifuged at 3000 r / min for 10 min and then mixed evenly to obtain the blank matrix.

[0035] 4. Clinical quality control samples and calibration standards: Precisely pipette 10 μL of the quality control and calibration curve working solutions into 1500 μL of the blank matrix to obtain high, medium, and low level clinical quality control samples of paraquat and diquat with mass concentrations of 5 μg / mL, 2 μg / mL, and 0.5 μg / mL respectively. Similarly, prepare serum and urine matrix calibration standards of 0.2 μg / mL, 0.4 μg / mL, 0.8 μg / mL, 1.6 μg / mL, 3.2 μg / mL, and 6.4 μg / mL in sequence. Note: (1) The pipette should be blown and aspirated at least 3 times repeatedly to ensure that the quality control solution is completely dissolved in the matrix;

[0036] (2) The quality control samples and calibration standards must be vortexed for 20 s before freezing to ensure that the analytes are evenly mixed in the matrix;

[0037] (3) They should be stored at a temperature below -60 °C and the storage time should not exceed 14 days.

[0038] Instruments and Methods

[0039] 1. Instrumentation: Waters e2695 high performance liquid chromatograph equipped with a 2489 UV / Vis detector (Waters Corporation, USA); AB135-5 electronic balance (Mettler Toledo, Shanghai); KDC-1044 low-speed centrifuge (Anhui Zhongke Zhongjia Scientific Instrument Co., Ltd.); Pipettes of various specifications (Eppendorf, Germany).

[0040] 2. Analytical conditions: Agilent HILIC plus (2.1 mm × 100 mm, 3.5 μm) was used as the stationary phase, 150 mM ammonium acetate (pH = 4) solution (A) and acetonitrile (B) were used as the mobile phase (35:65, v / v), and isocratic elution was performed. The flow rate was 0.6 mL / min, the column temperature was 35 °C, the injection volume was 5 μL, and the detection wavelengths (λ) of diquat and paraquat were 310 nm and 254 nm respectively.

[0041] 3. Sample pretreatment

[0042] 3.1 After the clinical quality control products and calibration products are naturally thawed, dilute them with deionized water by a factor of two; enrich the target components through equilibration, sample loading, rinsing, and elution. Equilibration: Alternately rinse the solid-phase extraction cartridge with 3 mL of methanol and 3 mL of deionized water; Sample loading: Load the diluted sample into the equilibrated extraction cartridge and wait for the sample to flow out naturally; Rinsing: Alternately rinse with 3 mL of deionized water and 3 mL of methanol, and dry by negative pressure; Elution: Add acetonitrile-formic acid solution (9:1, v / v) to disrupt the reverse phase and cation exchange interactions to elute the target components. Collect the eluate in a standard injection vial for analysis on the instrument.

[0043] 4. Methodological investigation

[0044] Refer to the guiding principles in Section 9012 of the Chinese Pharmacopoeia (2020) (Volume IV) to conduct methodological verification on the established method, including: selectivity, standard curve, precision, accuracy, stability, etc.

[0045] 4.1 Specificity: Pretreat the blank matrix, calibration product at the lower limit of quantitation, and patient blood and urine according to the "Sample Pretreatment" section, and perform the determination on the instrument according to the "Analysis Conditions" section. The results are as Figure 1 . Endogenous and exogenous components in serum and urine did not interfere with the chromatographic quantification of diquat and paraquat, indicating that the detection method has good specificity.

[0046] 4.2 Linear range: Pretreat the calibration product according to the "Sample Pretreatment" section, and perform the determination on the instrument according to the "Analysis Conditions" section. Using 1 / Y 2 as the weighting factor, perform linear regression on the analyte concentration and analyte response to obtain the following weighted standard curves: Y = 30624.67 (±2560.46)X - 12.27 (±658.03) (paraquat in serum matrix); Y = 26012 (±1462.33)X - 551.77 (±558.12) (paraquat in urine matrix); Y = 25050.67 (±2245.33)X - 293.7 (±643.94) (diquat in serum matrix); Y = 21422.67 (±845.23)X - 434.73 (±211.16) (diquat in urine matrix). The results show that paraquat and diquat in serum and urine matrices have a good linear relationship in the range of 0.2 μg / mL to 6.4 μg / mL (R 2 > 0.99).

[0047] 4.3 Recovery experiment Take clinical quality control samples, repeat each level 5 times, and perform pretreatment according to the "Sample Pretreatment" section; as a control, take blank matrix, perform pretreatment according to the "Sample Pretreatment" section, and then add corresponding concentration quality control working solutions respectively. Each concentration is also repeated 5 times. Measure on the machine according to the "Analysis Conditions" section and calculate the recovery rate of the analytical method. The results show that the recoveries of diquat in serum and urine are 83.3%-87.3% and 74.4%-80.6% respectively; the recoveries of paraquat in serum and urine are 83.7%-89.8% and 82.8%-88.8% respectively. The recovery rate of the established method is relatively high and the reproducibility is good (the results are shown in Table 1).

[0048] 4.4 Accuracy and precision tests: Take clinical quality control samples, repeat each level 5 times, perform pretreatment according to the "Sample Pretreatment" section, measure on the machine according to the "Analysis Conditions" section and calculate the within-run precision: measure 3 batches within the 1st day; between-run precision: measure 1 batch every day within 3 days. The results show that the calculated values of the lowest quantitation limit and three-level quality control samples are all within ±15% of their theoretical labeled values, and the relative standard deviations (RSD) within-run and between-run are both less than 15%, meeting the requirements of the "Pharmacopoeia of the People's Republic of China 2020 Edition" for the methodological verification of biological sample analysis (the results are shown in Table 1 and Table 2).

[0049] 4.5 Stability test Take clinical quality control samples, repeat each level 5 times, perform pretreatment according to the "Sample Pretreatment" section, measure on the machine according to the "Analysis Conditions" section to evaluate the stability of the measurement results of this method under different operating conditions: measure the stability of samples placed at room temperature for 6 h, placed in a -25°C refrigerator for 14 days, placed in the auto sampler of the treated samples for 24 h, and repeatedly frozen and thawed 3 times respectively. The results show that diquat and paraquat are stable in serum and urine matrices during short-term placement (except for diquat in urine), repeated freezing and thawing, and placement after treatment; the results of short-term stability and long-term stability suggest that diquat is prone to degradation in urine, and clinical urine samples should be processed immediately and measured within 24 h to ensure the reliability of the results. (The results are shown in Tables 3 to 6).

[0050] Table 1(1) Within-run accuracy, precision and recovery rate of diquat and paraquat in serum and urine matrices

[0051]

[0052]

[0053] Table 1(2) Within-run accuracy, precision and recovery rate of diquat and paraquat in serum and urine matrices

[0054]

[0055] a. The measured concentration is expressed as mean ± standard deviation, with the unit of μg / mL; b. CV: coefficient of variation.

[0056] Table 2(1) Between-batch accuracy and precision of diquat and paraquat in serum and urine matrices

[0057]

[0058] Table 2(2) Between-batch accuracy and precision of diquat and paraquat in serum and urine matrices

[0059]

[0060]

[0061] a. The measured concentration is expressed as mean ± standard deviation, with the unit of μg / mL; b. CV: coefficient of variation.

[0062] Table 3(1) Short-term stability of diquat and paraquat in serum and urine matrices at room temperature (25°C × 6 h)

[0063]

[0064] Table 3(2) Short-term stability of diquat and paraquat in serum and urine matrices at room temperature (25°C × 6 h)

[0065]

[0066] a. The measured concentration is expressed as mean ± standard deviation, with the unit of μg / mL; b. CV: coefficient of variation.

[0067] Table 4(1) Long-term stability of diquat and paraquat in serum and urine matrices (-25°C × 14 d)

[0068]

[0069]

[0070] Table 4(2) Long-term stability of diquat and paraquat in serum and urine matrices (-25°C × 14 d)

[0071]

[0072] a. The measured concentration is expressed as mean ± standard deviation, with the unit of μg / mL; b. CV: coefficient of variation

[0073] Table 5(1) Stability of diquat and paraquat in treated serum and urine matrices in the auto-sampler (24 h)

[0074]

[0075] Table 5(2) Stability of diquat and paraquat in processed serum and urine matrices when placed in an auto-sampler (24 h)

[0076]

[0077] a. The measured concentration is expressed as mean ± standard deviation, with the unit of μg / mL; b. CV: coefficient of variation

[0078] Table 6(1) Stability of diquat and paraquat in serum and urine matrices after three freeze-thaw cycles

[0079]

[0080] Table 6(2) Stability of diquat and paraquat in serum and urine matrices after three freeze-thaw cycles

[0081]

[0082] a. The measured concentration is expressed as mean ± standard deviation, with the unit of μg / mL; b. CV: coefficient of variation

[0083] Example 2: Detection of serum and urine specimens from clinically suspected diquat or paraquat poisoning cases

[0084] (1) Pretreatment of serum and urine samples

[0085] Collect venous blood from the patient using a vacuum clot activator tube and centrifuge at low speed (1800 g × 5 min) to separate the serum; collect midstream urine from the patient using a urine sediment tube and centrifuge at the above-mentioned centrifugal force to separate the supernatant.

[0086] Take 1500 μL of serum or urine sample and dilute it with deionized water in equal proportion. Load the diluted sample onto a solid-phase extraction column that has been pre-activated (washed with 3 mL of methanol and 3 mL of deionized water). After the sample has drained naturally, add 3 mL of deionized water and 3 mL of methanol for rinsing. After vacuum drying, add acetonitrile-formic acid solution (9:1, v / v) to the small column for elution. Collect the eluate in an injection vial for analysis on the machine.

[0087] (2) Quantitative analysis

[0088] The mobile phase consists of 150 mM ammonium acetate (A) and acetonitrile (B) (35:65, the pH of mobile phase A is adjusted to 4.0 with formic acid), isocratic elution, and the flow rate is 0.6 mL / min; the chromatographic column is Agilent HILIC plus (2.1 mm × 100 mm, 3.5 μm), the column temperature is 35 °C; the detection wavelengths (λ) of diquat and paraquat are 310 nm and 254 nm respectively, and the injection volume is 5 μL. The Empower workstation outputs the original signal peak area, and the regression equation is fitted using Graphpad prism 10.0 (the weight coefficient is 1 / Y 2 ), and the determination results can be calculated by the external standard method.

[0089] The method provided by the present invention was used to detect 5 outpatients or inpatients suspected of paraquat / diquat poisoning, with a total of 3 serum and 3 urine specimens. Among them, diquat was only detected in 1 serum sample, paraquat was only detected in 1 serum sample, and neither paraquat nor diquat was detected in 1 serum sample; diquat was only detected in 1 urine sample, paraquat was only detected in 1 urine sample, and neither paraquat nor diquat was detected in 1 urine sample. The results are shown in Table 7. Compared with Zhang *yun, other patients with diquat or paraquat poisoning showed acute liver and kidney function damage, and the pulmonary fibrosis in Yang *you was also in line with the characteristics of acute paraquat poisoning. The clinical manifestations of the patients were basically consistent with the detection results of this method.

[0090] Table 7 Detection results of diquat and paraquat in serum or urine samples of different patients

[0091]

[0092] The above are only preferred embodiments for explaining the present invention, and are not intended to limit the present invention in any form. Therefore, any modification or change to the present invention made under the same inventive spirit should still be included in the scope intended to be protected by the present invention.

Claims

1. A high performance liquid chromatography method for determining the blood and urine concentrations of diquat and paraquat, characterized in that: The following steps are involved: Step 1: Serum and urine sample preparation The patient's venous blood is collected by vacuum coagulation tube, and the serum is separated by centrifugation. After the urine is collected, the supernatant is centrifuged to obtain the sample to be tested; the sample to be tested is diluted with deionized water, and the diluted sample is loaded into a pre-activated solid phase extraction column. After the sample flows out naturally, deionized water and methanol are added for elution respectively. After vacuum drying, acetonitrile-formic acid solution is added to the solid phase extraction column for elution; the eluate is collected in a sample injection bottle for analysis on the machine; Step 2: HILIC-UV detection: Chromatographic conditions: The mobile phase consisted of A: 150 mM ammonium acetate and B: acetonitrile, isocratic elution, flow rate 0.6 mL / min; the chromatographic column was Agilent HILIC plus, column temperature 35 °C; the detection wavelengths of diquat and paraquat were 310 nm and 254 nm, respectively, and the injection volume was 5 μL; (3) Quantitative analysis The original peak area was output by Empower software developed by Waters Corporation, and the regression equation was fitted and the weight coefficient was selected using Graphpad prism 10.0, and the determination results were calculated by the external standard method.

2. The high performance liquid chromatography method for determining the blood and urine concentrations of diquat and paraquat according to claim 1, characterized in that: The centrifugal process parameters are: 1500-2000g×3-8min.

3. The high performance liquid chromatography method for determining the blood and urine concentrations of diquat and paraquat according to claim 1, characterized in that: The volume ratio of A to B was 35:65; the pH value of A was adjusted to 4.0 with formic acid.

4. The high performance liquid chromatography method for determining the blood and urine concentrations of diquat and paraquat according to claim 1, characterized in that: Chromatographic column: 2.1mm×100mm, 3.5μm.

5. The high performance liquid chromatography method for determining the blood and urine concentrations of diquat and paraquat according to claim 1, characterized in that: The volume ratio of acetonitrile to formic acid in the acetonitrile-formic acid solution is 9:1.

Images