Solid-phase extraction-ultra-high performance liquid chromatography-tandem mass spectrometry rapid determination method for multiple antibiotics in human urine

Abstract

The invention relates to a solid phase extraction-ultra-high performance liquid chromatography-tandem mass spectrometry rapid determination method for multiple antibiotics in human urine, which comprises the following steps: adding a mixed internal standard working solution, an acetic acid-ammonium acetate buffer solution and Roman snail beta-glucuronidase into a human urine sample, uniformly mixing, and carrying out enzymolysis in a water bath; then enriching, purifying and concentrating the urine: introducing the pretreated urine into a small HLB solid-phase extraction column activated by using methanol, high-purity water and a sodium hydroxide-monopotassium phosphate buffer solution, sequentially leaching by using the sodium hydroxide-monopotassium phosphate buffer solution and the high-purity water, eluting by using acetonitrile, carrying out weak nitrogen blowing on an eluent in a water bath until the eluent is nearly dried, and collecting the eluent; carrying out volume recovery by utilizing a methanol water solution, carrying out vortex oscillation and then centrifuging to obtain a human urine concentrated sample; and finally, carrying out UPLC-MS / MS detection on the human urine concentrated sample. Compared with the prior art, the method has the advantages of low detection limit, small detection error, short detection time, simplicity in operation, high experimental efficiency and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of urine detection, and in particular to a rapid determination method for multiple antibiotics in human urine by solid-phase extraction-ultra high performance liquid chromatography-tandem mass spectrometry. Background Art

[0002] Humans can be exposed to antibiotics through drugs, food, drinking water and other routes. Long-term exposure to low-dose antibiotics can lead to the emergence of new antibiotic-resistant bacteria, and the antibiotics to which humans are exposed can increase the bioavailability of pesticides by interfering with the intestinal flora, and may further increase the risk of pesticide exposure. In addition, exposure to antibiotics can affect the composition and function of the host flora, and may increase the risks of obesity, diabetes, neurological diseases and asthma. However, the data on the levels of antibiotic exposure in the population are not yet sufficient, which has caused an important obstacle to the health risk assessment of antibiotic exposure. Existing studies mostly use drug prescriptions or questionnaires to measure the clinical exposure to antibiotics, but ignore the exposure to antibiotics from food or the environment, so the true exposure level of antibiotics will be underestimated. There are also some studies that have evaluated the exposure to antibiotics in the environment and food, but external exposure is often difficult to accurately assess and cannot truly reflect the actual exposure level of the human body. By using human biomarkers, the actual internal exposure levels of chemicals from all routes can be analyzed, which may help to improve risk assessment. Experimental findings show that the antibiotics that enter the human body are excreted in urine in the form of prototypes or glucuronic acid conjugates in different proportions, and urine samples are easier to collect than other biological materials such as blood or tissues. Therefore, urinary antibiotic residues may become a scientifically feasible exposure biomarker.

[0003] So far, there is no unified standard for the detection of antibiotics in human urine globally. Although chromatography, immunoassay and electrochemical methods have been preliminarily used to detect antibiotics in human urine, due to the complex composition of urine and strong matrix interference, the vast majority of methods can detect only a small number of antibiotic types, and are complicated in operation and long in detection time, making it difficult to meet the needs of biological monitoring of large-scale populations. Therefore, it is of great significance to establish a high-throughput method that can effectively separate, enrich and quantitatively detect multiple trace antibiotics in human urine, and to study the characteristics of internal exposure to antibiotics in the population for the supervision of antibiotic use and the health risk assessment of the population.

[0004] A study titled "Simultaneous determination of 16 antibiotics in urine by ultra-high performance liquid chromatography-tandem mass spectrometry" published by Chen Cong et al. in the Journal of Forensic Medicine in 2011 analyzed 16 antibiotics in urine using isotope internal standard quantification and the methods of solid-phase extraction (SPE) and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS). However, the types of antibiotics detected by this method are relatively few, and they are all β-lactam antibiotics, without detecting other types of antibiotics, especially those widely used in humans and animals with relatively high detection rates and concentrations. Therefore, it is of great significance to expand the types of target substances to be detected. In addition, β-glucuronidase was not added for hydrolysis in the pretreatment of this method, ignoring the antibiotics in the conjugated form, which may lead to lower detection results or missed detections. Moreover, the limits of detection (LOD) and quantification (LOQ) of antibiotics in this method are 0.05 - 10.0 ng / mL and 0.25 - 20.0 ng / mL respectively. The LOD and LOQ of most antibiotics are relatively high, and the sensitivity is not ideal enough to meet the detection requirements of trace antibiotics in urine. Improving the sensitivity of the detection method is of great value for identifying antibiotic residues in urine and health risk assessment.

[0005] A study titled "Rapid and sensitive screening and selective quantification of antibiotics in human urine by two-dimensional ultraperformance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry" published by Hexing Wang et al. in "Anal Bioanal Chem" in 2014 analyzed 14 antibiotics in six categories in urine using isotope internal standard quantification and the methods of SPE and UPLC-MS / MS. Although this method used a 96-well HLB solid-phase extraction plate to improve the pretreatment efficiency, it only detected and studied 14 target compounds, ignoring most of the antibiotics used in humans, animals, and both. These antibiotics are often absorbed by the human body through various channels and were detected in the samples of this study. And the LOD and LOQ ranges of this method are 0.04 - 1.99 ng / mL and 0.14 - 6.65 ng / mL respectively. The LOD and LOQ are relatively high, and the sensitivity is not ideal.

[0006] Patent Publication No. CN113466381B discloses a method for determining antibiotics in human urine by solid-phase extraction-high performance liquid chromatography-tandem mass spectrometry. In this method, the pretreated human urine sample is processed by solid-phase extraction to obtain a concentrated human urine sample, which is then detected by high performance liquid chromatography-tandem mass spectrometry. However, the average pH of the urine in this method is about 6.0, which is weakly acidic. After adding acetic acid-ammonium acetate buffer solution in the pretreatment, the sample environment is in an acidic state, which is likely to inhibit the ionization of some substances or cause hydrolysis of some compounds, resulting in losses. Moreover, the isocratic elution method is adopted, and the solubility, resolution and peak shape of different compounds are poor. Patent Publication No. CN114509520A discloses a method for determining antibiotics in human urine by solid-phase extraction-high performance liquid chromatography-tandem mass spectrometry. By combining the pretreatment method of solid-phase extraction with the method of liquid chromatography-mass spectrometry, 18 antibiotic drugs in five categories in human urine are detected simultaneously. First, the urine sample is thawed and centrifuged to obtain the supernatant. The supernatant is taken and EDTA-2Na buffer solution, β-glucuronidase aqueous solution and mixed standard stock solution are added, and hydrolysis is carried out overnight. Then, a solid-phase extraction cartridge is used to purify, extract and enrich 18 target substances to be detected in the urine sample. Then, a high performance liquid chromatography-mass spectrometry is used as the detection platform to qualitatively and quantitatively detect the target substances in the urine. However, the types of antibiotics that can be detected by this method are few and the detection limit is relatively high. Summary of the Invention

[0007] The purpose of the present invention is to overcome the defects of the above-mentioned existing technologies and provide a rapid determination method for multiple antibiotics in human urine by solid-phase extraction-ultra high performance liquid chromatography-tandem mass spectrometry.

[0008] The purpose of the present invention can be achieved by the following technical solutions:

[0009] A rapid determination method for multiple antibiotics in human urine by solid-phase extraction-ultra high performance liquid chromatography-tandem mass spectrometry includes the following steps:

[0010] A1. Urine pretreatment: Add a mixed internal standard working solution, acetic acid-ammonium acetate buffer solution, and Helix pomatia β-glucuronidase to the human urine sample, mix well, and perform enzymatic hydrolysis in a water bath.

[0011] A2. Urine enrichment, purification and concentration: Pass the urine obtained in step A1 through an HLB solid-phase extraction cartridge activated with methanol, high-purity water, and sodium hydroxide-potassium dihydrogen phosphate buffer solution, then wash it successively with sodium hydroxide-potassium dihydrogen phosphate buffer solution and high-purity water, and dry it by suction under vacuum conditions. Elute with acetonitrile, weakly nitrogen-blow the eluate to near dryness in a water bath, then reconstitute it with methanol aqueous solution, vortex and centrifuge to obtain a concentrated human urine sample.

[0012] A3. Ultra Performance Liquid Chromatography-tandem Mass Spectrometry (UPLC-MS / MS) Detection: The concentrated human urine sample obtained in step A2 is subjected to UPLC-MS / MS for the detection of antibiotics. The antibiotics to be detected include tetracycline, oxytetracycline, chlortetracycline, doxycycline, ciprofloxacin, ofloxacin, norfloxacin, pefloxacin, enrofloxacin, danofloxacin, difloxacin, lomefloxacin, azithromycin, roxithromycin, clarithromycin, tilmicosin, trimethoprim, sulfadiazine, sulfamethoxazole, sulfamerazine, acetylated sulfamethoxazole, acetylated sulfamerazine, chloramphenicol, thiamphenicol, florfenicol.

[0013] Further, in step A1, the volume ratio of the acetic acid-ammonium acetate buffer solution, Roman snail β-glucuronidase, and human urine sample is (200 - 400):(15 - 25):1. Even further, in step A1, the volume ratio of the acetic acid-ammonium acetate buffer solution to the human urine sample is (300 - 400):1, preferably 400:1, to adapt to the optimal pH of the Roman snail β-glucuronidase.

[0014] Further, in step A1, the concentration of the mixed internal standard working solution is 50 or 100 μg / L, the pH of the acetic acid-ammonium acetate buffer solution is 4.4 - 4.6, and the enzyme activity of the Roman snail β-glucuronidase is greater than or equal to 100000 units / mL.

[0015] Further, in step A1, the water bath enzymatic hydrolysis temperature is 30 - 40°C, and the water bath enzymatic hydrolysis time is 7 - 12 h. Even further, the water bath enzymatic hydrolysis temperature is 35 - 39°C, preferably 37°C.

[0016] Further, in step A2, the volume ratio of the HLB solid phase extraction small column to methanol, high-purity water, and sodium hydroxide-potassium dihydrogen phosphate buffer solution used for activation is 3:(1 - 3):(1 - 3):(1 - 3); even further, the volume ratio of the HLB solid phase extraction small column to methanol, high-purity water, and sodium hydroxide-potassium dihydrogen phosphate buffer solution used for activation is 3:2:2:2.

[0017] Further, the flow rate of the urine passing through the activated HLB solid phase extraction small column is 1 - 5 mL / min.

[0018] Further, in step A2, the volume ratio of the HLB solid-phase extraction column to the sodium hydroxide-potassium dihydrogen phosphate buffer solution and high-purity water used for elution is 3:(1-2):(1-2). More specifically, the volume ratio of the HLB solid-phase extraction column to the sodium hydroxide-potassium dihydrogen phosphate buffer solution and high-purity water used for elution is 3:1:2.

[0019] Further, in step A2, the volume ratio of the HLB solid-phase extraction column to acetonitrile is 3:(2-4) mL. More specifically, in step A2, the volume ratio of the HLB solid-phase extraction column to acetonitrile is 1:1 mL.

[0020] Further, in step A2, the volume ratio of the methanol aqueous solution to the human urine sample is 1:2 or 1:4, which is specifically determined according to the concentration multiple and dissolution volume of the human urine sample.

[0021] Further, in step A2, the water bath temperature is 30-45 °C, the rotation speed of the vortex is 1000-3000 rpm, the rotation speed of the centrifugation is 8000-12000 rpm, and the centrifugation time is 2-5 min, and the purpose is to wash away impurities. More specifically, the water bath temperature is 35-39 °C, preferably 37 °C.

[0022] Further, in step A3, the parameters of the ultra-high performance liquid chromatography in the UPLC-MS / MS detection are as follows: the chromatographic column is a T3 chromatographic column, 100 mm × 2.1 mm, 1.8 μm; mobile phase A is a methanol-acetonitrile solution, mobile phase B is a formic acid-aqueous solution, and a gradient elution method is adopted; the flow rate is 0.5 mL / min, the column temperature is 35 °C, and the injection volume is 10 or 15 μL.

[0023] Further, the volume concentration of methanol in the methanol-acetonitrile solution is 35%-45%, and the volume concentration of formic acid in the formic acid-aqueous solution is 0.1%-0.3%. More specifically, the volume concentration of methanol in the methanol-acetonitrile solution is 40%, and the volume concentration of formic acid in the formic acid-aqueous solution is 0.2%.

[0024] More specifically, the specific conditions of the gradient elution method are as follows:

[0025] 0-1.0 min, 10% mobile phase A + 90% mobile phase B;

[0026] 1.0-7.0 min, 98% mobile phase A + 2% mobile phase B;

[0027] 7.0-9.5 min, 98% mobile phase A + 2% mobile phase B;

[0028] 9.5-9.8 min, 10% mobile phase A + 90% mobile phase B;

[0029] 9.8 to 11.5 min, 10% mobile phase A + 90% mobile phase B.

[0030] Further, in step A3, the parameters of the mass spectrometry in the HPLC-MS / MS detection are as follows: the ionization mode is simultaneous scanning of the electrospray positive ion source and the electrospray negative ion source, the ion source voltage is 4000 - 5000 V, the temperature is 300 - 500 °C, the curtain gas is 20 - 40 psi, the nebulizing gas is 50 - 60 psi, the auxiliary heating gas is 50 - 60 psi, and the monitoring mode is the multiple reaction monitoring mode.

[0031] Compared with the prior art, the present invention has the following advantages:

[0032] (1) Humans can be exposed to antibiotics through drug use, food intake, drinking water, living environment, etc. Previous studies have found that 54 antibiotics have been detected in human urine. Among the above 25 antibiotics, most have been reported to have a high detection rate and detection concentration. However, the interaction between substances in the mixed system will produce matrix effects, and the matrix effects have not been well eliminated or weakened. In addition, the above 25 antibiotics have a high usage frequency in population surveys and veterinary antibiotic lists, and pose a relatively large potential health risk to the population. The present invention optimizes the mass spectrometry parameters, chromatographic conditions and pretreatment methods, adopts the isotope internal standard method, and establishes a method for simultaneously detecting 25 kinds of antibiotic residues including tetracyclines, macrolides, fluoroquinolones, phenols and sulfonamides in human urine by solid phase extraction-ultra high performance liquid chromatography-tandem mass spectrometry, showing certain advantages in aspects such as sample pretreatment, mass spectrometry conditions, method verification and actual sample detection.

[0033] (2) The present invention greatly reduces the detection error. By adding β-glucuronidase, the conjugated antibiotics are hydrolyzed. The enzyme-hydrolyzed urine sample increases the chance of detecting residual antibiotics and can more accurately complete the qualitative and quantitative evaluation of residual antibiotics, providing technical support for the discovery of more trace antibiotic residues.

[0034] (3) During all pretreatment processes, experimental conditions need to be considered, including the addition of reagents and the pH of the samples. Inappropriate sample pH may cause ionization and degradation of certain antibiotics. Sulfonamides (basic compounds) are prone to ionization under acidic conditions; tetracycline is unstable at pH < 2.0. The average pH of urine is about 6.0, which is weakly acidic. After adding acetic acid-ammonium acetate buffer solution during pretreatment, the sample environment becomes acidic, which is likely to inhibit the ionization of certain substances or cause hydrolysis of some compounds, resulting in losses. In the present invention, sodium hydroxide-potassium dihydrogen phosphate buffer solution is added during the activation and elution of the HLB solid-phase extraction column, increasing the pH, further enhancing the ionization of the analytes, reducing hydrolysis, and thus improving the detection rate.

[0035] (4) The present invention uses UPLC-MS / MS, which has extremely high sensitivity and detection rate, further improving the sensitivity and detection efficiency. The selection and mixing ratio of the mobile phase in the liquid chromatography system have an important impact on the ionization of target compounds. Gradient elution can enhance the ionization of compounds as much as possible according to the heterogeneity of the compounds, improve the peak intensity and peak shape. By continuously optimizing the mass spectrometry conditions, while ensuring the stability of the spiked recovery rate, all 25 antibiotics in five categories can be detected within one method, compressing the time of the entire detection method as much as possible. Finally, all 25 antibiotic substances can elute within 11.5 minutes, and the quality of the peak shape diagram is relatively high.

[0036] (5) After the method of the present invention is established, it is verified. The 25 antibiotics have a good linear relationship within the measurement range, ensuring the stability of the recovery rate, and each target substance has a low detection limit, with high accuracy and good sensitivity.

[0037] (6) After the method verification of the present invention, the detection of human urine samples is carried out. This method is simple to operate, and the test results are similar to those of similar studies and consistent with the actual situation, providing a more sensitive and convenient technical method for the rapid detection of large sample sizes and greatly improving the experimental efficiency. Description of the Drawings

[0038] Figure 1 Ion current diagram of ciprofloxacin shown in Example 1;

[0039] Figure 2 Ion current diagram of ofloxacin shown in Example 1;

[0040] Figure 3 Ion current diagram of norfloxacin shown in Example 1;

[0041] Figure 4 Ion current diagram of pefloxacin shown in Example 1;

[0042] Figure 5Ion flow diagram of enrofloxacin shown in Example 1;

[0043] Figure 6 Ion flow diagram of danofloxacin shown in Example 1;

[0044] Figure 7 Ion flow diagram of difloxacin shown in Example 1;

[0045] Figure 8 Ion flow diagram of lomefloxacin shown in Example 1;

[0046] Figure 9 Ion flow diagram of tetracycline shown in Example 1;

[0047] Figure 10 Ion flow diagram of oxytetracycline shown in Example 1;

[0048] Figure 11 Ion flow diagram of chlortetracycline shown in Example 1;

[0049] Figure 12 Ion flow diagram of doxycycline shown in Example 1;

[0050] Figure 13 Ion flow diagram of azithromycin shown in Example 1;

[0051] Figure 14 Ion flow diagram of roxithromycin shown in Example 1;

[0052] Figure 15 Ion flow diagram of clarithromycin shown in Example 1;

[0053] Figure 16 Ion flow diagram of thiamphenicol shown in Example 1;

[0054] Figure 17 Ion flow diagram of tilmicosin shown in Example 1;

[0055] Figure 18 Ion flow diagram of florfenicol shown in Example 1;

[0056] Figure 19 Ion flow diagram of trimethoprim shown in Example 1;

[0057] Figure 20 Ion flow diagram of sulfadiazine shown in Example 1;

[0058] Figure 21 Ion flow diagram of sulfamethoxazole shown in Example 1;

[0059] Figure 22 Ion flow diagram of sulfamerazine shown in Example 1;

[0060] Figure 23 Ion current diagram of acetylated sulfamethoxazole shown in Example 1;

[0061] Figure 24 Ion current diagram of acetylated sulfamerazine shown in Example 1;

[0062] Figure 25 Ion current diagram of chloramphenicol shown in Example 1;

[0063] Figure 26 Ion current diagram of D4-sulfamethoxazole shown in Example 1;

[0064] Figure 27 Ion current diagram of D8-ciprofloxacin shown in Example 1;

[0065] Figure 28 Ion current diagram of D6-tetracycline shown in Example 1;

[0066] Figure 29 Ion current diagram of D3-azithromycin shown in Example 1;

[0067] Figure 30 Ion current diagram of D7-roxithromycin shown in Example 1;

[0068] Figure 31 Ion current diagram of D5-chloramphenicol shown in Example 1;

[0069] Figure 32 Ion current diagram of 25 antibiotics shown in Example 1 (c = 50 ug / L);

[0070] Figure 33 Total ion current diagram of 25 antibiotics shown in Example 1 (c = 50 ug / L). Detailed implementation mode

[0071] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. This embodiment is implemented on the premise of the technical solution of the present invention, and the detailed implementation mode and specific operation process are given. However, the protection scope of the present invention is not limited to the following embodiments. Based on the given embodiments, all other embodiments obtained by those of ordinary skill in the art without creative work belong to the scope of protection of this application.

[0072] Unless otherwise specified, the reagents, methods, instruments and equipment used in the present invention are conventional reagents, methods, instruments and equipment in the art.

[0073] Ultra-high performance liquid chromatograph (Waters, UK), equipped with a mass spectrometer (ABSCIEX QTRAP 6500+, USA); solid phase extraction instrument (ANPEL, China); nitrogen evaporator (LabTech, China); high-purity water instrument (Milli-Q, USA); Oasis HLB solid phase extraction cartridge 3cc, 60mg (Waters, USA); 0.7μm glass fiber filter membrane (Whatman, USA); chromatographic column: ACQUITY UPLC T3 (100mm×2.1mm, 1.8μm).

[0074] Reference standards: (1) Fluoroquinolones: Ofloxacin (CAS No.: 82419-36-1), Ciprofloxacin (CAS No.: 93107-08-5), Norfloxacin (CAS No.: 70458-96-7), Enrofloxacin (CAS No.: 93106-60-6), Pefloxacin (CAS No.: 70458-95-6), Difloxacin (CAS No.: 91296-86-5), Danofloxacin (CAS No.: 119478-55-6), Lomefloxacin (CAS No.: 98079-52-8). (2) Tetracyclines: Doxycycline (CAS No.: 24390-14-5), Tetracycline (CAS No.: 64-75-5), Oxytetracycline (CAS No.: 2058-46-0), Chlortetracycline (CAS No.: 64-72-2). (3) Macrolides: Clarithromycin (CAS No.: 81103-11-9), Azithromycin (CAS No.: 83905-01-5), Roxithromycin (CAS No.: 80214-83-1), Tilmicosin (CAS No.: 108050-54-0). (4) Sulfonamides: Sulfadiazine (CAS No.: 68-35-9), Trimethoprim (CAS No.: ), Sulfamerazine (CAS No.: 127-79-7), Sulfamethoxazole (CAS No.: 723-46-6), N-Acetylsulfamerazine (CAS No.: 100-90-3), N-Acetylsulfamethoxazole (CAS No.: 21312-10-7). (5) Phenols: Chloramphenicol (CAS No.: 56-75-7), Florfenicol (CAS No.: 73231-34-2), Thiamphenicol (CAS No.: 15318-45-3). All were purchased from Dr. Ehrenstorfer GmbH, Germany.

[0075] Reagents: Methanol (chromatographic grade, USA, Supelco), acetonitrile (chromatographic grade, USA, Supelco), formic acid (chromatographic grade, China, Aladdin), sodium hydroxide-potassium dihydrogen phosphate buffer solution (pH = 8.00 ± 0.02, China, Sinopharm), acetic acid-ammonium acetate buffer solution (pH = 4.50 ± 0.10, China, Sinopharm), Helix pomatia β-glucuronidase (Type HP-2, aqueous solution, ≥100,000 units / mL, USA, Sigma).

[0076] Example 1

[0077] A rapid determination method for multiple antibiotics in human urine by solid-phase extraction-ultra-high performance liquid chromatography-tandem mass spectrometry, comprising the following steps:

[0078] (1) Preparation of solutions

[0079] Accurately weigh 10 mg of each standard product, dissolve it with methanol and make up the volume to 10 mL in a brown volumetric flask. The standard stock solution is stored in a -20 °C refrigerator. Appropriately draw the standard stock solutions of 25 antibiotics, dilute them with methanol-water (V:V = 1:1) solution to prepare a mixed standard working solution with a total concentration of 1 mg / L, and store it in a 4 °C refrigerator for standby.

[0080] (2) Pretreatment of urine

[0081] Accurately measure 1 mL of human urine sample, add 50 μL of 100 μg / L mixed internal standard working solution, 400 μL of acetic acid-ammonium acetate buffer solution, and 15 μL of Helix pomatia β-glucuronidase respectively. After mixing well, enzymatically hydrolyze overnight (7 hours) in a 37 °C water bath.

[0082] (3) Enrichment, purification and concentration of urine

[0083] Use a solid-phase extraction instrument for sample enrichment and purification. Activate the HLB solid-phase extraction column with 2 mL of methanol, 2 mL of high-purity water and 2 mL of sodium hydroxide-potassium dihydrogen phosphate buffer solution in sequence, and dry it under vacuum conditions; pass the activated HLB solid-phase extraction column at a flow rate of 5 mL / min to enrich the urine sample. After the sample loading is completed, wash the HLB solid-phase extraction column with 1 mL of sodium hydroxide-potassium dihydrogen phosphate buffer solution and 2 mL of high-purity water in sequence, then elute with 3 mL of acetonitrile. The eluate is gently blown to dry in a 37 °C water bath under weak nitrogen, reconstitute with 0.5 mL of 50% methanol aqueous solution, vortex and oscillate, and centrifuge at 10,000 r for 2 minutes for measurement.

[0084] (4) UPLC-MS / MS detection

[0085] Ultra-high performance liquid chromatography tandem mass spectrometry was used to detect antibiotics in concentrated human urine samples. The antibiotics detected included tetracycline, oxytetracycline, chlortetracycline, doxycycline, ciprofloxacin, ofloxacin, norfloxacin, pefloxacin, enrofloxacin, danofloxacin, difloxacin, lomefloxacin, azithromycin, roxithromycin, clarithromycin, tilmicosin, trimethoprim, sulfadiazine, sulfamethoxazole, sulfamerazine, acetylated sulfamethoxazole, acetylated sulfamerazine, chloramphenicol, thiamphenicol, and florfenicol.

[0086] The parameters of ultra-high performance liquid chromatography in UPLC-MS / MS detection were as follows: the chromatographic column was ACQUITY UPLC T3 (100 mm × 2.1 mm, 1.8 μm); the column temperature was 35 °C; the flow rate was 0.35 mL / min; the injection volume was 10 μL. Mobile phase A: 40% methanol-acetonitrile solution, mobile phase B: 0.2% formic acid-aqueous solution. The gradient elution method was adopted, and the specific conditions are shown in Table 1.

[0087] Table 1 Gradient elution conditions

[0088]

[0089] The parameters of mass spectrometry in HPLC-MS / MS detection were as follows: the ionization modes were electrospray positive ion source (ESI + ) and electrospray negative ion source (ESI - ) for simultaneous scanning. The ion source voltage was 4500 V, the temperature was 400 °C, the curtain gas was 30 psi, the spray gas was 55 psi, the auxiliary heating gas was 55 psi, and the monitoring mode was multiple reaction monitoring mode.

[0090] The mass spectrometry parameters of 25 antibiotic drugs and 6 internal standard substances are shown in Table 2 and Figures 1 to 31 .

[0091] Table 2 Mass spectrometry parameters of 25 antibiotic drugs and 6 internal standard substances

[0092]

[0093]

[0094] This method was verified, standard curves and method detection limits were established, the accuracy, precision, and matrix effects of the method were verified, and actual samples were detected.

[0095] 1.1 Standard curves and method detection limits

[0096] An internal standard calibration curve (2, 5, 10, 20, 50, 100, 150, 200 ng / mL) was made using the isotopic compounds of antibiotics as quantitative internal standards. By the method of stepwise dilution of the standard solution, when the signal-to-noise ratios of the chromatographic peaks were not less than 3 and 10 respectively, they were determined as the instrument detection limit and the instrument quantification limit. According to the parameters of the instrument detection limit (Instrument detection limits, IDL) and the quantification limit (Instrument quantification limits, IQL), combined with the concentration factor and the spike recovery rate, the method detection limit (Method detection limits, MDL) and the method quantification limit (Method quantification limits, MQL) of each target antibiotic were calculated. The method detection limit (MDL) and the method quantification limit (MQL) were calculated using formula (1).

[0097] MDL = IDL / (Rec × Cf) × 100% Formula (1)

[0098] Among them, MDL represents the method detection limit, IDL represents the instrument detection limit, Rec represents the average spike recovery rate, and Cf represents the dilution factor.

[0099] Taking the peak areas of the antibiotics and their internal standards as the ordinate and the concentrations of the antibiotics as the abscissa for linear regression, 18 antibiotics showed good linearity in the range of 2 - 200 ng / mL, 5 antibiotics showed good linearity in the range of 2 - 150 ng / mL, and 2 antibiotics showed good linearity in the range of 2 - 100 ng / mL. The MDL was 0.0003 - 0.0509 ng / mL, and the MQL was 0.0009 - 0.1696 ng / mL. The linear equations, correlation coefficients, method detection limits and method quantification limits of each target analyte are shown in Table 3. The ion chromatograms and total ion chromatograms of 25 antibiotics are as Figure 32 and 33 shown.

[0100] Table 3 Linear parameters, method detection limits and method quantification limits of 25 antibiotic drugs

[0101]

[0102]

[0103] 1.2 Accuracy, precision and matrix effect of the method

[0104] Intraday precision and interday precision were verified by injecting samples at three standard points of 10, 50, and 100 ng / mL six times continuously within one day and repeating the operation continuously for three days. Six human urine samples were spiked and recovered at three spiked levels of 10, 50, and 100 ng / mL, and determined in parallel six times. The average spiked recoveries (adding standard recovery, Rec) of low, medium, and high levels of each target antibiotic were calculated. Matrix Effects (ME) were obtained by comparing the peak areas obtained by UHPLC-MS / MS analysis of the target compounds added at each mass concentration after extraction with blank urine with the peak areas directly obtained from the standard solutions of the corresponding mass concentrations. As shown in Tables 4 and 5, the average spiked recoveries of 25 antibiotics at three different concentration levels were 60.79% - 125.56%, the interday precision was 3.11% - 12.39%, and the intraday precision was 2.87% - 11.78%. In this study, the matrix effects of 25 target antibiotics were evaluated at three standard points of 10, 50, and 100 ng / mL. The results showed that the average matrix effects at the three concentration levels were 63.09% - 136.58%. The good spiked recoveries of the above 25 antibiotics indicated that the matrix effects could be corrected by isotope internal standards.

[0105] Table 4 Intraday precision and interday precision of 25 antibiotics

[0106]

[0107]

[0108] Table 5 Spiked recoveries and matrix effects of 25 antibiotics

[0109]

[0110]

[0111] 1.3 Detection of actual samples

[0112] From July to August 2020, 1,426 early morning urine samples were collected from residents in two rural communities, namely D County in Zhejiang Province and H City in Jiangsu Province. During sample collection, the samples were stored away from light, transported to the laboratory under cold chain conditions, and aliquoted and frozen at -80°C in the refrigerator within 12 hours. The urine samples were analyzed using the established optimized protocol. After pretreatment, an ultra-high performance liquid chromatography-tandem mass spectrometry instrument was used to detect 25 target antibiotics. The results are shown in Tables 6 - 8. A total of 24 antibiotics were detected, with the detection rate ranging from 0.14% to 40.11%, and the total detection rate being 73.91%. These included 4 tetracycline antibiotics, 8 fluoroquinolone antibiotics, 3 macrolide antibiotics, 6 sulfonamide antibiotics, and 3 phenolic antibiotics. The overall detection rates from high to low were: ofloxacin (40.11%), azithromycin (19.14%), ciprofloxacin (15.01%), norfloxacin (13.74%), danofloxacin (8.77%), enrofloxacin (8.49%), N-acetylsulfamethazine (5.26%), trimethoprim (3.58%), clarithromycin (2.38%), doxycycline (2.24%), pefloxacin (2.17%), roxithromycin (2.1%), tetracycline (1.68%), sulfadiazine (1.61%), chlortetracycline (1.54%), acetylsulfamethoxazole (1.05%), lomefloxacin (0.84%), sulfamethoxazole (0.77%), oxytetracycline (0.7%), difloxacin (0.56%), sulfamethazine (0.28%), chloramphenicol (0.28%), thiamphenicol (0.21%), and florfenicol (0.14%). This indicates that antibiotic exposure is widespread in the human body.

[0113] The total detection rate of 73.91% is close to the detection rates of 582 school-age children in Shanghai in 2016 (79.6%) and 31 preschool children in Hong Kong, China in 2017 (77.4%); however, it is higher than that of children (51.9 - 58.3%), pregnant women (41.6%), adults (45.9%) in East China (Shanghai, Zhejiang Province, Jiangsu Province) from 2015 to 2021 and children in the Qinghai-Tibet Plateau in 2020 (58.6%); and lower than that of pregnant women in East China (Shanghai, Anhui) in 2020 (93.6 - 98.6%), the elderly in Anhui Province (93.0%) and the general population in Anhui Province (92.0%). From the perspective of categories and uses, fluoroquinolone antibiotics and antibiotics shared by humans and animals contribute the most to the detection of antibiotics, which is also consistent with previous studies. For individual antibiotics, this study found that the top three detection rates were ofloxacin (40.11%), azithromycin (19.14%), and ciprofloxacin (15.01%). The detection rate of ofloxacin is close to the reports of pregnant women in Shanghai in 2020 (39.2%) and 2021 (39.3%) and pregnant women in Anhui Province in 2020 (38.5%). The detection rate of azithromycin is similar to the detection rates reported by the elderly in Anhui Province in 2021 (18.2%) and children in Shanghai in 2015 (16.4%). The detection rate of ciprofloxacin is consistent with the study of Wang et al. on adults in Shanghai in 2018 (15.2%) and is also close to several other studies in East China (11.3 - 16.5%). Ofloxacin and ciprofloxacin are commonly used antibiotics shared by humans and animals, and azithromycin is a commonly used antibiotic only for humans, which to a certain extent indicates that animal-derived food and drug use are the main causes of antibiotic exposure.

[0114] Table 6 Detection rates (%) and detected concentrations (ng / mL) of 25 antibiotics in urine of rural adults in East China

[0115]

[0116]

[0117] Table 7 Detection rates (%) and detected concentrations (ng / mL) of 25 antibiotics in urine of adults in County D

[0118]

[0119] Table 8 Detection rates (%) and detected concentrations (ng / mL) of 25 antibiotics in urine of rural adults in City H

[0120]

[0121] Note: a Sum of antibiotic concentrations in the corresponding category of an individual; b Sum of all antibiotic concentrations;c n (%), the number of positive samples detected (detection rate); d Volume-based concentration value, μg / L; e Due to similar antibacterial mechanisms, trimethoprim is classified into sulfonamides; <LOD, below the detection limit.

[0122] PHA, preferred as human antibiotics; PVA, preferred as veterinary antibiotics; HA / VA, human and veterinary antibiotics.

[0123] The detection method of the present invention optimizes the mass spectrometry parameters, chromatographic conditions and pretreatment methods, adopts the isotope internal standard method, and establishes a method for simultaneously detecting 25 kinds of antibiotic residues including tetracyclines, macrolides, fluoroquinolones, phenols and sulfonamides in human urine by solid phase extraction-ultra high performance liquid chromatography-tandem mass spectrometry, and applies it to the detection of antibiotic residues in urine of large-scale populations in two rural communities in Zhejiang Province and Jiangsu Province. From the above results, it can be seen that the 25 kinds of antibiotics have low detection limits, small detection errors, short detection times, simple operations and high experimental efficiencies.

[0124] Example 2

[0125] A rapid determination method for multiple antibiotics in human urine by solid phase extraction-ultra high performance liquid chromatography-tandem mass spectrometry, comprising the following steps:

[0126] (1) Preparation of solutions

[0127] Accurately weigh 10 mg of each standard product, dissolve it with methanol and make up the volume to 10 mL in a brown volumetric flask. The standard stock solution is stored in a refrigerator at -20 °C. Appropriately draw the standard stock solutions of 25 kinds of antibiotics, dilute them with methanol-water (V:V = 1:1) solution to prepare a mixed standard working solution with a total concentration of 1 mg / L, and store it in a refrigerator at 4 °C for later use.

[0128] (2) Pretreatment of urine

[0129] Accurately measure 1 ml of human urine sample, accurately measure 1 ml of human urine sample, add 50 μL of 100 μg / L mixed internal standard working solution, 200 μL of acetic acid-ammonium acetate buffer solution, and 15 μL of Roman snail β-glucuronidase respectively. After mixing well, enzymatically hydrolyze at 37 °C overnight (7 hours).

[0130] (3) Enrichment, purification and concentration of urine

[0131] The sample was enriched and purified using a solid-phase extraction instrument. The HLB solid-phase extraction cartridge was activated successively with 2 mL of methanol, 2 mL of high-purity water, and 2 mL of sodium hydroxide-potassium dihydrogen phosphate buffer solution, and then dried under vacuum conditions. The activated HLB solid-phase extraction cartridge was passed through at a flow rate of 5 mL / min to enrich the urine sample. After the sample loading was completed, the HLB solid-phase extraction cartridge was rinsed successively with 1 mL of sodium hydroxide-potassium dihydrogen phosphate buffer solution and 2 mL of high-purity water, and then eluted with 3 mL of acetonitrile. The eluate was gently blown to dry under nitrogen at 37 °C, reconstituted with 0.5 mL of 50% methanol aqueous solution, vortexed, and centrifuged at 10000 r for 2 minutes for determination.

[0132] (4) UPLC-MS / MS detection

[0133] Ultra-high performance liquid chromatography-tandem mass spectrometry was used to detect antibiotics in concentrated human urine samples. The antibiotics detected included tetracycline, oxytetracycline, chlortetracycline, doxycycline, ciprofloxacin, ofloxacin, norfloxacin, pefloxacin, enrofloxacin, danofloxacin, difloxacin, lomefloxacin, azithromycin, roxithromycin, clarithromycin, tilmicosin, trimethoprim, sulfadiazine, sulfamethoxazole, sulfamerazine, acetylated sulfamethoxazole, acetylated sulfamerazine, chloramphenicol, thiamphenicol, and florfenicol. The parameters of ultra-high performance liquid chromatography in UPLC-MS / MS detection were as follows: the chromatographic column was ACQUITY UPLC T3 (100 mm × 2.1 mm, 1.8 μm); the column temperature was 35 °C; the flow rate was 0.35 mL / min; the injection volume was 10 μL. Mobile phase A: 40% methanol-acetonitrile solution, mobile phase B: 0.2% formic acid-aqueous solution, and gradient elution was used. The specific conditions are shown in Table 1. The parameters of mass spectrometry in HPLC-MS / MS detection were as follows: ionization was performed by electrospray positive ion source (ESI + ) and electrospray negative ion source (ESI - ) simultaneously, the ion source voltage was 4500 V, the temperature was 400 °C, the curtain gas was 30 psi, the spray gas was 55 psi, the auxiliary heating gas was 55 psi, and the monitoring mode was multiple reaction monitoring mode.

[0134] Six human urine samples were taken for spike recovery tests at three spiked levels of 10, 50, and 100 ng / mL, and parallel determination was performed 6 times. The medium, low, high, and average spike recoveries of each target antibiotic were calculated, and the average spike recoveries of 25 antibiotics at three different concentration levels were 47.62% - 281.91%.

[0135] Example 3

[0136] A rapid determination method for multiple antibiotics in human urine by solid-phase extraction-ultra-high performance liquid chromatography-tandem mass spectrometry, comprising the following steps:

[0137] (1) Preparation of solutions

[0138] Accurately weigh 10 mg of each standard product, dissolve it with methanol and make up the volume to 10 mL in a brown volumetric flask. The standard stock solution is stored in a refrigerator at -20 °C. Appropriately pipette the standard stock solutions of 25 antibiotics, dilute them with methanol-water (V:V = 1:1) solution to prepare a mixed standard working solution with a total concentration of 1 mg / L, and store it in a refrigerator at 4 °C for standby.

[0139] (2) Pretreatment of urine

[0140] Accurately measure 1 mL of human urine sample, add 50 μL of 100 μg / L mixed internal standard working solution, 400 μL of acetic acid-ammonium acetate buffer solution, and 15 μL of Helix pomatia β-glucuronidase. After thorough mixing, enzymatically hydrolyze overnight (7 hours) in a water bath at 37 °C.

[0141] (3) Enrichment, purification and concentration of urine

[0142] Use a solid-phase extraction instrument for sample enrichment and purification. Activate the HLB solid-phase extraction cartridge with 2 mL of methanol, 2 mL of high-purity water and 2 mL of sodium hydroxide-potassium dihydrogen phosphate buffer solution in sequence, and dry it under vacuum conditions; pass the activated HLB solid-phase extraction cartridge at a flow rate of 5 mL / min to enrich the urine sample. After the sample loading is completed, wash the HLB solid-phase extraction cartridge with 2 mL of sodium hydroxide-potassium dihydrogen phosphate buffer solution and 2 mL of high-purity water in sequence, then elute with 3 mL of acetonitrile. The eluate is gently nitrogen blown to dryness in a water bath at 37 °C, reconstitute with 0.5 mL of 50% methanol aqueous solution, vortex and oscillate, and centrifuge at 10000 r for 2 minutes for measurement.

[0143] (4) UPLC-MS / MS detection

[0144] Ultra-high performance liquid chromatography tandem mass spectrometry was used to detect antibiotics in concentrated human urine samples. The antibiotics detected included tetracycline, oxytetracycline, chlortetracycline, doxycycline, ciprofloxacin, ofloxacin, norfloxacin, pefloxacin, enrofloxacin, danofloxacin, difloxacin, lomefloxacin, azithromycin, roxithromycin, clarithromycin, tilmicosin, trimethoprim, sulfadiazine, sulfamethoxazole, sulfamerazine, acetylated sulfamethoxazole, acetylated sulfamerazine, chloramphenicol, thiamphenicol, and florfenicol. The parameters of ultra-high performance liquid chromatography in the UPLC-MS / MS detection were as follows: the chromatographic column was ACQUITY UPLC T3 (100 mm × 2.1 mm, 1.8 μm); the column temperature was 35 °C; the flow rate was 0.35 mL / min; the injection volume was 10 μL. Mobile phase A: 40% methanol-acetonitrile solution, mobile phase B: 0.2% formic acid-aqueous solution, and gradient elution was used. The specific conditions are shown in Table 1. The parameters of the mass spectrometry in the HPLC-MS / MS detection were as follows: the ionization modes were electrospray positive ion source (ESI + ) and electrospray negative ion source (ESI - ) for simultaneous scanning. The ion source voltage was 4500 V, the temperature was 400 °C, the curtain gas was 30 psi, the spray gas was 55 psi, the auxiliary heating gas was 55 psi, and the monitoring mode was multiple reaction monitoring mode.

[0145] Six human urine samples were spiked and recovered at three spiked levels of 10, 50, and 100 ng / mL, and determined in parallel 6 times. The medium, low, high, and average spiked recoveries of each target antibiotic were calculated. The average spiked recoveries of 25 antibiotics at three different concentration levels were 56.32% - 141.88%.

[0146] Example 4

[0147] A rapid determination method for multiple antibiotics in human urine by solid-phase extraction-ultra-high performance liquid chromatography-tandem mass spectrometry, comprising the following steps:

[0148] (1) Preparation of solutions

[0149] Accurately weigh 10 mg of each standard product, dissolve it with methanol and make up the volume to 10 mL in a brown volumetric flask. The standard stock solution was stored in a -20 °C refrigerator. Appropriate amounts of the standard stock solutions of 25 antibiotics were respectively taken and diluted with methanol-water (V:V = 1:1) solution to prepare a mixed standard working solution with a total concentration of 1 mg / L, which was stored in a 4 °C refrigerator for later use.

[0150] (2) Pretreatment of urine

[0151] Accurately measure 1 mL of human urine sample, add 50 μL of 100 μg / L mixed internal standard working solution, 400 μL of acetic acid-ammonium acetate buffer solution, and 15 μL of Helix pomatia β-glucuronidase respectively. After mixing well, incubate at 37 °C in a water bath overnight (7 hours).

[0152] (3) Urine enrichment, purification and concentration

[0153] Use a solid-phase extraction instrument for sample enrichment and purification. Activate the HLB solid-phase extraction cartridge with 2 mL of methanol, 2 mL of high-purity water, and 2 mL of sodium hydroxide-potassium dihydrogen phosphate buffer solution in sequence, and dry it under vacuum conditions; pass the activated HLB solid-phase extraction cartridge at a flow rate of 5 mL / min to enrich the urine sample. After the sample loading is completed, wash the HLB solid-phase extraction cartridge with 1 mL of sodium hydroxide-potassium dihydrogen phosphate buffer solution and 2 mL of high-purity water in sequence, then elute with 2 mL of acetonitrile. The eluate is gently blown to dry in a 37 °C water bath, reconstitute with 0.5 mL of 50% methanol aqueous solution, vortex and oscillate, and centrifuge at 10000 r for 2 minutes for measurement.

[0154] (4) UPLC-MS / MS detection

[0155] The concentrated human urine sample is subjected to ultra-high performance liquid chromatography tandem mass spectrometry for the detection of antibiotics. The detected antibiotics include tetracycline, oxytetracycline, chlortetracycline, doxycycline, ciprofloxacin, ofloxacin, norfloxacin, pefloxacin, enrofloxacin, danofloxacin, difloxacin, lomefloxacin, azithromycin, roxithromycin, clarithromycin, tilmicosin, trimethoprim, sulfadiazine, sulfamethoxazole, sulfamerazine, acetylated sulfamethoxazole, acetylated sulfamerazine, chloramphenicol, thiamphenicol, florfenicol. The parameters of ultra-high performance liquid chromatography in UPLC-MS / MS detection are as follows: the chromatographic column is ACQUITY UPLC T3 (100 mm × 2.1 mm, 1.8 μm); the column temperature is 35 °C; the flow rate is 0.35 mL / min; the injection volume is 10 μL. Mobile phase A: 40% methanol-acetonitrile solution, mobile phase B: 0.2% formic acid-aqueous solution, and gradient elution method is adopted. The specific conditions are shown in Table 1. The parameters of mass spectrometry in HPLC-MS / MS detection are as follows: the ionization modes are electrospray positive ion source (ESI + ) and electrospray negative ion source (ESI - ) for simultaneous scanning. The ion source voltage is 4500 V, the temperature is 400 °C, the curtain gas is 30 psi, the spray gas is 55 psi, the auxiliary heating gas is 55 psi, and the monitoring mode is multiple reaction monitoring mode.

[0156] Six human urine samples were spiked and recovered at three spiking levels of 10, 50, and 100 ng / mL, and determined in parallel six times. The medium, low, high, and average spiked recoveries of each target antibiotic were calculated, and the average spiked recoveries of 25 antibiotics at three different concentration levels were 33.25% - 139.15%.

[0157] Example 5

[0158] A rapid determination method of multiple antibiotics in human urine by solid-phase extraction-ultra high performance liquid chromatography-tandem mass spectrometry, comprising the following steps:

[0159] (1) Preparation of solutions

[0160] Accurately weigh 10 mg of each standard product, dissolve it with methanol and make up the volume to 10 mL in a brown volumetric flask. The standard stock solution is stored in a -20 °C refrigerator. Appropriately draw the standard stock solutions of 25 antibiotics, dilute them with methanol-water (V:V = 1:1) solution to prepare a mixed standard working solution with a total concentration of 1 mg / L, and store it in a 4 °C refrigerator for standby.

[0161] (2) Pretreatment of urine

[0162] Accurately measure 1 mL of human urine sample, add 50 μL of 100 μg / L mixed internal standard working solution, 400 μL of acetic acid-ammonium acetate buffer solution, and 15 μL of Helix pomatia β-glucuronidase respectively. After mixing well, enzymatically hydrolyze overnight (7 hours) in a 37 °C water bath.

[0163] (3) Enrichment, purification and concentration of urine

[0164] Use a solid-phase extraction instrument to enrich and purify the sample. Activate the HLB solid-phase extraction small column with 2 mL of methanol, 2 mL of high-purity water, and 2 mL of sodium hydroxide-potassium dihydrogen phosphate buffer solution in sequence, and dry it under vacuum conditions; pass the activated HLB solid-phase extraction small column at a flow rate of 5 mL / min to enrich the urine sample. After the sample loading is completed, wash the HLB solid-phase extraction small column with 1 mL of sodium hydroxide-potassium dihydrogen phosphate buffer solution and 2 mL of high-purity water in sequence, then elute with 3 mL of acetonitrile. The eluate is gently nitrogen blown to dry in a 37 °C water bath, reconstitute with 0.25 mL of 50% methanol aqueous solution, vortex and oscillate, and centrifuge at 10000 r for 2 minutes for determination.

[0165] (4) UPLC-MS / MS detection

[0166] Ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS / MS) was used to detect antibiotics in concentrated human urine samples. The antibiotics detected included tetracycline, oxytetracycline, chlortetracycline, doxycycline, ciprofloxacin, ofloxacin, norfloxacin, pefloxacin, enrofloxacin, danofloxacin, difloxacin, lomefloxacin, azithromycin, roxithromycin, clarithromycin, tilmicosin, trimethoprim, sulfadiazine, sulfamethoxazole, sulfamerazine, acetylated sulfamethoxazole, acetylated sulfamerazine, chloramphenicol, thiamphenicol, and florfenicol. The parameters of ultra-high performance liquid chromatography in UPLC-MS / MS detection were as follows: the chromatographic column was ACQUITY UPLC T3 (100 mm × 2.1 mm, 1.8 μm); the column temperature was 35°C; the flow rate was 0.35 mL / min; the injection volume was 10 μL. Mobile phase A: 40% methanol-acetonitrile solution, mobile phase B: 0.2% formic acid-aqueous solution. The gradient elution method was adopted, and the specific conditions are shown in Table 1. The parameters of mass spectrometry in HPLC-MS / MS detection were as follows: the ionization modes were electrospray positive ion source (ESI + ) and electrospray negative ion source (ESI - ) for simultaneous scanning. The ion source voltage was 4500 V, the temperature was 400°C, the curtain gas was 30 psi, the spray gas was 55 psi, the auxiliary heating gas was 55 psi, and the monitoring mode was multiple reaction monitoring mode.

[0167] Six human urine samples were spiked and recovered at three spiked levels of 10, 50, and 100 ng / mL, and determined in parallel 6 times. The medium, low, high, and average spiked recoveries of each target antibiotic were calculated. The average spiked recoveries of 25 antibiotics at three different concentration levels were 52.61% - 133.33%.

[0168] Comparative Example 1

[0169] Compared with Example 2, most of them were the same, except that the urine pretreatment in step (2) was adjusted to: accurately measure 1 ml of human urine sample, add 50 μL of 100 μg / L mixed internal standard working solution, mix well, and enzymatically hydrolyze overnight (7 hours) in a 37°C water bath. Six human urine samples were spiked and recovered at three spiked levels of 10, 50, and 100 ng / mL, and determined in parallel 6 times. The medium, low, high, and average spiked recoveries of each target antibiotic were calculated. The average spiked recoveries of 25 antibiotics at three different concentration levels were 0 - 1435.34%.

[0170] Comparative Example 2

[0171] A rapid determination method for multiple antibiotics in human urine by solid-phase extraction-ultra-high performance liquid chromatography-tandem mass spectrometry, comprising the following steps:

[0172] (1) Preparation of solutions

[0173] Accurately weigh 10 mg of each standard, dissolve it in methanol to a 10 mL brown volumetric flask, and store the standard stock solution in a -20°C refrigerator. Take appropriate amounts of the standard stock solutions of 25 antibiotics, dilute them with methanol-water (V:V=1:1) solution, and prepare a mixed standard working solution with a total concentration of 1 mg / L, store it in a 4°C refrigerator for later use.

[0174] (2) Urine pretreatment

[0175] Accurately measure 1 ml of human urine sample, add 50 uL of 100ug / L mixed internal standard working solution, 400 uL of acetic acid-ammonium acetate buffer solution, and 15 uL of Roman snail β-glucuronidase, mix thoroughly, and perform enzymolysis in a 37°C water bath overnight (7 hours).

[0176] (3) Urine enrichment, purification and concentration

[0177] Use a solid phase extractor to enrich and purify the sample. Use 2mL of methanol to activate the HLB solid phase extraction column and blow it dry under vacuum conditions. Pass the activated HLB solid phase extraction column at a flow rate of 5mL / min to enrich the urine sample. After the loading is completed, rinse the HLB solid phase extraction column with 1mL of sodium hydroxide-potassium dihydrogen phosphate buffer solution and 2mL of high-purity water in turn, and then use 3mL of acetonitrile for elution. Blow the eluate to dryness with weak nitrogen in a 37°C water bath, reconstitute it with 0.5mL of 50% methanol aqueous solution, vortex and oscillate, then centrifuge at 10000r for 2 minutes for testing.

[0178] (4) UPLC-MS / MS detection

[0179] Human urine concentrated samples were subjected to ultra-high performance liquid chromatography tandem mass spectrometry to detect antibiotics, including tetracycline, oxytetracycline, chlortetracycline, doxycycline, ciprofloxacin, ofloxacin, norfloxacin, pefloxacin, enrofloxacin, danofloxacin, difloxacin, lomefloxacin, azithromycin, roxithromycin, clarithromycin, tilmicosin, trimethoprim, sulfadiazine, sulfamethoxazole, sulfamethoxazole, acetylated sulfamethoxazole, acetylated sulfamethoxazole, chloramphenicol, thiamphenicol, and florfenicol. The parameters of ultra-high performance liquid chromatography in UPLC-MS / MS detection were as follows: chromatographic column: ACQUITY UPLC T3 (100 mm × 2.1 mm, 1.8 μm); column temperature: 35 °C; flow rate: 0.35 mL / min; injection volume: 10 μL. Mobile phase A: 40% methanol-acetonitrile solution, mobile phase B: 0.2% formic acid-water solution, gradient elution method was used, and the specific conditions are shown in Table 1. The mass spectrometry parameters in HPLC-MS / MS detection are: the ionization mode is electrospray positive ion source (ESI + ) and electrospray ionization source (ESI -)Simultaneous scanning, with the ion source voltage at 4500 V, the temperature at 400 °C, the curtain gas at 30 psi, the nebulizing gas at 55 psi, the auxiliary heating gas at 55 psi, and the monitoring mode being the multiple reaction monitoring mode.

[0180] Six human urine samples were spiked and recovered at three spiking levels of 10, 50, and 100 ng / mL, determined in parallel 6 times, and the medium, low, high, and average spiking recoveries of each target antibiotic were calculated. The average spiking recoveries of 25 antibiotics at three different concentration levels were 1.85% - 1257.95%.

[0181] Comparative Example 3

[0182] Compared with Comparative Example 2, most of them are the same. Except in the process of urine enrichment, purification, and concentration in step (3), a solid-phase extraction instrument was used for sample enrichment and purification, and an HLB solid-phase extraction small column was activated successively with 2 mL of methanol and 2 mL of high-purity water. Six human urine samples were spiked and recovered at three spiking levels of 10, 50, and 100 ng / mL, determined in parallel 6 times, and the medium, low, high, and average spiking recoveries of each target antibiotic were calculated. The average spiking recoveries of 25 antibiotics at three different concentration levels were 33.25% - 132.78%.

[0183] Comparative Example 4

[0184] Compared with Comparative Example 2, most of them are the same. Except in the process of urine enrichment, purification, and concentration in step (3), a solid-phase extraction instrument was used for sample enrichment and purification, and an HLB solid-phase extraction small column was activated successively with 2 mL of methanol and 2 mL of sodium hydroxide-potassium dihydrogen phosphate buffer solution. Six human urine samples were spiked and recovered at three spiking levels of 10, 50, and 100 ng / mL, determined in parallel 6 times, and the medium, low, high, and average spiking recoveries of each target antibiotic were calculated. The average spiking recoveries of 25 antibiotics at three different concentration levels were 36.24% - 118.66%.

[0185] Comparative Example 5

[0186] Compared with Example 3, most of them are the same. Except in the process of urine enrichment, purification, and concentration in step (3), after sample loading, the HLB solid-phase extraction small column was rinsed with 2 mL of high-purity water. Six human urine samples were spiked and recovered at three spiking levels of 10, 50, and 100 ng / mL, determined in parallel 6 times, and the medium, low, high, and average spiking recoveries of each target antibiotic were calculated. The average spiking recoveries of 25 antibiotics at three different concentration levels were 33.11% - 240.67%.

[0187] Comparative Example 6

[0188] Compared with Example 3, most of them are the same. Except that in the process of urine enrichment, purification and concentration in step (3), after the sample loading is completed, the HLB solid-phase extraction column is rinsed with 2 mL of methanol. Six human urine samples were taken for spiked recovery tests at 3 spiked levels of 10, 50, and 100 ng / mL respectively, and determined in parallel 6 times. The medium, low, high, and average spiked recoveries of each target antibiotic were calculated, and the average spiked recoveries of 25 antibiotics at 3 different concentration levels were 11.31% - 340.26%.

[0189] Comparative Example 7

[0190] Compared with Example 3, most of them are the same. Except that in the process of urine enrichment, purification and concentration in step (3), after the sample loading is completed, the HLB solid-phase extraction column is rinsed with 2 mL of 30% methanol aqueous solution. Six human urine samples were taken for spiked recovery tests at 3 spiked levels of 10, 50, and 100 ng / mL respectively, and determined in parallel 6 times. The medium, low, high, and average spiked recoveries of each target antibiotic were calculated, and the average spiked recoveries of 25 antibiotics at 3 different concentration levels were 24.83% - 350.55%.

[0191] Comparative Example 8

[0192] Compared with Example 3, most of them are the same. Except that in the process of urine enrichment, purification and concentration in step (3), after the sample loading is completed, the HLB solid-phase extraction column is rinsed with 2 mL of sodium hydroxide-potassium dihydrogen phosphate buffer solution. Six human urine samples were taken for spiked recovery tests at 3 spiked levels of 10, 50, and 100 ng / mL respectively, and determined in parallel 6 times. The medium, low, high, and average spiked recoveries of each target antibiotic were calculated, and the average spiked recoveries of 25 antibiotics at 3 different concentration levels were 43.98% - 178.47%.

[0193] Comparative Example 9

[0194] Compared with Example 4, most of them are the same. Except that in the process of urine enrichment, purification and concentration in step (3), 2 mL of methanol is used for elution. Six human urine samples were taken for spiked recovery tests at 3 spiked levels of 10, 50, and 100 ng / mL respectively, and determined in parallel 6 times. The medium, low, high, and average spiked recoveries of each target antibiotic were calculated, and the average spiked recoveries of 25 antibiotics at 3 different concentration levels were 33.25% - 139.15%.

[0195] The average spiked recoveries of 25 antibiotics at 3 different concentration levels in Examples 1 - 5 and Comparative Examples 1 - 9 were summarized, as shown in Table 9 specifically.

[0196] Summary Table of Average Spike Recovery Rates of 25 Antibiotics at 3 Different Concentration Levels under Different Conditions

[0197]

[0198]

[0199] It can be seen from the table that for a rapid determination method of multiple antibiotics in human urine by solid-phase extraction-ultra-high performance liquid chromatography-tandem mass spectrometry, in the urine pretreatment in step (2), adding 400 μL of acetic acid-ammonium acetate buffer solution and 15 μL of Roman snail β-glucuronidase for overnight treatment, and in the urine enrichment, purification and concentration in step (3), activating the HLB solid-phase extraction column with 2 mL of methanol, 2 mL of high-purity water, and 2 mL of sodium hydroxide-potassium dihydrogen phosphate buffer solution, rinsing the HLB solid-phase extraction column with 1 mL of sodium hydroxide-potassium dihydrogen phosphate buffer solution and 2 mL of high-purity water, eluting with 3 mL of acetonitrile, and reconstituting with 0.5 mL of 50% methanol aqueous solution has the best effect. The average spike recovery rates of 25 antibiotics at 3 different concentration levels are 63.09% - 136.58%.

[0200] Although the present invention has been described in detail above with general descriptions, specific embodiments and experiments, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. A rapid determination method for multiple antibiotics in human urine by solid-phase extraction-ultra high performance liquid chromatography-tandem mass spectrometry, characterized in that, it comprises the following steps: A1. Urine pretreatment: Add the mixed internal standard working solution, acetic acid-ammonium acetate buffer solution, and Helix pomatia β-glucuronidase to the human urine sample, mix well and enzymatically hydrolyze in a water bath; A2. Urine enrichment, purification and concentration: Pass the urine obtained in step A1 through an HLB solid-phase extraction small column activated with methanol, high-purity water, and sodium hydroxide-potassium dihydrogen phosphate buffer solution, then wash successively with sodium hydroxide-potassium dihydrogen phosphate buffer solution and high-purity water, and dry under vacuum. Elute with acetonitrile, weakly nitrogen-blow the eluate to nearly dry in a water bath, then reconstitute with methanol aqueous solution, vortex and centrifuge to obtain a concentrated human urine sample; A3. UPLC-MS / MS detection: Perform ultra high performance liquid chromatography-tandem mass spectrometry on the concentrated human urine sample obtained in step A2 to detect antibiotics, and the detected antibiotics include tetracycline, oxytetracycline, chlortetracycline, doxycycline, ciprofloxacin, ofloxacin, norfloxacin, pefloxacin, enrofloxacin, danofloxacin, difloxacin, lomefloxacin, azithromycin, roxithromycin, clarithromycin, tilmicosin, trimethoprim, sulfadiazine, sulfamethoxazole, sulfamerazine, acetylated sulfamethoxazole, acetylated sulfamerazine, chloramphenicol, thiamphenicol, florfenicol.

2. The rapid determination method for multiple antibiotics in human urine by solid-phase extraction-ultra high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, in step A1, the volume ratio of the acetic acid-ammonium acetate buffer solution, Helix pomatia β-glucuronidase and human urine sample is (200-400):(15-25):

1.

3. The rapid determination method for multiple antibiotics in human urine by solid-phase extraction-ultra high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, in step A1, the concentration of the mixed internal standard working solution is 50 or 100 μg / L, the pH of the acetic acid-ammonium acetate buffer solution is 4.4-5.0, and the enzyme activity of Helix pomatia β-glucuronidase is greater than or equal to 100000 units / mL.

4. The rapid determination method for multiple antibiotics in human urine by solid-phase extraction-ultra high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, in step A1, the water bath enzymatic hydrolysis temperature is 30-40 °C, and the water bath enzymatic hydrolysis time is 7-12 h.

5. The rapid determination method for multiple antibiotics in human urine by solid-phase extraction-ultra high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, in step A2, the volume ratio of the HLB solid-phase extraction small column to the methanol, high-purity water, and sodium hydroxide-potassium dihydrogen phosphate buffer solution used for activation is 3:(1-3):(1-3):(1-3); the volume ratio of the HLB solid-phase extraction small column to the sodium hydroxide-potassium dihydrogen phosphate buffer solution and high-purity water used for washing is 3:(1-2):(1-2); The volume ratio of the HLB solid-phase extraction cartridge to acetonitrile is 3:(2 - 4) mL; The volume ratio of the methanol aqueous solution to the human urine sample is 1:2 or 1:

4.

6. A rapid determination method for multiple antibiotics in human urine by solid-phase extraction-ultra high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that in step A2, the water bath temperature is 30 - 45 °C, and the centrifugation speed is 8000 - 12000 rpm.

7. A rapid determination method for multiple antibiotics in human urine by solid-phase extraction-ultra high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that in step A3, the parameters of ultra high performance liquid chromatography in UPLC-MS / MS detection are: the chromatographic column is a T3 chromatographic column, 100 mm × 2.1 mm, 1.8 μm; mobile phase A is a methanol-acetonitrile solution, mobile phase B is a formic acid-aqueous solution, and gradient elution is adopted; the flow rate is 0.5 mL / min, the column temperature is 35 °C, and the injection volume is 10 or 15 μL.

8. A rapid determination method for multiple antibiotics in human urine by solid-phase extraction-ultra high performance liquid chromatography-tandem mass spectrometry according to claim 7, characterized in that the volume concentration of methanol in the methanol-acetonitrile solution is 35% - 45%, and the volume concentration of formic acid in the formic acid-aqueous solution is 0.1% - 0.3%.

9. A rapid determination method for multiple antibiotics in human urine by solid-phase extraction-ultra high performance liquid chromatography-tandem mass spectrometry according to claim 7, characterized in that the specific conditions of the gradient elution method are: 0 - 1.0 min, 10% mobile phase A + 90% mobile phase B; 1.0 - 7.0 min, 98% mobile phase A + 2% mobile phase B; 7.0 - 9.5 min, 98% mobile phase A + 2% mobile phase B; 9.5 - 9.8 min, 10% mobile phase A + 90% mobile phase B; 9.8 - 11.5 min, 10% mobile phase A + 90% mobile phase B.

10. A rapid determination method for multiple antibiotics in human urine by solid-phase extraction-ultra high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that in step A3, the parameters of mass spectrometry in UPLC-MS / MS detection are: the ionization mode is simultaneous scanning of electrospray positive ion source and electrospray negative ion source, the ion source voltage is 4000 - 5000 V, the temperature is 300 - 500 °C, the curtain gas is 20 - 40 psi, the spray gas is 50 - 60 psi, the auxiliary heating gas is 50 - 60 psi, and the monitoring mode is multiple reaction monitoring mode.

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