Method for detecting urea concentration in multiple biological matrixes by adopting high performance liquid chromatography-mass spectrometry

Abstract

The invention discloses a method for detecting the concentration of urea in various biological matrixes by adopting a high performance liquid chromatography-mass spectrometry method, and relates to the field of urea concentration detection. According to the detection method for detecting the urea concentration in the multiple biological matrixes by adopting a high performance liquid chromatography-mass spectrometry method, water is used as a blank matrix, a biological matrix sample is subjected to protein precipitation treatment, based on an API5000 platform, an APCI source is adopted, a C18 bonded phase mode is adopted for a liquid phase, the urea concentration in plasma / serum / BALF is detected, the linear range is 0.500-20.0 mu g / mL, and the detection result is accurate. The sensitivity is greatly improved, and the detection requirements of clinical samples can be completely met.

Description

Technical Field

[0001] The invention relates to the field of urea concentration detection, and in particular to a method for detecting urea concentrations in various biological matrices by using a high performance liquid chromatography-mass spectrometry method. Background Art

[0002] In the study of human respiratory diseases, bronchoalveolar lavage fluid (BALF) of human subjects is recognized to provide information on drug exposure distribution in the lower respiratory tract. BALF is currently the most commonly used method for detecting drug concentrations in ELF and AM. The volume and drug concentration of BALF are relatively easy to obtain, but the natural volume of bronchoalveoli is difficult to assess due to individual differences, health status, age and other factors.

[0003] Urea diffuses freely between different body components and is evenly distributed throughout the body. Urea has a low turnover rate and its concentration is fairly stable over 24 hours. In addition, urea concentration is believed to be minimally affected by disease state. Therefore, it is generally believed that urea concentration is consistent in different biological matrices from the same subject, including nasal secretions, exhaled blood (EBC), mucosal lining fluid (MLF), lung epithelial lining fluid (ELF), bronchoalveolar lavage fluid (BALF), plasma or serum, etc. Scholars use urea as a widely used dilution marker for standardizing biological sampling volumes. By measuring the urea concentration in plasma and body fluids, they are defined as C plasma and C sample fluid , the original physiological matrix volume (V native ), the calculation formula is V native = (C sample fluid / C plasma ) * V sample fluid By measuring the concentration of the study drug in BALF, multiplying by V BALF / V native , the exposure of the research drug in the bronchoalveoli can be obtained, providing reference data for the evaluation of drug effectiveness and administration methods.

[0004] The detection methods of urea in different biological matrices are reported in the following literature: 1) Colorimetric method: urea is acted upon by enzymes to form products, which react with the probe to produce color. The absorbance is proportional to the concentration of urea in the solution. This detection scheme is completed using commercial kits. This method is limited in sensitivity. At the same time, the detection results vary greatly with the manufacturer and batch of the test kit, and the detection method is not easy to transfer or reproduce.

[0005] 2) There are a few reports on the LC-MS method, but it still has some shortcomings: Due to its small molecular weight, urea is easily affected by interference from other ion pairs in mass spectrometry detection. In order to improve sensitivity and selectivity, literature uses derivatization, and the sample pretreatment process is complicated and time-consuming; In the prior art, alternative matrices are used to prepare standard curves and quality control samples, which simplifies the sample pretreatment steps. However, the sensitivity is difficult to meet the detection requirements of multiple matrix samples, and the detection method still has challenges such as high baseline noise, matrix effect, and mismatch of multiple biological matrices. The existing methods using alternative analytes bring challenges to the interpretation of test results, the linear range of the method, and the cost of testing; There is a literature that establishes a LC-MS coupling method. After salting-out assisted liquid-liquid extraction of biological samples, the concentration of urea in plasma, serum or ELF is detected simultaneously based on the API5500 platform, using an ESI source and a Hilic mode for liquid phase, with a linear range of 1~50 μg / mL. This method still faces challenges in the application process: for example, high baseline, method sensitivity defects, and Hilic mode is prone to fluctuations.

[0006] In order to better support the increasing number of pharmacokinetic studies in respiratory diseases, a convenient and universal determination method is urgently needed for the detection and analysis of urea concentration in different biological matrices. Summary of the invention

[0007] Purpose of the invention: The purpose of the present invention is to provide an accurate, reliable, simple and efficient method for detecting the concentration of urea in various biological matrices by high performance liquid chromatography-mass spectrometry. The concentration of urea in different types of biological matrices can be detected, the method is easy to transfer and reproduce, and can meet the detection needs of clinical samples.

[0008] Technical solution: The present invention provides a detection method for detecting the urea concentration in various biological matrices by high performance liquid chromatography-mass spectrometry. Water is used as a blank matrix, and the biological matrix sample is subjected to protein precipitation treatment. Based on the API5000 platform, an APCI source is used, and a C18 bonded phase mode is used for liquid phase to detect the concentration of urea in plasma, serum or ELF. The linear range is 0.500-20.0 μg / mL, and the sensitivity is further improved, which can fully meet the detection needs of clinical samples.

[0009] The relevant parameters and processing of this method are as follows: Analytes Urea Internal Standard <![CDATA[[ 13 C; 15 N2]-urea ([ 13 C; 15 N2]-Urea, Urea-IS)]]> Analytical methods LC-MS / MS quantitative analysis method Biological matrix and source of samples Real matrix: human plasma or human serum or human bronchoalveolar lavage fluid (BALF); alternative matrix: ultrapure water Anticoagulants (if any) <![CDATA[Human plasma is K2EDTA; human serum and human bronchoalveolar lavage fluid are without anticoagulant]]> Sample pretreatment method Protein precipitation Sample quantity 50.0 µL Injection volume 5.00 µL Quantitative indicators Peak area ratio of analyte to internal standard Regression equation Linear fitting, the equation is y=ax+b Weight coefficient <![CDATA[1 / x 2 ]]> Linear range 0.500~20.0 µg / mL Standard curve concentration points 0.500, 1.00, 2.00, 4.00, 8.00, 10.0, 16.0, 20.0 µg / mL Surrogate matrix quality control sample concentration points 0.500 (LLOQ QC), 1.50 (LQC), 3.00 (MQC-1), 6.00 (MQC-2), 15.0 (HQC) µg / mL Real matrix quality control sample concentration points Human plasma or human serum: LQC-P background + 90.0 µg / mL, MQC-1-P background + 150 µg / mL, MQC-2-P background + 450 µg / mL and HQC-P background + 675 µg / mL, all diluted 50 times with alternative matrix before processing for detection and analysis; human bronchoalveolar lavage fluid: LQC-B background + 1.50 µg / mL, MQC-1-B background + 3.00 µg / mL, MQC-2-B background + 6.00 µg / mL and HQC-B background + 15.0 µg / mL. The technical method and steps of this embodiment are as follows: (1) Solvent preparation The reagent volumes listed in this analytical procedure can be adjusted proportionally as needed.

[0010] 1) Mobile phase A (MPA): an aqueous solution containing 0.1% (v / v) formic acid and 10 mM ammonium acetate 2) Mobile phase B (MPB): acetonitrile solution containing 0.1% (v / v) formic acid 3) Needle washing solution (NW): methanol: acetonitrile: isopropanol: ultrapure water solution, prepared in a volume ratio of 1:1:1:1, add 0.1% (v / v) trifluoroacetic acid 4) Solution A: 5.0% (v / v) methanol in water 5) Solution B: 1.0 M ammonium acetate aqueous solution (2) Liquid phase conditions are as follows: Analytes Urea Chromatographic columns Xselect HSS T3 2.5 μm, 4.6×100 mm Mobile phase A: Aqueous solution containing 0.1% (v / v) formic acid and 10 mM ammonium acetate Mobile phase B: Acetonitrile solution containing 0.1% (v / v) formic acid Needle wash solution Methanol: acetonitrile: isopropanol: ultrapure water solution was prepared in a volume ratio of 1:1:1:1, and 0.1% (v / v) trifluoroacetic acid was added. Automatic sampler needle wash parameters Rinsing Volume:200 μL Automatic sampler needle wash parameters Rinsing Speed: 35 μL / sec Automatic sampler needle wash parameters Rinse Dip Time: 2 sec Automatic sampler needle wash parameters Rinse Mode: Before and after aspiration Autosampler temperature <![CDATA[4 o C]]> Column temperature <![CDATA[40 o C]]> Injection volume 5.00 µL Initial flow rate 0.700 mL / min Switching valve 0~0.9 min and 2.2 ~4.0 min enter the waste liquid, 0.9~2.2 min enters the MS Expected retention time <![CDATA[Urea: 1.57 min;[ 13 C; 15 N2]-Urea: 1.57 min]]> Continued: Gradient elution program: Time (min) Module Event %B Flow rate (mL / min) 0.01 Pumps Pump B Conc. 5 0.700 2.00 Pumps Pump B Conc. 5 0.700 2.01 Pumps Pump B Conc. 95 0.700 3.00 Pumps Pump B Conc. 95 0.700 3.01 Pumps Pump B Conc. 5 0.700 4.00 Controller Stop - 0.700

[0011] (3) Mass spectrometry conditions are as follows: Analytes Urea Mass spectrometry parameters Parameter settings Ion spray voltage 4500 V temperature 550 ºC <![CDATA[Gas 1 (GS1, N2)]]> 45 psi <![CDATA[Gas 2 (GS2, N2)]]> 45 psi Sprayer current (NC) 3.0 <![CDATA[Curtain gas (CUR, N2)]]> 45 psi <![CDATA[Collision gas (CAD, N2)]]> 12 Ion source APCI Detection method Positive ion mode Scanning method Multiple ion reaction monitoring (MRM) method Mass spectrometry acquisition time 4.00 min Q1 / Q3 resolution Unit / unit Pause between mass ranges 5.007 ms (4) Preparation of stock solution and working solution: Accurately weigh the urea reference standard substance in a transparent plastic tube. After conversion by correction factor, add an appropriate amount of 5.0% (v / v) methanol aqueous solution to prepare a 40.0 mg / mL urea standard curve and quality control sample stock solution. Store in a -10~-30 ºC refrigerator for use.

[0012] Use 5.0% (v / v) methanol aqueous solution as diluent to prepare urea standard curve and quality control sample working solutions in transparent plastic bottles. The concentrations of urea standard curve sample working solutions are: 10.0, 20.0, 40.0, 80.0, 160, 200, 320, 400 µg / mL. The concentrations of urea quality control sample working solutions are: 1) human plasma or human serum: 1800, 3000, 9000, 13500 µg / mL; 2) human bronchoalveolar lavage fluid: 10.0, 30.0, 60.0, 120, 300 µg / mL.

[0013] (5) Preparation of internal standard stock solution and working solution In a transparent plastic bottle, accurately weigh [ 13 C; 15 N2]-urea reference standard material, after correction factor conversion, was added with an appropriate amount of 5.0% (v / v) methanol aqueous solution to prepare 1.00 mg / mL [ 13 C; 15 N2]-urea internal standard stock solution was stored in a refrigerator at -10~-30℃.

[0014] Take the internal standard stock solution and dilute it with 5.0% (v / v) methanol aqueous solution in a transparent plastic bottle to prepare an internal standard working solution with a concentration of 20000 ng / mL, and store it in a refrigerator at -10~-30℃.

[0015] (6) Preparation of standard curve samples and quality control samples The concentrations of urea standard curve samples were 0.500, 1.00, 2.00, 4.00, 8.00, 10.0, 16.0, and 20.0 µg / mL, and the standard curve samples were prepared by adding the analyte working solution to the surrogate matrix (ultrapure water).

[0016] Quality control samples include quality control samples prepared with alternative matrices (ultrapure water) and quality control samples prepared with real matrices (blank human plasma or human serum or blank human bronchoalveolar lavage fluid). Quality control samples prepared with alternative matrices are obtained by adding analytes of corresponding concentrations to the alternative matrices; quality control samples prepared with real matrices need to take endogenous concentrations into account, which is affected by the background concentration of the analyte. Quality control samples with real matrices are obtained by adding analytes of appropriate concentrations to the real matrices.

[0017] Concentration of urea quality control samples: The quality control samples prepared with surrogate matrix included 5 concentration levels, namely 0.500 (LLOQ QC), 1.50 (LQC), 3.00 (MQC-1), 6.00 (MQC-2), and 15.0 (HQC) µg / mL; the quality control samples prepared with human plasma or human serum real matrix included 4 concentration levels, LQC-P (background + 90.0 µg / mL), MQC-1-P (background + 150 µg / mL), MQC-2-P (background + 450 µg / mL) and HQC-P (background + 675 µg / mL). The quality control samples prepared with human plasma or human serum real matrix were diluted 50 times with surrogate matrix and then processed for detection and analysis; the quality control samples prepared with human bronchoalveolar lavage fluid real matrix included 4 concentration levels, LQC-B (background + 1.50 µg / mL), MQC-1-B (background + 3.00 µg / mL), MQC-2-B (background + 6.00 µg / mL), and HQC-B (background + 15.0 µg / mL).

[0018] (7) Data processing Using Watson LIMS TM 7.5 SP1 (Thermo Scientific Inc.) with Linear, Weighting factor = 1 / x 2 The model processed the data and reported the results in μg / mL with 3 significant figures. For % Bias and % CV, 1 decimal place was retained.

[0019] Compared with the prior art, the present invention has the following beneficial effects: 1. The method of the present invention does not rely on a kit, does not require a replacement analyte, has a low detection cost, stable detection results, and a guaranteed detection time limit; 2. The instrument detection method has good reproducibility and can successfully complete the methodological transfer between different detection platforms (API 5500, API6500) and different laboratories; 3. The concentration range of urea in BALF is 0.500~10.0 μg / mL. The sensitivity of this method is further improved compared with previous literature and can fully meet the needs of sample testing. This method optimizes the peak shape and interference by screening chromatographic columns and optimizing chromatographic gradients, optimizes pretreatment and mass spectrometry parameters to improve mass spectrometry response, and screens ion sources to improve response and reduce baseline. The sensitivity of the method is further improved compared with the existing technology. According to the test results of clinical samples, the concentration range of urea in BALF is 0.500~10.0 μg / mL. Therefore, the optimized method can fully meet the needs of sample testing. 4. Alternative matrices are easy to obtain, sample pretreatment is simple and efficient, reproducible and highly operable; 5. The method of the present invention has been applied to the detection and analysis of clinical samples. The project implementation complies with the principles of Good Laboratory Practice (GLP), Good Clinical Practice (GCP), NMPA Guidelines for Laboratory Management of Biological Sample Analysis in Clinical Trials of Drugs (Trial) (National Medicine Supervision

[2011] No. 482), Pharmacopoeia of the People's Republic of China (2020 Edition) Part IV 9012: Guidelines for Validation of Quantitative Analysis Methods for Biological Samples and ICH M10: Validation of Bioanalytical Methods and Sample Analysis. The research results are accurate, complete and reliable, providing sufficient data support for drug research and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Urea is the analyte and the internal standard [ 13 C; 15 N2]-urea ion scan, a is the urea parent ion, b is [ 13 C; 15 N2]-urea parent ion, c is the urea daughter ion, d is [ 13 C; 15 N2]-urea daughter ion; Figure 2 is the spectrum of blank matrix, a is the double blank sample, and b is the zero concentration sample; Figure 3It is the spectrum of the standard curve sample with lower limit of quantitation; Figure 4 The spectrum of the quantitative upper limit standard curve sample; Figure 5 It is a standard curve graph. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be further described below.

[0022] Example 1: Alveolar lavage fluid experiment 1. Materials and Instruments 1.1 Object under test The analyte in this example is urea (Urea), with a purity of 100% (Shanghai Zhenzhun Biotechnology Co., Ltd.).

[0023] 1.2 Reference Standards 1.2.1 Reference standard substances and internal standard information project Reference Internal Standard name Urea <![CDATA[[ 13 C; 15 N2]-urea ([ 13 C; 15 N2]-Urea, Urea-IS)]]> source Shanghai Zhenzhun Biotechnology Co., Ltd. Shanghai Zhenzhun Biotechnology Co., Ltd. Purity / content 100% 99.10% The following are the actual preparation steps in the project. The volumes of the listed reagents can be adjusted proportionally as needed.

[0024] (1) Mobile phase A (MPA): an aqueous solution containing 0.1% (v / v) formic acid and 10 mM ammonium acetate Take 1000 mL of ultrapure water in a reagent bottle, add 1.00 mL of formic acid and 10.0 mL of 1.0 M ammonium acetate aqueous solution, and mix well.

[0025] (2) Mobile phase B (MPB): acetonitrile solution containing 0.1% (v / v) formic acid Take 1000 mL of acetonitrile in a reagent bottle, add 1.00 mL of formic acid, and mix well.

[0026] (3) Needle washing solution (NW): methanol: acetonitrile: isopropanol: ultrapure water solution, prepared in a volume ratio of 1:1:1:1, and 0.1% (v / v) trifluoroacetic acid was added Take 250 mL each of methanol, acetonitrile, isopropanol, and ultrapure water into a reagent bottle, add 1.00 mL of trifluoroacetic acid, and mix well.

[0027] (4) Solution A: 5.0% (v / v) methanol in water Take 25.0 mL of methanol and 475 mL of ultrapure water in a reagent bottle and mix well.

[0028] (5) Solution B: 1.0 M ammonium acetate aqueous solution Weigh about 3854.08 mg of ammonium acetate powder, add 50.0 mL of ultrapure water, and mix well.

[0029] 1.3 Main instruments name describe Mass spectrometry API 5000, SCIEX Liquid chromatography LC-20AD, Shimadzu 1.4 Analysis conditions 1.4.1 Chromatographic conditions Chromatographic columns Xselect HSS T3 2.5 μm, 4.6×100 mm Mobile phase A: Aqueous solution containing 0.1% (v / v) formic acid and 10 mM ammonium acetate Mobile phase B: Acetonitrile solution containing 0.1% (v / v) formic acid Needle wash solution Methanol: acetonitrile: isopropanol: ultrapure water solution was prepared in a ratio of 1:1:1:1, and 0.1% (v / v) trifluoroacetic acid was added. Automatic sampler needle wash parameters Rinsing Volume:200 μLRinsing Speed: 35 μL / secRinse Dip Time: 2 secRinseMode: Before and after aspiration Autosampler temperature <![CDATA[4 o C]]> Column temperature <![CDATA[40 o C]]> Injection volume 5.00 µL Initial flow rate 0.700 mL / min Switching valve 0~0.9 min and 2.2 ~4.0 min enter the waste liquid, 0.9~2.2 min enters the MS Expected retention time <![CDATA[Urea: 1.57 min;[ 13 C; 15 N2]-Urea: 1.57 min]]> Continued: Gradient elution program: Time (min) Module Event %B Flow rate (mL / min) 0.01 Pumps Pump B Conc. 5 0.700 2.00 Pumps Pump B Conc. 5 0.700 2.01 Pumps Pump B Conc. 95 0.700 3.00 Pumps Pump B Conc. 95 0.700 3.01 Pumps Pump B Conc. 5 0.700 4.00 Controller Stop - 0.700

[0030] 1.4.2 Mass spectrometry conditions Mass spectrometry parameters Parameter settings Ion spray voltage 4500 V temperature 550 ºC <![CDATA[Gas 1 (GS1, N2)]]> 45 psi Sprayer current (NC) 3.0 <![CDATA[Curtain gas (CUR, N2)]]> 45 psi <![CDATA[Collision gas (CAD, N2)]]> 12 Ion source APCI Detection method Positive ion mode Scanning method Multiple ion reaction monitoring (MRM) method Mass spectrometry acquisition time 4.00 min Q1 / Q3 resolution Unit / unit Pause between mass ranges 5.007 ms Ion pairs and parameters: Compound Ion pair () ScanTime() DP() CE() EP() CXP() Urea 61.100→44.100 100 50 26 10 15 Urea-IS 64.000→46.000 100 50 26 10 15 1.5 Methodological Validation Results The method validation results of this example show that the quantitative range of the standard curve is: 0.500 ~ 20.0 µg / mL, and the LLOQ and ULOQ are 0.500 µg / mL and 20.0 µg / mL respectively; the accuracy and precision validation results show that the precision and accuracy of the method are within the acceptable range. Sensitivity, residue and selectivity all meet the acceptable standards. The alveolar lavage fluid samples and surrogate matrix samples are stable after freezing and thawing 6 times at -10 ~ -30 ℃ and -60 ~ -90 ℃, placing under room temperature white light conditions for 24 hours, placing under wet ice white light conditions for 18 hours, storing at -10 ~ -30 ºC for 87 days, and storing at -60 ~ -90 ºC for 87 days; the sample remains stable after being placed in the autosampler for 55.2 hours after extraction; the repeatability of the sample extraction and re-injection after being placed in the autosampler for 182.7 hours meets the acceptable standard. The maximum number of samples in the analysis batch is 210 injections. The background concentration of real mixed human bronchoalveolar lavage fluid samples was between 1.48-1.66 µg / mL.

[0031] 2 Methodological validation content, methods and acceptance criteria Methodological validation projects include: selectivity, standard curve, sensitivity, precision and accuracy, extraction recovery, matrix effect, residual effect and stability. The stability study includes long-term freezing of biological samples, repeated freezing and thawing, biological sample pretreatment process, stability under storage conditions after preparation, stability of analyte stock solution / analyte working solution / isotope internal standard working solution (room temperature, white light placement, long-term storage). Specific inspection parameters and acceptance criteria are shown in the table below.

[0032] Verify Parameters method Acceptance criteria Selectivity Double blank samples were prepared using the surrogate matrix (n=1), and double blank samples were prepared using 6 individual blank authentic matrices from different sources (n=1). At the retention time of the analyte and the internal standard in the double blank samples of the surrogate matrix, the peak area of ​​the interfering peak is no greater than 20.0% of the average peak area of ​​the analyte in the LLOQ standard curve samples that meet the acceptance criteria, and 5.0% of the average peak area of ​​the internal standard in the LLOQ standard curve samples that meet the acceptance criteria. In the double blank samples of the real matrix, since the analyte is an endogenous substance, there is no specific acceptance criterion for the individual blank real matrices of 6 different sources at the retention time of the analyte. At the retention time of the internal standard, the peak area of ​​the interfering peak is no greater than 5.0% of the average peak area of ​​the internal standard in the LLOQ standard curve samples that meet the acceptance criteria in the batch. The blank matrices of all normal individuals should meet the above standards. If they do not meet the above standards, the cause of failure needs to be evaluated, and the test should be repeated or more individuals should be added for evaluation. Selectivity Interference of analytes on internal standards: Samples at ULOQ concentrations were prepared using alternative matrices (n ≥ 3). The extraction process was the same as the normal method, except that the internal standard working solution was replaced by the corresponding solution without internal standard. In this sample, the average peak area of ​​the interfering peak at the internal standard retention time is no greater than 5.0% of the average peak area of ​​the internal standard of all LLOQ standard curve samples that meet the acceptance criteria. Selectivity Interference of internal standard on the analyte: Through zero-concentration sample investigation, internal standard is added to the alternative matrix to extract and process it into zero-concentration sample (Zero Blank, n≥3). In this sample, the average peak area of ​​the interfering peak at the retention time of the analyte is not greater than 20.0% of the average peak area of ​​the analyte in the LLOQ standard curve samples that meet the acceptance criteria in this analytical batch. Continued Verify Parameters method Acceptance criteria Standard curve In the method validation, the standard curve samples need to be freshly prepared using alternative matrices. Each standard curve contains 1 double blank (DoubleBlank), 1 zero concentration sample (Zero Blank) and 8 non-zero standard curve samples, each sample is n=2. One set of standard curve samples is distributed at the beginning of the analysis batch sequence, and the other set is located at the end of the analysis batch. The concentrations of Urea standard curve samples are 0.500, 1.00, 2.00, 4.00, 8.00, 10.0, 16.0, and 20.0 µg / mL. <![CDATA[When the deviation of the standard curve sample exceeds the range of ±15.0% of the theoretical concentration (±20.0% for the lower limit of quantification), or there are clear reasons for failure, such as abnormal instrument status or clearly recorded sample handling errors, this standard curve sample will be excluded and not participate in the regression calculation of the standard curve. The exclusion order starts from the point with the largest deviation. After exclusion, re-regression analysis is performed, and then the next point with the largest deviation is excluded. Repeat this process until the remaining points all meet the acceptance criteria. At least 75% of the non-zero points must satisfy the deviation within the range of ±15.0% of the theoretical value, and the lower limit of quantification sample within the range of ±20.0% of the theoretical value, and the linear fitting degree R]] 2 is not less than 0.9800; in the method validation, at least 50% of the samples at the LLOQ and ULOQ concentrations meet the above requirements. At the retention times of the analyte and internal standard in the alternative matrix double blank sample, the peak area of the interfering peak is not greater than 20.0% of the average analyte peak area of the LLOQ standard curve samples that meet the acceptance criteria, and 5.0% of the average internal standard peak area of the LLOQ standard curve samples that meet the acceptance criteria. At the retention time of the analyte in the zero concentration sample, the peak area of the interfering peak is not greater than 20.0% of the average analyte peak area of the LLOQ standard curve samples that meet the acceptance criteria.]]> Continued Verify Parameters method Acceptance criteria Accuracy and precision Accuracy and precision experiments need to examine at least three analytical batches and be carried out for at least 2 days. Accuracy and precision experiment quality control samples must be freshly prepared on the same day. Quality control samples include quality control samples prepared with surrogate matrices and quality control samples prepared with real matrices. Quality control samples prepared with surrogate matrices contain 5 concentration levels, with at least 5 samples for each concentration, and are obtained by adding the corresponding concentration of analytes to the surrogate matrix; quality control samples prepared with real matrices contain 4 concentration levels, with at least 5 samples for each concentration. The preparation of quality control samples for real matrices needs to take endogenous concentrations into account, and is obtained by adding the corresponding concentration of analytes to the real matrix. Each accuracy and precision analysis batch contains 5 concentration levels of surrogate matrices and 4 concentration levels of real matrix quality control samples. For routine analytical batches (analytical batches not used to examine accuracy and precision), at least surrogate matrix LQC, MQC-1, MQC-2 and HQC samples and real matrix LQC, MQC-1, MQC-2, HQC samples are included, for a total of 2 sets of quality control samples of eight concentrations. For alternative matrix quality control samples, the intra-batch and inter-batch precision at each concentration level should be ≤ 15.0%, and the lower limit of quantification should be ≤ 20.0%; the intra-batch and inter-batch accuracy deviation at each concentration level should not exceed ±15.0% of the theoretical value, and the lower limit of quantification should not exceed ±20.0% of the theoretical value. For real matrix quality control samples, the introduction of background concentration in the real matrix needs to be considered, so the background concentration of the real matrix used will be quantified (pre-treatment is the same as zero concentration sample, named Zero-B, n≥3), and when calculating the real matrix quality control samples, the concentration is back-calculated by the background subtraction method and the accuracy is calculated. The background subtraction method formula is as follows (all verification samples containing real matrix in this project are calculated according to this formula after the concentration is back-calculated and their accuracy deviation is calculated): Unless there is a clearly recorded chromatographic or instrument error, or a sample processing error, the results of all quality control samples should be included in the calculation of intra-batch and inter-batch accuracy and precision. For routine analysis batches, the accuracy deviation of all quality control samples is within ±15.0% of the theoretical value, at least 2 / 3 of the quality control samples meet the above requirements, and at least 50% of the quality control samples at each concentration level meet the above requirements. In the above table: Accuracy deviation (%) = Continued Verify Parameters method Acceptance criteria Sensitivity The lower limit of quantitation (LLOQ) represents the sensitivity of the method. The LLOQ can be assessed as part of accuracy and precision experiments. The response of the analyte in the LLOQ standard curve sample that meets the acceptance criteria is not less than 5 times the average response of the analyte in the zero-concentration sample in the analytical batch. The accuracy deviation of the LLOQ standard curve sample should not exceed ±20.0% of the theoretical value (calculated using the data in the analytical batch of inter-batch accuracy and precision, at least 3 analytical batches, 2 replicates for each analytical batch), and the precision should be ≤20.0% (calculated using the data in the analytical batch of inter-batch accuracy and precision, at least 3 analytical batches, 2 replicates for each analytical batch). Continued Verify Parameters method Acceptance criteria Matrix Effects Matrix effects were evaluated using six batches of authentic matrix from different individuals (n = 1). The analyte working solutions were spiked into authentic individual matrices to prepare samples at the MQC-2 and HQC levels, and the quantitative background concentration was performed in the analytical batch (for each batch (n ≥ 3)). The average accuracy deviation of the normal blank matrix for each batch at each concentration level should be within ±15.0% of the theoretical concentration. The precision of each batch of matrix should not be greater than 15.0%. The normal blank matrix should meet the acceptance criteria. If it does not meet the above criteria, the cause of failure needs to be evaluated, the test should be repeated, or more individuals should be added for evaluation. Continued Verify Parameters method Acceptance criteria Extraction recovery Recovery tests should be performed on mixed authentic matrix samples (n ≥ 5) at two concentration levels (MQC-2, HQC) and surrogate matrix samples (n ≥ 5) at three concentration levels (LQC, MQC-2, HQC) by comparing the peak areas of analytes and internal standards in extracted samples with those in unextracted samples (spiking the blank samples with the corresponding concentrations of analytes and internal standards after extraction). The analyte recovery in authentic matrix is ​​calculated from the results at two concentration levels (MQC-2, HQC), the analyte recovery in surrogate matrix is ​​calculated from the results at three concentration levels (LQC, MQC-2, HQC). The recovery of internal standard in authentic matrix is ​​calculated from the results of the internal standard response in authentic matrix QC samples (MQC-2, HQC), and the recovery of internal standard in surrogate matrix is ​​calculated from the results of the internal standard response in surrogate matrix QC samples (LQC, MQC-2, HQC). The extraction recovery %CV of the quality control sample at each concentration level is ≤ 15.0%, and the extraction recovery %CV of the three concentration levels is ≤ 15.0%. If the acceptance criteria are not met, the extraction recovery can be calculated using the results of the internal standard calibration (analyte / internal standard peak area ratio). The following formula can be used to calculate the recovery of the analyte in the real matrix. The mixed real matrix background concentration should have been quantified or quantified during the analytical batch. In the above table: Extraction recovery rate (%) = The following formula was used to calculate the recovery of the analyte in the surrogate matrix: Extraction recovery rate (%) = extracted sample peak area / average peak area of ​​unextracted samples × 100.

[0033] Continued Verify Parameters method Acceptance criteria Stability study in real matrix For the investigation of urea stability in real human matrix, two concentration level quality control samples (LQC and HQC) should be prepared in large volume and aliquoted, and the aliquoted stability samples should be used to evaluate benchtop (wet ice white light and room temperature white light) stability, freeze-thaw cycles and long-term stability. Due to the influence of background concentration, LQC samples can be replaced by real matrix without additional addition of analyte working solution. Zero time samples (T0) should be extracted and processed on the day of preparation (n≥3) to prove the accuracy of stability sample preparation. Biological matrix stability samples must be frozen for at least 24 hours before evaluating their short-term stability. In order to facilitate the subsequent sample analysis, the stability of the analyte in the alternative matrix can also be investigated at the same time, including wet ice and room temperature stability, freeze-thaw cycles and long-term stability, etc., using two concentration level quality control samples (LQC and HQC) for investigation. For alternative matrix stability samples, the deviation of the mean stability concentration of each concentration level from the theoretical concentration is within ±15.0%, and the precision is ≤ 15.0%. For the real matrix, the stability samples at T0 have no deviation acceptance criteria, and the precision is ≤ 15.0%; for stability samples at other times, the deviation of the mean stability concentration of each concentration level from the mean concentration of the stability samples at T0 is within ±15.0%, and the precision is ≤ 15.0%. The number of freeze-thaw cycles, wet ice white light, and room temperature white light conditions for short-term and long-term storage stability can be adjusted according to experimental needs without the need to write additional amendments to the method validation plan. Continued Verify Parameters method Acceptance criteria Sample stability after extraction The surrogate matrix and real matrix quality control samples of LQC and HQC (n≥3) were extracted and stored in an autosampler at 4°C. After reaching the target time, the standard curve and quality control samples were freshly prepared and extracted according to the analysis procedure. The stored samples were analyzed together with the freshly prepared standard curve and quality control samples. The extracted and stored quality control samples were quantified by the freshly prepared standard curve samples. The time calculation method for the stability is: the time when the sample in the autosampler is placed in the autosampler is 0 time, and the time from 0 time to the completion of the injection of the last sample of the analysis batch is the stability time after the sample extraction. In the quality control samples prepared with the stored real matrix and surrogate matrix, the deviation between the average concentration and the theoretical concentration was within ±15.0%, and the precision was ≤ 15.0%. Continued Verify Parameters method Acceptance criteria Repeatability of sample extraction and re-injection After the first injection of an analysis batch containing an accuracy and precision (A&P) experiment is completed, the sample is placed in the autosampler at 4°C for a period of time and then re-injected and analyzed to examine the reproducibility of the injector. The time interval between the first analysis of the first sample in the analysis batch and the second first sample analysis is used to calculate the time interval of injection reproducibility. If both injections of the same batch meet the accuracy and precision batch acceptance criteria, then the results are reproducible after the sample has been in the autosampler for a period of time.

[0034] 3 Methodological validation results 3.1 Selectivity In the surrogate matrix double blank sample, the interference on both the analyte and the internal standard was 0.0%; in the surrogate matrix, the interference of the analyte on the internal standard was 0.0%, and the maximum interference of the internal standard on the analyte was 2.8%. The selectivity results all met the acceptance criteria.

[0035] In real matrix samples, since urea is an endogenous substance, there is no specific acceptance criterion at the retention time of the analyte for 6 individual blank human bronchoalveolar lavage fluids from different sources, and there is no interference at the retention time of the internal standard.

[0036] 3.2 Sensitivity The sensitivity of 0.500 μg / mL met the acceptance criteria.

[0037] 3.3 Matrix effects At the MQC-2 concentration, the coefficients of variation of normal bronchoalveolar lavage fluid (6 different individuals) were 1.1%, 0.2%, 1.6%, 2.9%, 3.8%, and 2.5%, respectively, and the deviations were -0.7%, -1.3%, -1.7%, 2.2%, -4.5%, and -1.0%, respectively, with an overall variation %CV of 2.8%, all of which met the acceptance criteria.

[0038] At HQC concentration, the coefficients of variation of normal bronchoalveolar lavage fluid (6 different individuals) were 1.2%, 1.4%, 1.0%, 3.3%, 1.8%, and 1.7%, respectively, and the deviations were -5.3%, -2.0%, -1.3%, -6.0%, -0.7%, and -2.0%, respectively, with an overall variation %CV of 2.7%, all of which met the acceptance criteria.

[0039] 3.4 Standard curve The coefficient of variation of the slope of the standard curve is 3.5%. In all test batches, the relative error of 8 standard curve samples at different concentration levels ranges from -2.6% to 1.5%. The standard curve passed well.

[0040] 3.5 Accuracy and precision The accuracy and precision validation included three analytical batches, each of which contained quality control samples of five concentrations (0.500, 1.50, 3.00, 6.00, and 15.0 μg / mL) prepared in surrogate matrix and quality control samples of four concentrations (background + 1.50, background + 3.00, background + 6.00, and background + 15.0 μg / mL) prepared in real matrix, with six replicates at each concentration level. The accuracy and precision of the analytical batches met the acceptance criteria.

[0041] The precision of the method was expressed as the percentage of the coefficient of variation (CV% = [(standard deviation / mean) × 100%]); the accuracy of the method was expressed as the percentage of the relative error (RE% = [(mean observed concentration - nominal concentration) / nominal concentration] ´100%).

[0042] The results of intra-batch and inter-batch accuracy and precision verification are shown in the following table.

[0043] Surrogate Matrix Quality Control Sample Accuracy and Precision Maximum precision (%CV) Accuracy (deviation from theoretical concentration) (%) range Intra-batch precision and accuracy (all concentration controls except LLOQ QC) 8.1 -1.7 ~ 4.7 Intra-batch precision and accuracy (LLOQ QC) 14.2 -11.2 ~ 9.6 Inter-assay precision and accuracy (all concentrations except LLOQ QC) 6.1 -1.0 ~ 1.3 Batch-to-batch precision and accuracy (LLOQ QC) 14.1 1.8 Accuracy and precision of real matrix quality control samples Maximum precision (%CV) Accuracy (deviation from theoretical concentration) (%) range Intra-batch precision and accuracy (all concentration controls except LLOQ QC) 13.0 -0.7 ~ 9.3 Intra-batch precision and accuracy (LLOQ QC) NA NA Inter-assay precision and accuracy (all concentrations except LLOQ QC) 11.0 2.0 ~ 6.7 3.6 Extraction recovery The results of the extraction recovery are shown in the following table. Urea and [ 13 C; 15 The recovery results of N2-urea met the acceptance criteria.

[0044] Extraction recoveries in alternative matrices Precision (%CV) Extracted samples Low concentration Medium concentration -2 High concentration Average recovery rate of urea extraction of analytes % 97.1 94.6 96.8 Coefficient of variation of urea extraction recovery of the analyte %CV 9.1 3.4 3.7 Coefficient of variation of total urea extraction recovery of the analyte %CV 5.8 Internal standard urea-IS total extraction recovery value% 92.7 Coefficient of variation of total extraction recovery of internal standard urea-IS %CV 5.5 Extraction recoveries in real matrices Precision (%CV) Extracted samples Medium concentration -2 High concentration Average recovery rate of urea extraction of analytes % 98.6 106.1 Coefficient of variation of urea extraction recovery of the analyte %CV 3.8 5.4 Coefficient of variation of total urea extraction recovery of the analyte %CV 5.9 Internal standard urea-IS total extraction recovery value% 99.2 Coefficient of variation of total extraction recovery of internal standard urea-IS %CV 4.2

[0045] 3.7 Stability Stability of stock solution of analyte (difference%, %CV) Transparent plastic tube, placed in 5.0% (v / v) methanol in water at room temperature for 24 hours: 0.2%, 3.7% Stability of stock solution of analyte (difference%, %CV) Transparent plastic tube, stored in 5.0% (v / v) methanol aqueous solution at -10 ~ -30℃ for 92 days: 1.1%, 2.7%. Stability of working solution of the test substance (difference%, %CV) Transparent plastic bottle, placed in 5.0% (v / v) methanol-water solution at room temperature for 24 hours: -0.5%, 2.7%; -1.9%, 1.7% Stability of working solution of the test substance (difference%, %CV) Transparent plastic bottles, stored in 5.0% (v / v) methanol aqueous solution at -10 ~ -30℃ refrigerator for 92 days: -2.8%, 1.9%; -0.3%, 3.0% Internal standard working solution stability (difference%, %CV) Transparent plastic bottles, stored in 5.0% (v / v) methanol-water solution at room temperature for 24 hours: -1.5%, 3.0% Internal standard working solution stability (difference%, %CV) Transparent plastic bottle, stored in 5.0% (v / v) methanol aqueous solution at -10 ~ -30℃ refrigerator for 92 days: -0.2%, 2.2% Stability during biological sample preparation (% difference, %CV) The stability of human bronchoalveolar lavage fluid samples prepared with authentic matrix after storage under wet ice conditions for 18 hours LQC-B: 0.6%, 3.1% HQC-B: 0.0%, 1.1% The stability of human bronchoalveolar lavage fluid samples prepared with authentic matrix after storage under room temperature conditions for 24 hours LQC-B: -11.3%, 7.2% HQC-B: 0.0%, 1.1% The stability of samples prepared with surrogate matrix after storage under wet ice conditions for 18 hours LQC: -2.0%, 7.6% HQC: -4.0%, 1.4% The stability of samples prepared with surrogate matrix after storage under room temperature conditions for 24 hours LQC: -4.7%, 4.8% HQC: -4.7%, 0.7%. Stability of samples after extraction (% difference, %CV) After pretreatment, the sample was placed in the autosampler (4°C) for 55.2 hours. LQC: 0.0%, 3.3% HQC: -2.0%, 2.0% LQC-B: 4.6%, 10.1% HQC-B: 0.7%, 1.3% Freeze-thaw stability (% difference, %CV) Human bronchoalveolar lavage fluid samples prepared with real matrix were frozen and thawed 6 times at -10 ~ -30℃LQC-B: 8.8%, 8.2%HQC-B: 0.0%, 1.1%Human bronchoalveolar lavage fluid samples prepared with real matrix were frozen and thawed 6 times at -60 ~ -90℃LQC-B: 2.5%, 3.0%HQC-B: 0.0%, 1.1%Samples prepared with alternative matrix were frozen and thawed 6 times at -10 ~ -30℃LQC: -2.0%, 4.4%HQC: -2.0%, 1.4%Samples prepared with alternative matrix were frozen and thawed 6 times at -60 ~ -90℃LQC: -6.7%, 2.7%HQC: -2.7%, 1.4% Long-term stability of analytes in biological matrices (%Difference, CV%) Human bronchoalveolar lavage fluid samples prepared with real matrix were placed at -10 ~ -30℃ for 87 daysLQC-B: -3.1%, 9.8%HQC-B: 0.0%, 1.1%Human bronchoalveolar lavage fluid samples prepared with real matrix were placed at -60 ~ -90℃ for 87 daysLQC-B: -12.6%, 11.2%HQC-B: 0.0%, 1.1%Samples prepared with alternative matrix were placed at -10 ~ -30℃ for 87 daysLQC: -5.3%, 6.5%HQC: -5.3%, 3.5%Samples prepared with alternative matrix were placed at -60 ~ -90℃ for 87 daysLQC: -2.0%, 5.5%HQC: -1.3%, 0.4% Autosampler injection reproducibility The sample was placed in the autosampler (4°C) for 182.7 hours and the results were reproducible.

[0046] 4 Conclusion The results of method validation showed that the quantitative range of the standard curve was 0.500 ~ 20.0 µg / mL, and the LLOQ and ULOQ were 0.500 µg / mL and 20.0 µg / mL, respectively. The accuracy and precision validation results showed that the precision and accuracy of the method were within the acceptable range. The extraction recovery, sensitivity, selectivity and matrix effect all met the acceptance criteria. The bronchoalveolar lavage fluid samples and surrogate matrix samples were stable after freezing and thawing for 6 times at -10 ~ -30℃ and -60 ~ -90℃, placed under white light at room temperature for 24 hours, placed under white light on wet ice for 18 hours, stored at -10 ~ -30ºC for 87 days, and stored at -60 ~ -90ºC for 87 days. After the sample was extracted and placed in the autosampler for 55.2 hours, it was quantified by the freshly prepared and processed standard curve, and the stability results met the acceptance criteria. After being placed in the autosampler for 182.7 hours, the repeatability of the sample after extraction and re-injection met the acceptance criteria.

[0047] This method uses [ 13 C; 15 N2]-urea was used as the internal standard compound (IS). The analyte urea and the internal standard [ 13 C; 15 N2]-urea. The samples were analyzed by LC-MS / MS system, which included Shimadzu LC-20AD high performance liquid chromatography system, Xselect HSS T3 2.5 μm, 4.6*100 mm and API 5000, SCIEX mass spectrometer. The mass spectrometer used positive atmospheric pressure chemical ionization (APCI) mode to monitor the analytes urea and [ 13 C; 15 N2]-urea parent ion and product ion mass-to-charge ratio ( m / z ), were 61.100→44.100 and 64.000→46.000 respectively. This method was used to determine the analytes urea and [ 13 C; 15 N2]-urea is specific, and the standard curve is linear in the concentration range of 0.500~20.0 µg / mL.

[0048] Example 2: Serum Example The difference between this embodiment and embodiment 1 is that the biological matrix used in this embodiment is serum. During the detection process, the quality control and clinical unknown samples are diluted 50 times with the alternative matrix and then detected. The other operations and procedures are the same as those in embodiment 1. According to the literature records and some laboratory data, the urea concentration in plasma and serum matrices is consistent, so this method is also applicable to the detection of plasma samples. The results and analysis are as follows.

[0049] 1.1 Selectivity, sensitivity, and calibration curve The contents and data of selectivity, sensitivity and standard curve are the same as those in Example 1.

[0050] 1.2 Matrix Effect At the MQC-2-P concentrations, the coefficients of variation of normal serum (six different individuals) were 2.1%, 7.7%, 4.1%, 4.7%, 3.5%, and 7.8%, and the deviations were 10.0%, -3.5%, 1.5%, 1.0%, 2.5%, and 8.0%, respectively, all meeting the acceptance criteria.

[0051] At the HQC-P concentration, the coefficients of variation of normal serum (six different individuals) were 2.2%, 4.5%, 5.2%, 1.9%, 6.6%, and 4.4%, and the deviations were 3.8%, -1.5%, 3.5%, 1.0%, -2.0%, and -3.3%, respectively, which all met the acceptance criteria.

[0052] The coefficients of variation of the MQC-2-P and HQC-P quality control samples of hyperlipidemia were 3.2% and 2.5%, respectively, and the deviations were 1.0% and 3.0%, respectively, which met the acceptance criteria.

[0053] The coefficients of variation of MQC-2-P and HQC-P quality control samples of hemolytic serum were 8.9% and 9.5%, respectively, and the deviations were -5.5%, 0.5%, which meets the acceptance criteria.

[0054] 1.3 Accuracy and precision The accuracy and precision validation included three analytical batches, each of which contained quality control samples of five concentrations (0.500, 1.50, 3.00, 6.00, and 15.0 μg / mL) prepared in surrogate matrix and quality control samples of four concentrations (background + 90.0, background + 150, 450, and background + 675 μg / mL) prepared in real matrix, with six replicates at each concentration level. The accuracy and precision of the analytical batches met the acceptance criteria.

[0055] The intra-batch and inter-batch accuracy and precision results of the surrogate matrix are the same as those of Example 1, and the intra-batch and inter-batch accuracy and precision results of the real matrix are shown in the following table.

[0056] Accuracy and precision of real matrix quality control samples Maximum precision (%CV) Accuracy (deviation from theoretical concentration) (%) range Intra-batch precision and accuracy (all concentrations except LLOQ) 7.8 -1.5 ~ 6.5 Intra-batch precision and accuracy (LLOQ) NA NA Inter-assay precision and accuracy (all concentrations except LLOQ) 6.3 1.2 ~ 4.3 1.4 Extraction recovery The results of the extraction recovery of the surrogate matrix are the same as those of Example 1. The results of the extraction recovery of the real matrix are shown in the following table. Urea and [ 13 C; 15 The recovery results of N2-urea met the acceptance criteria.

[0057] Extraction recoveries in real matrices Precision (%CV) Extracted samples Medium concentration -2 High concentration Average recovery rate of urea extraction of analytes % 92.8 95.6 Coefficient of variation of urea extraction recovery of the analyte %CV 2.6 1.0 Coefficient of variation of total urea extraction recovery of the analyte %CV 2.1 Internal standard urea-IS total extraction recovery value% 94.9 Coefficient of variation of total extraction recovery of internal standard urea-IS %CV 2.2

[0058] 1.5 Stability Stability of working solution of the test object (difference%, %CV) Transparent plastic bottle, placed in 5% (v / v) methanol-water solution at room temperature for 24 hours: -1.0%, 3.7%; -0.3%, 1.8% Stability of working solution of the test object (difference%, %CV) Transparent plastic bottles, stored in 5% (v / v) methanol aqueous solution at -10 ~ -30℃ refrigerator for 99 days: -2.6%, 2.4%; 1.3%, 1.8% Stability during biological sample pretreatment (accuracy deviation RE%, CV%) Human serum samples prepared with real matrix were stored under wet ice for 17 hours. Stability LQC-P: 2.0%, 2.7% HQC-P: -1.4%, 2.0% Human serum samples prepared with real matrix were stored under room temperature for 17 hours. Stability LQC-P: 2.0%, 1.6% HQC-P: 0.3%, 2.3% Sample stability after preparation (accuracy deviation RE%, CV%) After sample pretreatment, the autosampler (4°C) was placed for 45.6 hours. LQC-P: 5.2%, 3.5% HQC-P: 7.8%, 3.7% Freeze-thaw stability (accuracy deviation RE%, CV%) Human serum samples prepared with real matrix were frozen and thawed 6 times at -10 ~ -30℃LQC-P: 1.9%, 5.5%HQC-P: -5.0%, 1.0%Human serum samples prepared with real matrix were frozen and thawed 6 times at -60 ~ -90℃LQC-P: 1.7%, 5.6%HQC-P: -0.9%, 2.5% Long-term stability of the analyte in biological matrices (RE%, CV%) Human serum samples prepared with real matrix were placed at -10 ~ -30℃ for 77 daysLQC-P: 3.7%, 3.9%HQC-P: -0.6%, 1.8%Human serum samples prepared with real matrix were placed at -60 ~ -90℃ for 77 daysLQC-P: 4.8%, 2.5%HQC-P: -4.5%, 2.0% Injection reproducibility The sample was placed in the autosampler (4°C) for 69.8 hours and the results were reproducible.

[0059] 2 Conclusion The results of method validation showed that the quantitative range of the standard curve was 0.500 ~ 20.0 µg / mL. The plasma or serum quality control and clinical unknown samples were diluted 50 times with alternative matrix and tested. The accuracy and precision validation results of the method showed that they were within the acceptable range. The extraction recovery, sensitivity, residue, selectivity and matrix effect all met the acceptance criteria. The serum samples were stable after being frozen and thawed 6 times at -10 ~ -30℃ and -60 ~ -90℃, placed under white light at room temperature for 17 hours, placed under white light on wet ice for 17 hours, stored at -10 ~ -30ºC for 77 days, and stored at -60 ~ -90ºC for 77 days. After the sample was extracted and placed in the autosampler for 45.6 hours, it was quantified by the freshly prepared and processed standard curve, and the stability results met the acceptance criteria. After being placed in the autosampler for 69.8 hours, the repeatability of the sample extraction and re-injection met the acceptance criteria. The maximum number of samples in the analysis batch was 117 injections.

[0060] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any technician in the relevant technical field, without departing from the scope of the technical solution of the present invention, makes any form of equivalent replacement or modification to the technical solution and technical content disclosed in the present invention, which does not depart from the content of the technical solution of the present invention and still falls within the protection scope of the present invention.

Claims

1. A method for detecting urea concentration in various biological matrices using high performance liquid chromatography-mass spectrometry, characterized in that: Water was used as blank matrix, and biological matrix samples were treated with protein precipitation. Based on API5000 platform, APCI source was used, and C18 bonded phase mode was used for liquid phase, with a linear range of 0.500~20.0 µg / mL.

2. The method for detecting urea concentration in various biological matrices by high performance liquid chromatography-mass spectrometry according to claim 1, characterized in that: The biological matrix sample includes human plasma, human serum or human bronchoalveolar lavage fluid.

3. The method for detecting urea concentration in various biological matrices by high performance liquid chromatography-mass spectrometry according to claim 1, characterized in that: Preparation method of detection solvent: mobile phase A: aqueous solution containing 0.1% (v / v) formic acid and 10 mM ammonium acetate; mobile phase B: acetonitrile solution containing 0.1% (v / v) formic acid; Needle washing solution: methanol: acetonitrile: isopropanol: ultrapure water solution, prepared in a volume ratio of 1:1:1:1, then add 0.1% (v / v) trifluoroacetic acid; Solution A: 5.0% (v / v) methanol aqueous solution; Solution B: 1.0 M ammonium acetate aqueous solution.

4. The method for detecting urea concentration in various biological matrices by high performance liquid chromatography-mass spectrometry according to claim 1, characterized in that: The concentrations of urea standard curve sample working solutions are: 10.0, 20.0, 40.0, 80.0, 160, 200, 320, 400 µg / mL; the concentrations of urea quality control sample working solutions are: 1) human plasma or human serum: 1800, 3000, 9000, 13500 µg / mL; 2) human bronchoalveolar lavage fluid: 10.0, 30.0, 60.0, 120, 300 µg / mL.

5. The method for detecting urea concentration in various biological matrices by high performance liquid chromatography-mass spectrometry according to claim 1, characterized in that: The adopted 13 C; 15 N2]-urea internal standard stock solution concentration was 1.00 mg / mL, and the internal standard working solution concentration was 20000 ng / mL.

6. The method for detecting urea concentration in various biological matrices by high performance liquid chromatography-mass spectrometry according to claim 1, characterized in that: The concentrations of urea standard curve samples were 0.500, 1.00, 2.00, 4.00, 8.00, 10.0, 16.0, and 20.0 µg / mL; Concentration of urea quality control samples: The quality control samples prepared with surrogate matrix included 5 concentration levels, namely 0.500, 1.50, 3.00, 6.00, and 15.0 µg / mL; the quality control samples prepared with real human plasma or human serum matrix included 4 concentration levels, namely LQC-P background + 90.0 µg / mL, MQC-1-P background + 150 µg / mL, MQC-2-P background + 450 µg / mL, and HQC-P background + 675 µg / mL. The quality control samples prepared with real human plasma or human serum matrix were diluted 50 times with surrogate matrix and then processed for detection and analysis; the quality control samples prepared with real human bronchoalveolar lavage fluid matrix included 4 concentration levels, namely LQC-B background + 1.50 µg / mL, MQC-1-B background + 3.00 µg / mL, MQC-2-B background + 6.00 µg / mL, and HQC-B background + 675 µg / mL. Background +15.0 µg / mL.

7. The method for detecting urea concentration in various biological matrices by high performance liquid chromatography-mass spectrometry according to claim 1, characterized in that: Testing instruments used: The mass spectrometer used was API 5000, SCIEX; the liquid chromatography used was LC-20AD, Shimadzu; the autosampler used was RackChanger II sample rack switching device and SIL-20AC HT autosampler.

8. The method for detecting urea concentration in various biological matrices by high performance liquid chromatography-mass spectrometry according to claim 1, characterized in that: The liquid phase conditions of the detection method are: The analyte was urea; the chromatographic column was Xselect HSS T3 2.5 µm, 4.6×100 mm; the mobile phase was A: an aqueous solution containing 0.1% (v / v) formic acid and 10 mM ammonium acetate; B: an acetonitrile solution containing 0.1% (v / v) formic acid; the autosampler temperature was 4 o C; column temperature is 40 o C; injection volume was 5.00 µL; initial flow rate was 0.700 mL / min; Gradient elution program: 。 9. The method for detecting urea concentration in various biological matrices by high performance liquid chromatography-mass spectrometry according to claim 1, characterized in that: The mass spectrometry conditions for the detection method were: The ion spray voltage was 4500 V and the temperature was 550 o C; Gas 1 is 45 psi; Gas 2 is 45 psi; Nebulizer current is 3.0; Curtain gas is 45 psi; The collision gas is 12; the ion source is APCI; the detection mode is the positive ion mode; the scanning mode is the multiple ion reaction monitoring mode; the mass spectrometry acquisition time is 4.00 min; the Q1 / Q3 resolution is Unit / unit; the interval time is 5.007 ms; the gas 1, gas 2, curtain gas, and collision gas are all nitrogen.

10. The method for detecting urea concentration in various biological matrices by high performance liquid chromatography-mass spectrometry according to claim 1, characterized in that: Ion pairs and parameters of the detection method: 。

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