A detection method for detecting the urea concentration in multiple biological matrices by using high performance liquid chromatography-mass spectrometry

Abstract

The present invention discloses a detection method for detecting the urea concentration in multiple biological matrices by using high performance liquid chromatography - mass spectrometry, which relates to the field of urea concentration detection. The detection method for detecting the urea concentration in multiple biological matrices by using high performance liquid chromatography - mass spectrometry provided by the present invention uses water as a blank matrix, performs protein precipitation treatment on the biological matrix sample, and based on the API5000 platform, uses the APCI source, and the liquid phase uses the C18 bonded phase mode to detect the urea concentration in plasma / serum / BALF. The linear range is 0.500 - 20.0 μg / mL, and the sensitivity is greatly improved, which can fully meet the detection requirements of clinical samples.

Description

Technical Field

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

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

[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 quite stable within 24 hours. In addition, the urea concentration is considered to be minimally affected by disease states. Therefore, it is generally believed that the urea concentration is consistent in different biological matrices from the same subject, including nasal secretions, exhaled breath condensate (EBC), mucosal lining fluid (MLF), epithelial lining fluid (ELF), bronchoalveolar lavage fluid (BALF), plasma or serum, etc. Scholars use urea as a widely used dilution marker to standardize the volume of biological sampling. By measuring the urea concentrations in plasma and body fluids, defined as C plasma and C sample fluid , the volume of the native physiological matrix (V native ) can be calculated. The calculation formula is V native = (C sample fluid / C plasma ) * V sample fluid . By detecting the concentration of the research drug in BALF and multiplying it by V BALF / V native , the exposure amount of the research drug in bronchoalveoli can be obtained, providing reference data for the evaluation of drug efficacy and the administration method.

[0004] Literature reports on the detection methods of urea in different biological matrices include:

[0005] 1) Colorimetric method: Urea is acted on by an enzyme to form a product, which reacts with a probe to produce a color. The absorbance is proportional to the urea concentration in the solution. This detection scheme is completed using a commercial kit. This method is limited in sensitivity. At the same time, the detection results are greatly affected by the manufacturer and batch of the detection kit, and the detection method is not easy to transfer or reproduce.

[0006] 2) LC-MS coupling method: There are a few reports, but there are still deficiencies:

[0007] Due to its very small molecular weight, urea is prone to interference from other ion pairs in mass spectrometry detection. To improve sensitivity and selectivity, the literature uses derivatization, but the sample pretreatment process is complex and time-consuming;

[0008] In the prior art, alternative matrices are used to prepare standard curves and quality control samples, which simplifies the sample pretreatment steps. However, it is difficult to meet the detection requirements of multiple matrix samples in terms of sensitivity, and there are still challenges such as high baseline noise, matrix effects, and mismatches in the performance of multiple biological matrices in the detection method;

[0009] In the prior art, the method of using alternative analytes brings challenges to the interpretation of detection results, the linear range of the method, and detection costs;

[0010] Some literature has established an LC-MS coupling method. After salting-out assisted liquid-liquid extraction of biological samples, based on the API5500 platform, using the ESI source and the Hilic mode for liquid phase, the concentration of urea in plasma or serum or ELF is detected simultaneously, and the linear range is 1-50 μg / mL. This method still faces challenges in the application process, such as high baseline, defects in method sensitivity, and easy fluctuations in the Hilic mode.

[0011] To better support the increasing number of pharmacokinetic studies on respiratory diseases, there is an urgent need for a convenient and universal determination method for detecting and analyzing the concentration of urea in different biological matrices. Summary of the Invention

[0012] Object of the Invention: The object of the present invention is to provide an accurate, reliable, simple and efficient detection method for detecting the concentration of urea in multiple biological matrices by using high performance liquid chromatography-mass spectrometry coupling method. It can detect the concentration of urea in different types of biological matrices, and the method is easy to transfer and reproduce, and can meet the detection requirements of clinical samples.

[0013] Technical Solution: The detection method provided by the present invention for detecting the concentration of urea in multiple biological matrices by using high performance liquid chromatography-mass spectrometry coupling method uses water as a blank matrix, performs protein precipitation treatment on biological matrix samples, based on the API5000 platform, uses the APCI source, and uses the C18 bonded phase mode for liquid phase to detect the concentration of urea in plasma or serum or ELF. The linear range is 0.500-20.0 μg / mL, and the further improvement of sensitivity can fully meet the detection requirements of clinical samples.

[0014] The relevant parameters of this method are processed as follows:

[0015] Analyte Urea Internal Standard <![CDATA 13 C; 15 N2]-Urea ( 13 C; 15 N2]-Urea, Urea-IS)]]> Analytical Method LC-MS / MS Quantitative Analysis Method Biological Matrix and Source of Samples Authentic Matrix: Human plasma or human serum or bronchoalveolar lavage fluid (BALF); Surrogate Matrix: Ultra-pure water Anticoagulant (if any) <![CDATA[Human plasma is K2EDTA; human serum and human bronchoalveolar lavage fluid are without anticoagulants]]> Sample Pretreatment Method Protein Precipitation Sample Quantity 50.0 µL Injection Volume 5.00 µL Quantitative Index Peak Area Ratio of Analyte to Internal Standard Regression Equation Linear Fitting, Equation Expression: y = ax + b Weighting 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 QC Sample Concentration Points 0.500 (LLOQ QC), 1.50 (LQC), 3.00 (MQC-1), 6.00 (MQC-2), 15.0 (HQC) µg / mL Authentic Matrix QC Sample Concentration Points Human plasma or human serum: LQC-P baseline + 90.0 µg / mL, MQC-1-P baseline + 150 µg / mL, MQC-2-P baseline + 450 µg / mL, and HQC-P baseline + 675 µg / mL, all diluted 50-fold with surrogate matrix before processing and detection analysis; Bronchoalveolar lavage fluid: LQC-B baseline + 1.50 µg / mL, MQC-1-B baseline + 3.00 µg / mL, MQC-2-B baseline + 6.00 µg / mL, and HQC-B baseline + 15.0 µg / mL.

[0016] The technical method and steps of this embodiment are as follows:

[0017] (1) Solvent Preparation

[0018] The reagent volumes listed in this analysis procedure can be adjusted proportionally as needed.

[0019] 1) Mobile phase A (MPA): An aqueous solution containing 0.1% (v / v) formic acid and 10 mM ammonium acetate

[0020] 2) Mobile phase B (MPB): An acetonitrile solution containing 0.1% (v / v) formic acid

[0021] 3) Needle wash solution (NW): A solution of methanol:acetonitrile:isopropanol:ultrapure water, prepared in a volume ratio of 1:1:1:1, and then 0.1% (v / v) trifluoroacetic acid is added.

[0022] 4) Solution A: A 5.0% (v / v) aqueous methanol solution

[0023] 5) Solution B: An aqueous solution of 1.0 M ammonium acetate

[0024] (2) The liquid phase conditions are as follows in the table:

[0025] Analyte Urea Chromatographic Column 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: Ultra-pure water solution, prepared according to the volume ratio of 1:1:1:1, and then added with 0.1% (v / v) trifluoroacetic acid Autosampler Needle Wash Parameters Rinsing Volume: 200 μL Autosampler Needle Wash Parameters Rinsing Speed: 35 μL / sec Autosampler Needle Wash Parameters Rinse Dip Time: 2 sec Autosampler 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 Waste for 0~0.9 min and 2.2~4.0 min, enter MS for 0.9~2.2 min Expected Retention Time <![CDATA[Urea: 1.57 min; 13 C; 15 N2]-Urea: 1.57 min]]>

[0026] Continued:

[0027] Gradient elution program:

[0028] 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

[0029] (3) The mass spectrometry conditions are as follows in the table:

[0030] Analyte Urea Mass spectrometry parameters Parameter settings Ion spray voltage 4500 V Temperature 550 ºC <![CDATA[Gas 1 (GS1, N2)]]> 45 p.s.i. <![CDATA[Gas 2 (GS2, N2)]]> 45 p.s.i. Sprayer current (NC) 3.0 <![CDATA[Curtain air gas (CUR, N2)]]> 45 p.s.i <![CDATA[Collision gas (CAD, N2)]]> 12 Ion source APCI Detection mode Positive ion mode Scanning mode Multiple reaction monitoring (MRM) mode Mass spectrometry acquisition duration 4.00 min Q1 / Q3 resolution Unit / unit Intermittent time (Pause between mass ranges) 5.007 ms

[0031] (4) Preparation of stock solutions and working solutions:

[0032] Precisely weigh the urea reference standard substance in a transparent plastic tube. After conversion by the correction factor, add an appropriate amount of 5.0% (v / v) aqueous methanol solution to prepare a urea standard curve and quality control sample stock solution at a concentration of 40.0 mg / mL, and store it in a refrigerator at -10~-30 ºC for later use.

[0033] Use 5.0% (v / v) aqueous methanol solution as the dilution solvent to prepare the urea standard curve and quality control sample working solutions in a transparent plastic bottle. The concentrations of the urea standard curve sample working solutions are: 10.0, 20.0, 40.0, 80.0, 160, 200, 320, 400 µg / mL. The concentrations of the 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.

[0034] (5) Preparation of internal standard stock solution and working solution

[0035] Precisely weigh 13 C; 15 [[13C; 13 15N2]-urea reference standard substance. After conversion by the correction factor, add an appropriate amount of 5.0% (v / v) methanol aqueous solution to prepare a 1.00 mg / mL 15 [[13C;

[0036] 15N2]-urea internal standard stock solution, and store it in a refrigerator at -10~-30 °C.

[0037] (6) Preparation of standard curve samples and quality control samples

[0038] The concentrations of urea standard curve samples are 0.500, 1.00, 2.00, 4.00, 8.00, 10.0, 16.0, 20.0 μg / mL. The standard curve samples are prepared by adding the analyte working solution to the surrogate matrix (ultrapure water).

[0039] Quality control samples include quality control samples prepared with the surrogate matrix (ultrapure water) and quality control samples prepared with the real matrix (blank human plasma or human serum or blank human bronchoalveolar lavage fluid). The quality control samples prepared with the surrogate matrix are obtained by adding the analyte at the corresponding concentration to the surrogate matrix; when preparing the quality control samples of the real matrix, the endogenous concentration needs to be taken into account. Affected by the background concentration of the analyte, the quality control samples of the real matrix are obtained by adding an appropriate concentration of the analyte to the real matrix.

[0040] Quality control sample concentrations of urea: Quality control samples prepared with surrogate matrix contain 5 concentration levels, namely 0.500 (LLOQ QC), 1.50 (LQC), 3.00 (MQC-1), 6.00 (MQC-2), 15.0 (HQC) μg / mL; Quality control samples prepared with human plasma or human serum authentic matrix contain 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). Quality control samples prepared with human plasma or human serum authentic matrix are diluted 50-fold with surrogate matrix and then processed for detection and analysis; Quality control samples prepared with human bronchoalveolar lavage fluid authentic matrix contain 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).

[0041] (7) Data processing

[0042] Use Watson LIMS TM 7.5 SP1 (Thermo Scientific Inc.), adopt Linear, Weighting factor = 1 / x 2 The model processes the data. The concentration unit used when reporting the results is μg / mL, retaining 3 significant figures. For % Bias and % CV, retain 1 decimal place after the decimal point.

[0043] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0044] 1. The method of the present invention does not rely on kits, does not require surrogate analytes, has low detection costs, stable detection results, and guaranteed detection time limits;

[0045] 2. The instrument detection method has good reproducibility and can successfully complete method transfer on different detection platforms (API 5500, API6500) and in different laboratories;

[0046] 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, which can fully meet the sample measurement requirements. This method optimizes the peak shape and improves interference by screening the chromatographic column and optimizing the chromatographic gradient, improves the mass spectrometry response by optimizing the pretreatment and mass spectrometry parameters, and screens the ion source to improve the response and reduce the baseline. The sensitivity of the method is further improved compared with the existing technology. According to the detection 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 sample measurement requirements;

[0047] 4. The alternative matrix is easily available, the sample pretreatment is simple and efficient, the reproducibility is good, and the operability is high;

[0048] 5. The method of the present invention has been applied to the detection and analysis of clinical samples. The project implementation follows the principles of Good Laboratory Practice (GLP), Good Clinical Practice (GCP), the Guidelines for the Management of Bioanalytical Laboratories in Drug Clinical Trials (Trial Implementation) (State Drug Administration Notice

[2011] No. 482) of the NMPA, Section IV 9012 of the Pharmacopoeia of the People's Republic of China (2020 Edition): Guidelines for the Validation of Quantitative Analysis Methods for Biological Samples, and the requirements of 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. Description of the Drawings

[0049] Figure 1 For the analyte urea and internal standard 13 C; 15 N2]-urea ion scan diagram, 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;

[0050] Figure 2 For the spectrum of the blank matrix, a is the double blank sample, b is the zero concentration sample;

[0051] Figure 3 For the spectrum of the lower limit of quantification standard curve sample;

[0052] Figure 4 For the spectrum of the upper limit of quantification standard curve sample;

[0053] Figure 5 For the standard curve graph. Detailed Embodiments

[0054] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be further described below.

[0055] Example 1: Alveolar lavage fluid experiment

[0056] 1 Materials and instruments

[0057] 1.1 Analyte

[0058] The analyte in this example is urea, with a purity of 100% (Shanghai Zhenzhun Biotechnology Co., Ltd.).

[0059] 1.2 Control reference substance

[0060] 1.2.1 Information of control reference substance and internal standard

[0061] Item Reference substance Internal standard Name Urea <![CDATA 13 C; 15 N2]-Urea ( 13 C; 15 N2]-Urea, Urea-IS)]]> Source Shanghai Zhenzhu Biotechnology Co., Ltd. Shanghai Zhenzhu Biotechnology Co., Ltd. Purity / Content 100% 99.10%

[0062] The following are the actual preparation steps in the project. The listed reagent volumes can be adjusted proportionally as needed.

[0063] (1) Mobile phase A (MPA): Aqueous solution containing 0.1% (v / v) formic acid and 10 mM ammonium acetate

[0064] 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 aqueous ammonium acetate solution, and mix well.

[0065] (2) Mobile phase B (MPB): Acetonitrile solution containing 0.1% (v / v) formic acid

[0066] Take 1000 mL of acetonitrile in a reagent bottle, add 1.00 mL of formic acid, and mix well.

[0067] (3) Needle wash solution (NW): Methanol:acetonitrile:isopropanol:ultrapure water solution, prepared according to a volume ratio of 1:1:1:1, and then add 0.1% (v / v) trifluoroacetic acid

[0068] Take 250 mL each of methanol, acetonitrile, isopropanol, and ultrapure water in a reagent bottle, add 1.00 mL of trifluoroacetic acid, and mix well.

[0069] (4) Solution A: 5.0% (v / v) methanol aqueous solution

[0070] Take 25.0 mL of methanol and 475 mL of ultrapure water in a reagent bottle, and mix well.

[0071] (5) Solution B: 1.0 M aqueous ammonium acetate solution

[0072] Weigh about 3854.08 mg of ammonium acetate powder, add 50.0 mL of ultrapure water, and mix well.

[0073] 1.3 Main Instruments

[0074] Name Description Mass spectrometry API 5000, SCIEX Liquid chromatography LC-20AD, Shimadzu

[0075] 1.4 Analytical Conditions

[0076] 1.4.1 Chromatographic Conditions

[0077] Chromatographic column 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:Ultra-pure aqueous solution, prepared in a ratio of 1:1:1:1, and then 0.1% (v / v) trifluoroacetic acid is added Automatic sampler needle wash parameters Rinsing Volume:200 μLRinsing Speed: 35 μL / secRinse Dip Time: 2secRinse Mode: Before and after aspiration Automatic sampler 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 Waste for 0~0.9 min and 2.2 ~4.0 min, enter MS for 0.9~2.2 min Expected retention time <![CDATA[Urea: 1.57 min; 13 C; 15 N2]-Urea: 1.57 min]]>

[0078] Continued:

[0079] Gradient Elution Program:

[0080] 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

[0081] 1.4.2 Mass Spectrometry Conditions

[0082] Mass spectrometry parameters Parameter settings Ion spray voltage 4500 V Temperature 550 ºC <![CDATA[Gas 1 (GS1, N2)]]> 45 p.s.i. Sprayer current (NC) 3.0 <![CDATA[Curtain gas (CUR, N2)]]> 45 p.s.i <![CDATA[Collision gas (CAD, N2)]]> 12 Ion source APCI Detection mode Positive ion mode Scanning mode Multiple reaction monitoring (MRM) mode Mass spectrometry acquisition duration 4.00 min Q1 / Q3 resolution Unit / unit Intermittent time (Pause between mass ranges) 5.007 ms

[0083] Ion Pair and Parameters:

[0084] Compound Ion pair() Scanning time() 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

[0085] 1.5 Results of Methodology Validation

[0086] The results of the methodology validation in 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 results of the accuracy and precision validation show that the precision and accuracy of this method are within the acceptable range. The sensitivity, residue, and selectivity all meet the acceptable standards. The alveolar lavage fluid samples and surrogate matrix samples are stable after being frozen and thawed 6 times at -10 to -30 °C and -60 to -90 °C, placed at room temperature under white light for 24 hours, placed at wet ice under white light for 18 hours, stored at -10 to -30 °C for 87 days, and stored at -60 to -90 °C for 87 days; the samples remain stable after being placed in the autosampler for 55.2 hours after extraction; the repeatability of the samples after being placed in the autosampler for 182.7 hours and then injected again after extraction meets the acceptable standards. The maximum number of samples in an analytical batch is 210 injections. The background concentration of the real mixed human alveolar lavage fluid samples is between 1.48 - 1.66 μg / mL.

[0087] 2 Contents, Methods, and Acceptance Criteria of Methodology Validation

[0088] The methodological verification items include: selectivity, standard curve, sensitivity, precision and accuracy, extraction recovery rate, matrix effect, residual effect, and stability. Among them, the stability study includes the long-term freezing storage of biological samples, repeated freezing and thawing, the stability of biological samples during the pretreatment process, the stability under the storage conditions after preparation, and the stability of the analyte stock solution / analyte working solution / isotope internal standard working solution (at room temperature, placed under white light, long-term storage). The specific inspection parameters and acceptance criteria are shown in the following table.

[0089] Verification parameters Method Acceptance criteria Selectivity Prepare double blank samples (n = 1) using alternative matrices and prepare double blank samples (n = 1) using 6 different sources of individual blank true matrices. At the retention times of the analyte and the internal standard in the double blank samples of the alternative matrix, the peak area of the interfering peak is not greater than 20.0% of the average peak area of the analyte in the LLOQ standard curve samples meeting the acceptance criteria, and 5.0% of the average peak area of the internal standard in the LLOQ standard curve samples meeting 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 at the retention time of the analyte for 6 individual blank real matrices from different sources. At the retention time of the internal standard, the peak area of the interfering peak is not greater than 5.0% of the average peak area of the internal standard in the LLOQ standard curve samples meeting the acceptance criteria in this batch. The blank matrices of all normal individuals should meet the above criteria. If they do not meet the above criteria, the reasons for failure need to be evaluated, and the test should be repeated or more individuals should be added for evaluation. Selectivity Interference of the analyte on the internal standard: Using the alternative matrix, prepare samples at the ULOQ concentration (n≥3). Except that the internal standard working solution is replaced by the corresponding solution without the internal standard, the extraction process is the same as the normal method. In this sample, at the retention time of the internal standard, the average peak area of the interfering peak is not greater than 5.0% of the average peak area of the internal standard in all LLOQ standard curve samples meeting the acceptance criteria. Selectivity Interference of the internal standard on the analyte to be measured: Investigated through zero-concentration samples. Add the internal standard to the alternative matrix and process it into zero-concentration samples (ZeroBlank, n≥3). In this sample, at the retention time of the analyte, the average peak area of the interfering peak is not greater than 20.0% of the average peak area of the analyte in the LLOQ standard curve samples meeting the acceptance criteria in this analysis batch.

[0090] Continued table

[0091] Verification parameters Method Acceptance criteria Standard curve In the method validation, the standard curve samples need to be freshly prepared using the alternative matrix. Each standard curve contains 1 double blank, 1 zero-concentration sample, and 8 non-zero-point standard curve samples, with n = 2 for each sample. 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 the Urea standard curve samples are 0.500, 1.00, 2.00, 4.00, 8.00, 10.0, 16.0, 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 the 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 peak area of the analyte in the LLOQ standard curve sample that meets the acceptance criteria, and 5.0% of the average peak area of the internal standard in the LLOQ standard curve sample that meets 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 peak area of the analyte in the LLOQ standard curve sample that meets the acceptance criteria.]]>

[0092] Continued table

[0093] Verification parameters Method Acceptance criteria Accuracy and precision The accuracy and precision experiments should be conducted for at least three analytical batches and over at least 2 days. The quality control (QC) samples for the accuracy and precision experiments should be freshly prepared on the same day. The QC samples include QC samples prepared with surrogate matrix and QC samples prepared with real matrix. The QC samples prepared with surrogate matrix contain 5 concentration levels, with at least 5 samples for each concentration level, which are obtained by adding the corresponding concentration of the analyte to the surrogate matrix; the QC samples prepared with real matrix contain 4 concentration levels, with at least 5 samples for each concentration level. When preparing the QC samples with real matrix, the endogenous concentration needs to be taken into account, and they are obtained by adding the corresponding concentration of the analyte to the real matrix. Each analytical batch for accuracy and precision contains QC samples with 5 concentration levels of surrogate matrix and 4 concentration levels of real matrix. For the routine analytical batches (the analytical batches not used for investigating accuracy and precision), they should include at least two sets of QC samples with eight concentrations, namely surrogate matrix LQC, MQC-1, MQC-2, and HQC samples, and real matrix LQC, MQC-1, MQC-2, and HQC samples. For the QC samples with surrogate matrix, the within-batch and between-batch precision at each concentration level should be ≤ 15.0%, and the precision at the lower limit of quantitation (LLOQ) should be ≤ 20.0%; the accuracy deviation within-batch and between-batch at each concentration level should not exceed ±15.0% of the theoretical value, and the accuracy deviation at the LLOQ should not exceed ±20.0% of the theoretical value. For the QC samples with real matrix, since the introduction of the background concentration in the real matrix needs to be considered, the background concentration of the real matrix used will be quantified (the pretreatment is the same as that of the zero-concentration samples, named Zero-B, n≥3), and when calculating the accuracy of the QC samples with real matrix, the concentration will be back-calculated by the background subtraction method and then the accuracy will be calculated. The formula for the background subtraction method is as follows (all the validation samples containing real matrix in this project will calculate their accuracy deviation after back-calculating the concentration according to this formula): Unless there are clearly recorded chromatographic or instrumental errors, or sample handling errors, the results of all QC samples should be included in the calculation of within-batch and between-batch accuracy and precision. For the routine analytical batches, the accuracy deviation of all QC samples is within ±15.0% of the theoretical value, at least 2 / 3 of the QC samples meet the above requirements, and at least 50% of the QC samples at each concentration level meet the above requirements.

[0094] In the above table: Accuracy deviation (%) =

[0095]

[0096] Continued table

[0097] Validation parameters Method Acceptance criteria Sensitivity The lower limit of quantitation (LLOQ) represents the sensitivity of the method. The evaluation of LLOQ can be part of the accuracy and precision experiments. The response of the analyte in the LLOQ standard curve samples that meet the acceptance criteria compared to the average response of the analyte in the zero-concentration samples in the same analytical batch is not less than 5 times. The accuracy deviation of the LLOQ standard curve samples should not exceed ±20.0% of the theoretical value (calculated using the data from the analytical batches for between-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 from the analytical batches for between-batch accuracy and precision, at least 3 analytical batches, 2 replicates for each analytical batch).

[0098] Continued table

[0099] Validation parameters Method Acceptance criteria Matrix effect Six batches of real matrices from different individuals (n = 1) were used to evaluate matrix effects. The analyte working solution was added to the real individual matrices to prepare samples at the MQC-2 and HQC levels, and the background concentration was quantified in each analytical batch (n≥3 for each batch number). The average accuracy deviation of the normal blank matrix for each concentration level and each batch 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 the above criteria are not met, the reasons for failure need to be evaluated, and the test should be repeated or more individuals should be added for evaluation.

[0100] Continued table

[0101] Verification parameters Method Acceptance criteria Extraction recovery The recovery test should be performed on mixed real 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). The peak areas of the analyte and internal standard in the extracted samples were compared with those in the unextracted samples (adding the corresponding concentration of analyte and internal standard to the extracted blank samples). The recovery of the analyte in the real matrix was calculated from the results at two concentration levels (MQC-2, HQC), and the recovery of the analyte in the surrogate matrix was calculated from the results at three concentration levels (LQC, MQC-2, HQC). The recovery of the internal standard in the real matrix was calculated from the results of the internal standard response in the real matrix quality control samples (MQC-2, HQC), and the recovery of the internal standard in the surrogate matrix was calculated from the results of the internal standard response in the surrogate matrix quality control samples (LQC, MQC-2, HQC). The %CV of the extraction recovery of the quality control samples at each concentration level ≤ 15.0%, and the %CV of the extraction recovery at three concentration levels ≤ 15.0%. If the acceptance criteria are not met, the results corrected by the internal standard (analyte / internal standard peak area ratio) can be used to calculate the extraction recovery. The following formula can be used to calculate the recovery of the analyte in the real matrix, and the background concentration of the mixed real matrix should have been quantified or quantified concomitantly in the analytical batch.

[0102] In the above table:

[0103]

[0104] Extraction recovery rate (%) =

[0105]

[0106] The following formula is used to calculate the recovery rate of the analyte in the surrogate matrix:

[0107] Extraction recovery rate (%) = Peak area of the extracted sample / Average peak area of the unextracted sample × 100.

[0108] Continued table

[0109] Verification parameters Method Acceptance criteria Investigation of stability in real matrix For the investigation of the stability of urea in human real matrix, quality control samples at two concentration levels (LQC and HQC) should be prepared in large volumes and aliquoted. The aliquoted stability samples were used to evaluate the stability on the laboratory bench (wet ice white light and room temperature white light), freeze-thaw cycles and long-term stability. Due to the influence of the background concentration, the LQC samples can be replaced by real matrices without additional addition of the analyte working solution. The zero-time samples (T0) should be extracted on the day of preparation (n≥3) to prove the accuracy of the preparation of the stability samples. The biological matrix stability samples must be frozen for at least 24 hours before evaluating their short-term stability. For the convenience of later sample analysis, the stability of the analyte in the surrogate matrix can also be investigated, including wet ice and room temperature stability, freeze-thaw cycles and long-term stability, etc., using quality control samples at two concentration levels (LQC and HQC). For surrogate matrix stability samples, the deviation of the average stability concentration from the theoretical concentration at each concentration level is within ±15.0%, and the precision ≤ 15.0%. For real matrix, there is no deviation acceptance criterion for stability samples at T0, and the precision ≤ 15.0%; for stability samples at other times, the deviation of the average stability concentration at each concentration level from the average concentration of stability samples at T0 is within ±15.0%, and the precision ≤ 15.0%. The number of freeze-thaw cycles, and the time points of short-term and long-term storage stability under wet ice white light and room temperature white light conditions can be adjusted according to experimental requirements without additional amendment to the method validation plan.

[0110] Continued table

[0111] Verification parameters Method Acceptance criteria Stability after sample extraction Extract surrogate matrix and real matrix quality control samples of LQC and HQC (n≥3) and store them in the autosampler at 4°C. After reaching the target time, freshly prepare the standard curve and quality control samples and perform extraction according to the analysis protocol, and analyze the stored samples together with the freshly prepared standard curve and quality control samples. The quality control samples extracted and stored are quantified by the freshly prepared standard curve samples. The time calculation method for this stability is: the time when the sample is placed in the autosampler is the 0 moment, and the time from the 0 moment to the end of injection of the last sample in this analysis batch is used as the stability time after sample extraction. For the quality control samples prepared from the stored real matrix and surrogate matrix, the deviation of the average concentration from the theoretical concentration is within ±15.0%, and the precision ≤ 15.0%.

[0112] Continued table

[0113] Verification parameters Method Acceptance criteria Repeatability of re-injection after sample extraction After the first injection of an analysis batch containing accuracy and precision (A&P) experiments is completed, place it in the 4°C autosampler for a period of time and then re-inject for analysis to examine the reproducibility of the autosampler. The time interval between the first analysis time of the first sample in the analysis batch and the second analysis time of the first sample is used to calculate the time interval for injection reproducibility. If the results of the two injections in this analysis batch both meet the acceptance criteria for the accuracy and precision analysis batch, it can be shown that the results are reproducible after the samples in the autosampler are placed for a period of time.

[0114] 3 Results of methodological validation

[0115] 3.1 Selectivity

[0116] In the alternative matrix double blank samples, the interferences on the analyte and the internal standard were both 0.0%; in the alternative 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 acceptable criteria.

[0117] In the real matrix samples, since urea is an endogenous substance, for the individual blank human bronchoalveolar lavage fluids from 6 different sources, there is no specific acceptance criterion at the retention time of the analyte, and there is no interference at the retention time of the internal standard.

[0118] 3.2 Sensitivity

[0119] The sensitivity of 0.500 μg / mL met the acceptable criteria.

[0120] 3.3 Matrix effect

[0121] At the MQC-2 concentration, the coefficients of variation of normal bronchoalveolar lavage fluids (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. The overall coefficient of variation %CV was 2.8%, all of which met the acceptance criteria.

[0122] At the HQC concentration, the coefficients of variation of normal bronchoalveolar lavage fluids (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. The overall coefficient of variation %CV was 2.7%, all of which met the acceptance criteria.

[0123] 3.4 Standard curve

[0124] The coefficient of variation of the standard curve slope was 3.5%. In all detection batches, for the standard curve samples at 8 different concentration levels, the relative error % range was -2.6% to 1.5%. The standard curve passed well.

[0125] 3.5 Accuracy and precision

[0126] Accuracy and precision verification included 3 analytical batches, each analytical batch contained quality control samples at 5 concentrations (0.500, 1.50, 3.00, 6.00, and 15.0 μg / mL) prepared with surrogate matrix and quality control samples at 4 concentrations (background + 1.50, background + 3.00, background + 6.00, and background + 15.0 μg / mL) prepared with real matrix. Six replicates were performed for each concentration level. The accuracy and precision of the analytical batches both met the acceptance criteria.

[0127] 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 relative error (RE% = [(mean observed concentration - nominal concentration) / nominal concentration] ´ 100%).

[0128] The results of within - batch and between - batch accuracy and precision verification are shown in the following table.

[0129] Accuracy and precision of surrogate matrix quality control samples Maximum precision (%CV) Accuracy (deviation from theoretical concentration) (%) range Intra-batch precision and accuracy (for all concentration quality controls except LLOQ QC) 8.1 -1.7 ~ 4.7 Intra-batch precision and accuracy (LLOQ QC) 14.2 -11.2 ~ 9.6 Inter-batch precision and accuracy (for all concentration quality controls except LLOQ QC) 6.1 -1.0 ~ 1.3 Inter-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 (for all concentration quality controls except LLOQ QC) 13.0 -0.7 ~ 9.3 Intra-batch precision and accuracy (LLOQ QC) NA NA Inter-batch precision and accuracy (for all concentration quality controls except LLOQ QC) 11.0 2.0 ~ 6.7

[0130] 3.6 Extraction recovery

[0131] The results of extraction recovery are shown in the following table. The recovery results of urea and 13 C; 15

[0132] Extraction recovery of surrogate matrix Precision (%CV) of extracted samples: low concentration, medium concentration - 2, high concentration Mean % of extraction recovery rate of analyte urea 97.1 94.6 96.8 Coefficient of variation %CV of extraction recovery rate of analyte urea 9.1 3.4 3.7 Coefficient of variation %CV of total extraction recovery rate of analyte urea 5.8 Value % of total extraction recovery rate of internal standard urea - IS 92.7 Coefficient of variation %CV of total extraction recovery rate of internal standard urea - IS 5.5 Extraction recovery rate of real matrix Precision (%CV) Concentration in the extracted samples - 2 High concentration Mean % of extraction recovery rate of analyte urea 98.6 106.1 Coefficient of variation %CV of extraction recovery rate of analyte urea 3.8 5.4 Coefficient of variation %CV of total extraction recovery rate of analyte urea 5.9 Value % of total extraction recovery rate of internal standard urea - IS 99.2 Coefficient of variation %CV of total extraction recovery rate of internal standard urea - IS 4.2

[0133] 3.7 Stability

[0134] Stability of analyte stock solution (Difference %, %CV) Transparent plastic tube, placed at room temperature for 24 hours in 5.0% (v / v) methanol aqueous solution: 0.2%, 3.7% Stability of analyte stock solution (Difference %, %CV) Transparent plastic tube, stored at -10 ~ -30°C for 92 days in 5.0% (v / v) methanol aqueous solution: 1.1%, 2.7%. Stability of analyte working solution (Difference %, %CV) Transparent plastic bottle, placed at room temperature for 24 hours in 5.0% (v / v) methanol aqueous solution: -0.5%, 2.7%; -1.9%, 1.7% Stability of analyte working solution (Difference %, %CV) Transparent plastic bottle, stored at -10 ~ -30°C in a refrigerator for 92 days in 5.0% (v / v) methanol aqueous solution: -2.8%, 1.9%; -0.3%, 3.0% Stability of internal standard working solution (Difference %, %CV) Transparent plastic bottle, stored at room temperature for 24 hours in 5.0% (v / v) methanol aqueous solution: -1.5%, 3.0% Stability of internal standard working solution (Difference %, %CV) Transparent plastic bottle, stored at -10 ~ -30°C in a refrigerator for 92 days in 5.0% (v / v) methanol aqueous solution: -0.2%, 2.2% Stability during the pretreatment process of biological samples (Difference %, %CV) Stability of human bronchoalveolar lavage fluid samples prepared with real matrix stored under wet ice conditions for 18 hours LQC - B: 0.6%, 3.1% HQC - B: 0.0%, 1.1% Stability of human bronchoalveolar lavage fluid samples prepared with real matrix stored at room temperature for 24 hours LQC - B: -11.3%, 7.2% HQC - B: 0.0%, 1.1% Stability of samples prepared with surrogate matrix stored under wet ice conditions for 18 hours LQC: -2.0%, 7.6% HQC: -4.0%, 1.4% Stability of samples prepared with surrogate matrix stored at room temperature for 24 hours LQC: -4.7%, 4.8% HQC: -4.7%, 0.7%. Stability after sample extraction (Difference %, %CV) Samples placed in an autosampler (4°C) for 55.2 hours after pretreatment 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 authentic matrix were frozen and thawed 6 times at -10 to -30 °C. LQC-B: 8.8%, 8.2%; HQC-B: 0.0%, 1.1%. Human bronchoalveolar lavage fluid samples prepared with authentic matrix were frozen and thawed 6 times at -60 to -90 °C. LQC-B: 2.5%, 3.0%; HQC-B: 0.0%, 1.1%. Samples prepared with surrogate matrix were frozen and thawed 6 times at -10 to -30 °C. LQC: -2.0%, 4.4%; HQC: -2.0%, 1.4%. Samples prepared with surrogate matrix were frozen and thawed 6 times at -60 to -90 °C. LQC: -6.7%, 2.7%; HQC: -2.7%, 1.4%. Long-term stability of analytes in biological matrix (% difference, CV%) Human bronchoalveolar lavage fluid samples prepared with authentic matrix were stored at -10 to -30 °C for 87 days. LQC-B: -3.1%, 9.8%; HQC-B: 0.0%, 1.1%. Human bronchoalveolar lavage fluid samples prepared with authentic matrix were stored at -60 to -90 °C for 87 days. LQC-B: -12.6%, 11.2%; HQC-B: 0.0%, 1.1%. Samples prepared with surrogate matrix were stored at -10 to -30 °C for 87 days. LQC: -5.3%, 6.5%; HQC: -5.3%, 3.5%. Samples prepared with surrogate matrix were stored at -60 to -90 °C for 87 days. LQC: -2.0%, 5.5%; HQC: -1.3%, 0.4%. Autosampler injection reproducibility The samples were placed in the autosampler (4 °C) for 182.7 hours, and the results were reproducible

[0135] 4 Conclusion

[0136] ​The results of methodological 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 results of accuracy and precision validation indicated that the precision and accuracy of this method were within the acceptable range. The extraction recovery, sensitivity, selectivity, and matrix effect all met the acceptable standards. The bronchoalveolar lavage fluid samples and surrogate matrix samples were stable after being frozen and thawed 6 times at -10 to -30°C and -60 to -90°C, placed at room temperature under white light for 24 hours, placed at wet ice under white light for 18 hours, stored at -10 to -30°C for 87 days, and stored at -60 to -90°C for 87 days. After the samples were extracted and placed in the autosampler for 55.2 hours, they were quantified by the freshly prepared and processed standard curve, and the stability results met the acceptable standards. The repeatability of the samples after extraction and reinjection after being placed in the autosampler for 182.7 hours met the acceptable standards.

[0137] This method uses 13 C; 15 N2]-urea as the internal standard compound (IS), and the analyte urea and the internal standard 13 C; 15 N2]-urea are extracted from human bronchoalveolar lavage fluid samples by the protein precipitation method. The samples are analyzed using an LC-MS / MS system, which includes a Shimadzu LC-20AD high-performance liquid chromatography system, Xselect HSS T3 2.5 μm, 4.6 * 100 mm, and an API 5000, SCIEX mass spectrometer. The mass spectrometer monitors the precursor and product ion mass-to-charge ratios ( 13 C; 15 N2]-urea) of the analyte urea and m / z ), which are 61.100 → 44.100 and 64.000 → 46.000, respectively. This method is specific for the determination of the analyte urea and 13 C; 15 N2]-urea in human bronchoalveolar lavage fluid, and the standard curve is linear in the concentration range of 0.500 - 20.0 μg / mL.

[0138] Example 2: Serum Example

[0139] The difference between this example and Example 1 is that the biological matrix in this example is serum. During the detection process, the quality control and clinical unknown samples are diluted 50 times with the surrogate matrix before detection, and other operations are the same as those in Example 1. According to the literature records and some laboratory data, the urea concentrations in plasma and serum matrices are the same. Therefore, this method is also applicable to the detection of plasma samples. The results and analysis are as follows.

[0140] 1.1 Selectivity, Sensitivity, and Calibration Curve

[0141] The content and data of selectivity, sensitivity, and calibration curve are the same as those in Example 1.

[0142] 1.2 Matrix Effect

[0143] At the MQC-2-P concentration, the coefficients of variation of normal sera (6 different individuals) were 2.1%, 7.7%, 4.1%, 4.7%, 3.5%, and 7.8% respectively, and the deviations were 10.0%, -3.5%, 1.5%, 1.0%, 2.5%, and 8.0% respectively, all meeting the acceptance criteria.

[0144] At the HQC-P concentration, the coefficients of variation of normal sera (6 different individuals) were 2.2%, 4.5%, 5.2%, 1.9%, 6.6%, and 4.4% respectively, and the deviations were 3.8%, -1.5%, 3.5%, 1.0%, -2.0%, and -3.3% respectively, all meeting the acceptance criteria.

[0145] For the MQC-2-P and HQC-P quality control samples in hyperlipidemic sera, the coefficients of variation were 3.2% and 2.5% respectively, and the deviations were 1.0% and 3.0% respectively, meeting the acceptance criteria.

[0146] For the MQC-2-P and HQC-P quality control samples in hemolyzed sera, the coefficients of variation were 8.9% and 9.5% respectively, and the deviations were -5.5% and

[0147] 0.5% respectively, meeting the acceptance criteria.

[0148] 1.3 Accuracy and Precision

[0149] The verification of accuracy and precision included 3 analytical batches. Each analytical batch contained quality control samples at 5 concentrations (0.500, 1.50, 3.00, 6.00, and 15.0 μg / mL) prepared with surrogate matrix and 4 concentrations (background + 90.0, background + 150, 450, and background + 675 μg / mL) prepared with real matrix. Six replicates were performed for each concentration level. The accuracy and precision of the analytical batches all met the acceptance criteria.

[0150] The intra-batch and inter-batch accuracy and precision results for the surrogate matrix were the same as those in Example 1. The intra-batch and inter-batch accuracy and precision results for the real matrix are shown in the following table.

[0151] Accuracy and precision of authentic matrix quality control samples Maximum precision (%CV) Accuracy (deviation from the theoretical concentration) (%) range Intra-batch precision and accuracy (for all concentration quality controls except LLOQ) 7.8 -1.5 ~ 6.5 Intra-batch precision and accuracy (LLOQ) NA NA Inter-batch precision and accuracy (for all concentration quality controls except LLOQ) 6.3 1.2 ~ 4.3

[0152] 1.4 Extraction Recovery

[0153] The extraction recovery results of the surrogate matrix are the same as those in Example 1. The extraction recovery results of the real matrix are shown in the following table. The recovery results of urea and 13 C; 15 N2]-urea meet the acceptable criteria.

[0154] Extraction recovery of authentic matrix Precision (%CV) in the extracted samples, concentration -2 high concentration Mean % of extraction recovery of analyte urea 92.8 95.6 Coefficient of variation %CV of extraction recovery of analyte urea 2.6 1.0 Coefficient of variation %CV of total extraction recovery of analyte urea 2.1 Value % of total extraction recovery of internal standard urea-IS 94.9 Coefficient of variation %CV of total extraction recovery of internal standard urea-IS 2.2

[0155] 1.5 Stability

[0156] Stability of analyte working solution (% difference, %CV) In clear plastic bottles, placed in 5% (v / v) methanol aqueous solution at room temperature for 24 hours: -1.0%, 3.7%; -0.3%, 1.8% Stability of analyte working solution (% difference, %CV) In clear plastic bottles, stored in 5% (v / v) methanol aqueous solution in a refrigerator at -10 to -30 °C for 99 days: -2.6%, 2.4%; 1.3%, 1.8% Stability during the pretreatment process of biological samples (accuracy deviation RE%, CV%) Stability of human serum samples prepared with real matrix stored under wet ice conditions for 17 hours. LQC-P: 2.0%, 2.7%; HQC-P: -1.4%, 2.0%. Stability of human serum samples prepared with real matrix stored at room temperature for 17 hours. LQC-P: 2.0%, 1.6%; HQC-P: 0.3%, 2.3%. Stability of samples after preparation (accuracy deviation RE%, CV%) Samples placed in the autosampler (4°C) for 45.6 hours after pretreatment. 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 freeze-thawed 6 times at -10 to -30°C. LQC-P: 1.9%, 5.5%; HQC-P: -5.0%, 1.0%. Human serum samples prepared with real matrix were freeze-thawed 6 times at -60 to -90°C. LQC-P: 1.7%, 5.6%; HQC-P: -0.9%, 2.5%. Long-term stability of analytes in biological matrix (accuracy deviation RE%, CV%) Human serum samples prepared with real matrix were placed at -10 to -30°C for 77 days. LQC-P: 3.7%, 3.9%; HQC-P: -0.6%, 1.8%. Human serum samples prepared with real matrix were placed at -60 to -90°C for 77 days. LQC-P: 4.8%, 2.5%; HQC-P: -4.5%, 2.0%. Injection reproducibility The samples were placed in the autosampler (4°C) for 69.8 hours, and the results were reproducible.

[0157] 2 Conclusion

[0158] The results of the methodology verification show that the quantitative range of the standard curve is: 0.500 - 20.0 µg / mL. Plasma or serum quality control and clinical unknown samples are diluted 50-fold with the surrogate matrix and then detected. The verification results of the accuracy and precision of the method show that they are within the acceptable range. The extraction recovery, sensitivity, residue, selectivity, and matrix effect all meet the acceptable criteria. Serum samples are stable under the conditions of freezing and thawing 6 times at -10 to -30 °C and -60 to -90 °C, placing at room temperature under white light for 17 hours, placing at wet ice under white light for 17 hours, storing at -10 to -30 °C for 77 days, and storing at -60 to -90 °C for 77 days; after the samples are extracted and placed in the autosampler for 45.6 hours, they are quantified by the freshly prepared and processed standard curve, and the stability results meet the acceptable criteria. The repeatability of the samples after extraction and injection again after being placed in the autosampler for 69.8 hours meets the acceptable criteria. The maximum number of samples in an analytical batch is 117 injections.

[0159] The above are only the preferred embodiments of the present invention and do not impose any limitation on the present invention. Any person skilled in the art, without departing from the scope of the technical solution of the present invention, makes any form of equivalent substitution or modification and other changes to the technical solution and technical content disclosed in the present invention, which are all within the content of the technical solution of the present invention and still fall within the protection scope of the present invention.

Claims

1. A detection method for detecting the urea concentration in multiple biological matrices by using high performance liquid chromatography - mass spectrometry, characterized in that, Using water as the blank matrix, the biological matrix samples were treated by protein precipitation. Based on the API 5000 platform, with APCI source, the liquid phase was in C18 bonded phase mode, and the linear range was 0.500 - 20.0 µg / mL. The biological matrix samples include human plasma or human serum or human bronchoalveolar lavage fluid. The chromatographic column used was Xselect HSS T3 2.5 µm. The surrogate matrix was ultrapure water. The mobile phase: A: aqueous solution containing 0.1% formic acid and 10 mM ammonium acetate; B: acetonitrile solution containing 0.1% formic acid. The temperature of the autosampler was 4°C; the column temperature was 40°C; the injection volume was 5.00 µL; the initial flow rate was 0.700 mL / min. Gradient elution program:

2. The detection method for detecting the urea concentration in multiple biological matrices by using high performance liquid chromatography-mass spectrometry according to claim 1, characterized in that, Preparation method of the detection solvents: Mobile phase A: aqueous solution containing 0.1% formic acid and 10 mM ammonium acetate; Mobile phase B: acetonitrile solution containing 0.1% formic acid; Needle wash solution: methanol:acetonitrile:isopropanol:ultrapure water solution, prepared according to the volume ratio of 1:1:1:1, and then added with 0.1% trifluoroacetic acid by volume; Solution A: 5.0% methanol aqueous solution; Solution B: 1.0 M ammonium acetate aqueous solution.

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

4. The detection method for detecting the urea concentration in a variety of biological matrices by using high performance liquid chromatography-mass spectrometry according to claim 1, characterized in that, Adopted 13 C; 15 The concentration of the [C; 15 N2]-urea internal standard stock solution is 1.00 mg / mL, and the concentration of the internal standard working solution is 20,000 ng / mL.

5. The detection method for detecting the urea concentration in a variety of biological matrices by using high performance liquid chromatography-mass spectrometry according to claim 1, characterized in that, The concentrations of the urea standard curve samples were 0.500, 1.00, 2.00, 4.00, 8.00, 10.0, 16.0, 20.0 µg / mL. The concentrations of the urea quality control samples: The quality control samples prepared with the surrogate matrix contained 5 concentration levels, which were 0.500, 1.50, 3.00, 6.00, 15.0 µg / mL respectively; The quality control samples prepared with the real matrix of human plasma or human serum contained 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 the real matrix of human plasma or human serum were all diluted 50 times with the surrogate matrix and then processed for detection and analysis; The quality control samples prepared with the real matrix of human bronchoalveolar lavage fluid contained 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.

6. The detection method for detecting the urea concentration in multiple biological matrices by using high performance liquid chromatography-mass spectrometry according to claim 1, characterized in that, The detection instrument used was: The mass spectrometry used API 5000, SCIEX; the liquid chromatography used LC-20AD, Shimadzu; the auto sampler used RackChanger II sample rack switching device and SIL-20AC HT auto sampler.

7. The detection method for detecting the urea concentration in a variety of biological matrices by using high performance liquid chromatography-mass spectrometry according to claim 1, characterized in that, The analyte was urea.

8. The detection method for detecting the urea concentration in a variety of biological matrices by using high performance liquid chromatography-mass spectrometry according to claim 1, characterized in that, The mass spectrometry conditions of the detection method were as follows: The ion spray voltage was 4500 V; the temperature was 550 °C; Gas 1 was 45 p.s.i.; Gas 2 was 45 p.s.i.; the nebulizer current was 3.0; the curtain gas was 45 p.s.i; The collision gas was 12; the ion source was APCI; the detection mode was positive ion mode; the scanning mode was multiple reaction monitoring mode; the mass spectrometry acquisition duration was 4.00 min; the Q1 / Q3 resolution was Unit / unit; the dwell time was 5.007 ms; Gas 1, Gas 2, the curtain gas, and the collision gas were all nitrogen.

9. The detection method for detecting the urea concentration in multiple biological matrices by using 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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