A kit and method for detecting infliximab concentration

Abstract

The application provides a kit and a method for detecting infliximab concentration, and belongs to the technical field of substance detection. The kit comprises an internal standard, a working solution, a diluent, an enzyme cutting reagent, a termination solution, a mobile phase, a calibration sample and a quality control sample. The kit provided by the application can effectively shorten the preparation steps and time of the infliximab detection sample, has good sensitivity and specificity, and is suitable for clinical application.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of analytical detection, and particularly relates to a kit and method for detecting infliximab concentration. BACKGROUND

[0002] The statements in this section merely provide background information related to the present application and do not necessarily constitute the prior art.

[0003] Infliximab is a human-mouse chimeric monoclonal antibody that can specifically block tumor necrosis factor-α (TNF-α), and can be used in the treatment of rheumatoid arthritis, ankylosing spondylitis, psoriasis, Crohn's disease and ulcerative colitis. Some patients do not have a good improvement in the condition after receiving infliximab treatment, which may be due to a low drug concentration of infliximab in the blood or the generation of anti-drug antibodies. In 2017, the United States and Australia respectively issued the consensus on therapeutic drug monitoring (TDM) of inflammatory bowel disease, and in 2018, the Expert Consensus Opinion on Therapeutic Drug Monitoring of Inflammatory Bowel Disease in China pointed out that the effective concentration of infliximab is 3-7 μg / mL. Therefore, it is of great significance to accurately determine the blood drug concentration of infliximab in patients at regular intervals to guide doctors to develop a reasonable drug administration plan and timely adjust the treatment strategy of patients. In addition, the price of monoclonal antibody drugs is high, and the adjustment of the drug dosage and interval through TDM to avoid overuse of drugs can also help to reduce the economic burden of patients.

[0004] At present, the main methods for monitoring the blood drug concentration of infliximab include enzyme-linked immunosorbent assay (ELISA), fluorescence chromatography, colloidal gold method and high performance liquid chromatography mass spectrometry (LC-MS).

[0005] ELISA, fluorescence chromatography and colloidal gold method are all immunological detection methods based on the principle of antigen-antibody reaction. The main disadvantages of immunological methods for the determination of blood drug concentration of monoclonal antibodies are as follows: 1) susceptible to interference of anti-drug antibodies in the body, and lack of accuracy; 2) the results are greatly affected by the quality of antibodies in the kit, and cross-reactions may occur. Among them, fluorescence chromatography and colloidal gold method can only provide semi-quantitative or qualitative detection results, and cannot provide accurate quantification, and the sensitivity of colloidal gold method is low (patent application No. 201910586707.8). The LC-MS method is not affected by anti-drug antibodies in the body, and specific peptides of infliximab are selected for detection, and the results are highly specific. However, the LC-MS method reported for protein detection has the problems of long pretreatment time (incubation for several hours or overnight) and complicated operation (usually including multiple steps such as protein precipitation or antibody enrichment, denaturation, reduction, alkylation, enzymolysis, purification, etc.), which requires high technical requirements for experimenters and is not easy to carry out in clinical detection work. SUMMARY

[0006] In order to overcome the above-mentioned deficiencies of the prior art, the present application provides a kit and a method for detecting the concentration of infliximab.

[0007] In order to achieve the above-mentioned purpose, one or more embodiments of the present application provide the following technical solutions:

[0008] In a first aspect, a kit for detecting the concentration of infliximab is provided, the kit comprising: an internal standard, a working solution, a diluent, an enzyme digestion reagent, a termination solution, a mobile phase, a calibration standard, and a quality control standard; the internal standard is a stable isotope-labeled infliximab-specific peptide segment;

[0009] The working solution comprises working solution A containing buffer salt and working solution B containing denaturant;

[0010] The enzyme digestion reagent is a lyophilized powder containing trypsin;

[0011] The termination solution is an aqueous solution containing formic acid;

[0012] The mobile phase comprises mobile phase A, which is an aqueous solution containing formic acid, and mobile phase B, which is an acetonitrile solution containing formic acid;

[0013] The diluent comprises diluent A, which is pure water, and diluent B, which contains bovine serum.

[0014] In the specific embodiments of the present application, the stable isotope-labeled infliximab-specific peptide segment is SINSATHYAESVK(K→K 13 C6 15 N2); the concentration of the infliximab-specific peptide segment is 10-40 ng / mL.

[0015] In the specific embodiments of the present application, the buffer salt is ammonium bicarbonate buffer salt; the concentration of the buffer salt is 25-100 mM;

[0016] The denaturant is trifluoroethanol; the volume concentration of the denaturant is 20%-60%.

[0017] In the specific embodiments of the present application, the volume concentration of formic acid in the termination solution is 10%-50%; or,

[0018] The mobile phase A is an aqueous solution containing 0.1-0.2% formic acid; or,

[0019] The mobile phase B is an acetonitrile solution containing 0.1-0.2% formic acid.

[0020] In the specific embodiment of the present application, the volume ratio of the detection sample to the working solution, the enzyme cutting reagent, the termination solution in the kit is 1: (5-10): (7.5-15): (0.5-1).

[0021] In the specific embodiment of the present application, the dosage form of at least one of the internal standard, the calibrator and the quality control sample is a freeze-dried product.

[0022] In a second aspect, a method for detecting the concentration of infliximab is provided, comprising the following steps:

[0023] S1: sample pretreatment: add the working mixed solution containing the internal standard and the working solution to the sample, shake and incubate at 50-60 DEG C for 20-40 min; add the enzyme cutting reagent, shake and incubate at 35-38 DEG C for 50-70 min; add the termination solution and centrifuge at high speed;

[0024] S2: machine detection: take the supernatant to determine the liquid chromatography tandem mass spectrometry, bring the detection result into the standard curve, and obtain the concentration of the actual sample;

[0025] The method is not for the purpose of diagnosis and treatment of diseases.

[0026] In the specific embodiment of the present application, the non-essential steps of infliximab detection such as protein precipitation, antibody enrichment, reduction, alkylation, desalination purification and the like are removed; and all operations can be completed in a 1.5 mL centrifugal tube, greatly shortening the sample processing time, reducing the reagent cost and experimental operation difficulty, and being conducive to improving the repeatability and accuracy of the detection result.

[0027] In the specific embodiment of the present application, the method for obtaining the standard curve is:

[0028] The calibrator is prepared into a series of concentrations and the internal standard is added, and the liquid chromatography tandem mass spectrometry is determined, so that the concentration of the calibrator is taken as the independent variable xi, the ratio of the peak area of the corresponding concentration calibrator to the internal standard is taken as the dependent variable yi, and the standard curve is drawn.

[0029] In the specific embodiment of the present application, the calibration curve used for quantification is taken as the independent variable xi, and the peak area ratio of the corresponding concentration calibrator and the internal standard is taken as the dependent variable yi, and the linear regression equation y = bx + a and the correlation coefficient r are calculated. The calibrator is preferably distributed in a series of concentrations of 0.391-100 μg / mL, and the concentration points are preferably set to 0.391, 0.781, 1.563, 3.125, 6.25, 12.5, 25, 50 and 100 μg / mL.

[0030] In the specific embodiment of the present application, the determination method of the present application further comprises detecting a quality control sample, when the correlation coefficient r of the calibration curve is ≥0.9900, the detection result of the quality control sample is brought into the linear regression equation to calculate the concentration of the quality control sample. The quality control sample includes two concentrations of low-value quality control sample and high-value quality control sample. When the quality control sample result meets the expected requirement, the detection result of the actual sample is brought into the linear regression equation to obtain the concentration of the actual sample.

[0031] In the specific embodiment of the present application, the matrix of the calibration sample and the quality control sample in the determination method of the present application is serum or plasma.

[0032] In the specific embodiment of the present application, the conditions of the liquid chromatography tandem mass spectrometry are as follows:

[0033] The specifications of the chromatographic column are as follows: length 5-15 cm, diameter 1-4.6 mm, using C18 reversed-phase filler, particle size 1.6-3 μm;

[0034] The mobile phase A is an aqueous solution containing 0.1-0.2% formic acid; the mobile phase B is an acetonitrile solution containing 0.1-0.2% formic acid; the flow rate is 0.2-0.4 mL / min; the column temperature is 40-50 °C; the injection volume is 1-25 μL; the gradient elution is 0 min 2% B, 1.0 min 2% B, 6 min 65% B, 6.5 min 90% B, 7.5 min 90% B, 7.6 min 2% B, 8 min 2% B;

[0035] The mass spectrometry detection adopts triple quadrupole mass spectrometry or Orbitrap high-resolution mass spectrometry.

[0036] In the specific embodiment of the present application, when the mass spectrometry detection adopts triple quadrupole mass spectrometry, the ion source adopts electrospray ion source (H-ESI) in positive ion mode; the ion spray voltage is 3200-3800 V; the sheath gas is 30-50 Arb; the auxiliary gas is 8-12 Arb; the purge gas is 0.8-1.2 Arb; the ion transmission tube temperature is 280-320 °C; the atomization temperature is 300-340 °C; the scanning mode is multiple reaction monitoring (MRM / SRM);

[0037] The information of the analyte for detection and quantification is as follows:

[0038]

[0039] Or, when the mass spectrometry detection adopts Orbitrap high-resolution mass spectrometry, the ion source adopts H-ESI, the positive ion mode; the ion spray voltage is 3200-3800V; the sheath gas is 30-50Arb; the auxiliary gas is 8-12Arb; the purge gas is 0.8-1.2Arb; the ion transmission tube temperature is 280-320 DEG C; the atomization temperature is 300-340 DEG C; the fragmentation mode is high-energy collisional dissociation (HCD); the resolution is 30000 (200m / z); the scanning mode: parallel reaction monitoring (PRM)

[0040] The analyte information for detection and quantification is as follows:

[0041]

[0042] In the present application, the use of Orbitrap high-resolution mass spectrometry can provide accurate mass number, further improve the ability to accurately identify target substances from complex matrix, and simultaneously realize qualitative and quantitative.

[0043] In the specific embodiment of the present application, the linear range of the standard curve is 0.391-100 μg / mL;

[0044] In the specific embodiment of the present application, the standard curve is y=0.1780x-0.001198 or y=0.1372x+0.02039, respectively.

[0045] The above one or more technical solutions have the following beneficial effects:

[0046] The detection method provided by the present application simplifies the pretreatment process, optimizes the chromatography and mass spectrometry conditions, effectively shortens the sample processing time, improves the detection sensitivity, and is more conducive to clinical application.

[0047] Specifically, the pretreatment method provided by the present application takes no more than 2.5h (including 1.5h sample incubation time), which greatly shortens the processing time compared with the traditional protein spectrum detection pretreatment method, and can better meet the clinical detection demand.

[0048] The pretreatment process established by the present application only includes denaturation, enzyme digestion and termination reaction, all of which are liquid addition operations, and all operations can be completed in a 1.5mL centrifuge tube, which greatly reduces the experimental technical difficulty and detection cost, and is conducive to improving the repeatability and accuracy of the detection results.

[0049] The present application detects by using H-ESI ionization mode, improves the detection sensitivity by optimizing the chromatography and mass spectrometry parameters; the accuracy, precision and other methodological investigations meet the requirements of quantitative analysis; the present application can accurately quantify in the linear range of 0.391-100 μg / mL, and can be applied to the detection of blood drug concentration of infliximab.

[0050] The present application adopts the internal standard method to correct the whole experiment process, uses the standard curve to carry out quantitative analysis, and simultaneously uses low and high level quality control products to investigate the accuracy and effectiveness of the experiment, so as to avoid the distortion of the detection result.

[0051] The detection method provided by the present application can use the triple quadrupole mass spectrometry MRM / SRM method or the Orbitrap high-resolution mass spectrometry PRM method for detection.

[0052] The advantages of the additional aspects of the present application will be partially given in the following description, partially will become obvious from the following description, or will be understood through the practice of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0053] The drawings accompanying the specification of the present application are used to provide further understanding of the present application, the illustrative embodiments of the present application and the description thereof are used to explain the present application, and do not constitute improper limitation on the present application.

[0054] Figure 1 It is a detection principle schematic diagram of the detection method of the third embodiment of the present application, A: triple quadrupole mass spectrometry, B: Orbitrap high-resolution mass spectrometry.

[0055] Figure 2 It is a sample processing flowchart of the third embodiment of the present application.

[0056] Figure 3 It is an infliximab calibration curve diagram obtained by using the triple quadrupole mass spectrometry of the third embodiment of the present application.

[0057] Figure 4 It is an infliximab calibration curve diagram obtained by using the Orbitrap high-resolution mass spectrometry of the third embodiment of the present application.

[0058] Figure 5 It is an infliximab extraction ion chromatogram at the lowest point of the standard curve obtained by using the triple quadrupole mass spectrometry of the third embodiment of the present application.

[0059] Figure 6 It is an infliximab internal standard extraction ion chromatogram at the lowest point of the standard curve obtained by using the triple quadrupole mass spectrometry of the third embodiment of the present application.

[0060] Figure 7 It is an infliximab extraction ion chromatogram at the lowest point of the standard curve obtained by using the Orbitrap high-resolution mass spectrometry of the third embodiment of the present application.

[0061] Figure 8 The standard curve minimum point infliximab internal standard extracted ion chromatogram obtained by Orbitrap high-resolution mass spectrometry for Example 3 of the present application.

[0062] Figure 9 The comparison chart of detection results of different pretreatment operations for Comparative Example 1 of the present application. DETAILED DESCRIPTION

[0063] It should be noted that the following detailed description is exemplary in nature and is intended to provide further description of the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs.

[0064] It should be noted that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application.

[0065] In the present application, infliximab is purchased from Sigma Company, CAS number: 170277-31-3; and the heavy isotope-labeled infliximab specific peptide segment is purchased from Zhongpeishenghua Company.

[0066] In the present application, the mass spectrometry chromatography instrument is YS EXACT 9900MD high-performance liquid chromatography tandem mass spectrometry detection system and YS HR 1080MD liquid chromatography high-resolution mass spectrometry detection system from Shandong Yingsheng Biotechnology Co., Ltd.

[0067] Example 1

[0068] The present embodiment provides a kit for detecting the concentration of infliximab by LC-MS, which comprises:

[0069] Internal standard: freeze-dried product containing isotope-labeled infliximab specific peptide segment SINSATHYAESVK(K→K 13 C6 15 N2);

[0070] Working solution: including working solution A and working solution B, wherein the working solution A contains 100 mM ammonium bicarbonate, and the working solution B contains 60% trifluoroethanol (TFE);

[0071] Enzymatic digestion reagent: freeze-dried product containing trypsin;

[0072] Termination solution: 50% formic acid aqueous solution;

[0073] Mobile phase A: 0.2% formic acid aqueous solution;

[0074] Mobile phase B: 0.2% formic acid acetonitrile solution;

[0075] Calibrator: lyophilized product containing infliximab;

[0076] Quality control: lyophilized product containing infliximab.

[0077] Example 2

[0078] The present example provides a kit for detecting the concentration of infliximab by LC-MS. The composition and specifications of the kit are shown in Table 1.

[0079] Table 1: Composition of infliximab assay kit

[0080]

[0081] The final concentration is recommended as follows:

[0082] The working solution A contains 25 mM ammonium bicarbonate, and the working solution B contains 40% trifluoroethanol (TFE);

[0083] Termination solution: 30% formic acid in water;

[0084] Mobile phase A: 0.1% formic acid in water;

[0085] Mobile phase B: 0.1% formic acid in acetonitrile.

[0086] Example 3

[0087] The purpose of the present example is to provide a method for detecting the concentration of infliximab.

[0088] The detection principle of the detection method provided by the present application is as follows: a reagent containing a stable isotope internal standard specific peptide segment of infliximab is used to pretreat samples (including calibrators, quality controls, and actual samples), and then LC-MS is used to detect the pretreated samples. The sample first enters the liquid phase system, is separated by the chromatographic column, and is then introduced into the mass spectrometry system. The mass spectrometry system can be a triple quadrupole mass spectrometer or an Orbitrap high-resolution mass spectrometer.

[0089] Triple quadrupole mass spectrometer Figure 1 A): After ionization in the ion source of the mass spectrometry system, the sample enters the quadrupole mass analyzer, is separated according to the mass-to-charge ratio (m / z) of the ions, and the data are collected in the MRM / SRM mode. Q1 is used to screen specific parent ions, and the selected parent ions are introduced into the collision cell Q2 after passing through Q1 to undergo fragmentation. After the fragmentation of the parent ions, only specific daughter ions can pass through Q3 to enter the detector to form a mass spectrum signal, and the chromatogram and peak area of the specific daughter ions of infliximab specific peptide segments and internal standards in the detection sample are recorded.

[0090] Orbitrap high-resolution mass spectrometer Figure 1B) : After ionization in the ion source of the mass spectrometry system, enter the quadrupole mass analyzer, separate according to the mass-to-charge ratio (m / z) of the ions, collect data in PRM mode, Q1 is used to screen specific parent ions, and the screened parent ions are introduced into the collision cell Q2 to occur fragmentation, and then the Orbitrap is used to collect the secondary spectrum containing all fragment ions. The specific parent-daughter ion pair is used to quantify the target peptide segment, and the chromatogram and peak area of the specific daughter ion of the infliximab specific peptide segment and the internal standard in the sample are recorded.

[0091] The ratio of the peak area of the infliximab specific peptide segment to the internal standard in the sample is calculated, the target value concentration of the calibrator is taken as the abscissa, and the ratio of the peak area of the calibrator to the corresponding internal standard is taken as the ordinate. The standard curve is drawn and the linear equation is fitted, and the concentration of infliximab is calculated by substituting the peak area ratio of the infliximab specific peptide segment in the quality control sample and the actual sample into the standard curve equation. The specific principle diagram is shown in Figure 1 .

[0092] Specifically, the kit in Example Two is used for detection, including the following steps:

[0093] (I) Sample requirements:

[0094] 1. Sample type: human serum or plasma.

[0095] 2. Sample collection: prepared according to the standard clinical procedure (refer to the People's Republic of China Health Industry Standard WS / T 225-2002 "Collection and Processing of Blood Samples for Clinical Chemistry Testing").

[0096] 3. Transportation and storage: frozen (-18℃ or below) for 28 days, or refrigerated (2-8℃) for 7 days.

[0097] (II) Detection method:

[0098] 1. Internal standard and working solution preparation

[0099] (1) Preparation of high-concentration internal standard solution: take 1 mL of diluent A and add it to the internal standard tube, vortex for 30 s, centrifuge at 3000 rpm for 30 s, and prepare it immediately.

[0100] (2) Preparation of working solution: Calculate the volume of working solution according to the total number of experimental samples, and mix the three liquids thoroughly according to the ratio of working solution A: working solution B: high-concentration internal standard solution = 100:50:20, and prepare it immediately.

[0101] 2. Preparation of calibrator working solution

[0102] Precisely pipette 50 μL of diluent B into the calibration tube, vortex for 30 s, centrifuge at 3000 rpm for 30 s, and obtain S1 solution, which is prepared immediately before use; dilute S1 solution with diluent B in proportion to obtain S2-S9; the dilution method and corresponding theoretical concentration are shown in Table 2:

[0103] Table 2 Dilution of infliximab assay kit calibrators

[0104]

[0105] 3. Preparation of quality control working solution

[0106] Precisely pipette 50 μL of diluent B into the quality control I and II tubes, vortex for 30 s, centrifuge at 3000 rpm for 30 s, and obtain low and high value quality control solutions with concentrations of 2.0 and 20 μg / mL, respectively, which are prepared immediately before use.

[0107] 4. Preparation of enzyme digestion reagent

[0108] Take 15 mL of diluent A into the enzyme digestion reagent bottle, mix well, and store at 4℃ for standby, and the remaining can be aliquoted and stored frozen.

[0109] 5. Sample preparation

[0110] (1) Add 20 μL of sample to a 1.5 mL centrifuge tube;

[0111] (2) Add 170 μL of working solution to each tube, and incubate at 55℃ for 30 min with shaking;

[0112] (3) After the sample tube returns to room temperature, add 300 μL of enzyme digestion reagent to each tube, and incubate at 37℃ for 60 min with shaking;

[0113] (4) Add 10 μL of termination solution to each tube, vortex for 15 s, and centrifuge at 14000 rpm for 5 min;

[0114] (5) Transfer 100 μL of supernatant to a micro-injection bottle or injection plate for LC-MS detection;

[0115] (6) The calibration and quality control samples are treated in the same way as the samples.

[0116] 6. On-machine detection

[0117] Place the micro-injection bottle or injection plate into the automatic injection device, enable the application software, and establish a sample list.

[0118] (1) Liquid chromatography conditions

[0119] Chromatographic column: Hypersil GOLD TMC18 (2.1 x 100 mm, 1.9 μm); flow rate: 0.35 mL / min; column temperature: 45 °C; autosampler temperature: 8 °C; injection volume: 20 μL; gradient elution program as shown in Table 3:

[0120] Table 3 Liquid phase gradient elution program

[0121]

[0122] (2) Mass spectrometry conditions

[0123] As using triple quadrupole mass spectrometry MRM / SRM detection mode, the information of analytes for detection and quantification as shown in Table 4:

[0124] Table 4 Triple quadrupole mass spectrometry parameter settings

[0125]

[0126] As using Orbitrap high resolution mass spectrometry PRM detection mode, the information of analytes for detection and quantification as shown in Table 5:

[0127] Table 5 Orbitrap high resolution mass spectrometry parameter settings

[0128]

[0129] Wherein, the detection principle as shown in Figure 1 , and the sample processing procedure as shown in Figure 2 . It should be noted that the kit of the present application does not contain, but other necessary experimental materials including: 1.5 mL centrifuge tube, 10 μL / 100 μL / 200 μL / 1000 μL pipette and pipette tip, C18 chromatographic column, micro sample bottle or sample plate, constant temperature oscillator, high speed centrifuge, ultrasonic cleaner and vortex mixer.

[0130] 7. Data processing

[0131] With the concentration of the calibration as the independent variable xi, and the ratio of the peak area of the corresponding concentration calibration and the internal standard as the dependent variable yi, the standard curve is drawn, the linear regression equation y = bx + a and the correlation coefficient r are calculated; when the correlation coefficient r is greater than or equal to 0.9900, the detection results of the quality control are brought into the linear standard curve, and the concentration of the quality control is calculated; when the results of the quality control are within the expected range, the detection results of the sample are brought into the standard curve, and the concentration of the target substance of the sample is obtained.

[0132] Calibration curve linear evaluation:

[0133] Using the calibrators provided by the kit, and according to the preparation method of the calibrators in Example 3, a series of concentration calibrator working solutions were obtained. Taking the analyte concentration as the abscissa and the peak area ratio of the analyte to the internal standard as the ordinate, the linear regression equation y = bx + a was obtained, and the correlation coefficient r was calculated, which was required to be ≥0.9900.

[0134] The experimental results are shown in Table 6.

[0135] Table 6 Evaluation results of calibration curves of two mass spectrometers

[0136]

[0137] The correlation coefficient r of the calibration curve obtained by treating the sample using the kit method provided by the present application and detected by the triple quadrupole mass spectrometry MRM / SRM detection mode or the Orbitrap high-resolution mass spectrometry PRM detection mode was greater than 0.9900, and the calibration curve evaluation met the methodological requirements. The calibration curves obtained by the two mass spectrometers are shown in Figure 3 、 Figure 4 The target and internal standard ion chromatograms at the lowest point of the calibration curve are shown in Figures 5-8 From Figures 5-8 , it can be seen that the target and internal standard are well retained and separated, and the peak shape of the target and internal standard obtained by the two mass spectrometers is sharp and symmetrical, and the signal-to-noise ratio is high. The results of other performance verification tests using the two mass spectrometers also meet the methodological requirements.

[0138] Based on the above, the following examples only use Orbitrap high-resolution mass spectrometry detection data as preferred examples for display.

[0139] Precision evaluation:

[0140] Using the quality control samples provided by the kit, low and high value quality control samples were obtained according to the preparation method of the quality control samples in Example 3; 6 samples were prepared in parallel for each concentration, and each sample was detected once; 3 batches were continuously processed for 3 days; the batch and inter-batch precision was evaluated, and the coefficient of variation CV was used to represent.

[0141] The experimental results are shown in Table 7.

[0142] Table 7 Evaluation results of batch and inter-batch precision

[0143]

[0144]

[0145] The data in Table 7 show that the precision experimental data of 3 batches in succession, the batch and inter-batch precision CV values of infliximab high and low concentration quality control samples were all ≤5%, and the precision evaluation results met the methodological requirements.

[0146] Accuracy evaluation:

[0147] The normal human blank serum was used to prepare four different concentrations of samples within the linear range, and each concentration sample was processed in triplicate, and each sample was detected once. The accuracy of the detection results was represented by the recovery rate.

[0148] The experimental results are shown in Table 8.

[0149] Table 8: Recovery rate evaluation results of four levels

[0150]

[0151]

[0152] Table 8 shows that the detection recovery rates of infliximab samples at four concentrations are between 89.6% and 105.5%, meeting the requirements of 85.0%-115.0%; the CV of three repeated experiments is between 1.3% and 7.3%.

[0153] Quantitative detection limit evaluation:

[0154] The quantitative detection limit of the method was investigated by detecting the linear minimum point of the calibration curve; 10 samples were prepared in parallel, and each sample was detected once; the precision (CV) of the measurement results and the relative deviation (RE) of the measured value and the specified value were investigated.

[0155] The experimental results are shown in Table 9.

[0156] Table 9: Quantitative detection limit evaluation results

[0157]

[0158] Table 9 shows that the CV of the linear minimum point (LOQ) of infliximab is ≤20%, and the RE is ≤15%, indicating that the quantitative detection limit evaluation meets the methodological requirements.

[0159] Matrix effect evaluation:

[0160] Low and high concentration levels of infliximab standard solution and a certain concentration of internal standard solution were added to the reagent diluent B, normal human blank serum and pure solvent, respectively; each concentration sample was processed in triplicate, and each sample was determined once; the matrix effect was evaluated by the internal standard normalized matrix effect factor, and the specific calculation formula was as follows: internal standard normalized matrix effect factor = (analyte peak area / internal standard peak area in the presence of matrix) / (analyte peak area / internal standard peak area in the absence of matrix).

[0161] The results are shown in Table 10.

[0162] Table 10: Matrix effect evaluation evaluation results

[0163]

[0164] The data in Table 10 show that the matrix factor of infliximab internal standard at different spiked concentrations is between 0.95 and 0.99 using kit diluent B and human normal blank serum as the matrix. The results show that the matrix effect of the internal standard and the analyte is close, and the matrix effect of the target analyte that may occur can be compensated by using the internal standard correction, so as to not affect the accuracy and precision of the quantitative results, and the average result of the matrix effect meets the methodological requirements.

[0165] Residual evaluation:

[0166] The linear highest point of the calibration curve and the blank matrix sample were injected 5 times; by determining the peak area of infliximab and the internal standard in the blank matrix sample, and comparing with the detection results of the linear lowest point of the calibration curve, the influence of the residual on the accurate quantification of the target was investigated.

[0167] The experimental results are shown in Table 11:

[0168] Table 11 Residual experiment evaluation results

[0169]

[0170] Experimental summary:

[0171] The data in Table 11 show that the residual of infliximab is about 2.02%, and the residual of the internal standard is about 0.12%. According to the 2020 edition of the Chinese Pharmacopoeia, the guidance principles for the validation of biological sample quantitative analysis methods, in the blank matrix sample after the high concentration sample, the response of the interference peak at the retention time of the analyte should not exceed 20% of the response of the analyte in the sample at the lower limit of quantification; the response of the interference peak at the retention time of the internal standard should not exceed 5% of the response of the internal standard. The residual experiment evaluation results meet the methodological requirements.

[0172] By evaluating the calibration curve, precision, accuracy, quantitative detection limit, matrix effect and residual of the mass spectrometric detection method of infliximab, the results show that all the performance index verification results meet the methodological requirements, and the detection method established in the present application can meet the clinical blood sample detection requirements.

[0173] Comparative Example 1

[0174] In the prior art, the complete pretreatment process of LC-MS for detecting proteins includes the following six steps: ① protein precipitation or enrichment (such as methanol or Melon gel); ② denaturation (such as TFE); ③ reduction (such as DTT); ④ alkylation (such as IAA); ⑤ enzymatic digestion (such as trypsin); and ⑥ sample purification (such as C18 desalting). The complex operation steps require high technical requirements for the operator, and are prone to introduce large measurement bias. Moreover, the long processing time is difficult to meet the actual clinical application requirements.

[0175] In the present comparative example, different pretreatment processes are used, and the detection results are compared to determine which steps are necessary for infliximab detection, so as to simplify the operation steps and shorten the processing time.

[0176] The comparative test results are shown in Table 1. Figure 9 As shown in Table 1, the control refers to a process containing the above six steps, and the rest are respectively missing one step, and the detection is carried out by using the detection method in Example 3. The results show that removing the steps of protein precipitation, reduction, alkylation or desalting does not affect the quantitative detection results of infliximab. Therefore, the present application only retains the two necessary steps of denaturation and enzymatic digestion.

[0177] Although the specific embodiments of the present application are described above in combination with the accompanying drawings, it is not a limitation on the protection scope of the present application. It should be understood by those skilled in the art that various modifications or variations made on the basis of the technical solutions of the present application without creative labor are still within the protection scope of the present application.

Claims

1. A method of detecting infliximab concentration, characterized in that, The method comprises the following steps: S1: sample pretreatment: adding a working mixture containing an internal standard and a working solution to the sample, incubating at 50-60°C for 20-40 min, adding enzyme digestion reagent, incubating at 35-38°C for 50-70 min, and high-speed centrifugation after adding a termination solution; S2: instrument detection: taking the supernatant for liquid chromatography-mass spectrometry determination, bringing the detection result into a standard curve, and obtaining the concentration of the actual sample; The internal standard is a stable isotope labeled infliximab specific peptide segment SINSATHYAESVK (K→K 13 C6 15 N2); The working solution comprises working solution A containing a buffer salt and working solution B containing a denaturant; The buffer salt is ammonium bicarbonate buffer salt, and the concentration of the buffer salt is 25-100 mM; The denaturant is trifluoroethanol, and the volume concentration of the denaturant is 20%-60%; The method is not for the purpose of diagnosis and treatment of diseases; The conditions of the liquid chromatography-mass spectrometry are as follows: The chromatographic column is Hypersil GOLD™ C18, 2.1 x 100 mm, 1.9 μm; The mobile phase A is a water solution containing 0.1-0.2% formic acid, the mobile phase B is an acetonitrile solution containing 0.1-0.2% formic acid, the flow rate is 0.2-0.4 mL / min, the column temperature is 40-50°C, the injection amount is 1-25 μL, and the gradient elution is 0 min 2% B, 1.0 min 2% B, 6 min 65% B, 6.5 min 90% B, 7.5 min 90% B, 7.6 min 2% B, 8 min 2% B; The mass spectrometry detection adopts triple quadrupole mass spectrometry or Orbitrap high-resolution mass spectrometry; When the mass spectrometry detection adopts triple quadrupole mass spectrometry, the ion source adopts electrospray ion source in positive ion mode, the ion spray voltage is 3200-3800 V, the sheath gas is 30-50 Arb, the auxiliary gas is 8-12 Arb, the purge gas is 0.8-1.2 Arb, the ion transmission tube temperature is 280-320°C, the atomization temperature is 300-340°C, and the scanning mode is multiple reaction monitoring; Or, when the mass spectrometry detection adopts Orbitrap high-resolution mass spectrometry, the ion source adopts H-ESI in positive ion mode, the ion spray voltage is 3200-3800 V, the sheath gas is 30-50 Arb, the auxiliary gas is 8-12 Arb, the purge gas is 0.8-1.2 Arb, the ion transmission tube temperature is 280-320°C, the atomization temperature is 300-340°C, the fragmentation mode is high-energy collision fragmentation, the resolution is 30000 (200 m / z), and the scanning mode is parallel reaction monitoring.

2. A method of detecting infliximab concentration according to claim 1, wherein, The method for obtaining the standard curve is as follows: The calibrators are configured to have a series of concentrations and are added with an internal standard, and the standard curve is drawn by taking the calibrator concentration as the independent variable xi and taking the ratio of the peak area of the corresponding concentration calibrator to the internal standard as the dependent variable yi through liquid chromatography-mass spectrometry determination.

3. A method of detecting infliximab concentration according to claim 2, wherein, The linear range of the standard curve is 0.391-100 μg / mL.

4. A kit for detecting infliximab concentration, characterized by, The kit comprises an internal standard, a working solution, a diluent, enzyme digestion reagent, a termination solution, a mobile phase, calibrators, and quality control products; The internal standard is a stable isotope-labeled infliximab specific peptide segment. The working solution comprises a working solution A containing a buffer salt and a working solution B containing a denaturant; The enzyme digestion reagent is a lyophilized powder containing trypsin; The termination solution is an aqueous formic acid solution; The mobile phase comprises an aqueous formic acid solution mobile phase A and an acetonitrile solution containing formic acid mobile phase B; The dilution solution comprises pure water dilution solution A and dilution solution B containing bovine serum; Isotopically labeled versions of the infliximab-specific peptide segment are SINSATHYAESVK (K→K 13 C6 15 N2); the concentration of the infliximab-specific peptide segment is 10-40 ng / mL; The buffer salt is ammonium bicarbonate buffer salt; the concentration of the buffer salt is 25-100 mM; The denaturant is trifluoroethanol; the volume concentration of the denaturant is 20%-60%; The concentration of formic acid in the termination solution is 10%-50%; or, The mobile phase A is an aqueous solution containing 0.1-0.2% formic acid; or, The mobile phase B is an acetonitrile solution containing 0.1-0.2% formic acid.

5. The kit for detecting the concentration of infliximab according to claim 4, wherein, The volume ratio of the sample to the working solution, the enzyme digestion reagent, the termination solution in the kit is 1:(5-10):(7.5-15):(0.5-1); The dosage form of at least one of the internal standard, the calibrator and the quality control is a lyophilized product.

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