Mass spectrometric detection method for enriching serum human insulin by using specific antibody immunomagnetic beads

Abstract

The invention relates to the field of medical detection, in particular to a mass spectrometric detection method for enriching serum human insulin by using specific antibody immunomagnetic beads. According to the present invention, the human insulin in the serum is subjected to immune enrichment by using the magnetic beads coated with the insulin specific antibody, and the concentration level of the human insulin in the serum is accurately determined based on the accurate quantification technology of the isotope dilution liquid chromatography-tandem mass spectrometry platform in combination with the bracket method quantification mode. The pretreatment method provided by the invention is simple and convenient to operate, the absolute recovery rate is excellent, the sensitivity requirement is met, and the mass spectrometry method is an isotope diluent liquid chromatography-tandem mass spectrometry method capable of eliminating interference of structural analogues.

Description

Technical Field

[0001] The invention relates to the field of medical detection, and in particular to a mass spectrometry detection method for serum human insulin enriched with specific antibody immunomagnetic beads. Background Art

[0002] Human insulin is a polypeptide hormone secreted by pancreatic B cells, consisting of 51 amino acids and a molecular weight of 5808. Insulin is synthesized by pancreatic B cells in the form of single-chain preproinsulin, which is then immediately decomposed into proinsulin. Under the action of specific proteases, proinsulin is decomposed into insulin and C-peptide and enters the blood circulation. The main physiological function of serum insulin is to regulate the metabolism of sugar, fat, and protein. It is the only hormone in the body that lowers blood sugar. Absolute or relative deficiency of insulin secretion will lead to increased blood sugar. Therefore, human serum insulin content is one of the important means for clinical diagnosis of diabetes. Clinically, glucose-insulin release test is often used to monitor insulin release. Patients take glucose orally to stimulate pancreatic B cells to release insulin. By measuring the serum / plasma insulin levels at fasting and 0.5 hours, 1 hour, 2 hours, and 3 hours after taking sugar, the reserve function of pancreatic B cells can be understood, which is also helpful for the classification of diabetes and guiding treatment. The peak of insulin release in normal people occurs within 1 hour, and the peak value is 5-10 times the basal value. Type 1 diabetes is severely insulin deficient, with no significant increase in postprandial insulin. In the early stages of type 2 diabetes, fasting insulin levels are slightly higher or normal, but tend to decrease in the late stages. The peak of postprandial insulin secretion is often delayed for 2-3 hours. In the late stages, due to pancreatic B cell failure, the results are similar to those of type 1 diabetes. Therefore, a reliable insulin quantification method is of great significance for the diagnosis and treatment of diabetes.

[0003] At present, the quantitative methods for insulin are mainly divided into immunoassay and chromatographic analysis. Immunoassay includes enzyme-linked immunosorbent assay, chemiluminescent immunoassay, radioimmunoassay and chip immunoassay, etc.; chromatographic analysis includes high-performance liquid chromatography combined with ultraviolet detection, micellar electrokinetic capillary chromatography, liquid chromatography-tandem mass spectrometry, etc. Enzyme-linked immunosorbent assay and chemiluminescent immunoassay are the most common immunoassay methods for clinical detection of insulin. They have the advantages of high sensitivity, high throughput and high automation, but their specificity is poor, they are prone to cross-reactions, and they cannot distinguish between human insulin, insulin analogs and proinsulin. In addition, the cross-reactivity of exogenous insulin (insulin analogs) varies greatly in different assays, resulting in poor accuracy of insulin detection and difficulty in standardization.

[0004] Liquid chromatography-tandem mass spectrometry has the characteristics of high sensitivity and high specificity, and can overcome problems such as cross-reaction of analogs in immunological methods. Among them, isotope dilution liquid chromatography-tandem mass spectrometry has the highest traceability level, and the measured value can be directly traced back to SI units. At present, this technology is considered to be the first reference measurement principle for the establishment of reference measurement procedures in laboratory medicine standardization. The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Insulin Analysis Standardization Working Group clearly proposed that the reference method for human insulin detection should be established based on isotope dilution liquid chromatography-tandem mass spectrometry.

[0005] At present, there are two methods for liquid chromatography-mass spectrometry detection of human insulin: 1. "Bottom-up", that is, quantitative detection of characteristic peptides after enzymatic hydrolysis of insulin; this method synthesizes isotope-labeled insulin B chain and prepares it into a solution as an internal standard; prepares insulin standard and insulin sample solutions; takes insulin standard solution and sample solution and adds isotope-labeled insulin B chain respectively, mixes them, and then uses dithiothreitol reduction and iodoacetamide solution alkylation to decompose insulin in standard solution and sample solution into insulin B chain, and then performs mass spectrometry analysis after solid phase extraction; the method sensitivity LOQ can be as low as 12.4ng / g. 2. "Top-down", that is, quantitative detection of whole insulin; this method makes the sample undergo solid phase extraction (SPE) and high performance liquid chromatography (HPLC) enrichment and purification, and the amount of one or more ions determined by tandem mass spectrometry is used to determine the amount of insulin in the sample. The method LLOQ is 3μIU / mL, about 0.1ng / g.

[0006] The pretreatment methods for mass spectrometry detection of human insulin in biological samples mainly include protein precipitation combined with solid phase extraction, immune solid phase extraction, and immunomagnetic bead enrichment. The LOD of protein precipitation combined with mixed anion solid phase extraction for mass spectrometry quantitative methods of insulin in human plasma and five structurally similar methods is 50pg / mL; among the five pretreatment methods (protein precipitation combined with mixed anion solid phase extraction, ultrafiltration combined with mixed anion solid phase extraction, ultrafiltration combined with enzyme-linked immunoaffinity plate, protein precipitation mixed anion solid phase extraction combined with immunoaffinity column, protein precipitation ultrafiltration combined with enzyme-linked immunoaffinity plate), the LOQ of insulin after ultrafiltration combined with enzyme-linked immunoaffinity plate pretreatment is the lowest, which is 0.5ng / g, and the absolute recovery rate of this pretreatment is 22.6%.

[0007] In terms of analytical methods, the "bottom-up" detection of insulin requires enzymatic hydrolysis and reduction before it is processed. The steps are cumbersome and time-consuming, with low absolute recovery and detection sensitivity, which cannot meet the needs of clinical sample detection. The "top-down" detection of insulin requires a high recovery rate in the pretreatment to improve the detection sensitivity because insulin has a large molecular weight and low ionization efficiency at the mass spectrometer end. In addition, there is the problem of being unable to distinguish it from its structural analog lispro insulin. In the pretreatment, the protein precipitation combined with solid phase extraction pretreatment method is cumbersome, with a low absolute recovery rate, and the immunoaffinity column is expensive. Few of the currently reported insulin mass spectrometry quantitative methods have a sensitivity that can fully meet the needs of clinical sample detection. Summary of the invention

[0008] In view of this, this method aims to establish an isotope dilution liquid chromatography-tandem mass spectrometry method with simple pretreatment method, excellent absolute recovery rate, and sensitivity requirements, and the mass spectrometry method can exclude the interference of structural analogues.

[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0010] The present invention provides a method for detecting insulin in serum, comprising the following steps:

[0011] Step 1: Take serum, human insulin international standard material NIBSC11 / 212 and isotope internal standard (4-[D10]leucine insulin) to prepare blank matrix, standard working solution and internal standard working solution;

[0012] Step 2: Mix the standard working solution, the internal standard working solution and the blank matrix to prepare a standard solution;

[0013] Taking a sample to be tested, mixing it with the internal standard working solution and the blank matrix to prepare a sample solution;

[0014] Step 3: The standard solution and the sample solution are pre-treated and analyzed by isotope dilution liquid chromatography tandem mass spectrometry. The present invention uses the bracket method commonly used in the reference measurement procedure to process and calculate the results. According to the sample peak area ratio, the mass of the sample and the internal standard, the insulin content in the sample to be tested is obtained, and the sample concentration is calculated according to the following formula:

[0015]

[0016] Where:

[0017] CS——sample measurement result, in nanomoles per liter (nmol·L-1);

[0018] S MH——mass of standard solution added to high standard;

[0019] SC——concentration of standard solution;

[0020] I MH——The mass of internal standard solution added to high standard;

[0021] S ML——mass of standard solution added to low standard;

[0022] I ML——The mass of internal standard solution added to the low standard;

[0023] RH – response ratio of analyte to internal standard in high standard;

[0024] RL - Response ratio of analyte to internal standard in low standard

[0025] I MS – the mass of the internal standard solution in the sample;

[0026] WS - the mass of the sample added in the measurement;

[0027] DS——density of the sample to be tested;

[0028] M – molecular weight of the analyte;

[0029] P——Purity of standard.

[0030] In some specific embodiments of the present invention, the pretreatment in step 3 includes: taking the magnetic bead suspension and letting it stand to discard the supernatant, washing, mixing with the standard solution and the sample solution respectively, incubating, letting it stand to discard the supernatant, washing the magnetic beads, eluting, re-dissolving, and centrifuging to collect the supernatant.

[0031] In some specific embodiments of the present invention, the volume ratio of the standard solution to the magnetic bead suspension is 500:125;

[0032] The volume ratio of the sample solution to the magnetic bead suspension is 500:125;

[0033] In some specific embodiments of the present invention, the washing is performed using a PBS solution and water for one time each; the concentration of the PBS solution is 10 mM;

[0034] The volume ratio of the standard solution or the sample solution to the PBS solution is 550:0.5 or 500:0.5;

[0035] The volume ratio of the standard solution or the sample solution to the water is 550:0.5 or 500:0.5;

[0036] The cleaning time is 2 minutes.

[0037] In some specific embodiments of the present invention, the incubation condition is 25±5° C. for 30 min; and the standing time is 5 min.

[0038] In some specific embodiments of the present invention, the eluent used for elution is an aqueous solution with a volume percentage of 30% acetonitrile and 1% acetic acid;

[0039] The elution was performed twice, each time for 5 minutes;

[0040] The volume ratio of the standard solution or the sample solution to the eluent is 550:100 or 500:100.

[0041] In some specific embodiments of the present invention, the re-dissolving solution used for the re-dissolving is an aqueous solution with a volume percentage of 20% acetonitrile and 0.1% formic acid;

[0042] The volume ratio of the standard solution or the sample solution to the reconstitution solution is 550:75 or 500:75; and / or

[0043] The centrifugal condition is centrifugation at 15000 r / min for 10 min.

[0044] In some specific embodiments of the present invention, the pretreatment is: take 125 μL of magnetic bead suspension in a low protein adsorption centrifuge tube, place it on a magnetic plate, absorb the supernatant, add 0.5 mL of 10 mM PBS solution, mix for 2 minutes, place it on a magnetic plate, absorb the supernatant, add 0.5 mL of water, mix for 2 minutes, place it on a magnetic plate, and absorb the supernatant; add 550 μL of Q1 sample to which the internal standard has been added; incubate at 25±5°C for 15 to 60 minutes, place it on a magnetic plate, and take out the supernatant after 5 minutes; add 0.5 mL of 10 mM PBS solution to the centrifuge tube to wash the magnetic beads for 1 minute, and absorb the supernatant again; add 0.5 mL of water, mix for 2 minutes, place it on a magnetic plate, and absorb the supernatant; add 100 μL of 30% acetonitrile 1% acetic acid aqueous solution, mix well, and let it stand for 5 minutes, elute twice and combine the eluate, blow dry with nitrogen, and 75 μL The supernatant was reconstituted with 20% acetonitrile and 0.1% formic acid aqueous solution, centrifuged at 15000r / min for 10min, and the supernatant was transferred from a magnetic plate to a pretreated 96-well sample plate for liquid-mass analysis.

[0045] In some specific embodiments of the present invention, the mobile phase A of the chromatography is acetonitrile, and the mobile phase B is 0.2% formic acid aqueous solution; the gradient elution conditions are as follows:

[0046]

[0047] The chromatographic analysis time was 10 min, the injection volume was 20 μL, the flow rate was 0.2 mL / min, the column temperature was 40° C., and the autosampler temperature was 10° C.

[0048] In some specific embodiments of the present invention, the mass spectrometer is analyzed using electrospray ionization (ESI) positive ion mode and multiple reaction monitoring (MRM) mode, capillary voltage 2.84 kV, ion source temperature: 150 ° C, desolvation gas temperature: 550 ° C, desolvation gas flow: 1000 L / Hr;

[0049] The ion pairs used for quantification were detected as m / z 1162.223>225.932 (insulin), m / z1170.340>225.932 (insulin internal standard);

[0050] The ion pair for qualitative analysis was detected as m / z 1162.223>135.97 (insulin), and the ion pair for monitoring insulin lispro was m / z 1162.66>217.21 (insulin lispro).

[0051] In some schemes, pretreatment enrichment can be achieved by other types of magnetic particles such as ion exchange magnetic particles; in some schemes, one or more ions measured include insulin parent ions of 1162, 969, and 1453, and daughter ions include 226, 217, and 136; in some schemes, the mass spectrometer used, such as a high-resolution mass spectrometer, may achieve the same quantitative performance.

[0052] In some specific embodiments of the present invention, the chromatography uses a Fenome C18 (2.1*100mm, 2.6μm) chromatography column.

[0053] In some specific embodiments of the present invention, the blank matrix is ​​prepared by mixing human serum with an antibody magnetic particle suspension at a volume ratio of 500:125, incubating at 25±5°C for 15 min to remove insulin from the serum, letting it stand, collecting the supernatant to detect insulin, and a detection value of zero can be used as a blank matrix. If there is a signal value, the supernatant is taken and the above steps are repeated; and / or

[0054] The preparation method of the standard working solution is: take human insulin international standard material NIBSC11 / 212, prepare a stock solution by weighing with 0.1% formic acid 0.1% BSA solution, then dilute the intermediate dilution solution with 0.1% formic acid 0.1% BSA solution, and further dilute to obtain the standard working solution, the common working solution concentration is about 100 ng / g and 10 ng / g; and / or

[0055] The preparation method of the internal standard working solution is: take the isotope internal standard (4-[D10]leucine insulin) and dissolve it by weighing method using 0.1% formic acid and 0.1% BSA to obtain a stock solution, and dilute it to obtain the internal standard working solution. The commonly used internal standard working solution concentrations are 100 ng / g and 10 ng / g.

[0056] The present invention uses insulin-specific antibody-coated magnetic beads to immunoenrich human insulin in serum, and is based on an accurate quantitative technology of an isotope dilution liquid chromatography tandem mass spectrometry platform, combined with a bracket method quantitative method, to accurately determine the concentration level of human insulin in serum. The pretreatment method provided by the present invention is simple to operate, has an excellent absolute recovery rate, and meets sensitivity requirements, and the mass spectrometry method can eliminate the interference of structural analogs by isotope dilution liquid chromatography tandem mass spectrometry. In the mass spectrometry conditions of the detection method provided by the present invention, lispro insulin monitoring ion pairs (m / z 1162.66>217.21 (lispro insulin)) are added to effectively identify whether there is lispro insulin interference; the antibody coupled to the magnetic beads is an insulin-specific antibody, which has a greatly improved enrichment efficiency for insulin compared with non-specific antibodies; in the sample pre-treatment method, blank human serum is selected as the standard solution diluent in the preparation of the calibration solution to effectively reduce the matrix effect; a 96-well receiving plate is soaked in a 3% BSA solution for pre-saturation to reduce the non-specific adsorption of the target; the present invention also optimizes and screens the amount of magnetic particle suspension, room temperature incubation time, eluent organic solvent and acid content; in the liquid phase conditions, the type of mobile phase A, the type and content of mobile phase B modifier, and the chromatographic column model are optimized to make the detection method of the present invention more conducive to actual detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0058] Figure 1 Indicates the choice of chromatographic column;

[0059] Figure 2 Indicates the selection of organic phase; wherein A shows the chromatogram of the organic phase being methanol; B shows the chromatogram of the organic phase being acetonitrile;

[0060] Figure 3 The chromatograms are shown with acetonitrile as the organic phase and injected under different aqueous phase conditions;

[0061] Figure 4 The figure shows the optimization of mobile phase gradient; A is the chromatographic peak of gradient 1; B is the chromatographic peak of gradient 2;

[0062] Figure 5The figure above shows the mass spectrometry response of insulin and internal standard in samples under different magnetic particle dosages; the figure below shows the mass spectrometry response of internal standard in samples under different magnetic particle dosages;

[0063] Figure 6 The left figure shows the effect of equilibrium time on the insulin peak area, and the right figure shows the effect of equilibrium time on the internal standard peak area.

[0064] Figure 7 The left figure shows the response of insulin and internal standard under different cleaning solutions; the right figure shows the response of insulin under different cleaning solutions;

[0065] Figure 8 The insulin response is shown in Figure 1. The upper figure is the chromatogram after washing once with PBS solution; the lower figure is the chromatogram after washing twice with PBS solution.

[0066] Fig. 9 The responses of insulin and internal standard in different cleaning methods are shown; the upper figure is washed twice with PBS; the lower figure is washed once with PBS and once with water;

[0067] Fig.10 The responses of insulin and internal standard under different organic solvent contents in the eluent are shown;

[0068] Fig.11 The responses of insulin and internal standard under different acid contents of eluent are shown;

[0069] Fig.12 The standard curve linear diagram is shown; the upper diagram is the deviation diagram of each concentration point from the theoretical value; the lower diagram is the standard curve diagram;

[0070] Fig.13 Shown are chromatograms of 10 structural analogue standard solutions;

[0071] Fig.14 The chromatograms of 10 structural analogs spiked samples after pretreatment are shown;

[0072] Fig.15 Shows the insulin peak area in samples under different pretreatment conditions. DETAILED DESCRIPTION

[0073] The present invention discloses a mass spectrometry detection method for enriching serum human insulin with specific antibody immunomagnetic beads. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described by preferred embodiments, and relevant personnel can obviously change or appropriately change and combine the method and application described herein without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0074] The present invention is based on magnetic bead immunoenrichment pretreatment combined with isotope dilution liquid chromatography tandem mass spectrometry platform and is used to accurately determine the human insulin content in serum.

[0075] Materials and Methods

[0076] Material:

[0077] Human insulin international standard material NIBSC11 / 212 is derived from the World Health Organization International Standard (WHO International Standard). The mass of each bottle is 9.19 mg ± 0.05 mg. 4-[D10] Leucine-human insulin (isotope internal standard) was purchased from the Japan Peptide Research Institute. Methanol and formic acid were purchased from Merck, and the experimental water was homemade by the Millipore pure water system of this laboratory. The standard substance and internal standard were diluted with 0.1% formic acid 0.1% BSA solution, dispensed in low protein adsorption tubes, and taken individually to avoid repeated freezing and thawing. BSA was purchased from SIGMA, item number A9647.

[0078] Main instruments and consumables:

[0079] The instrument used in the present invention is an ACQUITY UPLC I-Class chromatogram tandem with a TQ-S mass spectrometer, equipped with a chromatographic column Fenomi C18 (2.1×100, 2.6 μm). A Mettler XPE205 electronic balance was used for the preparation of standard solutions and the weighing of serum samples and calibration samples. The anti-human insulin specific antibody magnetic particle suspension was derived from the insulin in-production kit of Antu Biotechnology Co., Ltd., batch number 20230928.

[0080] The raw materials and reagents used in the mass spectrometry detection method for enriching serum human insulin with specific antibody immunomagnetic beads provided by the present invention can all be purchased from the market.

[0081] The present invention will be further described below in conjunction with embodiments:

[0082] Example 1 Pretreatment method optimization:

[0083] Optimization of the dosage of magnetic particle suspension:

[0084] Different amounts of magnetic microparticle suspension were used to enrich 500 μL samples (human serum), and the serum insulin content before and after enrichment was determined by immunoassay, and the residual amount of serum insulin after enrichment was calculated. The results are shown in Table 1. As the amount of magnetic beads increased, the residual rate of the supernatant gradually decreased, and 125 μL of magnetic beads could almost completely enrich the target. Figure 5 The results are the mass spectrometry responses of insulin and internal standard in the sample after magnetic bead enrichment. The results show that the use of 125μL, 250μL, and 500μL magnetic beads has little effect on the target peak and internal standard peak area. The optimization result of the magnetic particle dosage is that the volume ratio of magnetic particle dosage: sample is 125:500.

[0085] Table 1 Residual insulin in serum after enrichment with different amounts of magnetic microparticles

[0086] Sample No. 62.5μL 125μL 250μL 500μL BY(10ng / g) 6.4％ 2.5％ 1.4％ 0.7％ Q1(0.4ng / g) 3.7％ 0.6％ 0.2％ 0.1％ Q2 (7.5ng / g) 5.7％ 1.5％ 0.6％ 0.3％ Q3(2ng / g) 3.9％ 0.7％ 0.3％ 0.2％

[0087] Comparison of cleaning methods before enrichment:

[0088] 500μL blank matrix was used to prepare standard solution, sample Q3 (concentration 7.1ng / g), and sample Q7 (concentration 0.90ng / g), and enriched with 125μL magnetic beads, respectively, with 2 parallels each; 3 groups of experiments were set up: 500μL PBS washing before enrichment, 500μL PBS washing and then 125μL PBS resuspension, and no washing. The response after PBS washing and resuspension was slightly higher, but the CV was larger. Considering that the magnetic bead antibody may have a loose binding part that is easy to cause shedding during the pretreatment process, PBS washing before enrichment was selected.

[0089] Table 2 Effect of different cleaning methods on response and CV

[0090]

[0091]

[0092] Comparison of washing times before enrichment:

[0093] 500μL blank matrix was used to prepare standard solution, sample Q5 (concentration 0.72ng / g), and sample Q7 (concentration 0.90ng / g), and enriched with 125μL magnetic beads, respectively, with 2 parallels each; 3 groups of experiments were set up before enrichment, with 500μL PBS and 500μL water for 1, 2, and 3 cycles of washing. There was no significant difference in response with different washing times, and the CV between tubes tended to increase with increasing the number of washing times. PBS and water were used for washing once in turn.

[0094] Table 3 Effect of different cleaning times on response and CV

[0095]

[0096] Optimization of incubation time:

[0097] 500 μL blank matrix was used to prepare standard solution (1 ng / mL), sample Q3 (concentration 7.1 ng / g), and sample Q7 (concentration 0.90 ng / g), and enriched with 125 μL magnetic beads, respectively, with 2 parallels each; the equilibrium time for magnetic particle enrichment at room temperature was set to 15 min, 30 min, 60 min, and 90 min for 4 groups of experiments. The results are shown in Figure 6 As shown: the responses of insulin and internal standard were slightly different in the incubation time range of 15 min to 90 min, and the equilibrium condition was selected as room temperature reaction for 30 min.

[0098] Selection of washing solution after magnetic bead enrichment:

[0099] 500 μL of blank matrix prepared standard solution (1 ng / mL), sample Q4 (concentration 12.2 ng / g), and sample Q7 (concentration 0.90 ng / g) were enriched with 125 μL of magnetic beads, and then the magnetic beads were washed with 0.5 mL of PBS buffer (PH7.4), TRIS-HCl buffer (PH7.4), and purified water, respectively, to compare the effects of different washing solutions. Figure 7 It showed that PBS solution had the highest washing response; Figure 8 Shown PBS solution washed twice ( Figure 8 Chromatogram below) than cleaning once ( Figure 8 The interfering peaks at the back end of the chromatogram (upper) are significantly reduced; Fig. 9 Shown PBS solution washed twice ( Fig. 9 chromatogram) and PBS solution and water once each ( Fig. 9 Considering that direct elution after washing with PBS solution may cause phosphate contamination of the instrument, PBS solution and water were selected for washing once each.

[0100] Optimization of organic solvent content in elution solvent:

[0101] The same standard solution and sample were eluted with acetonitrile 5% acetic acid aqueous solution with organic solvent content of 0%, 5%, 10%, 20%, and 30%, 75μL elution twice, the eluate was combined, and centrifuged at 15000rpm. The results are as follows Fig.10 As shown in Table 4, the response was highest at 30% acetonitrile content. The acetonitrile content (30%-70%) was further optimized. The results are shown in Table 4. The response was highest at 30% acetonitrile content. Considering that the high organic phase may elute more impurities, the acetonitrile content was selected to be 30%.

[0102] Table 4 Effect of different acetonitrile dosages on response and CV

[0103]

[0104]

[0105] Optimization of acid content in elution solution:

[0106] According to the above pretreatment steps, mixed serum samples 1 and 2 were processed separately, 2 in parallel; 30% acetonitrile aqueous solution with acetic acid content of 0%, 0.1%, 1%, 2%, and 5% was used for elution, 75 μL was used for elution twice, the eluate was combined, and centrifuged at 15000rpm; and standard working solution was prepared with eluate of different acid content for detection. The results are shown in Fig.11 As shown, the response is less affected by the acid content, and 1% acetic acid 30% acetonitrile solution is selected as the eluent.

[0107] Example 2 Liquid Phase Condition Optimization Process

[0108] Column selection:

[0109] The sample pre-treated under the optimized conditions provided in Example 1 has a significant chromatographic peak after separation by a Fenome C18 (2.1*100mm, 2.6μm) chromatographic column. The Fenome C18 (2.1*100mm, 2.6μm) chromatographic column is selected. Figure 1 shown.

[0110] Choice of mobile phase A (organic phase):

[0111] When acetonitrile is used as mobile phase A, the target peak and the interfering peak are more separated and the back-end interference is less. Acetonitrile is selected as the organic phase. Figure 2 shown.

[0112] Selection and content of mobile phase B (aqueous phase) modifier:

[0113] Acetonitrile was used as the organic phase and the samples were injected under different aqueous phase conditions.

[0114] When mobile phase B is 0.2% formic acid aqueous solution, the response is the highest. 0.2% formic acid aqueous solution is selected as the aqueous phase. Figure 3 shown.

[0115] Mobile phase gradient optimization: Gradient optimization was performed by preparing sample solution by magnetic particle enrichment.

[0116] Table 5 Gradient 1

[0117]

[0118] Table 6 Gradient 2

[0119]

[0120] like Figure 4As shown, after gradient optimization, the separation between the target peak and the back-end interference peak increases, the target peak signal-to-noise ratio S / N192 increases to S / N422, and gradient 2 is selected.

[0121] Example 3

[0122] Sample pretreatment method

[0123] Preparation of blank matrix (blank real human serum): human serum was added to the antibody magnetic particle suspension at a ratio of 500:125 (V / V), incubated at 25±5℃ for 15 min to remove insulin in the serum, and placed on a magnetic plate. The supernatant was tested for insulin on the immune platform Autobio A2000 and the mass spectrometry platform (this method) respectively. If the detection value is zero, it can be used as a blank matrix. If there is a signal value, the supernatant is treated once according to the above steps.

[0124] Preparation of standard working solution: Human insulin international standard material NIBSC11 / 212 is used to prepare the stock solution by weighing method with 0.1% formic acid 0.1% BSA solution, and then the intermediate dilution solution is diluted with 0.1% formic acid 0.1% BSA solution, and further diluted to obtain the insulin working solution. The commonly used working solution concentrations are about 100 ng / g and 10 ng / g.

[0125] Preparation of internal standard working solution: The isotope internal standard (4-[D10]leucine insulin) was dissolved by weighing method with 0.1% formic acid and 0.1% BSA to obtain a stock solution, which was then diluted to obtain the insulin internal standard working solution. The commonly used internal standard working solution concentrations are approximately 100 ng / g and 10 ng / g.

[0126] This method uses triple isotope dilution mass spectrometry, i.e. bracket method, for quantification. Before formal analysis and determination, a pre-experimental determination sample is added to obtain the approximate concentration of insulin. According to the initial concentration, the appropriate standard and internal standard working solution (100 ng / g or 10 ng / g) are determined, the internal standard volume is 50 μL, and according to the formula C1*m1=C2*m2 (C1: internal standard working solution concentration, ng / g; m1: internal standard working solution mass, about 50 mg; C2: sample insulin initial concentration, ng / g; m2: sample mass, mg), the appropriate sample dosage is calculated so that the ratio of insulin mass in the sample to internal standard mass is approximately 1:1.

[0127] The prepared standard working solution and internal standard working solution were weighed in the designed amount according to the standard to internal standard mass ratio of 0.9 and 1.1, and their accurate mass was weighed on the balance. The concentration of insulin in the sample was calculated based on the sample peak area ratio, the mass of the sample and the internal standard.

[0128] Take the sample with an initial concentration of 2.0ng / g as an example:

[0129] Preparation of calibration solution: Weigh an appropriate amount of 10 ng / g standard working solution, add 50 μL of internal standard working solution of the same concentration, add blank matrix to 550 μL, mix well, and perform the same pretreatment on the same sample (pretreatment according to the optimized conditions provided in Example 1).

[0130] Preparation of sample solution: According to the initial concentration of the sample to be tested (2.0 ng / g), weigh an appropriate amount of the sample to be tested (250 mg), add 50 μL of internal standard working solution; add blank matrix to the sample to make up to 550 μL, mix well and wait for pretreatment;

[0131] Pretreatment method: Take 125μL of magnetic bead suspension in a low protein adsorption centrifuge tube, place it on a magnetic plate, remove the supernatant, and add 0.5mL 10mM PBS solution, mix for 2 minutes, let it stand on the magnetic plate, absorb the supernatant, add 0.5mL water, mix for 2 minutes, let it stand on the magnetic plate, absorb the supernatant; add 550μL of the pre-prepared calibration solution to be processed and the sample solution to be tested respectively; incubate at 25±5℃ for 30 minutes, let it stand on the magnetic plate, and take out the supernatant after 5 minutes; add 0.5mL10mMPBS solution to the centrifuge tube to wash the magnetic beads for 1 minute, and absorb the supernatant again; add 0.5mL water, mix for 2 minutes, let it stand on the magnetic plate, and absorb the supernatant; add 100μL30% acetonitrile 1% acetic acid aqueous solution, mix well and let it stand for 5 minutes, elute twice and combine the eluate, blow dry with nitrogen, re-dissolve with 75μL20% acetonitrile 0.1% formic acid aqueous solution, centrifuge at 15000r / min for 10 minutes, transfer the supernatant from the magnetic plate to the pre-treated 96-well sample plate for liquid-mass analysis.

[0132] Pretreatment method of 96-well sample injection plate: soak in 3% BSA solution for 24 hours, wash with ultrapure water and dry for use.

[0133] Liquid phase method:

[0134] Mobile phase A was acetonitrile, and mobile phase B was 0.2% formic acid aqueous solution; the gradient elution conditions were shown in Table 7, the entire analysis time was 10 min, the injection volume was 20 μL, the flow rate was 0.2 mL / min, the column temperature was 40° C., and the autosampler temperature was 10° C.

[0135] Table 7 Insulin liquid phase conditions

[0136] Time (min) Flow rate (mL / min) ％B 0 0.2 80 1.0 0.2 80 6.5 0.2 60 7.0 0.2 60 7.5 0.2 2 8.0 0.2 2 8.2 0.2 80 10 0.2 80

[0137] Mass spectrometry method:

[0138] Electrospray ionization (ESI) positive ion mode and multiple reaction monitoring (MRM) mode were used for analysis, capillary voltage was 2.84 kV, ion source temperature was 150°C, desolvation gas temperature was 550°C, and desolvation gas flow was 1000 L / Hr. The ion pairs for quantitative detection were m / z 1162.223>225.932 (insulin), m / z 1170.340>225.932 (insulin internal standard); the ion pairs for qualitative detection were m / z 1162.223>135.97 (insulin), and the ion pairs for monitoring insulin lispro (m / z 1162.66>217.21 (insulin lispro)).

[0139] Example 4 Method Performance Verification

[0140] Linear:

[0141] Blank human serum (TRINA, batch number 23ESP290685) (purchased from TRINA, batch number 23ESP290685) was selected as the matrix, and the standard working solution was added to prepare the concentrations of 0.051, 0.105, 0.201, 0.480, 1.99, 3.84, 7.80, 19.37, 29.73, and 39.70 ng / g sample, respectively. The same amount of internal standard solution was added to each sample, and the sample was pretreated (pretreated according to the optimized conditions provided in Example 1). Each sample was injected twice continuously, and the response ratio mean (response) and the mass ratio were plotted to obtain a linear equation.

[0142] like Fig.12 As shown, the linear equation is: y=0.851346x+0.00297604, R2=0.999, and the linearity is good in the range of 0.051ng / g-39.7ng / g, meeting the linearity requirement (R2≥0.999).

[0143] Limit of detection and limit of quantification (LOD&LOQ):

[0144] A series of dilution samples (0.0244, 0.0383, 0.0488, 0.0737 ng / g) were prepared with blank human serum (TRINA, batch number 23ESP290685) as the matrix to assess the analytical sensitivity of the method. Three samples were processed in parallel for each level test sample. 500 μL of each sample was pre-treated (pre-treated according to the optimized conditions provided in Example 1) and then subjected to mass spectrometry analysis to calculate the deviation and variation from the theoretical value. The concentration when the average signal-to-noise ratio (S / N) was ≥3 was used as the detection limit of the method; the lowest concentration sample that met the conditions of S / N ≥10, total sample variation CV ≤20% (imprecision), and the deviation between the mean of the concentration determination and the theoretical concentration <15% (accuracy) was used as the quantitative limit of the method. The concentration of 0.0244 ng / g is now used as the detection limit of the method, and 0.0383 ng / g is used as the quantitative limit of the method.

[0145] Table 8 LOD & LOQ

[0146]

[0147] Spike recovery rate:

[0148] A low-value sample of 0.4 ng / g was selected as the basic spiked sample, and the standard working solution was added at concentrations of 1.000 ng / g, 5.07 ng / g, and 10.063 ng / g, respectively, and then pre-treated (pre-treated according to the optimized conditions provided in Example 1). The spiked recovery experiment tested 2 batches, each with 3 parallels, and the corresponding spiked recoveries were 99.5%, 100.2%, and 101.7%.

[0149] Table 9 Spiked recovery results

[0150]

[0151] Matrix Effects:

[0152] The matrix mixing experiment was used to investigate the relative matrix effect, and 5 serum samples were randomly selected. Five matrix samples were prepared, including matrix standard solution (blank human serum matrix, insulin concentration was 1 ng / g), serum matrix sample (serum sample), serum sample and matrix standard solution 20:80 mixture (V / V), serum sample and matrix standard solution 1:1 mixture (V / V), serum and matrix standard solution 80:20 mixture (V / V), and the same amount of internal standard solution was added. After sample pretreatment (pretreatment was performed according to the optimized conditions provided in Example 1), the samples were injected and analyzed, and two injections were made continuously. The mean response of the matrix standard solution is A, the mean response of the serum matrix sample (serum sample) is B, and the mean response of the 20:80 mixture of serum sample and matrix standard solution is C, then its relative matrix effect = [C-(0.8A+0.2B)] / (0.8A+0.2B)×100%; the mean response of the 1:1 mixture of serum sample and matrix standard solution is D, then its relative matrix effect = [D-(0.5A+0.5B)] / (0.5A+0.5B)×100%; the mean response of the 80:20 mixture of serum and matrix standard solution is E, then its relative matrix effect = [E-(0.2A+0.8B)] / (0.2A+0.8B)×100%.

[0153] The average relative matrix effect was evaluated by the mean deviation between the measured response value of the mixture and the theoretical response value. The results in Table 10 show that the average relative matrix response was -2.14% to 2.19%.

[0154] Table 10 Matrix response results

[0155]

[0156]

[0157] The matrix in Table 10 represents the matrix standard solution.

[0158] Interference: Interference experiment refers to verifying whether the common possible interference substances triglycerides, bilirubin and hemoglobin will affect the detection of human insulin, that is, adding high concentrations of the three substances to the sample and measuring the difference in sample concentration before and after addition. In order to avoid the influence of the positive and negative interferences on the results due to the competitive effect, samples with two concentration levels of 0.902ng / g and 9.269ng / g were selected, and high concentrations of hemoglobin, triglycerides and bilirubin were added to the samples, respectively, and the concentrations of the three added were 1000mg / dL, 1000mg / dL, and 20mg / dL, respectively. Each sample was treated with two blank samples with three interfering substances added and no addition, and the samples were pre-treated according to the optimized conditions disclosed in Example 1 and then sampled for quantitative analysis. The concentrations of the samples before and after addition were calculated respectively, and the recovery rate was calculated by the mean to indicate the degree of interference. The results showed that the recovery rate was between 99% and 105%, and the sample detection was not affected by the interference substances of triglycerides, bilirubin and hemoglobin.

[0159] Table 11 Interference test results

[0160]

[0161] Precision:

[0162] Samples at three concentration levels of 0.436ng / g, 2.003ng / g, and 11.449ng / g were selected for precision experiments. Samples at each concentration level were tested for 3 days, with the test repeated 5 times each day. The total variation results of the precision test samples at the three concentrations were 2.98%, 2.93%, and 1.74%, respectively.

[0163] Carryover pollution:

[0164] Considering the concentration range of human insulin in human serum, two groups of high and low value samples were prepared using blank human serum. The concentration of the high value sample was about 40ng / g, and the low value sample was about 50pg / g. 500μL of each sample was taken, and an equal amount of internal standard was added and mixed for pretreatment. The sample solution after pretreatment according to the optimized conditions disclosed in Example 1 was first repeatedly injected with low concentration samples, and then high concentration samples and low concentration samples were alternately injected. The injection order was low value L1, low value L2, high value H, and low value sample L3. Compare the low value second test result L2 and the low value sample test result L3 after the high value sample, and calculate the carryover contamination rate = (L3-L2) / (H-L2)*100%. The results are shown in Table 12, and the carryover contamination rates of the two groups of samples are less than 0.1%.

[0165] Table 12 Carryover contamination test results

[0166]

[0167] Example 5

[0168] 1. In the pretreatment method (pretreatment according to the optimized conditions provided in Example 1), the present invention uses magnetic beads coated with insulin-specific antibodies to enrich insulin in serum, and the enrichment efficiency is close to 100%, and the enrichment effect is excellent; the chemiluminescence immunoreagent is used to measure insulin in the serum before and after enrichment with specific antibody magnetic beads (the antibody is Zhengzhou Imeno Biotechnology Co., Ltd., batch number 2023070301; the magnetic beads are purchased from Merck, item number M9372) and non-specific antibody (the antibody is Zhengzhou Imeno Biotechnology Co., Ltd., batch number 2022073001; the magnetic beads are purchased from Merck, item number M9372) magnetic beads, and the residual insulin in the serum after enrichment is calculated to determine the enrichment effect of different antibody magnetic beads on serum insulin. The results in Table 13 show that the enrichment efficiency of specific antibody magnetic beads is more than 97.5%, which is significantly better than the enrichment effect of non-specific antibody magnetic beads.

[0169] Table 13 Results of different antibody magnetic beads enrichment efficiency

[0170]

[0171] 2. The magnetic bead immunoenrichment process was explored and optimized in detail, making the pre-treatment operation simple and the absolute recovery rate excellent;

[0172] Three different pretreatment methods were compared, and the enrichment effects of different pretreatments were compared by absolute sample recovery. By comparing the absolute recovery, the pretreatment method of enriching serum insulin with insulin-specific antibody magnetic particles was significantly better than the pretreatment method of protein precipitation combined with solid phase extraction.

[0173] Pretreatment method 1: protein precipitation combined with MCX solid phase extraction, blank serum sample 250μL, standard solution and internal standard concentration are 100ng / mL, add 5μL each, mix and add 600μL 1% acetic acid methanol acetonitrile (1:1), vortex mix at 2000rpm for 30min, centrifuge and take all supernatant, add 1.4mL 1% acetic acid aqueous solution in a 5mL centrifuge tube, mix and wait for treatment. MCX solid phase column is activated by 1mL methanol and 1mL water; load, rinse with 1mL 2% acetic acid aqueous solution, 1mL 2% acetic acid methanol solution, and drain; elute with 1.2mL methanol / ammonia (5:1), blow dry with nitrogen, re-dissolve with 75μL 30% acetonitrile 1% acetic acid aqueous solution, and transfer to a 96-well receiving plate. At the same time, 250 μL of blank matrix (without adding standard solution and internal standard) was pretreated, and 5 μL of standard working solution (100 ng / g) was diluted to 75 μL with reconstitution solution (30% acetonitrile 1% acetic acid aqueous solution), and the sample was injected for analysis. The response ratio of the two results was the absolute recovery rate of pretreatment method 1. As shown in Table 14, the absolute recoveries of insulin and internal standard in the sample after protein precipitation combined with MCX solid phase extraction were 43.7% and 41.2%, respectively.

[0174] Pretreatment method 2: 250 μL of blank serum sample, the concentration of standard solution and internal standard is 100 ng / mL, add 5 μL of each and mix well, add 600 μL of 1% acetic acid methanol acetonitrile (1:1), vortex and mix well at 2000rpm for 30 minutes, centrifuge and take all the supernatant, add 0.9mL of 5% ammonia solution in a 5mL centrifuge tube, and mix well. MAX plate: 0.2mL methanol, 0.2mL water; load, rinse: 0.2mL 5% ammonia solution, 0.2mL 5% methanol 1% acetic acid solution, drain; elution: 75 μL 30% acetonitrile 1% acetic acid solution reconstitute, mix well and load on the machine.

[0175] At the same time, 250 μL of blank matrix (without adding standard solution and internal standard) was pretreated, and 5 μL of standard working solution (100 ng / g) was diluted to 75 μL with reconstitution solution (30% acetonitrile 1% acetic acid aqueous solution), and the sample was injected for analysis. The response ratio of the two results was the absolute recovery rate of pretreatment method 2. As shown in Table 15, the absolute recovery rates of insulin and internal standard in the sample after protein precipitation combined with MAX solid phase extraction were 26.3% and 25.8%, respectively.

[0176] Table 14 Absolute recovery of pretreatment method 1

[0177]

[0178]

[0179] Table 15 Absolute recovery of pretreatment method 2

[0180]

[0181] Pretreatment method 3: Pretreatment method developed by the present invention

[0182] The blank matrix was prepared by the sample pretreatment method in Example 1.

[0183] Insulin in serum was enriched and removed by using insulin-specific antibody magnetic particles to prepare blank matrix, 5 μL of standard working solution (100 ng / g, prepared by the standard working solution in the sample pretreatment method in Example 1) was added to 500 μL of blank matrix, and 75 μL of eluent (30% acetonitrile 1% acetic acid aqueous solution) was injected for analysis after pretreatment; at the same time, 500 μL of blank matrix was pretreated (pretreated according to the optimized conditions provided in Example 1), and 5 μL of standard working solution (100 ng / g) was diluted to 75 μL with eluent (30% acetonitrile 1% acetic acid aqueous solution), and injected for analysis. The response ratio of the two results was the absolute recovery rate of pretreatment, as shown in Table 16. The absolute recovery rates of insulin and internal standard in the sample enriched by magnetic particles were 89.9% and 93.4%, respectively. By comparing the absolute recovery rates, the pretreatment method of enriching serum insulin with insulin-specific antibody magnetic particles was significantly better than the pretreatment method of protein precipitation combined with solid phase extraction.

[0184] Table 16 Absolute recovery of magnetic bead enrichment pretreatment

[0185]

[0186] 3. The solvent for preparing the standard substance and internal standard dilution is 0.1% formic acid 0.1% BSA solution.

[0187] It has been verified that the standard solution prepared with 0.1% formic acid aqueous solution showed a gradually decreasing response trend in the injection bottle. Since insulin and internal standards are polypeptide compounds that are easily adsorbed on the tube wall, the standard solution prepared with 0.1% formic acid aqueous solution is also prone to non-specific adsorption in the pipeline after entering the instrument, thus affecting the accuracy of the target. After the standard substance diluent was changed to 0.1% formic acid 0.1% BSA solution, the response of the standard solution on the instrument was significantly improved and stable.

[0188] Table 17 Response of target and internal standard under different standard solution dilutions

[0189]

[0190] 4. Since the structure of insulin drug lispro insulin differs from that of human insulin only in the order of two amino acids, B28 and B29, it is difficult to separate them under LC-MS conditions. During the exploration and development of specific LC-MS methods, it was found that lispro insulin has a specific ion pair m / z 1162.66>217.21 relative to human insulin. Therefore, the characteristic ion pair of lispro insulin (m / z 1162.66>217.21 (lispro insulin)) was added to the insulin LC-MS method to monitor whether there is interference from lispro insulin. The liquid phase conditions and mass spectrometry method were optimized and determined, and a detection method with excellent performance was developed.

[0191] Structural analog monitoring:

[0192] Since the structure of insulin drug lispro insulin is different from that of human insulin only in the order of two amino acids, B28 and B29, the separation degree is poor under liquid chromatography-mass spectrometry conditions. The specific substances are shown in Table 18. Lispro insulin has strong interference on both the qualitative and quantitative ions of insulin. The other 9 substances do not affect the accurate quantification of insulin due to the differences in characteristic ions. Fig.13 , Fig.14 By comparison, it can be seen that after the pretreatment of magnetic particle enrichment, there is no response of lispro insulin in serum in both the qualitative and quantitative ion channels of insulin, indicating that the pretreatment can eliminate the interference of lispro insulin. Therefore, 10 specific interferences can be separated from insulin and internal standards under chromatographic conditions, mass spectrometry conditions or pretreatment conditions, without affecting the accurate quantification of insulin.

[0193] Table 18

[0194]

[0195] 5. The sample injection plate was pre-saturated with a 96-well receiving plate soaked in 3% BSA solution to reduce nonspecific adsorption of the target. The same sample solution was added to a common 96-well plate and a 3% BSA saturated 96-well plate, and the samples were injected continuously on each plate to detect the target and internal standard responses and their changes. The results are shown in Table 19. The peak areas of insulin and internal standard in the sample solution in the 3% BSA saturated 96-well plate were significantly increased and the response was more stable.

[0196] Table 19 Effects of different injection plates on the response of standard substances and internal standards

[0197]

[0198]

[0199] 6. In the sample pretreatment method, the preparation of the calibration solution uses real blank human serum as the standard solution diluent, which reduces the quantitative deviation caused by the matrix effect. The effects of three blank matrix standard substance diluents on sample quantification were compared, namely 3% blank bovine serum albumin solution, blank hormone-free human serum, and blank real human serum. The experiment used three different blank matrix calibration solutions to calibrate the same samples and spiked samples, and calculated the spike recovery rate. The results are shown in Table 20. The spike recovery rate calculated by the blank real human serum calibration solution is about 100%, which is the best choice for the blank matrix.

[0200] Table 20 Effect of different matrix correction solutions on the fixed value

[0201]

[0202] 7. In terms of result processing and calculation, the present invention uses the bracket method commonly used in the reference measurement procedure to process and calculate the results, ensuring the accuracy and reliability of our calculation results;

[0203] 8. After the method was established, the present invention confirmed the performance of the method including magnetic bead enrichment efficiency, absolute recovery rate, linearity, sensitivity, spiked recovery rate, matrix effect, carryover contamination, etc., and all performances were excellent.

[0204] Comparison of magnetic bead enrichment method before and after optimization:

[0205] Before optimization (control group) plan: 500 μL of serum sample was placed in a 2 mL centrifuge tube, 50 μL of internal standard was added, and the mixture was vortexed at high speed for 20 s. 250 μL of magnetic bead suspension was added. After incubation at 500 rpm at room temperature for 1 h, the mixture was placed on a magnetic plate. After 5 min, the supernatant was taken out and the residual amount of the supernatant was analyzed by chemiluminescence immunoassay reagent. The results are shown in Table 21.

[0206] The enrichment scheme of this experiment is the optimized scheme: take 125 μL of magnetic bead suspension into a low-protein adsorption centrifuge tube, place it on a magnetic plate, absorb the supernatant, add 0.5 mL of 10 mM PBS solution, mix for 2 minutes, place it on a magnetic plate, absorb the supernatant, add 0.5 mL of water, mix for 2 minutes, place it on a magnetic plate, and absorb the supernatant; add 500 μL of serum sample and 50 μL of internal standard; incubate at 25±5°C for 15 minutes, place it on a magnetic plate, take out the supernatant after 5 minutes, and detect and analyze the residual amount of the supernatant according to the detection method described in Example 3. The results are shown in Table 22.

[0207] Table 21 Enrichment efficiency of control group

[0208]

[0209] Table 22 Enrichment efficiency of this experiment

[0210]

[0211] Example 6

[0212] The human serum sample S16 was tested for fixed value using the method established by the present invention.

[0213] Preparation of calibration solution: Weigh an appropriate amount of 10 ng / g standard working solution, add 50 μL of internal standard working solution of the same concentration, add blank matrix to 550 μL, mix well, and perform the same pretreatment on the same sample (pretreatment according to the optimized conditions provided in Example 1).

[0214] Preparation of sample solution: According to the initial concentration of the sample to be tested (5.58 ng / g), weigh an appropriate amount of the sample to be tested (101 mg), three times in parallel, add 50 μL of internal standard working solution; add blank matrix to the sample to make up to 550 μL, mix well for pretreatment, and test the sample for two batches in succession;

[0215] Pretreatment method: Take 125μL of magnetic bead suspension in a low protein adsorption centrifuge tube, place it on a magnetic plate, remove the supernatant, and add 0.5mL 10mM PBS solution, mix for 2 minutes, let it stand on the magnetic plate, absorb the supernatant, add 0.5mL water, mix for 2 minutes, let it stand on the magnetic plate, absorb the supernatant; add 550μL of the pre-prepared calibration solution to be processed and the sample solution to be tested respectively; incubate at 25±5℃ for 30 minutes, let it stand on the magnetic plate, and take out the supernatant after 5 minutes; add 0.5mL10mMPBS solution to the centrifuge tube to wash the magnetic beads for 1 minute, and absorb the supernatant again; add 0.5mL water, mix for 2 minutes, let it stand on the magnetic plate, and absorb the supernatant; add 100μL30% acetonitrile 1% acetic acid aqueous solution, mix well and let it stand for 5 minutes, elute twice and combine the eluate, blow dry with nitrogen, re-dissolve with 75μL20% acetonitrile 0.1% formic acid aqueous solution, centrifuge at 15000r / min for 10 minutes, transfer the supernatant from the magnetic plate to the pre-treated 96-well sample plate for liquid-mass analysis.

[0216] Liquid phase method:

[0217] Mobile phase A was acetonitrile, and mobile phase B was 0.2% formic acid aqueous solution; the gradient elution conditions were shown in Table 23, the entire analysis time was 10 min, the injection volume was 20 μl, the flow rate was 0.2 mL / min, the column temperature was 40°C, and the autosampler temperature was 10°C.

[0218] Table 23 Insulin liquid phase conditions

[0219]

[0220] Mass spectrometry method:

[0221] Electrospray ionization (ESI) positive ion mode and multiple reaction monitoring (MRM) mode were used for analysis, capillary voltage was 2.84 kV, ion source temperature was 150°C, desolvation gas temperature was 550°C, and desolvation gas flow was 1000 L / Hr. The ion pairs for quantitative detection were m / z 1162.223>225.932 (insulin), m / z 1170.340>225.932 (insulin internal standard); the ion pairs for qualitative detection were m / z 1162.223>135.97 (insulin), and the ion pairs for monitoring insulin lispro (m / z 1162.66>217.21 (insulin lispro)).

[0222] Measurement results:

[0223] Table 24 Measurement results of sample S16

[0224]

[0225] Example 7

[0226] The human serum sample W6 was tested for fixed value using the method established by the present invention.

[0227] Preparation of calibration solution: Weigh an appropriate amount of 100 ng / g standard working solution, add 50 μL of internal standard working solution of the same concentration, add blank matrix to 550 μL, mix well, and perform the same pretreatment on the same sample (pretreatment according to the optimized conditions provided in Example 1).

[0228] Preparation of sample solution: According to the initial concentration of the sample to be tested (31.5 ng / g), weigh an appropriate amount of the sample to be tested (63 mg), three times in parallel, add 50 μL of internal standard working solution; add blank matrix to the sample to make up to 550 μL, mix well for pretreatment, and test the sample for two batches in succession;

[0229] Pretreatment method: Take 125μL of magnetic bead suspension in a low protein adsorption centrifuge tube, place it on a magnetic plate, remove the supernatant, and add 0.5mL 10mM PBS solution, mix for 2 minutes, let it stand on the magnetic plate, absorb the supernatant, add 0.5mL water, mix for 2 minutes, let it stand on the magnetic plate, absorb the supernatant; add 550μL of the pre-prepared calibration solution to be processed and the sample solution to be tested respectively; incubate at 25±5℃ for 30 minutes, let it stand on the magnetic plate, and take out the supernatant after 5 minutes; add 0.5mL10mMPBS solution to the centrifuge tube to wash the magnetic beads for 1 minute, and absorb the supernatant again; add 0.5mL water, mix for 2 minutes, let it stand on the magnetic plate, and absorb the supernatant; add 100μL30% acetonitrile 1% acetic acid aqueous solution, mix well and let it stand for 5 minutes, elute twice and combine the eluate, blow dry with nitrogen, re-dissolve with 75μL20% acetonitrile 0.1% formic acid aqueous solution, centrifuge at 15000r / min for 10 minutes, transfer the supernatant from the magnetic plate to the pre-treated 96-well sample plate for liquid-mass analysis.

[0230] Liquid phase method:

[0231] Mobile phase A was acetonitrile, and mobile phase B was 0.2% formic acid aqueous solution; the gradient elution conditions were shown in Table 25, the entire analysis time was 10 min, the injection volume was 20 μl, the flow rate was 0.2 mL / min, the column temperature was 40°C, and the autosampler temperature was 10°C.

[0232] Table 25 Insulin liquid phase conditions

[0233]

[0234] Mass spectrometry method:

[0235] Electrospray ionization (ESI) positive ion mode and multiple reaction monitoring (MRM) mode were used for analysis, capillary voltage was 2.84 kV, ion source temperature was 150°C, desolvation gas temperature was 550°C, and desolvation gas flow was 1000 L / Hr. The ion pairs for quantitative detection were m / z 1162.223>225.932 (insulin), m / z 1170.340>225.932 (insulin internal standard); the ion pairs for qualitative detection were m / z 1162.223>135.97 (insulin), and the ion pairs for monitoring insulin lispro (m / z 1162.66>217.21 (insulin lispro)).

[0236] Measurement results:

[0237] Table 26 Test results of sample W6

[0238]

[0239] Example 8

[0240] Serum samples with concentrations of 1.0 ng / g, 10 ng / g, and 20 ng / g were prepared by spiked blank serum, and were recorded as sample 1, sample 2, and sample 3; the three pretreatment methods described in Part 2 of Example 5 (PPT+MAX, PPT+MCX, and magnetic particle immunoenrichment) were used to detect the three prepared samples with known concentrations. The fixed value results are shown in Table 27. The deviation between the fixed value result of magnetic particle immunoenrichment and the theoretical value was less than 3.75%, and the insulin peak area in the sample was significantly better than that of the other two pretreatment methods ( Fig.15 ), indicating that the method of the invention has good accuracy and high sensitivity.

[0241] Table 27 Sample determination results under different pre-treatment conditions

[0242]

[0243] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for detecting insulin in serum, characterized in that: The steps include: Step 1: Take serum, human insulin international standard material NIBSC11 / 212 and isotope internal standard (4-[D10]leucine insulin) to prepare blank matrix, standard working solution and internal standard working solution; Step 2: Mix the standard working solution, the internal standard working solution and the blank matrix to prepare a standard solution; Taking a sample to be tested, mixing it with the internal standard working solution and the blank matrix to prepare a sample solution; Step 3: The standard solution and the sample solution are pre-treated and analyzed by isotope dilution liquid chromatography tandem mass spectrometry, and the insulin content in the sample to be tested is obtained according to the sample peak area ratio and the mass of the sample and the internal standard.

2. The detection method according to claim 1, characterized in that The pretreatment in step 3 includes: taking the magnetic bead suspension and letting it stand to discard the supernatant, washing, mixing with the standard solution and the sample solution respectively, incubating, letting it stand to discard the supernatant, washing the magnetic beads, eluting, re-dissolving, and centrifuging to collect the supernatant.

3. The detection method according to claim 2, characterized in that The volume ratio of the standard solution to the magnetic bead suspension is 500:125; The volume ratio of the sample solution to the magnetic bead suspension is 500:

125.

4. The detection method according to claim 2 or 3, characterized in that: The washing was performed using PBS solution and water respectively, and the concentration of the PBS solution was 10 mM. The volume ratio of the standard solution or the sample solution to the PBS solution is 550:0.5 or 500:0.5; The volume ratio of the standard solution or the sample solution to the water is 550:0.5 or 500:0.5; The cleaning time is 2 minutes.

5. The detection method according to any one of claims 2 to 4, characterized in that: The incubation condition is 25±5° C. for 30 min; the standing time is 5 min.

6. The detection method according to any one of claims 2 to 5, characterized in that: The eluent used for the elution is an aqueous solution with a volume percentage of 30% acetonitrile and 1% acetic acid; The elution was performed twice, each time for 5 minutes; The volume ratio of the standard solution or the sample solution to the eluent is 550:100 or 500:

100.

7. The detection method according to any one of claims 2 to 6, characterized in that: The re-dissolving solution used in the re-dissolving is an aqueous solution with a volume percentage of 20% acetonitrile and 0.1% formic acid; The volume ratio of the standard solution or the sample solution to the reconstitution solution is 550:75 or 500:75; and / or The centrifugal condition is centrifugation at 15000 r / min for 10 min.

8. The detection method according to any one of claims 1 to 7, characterized in that: The mobile phase A of the chromatography was acetonitrile, and the mobile phase B was 0.2% formic acid aqueous solution; the gradient elution conditions were as follows: The chromatographic analysis time was 10 min, the injection volume was 20 μL, the flow rate was 0.2 mL / min, the column temperature was 40° C., and the autosampler temperature was 10° C.

9. The detection method according to any one of claims 1 to 8, characterized in that: The mass spectrometer was analyzed in electrospray ionization (ESI) positive ion mode and multiple reaction monitoring (MRM) mode, with a capillary voltage of 2.84 kV, an ion source temperature of 150° C., a desolvation gas temperature of 550° C., and a desolvation gas flow of 1000 L / Hr. The ion pairs used for quantification were detected as m / z 1162.223>225.932 (insulin), m / z1170.340>225.932 (insulin internal standard); The ion pair for qualitative analysis was detected as m / z 1162.223>135.97 (insulin), and the ion pair for monitoring insulin lispro was m / z 1162.66>217.21 (insulin lispro).

10. The detection method according to any one of claims 1 to 9, characterized in that: The preparation method of the blank matrix is: human serum is mixed with the antibody magnetic particle suspension at a volume ratio of 500:125, incubated at 25±5°C for 15 minutes to remove insulin in the serum, left to stand, and the supernatant is collected to detect insulin. If the detection value is zero, it can be used as a blank matrix. If there is a signal value, the supernatant is taken and the above steps are repeated; and / or The preparation method of the standard working solution is: take human insulin international standard material NIBSC11 / 212, prepare a stock solution by weighing with 0.1% formic acid 0.1% BSA solution, then dilute the intermediate dilution solution with 0.1% formic acid 0.1% BSA solution, and further dilute to obtain the standard working solution, the common working solution concentration is about 100 ng / g and 10 ng / g; and / or The preparation method of the internal standard working solution is: take the isotope internal standard (4-[D10]leucine insulin) and dissolve it by weighing method using 0.1% formic acid and 0.1% BSA to obtain a stock solution, and dilute it to obtain the internal standard working solution. The commonly used internal standard working solution concentrations are 100 ng / g and 10 ng / g.

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