Method for detecting semeglutide by liquid chromatography tandem mass spectrometry and detection kit

Abstract

The invention discloses a method for detecting semeglutide through liquid chromatography tandem mass spectrometry and a detection kit. The invention provides a method which can be rapidly applied to clinic for detecting maturity of the semeglutide in the sample for the first time, and an automatic magnetic bead pretreatment method is adopted, so that the labor force is saved, and the detection precision can be improved. In addition, the automation of the method can be further improved, the working efficiency is improved, and the requirements for professionals are reduced. The leucine in the semeglutide is subjected to d10 labeling, isotope labeling is adopted as an internal label, method calibration is carried out, the detection precision is good, and the method can be popularized and applied in clinical detection.

Description

Technical Field

[0001] The present invention relates to a liquid chromatography tandem mass spectrometry detection technology, and in particular to a method and a detection kit for detecting semaglutide by liquid chromatography tandem mass spectrometry. Background Art

[0002] Semaglutide (CAS No. 900463-68-2) is a long-acting GLP-1 analog used to treat type 2 diabetes. In addition to lowering blood sugar, semaglutide also has the effects of weight loss and cardiovascular protection.

[0003] The existing methods have low sensitivity. In addition, the existing methods use liraglutide as the internal standard for calibration instead of the isotope internal standard of semaglutide, which causes interference in the detection of semaglutide levels in patients using liraglutide. In addition, most of the commonly used pre-treatment methods are manual pre-treatment operations, which are time-consuming, have poor precision, and are not easy to standardize. The existing methods are mostly applied to animals and less to people. Therefore, it is urgent to develop an efficient method for detecting the content of semaglutide in patients. Summary of the invention

[0004] The purpose of the present invention is to provide a method and a detection kit for detecting semaglutide by liquid chromatography tandem mass spectrometry.

[0005] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides a method for detecting semaglutide by liquid chromatography tandem mass spectrometry, comprising the following steps: A. Prepare semaglutide standard solutions of different concentrations; B. Add the isotope internal standard to the standard solution and mix well, then add methanol (chromatographic grade), vortex and centrifuge, and take the supernatant; then extract with MAX magnetic beads, specifically including: adding ammonia water (to adjust the pH value) to the supernatant, mixing with MAX magnetic beads, then washing the magnetic beads with weak washing solution and strong washing solution in turn, and finally eluting with eluent, collecting samples for liquid chromatography tandem mass spectrometry detection; C. Replace the standard solution in step B with the sample to be tested and perform the test using the same method; D. According to the test results of the sample to be tested, the concentration of semaglutide in the sample to be tested is obtained by comparing with the standard curve.

[0006] The isotope internal standard is two leucines in semaglutide d 10 Labeled peptides.

[0007] The weak washing liquid is 1-10% (preferably 5%) ammonia water.

[0008] The strong washing liquid is prepared by mixing water, methanol and acetonitrile in a volume ratio of 1:(1-5):(1-5) (preferably a volume ratio of 1:2:2).

[0009] The eluent is prepared by mixing 0.01% to 15% (preferably 8%) formic acid aqueous solution, methanol and acetonitrile in a volume ratio of 1:(1-5):(1-5) (preferably a volume ratio of 1:2:2).

[0010] Furthermore, the reagent used to prepare the standard solution in step A is prepared by mixing a methanol solution containing 0.01% to 15% (preferably 2.5%) formic acid and acetonitrile in a volume ratio of 1: (1-5) (preferably a volume ratio of 1:1).

[0011] Further, step B is specifically as follows: add 5~50 uL 100~500ng / mL isotope internal standard solution and 100~500 uL methanol (chromatographic grade) to 50~200 uL standard solution, vortex for 10 minutes, and then centrifuge at 13000×g for 10 minutes, take 100~500 uL supernatant, add 100~500 uL 5%~10% ammonia water, and mix with 50~500uL 0.1~10mg / mL MAX magnetic bead solution, and then use 50~200 uL weak wash solution, 50~200 uL strong wash solution, and 50~200 uL elution solution for weak wash, strong wash and elution, and collect samples for machine detection.

[0012] Preferably, add 20 uL of 200ng / mL isotope internal standard solution and 300 uL of methanol to 100 uL of standard solution, vortex for 10 minutes, and then centrifuge at 13000×g for 10 minutes, take 300 uL of supernatant, add 300 uL of 10% ammonia water, and mix with 200 uL of 1mg / mL MAX magnetic bead solution, then use 200 uL of weak wash solution, 200 uL of strong wash solution, and 100 uL of elution solution for weak wash, strong wash and elution, collect samples and prepare for machine detection.

[0013] Wherein, the reagent used for preparing the isotope internal standard solution is 1%~100% (preferably 40%) acetonitrile.

[0014] The HPLC column used in step B is: ACQUITY TM Premier HSS T3 1.8um VanGuard TM FIT2.1mm×100mm Column.

[0015] The liquid chromatography detection conditions are: flow rate 0.3~0.5mL / min (preferably 0.3mL / min), column temperature 40℃; injection volume 5uL.

[0016] Gradient elution was performed according to the following program:

[0017] Wherein, mobile phase A: 0.1% to 0.5% formic acid aqueous solution, mobile phase B: acetonitrile containing 0% to 0.5% formic acid. Preferably, mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: acetonitrile.

[0018] Further, the conditions for mass spectrometry detection in step B are as follows: the mass spectrometer uses Waters Xevo TQ-S, an electrospray ionization source, positive mode acquisition, a capillary voltage of 0.8 kv, an ion source temperature of 150° C., a desolvation temperature of 600° C., a desolvation gas of 800 L / Hr, and a cone gas of 150 L / Hr. Masslynx software is used to complete data acquisition and qualitative and quantitative analysis.

[0019] The ion pairs generated in the mass spectrometer for the qualitative detection of semaglutide were: parent ion 1028.8 and daughter ion 529.8, parent ion 1028.8 and daughter ion 1109.3.

[0020] The ion pair used for quantitative detection of semaglutide was: parent ion 1028.8 and daughter ion 689.8.

[0021] The ion pair generated in the mass spectrometer for qualitative detection of the isotope internal standard is: parent ion 1034.3 and daughter ion 1150.9.

[0022] The ion pair used for quantitative detection of the isotope internal standard is: parent ion 1034.3 and daughter ion 530.3.

[0023] By using the method of the present invention, the linear detection range of semaglutide in the sample to be tested is 0.1-500 ng / mL.

[0024] The sample to be tested can be a blood sample from a patient, etc.

[0025] In a second aspect, the present invention provides a kit for detecting semaglutide by liquid chromatography tandem mass spectrometry, the kit comprising at least one of a semaglutide standard solution, an isotope internal standard, MAX magnetic beads, a weak wash solution, a strong wash solution, an eluent, and the like.

[0026] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: (I) The present invention provides for the first time a mature method for the detection of semaglutide in samples that can be quickly applied to clinical practice, using an automated magnetic bead pre-treatment method that saves labor while improving detection precision. In addition, it can further improve the automation of the method, increase work efficiency, and reduce the demand for professional technicians.

[0027] (ii) By treating leucine in semaglutide d 10 The isotope label was used as the internal standard for method calibration, and both quantitative and qualitative ion pairs were used for the first time.

[0028] (III) The method has good precision, with repeatability and intra-laboratory imprecision CV both less than 7%. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a chromatogram of the synthetic internal standard in a preferred embodiment of the present invention.

[0030] Figure 2 This is a mass spectrum of the synthetic internal standard in a preferred embodiment of the present invention.

[0031] Figure 3 This is the linear detection range of semaglutide in a preferred embodiment of the present invention.

[0032] Figure 4 This is a typical chromatogram of semaglutide (upper) and internal standard (lower) in a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0033] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0034] The MAX magnetic beads used in the following examples were purchased from Biosepur (Lot: B0903).

[0035] Example 1 Kit and method for detecting semaglutide by liquid chromatography tandem mass spectrometry

[0036] 1. Preparation of standard products: methanol, acetonitrile, water, formic acid (FA)

[0037] (1) Prepare a 2.5% FA solution in methanol:acetonitrile = 1:1 (volume ratio) for standard preparation.

[0038] (2) Dissolve 1 mg of semaglutide standard in 1 mL of an aqueous solution containing 1% FA and 20% acetonitrile to prepare a 1 mg / mL stock solution; then use a 2.5% FA methanol: acetonitrile = 1:1 (volume ratio) solution to prepare a 1 ug / mL standard solution as a stock solution; finally, use a 2.5% FA methanol: acetonitrile = 1:1 (volume ratio) solution to dilute the standard to 200 ng / mL.

[0039] (3) Preparation of standard solution (Table 1): Table 1

[0040] 2. Synthesis and preparation of internal standard

[0041] (2) Synthetic internal standard chromatogram Figure 1 .

[0042] (3) The mass spectrum of the synthetic internal standard is shown in Figure 2 .

[0043] (4) Preparation of internal standard solution: Dissolve 1 mg of internal standard in 1 mL of 40% acetonitrile to prepare a 1 mg / mL internal standard solution, which was then aliquoted and frozen at -80 °C as a stock solution.

[0044] Then, 1ug / mL internal standard solution was prepared using 40% acetonitrile as the stock solution; Finally, the 1ug / mL stock solution was further diluted to 200ng / mL using 40% acetonitrile for sample pretreatment.

[0045] 3. Liquid chromatography detection method

[0046] 3.1 Effects of different proportions of formic acid in the mobile phase, different proportions of formic acid in the solvent, and solvent types (methanol, acetonitrile, and water ratios) on carryover contamination.

[0047] (1) Comparing the mobile phase A (0.1% formic acid water to 0.4% formic acid water) and the mobile phase B (0.1% formic acid acetonitrile to 0.4% formic acid acetonitrile), it was found that adding different proportions of acid to the mobile phase had little effect on the removal of carryover interference. Finally, 0.1% formic acid water was selected as the mobile phase A and acetonitrile was selected as the mobile phase B.

[0048] (2) The effects of different proportions of formic acid in the solvent on carryover contamination were compared and 1% to 8% formic acid solutions were evaluated. The results showed that the carryover rate of 8% formic acid solution was less than 3%.

[0049] 3.2 Determination of liquid chromatography detection method

[0050] The HPLC column used is: ACQUITY TM Premier HSS T3 1.8um VanGuard TM FIT 2.1mm×100mm Column.

[0051] (1) Mobile phase A was 0.1% formic acid in water; mobile phase B was acetonitrile.

[0052] (2) The mobile phase flow rate and gradient are shown in Table 2: the flow rate is 0.3 mL / min, the column temperature is 40 °C, and the running time is 5 min.

[0053] Table 2

[0054] 4. Mass spectrometry

[0055] The mass spectrometer was Waters Xevo TQ-S, with an electrospray ionization source, positive mode acquisition, capillary voltage 0.8 kV, ion source temperature 150 ° C, desolvation temperature 600 ° C, desolvation gas: 800 L / Hr, cone gas: 150 L / Hr. Masslynx software was used to complete data acquisition and qualitative and quantitative analysis.

[0056] According to the properties of the compounds, select the appropriate acquisition mode, specifically: positive ion, ESI, and MRM mode (Table 3).

[0057] Table 3

[0058] 5. Optimization of pre-treatment methods

[0059] 5.1 Optimization of protein precipitation solvent: The effects of methanol, acetonitrile, 4% phosphoric acid and 0.1M zinc sulfate as protein precipitation solvents were compared. The results showed that methanol precipitation had the best protein extraction effect, and methanol was finally used for protein precipitation.

[0060] 5.2 Pre-treatment using MAX automated magnetic beads

[0061] The pKa of semaglutide is 2.74, which is an acidic compound. The suitable solid phase extraction plate is MAX, so MAX magnetic beads are selected to extract the analyte.

[0062] MAX magnetic beads extraction method optimization: According to the general extraction scheme of MAX for acidic compounds, activation / equilibrium → adding sample → weak wash (5% ammonia water is recommended) → strong wash (methanol) → elution (2% formic acid methanol)

[0063] The present invention is optimized on the basis of the original recommended scheme. Considering the characteristics of MAX magnetic bead fillers, alkaline solution is generally selected for sample loading and acidic solution for elution. Generally, 5% ammonia water is selected for weak washing, and the optimization space is relatively small. Therefore, the two steps of strong washing and elution are optimized separately.

[0064] 1) Optimization of MAX magnetic bead strong washing: methanol, acetonitrile, 80% methanol, 80% acetonitrile, methanol:acetonitrile = 1:1 (volume ratio), methanol:acetonitrile (ACN): water = 1:1:1 (volume ratio), water:methanol:acetonitrile = 1:2:2 (volume ratio) were investigated.

[0065] The results showed that when H2O:MeOH:ACN=1:2:2 (volume ratio) was used, the least amount of the analyte was washed off.

[0066] The peak areas of the analytes corresponding to different strong washing solvents are shown in Table 4.

[0067] Table 4

[0068] 2) Optimization of MAX magnetic bead elution: Considering the properties of the analyte and the use of MAX magnetic beads, the strong elution mainly examined the effect of different proportions of formic acid in the elution solvent on the analyte. 1% FA~8% FA aqueous solution: methanol: acetonitrile (1:2:2, volume ratio) was examined, and it was found that 8% FA aqueous solution had the best elution effect.

[0069] The peak areas of the analytes eluted with acids at different ratios are shown in Table 5.

[0070] Table 5

[0071] The final sample pre-treatment method is: 100 uL sample / standard solution was added with 20 uL 200ng / mL internal standard and 300uL methanol solution, vortexed for 10min, then centrifuged at 13000×g for 10min, 300uL supernatant was taken, and 300uL 10% ammonia solution was added. 200uL MAX magnetic beads with a concentration of 1mg / mL were first activated with 200uL ethanol, then balanced with 200uL water, and then the beads were mixed with the sample by magnetic attraction, and then 200uL 5% ammonia solution, 200uL water: methanol: acetonitrile = 1:2:2 (volume ratio), 100uL 8% FA aqueous solution: methanol: acetonitrile = 1:2:2 (volume ratio) were used for weak washing, strong washing and elution, and 5uL sample was tested.

[0072] In addition, the manual SPE method can also be used for sample pretreatment, using methanol and water to activate the SPE plate, then loading the sample, and using 5% ammonia water, water: methanol: acetonitrile = 1:2:2 (volume ratio), 8% FA aqueous solution: methanol: acetonitrile = 1:2:2 (volume ratio) for weak washing, strong washing, and elution, and finally collecting the sample for testing. In comparison, the MAX magnetic bead extraction method has better precision and requires less professional technicians, and is suitable for large-throughput sample testing.

[0073] 6. Performance Verification

[0074] 6.1 Linearity Verification

[0075] Semaglutide has a good linear correlation in the linear range of 0.5-200 ng / mL. r>0.990( Figure 3 ).

[0076] 6.2 Precision

[0077] Two samples of high and low concentrations were prepared, each sample was tested four times, and four batches were measured continuously. The intra-laboratory imprecision was 6.06%~6.98%.

[0078] 6.3 Recovery rate

[0079] The recovery rate was verified by using the spike recovery method, and the recovery rate was 82.5%~112.8%.

[0080] 6.4 Typical chromatogram

[0081] Typical chromatograms of semaglutide and internal standard are shown in Figure 2. Figure 4 shown.

[0082] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for detecting semaglutide by liquid chromatography tandem mass spectrometry, characterized in that: The following steps are involved: A. Prepare semaglutide standard solutions of different concentrations; B. Add the isotope internal standard to the standard solution and mix well, then add methanol, vortex and centrifuge, and take the supernatant; then use MAX magnetic beads for extraction, specifically including: add ammonia water to the supernatant, mix with MAX magnetic beads, then wash the magnetic beads with weak washing solution and strong washing solution in turn, and finally elute with eluent, collect samples for liquid chromatography tandem mass spectrometry detection; then, use the concentration of the standard solution as the horizontal axis and the ratio of the peak area of ​​the standard solution to the peak area of ​​the isotope internal standard as the vertical axis to draw a standard curve; C. Replace the standard solution in step B with the sample to be tested and perform the test using the same method; D. According to the test results of the sample to be tested, the concentration of semaglutide in the sample to be tested is obtained by comparing with the standard curve; The isotope internal standard is leucine in semaglutide d 10 labeled peptides; The weak washing liquid is 1-10% ammonia water; The strong washing solution is prepared by mixing water, methanol and acetonitrile in a volume ratio of 1:(1-5):(1-5); The eluent is prepared by mixing 0.01% to 15% formic acid aqueous solution, methanol and acetonitrile in a volume ratio of 1:(1-5):(1-5).

2. The method according to claim 1, characterized in that The reagent used to prepare the standard solution in step A is a mixture of a methanol solution containing 0.01% to 15% formic acid and acetonitrile in a volume ratio of 1:(1-5).

3. The method according to claim 1, characterized in that Step B is as follows: add 5~50 uL 100~500ng / mL isotope internal standard solution and 100~500 uL methanol to 50~200 uL standard solution, vortex for 10 minutes, then centrifuge at 13000×g for 10 minutes, take 100~500 uL supernatant, add 100~500 uL 5%~10% ammonia water, and mix with 50~500 uL 0.1~10mg / mL MAX magnetic bead solution, then use 50~200 uL weak wash solution, 50~200 uL strong wash solution, and 50~200 uL elution solution for weak wash, strong wash and elution, collect samples for machine detection; Among them, the reagent used to prepare the isotope internal standard solution is 1%~100% acetonitrile.

4. The method according to claim 1, characterized in that: The HPLC column used in step B is: ACQUITY TM Premier HSS T3 1.8um VanGuardFIT 2.1mm×100mm Column; The liquid chromatography detection conditions were: flow rate 0.3-0.5 mL / min, column temperature 40 °C; Gradient elution was performed according to the following program: Among them, mobile phase A: 0.1%~0.5% formic acid aqueous solution, mobile phase B: acetonitrile containing 0%~0.5% formic acid.

5. The method according to claim 1, characterized in that The conditions for mass spectrometry detection in step B are as follows: the mass spectrometer uses Waters Xevo TQ-S, an electrospray ionization source, positive mode acquisition, a capillary voltage of 0.8 kV, an ion source temperature of 150°C, a desolvation temperature of 600°C, a desolvation gas: 800 L / Hr, and a cone gas: 150 L / Hr.

6. The method according to claim 1, characterized in that The ion pairs generated in the mass spectrometer for the qualitative detection of semaglutide were: parent ion 1028.8 and daughter ion 529.8, parent ion 1028.8 and daughter ion 1109.3; The ion pair used for quantitative detection of semaglutide was: parent ion 1028.8 and daughter ion 689.

8.

7. The method according to claim 1, characterized in that The ion pairs generated in the mass spectrometer for qualitative detection of the isotope internal standard are: parent ion 1034.3 and daughter ion 1150.9; The ion pair used for quantitative detection of the isotope internal standard is: parent ion 1034.3 and daughter ion 530.

3.

8. The method according to any one of claims 1 to 7, characterized in that: The linear detection range of semaglutide is 0.1~500ng / mL.

9. A kit for detecting semaglutide by liquid chromatography tandem mass spectrometry, characterized in that: The kit comprises at least one of a semaglutide standard solution, an isotope internal standard, MAX magnetic beads, a weak washing solution, a strong washing solution, and an eluent; Wherein, the isotope internal standard, weak washing solution, strong washing solution and eluent are the same as those described in claim 1.

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