A new method for detecting lanreotide in human plasma by high performance liquid chromatography-tandem mass spectrometry

Abstract

The application discloses a new type of high-performance liquid chromatography-tandem mass spectrometry analysis method for detecting lanreotide in human plasma, enriches the lanreotide by using a liquid-liquid extraction method, and the quantitative lower limit is 0.05 ng / mL. By liquid-liquid extraction treatment, the interfering substances in human plasma can be removed, and the matrix effect is improved. The solvent composition and pH value of the liquid-liquid extraction are optimized, and the purpose of effective extraction is achieved. The lanreotide standard is used to prepare a standard curve, and the result is corrected by using the lanreotide isotope internal standard, and the detection result has good accuracy. Without additional preparation of antibody reagents, the operation is simple. The method has high sensitivity, specificity and reproducibility, and has many advantages compared with the traditional radioimmunoassay.

Description

TECHNICAL FIELD

[0001] The present application relates to the detection method of Lanreotide, in particular to a new high performance liquid chromatography-tandem mass spectrometry method for detecting Lanreotide in human plasma. BACKGROUND

[0002] Lanreotide is a somatostatin receptor antagonist, used for treating acromegaly and syndromes caused by neuroendocrine tumors. By detecting the concentration of Lanreotide in human plasma, it is of great significance to study the metabolism of the drug and establish the concentration-effect relationship of the drug.

[0003] In the past, radioimmunoassay was used to detect the concentration of Lanreotide in human plasma. Radioimmunoassay has the advantage of high sensitivity, but also has many shortcomings, resulting in great limitations in use.

[0004] The disadvantages of radioimmunoassay are: the reagents used are radioactive, causing radioactive pollution, and it is difficult for conventional laboratories to have the use conditions; the detection principle is antigen-antibody immune reaction, which requires the preparation of antibody reagents with high specificity, and the preparation process is complex and time-consuming; the half-life of the reagent is short, and the use cost is high; the specificity is poor, and it is difficult to distinguish the analyte and structural analogues; the linear range is narrow, and the sample needs to be diluted for detection, which is easy to cause detection error.

[0005] Therefore, there is an urgent need in the market for a better method for detecting Lanreotide. SUMMARY

[0006] The purpose of the present application is to provide a new high performance liquid chromatography-tandem mass spectrometry method for detecting Lanreotide in human plasma, which is accurate, reliable, high sensitivity, high specificity and reproducible.

[0007] Technical scheme: The new high performance liquid chromatography-tandem mass spectrometry method for detecting Lanreotide in human plasma provided by the present application uses liquid-liquid extraction method to enrich Lanreotide, and the lower limit of quantification is 0.05 ng / mL. The solvent for liquid-liquid extraction is ethyl acetate: isopropyl alcohol (90 / 10; v / v), 100 μL of 1 mol / L sodium carbonate solution is added to the plasma sample, and the standard curve is linear in the range of 0.0500-25.0 ng / mL.

[0008] 1. Control standard substance used for detection

[0009] Reference standard Internal standard Lanreotide acetate [2H8]-Lanreotide (Lanreotide-D8)

[0010] 2. Reagents prepared during the detection process, the volume of the listed reagents can be adjusted in proportion according to the needs.

[0011] 1) Mobile phase A (MPA): water solution containing 0.1% formic acid

[0012] Preparation method: Take 1000 mL of ultrapure water in a 1000 mL reagent bottle, add 1.00 mL of formic acid, mix well. Store at room temperature, valid for two weeks from the preparation date.

[0013] 2) Mobile phase B (MPB): methanol solution containing 0.1% formic acid

[0014] Preparation method: Take 1000 mL of methanol in a 1000 mL reagent bottle, add 1.00 mL of formic acid, mix well. Store at room temperature, valid for two weeks from the preparation date.

[0015] 3) Strong needle washing solution (SNW): 0.1% trifluoroacetic acid in methanol / acetonitrile / isopropanol / water (1 / 1 / 1 / 1; v / v / v / v)

[0016] Preparation method: Take 500 mL of methanol, 500 mL of acetonitrile, 500 mL of isopropanol, and 500 mL of ultrapure water into a 2000 mL reagent bottle, then add 2.00 mL of trifluoroacetic acid and mix well. Store at room temperature, valid for two weeks from the preparation date.

[0017] 4) Weak needle washing solution (WNW): 50% methanol aqueous solution

[0018] Preparation method: Take 500 mL of ultrapure water and 500 mL of methanol in a 1000 mL reagent bottle, mix well. Store at room temperature, valid for one month from the preparation date.

[0019] 5) Solution A: ethyl acetate / isopropanol (90 / 10, v / v)

[0020] Preparation method: Take 900 mL of ethyl acetate and 100 mL of isopropanol in a 1000 mL reagent bottle, mix well. Store at room temperature, valid for one month from the preparation date.

[0021] 6) Solution B: 1M aqueous sodium carbonate solution Preparation method: Take 105.99 g of sodium carbonate in a 1000 mL reagent bottle, add 1000 mL of ultrapure water, shake well to dissolve, mix well. Store at room temperature, valid for one month from the preparation date.

[0022] 7) Solution C: 30% acetonitrile aqueous solution containing 0.1% formic acid Preparation method: Take 70.0 mL of ultrapure water, 30.0 mL of acetonitrile, and 0.100 mL of formic acid in a 100 mL reagent bottle, mix well. Store at room temperature, valid for two weeks from the preparation date.

[0023] 8) Solution D: methanol / individual human plasma (K2EDTA) / water (50 / 0.2 / 50, v / v / v)

[0024] Preparation method: Take 50.0 mL methanol, 0.200 mL human plasma (K2EDTA) and 50.0 mL ultrapure water into a 100 mL reagent bottle, mix well. Store at room temperature, the effective period is two weeks from the preparation date.

[0025] 3. Instruments and equipment

[0026]

[0027] 4. Liquid chromatography conditions

[0028] 5. Mass spectrometry conditions

[0029] Mass spectrometry parameters Parameter settings Ionization voltage 5500V Temperature 550℃ Gas 1 (GS1, N2) 55 p.s.i. gas 2 (GS2, N2) 55 p.s.i. Gas curtain gas (CUR, N2) 45 p.s.i Collision gas (CAD, N2) High Ion source ESI Detection mode Positive mode Scan mode Multiple reaction monitoring (MRM) mode Q1 / Q3 resolution Unit / unit Pause between mass ranges 15.00 ms

[0030]

[0031]

[0032] 6. Preparation of analyte stock solution and working solution

[0033] In a low-adsorption plastic bottle, accurately weigh the lanreotide control standard material, after conversion by the correction factor, add an appropriate amount of dimethyl sulfoxide solution to prepare a 1.00 mg / mL lanreotide standard curve and quality control sample stock solution, store in a -10 to -30 °C refrigerator for later use. The standard curve sample working solution and quality control sample working solution should be prepared using analyte stock solutions from different sources.

[0034] Use methanol / human plasma (K2EDTA) / water (50 / 0.2 / 50, v / v / v) as dilution solvent, prepare the lanreotide standard curve and quality control sample working solution in a low-adsorption plastic bottle, store in a -10 to -30 °C refrigerator for later use. The concentration of the lanreotide standard curve sample working solution is: 1.00, 2.00, 5.00, 10.0, 50.0, 250, 400, 500 ng / mL. The concentration of the lanreotide quality control sample working solution is: 1.00, 3.00, 20.0, 200, 380 ng / mL.

[0035] 7. Preparation of internal standard stock solution and working solution

[0036] In a low adsorption plastic tube, accurately weigh Lanreotide-D8 internal standard control reference material, after conversion by correction factor, add dimethyl sulfoxide solution, prepare 1.00 mg / mL Lanreotide-D8 internal standard stock solution, store in -10 to -30 °C refrigerator. Take internal standard stock solution, dilute with methanol / individual human plasma (K2EDTA) / water (50 / 0.2 / 50, v / v / v) solution in a transparent plastic bottle to prepare internal standard working solution with a concentration of 10.0 ng / mL, store in -10 to -30 °C refrigerator.

[0037] 8. Preparation of standard curve samples and quality control samples

[0038] For method validation: standard curve samples and quality control samples need to be freshly prepared every day, and the corresponding working solution can be diluted with blank matrix in a plastic tube.

[0039] For sample analysis: if within the stability range, standard curve samples and quality control samples can be stored every day.

[0040] The concentration of Lanreotide standard curve samples is 0.0500, 0.100, 0.250, 0.500, 2.50, 12.5, 20.0, 25.0 ng / mL. The concentration of Lanreotide quality control samples: for method validation precision and accuracy, personnel and instrument validation analysis batch, respectively, 0.0500 (LLOQ QC), 0.150 (LQC), 1.00 (MQC-1), 10.0 (MQC-2), 19.0 (HQC) ng / mL, at least 5 repeats for each concentration, for other analysis batches of method validation, LLOQ QC does not need to be prepared, the concentration of quality control samples can be adjusted to at least 2 repeats for each concentration; for sample analysis, respectively, LQC (0.150 ng / mL), MQC-1 (1.00 ng / mL), MQC-2 (10.0 ng / mL), HQC (19.0 ng / mL), the concentration of quality control samples can be appropriately adjusted according to the detection needs, at least 2 repeats for each concentration.

[0041] 9. Sample processing method

[0042] (1) Add 50 μL of standard curve samples, quality control samples, plasma samples to be tested and blank samples to a 96-well deep well plate;

[0043] (2) Add 30 μL of 10.0 ng / mL Lanreotide-D8 internal standard working solution;

[0044] (3) Add 100 μL of 1M sodium carbonate aqueous solution to each well and shake well;

[0045] (4) Add 600 μL of ethyl acetate: isopropyl alcohol (90:10, v / v) extractant to each well;

[0046] (5) Transfer 400 μL of the supernatant to a new 96-well low adsorption plate, and dry under nitrogen;

[0047] (6) Add 100 μL of 30% acetonitrile aqueous solution containing 0.1% formic acid to each well;

[0048] (7) Mix well, and then analyze by sample injection.

[0049] The standard curve and the calculation method of the detection result are as follows:

[0050] Take 50 μL of the standard curve, quality control, the sample to be tested and the blank sample, and perform LC-MS / MS analysis according to the operation in the above-mentioned "sample processing method" in step 9, and record the chromatogram. Take the series of theoretical concentration values of lanreotide in the standard curve sample as the abscissa, and take the peak area ratio of lanreotide and the internal standard lanreotide-D8 as the ordinate. The linear regression equation obtained by weighted least squares regression is the 0.0500-25.0 ng / mL standard curve. The peak area ratio of lanreotide and the internal standard lanreotide-D8 in the unknown plasma sample is brought into the equation, and the blood drug concentration of lanreotide in the unknown sample is obtained.

[0051] Compared with the prior art, the beneficial effects of the present application are as follows:

[0052] 1. The high-performance liquid chromatography-tandem mass spectrometry method has high sensitivity, specificity and reproducibility, and has multiple advantages compared with the traditional radioimmunoassay method; a) no antibody reagent is needed b) no radioactive reagent is needed c) the specificity and reproducibility are better d) the linear range is wider;

[0053] 2. The high-performance liquid chromatography-tandem mass spectrometry method for lanreotide in human plasma is established for the first time;

[0054] 3. The sensitivity of the high-performance liquid chromatography-tandem mass spectrometry method is usually inferior to that of the radioimmunoassay method. In the establishment process of the present method, the liquid-liquid extraction method is used to fully enrich lanreotide, so that the sensitivity is comparable to that of the radioimmunoassay method, and the lower limit of quantification of the method is 0.05 ng / mL, which meets the detection requirements of clinical trials;

[0055] 4. Through liquid-liquid extraction treatment, the interfering substances in human plasma can be removed, and the matrix effect can be improved;

[0056] 5. Generally, polypeptide drugs have high polarity and are difficult to extract by liquid-liquid extraction. In the present method, the solvent composition and pH value of liquid-liquid extraction are fully optimized, so that effective extraction is achieved;

[0057] 6. Using methanol / individual human plasma (K2EDTA) / water (50 / 0.2 / 50, v / v / v) as diluent, the standard curve and quality control sample working solution of lanreotide were prepared in a low-adsorption plastic bottle, which achieved the purpose of preventing non-specific adsorption of the analyte on the container wall.

[0058] 7. The methodology used lanreotide standards to prepare a standard curve, and the results were corrected using lanreotide isotope internal standards. The detection results have good accuracy and precision.

[0059] 8. Tandem mass spectrometry, as a detector, has high sensitivity, specificity and reproducibility, and is the gold standard for detecting the blood concentration of peptide drugs such as lanreotide;

[0060] 9. This methodology can detect a sample in less than 4 minutes, which can meet the high-throughput requirements for biological sample detection in clinical research;

[0061] 10. This high-performance liquid chromatography-tandem mass spectrometry method does not require the preparation of additional antibody reagents and is simple to operate;

[0062] 11. This method does not require radioactive reagents, thus protecting the environment from radioactive contamination;

[0063] 12. Use methanol / individual human plasma (K2EDTA) / water (50 / 0.2 / 50, v / v / v) as a dilution solvent to protect lanreotide from nonspecific adsorption.

[0064] 13. The use of low-adsorption plates effectively reduces the non-specific adsorption of lanreotide;

[0065] 14. Standards were used to prepare the standard curve, and the results were corrected using isotope internal standards, resulting in good accuracy and reproducibility of the test results;

[0066] 15. The liquid-liquid extraction method is used to purify and enrich lanreotide, which effectively removes interfering substances, improves matrix effect, enriches analytes and improves detection sensitivity, and is low in cost.

[0067] 16. The solution composition, pH value, and volume ratio of liquid-liquid extraction have been fully optimized to achieve a satisfactory extraction recovery rate;

[0068] 17. Optimized liquid phase conditions enable the analyte lanreotide to be well separated from interfering peaks, thus improving the detection sensitivity;

[0069] 18. The mass spectrometry conditions have been optimized to achieve the best instrument response signal and a good signal-to-noise ratio;

[0070] 19. Adding a trace amount of human plasma when preparing the working solution helps prevent non-specific adsorption. BRIEF DESCRIPTION OF DRAWINGS

[0071] Figure 1 Blank sample chromatogram of lanreotide in human plasma;

[0072] Figure 2 Blank sample chromatogram of lanreotide in human plasma;

[0073] Figure 3 Lower limit of quantification (LLOQ) sample chromatogram of lanreotide in human plasma;

[0074] Figure 4 Upper limit of quantification (ULOQ) sample chromatogram of lanreotide in human plasma. DETAILED DESCRIPTION

[0075] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions of the present application will be further described below.

[0076] (I) The new high-performance liquid chromatography-tandem mass spectrometry method for detecting lanreotide in human plasma in the present embodiment is as follows:

[0077] 1. Control standard substances used in detection

[0078] Reference standard Internal standard Lanreotide acetate [2H8]-Lanreotide (Lanreotide-D8)

[0079] 2. Reagents prepared during detection, the volumes of the listed reagents can be adjusted in proportion as needed.

[0080] 1) Mobile phase A (MPA): 0.1% formic acid aqueous solution Preparation method example: take 1000 mL of ultrapure water in a 1000 mL reagent bottle, add 1.00 mL of formic acid, and mix well. Store at room temperature, and the effective period is two weeks from the preparation date.

[0081] 2) Mobile phase B (MPB): 0.1% formic acid methanol solution Preparation method example: take 1000 mL of methanol in a 1000 mL reagent bottle, add 1.00 mL of formic acid, and mix well. Store at room temperature, and the effective period is two weeks from the preparation date.

[0082] 3) Strong washing needle liquid (SNW): 0.1% trifluoroacetic acid in methanol / acetonitrile / isopropanol / water (1 / 1 / 1 / 1; v / v / v / v)

[0083] Preparation method example: take 500 mL of methanol, 500 mL of acetonitrile, 500 mL of isopropanol, and 500 mL of ultrapure water into a 2000 mL reagent bottle, and then add 2.00 mL of trifluoroacetic acid and mix well. Store at room temperature, and the effective period is two weeks from the preparation date.

[0084] 4) Weak wash needle (WNW): 50% methanol in water. Preparation method: Take 500 mL of ultrapure water and 500 mL of methanol in a 1000 mL reagent bottle, mix well. Store at room temperature, valid for one month from the preparation date.

[0085] 5) Solution A: Ethyl acetate / isopropyl alcohol (90 / 10; v / v)

[0086] Preparation method: Take 900 mL of ethyl acetate and 100 mL of isopropyl alcohol in a 1000 mL reagent bottle, mix well. Store at room temperature, valid for one month from the preparation date.

[0087] 6) Solution B: 1M sodium carbonate aqueous solution. Preparation method: Take 105.99 g of sodium carbonate in a 1000 mL reagent bottle, add 1000 mL of ultrapure water, shake well to dissolve, mix well. Store at room temperature, valid for one month from the preparation date.

[0088] 7) Solution C: 30% acetonitrile aqueous solution containing 0.1% formic acid. Preparation method: Take 70.0 mL of ultrapure water, 30.0 mL of acetonitrile and 0.100 mL of formic acid in a 100 mL reagent bottle, mix well. Store at room temperature, valid for two weeks from the preparation date.

[0089] 8) Solution D: Methanol / individual human plasma (K2EDTA) / water (50 / 0.2 / 50, v / v / v)

[0090] 9) Preparation method: Take 50.0 mL of methanol, 0.200 mL of individual human plasma (K2EDTA) and 50.0 mL of ultrapure water in a 100 mL reagent bottle, mix well. Store at room temperature, valid for two weeks from the preparation date.

[0091] 3. Instrumentation

[0092]

[0093] 4. Liquid chromatography conditions

[0094]

[0095] 5. Mass spectrometry conditions and ion parameters

[0096] Mass spectrometry parameters Parameter settings Ionization voltage 5500V Temperature 550℃ Gas 1 (GS1, N2) 55 p.s.i. gas 2 (GS2, N2) 55 p.s.i. Gas curtain gas (CUR, N2) 45 p.s.i Collision gas (CAD, N2) High Ion source ESI Detection mode Positive mode Scan mode Multiple reaction monitoring (MRM) mode Q1 / Q3 resolution Unit / unit Pause between mass ranges 15.00 ms

[0097] Ion pairs and parameters:

[0098]

[0099]

[0100] 6. Preparation of analyte stock solution and working solution

[0101] In low adsorption plastic bottle, accurately weigh the lanreotide control standard material, after conversion by correction factor, add appropriate amount of dimethyl sulfoxide solution, prepare 1.00 mg / mL lanreotide standard curve and quality control sample stock solution, store in -10 to -30 °C refrigerator for standby. The standard curve sample working solution and quality control sample working solution should be prepared using analyte stock solution from different sources.

[0102] Using methanol / human plasma (K2EDTA) / water (50 / 0.2 / 50, v / v / v) as dilution solvent, prepare lanreotide standard curve and quality control sample working solution in low adsorption plastic bottle, store in -10 to -30 °C refrigerator for standby. The concentration of lanreotide standard curve sample working solution is: 1.00, 2.00, 5.00, 10.0, 50.0, 250, 400, 500 ng / mL. The concentration of lanreotide quality control sample working solution is: 1.00, 3.00, 20.0, 200, 380 ng / mL.

[0103] 7. Preparation of internal standard stock solution and working solution

[0104] In low adsorption plastic tube, accurately weigh the lanreotide-D8 internal standard control standard material, after conversion by correction factor, add dimethyl sulfoxide solution, prepare 1.00 mg / mL lanreotide-D8 internal standard stock solution, store in -10 to -30 °C refrigerator. Take the internal standard stock solution and dilute it with methanol / individual human plasma (K2EDTA) / water (50 / 0.2 / 50, v / v / v) solution in a transparent plastic bottle to prepare an internal standard working solution with a concentration of 10.0 ng / mL, store in -10 to -30 °C refrigerator.

[0105] 8. Preparation of standard curve sample and quality control sample

[0106] For method validation: the standard curve sample and quality control sample need to be freshly prepared every day, dilute the corresponding working solution with blank matrix in a plastic tube.

[0107] For sample analysis: If within the stability range, daily standard curve samples and quality control samples can be stored. The concentrations of the standard curve samples of lantus are 0.0500, 0.100, 0.250, 0.500, 2.50, 12.5, 20.0, 25.0 ng / mL. The concentrations of the quality control samples of lantus are 0.0500 (LLOQ QC), 0.150 (LQC), 1.00 (MQC-1), 10.0 (MQC-2), 19.0 (HQC) ng / mL, at least 5 replicates for each concentration for method validation precision and accuracy, personnel and instrument validation analysis batch, no LLOQ QC is needed for other analysis batch for method validation, the concentrations of the quality control samples can be adjusted to at least 2 replicates for each concentration; for sample analysis, the concentrations of the quality control samples are LQC (0.150 ng / mL), MQC-1 (1.00 ng / mL), MQC-2 (10.0 ng / mL), HQC (19.0 ng / mL), the concentrations of the quality control samples can be adjusted according to the detection needs, at least 2 replicates for each concentration.

[0108] 9. Sample processing method

[0109] (8) Add 50 μL of standard curve samples, quality control samples, plasma samples to be tested and blank samples to a 96-well deep well plate, respectively

[0110] (9) Add 30 μL of 10.0 ng / mL lantus-D8 internal standard working solution

[0111] (10) Add 100 μL of 1M sodium carbonate aqueous solution to each well and shake well

[0112] (11) Add 600 μL of ethyl acetate: isopropyl alcohol (90:10, v / v) extraction solution to each well

[0113] (12) Transfer 400 μL of the supernatant to a new 96-well low adsorption plate and dry it under nitrogen

[0114] (13) Add 100 μL of 0.1% formic acid in 30% acetonitrile aqueous solution to each well

[0115] (14) After mixing, inject and analyze.

[0116] The standard curve and the calculation method of the detection results are as follows:

[0117] Take 50 μL standard curve, quality control, the sample to be measured and blank sample, according to the invention in the "sample processing method" under the operation of the 9th step, LC-MS / MS analysis, record chromatogram. In the standard curve sample lanreotide a series of theoretical concentration values as the abscissa, with lanreotide and internal standard lanreotide-D8 peak area ratio for the ordinate, with weighted least squares regression operation, the linear regression equation obtained is 0.0500-25.0 ng / mL standard curve. The unknown plasma sample of lanreotide and internal standard lanreotide-D8 peak area ratio into the equation, namely the blood concentration of lanreotide in the unknown sample.

[0118] The method described herein for determining the concentration of lanreotide in human plasma, a complete methodological validation, the results are as follows:

[0119] (II) Methodological validation

[0120] (1) selectivity

[0121]

[0122] The results of the selectivity experiment:

[0123] (2) the interference of the test substance on the stable isotope internal standard

[0124]

[0125]

[0126] The interference of the test substance on the internal standard

[0127]

[0128] Conclusion: the interference of the test substance on the stable isotope internal standard meets the acceptance criteria

[0129] (3) the interference of the internal standard on the test substance

[0130]

[0131]

[0132] The interference of the internal standard on the test substance

[0133]

[0134] (4) standard curve statistics

[0135]

[0136]

[0137]

[0138] The series of theoretical concentration values of lanreotide in the standard curve sample were taken as the abscissa, and the ratio of the peak area of lanreotide and the internal standard lanreotide-D8 was taken as the ordinate. The linear regression equation obtained by regression operation with weighted least squares was the 0.0500-25.0 ng / mL standard curve. The ratio of the peak area of lanreotide and the internal standard lanreotide-D8 of the unknown plasma sample was brought into the equation, and the blood drug concentration of lanreotide in the unknown sample was obtained.

[0139] (5) Precision and accuracy

[0140]

[0141]

[0142] (6) Accuracy and precision control sample statistics

[0143]

[0144] Conclusion: The accuracy and precision control sample meets the acceptance criteria

[0145] (7) Extraction recovery rate

[0146]

[0147] Recovery rate of the test substance (calculated by the area ratio of the test substance)

[0148]

[0149] Conclusion: The recovery rate of the test substance lanreotide meets the acceptance criteria

[0150] Internal standard recovery rate (calculated by the area ratio of the test substance)

[0151]

[0152]

[0153] Conclusion: The internal standard recovery rate meets the acceptance criteria

[0154] (8) Matrix effect

[0155]

[0156] Matrix effect (calculated by the detection concentration)

[0157]

[0158]

[0159]

[0160] Conclusion: Matrix effect meets acceptance criteria for hemolyzed matrix effect

[0161]

[0162]

[0163] High-fat matrix effect

[0164]

[0165] Conclusion: High-fat matrix effect meets acceptance criteria (9) Dilution reliability

[0166]

[0167] Dilution reliability

[0168]

[0169] Conclusion: Dilution verification meets acceptance criteria (10) Stability

[0170]

[0171] Matrix stability samples (-10 to -30 °C, 4 freeze-thaw cycles) Analyte: Lanreotide

[0172]

[0173] Conclusion: Matrix stability (-10 to -30 °C, 4 freeze-thaw cycles) meets acceptance criteria

[0174] Note:

[0175] -: Not applicable.

[0176] Acceptance criteria: The deviation of the mean concentration of the stability samples at each concentration level from the theoretical concentration is within ± 15.0%, and the %CV is ≤ 15.0% Matrix stability samples (-60 to -90 °C, 4 freeze-thaw cycles) Analyte: Lanreotide

[0177]

[0178] Conclusion: Matrix stability (-60 to -90 °C, 4 freeze-thaw cycles) meets acceptance criteria

[0179] Note:

[0180] -: Not applicable.

[0181] Acceptance Criteria: The average concentration of the stability samples at each concentration level was within ± 15.0 % of the theoretical concentration, and the %CV was ≤ 15.0 % Matrix stability samples (-10 to -30 °C, 142 days) Analyte: Lanreotide

[0182]

[0183] Conclusion: Matrix stability (142 days) met acceptance criteria

[0184] -: Not applicable.

[0185] Matrix stability samples (-60 to -90 °C, 142 days) Analyte: Lanreotide

[0186]

[0187] Conclusion: Matrix stability (142 days) met acceptance criteria

[0188] Whole blood stability

[0189]

[0190]

[0191] Conclusion: 2h whole blood stability met acceptance criteria (11) Post-sample extraction stability

[0192]

[0193] Post-sample extraction stability: 129.6h Analyte: Lanreotide

[0194]

[0195]

[0196] Conclusion: Post-sample extraction stability met acceptance criteria

[0197] Comparative Example:

[0198] The following examples show the advantages of the present application over the prior art:

[0199]

[0200] The above merely describes the preferred embodiments of the present application and does not limit the present application in any way. Any person skilled in the art can make any form of equivalent replacement or modification to the technical solutions and technical contents disclosed by the present application without departing from the scope of the technical solutions of the present application, and such changes still belong to the protection scope of the present application.

Claims

1. A novel high performance liquid chromatography-tandem mass spectrometry method for detecting lanreotide in human plasma, characterized in that, The liquid-liquid extraction method was used to enrich Lanreotide, and the lower limit of quantification was 0.05 ng / mL. The solvent for liquid-liquid extraction was ethyl acetate: isopropyl alcohol (90 / 10; v / v). 100 μL of 1 mol / L sodium carbonate solution was added to the plasma sample. The standard curve was linear in the range of 0.0500-25.0 ng / mL, Characterized in that the liquid chromatography conditions are: Mass spectrometry conditions: Ion pairs and parameters: Sample processing method: (1) 50 μL of standard curve sample, quality control sample, plasma sample to be tested and blank sample were added to the 96-well deep well plate, respectively; (2) 30 μL of 10.0 ng / mL Lanreotide-D8 internal standard working solution was added; (3) 100 μL of 1M sodium carbonate aqueous solution was added to each well, and shaken to mix well; (4) 600 μL of ethyl acetate: isopropyl alcohol (90:10, v / v) extract was added to each well; (5) 400 μL of the above supernatant was transferred to a new 96-well low adsorption plate, and nitrogen was blown to dryness; (6) 100 μL of 0.1% formic acid in 30% acetonitrile aqueous solution was added to each well; (7) After mixing, it was ready for sample analysis.

2. The novel high performance liquid chromatography-tandem mass spectrometry method for detecting lanreotide in human plasma according to claim 1, characterized in that, Standard substances used for detection: Internal standard of control standard substance Lanreotide acetate [2H8]-Lanreotide (Lanreotide-D8).

3. The novel high performance liquid chromatography-tandem mass spectrometry method for the detection of lanreotide in human plasma as claimed in claim 1, wherein, Reagents prepared during detection: 1) Mobile phase A MPA: 0.1% formic acid in water; 2) Mobile phase B MPB: 0.1% formic acid in methanol; 3) Strong washing needle solution SNW: 0.1% trifluoroacetic acid in methanol / acetonitrile / isopropyl alcohol / water (1 / 1 / 1 / 1; v / v / v / v) solution; 4) Weak washing needle solution WNW: 50% methanol aqueous solution; 5) Solution A: ethyl acetate / isopropyl alcohol (90 / 10; v / v); 6) Solution B: 1M sodium carbonate aqueous solution; 7) Solution C: 0.1% formic acid in 30% acetonitrile aqueous solution; 8) Solution D: methanol / individual human plasma (K2EDTA) / water (50 / 0.2 / 50, v / v / v).

4. The novel high performance liquid chromatography-tandem mass spectrometry method for detecting lanreotide in human plasma according to claim 1, characterized in that, Instrument used: Mass spectrum 6500+, Sciex Liquid chromatography LC-30AD, Shimadzu.

5. The novel high performance liquid chromatography-tandem mass spectrometry method for the detection of lanreotide in human plasma as claimed in claim 1, wherein, Preparation of analyte stock solution and working solution: after the correction factor of lanreotide control standard substance was converted, dimethyl sulfoxide solution was added to prepare 1.00 mg / mL of lanreotide standard curve and quality control sample stock solution; methanol / human plasma (K2EDTA) / water (50 / 0.2 / 50, v / v / v) was used as dilution solvent to prepare lanreotide standard curve and quality control sample working solution. The concentration of lanreotide standard curve sample working solution was: 1.00, 2.00, 5.00, 10.0, 50.0, 250, 400, 500 ng / mL, and the concentration of lanreotide quality control sample working solution was: 1.00, 3.00, 20.0, 200, 380 ng / mL; Preparation of internal standard stock solution and working solution: Take Lanreotide-D8 internal standard control standard material, add dimethyl sulfoxide solution to prepare 1.00 mg / mL Lanreotide-D8 internal standard stock solution, and take the internal standard stock solution to dilute with methanol / individual human plasma (K2EDTA) / water (50 / 0.2 / 50, v / v / v) solution to prepare an internal standard working solution with a concentration of 10.0 ng / mL; Preparation of standard curve samples and quality control samples: the concentration of Lanreotide standard curve samples is 0.0500, 0.100, 0.250, 0.500, 2.50, 12.5, 20.0, 25.0 ng / mL; the concentration of Lanreotide quality control samples is 0.0500 (LLOQ QC), 0.150 (LQC), 1.00 (MQC-1), 10.0 (MQC-2), 19.0 (HQC) ng / mL; for sample analysis, LQC (0.150 ng / mL), MQC-1 (1.00 ng / mL), MQC-2 (10.0 ng / mL), and HQC (19.0 ng / mL).

6. The novel high performance liquid chromatography-tandem mass spectrometry method for the detection of lanreotide in human plasma as claimed in claim 1, wherein, The standard curve and the calculation method of the detection results are as follows: take the prepared standard curve samples, quality control samples, samples to be tested, and blank samples, respectively, for LC-MS / MS analysis, record the chromatogram, take the series of theoretical concentration values of Lanreotide in the standard curve samples as the abscissa, take the peak area ratio of Lanreotide and internal standard Lanreotide-D8 as the ordinate, and perform regression operation by weighted least squares method to obtain the linear regression equation, which is the 0.0500-25.0 ng / mL standard curve. The peak area ratio of Lanreotide and internal standard Lanreotide-D8 in the unknown plasma sample is substituted into the equation to obtain the blood drug concentration of Lanreotide in the unknown sample.

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