Novel high performance liquid chromatography-tandem mass spectrometry analysis method for detecting lanreotide in human plasma

Abstract

The invention discloses a novel high performance liquid chromatography-tandem mass spectrometry analysis method for detecting lanreotide in human plasma, which is characterized in that the lanreotide is enriched by using a liquid-liquid extraction method, and the quantification lower limit is 0.05 ng / mL. Through liquid-liquid extraction treatment, interfering substances in human plasma can be removed, and the matrix effect is improved. The solvent components and the pH value of liquid-liquid extraction are optimized, and the purpose of effective extraction is achieved. A lanreotide standard product is used for preparing a standard curve, result correction is carried out through a lanreotide isotope internal standard, and the detection result has good accuracy. No extra antibody reagent needs to be prepared, and the operation is simple. The method provided by the invention has high sensitivity, specificity and reproducibility, and has multiple advantages compared with the traditional radioactive immunoassay.

Description

Technical Field

[0001] The present invention relates to a method for detecting lanreotide, and in particular to a novel high performance liquid phase-tandem mass spectrometry analysis method for detecting lanreotide in human plasma. Background Art

[0002] Lanreotide is a somatostatin receptor antagonist used to treat acromegaly and syndromes caused by neuroendocrine tumors. Detecting the concentration of lanreotide in human plasma is of great significance for studying the metabolism of drugs and establishing the relationship between drug concentration and drug efficacy.

[0003] In the past, radioimmunoassay was used to measure the concentration of lanreotide in human plasma. While radioimmunoassay has the advantage of high sensitivity, it also has many disadvantages, which leads to great limitations in its use.

[0004] The disadvantages of radioimmunoassay are: the reagents used are radioactive and produce radioactive contamination, making it difficult for conventional laboratories to use them; the detection principle is an antigen-antibody immune reaction, which requires the preparation of highly specific antibody reagents, and the preparation process is complex and time-consuming; the half-life of the reagents is short and the cost of use is high; the specificity is poor, cross-reactions are likely to occur, and it is difficult to distinguish between the analyte and structural analogs; the linear range is narrow, and the sample needs to be diluted before testing, which can easily cause detection errors.

[0005] Therefore, a better method for detecting lanreotide is urgently needed in the market. Summary of the invention

[0006] Purpose of the invention: The purpose of the present invention is to provide a novel high performance liquid chromatography-tandem mass spectrometry method for detecting lanreotide in human plasma that is accurate, reliable, highly sensitive, highly specific and reproducible.

[0007] Technical solution: The novel high performance liquid phase-tandem mass spectrometry analysis method for detecting lanreotide in human plasma described in the present invention uses liquid-liquid extraction to enrich lanreotide, with a lower limit of quantification of 0.05 ng / mL. The solvent for liquid-liquid extraction is ethyl acetate:isopropanol (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. Reference standard substances used in testing

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

[0010] 2. The volumes of reagents prepared during the test can be adjusted proportionally as needed.

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

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

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

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

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

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

[0017] 4) Weak needle wash solution (WNW): 50% methanol in water

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

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

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

[0021] 6) Solution B: 1M sodium carbonate aqueous solution Preparation method: Weigh 105.99g of sodium carbonate into a 1000mL reagent bottle, add 1000mL of ultrapure water, shake thoroughly to dissolve, and mix evenly. Store at room temperature, the validity period is one month from the date of preparation.

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

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

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

[0025] 3. Instruments and Equipment

[0026]

[0027] 4. Liquid chromatography conditions

[0028] 5. Mass spectrometry conditions

[0029] Mass spectrometry parameters Parameter settings Ion spray voltage 5500V temperature 550℃ <![CDATA[Gas 1 (GS1, N2)]]> 55p.si <![CDATA[Gas 2 (GS2, N2)]]> 55p.si <![CDATA[Curtain air gas (CUR, N2)]]> 45p.si <![CDATA[Collision gas (CAD, N2)]]> High Ion source ESI Detection method Positive ion mode Scanning method Multiple ion reaction monitoring (MRM) method Q1 / Q3 Resolution Unit / unit Pause between intervals 15.00ms

[0030]

[0031]

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

[0033] In a low-absorption plastic bottle, accurately weigh the lanreotide reference standard substance, and after the correction factor is converted, add an appropriate amount of dimethyl sulfoxide solution to prepare a 1.00 mg / mL lanreotide standard curve and quality control sample stock solution, and store it in a -10 to -30°C refrigerator for use. The standard curve sample working solution and the 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 to prepare the standard curve and quality control sample working solutions of lanreotide in low adsorption plastic bottles and store them in a refrigerator at -10 to -30°C for later use. The concentrations of the lanreotide standard curve sample working solutions are: 1.00, 2.00, 5.00, 10.0, 50.0, 250, 400, 500 ng / mL. The concentrations of the lanreotide quality control sample working solutions are: 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-absorption plastic tube, accurately weigh the lanreotide-D8 internal standard reference standard substance, after correction factor conversion, add dimethyl sulfoxide solution to prepare 1.00 mg / mL lanreotide-D8 internal standard stock solution, and store it in a refrigerator at -10 to -30°C. 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, and store it in a refrigerator at -10 to -30°C.

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

[0038] For method validation: Standard curve samples and quality control samples should be prepared freshly every day by diluting the corresponding working solutions with blank matrix in plastic tubes.

[0039] For sample analysis: Daily standard curve samples and quality control samples may be used from storage if within stability range.

[0040] The concentrations of lanreotide standard curve samples were 0.0500, 0.100, 0.250, 0.500, 2.50, 12.5, 20.0, and 25.0 ng / mL. The concentration of quality control samples of lanreotide: for the analysis batches of methodological validation precision and accuracy, personnel and instrument validation, they are 0.0500 (LLOQ QC), 0.150 (LQC), 1.00 (MQC-1), 10.0 (MQC-2), 19.0 (HQC) ng / mL, respectively, with at least 5 replicates for each concentration. For other analysis batches of methodological validation, there is no need to prepare LLOQ QC, and each concentration of the quality control sample can be adjusted to at least 2 replicates; for sample analysis, they are LQC (0.150 ng / mL), MQC-1 (1.00 ng / mL), MQC-2 (10.0 ng / mL), HQC (19.0 ng / mL), respectively. The concentration of the quality control sample can be appropriately adjusted according to the detection needs, and each concentration should be repeated at least 2 times.

[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 1 M sodium carbonate aqueous solution to each well and shake to mix;

[0045] (4) Add 600 μL of ethyl acetate:isopropanol (90:10, v / v) extraction solution to each well;

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

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

[0048] (7) After mixing, prepare for sample injection and analysis.

[0049] The standard curve and test result calculation method are as follows:

[0050] Take 50 μL of the standard curve, quality control, plasma sample to be tested and blank sample respectively, and perform LC-MS / MS analysis according to the operation under the "Sample Processing Method" in the above step 9, and record the chromatogram. Take a series of theoretical concentration values ​​of lanreotide in the standard curve sample as the horizontal coordinate, take the ratio of the peak area of ​​lanreotide and the internal standard lanreotide-D8 as the vertical coordinate, and use the weighted least squares method to perform regression operation. The linear regression equation obtained is the 0.0500-25.0 ng / mL standard curve. Substitute the ratio of the peak area of ​​lanreotide and the internal standard lanreotide-D8 of the unknown plasma sample into the equation to obtain the blood concentration of lanreotide in the unknown sample.

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] 1. The high performance liquid chromatography-tandem mass spectrometry method is used, which has high sensitivity, specificity and reproducibility. Compared with the traditional radioimmunoassay, it has many advantages: a) no antibody reagents are required b) no radioactive reagents are required c) better specificity and reproducibility d) wider linear range;

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

[0054] 3. The sensitivity of the HPLC-MS / MS method is usually inferior to that of the radioimmunoassay. In the process of establishing this method, the lanreotide was fully enriched by using the liquid-liquid extraction method, achieving a sensitivity comparable to that of the radioimmunoassay. The quantitative limit of the methodology was 0.05 ng / mL, which met the needs of clinical trial detection.

[0055] 4. Liquid-liquid extraction can remove interfering substances in human plasma and improve the matrix effect;

[0056] 5. Peptide drugs are usually highly polar and difficult to extract by liquid-liquid extraction. This method fully optimizes the solvent composition and pH value of liquid-liquid extraction to achieve the purpose of effective extraction;

[0057] 6. Methanol / individual human plasma (K2EDTA) / water (50 / 0.2 / 50, v / v / v) was used as the diluent to prepare the standard curve and quality control sample working solution of lanreotide in a low-absorption plastic bottle, thereby preventing the nonspecific adsorption of the analyte to the container wall;

[0058] 7. Methodology: Lanreotide standard was used to prepare the standard curve, and the results were calibrated by Lanreotide isotope internal standard. The test 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 blood concentrations of peptide drugs such as lanreotide;

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

[0061] 10. This HPLC-MS / MS method does not require additional preparation of antibody reagents and is simple to operate;

[0062] 11. This method does not require radioactive reagents, protecting the environment from being polluted by radioactive substances;

[0063] 12. Methanol / individual human plasma (K2EDTA) / water (50 / 0.2 / 50, v / v / v) was used as the diluent to protect lanreotide from nonspecific adsorption;

[0064] 13. Use low adsorption plates to effectively reduce the nonspecific adsorption of lanreotide;

[0065] 14. Standard products were used to prepare the standard curve, and the results were corrected by isotope internal standards. The test results had good accuracy and reproducibility;

[0066] 15. The liquid-liquid extraction method is used to purify and enrich lanreotide, which effectively removes interferences, improves matrix effects, enriches the analyte and improves the sensitivity of detection at a low 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. The liquid phase conditions were optimized to separate the analyte lanreotide from the interfering peaks, thus improving the sensitivity of the detection.

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

[0070] 19. Add a small amount of human plasma when preparing the working solution to prevent nonspecific adsorption. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 Chromatogram of double blank samples of lanreotide in human plasma;

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

[0073] Figure 3 Sample chromatogram of the lower limit of quantification (LLOQ) of lanreotide in human plasma;

[0074] Figure 4 Sample chromatogram of the upper limit of quantitation (ULOQ) of lanreotide in human plasma. DETAILED DESCRIPTION

[0075] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be further described below.

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

[0077] 1. Reference standard substances used in testing

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

[0079] 2. The volumes of reagents prepared during the test can be adjusted proportionally as needed.

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

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

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

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

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

[0085] 5) Solution A: ethyl acetate / isopropanol (90 / 10; v / v)

[0086] Preparation method: Take 900mL of ethyl acetate and 100mL of isopropanol in a 1000mL reagent bottle and mix well. Store at room temperature. The validity period is one month from the date of preparation.

[0087] 6) Solution B: 1M sodium carbonate aqueous solution Preparation method: Weigh 105.99g of sodium carbonate into a 1000mL reagent bottle, add 1000mL of ultrapure water, shake thoroughly to dissolve, and mix evenly. Store at room temperature, the validity period is one month from the date of preparation.

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

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

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

[0091] 3. Instruments and Equipment

[0092]

[0093] 4. Liquid chromatography conditions

[0094]

[0095] 5. Mass spectrometry conditions and ion parameters

[0096] Mass spectrometry parameters Parameter settings Ion spray voltage 5500V temperature 550℃ <![CDATA[Gas 1 (GS1, N2)]]> 55p.si <![CDATA[Gas 2 (GS2, N2)]]> 55p.si <![CDATA[Curtain air gas (CUR, N2)]]> 45p.si <![CDATA[Collision gas (CAD, N2)]]> High Ion source ESI Detection method Positive ion mode Scanning method Multiple ion reaction monitoring (MRM) method Q1 / Q3 Resolution Unit / unit Pause between intervals 15.00ms

[0097] Ion pairs and parameters:

[0098]

[0099]

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

[0101] In a low-absorption plastic bottle, accurately weigh the lanreotide reference standard substance, and after the correction factor is converted, add an appropriate amount of dimethyl sulfoxide solution to prepare a 1.00 mg / mL lanreotide standard curve and quality control sample stock solution, and store it in a -10 to -30°C refrigerator for use. The standard curve sample working solution and the quality control sample working solution should be prepared using analyte stock solutions from different sources.

[0102] Use methanol / human plasma (K2EDTA) / water (50 / 0.2 / 50, v / v / v) as dilution solvent to prepare the standard curve and quality control sample working solutions of lanreotide in low adsorption plastic bottles and store them in a refrigerator at -10 to -30°C for later use. The concentrations of the lanreotide standard curve sample working solutions are: 1.00, 2.00, 5.00, 10.0, 50.0, 250, 400, 500 ng / mL. The concentrations of the lanreotide quality control sample working solutions are: 1.00, 3.00, 20.0, 200, 380 ng / mL.

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

[0104] In a low-absorption plastic tube, accurately weigh the lanreotide-D8 internal standard reference standard substance, after correction factor conversion, add dimethyl sulfoxide solution to prepare 1.00 mg / mL lanreotide-D8 internal standard stock solution, and store it in a refrigerator at -10 to -30°C. 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, and store it in a refrigerator at -10 to -30°C.

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

[0106] For method validation: Standard curve samples and quality control samples should be prepared freshly every day by diluting the corresponding working solutions with blank matrix in plastic tubes.

[0107] For sample analysis: If within the stability range, daily standard curve samples and quality control samples can be used for storage. The concentrations of lanreotide standard curve samples are 0.0500, 0.100, 0.250, 0.500, 2.50, 12.5, 20.0, 25.0 ng / mL. The concentration of quality control samples of lanreotide: for the analysis batches of methodological validation precision and accuracy, personnel and instrument validation, they are 0.0500 (LLOQ QC), 0.150 (LQC), 1.00 (MQC-1), 10.0 (MQC-2), 19.0 (HQC) ng / mL, respectively, with at least 5 replicates for each concentration. For other analysis batches of methodological validation, there is no need to prepare LLOQ QC, and each concentration of the quality control sample can be adjusted to at least 2 replicates; for sample analysis, they are LQC (0.150 ng / mL), MQC-1 (1.00 ng / mL), MQC-2 (10.0 ng / mL), HQC (19.0 ng / mL), respectively. The concentration of the quality control sample can be appropriately adjusted according to the detection needs, and each concentration should be repeated at least 2 times.

[0108] 9. Sample processing method

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

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

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

[0112] (11) Add 600 μL of ethyl acetate:isopropanol (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 blow dry with nitrogen.

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

[0115] (14) After mixing, the mixture is ready for sampling and analysis.

[0116] The standard curve and test result calculation method are as follows:

[0117] Take 50 μL of the standard curve, quality control, plasma sample to be tested and blank sample respectively, and perform LC-MS / MS analysis according to the "Sample Processing Method" in step 9 of the invention content, and record the chromatogram. A series of theoretical concentration values ​​of lanreotide in the standard curve sample are used as the abscissa, and the ratio of the peak area of ​​lanreotide and the internal standard lanreotide-D8 is used as the ordinate. The weighted least squares method is used for regression operation, and the linear regression equation obtained is 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 is substituted into the equation to obtain the blood concentration of lanreotide in the unknown sample.

[0118] A complete methodological validation of the method described in this article for determining lanreotide concentrations in human plasma was performed with the following results:

[0119] (II) Methodological Validation

[0120] (1) Selectivity

[0121]

[0122] Results of the selective experiment:

[0123] (2) Interference of the analyte on the stable isotope internal standard

[0124]

[0125]

[0126] Interference results of the analyte on the internal standard

[0127]

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

[0129] (3) Interference of internal standard with the analyte

[0130]

[0131]

[0132] Interference of internal standard on analytes

[0133]

[0134] (4) Standard curve statistics

[0135]

[0136]

[0137]

[0138] A series of theoretical concentration values ​​of lanreotide in the standard curve samples are used as the horizontal coordinates, and the ratio of the peak area of ​​lanreotide to the internal standard lanreotide-D8 is used as the vertical coordinate. The weighted least square method is used for regression calculation, and the linear regression equation obtained is the 0.0500-25.0ng / mL standard curve. The ratio of the peak area of ​​lanreotide in the unknown plasma sample and the internal standard lanreotide-D8 is substituted into the equation to obtain the blood concentration of lanreotide in the unknown sample.

[0139] (5) Precision and accuracy

[0140]

[0141]

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

[0143]

[0144] Conclusion: The accuracy and precision quality control samples meet the acceptance criteria

[0145] (7) Extraction recovery rate

[0146]

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

[0148]

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

[0150] Internal standard recovery rate (calculated as the area ratio of the analyte)

[0151]

[0152]

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

[0154] (8) Matrix effect

[0155]

[0156] Matrix effect (calculated based on detection concentration)

[0157]

[0158]

[0159]

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

[0161]

[0162]

[0163] High-fat matrix effect

[0164]

[0165] Conclusion: The matrix effect of high fat content meets the acceptance criteria (9) Dilution reliability

[0166]

[0167] Dilution reliability

[0168]

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

[0170]

[0171] Matrix stability sample (-10~-30℃, 4 freeze-thaw cycles) Analyte: Lanreotide

[0172]

[0173] Conclusion: The matrix stability (-10~-30℃, 4 freeze-thaw cycles) meets the acceptance criteria

[0174] Note:

[0175] -:not applicable.

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

[0177]

[0178] Conclusion: The matrix stability (-60~-90℃, 4 freeze-thaw cycles) meets the acceptance criteria

[0179] Note:

[0180] -:not applicable.

[0181] Acceptance criteria: The deviation of the average concentration of each stability sample at each concentration level from the theoretical concentration is within ±15.0%, and %CV≤15.0% Matrix stability sample (-10~-30℃, 142 days) Analyte: Lanreotide

[0182]

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

[0184] -:not applicable.

[0185] Matrix stability sample (-60~-90℃, 142 days) Analyte: Lanreotide

[0186]

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

[0188] Whole blood stability

[0189]

[0190]

[0191] Conclusion: 2h whole blood stability meets the acceptance criteria (11) Stability after sample extraction

[0192]

[0193] Stability after sample extraction: 129.6h Analyte: Lanreotide

[0194]

[0195]

[0196] Conclusion: The stability of the sample after extraction meets the acceptance criteria

[0197] Comparative Example:

[0198] The following lists the relatively close prior arts and compares them with the present invention. The results show that the present invention has relatively prominent advantages, as shown in the following table:

[0199]

[0200] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any technician in the relevant technical field, without departing from the scope of the technical solution of the present invention, makes any form of equivalent replacement or modification to the technical solution and technical content disclosed in the present invention, which does not depart from the content of the technical solution of the present invention and still falls within the protection scope of the present invention.

Claims

1. A novel high performance liquid chromatography-tandem mass spectrometry method for detecting lanreotide in human plasma, characterized in that: Lanreotide was enriched using liquid-liquid extraction with a lower limit of quantification of 0.05 ng / mL. The solvent for liquid-liquid extraction was ethyl acetate:isopropanol (90 / 10; v / v). 100 μL of 1 mol / L sodium carbonate solution was added to the plasma sample, and the standard curve was linear in the range of 0.0500-25.0 ng / mL.

2. The novel high performance liquid phase-tandem mass spectrometry analysis method for detecting lanreotide in human plasma according to claim 1, characterized in that: Standard substances used in testing: Reference Standard Internal Standard Lanreotide acetate (Lanreotide acetate) [2H8]-Lanreotide (Lanreotide-D8).

3. The novel high performance liquid phase-tandem mass spectrometry analysis method for detecting lanreotide in human plasma according to claim 1, characterized in that: Reagents prepared during the test: 1) Mobile phase A MPA: aqueous solution containing 0.1% formic acid; 2) Mobile phase B MPB: methanol solution containing 0.1% formic acid; 3) Strong needle wash solution SNW: a solution containing 0.1% trifluoroacetic acid in methanol / acetonitrile / isopropanol / water (1 / 1 / 1 / 1; v / v / v / v); 4) Weak needle wash solution WNW: 50% methanol in water; 5) Solution A: ethyl acetate / isopropanol (90 / 10; v / v); 6) Solution B: 1M sodium carbonate aqueous solution; 7) Solution C: 30% acetonitrile aqueous solution containing 0.1% formic acid; 8) Solution D: methanol / individual human plasma (K2EDTA) / water (50 / 0.2 / 50, v / v / v).

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

5. The novel high performance liquid chromatography-tandem mass spectrometry analysis method for detecting lanreotide in human plasma according to claim 1, characterized in that: Liquid chromatography conditions:

6. The novel high performance liquid chromatography-tandem mass spectrometry analysis method for detecting lanreotide in human plasma according to claim 1, characterized in that: Mass spectrometry conditions:

7. The novel high performance liquid chromatography-tandem mass spectrometry analysis method for detecting lanreotide in human plasma according to claim 1, characterized in that: Ion pairs and parameters:

8. The novel high performance liquid chromatography-tandem mass spectrometry analysis method for detecting lanreotide in human plasma according to claim 1, characterized in that: Preparation of analyte stock solution and working solution: After the correction factor of lanreotide reference standard substance was converted, dimethyl sulfoxide solution was added to prepare 1.00 mg / mL lanreotide standard curve and quality control sample stock solution; methanol / human plasma (K2EDTA) / water (50 / 0.2 / 50, v / v / v) was used as the dilution solvent to prepare lanreotide standard curve and quality control sample working solutions. The concentrations of lanreotide standard curve sample working solutions were: 1.00, 2.00, 5.00, 10.0, 50.0, 250, 400, 500 ng / mL, and the concentrations of lanreotide quality control sample working solutions were: 1.00, 3.00, 20.0, 200, 380 ng / mL; Preparation of internal standard stock solution and working solution: Take lanreotide-D8 internal standard reference standard substance, add dimethyl sulfoxide solution to prepare 1.00 mg / mL lanreotide-D8 internal standard stock solution, take the internal standard stock solution and dilute it 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 concentrations of lanreotide standard curve samples are 0.0500, 0.100, 0.250, 0.500, 2.50, 12.5, 20.0, and 25.0 ng / mL; the concentrations of lanreotide quality control samples are: 0.0500 (LLOQQC), 0.150 (LQC), 1.00 (MQC-1), 10.0 (MQC-2), and 19.0 (HQC) ng / mL; for sample analysis, they are LQC (0.150 ng / mL), MQC-1 (1.00 ng / mL), MQC-2 (10.0 ng / mL), and HQC (19.0 ng / mL).

9. The novel high performance liquid chromatography-tandem mass spectrometry analysis method for detecting lanreotide in human plasma according to claim 1, characterized in that: Sample processing method: (1) Add 50 μL of standard curve sample, quality control sample, plasma sample to be tested, and blank sample to a 96-well deep-well plate; (2) Add 30 μL of 10.0 ng / mL lanreotide-D8 internal standard working solution; (3) Add 100 μL of 1 M sodium carbonate aqueous solution to each well and shake to mix; (4) Add 600 μL of ethyl acetate:isopropanol (90:10, v / v) extraction solution to each well; (5) Transfer 400 μL of the supernatant to a new 96-well low-adsorption plate and blow dry with nitrogen gas; (6) Add 100 μL of 30% acetonitrile aqueous solution containing 0.1% formic acid to each well; (7) After mixing, prepare for sample injection and analysis.

10. The novel high performance liquid chromatography-tandem mass spectrometry analysis method for detecting lanreotide in human plasma according to any one of claims 1 to 9, characterized in that: The standard curve and the method for calculating the test results are as follows: 50 μL of the standard curve, quality control, plasma sample to be tested and blank sample are taken respectively, and LC-MS / MS analysis is performed according to the operation of claim 9, and the chromatogram is recorded, and a series of theoretical concentration values ​​of lanreotide in the standard curve sample are used as the horizontal coordinate, and the ratio of the peak area of ​​lanreotide and the internal standard lanreotide-D8 is used as the vertical coordinate, and the weighted least squares method is used for regression operation. The linear regression equation obtained is the 0.0500-25.0 ng / mL standard curve, and the ratio of the peak area of ​​lanreotide in the unknown plasma sample and the internal standard lanreotide-D8 is substituted into the equation to obtain the blood concentration of lanreotide in the unknown sample.

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