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

Abstract

This invention discloses a novel high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) method for detecting abaloidin in human plasma. Teriparatide is used as the internal standard analyte. Solid-phase extraction is employed to extract both abaloidin and teriparatide from plasma samples. The standard curve is linear in the range of 0.010–1.00 ng / mL. During method validation, selectivity, matrix effect, recovery, sensitivity, linearity, accuracy and precision, stability, and dilution reliability were evaluated and met the standards. This method is specific for the determination of abaloidin and teriparatide in human plasma, and the standard curve is linear in the range of 0.010–1.00 ng / mL. The limit of quantitation is 0.01 ng / mL. It exhibits good accuracy, reliability, high sensitivity, high specificity, and reproducibility.

Description

Technical Field

[0001] This invention relates to a method for detecting abalopeptide, specifically to a novel high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) method for detecting abalopeptide in human plasma. Background Technology

[0002] Abalotropic peptide is an analogue of parathyroid hormone protein that selectively activates the parathyroid hormone type 1 receptor signaling pathway, promoting bone formation and increasing bone density. It is used to treat postmenopausal women at risk of osteoporosis. Measuring the concentration of abalotropic peptide in human plasma is of great significance for studying drug metabolism and establishing drug concentration-pharmacological relationships.

[0003] Previously, radioimmunoassay was used to determine the concentration of abalopeptide in human plasma. While radioimmunoassay has the advantage of high sensitivity, it also has many drawbacks, leading to significant limitations in its application.

[0004] Disadvantages of radioimmunoassay:

[0005] 1. The reagents used are radioactive, causing radioactive pollution, and conventional laboratories are unlikely to have the necessary conditions for their use;

[0006] 2. The detection principle is based on antigen-antibody immune reaction, which requires the preparation of highly specific antibody reagents, and the preparation process is complex and time-consuming;

[0007] 3. The reagent has a short half-life and high usage cost;

[0008] 4. It has poor specificity, is prone to cross-reaction, and is difficult to distinguish between the analyte and structural analogues;

[0009] 5. Narrow linear range, requiring sample dilution before detection, which can easily lead to detection errors;

[0010] Existing technology:

[0011] (Clinical Drug Investigation (2021) 41:277–285 https: / / doi.org / 10.1007 / s40261-021-01008-7) disclosed a radioimmunoassay for abalotide, but it has low sensitivity, causes environmental pollution, requires labeling with radioactive elements (I-125), and special antibody reagents (which must not cross-react with natural PTH or PTHrP, so preparation and acquisition are relatively difficult). The preparation and acquisition of the reagents are quite challenging.

[0012] Therefore, there is an urgent need in the market for a better method for detecting abalopeptides. Summary of the Invention

[0013] Purpose of the invention: To address the shortcomings of existing detection technologies, this invention provides a novel high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) method for the accurate, reliable, highly sensitive, highly specific, and reproducible detection of abalopeptide in human plasma.

[0014] Technical solution: The novel high-performance liquid chromatography-tandem mass spectrometry method for detecting abaloidin in human plasma described in this invention uses teriparatide as the internal standard analyte. The analyte abaloidin and the internal standard teriparatide are extracted from the plasma sample by solid-phase extraction. The standard curve is linear in the range of 0.010 to 1.00 ng / mL.

[0015] 1. Reference standards used in the test

[0016] Abalopeptide Standard Teriparatide

[0017] 2. The volumes of the reagents prepared during the testing process can be adjusted proportionally as needed.

[0018] 1) MPA: Contains an aqueous solution of 0.1% formic acid.

[0019] Preparation method: Add 1000mL of ultrapure water to a 1000mL reagent bottle, add 1.0mL of formic acid, and mix well. Store at room temperature. Shelf life is two weeks from the date of preparation.

[0020] 2) MPB: a solution containing 0.1% formic acid and acetonitrile.

[0021] Preparation method: Take 1000 mL of acetonitrile and add 1.0 mL of formic acid to a 1000 mL reagent bottle, then mix well. Store at room temperature. Shelf life is two weeks from the date of preparation.

[0022] 3) Needle Wash: A 50% methanol aqueous solution containing 0.1% formic acid.

[0023] Preparation method: Add 500 mL of methanol and 500 mL of ultrapure water to a 1000 mL reagent bottle, add 1.0 mL of formic acid, and mix well. Store at room temperature. Shelf life is one month from the date of preparation.

[0024] mass spectrometry API7500,AppliedBiosystems / Sciex Liquid chromatography LC-40, Shimadzu Automatic sampler RackChanger II Sample Rack Switching Device SIL-20ACHT Autosampler Data acquisition software SciexOS software Data processing software Microsoft Office 2013 or other versions <![CDATA[WatsonLIMS TM 7.5SP1,Thermo]]>

[0025] 3. Instruments and equipment

[0026] 4. Liquid Chromatography Conditions

[0027]

[0028]

[0029] 5. Mass spectrometry conditions

[0030]

[0031]

[0032] 6. Preparation of analyte stock solutions

[0033] Accurately weigh the abalopeptide reference standard, adjust the concentration by the correction factor, add an appropriate amount of DMSO, and prepare abalopeptide stock solution with a concentration of 1.00 mg / mL. Store the stock solution in a refrigerator at -10 to -30°C until use.

[0034] 7. Preparation of internal standard stock solution and working solution

[0035] Accurately weigh teriparatide reference standard, adjust the concentration using a correction factor, and add an appropriate amount of DMSO to prepare teriparatide stock solutions with a concentration of 1.00 mg / mL. Store at -10 to -30°C for later use. Take 10 μL of teriparatide stock solution and add it to 1000 mL of methanol-water mixture (50:50, v / v) containing 0.1% human plasma.

[0036] 8. Preparation of standard curve samples and quality control samples

[0037] Roche's cOmplete was added to human blood plasma at a ratio of 1:99. TM The final concentration of the protease inhibitor in plasma was 0.2 mg / mL.

[0038] Using human plasma for sequential dilution, the standard curve contained eight different concentration points, with abalotropic peptide concentrations of 0.010, 0.020, 0.050, 0.100, 0.200, 0.400, 0.800, and 1.00 ng / mL.

[0039] By using human plasma for sequential dilution, the quality control samples contained four different concentrations of abalopeptide: 0.010 (LLOQ QC), 0.030 (LQC), 0.400 (MQC), and 0.750 (HQC) ng / mL.

[0040] For method validation precision and accuracy, and personnel and instrument validation analysis batches, each accuracy and precision analysis batch shall contain 4 quality control samples at 4 concentration levels, with at least 5 replicates for each concentration. For other analytical batches and sample analyses for method validation, the quality control samples shall include at least three concentration levels of quality control samples: LQC, MQC and HQC, with at least 2 replicates for each concentration.

[0041] 9. Sample processing methods

[0042]

[0043]

[0044] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0045] 1) A high-performance liquid chromatography-tandem mass spectrometry method for the determination of abalopeptide in human plasma was established for the first time;

[0046] 2) This method overcomes the poor sensitivity of high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) and achieves extremely high sensitivity. The sensitivity of this invention is 10 pg / ml, while the sensitivity of existing technologies is 20 pg / ml, representing a doubling of the sensitivity. The method's limit of quantitation is 0.01 ng / mL, meeting the requirements for clinical trial detection.

[0047] 3) Through simple solid-phase extraction, interfering substances in human plasma can be removed, analytes can be enriched, and matrix effects can be improved;

[0048] 4) Adding protease inhibitors to plasma samples stabilizes the analytes;

[0049] 5) The methodology uses standards to prepare standard curves and internal standards to correct results, resulting in good accuracy of the test results;

[0050] 6) 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 abalopeptide;

[0051] 7) This methodology takes less than 5 minutes to test a sample, which can meet the high-throughput requirements for biological sample testing in clinical research;

[0052] 8) This high-performance liquid chromatography-tandem mass spectrometry method does not require the preparation of additional antibody reagents and is simple to operate;

[0053] 9) This method does not require radioactive reagents, thus protecting the environment from radioactive contamination;

[0054] 10) No antibody reagents or radioactive reagents required; better specificity and reproducibility; wider linear range.

[0055] 11) The matrix for preparing the standard curve used plasma with added protease inhibitors to protect abalotide from degradation by endogenous proteases in human plasma. Standards were used to prepare the standard curve, and teriparatide was used as an internal standard for result correction. The test results showed good accuracy and reproducibility.

[0056] 12) Solid-phase extraction was used to purify and enrich abalopeptide, which effectively removed interfering substances, improved matrix effect, enriched the analyte, and improved detection sensitivity.

[0057] 13) The loading, elution, and elution solvents for solid-phase extraction were fully optimized to achieve satisfactory extraction recovery rates;

[0058] 14) The liquid phase conditions have been optimized to ensure that the analyte abalopeptide is well separated from the interfering peaks, thus improving the detection sensitivity; the mass spectrometry conditions have been optimized to achieve the best instrument response signal and a good signal-to-noise ratio.

[0059] 15) Adding a trace amount of human plasma when preparing the working solution helps prevent non-specific adsorption;

[0060] 16) Using low-adsorption plates effectively reduces the non-specific adsorption of abalopeptide. Attached Figure Description

[0061] Figure 1 This is a Q1 scan of abalopeptide;

[0062] Figure 2 This is a Q3 scan of abalopeptide;

[0063] Figure 3 Chromatogram of abalopeptide in human plasma with two blank samples;

[0064] Figure 4 The chromatogram of abalopeptide in a single blank sample in human plasma;

[0065] Figure 5 This is a chromatogram of abaloxetine at the lower limit of quantification (LLOQ) in human plasma. Detailed Implementation

[0066] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of the present invention will be further described below.

[0067] (I) The detection method in this embodiment is as follows:

[0068] 1. Reference standards used in the test

[0069] Abalopeptide Standard Teriparatide

[0070] 2. The volumes of the reagents prepared during the testing process can be adjusted proportionally as needed.

[0071] 4) MPA: Contains a 0.1% formic acid aqueous solution.

[0072] Preparation method: Add 1000mL of ultrapure water to a 1000mL reagent bottle, add 1.0mL of formic acid, and mix well. Store at room temperature. Shelf life is two weeks from the date of preparation.

[0073] 5) MPB: a solution containing 0.1% formic acid and acetonitrile.

[0074] Preparation method: Take 1000 mL of acetonitrile and add 1.0 mL of formic acid to a 1000 mL reagent bottle, then mix well. Store at room temperature. Shelf life is two weeks from the date of preparation.

[0075] 6) Needle Wash: A 50% methanol aqueous solution containing 0.1% formic acid.

[0076] Preparation method: Add 500 mL of methanol and 500 mL of ultrapure water to a 1000 mL reagent bottle, add 1.0 mL of formic acid, and mix well. Store at room temperature. Shelf life is one month from the date of preparation.

[0077] 3. Instruments and equipment

[0078]

[0079]

[0080] 4. Liquid Chromatography Conditions

[0081]

[0082] 5. Mass spectrometry conditions

[0083]

[0084]

[0085]

[0086] 6. Preparation of analyte stock solutions

[0087] Accurately weigh the abalopeptide reference standard, adjust the concentration by the correction factor, add an appropriate amount of DMSO, and prepare abalopeptide stock solution with a concentration of 1.00 mg / mL. Store the stock solution in a refrigerator at -10 to -30°C until use.

[0088] 7. Preparation of internal standard stock solution and working solution

[0089] Accurately weigh teriparatide reference standard, adjust the concentration using the correction factor, and add an appropriate amount of DMSO to prepare teriparatide stock solutions with a concentration of 1.00 mg / mL. Store the stock solutions at -10 to -30°C until use. Take 10 μL of the teriparatide stock solution and add it to 1000 mL of methanol-water solution (50:50, v / v) containing 0.1% human plasma.

[0090] 8. Preparation of standard curve samples and quality control samples

[0091] Roche's cOmplete was added to human blood plasma at a ratio of 1:99. TMThe final concentration of the protease inhibitor in plasma was 0.2 mg / mL.

[0092] Using human plasma for sequential dilution, the standard curve contained eight different concentration points, with abalotropic peptide concentrations of 0.010, 0.020, 0.050, 0.100, 0.200, 0.400, 0.800, and 1.00 ng / mL.

[0093] By using human plasma for sequential dilution, the quality control samples contained four different concentrations of abalopeptide: 0.010 (LLOQ QC), 0.030 (LQC), 0.400 (MQC), and 0.750 (HQC) ng / mL.

[0094] For method validation precision and accuracy, and personnel and instrument validation analysis batches, each accuracy and precision analysis batch shall contain 4 quality control samples at 4 concentration levels, with at least 5 replicates for each concentration. For other analytical batches and sample analyses for method validation, the quality control samples shall include at least three concentration levels of quality control samples: LQC, MQC and HQC, with at least 2 replicates for each concentration.

[0095] 9. Sample processing methods

[0096] 1. Take the connecting tube and add the sample as described below: For double blank samples and zero-concentration samples, add 300 μL of mixed blank matrix; for standard curve samples, quality control samples, test samples, and system suitability samples, add 300 μL of the corresponding blank matrix sample.

[0097] 2. For zero blank samples, zero concentration samples, standard curve samples, quality control samples, test samples, and system suitability samples, add 10.0 μL of internal standard working solution (10.0 ng / mL). For double blank samples, add 10.0 μL of internal standard preparation solvent (50% methanol-water containing 0.1% human plasma) and centrifuge at 3000g for 1 min.

[0098] 3. Add 200 μL of methanol / water (40 / 60; v / v) solution to each well;

[0099] 4. Shake at 2500 rpm for 5 minutes;

[0100] 5. Centrifuge at 3500g for 5 minutes at 4℃;

[0101] 6. Take a new 96-well plate, take 450 μL of the supernatant from step 5 above, and then add 550 μL of water;

[0102] 7. Shake at 800 rpm for 10 minutes;

[0103] 8. Take the Oasis HLB uElution solid phase extraction plate and perform solid phase extraction:

[0104] a) → Activation: Activate with 200uL of methanol;

[0105] b) → Balance: Balance with 200uL of ultrapure water;

[0106] c) → Sample loading: Transfer 800 μL of the diluted supernatant onto the extraction plate in two separate transfers of 400 μL each time. Adjust the pressure valve to allow the sample to slowly pass through the solid-phase extraction plate.

[0107] d) → Rinse once with 1:250uL ultrapure water;

[0108] e) → Clean once with 2:250uL methanol / water (10 / 90, v / v);

[0109] f) → Elution: 25.0 μL acetonitrile / trifluoroacetic acid / water (75 / 1 / 25, w / w / v, elute three times) + added to each well;

[0110] 50.0 μL of ultrapure water was mixed evenly, and this step was received using a 96-well low-absorption plate.

[0111] g) → Centrifuge at 3500g for 1 min at 4℃ for the detection of abalopeptide.

[0112] Using an LC-MS / MS instrument, the analysis was performed according to "4. Liquid Chromatography Conditions" and "5. Mass Spectrometry Conditions". The injection volume for each sample was 5 μL, and the chromatograms were recorded.

[0113] Using the theoretical concentrations of abaloidin in the standard curve samples as the x-axis and the ratio of the peak areas of abaloidin to the internal standard teriparatide as the y-axis, a weighted least squares regression was performed to obtain the linear regression equation, which forms the standard curve for 0.010–1.00 ng / mL. Substituting the ratio of the peak areas of abaloidin to the internal standard teriparatide in the unknown plasma sample into the equation yields the blood concentration of abaloidin in the unknown sample.

[0114] (II) Methodological Validation

[0115] (1) Method:

[0116]

[0117]

[0118]

[0119]

[0120] (2) Results:

[0121] 1. Selectivity

[0122] The maximum interference effect at the analyte retention time was 0.0% for blank plasma from six different individuals, one hemolyzed matrix, and one hyperlipidemic matrix, with each individual preparing a double blank. This was less than 20.0% of the average peak area of ​​the analyte in the LLOQ standard curve samples that met the acceptance criteria. The maximum interference effect at the internal standard retention time was also 0.0%, less than 5.0% of the average peak area of ​​the internal standard in all standard curve samples and quality control samples that met the acceptance criteria.

[0123] Selectivity study of individuals from different sources, hemolyzed matrix, and hyperlipidemic matrix

[0124]

[0125] The interference of the analyte on the internal standard was 0.0% in the ULOQ sample of abalopeptide without internal standard at the retention time of the internal standard, and the peak area of ​​the interference peak was less than 5.0% of the average peak area of ​​the internal standard in all standard curve samples and quality control samples that met the acceptance criteria.

[0126] Interference of the analyte to the internal standard

[0127]

[0128] The interference of the internal standard on the analyte was 0.0% at the retention time of the analyte in the zero-concentration sample of the analytical batch, and the peak area of ​​the interference peaks was less than 20.0% of the average peak area of ​​the analyte in the LLOQ standard curve samples that met the acceptance criteria in the analytical batch.

[0129] Interference of internal standard with analyte

[0130]

[0131] 2. Matrix effect

[0132] The matrix effects of normal matrix as well as those of hyperlipidemic and hemolytic matrix met the acceptance criteria.

[0133] Normal individual matrix (LQC concentration)

[0134]

[0135]

[0136] Note: "-" is not applicable.

[0137] Normal individual matrix (HQC concentration)

[0138]

[0139]

[0140] Note: "-" is not applicable.

[0141] 3. Hemolytic matrix

[0142]

[0143] Note: "-" is not applicable.

[0144] 4. High-fat matrix

[0145]

[0146] Note: "-" is not applicable

[0147] 5. Standard Curve

[0148] The standard curve results meet the acceptance criteria.

[0149]

[0150] The standard curve results meet the acceptance criteria.

[0151]

[0152]

[0153] 6. Accuracy and precision

[0154] Accuracy and precision experiments must be conducted on at least three independent analytical batches over a period of at least two days. Quality control samples for accuracy and precision experiments must be freshly prepared on the same day. Each accuracy and precision analytical batch must contain quality control validation samples at five concentration levels: 0.010 (LLOQ QC), 0.030 (LQC), 0.400 (MQC), and 0.750 (HQC) ng / mL, with at least five replicates for each concentration.

[0155] The precision of the method is expressed as a percentage of the coefficient of variation (CV% = [(standard deviation / mean) × 100%]); the accuracy of the method is expressed as a percentage of the relative error (RE% = [(mean observed concentration - nominal concentration) / nominal concentration] × 100%).

[0156] Intra-batch and inter-batch accuracy and precision meet the acceptance criteria for methodology validation.

[0157] Intra-batch and inter-batch accuracy and precision results.

[0158]

[0159]

[0160] 7. Extraction recovery rate

[0161] The extraction and recovery rate of the analyte met the acceptable standard.

[0162]

[0163] The dilution reliability sample (5.00 ng / mL) was diluted 10-fold with mixed human plasma, and the results met the acceptance criteria.

[0164]

[0165]

[0166] Plasma samples remained stable after five freeze-thaw cycles (at room temperature) at -10 to -30°C and five freeze-thaw cycles (at room temperature) at -60 to -90°C.

[0167]

[0168] Plasma samples stored at -10 to -30°C for 135 days and at -60 to -90°C for 135 days showed acceptable stability.

[0169]

[0170] The aim of this study was to validate the methodology for the quantitative determination of abalopeptide concentration in human plasma using liquid chromatography-mass spectrometry (LC-MS / MS) to support clinical research on abalopeptide. During method validation, selectivity, matrix effect, recovery, sensitivity, linearity, accuracy and precision, stability, and dilution reliability were evaluated.

[0171] This method validation experiment established a liquid chromatography-tandem mass spectrometry (LC-MS / MS) method for determining the concentration of abaloidin in human plasma. The method uses teriparatide as the internal standard analyte (IS), and employs solid-phase extraction to extract the analyte abaloidin and the internal standard teriparatide from plasma samples. The method is specific for the determination of abaloidin and teriparatide in human plasma, and the standard curve is linear in the range of 0.010–1.00 ng / mL. Plasma samples remained stable after five freeze-thaw cycles (at room temperature) at -10 to -30°C and five freeze-thaw cycles (at room temperature) at -60 to -90°C. Plasma samples stored at -10 to -30°C for 135 days and at -60 to -90°C for 135 days showed acceptable stability.

[0172] The above are merely preferred embodiments of the present invention and do not constitute any limitation on the present invention. Any equivalent substitutions or modifications made by those skilled in the art to the technical solutions and content disclosed in the present invention without departing from the scope of the present invention shall be deemed to have remained within the protection scope of the present invention.

Claims

1. A high-performance liquid chromatography-tandem mass spectrometry method for detecting abalopeptide in human plasma, characterized in that, Teriparatide was used as an internal standard. The analyte abalotropic peptide and the internal standard teriparatide were extracted from plasma samples by solid-phase extraction. The standard curve was linear in the range of 0.010 to 1.00 ng / mL. The instruments used for the detection were: API 7500 mass spectrometer and LC-40 liquid chromatograph. The liquid chromatography conditions for the detection were as follows: ACE C18 column, 50*2.1mm, 3μm; mobile phase A was an aqueous solution containing 0.1% formic acid, and mobile phase B was an acetonitrile solution containing 0.1% formic acid. The mass spectrometry conditions for the detection were as follows: ion ejection voltage 5000V, temperature 550℃, gas 1 GS1 was N2 at a pressure of 55 p.si; gas 2 GS2 was N2 at a pressure of 55 psi; curtain gas CUR was N2 at a pressure of 45 psi; collision gas CAD was N2 at a pressure of 12 psi; ion source ESI; detection mode positive ion mode; scanning mode multi-ion reaction monitoring. The monitored ion pairs were: abalopeptide 566.7→632.8, teriparatide 687.4→787.

4. The solid-phase extraction includes: using an Oasis HLB uElution solid-phase extraction plate, with acetonitrile / trifluoroacetic acid / water as the eluent; The preparation of the standard curve includes adding a protease inhibitor to human plasma.

2. The method according to claim 1, characterized in that, The solid-phase extraction uses Oasis HLB uElution plates and includes activation, equilibration, loading, washing, and elution steps.

3. The method according to claim 1, characterized in that, The preparation of the standard curve includes: sequentially diluting the abalopeptide stock solution with plasma containing the inhibitor to obtain a standard curve sample with at least 8 concentration points.

4. The method according to claim 1, characterized in that, The sample processing method includes: adding internal standard working solution to plasma sample, followed by protein precipitation and dilution, solid phase extraction, and analysis of the resulting eluent by LC-MS / MS to calculate the concentration of abalopeptide using the internal standard method.

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