Method for detecting estradiol 17 beta-D glucuronic acid in cell lysis buffer

Abstract

The invention belongs to the field of quantitative analysis of drug concentration of a cell lysis solution, and particularly discloses a method for detecting estradiol 17 beta-D glucuronic acid (Estradil 17-(beta-D-Glucuronide)) in the cell lysis solution. According to the method, the concentration of the estradiol 17 beta-D glucuronic acid in the cell lysis buffer can be accurately quantified through specific sample treatment and liquid chromatography and mass spectrometry conditions, the sensitivity is high, the detection limit is low, a novel method capable of being practically popularized and applied is provided for pharmacokinetic research of the estradiol 17 beta-D glucuronic acid, and the method has popularization and application values.

Description

Technical Field

[0001] This invention relates to the field of quantitative analysis of drug concentration in cell lysates, and specifically to a method for detecting estradiol 17β-D glucuronic acid in cell lysates. Background Technology

[0002] Organic anion transport peptides OATP1B1 and OATP1B3 belong to the solute carrier (SLC) family and are mainly expressed on the basolateral membrane of hepatocytes. They are responsible for mediating the hepatic uptake of various endogenous substances and drugs. As the rate-limiting step in drug metabolism in the liver, their transport efficiency affects the total drug clearance rate and is a key aspect in pharmacokinetic drug interaction studies. Therefore, establishing reliable in vitro functional evaluation models is of great significance for accurately predicting drug interactions and metabolic characteristics.

[0003] Estradiol 17-(β-D-Glucuronide) (E217G), a major metabolite of estradiol, is a specific substrate for OATP1B1 and OATP1B3. Accumulation of this substance in hepatocytes can induce cholestatic liver injury and is considered an important pathological factor in estrogen-related liver diseases such as intrahepatic cholestasis of pregnancy. Monitoring its intracellular concentration helps assess the degree of liver dysfunction and related disease activity. Based on this mechanism, E217G has become a landmark probe substrate for evaluating the function of OATP1B1 / OATP1B3. By quantitatively detecting its concentration in OATP1B1 / OATP1B3 cells and calculating the uptake rate, the transport activity in in vitro models can be effectively verified, thus providing a standardized functional evaluation tool for hepatic pharmacokinetic studies and the elucidation of cholestatic toxicity mechanisms.

[0004] However, there are currently no reported analytical methods for detecting estradiol 17β-D-glucuronide concentration in OATP1B1 / OATP1B3 cells. To assess transporter functional status and predict the risk of drug-induced liver injury, it is necessary to provide a method for detecting estradiol 17β-D-glucuronide concentration in cell lysates to fill the current technological gap and meet the urgent needs of scientific research and preclinical evaluation. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a method for detecting estradiol 17β-D glucuronic acid in cell lysates. This method utilizes UPLC-MS / MS liquid chromatography-mass spectrometry and includes the following steps:

[0006] (1) Establishment of the standard curve

[0007] a. Preparation of working solutions for a series of concentration standard curves: Take estradiol 17β-D glucuronic acid standard material, dissolve it in dimethyl sulfoxide to obtain estradiol 17β-D glucuronic acid standard curve stock solution; take estradiol 17β-D glucuronic acid standard curve stock solution and dilute it with acetonitrile aqueous solution to prepare standard curve working solution.

[0008] Take the standard curve working solution and add blank matrix to prepare standard curve samples. Add internal standard working solution to the standard curve samples, mix well, transfer to protein precipitation plate, extract under positive pressure, and take the filtrate and mix with pure water to obtain the standard curve test sample.

[0009] b. Take the standard curve test samples and inject them into the UPLC-MS / MS liquid chromatography-mass spectrometry instrument, detect the peak area, and fit the regression curve of estradiol 17β-D glucuronic acid.

[0010] The chromatographic conditions are as follows:

[0011] Chromatographic column: T3 column; mobile phase A: water, mobile phase B: acetonitrile; gradient elution program:

[0012]

[0013] Mass spectrometry conditions: ESI ion source - Detection mode: Negative ion multiple reaction monitoring;

[0014] (2) Detection of unknown sample concentration:

[0015] c. Unknown sample processing

[0016] Take an unknown sample, add internal standard working solution, mix well, transfer to a protein precipitation plate, extract under positive pressure, take the filtrate and mix with pure water to obtain the sample to be tested;

[0017] d. Detection of the sample to be tested

[0018] Take the sample to be tested obtained in step c, inject it into the UPLC-MS / MS liquid chromatography-mass spectrometry instrument, and detect it under the same conditions as in step b. The concentration of estradiol 17β-D glucuronic acid in the sample to be tested is obtained according to the standard curve in step (1).

[0019] The unknown sample was a cell lysate obtained by lysing cells incubated with estradiol 17β-D glucuronic acid solution using pure water via liquid nitrogen freeze-thaw lysis.

[0020] Furthermore, step (2) of the concentration detection process also includes a quality control procedure, the specific steps of which are as follows:

[0021] Estradiol 17β-D-glucuronic acid standard was dissolved in dimethyl sulfoxide to obtain estradiol 17β-D-glucuronic acid quality control stock solution. The obtained estradiol 17β-D-glucuronic acid quality control stock solution was diluted with acetonitrile aqueous solution to obtain quality control working solution. The quality control working solution was taken and a blank matrix was added to prepare a quality control sample. The internal standard working solution was added to the quality control sample, mixed well, and transferred to a protein precipitation plate. Positive pressure extraction was performed, and the filtrate was mixed with pure water and injected into a UPLC-MS / MS liquid chromatography-mass spectrometry instrument as a quality control for the sample detection process.

[0022] The volume ratio of the quality control working solution, blank matrix, and internal standard working solution is 1:9:40;

[0023] The concentration range of estradiol 17β-D glucuronic acid in the quality control working solution is 20–15000 nM.

[0024] Furthermore, the volume ratio of the filtrate to pure water is 1:1.

[0025] Further, the volume ratio of the standard curve working solution, blank matrix, and internal standard working solution in step a is 1:9:40; the concentration range of estradiol 17β-D glucuronic acid in the standard curve working solution is 20–20000 nM.

[0026] Furthermore, the blank matrix is ​​a cell lysate obtained by freezing and thawing cells with pure water using liquid nitrogen.

[0027] Furthermore, the internal standard working solution is prepared by dissolving toluenebutyrate standard substance in dimethyl sulfoxide and then diluting it with acetonitrile to obtain a 0.5 ng / mL toluenebutyrate internal standard working solution.

[0028] Furthermore, the proportion of the acetonitrile aqueous solution is 50%.

[0029] Further, the chromatographic column in step b is an ACQUITYUPLC-HSS T3, 1.8 μm, 2.1 × 50 mm, Waters; the injection volume is 4 μL; the column temperature is 40℃; and the flow rate is 0.4 mL / min.

[0030] Further, in step b, the mass spectrometry conditions include: scanning time 4 min; capillary voltage 2.5 kV; ion source temperature 150 °C; desolvent gas temperature 550 °C; cone gas flow rate 150 L / h; desolvent gas flow rate 1000 L / h; and nebulizer gas pressure 6.0 bar.

[0031] Furthermore, in the mass spectrometry conditions, the detection ion pair of estradiol 17β-D glucuronic acid is m / z 446.92→m / z 271.15, the cone voltage is 48V, and the collision energy is 28eV; the detection ion pair of tolbutamide is m / z 268.91→m / z 170.06, the cone voltage is 28V, and the collision energy is 16eV.

[0032] The cells described in this invention are cells transfected with human OATP1B1 or OATP1B3.

[0033] This invention provides a method for detecting estradiol 17β-D-glucuronic acid in cell lysates. Through specific sample processing and chromatographic / mass spectrometric conditions, it accurately quantifies estradiol 17β-D-glucuronic acid in cell lysates obtained by freeze-thawing cells in pure water using liquid nitrogen. The method exhibits high sensitivity and a low detection limit. Furthermore, this invention accurately quantifies estradiol 17β-glucuronic acid (E217G) in OATP1B1 / OATP1B3 cell uptake experiments. By precisely measuring the concentration of E217G in cell lysates and calculating its uptake rate, it provides a standardized in vitro drug evaluation system for in vitro pharmacokinetic studies, accurately assessing the functional activity of the OATP1B1 and OATP1B3 transporters.

[0034] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0035] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0036] Figure 1 Chromatograms of estradiol 17β-D glucuronic acid and internal standard in blank sample;

[0037] Figure 2 Chromatograms of estradiol 17β-D glucuronic acid and internal standard in zero-concentration sample;

[0038] Figure 3 Chromatograms of estradiol 17β-D glucuronic acid and internal standard in the sample (STD1) at the lowest point of the standard curve;

[0039] Figure 4 Chromatograms of estradiol 17β-D glucuronic acid and internal standard in the sample (STD7) at the highest point of the standard curve;

[0040] Figure 5Chromatograms of estradiol 17β-D glucuronic acid and internal standard in the carryover residue;

[0041] Figure 6 Typical standard curve for detecting estradiol 17β-D glucuronic acid in cell lysates using UPLC-MS / MS. Detailed Implementation

[0042] Example 1: Detection of estradiol 17β-D glucuronic acid concentration in cell lysate

[0043] (I) Solution preparation

[0044] ①Standard curve stock solution and quality control stock solution

[0045] Take estradiol 17β-D glucuronic acid standard material and add dimethyl sulfoxide to prepare a 10 mM estradiol 17β-D glucuronic acid standard curve / quality control stock solution;

[0046] ② Internal standard working solution

[0047] Prepare a 1 mg / mL tolbutamide internal standard stock solution by adding dimethyl sulfoxide to tolbutamide standard material;

[0048] Take the tolbutamide internal standard stock solution and dilute it with acetonitrile to prepare a 0.5 ng / mL tolbutamide internal standard working solution;

[0049] ③ Blank substrate: Cells were lysed using pure water via liquid nitrogen freeze-thaw cycles, and the lysates were collected;

[0050] ④ Unknown samples: Cells incubated with estradiol 17β-D glucuronic acid solution were lysed with pure water by liquid nitrogen freeze-thaw, and the lysate was collected;

[0051] Note: The cells in the blank matrix should be the same as the cells in the unknown sample. Specifically, the cells used should be cells transfected with human OATP1B1 or OATP1B3.

[0052] (II) Establishment of the standard curve

[0053] a. Preparation of a series of concentration standard curve samples:

[0054] Take the stock solution of estradiol 17β-D glucuronic acid standard curve and dilute it with 50% acetonitrile aqueous solution to prepare the working solution of estradiol 17β-D glucuronic acid standard curve with a concentration range of 20-20000 nM.

[0055] Take 5 μL of standard curve working solution and add 45 μL of blank matrix to mix well to obtain standard curve samples; add 200 μL of internal standard working solution to 50 μL of standard curve samples, vortex mix for 5 min, and then transfer all to protein precipitation plate. Extract under positive pressure to another new 96-well plate, take 200 μL of filtrate, add 200 μL of pure water, mix well, and the standard curve test sample is obtained.

[0056] b. Inject the standard curve test samples into the UPLC-MS / MS liquid chromatography-mass spectrometry instrument and detect the peak area. Plot the concentration of estradiol 17β-D glucuronic acid in the test samples of the standard curve as the abscissa (X) and the ratio of the peak area of ​​estradiol 17β-D glucuronic acid to the internal standard as the ordinate (Y). Use the weighted least squares method (W = 1 / X) to calculate the peak area. 2 Regression calculations are performed to fit the regression equation, which is the standard curve.

[0057] The chromatographic conditions are as follows:

[0058] The chromatographic column was an ACQUITY UPLC-HSS T3, 1.8 μm, 2.1 × 50 mm, Waters; column temperature 40℃; flow rate 0.4 mL / min; pure water was used as mobile phase A, acetonitrile as mobile phase B, and the gradient elution program was as follows:

[0059]

[0060] Mass spectrometry conditions: Ion source: Electrospray ionization source (ESI) - Scanning time: 4.0 min; Capillary voltage: 2.5 kV; Ion source temperature: 150℃; Desolvent gas temperature: 550℃; Cone gas flow rate: 150 L / h; Desolvent gas flow rate: 1000 L / h; Nebulizing gas pressure: 6.0 bar; Detection mode: Negative ion multiple reaction monitoring; Estradiol 17β-D glucuronic acid detection ion pair m / z 446.92→m / z 271.15, cone voltage 48 V, collision energy 28 eV; Tolbutamide detection ion pair m / z 268.91→m / z 170.06, cone voltage 28 V, collision energy 16 eV;

[0061] (III) Sample processing and testing

[0062] c. Handling of unknown samples and quality control samples

[0063] Take 50 μL of the unknown sample into a 96-well plate, add 200 μL of internal standard working solution, vortex mix for 5 min, then transfer the whole sample to a protein precipitation plate, extract under positive pressure into a new 96-well plate, take 200 μL of filtrate, add 200 μL of pure water, mix well, and the unknown sample to be tested is obtained.

[0064] Take estradiol 17β-D glucuronic acid quality control stock solution and dilute it with 50% acetonitrile aqueous solution to prepare estradiol 17β-D glucuronic acid quality control working solution with a concentration range of 20-15000 nM; take 5 μL of quality control working solution and add 45 μL of blank matrix to obtain quality control samples; add 200 μL of internal standard working solution to 50 μL of quality control samples, vortex mix for 5 min, and then transfer the whole solution to a protein precipitation plate. Extract the solution under positive pressure to another new 96-well plate, take 200 μL of filtrate, add 200 μL of pure water, mix well, and the quality control test sample is obtained.

[0065] d. Sample testing

[0066] Take the quality control sample and the unknown sample respectively, inject them into the UPLC-MS / MS liquid chromatography-mass spectrometry instrument, and detect them under the same conditions as in step b. Use the quality control sample as the quality control of the sample detection process to monitor the deviations in the detection process. The concentration of estradiol 17β-D glucuronic acid in the unknown sample is obtained according to the regression curve fitted in step (ii).

[0067] The following experimental examples illustrate the beneficial effects of the present invention.

[0068] Experimental Example 1: Methodological Validation of the Invention

[0069] 1. Method Overview

[0070] Proteins were precipitated with acetonitrile before analysis. The standard curve was obtained using the weighted least squares method (W = 1 / X). 2 Regression calculations were performed, and quantification was achieved by comparing the peak area of ​​the analyte with the peak area of ​​the internal standard. Relevant information is shown in Table 1 below.

[0071] Table 1. Information related to the detection method

[0072]

[0073] 2. Instruments and Chromatography-Mass Spectrometry Conditions

[0074] 2.1 Main Instruments and Equipment

[0075]

[0076] 2.2 Chromatographic conditions

[0077]

[0078]

[0079] Gradient elution program

[0080]

[0081] 2.3 Mass Spectrometry Conditions

[0082]

[0083] 3. Preparation of standard substances, reagents, solutions and blank matrix 3.1 Analyte standard substance Estradiol 17-(β-D-Glucuronide), source Apex Bio.

[0084] 3.2 Internal standard (IS) reference material: tolbutamide, purity 99.9%, source: Sigma-Aldrich.

[0085] 3.3 Main reagents: Pure water: Grade I water, Veolia water purifier, colorless and clear liquid.

[0086] 3.4 Blank matrix

[0087] Cell lysis buffer.

[0088] 4. Preparation of stock solutions, working solutions and samples

[0089] 4.1 Preparation of a stock solution of approximately 10 mM estradiol 17β-D glucuronic acid

[0090] Take Estradiol 17-(β-D-Glucuronide) standard material, add dimethyl sulfoxide, and prepare a 10 mM estradiol 17β-D glucuronic acid standard curve / quality control stock solution.

[0091] 4.2 Preparation of working solution

[0092] Prepare the standard curve working solution and quality control working solution according to Tables 2-3. The diluent is a 50% acetonitrile aqueous solution.

[0093] Table 2 Preparation of working solutions for standard curves

[0094]

[0095] Table 3 Preparation of Quality Control Working Solution

[0096]

[0097] 4.3 Matrix Sample Preparation

[0098] Prepare the standard curve and quality control samples according to Tables 4-5, and mix them thoroughly after preparation.

[0099] Table 4 Preparation of Standard Curve Samples

[0100]

[0101]

[0102] Table 5 Preparation of quality control samples

[0103]

[0104] In this study, the blank matrix consisted of pure water, which was used to lyse human OATP1B1 or OATP1B3 cells via liquid nitrogen freeze-thaw lysis, and the cell lysates were collected.

[0105] 4.4 Preparation of internal standard stock solution and working solution

[0106] 4.4.1 Preparation of 1 mg / mL tolbutamide internal standard stock solution

[0107] Take 10 mg of tolbutamide standard material into a polypropylene tube, add dimethyl sulfoxide, and prepare a 1 mg / mL tolbutamide internal standard stock solution.

[0108] 4.4.2 Preparation of 1 μg / mL tolbutamide internal standard intermediate solution

[0109] Pipette 100 μL of 1 mg / mL tolbutamide internal standard stock solution into a polypropylene tube, add 99.9 mL of acetonitrile, and mix well to obtain 1 μg / mL tolbutamide internal standard intermediate solution.

[0110] 4.4.3 Preparation of 0.5 ng / mL tolbutamide internal standard working solution

[0111] Pipette 100 μL of the internal standard intermediate solution into the internal standard bottle, add 199.9 mL of acetonitrile, and mix well to obtain the final solution.

[0112] 5. Sample processing

[0113] (1) Add 200 μL of acetonitrile to 50 μL of blank sample and vortex mix for about 5 min;

[0114] (2) Add 200 μL of internal standard working solution to all samples except Blank sample, and vortex mix for about 5 min;

[0115] (3) All were transferred to a protein precipitation plate and extracted under positive pressure into a new 96-well plate;

[0116] (4) Take 200 μL of filtrate into a new 96-well plate, add 200 μL of pure water, mix well and then analyze.

[0117] 6. Preparation of stability samples of stock solution and working solution

[0118] 6.1 Preparation method of stability sample for estradiol 17β-D glucuronic acid stock solution:

[0119] (1) Take 20 μL of stock solution and add 9980 μL of acetonitrile to dilute it to a solution containing estradiol 17β-D glucuronic acid at a concentration of 20 μM;

[0120] (2) Take 10 μL of (1), add 990 μL of acetonitrile, and mix well;

[0121] (3) Take 50 μL of (2), add 200 μL of internal standard working solution, shake to mix, then take 200 μL of the solution, add 200 μL of pure water, shake to mix, and then inject for analysis.

[0122] 6.2 Preparation method of working solution stability sample for estradiol 17β-D glucuronic acid:

[0123] (1) Take 20 μL of the working solution at the highest point of the standard curve and add 980 μL of 50% acetonitrile aqueous solution;

[0124] (2) Take 50 μL of (1), add 200 μL of internal standard working solution, shake to mix, then take 200 μL of the solution, add 200 μL of pure water, shake to mix, and then inject for analysis.

[0125] (3) Take 50 μL of the working solution at the lowest point of the standard curve, add 200 μL of the internal standard working solution, shake to mix, then take 200 μL of the solution, add 200 μL of pure water, shake to mix, and then inject for analysis.

[0126] 7. Data Processing

[0127] Deviation (%) = (Measured value - Theoretical value) ÷ Theoretical value × 100%

[0128]

[0129] Accuracy is expressed as bias, while precision is expressed as relative standard deviation (RSD) or coefficient of variation (CV).

[0130] Peak area is reported as an integer and used in calculations with full precision; concentration is retained to three significant figures, and bias, coefficient of variation (CV), and ratio are retained to one decimal place.

[0131] 8. Verification Results

[0132] 8.1 Linear Range of the Standard Curve

[0133] Prepare two samples for each standard curve, containing seven concentrations, and establish the standard curve. Plot the concentration (C) of the analyte in the biological sample on the x-axis (X) and the ratio of the analyte peak area to the internal standard peak area on the y-axis (Y), respectively. Use the weighted least squares method (W = 1 / X). 2Regression calculations are performed to fit a regression equation, which becomes the standard curve. The concentrations of the standard curve samples are calculated using the regression equation. Blank and zero-concentration samples are not included in the calculation. The deviation of their recalculated concentrations [(calculated value - theoretical value) / theoretical value × 100%] should be within ±15% (±20% for LLOQ). At least 75% of the samples, and at least 50% of each concentration, should meet the above requirements. Unqualified samples should be discarded, and regression calculations should be performed again.

[0134] The results showed that estradiol 17β-D-glucuronic acid exhibited good linearity within the sample concentration range of 2.00–2000 nM, and the results met the requirements. A typical chromatogram of estradiol 17β-D-glucuronic acid in cell lysates is shown below. Figures 1-4 Typical standard curves are shown below. Figure 6 .

[0135] 8.2 Residue

[0136] Inject a blank sample after the STD7 sample for analysis. The residual peak area of ​​the analyte should not exceed 20% of the average peak area of ​​the analyte in the qualified STD1 sample, and the residual peak area of ​​the internal standard should not exceed 5% of the average peak area of ​​the internal standard in the qualified STD1 sample.

[0137] The results showed that the residue of estradiol 17β-D glucuronic acid was less than 0.0%, and the residue of the internal standard was less than 0.1%, both meeting the requirements. A typical chromatogram is shown below. Figure 5 .

[0138] 8.3 Accuracy and Precision

[0139] Prepare six LLOQ, six low-, six medium-, and six high-concentration quality control samples (QC). Calculate the concentration of the QC samples using the standard curve for each batch, and then determine the precision and accuracy of the calculation method (prepare and measure three batches). The intra-batch and inter-batch precision (RSD) of the low-, medium-, and high-concentration QC samples should not exceed 15%, and the LLOQ should not exceed 20%. The accuracy (Bias) of the average value of the low-, medium-, and high-concentration QC samples should be within ±15%, and the LLOQ should be within ±20%. Simultaneously, at least two-thirds of the quality control samples, and at least 50% of the samples at the same concentration level, should meet the requirement that the accuracy (Bias) of the low-, medium-, and high-concentration QC samples is within ±15%, and the LLOQ is within ±20%. The signal-to-noise ratio of the LLOQ sample should be ≥5.

[0140] The results showed that the intra-batch precision (RSD) of estradiol 17β-D-glucuronic acid in the LLOQ was within 13.6%, and the mean accuracy (Bias) was within ±14.6%. The intra-batch precision (RSD) of low, medium, and high concentration QC samples was within 13.4%, and the mean accuracy (Bias) was within ±11.3%. The inter-batch precision (RSD) of the LLOQ was 12.1%, and the mean accuracy (Bias) was -6.9%. The inter-batch precision (RSD) of low, medium, and high concentration QC samples was within 11.0%, and the mean accuracy (Bias) was within ±10.6%. All results met the requirements. The signal-to-noise ratio (S / N) of the LLOQ samples in both precision and accuracy was >5, indicating that the results met the requirements.

[0141] 8.4 Stability of Stock Solution and Working Solution

[0142] The stability of estradiol 17β-D glucuronic acid stock solution was investigated after 143 days of storage at -25 to -15°C, and the stability of estradiol 17β-D glucuronic acid working solution was investigated after 42 days of storage at 2 to 8°C. The precision (RSD) of comparing the average peak area of ​​the stock or working solution stored for a period of time with the average peak area of ​​the freshly prepared solution should be within 10%, and the ratio of the two response values ​​should be within 100 ± 10%.

[0143] The results showed that estradiol 17β-D glucuronic acid was stable for 143 days when stored at -25 to -15℃; the working solution of estradiol 17β-D glucuronic acid was stable for 42 days when stored at 2 to 8℃. The precision (RSD) of all stable samples was within 10%, and the ratio of average peak area was within 100 ± 10%, all of which met the requirements.

[0144] 8.5 Stability of treated samples

[0145] After pretreatment, the samples of low and high concentrations (n=5) were stored in a sample manager (4-10℃) for about 73.2 hours before injection. The precision (RSD) of each concentration was calculated using a freshly prepared standard curve, and the mean deviation should be within ±15%.

[0146] The results showed that the samples were stable after being placed in the sample manager (4-10℃) for about 73.2 hours after treatment. The RSDs of the low and high concentrations of estradiol 17β-D glucuronic acid were 7.7% and 2.1%, respectively, with mean deviations of -10.6% and 7.9%, respectively. All results met the requirements.

[0147] 8.6 Stability of samples before treatment

[0148] The stability of low- and high-concentration samples (n=5) was examined, including stability after 3 freeze-thaw cycles and stability after long-term freezing at -66℃ for 7 days. The concentration of the analyte in the samples was calculated based on the standard curve, and the precision (RSD) of each concentration did not exceed 15%, with a mean deviation within ±15%.

[0149] The results showed that the low and high concentration samples of estradiol 17β-D glucuronic acid were stable after three freeze-thaw cycles, with RSDs of 5.9% and 0.8%, respectively, and mean deviations of 13.2% and 2.8%, respectively. The low and high concentration samples were also stable after long-term freezing at -66℃ for 7 days, with RSDs of 3.1% and 1.0%, respectively, and mean deviations of 4.7% and 3.7%, respectively. All results met the requirements.

[0150] 8.7 Analysis and critique

[0151] 8.7.1 Quality control samples

[0152] Three quality control samples (low, medium, and high concentrations) must be measured concurrently, with at least two samples per batch. The number of quality control samples must be no less than 5% of the total number of unknown samples. At least 67% of the quality control samples and at least 50% of the samples at each concentration must meet the deviation within ±15%. If the acceptance criteria are not met, the batch of results should be rejected.

[0153] The results showed that all qualified analytical batches of low, medium, and high concentration quality control samples met the acceptance criteria.

[0154] 8.7.2 Blank samples and zero-concentration samples

[0155] For analytical batches, the peak area of ​​the blank sample must be less than 20% of the average peak area of ​​the lowest point of the acceptable standard curve and less than 5% of the analyte peak area. For zero-concentration samples, the peak area must be less than 20% of the analyte peak area compared to the average peak area of ​​the lowest point of the acceptable standard curve. If any of these conditions are not met, the batch results are invalid.

[0156] The results showed that the peak area of ​​estradiol 17β-D glucuronic acid measured in the Blank sample was less than 0.0% of the average peak area of ​​the lowest point of the qualified standard curve; the peak area of ​​the internal standard measured in the Blank sample was less than 0.2% of the average peak area of ​​the lowest point of the qualified standard curve; and the peak area of ​​estradiol 17β-D glucuronic acid measured in the Zero sample was less than 0.0% of the average peak area of ​​the lowest point of the qualified standard curve. All results met the requirements.

[0157] In summary, the UPLC-MS / MS analytical method for detecting estradiol 17β-D glucuronic acid in cell lysates of the present invention has high specificity, accuracy, and reproducibility. It can be used to detect the concentration of estradiol 17β-D glucuronic acid in cell lysates to examine the normal uptake function of OATP1B1 and OATP1B3 cells in in vitro pharmacokinetic studies.

Claims

1. A method for detecting estradiol 17β-D glucuronic acid in cell lysates, characterized in that: It is detected using a UPLC-MS / MS liquid chromatography-mass spectrometry system, and specifically includes the following steps: (1) Establishment of the standard curve a. Preparation of working solutions for a series of concentration standard curves: Take estradiol 17β-D glucuronic acid standard material, dissolve it in dimethyl sulfoxide to obtain estradiol 17β-D glucuronic acid standard curve stock solution; take estradiol 17β-D glucuronic acid standard curve stock solution and dilute it with acetonitrile aqueous solution to prepare standard curve working solution. Take the standard curve working solution and add blank matrix to prepare standard curve samples. Add internal standard working solution to the standard curve samples, mix well, transfer to protein precipitation plate, extract under positive pressure, and take the filtrate and mix with pure water to obtain the standard curve test sample. b. Take the standard curve test samples and inject them into the UPLC-MS / MS liquid chromatography-mass spectrometry instrument, detect the peak area, and fit the regression curve of estradiol 17β-D glucuronic acid. The chromatographic conditions are as follows: Chromatographic column: T3 column; mobile phase A: water, mobile phase B: acetonitrile; gradient elution program: Mass spectrometry conditions: ESI ion source - Detection mode: Negative ion multiple reaction monitoring; (2) Detection of unknown sample concentration: c. Unknown sample processing Take an unknown sample, add internal standard working solution, mix well, transfer to a protein precipitation plate, extract under positive pressure, take the filtrate and mix with pure water to obtain the sample to be tested; d. Detection of the sample to be tested Take the sample to be tested obtained in step c, inject it into the UPLC-MS / MS liquid chromatography-mass spectrometry instrument, and detect it under the same conditions as in step b. The concentration of estradiol 17β-D glucuronic acid in the sample to be tested is obtained according to the standard curve in step (1). The unknown sample was a cell lysate obtained by lysing cells incubated with estradiol 17β-D glucuronic acid solution using pure water via liquid nitrogen freeze-thaw lysis.

2. The method according to claim 1, characterized in that: Step (2) of the concentration detection process also includes a quality control procedure, the specific steps of which are as follows: Estradiol 17β-D-glucuronic acid standard was dissolved in dimethyl sulfoxide to obtain estradiol 17β-D-glucuronic acid quality control stock solution. The estradiol 17β-D-glucuronic acid quality control stock solution was diluted with acetonitrile aqueous solution to obtain quality control working solution. The quality control working solution was mixed with blank matrix to prepare quality control sample. The internal standard working solution was added to the quality control sample, mixed well, and transferred to protein precipitation plate. Positive pressure extraction was performed, and the filtrate was mixed with pure water and injected into UPLC-MS / MS liquid chromatography-mass spectrometry instrument as quality control of the sample detection process. The volume ratio of the quality control working solution, blank matrix, and internal standard working solution is 1:9:40; The concentration range of estradiol 17β-D glucuronic acid in the quality control working solution is 20–15000 nM.

3. The method according to claim 1 or 2, characterized in that: The volume ratio of the filtrate to pure water is 1:

1.

4. The method according to claim 1, characterized in that: The volume ratio of the standard curve working solution, blank matrix, and internal standard working solution in step a is 1:9:40; the concentration range of estradiol 17β-D glucuronic acid in the standard curve working solution is 20–20000 nM.

5. The method according to claim 1, 2 or 4, characterized in that: The blank matrix is ​​a cell lysate obtained by freezing and thawing cells with pure water using liquid nitrogen.

6. The method according to claim 1, 2 or 4, characterized in that: The internal standard working solution is prepared by dissolving toluenebutyrate standard material in dimethyl sulfoxide and then diluting it with acetonitrile to obtain a 0.5 ng / mL toluenebutyrate internal standard working solution.

7. The method according to claim 1 or 2, characterized in that: The proportion of the acetonitrile aqueous solution is 50%.

8. The method according to claim 1, characterized in that: The chromatographic column used in step b was an ACQUITYUPLC-HSS T3, 1.8 μm, 2.1 × 50 mm, Waters; the injection volume was 4 μL; the column temperature was 40℃; and the flow rate was 0.4 mL / min.

9. The method according to claim 1, characterized in that: The mass spectrometry conditions described in step b are as follows: scan time 4 min; capillary voltage 2.5 kV; ion source temperature 150 °C; desolvent gas temperature 550 °C; cone gas flow rate 150 L / h; desolvent gas flow rate 1000 L / h; nebulizer gas pressure 6.0 bar.

10. The method according to claim 9, characterized in that: In the specified mass spectrometry conditions, the detection ion pair for estradiol 17β-D glucuronic acid was m / z 446.92→m / z 271.15, the cone voltage was 48V, and the collision energy was 28eV; the detection ion pair for tolbutamide was m / z 268.91→m / z 170.06, the cone voltage was 28V, and the collision energy was 16eV.

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