Method for detecting silodosin in biological sample and application of silodosin in pharmacokinetics

Abstract

The invention discloses a method for detecting silodosin in a biological sample and application of silodosin in pharmacokinetics, and belongs to the technical field of medicines. Aiming at the problems of unstable chiral center and chemical property of silodosin and long time, low sensitivity and the like of an existing analysis method, the invention adopts an LC-MS / MS technology, combines toluenesulfobutyl urea as an internal standard substance, and realizes rapid and high-sensitivity detection through pretreatment of a biological sample, preparation of a test solution and establishment of a standard curve. According to the method, the analysis time is only 2.5 minutes, the lower quantitative limit of silodosin in plasma reaches 1.00 ng / mL, the tissue sample reaches 20.0 ng / g, and the method is high in sensitivity, small in matrix effect and excellent in accuracy and precision. The method is suitable for detecting the concentration of silodosin in rat plasma and various tissues (such as liver, fat and the like), is successfully applied to pharmacokinetic research, fills the technical blank of silodosin biological sample detection, and provides an efficient and reliable analysis means for preclinical and clinical research.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a method for detecting silodosin in a biological sample and an application thereof in pharmacokinetics. Background Art

[0002] Silodosin is an α-1 adrenergic receptor antagonist primarily used to treat lower urinary tract symptoms (LUTS) associated with benign prostatic hyperplasia (BPH). Its chemical name is (R)-(-)-1-(3-hydroxypropyl)-5-[2-[2-[2-(2,2,2-trifluoroethoxy)phenoxy]ethylamino]propyl]-7-carbamoylindoline, with a molecular weight of 495.53. It belongs to the phenylamine class of compounds. Silodosin's chemical structure has a chiral center, which makes it chemically unstable and susceptible to hydrolysis in biological matrices (such as plasma and tissues), posing a challenge to the quantitative analysis of silodosin in biological samples. Pharmacokinetic studies are crucial for drug development and clinical application. However, the detection of silodosin faces multiple technical challenges: first, its low concentration in biological matrices requires highly sensitive detection methods; second, the presence of chiral centers can lead to interference from metabolites, necessitating highly specific analytical methods; and finally, existing analytical methods generally suffer from long analysis times, limiting their application in preclinical and clinical studies that require rapid processing of large numbers of samples.

[0003] At present, the field of drug analysis has developed a variety of technologies for the quantitative detection of compounds in biological samples, among which LC-MS / MS has become the preferred technology in drug metabolism and pharmacokinetic research due to its high sensitivity, high selectivity and multi-component analysis capabilities. However, for the biological sample detection of silodosin, the existing technology still has deficiencies. In the literature, the analytical method of silodosin mostly focuses on the content determination of pharmaceutical preparations or simple in vitro experiments, lacking a systematic biological sample detection method. For example, CN109580853A discloses a silodosin drug content determination method based on HPLC, but this method is mainly used for pharmaceutical preparation quality control, and the analysis time is long (reaching 45 minutes), and it is not optimized for the complex interference in the biological matrix, and it is difficult to meet the demand for high throughput and rapid analysis in pharmacokinetic research. In addition, the internal standard used in the existing method usually has a complex structure, such as some isotope-labeled compounds, which is not only costly, but also has poor versatility in different biological matrices, limiting its application in multiple tissue samples. Summary of the Invention

[0004] In response to the shortcomings of existing technologies for silodosin detection in biological samples, the present invention aims to establish a method for detecting silodosin in biological samples with high sensitivity, good selectivity, fast analysis speed, minimal matrix effect, simple operation, and good reproducibility, and to apply this method to the study of the pharmacokinetics of silodosin in vivo. To this end, the present invention provides a detection method based on LC-MS / MS. By optimizing the gradient elution procedure, the analysis time is shortened to 2.5 minutes, significantly improving the analysis efficiency, making it suitable for scenarios requiring rapid processing of large numbers of samples.

[0005] To achieve the above objectives, the present invention adopts the following technical solutions.

[0006] A method for detecting silodosin in a biological sample is performed by liquid chromatography-tandem mass spectrometry. The method comprises the following steps: (1) biological sample pretreatment: taking a biological sample, treating it appropriately, and mixing it to obtain a biological sample solution; (2) preparing a biological sample test solution: transferring the biological sample solution, adding an organic solvent containing an internal standard, vortexing and mixing it, and centrifuging to obtain a supernatant to obtain a biological sample test solution; (3) preparing a series of silodosin standard test solutions for a standard curve: transferring a series of silodosin standard working solutions diluted with a blank biological sample solution, adding an organic solvent containing an internal standard, vortexing and mixing it, and centrifuging to obtain a supernatant to obtain a series of silodosin standard test solutions for a standard curve; and (4) liquid chromatography-tandem mass spectrometry analysis: testing and analyzing the biological sample test solution and the series of silodosin standard test solutions for a standard curve by liquid chromatography-tandem mass spectrometry.

[0007] Preferably, the biological sample in step (1) is a plasma sample or a tissue sample, and the tissue sample includes fat, testicle, brain, skeletal muscle, inguinal lymph node, prostate, spleen, heart, bladder, kidney, adrenal gland, thyroid, trachea, liver or lung.

[0008] Preferably, the anticoagulant used in the biological sample pretreatment in step (1) includes heparin, sodium citrate or EDTA-K2.

[0009] Preferably, the internal standard substance in step (2) is tolbutamide, verapamil or warfarin sodium.

[0010] Preferably, the organic solvent in step (2) is acetonitrile or methanol.

[0011] Preferably, the liquid chromatography conditions of the liquid chromatography tandem mass spectrometry described in step (4) include: chromatographic column: octadecylsilane bonded silica gel as the filler; mobile phase: mobile phase A is an acid-containing aqueous solution, and mobile phase B is an acid-containing acetonitrile solution; the acid is formic acid, acetic acid or trifluoroacetic acid, and the volume percentage of the acid is 0.1%-0.5%; injection volume: 2-10 μL; column temperature: 20-60°C; flow rate: 0.5-1.5 mL / min; injector temperature: 4-40°C.

[0012] Preferably, the mass spectrometry conditions described in step (4) include: ion source: electrospray ion source; monitoring mode: positive ion multiple reaction monitoring mode; curtain gas: 30 psi; collision activated dissociation: 9 psi; ion source temperature: 500°C; nebulizing gas: 45-60 psi; auxiliary heating gas: 45-60 psi; ion source voltage: 5100-5800 V.

[0013] Preferably, the mass spectrometry conditions described in step (4) further include: for silodosin, the ion pair used for quantitative analysis is: m / z 496.065 to 261.200; for the internal standard tolbutamide, the ion pair used for quantitative analysis is: m / z 271.100 to 154.800.

[0014] The method for detecting silodosin in biological samples is used in the pharmacokinetics study of silodosin. The administration methods of silodosin include intravenous injection, oral administration, inhalation or airway instillation.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] 1. The analysis efficiency is significantly improved: the present invention adopts an optimized LC-MS / MS detection method, through The HSST3 column and a 0.1% formic acid-water / acetonitrile mobile phase, combined with a gradient elution procedure, shortened the single analysis time to 2.5 minutes, significantly improving analytical efficiency compared to existing technologies. This high-throughput feature is particularly suitable for scenarios requiring rapid processing of large numbers of samples in pharmacokinetic studies.

[0017] 2. Highly versatile and low-cost internal standard: This invention innovatively uses tolbutamide as an internal standard. It features a simple structure, low cost, and high chemical stability. It effectively separates silodosin under chromatographic and mass spectrometric conditions and is compatible with a variety of biological matrices. Compared to isotope-labeled internal standards commonly used in the prior art, tolbutamide not only reduces testing costs but also demonstrates excellent versatility and reproducibility, ensuring the reliability of analytical results across samples from diverse matrices.

[0018] 3. High Sensitivity and Low Limit of Quantitation: The method achieves a lower limit of quantitation (LLOQ) of 1.00 ng / mL in plasma and 20.0 ng / g in tissue samples. This high sensitivity meets the requirements for detecting trace amounts of silodosin in pharmacokinetic studies, which is particularly important when investigating novel routes of administration, such as low-dose administration or inhalation.

[0019] 4. Excellent specificity and stability: By optimizing mass spectrometry ion pairs and chromatographic conditions, this method effectively reduces interference from endogenous substances and metabolites, ensuring excellent detection specificity. Silodosin exhibits excellent stability in biological samples, with precision and accuracy deviations less than 15%, meeting bioanalysis requirements.

[0020] 5. Low matrix effects and stable recoveries: This method utilizes a simplified acetonitrile protein precipitation pretreatment method, combined with liquid-liquid extraction technology, to significantly reduce matrix effects in complex biological matrices (such as tissue homogenates) and achieve stable recoveries. This pretreatment method is simple to operate, time-efficient, and suitable for efficient processing of a variety of biological matrices, ensuring the accuracy and reproducibility of test results.

[0021] 6. Broad application value: The method of the present invention is not only suitable for routine plasma sample analysis, but also for tissue samples such as liver, kidney, and fat. It is particularly important in the pharmacokinetic studies of emerging drug delivery methods such as inhalation. This method can quickly and accurately determine the concentration distribution of silodosin in target organs, providing important technical support for optimizing drug delivery regimens and evaluating drug efficacy and safety. It has significant application value in preclinical and clinical research.

[0022] In summary, the present invention overcomes the shortcomings of the existing technologies in terms of analysis time, sensitivity, specificity, matrix effect, and internal standard versatility by optimizing LC-MS / MS analysis conditions, internal standard selection, and sample pretreatment methods. A rapid, sensitive, specific, and universal method for the detection of silodosin is established, providing an efficient and reliable analytical tool for its pharmacokinetic studies. The method has significant technical advantages and broad application prospects. DETAILED DESCRIPTION

[0023] The present invention is described in detail below through specific examples. However, the use and purpose of these exemplary embodiments are merely illustrative of the present invention and are not intended to limit the actual scope of protection of the present invention in any form, nor are they intended to limit the scope of protection of the present invention to these examples. For parameter ranges not mentioned, intermediate values ​​are selected. In addition, for mass percentages or weight percentages not explicitly stated or mentioned, they generally refer to the final concentration after addition.

[0024] This method is suitable for measuring silodosin content in various rat tissues and organs, including plasma, fat, testes, brain, skeletal muscle, inguinal lymph nodes, prostate, spleen, heart, bladder, kidney, adrenal gland, thyroid gland, trachea, and lung. The following detailed description uses plasma and liver as examples; the processing methods for other tissues are similar and are not repeated here.

[0025] Example 1: Detection of Silodosin in Biological Samples—Selective Methodology Investigation

[0026] Six different rat blank plasma and liver tissue samples were used for selectivity testing. Silodosin was used as the target analyte in these samples, and tolbutamide was used as the internal standard. LC-MS / MS was used to determine the concentration of silodosin in these samples.

[0027] (1) The method comprises the following steps:

[0028] Step 1: Biological sample pretreatment

[0029] 1. Preparation of plasma blank biological sample solution

[0030] Whole blood samples from rats not given silodosin were collected and placed in EDTA-K2 blood collection tubes. After mixing, the samples were centrifuged at 3500 rpm at 4°C for 10 min, and the upper plasma was aspirated to obtain a plasma blank biological sample solution.

[0031] 2. Preparation of blank biological sample solution of liver tissue

[0032] A liver tissue sample of a rat not given silodosin was taken, and homogenization medium (10% homogenate) was added at a ratio of weight (g): homogenization medium (mL) = 1:9, and the liver tissue blank biological sample solution was obtained by mechanical homogenization.

[0033] Step 2: Preparation of biological sample test fluid

[0034] 1. Preparation of plasma blank biological sample test solution

[0035] Take a certain volume of plasma blank biological sample solution, add acetonitrile solution at a volume ratio of 1:3, vortex mix, and centrifuge at 12000 rpm for 10 min at 4°C to obtain the plasma blank biological sample test solution.

[0036] 2. Preparation of liver tissue blank biological sample test solution

[0037] A certain volume of liver tissue blank biological sample solution was taken, and acetonitrile solution was added at a volume ratio of 1:3. The solution was vortexed and mixed, and centrifuged at 12000 rpm for 10 min at 4°C. The supernatant was taken to obtain the liver tissue blank biological sample test solution.

[0038] Step 3: Preparation of Silodosin Series Standard Test Solutions for Standard Curve

[0039] 1. Preparation of Silodosin Series Standard Test Solutions for Plasma Standard Curve

[0040] Accurately pipette a series of silodosin standard working solutions at standard concentrations of 50.0, 250, 500, 5000, 10000, 25000, 40000, and 50000 ng / mL and dilute them with the plasma blank biological sample solution to prepare a series of plasma samples containing silodosin standard concentrations of 2.00, 5.00, 10.0, 100, 200, 500, 800, and 1000 ng / mL. A certain volume of plasma sample from the series of standard curves was added to an acetonitrile solution containing bensulfuronamide as the internal standard (internal standard working solution) at a 1:3 volume ratio. Vortex mix thoroughly, centrifuge at 12000 rpm for 10 min at 4°C, and remove the supernatant to obtain the series of silodosin standard test solutions for the plasma standard curve.

[0041] 2. Preparation of Silodosin Series Standard Test Solutions for Liver Tissue Standard Curve

[0042] Accurately pipette a series of silodosin standard working solutions at standard concentrations of 50.0, 250, 500, 5000, 10000, 25000, 40000, and 50000 ng / mL and dilute them with liver tissue blank biological sample solution to prepare a series of liver tissue biological samples containing silodosin standard concentrations of 2.00, 5.00, 10.0, 100, 200, 500, 800, and 1000 ng / mL. Take a certain volume of liver tissue sample from the series of standard curves and add acetonitrile solution containing bensulfuronamide internal standard (internal standard working solution) at a volume ratio of 1:3. Vortex mix, centrifuge at 12000 rpm at 4°C for 10 min, and collect the supernatant to obtain the silodosin series standard test solution for the liver tissue standard curve.

[0043] Step 4: Test and analyze using LC-MS / MS

[0044] The plasma blank biological sample test solution and the liver tissue blank biological sample test solution obtained in step 2, and the plasma standard curve obtained in step 3 using the silodosin series standard test solution and the liver tissue standard curve using the silodosin series standard test solution, were tested and analyzed using liquid chromatography tandem mass spectrometry LC-MS / MS.

[0045] Chromatographic conditions are as follows: Chromatographic column: HSS T32.5μm; mobile phase A: 0.1% formic acid-water solution; mobile phase B: 0.1% formic acid-acetonitrile solution; column temperature: 40℃; flow rate: 0.5mL / min; injection volume: 3μL; analysis time: 2.5min.

[0046] The gradient elution program is shown in Table 1 below:

[0047] Table 1 Gradient elution program parameters

[0048] Time (min) Flow rate (mL / min) A% (aqueous phase) B% (organic phase) 0.00 0.50 90 10 0.20 0.50 90 10 1.80 0.50 10 90 2.20 0.50 10 90 2.21 0.50 90 10 2.50 0.50 90 10

[0049] Mass spectrometry conditions:

[0050] The mass spectrometry parameters are shown in Table 2 below:

[0051] Table 2 Mass spectrometry parameters

[0052]

[0053]

[0054] (2) The results are as follows:

[0055] Table 3 Plasma selectivity investigation results

[0056]

[0057] Table 4 Liver tissue selectivity investigation results

[0058]

[0059] As shown in Tables 3 and 4, the interference effect of endogenous substances in both plasma and liver tissue on the internal standard, tolbutamide, was 0.00%. The maximum interference effect on the analyte, silodosin, was only 5.16%, well below the limit specified in the bioanalysis method validation guidelines. These results demonstrate that the LC-MS / MS detection method provided by the present invention is highly selective for the determination of silodosin and the internal standard, tolbutamide, in plasma and liver tissue samples, meeting the requirements for biological sample testing.

[0060] Example 2 Detection Method of Silodosin in Biological Samples - Linear Methodology Investigation

[0061] (1) The method comprises the following steps:

[0062] Step 1: Preparation of zero-concentration biological sample test solution

[0063] 1. Preparation of plasma blank biological sample solution: same as step 1 in Example 1.

[0064] 2. Preparation of blank liver tissue biological sample solution: same as step 1 in Example 1.

[0065] 3. Preparation of zero-concentration plasma biological sample test solution: Take a certain volume of plasma blank biological sample solution, add acetonitrile solution containing bensulfuronamide internal standard (internal standard working solution) at a volume ratio of 1:3, vortex mix, centrifuge at 12000 rpm at 4°C for 10 min, and collect the supernatant to obtain the zero-concentration plasma biological sample test solution.

[0066] 4. Preparation of zero-concentration liver tissue biological sample test solution: Take a certain volume of liver tissue blank biological sample solution, add acetonitrile solution containing benzylbutamide internal standard (internal standard working solution) at a volume ratio of 1:3, vortex mix, centrifuge at 12000 rpm at 4°C for 10 min, and collect the supernatant to obtain the zero-concentration liver tissue sample test solution.

[0067] Step 2: Preparation of Silodosin Standard Test Solution Series for Standard Curve

[0068] 1. Preparation of Silodosin series standard test solutions for plasma standard curve: The method is the same as step 3 of Example 1.

[0069] 2. Preparation of a series of silodosin standard test solutions for liver tissue standard curve: The method is the same as step 3 of Example 1.

[0070] Step 3: Test and analyze using LC-MS / MS

[0071] The obtained zero-concentration plasma biological sample test solution and the zero-concentration liver tissue biological sample test solution, and the obtained plasma standard curve using the silodosin series standard test solution or the obtained liver tissue standard curve using the silodosin series standard test solution, were tested and analyzed using liquid chromatography tandem mass spectrometry LC-MS / MS, using the same method as step 4 in Example 1.

[0072] (2) Results

[0073] Table 5 Plasma linearity test results

[0074]

[0075]

[0076] Table 6 Results of linear investigation of liver tissue

[0077]

[0078] The internal standard method uses the chromatographic response ratio of the analyte silodosin to the internal standard tolbutamide as the ordinate, and uses the weighted (W = 1 / x2) least squares method to perform linear regression between the concentration of the analyte in the biological sample (x) and the response ratio (y). The linear range of the plasma biological sample is 1-1000 ng / mL, the minimum limit of quantification is 1 ng / mL, and the linear correlation coefficient R 2≥0.995. The linear range of liver tissue biological samples is 2-1000 ng / mL, the minimum limit of quantification is 2 ng / mL, and the linear correlation coefficient R 2 ≥0.995. The accuracy deviation range of this method is 0.0025-0.06 (0.25%-6.0%), which meets the requirements of the bioanalytical method validation guidelines and shows that the method has excellent accuracy throughout the linear range. The LC-MS / MS detection method provided by the present invention has excellent linear performance and sensitivity for the determination of silodosin in plasma (1.00-1000 ng / mL) and liver tissue (2.00-1000 ng / mL) samples, meeting the needs of biological sample detection.

[0079] Example 3 Detection Method of Silodosin in Biological Samples - Accuracy and Precision Methodology Investigation

[0080] (1) The method comprises the following steps

[0081] Step 1: Biological sample pretreatment

[0082] 1. Preparation of plasma blank biological sample solution: The method is the same as step 1 of Example 1.

[0083] 2. Preparation of blank liver tissue biological sample solution: The method is the same as step 1 of Example 1.

[0084] 3. Preparation of Silodosin Series Quality Control Plasma Biological Sample Solutions: Take Silodosin Series Quality Control Working Solutions with concentrations of 50.0 ng / mL, 150 ng / mL, 15,000 ng / mL, and 37,500 ng / mL, and dilute them with plasma blank biological sample solutions to prepare Silodosin Series Quality Control Plasma Biological Sample Solutions with silodosin concentrations of 1.00 ng / mL (LLOQ), 3.00 ng / mL (LQC), 300 ng / mL (MQC), and 750 ng / mL (HQC), respectively.

[0085] 4. Preparation of silodosin quality control liver tissue biological sample solutions: Take silodosin quality control working solutions with concentrations of 100 ng / mL, 300 ng / mL, 15,000 ng / mL, and 37,500 ng / mL, and dilute them with plasma blank biological sample solutions to prepare silodosin quality control plasma biological sample solutions with silodosin concentrations of 2.00 ng / mL (LLOQ), 6.00 ng / mL (LQC), 300 ng / mL (MQC), and 750 ng / mL (HQC), respectively.

[0086] Step 2: Preparation of biological sample test fluid

[0087] 1. Preparation of Silodosin Series Quality Control Plasma Biological Sample Test Solution

[0088] A certain volume of 1.00 ng / mL (LLOQ), 3.00 ng / mL (LQC), 300 ng / mL (MQC), and 750 ng / mL (HQC) silodosin series quality control plasma biological sample solution was taken, and an acetonitrile solution containing bensulfuronamide internal standard (internal standard working solution) was added at a volume ratio of 1:3. After vortex mixing, the solution was centrifuged at 12000 rpm at 4°C for 10 min, and the supernatant was obtained to obtain the silodosin series quality control plasma biological sample test solution.

[0089] 2. Preparation of Silodosin Series Quality Control Liver Tissue Biological Sample Test Solution

[0090] A certain volume of 2.00 ng / mL (LLOQ), 6.00 ng / mL (LQC), 300 ng / mL (MQC), and 750 ng / mL (HQC) silodosin series quality control liver tissue biological sample solution was taken, and an acetonitrile solution containing bensulfuronamide internal standard (internal standard working solution) was added at a volume ratio of 1:3. After vortex mixing, the solution was centrifuged at 12000 rpm at 4°C for 10 min, and the supernatant was obtained to obtain the silodosin series quality control liver tissue biological sample test solution.

[0091] Step 3: Preparation of Silodosin Series Standard Test Solutions for Standard Curve

[0092] 1. Preparation of a series of silodosin standard test solutions for plasma standard curve: The method is the same as step 3 of Example 1.

[0093] 2. Preparation of a series of silodosin standard test solutions for liver tissue standard curve: The method is the same as step 3 of Example 1.

[0094] Step 4: Test and analyze using LC-MS / MS

[0095] The obtained silodosin series quality control plasma biological sample test solution and the silodosin series quality control liver tissue biological sample test solution, as well as the obtained plasma standard curve using the silodosin series standard test solution or the liver tissue standard curve using the silodosin series standard test solution, were tested and analyzed using liquid chromatography tandem mass spectrometry LC-MS / MS, using the same method as step 4 in Example 1.

[0096] (2) Results

[0097] The intra- and inter-batch precision and accuracy were investigated; the actual concentrations were calculated using the linear regression equation, and the measured mean, deviation, and precision (%CV) for each concentration were calculated. The results are shown in Tables 7 and 8.

[0098] Table 7 Results of plasma accuracy and precision investigation

[0099]

[0100]

[0101] Note: SD is standard deviation; %CV is precision.

[0102] Table 8 Results of liver tissue accuracy and precision inspection

[0103]

[0104]

[0105]

[0106] Note: SD is standard deviation; %CV is precision; NA means no valid value; * means the data is outlier data and is not included in the calculation.

[0107] As shown in Tables 7 and 8, accuracy deviations were below 15% for all batches and concentration levels. Although individual LQCs and MQCs approached the upper limit in some batches, overall performance was excellent. Furthermore, both intra- and inter-batch precision were less than 15% for all batches and concentration levels, demonstrating good reproducibility. These results demonstrate that this method offers high accuracy, precision, and reproducibility, making it suitable for batch biological sample analysis.

[0108] Example 4 Detection Method of Silodosin in Biological Samples - Matrix Effect and Recovery Methodology

[0109] (1) The method comprises the following steps

[0110] Step 1: Biological sample pretreatment

[0111] 1. Preparation of plasma blank biological sample solution: The method is the same as step 1 of Example 1.

[0112] 2. Preparation of Silodosin series quality control plasma biological sample solution: The method is the same as step 2 of Example 3.

[0113] Step 2: Preparation of biological sample test fluid

[0114] 1. Preparation of extracted silodosin series quality control plasma test solutions

[0115] A certain volume of silodosin series quality control plasma biological sample solution containing silodosin concentrations of 3.00, 300, and 750 ng / mL was taken, and an acetonitrile solution containing bensulfuronamide internal standard (internal standard working solution) was added at a volume ratio of 1:3. The solution was vortexed and centrifuged at 12000 rpm for 10 min at 4°C. The supernatant was collected to obtain the extracted silodosin series quality control plasma sample test solution.

[0116] 2. Preparation of unextracted Silodosin quality control plasma test solution

[0117] A certain volume of plasma blank biological sample solution was taken, acetonitrile was added at a volume ratio of 1:3, the solution was vortexed and centrifuged at 12000 rpm for 10 min at 4°C, and the supernatant was collected. Silodosin series quality control working solutions with concentrations of 150, 15000, and 37500 ng / mL and acetonitrile solution containing bensulfuronamide internal standard were added to the supernatant at a ratio of 50:1, the solution was vortexed and centrifuged at 12000 rpm for 10 min at 4°C, and the supernatant was collected to obtain the unextracted silodosin series quality control plasma sample test solution.

[0118] 3. Preparation of Silodosin Series Quality Control Sample Test Solution for Solution Samples

[0119] A series of silodosin quality control working solutions with concentrations of 150, 15,000, and 37,500 ng / mL were taken and diluted with water to prepare a series of solution samples with silodosin concentrations of 3.00, 300, and 750 ng / mL, respectively. A certain volume of solution was added to an acetonitrile solution containing bensulfuronamide internal standard (internal standard working solution) at a volume ratio of 1:3, vortexed and mixed, and centrifuged at 12,000 rpm for 10 min at 4°C to obtain the supernatant to obtain the silodosin series quality control sample test solution of the solution sample.

[0120] Step 3: Preparation of Silodosin Series Standard Test Solutions for Standard Curve

[0121] Preparation of a series of silodosin standard test solutions for plasma standard curve: The method is the same as step 3 of Example 1.

[0122] Step 4: Test and analyze using LC-MS / MS

[0123] The obtained extracted silodosin series quality control plasma sample test solution, the unextracted silodosin series quality control plasma sample test solution, the solution sample silodosin series quality control sample test solution, and the obtained plasma standard curve were tested and analyzed using liquid chromatography tandem mass spectrometry LC-MS / MS, using the same method as step 4 in Example 1.

[0124] (2) Results

[0125] Table 9 Investigation of plasma matrix effect

[0126]

[0127]

[0128]

[0129] Note: SD is standard deviation; %CV is precision; NA means no valid value.

[0130] Table 10 Investigation of the recovery rate of plasma analytes

[0131]

[0132]

[0133]

[0134] Note: Mean is the mean; SD is the standard deviation; %CV is the precision; NA means no valid value.

[0135] The results in Table 9 show that the average accuracy deviations for the low and high concentrations of silodosin quality control samples in plasma from six different individuals ranged from -7.78% to 4.62%, with coefficients of variation ranging from 0.23% to 8.93%. The results in Table 10 indicate that the overall coefficient of variation for the recoveries of low, medium, and high concentrations was 7.55%, and the overall coefficient of variation for the recoveries of the internal standard at low, medium, and high concentrations was 1.47%. These results demonstrate that this method exhibits no matrix effects, and that the extraction recoveries meet the requirements, satisfying the requirements for biological sample analysis.

[0136] Example 5 Detection Method of Silodosin in Biological Samples - Stability Methodology Investigation

[0137] (1) The method comprises the following steps

[0138] Step 1: Biological sample pretreatment

[0139] 1. Pretreatment of simulated plasma biological samples

[0140] Preparation of plasma blank biological sample solution: same as step 1 of Example 1.

[0141] 2. Preparation of plasma biological sample stability solution: Take a certain volume of 150 and 37500 ng / mL silodosin quality control working solution and blow it dry with nitrogen flow at room temperature. Then add plasma blank biological sample solution at a volume ratio of 1:50 to reconstitute and prepare simulated plasma biological sample stability solution.

[0142] The simulated plasma biological sample stability solution was stored at <-65°C for 12 days. Three replicates were prepared for each concentration. After the storage condition ended, the solution was immediately tested.

[0143] Step 2: Preparation of biological sample test fluid

[0144] Preparation of plasma biological sample stability test solution: Quantitatively pipette the plasma biological sample stability solution, add acetonitrile solution containing bensulfuronamide internal standard (internal standard working solution) at a volume ratio of 1:3, vortex mix, centrifuge at 12000 rpm at 4°C for 10 min, and collect the supernatant to obtain the plasma biological sample stability test solution.

[0145] Step 3: Preparation of Silodosin Series Standard Test Solutions for Standard Curve

[0146] Preparation of a series of silodosin standard test solutions for plasma standard curve: The method is the same as step 3 of Example 1.

[0147] Step 4: Test and analyze using LC-MS / MS

[0148] The plasma biological sample stability test solution and the silodosin series standard test solution were analyzed by liquid chromatography tandem mass spectrometry (LC-MS / MS) in the same manner as step 4 of Example 1.

[0149] (2) Results

[0150] Table 11 Stability of plasma biological samples after preparation and storage at <-65°C for 12 days

[0151]

[0152] Note: SD is standard deviation; %CV is precision; NA means no valid value.

[0153] The experimental results showed that the average accuracy deviation of the samples did not exceed the test standard requirement of ±15%, and the %CV (precision) of the stability sample values ​​at all concentration levels did not exceed the test standard requirement of 15.0%. The results showed that the stability of silodosin in biological samples was good when the low- and high-concentration quality control samples were stored at <-65°C for 12 days, and the stability met the test requirements.

[0154] Example 6 Application of the Detection Method of Silodosin in Biological Samples to Silodosin Pharmacokinetics (Plasma)

[0155] After rats were administered with silodosin, plasma was collected before administration (0 h) and at 0.05, 0.083, 0.167, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h after administration, and the plasma silodosin concentration was determined by LC-MS / MS.

[0156] (1) The method comprises the following steps

[0157] Step 1: Plasma biological sample pretreatment

[0158] 1. Preparation of plasma blank biological sample solution: The method is the same as step 1 of Example 1.

[0159] 2. Preparation of plasma biological sample solution: Whole blood samples of rats were collected at different time points after administration of silodosin. The fresh whole blood samples were placed in a blood collection tube containing EDTA-K2. After mixing evenly, the samples were centrifuged at 3500 rpm at 4°C for 10 minutes. The supernatant was aspirated to obtain the plasma biological sample solution.

[0160] Step 2: Preparation of plasma biological sample test solution

[0161] Take a certain volume of plasma biological sample solution, add acetonitrile solution containing bensulfuronamide internal standard (internal standard working solution) at a volume ratio of 1:3, vortex mix, centrifuge at 12000 rpm at 4°C for 10 minutes, and take the supernatant to obtain the plasma biological sample test solution.

[0162] Step 3: Preparation of Silodosin Series Standard Test Solutions for Standard Curve

[0163] The method is the same as step 3 in Example 1.

[0164] Step 4: Test and analyze using LC-MS / MS method:

[0165] The plasma biological sample test solution obtained in step 2 and the plasma standard curve obtained in step 3 were tested and analyzed using a series of silodosin standard test solutions using liquid chromatography tandem mass spectrometry (LC-MS / MS). The chromatographic and mass spectrometry conditions were the same as those in step 4 of Example 1.

[0166] (2) Drawing of the standard curve

[0167] The standard curve obtained in step 3 was tested and analyzed using a series of silodosin standard test solutions using liquid chromatography tandem mass spectrometry (LC-MS / MS). The results are shown in Tables 12 and 13.

[0168] Table 12 Silodosin standard curve results

[0169]

[0170] Note: * indicates that the data is an outlier and does not participate in the calculation; NA indicates no valid value.

[0171] Table 13 Silodosin standard curve parameters

[0172] Slope Intercept R-Squared LLOQng / mL ULOQng / mL 0.00731 0.00499 0.9973 1.00 1000

[0173] (III) Determination of a series of quality control test solutions

[0174] The method is the same as Example 3.

[0175] Table 14 Silodosin quality control results

[0176]

[0177] Note: * indicates that the data is an outlier and does not participate in the calculation; NA indicates no valid value.

[0178] (IV) Determination of silodosin concentration in plasma after administration of silodosin to rats

[0179] The standard curve method was used to deduce the concentration values ​​of silodosin in plasma biological samples at different administration time points (within the range of the standard curve).

[0180] Table 15 Silodosin concentration in plasma after administration

[0181]

[0182] Note: SD is standard deviation; BQL means the sample concentration is below the limit of quantification; NA means no valid value.

[0183] Table 16 Individual pharmacokinetic parameters of rats after administration of silodosin

[0184] parameter <![CDATA[t 1 / 2 ]]> <![CDATA[T max ]]> <![CDATA[C max ]]> <![CDATA[AUC last ]]> unit h h ng / mL h*ng / mL plasma 1.13 0.0932 650 454

[0185] The linear range of silodosin in plasma was 1.00~1000 ng / mL, with good linearity (R 2 ≥0.995), and both precision and accuracy met the standards. After administration of silodosin to rats, the average time for silodosin concentration in plasma reached its peak was 0.0932 hours, after which it was eliminated and below the limit of quantification by 12 hours.

[0186] Example 7 Application of the Detection Method of Silodosin in Biological Samples in the Pharmacokinetics of Silodosin (Liver)

[0187] After rats were administered with silodosin, the livers were collected at different time points after administration, and the concentration of silodosin in the tissues was determined by LC-MS / MS.

[0188] (1) The method comprises the following steps

[0189] Step 1: Pretreatment of liver tissue biological samples

[0190] 1. Preparation of blank liver tissue biological sample solution: The method is the same as step 1 of Example 1.

[0191] 2. Preparation of liver biological sample solution: Rat liver tissue samples were collected 0.015, 1, and 4 hours after administration of silodosin. Homogenization medium (10% homogenate) was added at a ratio of weight (g): homogenization medium (mL) = 1:9, and mechanical homogenization was performed to obtain liver tissue blank biological sample solution.

[0192] Step 2: Preparation of liver tissue biological sample test fluid

[0193] A certain volume of liver tissue biological sample solution was taken, and an acetonitrile solution containing bensulfuronamide internal standard (internal standard working solution) was added at a volume ratio of 1:3. The solution was vortexed and centrifuged at 12000 rpm for 10 min at 4°C. The supernatant was taken to obtain the liver tissue biological sample test solution.

[0194] Step 3: Preparation of Silodosin Series Standard Test Solutions for Standard Curve

[0195] Preparation of a series of silodosin standard test solutions for the liver tissue standard curve: The method is the same as step 3 of Example 1.

[0196] Step 4: Test and analyze using LC-MS / MS method:

[0197] The liver tissue biological sample test solution obtained in step 2 and the liver tissue standard curve obtained in step 3 were tested and analyzed using liquid chromatography tandem mass spectrometry (LC-MS / MS) with silodosin series standard test solutions. The chromatographic and mass spectrometric conditions were the same as those in step 4 of Example 1.

[0198] (2) Drawing of the standard curve

[0199] The standard curve obtained in step 3 was tested and analyzed using a series of silodosin standard test solutions using liquid chromatography tandem mass spectrometry (LC-MS / MS). The results are shown in Tables 17 and 18.

[0200] Tissue concentration (ng / g) = tissue homogenate concentration (ng / mL) × dilution factor ÷ 1g / mL (tissue density is calculated as 1g / mL)

[0201] Table 17 Silodosin standard curve results

[0202]

[0203] Note: * indicates that the data is an outlier and does not participate in the calculation; NA indicates no valid value.

[0204] Table 18 Silodosin standard curve parameters

[0205] Slope intercept <![CDATA[R 2 ]]> LLOQ (ng / g) ULOQ (ng / g) 0.000596 0.00102 0.9958 20.0 10000

[0206] (III) The determination method of the series of quality control test solutions is the same as that in Example 3

[0207] Table 19 Silodosin quality control results

[0208]

[0209] (IV) Determination of silodosin concentration in liver tissue of rats after administration of silodosin The standard curve method was used to derive the silodosin concentration values ​​in biological samples at different administration time points.

[0210] Table 20 Silodosin concentration in liver tissue after administration

[0211]

[0212]

[0213] Note: SD is standard deviation; BQL means the sample concentration is below the limit of quantification.

[0214] Table 21 Pharmacokinetic parameters of silodosin in liver tissue after administration

[0215] parameter <![CDATA[T max ]]> <![CDATA[C max ]]> <![CDATA[AUC last ]]> unit hr ng / g hr*ng / g liver 0.150 967 709

[0216] The linear range of silodosin in liver tissue was 20-10000 ng / g, with good linearity (R 2 ≥0.99), and the precision and accuracy met the standards. After administration of silodosin to rats, the average time for silodosin concentration in the liver was 0.15 hours, after which it was eliminated. By 4 hours, most test results were below the limit of quantification.

[0217] Conclusion: This method is rapid, sensitive and specific, and is suitable for the accurate determination of silodosin concentrations in rat plasma and tissues and for the study of its pharmacokinetic profile.

[0218] The above content is a further detailed description of the present invention in conjunction with specific implementation methods. It cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, they can make several simple deductions or substitutions without departing from the concept of the present invention, which should be regarded as falling within the scope of protection determined by the claims submitted for the present invention.

Claims

1. A method for detecting silodosin in a biological sample, characterized in that: Liquid chromatography tandem mass spectrometry was used for detection, and the detection method included the following steps: (1) Biological sample pretreatment: Take the biological sample, treat it appropriately, and mix it to obtain a biological sample solution; (2) Preparation of biological sample test solution: Pipette the biological sample solution, add the organic solvent containing the internal standard, vortex mix, centrifuge to obtain the supernatant, and obtain the biological sample test solution; (3) Preparation of silodosin series standard test solutions for the standard curve: Pipette the silodosin series standard working solution diluted with the blank biological sample solution, add the organic solvent containing the internal standard, vortex mix, and centrifuge to obtain the supernatant to obtain the silodosin series standard test solution for the standard curve; (4) Liquid chromatography tandem mass spectrometry analysis: The biological sample test solution and the standard curve were tested and analyzed using a series of silodosin standard test solutions and liquid chromatography tandem mass spectrometry.

2. The method for detecting silodosin in a biological sample according to claim 1, wherein: The biological sample described in step (1) is a plasma sample or a tissue sample, and the tissue sample includes fat, testicle, brain, skeletal muscle, inguinal lymph node, prostate, spleen, heart, bladder, kidney, adrenal gland, thyroid gland, trachea, liver or lung.

3. The method for detecting silodosin in a biological sample according to claim 1, wherein: The anticoagulant used in the biological sample pretreatment described in step (1) includes heparin, sodium citrate or EDTA-K2.

4. The method for detecting silodosin in a biological sample according to claim 1, wherein: The internal standard substance described in step (2) is tolbutamide, verapamil or warfarin sodium.

5. The method for detecting silodosin in a biological sample according to claim 1, wherein: The organic solvent described in step (2) is acetonitrile or methanol.

6. The method for detecting silodosin in a biological sample according to claim 1, wherein: The liquid chromatography conditions of the liquid chromatography tandem mass spectrometry described in step (4) include: chromatographic column: octadecylsilane bonded silica gel as the filler; mobile phase: mobile phase A is an acid-containing aqueous solution, and mobile phase B is an acid-containing acetonitrile solution; the acid is formic acid, acetic acid or trifluoroacetic acid, and the volume percentage of the acid is 0.1%-0.5%; injection volume: 2-10 μL; column temperature: 20-60°C; flow rate: 0.5-1.5 mL / min; injector temperature: 4-40°C.

7. The method for detecting silodosin in a biological sample according to claim 1, wherein: The mass spectrometry conditions described in step (4) include: ion source: electrospray ion source; monitoring mode: positive ion multiple reaction monitoring mode; curtain gas: 30 psi; collision activated dissociation: 9 psi; ion source temperature: 500°C; nebulizing gas: 45-60 psi; auxiliary heating gas: 45-60 psi; ion source voltage: 5100-5800 V.

8. The method for detecting silodosin in a biological sample according to claim 7, wherein: The mass spectrometry conditions described in step (4) also include: for silodosin, the ion pair used for quantitative analysis is: m / z 496.065 to 261.200; for the internal standard tolbutamide, the ion pair used for quantitative analysis is: m / z 271.100 to 154.

800.

9. Use of the method for detecting silodosin in a biological sample according to any one of claims 1 to 8 in a study on the pharmacokinetics of silodosin.

10. Application of the method for detecting silodosin in a biological sample according to claim 9 in a study of the pharmacokinetics of silodosin, characterized in that: Silodosin can be administered intravenously, orally, by inhalation, or by airway instillation.

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