CN110927307A - Method for determining concentration of tadalafil in blood plasma by liquid chromatography-mass spectrometry - Google Patents
Method for determining concentration of tadalafil in blood plasma by liquid chromatography-mass spectrometry Download PDFInfo
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Abstract
The invention discloses a method for determining the concentration of tadalafil in blood plasma by liquid chromatography-mass spectrometry, which adopts a liquid chromatography-mass spectrometry system to determine, and comprises the steps of firstly taking a sample to be determined, adding a certain amount of mixed organic solvent for extraction, pretreating, separating by a chromatographic column, and detecting by a mass spectrometer. The method is rapid, accurate, high in sensitivity and simple and convenient to operate, and provides a basis for measuring the blood concentration of tadalafil; the linear range of the plasma standard curve of the method is 1-600 ng/mL, the precision RSD in batch and between batches is less than +/-15%, and the method is suitable for measuring the concentration of tadalafil in plasma.
Description
Technical Field
The invention belongs to the technical field of medicines, particularly relates to a method for determining a medicine, and particularly relates to a method for determining the concentration of tadalafil in blood plasma by liquid chromatography-mass spectrometry.
Background
The chemical name of tadalafil (Tadalafi) is: (6R-12aR) -6- (1, 3-Benzodioxazin-5-yl) -2-methyl-2, 3, 6, 7, 12, 12 a-hexahydropyrazino [1', 2' -1, 6] -pyrido [3, 4-b ] indole-1, 4-dione, a selective reversible inhibitor of cyclic guanosine monophosphate (cGMP) -specific phosphodiesterase 5(PDE5), was first developed by Kulanin Schker (GSK) and subsequently assigned to ICOS, and was later developed by a combination of ICOS and Gift (EliLillv). Approved by the FDA in 2003, is marketed in the united states as a drug for the treatment of Male Erectile Dysfunction (MED). When sexual stimulation results in local nitric oxide release, PDE5 is inhibited by tadalafil, resulting in elevated cGMP levels in the corpora cavernosa, which leads to smooth muscle relaxation, blood flow into penile tissue, and erection, e.g., asexual stimulation. In 2013, the present (EliLillv) introduced in china a daily small dose of 5mg oral dosage form of huili (tadalafil) with the same therapeutic effect at a price of only one third of that of wanaike (cedilafil). The Chinese imitation drug market has strong field intensity demand, and the economic burden can be reduced by the drug demand which is cheap and has good effect and quality. The method has wide prospect when being used for developing a biological detection method of tadalafil and being applied to the evaluation of the pharmaceutical imitation consistency of tadalafil. The chemical structural formula of tadalafil is as follows:
in the evaluation of the imitation drug consistency, how to determine better and more stable bioequivalence evaluation is very important for the detection and analysis of the drug. The tadalafil biological sample has complex components and large matrix interference, and meanwhile, the sample is difficult to obtain and has small quantity, and the concentration of an analyte is usually very low, so that the requirements on the technology for separating and analyzing the biological sample are higher and higher, and a method for quickly determining the content of tadalafil with high sensitivity, high precision and high selectivity is urgently needed to be established.
Disclosure of Invention
The invention aims to provide a method for determining the concentration of tadalafil in plasma by liquid chromatography-mass spectrometry, which can improve the sensitivity, precision, selectivity and speed of detection.
In order to realize the aim, the method for determining the concentration of tadalafil in plasma by liquid chromatography-mass spectrometry comprises the following steps of:
(1) pretreatment of a plasma sample:
plasma with K2EDTA as anticoagulant, tadalafil-d 3 as internal standard; adding 100 μ L of plasma sample into labeled glass test tubeAdding 5 mu L of the mixture in a volume ratio of 1: 1, adding 20 mu L of 0.2 ng/mu L internal standard tadalafil-d 3 solution after uniformly mixing, adding 20 mu L of 1mol/L dipotassium hydrogen phosphate after uniformly mixing, adding 3mL of methyl tert-butyl ether into a test tube after uniformly mixing, vortex mixing for 1min, centrifuging for 5min at 20 ℃ and 3000rpm, taking an organic solution layer and a label, transferring the organic solution layer and the label to another clean test tube, carrying out evaporation drying for at least 15min at 35 ℃ until the test tube is completely dried under pure air flow, and redissolving 1000 mu L of organic solution in the test tube, wherein the organic solution is methanol: water: formic acid is mixed according to a volume ratio of 55: 45: 0.5, uniformly mixing the obtained mixture in a vortex mode for 1min, transferring the solution in the test tube to a 96-deep-hole plate, and taking the solution as a test sample to be detected;
(2) and (3) sample measurement:
injecting 10 mu L of test sample into a high performance liquid chromatography-tandem mass spectrometer, detecting chromatographic peaks of tadalafil and internal standard tadalafil-d 3 in the sample, and calculating the concentration of tadalafil in the plasma sample according to the chromatographic peaks;
(3) liquid chromatography determination, conditions are as follows:
a chromatographic column: agilent ZORBAX XDB-C18, 5 μm, column specification 50X 2.1 mm; temperature of the chromatographic column: 40 ℃; mobile phase A: the volume percentage of water/formic acid is 100/0.5; mobile phase B: the volume percentage of methanol/formic acid is 100/0.5; washing liquid: the methanol/water volume percentage was 50/50; the autosampler temperature was 15 ℃; gradient elution with flow rate of 0.4mL/min, sample size of 10 μ L, and analysis time of 3.5 min;
(4) mass spectrometry under the conditions:
the ion source is an electrospray ion source, the spraying voltage is 5000V, the atomizing temperature is 500 ℃, the pressure of spraying air is 55Psi, the pressure of auxiliary heating air is 55Psi, the pressure of air curtain air is 30Psi, the pressure of collision air is Low, and the declustering voltage is tadalafil and tadalafil-d 3 of 56eV respectively; tadalafil and tadalafil-d 3 with collision cell inlet voltages of 10eV, respectively; tadalafil and tadalafil-d 3 having collision voltages of 15eV, respectively; tadalafil and tadalafil-d 3 with collision cell exit voltages of 20eV, respectively; detecting in a positive ion mode; the scanning mode is multiple reaction monitoring; the ion reactions for quantitative analysis were: m/z390.3 → m/z268.1, which is tadalafil; and m/z393.3 → m/z271.1, which is tadalafil-d 3.
Preferably, the gradient elution in step (3) is performed by the following procedure:
| total time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 45 | 55 |
| 0.4 | 45 | 55 |
| 0.5 | 10 | 90 |
| 1.7 | 10 | 90 |
| 1.8 | 45 | 55 |
Preferably, in the step (2), the concentration of tadalafil in the plasma sample is calculated by taking the peak area ratio of tadalafil and internal standard tadalafil-d 3 into a standard curve equation by using an internal standard method.
Preferably, the establishment of the standard curve equation comprises the following steps:
taking 10 parts of 100 mu L blank plasma, placing the blank plasma in a labeled glass test tube, adding 5 mu L of tadalafil solution with the concentration of 0.02 ng/mu L, 0.04 ng/mu L, 0.1 ng/mu L, 0.2 ng/mu L, 0.6 ng/mu L, 1.6 ng/mu L, 4 ng/mu L and 12 ng/mu L to the lowest quantitative lower limit sample, the standard sample 1, the standard sample 2, the standard sample 3, the standard sample 4, the standard sample 5, the standard sample 6 and the highest quantitative upper limit sample in the form of stock solution, respectively adding 5 mu L of the blank plasma with the volume ratio of 1: 1 to a blank sample and a zero-concentration sample, respectively adding 20 mu L of 0.2ng/uL internal standard tadalafil-d 3 solution into the lowest quantitative lower limit sample, the standard 1, the standard 2, the standard 3, the standard 4, the standard 5, the standard 6, the highest quantitative upper limit sample and the zero-concentration sample after uniformly mixing, and adding 20 mu L of the internal standard tadalafil-d 3 solution into the blank sample according to the volume ratio of 1: 1, after uniformly mixing, respectively adding 20 mu L of 1mol/L dipotassium hydrogen phosphate into 10 samples, after uniformly mixing, adding 3mL of methyl tert-butyl ether into a test tube, carrying out vortex mixing for 1min, centrifuging for 5min at 20 ℃ at 3000rpm, taking an organic solution layer and a label, transferring the organic solution layer and the label to another clean test tube, carrying out evaporation drying at 35 ℃ for at least 15min under the flow of pure air until the test tube is completely dried, and redissolving 1000 mu L of organic solution in the test tube, wherein the organic solution is methanol: water: formic acid is mixed according to a volume ratio of 55: 45: 0.5, uniformly mixing the obtained mixture in a vortex mode for 1min, transferring the solution in the test tube into a 96-deep-hole plate, and respectively using the solution as 10 standard samples to be detected;
and respectively injecting 10 mu L of standard sample into a high performance liquid chromatography-tandem mass spectrometer, detecting chromatographic peaks of tadalafil in the sample and internal standard tadalafil-d 3, and obtaining a standard curve according to the chromatographic peaks so as to calculate the concentration of tadalafil in the plasma.
Compared with the prior art, the invention has the following advantages:
(1) the pretreatment method is simple and convenient, two-step organic solution extraction is adopted, and the method is suitable for conventional determination;
(2) the specificity is strong: under the chromatographic conditions adopted in the experiment, the retention time of tadalafil is about 1.342min, and the retention time of internal standard tadalafil-d 3 is about 1.328 min. The Tadalafil and the internal standard Tadalafil-d 3 have good peak shapes, no interference of miscellaneous peaks in measurement and stable base line;
(3) the sensitivity is high: the minimum limit of plasma quantification is 1ng/mL, the concentration of tadalafil in the plasma can be accurately measured, the sensitivity is high, and the specificity is strong;
(4) the method is rapid, accurate, high in sensitivity and simple and convenient to operate, and provides a basis for measuring the blood concentration of tadalafil. The linear range of the plasma standard curve of the method is 1-600 ng/mL, and the precision RSD in batch and between batches is less than +/-15%.
Drawings
FIG. 1 is a standard graph of tadalafil in human plasma by HPLC-MS/MS;
FIG. 2 is a HPLC-MS/MS graph of human blank plasma;
FIG. 3 is a HPLC-MS/MS graph of human blank plasma with addition of tadalafil and tadalafil-d 3;
FIG. 4 is a HPLC-MS/MS chart of plasma samples with the addition of internal standard tadalafil-d 3 after oral administration of tadalafil drug to healthy subjects.
Detailed Description
The present invention will be described in further detail with reference to examples.
Example (b): human K2Determination of tadalafil concentration in EDTA plasma
First, experimental material and analytical equipment
Tadalafil (analyte): pharmaceutical Convention or same, higher-grade standard
Tadalafil-d 3 (internal standard): simson Pharma or same, higher-grade standard
The reagents used are shown in table 1 below:
TABLE 1 details of reagents
Note: the same or higher level of reagents may also be used
The analytical equipment used is shown in table 2 below:
TABLE 2 details of the devices used
| Assembly | Model number | Manufacturer(s) |
| Binary pump (Binary pump) | AC Pump | AB SCIEX |
| Degasser (deaerator) | Degasser | AB SCIEX |
| Column oven (constant temperature Column box) | AC Columnoven | AB SCIEX |
| Autosampler (automatic sampler) | AC Autosampler | AB SCIEX |
| Sample rack | Rack Changer | AB SCIEX |
| Mass spectrometer | QTRAPTM 6500+ | AB SCIEX |
| Data processor | Analyst 1.6.3(software) | AB SCIEX |
The same LC/MS system may also be used.
Second, liquid condition
1. Conditions of liquid chromatography
A chromatographic column: agilent ZORBAX XDB-C18, 5 μm, column specification 50X 2.1 mm; temperature of the chromatographic column: 40 ℃; mobile phase A: the volume percentage of water/formic acid is 100/0.5; mobile phase B: the volume percentage of methanol/formic acid is 100/0.5; washing liquid: the methanol/water volume percentage was 50/50; the autosampler temperature was 15 ℃; gradient elution with flow rate of 0.4mL/min, sample size of 10 μ L, and analysis time of 3.5 min;
TABLE 3 gradient elution procedure
| Step (ii) of | Total time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 1 | 0 | 45 | 55 |
| 2 | 0.4 | 45 | 55 |
| 3 | 0.5 | 10 | 90 |
| 4 | 1.7 | 10 | 90 |
| 5 | 1.8 | 45 | 55 |
2. Conditions of Mass Spectrometry
The ion source is an electrospray ion source, the spraying voltage is 5000V, the atomizing temperature is 500 ℃, the pressure of spraying air is 55Psi, the pressure of auxiliary heating air is 55Psi, the pressure of air curtain air is 30Psi, the pressure of collision air is Low, and the declustering voltage is tadalafil and tadalafil-d 3 of 56eV respectively; tadalafil and tadalafil-d 3 with collision cell inlet voltages of 10eV, respectively; tadalafil and tadalafil-d 3 having collision voltages of 15eV, respectively; tadalafil and tadalafil-d 3 with collision cell exit voltages of 20eV, respectively; detecting in a positive ion mode; the scanning mode is multiple reaction monitoring;
the ion reactions for quantitative analysis were: m/z390.3 → m/z268.1, which is tadalafil; and m/z393.3 → m/z271.1, which is tadalafil-d 3.
Third, the experimental process
1. Preparation of Tadalafil standard solution
Preparing a tadalafil standard solution: 0.001g of tadalafil (analyte) was precisely weighed, placed in a 10mL volumetric flask, and added with a volume ratio of 1: dissolving the acetonitrile aqueous solution of 1, fixing the volume to scale, shaking up to obtain 100 ng/mu L tadalafil stock solution, and mixing the solution according to the volume ratio of 1: 1, sequentially diluting the acetonitrile aqueous solution to prepare a tadalafil standard solution, wherein the specific dilution concentration is shown in the following table 4:
TABLE 4 Tadalafil Standard solution preparation concentration
| Source solution (ng/. mu.L) | Volume of source solution (μ L) | Final volume (mL) | Final concentration (ng/. mu.L) |
| 100a | 1200 | 10 | 12 |
| 100a | 400 | 10 | 4 |
| 100a | 160 | 10 | 1.6 |
| 100a | 60 | 10 | 0.6 |
| 100a | 20 | 10 | 0.2 |
| 4 | 250 | 10 | 0.1 |
| 4 | 100 | 10 | 0.04 |
| 4 | 50 | 10 | 0.02 |
| 4 | 150 | 10 | 0.06 |
a: prepared directly from tadalafil (analyte)
The tadalafil standard solution is stored in plastic containers and refrigerators (4 ℃) when not in use, and the volume can be increased or decreased proportionally as required.
2. Preparation of standard solution of tadalafil-d 3 internal standard
Preparation of standard solution of tadalafil-d 3 internal standard: 0.001g of tadalafil-d 3 (internal standard) was precisely weighed, placed in a 10mL volumetric flask, and added with a solution of 1: dissolving the acetonitrile aqueous solution of 1, fixing the volume to scale, shaking up to obtain 100 ng/mu L tadalafil-d 3 stock solution, and mixing the stock solution with the volume ratio of 1: 1 to prepare an internal standard solution of tadalafil-d 3 with the concentration of 0.2 ng/. mu.L, wherein the specific dilution concentration is shown in the following table 5:
TABLE 5 Tadalafil-d 3 Standard solution preparation concentration
| Source solution (ng/. mu.L) | Volume of source solution (μ L) | Final volume (mL) | Final concentration (ng/. mu.L) |
| 100a | 200 | 100 | 0.2b |
a: prepared directly from tadalafil-d 3 (internal standard)
b: for sample preparation procedures
The standard solution of tadalafil-d 3 internal standard was stored in plastic containers and a refrigerator (4 ℃) when not in use, and the volume was increased or decreased proportionally as required.
3. Linear test
Putting the blank plasma into a water bath at room temperature for unfreezing; transferring 10 parts of 100 mu L of blank plasma into a 96-deep-well plate (each standard curve sample, blank sample-00 and zero concentration sample-0), respectively and precisely adding 5 mu L of tadalafil standard solution or diluted solution with different concentrations according to the list in the following table 6 to prepare each sample, uniformly mixing the samples to prepare the drug-containing plasma with different concentrations, and carrying out the operation according to 'plasma sample pretreatment'. Calculate himThe ratio Y (Y ═ As/Ai) of the area As of dalafei peak and the area Ai of tadalafil-d 3 internal standard was used for the regression calculation of the blood concentration X by the peak area ratio Y, and the results are shown in FIG. 1 and Table 7. The average ratio Y is used for carrying out regression calculation on the blood concentration X to obtain a regression equation Y which is 0.0263X-0.000680, r is 0.9995, and a weight coefficient W which is 1/X2The lowest quantitative limit of the blood concentration of tadalafil measured by the method is as follows: 10 ng/mL.
TABLE 6 Tadalafil Standard Curve formulation concentration
b: diluted solution of analyte: ACN/H2O＝50/50
TABLE 7 Standard Curve of tadalafil in human plasma by HPLC-MS/MS method (n ═ 10)
4. Accuracy and precision
Putting the blank plasma into a water bath at room temperature for unfreezing; transfer appropriate volume of blank plasma to appropriate container and add tadalafil standard solution to prepare 5 different concentration drug-containing plasma quality control samples (LLOQ, QL, QLM, QM, QH) and a running standard curve, according to the "plasma sample pretreatment" procedure, quality control sample preparation is as shown in table 8 below. Making one batch and one following standard curve every day, continuously making 3 days, making 6 samples for each concentration of the first batch and the second batch, making 14 samples for each concentration of the third batch, calculating the ratio Y of the tadalafil peak area As and the internal standard tadalafil-d 3 peak area Ai, substituting the ratio Y into the standard curve on the day to obtain the measured concentration, calculating the precision between batches and the measured concentration according to the measured concentration, wherein the ratio of the measured concentration to the added concentration is the accuracy, and the result is shown in Table 9. The result shows that the precision and accuracy of the tadalafil plasma sample in and among batches are less than +/-15 percent and meet the requirements.
TABLE 8 quality control sample preparation concentration
a: final volume is source solution volume + plasma volume
Sufficient volume was dispensed into the labeled sample vials as required for each assay batch and stored at the theoretical temperature-80 ℃. The volume may be scaled up or down as desired.
TABLE 9 Intra-batch, inter-batch precision and accuracy of tadalafil determination in plasma by HPLC-MS/MS method
a, the concentration exceeds the standard; sample preparation Defect of PSD
Note: the data of the evaluation results are from the data of 3 batches of 26 groups of quality control samples in table 9.
5. Interference
Six different blank plasma samples are respectively from different healthy human bodies, and the six different blank plasma samples are prepared and analyzed in the same analysis batch according to the sample preparation steps to evaluate the interference of the different blank plasma on the tadalafil analyte and the internal standard tadalafil-d 3.
After the preparation and analysis of the blank healthy human plasma samples from six different sources, the interference peak responses at the retention time of tadalafil are all lower than 20.0% of the tadalafil response of the quantitative lower limit sample in the standard curve of the analysis batch, and the results are shown in table 10. The result shows that the analysis method has specificity to the analysis of tadalafil.
After analysis of the six different source blank healthy human plasma samples, the interference peak responses at retention times consistent with internal standard tadalafil-d 3 were all less than 5.0% of the internal standard tadalafil-d 3 response of the lower quantitative limit sample in the standard curve of the assay lot, see table 11 in the appendix. The results show that the assay is selective for the assay of internal standard tadalafil-d 3.
TABLE 10 comparison of interference data of blank healthy human plasma from six different sources on tadalafil analytes
a: analyte peak area (selective sample)/analyte peak area (LLOQ of standard curve). times.100.0% to 20.0%
b: the area peak area is considered zero when "no significant peak can be integrated (or no peak)" or "the retention time of the peak area does not match the retention time of the analyte in the sample".
TABLE 11 comparison of interfering data of blank healthy human plasma from six different sources against internal standard tadalafil-d 3
a: the area of the peak of the analyte (selective sample)/the area of the peak of the internal standard (LLOQ of the standard curve) multiplied by 100.0 percent is less than or equal to 5.0 percent
b: the area peak area is considered zero when "no significant peak can be integrated (or no peak)" or "the retention time of the peak area does not match the retention time of the analyte in the sample".
As can be seen from tables 10 and 11, blank plasma from different human bodies did not interfere with the detection results of tadalafil. Therefore, the method can be used for detecting the concentration of tadalafil in different human plasmas.
6. Detection of human plasma samples
(1) Taking blank plasma of a person to which tadalafil is not administered, precisely adding 100 mu L of blank plasma sample into a labeled glass test tube, and adding 40 mu L of blank plasma sample in a volume ratio of 1: 1, adding 20 mu L of 1mol/L dipotassium phosphate after uniformly mixing, adding 3mL of methyl tert-butyl ether into a test tube after uniformly mixing, carrying out vortex mixing for 1min, centrifuging for 5min at 3000rpm at 20 ℃, taking an organic solution layer and a label, transferring the organic solution layer and the label to another clean test tube, carrying out evaporation drying for at least 15min at 35 ℃ under a pure air flow until the test tube is completely dried, and redissolving 1000 mu L of organic solution in the test tube, wherein the organic solution is methanol: water: formic acid is mixed according to a volume ratio of 55: 45: 0.5 of the resulting mixture, vortex for 1min, transfer the solution in the tube to a 96-deep well plate, take 10. mu.L of sample for LC-MS/MS analysis, the results are shown in FIG. 2.
(2) Taking blank plasma of a person to which tadalafil is not given, precisely adding 100 mu L of blank plasma sample into a glass test tube with a label, adding 20 mu L of tadalafil standard solution with the concentration of 0.2ng/uL, uniformly mixing, adding 20 mu L of 0.2ng/uL internal standard tadalafil-d 3 solution, uniformly mixing, adding 20 mu L of 1mol/L dipotassium hydrogen phosphate, uniformly mixing, adding 3mL of methyl tert-butyl ether into the test tube, vortex mixing for 1min, centrifuging for 5min at 20 ℃ and 3000rpm, taking an organic solution layer and the label, transferring the organic solution layer and the label to another clean test tube, carrying out evaporation drying at 35 ℃ for at least 15min under pure air flow, and dissolving 1000 mu L of organic solution in the test tube, wherein the organic solution is methanol: water: formic acid is mixed according to a volume ratio of 55: 45: 0.5 mixing the resulting mixture, vortex mixing for 1min, transferring the solution in the tube to a 96 deep well plate, taking 10uL sample for LC-MS/MS analysis, the results are shown in FIG. 3.
(3) Collecting plasma of healthy subjects after oral administration of tadalafil medicaments, precisely adding 100 mu L of collected human plasma samples into a glass test tube stuck with a label, and adding 5uL of the plasma samples in a volume ratio of 1: 1, adding 20uL of 0.2 ng/muL internal standard tadalafil-d 3 solution after uniformly mixing, adding 20uL of 1mol/L dipotassium hydrogen phosphate after uniformly mixing, adding 3mL of methyl tert-butyl ether into a test tube after uniformly mixing, vortex mixing for 1min, centrifuging for 5min at 3000rpm at 20 ℃, taking an organic solution layer and a label, transferring the organic solution layer and the label to another clean test tube, carrying out evaporation drying for at least 15min at 35 ℃ under a pure air flow until the test tube is completely dried, and redissolving 1000 muL of organic solution in the test tube, wherein the organic solution is methanol: water: formic acid is mixed according to a volume ratio of 55: 45: 0.5 of the resulting mixture, vortex for 1min, transfer the solution in the tube to a 96-deep well plate, take 10. mu.L of sample for LC-MS/MS analysis, the results are shown in FIG. 4.
In conclusion, the invention provides a simple and convenient method for determining the concentration of tadalafil in blood plasma by a pretreatment method, adopts a two-step organic solution extraction method, and is suitable for conventional determination; meanwhile, under the chromatographic conditions adopted in the experiment, the retention time of tadalafil is about 1.342min, the retention time of internal standard tadalafil-d 3 is about 1.328min, the peak shapes of tadalafil and internal standard tadalafil-d 3 are good, the determination is free of the interference of miscellaneous peaks, and the base line is stable; the method has high specificity, can accurately measure the concentration of tadalafil in blood plasma, and has high sensitivity, and the minimum limit of quantitation of the blood plasma is 1 ng/mL; meanwhile, the method is rapid, accurate, high in sensitivity and simple and convenient to operate, and provides a basis for measuring the blood concentration of tadalafil. The linear range of the plasma standard curve of the method is 1-600 ng/mL, and the precision RSD in batch and between batches is less than +/-15%.
Claims (4)
1. A method for determining the concentration of tadalafil in plasma by liquid chromatography-mass spectrometry is characterized by comprising the following steps: the plasma sample is pretreated and then the concentration of the plasma sample is detected by high performance liquid chromatography-tandem mass spectrometry, and the specific method comprises the following steps:
(1) pretreatment of a plasma sample:
plasma with K2EDTA as anticoagulant, tadalafil-d 3 as internal standard; to a labeled glass tube, 100. mu.L of plasma sample was added, and 5. mu.L of a 1: 1, adding 20 mu L of 0.2 ng/mu L internal standard tadalafil-d 3 solution after uniformly mixing, adding 20 mu L of 1mol/L dipotassium hydrogen phosphate after uniformly mixing, adding 3mL of methyl tert-butyl ether into a test tube after uniformly mixing, vortex mixing for 1min, centrifuging for 5min at 20 ℃ and 3000rpm, taking an organic solution layer and a label, transferring the organic solution layer and the label to another clean test tube, carrying out evaporation drying for at least 15min at 35 ℃ under pure air flow until the test tube is completely dried, redissolving 1000 mu L of organic solution in the test tube, wherein the organic solution is AAlcohol: water: formic acid is mixed according to a volume ratio of 55: 45: 0.5, uniformly mixing the obtained mixture in a vortex mode for 1min, transferring the solution in the test tube to a 96-deep-hole plate, and taking the solution as a test sample to be detected;
(2) and (3) sample measurement:
injecting 10 mu L of test sample into a high performance liquid chromatography-tandem mass spectrometer, detecting chromatographic peaks of tadalafil and internal standard tadalafil-d 3 in the sample, and calculating the concentration of tadalafil in the plasma sample according to the chromatographic peaks;
(3) liquid chromatography determination, conditions are as follows:
a chromatographic column: agilent ZORBAX XDB-C18, 5 μm, column specification 50X 2.1 mm; temperature of the chromatographic column: 40 ℃; mobile phase A: the volume percentage of water/formic acid is 100/0.5; mobile phase B: the volume percentage of methanol/formic acid is 100/0.5; washing liquid: the methanol/water volume percentage was 50/50; the autosampler temperature was 15 ℃; gradient elution with flow rate of 0.4mL/min, sample size of 10 μ L, and analysis time of 3.5 min;
(4) mass spectrometry under the conditions:
the ion source is an electrospray ion source, the spraying voltage is 5000V, the atomizing temperature is 500 ℃, the pressure of spraying air is 55Psi, the pressure of auxiliary heating air is 55Psi, the pressure of air curtain air is 30Psi, the pressure of collision air is Low, and the declustering voltage is tadalafil and tadalafil-d 3 of 56eV respectively; tadalafil and tadalafil-d 3 with collision cell inlet voltages of 10eV, respectively; tadalafil and tadalafil-d 3 having collision voltages of 15eV, respectively; tadalafil and tadalafil-d 3 with collision cell exit voltages of 20eV, respectively; detecting in a positive ion mode; the scanning mode is multiple reaction monitoring;
the ion reactions for quantitative analysis were: m/z390.3 → m/z268.1, which is tadalafil; and m/z393.3 → m/z271.1, which is tadalafil-d 3.
2. The method for determining the concentration of tadalafil in plasma by LC-MS as claimed in claim 1, wherein: the gradient elution procedure in the step (3) is as follows:
3. the method for determining the concentration of tadalafil in plasma by LC-MS as claimed in claim 1 or 2, wherein: in the step (2), an internal standard method is adopted, and the concentration of tadalafil in the plasma sample is calculated by substituting the peak area ratio of tadalafil and internal standard tadalafil-d 3 into a standard curve equation.
4. The method for determining the concentration of tadalafil in plasma by LC-MS as claimed in claim 3, wherein: the establishment of the standard curve equation comprises the following steps:
taking 10 parts of 100 mu L blank plasma, placing the blank plasma in a labeled glass test tube, adding 5 mu L of tadalafil solution with the concentration of 0.02 ng/mu L, 0.04 ng/mu L, 0.1 ng/mu L, 0.2 ng/mu L, 0.6 ng/mu L, 1.6 ng/mu L, 4 ng/mu L and 12 ng/mu L to the lowest quantitative lower limit sample, the standard sample 1, the standard sample 2, the standard sample 3, the standard sample 4, the standard sample 5, the standard sample 6 and the highest quantitative upper limit sample in the form of stock solution, respectively adding 5 mu L of the blank plasma with the volume ratio of 1: 1 to a blank sample and a zero-concentration sample, respectively adding 20 mu L of 0.2ng/uL internal standard tadalafil-d 3 solution into the lowest quantitative lower limit sample, the standard 1, the standard 2, the standard 3, the standard 4, the standard 5, the standard 6, the highest quantitative upper limit sample and the zero-concentration sample after uniformly mixing, and adding 20 mu L of the internal standard tadalafil-d 3 solution into the blank sample according to the volume ratio of 1: 1, after uniformly mixing, respectively adding 20 mu L of 1mol/L dipotassium hydrogen phosphate into 10 samples, after uniformly mixing, adding 3mL of methyl tert-butyl ether into a test tube, carrying out vortex mixing for 1min, centrifuging for 5min at 20 ℃ at 3000rpm, taking an organic solution layer and a label, transferring the organic solution layer and the label to another clean test tube, carrying out evaporation drying at 35 ℃ for at least 15min under the flow of pure air until the test tube is completely dried, and redissolving 1000 mu L of organic solution in the test tube, wherein the organic solution is methanol: water: formic acid is mixed according to a volume ratio of 55: 45: 0.5, uniformly mixing the obtained mixture in a vortex mode for 1min, transferring the solution in the test tube into a 96-deep-hole plate, and respectively using the solution as 10 standard samples to be detected;
and respectively injecting 10 mu L of standard sample into a high performance liquid chromatography-tandem mass spectrometer, detecting chromatographic peaks of tadalafil in the sample and internal standard tadalafil-d 3, and obtaining a standard curve according to the chromatographic peaks so as to calculate the concentration of tadalafil in the plasma.
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