CN114965781A - LC-MS/MS (liquid chromatography-mass spectrometry/mass spectrometry) determination method for penfluridol in serum - Google Patents
LC-MS/MS (liquid chromatography-mass spectrometry/mass spectrometry) determination method for penfluridol in serum Download PDFInfo
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Abstract
The invention provides an LC-MS/MS (liquid chromatography-mass spectrometry/mass spectrometry) determination method for penfluridol in serum, and relates to the technical field of physicochemical detection. The LC-MS/MS method for determining the penfluridol in the serum comprises the steps of firstly adding a protein precipitator mixed with an internal standard into a test sample, and then centrifuging to obtain a supernatant to obtain a sample to be measured; then performing LC-MS/MS analysis on the penfluridol in the sample to be detected in a positive ion mode; and obtaining the content of the penfluridol in the serum according to a standard curve of the penfluridol in the serum. The determination method comprises the steps of adding an organic solvent to precipitate proteins, precipitating the proteins to remove most of the proteins in a biological sample, centrifuging to obtain a supernatant, and performing LC-MS/MS analysis by adopting a multiple reaction monitoring positive ion mode. Meanwhile, memantine-D6 is used as an internal standard, and high-flux qualitative and quantitative detection of penfluridol is realized.
Description
Technical Field
The invention relates to the technical field of physical and chemical detection, in particular to an LC-MS/MS (liquid chromatography-mass spectrometry/mass spectrometry) determination method for penfluridol in serum.
Background
The penfluridol (penfluridol) is a diphenylbutylpiperidine derivative, is the first clinically-applied oral long-acting anti-psychotic medicament, can be used for treating acute and chronic schizophrenia, has effective disease courses, and has a plasma half-life of 24-48 h. The main adverse reactions of the medicine, such as paralysis agitans, akathisia, dystonia, uneasiness and the like, are toxic events caused by mistaken taking reported in documents in recent years.
At present, few reports are reported on methods for determining penfluridol in biological samples at home and abroad, mainly including a Gas Chromatography (GC), a gas chromatography-mass spectrometry (GC/MS) and a liquid chromatography-mass spectrometry combined method (LC-MS/MS), and the literature reports that the sensitivity and specificity of an HPLC method are relatively low, so that the method cannot be suitable for monitoring the blood concentration with high flux requirement. The GC-MS method uses a solid phase extraction method for sample pretreatment, the operation is complicated, the required time is long, the use amount of an organic solvent is large, the sample use amount is large during measurement, 3mL of serum is usually required, and the analysis time is 18 min. The LC-MS/MS pretreatment is a multi-purpose liquid-liquid extraction or solid-phase extraction method, and the extraction process is complex.
Therefore, it is necessary and urgent to develop a method for measuring LC-MS/MS of penfluridol in serum, which uses an internal standard method for detection and does not require a complicated pretreatment extraction process.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first object of the present invention is to provide an LC-MS/MS measurement method of penfluridol in serum, which comprises precipitating proteins by adding an organic solvent, removing most of the proteins in a biological sample by precipitating the proteins, centrifuging the supernatant, and performing LC-MS/MS analysis using a multiple reaction monitoring negative ion mode. Meanwhile, Memantine-D6 (Memantine-D6Hydrochloride) is used as an internal standard to realize high-flux qualitative and quantitative detection of penfluridol.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
the invention provides an LC-MS/MS (liquid chromatography-mass spectrometry/mass spectrometry) determination method of penfluridol in serum, which comprises the following steps:
(a) and sample pretreatment: adding a protein precipitator mixed with an internal standard into a test sample, and centrifuging to obtain a supernatant to obtain a sample to be tested;
the protein precipitator mixed with the internal standard is mainly prepared by diluting memantine-D6 methanol solution with acetonitrile;
(b) performing LC-MS/MS analysis on the penfluridol in the sample to be detected in a positive ion mode, and collecting detection data; and obtaining the content of the penfluridol in the serum according to the standard curve of the penfluridol in the serum.
Further, the concentration of the memantine-D6 methanol solution is 0.01-1 mg/mL, preferably 0.1 mg/mL;
preferably, the concentration of the memantine-D6 internal standard is 1-100 ng/mL, and is preferably 12 ng/mL.
Further, the test sample is plasma or serum of a subject to be tested.
Further, the step (a) includes the steps of:
adding 100 mu L of test sample solution into 200 mu L of internal standard working solution and uniformly mixing at 1200rpm for 10 minutes; centrifuging at 4000rpm/min for 10 min, and removing 100 mu L of supernatant; and then centrifuging the removed supernatant at 4000rpm/min for 5 minutes again to obtain a sample to be detected.
Further, the liquid chromatography conditions of the LC-MS/MS analysis include:
liquid phase analysis chromatographic column: XBridge BEH C182.5 μm 3.0 x 50 mm;
the column temperature is 30-50 ℃, and the sample injection amount is 1-20 mu L;
preferably, the column temperature is 40 ℃; the sample size was 5. mu.L.
Further, the liquid chromatography gradient elution procedure is:
the phase A is 0.05-0.25% formic acid-water solution, and the phase B is 0.05-0.25% formic acid-methanol water solution;
preferably, phase a is a 0.1% formic acid-water solution, phase B is a 0.1% formic acid-methanol water solution;
the gradient elution procedure was:
further, the mass spectrum detection of the LC-MS/MS analysis is detection in a negative ion scanning mode and a multiple reaction monitoring mode;
the ion source parameters for mass spectrometry detection include:
further, the standard curve of the penfluridol in the serum in the step (B) is established according to the following method:
preparing a pentafluridol standard working solution, mixing the pentafluridol standard working solution with an internal standard solution, and centrifuging to obtain a supernatant; performing LC-MS/MS analysis and detection on the supernatant of the standard working solution of penfluridol to obtain chromatographic and mass spectrometric data of the penfluridol in serum;
and performing regression analysis on the chromatographic data and chromatographic peak area of the penfluridol according to an internal standard method to the concentration of the standard working solution of the penfluridol, thereby obtaining a standard curve of the penfluridol in the serum.
Further, the standard curve of penfluridol in serum is as follows:
y＝0.00756x+0.00064(r＝0.9967)。
further, the prepared plasma of the pentafluridol standard working solution is the serum or the plasma of an animal;
the animal serum or plasma includes pig serum or plasma, sheep serum or plasma, horse serum or plasma, and cattle serum or plasma, preferably calf serum or plasma.
Compared with the prior art, the invention has the beneficial effects that:
the LC-MS/MS determination method of penfluridol in serum comprises the steps of firstly mixing a test sample solution into an internal standard solution, and then centrifuging to obtain a supernatant to obtain a sample to be measured; then performing LC-MS/MS analysis on the penfluridol in the sample to be detected in a positive ion mode, and collecting detection data; and obtaining the content of the penfluridol in the serum according to the standard curve of the penfluridol in the serum. Wherein the internal standard solution is mainly prepared by diluting a memantine-D6 methanol solution with acetonitrile; the determination method comprises the steps of adding an organic solvent to precipitate proteins, precipitating the proteins to remove most of the proteins in a biological sample, centrifuging to obtain a supernatant, and performing LC-MS/MS analysis by adopting a multiple reaction monitoring negative ion mode. Meanwhile, Memantine-D6 (Memantine-D6Hydrochloride) is used as an internal standard to realize high-throughput qualitative and quantitative detection of penfluridol.
Experiments show that the LC-MS/MS method is adopted to rapidly determine the concentration of the penfluridol in human serum, and the method is specificGood specificity and high analysis sensitivity, and the LOB concentration reaches 0.04 ng/mL; the accuracy is high, and the recovery rates of low, medium and high concentration samples are all within 85-115%; the variation coefficients (% CV) of the imprecision degree of the low, medium and high concentration quality control samples in batches and between batches are all within 15 percent; the deviation of the lowest concentration point of the graticule is within +/-20.0 percent, the deviation of the other concentration points is within +/-15.0 percent, and a linear regression fitting constant R 2 Greater than 0.99; the matrix effect is within the range of +/-20%; residue tests show that the peak areas of the blank matrixes are all smaller than the peak area of the lowest point of the marked line by 20.0 percent, and the internal standard does not exceed 5 percent; all meet the acceptance criteria.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a standard graph of penfluridol in serum provided in example 1 of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The technical solution of the present invention will be further described with reference to the following examples.
Example 1 standard working curve plotting and calculation:
firstly, material information and solution preparation:
(1) and material information:
(2) pentafluridol and internal standard stock solution configuration:
(3) and preparing a pentafluridol standard working solution:
note: because human samples are difficult to obtain, the invention adopts calf plasma as a substitute matrix of human serum to prepare various concentrations of a standard curve.
Gradient solution:
(4) and preparing internal standard working solution:
secondly, preparing a penfluridol standard detection sample:
(1) and respectively transferring 100 mu L of the standard working solution of Penfluridol C1-C6, respectively adding the standard working solution into a new 96-well plate, adding 200 mu L of internal standard working solution, and uniformly mixing at 1200rpm for 10 minutes.
(2) And placing the mixed sample in a centrifuge for centrifugation at 4000rpm for 10 minutes. 100 μ L of the supernatant was removed in a new 96-well plate and centrifuged at 4000rpm for 5 minutes.
(3) And taking 5 mu L of the treated standard working solutions of the pentafluridol C1-C6 respectively for LC-MS/MS analysis.
Thirdly, chromatographic detection conditions:
liquid phase analysis chromatographic column: XBridge BEH C182.5 μm 3.0 x 50 mm;
column temperature: 40 ℃; sample introduction amount: 5 μ L.
The liquid chromatography gradient elution procedure is as follows: phase A is 0.1% formic acid-water solution, phase B is 0.1% formic acid-methanol water solution;
the gradient elution procedure was:
fourthly, mass spectrum detection conditions:
ion information:
note: PFD-1, PFD-2, PFD-3 and PFD-4 are four ion channels, and the other ion channels are quantitative ion pairs and qualitative ion pairs.
PFD: pentafluridol; MM-D6: memantine-D6; quantitation of ion pairs.
Ion source parameters:
| item | Parameter(s) |
| Ion source | Electrospray ion source |
| Detection mode | Positive ion |
| Scanning mode | Multiple reaction monitoring |
| CUR (air curtain) | 35psi |
| CAD (air blast) | 8 |
| IS (ionization voltage) | 5500(V) |
| TEM (temperature) | 600(℃) |
| GS1 (spray) | 50psi |
| GS2 (auxiliary heating gas) | 50psi |
。
Drawing a standard curve and calculating a result:
respectively analyzing the standard working solutions of the pentafluridol with the concentration of C1-C6, and performing regression analysis on the corresponding concentration of the peak area of the chromatographic peak of the pentafluridol according to an internal standard method to obtain a standard working curve; the specific standard working curve is as follows:
y＝0.00756x+0.00064(r＝0.9967)。
FIG. 1 is a standard graph of penfluridol in serum provided in this example.
Example 2 test method validation:
the detection method in this example verifies that the solution used and the parameters of the LC-MS/MS detection are the same as in example 1.
Firstly, solution preparation:
preparing a quality control product working solution:
high, medium and low concentration sample configuration:
| sample (I) | LQC | MQC | HQC |
| H0 volume (ul) | 3976 | 2400 | 1000 |
| C6 volume (ul) | 24 | 1600 | 3000 |
| Concentration (ng/ml) | 1.2 | 80 | 150 |
| Total volume (ul) | 4000 | 4000 | 4000 |
Preparing an accurate working solution:
sample configuration with high, medium and low concentration accuracy:
| sample (I) | LQC | MQC | HQC |
| H0 volume (ul) | 12 | 800 | 1500 |
| C6 volume (ul) | 1988 | 1200 | 500 |
| Concentration (ng/ml) | 1.2 | 80 | 150 |
| Total volume (ul) | 2000 | 2000 | 2000 |
Secondly, imprecision verification:
(1) internal imprecision:
the verification method comprises the following steps: pre-treating 100 μ L of low, medium and high concentration imprecise working solution samples (concentration: 1.2ng/mL, 80ng/mL, 150ng/mL, respectively); 5 samples per concentration level were run in parallel.
Acceptance criteria: precision is assessed by the coefficient of variation (% CV), which should be within 15.0%.
(2) Batch imprecision:
the verification method comprises the following steps: pre-treating 100 μ L of low, medium and high concentration imprecise working solution samples (concentration: 1.2ng/mL, 80ng/mL, 150ng/mL, respectively); 5 samples per concentration level were run in parallel, with a validation period of 5 days, one batch per day.
Acceptance criteria: precision is assessed by the coefficient of variation (% CV), which should be within 15.0%.
The specific validation data is as follows:
from the above, the variation coefficients (% CV) of the low, medium and high concentration in-batch and inter-batch imprecisions were all within 15%, and satisfied the acceptance criteria.
Thirdly, verifying the accuracy:
the verification method comprises the following steps:
adding a known high-level test object (A) into low-concentration serum B, wherein the volume ratio of the added test object A to the serum B is not more than 1: 9, repeat the test 3 times each and average the value.
The recovery rate can be calculated by referring to a formula, and the average recovery rate of each sample is within the range of 85 percent and 115 percent.
In the formula:
r-recovery rate;
c, average value of detection concentration after adding the solution A into the solution B;
v0-volume of liquid B;
vs — volume of liquid a;
c0-average value of concentration of the solution B;
Cs-A solution concentration.
The specific validation data is as follows:
as can be seen from the above table, the recovery rates of the low, medium and high concentration samples are all within 85-115%, and meet the acceptance standard.
Fourthly, verifying analysis sensitivity:
LOB verification method: repeating 4 blank substrates and 4 samples of C1 each day for 5 days, calculating peak Area means of the blank samples and C1 samples, LOB Area being defined as 20 blank sample peak Area means +3SD, concentration being calculated as: LOB Area/C1Area C1, units as C1;
acceptance criteria: and (4) NA.
And (3) calculating a result of data:
fifthly, linear verification:
the verification method comprises the following steps: a certain amount of compound working solution (the concentration is respectively C1, C2, C3, C4, C5 and C6) is pre-treated for 5 days continuously.
Acceptance criteria: the deviation of the lowest concentration point should be within. + -. 20.0%, the deviation of the remaining concentration points should be within. + -. 15.0%, and the CV should be within 20.0%. Linear regression: r is 2 ≥0.99。
And (3) calculating the result of data:
and (4) conclusion: the deviation of the lowest concentration point is within plus or minus 20.0 percent, and the deviation of the other concentration points is within plus or minus 15.0 percent, so that the receiving standard is met; linear regression: r 2 > 0.99, meeting the acceptance criteria.
Sixthly, matrix effect:
the verification method comprises the following steps: high-concentration samples and low-concentration samples (the concentrations are respectively 1.2ng/mL and 150ng/mL) are prepared by adopting serum, pure solvent samples with the same concentration are prepared, each concentration is repeatedly measured for 3 times, and the sample concentration mean value is respectively calculated.
Acceptance criteria: relative matrix deviation (%) - (plasma sample mean-serum background mean-pure solvent sample mean)/pure solvent sample mean (%), matrix deviation < ± 20%.
And (3) calculating a result of data:
and (4) conclusion: the matrix effect is less than +/-20 percent and meets the acceptance standard.
Seventhly, residue evaluation:
respectively taking a certain amount of residual working solution sample (C6) and a blank solution for pretreatment; the residue was evaluated by injecting a sample of residual working solution first, followed by 3 consecutive injections of blank solution, and by repeated experiments of 5 analytical batches.
Acceptance criteria: the peak area of the compound of the blank matrix is less than 20.0% of the peak area of the C1 compound, and the peak area of the internal standard of the blank matrix is less than 5% of the peak area of the C1 internal standard.
And (3) calculating a result of data:
and (4) conclusion: the peak areas of the blank substrates are all less than 20.0% of the peak area of C1, and the acceptance standard is met. The internal standard does not exceed 5 percent and meets the acceptance standard.
According to the analysis, the LC-MS/MS method is adopted to rapidly determine the concentration of the penfluridol in human serum, the method is good in specificity, strong in specificity and high in analysis sensitivity, and the LOB concentration reaches 0.04 ng/mL; the accuracy is high, and the recovery rates of low, medium and high concentration samples are all within 85-115%; the variation coefficients (% CV) of the imprecision degree of the low, medium and high concentration quality control samples in batches and between batches are all within 15 percent; the deviation of the lowest concentration point of the graticule is within +/-20.0 percent, the deviation of the other concentration points is within +/-15.0 percent, and the linear regression fitting constant R2 is more than 0.99; the matrix effect is within the range of +/-20%; residue tests show that the peak areas of the blank matrixes are all smaller than the peak area of the lowest point of the marked line by 20.0 percent, and the internal standard does not exceed 5 percent; all meet the acceptance criteria.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. An LC-MS/MS measurement method of penfluridol in serum, which is characterized by comprising the following steps:
(a) and sample pretreatment: adding a protein precipitator mixed with an internal standard into a test sample, and centrifuging to obtain a supernatant to obtain a sample to be tested;
the protein precipitator mixed with the internal standard is mainly prepared by diluting a memantine-D6 methanol solution with acetonitrile;
(b) performing LC-MS/MS analysis on the penfluridol in the sample to be detected in a positive ion mode, and collecting detection data; and obtaining the content of the penfluridol in the serum according to the standard curve of the penfluridol in the serum.
2. The LC-MS/MS method for determining pentafluridon in serum according to claim 1, wherein the concentration of the memantine-D6 methanol solution is 0.01-1 mg/mL, preferably 0.1 mg/mL;
preferably, the concentration of the memantine-D6 internal standard is 1-100 ng/mL, and is preferably 12 ng/mL.
3. The method for LC-MS/MS measurement of penfluridol in serum according to claim 1, wherein the test sample is plasma or serum of a subject.
4. The method for LC-MS/MS determination of penfluridol in serum according to claim 1, wherein said step (a) comprises the steps of:
adding 100 mu L of test sample solution into 200 mu L of internal standard working solution and uniformly mixing for 10 minutes at 1200 rpm; then centrifuging at 4000rpm/min for 10 minutes, and removing 100 mu L of supernatant; and then centrifuging the removed supernatant at 4000rpm/min for 5 minutes again to obtain a sample to be detected.
5. The LC-MS/MS method for measuring penfluridol in serum according to claim 1, wherein the liquid chromatography conditions for the LC-MS/MS analysis include:
liquid phase analysis chromatographic column: XBridge BEH C182.5 μm 3.0 x 50 mm;
the column temperature is 30-50 ℃, and the sample injection amount is 1-20 mu L;
preferably, the column temperature is 40 ℃ and the sample size is 5. mu.L.
6. The LC-MS/MS method for determining penfluridol in serum according to claim 5, wherein said liquid chromatography gradient elution procedure is:
the phase A is 0.05-0.25% formic acid-water solution, and the phase B is 0.05-0.25% formic acid-methanol water solution;
preferably, phase a is a 0.1% formic acid-water solution, phase B is a 0.1% formic acid-methanol water solution;
the gradient elution procedure was:
7. the method for LC-MS/MS measurement of penfluridol in serum according to claim 1, characterized in that the mass spectrometric detection of the LC-MS/MS analysis is a positive ion scanning mode, a multiple reaction monitoring mode detection;
the ion source parameters for mass spectrometry detection include:
8. the method for LC-MS/MS measurement of penfluridol in serum according to claim 1, wherein the standard curve of penfluridol in serum in step (B) is established by the following method:
preparing a pentafluridol standard working solution, mixing the pentafluridol standard working solution with an internal standard solution, and centrifuging to obtain a supernatant; performing LC-MS/MS analysis and detection on the supernatant of the standard working solution of penfluridol to obtain chromatographic and mass spectrometric data of the penfluridol in serum;
and carrying out regression analysis on the concentration of the standard working solution of the penfluridol according to the chromatographic data and the chromatographic peak area of the penfluridol by an internal standard method to obtain a standard curve of the penfluridol in the serum.
9. The method for LC-MS/MS measurement of penfluridol in serum according to claim 8, wherein the standard curve of penfluridol in serum is:
y＝0.00756x+0.00064(r＝0.9967)。
10. the LC-MS/MS assay method for penfluridol in serum according to claim 8, characterized in that the prepared plasma of the standard working solution of penfluridol is animal serum or plasma;
the animal serum or plasma includes pig serum or plasma, sheep serum or plasma, horse serum or plasma, and cattle serum or plasma, preferably calf serum or plasma.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104546784A (en) * | 2014-12-31 | 2015-04-29 | 康普药业股份有限公司 | Penfluridol tablet composition and preparation method thereof |
| WO2016176726A1 (en) * | 2015-05-01 | 2016-11-10 | Griffith University | Diagnostic methods |
| CN114414707A (en) * | 2022-03-30 | 2022-04-29 | 北京和合医学诊断技术股份有限公司 | Method and kit for detecting 19 drugs and metabolites thereof in blood by liquid chromatography tandem mass spectrometry |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104546784A (en) * | 2014-12-31 | 2015-04-29 | 康普药业股份有限公司 | Penfluridol tablet composition and preparation method thereof |
| WO2016176726A1 (en) * | 2015-05-01 | 2016-11-10 | Griffith University | Diagnostic methods |
| CN114414707A (en) * | 2022-03-30 | 2022-04-29 | 北京和合医学诊断技术股份有限公司 | Method and kit for detecting 19 drugs and metabolites thereof in blood by liquid chromatography tandem mass spectrometry |
Non-Patent Citations (4)
| Title |
|---|
| CHEN C 等: "Metabolomics‐based parallel discovery of xenobiotics and induced endogenous metabolic dysregulation in clinical toxicology", BIOMEDICAL CHROMATOGRAPHY, vol. 33, pages 1 - 10 * |
| 严慧 等: "LC-MS/MS 分析人全血中五氟利多", 中国法医学杂志, vol. 26, no. 04, pages 285 - 287 * |
| 傅强 等: "现代药物分离与分析技术", 西安交通大学出版社, pages: 305 - 306 * |
| 贾晶莹 等: "液相色谱-串联质谱法同时测定7种抗抑郁类和5种抗精神病类药物的血药浓度", 中国药物应用与监测, vol. 7, no. 05, pages 272 - 275 * |
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