CN118348170B - Method for detecting milabalin in blood plasma by HPLC-MS/MS method - Google Patents
Method for detecting milabalin in blood plasma by HPLC-MS/MS method Download PDFInfo
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- RZVAJINKPMORJF-UHFFFAOYSA-N Acetaminophen Chemical compound CC(=O)NC1=CC=C(O)C=C1 RZVAJINKPMORJF-UHFFFAOYSA-N 0.000 claims abstract description 38
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G01N30/02—Column chromatography
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Abstract
The invention discloses a method for detecting milabalin in blood plasma by an HPLC-MS/MS method. The method adopts a protein precipitation method for pretreatment; carrying out gradient elution under the chromatographic condition by adopting Agela Venusil MP C (2) chromatographic column and using 5mM ammonium acetate aqueous solution and acetonitrile as mobile phases; the mass spectrum condition adopts electrospray ionization source (ESI) tandem mass spectrum, and takes acetaminophen as an internal standard for quantitative detection. The method has the advantages of simple operation, high analysis speed, high sensitivity and proper linear range, and meets the requirement of mass sample analysis in clinical research.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a method for detecting milabalin in blood plasma by an HPLC-MS/MS method.
Background
Minobaclin is an α2δ ligand that inhibits excitatory neurotransmitter release by specifically binding to the α2δ subunit of voltage-gated calcium channels, reducing calcium influx at presynaptic terminals, thereby alleviating pain. In 2019, 1 month, the product was first marketed in japan for the treatment of Peripheral Neuropathic Pain (PNP).
Therapeutic drug monitoring and drug-mimetic clinical studies require the measurement of the blood concentration of a subject or patient to study its pharmacokinetic behavior. In order to accelerate the clinical application of milabalin, a simple, accurate, rapid and highly sensitive bioanalytical method is needed. Currently Naotoshi Yamamura et al （Pharmacokinetics and metabolism of mirogabalin, a novel α2δ ligand, in rats and monkeys[J]. Xenobiotica. 2022(52),54-64） examined the pharmacokinetic and metabolic profile of rats and monkeys following gastric lavage with radiolabeled 14 C-milabalin. But the method has narrow applicability, is not an HPLC-MS/MS method widely used in the industry, and is not suitable for a simple, accurate, rapid and high-sensitivity biological analysis scene.
Up to now, no related literature and patent report on methods for detecting milabalin in plasma by HPLC-MS/MS method, so the analytical method of the study is original. In view of the blank of the existing analysis method, an analysis method which is simple in pretreatment operation and compatible with sensitivity, accuracy and analysis speed needs to be developed so as to be suitable for accurately analyzing a large number of samples in clinical research.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide a method for detecting milabalin in blood plasma by an HPLC-MS/MS method.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a method for detecting milabalin in blood plasma by an HPLC-MS/MS method, comprising the steps of:
(1) Pretreatment: adding acetonitrile precipitated protein into a plasma sample, centrifuging, taking supernatant, and diluting with water to obtain a sample;
(2) Detecting the pretreated plasma sample by HPLC-MS/MS, and determining the content of the milabalin by an internal calibration method, wherein:
Chromatographic conditions:
chromatographic column: agela Venusil MP C18 (2) chromatography column;
Mobile phase a: 5mM ammonium acetate in water, mobile phase B: acetonitrile;
Gradient elution procedure: 0-1.4 min: mobile phase B was 40%;1.4-1.5 min: mobile phase B is 40% -90%; 1.5-2.5 min: mobile phase B was 90%;2.5-2.6 min: mobile phase B is 90% -40%; 2.6-3.0min: mobile phase B was 40%;
column temperature: 35-45 ℃;
Mass spectrometry conditions:
ion source: an electrospray ion source; scanning positive ions; monitoring multiple reactions; the injection voltage is 5500V; the ion source temperature was 550 ℃.
In some embodiments, an internal standard solution is added into a plasma sample, and a test sample containing an internal standard substance is obtained through treatment; the internal standard solution is obtained by dissolving acetaminophen in methanol and diluting with acetonitrile-water (50:50, v/v). In some embodiments, the internal standard solution is added in an amount of 0.5 to 2 times the plasma sample, preferably in an equal volume.
In some embodiments, in step (1), the volume of the plasma sample is 30-100 μl, preferably 40-60 μl, for example 50 μl. In some embodiments, the amount of milbelin in the plasma sample is 1.00-500 ng/mL.
In some embodiments, in step (1), the content of internal standard substance in the test sample is 10-40 ng/mL, preferably 30 ng/mL.
In some embodiments, in step (1), the volume ratio of plasma sample to acetonitrile is 1 (3-7), preferably 1 (4-6).
In some embodiments, in the step (1), the supernatant obtained after centrifugation is diluted by adding water, so that the sample is not easy to volatilize, and the preservation and detection accuracy of the sample are facilitated; the amount of water added is 0.5-2 times the volume of the supernatant, preferably the amount of water added is equal to the volume of the supernatant.
In some embodiments, in step (2), the column temperature is 40 ℃; the flow rate of the mobile phase is 0.7 mL/min; the sample injection amount is 10 mu L.
In some embodiments, in the mass spectrometry conditions of step (2), gas1 (Gas 1): 55 psi, gas2 (Gas 2): 55 psi, curtain Gas (Curtain Gas): 35 psi, collision induced dissociation: 6 psi, residence time: 300ms.
In some embodiments, in the mass spectrometry conditions of step (2), milabalin quantitatively analyzes ion pairs: m/z 210.2.133.0, collision energy: 20 eV, declustering voltage: 100 V, V; quantitative analysis of ion pairs for acetaminophen: 152.1 cube 110.0, collision energy: 21 eV, declustering voltage: 100 V is provided.
The beneficial effects of the invention are as follows:
1. The method has the characteristics of simple pretreatment operation, can analyze by only one-step extraction and one-step dilution, and is easy to preserve after the extraction; the method has high analysis speed (the analysis time is only 3 min), and is suitable for analyzing a large amount of clinical research samples.
2. In clinical studies, the improvement of the sensitivity of the detection method can reduce the dosage of the subjects and increase the safety of the clinical study. The method disclosed by the invention has the advantages of higher sensitivity and low plasma dosage (the plasma dosage can be only 50.0 mu L), the lower limit of the milobulin ration is 1.00 ng/mL, the detection limit is calculated to be 0.200 ng/mL, and the amount of an object to be detected on the detection limit column is calculated to be 0.125 pg according to the dilution multiple 16 and the sample injection amount 10 mu L.
3. The invention has reasonable linear range selection (the linear range is 1.00-500 ng/mL), and can more accurately measure the concentration of the low-content medicine.
The foregoing description is only an overview of the present invention, and is intended to provide a better understanding of the present invention, as it is embodied in the following description, with reference to the preferred embodiments of the present invention and the accompanying drawings.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a mass spectrum of the ion scan of the product of Milopolin in the process of the invention.
FIG. 2 is an ion flow chromatogram of Milobulin and internal standard acetaminophen in human blank plasma (upper: acetaminophen, lower: milobulin).
FIG. 3 is an ion flow chromatogram of Milopaline and the internal standard acetaminophen in the lower limit of human plasma quantification (1.00 ng/mL) (upper: acetaminophen, lower: milopaline).
FIG. 4 is an ion flow chromatogram of Milopaline and the internal standard acetaminophen in the upper limit of human plasma quantification (500 ng/mL) (upper: acetaminophen, lower: milopaline).
FIG. 5 is an ion flow chromatogram of milabalin and internal standard acetaminophen (upper: acetaminophen, lower: milabalin) when taken in healthy subjects with milabalin 1 h.
Fig. 6 is a graph of blood concentration versus time for a healthy subject taken with 10 mg m of ribavirin.
Detailed Description
The present invention will be described in further detail with reference to the following specific examples and drawings, to which the present invention is not limited. Variations and advantages that would occur to one skilled in the art are included in the invention without departing from the spirit and scope of the inventive concept, and the scope of the invention is defined by the appended claims. The procedures, conditions, reagents, experimental methods, etc. for carrying out the present invention are common knowledge and common knowledge in the art, except for those specifically mentioned below, and the present invention is not particularly limited.
The development of methods for detecting drug concentration in plasma by liquid chromatography-tandem mass spectrometry can be generally divided into three parts, i.e., an extraction method (i.e., a pretreatment method), a liquid chromatography method and a mass spectrometry method. The present invention addresses the shortcomings of the prior art by starting with the above three aspects to establish an analytical method.
The dosage of the plasma in the method can be only 50.0 mu L, and the method is suitable for biological analysis in clinical research; the pretreatment method selects a protein precipitation method, has better reproducibility on the milabalin, and has the advantages of simple operation, short extraction time and no time-consuming concentration step. The kit is matched with a 96-well plate for use, and is suitable for pretreatment of high-flux samples in clinical researches.
In the embodiment of the invention, agela Venusil MP C (2) chromatographic columns are adopted for chromatographic separation, and the chromatographic columns have better retention of objects to be detected and internal standards and sharp peak shapes. The mobile phase adopted in chromatographic analysis is 5mM ammonium acetate solution, which is favorable for maintaining the stability of pH, and the added ammonium acetate can be used as an improver to improve the peak shape and improve the integration accuracy. The instrument analysis flux is higher in the mode, the chromatographic running time is only 3.0 min, the detection is rapid, and the method is suitable for mass sample analysis in clinical research.
Description of the abbreviations
| Abbreviations (abbreviations) | Full text | Chinese description |
| LLOQ | lower limit of quantification | Lower limit of quantification |
| LQC | low quality control | Low quality control |
| AMQC | accessorial Middle Quality Control | Quality control of secondary medium concentration |
| MQC | medium quality control | Medium quality control |
| HQC | high quality control | High quality control |
| RSD | relative standard deviation | Relative standard deviation |
| RE | relative Error | Relative deviation of |
Examples
1 Material
1.1 Instruments
Chromatograph: LC-30AD flash liquid chromatography system, shimadzu corporation, japan.
Mass spectrometer: 5500 triple quadrupole tandem mass spectrometer equipped with electrospray ionization source (ESI) Sciex company, canada.
The data processing adopts software: analyst (version 1.6.3), sciex, canada.
Centrifuge: TGL-16RC bench centrifuge, shandong Bai European medical science and technology Co.
Analytical balance: BCE55I-10CN analytical balance, beijing certolis instruments limited.
1.2 Control and reagent
Milobulin is available from CATO. Methanol (HPLC grade), acetonitrile (HPLC grade) were purchased from Sigma, usa. Ammonium acetate (HPLC grade) was purchased from ROE company. Distilled water, drohent.
2 Method
2.1 Preparation of solutions and samples
Standard series of samples: accurately weighing a proper amount of each milbelin reference substance, respectively dissolving with methanol, and fixing volume to prepare Cheng Miluo barlin stock solution of about 1.00 mg/mL. And precisely sucking a proper amount of each stock solution, and gradually diluting with human blank plasma to obtain mixed standard series samples, wherein the concentration range of the milabalin is 1.00-500 ng/mL.
Quality control sample: the 3 concentration levels of Milobaline mix quality control samples were prepared using a method similar to the standard series of samples. The lower limit concentration of quantification was 1.00 ng/mL, the low-quality control (low quality control, LQC) concentration was 3.00 ng/mL, the medium-quality control (medium quality control, MQC) concentration was 150 ng/mL, and the high-quality control (high quality control, HQC) concentration was 400 ng/mL.
Internal standard solution: accurately weighing acetaminophen reference substance, dissolving with methanol, and fixing volume to obtain internal standard stock solution with concentration of about 1.00 mg/mL. Precisely sucking a proper amount of the internal standard stock solution, and adding acetonitrile: dilution with water (50:50, v/v) gave an internal standard solution with a acetaminophen concentration of 30 ng/mL.
2.2 Plasma sample treatment
1) Adding 50.0 mu L of plasma sample, 50.0 mu L of internal standard solution (the concentration of acetaminophen is 30 ng/mL) and 300 mu L of acetonitrile into a 96-well plate;
2) Vortex mixing, centrifuging 10 min (4 ℃,3900 rpm);
3) Taking 150 mu L of supernatant into another clean 96-well plate;
4) And adding 150 mu L of water, and uniformly mixing by vortex.
2.3 Chromatography and Mass Spectrometry conditions
Chromatographic conditions:
| Chromatographic column: | Agela Venusil MP C18 (2), 5 μm, 4.6X100 mm (Agela Co.) |
| Mobile phase: | Phase A: 5mM ammonium acetate aqueous solution B phase: acetonitrile gradient elution: 0-1.4 min:50% of phase B is 1.40-1.50 min: the phase B is increased from 50% to 90% 1.5-2.5 min:90% of phase B is 2.50-2.60 min: phase B was reduced from 90% to 50%3.00min: stop elution time: 3.00min |
| Flow rate: | 0.7000 mL/min |
| sample injection amount: | 10.00 µL |
| Autoinjector temperature: | 4°C |
| Column temperature: | 40°C |
Mass spectrometry conditions:
| ion source | Electrospray ion source (ESI) |
| Injection voltage | 5500 V |
| Spray Gas (Gas 1) | 55 psi |
| Auxiliary Gas (Gas 2) | 55 psi |
| Detection mode | Positive ions |
| Ion source temperature | 550℃ |
| Air Curtain Gas (Curtain Gas) | 35 psi |
| Residence time | 300 ms |
| For quantitative analysis of ion pairs | Milobalin m/z 210.2 [ 133.0,CE 20 eV, DP ] 100 [ V ] acetaminophen m/z 152.1 [ 110.0,CE 21 eV,DP 100V ] |
The scanning mass spectrum of the milabalin product ion is shown in figure 1.
2.4 Methodological verification
The method is verified according to the guidelines of Chinese pharmacopoeia 9012, and the contents include stability, selectivity, linearity, accuracy, precision, recovery rate, matrix effect and the like.
Selectivity is as follows:
and taking blank plasma with six different sources, and carrying out sample injection analysis after processing the prepared quantitative lower limit samples. The peak area of the chromatographic co-outflow interfering substance is required to be smaller than 20% of the peak area of the quantitative lower limit object to be detected and smaller than 5% of the peak area of the internal standard.
Standard curve:
And (3) carrying out a linear regression equation (weight factor W=1/x 2) calculated by regression analysis by taking the physical concentration to be measured as an abscissa (x) and the peak area ratio of the object to be measured to the internal standard as an ordinate (y). The method verifies that each analysis batch is double-sample analyzed on the standard curve sample.
Precision and accuracy:
Six samples of each of the 4 concentration quality control samples were measured. The precision of the quantitative lower limit in-batch and between-batch is acceptable by calculating the Relative Standard Deviation (RSD) of less than 20%, and the accuracy is acceptable by calculating the relative deviation (RE) of-20%. The precision of each component of QC samples with the other concentration levels in batches is required to be less than 15% and acceptable, and the accuracy is within-15%.
Stability:
When the stability of each analyte in a plasma sample was examined, LQC and HQC were placed in an environment at room temperature and ultra-low temperature (-60 ℃ or below), and six sample analyses were performed after the completion of the placement. Short-term room temperature placement 24 h stability was examined.
Recovery rate:
Taking 50.0 mu L of blank plasma, adding the solution of the object to be detected and the internal standard solution after extraction (without adding the internal standard solution), leading the final concentration to be the same as LQC, MQC and HQC, and carrying out sample injection measurement. And 6 parts of LQC, MQC and HQC are extracted, and sample injection is carried out for measurement. The extraction recovery was calculated as the ratio of peak areas of the 2 treatments.
Matrix effect:
Six normal blank plasmas from different sources are taken, low-concentration and high-concentration quality control plasma samples are respectively prepared, and 3-sample analysis is carried out on each concentration. And measured as a standard curve for normal plasma formulation.
2.5 Clinical study
The concentration of the milabalin in the blood plasma sample of the clinical study is analyzed by the established method and is used for the human pharmacokinetic study of the milabalin. 1 healthy subject is given 10 mg milobalin tablets, venous blood is collected at different time points in the period of pre-administration (0 h) and post-administration 12 h, 3-4 mL of venous blood is taken respectively, the venous blood is placed in an K 2 EDTA anticoagulation centrifuge tube, and blood plasma is separated after centrifugation (1700 g,4 ℃ C.) of 10 min, and the venous blood is stored in an ultralow temperature refrigerator at-60 ℃ or below.
3 Results and discussion
3.1 Methodological verification
Selectivity of
As shown in fig. 3-5, the milabalin and acetaminophen retention times were about 1.94 and 1.80 min with no co-current interference peaks at the retention times.
Standard curve
The linear range of the Milopolin in the plasma samples of the Milopolin clinical study is 1.00-500 ng/mL respectively. The standard curve typical linear regression equation of the object to be measured is respectively:
Milobalin: y=0.00458x+0.000332 (r= 0.9986);
Detection limit
The concentration of the milabalin in the lower limit sample of quantification is 1.00 ng/mL, and the signal to noise ratio is about 15 respectively. The detection limits were calculated to be 0.200 ng/mL for a signal to noise ratio of 5. The amount of the object to be detected on the column is calculated to be 0.125 pg according to the dilution multiple 16 and the sample injection amount 10 MuL.
Precision and accuracy
The results of precision and accuracy meet the acceptance criteria, and the results are shown in Table 1.
TABLE 1 determination of the precision and accuracy of Milobulin in human plasma
Recovery rate
LQC, MQC, and HQC concentration levels: the extraction recovery rates of milabalin are 95.6%, 96.0% and 96.5%, respectively; the recovery rates of acetaminophen were 100.3%, respectively.
Matrix effect
CV% measured value of each source per concentration QC sample under the concentration level of LQC and HQC is less than or equal to 15.0%; the average RE% measured per concentration of QC sample from each source is not more than +/-15.0%, and the results show that the matrix effect does not interfere with the accuracy of the analysis of the analyte.
Plasma stability investigation
Low (3.00 ng/mL) and high concentration (400 ng/mL) milbalin plasma stability samples were formulated and tested for differences from theoretical after 24h and ultra-low temperature (-60 ℃ below) placement at room temperature, as shown in table 2 below, and the results demonstrate 24h stability at room temperature and 20 days stability at ultra-low temperature (-60 ℃ below) placement in plasma.
TABLE 2 Milobulin in human plasma stability at 24h at room temperature and ultra-low temperature for 20 days
4 Human pharmacokinetic study
The validated method was used to analyze the plasma for milabalin to evaluate the pharmacokinetic profile of milabalin. 1 healthy subject orally administrates 10 mg milabalin tablets for a single time, the plasma drug concentration-time curve is shown in fig. 6, the sensitivity of the detection method can completely describe the pharmacokinetic properties of milabalin, the selection of the linear range is close to the concentration level of an actual sample, and the measurement accuracy is high.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, and it should be noted that it is possible for those skilled in the art to make several improvements and modifications without departing from the technical principle of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.
Claims (3)
1. A method for detecting milabalin in blood plasma by an HPLC-MS/MS method, comprising the steps of:
(1) Pretreatment: adding an internal standard solution into a plasma sample, adding acetonitrile to precipitate protein into the plasma sample, centrifuging, taking supernatant, adding water to dilute to obtain a sample, wherein the internal standard solution is obtained by dissolving acetaminophen in methanol, diluting with acetonitrile-water, 50:50 v/v, wherein the addition amount of the internal standard solution is 0.5-2 times that of the plasma sample, and the volume of the plasma sample is 30-100 mu L; the content of the milbelin in the plasma sample is 1.00-500 ng/mL, and the content of the internal standard substance in the test sample is 10-40 ng/mL;
(2) Detecting the pretreated plasma sample by HPLC-MS/MS, and determining the content of the milabalin by an internal calibration method, wherein:
Chromatographic conditions:
chromatographic column: agela Venusil MP C18 (2) chromatography column;
Mobile phase a: 5mM ammonium acetate in water, mobile phase B: acetonitrile;
Gradient elution procedure: 0-1.4 min: mobile phase B was 40%;1.4-1.5 min: mobile phase B is 40% -90%; 1.5-2.5 min: mobile phase B was 90%;2.5-2.6 min: mobile phase B is 90% -40%; 2.6-3.0min: mobile phase B was 40%;
The flow rate of the mobile phase is 0.7 mL/min;
The sample injection amount is 10.0 mu L;
column temperature: 40 ℃;
Mass spectrometry conditions:
Ion source: an electrospray ion source; scanning positive ions; monitoring multiple reactions; the injection voltage is 5500V; the temperature of the ion source is 550 ℃; gas 1:55 psi, gas 2:55 psi, gas curtain gas: 35 psi, collision induced dissociation: 6 psi, residence time: 300ms; quantitative analysis of ion pairs by milabalin: m/z 210.2.133.0, collision energy: 20 eV, declustering voltage: 100 V, V; quantitative analysis of ion pairs for acetaminophen: 152.1 cube 110.0, collision energy: 21 eV, declustering voltage: 100 V is provided.
2. The method for detecting milabalin in blood plasma by HPLC-MS/MS according to claim 1, characterized in that in step (1), the volume ratio of the blood plasma sample to acetonitrile is 1 (3-7).
3. The method for detecting milabalin in plasma by HPLC-MS/MS according to claim 1, characterized in that in step (1), the amount of water added in the dilution with water is 0.5-2 times the volume of the supernatant.
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