CN111413439A - Method for determining metformin in blood plasma by rapid hydrophilic interaction chromatography-tandem mass spectrometry - Google Patents
Method for determining metformin in blood plasma by rapid hydrophilic interaction chromatography-tandem mass spectrometry Download PDFInfo
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Abstract
The invention discloses a method for determining metformin in blood plasma by using a rapid hydrophilic interaction chromatography-tandem mass spectrometry method, belonging to the technical field of pharmaceutical analysis. The invention aims at the hydrophilic property of metformin, establishes a method for determining metformin in human plasma by using a rapid hydrophilic interaction chromatography-tandem mass spectrometry method, and uses a plasma sampleAfter the protein precipitation method, L una HI L IC chromatographic column is adopted for rapid isochromatic separation, and the ion reaction is monitored by mass spectrumm/z130.3 → 71.1 (metformin) andm/z136.2 → 77.1 (internal standard metformin-d 6). The method can realize the rapid and sensitive detection of the metformin in the blood plasma; and the dosage of blood plasma is less, the pretreatment of the sample is simple, and the method can be applied to the bioequivalence of the metformin hydrochloride controlled release tablet.
Description
Technical Field
The invention relates to the technical field of drug analysis, in particular to a method for determining metformin in blood plasma by using a rapid hydrophilic interaction chromatography-tandem mass spectrometry method.
Background
Metformin is a biguanide oral hypoglycemic agent, has the effects of improving the blood sugar tolerance of type 2 diabetes patients and reducing the basal blood sugar and the postprandial blood sugar, and is widely used for treating type II diabetes clinically.
According to the relevant regulations of the imitation drug quality and the consistency evaluation of the curative effect, the consistency evaluation of the oral solid preparation imitation drug needs to be accelerated. Therefore, many clinical institutions in China have conducted studies on the bioequivalence of metformin. To support bioequivalence studies, a rapid, stable, accurate assay is needed to determine the concentration of metformin in a subject's plasma. The liquid chromatography-tandem mass spectrometry method is widely applied to the concentration determination of the metformin in a biological sample due to the characteristics of good selectivity, high sensitivity and the like. However, due to the strong polarity and weak retention on reverse phase chromatography, metformin often requires a higher aqueous phase ratio or longer chromatographic separation to enhance its chromatographic retention, thereby reducing the detection flux. In addition, the existing detection method of metformin has the defects of large plasma dosage, complex pretreatment operation and the like.
Disclosure of Invention
In view of the prior art, the invention aims to provide a method for determining metformin in plasma by using a rapid hydrophilic interaction chromatography-tandem mass spectrometry method. The method can realize the rapid and sensitive detection of the metformin in the blood plasma; and the dosage of the blood plasma is less, and the pretreatment of the sample is simple.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for determining metformin in plasma by using a rapid hydrophilic interaction chromatography-tandem mass spectrometry method comprises the following steps:
(1) pretreating a plasma sample;
(2) determining the concentration of the metformin in the plasma sample pretreated in the step (1) by adopting a rapid hydrophilic interaction chromatography-tandem mass spectrometry method;
the fast hydrophilic interaction chromatography-tandem mass spectrometry comprises chromatography conditions and mass spectrometry conditions, wherein:
the chromatographic conditions include: acetonitrile-water-formic acid with the volume ratio of 90:10:0.2 is taken as a mobile phase, and the mobile phase also contains 5mM ammonium acetate; isocratic elution, with elution time of 2.5 min;
the mass spectrometry conditions include: an ion source: detecting positive ions by electrospray ionization; temperature of the sprayed gas: 400 ℃; atomizing: 50 psi; heating auxiliary gas: 50 psi; air curtain air: 30 psi; collision gas: 6 psi; spray voltage (IS): 4500V; scanning time: 150 ms; the scanning mode is as follows: multiple Reaction Monitoring (MRM).
Preferably, in the step (1), the method for pretreating the plasma sample comprises: adding the internal standard solution into the plasma sample, adding a methanol-acetonitrile mixed solution with the volume ratio of 1:1, carrying out vortex oscillation for 2-3min, and centrifuging at the speed of 4500 rpm for 10 min at the temperature of 4 ℃.
More preferably, the internal standard solution is 500 ng/m L Metformin hydrochloride-d 6 (Metformin-d 6) solution, and the volume ratio of the internal standard solution to the plasma sample is 1: 1.
More preferably, the volume ratio of the methanol-acetonitrile mixture to the plasma sample is 8: 1.
Preferably, in the step (2), the chromatographic conditions further comprise that the analytical column is an L una HI L IC chromatographic column, the flow rate is 0.9m L/min, then the flow rate is 0.45 m L/min, and the flow rate is divided into a mass spectrum, and the sample amount is 1.0 mu L.
Preferably, in step (2), the mass spectrometry conditions further include: the ionic reactions used for quantification were respectively:m/z130.3 → 71.1 (metformin) andm/z136.2.2 → 77.1 (metformin-d 6); the collision energy was 30 eV.
The invention has the beneficial effects that:
(1) the method is used for determining the metformin in human plasma by the rapid hydrophilic interaction chromatography-tandem mass spectrometry, aiming at the hydrophilic property of the metformin, a plasma sample can be directly subjected to sample injection analysis after being treated by a protein precipitation method, the sample pretreatment is simple, and the sample injection amount only needs 1.0 mu L to obtain a chromatographic peak with an ideal signal-to-noise ratio.
(2) The invention optimizes the chromatographic conditions, adopts hydrophilic interaction chromatography and enhances the chromatographic retention of the metformin. The high-flow-rate isocratic elution is matched with an elution mode of split flow after the column, so that the chromatographic analysis flux is greatly improved, and the chromatographic analysis time is only 2.5 min.
(3) The chromatographic condition optimized by the method has excellent separation capability, can effectively reduce the interference of a sample matrix during mass spectrum detection, has an inhibiting effect on the separation and the ionization of a group to be detected, and effectively improves the detection sensitivity.
Drawings
FIG. 1: [ M + H ] of metformin (A) and metformin-d6 (B)]+A scan of the product ions of (a).
FIG. 2: typical chromatograms for detecting metformin (I) and metformin-d6 (II) in plasma; a: blank plasma; b: quantifying a lower limit concentration plasma sample; c: healthy subjects took a plasma sample 4 hours after oral administration of 1000 mg of the metformin hydrochloride controlled-release tablet.
FIG. 3: plasma concentration-time profile of subjects after oral administration of 1000 mg of metformin hydrochloride controlled release tablet test or reference formulations on an empty stomach.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
As described in the background section, since metformin has a small molecular weight and contains a plurality of basic nitrogen atoms with strong polarity, it has strong hydrophilicity, so that conventional reversed phase chromatography is weak in retention, difficult to separate from endogenous substances in a short time, and further susceptible to endogenous matrix, generating interference peaks or causing mass spectrum signal fluctuation. The chromatographic retention can be enhanced by reducing the organic phase ratio and increasing the chromatographic run time. But longer chromatographic run times result in broadening of chromatographic peak profiles or reduced analytical throughput. In addition, in the currently reported metformin detection method, the pretreatment of a plasma sample is complicated, and the amount of plasma is large. Therefore, the detection of the concentration of metformin in plasma remains a technical difficulty in the art.
Based on this, the present inventors have conducted the following intensive studies on the detection of metformin concentration in plasma:
the principle of the hydrophilic interaction chromatography (HI L IC) is that in a high-proportion organic phase chromatography system, a water-rich layer is formed on the surface of a chromatographic column silica gel, and a compound with strong polarity is easy to interact with the water-rich layer to generate chromatographic retention.
Secondly, the selection of a chromatographic mobile phase system is the key of the separation effect, acetonitrile-water-formic acid (v/v/v, 90:10: 0.2) (containing 5mM ammonium acetate) is selected as a mobile phase, wherein the acetonitrile can ensure that the metformin is kept stronger, the peak shape is sharper, the pressure of a chromatographic system is lower and is suitable for a conventional high performance liquid chromatograph, 5mM ammonium acetate is added into the mobile phase to reduce a chromatographic baseline and improve the signal to noise ratio, formic acid is added into the mobile phase to reduce the secondary interaction of silica gel and the metformin and starts to improve the peak shape, and a chromatographic elution mode is isocratic elution, the chromatographic system is relatively stable in the mode, the operation time is shorter than that of gradient elution, the flow rate of the mobile phase is relatively higher and is 0.90 m L/min, the high flow rate can effectively shorten the chromatographic analysis time and improve the flux, the chromatographic operation time of the method is only 2.5min, and in order to avoid the influence on the ionization efficiency due to the high flow rate, a post-column split-flow method is adopted, and the mobile phase.
Thirdly, the metformin structure has a plurality of basic nitrogen atoms, and [ M + H ] is easy to generate under a positive ion detection mode]+Peak(s). Metformin and metformin-d6 form [ M + H ]]+The mass-to-charge ratios of the peaks are respectivelym/z130.3 andm/z136.2。the ions are generated after collision induced dissociation, and the product ions with the best signal-to-noise ratio are respectivelym/z71.1 andm/z77.1, selecting the ions as mass spectrum detection ions. The collision-induced dissociation diagram and the putative cleavage pathway are shown in FIG. 1.
In addition, the pretreatment mode of the plasma sample is optimized and studied, a protein precipitation method is adopted, after the 50 mu L plasma sample is subjected to protein precipitation, the proportion of an organic phase in an extracting solution is close to that of a hydrophilic interaction chromatographic system, so that a direct sample injection mode can be adopted, and the sample injection amount only needs 1.0 mu L to obtain a chromatographic peak with an ideal signal-to-noise ratio.
In conclusion, the invention establishes a method for determining the metformin in human plasma by using a rapid hydrophilic interaction chromatography-tandem mass spectrometry method aiming at the hydrophilic property of the metformin. Plasma samples were processed by the precipitated protein method and directly injected for analysis. The metformin is remarkably retained on the hydrophilic interaction chromatogram, so that endogenous substance interference is avoided. After chromatographic separation, the sample is detected by a tandem quadrupole mass spectrometer. The method can be applied to the bioequivalence research of the metformin hydrochloride controlled release tablet after verification.
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
The test materials used in the examples of the present invention, which were not specifically described, were all those conventional in the art and commercially available. Wherein:
metformin hydrochloride (batch number: 100664-201604, purity: 100%) was purchased from China institute for food and drug testing, metformin hydrochloride-d 6 (batch number: 4-E L Z-101-5, chemical purity 97.0%, isotopic purity 99.8%) was purchased from TRC, Canada, both chromatographic grade methanol and acetonitrile were purchased from Sigma, German, Fluka, America, chromatographic grade ammonium acetate was purchased from ROE, and deionized water was prepared from Millipore pure water instruments, France.
API model 4000 triple quadrupole tandem mass spectrometer from Sciex, Canada and analysis 1.6.3 data processing software, L C-20 high performance liquid chromatography system from Shimadzu, Japan, including DGU-20A3 degasser, L C-20AD infusion pump, SI L-20 AC autosampler, CTO-20A column incubator, L una HI L IC column (3.0 × 100 mm, 3.0 μm, Philon, USA).
Example 1: rapid hydrophilic interaction chromatography-tandem mass spectrometry method for determining metformin in plasma
1. Plasma sample pretreatment:
adding 50.0 mu L plasma sample into a 96-well plate, adding 50.0 mu L Metformin-d6, and controlling the concentration to be 500 ng.mL−1Adding 400 mu L methanol-acetonitrile (1: 1, v/v) into the internal standard solution, carrying out vortex oscillation for 2 min, centrifuging at the speed of 4500 rpm for 10 min at the temperature of 4 ℃, and taking 1.0 mu L of supernatant to carry out L C-MS/MS analysis.
2. Preparation of standard series samples and quality control samples:
weighing two parts of metformin hydrochloride standard substance, using methanol to fix the volume and dissolving to obtain metformin stock solution with the concentration of 0.992 mg/m L and 1.01mg/m L respectively, using one part of metformin stock solution to prepare standard series solution, using one part of metformin stock solution to prepare Quality Control (QC) solution, using methanol-water (1: 1, v/v) to dilute the metformin stock solution and the QC stock solution to obtain metformin standard series working solution and quality control solution, using human blank plasma to dilute the standard series working solution and quality control solution to obtain plasma concentration of 10.0, 20.0, 50.0, 150, 500, 750, 1000 and 1200ng respectively.mL−1The standard curve series samples, and the plasma concentration were 10.0 ng each.mL−1(lower limit of quantitation) 25.0 ng.mL−1、100 ng.mL−1、900 ng.mL−1And (5) controlling the quality of the sample.
3. L C-MS/MS analysis:
3.1 chromatographic conditions:
an analytical column L una HI L IC chromatographic column (3.0 × 100 mM, 3.0 μm, Philomen, USA), a mobile phase of acetonitrile-water-formic acid (v/v/v, 90:10: 0.2) (containing 5mM ammonium acetate), isocratic elution for 2.5min, a flow rate of 0.9m L/min, a flow rate of 0.45 m L/min divided into mass spectra, and a sample injection amount of 1.0 μ L.
3.2 Mass Spectrometry conditions:
an ion source: detecting positive ions by electrospray ionization; temperature of the sprayed gas: 400 ℃; atomizing: 50 psi; heating auxiliary gas: 50 psi; air curtain air: 30 psi; collision gas: 6 psi; spray voltage (IS): 4500V; scanning time: 150 ms; the scanning mode is as follows: multiple Reaction Monitoring (MRM). The ionic reactions used for quantification were respectively:m/z130.3 → 71.1 (metformin) andm/z136.2.2 → 77.1 (metformin-d 6); the collision energy was 30 eV.
Example 2: methodology validation
The methodology of the assay of example 1 was verified as follows:
1. and (3) selectivity:
the selectivity of the quantitative lower limit sample evaluation method prepared by blank plasma, hemolytic plasma and hyperlipemia plasma of 6 individuals with different sources and the blank plasma is analyzed.
The results show that the endogenous substances do not interfere with the measurement of metformin and metformin-d 6. A typical chromatogram is shown in FIG. 2.
2. Standard curve:
taking the theoretical concentration of the metformin as the abscissa (x), the metformin and the metformin-d6The peak area ratio of (a) is the ordinate (y), a linear regression equation is calculated by regression analysis, and the weight factor W =1/x2The range of the plasma concentration of the metformin is 10.0-1200 ng.mL−1The internal linearity is good, and the value of the correlation coefficient r is not less than 0.9978. A typical standard curve regression equation is: y =0.00115x + 0.000967 (r = 0.9983).
3. Precision and accuracy:
the method verifies that of the three analytical batches, 6 samples each from the lower limit sample and low, medium and high concentration level QC samples were checked for each batch, and calculates the precision and accuracy within each batch (n = 6) and between batches (n = 18), with the results shown in table 1.
Table 1: precision and accuracy in determining metformin plasma concentration
4. And (3) recovery rate:
the extraction recovery rates of the metformin under the low, medium and high quality control concentration levels are close to 113.9%, 98.9% and 104.1%, respectively. The recovery of the internal standard was 108.8%.
5. Matrix effect:
in the test, the matrix effect of the metformin in low and high quality control concentration levels in blank plasma of 6 different sources is inspected, the mean values of internal standard normalized matrix effect factors are respectively 102.8 percent and 108.8 percent, the blank plasma of different sources has no obvious difference, and the precision is less than 4.0 percent. And (3) determining the low and high concentration quality control samples prepared from the high fat plasma and the hemolytic plasma for evaluating the matrix effect of the high fat and the hemolytic plasma.
The result shows that the high-fat and hemolytic plasma does not influence the measurement of the concentration of the metformin, and the relative deviation between the measured value and the theoretical value is-5.6% -4.1%. Therefore, under the detection conditions of the experiment, the influence of the matrix effect can be ignored.
6. Stability:
the stability of metformin in different matrices and storage conditions was investigated in this experiment. The results are shown in Table 2.
Table 2: stability of metformin under different conditions
In the whole blood stability test, the theoretical value is the peak area ratio of metformin to metformin-d6 in a fresh sample, and the measured value is the peak area ratio of metformin to metformin-d6 in the sample after being placed at room temperature.
The results show that: metformin is stable under different conditions.
7. And (4) residual:
the method verifies and inspects the residues of the object to be detected and the internal standard in the sample detection process. The results show that the percentage of the area of the metformin peak in the blank matrix to the area of the lower limit peak of the corresponding analysis batch is less than 10.5 percent, and the residue of the metformin-d6 is 0.0 percent, which all meet the requirements.
Example 3: clinical sample testing
The test of the bioequivalence of the metformin hydrochloride controlled release tablet to human bodies is carried out in Suzhou science and technology city hospital. The ethical committee approved the inclusion of 8 healthy subjects. Subjects and two cycles, respectively, take a 1000 mg tablet of test preparation (T) or reference preparation (R) of metformin hydrochloride controlled release tablets orally on an empty stomach, and blood samples are collected at 0 h (within 1 h before administration) and 0.5, 1.0, 1.5, 2.0, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 8.0, 10.0, 12.0, 16.0, 24.0, 36.0 and 48.0 h after administration, and after plasma is obtained by separation, the concentration of metformin in plasma is measured by using an established method. The mean plasma concentration-time curve of the subjects is shown in figure 3.
In combination with the above embodiments, it can be seen that: the invention adopts hydrophilic interaction chromatography, and enhances the chromatographic retention of the metformin. The high-flow-rate isocratic elution is matched with an elution mode of split flow after the column, so that the chromatographic analysis flux is greatly improved, and the chromatographic analysis time is only 2.5 min. The pretreatment of the plasma sample adopts a protein precipitation method. After the plasma sample is subjected to protein precipitation treatment, the extracting solution can be directly subjected to sample injection analysis, and the operation is simple and convenient. The solvent system of the extracting solution and the hydrophilic interaction chromatographic system of the high organic phase have good compatibility, and the peak shape is sharp and symmetrical. The method can be successfully applied to the determination of the concentration of the metformin in the plasma sample of a subject in the human body bioequivalence research of the metformin hydrochloride controlled release tablet.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (6)
1. A method for determining metformin in plasma by using a rapid hydrophilic interaction chromatography-tandem mass spectrometry method is characterized by comprising the following steps:
(1) pretreating a plasma sample;
(2) determining the concentration of the metformin in the plasma sample pretreated in the step (1) by adopting a rapid hydrophilic interaction chromatography-tandem mass spectrometry method;
the fast hydrophilic interaction chromatography-tandem mass spectrometry comprises chromatography conditions and mass spectrometry conditions, wherein:
the chromatographic conditions include: acetonitrile-water-formic acid with the volume ratio of 90:10:0.2 is taken as a mobile phase, and the mobile phase also contains 5mM ammonium acetate; isocratic elution, with elution time of 2.5 min;
the mass spectrometry conditions include: an ion source: detecting positive ions by electrospray ionization; temperature of the sprayed gas: 400 ℃; atomizing: 50 psi; heating auxiliary gas: 50 psi; air curtain air: 30 psi; collision gas: 6 psi; spray voltage (IS): 4500V; scanning time: 150 ms; the scanning mode is as follows: and (5) monitoring multiple reactions.
2. The method according to claim 1, wherein in step (1), the plasma sample is pretreated in step (1) by: adding the internal standard solution into the plasma sample, adding a methanol-acetonitrile mixed solution with the volume ratio of 1:1, carrying out vortex oscillation for 2-3min, and centrifuging at the speed of 4500 rpm for 10 min at the temperature of 4 ℃.
3. The method of claim 2, wherein the internal standard solution is 500 ng/m L metformin hydrochloride-d 6 solution and the volume ratio of the internal standard solution to the plasma sample is 1: 1.
4. The method of claim 2, wherein the methanol-acetonitrile mixture is added to the plasma sample at a volume ratio of 8: 1.
5. The method according to claim 1, wherein in the step (2), the chromatographic conditions further comprise that the analytical column is an L una HI L IC chromatographic column, the flow rate is 0.9m L/min, then the flow rate is 0.45 m L/min, and the flow rate is further divided into a mass spectrum, and the sample amount is 1.0 mu L.
6. The method of claim 1, wherein in step (2), the mass spectrometry conditions further comprise: the ionic reactions used for quantification were respectively:m/z130.3 → 71.1 (metformin) andm/z136.2.2 → 77.1 (metformin-d 6); the collision energy was 30 eV.
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