CN109946408B - Detection method for measuring phillyrin, metabolite aglycone glucuronic acid conjugate and aglycone sulfuric acid conjugate in human plasma - Google Patents
Detection method for measuring phillyrin, metabolite aglycone glucuronic acid conjugate and aglycone sulfuric acid conjugate in human plasma Download PDFInfo
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Abstract
The invention relates to a detection method for measuring phillyrin, metabolite aglycone glucuronic acid conjugate and aglycone sulfuric acid conjugate in human plasma, which analyzes the phillyrin, metabolite aglycone glucuronic acid conjugate and aglycone sulfuric acid conjugate in the plasma through LC-MS/MS. The method preferably adopts a protein precipitation pretreatment method, takes teniposide as an internal standard, adopts a Gemini-C18 column, performs gradient elution, and performs electrospray ionization source (ESI) tandem mass spectrometry detection. The invention has strong specificity and selectivity, high sensitivity, rapid detection, small sample consumption, simplicity, reliability, high flux and controllable conditions, and meets the analysis requirements of clinical large-batch samples.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a detection method for measuring phillyrin, metabolite aglycone glucuronic acid conjugate and aglycone sulfuric acid conjugate in human plasma.
Background
Fructus forsythiae is a dry fruit of forsythia, a plant of the family Oleaceae, is mainly produced in Shanxi, Henan, Shaanxi and other places in China, and is one of the commonly used heat-clearing and toxicity-removing medicines. The chemical component of fructus forsythiae is mainly phillyrin. It has the functions of resisting bacteria, resisting infection, clearing away heat, resisting virus and resisting oxidation, and has clinical research value. The pharmacokinetic study is one of the main contents of the clinical study of innovative drugs. Pharmacokinetic studies require the measurement of the concentration of drugs and their metabolites in the biological matrix in vivo after administration. Early studies indicate that the metabolites of phillyrin in the human body are aglycone sulfate conjugate and aglycone glucuronic acid conjugate. According to relevant regulations, the pharmacokinetic characteristics of phillyrin and two metabolites thereof in the body are required to be evaluated. Therefore, there is a need for an assay to measure the above three substances in human plasma.
At present, the analysis method for determining the concentration or the content of the phillyrin mainly comprises a liquid chromatography-ultraviolet method, a liquid chromatography-mass spectrometry method and a liquid chromatography-tandem mass spectrometry (LC-MS/MS). The detection methods are mainly used for detecting the content of phillyrin in traditional Chinese medicine preparations and traditional Chinese medicinal materials. The assay method is less sensitive, typically on the order of μ g/mL to mg/mL, while the in vivo drug concentration is typically on the order of ng/mL. According to previous research, the structural formula of phillyrin in human plasma is shown in figure 1, and the sensitivity of a concentration analysis method at least reaches 1.00 ng/mL. The above detection methods do not meet the requirements of in vivo pharmacokinetic studies. Other studies have used liquid chromatography-tandem mass spectrometry to determine the concentration of forsythin in animals. However, the concentration of drugs in preclinical animals is usually much higher than in humans. Therefore, the sensitivity of these detection methods is often not satisfactory. In addition, ethical issues are involved in clinical studies, and sample collection volumes cannot be too large. Due to the requirements of the existing regulations, a part of collected blood plasma needs to be backed up, less blood plasma is used for detecting concentration, and repeated detection, reanalysis of detected samples and other repeated detection needs to be met. It is therefore desirable that the detection method use a small sample volume, typically not exceeding 200. mu.L. The detection method in the preclinical research has large sample usage amount.
The number of samples in clinical studies is usually large and requires high throughput of the assay. The detection time of the instrument is as short as possible. So as to rapidly obtain data, guide the subsequent clinical trial design and accelerate the research and development speed of new drugs.
At present, no related technology reports to detect major metabolites of phillyrin in human bodies. None of the known techniques can be used for clinical pharmacokinetic studies.
The method for determining phillyrin and a plurality of traditional Chinese medicine active ingredients in rat plasma by adopting liquid chromatography-tandem mass spectrometry is established in 2015. The sensitivity of the analysis method is high and can reach about 1.5ng/mL, but the operation time of the chromatography is 13 minutes, and the structural formula of the phillyrin-human in-vivo metabolite aglycone-sulfuric acid conjugate can not be determined and can not be shown in figure 3, and the structural formula of the aglycone-glucuronic acid conjugate can be shown in figure 2. Is not suitable for clinical research.
An LC-MS/MS method for determining paeoniflorin and phillyrin in rat plasma is established in Tianxi, which is 2016. The sensitivity of the analysis method for determining the phillyrin is low, and the limit of quantification is 5.00 ng/mL. The plasma volume was approximately 0.2 ml. The running time of the chromatogram is longer than 20 minutes, and the metabolites aglycon sulfuric acid conjugate and aglycon glucuronic acid conjugate in the phillyrin human body can not be measured, so that the requirement of clinical research is not met.
An LC-MS/MS method established by Liuxi hucho in 2012 is used for determining phillyrin in rat bile, and the analysis method is low in phillyrin determination sensitivity and has the limit of quantitation of 58.0 ng/mL. The sample was used in a large amount, 0.5ml of bile was used. The chromatography time was 15 min. Meanwhile, the Liuxi hucho also establishes an HPLC-MS method for measuring the concentration of the phillyrin in the plasma of the rat. The lower limit of quantitation was 11.0ng/mL, the sample volume was large, and 0.2 mL of plasma was used.
None of the above methods can determine the metabolites aglycon sulfate conjugate and aglycon glucuronic acid conjugate in phillyrin human body, and the sensitivity is low, which is not suitable for the requirement of clinical research.
In summary, the prior art has the following disadvantages:
the prior art cannot determine phillyrin-glucuronic acid conjugate and phillyrin-sulfuric acid conjugate which are metabolites in a human body.
The prior art has the defects of large sample dosage, low sensitivity and long detection chromatographic time when detecting the phillyrin, and can not meet the requirements of clinical pharmacokinetic research.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide a detection method for measuring phillyrin, metabolite aglycone glucuronic acid conjugate and aglycone sulfuric acid conjugate in human plasma.
In order to achieve the purpose, the invention adopts the following technical scheme:
the detection method for measuring phillyrin, metabolite aglycone glucuronic acid conjugate and aglycone sulfuric acid conjugate in human plasma comprises the following steps:
pretreating, collecting plasma, adding an internal standard solution, adding methanol, carrying out vortex flow and centrifugation, taking supernatant, carrying out blow-drying concentration on the supernatant in nitrogen flow, dissolving residues by using a double solvent, and carrying out vortex mixing to obtain a sample to be detected;
and (2) performing chromatography, namely performing liquid chromatography separation on a sample to be detected, performing gradient elution by adopting a Gemini-C18 column, and performing gradient elution on a mobile phase A: the volume ratio of the mobile phase B is 85:15, and at 3.8min, the volume ratio of the mobile phase A: the volume ratio of the mobile phase B is 43:57, and at 3.9min, the volume ratio of the mobile phase A: the volume ratio of the mobile phase B is 5:95, and at 4.7min, the volume ratio of the mobile phase A: the volume ratio of the mobile phase B is 5:95, and the volume ratio of the mobile phase A to the mobile phase B is within 4.8 min: the volume ratio of the mobile phase B is 85:15, meanwhile, the flow rate is 0.5mL/min at 0min, the flow rate is 0.5mL/min at 3.90min, the flow rate is 0.8mL/min at 3.91min, the flow rate is 0.8mL/min at 4.70min, the flow rate is 0.5mL/min at 4.71min, the column temperature is 40 ℃, the temperature of an autosampler is 4 ℃, the sample injection amount is 2.0 mu L, the mobile phase A is one ten thousandth of ammonia water solution, and the mobile phase B is a mixed solution of methanol and acetonitrile;
mass spectrometry using an electrospray ion source, negative ion detection, spray voltage-4500V, endogenous Gas 1(Gas1)60psi, Gas 2(Gas2)60psi, Curtain Gas (Curtain Gas)30psi, ion source temperature 400 ℃, collision induced dissociation 8psi, residence time 100ms, phillyrin quantitative analysis ion pair m/z 371.1 (intrasource fragment ion) → 356.1, Collision Energy (CE) -29eV, declustering voltage (DP) -70V, aglycon glucuronic acid conjugate quantitative analysis ion pair m/z 547.2 → 356.0, Collision Energy (CE) -51eV, declustering voltage (DP) -20V, aglycon sulfuric acid conjugate quantitative analysis ion pair m/z 451.2 → 356.2, energy Collision (CE) -30eV, declustering voltage (DP) -50V, teniposide quantitative analysis ion pair m/z655.3 → 112.9, collision Energy (CE) -24eV, declustering voltage (DP) -85V.
Preferably, in the step of preprocessing, 100 μ L of a plasma sample is taken, 50.0 μ L of an internal standard solution is added, 300 μ L of methanol is added, 300 μ L of supernatant is taken after vortex and centrifugation, the supernatant is dried and concentrated in nitrogen flow, residues are dissolved in 120 μ L of a re-solvent, and the sample to be detected is obtained after vortex mixing.
Preferably, in the detection method for detecting phillyrin, metabolite aglycone glucuronic acid conjugate and aglycone sulfuric acid conjugate in human plasma, the internal standard solution is teniposide solution.
Preferably, the method for detecting phillyrin, metabolite aglycone glucuronic acid conjugate and aglycone sulfate conjugate in human plasma is characterized in that negative ions are adopted for detecting mass spectrum, precursor ions or parent ions of phillyrin are used as source internal schizonts, and the mass spectrum is m/z 371.1.
Preferably, in the detection method for detecting phillyrin, metabolite aglycone glucuronic acid conjugate and aglycone sulfuric acid conjugate in human plasma, ammonia water is used as a mobile phase A additive of a Gemini-C18 column in the chromatography step.
By the scheme, the invention at least has the following advantages:
1. the invention has high sensitivity, the lower limit concentration of the phillyrin, the aglycone sulfate conjugate and the aglycone glucuronic acid conjugate is 1.00/2.50/5.00ng/mL, and the pharmacokinetic properties can be completely described.
2. The sample dosage of the invention is only 100 mu L, which is suitable for clinical research.
3. The invention has fast detection speed, and the detection time is 5.5min, thus being suitable for the analysis of large-batch clinical research samples.
4. The invention can simultaneously detect phillyrin and the main metabolite aglycone glucuronic acid conjugate in human body, and can meet the requirement of clinical pharmacokinetics research of phillyrin and metabolite thereof.
The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following detailed description is given 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 needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 shows the chemical structure of forsythin.
FIG. 2 is the chemical structural formula of aglycone glucuronic acid conjugate.
FIG. 3 is a chemical structural formula of aglycone sulfate conjugate.
FIG. 4 is a chemical structural formula of teniposide.
FIG. 5 is a scanning mass spectrum of phillyrin ion.
FIG. 6 is a scanning mass spectrum of an ion of an aglycone glucuronic acid conjugate.
FIG. 7 is a scanning mass spectrum of the ion of the aglycone sulfuric acid conjugate.
FIG. 8 is a scanning mass spectrum of teniposide ion.
FIG. 9 is an MRM chromatogram of phillyrin in blank plasma (top), lower limit of quantitation (middle), and after single administration of phillyrin capsules to healthy subjects (bottom).
Figure 10 is a MRM chromatogram of aglycone sulfate conjugates in blank plasma (top), lower limit of quantitation (middle), and after a single administration of phillyrin capsules to healthy subjects (bottom).
Figure 11 is an MRM chromatogram of aglycon glucuronic acid conjugates in blank plasma (top), lower limit of quantitation (middle) and after a single administration of phillyrin capsules to healthy subjects (bottom).
Figure 12 is an MRM chromatogram of internal teniposide internal standard on blank plasma (top), lower limit of quantitation (middle), and after single administration of phillyrin capsules to healthy subjects (bottom).
FIG. 13 is a plot of mean drug concentration versus time for a single oral dose of 400mg phillyrin capsules in 8 healthy subjects.
Detailed Description
The present invention will be described in further detail with reference to the following specific examples and drawings, and the present invention is not limited to the following examples. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected. The procedures, conditions, reagents, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.
The development of a method for detecting the concentration of a drug in blood plasma by liquid chromatography-tandem mass spectrometry can be generally divided into three parts, namely an extraction method (namely a pretreatment method), a liquid chromatography method and a mass spectrometry method. The invention aims at the defects of the prior art and sets up a detection method from the three aspects.
Examples
Pretreatment
The method uses the plasma with the dosage of only 100 mu L, and the extraction method adopts a protein precipitation method, has higher recovery rate on polar substances, has simple operation and short extraction time, and is highly suitable for high-throughput sample pretreatment in clinical research.
The specific pretreatment method comprises the following steps:
1. 1.5mL of EP tube with 100. mu.L of plasma sample;
2. adding 50.0 μ L of internal standard solution (concentration of teniposide is 1000 ng/mL);
3. adding 300 mu L of methanol; vortex for 2min, centrifuge for 5min (14000 rpm);
4. taking 300 mu L of supernatant, drying and concentrating in nitrogen flow;
5. dissolving the residue with 120 μ L of complex solvent, mixing with vortex, and performing LC-MS/MS analysis in an automatic sample injector;
6. the injection volume was 2.0. mu.L.
Mass spectrometry
Using an electrospray ion source, negative ion detection, spray voltage-4500V, endogenous Gas 1(Gas1)60psi, Gas 2(Gas2)60psi, Curtain Gas (Curtain Gas)30psi, ion source temperature 400 ℃, collision induced dissociation 8psi, residence time 100ms, forsythin quantitation ion pair m/z 371.1 → 356.1, Collision Energy (CE) -29eV, declustering voltage (DP) -70V, aglycon glucuronic acid conjugate quantitation ion pair m/z 547.2 → 356.0, Collision Energy (CE) -51eV, declustering voltage (DP) -20V, aglycon sulfuric acid conjugate quantitation ion pair m/z 451.2 → 356.2, Collision Energy (CE) -30eV, declustering voltage (DP) -50V, teniposide (see FIG. 4) quantitation ion pair m/z655.3 → 112.9, Collision Energy (CE) -24eV, declustering voltage (DP) -85V.
As shown in fig. 5, 6, 7 and 8, fig. 5 is a phillyrin ion scanning mass spectrum, fig. 6 is an aglycon glucuronic acid conjugate ion scanning mass spectrum, fig. 7 is an aglycon sulfuric acid conjugate ion scanning mass spectrum, fig. 8 is a teniposide ion scanning mass spectrum, the aglycon sulfuric acid conjugate and the aglycon glucuronic acid conjugate contain acidic groups, and higher and significant precursor ions can be generated under a mass spectrum negative ion detection mode, namely m/z 451.2 and m/z 547.2 respectively. Product ion scan (CID) was performed on the above ions, with a major fragment ion m/z of 356.2, as a product ion member for quantitative analysis of aglycon sulfate conjugate and aglycon glucuronic acid monitoring. The phillyrin structure has no alkaline nitrogen atoms and more hydroxyl groups, and the response in the positive ion detection mode is lower than that in the negative ion mode. The excimer precursor ion generated by the phillyrin in the negative ion mode is m/z 533.2, the structure contains glycosyl, and obvious neutral loss occurs in the ionization process, so that neutral loss ion m/z 371.1 is generated. The ion intensity of m/z 371.1 is far higher than that of m/z 533.2, so that mass spectrum response can be greatly improved by selecting neutral lost ions as precursor ions for quantitative detection, and the sensitivity of an analysis method is further improved. Product ion scan (CID) was performed on m/z 371.1, the major fragment ion m/z was also 356.2.
Chromatography
Separating the liquid chromatogram of the sample to be detected, adopting a Gemini-C18 column, carrying out gradient elution, and carrying out the following steps of 1min on a mobile phase A: the volume ratio of the mobile phase B is 85:15, and at 3.8min, the volume ratio of the mobile phase A: the volume ratio of the mobile phase B is 43:57, and at 3.9min, the volume ratio of the mobile phase A: the volume ratio of the mobile phase B is 5:95, and at 4.7min, the volume ratio of the mobile phase A: the volume ratio of the mobile phase B is 5:95, and the volume ratio of the mobile phase A to the mobile phase B is within 4.8 min: the volume ratio of the mobile phase B is 85:15, meanwhile, the flow rate is 0.5mL/min at 0min, the flow rate is 0.5mL/min at 3.90min, the flow rate is 0.8mL/min at 3.91min, the flow rate is 0.8mL/min at 4.70min, the flow rate is 0.5mL/min at 4.71min, the column temperature is 40 ℃, the autosampler temperature is 4 ℃, the sample injection amount is 2.0 mu L, the mobile phase A is ten thousandth of ammonia water solution, the mobile phase B is methanol: the volume ratio of acetonitrile is 2:1, and the length of a Gemini-C18 column is 50mm, and the diameter is 2.0 mm.
The phillyrin, the aglycone glucuronic acid conjugate and the aglycone sulfuric acid conjugate have strong polarity and are difficult to retain, so the method adopts a gradient elution mode, and the initial flowing phase ratio is low, thereby being beneficial to chromatographic retention. The three components to be detected and the internal standard have different polarities, and the gradient elution can ensure that all the components can be eluted in a short time. The ionization mode of negative ions is adopted during mass spectrometry detection, and the generation of the negative ions can be promoted by adding ammonia water in the mobile phase, so that the mass spectrometry response is improved. The mobile phase is alkaline due to the addition of ammonia water, and the invention adopts a Gemini-C18 column, has wide pH application range and can tolerate the alkaline mobile phase. The running time of the chromatogram is only 5.5min, and the detection is fast and suitable for detecting a large batch of samples in clinical research.
The first embodiment is as follows:
description of abbreviations
1 Material
1.1 instruments
Chromatograph: LC-30AD flash liquid chromatography system, Shimadzu, Japan.
Mass spectrometry: model 6500 triple quadrupole tandem mass spectrometer equipped with electrospray ionization source (Turbo Ion Spray) Sciex, Canada.
The data processing adopts software: analyst (version 1.6.3), Sciex, Canada.
A centrifuge: model Her μ Le Z2326K bench centrifuge, Haemomer, Germany.
Analytical balance: analytical balance model CD225D, beijing sidoris instruments ltd.
1.2 control and reagents
Phillyrin (94.9% content) was purchased from the institute of food and drug testing, china. The aglycone glucuronic acid conjugate (with the purity of 96.6 percent) and the aglycone sulfuric acid conjugate (with the purity of 99.5 percent) are self-made. Teniposide was purchased from Tokyo Chemical Industry CO., LTD. Methanol (HPLC grade), acetonitrile (HPLC grade) were purchased from Sigma company, usa. 25% ammonia (HPLC grade) was purchased from Merck, Germany. Deionized water (18.2m Ω, TOC. ltoreq.50 ppb) was prepared from a Milli-Q ultrapure water system, France.
2 method
2.1 preparation of solutions and samples
Standard series of samples: accurately weighing appropriate amount of each reference substance, dissolving with methanol respectively, and diluting to constant volume to obtain stock solution with phillyrin, aglycone glucuronic acid conjugate and aglycone sulfuric acid conjugate concentration of about 1.00 mg/mL. Precisely sucking appropriate amount of respective stock solution, diluting with human blank plasma step by step to obtain mixed standard series samples, wherein the concentration ranges of phillyrin, aglycone glucuronic acid conjugate and aglycone sulfuric acid conjugate are 1.00-1000, 2.50-2500 and 5.00-5000ng/mL respectively.
Quality control of the sample: five concentration level mixed quality control samples of phillyrin, aglycone glucuronic acid conjugate and aglycone sulfuric acid conjugate are prepared by a method similar to that of a standard series of samples. The lower limit concentration of the quantification is 1.00/2.50/5.00ng/mL, the concentration of Low Quality Control (LQC) is 3.00/7.50/15.0, the concentration of Medium Quality Control (MQC) is 15.0/37.5/75.0ng/mL, the concentration of Medium High Quality Control (MHQC) is 60.0/150/300, and the concentration of High Quality Control (HQC) is 800/2000/4000 ng/mL.
Internal standard solution: the teniposide reference substance is precisely weighed, dissolved by methanol and subjected to constant volume to prepare internal standard stock solution with the concentration of about 1.00 mg/mL. Precisely sucking appropriate amount of the internal standard stock solutions, adding methanol: the internal standard solutions were obtained at a concentration of 1000ng/mL by dilution with water (50:50, v/v).
2.2 plasma sample treatment
100 μ L of plasma sample was added to a 1.5mL EP tube, 50.0 μ L of internal standard solution (concentration of teniposide is 1000ng/mL) was added, and 300 μ L of methanol was added; vortex for 2min, centrifuge for 5min (14000rpm), take 300. mu.L of supernatant and blow dry in nitrogen stream for concentration. The residue was dissolved in 120. mu.L of a double solvent, vortexed, mixed, and placed in an autosampler for LC-MS/MS analysis with a sample volume of 2.0. mu.L.
2.3 chromatographic and Mass Spectrometry conditions
Chromatographic conditions
A chromatographic column: Gemini-C18, 110A, 5 μm, 2.0X 50mm, Philomen, USA.
Pre-column: krudkatcher ultra, Philomen, USA.
Mobile phase A: one ten thousandth of aqueous ammonia solution.
Mobile phase B: methanol: acetonitrile (2:1, v/v).
The elution is carried out at an equal rate,
column temperature: at 40 ℃.
Flow rate:
sample introduction amount: 2.0. mu.L.
Autosampler temperature: 4 ℃ is prepared.
Switching valve program: 0-1.5 min, A position; 1.5-4.6 min, B position; 4.6-5.5 min, A position.
Conditions of Mass Spectrometry
Using an electrospray ion source, negative ion detection, spray voltage-4500V, endogenous Gas 1(Gas1)60psi, Gas 2(Gas2)60psi, Curtain Gas (Curtain Gas)30psi, ion source temperature 400 ℃, collision induced dissociation 8psi, residence time 100ms, forsythin quantitation ion pair m/z 371.1 → 356.1, Collision Energy (CE) -29eV, declustering voltage (DP) -70V, aglycon glucuronic acid conjugate quantitation ion pair m/z 547.2 → 356.0, Collision Energy (CE) -51eV, declustering voltage (DP) -20V, aglycon sulfuric acid conjugate quantitation ion pair m/z 451.2 → 356.2, Collision Energy (CE) -30eV, declustering voltage (DP) -50V, teniposide quantitation ion pair m/z655.3 → 112.9, Collision Energy (CE) -24eV, declustering voltage (DP) -85V.
2.4 methodological validation
The methodology of the method was verified according to the U.S. FDA guidelines, including stability, selectivity, linearity, accuracy, precision, recovery matrix effect, etc.
Selectivity is
Six blank blood plasma with different sources and respectively prepared quantitative lower limit samples are taken for processing and then sample injection analysis is carried out. The peak area of the chromatogram co-outflow interferent is required to be smaller than 1/5 of the peak area of the quantitative lower limit analyte and smaller than 1/20 of the peak area of the internal standard.
Standard curve
Linear regression equation (weight factor W is 1/x) calculated by regression analysis with the concentration of the physical theory to be measured as abscissa (x) and the peak area ratio of the substance to be measured to the internal standard substance as ordinate (y)2). The method verifies that each analysis batch is analyzed against a double sample of standard curve samples.
Precision and accuracy
The method verifies that each analysis batch determines six samples of five concentration quality control samples. Quantitative lower limit intra-and inter-batch precision is acceptable at less than 20% as calculated by Relative Standard Deviation (RSD) and accuracy is acceptable at between-20% and 20% as calculated by relative deviation (RE). The precision of the QC samples of other concentration levels in each component batch and between batches is required to be less than 15 percent to be acceptable, and the precision is between-15 percent and 15 percent to be acceptable.
Stability of
And (3) when the stability of each object to be detected in the plasma sample is inspected, placing the LQC and the HQC in different temperatures and environments, and analyzing the three samples after the placing is finished. A total of four placement conditions were examined, which were: standing at room temperature for 16h, extracting, placing in a sample holder for 97h, and performing 5 freeze-thaw cycles (from-75 + -10 deg.C to ice room temperature), and standing at 75 + -5 deg.C for 194 days.
Recovery rate
Taking blank plasma 50.0 μ L, adding the solution to be measured and the internal standard solution after extraction (without adding the internal standard solution) to make the final concentration the same as LQC, MQC and HQC, and carrying out sample injection determination. And 6 parts of each of LQC, MQC and HQC are extracted, and the sample injection and the determination are carried out. The extraction recovery rate was calculated from the peak area ratios of the 2 treatments.
Matrix effect
Taking 6 blank plasma with different sources, extracting (without adding an internal standard solution), taking all acetonitrile layer liquid, adding a solution to be measured and an internal standard solution with the same concentration as that of LQC and HQC, mixing by vortex, and measuring. And treating with deionized water instead of blood plasma by the above method. The ratio of the peak areas obtained by the two methods is used for calculating the matrix factor, and the matrix effect is evaluated by the RSD of the matrix factor, wherein the matrix factor is acceptable when the ratio is less than 15%.
2.5 clinical study
The established method is applied to determine the concentration of the phillyrin and metabolites in the blood plasma, and the phillyrin is used for human pharmacokinetics research. The clinical study was approved by the hospital ethics committee, and subjects were informed of trial risk prior to the trial and voluntarily signed an informed consent. 400mg phillyrin capsules were administered to 8 healthy subjects. 4mL of blood is taken from vein before administration (0h), 0.25h, 0.5h, 0.75h, 1h, 1.5h, 2h, 2.5h, 3h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 24h and 36h after administration, the blood plasma is separated after centrifugation (3000rpm, 4 ℃) for 10min, and the blood plasma is preserved at the temperature of minus 75 +/-10 ℃.
3 results and discussion
3.1 methodological validation
Selectivity of the process
As shown in FIGS. 9 to 12, the retention times of phillyrin, aglycone glucuronic acid conjugate and aglycone sulfuric acid conjugate were about 3.6, 2.3 and 2.7min, respectively, and there was no co-efflux interference peak at the retention time.
Standard curve
The linear ranges of phillyrin, aglycone glucuronic acid conjugate and aglycone sulfuric acid conjugate in human plasma are respectively 1.00-1000ng/mL, 2.50-2500ng/mL and 5.00-5000 ng/mL. The linear average regression equation (first batch verified by methodology) of the standard curve of the object to be tested is respectively as follows:
forsythin, y is 0.0125x + 0.000729;
aglycone glucuronic acid conjugate y 0.00798x + -0.000245;
the aglycone sulfuric acid conjugate is y-0.00505 x + 0.0012.
Precision and accuracy of the method
The results of precision accuracy were in accordance with the accepted standards and are shown in Table 1.
TABLE 1 determination of precision and accuracy of phillyrin, aglycone glucuronic acid conjugate, aglycone sulfuric acid conjugate in human plasma
Recovery rate of treatment
LQC, MQC and HQC concentration levels: the extraction recovery rates of the phillyrin are 76.3%, 73.2% and 72.4% respectively; the extraction recovery rates of the aglycon glucuronic acid conjugate are 77.3%, 73.2% and 73.4% respectively; the extraction recovery rates of the aglycon sulfuric acid conjugate are 80.4%, 78.0% and 75.2% respectively; the recovery rate of teniposide is 66.2%.
Matrix effect
The matrix factors of phillyrin at the concentration levels of LQC and HQC are 121.6% and 120.7% respectively, and the RSD is 4.4% and 5.2% respectively; the aglycon glucuronic acid conjugate matrix factor is 176.5 percent and 170.3 percent respectively, and the RSD is 4.9 percent and 4.6 percent respectively; the aglycone sulfuric acid conjugate matrix factor is 143.0% and 126.1%, and the RSD is 4.6% and 4.3%, respectively. The above results indicate that the matrix does not interfere with the quantitative analysis of the analyte.
Plasma stability study
The results of the plasma stability tests are shown in table 2. The results show that under the investigation conditions, phillyrin, aglycone glucuronic acid conjugate and aglycone sulfuric acid conjugate are stable.
Table 2 stability of phillyrin, aglycone glucuronic acid conjugate, and aglycone sulfate conjugate in human plasma (n ═ 6).
4 human pharmacokinetics study
The verified method is used for simultaneously and quantitatively detecting the phillyrin, the aglycone glucuronic acid conjugate and the aglycone sulfuric acid conjugate in the blood plasma so as to evaluate the pharmacokinetic characteristics. 8 healthy subjects orally take 400mg phillyrin capsules in a single time. The mean plasma drug concentration-time curve is shown in figure 13. The detection method sensitivity can completely describe the pharmacokinetic characteristics of the phillyrin and the metabolites thereof.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, it should be noted that, for those skilled in the art, many modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.
Claims (2)
1. The detection method for measuring phillyrin, metabolite aglycone glucuronic acid conjugate and aglycone sulfate conjugate in human plasma is characterized by comprising the following steps:
pretreating, collecting plasma, adding a teniposide solution, adding methanol, carrying out vortex flow and centrifugation, taking supernatant, drying and concentrating in nitrogen flow, dissolving residues with a double solvent, and carrying out vortex flow and uniform mixing to obtain a sample to be detected;
and (2) performing chromatography, namely performing liquid chromatography separation on a sample to be detected, performing gradient elution by adopting a Gemini-C18 column, and performing gradient elution on a mobile phase A: the volume ratio of the mobile phase B is 85:15, and at 3.8min, the volume ratio of the mobile phase A: the volume ratio of the mobile phase B is 43:57, and at 3.9min, the volume ratio of the mobile phase A: the volume ratio of the mobile phase B is 5:95, and at 4.7min, the volume ratio of the mobile phase A: the volume ratio of the mobile phase B is 5:95, and the volume ratio of the mobile phase A to the mobile phase B is within 4.8 min: the volume ratio of the mobile phase B is 85:15, meanwhile, the flow rate is 0.5mL/min at 0min, the flow rate is 0.5mL/min at 3.90min, the flow rate is 0.8mL/min at 3.91min, the flow rate is 0.8mL/min at 4.70min, the flow rate is 0.5mL/min at 4.71min, the column temperature is 40 ℃, the temperature of an autosampler is 4 ℃, the sample injection amount is 2.0 mu L, the mobile phase A is one ten thousandth of ammonia water solution, and the mobile phase B is a mixed solution of methanol and acetonitrile;
mass spectrometry using electrospray ion source, negative ion detection, ejection voltage-4500V, endogenous Gas 1(Gas1)60psi, Gas 2(Gas2)60psi, Curtain Gas (Curtain Gas)30psi, ion source temperature 400 deg.C, collision induced dissociation 8psi, residence time 100ms, forsythin quantitation ion pair m/z 371.1 → 356.1, Collision Energy (CE) -29eV, declustering voltage (DP) -70V, aglycon glucuronic acid conjugate quantitation ion pair m/z 547.2 → 356.0, Collision Energy (CE) -51eV, declustering voltage (DP) -20V, aglycon sulfuric acid conjugate quantitation ion pair m/z 451.2 → 356.2, Collision Energy (CE) -30eV, declustering voltage (DP) -50V, teniposide quantitation ion pair m/z655.3 → 112.9, Collision Energy (CE) -24eV, declustering voltage (DP) -85V.
2. The detection method for detecting phillyrin, metabolite aglycone glucuronic acid conjugate and aglycone sulfate conjugate in human plasma according to claim 1, which is characterized in that: and in the pretreatment step, 100 mu L of plasma sample is taken, 50.0 mu L of teniposide solution is added, 300 mu L of methanol is added, 300 mu L of supernatant is taken after vortex flow and centrifugation, blow-drying and concentration are carried out on the supernatant in nitrogen flow, residues are dissolved in 120 mu L of complex solvent, and the sample to be detected is obtained after vortex mixing.
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