CN115219616A - Method for determining concentration of endogenous substances including coenzyme Q10 in biological sample based on liquid chromatography-mass spectrometry technology - Google Patents

Method for determining concentration of endogenous substances including coenzyme Q10 in biological sample based on liquid chromatography-mass spectrometry technology Download PDF

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CN115219616A
CN115219616A CN202210736726.6A CN202210736726A CN115219616A CN 115219616 A CN115219616 A CN 115219616A CN 202210736726 A CN202210736726 A CN 202210736726A CN 115219616 A CN115219616 A CN 115219616A
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concentration
coenzyme
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plasma
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王祥艳
唐晓荞
郏自明
樊柏林
黎炎梅
张寅静
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Hubei Provincial Center For Disease Control And Prevention (hubei Academy Of Preventive Medicine)
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Abstract

The invention discloses a method for determining the concentration of endogenous substances including coenzyme Q10 in a biological sample based on a liquid chromatography-mass spectrometry technology, which comprises the steps of adding an endogenous reference substance into a normal matrix to prepare a correction standard sample, calculating to obtain the background of the endogenous substances in the normal matrix in the preparation correction standard sample, then carrying out back calculation on the background and the concentration of the added endogenous reference substance to obtain a standard curve, and calculating to obtain the concentration of the endogenous substances in a sample to be detected by using the standard curve. In addition, the method with small sample volume, high sensitivity, high accuracy and high selectivity is successfully established for the concentration detection of the coenzyme Q10 in the rat plasma by adopting the method, the sample volume is only 1 mu L, the data acquisition time is only 3.6min, and a foundation is provided for the in vivo pharmacokinetic research of the coenzyme Q10.

Description

Method for determining concentration of endogenous substances including coenzyme Q10 in biological sample based on liquid chromatography-mass spectrometry technology
Technical Field
The invention relates to the fields of biotechnology and analytical chemistry, in particular to a method for determining the concentration of endogenous substances including coenzyme Q10 based on a liquid chromatography-mass spectrometry technology.
Background
Liquid mass spectrometry is widely used in quantitative detection of drugs and their metabolites in biological samples, and the conventional detection objects are usually exogenous substances and do not exist in biological matrixes. Researchers add a target substance to be measured into a blank matrix to prepare a quantitative standard curve sample and a quality control sample simulation actual sample, and measure the concentration of a compound in a biological sample through a standard curve. However, when analyzing endogenous substances in a biological sample, it is difficult to find a blank matrix corresponding to the biological sample, and without the blank matrix, the actual sample cannot be simulated, so that various risks cannot be avoided, and the detection result may be unreliable. For clinical biological sample analysis, the medicine is safe for administration. The accurate quantification of endogenous substances is a century-old problem.
Coenzyme Q10 is an endogenous fat-soluble quinone compound, and widely exists in human bodies and some mammals. The coenzyme Q10 has multiple effects of resisting oxidation, eliminating free radicals, improving human immunity, resisting aging, relieving fatigue, protecting cardiovascular and the like. The compound has been widely used as an additive in cosmetics and foods in recent years, and has been researched and found to have the effects of resisting Parkinson, treating infantile myocarditis, controlling recurrent oral ulcer, improving heart failure, relieving chest distress and the like in the field of clinical treatment. However, coenzyme Q10 belongs to a lipid-soluble endogenous compound, the blank matrix is more difficult to obtain and interfere with endogenous or other sources, and the detection of the biological sample has unique technical difficulties.
The quantitative analysis of biological samples of endogenous compounds currently mainly comprises four strategies, namely a standard addition method, a background subtraction method, a substitute matrix (simplified matrix, artificial matrix or purified matrix) method and a substitute analyte method. The standard addition method requires a single standard curve for each test sample, and is suitable for analysis of only large volume of small sample that can be collected, due to the large amount of sample used. The background subtraction method is to obtain the ratio (R) of peak response values in the blank matrix blk ) The peak response ratio obtained by sample analysis is R, the background in the blank matrix is deducted, and the adjusted peak response value R' = R-R is used blk The standard curve was linearly regressed with the adjusted peak response ratio R' as the Y value and the concentration of the added standard curve sample as X. Since the adjusted Y value is used for regression, the background subtraction method requires additional data processing software to perform linear regression and sample data processing, and the regression equation is fitted after background subtraction is performed by using excel software or sps statistical analysis software, so that the sample concentration cannot be directly obtained from instrument software, the statistical analysis software performs additional calculation, and the data processing and analysis method is very complex. In the research of UPLC-MS/MS quantitative analysis method of coenzyme Q10, the inventors used light-irradiated coenzyme Q10 and 4% Bovine Serum Albumin (BSA) as substitute matrices, respectively, and found that the substitute matrix of light-irradiated coenzyme Q10 had a fluctuation in response of internal standard in the analysis of actual test samples, and that the internal standard had a different effect on the light-irradiated coenzyme Q10 substitute matrix from that of the test samples, whereas the liquid-liquid extraction method using 4 BSA as substitute matrix resulted in too low extraction recovery, requiring several extractions and concentrations to improve the recovery, the processing method was complicated, and when 4 BSA was used as substitute matrix in the protein precipitation treatment, the extraction method was complicated from the actual sampleThere is a phenomenon that the extraction recovery rate is not uniform. Alternative analyte methods, which introduce compounds not present in the body, need to demonstrate a constant ratio to endogenous substances, and also demonstrate similar matrix effects and recovery rates of the alternative to the analyte, which in practice is more complex and more expensive than other methods.
Therefore, there is a need to develop a liquid-phase mass spectrometry detection method for endogenous substances (coenzyme Q10) in biological samples, which has the advantages of small matrix effect influence, small interference, small sampling amount, high sensitivity, reliability and low cost, and provides a basis for pharmacokinetic research of the coenzyme Q10 and even all endogenous compounds.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provides a method for determining the concentration of endogenous substances including coenzyme Q10 in a biological sample based on a liquid chromatography-mass spectrometry technology.
In order to realize the purpose, the invention adopts the technical scheme that:
a method for determining the concentration of endogenous substances in a biological sample based on a liquid chromatography-mass spectrometry (LC-MS) technology comprises a quantitative analysis step, wherein an endogenous reference substance is added into a real substrate containing the endogenous substances to prepare a calibration standard sample, the concentration X1 of the endogenous reference substance in the added calibration standard sample is used as a horizontal coordinate, the chromatographic peak area ratio Y1 of an analyte to an internal standard is used as a vertical coordinate, and the concentration X1 of the endogenous reference substance added into the calibration standard sample and the peak area ratio Y1 are subjected to linear regression by using a weighted least square method to obtain a regression equation Y1= a + bX1, so that the equation obtains the background value of the real substrate used for preparing a calibration standard sample curve as c = -a/b |; and then using the total concentration X2 of endogenous substances in the calibration standard sample as a horizontal coordinate, wherein the total concentration of the endogenous substances = the background value of a real matrix for preparing a calibration standard sample curve + the concentration of an added endogenous reference substance, the chromatographic peak area ratio Y2 of an analyte to an internal standard is a vertical coordinate, performing linear regression by using a weighted least square method according to the total concentration X2 of the endogenous substances in the calibration standard sample and the chromatographic peak area ratio Y2, and obtaining a regression equation Y2= A + BX2, namely a standard curve, thereby calculating the concentration of the endogenous substances in the biological sample to be detected.
Further, the theoretical concentration value of the quality control sample is the sum of the background concentration of the real matrix for preparing the quality control sample and the concentration of the endogenous reference substance added into the quality control sample, so as to calculate the accuracy of the quality control sample.
According to the method, the normal matrix is added with the endogenous reference substance to prepare the correction standard sample, the background of the endogenous substance in the normal matrix in the prepared correction standard sample is calculated firstly, then the background and the concentration of the added endogenous reference substance are used for carrying out back calculation to obtain a standard curve, and the concentration of the endogenous substance in the sample to be detected is calculated and obtained by the standard curve. The method can be used for quantitative detection and analysis of various endogenous compounds in a biological sample, effectively eliminates matrix effect, has no interference, has small sampling amount and high accuracy, does not need additional data software for regression, directly samples on an instrument, can directly obtain a sample concentration result on the instrument software, does not need additional data processing software for processing, and is simple and easy to operate.
The invention also specifically discloses a method for determining the concentration of coenzyme Q10 in blood plasma based on the LC-MS technology, which comprises the following steps: respectively mixing the working solutions of the series coenzyme Q10 standard curves with blank plasma to obtain a calibration standard sample, and injecting the calibration standard sample into UPLC-MS/MS for analysis after pretreatment; adding an internal standard working solution into the plasma to be detected, pretreating, and injecting into UPLC-MS/MS for analysis; firstly, taking the concentration X1 of coenzyme Q10 added into a calibration standard sample as an abscissa, taking the chromatographic peak area ratio Y1 of the coenzyme Q10 and an internal standard as an ordinate, and carrying out linear regression on the concentration X1 of the coenzyme Q10 added into the calibration standard sample and the peak area ratio Y1 by using a weighted least square method to obtain a regression equation Y1= a + bX1, wherein the equation obtains a background value of blank plasma for preparing the calibration standard sample as c = -a/b |; and then, taking the total concentration of the coenzyme Q10 in the corrected standard sample as a horizontal coordinate, taking the total concentration of the coenzyme Q10 = the background value of blank plasma for preparing the corrected standard sample + the concentration of the added coenzyme Q10, taking a chromatographic peak area ratio Y2 of an analyte and an internal standard as a vertical coordinate, performing linear regression by using a weighted least square method and the total concentration X2 and the peak area ratio Y2 of the coenzyme Q10 in the corrected standard sample, and obtaining a regression equation Y2= A + BX2, namely a standard curve, and calculating the concentration of the coenzyme Q10 in the plasma to be detected by using the standard curve.
Compared with the alternative analyte method, the method does not need to search for the alternative analyte which does not exist in the organism but is proved to have a constant proportional relation with the endogenous substance, does not need a complex verification process, does not need lower cost, can adopt the real matrix consistent with the biological sample, and can effectively eliminate the difference of matrix effect and recovery rate. Compared with the alternative matrix method, the coenzyme Q10 of the analyte is unstable due to light, the alternative matrix obtained by removing the coenzyme Q10 in the real matrix through light irradiation is used, and due to the difference of the matrixes, the problem that the internal standard fluctuates when the sample is processed after the sample is compared with the matrix analysis of an actual biological sample, so that the matrix effect and the extraction recovery rate are different exists, and the coenzyme Q10 is not suitable for the analysis of the coenzyme Q10 after the coenzyme Q10 is removed through light irradiation. Also, 4-percent BSA was used as a substitute matrix, which also resulted in a low extraction yield. Compared with the standard addition method, the biological analysis amount is only 100 microliter or less in the actual experiment due to the limitation of the sample amount (sample volume and sample number), and is generally not as large as the sample amount, so the standard addition method is not suitable for the analysis of trace large amount samples. Compared with the background deduction method, the method and the background deduction method both adopt real matrixes, but the background deduction method needs additional data processing software to perform linear regression and sample data processing, after background deduction is performed by using excel software or statistical analysis software such as a sps, a regression equation is fitted, the sample concentration cannot be directly obtained from instrument software, the statistical analysis software calculates additionally, and the data processing and analysis method is extremely complex. And further mixing the series of quality control working solutions with blank plasma to obtain a quality control sample, pretreating, and injecting into UPLC-MS/MS for analysis, wherein the theoretical concentration value of the coenzyme Q10 in the quality control sample is the sum of the background concentration of the blank plasma for preparing the quality control sample and the concentration of the coenzyme Q10 added in the quality control sample.
Preparing a working solution: weighing two parts of a coenzyme Q10 reference substance, respectively adding isopropanol to dissolve and dilute the two parts to prepare coenzyme Q10 reference substance stock solutions S01 and S02; taking a reference substance stock solution S01, and diluting with methanol to obtain a series of standard curve working solutions with concentration; taking a reference substance stock solution S02, and diluting with methanol to obtain a series of quality control working solutions with concentration;
preparing an internal standard solution: weighing coenzyme Q10-d9, dissolving the coenzyme Q10-d9 with isopropanol to prepare coenzyme Q10-d9 stock solution, and diluting the stock solution with methanol to prepare internal standard working solution.
The pretreatment mode is as follows: taking a plasma sample, adding an internal standard working solution, adding isopropanol in a volume ratio of 9: ethyl acetate, vortex and mix evenly; centrifuging, taking supernatant, adding methanol, mixing uniformly, and placing in a sample injection tube.
Chromatographic conditions are as follows: mobile phase B:0.1% formic acid methanol, elution mode: isocratic elution with a flow rate of 0.6 mL/min -1 The sample size is 1 μ L, and the chromatographic column is ACQUITY UPLC-BEH C 18 Column, column temperature 40 ℃, autosampler wash solution: methanol: isopropyl alcohol: acetonitrile: the volume ratio of water is 1:1:1.
Mass spectrum conditions: an electrospray ion source is adopted, a positive ion mode is adopted, and a scanning mode is multi-reaction monitoring (MRM); ion source parameters: capillary voltage 2.5kV, taper hole voltage 20V, ion source temperature 150 ℃, desolventizing temperature: air flow rate of 150 L.h at 400 ℃ and conical hole -1 The desolventizing gas flow rate is 800 L.h -1 Atomizing gas pressure 6.0Bar, collision gas flow rate 0.15mL/min -1 The data acquisition time is 3.60min; the quantitative ion pair of coenzyme Q10 is [ M + H] + M/z863.75 → 197.00, collision energy of 40eV, and quantitative ion pair of internal standard coenzyme Q10-d9 of [ M + H ]] + m/z 872.70 → 205.90, collision energy 40eV.
Compared with the prior art, the invention has the beneficial effects that:
1. the method adopts the normal matrix to prepare the standard yeast and quality control, better simulates the real condition of the actual biological sample, calculates the background of endogenous substances (coenzyme Q10) in the normal matrix in the preparation of the standard yeast and quality control by utilizing the principle of a standard addition method, and simultaneously uses the background and the concentration of an added endogenous reference substance to calculate a standard curve to measure the concentration of the endogenous substances (coenzyme Q10) in the sample to be measured.
2. The UPLC-MS/MS determination method for the blood concentration of the endogenous substance coenzyme Q10 in the rat plasma, which is disclosed by the invention, is good in peak shape, high in sensitivity, strong in specificity, wide in analysis range, good in stability, short in determination period, simple and convenient to operate and low in cost, is researched and established.
3. The invention adopts isopropanol to dissolve methanol for dilution to prepare working solution, and a plasma sample is processed by isopropanol: ethyl acetate (9 -1 The sample injection amount is only 1 mu L, the data acquisition time is only 3.6min, the volume of the blood plasma for detection is small, the recovery rate is high, the matrix effect is avoided, the acquisition time is short, and a foundation can be provided for the in-vivo pharmacokinetic study of the substance.
Drawings
FIG. 1 is a chromatogram of coenzyme Q10 and coenzyme Q10-d9 (blank plasma A. Blank reagent C. Blank plasma added with coenzyme Q10 reference and internal standard coenzyme Q10-d 9D. Intragastric lavage plasma sample added with internal standard treatment chromatogram);
FIG. 2 is a graph showing the average drug effect of rats after gavage with 5% coenzyme Q10 self-microemulsion and 10% coenzyme Q10 microparticles (see
Figure BDA0003716165130000051
n＝6)。
Fig. 3 is a Y2= a + BX2 standard curve.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it is to be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
The materials used in the present invention:
animals: male SD rats, SPF grade, 12, body weight 180-220 g, provided by the Experimental animals research center in Hubei province.
Reagent: isopropyl alcohol: chromatographically pure, merck, batch number: k53060340107; formic acid: chromatographically pure, alatin, batch number: e2022005; acetonitrile: chromatographic purity, fisher Scientific, lot number: 186350; methanol: chromatographic purity, fisher Scientific, lot number: 211769; ethyl acetate: chromatographic purity, fisher Scientific, lot number: 179268; coenzyme Q10 reference substance, purity 99.1%, batch number: 1120070107,5% coenzyme Q10 self-microemulsion and 10% coenzyme Q10 microparticles, both provided by Xinhe GmbH, zhejiang province, inc.; coenzyme Q10-d9 reference substance, purity 98.2%, batch number: MBBD0885.
The instrument comprises the following steps: waters ACQUITY UPLC I-Class model ultra high performance liquid chromatograph (Waters corporation, USA), waters Xevo TQ-S triple quadrupole mass spectrometer and UNIFI data processing software (Waters corporation, USA).
The invention discloses a method for determining coenzyme Q10 concentration in rat plasma by UPLC-MS/MS, which comprises the following steps:
1 test
1.1 chromatographic and Mass Spectrometry conditions
Chromatographic conditions are as follows: the chromatographic column is ACQUITY UPLC-BEH C 18 Column (2.1 mm 50mm,1.7 μm), isocratic elution, mobile phase: 0.1% formic acid methanol at a flow rate of 0.6 mL/min -1 The temperature of the column oven was 40 ℃ and the injection volume was 1. Mu.L.
Mass spectrum conditions: ESI ion source, positive ion mode, multiple Reaction Monitoring (MRM) mode; ion source parameters: capillary Voltage (Capillary Voltage): 2.5Kv; cone voltage (Sample Cone): 20V, and (3); ion Source temperature (Source temperature): 150 ℃; desolvation temperature (Desolvation temperature): 400 ℃; cone orifice airflow rate (Cone gas flow): 150L/h; desolvation gas flow rate (Desolvation gas flow): 800L/h; atomising gas pressure (Nebulizer gas pressure): 6.0Bar; primary mass spectrometry collision energy (MS collision energy): 4eV; secondary mass spectrometry collision energy (MSMS collision energy): 40eV; collision gas flow rate (Collision gas flow): 0.15mL/min; time of data acquisition3.60min, the ion pair of coenzyme Q10 and coenzyme Q10-d9 is [ M + H] + M/z863.75 → 197.00 and m/z 872.70 → 205.90 respectively, the collision energy is 40eV.
1.2 preparation of working solution
Preparation of analyte control working solution: a certain amount of coenzyme Q10 was weighed and placed in two brown glass bottles, and the weights were recorded and marked as S01, S02, respectively. Calculating the true weight of the analyte according to the purity, moisture, salification or not of coenzyme Q10, adding a proper amount of isopropanol to obtain a stock solution with the final concentration of 0.60mg/mL, and mixing by vortex. Taking a proper amount of coenzyme Q10 stock solution S01, and sequentially diluting with methanol to obtain series standard curve working solutions with the concentrations of 1.60, 3.20, 8.00, 16.0, 32.0, 64.0, 128 and 160 mu g/mL; taking a proper amount of coenzyme Q10 stock solution S02, and sequentially diluting with methanol to obtain quality control working solutions with the concentrations of 1.60, 4.80, 24.0 and 120 mu g/mL.
Preparing an internal standard solution: weighing 1mg of coenzyme Q10-d9 reference substance, adding an appropriate volume of isopropanol, and preparing an internal standard stock solution with the final concentration of 0.60 mg/mL. And precisely measuring an appropriate amount of internal standard stock solution, and diluting the internal standard stock solution into an internal standard working solution with the concentration of 1 mu g/mL by using methanol.
1.3 animal Experimental methods
12 male Sprague Dawley rats. The groups were randomized into 2 groups of 6 individuals. Fasted for 12h (without water deprivation) before dosing and the rats were allowed free access to food 4h after dosing. The first group was gavage with 5% coenzyme Q10 self-microemulsions at a dosing dose of 21mg/kg and the second group was gavage with 10% coenzyme Q10 microparticles at the same dosing dose. Collecting blood of 0.3mL from jugular vein plexus according to blood collection time points of 0h, 1h, 2h, 4h, 6h, 8h, 12h, 24h, 36h and 48h, centrifuging for 10min under the condition of 3000rpm/min by using EDTA-K2 as anticoagulant, and collecting supernatant plasma for preservation at minus 80 +/-10 ℃.
1.4 plasma sample treatment
Plasma samples were taken at 40 μ L, 25 μ L of internal standard working solution (1 μ g/mL) was added, followed by 400 μ L of isopropanol: ethyl acetate (9. Centrifuging at 12000rpm in a4 deg.C centrifuge for 10min; and taking 100 mu L of supernatant, adding 200 mu L of methanol, mixing uniformly, and carrying out UPLC-MS/MS on-machine analysis.
1.5 methodological examination
The specificity is as follows: and (3) processing a blank reagent, a zero-concentration sample (a blank sample only added with an internal standard) and a quantitative upper limit plasma sample without the internal standard according to steps, and then analyzing to investigate the influence degree of interference peaks in the blank reagent on the peak area integrals of the analyte and the internal standard. Standard curve and lower limit of quantitation: 10 μ L of serial standard curve working solutions are mixed with 190 μ L of blank rat plasma to prepare standard curve plasma samples (calibration standard samples) with the concentrations of 80, 160, 400, 800, 1600, 3200, 6400 and 8000ng/mL, and the standard curve plasma samples are processed according to a plasma sample processing method and then analyzed. The coenzyme Q10 concentration in the calibration standard sample added is used as the abscissa, the ratio of the chromatographic peak area of the analyte (CoQ 10) to the internal standard (coenzyme Q10-d 9) is used as the ordinate, and the weight is used (W = 1/x) 2 ) Least squares to correct for the concentration of coenzyme Q10 control added to the standards (X) 1 ) To peak area ratio (Y) 1 ) Linear regression was performed, resulting in regression equation (Y) 1 ＝a+bX 1 ) That is, from this equation, the background value of the blank matrix used to formulate the standard curve is c = | -a/b |, the total coenzyme Q10 concentration in the calibration standard (background value of the matrix from which the standard curve is formulated + concentration of the calibration standard) is plotted on the abscissa, the ratio of the chromatographic peak areas of the analyte (CoQ 10) and the internal standard (coenzyme Q10-d 9) is plotted on the ordinate, and the weights (W =1/x |) 2 ) Least squares method to correct the total concentration of coenzyme Q10 (X2) in the standards to the peak area ratio (Y) 2 ) Linear regression was performed, resulting in regression equation (Y) 2 ＝A+BX 2 ) I.e. the standard curve. And the coenzyme Q10 concentration in the blood plasma of the rat to be detected is calculated back according to the standard curve to the concentration of the calibration standard sample. Precision and accuracy: respectively mixing 10 μ L of quality control working solution and 190 μ L of blank rat plasma to obtain quantitative lower limit quality control (LLOQ QC) with concentration of 80ng/mL, low concentration quality control (LQC) with concentration of 240ng/mL, medium concentration quality control (MQC) with concentration of 1200ng/mL and low concentration quality control (HQC) with concentration of 6000 ng/mL. The quality control samples are processed according to the plasma sample processing mode and then are subjected to sample injection analysis, and in 3 analysis batches with precision and accuracy, each concentration quality control sample in each analysis batch is processed in at least 2 days6 replicates were prepared. To examine the precision and accuracy within and between batches.
Extraction recovery rate: in one analysis batch, the peak areas of the extracted low, medium and high concentration quality control samples prepared by blank plasma are compared with the peak areas of pure solutions prepared by adding working solutions for preparing the low, medium and high quality control samples and internal standard working solutions into the extracted blank plasma respectively for evaluation. The method comprises the following specific steps: coenzyme Q10 working solution and internal standard working solution I03 (1000 ng/mL) with the concentrations of 4.80, 24.0 and 120 mu g/mL are precisely sucked, diluted by methanol and prepared into three concentrations of re-solutions with the analyte concentrations of 10.32, 51.61 and 258.06ng/mL and the internal standard concentration of 26.88ng/mL respectively, so as to prepare recovery rate evaluation samples. Processing the blank matrix according to a plasma sample processing method (replacing the internal standard by 25 mu L of methanol), respectively adding the 3 complex solutions in the last step without adding 200 mu L of methanol, preparing 3 parallel samples at each concentration to obtain a recovery rate evaluation sample REC sample, and measuring the analyte peak area (A1) and the internal standard peak area (A2) in each concentration recovery rate evaluation sample by sample injection. Simultaneously measuring the peak area (A3) of coenzyme Q10 in the blank matrix and simultaneously measuring the peak area (A4) of the analyte and the peak area (A5) of the internal standard in the low, medium and high concentration quality control samples respectively, so that the recovery rate of the analyte is (A4-A3)/A1 x 100%; the recovery of the internal standard was A5/A2 x 100%.
Matrix effect: the method comprises the steps of respectively adopting 6 matrixes from different sources, adding pure solutions prepared from working solutions for preparing low, medium and high quality control samples and internal standard working solutions after sample treatment, preparing matrix effect evaluation samples (MER), preparing pure solution control samples (MEP) containing no matrix extract and having the same concentration, and evaluating the influence of matrix effects on accurate quantitative analysis of analytes by calculating matrix factors normalized by the internal standards in each sample. The method comprises the following specific steps: coenzyme Q10 working solution and internal standard working solution I03 (1000 ng/mL) with the concentrations of 4.80, 24.0 and 120 mu g/mL respectively are precisely sucked, and are diluted by methanol to respectively prepare compound solutions with three concentrations, namely 10.32, 51.61 and 258.06ng/mL of analyte concentration and 26.88ng/mL of internal standard concentration. Matrix-containing samples blank matrices from 6 different sources were processed according to the plasma sample processing method (internal standard was replaced with 25. Mu.L of methanol), and 200. Mu.L of the three complex solutions were added in the last step instead of 200. Mu.L of methanol, and 1 replicate was prepared using matrices from 6 different sources per concentration. Non-matrix samples blank reagents were processed according to plasma samples (internal standard was replaced with 25. Mu.L of methanol) and 200. Mu.L of each of the three above-mentioned double solutions was added in the final step instead of 200. Mu.L of methanol, to prepare 3 replicates per concentration. The evaluation of the matrix effect was done by calculating the matrix factor of the analyte and the internal standard. Matrix factors for the analyte and internal standard were calculated by calculating the ratio of the peak area in the presence of matrix to the corresponding peak area without matrix, respectively. The formula for calculating the matrix factor is as follows:
Figure BDA0003716165130000081
the internal standard normalized matrix factor is calculated as follows:
Figure BDA0003716165130000082
and calculating the normalized matrix factor, wherein the closer the value is to 1, the closer the matrix effect of the internal standard and the analyte is, the matrix effect which may occur to the analyte in any sample can be compensated, and the matrix effect does not influence the final accurate quantitative analysis. Stability: the low-concentration quality control sample and the high-concentration quality control sample are prepared according to the method. The stability of the sample was examined by storage at room temperature for 27h, storage at a sampler temperature after pretreatment for 8d, storage at 80 ℃ for 77d, and repeated freeze thawing for 5 times.
2 results of
2.1 methodological validation
The specificity is as follows: the retention time of coenzyme Q10 and coenzyme Q10-d9 of the internal standard are 3.02min and 2.98min respectively, and since coenzyme Q10 is an endogenous compound, the coenzyme Q10 is contained in a certain amount in blank plasma. Coenzyme Q10 and internal standard Q10-d9 are not detected in the blank reagent sample, which indicates that the blank reagent has no interference to the analyte and the internal standard, and coenzyme Q10 is not detected in the zero concentration sample (CTL-0), which indicates that the internal standard Q10-d9 has no interference to the peak emergence of the analyte coenzyme Q10; coenzyme Q10-d9 is not detected in the quantitative upper limit sample without the internal standard, which shows that the coenzyme Q10 of the analyte has no interference to the peak generation of the internal standard Q10-d9, and the result is shown in figure 1.
Standard curve and lower limit of quantitation: each standard curve has at least 75% and the accuracy deviation of the calculated concentration and the marked concentration of at least 6 calibration standards is not more than +/-15% (LLOQ is not more than +/-20%), and the correlation coefficient R2 of each standard curve is more than or equal to 0.990261. The standard curve is shown to be linear well within the range of 80.0-8000 ng/mL. The results are shown in FIG. 3.
Precision and accuracy: the intra-batch/inter-batch average accuracy deviation range of the LQC, MQC and HQC quality control samples is-2.5% -11.0%/0.1% -8.4%, and the precision is 8.7% and 6.1% respectively at the maximum; the quantitative lower limit quality control sample (LLOQ QC) has an intra-batch average accuracy deviation of 3.3-13.3%, an inter-batch accuracy deviation of 7.7%, and intra/inter-batch maximum precision values of 3.3% and 5.9%, and the deviations of the quality control samples of all concentrations do not exceed +/-15%, and the RSD is less than or equal to 15%. The method shows that the precision and accuracy of the quality control samples of various concentrations are good in intra-batch precision and inter-batch precision. The results are shown in Table 1.
TABLE 1 results of the investigation of the accuracy and precision of coenzyme Q10 in rat plasma
Figure BDA0003716165130000091
Extraction recovery rate: the average extraction recovery rate of coenzyme Q10 in the low, medium and high concentration quality control samples is 88.3 percent, the RSD is 8.1 percent, the average extraction recovery rate of the internal standard is 96.0 percent, and the RSD is 3.7 percent. The detailed results are shown in table 2, and the average recovery rate of each concentration quality control sample has no concentration-related trend. The results show that the recovery of analyte and internal standard does not affect the accuracy of the quantitative analysis.
Matrix effect: the calculated matrix factor means/Relative Standard Deviation (RSD) of the low, medium and high concentration analytes for 6 different sources of matrix normalized by the internal standard were 0.997/1.3%, 0.995/0.8% and 0.996/0.01%, respectively. Detailed results are shown in table 2, relative standard deviations of internal standard normalized matrix factors in 6 matrices from different sources do not exceed 15%, indicating that matrix effects do not affect the quantitative analysis of analytes.
Table 2 recovery and matrix effect of coenzyme Q10 in rat plasma: (
Figure BDA0003716165130000092
n＝6)
Figure BDA0003716165130000093
Figure BDA0003716165130000101
Stability: the results are shown in Table 3, and the samples were stable under the above conditions and met the analytical measurements.
Table 3 stability study table-3 stability of coenzyme Q10 in rat plasma (n = 6)
Figure BDA0003716165130000102
2.2 application of methodology
The mean blood concentration-time curve of the coenzyme Q10 after the rat is subjected to intragastric administration by adopting 5 percent of coenzyme Q10 self-microemulsion and 10 percent of coenzyme Q10 particles is shown in figure 2. The results of the main parameters of pharmacokinetics are shown in table 4, and the relative bioavailability of 5 percent of coenzyme Q10 self-microemulsion to 10 percent of coenzyme Q10 micro-particles oral administration is 127.16 percent according to the calculation of the pharmacokinetics parameters. The result shows that the method can be applied to the pharmacokinetics research of the coenzyme Q10 in the rat body and the research of the relative bioavailability of different preparations (such as self-microemulsion and microparticles).
TABLE 4 pharmacokinetic parameters after gavage of 5% coenzyme Q10 self-microemulsion and 10% coenzyme Q10 microparticles in rats ((C))
Figure BDA0003716165130000103
n＝6)
Figure BDA0003716165130000104
Figure BDA0003716165130000111
The UPLC-MS/MS method for measuring the blood concentration of endogenous substance coenzyme Q10 in rat plasma, which is established by the invention, has the advantages of high sensitivity, strong specificity, wide analysis range, good stability, short measurement period, rapidness, convenience, capability of accurately quantifying the concentration of coenzyme Q10 in a complex biological matrix through various index requirements verified by methodology, small volume of plasma for detection, high recovery rate, no matrix effect, wide linear range, simplicity in operation and short acquisition time, can provide a basis for the research of pharmacokinetics in vivo of the substance, and is favorable for the popularization of clinical high-flux detection. Meanwhile, the concentration of the endogenous coenzyme Q10 in the matrix is obtained by using the same set of processing standard curve samples, and then the total concentration of the coenzyme Q10 in the actual sample is calculated.
The method of the invention overcomes the following technical difficulties: (1) coenzyme Q10 is an endogenous compound with high fat solubility, because the acquisition of a blank matrix is difficult to interfere with endogenous or other sources, the actual sample cannot be simulated without the blank matrix, and various risks (such as different recovery rates, matrix effect and the like) can cause unreliable detection results. The four common methods for detecting endogenous compounds have respective defects, and the invention adopts a unique method to better overcome the defects of the four conventional methods for detecting endogenous compounds and solve the problem of the quantification of endogenous substances. (2) The concentration of coenzyme Q10 in blood plasma is generally lower, the requirement on the sensitivity of a high performance liquid chromatography-tandem mass spectrometry method is higher, the lower limit of the quantification of the method is 80ng/mL, and the clinical treatment of the coenzyme Q10 is assisted. (2) The invention adopts isopropanol to dissolve and methanol to dilute to prepare working solution, and adopts isopropanol: the ethyl acetate is used as a pretreatment mode of a plasma sample, so that the complexity of the quantitative determination of a clinically unknown sample is obviously reduced, the quantitative analysis of a high-throughput biological sample can be realized, and the possible system errors are also reduced to the maximum extent. (3) Conventionally, the detection sensitivity and accuracy can be improved by the large sample injection amount, the sample injection amount is greatly influenced by noise, but the pharmacokinetic test is carried out by adopting a mouse, the blood sampling is very limited, the sample injection amount required by the invention is only 40 microliters, and the sample injection amount is only 1uL, so that the detection accuracy can be ensured, and the rat even clinical invasive blood sampling links can be reduced to the maximum extent. (4) The invention adopts the following components in a volume ratio of 1:1, methanol: isopropyl alcohol: acetonitrile: the water is used as the cleaning solution of the automatic sample injector, so that the residual effect can be effectively eliminated.
In addition, the inventor treats the plasma sample by a conventional method in the process of establishing the method of the invention, and finds that the conventional method has various problems and cannot meet the requirement of detecting the coenzyme Q10 concentration by high performance liquid mass spectrometry, and the method is simply listed as follows:
1, MD1: adding 25 mu L of internal standard into 40 mu L of plasma, adding 200 mu L of ACN and 600 mu L of n-hexane, drying 400 mu L of upper nitrogen, adding 200 mu L of 10% isopropanol acetonitrile solution for redissolution, and injecting.
The problems that exist are that: the extraction recovery rate is 13-18.9%, and the recovery rate is low.
And MD2: to 40. Mu.L of plasma was added 25. Mu.L of internal standard followed by 200. Mu.L of methanol: precipitating with isopropanol (1, v;
the problems that exist are that: the extraction recovery rate is about 20 percent, and the recovery rate is low.
And (3) MD: adding 25 mul of internal standard into 40 mul of plasma, adding 200 mul of methanol and 600 mul of normal hexane, drying 200 mul of upper layer nitrogen, adding 200 mul of methanol solution for redissolution, and injecting.
The problems exist: the recovery rate is between 30 and 49.5 percent, and the recovery rate is low.
And MD4: adding 25 mul of internal standard into 40 mul of plasma, adding 600 mul of normal hexane, drying 200 mul of upper layer nitrogen, adding 200 mul of isopropanol: methanol (1.
There are problems: the internal standard fluctuates widely in the actual sample and there is a matrix effect.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims (10)

1. The method for determining the concentration of endogenous substances in a biological sample based on the liquid chromatography-mass spectrometry technology is characterized by comprising a quantitative analysis step, wherein an endogenous reference substance is added into a real matrix containing the endogenous substances to prepare a calibration standard sample, the concentration X1 of the endogenous reference substance in the added calibration standard sample is taken as a horizontal coordinate, the chromatographic peak area ratio Y1 of an analyte to an internal standard is taken as a vertical coordinate, and the concentration X1 of the endogenous reference substance added into the calibration standard sample and the peak area ratio Y1 are subjected to linear regression by using a weighted least square method to obtain a regression equation Y1= a + bX1, so that the equation obtains the background value of the real matrix for preparing a calibration standard sample curve as c = -a/b |; and then using the total concentration X2 of endogenous substances in the calibration standard sample as a horizontal coordinate, wherein the total concentration of the endogenous substances = the background value of a real matrix for preparing a calibration standard sample curve + the concentration of an added endogenous reference substance, the chromatographic peak area ratio Y2 of an analyte to an internal standard is a vertical coordinate, performing linear regression by using a weighted least square method according to the total concentration X2 of the endogenous substances in the calibration standard sample and the chromatographic peak area ratio Y2, and obtaining a regression equation Y2= A + BX2, namely a standard curve, thereby calculating the concentration of the endogenous substances in the biological sample to be detected.
2. The method for determining the concentration of an endogenous substance in a biological sample according to claim 1, further comprising calculating the accuracy of the quality control sample by adding the theoretical concentration value of the quality control sample to the sum of the background concentration of the real substrate for preparing the quality control sample and the concentration of the endogenous control substance in the quality control sample.
3. The method for determining the concentration of coenzyme Q10 in blood plasma based on the liquid chromatography-mass spectrometry technology is characterized by comprising the following steps: respectively mixing a series of coenzyme Q10 standard curve working solutions with blank plasma to obtain a correction standard sample, and injecting the correction standard sample into a liquid phase mass spectrometer for analysis after pretreatment; adding an internal standard working solution into the plasma to be detected, pretreating, and injecting into a liquid phase mass spectrometer for analysis; firstly, taking the concentration X1 of coenzyme Q10 added into a calibration standard sample as an abscissa, taking the chromatographic peak area ratio Y1 of the coenzyme Q10 and an internal standard as an ordinate, and carrying out linear regression on the concentration X1 of the coenzyme Q10 added into the calibration standard sample and the peak area ratio Y1 by using a weighted least square method to obtain a regression equation Y1= a + bX1, wherein the equation obtains a background value of blank plasma for preparing the calibration standard sample as c = -a/b |; and then, taking the total concentration of the coenzyme Q10 in the corrected standard sample as a horizontal coordinate, taking the total concentration of the coenzyme Q10 = the background value of blank plasma for preparing the corrected standard sample + the concentration of the added coenzyme Q10, taking a chromatographic peak area ratio Y2 of an analyte and an internal standard as a vertical coordinate, performing linear regression by using a weighted least square method and the total concentration X2 and the peak area ratio Y2 of the coenzyme Q10 in the corrected standard sample, and obtaining a regression equation Y2= A + BX2, namely a standard curve, and calculating the concentration of the coenzyme Q10 in the plasma to be detected by using the standard curve.
4. The method for measuring the concentration of coenzyme Q10 in plasma based on LC-MS technique according to claim 3,
preparing a working solution: weighing two parts of a coenzyme Q10 reference substance, respectively adding isopropanol to dissolve and dilute, and preparing coenzyme Q10 reference substance stock solutions S01 and S02; taking a reference substance stock solution S01, and diluting with methanol to obtain a series of standard curve working solutions with concentration; taking a reference substance stock solution S02, and diluting with methanol to obtain a series of quality control working solutions with concentration;
preparing an internal standard solution: weighing coenzyme Q10-d9, dissolving the coenzyme Q10-d9 with isopropanol to prepare coenzyme Q10-d9 stock solution, and diluting the stock solution with methanol to prepare internal standard working solution.
5. The method for determining the concentration of coenzyme Q10 in plasma based on LC-MS technology according to claim 3, wherein the pretreatment mode is: taking a plasma sample, adding an internal standard working solution, adding isopropanol in a volume ratio of 9: ethyl acetate, vortex and mix evenly; centrifuging, taking supernatant, adding methanol, mixing uniformly, and placing in a sample tube.
6. The method for determining the concentration of coenzyme Q10 in plasma based on LC-MS technology according to claim 3, wherein the chromatographic conditions are as follows: mobile phase B:0.1% formic acid methanol, elution mode: isocratic elution with a flow rate of 0.6 mL/min -1 The sample size was 1. Mu.L.
7. The method for determining the concentration of coenzyme Q10 in blood plasma based on the liquid chromatography-mass spectrometry technology as claimed in claim 4, wherein the series of quality control working solutions are mixed with blank plasma respectively to obtain quality control samples, and the quality control samples are injected into a liquid phase mass spectrometer for analysis after pretreatment, and the theoretical concentration value of coenzyme Q10 in the quality control samples is the sum of the background concentration of the blank plasma for preparing the quality control samples and the concentration of the coenzyme Q10 added in the quality control samples.
8. The method for determining the concentration of coenzyme Q10 in blood plasma based on the LC-MS technology according to claim 6, wherein the chromatographic conditions further comprise: the chromatographic column is ACQUITY UPLC-BEH C 18 Column, column temperature 40 ℃, autosampler wash solution: methanol: isopropyl alcohol: acetonitrile: the volume ratio of water is 1:1:1.
9. The method for determining the concentration of coenzyme Q10 in plasma based on the LC-MS technology according to claim 3, wherein the mass spectrometry conditions are as follows: an electrospray ion source is adopted, a positive ion mode is adopted, and a scanning mode is multi-reaction monitoring (MRM); ion source parameters: capillary voltage 2.5kV, taper hole voltage 20V, ion source temperature 150 ℃, desolventizing temperature: air flow rate of 150 L.h at 400 ℃ and conical hole -1 The desolventizing gas flow rate is 800 L.h -1 Atomizing gas pressure 6.0Bar, collision gas flow rate 0.15mL/min -1 And the data acquisition time is 3.60min.
10. The LC-MS-based technique of claim 9A method for measuring the concentration of coenzyme Q10 in plasma by a technique, characterized in that the quantitative ion pair of coenzyme Q10 is [ M + H ]] + M/z863.75 → 197.00, collision energy of 40eV, and quantitative ion pair of internal standard coenzyme Q10-d9 of [ M + H ]] + m/z 872.70 → 205.90, collision energy 40eV.
CN202210736726.6A 2022-06-27 2022-06-27 Method for determining concentration of endogenous substances including coenzyme Q10 in biological sample based on liquid chromatography-mass spectrometry technology Pending CN115219616A (en)

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