CN115219616B - 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 PDFInfo
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Abstract
The invention discloses a method for measuring the concentration of endogenous substances including coenzyme Q10 in a biological sample based on a liquid chromatography-mass spectrometry (LC-MS) technology, which comprises the steps of adding an endogenous reference substance into a normal substrate to prepare a calibration standard sample, calculating to obtain the background of the endogenous substances in the normal substrate in the calibration standard sample, then calculating back to obtain a standard curve by using the background and the concentration of the added endogenous reference substance, and calculating to obtain the concentration of the endogenous substances in the sample to be measured by using the standard curve. In addition, the method successfully establishes a method with small sample injection amount, high sensitivity, high accuracy and high selectivity for detecting the concentration of the coenzyme Q10 in the rat plasma, the sample injection amount is only 1 mu L, the data acquisition time is only 3.6min, and a foundation is provided for the in vivo pharmacokinetics study of the coenzyme Q10.
Description
Technical Field
The invention relates to the fields of biotechnology and analytical chemistry, in particular to a method for measuring the concentration of endogenous substances including coenzyme Q10 based on a liquid chromatography-mass spectrometry technology.
Background
The liquid phase mass spectrometry technology is widely used for quantitative detection of drugs and metabolites thereof in biological samples, and conventional detection objects are usually exogenous substances and do not exist in biological matrixes. Researchers add target test substances into a blank matrix to prepare quantitative standard curve samples and quality control samples to simulate actual samples, and the concentration of the compound in biological samples is quantified through the standard curve. However, when analyzing endogenous substances in biological samples, it is difficult to find a blank matrix corresponding to the biological samples, and the actual samples cannot be simulated without the blank matrix, so that various risks cannot be avoided, and the detection result may be unreliable. For clinical biological sample analysis, the medical administration is safe. Accurate quantification of endogenous substances has become a century difficult problem.
Coenzyme Q10 is an endogenous, fat-soluble quinone compound that is widely found in humans and some mammals. Coenzyme Q10 has the effects of resisting oxidation, scavenging free radicals, improving immunity, resisting aging, relieving fatigue, protecting cardiovascular system, etc. In recent years, the compound is widely used as an additive in cosmetics and foods, and researches show that the compound has the effects of resisting Parkinson, treating pediatric myocarditis, controlling recurrent oral ulcer, improving heart failure, relieving chest distress and the like in the clinical treatment field. However, coenzyme Q10 belongs to an endogenous compound with large lipid solubility, a blank matrix is more difficult to obtain, and interference from endogenous or other sources is caused, so that detection of biological samples has unique technical difficulties.
The quantitative analysis of biological samples of endogenous compounds currently mainly has four strategies, namely a standard addition method, a background deduction method, a matrix replacement (simplified matrix, artificial matrix or purified matrix) method and a test object replacement method. The standard addition method requires a single standard curve for each test sample, and is very large in test sample consumption and only suitable for analysis of the test samples with a large collectable volume and a small number. The background subtraction method is to obtain the ratio (R blk) of the peak response value in the blank matrix, the peak response ratio obtained by sample analysis is R, the background in the blank matrix is subtracted, the adjusted peak response value R '=R-R blk is used for the standard curve, and the adjusted peak response ratio R' is used as the Y value and the concentration of the added standard curve sample is X for linear regression. Because the adjusted Y value is used for regression, the background deduction method needs additional data processing software for linear regression and sample data processing, the regression equation is fitted after the background is deducted by using excel software or spss statistical analysis software, the sample concentration cannot be directly obtained from instrument software, the statistical analysis software additionally calculates, and the data processing analysis method is extremely complex. In the method for replacing matrix by adopting a replacement matrix instead of a test sample matrix, the complexity of the replacement matrix is greatly different, the difference of corresponding difference of instruments and the difference of extraction recovery rate are caused by different matrixes, in the research process of the UPLC-MS/MS quantitative analysis method of coenzyme Q10, the inventor uses photo-removed coenzyme Q10 and 4% Bovine Serum Albumin (BSA) as the replacement matrixes respectively, finds out that the photo-removed coenzyme Q10 replacement matrixes exist in the actual test sample analysis, considers that the phenomenon that the extraction recovery rate is too low when the internal standard is different from the matrix effect of the test sample in the photo-removed coenzyme Q10 replacement matrixes, and the phenomenon that the extraction recovery rate is too low when the sample is treated by a liquid-liquid extraction method by using 4% BSA as the replacement matrix, the extraction concentration is needed to improve the recovery rate several times, the treatment method is complicated, and the phenomenon that the extraction recovery rate is inconsistent with the actual sample when the 4% BSA is treated by protein precipitation as the replacement matrix. The surrogate analyte method needs to prove that the surrogate analyte has similar matrix effect and recovery rate with the analyte and is more complex and more expensive than other methods in practical application because the surrogate analyte method introduces the compound which does not exist in the organism and has a constant specific relationship with the endogenous substance.
Therefore, it is necessary to research a liquid-phase mass spectrometry detection method of endogenous substances (coenzyme Q10) in biological samples, which has the advantages of small influence of matrix effect, small interference, small sampling amount, high sensitivity, reliability and low cost, and provides a foundation for the pharmacokinetic research of coenzyme Q10 and even all endogenous compounds.
Disclosure of Invention
The invention aims at solving the problems existing in the prior art and provides a method for measuring the concentration of endogenous substances including coenzyme Q10 in a biological sample based on a liquid chromatography-mass spectrometry technology.
In order to achieve the above purpose, the invention adopts the following technical scheme:
The method for determining the concentration of the endogenous substances in the biological sample based on the liquid chromatography-mass spectrometry technology comprises the steps of preparing a correction standard sample after adding an endogenous reference substance into a real matrix containing the endogenous substances, wherein the concentration X1 of the endogenous reference substance in the added correction standard sample is firstly used as an abscissa, the concentration X1 of the endogenous reference substance added into the correction standard sample and the peak area ratio Y1 of the internal standard sample are used as an ordinate, and a weighted least square method is used for carrying out linear regression on the concentration X1 of the endogenous reference substance added into the correction standard sample and the peak area ratio Y1, so that a regression equation Y1=a+bx1 is obtained, and the background value of the real matrix for preparing a correction standard sample curve is c= -a/b; and then the total concentration X2 of the endogenous substances in the calibration standard sample is taken as an abscissa, the total concentration of the endogenous substances = the background value of the real matrix for preparing the calibration standard sample curve + the concentration of the added endogenous reference substance, the chromatographic peak area ratio Y2 of the analyte and the internal standard is taken as an ordinate, the weighted least square method is used for carrying out linear regression on the total concentration X2 of the endogenous substances in the calibration standard sample and the peak area ratio Y2, and the regression equation Y2 = A + BX2 is taken as a standard curve, so that the concentration of the endogenous substances in the biological sample to be measured is calculated.
Further, the method also comprises that 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.
The method can effectively eliminate the interference of endogenous analytes contained in the blank matrix, and has obvious advantages compared with the traditional standard addition method, background deduction method, matrix replacement method and object replacement method. The method can be used for quantitative detection and analysis of various endogenous compounds in biological samples, effectively eliminates matrix effect, has no interference, small sampling amount and high accuracy, does not need additional data software to carry out regression, directly carries out sample injection on an instrument, directly can obtain a sample concentration result on the instrument software, does not need additional data processing software to process, and has simple and easy operation.
The invention also specifically discloses a method for measuring the concentration of coenzyme Q10 in blood plasma based on a liquid chromatography-mass spectrometry technology, which comprises the following steps: mixing serial coenzyme Q10 standard curve working solutions with blank plasma to obtain correction standard samples, preprocessing, and injecting into UPLC-MS/MS for analysis; adding an internal standard working solution into plasma to be tested, preprocessing, and then injecting into UPLC-MS/MS for analysis; firstly, using the concentration X1 of coenzyme Q10 added in a correction standard sample as an abscissa, using the chromatographic peak area ratio Y1 of the coenzyme Q10 and an internal standard as an ordinate, and using a weighted least square method to carry out linear regression on the concentration X1 of the coenzyme Q10 added in the correction standard sample and the peak area ratio Y1 to obtain a regression equation Y1=a+bX1, wherein the background value of blank plasma for preparing the correction standard sample is c= -a/b; and then the total concentration of the coenzyme Q10 in the correction standard sample is taken as an abscissa, the total concentration of the coenzyme Q10 = the background value of blank plasma for preparing the correction standard sample + the concentration of the added coenzyme Q10, the chromatographic peak area ratio Y2 of the analyte and the internal standard is taken as an ordinate, the weighted least square method is used for carrying out linear regression on the total concentration X2 of the coenzyme Q10 in the correction standard sample and the peak area ratio Y2, the regression equation Y2 = A + BX2 is obtained, namely a standard curve, and the concentration of the coenzyme Q10 in the plasma to be detected is calculated by the standard curve.
Compared with the alternative analyte method, the invention does not need to search for the alternative analyte which does not exist in the organism but has a constant proportion relation with the endogenous substances through verification, does not need a complex verification process, and can adopt a real matrix consistent with the biological sample without lower cost, thereby effectively eliminating the difference of matrix effect and recovery rate. Compared with the alternative matrix method, the analyte coenzyme Q10 has the problems that the light is unstable, the alternative matrix obtained by removing the coenzyme Q10 in the real matrix by using light is different from the matrix, the sample injection after sample treatment has internal standard fluctuation compared with the matrix analysis of the actual biological sample, the matrix effect and the extraction recovery rate are different, and the coenzyme Q10 is not applicable to the analysis of the coenzyme Q10 by using light. At the same time, 4% BSA is used as a substitute matrix, which is also a problem of low extraction recovery rate. Compared to standard addition methods, standard addition methods are not suitable for micro-scale and large-volume sample analysis because of the limitations of the sample volume (sample volume and sample number), the bioanalytical sample volume in practical experiments is only 100 microliters or less, and generally less large-volume sample volumes. 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 process linear regression and sample data, and after the background is deducted by using excel software or spss statistical analysis software, a regression equation is fitted, the sample concentration cannot be directly obtained from instrument software, the statistical analysis software additionally calculates, and the data processing analysis method is extremely complex. Further, the method further comprises the steps of respectively mixing a series of quality control working solutions with blank plasma to obtain quality control samples, preprocessing the quality control samples, injecting the quality control samples into UPLC-MS/MS for analysis, wherein 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 coenzyme Q10 added into the quality control samples.
Preparing a working solution: weighing two parts of coenzyme Q10 reference substances, respectively adding isopropanol to dissolve and dilute the two parts of coenzyme Q10 reference substances 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 a concentration;
Preparing an internal standard solution: weighing coenzyme Q10-d9, dissolving the coenzyme Q10-d9 into a coenzyme Q10-d9 stock solution by isopropanol, and diluting the stock solution by methanol to prepare an internal standard working solution.
The pretreatment mode is as follows: taking a plasma sample, adding an internal standard working solution, and adding isopropanol with the volume ratio of 9:1: ethyl acetate, vortex mixing; centrifuging, adding methanol into the supernatant, mixing, and placing into a sample tube.
Chromatographic conditions: mobile phase B:0.1% methanolic formate, elution mode: isocratic elution, flow rate 0.6mL min -1, sample injection amount 1 μL, chromatographic column ACQUITY UPLC-BEH C 18 column, column temperature 40 ℃, automatic sample injector cleaning solution: methanol: isopropyl alcohol: acetonitrile: the volume ratio of water is 1:1:1:1.
Mass spectrometry conditions: adopting an electrospray ion source, a positive ion mode and a scanning mode of multi-reaction monitoring (MRM); ion source parameters: capillary voltage 2.5kV, taper hole voltage 20V, ion source temperature 150 ℃, solvent removal temperature: 400 ℃,150 L.h -1 of taper hole air flow rate, 800 L.h -1 of desolvation air flow rate, 6.0Bar of atomization gas pressure, 0.15 mL.min -1 of collision air flow rate and 3.60min of data acquisition time; the quantitative ion pair of coenzyme Q10 is [ M+H ] + M/z 863.75- & gt 197.00, the collision energy is 40eV, and the quantitative ion pair of internal standard coenzyme Q10-d9 is [ M+H ] + M/z 872.70- & gt 205.90, and the collision energy is 40eV.
Compared with the prior art, the invention has the beneficial effects that:
1. The invention adopts the normal matrix to prepare standard curve 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 standard curve and quality control by using the principle of a standard addition method, and simultaneously uses the background and the concentration of the added endogenous reference substance to calculate the standard curve back to determine the concentration of the endogenous substances (coenzyme Q10) in the sample to be measured.
2. The invention researches and establishes a UPLC-MS/MS measuring method of the blood concentration of the endogenous substance coenzyme Q10 in the rat plasma, which has the advantages of good peak shape, high sensitivity, strong specificity, wide analysis range, good stability, short measuring period, simple and convenient operation and low cost.
3. The invention adopts isopropanol to dissolve methanol to dilute and prepare working solution, and plasma samples are subjected to isopropanol: ethyl acetate (9:1, v:v) protein precipitation treatment, using coenzyme Q10-d9 as an internal standard, using 0.1% methanolic formate solution as a mobile phase, performing isocratic elution, wherein the flow rate is 0.60 mL-min -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, no matrix effect exists, the acquisition time is short, and the method can provide a basis for in vivo pharmacokinetics research of the substance.
Drawings
FIG. 1 is a chromatogram of coenzyme Q10 and coenzyme Q10-d9 (A. Blank plasma B. Blank reagent C. Blank plasma with coenzyme Q10 control and internal standard coenzyme Q10-d 9D. Lavage plasma sample with internal standard treatment);
FIG. 2 graph of average drug time after gavage of rats with 5% coenzyme Q10 self-microemulsion and 10% coenzyme Q10 microparticles n＝6)。
Fig. 3 is a y2=a+bx2 standard curve.
Detailed Description
The following description of the embodiments of the present invention will be made more apparent and fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown. All other embodiments, which can be made by one of ordinary skill in the art without undue burden on the person of ordinary skill in the art based on embodiments of the present invention, are within the scope of the present invention.
The materials used in the invention are as follows:
animals: male SD rats, grade SPF, 12, weighing 180-220 g, offered by Experimental animal research center in Hubei province.
Reagent: isopropyl alcohol: chromatographic purity, merck, lot number: k53060340107; formic acid: chromatographic purity, aladine, lot 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 control, purity 99.1%, lot number: 1120070107,5% of coenzyme Q10 self-microemulsion and 10% of coenzyme Q10 microparticles, both supplied by Zhejiang New and Chengji Co., ltd; coenzyme Q10-d9 control, purity 98.2%, lot number: MBBD0885.
Instrument: waters ACQUITY UPLC I-Class ultra-high performance liquid chromatograph (Waters, USA), waters Xevo TQ-S triple quadrupole mass spectrometer, and UNIFI data processing software (Waters, USA) were provided.
The invention relates to a method for measuring the concentration of coenzyme Q10 in rat plasma by UPLC-MS/MS, which comprises the following steps:
1 test
1.1 Chromatography and Mass Spectrometry conditions
Chromatographic conditions: the column was an ACQUITY UPLC-BEH C 18 column (2.1 mm. Times.50 mm,1.7 μm), isocratic elution, mobile phase: 0.1% methanol formate, flow rate 0.6mL min -1, column oven temperature 40℃ sample volume 1. Mu.L.
Mass spectrometry conditions: ESI ion source, positive ion mode, multiple Reaction Monitoring (MRM) mode; ion source parameters: capillary voltage (CAPILLARY VOLTAGE): 2.5Kv; taper hole voltage (Sample Cone): 20V; ion source temperature (Source temperature): 150 ℃; desolvation temperature (Desolvation temperature): 400 ℃; taper hole gas flow rate (Cone gas flow): 150L/h; desolvation gas flow rate (Desolvation gas flow): 800L/h; atomization gas pressure (Nebulizer gas pressure): 6.0Bar; primary mass spectral collision energy (MS collision energy): 4eV; secondary mass spectral collision energy (MSMS collision energy): 40eV; collision gas flow rate (Collision gas flow): 0.15mL/min; the data acquisition time is 3.60min, the ion pair of the coenzyme Q10 and the coenzyme Q10-d9 is [ M+H ] +, the ion pair is M/z 863.75- & gt 197.00 and M/z 872.70- & gt 205.90 respectively, and the collision energy is 40eV.
1.2 Preparation of working solution
Preparing an analyte control working solution: a certain amount of coenzyme Q10 was weighed separately and placed in two brown glass bottles, and weights were recorded, labeled S01, S02, respectively. The real weight of the analyte is calculated according to the purity, the moisture, whether salt formation and the like of the coenzyme Q10, and a proper amount of isopropanol is added to prepare the final concentration of the stock solution of 0.60mg/mL, and vortex mixing is carried out. Taking a proper amount of coenzyme Q10 stock solution S01, and sequentially diluting with methanol to obtain a series of standard curve working solutions with the concentrations of 1.60, 3.20, 8.00, 16.0, 32.0, 64.0, 128 and 160 mug/mL; and 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 mug/mL.
Preparing an internal standard solution: 1mg of coenzyme Q10-d9 reference substance is weighed, and an appropriate volume of isopropanol is added to prepare an internal standard stock solution with the final concentration of 0.60 mg/mL. An appropriate amount of internal standard stock solution was precisely measured and diluted with methanol to give an internal standard working solution with a concentration of 1. Mu.g/mL.
1.3 Animal experiment method
12 Male Sprague Dawley rats. The cells were randomly divided into 2 groups of 6 cells. The rats were fasted for 12h (no water) prior to dosing and allowed free feeding after 4h of dosing. The first group was gavaged at a dose of 21mg/kg with 5% coenzyme Q10 from the microemulsion and the second group was gavaged at the same dose with 10% coenzyme Q10 particles. Taking blood from the jugular vein Cong Caixie 0.3.3 mL according to blood sampling time points of 0h, 1h, 2h, 4h, 6h, 8h, 12h, 24h, 36h and 48h, centrifuging for 10min under the condition that the anticoagulant is EDTA-K2 and 3000rpm/min, taking supernatant plasma, and preserving at-80+/-10 ℃.
1.4 Plasma sample treatment
40. Mu.L of plasma sample was taken, 25. Mu.L of internal standard working solution (1. Mu.g/mL) was added, followed by 400. Mu.L of isopropanol: the ethyl acetate (9:1, v:v) was subjected to protein precipitation treatment and vortexed. Centrifuging at 12000rpm in a centrifuge at 4deg.C for 10min; 100. Mu.L of the supernatant was mixed with 200. Mu.L of methanol, and then analyzed by UPLC-MS/MS.
1.5 Methodology investigation
Specialization: and (3) taking a blank reagent, a zero-concentration sample (only the blank sample with the internal standard is added), and analyzing the quantitative upper limit plasma sample without the internal standard after the step treatment so as to examine the influence degree of interference peaks in the blank reagent on the area integration of the analyte and the internal standard peak. Standard curve and lower limit of quantification: 10 mu L of a series of standard curve working solutions are mixed with 190 mu L of blank rat plasma to prepare standard curve plasma samples (correction standard samples) with the concentrations of 80, 160, 400, 800, 1600, 3200, 6400 and 8000ng/mL respectively, and the standard curve plasma samples are processed according to a plasma sample processing method and then analyzed. Firstly, using the concentration of coenzyme Q10 in the added calibration standard sample as an abscissa, using the chromatographic peak area ratio of the analyte (CoQ 10) and the internal standard (coenzyme Q10-d 9) as an ordinate, using a weighted (W=1/X 2) least square method to carry out linear regression on the concentration (X 1) of the added coenzyme Q10 control substance in the calibration standard sample and the peak area ratio (Y 1), The regression equation (Y 1＝a+bX1) obtained is, whereby the equation can be used to formulate the blank matrix for the standard curve with a background value of c= -a/b|, and then the total concentration of coenzyme Q10 in the calibration standard (background value of matrix for formulation of the standard curve + concentration of calibration standard) is used as abscissa, the chromatographic peak area ratio of analyte (CoQ 10) to internal standard (coenzyme Q10-d 9) is used as ordinate, the weighted (W=1/X 2) least square method is used to perform linear regression of the total concentration of coenzyme Q10 in the calibration standard (X2) to peak area ratio (Y 2), the regression equation (Y 2＝A+BX2) obtained is the standard curve. And the concentration of the coenzyme Q10 in the plasma of the rat to be detected is calculated back according to the concentration of the calibration standard sample. Precision and accuracy: the plasma of blank rats with the concentration of 80ng/mL, the low concentration and the quality control (LQC) with the concentration of 240ng/mL, the medium concentration and the quality control (MQC) with the concentration of 1200ng/mL and the low concentration and the quality control (HQC) with the concentration of 6000ng/mL are respectively obtained by mixing 10 mu L of the quality control working solution and 190 mu L of the plasma of blank rats. The quality control samples are processed according to a plasma sample processing mode and then are subjected to sample injection analysis, and in 3 precision and accuracy analysis batches processed in at least 2 days, 6 parallel samples are prepared for each concentration of the quality control samples in each analysis batch. to examine the precision and accuracy of the batch and the batch.
Extraction recovery rate: in one analysis batch, the peak areas after extraction of low, medium and high concentration quality control samples prepared from blank plasma are compared with the peak areas in pure solutions prepared from working solutions for preparing low, medium and high quality control samples and internal standard working solutions respectively added into the extracted blank plasma. The method comprises the following steps: coenzyme Q10 working solution and internal standard working solution I03 (1000 ng/mL) with the concentration of 4.80, 24.0 and 120 mug/mL are precisely sucked, and are diluted by methanol to prepare three complex solutions with the analyte concentration 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. The blank substrate is treated according to a plasma sample treatment method (an internal standard is replaced by 25 mu L of methanol), 200 mu L of methanol is not added in the last step, 3 parallel samples are prepared for each concentration, namely a recovery rate evaluation sample REC sample is obtained, and the recovery rate evaluation sample of each concentration is obtained by sampling to obtain the peak area (A1) of an analyte and the peak area (A2) of the internal standard. 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, wherein the analyte recovery rate is (A4-A3)/A1 x 100%; the recovery rate of the internal standard is A5/A2 which is 100 percent.
Matrix effect: after sample treatment, adding pure solution prepared from working solution for preparing low, medium and high quality control samples and internal standard working solution, preparing matrix effect evaluation sample (MER), preparing pure solution control sample (MEP) with the same concentration and without matrix extract, and evaluating the influence of matrix effect on accurate quantitative analysis of analytes by calculating internal standard normalized matrix factors in each sample. The method comprises the following steps: the coenzyme Q10 working solution and the internal standard working solution I03 (1000 ng/mL) with the concentration of 4.80, 24.0 and 120 mug/mL are precisely sucked, and diluted by methanol to prepare three complex solutions with the analyte concentrations of 10.32, 51.61 and 258.06ng/mL and the internal standard concentrations of 26.88ng/mL respectively. The substrate-containing samples were treated with 6 blank substrates of different sources according to the plasma sample treatment method (the internal standard was replaced with 25. Mu.L of methanol), and 200. Mu.L of methanol was not added in the final step, but 200. Mu.L of each of the above three complex solutions was added, and 1 parallel was prepared with 6 substrates of different sources for each concentration. The blank reagent was taken without the matrix sample and treated as a plasma sample (the internal standard was replaced with 25 μl of methanol), and 200 μl of methanol was not added in the last step, but 200 μl of each of the three complex solutions was added, and 3 replicates were prepared for each concentration. Evaluation of matrix effects was accomplished by calculation of matrix factors for the analyte and internal standard. The matrix factors for the analyte and internal standard are calculated separately by calculating the ratio of the peak area in the presence of the matrix to the corresponding peak area without matrix. The calculation formula of the matrix factor is as follows:
The calculation formula of the internal standard normalized matrix factor is as follows:
The normalized matrix factor is calculated and if its value is closer to 1, it is stated that the matrix effect of the internal standard and the analyte is closer, and the possible matrix effect of the analyte in any sample can be compensated, the matrix effect does not affect 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 is examined respectively, wherein the sample is stored for 27 hours at room temperature, the sample injector is stored for 8 days at 80 ℃ after pretreatment, and the sample injector is stored for 77 days and repeatedly frozen and thawed for 5 times.
2 Results
2.1 Methodological verification
Specialization: the retention times of coenzyme Q10 and internal standard coenzyme Q10-d9 were 3.02min and 2.98min, respectively, and since coenzyme Q10 is an endogenous compound, it contained a certain amount of coenzyme Q10 in the blank plasma. The coenzyme Q10 and the internal standard Q10-d9 are not detected in the blank reagent sample, which shows that the blank reagent has no interference to the analyte and the internal standard, and the coenzyme Q10 is not detected in the zero concentration sample (CTL-0), which shows that the internal standard Q10-d9 has no interference to the peak of the analyte coenzyme Q10; no coenzyme Q10-d9 was detected in the upper limit sample without the addition of the internal standard, which indicates that analyte coenzyme Q10 was not interfering with the peak of internal standard Q10-d9, and the results are shown in FIG. 1.
Standard curve and lower limit of quantification: the accuracy deviation of the calculated concentration and the marked concentration of at least 6 correction standard samples is not more than +/-15 percent (LLOQ is not more than +/-20 percent) and the correlation coefficient R2 of each standard curve is more than or equal to 0.990261. The standard curve shows good linearity in the range of 80.0-8000 ng/mL. The results are shown in FIG. 3.
Precision and accuracy: the average accuracy deviation range of LQC, MQC, HQC quality control samples in batch/batch is-2.5% -11.0%/0.1% -8.4%, and the precision is 8.7% and 6.1% respectively; the average accuracy deviation in the batch of the quantitative lower limit quality control sample (LLOQ QC) is 3.3% -13.3%, the accuracy deviation between batches is 7.7%, the maximum value of the precision between batches is 3.3% and 5.9%, the deviation of the quality control samples with the concentrations is not more than +/-15%, and the RSD is not more than 15%. The method shows that the precision and accuracy of the quality control samples with various concentrations are good in batch and batch-to-batch. The results are shown in Table 1.
TABLE 1 results of investigation of precision and accuracy of coenzyme Q10 within and between batches in rat plasma
Extraction recovery rate: the average extraction recovery rate of coenzyme Q10 in the low, medium and high concentration quality control samples is 88.3%, the RSD is 8.1%, the average extraction recovery rate of the internal standard is 96.0%, and the RSD is 3.7%. The detailed results are shown in Table 2, and the average recovery rate of each concentration quality control sample does not have a concentration-related trend. The results indicate that the recovery of analyte and internal standard does not affect the accuracy of the quantitative analysis.
Matrix effect: the medium factor mean/Relative Standard Deviation (RSD) of the low, medium and high concentrations of analyte calculated for the 6 different source matrices was 0.997/1.3%, 0.995/0.8% and 0.996/0.01%, respectively, normalized by the internal standard. The detailed results are shown in Table 2, where the relative standard deviation of internal standard normalized matrix factors in 6 matrices of different sources is no more than 15%, indicating that the matrix effect does not affect the quantitative analysis of the analytes.
TABLE 2 recovery and matrix Effect of coenzyme Q10 in rat plasman＝6)
Stability: the results are shown in Table 3, and the samples are stable under the above conditions and meet the analysis and measurement requirements.
Table 3 stability study table-3 stability of coenzyme Q10 in rat plasma (n=6)
2.2 Methodological applications
Average blood concentration-time curves of rats after gavage with 5% coenzyme Q10 from microemulsion and 10% coenzyme Q10 microparticles, respectively, are shown in FIG. 2. The results of the main parameters of the pharmacokinetics are shown in Table 4, and the oral relative bioavailability of 5% of coenzyme Q10 from the microemulsion relative to 10% of coenzyme Q10 particles is calculated to be 127.16% according to the parameters of the pharmacokinetics. The results show that the method can be applied to the research on the pharmacokinetics of coenzyme Q10 in rats and the research on the relative bioavailability of different preparations (such as self-microemulsion and microparticles).
TABLE 4 pharmacokinetic parameters after lavage of 5% coenzyme Q10 from microemulsion and 10% coenzyme Q10 microparticles in ratsn＝6)
The UPLC-MS/MS method for determining the blood concentration of the coenzyme Q10 in the rat plasma, which is established by the invention, has the advantages of high sensitivity, strong specificity, wide analysis range, good stability, short determination period, rapidness, convenience and convenience, can accurately quantify the concentration of the coenzyme Q10 in the complex biological matrix by passing each index requirement verified by methodology, has the advantages of small volume of the plasma for detection, high recovery rate, no matrix effect, wide incoming line range, simple operation and short acquisition time, can provide a basis for the in vivo pharmacokinetics study of the substance, and is favorable for popularization of clinical high-throughput detection. And simultaneously, the same set of standard curve samples are treated to obtain the concentration of endogenous coenzyme Q10 in the matrix, so that 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 large lipid solubility, and the endogenous compound is difficult to obtain a blank matrix and interfere with endogenous or other sources, so that an actual sample cannot be simulated without the blank matrix, and various risks (such as different recovery rates, matrix effects and the like) may lead to unreliable detection results. The four common methods for detecting the endogenous compounds have respective defects, and the invention adopts a unique method to better overcome the defects of the four conventional methods for detecting the endogenous compounds and solve the problem of endogenous substance quantification. ② The concentration of coenzyme Q10 in blood plasma is generally lower, the sensitivity requirement on a high performance liquid chromatography tandem mass spectrometry method is higher, the lower limit of quantification of the method is 80ng/mL, and the clinical treatment of the assistance coenzyme Q10 is realized. ② The invention adopts isopropanol to dissolve and methanol to dilute and prepare working solution, and adopts isopropanol: the ethyl acetate is used as a pretreatment mode of the plasma sample, so that the complexity of quantitative measurement of a clinically unknown sample is remarkably reduced, high-flux quantitative analysis of biological samples can be realized, and meanwhile, possible systematic errors are reduced to the greatest extent. ③ Conventionally, the sensitivity and accuracy of detection can be improved due to the fact that the sample injection amount is large, the sample injection amount is small and is greatly influenced by noise, but in a pharmacokinetic test, a mouse is adopted for the test, a blood sampling sample is very limited, the required sample amount is small and only 40 microliters, the sample injection amount is only 1uL, the detection accuracy can be ensured, and even a clinical invasive blood sampling link of a rat is reduced to the greatest extent. ④ The volume ratio of the invention is 1:1: methanol 1:1: isopropyl alcohol: acetonitrile: the water is used as the cleaning solution of the automatic sampler, so that the residual effect can be effectively eliminated.
In addition, the inventors treated the plasma sample by conventional methods in the process of establishing the method of the present invention, found that the conventional methods have various problems, and cannot meet the requirements of high performance liquid chromatography-mass spectrometry for detecting the concentration of coenzyme Q10, and the following are listed briefly:
MD1: after adding 25. Mu.L of internal standard into 40. Mu.L of plasma, 200. Mu.L of ACN and 600. Mu.L of n-hexane are added, 400. Mu.L of upper layer nitrogen is taken for drying, 200. Mu.L of 10% isopropanol acetonitrile solution is added for redissolution and then sample injection is carried out.
The problems are that: the extraction recovery rate is 13-18.9% and the recovery rate is low.
MD2: to 40 μl of plasma was added 25 μl of internal standard followed by 200 μl of methanol: precipitating isopropanol (1:1, v:v), centrifuging, taking 100 μl of supernatant, adding 100 μl of methanol, mixing, and introducing sample;
the problems are that: the extraction recovery rate is about 20%, and the recovery rate is low.
MD3: adding 25 mu L of internal standard into 40 mu L of plasma, adding 200 mu L of methanol and 600 mu L of n-hexane, taking 200 mu L of upper nitrogen gas for drying, adding 200 mu L of methanol solution for redissolution, and then injecting.
There are problems: the recovery rate is between 30% and 49.5%, and the recovery rate is low.
MD4: after adding 25. Mu.L of internal standard to 40. Mu.L of plasma, 600. Mu.L of n-hexane was added, 200. Mu.L of upper nitrogen was blown dry, and 200. Mu.L of isopropanol was added: and (3) re-dissolving the methanol (1:9, v:v) solution, and then injecting the sample.
There are problems: the internal standard fluctuates greatly in the actual sample, and there is a matrix effect.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein 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 is characterized by comprising the steps of quantitatively analyzing, namely adding an endogenous reference substance into a real matrix containing the endogenous substance to prepare a correction standard sample, firstly using the concentration X1 of the endogenous reference substance in the added correction standard sample as an abscissa, using the chromatographic peak area ratio Y1 of an analyte and an internal standard as an ordinate, and using a weighted least square method to carry out linear regression on the concentration X1 of the endogenous reference substance in the correction standard sample and the peak area ratio Y1, so as to obtain a regression equation Y1=a+bx1, wherein the regression equation obtains a background value of the real matrix for preparing a correction standard curve as c= -a/b; and then the total concentration X2 of the endogenous substances in the calibration standard sample is taken as an abscissa, the total concentration of the endogenous substances = the background value of the real matrix for preparing the calibration standard sample curve + the concentration of the added endogenous reference substance, the chromatographic peak area ratio Y2 of the analyte and the internal standard is taken as an ordinate, the weighted least square method is used for carrying out linear regression on the total concentration X2 of the endogenous substances in the calibration standard sample and the peak area ratio Y2, and the regression equation Y2 = A + BX2 is taken as a standard curve, so that the concentration of the endogenous substances in the biological sample to be measured is calculated.
2. The method of claim 1, further comprising calculating the accuracy of the quality control sample by adding the sum of the background concentration of the real substrate for preparing the quality control sample and the concentration of the endogenous reference substance added to the quality control sample to the theoretical concentration value of the quality control sample.
3. A method for determining the concentration of coenzyme Q10 in plasma based on a liquid chromatography-mass spectrometry technique, comprising: mixing serial coenzyme Q10 standard curve working solutions with blank plasma to obtain correction standard samples, preprocessing, and injecting into a liquid phase mass spectrometer for analysis; adding an internal standard working solution into plasma to be detected, preprocessing, and injecting the plasma to be detected into a liquid phase mass spectrometer for analysis; firstly, using the concentration X1 of coenzyme Q10 added in a correction standard sample as an abscissa, using the chromatographic peak area ratio Y1 of the coenzyme Q10 and an internal standard as an ordinate, and using a weighted least square method to carry out linear regression on the concentration X1 of the coenzyme Q10 added in the correction standard sample and the peak area ratio Y1 to obtain a regression equation Y1=a+bX1, wherein the background value of blank plasma for preparing the correction standard sample is c= -a/b; and then the total concentration of the coenzyme Q10 in the correction standard sample is taken as an abscissa, the total concentration of the coenzyme Q10 = the background value of blank plasma for preparing the correction standard sample + the concentration of the added coenzyme Q10, the chromatographic peak area ratio Y2 of the analyte and the internal standard is taken as an ordinate, the weighted least square method is used for carrying out linear regression on the total concentration X2 of the coenzyme Q10 in the correction standard sample and the peak area ratio Y2, the regression equation Y2 = A + BX2 is obtained, namely a standard curve, and the concentration of the coenzyme Q10 in the plasma to be detected is calculated by the standard curve.
4. The method for determining the concentration of coenzyme Q10 in plasma based on the liquid chromatography-mass spectrometry technique according to claim 3,
Preparing a working solution: weighing two parts of coenzyme Q10 reference substances, respectively adding isopropanol to dissolve and dilute the two parts of coenzyme Q10 reference substances 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 a concentration;
Preparing an internal standard solution: weighing coenzyme Q10-d9, dissolving the coenzyme Q10-d9 into a coenzyme Q10-d9 stock solution by isopropanol, and diluting the stock solution by methanol to prepare an internal standard working solution.
5. The method for determining the concentration of coenzyme Q10 in blood plasma based on the liquid chromatography-mass spectrometry technique according to claim 3, wherein the pretreatment mode is as follows: taking a plasma sample, adding an internal standard working solution, and adding isopropanol with the volume ratio of 9:1: ethyl acetate, vortex mixing; centrifuging, adding methanol into the supernatant, mixing, and placing into a sample tube.
6. The method for determining the concentration of coenzyme Q10 in plasma based on the liquid chromatography-mass spectrometry technique according to claim 3, wherein the chromatographic conditions are as follows: mobile phase B:0.1% methanolic formate, elution mode: isocratic elution, flow rate 0.6mL min -1, sample injection amount 1. Mu.L.
7. The method for measuring the concentration of coenzyme Q10 in blood plasma based on the liquid chromatography-mass spectrometry according to claim 4, wherein the series of quality control working solutions are respectively mixed with blank blood plasma to obtain quality control samples, the quality control samples are pretreated and then are injected into a liquid phase mass spectrometer for analysis, and the theoretical concentration value of coenzyme Q10 in the quality control samples is the sum of the background concentration of the blank blood plasma of the quality control samples and the concentration of coenzyme Q10 added into the quality control samples.
8. The method of determining the concentration of coenzyme Q10 in plasma based on the liquid chromatography-mass spectrometry technique according to claim 6, wherein the chromatographic conditions further comprise: the chromatographic column is an ACQUITY UPLC-BEH C 18 column, the column temperature is 40 ℃, and the solution is washed by an automatic sampler: methanol: isopropyl alcohol: acetonitrile: the volume ratio of water is 1:1:1:1.
9. The method for determining the concentration of coenzyme Q10 in plasma based on the liquid chromatography-mass spectrometry technique according to claim 3, wherein the mass spectrometry conditions are as follows: adopting an electrospray ion source, a positive ion mode and a scanning mode of multi-reaction monitoring (MRM); ion source parameters: capillary voltage 2.5kV, taper hole voltage 20V, ion source temperature 150 ℃, solvent removal temperature: 400 ℃, 150 L.h -1 of taper hole air flow rate, 800 L.h -1 of desolvation air flow rate, 6.0Bar of atomization air pressure, 0.15 mL.min -1 of collision air flow rate and 3.60min of data acquisition time.
10. The method for measuring the concentration of coenzyme Q10 in blood plasma based on the liquid chromatography-mass spectrometry according to claim 9, wherein the quantitative ion pair of coenzyme Q10 is [ M+H ] + M/z 863.75.fwdarw. 197.00, the collision energy is 40eV, and the quantitative ion pair of internal standard coenzyme Q10-d9 is [ M+H ] + M/z 872.70.fwdarw. 205.90, and the collision energy is 40eV.
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