Method for detecting oxidized coenzyme Q10 in serum by HPLC-MS (High Performance Liquid Chromatography-Mass Spectrometer) combination

Abstract

The invention relates to a method for detecting oxidized coenzyme Q10 in serum through HPLC-MS (High Performance Liquid Chromatography-Mass Spectrometry) combination, and relates to the technical field of oxidized coenzyme Q10 detection. The oxidized coenzyme Q10 is detected through HPLC-MS combination, the serum is subjected to treatment such as supercritical fluid extraction and the like, then sample introduction is performed, a specific mobile phase and elution conditions are set, and the content of the oxidized coenzyme Q10 in the serum is detected. The detection precision and accuracy of the oxidized coenzyme Q10 are improved, and the recovery rate of the oxidized coenzyme Q10 is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of oxidized coenzyme Q10 detection. Specifically, the present invention relates to a method for detecting oxidized coenzyme Q10 in serum by HPLC-MS coupling. Background Art

[0002] Coenzyme Q10 is the only lipophilic antioxidant that can be de novo synthesized by cells and has an enzymatic mechanism to regenerate its reduced form, ubiquinol. Dietary coenzyme Q10 is enzymatically reduced to lipophilic ubiquinol, and approximately 75% of the coenzyme Q10 detected in human serum exists in the reduced form of ubiquinol. Ubiquinol is the main form for coenzyme Q10 to exert antioxidant activity. It protects mitochondrial proteins and DNA from oxidative damage by inhibiting lipid peroxidation in biological membranes.

[0003] Coenzyme Q10 (CoQ10) or ubiquinone is well-known for its key role as an electron and proton carrier in mitochondrial bioenergetics; later studies have shown that it exists in other cell membranes and plasma, and its antioxidant effects have been extensively studied. These two functions form the basis for supporting the clinical indications of CoQ10. In addition, recent data indicate that coenzyme Q10 affects the expression of genes involved in human cell signaling, metabolism, and transport, and some of the effects of coenzyme Q10 supplementation may be due to this property. Coenzyme Q10 deficiency is associated with autosomal recessive mutations, mitochondrial diseases, and aging.

[0004] Many neurodegenerative diseases, diabetes, cancer, fibromyalgia, muscle, and cardiovascular diseases are associated with low CoQ10 levels. CoQ10 treatment does not cause serious adverse reactions in humans, and new formulations have been developed to increase the absorption and tissue distribution of CoQ10. Oral coenzyme Q10 treatment is a common mitochondrial energizer and antioxidant strategy in many diseases and may have significant benefits for symptoms.

[0005] However, the existing methods for detecting coenzyme Q10 in the prior art have disadvantages such as being vulnerable to matrix interference, being easily oxidized, having large result differences, insufficient sensitivity, and low throughput, thus limiting the accurate determination of coenzyme Q10 and the accurate health assessment of the coenzyme Q10 nutritional status in the human body.

[0006] Therefore, it is an urgent technical problem for those skilled in the art to develop an accurate, rapid, and high-precision method for determining coenzyme Q10. Summary of the Invention

[0007] The object of the present invention is to provide a method for accurately and rapidly detecting oxidized coenzyme Q10.

[0008] For the above purposes, on the one hand, the present invention provides a method for detecting oxidized coenzyme Q10 in serum by HPLC-MS. First, oxidized coenzyme Q10 in serum is separated from interfering components by high-performance liquid chromatography, and then the mass-to-charge ratios of oxidized coenzyme Q10 and its corresponding internal standard are detected by mass spectrometry. According to the internal standard method for quantitative analysis, the content of oxidized coenzyme Q10 is obtained;

[0009] Among them, the high-performance liquid chromatography method is gradient elution and separation of oxidized coenzyme Q10;

[0010] The high-performance liquid chromatography conditions are as follows:

[0011] Chromatographic column: reversed-phase chromatographic column;

[0012] Mobile phase: Mobile phase A includes ultrapure water containing 0.05% formic acid and 0.02% trifluoroacetic acid; Mobile phase B includes a mixture of methanol, acetonitrile, and isopropanol;

[0013] The gradient elution program is as follows:

[0014] Time Mobile Phase A Mobile Phase B 0 min 30％ 70％ 6 min 5％ 95％ 8 min 5％ 95％ 10 min 30％ 70％ .

[0015] In the above method, the volume ratio of methanol, acetonitrile, and isopropanol in mobile phase B is 60:30:10.

[0016] In the above method, the high-performance liquid chromatography conditions include:

[0017] Mobile phase flow rate: 0.4 - 0.5 mL / min;

[0018] Column temperature: 40 - 55 °C;

[0019] Injection volume: 1 - 20 μL.

[0020] Preferably, the mobile phase flow rate is 0.5 mL / min;

[0021] Preferably, the column temperature is 45 °C;

[0022] Preferably, the injection volume is 5 - 10 μL.

[0023] In the above method, the reversed-phase chromatographic column is a C18 alkylsilyl-bonded silica gel column;

[0024] The diameter of the reversed-phase chromatographic column is 2.1 mm, the length is 50 mm, and the particle size is 3 μm.

[0025] In the above method, the conditions of the mass spectrometry are:

[0026] Under the atmospheric pressure chemical ionization (APCI) mode, positive ion mode scanning was performed using multiple reaction monitoring (MRM); the spray current was 3 μA; the ion source temperature was 150 °C; the nebulizer gas temperature was 450 °C, the spray gas GAS1 was 45 psi, the collision gas CAD was 7 psi, and the curtain gas CUR was 25 psi.

[0027] In the above method, the serum was injected after being processed. The processing steps were as follows:

[0028] 1) Add an oxidant to the serum for oxidation, and then add a diluent for dilution to obtain the diluted serum;

[0029] 2) Add a precipitant to the diluted serum obtained in step 1), vortex to precipitate proteins, and take the supernatant after centrifugation;

[0030] 3) Dry the supernatant obtained in step 2), load it into a supercritical fluid extraction cell, set the pressure, time, and temperature, start the extraction, rinse the collection tube with the collection solvent, combine the extraction solutions, dry them, dissolve them with a mixed solution of methanol and acetonitrile, vortex, and filter to obtain the processed serum.

[0031] In the above method, in step 1), the diluent was an ethanol solution containing an internal standard, and the oxidant was a 1,4-benzoquinone ethanol solution; in step 2), the precipitant was perchloric acid.

[0032] In the above method, in step 3), the mobile phase for extraction was CO2, the flow rate was 2 - 4 mL / min, and the entrainer was methanol with a volume ratio of 5 - 7%.

[0033] In the above method, in step 3), the pressure for extraction was 25 - 30 MPa, the temperature was 40 - 50 °C, and the time was 20 - 30 min.

[0034] In the above method, in step 3), the collection solvent was a mixed solution of n-hexane and isopropanol with a volume ratio of 9:1.

[0035] In the above method, the preparation method of the internal standard ethanol solution was: weigh 1 mg of the isotopic internal standard oxidized coenzyme Q10-D6, add pure ethanol to completely dissolve it, and prepare an isotopic internal standard mother solution with a concentration of 0.1 mg / mL; then prepare an isotopic internal standard solution containing 1 μg / mL oxidized coenzyme Q10-D6 with pure ethanol; add 5 mL of ethanol and mix evenly to obtain the internal standard ethanol solution.

[0036] The second aspect of the present invention provides the use of any of the above methods, and the use is for the application in the preparation of a system for detecting oxidized coenzyme Q10 in serum samples.

[0037] As can be seen from the above technical solutions, the present invention uses the HPLC-MS method to determine oxidized coenzyme Q10 in human serum. The method has the characteristics of high sensitivity and strong specificity. Quantitative determination using the isotope internal standard method can greatly eliminate matrix effects and the interference of other components, is not affected by the sample treatment process, is not affected by the analyte, and can achieve accurate quantification. At the same time, the methods of liquid-liquid extraction and derivatization can use fewer blood samples to quickly and efficiently detect samples, and the treatment is convenient.

[0038] The present invention has investigated the spiked recovery rate of oxidized coenzyme Q10 in serum for three days, all of which are between 85% and 115%, meeting the requirements. At the same time, the accuracy and precision have been investigated. The reproducibility results of the method show that the intra-day precision, inter-day precision, and RSD of oxidized coenzyme Q10 in serum are less than 15%, and the accuracy is between 85% and 115%. The reproducibility of the method is good.

[0039] Compared with other LC-MS / MS methods, the method of the present invention has higher sensitivity, requires less sample volume, can complete the detection of oxidized coenzyme Q10 within a short time, and can be used for the detection of oxidized coenzyme Q10 in clinical serum.

[0040] The present invention pre-treats serum samples, improving the morphological controllability, anti-interference ability, and operation stability of coenzyme Q10. The oxidant promotes morphological stabilization, and the quinone ring structure of oxidized coenzyme Q10 is more easily ionized, increasing the mass spectrometry signal intensity. The perchloric acid precipitation of proteins has a high recovery rate, has no significant impact on the stability of oxidized coenzyme Q10 and the internal standard, reduces serum matrix interference, reduces chromatographic column contamination, and improves detection accuracy. Compared with traditional liquid-liquid extraction, supercritical fluid extraction can improve extraction selectivity, is suitable for the enrichment of the lipophilic target coenzyme Q10, reduces the use of organic solvents, and the limited extraction temperature and pressure are conducive to improving extraction efficiency and balancing the CO2 diffusion ability and the stability of thermosensitive targets.

[0041] Mobile phase A of the present invention adopts a dual-acid design. Formic acid can adjust the pH to be acidic, enhancing the retention of oxidized coenzyme Q10 on the reversed-phase chromatographic column; the strong ion pair effect of trifluoroacetic acid can inhibit the secondary interaction between the target and the stationary phase, reducing peak tailing, and trifluoroacetic acid avoids the negative impact on the mass spectrometry signal at low concentrations. There is a synergistic effect in the organic phase of mobile phase B. Methanol provides medium elution strength, balancing the retention time and separation efficiency; acetonitrile enhances the elution ability and improves the peak shape, reducing the column pressure; isopropanol is used as a strong elution solvent, enhancing the selectivity for oxidized coenzyme Q10, shortening the gradient time, and significantly improving the resolution for lipophilic interfering substances such as cholesterol esters. By limiting the ratio of the three, the separation of substances with different hydrophobicity differences is optimized.

[0042] In summary, the present invention has developed a method for detecting serum coenzyme Q10 by HPLC-MS. This method is not restricted by region and time, the samples are convenient for storage, and the method of the present invention has the advantages of high detection sensitivity and precision, good repeatability and stability, strong specificity, the ability to distinguish interference generated by other substances in the body and reduce the false positive rate and false negative rate, and low sample consumption. Moreover, samples such as serum can be injected for detection after simple pretreatment, with a short analysis time, fast speed, low analysis cost, and not affected by the oxidation of the analyte, and can be used for rapid large-scale and high-throughput detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 FIG. 6 is an HPLC chromatogram of oxidized coenzyme Q10 in a serum sample, where the retention time of 1.43 min is the peak of oxidized coenzyme Q10.

[0044] Figure 2 FIG. 10 is an HPLC chromatogram of oxidized coenzyme Q10-D6 in a serum sample, where the retention time of 1.42 min is the peak of oxidized coenzyme Q10-D6. DETAILED DESCRIPTION OF THE INVENTION

[0045] The embodiments of the present invention provide a method for detecting oxidized coenzyme Q10 in serum by high performance liquid chromatography coupled with mass spectrometry:

[0046] First, oxidized coenzyme Q10 in serum is separated from interfering components by high performance liquid chromatography, and then the mass-to-charge ratio of oxidized coenzyme Q10 and its corresponding internal standard is detected by mass spectrometry, and the content of oxidized coenzyme Q10 is obtained according to quantitative analysis by the internal standard method;

[0047] Among them, the high performance liquid chromatography method is gradient elution and separation of oxidized coenzyme Q10;

[0048] The high performance liquid chromatography conditions include the following:

[0049] Chromatographic column: reverse phase chromatographic column;

[0050] Mobile phase: Mobile phase A includes ultrapure water containing 0.05% formic acid and 0.02% trifluoroacetic acid; Mobile phase B includes a mixed solution of methanol, acetonitrile, and isopropanol;

[0051] The program of the gradient elution is as follows:

[0052] Table 1 High performance liquid chromatography gradient elution conditions

[0053] Time Mobile Phase A Mobile Phase B 0 min 30％ 70％ 6 min 5％ 95％ 8 min 5％ 95％ 10 min 30％ 70％ 。

[0054] The volume ratio of methanol, acetonitrile, and isopropanol in the mobile phase B is 60:30:10.

[0055] The chromatographic conditions of the high performance liquid chromatography include:

[0056] Mobile phase flow rate: 0.5 mL / min;

[0057] Column temperature: 45 °C;

[0058] Injection volume: 5 μL.

[0059] The reverse chromatographic column is a C18 alkylsilyl-bonded silica gel column;

[0060] The diameter of the reverse chromatographic column is 2.1 mm, the length is 50 mm, and the particle size is 3 μm.

[0061] Materials used in the present invention:

[0062] Sample: Human serum sample.

[0063] (1) Instruments: AB Sciex 4500MD triple quadrupole mass spectrometer (AB Sciex); LC-20ADXR liquid chromatography system (equipped with an auto sampler, SHIMADZU); H1850R high-speed bench centrifuge (Hunan Xiangyi); multi-tube vortex mixer (MTV-100, Hangzhou Ausheng); adjustable pipette (Eppendorf 0.5 - 10 μL, 10 - 100 μL, 100 - 1000 μL); measuring cylinder.

[0064] (2) Reagents and consumables: HPLC-grade methanol (Fisher, USA); HPLC-grade 1-propanol (Merck, USA); 1,4-benzoquinone (Maclean, China); ethanol (Maclean, China); chromatographic column: Luna Omega 3 μm C18, 2.1 * 50 mm, 3 μm (phenomenex).

[0065] (3) Standards: Oxidized coenzyme Q10 and its corresponding isotope internal standards oxidized coenzyme Q10 and oxidized coenzyme Q10-D6 were purchased from Cerllient Corporation.

[0066] (4) Quality control products: Blank serum containing oxidized coenzyme Q10, namely QC (LLOQ), QC (LQC), and QC (HQC).

[0067] Mass spectrometry method:

[0068] In the atmospheric pressure chemical ionization (APCI) mode, positive ion mode scanning was performed using multiple reaction monitoring (MRM); the spray current was 3 μA; the ion source temperature was 150 °C; the nebulizer gas temperature was 450 °C, the spray gas GAS1 was 45 psi, the collision gas CAD was 7 psi, and the curtain gas CUR was 25 psi; Coenzyme Q10 in oxidized form and its corresponding isotope internal standard compound were monitored simultaneously. The mass spectrometry acquisition parameters for each target analyte are shown in Table 2.

[0069] Table 2 Mass Spectrometry Parameters

[0070]

[0071] Among them: Conezyme-Q10 is Coenzyme Q102 in oxidized form;

[0072] Conezyme-Q10-IS is the isotope internal standard Coenzyme Q10-D6 of Conezyme-Q10 in oxidized form.

[0073] Example 1

[0074] A method for detecting Coenzyme Q10 in oxidized form in serum by HPLC-MS, the steps include:

[0075] Step 101: Preparation of standard products: Weigh 1 mg of Coenzyme Q10 standard product into a volumetric flask, add pure ethanol until completely dissolved, and prepare a standard product stock solution with a concentration of 1000 μg / mL; then dilute the standard product stock solution with pure ethanol to prepare a mixed standard WS07 solution (see Table 3 for details), and set aside.

[0076] Table 3 Preparation of Standard Solution WS07

[0077]

[0078] Step 102: Using WS07 as the first high-concentration point, and then gradually diluting it with blank instead of matrix to S07 (see Table 4 for details). The concentrations of each calibration curve point are listed in Table 4.

[0079] Table 4 Preparation and Concentration of Calibration Curve

[0080]

[0081] Step 103: Preparation of QC: Weigh 1 mg of Coenzyme Q10 standard product into a volumetric flask, add pure ethanol until completely dissolved, and prepare a standard product stock solution with a concentration of 1000 μg / mL; then dilute the standard product stock solution with pure ethanol to prepare a mixed standard WS07 solution (see Table 3 for details), and set aside.

[0082] Step 104: Replace the matrix with blank. Dilute WS07 according to Table 5 to prepare QC(SLLOQ), QC(SLQC), and QC(SHQC).

[0083] Table 5 Preparation and Concentration of QC Solutions

[0084]

[0085] Step 105: Preparation of the internal standard solution. Weigh 1 mg of isotopically labeled internal standard coenzyme Q10-D6 into a 10 mL brown volumetric flask, add absolute ethanol to completely dissolve it, and prepare an isotopically labeled internal standard stock solution with a concentration of 0.1 mg / mL. Then, dilute each of the above isotopically labeled internal standard stock solutions with absolute ethanol solution to prepare an isotopically labeled internal standard solution containing 1 μg / mL of coenzyme Q10-D6.

[0086] Step 106: Preparation of the ethanol solution containing the internal standard. Take 1000 μL of the above isotopically labeled internal standard solution, add 5 mL of ethanol, and mix well to obtain the ethanol solution containing the internal standard.

[0087] Step 107: Preparation of the oxidant. Weigh 10 mg of 1,4-benzoquinone into a volumetric flask, add 1 mL of ethanol to completely dissolve it, dilute it to 20 mL with ethanol, and mix well to obtain the oxidant.

[0088] Step 108: Sample treatment for the calibration curve. Take 50 μL of the calibration curve serum sample, add 50 μL of the oxidant and 50 μL of the ethanol solution containing the internal standard to the serum, vortex for 3 min, then add 20 μL of 20% perchloric acid solution, vortex for 30 s to precipitate proteins, centrifuge at 4°C and 12000 rpm for 5 min, take the supernatant, dry it, load it into the supercritical fluid extraction cell, set the pressure at 30 MPa, the temperature at 50°C, and the time at 30 min, start the extraction, the mobile phase is CO2, the flow rate is 4 mL / min, the entrainer is methanol with a volume ratio of 7%, rinse the collection tube with the collection solvent, the collection solvent is a mixture of n-hexane and isopropanol with a volume ratio of 9:1, combine the extraction solutions, dry them, dissolve them with a mixture of methanol and acetonitrile, vortex, and filter through a 0.22 μm filter membrane to obtain the treated serum.

[0089] Step 109: QC Sample Processing: Take 50 μL of QC serum sample, add 50 μL of oxidant and 50 μL of internal standard ethanol solution to the serum, vortex for 3 min, then add 20 μL of 20% perchloric acid solution, vortex for 30 s to precipitate proteins, centrifuge at 4°C and 12,000 rpm for 5 min, take the supernatant, dry it, load it into the supercritical fluid extraction cell, set the pressure at 30 MPa, the temperature at 50°C, and the time at 30 min, start the extraction, the mobile phase is CO2, the flow rate is 4 mL / min, the entrainer is methanol with a volume ratio of 7%, rinse the collection tube with the collection solvent, the collection solvent is a mixture of n-hexane and isopropanol with a volume ratio of 9:1, combine the extraction solutions, dry them, dissolve them with a mixture of methanol and acetonitrile, vortex, and filter through a 0.22 μm filter membrane to obtain the processed serum.

[0090] Step 110: Serum Sample Processing: Take 50 μL of serum sample, add 50 μL of oxidant and 50 μL of internal standard ethanol solution to the serum, vortex for 3 min, then add 20 μL of 20% perchloric acid solution, vortex for 30 s to precipitate proteins, centrifuge at 4°C and 12,000 rpm for 5 min, take the supernatant, dry it, load it into the supercritical fluid extraction cell, set the pressure at 30 MPa, the temperature at 50°C, and the time at 30 min, start the extraction, the mobile phase is CO2, the flow rate is 4 mL / min, the entrainer is methanol with a volume ratio of 7%, rinse the collection tube with the collection solvent, the collection solvent is a mixture of n-hexane and isopropanol with a volume ratio of 9:1, combine the extraction solutions, dry them, dissolve them with a mixture of methanol and acetonitrile, vortex, and filter through a 0.22 μm filter membrane to obtain the processed serum, and inject 5 μL for analysis.

[0091] Step 115: Calculate Results: Use Analyst 1.6.2 for data processing, generate a standard curve by linear regression weighted 1 / x or 1 / x2, where x is the spiked concentration of oxidized coenzyme Q10, and y is the ratio of the peak area of oxidized coenzyme Q10 to the internal standard peak signal. The results are shown in Figure 1 and Figure 2 .

[0092] Figure 1 is the HPLC chromatogram of oxidized coenzyme Q10 in the serum sample; Figure 2 is the HPLC chromatogram of oxidized coenzyme Q10-D6 in the serum sample. It can be seen from Figure 1 and Figure 2 that in the detection method, the peak shapes of the standard product of oxidized coenzyme Q10 and the serum sample are symmetrical, and there is no interference from impurity peaks, indicating that good detection can be obtained under these conditions.

[0093] Example 2: Plot the calibration curve

[0094] The isotope internal standard quantification method was adopted, and Analyst 1.6.2 was used for data processing. A standard curve was generated by linear regression weighted by 1 / x or 1 / x2, where x was the spiked concentration of oxidized coenzyme Q10, and y was the ratio of the peak area of oxidized coenzyme Q10 to the peak area of the internal standard. A calibration curve was established, and the concentration of the oxidized coenzyme Q10 analyte in the serum was calculated. The linear fitting equations of oxidized coenzyme Q10 within their respective concentration ranges showed good linearity, with a correlation coefficient above 0.997. See Table 6 for details.

[0095] Table 6 Linear Range of Oxidized Coenzyme Q10

[0096] Compound Linear Range (ng / mL) Linear Equation Correlation Coefficient 25-OHVD3 50-5000 y = 0.00331 * x - 0.0207 0.9991

[0097] Example 3 Investigation of Spike Recovery

[0098] One serum sample was randomly selected. One of them was not spiked with the standard, and the other two were spiked with QC standards at two concentrations. They were repeatedly processed and measured 6 times in the same steps. The concentration of oxidized coenzyme Q10 was quantitatively determined by the isotope internal standard method, and the recovery results were calculated. Three batches were repeated. See Table 7. The results showed that the spike recovery results of serum oxidized coenzyme Q10 were between 85% and 115%, all meeting the requirements.

[0099] Table 7 Spike Recovery Results of Serum Oxidized Coenzyme Q10 (unit: ng / mL)

[0100]

[0101] Example 4 Accuracy and Precision Test

[0102] Six batches of serum quality control samples were repeatedly processed within one day and for 3 days. The concentration of oxidized coenzyme Q10 was quantitatively determined by the isotope internal standard method. The within-batch and between-batch precision and accuracy were statistically analyzed for three consecutive days. The calculation results are shown in Table 8 and Table 9. The results showed that the within-batch / between-batch accuracy results were between 85% and 115%, and the RSD values were less than 15%, meeting the requirements.

[0103] Table 8 Within-Batch Precision and Accuracy (n = 6)

[0104]

[0105]

[0106] Table 9 Between-Batch Precision and Accuracy (n = 18)

[0107]

[0108] Comparative Example 1 used the method of Example 1, with the difference that when the serum was treated, no oxidant was added. The recovery rate of oxidized coenzyme Q10 was only 75.61%. It can be seen that the lack of oxidant led to unstable morphology of coenzyme Q10 and a significant decrease in the recovery rate.

[0109] Comparative Example 2 used the method of Example 1, with the difference that when the serum was treated, perchloric acid was not added or replaced with other precipitants, as shown in Table 10. It can be seen that the insufficient miscibility of methanol and acetonitrile with the internal standard led to a decrease in the recovery rate and an aggravation of the matrix effect. The recovery rate of perchloric acid for protein precipitation was significantly better than that of methanol and acetonitrile, which could reduce column contamination and extend the column life.

[0110] Table 10 Recovery rates of oxidized coenzyme Q10 under different precipitants

[0111] Group Recovery Rate % Without Perchloric Acid 55.48 Acetonitrile 83.12 Methanol 76.64

[0112] Comparative Example 3 used the method of Example 1, with the difference that when the serum was treated, supercritical fluid extraction was not performed in step 3), and it was replaced with: adding n-hexane or ethyl acetate or dichloromethane to the supernatant obtained in step 2), vortexing, centrifuging at 4°C and 12,000 rpm for 5 min, taking the supernatant, drying, redissolving the residue with a mixed solution of methanol and acetonitrile, vortexing for 3 min, and filtering through a 0.22 μm filter membrane to obtain the treated serum. As shown in Table 11. It can be seen that compared with traditional liquid-liquid extraction, supercritical fluid extraction can improve extraction selectivity, is suitable for the enrichment of the lipophilic target coenzyme Q10, and reduces the use of organic solvents.

[0113] Table 11 Recovery rates of oxidized coenzyme Q10 under different extractants

[0114] Group Recovery Rate % n-Hexane 83.49 Ethyl Acetate 80.71 Dichloromethane 72.35

[0115] Comparative Example 4 used the method of Example 1, with the difference in the mobile phase, including: Group 1: Phase A, 0.02% aqueous trifluoroacetic acid solution, Phase B, a mixed solution of methanol, acetonitrile, and isopropanol (volume ratio 60:30:10). Group 2: Phase A, an aqueous solution containing 0.05% formic acid and 0.02% trifluoroacetic acid, Phase B, a mixed solution of methanol and acetonitrile (volume ratio 60:40). Group 3: Phase A, 0.05% aqueous formic acid solution, Phase B, a mixed solution of methanol, acetonitrile, and isopropanol (volume ratio 60:30:10). The results are shown in Table 12. It can be seen that the mobile phase of the present invention reduced the retention time, and isopropanol significantly improved the separation of interfering substances such as cholesterol esters, avoided co-elution, reduced the use of a high proportion of acetonitrile, and was more environmentally friendly.

[0116] Table 12 Effects of different mobile phases

[0117] Group Retention Time min Resolution (vs Cholesterol Ester) Example 1 5.5 3.5 Group 1 6.2 2.3 Group 2 5.7 2.5 Group 3 5.9 2.9

[0118] The various embodiments in this specification are described in a progressive manner. Each embodiment focuses on the differences from other embodiments. For the same or similar parts among the various embodiments, reference may be made to each other.

[0119] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the broadest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for detecting oxidized coenzyme Q10 in serum by HPLC-MS, characterized in that: First, the oxidized coenzyme Q10 in the serum is separated from the interfering components by high performance liquid chromatography, and then the charge-to-mass ratio of the oxidized coenzyme Q10 and its corresponding internal standard is detected by mass spectrometry. The content of oxidized coenzyme Q10 is obtained by quantitative analysis based on the internal standard method. Wherein, the high performance liquid chromatography method is to perform gradient elution and separation on oxidized coenzyme Q10; The HPLC conditions include the following: Chromatographic column: reverse phase column; Mobile phase: Mobile phase A includes ultrapure water containing 0.05% formic acid and 0.02% trifluoroacetic acid; mobile phase B includes a mixture of methanol, acetonitrile, and isopropanol; The procedure of the gradient elution is: 。 2. The method for detecting oxidized coenzyme Q10 in serum by HPLC-MS according to claim 1, characterized in that: The volume ratio of methanol, acetonitrile and isopropanol in the mobile phase B is 60:30:

10.

3. The method for detecting oxidized coenzyme Q10 in serum by HPLC-MS according to claim 1, characterized in that: The chromatographic conditions of the high performance liquid chromatography include: Mobile phase flow rate: 0.4-0.5 mL / min; Column temperature: 40~55℃; Injection volume: 1~20μL.

4. The method for detecting oxidized coenzyme Q10 in serum by HPLC-MS according to claim 1, characterized in that: The reverse chromatographic column is a C18 alkylsilane bonded silica gel column; The reverse phase chromatography column has a diameter of 2.1 mm, a length of 50 mm, and a particle size of 3 μm.

5. The method for detecting oxidized coenzyme Q10 in serum by HPLC-MS according to claim 1, characterized in that: The conditions of the mass spectrometry are: In atmospheric pressure chemical ionization APCI mode, multiple reaction monitoring MRM was used for positive ion mode scanning; the spray current was 3 μA; the ion source temperature was 150°C; the nebulizer gas temperature was 450°C, the spray gas GAS1 was 45 psi, the collision gas CAD was 7 psi, and the curtain gas CUR was 25 psi.

6. The method for detecting oxidized coenzyme Q10 in serum by HPLC-MS according to claim 5, characterized in that: The serum is injected after being processed, and the processing steps are as follows: 1) adding an oxidant to the serum for oxidation, and then adding a diluent to dilute it to obtain diluted serum; 2) adding a precipitant to the diluted serum obtained in step 1), vortexing to precipitate protein, and collecting the supernatant after centrifugation; 3) drying the supernatant obtained in step 2), loading it into a supercritical fluid extraction cell, setting the pressure, time and temperature, starting the extraction, flushing the collection tube with a collection solvent, combining the extracts, drying them, dissolving them with a mixture of methanol and acetonitrile, vortex filtering, and obtaining the treated serum.

7. The method for detecting oxidized coenzyme Q10 in serum by HPLC-MS according to claim 6, characterized in that: In step 1), the diluent is an ethanol solution containing an internal standard, and the oxidant is a 1,4-p-benzoquinone ethanol solution; in step 2), the precipitant is perchloric acid.

8. The method for detecting oxidized coenzyme Q10 in serum by HPLC-MS according to claim 6, characterized in that: In step 3), the extraction pressure is 25-30 MPa, the temperature is 40-50° C., and the time is 20-30 min.

9. The method for detecting oxidized coenzyme Q10 in serum by HPLC-MS according to claim 7, characterized in that: The preparation method of the internal standard ethanol solution is as follows: weigh 1 mg of the isotope internal standard oxidized coenzyme Q10-D6, add pure ethanol to completely dissolve it, and prepare an isotope internal standard mother solution with a concentration of 0.1 mg / mL; then use pure ethanol to prepare an isotope internal standard solution containing 1 μg / mL oxidized coenzyme Q10-D6; add 5 mL of ethanol, mix well, and obtain the internal standard ethanol solution.

10. The use of the method according to any one of claims 1 to 9, characterized in that: The application is application in preparing a system for detecting oxidized coenzyme Q10 in serum samples.

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