Detection method and application of coenzyme Q10

Abstract

The invention provides a detection method and application of coenzyme Q10, and relates to the technical field of medicine detection. The detection method comprises the following steps: detecting a to-be-detected sample by adopting two-dimensional liquid chromatography; the two-dimensional liquid chromatography detection comprises the following steps: after sample introduction, separating a sample in a one-dimensional chromatographic column by using a one-dimensional mobile phase; abandoning the one-dimensional eluent until the coenzyme Q10 appears a peak, switching a flow path, and transferring the one-dimensional eluent to a two-dimensional chromatographic column; simultaneously replacing the two-dimensional mobile phase to elute the one-dimensional chromatographic column and the two-dimensional chromatographic column, and detecting a two-dimensional eluent by using a detector; a one-dimensional chromatographic column adopts a C18 chromatographic column, and a one-dimensional mobile phase comprises water, methanol and ethanol; a two-dimensional chromatographic column adopts a C18 chromatographic column, and a two-dimensional mobile phase comprises methanol and ethanol. The detection method relieves the problems of high detection cost and / or high detection operation difficulty of the coenzyme Q10 in the prior art.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical detection, and particularly to a detection method and application of coenzyme Q10. Background Art

[0002] Coenzyme Q10 (Coenzyme Q10, abbreviated as CoQ10) is a vitamin-like substance present in the human body and is also known as ubiquinone. It plays an important role in the mitochondria of human cells and is involved in energy metabolism and antioxidant processes. Coenzyme Q10 can be used for cardiovascular diseases, neurodegenerative diseases, diabetes, and improving fertility. Monitoring the content of coenzyme Q10 in subjects, understanding the content of endogenous coenzyme Q10 in subjects, or the metabolism of exogenously supplemented coenzyme Q10 in subjects helps to study mitochondrial function, cellular antioxidant capacity, and the mechanism of action of coenzyme Q10 in alleviating diseases.

[0003] The existing detection methods of coenzyme Q10 mainly include: (1) High-performance liquid chromatography-ultraviolet absorption method. This method is widely used, but has low sensitivity and cannot inject large volumes of samples. Therefore, liquid-liquid extraction is usually required to concentrate the samples, and the sample pretreatment process is relatively cumbersome; the sample matrix is complex and there are interference problems. (2) Liquid chromatography-tandem mass spectrometry method. This method has high sensitivity and strong specificity, but the instruments and isotope internal standards are expensive, and the maintenance cost is high, and high requirements are imposed on the operators. (3) High-performance liquid chromatography-electrochemical method. This method has high sensitivity, but the lipid substances in plasma will passivate the electrodes of the electrochemical detector and shorten the service life, resulting in an increase in maintenance costs. Therefore, how to reduce the detection cost of coenzyme Q10 and the operation difficulty of detection is an issue to be solved at present.

[0004] In view of this, the present invention is specifically proposed. Summary of the Invention

[0005] The object of the present invention is to provide a detection method and application of coenzyme Q10 to alleviate the problems of high detection cost and / or high detection operation difficulty of coenzyme Q10 in the prior art.

[0006] To solve the above technical problems, the present invention specifically adopts the following technical solutions: In the first aspect, a detection method of coenzyme Q10 is provided, and the detection method includes detecting a sample to be tested by using two-dimensional liquid chromatography; The two-dimensional liquid chromatography detection includes, after injection, the sample is first separated using a one-dimensional mobile phase on a one-dimensional chromatographic column; the one-dimensional eluate is discarded and the flow path is switched before the peak of coenzyme Q10 appears, so that the one-dimensional eluate is transferred to a two-dimensional chromatographic column; at the same time, the two-dimensional mobile phase is replaced to elute the one-dimensional chromatographic column and the two-dimensional chromatographic column, and the two-dimensional eluate is detected using a detector; The one-dimensional chromatographic column uses a C18 chromatographic column, and the one-dimensional mobile phase includes water, methanol, and ethanol; the two-dimensional chromatographic column uses a C18 chromatographic column, and the two-dimensional mobile phase includes methanol and ethanol.

[0007] In a second aspect, there is provided an application of the method for detecting coenzyme Q10 described in the first aspect in the preparation of products for detecting the pharmacokinetics of coenzyme Q10; or an application in the preparation of products for detecting the content of endogenous coenzyme Q10 in a subject.

[0008] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses two-dimensional liquid chromatography to detect coenzyme Q10, including introducing the analyte into a one-dimensional system, performing preliminary separation on a one-dimensional chromatographic column, transferring the target substance to the two-dimensional system by the central cutting method, and further separating and detecting on a two-dimensional chromatographic column. The present invention selects a C18 chromatographic column as the one-dimensional chromatographic column, and selecting a C18 chromatographic column as the two-dimensional chromatographic column can achieve the purposes of impurity removal, enrichment, and separation, so that the target substance is not interfered. The detection method provided by the present invention has high sensitivity, strong specificity, low cost, and stable and reliable test results, and both the precision and accuracy can meet the requirements. In a preferred embodiment, the separation and detection of coenzyme Q10 and the flushing of the chromatographic column can be completed within 16 minutes. The accuracy and precision are relatively high, and it can be used for the quantitative analysis of coenzyme Q10, providing a reliable detection method for the concentration monitoring of coenzyme Q10.

[0009] The present invention using two-dimensional liquid chromatography to detect coenzyme Q10 also has the following advantages: The combination of the two chromatographic columns can greatly reduce the interference of drugs or endogenous substances on the analyte; The two-dimensional liquid chromatography can perform online impurity removal, online enrichment, online transfer, and online analysis and detection for all processes, with a higher degree of automation; The detection cost of two-dimensional liquid chromatography is low, and it does not require expensive instruments and isotope internal standards of liquid chromatography-tandem mass spectrometry, and the maintenance cost is low, reducing the detection cost; The operation of two-dimensional liquid chromatography is simple, and it does not require high professional requirements for operators as in liquid chromatography-tandem mass spectrometry. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0011] Figure 1 It is a schematic diagram of the flow path connection of the six-port valve at positions 1→6 in Example 1; Figure 2 It is a schematic diagram of the flow path connection of the six-port valve at positions 1→2 in Example 1; Figure 3 It is the typical spectrum of coenzyme Q10 in Example 2; Figure 4 It is the spectrum of coenzyme Q10 with three one-dimensional mobile phases in Example 4, where green is the first kind, blue is the second kind, and red is the third kind; Figure 5 It is the spectrum of coenzyme Q10 at different flow rates in the two-dimensional mobile phase in Example 4, where green is the flow rate of 0.5 mL / min, blue is the flow rate of 0.6 mL / min, and red is the flow rate of 0.7 mL / min. Detailed implementation manners

[0012] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0013] It should be noted that: In this article, if there is no special description, all the implementation manners and preferred implementation methods mentioned in this article can be combined with each other to form a new technical solution; all the technical features and preferred features mentioned in this article can be combined with each other to form a new technical solution; the various components or their preferred components involved can be combined with each other to form a new technical solution.

[0014] In this article, unless otherwise stated, each reaction or operation step can be carried out in sequence or not in sequence. Preferably, the reaction methods in this article are carried out in sequence.

[0015] In this article, "and / or" is used to indicate that either or both of the described situations may occur. For example, A and / or B includes (A and B) and (A or B).

[0016] In this article, unless otherwise stated, "optionally", "optional", "optional" or "optional" means that the subsequent described event or environment can but does not have to occur, and this description includes the occasions where the event or environment occurs or does not occur.

[0017] In this article, the term "comprising" or "including" means including the described elements, integers or steps, but does not exclude any other elements, integers or steps.

[0018] In this text, the expressions "separately independent", "each... independently selected from", "... separately independently selected from", and "... independently selected from" can be interchanged and should be understood in a broad sense. They refer to the range or options that each member in a group of variables or components can be independently selected, that is, the selection of each variable or component is independent and not affected by the selection of other variables or components.

[0019] Unless otherwise specified, the professional and scientific terms used in this text have the same meanings as those familiar to persons skilled in the art. In addition, any methods or materials similar or equivalent to the described content can also be applied to the present invention.

[0020] The present invention provides a method for detecting coenzyme Q10, and the detection method uses two-dimensional liquid chromatography to detect a sample to be tested.

[0021] Two-dimensional liquid chromatography (2D-LC) connects two chromatographic columns with different separation mechanisms to the first and second dimensions of the system respectively. The sample passes through the chromatographic column in the first dimension and is switched into the second-dimensional chromatographic column and detector after concentration, enrichment or cutting. Two-dimensional liquid chromatography usually uses two different separation mechanisms to analyze the sample, aiming to obtain different separations in the first and second dimensions, that is, using the different selectivities of the two chromatographic columns for the sample, or separating the sample according to different characteristics such as molecular size, isoelectric point, hydrophilicity, charge, special intermolecular interaction, etc., to perform different separations on complex mixtures. The present invention adopts the central cutting method to transfer the effluent in a specific time period in the one-dimensional chromatographic column to the two-dimensional chromatographic column for further separation, which specifically includes the following steps: the sample is first separated in the one-dimensional chromatographic column using the one-dimensional mobile phase; from the start of elution from the one-dimensional chromatographic column until before the peak of coenzyme Q10 appears, the one-dimensional eluent is discharged as waste from the chromatographic system, and the flow path is switched before the peak of coenzyme Q10 appears, so that the one-dimensional eluent is transferred to the two-dimensional chromatographic column, and at the same time, the two-dimensional mobile phase is replaced to elute the one-dimensional chromatographic column and the two-dimensional chromatographic column in turn, and the two-dimensional eluent is detected using a detector. In this text, the one-dimensional eluent refers to the eluent flowing out of the one-dimensional chromatographic column, and the two-dimensional eluent refers to the eluent flowing out of the two-dimensional chromatographic column.

[0022] The present invention does not limit the device for constructing the flow path, the flow path and the switching of the chromatographic column in the two-dimensional liquid chromatography detection. In some embodiments, the switching of the flow path and the chromatographic column is achieved through a six-port valve, and the schematic diagram is as Figure 1 and Figure 2 shown. Figure 1It shows the flow path connection mode before the flow path switching when the one-dimensional eluent does not enter the two-dimensional chromatographic column. In this mode, the six-way valve ports 1 and 6 are connected, ports 2 and 3 are connected, and ports 4 and 5 are connected. The analyte is introduced into the one-dimensional system through an autosampler. The one-dimensional mobile phase is introduced into the one-dimensional chromatographic column by a one-dimensional pump, and the auxiliary mobile phase is introduced into the one-dimensional chromatographic column by an auxiliary pump. The sample, one-dimensional mobile phase, and auxiliary mobile phase pass through ports 1 to 6 to the one-dimensional chromatographic column, and the one-dimensional eluent is discharged from the chromatographic system as waste liquid. When the flow path is switched before the peak of coenzyme Q10 appears, the flow path connection mode after switching is as shown in Figure 2 shown. In this mode, the six-way valve ports 1 and 2 are connected, ports 3 and 4 are connected, and ports 5 and 6 are connected. The two-dimensional mobile phase is introduced into the one-dimensional chromatographic column through ports 5 to 6. The one-dimensional eluent enters the two-dimensional chromatographic column through ports 3 to 4 and finally enters the detector for detection.

[0023] In the detection method of coenzyme Q10 provided by the present invention, the one-dimensional chromatographic column uses a C18 chromatographic column, and the one-dimensional mobile phase includes water, methanol, and ethanol; the two-dimensional chromatographic column uses a C18 chromatographic column, and the two-dimensional mobile phase includes methanol and ethanol.

[0024] In an optional embodiment, the specifications of the one-dimensional chromatographic column are: C18 chromatographic column, 4.6 mm×50 mm, packing particle size 5 μm. Preferably, it is a Chromai Lotus EC C2 chromatographic column with specifications of 4.6 mm×50 mm, 5 μm.

[0025] In an optional embodiment, the specifications of the two-dimensional chromatographic column are: C18 chromatographic column, 3.0 mm×100 mm, packing particle size 2.7 μm. Preferably, it is a Chromai Lotus AC C3 chromatographic column with specifications of 3.0 mm×100 mm, 2.7 μm.

[0026] In an optional embodiment, starting from the start of elution with the one-dimensional mobile phase, the one-dimensional mobile phase uses gradient elution, and the gradient elution program is as follows: At 0 min, pure water is 20~80% v / v (such as but not limited to 20, 30, 40, 50, 60, 70, or 80% v / v), methanol is 10~40% v / v (such as but not limited to 10, 15, 20, 25, 30, 35, or 40% v / v), ethanol is 10~40% v / v (such as but not limited to 10, 15, 20, 25, 30, 35, or 40% v / v), and the total of pure water, methanol, and ethanol is 100% v / v; 0.8 min, pure water is 20 - 80% v / v (such as but not limited to 20, 30, 40, 50, 60, 70 or 80% v / v), methanol is 10 - 40% v / v (such as but not limited to 10, 15, 20, 25, 30, 35 or 40% v / v), ethanol is 10 - 40% v / v (such as but not limited to 10, 15, 20, 25, 30, 35 or 40% v / v), and the total of pure water, methanol and ethanol is 100% v / v; 4.0 min, pure water is 20% v / v, methanol is 40% v / v, ethanol is 40% v / v; From 4.01 min to the replacement of the two - dimensional mobile - phase elution, pure water is 10% v / v, methanol is 45% v / v, ethanol is 45% v / v.

[0027] In an alternative embodiment, starting from the start of the elution with the one - dimensional mobile phase, the one - dimensional mobile phase uses gradient elution, and the gradient elution program is as follows: 0 min, pure water is 50% v / v, methanol is 25% v / v, ethanol is 25% v / v; 0.8 min, pure water is 50% v / v, methanol is 25% v / v, ethanol is 25% v / v; 4.0 min, pure water is 20% v / v, methanol is 40% v / v, ethanol is 40% v / v; From 4.01 min to the replacement of the two - dimensional mobile - phase elution, pure water is 10% v / v, methanol is 45% v / v, ethanol is 45% v / v.

[0028] In an alternative embodiment, starting from the start of the elution with the one - dimensional mobile phase, the one - dimensional mobile phase uses gradient elution, and the gradient elution program is as follows: 0 min, pure water is 80% v / v, methanol is 10% v / v, ethanol is 10% v / v; 0.8 min, pure water is 80% v / v, methanol is 10% v / v, ethanol is 10% v / v; 4.0 min, pure water is 20% v / v, methanol is 40% v / v, ethanol is 40% v / v; From 4.01 min to the replacement of the two - dimensional mobile - phase elution, pure water is 10% v / v, methanol is 45% v / v, ethanol is 45% v / v.

[0029] In an alternative embodiment, starting from the start of the elution with the one - dimensional mobile phase, the one - dimensional mobile phase uses gradient elution, and the gradient elution program is as follows: 0 min, pure water is 20% v / v, methanol is 40% v / v, ethanol is 40% v / v; 0.8 min, pure water is 20% v / v, methanol is 40% v / v, ethanol is 40% v / v; 4.0 min, pure water is 20% v / v, methanol is 40% v / v, ethanol is 40% v / v; From 4.01 min until the two - dimensional mobile phase is changed for elution, pure water is 10% v / v, methanol is 45% v / v, ethanol is 45% v / v.

[0030] In an alternative embodiment, the flow path is switched 10 - 20 s before the peak of coenzyme Q10 appears, so that the one - dimensional eluent is transferred to the two - dimensional chromatographic column.

[0031] In an alternative embodiment, starting from the start of elution with the one - dimensional mobile phase, the flow path is switched at 6.5 min, so that the one - dimensional eluent is transferred to the two - dimensional chromatographic column.

[0032] In an alternative embodiment, in order to facilitate the next injection analysis, the one - dimensional mobile phase must be restored to the proportion of the initial mobile phase at 0 min in advance. Therefore, starting from the start of elution with the one - dimensional mobile phase, the one - dimensional mobile phase gradient elution program is as follows: 0 min, pure water is 20 - 80% v / v, methanol is 10 - 40% v / v, ethanol is 10 - 40% v / v; 0.8 min, pure water is 20 - 80% v / v, methanol is 10 - 40% v / v, ethanol is 10 - 40% v / v; 4.0 min, pure water is 20% v / v, methanol is 40% v / v, ethanol is 40% v / v; 4.01 min, pure water is 10% v / v, methanol is 45% v / v, ethanol is 45% v / v; 9.0 min, pure water is 10% v / v, methanol is 45% v / v, ethanol is 45% v / v; 9.01 min, pure water is 20 - 80% v / v, methanol is 10 - 40% v / v, ethanol is 10 - 40% v / v; 13.0 min, pure water is 20 - 80% v / v, methanol is 10 - 40% v / v, ethanol is 10 - 40% v / v.

[0033] In an alternative embodiment, the flow rate of the one - dimensional eluent is 0.7 mL / min before 0.8 min, and the initial flow rate gradient increases to 1.5 mL / min from 0.8 - 4 min.

[0034] In an alternative embodiment, an auxiliary mobile phase is introduced into the one-dimensional chromatographic column at the beginning of the one-dimensional chromatographic column. Starting from the start of elution with the one-dimensional mobile phase, the auxiliary mobile phase is eluted within 0 to 0.8 minutes, and the auxiliary mobile phase includes water. The introduction of the auxiliary mobile phase can increase the injection volume. By using the large-volume injection function, it alleviates the problems of low sensitivity, inability to perform large-volume injection, and the need for liquid-liquid extraction to concentrate samples in the traditional high-performance liquid chromatography-ultraviolet absorption method. It can greatly improve the detection sensitivity and directly detect the sample to be tested without enrichment. At the same time, increasing the injection volume shortens the sample pretreatment procedure, eliminating operations such as extraction with organic reagents, nitrogen blowing dry, and reconstitution with organic solvents in the sample pretreatment method without liquid-liquid extraction, reducing the errors that may be caused by human factors and improving work efficiency.

[0035] In an alternative embodiment, the flow rate of the auxiliary mobile phase is 1 mL / min.

[0036] In an alternative embodiment, starting from the start of elution with the two-dimensional mobile phase, the two-dimensional mobile phase uses isocratic elution, with methanol at 40% v / v and ethanol at 60% v / v.

[0037] In an alternative embodiment, the flow rate of the two-dimensional mobile phase is 0.5 to 0.7 mL / min, such as but not limited to 0.5, 0.6, or 0.7 mL / min.

[0038] In an alternative embodiment, the detector in the detection method is an ultraviolet detector, and the detection wavelength is 275 nm.

[0039] In an alternative embodiment, the ultraviolet detector is a UV-VIS detector (Ultraviolet-Visible Detector).

[0040] In an alternative embodiment, the injection volume is 100 to 1000 μL, preferably 500 μL.

[0041] In an alternative embodiment, the column temperature of the one-dimensional chromatographic column is 35 to 45 °C, preferably 40 °C.

[0042] In an alternative embodiment, the column temperature of the two-dimensional chromatographic column is 35 to 45 °C, preferably 40 °C.

[0043] In an alternative embodiment, the detection method includes performing two-dimensional liquid chromatography detection after removing proteins from the sample to be tested.

[0044] In an alternative embodiment, a protein precipitant is used to remove proteins from the sample to be tested. By adopting a simple protein precipitation pretreatment method, a sample such as a blood sample (e.g., a plasma or serum sample) can be directly tested on a machine after pretreatment. This avoids the use of excessive organic reagents such as ethyl acetate and n-hexane in the high-performance liquid chromatography-ultraviolet absorption method.

[0045] In an alternative embodiment, the protein precipitant includes, but is not limited to, organic acids (e.g., trichloroacetic acid), inorganic salts (e.g., sulfates or zinc sulfate), or organic solvents (e.g., acetonitrile, acetone, methanol, ethanol, isopropanol, or n-propanol), and n-propanol is preferred.

[0046] In an alternative embodiment, removing proteins from the sample to be tested includes fully mixing the sample to be tested with a protein precipitant, centrifuging, and taking the supernatant for two-dimensional liquid chromatography detection.

[0047] In an alternative embodiment, the detection method for coenzyme Q10 further includes obtaining a chromatogram and calculating the content of coenzyme Q10 in the sample to be tested by the external standard method.

[0048] In an alternative embodiment, a standard curve is constructed by detecting coenzyme Q10 calibrators with known series concentrations. The concentrations of the coenzyme Q10 calibrators with series concentrations are distributed from 100 to 3000 ng / mL, and the concentration points are preferably set at 100 ng / mL, 300 ng / mL, 600 ng / mL, 1000 ng / mL, 2000 ng / mL, and 3000 ng / mL.

[0049] In an alternative embodiment, the matrix of the calibrator is blank plasma.

[0050] In an alternative embodiment, the detection method for coenzyme Q10 further includes detecting a quality control sample to evaluate the detection results.

[0051] In an alternative embodiment, the quality control sample includes one or more of a low-value quality control sample containing 300 ng / mL coenzyme Q10, a medium-value quality control sample containing 1000 ng / mL coenzyme Q10, and a high-value quality control sample containing 2000 ng / mL coenzyme Q10.

[0052] In an alternative embodiment, the matrix of the quality control sample is blank plasma.

[0053] The detection method of coenzyme Q10 provided by the present invention, the sources of samples include but are not limited to foods, drugs, biological products or samples isolated from organisms, such as blood isolated from organisms (such as whole blood, serum or plasma), body fluids (such as saliva, urine, cerebrospinal fluid, pleural effusion or peritoneal effusion), tissues or cells, etc. The organism can be a mammal or an animal model prepared by artificial intervention. For example, it can be but is not limited to mice, rats, guinea pigs, hamsters, rabbits, ferrets, cats, dogs, goats, sheep, cows, pigs, horses, monkeys or humans. It should be noted that the detection method of coenzyme Q10 provided by the present invention is for non-diagnostic and non-therapeutic purposes. Specifically, for example, for samples from the above-mentioned mammals, the detection method of the present invention is used to provide experimental results, which are further used for the pharmacokinetic study of coenzyme Q10; or to detect the content of coenzyme Q10 in foods or drugs to achieve quality control or impurity detection, etc. Knowing the content of coenzyme Q10 in the sample to be tested does not mean that the result can be directly directed to the diagnosis result of the disease. Therefore, the detection method of coenzyme Q10 provided by the present invention is for non-diagnostic and non-therapeutic purposes.

[0054] In the second aspect, there is provided an application of the detection method of coenzyme Q10 described in the first aspect in the preparation of a product for detecting the pharmacokinetics of coenzyme Q10; or an application in the preparation of a product for detecting the content of endogenous coenzyme Q10 in a subject.

[0055] In an optional embodiment, the application includes an application in the preparation of a product for detecting the pharmacokinetics of orally administered coenzyme Q10 in humans.

[0056] The present invention will be further illustrated by specific examples below. However, it should be understood that these examples are only for more detailed illustration and should not be construed as limiting the present invention in any form.

[0057] Example 1 This example provides a detection method of coenzyme Q10: 1. Reagents and instruments: (1) Mobile phase for one-dimensional separation: pure water, methanol and ethanol; (2) Mobile phase for two-dimensional separation: methanol and ethanol; (3) Auxiliary mobile phase: pure water; (4) Protein precipitant: n-propanol; (5) Calibrator solution: a solution of coenzyme Q10 with known series of concentrations prepared from blank plasma. The concentrations of coenzyme Q10 in each calibrator solution are as follows: the concentration of solution L1 is 100 ng / mL; the concentration of solution L2 is 300 ng / mL; the concentration of solution L3 is 600 ng / mL; the concentration of solution L4 is 1000 ng / mL; the concentration of solution L5 is 2000 ng / mL; the concentration of solution L6 is 3000 ng / mL; blank plasma solution L0.

[0058] (6)Quality control product solution: A coenzyme Q10 solution with a known concentration prepared from blank plasma. The concentrations of coenzyme Q10 in each quality control product solution are as follows: the low-concentration quality control product solution is 300 ng / mL; the medium-concentration quality control product solution is 1000 ng / mL; the high-concentration quality control product solution is 2000 ng / mL.

[0059] (7)Instruments: Chromai Voyager two-dimensional liquid chromatography system; Xiangyi H2050R high-speed centrifuge; DLAB MX-S vortex mixer; Eppendorf adjustable pipette; glassware, etc.

[0060] 2. Pretreatment: (1)Put all mobile phases into the corresponding pipelines, rinse and equilibrate the chromatographic column for testing.

[0061] (2)Take 300 μL of the calibration product solution respectively, add 900 μL of the protein precipitant, vortex for 1 - 2 min, centrifuge at high speed for 8 - 10 min, and take 900 μL of the supernatant, which can be directly injected into the instrument.

[0062] In the same way, take 300 μL of the quality control product solution, add 900 μL of the protein precipitant respectively, vortex for 1 - 2 min, centrifuge at high speed for 8 - 10 min, and take 900 μL of the supernatant, which can be directly injected into the instrument.

[0063] In the same way, take 300 μL of the sample to be tested, add 900 μL of the protein precipitant respectively, vortex for 1 - 2 min, centrifuge at high speed for 8 - 10 min, and take 900 μL of the supernatant, which can be directly injected into the instrument.

[0064] 3. Liquid chromatography conditions:

[0065] Example 2 Set the parameters of the liquid chromatography system according to the conditions of Example 1, and prepare each test solution, calibration product solution and quality control product solution.

[0066] (1)Establish a linear standard curve, test the sample to be tested, and the typical chromatogram is as Figure 3 shown, and the linear regression equation and linear correlation coefficient are shown in Table 1.

[0067] Table 1: Linear regression equation and linear correlation coefficient of coenzyme Q10

[0068] (2)Accuracy The accuracy of the method was evaluated by the spike recovery test. The standard substance with a concentration of 1000 ng / mL was added to the plasma samples, and the results were compared with the theoretical concentration. As shown in Table 2, the spike recoveries were in the range of 95.0% - 105.5%.

[0069] Table 2: Accuracy of Coenzyme Q10

[0070] (3)Precision The standard substance with a concentration of 3000 ng / mL was added to the plasma samples. After pretreatment, the samples were injected six times, and the precision of the peak areas was statistically analyzed. The test results are shown in Table 3. The CV of the peak areas was 0.6%, indicating good precision.

[0071] Table 3: Precision of Coenzyme Q10

[0072] Example 3 Some samples collected from the outpatient department of a certain hospital were tested using the method of Example 1, and the content test results are as follows.

[0073] Table 4: Test of Some Samples from the Outpatient Department of a Certain Hospital

[0074] Example 4 1) Experiments were conducted on different ratios of the initial aqueous phase and organic phase in the one-dimensional mobile phase of the detection method for coenzyme Q10 provided in Example 1, and the following three settings were made respectively. The other parameters were the same as those in Example 1:

[0075] The results are as Figure 4 , It can be seen that when the setting range of the initial aqueous phase ratio in the one-dimensional mobile phase is 20% - 80%, there is no significant impact on the two-dimensional results, and satisfactory peak shapes and retention times can be obtained. The second setting is preferred in the present invention.

[0076] 2) Experiments were conducted on different flow rates in the two-dimensional mobile phase of the detection method for coenzyme Q10 provided in Example 1, and the following three settings were made respectively: 0.5 mL / min, 0.6 mL / min, and 0.7 mL / min. The other parameters were the same as those in Example 1.

[0077] The results are as Figure 5 , It can be seen that within the range of 0.5 - 0.7 mL / min for the flow rate of the two-dimensional mobile phase, the peak shapes and retention times in two dimensions are different. As the flow rate increases, the peak width becomes narrower and the retention time becomes shorter. The flow rate of 0.7 mL / min is preferred in the present invention.

[0078] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A detection method for coenzyme Q10, characterized in that, It includes detecting the sample to be tested by two-dimensional liquid chromatography; The two-dimensional liquid chromatography detection includes, after injection, the sample is first separated by a one-dimensional mobile phase in a one-dimensional chromatographic column; discard the one-dimensional eluate and switch the flow path before the peak of coenzyme Q10 appears, so that the one-dimensional eluate is transferred to the two-dimensional chromatographic column; at the same time, replace the two-dimensional mobile phase to elute the one-dimensional chromatographic column and the two-dimensional chromatographic column, and use a detector to detect the two-dimensional eluate; The one-dimensional chromatographic column uses a C18 chromatographic column, and the one-dimensional mobile phase includes water, methanol and ethanol; the two-dimensional chromatographic column uses a C18 chromatographic column, and the two-dimensional mobile phase includes methanol and ethanol.

2. The detection method of coenzyme Q10 according to claim 1, characterized in that, The specifications of the one-dimensional chromatographic column are: C18 chromatographic column, 4.6 mm×50 mm, packing particle size 5 μm; and / or, the specifications of the two-dimensional chromatographic column are: C18 chromatographic column, 3.0 mm×100 mm, packing particle size 2.7 μm.

3. The detection method of coenzyme Q10 according to claim 1, wherein, Starting from the start of elution with the one-dimensional mobile phase, the one-dimensional mobile phase uses gradient elution, and the gradient elution program is as follows: At 0 min, pure water is 20~80%v / v, methanol is 10~40%v / v, ethanol is 10~40%v / v, and the total of pure water, methanol and ethanol is 100%v / v; At 0.8 min, pure water is 20~80%v / v, methanol is 10~40%v / v, ethanol is 10~40%v / v, and the total of pure water, methanol and ethanol is 100%v / v; At 4.0 min, pure water is 20%v / v, methanol is 40%v / v, ethanol is 40%v / v; From 4.01 min to the replacement of the two-dimensional mobile phase for elution, pure water is 10%v / v, methanol is 45%v / v, ethanol is 45%v / v; and / or, The flow rate of the one-dimensional eluate is 0.7 mL / min before 0.8 min, and the initial flow rate gradient increases to 1.5 mL / min from 0.8 to 4 min.

4. The detection method of coenzyme Q10 according to claim 3, characterized in that, Starting from the start of elution with the one-dimensional mobile phase, switch the flow path at 6.5 min to transfer the one-dimensional eluate to the two-dimensional chromatographic column.

5. The detection method of coenzyme Q10 according to claim 1, characterized in that, At the same time as the start of the one-dimensional chromatographic column, an auxiliary mobile phase is introduced into the one-dimensional chromatographic column. Starting from the start of elution with the one-dimensional mobile phase, the auxiliary mobile phase is eluted from 0 to 0.8 min, and the auxiliary mobile phase includes water; and / or; the flow rate of the auxiliary mobile phase is 1 mL / min.

6. The detection method of coenzyme Q10 according to claim 1, characterized in that, Starting from the start of elution with the two-dimensional mobile phase, the two-dimensional mobile phase uses isocratic elution, methanol is 40%v / v, ethanol is 60%v / v; and / or, the flow rate of the two-dimensional mobile phase is 0.5~0.7 mL / min.

7. The detection method of coenzyme Q10 according to claim 1, characterized in that The detector is an ultraviolet detector, and the detection wavelength is 275 nm; and / or, the injection volume is 100~1000 μL, and / or, the column temperatures of the one-dimensional chromatographic column and the two-dimensional chromatographic column are independently 35~45 °C.

8. The detection method of coenzyme Q10 according to any one of claims 1 to 7, characterized in that, It includes removing the protein in the sample to be tested and then performing two-dimensional liquid chromatography detection.

9. The detection method of coenzyme Q10 according to any one of claims 1 to 7, characterized in that, It also includes obtaining a chromatogram and calculating the content of coenzyme Q10 in the sample to be tested by the external standard method.

10. The application of the detection method of coenzyme Q10 according to any one of claims 1~9 in the preparation of products for detecting the pharmacokinetics of coenzyme Q10; or the application in the preparation of products for detecting the content of endogenous coenzyme Q10 in a subject.

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