Method for detecting coenzyme q10 and application thereof

Abstract

The application provides a coenzyme Q10 detection method and application thereof, and relates to the technical field of medical detection. The detection method comprises detecting a to-be-detected sample by using two-dimensional liquid chromatography. The two-dimensional liquid chromatography detection comprises the following steps: after sample injection, the sample is separated by using a one-dimensional mobile phase in a one-dimensional chromatographic column; before the coenzyme Q10 elution, the one-dimensional eluent is discarded, the flow path is switched, and the one-dimensional eluent is transferred to a two-dimensional chromatographic column; meanwhile, the two-dimensional mobile phase is changed to elute the one-dimensional chromatographic column and the two-dimensional chromatographic column, and a detector is used to detect the two-dimensional eluent; the one-dimensional chromatographic column is a C18 chromatographic column, the one-dimensional mobile phase comprises water, methanol and ethanol; the two-dimensional chromatographic column is a C18 chromatographic column, and the two-dimensional mobile phase comprises methanol and ethanol. The detection method can alleviate the problems of high detection cost and / or high detection operation difficulty of the coenzyme Q10 in the prior art.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical testing technology, and in particular to a method for detecting coenzyme Q10 and its application. Background Technology

[0002] Coenzyme Q10 (CoQ10 for short) is a vitamin-like substance found in the human body, also known as ubiquinone. It plays a crucial role in the mitochondria of human cells, participating in energy metabolism and antioxidant processes. Coenzyme Q10 can be used to treat cardiovascular diseases, neurodegenerative diseases, diabetes, and improve fertility. Monitoring the levels of coenzyme Q10 in subjects, understanding the levels of endogenous coenzyme Q10 or the metabolism of exogenously supplemented coenzyme Q10, helps in studying mitochondrial function, cellular antioxidant capacity, and the mechanism of action of coenzyme Q10 in alleviating symptoms.

[0003] The existing methods for detecting coenzyme Q10 mainly include: (1) High performance liquid chromatography-ultraviolet absorption method, which is widely used, but has low sensitivity and cannot be used for large-volume injection. Therefore, liquid-liquid extraction is usually required to concentrate the sample, and the sample pretreatment process is relatively complicated. The sample matrix is ​​complex and there is interference. (2) Liquid chromatography-tandem mass spectrometry method, which has high sensitivity and strong specificity, but the instrument and isotope internal standard are expensive, and the maintenance cost is high, and the requirements for operators are high. (3) High performance liquid chromatography-electrochemical method, which has high sensitivity, but the lipids in the plasma will passivate the electrochemical detector electrode and shorten its lifespan, resulting in increased maintenance costs. Therefore, how to reduce the detection cost of coenzyme Q10 and reduce the operation difficulty of detection is a problem that needs to be solved.

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

[0005] The purpose of this invention is to provide a method for detecting coenzyme Q10 and its application, so as to alleviate the problems of high detection cost and / or high difficulty of detection operation in the prior art.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0007] In a first aspect, a method for detecting coenzyme Q10 is provided, the method comprising detecting the sample to be tested using two-dimensional liquid chromatography;

[0008] The two-dimensional liquid chromatography detection includes the following steps: after injection, the sample is first separated on a one-dimensional column using a one-dimensional mobile phase; the one-dimensional eluent is discarded until the coenzyme Q10 peak is reached, then the flow path is switched to transfer the one-dimensional eluent to the two-dimensional column; at the same time, the two-dimensional mobile phase is replaced to elute both the one-dimensional and two-dimensional columns, and the two-dimensional eluent is detected using a detector.

[0009] The one-dimensional chromatographic column uses a C18 column, and the one-dimensional mobile phase includes water, methanol, and ethanol; the two-dimensional chromatographic column uses a C18 column, and the two-dimensional mobile phase includes methanol and ethanol.

[0010] Secondly, the method for detecting coenzyme Q10 described in the first aspect is provided for use in the preparation of products for detecting the pharmacokinetic properties of coenzyme Q10; or for use in the preparation of products for detecting the endogenous coenzyme Q10 content in subjects.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] This invention employs two-dimensional liquid chromatography (HPLC) for the detection of coenzyme Q10. The method includes introducing the analyte into a one-dimensional system for preliminary separation on a one-dimensional column, transferring the target analyte to a two-dimensional system using a center-cutting method, and further separation and detection on a two-dimensional column. This invention uses a C18 column as the one-dimensional column; using a C18 column as the two-dimensional column effectively removes impurities, enriches, and separates the analyte, ensuring it remains unaffected by interference. The detection method provided by this invention exhibits high sensitivity, strong specificity, low cost, and stable and reliable test results, with both precision and accuracy meeting requirements. In a preferred embodiment, the separation, detection, and column rinsing of coenzyme Q10 can be completed within 16 minutes. With high accuracy and precision, it can be used for the quantitative analysis of coenzyme Q10, providing a reliable detection method for monitoring coenzyme Q10 concentration.

[0013] The present invention, employing two-dimensional liquid chromatography (2D-LC) for the detection of coenzyme Q10, offers the following advantages: The use of two chromatographic columns can significantly reduce interference from drugs or endogenous substances on the analyte; 2D-LC allows for online impurity removal, online enrichment, online transfer, and online analysis, resulting in a higher degree of automation; 2D-LC has low detection costs, eliminating the need for expensive instruments and isotopic internal standards required by LC-tandem mass spectrometry (LC-MS / MS), thus reducing maintenance costs and overall detection expenses; 2D-LC is simple to operate, unlike LC-MS / MS which requires highly specialized personnel. Attached Figure Description

[0014] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0015] Figure 1 This is a schematic diagram of the flow path connection of the six-way valve in positions 1 to 6 in Example 1;

[0016] Figure 2 This is a schematic diagram of the flow path connection of the six-way valve in position 1→2 in Example 1;

[0017] Figure 3 This is a typical spectrum of coenzyme Q10 in Example 2;

[0018] Figure 4 The images show the spectra of coenzyme Q10 in the three one-dimensional mobile phases in Example 4, where green represents the first type, blue represents the second type, and red represents the third type.

[0019] Figure 5 The image shows the coenzyme Q10 spectra at different flow rates in the two-dimensional mobile phase in Example 4, where green represents a flow rate of 0.5 mL / min, blue represents a flow rate of 0.6 mL / min, and red represents a flow rate of 0.7 mL / min. Detailed Implementation

[0020] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] It should be noted that:

[0022] Unless otherwise specified, all embodiments and preferred embodiments mentioned herein can be combined to form new technical solutions; all technical features and preferred features mentioned herein can be combined to form new technical solutions; and the components involved or their preferred components can be combined to form new technical solutions.

[0023] In this document, unless otherwise stated, the various reactions or operational steps may be performed sequentially or not in sequence. Preferably, the reaction methods described herein are performed sequentially.

[0024] In this document, “and / or” is used to indicate that one or both of the situations described may occur, for example, A and / or B includes (A and B) and (A or B).

[0025] In this document, unless otherwise stated, “optional,” “optional,” “optional,” or “optional” means that the event or situation described below may, but does not have to, occur, including the circumstances in which the event or situation may or may not occur.

[0026] In this document, the terms “comprising” or “including” mean that the stated elements, integers or steps are included, but do not exclude any other elements, integers or steps.

[0027] In this article, "separately independent", "each...independently selected" and "...separately independently selected" and "...independently selected" are interchangeable and should all be interpreted broadly. They refer to the range or options that each member of a set of variables or components can choose independently, that is, the choice of each variable or component is independent and is not affected by the choice of other variables or components.

[0028] Unless otherwise stated, the technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods or materials similar to or equivalent to those described herein may also be used in this invention.

[0029] This invention provides a method for detecting coenzyme Q10, wherein the detection method uses two-dimensional liquid chromatography to detect the sample to be tested.

[0030] Two-dimensional liquid chromatography (2D-LC) connects two chromatographic columns with different separation mechanisms in the first and second dimensions of the system, respectively. After passing through the first-dimensional column, the sample is concentrated, enriched, or fragmented before being switched to the second-dimensional column and detector. 2D-LC typically employs two different separation mechanisms to analyze samples, aiming to achieve different separations in the first and second dimensions. This means utilizing the different selectivities of the two columns, or different characteristics of the sample such as molecular size, isoelectric point, hydrophilicity, charge, or specific intermolecular interactions, to separate complex mixtures. This invention employs a center-cutting method to transfer the elution from a one-dimensional chromatographic column at a specific time point to a two-dimensional chromatographic column for further separation. Specifically, the steps include: the sample is first separated on a one-dimensional chromatographic column using a one-dimensional mobile phase; from the start of elution on the one-dimensional column until the coenzyme Q10 peak, the one-dimensional eluent is discharged from the chromatographic system as waste; before the coenzyme Q10 peak, the flow path is switched to transfer the one-dimensional eluent to the two-dimensional chromatographic column, while simultaneously replacing the two-dimensional mobile phase to elute the one-dimensional and two-dimensional columns sequentially; the two-dimensional eluent is detected using a detector. In this text, one-dimensional eluent refers to the eluent flowing from the one-dimensional chromatographic column, and two-dimensional eluent refers to the eluent flowing from the two-dimensional chromatographic column.

[0031] This invention does not limit the apparatus for constructing flow paths and switching flow paths and columns in two-dimensional liquid chromatography detection. In some embodiments, a six-way valve is used to switch the flow path and column, as shown in the schematic diagram. Figure 1 and Figure 2 As shown. Figure 1The flow path connection is shown before the flow path switch, when the one-dimensional eluent does not enter the two-dimensional column. In this mode, ports 1 and 6 of the six-way valve are connected, ports 2 and 3 are connected, and ports 4 and 5 are connected. The analyte is introduced into the one-dimensional system via an autosampler. The one-dimensional mobile phase is introduced into the one-dimensional column by a one-dimensional pump, and the auxiliary mobile phase is introduced into the one-dimensional column via an auxiliary pump. The sample, one-dimensional mobile phase, and auxiliary mobile phase all pass through ports 1 to 6 into the one-dimensional column. The one-dimensional eluent is discharged from the chromatographic system as waste. When the flow path is switched before the coenzyme Q10 peak, the flow path connection after the switch is as follows: Figure 2 As shown, in this mode, ports 1 and 2 of the six-way valve 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.

[0032] In the coenzyme Q10 detection method provided by the present invention, the one-dimensional chromatographic column is a C18 chromatographic column, and the one-dimensional mobile phase includes water, methanol and ethanol; the two-dimensional chromatographic column is a C18 chromatographic column, and the two-dimensional mobile phase includes methanol and ethanol.

[0033] In an optional embodiment, the one-dimensional chromatographic column specifications are: C18 column, 4.6 mm × 50 mm, with a packing particle size of 5 μm. Preferably, a Chromai Lotus EC C2 column with specifications of 4.6 mm × 50 mm and 5 μm is used.

[0034] In an optional embodiment, the two-dimensional chromatographic column has the following specifications: a C18 column, 3.0 mm × 100 mm, with a packing particle size of 2.7 μm, preferably a Chromai Lotus AC C3 column, with specifications of 3.0 mm × 100 mm and 2.7 μm.

[0035] In an optional implementation, starting with elution from the one-dimensional mobile phase, gradient elution is used for the one-dimensional mobile phase, and the gradient elution procedure is as follows:

[0036] 0 min, pure water is 20~80% v / v (e.g., but not limited to 20, 30, 40, 50, 60, 70 or 80% v / v), methanol is 10~40% v / v (e.g., but not limited to 10, 15, 20, 25, 30, 35 or 40% v / v), ethanol is 10~40% v / v (e.g., 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;

[0037] 0.8 min, pure water at 20~80% v / v (e.g., but not limited to 20, 30, 40, 50, 60, 70 or 80% v / v), methanol at 10~40% v / v (e.g., but not limited to 10, 15, 20, 25, 30, 35 or 40% v / v), ethanol at 10~40% v / v (e.g., but not limited to 10, 15, 20, 25, 30, 35 or 40% v / v), and the total of pure water, methanol and ethanol at 100% v / v;

[0038] 4.0 min, pure water 20% v / v, methanol 40% v / v, ethanol 40% v / v;

[0039] 4.01 min until the two-dimensional mobile phase was replaced for elution. Pure water was 10% v / v, methanol was 45% v / v, and ethanol was 45% v / v.

[0040] In an optional implementation, starting with elution from the one-dimensional mobile phase, gradient elution is used for the one-dimensional mobile phase, and the gradient elution procedure is as follows:

[0041] 0 min, pure water 50% v / v, methanol 25% v / v, ethanol 25% v / v;

[0042] 0.8 min, pure water 50% v / v, methanol 25% v / v, ethanol 25% v / v;

[0043] 4.0 min, pure water 20% v / v, methanol 40% v / v, ethanol 40% v / v;

[0044] 4.01 min until the two-dimensional mobile phase was replaced for elution. Pure water was 10% v / v, methanol was 45% v / v, and ethanol was 45% v / v.

[0045] In an optional implementation, starting with elution from the one-dimensional mobile phase, gradient elution is used for the one-dimensional mobile phase, and the gradient elution procedure is as follows:

[0046] 0 min, pure water 80% v / v, methanol 10% v / v, ethanol 10% v / v;

[0047] 0.8 min, pure water 80% v / v, methanol 10% v / v, ethanol 10% v / v;

[0048] 4.0 min, pure water 20% v / v, methanol 40% v / v, ethanol 40% v / v;

[0049] 4.01 min until the two-dimensional mobile phase was replaced for elution. Pure water was 10% v / v, methanol was 45% v / v, and ethanol was 45% v / v.

[0050] In an optional implementation, starting with elution from the one-dimensional mobile phase, gradient elution is used for the one-dimensional mobile phase, and the gradient elution procedure is as follows:

[0051] 0 min, pure water 20% v / v, methanol 40% v / v, ethanol 40% v / v;

[0052] 0.8 min, pure water 20% v / v, methanol 40% v / v, ethanol 40% v / v;

[0053] 4.0 min, pure water 20% v / v, methanol 40% v / v, ethanol 40% v / v;

[0054] 4.01 min until the two-dimensional mobile phase was replaced for elution. Pure water was 10% v / v, methanol was 45% v / v, and ethanol was 45% v / v.

[0055] In an optional implementation, the flow path is switched 10-20 seconds before the coenzyme Q10 peak to transfer the one-dimensional eluent to the two-dimensional chromatographic column.

[0056] In an optional implementation, the flow path is switched at 6.5 min, starting with elution from the one-dimensional mobile phase, to transfer the one-dimensional eluent to the two-dimensional column.

[0057] In an optional implementation, to facilitate the next injection analysis, the one-dimensional mobile phase must be restored to the initial mobile phase ratio at 0 minutes in advance. Therefore, starting with the elution of the one-dimensional mobile phase, the one-dimensional mobile phase gradient elution procedure is as follows:

[0058] 0 min, pure water 20~80% v / v, methanol 10~40% v / v, ethanol 10~40% v / v;

[0059] 0.8 min, pure water 20~80% v / v, methanol 10~40% v / v, ethanol 10~40% v / v;

[0060] 4.0 min, pure water 20% v / v, methanol 40% v / v, ethanol 40% v / v;

[0061] 4.01 min, pure water 10% v / v, methanol 45% v / v, ethanol 45% v / v;

[0062] 9.0 min, pure water 10% v / v, methanol 45% v / v, ethanol 45% v / v;

[0063] 9.01 min, pure water 20~80% v / v, methanol 10~40% v / v, ethanol 10~40% v / v;

[0064] 13.0 min, pure water 20~80% v / v, methanol 10~40% v / v, ethanol 10~40% v / v.

[0065] In an optional implementation, the flow rate of the one-dimensional eluent is 0.7 mL / min for the first 0.8 min, and the initial flow rate gradient is increased to 1.5 mL / min from 0.8 to 4 min.

[0066] In an optional implementation, an auxiliary mobile phase is introduced into the one-dimensional chromatographic column simultaneously during the initial stage of entry. Starting with the elution of the one-dimensional mobile phase, the auxiliary mobile phase elutes over 0–0.8 minutes. The auxiliary mobile phase includes water. Introducing the auxiliary mobile phase increases the injection volume, enabling large-volume injection and alleviating the problems of low sensitivity, inability to perform large-volume injection, and the need for liquid-liquid extraction to concentrate samples inherent in traditional high-performance liquid chromatography-ultraviolet absorption methods. This significantly improves detection sensitivity, allowing direct detection without the need for sample enrichment. Simultaneously, increasing the injection volume shortens the sample pretreatment process. Sample pretreatment methods that eliminate the need for liquid-liquid extraction, which involves extraction with organic reagents, nitrogen drying, and reconstitution with organic solvents, reduce potential human error and improve work efficiency.

[0067] In an optional implementation, the auxiliary mobile phase flow rate is 1 mL / min.

[0068] In an optional implementation, the elution begins with a two-dimensional mobile phase, which is eluted isocratically with methanol at 40% v / v and ethanol at 60% v / v.

[0069] In an optional embodiment, the flow rate of the two-dimensional mobile phase is 0.5 to 0.7 mL / min, for example, but not limited to 0.5, 0.6 or 0.7 mL / min.

[0070] In an optional embodiment, the detector in the detection method is an ultraviolet detector with a detection wavelength of 275 nm.

[0071] In an optional embodiment, the ultraviolet detector is a UV-VIS detector (Ultraviolet-Visible Detector).

[0072] In an optional embodiment, the injection volume is 100~1000 μL, preferably 500 μL.

[0073] In an optional embodiment, the column temperature of the one-dimensional chromatographic column is 35~45 ℃, preferably 40 ℃.

[0074] In an optional embodiment, the column temperature of the two-dimensional chromatographic column is 35~45 ℃, preferably 40 ℃.

[0075] In an optional embodiment, the detection method includes removing proteins from the sample to be tested and then performing two-dimensional liquid chromatography detection.

[0076] In an optional implementation, a protein precipitant is used to remove proteins from the sample. This simple protein precipitation pretreatment method allows blood samples (e.g., plasma or serum samples) to be directly tested after pretreatment. It avoids the excessive use of organic reagents such as ethyl acetate and n-hexane in high-performance liquid chromatography-ultraviolet absorption methods.

[0077] In optional embodiments, 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), preferably n-propanol.

[0078] In an optional embodiment, removing protein from the sample to be tested includes thoroughly mixing the sample to be tested with a protein precipitant, centrifuging, and then taking the supernatant for two-dimensional liquid chromatography detection.

[0079] In an optional embodiment, the method for detecting coenzyme Q10 further includes obtaining a chromatogram and calculating the coenzyme Q10 content in the sample to be tested using the external standard method.

[0080] In an optional implementation, a standard curve is constructed by detecting a series of known concentrations of coenzyme Q10 calibrators, wherein the concentrations of the coenzyme Q10 calibrators range from 100 to 3000 ng / mL, and the concentration points are preferably set to 100 ng / mL, 300 ng / mL, 600 ng / mL, 1000 ng / mL, 2000 ng / mL, and 3000 ng / mL.

[0081] In an optional embodiment, the matrix of the calibrator is blank plasma.

[0082] In an optional embodiment, the method for detecting coenzyme Q10 further includes detecting quality control samples to evaluate the detection results.

[0083] In an optional embodiment, the quality control product includes one or more of the following: a low-value quality control product containing 300 ng / mL coenzyme Q10, a medium-value quality control product containing 1000 ng / mL coenzyme Q10, and a high-value quality control product containing 2000 ng / mL coenzyme Q10.

[0084] In an optional embodiment, the matrix of the quality control sample is blank plasma.

[0085] The coenzyme Q10 detection method provided by this invention allows for samples from sources including, but not limited to, food, pharmaceuticals, biological products, or samples isolated from organisms. Examples include blood (e.g., whole blood, serum, or plasma), body fluids (e.g., saliva, urine, cerebrospinal fluid, pleural effusion, or ascites), tissues, or cells isolated from organisms. The organism can be a mammal or an artificially created animal model, such as, but 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 coenzyme Q10 detection method provided by this invention is not for diagnostic or therapeutic purposes. Specifically, for example, samples derived from the aforementioned mammals are used to provide experimental results for further coenzyme Q10 pharmacokinetic studies; or to detect the coenzyme Q10 content in food or pharmaceuticals for quality control or impurity detection. Knowing the coenzyme Q10 content in the sample does not mean that the result directly points to a disease diagnosis; therefore, the coenzyme Q10 detection method provided by this invention is not for diagnostic or therapeutic purposes.

[0086] Secondly, the method for detecting coenzyme Q10 described in the first aspect is provided for use in the preparation of products for detecting the pharmacokinetic properties of coenzyme Q10; or for use in the preparation of products for detecting the endogenous coenzyme Q10 content in subjects.

[0087] In an optional embodiment, the application includes its use in the preparation of products for the detection of pharmacokinetic parameters of human oral coenzyme Q10.

[0088] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0089] Example 1

[0090] This embodiment provides a method for detecting coenzyme Q10:

[0091] 1. Reagents and instruments:

[0092] (1) One-dimensional mobile phase: pure water, methanol and ethanol;

[0093] (2) Two-dimensional mobile phase: methanol and ethanol;

[0094] (3) Auxiliary mobile phase: pure water;

[0095] (4) Protein precipitant: n-propanol;

[0096] (5) Calibration solutions: Coenzyme Q10 solutions of known concentrations prepared from blank plasma. The concentrations of coenzyme Q10 in each calibrator solution are as follows: Solution L1 concentration is 100 ng / mL; Solution L2 concentration is 300 ng / mL; Solution L3 concentration is 600 ng / mL; Solution L4 concentration is 1000 ng / mL; Solution L5 concentration is 2000 ng / mL; Solution L6 concentration is 3000 ng / mL; Blank plasma solution L0.

[0097] (6) Quality control solution: Coenzyme Q10 solution of known concentration prepared from blank plasma. The concentration of coenzyme Q10 in each quality control solution is as follows: 300 ng / mL for low concentration quality control solution; 1000 ng / mL for medium concentration quality control solution; and 2000 ng / mL for high concentration quality control solution.

[0098] (7) Instruments: Chromai Voyager two-dimensional liquid chromatography system; Xiangyi H2050R high-speed centrifuge; DLAB MX-S vortex apparatus; Eppendorf adjustable pipette; glassware, etc.

[0099] 2. Pre-processing:

[0100] (1) Place all mobile phases into the corresponding pipelines, flush and equilibrate the column, and prepare for analysis.

[0101] (2) Take 300 µL of the calibrator solution and add 900 µL of protein precipitant. Vortex for 1-2 min, centrifuge at high speed for 8-10 min, and take 900 µL of the supernatant. It can be directly used on the instrument.

[0102] Using the same method, take 300 µL of the quality control solution, add 900 µL of 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 used on the instrument.

[0103] Using the same method, take 300 µL of the sample to be tested, add 900 µL of 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 used on the instrument.

[0104] 3. Liquid chromatography conditions:

[0105]

[0106] Example 2

[0107] Set the parameters of the liquid chromatography system according to the conditions in Example 1, and prepare the test solutions, calibrator solutions, and quality control solutions.

[0108] (1) Establish a linear standard curve and test the sample to be tested. Typical spectra are shown below. Figure 3 As shown in Table 1, the linear regression equation and the linear correlation coefficient are shown in Table 1.

[0109] Table 1: Linear regression equation and linear correlation coefficient of coenzyme Q10

[0110]

[0111] (2) Accuracy

[0112] The accuracy of the method was evaluated using a spiked recovery test. A standard at a concentration of 1000 ng / mL was added to plasma samples and compared with the theoretical concentration. The results are shown in Table 2, with spiked recoveries ranging from 95.0% to 105.5%.

[0113] Table 2: Accuracy of Coenzyme Q10

[0114]

[0115] (3) Precision

[0116] A standard at a concentration of 3000 ng / mL was added to the plasma sample. After pretreatment, the sample was injected six times, and the precision of the peak area was calculated. The test results are shown in Table 3. The peak area CV was 0.6%, indicating good precision.

[0117] Table 3: Precision of Coenzyme Q10

[0118]

[0119] Example 3

[0120] Samples collected from a hospital outpatient department were tested using the method described in Example 1, and the content test results are as follows.

[0121] Table 4: Sample Test Results from Outpatient Department of a Hospital

[0122]

[0123] Example 4

[0124] 1) Experiments were conducted on the coenzyme Q10 detection method provided in Example 1 using different ratios of initial aqueous and organic phases in the one-dimensional mobile phase. Three settings were implemented as follows, with all other parameters remaining the same as in Example 1:

[0125]

[0126] The results are as follows Figure 4It can be seen that when the initial aqueous phase ratio of the one-dimensional mobile phase is set in the range of 20%-80%, it has no significant impact on the two-dimensional results, and satisfactory peak shape and retention time can be obtained. The second setting is preferred in this invention.

[0127] 2) Experiments were conducted on different flow rates in the two-dimensional mobile phase of the coenzyme Q10 detection method provided in Example 1. The following three settings were made: 0.5 mL / min, 0.6 mL / min and 0.7 mL / min, and the remaining parameters were the same as in Example 1.

[0128] The results are as follows Figure 5 It can be seen that the peak shape and retention time of the two-dimensional mobile phase are different when the flow rate is in the range of 0.5~0.7 mL / min. With the increase of flow rate, the peak width is narrower and the retention time is shorter. The preferred flow rate of the present invention is 0.7 mL / min.

[0129] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions 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 method for detecting coenzyme Q10, characterized in that, This includes using n-propanol to remove proteins from plasma followed by two-dimensional liquid chromatography detection; The two-dimensional liquid chromatography detection includes the following steps: after injection, the sample is first separated on a one-dimensional column using a one-dimensional mobile phase; the one-dimensional eluent is discarded until the coenzyme Q10 peak is reached, then the flow path is switched to transfer the one-dimensional eluent to the two-dimensional column; at the same time, the two-dimensional mobile phase is replaced to elute both the one-dimensional and two-dimensional columns, and the two-dimensional eluent is detected using a detector. The one-dimensional chromatographic column uses a C18 column, and the one-dimensional mobile phase consists of water, methanol, and ethanol; the two-dimensional chromatographic column uses a C18 column, and the two-dimensional mobile phase consists of methanol and ethanol. Starting with elution from a one-dimensional mobile phase, gradient elution is used for the one-dimensional mobile phase. The gradient elution procedure is as follows: 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; 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; 4.0 min, pure water 20% v / v, methanol 40% v / v, ethanol 40% v / v; 4.01 min until the two-dimensional mobile phase is replaced for elution: pure water at 10% v / v, methanol at 45% v / v, and ethanol at 45% v / v. Starting with elution from the two-dimensional mobile phase, isocratic elution was used in the two-dimensional mobile phase, with methanol at 40% v / v and ethanol at 60% v / v. At the same time as entering the initial stage of the one-dimensional chromatography column, an auxiliary mobile phase is introduced into the one-dimensional chromatography column. Taking the start of elution of the one-dimensional mobile phase as the starting point, the auxiliary mobile phase elutes for 0~0.8 min. The auxiliary mobile phase is water. The one-dimensional chromatographic column is a Chromai Lotus EC C2 column; The two-dimensional chromatographic column is a Chromai Lotus AC C3 column; The specifications of the one-dimensional chromatographic column are: C18 column, 4.6mm×50mm, packing particle size 5μm; and / or, the specifications of the two-dimensional chromatographic column are: C18 column, 3.0mm×100mm, packing particle size 2.7μm; The flow rate of the one-dimensional eluent was 0.7 mL / min before 0.8 min, and the initial flow rate gradient increased to 1.5 mL / min from 0.8 to 4 min. Starting with elution from the one-dimensional mobile phase, the flow path was switched at 6.5 min to transfer the one-dimensional eluent to the two-dimensional column. The auxiliary mobile phase flow rate is 1 mL / min; The flow rate of the two-dimensional mobile phase was 0.5~0.7 mL / min; The detector is an ultraviolet detector with a detection wavelength of 275 nm; and / or, the injection volume is 100~1000 μL; and / or, the column temperature of the one-dimensional column and the two-dimensional column are independently 35~45℃.

2. The method for detecting coenzyme Q10 according to claim 1, characterized in that, It also includes obtaining chromatograms and calculating the coenzyme Q10 content in the sample to be tested using the external standard method.

3. The application of the coenzyme Q10 detection method according to claim 1 or 2 in the preparation of products for coenzyme Q10 pharmacokinetic detection; or in the preparation of products for the detection of endogenous coenzyme Q10 content in subjects.

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