Method for Determining the Concentration of Coenzyme Q in a Biological Matrix 10 ​

Abstract

The present invention relates to a method for determining coenzyme Q in a biological matrix 10 The method includes the following steps: mixing a peripheral blood sample with a diluent, then adding an oxidant to carry out an oxidation reaction. After the oxidation reaction ends, adding an alcohol solvent to the reaction solution of the oxidation reaction to carry out a protein precipitation reaction and extract coenzyme Q 10 , obtaining a suspension with an absorbance of 2.5 - 3.5, centrifuging the suspension to obtain a supernatant; using liquid chromatography - mass spectrometry to determine the concentration of coenzyme Q in the supernatant 10 ; converting the concentration of coenzyme Q in the supernatant to the concentration of coenzyme Q in the peripheral blood sample 10 ; linearly fitting the concentrations of coenzyme Q in multiple groups of existing peripheral blood with the concentrations of coenzyme Q in the biological matrix to obtain a correlation equation; obtaining the concentration of coenzyme Q in the biological matrix according to the concentration of coenzyme Q in the peripheral blood sample and the correlation equation. The method can detect the concentration of coenzyme Q in the biological matrix with a minimum of only 2 μL of peripheral blood sample 10 ; linearly fitting the concentrations of coenzyme Q in multiple groups of existing peripheral blood with the concentrations of coenzyme Q in the biological matrix to obtain a correlation equation; obtaining the concentration of coenzyme Q in the biological matrix according to the concentration of coenzyme Q in the peripheral blood sample and the correlation equation. The method can detect the concentration of coenzyme Q in the biological matrix with a minimum of only 2 μL of peripheral blood sample 10 ; linearly fitting the concentrations of coenzyme Q in multiple groups of existing peripheral blood with the concentrations of coenzyme Q in the biological matrix to obtain a correlation equation; obtaining the concentration of coenzyme Q in the biological matrix according to the concentration of coenzyme Q in the peripheral blood sample and the correlation equation. The method can detect the concentration of coenzyme Q in the biological matrix with a minimum of only 2 μL of peripheral blood sample 10 ; linearly fitting the concentrations of coenzyme Q in multiple groups of existing peripheral blood with the concentrations of coenzyme Q in the biological matrix to obtain a correlation equation; obtaining the concentration of coenzyme Q in the biological matrix according to the concentration of coenzyme Q in the peripheral blood sample and the correlation equation. The method can detect the concentration of coenzyme Q in the biological matrix with a minimum of only 2 μL of peripheral blood sample 10 ; linearly fitting the concentrations of coenzyme Q in multiple groups of existing peripheral blood with the concentrations of coenzyme Q in the biological matrix to obtain a correlation equation; obtaining the concentration of coenzyme Q in the biological matrix according to the concentration of coenzyme Q in the peripheral blood sample and the correlation equation. The method can detect the concentration of coenzyme Q in the biological matrix with a minimum of only 2 μL of peripheral blood sample 10 ; linearly fitting the concentrations of coenzyme Q in multiple groups of existing peripheral blood with the concentrations of coenzyme Q in the biological matrix to obtain a correlation equation; obtaining the concentration of coenzyme Q in the biological matrix according to the concentration of coenzyme Q in the peripheral blood sample and the correlation equation. The method can detect the concentration of coenzyme Q in the biological matrix with a minimum of only 2 μL of peripheral blood sample 10 .

Description

Technical Field

[0001] The present invention relates to the technical field of biotechnology and analytical chemistry, in particular to the determination of coenzyme Q in biological matrix 10 Concentration method. Background Art

[0002] Coenzyme Q 10 (CoQ 10 ) is a highly lipophilic molecule that occurs naturally in the membranes of human cells and is mainly divided into reduced forms (CoQ 10 H2) and oxidized forms. More than 90% of coenzyme Q in serum and biological tissues 10 CoQ 10 The presence of H2 can scavenge peroxyl radicals and reduce α-tocopheryl radicals to protect low-density lipoprotein from lipid peroxidation. Therefore, monitoring the coenzyme Q 10 The concentration is of great significance for maintaining human health and evaluating the diagnosis and treatment methods and prognosis of patients with heart failure, diabetes, and those taking statins.

[0003] Coenzyme Q in traditional biological matrices 10 The detection method usually uses an oxidant to oxidize the CoQ 10 H2 is completely oxidized to coenzyme Q 10 , and then analyzed by liquid chromatography-electrochemical (ECD), liquid chromatography-ultraviolet (UV), liquid chromatography-fluorescence (FL), liquid chromatography-chemiluminescence (CL) and other detection methods. However, the sensitivity of UV, electrochemistry, fluorescence, and chemiluminescence is low, resulting in a large amount of sample used in biological matrices. Therefore, it is urgent to develop a detection method with extremely low sample usage to meet the detection needs. Summary of the invention

[0004] Based on this, it is necessary to provide a method for determining coenzyme Q in biological matrix to solve the above problems. 10 The concentration method can greatly reduce the sample volume, and only 2 μL of peripheral blood sample is needed to detect coenzyme Q in biological matrix. 10 concentration.

[0005] The invention discloses a method for determining coenzyme Q in biological matrix 10 The concentration method comprises the following steps:

[0006] The peripheral blood sample is mixed with a diluent, and then an oxidant is added to perform an oxidation reaction. After the oxidation reaction is completed, an alcohol solvent is added to the reaction solution of the oxidation reaction to perform a protein precipitation reaction and extract coenzyme Q 10 , after vortexing, a suspension with an absorbance of 2.5-3.5 is obtained, and the suspension is centrifuged to obtain a supernatant;

[0007] Determine the concentration of coenzyme Q in the supernatant using liquid chromatography-mass spectrometry 10 ;

[0008] Convert the concentration of coenzyme Q in the supernatant 10 to the concentration of coenzyme Q in the peripheral blood sample 10 ;

[0009] Perform linear fitting on the concentrations of coenzyme Q in multiple groups of existing peripheral blood 10 and the concentrations of coenzyme Q in the biological matrix 10 to obtain a correlation equation, wherein the peripheral blood and the biological matrix in the same group are from the same living body, and the peripheral blood, the biological matrix and the peripheral blood sample are from the same species;

[0010] According to the concentration of coenzyme Q in the peripheral blood sample 10 and the correlation equation, obtain the concentration of coenzyme Q in the biological matrix 10 ;

[0011] In one embodiment, the step of mixing the peripheral blood sample with the diluent satisfies at least one of the following conditions:

[0012] (1) The volume of the peripheral blood sample is 2 μL - 300 μL;

[0013] (2) The diluent includes at least one of water, methanol, isopropanol, aqueous solution of formic acid, methanol solution of formic acid or isopropanol solution of formic acid. When the diluent is selected from aqueous solution of formic acid, methanol solution of formic acid or isopropanol solution of formic acid, the mass fraction of formic acid in the diluent is 0.08% - 0.12%;

[0014] (3) The volume ratio of the peripheral blood sample to the diluent is 1:1 - 1:5.

[0015] In one embodiment, the step of adding an oxidant and mixing for an oxidation reaction satisfies at least one of the following conditions:

[0016] (a) The oxidant includes p-benzoquinone;

[0017] (b) In the step of performing the oxidation reaction, the reaction temperature is 20°C - 35°C and the reaction time is 2 min - 5 min.

[0018] In one embodiment, the step of adding an alcohol solvent to the reaction solution of the oxidation reaction to perform a protein precipitation reaction and extract coenzyme Q 10 satisfies at least one of the following conditions:

[0019] (α) The alcohol solvent includes at least one of methanol, ethanol, or isopropanol;

[0020] (β) The mass ratio of the reaction solution to the alcohol solvent is 1:2 - 1:10;

[0021] (γ) The reaction temperature of the precipitation reaction is 20°C - 35°C, and the reaction time is 2 min - 5 min.

[0022] In one embodiment, in the step of vortex oscillation, the frequency of vortex oscillation is 1000 rpm - 2500 rpm, and the time is 10 min - 20 min.

[0023] In one embodiment, the biological matrix is selected from serum, plasma, or whole blood.

[0024] In one embodiment, converting the concentration of coenzyme Q in the supernatant 10 to the concentration of coenzyme Q in the peripheral blood sample 10 includes the steps of:

[0025] Calculating the total mass of coenzyme Q in the supernatant according to the concentration of coenzyme Q 10 in the supernatant and the total volume of the supernatant; and 10 Calculating the concentration of coenzyme Q

[0026] in the peripheral blood sample according to the total mass of coenzyme Q 10 in the supernatant and the volume of the peripheral blood sample. 10

[0027] In one embodiment, when the biological matrix is serum, the correlation equation is Y = K×X - B, where Y represents the concentration of coenzyme Q 10 in serum, X represents the concentration of coenzyme Q 10 in peripheral blood, K is 1.4 - 2.0, and B is 150 - 280.

[0028] In one embodiment, in the step of determining the concentration of coenzyme Q 10 in the supernatant by liquid chromatography - mass spectrometry, the liquid chromatography satisfies at least one of the following conditions:

[0029] (Ⅰ) The liquid chromatography uses a reverse C18 column;

[0030] (Ⅱ) The column temperature of the liquid chromatography is 30°C - 40°C;

[0031] (Ⅲ) The liquid chromatography is eluted with a mixed solution of methanol, isopropanol, and formic acid as the mobile phase;

[0032] (IV) The mobile phase in the liquid chromatography is 0.6 mL / min - 1.2 mL / min;

[0033] (V) The injection volume of the liquid chromatography is 2 μL - 30 μL.

[0034] In one embodiment, in the liquid chromatography - mass spectrometry technique, the mass spectrometry uses an electrospray ionization source, a multiple reaction monitoring positive ion detection mode, the spray voltage is 4500 V - 5500 V, the temperature is 450 °C - 550 °C, the nebulizer pressure is 50 psi - 55 psi, the auxiliary heating gas is 50 psi - 55 psi, and the curtain gas is 20 psi - 25 psi.

[0035] The method for determining coenzyme Q 10 concentration provided by the present invention, first, using a peripheral blood sample as a raw material, before performing protein precipitation on the peripheral blood sample, the peripheral blood sample is first diluted and then oxidized, and the absorbance (OD value) of the suspension after protein precipitation is controlled between 2.5 - 3.5, thereby avoiding agglomeration of the peripheral blood sample during the oxidation process, and further enabling CoQ 10 H2 in the peripheral blood sample to be completely oxidized into the oxidized form of coenzyme Q 10 , and it can also be well extracted and retained in the supernatant. Secondly, the concentration of coenzyme Q 10 is determined by liquid chromatography - mass spectrometry technique, and the detection sensitivity is greatly improved, and the concentration of coenzyme Q 10 in the supernatant can be accurately obtained. Thirdly, the concentrations of coenzyme Q 10 in multiple groups of existing peripheral blood samples are linearly fitted with the concentration of coenzyme Q 10 in the biological matrix to obtain a correlation equation, and the concentration of coenzyme Q 10 in the biological matrix is calculated according to the concentration of coenzyme Q 10 in the supernatant and this correlation equation. Thus, the method provided by the present invention greatly reduces the sample consumption, and the concentration of coenzyme Q 10 in the biological matrix can be detected with a minimum of only 2 μL of peripheral blood sample. In addition, the raw material peripheral blood sample of this method is convenient and fast to collect, greatly reducing the time cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments recorded in the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0037] Figure 1For coenzyme Q in peripheral blood 10 Concentration and the correlation curve of coenzyme Q in biological matrix 10 Concentration;

[0038] Figure 2 For the chromatogram when the injection concentration is 10 ng / mL coenzyme Q during the limit of quantitation test 10 ;

[0039] Figure 3 For the chromatogram when the injection concentration is 1 ng / mL coenzyme Q during the limit of quantitation test 10 ;

[0040] Figure 4 For the chromatogram when the injection concentration is 0.2 ng / mL coenzyme Q during the limit of quantitation test 10 ; Detailed implementation mode

[0041] For the convenience of understanding the present invention, the present invention will be described in more detail below. However, it should be understood that the present invention can be implemented in many different forms and is not limited to the embodiments or examples described herein. On the contrary, the purpose of providing these embodiments or examples is to make the understanding of the disclosure content of the present invention more thorough and comprehensive.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments or examples and are not intended to limit the present invention.

[0043] The present invention provides a method for determining the concentration of coenzyme Q in biological matrix 10 , comprising the following steps:

[0044] S10. Mix the peripheral blood sample with a diluent, then add an oxidant to carry out an oxidation reaction. After the oxidation reaction ends, add an alcohol solvent to the reaction solution of the oxidation reaction to carry out a protein precipitation reaction and extract coenzyme Q 10 , to obtain a suspension with an absorbance of 2.5 - 3.5, and centrifuge the suspension to obtain a supernatant;

[0045] S20. Use liquid chromatography - mass spectrometry technology to determine the concentration of coenzyme Q in the supernatant 10 ;

[0046] S30. Convert the concentration of coenzyme Q in the supernatant 10 to the concentration of coenzyme Q in the peripheral blood sample 10 ;

[0047] S40. For multiple groups of existing coenzyme Q in peripheral blood 10The concentration of coenzyme Q in the biological matrix 10 The concentrations of the peripheral blood samples and the biological matrix samples were linearly fitted to obtain the correlation equation, wherein the peripheral blood samples and the biological matrix samples of the same group were derived from the same organism, and the peripheral blood samples, the biological matrix samples and the peripheral blood samples were derived from the same species;

[0048] S50, based on the concentration of coenzyme Q 10 The concentration of coenzyme Q in the biological matrix is ​​obtained by the correlation equation 10 concentration.

[0049] The present invention provides a method for determining coenzyme Q in biological matrix 10 In the concentration method, each step cooperates with each other, greatly reducing the amount of sample required. Only 2 μL of peripheral blood is needed to detect coenzyme Q in biological matrix. 10 The concentration, specifically:

[0050] In step S10, peripheral blood is used as a raw material. Before protein precipitation is performed on the peripheral blood, the peripheral blood is diluted and then oxidized, and the OD value of the suspension after protein precipitation is controlled to be between 2.5 and 3.5, including but not limited to 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4 or 3.5, thereby avoiding aggregation of peripheral blood during oxidation, thereby reducing the CoQ in the peripheral blood. 10 H2 is not only completely oxidized to the oxidized form of coenzyme Q 10 , and can be completely retained in the supernatant.

[0051] It should be noted that the absorbance in the present invention refers to the absorbance of the suspension at a wavelength of 490 nm.

[0052] In one embodiment, the volume of the peripheral blood sample is 2μL-300μL, including but not limited to 2μL, 10μL, 25μL, 100μL, 150μL, 200μL, 250μL or 300μL. Preferably, the volume of the peripheral blood sample is 2μL-50μL, and more preferably 10μL-50μL.

[0053] In one embodiment, the diluent comprises at least one of water, methanol, isopropanol, an aqueous solution of formic acid, a methanol solution of formic acid, or an isopropanol solution of formic acid. When the diluent is selected from an aqueous solution of formic acid, a methanol solution of formic acid, or an isopropanol solution of formic acid, the mass fraction of formic acid in the diluent is 0.08% - 0.12%. Preferably, the diluent is selected from water, a mixture of water and methanol, or a mixture of methanol and isopropanol. Further preferably, when the diluent is a mixture of water and methanol, the volume ratio of water to methanol is 1:1.5 - 1.5:1, including but not limited to 1:1.5, 1:1.4, 1:1.3, 1:1.2, 1:1.1, 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, or 1.5:1. When the diluent is a mixture of methanol and isopropanol, the volume ratio of methanol to isopropanol is 10:1 - 8:1, including but not limited to 10:1, 9:1, or 8:1.

[0054] In one embodiment, the volume ratio of the peripheral blood sample to the diluent is 1:1 - 1:10, including but not limited to 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. To have a more excellent peak area response in liquid chromatography - mass spectrometry, and at the same time, better avoid agglomeration of peripheral blood during the oxidation process, preferably, the volume ratio of the peripheral blood sample to the diluent is 1:1 - 1:5.

[0055] In one embodiment, in the step of adding an oxidant and mixing for an oxidation reaction, the oxidant includes p - benzoquinone. Since the sample amount required in the present invention is extremely low, the amount of the oxidant used is small. Even when the amount of the peripheral blood sample is 2 μL - 10 μL, only the oxygen in the air as the oxidant is required to completely oxidize CoQ 10 H2 to the oxidized form of coenzyme Q 10 , in order to more completely oxidize CoQ 10 H2 to the oxidized form of coenzyme Q 10 , the amount of the oxidant used is preferably 8 μg - 20 μg per sample. Further, in the step of carrying out the oxidation reaction, the reaction temperature is 20°C - 35°C, and the reaction time is 2 min - 5 min, so that the oxidation reaction proceeds sufficiently.

[0056] In one embodiment, in the step of adding an alcohol solvent to the reaction solution of the oxidation reaction for a precipitation reaction, the alcohol solvent includes at least one of methanol, ethanol, or isopropanol.

[0057] In one embodiment, the mass ratio of the reaction solution to the alcohol solvent is 1:2 - 1:10, including but not limited to 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10, so as to better avoid the matrix effect.

[0058] In one embodiment, the reaction temperature of the precipitation reaction is 20°C - 35°C, and the reaction time is 2 min - 5 min.

[0059] In one embodiment, vortex oscillation is performed, and the frequency of the vortex oscillation is 1000 rpm - 2500 rpm, and the time is 10 min - 20 min.

[0060] Step S20 measures the concentration of coenzyme Q by liquid chromatography - mass spectrometry 10 , and the detection sensitivity is greatly improved, and the concentration of coenzyme Q in the supernatant can be accurately obtained 10 .

[0061] In one embodiment, the liquid chromatography uses a reverse C 18 column; the column temperature of the liquid chromatography is 30°C - 40°C; the liquid chromatography is eluted with a mixed solution of methanol, isopropanol, and formic acid as the mobile phase; the flow rate of the mobile phase in the liquid chromatography is 0.6 mL / min - 1.2 mL / min; the injection volume of the liquid chromatography is 2 μL - 30 μL, so as to have a more excellent separation effect.

[0062] In one embodiment, in the liquid chromatography - mass spectrometry technology, the mass spectrometry uses an electrospray ionization source, a multiple reaction monitoring positive ion detection mode, the spray voltage is 4500 V - 5500 V, the temperature is 450°C - 550°C, the nebulizer pressure is 50 psi - 55 psi, the auxiliary heating gas is 50 psi - 55 psi, and the curtain gas is 20 psi - 25 psi, so as to have a higher detection sensitivity.

[0063] Step S30 converts the concentration of coenzyme Q in the supernatant 10 to the concentration of coenzyme Q in the peripheral blood sample, and step S40 linearly fits the concentrations of coenzyme Q in multiple groups of existing peripheral blood 10 with the concentrations of coenzyme Q in the biological matrix to obtain a correlation equation; step S50 substitutes the peripheral blood sample concentration obtained in step S30 into the correlation equation obtained in step S40 to finally obtain the concentration of coenzyme Q in the biological matrix 10 with the concentrations of coenzyme Q in the biological matrix 10 . Thus, the method provided by the present invention greatly reduces the sample consumption, and only 2 μL of peripheral blood sample is required at the lowest to detect the concentration of coenzyme Q in the biological matrix 10 . 10

[0064] In one embodiment, step S30 includes the following steps:

[0065] S301. According to the concentration of coenzyme Q in the supernatant 10 and the total volume of the supernatant, calculate the coenzyme Q in the supernatant10 total mass; and

[0066] S302. Calculate the concentration of coenzyme Q in the peripheral blood sample based on 10 the total mass of coenzyme Q in the supernatant and the volume of the peripheral blood sample. 10 concentration.

[0067] In step S40, the concentrations of coenzyme Q 10 in multiple groups of existing peripheral blood and the concentrations of coenzyme Q 10 in the biological matrix are from different living organisms of the same species, while the concentrations of coenzyme Q 10 in the same group of peripheral blood and the concentrations of coenzyme Q 10 in the biological matrix need to be from the same living organism.

[0068] In order to make the correlation equation more accurate, in one embodiment, the concentrations of coenzyme Q 10 in more than 30 groups of existing peripheral blood and the concentrations of coenzyme Q 10 in the biological matrix are used for linear fitting. Preferably, the concentrations of coenzyme Q 10 in 30 - 600 groups of existing peripheral blood and the concentrations of coenzyme Q 10 in the biological matrix are used for linear fitting, and then the concentrations of coenzyme Q 10 in 20 - 120 groups of existing peripheral blood and the concentrations of coenzyme Q 10 in the biological matrix are used to verify the fitting effect of the correlation equation.

[0069] In one embodiment, the biological matrix includes but is not limited to serum, plasma or whole blood; it depends on actual needs.

[0070] In one embodiment, when the biological matrix is serum, the correlation equation is Y = K×X - B, where Y represents the concentration of coenzyme Q 10 in serum, X represents the concentration of coenzyme Q 10 in peripheral blood, K is 1.4 - 2.0, and B is 150 - 280; including but not limited to, K is 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0, and B is 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275 or 280.

[0071] In step S50, substitute the concentration of coenzyme Q 10 in the supernatant into the correlation equation, and the concentration of coenzyme Q 10 in the biological matrix can be obtained.

[0072] In addition, the collection of peripheral blood, which is the raw material of this method, is convenient and fast, greatly reducing the time cost.

[0073] Hereinafter, the method for determining coenzyme Q in a biological matrix will be further described by the following specific examples. 10 concentration will be further described. Example 1

[0074] Mix 10 μL of the peripheral blood sample with 20 μL of water, vortex for 1 min, add 20 μL of p-benzoquinone solution (weigh 4.0 mg of p-benzoquinone and dissolve it in 10 mL of methanol) for oxidation reaction. The oxidation reaction temperature is 25 °C and the oxidation reaction time is 2 min. After the oxidation reaction, add 200 μL of the methanol solution with internal standard and 80 μL of methanol to the reaction solution of the oxidation reaction in sequence for protein precipitation reaction and extraction of coenzyme Q 10 , the reaction temperature is 25 °C and the reaction time is 2 min, then vortex, the frequency of vortex is 1000 rpm and the time is 10 min to obtain a suspension. Centrifuge the suspension to obtain a supernatant.

[0075] Use liquid chromatography-mass spectrometry to determine the concentration of coenzyme Q in the supernatant 10 The concentration of coenzyme Q in the supernatant 10 is 15 ng / mL; among them, the conditions of liquid chromatography are: mobile phase of high-pressure pump A in the liquid chromatography system: none, mobile phase of high-pressure pump B in the liquid chromatography system: methanol / isopropanol / formic acid = 90 / 10 / 0.1, flow rate: 1 mL / min, chromatographic column: Phenomenex KinetexC 18 (3.0×50 mm, 2.6 μm), column temperature: 40 °C, injection volume: 5 μL, gradient elution parameters are as follows: isocratic elution with 100% mobile phase B, retention time of coenzyme Q 10 : 2.12 min;

[0076] The conditions of mass spectrometry are: the mass spectrometry uses an electrospray ion source, multiple reaction monitoring positive ion detection mode, spray voltage is 5500 V, temperature is 550 °C, nebulizer pressure is 55 psi, auxiliary heating gas is 55 psi, and curtain gas is 25 psi.

[0077] According to the concentration of coenzyme Q in the supernatant 10 and the total volume of the supernatant 250 μL, calculate the total mass of coenzyme Q in the supernatant 10 is 3.75 ng. Divide the total mass of coenzyme Q in the supernatant 10 by the volume of the peripheral blood sample to calculate the concentration of coenzyme Q in the peripheral blood sample 10 is 375 ng / mL.

[0078] AsFigure 1 As shown, the concentrations of coenzyme Q in 60 existing peripheral blood samples 10 and the concentrations of coenzyme Q in 60 existing serum samples 10 were linearly fitted. The peripheral blood and serum in the same group were from the same living body, and the peripheral blood, serum, and peripheral blood samples were from the same species. The correlation equation was Y = K×X - B, where K = 1.79 and B = 264. Y represents the concentration of coenzyme Q in serum 10 and X represents the concentration of coenzyme Q in peripheral blood 10 . Then, using the existing concentration of coenzyme Q in the above-mentioned peripheral blood 10 , and the concentration of coenzyme Q in serum 10 and the concentration of coenzyme Q in peripheral blood calculated by the correlation equation, 10 the fitting effect of the correlation equation was verified. The verification results are shown in Table 1. Taking No. 1 as an example, the concentration of coenzyme Q in the serum sample of No. 1 10 was 1888.1 ng / mL. Substituting it into the correlation equation, the calculated concentration of coenzyme Q in peripheral blood 10 was 1202.3 ng / mL. Compared with the directly detected concentration of 1334.5 ng / mL in the peripheral blood sample, the bias was -9.9%.

[0079] According to the concentration of coenzyme Q in the peripheral blood sample 10 of 375 ng / mL and the above correlation equation, the concentration of coenzyme Q in the biological matrix 10 was 407.3 ng / mL. To verify the accuracy of this method, the serum of the same living body as the peripheral blood sample was collected, and the concentration of coenzyme Q in the serum was detected by the liquid chromatography-mass spectrometry technology provided in Example 1 10 . The detection result was 430.2 ng / mL, and the bias was 5.3%.

[0080] Table 1

[0081]

[0082] Example 2

[0083] Example 2 was carried out with reference to Example 1, except that the method for preparing the supernatant was different. The method for preparing the supernatant in Example 2 included the following steps:

[0084] Mix 10 μL of peripheral blood sample with 10 μL of water, vortex for 1 min, add 20 μL of p-benzoquinone methanol solution (weigh 4.0 mg of p-benzoquinone and dissolve it in 10 mL of methanol) for oxidation reaction. The oxidation reaction temperature is 25 °C and the oxidation reaction time is 2 min. After the oxidation reaction, add 200 μL of methanol solution with internal standard and 90 μL of methanol to the reaction solution of the oxidation reaction in sequence for protein precipitation reaction and extraction of coenzyme Q 10 , the reaction temperature of the precipitation reaction is 25 °C and the reaction time is 2 min, then vortex, the frequency of vortex is 1000 rpm and the time is 10 min to obtain a suspension, and centrifuge the suspension to obtain a supernatant.

[0085] The coenzyme Q in peripheral blood obtained by testing in Example 2 10 has a concentration of 373 ng / mL. According to the concentration of coenzyme Q10 in the peripheral blood sample, which is 373 ng / mL, and the correlation equation, the concentration of coenzyme Q 10 in the biological matrix is 403.7 ng / mL.

[0086] Example 3

[0087] Example 3 is carried out with reference to Example 1, the difference is that the preparation method of the supernatant is different. The preparation method of the supernatant in Example 3 includes the following steps:

[0088] Mix 10 μL of peripheral blood sample with 50 μL of water, vortex for 1 min, add 20 μL of p-benzoquinone methanol solution (weigh 4.0 mg of p-benzoquinone and dissolve it in 10 mL of methanol) for oxidation reaction. The oxidation reaction temperature is 25 °C and the oxidation reaction time is 2 min. After the oxidation reaction, add 200 μL of methanol solution with internal standard and 50 μL of methanol to the reaction solution of the oxidation reaction in sequence for protein precipitation reaction and extraction of coenzyme Q 10 , the reaction temperature of the precipitation reaction is 25 °C and the reaction time is 2 min, then vortex, the frequency of vortex is 1000 rpm and the time is 10 min to obtain a suspension, and centrifuge the suspension to obtain a supernatant.

[0089] The coenzyme Q in peripheral blood obtained by testing in Example 3 10 has a concentration of 463 ng / mL. According to the concentration of coenzyme Q 10 in the peripheral blood sample, which is 463 ng / mL, and the correlation equation, the concentration of coenzyme Q 10 in the biological matrix is 564.8 ng / mL.

[0090] Example 4

[0091] Example 4 was carried out with reference to Example 1, except that the preparation method of the supernatant was different. The preparation method of the supernatant in Example 4 included the following steps:

[0092] Mix 10 μL of peripheral blood sample with 100 μL of water, vortex for 1 min, add 20 μL of p-benzoquinone methanol solution (weigh 4.0 mg of p-benzoquinone and dissolve it in 10 mL of methanol) for oxidation reaction. The oxidation reaction temperature is 25 °C and the oxidation reaction time is 2 min. After the oxidation reaction, add 200 μL of methanol with internal standard to the reaction solution of the oxidation reaction for protein precipitation reaction and extract coenzyme Q 10 , the reaction temperature of the precipitation reaction is 25 °C and the reaction time is 2 min. Then carry out vortex oscillation, the frequency of vortex oscillation is 1000 rpm and the time is 10 min to obtain a suspension. Centrifuge the suspension to obtain the supernatant.

[0093] The concentration of coenzyme Q in the peripheral blood tested in Example 4 10 was 433 ng / mL. According to the concentration of coenzyme Q10 in the peripheral blood sample of 433 ng / mL and the correlation equation, the concentration of coenzyme Q in the biological matrix 10 was 511.1 ng / mL.

[0094] Example 5

[0095] Example 5 was carried out with reference to Example 1, except that the preparation method of the supernatant was different. The preparation method of the supernatant in Example 5 included the following steps:

[0096] Mix 10 μL of peripheral blood sample with 20 μL of pure methanol, vortex for 1 min, add 20 μL of p-benzoquinone methanol solution (weigh 4.0 mg of p-benzoquinone and dissolve it in 10 mL of methanol) for oxidation reaction. The oxidation reaction temperature is 25 °C and the oxidation reaction time is 2 min. After the oxidation reaction, add 200 μL of methanol solution with internal standard and 80 μL of methanol to the reaction solution of the oxidation reaction for protein precipitation reaction and extract coenzyme Q 10 , the reaction temperature of the precipitation reaction is 25 °C and the reaction time is 2 min. Then carry out vortex oscillation, the frequency of vortex oscillation is 1000 rpm and the time is 10 min to obtain a suspension. Centrifuge the suspension to obtain the supernatant.

[0097] The concentration of coenzyme Q in the peripheral blood tested in Example 5 10 was 289 ng / mL. According to the concentration of coenzyme Q10 in the peripheral blood sample of 289 ng / mL and the correlation equation, the concentration of coenzyme Q in the biological matrix 10 was 253.3 ng / mL.

[0098] Example 6

[0099] Example 6 was carried out with reference to Example 1, except that the method for preparing the supernatant was different. The method for preparing the supernatant in Example 6 included the following steps:

[0100] Mix 10 μL of peripheral blood sample with 20 μL of a mixed solution of methanol and isopropanol (the volume ratio of methanol to isopropanol is 9:1), vortex for 1 min, add 20 μL of p-benzoquinone methanol solution (weigh 4.0 mg of p-benzoquinone and dissolve it in 10 mL of methanol) for oxidation reaction. The oxidation reaction temperature is 25°C and the oxidation reaction time is 2 min. After the oxidation reaction, add 200 μL of methanol solution with internal standard and 80 μL of methanol to the reaction solution of the oxidation reaction in sequence for protein precipitation reaction and extraction of coenzyme Q 10 , the reaction temperature of the precipitation reaction is 25°C and the reaction time is 2 min, then carry out vortex oscillation, the frequency of vortex oscillation is 1000 rpm and the time is 10 min to obtain a suspension, and centrifuge the suspension to obtain the supernatant.

[0101] The coenzyme Q in peripheral blood tested in Example 6 10 The concentration is 369 ng / mL. According to the concentration of coenzyme Q10 in the peripheral blood sample of 369 ng / mL and the said correlation equation, the concentration of coenzyme Q in the biological matrix 10 is 396.5 ng / mL.

[0102] Example 7

[0103] Example 7 was carried out with reference to Example 1, except that the method for preparing the supernatant was different. The method for preparing the supernatant in Example 7 included the following steps:

[0104] Mix 10 μL of peripheral blood sample with 20 μL of a mixed solution of methanol and water (the volume ratio of methanol to water is 1:1), vortex for 1 min, add 20 μL of p-benzoquinone methanol solution (weigh 4.0 mg of p-benzoquinone and dissolve it in 10 mL of methanol) for oxidation reaction. The oxidation reaction temperature is 25°C and the oxidation reaction time is 2 min. After the oxidation reaction, add 200 μL of methanol solution with internal standard and 80 μL of methanol to the reaction solution of the oxidation reaction in sequence for protein precipitation reaction and extraction of coenzyme Q 10 , the reaction temperature of the precipitation reaction is 25°C and the reaction time is 2 min, then carry out vortex oscillation, the frequency of vortex oscillation is 1000 rpm and the time is 10 min to obtain a suspension, and centrifuge the suspension to obtain the supernatant.

[0105] The coenzyme Q in peripheral blood tested in Example 7 10The concentration is 403 ng / mL. Based on the concentration of coenzyme Q10 in the fingertip blood sample, which is 403 ng / mL, and the said correlation equation, the concentration of coenzyme Q in the biological matrix is obtained. 10 The concentration is 457.4 ng / mL.

[0106] Comparative Example 1

[0107] Comparative Example 1 was carried out with reference to Example 1, except that the substance injected in Comparative Example 1 was water.

[0108] Comparative Example 2

[0109] Comparative Example 2 was carried out with reference to Example 1, except that the substance injected in Comparative Example 2 was methanol.

[0110] Comparative Example 3

[0111] Comparative Example 3 was carried out with reference to Example 1, except that the method for preparing the supernatant was different. The method for preparing the supernatant in Comparative Example 3 included the following steps:

[0112] 10 μL of the fingertip blood sample was added to 20 μL of the p-benzoquinone methanol solution (4.0 mg of p-benzoquinone was weighed and dissolved in 10 mL of methanol) for an oxidation reaction. The oxidation reaction temperature was 25 °C, and the oxidation reaction time was 2 min. After the oxidation reaction ended, 200 μL of the methanol solution with an internal standard and 100 μL of methanol were successively added to the reaction solution of the oxidation reaction for a protein precipitation reaction and extraction of coenzyme Q 10 , the reaction temperature of the precipitation reaction was 25 °C, and the reaction time was 2 min. After the protein precipitation reaction, the reaction solution of the protein precipitation reaction was vortexed. The frequency of the vortex oscillation was 1000 rpm, and the time was 10 min to obtain a suspension. The suspension was centrifuged to obtain the supernatant.

[0113] The concentration of coenzyme Q in the fingertip blood tested in Comparative Example 3 10 is 259 ng / mL. Based on the concentration of coenzyme Q10 in the fingertip blood sample, which is 259 ng / mL, and the said correlation equation, the concentration of coenzyme Q in the biological matrix is obtained. 10 The concentration is 199.6 ng / mL.

[0114] Test Example 1

[0115] The supernatants prepared in Examples 1-7 and Comparative Examples 1-3 were placed on an enzyme-linked immunosorbent assay (ELISA) reader, and the absorbance (OD value) was detected at a wavelength of 490 nm. The detection results are shown in Table 2. In Table 2, the mean value and SD (standard deviation) of 3 parallel detections are shown, and n = 3 represents 3 parallel detections. The peak areas and recovery rates of coenzyme Q 10 and its internal standard in the supernatants of Comparative Example 3 and Examples 1-7 are shown in Table 3, and n = 3 represents 3 parallel detections.

[0116] Table 2

[0117]

[0118] As can be seen from the table, the OD values of Comparative Example 1 (blank - pure water group) and Comparative Example 2 (blank - methanol group) are only about 0.04 at a wavelength of 490 nm, and the OD value of Comparative Example 3 (undiluted group) is only 1.16, which is much lower than that of Examples 1 - 7. Thus, it can be known that adding a diluent to the peripheral blood sample for dilution can enable coenzyme Q in the peripheral blood sample 10 to be retained in the supernatant, rather than being unable to vortex well and extract the relevant target coenzyme Q after adding an oxidant 10 .

[0119] Table 3

[0120]

[0121] As can be seen from the table, if the peripheral blood sample is not diluted, the detection recovery rate will only be 66.2%, which cannot meet the methodological requirements of the detection. If the sample is only diluted 1:2 with pure methanol, the recovery rate will also decrease to 73.9%. If the dilution ratio with water is too high, reaching a dilution ratio of 1:5 or 1:10, then coenzyme Q 10 and the peak area response of its internal standard will become worse, and the response will be 1 - 2 orders of magnitude lower than that of the method of diluting 1:2 with water, affecting the LOQ (limit of quantification) of the detection method

[0122] Example 8

[0123] Example 8 was carried out with reference to Example 1, except that the volume of the peripheral blood sample was different and the method for preparing the supernatant was different. The method for preparing the supernatant of Example 8 included the following steps:

[0124] Mix 25 μL of the peripheral blood sample with 25 μL of water, vortex for 1 min, add 50 μL of p - benzoquinone methanol solution (weigh 4.0 mg of p - benzoquinone and dissolve it in 10 mL of methanol) for oxidation reaction. The oxidation reaction temperature is 25 °C, and the oxidation reaction time is 2 min. After the oxidation reaction ends, sequentially add 400 μL of the methanol solution with internal standard and 100 μL of methanol to the reaction solution of the oxidation reaction for protein precipitation reaction and extract coenzyme Q 10 , the reaction temperature of the precipitation reaction is 25 °C, and the reaction time is 2 min. After the protein precipitation reaction, vortex the protein precipitation reaction solution. The vortex frequency is 1000 rpm and the time is 10 min to obtain a suspension. Centrifuge the suspension to obtain the supernatant

[0125] Coenzyme Q in the peripheral blood tested in Example 810 The concentration of CoQ10 is 375 ng / mL. Based on the concentration of CoQ10 in the fingertip blood sample, which is 375 ng / mL, and the above-mentioned correlation equation, the concentration of CoQ in the biological matrix is 10 407.3 ng / mL.

[0126] Example 9

[0127] Example 9 was carried out with reference to Example 1, except that the volume of the fingertip blood sample was different and the method for preparing the supernatant was different. The method for preparing the supernatant in Example 9 included the following steps:

[0128] Mix 25 μL of the fingertip blood sample with 50 μL of water, vortex for 1 min, add 50 μL of p-benzoquinone methanol solution (weigh 4.0 mg of p-benzoquinone and dissolve it in 10 mL of methanol) for oxidation reaction. The oxidation reaction temperature is 25 °C and the oxidation reaction time is 2 min. After the oxidation reaction is completed, add 400 μL of the internal standard-containing methanol solution and 75 μL of methanol to the reaction solution of the oxidation reaction in sequence for protein precipitation reaction and extraction of CoQ 10 , the reaction temperature of the precipitation reaction is 25 °C and the reaction time is 2 min. After the protein precipitation reaction, vortex the protein precipitation reaction solution. The vortex frequency is 1000 rpm and the time is 10 min to obtain a suspension. Centrifuge the suspension to obtain the supernatant.

[0129] The concentration of CoQ in the fingertip blood tested in Example 9 is 10 385 ng / mL. Based on the concentration of CoQ10 in the fingertip blood sample, which is 385 ng / mL, and the above-mentioned correlation equation, the concentration of CoQ in the biological matrix is 10 425.2 ng / mL.

[0130] Example 10

[0131] Example 10 was carried out with reference to Example 1, except that the volume of the fingertip blood sample was different and the method for preparing the supernatant was different. The method for preparing the supernatant in Example 10 included the following steps:

[0132] Mix 25 μL of the fingertip blood sample with 125 μL of water, vortex for 1 min, add 50 μL of p-benzoquinone methanol solution (weigh 4.0 mg of p-benzoquinone and dissolve it in 10 mL of methanol) for oxidation reaction. The oxidation reaction temperature is 25 °C and the oxidation reaction time is 2 min. After the oxidation reaction is completed, add 400 μL of the internal standard-containing methanol solution to the reaction solution of the oxidation reaction for protein precipitation reaction and extraction of CoQ 10, the reaction temperature of the precipitation reaction is 25 °C, the reaction time is 2 min. After the protein precipitation reaction, the protein precipitation reaction solution is vortexed. The frequency of vortexing is 1000 rpm and the time is 10 min to obtain a suspension. The suspension is centrifuged to obtain a supernatant.

[0133] The concentration of coenzyme Q in the peripheral blood obtained by testing in Example 10 10 is 454 ng / mL. According to the concentration of coenzyme Q10 in the peripheral blood sample, which is 454 ng / mL, and the correlation equation, the concentration of coenzyme Q in the biological matrix 10 is 548.7 ng / mL.

[0134] Example 11

[0135] Example 11 was carried out with reference to Example 1, except that the volume of the peripheral blood sample was different and the method for preparing the supernatant was different. The method for preparing the supernatant in Example 11 includes the following steps:

[0136] Mix 25 μL of the peripheral blood sample with 50 μL of a mixture of methanol and water (the volume ratio of methanol to water is 1:1), vortex for 1 min, add 50 μL of a p-benzoquinone methanol solution (weigh 4.0 mg of p-benzoquinone and dissolve it in 10 mL of methanol) for an oxidation reaction. The oxidation reaction temperature is 25 °C and the oxidation reaction time is 2 min. After the oxidation reaction, add 400 μL of the methanol solution with an internal standard and 125 μL of methanol to the reaction solution of the oxidation reaction in sequence for a protein precipitation reaction and extract coenzyme Q 10 , the reaction temperature of the precipitation reaction is 25 °C, the reaction time is 2 min. After the protein precipitation reaction, the protein precipitation reaction solution is vortexed. The frequency of vortexing is 1000 rpm and the time is 10 min to obtain a suspension. The suspension is centrifuged to obtain a supernatant.

[0137] The concentration of coenzyme Q in the peripheral blood obtained by testing in Example 11 10 is 393 ng / mL. According to the concentration of coenzyme Q10 in the peripheral blood sample, which is 393 ng / mL, and the correlation equation, the concentration of coenzyme Q in the biological matrix 10 is 439.5 ng / mL.

[0138] Comparative Example 4

[0139] Comparative Example 4 was carried out with reference to Example 1, except that the volume of the peripheral blood sample was different and the method for preparing the supernatant was different. The method for preparing the supernatant in Comparative Example 4 includes the following steps:

[0140] 25 μL of peripheral blood sample was oxidized with 50 μL of p-benzoquinone methanol solution (4.0 mg of p-benzoquinone was weighed and dissolved in 10 mL of methanol) at 25 °C for 2 min. After the oxidation reaction, 400 μL of methanol solution with internal standard and 125 μL of methanol were added to the reaction solution for protein precipitation and extraction of coenzyme Q 10 The reaction temperature of the precipitation reaction is 25°C, and the reaction time is 2 min. After the protein precipitation reaction, the protein precipitation reaction solution is vortexed at a frequency of 1000 rpm for 10 min to obtain a suspension, which is then centrifuged to obtain a supernatant.

[0141] Comparative Example 4: Peripheral blood coenzyme Q 10 The concentration of coenzyme Q10 in the peripheral blood sample is 156 ng / mL and the correlation equation is obtained. 10 The concentration is 15.2ng / mL. It can be seen that if the pretreatment is improper, the test results will deviate from the actual value, and the conversion by formula will also be invalid.

[0142] Test Example 2

[0143] Refer to Test Example 1 to test the coenzyme Q in the supernatant. 10 The peak areas and recoveries of the internal standards are shown in Table 4. In Table 4, n=3 represents three parallel tests.

[0144] Table 4

[0145]

[0146] It can be seen from the table that if no dilution is performed during the pretreatment process, the recovery rate of the 25 μL peripheral blood sample will be lower than that of the 10 μL peripheral blood sample in Example 7, which is only 39.8%. In addition, if the dilution ratio of water is too high, reaching a dilution ratio of 1:5, the coenzyme Q 10 The peak area response of the internal standard is poor, and the response will be at least one order of magnitude lower than the method of diluting with water 1:2, which affects the LOQ (limit of quantification) of the method and is not preferred. Therefore, it is preferred to dilute the peripheral blood sample with 1-2 times the volume of water, or a mixture of methanol and water, so as to more accurately determine the coenzyme Q in the peripheral blood sample. 10 The concentration of coenzyme Q 10 concentration.

[0147] The detection method is demonstrated as follows:

[0148] (1) Evaluation of the limit of quantification of liquid chromatography-mass spectrometry:

[0149] Prepare coenzyme Q solutions with different concentrations 10 and measure them using the liquid chromatography-mass spectrometry technique in Example 1. As Figure 2 shown, when the concentration of the coenzyme Q 10 solution is 10 ng / mL, the signal-to-noise ratio is 498. As Figure 3 shown, when the concentration of the coenzyme Q 10 solution is 1 ng / mL, the signal-to-noise ratio still remains above 60. When the concentration of the coenzyme Q 10 solution is 0.2 ng / mL, as Figure 4 shown, the signal-to-noise ratio is 16.1, still meeting the standard of the quantitative limit with a signal-to-noise ratio of 10:1, meaning that the LOQ (limit of quantification) of this method reaches the level of 0.2 ng / mL.

[0150] (2) Evaluation of inter-day precision and intra-day precision:

[0151] Take peripheral blood samples with known concentrations, prepare the supernatant according to the method in Example 1, and evaluate the intra-day and inter-day precision and accuracy. For intra-day evaluation, six parallel detections were carried out at three levels, and for inter-day evaluation, six parallel detections at three levels were carried out for three consecutive days. The test results of intra-day precision are shown in Table 5, where n = 6 represents six parallel detections, and the test results of inter-day precision are shown in Table 6, where n = 18 represents six parallel detections for three consecutive days. As can be seen from Table 5 and Table 6, the recoveries of coenzyme Q 10 at the three levels are all between 98.4% - 106.1%, and the precision CVs are all between 1.7% - 3.2%, meeting the quantitative requirements for biological sample analysis.

[0152] Table 5

[0153]

[0154] Table 6

[0155]

[0156] (3) Carryover contamination evaluation:

[0157] Prepare an ethanol solution of coenzyme Q 10 at 4000 ng / mL and an ethanol solution of coenzyme Q 10 at 1000 ng / mL, and inject samples according to the liquid chromatography-mass spectrometry technique in Example 1. After injecting the ethanol solution of coenzyme Q 10 at 4000 ng / mL, inject a blank sample, and then inject the ethanol solution of coenzyme Q 10Taking the 25% value (6.3E+03) of the response to the ethanol solution as the standard, three tests were conducted. As shown in Table 7, the responses in the blank samples were much smaller than this value, indicating that there was no problem of carry-over contamination interfering with the accuracy of the results between each sample test.

[0158] Table 7

[0159]

[0160] (4)Matrix effect evaluation:

[0161] Taking high-concentration peripheral blood samples and low-concentration peripheral blood samples with known concentrations, five samples were prepared according to the mass ratios of 5:0 (5 parts by mass of low-concentration peripheral blood sample, 5L), 4:1 (4 parts by mass of low-concentration peripheral blood sample and 1 part by mass of high-concentration peripheral blood sample, 4L + 1H), 2.5:2.5 (2.5 parts by mass of low-concentration peripheral blood sample and 2.5 parts by mass of high-concentration peripheral blood sample, 2.5L + 2.5H), 1:4 (1 part by mass of low-concentration peripheral blood sample and 4 parts by mass of high-concentration peripheral blood sample, 1L + 4H), 0:5 (5 parts by mass of high-concentration peripheral blood sample, 5H). The supernatant was prepared with reference to the method of Example 1, and coenzyme Q in the peripheral blood was measured. 10 The concentration was measured, and the measurement results are shown in Table 8. In Table 8, n = 3 represents three parallel tests. As can be seen from Table 8, since the sample volume can be made 10 μL or even lower during the preparation of the supernatant, and it is diluted more than 20 times with solvents such as methanol, the matrix effect of the solution during injection can be ignored. The biases at three concentrations of 4L + 1H, 2.5L + 2.5H, and 1L + 4H are in the range of 3.3% - 5.9%, which belongs to the negligible category.

[0162] Table 8

[0163]

[0164] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0165] The above-described embodiments only represent several implementation manners of the present invention. Their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent should be subject to the appended claims.

Claims

1. A method for determining coenzyme Q in biological matrices 10 The concentration method is characterized in that The following steps are involved: The peripheral blood sample is mixed with a diluent, and then an oxidant is added to perform an oxidation reaction. After the oxidation reaction is completed, an alcohol solvent is added to the reaction solution of the oxidation reaction to perform a protein precipitation reaction and extract coenzyme Q 10 , after vortexing, a suspension with an absorbance of 2.5-3.5 is obtained, and the suspension is centrifuged to obtain a supernatant; Determination of coenzyme Q in the supernatant by liquid chromatography-mass spectrometry 10 concentration; Coenzyme Q in the supernatant 10 The concentration of coenzyme Q in the peripheral blood sample is converted to 10 concentration; Multiple groups of peripheral blood coenzyme Q 10 The concentration of coenzyme Q in the biological matrix 10 The concentration of is linearly fitted to obtain a correlation equation, wherein the peripheral blood and the biological matrix of the same group are derived from the same organism, the biological matrix and the peripheral blood sample are derived from the same species, and the biological matrix is ​​selected from serum, plasma or whole blood; According to the coenzyme Q 10 The concentration of coenzyme Q in the biological matrix is ​​obtained by using the correlation equation. 10 concentration.

2. The method for determining coenzyme Q in biological matrix according to claim 1 10 The concentration method is characterized in that The step of mixing the peripheral blood sample with the diluent satisfies at least one of the following conditions: (1) The volume of the peripheral blood sample is 2 μL-300 μL; (2) The diluent includes at least one of water, methanol, isopropanol, an aqueous solution of formic acid, a methanol solution of formic acid or an isopropanol solution of formic acid. When the diluent is selected from an aqueous solution of formic acid, a methanol solution of formic acid or an isopropanol solution of formic acid, the mass fraction of formic acid in the diluent is 0.08%-0.12%; (3) The volume ratio of the peripheral blood sample to the diluent is 1:1-1:

5.

3. The method for determining coenzyme Q in biological matrix according to claim 1 10 The concentration method is characterized in that The step of adding an oxidant and mixing for oxidation reaction satisfies at least one of the following conditions: (a) the oxidizing agent comprises p-benzoquinone; (b) In the step of performing the oxidation reaction, the reaction temperature is 20°C-35°C, and the reaction time is 2 min-5 min.

4. The method for determining coenzyme Q in a biological matrix according to any one of claims 1 to 3. 10 The concentration method is characterized in that Add an alcohol solvent to the reaction solution of the oxidation reaction to perform a protein precipitation reaction and extract coenzyme Q 10 The steps meet at least one of the following conditions: (a) the alcohol solvent comprises at least one of methanol, ethanol or isopropanol; (β) the mass ratio of the reaction solution to the alcohol solvent is 1:2-1:10; (γ) The reaction temperature of the precipitation reaction is 20°C-35°C, and the reaction time is 2min-5min.

5. The method for determining coenzyme Q in a biological matrix according to any one of claims 1 to 3. 10 The concentration method is characterized in that In the step of vortexing, the frequency of vortexing is 1000 rpm-2500 rpm, and the time is 10 min-20 min.

6. The method for determining coenzyme Q in a biological matrix according to any one of claims 1 to 3. 10 The concentration method is characterized in that The biological matrix is ​​selected from serum, plasma or whole blood.

7. The method for determining coenzyme Q in a biological matrix according to any one of claims 1 to 3. 10 The concentration method is characterized in that Coenzyme Q in the supernatant 10 The concentration of coenzyme Q in the peripheral blood sample is converted to 10 The steps of the concentration include: According to the coenzyme Q 10 The concentration of coenzyme Q in the supernatant was calculated based on the total volume of the supernatant. 10 The total mass of According to the coenzyme Q 10 The total mass of the peripheral blood sample and the volume of the peripheral blood sample are used to calculate the coenzyme Q 10 concentration.

8. The method for determining coenzyme Q in a biological matrix according to any one of claims 1 to 3. 10 The concentration method is characterized in that When the biological matrix is ​​serum, the correlation equation is Y=K×XB, where Y represents the coenzyme Q in serum. 10 The concentration of X represents the concentration of coenzyme Q in peripheral blood. 10 The concentration of K is 1.4-2.0 and B is 150-280.

9. The method for determining coenzyme Q in a biological matrix according to any one of claims 1 to 3. 10 The concentration method is characterized in that Determination of coenzyme Q in the supernatant by liquid chromatography-mass spectrometry 10 In the step of determining the concentration of the liquid chromatogram, the liquid chromatogram satisfies at least one of the following conditions: (I) The liquid chromatography uses a reverse phase C18 column; (II) The column temperature of the liquid chromatography is 30°C-40°C; (III) the liquid chromatography is eluted with a mixture of methanol, isopropanol and formic acid as the mobile phase; (IV) The mobile phase in the liquid chromatography is 0.6 mL / min-1.2 mL / min; (V) The injection volume of the liquid chromatography is 2 μL-30 μL.

10. The method for determining coenzyme Q in a biological matrix according to any one of claims 1 to 3. 10 The concentration method is characterized in that In the liquid chromatography-mass spectrometry technology, the mass spectrometer adopts an electrospray ion source, a multiple ion reaction positive ion detection mode, a spray voltage of 4500V-5500V, a temperature of 450℃-550℃, a nebulizer pressure of 50psi-55psi, an auxiliary heating gas of 50psi-55psi, and a curtain gas of 20psi-25psi.

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