A method for detecting retinol, all-trans retinoic acid, 9-cis retinoic acid and all-trans retinal based on HPLC-MS / MS

Abstract

The present invention discloses a method for detecting retinol, all-trans retinoic acid, 9-cis retinoic acid and all-trans retinal based on HPLC-MS / MS. The method comprises the following steps: treating a blood sample to obtain a test sample; using HPLC-MS / MS to detect a standard curve working solution added with an internal standard solution to obtain a standard curve equation corresponding to a target analyte; using HPLC-MS / MS to detect the test sample added with the internal standard solution; and calculating the content of the target analyte in the test sample using the standard curve equation; the HPLC-MS / MS detection uses an ACE PFP column as the chromatographic column; mobile phase A: methanol: acetonitrile: water: formic acid = 300:400:300:0.1; and mobile phase B: methanol: acetonitrile: water: formic acid = 300:550:150:0.1. The detection method of the present invention can achieve good liquid phase separation for all-trans retinoic acid and 9-cis retinoic acid, can simultaneously detect retinol, all-trans retinoic acid, 9-cis retinoic acid, and all-trans retinal, achieves one-shot sampling and produces results, has strong specificity, and has a low detection limit.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and in particular relates to a method for detecting retinol, all-trans retinoic acid, 9-cis retinoic acid and all-trans retinal based on HPLC-MS / MS. Background Art

[0002] Vitamin A compounds play an important role in regulating and controlling the proliferation and differentiation of various normal tissues and epithelial cells, growth and development, nervous system function, and internal homeostasis. Retinol, all-trans retinoic acid, 9-cis retinoic acid, and all-trans retinal are functional substances in the retinol metabolic pathway. Simultaneous detection of these four substances is crucial for studying their physiological functions.

[0003] There are two major difficulties in detecting these four substances at the same time:

[0004] 1. Huge difference in concentration: The concentration of retinol is as high as 1ug / ml, while the concentration of retinal and retinoic acid is very low, with retinal only around 0.3ng / ml. This makes it difficult to get results with one injection.

[0005] 2. All-trans retinoic acid and 9-cis retinoic acid are cis-trans isomers and cannot be separated in mass spectrometry, making detection difficult. Summary of the Invention

[0006] In response to the current technical problems, the present invention provides a method for detecting retinol, all-trans retinoic acid, 9-cis retinoic acid and all-trans retinal based on HPLC-MS / MS, which enables a group of cis and trans isomers of all-trans retinoic acid and 9-cis retinoic acid to be well separated in the liquid phase, and achieves the result of retinol, all-trans retinoic acid, 9-cis retinoic acid and all-trans retinal with huge concentration differences with a single injection.

[0007] In order to achieve the above object, the present invention adopts the following technical means:

[0008] The present invention provides a method for detecting retinol, all-trans retinoic acid, 9-cis retinoic acid and all-trans retinal based on HPLC-MS / MS, the detection method comprising the following steps:

[0009] (1) Prepare the standard stock solution, standard curve working solution and internal standard solution of the target analyte;

[0010] (2) The blood sample is processed to obtain a test sample;

[0011] (3) Using HPLC-MS / MS to detect the standard curve working solution with the internal standard solution added, the peak area ratio of the chromatographic peak of the target analyte to the corresponding isotope internal standard is used as the ordinate, and the concentration of the target analyte in the standard curve working solution is used as the abscissa, respectively fitting to obtain the standard curve equation corresponding to the target analyte, using HPLC-MS / MS to detect the test sample with the internal standard solution added, and calculating the content of the target analyte in the test sample according to the corresponding standard curve equation; the chromatographic conditions for HPLC-MS / MS detection are:

[0012] Chromatographic column: ACE PFP column (100x2.1 mm, 2.6 μm);

[0013] Mobile phase A: methanol: acetonitrile: water: formic acid = 300:400:300:0.1;

[0014] Mobile phase B: methanol: acetonitrile: water: formic acid = 300:550:150:0.1;

[0015] Strong wash solution: 80% methanol;

[0016] The flow rate was 0.3 mL / min, the column temperature was 30 °C, and the injection volume was 5 μL;

[0017] A gradient elution method is used, and the gradient elution method is as follows: the volume fraction of mobile phase A + the volume fraction of mobile phase B = 100%; wherein:

[0018] The volume fraction of mobile phase A decreased from 15% to 0 during 0-3 min;

[0019] The volume fraction of mobile phase A was maintained at 0 for 3–4 min;

[0020] 4-4.1 min: the volume fraction of mobile phase A increased from 0% to 15%;

[0021] The volume fraction of mobile phase A was maintained at 15% from 4.1 to 5 min and then stopped at 5.2 min.

[0022] Furthermore, the mass spectrometry conditions for the HPLC-MS / MS detection are:

[0023] Ion source: electrospray ionization source, positive ion mode;

[0024] Ion spray voltage (IS): 4500 V;

[0025] Ion source temperature (TEM): 450°C;

[0026] Ion source nebulizer gas (GS1): 40 psi;

[0027] Ion source heating auxiliary gas (GS2): 45psi;

[0028] Curtain gas (CG): 40psi;

[0029] Scan mode: MRM, MRM parameter conditions are as follows:

[0030] Retinol: Q1, 269 (m / z); Q3, 93 (m / z); T, 60 (m sec); DP, 50 (V); EP, 7 (V); CE, 15 (V); CXP, 12 (V);

[0031] Cis-retinoic acid: Q1, 301 (m / z); Q3, 122.8 (m / z); T, 60 (m sec); DP, 60 (V); EP, 10 (V); CE, 20.91 (V); CXP, 20 (V);

[0032] All-trans retinoic acid: Q1, 301 (m / z); Q3, 123.1 (m / z); T, 60 (m sec); DP, 60 (V); EP, 8 (V); CE, 19.66 (V); CXP, 15 (V);

[0033] Retinal: Q1, 285 (m / z); Q3, 161 (m / z); T, 60 (m sec); DP, 60 (V); EP, 10 (V); CE, 12.04 (V); CXP, 20 (V);

[0034] Retinol-d4: Q1, 273 (m / z); Q3, 93.8 (m / z); T, 60 (m sec); DP, 70 (V); EP, 7 (V); CE, 24 (V); CXP, 12 (V);

[0035] All-trans retinoic acid-d6: Q1, 307.7 (m / z); Q3, 123 (m / z); T, 60 (m sec); DP, 62 (V); EP, 10 (V); CE, 18 (V); CXP, 20 (V).

[0036] Furthermore, the standard stock solutions of the target analytes include retinol standard stock solution, all-trans retinoic acid standard stock solution, 9-cis retinoic acid standard stock solution, all-trans retinal standard stock solution, retinol-d4 internal standard stock solution and all-trans retinoic acid-d6 internal standard stock solution, which are prepared by dissolving the standard sample or internal standard sample of the target analyte in methanol solvent, and the concentration of each is 1 mg / ml.

[0037] Furthermore, the standard curve working solution includes several designed concentration gradients obtained by serial dilution of W1 using a blank matrix, wherein W1 is prepared by dissolving 41.9 μL of 477.85 μg / ml retinol, 100 μL of 10 μg / ml all-trans retinoic acid, 20 μL of 10 μg / ml 9-cis retinoic acid, and 20 μL of 10 μg / ml retinal in 9818.1 μL of 5% BSA; in the standard curve working solution, the concentration range of retinol is 10-2000 ng / ml, the concentration range of all-trans retinoic acid is 0.5-100 ng / ml, the concentration range of 9-cis retinoic acid is 0.1-20 ng / ml, and the concentration range of retinal is 0.1-20 ng / ml.

[0038] Furthermore, the internal standard solution was prepared by adding 10 μL of 1 mg / mL retinoic acid-d6 internal standard and 420 μL of 1 mg / mL retinol-d4 internal standard into 16 ml of methanol.

[0039] Furthermore, the sample to be tested is obtained by placing a blood sample in a clean sample tube a, adding 1000 μL of methyl tert-butyl ether, mixing thoroughly, centrifuging at 4°C, aspirating the supernatant into a sample tube b, blowing dry with nitrogen gas at room temperature in the dark, adding 80% methanol solution to dissolve, mixing thoroughly, and centrifuging. The supernatant is the sample to be tested.

[0040] Furthermore, a quality control step is included. The quality control products used for quality control include high-concentration quality control products, medium-concentration quality control products and low-concentration quality control products. The high-concentration quality control is prepared by mixing 800 μL W1 and 200 μL blank serum matrix, the medium-concentration quality control is prepared by mixing 100 μL W1 and 900 μL blank serum matrix, and the low-concentration quality control sample is prepared by mixing 20 μL W1 and 1980 μL blank serum matrix.

[0041] Furthermore, the high-concentration quality control product contains: retinol at a concentration of 1600 ng / mL, all-trans retinoic acid at a concentration of 80 ng / mL, 9-cis retinoic acid at a concentration of 16 ng / mL, and retinal at a concentration of 16 ng / mL;

[0042] The medium-concentration quality control product contains: retinol at a concentration of 200 ng / mL, all-trans retinoic acid at a concentration of 10 ng / mL, 9-cis retinoic acid at a concentration of 2 ng / mL, and retinal at a concentration of 2 ng / mL;

[0043] The low-concentration quality control contains: retinol at a concentration of 40 ng / mL, all-trans retinoic acid at a concentration of 2 ng / mL, 9-cis retinoic acid at a concentration of 0.4 ng / mL, and retinal at a concentration of 0.4 ng / mL.

[0044] Beneficial effects of the present invention

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] 1. Based on the high sensitivity and specificity of HPLC-MS / MS, retinol, all-trans retinoic acid, 9-cis retinoic acid, and all-trans retinal are detected. By optimizing the pretreatment and mass spectrometry methods simultaneously, retinol, all-trans retinoic acid, 9-cis retinoic acid, and all-trans retinal with huge concentration differences can be detected with a single injection. This method only takes 5.2 minutes, which saves time for experiments with large sample sizes.

[0047] 2. The cis-trans isomers of all-trans retinoic acid and 9-cis retinoic acid are well separated in the liquid phase to obtain two independent peaks, which can be detected simultaneously. This allows us to monitor both vitamin A and its metabolic pathway, providing more possibilities for exploring the overall metabolism of vitamin A.

[0048] 3. The chromatographic column type is highly compatible with other compounds. This method uses reversed-phase chromatography, which is consistent with the column type of other antibiotics. It does not need to be replaced or balanced, saving time.

[0049] 4. Sample pretreatment is very simple, and no other substances are added to change the polarity of the substance. Instead, through reverse thinking, the sample detection is carried out by adjusting the liquid phase to the forward use of the reverse chromatographic column. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 shows a linear relationship diagram of retinol of the present invention;

[0051] Figure 2 Shown is a linear relationship diagram of 9-cis retinoic acid of the present invention;

[0052] Figure 3 Shown is a linear relationship diagram of all-trans retinoic acid of the present invention;

[0053] Figure 4 Shown is a linear relationship diagram of retinal of the present invention;

[0054] Figure 5 The detection spectrum of the sample of Example 1 of the present invention is shown; in the figure, blue is retinol, the peak in front of green is 9-cis retinoic acid, the peak behind green is all-trans retinoic acid, and red is retinal;

[0055] Figure 6 The liquid phase separation diagram of all-trans retinoic acid and 9-cis retinoic acid in Example 1 of the present invention is shown; in the figure, the peak time of 9-cis retinoic acid is 3.73, and the peak time of all-trans retinoic acid is 4.02;

[0056] Figure 7The liquid phase diagram shows that the all-trans retinoic acid and 9-cis retinoic acid solutions of Example 2 of the present invention are indistinguishable, and the front time is 3.26;

[0057] Figure 8 The liquid phase diagram shows that the all-trans retinoic acid and 9-cis retinoic acid solutions of Example 3 of the present invention are indistinguishable, and the front time is 3.15;

[0058] Figure 9 The liquid phase diagram of Example 4 of the present invention shows that all-trans retinoic acid and 9-cis retinoic acid solutions are indistinguishable.

[0059] The forward time is 3.6; DETAILED DESCRIPTION

[0060] Unless otherwise indicated, implied from the context, or customary in the art, all parts and percentages in this application are based on weight, and the test and characterization methods used are current as of the filing date of this application. Where applicable, the contents of any patents, patent applications, or publications referred to in this application are incorporated herein by reference in their entirety, and their equivalent patent families are also incorporated by reference, especially with respect to definitions of synthetic techniques, product and processing designs, polymers, comonomers, initiators, or catalysts disclosed in these documents in the art. If the definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition of the term provided in this application shall prevail.

[0061] Numerical ranges in this application are approximate values, so unless otherwise stated, they may include numerical values ​​outside the scope. Numerical ranges include all numerical values ​​from the lower limit to the upper limit increased by 1 unit, provided that there is an interval of at least 2 units between any lower value and any higher value. For example, if the description component, physical or other properties (such as molecular weight, melt index, etc.) is 100 to 1000, it means that all individual numerical values ​​are clearly enumerated, such as 100, 101, 102, etc., and all subranges, such as 100 to 166, 155 to 170, 198 to 200, etc. For the scope comprising a numerical value less than 1 or comprising a fraction greater than 1 (such as 1.1, 1.5, etc.), 1 unit is appropriately considered as 0.0001, 0.001, 0.01 or 0.1. For the scope comprising a single digit less than 10 (such as 1 to 5), 1 unit is usually considered as 0.1. These are only specific examples of what is intended, and all possible combinations of numerical values ​​between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application.

[0062] When used with respect to chemical compounds, unless expressly stated otherwise, the singular includes all isomeric forms and vice versa (e.g., "hexane" includes all isomers of hexane, individually or collectively). In addition, nouns using "a," "an," or "the" also include their plural forms unless expressly stated otherwise.

[0063] The terms "comprising", "including", "having" and their derivatives do not exclude the presence of any other components, steps or processes, and are irrelevant to whether these other components, steps or processes are disclosed in this application. To eliminate any doubt, all compositions using the terms "comprising", "including", or "having" in this application may include any additional additives, excipients or compounds unless expressly stated otherwise. In contrast, the term "essentially consisting of" excludes any other components, steps or processes from the scope of any subsequent description of the term, except those necessary for operational performance. The term "consisting of" does not include any components, steps or processes that are not specifically described or listed. Unless expressly stated otherwise, the term "or" refers to the listed members alone or in any combination.

[0064] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below with reference to the embodiments.

[0065] Example

[0066] The following examples are provided to illustrate preferred embodiments of the present invention. Those skilled in the art will appreciate that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to practice the present invention and, therefore, can be considered preferred embodiments of the present invention. However, those skilled in the art will appreciate from this disclosure that many modifications may be made to the specific embodiments disclosed herein while still achieving the same or similar results without departing from the spirit or scope of the present invention.

[0067] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs, and the disclosures herein and the materials they cite are hereby incorporated by reference. Those skilled in the art will recognize or be able to ascertain, through routine experimentation, many technical equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0068] Example 1 Detection of Retinol, All-trans Retinoic Acid, 9-cis Retinoic Acid, and All-trans Retinal in Serum Samples I. Preparation of Standard Solutions

[0069] Preparation of single-label stock solutions: Prepare single-label stock solutions according to the preparation method in Table 1. After preparation, seal and store in a -80℃ refrigerator.

[0070] Table 1 Preparation of single standard stock solution

[0071]

[0072] 2. Preparation of the highest point of retinol substances (W1): Prepare the highest point solution of retinol substances according to the preparation method in Table 2, and prepare W1 and mix well for later use.

[0073] Table 2 Preparation of the highest point (W1) of retinol substances

[0074]

[0075] 3. Preparation of internal standard solution of retinol

[0076] Take 10 μL of 1 mg / mL all-trans retinoic acid-d6 internal standard solution and 420 μL of 1 mg / mL retinol-d4 internal standard solution, add 16 ml of methanol to prepare an internal standard mixed solution, mix well and set aside.

[0077] Note: Calculate the exact concentration based on the actual weighing result and purity.

[0078] 4. Preparation of standard curve working solution

[0079] After the highest point (W1) of retinol substances is fully mixed, the standard curve working solution samples of various concentrations are obtained by stepwise dilution. The preparation method is shown in Table 3. After preparation, 100 μL aliquots are packaged and sealed and stored in a -80°C refrigerator.

[0080] Table 3 Configuration of standard curve working solution

[0081]

[0082] 5. Preparation of Quality Control Samples

[0083] Dilute W1 with blank matrix to prepare high-concentration quality control (HQC), medium-concentration quality control (MQC), and low-concentration quality control (LQC) samples. The preparation methods are shown in Table 4, and the concentrations of the standard curve quality control samples are shown in Table 5. After preparation, dispense 100 μL of each sample into 1.5 mL centrifuge tubes and prepare them immediately before use.

[0084] Table 4 Quality control sample preparation

[0085] HQC MQC LQC Addition volume (μL) 800W1 100W1 20W1 Blank serum matrix (μL) 200 900 1980

[0086] Table 5 Concentrations of standard curves and quality control samples (ng / ml)

[0087]

[0088] Note: The actual concentration is subject to the measured target value.

[0089] 6. Sample Processing

[0090] (1) Blood sample collection

[0091] If collecting plasma or serum specimens, use a vacuum negative pressure blood collection tube containing EDTA anticoagulant (purple) to collect plasma, and a vacuum negative pressure blood collection tube with a yellow cap (separation gel) to collect serum;

[0092] If random blood is collected, the plasma or serum must be separated by centrifugation (3500 rpm, 8 min) within 2 hours and stored after aliquoting;

[0093] Plasma / serum is stored at -80℃ for a long time. When processing, it should be taken out from -80℃ and placed at -20℃ for about 30 minutes, and then transferred to 4℃ for about 1 hour to dissolve.

[0094] (2) Sample processing

[0095] Use 1.5mL Eppendorf tubes for sample preparation, accurately measure 100μL of standard curve samples, quality control samples, and human serum samples and place them in clean centrifuge tubes; add 20μL of internal standard solution, mix thoroughly for 2 minutes, then add 1000μL of methyl tert-butyl ether, mix thoroughly for 5 minutes, and centrifuge at 12000rpm for 5 minutes at 4℃; draw 800μL of supernatant into a 1.5ml centrifuge tube, and blow it dry with nitrogen at room temperature in the dark (about 10 minutes), add 100μL of reconstitution solution, mix for 2 minutes, centrifuge for 3 minutes, transfer the supernatant to a 96-well plate, and inject after writing the sequence.

[0096] (3) Reconstitution solution

[0097] Add 20 ml of water to 80 ml of methanol and mix well.

[0098] 7. HPLC-MS / MS detection conditions

[0099] (1) Chromatographic conditions

[0100] Chromatographic column: ACE PFP column (100x2.1 mm, 2.6 μm);

[0101] Mobile phase A: methanol: acetonitrile: water: formic acid = 300:400:300:0.1;

[0102] Mobile phase B: methanol: acetonitrile: water: formic acid = 300:550:150:0.1;

[0103] Strong wash solution: 80% methanol;

[0104] The flow rate was 0.3 mL / min, the column temperature was 30 °C, and 5 μL was injected.

[0105] Gradient elution was used, and the elution gradient settings are shown in Table 6;

[0106] Table 6 Liquid phase elution gradient

[0107]

[0108] (2) Mass spectrometry conditions

[0109] Ion source: electrospray ionization source, positive ion mode;

[0110] IonSpary Voltage(IS): 4500V;

[0111] Temperature (TEM): 450°C;

[0112] Ion Source Gas 1(GS1): 40psi;

[0113] Ion Source Gas 2(GS2): 45psi;

[0114] Curtain Gas: 40psi;

[0115] Scan mode: MRM. The MRM parameter conditions are shown in Table 7.

[0116] Table 7 MRM quantitative detection product ion parameters

[0117]

[0118] In the above table, Q1 is the parent ion; Q3 is the daughter ion; T is the dwell time; DP is the declustering voltage; EP is the injection voltage, CE is the collision voltage; and CXP is the collision cell ejection voltage.

[0119] 8. Results

[0120] (1) Standard curve

[0121] The prepared standard curve working solutions of 9 retinol substances with known concentrations of W1-W9 were tested. The chromatographic and mass spectrometric parameters were set as described above. The internal standard quantification method was used, with the concentration of the standard curve working solution as the X-axis and the chromatographic peak area ratio of the standard curve working solution to the internal standard solution as the Y-axis. The corresponding standard curve equation is shown in Table 8 below, and the corresponding standard curve graph is shown in Figure 1 、 Figure 2 、 Figure 3 、 Figure 4 shown.

[0122] Table 8 Standard curve equations for retinols

[0123] project Linear equations Correlation coefficient r Weighting Factor Standard curve Retinol y=0.000312958x-6.83048e-5 0.998 1 / x2 Figure 1 9-cis retinoic acid y=0.06584x+0.05932 0.996 1 / x2 Figure 2 All-trans retinoic acid y=0.08937x+0.01115 0.998 1 / x Figure 3 Retinaldehyde y=0.22680x+0.00973 0.997 1 / x2 Figure 4

[0124] Conclusion: According to the above table and Figures 1 to 4 It can be seen that retinol, 9-cis retinoic acid, all-trans retinoic acid and retinal have good linear relationships within the linear range.

[0125] (2) Detection patterns and results

[0126] Three serum samples were tested using the method described in Example 1. 5 μL of the test sample was taken and tested using HPLC-MS / MS. Figure 5 and Figure 6 The detection spectrum of one sample is shown, and the detection results of the test substances in the three samples are shown in Table 9.

[0127] Table 9 Test results of the tested substances in three samples

[0128]

[0129] Conclusion: Under the detection conditions provided in this example, retinol, 9-cis retinoic acid, all-trans retinoic acid, and retinal can be separated and detected by HPLC-MS / MS. The peak ejaculation time of all-trans retinoic acid is 4.02, and the peak ejaculation time of 9-cis retinoic acid is 3.73. All-trans retinoic acid and 9-cis retinoic acid are completely separated by liquid phase. The liquid phase peak ejaculation times of retinol and 9-cis retinoic acid are both 3.73. Since the ion pairs of retinol and 9-retinoic acid are completely different, they are ultimately detected and differentiated by mass spectrometry.

[0130] (2) Linear range and detection limit

[0131] The injection was repeated 6 times, and the detection limit was determined when the peak height reached a concentration 3 times higher than the signal height.

[0132] Table 10 Linear range detection limit and detection limit

[0133] Serial number project English abbreviation Linear range (ng / ml) Detection limit (ng / ml) 1 Retinol Retinol 10-2000 0.5 2 All-trans retinoic acid Retinoic 0.5-100 0.03 3 9-cis retinoic acid 9-cis-Retinoic 0.1-20 0.03 4 Retinaldehyde Retinal 0.1-20 0.03

[0134] Conclusion: The linear range of retinol is 10-2000 ng / ml, and the lowest quantitative limit of blood concentration is 0.5 ng / ml; the linear range of all-trans retinoic acid is 0.5-100 ng / ml, and the lowest quantitative limit of blood concentration is 0.03 ng / ml; the linear range of 9-cis retinoic acid is 0.1-20 ng / ml, and the lowest quantitative limit of blood concentration is 0.03 ng / ml; the linear range of all-trans retinoic acid is 0.1-20 ng / ml, and the lowest quantitative limit of blood concentration is 0.03 ng / ml.

[0135] (3) System adaptability, precision and stability

[0136] System suitability: Repeat the injection 6 times to obtain the retention time of the peak area, the CV value of the internal standard retention time and concentration.

[0137] Precision: Intra-batch precision: Prepare six samples of the same concentration, pre-treat them, and then load them onto the instrument. Calculate the precision between the six samples. Inter-batch precision: Perform intra-batch precision testing on three consecutive days. Calculate the precision between the three days, which is called inter-batch precision.

[0138] 24h stability: After testing the sample concentration, leave the sample for 24 hours and test the sample concentration again to compare the sample deviation.

[0139] Table 11 System suitability, precision and stability verification data

[0140] project Require Retinol All-trans retinoic acid 9-cis retinoic acid Retinaldehyde System suitability (RT) <5％ 0.5％ 0.4％ 0.5％ 0.3％ System adaptability (IS-RT) <5％ 0.5％ 0.4％ 0.4％ 0.4％ System adaptability <5％ 2.8％ 3.0％ 4.2％ 3.3％ Residue <20％ 1.3％ 0％ 12.4％ 2.3％ Intra-batch precision <15％ 4.5 3.2 5.2 4.3 Inter-batch precision <15％ 3.2 4.5 6.6 5.1 24h stability <15％ 4.8 6.9 8.2 12.1

[0141] The above results show that the system suitability, residue, and precision of retinoic acid, all-trans retinoic acid, 9-cis retinoic acid, and retinal all meet the relevant requirements of: FDA: Bioanalytical Method Validation-Guidance for Industry; CLSI C62-A: A New Standard for Clinical Mass Spectrometry; and Chinese Medical Doctor Association Branch of Laboratory Physicians: Guidelines for the Development and Validation of Clinical Detection Methods Using Liquid Chromatography Tandem Mass Spectrometry.

[0142] Example 2 Mobile phase screening

[0143] During the experimental exploration, three mobile phases were selected:

[0144] (1) Mobile phase A: 0.1% formic acid in water, mobile phase B: 0.1% formic acid in methanol;

[0145] (2) Mobile phase A: 0.1% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile;

[0146] (3) Mobile phase A: methanol: acetonitrile: water: formic acid = 300:400:300:0.1; Mobile phase B: methanol: acetonitrile: water: formic acid = 300:550:150:0.1;

[0147] The results showed that under the three mobile phases, the four analytes all produced peaks within the appropriate range. In comparison, the third mobile phase A and mobile phase B had the smallest polarity difference, which allowed for maximum gradient adjustment. This was because the detection scheme for the four substances used mobile phase A: methanol: acetonitrile: water: formic acid = 300:400:300:0.1; mobile phase B: methanol: acetonitrile: water: formic acid = 300:550:150:0.1.

[0148] Example 3 Liquid Phase Separation Optimization 1

[0149] Other conditions are the same as in Example 1, except that:

[0150] Chromatographic column: Phenomenex Kinetex C18 column (100x2.1 mm, 2.6 μm);

[0151] Liquid phase conditions: flow rate 0.3 mL / min, column temperature 30 °C, injection 5 μL;

[0152] Gradient elution was used, and the elution gradient settings are shown in Table 12;

[0153] Table 12 Liquid phase elution gradient

[0154]

[0155] The results showed that the liquid phase spectrum was as follows: Figure 7 As shown in the figure, the peak times of all-trans retinoic acid and 9-cis retinoic acid are both 3.26, and they cannot be separated and detected.

[0156] Example 4 Liquid Phase Separation Optimization 2

[0157] Other conditions are the same as in Example 1, except that:

[0158] Chromatographic column: Phenomenex Kinetex C18 column (100x2.1 mm, 2.6 μm);

[0159] Liquid phase conditions: flow rate 0.3 mL / min, column temperature 30 °C, injection 5 μL;

[0160] Gradient elution was used, and the elution gradient settings are shown in Table 13;

[0161] Table 13 Liquid phase elution gradient

[0162]

[0163] The results showed that the liquid phase spectrum was as follows: Figure 8 As shown, the peak times of all-trans retinoic acid and 9-cis retinoic acid are both 3.15, and they cannot be separated and detected.

[0164] Example 5 Liquid Phase Separation Optimization 3

[0165] Other conditions are the same as in Example 1, except that:

[0166] Chromatographic column: Phenomenex Kinetex C18 column (100x2.1 mm, 2.6 μm);

[0167] Liquid phase conditions: flow rate 0.3 mL / min, column temperature 30 °C, injection 5 μL;

[0168] Gradient elution was used, and the elution gradient settings are shown in Table 14;

[0169] Table 14 Liquid phase elution gradient

[0170]

[0171] The results showed that the liquid phase spectrum was as follows: Figure 9 As shown in the figure, the peak times of all-trans retinoic acid and 9-cis retinoic acid are both 3.6, and they cannot be separated and detected.

[0172] Example 6 Mass Spectrum Optimization

[0173] Other conditions are the same as those in Example 1, except that: cone voltage is 80 V, collision energy is 26.7 V, and curtain gas is 30.

[0174] The results showed that according to the human physiological reference range, the retinol concentration curve reached 10-2000 ng / ml, and the 9-cis retinoic acid and retinal concentrations were 0.1-20 ng / ml. Under the conditions of Example 6, the retinol high point was oversaturated, the response was too high, and the linear relationship could only reach 0.89, which did not meet the requirement of 0.99. The linearity of the retinol standard curve was not good.

[0175] The cone voltage was adjusted from the optimal 80 V to 50 V; the collision energy was adjusted from 26.7 V to 15 V; and the curtain gas was adjusted from 30 to 40 to obtain the optimized mass spectrometry conditions, as shown in Example 1. Finally, the response of retinol decreased by 20 times, and the linear relationship reached 0.998.

[0176] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A method for detecting retinol, all-trans retinoic acid, 9-cis retinoic acid, and all-trans retinal based on HPLC-MS / MS, characterized by: The following steps are involved: (1) Prepare the target analyte standard stock solution, standard curve working solution, and internal standard solution; (2) The blood sample is processed to obtain the test sample; (3) Using HPLC-MS / MS to detect the standard curve working solution with the internal standard solution added, the chromatographic peak area ratio of the target analyte to the corresponding isotope internal standard is used as the ordinate, and the concentration of the target analyte in the standard curve working solution is used as the abscissa, respectively, to fit the standard curve equation corresponding to the target analyte, and using HPLC-MS / MS to detect the test sample with the internal standard solution added, the content of the target analyte in the test sample is calculated according to the corresponding standard curve equation; wherein, the chromatographic conditions for HPLC-MS / MS detection are: Chromatographic column: ACE PFP column, 100 x 2.1 mm, 2.6 μm; Mobile phase A: methanol: acetonitrile: water: formic acid = 300:400:300:0.1; Mobile phase B: methanol: acetonitrile: water: formic acid = 300:550:150:0.1; Strong wash solution: 80% methanol; The flow rate was 0.3 mL / min, the column temperature was 30°C, and the injection volume was 5 μL; The gradient elution method is as follows: the volume fraction of mobile phase A + the volume fraction of mobile phase B = 100%; where: From 0 to 3 min, the volume fraction of mobile phase A decreased from 15% to 0; The volume fraction of mobile phase A was maintained at 0 for 3–4 min; 4-4.1 min: the volume fraction of mobile phase A increased from 0% to 15%; The volume fraction of mobile phase A was maintained at 15% from 4.1 to 5 min and stopped at 5.2 min.

2. The method for detecting retinol, all-trans retinoic acid, 9-cis retinoic acid and all-trans retinal based on HPLC-MS / MS according to claim 1, characterized in that: The mass spectrometry conditions for the HPLC-MS / MS detection are: Ion source: electrospray ionization source, positive ion mode; Ion spray voltage, IS: 4500 V; Ion source temperature, TEM: 450 °C; Ion source nebulizer gas, GS1: 40 psi; Ion source heating auxiliary gas, GS2: 45 psi; Curtain gas, CG: 40 psi; Scan mode: MRM, MRM parameter conditions are as follows: Retinol: Q1, 269 m / z; Q3, 93 m / z; T, 60 m sec; DP, 50 V; EP, 7 V; CE, 15 V; CXP, 12 V; 9-cis-retinoic acid: Q1, 301 m / z; Q3, 122.8 m / z; T, 60 m sec; DP, 60 V; EP, 10 V; CE, 20.91 V; CXP, 20 V; All-trans retinoic acid: Q1, 301 m / z; Q3, 123.1 m / z; T, 60 m sec; DP, 60 V; EP, 8 V; CE, 19.66V; CXP, 15 V; Retinal: Q1, 285 m / z; Q3, 161 m / z; T, 60 m sec; DP, 60 V; EP, 10 V; CE, 12.04 V; CXP, 20V; Retinol-d4: Q1, 273 m / z; Q3, 93.8 m / z; T, 60 m sec; DP, 70 V; EP, 7V; CE, 24V; CXP, 12V; All-trans retinoic acid-d6: Q1, 307.7 m / z; Q3, 123 m / z; T, 60 m sec; DP, 62 V; EP, 10 V; CE, 18 V; CXP, 20 V.

3. The method for detecting retinol, all-trans retinoic acid, 9-cis retinoic acid and all-trans retinal based on HPLC-MS / MS according to claim 1, characterized in that: The standard stock solutions of the target analytes include retinol standard stock solution, all-trans retinoic acid standard stock solution, 9-cis retinoic acid standard stock solution and all-trans retinal standard stock solution, retinol-d4 internal standard stock solution and all-trans retinoic acid-d6 internal standard stock solution, which are prepared by dissolving the standard sample or internal standard sample of the target analyte in methanol solvent, and the concentration of each is 1 mg / mL.

4. The method for detecting retinol, all-trans retinoic acid, 9-cis retinoic acid and all-trans retinal based on HPLC-MS / MS according to claim 1, characterized in that: The standard curve working solution includes several designed concentration gradients obtained by serial dilution of W1 using a blank matrix, wherein W1 is prepared by dissolving 41.9 μL of 477.85 μg / mL retinol, 100 μL of 10 μg / mL all-trans retinoic acid, 20 μL of 10 μg / mL 9-cis retinoic acid, and 20 μL of 10 μg / mL retinal in 9818.1 μL of 5% BSA; in the standard curve working solution, the concentration range of retinol is 10-2000 ng / mL, the concentration range of all-trans retinoic acid is 0.5-100 ng / mL, the concentration range of 9-cis retinoic acid is 0.1-20 ng / mL, and the concentration range of retinal is 0.1-20 ng / mL.

5. The method for detecting retinol, all-trans retinoic acid, 9-cis retinoic acid and all-trans retinal based on HPLC-MS / MS according to claim 1, characterized in that: The internal standard solution was prepared by adding 10 μL of 1 mg / mL retinoic acid-d6 internal standard and 420 μL of 1 mg / mL retinol-d4 internal standard to 16 ml of methanol.

6. The method for detecting retinol, all-trans retinoic acid, 9-cis retinoic acid and all-trans retinal based on HPLC-MS / MS according to claim 1, characterized in that: The test sample is obtained by placing a blood sample in a clean sample tube a, adding 1000 μL of methyl tert-butyl ether, mixing thoroughly, centrifuging at 4°C, and aspirating the supernatant into a sample tube b. The sample is dried under nitrogen at room temperature in the dark, and then re-dissolved in 80% methanol solution. The sample is mixed and centrifuged. The supernatant is the test sample.

7. The method for detecting retinol, all-trans retinoic acid, 9-cis retinoic acid and all-trans retinal based on HPLC-MS / MS according to claim 4, characterized in that: Quality control steps are also included. The quality control samples used for quality control include high-concentration quality control, medium-concentration quality control, and low-concentration quality control. The high-concentration quality control is prepared by mixing 800 µL W1 and 200 µL blank serum matrix, the medium-concentration quality control is prepared by mixing 100 µL W1 and 900 µL blank serum matrix, and the low-concentration quality control sample is prepared by mixing 20 µL W1 and 1980 µL blank serum matrix.

8. The method for detecting retinol, all-trans retinoic acid, 9-cis retinoic acid and all-trans retinal based on HPLC-MS / MS according to claim 7, characterized in that: The high-concentration quality control product contains: 1600 ng / mL retinol, 80 ng / mL all-trans retinoic acid, 16 ng / mL 9-cis retinoic acid, and 16 ng / mL retinal; the medium-concentration quality control product contains: 200 ng / mL retinol, 10 ng / mL all-trans retinoic acid, 2 ng / mL 9-cis retinoic acid, and 2 ng / mL retinal; the low-concentration quality control product contains: 40 ng / mL retinol, 2 ng / mL all-trans retinoic acid, 0.4 ng / mL 9-cis retinoic acid, and 0.4 ng / mL retinal.

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