CN115436502A

CN115436502A - Method for detecting concentration of folic acid and derivatives thereof in blood plasma based on LC-MS/MS technology

Info

Publication number: CN115436502A
Application number: CN202210923639.1A
Authority: CN
Inventors: 仇峰; 成永之; 连增林
Original assignee: LIANYUNGANG JINKANG HEXIN PHARMACEUTICAL CO Ltd
Current assignee: LIANYUNGANG JINKANG HEXIN PHARMACEUTICAL CO Ltd
Priority date: 2022-08-02
Filing date: 2022-08-02
Publication date: 2022-12-06

Abstract

The invention discloses a method for detecting concentration of folic acid and derivatives thereof in blood plasma based on LC-MS/MS technology, which comprises the following steps: preparing a mixed standard solution from folic acid, dihydrofolic acid, tetrahydrofolic acid and 5-methyltetrahydrofolic acid, adding the mixed standard solution into blank plasma, adding a stabilizer, adding an internal standard working solution, performing vortex and centrifugation, mixing a supernatant with the stabilizer, performing LC-MS/MS detection, and performing linear regression on the concentration of a substance to be detected according to the peak area ratio of the substance to be detected to the internal standard peak area to obtain a standard curve; and adding methanol into the plasma to be detected, adding an internal standard working solution and a stabilizer, carrying out vortex and centrifugation, mixing the supernatant with the stabilizer, carrying out LC-MS/MS detection, recording peak areas corresponding to the object to be detected and the internal standard substance, calculating a peak concentration ratio, and substituting the peak concentration ratio into a standard curve to obtain the concentration of the object to be detected. The invention solves the problem that the folic acid components in the biological sample are unstable in analysis, and improves the accuracy and reliability.

Description

Method for detecting concentration of folic acid and derivatives thereof in blood plasma based on LC-MS/MS technology

Technical Field

The invention relates to a method for detecting concentrations of different forms of folic acid in blood plasma based on an LC-MS/MS (liquid chromatography-mass spectrometry) technology, in particular to a method for simultaneously determining concentrations of folic acid, dihydrofolic acid, tetrahydrofolic acid and 5-methyltetrahydrofolic acid in blood plasma based on an LC-MS/MS technology.

Background

Folate (also known as vitamin B9, folic acid, FA) is a water-soluble vitamin that is important for DNA synthesis, RNA transcription, methionine synthesis from homocysteine, and various other chemical reactions involved in cellular metabolism. Especially folic acid is important for the period of frequent cell division and growth, and the intake of sufficient folic acid during pregnancy, lactation and infancy is essential for the health and normal growth of mother and infant. Humans cannot synthesize folate and rely on supplementation to maintain their normal levels. After entering the body, folic acid can be converted into Dihydrofolic acid (DHFA) under the action of Dihydrofolic acid reductase, and further converted into Tetrahydrofolic acid (THFA); under the action of serine hydroxymethyltransferase, tetrahydrofolic acid can be activated into 5,10-methylenetetrahydrofolic acid (METHF), and the reaction is reversible; under the action of methylenetetrahydrofolate reductase, 5, 10-methylenetetrahydrofolate is converted into 5-Methyltetrahydrofolate (MTHF). Therefore, folic acid is a generic term for a class of compounds that are similar in chemical structure to those described above. Different forms of folic acid have unique biological functions, and whether the state of folic acid of a patient is clinically evaluated or the different forms of folic acid compounds are best determined by scientific research, the use of folic acid determination is limited due to the complicated sample preparation process and the instability of reducing folic acid.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is used as a high-order analysis method for analyzing folic acid, and different folic acid forms can be studied more deeply than a common microbiological assay or an immune protein method. However, accurate measurement of a series of metabolites still has great difficulty, and firstly folic acid is very unstable and easily lost in the sample preparation process, and in addition, different folic acid metabolites can be converted, so that it is difficult to determine whether the measured concentration is consistent with the concentration and proportion of the corresponding folic acid form in the sample before treatment.

Folic acid is an artificially synthesized compound, unlike other forms of folic acid compounds, which do not exist in the natural world, have good stability, but need to be finally reduced to 6S-5-methyltetrahydrofolic acid in the body to be absorbed and metabolized by cells. Folic acid in nature is an amphoteric unstable compound, and changes in ionic form along with changes in pH value, and particularly, tetrahydrofolic acid, dihydrofolic acid, 5,10-methylenetetrahydrofolic acid and 5-methyltetrahydrofolic acid are extremely sensitive to pH value. Since folic acid may be converted or degraded during the course of the analysis, most LC-MS/MS methods focus on measuring folic acid in certain isoforms that are more stable.

Nelson et al simultaneously measure homocysteine, 5-methyltetrahydrofolate and folate levels in serum for the first time using liquid chromatography tandem mass spectrometry. Nandania et al describe for the first time a measurement method for measuring different forms of folic acid in a biological sample. However, the above method is time-consuming and labor-consuming in preparing the analysis sample, the related folic acid compounds are folic acid, 5-methyltetrahydrofolic acid, tetrahydrofolic acid, dihydrofolic acid, and 5,10-methylenetetrahydrofolic acid, the above compounds are unstable and interconvert, and the long preparation process causes the loss or conversion of the above components after the sample preparation, thereby affecting the accuracy of the final result.

Disclosure of Invention

Based on the defects of the technical scheme, the invention aims to provide the LC-MS/MS detection method which has high accuracy and short time required for sample preparation and measurement and can detect 5-methyltetrahydrofolate, tetrahydrofolate, dihydrofolate and folic acid in plasma at high flux.

In order to achieve the above purpose, the invention provides the following technical scheme:

a method for detecting the concentration of folic acid and derivatives thereof in blood plasma based on LC-MS/MS technology is used for simultaneously determining the concentrations of folic acid, dihydrofolic acid, tetrahydrofolic acid and 5-methyltetrahydrofolic acid in the blood plasma, and comprises the following steps:

step (1), preparation of a standard curve: preparing folic acid, dihydrofolic acid, tetrahydrofolic acid and 5-methyltetrahydrofolic acid into mixed standard solutions with different concentrations, adding the mixed standard solutions into blank plasma and adding a stabilizer, uniformly mixing, adding an internal standard working solution, performing vortex, centrifuging, taking supernate and mixing the supernate with the stabilizer to obtain a standard curve sample solution, feeding an LC-MS/MS system, performing LC-MS/MS detection according to a liquid chromatography-mass spectrometry combined detection condition, recording the corresponding peak area of an object to be detected, and performing linear regression on the concentration of the object to be detected and the internal standard peak area according to the ratio of the object to be detected and the internal standard peak area to obtain a corresponding standard curve;

step (2), determination of folic acid, dihydrofolic acid, tetrahydrofolic acid and 5-methyltetrahydrofolic acid in blood plasma to be tested: adding methanol into blood plasma to be detected, adding an internal standard working solution and a stabilizer, performing vortex and centrifugation, and mixing supernatant with the stabilizer; a sample injection LC-MS/MS system performs LC-MS/MS detection according to the detection conditions of liquid chromatography-mass spectrometry, records peak areas corresponding to the object to be detected and the internal standard substance, calculates peak concentration ratio, substitutes the peak concentration ratio into a standard curve, and calculates to obtain the concentration of the object to be detected;

wherein, the detection conditions of the liquid chromatogram-mass spectrum combination are as follows:

liquid chromatography conditions: a C18 reverse phase chromatography column; mobile phase A:0.1% aqueous formic acid, mobile phase B:0.1% formic acid in methanol; and (3) an elution mode: starting elution by isocratic gradient, eluting by 95% -98% of mobile phase A for not less than 0.5min, then increasing the proportion of mobile phase B to 100% in not more than 1min, continuously eluting by 100% of mobile phase B for not less than 3min, and adjusting the proportion of mobile phase to initial gradient; flow rate: 0.400-0.600 mL/min;

mass spectrum conditions: ESI source is adopted, and detection is performed in a negative ion MRM mode.

The stabilizer is a mixed aqueous solution of Dithiothreitol (DTT) and ascorbic acid (vitamin C, vc), and the concentration of the dithiothreitol and the ascorbic acid in the stabilizer is not less than 5mg/mL. The stabilizer is prepared by the following steps: taking a proper amount of dithiothreitol and a proper amount of ascorbic acid, dissolving the dithiothreitol and the ascorbic acid in a proper amount of water, and preparing a mixed aqueous solution as a stabilizer.

The internal standardAs a solution, sodium phenytoin or an isotope label C ¹³ The-5-methyltetrahydrofolic acid is used as an internal standard substance, dithiothreitol and ascorbic acid are added, and an internal standard solution prepared from methanol is adopted. In the internal standard working solution, the concentration of an internal standard substance is 60-80 mug/mL, the concentration of dithiothreitol is 1mg/mL, and the concentration of ascorbic acid is 1mg/mL.

Preferably, the liquid chromatography conditions are: an Agilent Zorbax XDB C18 chromatographic column with specification of 50mm × 2.1mm,3.5 μm; column temperature: room temperature; mobile phase A:0.1% aqueous formic acid; mobile phase B:0.1% formic acid in methanol; and (3) an elution mode: eluting with 98% mobile phase A for 0.5min, then increasing the proportion of mobile phase B to 100% within 0.5min, eluting with 100% mobile phase B for 3min, adjusting the mobile phase proportion to initial gradient within 0.1min, and continuously balancing the chromatographic column for 5min; flow rate: 0.400mL/min; sample introduction amount: 5 μ L.

More preferably, the elution method is as follows: 0.00-0.50min,2% mobile phase B;0.50-1.00min,2% -100% of mobile phase B;1.00-4.00min,100% mobile phase B;4.00-4.01min,100% -2% of mobile phase B;4.01-5.00min,2% of mobile phase B.

Preferably, the mass spectrometry conditions are as follows: ESI ionization source, MRM detection mode of anion scanning, gas curtain gas and atomization gas both use nitrogen, the set values are 20 and 10psi respectively; the source spray Voltage (IonSpray Voltage) in the negative ion mode was set to-4500V, ion pair of folic acid: m/z 440 → 311, impact energy (CE) -27eV; ion pair of dihydrofolic acid: m/z 442 → 176, collision Energy (CE) 33eV; ion pair of tetrahydrofolic acid: m/z 458 → 286, collision Energy (CE) 33eV; ion pair of 5-methyltetrahydrofolate: m/z is 458 → 329 and Collision Energy (CE) is-36 eV.

In the step (1), the preparation of the mixed standard solution comprises the following steps: dissolving folic acid, dihydrofolic acid, tetrahydrofolic acid and 5-methyltetrahydrofolic acid reference substances by using a proper amount of DMSO (dimethylsulfoxide), and respectively preparing folic acid reference substance stock solution with the concentration of 1.00mg/ml, dihydrofolic acid reference substance stock solution with the concentration of 1.00mg/ml, tetrahydrofolic acid reference substance stock solution with the concentration of 1.00mg/ml and 5-methyltetrahydrofolic acid reference substance stock solution with the concentration of 1.00 mg/ml; mixing the 4 reference stock solutions, and diluting with methanol to obtain mixed standard solutions with different concentrations.

The concentration of dithiothreitol and ascorbic acid in the standard curve sample solution is not less than 0.9mg/mL.

Preferably, the preparation method of the standard curve sample solution comprises the following steps: taking 50 mu L of blank plasma, adding 5 mu L of mixed standard working solution, vortexing for 5-10 s, adding 5 mu L of stabilizer, vortexing for 10s, adding 300 mu L of internal standard working solution, vortexing for no less than 1min, centrifuging at 12000rpm for no less than 10min, taking supernate, and uniformly mixing 20 mu L of supernate with 80 mu L of stabilizer to ensure that the concentration of dithiothreitol and ascorbic acid in the standard curve sample solution is no less than 0.9mg/mL.

More preferably, the method for preparing the sample solution for preparing the standard curve comprises: taking 50 mu L of blank plasma, adding 5 mu L of mixed standard working solution, swirling for 5-10 s, adding 5 mu L of stabilizer with dithiothreitol and ascorbic acid concentration of 5mg/mL, swirling for 10s, adding 300 mu L of internal standard working solution with dithiothreitol and ascorbic acid concentration of 60-80 mu g/mL, swirling for no less than 1min, and centrifuging at 12000rpm for no less than 10min, taking supernatant, and uniformly mixing 20 mu L of supernatant with 80 mu L of stabilizer with dithiothreitol and ascorbic acid concentration of 1mg/mL.

In the step (2), preferably, the concentration of the dithiothreitol and the ascorbic acid in the supernatant is not less than 0.9mg/mL, and the concentration of the internal standard substance in the supernatant is 5-20 mu g/mL.

Preferably, 50 mu L of plasma to be detected is taken, 5 mu L of methanol is added, vortex is carried out for 5-10 s, 5 mu L of stabilizer with the concentration of dithiothreitol and ascorbic acid being 5mg/mL is added, vortex is carried out for 10s, 300 mu L of internal standard working solution with the concentration of dithiothreitol and ascorbic acid being 60-80 mu g/mL and the concentration of dithiothreitol and ascorbic acid being 1mg/mL is added, vortex is not less than 1min, centrifugation at 12000rpm is not less than 10min, 20 mu L of supernatant is taken and is uniformly mixed with 80 mu L of stabilizer, so that the concentration of dithiothreitol and ascorbic acid in the sample solution is not less than 0.9mg/mL.

The blank plasma refers to plasma without exogenous folic acid introduced. The plasma sample to be detected refers to the plasma without introducing exogenous folic acid or with introduced exogenous folic acid.

The blank plasma or the plasma to be detected is as follows: after blood is taken, the blood plasma obtained by centrifugation at 2-6 ℃ is preserved at the temperature not higher than-20 ℃.

It is another object of the present invention to provide the use of the method of the present invention for determining the concentration of folic acid and derivatives thereof in plasma.

Compared with the prior art, the invention has the beneficial effects that:

the shorter the chromatographic separation time, the more beneficial the detection accuracy for LC-MS/MS, and the influence of flow on the stability of the sample is also important, the inventors evaluated the stability of folic acid in sample treatment and determined the optimal method conditions. To minimize the presence of matrix compounds in plasma and to ensure selectivity of the process, the inventors did not choose to purify the sample by Solid Phase Extraction (SPE), but rather chose to precipitate the proteins with organic solvents and to minimize the precipitation process. The inventor finds that related impurities can seriously interfere the content of the dihydrofolic acid under the ion channel of the dihydrofolic acid, and endogenous substances and target compounds have certain separation degree within the total detection time of 5min by optimizing chromatographic elution conditions.

The invention solves the problem that the folic acid components in the biological sample are unstable in the analysis process, and improves the accuracy and reliability of the analysis method; compared with the prior art, the method has the advantages of less sample consumption, high sensitivity, high detection flux, greatly improved result accuracy and capability of truly reflecting the actual conditions of different folic acid metabolites in the sample.

Drawings

FIG. 1 is the MS2 product ion spectrum of Folic Acid (FA).

FIG. 2 is the MS2 product ion spectrum of Dihydrofolate (DHFA).

FIG. 3 is an ion spectrum of the MS2 product of tetrahydrofolic acid (THFA).

FIG. 4 is an MS2 product ion spectrum of 5-methyltetrahydrofolate (5-MTHF).

FIG. 5 IS a typical chromatogram of Folic Acid (FA), dihydrofolic acid (DHFA), tetrahydrofolic acid (THFA), 5-methyltetrahydrofolic acid (5-MTHF), and sodium phenytoin (IS) in mouse blank plasma; wherein, fig. 5A is a typical chromatogram of folic acid in mouse blank plasma, fig. 5B is a typical chromatogram of dihydrofolic acid in mouse blank plasma, fig. 5C is a typical chromatogram of tetrahydrofolic acid in mouse blank plasma, fig. 5D is a typical chromatogram of 5-methyltetrahydrofolic acid in mouse blank plasma, and fig. 5E is a typical chromatogram of phenytoin sodium in mouse blank plasma.

FIG. 6 IS a typical chromatogram of Folate (FA), dihydrofolate (DHFA), tetrahydrofolate (THFA), 5-methyltetrahydrofolate (5-MTHF), and sodium phenytoin (IS) in plasma of mice after addition of a control; wherein, fig. 6A is a typical chromatogram of folic acid in blood plasma of a mouse after a control is added, fig. 6B is a typical chromatogram of dihydrofolic acid in blood plasma of a mouse after a control is added, fig. 6C is a typical chromatogram of tetrahydrofolic acid in blood plasma of a mouse after a control is added, fig. 6D is a typical chromatogram of 5-methyltetrahydrofolic acid in blood plasma of a mouse after a control is added, and fig. 6E is a typical chromatogram of phenytoin sodium in blood plasma of a mouse after a control is added.

Fig. 7 is a plasma concentration-time curve (n = 8) of Folate (FA), dihydrofolate (DHFA) and 5-methyltetrahydrofolate (5-MTHF) after oral administration of folate to mice.

Detailed Description

The detailed description of the embodiments of the present invention is not intended to limit the scope of the technical solutions of the present invention as claimed, and embodiments obtained by a person skilled in the art without creative efforts based on the embodiments of the present invention should fall within the scope of the present invention.

Instruments and reagents

The applied biosystems liquid mass spectrometry system API 6500Qtrap (containing Agilent 1290 liquid chromatograph, ESI and APCI interface ion source, analysis Software 1.6.3 chromatographic workstation); IKA Vortex oscillator model 2 (Aika instruments, inc.), 3K15 low temperature high speed centrifuge (Sigma, USA), KQ-500E ultrasonic cleaner (Kunshan ultrasonic instruments, inc.), DV215CD electronic balance (Aohaus instruments, inc.), CP224C electronic balance (Aohaus instruments, inc.).

The reference information is shown in table 1; the selected negative ion internal standard is phenytoin sodium (batch BNV229, purity 99.97%, available from Shanghai Bigdi medicine science and technology Co., ltd.); dithiothreitol (DTT, 99.0% pure, batch No. C12084305, available from Shanghai Michelin Biochemical technology, inc.); ascorbic acid (vitamin C, vc, 99.0% purity, batch No. M19518023, available from shanghai mclin biochemistry technologies, ltd); chromatographically pure acetonitrile (available from Fisher corporation); chromatographically pure methanol (MeOH, available from Fisher corporation); formic acid (HCOOH, available from Sigma); acetic acid (available from Sigma); purified water (purchased from Hangzhou child haha Co., ltd.).

Example 1

According to the stability result of the preliminary experiment, DTT and ascorbic acid are jointly used as the stabilizer, so that formaldehyde generated by degradation of tetrahydrofolic acid can be effectively reduced. DTT eliminates formaldehyde that may be present in the solvent, and free formaldehyde causes the conversion of tetrahydrofolic acid to 5,10-methylenetetrahydrofolic acid.

Preparing a reference substance stock solution: taking appropriate amount of the dihydrofolic acid, the tetrahydrofolic acid and the 5-methyltetrahydrofolic acid as reference substances, precisely weighing, dissolving in DMSO, and respectively preparing into a dihydrofolic acid reference substance stock solution with a concentration of 1.00mg/mL, a tetrahydrofolic acid reference substance stock solution with a concentration of 1.00mg/mL and a 5-methyltetrahydrofolic acid reference substance stock solution with a concentration of 1.00 mg/mL.

Preparing a stabilizer: taking a proper amount of DTT and Vc reference substances, precisely weighing, dissolving with a proper amount of water, and preparing into a mixed aqueous solution with the DTT concentration of 1.00mg/mL and the Vc concentration of 1.00 mg/mL.

Preparation of a mobile phase: the acidic pH of the mobile phase may affect the stability of folic acid, and thus mobile phase a '(the volume ratio of water to formic acid = 100.1), mobile phase B' (the volume ratio of water to acetic acid = 100.1), mobile phase C (the volume ratio of methanol to formic acid =100 = 0.1.

Taking 5 μ L of each control stock solution, adding 80 μ L of stabilizer, dissolving in 1.5mL of mobile phase respectively, sealing and storing at room temperature (25 deg.C), and measuring the mobile phase solution containing unstable folic acid by HPLC-MS/MS technique at 0min and 60 min.

Chromatographic conditions, RP-C18 column (100 × 4.6mm,5 μm), mobile phase a (0.1% formic acid in water) and mobile phase B (0.1% formic acid in acetonitrile), elution conditions of 0.00-1.00min (1-10% mobile phase B), 1.00-3.00min (10-22% mobile phase B), 3.00-5.00min (22-28% mobile phase B), 5.00-6.00min (28-100% mobile phase B), 5.50-8.00min (100% mobile phase B), 8.00-12.00 (1% mobile phase B), flow rate: 0.4mL/min; the sample size was 10. Mu.L.

The results are shown in table 2, which shows that the stability of the reduced folic acid is not greatly affected by the acetonitrile and methanol systems, that the stability of tetrahydrofolic acid is slightly better than that of water-acetic acid in the water-formic acid system, and that formic acid is the preferred acid in the chromatographic mobile phase system.

Example 2 chromatographic conditions after sample treatment

Various complex components of folic acid are present in serum, and serum samples need to be purified prior to LC-MS/MS analysis in order to minimize the presence of other matrix compounds in serum to ensure selectivity of the method. Currently, serum samples are typically purified using solid phase extraction columns (SPE) with acetonitrile or methanol and buffer as eluents to obtain purified serum samples for LC-MS/MS analysis. Another treatment is protein precipitation with an organic solvent (e.g., methanol or acetonitrile), followed by drying with nitrogen and reconstitution in a mobile phase.

The sample handling methods described above all increase the residence time due to folic acid instability, resulting in folic acid loss. Therefore, the invention adopts an organic solvent precipitation method, but does not carry out drying and redissolving, thereby shortening the sample processing time to the maximum extent. In order to reduce the interference of plasma endogenous substances on target components, the measurement of the blank plasma and the plasma solution containing a target control substance is carried out, and the proportion of the mobile phase is adjusted.

Plasma collection

Mice were fasted for 12h and had free access to water. About 300 mu L of blood is collected from the orbit, the blood is placed in an EP tube which is pre-filled with 20 mu L of heparin sodium solution with the concentration of 250U/mL and dried, the EP tube is immediately placed on ice, the blood sample is centrifuged for 10min at the temperature of 4 ℃ and 5000rpm, and supernatant fluid, namely blank plasma, is collected and transferred to a 1.5mL centrifuge tube and is stored in a refrigerator at the temperature of 20 ℃ below zero.

Solution preparation

Taking appropriate amount of FA, DHFA, THFA and MTHF reference substances, precisely weighing, respectively placing in a 1.5mLEP tube, dissolving with appropriate amount of DMSO, and respectively preparing into folic acid reference substance stock solution with concentration of 1.00mg/mL, dihydrofolic acid reference substance stock solution with concentration of 1.00mg/mL, tetrahydrofolic acid reference substance stock solution with concentration of 1.00mg/mL, and 5-methyltetrahydrofolic acid reference substance stock solution with concentration of 1.00 mg/mL. Taking appropriate amount of the above reference stock solutions, mixing, adding methanol, and diluting to obtain series mixed standard solutions with different concentrations.

Taking a proper amount of DTT and Vc reference substances, precisely weighing, dissolving with a proper amount of water respectively, and preparing DTT standard stock solution with the concentration of 10.0mg/mL and Vc standard stock solution with the concentration of 10.0 mg/mL. Accurately measuring appropriate amounts of DTT standard stock solution and Vc standard stock solution respectively, mixing, adding water to dilute to obtain a mixed aqueous solution (stabilizer C) with DTT and Vc concentrations of 1.00mg/mL and a mixed aqueous solution (stabilizer B) with DTT and Vc concentrations of 5.00 mg/mL.

Taking a proper amount of phenytoin sodium reference substance, precisely weighing, dissolving with a proper amount of DMSO, and preparing the standard stock solution of phenytoin sodium with the concentration of 1.00 mg/mL. Precisely measuring a proper amount of phenytoin sodium standard stock solution, adding DTT standard stock solution and Vc standard stock solution, and adding methanol to dilute until the phenytoin sodium concentration is 80 mu g/mL and the DTT and Vc concentrations are both 1mg/mL, namely, the internal standard working solution.

Sample processing

Treatment of blank plasma: taking 50 mu L of mouse blank plasma, vortexing for 10s, adding 300 mu L of phenytoin sodium methanol solution with phenytoin sodium concentration of 80 mu g/mL, vortexing for 1min, centrifuging at 12000rpm for 10min, taking 20 mu L of supernatant, mixing the supernatant with 80 mu L of stabilizer C (mixed aqueous solution of DTT and Vc, the concentration of DTT and Vc is both 1 mg/mL), and taking 5 mu L for LC-MS/MS quantitative analysis.

Treatment of plasma containing the target compound: taking 50 mu L of mouse blank plasma, adding 5 mu L of mixed standard solution with each concentration, swirling for 10s, adding 5 mu L of stabilizer B (mixed aqueous solution of DTT and Vc, the concentration of DTT and Vc is both 5 mg/mL), swirling for 10s, adding 300 mu L of phenytoin methanol solution (the concentration of phenytoin sodium is 80 mu g/mL, the concentration of DTT and Vc is both 1 mg/mL), swirling for 1min, centrifuging at 12000rpm for 10min, taking 20 mu L of supernate, uniformly mixing the supernate with 80 mu L of stabilizer C (mixed aqueous solution of DTT and Vc, the concentration of DTT and Vc is both 1 mg/mL) to obtain standard curve sample solution, and taking 5 mu L for LC-MS/MS quantitative analysis.

MS detection conditions

Nitrogen Gas was used for both Curtain Gas (Curtain Gas) and nebulised Gas (Ion Source Gas 1) using ESI ionization Source, negative Ion scanning MRM detection mode, with settings of 20 and 10psi, respectively. The source injection Voltage (IonSpray Voltage) in the negative ion mode was set to-4500V, and the residence time of each channel in the MRM mode was set to 50ms. See table 3 for specific mass spectrometric detection conditions. The individual analytes MS are shown in FIGS. 1-4.

Searching for chromatographic condition

The column was an Agilent Zorbax XDB C18 column (50 mm. Times.2.1 mm,3.5 μm), mobile phase A0.1% formic acid-water, mobile phase B0.1% formic acid-methanol, and gradient elution was performed from initial gradient 98% mobile phase A to 20% mobile phase A to examine whether the target compound and the endogenous substance in plasma affected each other. Because the treatment method is simplified into methanol precipitation and supernatant taking, and the methanol precipitation and supernatant taking are not evaporated to dryness for redissolution, the decomposition of the folic acid is reduced, but the interfering substances existing in the sample are increased. The result shows that the endogenous substances in the plasma mainly interfere with the DHFA, and the endogenous interfering components are separated from target components such as the DHFA by adjusting the elution of a chromatographic mobile phase.

The chromatographic conditions select 95% -98% of mobile phase A elution for 0.5-1min, which can preferentially elute most impurities interfering with DHFA. Optimizing the chromatographic elution time, and determining the optimized chromatographic conditions: agilent Zorbax XDB C18 chromatography column (50 mm × 2.1mm,3.5 μm), mobile phase a 0.1% formic acid-water, mobile phase B0.1% formic acid-methanol, elution conditions 0.00-0.50min (2% mobile phase B), 0.50-1.00min (2% -100% mobile phase B), 1.00-4.00min (100% mobile phase B), 4.00-4.01min (100% -2% mobile phase B), 4.01-5.00min (2% mobile phase B), flow rate: 0.4mL/min; the sample size is 5 mu L; column temperature: and (4) room temperature.

Under the optimized chromatographic conditions, the chromatogram of the blank plasma is shown in FIG. 5, and the chromatogram of different target compounds is shown in FIG. 6. As shown, endogenous substances in plasma did not interfere with the assay of 4 folic acid and 4 components and the internal standard phenytoin sodium did not interfere with each other.

A series of mixed standard solutions were taken, treated as in this example "treatment of plasma containing the target compound", and then subjected to sample injection measurement under optimized chromatographic conditions. The concentration is linearly regressed by the ratio of each component to the peak area of the corresponding internal standard, and the weight factor is 1/x ² And obtaining a corresponding standard curve. The standard curves, linear ranges for the 4 components are shown in table 4.

Under this method, the accuracy and precision of the sample are good (see table 5), and the Matrix effect (Matrix effect) and Recovery (Recovery) are also satisfactory (see table 6).

Example 3 metabolism of Folic acid in mice

LC-MS/MS detection conditions

Nitrogen Gas was used for both Curtain Gas (Curtain Gas) and nebulised Gas (Ion Source Gas 1) using ESI ionization Source, negative Ion scanning MRM detection mode, with settings of 20 and 10psi, respectively. The source injection Voltage (IonSpray Voltage) in the negative ion mode was set to-4500V, and the residence time of each channel in the MRM mode was set to 50ms. See table 3 for specific mass spectrometric detection conditions.

Chromatographic conditions are as follows: agilent Zorbax XDB C18 column (50 mm × 2.1mm,3.5 μm), mobile phase a 0.1% formic acid-water, mobile phase B0.1% formic acid-methanol, elution conditions: 0.00-0.50min (2% mobile phase B), 0.50-1.00min (2% -100% mobile phase B), 1.00-4.00min (100% mobile phase B), 4.00-4.01min (100% -2% mobile phase B), 4.01-5.00min (2% mobile phase B), flow rate: 0.4mL/min; the sample size was 5. Mu.L. Column temperature: and (4) room temperature.

80 ICR male mice, 18-22g in weight, 6-8 weeks of week age, were assigned 10 time points (0 min before administration, 0.25h, 0.5h, 1, 2h, 4h, 6h, 8h, 12h, 24h after administration), 8 at each time point. Before administration, animals are adapted to standard environment for 2 days, on the test day, proper amount of Folic Acid (FA) is taken and added with water to prepare solution with the concentration of 0.212mg/mL, the administration volume is 10mL/kg, and the administration is performed by gastric lavage.

Collection of plasma samples

Mice were fasted for 12h before administration and had free access to water. Before (0 min) gastric lavage administration of mice, about 300 mu L of blood is taken from the orbit 0.25h, 0.5h, 1 h, 2h, 4h, 6h and 8h after administration, the blood is placed in an EP tube which is pre-filled with 20 mu L of heparin sodium solution with the concentration of 250U/mL and dried, the blood sample is immediately placed on ice, the blood sample is centrifuged for 10min at 5000rpm and 4 ℃, supernatant fluid, namely blood plasma, is taken, the blood sample is transferred to a 1.5mL centrifuge tube and is stored in a refrigerator at 20 ℃ below zero.

Preparation of the solution

Taking a proper amount of phenytoin sodium reference substance, precisely weighing, dissolving with a proper amount of DMSO, and preparing the standard stock solution of phenytoin sodium with the concentration of 1.00 mg/mL. Precisely measuring a proper amount of phenytoin sodium standard stock solution, adding DTT standard stock solution and Vc standard stock solution, and adding methanol to dilute to a phenytoin sodium methanol solution with phenytoin sodium concentration of 80 mu g/mL and both DTT concentration and Vc concentration of 1mg/mL.

Sample handling and assay

Plasma samples at different time points were processed as follows:

taking 50 mu L of mouse blank plasma (plasma sampled at 0 min), adding 5 mu L of mixed standard solution with each concentration, swirling for 10s, adding 5 mu L of stabilizer B (mixed aqueous solution of DTT and Vc, the concentrations of DTT and Vc are both 5 mg/mL), swirling for 10s, adding 300 mu L of phenytoin sodium methanol solution with the concentration of 80 mu g/mL (simultaneously containing DTT and Vc, the concentrations of DTT and Vc are both 1 mg/mL), swirling for 1min, centrifuging for 10min at 12000rpm, taking 20 mu L of supernate, mixing with 80 mu L of stabilizer C (mixed aqueous solution of DTT and Vc, the concentrations of DTT and Vc are both 1 mg/mL) to obtain standard curve sample solution, taking 5 mu L of sample injection, and carrying out LC-MS/MS quantitative analysis. The concentrations of the components were linearly regressed by the peak area ratios of each component to the corresponding internal standard to obtain the corresponding standard curves for the 4 compounds.

Taking 50 mu L of mouse plasma (plasma sampled at 0.25h, 0.5h, 1, 2h, 4h, 6h, 8h, 12h and 24h after administration), adding 5 mu L of methanol, vortexing for 10s, adding 5 mu L of stabilizer B (mixed aqueous solution of DTT and Vc, the concentration of DTT and Vc is 5 mg/mL), vortexing for 10s, adding 300 mu L of phenytoin sodium methanol solution with 80 mu g/mL concentration (containing DTT and Vc simultaneously, the concentration of DTT and Vc is 1 mg/mL), vortexing for 1min, centrifuging for 10min at 12000rpm, taking 20 mu L of supernatant, mixing with 80 mu L of stabilizer C (mixed aqueous solution of DTT and Vc, the concentration of DTT and Vc is 1 mg/mL), taking 5 mu L of sample injection, and carrying out LC-MS/MS quantitative analysis. And recording peak areas corresponding to the object to be measured and the internal standard substance, calculating the peak area ratio of the object to be measured to the peak area of the internal standard substance, substituting the peak area ratio of the object to be measured to the peak area ratio of the internal standard substance into the standard curve, and calculating the corresponding concentration of the object to be measured.

The results are shown in Table 7, FIG. 7, showing: after taking folic acid, folic acid is converted into active dihydrofolic acid and 5-methyltetrahydrofolic acid in vivo, and folic acid is completely metabolized after 24 hours.

Claims

1. A method for detecting the concentration of folic acid and derivatives thereof in blood plasma based on LC-MS/MS technology is characterized in that: the method for simultaneously measuring the concentrations of folic acid, dihydrofolic acid, tetrahydrofolic acid and 5-methyltetrahydrofolic acid in blood plasma comprises the following steps:

step (1), preparation of a standard curve: preparing folic acid, dihydrofolic acid, tetrahydrofolic acid and 5-methyltetrahydrofolic acid into mixed standard solutions with different concentrations, adding the mixed standard solutions into blank plasma and adding a stabilizer, uniformly mixing, adding an internal standard working solution, performing vortex, centrifuging, taking supernate and mixing the supernate with the stabilizer to obtain a standard curve sample solution, feeding an LC-MS/MS system, performing LC-MS/MS detection according to a liquid chromatography-mass spectrometry combined detection condition, recording corresponding peak areas of an object to be detected, and performing linear regression on the concentration of the object to be detected and the internal standard peak areas according to the ratio of the peak areas of the object to be detected to obtain a corresponding standard curve;

step (2), determination of folic acid, dihydrofolic acid, tetrahydrofolic acid and 5-methyltetrahydrofolic acid in blood plasma to be detected: adding methanol into blood plasma to be detected, adding an internal standard working solution and a stabilizer, performing vortex and centrifugation, and mixing supernatant with the stabilizer; a sample injection LC-MS/MS system performs LC-MS/MS detection according to the detection conditions of liquid chromatography-mass spectrometry, records peak areas corresponding to the object to be detected and the internal standard substance, calculates peak concentration ratio, substitutes the peak concentration ratio into a standard curve, and calculates to obtain the concentration of the object to be detected;

wherein, the detection conditions of the liquid chromatography-mass spectrometry are as follows:

liquid chromatography conditions: a C18 reverse phase chromatography column; a mobile phase A:0.1% aqueous formic acid, mobile phase B:0.1% formic acid in methanol; and (3) an elution mode: starting elution by isocratic gradient, eluting by 95% -98% of mobile phase A for not less than 0.5min, then increasing the proportion of mobile phase B to 100% within not more than 1min, continuously eluting by 100% of mobile phase B for not less than 3min, and adjusting the proportion of mobile phase to initial gradient; flow rate: 0.400-0.600 mL/min;

mass spectrum conditions: detecting by adopting an ESI source and an anion MRM mode;

the internal standard working solution is phenytoin sodium or isotope label C ¹³ -5-methyltetrahydrofolic acid is used as an internal standard substance, dithiothreitol and ascorbic acid are added, and internal standard solution prepared by methanol is adopted.

2. The method for detecting the concentration of folic acid and derivatives thereof in plasma based on LC-MS/MS technology according to claim 1, characterized in that: the stabilizer is a mixed aqueous solution of dithiothreitol and ascorbic acid.

3. The method for detecting the concentration of folic acid and derivatives thereof in plasma based on LC-MS/MS technique according to claim 1, characterized in that: in the internal standard working solution, the concentration of an internal standard substance is 60-80 mug/mL, the concentration of dithiothreitol is 1mg/mL, and the concentration of ascorbic acid is 1mg/mL.

4. The method for detecting the concentration of folic acid and derivatives thereof in plasma based on LC-MS/MS technique according to claim 1, characterized in that: the liquid chromatography conditions are as follows: an Agilent Zorbax XDB C18 chromatographic column with specification of 50mm × 2.1mm,3.5 μm; column temperature: room temperature; mobile phase A:0.1% aqueous formic acid; and (3) mobile phase B:0.1% formic acid in methanol; an elution mode: eluting with 98% mobile phase A for 0.5min, then increasing the proportion of mobile phase B to 100% within 0.5min, eluting with 100% mobile phase B for 3min, adjusting the mobile phase proportion to initial gradient within 0.1min, and continuously balancing the chromatographic column for 5min; flow rate: 0.400mL/min; sample injection amount: 5 μ L.

5. The method for detecting the concentration of folic acid and its derivatives in plasma based on LC-MS/MS technique according to claim 1 or 4, characterized in that: the elution mode is as follows: 0.00-0.50min,2% mobile phase B;0.50-1.00min,2% -100% of mobile phase B;1.00-4.00min,100% mobile phase B;4.00-4.01min,100% -2% of mobile phase B;4.01-5.00min,2% of mobile phase B.

6. The method for detecting the concentration of folic acid and derivatives thereof in plasma based on LC-MS/MS technique according to claim 1, characterized in that: the mass spectrum conditions are as follows: ESI ionization source, MRM detection mode of anion scanning, gas curtain gas and atomization gas both use nitrogen, the set values are 20 and 10psi respectively; the source spray voltage in negative ion mode was set to-4500V, ion pair of folic acid: m/z 440 → 311, collision energy-27 eV; ion pair of dihydrofolic acid: m/z is 442 → 176, collision energy is-33 eV; ion pair of tetrahydrofolic acid: m/z 458 → 286, collision energy-33 eV; ion pair of 5-methyltetrahydrofolate: m/z is 458 → 329 and the collision energy is-36 eV.

7. The method for detecting the concentration of folic acid and derivatives thereof in plasma based on LC-MS/MS technique according to claim 1, characterized in that: in the step (1), the preparation of the mixed standard solution comprises the following steps: dissolving folic acid, dihydrofolic acid, tetrahydrofolic acid and 5-methyltetrahydrofolic acid reference substances in a proper amount of DMSO (dimethyl sulfoxide), and respectively preparing a folic acid reference substance stock solution with the concentration of 1.00mg/ml, a dihydrofolic acid reference substance stock solution with the concentration of 1.00mg/ml, a tetrahydrofolic acid reference substance stock solution with the concentration of 1.00mg/ml and a 5-methyltetrahydrofolic acid reference substance stock solution with the concentration of 1.00 mg/ml; mixing the 4 reference substance stock solutions, and performing serial dilution with methanol to obtain mixed standard solutions with different concentrations;

preparation method of standard curve sample solution: taking 50 mu L of blank plasma, adding 5 mu L of mixed standard working solution, vortexing for 5-10 s, adding 5 mu L of stabilizer, vortexing for 10s, adding 300 mu L of internal standard working solution, vortexing for no less than 1min, centrifuging at 12000rpm for no less than 10min, taking supernate, and uniformly mixing 20 mu L of supernate with 80 mu L of stabilizer to ensure that the concentration of dithiothreitol and ascorbic acid in the standard curve sample solution is no less than 0.9mg/mL.

8. The method for detecting the concentration of folic acid and derivatives thereof in plasma based on LC-MS/MS technique according to claim 1, characterized in that: in the step (2), the concentrations of dithiothreitol and ascorbic acid in the supernatant are not less than 0.9mg/mL, and the concentration of the internal standard substance in the supernatant is 5-20 mug/mL.

9. The method for detecting the concentration of folic acid and its derivatives in plasma based on LC-MS/MS technique according to claim 1 or 8, characterized in that: taking 50 mu L of plasma to be detected, adding 5 mu L of methanol, whirling for 5-10 s, adding 5 mu L of stabilizer with dithiothreitol and ascorbic acid both having concentration of 5mg/mL, whirling for 10s, adding 300 mu L of internal standard working solution with internal standard substance concentration of 60-80 mu g/mL and dithiothreitol and ascorbic acid both having concentration of 1mg/mL, whirling for not less than 1min, centrifuging at 12000rpm for not less than 10min, taking 20 mu L of supernatant, and mixing with 80 mu L of stabilizer uniformly, so that the concentration of dithiothreitol and ascorbic acid in the sample solution is not less than 0.9mg/mL.

10. Use of the method of claim 1 for determining the concentration of folic acid and derivatives thereof in plasma.