CN115902048A

CN115902048A - Method for detecting water-soluble vitamins in serum by methyl derivatization-high performance liquid chromatography tandem mass spectrometry

Info

Publication number: CN115902048A
Application number: CN202310027863.7A
Authority: CN
Inventors: 成晓亮; 周亚飞; 李美娟
Original assignee: Nanjing Pinsheng Medical Technology Co ltd; Shanghai Ammonia Biotechnology Co ltd; Nanjing Pinsheng Medical Laboratory Co ltd
Current assignee: Nanjing Pinsheng Medical Technology Co ltd; Shanghai Ammonia Biotechnology Co ltd; Nanjing Pinsheng Medical Laboratory Co ltd
Priority date: 2023-01-09
Filing date: 2023-01-09
Publication date: 2023-04-04

Abstract

The invention relates to a method for detecting water-soluble vitamins in serum by methyl derivatization-high performance liquid chromatography tandem mass spectrometry, which mixes a serum sample with mixed internal standard solutions of all objects to be detected, only adopts a method of protein precipitation and derivatization pretreatment, has simple derivatization reaction, high sensitivity, small sample dosage and strong specificity, can simultaneously detect 3 water-soluble vitamins within 5 minutes, and can be used for clinical diagnosis and health evaluation of the water-soluble vitamins in the serum.

Description

Method for detecting water-soluble vitamins in serum by methyl derivatization-high performance liquid chromatography tandem mass spectrometry

Technical Field

The invention belongs to the technical field of blood detection, and particularly relates to a method for detecting water-soluble vitamins in serum by methyl derivatization-high performance liquid chromatography tandem mass spectrometry.

Background

Water-soluble vitamins are a group of vitamins that are soluble in water, often being a component of coenzymes or prosthetic groups, including the B vitamins that play an important role in the catalysis of enzymes. For example, vitamins include vitamin B7 (Biotin, VB 7), vitamin B9 (folic acid, VB 9), and niacin (Nicotinic acid, NA).

Vitamin B7, also known as vitamin H, biotin or coenzyme R, is a water-soluble vitamin belonging to the B group, is a prosthetic group of various hydroxylases, is an indispensable substance for normal metabolism of fats and proteins, and is also a nutrient necessary for maintaining normal growth, development and health. The main manifestations of biotin deficiency are mainly skin symptoms, such as dermatitis, eczema, anorexia, mild anemia and alopecia. The regulation of blood glucose can be improved by biotin in diabetic patients.

Nicotinic acid, also known as vitamin B3, helps to promote the health of the digestive system, improve gastrointestinal dysfunction and diarrhea; helps to reduce cholesterol and triglyceride levels in the blood; it is also used for improving stomatitis and preventing halitosis.

Vitamin B9, also called folic acid, is an important vitamin that, together with B12, contributes to red blood cell formation and reduces anemia. The lack of folic acid in pregnant women can lead to spina bifida and a brain-free abnormality in the fetus. In addition, folic acid also helps to keep normal homocysteine level in blood (an important index for measuring heart disease), and reduces the occurrence of heart disease.

At present, there are many detection methods related to water-soluble vitamins, and besides traditional microbiological detection methods, ELISA, radioimmunoassay methods, liquid chromatography-ultraviolet detection (LC-UV) and liquid chromatography tandem mass spectrometry (LC-MS/MS) are also increasingly applied to the detection of vitamins. However, in the detection of biological samples, some water-soluble vitamins containing carboxylic acid groups often have poor sensitivity due to matrix effect and high background noise, so that the detection requirements of clinics cannot be met. Through prior attempts at technology, carboxylic acid groups are generally susceptible to deprotonation and negative charge under ESI ionization conditions, but vitamin B7 is less sensitive in both positive and negative ion detection modes.

There are also documents in which 2-nitrophenylhydrazine is used to perform a derivatization reaction on VB7 to improve the detection sensitivity (Journal of Chromatography a, 1142 (2007) 231-235), but the derivatization reaction is relatively complex, the types of reagents used are many, and the addition of a large amount of reagents inevitably dilutes the detection concentration of an object to be detected, which is unfavorable for trace detection.

Disclosure of Invention

The invention aims to provide a method for detecting water-soluble vitamins in serum by methyl derivatization-high performance liquid chromatography tandem mass spectrometry on the basis of the prior art.

The technical scheme of the invention is as follows:

a method for detecting water-soluble vitamins in serum by methyl derivatization-high performance liquid chromatography tandem mass spectrometry is disclosed, wherein the water-soluble vitamins are respectively: vitamin B7 (VB 7), vitamin B9 (VB 9) and Niacin (NA);

the isotope internal standard substances corresponding to the water-soluble vitamins are respectively as follows: vitamin B7-d4 (VB 7-d 4), vitamin B9-d4 (VB 9-d 4) and niacin-d 4 (NA-d 4);

adopting high performance liquid chromatography tandem mass spectrometry to detect water-soluble vitamins in preprocessed serum, utilizing the high performance liquid chromatography to separate a target object to be detected from interfering components in a serum matrix, detecting the response of the mass-to-charge ratio of the target object to be detected and a corresponding isotope internal standard thereof through a mass spectrometer, quantifying by using an isotope internal standard method, and respectively calculating the content of 3 water-soluble vitamins, wherein the specific chromatographic conditions are as follows:

(1) High performance liquid chromatography conditions:

a mobile phase A:0.01 to 0.5% aqueous formic acid solution; and (3) mobile phase B: methanol;

the chromatographic column is as follows: agilent EC C18;

and (3) performing gradient elution by adopting the mobile phase A and the mobile phase B as a mixed mobile phase, wherein the gradient elution process is as follows: uniformly and gradually changing the volume ratio of the mobile phase A to the mobile phase B from 80 to 30 within 0-1.0 min; in 1.0-2.5 minutes, the volume ratio of the mobile phase A to the mobile phase B is uniformly graded from 30; the volume ratio of the mobile phase A to the mobile phase B is 2; in 3.0-3.1 minutes, the volume ratio of the mobile phase A to the mobile phase B is uniformly graded from 2; the volume ratio of the mobile phase A to the mobile phase B is 80;

(2) Mass spectrum conditions:

in an electrospray ionization mode, carrying out positive and negative switching scanning by adopting multi-reaction monitoring; the capillary voltage is 3.5kV, and the nozzle voltage is 100V; the temperature of the drying gas is 300 ℃, the temperature of the sheath gas is 325 ℃, the flow rate of the drying gas is 7L/min, the flow rate of the sheath gas is 11L/min, and the pressure of the atomizing gas is 30 psi; simultaneously, 3 water-soluble vitamins and their corresponding isotope internal standards were monitored.

In chromatography, the choice of the chromatographic column is important and the requirements for the chromatographic column: high column efficiency, good selectivity, high analysis speed and the like. According to the invention, 0.01 to 0.5% formic acid water solution-methanol is used as a mobile phase, the chromatographic column is Agilent EC C18, endogenous substances do not interfere the determination of a sample under the coordination of other conditions, the sensitivity is high, the specificity is strong, the cost is low, the separation and the detection can be completed within 5.0 min, and the precision and the accuracy meet the requirements. In a preferred embodiment, the column has a length of 50mm, a diameter of 3.0mm and a packing particle size of 2.7. Mu.m. For example, the column was an Agilent EC C18 (2.7 μm,3.0mm x 50mm).

In order to improve the chromatographic separation selectivity, it may be considered to adjust the polarity of the mobile phase. The invention adds formic acid into the mobile phase A, which can effectively improve the ionization efficiency of some target compounds, and under the coordination of other conditions, compared with the method for detecting water-soluble vitamins by adopting an LC-MS/MS method in the prior art, the invention only adopts a method of protein precipitation and derivatization pretreatment, and has the advantages of simple derivatization reaction, higher sensitivity, small sample dosage, strong specificity and low cost, and 3 water-soluble vitamins can be simultaneously detected within 5 minutes. In a preferable embodiment, the mobile phase A is 0.05 to 0.15% formic acid aqueous solution without affecting the effect of the present invention. In a more preferred embodiment, mobile phase a is 0.1% aqueous formic acid.

When the internal standard method is adopted, the selection of the internal standard substance is very important work. The ideal internal standard should be capable of being added to the sample in an accurate, known amount, and have substantially the same or as consistent as possible physicochemical properties, chromatographic behavior, and response characteristics as the sample being analyzed; under chromatographic conditions, the internal standard must be sufficiently separated from the components of the sample. The invention respectively adopts vitamin B7 (VB 7), vitamin B9 (VB 9) and Nicotinic Acid (NA) as internal standards, the deuterated internal standards and the substance to be measured have the same retention time, chemical property and matrix effect, and the reproducibility and accuracy of the measurement of the water-soluble vitamins in serum are better.

In a preferred embodiment, the flow rate is 0.2 to 0.5 mL/min, preferably 0.4 mL/min.

Further, the column temperature is from 35 to 45 ℃, preferably 40 ℃.

Further, the sample injection volume is 1 to 10 μ L, preferably 2 μ L.

In a preferred scheme, a method for detecting water-soluble vitamins in serum by adopting methyl derivatization-high performance liquid chromatography tandem mass spectrometry has the following specific chromatographic conditions:

(1) Ultra-high performance liquid chromatography conditions:

mobile phase A:0.1% aqueous formic acid; and (3) mobile phase B: methanol;

the chromatographic column comprises: agilent EC C18 (2.7 μm,3.0mm x 50mm);

and (3) performing gradient elution by adopting the mobile phase A and the mobile phase B as a mixed mobile phase, wherein the gradient elution process is as follows: the volume ratio of the mobile phase A to the mobile phase B is uniformly graded from 80 to 30 within 0 to 1.0 minutes; in 1.0-2.5 minutes, the volume ratio of the mobile phase A to the mobile phase B is uniformly graded from 30; the volume ratio of the mobile phase A to the mobile phase B is 2; in 3.0-3.1 minutes, the volume ratio of the mobile phase A to the mobile phase B is uniformly graded from 2; the volume ratio of the mobile phase A to the mobile phase B is 80 within 3.1-5.0 minutes; the specific gradient elution mode is shown in Table 1, the flow rate is 0.4 mL/min, the column temperature is 40 ℃, and the sample injection volume is 2 muL.

TABLE 1 mobile phase gradient elution parameters

(2) Mass spectrum conditions:

in an electrospray ionization (ESI) mode, adopting multi-reaction monitoring (MRM) to perform positive and negative switching scanning; the capillary voltage is 3.5kV, and the nozzle voltage is 100V; the temperature of the drying gas is 300 ℃, the temperature of the sheath gas is 325 ℃, the flow rate of the drying gas is 7L/min, the flow rate of the sheath gas is 11L/min, and the pressure of the atomizing gas is 30 psi; and 3 water-soluble vitamins and corresponding isotope internal standards thereof are monitored simultaneously, and the mass spectrum acquisition parameters of each target object to be detected are shown in table 2.

TABLE 2 Water soluble vitamin Profile parameters

Wherein the serum is human or animal serum.

For the purposes of the present invention, the pretreated serum is prepared as follows: adding the mixed internal standard working solution into serum, adding a protein precipitator after vortex, taking supernatant after oscillation centrifugation, drying the supernatant by nitrogen, adding a derivatization reagent into the supernatant, carrying out derivatization reaction for 10-60 min at 40-80 ℃ under the condition of keeping out of the sun, cooling the obtained reaction solution to room temperature, adding an ammonia water solution for neutralization, carrying out oscillation centrifugation again, and taking the supernatant for sample injection.

Wherein, the protein precipitant is methanol or acetonitrile, preferably methanol.

Further, the derivatization reagent is a mixed solution of hydrochloric acid and methanol, and preferably, the volume ratio of the hydrochloric acid to the methanol in the mixed solution is 1 to 4 to 10; more preferably, the volume ratio of hydrochloric acid to methanol in the mixed solution is 1:9.

Further, the aqueous ammonia solution is a 40 to 80% aqueous ammonia solution, preferably a 50% aqueous ammonia solution.

In a preferred embodiment, the pre-treated serum is prepared as follows: putting 50 mu L of serum into a 1.5 mL centrifuge tube, adding 20 mu L of mixed internal standard working solution, adding 180 mu L of methanol after vortex, and centrifuging for 4-10min at the rotation speed of 1400-15000 r/min and at the temperature of 4 ℃ after oscillation; drying 150 muL of supernatant under nitrogen at 37 ℃, adding 100 muL of mixed solution of hydrochloric acid 1:9 and methanol in volume ratio, uniformly mixing in a vortex mode, performing derivatization reaction at 50 ℃ for 20 min under a lightproof condition, cooling obtained reaction liquid to room temperature, adding 20 muL of 50% ammonia water for redissolution, oscillating again, centrifuging at the rotating speed of 1400 to 15000 r/min for 1 to 5min under the condition of 4 ℃, and taking the supernatant for sample injection.

In a more preferred embodiment, the pre-treated serum is prepared as follows: putting 50 mu L of serum into a 1.5 mL centrifuge tube, adding 20 mu L of mixed internal standard working solution, adding 180 mu L of methanol 5s after vortex, and centrifuging for 5min at the rotation speed of 14500 r/min and 4 ℃; drying 150 muL of supernatant under nitrogen at 37 ℃, adding 100 muL of mixed solution of hydrochloric acid 1:9 in volume ratio and methanol into the supernatant, uniformly mixing the mixture for 5s in a vortex mode, performing derivatization reaction for 20 min at 50 ℃ under a light-tight condition, cooling the obtained reaction solution to room temperature, adding 20 muL of 50% ammonia water for neutralization, after oscillation again, centrifuging the mixture for 3min at the rotation speed of 14500 r/min and at 4 ℃, taking 70 muL of supernatant, and filling the supernatant into a sample inlet bottle containing an inner insertion tube for detection, wherein the sample inlet amount is 2 muL.

In one embodiment, the mixed internal standard working solution is prepared as follows:

weighing each isotope internal standard substance, vitamin B7-d4, vitamin B9-d4 and nicotinic acid-d 4, respectively adding methanol aqueous solution to completely dissolve, and preparing isotope stock solutions with the concentration of 1 mug/mL vitamin B7-d4, 10 mug/mL vitamin B9-d4 and 10 mug/mL nicotinic acid-d 4;

preparing isotope mixed internal standard SI solution containing 5 ng/mL vitamin B7-d4, 20 ng/mL vitamin B9-d4 and 200 ng/mL nicotinic acid-d 4 by using methanol aqueous solution;

and taking 100 muL of SI solution, adding 900 muL of methanol, and uniformly mixing to obtain a mixed internal standard working solution.

When preparing the mixed internal standard working solution, the methanol aqueous solution is 60 to 90% methanol aqueous solution, and preferably 80% methanol aqueous solution.

In a preferred embodiment, the mixed internal standard working solution is prepared according to the following method: accurately weighing about 3-5 mg isotope internal standard substances in a 5 mL centrifuge tube (standard substances with the specification below 3 mg are not required to be weighed and are completely dissolved), preparing isotope stock solutions in the following table 3 by using 80% methanol water solution, preparing isotope mixed internal standard SI solutions (detailed in the table 3) from the stock solutions with various concentrations by using 80% methanol water solution, finally taking 100 mu L of SI solution, adding 900 mu L of methanol, and uniformly mixing to obtain mixed internal standard working solution. Wherein the mixed internal standard working solution contains 0.5 ng/mL vitamin B7-d4, 2 ng/mL vitamin B9-d4 and 20 ng/mL nicotinic acid-d 4.

TABLE 3 preparation of SI solution as internal mixed standard

In one embodiment, the standard is prepared by the following steps:

weighing each standard substance to be detected, including vitamin B7, vitamin B9 and nicotinic acid, respectively adding a methanol aqueous solution to completely dissolve the standard substances, and preparing standard substance stock solutions with the concentrations of the vitamin B7 of 25 mug/mL, the vitamin B9 of 100 mug/mL and the nicotinic acid of 100 mug/mL;

preparing the stock solution of each standard substance into a mixed standard S0 solution containing 50 ng/mL vitamin B7, 200 ng/mL vitamin B9 and 2000 ng/mL nicotinic acid by using a methanol water solution;

preparing the mixed standard S0 solution into eight calibrator solutions with different concentration levels by using a blank matrix, wherein the eight concentration points of the calibrator solution are as follows:

the concentration of the vitamin B7 is 0.01 ng/mL, 0.02 ng/mL, 0.05 ng/mL, 0.1 ng/mL, 0.25 ng/mL, 0.5 ng/mL, 1.25 ng/mL and 2.5 ng/mL in sequence;

the concentration of the vitamin B9 is 0.04 ng/mL, 0.08 ng/mL, 0.2 ng/mL, 0.4 ng/mL, 1 ng/mL, 2 ng/mL, 5 ng/mL and 10 ng/mL in sequence;

the concentration of the nicotinic acid is 0.4 ng/mL, 0.8 ng/mL, 2 ng/mL, 4 ng/mL, 10 ng/mL, 20 ng/mL, 50 ng/mL and 100 ng/mL in sequence.

In the preparation of the standard, the aqueous methanol solution is 60 to 90% aqueous methanol solution, preferably 80% aqueous methanol solution. The blank matrix is formic acid-methanol solution; preferably 0.05 to 0.2% formic acid-methanol solution; more preferably 0.1% formic acid in methanol.

In a preferred embodiment, the standard is prepared according to the following steps:

accurately weighing about 3-5 mg standard substance powder in a 5 mL centrifuge tube (standard substances with specification below 3 mg are not required to be weighed and are completely dissolved), preparing standard substance stock solution in the following table by using 80% methanol aqueous solution, preparing mixed standard S0 solution (detailed in table 4) by using 80% methanol aqueous solution for stock solution with various concentrations, and uniformly mixing for later use.

TABLE 4 preparation of Mixed Standard solution S0

mu.L of the mixed standard solution S0 to 1.5 mL was added to a centrifuge tube, 380. Mu.L of 0.1% formic acid-methanol solution was added to obtain the first high concentration point S8, and the mixture was further diluted stepwise to S1 (see Table 5 for details), wherein the concentrations of the respective standard points are listed in Table 5.

TABLE 5 preparation and concentration of standard

(Note: concentration units are ng/mL)

Taking 50 mu L of each concentration point sample, adding 20 mu L of mixed internal standard working solution into the sample, adding 180 mu L of methanol 5s after vortex, and centrifuging for 5min at the rotating speed of 14500 r/min and at 4 ℃; drying 150 muL of supernatant under nitrogen at 37 ℃, adding 100 muL of mixed solution of hydrochloric acid 1:9 in volume ratio and methanol into the supernatant, uniformly mixing the mixture for 5s in a vortex mode, performing derivatization reaction for 20 min at 50 ℃ under a light-tight condition, cooling the obtained reaction solution to room temperature, adding 20 muL of 50% ammonia water for neutralization, after oscillation again, centrifuging the mixture for 3min at the rotation speed of 14500 r/min and at 4 ℃, taking 70 muL of supernatant, and filling the supernatant into a sample inlet bottle containing an inner insertion tube for detection, wherein the sample inlet amount is 2 muL.

The concentration of the aqueous methanol solution or the aqueous formic acid-methanol solution referred to in the present invention generally refers to a volume concentration.

The invention also comprises the preparation of quality control products, wherein the quality control products are blank serum matrix solution containing 3 water-soluble vitamins, and the blank serum matrix solution is divided into low, medium and high concentrations, namely QC (L), QC (M) and QC (H). Wherein, the first and the second end of the pipe are connected with each other,

QC (L) is the above-mentioned mixed standard S0 solution diluted to 2000 times with blank serum matrix solution;

QC (M) is the above mixed standard S0 solution diluted to 200 times with blank serum matrix solution;

QC (H) is the above mixed standard S0 solution diluted 20-fold with blank serum matrix solution.

In a preferred embodiment, the quality control product is prepared according to the following method: and (3) preparing the mixed standard S0 solution into QC (L), QC (M) and QC (H) with three different concentrations by using 0.05 to 0.2% formic acid-methanol aqueous solution.

In a more preferred embodiment, the quality control material is prepared by the following method: the mixed standard S0 solution is prepared into QC (L), QC (M) and QC (H) with three different concentrations by using 0.1% formic acid-methanol aqueous solution.

QC (L) includes: 0.025 ng/mL vitamin B7 (VB 7), 0.1 ng/mL vitamin B9 (VB 9) and 1 ng/mL Niacin (NA).

QC (M) includes: 0.25 ng/mL vitamin B7 (VB 7), 1 ng/mL vitamin B9 (VB 9) and 10 ng/mL Niacin (NA).

QC (H) includes: 2.5 ng/mL vitamin B7 (VB 7), 10 ng/mL vitamin B9 (VB 9) and 100 ng/mL Niacin (NA).

The invention establishes a simple, efficient and reliable method for detecting the water-soluble vitamins in the serum, and compared with the prior art, the method can better meet the requirements of clinical application.

By adopting the technical scheme of the invention, the advantages are as follows:

the method for detecting the water-soluble vitamins in the serum provided by the invention has the advantages that the serum sample is mixed with the mixed internal standard solution of all substances to be detected, only the methods of protein precipitation and derivatization pretreatment are adopted, the derivatization reaction is simple, the sensitivity is high, the sample dosage is small, the specificity is strong, 3 water-soluble vitamins can be simultaneously detected within 5 minutes, and the method can be used for clinical diagnosis and health assessment of the water-soluble vitamins in the serum.

Drawings

FIG. 1 is a selective ion flow chromatogram of 3 water-soluble vitamin standards;

FIG. 2 is a selective ion flow chromatogram of 3 water-soluble vitamins in a serum sample.

Detailed Description

In order to understand the technical scheme of the invention more clearly, the invention is further illustrated by the following examples, which are not intended to limit the invention in any way.

Example 1:

1. experimental materials and instruments

1. Material

Methodology samples from the study were obtained from serum samples collected from the heart hospital, wuhan Asia, 2022 years 6 months out-patient.

(1) The instrument comprises the following steps: qlife Lab 9000plus triple quadrupole mass spectrometer (department of medical); a Qlife Lab 9000 ultra-high performance liquid chromatography system (G7167A autosampler for medical care); the system working software is MS quantitative analysis 10.0; SCILOGEX D2012 high speed bench top centrifuge (usa); ultra pure water meter (ELGA LabWater, uk); multi-tube Vortex mixer (Vortex genie2, usa); an adjustable pipette (Eppendorf 0.5 to 10 μ L,10 to 100 μ L,100 to 1000 μ L); glassware, graduated cylinders, etc.

(2) Reagent consumables: MS grade methanol (Fisher, usa); HPLC grade methanol (Honeywell, usa); AR grade hydrochloric acid (36.0 to 38.0%, chinese medicine); trace levels of ammonia (Fisher, usa); column Agilent EC C18,2.7 μm,3.0mm x 50mm (Agilent, usa).

(3) And (3) standard substance: VB7, VB9 and NA standards were purchased from Sigma and the corresponding isotopic standards were purchased from TRC.

2. Liquid condition

(1) Chromatographic conditions are as follows: mobile phase A:0.1% formic acid-water solution; mobile phase B: methanol. The chromatographic column is Agilent EC C18,2.7 μm,3.0mm 50mm, and adopts gradient elution mode, and the specific gradient process is detailed in Table 1. The flow rate is 0.4 mL/min, the column temperature is 40 ℃, and the sample injection volume is 2 muL.

(2) Mass spectrum conditions: performing positive ion mode scanning in an electrospray ionization (ESI) mode by using Multiple Reaction Monitoring (MRM); the capillary voltage is 3.5kV, and the nozzle voltage is 100V; the temperature of the drying gas is 300 ℃, the temperature of the sheath gas is 325 ℃, the flow rate of the drying gas is 7L/min, the flow rate of the sheath gas is 11L/min, and the pressure of the atomizing gas is 30 psi; and 3 kinds of water-soluble vitamins and corresponding isotope internal standards thereof are monitored at the same time, and the mass spectrum acquisition parameters of each target object to be detected are shown in table 2.

3. Procedure of experiment

(1) Preparing a standard substance:

(2) mu.L of the mixed standard S0 solution was put into a 1.5 mL centrifuge tube, 380. Mu.L of 0.1% formic acid-methanol solution was added to obtain the first high concentration point S8, and the mixture was further diluted stepwise to S1 (see Table 5 for details), and the concentrations of the respective standard points are listed in Table 5.

(3) Preparing a mixed internal standard working solution:

(4) Accurately weighing about 3-5 mg isotope internal standard substances 5 mL centrifuge tubes (standard substances with the specification below 3 mg are not required to be weighed and are completely dissolved), preparing isotope stock solutions in the following table by using 80% methanol aqueous solution, and preparing isotope mixed internal standard SI solutions (see table 3 for details) by using 80% methanol aqueous solution for stock solutions with various concentrations.

And then taking 100 muL of SI solution, adding 900 muL of methanol, and uniformly mixing to obtain the mixed internal standard working solution. Wherein the mixed internal standard working solution contains 0.5 ng/mL vitamin B7-d4, 2 ng/mL vitamin B9-d4 and 20 ng/mL nicotinic acid-d 4.

(3) Preparing a quality control product:

the mixed standard S0 solution is prepared into QC (L), QC (M) and QC (H) with three different concentrations by using 0.1% formic acid-methanol aqueous solution.

(4) Sample processing

1) Pretreatment of a standard product: taking 50 mu L of each concentration point sample, putting the 50 mu L of each concentration point sample into a 1.5 mL centrifuge tube, adding 20 mu L of mixed internal standard working solution into the centrifuge tube, adding 180 mu L of methanol 5s after vortex, and centrifuging for 5min at the rotating speed of 14500 r/min and at 4 ℃; drying 150 muL of supernatant under nitrogen at 37 ℃, adding 100 muL of mixed solution of hydrochloric acid 1:9 in volume ratio and methanol into the supernatant, uniformly mixing the mixture for 5s in a vortex mode, performing derivatization reaction for 20 min at 50 ℃ under a light-tight condition, cooling the obtained reaction solution to room temperature, adding 20 muL of 50% ammonia water for neutralization, after oscillation again, centrifuging the mixture for 3min at the rotation speed of 14500 r/min and at 4 ℃, taking 70 muL of supernatant, and filling the supernatant into a sample inlet bottle containing an inner insertion tube for detection, wherein the sample inlet amount is 2 muL.

2) Pretreatment of a serum sample: putting 50 mu L of serum into a 1.5 mL centrifuge tube, adding 20 mu L of mixed internal standard working solution, adding 180 mu L of methanol 5s after vortex, and centrifuging for 5min at the rotation speed of 14500 r/min and 4 ℃; drying 150 muL of supernatant under nitrogen at 37 ℃, adding 100 muL of mixed solution of hydrochloric acid 1:9 in volume ratio and methanol into the supernatant, uniformly mixing the mixture for 5s in a vortex mode, performing derivatization reaction for 20 min at 50 ℃ under a light-tight condition, cooling the obtained reaction solution to room temperature, adding 20 muL of 50% ammonia water for neutralization, after oscillation again, centrifuging the mixture for 3min at the rotation speed of 14500 r/min and at 4 ℃, taking 70 muL of supernatant, and filling the supernatant into a sample inlet bottle containing an inner insertion tube for detection, wherein the sample inlet amount is 2 muL.

3) Pretreatment of quality control products: respectively taking 50 mu L of quality control solution QC (L), QC (M) and QC (H) in a 1.5 mL centrifuge tube, and then consistent with the pretreatment of a serum sample, which is not described herein again.

4. Method verification

In the detection method of the invention, the peak shapes of the 3 water-soluble vitamin standards are symmetrical with those of the serum sample, and there is no interference of miscellaneous peaks, which indicates that good detection can be obtained under the condition, and fig. 1 is a selective ion flow chromatogram of the 3 water-soluble vitamin standards; FIG. 2 is a selective ion flow chromatogram of 3 water-soluble vitamins in a serum sample. It should be noted that, as known in the art, in fig. 2, when analyzing each vitamin in a serum sample, an ion flow chromatogram is selected by mass spectrometry, and when 259.1 (a parent ion mass spectrum parameter of VB 7) is input, two peaks appear in the chromatogram, namely peaks appearing at 2.6min (1 peak) and 3.18min (VB 7 peak), and by comparing peak times of the chromatogram, the 1 peak can be determined as an interference peak of VB7, because the matrix in the serum sample is complex and an interference substance with a molecular weight of 259.1 also exists, so that when a mass spectrum of 259.1 is adjusted, two peaks appear. The method carries out quantification through mass spectrum signal intensity, so even if the 1 peak part is overlapped with the NA peak, the quantification of the NA is not influenced at all, and meanwhile, the 1 peak is well separated from the VB7 and has no influence on the quantification of the VB 7.

1. Comparison of results before and after derivatization

The chromatographic behavior before and after derivatization is compared (see Table 6 in detail), and experiments are carried out by adopting a VB7 standard substance of 10 ng/mL, a VB9 standard substance of 10 ng/mL and an NA standard substance of 10 ng/mL.

Wherein the pretreatment process before derivatization of the VB7 standard product is as follows: 50 mu L of VB7 standard substance with the concentration of 10 ng/mL is taken to be placed in a 1.5 mL centrifuge tube, 20 mu L of mixed internal standard working solution is added into the centrifuge tube, after vortex, 5s, 180 mu L of methanol is added, after oscillation, the centrifuge tube is centrifuged for 5min at the rotating speed of 14500 r/min and the temperature of 4 ℃; and (4) taking 150 mu L of supernatant liquid, and filling the supernatant liquid into an injection bottle containing an inner insertion tube for detection, wherein the injection amount is 2 mu L.

The pretreatment process before derivatization of the other 2 samples (10 ng/mL VB9 standard and 10 ng/mL NA standard) was the same as that of the VB7 standard.

The pretreatment process after the derivatization of the VB7 standard product is as follows: 50 mu L of VB7 standard substance with the concentration of 10 ng/mL is taken to be placed in a 1.5 mL centrifuge tube, 20 mu L of mixed internal standard working solution is added into the centrifuge tube, after vortex, 5s, 180 mu L of methanol is added, after oscillation, the centrifuge tube is centrifuged for 5min at the rotating speed of 14500 r/min and the temperature of 4 ℃; drying 150 mu L of supernatant under nitrogen at 37 ℃, adding 100 mu L of mixed solution of hydrochloric acid 1:9 in volume ratio and methanol, vortex and mixing uniformly for 5s, carrying out derivatization reaction at 50 ℃ for 20 min under a light-tight condition, cooling obtained reaction liquid to room temperature, adding 20 mu L of 50% ammonia water for neutralization, after oscillation again, centrifuging at the rotation speed of 14500 r/min and at 4 ℃ for 3min, putting 70 mu L of supernatant into a sample introduction bottle containing an inner insertion tube for detection, and carrying out sample introduction by 2 mu L.

The pretreatment process after derivatization of the other 2 samples (10 ng/mL VB9 standard and 10 ng/mL NA standard) was the same as that of the VB7 standard.

The VB7 standard, the VB9 standard and the NA standard before and after derivatization are detected according to the chromatographic conditions and mass spectrum conditions disclosed in the liquid mass conditions, and the results show that: after the retention time of the 3 water-soluble vitamins and the internal standards thereof after derivatization is delayed, the peak area response of VB7 and VB9 is greatly improved, the peak area of NA is not improved, but the peak shape of NA is improved and the anti-interference capability is also enhanced.

TABLE 6 comparison of chromatographic behavior before and after derivatization of water-soluble vitamins

2. Serum sample testing

12 cases of human serum samples of healthy people are randomly selected, treated according to the sample pretreatment method and subjected to computer detection, the measured results are shown in the following table 7, and the results show that the VB7 and VB9 content in individual samples is extremely low, so that the improvement of the sensitivity has great significance for clinical detection.

TABLE 7 concentration of water-soluble vitamins in actual serum samples (unit: ng/mL)

Sample numbering	VB7	VB9	NA
				P01	0.024	0.324	20.267
P02	0.038	0.178	32.483
				P03	0.149	1.021	55.884
P04	0.012	0.098	8.294
				P05	0.048	0.029	40.921
P06	0.059	0.045	20.254
				P07	0.278	0.997	32.487
P08	0.872	0.129	29.823
				P09	0.018	0.059	10.638
P10	0.489	0.552	32.554
				P11	0.982	0.732	45.351
P12	0.593	0.239	38.943

3. Standard curve of

By adopting an isotope internal standard quantitative method and using MS quantitative analysis 10.0 software, a calibration curve is established by taking the concentration ratio of the standard substance to the internal standard substance as an X axis and the peak area ratio of the standard substance to the internal standard substance as a Y axis, and the concentration of the substance to be measured is calculated. The following linear fit equations for the 3 water-soluble vitamins over their respective concentration ranges were found to be good, with correlation coefficients above 0.995, as detailed in table 8.

TABLE 8 Retention time and Linear Range of the Water-soluble vitamins

4. Minimum limit of quantitation

The lowest limit of quantitation (LLOQ), which is the lowest point of the normalized range, also reflects the sensitivity of the method. The content of the water-soluble vitamins in human bodies is low, the requirements on the sensitivity and the specificity of the method are high, the method is optimized and examined, the lowest limit of quantitation (LLOQ) at present basically meets the sensitivity requirement of simultaneous detection of 3 water-soluble vitamins, and the concentration of the LLOQ is specifically shown in a table 9.

TABLE 9 quantitative lower limit data sheet

5. Spiking recovery survey

Mixed standard S0 solution with known concentration is added into the mixed serum to prepare three concentrations of low concentration and medium concentration, the three concentrations are processed for 5 times in parallel, and the recovery rate result is calculated and shown in a table 10. The result shows that the standard recovery rate of the 3 water-soluble vitamins in the serum is between 85 and 115 percent, and the recovery rate meets the requirement.

TABLE 10 results of recovery of spiked 3 water-soluble vitamins in serum (in ng/mL)

6. Precision test

Taking serum quality control samples, repeatedly treating 6 batches of the serum quality control samples in one day for 3 days, quantitatively measuring the concentration of 3 water-soluble vitamins by using an isotope internal standard method, continuously counting the precision in each batch every three days, and obtaining the calculation result shown in a table 11; the results of calculating the precision between batches are shown in Table 11, for 3 batches within three days. The results show that the precision test results of 3 kinds of water-soluble vitamins in the serum are all below 15 percent in batches and between batches, and the requirements are met.

TABLE 11 precision test results (in ng/mL) within and between lots

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5. Discussion of the related Art

According to the invention, the carboxyl groups of VB7, VB9 and NA are modified by adopting an HPLC-MS/MS and a methyl derivatization method, and the ionization efficiency is improved by the methyl derivatization method through comparing the chromatographic behaviors of compounds in an undivided method, so that the sensitivity of VB7 and VB9 is effectively improved, and although the sensitivity of NA is not improved, the peak type and the anti-interference capability are improved. Experimental results show that the methyl derivatization can effectively improve the sensitivity of the substance to be detected, and the derivatization reaction is complete and efficient. Meanwhile, the isotope internal standard method is adopted for quantification, so that the matrix interference can be greatly eliminated, the influence of the conditions such as a pretreatment process, a sample loading volume and flow is avoided, and accurate quantification can be achieved.

The method considers that the standard recovery rates of the derived 3 water-soluble vitamins in serum are all 85-115 percent and meet the requirements; the reproducibility result of the method shows that the precision of 3 water-soluble vitamins in the blood serum after derivatization is within 15 percent in batch and between batches, and the method has good reproducibility.

Compared with other LC-MS/MS methods, the method has higher sensitivity and specificity, simple derivatization reaction, can simultaneously detect 3 water-soluble vitamins within 5 minutes, and can meet the diagnosis requirement and health assessment of clinical serum water-soluble vitamins.

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it should be understood by those of ordinary skill in the art that: modifications of the technical solutions described in the foregoing embodiments are still possible, or some technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting water-soluble vitamins in serum by methyl derivatization-high performance liquid chromatography tandem mass spectrometry,

the water-soluble vitamins are respectively: vitamin B7, vitamin B9 and niacin;

the isotope internal standard substances corresponding to the water-soluble vitamins are respectively as follows: vitamin B7-d4, vitamin B9-d4 and niacin-d 4;

adopting high performance liquid chromatography tandem mass spectrometry to detect water-soluble vitamins in pretreated serum, utilizing high performance liquid chromatography to separate a target substance to be detected from interfering components in a serum matrix, detecting the response of the mass-to-charge ratio of the target substance to be detected and a corresponding isotope internal standard by a mass spectrometer, quantifying by using an isotope internal standard method, and respectively calculating the content of 3 water-soluble vitamins, wherein the specific chromatographic conditions are as follows:

(1) High performance liquid chromatography conditions:

a mobile phase A:0.01 to 0.5% aqueous formic acid solution; mobile phase B: methanol;

the chromatographic column is as follows: agilent EC C18;

and (3) performing gradient elution by adopting the mobile phase A and the mobile phase B as a mixed mobile phase, wherein the gradient elution process is as follows: uniformly and gradually changing the volume ratio of the mobile phase A to the mobile phase B from 80 to 30 within 0-1.0 min; in 1.0-2.5 minutes, the volume ratio of the mobile phase A to the mobile phase B is uniformly graded from 30; the volume ratio of the mobile phase A to the mobile phase B is 2; in 3.0-3.1 minutes, the volume ratio of the mobile phase A to the mobile phase B is uniformly graded from 2; the volume ratio of the mobile phase A to the mobile phase B is 80 within 3.1-5.0 minutes;

(2) Mass spectrum conditions:

2. The method for detecting water-soluble vitamins in serum according to claim 1, wherein the pretreated serum is prepared according to the following method: adding the mixed internal standard working solution into serum, adding a protein precipitator after vortex, taking supernatant after oscillation and centrifugation, drying the supernatant by nitrogen, adding a derivatization reagent into the supernatant, carrying out derivatization reaction for 10-60 min at 40-80 ℃ under the condition of keeping out of the sun, cooling the obtained reaction solution to room temperature, adding an ammonia water solution for neutralization, carrying out oscillation and centrifugation again, and taking the supernatant for sample injection.

3. The method for detecting water-soluble vitamins in serum according to claim 2, wherein the protein precipitating agent is methanol or acetonitrile; the derivatization reagent is a mixed solution of hydrochloric acid and methanol; the ammonia water solution is 40-80% ammonia water solution.

4. The method for detecting water-soluble vitamins in serum according to claim 3, wherein the protein precipitating agent is methanol; the derivatization reagent is a mixed solution of hydrochloric acid and methanol with the volume ratio of 1 to 4 to 10; the ammonia water solution is 50% ammonia water solution.

5. The method for detecting water-soluble vitamins in serum according to claim 4, wherein the derivatization reagent is a mixed solution of hydrochloric acid and methanol with a volume ratio of 1:9.

6. The method for detecting water-soluble vitamins in serum according to claim 5, wherein the pretreated serum is prepared according to the following method: putting 50 mu L of serum into a 1.5 mL centrifuge tube, adding 20 mu L of mixed internal standard working solution, adding 180 mu L of methanol after vortex, and centrifuging for 4-10min at the rotation speed of 1400-15000 r/min and at the temperature of 4 ℃ after oscillation; drying 150 muL of supernatant under nitrogen at 37 ℃, adding 100 muL of mixed solution of hydrochloric acid 1:9 and methanol in volume ratio, uniformly mixing in a vortex manner, performing derivatization reaction at 50 ℃ for 20 min under a dark condition, cooling the obtained reaction liquid to room temperature, adding 20 muL of 50% ammonia water for neutralization, oscillating again, centrifuging at the rotation speed of 1400 to 15000 r/min for 1 to 5min under the condition of 4 ℃, and taking the supernatant for sample injection.

7. The method for detecting water-soluble vitamins in serum according to claim 6, wherein the mixed internal standard working solution is prepared according to the following method:

preparing isotope mixed internal standard SI solution containing 5 ng/mL vitamin B7-d4, 20 ng/mL vitamin B9-d4 and 200 ng/mL nicotinic acid-d 4 by using methanol water solution;

8. The method for detecting water-soluble vitamins in serum according to claim 7, wherein the standard substance is prepared by the following steps:

weighing each standard substance to be detected, including vitamin B7, vitamin B9 and nicotinic acid, respectively adding a methanol aqueous solution for complete dissolution, and preparing standard substance stock solutions with the concentrations of the vitamin B7, the vitamin B9 and the nicotinic acid being 25 mug/mL, 100 mug/mL;

preparing the standard substance stock solution into a mixed standard S0 solution containing 50 ng/mL vitamin B7, 200 ng/mL vitamin B9 and 2000 ng/mL nicotinic acid by using a methanol aqueous solution;

preparing the mixed standard S0 solution into eight calibrator solutions with different concentration levels by using a blank matrix, wherein the eight concentration points of the calibrator solutions are as follows:

9. The method for detecting water-soluble vitamins in serum according to claim 7 or 8, wherein the methanol aqueous solution is 60-90% methanol aqueous solution; the blank matrix is formic acid-methanol solution.

10. The method for detecting water-soluble vitamins in serum according to claim 9, wherein the methanol aqueous solution is 80% methanol aqueous solution; the blank matrix is 0.05 to 0.2 percent formic acid-methanol solution.

11. The method for detecting water-soluble vitamins in serum according to claim 10, wherein the blank matrix is 0.1% formic acid-methanol solution.

12. The method for detecting water-soluble vitamins in serum according to claim 1, wherein the serum is human or animal serum.

13. The method for detecting water-soluble vitamins in serum according to claim 1, wherein the mobile phase A is 0.05-0.15% formic acid aqueous solution; the length of the chromatographic column is 50mm, the diameter is 3.0mm, and the grain diameter of the filler is 2.7 mu m.

14. The method for detecting water-soluble vitamins in serum according to claim 13, wherein the mobile phase A is 0.1% formic acid aqueous solution.

15. The method for detecting water-soluble vitamins in serum according to claim 14, wherein the high performance liquid chromatography conditions comprise: the flow rate is 0.2 to 0.5 mL/min; the column temperature is 35 to 45 ℃; the sample introduction volume is 1 to 10 mu L.

16. The method of claim 15, wherein the high performance liquid chromatography conditions comprise: the flow rate is 0.4 mL/min; the column temperature was 40 ℃; the sample injection volume is 2 muL.