CN113917018A

CN113917018A - Precipitator and method for simultaneously detecting water-soluble vitamins and metabolites

Info

Publication number: CN113917018A
Application number: CN202111166718.4A
Authority: CN
Inventors: 谢永明; 张子豪
Original assignee: Shanghai Applied Protein Technology Co Ltd
Current assignee: Shanghai Applied Protein Technology Co Ltd
Priority date: 2021-09-30
Filing date: 2021-09-30
Publication date: 2022-01-11
Anticipated expiration: 2041-09-30
Also published as: CN113917018B

Abstract

The invention provides a precipitator and a method for simultaneously detecting water-soluble vitamins and metabolites, which belong to the technical field of vitamin detection, wherein the precipitator consists of trichloroacetic acid with the final concentration of 7%, zinc sulfate with the final concentration of 0.2M, vitamin C with the final concentration of 3% and water, the detection method adopts liquid phase mass spectrum-tandem mass spectrum for detection, the pH value range of a sample detected on a computer is 2.2-2.8, and 0.2% formic acid and 2mM ammonium formate are added into a mobile phase A, B of the liquid phase mass spectrum, PLP, 5M-THF and MMA can be combined with other water-soluble vitamins for detection, and SPE or derivation and other methods are not needed, and the pretreatment process is simple, convenient and easy to operate.

Description

Precipitator and method for simultaneously detecting water-soluble vitamins and metabolites

Technical Field

The invention belongs to the technical field of vitamin detection, and particularly relates to a method for simultaneously detecting water-soluble vitamins and metabolites, and a precipitator suitable for the method.

Background

Vitamins are a kind of trace organic substances indispensable for maintaining normal physiological functions of human beings and animals, and play an important role in the growth, metabolism and development processes of human bodies. Essential vitamins for the human body can be divided into two categories: the vitamin B group mainly comprises thiamine (VB1), riboflavin (VB2), nicotinamide, nicotinic acid (VB3), pantothenic acid (VB5), pyridoxine (VB6), biotin (VB7), folic acid (VB9), cobalamin (VB12) and the like. They are synergistic, regulate metabolism, maintain skin and muscle health, enhance immune and nervous system function, promote cell growth and division (including promoting the production of red blood cells, preventing anemia). Vitamin B deficiency refers to a condition resulting from a deficiency in vitamin B. The common symptoms include beriberi, angular stomatitis, oral ulcer and the like.

Different vitamins exist in different forms in vivo, and even most of the time, multiple forms coexist, so that the selection of a vitamin detection marker having clinical diagnostic significance is critical. For example, vitamin B6, also known as pyridoxine, includes three subtypes pyridoxine, pyridoxal, pyridoxamine and their phosphate derivatives. However, pyridoxal phosphate (PLP) in serum is the main active form, accounting for about 70% -90% of the main metabolite of vitamin B6, is also an important coenzyme in the metabolic processes of amino acids, sugars and lipids, participates in hundreds of enzyme-catalyzed reactions, is necessary for maintaining the health of the nervous system and the immune system, and plays an important role in maintaining the normal level of homocysteine in blood. Vitamin B9, also known as folic acid. However, the major form in serum is 5-methyltetrahydrofolate (Levomefolic Acid, 5M-THF). Vitamin B12, also known as cobalamin, is a functional indicator of Methylmalonic acid (MMA). Therefore, for the three vitamins of VB6, VB9 and VB12, the detection of pyridoxal 5-phosphate, 5-methyltetrahydrofolic acid and methylmalonic acid in serum is of more practical clinical significance.

The detection methods of water-soluble vitamins are various, and mainly include a radioimmunoassay, a chemiluminescence method, a high performance liquid chromatography, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the like. Although the chemiluminescence method can realize high speed and high flux, the specificity is low, and the chemiluminescence method is easy to generate cross reaction with autoantibodies and specific antibodies. Moreover, the capture efficiency of different brands of reagents to target compounds may be different, the comparability of detection results of different platforms is poor, and false positive or false negative often appears in the immunological method detection for the lacking and infant population. High performance liquid or gas chromatography is low in sensitivity and cannot achieve high throughput with liquid phase separation requiring a long time, and is difficult to use for routine clinical examination. The liquid chromatography-tandem mass spectrometry technology is gradually concerned by clinical examination experts with the advantages of small sample size, rapidness, high sensitivity, high specificity, capability of detecting various compounds simultaneously and the like. Mass spectrometry is an analytical method for measuring the mass-to-charge ratio (mass-to-charge ratio) of ions. In the countries of the Europe and America, mass spectrometry technology, as a gold standard method for small molecule analysis in biological samples recognized by the medical industry society, has been widely applied to clinical research and diagnosis by authoritative detection institutions abroad, including Mayo clinical, Quest diagnostics, Labcorp, and the like.

Methods for simultaneously measuring multiple water-soluble vitamins by using LC-MS/MS are reported, but due to the particularity of active metabolites of partial water-soluble vitamins (including pyridoxal 5-phosphate, 5-methyltetrahydrofolate and methylmalonic acid), the substances are difficult to combine with other water-soluble compounds for detection. In their respective detection reports, pyridoxal 5-phosphate requires an acid precipitation method, while methylmalonic acid requires a separately derived method, and 5-methyltetrahydrofolic acid, a compound, faces stability tests, which greatly increases the difficulty of combined detection. Therefore, there is no report of the combined detection of these active metabolites together with other water-soluble vitamins.

Disclosure of Invention

Based on the problems in the prior art, the invention provides the precipitator and the method for simultaneously detecting the water-soluble vitamins and metabolites, the PLP, the 5M-THF and the MMA can be combined with other water-soluble vitamins for detection, methods such as SPE or derivation and the like are not needed, and the pretreatment process is simple, convenient and easy to operate.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

a precipitator for simultaneously detecting water-soluble vitamins and metabolites comprises trichloroacetic acid, zinc sulfate and vitamin C.

According to the scheme, the precipitator for simultaneously detecting the water-soluble vitamins and the metabolites consists of trichloroacetic acid with the final concentration of 7%, zinc sulfate with the final concentration of 0.2M, vitamin C with the final concentration of 3% and water.

Trichloroacetic acid, zinc sulphate and vitamin C, each of which exerts a different effect:

firstly, TCA with a certain concentration not only has good effect of sediment protein, but also is an ion pair reagent, so that certain amphoteric compounds can be well reserved on a column; however, it is not preferable that the concentration of TCA is larger, because too much TCA results in suppression of the signal from MMA. The concentration required therefore requires finding an equilibrium between the two.

Secondly, the purpose of ZnSO4 is still to precipitate protein, TCA and ZnSO4 are selected for composite use, the effect of protein precipitation is enhanced, mainly because PLP and serum albumin have a very strong combination, if the protein precipitation is insufficient, the value of PLP detected in the final quantitative result is low, and the detection result is inaccurate;

also the concentration of ZnSO4 needed to be found as too much ZnSO4 inhibited the signal of PLP and other water soluble vitamins.

Finally VC is a common reducing agent and its use is intended to ensure the stability of the substance 5M-THF.

The invention also provides a method for simultaneously detecting the water-soluble vitamins and the metabolites, which is characterized in that the precipitator is added into the sample for precipitation in the sample pretreatment process, and the water-soluble vitamins and the metabolites in the sample are simultaneously detected by liquid chromatography-tandem mass spectrometry.

According to the scheme, the pH value range of the upper computer sample of the liquid chromatography-tandem mass spectrum is 2.2-2.8.

According to the scheme, 0.2% formic acid and 2mM ammonium formate are added into the mobile phase A and the mobile phase B of the liquid chromatography-tandem mass spectrometry, and most methods for detecting MMA adopt derivative methods because two major problems of low response and poor peak shape are met by directly testing the substances. MMA is very sensitive to pH in the solvent, too low a pH leads to peak splitting and too high a pH leads to large peak broadening. Repeated tests show that the pH value of the finally-machined sample is in a range of 2.2-2.8, good peak shape and response can be obtained on a liquid phase mass spectrum, and the peak shape is found to be optimal and the peak broadening is found to be minimum after 0.2% formic acid and 2mM ammonium formate are added into the mobile phase A, B.

According to the scheme, the method for simultaneously detecting the water-soluble vitamins and the metabolites comprises the following detailed steps:

step S1, preparing a calibrator solution, a quality control solution and an internal standard working solution with series concentrations;

step S2, respectively adding internal standard working solution into the calibrator solution, the quality control solution and the sample to be tested with the series of concentrations, and carrying out vortex mixing;

step S3, adding the precipitant of

claim

1 or 2 into each sample mixed in step S2, shaking at 1800-14000 rpm, incubating the shaken sample on ice, centrifuging the incubated sample at 10000-14000rpm and 4 ℃, and taking the supernatant;

step S4, adding NaOH to each supernatant obtained in step S3 to adjust the pH value to be within the range of 2.2-2.8, performing instant centrifugation on the supernatant after the pH value is adjusted by using the conditions of 1000-1500rpm and 4 ℃, and taking the supernatant after the centrifugation as an upper machine sample;

and step S5, taking the computer-loaded sample in the step S4 for LC-MS/MS analysis.

According to the above scheme, the conditions of the liquid chromatography are as follows:

mobile phase: phase A: an aqueous solution containing 0.2% formic acid, 2mM ammonium formate; phase B: a methanol solution containing 0.2% formic acid, 2mM ammonium formate;

elution gradient:

time (min)	Flow rate (mL/min)	Mobile phase A (%)	Mobile phase B (%)
				0.00	0.30	100	0
2.00	0.30	100	0
				4.00	0.30	50	50
5.50	0.30	10	90
				6.50	0.30	10	90
7.00	0.30	100	0
				8.50	0.30	100	0

Column temperature: at 40 ℃.

According to the scheme, the mass spectrum adopts a method of simultaneously scanning positive ions and negative ions, and the ion source parameters are as follows:

positive ion, ion source: electrospray positive ion source (ESI); the detection mode is as follows: multiple Reaction Monitoring (MRM); ion source Temperature (TEM): 400 ℃; atomizing Gas (Gas 1): 55 psi; auxiliary Gas (Gas2) 55 psi; air curtain Gas (Gurtain Gas) 35 psi; electrospray voltage: 5500V;

negative ions, ion source: electrospray negative ion source (ESI); the detection mode is as follows: multiple Reaction Monitoring (MRM); ion source Temperature (TEM): 400 ℃; atomizing Gas (Gas 1): 55 psi; auxiliary Gas (Gas2) 55 psi; air curtain Gas (Gurtain Gas):35 psi; electrospray voltage: 4500V.

The invention has the beneficial effects that: the method for combining and detecting the three active metabolites and other water-soluble vitamins is realized, and a complex solid phase extraction column or a derivative method is not needed, so that the detection cost can be reduced, and the operation difficulty of experimenters is reduced.

Drawings

Fig. 1 is a standard graph of VB 1.

Fig. 2 is a standard graph of VB 2.

FIG. 3 is a standard graph of VB3 (Nicotinamide).

Fig. 4 is a standard graph of VB 5.

Fig. 5 is a standard graph of VB6 (PLP).

Fig. 6 is a standard graph of VB 7.

FIG. 7 is a standard graph of VB9 (5M-THF).

FIG. 8 is a standard graph of VB12 (MMA).

FIG. 9 is a LLOQ (0.05ng/mL) chromatogram of VB1 and a chromatogram of internal standard VB1-13C 4.

FIG. 10 is a LLOQ (0.25ng/mL) chromatogram of VB2 and chromatograms of internal standards VB2-13C4,15N 2.

FIG. 11 is a LLOQ (5ng/mL) chromatogram of VB3(Nicotinamide) and a chromatogram of internal standard VB3-13C 6.

FIG. 12 is a LLOQ (2.5ng/mL) chromatogram of VB5 and chromatograms of internal standards VB5-13C6,15N 2.

FIG. 13 is a LLOQ (5ng/mL) chromatogram of VB6(PLP) and a chromatogram of internal standard VB 6-D3.

FIG. 14 is a LLOQ (0.05ng/mL) chromatogram of VB7 and a chromatogram of internal standard VB 7-D4.

FIG. 15 is a LLOQ (0.5ng/mL) chromatogram of VB9(5M-THF) and a chromatogram of internal standard VB 9-D3.

FIG. 16 is a LLOQ (10ng/mL) chromatogram of VB12(MMA) and a chromatogram of internal standard VB 12-D3.

The figures are LC-MS/MS detection analysis result graphs which are result displays in the embodiment, characters in the graphs are result displays, and the results change according to the result of each detection analysis, namely the characters in the graphs are irrelevant to whether the detection method provided by the invention can be repeatedly implemented, and the characters in the graphs are unclear, so that a person skilled in the art can repeatedly implement the detection method provided by the invention.

Detailed Description

The technical solution of the present invention will be described below with reference to the specific embodiments and the accompanying drawings.

The first embodiment is as follows: a precipitant for simultaneously detecting water-soluble vitamins and metabolites is provided.

A precipitator for simultaneously detecting water-soluble vitamins and metabolites comprises trichloroacetic acid with a final concentration of 7%, zinc sulfate with a final concentration of 0.2M, vitamin C with a final concentration of 3% and water.

Procedure for preparation of 20ml precipitant: weighing 1.15g of ZnSO₄*7H₂O, 1.40g TCA and 0.6g VC were dissolved by vortexing with 20mL ultrapure water, placed in a brown glass bottle and stored in a refrigerator at 4 ℃.

Example two: a method for simultaneously detecting water-soluble vitamins and metabolites is provided.

A method for simultaneously detecting water-soluble vitamins and metabolites is characterized in that in the sample pretreatment process, the precipitator in the embodiment is added into a sample for precipitation, and the water-soluble vitamins and the metabolites in the sample are simultaneously detected through liquid chromatography-tandem mass spectrometry; the pH value range of the computer sample of the liquid chromatogram-tandem mass spectrum is 2.2-2.8; both mobile phase a and mobile phase B of the liquid chromatography-tandem mass spectrometry were supplemented with 0.2% formic acid and 2mM ammonium formate.

The preparation process of the reagent solution related to the detection pretreatment is as follows:

7% TCA (containing 0.2M ZnSO)₄And 3% VC) precipitant preparation: weighing 1.15g of ZnSO₄*7H₂O, 1.40g TCA and 0.6gVC were dissolved by vortexing with 20mL ultrapure water, placed in a brown glass bottle and stored in a refrigerator at 4 ℃.

Preparing a 2M NaOH solution: 4g NaOH was weighed out and dissolved in 50mL water and stored at room temperature.

Vitamin serum preparation:

20g of activated carbon was weighed, rinsed twice with ultrapure water, and 20mL of a purchased blank human serum substrate was added and shaken overnight at 37 ℃. After the night, the mixture is subpackaged into 2mL EP tubes, centrifuged at 13000rpm for 30min, taken out the supernatant, the centrifugation step is repeated, and the centrifuged supernatant is taken out and combined to obtain the vitamin-free serum.

A method for simultaneously detecting water-soluble vitamins and metabolites comprises the following detailed steps:

step S1, preparing a calibrator (standard) solution, a quality control solution and an internal standard working solution with series concentrations:

step S11, accurately weighing each water-soluble vitamin standard, adding methanol (containing 1% VC) to dissolve, mixing all vitamin standard solutions together, and diluting the mixed solution with 50% methanol (containing 1% VC) to prepare a series of working solutions, wherein the series of concentrations are as follows:

step S12: diluting the working solution prepared in the step S11 by using vitamin serum for 20 times to prepare a final standard curve solution with the following concentration:

step S2, respectively taking 200-1.5 mL of each EP tube of the sample to be tested, the calibrator solution and the quality control solution, adding 10-L of the internal standard working solution (the concentration of vitamin in the internal standard working solution is the same as that of 4 in the standard working solution), and carrying out vortex mixing for 15S;

step S3, adding 100 mu L of the precipitator in the first embodiment into each sample mixed in the step S2, placing an EP tube on a shaker at 2000rpm for shaking for 5min, placing the shaken sample on ice for hatching for 15min, placing the hatched sample in a high-speed centrifuge at 12000rpm at 4 ℃ for 10min, and taking supernatant;

s4, placing 150 mu L of each supernatant obtained in the S3 into a 96-pore plate, adding 2M NaOH to adjust the pH value to be within the range of 2.2-2.8, placing the 96-pore plate into an oscillator, oscillating at 1300rpm for 2min, centrifuging at 4 ℃ at 4000rpm for 5min, and taking the supernatant as an upper computer sample;

and step S5, taking the computer-loaded sample in the step S4 to perform LC-MS/MS analysis, and selecting a multi-reaction monitoring MRM scanning mode by adopting an AB SCEIX API 4500 system.

As a preferred example, the conditions of the liquid chromatography are as follows:

and (3) analyzing the column: waters xselectricity HSS T3, 2.1 × 100mm 2.5 μm;

sample introduction amount: 10 mu L of the solution;

elution gradient:

Column temperature: at 40 ℃.

As a preferred embodiment, the mass spectrum adopts a method of simultaneously scanning positive ions and negative ions, and the ion source parameters are as follows:

positive ion, ion source: an electrospray positive ion source; the detection mode is as follows: multiple Reaction Monitoring (MRM); ion source Temperature (TEM): 400 ℃; atomizing Gas (Gas 1): 55 psi; auxiliary Gas (Gas2) 55 psi; air curtain Gas (Gurtain Gas) 35 psi; electrospray voltage: 5500V;

qualitative ions, quantitative ions, fragmentation voltage and collision energy are as follows (· quantitative ions):

negative ions, ion source: an electrospray negative ion source; the detection mode is as follows: multiple Reaction Monitoring (MRM); ion source Temperature (TEM): 400 ℃; atomizing Gas (Gas 1): 55 psi; auxiliary Gas (Gas2) 55 psi; air curtain Gas (Gurtain Gas):35 psi; electrospray voltage: -4500V;

Q1	Q3	Dwell time	Name	DP/V	EP	CE/V	CXP
								117.2	73.1	20	MMA	-15	-10	-12	-10
116.8	55.1	20	MMA-2*	-35	-12	-34	-10
								120.1	76.1	20	MMA-IS	-10	-10	-13	-10
120.1	58.1	20	MMA-IS-1*	-35	-12	-34	-10

the standard curve was established by the internal standard method, as shown in fig. 1-16, and the results show that:

VB1 has good linear relation in the range of 0.05-10ng/mL,

VB2 was well linearly related in the range of 0.25-50 ng/mL.

VB3(Nicotinamide) was linearly related well in the range of 5-1000 ng/mL.

VB5 was well linearly related in the range of 2.5-500 ng/mL.

VB6(PLP) was linearly related well in the range of 5-1000 ng/mL.

VB7 was linearly related well in the range of 0.05-10 ng/mL.

VB9(5M-THF) was linearly well-correlated in the range of 0.5-100 ng/mL.

VB12(MMA) was linearly related well in the range of 10-1000 ng/mL.

The performance index of the method provided by the invention is verified for three days, and the result is as follows:

the present invention is provided by the above embodiments only for illustrating and not limiting the technical solutions of the present invention, and although the above embodiments describe the present invention in detail, those skilled in the art should understand that: modifications and equivalents may be made thereto without departing from the spirit and scope of the invention and any modifications and equivalents may fall within the scope of the claims.

Claims

1. A precipitator for simultaneously detecting water-soluble vitamins and metabolites is characterized by comprising trichloroacetic acid, zinc sulfate and vitamin C.

2. The precipitator for simultaneous detection of water-soluble vitamins and metabolites according to claim 1, consisting of trichloroacetic acid at a final concentration of 7%, zinc sulfate at 0.2M, vitamin C at 3% and water.

3. A method for simultaneously detecting water-soluble vitamins and metabolites, characterized in that, in the sample pretreatment process, the precipitant according to claim 1 or 2 is added to the sample for precipitation, and the water-soluble vitamins and metabolites in the sample are simultaneously detected by liquid chromatography-tandem mass spectrometry.

4. The method according to claim 3, wherein the pH of the sample obtained from the mass spectrometer is in the range of 2.2-2.8.

5. The method of claim 4, wherein 0.2% formic acid and 2mM ammonium formate are added to both mobile phase A and mobile phase B of the LC-MS.

6. The method for simultaneously detecting water-soluble vitamins and metabolites according to claim 5, comprising the following detailed steps:

step S3, adding the precipitant of claim 1 or 2 into each sample mixed in step S2, shaking at 1800-14000 rpm, incubating the shaken sample on ice, centrifuging the incubated sample at 10000-14000rpm and 4 ℃, and taking the supernatant;

7. The method for simultaneously detecting water-soluble vitamins and metabolites as claimed in claim 5 or 6, wherein the conditions of the liquid chromatography are as follows:

elution gradient:

Column temperature: at 40 ℃.

8. The method according to claim 5 or 6, wherein the mass spectrometry is performed by simultaneous scanning of positive and negative ions, and the ion source parameters are as follows:

positive ion, ion source: an electrospray positive ion source; the detection mode is as follows: monitoring multiple reactions; ion source temperature: 400 ℃; atomizing: 55 psi; auxiliary gas 55 psi; 35psi air curtain air; electrospray voltage: 5500V;

negative ions, ion source: an electrospray negative ion source; the detection mode is as follows: monitoring multiple reactions; ion source temperature: 400 ℃; atomizing: 55 psi; auxiliary gas 55 psi; air curtain air: 35 psi; electrospray voltage: 4500V.