CN106770802B - Method and kit for simultaneously detecting multiple vitamins in dry blood filter paper sheet - Google Patents

Abstract

The invention discloses a method for simultaneously detecting multiple vitamins in a dried blood filter paper sheet, which comprises the following steps: (1) mixing the dry blood filter paper sheet specimen with the extracting solution, oscillating, incubating, centrifuging, and taking supernatant; (2) uniformly mixing the supernatant obtained in the step (1) with a mobile phase and internal standard mixed solution, detecting by adopting a tandem mass spectrometry, and comparing the ionic strength of the internal standard of the vitamin and the vitamin to obtain the content of the vitamin in the serum or the plasma to be detected; in addition, the invention also discloses a kit for simultaneously detecting multiple vitamins in the dried blood filter paper sheet, which comprises internal standard mixed liquor, extracting solution, mobile phase and probe washing liquor. The method can extract various water-soluble and fat-soluble vitamins from the dry blood filter paper sheet, and simultaneously quantify more than ten vitamins in 1 specimen within 2-3 min by using a tandem mass spectrometer, so that the detection period is obviously shortened, and the detection result of the vitamins has higher precision and accuracy.

Description

Method and kit for simultaneously detecting multiple vitamins in dry blood filter paper sheet

Technical Field

The invention relates to a vitamin detection technology, in particular to a method and a kit for simultaneously detecting multiple vitamins in a dried blood filter paper sheet.

Background

Vitamins (vitamin) are a class of organic substances that must be obtained from food in order for humans and animals to maintain normal physiological functions, and play an important role in the growth and metabolic processes of the human body. The deficiency of vitamins in the body causes metabolic disorders and various diseases, which are collectively called vitamin deficiency. At present, vitamins can be divided into two categories, namely fat-soluble vitamins and water-soluble vitamins according to solubility, wherein the water-soluble vitamins comprise B vitamins, vitamin C and the like; fat-soluble vitamins include vitamin A, D, E, K, and the like.

Among fat-soluble vitamins, vitamin A is a main component of photosensitive substances of photosensitive cells in retina, and the deficiency of vitamin A can cause xerophthalmia, and for adults, the deficiency of vitamin A can easily cause nyctalopia; vitamin D (VitaminD, VitD) has important significance to human health, especially children health, VitD deficient rickets are one of four diseases which are mainly prevented and treated by children in China, and the clinical findings show that the VitD deficient rickets can influence the functions of tissues and organs such as nerves, muscles, hematopoiesis, immunity and the like besides bone lesions; the vitamin E has the function of antioxidation, can prevent the self oxidation of unsaturated fatty acid, thereby having the function of protecting a biological membrane and preventing the membrane from being damaged due to the hardening of unsaturated fatty acyl in the biological membrane due to oxidation; vitamin K plays an important role in normal blood coagulation, and the lack of vitamin K can cause slow blood coagulation and even cause heavy bleeding.

The water soluble vitamins mainly include B vitamins and vitamin C, such as B₁(thiamine), B₂(Riboflavin), B₅(pantothenic acid), B₆(pyridoxal, etc.), B₁₂(cobalamin), nicotinic acid (niacin), biotin, folic acid, and the like; the B vitamins influence the metabolism of substances in the body by constituting coenzymes, and play an important role in cellular respiration.

The earliest detection of vitamins was used in general additive foods such as infant formula, cereal products and fruit juices. European Directive 2002/46EC enacts a series of regulations regarding the identification of food additives and the detection of labeled vitamin content in vitamin-supplemented food products. National standards GB 5413-2010 in China also have clear regulations on vitamin contents in infant formula food and dairy products, and methods related to the standards include a microbiological method and a high performance liquid chromatography. Regarding the detection of vitamins, the traditional method is a microbiological method, which has high sensitivity and accurate result, but the method has many limitations: the whole experiment period is long, the repeatability of batch detection results is poor, the detection result error is large, and only single vitamin type can be detected each time; recently, the development of the liquid phase tandem mass spectrometry technology enables the detection of vitamins to enter a brand new field, and in recent years, the occupancy rate of the tandem mass spectrometry technology in the domestic medical market is higher and higher, so that the tandem mass spectrometry is popularized in the diagnosis of clinical diseases.

At present, only a single kind of vitamin method for measuring human serum is used in China, and serum is liquid in the transportation process, needs to be contained in a container and needs to be stabilized within a certain temperature range, so that some containers are made of glass and are fragile, and meanwhile, the containers occupy more space; and the serum is maintained in a certain temperature range, constant temperature devices such as a refrigerator need to be purchased, and the refrigerator occupies a large space, so that the serum transportation cost is too high, and the number of transportation in each time is limited.

Disclosure of Invention

Based on this, the invention aims to overcome the defects of the prior art and provide a method and a kit for simultaneously detecting multiple vitamins in a dry blood filter paper sheet.

In order to achieve the purpose, the invention adopts the technical scheme that: a method for simultaneously detecting multiple vitamins in a dried blood filter paper sheet comprises the following steps:

(1) mixing the dry blood filter paper sheet specimen with the extracting solution, oscillating, incubating, centrifuging, and taking supernatant;

(2) and (2) uniformly mixing the supernatant obtained in the step (1) with the mobile phase and internal standard mixed solution, detecting by adopting a tandem mass spectrometry, and comparing the ionic strength of the vitamin and the internal standard of the vitamin to obtain the content of the vitamin in the serum or the plasma to be detected. It should be noted that the tandem mass spectrometry in the present invention employs a liquid chromatography tandem mass spectrometer.

Preferably, the extracting solution in the step (1) comprises the following components in percentage by volume: 84.75-98.95% of methanol, 1-15% of water and 0.05-0.25% of formic acid; or the extracting solution comprises the following components in percentage by volume: 20-79% of ethanol, 20-70% of methanol and 1-10% of water.

Preferably, the collection method of the dried blood filter paper sheet specimen in the step (1) comprises the following steps: dripping fresh whole blood onto quantitative filter paper to make the blood drop distribute uniformly, air drying the filter paper, sealing and storing for use.

Preferably, the chromatographic conditions in step (2):

the mobile phase comprises the following components in percentage by volume: 79.64-97.989% of methanol, 2-20% of water, 0.01-0.35% of formic acid and 0.001-0.01% of at least one of perfluorobutyric acid and trifluoroacetic acid; or the mobile phase comprises the following components in percentage by volume: 79.64-97.989% of acetonitrile, 2-20% of water, 0.01-0.35% of formic acid and 0.001-0.01% of at least one of perfluorobutyric acid and trifluoroacetic acid; or the mobile phase comprises the following components in percentage by volume: 49.71-84.989% of methanol, 15-50% of acetonitrile, 0.01-0.28% of formic acid and 0.001-0.01% of at least one of perfluorobutyric acid and trifluoroacetic acid;

the probe washing liquid is an acetonitrile aqueous solution with the volume concentration of 50-80%, or the probe washing liquid is a methanol aqueous solution with the volume concentration of 50-80%. It should be noted that the probe wash solution functions to equilibrate the mobile phase before and after the assay, aid the assay, wash the probe, and avoid the effect of the pre-sample on the post-sample assay.

Meanwhile, the invention also provides a kit for simultaneously detecting multiple vitamins in a dried blood filter paper sheet, which comprises: internal standard mixed liquor, extracting solution, mobile phase and probe washing liquor.

Preferably, the extracting solution comprises the following components in percentage by volume: 84.75-98.95% of methanol, 1-15% of water and 0.05-0.25% of formic acid; or the extracting solution comprises the following components in percentage by volume: 20-79% of ethanol, 20-70% of methanol and 1-10% of water.

Preferably, the mobile phase comprises the following components in volume percent: 79.64-97.989% of methanol, 2-20% of water, 0.01-0.35% of formic acid and 0.001-0.01% of at least one of perfluorobutyric acid and trifluoroacetic acid; or the mobile phase comprises the following components in percentage by volume: 79.64-97.989% of acetonitrile, 2-20% of water, 0.01-0.35% of formic acid and 0.001-0.01% of at least one of perfluorobutyric acid and trifluoroacetic acid; or the mobile phase comprises the following components in percentage by volume: 49.71-84.989% of methanol, 15-50% of acetonitrile, 0.01-0.28% of formic acid and 0.001-0.01% of at least one of perfluorobutyric acid and trifluoroacetic acid.

Preferably, the probe washing liquid is an acetonitrile aqueous solution with a volume concentration of 50-80%, or the probe washing liquid is a methanol aqueous solution with a volume concentration of 50-80%.

Preferably, the internal standard mixed liquor comprises a vitamin B1 internal standard, a folic acid internal standard, a vitamin A internal standard, a 25 hydroxy vitamin D2 internal standard, a 25 hydroxy vitamin D3 internal standard, a vitamin E internal standard and a vitamin K1 internal standard.

In conclusion, the beneficial effects of the invention are as follows:

(1) according to the method, vitamin molecules are cracked into the sub-ions with different mass-to-charge ratios through voltage, the number of the sub-ions with different mass-to-charge ratios is scanned, the concentration of the vitamin to be detected is calculated by referring to the concentration of a known vitamin internal standard, the content of the water-soluble vitamin and the content of the fat-soluble vitamin in the dry blood filter paper sheet can be detected at the same time, and the method and the kit for detecting the vitamin are the first example in China, so that the precision and the accuracy are higher, the sample to be detected is easy to transport, and the cost is lower;

(2) the method can extract various water-soluble and fat-soluble vitamins from the dried blood filter paper sheet, simultaneously quantify more than ten vitamins in 1 sample within 2-3 min by using a tandem mass spectrometer, and can evaluate and monitor the vitamin level in a human body through analysis, so that the diagnosis and treatment effect evaluation of vitamin-related diseases are carried out, and the detection period is obviously shortened;

(3) the method of the invention uses quantitative filter paper to prepare a clinically collected whole blood sample into a dry blood filter paper sheet sample, the dry blood filter paper sheet is easy to quantify a trace sample and is just matched with a tandem mass spectrometry method with extremely high sensitivity, the dry blood filter paper sheet is easy to store and transport, and the extracting solution in the dry blood filter paper sheet sample can directly denature protein in the dry blood filter paper sheet sample, thereby avoiding the complicated step of removing the protein from the liquid sample.

Drawings

FIG. 1 is the total ion diagram obtained in example 1;

FIG. 2 is a mass spectrum of each component obtained in example 1 after scanning.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples.

Example 1 method for simultaneous detection of multiple vitamins in a sheet of dried blood filter paper

The method comprises the following main experimental steps:

(1) preparing an internal standard working solution: isotope internal standards and concentrations of various vitamins provided by Cambridge isotope laboratories in America are adopted; and calculating the amount of the internal standard working solution according to the current experimental sample amount. And mixing the various internal standards to obtain high-concentration internal standard mixed liquor, diluting the high-concentration internal standard mixed liquor by adopting a mobile phase according to the proportion of 1:100 when the internal standard mixed liquor is used, and fully and uniformly mixing to obtain the internal standard working solution.

(2) Collecting a sample to be detected: and (3) dripping fresh whole blood onto a quantitative filter paper sheet, enabling the blood drops to be distributed uniformly naturally, hanging and airing for no less than 4 hours to obtain a dry blood filter paper sheet specimen, and sealing and storing at 2-8 ℃ for later use.

(3) The analysis process comprises the following steps:

1. taking a dried blood filter paper specimen, using a perforating pliers to punch 10-20 blood spots with the diameter of 3.2 mm, putting the blood spots into a centrifuge tube, adding 200-500 microliters of extracting solution, and incubating for 45 minutes at the temperature of 27-30 ℃ under the vibration frequency oscillation of 650-750 rpm;

2. centrifuging, adding all the supernatant into a 96-well plate, blow-drying with a nitrogen blow-drying apparatus, re-dissolving 100 microliters of mobile phase, simultaneously adding 50 microliters of mixed internal standard working solution, covering with an aluminum foil sealing film to reduce volatilization, oscillating and mixing uniformly;

3. and (3) putting the 96-well plate into an automatic sample injector of a tandem mass spectrometry system, starting application software, establishing a sample list, and selecting a correct internal standard concentration file and a collection method to detect the content of multiple vitamins in the dry blood filter paper sheet.

(4) And (4) calculating a result:

software in the tandem mass spectrometry system can calculate the detection result by comparing the ionic strength of the analyte and the internal standard, give the concentration of each vitamin and generate a data report.

(5) Note that:

1. immediately putting the isotope standard substance into a refrigerator at 2-8 ℃ for storage after the isotope standard substance is used up;

2. when the microporous plate is wrapped by the aluminum foil, the microporous plate must be tightly wrapped;

3. if the reagent has been poured from the reagent bottle, it cannot be used again.

II, main reagents:

the reagents used in this example include an extract, a mobile phase and a probe wash solution, and the specific reagent formula in this example is:

(1) internal standard: isotope internal standards and concentrations of each vitamin provided by Cambridge isotope laboratories in the United states were selected and are shown in the following table:

name (R)	English name of commodity	Concentration of
			Vitamin B1 internal standard	Thiamine～d3Hydrochloride	30nmol/L
Internal standard of folic acid	Folic Acid～d2	4nmol/L
			Vitamin A internal standard	Vitamin A～d5Acetate	1.5μmol/L
25 hydroxy vitamin D2 internal standard	25～Hydroxyvitamin D2～[d6]	10nmol/L
			25 hydroxy vitamin D3 internal standard	25～Hydroxyvitamin D3～～[d6]	30nmol/L
Vitamin E internal standard	α～Tocopherol～d6	6μg/mL
			Vitamin K1 internal standard	Vitamin K1～[d7])	1.5ng/mL

(2) Extracting solution:

one of the following formulations was used:

the formula I is as follows: the composite material comprises the following components in percentage by volume: 84.75-98.95% of methanol, 1-15% of water and 0.05-0.25% of formic acid;

and a second formula: the composite material comprises the following components in percentage by volume: 20-79% of ethanol, 20-70% of methanol and 1-10% of water.

(3) Mobile phase:

the mobile phase has three formulas which can be respectively combined with the two extracting solution formulas in a cross way, and the following three different formulas have no influence on the accuracy and precision of the final detection result.

The formula I is as follows: the composite material comprises the following components in percentage by volume: 79.64-97.989% of methanol, 2-20% of water, 0.01-0.35% of formic acid and 0.001-0.01% of at least one of perfluorobutyric acid and trifluoroacetic acid, and after being uniformly mixed, the mixture is sealed and subjected to ultrasonic degassing for 15-20 minutes at normal temperature;

and a second formula: the composite material comprises the following components in percentage by volume: 79.64-97.989% of acetonitrile, 2-20% of water, 0.01-0.35% of formic acid and 0.001-0.01% of at least one of perfluorobutyric acid and trifluoroacetic acid, and after being uniformly mixed, the mixture is sealed and subjected to ultrasonic degassing for 15-20 minutes at normal temperature;

and the formula III: the composite material comprises the following components in percentage by volume: 49.71-84.989% of methanol, 15-50% of acetonitrile, 0.01-0.28% of formic acid and 0.001-0.01% of at least one of perfluorobutyric acid and trifluoroacetic acid, and sealing and ultrasonically degassing for 15-20 minutes at normal temperature after uniformly mixing.

(4) Probe washing solution: 1. 50-80% acetonitrile in water; 2. 50-80% methanol aqueous solution.

(5) Note that:

1. the proportions of acetonitrile, water, formic acid and methanol in the public extracting solution and mobile phase can be adjusted in corresponding ranges, and corresponding parameters need to be adjusted on a mass spectrometer, so that the accuracy and precision of the detected vitamin content result are not influenced;

2. all reagents are in chromatographic grade;

3. the water used for preparation is ultrapure water filtered by a triple-distilled water or water purifier, the resistance of the ultrapure water is more than or equal to 18 MOmega or the conductance of the ultrapure water is less than 5us/cm, and the PH value is 7.0 +/-0.2;

4. and (3) according to the formulas of the extracting solution and the mobile phase, the parent ions and the daughter ions of each vitamin in a series mass spectrometer model TQD and a model AB3200, and mass spectrum conditions.

Thirdly, mass spectrum parameters:

the set of reference mass spectra conditions on the tandem mass spectrometer waters TQD according to the extract and mobile phase formulations are as follows:

the reference mass spectrometry conditions according to the set up of the extract and mobile phase formulations on the tandem mass spectrometer AB3200 are as follows:

it should be noted that, the parent ions in the above parameters are fixed, and when the mass spectrum parameters of different machines or machines of the same brand are changed, the daughter ions may be changed.

Wherein, the liquid phase pump gradient and the mass spectrometer conditions are as follows:

it should be noted that, because each machine is installed and debugged, the precision of the machine is different, the above parameters are only used for reference, and the specific debugging parameters are slightly changed by engineers.

As shown in fig. 1, the abscissa of the total ion graph obtained by the method of this embodiment represents time, and the ordinate represents total ion number, and the graph represents total scanned ion number of all ions to be detected, and it can be determined whether the total ion number meets the detection requirement, and if not, the result accuracy is affected.

As shown in fig. 2, the mass spectrum of each scanned component obtained by the method of the present embodiment has an abscissa representing the mass-to-charge ratio of parent ions (number of monitoring in real time, not arranged by size) and an ordinate representing the ion intensity, and represents the ion number of different detection substances, from which the vitamin concentration to be detected can be calculated.

Example 2 kit for simultaneous detection of multiple vitamins in dried blood filter paper sheet

According to different blood samples, reagent proportion and tandem mass spectrometry conditions are respectively set, the kit can measure 11 water-soluble vitamins and 7 fat-soluble vitamins at one time, and the measured vitamins are shown in the following table:

water-soluble vitamins	Fat-soluble vitamin
		Nicotinic acid amine	Vitamin A
Nicotinic acid	Vitamin D2
		Vitamin B1	Vitamin D3
Vitamin B2	25-hydroxy vitamin D2
		Pyridoxine hydrochloride	25-Hydroxyvitamin D3
Pyridoxamine hydrochloride	Vitamin E
		Pyridoxal hydrochloride	Vitamin K1
Folic acid
		Pantothenic acid
Biotin
		Vitamin B12

The kit of this embodiment comprises: the probe washing solution comprises internal standard substances, the internal standard substances adopt isotope internal standards of various vitamins and concentrations thereof provided by Cambridge isotope laboratories in America, and the extracting solution and the mobile phase can be mutually crossed and matched for use.

(1) Use of internal standard mixture:

the method comprises the steps of adopting isotope internal standards and concentrations of various vitamins provided by Cambridge isotope laboratories, mixing the internal standards to obtain high-concentration internal standard mixed liquor, diluting the high-concentration internal standard mixed liquor by using a mobile phase according to a required proportion when the internal standard mixed liquor is used, and fully and uniformly mixing to obtain internal standard working solution.

(2) The mobile phase has three formulas which can be respectively combined with the two extracting solution formulas in a cross way, the three different formulas have no influence on the accuracy and precision of the final detection result, and one of the following formulas is adopted by the mobile phase:

the formula I is as follows: the composite material comprises the following components in percentage by volume: 79.64-97.989% of methanol, 2-20% of water, 0.01-0.35% of formic acid and 0.001-0.01% of at least one of perfluorobutyric acid and trifluoroacetic acid, and after being uniformly mixed, the mixture is sealed and subjected to ultrasonic degassing for 15-20 minutes at normal temperature;

and a second formula: the composite material comprises the following components in percentage by volume: 79.64-97.989% of acetonitrile, 2-20% of water, 0.01-0.35% of formic acid and 0.001-0.01% of at least one of perfluorobutyric acid and trifluoroacetic acid, and after being uniformly mixed, the mixture is sealed and subjected to ultrasonic degassing for 15-20 minutes at normal temperature;

and the formula III: the composite material comprises the following components in percentage by volume: 49.71-84.989% of methanol, 15-50% of acetonitrile, 0.01-0.28% of formic acid and 0.001-0.01% of at least one of perfluorobutyric acid and trifluoroacetic acid, and sealing and ultrasonically degassing for 15-20 minutes at normal temperature after uniformly mixing.

(3) The extracting solution adopts one of the following formulas:

the formula I is as follows: the composite material comprises the following components in percentage by volume: 84.75-98.95% of methanol, 1-15% of water and 0.05-0.25% of formic acid;

and a second formula: the composite material comprises the following components in percentage by volume: 20-79% of ethanol, 20-70% of methanol and 1-10% of water.

(4) Probe washing solution: one of the following may be employed:

1.50-80% acetonitrile in water; 2.50-80% methanol aqueous solution.

The method of example 1 can be used in combination with a tandem mass spectrometer (MS/MS measuring instruments of AB and WATERS) commonly used in China and the kit of example 2 to simultaneously detect a plurality of water-soluble vitamins and fat-soluble vitamins in a dry blood filter paper sheet through one experiment, and the detection result has high precision and accuracy.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims (3)

1. A method for simultaneously detecting multiple fat-soluble and water-soluble vitamins in a dried blood filter paper sheet is characterized by comprising the following steps:

(1) mixing the dry blood filter paper sheet specimen with the extracting solution, oscillating, incubating, centrifuging, and taking supernatant;

(2) uniformly mixing the supernatant obtained in the step (1) with a mobile phase and internal standard mixed solution, detecting by adopting a tandem mass spectrometry, and comparing the ionic strength of vitamins and internal standards of the vitamins to obtain the content of the vitamins in the dry blood filter paper to be detected; wherein,

the extracting solution in the step (1) comprises the following components in percentage by volume: 84.75-98.95% of methanol, 1-15% of water and 0.05-0.25% of formic acid; or the extracting solution comprises the following components in percentage by volume: 20-79% of ethanol, 20-70% of methanol and 1-10% of water;

the chromatographic conditions in the step (2) comprise: the mobile phase comprises the following components in percentage by volume: 79.64-97.989% of acetonitrile, 2-20% of water, 0.01-0.35% of formic acid and 0.001-0.01% of perfluorobutyric acid; or the mobile phase comprises the following components in percentage by volume: 49.71-84.989% of methanol, 15-50% of acetonitrile, 0.01-0.28% of formic acid and 0.001-0.01% of perfluorobutyric acid;

the multiple fat-soluble vitamins are vitamin A, vitamin D2, vitamin D3, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, vitamin E and vitamin K1, and the multiple water-soluble vitamins are niacinamide, nicotinic acid, vitamin B1, vitamin B2, pyridoxine hydrochloride, pyridoxamine hydrochloride, pyridoxal hydrochloride and folic acid;

the chromatographic conditions in the step (2) comprise: the probe washing liquid is an acetonitrile aqueous solution with the volume concentration of 50-80%, or the probe washing liquid is a methanol aqueous solution with the volume concentration of 50-80%;

the internal standard mixed liquid comprises a vitamin B1 internal standard, a folic acid internal standard, a vitamin A internal standard, a 25-hydroxyvitamin D2 internal standard, a 25-hydroxyvitamin D3 internal standard, a vitamin E internal standard and a vitamin K1 internal standard.

2. The method for simultaneously detecting multiple fat-soluble and water-soluble vitamins in a dried blood filter paper sheet according to claim 1, wherein the collection method of the dried blood filter paper sheet specimen in the step (1) comprises the following steps: dripping fresh whole blood onto quantitative filter paper to make the blood drop distribute uniformly, air drying the filter paper, sealing and storing for use.

3. A kit for simultaneously detecting multiple fat-soluble and water-soluble vitamins in a dried blood filter paper sheet is characterized by comprising: the kit comprises an internal standard mixed solution, an extracting solution, a mobile phase and a probe washing solution, wherein the extracting solution comprises the following components in percentage by volume: 84.75-98.95% of methanol, 1-15% of water and 0.05-0.25% of formic acid; or the extracting solution comprises the following components in percentage by volume: 20-79% of ethanol, 20-70% of methanol and 1-10% of water; the mobile phase comprises the following components in percentage by volume: 79.64-97.989% of acetonitrile, 2-20% of water, 0.01-0.35% of formic acid and 0.001-0.01% of perfluorobutyric acid; or the mobile phase comprises the following components in percentage by volume: 49.71-84.989% of methanol, 15-50% of acetonitrile, 0.01-0.28% of formic acid and 0.001-0.01% of perfluorobutyric acid;

the multiple fat-soluble vitamins are vitamin A, vitamin D2, vitamin D3, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, vitamin E and vitamin K1, and the multiple water-soluble vitamins are niacinamide, nicotinic acid, vitamin B1, vitamin B2, pyridoxine hydrochloride, pyridoxamine hydrochloride, pyridoxal hydrochloride and folic acid;

the probe washing liquid is an acetonitrile aqueous solution with the volume concentration of 50-80%, or the probe washing liquid is a methanol aqueous solution with the volume concentration of 50-80%; the internal standard mixed liquid comprises a vitamin B1 internal standard, a folic acid internal standard, a vitamin A internal standard, a 25-hydroxyvitamin D2 internal standard, a 25-hydroxyvitamin D3 internal standard, a vitamin E internal standard and a vitamin K1 internal standard.

Images