CN113138236A - Method for quantitatively detecting vitamin B12 in serum and pretreatment kit - Google Patents

Abstract

The invention relates to the technical field of trace compound detection, in particular to a method for quantitatively detecting vitamin B12 in serum and a pretreatment kit. The detection method comprises the following steps: pretreating a serum sample by adopting a method of methanol and isopropanol precipitation and nitrogen blowing redissolution; preparing reference solutions of cyanocobalamin, mecobalamin, hydroxycobalamin and cobamamide; and (3) carrying out HPLC-MS/MS detection on the pretreated serum sample and a prepared reference solution, wherein the mixed solution of methanol and isopropanol is methanol: the volume ratio of the isopropanol is 9: 1. The method for determining the concentrations of cyanocobalamin, mecobalamin, hydroxycobalamin and cobamamide in serum established in the embodiment of the invention has high detection sensitivity, low detection limit and good accuracy, can be used for clinically monitoring the concentration of VB12 family, more truly reflects the content of vitamin B12 in human serum, and can more clearly and accurately assist in guiding medication in the aspect of monitoring the concentration of a medicament.

Description

Method for quantitatively detecting vitamin B12 in serum and pretreatment kit

Technical Field

The invention relates to the technical field of trace compound detection, in particular to a method for quantitatively detecting vitamin B12 in serum and a pretreatment kit.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Vitamin B12 is also called cobalamin, is the only vitamin containing metal elements, vitamin B12 in the nature is synthesized by microorganisms, and higher animals and plants cannot produce vitamin B12. Vitamin B12 is the only vitamin that needs the help of intestinal secretions (endogenous factors) to be absorbed. Some people suffer from pernicious anemia due to gastrointestinal abnormalities, lack of this endogenous factor, and even adequate dietary sources. Vitamin b12 is substantially absent from vegetable foods and remains in the intestinal tract for a long period of time, approximately three hours (most water-soluble vitamins require only a few seconds) to be absorbed. The main physiological functions of vitamin B12 are to participate in the production of bone marrow red blood cells, prevent pernicious anemia, and prevent the destruction of cerebral nerves.

Members of the vitamin B12 family mainly include: cyanocobalamin, hydroxycobalamin, cobamamide and mecobalamin. They also have different meanings in clinical applications.

(1) Cyanocobalamin

Cyanocobalamin is required to be converted into mecobalamin and cobamamide in vivo to take effect, has the best stability, and has the absorption speed in vivo at the head of four, although the cyanocobalamin cannot be directly utilized by the human body, the efficacy of the cyanocobalamin is no less than that of the direct use of the cobamamide.

(2) Mecobalamin

The mecobalamin has the absorption speed in a human body only second to that of cyanocobalamin, has good transmissibility on nerve tissues, can promote nucleic acid-protein-fat metabolism and repair damaged nerve tissues, and has obvious curative effects on peripheral neuropathy such as nerve disorder caused by diabetes, polyneuritis and the like, particularly on numbness, pain and paralysis in clinic. In the aspect of treating peripheral neuropathy, the clinical application safety and curative effect of mecobalamin are superior to those of cobamamide and cyanocobalamin.

2013 diagnosis and treatment consensus on diabetic peripheral neuropathy recommends that supplementing B vitamins can improve diabetic neuropathy, and recommends mecobalamin as adjuvant therapy for neurotrophic rescue therapy.

(3) Hydroxycobalamins

Hydroxocobalamin is known as long-acting cobalamin because of its good water solubility and slow metabolic rate in urine. Although the absorption speed of the medicine is not as fast as that of cyanocobalamine and mecobalamin, the medicine can reach higher concentration in eyes, is beneficial to relieving visual fatigue and nourishing optic nerves, and therefore, the medicine has certain curative effect on tobacco toxicity amblyopia.

In addition, hydroxycobalamin is capable of rapidly binding to free cyanide groups in aqueous solution to form non-toxic cyanocobalamin, and has therefore been approved by many countries for use in the treatment of known or suspected cyanide poisoning.

(4) Cobamamide as a stabilizer

Cobamamide is internationally used as a biochemical reagent and a test reagent, and is currently only collected as a medicine by the Chinese pharmacopoeia. Cobamamide has been used in the chinese market for many years with essentially the same effect as other family members.

The reference book divides cobamamide into blood system medicines, which can be used for treating megaloblastic anemia, malnutritional anemia, gestational anemia, and leukopenia caused by radioactive rays and chemotherapy drugs. Cobamamide can increase the hemoglobin content in vivo, improve anemia, and has obvious curative effect.

The determination of serum vitamin B12 is the most straightforward method of identification. At present, the serum detection method mainly comprises a chemiluminescence method, a liquid chromatogram-tandem mass spectrometry method, a liquid chromatogram-inductively coupled plasma method and the like. Wherein the liquid chromatography-tandem mass spectrometry only detects the cyanocobalamin in the VB12 family singly and does not detect the quadruple substances in the VB12 family simultaneously,

the chemiluminescence method applied in the market can be used for measuring the vitamin B12 family, and the application principle is enzyme-linked immunity. The liquid chromatography-inductively coupled plasma method can also detect vitamin B12 family, and the principle is to detect cobalt element contained in vitamin B12 family. The application of the liquid chromatography-tandem mass spectrometry method to singly detect the cyanocobalamin in the VB12 family has certain one-sidedness, and cannot truly reflect the content of VB12 in a human body. According to the invention, mecobalamin, hydroxycobalamin and cobamamide are converted into cyanocobalamin to detect total VB12, other members of a VB12 family are not directly quantified by the method, and the problem of conversion efficiency in the process of converting the cyanocobalamin is not considered, so that inaccurate quantification is caused. And cyanide used in the conversion process is extremely toxic and is not suitable for being used as a conventional detection method.

Disclosure of Invention

In order to solve the technical problems in the prior art, the invention establishes a method for measuring the concentrations of cyanocobalamin, mecobalamin, hydroxycobalamin and cobamamide in serum by a high performance liquid chromatography-mass spectrometry (HPLC-MS/MS) combined method, can be used for clinically monitoring the concentration of VB12 family, more truly reflecting the content of vitamin B12 in human serum, can more clearly and accurately guide medication in the aspect of monitoring the concentration of a medicament, and can more intuitively adjust the dosage of the medicament and evaluate the curative effects of different medicaments by respectively quantifying the cyanocobalamin, the mecobalamin, the hydroxycobalamin and the cobamamide.

In order to achieve the above object, the technical solution of the present invention is as follows:

in a first aspect of the invention, there is provided a method for quantitatively detecting vitamin B12 in serum, the method comprising:

pretreating a serum sample by adopting a method of methanol and isopropanol precipitation and nitrogen blowing redissolution; preparing reference solutions of cyanocobalamin, mecobalamin, hydroxycobalamin and cobamamide;

and (3) carrying out HPLC-MS/MS detection on the pretreated serum sample and the prepared reference substance solution.

The inventor finds that only a single detection of cyanocobalamin in a VB12 family has certain one-sidedness when the content of VB12 in a human body is detected by HPLC-MS/MS, and the content of VB12 in the human body cannot be truly reflected; the method for detecting the total VB12 by converting mecobalamin, hydroxycobalamin and cobamamide into cyanocobalamin does not consider the problem of conversion efficiency when the cyanocobalamin is converted, and also causes inaccurate quantification.

The invention provides a method for detecting vitamin B12 family concentration by high performance liquid chromatography-tandem mass spectrometry for the first time, which can detect the drug concentration of cyanocobalamin, mecobalamin, hydroxycobalamin and cobamamide in serum, and can also reflect the nutritional status of vitamin B12 in human body by using the index, thus solving the problem of single detection of the sheet surface property of vitamin B12 represented by cyanocobalamin by mass spectrometry.

The invention needs to overcome two technical difficulties, one is that the VB12 family has low content in human body and is difficult to detect, the inventor compares several methods of protein precipitation, liquid-liquid extraction, solid-phase extraction and the like, integrates the extraction efficiency of four substances, and finally selects a method of methanol and isopropanol mixed solution precipitation and nitrogen blowing redissolution for extraction, so that the detection requirements can be met; secondly, due to the fact that VB12 family structures are similar, the sizes of fragmented daughter ion fragments are the same, the sizes of parent ions are very close, in order to avoid mutual interference on a mass spectrum, four substances need to be separated on a liquid phase, and after a plurality of different chromatographic columns, mobile phase compositions and liquid phase gradient adjustment are tried, the four substances are finally separated.

The method for determining the concentrations of cyanocobalamin, mecobalamin, hydroxycobalamin and cobamamide in serum established by the invention can be used for clinically monitoring the concentration of VB12 family, more truly reflecting the content of vitamin B12 in human serum, and can more clearly and accurately assist in guiding medication in the aspect of monitoring the concentration of a medicament. After the serum is extracted, ion pairs of cyanocobalamin, mecobalamin, hydroxycobalamin and cobamamide are scanned by mass spectrometry to carry out concentration quantification, and the method has the advantages of high detection sensitivity, low detection limit and good accuracy.

In a second aspect of the present invention, there is provided a pretreatment kit for detecting vitamin B12 in serum, the kit comprising a mixed solution of methanol and isopropanol.

The specific embodiment of the invention has the following beneficial effects:

the problem that in the prior art, when the liquid chromatography-tandem mass spectrometry is used for detecting the content of VB12 in a human body, only cyanocobalamin in a VB12 family is singly detected, the content of VB12 in the human body cannot be truly reflected due to certain one-sidedness is solved, and the quantification is more accurate. In addition, no virulent cyanide is used in the detection process, so that the detection method is safer;

the cyanocobalamin, the mecobalamin, the hydroxycobalamin and the cobamamide are respectively subjected to quantitative analysis, so that the dosage of the medicine can be more intuitively adjusted and the curative effects of different medicines can be evaluated in the aspect of monitoring the blood medicine concentration to guide the medicine application;

the problems that the VB12 family is low in content in a human body and difficult to detect are solved by preprocessing the serum sample, and VB12 in the serum sample is extracted by a method of methanol and isopropanol mixed solution precipitation and nitrogen blowing redissolution, so that the detection requirement can be met;

VB12 family structure is similar, the size of the broken daughter ion fragment is the same, the size of the parent ion is very close, the implementation mode of the invention separates four substances of cyanocobalamin, mecobalamin, hydroxycobalamin and adenosylcobalamin by the composition of chromatographic columns and mobile phases and the adjustment of liquid phase gradient, and avoids mutual interference on mass spectrum;

the method for determining the concentrations of cyanocobalamin, mecobalamin, hydroxycobalamin and cobamamide in serum established in the embodiment of the invention has high detection sensitivity, low detection limit and good accuracy, can be used for clinically monitoring the concentration of VB12 family, more truly reflects the content of vitamin B12 in human serum, and can more clearly and accurately assist in guiding medication in the aspect of monitoring the concentration of a medicament.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a chromatogram of cyanocobalamin, methylcobalamin, hydroxocobalamin and cobamamide from the vitamin B12 family according to an embodiment of the present invention;

FIG. 2 is a graph showing the standard curves of cyanocobalamin, methylcobalamin, hydroxocobalamin and cyanocobalamin in the vitamin B12 family in accordance with the example of the present invention.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

In one embodiment of the present invention, there is provided a method for quantitatively detecting vitamin B12 in serum, the method comprising:

pretreating a serum sample by adopting a method of methanol and isopropanol precipitation and nitrogen blowing redissolution; preparing reference solutions of cyanocobalamin, mecobalamin, hydroxycobalamin and cobamamide;

and (3) carrying out HPLC-MS/MS detection on the pretreated serum sample and the prepared reference substance solution.

In a specific embodiment, the serum sample is pretreated by the following steps:

adding an internal standard solution into a serum sample, adding a mixed solution of methanol and isopropanol, blowing the supernatant solution by using nitrogen, redissolving by using a methanol solution, transferring the redissolved solution into a sample plate, and carrying out HPLC-MS/MS analysis, wherein all operations are carried out under the condition of keeping out of the sun.

Preferably, the internal standard solution is a cyanocobalamine-d 7 solution, and further preferably, the concentration of the cyanocobalamine solution is 0.5-1.5 mu g/mL; when in use, the mixture is diluted to the required concentration by methanol; the isotope labeled cyanocobalamin is used as an internal standard, so that the error caused in the experimental process can be reduced, and the accuracy is improved.

In this series of examples, the mixed solution of methanol and isopropanol was methanol: mixed solution with the volume ratio of isopropanol of 8-10: 1; preferably, the ratio of methanol: the volume ratio of isopropanol is 9: 1.

In the series of embodiments, the methanol solution is 40-60% methanol aqueous solution by volume percentage;

in this series of examples, all manipulations of the serum sample pretreatment process were performed under light-shielding conditions.

In one particular embodiment, the control solution is formulated as follows: dissolving a cyanocobalamine standard substance by using water to prepare a stock solution with the final concentration of 1 mg/mL; dissolving the mecobalamin standard substance with methanol to prepare stock solution with the final concentration of 1 mg/mL; dissolving the standard substance of hydroxycobalamin with water to prepare stock solution with the final concentration of 1 mg/mL; the cobamamide standard was dissolved in methanol to prepare a stock solution with a final concentration of 1 mg/mL.

In the series of examples, the preparation process of the reference solution was performed in the dark.

In a specific embodiment, the liquid chromatography column is a pentafluorophenyl packing column, preferably a femomei Kinetex F5 column (2.1mm x 100mm, 2.7 μm);

in a specific embodiment, the mobile phase a of the liquid chromatography is an aqueous solution containing 0.1-0.2%% formic acid; the mobile phase B is a methanol solution containing 0.1-0.2% of formic acid;

in the series of embodiments, the gradient elution procedure of the liquid chromatography is 0-1.0 min, 1% B, 3.0-6.0 min, 98% B, 6.5-9 min, 1% B;

in the series of embodiments, the column temperature is 35-45 ℃; the flow rate is 0.3-0.5 mL/min.

In one particular embodiment, the mass spectrometry conditions are: an electrospray ESI source positive ion mode is adopted, the ion source temperature is 500-.

In a second aspect of the present invention, there is provided a pretreatment kit for detecting vitamin B12 in serum, the kit comprising a mixed solution of methanol and isopropanol;

preferably, the volume ratio of the methanol to the isopropanol is 8-10: 1, and more preferably 9: 1.

The invention will be further explained and illustrated with reference to specific examples.

Example 1

Cyanocobalamin (control), purity: 99 percent; mecobalamin (control), purity 97%; hydroxycobalamin (control), purity: 96 percent; cobamamide (control), purity: 98 percent; cyanocobalamin-d 7 (internal standard), purity: 99 percent.

(1) Preparation of an internal Standard solution

Preparing cyanocobalamine-d 7 into a solution of 1 mu g/mL by using isotope labeled cyanocobalamine as an internal standard and methanol; for use, dilute to the desired concentration with methanol.

(2) Preparing reference substance solution

Dissolving a cyanocobalamine standard substance by using water to prepare a stock solution with the final concentration of 1 mg/mL; dissolving the mecobalamin standard substance with methanol to prepare stock solution with the final concentration of 1 mg/mL; dissolving the standard substance of hydroxycobalamin with water to prepare stock solution with the final concentration of 1 mg/mL; the cobamamide standard was dissolved in methanol to prepare a stock solution with a final concentration of 1 mg/mL. The whole process is carried out in a dark place.

(3) Pretreatment of serum samples

a. Optimization of serum sample pretreatment conditions

Compared with pretreatment methods such as protein precipitation, liquid-liquid extraction, solid-phase extraction and the like, the method can extract four substances such as cyanocobalamine, mecobalamine, hydroxycobalamin and cobamamide, is a methanol precipitation method, and on the basis, the inventor optimizes an extraction reagent and finally determines that the mixed solution (8-10: 1, v/v) of methanol and isopropanol has the best effect.

b. Pretreatment of serum samples using optimized conditions

A serum sample of 200. mu.L was added to a brown glass vial, and an internal standard solution (1. mu.g/mL) of 1. mu.L was added, vortexed for 30s, and methanol was added: 600 μ L of isopropanol (9:1, v/v), vortexing for 5min, centrifuging for 5min, taking the supernatant solution to another glass bottle, drying with nitrogen, redissolving with 75 μ L of 50% methanol (methanol: water ═ 1: 1, v/v), transferring into a sample plate after redissolution, and performing HPLC-MS/MS analysis, wherein all operations are performed under dark conditions.

(4) HPLC-MS/MS detection

The instrument comprises the following steps: YS EXT 9900MD high performance liquid chromatography tandem mass spectrometry detection system.

a. Optimization selection of chromatographic column:

in order to separate cyanocobalamin, methylcobalamin, hydroxocobalamin and cobamamide, the inventors tried several different columns, of which the types were femomei Kinetex C18 column (2.1mm × 100mm, 2.7 μm), femomei Kinetex F5 column (2.1mm × 100mm, 2.7 μm), femomei Kinetex polar C18 column (2.1mm × 100mm, 2.7 μm), respectively, and the retention times of the respective substances were as follows. A pentafluorophenyl packed column was found to separate the four materials, the column model was a Heinomei Kinetex F5 column (2.1 mm. times.100 mm, 2.7 μm).

Retention time (min)	C18	F5	polor C18
				Cyanocobalamin	2.20	4.81	3.77
Mecobalamin	2.34	5.57	3.80
				Hydroxycobalamins	2.12	4.01	3.42
Cobamamide as a stabilizer	2.42	5.03	3.61

b. HPLC-MS/MS detection with optimally selected chromatography columns

Chromatographic conditions are as follows: a Fenomei Kinetex F5 column (2.1 mm. times.100 mm, 2.7 μm); mobile phase A: an aqueous solution containing 0.1% formic acid; mobile phase B: 0.1% formic acid in methanol; column temperature: 40 ℃; flow rate: 0.4 mL/min. Gradient elution: 0-1.0 min, 1% of B, 3.0-6.0 min, 98% of B, 6.5-9 min and 1% of B.

As shown in figure 1, the retention time of cyanocobalamin is 4.81min, the retention time of mecobalamin is 5.57min, the retention time of hydroxycobalamin is 4.01min, the retention time of adenosylcobalamin is 5.03min, the chromatographic peaks of four cobalamins are separated, and the substances do not interfere with each other.

Mass spectrum conditions: the positive ion mode of an electrospray ESI source is adopted, the temperature of the ion source is 550 ℃, and the positive ion voltage is 3500V. The vitamin B12 family mass spectrometry scan ion pairs and voltages are shown in table 1.

TABLE 1 vitamin B12 family Mass Spectroscopy scanning ion pairs and Voltage

Substance(s)	Parent ion	Daughter ions	Lens voltage	Collision voltage
					Cyanocobalamin	678.5	147	45	40
Mecobalamin	673	147	60	48
					Hydroxycobalamins	665	147	80	38
Cobamamide as a stabilizer	790.5	147	85	50
					Cyanocobalamin internal standard	682	154	45	42

Example 2

The feasibility of the method of HPLC-MS established in example 1 was examined:

(1) linear relationship and detection limit, quantitation limit: the cyanocobalamine, mecobalamin, hydroxycobalamin, adenosylcobalamin standard solution and Bovine Serum Albumin (BSA) are accurately prepared into a mixed solution of 10ng/mL, and a series of standard curve concentrations with different concentrations are diluted by a BSA multiple ratio. The extraction method is the same as that of a sample, the peak area ratios of the four substances and the cyanocobalamin internal standard are used as vertical coordinates, the concentration is used as horizontal coordinates, a calibration curve is constructed, and a regression equation and a correlation coefficient are obtained. The detection limit and the quantitative limit of each substance in the vitamin B12 family are obtained by taking the concentration corresponding to the signal-to-noise ratio (S/N) being more than or equal to 3 as the detection limit and the concentration corresponding to the signal-to-noise ratio (S/N) being more than or equal to 10 as the quantitative limit, and the results are shown in Table 2.

TABLE 2 Linear equation, detection limit and quantification limit of the method for determining vitamin B12 family content in human serum

(2) Extraction recovery rate: clinical samples were selected and divided into 3 aliquots of equal volume, 200 μ L each. Adding 10 μ L of blank reagent (methanol chromatogram grade or above) containing no substance to be detected into 1 sample as basic sample; and adding 10 μ L of standard solutions of the substances to be detected with different concentrations into the other 2 samples respectively, wherein the concentrations of the standard solutions in the added samples are shown in Table 3, and preparing 2 recovered samples with different addition concentrations. After the standard liquid is added, the object to be measured in the low-level sample is required to be near the middle value of the reference range, and the object to be measured in the high-level sample reaches the upper limit of the reference range. The extraction recovery rate of cyanocobalamin, mecobalamin, hydroxycobalamin and cobamamide under different concentrations has accurate result and good reproducibility.

TABLE 3 extraction recovery of the method for determining vitamin B12 family content in human serum

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. A method for quantitatively detecting vitamin B12 in serum, comprising:

pretreating a serum sample by adopting a method of methanol and isopropanol precipitation and nitrogen blowing redissolution; preparing reference solutions of cyanocobalamin, mecobalamin, hydroxycobalamin and cobamamide;

and (3) carrying out HPLC-MS/MS detection on the pretreated serum sample and the prepared reference substance solution.

2. The method for quantitatively detecting vitamin B12 in serum as claimed in claim 1, wherein the step of pretreating the serum sample comprises:

adding the serum sample into the internal standard solution, adding the mixed solution of methanol and isopropanol, blowing the supernatant solution by using nitrogen, redissolving by using a methanol solution, transferring the redissolved solution into a sample plate, and carrying out HPLC-MS/MS analysis.

3. The method for quantitatively detecting vitamin B12 in serum according to claim 2, wherein the internal standard solution is cyanocobalamin-d 7 solution.

4. The method for quantitatively detecting vitamin B12 in serum according to claim 2, wherein the mixed solution of methanol and isopropanol is methanol: mixed solution with the volume ratio of isopropanol of 8-10: 1; preferably, the volume ratio is 9: 1.

5. The method for quantitatively detecting vitamin B12 in serum according to claim 2, wherein the methanol solution is 40-60% by volume methanol aqueous solution.

6. The method for quantitatively detecting vitamin B12 in serum according to claim 1, wherein the control solution is prepared by: dissolving a cyanocobalamine standard substance and a hydroxycobalamin standard substance respectively with water to prepare stock solutions; dissolving mecobalamin standard and cobamamide standard with methanol respectively to prepare stock solutions.

7. The method for quantitatively detecting vitamin B12 in serum as claimed in claim 1, wherein the liquid chromatography column is a pentafluorophenyl packing material, preferably a Fenomei Kinetex F5 column, 2.1mm x 100mm, 2.7 μm.

8. The method for quantitatively detecting vitamin B12 in serum as claimed in claim 1, wherein the mobile phase A of the liquid chromatography is an aqueous solution containing 0.1-0.2% formic acid; the mobile phase B is a methanol solution containing 0.1-0.2% of formic acid;

alternatively, the gradient elution procedure for liquid chromatography is: 0-1.0 min, 1% of B, 3.0-6.0 min, 98% of B, 6.5-9 min and 1% of B;

or the column temperature is 35-45 ℃; the flow rate is 0.3-0.5 mL/min.

9. The method for the quantitative determination of vitamin B12 in serum of claim 1, wherein the mass spectrometric conditions are: an electrospray ESI source positive ion mode is adopted, the ion source temperature is 500-.

10. A pretreatment kit for detecting vitamin B12 in serum, which is characterized by comprising a mixed solution of methanol and isopropanol;

preferably, the volume ratio of the methanol to the isopropanol is 8-10: 1, and more preferably 9: 1.

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