CN113607854A - Method and detection kit for simultaneously detecting multiple vitamins - Google Patents
Method and detection kit for simultaneously detecting multiple vitamins Download PDFInfo
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Abstract
The invention relates to the technical field of chemical analysis and detection, in particular to a method and a detection kit for simultaneously detecting multiple vitamins. A method for simultaneously detecting multiple vitamins comprising: pretreating a blood sample by using an extraction liquid, then mixing isotope internal standard mixed liquor of a vitamin to be detected with the pretreated blood sample, and then detecting by adopting a liquid chromatography tandem mass spectrometry, wherein the extraction liquid comprises alcohol-water solution of TCA with the mass percent of 1-10%. The detection speed is high, the detection of 15 vitamins can be basically realized within 5 minutes, and the detection result is accurate.
Description
Cross Reference to Related Applications
The present application claims priority of chinese patent application No. CN202110526613.9 entitled "method for simultaneously detecting multiple vitamins and detection kit" filed by chinese patent office on 14/05/2021, the entire contents of which are incorporated herein by reference.
Technical Field
The invention relates to the technical field of chemical analysis and detection, in particular to a method and a detection kit for simultaneously detecting multiple vitamins.
Background
Vitamins are small molecular organic substances necessary for maintaining normal physiological functions of human bodies and specific metabolic reactions in cells, and are basic nutrient substances necessary for human health. It is in small demand in the body, is mainly supplied by food, and plays an important role in regulating the metabolic processes of substances.
At present, it is known that human body contains various vitamins, and the vitamins are classified into two major classes of fat-soluble vitamins and water-soluble vitamins according to their polarity. The water-soluble vitamins comprise B vitamins and vitamin C, wherein the B vitamins are a general name for a class of vitamins with similar functions, structures and physical and chemical properties, all the vitamins in the structure contain nitrogen, are easy to dissolve in water, mainly participate in the synthesis and catabolism processes of various substances in vivo in the form of coenzyme, and have close relation with the formation of blood cells and the release of energy. The vitamin B deficiency is closely related to the occurrence and development of various diseases, and can cause skin diseases, hypomnesis, hypodynamia, insomnia, psychasthenia and the like. For example, VB1 deficiency is commonly called beriberi, VB2 deficiency is also called 'orogenital syndrome', VB6 deficiency can cause seborrheic dermatitis, VB9 deficiency can cause hyperhomocysteinemia, fetal neural tube malformation, VC deficiency can cause scurvy, and the resistance of human bodies is reduced. Therefore, the method for monitoring the content of the water-soluble vitamins in the human body is beneficial to evaluating the nutritional status of the human body and monitoring the diagnosis and treatment process of diseases, and has very important clinical significance.
The clinical detection method of water soluble vitamins in human body includes immunoassay, liquid chromatography-tandem mass spectrometry, etc. The immunoassay method is a method for determining the content of a substance to be detected in a sample by using the substance to be detected as an antigen or an antibody by utilizing the immunological principle, is generally simple and convenient to operate, does not have a complicated sample pretreatment step, but does not have the specific analysis and high-throughput detection of the substance to be detected; the liquid chromatography realizes separation and detection through interaction between an analyte and a mobile phase and a stationary phase, and has better separation capability and analysis specificity compared with an immunoassay method; the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method combines the chromatographic separation characteristic and the qualitative function of mass spectrometry, is more accurate in quantitative analysis of complex mixtures, can realize high-throughput one-time test detection of multiple vitamins, and has the problems of long time, inaccurate detection effect and the like even if the liquid chromatography-tandem mass spectrometry is adopted for vitamin detection.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a method and a detection kit for simultaneously detecting multiple vitamins. The embodiment of the invention provides a novel method for detecting multiple vitamins, which has high detection speed, can basically realize the detection of 15 vitamins within 5 minutes, and has accurate detection result.
The invention is realized by the following steps:
in a first aspect, the present invention provides a method for simultaneously detecting multiple vitamins, comprising: pretreating a blood sample by using an extraction liquid, then mixing isotope internal standard mixed liquor of a vitamin to be detected with the pretreated blood sample, and then detecting by adopting a liquid chromatography tandem mass spectrometry, wherein the extraction liquid comprises alcohol-water solution of TCA with the mass percent of 1-10%.
In a second aspect, the present invention provides a detection kit for simultaneously detecting multiple vitamins, which comprises an extract package, an internal standard mixed solution package of the vitamin to be detected, and a mobile phase solvent package for liquid chromatography, which are required by the method for simultaneously detecting multiple vitamins according to the foregoing embodiments.
The invention has the following beneficial effects: according to the embodiment of the invention, the blood sample is pretreated by adopting the 1-10% TCA alcohol-water solution, so that multiple vitamins in the blood sample can be effectively extracted, the detection effect of the multiple vitamins is further improved, and meanwhile, the detection time is shortened.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a graph showing the results of elution gradient 1 provided in test example 1 of the present invention;
FIG. 2 is a graph showing the results of elution gradient 2 provided in test example 1 of the present invention;
FIG. 3 is a graph showing the results of mobile phase 1 provided in test example 2 of the present invention;
FIG. 4 is a graph showing the results of mobile phase 2 provided in test example 2 of the present invention;
FIG. 5 is a graph showing the results of mobile phase 3 provided in test example 2 of the present invention;
FIG. 6 is a graph showing the results of detection provided in test example 3 of the present invention;
FIG. 7 is a graph showing the results of detection provided in test example 4 of the present invention;
FIG. 8 is a comparison chromatogram and standard curve linear plot of B1 and an internal standard provided in test example 5 of the present invention;
FIG. 9 is a comparison chromatogram and standard curve linear plot of B2 and an internal standard provided in test example 5 of the present invention;
FIG. 10 is a comparison chromatogram and standard curve linear plot of B3 and an internal standard provided in test example 5 of the present invention;
FIG. 11 shows B3-NH supplied in test example 5 of the present invention2Comparing the chromatogram with an internal standard and a standard curve linear graph;
FIG. 12 is a comparison chromatogram and standard curve line plot of B5 and an internal standard provided in test example 5 of the present invention;
FIG. 13 is a comparison chromatogram and standard curve line plot of B6-OH and internal standard provided in test example 5 of the present invention;
FIG. 14 is a comparison chromatogram and standard curve line plot of B6-COOH and internal standard provided in test example 5 of the present invention;
FIG. 15 is a comparison chromatogram and standard curve line plot of B6-CHO and internal standard provided in test example 5 of the present invention;
FIG. 16 shows B6-NH supplied in test example 5 of the present invention2Comparing the chromatogram with an internal standard and a standard curve linear graph;
FIG. 17 is a comparison chromatogram and standard curve line plot of B7 with an internal standard provided in test example 5 of the present invention;
FIG. 18 is a comparison chromatogram and standard curve line plot of B9 with an internal standard provided in test example 5 of the present invention;
FIG. 19 is a comparison chromatogram and standard curve line plot of 5-MTHF and internal standard provided in test example 5 of the present invention;
FIG. 20 is a comparison chromatogram and standard curve line plot of B12 with an internal standard provided in test example 5 of the present invention;
FIG. 21 is a comparison chromatogram and standard curve linearity of VC and internal standard provided in test example 5 of the present invention;
FIG. 22 is a comparison chromatogram and standard curve linear plot of B13 and an internal standard provided in test example 5 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The embodiment of the invention provides a method for simultaneously detecting multiple vitamins, which comprises the following steps:
pretreating the blood sample by using an extraction liquid to precipitate the protein in the blood sample, wherein the extraction liquid comprises 1-10% by mass of aqueous alcohol solution of TCA, and the mass content of TCA in the aqueous alcohol solution is 1-10%. The extraction liquid can ensure the precipitation effect of protein, is favorable for improving the extraction effect of each vitamin, and improves the detection accuracy. If the extraction liquid is changed, for example, the pure alcohol solvent or the alcohol-acetonitrile solvent is changed to reduce the extraction effect of the vitamin, and then the accuracy of the detection effect is influenced.
Further, the alcohol water solution is a miscible solution of monohydric alcohol and water, and is an alcohol solution with the volume percentage of 35-75%; that is, the alcohol content of the alcohol aqueous solution is 35 to 75% by volume, preferably 50% by volume, and the monohydric alcohol includes methanol, but monohydric alcohols such as ethanol may also be used.
Further, the volume ratio of the extraction liquid to the blood sample is 1:1-4:1, and the blood sample comprises a plasma sample or a serum sample, and the sample can be a human blood sample or an animal blood sample.
Then, mixing the isotope internal standard mixed solution of the vitamin to be detected with the blood sample after pretreatment, whereinThe isotope internal standard mixed solution is isotope internal standard solution corresponding to vitamin to be detected, for example, a plurality of vitamins are water-soluble vitamins and include B1, B2, B3 and B3-NH2、B5、B6-OH、B6-COOH、B6-CHO、B6-NH2And at least 9 of B7, B9,5-MTHF, B12, VC and B13, the corresponding isotope internal standard mixed solution is the isotope internal standard mixed solution of the vitamins corresponding to the 9, 10, 11, 12, 13, 14 or 15 cases to be detected. The vitamin standard substance to be detected and the corresponding isotope internal standard information are as follows:
it should be noted that other suppliers may be used, and this is merely an example and does not necessarily have to be the pure vitamin available to the supplier.
And then, detecting by using a liquid chromatography tandem mass spectrometry, wherein the conditions of the liquid chromatography comprise: a chromatographic column: agela Venusil MP C18(3 μm,3.0x30 mm), mobile phase a: 2-10mM ammonium acetate in water; mobile phase B: 2-10mM ammonium acetate alcohol solution (for example, a mixed solution of ammonium acetate and methanol, and the concentration of ammonium acetate in the mixed solution is 2-10mM), wherein the ammonium acetate alcohol solution contains 0.05-0.15% by volume of formic acid; the elution conditions included: 0.0-0.5min, 99% A, 0.5-2.6min, 10% A, 2.6-3.6min, 1% A, 3.6-4.5min, 99% A; the flow rate is 0.3-0.4 mL/min; the sample amount is 2-10 μ L. The adoption of the mobile phase and the elution process is beneficial to the separation of each vitamin, the accuracy of the detection result is improved, and the detection time is also beneficial to be shortened.
Further, mass spectrometry conditions: positive ion electrospray ionization multi-ion reaction monitoring, specifically, gas curtain gas: 20.0kPa, ion source temperature: 150 ℃, desolvation temperature: 550 ℃, desolventizing gas flow rate: 800L/hr, spray voltage: 5.5kV, taper hole voltage: 30V, taper hole airflow: 30L/hr. Wherein, the MRM mass spectrum parameters of each vitamin are shown in the table 1:
TABLE 1
The detection process specifically comprises the following steps: and injecting the sample to be detected mixed with the isotope internal standard mixed solution into a liquid chromatograph, introducing the sample into a mass spectrometer after chromatographic separation, and ionizing the sample in an ion source to form charged ions. Under the action of an electric field, the light is focused into a triple quadrupole mass analyzer. In the first-stage quadrupole (Q1), charged ions are separated according to the mass-to-charge ratio, parent ions are screened out, then the parent ions enter the second-stage quadrupole (Q2), fragment ions are formed through fragmentation under the action of collision gas and collision energy, the generated fragment ions enter the third-stage quadrupole (Q3), the target ions are screened out according to the mass-to-charge ratio, finally the target ions enter a detector, and signals are generated to obtain a detection result. And then, the detection result is brought into a calibration curve and a linear regression equation, so that the measurement values of the multiple water-soluble vitamins in the sample to be detected are calculated. The calibration curve and the linear regression equation are obtained in the same way as the prior art internal standard method, and the details are not described here.
Further, an embodiment of the present invention further provides a detection kit for simultaneously detecting multiple vitamins, which includes an extract package capable of implementing the method for simultaneously detecting multiple vitamins, an internal standard mixed solution package of a vitamin to be detected, and a mobile phase solvent package for liquid chromatography, wherein components and contents of the extract package, the internal standard mixed solution package of the vitamin to be detected, and the mobile phase solvent package for liquid chromatography are consistent with those of corresponding substances used in the method.
The vitamin combination test device further comprises a calibrator mixed liquid bag of the vitamin to be tested and a multi-level quality control product mixed liquid bag of the vitamin to be tested, and similarly, the combination of the vitamins in the calibrator mixed liquid bag and the multi-level quality control product mixed liquid bag is the same as that of the vitamin to be tested.
Further, it also includes a diluent bag; the diluent pack includes: PBS.
Further, the internal standard mixed solution package of the vitamin to be tested, the calibrator mixed solution package of the vitamin to be tested, and the multi-level quality control product mixed solution of the vitamin to be tested further include a protective reagent capable of protecting the water-soluble vitamin, for example, 2 to 10mg/ml of citric acid, but it is understood that other protective reagents, for example, DTT, may also be used.
The term "package" in the kit merely means that the corresponding solution or substance is placed alone, and does not necessarily mean that the corresponding solution or substance is packaged in bags, and may be in bottles or other individual packages.
For example, the components of the kit and the like are shown in table 2:
TABLE 2
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
The embodiment provides a method for simultaneously detecting multiple vitamins, which comprises the following steps:
the instrument comprises the following steps: tandem mass spectrometry system: AB Sciex Jasper HPLC-AB4500 Mass Spectroscopy; vortex oscillator, low temperature high speed centrifuge, pipettor and gun head, sample preparation EP pipe, 96-well inlet plate and plate pad.
And (3) standard substance: b1: about 25 ng/mL; b2: about 100 ng/mL; b3: about 200 ng/mL; B3-NH2: about 100 ng/mL; b5: about 250 ng/mL; B6-OH: about 25 ng/mL; B6-COOH: about 50 ng/mL; B6-CHO: about 200 ng/mL; B6-NH2: about 50 ng/mL; b7: about 50 ng/mL; b9: about 100 ng/mL; 5-MTHF: about 100 ng/mL; b12: about50 ng/mL; VC: about 50 μ g/mL; b13: about 100 ng/mL;
quality control product-1: b1: about 18.75 ng/mL; b2: about 75 ng/mL; b3: about 150 ng/mL; B3-NH2: about 75 ng/mL; b5: about 187.5 ng/mL; B6-OH: about 18.75 ng/mL; B6-COOH: about 37.5 ng/mL; B6-CHO: about 150 ng/mL; B6-NH2: about 37.5 ng/mL; b7: about 37.5 ng/mL; b9: about 75 ng/mL; 5-MTHF: about 75 ng/mL; b12: about 37.5 ng/mL; VC: about 37.5 μ g/mL; b13: about 75 ng/mL;
quality control product-2: b1: about 1.5 ng/mL; b2: about 6 ng/mL; b3: about 12 ng/mL; B3-NH2: about 6 ng/mL; b5: about 15 ng/mL; B6-OH: about 1.5 ng/mL; B6-COOH: about 3 ng/mL; B6-CHO: about 12 ng/mL; B6-NH2: about 3 ng/mL; b7: about 3 ng/mL; b9: about 6 ng/mL; 5-MTHF: about 6 ng/mL; b12: about 3 ng/mL; VC: about 3 μ g/mL; b13: about 6 ng/mL;
and isotopic internal standards thereof.
Obtaining a standard curve: with the concentration of the calibration sample as the independent variable xiTaking the peak area ratio of the corresponding external standard and internal standard as a dependent variable yiAnd calculating a linear regression equation y as ax + b and a correlation coefficient r, wherein r is equal to or more than 0.99.
Analyzing quality control sample data: when r of the calibration curve is more than or equal to 0.990, substituting the signal intensity of the quality control product into a regression equation to obtain the concentration of the quality control sample;
sample data analysis: when the determination result of the quality control product is within the allowable range, extracting a plurality of water-soluble vitamins in the actual clinical sample, performing LC-MS/MS determination to obtain the determination value, and calculating the concentrations of the plurality of water-soluble vitamins in the clinical sample through a calibration curve linear regression equation;
conditions of liquid chromatography: a chromatographic column: agela Venusil MP C18(3 μm,3.0x30 mm), mobile phase a: 5mM ammonium acetate in water; mobile phase B: 5mM ammonium acetate-methanol solution, wherein the ammonium acetate-methanol solution contains 0.10 volume percent of formic acid; the elution conditions included: 0.0-0.5min, 99% A, 0.5-2.6min, 10% A, 2.6-3.6min, 1% A, 3.6-4.5min, 99% A; the flow rate is 0.4 mL/min; the sample amount is 2-10 μ L.
Mass spectrum conditions: air curtain air: 20.0kPa, ion source temperature: 150 ℃, desolvation temperature: 550 ℃, desolventizing gas flow rate: 800L/hr, spray voltage: 5.5kV, taper hole voltage: 30V, taper hole airflow: 30L/hr. The MRM mass spectrum parameters of each vitamin are shown in Table 1.
Preparation of a detection sample: taking the mixed water-soluble vitamin calibrator, and performing gradient dilution by taking a PBS buffer solution as a diluent to prepare a calibration curve working solution C1-C6 (table 3); taking calibration curve working solution C1-C6, putting 100 microliter of quality control samples QC-1 and QC-2 or clinical serum samples contained in the kit into an EP tube, sequentially adding 10 microliter of internal standard IS and 150 microliter of extraction liquid, carrying out vortex oscillation on all samples for 3-5 minutes, and centrifuging for 5-10 minutes at 4 ℃ at 2000-4000 g; after centrifugation, transferring 100-200 mu L of supernatant into a 96-hole U-shaped plate, and tightly sealing by using a plate gasket;
TABLE 3
| Analyte | C6 | C5 | C4 | C3 | C2 | C1 |
| B1(ng/mL) | 0.5 | 1 | 2.5 | 5 | 12.5 | 25 |
| B2(ng/mL) | 2 | 4 | 10 | 20 | 50 | 100 |
| B3(ng/mL) | 4 | 8 | 20 | 40 | 100 | 200 |
| B3-NH2(ng/mL) | 2 | 4 | 10 | 20 | 50 | 100 |
| B5(ng/mL) | 5 | 10 | 25 | 50 | 125 | 250 |
| B6-OH(ng/mL) | 0.5 | 1 | 2.5 | 5 | 12.5 | 25 |
| B6-COOH(ng/mL) | 1 | 2 | 5 | 10 | 25 | 50 |
| B6-CHO(ng/mL) | 4 | 8 | 20 | 40 | 100 | 200 |
| B6-NH2(ng/mL) | 1 | 2 | 5 | 10 | 25 | 50 |
| B7(ng/mL) | 1 | 2 | 5 | 10 | 25 | 50 |
| B9(ng/mL) | 2 | 4 | 10 | 20 | 50 | 100 |
| 5-MTHF(ng/mL) | 2 | 4 | 10 | 20 | 50 | 100 |
| B12(ng/mL) | 1 | 2 | 5 | 10 | 25 | 50 |
| VC(μg/mL) | 1 | 2 | 5 | 10 | 25 | 50 |
| B13(ng/mL) | 2 | 4 | 10 | 20 | 50 | 100 |
Sample detection: and (3) placing the sealed 96-hole U-shaped plate in a liquid chromatography tandem mass spectrometer, setting a sample volume of 2-10 mu L, detecting, and substituting the result into a regression equation to calculate to obtain a result.
The serum samples to be detected are from Beijing Haoxisi Biotech institute, 79 examples of the serum samples to be detected are detected according to the method, and the detection results are as follows:
test example 1
Evaluation of the Effect of Using different gradient elution parameter settings on the detection methods provided by embodiments of the invention
1) Preparing a sample to be tested: taking a water-soluble vitamin mixed solution containing: b1, B2, B3-NH2B5, B6-COOH, B6-CHO, B7, B9 and 5-MTHF, the concentrations of which are respectively 50 ng/mL;
2) preparing a sample to be tested: taking 100 mu L of a sample to be detected, putting the sample into an EP tube, and sequentially adding 10 mu L of internal standard IS and 150uL of extract liquor, wherein the extract liquor IS 10% of TCA methanol-water mixed solution. All samples were vortexed for 3-5 minutes and centrifuged at 2000-4000g for 5-10 minutes at 4 ℃; after centrifugation, transferring 100-200 mu L of supernatant into a 96-hole U-shaped plate, and tightly sealing by using a plate gasket;
3) elution gradient 1 was set: 0.0-0.5min, 99% A, 0.5-2.6min, 10% A, 2.6-3.6min, 1% A, 3.6-4.5min, 99% A; the flow rate is 0.4 mL/min;
4) setting elution gradient 2: 0.0-0.5min, 99% A, 0.5-3.0min, 10% A, 3.0-4.0min, 99% A; flow rate 0.3mL/min
5) Sample detection: and (3) placing the sealed 96-hole U-shaped plate in a liquid chromatography tandem mass spectrometer, setting the sample volume of 2-10 mu L, and detecting.
The detection results are shown in fig. 1 and fig. 2, wherein fig. 1 is the result of elution gradient 1; FIG. 2 shows the result of elution gradient 2, from which it can be seen that, when elution gradient 2 is used, the overall retention, separation degree and sensitivity of water-soluble vitamins are inferior to those of elution gradient 1, wherein B6-CHO, B6-COOH and B7 have almost no retention, and in some retained analytes, the sensitivity of B1, B5, B9 and 5-MTHF is greatly reduced compared with the result of elution gradient 1, which indicates that the separation effect can be effectively ensured by using the elution method of the embodiment of the present invention, and thus the detection accuracy is ensured.
Test example 2
Evaluation of the Effect of Using different elutions on the detection method provided in the examples of the invention
1) Preparing a sample to be tested: taking a water-soluble vitamin mixed solution containing: b1, B2, B3, B3-NH2、B5、B6-OH、B6-COOH、B6-CHO、B6-NH2B7, B9,5-MTHF, B12, VC and B13, the concentration is 50ng/mL respectively;
2) preparing a sample to be tested: taking 100 mu L of a sample to be detected, putting the sample into an EP tube, and sequentially adding 10 mu L of internal standard IS and 150uL of extract liquor, wherein the extract liquor IS 10% of TCA methanol-water mixed solution. All samples were vortexed for 3-5 minutes and centrifuged at 2000-4000g for 5-10 minutes at 4 ℃; after centrifugation, transferring 100-200 mu L of supernatant into a 96-hole U-shaped plate, and tightly sealing by using a plate gasket;
3) preparing a mobile phase 1: taking 500mL of purified water, adding 192mg of ammonium acetate (analytical grade), and ultrasonically degassing for later use; taking 500mL of methanol (analytical grade), adding 500uL of formic acid and 192mg of ammonium acetate, and performing ultrasonic degassing, wherein the mobile phase A is a 5mM ammonium acetate aqueous solution, and the mobile phase B is a 5mM ammonium acetate methanol solution and contains 0.10% by volume of formic acid; standby;
4) preparing a mobile phase 2: taking 500mL of purified water, adding 192mg of ammonium acetate (analytical grade), and ultrasonically degassing for later use; taking 500mL of acetonitrile (analytical grade), adding 500uL of formic acid and 192mg of ammonium acetate, and performing ultrasonic degassing, wherein the mobile phase A is a 5mM ammonium acetate aqueous solution, and the mobile phase B is a 5mM ammonium acetate acetonitrile solution and contains 0.10% by volume of formic acid; standby;
5) preparing a mobile phase 3: taking 500mL of purified water, adding 192mg of ammonium formate (analytical grade), and ultrasonically degassing for later use; taking 500mL of methanol (analytical grade), adding 500uL of formic acid and 192mg of ammonium formate, and performing ultrasonic degassing, wherein the mobile phase A is a 5mM ammonium formate aqueous solution, and the mobile phase B is a 5mM ammonium formate methanol solution and contains 0.10% by volume of formic acid; standby;
then, the test was carried out under the conditions and by the method of example 1. The detection results are shown in fig. 3-5, fig. 3 is a detection result graph of the mobile phase 1, and fig. 4 is a detection result graph of the mobile phase 2; fig. 5 is a graph showing the detection results of the mobile phase 3. From these results, it was found that when ammonium acetate was used as the mobile phase buffer salt and methanol was used as the solvent for mobile phase B, the retention, separation effect, chromatographic profile and sensitivity of the whole water-soluble vitamins were superior to those when ammonium formate was used as the mobile phase buffer salt or acetonitrile was used as the solvent for mobile phase B.
Test example 3
Evaluation of the Effect of Using different extracts on the detection methods provided by the embodiments of the invention
1) Preparing a sample to be tested: taking concentrated solution of each water-soluble vitamin with the concentration of 1 mg/mL; b1, B2, B3, B3-NH2、B5、B6-OH、B6-COOH、B6-CHO、B6-NH2B7, B9,5-MTHF, B12, VC and B13, adding blank serum which is adsorbed and treated by active carbon to prepare B1, B2, B3 and B3-NH with the concentration of 50ng/mL2、B5、B6-OH、B6-COOH、B6-CHO、B6-NH2Serum standard samples of B7, B9,5-MTHF, B12, VC and B13;
a mixed water-soluble vitamin calibrator was taken and subjected to gradient dilution using PBS buffer as a diluent to prepare calibration curve working solutions C1-C6 (see table 3 of example 1):
2) preparing a sample to be tested 1: taking the prepared serum standard sample, putting 100 microliter of calibration curve working solution C1-C6 and each 100 microliter into an EP tube, and sequentially adding 10 microliter of internal standard substance IS and 300 microliter of extract liquor, wherein the extract liquor IS pure methanol solution. All samples were vortexed for 3-5 minutes and centrifuged at 2000-4000g for 5-10 minutes at 4 ℃; after centrifugation, transferring 100-200 mu L of supernatant into a 96-hole U-shaped plate, and tightly sealing by using a plate gasket;
3) preparing a sample to be tested 2: taking the prepared serum standard sample, putting 100 microliter of calibration curve working solution C1-C6 and 100 microliter of each in an EP tube, and sequentially adding 10 microliter of internal standard substance IS and 300 microliter of extract liquor, wherein the extract liquor IS a methanol-acetonitrile mixed solution (the volume ratio IS 1: 1). All samples were vortexed for 3-5 minutes and centrifuged at 2000-4000g for 5-10 minutes at 4 ℃; after centrifugation, transferring 100-200 mu L of supernatant into a 96-hole U-shaped plate, and tightly sealing by using a plate gasket;
4) preparing a sample to be tested 3: taking the prepared serum standard sample, calibrating curve working solution C1-C6, 100 uL each in an EP tube, and sequentially adding 10 uL of internal standard IS and 150uL of extract liquor, wherein the extract liquor IS 10% TCA methanol-water mixed solution. All samples were vortexed for 3-5 minutes and centrifuged at 2000-4000g for 5-10 minutes at 4 ℃; after centrifugation, transferring 100-200 mu L of supernatant into a 96-hole U-shaped plate, and tightly sealing by using a plate gasket;
then, the test was carried out under the conditions and by the method of example 1. The results of the test are shown in FIG. 6. When the theoretical values of various substances to be detected in a serum standard product are compared, B6-CHO can not be effectively measured from a serum sample when methanol or a methanol/acetonitrile mixed solution is used for extracting various water-soluble vitamins in the serum sample, and VC is insufficient in extraction efficiency. When 10% TCA methanol-water mixed solution is used for extraction, 15 water-soluble vitamins can be extracted from a sample, wherein the extraction rate of B6-CHO and VC is better than that of methanol or methanol/acetonitrile mixed solution, but the extraction rate of B9,5-MTHF and B12 is slightly lower than that of methanol or methanol/acetonitrile mixed solution. Considering the comprehensive extraction efficiency for 15 water-soluble vitamins, the extraction liquid is preferably TCA methanol water mixed solution.
Test example 4
Evaluation of the Effect of TCA methanol aqueous solutions of different concentrations on the detection method provided by the embodiments of the present invention
Taking the prepared serum standard sample, putting 100 microliter of calibration curve working solution C1-C6 (same as above) and 100 microliter of each in an EP tube, and sequentially adding 10 microliter of internal standard substance IS and 150 microliter of extract liquor, wherein the extract liquor IS respectively 3 percent, 6 percent, 9 percent and 15 percent of TCA methanol-water mixed solution. All samples were vortexed for 3-5 minutes and centrifuged at 2000-4000g for 5-10 minutes at 4 ℃; after centrifugation, transferring 100-200 mu L of supernatant into a 96-hole U-shaped plate, and tightly sealing by using a plate gasket;
then, the test was carried out under the conditions and by the method of example 1. The results of the measurements are shown in FIG. 7. When the theoretical values of all the substances to be detected in the serum standard are compared, the comprehensive extraction effect of the water-soluble vitamins in the 15 th sample is found to be the best when the 6% TCA methanol water mixed solution is used for extracting various water-soluble vitamins in the serum sample.
Verification example 5
The assay was carried out using the kit of components shown in Table 2, and the operation was the same as in example 1, except that the detection reagent used, for example, extraction was also a solution pack in the kit, specifically,
preparing a sample to be tested: taking 100 mu L of each of calibration curve working solution C1-C6 and multi-level quality control samples QC-1 and QC-2 (refer to example 1) to an EP tube, sequentially adding 10 mu L of internal standard IS and 150uL of extraction liquid contained in the kit, carrying out vortex oscillation on all samples for 3-5 minutes, and centrifuging for 5-10 minutes at 4 ℃ under 2000-4000 g; after centrifugation, transferring 100-200 mu L of supernatant into a 96-hole U-shaped plate, and tightly sealing by using a plate gasket;
then, the test was carried out under the conditions and by the method of example 1. As can be seen from FIGS. 8 to 22, the results obtained in the kit B1 were in good linear relationship in the range of 0.5 to 25ng/ml, and R was found to be good in the range of from 8 to 222Not less than 0.99; kit determination B2 showed good linearity in the range of 2-100ng/ml, R2Not less than 0.99; the kit measures B3, the linear relation is good in the range of 4-200ng/ml, R2Not less than 0.99; kit for determining B3-NH2Has a good linear relationship in the range of 2-100ng/ml, R2Not less than 0.99; the kit measures B5, has good linear relation in the range of 5-250ng/ml,R2not less than 0.99; the kit is used for measuring B6-OH, the linear relation is good in the range of 0.5-25ng/ml, and R is2Not less than 0.99; the kit measures B6-CHO, the linear relation is good in the range of 4-200ng/ml, R2Not less than 0.99; kit for determining B6-NH2Has a good linear relationship in the range of 1-50ng/ml, R2Not less than 0.99; the kit measures B7, the linear relation is good in the range of 1-50ng/ml, R2Not less than 0.99; the kit measures B9, the linear relation is good in the range of 2-100ng/ml, R2Not less than 0.99; the kit is used for measuring 5-MTHF, the linear relation is good in the range of 2-100ng/ml, and R is2Not less than 0.99; the kit measures B12, the linear relation is good in the range of 2-100ng/ml, R2Not less than 0.99; the VC is measured by the kit, the linear relation is good in the range of 1-50 mu g/mL, R is2Not less than 0.99; the kit measures B13, the linear relation is good in the range of 2-100ng/ml, R2Not less than 0.99; the kit is used for measuring B6-COOH, the linear relation is good in the range of 1-50ng/ml, R2≥0.99。
Meanwhile, preparing low-value and high-value quality control products by using a multi-level quality control solution prepared by the kit, preparing 6 parts of each concentration in parallel, and respectively calculating the average value and standard deviation SD of the measured values; each assay lot was prepared and tested 1 time to convert the concentration of the quality control substance when the standard curve was prepared for a new lot, 3 consecutive lots, and the precision between lots was evaluated by calculating the Relative Standard Deviation (RSD) and the accuracy between lots was evaluated by the relative deviation (RE). The results are shown in tables 3-17:
TABLE-3B 1 results of the evaluation of the precision within and between batches
TABLE 4B2 results of the evaluation of the precision within and between batches
TABLE 5B3 results of the evaluation of the precision within and between batches
TABLE 6B3-NH2Results of Intra-and inter-batch precision evaluation
TABLE 7B5 results of the evaluation of the precision within and between batches
TABLE 8B6-OH results of the evaluation of the precision within and between batches
TABLE 9B6-COOH results of the evaluation of the precision within and between batches
TABLE 10B 6-results of the evaluation of the precision within and between the CHO batches
TABLE 11B6-NH2Results of Intra-and inter-batch precision evaluation
TABLE 12B7 results of the evaluation of the precision within and between batches
TABLE 13B9 results of the evaluation of the precision within and between batches
TABLE 145 MTHF Intra and Inter batch precision evaluation results
TABLE-15B 12 results of the evaluation of the precision within and between batches
TABLE-16 VC evaluation results of Intra-and inter-batch precision
TABLE 17B13 results of the evaluation of the precision within and between batches
From tables 3-17, the precision and accuracy experimental data of 3 consecutive batches, B1, B2, B3, B3-NH, can be found2、B5、B6-OH、B6-COOH、B6-CHO、B6-NH2The precision RSD of B7, B9,5-MTHF, B12, VC and B13 in batch and between batches and the accuracy of the RSD between the batches are all within +/-15 percent, and the requirement of methodology evaluation is met.
Verification example 6
In order to evaluate the possible result deviation caused by the matrix effect when the PBS buffer salt is substituted for the matrix in the detection of the serum/plasma sample, the possible matrix effect of the kit in the using process is also researched correspondingly.
The matrix effect of endogenous substances can be assessed by spiking recovery. The ratio of the concentration difference value and the theoretical value of a sample obtained by adding low-concentration and high-concentration quality control sample working solution into a blank serum matrix with vitamin background removed by activated carbon adsorption and a sample obtained by adding no standard is adopted for evaluation, 6 parallel samples are prepared for each concentration sample, the detection is carried out according to the conditions and the method of the example 1, and the detection results are as follows:
according to the table, the two quality-controlled labeling recovery rates of the 15 water-soluble vitamins to be detected are between 85% and 115%, which shows that when the PBS buffer solution is used as the substitute matrix, the PBS buffer solution has interchangeability with the actual human serum matrix, and the deviation of the result of the detection method cannot be caused.
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 simultaneously detecting multiple vitamins, comprising: pretreating a blood sample by using an extraction liquid, then mixing isotope internal standard mixed liquor of a vitamin to be detected with the pretreated blood sample, and then detecting by adopting a liquid chromatography tandem mass spectrometry, wherein the extraction liquid comprises alcohol-water solution of TCA with the mass percent of 1-10%.
2. The method for simultaneously detecting multivitamins according to claim 1, wherein the alcohol aqueous solution is an alcohol solution with a volume percentage of 35-75%;
preferably, the aqueous alcohol solution is a miscible solution of monohydric alcohol and water; preferably, the monohydric alcohol comprises methanol.
3. The method for simultaneously detecting multivitamins according to claim 1, wherein the conditions of the liquid chromatography include: mobile phase A: 2-10mM ammonium acetate in water; mobile phase B: 2-10mM of ammonium acetate alcoholic solution, wherein the ammonium acetate alcoholic solution contains 0.05-0.15% of formic acid by volume percentage; the elution conditions included: 0.0-0.5min, 99% A, 0.5-2.6min, 10% A, 2.6-3.6min, 1% A, 3.6-4.5min, 99% A; the flow rate is 0.3-0.4 mL/min; the sample amount is 2-10 mu L;
preferably, the ammonium acetate alcohol solution is a mixed solution of ammonium acetate and a monohydric alcohol, preferably, the monohydric alcohol comprises methanol;
preferably, the mass spectrometry conditions are: air curtain air: 20.0kPa, ion source temperature: 150 ℃, desolvation temperature: 550 ℃, desolventizing gas flow rate: 800L/hr, spray voltage: 5.5kV, taper hole voltage: 30V, taper hole airflow: 30L/hr.
4. The method for simultaneously detecting multiple vitamins according to any one of claims 1-3, wherein said blood sample comprises a plasma sample or a serum sample.
5. The method for simultaneously detecting multiple vitamins according to any one of claims 1-3, wherein the volume ratio of the extraction liquid to the blood sample is 1:1-4: 1.
6. The method for simultaneously detecting multiple vitamins according to any one of claims 1-3, wherein multiple of said vitamins are water-soluble vitamins,
preferably, the plurality of vitamins comprises B1, B2, B3, B3-NH2、B5、B6-OH、B6-COOH、B6-CHO、B6-NH2At least 9 of B7, B9,5-MTHF, B12, VC and B13;
preferably, the isotope internal standard mixed liquor is B1, B2, B3 and B3-NH to be detected2、B5、B6-OH、B6-COOH、B6-CHO、B6-NH2A mixed solution of at least 9 isotopes selected from the group consisting of B7, B9,5-MTHF, B12, VC and B13.
7. A detection kit for simultaneously detecting multiple vitamins, which is characterized by comprising an extract bag required by the method for simultaneously detecting multiple vitamins according to claim 1, an internal standard mixed solution bag of the vitamin to be detected and a mobile phase solvent bag used for liquid chromatography.
8. The detection kit according to claim 7, further comprising a calibrator mixture package for the vitamin to be tested and a multi-level quality control material mixture package for the vitamin to be tested.
9. The detection kit of claim 8, wherein the calibrator mixture package comprises B1, B2, B3, B3-NH to be detected2、B5、B6-OH、B6-COOH、B6-CHO、B6-NH2At least 9 of B7, B9,5-MTHF, B12, VC, and B13;
preferably, the calibrator mixture pack comprises 2 to 10mg/ml of citric acid;
preferably, the multi-level quality control product mixed liquid bag of the vitamin to be detected comprises B1, B2, B3 and B3-NH to be detected2、B5、B6-OH、B6-COOH、B6-CHO、B6-NH2Quality control materials of at least 9 of B7, B9,5-MTHF, B12, VC and B13;
preferably, the calibrator mixture pack contains 2 to 10mg/ml of citric acid.
10. The test kit of claim 7, further comprising a diluent pack;
preferably, the diluent pack comprises: PBS.
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| CN116559335A (en) * | 2023-06-20 | 2023-08-08 | 山东英盛生物技术有限公司 | Method for simultaneously detecting multiple water-soluble vitamins in human serum based on LC-MS |
| CN117099774A (en) * | 2023-10-24 | 2023-11-24 | 西湖维泰(杭州)诊断技术有限公司 | Preservation and detection methods of nicotinic acid metabolite blood sample, kit and application of nicotinic acid metabolite blood sample |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116559335A (en) * | 2023-06-20 | 2023-08-08 | 山东英盛生物技术有限公司 | Method for simultaneously detecting multiple water-soluble vitamins in human serum based on LC-MS |
| CN117099774A (en) * | 2023-10-24 | 2023-11-24 | 西湖维泰(杭州)诊断技术有限公司 | Preservation and detection methods of nicotinic acid metabolite blood sample, kit and application of nicotinic acid metabolite blood sample |
| CN117099774B (en) * | 2023-10-24 | 2024-03-15 | 西湖维泰(杭州)诊断技术有限公司 | Preservation and detection methods of nicotinic acid metabolite blood sample, kit and application of nicotinic acid metabolite blood sample |
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