CN113390975A

CN113390975A - Sample pretreatment method for detecting fat-soluble vitamins in serum by high performance liquid chromatography tandem mass spectrometry

Info

Publication number: CN113390975A
Application number: CN202110241142.7A
Authority: CN
Inventors: 曾珊珊; 张琦; 王媛; 韩宝玉; 刘鹏云; 刘华芬
Original assignee: Hangzhou Calibra Diagnostics Co ltd
Current assignee: Hangzhou Calibra Diagnostics Co ltd
Priority date: 2021-03-04
Filing date: 2021-03-04
Publication date: 2021-09-14

Abstract

The invention provides a sample pretreatment method for detecting fat-soluble vitamins in serum by high performance liquid chromatography tandem mass spectrometry, which adopts a specific protein precipitator and is mixed with an internal standard working solution to form an internal standard solution, thereby simply and efficiently completing sample pretreatment, and the recovery rates of the fat-soluble vitamins A, E, K1 and K2 in a serum sample can be obviously improved without any additional operations such as sample enrichment and the like, the cost of the sample pretreatment is reduced, the sensitivity of the detection of the fat-soluble vitamins in the serum is greatly improved, and the accuracy and the stability of a detection result can be ensured. The invention also provides the application of the ammonium acetate in preparing the protein precipitator.

Description

Sample pretreatment method for detecting fat-soluble vitamins in serum by high performance liquid chromatography tandem mass spectrometry

Technical Field

The invention relates to the technical field of chemical analysis, in particular to a sample pretreatment method for detecting fat-soluble vitamins in serum by high performance liquid chromatography tandem mass spectrometry.

Background

Vitamin A (VA), also known as retinol, has the functions of maintaining normal vision, maintaining epithelial tissues and promoting normal growth and development of bones. Insufficient VA in the human body can lead to nutritional deficiencies such as dry eye, nyctalopia and hyperkeratosis of the skin. 25-hydroxy vitamin D is an important factor for regulating calcium and phosphorus metabolism of a human body, coordinates mobilization or deposition of bone calcium and absorption or excretion of urine calcium, maintains the stability of blood calcium level in vivo, and is important for life activities such as bone development molding, muscle nerve conduction effect, cell activity, information transmission and the like. Deficiency in children manifests as rickets, and deficiency in adults manifests as osteomalacia, osteoporosis; excess causes increased calcium absorption, resulting in hypercalcemia. Vitamin e (ve) is a group of tocopherols which exist as stereoisomers, with the most active as alpha-tocopherol. VE can promote sex hormone secretion, improve male and female fertility, prevent free radical or oxidant from damaging cell membrane, and has physiological effects of preventing atherosclerosis, cardiovascular system diseases and climacteric syndrome, and abnormal pregnancy caused by testis atrophy and epithelial cell degeneration in absence of VE. The main physiological action of vitamin K is to participate in the synthesis of prothrombin and coagulation factors, the human body lacks VK, which can lead to the prolongation of coagulation time, serious patients have bleeding tendency and even have the risk of intracranial bleeding under the influence of certain factors (such as infection, diarrhea and the like), wherein, the vitamin K1 is a necessary substance for liver synthesis factors II, VII, IX and X, and is clinically applied to the prevention and treatment of prothrombin syndrome, vitamin K1 deficiency, neonatal spontaneous hemorrhage, hemorrhage caused by obstructive jaundice, biliary fistula, chronic diarrhea and the like, and hypoprothrombinemia caused by coumarins, sodium salicylate and the like; vitamin K2 is the only bioactive form of vitamin K, is often used for accelerating blood coagulation, maintaining blood coagulation time, and treating hemorrhage due to vitamin K deficiency, and is also reported to be used in other health care approaches. Thus, detection of a fat soluble vitamin can assess the nutritional status of the fat soluble vitamin in the patient. Has auxiliary diagnosis significance for clinical judgment, treatment management and physiological evaluation of fat-soluble vitamin deficiency or excess.

The existing sample pretreatment method for detecting fat-soluble vitamins in serum by high performance liquid chromatography tandem mass spectrometry mainly adopts a liquid-liquid extraction method (such as CN106504947A) and a solid-liquid extraction method (such as CN110763788A), and needs a plurality of steps of dilution, multiple extraction, rotary evaporation, nitrogen blow-drying, purification and the like, so that the process is complicated, a large amount of manual operation is needed, the liquid-liquid extraction is difficult to realize high flux and automation, and although the solid-liquid extraction can realize high flux, the sample extraction process is mainly completed by an SLE plate, and the consumable cost is high.

At present, there are also many related patent applications for sample pretreatment in which a protein precipitation method is adopted to determine fat-soluble vitamin samples in serum and plasma, such as CN111999397A and CN110487943B, but the problems of low recovery rate and low sensitivity generally exist, and some applications even after protein precipitation require further extraction to perform subsequent detection and analysis, so that clinical detection requires more manual operations, and is not favorable for clinical popularization and use.

Therefore, a pretreatment method of the fat-soluble vitamin sample, which has the advantages of simple operation, high treatment efficiency, high recovery rate, low cost, small manual workload and the like, is urgently needed, and can overcome the defects of the existing method, so that the method for detecting the fat-soluble vitamin in the serum by the high performance liquid chromatography-tandem mass spectrometry has higher sensitivity, the detection process is simpler and more efficient, and the accuracy and the stability of the detection result can be ensured.

Disclosure of Invention

In order to solve the problems, the invention provides a sample pretreatment method for detecting fat-soluble vitamins in serum by high performance liquid chromatography tandem mass spectrometry, which adopts a specific protein precipitator and mixes the protein precipitator and an internal standard substance working solution into an internal standard solution, so that the sample pretreatment is simply and efficiently completed, the operations such as any additional sample enrichment and the like are not needed, the recovery rate of the fat-soluble vitamins in a serum sample can be obviously improved, the cost of the sample pretreatment is reduced, the sensitivity of the detection of the fat-soluble vitamins in the serum is greatly improved, and the accuracy and the stability of a detection result can be ensured.

In one aspect, the invention provides a protein precipitant for extracting fat soluble vitamins from serum, comprising methanol, acetonitrile and isopropanol.

The fat-soluble vitamin provided by the invention is any one or more of vitamin A, 25-hydroxy vitamin D2, 25-hydroxy vitamin D3, vitamin E, vitamin K1 and vitamin K2. The six fat-soluble vitamins are detected by a high performance liquid chromatography tandem mass spectrometry method, the six fat-soluble vitamins are grouped according to similar retention time, and the mass spectrometry is subjected to sectional acquisition at different time, so that the detection sensitivity of vitamin K1 and vitamin K2 can be further improved.

A large number of experiments prove that the protein precipitant prepared by mixing methanol, acetonitrile and isopropanol has very high selectivity and solubility to fat-soluble vitamins in a sample, completely does not need the enrichment processes of nitrogen blowing and the like after freeze drying or liquid-liquid extraction, can directly carry out sample injection detection after separating precipitate impurities, and can obviously improve the detection sensitivity.

Further, the protein precipitator also comprises an additive, and the additive is any one or more of formic acid, ammonium fluoride, ammonium formate, ammonium acetate and 2, 6-di-tert-butyl-p-cresol.

The additive is added into the protein precipitator, so that a synergistic effect can be exerted, and the extraction yield of fat-soluble vitamins in a sample is further improved.

Further, the additive is ammonium acetate.

Research proves that when the additive is acetic acid, the synergistic effect is most obvious, and the extraction yield of the fat-soluble vitamins in the sample is highest.

Further, the volume ratio of the methanol to the acetonitrile to the isopropanol is 5-75% to 5-75%, and the content of the ammonium acetate is 10-50 mM.

Further, the volume ratio of the methanol to the acetonitrile to the isopropanol is 70-75% to 15-25% to 5-10%, and the content of the ammonium acetate is 40-50 mM.

In another aspect, the present invention provides an internal standard solution for extracting lipid-soluble vitamins from serum, comprising an internal standard working solution and a protein precipitant as described above; the internal standard working solution comprises an internal standard of the fat-soluble vitamin to be detected.

In the prior sample pretreatment process, an internal standard solution and a pretreatment reagent are separately prepared and added and mixed twice, so that more manual operations are brought to the sample pretreatment process.

Through a large number of experiments, research groups surprisingly find that a protein precipitator containing methanol, acetonitrile, isopropanol and ammonium acetate is directly mixed with internal standard working solution to form an optimized internal standard solution system, the internal standard solution system is added into a sample and uniformly mixed, the internal standard solution system can play a role of internal standard, and can also realize the purification and extraction of target substances in the sample, the internal standard solution system has very high selectivity and solubility on fat-soluble vitamins in the sample, the enrichment processes such as freeze drying or nitrogen blowing after liquid-liquid extraction are completely not needed, the sample injection detection can be directly carried out after precipitate impurities are separated, the detection sensitivity can be obviously improved, the operation process is simplified, and the used reagents are conventional chemical reagents with lower cost, so the detection cost is reduced.

The stability evaluation of the prepared internal standard solution proves that the internal standard solution provided by the invention is very stable, can be stored for a long time, can be taken and used at any time and is very convenient.

Further, the internal standard working solution contains an internal standard of the fat-soluble vitamin to be detected.

Further, the internal standard working solution is an isotope internal standard solution containing one or more of VA1-d6, VD3-d6, VD2-d6, VK1-d4 and VE-d 6.

Wherein VA1-D6 is an internal standard of vitamin A, VD3-D6 is an internal standard of vitamin D3, VD2-D6 is an internal standard of vitamin D2, VK1-D4 is an internal standard of vitamin K1 and vitamin K2, and VE-D6 is an internal standard of vitamin E.

Further, the volume ratio of the internal standard substance working solution to the protein precipitator is 1: 124.

A large number of experiments prove that the internal standard solution system provided by the invention can be stably stored for more than two years at room temperature.

The formula and the proportional relation of the internal standard solution or the protein precipitator provided by the invention have higher contribution to the selectivity and the solubility of fat-soluble vitamins in a sample and the improvement of liquid quality parameters, and also make a great contribution to the improvement of the sensitivity of a detection method.

In another aspect, the invention provides a sample pretreatment method for detecting fat-soluble vitamins in serum by high performance liquid chromatography tandem mass spectrometry, which comprises the step of carrying out sample pretreatment by using the internal standard solution, wherein the volume ratio of the sample to the protein precipitator or the internal standard solution is 1: 3-1: 7.

Further, the sample pretreatment method comprises the following steps of 1): and (3) putting 50 mu L of sample into a 96-well plate, adding 250 mu L of internal standard solution, uniformly mixing by vortex, oscillating at 600rpm for 10min, performing centrifugal separation, and putting 200 mu L of supernatant into a low-adsorption 96-well plate to be detected.

Or the sample pretreatment method comprises the step 2): and (3) putting 50 mu L of sample into a 96-well plate, adding 2 mu L of internal standard substance working solution, adding 248 mu L of protein precipitator, uniformly mixing by vortex, shaking at 600rpm for 10min, carrying out centrifugal separation, and putting 200 mu L of supernatant into a low-adsorption 96-well plate to be detected.

The sample pretreatment method provided by the invention has the advantages that the sample dosage is less and is only 50uL, and the clinical blood sampling difficulty is reduced.

The low adsorption 96-well plate is used for sample injection, so that the risk of fat-soluble vitamins being adsorbed by the sample injection plate can be reduced, and the precision and accuracy of the detection result are improved.

A large number of researches prove that on the basis of an optimized internal standard solution system or a protein precipitator system, the low-adsorption sample injection plate is used, so that the risk of target substances being adsorbed by the sample injection plate can be reduced, the stability of the extracted fat-soluble vitamins is improved, and the precision and the accuracy of detection results are ensured.

In another aspect, the invention provides the use of ammonium acetate for preparing a protein precipitant for sample pretreatment in high performance liquid chromatography tandem mass spectrometry detection of fat-soluble vitamins in serum; the fat-soluble vitamin is one or more of vitamin A, vitamin E, vitamin K1, and vitamin K2.

In another aspect, the invention provides a method for detecting fat-soluble vitamins in serum by high performance liquid chromatography tandem mass spectrometry, which comprises sample preparation, sample pretreatment and sample detection; the sample pretreatment is performed by the method of sample pretreatment described above.

Further, the sample preparation comprises preparation of standard samples, quality control products and internal standard solutions (or internal standard working solutions and protein precipitants).

The standard sample is as follows: the solution containing any one or more of vitamin A, 25-hydroxy vitamin D2, 25-hydroxy vitamin D3, vitamin E, vitamin K1 and vitamin K2 with standard concentration is prepared by adopting negative blank human serum as a matrix;

the preparation of the standard sample comprises the following steps: negative blank human serum is used as a blank matrix, and any one or more standard solutions of vitamin A, 25-hydroxy vitamin D2, 25-hydroxy vitamin D3, vitamin E, vitamin K1 and vitamin K2 are added to prepare a series of fat-soluble vitamin standards with known concentrations.

The quality control samples are as follows: serum matrix samples containing concentrations at three different levels, low, medium and high; the preparation of the quality control product comprises the following steps: serum is used as a matrix, and any one or more standard solutions of vitamin A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, vitamin E, vitamin K1 and vitamin K2 are added to prepare low (L), medium (M) and high (H) concentration fat-soluble vitamin quality control samples.

The negative blank human serum is adopted to prepare the standard substance and the serum is adopted to prepare the quality control substance, so that the matrix effect in the detection of the human serum sample is reduced, and the accuracy and the reliability of the standard curve are improved.

Further, the sample detection comprises high performance liquid chromatography and tandem mass spectrometry detection, and gradient elution is adopted; tandem mass spectrometry, using atmospheric pressure chemical ionization ion source (APCI) and positive ion multiple reaction monitoring scan mode (MRM), and segmented acquisition according to retention time.

The eluent of the liquid chromatogram comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is 0.1% formic acid aqueous solution, and the mobile phase B is 0.1% formic acid methanol solution. When the mobile phase A is 0.1% formic acid aqueous solution and the mobile phase B is 0.1% formic acid methanol solution for detection, the sensitivity is high, and when 50uL serum is used, the standard curve minimum concentration point (S1) of 6 fat-soluble vitamins completely meets the requirement of minimum quantification.

The gradient elution time is 7min, and the gradient elution program is as follows:

time (min)	Mobile phase A%	Mobile phase B%	Flow rate (ml/min)
				0.00	60.0	40.0	0.70
4.50	10.0	90.0	0.70
				5.00	0.0	100.0	0.70
6.70	0.0	100.0	0.70
				6.71	60.0	40.0	0.70
7.00	60.0	40.0	0.70

By adopting the elution conditions, the interference of serum matrix on the detection of 6 fat-soluble vitamins can be eliminated, and the detection accuracy is ensured. The detection time is short, the whole detection time is about 7.0min, the sample detection time is shortened, the specific gradient elution program is adopted for gradient elution, the sample separation effect is ensured, the simultaneous accurate detection of 6 fat-soluble vitamins is realized through mass spectrum, the detection time of a single sample is greatly shortened, the analysis cost is effectively reduced, the reporting range is wide, and the serum samples with abnormal concentrations of the 6 fat-soluble vitamins can be accurately analyzed.

Further, the high performance liquid chromatography conditions are as follows: the chromatographic column is a C18 chromatographic column, the flow rate of a mobile phase is 0.70mL/min, and the temperature of the column is 40 ℃.

Further, the mass spectrum conditions are as follows:

further, the mass spectrum conditions are as follows: the parent ion/daughter ion to mass-to-charge ratios (m/z) of the 6 fat-soluble vitamins and their internal standards used for the detection are shown in the following table:

further, the specific mass spectrum sectional acquisition mode is as follows: vitamin A, 25-hydroxyvitamin D2, and 25-hydroxyvitamin D3 as the first group, and vitamin E, vitamin K1, and vitamin K2 as the second group; or vitamin A, 25-hydroxy vitamin D2, 25-hydroxy vitamin D3, and vitamin E as the first group, and vitamin K1 and vitamin K2 as the second group.

Further, the method also comprises sample analysis, and the main steps are as follows: drawing a standard curve, calculating the recovery rate, the matrix effect and the precision, and calculating the concentration of the fat-soluble vitamin in the human serum sample to be detected.

The invention has the following beneficial effects:

(1) the protein precipitator is provided, when being used for pretreatment of serum sample fat-soluble vitamin detection, the recovery rate of fat-soluble vitamins (especially vitamin A, E, K1 and K2) can be obviously improved, and the pretreated sample is more stable, so that the sensitivity of high performance liquid chromatography tandem mass spectrometry detection of the fat-soluble vitamins in the serum is greatly improved, the detection process is simpler and more efficient, and the detection result is more accurate and stable;

(2) the novel internal standard solution system is provided, a protein precipitator and an internal standard working solution are mixed into the internal standard solution, the internal standard solution system can play a role of an internal standard, the purification and extraction of target substances in a sample can be realized, the selectivity and the solubility of fat-soluble vitamins (particularly vitamins A, E, K1 and K2) in the sample are very high, the enrichment processes such as nitrogen blowing and the like after freeze drying or liquid-liquid extraction are completely not needed, the direct sample injection detection can be carried out after separating precipitate impurities, the detection sensitivity can be obviously improved, the operation is further simplified, and the labor cost is saved;

(3) the reagents used by the protein precipitator or the internal standard solution prepared by the invention are conventional chemical reagents with lower cost, and the sample pretreatment cost is lower, so that the detection cost of fat-soluble vitamins in serum by high performance liquid chromatography tandem mass spectrometry is reduced.

(3) The internal standard solution provided by the invention is very stable, can be stored for a long time, can be taken and used at any time, is very convenient, and enables the detection process to be simpler, more convenient and more efficient;

(4) on the basis of an optimized internal standard solvent system, the low-adsorption sample injection plate is used, so that the risk of target substances being adsorbed by the sample injection plate can be reduced, the stability of the fat-soluble vitamins after extraction is improved, and the precision and the accuracy of a detection result are ensured;

(5) the negative blank human serum is adopted to prepare the standard substance and the serum is adopted to prepare the quality control substance, so that the matrix effect in the detection of the human serum sample is reduced, and the accuracy and the reliability of the standard curve are improved;

(6) the mobile phase A is 0.1% formic acid water solution, and the mobile phase B is 0.1% formic acid methanol solution, so that the sensitivity is high;

(7) the detection time is short, the whole detection time is about 7.0min, a specific gradient elution program is adopted for gradient elution, the sample separation effect is ensured, the detection sensitivity of vitamin K1 and K2 is further improved by mass spectrum detection in different time periods, the simultaneous accurate detection of 6 fat-soluble vitamins is realized, the detection time of a single sample is greatly shortened, and the analysis cost is effectively reduced;

(8) the dosage of the sample is less, and is only 50uL, so that the clinical blood sampling difficulty is reduced;

(9) the reporting range is wide, and serum samples with abnormal concentrations of 6 fat-soluble vitamins can be accurately analyzed;

(10) the sensitivities of vitamin A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, vitamin E, vitamin K1 and vitamin K2 can reach 0.4ng/mL, 0.7ng/mL, 0.3ng/mL, 3ng/mL, 10pg/mL and 6pg/mL respectively.

Drawings

FIG. 1 is a test pattern obtained by testing a sample to be tested prepared according to the standard S1 series concentration and by using negative blank human serum as a matrix in example 1

Detailed Description

The invention will be described in further detail below with reference to the drawings and examples, which are intended to facilitate the understanding of the invention without limiting it in any way. The reagents used in this example were all known products and were obtained by purchasing commercially available products.

Example 1: sample preparation, pretreatment, detection and analysis

First, sample preparation

1. Preparation of standard curve and quality control sample

Preparing a mixed solution from vitamin A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, vitamin E, vitamin K1 and vitamin K2 standard substances, using the mixed solution as a stock solution of a standard working solution and a quality control working solution, mixing the stock solution with negative blank human serum and negative serum according to a volume ratio of 1:49 respectively, and preparing a standard curve and a quality control sample.

The 6 fat-soluble vitamins have 10 series concentrations (S1-S10) in the standard, as shown in table 1:

TABLE 1, 10 series concentrations of 6 fat-soluble vitamins in the standard (S1-S10)

ng/ml	Vitamin A	25 hydroxy vitamin D2	25 hydroxy vitamin D3	Vitamin E	Vitamin K1	Vitamin K2
							S1	40	2	3	500	0.10	0.10
S2	60	3	4.5	750	0.15	0.15
							S3	100	5	7.5	1250	0.25	0.25
S4	160	8	12	2000	0.40	0.40
							S5	200	10	15	2500	0.50	0.50
S6	400	20	30	5000	1.00	1.00
							S7	600	30	45	7500	1.50	1.50
S8	1000	50	75	12500	2.50	2.50
							S9	1600	80	120	20000	4.00	4.00
S10	2000	100	150	25000	5.00	5.00

The 6 fat-soluble vitamins have three series of concentrations of low (L), medium (M) and high (H) in the quality control product, as shown in the table 2:

table 2, three series of concentrations of 6 fat-soluble vitamins in quality control material

ng/ml	Vitamin A	25 hydroxy vitamin D2	25 hydroxy vitamin D3	Vitamin E	Vitamin K1	Vitamin K2
							L	144	7.2	10.8	1800	0.36	0.36
M	480	24.0	36.0	6000	1.20	1.20
							H	1440	72.0	108.0	18000	3.60	3.60

2. Preparation of internal standard substance working solution

Preparing mixed internal standard working solution, wherein the concentrations of vitamin A-D6, 25-hydroxyvitamin D2-D3, 25-hydroxyvitamin D3-D6, vitamin E-D6 and vitamin K-D4 are respectively 3.0, 1.0, 20 and 0.025 mu g/mL.

3. Preparation of protein precipitant

750ml of methanol, 150ml of acetonitrile and 100ml of isopropanol were measured respectively to prepare a mixed solution containing 75% of methanol, 15% of acetonitrile and 10% of isopropanol, and 3.85g of ammonium acetate was weighed to prepare a protein precipitant containing 50mM of ammonium acetate, 5% of methanol, 15% of acetonitrile and 10% of isopropanol.

Although ammonium acetate is used as the additive in this embodiment, multiple experiments prove that the additive can also be formic acid, ammonium fluoride, ammonium formate or 2, 6-di-tert-butyl-p-cresol, and can also exert a synergistic effect of improving the extraction rate of the fat-soluble vitamins in the sample.

4. Preparation of internal standard solution

Adding 2ml of internal standard substance working solution into 248ml of protein precipitator to prepare the internal standard substance working solution with the protein precipitator in a ratio of 1:124 internal standard solution.

In the step, the internal standard substance working solution and the protein precipitator are directly mixed according to the ratio of 1:124 times the ratio was added to the sample.

Secondly, sample pretreatment

The samples comprise human serum to be detected, a standard substance and a quality control substance, and are processed by adopting the following method:

(1) adding 50 mu L of standard substance/quality control substance/human serum to be detected into a 96-hole deep-hole plate;

(2) adding 250 μ L of internal standard solution (or internal standard working solution and protein precipitant), and shaking at 1000rpm for 10 min;

(3) the sample was tested by centrifugation at 4,000rpm for 10min and 200. mu.L of supernatant was taken in a clean low adsorption 96 well plate.

Third, sample detection

Taking 40 mu L of sample, and analyzing by a liquid chromatography-mass spectrometry combined system, wherein the specific analysis conditions are as follows:

liquid chromatography tandem mass spectrometry system: AB SCIEX Triple Quad 4500 MD; a chromatographic column: phenomenex Luna C18(3 μm, 50X 2.0 mm); mobile phase A: 0.1% aqueous formic acid; mobile phase B: 0.1% formic acid methanol; flow rate: 0.7 mL/min; column temperature: 40 ℃; sample injector temperature: 15 ℃; sample introduction amount: 40 μ L.

The elution gradient is shown in table 3:

TABLE 3 elution gradient

The retention times for the 6 vitamins were as follows: the retention time of vitamin A is 2.91min, the retention time of 25 hydroxy vitamin D2 is 2.83min, the retention time of 25 hydroxy vitamin D3 is 2.76min, the retention time of vitamin E and vitamin K2 is 5.99min, and the retention time of vitamin K is 6.75 min.

As shown in fig. 1, after the sample is separated by the ultra-high pressure liquid chromatography, different fat-soluble vitamins peak at different elution times and are detected by a mass spectrometry selective reaction monitoring mode, so as to detect the content of the vitamins.

The 6 fat-soluble vitamins separated from the liquid chromatogram enter a mass spectrum for detection, the content of the 6 fat-soluble vitamins is detected by adopting an atmospheric pressure chemical ion source (APCI) and a multi-reaction monitoring scanning mode (MRM), and a standard curve graph is drawn.

The mass spectrum sectional acquisition mode is as follows: vitamin A, 25-hydroxyvitamin D2, and 25-hydroxyvitamin D3 are in the first group, and vitamin E, vitamin K1, and vitamin K2 are in the second group.

The mass spectrometric detection conditions are shown in table 4:

TABLE 4 Mass Spectrometry detection conditions

The parent ion/daughter ion to mass-to-charge ratio, the positive ion mode optimized declustering voltage, the collision chamber ejection voltage, and the like of each analyte are shown in table 5:

TABLE 5 parent ion/daughter ion to Mass/Charge ratio of analyte

Detection of the fat-soluble vitamin can be determined by monitoring the detected ion pairs by selective reaction, and the corresponding retention times, and quantified by internal standards for various fat-soluble vitamins.

After the sample is separated by liquid chromatography, different fat-soluble vitamins peak at different elution times and are detected by a mass spectrum multi-reaction monitoring mode, so that the content of the vitamins is detected. According to the series of concentrations of the standard sample S1, a sample to be detected is prepared by taking negative blank human serum as a matrix for detection, and the detection map is shown in figure 1. As can be seen from fig. 1, according to the method provided in this example, 6 fat-soluble vitamins can be simultaneously and accurately detected.

Fourth, data processing and analysis

1. Drawing a standard curve

The standard curve graph uses the concentration of 6 fat-soluble vitamin standard substances as a horizontal coordinate, uses the peak area ratio of each internal standard of the 6 fat-soluble vitamins as a vertical coordinate, and performs linear regression to obtain a standard curve, wherein the standard curve and the correlation coefficient are shown in table 6.

TABLE 6 regression equation of standard curve and correlation coefficient

Substance(s)	Linear and linear correlation coefficient
		VA	y＝0.05055*x+0.09145(r＝0.99845)
25(OH)VD2	y＝0.04916*x+0.1723(r＝0.99906)
		25(OH)VD3	y＝0.05218*x+0.27584(r＝0.99920)
VE	y＝0.04822*x+0.13845(r＝0.99898)
		VK1	y＝0.05207*x+0.19085(r＝0.99553)
VK2	y＝0.05064*x+0.2871(r＝0.99721)

2. Computational accuracy, precision and matrix effects

(1) Accuracy & precision

The peak area ratios of the substance to be measured and the internal standard in the low, medium and high concentration quality control samples (L, M, H) are substituted into the established 6 respective vitamin standard curves to calculate the concentration of 6 fat-soluble vitamins in the quality control samples, and then the accuracy and precision result of each quality control sample of at least 3 analysis batches are calculated, wherein the acceptance standard is that the accuracy between the measured value mean value and the theoretical value is between 85.0 and 115.0 percent, and the precision (CV) is less than or equal to 15 percent. Three precision tests show that the accuracy of three concentrations (L, M, H) is between 91.2% and 110.5%, and the batch precision (CV) is between 1.46% and 9.25%, which meets the requirements.

(2) Matrix effect

The matrix effect is obtained by comparing the response of pure solution samples with quality control levels of various concentrations with the response of the to-be-detected object after matrix blank and serum blank are correspondingly added (the background response needs to be subtracted from the matrix and serum samples), and when the matrix effect is between 85% and 115%, the matrix has negligible influence on the determination of the to-be-detected object. If matrix effects exist, the matrix effects of quality control levels of various concentrations should be close. The internal standard normalized matrix factor is between 95% and 106%, and the CV is between 1.75% and 8.76%, which meets the requirement.

3. Calculating the concentration of 6 fat-soluble vitamins in the human serum sample to be tested

And (3) detecting by mass spectrometry to obtain the ratio of the fat-soluble vitamin to the internal standard, substituting the ratio into a quantitative correction equation, and calculating to obtain the content of the fat-soluble vitamin in the serum.

Example 2: comparison of long-term standing stability evaluation results of different internal standard solvent systems

This example prepares an internal standard solution containing 6 internal standards of fat-soluble vitamins according to the preparation method of internal standard solution provided in example 1, and different protein precipitant components shown in Table 7 are adopted to respectively perform stability tests, wherein the stability test means that the internal standard solution is placed in a thermostat with the temperature of 48 ℃ for six months, whether the internal standard solution has abnormal phenomena such as turbidity, deterioration and the like is observed, whether the internal standard solution subjected to stability test is different from a newly prepared internal standard solution is compared, by the detection method provided in example 1, it was examined whether or not concentration deviation of the internal standard component occurred therein, and whether the recovery rate of the target object to be detected deviates after the sample pretreatment is carried out is analyzed by a liquid chromatography-mass spectrometry system at the lowest concentration point (S1) of the standard curve, since the results of the investigation of each fat-soluble vitamin are basically consistent, the deviation of the concentration and the extraction rate of the internal standard of vitamin K1 with the lowest content is only demonstrated by way of example; meanwhile, the quality guarantee period of long-term storage at room temperature is also considered; the results of the examination are shown in Table 7.

TABLE 7 comparison of Long-term storage stability evaluation results for different internal standard solvent systems

As can be seen from table 7, when the internal standard solution contains four components of ammonium acetate, methanol, acetonitrile and isopropanol, the constructed internal standard solution system is most stable, after six months in a thermostat at 48 ℃, the internal standard solution system is almost indistinguishable from a newly prepared internal standard solution, and the quality guarantee period is more than two years; when the internal standard solution only contains ammonium acetate, methanol and acetonitrile or methanol, acetonitrile and isopropanol, the stability is obviously reduced, and the deviation is rapidly increased when the internal standard solution is detected with a newly prepared internal standard solution; when the internal standard solution only contains methanol and acetonitrile or methanol and acetonitrile, the stability is poor, and even the internal standard solution needs to be prepared and used immediately, so that the pretreatment process of the sample is complicated.

The internal standard solution provided by the invention is very stable in system, the internal standard solution is only needed to be added into the sample during sample pretreatment, the pretreatment can be completed by one-step mixing, the internal standard solution can be prepared in advance and taken at any time, the method is very convenient, the pretreatment steps are greatly simplified, the method has extremely obvious superiority, great convenience is brought to clinical detection of fat-soluble vitamins in serum, and the method is very worthy of popularization and application.

Example 3: comparison of detection results after sample pretreatment by using different protein precipitants

In this example, according to the preparation method of the protein precipitant provided in example 1, different protein precipitants shown in table 8 are used, and after sample preparation and pretreatment are completed according to the steps provided in example 1, the peak areas of 5 fat-soluble vitamins in the S1 sample are determined by performing gc-ms analysis on the lowest concentration point (S1) of the standard curve (as shown in table 8):

TABLE 8 comparison of test results after pretreatment of samples with different protein precipitants

As can be seen from table 8, when the protein precipitant provided in this example is 50mM ammonium acetate (75% methanol + 15% acetonitrile + 10% isopropanol), the peak areas of detected vitamins A, E, K1 and K2 are significantly increased, and although the peak areas of vitamin D2 and D3 are slightly decreased, the protein precipitant has positive significance for detecting vitamins A, E, K1 and K2, especially for detecting vitamins K1 and K2 with low concentration levels in human serum samples, and for simultaneously detecting 6 fat-soluble vitamins including vitamins K1 and K2.

The reference interval of the clinical detection of vitamin K is only 0.13-1.39ng/mL, so the requirements on the sample pretreatment recovery rate and the detection sensitivity of the detection method are very high, and the detection sensitivity of vitamin K1 and K2 needs to be particularly concerned in the method for simultaneously detecting 6 fat-soluble vitamins. Although some existing sample pretreatment methods can simultaneously detect multiple fat-soluble vitamins in a sample, the recovery rates of the vitamins K1 and K2 are low, so that the detection sensitivity of the vitamins K1 and K2 is limited, and it is difficult to accurately judge whether 6 fat-soluble vitamins containing vitamin K1 and K2 are deficient clinically.

As can be seen from table 8, the extraction rate of vitamin K from pure methanol is low, and cannot meet the sensitivity requirement of clinical detection, and the extraction efficiency of vitamin K can be significantly improved by using acetonitrile or a mixed solution of methanol and acetonitrile. Because the substances to be detected are fat-soluble vitamins, isopropanol is introduced into an extraction system according to the principle of similarity and compatibility, so that the extraction rates of the vitamins A, E, K1 and K2 can be further improved; meanwhile, on the basis, ammonium acetate, ammonium formate or 2, 6-di-tert-butyl-p-cresol are added, so that an obvious synergistic effect can be achieved, and the extraction rate of the vitamin A, E and K, particularly the synergistic effect of the ammonium acetate, is obvious; the optimal choice of protein precipitant is therefore 50mM ammonium acetate (75% methanol + 15% acetonitrile + 10% isopropanol).

By adopting the protein precipitator provided by the embodiment, the pretreatment operation is simplified, the recovery rates of the vitamins A, E and K (particularly the vitamins K1 and K2 with the lowest content) in human serum are obviously improved, the subsequent detection result of the high performance liquid chromatography tandem mass spectrometry is more accurate, the sensitivity is higher, and the detection capability of the protein precipitator on various fat-soluble vitamins in a blood sample can simultaneously meet the actual requirements of a clinical reference interval. After the protein precipitant 50mM ammonium acetate (75% methanol + 15% acetonitrile + 10% isopropanol) provided by the embodiment is used for pretreatment, and subsequent high performance liquid chromatography tandem mass spectrometry detection verifies that the sensitivities of vitamin A, 25 hydroxyvitamin D2, 25 hydroxyvitamin D3, vitamin E, vitamin K1 and vitamin K2 can reach 0.4ng/mL, 0.7ng/mL, 0.3ng/mL, 3ng/mL, 10pg/mL and 6pg/mL respectively.

Example 4: influence of proportion relation of components in protein precipitant on detection result

In this example, according to the preparation method of the protein precipitant provided in example 1, components of the protein precipitant include ammonium acetate, methanol, acetonitrile, and isopropanol, and after sample preparation and pretreatment are completed according to the different proportional relationships shown in table 9 and the steps provided in example 1, peak areas of 6 fat-soluble vitamins in the S1 sample measured by analysis of a gc-ms system at the lowest concentration point (S1) of the standard curve are shown in table 9:

TABLE 9 comparison of the test results after pretreatment of samples with protein precipitants of different component ratios

As shown in Table 9, when the concentration of ammonium acetate is as low as 20mM, the extraction rate of the protein precipitant for the fat-soluble vitamin is reduced to a certain extent, and therefore the concentration of ammonium acetate should preferably be maintained at 40-50 mM; similarly, when the content of the methanol is as high as 80% or as low as 15%, the extraction rate of the protein precipitator on the fat-soluble vitamins is reduced to different degrees; when the acetonitrile or the isopropanol is too high or too low, the extraction rate of the fat-soluble vitamins is also reduced to different degrees; therefore, a large number of experiments prove that the component proportion of the protein precipitator is preferably 40-50 mM ammonium acetate (70-75% of methanol, 15-25% of acetonitrile and 5-10% of isopropanol).

Example 5: influence of volume ratio of sample to internal standard solution on detection result

In this embodiment, according to the preparation method of the internal standard solution provided in example 1, an internal standard solution containing 6 internal fat-soluble vitamins is prepared, where system components of the internal standard solution include 50mM ammonium acetate (75% methanol + 15% acetonitrile + 10% isopropanol), different volume ratios of the internal standard solution and the sample to be detected shown in table 10 are used, sample preparation and pretreatment are completed according to the method provided in example 1, a lowest concentration point (S1) of a standard curve is analyzed by a hplc-ms system, and peak areas of the 6 fat-soluble vitamins in an S1 sample are shown in table 10:

TABLE 10 comparison of the results of the measurements of the volume ratios of the different samples to the internal standard solution

As can be seen from table 10, different contents of the internal standard solution and the sample in different volume ratios also have a significant effect on the extraction rate of 6 fat-soluble vitamins in the sample: when the volume ratio of the internal standard solution to the sample is 1:1, the feeding amount of the precipitator is not enough to completely precipitate the protein, the extraction efficiency of the fat-soluble vitamin to be detected is reduced, the impurity separation effect is poor, and the detection process is obviously influenced by the matrix effect, so that the detection sensitivity is influenced; when the volume ratio of the internal standard solution to the sample is 1:9, the sample is excessively diluted, so that the response value is lowered, thereby affecting the detection sensitivity. Therefore, the volume ratio of the sample to the internal standard solution is preferably 1: 3-1: 7, and most preferably 1: 5.

Example 6: comparison of stability results of different internal standard solution systems and sample feeding plates

In this embodiment, an internal standard solution containing 6 internal standards of fat-soluble vitamins and a protein precipitant is prepared according to the method for preparing an internal standard solution provided in example 1, different internal standard solution system components shown in table 11 are adopted, the sample preparation and pretreatment are completed according to the method provided in example 1, and the internal standard solution is placed in a sample injector for more than 12 hours, and subjected to liquid chromatography-mass spectrometry, so as to detect vitamin K1 in a clinical sample, wherein the concentration deviation from the direct sample injection after the treatment is shown in table 11:

TABLE 11 comparison of stability results for different internal standard solution systems and run-in panels

Vitamin K1 is the least polar analyte of 6 fat-soluble vitamins and has strong adsorbability. When the sample is extracted and separated from the serum matrix by the internal standard solution, the sample can be gradually adsorbed on the surface of the sample injection plate, so that the sensitivity and the accuracy of detection are influenced.

As can be seen from table 11, when a protein precipitant (70% methanol + 25% acetonitrile + 5% isopropanol +50mM ammonium acetate) having good solubility to vitamin K1 is used, the risk of vitamin K1 being adsorbed by the sample injection plate can be weakened to some extent, so that the concentration deviation of vitamin K1 detection by the protein precipitant is significantly reduced; and then, comparing different sample feeding plates, it can be found that the stability of the vitamin K1 detection result can be remarkably improved by storing the extracted sample feeding solution by using a low adsorption plate, so that the low adsorption sample feeding plate is preferred.

Although the present invention is disclosed above, the present invention is not limited thereto. Various changes and modifications may be effected therein by one skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A protein precipitant for extracting liposoluble vitamins from blood serum is characterized by comprising methanol, acetonitrile and isopropanol.

2. The protein precipitant according to claim 1, further comprising an additive, wherein the additive is any one or more of formic acid, ammonium fluoride, ammonium formate, ammonium acetate, and 2, 6-di-tert-butyl-p-cresol.

3. The protein precipitant of claim 1 wherein said additive is ammonium acetate.

4. The protein precipitant according to claim 2, wherein the volume ratio of methanol, acetonitrile and isopropanol is 5-75%: 5-75%, and the content of ammonium acetate is 10-50 mM.

5. The protein precipitant according to claim 3, wherein the volume ratio of methanol, acetonitrile and isopropanol is 70-75%: 15-25%: 5-10%, and the content of ammonium acetate is 40-50 mM.

6. An internal standard solution for extraction of fat soluble vitamins from serum, comprising an internal standard working solution and a protein precipitant according to any one of claims 1 to 5; the internal standard working solution comprises an internal standard of the fat-soluble vitamin to be detected.

7. The internal standard solution of claim 5, wherein the volume ratio of the internal standard working solution to the protein precipitant is 1: 124.

8. A sample pretreatment method for detecting fat-soluble vitamins in serum by high performance liquid chromatography-tandem mass spectrometry is characterized in that the protein precipitator in any one of claims 1-5 or the internal standard solution in claim 6 or 7 is adopted for sample pretreatment, wherein the volume ratio of the sample to the protein precipitator or the internal standard solution is 1: 3-1: 7.

9. The sample pretreatment method according to claim 7, comprising the step 1): putting 50 mu L of sample into a 96-well plate, adding 250 mu L of internal standard solution, uniformly mixing by vortex, oscillating at 600rpm for 10min, performing centrifugal separation, putting 200 mu L of supernatant into a low-adsorption 96-well plate, and waiting for detection;

or comprising step 2): and (3) putting 50 mu L of sample into a 96-well plate, adding 2 mu L of internal standard substance working solution, adding 248 mu L of protein precipitator, uniformly mixing by vortex, shaking at 600rpm for 10min, carrying out centrifugal separation, and putting 200 mu L of supernatant into a low-adsorption 96-well plate to be detected.

10. The application of ammonium acetate in preparing the protein precipitant is characterized in that the protein precipitant is used for sample pretreatment of high performance liquid chromatography tandem mass spectrometry detection of fat-soluble vitamins in serum, wherein the fat-soluble vitamins are one or more of vitamin A, vitamin E, vitamin K1 and vitamin K2.