CN112924606A

CN112924606A - Method for simultaneously detecting vitamin A and 25-hydroxy vitamin D in human serum

Info

Publication number: CN112924606A
Application number: CN201911233948.0A
Authority: CN
Inventors: 肖玲; 程雅婷; 赵蓓蓓; 董衡; 佘旭辉; 徐玉兵
Original assignee: SHANGHAI KINGMED MEDICAL DIAGNOSTICS INSTITUTE CO LTD; Guangzhou Kingmed Diagnostics Central Co Ltd
Current assignee: SHANGHAI KINGMED MEDICAL DIAGNOSTICS INSTITUTE CO LTD; Guangzhou Kingmed Diagnostics Central Co Ltd
Priority date: 2019-12-05
Filing date: 2019-12-05
Publication date: 2021-06-08

Abstract

The invention relates to a method for simultaneously detecting vitamin A and 25-hydroxy vitamin D in human serum, which comprises the following steps: preparing a standard solution; preparing mixed internal standard liquid; preparation of a sample to be tested: adding water into the standard solution and the human serum sample to be detected, mixing, then adding the mixed internal standard solution, and performing protein precipitation; extracting with n-hexane, taking the upper layer extract, removing the solvent, and re-dissolving to obtain a standard sample to be detected and a human serum sample to be detected respectively; and injecting the sample to be detected into a liquid chromatogram tandem mass spectrometer for detection. The method can give consideration to the sensitivity of 25-hydroxyvitamin D and the problem of signal saturation of a high-concentration vitamin A sample detector, realizes simultaneous detection of vitamin A and 25-hydroxyvitamin D in human serum, and has the advantages of high detection efficiency, low cost, accurate detection result and no relative matrix effect.

Description

Method for simultaneously detecting vitamin A and 25-hydroxy vitamin D in human serum

Technical Field

The invention relates to the technical field of detection, in particular to a method for simultaneously detecting vitamin A and 25-hydroxy vitamin D in human serum.

Background

Vitamin A (vitamin A), also called retinol (retinol) or anti-xerophthalmia factor, is an unsaturated monohydric alcohol with alicyclic ring, including two vitamins A1 and A2 from animal food sources, which are substances with retinol bioactivity.

Vitamin D is a fat-soluble steroid derivative, including vitamin D3 (cholecalciferol) of animal origin and vitamin D2 (ergocalciferol) of vegetable origin. 25-hydroxy vitamin D is one of the most abundant, most stable and longest half-life of serum multivitamin D metabolites, and is a good index for reflecting the level of vitamin D in vivo, and the serum level can represent the nutritional state of vitamin D in the body.

The content difference between vitamin A and 25-hydroxy vitamin D in vivo is large, and if the detection is carried out simultaneously, the problems of sensitivity of 25-hydroxy vitamin D and signal saturation of a high-concentration vitamin A sample detector are difficult to be considered. Therefore, at present, vitamin A and 25-hydroxy vitamin D need to be pretreated twice and tested on a computer twice, wherein the vitamin A is mainly tested by high performance liquid chromatography, and the 25-hydroxy vitamin D is tested by high performance liquid chromatography tandem mass spectrometry.

In some methods, a method for simultaneously detecting vitamin a and 25-hydroxyvitamin D in blood by using a high performance liquid chromatography-tandem mass spectrometry method is tried, but the method is difficult to accurately react the content of vitamin a and 25-hydroxyvitamin D in a sample, and the main reason is that:

1) the matrix of the used calibration curve is inconsistent with the matrix of the sample, the calibration curve is not pretreated as the sample, the method does not refer to the verification related to matrix effect, and some endogenous substances, such as phospholipid, salts, protein and the like, exist in the biological matrix and interfere with the ionization process, possibly have ion inhibition or ion enhancement influence on the high performance liquid chromatography tandem mass spectrometry, namely the matrix effect, and the matrix effect influences the accuracy of the method and is one of the main factors for inaccurate results of the liquid chromatography mass spectrometry;

2) the vitamin A, 25-hydroxyvitamin D2 and 25-hydroxyvitamin D33 compounds did not achieve chromatographic separation.

Therefore, how to provide a method which can simultaneously detect vitamin A and 25-hydroxy vitamin D of human serum, has accurate detection result and no matrix effect is an urgent technical problem to be solved.

Disclosure of Invention

Based on this, the present invention provides a method for simultaneously detecting vitamin a and 25-hydroxyvitamin D in human serum. The method can give consideration to the sensitivity of 25-hydroxyvitamin D and the problem of signal saturation of a high-concentration vitamin A sample detector, realizes simultaneous detection of vitamin A and 25-hydroxyvitamin D in human serum, and has the advantages of high detection efficiency, low cost, accurate detection result and no relative matrix effect.

A method for simultaneously detecting vitamin a and 25-hydroxyvitamin D in human serum comprising the steps of:

preparation of standard solution: respectively taking a vitamin A standard substance, a 25-hydroxyvitamin D2 standard substance and a 25-hydroxyvitamin standard substance, dissolving the vitamin A standard substance, the 25-hydroxyvitamin D2 standard substance and the 25-hydroxyvitamin standard substance by using a solvent, and adding an aqueous solution of bovine serum albumin to prepare solutions with different concentrations to obtain standard solutions;

preparing a mixed internal standard solution: dissolving a vitamin A internal standard substance, a 25-hydroxyvitamin D2 internal standard substance and a 25-hydroxyvitamin D3 internal standard substance by using a solvent to obtain a mixed internal standard solution;

preparation of a sample to be tested: adding water into the standard solution and the human serum sample to be detected, mixing, then adding the mixed internal standard solution, and performing protein precipitation; extracting with n-hexane, taking the upper layer extract, removing the solvent, and re-dissolving to obtain a standard sample to be detected and a human serum sample to be detected respectively;

and (3) detection: injecting the standard sample to be detected into a liquid chromatogram tandem mass spectrometer for detection, and constructing a standard curve; injecting the human serum sample to be detected into a liquid chromatogram tandem mass spectrometer for detection, and comparing a detection map with the standard curve; the liquid chromatogram adopts a mobile phase A which is a methanoic acid solution with the volume concentration of 0.08-0.12%, a mobile phase B which is a methanoic acid solution with the volume concentration of 0.08-0.12%, and the adopted elution mode is gradient elution.

In one embodiment, the aqueous solution of Bovine Serum Albumin (BSA) has a mass concentration of 3-5%.

In one embodiment, in the preparation of the sample to be detected, the redissolved solvent is a mixed solution of methanol and water in a volume ratio of 65-75: 35-25.

In one embodiment, in the preparation of the sample to be tested, the method for removing the solvent is as follows: and drying the glass by nitrogen at the temperature of 15-30 ℃.

In one embodiment, the vitamin a internal standard is D5-vitamin a, the 25-hydroxyvitamin D2 internal standard is D6-25-hydroxyvitamin D2, and the 25-hydroxyvitamin D3 internal standard is D6-25-hydroxyvitamin D3; in the mixed internal standard solution, the concentration of the D5-vitamin A is 75-85 ng/mL, the concentration of the D6-25-hydroxyvitamin D2 is 45-55 ng/mL, and the concentration of the D6-25-hydroxyvitamin D3 is 55-65 ng/mL.

In one embodiment, the protein precipitation is performed by using a protein precipitating agent which is an acetonitrile solution of 2, 6-di-tert-butyl-p-cresol and estriol.

In one embodiment, the protein precipitant contains 2, 6-di-tert-butyl-p-cresol 0.03-0.05 wt% and estriol 0.8-1.2 mg/L.

In one embodiment, the liquid chromatography employs a gradient elution method comprising:

the volume ratio of the mobile phase A is increased from 70% to 80% in 0-1 min;

keeping the volume ratio of the mobile phase A at 80% for 1-2.9 min;

2.9-3 min, wherein the volume ratio of the mobile phase A is increased from 80% to 94%;

keeping the volume ratio of the mobile phase A at 94% for 3-4.5 min;

4.5-4.6 min, and reducing the volume ratio of the mobile phase A from 94% to 70%;

and 4.6-5.1 min, and keeping the volume ratio of the mobile phase A at 70%.

In one embodiment, the chromatographic column used in the liquid chromatography is a C18 chromatographic column, the column temperature of the chromatographic column is 53-57 ℃, and the flow rate of the mobile phase is 0.4-0.6 ml/min.

In one embodiment, the mass spectrum is detected using electrospray ionization (ESI), multiple reaction detection (MRM) scanning in positive ion mode.

In one embodiment, the parameters of the Multiple Reaction Monitoring (MRM) scan are as follows:

the quantitative ion pair of vitamin A: the declustering voltage (DP) is 100V, the inlet voltage (EP) is 10V, the impact voltage (CE) is 22V, and the outlet voltage (CXP) is 11V;

quantitative ion pair of the 25-hydroxyvitamin D2: the declustering voltage (DP) is 130V, the inlet voltage (EP) is 9V, the impact voltage (CE) is 12V, and the outlet voltage (CXP) is 13V;

quantitative ion pair of the 25-hydroxyvitamin D3: the declustering voltage (DP) is 120V, the inlet voltage (EP) is 10V, the impact voltage (CE) is 12V, and the outlet voltage (CXP) is 14V;

quantitative ion pair of the vitamin A internal standard substance: the declustering voltage (DP) is 100V, the inlet voltage (EP) is 10V, the impact voltage (CE) is 22V, and the outlet voltage (CXP) is 11V;

quantitative ion pair of the 25-hydroxyvitamin D2 internal standard: the declustering voltage (DP) is 130V, the inlet voltage (EP) is 9V, the impact voltage (CE) is 12V, and the outlet voltage (CXP) is 14V;

quantitative ion pair of the 25-hydroxyvitamin D3 internal standard: the declustering voltage (DP) was 120V, the entrance voltage (EP) was 7V, the impact voltage (CE) was 12V, and the exit voltage (CXP) was 13V.

In one embodiment, the detection conditions of the mass spectrum include: the air curtain air (CUR) is 38-42 psi; the voltage (IS) of the electrospray needle IS 4400-4600V; the heating gas stability (TEM) is 440-460 ℃; the heating gas (GS1) is 43-47 psi, and the auxiliary heating gas (GS2) is 48-52 psi; the collision gas (CAD) is 5-10 psi.

Compared with the prior art, the invention has the following beneficial effects:

according to the method, the BSA aqueous solution is used as a matrix of a standard sample, namely a calibration curve matrix, the standard sample and a human blood sample are subjected to the same pretreatment, the problem of matrix effect can be better avoided, the pretreatment process of the sample to be detected is optimized, the sensitivity of 25-hydroxyvitamin D is improved, the vitamin A signal is not saturated, and on the basis, the vitamin A, the 25-hydroxyvitamin D2 and the 25-hydroxyvitamin D3 can be effectively separated by combining a proper liquid chromatography mobile phase and an elution mode. Therefore, the steps are integrated, the simultaneous detection of the human serum vitamin A and the 25-hydroxy vitamin D is realized, the detection efficiency is high, the cost is low, the detection result is accurate, and the relative matrix effect is avoided.

Drawings

FIG. 1 is a total ion flow chromatogram of VA, 25OHD2, 25OHD3 detection in a serum sample according to one embodiment of the invention;

FIG. 2 is a graph of vitamin A standards according to one embodiment of the present invention;

FIG. 3 is a graph of a standard 25-hydroxyvitamin D2 graph according to one embodiment of the present invention;

FIG. 4 is a graph of a standard 25-hydroxyvitamin D3 graph according to one embodiment of the present invention;

FIG. 5 is a total ion flow graph for different mobile phase ratios (18%, 20%, 21%, 22%, or 25% for mobile phase B, respectively);

figure 6 is an ion flow graph showing the lower limit of 25-hydroxyvitamin D quantitation and the upper limit of vitamin a quantitation.

Detailed Description

The present invention will be described in further detail with reference to specific examples. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

Example 1

The embodiment provides a method for simultaneously detecting vitamin A and 25-hydroxyvitamin D in human serum by adopting a liquid chromatography-tandem mass spectrometer, which comprises the following steps:

(1) preparation of standard solution:

1.1, 30% ethanol water: 300mL of absolute ethanol and 700mL of ultrapure water are weighed by a measuring cylinder and uniformly mixed to prepare a 30% ethanol water solution.

1.2, 4% BSA formulation: accurately weighing 40g of BSA, dissolving in 1L of water, and carrying out ultrasonic treatment at room temperature (15-30 ℃) until the BSA is completely dissolved.

1.3, preparing a standard stock solution:

a. preparing a vitamin A stock solution:

accurately weighing 10.0mg (purity of 95%) retinol standard substance with one hundred thousand parts of balance, adding into brown reagent bottle containing 100ml 0.04% BHT ethanol solution, vortex dissolving, mixing, and storing at-80 deg.C or below.

b. Preparation of 25-hydroxyvitamin D3(25OHD3) stock solution: adding anhydrous ethanol with corresponding milliliter volume according to the milligram number on a freeze-dried powder tube in a ratio of 1:0.5, uniformly mixing and dissolving, wherein the concentration is 2.0mg/mL, and storing for later use at the temperature of less than or equal to-20 ℃ for 3 years.

c. Preparation of 25-hydroxyvitamin D2(25OHD2) stock solution: adding anhydrous ethanol with corresponding milliliter volume according to the milligram number on a freeze-dried powder tube in a ratio of 1:0.5, uniformly mixing and dissolving, wherein the concentration is 2.0mg/mL, and storing for later use at the temperature of less than or equal to-20 ℃ for 3 years.

1.4, preparing an intermediate stock solution:

a. preparation of 25-hydroxy vitamin D3 intermediate stock solution: sucking 62.5 μ L25 OHD3 stock solution into a 10mL volumetric flask, adding absolute ethanol to constant volume, mixing, storing at-20 deg.C or below with concentration of 12.5 μ g/mL, and keeping for use for 1 year.

b. 25-hydroxy vitamin D2 intermediate stock solution: sucking 62.5 μ L25 OHD2 stock solution into a 10mL volumetric flask, adding absolute ethanol to constant volume, mixing, storing at-20 deg.C or below with concentration of 12.5 μ g/mL, and keeping for use for 1 year.

1.5, preparation of standard solution for standard working curve: accurately sucking standard stock solutions or intermediate stock solutions with different volumes into a volumetric flask of 100ml, fixing the volume by using a 4% BSA blank matrix, and shaking up to obtain a standard solution for a standard working curve, wherein:

the concentration of vitamin A curve points is as follows: 3.71, 7.42, 14.84, 29.69, 59.38, 118.75, 237.5, 475, 950 ng/ml;

the concentration of the 25-hydroxy vitamin D3 at the curve points is as follows: 1.88, 3.75, 7.50, 15.0, 30.0, 60.0, 120.0 ng/ml;

the concentration of the 25-hydroxy vitamin D2 at the curve points is as follows: 1.88, 3.75, 7.50, 15.0, 30.0, 60.0, 120.0 ng/ml.

(2) Preparing a mixed internal standard solution:

2.1, preparing an internal standard stock solution:

d6-25OHD2 stock solution: adding anhydrous ethanol with the corresponding milliliter volume according to the milligram number on a freeze-dried powder tube in a ratio of 1:1, uniformly mixing and dissolving, wherein the concentration is 1.0mg/mL, and storing at the temperature of less than or equal to-20 ℃ for later use.

d6-25OHD3 stock solution: adding anhydrous ethanol with the corresponding milliliter volume according to the milligram number on a freeze-dried powder tube in a ratio of 1:1, uniformly mixing and dissolving, wherein the concentration is 1.0mg/mL, and storing at the temperature of less than or equal to-20 ℃ for later use.

d 5-vitamin a stock solution: adding anhydrous ethanol containing 0.04% BHT in a corresponding milliliter volume according to the milligram on the freeze-dried powder tube in a ratio of 1:1, uniformly mixing and dissolving, wherein the concentration is 1.0mg/mL, and storing at the temperature of less than or equal to-80 ℃ for later use.

2.2, preparing a mixed internal standard solution: 50 μ L of d6-25OHD2 stock solution (1.0mg/mL), 60 μ L of d6-25OHD3 stock solution (1.0mg/mL), 80 μ L of d 5-vitamin A stock solution (1.0mg/mL) and 1mL of estriol (1.0mg/mL) were respectively sucked and added to 1L of acetonitrile containing 0.04% BHT, mixed well and stored at 2-8 ℃ for later use.

(3) Preparation of a sample to be tested:

3.1, taking the standard solution and the mixed internal standard solution out of a refrigerator, and balancing the standard solution and the mixed internal standard solution together with a human serum sample to be detected to room temperature;

3.2, respectively taking 100 mu L of standard solution and the human serum sample to be detected for synchronous treatment: placing in 2mL centrifuge tube, adding 100 μ L deionized water, and vortex mixing for 1 min; adding 200 μ L mixed internal standard solution, vortex mixing for 3min for protein precipitation; adding 700 μ L of 0.04% BHT n-hexane solution, vortex mixing for 10min, extracting, and centrifuging at 10000rpm and 25 deg.C for 5 min; transferring 550 mu L of the n-hexane layer into a 1.5mL centrifuge tube, and drying by nitrogen (at room temperature of 15-30 ℃); adding 100 μ L of 70% methanol water (containing 0.01% estriol and 0.04% BHT) into the dried centrifugal tube, vortex vibrating for 3min, and centrifuging at 10000rpm and 25 deg.C for 5 min; and respectively obtaining standard solutions with different concentration points and human serum samples to be detected, and storing the standard solutions and the human serum samples in brown bottles.

(4) And (3) detection:

4.1, standard curve construction: and respectively injecting 70ul of standard solutions with different concentration points into a liquid chromatography tandem mass spectrometer for detection, and constructing a standard curve.

4.2, injecting 60 mu L of human serum sample to be detected into a liquid chromatogram tandem mass spectrometer for detection; the chromatographic conditions were as follows:

the conditions of the liquid chromatography tandem mass spectrometer of items 4.3, "4.1" and "4.2" are as follows:

chromatographic conditions are as follows: a chromatographic column: 2.7um 2.1mm 50C 18; column temperature of the chromatographic column: 55 ℃; sample introduction amount: 10 mu L of the solution; flow rate: 0.5 ml/min;

mobile phase: phase A is methanol containing formic acid with a volume concentration of 0.1%, and phase B is water containing formic acid with a volume concentration of 0.1%;

gradient elution was used, the gradient being shown in the liquid phase separation conditions (volume ratio) of table 1.

TABLE 1 liquid phase separation conditions

The mass spectrum detection adopts an electrospray ionization (ESI) and a multi-reaction detection (MRM) scanning mode in a positive ion mode. The mass spectrometry conditions include:

air curtain air CUR: 40.00 psi; electrospray needle voltage IS: 4500.00V; heating gas stabilization TEM: 450.00 deg.C; heating gas GS 1: 45 psi; auxiliary heating gas GS 2: 50 psi; collision gas CAD: 7 psi;

multiple Reaction Monitoring (MRM) scan mode parameters are shown in table 2.

TABLE 2 multiple reaction monitoring mode parameters

(5) And (3) detection results:

5.1 qualitative judgment:

the retention time of the vitamin A, 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 ion pairs in the sample is compared with that of the corresponding standard to serve as a qualitative basis, and the retention time of each compound is as follows:

the retention time of vitamin A is 2.55min, and the retention time of d5-VA is 2.52 min; the retention time of 25-hydroxyvitamin D2 is 2.34min, and the retention time of D6-25-hydroxyvitamin D2 is 2.32 min; the retention time of 25-hydroxyvitamin D3 was 2.16min, and the retention time of D6-25-hydroxyvitamin D3 was 2.14 min.

The total ion current chromatogram of the three compounds is shown in figure 1, and the vitamin A, the 25-hydroxyvitamin D2 and the 25-hydroxyvitamin D3 realize chromatographic separation.

The presence of vitamin a, 25-hydroxyvitamin D2, and 25-hydroxyvitamin D3 was judged by the relative retention times of vitamin a, 25-hydroxyvitamin D2, and 25-hydroxyvitamin D3 and the abundance ratio of the detected quantitative ion pair: under the same test condition, the retention time of the mass chromatographic peak of the substance to be analyzed in the detection sample is consistent with that of the corresponding substance in the standard solution; if the deviation between the relative abundance ratio of the selected detection ion pair in the chromatogram of the detection sample and the relative abundance ratio of the ion pair of the standard solution with the corresponding concentration does not exceed the range specified in table 3, the presence of the corresponding analyte in the sample can be determined.

TABLE 3 qualitative determination of maximum permissible error in relative abundance

5.2 quantitative determination: and (3) quantifying by adopting an internal standard curve method, and calculating the content of the substance to be detected in the sample according to the peak area ratio of the substance to be detected and the internal standard substance.

Establishing an internal standard curve by using the peak area ratio of the to-be-detected substance to the internal standard substance in the standard solution detection results of different concentration points, wherein the standard curve of the vitamin A is shown in figure 2, the equation of the curve is that y is 0.32499x +0.23722, and r is 0.99975; the standard curve of 25-hydroxyvitamin D3 is shown in FIG. 3, and the equation of the curve is 0.00980x +6.81945e^-5R is 0.99996; the standard curve of 25-hydroxyvitamin D2 is shown in fig. 4, and the equation of the curve is that y is 0.01432x +0.00896, and r is 0.99994; and then calculating the concentration of the substance to be detected in the human serum sample to be detected by using the standard curve.

The peak area of vitamin A measured by the human serum sample to be detected in the embodiment is 7.05E +06, the peak area of the internal standard substance is 8.51E +04, and the concentration of the vitamin A in the human serum sample to be detected in the embodiment is calculated to be 254.16 ng/ml; the peak area of 25-hydroxyvitamin D3 is 7.81E +05, the peak area of the internal standard substance is 4.47E +06, and the concentration of 25-hydroxyvitamin D3 in the human serum sample to be detected in the embodiment is 14.89ng/ml through calculation; the peak area of 25-hydroxyvitamin D2 is 1.72E +05, the peak area of the internal standard substance is 1.57E +06, and the concentration of 25-hydroxyvitamin D2 in the human serum sample to be tested in the embodiment is 7.73ng/ml through calculation.

Example 2

The method described in example 1 was validated for condition examination and methodology.

Firstly, condition investigation:

(1) matrix investigation of standard solutions

30% ethanol water and 4% BSA are respectively adopted as standard solution matrixes, wherein the signal response of the 25-hydroxy vitamin D internal standard in the 30% ethanol water is inconsistent with that in a human serum sample to be detected, a matrix effect may exist, and the signal response in the 4% BSA matrix is consistent with that in the human serum sample to be detected, and specifically shown in the following table 4, so 4% BSA is selected as a calibration curve matrix.

TABLE 4 Signal response (cps) in different matrices as internal standard

	d6-25OHD3	d6-25OHD2	d5-VA
				30% ethanol water	1.74E+06	8.53E+05	/
4％BSA	5.14E+06	1.73E+06	7.10E+04
				Human serum sample to be tested	5.07E+06	1.75E+06	7.17E+04

(2) Selection of isotopic internal standards

Because d6-25OHD3 is easier to obtain than d5-VA and has better stability than d5-VA, d6-25OHD3 and d5-VA are selected as alternative internal standards of vitamin A, and the relative matrix effect is verified on the internal standards.

Collecting 8 patient samples with different concentrations and different sources; dissolving standard solutions of different concentration levels using a matrix of 4% BSA (in deionized water) formulated standard curve; respectively mixing the standard solution and 8 patient matrix samples from different sources according to different proportions to obtain mixed samples; the standard solution, the patient sample and the mixed sample were each processed in parallel in 3 samples and tested on the machine (the processing method and the testing method were the same as those in example 1). The method comprises the following steps: the response value of the mixed sample (patient sample/standard solution) should differ by less than 20% from the mean response value of the patient sample and the standard solution, indicating no relative matrix effect.

The verification results of d6-25OHD3 as the internal standard matrix effect are shown in the following table 5, the results are acceptable, but positive deviation exists, d5-VA is internal standard matrix effect data shown in the following table 7, the deviation is smaller, the results are better than those of d6-25OHD3 as the internal standard, and d5-VA is used as the internal standard.

TABLE 5-1 substrate Effect results (before mixing)

TABLE 5-2 VA matrix Effect results (post-mix d6-25OHD3 as internal standard)

(3) Screening for suitable protein precipitating agents

Ethanol and ethanol containing isotope internal standard solution are respectively adopted: carrying out protein precipitation by acetonitrile (1:1) and acetonitrile, observing the effects of precipitated proteins of vitamin A and vitamin D, carrying out extraction, blow-drying and redissolving of the precipitated proteins of the sample, and carrying out ethanol and ethanol: and (2) centrifuging the acetonitrile (1:1) protein precipitation sample, floating a small amount of white suspended matters on the surface of the supernatant, wherein the supernatant is slightly turbid, and the acetonitrile protein precipitation sample is clear and has no suspended matters after the steps, so that the acetonitrile solution containing the isotope internal standard, 0.1% estriol and 0.04% BHT is determined as the protein precipitant.

(4) Determination of suitable drying temperature of nitrogen

The obtained supernatant is respectively dried by nitrogen at room temperature (15-30 ℃), at 40 ℃, at 50 ℃ and at 60 ℃, the response of internal standard signals under different conditions is shown in the following table 6, the internal standard signal is the highest at room temperature, and the drying at room temperature is finally determined; (d5-VA reagent is relatively difficult to obtain and thus is not involved in validation).

TABLE 6

	d6-25OHD3	d6-25OHD2
			At room temperature	5.2E+06	1.8E+05
40℃	4.0E+06	1.5E+06
			50℃	2.5E+06	1.0E+06
60℃	1.8E+06	0.6E+06

(5) Optimization of chromatographic conditions

The proportion of the mobile phase B at 1min is adjusted to be 18%, 20%, 21%, 22% and 25%, the total ion flow diagram is shown in figure 5, the separation degree is better than that of the mobile phase B when the proportion of the mobile phase B is 20%, and the proportion of the mobile phase B at 1min is selected to be 20%.

(6) Sensitivity of 25-hydroxyvitamin D and saturation of high concentration sample signals of vitamin A

Selecting a 25-hydroxyvitamin D quantitative lower limit concentration sample and a vitamin A quantitative upper limit concentration sample, processing the samples, and performing machine detection, wherein the signal response value of the 25-hydroxyvitamin D2 quantitative lower limit concentration sample is 4.6E +04cps, the signal-to-noise ratio is 116.6 (shown in figure 6a), the signal response value of the 25-hydroxyvitamin D3 quantitative lower limit concentration sample is 1.2E +05cps, and the signal-to-noise ratio is 111.5 (shown in figure 6b), so that the requirement that the quantitative lower limit signal-to-noise ratio is greater than 10 is met, namely the sensitivity of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 meets the requirement; the signal response value of the vitamin A quantitative upper limit concentration sample is 2.0E +07cps, the target analyte peak type is normal, no flat peak and other conditions occur (see figure 6c), the linearity of the vitamin A standard curve is good (see figure 2), and the problem of detector signal saturation does not occur.

Second, methodology verification

The methodology described in example 1 was validated from matrix effects, reproducibility precision, period precision and accuracy.

1. Relative matrix Effect test

a. Collecting 8 patient samples with different concentrations and different sources; dissolving standard solutions of different concentration levels using a matrix of 4% BSA (in deionized water) formulated standard curve; respectively mixing the standard solution and 8 patient matrix samples from different sources according to different proportions to obtain mixed samples; the standard solution, the patient sample and the mixed sample were each processed in parallel in 3 samples and tested on the machine (the processing method and the testing method were the same as those in example 1). The method comprises the following steps: the response value of the mixed sample (patient sample/standard solution) should differ by less than 20% from the mean response value of the patient sample and the standard solution, indicating no relative matrix effect. The results of the relative matrix effect are shown in tables 7 to 8 (unit: ng/mL), which indicates that there is no relative matrix effect and the requirement is satisfied.

TABLE 7-1 VA matrix Effect results (before mixing)

TABLE 7-225 OHD2 matrix Effect results (before mixing)

TABLE 7-325 OHD3 matrix Effect results (before mixing)

TABLE 8-1 VA matrix Effect results (after mixing)

TABLE 8-225 OHD2 substrate Effect results (after mixing)

TABLE 8-325 OHD3 substrate Effect results (after mixing)

2. Precision experiment

2.1 repeatability precision experiment:

a. preparing patient serum samples with low, medium and high levels of concentration, and verifying repeatability and precision;

b. each sample of concentration level is processed in parallel by 20 samples, and each sample is injected for 1 time;

c. calculating the mean value X, the standard deviation SD and the coefficient of variation CV of the detection result, wherein the CV is required to be less than 1/4 ALE (6.25%);

d. TABLE 9-1 VA, 25OHD₂And 25OHD₃The detection data show that the CV is less than 6.25 percent and meets the requirement.

TABLE 9-1 repeatability precision data Experimental data (Unit: ng/mL)

2.2 duration precision experiment:

a. levels I and II of ClinChek quality control products (RECIPE, Germany) are taken as low-concentration and high-concentration samples, level II is used for preparing medium-concentration samples by double dilution, and the precision in the period is verified;

b. 2 groups of data are respectively detected from each level of sample, and the data are continuously detected for 10 days;

c. calculating the mean value X, standard deviation SD and coefficient of variation CV of each horizontal detection result, wherein the CV is required to be less than 1/3ALE (8.3%);

d. table 9-2 shows the precision data of the period, and the detection data shows that the CV values are less than 8.3% and meet the requirement.

TABLE 9-2 precision experimental data (unit: ng/mL)

2.3 precision results

The precision experiment verification result is shown in table 9-3, the repeatability precision of the method is less than 6.25%, the period precision is less than 8.3%, and the detection requirement is met.

TABLE 9-3 summary of precision test results (unit: ng/mL)

3. Analytical sensitivity and Linear Range

3.1 method quantitative limits and Linear Range

a. Preparing a 4% BSA blank matrix and standard curve points;

b. each concentration curve point is processed in parallel for 6 times and is detected for 2 times respectively;

c. calculating the average value, CV and recovery rate of each concentration sample;

d. method determination criteria for quantitative limits: the lowest concentration point with CV less than 20% and recovery in the range of 85% -115% was taken as the limit of quantitation concentration, LOQ;

e. determination of linear range criteria: CV is less than 20%, recovery rate is in the range of 85% -115%, and regression curve R is drawn by the ratio of theoretical concentration to actual signal response peak area²>0.98, namely, the requirement of linear range is met;

f. verification of experimental data As shown in Table 10-1 and FIGS. 2-4, LOQ of VA was 3.71ng/mL, the linear range was 3.71-950ng/mL, LOQ of 25OHD2 and 25OHD3 was 1.88ng/mL, and the linear range was 1.88-120.0 ng/mL.

TABLE 10-1 method quantitative limits Experimental data

3.2 method detection limits

a. Collecting patient samples with the concentrations of the substances to be detected being near the quantitative limit LOQ;

b. the patient samples were treated in parallel for 12 tests each time;

c. calculating the mean, SD and method detection Limit (LOD) of each concentration sample;

d. as shown in Table 10-2, the LOD of VA was 1.31ng/mL, that of 25OHD2 was 0.26ng/mL, and that of 25OHD3 was 0.11 ng/mL.

TABLE 10-2 detection limit test data (unit: ng/mL)

3.3 conclusion

The analytical sensitivity and linear range results for VA, 25OHD2 and 25OHD3 are summarized in table 11.

TABLE 11 summary of analytical sensitivity and Linear Range results

4. Experiment of accuracy

a. Preparing low, medium and high concentration samples, and performing a standard recovery rate experiment;

b. respectively detecting 3 un-labeled and labeled samples which are processed in parallel, and calculating the result recovery rate of the labeled samples, wherein the recovery rate is in the range of 85-115%, and the method is considered to be accurate;

c. the results of the recovery test are shown in Table 12. The results show that the recovery rate of the method is between 85 and 115 percent, and the method meets the requirements.

TABLE 12-1 vitamin A spiked recovery data

TABLE 12-225 OHD2 normalized recovery data

TABLE 12-325 OHD3 normalized recovery data

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A method for simultaneously detecting vitamin a and 25-hydroxyvitamin D in human serum comprising the steps of:

2. The method according to claim 1, wherein the aqueous solution of bovine serum albumin has a mass concentration of 3 to 5% of bovine serum albumin.

3. The method according to claim 1, wherein in the preparation of the sample to be tested, the redissolved solvent is a mixed solution of methanol and water in a volume ratio of 65-75: 35-25.

4. The method according to claim 1, wherein in the preparation of the sample to be tested, the solvent is removed by: and drying the glass by nitrogen at the temperature of 15-30 ℃.

5. The method of claim 1, wherein the vitamin a internal standard is D5-vitamin a, the 25-hydroxyvitamin D2 internal standard is D6-25-hydroxyvitamin D2, the 25-hydroxyvitamin D3 internal standard is D6-25-hydroxyvitamin D3; in the mixed internal standard solution, the concentration of the D5-vitamin A is 75-85 ng/mL, the concentration of the D6-25-hydroxyvitamin D2 is 45-55 ng/mL, and the concentration of the D6-25-hydroxyvitamin D3 is 55-65 ng/mL.

6. The method of claim 1, wherein the protein precipitation is carried out using a protein precipitating agent comprising a solution of 2, 6-di-tert-butyl-p-cresol and estriol in acetonitrile.

7. The method according to claim 6, wherein the protein precipitant contains 2, 6-di-tert-butyl-p-cresol at a concentration of 0.03 to 0.05% by mass and estriol at a concentration of 0.8 to 1.2mg/L by mass.

8. The method according to any one of claims 1 to 7, wherein the liquid chromatography is performed using a gradient elution method comprising:

the volume ratio of the mobile phase A is increased from 70% to 80% in 0-1 min;

keeping the volume ratio of the mobile phase A at 80% for 1-2.9 min;

keeping the volume ratio of the mobile phase A at 94% for 3-4.5 min;

and 4.6-5.1 min, and keeping the volume ratio of the mobile phase A at 70%.

9. The method according to any one of claims 1 to 7, wherein the liquid chromatography uses a C18 column as a chromatographic column, the temperature of the chromatographic column is 53-57 ℃, and the flow rate of the mobile phase is 0.4-0.6 ml/min.

10. The method of any one of claims 1 to 7, wherein the mass spectrometry is detected using electrospray ionization, multiple reaction detection scanning in positive ion mode.

11. The method of claim 10, wherein the parameters of the multiple reaction monitoring scan are as follows:

the quantitative ion pair of vitamin A: the cluster removing voltage is 100V, the inlet voltage is 10V, the collision voltage is 22V, and the outlet voltage is 11V;

quantitative ion pair of the 25-hydroxyvitamin D2: the cluster removing voltage is 130V, the inlet voltage is 9V, the collision voltage is 12V, and the outlet voltage is 13V;

quantitative ion pair of the 25-hydroxyvitamin D3: the cluster removing voltage is 120V, the inlet voltage is 10V, the collision voltage is 12V, and the outlet voltage is 14V;

quantitative ion pair of the vitamin A internal standard substance: the cluster removing voltage is 100V, the inlet voltage is 10V, the collision voltage is 22V, and the outlet voltage is 11V;

quantitative ion pair of the 25-hydroxyvitamin D2 internal standard: the cluster removing voltage is 130V, the inlet voltage is 9V, the collision voltage is 12V, and the outlet voltage is 14V;

quantitative ion pair of the 25-hydroxyvitamin D3 internal standard: the declustering voltage was 120V, the entrance voltage was 7V, the collision voltage was 12V, and the exit voltage was 13V.

12. The method of any one of claims 1-7, wherein the detection conditions for mass spectrometry comprise: the air curtain air pressure is 38-42 psi; the voltage of the electrospray needle is 4400-4600V; the heating gas is stabilized to 440-460 ℃; the heating gas is 43-47 psi, and the auxiliary heating gas is 48-52 psi; the collision gas is 5-10 psi.