CN111983071A - Mass spectrum detection method for vitamin D in peripheral trace blood - Google Patents

Mass spectrum detection method for vitamin D in peripheral trace blood Download PDF

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CN111983071A
CN111983071A CN202010813621.7A CN202010813621A CN111983071A CN 111983071 A CN111983071 A CN 111983071A CN 202010813621 A CN202010813621 A CN 202010813621A CN 111983071 A CN111983071 A CN 111983071A
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hydroxyvitamin
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余晓丹
焦先婷
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Shanghai Childrens Medical Center Affiliated to Shanghai Jiaotong University School of Medicine
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/86—Signal analysis
    • G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information

Abstract

The invention relates to a mass spectrometric detection method for vitamin D in peripheral trace blood, which comprises the steps of collecting a serum sample, preparing detection materials and chemicals, calibrating a standard, carrying out intravenous serum and trace serum 25-hydroxyvitamin D detection, carrying out data analysis, evaluating the analytical performance of an HPLC-MS/MS method, verifying the result of the intravenous and trace blood HPLC-MS/MS analysis method, and comparing the 25-hydroxyvitamin D level in the intravenous and trace blood and the 25-hydroxyvitamin D concentration result in the intravenous and trace blood sample. Has the advantages that: simultaneously collecting trace blood and macroblood of the same object, simultaneously detecting the levels of 25-hydroxyvitamin D in the macroblood and the trace blood by using a high performance liquid chromatography-tandem mass spectrometry method, and analyzing the correlation and the conversion coefficient of the 25-hydroxyvitamin D in the macroblood and the macroblood by using a linear regression analysis method, thereby obtaining a conversion formula; the concentration of the 25-hydroxyvitamin D in the constant blood and the trace blood can be converted into each other through a formula; provides reliable basis for replacing the constant blood with the trace blood to carry out VitD detection clinically.

Description

Mass spectrum detection method for vitamin D in peripheral trace blood
Technical Field
The invention relates to the technical field of peripheral trace blood vitamin D detection, in particular to a mass spectrometric detection method for peripheral trace blood vitamin D.
Background
Vitamin D is a fat-soluble vitamin, having the function of steroid hormones. Vitamin d (vitd) deficiency is a major public health problem facing the health of children.
The research shows that: the deficiency rate of VitD deficiency of the infants of 6-12 months old is as high as 35-40%. The diagnostic criteria for vitamin D deficiency are based on the concentration of 25-hydroxyvitamin D in serum. At present, the liquid chromatography-tandem mass spectrometry method is internationally accepted as the most reliable method for detecting 25-hydroxyvitamin D, has high sensitivity, specificity and accuracy, and can simultaneously determine the concentration of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in serum.
Clinically, the serum VitD needs to collect venous blood (constant blood), because the blood vessels of the children of low age, especially the neonates and the infants are thin, the venous blood is crying and screaming in the conventional venous blood collecting process, the compliance is poor, parents worry about the age of the children and are not willing to draw 2mL of venous blood at one time, and the conventional venous blood collection is difficult. Therefore, the blood sampling device adopts trace blood for detection, can relieve the pain of the children in the blood sampling process, and improves the compliance of patients and parents.
However, the micro blood collection is affected by factors such as the depth of a blood collection part, tissue fluid interference and the like, and has a difference from a detection result of a constant blood, so that a micro blood method cannot be used for detecting 25-hydroxyvitamin D at present.
The foregoing description is provided for general background information and is not admitted to be prior art.
Disclosure of Invention
The invention aims to provide a mass spectrometric detection method for vitamin D in peripheral trace blood, which establishes a new method for measuring 25-hydroxyvitamin D in finger trace blood by an HPLC-MS/MS method on the basis of measuring 25-hydroxyvitamin D in venous constant serum by the traditional HPLC-MS/MS method; the method is simple, convenient and quick, and has high sensitivity, less interference, and good accuracy and reproducibility; provides reliable basis for replacing the constant blood with the trace blood to carry out VitD detection clinically.
The invention provides a mass spectrometric detection method of peripheral trace blood vitamin D, which comprises the following steps:
(1) collecting a serum sample:
a venous blood sample is punctured into a test tube containing a coagulation activator through a vein; placing fingertip blood into an EP test tube, adding anticoagulant ethylenediamine tetraacetic acid (EDTA) into the EP test tube, separating serum, and storing at-20 deg.C in dark place;
(2) preparation of detection materials and chemicals:
25-hydroxyvitamin D2 (100. mu.g/mL, purity 98%), 25-hydroxyvitamin D3 (100. mu.g/mL, purity 98%), 25-hydroxyvitamin D2-D3 (internal standard of 25-hydroxyvitamin D2, 100. mu.g/mL, purity 98%), 25-hydroxyvitamin D3-D6 (internal standard of 25-hydroxyvitamin D3, 0.5mg, purity 95%), formic acid, standard substances (SRM 2972, SRM972 a), organic solvents n-hexane and methanol (HPLC grade), zinc sulfate (ZnSO 4);
(3) calibration standard:
preparing mixed calibration standards at methanol concentrations of 6.25/6.25, 12.5/12.5, 25/25, 50/50, 125/125, 250/250 and 500/500nmol/L (25-hydroxyvitamin D2/25-hydroxyvitamin D3), each batch of calibration standard being treated as a sample;
(4) the detection method of the 25-hydroxy vitamin D in the venous serum and the trace serum comprises the following steps:
the concentration of 25-hydroxyvitamin D in the serum is quantitatively measured by adopting a high performance liquid chromatography-tandem mass spectrometry method, and a detection instrument is a high performance liquid chromatography-tandem mass spectrometer;
(5) and (3) data analysis:
adopting an HPLC-MS/MS method to carry out 25-hydroxy vitamin D detection on the blood serum samples of venous blood and trace blood; carrying out statistical analysis on the mean deviation of the two methods; assessing the consistency between 25-hydroxyvitamin D states;
(6) evaluation of analytical Performance of HPLC-MS/MS method:
fully separating vitamin D metabolites from an internal standard by HPLC-MS/MS chromatograms of a venous serum sample and a peripheral trace blood sample by adopting an HPLC-MS/MS technology;
(7) the result of the vein and trace blood HPLC-MS/MS analysis method is verified;
(8) levels of 25-hydroxyvitamin D in the vein and trace blood:
comparing the change in the concentration of 25-hydroxyvitamin D in three venous blood groups and in the trace blood group, the amount of 25-hydroxyvitamin D was equal to the sum of the amounts of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2.
(9) Comparing the results of the concentration of 25-hydroxyvitamin D in the vein and trace blood samples:
the consistency of the results of the two samples is further tested by a Bland-Altman method, and conversion models of the two samples are obtained through statistics: log (corrected trace 25-hydroxyvitamin D) ═ 0.01049+1.06692 log (trace 25-hydroxyvitamin D), log (corrected trace 25-hydroxyvitamin D3) ═ 0.02864+1.05947 log (trace 25-hydroxyvitamin D3); converting the results of the trace blood 25(OH) D and 25(OH) D3 according to the formula;
(10) comparative analysis of corrected 25-hydroxyvitamin D and 25-hydroxyvitamin D3 results:
there was no difference between the venous blood 25-hydroxyvitamin D and corrected minim blood 25-hydroxyvitamin D mean values in the three groups, and the three groups of data were combined.
Further, the venous serum detection instrument in the step (4) is a high performance liquid chromatography tandem mass spectrometer with the model of Shimadzu 8040; the mass spectrometer setting conditions are as follows: the atomizer temperature was 350 ℃, the atomizing gas flow was 6L/min, the atomizing gas pressure was 35psi, and the DL tube temperature was 150 ℃.
Further, the trace serum detection instrument in the step (4) is a high performance liquid chromatography tandem mass spectrometer with the model of AB Sciex 4500 MD; the mass spectrometer setting conditions are as follows: the ion source temperature is 400 ℃, the spray gas pressure is 70psi, the gas curtain gas pressure is 20psi, and the ionization voltage is 4500V
The invention discloses a mass spectrum detection method of peripheral trace blood vitamin D, which comprises the steps of simultaneously collecting trace blood and macroblood of the same object, simultaneously detecting the levels of 25-hydroxyvitamin D in the macroblood and the macroblood by using a high performance liquid chromatography-tandem mass spectrometry method, and analyzing the correlation and the conversion coefficient of 25-hydroxyvitamin D in the macroblood and the macroblood by using a linear regression analysis method, thereby obtaining a log (corrected trace 25-hydroxyvitamin D) of 0.01049+1.06692 log (trace 25-hydroxyvitamin D), and a log (corrected trace 25-hydroxyvitamin D3) of 0.02864+1.05947 log (trace 25-hydroxyvitamin D3) formula; thus the concentration of the 25-hydroxy vitamin D in the constant blood and the trace blood can be converted into each other by a formula; provides reliable basis for replacing the constant blood with the trace blood to carry out VitD detection clinically.
Drawings
FIG. 1 is an HPLC-MS/MS chromatogram of an intravenous serum sample according to an embodiment of the present invention.
FIG. 2 is an HPLC-MS/MS chromatogram of a peripheral blood sample in a trace amount according to an embodiment of the present invention.
FIG. 3 is a scatter plot of the results of the Bland-Altman consistency test for two samples of 25-hydroxyvitamin D and 25-hydroxyvitamin D3 according to embodiments of the present invention.
FIG. 4 is a graph showing the correlation between intravenous 25-hydroxyvitamin D/25-hydroxyvitamin D3 and corrected trace blood 25-hydroxyvitamin D/25-hydroxyvitamin D3 in accordance with an embodiment of the present invention.
FIG. 5 is a ROC curve analysis plot of intravenous 25-hydroxyvitamin D/25-hydroxyvitamin D3 concentration versus corrected microvessel 25-hydroxyvitamin D/25-hydroxyvitamin D3 for the examples of the present invention.
Detailed Description
The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
The terms first, second, third, fourth and the like in the description and in the claims of the present invention are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order.
The invention aims to provide a mass spectrometric detection method for vitamin D in peripheral trace blood, which establishes a new method for measuring 25(OH) D in finger trace blood by an HPLC-MS/MS method on the basis of measuring 25(OH) D in venous constant serum by the traditional HPLC-MS/MS method; the method is simple, convenient and quick, and has high sensitivity, less interference, and good accuracy and reproducibility; provides reliable basis for replacing the constant blood with the trace blood to carry out VitD detection clinically.
Collecting a serum sample:
a venous blood sample is punctured into a test tube containing a coagulation activator through a vein; the trace blood collected from the finger is squeezed into an EP tube containing the anticoagulant ethylenediaminetetraacetic acid (EDTA); the serum was separated and stored at-20 ℃ in the dark.
Detection materials and chemicals:
25(OH) D2 (100. mu.g/mL, purity 98%), 25(OH) D3 (100. mu.g/mL, purity 98%), 25(OH) D2-D3 (internal standard of 25(OH) D2, 100. mu.g/mL, purity 98%), 25(OH) D3-D6 (internal standard of 25(OH) D3, 0.5mg, purity 95%) and formic acid. Standard substances SRM2972 and SRM972 a; organic solvents of n-hexane, methanol (high performance liquid chromatography grade) and zinc sulfate (ZnSO 4).
Calibration standard:
preparing mixed calibration standards at methanol concentrations of 6.25/6.25, 12.5/12.5, 25/25, 50/50, 125/125, 250/250 and 500/500nmol/L (25(OH) D2/25(OH) D3); each batch of calibrant was treated as a sample.
Intravenous serum 25- (OH) D detection method:
the concentration of 25- (OH) D in serum is quantitatively determined by adopting a high performance liquid chromatography-tandem mass spectrometry method, and a detection instrument is a high performance liquid chromatography-tandem mass spectrometer (model: Shimadzu 8040). Collecting venous serum, and extracting effective components from 100uL sample after pretreatment such as protein removal, extraction, purification and the like. The mass spectrometer was equipped with an ESI source using positive ion scanning mode, and mass spectrum MRM ion monitoring method was used to select precursor ions (m/z401.3 for 25OHD3, m/z 413.3 for 25OHD2, and m/z 407.3for 25(OH) D3-D6, m/z 416.3for 25(OH) D2-D3) to further generate specific product ions for data analysis. As shown in the following table (parameter table for venous blood reaction monitoring acquisition based on HPLC-MS/MS method):
Figure BDA0002631895190000051
Figure BDA0002631895190000061
the mass spectrometer setting conditions are as follows: the atomizer temperature was 350 ℃, the atomizing gas flow was 6L/min, the atomizing gas pressure was 35psi, and the DL tube temperature was 150 ℃.
Making a standard curve equation according to the data of the standard substance detected by the instrument, and obtaining the relative standard deviation<15 percent. Calculating the contents of the quality control sample and the samples to be detected, namely 25- (OH) D3 and 25- (OH) D2 according to a standard curve equation, and calculating the quality control range
Figure BDA0002631895190000063
And judging whether the quality control result is qualified by the multi-rule quality control method, wherein each batch of detection samples are subjected to at least double quality control. 25- (OH) D3 within and between batches<5%, 25- (OH) D2 within and between batches<5％。
The detection method of trace blood 25- (OH) D comprises the following steps:
the concentration of 25- (OH) D in trace blood is quantitatively determined by high performance liquid chromatography tandem mass spectrometry, and the detection instrument is a high performance liquid chromatography tandem mass spectrometer (model: AB Sciex 4500 MD). Collecting micro finger blood, and removing protein and impurities from 20uL of the sample to extract effective components. The mass spectrometer was equipped with an ESI source using positive ion scanning mode, and mass spectrum MRM ion monitoring method was used to select precursor ions (m/z401.3 for 25OHD3, m/z 413.3 for 25OHD2, and m/z 407.3for 25(OH) D3-D6, m/z 416.3for 25(OH) D2-D3) to further generate specific product ions for data analysis. The following table (parameter table for trace blood reaction monitoring collection based on HPLC-MS/MS method) shows:
Figure BDA0002631895190000062
the mass spectrometer setting conditions are as follows: the ion source temperature was 400 ℃, the spray gas pressure was 70psi, the gas curtain pressure was 20psi, and the ionization voltage was 4500V.
Making a standard curve equation according to the data of the standard substance detected by the instrument, and obtaining the relative standard deviation<15 percent. Calculating the contents of the quality control sample and the samples to be detected, namely 25- (OH) D3 and 25- (OH) D2 according to a standard curve equation, and calculating the quality control range
Figure BDA0002631895190000071
Judging whether the quality control result is qualified by the multi-rule quality control method, and performing at least dual-quality control on each batch of detection samples. 25- (OH) D3 within and between batches<10%, 25- (OH) D2 within and between batches<10％。
And (3) data analysis:
the serum sample is subjected to 25(OH) D detection by two methods of HPLC-MS/MS. The mean deviation of both methods was statistically analyzed by Bland-Altman. Cohen's kappa was used to evaluate the consistency between 25(OH) D states (consistency: <0.4, poor; 0.4-0.75, moderate to good; greater than 0.75, good). P <0.05 was statistically significant. All data analyses applied empower (r) and GraphPad statistical software.
Evaluation of analytical Performance of HPLC-MS/MS method:
FIG. 1 shows an HPLC-MS/MS chromatogram of an intravenous serum sample, using HPLC-MS/MS techniques, with full separation of vitamin D metabolites from the internal standard, with 25(OH) D3 eluting at 1.50min and the internal standard at 1.45 min; while 25(OH) D2 eluted at 1.60min, which is indicated by a 1.61min bar. Potential interference 3-epi-25(OH) D elutes at 1.00-1.25min or 2.00min, and detection of 25(OH) D2 and 25(OH) D3 did not produce interference.
FIG. 2 shows an HPLC-MS/MS chromatogram of a peripheral trace blood sample, wherein vitamin D metabolites and internal standards are sufficiently separated by HPLC-MS/MS technique, 25(OH) D3 is eluted at 1.40min, and the internal standards are eluted at 1.40 min; while 25(OH) D2 eluted at 1.50min, its internal standard also eluted at 1.50 min. Potential interference 3-epi-25(OH) D elutes at 1.00-1.25min or 2.00min, and detection of 25(OH) D2 and 25(OH) D3 did not produce interference.
The result of the vein and trace blood HPLC-MS/MS analysis method is verified:
for the venous serum samples, the limit of detection (LOD) of 25(OH) D3 is 0.01ng/ml, and the limit of detection of 25(OH) D2 is 0.05 ng/ml; the limit of quantitation (LOQ) of 25(OH) D3 is 1.5ng/mL, the LOQ of 25(OH) D2 is 1.5ng/mL, and when 25(OH) D2 is in the range of 0.8ng/mL to 50.0ng/mL, the linearity is good, and the correlation coefficient R2> 0.99. When 25(OH) D3 is in the range of 2.5 ng/mL-160.0 ng/mL, linearity is good, and the correlation coefficient R2 is greater than 0.99. In the assay of 25(OH) D3, three concentrations, 10ng/mL, 40ng/mL and 160ng/mL, were set as quality controls. In the determination of 25(OH) D2, three concentrations of 5, 10 and 40ng/mL were used for quality control. The accuracy recovery (%) of the two compounds is within a reasonable range of 85-115%, and the accuracy is 3.87% and 4.91% respectively. The following table (validation results table for intravenous serum sample HPLC-MS/MS detection method) shows:
Figure BDA0002631895190000081
for a trace blood sample, the LOD of 25(OH) D3 is 0.01ng/ml, and the detection limit of 25(OH) D2 is 0.05 ng/ml; LOQ of 25(OH) D3 is 5ng/mL, quantitation limit of 25(OH) D2 is 0.8ng/mL, linearity is good when 25(OH) D2 is in the range of 0.10ng/mL to 12.50ng/mL, correlation coefficient R2> 0.99. When 25(OH) D3 is in the range of 0.31ng/mL to 40.0ng/mL, linearity is good, and the correlation coefficient R2 is greater than 0.99. In the determination of 25(OH) D3, three concentrations of 1.25ng/mL, 5.00ng/mL and 20.00ng/mL were set as quality controls. In the determination of 25(OH) D2, three concentrations of 0.39, 1.56 and 6.25ng/mL were used for quality control. The accuracy recovery (%) of the two compounds is in a reasonable range of 85-115%, and the accuracy is 1.65% and 45.32% respectively. The following table (validation results table for the trace serum sample HPLC-MS/MS detection method) shows:
Figure BDA0002631895190000082
Figure BDA0002631895190000091
venous and trace blood 25(OH) D levels:
three groups of venous blood and minute blood were compared for changes in 25(OH) D concentration. The amount of 25(OH) D is equal to the sum of the amounts of 25(OH) D3 and 25(OH) D2. The mean values of 25(OH) D and 25(OH) D3 in venous and capillary blood differed between the three groups. The following table (results and precision comparison table for venous and microvessel 25(OH) D/25(OH) D3):
Figure BDA0002631895190000092
since 92% of the samples contained less than 0.1ng/mL of 25(OH) D2, we did not include this analyte in the subsequent statistical analysis.
Comparison of the results of 25(OH) D concentration in venous and microvolume blood samples:
the consistency of the results from the two samples was further tested by the Bland-Altman method (see fig. 3), with the 95% consistency limit (-4.36, 12.75) for the 25(OH) D concentration of the two samples, with a bias of 4.196 for the results and a bias of SD of 4.365, indicating better consistency of the results from the two samples. Likewise, the 95% consistency limit for the 25(OH) D3 concentration therein was (-4.01, 12.40). Further statistics results in two transformation models for the samples: log (corrected trace 25(OH) D) ═ 0.01049+1.06692 log (trace 25(OH) D), log (corrected trace 25(OH) D3) ═ 0.02864+1.05947 log (trace 25(OH) D3). The results of trace blood 25(OH) D and 25(OH) D3 were transformed according to the formula.
Comparison of corrected 25(OH) D and 25(OH) D3 results:
there were no differences between groups in venous 25(OH) D versus corrected miniblood 25(OH) D mean in the three groups, as shown in the following table (venous 25(OH) D/25(OH) D3 versus corrected capillary 25(OH) D/25(OH) D3 results):
Figure BDA0002631895190000101
three sets of data are combined. Further analysis of the correlation of vein 25(OH) D and 25(OH) D3 with corrected microvessel 25(OH) D and 25(OH) D3 was 0.7941 and 0.8103, respectively (see fig. 4), and ROC curve statistics analyzed the AUC for the two 25(OH) D and 25(OH) D3 was 0.9367 and 0.9565, respectively (see fig. 5). The analysis of the results proves that the HPLC-MS/MS has good accuracy and sensitivity in detecting the trace blood 25(OH) D.
Based on the above description, the present invention has the following advantages:
1. simultaneously collecting trace blood and macroblood of the same object, simultaneously detecting the levels of the macroblood and the macroblood 25(OH) D by using a high performance liquid chromatography-tandem mass spectrometry method, and analyzing the correlation and the conversion coefficient of the macroblood and the macroblood 25(OH) D by using a linear regression analysis method, thereby obtaining a log (corrected trace 25(OH) D) is 0.01049+1.06692 log (trace 25(OH) D), and log (corrected trace 25(OH) D3) is 0.02864+1.05947 log (trace 25(OH) D3); thus the constant blood and trace blood 25(OH) D concentrations can be converted to each other by a formula; provides reliable basis for replacing the constant blood with the trace blood to carry out VitD detection clinically.
2. The method establishes a novel method for measuring 25(OH) D in finger trace blood by using an HPLC-MS/MS method on the basis of measuring 25(OH) D in vein constant serum by using HPLC-MS/MS in the prior art, and the method is simple, convenient, rapid, high in sensitivity, less in interference and good in accuracy and reproducibility.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

Claims (3)

1. A mass spectrometric detection method for peripheral trace blood vitamin D is characterized by comprising the following steps:
(1) collecting a serum sample:
a venous blood sample is punctured into a test tube containing a coagulation activator through a vein; placing fingertip blood into an EP test tube, adding anticoagulant ethylenediamine tetraacetic acid (EDTA) into the EP test tube, separating serum, and storing at-20 deg.C in dark place;
(2) preparation of detection materials and chemicals:
25-hydroxyvitamin D2 (100. mu.g/mL, purity 98%), 25-hydroxyvitamin D3 (100. mu.g/mL, purity 98%), 25-hydroxyvitamin D2-D3 (internal standard of 25-hydroxyvitamin D2, 100. mu.g/mL, purity 98%), 25-hydroxyvitamin D3-D6 (internal standard of 25-hydroxyvitamin D3, 0.5mg, purity 95%), formic acid, standard substances (SRM 2972, SRM972 a), organic solvents n-hexane and methanol (HPLC grade), zinc sulfate (ZnSO 4);
(3) calibration standard:
preparing mixed calibration standards at methanol concentrations of 6.25/6.25, 12.5/12.5, 25/25, 50/50, 125/125, 250/250 and 500/500nmol/L (25-hydroxyvitamin D2/25-hydroxyvitamin D3), each batch of calibration standard being treated as a sample;
(4) the detection method of the 25-hydroxy vitamin D in the venous serum and the trace serum comprises the following steps:
the concentration of 25-hydroxyvitamin D in the serum is quantitatively measured by adopting a high performance liquid chromatography-tandem mass spectrometry method, and a detection instrument is a high performance liquid chromatography-tandem mass spectrometer;
(5) and (3) data analysis:
adopting an HPLC-MS/MS method to carry out 25-hydroxy vitamin D detection on the blood serum samples of venous blood and trace blood; carrying out statistical analysis on the mean deviation of the two methods; assessing the consistency between 25-hydroxyvitamin D states;
(6) evaluation of analytical Performance of HPLC-MS/MS method:
fully separating vitamin D metabolites from an internal standard by HPLC-MS/MS chromatograms of a venous serum sample and a peripheral trace blood sample by adopting an HPLC-MS/MS technology;
(7) the result of the vein and trace blood HPLC-MS/MS analysis method is verified;
(8) levels of 25-hydroxyvitamin D in the vein and trace blood:
comparing the change in the concentration of 25-hydroxyvitamin D in three venous blood groups and in the trace blood group, the amount of 25-hydroxyvitamin D was equal to the sum of the amounts of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2.
(9) Comparing the results of the concentration of 25-hydroxyvitamin D in the vein and trace blood samples:
and further checking the consistency of the results of the two samples, and counting to obtain conversion models of the two samples: log (corrected trace 25-hydroxyvitamin D) ═ 0.01049+1.06692 log (trace 25-hydroxyvitamin D), log (corrected trace 25-hydroxyvitamin D3) ═ 0.02864+1.05947 log (trace 25-hydroxyvitamin D3); converting the results of the trace blood 25(OH) D and 25(OH) D3 according to the formula;
(10) comparative analysis of corrected 25-hydroxyvitamin D and 25-hydroxyvitamin D3 results:
there was no difference between the venous blood 25-hydroxyvitamin D and corrected minim blood 25-hydroxyvitamin D mean values in the three groups, and the three groups of data were combined.
2. The method for detecting vitamin D in peripheral trace blood by mass spectrometry as claimed in claim 1, wherein the venous serum detection instrument in step (4) is a high performance liquid chromatography tandem mass spectrometer of type Shimadzu 8040; the mass spectrometer setting conditions are as follows: the atomizer temperature was 350 ℃, the atomizing gas flow was 6L/min, the atomizing gas pressure was 35psi, and the DL tube temperature was 150 ℃.
3. The method for detecting peripheral trace blood vitamin D mass spectrum according to claim 1, wherein the trace serum detection instrument in the step (4) is a high performance liquid chromatography tandem mass spectrometer with the model AB Sciex 4500 MD; the mass spectrometer setting conditions are as follows: the ion source temperature was 400 ℃, the spray gas pressure was 70psi, the gas curtain pressure was 20psi, and the ionization voltage was 4500V.
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CN113866255A (en) * 2021-10-01 2021-12-31 北京毅新博创生物科技有限公司 Inductively coupled plasma mass spectrometry detection of 10 elements in peripheral blood
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Application publication date: 20201124