CN116381096A - Method for detecting concentration of active metabolite thiamine diphosphate of vitamin B1 in whole blood - Google Patents

Method for detecting concentration of active metabolite thiamine diphosphate of vitamin B1 in whole blood Download PDF

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CN116381096A
CN116381096A CN202310407993.3A CN202310407993A CN116381096A CN 116381096 A CN116381096 A CN 116381096A CN 202310407993 A CN202310407993 A CN 202310407993A CN 116381096 A CN116381096 A CN 116381096A
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赵福荣
王琳
李莹
王璐
崔颖
蒋玉新
关丽萍
曹云峰
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Dalian Boyuan Medical Technology Co ltd
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Abstract

The invention provides a method for detecting the concentration of vitamin B1 (VB 1) in whole blood, namely Thiamine Diphosphate (TDP), an active metabolite. The method is characterized in that protein precipitation pretreatment is carried out, a liquid chromatography tandem mass spectrometry (LC-MS/MS) is used for analyzing a target object, and a standard curve is established through an internal standard method to calculate the TDP concentration of VB1 in whole blood, so that a method for more comprehensively detecting the TDP concentration of active metabolite of VB1 in whole blood is realized. The method has the advantages of high sensitivity, high flux, simplicity, convenience and high extraction recovery rate, meets the quantitative requirements of various mass spectrometry equipment, and can be applied to clinical detection of the TDP concentration.

Description

Method for detecting concentration of active metabolite thiamine diphosphate of vitamin B1 in whole blood
Technical Field
The invention belongs to the technical field of inspection and analysis, and particularly relates to a method for detecting concentration of active metabolite Thiamine Diphosphate (TDP) of vitamin B1 (VB 1) in whole blood.
Background
Vitamin B1 (Vitamin B1, thiamin) is also known as thiamine and may also be referred to as anti-neuritis. Belongs to the water-soluble vitamins and is utilized in the form of salt. Mainly in 4 forms, respectively: free thiamine, thiamine Monophosphate (TMP), thiamine Diphosphate (TDP) and Thiamine Triphosphate (TTP). LC/MS-MS analysis of TDP in whole blood is the most sensitive, specific and accurate method to determine thiamine nutritional status and is a reliable indicator of whole body reserves. Serum or plasma thiamine detection has poor sensitivity and specificity, and the thiamine content in the plasma is less than 10%. Therefore, there is a need for developing a method for detecting the concentration of TDP in whole blood of vitamin B1 to evaluate the concentration of vitamin B1 in vivo.
Chinese patent (application No. 202010172197.2) in 2020 discloses a method for detecting a kit of 9 water-soluble vitamins in serum by using an ultra-high performance liquid chromatography tandem mass spectrometry technology, chinese patent (application No. 201911234804.7) in 2019, a method for rapidly detecting vitamin B1 and vitamin C concentrations based on a liquid chromatography tandem mass spectrometry technology is disclosed, both of which detect the concentration of thiamine in serum instead of thiamine diphosphate, which is an active metabolite in vivo, and the concentration of plasma or serum thiamine reflects the latest intake instead of body storage; whole blood is the preferred sample for thiamine evaluation.
Disclosure of Invention
The invention aims to provide a method for detecting the concentration of TDP in whole blood. The method comprises the steps of carrying out protein precipitation pretreatment, adding a pH regulator to obtain a sample injection solution, and analyzing a target object by using liquid chromatography tandem mass spectrometry (LC-MS/MS), wherein the liquid analysis time required by a single sample is only 1.5min, so that the detection flux is greatly increased; the pretreatment is simple, the operation is easy, the extraction recovery rate is high, the sensitivity is high, no obvious matrix effect exists, the quantitative requirements of mass spectrum detection equipment of multiple models are met, and the method can be applied to clinical detection work.
The method for detecting the TDP concentration in whole blood provided by the invention comprises the following steps:
carrying out pretreatment on a whole blood sample containing TDP to be detected to obtain a target sample solution;
detecting the TDP of the target sample solution, and calculating the concentration of the TDP in the whole blood sample.
Wherein, the operation of preprocessing the whole blood sample containing TDP to obtain the target sample solution is as follows:
collecting a whole blood sample containing TDP to be detected, adding an internal standard solution after freeze thawing cycle, adding a precipitant, vortex shaking, vortex mixing uniformly, centrifuging, taking supernatant, adding a pH regulator, and mixing uniformly to obtain a target sample solution.
The step of testing the TDP of the target sample solution and calculating the concentration of TDP in the whole blood sample includes:
preparing standard curve sample solutions containing different TDPs with known concentrations, and drawing a standard curve of the standard curve sample solutions;
testing the target sample solution by using high performance liquid chromatography-tandem mass spectrometry, and obtaining the TDP content in the target sample solution according to the standard curve, thereby obtaining the TDP content in the whole blood sample containing the TDP to be tested;
the step of configuring standard curve sample solutions containing different known concentrations of TDP and drawing a standard curve of the standard curve sample solutions includes:
firstly, respectively preparing standard curve samples containing different TDPs with known concentrations, adding an internal standard solution, carrying out vortex vibration, adding a precipitator, carrying out vortex mixing, centrifuging, taking supernatant, adding a pH regulator, and carrying out uniform mixing to obtain a series of standard curve sample solutions containing different TDPs with known concentrations;
and secondly, testing the standard curve sample solution by using high performance liquid chromatography-tandem mass spectrometry, calculating the area ratio of TDP and internal standard peak, and establishing a standard curve by an internal standard method.
In the method, the internal standard solution is TDP-d3 solution, and the concentration of the internal standard solution is 0.8-1.6nmol/L;
the volume ratio of the whole blood sample or standard curve sample containing TDP to be measured to the internal standard solution is 10:1, a step of;
aqueous ammonia solution with the concentration of 10% -40% of the pH regulator;
the volume ratio of the whole blood sample or standard curve sample containing TDP to be measured to the pH regulator is 8:1-9:1
The volume ratio of the whole blood sample or standard curve sample containing TDP to be measured to the precipitation solution is 1:0.6-1:3, a step of;
the precipitation solution is trichloroacetic acid, perchloric acid and metaphosphoric acid solution;
the concentration of the precipitation solution is 3% -10%;
adding the pH regulator and the precipitation solution, and then carrying out vortex mixing for 5min at 2000 rpm;
the centrifugation conditions may be 15000g,4℃and 5 minutes;
the diluent can be specifically an aqueous solution with pH of 2-4;
the blank matrix of the TDP standard curve is a mixed aqueous solution of 0.01M PBS and Bovine Serum Albumin (BSA);
the concentration of Bovine Serum Albumin (BSA) is 4% -5%;
the liquid chromatography conditions were: chromatographic column: waters, atlantis Premier BEH C182.5 μm 2.1X100 mm; column temperature 40 ℃; the temperature of the sample injector is 4 ℃; mobile phase a was 0.05% aqueous ammonia (containing 1mmol/L ammonium formate), B was acetonitrile, isocratic elution: 0-1.5min, mobile phase B5%; the flow rate is 0.3mL/min; the sample injection amount was 3. Mu.L.
The mass spectrum parameters are: esi+; air curtain gas (CUR): 30psi; the method comprises the steps of carrying out a first treatment on the surface of the Spray voltage (IS) 5500V; temperature (TEM) 400 ℃; atomizing Gas (Gas 1) at 50psi; auxiliary heating Gas (Gas 2): 50psi; multiple reaction detection (MRM);
TABLE 1TDP specific Mass Spectrometry parameters
Figure BDA0004182182750000031
* To quantify ions
The TDP quantitative range of the standard curve is as follows: 10nmol/L to 1000nmol/L.
The invention has the beneficial effects that:
(1) The invention starts from the clinical practical requirement and adopts a pretreatment mode of protein precipitation. The method is stronger in operability, meets clinical requirements, establishes a method for simultaneously qualitatively and quantitatively, is favorable for unifying detection standards, has better sensitivity, has a lower limit of 10nmol/L, and can be suitable for detection requirements of various mass spectrometry equipment; the clinical application and scientific research cost is greatly reduced;
(2) The liquid analysis time is only 1.5min, so that baseline separation can be achieved, the interference of mixed peaks is avoided, and the detection flux of a clinical laboratory is greatly increased.
The detection method meets the verification requirement, and the stability of the standard yeast solution is inspected, so that the detection method meets the clinical requirement.
According to the invention, from the clinical practical requirement, protein precipitation and pH regulator pretreatment are adopted, the LC-MS/MS analysis time is 1.5min, and the detection flux is greatly increased; meanwhile, the method meets the method requirements of '2020 edition of Chinese pharmacopoeia-9012 biological sample quantitative analysis method verification guiding principle' and 'liquid chromatography-mass spectrum clinical application suggestion (guide and consensus' (Chinese journal of inspection medical science, 2017,40 (10): 770-779)), has high extraction recovery rate and high sensitivity, meets the quantitative requirements of mass spectrum detection equipment of multiple types, and can be applied to clinical practical detection work.
Drawings
FIG. 1 is a chemical formula of TDP;
FIG. 2 is a chromatogram of TDP and its internal standard in whole blood;
FIG. 3 is a chromatogram of Condition 2 TDP;
FIG. 4 is a chromatogram of Condition 3 TDP;
FIG. 5 is a chromatogram of Condition 4 TDP;
FIG. 6 is a chromatogram of Condition 5 TDP;
FIG. 7 is a chromatogram of Condition 6 TDP;
FIG. 8 is a chromatogram of Condition 6 TDP;
Detailed Description
The preferred embodiments of the present invention will be described below with reference to the accompanying drawings, it being understood that the preferred embodiments described herein are for illustration and explanation of the present invention only, and are not intended to limit the present invention.
Example 1 establishment and verification of method for detecting TDP in Whole blood
1. Materials and reagents
Cocarboxylase (TDP) (source leaf organism, purity not less than 97%), thiamine diphosphate-d 3 (TDP-d 3) (TRC, purity not less than 95%); 10×0.01m pbs (Solarbio), trichloroacetic acid (analytical grade, komiou), acetonitrile (chromatographic purity, 99.9%, fishr), water (ultrapure water), hydrochloric acid (komiou), ammonia (HPLC grade, sigma), bovine Serum Albumin (BSA) (Solarbio), ammonium formate (Sigma).
2. Apparatus and device
LCMS-4500MD liquid chromatograph-tandem mass spectrometer with triple quadrupole tandem mass spectrometer as detector; one ten thousandth of electronic analytical balance; a MiliQ pure water meter; a vortex mixer; a low temperature high speed centrifuge; medical low-temperature preservation box.
3. Preparing a standard curve and a quality control basic working solution:
preparing various series of standard curves and quality control working solutions according to requirements, and uniformly mixing by vortex to prepare standard curves and quality control samples with different concentrations. Finally, standard curve concentrations and quality control concentrations are shown in table 2 below:
TABLE 2 final concentration and formulation of standard curve and quality control whole blood samples
TDP Concentration (mu mol/L) Formulation source Stock solution (mu L) 0.01MHCL(μL)
WS A 0.10 WS E 20 300
WS B 0.20 WS E 20 140
WS C 0.40 WS E 60 180
WS D 0.80 WS E 60 60
WS E 1.60 WS I 64 336
WS F 3.20 WS I 64 136
WS G 4.00 WS I 60 90
WS H 5.00 WS I 60 60
WS I 10.00 TDP：250μmol/L 30 720
WS QC LLOQ 0.10 WS QC M 30 390
WS QC L 0.25 WS QC M 75 345
WS QC M 1.40 WS QC H 84 396
WS QC H 8.00 TDP：250μmol/L 24 726
4. Standard curve, quality control whole blood sample pretreatment process:
standard curve, quality control whole blood sample preparation, 180 μl of blank matrix (0.01 m pbs+4% bsa) was taken, 20 μl of working fluid was added, and vortexed for use.
The whole blood sample preparation comprises the steps of taking 200 mu L of sample, placing the sample into a medical low-temperature preservation box for more than or equal to 15min, and then swirling the sample at room temperature for 5-10min, and performing vortex oscillation for later use.
The precipitation process comprises the steps of taking a standard curve or whole blood sample, adding 20 mu L of internal standard working solution, carrying out vortex oscillation at 2000rpm for 5min, adding 200 mu L of precipitant, carrying out vortex oscillation at 2000rpm 5min,15000g,4 ℃, centrifuging for 5min, taking 60 mu L of supernatant solution, adding 25 mu L of pH regulator, carrying out vortex oscillation at 2000rpm for 30s, and taking 60 mu L of supernatant for sample injection analysis.
5. Detection of TDP: the following chromatographic and mass spectrometric conditions were used in this experiment:
the liquid chromatography conditions were: chromatographic column: waters, atlantis Premier BEH C, 2.5 μm 2.1X100 mm; column temperature 40 ℃; the temperature of the sample injector is 4 ℃; mobile phase a was 0.05% aqueous ammonia (containing 1mmol/L ammonium formate), B was acetonitrile, isocratic elution: 0-1.5min, mobile phase B5%; the flow rate is 0.3mL/min; the sample injection amount was 3. Mu.L.
The mass spectrum parameters are: esi+; air curtain gas (CUR): 30psi; the method comprises the steps of carrying out a first treatment on the surface of the Spray voltage (IS) 5500V; temperature (TEM) 400 ℃; atomizing Gas (Gas 1) at 50psi; auxiliary heating Gas (Gas 2): 50psi; multiple reaction detection (MRM) (see table 1);
6. methodology evaluation
6.1 the invention is verified according to the '2020 edition of Chinese pharmacopoeia-9012 biological sample quantitative analysis method verification guiding principle', liquid chromatography-mass spectrometry clinical application suggestion (guidance and consensus) '(Chinese journal of inspection, 2017,40 (10): 770-779) and CNAS-GL037 clinical chemistry quantitative inspection program performance verification guidance': including specificity and selection, linear range, sensitivity, precision, accuracy (labeled recovery), matrix effects, and stability.
(1) Specificity and Selectivity investigation
After the double blank, single blank and LLOQ samples are processed according to the items of 4, standard curve and quality control whole blood sample pretreatment process, LC-MS/MS analysis is carried out, and the results show that under the experimental conditions, each object to be detected and the internal standard peak shape are good, and the specificity is higher. The chromatographic peak has no interference of impurity peak at both sides, and meets the requirement that the area of no background peak or interference peak is less than 20% of the area of the lower limit peak of the analyte quantification and less than 5% of the area of the internal standard peak.
TABLE 3TDP Selectivity and specificity
Figure BDA0004182182750000071
(2) Linear range
After the standard curve whole blood sample is processed according to the items of 4, standard curve and quality control whole blood sample pretreatment process, LC-MS/MS analysis is carried out, the weight linear regression is carried out on the drug concentration X according to the ratio Y (Y=As/Ai) of the peak area As of each drug to the peak area Ai of the internal standard, the standard curve is fitted, and the weight is 1/X 2 (X is the concentration value), standard curve expressed as y=slope x+interceptSee table 4.
TABLE 4 Standard Curve and Linear Range
Figure BDA0004182182750000072
(3) Precision and accuracy
After the quality control samples are processed according to the items of 4, standard curve and quality control whole blood sample pretreatment process, LC-MS/MS analysis is carried out, 6 parts of each concentration are carried out in parallel in each batch, three days are continuously carried out, and one batch is carried out every day. The ratio Y of the drug peak area As to the internal standard peak area Ai (y=as/Ai) was substituted into the current day standard curve to calculate the sample concentration, thereby calculating the precision (CV <15% qualified) and accuracy (85% -115% qualified) between the batches, and the respective drug precision and accuracy data are shown in table 5.
TABLE 5 precision and accuracy
Figure BDA0004182182750000073
(4) Matrix effect
Preparing a pure solution sample: a1.5 mL EP tube was taken, 40. Mu.L of WS QC solution was added, 360. Mu.L of 0.01M HCL solution was added, and vortexed for 30 seconds. And (3) performing liquid chromatography-tandem mass spectrometry analysis on the supernatant according to the process operation of the pretreatment process of the whole blood sample with the quality control of 4, the standard curve. 6 parts were made in parallel. Calculating the average value y of the ratio of the object to be detected to the internal standard response value in the pure solution sample s 。
Whole blood sample preparation: a1.5 mL EP tube was used, and 200. Mu.L of 6 different human whole blood was taken. And (3) performing liquid chromatography-tandem mass spectrometry analysis on the supernatant according to the process operation of the pretreatment process of the whole blood sample with the quality control of 4, the standard curve. Calculating the average value y of the ratio of the object to be detected to the internal standard response value in the whole blood sample x 。
Whole blood: pure solution mixture (3:7, v/v): a1.5 mL EP tube was taken, 60. Mu.L and 140. Mu.L of each of the above two samples were taken, and vortexed for 30 seconds. Operating according to the sample pretreatment process, collecting supernatant, and performing liquid chromatography-tandem mass spectrometryAnd (5) analyzing. Calculating the average value 7:3y of the ratio of the to-be-detected object to the internal standard response value in the mixture sample s+x 。
Whole blood: pure solution mixture (7:3, v/v): a1.5 mL EP tube was taken, 140. Mu.L and 60. Mu.L of each of the two samples were taken, and vortexed for 30 seconds. The process is operated according to the sample pretreatment process, and the supernatant is taken for liquid chromatography-tandem mass spectrometry analysis. Calculating the average value of the ratio of the to-be-detected object to the internal standard response value in the mixture sample to 3:7y s+x 。
Corresponding each group of y x ，y s Y s+x Substitution formula: deviation (%) =1- (y) s+x )/(y x *0.3+y s *0.7 100% x, deviation (%) =1- (y) s+x )/(y x *0.7+y s *0.3 Calculating the relative matrix effect deviation of the object to be detected by the X100 percent. The results are shown in Table 6:
TABLE 6 matrix Effect
Figure BDA0004182182750000081
(5) Sample stability investigation
Adopting QC whole blood samples with low concentration and high concentration, wherein each concentration is 4 parallel samples, substituting the samples into the standard curve along with the current day to calculate each drug concentration after analysis, and respectively inspecting the stability of the samples at room temperature for 18 hours, at 2-8 ℃ for 18 hours, for three times of freeze thawing and circulation, at-20 ℃ for 15 days, at-70 ℃ for 15 days and at 4 ℃ for 48 hours by an automatic sampler after sample treatment, wherein the results are shown in Table 7;
TABLE 7 stability
Figure BDA0004182182750000091
(6) Investigation of working fluid stability
Working fluid to be inspected: and respectively storing the working solution WS A and WS I in a dark place at the room temperature for 18 hours and at the temperature of minus 70 ℃ for 20 days after subpackaging. On the day of investigation, the working solutions are placed to room temperature, 10 mu L of the working solution is taken respectively, 10 mu L of the internal standard working solution is added, 90 mu L of the diluent is added, vortex mixing is carried out for 30s, 100 mu L of the precipitant is added, vortex mixing is carried out for 5min, 60 mu L of the supernatant is taken, 25 mu L of the pH regulator is added, vortex is carried out for 30s, and sample injection analysis is carried out on the supernatant. 6 parts per concentration were prepared in parallel.
And (3) comparing the basic working solution: working fluids WS a and WS I were freshly prepared as control solutions. Respectively taking 10 mu L of working solution, adding 10 mu L of internal standard working solution, adding 90 mu L of diluent, mixing for 30 seconds by vortex, adding 100 mu L of precipitant, mixing for 5 minutes by vortex, taking 60 mu L of supernatant, adding 25 mu L of pH regulator, mixing for 30 seconds by vortex, and taking supernatant for sample injection analysis. 6 parts per concentration were prepared in parallel.
The ratio y of the peak area As of the object to be measured to the internal standard peak area Ai is calculated (y=as/Ai). And (5) performing t-test on the ratio obtained by the two groups of calculation, and calculating the P value. The calculation results are shown in Table 8.
TABLE 8 working fluid stability
Figure BDA0004182182750000101
The requirements of the quantitative analysis method verification guidelines of biological samples in the Chinese pharmacopoeia of 2020 edition, the-9012 biological samples, the liquid chromatography-mass spectrum clinical application suggestions (guidelines and consensus) of China journal of inspection medicine, 2017,40 (10): 770-779) and the clinical chemistry quantitative inspection program performance verification guidelines of CNAS-GL037 are met, and the quantitative analysis method can be applied to detection of TDP.
Example 2 screening of TDP liquid phase conditions
1. Materials and reagents
Cocarboxylase (TDP) (source leaf organism, purity not less than 97%), thiamine diphosphate-d 3 (TDP-d 3) (TRC, purity not less than 95%); 10×0.01m pbs (Solarbio), trichloroacetic acid (analytical grade, komiou), acetonitrile (chromatographic purity, 99.9%, fishr), water (ultrapure water), hydrochloric acid (komiou), ammonia (HPLC grade, sigma), bovine Serum Albumin (BSA) (Solarbio), ammonium formate (Sigma).
2. Apparatus and device
LCMS-4500MD liquid chromatograph-tandem mass spectrometer with triple quadrupole tandem mass spectrometer as detector; one ten thousandth of electronic analytical balance; a MiliQ pure water meter; a vortex mixer; a low temperature high speed centrifuge; medical low-temperature preservation box.
3. Detection of TDP: the following chromatographic and mass spectrometric conditions were used in this experiment:
the liquid chromatography conditions were: chromatographic column: waters, atlantis Premier BEH C, 2.5 μm 2.1X100 mm; column temperature 40 ℃; the temperature of the sample injector is 4 ℃; mobile phase B was acetonitrile, isocratic elution: 0-1.5min, mobile phase B5%; the flow rate was 0.3mL/min.
The mass spectrum parameters are: esi+; air curtain gas (CUR): 30psi; the method comprises the steps of carrying out a first treatment on the surface of the Spray voltage (IS) 5500V; temperature (TEM) 400 ℃; atomizing Gas (Gas 1) at 50psi; auxiliary heating Gas (Gas 2): 50psi; multiple reaction detection (MRM) (see table 1);
4. TDP pretreatment process
200 mu L of sample is taken, placed in a medical low-temperature preservation box for more than or equal to 15min, vortexed for 5-10min at room temperature, 20 mu L of internal standard working solution is added, vortexing for 2000rpm for 5min, 200 mu L of precipitant is added, vortexing for 2000rpm 5min,15000g,4 ℃, centrifugation is carried out for 5min, 60 mu L of supernatant solution is taken, 25 mu L of pH regulator is added, vortexing for 2000rpm for 30s, and 60 mu L of supernatant is taken for sample injection analysis.
5. Comparison of TDP liquid phase conditions
(1) Condition 1: mobile phase A was 0.05% ammonia (containing 1mmol/L ammonium formate); the sample amount was 3. Mu.L (see FIG. 2 for the results).
(2) Condition 2: mobile phase A is 0.05% ammonia water; the sample amount was 3. Mu.L (see FIG. 3 for the results).
(3) Condition 3: mobile phase a was 0.02% formic acid (containing 0.5mmol/L ammonium formate); the sample amount was 3. Mu.L (see FIG. 4 for the results).
(4) Condition 4: mobile phase a was 0.02% formic acid; the sample amount was 3. Mu.L (see FIG. 5 for the results).
(5) Condition 5: mobile phase A was 0.05% ammonia (containing 1mmol/L ammonium formate); the sample injection amount was 1. Mu.L.
(results see FIG. 6)
(6) Condition 6: the gradient of mobile phase is (0-1 min, mobile phase B is 5%, 1-1.1min, mobile phase B is raised from 5% to 60%, 1.1-1.5min, mobile phase B is 60%, 1.5-1.6min, mobile phase B is lowered from 60% to 5%, 1.6-2.0min, mobile phase B is 5%). (results the first needle for injection is shown in FIG. 7 and the second needle for injection is shown in FIG. 8)
5. Analysis of results
Comparing 6 different liquid phase conditions, and detecting a whole blood sample, wherein the result shows that the peak shape of the mobile phase A of the condition 2 is widened when ammonium formate solution is not added; condition 3, improved peak shape, peak tailing and poor reproducibility; the object to be detected does not form a peak shape under the condition 4; condition 5 showed 2 peaks; the mobile phase is changed into gradient from isocratic under the condition 6, when the second needle is sampled from the graph, the pH condition of the sample is obviously not balanced, and if the pH condition of the sample is required to be balanced and the sampling time is required to be far longer than 1.5min; under the alkaline system, the sample injection amount of the sample to be detected is more than or equal to 3 mu L, and the sample to be detected has symmetrical single peak, good reproducibility and high sensitivity, and meets the detection requirement.
Example 3 screening of TDP blank matrices
1. Materials and reagents
Cocarboxylase (TDP) (source leaf organism, purity not less than 97%), thiamine diphosphate-d 3 (TDP-d 3) (TRC, purity not less than 95%); 10×0.01m pbs (Solarbio), trichloroacetic acid (analytical grade, komiou), acetonitrile (chromatographic purity, 99.9%, fishr), water (ultrapure water), hydrochloric acid (komiou), ammonia (HPLC grade, sigma), bovine Serum Albumin (BSA) (Solarbio), ammonium formate (Sigma).
2. Apparatus and device
LCMS-4500MD liquid chromatograph-tandem mass spectrometer with triple quadrupole tandem mass spectrometer as detector; one ten thousandth of electronic analytical balance; a MiliQ pure water meter; a vortex mixer; a low temperature high speed centrifuge; medical low-temperature preservation box.
3. TDP pretreatment process:
standard curve, quality control whole blood sample preparation, 180 μl of blank matrix is taken, 20 μl of working solution is added, and vortex oscillation is carried out for standby.
The whole blood sample preparation comprises the steps of taking 200 mu L of sample, placing the sample into a medical low-temperature preservation box for more than or equal to 15min, and then swirling the sample at room temperature for 5-10min, and performing vortex oscillation for later use.
The precipitation process comprises the steps of taking a standard curve or whole blood sample, adding 20 mu L of internal standard working solution, carrying out vortex oscillation for 2000rpm for 5min, adding 200 mu L of precipitant, carrying out vortex oscillation for 2000rpm 5min,15000g,4 ℃, centrifuging for 5min, taking 60 mu L of supernatant solution, adding 25 mu L of pH regulator, carrying out vortex oscillation for 2000rpm for 30s, and taking 60 mu L of supernatant for sample injection analysis.
4. Detection of TDP: the following chromatographic and mass spectrometric conditions were used in this experiment:
the liquid chromatography conditions were: chromatographic column: waters, atlantis Premier BEH C, 2.5 μm 2.1X100 mm; column temperature 40 ℃; the temperature of the sample injector is 4 ℃; mobile phase B was acetonitrile, isocratic elution: 0-1.5min, mobile phase A of 0.05% ammonia (containing 1mmol/L ammonium formate) and mobile phase B of 5%; the flow rate is 0.3mL/min; the sample injection amount was 3. Mu.L.
The mass spectrum parameters are: esi+; air curtain gas (CUR): 30psi; the method comprises the steps of carrying out a first treatment on the surface of the Spray voltage (IS) 5500V; temperature (TEM) 400 ℃; atomizing Gas (Gas 1) at 50psi; auxiliary heating Gas (Gas 2): 50psi; multiple reaction detection (MRM) (see table 1);
comparison of blank matrices (results see Table 9)
(1) Blank substrate 1:0.01M PBS+4% BSA;
(2) Blank matrix 2:0.01M PBS;
TABLE 9 retention times for different blank matrices
Figure BDA0004182182750000131
5. Analysis of results
Comparison of two different blank matrices, the sample was selected as a single human whole blood sample, and the results show that blank matrix 1 as a blank matrix did not interfere with the retention time of human sample detection. Thus, blank matrix 1 was selected.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

Claims (7)

1. A method for detecting the concentration of vitamin B1 (VB 1) in whole blood as an active metabolite Thiamine Diphosphate (TDP), said method comprising the steps of:
pre-treating a whole blood sample containing VB1 to be detected to obtain a target sample solution;
testing the TDP of the target sample solution, and calculating the concentration of the TDP in the whole blood sample;
wherein, the operation of preprocessing the whole blood sample containing VB1 to obtain the target sample solution is as follows: collecting a whole blood sample containing vitamin B1 to be detected, adding an internal standard solution after freeze thawing cycle, vortex oscillating, adding a precipitator, vortex mixing uniformly, centrifuging, taking supernatant, adding a pH regulator, and mixing uniformly to obtain a target sample solution.
2. The method according to claim 1, characterized in that: the step of testing the TDP of the target sample solution and calculating the concentration of TDP in the whole blood sample includes:
preparing standard curve sample solutions containing different TDPs with known concentrations, and drawing a standard curve of the standard curve sample solutions;
and testing the target sample solution by using high performance liquid chromatography-tandem mass spectrometry, and obtaining the TDP content in the target sample solution according to the standard curve, thereby obtaining the TDP content in the whole blood sample containing the TDP to be tested.
3. The method according to claim 1 or 2, characterized in that: the step of configuring standard curve sample solutions containing different known concentrations of TDP and drawing a standard curve of the standard curve sample solutions includes:
firstly, respectively preparing standard curve samples containing different TDPs with known concentrations, adding an internal standard solution, carrying out vortex vibration, adding a precipitator, carrying out vortex mixing, centrifuging, taking supernatant, adding a pH regulator, and carrying out uniform mixing to obtain a series of standard curve sample solutions containing different TDPs with known concentrations;
and secondly, testing the standard curve sample solution by using high performance liquid chromatography-tandem mass spectrometry, calculating the area ratio of TDP and internal standard peak, and establishing a standard curve by an internal standard method.
4. A method according to any one of claims 1-3, characterized in that: the internal standard solution is TDP-d3 solution, and the concentration of the internal standard solution is 0.8-1.6nmol/L;
the volume ratio of the whole blood sample or standard curve sample containing TDP to be measured to the internal standard solution is 10:1, a step of;
aqueous ammonia solution with the concentration of 10% -40% of the pH regulator;
the volume ratio of the whole blood sample or the standard curve sample containing TDP to be detected and the pH regulator is 8:1-9:1.
5. The method according to any one of claims 1-4, wherein: the volume ratio of the whole blood sample or standard curve sample containing TDP to be measured to the precipitation solution is 1:0.6-1:3, a step of;
the precipitation solution is trichloroacetic acid, perchloric acid and metaphosphoric acid solution;
the concentration of the precipitation solution is 3% -10%;
the pH adjustor and the precipitant were added and vortexed at 2000rpm for 5min.
6. The method according to any one of claims 1-5, wherein: the liquid chromatography conditions were: chromatographic column: waters, atlantis Premier BEH C182.5 μm 2.1X100 mm; column temperature 40 ℃; the temperature of the sample injector is 4 ℃; mobile phase a was 0.05% ammonia (containing 1mmol/L ammonium formate) and B was acetonitrile, isocratic elution: 0-1.5min, mobile phase B5%; the flow rate is 0.3mL/min; the sample injection amount was 3. Mu.L.
7. The method according to any one of claims 1-6, wherein: the mass spectrum parameters are: ionization mode: esi+; air curtain gas (CUR): 30psi; the method comprises the steps of carrying out a first treatment on the surface of the Spray voltage (IS) 5500V; temperature (TEM) 400 ℃; atomizing Gas (Gas 1) at 50psi; auxiliary heating Gas (Gas 2): 50psi; multiple reaction detection (MRM).
CN202310407993.3A 2023-04-17 2023-04-17 Method for detecting concentration of active metabolite thiamine diphosphate of vitamin B1 in whole blood Pending CN116381096A (en)

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