CN107478761A - 25 hydroxy-vitamine D in serum2/D3The parallel liquid chromatography mass combination detection method of content of dual flow path - Google Patents
25 hydroxy-vitamine D in serum2/D3The parallel liquid chromatography mass combination detection method of content of dual flow path Download PDFInfo
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Abstract
The invention discloses 25 hydroxy-vitamine Ds in serum2/D3The parallel liquid chromatography mass spectrometric combination detection method of content of dual flow path, including step：(1) sample pre-treatments；(2) analysis detection is carried out using liquid chromatography tandem mass spectrometry, wherein, liquid chromatogram carries out double samples using the first parallel liquid phase liquid phase parallel with second and alternately analyzed, another parallel liquid phase washes balance while any parallel liquid phase analysis, during any parallel liquid phase data acquisition, another parallel liquid phase immediately begins to gathered data, to realize that continuous data gathers.The present invention greatly shortens analysis detection time, reduces equipment and analysis cost, improves detection efficiency and economic benefit.
Description
Technical field
The present invention relates to a kind of detection method, and in particular to 25-hydroxy-vitamin D in a kind of serum2/D3Dual flow path is parallel
Liquid chromatography mass is combined detection method of content.Belong to technical field of analysis and detection.
Background technology
Vitamin D is a kind of fat-soluble hormone, and to human skeleton health, balance adjustment plays important inside calcium, phosphorus
Effect, in addition vitamin D also have extensive non-bone effect, with the disease such as angiocardiopathy, immunological diseases, diabetes and tumour
Disease is closely related.The shortage of vitamin D can cause impaired skeletal development, and the onset risk increase of all kinds of chronic diseases, such as
Rheumatoid arthritis, cartilaginification, coronary heart disease, diabetes and tumour etc..Mainly there are two approach in body vitamin D source,
First, the vitamin D taken in by meals approach2(yeast or fungi) and vitamin D3(fish, cod-liver oil or yolk), second, body
The 7- dehydrocholesterols in skin are converted into vitamin D through illumination3。
Vitamin D can be converted into 25-hydroxy-vitamin D (25-OHD) in liver, so as to by body transport or storage, because
This internal 25-OHD content can effectively reflect body vitamin D nutritional status.In addition, 25-OHD stablizes in vivo, half-life period
Long, its concentration level is directly related with internal vitamin D content, therefore 25-OHD can use as a kind of main biomarker
In embodiment body vitamin D level.
At present, 25-OHD detection method has a variety of, including immune detection, HPLC methods and LC-MS/MS methods etc..Wherein,
LC-MS/MS methods have the characteristics that high specificity, the degree of accuracy is high, analysis time is short, it is considered to be evaluation body vitamin D nutrition
" goldstandard " of situation.But existing conventional LC-MS/MS methods detection time length, detection device use cost are higher.
The content of the invention
The purpose of the present invention is to overcome above-mentioned the deficiencies in the prior art, there is provided 25-hydroxy-vitamin D in a kind of serum2/
D3Detection method of content.
To achieve the above object, the present invention uses following technical proposals：
25-hydroxy-vitamin D in serum2/D3Detection method of content, including step：
(1) sample pre-treatments：Saturation solution of zinc sulfate and second are sequentially added into the blood serum sample for adding internal standard working solution
Nitrile, shake, stand, add n-hexane, concussion, high speed centrifugation, take upper organic layer, dry up, redissolution is made into methanol solution；
(2) carry out analysis detection using liquid chromatography tandem mass spectrometry, wherein, liquid chromatogram using the first parallel liquid phase and
Second parallel liquid phase carries out double samples and alternately analyzed, and another parallel liquid phase washes balance while any parallel liquid phase analysis, any
During parallel liquid phase data acquisition, another parallel liquid phase immediately begins to gathered data, to realize that continuous data gathers, mobile phase
Including A phases and B phases, A phases are the aqueous formic acid of mass concentration 0.1%, B phases for the formic acid containing mass concentration 0.1% and
The methanol solution of 2mmol/L ammonium acetates；Analyze and wash the gradient elution time-program(me) difference of balance as shown in Table 1 and Table 2.
The gradient elution time-program(me) that table 1. is analyzed
Table 2. washes the gradient elution time-program(me) of balance
Preferably, the specific method of step (1) is：100 μ L blood serum samples are taken in 1.5mL centrifuge tubes, are added in 10 μ L
Working solution is marked, is fully mixed；Add the μ L of saturation solution of zinc sulfate 100：Add 200 μ L acetonitriles；Acutely concussion 30 seconds, room temperature is quiet
Put 15 minutes；1mL n-hexanes are added, acutely concussion 30 seconds, more than 14000g is centrifuged 5 minutes；The μ L of the superiors' organic layer 600 are taken,
Nitrogen dries up at room temperature, adds the μ L of 75% methanol solution 200, fully mixes 30 seconds.
It is further preferred that the compound method of the internal standard working solution is as follows：It is 1000 μ g/mL's with methanol compound concentration
D6-25-OHD2And D6-25-OHD3Mixing internal standard storing solution, again with methanol be diluted to concentration be 25ng/mL internal standard work
Liquid.
Preferably, the liquid chromatogram in step (2) uses Shimadzu Nexera MX systems.
Preferably, the mass spectrum in step (2) uses triple quadrupole mass spectrometer.
Preferably, the liquid phase chromatogram condition in step (2) is as follows：
Chromatographic column：Shim-pack XR-C8 (2.0mm I.D. × 50mm L., 2.2 μm)
Flow velocity：0.4mL/min
Column temperature：25℃
Sample size：20μL
Automatic sampler temperature：4℃
The Vavle switching time：0.15min..
Preferably, the Mass Spectrometry Conditions in step (2) are as follows：
Table 3.MRM parameters
Beneficial effects of the present invention：
The present invention utilizes Shimadzu Nexera MX Cascade System triple quadrupole mass spectrometers, establishes 25-OHD in serum2/D3
Quantitative analysis method, greatly shorten analysis detection time, improve detection sensitivity, be a kind of supper-fast, high flux,
The good detection method of high sensitivity, accuracy.In addition, this method not only reduces equipment and analysis cost, detection effect is improved
Rate and economic benefit, the use of the reagent with toxicity and environmental pollution such as organic reagent is also reduced, it is undoubtedly and a kind of low
Cost, high efficiency, more green detection method.
Brief description of the drawings
Fig. 1 a and Fig. 1 b are lower limit of quantitation 0.39ng/mL 25-OHD respectively2With 1.25ng/mL 25-OHD3Chromatogram；
Fig. 2 a and Fig. 2 b are 25-OHD respectively2And 25-OHD3Standard curve；
Fig. 3 is 25-OHD2Detection limit chromatogram；
Fig. 4 is 25-OHD3Detection limit chromatogram；
Fig. 5 is single liquid phase process 25-OHD20.39ng/mL chromatograms；
Fig. 6 is single liquid phase process 25-OHD31.25ng/mL chromatogram.
Embodiment
The present invention will be further elaborated with reference to the accompanying drawings and examples, it should which explanation, the description below is only
It is to explain the present invention, its content is not defined.
Embodiment：
25-hydroxy-vitamin D in serum2/D3Detection method of content, including step：
(1) sample pre-treatments：100 μ L blood serum samples are taken in 1.5mL centrifuge tubes, add 10 μ L internal standard working solutions, fully
Mix；Add the μ L of saturation solution of zinc sulfate 100；Add 200 μ L acetonitriles；Acutely concussion 30 seconds, are stored at room temperature 15 minutes；Add
1mL n-hexanes, acutely concussion 30 seconds, more than 14000g are centrifuged 5 minutes；The μ L of the superiors' organic layer 600 are taken, nitrogen blows at room temperature
It is dry, the μ L of 75% methanol solution 200 are added, are fully mixed 30 seconds.The compound method of internal standard working solution is as follows：With methanol compound concentration
For 100 μ g/mL D6-25-OHD2And D6-25-OHD3Mixing internal standard storing solution, it is 25ng/mL that again with methanol, which is diluted to concentration,
Internal standard working solution；
The preparation of standard working solution：25-OHD is prepared with methanol2And 25-OHD3Hybrid standard storing solution, then with 75% first
Hybrid standard stock solution is diluted to 25-OHD by alcohol (0.1% formic acid) step by step2Concentration be 0.390625ng/mL,
0.78125ng/mL, 1.5625ng/mL, 3.125ng/mL, 6.25ng/mL, 12.5ng/mL, 25ng/mL and 50ng/mL, 25-
OHD3Concentration is 1.25ng/mL, 2.5ng/mL, 5ng/mL, 10ng/mL, 20ng/mL, 40ng/mL, 80ng/mL, 160ng/mL
Standard liquid.
(2) analysis detection is carried out using liquid chromatogram-triple tandem quadrupole mass spectrography, wherein, liquid chromatogram uses first
Parallel with second liquid phase of parallel liquid phase carries out double samples and alternately analyzed, and another parallel liquid phase is washed while any parallel liquid phase analysis
Balance, during any parallel liquid phase data acquisition, another parallel liquid phase immediately begins to gathered data, to realize that continuous data is adopted
Collection, mobile phase include A phases and B phases, and A phases are the aqueous formic acid of mass concentration 0.1%, and B phases are the first containing mass concentration 0.1%
The methanol solution of acid and 2mmol/L ammonium acetates；Liquid phase chromatogram condition is as follows：
Chromatographic column：Shim-pack XR-C8 (2.0mm I.D. × 50mm L., 2.2 μm)
Flow velocity：0.4mL/min
Column temperature：25℃
Sample size：20μL
Automatic sampler temperature：4℃
The Vavle switching time：0.15min..
Analyze and wash the gradient elution time-program(me) difference of balance as shown in Table 1 and Table 2.
The gradient elution time-program(me) that table 1. is analyzed
Table 2. washes the gradient elution time-program(me) of balance
Mass Spectrometry Conditions are as follows：
Table 3.MRM parameters
Fig. 1 a and Fig. 1 b respectively illustrate lower limit of quantitation 0.39ng/mL 25-OHD2With 1.25ng/mL 25-OHD3Chromatogram
Figure.
Standard curve is established using internal standard method.As knowable to Fig. 2 a, Fig. 2 b and table 4,25-OHD2In 0.39~50ng/mL
Linear concentration in the range of, 25-OHD3Good in 1.25~160ng/mL linear dependences, correlation coefficient r is respectively 0.9998
With 0.9998, accuracy range is respectively 97.6~105.2% and 99.5~103.8%.
The standard curve result of table 4.
By 25-OHD2With 25-OHD3Standard curve minimum point diluted to obtain signal to noise ratio step by step with dilution when being about 3, to obtain
To detection limit, concentration is respectively 0.039ng/mL and 0.125ng/mL, and chromatogram is shown in Fig. 3 and Fig. 4.
On the determination of lower limit of quantitation, 25-OHD in the veracity and precision and human body of lower limit of quantitation is considered
Actual content scope, empirical tests, 25-OHD2With 25-OHD3Lower limit of quantitation when being respectively 0.39ng/mL with 1.25ng/mL, more
The accurate and effective of detection can be ensured by adding.Chromatogram is shown in Fig. 1 a and Fig. 1 b.
1 precision is verified
1.1 withinday precision
Take respectively processing after and basic, normal, high three concentration of concentration known quality-control sample, 25-OHD2That is LQC=
2.439ng/mL, MQC=9.367ng/mL and HQC=30.654ng/mL, 25-OHD3That is LQC=5.297ng/mL, MQC=
26.277ng/mL and HQC=79.798ng/mL.For sample after analysis, data statistic analysis obtains the coefficient of variation (CV%) result
As shown in table 5.
The withinday precision of table 5. investigates result (n=6)
1.2 day to day precision
Take processing after and basic, normal, high three concentration of concentration known quality-control sample, 25-OHD2That is LQC=2.439ng/
ML, MQC=9.367ng/mL and HQC=30.654ng/mL, 25-OHD3That is LQC=5.297ng/mL, MQC=26.277ng/
ML and HQC=79.798ng/mL, determine 6 times respectively daily, METHOD FOR CONTINUOUS DETERMINATION 3 days.After analysis, data statistic analysis obtains sample
It is as shown in table 6 to the coefficient of variation (CV%) result.
The day to day precision of table 6. investigates result (n=18)
2 accuracy validations
The degree of accuracy in 2.1 batches
Take respectively processing after and basic, normal, high three concentration of concentration known quality-control sample, 25-OHD2That is LQC
(2.439ng/mL), MQC (9.367ng/mL) and HQC (30.654ng/mL), 25-OHD3 are LQC=5.297ng/mL, MQC=
26.277ng/mL and HQC=79.798ng/mL.Sample is after analysis, and data statistic analysis obtains the degree of accuracy (%), as a result such as
Shown in table 7 and table 8.
The 25-OHD of table 7.2(ng/mL) accuracy validation result in criticizing
The 25-OHD of table 8.3(ng/mL) accuracy validation result in criticizing
Accuracy validation between 2.2 batches
Take processing after and basic, normal, high three concentration of concentration known quality-control sample, 25-OHD2That is LQC=2.439ng/
ML, MQC=9.367ng/mL and HQC=30.654ng/mL, 25-OHD3That is LQC=5.297ng/mL, MQC=26.277ng/
ML and HQC=79.798ng/mL, determine 6 times respectively daily, METHOD FOR CONTINUOUS DETERMINATION 3 days.After analysis, data statistic analysis obtains sample
To the degree of accuracy (%) result as shown in Table 9 and Table 10.
The 25-OHD of table 9.2(ng/mL) accuracy validation result between criticizing
The 25-OHD of table 10.3(ng/mL) accuracy validation result between criticizing
3 clinical sample measurement results
Obtain altogether and be proved for the normal human serum sample of 25-hydroxy-vitamin D content 8, after pre-treatment respectively to enter
Row measure, as a result as shown in table 11.Normally the total content scope of 25-hydroxy-vitamin D is 10-60ng/mL in human body, respectively
Clinical sample determines numerical value in normal range (NR), therefore this method 25-hydroxy-vitamin D content suitable for clinical serum sample
Accurate quantitative analysis.
The human serum clinical sample testing result of table 11.
Comparative example
Step (2) carries out analysis detection using single liquid chromatography tandem mass spectrometry, wherein single liquid phase gradient elution time journey
Sequence is shown in Table 12, remaining same embodiment.
The solution A phase gradient elution time program of table 12.
Fig. 5 and Fig. 6 shows single liquid phase process 25-OHD2And 25-OHD3Chromatogram, its test limit (sensitivity) is obvious
It is poorer than embodiment.
Although above-mentioned the embodiment of the present invention is described with reference to accompanying drawing, model not is protected to the present invention
The limitation enclosed, on the basis of technical scheme, those skilled in the art need not pay creative work and can do
The various modifications or deformation gone out are still within protection scope of the present invention.
Claims (7)
1. 25-hydroxy-vitamin D in serum2/D3Detection method of content, it is characterised in that including step：
(1) sample pre-treatments：Saturation solution of zinc sulfate and acetonitrile are sequentially added into the blood serum sample for adding internal standard working solution, is shaken
Swing, stand, add n-hexane, shake, centrifugation, take upper organic layer, dry up, redissolution is made into methanol solution；
(2) analysis detection is carried out using liquid chromatography tandem mass spectrometry, wherein, liquid chromatogram uses the first parallel liquid phase and second
Parallel liquid phase carries out double samples and alternately analyzed, and another parallel liquid phase washes balance while any parallel liquid phase analysis, any parallel
During liquid phase data acquisition, another parallel liquid phase immediately begins to gathered data, and to realize that continuous data gathers, mobile phase includes
A phases and B phases, A phases are the aqueous formic acid of mass concentration 0.1%, and B phases are formic acid and 2mmol/L second containing mass concentration 0.1%
The methanol solution of sour ammonium；Analyze and wash the gradient elution time-program(me) difference of balance as shown in Table 1 and Table 2.
The gradient elution time-program(me) that table 1. is analyzed
Table 2. washes the gradient elution time-program(me) of balance
2. detection method according to claim 1, it is characterised in that the specific method of step (1) is：Take 100 μ L serum
Sample adds 10 μ L internal standard working solutions, fully mixed in 1.5mL centrifuge tubes；Add the μ L of saturation solution of zinc sulfate 100；Add
Enter 200 μ L acetonitriles；Acutely concussion 30 seconds, are stored at room temperature 15 minutes；1mL n-hexanes are added, acutely concussion 30 seconds, more than 14000g
Centrifugation 5 minutes；The μ L of the superiors' organic layer 600 are taken, nitrogen dries up at room temperature, adds the μ L of 75% methanol solution 200, fully mixes 30
Second.
3. detection method according to claim 2, it is characterised in that the compound method of the internal standard working solution is as follows：With
Methanol compound concentration is 1000 μ g/mL D6-25-OHD2And D6-25-OHD3Mixing internal standard storing solution, again with methanol is diluted to
Concentration is 25ng/mL internal standard working solution.
4. detection method according to claim 1, it is characterised in that the liquid chromatogram in step (2) uses Shimadzu
Nexera MX systems.
5. detection method according to claim 1, it is characterised in that the mass spectrum in step (2) uses triple quadrupole bar matter
Spectrometer.
6. detection method according to claim 1, it is characterised in that the liquid phase chromatogram condition in step (2) is as follows：
Chromatographic column：Shim-packXR-C8
Flow velocity：0.4mL/min
Column temperature：25℃
Sample size：20μL
Automatic sampler temperature：4℃
The Vavle switching time：0.15min..
7. detection method according to claim 1, it is characterised in that the Mass Spectrometry Conditions in step (2) are as follows：
Table 3.MRM parameters
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| CN115267015A (en) * | 2022-05-26 | 2022-11-01 | 天津国科医工科技发展有限公司 | Sample pretreatment composition for mass spectrometry detection, application and pretreatment method |
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