CN106885867A - Five kinds of methods of ox sulphur conjugated bile acidses in high performance liquid chromatography tandem mass spectrum detection serum - Google Patents
Five kinds of methods of ox sulphur conjugated bile acidses in high performance liquid chromatography tandem mass spectrum detection serum Download PDFInfo
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Abstract
The invention discloses five kinds of methods of ox sulphur conjugated bile acidses in a kind of utilization high performance liquid chromatography tandem mass spectrum detection serum, comprise the following steps：The preparation of standard items, high performance liquid chromatography separation, tandem mass spectrum detection; the detection of ox sulphur conjugated bile acidses in serum, qualitative and accurate quantitative determination serum includes tauroursodeoxycholic acid, taurocholate; Irish moss (chondrux); tauroursodeoxycholic acid, five kinds of ox sulphur conjugated bile acidses of taurolithocholic acid, detection time is short; efficiency high; detection sensitivity is high, and specificity is good, with low cost.
Description
Technical field
Five kinds of ox sulphur knots are detected the present invention relates to the detection field of bile acid, more particularly to high performance liquid chromatography tandem mass spectrum
The method of mould assembly bile acid.
Background technology
Ox sulphur conjugated bile acidses refer to that bile acid is combined with amido link (abbreviation peptide bond) with taurine, are combined as ox sulphur
Type bile acid, including taurocholate (taurocholic acid, TCA), Irish moss (chondrux)
(taurochenodeoxycholic, TCDCA), taurolithocholic acid (taurolithocholic acid, TLCA), ox sulphur deoxidation
Cholic acid (taurodesoxycholic acid, TDCA), Tauro ursodesoxy cholic acid (tauroursodeoxycholic acid,
TUDCA)。
Ox sulphur conjugated bile acidses are soluble in water, and this is due to, both containing hydrophilic hydroxyl and carboxyl, containing in its molecule again
Have a hydrophobic methyl, and the different group of both properties Total enumeration, in the both sides of cyclopentanoperhy drophenanthrene core, makes molecule point again
It is " hydrophilic " and " hydrophobic " two sides.Therefore ox sulphur conjugated bile acidses have strong emulsifying agent function, make grease emulsifying in enteric cavity
Into particulate, it is easy to lipid to digest and assimilate with (lipase) contact area of lipase in digestive juice to increase grease.Ox sulphur is combined
Type bile acid in human body in addition to the main component as chylomicron, it or in vivo cholesterol eliminate an important way
Footpath, the product of metabolism can be discharged with bile in liver.Ox sulphur conjugated bile acidses participate in fat circulation, and phosphatide forms micro-
Grain, and form the intestinal absorption that emulsion helps aliphatic acid and liposoluble vitamin.Although ox sulphur conjugated bile acidses are liver
Intestines are circulated, and content is very low in body circulation and urine, but when there is disease in the liver and gallbladder and cause bile acid excretion obstacle, in liver,
Content is significantly raised in blood and urine.Ox sulphur conjugated bile acidses level is often by as cholestasia in urine and blood
The biomarker of property disease.Ox sulphur conjugated bile acidses are not only adjusted in the molecular signal as endocrine and paracrine function
The internal level of its own is controlled, energy ezpenditure, glucose and lipid-metabolism, the signal transmission of thyroid hormone and thin is also participated in
Born of the same parents are immunized.Therefore detection detects the internal level of each ox sulphur conjugated bile acids rather than simply quantifies TBA and contains
Amount, examination, diagnosis and differential diagnosis for liver and gall and intestines problem have important value.
Separate and quantitative ox sulphur conjugated bile acidses are a challenges, because the biochemical character of this kind of compound is different, have
Also there is isomer, and their contents in human body are relatively low.Current common detection methods are enzymatic cycling assay for detecting lifes
Bile acid in reason solution, although operation is relatively simple, but the method can only detect TBA content, it is impossible to accomplish to determine
The difference of different ox sulphur conjugated bile acidses；Gas-chromatography and gas chromatography mass spectrometry need complexity when ox sulphur conjugated bile acidses are detected
Sample preparation, derivatization, ox sulphur conjugated bile acidses need to be hydrolyzed into non-binding type can just be analyzed；High performance liquid chromatography
Ox sulphur conjugated bile acidses of the series connection UV-detector in detection of complex bio-matrix can cause sensitivity and specificity not enough；
Fluoroscopic examination rule needs complicated Derivative and can not promote.
The content of the invention
In order to overcome the deficiencies in the prior art, it is an object of the invention to provide a kind of inspection of high performance liquid chromatography tandem mass spectrum
Survey five kinds of methods of ox sulphur conjugated bile acidses in serum, ox sulphur conjugated bile acidses detection complex operation, sensitivity and special
Property not enough problem.
The purpose of the present invention is realized using following technical scheme：
Five kinds of methods of ox sulphur conjugated bile acidses in high performance liquid chromatography tandem mass spectrum detection serum, including following step
Suddenly：
(1) preparation of standard items：Ox sulphur conjugated bile acidses standard items are standby to several various concentrations with dilution in acetonitrile
With, in the standard items of each concentration add equivalent containing target acetonitrile solution in concentration known, supernatant is taken after high speed centrifugation,
After supernatant is freezed using centrifugal concentrating refrigeration system, it is centrifuged after adding a certain amount of aqueous solution containing acetonitrile, takes supernatant
Analyzed for high performance liquid chromatography tandem mass spectrum loading in 96 orifice plates.
(2) high performance liquid chromatography separation：Using ultrahigh pressure liquid phase chromatogram and corresponding mobile phase, in reverse C18 analyses
5 kinds of ox sulphur conjugated bile acidses are separated on post；
(3) tandem mass spectrum detection：5 kinds of ox sulphur conjugated bile acidses after being separated in chromatogram enter into mass spectrum and are detected,
5 kinds of ox sulphur conjugated bile acidses are specifically detected using the multiple reaction monitoring mode in triple level Four bar mass spectrums, according to letter
Number noise ratio is calculated quantitative determination limit and identification test limit, is drawn according to ratio and known standard items and internal standard concentration value
Canonical plotting simultaneously obtains quantitative correction equation；
(4) in serum ox sulphur conjugated bile acidses detection：Serum sample is taken, adding the acetonitrile solution of containing the internal standard carries out egg
Be centrifuged for the first time after white precipitation, fully precipitation, pipette serum supernatant using centrifugal concentrating refrigeration system it is lyophilized after, add certain
It is centrifuged after the aqueous solution containing acetonitrile of amount, supernatant is taken in standby on 96 orifice plates, for high performance liquid chromatography tandem mass spectrum
Sample is analyzed；
The same step of high performance liquid chromatography tandem mass spectrum loading analytical procedure (2), step (3), liquid chromatography mass is detected
To ox sulphur conjugated bile acidses and interior target ratio, ratio is updated to quantitative correction equation, is calculated ox sulphur knot in serum
The content of mould assembly bile acid.
Further, it is interior in the step (1) to be designated as deuterated cholic acid, deuterated ursodesoxycholic acid, it is described containing in concentration known
Target acetonitrile solution is that the deuterated Bile acid concentrations of internal standard are 76nmol/L, and the deuterated ursodesoxycholic acid diluted concentration of internal standard is
126nmol/L。
Further, the ox sulphur conjugated bile acidses concentration range of several various concentrations is in the step (1)
2nmol/L~6 μm ol/L.
Further, high performance liquid chromatography separation condition is in the step (2)：Chromatographic column：Waters ACQUITY
UPLCC18 Column:pore size1.7 μm of particle size, 2.1mm × 50mm；cat.#
186002350IVD；Chromatographic column column temperature：45℃；Sample size：10μL；Flow velocity：A phases are 0.1% aqueous formic acid, i.e. formic acid mixed
The volume accounting in liquid is closed for 0.1%, B phases are 0.1% formic acid-acetonitrile：Methyl alcohol=3：The mixed liquor of 1, i.e. formic acid and acetonitrile with
The volume ratio of methyl alcohol is 3:1, wherein formic acid volume accounting in the mixed liquor of formic acid and acetonitrile is 0.1%.
Further, in the step (3) in Mass Spectrometer Method condition, the detection parameter of each material first uses standard items
Optimization, then using the multiple reaction monitoring mode in triple level Four bar mass spectrums and the specific mass spectrometry parameters for having optimized specificity
Ground detects five kinds of ox sulphur conjugated bile acidses, and the MS detection parameters are：It is electron spray pin voltage：3.0kV, goes solvent stream
Speed：800L/h, goes solvent gas temperature：400 DEG C, taper hole gas velocity：50L/h, is detected as negative ion mode, multiple reaction monitoring.
Further, centrifugal condition is twice in the step (1)：Refrigerated centrifuge at 4 DEG C of 18000g centrifugal force from
Heart 5min.
Further, serum and the volume ratio of containing the internal standard acetonitrile solution are 1 in the step (4):3.
Further, serum is behaved or animal blood serum in the step (4).
Compared to existing technology, the beneficial effects of the present invention are：Albumen precipitation is carried out using acetonitrile, liquid phase is used after extraction
The triple level Four bar mass spectrograph detections of chromatographic tandem, pre-treatment step is simple, can effectively remove serum matrix interference, specificity
It is good；High performance liquid chromatography tandem mass spectrum instrument detected, at the same it is qualitative and precisely quantify serum and include tauroursodeoxycholic acid,
5 kinds of ox sulphur conjugated bile acidses such as taurocholate, Irish moss (chondrux), tauroursodeoxycholic acid, taurolithocholic acid, detection time
Short, flux is high, and detection sensitivity is high, and specificity is good, with low cost.
Brief description of the drawings
The schematic flow sheet of Fig. 1, detection method of the invention；
The absolute retention time of Fig. 2, each ox sulphur conjugated bile acidses and interior target chromatography eluant appearance；
Fig. 3, taurocholate standard curve；
Fig. 4, taurolithocholic acid standard curve；
Fig. 5, tauroursodeoxycholic acid standard curve；
Fig. 6, tauroursodeoxycholic acid standard curve；
Fig. 7, Irish moss (chondrux) standard curve.
Specific embodiment
Below, with reference to accompanying drawing and specific embodiment, the present invention is described further：
5 kinds of ox sulphur conjugated bile acidses (taurocholate, taurolithocholic acid, tauroursodeoxycholic acid, tauroursodeoxycholic acid,
Irish moss (chondrux)) standard items and isotope marks internal standard (deuterated cholic acid, deuterated ursodesoxycholic acid) purchase in
Sigma Aldriches.Standard items and internal standard are dissolved with acetonitrile first, are then stored in -80 DEG C, it is necessary to carry out reality
During sample detection, it is 76nmol/L, the deuterated ursodesoxycholic acid dilution of internal standard by the deuterated cholic acid diluted concentration of internal standard to recycle acetonitrile
Concentration is 126nmol/L, used as working solution.
(1) prepared by standard items：The ox sulphur conjugated bile acidses acetonitrile solution for taking 9 various concentrations of 0.1mL respectively is arrived
In 1.5mL EP pipes, then the deuterated cholic acid of containing the internal standard of 0.3mL Cord bloods is separately added into, deuterated ursodesoxycholic acid acetonitrile solution,
After being sufficiently mixed with vortex mixer, with the deuterated bile acid acetonitrile solution of the liquid-transfering gun transferase 10 .3mL containing the internal standards of 1mL in addition
One 1.5mL EP pipe, is then freezed with centrifugal concentrating refrigeration system；
After adding a certain amount of aqueous solution containing acetonitrile after lyophilized, blending ingredients using refrigerated centrifuge 18000g from
5min is centrifuged at 4 DEG C of mental and physical efforts, takes supernatant and is analyzed for Liquid Chromatography-Tandem Mass Spectrometry loading in 96 orifice plates；
(2) high performance liquid chromatography separation：Using ultrahigh pressure liquid phase chromatogram and corresponding mobile phase, in reverse C18 analyses
Chromatography eluant separation is carried out to ox sulphur conjugated bile acidses in sample supernatant on post, by controlling elution requirement, 5 kinds is isolated
Ox sulphur conjugated bile acidses；
Liquid phase chromatogram condition is：Chromatographic column：Waters ACQUITY UPLCC18 Column:pore sizeparticle size 1.7μm,2.1mm×50mm；cat.#186002350IVD；Chromatographic column column temperature：45℃；Sample introduction
Amount：10μL；Flow velocity：0.4mL/min；Flowing phase composition：A phases are the volume of 0.1% aqueous formic acid, i.e. formic acid in mixed liquor
Accounting is that 0.1%, B phases are 0.1% formic acid-acetonitrile：Methyl alcohol=3：The mixed liquor of 1, i.e. formic acid and acetonitrile and the volume ratio of methyl alcohol
It is 3:1, wherein formic acid volume accounting in the mixed liquor of formic acid and acetonitrile is 0.1%.
Gradient is as shown in table 1 below or table 2：
Table 1
Table 2
| Sequence number | Time (min) | Flow velocity (mL/min) | A% | B% | Curve values |
| 1 | - | 0.4 | 65 | 35 | - |
| 2 | 2.0 | 0.4 | 57 | 43 | 6 |
| 3 | 3.5 | 0.4 | 54 | 46 | 6 |
| 4 | 5.0 | 0.4 | 41 | 59 | 6 |
| 5 | 7.0 | 0.4 | 41 | 59 | 6 |
| 6 | 8.7 | 0.4 | 34 | 66 | 6 |
| 7 | 10.7 | 0.4 | 2 | 98 | 1 |
| 8 | 11.3 | 0.4 | 65 | 35 | 1 |
(3) Mass Spectrometer Method and standard curve processed：The 5 kinds of ox sulphur conjugated bile acidses isolated in liquid chromatogram enter into three
Weigh level Four bar mass spectrum to be detected, 5 kinds of oxen are specifically detected using the multiple reaction monitoring mode in triple level Four bar mass spectrums
The content of sulphur conjugated bile acidses, and draw canonical plotting processed；
According to different chromatographic elution times, different detection window and parameters, the detection ginseng of each material are set
Number is first optimized with standard items, then using the multiple reaction monitoring mode in triple level Four bar mass spectrums and optimized it is specific
Mass spectrometry parameters specifically detect five kinds of ox sulphur conjugated bile acidses.
Mass spectrum is Waters Xevo TQD IVD (Waters, Milford, MA)；Mass Spectrometer Method condition is as follows：Electron spray
Pin voltage：3.0kV, removes solvent gas flow velocity：800L/h, goes solvent gas temperature：400 DEG C, taper hole gas velocity：50L/h, is detected as bearing
Ion mode, multiple reaction monitoring, the specific reactive ion of each determinand to, residence time, taper hole voltage, collision energy etc.
(different type instrument, collision energy, taper hole voltage value are different, it is necessary to independent optimization) as shown in the table：
Table 3
By screening twice, i.e., first level Four bar carries out specific parent ion screening, second level Four for multiple reaction monitoring
Bar carries out parent ion fragmentation generation daughter ion, and the 3rd level Four bar carries out specific daughter ion screening, special with extraordinary detection
The opposite sex.Can be by the ion stream that selects reaction monitoring to detect, and corresponding retention time determines ox sulphur mating type courage
The detection of juice acid, recycles the deuterated cholic acid internal standard of addition known quantity, deuterated ursodesoxycholic acid internal standard to be quantified.
As shown in Fig. 2 sample is by the way that after ultrahigh pressure liquid phase chromatographic isolation, different ox sulphur conjugated bile acidses are washed in difference
De- time appearance, and arrived by mass spectrum selection reaction monitoring mode detection, the material detected from after arriving first is respectively：Ox sulphur bear
Deoxycholic acid, taurocholate, Irish moss (chondrux), tauroursodeoxycholic acid, deuterated ursodesoxycholic acid, deuterated cholic acid, ox sulphur stone
Cholic acid, deuterated cholic acid retention time is 3.87min, and deuterated ursodesoxycholic acid retention time is 3.58min, when taurocholate retains
Between be 1.63min, taurolithocholic acid retention time be 4.79min, tauroursodeoxycholic acid retention time be 3.08min, ox sulphur bear
Deoxycholic acid retention time is 1.38min, and Irish moss (chondrux) retention time is 2.72min.
Canonical plotting is shown in Fig. 3 to Fig. 7, and test limit is defined as signal to noise ratio>3, quantitative limit is defined as signal to noise ratio>10, retain
Time is the absolute retention time of chromatography eluant appearance, and the coefficient R 2 of all detection materials is equal>0.99, every kind of bile acid
Retention time, detection limit, quantitative limit, the range of linearity and quantitative correction equation difference are as shown in Figure 4；By the precision of three days
Degree test, including basic, normal, high three concentration, in a few days day to day precision RSD is respectively less than 15%, as shown in figure 5, showing detection knot
Fruit is accurate, repeats.
Five kinds of retention time, test limit, quantitative limit, the range of linearity, linear equation, phase relations of ox sulphur conjugated bile acidses
Number is as follows：
Table 4
Five kinds of ox sulphur conjugated bile acidses it is in a few days as follows with day to day precision data：
Table 5
(4) in serum ox sulphur conjugated bile acidses detection：0.1mL blood serum samples are taken in 1.5mL EP pipes, is added
The deuterated cholic acid internal standard of containing the internal standard of 0.3mL Cord bloods, deuterated ursodesoxycholic acid acetonitrile solution is sufficiently mixed with vortex mixer
Afterwards, with the liquid-transfering gun deuterated cholic acid internal standard of transferase 10 .3mL containing the internal standards of 1mL, the bile acid acetonitrile solution of deuterated ursodesoxycholic acid is arrived
Another 1.5mL EP is managed, then lyophilized with vacuum freeze dryer；
After adding a certain amount of aqueous solution containing acetonitrile after lyophilized, blending ingredients using refrigerated centrifuge 18000g from
5min is centrifuged at 4 DEG C of mental and physical efforts, takes supernatant and is analyzed for Liquid Chromatography-Tandem Mass Spectrometry loading in 96 orifice plates；
Liquid Chromatography-Tandem Mass Spectrometry analyzes same step (2) and (3), and Mass Spectrometer Method obtains ox sulphur conjugated bile acidses and internal standard
Ratio, ratio is updated to quantitative correction equation, be calculated the content of ox sulphur conjugated bile acidses in serum.
It will be apparent to those skilled in the art that technical scheme that can be as described above and design, make other various
It is corresponding to change and deformation, and all these change and deformation should all belong to the protection domain of the claims in the present invention
Within.
Claims (7)
1. high performance liquid chromatography tandem mass spectrum detects five kinds of methods of ox sulphur conjugated bile acidses in serum, it is characterised in that bag
Include following steps：
(1) preparation of standard items：Ox sulphur conjugated bile acidses standard items are standby to several various concentrations with dilution in acetonitrile, often
Equivalent is added in the standard items of individual concentration contains target acetonitrile solution in concentration known, the ox sulphur conjugated bile acidses acetonitrile
The concentration range of solution be 2nmol/L~6 μm ol/L, it is described in be designated as deuterated cholic acid, deuterated ursodesoxycholic acid, after high speed centrifugation
Supernatant is taken, after supernatant is freezed using centrifugal concentrating refrigeration system, is centrifuged after adding a certain amount of aqueous solution containing acetonitrile,
Supernatant is taken to be analyzed for high performance liquid chromatography tandem mass spectrum loading in 96 orifice plates；
(2) high performance liquid chromatography separation：Using ultrahigh pressure liquid phase chromatogram and corresponding mobile phase, on reverse C18 analytical columns
Five kinds of ox sulphur conjugated bile acidses are separated；
(3) tandem mass spectrum detection：Five kinds of ox sulphur conjugated bile acidses after being separated in chromatogram enter into mass spectrum and are detected, utilize
Multiple reaction monitoring mode in triple level Four bar mass spectrums specifically detects five kinds of ox sulphur conjugated bile acidses, is made an uproar according to signal
Signal to noise ratio is calculated quantitative determination limit and identification test limit, and standard is drawn according to ratio and known standard items and internal standard concentration value
Curve map simultaneously obtains quantitative correction equation；
(4) in serum ox sulphur conjugated bile acidses detection：Serum sample is taken, is added the acetonitrile solution of containing the internal standard to carry out albumen and is sunk
Form sediment, be fully centrifuged for the first time after precipitation, pipette serum supernatant using centrifugal concentrating refrigeration system it is lyophilized after, add a certain amount of
It is centrifuged after the aqueous solution containing acetonitrile, supernatant is taken in standby on 96 orifice plates, for high performance liquid chromatography tandem mass spectrum loading point
Analysis；
The same step of high performance liquid chromatography tandem mass spectrum loading analytical procedure (2), step (3), liquid chromatography mass detection obtain ox
Sulphur conjugated bile acidses and interior target ratio, quantitative correction equation is updated to by ratio, is calculated ox sulphur mating type in serum
The content of bile acid.
2. five kinds of ox sulphur conjugated bile acidses in high performance liquid chromatography tandem mass spectrum detection serum according to claim 1
Method, it is characterised in that be containing target acetonitrile solution in concentration known described in step (1)：The deuterated Bile acid concentrations of internal standard are
76nmol/L, the deuterated ursodesoxycholic acid diluted concentration of internal standard is 126nmol/L.
3. five kinds of ox sulphur conjugated bile acidses in high performance liquid chromatography tandem mass spectrum detection serum according to claim 1
Method, it is characterised in that high performance liquid chromatography separation condition is in the step (2)：Chromatographic column：Waters ACQUITY
UPLCC18Column:pore size1.7 μm of particle size, 2.1mm × 50mm；cat.#
186002350IVD；Chromatographic column column temperature：45℃；Sample size：10μL；Flow velocity：0.4mL/min；Flowing phase composition：A phases are 0.1%
Aqueous formic acid, B phases are 0.1% formic acid-acetonitrile：Methyl alcohol=3：1 solution.
4. five kinds of ox sulphur conjugated bile acidses in high performance liquid chromatography tandem mass spectrum detection serum according to claim 1
Method, it is characterised in that in the step (3) in Mass Spectrometer Method condition, the Mass Spectrometer Method of each cow-bezoar conjugated bile acids
Parameter is first optimized with standard items, then using the multiple reaction monitoring mode in triple level Four bar mass spectrums and the spy for having optimized
Determine mass spectrometry parameters and specifically detect five kinds of ox sulphur conjugated bile acidses, the MS detection parameters are：It is electron spray pin voltage：
3.0kV, removes solvent gas flow velocity：800L/h, goes solvent gas temperature：400 DEG C, taper hole gas velocity：50L/h, is detected as anion mould
Formula, multiple reaction monitoring.
5. five kinds of ox sulphur conjugated bile acidses in high performance liquid chromatography tandem mass spectrum detection serum according to claim 1
Method, it is characterised in that centrifugal condition is twice in the step (1)：Refrigerated centrifuge is centrifuged at 4 DEG C of 18000g centrifugal force
5min。
6. the high performance liquid chromatography tandem mass spectrum according to claim 1 detects five kinds of sides of sequestered bile acid in serum
Method, it is characterised in that serum and the volume ratio of containing the internal standard acetonitrile solution are 1 in the step (4):3.
7. the high performance liquid chromatography tandem mass spectrum according to claim 1 detects five kinds of sides of sequestered bile acid in serum
Method, it is characterised in that serum is behaved or animal blood serum in the step (4).
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| CN107356694A (en) * | 2017-07-21 | 2017-11-17 | 杭州汉库医学检验所有限公司 | A kind of method of 15 kinds of bile acids in efficient detection blood |
| CN107843671A (en) * | 2017-12-21 | 2018-03-27 | 杭州佰勤医疗器械有限公司 | A kind of detection method of the high flux Liquid Chromatography-Tandem Mass Spectrometry of high flux Liquid Chromatography-Tandem Mass Spectrometry |
| CN111474256A (en) * | 2020-04-16 | 2020-07-31 | 上海中科新生命生物科技有限公司 | HP L C-MSMS-based quantitative analysis method for bile acid in feces |
| CN114923995A (en) * | 2022-04-21 | 2022-08-19 | 中山大学 | Guangzhou angiostrongyliasis diagnosis biomarker and application thereof |
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