CN109298084A - Kit for detecting oleic acid concentration in serum or plasma, preparation method and application thereof - Google Patents
Kit for detecting oleic acid concentration in serum or plasma, preparation method and application thereof Download PDFInfo
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- CN109298084A CN109298084A CN201810810874.1A CN201810810874A CN109298084A CN 109298084 A CN109298084 A CN 109298084A CN 201810810874 A CN201810810874 A CN 201810810874A CN 109298084 A CN109298084 A CN 109298084A
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- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 title claims abstract description 124
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 title claims abstract description 123
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 title claims abstract description 123
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 title claims abstract description 123
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 title claims abstract description 123
- 239000005642 Oleic acid Substances 0.000 title claims abstract description 123
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 title claims abstract description 123
- 210000002966 serum Anatomy 0.000 title claims abstract description 52
- 238000002360 preparation method Methods 0.000 title claims abstract description 20
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 69
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 64
- 238000000034 method Methods 0.000 claims abstract description 35
- 230000000155 isotopic effect Effects 0.000 claims abstract description 32
- 239000000243 solution Substances 0.000 claims abstract description 30
- 238000001514 detection method Methods 0.000 claims abstract description 28
- 239000011550 stock solution Substances 0.000 claims abstract description 27
- 239000007788 liquid Substances 0.000 claims abstract description 26
- 210000002381 plasma Anatomy 0.000 claims abstract description 21
- 238000000605 extraction Methods 0.000 claims abstract description 17
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 15
- 229940098773 bovine serum albumin Drugs 0.000 claims abstract description 15
- 239000012895 dilution Substances 0.000 claims abstract description 11
- 238000010790 dilution Methods 0.000 claims abstract description 11
- 239000013558 reference substance Substances 0.000 claims abstract description 10
- 229910021642 ultra pure water Inorganic materials 0.000 claims abstract description 10
- 239000012498 ultrapure water Substances 0.000 claims abstract description 10
- 235000021588 free fatty acids Nutrition 0.000 claims abstract description 8
- 239000000203 mixture Substances 0.000 claims abstract description 6
- 239000000047 product Substances 0.000 claims description 18
- 239000012224 working solution Substances 0.000 claims description 16
- 238000004458 analytical method Methods 0.000 claims description 15
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- 150000002500 ions Chemical class 0.000 claims description 7
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- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 2
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- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
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- BNRRFUKDMGDNNT-JQIJEIRASA-N (e)-16-methylheptadec-2-enoic acid Chemical compound CC(C)CCCCCCCCCCCC\C=C\C(O)=O BNRRFUKDMGDNNT-JQIJEIRASA-N 0.000 description 1
- HVCOBJNICQPDBP-UHFFFAOYSA-N 3-[3-[3,5-dihydroxy-6-methyl-4-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyoxan-2-yl]oxydecanoyloxy]decanoic acid;hydrate Chemical compound O.OC1C(OC(CC(=O)OC(CCCCCCC)CC(O)=O)CCCCCCC)OC(C)C(O)C1OC1C(O)C(O)C(O)C(C)O1 HVCOBJNICQPDBP-UHFFFAOYSA-N 0.000 description 1
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- YPMOAQISONSSNL-UHFFFAOYSA-N 8-hydroxyoctyl 2-methylprop-2-enoate Chemical compound CC(=C)C(=O)OCCCCCCCCO YPMOAQISONSSNL-UHFFFAOYSA-N 0.000 description 1
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- SIARJEKBADXQJG-LFZQUHGESA-N stearoyl-CoA Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)CCCCCCCCCCCCCCCCC)O[C@H]1N1C2=NC=NC(N)=C2N=C1 SIARJEKBADXQJG-LFZQUHGESA-N 0.000 description 1
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Classifications
-
- 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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention provides a kind of for detecting the kit of oleic acid concentration in serum or blood plasma, including mobile phase A, Mobile phase B, reference substance stock solution, Isotopic Internal Standard object stock solution, specimen extraction liquid and dilution.Mobile phase A is ultrapure water, and Mobile phase B is the solution that methanol and acetonitrile forms according to volume ratio 1:1, and reference substance stock solution is the oleic acid solutions of 20mg/mL, isotopic label stock solution for 20mg/mL oleic acid-[13C5] solution, specimen extraction liquid is containing methanol, acetonitrile according to isometric solution than composition of 1:1, and dilution is the bovine serum albumin(BSA) without free fatty acid, and the concentration of bovine serum albumin(BSA) is 50mg/mL.The present invention also provides the preparation methods of mentioned reagent box, and using the method for above-mentioned kit detection oleic acid concentration.The present invention be it is a kind of quickly, stablize, reliable diagnosis, treatment and the diseases related effective tool of curative effect monitoring oleic acid metabolic disorder.
Description
Technical field:
The invention belongs to field of biological detection, are related to a kind of detection kit, specifically come be it is a kind of for detect serum or
The kit and its preparation method and application of oleic acid (Oleic acid, OA) concentration in person's blood plasma.
Background technique:
Oleic acid (Oleic acid, OA) is a kind of single unsaturation Omega-9 fatty acid, molecular formula C18H34O2.Human body blood
Oleic acid in liquid is divided into exogenous oleic acid and endogenous oleic acid, and exogenous oleic acid derives from diet regimen, and endogenous oleic acid passes through
Stearyl-coenzyme A desaturase (stearoyl-coenzyme Adesaturase, SCD) is catalyzed saturated fatty acid and generates.Oleic acid
It is the main component in free fatty acid (free fatty acid, FFA), when glycogen exhausts, adipose tissue can decompose neutral fats
Fat becomes free fatty acid and uses to serve as the energy.Under normal circumstances, FFA is three big energy substances, sugar, protein and fatty generation
The important ring thanked, with nonalcoholic fatty liver (nonalcoholic fatty liver disease, NAFLD), artery congee
The occurrence and development of the metabolic diseases such as sample hardening and diabetes are closely related.Research finds that OA can be by interfering pancreas in liver cell
Island element receptor substrate phosphorylation, activation phosphatidylinositols 3 etc., cause glucose uptake or Glycogen synthesis to decline, i.e. generation liver is thin
Born of the same parents' insulin resistance.Confirming that blood plasma fatty acid increases in short term in animal experiments can cause rat liver and periphery insulin to support
It is anti-, wherein the effect of induction liver insulin resistance is significantly stronger than polyunsaturated fat using OA as the monounsaturated fatty acids of representative
Acid.It is most that liver cell/Insulin Resistance of Rats model is prepared to induce with OA in the related academic paper in the past few years delivered.
Insulin resistance (Insulin Resistance, IR) refers to insulin target tissue, including liver, muscle and fat
It organizes to reduce the sensibility of insulin, is a variety of metabolic diseases, including dyslipidemia, hypertension, central obesity, 2 types
Diabetes (Type 2 Diabetes, T2D), the common pathophysiological basis of nonalcoholic fatty liver (NAFLD).In China and
It is explicitly pointed out in American-European NAFLD practice guidelines, NAFLD is a kind of metabolic stress liver closely related with IR and inheritance susceptible
Dirty damage, in addition to cardiovascular and cerebrovascular diseases, diabetes B, NAFLD is also important one of the public health problem of 2l th Century, also
It is the chronic liver disease problem that China more and more payes attention to.But regrettably NAFLD is so far without effective laboratoary markers.
OA is internal triglyceride hydrolysis product, be induced insulin resist, particularly liver insulin resistance it is important because
Element shows that plasma/serum OA level is likely to become the mark that the early warning of IR and/or NAFLD, early diagnosis occurs in body
Object.OA is a kind of monounsaturated fatty acids, and conventional method is difficult to quantitative detection.It is at home and abroad based on ultra performance liquid chromatography at present
There is not been reported for the method and kit of tandem mass spectrum (UHPLC-MS/MS) quantitative detection OA.
Summary of the invention:
For above-mentioned technical problem in the prior art, the present invention provides a kind of ultra performance liquid chromatography tandem mass spectrums
(UHPLC-MS/MS) technology is used to detect the kit and method of OA concentration in serum or blood plasma, and described is this for examining
Surveying the kit of OA concentration and method in serum or blood plasma will solve to detect OA concentration in serum or blood plasma in the prior art
Method it is complicated, the not high technical problem of accuracy.
The present invention provides a kind of for detecting the kit of oleic acid concentration in serum or blood plasma, including mobile phase A, stream
Dynamic phase B, reference substance stock solution, Isotopic Internal Standard object stock solution, specimen extraction liquid and dilution, the mobile phase A are ultrapure
Water, the Mobile phase B are the solution that methanol and acetonitrile are formed according to volume ratio 1:1, and the reference substance stock solution is 20mg/
The oleic acid solutions of mL, the Isotopic Internal Standard object stock solution be 20mg/mL OA- [13C5] solution, the specimen extraction liquid
For the solution that methanol and acetonitrile are formed according to volume ratio 1:1, the dilution is the bovine serum albumin without free fatty acid
White, the concentration of the bovine serum albumin(BSA) is 50mg/mL.
The present invention also provides the preparation method of the kit of oleic acid concentration in a kind of above-mentioned detection serum or blood plasma,
Include the following steps:
1) the step of preparation mobile phase A, ultrapure water ultrasonic degassing is spare；
2) the step of preparation Mobile phase B, methanol is added in acetonitrile, and the volume ratio of methanol and acetonitrile is 1:1,
It is uniformly mixed and ultrasonic degassing is spare；
3) prepare reference substance stock solution the step of, oleic acid standard items are weighed, methanol and second is added with Autosampler
The solution that nitrile is formed according to volume ratio 1:1, the concentration of oleic acid is 20mg/mL after addition, and -20 DEG C of refrigerators are placed in after being completely dissolved
In save backup；
4) prepare Isotopic Internal Standard object stock solution the step of, weigh OA- [13C5] methanol and acetonitrile are added to according to body
The solution that is formed than 1:1 of product, OA- after addition [13C5] concentration be 20mg/mL, saved in -20 DEG C of refrigerators after being completely dissolved standby
With；
5) prepare specimen extraction liquid the step of, methanol and acetonitrile are taken, forms solution according to volume ratio 1:1, mixes, is standby
With；
6) prepare dilution the step of, bovine serum albumin(BSA) is weighed, the bovine serum albumin(BSA) is free of free rouge
Fat acid, bovine serum albumin(BSA) is added in phosphate buffered saline solution, so that the concentration of bovine serum albumin(BSA) is 50mg/mL.
The present invention also provides the method using OA concentration in above-mentioned kit detection serum or blood plasma, including it is as follows
Step:
1) prepare standard working solution and quality-control product the step of；Reference substance stock solution is added in dilution, is diluted to
10, in addition the standard working solution of 25,50,100,500,1000 μ g/mL is configured to the solution of 25,500 μ g/mL as Quality Control
Product.
2) prepare Isotopic Internal Standard object working solution the step of, with specimen extraction liquid that Isotopic Internal Standard object stock solution is dilute
It is interpreted into the working solution of 100 μ g/mL, is deposited in spare in 4 DEG C of refrigerators.
3) a specimen extraction liquid step of the preparation containing Isotopic Internal Standard object takes Isotopic Internal Standard working solution and sample to extract
Liquid is taken to mix by the volume ratio of 1:14.
4) prepare sample the step of, sample are filtered after equilibrium at room temperature 1 hour using 0.22 μm of disposable filter,
Then it accurately pipettes the filtered sample of 10 μ L to be added in 1.5mL EP pipe, be added above-mentioned 3) containing the specimen extraction of Isotopic Internal Standard
Liquid 100 μ L, vortex oscillation 2min are centrifuged 20 minutes with the revolving speed of 10000rpm, and transfer supernatant is dedicated to liquid chromatographic detection
In 96 orifice plates.All standard curves and quality-control product are according to sample process.
5) the step of ultra performance liquid chromatography analysis, chromatographic parameter are as follows: the mobile phase A of ultrapure water composition；Methanol and
The Mobile phase B solution that acetonitrile is formed according to volume ratio 1:1；Gradient elution, B phase are initiated with 5%, and flow velocity is 300 μ l/min, sample introduction
Volume is 5 μ l.OA and interior target retention time are 3.11min, analysis time 12min.
6) mass spectrometry procedure, mass spectrometry parameters: autosampler temperature is 4 DEG C, and column temperature is 40 DEG C, uses electron spray
Ionization source negative ion mode and multiple-reaction monitoring pattern analysis, OA and Isotopic Internal Standard OA-13C5Ion mass-to-charge ratio (the m/ of detection
It z) is 281.3/281.3,286.3/286.3.Atomization gas, atomization auxiliary heating gas, gas curtain gas and collision gas be respectively 55psi,
55psi, 40psi and 6psi, atomization auxiliary heating gas heating temperature is 450 DEG C.Go cluster voltage, entrance potential, exit potential and
Spray voltage is respectively -100V, -10V, -5V and -4500V.OA and OA- [13C5] collision energy be -30V.
7) by the standard solution of step 4) preparation successively from low concentration to high concentration sample introduction, each hybrid standard product solution
It continuous sample introduction 3 times, is analyzed using step 5) and the method for the Liquid Chromatography-Tandem Mass Spectrometry of step 6).With oleic acid and Isotopic Internal Standard
OA-[13C5] concentration ratio is X-axis, the peak area ratio of oleic acid and Isotopic Internal Standard is Y-axis, carries out linear regression, obtains the mark of oleic acid
Directrix curve.
8) using step 5) and step 6) Liquid Chromatography-Tandem Mass Spectrometry method analytical procedure 4) sample to be tested and matter
Control product calculate concentration of specimens according to standard curve.
Kit provided by the invention and chromatography, mass spectral analysis parameter, measuring the OA range of linearity in serum or blood plasma is 10
~1000 μ g/mL, betweenrun precision≤2.8%, batch interior CV≤3.5%.4 DEG C of kit place for 24 hours, five days and after ten days, together
For the concentration of one group of sample OA compared with initial value, error is 0~5%, shows that kit and sample stability are good.By preliminary
Clinical detection assays, 50 health adult's Diagnostic Value of Fasting Serum OA term of reference are 113.2 ± 58 μ g/mL, and 100 NAFLD patients are empty
Abdomen serum levels are respectively 328.1 ± 196.7 μ g/mL, and 66 simple T2DM and 43 T2DM merging NAFLD patients are respectively
407.2 ± 202.9 μ g/mL, 453.8 ± 241.6 μ g/mL are all remarkably higher than healthy control group, and T2DM, T2DM merge
NAFLD group serum OA level is significantly higher than simple NAFLD group.In 50 physical examination of healthy population, serum OA level and weight,
HOMA-IR is positively correlated (P < 0.05) in significant.It is separately analyzed by case group, serum OA level and T2DM patient FPG, HOMA-IR
It is positively correlated in significant, is positively correlated with NAFLD patient FPG, INS, HOMA-IR.Serum OA level is examined with HOMA-IR auxiliary
The concordance rate of disconnected insulin resistance is up to 65.3%.Preliminary clinical analysis confirm serum OA auxiliary judgment IR AUC value be o.689,
Then AUC value rises to 0.806 to joint fasting blood-glucose (FPG), and the AUC value of serum OA auxiliary judgment NAFLD is 0.738, combines blood
Then AUC value rises to 0.774. and shows that dynamic monitoring serum promoting blood circulation slurry OA level is supported in auxiliary diagnosis body insulin clear ALT value
There is important clinical value in anti-and NAFLD and the judgement of its state of an illness.
The present invention is compared with prior art, and technological progress is significant.The present invention is a kind of quick, stable, reliable
It diagnoses, the effective tool that treatment and curative effect monitoring oleic acid metabolic disorder are diseases related, is suitable for routine clinical and large sample stream
Row disease learns the quantitative detecting method of investigation.
Detailed description of the invention:
Fig. 1, which is shown, carries out gradient elution with mobile phase A, B, and OA and interior target retention time are 3.11min, when analysis
Between be 12min.
Fig. 2 is blank sample detection figure, it is seen that is existed at the noiseless peak in OA and internal standard channel.
Fig. 3 shows the detection range of linearity of oleic acid (OA).
Fig. 4 display oleic acid is independent and combines the ROC curve of fasting blood-glucose auxiliary judgment insulin resistance
Fig. 5 display oleic acid is independent and combines the ROC curve of serum transaminase auxiliary judgment NAFLD
Specific embodiment:
The ultra performance liquid chromatography tandem mass spectrum (UHPLC-MS/MS) of 1 serum of embodiment or blood plasma oleic acid (OA) content
The foundation of detection method
One, instrument: 1290 UPLC of high performance liquid chromatograph Agilent (Agilent, USA), series connection triple quadrupole bar matter
Spectrometer AB API 5000 (AB sciex, USA), METTLER TOLEDO balance (Max=220g, d=0.1mg)
(METTLER, Germany), the narrow diameter column (2.1mm*100mm*1.8 μm) of SB-C18 quick separating high throughput (Agilent,
USA), Eppendorf 5424R low-temperature and high-speed centrifuge (Eppendorf, Germany).
Two, Commercial reagents: oleic acid standard items are purchased from sigma company, purity 98.0%；Oleic acid isotopic label OA-
[13C5] it is purchased from IsoSciences company, isotopes concentration is 1030 ± 1 μ g/mL；Methanol (LC//MS/MS grades), acetonitrile
(LC//MS/MS grades), formic acid (LC//MS/MS grades) are purchased from Fisher company of the U.S..Test water is by Milli-Q
Integral ultrapure water machine (Germany, Merck Mi Libo, MERCK MILLIPORE) preparation.
Three, mobile phase is prepared:
1. mobile phase A: ultrapure water
2. Mobile phase B: (volume ratio) 50% methanol: 50% acetonitrile solution
Four, the preparation of stock solution and working stamndard liquid
The preparation of 1.20mg/mL OA pure material stock solution: OA 200mg, precise to 0.1mg are weighed.It is shifted
Into 10mL volumetric flask, 10mL, 0.1% sodium azide aqueous solution is added with Autosampler.Conical flask is gently shaken, makes it completely
Dissolution.Bottleneck is sealed with sealed membrane, refrigerator is placed in 4 hours, stock solution is respectively charged into 1.5mLEp pipe after taking-up, sealing is simultaneously
It is stored in -20 DEG C of refrigerators.
2.20mg/mL OA-[13C5] Isotopic Internal Standard object stock solution preparation: method is matched with OA pure material stock solution
System.
Five, the preparation of standard working solution and quality-control product:
Standard items stock solution, dilution are added in the bovine serum albumin(BSA) (50mg/mL in PBS) without free fatty acid
Standard working solution at 10,25,50,100,500,1000 μ g/mL makees standard curve, is in addition configured to the molten of 25,500 μ g/mL
Liquid is as quality-control product.Methanol: Isotopic Internal Standard object stock solution is diluted to the working solution of 100 μ g/mL by acetonitrile (1:1) solution, so
Afterwards again with methanol: acetonitrile (1:1) is mixed with volume ratio 1:14 ratio, prepare the sample extraction liquid containing internal standard compound.
Six, experimental procedure:
1. the preparation of blood serum sample
It takes 10uL test serum or plasma sample, standard working solution, quality-control product, after equilibrium at room temperature 1h, 100uL is added and contains
The specimen extraction liquid of Isotopic Internal Standard, vortex oscillation 2min are centrifuged 10 minutes with the revolving speed of 10000g, and transfer supernatant liquor is extremely
In dedicated 96 orifice plate of liquid chromatographic detection.All standard curves and quality-control product are according to sample process.
2. chromatographic condition
Mobile phase A group becomes ultra-pure water solution, and B is methanol acetonitrile solution (volume ratio 1:1), and gradient elution, B phase originates
It is 5%, flow velocity is 300 μ l/min, and sampling volume is 5 μ l.OA and interior target retention time are 3.11min, and analysis time is
12min。
3. Mass Spectrometry Conditions
Autosampler temperature is 4 DEG C, and column temperature is 40 DEG C, uses electrospray ionisation source negative ion mode and multiple-reaction monitoring
Pattern analysis, OA and Isotopic Internal Standard OA- [13C5] detection ion mass-to-charge ratio (m/z) be 281.3/281.3,286.3/
286.3.Atomization gas, atomization auxiliary heating gas, gas curtain gas and collision gas are respectively 55psi, 55psi, 40psi and 6psi, atomization
Auxiliary heating gas heating temperature is 450 DEG C.Go cluster voltage, entrance potential, exit potential and spray voltage be respectively -100V, -
10V, -5V and -4500V.OA and OA- [13C5] collision energy be -30V.
4. methodology validation
1) standard curve and quantitative limit
Requirement for quantitative limit (lower limit of quantification, LLOQ) is accuracy 80~
Between 120%, precision (coefficient of variation CV)≤20%.Investigation method is by standard working solution successively from low concentration to highly concentrated
Spend sample introduction, each standard solution continuous sample introduction 3 times, with OA and Isotopic Internal Standard object OA- [13C5] concentration ratio be X-axis, OA with
Isotopic Internal Standard OA- [13C5] peak area ratio be Y-axis, carry out linear regression, obtain the standard curve of OA, related coefficient (r) and
The range of linearity.
The LLOQ for investigating OA using gradually diluted method with minimum concentration standard working solution, by signal-to-noise ratio S/N
(Signal/Noise) LLOQ of the standard concentration as untested compound when being 10.
2) precision (in batch, between criticizing) and accuracy
Under normal circumstances, the acceptable standard of imprecision is CV < 20% within 3 times of LLOQ concentration；Remaining concentration point CV
< 15%.Investigation method is that compound concentration is the OA quality-control product of 25,500 μ g/mL respectively in blank serum, replication 6 batches,
The each sample replication of every batch of 3 times calculates CV to evaluate betweenrun precision, calculates relative error to evaluate accuracy.It is another right
20 pipe of serum sample parallel processing of two concentration is to evaluate withinrun precision.
3) stability
The stability of sample may influence testing result, since experiment sample is generally stored in 4 DEG C of refrigerators or is placed in liquid phase
In sampling system (4 DEG C), therefore this experiment is dense after saving one day, five days and ten days by the sample for being detected on 4 DEG C of preservations
Degree, and to observe its stability compared with initial value.
4) pollution is carried
It first measures 10 μ g/mL of low concentration sample 10 times, then measures 1000 μ g/mL of high concentration sample 10 times, then measure low
10 μ of concentration samples g/mL10 times analyzes the statistical difference between two batch low concentration sample mean values.Seven, experimental result:
The foundation of 1.UPLC-MS/MS technology measurement OA method
Detect OA when, ion mass-to-charge ratio (m/z) be 281.3/281.3, measurement Isotopic Internal Standard OA- [13C5] when, selection
Ion mass-to-charge ratio 286.3/286.3.Utilize super aqueous solution and 50% methanol: 50% acetonitrile solution is mobile phase, when the reservation of OA
Between be 3.11min, analysis time be 12min (see Fig. 1).Fig. 2 is blank sample detection figure, it is seen that in the equal nothing of OA and internal standard channel
Interference Peaks exist.
2. evaluation of methodology
1) standard curve and quantitative limit
Using OA and interior target concentration ratio as X-axis, OA and interior target peak area ratio are Y-axis, carry out linear regression, obtain the side of recurrence
Journey is y=0.7009x+0.2158, r=0.9940, shows OA linear relationship within the scope of 10~1000 μ g/mL serum-concentrations
Well, the LLOQ of method is 10 μ g/mL, and the range of linearity and quantitative limit are all satisfied the detection to human serum sample.It is shown in Table 1, figure
3。
The standard curve and related coefficient of 1 OA of table
2) precision (in batch, between criticizing) and accuracy
Withinrun precision CV≤3.5%, betweenrun precision≤2.8% of this method, method precision are good；Relative error≤
3.7%, illustrate that accuracy is good, is suitble to the measurement of serum sample.It the results are shown in Table 2,3.
2 batches of accuracy of table, precision
3 withinrun precision of table, instrument precision
3) stability
Observe 3 scrum samples placed at 4 DEG C for 24 hours, behind five days and ten days OA concentration compared with initial value, error
It is 0~5%, shows that sample stability is good.It the results are shown in Table 4
- 4 stability result of table
4) pollution is carried
It first measures 10 μ g/mL of low concentration sample 10 times, then measures 1000 μ of high concentration sample g/mL10 times, then measure low dense
It spends 10 μ g/mL of sample 10 times, no difference of science of statistics between two batch low concentration sample mean values.It is shown in Table -5.
Table -5 carries pollution result
Embodiment 2OA detects clinical principium application
One, research object
Serum sample collection is completed in Shanghai East Hospital.
NAFLD group: subject between in December, 2016 to 2 months 2017 in the institute, make a definite diagnosis by department of traditional Chinese medicine fatty liver disease that calls for specialized treatment outpatient service
It is patient 100 of NAFLD, wherein male 42, female 58, average age 55.6 years old.
Diagnostic criteria:
(1) it is less than 140g/ weeks (women < 70g/ week) without history of drinking history or equivalent amount of alcohol of drinking；
(2) virus hepatitis, drug-induced liver disease, total parenteral nutrition, hepatolenticular degeneration, autoimmune liver disease except
Etc. the specified disease that can lead to fatty liver；
(3) liver ultrasound meets the diagnostic criteria of diffusivity fatty liver；
(4) serum alanine aminotransferase (ALT) or aspartate amino transferase (AST), γ-glutamyl
Transferase (GGT) persistently increases half a year or more.
T2DM group: in the division of endocrinology of institute, outpatient service is diagnosed as T2DM's to subject between in December, 2016 to 2 months 2017
Patient 109, wherein with NAFLD43, average age 57.2 years old.
T2DM diagnostic criteria:
(1) diabetic symptom (more drinks, more food, diuresis, weight loss)+random blood sugar >=11.1mmol/L；
(2)FPG≥7.0mmol/L；
(3) in OGTT experiment, 2hPG >=11.1mmol/L.
Meet any of the above one, after either method check confirmation, diagnosis is set up.
Normal group: the same period is in physical examination of healthy population 50 of Shanghai East Hospital's medical center, wherein male 22,
Female 28, average age 53.8 years old.It is normal to specify FPG, Liver and kidney function, aglycosuria medical history, without infectious diseases are no pernicious swollen
Tumor, no surgery trauma history.
It is stored in after serum sample collection spare in -20 DEG C of refrigerators.
Two, research method
1, steady state method assesses insulin resistance
According to the blood glucose level and insulin level under base state, steady-state model method (HOMA insulin is utilized
Resistance, HOMA-IR) evaluation insulin resistance.
HOMA-IR=fasting insulin (1U/mL) × fasting blood-glucose (mmol/L)/22.5.
2, instrument and material
With embodiment 1
3, the detection of sample OA
Above-mentioned all serum samples are tested according to the concentration of operating procedure shown in embodiment 1, and every batch of detection is all provided with 10
~100 μ g/mL standard curves and blank sample, Quality Control sample.
4, routine biochemistry Indexs measure: using German Roche Diagnistics' Products Co., Ltd's automatic clinical chemistry analyzer and its mating examination
Agent, according to the above-mentioned sample blood glucose of clinical examination conventional detection, insulin (In), cholesterol, triglyceride, low-density lipoprotein
(LDL), high-density lipoprotein (HDL), C reactive protein (CRP), using Japanese TOSOH G8 type glycolated hemoglobin analysis
And matched reagent detection glycosylated hemoglobin (HbA1c).
Three, it statisticallys analyze
It is for statistical analysis to data using SPSS19.0 software.Data are with mean ± standard deviationForm be in
It is existing.Comparison among groups use independent samples t test, and correlation analysis uses pearson method, and the comparison of area under the curve uses Z test,
Significance is P less than 0.05.
Four, interpretation of result
4.1 subject's relevant parameters
4.1.1 Clinical symptoms
Subject's Clinical symptoms is shown in Table 6, T2DM group and NAFLD group except average weight is slightly larger compared with Normal group, and difference has
Statistical significance (P < 0.05) outside, three groups of equal no significant differences on height, age and BMI.NAFLD group ALT and CREA is high
In Normal group, difference is statistically significant (P < 0.05), and although GGT content is not statistically significant, but due to fatty liver
Group GGT data are more dispersed, from the point of view of average value, are much higher than Normal group (51.08 ± 78.56 and 27.13 ± 24.77),
FPG and INS is above healthy control group, and difference is statistically significant (P < 0.05).T2DM group ALT, GGT, FPG and INS is high
In Normal group, difference is statistically significant (P < 0.05).For NAFLD group compared with T2DM group, the latter FPG and INS is obviously high
In the former, difference is statistically significant (P < 0.05).Wherein T2DM merges NAFLD group compared with nonjoinder NAFLD, indices
Without significant difference, but the mean value of the former ALT, GGT, FPG and INS are higher than the latter, and T2DM is prompted to merge NAFLD person's glycolipid generation
It thanks, liver enzyme level is easier to deteriorate.
- 6 subject's Clinical symptoms of table and biochemical indicator Testing index
Note: T2DM is divided to nonjoinder NAFLD and merges two subgroup of NAFLD, but when for statistical analysis with T2DM it is whole into
Row compares.P*, P#, P$Respectively represent the t inspection between NAFLD and normal control, T2DM and normal control and NAFLD and T2DM
It tests.Each index unit is respectively as follows: height/m, weight/kg, BMI (kg/m2), AST (U/L), ALT (U/L), GGT (U/L), ALP
(U/L), LDH (U/L), CREA (μm ol/L), BUN (mmol/L), UA (μm ol/L), TC (mmol/L), TG (mmol/L), HDL
(mmol/L), LDL (mmol/L), FPG (mmol/L), INS (U/mL).
4.1.2 serum OA is horizontal and insulin resistance is assessed
- 7 are shown in Table, compared with normal control, T2DM group and NAFLD group OA content obviously increase, and T2DM group ratio NAFLD
Group OA content is high, and difference is statistically significant (P < 0.05), and wherein T2DM group merges NAFLD (453.8 ± 241.6 μm of ol/L) ratio
Nonjoinder NAFLD person (407.2 ± 202.9 μm of ol/L) OA content is high, but no significant difference (P > 0.05).Using steady
State method calculates insulin resistance index IR, and T2DM group HOMA-IR is significantly higher than control group (P < 0.001), but merges in T2DM group
NAFLD and nonjoinder NAFLD person HOMA-IR is without significant difference (P < 0.05), while NAFLD group HOMA-IR and control group are without aobvious
Sex differernce is write, therefrom it can be found that serum OA level is able to reflect NAFLD compared with HOMA-IR.
- 7 steady state method of table assesses insulin resistance
Note: a:T2DM group contains merging NAFLD group (n=43) and nonjoinder NAFLD group (n=66), corresponding HOMA-IR
For 7.45 ± 5.45 and 6.77 ± 6.2, two groups of no difference of science of statistics (p < 0.05).P*, P#, P$Respectively represent NAFLD with it is normal right
It is examined according to the t between, T2DM and normal control and NAFLD and T2DM.
4.1.3. the correlation analysis of serum OA level and insulin resistance
Using OA as dependent variable, pearson correlation point is carried out using age, BMI, weight, FPG, INS, HOMA-IR as independent variable
Analysis, the results are shown in Table shown in -8.Serum OA is horizontal as the result is shown, in healthy control group with weight and HOMA-IR conspicuousness positive
Close, be positively correlated in T2DM group with HOMA-IR and FPG conspicuousness, in NAFLD group with the significant positive of HOMA-IR, FPG and INS
It closes.The relationship of OA and insulin resistance are further analyzed, table -9,10 serum OA are consistent with HOMA-IR evaluation insulin resistance
Property up to 65.3%,
The correlation of table -8 serum OA level and IR index of correlation
The correlation of table -9 serum OA level and insulin resistance
Table 10. judges the consistency analysis of IR respectively with HOMA-IR, OA
Diagnose consistency 65.3%
4.1.4 serum OA level is for evaluating insulin resistance and the value analysis of NAFLD
ROC (Receiver operating characteristic) curve (figure -4) is drawn, is analyzed when to IR
When, the AUC (area-under-the concentration curve) of OA is 0.689, determines OA by the size of youden index
The cut-off value of auxiliary judgment IR is 235.8 μm of ol/mL, and sensitivity 70.4%, specificity is 63%.When joint FPG is to IR
When being diagnosed, AUC increases to 0.806, and difference is statistically significant (P < 0.05).
When analyzing NAFLD, as shown in figure -5, the AUC of OA is that 0.738, cut-off value is 250.6, sensitive
Degree is 63%, and specificity is 81.7%.When OA joint ALT diagnoses NAFLD, AUG increases to 0.774, and difference has system
Meter learns meaning (P < 0.05).
Conclusion: serum (blood plasma) the OA detection kit and detection method established using the present invention, it is determined that it was detected
Sensitivity, accuracy, repeatability and the range of linearity.259 (109 T2DM patients, 100 are analyzed in clinical principium application
NAFLD and 50 normal control of example) OA content in fasting blood, discovery T2DM, NAFLD patients serum OA level is above health
Control, and T2DM group is higher than NAFLD group.It is poor with healthy control group that the HOMA-IR that stable state Evaluation Method obtains is able to reflect T2DM
Not, but with NAFLD group no difference of science of statistics.T2DM group serum OA level is positively correlated with FPG and HOMA-IR, in NAFLD group
Serum OA level is positively correlated with FPG, INS and HOMA-IR.Serum OA level and HOMA-IR auxiliary diagnosis insulin resistance
Concordance rate up to 65.3%, illustrate that serum (blood plasma) OA level is the blood plasma marker object that an early stage can directly judge IR.This
Outside, for NAFLD auxiliary diagnosis, the AUC of OA is 0.738, when setting cut-off value is 250.6 μm of ol/L, diagnoses NAFLD's
Sensitivity is 63%, and specificity is 81.7%, and when OA joint ALT diagnoses NAFLD, AUG increases to 0.774.Show to move
State monitoring serum OA, ALT and HOMA-IR level variation in the disease progression judgement of auxiliary NAFLD there is important clinic to answer
With value.
Significance of the invention is disclosed serum (blood plasma) OA ultra performance liquid chromatography tandem mass spectrum
(UHPLC-MS/MS) detection method, OA changes of contents in dynamic Quantitative Monitoring blood, for the morning of insulin resistance, NAFLD
Phase Prevention Research has established experiment basis.The present invention be it is a kind of quickly, stablize, reliable diagnosis, treatment and examination of curative effect oleic acid
(OA) diseases related effective tool is suitable for routine clinical and large sample epidemiological survey.
Claims (3)
1. a kind of for detecting the kit of oleic acid concentration in serum or blood plasma, it is characterised in that: including mobile phase A, flowing
Phase B, reference substance stock solution, Isotopic Internal Standard object stock solution, specimen extraction liquid and dilution, the mobile phase A are ultrapure water,
The Mobile phase B is the solution that methanol and acetonitrile are formed according to volume ratio 1:1, and the reference substance stock solution is 20mg/mL
Oleic acid solutions, the Isotopic Internal Standard object stock solution be 20mg/mL OA- [13C5] solution, the specimen extraction liquid is
The solution that methanol and acetonitrile are formed according to volume ratio 1:1, the dilution are the bovine serum albumin(BSA) without free fatty acid,
The concentration of the bovine serum albumin(BSA) is 50mg/mL.
2. the preparation method of the kit of oleic acid concentration, feature in a kind of detection serum described in claim 1 or blood plasma
It is to include the following steps:
1) the step of preparation mobile phase A, ultrapure water ultrasonic degassing is spare；
2) the step of preparation Mobile phase B, methanol is added in acetonitrile, and the volume ratio of methanol and acetonitrile is 1:1, and mixing is equal
Even and ultrasonic degassing is spare；
3) prepare reference substance stock solution the step of, oleic acid standard items are weighed, methanol is added with Autosampler and acetonitrile is pressed
According to the solution of volume ratio 1:1 composition, the concentration of oleic acid is 20mg/mL after addition, is placed in -20 DEG C of refrigerators and protects after being completely dissolved
It deposits spare；
4) prepare Isotopic Internal Standard object stock solution the step of, weigh OA- [13C5] methanol and acetonitrile are added to according to volume ratio
The solution of 1:1 composition, OA- after addition [13C5] concentration be 20mg/mL, saved backup in -20 DEG C of refrigerators after being completely dissolved；
5) prepare specimen extraction liquid the step of, methanol and acetonitrile are taken, forms solution according to volume ratio 1:1, mixes, is spare；
6) prepare dilution the step of, weighs bovine serum albumin(BSA), and the bovine serum albumin(BSA) is free of free fatty acid,
Bovine serum albumin(BSA) is added in phosphate buffered saline solution, so that the concentration of bovine serum albumin(BSA) is 50mg/mL.
3. using the method for oleic acid concentration in kit described in claim 1 detection serum or blood plasma, it is characterised in that packet
Include following steps:
1) prepare standard working solution and quality-control product the step of；Reference substance stock solution is added in dilution, be diluted to 10,
25, the standard working solution of 50,100,500,1000 μ g/mL makees standard curve, be in addition configured to 25 μ g/mL, 500 μ g/mL it is molten
Liquid is as quality-control product；
2) prepare Isotopic Internal Standard object working solution the step of, Isotopic Internal Standard object stock solution is diluted to specimen extraction liquid
The working solution of 100 μ g/mL is deposited in spare in 4 DEG C of refrigerators；
3) a specimen extraction liquid step of the preparation containing Isotopic Internal Standard object, takes Isotopic Internal Standard working solution and specimen extraction liquid
1:14 ratio mixes by volume, deposits in spare in 4 DEG C of refrigerators；
4) prepare sample the step of, sample are filtered, then after equilibrium at room temperature 1 hour using 0.22 μm of disposable filter
It accurately pipettes the filtered sample of 10 μ L to be added in 1.5mL EP pipe, the specimen extraction liquid of the above-mentioned 3) object containing Isotopic Internal Standard is added
100 μ L, vortex oscillation 2min are centrifuged 20 minutes with the revolving speed of 10000rpm, transfer supernatant to liquid chromatographic detection dedicated 96
In orifice plate, all standard curves and quality-control product are according to sample process；
5) a step of use ultra performance liquid chromatography is analyzed, chromatographic parameter are as follows: flowing phase composition is A ultrapure water, and B is methanol
Solution with acetonitrile according to product ratio for 1:1 composition, gradient elution, B phase are initiated with 5%, and flow velocity is 300 μ l/min, sampling volume
For 5 μ l；
6) mass spectrometry procedure, mass spectrometry parameters: autosampler temperature is 4 DEG C, and column temperature is 40 DEG C, uses electrospray ionisation
Source negative ion mode and multiple-reaction monitoring pattern analysis, OA and Isotopic Internal Standard OA- [13C5] detection ion mass-to-charge ratio be
281.3/281.3,286.3/286.3, atomization gas, atomization auxiliary heating gas, gas curtain gas and collision gas be respectively 55psi,
55psi, 40psi and 6psi, Gas2 heating temperature are 450 DEG C；Go cluster voltage, entrance potential, exit potential and spray voltage point
It Wei not -100V, -10V, -5V and -4500V；
7) by the standard solution of step 4) preparation successively from low concentration to high concentration sample introduction, each hybrid standard product solution is continuous
It sample introduction 3 times, is analyzed using step 5) and the method for the Liquid Chromatography-Tandem Mass Spectrometry of step 6)；With oleic acid and Isotopic Internal Standard object
OA-[13C5] concentration ratio is X-axis, the peak area ratio of oleic acid and Isotopic Internal Standard is Y-axis, carries out linear regression, obtains the mark of oleic acid
Directrix curve；
8) using step 5) and step 6) Liquid Chromatography-Tandem Mass Spectrometry method analytical procedure 4) sample to be tested and quality-control product,
Concentration of specimens is calculated according to standard curve.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115078598A (en) * | 2022-05-05 | 2022-09-20 | 天津国科医工科技发展有限公司 | Kit for directly sampling and testing blood concentration sample and application |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120040383A1 (en) * | 2010-08-12 | 2012-02-16 | Wei Jia | Methods and Kits Relating To Metabolite Biomarkers For Colorectal Cancer |
| CN102449161A (en) * | 2009-03-31 | 2012-05-09 | 梅塔博隆公司 | Biomarkers related to insulin resistance and methods using the same |
| CN104777242A (en) * | 2014-01-14 | 2015-07-15 | 中国科学院大连化学物理研究所 | Combined markers, kit and system for diagnosis of polycystic ovarian syndrome |
| WO2017210097A1 (en) * | 2016-06-02 | 2017-12-07 | Metabolon, Inc. | Mass spectrometry method for detection and quantitation of metabolites |
| CN109060977A (en) * | 2018-07-13 | 2018-12-21 | 深圳市绘云生物科技有限公司 | Biomarker and kit and application method for liver fibrosis and liver cirrhosis diagnosis |
-
2018
- 2018-07-23 CN CN201810810874.1A patent/CN109298084A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102449161A (en) * | 2009-03-31 | 2012-05-09 | 梅塔博隆公司 | Biomarkers related to insulin resistance and methods using the same |
| US20120040383A1 (en) * | 2010-08-12 | 2012-02-16 | Wei Jia | Methods and Kits Relating To Metabolite Biomarkers For Colorectal Cancer |
| CN104777242A (en) * | 2014-01-14 | 2015-07-15 | 中国科学院大连化学物理研究所 | Combined markers, kit and system for diagnosis of polycystic ovarian syndrome |
| WO2017210097A1 (en) * | 2016-06-02 | 2017-12-07 | Metabolon, Inc. | Mass spectrometry method for detection and quantitation of metabolites |
| CN109060977A (en) * | 2018-07-13 | 2018-12-21 | 深圳市绘云生物科技有限公司 | Biomarker and kit and application method for liver fibrosis and liver cirrhosis diagnosis |
Non-Patent Citations (2)
| Title |
|---|
| XUAN-MAI T. PERSSON等: "Rapid measurement of plasma free fatty acid concentration and isotopic enrichment using LC/MS", 《JOURNAL OF LIPID RESEARCH》 * |
| 居文政等: "液相色谱-质谱测定鸦胆子油脂肪乳注射液在癌症患者体内的血药浓度 ", 《中国临床药理学杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115078598A (en) * | 2022-05-05 | 2022-09-20 | 天津国科医工科技发展有限公司 | Kit for directly sampling and testing blood concentration sample and application |
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