CN116858646B - Quality control product preparation and application method for non-target metabolism detection - Google Patents
Quality control product preparation and application method for non-target metabolism detection Download PDFInfo
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- 238000003908 quality control method Methods 0.000 title claims abstract description 65
- 238000000034 method Methods 0.000 title claims abstract description 38
- 238000001514 detection method Methods 0.000 title claims abstract description 34
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 230000004060 metabolic process Effects 0.000 title abstract description 8
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 47
- 239000000243 solution Substances 0.000 claims abstract description 32
- WWZKQHOCKIZLMA-UHFFFAOYSA-N octanoic acid Chemical compound CCCCCCCC(O)=O WWZKQHOCKIZLMA-UHFFFAOYSA-N 0.000 claims abstract description 11
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 claims abstract description 10
- UOQHWNPVNXSDDO-UHFFFAOYSA-N 3-bromoimidazo[1,2-a]pyridine-6-carbonitrile Chemical compound C1=CC(C#N)=CN2C(Br)=CN=C21 UOQHWNPVNXSDDO-UHFFFAOYSA-N 0.000 claims abstract description 9
- FTNIPWXXIGNQQF-UHFFFAOYSA-N UNPD130147 Natural products OC1C(O)C(O)C(CO)OC1OC1C(CO)OC(OC2C(OC(OC3C(OC(OC4C(OC(O)C(O)C4O)CO)C(O)C3O)CO)C(O)C2O)CO)C(O)C1O FTNIPWXXIGNQQF-UHFFFAOYSA-N 0.000 claims abstract description 9
- BHQCQFFYRZLCQQ-OELDTZBJSA-N cholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 BHQCQFFYRZLCQQ-OELDTZBJSA-N 0.000 claims abstract description 9
- 229940099563 lactobionic acid Drugs 0.000 claims abstract description 9
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- FJCUPROCOFFUSR-GMMZZHHDSA-N maltopentaose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O[C@H]([C@H](O)CO)[C@H](O)[C@@H](O)C=O)O[C@H](CO)[C@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O[C@@H]3[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O3)O)[C@@H](CO)O2)O)[C@@H](CO)O1 FJCUPROCOFFUSR-GMMZZHHDSA-N 0.000 claims abstract description 9
- 230000008569 process Effects 0.000 claims abstract description 9
- BHQCQFFYRZLCQQ-UHFFFAOYSA-N (3alpha,5alpha,7alpha,12alpha)-3,7,12-trihydroxy-cholan-24-oic acid Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)C(O)C2 BHQCQFFYRZLCQQ-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000004380 Cholic acid Substances 0.000 claims abstract description 8
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- PLSAJKYPRJGMHO-UHFFFAOYSA-N ursolic acid Natural products CC1CCC2(CCC3(C)C(C=CC4C5(C)CCC(O)C(C)(C)C5CCC34C)C2C1C)C(=O)O PLSAJKYPRJGMHO-UHFFFAOYSA-N 0.000 claims abstract description 8
- XUIIKFGFIJCVMT-LBPRGKRZSA-N L-thyroxine Chemical compound IC1=CC(C[C@H]([NH3+])C([O-])=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-LBPRGKRZSA-N 0.000 claims abstract description 7
- 239000007864 aqueous solution Substances 0.000 claims abstract description 7
- 229960004799 tryptophan Drugs 0.000 claims abstract description 7
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- 230000014759 maintenance of location Effects 0.000 claims abstract description 6
- WCGUUGGRBIKTOS-GPOJBZKASA-N (3beta)-3-hydroxyurs-12-en-28-oic acid Chemical compound C1C[C@H](O)C(C)(C)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@@]5(C(O)=O)CC[C@@H](C)[C@H](C)[C@H]5C4=CC[C@@H]3[C@]21C WCGUUGGRBIKTOS-GPOJBZKASA-N 0.000 claims abstract description 5
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 claims abstract description 5
- 239000012452 mother liquor Substances 0.000 claims abstract description 5
- 229940096998 ursolic acid Drugs 0.000 claims abstract description 5
- LUEWUZLMQUOBSB-OUBHKODOSA-N maltotetraose Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@H](CO)O[C@@H](O[C@@H]2[C@@H](O[C@@H](O[C@@H]3[C@@H](O[C@@H](O)[C@H](O)[C@H]3O)CO)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O LUEWUZLMQUOBSB-OUBHKODOSA-N 0.000 claims abstract 2
- 150000002500 ions Chemical class 0.000 claims description 15
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 9
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 5
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 claims description 5
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 4
- 239000005695 Ammonium acetate Substances 0.000 claims description 4
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- 239000000047 product Substances 0.000 description 7
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- 239000000126 substance Substances 0.000 description 4
- MUCRYNWJQNHDJH-UHFFFAOYSA-N Ursonic acid Natural products C1CC(=O)C(C)(C)C2CCC3(C)C4(C)CCC5(C(O)=O)CCC(C)C(C)C5C4=CCC3C21C MUCRYNWJQNHDJH-UHFFFAOYSA-N 0.000 description 3
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- NMELTECMHKKXLF-YNMYPSOUSA-N (3s,4r,5r)-3,4,5,6-tetrahydroxy-1-[(2r,3r,4s,5r,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyhexan-2-one Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C(=O)CO[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O NMELTECMHKKXLF-YNMYPSOUSA-N 0.000 description 1
- 241000222336 Ganoderma Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- OBETXYAYXDNJHR-UHFFFAOYSA-N alpha-ethylcaproic acid Natural products CCCCC(CC)C(O)=O OBETXYAYXDNJHR-UHFFFAOYSA-N 0.000 description 1
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- ACXGEQOZKSSXKV-UHFFFAOYSA-N octanoic acid Chemical compound CCCCCCCC(O)=O.CCCCCCCC(O)=O ACXGEQOZKSSXKV-UHFFFAOYSA-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
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/38—Diluting, dispersing or mixing samples
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8665—Signal analysis for calibrating the measuring apparatus
-
- 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
-
- 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
- G01N2030/042—Standards
- G01N2030/047—Standards external
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- 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)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention belongs to the field of analysis and detection, and provides a quality control product preparation and application method for non-target metabolism detection, which comprises the following steps: taking 50-60 mu L of n-octanoic acid, tryptophan, cholic acid, ursolic acid, maltotetraose, L-thyroxine, lactulose, lactobionic acid and maltopentaose solution with the mother liquor concentration of 10-15 mu g/mL, adding 650-700 mu L of 50-60% methanol aqueous solution, and uniformly mixing to obtain the mixed standard quality control solution with the concentration of 0.5-0.6 mu g/mL. The method of the invention uses the deviation of the peak area variation coefficient (CV%) and the Retention Time (RT) of the quality control of the mixed standard to judge whether the whole set of detection system is stable in the operation process, and the numerical variation is visual in observation and simple in judgment. The method can be used as a long-term quality control monitoring instrument and a system state, is not limited by items and sample types, and is suitable for all instruments and sample types.
Description
Technical Field
The invention belongs to the field of analysis and detection, and particularly relates to a quality control product preparation and application method for non-target metabolism detection.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
Metabonomics is an emerging technology of genomics, transcriptomics, proteomics that appears after genomics, and is an important component of systems biology. Metabonomics mainly examines the change of metabolites or the change of the metabolites with time after a biological system (cells, tissues and the like) is stimulated or disturbed, and the biological process participated by the different metabolites is researched by screening the different metabolites of an experimental group and a control group, so that the living activity mechanism participated by the different metabolites is revealed.
Metabolomics can reflect the current physiological state of an organism more directly and accurately than genomics, transcriptomics and proteomics. If say genomics and proteomics tell you what might happen, then metabonomics tells you what does happen.
The non-target metabolism is a common metabonomics research method, is used for analyzing metabolic pathways and metabolic networks, and has wide application in the fields of clinical diseases, biological medicines, agriculture, forestry, livestock, marine aquatic products and the like.
In the detection process, whether the whole set of detection system is stable in the operation process can influence the accuracy of the search result, and currently, a quality control method is adopted to judge the stability of the detection result. However, the existing quality control method has the following problems:
1. the stability of the detection result cannot be judged by using specific numerical values, and only subjective judgment can be made by using the overlapping condition of the superimposed graphs.
2. The existing quality control method is different in projects, the sample type can be changed, and the stability of the instrument in different periods can not be recorded and monitored for a long time.
Disclosure of Invention
In order to solve the problems, the invention provides a quality control product preparation and application method for non-target metabolism detection.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
in a first aspect of the present invention, there is provided a method of formulating a quality control for use in non-target metabolic assays, comprising:
taking n-Octanoic acid (Octanoic acid) and tryptophan with mother liquor concentration of 10-15 mug/mL
(L-trytophan), cholic acid (Cholic acid), ursolic acid (Ursonic acid), maltotetraose (Maltotetraose), L-Thyroxine (L-Thyroxine), lactuLose (Lactulose), lactobionic acid (Lactobionic acid) and Maltopentaose (Maltopentaose) 50-60 mu L are added with 650-700 mu L of 50-60% methanol aqueous solution, and mixed evenly to prepare the mixed standard quality control solution with the concentration of 0.5-0.6 mu g/mL.
In a second aspect of the present invention, there is provided a method for evaluating the stability of instruments and systems used in non-target metabolic detection, comprising:
preparing a mixed label quality control solution according to the method;
preparing a mixed sample quality control solution;
preprocessing a sample, performing liquid chromatography-mass spectrometry detection, and collecting detection data;
judging whether the whole set of detection system is stable in the operation process according to the deviation of the peak area variation coefficient CV% and the retention time RT of the quality control of the mixed standard, and obtaining the product.
In a third aspect, the invention provides a kit for preparing a quality control product in non-target metabolism detection, which contains the mixed standard quality control solution prepared by the preparation method.
The beneficial effects of the invention are that
(1) The method of the invention uses the deviation of the peak area variation coefficient (CV%) and the Retention Time (RT) of the quality control of the mixed standard to judge whether the whole set of detection system is stable in the operation process, and the numerical variation is visual in observation and simple in judgment.
(2) The method can be used as a long-term quality control monitoring instrument and a system state, is not limited by items and sample types, and is suitable for all instruments and sample types.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a map of positive mode Maltopentaose (1.37 min), L-gyroxin (11.05 min), lactulose (12.49 min), lactobionic acid (14.45 min);
FIG. 2 is a graph of negative mode Maltotetraose (1.35 min), L-tryptophane (5.54 min), octanoc acid (10.53 min), L-tyroxin (11.21 min), cholic acid (11.87 min), ursonic acid (14.10 min);
fig. 3 is a sequence chart of the discharging machine of example 1.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
A method of formulating a quality control for use in non-target metabolic assays, comprising:
taking 50-60 mu L of n-octanoic acid, tryptophan, cholic acid, ursolic acid, maltotetraose, L-thyroxine, lactulose, lactobionic acid and maltopentaose solution with the mother liquor concentration of 10-15 mu g/mL, adding 650-700 mu L of 50-60% methanol aqueous solution, and uniformly mixing to obtain the mixed standard quality control solution with the concentration of 0.5-0.6 mu g/mL.
An evaluation method for stability of instruments and systems in non-target metabolism detection, comprising:
preparing a mixed label quality control solution according to the method;
preparing a mixed sample quality control solution;
preprocessing a sample, performing liquid chromatography-mass spectrometry detection, and collecting detection data;
judging whether the whole set of detection system is stable in the operation process according to the deviation of the peak area variation coefficient CV% and the retention time RT of the quality control of the mixed standard, and obtaining the product.
In some embodiments, comprising:
transferring 100-120 mu L of sample, adding into a centrifuge tube, adding 5-6 times of methanol into the centrifuge tube, mixing, precipitating to remove protein, standing in an ice bath for 5-10 min, centrifuging for the first time at 4-4.5 ℃, transferring 500-600 mu L of supernatant, adding 1/2-1/2 of ultrapure water, namely 250 mu L, mixing, centrifuging for the second time at 4-4.5 ℃, and transferring the supernatant to an upper sample tube for standby.
In some embodiments, the specific conditions of the first centrifugation are 10000-12000 rpm for 20-25 min.
In some embodiments, the specific conditions for the secondary centrifugation are 10000-12000 rpm for 5-8 min.
In some embodiments, the detection conditions for liquid chromatography are a chromatographic column: thermo Hypesil Gold column temperature: 38-40 ℃, flow rate: 0.3-0.5 mL/min.
In some embodiments, the positive mode: mobile phase a:0.1% formic acid in water;
mobile phase B: methanol;
negative mode: mobile phase a:5mM ammonium acetate in water;
mobile phase B: methanol.
In some embodiments, the chromatographic gradient elution procedure: 0-1.5 min, the proportion of mobile phase A is 98%;
1.5-12 min, the proportion of the mobile phase A is reduced from 98% to 0;
for 12-14 min, the ratio of mobile phase A is kept to be 0;
14-14.1 min, the proportion of mobile phase A is increased from 0 to 98%;
the proportion of mobile phase A is kept at 98% for 14.1-17 min.
In some embodiments, the scan range is selected from m/z100-1500;
ion source: an H-ESI source;
positive Ion (3500V);
negative Ion (Negative Ion): 3000V;
sheath Gas (Sheath Gas) (: 40Arb;
auxiliary Gas (Aux Gas): 10Arb;
sweep Gas (Sweep Gas): 1Arb;
ion transport tube temperature (Ion Transfer Tube Temp) 320 ℃;
atomizer temperature (Vaporizer Temp): 300 ℃.
In some embodiments, the overall machine-on sequence is split mode machine-on first positive/negative mode and then on the other mode;
the machine arranging sequence comprises the following steps: 3-needle blank solvent solution+3-needle mixed sample quality control+3-needle mixed sample quality control+10 samples of the first group+1-needle mixed sample quality control+1-needle blank solvent solution+1-needle mixed sample quality control+10 samples of the second group+1-needle mixed sample quality control+1-needle blank solvent solution+1-needle mixed sample quality control … …
The mixed label quality control method can also be used for other detection such as targeting detection, quasi-targeting/wide targeting and the like.
The invention will now be described in further detail with reference to the following specific examples, which should be construed as illustrative rather than limiting.
Example 1
1. Instrument for measuring and controlling the intensity of light
| Sequence number | Instrument type | Model number | Branding |
| 1 | Mass spectrometer | YH HR 1080MD | English Cheng Shengwu |
| 2 | Table type high-speed refrigerated centrifuge | H2050R | Hunan instrument |
| 3 | Ultrasonic cell grinder | JY92-IIN | Ningbo new glossy ganoderma |
| 4 | Vortex oscillator | Vortex-Genie 2(560E) | Scientific Industries |
| 5 | Vacuum centrifugal concentrator | Auto R1-Plus | Ji Aim |
| 6 | Electronic balance | AS 60/220.R2 | RADWAG |
2. Reagent(s)
| Sequence number | Reagent(s) | Purity of | CAS number | Branding |
| 1 | Methanol | LC-MS Grade | 67-56-1 | Thermo Fisher |
| 2 | Formic acid | LC-MS Grade | 64-18-6 | Thermo Fisher |
| 3 | Ammonium acetate | LC-MS Grade | 631-61-8 | Thermo Fisher |
| 4 | Water and its preparation method | Ultrapure water | - | Chen's disease |
| 5 | Octanoic acid | 99％ | 124-07-2 | Selleck |
| 6 | L-Tryptophan | 99％ | 73-22-3 | Selleck |
| 7 | Cholic acid | 99％ | 81-25-4 | Selleck |
| 8 | Ursonic acid | 99％ | 6246-46-4 | Selleck |
| 9 | Maltotetraose | 99％ | 34612-38-9 | Selleck |
| 10 | L-Thyroxine | 99％ | 51-48-9 | Selleck |
| 11 | LactuLose | 99％ | 4618-18-2 | Selleck |
| 12 | Lactobionic acid | 99％ | 96-82-2 | Selleck |
| 13 | Maltopentaose | 99％ | 1668-09-3 | Selleck |
3. Solution preparation
1. Preparation of mixed standard quality control solution
And respectively and accurately transferring 50 mu L of n-octanoic acid, tryptophan, cholic acid, ursolic acid, maltotetraose, L-thyroxine, lactulose, lactobionic acid and maltopentaose with the mother liquor concentration of 10 mu g/mL, adding 650 mu L of 50% methanol aqueous solution, and uniformly mixing to obtain the mixed standard quality control solution with the concentration of 0.5 mu g/mL.
2. Preparation of mixed sample quality control solution
And respectively and accurately transferring 50 mu L of samples, and uniformly mixing to prepare the mixed sample quality control solution.
The sample is blood plasma, and the specific collection method is as follows:
blood is collected by 2mL according to the current venous blood collection method, the blood is slowly injected into an anticoagulation tube, and is centrifuged for 15min at 3000rpm and 4 ℃, and the upper plasma layer is taken into a centrifuge tube with 1.5mL for standby.
4. Sample pretreatment
Transferring 100 μl sample, adding into 1.5mL centrifuge tube, adding 5 times volume total 500 μl methanol (i.e. methanol/water volume ratio is 4/1) into the 1.5mL centrifuge tube, mixing, precipitating to remove protein, standing in ice bath for 5min, centrifuging at 10000rpm for 20min at 4deg.C, transferring 500 μl of supernatant solution, adding 1/2 volume ultrapure water (i.e. 250 μl), mixing, centrifuging at 10000rpm for 5min at 4deg.C, and collecting supernatant solution to the upper sample tube for standby.
5. Instrument parameters
1. Chromatographic parameters
Chromatographic column: thermo Hypesil Gold (2.1. Times.100 mm,1.9 μm)
Column temperature: 40 DEG C
Flow rate: 0.3mL/min
Positive mode: mobile phase a:0.1% formic acid aqueous solution
Mobile phase B: methanol
Negative mode: mobile phase a:5mM ammonium acetate aqueous solution
Mobile phase B: methanol
Chromatographic gradient elution procedure: 0-1.5 min, the proportion of mobile phase A is 98%;
1.5-12 min, the proportion of the mobile phase A is reduced from 98% to 0;
for 12-14 min, the ratio of mobile phase A is kept to be 0;
14-14.1 min, the proportion of mobile phase A is increased from 0 to 98%;
the proportion of mobile phase A is kept at 98% for 14.1-17 min.
2. Mass spectral parameters
The scanning range is selected from m/z100-1500;
ion source: H-ESI source
Positive ions 3500V;
negative ion of 3000V
Sheath gas 40Arb
Auxiliary gas 10Arb
Purge gas 1Arb
The temperature of the ion transmission tube is 320 DEG C
The atomizer temperature is 300 DEG C
6. Sequencing of machine
The whole machine-loading sequence is divided modes of machine loading, namely a positive mode/a negative mode of machine loading firstly, and then another mode of machine loading;
sequencing: the specific examples of the 3-needle blank solvent solution+3-needle mixed label quality control+3-needle mixed sample quality control+10 samples of the first group+1-needle mixed sample quality control+1-needle blank solvent solution+1-needle mixed label quality control+10 samples of the second group+1-needle mixed sample quality control+1-needle blank solvent solution+1-needle mixed label quality control … … are shown in fig. 3.
7. 100 sample detection data results
1. Positive mode mixed label quality control result
2. Positive mode mixed label quality control result information
| No. | Compound—POS | MS1 | RT(min) | Area(CV％) | RT(max-min) |
| 1 | Lactulose | 343.2957 | 12.49 | 5.14 | 0.01 |
| 2 | Lactobionic acid | 359.3158 | 14.58 | 9.93 | 0.03 |
| 3 | L-Thyroxine | 777.6951 | 11.05 | 4.31 | 0.02 |
| 4 | Maltopentaose | 829.2824 | 1.37 | 6.08 | 0.01 |
3. Negative mode mixed label quality control result
4. Negative mode mixed label quality control result information
The method comprises the steps that 4 substances are selected in a positive mode, 6 substances are selected in a negative mode, mixed and prepared into mixed standard quality control solution, and in the process of sample on-machine detection, the mixed standard quality control solution and the traditional mixed standard quality control solution are arranged together, and a group of mixed standard quality control and mixed standard quality control are inserted into every 10 samples;
the mixed standard quality control of the positive mode 4 substances and the negative mode 6 substances is characterized in that the peak area CV% is less than 15% in the detection process of 100 samples, the retention time deviation is less than 0.1min, and the result is stable and reliable.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method of formulating a quality control for use in non-target metabolic assays, comprising:
taking 50-60 mu L of n-octanoic acid, tryptophan, cholic acid, ursolic acid, maltotetraose, L-thyroxine, lactulose, lactobionic acid and maltopentaose solution with the mother liquor concentration of 10-15 mu g/mL, adding 650-700 mu L of 50-60% methanol aqueous solution, and uniformly mixing to obtain the mixed standard quality control solution with the concentration of 0.5-0.6 mu g/mL.
2. A method for evaluating the stability of an instrument and system for use in non-target metabolic detection, comprising:
preparing a mixed label quality control solution according to the method of claim 1;
preparing a mixed sample quality control solution;
preprocessing a sample, performing liquid chromatography-mass spectrometry detection, and collecting detection data;
and judging whether the whole set of detection system is stable in the operation process according to the deviation of the peak area variation coefficient CV% and the retention time RT of the quality control of the mixed standard.
3. The evaluation method according to claim 2, comprising:
transferring 100-120 mu L of sample, adding into a centrifuge tube, adding 5-6 times of methanol into the centrifuge tube, mixing, precipitating to remove protein, standing in an ice bath for 5-10 min, centrifuging for the first time at 4-4.5 ℃, transferring 500-600 mu L of supernatant solution after centrifugation, adding 250 mu L of ultrapure water, mixing, centrifuging for the second time at 4-4.5 ℃, and transferring supernatant solution into an upper sample tube for standby.
4. The method according to claim 3, wherein the specific condition of the first centrifugation is 10000 to 12000rpm for 20 to 25 minutes;
or, the specific condition of the secondary centrifugation is 10000-12000 rpm, and the centrifugation is carried out for 5-8 min.
5. The method according to claim 2, wherein the detection conditions of the liquid chromatography are a column: thermo Hypesil Gold column temperature: 38-40 ℃, flow rate: 0.3-0.5 mL/min.
6. The evaluation method according to claim 2, wherein the positive mode: mobile phase a:0.1% formic acid in water;
mobile phase B: methanol;
negative mode: mobile phase a:5mM ammonium acetate in water;
mobile phase B: methanol.
7. The method of evaluation of claim 2, wherein the chromatographic gradient elution procedure: 0-1.5 min, the proportion of mobile phase A is 98%;
1.5-12 min, the proportion of the mobile phase A is reduced from 98% to 0;
for 12-14 min, the ratio of mobile phase A is kept to be 0;
14-14.1 min, the proportion of mobile phase A is increased from 0 to 98%;
the proportion of mobile phase A is kept at 98% for 14.1-17 min.
8. The method of evaluation of claim 2, wherein the scan range is selected from m/z100-1500;
ion source: an H-ESI source;
positive ions 3500V;
negative ions of 3000V;
sheath gas 40Arb;
auxiliary gas 10Arb;
1Arb of purge gas;
the temperature of the ion transmission tube is 320 ℃;
the atomizer temperature was 300 ℃.
9. The evaluation method according to claim 2, wherein the overall machine-on sequence is a split mode machine-on first a positive/negative mode and then another mode;
sequencing: 3-needle blank solvent solution+3-needle mixed label quality control+3-needle mixed sample quality control+10 samples of the first group+1-needle mixed sample quality control+1-needle blank solvent solution+1-needle mixed label quality control+10 samples of the second group+1-needle mixed sample quality control+1-needle blank solvent solution+1-needle mixed label quality control.
10. A kit for quality control preparation in non-target metabolic detection, comprising the mixed standard quality control solution prepared by the method of claim 1.
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