CN114624375B - Method for detecting content of carboxymethyl lysine in potato chips - Google Patents
Method for detecting content of carboxymethyl lysine in potato chips Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 46
- 235000013606 potato chips Nutrition 0.000 title claims abstract description 30
- RRUYWEMUWIRRNB-LURJTMIESA-N (2s)-6-amino-2-[carboxy(methyl)amino]hexanoic acid Chemical compound OC(=O)N(C)[C@H](C(O)=O)CCCCN RRUYWEMUWIRRNB-LURJTMIESA-N 0.000 title claims abstract description 7
- 239000000243 solution Substances 0.000 claims abstract description 56
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims abstract description 38
- 239000007788 liquid Substances 0.000 claims abstract description 25
- 238000002156 mixing Methods 0.000 claims abstract description 20
- 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 abstract description 19
- 235000019253 formic acid Nutrition 0.000 claims abstract description 19
- 239000006228 supernatant Substances 0.000 claims abstract description 19
- 230000007062 hydrolysis Effects 0.000 claims abstract description 15
- 238000006460 hydrolysis reaction Methods 0.000 claims abstract description 15
- 108091005804 Peptidases Proteins 0.000 claims abstract description 14
- 239000004365 Protease Substances 0.000 claims abstract description 14
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims abstract description 14
- 230000002378 acidificating effect Effects 0.000 claims abstract description 14
- 238000002414 normal-phase solid-phase extraction Methods 0.000 claims abstract description 14
- 239000003480 eluent Substances 0.000 claims abstract description 13
- 238000005238 degreasing Methods 0.000 claims abstract description 12
- 239000008055 phosphate buffer solution Substances 0.000 claims abstract description 12
- 238000001035 drying Methods 0.000 claims abstract description 9
- 238000001914 filtration Methods 0.000 claims abstract description 9
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- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 claims abstract description 5
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- 150000002500 ions Chemical class 0.000 claims description 42
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 24
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 23
- 239000007789 gas Substances 0.000 claims description 16
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 12
- 229910052757 nitrogen Inorganic materials 0.000 claims description 11
- 239000012071 phase Substances 0.000 claims description 10
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 claims description 8
- 238000010828 elution Methods 0.000 claims description 8
- 238000001819 mass spectrum Methods 0.000 claims description 8
- 238000001291 vacuum drying Methods 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
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- 239000007924 injection Substances 0.000 claims description 6
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- HQVFCQRVQFYGRJ-UHFFFAOYSA-N formic acid;hydrate Chemical compound O.OC=O HQVFCQRVQFYGRJ-UHFFFAOYSA-N 0.000 claims description 5
- 230000003301 hydrolyzing effect Effects 0.000 claims description 5
- 238000002013 hydrophilic interaction chromatography Methods 0.000 claims description 5
- 102000004190 Enzymes Human genes 0.000 claims description 4
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- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 2
- OQVOZFPJUCUPLW-LURJTMIESA-N N-(1-Carboxymethyl)-L-lysine Chemical class NCCCC[C@@H](C(O)=O)NCC(O)=O OQVOZFPJUCUPLW-LURJTMIESA-N 0.000 claims description 2
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 2
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- 229910001873 dinitrogen Inorganic materials 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 6
- 238000001514 detection method Methods 0.000 description 42
- 230000000052 comparative effect Effects 0.000 description 21
- MKYBYDHXWVHEJW-UHFFFAOYSA-N N-[1-oxo-1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propan-2-yl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(C(C)NC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 MKYBYDHXWVHEJW-UHFFFAOYSA-N 0.000 description 13
- 238000012360 testing method Methods 0.000 description 13
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 11
- 238000010811 Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Methods 0.000 description 10
- -1 BEH Amide Chemical class 0.000 description 8
- 235000013305 food Nutrition 0.000 description 8
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
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- 235000002595 Solanum tuberosum Nutrition 0.000 description 2
- 244000061456 Solanum tuberosum Species 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 239000003708 ampul Substances 0.000 description 2
- GHQPBDDZGPAVJP-UHFFFAOYSA-N azanium;methanol;hydroxide Chemical compound N.O.OC GHQPBDDZGPAVJP-UHFFFAOYSA-N 0.000 description 2
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- IRXSLJNXXZKURP-UHFFFAOYSA-N fluorenylmethyloxycarbonyl chloride Chemical compound C1=CC=C2C(COC(=O)Cl)C3=CC=CC=C3C2=C1 IRXSLJNXXZKURP-UHFFFAOYSA-N 0.000 description 2
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- 229920006393 polyether sulfone Polymers 0.000 description 2
- 229920001155 polypropylene Polymers 0.000 description 2
- 235000012015 potatoes Nutrition 0.000 description 2
- 230000007065 protein hydrolysis Effects 0.000 description 2
- 230000002797 proteolythic effect Effects 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 229910000033 sodium borohydride Inorganic materials 0.000 description 2
- 239000012279 sodium borohydride Substances 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- MTJGVAJYTOXFJH-UHFFFAOYSA-N 3-aminonaphthalene-1,5-disulfonic acid Chemical compound C1=CC=C(S(O)(=O)=O)C2=CC(N)=CC(S(O)(=O)=O)=C21 MTJGVAJYTOXFJH-UHFFFAOYSA-N 0.000 description 1
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- 101710117545 C protein Proteins 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
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- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- CXZGQIAOTKWCDB-UHFFFAOYSA-N perfluoropentanoic acid Chemical compound OC(=O)C(F)(F)C(F)(F)C(F)(F)C(F)(F)F CXZGQIAOTKWCDB-UHFFFAOYSA-N 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000006920 protein precipitation Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
Classifications
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- 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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
-
- 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/045—Standards internal
-
- 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
- G01N2030/062—Preparation extracting sample from raw material
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- 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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
- G01N2030/8818—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving amino acids
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- 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 discloses a method for detecting the content of carboxymethyl lysine in potato chips, which comprises the following steps: weighing the sample and degreasing; preparing an acidic protease solution by using a phosphate buffer solution; adding an acidic protease solution into a centrifuge tube filled with the degreased sample, adding an isotope internal standard, and finally sealing the centrifuge tube, and uniformly mixing by vortex to hydrolyze the sample; centrifuging to collect supernatant after hydrolysis is completed, leaching residues in the sample by using formic acid solution and centrifuging to collect supernatant; mixing and filtering the hydrolyzed supernatant and the supernatant extracted by the formic acid solution to obtain a pretreated sample liquid; extracting and purifying the pretreated sample liquid by adopting an MCX solid phase extraction column to obtain eluent, drying, redissolving and filtering the eluent to obtain a solution to be detected; and detecting the solution to be detected by adopting an ultra-high performance liquid chromatography-tandem mass spectrometry method, and calculating the content of CML by using an internal standard curve. The invention has the advantages of simple operation, high accuracy, good sensitivity and high precision.
Description
Technical Field
The invention relates to the technical field of food detection, in particular to carboxymethyl lysine (N) in potato chips ε -carboxymethyl, CML) content.
Background
Most foods can generate CML in the hot processing process, and the CML can accumulate in a plurality of different tissues and organs after entering a human body through food media, and can directly influence the functions of the tissues and organs after reaching a certain quantity, so that pathological changes of the organism are caused. CML is closely related to the occurrence of many diseases in the human body, and can promote the development of diseases such as diabetes, nephropathy, atherosclerosis and the like and the rapid aging of organs of the human body. Potatoes contain rich starch, protein and other CML-producing precursor substances, and a large amount of CML is produced during food processing, particularly deep processing such as frying and baking, by using potatoes as raw materials.
Conventional methods for detecting the CML content mainly include Enzyme-linked immunosorbent assay (Enzyme-Linked Immunosorbent Assay, ELISA), gas chromatography-mass spectrometry (Gas Chromatography-Mass Spectrometry, GC-MS), reversed-Phase-high performance liquid chromatography (RP-HPLC), liquid chromatography-mass spectrometry (Liquid Chromatography-Mass Spectrometry, LC-MS), and fluorescence sensing detection methods, and these methods have certain limitations at present. Because the sample matrix is complex, the ELISA method is easy to be interfered by impurities, the specific antibody is difficult to select, and the quantitative analysis result is unreliable. In both the GC-MS method and the RP-HPLC method, complicated pre-column derivatization treatment is required for the sample in order to improve the detection sensitivity and reduce the detection limit. In the LC-MS method, if the flow pattern of the mobile phase is changed due to the "column-out effect", retention and diffusion of the sample can significantly widen the chromatographic peak, resulting in a decrease in column efficiency. Although the fluorescence sensing detection method is simple in pretreatment, rapid, efficient and high in sensitivity, the detection limit (Limit of Detection, LOD) of the method is high, and the detection capability of trace CML in food is still to be further improved. In addition, since CML is at C 18 The retention of the column is poor, and it is necessary to add a trifluoroacetic acid or a nonafluorovaleric acid plasma pair reagent to the mobile phase to increase the retention, but these reagents increase the retention time and simultaneously cause a decrease in detection sensitivity due to inhibition of ionization efficiency, and it is difficult to clean the column and cause damage to the column. At present, no standard method for detecting the CML content in food is recommended at home and abroad.
Chinese patent CN103293243 discloses a method for detecting CML content in food, and specifically discloses pretreatment steps such as degreasing, sodium borohydride reduction, trichloroacetic acid precipitation of protein, hydrolysis of 110 ℃ protein hydrochloric acid (6.0 mol/L), derivatization of 9-fluorenylmethylchloroformate, solid phase extraction, and redissolution. However, this method is time-consuming, cumbersome to operate, and causes environmental pollution and corrosion of equipment in the laboratory due to the high concentration and temperature of hydrochloric acid used for proteolysis. Research and development of a new method for detecting CML content, which is simple and convenient to operate, environment-friendly, accurate and rapid, is urgent.
Disclosure of Invention
Aiming at the problems of long time consumption, complicated operation steps, serious pollution to the environment by concentrated acid, serious corrosion to instruments and the like in the prior art, the invention aims to provide a detection method for the CML content in the potato chips, which has obvious advantages compared with the existing method.
The invention is realized in the following way:
carboxymethyl lysine (N) in potato chips ε -carboxymethyl, CML) content detection method, comprising the following steps:
(1) Weighing a sample and degreasing the sample;
(2) Preparing an acidic protease solution by using a phosphate buffer solution;
(3) Adding an acidic protease solution into a centrifuge tube filled with the degreased sample, then adding an isotope internal standard, and finally sealing the centrifuge tube, and uniformly mixing by vortex to hydrolyze the sample;
(4) Centrifuging to collect supernatant after sample hydrolysis is completed, leaching residues in the sample with formic acid solution and centrifuging to collect supernatant; mixing and filtering the supernatant after sample hydrolysis and the supernatant after formic acid solution leaching to obtain a pretreated sample liquid;
(5) Carrying out solid phase extraction purification on the pretreated sample liquid by adopting an MCX solid phase extraction column to obtain an eluent, and then drying, redissolving and filtering the eluent to obtain a solution to be detected;
(6) And detecting the solution to be detected by adopting an ultra-high performance liquid chromatography-tandem mass spectrometry (Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry, UPLC-MS/MS), and calculating the content of CML by using an internal standard curve.
The degreasing step in the step (1) is as follows: grinding potato chips to particle size of 0.4-0.5-mm, adding n-hexane into the potato chips, mixing, centrifuging at 3500-4500r/min for 2-4min, discarding n-hexane, degreasing with n-hexane for 3 times, and drying with nitrogen at 35-40deg.C to volatilize and remove n-hexane.
The phosphate buffer solution in the step (2) is prepared from 0.15 to 0.25mol/L H 3 PO 4 Solution and NaH with concentration of 0.15-0.25mol/L 2 PO 4 The phosphate buffer solution is prepared by the solution, and the pH value of the phosphate buffer solution is 2-4.
The enzyme activity of the acid protease in the step (2) is 45000-55000U/g, and the concentration of the acid protease in the solution is 0.035-0.045 g/mL.
Adding an acidic protease solution into a centrifuge tube containing a degreased sample in the step (3), wherein the content of the acidic protease in the sample is 4500-5500U/g; the isotopic internal standard of CML is deuterated carboxymethyl lysine (N ε -carboxymethyllysine-D 4 ，CML-D 4 ) The concentration is 4.0-6.0 mug/mL.
And (3) sealing the centrifuge tube, and then uniformly mixing by vortex, hydrolyzing 10-14 and h in a water bath at the temperature of 35-40 ℃ for 1 time every 25-35min, so that the hydrolysis is uniform.
The centrifugal speed in the step (4) is 3500-4500r/min, and the centrifugal time is 3-5min.
The volume concentration of the formic acid solution in the step (4) is 4-6.0%, and the residual in the sample is leached by the formic acid solution and then centrifuged for 3-5min at 3500-4500r/min, and the process is repeated for at least 3 times.
The purification method in the step (5) comprises the following steps: after pretreatment is carried out on the MCX solid phase extraction column, the pretreated sample obtained in the step (4) is loaded, and then is leached by formic acid water solution with the volume concentration of 4% -6.0%, and is dried by vacuum pumping; eluting with methanol, and vacuum drying; discarding all effluent; eluting with 12-18% ammonia water and methanol solution, and vacuum drying; collecting eluent, drying the eluent with nitrogen at 35-40 ℃, and re-dissolving with ultrapure water to obtain purified liquid to be tested.
The liquid chromatography conditions in the step (6) are as follows: an ACQUITY ultra high performance liquid chromatograph; chromatographic column: ACQUITY BEH Amide HILIC column (2.1X100 mm,1.7μm); protective column: ACQUITY BEH Amide Vanguard column (1.7 μm); column temperature: 35 ℃; sample injection amount: 5. mu L; run time: 7.0min; mobile phase: phase A is 0.1% formic acid aqueous solution, and phase B is acetonitrile; gradient elution was performed at 0.30mL/min with the following elution parameters: 0-2.0min 10% A, 2.0-6.0min 20% A, 6.0-6.1min 90% A, and6.1-7.0min10% A;
the mass spectrometry conditions were: XEVO TQ triple quadrupole tandem mass spectrometer; ion source mode: ESI (electronic service provider interface) + The method comprises the steps of carrying out a first treatment on the surface of the Monitoring mode: MRM; capillary voltage: 2.0 A kV; ion source temperature: 150 ℃; desolventizing gas temperature: 350 ℃; desolventizing gas flow rate: 650L/h; taper hole air flow rate: 50L/h; the desolventizing gas and the taper hole gas are nitrogen; the collision gas is argon, CML and CML-D 4 The mass spectrum parameters of (2) are as follows: CML m/z 205.22-m/z 84.00 qualitative, m/z 205.22-m/z 130.00 quantitative; CML-D 4 m/z 209.00-m/z 87.70.
Compared with the prior art, the method avoids the use of high-concentration hydrochloric acid and high-temperature hydrolysis of protein, and adopts 40 ℃ acid protease for hydrolysis, so that the method is environment-friendly to a laboratory and can not cause corrosion to instruments and equipment. The enzyme hydrolysis temperature is greatly reduced compared with the hydrolysis temperature of hydrochloric acid, and the CML content regenerated from the intermediate product at the temperature is extremely low, so that the operation step of reducing the intermediate product by sodium borohydride is also omitted. The invention adopts HILIC column to replace C 18 The chromatographic column has a retention time of 4.68 min, and the sample pretreatment step thus avoids the use of ion pair reagents that affect the detection sensitivity and damage the chromatographic column, and cumbersome 9-fluorenylmethyl chloroformate derivatization operations. The invention is especially suitable for detecting the CML content in foods such as potato chips and the like with higher combined CML and lower free CML. The invention has the characteristics of simple and convenient operation method, high accuracy, good sensitivity and high precision.
Drawings
The internal standard curve of the CML of fig. 1.
FIG. 2 CML and CML-D 4 Is a primary mass spectrum of (a).
FIG. 3 CML and CML-D 4 Is a secondary mass spectrum of (c).
Fig. 4 total ion flow chromatogram of CML standard.
Figure 5 quantitative daughter ion flow chromatograms of CML standards.
FIG. 6 CML-D 4 Internal standard ion flow chromatograms.
FIG. 7 is a CML quantitative daughter ion flow chromatogram of sample 1 in example 1.
FIG. 8 CML-D of sample 1 in example 1 4 Internal standard ion flow chromatograms.
FIG. 9 CML quantitative daughter ion flow chromatogram for sample 2 in example 1.
FIG. 10 CML-D of sample 2 in example 1 4 Internal standard ion flow chromatograms.
FIG. 11 CML quantitative daughter ion flow chromatogram for sample 3 in example 1.
FIG. 12 CML-D of sample 3 in example 1 4 Internal standard ion flow chromatograms.
FIG. 13 is a CML quantitative daughter ion flow chromatogram of sample 4 in example 1.
FIG. 14 CML-D of sample 4 in example 1 4 Internal standard ion flow chromatograms.
FIG. 15 CML quantitative daughter ion flow chromatogram for sample 5 in example 1.
FIG. 16 CML-D of sample 5 of example 1 4 Internal standard ion flow chromatograms.
FIG. 17 is a CML quantitative daughter ion flow chromatogram of sample 1 of comparative example 1.
FIG. 18 CML-D of sample 1 of comparative example 1 4 Internal standard ion flow chromatograms.
FIG. 19 is a CML quantitative daughter ion flow chromatogram of sample 2 of comparative example 1.
FIG. 20 CML-D of sample 2 in comparative example 1 4 Internal standard ion flow chromatograms.
FIG. 21 is a CML quantitative daughter ion flow chromatogram of sample 3 in comparative example 1.
FIG. 22 CML-D of sample 3 of comparative example 1 4 Internal standard ion flow chromatograms.
FIG. 23 is a CML quantitative daughter ion flow chromatogram of sample 4 of comparative example 1.
FIG. 24 CML-D of sample 4 of comparative example 1 4 Internal standard ion flow chromatograms.
FIG. 25 is a CML quantitative daughter ion flow chromatogram of sample 5 of comparative example 1.
FIG. 26 CML-D of sample 5 of comparative example 1 4 Internal standard ion flow chromatograms.
Detailed Description
Example 1
5 kinds of potato chips with different brands are respectively pretreated by the following treatment methods:
(1) Weighing a sample and degreasing the sample: a sample of potato chips 0.10 g (to the nearest 1.0 mg) ground to a particle size of about 0.5mm was weighed and placed in a polypropylene centrifuge tube having a volume of 10 mL. Adding 5.0. 5.0 mL n-hexane into the sample, fully vortex mixing, centrifuging at 4000 r/min for 3.0 min, and discarding an n-hexane layer; the degreasing process of the normal hexane is carried out for 3 times to thoroughly remove fat, and finally the sample is dried by blowing nitrogen at 40 ℃ to volatilize and remove the normal hexane, so as to obtain a degreased sample;
(2) Preparing an acidic protease solution by using a phosphate buffer solution: with H at a concentration of 22.0. 22.0 mL at 0.20 mol/L 3 PO 4 Solution and NaH with concentration of 900.0. 900.0 mL of 0.20 mol/L 2 PO 4 Preparing a phosphate buffer solution with the pH value of 3.0; taking 1.00 g (accurate to 1.0 mg) acid protease (with the enzyme activity of 50000U/g) and dissolving by using the phosphate buffer solution, and fixing the volume to a volumetric flask of 25 mL to prepare an acid protease solution with the concentration of 0.04 g/mL;
(3) Adding an acidic protease solution to a centrifuge tube containing the defatted sample, followed by the addition of an isotopic internal standard CML-D 4 Vortex mixing after sealing the centrifuge tube to hydrolyze the sample: adding 250 mu L of the acidic protease solution with the concentration of 0.04 g/mL into a centrifuge tube containing the degreased sample, and simultaneously adding 5 mu L of the acidic protease solution0. CML-D with concentration of [ mu ] L of 5.0 mug/mL 4 Isotopic internal standard solution to make CML-D in final solution to be tested 4 Is 10.0 ng/mL. Finally add H 3 PO 4 -NaH 2 PO 4 Phosphate buffer, make up the total volume of liquid in the centrifuge tube to 4.0. 4.0 mL. Sealing the centrifuge tube, mixing uniformly by vortex, hydrolyzing 12 h in water bath at 40 ℃, mixing uniformly by vortex for 1 time every 30.0 min, and hydrolyzing the sample uniformly;
(4) Centrifuging after the sample hydrolysis is completed to collect supernatant, leaching residues in the sample with a 5.0% formic acid solution and centrifuging to collect supernatant; mixing and filtering the supernatant after sample hydrolysis and the supernatant after formic acid solution leaching to obtain a pretreated sample liquid: after hydrolysis, centrifuging the centrifuge tube at 4000 r/min for 3 min, and collecting supernatant; then adding 5.0 volumes mL volume concentration of 5.0% formic acid aqueous solution to leach residues, carrying out vortex mixing, centrifuging at 4000 r/min for 3 min, and collecting supernatant; leaching 3 times with 5.0 mL concentration 5.0% formic acid water solution; all supernatants from 4 centrifugations were collected in 25 mL volumetric flasks and fixed to 25 mL with 5.0% formic acid in water; filtering the solution with filter paper for 1 time after the volume is fixed to remove impurities with larger particles, and obtaining filtrate which is a pretreatment sample liquid;
(5) Carrying out solid phase extraction and purification on the pretreated sample liquid by adopting an MCX solid phase extraction column to obtain eluent: after the MCX solid phase extraction column is activated by 3.0 mL methanol and is balanced by 3.0 mL water, 1.0 mL pretreated sample liquid passes through the column at a speed of 1-2 drops/second, and after the sample liquid completely flows out, the sample liquid is leached by 3.0 mL formic acid water solution with a concentration of 5.0 percent, and is vacuumized and dried; eluting with 3.0. 3.0 mL methanol, and vacuum drying; the whole effluent was discarded. Finally, eluting with 5.0. 5.0 mL concentration 15.0% ammonia water methanol solution, vacuum drying, and collecting the eluting effluent with a test tube. Concentrating and drying the eluent by blowing nitrogen at 40 ℃, dissolving residues in a test tube by using 1.0 mL water, mixing uniformly by vortex, and putting the mixture into a sample injection vial of 2.0 mL through a 0.22 mu m polyether sulfone needle filter to obtain a solution to be tested;
the solution to be tested is treated by ultra-high performance liquid chromatography-tandem mass spectrometry (Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry, UPLC-MS/MS)Detection was performed by CML-D 4 And calculating the content of CML by using an isotope internal standard curve.
The liquid chromatography conditions are: an ACQUITY ultra high performance liquid chromatograph; chromatographic column: ACQUITY BEH Amide HILIC column (2.1X100 mm,1.7μm); protective column: ACQUITY BEH Amide Vanguard column (1.7 μm); column temperature: 35 ℃; sample injection amount: 5. mu L; run time: 7.0min; mobile phase: phase A is 0.1% formic acid aqueous solution, and phase B is acetonitrile; gradient elution was performed at 0.30mL/min with the following elution parameters: 0-2.0min 10% A, 2.0-6.0min 20% A, 6.0-6.1min 90% A, and6.1-7.0min10% A.
The mass spectrometry conditions were: XEVO TQ triple quadrupole tandem mass spectrometer; ion source mode: ESI (electronic service provider interface) + The method comprises the steps of carrying out a first treatment on the surface of the Monitoring mode: MRM; capillary voltage: 2.0 A kV; ion source temperature: 150 ℃; desolventizing gas temperature: 350 ℃; desolventizing gas flow rate: 650L/h; taper hole air flow rate: 50L/h; the desolventizing gas and the taper hole gas are nitrogen; the collision gas is argon, CML and CML-D 4 The mass spectrum parameters of (2) are as follows: CML m/z 205.22-m/z 84.00 qualitative, m/z 205.22-m/z 130.00 quantitative; CML-D 4 m/z 209.00-m/z 87.70.
The samples 1 to 5 were examined using the UPLC-MS/MS detection system described above, and the CML quantitative sub-ion-flow chromatograms of FIGS. 7, 9, 11, 13 and 15 were obtained, corresponding to the CML-D of FIGS. 8, 10, 12, 14 and 16 4 Internal standard ion flow chromatograms. The effective peak area value in the CML quantitative sub-ion flow chromatogram is calculated by integrating software of a detection system on a computer: sample 1 was 6287.56 mv s, sample 2 was 9072.55 mv s, sample 3 was 13713.73 mv s, sample 4 was 8533.08 mv s, and sample 5 was 8039.60 mv s; CML-D in each sample 4 The effective peak area values of the internal standard sub-ion chromatograms are: 3509.42 mv ·s for sample 1, 1531.73 mv ·s for sample 2, 2893.97 mv ·s for sample 3, 3122.41 mv ·s for sample 4, and 3340.80 mv ·s for sample 5.
The CML content in the sample was calculated taking sample 1 as an example:
firstly, carrying out methodology verification of a detection method to obtain an internal standard curve, wherein the specific process is as follows:accurately transferring a series of CML standard solutions with volume concentration of 100.0 ng/mL, and adding 50.0 mu L of 200.0 ng/mL of CML-D respectively 4 Internal standard solutions formulated as a series of CML standard solutions at concentrations of 1.0, 2.0, 5.0, 10.0, 20.0 and 50.0 ng/mL, respectively, each 1.0 mL, CML-D in each concentration gradient of the series of CML standard solutions 4 The content of the internal standard substance is 10.0 ng/mL, the vortex is uniform, and the content of CML is measured by adopting a UPLC-MS/MS method. The CML concentration X is plotted on the ordinate and abscissa with the response value Y (see formula 1) as an internal standard curve of CML as shown in fig. 1. The detection limit (Limit of Detection, LOD) and the quantification limit (Limit of Quantification, LOQ) were calculated based on the signal-to-Noise ratio (Signal intensity S, noise N; S/N) values of the CML quantitative daughter ion chromatogram at the lowest concentration (1.0 ng/mL) in the standard curve. The CML concentration corresponding to S/n=3 is the LOD of the method, and the CML concentration corresponding to S/n=10 is the LOQ of the method.
Equation 1.
The linear range of the CML content detection method is 1.0-50.0 ng/mL.
The LOD of the CML content detection method is 0.038 ng/mL, and the LOQ is 0.127 ng/mL.
The internal standard curve of the CML content detection method is shown in figure 1, and the regression equation and the correlation coefficient of the curve are as follows: y=2.4359 x-1.1831, r 2 =0.9989。
The CML and the CML-D are obtained while the detection method is validated in methodology 4 The primary (parent ion) mass spectrum of (C) is shown in FIG. 2, CML and CML-D 4 The secondary (sub-ion) mass spectrum of the CML standard substance is shown in figure 3, the total ion flow chromatogram of the CML standard substance is shown in figure 4, the quantitative sub-ion flow chromatogram of the CML standard substance is shown in figure 5, and the CML-D is shown in figure 5 4 The internal standard sub-ion flow chromatogram is shown in fig. 6.
CML quantitative ion chromatographic peak area of sample 1 is 6287.56 mv.s, CML-D 4 The internal standard ion chromatographic peak area is 3509.42 mv.s. Then according toCalculating the response value in sample 1, +.>The response value is brought into an internal standard curve regression equation Y= 2.4359.X-1.1831, X=7.84 ng/mL, and the CML content in the sample is X.25.10/1000, namely 1.96 mug/g.
The concentration of CM L in samples 2-5 was calculated using the same method as described above, and the specific results are shown in Table 1.
TABLE 1 detection results of CML content in potato chips of example 1
Evaluation of accuracy of detection method:
accuracy refers to the degree to which the average of the results of multiple measurements of the detection method is close to the true value, typically expressed in terms of recovery. In the enzymatic hydrolysis step of sample 3, 3 CML standard solutions with different concentration levels and CML-D with a concentration of 5.0 μg/mL of 50 [ mu ] L were added respectively according to the addition amounts shown in Table 2 4 The isotopic internal standard solution was repeatedly assayed 3 times and the average recovery and relative standard deviation (Relative Standard Deviation, RSD) for each addition level were calculated as shown in table 2. As can be seen from Table 2, the recovery rate of the detection method is 84.26% -104.40%, and the RSD is 3.26% -5.74%. For accuracy in methodological verification, the generally acceptable recovery rate ranges from 80.00% to 120.00%, and the acceptable RSD range is below 10.00%, so that the recovery rate meets the requirement, the accuracy is higher, and the quantitative detection requirement of the CML content in the potato chips can be met.
TABLE 2 labeled recovery for CML content detection in potato chips
Evaluation of precision of detection method:
repeatability and parallelism tests are used to verify the precision of the detection method, generally indicated by RSD. The repeatability test is to perform repeated measurement 6 times on the same sample of one sample in a short time, and calculate the RSD between the detection results. The parallelism test was performed on 6 parallel samples of the same sample, RSD was calculated between the detection results, and the repeatability test and the parallelism test were verified for sample 2. The RSD of the repeatability test of sample 2 was 4.19% as seen in table 3, and the RSD of the parallelism test of sample 2 was 8.57% as seen in table 4. RSD values are below 10.00%, and the method has good precision, and can meet the quantitative detection requirement of CML content in the potato chips.
TABLE 3 repeatability test results of CML content detection in potato chips
TABLE 4 parallelism test results of CML content detection in Potato chips
Comparative example 1
The potato chips used in this comparative experiment were identical to the samples used in example 1 and the CML content of the samples was measured using the UPLC-MS/MS measurement conditions identical to those used in example 1, except that the conventional proteolytic hydrolysis of protein hydrochloric acid was used instead of the proteolytic hydrolysis in example 1.
Sample pretreatment method:
(1) Weighing a sample and degreasing the sample: a sample of potato chips 0.10 g (to the nearest 1.0 mg) ground to a particle size of about 0.5mm was weighed and placed into a polypropylene centrifuge tube having a volume of 10 mL. Adding 5.0. 5.0 mL n-hexane into the sample, fully vortex mixing, centrifuging at 4000 r/min for 3.0 min, and discarding an n-hexane layer; the degreasing process of the normal hexane is carried out for 3 times to thoroughly remove fat, and finally the sample is dried by blowing nitrogen at 40 ℃ to volatilize and remove the normal hexane, so as to obtain a degreased sample;
(2) Transferring the degreased sample into ampoule bottle with volume of 10 mL, and adding CML-D with concentration of 5.0 μg/mL of 50 [ mu ] L 4 Isotopic internal standard solution to make CML-D in final solution to be tested 4 Is 10.0 ng/mL. Finally, 6.0mol/L HCl solution was added to make up the total volume of the liquid in the ampoule to 4.0. 4.0 mL. Vortex evenly and charge N 2 Sealing, hydrolyzing 24 h at 110deg.C;
(3) After the hydrolysis was completed, the hydrolysate was cooled to room temperature, the pH was adjusted to 3.0 with 6.0mol/L NaOH solution, and then the volume was adjusted to 25. 25 mL with 5.0% formic acid aqueous solution. Filtering the solution with filter paper for 1 time after the volume is fixed to remove impurities with larger particles, and obtaining filtrate which is a pretreatment sample liquid;
(4) And (3) carrying out solid phase extraction and purification on the pretreated sample liquid by adopting an MCX solid phase extraction column. After the MCX solid phase extraction column is activated by 3.0 mL methanol and is balanced by 3.0 mL water, 1.0 mL pretreated sample liquid passes through the column at a speed of 1-2 drops/second, and after the sample liquid completely flows out, the sample liquid is leached by 3.0 mL formic acid water solution with a concentration of 5.0 percent, and is vacuumized and dried; eluting with 3.0. 3.0 mL methanol, and vacuum drying; the whole effluent was discarded. Finally, eluting with 5.0. 5.0 mL concentration 15.0% ammonia water methanol solution, vacuum drying, and collecting the eluting effluent with a test tube. Concentrating and drying the eluent by blowing nitrogen at 40 ℃, dissolving residues in a test tube by using 1.0 mL water, mixing uniformly by vortex, and putting the mixture into a sample injection vial of 2.0 mL through a 0.22 mu m polyether sulfone needle filter to obtain a solution to be tested;
detecting the solution to be detected by UPLC-MS/MS through CML-D 4 And calculating the content of CML by using an isotope internal standard curve.
The liquid chromatography conditions are: an ACQUITY ultra high performance liquid chromatograph; chromatographic column: ACQUITY BEH Amide HILIC column (2.1X100 mm,1.7μm); protective column: ACQUITY BEH Amide Vanguard column (1.7 μm); column temperature: 35 ℃; sample injection amount: 5. mu L; run time: 7.0min; mobile phase: phase A is 0.1% formic acid aqueous solution, and phase B is acetonitrile; gradient elution was performed at 0.30mL/min with the following elution parameters: 0-2.0min 10% A, 2.0-6.0min 20% A, 6.0-6.1min 90% A, and6.1-7.0min10% A.
The mass spectrometry conditions were: XEVO TQ triple quadrupole tandem mass spectrometer; ion source mode: ESI (electronic service provider interface) + The method comprises the steps of carrying out a first treatment on the surface of the Monitoring mode: MRM; capillary voltage: 2.0 A kV; ion source temperature: 150 ℃; desolventizing gas temperature: 350 ℃; desolventizing gas flow rate: 650L/h; taper hole air flow rate: 50L/h; the desolventizing gas and the taper hole gas are nitrogen; the collision gas is argon, CML and CML-D 4 The mass spectrum parameters of (2) are as follows: CML m/z 205.22-m/z 84.00 qualitative, m/z 205.22-m/z 130.00 quantitative; CML-D 4 m/z 209.00-m/z 87.70.
Detection result:
samples of 5 different brands of potato chips identical to example 1 were pretreated in the pretreatment methods (1) - (4) of the samples of comparative example 1, and the CML content was measured using the UPLC-MS/MS measurement conditions exactly identical to those of example 1, and the measurement results are shown in Table 5. FIGS. 17, 19, 21, 23 and 25 are CML quantitative sub-ion flow chromatograms of comparative example 1, and FIGS. 18, 20, 22, 24 and 26 are CML-D of comparative example 1 4 Internal standard ion flow chromatograms. The CML content of the sample of comparative example 1 was obtained by the calculation method of example 1 and is shown in table 5. As can be seen from a comparison of tables 1 and 5, the CML-quantitative ion and CML-D of the samples 1 to 5 in example 1 4 The peak areas of the internal standard ion were larger than those of the corresponding sample in comparative example 1, and as can be seen from the chromatograms of FIGS. 7 to 26, CML quantifies the ion and CML-D in example 1 4 The internal standard sub-ion chromatographic peak was also better shaped than in comparative example 1 and the baseline noise was small. The results of the detection of the other samples in example 1 were higher than those of comparative example 1 except that the detection of sample 1 in example 1 was slightly lower than that of comparative example 1. The method for detecting the CML content is time-saving, environment-friendly and good in accuracy and precision.
TABLE 5 detection of CML content in chips of comparative example 1
Because the UPLC-MS/MS detection system has higher requirement on sample introduction, the sample pretreatment method is also applicable to LC-MS, LC-MS/MS and HPLC-MS/MS detection systems, and is naturally also applicable to pretreatment of samples by other detection methods.
The above-described series of matters are merely specific descriptions of possible embodiments of the present invention, and are not intended to limit the scope of the invention. Any changes or substitutions without inventive effort are intended to be within the scope of the present invention.
Claims (7)
1. The method for detecting the content of the carboxymethyl lysine in the potato chips is characterized by comprising the following steps of:
(1) Weighing a sample and degreasing the sample;
(2) Preparing an acidic protease solution by using a phosphate buffer solution;
(3) Adding an acidic protease solution into a centrifuge tube filled with the degreased sample, then adding an isotope internal standard, and finally sealing the centrifuge tube, and uniformly mixing by vortex to hydrolyze the sample;
(4) Centrifuging to collect supernatant after sample hydrolysis is completed, leaching residues in the sample with formic acid solution and centrifuging to collect supernatant; mixing and filtering the supernatant after sample hydrolysis and the supernatant after formic acid solution leaching to obtain a pretreated sample liquid;
(5) Carrying out solid phase extraction purification on the pretreated sample liquid by adopting an MCX solid phase extraction column to obtain an eluent, and then drying, redissolving and filtering the eluent to obtain a solution to be detected;
(6) Detecting a solution to be detected by adopting an ultra-high performance liquid chromatography-tandem mass spectrometry, and calculating the content of carboxymethyl lysine CML by an internal standard curve;
the phosphate buffer solution in the step (2) is prepared from 0.15 to 0.25mol/L H 3 PO 4 Solution and NaH with concentration of 0.15-0.25mol/L 2 PO 4 The phosphate buffer solution is prepared by solution, and the pH value of the phosphate buffer solution is 3-4;
the enzyme activity of the acid protease in the step (2) is 45000-55000U/g, and the concentration of the acid protease in the solution is 0.035-0.045g/mL;
the purification method in the step (5) comprises the following steps: after pretreatment is carried out on the MCX solid phase extraction column, the pretreated sample obtained in the step (4) is loaded, and then is leached by formic acid water solution with the volume concentration of 4% -6.0%, and is dried by vacuum pumping; eluting with methanol, and vacuum drying; discarding all effluent; eluting with 12-18% ammonia water and methanol solution, and vacuum drying; collecting eluent, drying the eluent with nitrogen at 35-40 ℃, and re-dissolving with ultrapure water to obtain purified liquid to be tested.
2. The method for detecting the CML level in potato chips of claim 1, wherein: the degreasing step in the step (1) is as follows: grinding the potato chips to a particle size of 0.4-0.5mm, adding n-hexane into the potato chips, mixing by vortex, centrifuging at 3500-4500r/min for 2-4min, discarding n-hexane, performing the n-hexane degreasing process for 3 times, and finally drying the potato chips at 35-40deg.C with nitrogen gas to volatilize and remove n-hexane.
3. The method for detecting the CML level in potato chips of claim 1, wherein: adding an acidic protease solution into a centrifuge tube containing a degreased sample in the step (3), wherein the content of the acidic protease in the sample is 4500-5500U/g; the isotopic internal standard of CML is deuterated carboxymethyl lysine CML-D 4 The concentration is 4.0-6.0 mug/mL.
4. A method for detecting the CML level in potato chips according to claim 3, wherein: and (3) sealing the centrifuge tube, and then uniformly mixing by vortex, hydrolyzing in a water bath at the temperature of 35-40 ℃ for 10-14h, and uniformly mixing by vortex for 1 time every 25-35min to uniformly hydrolyze.
5. The method for detecting the CML level in potato chips of claim 1, wherein: the centrifugal speed in the step (4) is 3500-4500r/min, and the centrifugal time is 3-5min.
6. The method for detecting the CML level in potato chips of claim 5, wherein: and (3) the volume concentration of the formic acid solution in the step (4) is 4-6.0%, and the residual in the sample is leached by the formic acid solution and then centrifuged for 3-5min at 3500-4500r/min, and the process is repeated for at least 3 times.
7. The method for detecting the CML content in the potato chips of claim 1, wherein: the liquid chromatography conditions in the step (6) are as follows: an ACQUITY ultra high performance liquid chromatograph; chromatographic column: ACQUITYBEHAmide HILIC column 2.1X100 mm,1.7 μm; protective column: the ACQUITYBEEHAmideVanguard column is 1.7 μm; column temperature: 35 ℃; sample injection amount: 5. Mu.L; run time: 7.0min; mobile phase: phase A is 0.1% formic acid aqueous solution, and phase B is acetonitrile; gradient elution was performed at 0.30mL/min with the following elution parameters: 0-2.0min 10% A, 2.0-6.0min 20% A, 6.0-6.1min 90%A,and6.1-7.0min10% A;
the mass spectrometry conditions were: a xevatq triple quadrupole tandem mass spectrometer; ion source mode: ESI (electronic service provider interface) + The method comprises the steps of carrying out a first treatment on the surface of the Monitoring mode: MRM; capillary voltage: 2.0kV; ion source temperature: 150 ℃; desolventizing gas temperature: 350 ℃; desolventizing gas flow rate: 650L/h; taper hole air flow rate: 50L/h; the desolventizing gas and the taper hole gas are nitrogen; the collision gas is argon, CML and CML-D 4 The mass spectrum parameters of (2) are as follows: CMLm/z 205.22-m/z 84.00 qualitative, m/z 205.22-m/z 130.00 quantitative; CML-D 4 m/z 209.00-m/z 87.70.
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