CN106124641A - Carboxymethyl-lysine and the method for lysopine in a kind of synchronous detecting green tea - Google Patents

Carboxymethyl-lysine and the method for lysopine in a kind of synchronous detecting green tea Download PDF

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CN106124641A
CN106124641A CN201610317577.4A CN201610317577A CN106124641A CN 106124641 A CN106124641 A CN 106124641A CN 201610317577 A CN201610317577 A CN 201610317577A CN 106124641 A CN106124641 A CN 106124641A
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lysine
lysopine
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carboxymethyl
green tea
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CN106124641B (en
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王彬
刘婧
田野
曾丽珍
刘青茹
周春江
蒋玉兰
唐靖
邸胜达
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HANGZHOU TEA INST ALL-CHINA SUPPLY AND MARKETING GENERAL COOPERATIVE
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HANGZHOU TEA INST ALL-CHINA SUPPLY AND MARKETING GENERAL COOPERATIVE
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    • 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
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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
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Abstract

The present invention relates to technical field of analysis and detection, can affect the problem of detection method for there is different material substrate in solution at present detection CML and CEL method, the present invention proposes carboxymethyl-lysine and the method for lysopine in a kind of synchronous detecting green tea, with d4CML and d4CEL is internal standard, uses more options reaction monitoring pattern, is proved by methodology, construct the quantitative analysis method of CML and CEL in Simultaneous Determination green tea, provides basis for the monitoring and control of AGEs in Folium Camelliae sinensis.The detection method accuracy of the present invention, repeatability, stability are preferable, are suitable to the actually detected of CML and CEL in green tea.

Description

Carboxymethyl-lysine and the method for lysopine in a kind of synchronous detecting green tea
Technical field
The present invention relates to technical field of analysis and detection, relate in particular to carboxymethyl in a kind of synchronous detecting green tea and rely Propylhomoserin and the method for lysopine.
Background technology
China's tea place area, tea yield occupy the first in the world, are Tea Industry big countries.Tea kinds is many, and endoplasm becomes Dividing abundant, manufacturing procedure is various, and in tea making process, endoplasm becomes branch that complicated chemical change occurs, and is easily generated late The potential hazard things such as phase glycosylation end products (Advanced Glycation End Products, AGEs), Tea Processing process safety has become the new focus that quality and safety of tea field is paid close attention to.AGEs is Maillard reaction One of product, be the complicated compound of a class formation, have irreversibility, bridging property, be difficult to be degraded, The characteristics such as structural heterogeneity, enzyme are stable.The primary product identified at present has carboxymethyl-lysine (N-(carboxymethyl) lysine, CML), lysopine (N-(carboxyethyl) lysine, CEL) Deng kind more than 20, wherein, CML and CEL is common two kind, is considered and diabetes, nephropathy, aging Deng generation and develop relevant.Currently reported employing LC-MS/MS analyzes method and measures in food substrate CML and CEL, this method pretreatment is relatively easy, and detection speed is fast, favorable reproducibility, good stability, but Different material substrate can affect the suitability of method.
Summary of the invention
Asking of detection method can be affected for solution at present detection CML and CEL method exist different material substrate Topic, the present invention proposes carboxymethyl-lysine and the method for lysopine in a kind of synchronous detecting green tea, this The detection method accuracy of invention, repeatability, stability are preferable, are suitable to the reality of CML and CEL in green tea Detection.
The present invention is achieved by the following technical solutions: carboxymethyl-lysine and carboxylic in a kind of synchronous detecting green tea The method of ethyl lysine, described detection method is:
(1) to tea-leaf power sample ungrease treatment, sodium borate buffer liquid and boron after then being dried up by sample, it are sequentially added into Sodium hydride solution, at 0-6 DEG C of reductase 12-10h, preferably 8h, adds chloroform and methyl alcohol mixed liquor, centrifugal The backward precipitate obtained adds acid solution, hydrolysis, after completing, hydrolyzation sample is carried out constant volume, filtration And collect filtrate；
Described ungrease treatment step is: centrifugal after adding normal hexane vibration in tea-leaf power sample, discards molten Agent, repeated several times, wherein centrifugation rate is 5000-12000rpm, and centrifugation time is 4-10min.Just own Alkane act as solvent, usage amount is the amount making Folium Camelliae sinensis be totally submerged in solvent.
Described sodium borate buffer liquid concentration is 0.1-0.3mol/L, and sodium borohydride concentration is 0.8-1.2mol/L, PH regulator is 8.5-10.0, described tea-leaf power sample, sodium borate buffer liquid and the weight of sodium borohydride solution Volume ratio is 1g: 8-12mL: 4-6mL.
As preferably, in chloroform and methyl alcohol mixed liquor, chloroform is 1-3: 1 with the volume ratio of methanol.
As preferably, in hydrolysis, acid solution is the HCl solution of 4-8mol/L selected from concentration, more preferably The HCl solution of 6mol/L；Hydrolysis condition is reaction 12-30h at 100-120 DEG C, more preferably exists 24h is reacted at 110 DEG C.
(2) take filtrate, be quantitatively adding isotope labelling carboxymethyl-lysine and isotope labelling lysopine Standard solution, obtains pretreatment sample；
In described filtrate, the interpolation concentration of isotope labelling carboxymethyl-lysine is 10-200ng/mL, isotope The interpolation concentration of labelling lysopine is 10-200ng/mL.As preferably, isotope in described filtrate The interpolation concentration of labelling carboxymethyl-lysine is 40-100ng/mL, the interpolation of isotope labelling lysopine Concentration is 40-100ng/mL.
(3) purify: use solid-phase extraction column that pretreatment sample is carried out purified treatment, obtain testing sample；
Described purification method is: after solid-phase extraction column is carried out pretreatment, pre-place step (2) obtained Reason sample carries out loading, and hydrochloric acid solution and ultra-pure water drip washing with concentration is 0.08-0.15mol/L enter the most successively Row loading is adsorbed, then carries out sample elution with the ammonia water-methanol solution drip washing that volumetric concentration is 4-6%, and collection is washed De-liquid, dries up, and ultra-pure water redissolves, and obtains liquid to be measured；Described hydrochloric acid solution, ultra-pure water, the volume of ammonia Ratio is 1: 0.5～2: 0.5～2.
The flow velocity when absorption of described loading, loading, eluting can be 0.3-0.8mL/min, preferably 0.5mL/min. 0.5-1.5mL pure water is used to redissolve, excessively the filter membrane of 0.22 μm ,-20 DEG C of preservations, to be checked.
As preferably, the preprocess method of described solid-phase extraction column can be: successively use 2-4mL methanol and 2-4mL concentration is that the hydrochloric acid solution of 0.08-0.15mol/L carries out drip washing, flow velocity 0.5-1.5 to solid-phase extraction column mL/min。
(4) use high performance liquid chromatography tandem mass spectrum method that testing sample is detected, calculate carboxylic first by internal standard method Base lysine and the content of lysopine.
High-efficient liquid phase chromatogram determining condition is: mobile phase A includes that concentration of volume percent is 0.1% formic acid solution With concentration of volume percent be 99.9% concentration be the ammonium acetate of 8-12mmol/L, Mobile phase B is acetonitrile, ladder Degree elution program, flow velocity 0.2mL/min, sample size 10 μ L, column temperature 38-42 DEG C, described gradient elution Program is:
Mass Spectrometry Conditions is:
Ionization mode is ESI ion source；Scan mode is for just to ionize；Detection mode is polyion reaction monitoring, touches Hitting energy is 20eV；
Ion source condition: be dried temperature: 325C；Dry gas stream speed: 5L/min；Atomization gas pressure: 45psi； Sheath temperature: 350 DEG C；Sheath gas velocity: 11L/min；Capillary voltage: 3000V, wherein:
The present invention is directed to green tea medium characteristics, optimize sample-pretreating method, with d4-CML and d4-CEL be Internal standard, is used more options reaction monitoring (MRM) pattern, is proved by methodology, construct Simultaneous Determination The quantitative analysis method of CML and CEL in green tea, provides basis for the monitoring and control of AGEs in Folium Camelliae sinensis.
Compared with prior art, the invention has the beneficial effects as follows: detection method accuracy, repeatability, stability Preferably, the actually detected of CML and CEL in green tea is suitable to.
Accompanying drawing explanation
Fig. 1 is embodiment CML canonical plotting；
Fig. 2 is embodiment CEL canonical plotting；
Fig. 3 is CML, d in the mixed mark of embodiment4-CML, CEL and d4The MRM mass spectrum of-CEL；
Fig. 4 is CML, d in embodiment green tea sample4-CML, CEL and d4The MRM mass spectrum of-CEL.
Specific implementation method
Below by embodiment, the present invention is described in further details, in embodiment raw materials used the most commercially available or Prepare by conventional method.
CML standard substance (purity ＞ 98%) and d in embodiment4-CML Isotopic Internal Standard (purity 99%), CEL standard substance (purity ＞ 98.6%) and d4-CEL Isotopic Internal Standard (purity 98.6%)；
Instrument is: Agilent 6460 Triple Quad LC-MS (triplex tandem quadrupole rod LC-MS Instrument), Agilent Zorbax SBC18Post (150mm × 2.1mm, 3.5 μm)；SPE solid-phase extraction column Cleanert PCX (60mg, 3mL, Tianjin Beaune Ai Jieer company limited)；Pulverizer, electronic balance, at a high speed freezing Centrifuge, baking oven etc. are laboratory conventional instrument or equipment.
Embodiment 1
(1) extract: weigh 50mg tea-leaf power sample and be placed in 10mL centrifuge tube, add 2mL normal hexane, Acutely after vibration 3min, it is centrifuged 5min in 10000rpm, discards solvent, repeat above step 2 times. After normal hexane defat, sample utilize nitrogen dry up to recovering powder.1mL is added in sample Na2B4O7Buffer (0.2mol/L) and 0.5mL NaBH4(1mol/L prepares with 0.1mol/LNaOH), Regulation pH=9.2, in 4 DEG C of reduction overnight (about 8h).Then, 1mL chloroform/methanol (2: 1, v/v) is added, in After 10000rpm is centrifuged 10min, abandoning supernatant；5mL HCI (6 is added in the precipitate obtained Mol/L), it is placed in 110 DEG C of baking ovens, hydrolyzes 12h.After having hydrolyzed, hydrolyzation sample is taken out and is cooled to Room temperature (25 DEG C), is settled to 10mL with ultra-pure water, then filters with qualitative filter paper and collect filtrate.
(2) accurately measure 0.4mL filtrate, add 0.4 μ g/mL d4-CML and 0.4 μ g/mL d4-CEL internal standard is each 100μL。
(3) purify: SPE post pretreatment: 3mL methanol, the HCl of 3mL 0.1M, flow velocity is 1mL/min； Loading is adsorbed: with HCl, the 3mL ultra-pure water drip washing of 3mL 0.1M, flow velocity is 0.5mL/min；Sample Ammonia water-methanol (v/v) solution of eluting: 3mL 5%, flow velocity is 0.5mL/min, collects filtrate；Dry up: The N of 40 DEG C2Dry up；Redissolve to be checked: use 1mL ultra-pure water to redissolve, excessively the filter membrane of 0.22 μm ,-20 DEG C Preserve, to be checked.
(4) chromatographic condition: mobile phase A is 0.1% formic acid solution (containing the ammonium acetate of concentration 10mmol/L), Mobile phase B is acetonitrile, and gradient elution program is shown in Table 1.Flow velocity 0.2mL/min, sample size 10 μ L, column temperature 40℃。
Mass Spectrometry Conditions: Ionization mode: ESI ion source；Scan mode: just ionize；Scanning ion pair: CML (m/z 205-84.2), d4-CML (m/z 209.26-88.1), CEL (m/z 219.11-84.2), d4-CEL(m/z 223.28-88.1).Detection mode: polyion reaction monitoring (MRM), collision energy: 20eV, collision lures Leading solution ionization voltage: CML is 70V, d4-CML be 83V, CEL be 158V, d4-CEL is 78V. Ion source condition: be dried temperature: 325C；Dry gas stream speed: 5L/min；Atomization gas pressure: 45psi； Sheath temperature: 350 DEG C；Sheath gas velocity: 11L/min；Capillary voltage: 3000V.
Mass hunter liquid matter workstation software (Agilent company, the U.S.) is utilized to gather related data.
Table 1 gradient elution program
CML standard substance ultra-pure water is configured to concentration is 1mg/mL, d4-CML standard substance ultra-pure water Being configured to concentration is that to be configured to concentration be 1mg/mL to 0.05mg/mL, CEL standard substance ultra-pure water, d4It is 0.1mg/mL that-CEL standard substance ultra-pure water is configured to concentration, molten as deposit in-20 DEG C of Refrigerator stores Liquid.Compound concentration is 10,50,100,200 and CML and the CEL hybrid standard of 300ng/mL respectively Solution, d4-CML and d4-CEL is internal standard, and concentration is 40ng/mL.With standard specimen concentration/internal standard concentration as horizontal stroke Coordinate, standard specimen peak area/internal standard peak area is vertical coordinate, draws standard curve, seeks curve linear regression equation, Thus calculate the content of CML and CEL in sample.
Embodiment 2
(1) extract: weigh 50mg tea-leaf power (with embodiment 1) sample and be placed in 10mL centrifuge tube, add Enter 2mL normal hexane, acutely after vibration 3min, be centrifuged 10min in 5000rpm, discard solvent, repeat Above step 3 times.After normal hexane defat, sample utilize nitrogen dry up to recovering powder.In sample Add 1mL Na2B4O7Buffer (0.1mol/L) and 0.5mL NaBH4(0.8mol/L, with 0.1 Mol/LNaOH prepares), regulate pH=8.5, in 4 DEG C of reduction 10h.Then, addition 1mL chloroform/methanol (1: 1, V/v), after 5000rpm is centrifuged 10min, abandoning supernatant；5mL HCI is added in the precipitate obtained (4mol/L), it is placed in 100 DEG C of baking ovens, hydrolyzes 30h.After having hydrolyzed, hydrolyzation sample is taken out and cools down To room temperature (25 DEG C), it is settled to 10mL with ultra-pure water, then filters with qualitative filter paper and collect filtrate.
(2) accurately measure 0.4mL filtrate, add 0.4 μ g/mL d4-CML and 0.4 μ g/mL d4-CEL internal standard is each 200μL。
(3) purify: SPE post pretreatment: 3mL methanol, the HCl of 2mL 0.1M, flow velocity is 1mL/min； Loading is adsorbed: with HCl, the 3mL ultra-pure water drip washing of 3mL 0.1M, flow velocity is 0.5mL/min；Sample Ammonia water-methanol (v/v) solution of eluting: 3mL 5%, flow velocity is 0.5mL/min, collects filtrate；Dry up: The N of 40 DEG C2Dry up；Redissolve to be checked: use 1mL ultra-pure water to redissolve, excessively the filter membrane of 0.22 μm ,-20 DEG C Preserve, to be checked.
(4) chromatographic condition: mobile phase A is 0.1% formic acid solution (containing the ammonium acetate of concentration 10mmol/L), Mobile phase B is acetonitrile, and gradient elution program is shown in Table 1.Flow velocity 0.2mL/min, sample size 10 μ L, column temperature 40℃。
Mass Spectrometry Conditions: Ionization mode: ESI ion source；Scan mode: just ionize；Scanning ion pair: CML (m/z 205-84.2), d4-CML (m/z 209.26-88.1), CEL (m/z 219.11-84.2), d4-CEL(m/z 223.28-88.1).Detection mode: polyion reaction monitoring (MRM), collision energy: 20eV, collision lures Leading solution ionization voltage: CML is 70V, d4-CML be 83V, CEL be 158V, d4-CEL is 78V. Ion source condition: dry temperature: 325 DEG C；Dry gas stream speed: 5L/min；Atomization gas pressure: 45psi； Sheath temperature: 350 DEG C；Sheath gas velocity: 11L/min；Capillary voltage: 3000V.
Mass hunter liquid matter workstation software (Agilent company, the U.S.) is utilized to gather related data.
Table 2 gradient elution program
CML standard substance ultra-pure water is configured to concentration is 1mg/mL, d4-CML standard substance ultra-pure water Being configured to concentration is that to be configured to concentration be 1mg/mL, d to 0.05mg/mL, CEL standard substance ultra-pure water4-CEL It is 0.1mg/mL that standard substance ultra-pure water is configured to concentration, in-20 DEG C of Refrigerator stores as stock solution.Respectively Compound concentration is 10,50,100,200 and CML and the CEL mixed standard solution of 300ng/mL, d4-CML And d4-CEL is internal standard, and concentration is 40ng/mL.With standard specimen concentration/internal standard concentration as abscissa, face, standard specimen peak Long-pending/internal standard peak area is vertical coordinate, draws standard curve, seeks curve linear regression equation, thus calculate sample The content of middle CML and CEL.
Embodiment 3
(1) extract: weigh 50mg tea-leaf power (with embodiment 1) sample and be placed in 10mL centrifuge tube, add Enter 2mL normal hexane, acutely after vibration 3min, be centrifuged 4min in 12000rpm, discard solvent, repeat Above step 2 times.After normal hexane defat, sample utilize nitrogen dry up to recovering powder.In sample Add 1mL Na2B4O7Buffer (0.3mol/L) and 0.5mL NaBH4(1.2mol/L, with 0.1 Mol/LNaOH prepares), regulate pH=10, in 6 DEG C of reductase 12 h.Then, addition 1mL chloroform/methanol (3: 1, V/v), after 12000rpm is centrifuged 4min, abandoning supernatant；5mL HCI is added in the precipitate obtained (8mol/L), it is placed in 120 DEG C of baking ovens, hydrolyzes 12h.After having hydrolyzed, hydrolyzation sample is taken out and cools down To room temperature (25 DEG C), it is settled to 10mL with ultra-pure water, then filters with qualitative filter paper and collect filtrate.
(2) accurately measure 0.4mL filtrate, add 0.4 μ g/mL d4-CML and 0.4 μ g/mL d4-CEL internal standard is each 10μL。
(3) purify: SPE post pretreatment: 3mL methanol, the HCl of 5mL 0.1M, flow velocity is 1mL/min； Loading is adsorbed: with HCl, the 3mL ultra-pure water drip washing of 3mL 0.1M, flow velocity is 0.5mL/min；Sample Ammonia water-methanol (v/v) solution of eluting: 3mL 5%, flow velocity is 0.5mL/min, collects filtrate；Dry up: The N of 40 DEG C2Dry up；Redissolve to be checked: use 1mL ultra-pure water to redissolve, excessively the filter membrane of 0.22 μm ,-20 DEG C Preserve, to be checked.
(4) chromatographic condition: mobile phase A is 0.1% formic acid solution (containing the ammonium acetate of concentration 10mmol/L), Mobile phase B is acetonitrile, and gradient elution program is shown in Table 1.Flow velocity 0.2mL/min, sample size 10 μ L, column temperature 40℃。
Mass Spectrometry Conditions: Ionization mode: ESI ion source；Scan mode: just ionize；Scanning ion pair: CML (m/z 205-84.2), d4-CML (m/z 209.26-88.1), CEL (m/z 219.11-84.2), d4-CEL(m/z 223.28-88.1).Detection mode: polyion reaction monitoring (MRM), collision energy: 20eV, collision lures Leading solution ionization voltage: CML is 70V, d4-CML be 83V, CEL be 158V, d4-CEL is 78V. Ion source condition: dry temperature: 325 DEG C；Dry gas stream speed: 5L/min；Atomization gas pressure: 45psi； Sheath temperature: 350 DEG C；Sheath gas velocity: 11L/min；Capillary voltage: 3000V.
Mass hunter liquid matter workstation software (Agilent company, the U.S.) is utilized to gather related data.
Table 3 gradient elution program
CML standard substance ultra-pure water is configured to concentration is 1mg/mL, d4-CML standard substance ultra-pure water Being configured to concentration is that to be configured to concentration be 1mg/mL, d to 0.05mg/mL, CEL standard substance ultra-pure water4-CEL It is 0.1mg/mL that standard substance ultra-pure water is configured to concentration, in-20 DEG C of Refrigerator stores as stock solution.Respectively Compound concentration is 10,50,100,200 and CML and the CEL mixed standard solution of 300ng/mL, d4-CML And d4-CEL is internal standard, and concentration is 40ng/mL.With standard specimen concentration/internal standard concentration as abscissa, face, standard specimen peak Long-pending/internal standard peak area is vertical coordinate, draws standard curve, seeks curve linear regression equation, thus calculate sample The content of middle CML and CEL.
Embodiment 1～3 the results are shown in Table 4 and Fig. 1, Fig. 2 shown in, CML standard curve equation is Y=1.0911x+0.2838, R2=0.9990, CEL standard curve equation is y=0.2056x+0.0149, R2= The range of linearity of 0.9992, CML and CEL is 10-300ng/mL, coefficient R2All more than 0.999, Linear relationship is good.
Table 4 HPLC-MS/MS method detection the standard curve equation of CML and CEL, LOD and LOQ
The MRM mass spectrum of mixed mark and green tea sample as shown in Figure 3, Figure 4, as seen from the figure, mixes mark and sample The retention time of Plays product is all between 1.84-1.88min, and retention time is more consistent.According to CML and CEL low content sample chromatogram figure, is calculated by extrapolation and show that the LOD value of CML and CEL is about 2 Ng/mL, LOQ value is about 10ng/mL.
Test case 1: recovery of standard addition is tested
In green tea sample, add CML, CEL standard solution, be respectively provided with basic, normal, high three and add scalar, CML is set to 50,100,200ng/mL, CEL be set to 20,50,100ng/mL, use HPLC-MS/MS Before and after method mensuration mark-on, CML, CEL content in sample, calculates recovery of standard addition, the results are shown in Table 5.By table Understanding, under different mark-on levels, its recovery of standard addition is different.For CML, recovery of standard addition is at 79.07-88.29% Between；For CEL, recovery of standard addition is between 76.40-85.18%.
The recovery of standard addition of CML and CEL in table 5 HPLC-MS/MS method detection green tea
Test case 2: precision
Green tea sample is complicated due to substrate, CML and CEL content is relatively low, HPLC-MS/MS method detection CML It is shown in Table 6 with the Precision test result of CEL.As can be seen from the table, the in a few days RSD and in the daytime of CML RSD between 5.0-7.0%, the in a few days RSD of CEL and RSD is between 5.0-9.0%, all 10% in the daytime Within, meet precision requirement.
The precision of CML and CEL in table 6 HPLC-MS/MS method detection green tea
Application examples
Use this method that CML and CEL content in four kinds of green tea samples in Zhejiang and Liang Gechancha district, Yunnan is entered Row measures, and the results are shown in Table 7.Visible, in four kinds of samples, CML and CEL all has detection, nearly all sample Middle CML content is above CEL content, and CML average content reaches 207.01ng/g, CEL average content Reach 49.40ng/g, control to provide foundation for Tea Processing process potential hazard thing.
The content of CML and CEL in table 7 several green tea sample
The present invention constructs based on the method for CML and CEL in HPLC-MS/MS synchronous detecting green tea, Method validation result shows: CML and CEL is good in 10-300ng/mL concentration range internal linear, relevant Coefficients R2More than 0.999, LOD and LQD value is respectively 2ng/mL, 10ng/mL；Three mark-ons are dense Under degree, recovery of standard addition is between 76-89%；In a few days RSD and in the daytime RSD all within 10%, show The accuracy of this detection method, repeatability, stability are preferable, are suitable to CML and CEL in green tea actually detected.

Claims (9)

1. carboxymethyl-lysine and the method for lysopine in a synchronous detecting green tea, it is characterised in that institute The detection method stated is:
(1) to tea-leaf power sample ungrease treatment, sodium borate buffer liquid and boron after then being dried up by sample, it are sequentially added into Sodium hydride solution, at 0-6 DEG C of reductase 12-10h, adds chloroform and methyl alcohol mixed liquor, centrifugal backward obtains Precipitate adds acid solution, hydrolysis, after completing, hydrolyzation sample is carried out constant volume, filters and collect filtrate；
(2) take filtrate, be quantitatively adding isotope labelling carboxymethyl-lysine and isotope labelling lysopine Standard solution, obtains pretreatment sample；
(3) purify: use solid-phase extraction column that pretreatment sample is carried out purified treatment, obtain testing sample；
(4) use high performance liquid chromatography tandem mass spectrum method that testing sample is detected, calculate carboxylic first by internal standard method Base lysine and the content of lysopine.
Carboxymethyl-lysine and lysopine in a kind of synchronous detecting green tea the most according to claim 1 Method, it is characterised in that: step (1) ungrease treatment step is: will add normal hexane in tea-leaf power sample Being centrifuged after vibration, discard solvent, repeated several times, wherein centrifugation rate is 5000-12000rpm, time centrifugal Between be 4-10min.
Carboxymethyl-lysine and lysopine in a kind of synchronous detecting green tea the most according to claim 1 Method, it is characterised in that: the sodium borate buffer liquid concentration described in step (1) is 0.1-0.3mol/L, boron hydrogen Change na concn is 0.8-1.2mol/L, and pH regulator is 8.5-10.0, and described tea-leaf power sample, sodium borate delay The w/v rushing liquid and sodium borohydride solution is 1g: 8-12mL: 4-6mL.
Carboxymethyl-lysine and lysopine in a kind of synchronous detecting green tea the most according to claim 1 Method, it is characterised in that: in step (1), in chloroform and methyl alcohol mixed liquor, the volume ratio of chloroform and methanol is 1-3: 1。
Carboxymethyl-lysine and lysopine in a kind of synchronous detecting green tea the most according to claim 1 Method, it is characterised in that: in step (1), in hydrolysis, acid solution is the HCl of 4-8mol/L selected from concentration Solution, at 100-120 DEG C of hydrolysis 12-30h.
Carboxymethyl-lysine and lysopine in a kind of synchronous detecting green tea the most according to claim 1 Method, it is characterised in that: in step (2), in described filtrate, isotope labelling carboxymethyl-lysine adds Adding concentration is 10-200ng/mL, and the interpolation concentration of isotope labelling lysopine is 10-200ng/mL.
Carboxymethyl-lysine and lysopine in a kind of synchronous detecting green tea the most according to claim 1 Method, it is characterised in that: the purification method described in step (3) is: after solid-phase extraction column is carried out pretreatment, The pretreatment sample that step (2) obtains is carried out loading, is 0.08-0.15mol/L's by concentration the most successively Hydrochloric acid solution and ultra-pure water drip washing carry out loading absorption, then drench with the ammonia water-methanol solution that volumetric concentration is 4-6% Washing and carry out sample elution, collect eluent, dry up, ultra-pure water redissolves, and obtains liquid to be measured.
Carboxymethyl-lysine and lysopine in a kind of synchronous detecting green tea the most according to claim 1 Method, it is characterised in that: high-efficient liquid phase chromatogram determining condition is: mobile phase A includes concentration of volume percent Being the ammonium acetate of 0.1% formic acid solution and concentration 8-12mmol/L, Mobile phase B is acetonitrile, gradient elution journey Sequence, flow velocity 0.2mL/min, sample size 10 μ L, column temperature 38-42 DEG C, described gradient elution program is:
Carboxymethyl-lysine and lysopine in a kind of synchronous detecting green tea the most according to claim 1 Method, it is characterised in that: Mass Spectrometry Conditions:
Ionization mode is ESI ion source；Scan mode is for just to ionize；Detection mode is polyion reaction monitoring, touches Hitting energy is 20eV；
Ion source condition: be dried temperature: 325C；Dry gas stream speed: 5L/min；Atomization gas pressure: 45psi； Sheath temperature: 350 DEG C；Sheath gas velocity: 11L/min；Capillary voltage: 3000V, wherein:
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