CN104965040B - Free state and the detection method of combined state carboxymethyl-lysine in milk and milk products - Google Patents
Free state and the detection method of combined state carboxymethyl-lysine in milk and milk products Download PDFInfo
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Abstract
The present invention is free state and the detection method of combined state carboxymethyl-lysine in a kind of milk and milk products, isotopic dilution high performance liquid chromatography tandem ion-trap mass spectrography, free state and protein binding state carboxymethyl-lysine in detection milk and milk products.Present invention, avoiding the use of ion-pairing agent without any derivatization pre-treatment step, and have feature efficient, accurate, highly sensitive, that favorable reproducibility, the range of linearity are wide, its distinctive feature is had in terms of free state and protein binding state carboxymethyl-lysine content in analyzing milk and milk products, fast and accurately free state in milk and milk products and combined state carboxymethyl-lysine qualitative and quantitative analysis be can be carried out, free state and the detection of combined state carboxymethyl-lysine in milk and milk products extraordinary can be applied to.
Description
Technical field
The present invention relates to food inspection analysis technical field, be specifically related to a kind of isotopic dilution high performance liquid chromatography series connection
Ion trap mass spectrometry, free state and protein binding state carboxymethyl-lysine in detection milk and milk products.
Background technology
In food processing process, Maillard reaction is easily generated with the advanced glycation end products (AGEs) nuisance as representative.Carboxylic
Methyllysine, as the clear and definite AGEs mark of a kind of structure, is widely present in food.
Lysine has epsilon-amino, it is easy to become the carbonyl target spot of high reaction activity, therefore rich in the food of lysine
And biological tissue is more easy to Maillard reaction.And milk product is as the food of a kind of rich in proteins, not only lysine content
Abundant, the lactose content of reproducibility is the highest, and (lactose is easier to react with lysine than glucose and two kinds of reducing sugar of maltose
Generate carboxymethyl-lysine).Numerous researchs show, milk and milk powder produce because of Maillard reaction in heat treatment process
Carboxymethyl-lysine be significantly larger than other food categories.
The most also there is no carboxymethyl-lysine standard detecting method in the food of recommendation to put into effect.
Owing to the polarity of carboxymethyl-lysine is big, ionizing power strong, when cannot retain or retain in general C18 chromatographic column
Between the shortest.Ion-pairing agent such as trifluoroacetic acid (TFA), nine fluorine valeric acids (NFPA) need to be added in flowing mutually for solving this problem.
Ion-pairing agent but inhibits Ionization Efficiency while increasing retention time, reduces mass signal analog value, object
The retention time of chromatographic peak is the most unstable, finally reduces the sensitivity of detection method, is unfavorable for qualitative and quantitative analysis.Another
Aspect is difficult to rinse well from chromatographic column due to ion-pairing agent, chromatographic column is produced irreversible infringement, thus significantly contracts
In the service life of shorter chromatogram column, NFPA is also bigger to mass spectrometer infringement in addition.For avoiding the use of ion-pairing agent, 2013
The Chinese patent CN103293243A that November 3 announced utilizes 9-fluorenylmethyl chloroformate to spread out before carboxymethyl-lysine is carried out post
The raw retention time by carboxymethyl-lysine in C18 chromatographic column has extended to about 4.95min, but this adds pre-treatment
Complexity.The Chinese patent CN103616466A that on March 5th, 2014 announces uses hydrophilic chromatographic column (HILLIC) to carry out generation
Detecting carboxymethyl-lysine for C18 post, but retention time only has 3.47min, retention is not ideal yet.
HILLIC chromatographic column range is narrower, and the suitability is not strong, and the compound stronger just for small part hydrophilic has well
Retention, the use of C18 post is not universal.
Summary of the invention
The present invention provides free state and protein binding state carboxymethyl-lysine in a kind of milk and milk products fast and accurately
Detection method, can not only carry out the most qualitative point to free state in milk and milk products and protein binding state carboxymethyl-lysine
Analysis, it is also possible to carry out quantitative analysis.
Free state and the detection method of combined state carboxymethyl-lysine in a kind of milk and milk products, it is characterised in that by as follows
Testing conditions is carried out:
1, the pre-treatment of sample: solid milk powder sample accurately weighs 0.1g, adding the mixing of 1mL ultra-pure water vortex, to make sample molten
Liquid, liquid milk product directly measures 1mL；Prepare addition 2～3mL normal hexane in liquid to sample, be centrifuged with 4000r/min after shaking up
10min, discards normal hexane layer, weighs 2～3 times thoroughly to remove fat, skimmed milk and milk powder and can remove this step from；Add
The sodium borate buffer liquid of 1.5mL0.2mol/L, pH9.4,1mL sodium borohydride solution, reacts 6h at 4 DEG C；Add and double sample
60% solution of trichloroacetic acid of volume, 8000r/min, 4 DEG C of centrifugal 10min, cleer and peaceful protein precipitation in separation, after collecting respectively
Treat to process further；In supernatant, add the D4-carboxymethyl-lysine Isotopic Internal Standard of 100 μ L 0.2 μ g/mL, cross SPE column purification；
Above-mentioned albumen precipitation is added the hydrochloric acid of 4mL 6mol/L, is passed through nitrogen and protects 110 DEG C of hydrolysis 24h, hydrolyzed solution is diluted constant volume
To 5mL, in hydrolyzed solution, add the D4-carboxymethyl-lysine of 100 μ L 50 μ g/mL, upper Solid-Phase Extraction column purification；By HiCapt
MCX solid-phase extraction column uses 3mL methanol, 3mL water activation balance successively, after loading, then uses 3mL water, and 3mL methanol drip washing is finally used
5mL ammonia: the ammoniated methanol eluting of methanol=5:95, v/v, collects eluent, after nitrogen dries up, accurately adds 1mL ultra-pure water
Redissolve, after vortex mixing, cross 0.22 μm cellulose membrane.The structure chart of D4-carboxymethyl-lysine is as shown in Figure 7.
2, sample HPLC detection: HPLC condition be chromatographic column be Waters Symmetry C18,150mm × 4.5mm, 5
μm；Column temperature is 25 DEG C；Sample size is 10 μ L；Flowing selects mass percent to be 0.3% aqueous formic acid and methanol mutually；Flowing phase
Flow velocity is 200 μ L/min；Operation time: 12min；Using gradient elution mode to detect, the step of gradient elution mode is:
0～7 minute: 0.3% aqueous formic acid 5%, methanol 95%,
7～8 minutes: 0.3% aqueous formic acid 22%, methanol 78%,
8～12 minutes: 0.3% aqueous formic acid 5%, methanol 95%；
3, the Mass Spectrometer Method of sample: Mass Spectrometry Conditions be chromatographic column flow out component enter mass spectrometric flow velocity be 200 μ L/min,
Ion source is electron spray ESI+ pattern；Sheath gas velocity is 40arb；Secondary air speed is 10arb；Electron spray voltage is 4.5kv；Hair
Capillary temperature is 275 DEG C；Capillary voltage is 16V；Lens voltage is 45V；Carrier gas is nitrogen；Collision gas is helium；Use one
Level mass spectrum full scan mode detection pattern, 50～500m/z；Second order ms is selective enumeration method SRM pattern, carboxymethyl-lysine
Quasi-quasi-molecular ions is 205m/z；Fragments characteristic quasi-molecular ions is 130m/z and 80m/z；Separation width is 1.5m/z；Collision energy 25%.
4, the method confirmation of detection method: be 0.1～100ng/mL (containing interior by standard reserving solution dilution preparation mass concentration
Mark 20ng/mL) and the series standard solution of 0.25～20 μ g/mL (containing the internal standard 5 μ g/mL), with target components peak area with corresponding
The ratio Y of internal standard peak area mass concentration X to target components, draws free state and protein binding state carboxymethyl-lysine respectively
Standard curve regression equation and correlation coefficient.With the signal to noise ratio of testing compound chromatographic peak in mark-on sample equal to 3 (S/N=
3) the corresponding detection limit that mass concentration is method (LOD), mass concentration corresponding for S/N=10 is the quantitative limit (LOQ) of method.
The detection linear equation of two states carboxymethyl-lysine, the range of linearity, correlation coefficient (R2), detection limit and quantitative limit.
The range of linearity of described detection method is: the carboxymethyl-lysine 0.1～100ng/mL of free state, protein binding state
Carboxymethyl-lysine 0.25～20 μ g/mL.
The lowest detection of described detection method is limited to 0.03ng/kg, is quantitatively limited to 0.1ng/kg.
Described detection method is in order to detect the carboxymethyl-lysine standard curve Y=0.1X+0.0417, R of free state2=
0.9997, the carboxymethyl-lysine standard curve Y=0.28X+0.0177, R of protein binding state2=0.9999.
The invention has the beneficial effects as follows: the use that present invention, avoiding ion-pairing agent is located without before any derivatization
Reason step, and have feature efficient, accurate, highly sensitive, that favorable reproducibility, the range of linearity are wide, analyzing milk and milk products middle reaches
Amorph and protein binding state carboxymethyl-lysine content aspect have its distinctive feature.In milk and milk products, carboxymethyl-lysine is main
Existing with protein binding state form, content is in ppm level, and free state content is in ppb level.For guaranteeing two kinds of form carboxymethyl-lysines
Dosing accuracy on two orders of magnitude, saves internal standard usage amount, and the present invention takes the method for two quantitation curves, adds inspection
The range of linearity surveyed, improves dosing accuracy, the carboxymethyl-lysine standard curve Y=0.1X+0.0417, R of free state2=
0.9997, the carboxymethyl-lysine standard curve Y=0.28X+0.0177, R of protein binding state2=0.9999, show combined state
In ppm level, free state is good linear relationship at ppb level, peak area and sample size.
It is fixed that free state in milk and milk products and combined state carboxymethyl-lysine can be carried out by the present invention fast and accurately
Property, quantitative analysis, extraordinary can be applied to the detection of free state and combined state carboxymethyl-lysine in milk and milk products.
Figure of description
Fig. 1 carboxymethyl-lysine first mass spectrometric figure；
Fig. 2 is carboxymethyl-lysine second order ms figure；
Fig. 3 carboxymethyl-lysine standard substance chromatography of ions figure；
Fig. 4 is free state carboxymethyl-lysine detection chromatography of ions figure in milk sample；
Fig. 5 is protein binding state carboxymethyl-lysine detection chromatography of ions figure in milk sample；
The response rate bar diagram of Fig. 6 HiCapt MCX solid-phase extraction column；
The molecular structure of Fig. 7 D4-carboxymethyl-lysine.
Detailed description of the invention
In order to be better understood from technical scheme, describe, below in conjunction with embodiment, the technology that the present invention provides in detail
Scheme, but limit the present invention never in any form.
1, the pre-treatment of sample: solid milk powder sample accurately weighs 0.1g, adding the mixing of 1mL ultra-pure water vortex, to make sample molten
Liquid, liquid milk product directly measures 1mL；Prepare addition 2～3mL normal hexane in liquid to sample, be centrifuged with 4000r/min after shaking up
10min, discards normal hexane layer, weighs 2～3 times thoroughly to remove fat, skimmed milk and milk powder and can remove this step from；Add
The sodium borate buffer liquid of 1.5mL0.2mol/L, pH9.4,1mL sodium borohydride solution, reacts 6h at 4 DEG C；Add and double sample
60% solution of trichloroacetic acid of volume, 8000r/min, 4 DEG C of centrifugal 10min, cleer and peaceful protein precipitation in separation, after collecting respectively
Treat to process further；In supernatant, add the D4-carboxymethyl-lysine Isotopic Internal Standard of 100 μ L 0.2 μ g/mL, cross SPE column purification；
Above-mentioned albumen precipitation is added the hydrochloric acid of 4mL 6mol/L, is passed through nitrogen and protects 110 DEG C of hydrolysis 24h, hydrolyzed solution is diluted constant volume
To 5mL, in hydrolyzed solution, add the D4-carboxymethyl-lysine of 100 μ L 50 μ g/mL, upper Solid-Phase Extraction column purification；By HiCapt
MCX solid-phase extraction column uses 3mL methanol, 3mL water activation balance successively, after loading, then uses 3mL water, and 3mL methanol drip washing is finally used
5mL ammonia: the ammoniated methanol eluting of methanol=5:95, v/v, collects eluent, after nitrogen dries up, accurately adds 1mL ultra-pure water
Redissolve, after vortex mixing, cross 0.22 μm cellulose membrane.As can be seen from Figure 6 the response rate of MCX mixed type cation be 104%～
115%, show that MCX pillar has good adsorption cleaning ability to carboxymethyl-lysine.
2, sample HPLC detection: HPLC condition be chromatographic column be Waters Symmetry C18,150mm × 4.5mm, 5
μm；Column temperature is 25 DEG C；Sample size is 10 μ L；Flowing selects mass percent to be 0.3% aqueous formic acid and methanol mutually；Flowing phase
Flow velocity is 200 μ L/min；Operation time: 12min；Using gradient elution mode to detect, the step of gradient elution mode is:
0～7 minute: 0.3% aqueous formic acid 5%, methanol 95%,
7～8 minutes: 0.3% aqueous formic acid 22%, methanol 78%,
8～12 minutes: 0.3% aqueous formic acid 5%, methanol 95%；
3, the Mass Spectrometer Method of sample: it is 200 μ L/min that chromatographic column flow out component to enter mass spectrometric flow velocity, to retention time
Peak for about 5.7min carries out mass spectral analysis, uses electron spray (ESI) ionization, and cation scan pattern, sheath gas velocity is
40arb；Secondary air speed is 10arb；Electron spray voltage is 4.5kv；Capillary temperature is 275 DEG C；Capillary voltage is 16V；
Lens voltage is 45V；Carrier gas is nitrogen；Collision gas is helium；Use first mass spectrometric full scan mode detection pattern, sweep limits
50～500m/z, obtain the quasi-molecular ions of m/z 205；Again m/z 205 quasi-molecular ions is carried out second order ms scanning, selective enumeration method
SRM pattern, separation width is 1.5m/z；Collision energy 25%.In Fig. 1, m/z 205.02 is carboxymethyl-lysine quasi-quasi-molecular ions [M
+H]+；In Fig. 2, m/z 129.87 and m/z 83.80 is carboxymethyl-lysine second order ms fragments characteristic ion.
4, the method confirmation of detection method: in milk and milk products, carboxymethyl-lysine is mainly deposited with protein binding state form
, content is in ppm level, and free state content is in ppb level.For guarantee two kinds of form carboxymethyl-lysines on two orders of magnitude quantitatively
Accuracy, saves internal standard usage amount, and the present invention takes the method for two quantitation curves, adds the range of linearity of detection, improves
Dosing accuracy.
Accurately weigh 10.0mg carboxymethyl-lysine and 1.0mgD4-carboxymethyl-lysine standard substance are respectively placed in 10mL palm fibre
In color tolerance measuring bottle, with ultra-pure water dissolve be settled to groove, be configured to 1.0mg/mL carboxymethyl-lysine standard reserving solution and
0.1mg/mL D4-carboxymethyl-lysine internal standard storing solution.By carboxymethyl-lysine standard reserving solution dilution preparation 100 μ g/mL and
The standard working solution of 1000ng/mL.By D4-carboxymethyl-lysine standard reserving solution dilution preparation 50 μ g/mL and 200ng/mL mark
Quasi-working solution.From the beginning of 1000ng/mL carboxymethyl-lysine titer, accurately pipette volume with pipettor, according to multiple proportions gradient
The mode of dilution obtains 100,20,10,5,1,0.5, the series standard solution of 0.1ng/mL, respectively take 0.9mL standard solution in entering
In sample bottle, being separately added into the 200ng/mL D4-carboxymethyl-lysine vortex mixing of 0.1mL, upper machine measures；From 100 μ g/mL carboxylics
Methyllysine titer starts, and accurately measures volume with pipettor, obtain 20 according to the mode of multiple proportions gradient dilution, 10,5,
2.5, the series standard solution of 1,0.5,0.25 μ g/mL, respectively takes 0.9mL standard solution in sample injection bottle, is separately added into 0.1mL's
50 μ g/mL D4-carboxymethyl-lysine vortex mixings, upper machine measures.According to above-mentioned instrument testing conditions, with face, target components peak
Amass mass concentration X to target components of the ratio Y with corresponding internal standard peak area, draw free state and protein binding state carboxylic first respectively
The standard curve regression equation of base lysine and correlation coefficient.It is equal to the signal to noise ratio of testing compound chromatographic peak in mark-on sample
The mass concentration of 3 correspondences is the detection limit of method, and signal to noise ratio is equal to the quantitative limit that mass concentration is method of 10 correspondences.Two kinds of shapes
The detection linear equation of state carboxymethyl-lysine, the range of linearity, correlation coefficient (R2), detection limit and quantitative limit.With mark-on sample
The signal to noise ratio of middle testing compound chromatographic peak is equal to the detection limit that mass concentration is method of 3 correspondences, and signal to noise ratio is corresponding equal to 10
The quantitative limit that mass concentration is method.
The range of linearity of described detection method is: the carboxymethyl-lysine 0.1～100ng/mL of free state, protein binding state
Carboxymethyl-lysine 0.25～20 μ g/mL.
The lowest detection of described detection method is limited to 0.03ng/kg, is quantitatively limited to 0.1ng/kg.
Described detection method is in order to detect the carboxymethyl-lysine standard curve Y=0.1X+0.0417, R2=of free state
0.9997, the carboxymethyl-lysine standard curve Y=0.28X+0.0177, R2=0.9999 of protein binding state.
Can be seen that the inventive method detection the carboxymethyl-lysine range of linearity width of two states, dependency be good, detection
Limit and quantitative limit are low, show that this invention has higher sensitivity.
Fig. 3 is carboxymethyl-lysine standard substance chromatography of ions figure.By the condition optimized, use liquid chromatography mass spectrometric combination right
Carboxymethyl-lysine standard substance parent ion and selected daughter ion chromatogram effect of optimization are verified, result is as shown in Figure 3.
5, the mensuration of the detection method response rate: be separately added into 4 variable concentrations water according to addition shown in table one before Chu Liing
The D4-carboxymethyl-lysine internal standard of flat carboxymethyl-lysine and 20 μ g/kg, replication 3 times, calculate the flat of each mark-on level
The all response rate and relative standard deviations (RSD) are shown in Table 1.Being known by table 1, the response rate of this inventive method is 104%～115%, RSD
Less than 5.13%, it is seen that this law response rate is good, precision is high, can meet carboxymethyl-lysine detection by quantitative requirement in milk product.
Fig. 4 is free state carboxymethyl-lysine detection chromatography of ions figure in milk sample, enters adding target dairy-like product
The carboxymethyl-lysine detection of row free state and protein binding state, good separating effect, the most with this understanding carboxylic
Methyllysine and the response of D4-carboxymethyl-lysine are good, disturb without other impurity.
Fig. 5 is protein binding state carboxymethyl-lysine detection chromatography of ions figure in milk sample, to adding target dairy-like
Product carry out the carboxymethyl-lysine detection of free state and protein binding state, and good separating effect, as can be seen from the figure in this condition
Lower carboxymethyl-lysine and the response of D4-carboxymethyl-lysine are good, disturb without other impurity.
Table 1 is the recovery of standard addition (n=3) of carboxymethyl-lysine detection in milk product
The stability of carboxymethyl-lysine detection method in table 2 milk product
6, the mensuration of the detection method response rate: repeatability and precision: take same testing sample, in 1 day 6 different time
Between point detect, continuously monitoring 6 days, calculate day interpolation and the poorest RSD, the stability of investigation method.As shown in table 2,
The withinday precision RSD of the present invention is 4.22% and 2.75%, and accurate RSD is 10.57% and 7.88% in the daytime, it was demonstrated that method
Have good stability, meet quantitative analysis requirement.
7, different types of sample is detected by the method for the invention, sample is carried out being embodied as step 1
Pre-treatment, instrument detection liquid-phase condition is with reference to implementing step 2, and Mass Spectrometer Method condition is with reference to being embodied as step 3, and every part of sample is put down
Row measures three times.Result such as table 3 and table 4.
Table 3 different types of solid milk powder middle reaches amorph and protein binding state carboxymethyl-lysine testing result (n=3)
Free state and protein binding state carboxymethyl-lysine testing result (n=3) in table 4 variety classes liquid milk
The invention has the beneficial effects as follows: the use that present invention, avoiding ion-pairing agent is located without before any derivatization
Reason step, and have feature efficient, accurate, highly sensitive, that favorable reproducibility, the range of linearity are wide, analyzing milk and milk products middle reaches
Amorph and protein binding state carboxymethyl-lysine content aspect have its distinctive feature.In milk and milk products, carboxymethyl-lysine is main
Existing with protein binding state form, content is in ppm level, and free state content is in ppb level.For guaranteeing two kinds of form carboxymethyl-lysines
Dosing accuracy on two orders of magnitude, saves internal standard usage amount, and the present invention takes the method for two quantitation curves, adds inspection
The range of linearity surveyed, improves dosing accuracy, the carboxymethyl-lysine standard curve Y=0.1X+0.0417, R of free state2=
0.9997, the carboxymethyl-lysine standard curve Y=0.28X+0.0177, R of protein binding state2=0.9999, show combined state
In ppm level, free state is good linear relationship at ppb level, peak area and sample size.
It is fixed that free state in milk and milk products and combined state carboxymethyl-lysine can be carried out by the present invention fast and accurately
Property, quantitative analysis, extraordinary can be applied to the detection of free state and combined state carboxymethyl-lysine in milk and milk products.
Claims (4)
1. free state and the detection method of combined state carboxymethyl-lysine in a milk and milk products, it is characterised in that by following inspection
Survey condition is carried out:
HPLC condition be chromatographic column be Waters Symmetry C18,150mm × 4.5mm, 5 μm；Column temperature is 25 DEG C；Sample size
It is 10 μ L；Flowing selects mass percent to be 0.3% aqueous formic acid and methanol mutually；Flow rate of mobile phase is 200 μ L/min；Run
Time: 12min；Using gradient elution mode to detect, the step of gradient elution mode is:
0～7 minute: 0.3% aqueous formic acid 5%, methanol 95%,
7～8 minutes: 0.3% aqueous formic acid 22%, methanol 78%,
8～12 minutes: 0.3% aqueous formic acid 5%, methanol 95%；
Mass Spectrometry Conditions be chromatographic column flow out component enter mass spectrometric flow velocity be 200 μ L/min, ion source is electron spray ESI+ mould
Formula；Sheath gas velocity is 40arb；Secondary air speed is 10arb；Electron spray voltage is 4.5kv；Capillary temperature is 275 DEG C；Capillary
Tube voltage is 16V；Lens voltage is 45V；Carrier gas is nitrogen；Collision gas is helium；Use first mass spectrometric full scan mode detection
Pattern, 50～500m/z；Second order ms is selective enumeration method SRM pattern, and the quasi-quasi-molecular ions of carboxymethyl-lysine is 205m/z；Feature
Fragment ion peak is 130m/z and 80m/z；Separation width is 1.5m/z；Collision energy 25%.
Free state and the detection side of combined state carboxymethyl-lysine in a kind of milk and milk products the most according to claim 1
Method, it is characterised in that: described detection method also includes that the pre-treatment of sample, the pre-treating method of described sample are: solid milk powder
Sample accurately weighs 0.1g, adds the mixing of 1mL ultra-pure water vortex and makes sample solution, and liquid milk product directly measures 1mL；To sample
Prepare in liquid addition 2～3mL normal hexane, be centrifuged 10min with 4000r/min after shaking up, discard normal hexane layer, repeat 2～3 times with
Thoroughly remove fat, skimmed milk and milk powder and can remove this step from；Add the sodium borate buffer liquid of 1.5mL0.2mol/L, pH9.4,
1mL sodium borohydride solution, reacts 6h at 4 DEG C；Add and double 60% solution of trichloroacetic acid of sample volume, 8000r/min, 4
DEG C centrifugal 10min, cleer and peaceful protein precipitation in separation, treat to process further after collecting respectively；100 μ L 0.2 μ are added in supernatant
The D4-carboxymethyl-lysine Isotopic Internal Standard of g/mL, crosses SPE column purification；Above-mentioned albumen precipitation is added the salt of 4mL 6mol/L
Acid, is passed through nitrogen and protects 110 DEG C of hydrolysis 24h, hydrolyzed solution dilution is settled to 5mL, adds 100 μ L 50 μ g/mL in hydrolyzed solution
D4-carboxymethyl-lysine, upper Solid-Phase Extraction column purification；HiCapt MCX solid-phase extraction column is used 3mL methanol, 3mL water successively
Activation balance, after loading, then uses 3mL water, and 3mL methanol drip washing finally uses 5mL ammonia: the ammoniated methanol of methanol=5:95, v/v
Eluting, collects eluent, after nitrogen dries up, accurately adds 1mL ultra-pure water and redissolves, cross 0.22 μm cellulose membrane after vortex mixing.
Free state and the detection side of combined state carboxymethyl-lysine in a kind of milk and milk products the most according to claim 1
Method, it is characterised in that: the range of linearity of described detection method is: the carboxymethyl-lysine 0.1～100ng/mL of free state, albumen
The carboxymethyl-lysine 0.25～20 μ g/mL of combined state.
Free state and the detection side of combined state carboxymethyl-lysine in a kind of milk and milk products the most according to claim 1
Method, it is characterised in that: the lowest detection of described detection method is limited to 0.03ng/kg, is quantitatively limited to 0.1ng/kg.
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| CN106124641B (en) * | 2016-05-13 | 2019-01-29 | 中华全国供销合作总社杭州茶叶研究所 | A kind of synchronous method for detecting carboxymethyl-lysine and lysopine in green tea |
| CN107290452B (en) * | 2017-06-23 | 2022-04-15 | 江南大学 | Method for detecting content of bound heterocyclic amine |
| CN109932468A (en) * | 2019-03-21 | 2019-06-25 | 华南理工大学 | A kind of synchronous method for detecting free state CML and 5-HMF in liquid or semisolid flavouring |
| CN114624375B (en) * | 2020-12-12 | 2023-12-22 | 浙江工业职业技术学院 | Method for detecting content of carboxymethyl lysine in potato chips |
| CN113808674B (en) * | 2021-08-11 | 2023-09-08 | 天津科技大学 | Calculation method and application of Maillard reaction byproduct content of dairy product added with tea polyphenol |
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| CN103616466A (en) * | 2013-11-27 | 2014-03-05 | 上海海洋大学 | Detecting method of carboxymethyl lysine and application thereof |
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