CN101793642A - Separation and liquid chromatography column pre-column derivatization method of biogenic amine in soybean paste - Google Patents
Separation and liquid chromatography column pre-column derivatization method of biogenic amine in soybean paste Download PDFInfo
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Abstract
The invention discloses a separation and liquid chromatography column pre-column derivatization method of biogenic amine in a soybean paste, comprising the following steps of: (1) adding a trichloroacetic acid solution to the soybean paste, homogenizing, centrifuging and collecting a supernate; (2) adding normal hexane to the supernate, vibrating and taking out a lower layer trichloroacetic acid phase for later use after static layering; (3) adding an NaOH solution to the trichloroacetic acid phase, then adding an NaHCO3 solution for buffering, and mixing in a dansyl chloride acetone solution to carry out derivatization; and (4) removing dansyl chloride to terminate the reaction to obtain a derivative solution and filtering. The separation method has simple treatment process and low reagent consumption and avoids analyte loss caused by transfer. The soybean paste can reach detection demands after being separated and purified; undesired peak disturbance of a spectrogram is less and the determinand loss is low, and the recovery rate of the biogenic amine can reach more than 80 percent. The method determines the optimum condition for deriving the biogenic amine; dansyl chloride derivative products are stable and the detection sensitivity is high.
Description
Technical field
The present invention relates to the separation and the derivatization method of biogenic amine, relate in particular to from beans sauce separating bio amine and, belong to the separation and the detection range of biogenic amine the method for biogenic amine liquid chromatography column pre-column derivatization.
Background technology
Biogenic amine in the beans sauce mainly is that the decarboxylation of the amino acid decarboxylases that produces by the Institute of Micro-biology that wherein exists generates.The generation of biogenic amine needs the condition of 3 aspects: have the precursor substance-free amino acid that generates biogenic amine; Has the condition that can make amino acid generation decarboxylic reaction; The environment of suitable growth of microorganism.
The biogenic amine that generates owing to microbial action in the beans sauce sweat has histamine, putrescine, cadaverine, tryptamines, tyrasamine, β-phenyl ethylamine, spermine and spermidine etc.Be considered to carcinogen as the N-nitrosamine in the biogenic amine, have report at present about the bean products of high biogenic amine content.Korea S's beans sauce salt content very high (about 12% or higher), cause hypertension and kidney trouble possibly, therefore need to adopt the less salt fermentation technique, but low-salt conditions is more suitable in microbial growth than high salt condition, in the less salt sweat, produces more biogenic amine possibly.In beans sauce sweat, adopt irradiation technique can stop microbial growth, thereby effectively suppress the formation of biogenic amine.
In the beans sauce spontaneous fermentation process, the kind that relates to microorganism is a lot, and the formation of biogenic amine and microorganism have substantial connection in the beans sauce, but is not that the microorganism of all kinds all can produce amino acid decarboxylases, generates biogenic amine.Beans sauce sweat mainly comprises bacterium, saccharomycete, mould, and these microorganisms all might be relevant with the formation of biogenic amine.The protelytic ability difference of different microorganisms, general fungi is than the easy decomposing protein of bacterium.Can decomposing protein in the bacterium proteus, clostridium, sporeformer, pseudomonad and micrococcus etc. are arranged, they only just can form proteinase when a large amount of breeding.Microorganisms proteinase, decomposing protein form various small peptides, generate amino acid then under the peptase effect, and free amino acid is one of key factor that influences biogenic amine formation, and it has vital role to the formation of biogenic amine in the beans sauce.The protein content of soybean is higher, in beans sauce sweat, increase with fermentation time, increasing protein is degraded, the content of free amino acid, peptide molecule increases thereupon, these materials all can be used as the catalytic reaction substrate of the decarboxylase of Institute of Micro-biology's generation, thereby generate various biogenic amines.
Because most microbial growth temperature influences are very big, when being suitable for the temperature of growth of microorganism, can produce large number of biological amine.It is slow that low temperature produces the amine bacteria growing down, causes biogenic amine in food content lower.Storage temperature is high more, and biogenic amine content gathers way fast more.Because biogenic amine has potential toxicity, so the generation of biogenic amine must attract great attention in the beans sauce.
During biogenic amine in the analyzing and testing sample, at first be from matrix, to extract biogenic amine.Acid medium or organic phase medium are generally selected in the extraction of biogenic amine for use.Acid medium generally has hydrochloric acid, perchloric acid and trichloroacetic acid; Organic phase medium such as methyl alcohol, acetone, acetonitrile, methylene chloride etc., mixed liquors such as also useful acetonitrile-perchloric acid, methylene chloride-perchloric acid are as the extraction medium of biogenic amine.The extraction of biogenic amine in meat, aquatic products and the vegetable food, the general concentration that adopts is that the hydrochloric acid of 0.1～0.5mol/L, the perchloric acid of 0.4～0.6mol/L or 5～6% acid mediums such as trichloroacetic acid extract, because 5% trichloroacetic acid is better than hydrochloric acid to the sedimentation effect of protein in these samples, when therefore extracting the biogenic amine in the higher sample of protein content, the use of trichloroacetic acid is more.But generally adopt 0.1mol/L hydrochloric acid effect better for the extraction of biogenic amine in the cheese.
Food substrate composition more complicated, other become to grade often to contain protein, free amino acid, fat, carbohydrate, polyphenol compound and some.The existence of these compositions can influence the effective separation and the accurate qualitative, quantitative of biogenic amine, also can reduce the sensitivity of checkout equipment simultaneously, has the danger of stopping up chromatographic column.When therefore using high performance liquid chromatography detection of biological amine, before derivatization, also need the extract of biogenic amine in the foodstuff samples of matrix complexity is carried out purified treatment.The purification method of biogenic amine mainly contains two kinds at present: a kind of is to adopt Solid-Phase Extraction (SPE) method; Another kind is the liquid-liquid extraction method that adopts organic solvent.
The Solid-Phase Extraction method of decontamination of biological amine mainly adopts SPE C at present
18Post and reinforcing yin essence ion-exchange (SAX) post.SPE C
18Post belongs to non-polar column, can remove apolar substance.The liquid that contains biogenic amine need not to handle just can the direct C of mistake
18Post, polyphenol and apolar substance are retained on the post, reach the purpose of decontamination of biological amine.The SAX post is the reinforcing yin essence ion exchange column, and anionic species can be removed, and also can play the effect that concentrates and decolour simultaneously.The clean-up effect of SAX post is than SPE C
18Post is good.The pH of sample extracting solution is the key factor that influences clean-up effect when crossing the SAX post, and the pH of liquid food or extract is transferred to 8 (making polyphenol and amino acid be the negative ion attitude), and sample is through behind the post, and the biogenic amine of kation state is retained on the post.
Liquid-liquid extraction is that a kind of organic solvent of employing or several mixed organic solvents extract the biogenic amine in the sample, thereby reaches the purpose of purification.Though this method operation steps is more loaded down with trivial details, can obtain higher sensitivity.The basic step of liquid-liquid extraction is: sample extracting solution carries out saturated processing with salt earlier, transfers pH to alkalescence then, uses organic solvent (normal butyl alcohol, normal butyl alcohol-chloroform) extraction at last.Wherein the selection of the pH of the kind of saturated used salt, solution and organic solvent is the key factor that influences the liquid-liquid extraction effect.Salt can impel biogenic amine to enter organic phase, but Na
2CO
3Can change the pH of solution, so generally select for use NaCl as the saturated salt of using.PH influences the extraction efficiency of part biological amine, and the biogenic amine recovery was the highest when pH11.5 was thought in research.In the liquid-liquid extraction, stricter when utilizing trichloroacetic acid to extract Billy to the requirement of pH with HCl extraction biogenic amine.During extracting n-butyl alcohol, the recovering effect of histamine, tyrasamine is better, but the recovery of putrescine, cadaverine, spermine and spermidine is lower.Normal butyl alcohol-chloroform (1: 1, v/v) than the easy evaporate to dryness of normal butyl alcohol, but when handling milk fish, can produce the colloid that is difficult to separate.So general when handling dairy products to adopt normal butyl alcohol be extraction solvent, other kinds based food then adopt normal butyl alcohol-chloroform (1: 1, v/v) be extraction solvent.Because purification method is loaded down with trivial details, also there is the researcher only to use filter membrane (0.22～0.45 μ m) to handle, then derivatization or direct injected (deriving automatically) to fluid sample (as grape wine) or biological food amine extract.
Column front derivation agent commonly used has dansyl Cl, o-phthalaldehyde(OPA), dinitrobenzoyl chloride and fluorescamine etc., and research usefulness 9-fluorenyl methyl-chloro-carbonate and the phenyl isothiocyanate derivating agent as biogenic amine is also arranged.When selecting derivating agent,, derivatization process few with derivative products good stability, impurity simply is principle, but all derivative reagents all have certain limitation.Dinitrobenzoyl chloride derivant and phenyl isothiocyanate derivatives do not possess fluorescent characteristic, can not detect with fluorescence detector, can only UV-detector detect, and its method sensitivity is lower than other derivative reagents.9-fluorenyl methyl-chloroformate derivative has fluorescent characteristic, and sensitivity, stability are all fine, but it produces the multiple product from the monoamine to the polyamines, serious interference when detecting matrix complicated sample and multiple hybrid standard thing; The advantage of o-phthalaldehyde(OPA) be can with elementary amine a few minutes internal reaction generate stronger fluorescent material, lack because of the required time of deriving, realize robotization easily, before the general post, post-column derivation all adopts the O-phthalic aldehyde reagent; But O-phthalic aldehyde derivatives instability does not allow after deriving to purify, and derivatization conditions is had strict requirement, for avoiding interference, increases sensitivity, when being derivating agent with the o-phthalaldehyde(OPA), needs to add 2 mercapto ethanol.The dansyl Cl derivatization reaction time is longer, but product is stable, and 4 ℃ of preservations loss in month is no more than 10% in the dark place.Detection sensitivity is also higher, before the post, post-column derivation all can use.
Summary of the invention
Technical matters to be solved by this invention be to existing from beans sauce the various technologies or the condition of the liquid chromatography column pre-column derivatization method of the method for separating bio amine and biogenic amine be optimized and screen, obtain the complete method of separating bio amine from beans sauce of a cover and the liquid chromatography column pre-column derivatization method of biogenic amine, this method has separation efficiency height, derivatization effect advantages of higher.
Technical matters to be solved by this invention is achieved through the following technical solutions:
The separation of biogenic amine and liquid chromatography column pre-column derivatization method in the beans sauce may further comprise the steps: (1) adds trichloroacetic acid solution in beans sauce, and homogeneous is centrifugal, collects supernatant; (2) add normal hexane in supernatant, lower floor's trichloroacetic acid phase is taken out in vibration after the static layering, standby; (3) add NaOH solution to trichloroacetic acid in mutually, add NaHCO again
3The solution buffering is mixed the dansyl Cl acetone soln and is carried out derivative reaction; (4) ammoniacal liquor is removed the dansyl Cl cessation reaction, obtains the liquid of deriving; The liquid of deriving filters, promptly.
Preferably, the 5% trichloroacetic acid solution that in beans sauce, adds 1.5 times of beans sauce quality in the step (1); Described homogeneous is preferably homogeneous 3min under the 10000r/min rotating speed; The described centrifugal centrifugal 5min under the 3500r/min rotating speed that is preferably;
Preferably, in supernatant, add isopyknic normal hexane in the step (2);
Add 2mol/L NaOH solution to trichloroacetic acid in mutually in the step (3), wherein trichloroacetic acid is 5: 1 with the volume ratio of 2mol/L NaOH solution; Then add saturated NaHCO again
3Solution cushions, wherein, and saturated NaHCO
3Solution is 3: 10 with trichloroacetic acid volume ratio mutually;
Derivatization conditions described in the step (3) is preferably: derivating agent dansyl Cl acetone soln consumption is that 1.5 times of the trichloroacetic acid phase volume, the time of deriving are 40min, and the temperature of deriving is 40 ℃.
The ammoniacal liquor that adds 0.5 times of initial volume in the step (4) is removed the dansyl Cl cessation reaction, is settled to 25 times of initial volumes with acetonitrile at last; With the filtering with microporous membrane of 0.22 μ m, be used for liquid-phase chromatographic analysis and detect after the derivation process.
Separation method processing procedure of the present invention is simple, adopt the liquid-liquid extraction method of purification consumption reagent of micro-trichloroacetic acid-normal hexane few, and the analyte of having avoided being caused when shifting loses.Can reach the detection requirement behind isolation of purified of the present invention, spectrogram is mixed, and the peak disturbs less and the determinand loss is little, and the recovery of biogenic amine all can reach more than 80%.The present invention adopts dansyl Cl as derivating agent, the top condition of the biogenic amine of having determined to derive, and the dansyl Cl derivative products is stable, the detection sensitivity height.
Description of drawings
The HPLC-DAD chromatogram of empty white miso sample (a) of Fig. 1 and mark-on beans sauce sample (b).
The HPLC-FLD chromatogram of empty white miso sample (a) of Fig. 2 and mark-on beans sauce sample (b).
Fig. 3 dansyl Cl consumption is to the derive influence of effect of biogenic amine.
Fig. 4 time is to the derive influence of effect of biogenic amine.
Fig. 5 temperature is to the derive influence of effect of biogenic amine.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage of the present invention and characteristics will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment or replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall within the scope of protection of the present invention the details of technical solution of the present invention and form.
Embodiment 1
Take by weighing the Northeast's spontaneous fermentation soya sauce 4g in centrifuge tube, add the 5% trichloroacetic acid solution of 6mL, 10000r/min, homogeneous 3min.The centrifugal 5min of 3500r/min behind the homogeneous.Collect supernatant, residue repeats to extract 1 time with the trichloroacetic acid of 6mL 5%, merges extract, add the 6mL normal hexane again and remove lipid impurity, static layering behind the thermal agitation discards the normal hexane layer, add 6mL normal hexane repetitive operation 1 time again, take out lower floor's trichloroacetic acid phase after the layering.The dansyl Cl of 10mg is dissolved in the acetone of 1mL, and prepared fresh also keeps in Dark Place.The NaOH solution of 200 μ L, 2mol/L is added in the 1mL sample solution, adds the saturated NaHCO of 300 μ L then
3The solution buffering.The dansyl Cl solution that adds 1.5mL is put into 40 ℃ of constant temperature ovens insulations with mixed solution and is handled after the 40min, and the ammoniacal liquor that adds 100 μ L is removed the dansyl Cl cessation reaction, is settled to 5mL with acetonitrile at last.Organic membrane filtration with 0.22 μ m after the derivation process is used for analyzing and testing biogenic amine (detection method and testing result are seen test example 1).
Take by weighing and grind the product thick broad-bean sauce 6g of the Northeast, back in small, broken bits in centrifuge tube, add the 5% trichloroacetic acid solution of 12mL, 10000r/min, homogeneous 3min.The centrifugal 5min of 3500r/min behind the homogeneous.Collect supernatant, residue repeats to extract 1 time with the trichloroacetic acid of 12mL 5%, merges extract, add the 12mL normal hexane again and remove lipid impurity, static layering behind the thermal agitation discards the normal hexane layer, add 12mL normal hexane repetitive operation 1 time again, take out lower floor's trichloroacetic acid phase after the layering.The dansyl Cl of 10mg is dissolved in the acetone of 1mL, and prepared fresh also keeps in Dark Place.The NaOH solution of 400 μ L, 2mol/L is added in the 2mL sample solution, adds the saturated NaHCO of 600 μ L then
3The solution buffering.The dansyl Cl solution that adds 3mL is put into 40 ℃ of constant temperature ovens insulations with mixed solution and is handled after the 40min, and the ammoniacal liquor that adds 200 μ L is removed the dansyl Cl cessation reaction, is settled to 10mL with acetonitrile at last.Organic membrane filtration with 0.22 μ m after the derivation process is used for analyzing and testing biogenic amine (detection method and testing result are seen test example 1).
The seasoning soya sauce 10g that takes by weighing suitability for industrialized production adds the 5% trichloroacetic acid solution of 15mL, 10000r/min, homogeneous 3min in centrifuge tube.The centrifugal 5min of 3500r/min behind the homogeneous.Collect supernatant, residue repeats to extract 1 time with the trichloroacetic acid of 15mL 5%, merges extract, add the 15mL normal hexane again and remove lipid impurity, static layering behind the thermal agitation discards the normal hexane layer, add 15mL normal hexane repetitive operation 1 time again, take out lower floor's trichloroacetic acid phase after the layering.The dansyl Cl of 10mg is dissolved in the acetone of 1mL, and prepared fresh also keeps in Dark Place.The NaOH solution of 200 μ L, 2mol/L is added in the 1mL sample solution, adds the saturated NaHCO of 300 μ L then
3The solution buffering.The dansyl Cl solution that adds 1.5mL is put into 40 ℃ of constant temperature ovens insulations with mixed solution and is handled after the 40min, and the ammoniacal liquor that adds 100 μ L is removed the dansyl Cl cessation reaction, is settled to 5mL with acetonitrile at last.Organic membrane filtration with 0.22 μ m after the derivation process is used for analyzing and testing biogenic amine (detection method and testing result are seen test example 1).
Test the recovery, precision and the detectability of routine 1HPLC-DAD and HPLC-FLD method detection of biological amine
1, column front derivation
(1) dansyl Cl solution preparation: the dansyl Cl of 10mg is dissolved in the acetone of 1mL, and prepared fresh also keeps in Dark Place.
(2) step of deriving: the NaOH solution of 200 μ L, 2mol/L is added in 1mL standard solution and the sample solution, adds the saturated NaHCO of 300 μ L then
3The solution buffering.The dansyl Cl solution that adds 1.5mL is put into 40 ℃ of constant temperature ovens insulations with mixed solution and is handled after the 40min, and the ammoniacal liquor that adds 100 μ L is removed the dansyl Cl cessation reaction, is settled to 5mL with acetonitrile at last.With organic membrane filtration of 0.22 μ m, be used for analyzing and testing after the derivation process.
2, high performance liquid chromatography (HPLC) method detection of biological amine
Agilent ZORBAX XDB-C
18Post (4.6mm * 250mm, 5 μ m); The moving phase KH of 0.01mol/L
2PO
4(pH 3.5) and methyl alcohol carry out gradient elution, flow velocity 1.0mL/min, and the gradient elution program sees Table 1; Adopt diode array detector (DAD) and fluorescence detector (FLD) to measure respectively: the ultraviolet detection wavelength is 254nm, fluoroscopic examination excitation wavelength (Ex) 330nm, emission wavelength (Em) 465nm; Sample size 20 μ L; 30 ℃ of column temperatures.
Table 1 gradient elution program
The methodology checking of HPLC-DAD method detection of biological amine
The range of linearity of method
Add the mixed standard solution of 7 kinds of biogenic amines in 0.5～50mg/kg concentration range respectively, compound concentration is 0.5,1,5,25, the series standard solution of 50mg/kg, derives and detects according to above-mentioned.According to the average peak area of 7 kinds of biogenic amines (y, n=5) with the normal concentration of corresponding biogenic amine (x mg/kg) carries out linear regression, the drawing standard calibration curve, after Agilent workstation software analyzing and processing, the equation of linear regression of 7 kinds of biogenic amines sees Table 2.
The equation of linear regression of table 27 kind of biogenic amine and related coefficient (R2)
y：the?mean?peak?area?of?7?BAs(n＝5)；
x：mass?concentrations?of?7?BAs(mg/kg).
The recovery of method, precision and detection limit
Select the empty white miso sample that does not contain 7 kinds of components to be measured for use, add 1,10 and the mixed standard solution of 7 kinds of biogenic amines of three concentration levels of 50mg/kg respectively, handle sample by the method for setting up, analyzing and testing and calculate recovery rate and precision, record average recovery rate (n=5) scope between 76.3～110.2%, relative standard deviation is 3.3～9.1%.Empty white miso sample and mark-on beans sauce sample such as Fig. 1.
This test is added the minimum quality concentration point 0.5mg/kg of recovery test in standard, and the signal to noise ratio (S/N ratio) of various biogenic amines (S/N) is all greater than 10, and has the recovery preferably, thus definite each biogenic amine quantitatively be limited to 0.5mg/kg; With the detection limit of 3 times of signal to noise ratio (S/N ratio)s (S/N) computing method, the recovery of 7 kinds of biogenic amines, precision and detection limit see Table 3.
The interpolation recovery, precision and the detection limit (n=5) of table 37 kind of biogenic amine
aDetection limit: signal to noise ratio (S/N ratio) is 3
The methodology checking of HPLC-FLD method detection of biological amine
The range of linearity of method
Add the mixed standard solution of 7 kinds of biogenic amines in 0.1～50mg/kg concentration range respectively, compound concentration is 0.1,0.5,5,25, the series standard solution of 50mg/kg, derives and detects according to the method for setting up.According to the average peak area of 7 kinds of biogenic amines (y, n=5) with the normal concentration of corresponding biogenic amine (x mg/kg) carries out linear regression, the drawing standard calibration curve, after Agilent workstation software analyzing and processing, the equation of linear regression of 7 kinds of biogenic amines sees Table 4.
The equation of linear regression of table 47 kind of biogenic amine and related coefficient (R2)
y：the?mean?peak?area?of?7?BAs(n＝5)；
x：mass?concentrations?of?7?BAs(mg/kg).
The recovery of method, precision and detection limit
Select the empty white miso sample that does not contain 7 kinds of components to be measured for use, add 0.5,5 and the mixed standard solution of 7 kinds of biogenic amines of three concentration levels of 50mg/kg respectively, handle sample by the method for setting up, analyzing and testing and calculate recovery rate and precision, record 7 kinds of biogenic amine average recovery rates (n=5) scope between 74.7～105.1%, relative standard deviation is 2.1～9.7%.Mark-on beans sauce sample such as Fig. 2.
This test is added the minimum quality concentration point 0.1mg/kg of recovery test in standard, and the signal to noise ratio (S/N ratio) of various biogenic amines (S/N) is all greater than 10, and has the recovery preferably, thus definite each biogenic amine quantitatively be limited to 0.1mg/kg; With the detection limit of 3 times of signal to noise ratio (S/N ratio)s (S/N) computing method, the recovery of 7 kinds of biogenic amines, precision and detection limit see Table 5.
The interpolation recovery, precision and the detection limit (n=5) of table 57 kind of biogenic amine
aDetection limit: signal to noise ratio (S/N ratio) is 3
The optimization Test of test example 2 biogenic amine derivatization conditions
In the derivatization process of biogenic amine, factors such as the consumption of derivating agent, the temperature of deriving, the time of deriving all can influence the carrying out of derivatization reaction and the stability of derivative products, thereby influence detection sensitivity.This test adopts dansyl Cl as derivating agent, and derivatization conditions is optimized, and determines the derive top condition of biogenic amine of Dns-C1.
The selection of 1 derivating agent consumption
The volume ratio of derivating agent and biogenic amine standard solution (v/v, dansyl Cl concentration 10mg/mL) is selected respectively: 0.5,1,1.5,2, and other conditions are constant, derive as stated above and detect.Biogenic amine mixes mark concentration: 50mg/L.Do 3 parallel experiments for every group, get and measure the efficient of deriving that biogenic amine standard items peak area mean value is represented biogenic amine, result such as Fig. 3 for 3 times.
Fig. 3 shows that the derivating agent consumption is above after sample liquid is more than 1.5 times, and the efficient of deriving of biogenic amine no longer improves, and illustrates that derivating agent is saturated at this moment, there is no need to increase the derivating agent consumption again.Therefore, when this test determined to utilize Dns-C1 to derive, the derivating agent consumption was 1.5 o'clock with the sample volume ratio, can reach the desirable biogenic amine effect of deriving.
2, the derive selection of time
The biogenic amine time of deriving is set at respectively: 20min, 30min, 40min, 50min, 60min, other conditions remain unchanged, and derive as stated above and detect.Biogenic amine mixes mark concentration: 50mg/L.Do 3 parallel experiments for every group, calculate the mean value of 3 parallel experiments.Result such as Fig. 4.
Fig. 4 shows that when biogenic amine was derived 40min, the efficient of deriving reached mxm., and it is little to the effectiveness affects of deriving to continue to prolong the time of deriving.Therefore this test and Selection 40min derives the time as the best.
3, the derive selection of temperature
The biogenic amine temperature of deriving is respectively: room temperature, 30 ℃, 40 ℃, 50 ℃, 60 ℃, other conditions remain unchanged, and derive as stated above and measure.Biogenic amine mixes mark concentration: 50mg/L.Do 3 parallel experiments for every group, calculate the mean value of 3 parallel experiments.Result such as Fig. 5.
Fig. 5 shows, in this deriving method, tyrasamine, phenyl ethylamine, histamine and putrescine are bigger from room temperature to 40 efficiency change of ℃ deriving, and 40 ℃ of follow-up temperature of continuing rising are little to the effectiveness affects of deriving; Cadaverine, spermine and spermidine are in room temperature to 60 ℃ scope, and the efficiency change of deriving is little.When room temperature and 30 ℃ were derived, the liquid of deriving often had the yellow of depth inequality, yellow basic disappearance the in the time of 40 ℃.Therefore, 40 ℃ of this test and Selection optimum temperature of deriving for the biogenic amine dansyl Cl.
Claims (8)
1. the separation and the liquid chromatography column pre-column derivatization method of biogenic amine in the beans sauce, may further comprise the steps: (1) adds trichloroacetic acid solution in beans sauce, and homogeneous is centrifugal, collects supernatant; (2) add normal hexane in supernatant, lower floor's trichloroacetic acid phase is taken out in vibration after the static layering, standby; (3) add NaOH solution to trichloroacetic acid in mutually, add NaHCO again
3The solution buffering is mixed the dansyl Cl acetone soln and is carried out derivative reaction; (4) ammoniacal liquor is removed the dansyl Cl cessation reaction, obtains the liquid of deriving; The liquid of deriving filters, promptly.
2. according to described separation of claim 1 and liquid chromatography column pre-column derivatization method, it is characterized in that: the 5% trichloroacetic acid solution that in beans sauce, adds 1.5 times of beans sauce quality in the step (1).
3. according to described separation of claim 1 and liquid chromatography column pre-column derivatization method, it is characterized in that: the homogeneous described in the step (1) carries out under the following conditions: rotating speed 10000r/min, homogenizing time 3min; Described centrifugally carry out under the following conditions: rotating speed 3500r/min, centrifugation time 5min.
4. according to described separation of claim 1 and liquid chromatography column pre-column derivatization method, it is characterized in that: in supernatant, add isopyknic normal hexane in the step (2).
5. according to described separation of claim 1 and liquid chromatography column pre-column derivatization method, it is characterized in that: add 2mol/L NaOH solution to trichloroacetic acid in mutually in the step (3), wherein trichloroacetic acid is 5: 1 with the volume ratio of 2mol/L NaOH solution; Then add saturated NaHCO again
3Solution cushions, wherein, and saturated NaHCO
3Solution is 3: 10 with trichloroacetic acid volume ratio mutually.
6. according to described separation of claim 1 and liquid chromatography column pre-column derivatization method, it is characterized in that, derivatization conditions described in the step (3) is: derivating agent dansyl Cl acetone soln consumption is 1.5 times of trichloroacetic acid phase volume, and the time of deriving is 40min, and the temperature of deriving is 40 ℃.
7. according to described separation of claim 1 and liquid chromatography column pre-column derivatization method, it is characterized in that: the ammoniacal liquor that adds 0.5 times of initial volume in the step (4) is removed the dansyl Cl cessation reaction, is settled to 25 times of initial volumes with acetonitrile at last.
8. according to described separation of claim 1 and liquid chromatography column pre-column derivatization method, it is characterized in that: after the derivation process with the filtering with microporous membrane of 0.22 μ m.
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| CN101509901B (en) * | 2009-03-11 | 2012-01-25 | 徐州市产品质量监督检验所 | Method for analyzing origin of amino acid in brewed sauce |
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