CN109541058A - A kind of method of biogenic amine in detection pueraria lobata - Google Patents
A kind of method of biogenic amine in detection pueraria lobata Download PDFInfo
- Publication number
- CN109541058A CN109541058A CN201811372977.0A CN201811372977A CN109541058A CN 109541058 A CN109541058 A CN 109541058A CN 201811372977 A CN201811372977 A CN 201811372977A CN 109541058 A CN109541058 A CN 109541058A
- Authority
- CN
- China
- Prior art keywords
- biogenic amine
- solution
- pueraria lobata
- biogenic
- standard
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—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
- G01N2030/067—Preparation by reaction, e.g. derivatising the sample
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
A kind of ultra performance liquid chromatography method is established for the first time, for 9 kinds of medicinal organism amine in rapidly extracting and measurement integration of drinking and medicinal herbs product pueraria lobata.9 kinds of biogenic amines include tryptamines, phenyl ethylamine, putrescine, cadaverine, histamine, octopamine, tyrasamine, spermidine, spermine.Pueraria lobata sample smash after with 0.4 mol/L hydrochloric acid extraction, dansyl Cl pre-column derivatization is added ammonium hydroxide and terminates reaction, acetonitrile constant volume.Using UPLC chromatographic column, gradient elution is carried out using ammonium acetate and acetonitrile as mobile phase, is detected at wavelength 254nm using diode array detector, good linear relationship is presented in detection method.Sample recovery of standard addition 75.6~97.2%, relative standard deviation 0.12~5.32%；The method quantitative limit of 9 kinds of biogenic amines is 10mg/kg.This method is easy, quick, sensitive, can be used for measurement while 9 kinds of biogenic amine residual quantities in the of animal or plant natures food such as pueraria lobata.
Description
Technical field
The present invention relates to a kind of methods of biogenic amine in detection pueraria lobata, belong to detection technique field.
Background technique
Biogenic amine (BAS) is a kind of low molecule alkali compounds with bioactivity and nitrogen.They are mainly by corresponding
Amino acid is formed under decarboxylate of microorganisms, or is generated under the action of amino acid transaminase by aldehyde and ketone[1].Common biogenic amine
Including tryptamines, putrescine, tyrasamine, histamine, spermine, spermidine, phenyl ethylamine, chapter amine, cadaverine etc.[2].Ordinary circumstance biogenic amine is toxic
, when human body gathers to a certain extent, human body can be caused to be poisoned.They can cause headache, nausea,vomiting,diarrhea, the heart
Throb with fear, blood pressure, disordered breathing and other allergic reactions, it may threat to life when serious[3].In biogenic amine, histamine toxicity is most
Greatly, putrescine, cadaverine, spermine and spermidine can be reacted with nitrite generates nitrosamine, a kind of strong carcinogen[4].Biogenic amine
Be widely present in the food rich in protein and amino acid, as meat and meat products, tinned food, seafood, cheese, beer,
Grape wine plant and agricultural product[5-6].It has recently found that may also have the presence of biogenic amine in Chinese herbal medicine[1].Currently, some countries are
Regulation is proposed to the limitation of biogenic amine in food content.For example, in European Union[6]In, histamine limitation is 100mg/kg, and in beauty
State[7], histamine limitation is 50mg/kg in aquatic products；And in China, high histamine fish histamine limitation is 40mg/100g, other fishes
Analogued histamine limitation is 20mg/100g[8-9].Biogenic amine is showed no limitation regulation in domestic and international plant product, but finds in this research
The rhizome of pueraria lobata contains biogenic amine.Biogenic amine activity with higher, the effect of facilitating pueraria lobata.
However, micro biogenic amine is the intracorporal normal activity ingredient of people, there is important physiology function in biological cell
Energy[11].The identification of biogenic amine and Simultaneous Determination method in pueraria lobata are established, it is either still living from ingredient from the angle of food safety
The angle of property, is all very necessary.
The detection method of biogenic amine has GC-MS/MS[12], ion chromatography[13-14]、HPLC[15-17]、TLC[18]And HPLC-MS/
MS method[1][18], but pre-treatment step is more complicated, operating difficulties, and detection difficulty is larger.Meanwhile the measurement of nine kinds of biogenic amines
More than 30min.Existing national standard[19]It is the measurement for biogenic amine in food, but pre-treating method is sufficiently complex.Sample
Pre-process relatively cumbersome, including n-hexane degreasing, liquid-liquid extraction, pH are adjusted, nitrogen flushing, need a large amount of organic solvent,
This is harmful to the health of experimenter.
[1]Wu YH,Zhou S,Xu GM.Determination of eight biogenic amines in
animal-derived foodstuffs by high performance liquid chromatography-tandem
mass spectrometry without derivatization.Chinese Journal of Chromatography,
2013.31(2):111-116.
[2]Silla Santos MH.Biogenic amines:their importance in
foods.International Journal of Food Microbiology,1996,29(2/3):213-231.
[3]Wang GQ,Yu JS,Hu J,et al.Progress in Research on Biogenic Amines
in Foods.Food Science,2016,37(1):269-278.
[4]SHALABY A R.Significance of biogenic amines to food safety and
human health.Food Research International,1996,29(7):675-690.
[5]Liu J,Ren J,Sun KJ.Safety of Biogenic Amines in Foods.Food
Science,2013,34(5):322-326.
[6]Zhang Lu,Bian Yin-Bing,Xing Zeng-Tao et al.Advance in detecting
techniques of bioamines in agri-food and agri-products.Acta Agriculture
Shanghai,2011,27(3):135-139.
[7]European Commission(EC).Commission recommendation of 10 January
2003 concerning a coordinated programmer for the official control of
foodstuffs for 2003(2003/10/EC).
[8]U.S.Food and Drug Administration.CPG Sec.540.525 Decomposition and
histamine raw,frozen tuna and mahi-Mahi；canned tuna；and related species,
revised compliance policy guide,availability.
[9]GB 2733-2015 National food safety standards of fresh,frozen animal
aquatic products.
[10]NY 5073—2006 the pollution-free food Poisonous and harmful
material set limit to of aquatic products.
[11]Sun YN,Zhang N,Wang CL.Identification and Determination of
Biogenic amines in Evodia Rutaecarpa(Juss.)Benth by High
Performance Liquid Chromatography.Analytical Chemistry.2014,42(2):
273-277
Wen YZ,Fan WL,Xu Y.Identification of a variety of biogenic amines by
GC-MS method in China’s liquor.Liquor Making,2013,140(1):38-41.
[12]Zhao XY,Jiao X,Xia M,et al.Determination of five biogenic amines
in source water by ion chromatography.Chinese Journal of Chromatography,2009,
27(4):505-508.
[13]Zhou Y,Wang PY,Zhao H,et al.Determination of Biogenic Amines in
Frozen Meat Products by High Performance Ion-exchange Chromatography-
electrochemical.The Food Industry,2014,35(5):238-241.
[14]Hu Y,Huang ZY.Column before derivative HPLC determination of many
kinds of biogenic amines in fish at the same time and its change rule.Journal
of Chinese Institute of Food Science and Technology,2012,12(11):142-147.
[15]Zhai HL,Yang XQ,Hao SX,et al.Optimization of Operating Conditions
for HPLC Determination of Biogenic Amines.Food Science,2011,32(18):180-184.
[16]Zhang N,Wang CL,Hong BL,et al.Identification and determination of
Biogenic amines in Ephedrae Herba by RP-HPLC with precolumn
derivatization.Chin J Pharm Anal 2015,35(3):389-395.
[17]SHALABY A R.Simple,rapid and valid thin layer chromatographic
method for determining biogenic amines in foods.Food Chemistry,1999,65(1):
117-121.
[18]Ding T,Lv C,Liu H,et al.Determination of Eight Biogenic Amines in
Red Wines by Liquid Chromatography–Quadrupole/Electrostatic Field Orbit Trap
Mass Spectrometry.Journal of Instrumental Analysis,2014,33(1):27-32.
[19]GB 5009.208.National Standard of Food Safety of Determination of
biogenic amine in foods.
Summary of the invention
The method that this experiment establishes pre-column derivatization UPLC method while measuring 9 kinds of biogenic amines in pueraria lobata.Pass through UPLC system
System can realize preferably separation and shorter analysis time using the column of small grain size.Meanwhile pre-treatment step is simplified,
Rapid detection method for a variety of biogenic amines in the agricultural product vegetable food product such as pueraria lobata provides foundation.
Technical solution is:
The method of biogenic amine, includes the following steps: in a kind of detection pueraria lobata
Step 1, the preparation of biogenic amine singly mark solution: precise putrescine, histamine, tyrasamine, spermine, spermidine, phenyl ethylamine,
Chapter amine, cadaverine, tryptamines standard items, be put into volumetric flask；Hydrochloric acid constant volume is added；It is configured to Standard Stock solutions；
The preparation of biogenic amine mixed standard solution: step 2 takes each standard items of the got in step 1 of equivalent respectively
Standard Stock solutions after mixing, with hydrochloric acid constant volume, are configured to biogenic amine mixing stoste；
The preparation of biogenic amine hybrid working liquid: step 3 biogenic amine is mixed, setting concentration is made after stoste is diluted with water
Solution, as biogenic amine hybrid working liquid；
Biogenic amine standard solution derivatization: step 4 takes the biogenic amine hybrid working liquid of 0.5-2mL to move into 15mL centrifuge tube
In, 1.5mL saturated sodium carbonate solution and 1mL solution of dansyl chloride is then added；It is temperature controlled at 40 DEG C after closeing the lid
It places and shakes 1 hour in dynamic sieve, the ammonium hydroxide that 100 μ L are then added terminates reaction；Acetonitrile is added to adjust volume to 5mL.?
The centrifugation that 5 minutes are carried out under 4000r/min, finally with 0.22 μm of filter filtering supernatant；
Step 5, the preparation of sample solution: taking pueraria lobata sample to be detected, and hydrochloric acid solution is added and extracts, then is freezing
It is centrifuged in centrifugation, takes supernatant；Saturated sodium bicarbonate solution and dansyl Cl compound solution will be added in supernatant, on shaking table
It places and shakes, ammonium hydroxide stopped reaction is added, reaction solution adds dilution in acetonitrile, as sample solution；
Step 6, UPLC detection: the standard that the derivatization biogenic amine standard solution and step 5 obtained using step 4 is obtained
Solution is detected by UPLC, obtains the content of biogenic amine to be measured by external standard method.
In the step 1, the concentration of each Standard Stock solutions is 1mg/mL；The concentration 0.1mol/L of hydrochloric acid solution.
In the step 2, the concentration 0.1mol/L of hydrochloric acid solution；The 100 μ g/ml of concentration of biogenic amine mixing stoste.
In the step 4, solution of dansyl chloride refers to that concentration is the acetone soln of 10mg/mL.
In the step 5, the concentration of hydrochloric acid solution is 0.4mol/L.
In the step 5, the supernatant that refrigerated centrifuge obtains need through titanium peroxide adsorbent adsorption-edulcoration and then
The step of into subsequent addition saturated sodium bicarbonate solution and dansyl Cl compound solution.
In the step 6, the chromatographic column in UPLC is UPLC BEH C18 column, and the temperature of UPLC column is set in 35 DEG C.
Mobile phase is pumped with the rate of 0.4mL/min；9 kinds of biogenic amines are measured under 254nm wavelength using PDA detector.
In the step 6, mobile phase A is 5mmol/L ammonium acetate, and Mobile phase B is acetonitrile；Gradient elution is as follows:
Beneficial effect
This optimum experimental Pretreatment of sample is attempted to use ultra-performance liquid chromatography for the first time, establishes pueraria lobata
In 9 kinds of biogenic amine extracting methods and accurate quantitative analysis detection method.This research is also to be found for the first time in pueraria lobata both at home and abroad containing coloured
The biology amine component such as amine, phenyl ethylamine, putrescine, cadaverine, spermine.Under the chromatographic condition established, it can be detected in 8 minutes
Sample.9 kinds of biogenic amines are in good linear relationship (r > 0.999) within the scope of 0.1-20mg/L.The quantitative limit of 9 kinds of biogenic amines
It is 10mg/kg.Precision experiment shows this method favorable reproducibility.This method is easy, quick, accurate, reliable, is suitable for large quantities of
The quick detection of sample is measured, is mentioned quickly, accurately to detect the biogenic amine in other fresh plants or drug and the homologous food of food
Technical support is supplied.Also for integration of drinking and medicinal herbs product the effect of, composition Study provided effective detection means.
Detailed description of the invention
The common high performance liquid chromatography separation figure of 9 kinds of biogenic amines of Fig. 1 (0.2 μ g/mL)
9 kinds of biogenic amine ultra performance liquid chromatography separation figures (0.2 μ g/mL) of Fig. 2
The fresh pueraria lobata sample ultra performance liquid chromatography figure of Fig. 3
The fresh pueraria lobata sample of Fig. 4 adds mixed standard solution (200mg/kg) ultra performance liquid chromatography figure
Specific embodiment
Experimental material
Unless otherwise specified, all solvents are analytical-reagent grade.(micropore, the U.S.) deionized water is ultrapure by Milli-Q
Whole 10 (the Milli-Q of water dispenser；Millipore, Bedford, MA, USA) application study.Putrescine, histamine, tyrasamine, spermine, sub- essence
The standard of amine, phenyl ethylamine, chapter amine, cadaverine is to have purchased from doctor Ehrenstorfer (Germany), tryptamines purchased from peace spectrum (Shanghai,
China).The purity of putrescine, histamine levels, spermine and spermidine is 99；Tyrasamine is 99.5%, 97.5% phenyl ethylamine, and octopus is
96.5%, cadaverine is 99%, 98% tryptamines.
Dansyl Cl is purchased from ampere (Shanghai, China), purity 98%.The chromatographic purity of acetonitrile, acetone and ammonium acetate is purchased
From Tedia company (Tedia, the U.S.).Sodium bicarbonate, hydrochloric acid, perchloric acid and ammonium hydroxide are the analysis of chemical reagent Co., Ltd
It is pure.
Pueraria lobata plant sample is purchased from Guangxi medical market.
The preparation of standard solution
Biogenic amine singly mark solution: the putrescine of precise 50mg, histamine, tyrasamine, spermine, spermidine, phenyl ethylamine, chapter amine,
The standard of cadaverine, tryptamines is put into 50mL volumetric flask；0.1mol/L hydrochloric acid constant volume is added；1mg/mL Standard Stock solutions are configured to,
Refrigerator is then stored in 4 DEG C of materials in volumetric flask.
Nine kinds of biogenic amine mixed standard solutions: it is same that single standard solution of each biogenic amine of 1mL pipettes addition respectively
10mL volumetric flask adds 0.1mol/L hydrochloric acid constant volume, is configured to 100 μ g/ml biogenic amines mixing stoste.
Nine kinds of biogenic amine hybrid working liquid: life is made after being diluted with water in experimental needs, the mixed solution of biogenic amine
The various concentration of the hybrid standard working solution of object amine.
Biogenic amine standard solution derivatization
Suitable biogenic amine hybrid working liquid suction pipe is moved into 15mL centrifuge tube, 1.5mL unsaturated carbonate is then added
Sodium solution and 1mL solution of dansyl chloride (10mg/mL is dissolved in acetone).After closeing the lid, in 40 DEG C of temperature controlled dynamic sieve
It places and shakes 1 hour, the ammonium hydroxide that 100 μ L are then added terminates reaction.Acetonitrile is added to adjust volume to 5mL.In 4000r/
The centrifugation that 5 minutes are carried out under min, finally carries out liquid chromatogram with 0.22 μm of filter filtering supernatant.
Sample pre-treatments
In this experiment, pre-treatment step is optimized, sample is directly extracted, is then performed the derivatization.This reality
The effect of extracting for having studied the extractants such as water, perchloric acid solution, hydrochloric acid solution, methanol, acetonitrile is tested, focuses on and considers how by mentioning
Process is taken to extract biogenic amine in Chinese herbal medicine.The result shows that hydrochloric acid solution extraction effect is best, 9 kinds of the total of biogenic amine are returned
Yield highest.Since most of biogenic amines are not UV chromophoric groups, so they are derived before being detected.Herein
In method, common two kinds of derivative reagent o-phthalaldehyde (OPA) and dansyl Cl (Dns-Cl) are compared.The result shows that adjacent
Phthalaldehyde is short as the derivatization reagent reaction time, but derivatization unstable products, poor reproducibility.Dansyl Cl has preferable
Derivative effect, wherein derivatization conditions are easily controllable, derivative products stablize, favorable reproducibility.
Carry out pre-treatment using following methods: the homogeneous samples for weighing 2.00 ± 0.01g (are dispersed with Germany FLUKO FA25
Homogenizer), it is put into 50mL centrifuge tube, 20mL 0.4mol/L hydrochloric acid is added.Then, by sample homogeneous 2min, with Germany
SIGMA3-18K high speed freezing centrifuge (SIGMA) is centrifuged 10 minutes at 4 DEG C at 10000r/min.1mL supernatant moves to
In 15mL centrifuge tube.Then 1.50mL saturated sodium bicarbonate solution and 1mL dansyl Cl compound solution (10mg/mL, dissolution is added
In acetone).After closeing the lid, in 40 DEG C of shaking tables (ALT-032R intelligent temperature control shaking table, the limited public affairs of ChemStar instrument and equipment
Department) in place and shake, be protected from light 1h.Then the ammonium hydroxide of 100 μ L is added to terminate derivatization reaction.Reaction solution adds dilution in acetonitrile extremely
5mL is centrifuged 5min in 4000r/min, finally carries out ultra performance liquid chromatography analysis with supernatant after 0.22 μm of membrane filtration.
The comparison of 1 chromatogram analysis method of embodiment
In order to compare liquid phase chromatographic isolation effect, two systems have been used to be analyzed in this research.
One is Waters Acquity UPLC system (Waters, the U.S.), including binary solvent manager pump, sample
Manager and PDA (photodiode array) detector.With UPLC BEH C18 (1.7 μm, 2.1mm × 100mm) (WATES, beauty
State) nine kinds of biogenic amines of post separation.The temperature of UPLC column is set in 35 DEG C.Mobile phase is pumped with the rate of 0.4mL/min, and gradient is washed
De- as shown in table 1, mobile phase A is 5mmol/L ammonium acetate, and Mobile phase B is acetonitrile.Sampling volume is 5 μ L, under 254nm wavelength
Measure 9 kinds of biogenic amines.
1 gradient elution program of table
Another analysis system is 1260 system of Agilent HPLC (Agilent company of the U.S.), including G1311C
1260Quat pumps 13298 1260ALS automatic sampler of VL, G and G 1315D 1260DAD VL.With Agilent ZOBAX SB-
C18 (5 μm, 4.6mm × 250mm) (Agilent, the U.S.) nine kinds of biogenic amines of post separation.The temperature of HPLC column is 35 DEG C.Mobile phase
It is pumped with the rate of 1.0mL/min, mobile phase A is 10mmol/L ammonium acetate, and Mobile phase B is acetonitrile.Gradient elution: 0-25min:
45% mobile phase A adds 55% Mobile phase B, and 25-30min:5% mobile phase A adds 95% Mobile phase B, and sampling volume is 10 μ L,
The spectrum of 9 kinds of biogenic amines is measured under 254nm wavelength.
Using 1260 high performance liquid chromatograph of Agilent, chromatographic condition detection, color are carried out by standard GB/T 5009.208
Spectrum separation figure is shown in Fig. 1.It will be seen from figure 1 that the peak of 9 kinds of biogenic amines occurs taking around 27 minutes completely, one injection is completed
Need 35 minutes or more.
UPLC platform is with high pressure resistant, partial size is small, analysis speed is fast, good separating effect, high sensitivity, detection in contrast
The features such as efficiency doubles.The peak of 9 kinds of biogenic amines only needs can display completely (see Fig. 2) for about 5 minutes.Sample injection
It completes only to need 8 minutes, peak shape and resolution ratio are good, not by the interference at heterochromatic peak.Quick detection suitable for batch samples.Mark
The chromatogram of quasi- solution is as shown in Figure 2.From figure 2 it can be seen that the peak of 9 kinds of biogenic amines is clearly symmetrical, without the dry of heterochromatic peak
It disturbs, may be implemented to be kept completely separate.
The selection of 2 chromatographic column of embodiment
On the basis of the UPLC parameter of embodiment 1, comparative test is carried out to chromatographic column, difference is: having studied
Acquity UPLC BEH C18 (1.7 μm, 2.1 × 100mm) column and Acquity UPLC BEH shielding RP18 (1.7 μm, 2.1
× 100mm) column.When using Acquity UPLC BEH Shield RP18 column, the time ratio of the peak completion of 9 kinds of biogenic amines
Acquity UPLCBEH C18 column is short, but cadaverine and histamine are not completely separated.As use Acquity UPLC BEH C18
When column, the peak response value of 9 kinds of biogenic amines is higher than Acquity UPLC BEH Shield RP18 column.For this purpose, having selected UPLC
BEH C18 column.
The selection of 3 mobile phase of embodiment
On the basis of the UPLC parameter of embodiment 1, comparative test is carried out to mobile phase, difference is: comparing second
Nitrile-water (with embodiment 1), acetonitrile -10mmol/L ammonium acetate buffer (volume ratio 1:1) and three kinds of methanol-water (volume ratio 1:1)
Most widely used flow visualizing.It was found that using methanol-water as mobile phase, the chromatographic peak of phenyl ethylamine and putrescine, cadaverine and tyrasamine
It is not completely separated.When acetonitrile-water be mobile phase when, the retention time of cadaverine and histamine is not sufficiently stable, and due to retain when
Between drift, two components are not completely separated, and affect quantitative accuracy.Using acetonitrile-ammonium acetate buffer salting liquid stream
When dynamic phase system, each target peak shape is preferable, and symmetrically, anury phenomenon, no miscellaneous peak interference, completely, retention time is steady for component separation
It is fixed.Therefore, this research is using acetonitrile -10mmol/L ammonium acetate buffer salt solution as flow visualizing.
The verifying of 4 range of linearity of embodiment and detection limit
On the parameter basis of embodiment 1, the biogenic amine of compound concentration 0.1,0.5,1,5,10 and 20 μ g/ml mix mark
Quasi- solution is for detecting.Using biogenic amine mixed standard solution concentration as abscissa, peak area is ordinate, depicts standard song
Line.Linear equation and linearly dependent coefficient are as shown in table 2.
29 kinds of biogenic amine standard curves of table
Within the scope of 0.1~20 μ g/mL, the concentration of 9 kinds of biogenic amines and peak area in good linear relationship (r >
0.999)。
The 5 method rate of recovery of embodiment and precision
The biogenic amine mixed standard solution of various concentration is added in pueraria lobata sample.It is detected with this method.It goes forward side by side
The Standard entertion rate of recovery of having gone is tested with accurate.Shown in table 3 the result shows that, water of 9 kinds of biogenic amines in 20,50 and 200mg/kg
Occurring spike on flat, average recovery rate is 75.6%~97.2% (n=6), relative standard deviation (RSD) is 0.12%~
5.32% (n=6) meets requirement of the test method to the rate of recovery and precision.
Biogenic amine mark-on reclaims and precision result (n=6) in 3 pueraria lobata sample of table
The chromatogram of pueraria lobata sample and the chromatogram of additional standard are shown in Fig. 3 and Fig. 4.
The improvement of 6 sample pretreatment of embodiment
On the basis of embodiment 1, method adjusting is carried out to sample pretreatment, saturated sodium bicarbonate solution and pellet is being added
The processing of titanium oxide inspiration row adsorption-edulcoration, titanium oxide specific surface area 180m are added after sulfonyl chloride solution, in reaction solution2/
G, after the completion of adsorption-edulcoration, adsorbent is centrifugated, then supernatant is being shaken by 25 DEG C of adsorption temp, adsorption time 30min
It is handled in bed, subsequent step is the same as embodiment 1.The characterization of sample recovery rate and precision is carried out, as a result such as table 4.
Biogenic amine mark-on reclaims and precision result (n=6) in 4 pueraria lobata sample of table
As can be seen that the precision of detection can be improved, and keep the rate of recovery more smart after the processing of sample liquid adsorption-edulcoration
It is quasi-.
In addition, the present invention can also carry out amino modified processing using to the surfaces of titan oxide particles, can be improved pair
The repulsive force of biogenic amine, Selective adsorption are more preferable.By above-mentioned titanium oxide absorption particle and 2wt% aqueous solution of methanesulfonic according to
Weight ratio 1:100 mixing, is warming up to 70 DEG C of holding 2h, makes titan oxide particles surface active；Titan oxide particles are filtered out again, go from
After sub- water washing, drying, add into the ethanol solution of the 3-aminopropyltriethoxysilane of 10wt%, active oxidation titanium
The weight ratio of particle and ethanol solution is 1:90, reacts in 65 DEG C of holding 3h, product is filtered out, and successively uses ethyl alcohol and deionized water
Washing, drying, obtain amino modified titan oxide particles adsorbent.Sample solution is pre-processed according to same method, is saturated being added
After sodium bicarbonate solution and dansyl Cl compound solution, amino modified titan oxide particles adsorbent inspiration row is added in reaction solution
Adsorption-edulcoration processing, after the completion of adsorption-edulcoration, adsorbent is centrifugated by 25 DEG C of adsorption temp, adsorption time 30min, then
Supernatant is handled in shaking table, subsequent step is the same as embodiment 1.
Biogenic amine mark-on reclaims and precision result (n=6) in 5 pueraria lobata sample of table
As can be seen that using amino treated adsorbent to sample treatment, can effectively improve ingredient to be measured with it is miscellaneous
The separation property of matter, can be improved the precision of detection, and keep the rate of recovery more accurate.
The analysis of 5 actual sample of embodiment
12 different sources pueraria lobata samples are analyzed with the method originally studied and defined.The result of positive sample such as table
Shown in 6.
The content practical measurement result (mg/kg) of biogenic amine in 6 pueraria lobata sample of table
"/" indicates detection detection
It was found that containing tryptamines, phenyl ethylamine, putrescine, cadaverine, spermidine, spermine and histamine in pueraria lobata.Content is in 9.79mg/kg
Between~125mg/kg.This and Sun Yanni et al.[11]There are the conclusions of biogenic amine not to contradict in measurement evodia rutaecarpa.Pueraria lobata and Wu
The fruit of medicinal cornel belongs to rhizome plants, but between have the difference of species and Content of Biogenic Amines.
Claims (7)
1. a kind of method of biogenic amine in detection pueraria lobata, which comprises the steps of:
Step 1, the preparation of biogenic amine singly mark solution: precise putrescine, histamine, tyrasamine, spermine, spermidine, phenyl ethylamine, chapter
Amine, cadaverine, tryptamines standard items, be put into volumetric flask；Hydrochloric acid constant volume is added；It is configured to Standard Stock solutions；
The preparation of biogenic amine mixed standard solution: step 2 takes the standard of each standard items of the got in step 1 of equivalent respectively
Stock solution after mixing, with hydrochloric acid constant volume, is configured to biogenic amine mixing stoste；
The preparation of biogenic amine hybrid working liquid: step 3 biogenic amine is mixed, the molten of setting concentration is made after stoste is diluted with water
Liquid, as biogenic amine hybrid working liquid；
Biogenic amine standard solution derivatization: step 4 takes the biogenic amine hybrid working liquid of 0.5-2mL to move into 15mL centrifuge tube, so
After 1.5 mL saturated sodium carbonate solutions and 1 mL solution of dansyl chloride are added；It is temperature controlled dynamic at 40 DEG C after closeing the lid
It places and shakes 1 hour in sieve, the ammonium hydroxide that 100 μ L are then added terminates reaction；Acetonitrile is added to adjust volume to 5 mL,
The centrifugation that 5 minutes are carried out under 4000r/min, finally with 0.22 μm of filter filtering supernatant；
Step 5, the preparation of sample solution: taking pueraria lobata sample to be detected, and hydrochloric acid solution is added and extracts, then in refrigerated centrifuge
In be centrifuged, take supernatant；Saturated sodium bicarbonate solution and dansyl Cl compound solution will be added in supernatant, is placed on shaking table
And shake, ammonium hydroxide stopped reaction is added, reaction solution adds dilution in acetonitrile, as sample solution；
Step 6, UPLC detection: the standard that the derivatization biogenic amine standard solution and step 5 obtained using step 4 is obtained is molten
Liquid is detected by UPLC, obtains the content of biogenic amine to be measured by external standard method.
2. the method for biogenic amine in detection pueraria lobata according to claim 1, which is characterized in that each in the step 1
The concentration of Standard Stock solutions is 1 mg/mL；0.1 mol/L of concentration of hydrochloric acid solution.
3. the method for biogenic amine in detection pueraria lobata according to claim 1, which is characterized in that in the step 2, hydrochloric acid
0.1 mol/L of concentration of solution；The 100 μ g/ml of concentration of biogenic amine mixing stoste.
4. the method for biogenic amine in detection pueraria lobata according to claim 1, which is characterized in that in the step 4, red sulphur
Solution of acid chloride refers to that concentration is the acetone soln of 10mg/mL.
5. the method for biogenic amine in detection pueraria lobata according to claim 1, which is characterized in that in the step 5, hydrochloric acid
The concentration of solution is 0.4mol/L.
6. the method for biogenic amine in detection pueraria lobata according to claim 1, which is characterized in that in the step 6, UPLC
In chromatographic column be UPLC BEH C18 column, the temperature of UPLC column is set in 35 DEG C, and mobile phase is pumped with the rate of 0.4mL/min
It send；9 kinds of biogenic amines are measured under 254nm wavelength using PDA detector.
7. the method for biogenic amine in detection pueraria lobata according to claim 1, which is characterized in that in the step 6, flowing
Phase A is 5mmol/L ammonium acetate, and Mobile phase B is acetonitrile；Gradient elution is as follows:
。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811372977.0A CN109541058A (en) | 2018-11-19 | 2018-11-19 | A kind of method of biogenic amine in detection pueraria lobata |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811372977.0A CN109541058A (en) | 2018-11-19 | 2018-11-19 | A kind of method of biogenic amine in detection pueraria lobata |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109541058A true CN109541058A (en) | 2019-03-29 |
Family
ID=65847952
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811372977.0A Withdrawn CN109541058A (en) | 2018-11-19 | 2018-11-19 | A kind of method of biogenic amine in detection pueraria lobata |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109541058A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112433017A (en) * | 2020-12-09 | 2021-03-02 | 宁夏农林科学院枸杞科学研究所 | Method for detecting specific metabolites of spermidine lycium barbarum |
| CN113075196A (en) * | 2021-03-24 | 2021-07-06 | 昆明理工大学 | Method for rapidly detecting biogenic amine in food by surface enhanced Raman scattering |
| TWI751875B (en) * | 2020-12-31 | 2022-01-01 | 高雄醫學大學 | Method and kit for detecting biogenic amine |
| CN114324682A (en) * | 2021-12-17 | 2022-04-12 | 南通市疾病预防控制中心 | Method for measuring biogenic amine in sea fish can by using dispersed solid phase extraction adsorbent |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS60131457A (en) * | 1983-12-19 | 1985-07-13 | Shimadzu Corp | Device for analyzing bio-amine |
| CN101793881A (en) * | 2009-12-17 | 2010-08-04 | 东北农业大学 | Method for detecting biogenic amine in food |
| CN108562679A (en) * | 2018-04-17 | 2018-09-21 | 安徽润安信科检测科技有限公司 | The UPLC-MS/MS detection methods of eight kinds of biogenic amines in a kind of white wine |
-
2018
- 2018-11-19 CN CN201811372977.0A patent/CN109541058A/en not_active Withdrawn
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS60131457A (en) * | 1983-12-19 | 1985-07-13 | Shimadzu Corp | Device for analyzing bio-amine |
| CN101793881A (en) * | 2009-12-17 | 2010-08-04 | 东北农业大学 | Method for detecting biogenic amine in food |
| CN108562679A (en) * | 2018-04-17 | 2018-09-21 | 安徽润安信科检测科技有限公司 | The UPLC-MS/MS detection methods of eight kinds of biogenic amines in a kind of white wine |
Non-Patent Citations (3)
| Title |
|---|
| J.KIRSCHBAUM 等: "Determination of Biogenic Amines in Fermented Beverages and Vinegars by Pre-column Derivatization with and Reversed-Phase LC", 《CHROMATOGRAPHIA》 * |
| 曾立威 等: "超高效液相色谱法同时快速测定多种动物源食品中9种生物胺的含量", 《食品安全质量检测学报》 * |
| 王燕华 等: "UPLC法测定不同加工方式鹿茸中的生物胺成分", 《分析测试学报》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112433017A (en) * | 2020-12-09 | 2021-03-02 | 宁夏农林科学院枸杞科学研究所 | Method for detecting specific metabolites of spermidine lycium barbarum |
| TWI751875B (en) * | 2020-12-31 | 2022-01-01 | 高雄醫學大學 | Method and kit for detecting biogenic amine |
| CN113075196A (en) * | 2021-03-24 | 2021-07-06 | 昆明理工大学 | Method for rapidly detecting biogenic amine in food by surface enhanced Raman scattering |
| CN113075196B (en) * | 2021-03-24 | 2022-02-15 | 昆明理工大学 | Method for rapidly detecting biogenic amine in food by surface enhanced Raman scattering |
| CN114324682A (en) * | 2021-12-17 | 2022-04-12 | 南通市疾病预防控制中心 | Method for measuring biogenic amine in sea fish can by using dispersed solid phase extraction adsorbent |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109541058A (en) | A kind of method of biogenic amine in detection pueraria lobata | |
| CN108680682B (en) | Liquid chromatography-mass spectrometry combined use method capable of simultaneously determining 45 prohibited drugs in health food for people with hypertension, hyperlipidemia and hyperglycemia | |
| CN106124674B (en) | A kind of pre-treating method of quick measurement Pesticide Residues in Tea and quantitative analysis method | |
| RU2431829C1 (en) | Method for determination of chloramphenicol content in food products and sorbent for its implementation | |
| CN109061016A (en) | A kind of preparation method and application of the solid-phase extraction column of enriched biological amine | |
| CN106610410B (en) | Detection method of biogenic amine in fish and products thereof | |
| Rehwald et al. | Trace analysis of harman alkaloids in Passiflora incarnata by reversed‐phase high performance liquid chromatography | |
| CN105301134A (en) | Method for estimating in-vitro detoxification effect of mycotoxin detoxification agent by using liquid chromatography | |
| CN109212070A (en) | Gas chromatography that is a kind of while measuring four kinds of benzoic acid herbicides in cereal | |
| CN108982730A (en) | Common group method combination UPLC-MSMS detects the analysis method of Prochloraz metabolin in plant source agricultural product | |
| CN115184497A (en) | Method for measuring content of 2, 4-epibrassinolide in dendrobium officinale | |
| CN105510312B (en) | A kind of method for quick of orlistat | |
| CN108267515B (en) | Method for detecting content of D-amygdalin and/or L-amygdalin in substance to be detected | |
| Kakigi et al. | Analysis of terpene lactones in a Ginkgo leaf extract by high-performance liquid chromatography using charged aerosol detection | |
| CN108445128A (en) | A kind of method of carbamates determination of drug residues in birds, beasts and eggs | |
| CN109212108B (en) | Gas chromatography-mass spectrometry combined method for simultaneously determining three trace quinoline herbicides in rice and soybean | |
| CN109900825B (en) | Separation and detection method for mycotoxin generated in transportation process of corn in bulk grain container | |
| Hassan et al. | Application of low density miniaturized dispersive liquid-liquid extraction method for determination of formaldehyde in aqueous samples (water, fruit juice and Streptococcus vaccine) by HPLC-UV | |
| CN107966506B (en) | The detection method of N-ethylaniline content in a kind of Rubber & Rubber Products | |
| Gummow et al. | Paired-ion extraction and high-performance liquid chromatographic determination of diminazene in cattle plasma: a modified method | |
| CN114669280A (en) | Purification filler for sulfonamide residues and pretreatment method | |
| CN114609273A (en) | Based on solid phase extraction-UPC2Method for separating and determining florfenicol enantiomer and metabolite thereof in pork | |
| CN102095817A (en) | Method for measuring trigonelline hydrochloride in trigonella tincture | |
| Tsai et al. | Disposition of asarone after intravenous administration to rabbits assessed using HPLC | |
| CN109917053A (en) | A method of amanita hemolysin in detection mushroom |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WW01 | Invention patent application withdrawn after publication | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20190329 |