CN108414643A - The triple level four bars Mass Spectrometry detection methods of liquid chromatogram-of biogenic amine in a kind of cold freshly-slaughtered poultry - Google Patents
The triple level four bars Mass Spectrometry detection methods of liquid chromatogram-of biogenic amine in a kind of cold freshly-slaughtered poultry Download PDFInfo
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Abstract
The present invention relates to a kind of triple level four bars Mass Spectrometry detection methods of liquid chromatogram of biogenic amine in cold freshly-slaughtered poultry.It carries out in the following way：Extraction：Cold freshly-slaughtered poultry breast is weighed, is placed in centrifuge tube, extracting solution acetonitrile+methanol, vortex mixing is added, centrifugation pipettes supernatant for purification；Purification：After the activation of MCX solid-phase extraction columns, supernatant is transferred on solid phase extraction column, efflux is discarded, after aqueous formic acid and methanol elution, drained, eluted with ammonium hydroxide methanol；Eluent is through drying, and with initial flow phased soln, filter membrane is measured for HPLC MS/MS.The present invention establishes HPLC MS/MS methods to 6 kinds of biogenic amines in cold freshly-slaughtered poultry while the method quickly detected, the method can complete 6 kinds of chemical substances in 5min and analyze simultaneously, detection time is short, linear relationship is preferable, the method rate of recovery is high, has good stability, and meets analysis and requires.
Description
Technical field
The present invention relates to a kind of triple level four bars Mass Spectrometry detection methods of liquid chromatogram-of biogenic amine in cold freshly-slaughtered poultry, belonging to
Close analyte detection technical field.
Background technology
Biogenic amine is a kind of with bioactivity, amino-containing low molecular mass compound.All contain in most of food
Biogenic amine, these biogenic amines are mainly generated by microbial amino acid decarboxylation in amino acid decarboxylase.Appropriate biogenic amine is taken the photograph
Enter to promote growth, enhancing metabolic activity, immunity and removing free radical etc., but Excess free enthalpy biogenic amine can then cause head
The bad physiological reaction such as pain, abdominal cramps, vomiting.Its potential toxic effect and the indicative function of food freshness is caused
The concerns of a large number of researchers.
Cold freshly-slaughtered poultry refers to by inspection and quarantine qualification, and the chicken trunk after butchering carries out cooling treatment, and makes in vivo in 1 hour
Temperature is down to 0 DEG C~4 DEG C, then divides, modifies, packaging, and is remained in subsequent processing, circulation and retail link
In this temperature range, whole chicken or divided chicken of the shelf-life in 7 days.Since cold freshly-slaughtered poultry in processing, storage, transport and was sold
It is highly prone to the pollution of microorganism in journey, causes putrid and deteriorated, it is possible to create the biogenic amine of high concentration.Therefore, cold freshly-slaughtered poultry is monitored
The variation of middle biogenic amine is even more important.
Currently, the detection method of biogenic amine mainly has reversed-phased high performace liquid chromatographic（RP-HPLC）, gas chromatography
（GC）, thin-layered chromatography（TLC）Deng.Wherein liquid chromatography is to measure biogenic amine in food content main method, but this method
It needs to carry out before column or post-column derivation, it is complex for operation step, take time and effort, and derivative degree is also difficult to ensure, and meanwhile it is derivative anti-
It should be also possible to generate non-targeted derivative, these can all have an impact the Accurate Determining of biogenic amine.It is detected compared to others
Method, TLC methods have that equipment is simple, easy to operate, separating rate is fast, the advantages such as cheap, can effectively reduce measurement life
The testing cost of object amine, but the reproducibility of this method and precision are poor, compare Modern Analytical Instrument, and sensitivity is also one big scarce
It falls into.GC methods have that separative efficiency is high, analyze speed is fast, good selective, but due to measuring biogenic amine using method when is same
Sample needs derivatization treatment, time-consuming and laborious, and part biological amine is unstable at high temperature.And liquid chromatography tandem mass spectrometry, it will
Chromatography to the high separation capacity of complex sample with it is mass spectrographic it is highly selective, highly sensitive combine, and do not need at derivatization
Reason, while having the advantages that relative molecular mass and structural information can be provided, it is qualitative while being a variety of biogenic amines in cold freshly-slaughtered poultry
Effective convenient analysis method is provided with quantitative analysis.
Invention content
The present invention is in view of the foregoing drawbacks, and it is an object of the present invention to provide a kind of method is simple, can realize that the one kind quickly detected is cold fresh
The triple level four bars Mass Spectrometry detection methods of the liquid chromatogram-of biogenic amine in chicken.
The technical solution adopted by the present invention is thus：The triple level four bars matter of liquid chromatogram-of biogenic amine in a kind of cold freshly-slaughtered poultry
Spectrum detection method, which is characterized in that carry out in the following way：
Extraction：Cold freshly-slaughtered poultry breast is weighed, is placed in centrifuge tube, extracting solution acetonitrile+methanol, vortex mixing is added, centrifugation pipettes
Supernatant is for purification；
Purification：After the activation of MCX solid-phase extraction columns, supernatant is transferred on solid phase extraction column, efflux is discarded, it is water-soluble through formic acid
After liquid and methanol elution, drains, eluted with ammonium hydroxide methanol；Eluent is through drying, and with initial flow phased soln, filter membrane supplies
HPLC-MS/MS are measured.
In purifying step, WCX columns also can be used.
Chromatographic condition is：
Mobile phase A：0.1% aqueous formic acid；Mobile phase B：Acetonitrile；Flow velocity：0.2mL/min；Column temperature：30℃；Sample size：2uL；
Chromatographic column：Sepx BR-C18（2.1 × 150mm, 3 μm）.
Mass Spectrometry Conditions are：
Scan mode：Electron spray cation scans（ESI+）；Detection reaction：Multiple-reaction monitoring（MRM）；Ion source temperature：350
℃；Electron spray voltage：3500 V；Sheath atmospheric pressure：35 Arb；Assist gas pressure power：10Arb；Collision gas：Argon gas.
Mobile phase elution program is as follows：
| Time | 0.1% aqueous formic acid (A) | Acetonitrile (B) |
| 0 | 90 | 10 |
| 2 | 50 | 50 |
| 2.1 | 90 | 10 |
| 4 | 90 | 10 |
Qualitatively and quantitatively ion pair, collision energy：
| Compound | Qualitative ion pair（m/z） | Quota ion pair（m/z） | Collision gas energy/eV | Retention time/min |
| Putrescine Put | 30.56/88.96 | 72.25/88.96 | 20/6 | 1.17 |
| Cadaverine Cad | 69.26/103.01 | 86.17/103.01 | 14/6 | 1.18 |
| Histamine H is | 68.25/111.89 | 95.12/111.89 | 19/11 | 1.18 |
| Tyrasamine Tyr | 91.27/137.92 | 121.05/137.92 | 21/6 | 1.72 |
| Spermine Spm | 84.19/202.71 | 112.13/202.71 | 28/16 | 1.09 |
| Spermidine Spd | 72.22/145.95 | 112.14/145.95 | 14/12 | 1.09 |
With following standard solution：
6 kinds of biogenic amine standard reserving solutions：It is appropriate that 6 kinds of biogenic amine standard items are weighed respectively（It is accurate to 0.1mg）In the brown of 10mL
In volumetric flask, with appropriate 0.1% formic acid constant volume, 1mg/mL storing solutions are configured to, are kept in dark place in 4 DEG C, 6 months shelf-lifves；
6 kinds of biogenic amines are respectively putrescine（Put, C4H12N2）Standard items, cadaverine（Cad, C5H14N2）Standard items, histamine
（His, C5H9N3）Standard items, tyrasamine（Tyr, C8H11NO）Standard items, spermine（Spm, C10H26N4）Standard items, spermidine（Spd,
C7H19N3）Standard items.
The hybrid standard storing solution is diluted to 5 respectively with 0.1% formic acid, 10,20,50,100,200,500ng/mL
Hybrid standard working curve, using each characteristic ion mass chromatography peak area ratio as ordinate, concentration of standard solution is painted for abscissa
Standard curve processed.
It is an advantage of the invention that：This experiment establishes HPLC-MS/MS methods to the quickly inspection simultaneously of 6 kinds of biogenic amines in cold freshly-slaughtered poultry
The method of survey.Using the advantage of liquid chromatogram-triple quadrupole bar mass spectrometric hyphenated technique, liquid chromatogram and mass spectrum item is separately optimized
Part selects suitable ionization pattern, mass spectrometry parameters is optimized, and is selected respectively 6 kinds of biogenic amines of detection a pair of qualitative
Ion pair and quantitative from right, the final separation, extraction and confirmation realized to 6 kinds of chemical substances realizes their quantitative analysis.
The method can complete 6 kinds of chemical substances in 5min and analyze simultaneously, detection time is short, linear relationship is preferable, the method rate of recovery is high,
It has good stability, meets analysis and require.
Description of the drawings
Fig. 1 is the MBR chromatograms of the present invention.
Specific implementation mode
1 materials and methods
1.1 materials, instrument and reagent
Material：The cold freshly-slaughtered poultry of 80 ages in days is purchased from Jiangsu Li Hua Food Co., Ltd, about 2000g/ plumages.
Instrument：The triple level four bars mass spectrographs of Thermo TSQ MAX match Thermo ACCLEA high performance liquid chromatographs（The U.S.
Thermo companies）, N-EVAP nitrogen evaporators（Organomation companies of the U.S.）, solid-phase extraction device（Supelco companies of the U.S.）；
AB104-L electronic balances（Mei Tele companies of Switzerland）, 3-30K table model high speed centrifuges（German Sigma companies）
Reagent：Putrescine（Put, C4H12N2）Standard items, cadaverine（Cad, C5H14N2）Standard items, histamine（His, C5H9N3）Standard items,
Tyrasamine（Tyr, C8H11NO）Standard items, spermine（Spm, C10H26N4）Standard items, spermidine（Spd, C7H19N3）Standard items are purchased from
Sigma companies；Methanol, acetonitrile, formic acid, anhydrous sodium sulfate（It is HPLC grades, CNW companies）；Ultra-pure water（18.2MΩ.cm）
1.2 test method
1.2.1 chromatographic condition
Mobile phase A：0.1% aqueous formic acid；Mobile phase B：Acetonitrile；Flow velocity：0.2mL/min；Column temperature：30℃；Sample size：2uL；
Chromatographic column：Sepx BR-C18（2.1 × 150mm, 3 μm）；Gradient elution program is shown in Table 1.
1 eluent gradient of table elutes
| Time | 0.1% aqueous formic acid (A) | Acetonitrile (B) |
| 0 | 90 | 10 |
| 2 | 50 | 50 |
| 2.1 | 90 | 10 |
| 4 | 90 | 10 |
1.2.2 Mass Spectrometry Conditions
Scan mode：Electron spray cation scans（ESI+）；Detection reaction：Multiple-reaction monitoring（MRM）；Ion source temperature：350
℃；Electron spray voltage：3500 V；Sheath atmospheric pressure：35 Arb；Assist gas pressure power：10Arb；Collision gas：Argon gas.Qualitatively and quantitatively
The parameters such as ion pair, collision energy are shown in Table 2.
2 the MS detection parameters of table
| Compound | Qualitative ion pair（m/z） | Quota ion pair（m/z） | Collision gas energy/eV | Retention time/min |
| Putrescine Put | 30.56/88.96 | 72.25/88.96 | 20/6 | 1.17 |
| Cadaverine Cad | 69.26/103.01 | 86.17/103.01 | 14/6 | 1.18 |
| Histamine H is | 68.25/111.89 | 95.12/111.89 | 19/11 | 1.18 |
| Tyrasamine Tyr | 91.27/137.92 | 121.05/137.92 | 21/6 | 1.72 |
| Spermine Spm | 84.19/202.71 | 112.13/202.71 | 28/16 | 1.09 |
| Spermidine Spd | 72.22/145.95 | 112.14/145.95 | 14/12 | 1.09 |
The preparation of 1.3 standard solution
1.3.1 the preparation of standard reserving solution
6 kinds of biogenic amine standard reserving solutions：It is appropriate that 6 kinds of biogenic amine standard items are weighed respectively（It is accurate to 0.1mg）In the brown of 10mL
In volumetric flask, with appropriate 0.1% formic acid constant volume, 1mg/mL storing solutions are configured to, are kept in dark place in 4 DEG C, 6 months shelf-lifves.
1. the drafting of 3.2 standard curves
Hybrid standard storing solution is diluted to 5 respectively with 0.1% formic acid, 10,20,50,100, the mixing mark of 200,500ng/mL
Quasi- working curve, using each characteristic ion mass chromatography peak area ratio as ordinate, concentration of standard solution is that abscissa draws standard
Curve.
1.4 sample pre-treatments
Extraction：Cold freshly-slaughtered poultry breast 2.0g is weighed, is placed in centrifuge tube, extracting solution is added（Acetonitrile+methanol=9+1）10mL, it is anhydrous
Sodium sulphate is a little, vortex mixing, and 5min is centrifuged with 5000r/min at 4 DEG C.Residue after separation is handled with 10 mL extracting solutions again,
Vortex mixing centrifuges 5min at 4 DEG C with 5000r/min.Merge supernatant twice.5mL supernatants are pipetted for purification.
Purification：MCX solid-phase extraction columns（100mg, 3mL）After 3 mL methanol, the activation of 3 mL water, supernatant is transferred to solid phase
It extracts on pillar, discards efflux, after 3 mL, 2% aqueous formic acids and the elution of 3 mL methanol, drain, with 5% ammonium hydroxide first of 5mL
Alcohol elutes.Eluent is dried up through 30 DEG C of nitrogen, with 1mL initial flow phased solns, 0.22 μm of filter membrane is crossed, for HPLC-MS/MS
It measures.
The measurement of 1.5 rate of recovery and precision
Three horizontal TIANZHU XINGNAO Capsul experiments are carried out to cold freshly-slaughtered poultry sample, it is 10,50,100 μ g/kg to make actual interpolation amount in chicken,
Parallel determination 6 times carries out sample pre-treatments and measurement according to this method.Precision is to pass through relative standard deviation（RSD）Ratio
Value is assessed.
2 results and analysis
The optimization of 2.1 sample pre-treatments
2.1.1 the selection of extracts reagent
There is researcher to think that there are two bottlenecks in determination of biogenic amines analysis:When the complexity of sample substrate, second is that in sample
Biogenic amine actual concentrations are relatively low.The selection of Extraction solvent will have a direct impact on the rate of recovery of analysis method.This experiment uses second respectively
Nitrile, methanol, water, 0.1% formic acid, 0.1% formic acid methanol, acetonitrile+methanol, acetonitrile+methanol+formic acid extraction.The result shows that：Using
Pure acetonitrile or aqueous solvent extract, and the small molecules diamine such as cadaverine, putrescine has apparent absorption, rate of recovery mistake on SPE
It is low；It is extracted using pure methanol or aqueous solvent, since its strong hydrogen bonding acts on, biogenic amine will not adsorb on SPE, but many miscellaneous
Matter can not be adsorbed on SPE, and clean-up effect is not achieved；Final acetonitrile+methanol of choosing is Extraction solvent, can not only remove sample
Most of impurity in product, moreover it is possible to obtain the higher rate of recovery.
The ratio of acetonitrile+methanol when advanced optimizing extraction uses acetonitrile+methanol of different proportion respectively（1+1、1+9、
9+1、8+2、2+8）Solvent is tested.The result shows that with the raising of methanol component in Extraction solvent, time of putrescine and cadaverine
Yield increases rapidly；But when methanol content is excessively high, impurity content also rises, and the matrix effect of sample constantly enhances, impact analysis
Accuracy.When methanol content is 10%, each biogenic amine rate of recovery can reach analysis and require；Reach 20% in methanol content
When, tryptamines starts apparent matrix effect occur.In summary test result, final choice acetonitrile+methanol (9+1) are extraction
Solvent.
2.1.2 the selection of solid-phase extraction column
According to the characteristics of various SPE pillars and use condition, all objects in sample are purified.It compared herein weak
The clean-up effect of cation exchange column C18, WCX, MCX and HLB pillar.Purification method is：With 3mL methanol, 3mL deionized waters
After activating SPE pillars, efflux is discarded, the sample after above-mentioned centrifugation is added in pillar, after waiting samples to adsorb completely, then with
2% aqueous formic acids of 3mL cross column, discard efflux, then washed twice with 5% ammonium hydroxide methanol of 2.5mL, collect elution liquid nitrogen and blow
To doing, with methanol constant volume to 1mL.Research find in cold freshly-slaughtered poultry using 6 kinds of biogenic amines of MCX pillars pair clean-up effect it is best,
Blank recovery of standard addition（The average value of 6 kinds of biogenic amines）Take second place in 90% or more, WCX columns, HLB columns are worst.
2.1.3 the selection of eluent
The selection of eluent can influence the rate of recovery of biogenic amine in sample, be influenced on test result accuracy very big.Experiment uses
MCX solid phase extraction columns, sample cross column flow rate control in 1mL/min, have probed into four kinds of common eluents：Methanol, acetonitrile,
Dichloromethane and acetone mixture（1:1）, ammonium hydroxide methanol solution（5mL ammonium hydroxide, 95mL methanol）.The result shows that with ammonium hydroxide methanol
Mixed solution（5:95）The rate of recovery of elution, putrescine and tyrasamine is respectively 89.8%, 93.0%, the average recovery rate of six kinds of biogenic amines
88.0%~93.6%, and with dichloromethane and acetone mixture（1:1）, methanol and acetonitrile be when eluting, six kinds of biogenic amines it is flat
The equal rate of recovery is less than 80%, and when being eluted with methanol, the rate of recovery of tyrasamine and putrescine is only 18.7%, 24.6%.In addition dichloromethane
Alkane and acetone mixture（1:1）After elution nitrogen is blown, it is methanol and constant volume to 1.0 mL, ammonium hydroxide methyl alcohol mixed liquor to need to turn solvent
After elution without turn solvent can directly constant volume sample introduction, operation it is also simple.So experiment is final to choose ammonium hydroxide methanol solution（5:95）
As eluent.
The establishment of 2.2 Mass Spectrometry Conditions
First mass spectrometric analysis is carried out to 6 kinds of biogenic amines under electron spray positive ion mode（Q1 is scanned）Obtain molecular ion, i.e., it is female
Ion.Second mass analysis is carried out to the molecular ion of compound（Daughter ion scans）, obtain fragment ion information, i.e., son from
Then son optimizes the second order ms parameter of 6 kinds of biogenic amines so that fragments characteristic ionic strength is best when reaching maximum, is obtained
The second order ms figure of each compound；According to the fragment ion information that second order ms figure provides, select the qualitative of 6 kinds of biogenic amines and
Quota ion pair, multiple-reaction monitoring spectrogram are shown in Fig. 1.
The linear relationship of 2.3 methods and sensitivity
Linear test is done with biogenic amine standard mixed solution, is measured according to the method that this experiment is created, then with quantitative
The peak area of ion is ordinate（Y）, corresponding concentration of standard solution is abscissa（X）Make standard curve, and calculates gained
As a result the equation of linear regression and related coefficient of standard curve show square being all higher than for the coefficient R of 6 kinds of biogenic amines
0.99, illustrate that this method has good linear relationship (being shown in Table 3) in the concentration range of 5~500 ng/mL.With 3 times of noises
Than（S/N=3）Corresponding object content is as detection limit（LOD）, with 10 times of signal-to-noise ratio（S/N=10）Corresponding object content
As quantitative limit（LOQ）(the results are shown in Table 3).
3 linear relationship of table, detection limit（LODs）And quantitative limit（LOQs）
| Title | The range of linearity（ng/mL） | Calibration curve equation | Related coefficient（R2） | Detection limit LOD（μg/kg） | Quantitative limit LOQ（μg/kg） |
| Put | 5~500 | Y=8866.4X+2.7e5 | 0.995 | 0.40 | 1.20 |
| Cad | 5~500 | Y=113764X+4.0e5 | 0.991 | 0.30 | 0.90 |
| Tyr | 5~500 | Y=229123X+2.5e6 | 0.990 | 0.02 | 0.06 |
| His | 5~500 | Y=121985X+1.2e7 | 0.992 | 0.10 | 0.30 |
| Spd | 5~500 | Y=295.6X+1.2e5 | 0.996 | 0.10 | 0.30 |
| Spm | 5~500 | Y=297262X+0.9e5 | 0.997 | 0.10 | 0.30 |
The rate of recovery and precision of 2.4 methods
A cold freshly-slaughtered poultry chest muscle sample is chosen, the addition that 10,50,100 ug/kg, tri- concentration levels are carried out by this test method is recycled
And precision test（n=6）, test result is shown in Table 4.As it can be seen that the average recovery rate of 6 kinds of determinands 73.74%~102.35% it
Between, relative standard deviation（RSD）Between 1.23%~9.89%, the rate of recovery and precision of illustration method are good.
4 recovery of standard addition of table and relative standard deviation（n=6）
Claims (8)
1. the triple level four bars Mass Spectrometry detection methods of the liquid chromatogram-of biogenic amine in a kind of cold freshly-slaughtered poultry, which is characterized in that according to following
Mode carries out：
Extraction：Cold freshly-slaughtered poultry breast is weighed, is placed in centrifuge tube, extracting solution acetonitrile+methanol, vortex mixing is added, centrifugation pipettes
Supernatant is for purification；
Purification：After the activation of MCX solid-phase extraction columns, supernatant is transferred on solid phase extraction column, efflux is discarded, it is water-soluble through formic acid
After liquid and methanol elution, drains, eluted with ammonium hydroxide methanol；Eluent is through drying, and with initial flow phased soln, filter membrane supplies
HPLC-MS/MS are measured.
2. the triple level four bars Mass Spectrometry detection methods of liquid chromatogram-of biogenic amine in a kind of cold freshly-slaughtered poultry according to claim 1,
It is characterized in that, in purifying step, WCX columns also can be used.
3. the triple level four bars Mass Spectrometry detection methods of liquid chromatogram-of biogenic amine in a kind of cold freshly-slaughtered poultry according to claim 1,
It is characterized in that,
Chromatographic condition is：
Mobile phase A：0.1% aqueous formic acid；Mobile phase B：Acetonitrile；Flow velocity：0.2mL/min；Column temperature：30℃；Sample size：2uL；
Chromatographic column：Sepx BR-C18（2.1 × 150mm, 3 μm）.
4. the triple level four bars Mass Spectrometry detection methods of liquid chromatogram-of biogenic amine in a kind of cold freshly-slaughtered poultry according to claim 1,
It is characterized in that,
Mass Spectrometry Conditions are：
Scan mode：Electron spray cation scans（ESI+）；Detection reaction：Multiple-reaction monitoring（MRM）；Ion source temperature：350
℃；Electron spray voltage：3500 V；Sheath atmospheric pressure：35 Arb；Assist gas pressure power：10Arb；Collision gas：Argon gas.
5. the triple level four bars Mass Spectrometry detection methods of liquid chromatogram-of biogenic amine in a kind of cold freshly-slaughtered poultry according to claim 3,
It is characterized in that, mobile phase elution program is as follows：
。
6. the triple level four bars Mass Spectrometry detection methods of liquid chromatogram-of biogenic amine in a kind of cold freshly-slaughtered poultry according to claim 4,
It is characterized in that, qualitatively and quantitatively ion pair, collision energy：
。
7. the triple level four bars Mass Spectrometry detection methods of liquid chromatogram-of biogenic amine in a kind of cold freshly-slaughtered poultry according to claim 1,
It is characterized in that, with following standard solution：
6 kinds of biogenic amine standard reserving solutions：It is appropriate that 6 kinds of biogenic amine standard items are weighed respectively（It is accurate to 0.1mg）In the brown of 10mL
In volumetric flask, with appropriate 0.1% formic acid constant volume, 1mg/mL storing solutions are configured to, are kept in dark place in 4 DEG C, 6 months shelf-lifves；
6 kinds of biogenic amines are respectively putrescine（Put, C4H12N2）Standard items, cadaverine（Cad, C5H14N2）Standard items, histamine（His,
C5H9N3）Standard items, tyrasamine（Tyr, C8H11NO）Standard items, spermine（Spm, C10H26N4）Standard items, spermidine（Spd, C7H19N3）
Standard items.
8. the triple level four bars Mass Spectrometry detection methods of liquid chromatogram-of biogenic amine in a kind of cold freshly-slaughtered poultry according to claim 7,
It is characterized in that, the hybrid standard storing solution is diluted to 5 respectively with 0.1% formic acid, 10,20,50,100,200,500ng/
The hybrid standard working curve of mL, using each characteristic ion mass chromatography peak area ratio as ordinate, concentration of standard solution is horizontal seat
Plotting standard curve.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111122758A (en) * | 2019-12-26 | 2020-05-08 | 浙江工业大学 | Method for detecting spermidine content by high performance liquid-mass spectrometry |
| CN111537650A (en) * | 2020-06-10 | 2020-08-14 | 吉林大学 | Detection method of heterocyclic amine in sauced beef |
| CN111679017A (en) * | 2020-07-29 | 2020-09-18 | 梧州市食品药品检验所 | Method for measuring alkaloid content in gelsemium elegans |
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| CN111679017A (en) * | 2020-07-29 | 2020-09-18 | 梧州市食品药品检验所 | Method for measuring alkaloid content in gelsemium elegans |
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