CN105092756A - Method for rapid detection of dhurrin in Baijiu brewing raw material sorghum - Google Patents
Method for rapid detection of dhurrin in Baijiu brewing raw material sorghum Download PDFInfo
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Abstract
The invention relates to a method for rapid detection of dhurrin in Baijiu brewing raw material sorghum, and belongs to the Baijiu analysis detection technical field. The method is achieved by the steps: a dhurrin standard is detected by liquid-phase mass spectrometry to determine a characteristic ion and establish a multiple reaction monitoring (LC-MS/MS-MRM) method, the dhurrin in the Baijiu brewing raw material sorghum is qualitatively detected by the LC-MS/MS-MRM method, and a dhurrin quantitative standard curve is made for accurate quantification on the dhurrin in the Baijiu brewing raw material sorghum. 0.1% formic acid-water and acetonitrile are used as a mobile phase for gradient elution separation of the dhurrin in the sample, then tandem mass spectrometry detection is carried out, and next accurate quantification is carried out by the standard curve. The method is simple to operate, sensitive in detection, high in degree of automation, and high in specificity; with further use of the method, the dhurrin in different types of wine brewing sorghum is detected, and thus the Baijiu safety is better controlled.
Description
Technical field
The present invention relates to the method for dhurrin in a kind of quick detection white spirit brewing raw material Chinese sorghum, being specifically related to one utilizes Gao Xiao Ye Xiang Se Pu – Rat Feces by Electrospray Ionization Tandem Mass Spectrometry (LC – MS/MS – MRM) to detect the method for dhurrin in white spirit brewing raw material Chinese sorghum fast, belongs to Liquor Analysis detection technique field.
Background technology
China white wine is as one of large Spirit (brandy Brandy, whiskey Whisky, vodka Vodka, gin Gin, Rum Rum, mescal Tequila, sake Sake, China white wine Spirit) in the world eight, ethanol content compares vodka, whiskey, brandy high (usually ethanol volume 40%-55%).China white wine cereal is produced, as Chinese sorghum, or wheat, barley, corn, the potpourri of rice and Chinese sorghum, by mixed microorganism solid state fermentation, form with the distillation of Chinese tradition rice steamer bucket after fermentation, be a complicated opening system, which increase the difficulty in conjunction with state prussiate (dhurrin) in researching white spirit.For the research of dhurrin in the middle of white wine, focus mostly in detection method at present, and still not enough to the research of its synthesis and regulatory mechanism.White wine has multiple odor type, mainly contains giving off a strong fragrance, sauce perfume, delicate fragrance, meter Xiang at present, phoenix is fragrant, medicine is fragrant, fermented soya beans, salted or other wise is fragrant, sesame is fragrant, hold concurrently perfume, special type and white spirit 11 kinds of odor types.
Along with people increase the attention degree of food security, Ge Jiu enterprise more and more pays close attention to the safety index in white wine, so quantitatively seem particularly important to dhurrin.
At present, the domestic research about dhurrin in white spirit brewing raw material Chinese sorghum is little, to the detection method of dhurrin composition, mainly contains:
1, liquid phase chromatography (HPLC – DAD) and high performance liquid chromatography (UPLC – PDAD)
:efficient and superelevation liquid chromatography is applicable to analyzing difficult gasification, not volatile material, as materials such as the organic acid in wine, amino acid, biogenic amines, so liquid chromatography is used for detecting some safety index materials in white wine.
2, GC-MS(gas chromatography-mass spectrography) (GC – MS): Liquor Analysis many uses gas chromatography is in conjunction with mass spectrum at present, and the material of gas chromatographic analysis is volatile constituent mostly.Gas chromatography is mainly used in the separation of material, by Gas chromatographyMass spectrometry, carries out qualitative and quantitative analysis to the volatile ingredient in white wine, thus has more comprehensive understanding to white wine material composition.
3, high-temperature acid solution or enzyme process indirect detection: high-temperature acid solution be under sour environment heating make testing sample be hydrolyzed after hydrolysate detected and then method to object indirect detection.Enzyme process to be hydrolyzed the method that detects hydrolysate after reaction to object under optimum condition, is also the method for indirect detection object.
The present invention Gao Xiao used Ye Xiang Se Pu – Rat Feces by Electrospray Ionization Tandem Mass Spectrometry (LC – MS/MS – MRM) only needs the dhurrin in dialogue fermented glutinous rice liquor raw material Chinese sorghum simply to extract pre-service just can to carry out quantitatively fast and accurately, because dhurrin is likely the important as precursors forming unfavorable material in Spirit, and itself has toxicity, in order to the health of consumers in general is considered, the detection method setting up dhurrin in a set of fast quantification liquor-making raw material Chinese sorghum is imperative.
Summary of the invention
The object of this invention is to provide the method for dhurrin in a kind of quick detection white spirit brewing raw material Chinese sorghum, specifically utilize High Performance Liquid Chromatography-Electrospray Ionization Tandem Mass method (LC-MS/MS-MRM) to detect the method for dhurrin in white spirit brewing raw material Chinese sorghum fast.The present invention uses 0.1% formic acid-water and acetonitrile to carry out gradient elution as the dhurrin in flowing relative sample and is separated rear tandem mass spectrum detection, then is undertaken quantitatively by typical curve.
Technical scheme of the present invention, in a kind of quick detection white spirit brewing raw material Chinese sorghum, the method for dhurrin, the steps include:
(1) dhurrin standard items are detected to determine its characteristic ion and to set up LC – MS/MS – MRM by XevoTQ series connection quadrupole rod liquid phase mass spectrograph (WatersXevoTQMS, water generation science and technology Shanghai company limited):
A, with methyl alcohol preparation 200mg/L dhurrin standard items storing solution as maximum concentration, be diluted to 20mg/L as dhurrin standard solution with methyl alcohol; Put into 2mL sample bottle after getting the process of 1mL dhurrin standard solution filtering with microporous membrane and treat sample detection;
B, use XevoTQ series connection quadrupole rod liquid phase mass spectrograph detect dhurrin standard solution, liquid-phase condition: mobile phase A: 0.1% formic acid-water; Mobile phase B: acetonitrile; Flow velocity 0.3mL/min; Gradient: Mobile phase B 4min is linearly increased to 40% by 5%, then 1min is linearly increased to 100% by 40%, continue 1min, then 1min is linearly reduced to 5% by 100%; Chromatographic column: WatersAcquityC18column, 50mm × 2.1mm × 1.7 μm; Sample size 2 μ L; ESI ion gun; Positive ion mode; Capillary voltage 2.7kV, taper hole voltage 20V, collision energy 12V; Source temperature 120 DEG C, desolventizing temperature 300 DEG C; Desolventizing gas flow rate 700L/h, taper hole gas flow rate 50L/h, collision gas flow velocity 0.15mL/min;
C, according to testing result, dhurrin parent ion is
m/z329, feature daughter ion is
m/z180 and
m/z132, set up LC – MS/MS – MRM method, select
m/z329-180 and
m/z329-132 as qualitative ion pair,
m/z329-132 is as quota ion pair;
(2) the dhurrin qualitative detection in LC – MS/MS – MRM method dialogue fermented glutinous rice liquor raw material Chinese sorghum: the Chinese sorghum of getting after 5g grinds is positioned in beaker, add 20mL methyl alcohol, to spend the night after ultrasonic extraction 30min preservation, get next day extract be placed in the centrifugal 30min of 50mL centrifuge tube 12000rpm get supernatant filter, add the ultrapure water of volume ratio 1:1, use is got 1mL after revolving and steaming instrument evaporative removal 20mL methyl alcohol and is treated sample detection;
(3) dhurrin made in dhurrin quantitation curves dialogue fermented glutinous rice liquor raw material Chinese sorghum carries out accurate quantitative analysis:
D, standard curve making: 20mg/L dhurrin standard solution gradient dilution is become a series of dhurrin standard solution, take peak area as horizontal ordinate, concentration is ordinate drawing standard curve;
E, the pre-service of white spirit brewing raw material Chinese sorghum: get 5g grind after Chinese sorghum in beaker, add 20mL methyl alcohol, spend the night after ultrasonic extraction 30min, next day extract is placed in the centrifugal 30min of 50mL centrifuge tube 12000rpm, get the ultrapure water adding volume ratio 1:1 after supernatant filters, use is got 1mL after revolving and steaming instrument evaporative removal 20mL methyl alcohol and is treated sample introduction;
The quantitative test of dhurrin in f, white spirit brewing raw material Chinese sorghum: use LC – MS/MS – MRM method to detect sample, use typical curve to carry out quantitatively.
Beneficial effect of the present invention: the present invention uses 0.1% formic acid-water and acetonitrile to carry out gradient elution as dhurrin in flowing relative sample and is separated rear tandem mass spectrum detection, and by typical curve, in final dialogue fermented glutinous rice liquor raw material Chinese sorghum, dhurrin carries out accurate quantitative analysis.The present invention establishes a kind of method of quick detection dhurrin completely newly, simple to operate, detects sensitive and automaticity is high, and specificity is high.
Accompanying drawing explanation
Fig. 1 dhurrin standard items LC – MS/MS liquid phase figure.
Fig. 2 dhurrin standard items LC – MS/MS chromatogram.
Dhurrin in Fig. 3 LC – MS/MS – MRM legal detection liquor-making raw material Chinese sorghum.
Fig. 4 LC – MS/MS – MRM standard measure detects the typical curve of dhurrin.
Embodiment
Embodiment 1: the quantitative and qualitative analysis of dhurrin in different breweries liquor-making raw material Chinese sorghum
(1) detect dhurrin standard items by XevoTQ series connection quadrupole rod liquid phase mass spectrograph (WatersXevoTQMS) (water generation science and technology Shanghai company limited) determine its characteristic ion and set up LC – MS/MS – MRM method:
A, with methyl alcohol preparation 200mg/L dhurrin standard items storing solution as maximum concentration, be diluted to 20mg/L as standard solution with methyl alcohol.Put into 2mL sample bottle after getting the process of 1mL dhurrin standard solution filtering with microporous membrane and treat sample introduction.
B, use XevoTQ series connection quadrupole rod liquid phase mass spectrograph detect dhurrin standard solution, liquid-phase condition: mobile phase A: 0.1% formic acid-water; Mobile phase B: acetonitrile.Flow velocity 0.3mL/min; Gradient: Mobile phase B 4min is linearly increased to 40% by 5%, then 1min is linearly increased to 100% by 40%, continue 1min, then 1min is linearly reduced to 5% by 100%; Chromatographic column: WatersAcquityC18column, 50mm × 2.1mm × 1.7 μm; Sample size 2 μ L.ESI ion gun; Positive ion mode; Capillary voltage 2.7kV, taper hole voltage 20V, collision energy 12V; Source temperature 120 DEG C, desolventizing temperature 300 DEG C; Desolventizing gas flow rate 700L/h, taper hole gas flow rate 50L/h, collision gas flow velocity 0.15mL/min.Testing result as depicted in figs. 1 and 2.
C, according to Fig. 1 and Fig. 2, dhurrin parent ion is
m/z329, feature daughter ion is
m/z180 and
m/z132, set up LC – MS/MS – MRM method, select
m/z329-180 and
m/z329-132 as qualitative ion pair,
m/z329-132 is as quota ion pair.
(2) the dhurrin qualitative detection in LC – MS/MS – MRM method dialogue fermented glutinous rice liquor raw material Chinese sorghum: get Chinese sorghum after 5g grinds in beaker, add 20mL methyl alcohol, to spend the night after ultrasonic extraction 30min preservation, get extract next day and be placed in the ultrapure water adding volume ratio 1:1 after the centrifugal 30min of 50mL centrifuge tube 12000rpm gets supernatant fluid filtrate, use is got 1mL after revolving and steaming instrument evaporative removal 20mL methyl alcohol and is treated sample detection, and qualitative detection result as shown in Figure 3.
(3) making dhurrin quantitation curves carries out quantitatively to dhurrin in different breweries liquor-making raw material Chinese sorghum:
D, standard curve making: 20mg/L dhurrin standard solution gradient dilution is become a series of dhurrin standard solution, take peak area as horizontal ordinate, and concentration is ordinate drawing standard curve, and typical curve as shown in Figure 4.
The preparation of e, test sample: gather that Luzhou Old Cellar, a small bay in a river board are not grudged, Fenyang wine, pull well, Gu Jinggong, two-wheel and Hengshui white spirit 7 brewery's Chinese sorghum samples down.
F, the liquor-making raw material Chinese sorghum pre-service of different distilleries: get respectively 5g grind after Chinese sorghum in beaker, add 20mL methyl alcohol, spend the night after ultrasonic extraction 30min, next day extract is placed in the centrifugal 30min of 50mL centrifuge tube 12000rpm and gets the ultrapure water adding volume ratio 1:1 after supernatant filters, use is got 1mL after revolving and steaming instrument evaporative removal 20mL methyl alcohol and is treated sample introduction.
The quantitative test of dhurrin in g, white spirit brewing raw material Chinese sorghum: use LC – MS/MS – MRM method to detect sample, use typical curve to carry out quantitatively, testing result is as shown in table 1.
The content of dhurrin in table 1 different brewery liquor-making raw material Chinese sorghum
(4) the quantitatively evaluation of dhurrin method in white spirit brewing raw material Chinese sorghum:
Known by above-mentioned typical curve analysis diagram, the method has the very wide range of linearity for quantitative dhurrin, and to be on good terms (R in range of linearity internal linear
2>0.99), the scope of application of visible the method is wider.
In liquor-making raw material Chinese sorghum sample, add the dhurrin standard items of similar concentration, calculate the recovery of dhurrin quantivative approach, and investigated the accuracy of this quantivative approach by the relative standard deviation of repeated experiment, specifically as shown in table 2.
The recovery of table 2 dhurrin quantivative approach and accuracy (n=3)
The lower limit of the quantitative dhurrin of the method is lower than the content of dhurrin in sample, the method can be adopted to carry out quantitatively to the dhurrin in liquor-making raw material Chinese sorghum, and the method shows good accuracy (relative standard deviation is between 1.17% ~ 2.26%) and accuracy (recovery is between 93% ~ 97%), can meet the quantitative detection of dhurrin in liquor-making raw material Chinese sorghum completely.
Claims (1)
1. detect a method for dhurrin in white spirit brewing raw material Chinese sorghum fast, it is characterized in that step is:
(1) detect dhurrin standard items by XevoTQ series connection quadrupole rod liquid phase mass spectrograph determine its characteristic ion and set up LC – MS/MS – MRM:
A, with methyl alcohol preparation 200mg/L dhurrin standard items storing solution as maximum concentration, be diluted to 20mg/L as dhurrin standard solution with methyl alcohol; Put into 2mL sample bottle after getting the process of 1mL dhurrin standard solution filtering with microporous membrane and treat sample detection;
B, use XevoTQ series connection quadrupole rod liquid phase mass spectrograph detect dhurrin standard solution, liquid-phase condition: mobile phase A: 0.1% formic acid-water; Mobile phase B: acetonitrile; Flow velocity 0.3mL/min; Gradient: Mobile phase B 4min is linearly increased to 40% by 5%, then 1min is linearly increased to 100% by 40%, continue 1min, then 1min is linearly reduced to 5% by 100%; Chromatographic column: WatersAcquityC18column, 50mm × 2.1mm × 1.7 μm; Sample size 2 μ L; ESI ion gun; Positive ion mode; Capillary voltage 2.7kV, taper hole voltage 20V, collision energy 12V; Source temperature 120 DEG C, desolventizing temperature 300 DEG C; Desolventizing gas flow rate 700L/h, taper hole gas flow rate 50L/h, collision gas flow velocity 0.15mL/min;
C, according to testing result, dhurrin parent ion is
m/z329, feature daughter ion is
m/z180 and
m/z132, set up LC – MS/MS – MRM method, select
m/z329-180 and
m/z329-132 as qualitative ion pair,
m/z329-132 is as quota ion pair;
(2) the dhurrin qualitative detection in LC – MS/MS – MRM method dialogue fermented glutinous rice liquor raw material Chinese sorghum: the Chinese sorghum of getting after 5g grinds is positioned in beaker, add 20mL methyl alcohol, to spend the night after ultrasonic extraction 30min preservation, get extract next day to be placed in the centrifugal 30min of 50mL centrifuge tube 12000rpm and to get supernatant fluid filtrate, add the ultrapure water of volume ratio 1:1, use is got 1mL after revolving and steaming instrument evaporative removal 20mL methyl alcohol and is treated sample detection;
(3) dhurrin made in dhurrin quantitation curves dialogue fermented glutinous rice liquor raw material Chinese sorghum carries out accurate quantitative analysis:
D, standard curve making: 20mg/L dhurrin standard solution gradient dilution is become a series of dhurrin standard solution, take peak area as horizontal ordinate, concentration is ordinate drawing standard curve;
E, the pre-service of white spirit brewing raw material Chinese sorghum: get 5g grind after Chinese sorghum in beaker, add 20mL methyl alcohol, spend the night after ultrasonic extraction 30min, next day extract is placed in the centrifugal 30min of 50mL centrifuge tube 12000rpm, add the ultrapure water of volume ratio 1:1 after getting supernatant fluid filtrate, use is got 1mL after revolving and steaming instrument evaporative removal 20mL methyl alcohol and is treated sample introduction;
The quantitative test of dhurrin in f, white spirit brewing raw material Chinese sorghum: use LC – MS/MS – MRM method to detect sample, use typical curve to carry out quantitatively.
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| CN115166106A (en) * | 2021-11-26 | 2022-10-11 | 国家食品安全风险评估中心 | Method for detecting cyanide content in white spirit or white spirit fermentation process sample |
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| CN115166106A (en) * | 2021-11-26 | 2022-10-11 | 国家食品安全风险评估中心 | Method for detecting cyanide content in white spirit or white spirit fermentation process sample |
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