The Study On The Identification Of Technology After Drinking Beers

Objectives1. To find in the differential diagnosis of the characteristics of a substance in beers.2. To establish the method of LC-MS/MS for determination the characteristic substance inbeer.3. To establish the method of SPE on the characteristic substance in beer and by comparingsolid phase extraction column with liquid-liquid extraction,the best method can be recovered andchoosed .4. All kinds of fermented wine (including beer, wine, and liquor) were measured to confirmthe selected substances be used to identify, and it can provide scientific basis for forensicidentification for samples taken detection, result analysis of a various of cases by after drinking.5. After detection of the biological samples including the saliva, urine and blood ofvolunteers ,we can have a conclusion whether the volunteers had have drunk beer through thedetection of the characteristics .Methods1. In a variety of trace substances in fermented beers can be understand comphensivlythrough search of the new literature,and the characteristic substance can be initially identified.2. LC-MS/MS detection Optimization:The retention time (Rt) and multiple reactionmonitoring (MRM) can be used for the qualitative and quantitative analysis. two-three paris ofion were selected for the qualitative analysis, and one paris on the relatively high responses wasselected for quantitative analysis using the external standard method, by the peak area and theconcentration as the standard curve .3. The extraction method of optimization characteristics:the recovery rate on thecharacteristic substance was compared by the liquid-liquid extraction and the solid-phaseextraction method,and compareing the diferent solid-phase extraction column Waters Sep-PakC18 and proElut C18 6cc (1000mg). The most appropriate PH were selected under the highrecovery of cleaning solvent and eluent to do the best choice,and the establishment of solid phaseextraction method finaly.4. After drinking a variety of fermented wine, it can be inferred whether somebody havedrunk beer by the detection of human biological samples on the characteristics .5. Experimental Design Test subjects were that ten healthy volunteers was the prohibition before three days ,avoiding intense exercise, maintaining a good attitude, and they were randomly divided intothree experimental groups:the first group of six volunteers were drinking beer; the second groupwere drinking red wine; the last one were drinking liquor. those who make urine voluntarybefore drinking as the control, in one hour was uniform up to the maximum tolerated dosealcohol, while they can have drink side of eating meat and vegetables adequatiy . Since the startof the first swallow of wine to drink for two hours ,the venous blood 5 ~ 8mL and about 5mLsaliva was collected ,and volunteers had to urinate in the collected urine for the threetimes,making a detailed record of collection time, and finally all of the human biological sampleswas stored in the refrigerator in the -20;6. Pretreatment of biological samplesPreparation ofthe treatment:the samples, high-speed centrifugation after thawing,4000r/min speed centrifuge for 3 minutes, the supernatant obtained after centrifugation3mL, adding 50mM (PH of about 3) of ammonium formate buffer 3m, mixing spare ; winesamples by adding acid 40min ultrasound in addition to gas conditioning PH, place thespare.Activation and balance:take ProElut C18 6cc (1000mg) column, 5mL of methanolactivation, 4mL water balance, 2mL ammonium formate buffer re-activation of the flow rateof 3mL/min.Sampling:Flow the sample through column with 1mL/min.Washing:after washed by 6mL water, 50mM (PH = 3) 3mL ammonium formate bufferwashed, the flow rate of 2mL/min.Drying:drying the residual water by the ear suction ball in the column.Elution:elution with the eluent B ,the flow rate of 1mL/min.Collection:under nitrogen blowing 2min in the 40℃, using the retention time byLC-MS/MS analysis can be as the characteristic on the qualitative and quantitative analysis.7. Statistics:SPSS11.5 statistic software was used for data analysis and output wasmean±standard deviation ((X|-)±S ).Results1. Xanthohumol by HPLC / MS / MS analysis, the results show that in the range of 0.5 ~40.0μg/mL a linear relationship was good, and the correlation coefficient R2 was 0.9929, thelowest limit of detection (LOD) for the 1.0μg/mL.2. Iso-αacid anions by HPLC / MS / MS analysis, in the range of 0.1 ~ 31.163.0μg/mL thatshowed a linear relationship was excellent, and the correlation coefficient R2 was 0.9945. Iso-α-acid anion of standard solution made into the different concentration by the HPLC / MS /MS analysis. Test results showed the minimum detection limit (LOD) was for the 0.01μg/mL,signal to noise ratio (S / N) is 4.5.Conclusions1. According to textbooks and literature:xanthohumol found in hops is trace so that theonly source of it for people is beer.for this reason, we can consider that xanthohumol can be usedto distinguish types of beer and other liquor. There are the different content in a variety of beers,but the xanthohumol content is less than 0.1mg /L in the majority of commercial beers.At homeand abroad , the relevant reports was that the content of some beers detected is more than 1mg/L ,some even up 2mg/L.2. After the solid phase extraction and high performance liquid chromatography massspectrometry in parallel xanthohumol in beer analysis and testing, the results shows that wereconsistent with reports in the most beer in the xanthohumol content of 0.04 ~ 0.2mg/L , throughthe special beer processed may be higher.trace substances was insufficient to identify whetherthere are drunk after drinking beer.3. In the text, solid phase extraction and HPLC-MS/MS detection methods about theiso-α-acidfrom biological samples are established , high sensitivity, high recovery rate, and thereproducible method is accurate and easy to operate. the sample does not require derivativetreatment.the method is established for the rapid detection of biological samples.4. Detection the iso-α-acid content in beer , the content of the commercial beer varieswidely, it can be seen from this experiment that the iso-α-acid content in non-alcoholic beer isrelatively low, while high in the stout ,which is related to the brewing process and content addedhops .5. After man drinking the beer, the iso-α-acids the body have a great individual differences.The DCHA concentration ,in the first saliva collected after 1.5 hours drinking in the first time ,was higher than the second and third. Drinking urine is collected after 0.5 h, resulting in the firstfew levels in urine, iso-α-acids in the urine after the distribution is to increase the downwardtrend. When blood collection began 3.0 hours after drinking, the basic iso-α-acids detected withsaliva and urine reflects the fact that the same.6.To further clear of beer in the iso-α-acids in the human body absorption, distribution,excretion of the situation, the body needs to do a lot of validation experiments for the screeningof alcohol lay a solid foundation types.