Slide 1What is Cocaine and Benzoylecognine?ProblemImportanceSummary of techniquesTechnique chosen Capillary Electrophoresis /Mass SpectrometryCEExperiment Design 3Data analysis 3Sample spectrum 3ConclusionReferenceScreening of Cocaine and Its Metabolites in UrineDecember 9,2009Ho Yan YipChem 41011What is Cocaine and Benzoylecognine?Cocaine (COC) is a alkaloid that can be obtained from the leaves of the coca plants.It is one of the most abused drugs.Benzoylecognine (BEC) is one the major metabolites of cocaine.Cocaine BEC2ProblemWhat procedure would allow detection of BEC, and thus, cocaine in urine.Purpose• To know more methods of testing the presence of cocaine in urine other than the commonly used immunoassay method.Hypothesis• The presence of cocaine in urine can be confirmed with the presence of one of its metabolites, BEC, through appropriate testing methods. 3ImportanceAbusing cocaine can have many adverse effects, such as destroying central nervous system, decrease appetite, increase heart rate and blood pressure¹.Cocaine is a required analyte for the federal mandated and many general workplace in the United States.In Hong Kong, there were growing number of cases of teenagers doing drugs. 23 secondary schools have started drug screen students in December 2009. Urine samples from volunteering students would be tested. The school and the respective parents would be notified if the test was positive. There is a growing concern that if the screening is a violation of students' rights.4Summary of techniquesMethods Advantage DisadvantageGCWidely use in urine testingIf operated at elevated temperatures, artifacts may be produced at the injection port ³IRVery fast response, easy sample preparationOnly good for clean samples. The content of urine is too complex for IR analysis. BEC itself has 114 vibrational modes.FluorescenceAllow the quantitative determination of species in trace amountRequire a fluorescent compound as label5Technique chosenCapillary Electrophoresis /Mass SpectrometryAble to separate charged macromolecules, such as proteins and DNAHigh efficiency and resolving powerLow solvent consumptionSmall sample amountsSimple instrumentationWhen coupled with MS, more information can be given to the identity and structure of the analytes6CEP/ACE MDQ CE system (Beckman Coulter) 2,3This instrument was chosen because it can used in many labs and is fast Capillaries: fused-silica, id=50mm, total length: 80cmUse patented capillary liquid cooling system, more efficient than simple air cooling.Applied voltage= 1-30 kV, 1 - 10 kV for injectionsSample Temperature=5° C to 60° CComputer program: 32 Karat softwareElectrolyte:1mol/L formic acid aqueous solutionMS 3• LCQ Deca XP Max (Thermo Electron Corporation)• electrospray ionization (ESI) mass spectrometry• sheath gas = Nitrogen, flow rate of 3- 5 mL/min• nebulizing gas flow rate of 22-55 arb. Units• spray voltage =2-4 kV 7Experiment Design 3Standard: 1mg/mL BEC (or COC) in acetonitrile from Cerilant; Control: Blank urine; internal standard: 1mg/mL benzoylecognine isopropyl ester 500μL urine sample+50 μL internal standard+500 μL acetonitrileCentrifuged at 10,000rpm for 5 min1mL uppernatant CE autosampler+500 μL electrolyteCE8Data analysis 3BEC COCRecovery (%) 500 ng/mL86.0 83.0Precision(%RSD) 500 ng/mL9.5 9.7Retention Time ~6.46 min ~7.07minm/z 290.4 304.4S/N 10 3LOD (ng/mL) 250 100LOQ (ng/mL) 500 250%RSD= % relative standard deviation• electropherogram positive to BEC and/or COC, indicates two types of abuse• confirm presence of peaks by MS/MS• quantitation can be performed by other techniques, e.g. GC/MS9Sample spectrum 3Electropherogram of an urine samplePositive to BEC but not COCPositive to both BEC and COC 10ConclusionImproper use of cocaine has adverse effect on health- it’s better for parents and teachers to know if their teenagers are doing drug as early as possibleCE is a great method to screen the usage of illegal drugs- fast, accurate response- little sample needed- can screen lots of students in a short amount of time11Reference1. Crack Cocaine http://www.cocaine.org/2. Beckman Coulter http://www.beckmancoulter.com/products/Discipline/Life_Science_Research/pr_disc_gen_capelect.asp?bhcp=13. Da Costa, Jose Luiz; Tonin, Fernando Gustavo; Zanolli, Luiz Antonio; Chasin, Alice Aparecida da Matta; Tavares, Marina Franco Maggi; “Simple method for determination of cocaine and main metabolites in urine by CE coupled to MS.” Electrophoresis,2009,30(12),2238-2244.
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