1. Screening of Cocaine and Its Metabolites in Urine December 9,2009 Ho Yan Yip Chem 4101 1

2. What 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 BEC 2

3. Problem What 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. 3

4. Importance Abusing 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. 4

5. Summary of techniques MethodsAdvantageDisadvantage GC Widely use in urine testing If operated at elevated temperatures, artifacts may be produced at the injection port ³ IR Very fast response, easy sample preparation Only good for clean samples. The content of urine is too complex for IR analysis. BEC itself has 114 vibrational modes. Fluorescence Allow the quantitative determination of species in trace amount Require a fluorescent compound as label 5

6. Technique chosen Capillary Electrophoresis /Mass Spectrometry Able to separate charged macromolecules, such as proteins and DNA High efficiency and resolving power Low solvent consumption Small sample amounts Simple instrumentation When coupled with MS, more information can be given to the identity and structure of the analytes 6

7. CE P/ACE MDQ CE system (Beckman Coulter) 2,3 This instrument was chosen because it can used in many labs and is fast Capillaries: fused-silica, id=50mm, total length: 80cm Use patented capillary liquid cooling system, more efficient than simple air cooling. Applied voltage= 1-30 kV, 1 - 10 kV for injections Sample Temperature=5° C to 60° C Computer program: 32 Karat software Electrolyte:1mol/L formic acid aqueous solution MS MS 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 7

8. Experiment Design Experiment Design 3 Standard: 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 acetonitrile Centrifuged at 10,000rpm for 5 min 1mL uppernatant CE autosampler +500 μL electrolyte CE 8

9. Data analysis Data analysis 3 BECCOC Recovery (%) 500 ng/mL 86.083.0 Precision(%RSD) 500 ng/mL 9.59.7 Retention Time~6.46 min~7.07min m/z290.4304.4 S/N103 LOD (ng/mL)250100 LOQ (ng/mL)500250 %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/MS 9

10. Sample spectrum Sample spectrum 3 Electropherogram of an urine sample Positive to BEC but not COC Positive to both BEC and COC 10

11. Conclusion Improper 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 possible CE 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 time 11

12. Reference 1. Crack Cocaine http://www.cocaine.org/ 2. Beckman Coulter http://www.beckmancoulter.com/products/Discipline/Life_Science_Research/pr_disc_ge n_capelect.asp?bhcp=1 3. 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. 12