Screening of Cocaine and I ts M etabolites in Urine

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

2. 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. What is Cocaine and Benzoylecognine? Cocaine BEC

3. 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. Problem

4. 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 notifiedif the test was positive. There is a growing concernthat if the screening is a violation of students' rights. Importance

5. Summary of techniques

6. 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 Technique chosenCapillary Electrophoresis /Mass Spectrometry

7. 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 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 CE

8. Standard: 1mg/mL BEC (or COC) in acetonitrile from Cerilant; Control: Blank urine; internal standard: 1mg/mLbenzoylecognine isopropyl ester 500μL urine sample+50 μL internal standard+500 μL acetonitrile 1mL uppernatant Centrifuged at 10,000rpm for 5 min +500 μL electrolyte Experiment Design 3 CE autosampler CE

9. 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 Data analysis 3 %RSD= % relative standard deviation

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

11. 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 Conclusion

12. 1. Crack Cocaine http://www.cocaine.org/ 2. Beckman Coulter http://www.beckmancoulter.com/products/Discipline/Life_Science_Research/pr_disc_gen_capelect.asp?bhcp=1 3. DaCosta, Jose Luiz; Tonin, Fernando Gustavo; Zanolli, Luiz Antonio; Chasin, Alice AparecidadaMatta; 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. Reference