1. Caffeine as a Surrogate for Cocaine in an HPLC Forensic Experiment Ray A. Gross Jr, Indravadan Shah and Muhamed Jasarevic Department of Physical Sciences Prince George’s Community College Largo, MD 20774

3. Abstract A modern high performance liquid chromatography (HPLC) method for the identification and quantitative determination of caffeine as a surrogate for cocaine in colas and other caffeine containing beverages is described. Our HPLC protocol resolves caffeine in three minutes with a HP - 1100 HPLC system. A calibration curve is prepared from standard caffeine solutions, and commercially available beverages are assayed for caffeine. The procedure is suitable for use by sophomore- level students in a forensics oriented instrumental analysis course.

5. Introduction HPLC chromatography involves the separation of mixtures into individual components by passing a fluid (liquid) through a stationary phase. It is a highly efficient method, which involves a stationary phase and a mobile phase. The liquid samples to be analyzed by HPLC are injected directly into the column, where the solvent is pumped at high speeds and high pressures - up to 1500 lb/in 2 or more from glass or stainless steel reservoirs, each of which contains 500 mL or more solvent. The reservoirs are degassed to remove dissolved gases - usually oxygen and nitrogen - that interfere by forming bubbles in the detector system.

6. Physical properties of the caffeine Molecular formula: C 8 H 10 N 4 O 2 Molecular weight: 194.19 Chemical name: 1,3,7-trimethylxanthine Properties: soft, white crystal or powder. No smell. Test is slightly bitter. Melting point: 235 - 238 ° C Solubility: Soluble in chloroform and partially soluble in water and ethanol. Chemicals: HPLC grade water, HPLC grade methanol, HPLC grade acetonitrile, Caffeine for Reference Standard preparation, Samples - beverages

8. Analytical Conditions Instrument: HP-1100 HPLC Column: 4 X 125 ODS Hypersil 5 µm (C18 column) Mobile phase: A = HPLC Acetonitrile 40%, B = HPLC water and methanol (50:50) 60% Flow rate: 1.0 mL/min Elution: Isocratic UV detector: 270/20 nm; Reference - 360/80 nm standard cell Run time: 3 min Injection volume: 1.0 µL Reference Standard: Prepared as follows. (1 ppm = 1mg/L = 1µg/mL) 100 µg/mL, 200 µg/mL, 400 µg/mL, 600 µg/mL

9. Procedure HPLC grade acetonitrile, methanol, and water solvents constitute the mobile phase in the reservoir. Turn on the HPLC; Prime the pump using the 30 or 60 mL syringe; Adjust flow of solvent to 1.00mL/min. Let the HPLC run approximately 15 minutes before injecting the sample. Make sure the waste is coming into the waste bottle; Open the Caffeine analysis method. When ready mode is displayed, inject 1 µL of the reference standard and run the sample. HP chemstation automatically starts data acquisition (run each concentration twice for the reference standards). Run the sample analysis for each soft drink.

10. Results Calibration table and curve Run Concentration (µg/mL) Area (mAU’s) Average area Standard deviation %CV or RSD 1100.00100.1996.313.473.60 293.49 395.25 1200.00173.69162.7811.397.00 2150.97 3163.67 1400.00261.29267.586.912.58 2274.97 3266.48 1600.00362.40377.9814.543.85 2391.18 3380.35

11. Calibration curve

12. Results for the Samples Beverages Area, mAU’s ( y value) Area x dilution factor (10) Conc.mg/L (x value) Hansen’s energy 60.707607.071013.45 AMP energy 51.633516.33849.52 SoBe adrenaline 76.890768.901305.83 KMX energy drink 40.439404.39647.27 High potency energy venom 121.9591219.592119.92 Red Bull ( sugar free) 39.995399.95639.25 Red Bull 31.790317.90491.02 Coke17.195171.95227.33 Coca-cola11.287112.87120.59 Pepsi8.01680.1661.49

13. Conclusions The experiment and its results serve as a basis for classroom discussion, including a general discussion of method, accuracy of results, limitations of method, and economical aspects. These results showed good comparison with other techniques. HPLC is an accurate, reliable method for determining the caffeine content of many popular beverages. Various colas may serve as cocaine surrogates in an instrumental analysis course emphasizing forensic techniques.

14. References Glenda K. Ferguson. Quantitative HPLC Analysis of an Analgesic/Caffeine Formulation: Determination of Caffeine J. Chem. Educ. 1998 75 467. Glenda K. Ferguson Wesleyan College, Department of Chemistry, 4760 Forsyth Road, Macon, GA 312 National Soft Drink Association, Bunker & McWilliums, J Am Diet, 74: 28- 32, 1979 http://www.astro.umd.edu