Caffeine as a Surrogate Caffeine as a Surrogate for Cocaine in an for Cocaine in an

HPLC Forensic ExperimentHPLC Forensic Experiment

Ray A. Gross Jr, Indravadan Shah and Muhamed Ray A. Gross Jr, Indravadan Shah and Muhamed JasarevicJasarevic

Department of Physical SciencesDepartment of Physical SciencesPrince George’s Community CollegePrince George’s Community College

Largo, MD 20774Largo, MD 20774

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.

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/in2 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.

Physical properties of the Physical properties of the caffeinecaffeine

Molecular formula: C8H10N4O2

Molecular weight: 194.19Chemical name: 1,3,7-trimethylxanthineProperties: soft, white crystal or powder. No smell. Test is slightly bitter.Melting point: 235 - 238 ° CSolubility: 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

N

N

N

O

O

CH3

CH3H3C

Caffeine

Analytical ConditionsAnalytical ConditionsInstrument: 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/minElution: Isocratic UV detector: 270/20 nm; Reference - 360/80 nm standard cellRun time: 3 minInjection volume: 1.0 µLReference Standard: Prepared as follows. (1 ppm = 1mg/L = 1µg/mL)100 µg/mL, 200 µg/mL, 400 µg/mL, 600 µg/mL

Procedure Procedure HPLC grade acetonitrile, methanol, and water solvents HPLC grade acetonitrile, methanol, and water solvents constitute the mobile phase in the reservoir. constitute the mobile phase in the reservoir.

Turn on the HPLC; Prime the pump using the 30 or 60 Turn on the HPLC; Prime the pump using the 30 or 60 mL syringe; Adjust flow of solvent to 1.00mL/min. Let the mL syringe; Adjust flow of solvent to 1.00mL/min. Let the HPLC run approximately 15 minutes before injecting the HPLC run approximately 15 minutes before injecting the sample. Make sure the waste is coming into the waste sample. Make sure the waste is coming into the waste bottle; Open the Caffeine analysis method. bottle; Open the Caffeine analysis method.

When ready mode is displayed, inject 1 µL of the When ready mode is displayed, inject 1 µL of the reference standard and run the sample. HP chemstation reference standard and run the sample. HP chemstation automatically starts data acquisition (run each automatically starts data acquisition (run each concentration twice for the reference standards). concentration twice for the reference standards).

Run the sample analysis for each soft drink.Run the sample analysis for each soft drink.

Results Results Calibration table and curve

Run Run ConcentrationConcentration

(µg/mL)(µg/mL) Area (mAU’s) Area (mAU’s) Average Average

areaareaStandard deviationStandard deviation

%CV or RSD%CV or RSD

11 100.00100.00 100.19100.19 96.3196.31 3.473.47

3.603.6022 93.4993.49

33 95.2595.25

11 200.00200.00 173.69173.69 162.78162.78 11.3911.39

7.007.0022 150.97150.97

33 163.67163.67

11 400.00400.00 261.29261.29 267.58267.58 6.916.91

2.582.5822 274.97274.97

33 266.48266.48

11 600.00600.00 362.40362.40 377.98377.98 14.5414.54

3.853.8522 391.18391.18

33 380.35380.35

Calibration curveCalibration curveCaffeine RS

y = 0.5535x + 46.123

R2 = 0.9988

0

50

100

150

200

250

300

350

400

0 200 400 600 800

Concentration (PPM)

Are

a (

mA

U)

Results for the SamplesResults for the SamplesBeveragesBeverages Area, mAU’s Area, mAU’s

( y value)( y value)Area x dilutionArea x dilution

factor (10)factor (10) Conc.mg/L Conc.mg/L

(x value)(x value) Hansen’s energyHansen’s energy 60.70760.707 607.07607.07 1013.451013.45

AMP energyAMP energy 51.63351.633 516.33516.33 849.52849.52

SoBe adrenaline SoBe adrenaline 76.89076.890 768.90768.90 1305.831305.83

KMX energy drinkKMX energy drink 40.43940.439 404.39404.39 647.27647.27

High potency High potency energy venomenergy venom

121.959121.959 1219.591219.59 2119.922119.92

Red Bull ( sugar Red Bull ( sugar free)free)

39.99539.995 399.95399.95 639.25639.25

Red BullRed Bull 31.79031.790 317.90317.90 491.02491.02

CokeCoke 17.19517.195 171.95171.95 227.33227.33

Coca-colaCoca-cola 11.28711.287 112.87112.87 120.59120.59

PepsiPepsi 8.0168.016 80.1680.16 61.4961.49

Conclusions 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.

ReferencesReferences

Glenda K. FergusonGlenda K. Ferguson. Quantitative HPLC Analysis of an Analgesic/Caffeine . Quantitative HPLC Analysis of an Analgesic/Caffeine

Formulation: Determination of Caffeine Formulation: Determination of Caffeine J. Chem. Educ.J. Chem. Educ. 19981998 7575 467. 467.

Glenda K. FergusonGlenda K. FergusonWesleyan College, Department of Chemistry, 4760 Forsyth Road, Macon, Wesleyan College, Department of Chemistry, 4760 Forsyth Road, Macon, GA 312 GA 312

National Soft Drink Association, Bunker & McWilliums, J Am Diet, 74: 28-National Soft Drink Association, Bunker & McWilliums, J Am Diet, 74: 28-32, 197932, 1979

http://www.astro.umd.eduhttp://www.astro.umd.edu