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Research and Technology - Forensic Science Communications - October 2006

Research and Technology - Forensic Science Communications - October 2006

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October 2006 - Volume 8 - Number 4

Research and Technology

Identification and Estimation of Methaqualone in Toffee Samples Using Gas Chromatography-Mass Spectrometry, Fourier Transform Infrared Spectrometry, and High-Performance Thin-Layer Chromatography

Dilip Kumar Kuila
Junior Scientific Officer
Central Forensic Science Laboratory
Kolkata, India


Baijayanta Muhkopadhyay

Senior Scientific Assistant
Central Forensic Science Laboratory
Kolkata, India


S. C. Lahiri

Emeritus Fellow
Kalyani Government Engineering College
Kalyani, India

Abstract | Introduction | Materials and Methods | Results and Discussion | Acknowledgments | References

Abstract

An analysis was made of some Indian-brand seized toffee samples suspected to contain adulterants/hypnotic drugs and alcohol. Methaqualone was extracted from the toffee samples using 1 molar NaHCO3 solution and an EvidexII solid-phase extraction (SPE) cartridge. Methaqualone was identified using gas chromatography-mass spectrometry (GC-MS) and Fourier transform infrared (FTIR) techniques. Most of the toffee samples did not contain any of the tested analytes. Only one variety of toffee sample contained methaqualone, at 4.49 ± 0.05 mg (n = 3). Methaqualone was estimated using high-performance thin-layer chromatography (HPTLC) with a densitometer CD 60. The calibration plot for the estimation of methaqualone was based on linear regression analysis (y = 34.89x + 125.64; r2 = 0.998). The average recovery percentage of methaqualone was found to be 95 percent with relative standard deviations (RSDs) of 0.95–1.75 percent (n = 3).

Introduction

Methaqualone [2-methyl-3-(2-methylphenyl)-4(3H)-quinazolinone] is a synthetic sedative-hypnotic drug having local anesthetic and weak antihistaminic properties with a pattern of pharmacological effects similar to those of barbiturates. It is best known for its medical and recreational popularity in the 1970s (Seigel et al. 2000). The sedative-hypnotics are used to depress the activity of nerve cells, causing drowsiness. They relieve feelings of anxiety, tension, and mental stress. Higher doses of the drug suppress cardiovascular activity and cause general anesthesia, sleep, and unconsciousness. The drug is highly addictive in nature (Seigel et al. 2000). Because of its toxicity and abuse potential, methaqualone is sold under scheduled, or controlled, drugs.

In India, drug traffickers have been known to use diazepam, lorazepam, and other sedative-hypnotics in confectionaries, such as chocolate, toffee, hazmi (a type of toffee available in India), and chewing gum, which are liked and used by both children and adults. Cases have frequently been reported to criminal investigative authorities in which criminals rob fellow passengers in trains or buses of their belongings after rendering them unconscious with tea, coffee, biscuits, or toffee adulterated with sedative-hypnotics (Ghosh et al. 2004; Hugel 1984; U.S. Department of Justice, Drug Enforcement Administration 2003). Investigative agencies in India seized samples of suspected adulterated toffees with such names as rum, brandy, whisky, gin, and vodka and sent them to the Central Forensic Science Laboratory in Kolkata to analyze for the presence of drugs or alcohol.

Analytical techniques employed to identify methaqualone in various matrices include spectrometry (Kuila and Lahiri 2004), thin-layer chromatography (TLC), and high-performance liquid chromatography (HPLC). In the present work, instead of the usual procedure of dissolving the samples in methanol, a modified procedure was used to extract methaqualone from different toffee samples. Rapid analytical procedures such as gas chromatography-mass spectrometry (GC-MS), Fourier transform infrared (FTIR) spectrometry, and high-performance thin-layer chromatography (HPTLC)were used to identify and estimate methaqualone in the supplied toffee samples.

Materials and Methods

Toffee Samples

Suspected adulterated sugar toffees with the names of such alcoholic beverages as rum, brandy, whisky, gin, and vodka were received from various law enforcement agencies. The same types of toffee samples were procured from the local market. The procured samples and the experimental samples that had tested negative for the presence of methaqualone were used to conduct experiments to determine the recovery percentage of methaqualone.

The method involved dissolution of the toffee samples, as well as the analyte, and the extraction of methaqualone using an EvidexII solid-phase extraction (SPE) cartridge (Agilent Technologies, Palo Alto, California). Ten grams of each type of toffee sample was dissolved separately in 1 molar sodium bicarbonate solution (a biological buffer) and passed through phase-separation filter paper (Whatman, Springfield, England). The methaqualone and other organic residues, if any, present in the aqueous bicarbonate solution were extracted with chloroform and filtered through an EvidexII SPE cartridge after treating the solution with activated charcoal. The chloroform extract was dried over anhydrous sodium sulfate and evaporated to dryness. The dried mass was dissolved in 10 μL of methanol, and the methanolic solutions were used for experimental purposes.

The recovery studies were conducted by spiking the similar but non-methaqualone-containing toffee samples (rum, whisky, gin, and vodka) with standard methaqualone concentrations of 3 mg per 10 g, 5 mg per 10 g, and 10 mg per 10 g of toffee and extracted as described above.

Preliminary TLC experiments with acidified potassium iodoplatinate solution as the visualizing reagent gave positive tests for methaqualone in one of the samples. The samples were then subjected to further investigation.

Chloroform extracts of the toffee samples were also passed through GC and GC-FTIR for confirmation of alcohol present.

The chemicals used were of analytical grade. Solvents used were of UV grade or HPLC grade.

Instruments

(a) (i) GC-MS system—Finnigan Trace GC-MS system (Thermo Electron, Waltham, Massachusetts).
(ii) GC system—Finnigan Trace GC system.
(b) GC-MS column—PerkinElmer Elite-5ms (PerkinElmer, Wellesley, Massachusetts); column
length, 15 m; inner diameter, 0.32 mm.
(c) (i) FTIR system—PerkinElmer Spectrum GX FTIR spectrometer.
(ii) GC-FTIR system—PerkinElmer Clarus 500 gas chromatograph coupled with Spectrum GX FTIR.
(d) HPTLC system—Desaga HPTLC system with densitometer CD 60 and autosampler model AS 30 (Desaga, Wiesloch, Germany).
(e) Mettler analytical balance (Mettler-Toledo, Columbus, Ohio), model AE 240 (accuracy ± 0.01 mg).

Chromatographic Condition

(a) HPTLC plate—Merck precoated silica gel GF254 HPTLC plate (Merck & Co., Whitehouse Station, New Jersey); thickness, 0.25 mm; size, 20 cm x 20 cm.
(b) Mobile phases—(i) cyclohexane + toluene + diethylamine in a ratio of 75:20:15 (v/v) and (ii) methanol + ammonia in a ratio of 100:1.5 (v/v).
(c) Visualizing reagent—Ultraviolet light at 254 nm and acidified potassium iodoplatinate reagent.

Quantitative Estimation Using HPTLC

The quantitative estimation and recovery of the percentage of methaqualone were determined using the HPTLC technique (Sharma and Lahiri 2005a), for which the standard solution of methaqualone in methanol was prepared dissolving 10 mg ± 0.01 mg of methaqualone (Sigma Central Drug House, Kolkata, India, having purity >99%) in 10 μL of methanol to obtain 1mg/μL concentration. The standard solution of methaqualone (1 mg/μL) and the experimental solution were spotted by a Desaga autosampler (model AS30) at several positions on the HPTLC plate, keeping a 15-mm margin at the bottom and 20-mm margins each at the right and left sides. Each spot was given by a 3 μL/cycle with a break of 5 seconds between two cycles. The standard methaqualone solution was spotted at 10 positions having volumes of 3, 6, 9, 12, 15, 18, 21, 24, 27, and 30 μL. The experimental sample (extracted from case-specimen toffee) and spiked samples (similarly extracted) were spotted having a volume of 21 μL for each of the samples. The plates were developed up to 100 mm from the margin of the plates in a CAMAG developing chamber (CAMAG, Muttenz, Switzerland) using the solvent systems (mobile phases) described previously. The developed plates were air-dried and scanned with the densitometer of the HPTLC system using a 254-nm light beam (8 nm long and 1 nm wide) in reflectance mode. The area of chromatogram was plotted against amount.

Results and Discussion

The amount of methaqualone detected in the various toffee samples received in the laboratory from different law enforcement agencies is given in Table 1. Methaqualone is insoluble in water, but usually, methaqualone hydrochloride soluble in 1-in-65 water is used in the toffees. Methaqualone hydrochloride with a pKa value of 2.5 (Moffat 1986) is soluble in 1M NaHCO3 solution (pH ≈ 8). Sugar and other ingredients present in the toffee samples are also soluble in 1M NaHCO3 solution. Therefore, 1M NaHCO3 solution was used instead of the usual procedure of dissolving the samples in methanol.

Table 1: The amount of methaqualone detected in toffee samples received in the laboratory from various law enforcement agencies. Only the brandy sample tested positive for methaqualone.

Sample Number

Sample Name

Amount Received
in kg

Methaqualone Detected

Amount of Methaqualone Estimated

1

Rum

2

Absent


2

Brandy

1.5

Present

4.49 ± 0.05 mg in
10 g of toffee

3

Whisky

1.5

Absent


4

Gin

2

Absent


5

Vodka

1.5

Absent


Preliminary investigations and TLC experiments of most of the toffee samples gave negative tests for diacetylmorphine (heroin), diazepam, cocaine, diphenhydramine, methaqualone, and caffeine, but methaqualone was present in one of the toffee samples (brandy). The hundredth-of-retention factor (hRf) values of standard heroin, diazepam, cocaine, diphenhydramine, methaqualone, and caffeine, as well as those obtained from the brandy toffee sample, are given in Table 2.

Table 2: hRf Values of Standard Narcotics and Related Drugs in Different Mobile Phases


Compound

hRf Values

Mobile Phase (i)

Mobile Phase (ii)

Heroin

19

46

Diazepam

29

75

Cocaine

52

67

Diphenhydramine

56

55

Methaqualone

40

74

Caffeine

5

63

Sample of Toffee (Brandy)

40.5

74.5

The HPTLC technique is more advantageous than the conventional TLC technique because the particle sizes of the sorbent material are much smaller and the size distribution of these particles is much tighter in the HPTLC plate than the TLC plate. HPTLC plates are also thinner, and their surface is more uniform than that of conventional plates. These differences often can result in the use of a smaller sample volume, smaller solvent volume for the mobile phase, shorter solvent migration distance, and greater sensitivity/resolution for the detection of separated compounds
(Fenimore and Davis 1981).

The presence of methaqualone in the brandy toffee sample was confirmed by GC-MS and FTIR experiments (Sharma and Lahiri 2005b; Sharma et al. 2005). The TLC (11.39) and m/zm/z values of the standard sample of methaqualone and match the mass spectrum library data (Figure 1). values of the main peak of methaqualone (mol wt 250) [present in the toffee (brandy)] are 250 (M+ 251 due to isotopes). Other tentative species of fragment ions are given in parenthesis: 235 (M–CH3)+, 236 (M–CH3+ due to isotopes), 233 (M–OH)+, 144 (C6H4NCNCO+), 132 (C6H4N2CO+), 104 (C6H4CO+), 91 (C6H5N+), 76 (C6H4+), 77 (C6H4+ due to isotopes), 65 (C5H5+), 50 (C4H2+). The values agree with the

Figure 1: Mass Spectrum of Methaqualone from Brandy Toffee Sample Compared with Mass Spectrum from Library Data

Figure 2 shows the FTIR spectrum of the methaqualone extracted from the experimental sample using the KBr pellet method. The major bands of the spectrum are 1608, 1487, 1378, 1269, 778, and 703 cm–1, which match the IR library spectrum.

Figure 2: FTIR Spectrum of Methaqualone Recovered from Brandy Toffee Sample Compared with IR Spectrum from Library Data

Quantitation of the methaqualone in the brandy toffee sample was made using the HPTLC technique (Table 3). The area under the chromatogram (y) of standard methaqualone at various concentrations was plotted against amount (x) (Figure 3). Regression analysis was performed using the equation y = mx + b. The slope (m) and intercept (b) values equaled 34.89 and 125.64, respectively, with a regression coefficient (r2) of 0.998. The calibration curve was used to calculate the concentration of methaqualone present in the experimental solution (extract of brandy toffee sample). The amount of methaqualone present was found to be 4.49 ± 0.05 mg (n = 3) in 10 g of toffee labeled “brandy.” The average recoveries of methaqualone from the toffee samples were found to be 95 percent with relative standard deviations (RSDs) of 0.95–1.75 percent (n = 3) (Table 4).

Table 3: HPTLC Data for Estimation and Recovery of Methaqualone from Toffee Samples

Volume of Standard and Sample Solution Spotted on HPTLC Plate in μL

Measured Value of Area Using Densitometer CD 60 of HPTLC*

Amount of Methaqualone Spotted/Calculated in μg

3

260.2

3

6

343.4

6

9

425.9

9

12

542.6

12

15

635.4

15

18

732.2

18

21

827.1

21

24

965.4

24

27

1080.7

27

30

1200.4

30




21 (extract from toffee)

455.5

9.45

21 (extract from toffee)

459.6

9.57

21 (extract from toffee)

450.7

9.31




21 (spiked sample, 3 mg/10 g)

339.2

6.12

21 (spiked sample, 3 mg/10 g)

329.8

5.85

21 (spiked sample, 3 mg/10 g)

335.0

6.00




21 (spiked sample, 5 mg/10 g)

469.8

9.86

21 (spiked sample, 5 mg/10 g)

475.0

10.01

21 (spiked sample, 5 mg/10 g)

478.6

10.11




21 (spiked sample, 10 mg/10 g)

812.6

19.69

21 (spiked sample, 10 mg/10 g)

817.1

19.81

21 (spiked sample, 10 mg/10 g)

828.9

20.15

*Optical density times length, measured in milliextinction times millimeter

Figure 3: Calibration Curve of Methaqualone from HPTLC Data

Table 4: Recovery of Methaqualone from Toffee Samples

Weight of the Sample Taken (g)

Methaqualone Added (mg/10 g)

Methaqualone Found (mg/10 g)*

Recovery %

Average Recovery %

RSD %

10

3.0

2.85 ± 0.05

95

95

1.75

10

5.0

4.76 ± 0.05

95.2

1.05

10

10.0

9.47 ± 0.09

94.7

0.95

*Each value is the mean ± standard deviation; n = 3

GC and GC-FTIR showed the absence of alcohol in the supplied toffee. The toffees were named after popular alcoholic drinks only to mislead and lure customers, particularly juveniles, to buy the toffee, thinking it may contain alcohol. However, criminals frequently use methaqualone and other sedative-hypnotics to rob passengers on buses and trains, and passengers must be careful in accepting and consuming tea, coffee, and toffees from unknown persons while traveling.

Acknowledgments

The authors thank Dr. C. N. Bhattacharya, Director, Central Forensic Science Laboratory, and Dr. M. S. Rao, Chief Forensic Scientist, Directorate of Forensic Science, New Delhi, for their keen interest and encouragement.

References

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