Journal of Analytical Sciences, Methods and Instrumentation, 2018, 8, 1-15

http://www.scirp.org/journal/jasmi
ISSN Online: 2164-2753
ISSN Print: 2164-2745

HPTLC-MS as a Neoteric Hyphenated Technique

for Separation and Forensic Identification of
Drugs

Kanak Lata Verma1, Manoj Kumar2, Amar Pal Singh1

Regional Forensic Science Laboratory, Government of NCT of Delhi, New Delhi, India
1

Forensic Science Post Graduate Course, Department of Anthropology, University of Delhi, New Delhi, India
2

How to cite this paper: Verma, K.L., Ku-

Abstract
mar, M. and Singh, A.P. (2018) HPTLC-MS
as a Neoteric Hyphenated Technique for Drugs are traditionally been identified on basis of chromatograph-
Separation and Forensic Identification of
ic-spectroscopic hyphenated techniques in instrumental analysis. Gas chro-
Drugs. Journal of Analytical Sciences, Me-
thods and Instrumentation, 8, 1-15. matography (GC) and Liquid chromatography (LC) hyphenated with mass
https://doi.org/10.4236/jasmi.2018.81001 spectroscopy (MS) i.e. GC-MS and LC-MS give reliable and confirmatory re-
sults in drugs identification. In the present work the novel hyphenated tech-
Received: February 2, 2018
Accepted: March 26, 2018
nique High Performance Thin Layer Chromatography-Mass Spectroscopy
Published: March 29, 2018 (HPTLC-MS) has been used. This technique provides efficient, quick and
simple method for identification and separation of Narcotic drugs and psy-
Copyright © 2018 by authors and
chotropic substances. The drugs under study are Papaverine, Methadone,
Scientific Research Publishing Inc.
This work is licensed under the Creative Cocaine, Ketamine, Caffeine, Codeine, Diazepam, Thebaine, Heroin, Me-
Commons Attribution International thamphetamine, Carbamazepine, Morphine, Narcotine and Ephedrine. The
License (CC BY 4.0). present study comprising of sixteen drugs has been carried out on CAMAG
http://creativecommons.org/licenses/by/4.0/
HPTLC instrument with automatic sampling. Thin layer chromatography
Open Access
(TLC) plates were developed in various solvent systems, scanned under TLC
scanner and the results in terms of Retention Factor (Rf value) and UV spec-
trum (λmax) are presented in the manuscript. Using hyphenated technique of
HPTLC-MS (MS 2020 SHIMADZU) spots of these drugs from TLC plate was
lifted with CAMAG TLC-MS interface and confirmed by the mass spectrum
of the individual drugs by their m/z values thus delivering fast and accurate
confirmatory result on the TLC plate.

Keywords
Narcotic Drugs, Psychotropic Substances, HPTLC-MS, Chromatography,
Mass Spectroscopy, Hyphenated Techniques, Forensic Science

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K. L. Verma et al.

1. Introduction
India geographical location makes it proximal to drug problem as India is sand-
wiched between the world’s two largest areas of illicit opium production, the
golden crescent i.e. Pakistan, Afghanistan, Iran and the golden triangle i.e. Viet-
nam, Laos, Thailand, Myanmar. The proximity has traditionally been viewed as
a source of vulnerability, since it has made India both a destination and a transit
route for opiates, cannabis, amphetamines and synthetic drugs produced in
these regions [1]. This fact defines drug trafficking trends in the subcontinent.
International drug trafficking is not just a trade for financial purposes but pro-
vides funding for cross border terrorisms which is called drug terrorism. The
United Nations Conference for the Adoption of a Single Convention on Narcot-
ic Drugs met at United Nations Headquarters from 24 January to 25 March 1961
to reduce the number of international treaty organs exclusively concerned with
control of narcotic drugs, and to make provision for the control of the produc-
tion of raw materials of narcotic drugs. The United Nations Conference for the
Adoption of a Protocol on Psychotropic Substances met in Vienna from 11 Jan-
uary to 21 February 1971 and determined to prevent and combat abuse of such
substances and the illicit traffic to which it gives rise. United Nations Conven-
tion against Illicit Traffic in Narcotic Drugs and Psychotropic Substances, 1988
provides comprehensive measures against drug trafficking, including provisions
against money laundering and the diversion of precursor chemicals. It provides
for international cooperation through, for example, extradition of drug traffick-
ers, controlled deliveries and transfer of proceedings [2]. On part of India, gov-
ernment agencies seize tons of Heroin along with other narcotic drugs and psy-
chotropic substances under NDPS Act, 1985. During 2015, 1687 kgs of Opium,
1416 kg of Heroin, 94,403 kg of Ganja, 3349 kg of Hashish, 113 kg of Cocaine,
827 kg of Ephedrine/Pseudo-Ephedrine, 211 kg of Ketamine etc. were seized by
various agencies. Illicit Opium poppy cultivation spread over 3461 acres & illicit
cannabis cultivation spread over 818 acres were destroyed all over the country
[3]. The NDPS Act provides a strong system to prevent illegal drug trafficking
and prevention over clandestine laboratories in India. The NDPS (The Narcotic
Drugs and Psychotropic substances Act, 1985) act is provisions for the control
and regulation of operations relating to An Act to consolidate and amend the
law relating to narcotic drugs, to make stringent narcotic drugs and psychotrop-
ic substances [1], to provide for the forfeiture of property derived from, or used
in, illicit traffic in narcotic drugs and psychotropic substances, to implement the
provisions of the International Convention on Narcotic Drugs and Psychotropic
Substances and for matters connected therewith [4]. In spite of these stringent
national and international efforts and law, drug seizure data show that illegal
drug manufacturing in clandestine laboratories and trafficking is flourishing.
The penalty for manufacturing, illegal consumption, holding and trafficking de-
pends upon nature of substance seized from the culprit. So, complete characte-
rization and identification of seized Narcotics and Psychotropic drugs is of ut-

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 K. L. Verma et al.

most importance to penalise the culprit with proper punishment. Drugs identi-
fication and separation is traditionally being done by chromatography [5] which
was discovered by M. Tswett in 1906 and in 1938 Izmailov and Shreiber intro-
duced TLC with some modifications. Chromatography is “the separation of two
or more compounds by distribution between two phases. One is stationary phase
and other is mobile phase” [6]. These two phases can be solid-liquid, liq-
uid-liquid and gas-liquid. The principle behind the work of this method is that
different types of compounds with different solubility, adsorption and polarity to
the two phases by which they are partitioned. The compounds to be retained on
the stationary phase are more interacted with it while the compounds to be
moving carried along by the mobile phase. The rates of migration for each
component on the system is depends on the degree of the compounds of mixture
are adsorbed by the stationary phase and their degree of solubility on the mobile
phase. The stronger the adsorption by stationary phase, the slower the com-
pound will move with the mobile phase. Due to which the migration distance for
each substance is found to be different and the components of the mixtures get
separated. The migration distance of substances on TLC chromatograms are
generally fixed as Rf value. The types of chromatography is divided into few
types which are gas chromatography (GC), high performance liquid chromato-
graphy (HPLC), thin layer chromatography (TLC) and column chromatography
(CC). Different types of chromatography make the task of identification firm,
easy and reliable. Since in forensic science laboratories drugs are identified and
separated by TLC, HPTLC, GC-FID and known hyphenated techniques which
are GC-MS, LC-MS. Drugs are traditionally been identified on basis of chroma-
tographic-spectroscopic hyphenated techniques in instrumental analysis. Gas
chromatography and Liquid chromatography hyphenated with mass spectros-
copy i.e. GC-MS and LC-MS give very reliable results in drugs identification. In
the present work the novel hyphenated technique High Performance Thin Layer
Chromatography-Mass Spectroscopy (HPTLC-MS) has been used to develop a
new method for identification and separation of drugs with High Performance
Thin Layer chromatography hyphenated with Mass spectroscopy. This tech-
nique provides efficient, quick and simple method for identification and separa-
tion of Narcotic drugs and psychotropic substances. High Performance Thin
Layer Chromatography (HPTLC) is the most advanced form of TLC and com-
prises the use of chromatographic layers of utmost separation efficiency and the
employment of state of the art instrumentation for all steps in the procedure:
precise sample application, standardized reproducible chromatogram develop-
ment and software controlled evaluation. HPTLC is an entire concept that in-
cludes a widely standardized methodology based on a scientific facts as well as
the use of validated methods for quantitative and qualitative analysis. HPTLC
meets all quality requirements of today’s analytical labs. The possibility of visual
evaluation of the separated samples on the plate is one of the most valuable as-
pects of TLC [7]. Advantages of HPTLC include that the technique is simple to

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K. L. Verma et al.

learn, operate, several analysis works could be done on same time, is a fast and
economic technique. Visual detection possible as it is an open system and at the
same time there is availability of great range of stationary phases with unique se-
lectivity of mixture components. The plates do not require regeneration as they
are disposable. Availability to choose mobile phase is not restricted by low UV
transparency or the need of ultra-high purity. Corrosive and UV absorbing mo-
bile phase can be employed. There is no possibility of interference from previous
analysis as fresh stationary and mobile phase is used for each analysis, so no
carry over and no contamination. TLC scanner provides the output to a system
in terms of Retention factors of the spot, height and area of the peak for quanti-
fication and absorbance in UV-Visible spectrum [8] for substance identification.
UV and visible spectrophotometry find their primary application in quantitative
analysis. The scope of absorption spectroscopy can be extended significantly by
the use of colour reactions, often with a concomitant increase in sensitivity
and/or selectivity. Such reactions are used to modify the spectrum of an absorb-
ing molecule so that it can be detected in the visible region, well separated from
other interfering components in the UV spectrum. Moreover, chemical modifi-
cation can be used to transform an otherwise non-absorbing molecule into a sta-
ble derivative that possesses significant absorption [9]. The whole instrument
becomes very efficient for substance identification having output as mass spec-
trum of the substance when Mass Spectroscopy is hyphenated with the HPTLC
instrument and the qualitative analysis is confirmatory. A mass spectrometer
works by generating charged molecules or molecular fragments either in a high
vacuum or immediately before the sample enters the high-vacuum region. In-
struments typically maintain vacuums of about 10 - 6 mmHg, since ionised mo-
lecules have to be generated in the gas phase to be able to manipulate those using
magnetic or electrostatic fields. In classic mass spectrometry (MS) only one me-
thod could produce the charged gaseous molecules, but now quite a number of
alternatives are available. Once the molecules are charged and in the gas phase,
they can be manipulated by the application of either electric or magnetic fields to
enable the determination of their relative molecular mass and the relative mole-
cular mass of any fragments produced by the molecules breaking up [10] [11].
In the present work, drugs examined under HPTLC-MS (CAMAG instru-
ment) are Methadone, Papaverine, Cocaine, Ketamine, Phenobarbital, Caffeine,
Codeine, Acetaminophen, Diazepam, Thebaine, Heroin, Methamphetamine,
Carbamazepine, Narcotine, Morphine and Ephedrine. Most of these drugs are
often encountered in seized samples of drugs. These drugs are used illicitly in
various forms and through multiple routes. Serious risks include opioid abuse or
a decreased effort to breathe [12] [13] [14]. Ketamine, a phencyclidine deriva-
tive, is a unique dissociative anaesthetic that produces amnesia and intense
analgesia [15]. Phenobarbital is an inexpensive anticonvulsant, and will therefore
remain important in developing countries. In the developed world, where cost is
less of a determining factor, it is less attractive than its better-tolerated succes-

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 K. L. Verma et al.

sors [16]. Caffeine is one of the world’s most widely ingested chemicals, The first
use of coffee beans occurred in Ethiopia as far back as the ninth century [17].
Codeine is an alkaloid found in very low concentrations in opium; it is now de-
rived from morphine. Codeine is frequently administered in combination with
acetaminophen, butalbital, and caffeine [18] Acetaminophen (known as parace-
tamol) is the most commonly administered over-the-counter analgesic, [19]. The
second benzodiazepine to be marketed, diazepam, was approved for human use
in the early 1960s and has sedative, hypnotic, muscle relaxant and anticonvul-
sant properties [20]. Thebaine (paramorphine), also known as codeine methyl
enol ether, is an opiate alkaloid and at high doses, it causes convulsions similar
to strychnine poisoning [21]. Heroin is an opioid drug made from morphine, a
natural substance taken from the seed pod of the Asian opium poppy plant.
Some people mix heroin with crack cocaine, called a speedball [22]. Metham-
phetamine is a psycho-stimulant that is similar in chemical structure to amphe-
tamine but has more profound effects on the central nervous system. Like co-
caine, methamphetamine ingestion produces stimulation and similar feelings of
euphoria; however, methamphetamine has a longer duration of action (6 - 8 h
after a single dose) [23]. Carbamazepine is an anticonvulsant with structural si-
milarity to the cyclic antidepressants [24]. Narcotine is a naturally occurring,
opium-isoquinoline alkaloid that is related to papaverine. It acts centrally as a
cough suppressant and has actions and uses similar to dextromethorphan.
Morphine is a pain medication of the opiate type which is found naturally in a
number of plants and animals. It acts directly on the central nervous system
(CNS) to decrease the feeling of pain. Potentially serious side effects include a
decreased respiratory effort and low blood pressure. Morphine has a high poten-
tial for addiction and abuse. If the dose is reduced after long-term use, with-
drawal may occur. Common side effects include drowsiness, vomiting, and con-
stipation. Caution is advised when used during pregnancy or breast feeding, as
morphine will affect the baby [25]. Ephedrine is one of the most commonly used
non catecholamine sympathomimetic drugs in the preoperative period [26].
Thin layer chromatography (TLC) and gas chromatography-mass spectros-
copy (GC-MS) have been reportedly used to confirm the presence of heroin and
its metabolite 6-monoacetyl morphine. The presence of 6-monoacetyl morphine
determines definite heroin exposuree [27]. The application of high performance
thin layer chromatography-bioautography in tandem with mass spectrometry
was investigated for the rapid identification of antimicrobial and anti-quorum
sensing (anti-QS) compounds [28]. Simultaneous Quantification of Methadone,
Cocaine, Opiates, and Metabolites in Human Placenta by Liquid Chromatogra-
phy-Mass Spectroscopy has been done [29]. HPTLC method for simultanoeus
determination of alprazolam and methyl paraben in tablet dosage form has been
carried out by Sagar B. Wankhede et al. [30]. Hui Qiao et al. have reported a
Quantitative Determination of Methadone and its Metabolite (EDDP) in Dry
Blood Spot by LC-MSMS [31]. Ketamine popularly known as date-rape drug

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K. L. Verma et al.

abused in rave parties and cause of the crimes against the women and determi-
nation of ketamine and its residues in non-biological exhibits in cases related to
drug facilitated sexual assaults by LC-MS/MS has been reported [32].

2. Experimental Set-Up
To execute a new hyphenated technique for separation and forensic identifica-
tion of drugs with the following steps:
1) To find out solvent system using HPTLC for the following drugs
Methadone, Papaverine, Cocaine, Ketamine, Phenobarbital, Caffeine, Codeine
Acetaminophen, Diazepam, Thebaine, Heroin, Methamphetamine, Carbamaze-
pine, Narcotine, Morphine and Ephedrine.
2) To separate the drugs and find out the retention factor using HPTLC for
each drug in its respective solvent systems in particular conditions.
3) Qualitative analysis to find out maximum absorbance and UV spectrum for
each drug in its particular condition.
4) To confirm the identification of drugs by hyphenated mass technique by
obtaining its mass spectrum and analysis of the peaks obtained.

3. Materials and Methods

Chemicals And Reagents: Standard drugs include Methadone, Papaverine, Co-
caine, Ketamine, Phenobarbital, Caffeine, Acetaminophen, Diazepam, The
baine, Methamphetamine, Carbamazepine, and Ephedrine Heroin, Codeine,
Narcotine, Morphine (From Indian Pharmacopoeia Commission, Sigma-Aldrich
and Government Opium and Alkaloid Works, Neemuch, India).
Reagents of sample preparation—Millipore water, Ethanol, Methanol (HPLC
Grade), Chloroform (HPLC Grade), Reagents for Mobile phase prepara-
tion-Chloroform (HPLC Grade), Methanol (HPLC Grade), Ammonia, Ethyl
acetate (HPLC Grade),Carrier Solvent for MS interface-Methanol (HPLC
Grade), Acetonitrile (HPLC Grade), Pre coated 10 × 10 Silica gel 60 F254 TLC
plates, Micro-pipette, Spatula, Test tubes, TLC Development Chamber (Camag).

4. Extraction Method
1) Preparation of standard samples
Standard solutions of drugs were prepared using HPLC Grade solvents. Sol-
vent selection for standard solutions was done according to the solubility [33] of
drugs. Standard solutions of drugs Methadone, Cocaine, Phenobarbital, Code-
ine, Acetaminophen, Methamphetamine, Carbamazepine, Morphine and Ephe-
drine were taken in 5 ml volumetric flask and ethanol was added to each of the
volumetric flask and volume was extended up to 5 ml mark. Standard solutions
for Papaverine, Caffeine, Diazepam, Thebaine, Heroin and Narcotine were pre-
pared in 5 ml Chloroform of HPLC Grade. Ketamine standard was prepared in 5
ml Methanol of HPLC Grade.
2) Preparation of developing solvents (mobile phase)

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 K. L. Verma et al.

Three different Mobile phases were prepared for TLC development as shown
in Table 1. For selection of mobile phase drugs were categorized into two cate-
gories. One is basic and other is acidic.
a) Basic drugs
Methadone, Papaverine, Cocaine, Ketamine, Caffeine, Codeine, Diazepam,
Thebaine, Heroin , Methamphetamine, Carbamazepine, Narcotine, Morphine &
Ephedrine.
b) Acidic drugs
Phenobarbital & Acetaminophen.

5. Instrumental Methods
Analysis of the drugs was performed using CAMAG HPTLC instrument coupled
with MS (MS 2020 SHIMADZU) by CAMAG TLC-MS interface and system was
controlled with winCATS CAMAG HPTLC Software. The fully automatic sam-
ple application device used was ATS 4 (CAMAG Automated TLC Sampler 4)
fitted with a 25 μl Hamilton micro syringe and connected to a nitrogen supply.
Four narrow band spots of 6.0 mm length were applied above 8mm on pre coated
10 × 10 Silica gel 60 F254 TLC plate for each drug sample of different concentra-
tion (2 μl, 5 μl, 10 μl, 2 μl). Methanol was used as solvent and Nitrogen gas pro-
vided pressure for spraying the sample for application ensuring homogenous
distribution of the sample across the entire length of application zone. After ap-
plication plate was dried for 10 minutes at room temperature (25˚ Celsius).
1) Sample application
Sampling Type—Automatic Spraying, Instrument Used—CAMAG HPTLC
No. of spots—4 (2 μl, 5 μl, 10 μl, 2 μl), Band Length—6 mm, Plate Size—10 ×
10.
Solvent used—Methanol HPLC Grade, Pressure gas—Nitrogen, Drying—At
25˚C for 10 minutes, Starting spot position—8 mm, Solvent front position—80
mm.
2) Development
After precise sample application, the chromatograms were developed in their
respective solvent system as specified in Table 2. Development of HPTLC plate
was done in 10 × 10 twin trough chamber saturated with mobile phase for 15
minutes. Solvent front was moved to a migration distance of 80 mm of the plate
and plate was dried at room temperature. Three types of mobile phase were used
for better separation.

Table 1. Solvent system used for TLC development.

SOLVENT SYSTEM SOLVENT USED (10 ml)

A CHLOROFORM:METHANOL (9:1)

B METHANOL:AMMONIA (10:0.15)

C ETHYL ACETATE:METHANOL:AMMONIA (8.5:1:0.5)

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K. L. Verma et al.

Table 2. Drugs and their respective solvent system.

S. No. NAME OF THE DRUG SOLVENT SYSTEM*

1 METHADONE A&B

2 PAPAVERINE A

3 COCAINE A

4 KETAMINE A

5 PHENOBARBITAL C

6 CAFFEINE A

7 CODEINE A&B

8 ACETAMINOPHEN C

9 DIAZEPAM A

10 THEBAINE A

11 HEROIN A

12 METHAMPHETAMINE B

13 CARBAMAZEPINE A

14 NARCOTINE A

15 MORPHINE B

16 EPHEDRINE B

*Solvent systems: Refer to Table 1.

3) Documentation
Reproducibly acquire and preserve best quality images of TLC/HPTLC chro-
matograms under different illuminations this high-end imaging and documen-
tation system is now available. With its new digital CCD camera a maximum
resolution of 82 μm on the plate is obtained. For electronic image acquisition the
camera—like the human eye—captures polychromatic visible light. Under white
light illumination it is the light reflected from the layer background. Under
long-wavelength UV light (366 nm) it is the light emitted by fluorescent sub-
stances. When short-wavelength UV light (254 nm) is used, substances absorb-
ing UV 254 appear as dark zones, provided the layer contains a fluorescence in-
dicator. Chromatograms on the plate were visualised and photographed for do-
cumentation under UV 254 nm, UV 366 nm and in white light with 85 point
exposure. The chromatograms were saved and documented.
4) TLC scanner
WIN CATS controls the CAMAG TLC Scanner 4 and enables quantitative
evaluation of the generated densitometry data. To determine the substance con-
centration in a sample, five different quantification functions (e.g. linear and
polynomial) are available. Several scanning steps (e.g. scanning the plate after
development) and up to five different evaluations can be performed (single wa-
velength, multiple wavelengths or a combination of measurements in absorption
and fluorescence detection mode). TLC plate was scanned under TLC Scanner.

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 K. L. Verma et al.

Spots were localised by automatic scanning and Rf (Retention factor) of each

spot was calculated and substance assignment was done in the evaluation sys-
tem. After proper substance assignment TLC plate was further scanned under
UV (200 - 400 nm) using Deuterium lamp. The output from the spectrum for
each drug in UV light was obtained, analysed and recorded in Table 3.
5) Lifting spots with camag TLC-MS interface
Spot were marked after visualising under UV light and lifted with CAMAG
TLC-MS interface. Carrier solvent i.e. Methanol and Acetonitrile (HPLC Grade)
for drug was taken into source solvent container. The SCHIMADZU 2020 Mass
Spectrometer was controlled with Lab Solutions software. The chromatogram
zones are eluted from the HPTLC plate with suitable solvents as shown in Table
4 with the flow speed appropriate for the LC-MS system. The round elution head
is used for circular zones and the oval elution head for zones in the form of
bands. After extraction of the elute is either transferred online to the mass spec-
trometer or collected in a sample vial for further offline analysis. The CAMAG
TLC-MS Interface 2 features a modified elution head and an easily accessible,
exchangeable filter, arranged in front of the valve. Cleaning is facilitated as com-
pared to the previous version, making it highly efficient. By pushing a button,

Table 3. Retention factor and maximum absorbence of drug.

SOLVENT RETENTION λmax

S. No. NAME OF THE DRUG
SYSTEM* FACTOR (nm)

A 0.15 200
1 METHADONE
B 0.48 200

2 PAPAVERINE A 0.91 243

3 COCAINE A 0.44 234

4 KETAMINE A 0.85 200

5 PHENOBARBITAL C 0.63 200

6 CAFFEINE A 0.74 275

A 0.25 212
7 CODEINE
B 0.43 209

8 ACETAMINOPHEN C 0.68 246

9 DIAZEPAM A 0.93 231

10 THEBAINE A 0.39 285

11 HEROIN A 0.47 206

12 METHAMPHETAMINE B 0.31 205

13 CARBAMAZEPINE A 0.78 286

14 NARCOTINE A 0.87 217

15 MORPHINE B 0.41 210

16 EPHEDRINE B 0.36 205

*Solvent systems: Refer to Table 1.

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K. L. Verma et al.

Table 4. Drugs with their lifting solvent.

MOLECULAR LIFTING
IUPAC NAME EVENT
WEIGHT SOLVENT

6-Dimethylamino-4,4-diphenyl-3-heptanone 309.4 1 M

1-[(3,4-Dimethoxyphenyl)methyl]-6,7-dimethoxyisouinoline 339.4 1 M

Methyl-[1R-exo,exo)]-3-(benzoyloxy)-8-methyl-8-azabicyclo[2.2.1]-octane-2-carboxylate 303.4 1 M

2-(2-chlorophenyl)-2-(methylamino)cyclohexanone 237.7 1 M

5-Ethyl-5-phenyl-2,4,6(1H,3H,5H)-pyirimidinetrione 232.2 2 M

3,7-Didehydro-1,3,7-trimethyl-1H-purine-2,6-dione 194.2 2 M

(5α,6α)-7,8-Didehydro-4,5-epoxy-3-methoxy-17-methylmorphinan-6-ol monohydrate 294.4 1 M

N-(4-Hydroxyphenyl)acetamide 151.2 2 M

7-Chloro-1,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one 284.7 1 A

(5α)-6,7,8,14-Tetradehydro-4,5-epoxy-3,6-dimethoxy-17-methylmorphina 311.4 1 M

(5α,6α)-7,8-Didehydro-4,5-epoxy-17-methylmorphinan-3,6-dioldiacetate 369.4 1 A

(αS)-N,α-Dimethylbenzeneethanamine 149.2 2 M

5H-Dibenz[b,f]azepine-5-carboxamide 236.3 2 A

(3S)-6,7-Dimethoxy-3-[(5R)-5,6,7,8-tetrahydro-4-methoxy-6-methyl-1,3-dioxolo[4,5-g]isoquin
413.4 1 A
oline-5-yl]-1(3H)-isoenzofuranone

(5α,6α)-7,8-Didehydro-4,5-epoxy-3-methoxy-17-methylmorphinan-6-ol monohydrate 285.3 2 A

(αR)-α[(1S)-1-(Methylamino)ethyl]benzenemethanol hemihydrate 165.2 1 M

M = Methanol; A = Acetonitrile; Even 1: Positive Ionisation; Even: Negative Ionisation.

the elution path is cleaned of matrix particles with compressed air, increasing
the lifetime of the filter and preventing the system from becoming blocked.
These filters can be easily replaced without any modification to the elution head.
The flow rate for mobile solvent for lifting up the spot from TLC was kept at
0.5 ml per minute and flow of Nebulising gas was kept at 1.5 L/min. The ioniza-
tion mode was electrospray ionization with chemical ionization. DL Tempera-
ture was 250˚C and detector voltage was kept at 0.95 KV. Spectra were acquired
in according to the mass of drugs in mass/charge (m/z) ratio range. Detection of
ions was done at both events positive and negative ion mode. The base peak and
mass peak was observed and recorded in Table 5.

6. Results
Developed chromatograms were scanned under CAMAG TLC Scanner and
output was Chromatogramed and UV spectrum display was recorded in obser-
vations. From the chromatogram and UV spectra for all tracks of drugs the out-
put received was recorded in Table 3 in terms of Retention Factor (Rf value) and
maximum absorbance pertaining to the conditions followed in materials and
methods. The data received was sufficient for separation and identification of
each drug. On further processing, when the spot was lifted with Camag

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 K. L. Verma et al.

Table 5. MS peak output of drugs.

NAME OF THE MOLECULA Peak detected in

S. No. IUPAC NAME EVENT
DRUG R WEIGHT MS ( m/z ratio)

1 METHADONE 6-Dimethylamino-4,4-diphenyl-3-heptanone 309.4 1 310

1-[(3,4-Dimethoxyphenyl)methyl]-6,7-dimethoxyiso
2 PAPAVERINE 339.4 1 340
uinoline

Methyl-[1R-exo,exo)]-3-(benzoyloxy)-8-methyl-8-
3 COCAINE 303.4 1 326*, 304**
azabicyclo[2.2.1]-octane-2-carboxylate

4 KETAMINE 2-(2-chlorophenyl)-2-(methylamino)cyclohexanone 237.7 1 238

5 PHENOBARBITAL 5-Ethyl-5-phenyl-2,4,6(1H,3H,5H)-pyirimidinetrione 232.2 2 231

6 CAFFEINE 3,7-Didehydro-1,3,7-trimethyl-1H-purine-2,6-dione 194.2 2 219*, 193**

(5α,6α)-7,8-Didehydro-4,5-epoxy-3-methoxy-17-met
7 CODEINE 294.4 1 322*, 293**
hylmorphinan-6-ol monohydrate

8 ACETAMINOPHEN N-(4-Hydroxyphenyl)acetamide 151.2 2 182*, 150**

7-Chloro-1,3-dihydro-1-methyl-5-phenyl-2H-1,4-be
9 DIAZEPAM 284.7 1 339*, 285**
nzodiazepin-2-one

(5α)-6,7,8,14-Tetradehydro-4,5-epoxy-3,6dimethoxy-
10 THEBAINE 311.4 1 312
17-methylmorphina

(5α,6α)-7,8-Didehydro-4,5-epoxy-17-methylmorphin
11 HEROIN 369.4 1 411*, 369**
an-3,6-dioldiacetate
METHAMPHETAMI
12 (αS)-N,α-Dimethylbenzeneethanamine 149.2 2 149
NE
13 CARBAMAZEPINE 5H-Dibenz[b,f]azepine-5-carboxamide 236.3 2 291*237**

(3S)-6,7-Dimethoxy-3-[(5R)-5,6,7,8-tetrahydro-4-me
14 NARCOTINE thoxy-6-methyl-1,3-dioxolo[4,5-g]isoquinoline-5-yl]- 413.4 1 477*, 414**
1(3H)-isoenzofuranone

(5α,6α)-7,8-Didehydro-4,5-epoxy-3-methoxy-17-met
15 MORPHINE 285.3 2 316*, 284**
hylmorphinan-6-ol monohydrate

(αR)-α[(1S)-1-(Methylamino)ethyl]benzenemethanol
16 EPHEDRINE 165.2 1 166
hemihydrate

*Base peak; **Actual molecular mass peak.

TLC-MS interface, the Chromatogram and Mass spectrum was obtained as out-
put which was recorded in observations. From the chromatogram and Mass
Spectrum of all the drugs the output received was their base peak or mass peak
indicating the exact molecular mass of the substance which is recorded in Table
4. The data received was sufficient for confirmation of the identity of the drug as
it shows the exact molecular weight.
Each of the drugs was separated with good resolution using different mobile
phases. The aim of the study of separating the drugs was done using three suita-
ble solvent systems for all the sixteen drugs which would help in rapid screening
of these drugs exhibit received for chemical examination in the forensic science
laboratory for queries pertaining to identification and detection of all these
drugs. These sixteen drugs are the most frequently encountered during investi-

DOI: 10.4236/jasmi.2018.81001 11 Journal of Analytical Sciences, Methods and Instrumentation

K. L. Verma et al.

gation by toxicologists. The solvent system A is found to be most versatile which

could separate eleven of the sixteen drugs which are Methadone, Papaverine,
Cocaine, Ketamine, Caffeine, Codeine, Diazepam, Thebaine, Heroin, Metham-
phetamine, Carbamazepine, Narcotine, Morphine and Ephedrine. Solvent sys-
tem B was found to be best for good resolution for Methamphetamine, Mor-
phine and Ephedrine. Codeine and Methadone also gave good results in context
of the Rf values in solvent system B (0.43, 0.48) as compared to solvent system A
(0.25, 0.15) respectively. Solvent system C was best for separation of acidic drugs
i.e. Phenobarbital and Acetaminophen with Rf 0.63 and 0.68 respectively The
UV spectrum shown by the drugs and the maximum absorbance in the particu-
lar conditions makes it possible to identify each and every drug. The Rf and
λmax for all the drugs in their respective solvents is given in Table 3. In the
present study HPTLC system instrument has been used. Automated spotting,
detection and scanning of the TLC plate makes this technique excellent for rap-
id, efficient and reliable screening of drugs especially for the forensic science la-
boratory where large number of exhibits are to be examined with in a stipulated
period of time to counter the pendency of cases. The last and final aim of the
study was achieved as the mass data is observed which is tabulated in Table 5.
The analysis is done in two modes, positive ion mode i.e. event 1 and negative
ion mode i.e. event 2. In negative ion mode i.e. EVENT 2 with electrospray ioni-
sation (ESI) and atmospheric pressure chemical ionization (APCI) of drugs
showed major ions [M-H]- at m/z 231 for Phenobarbital, m/z 219 and m/z 193
for Caffeine, m/z 182 and m/z 150 for Acetaminophen, m/z 149 for Metham-
phetamine and m/z 316 and 284 for Morphine. Positive ion mode i.e. EVENT 1
with electrospray ionisation (ESI) and atmospheric pressure chemical ionization
(APCI) of drugs showed major ions [M-H]+ at m/z 310 for Methadone, m/z 340
for Papaverine, m/z 326 and 304 for Cocaine, m/z 238 for Ketamine, m/z 322
and 293 for Codeine, m/z 339 and 285 for Diazepam, m/z 312 for Thebaine, m/z
411 and 369 for Heroin, m/z 477 & 413 for Narcotine and 166 for Ephedrine.
Base peak of mass spectrum of drugs Methadone, Papaverine, Ketamine, Phe-
nobarbital, Thebaine, Methamphetamine and Ephedrine showed their actual
molecular mass with +1 or −1 addition to their base peak in terms of m/z. So, all
the drugs standards were analysed for both the events and appropriate event was
selected by the analysing peaks detected and actual molecular mass of the sub-
stance. Rest of the drugs which are Cocaine, Caffeine, Codeine, Acetaminophen,
Diazepam, Heroin, Carbamazepine, Narcotine and Morphine showed multiple
peaks along with the actual molecular mass of the drug.

7. Conclusions
The aims and objectives of the study have been addressed successfully. The
present study has been conducted in view of the drugs which are often encoun-
tered by the toxicologists and forensic chemical examiners during their routine
work. By using the HPTLC-MS technique the sixteen drugs which are Papave-

DOI: 10.4236/jasmi.2018.81001 12 Journal of Analytical Sciences, Methods and Instrumentation

 K. L. Verma et al.

rine, Methadone, Cocaine, Ketamine, Caffein, Codeine, Diazepam, Thebaine,

Heroin, Methamphetamine, Carbamazepine, Morphine, Narcotine and Ephe-
drine are successfully separated with high performance thin layer chromatogra-
phy and identified by their UV absorbance and mass spectrometry. The hyphe-
nated technique of HPTLC-MS is an exemplary technique where in scientist has
the opportunity to play with a number of solvent systems and optimise/validate
his method best suited to the compound in question with in a very short time. It
is a rapid method where spot can be directly lifted from developed TLC plate
and extracted using TLC-MS interface. The whole exercise is completed within
10 to 20 minutes. The compound in question is identified by confirmation of its
molecular weight m/z value. The base peak present other than the actual mole-
cular weight and the variation of ±1 is due to adduct formation of the drug sam-
ple with its solvent. The technique has opened door for scientists to move to-
wards economic and clean technology wherein minimum amount of solvents are
used. When the spot is directly lifted from the plate for its mass analysis, it re-
duces the chances of contamination and cross contamination which is major is-
sue with columns used in GC-MS technology.
The future course of action pertaining to this study will be to explore the pos-
sibility of developing the single common solvent system for all these drugs so
that the challenge before the forensic scientist pertaining to time constraint and
resources is addressed successfully. The drugs and substances other than these
sixteen could also be taken for the same study. The technique could be used for
the quantitative analysis in further course of the studies.

Acknowledgements
The authors gratefully acknowledge Director, Forensic Science Laboratory and
Head of Office Regional Forensic Science Laboratory, Chanakya Puri, Govern-
ment of National Capital Territory of Delhi, India.

Competing Interests
The authors declared that they have no competing interests.

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