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CHAPTER-IV

A HIGH THROUGHPUT ANALYTICAL

METHOD ON GC-NPD/MSD FOR

DETECTION OF STIMULANTS AND

NARCOTICS: THE APPLICABILITY OF

DUAL DETECTOR SYSTEM

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ABSTRACT

The use of stimulants and narcotics by the athletes is prevalent since ancient times and

has undergone improvements in last six decades in terms of addition of more number

of drugs in the groups and the criteria of regulations imposed by International Olympic

Committee (IOC) since 1967 and later by theWorld Anti-Doping Agency (WADA)

since 2004. These drugs are indeedbanned, only “in competition”, as they are effective

over a relatively short period of time andtherefore only if taken immediately priorto

the sportevent, they will cause a performance enhancing effect. Doping control

involves initial screening of suspicious samples for a prohibited drug followed by

more specific confirmatory method. The dope testing has to be accomplished in a

defined time period while fulfilling relevant technical criteria, therefore a simple, high

throughput & open analytical method allowing detection of maximum number of

analytes is choice of every anti-doping laboratory. Traditionally, stimulants and

narcotics were detected using separate injections on GC-NPD and GC-MSD, thereby

requiring two set of data for analysis. With the evolution of technique employing GC

equipped with NPD plus MSD (dual detector), the problem of double injection

requiring data analysis twice has been uprooted. The present work provides a

comprehensive, sensitive and selective GC-NPD/MSD method for the detection of 80

stimulants, narcotics & few other drugs of abuse excreted free in human urine. The

method utilizes the feasibility of combining both the detectors (MS & NPD) with one

GC producing dual data in a single run for fast & more reliable identification. The

sample preparation was performed by liquid-liquid extraction of alkalinised urine. The

limit of detection (LOD) for all substances was between 25-100 ng/ml. The method

has been successfully utilized for the testing of more than 11,400 samples since 2009

till date.

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Introduction

The class of stimulants has a direct stimulating effect on the central nervoussystem

(CNS), because theymimic the adrenaline activity and enhance the cardiac rhythm.

Stimulants represent one of the oldest classes of doping agents and have been usedto

increase performance, endurance, and stamina for centuries. However, in the context

of sport, the word stimulant usually refers to agents stimulating the CNS, affecting

mood, alertness, locomotion and appetite, or targeting the sympathetic nervous system

causing particularly cardiovascular actions& increasing the ability to concentrate. In

addition they may improve the faculty to exercise strenuously or produce a decreased

sensitivity to pain.[1]

The class of stimulants prohibited by the WADAcontains various agents with different

structural features.[2]

Many of these compoundsare derived from phenethylamine or

phenylpropanolamine core structures and represent drugs such as amphetamine,

methamphetamine, methylenedioxymethamphetamine (MDMA). Additional alkaloids

with stimulating properties are cocaine andstrychnine which bear entirely different

structures based on tropane and indolenuclei. Moreover, alkylamines such as

tuaminoheptane or 4-methylhexan-2-amineas well as designer substances such as the

hybrid of amphetamine and piracetamreferred to as carphedone were considered

relevant for doping controls.The first case of doping offence as per modern regulations

was recorded in 18th

century for abuse of cocaine in race walking competition. In late

19th century use of cocaine in this event was frequently mentioned along with new

achievements probably associated with features of cocaine.[3]

Since 19th

century,

stimulants have been a major problem in elite sports and numerous adverse

analyticalfindings (AAFs) has been annually reported by doping control laboratories

worldwide.

The narcotic analgesics, which are banned in sports, are represented by morphine and

itschemical and pharmacological analogues. They are derived from opium, which in

turn isderived from the poppy plant (papawersomnifereum).They act on the central

nervous system (CNS) & surroundings tissues by stimulating opioid receptorsand

reduce feelings of pain.Narcotic analgesics are abused in sports, and therefore the IOC

medical commission issued a ban on their use during the Olympic Games in

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1967.[4]

Due to the properties of narcotics allowing an athlete to compete even after a

musculoskeletal injury with a short & rapid onset of action, they are listed as banned

substances in competition but are permitted out of competition.

The class of stimulants consists of wide variety of compounds closely related to that of

endogenous cathecholamines. Several are phenylalkylamine derivatives with

substituent‟s in different positions: in the amine group (e.g. methamphetamine,

dimethamphetamine), in the phenyl ring (e.g. fenfluramine, methoxyphenamine) and

in (x and ~ carbon atoms of the side chain (methylephedrine, ephedrine [(x-hydroxy-

phenylethylamine]). Since, these are nitrogen-containing compounds, they are basic

(pKa 7-10) and volatilein nature. Structures of selected stimulants are depicted in

figure 4.1.

Figure 4.1: Structures of selected stimulants: ephedrine ( 1 ), methcathinone ( 2 ), strychnine( 3 ),

and cocaine ( 4 )

The narcotics include natural opium alkaloids (eg, morphine, codeine),

semisyntheticanalogs (eg, hydromorphone, oxymorphone,), synthetic compounds

(eg,meperidine, methadone, fentanyl), and the partial agonists (eg,butorphanol,

buprenorphine). Morphine has rigid pentacyclic structure which contains both an

acidic phenolic group and a basic tertiary amine functions. However,since the amine

functions is significantly more basic than phenol group is acidic, thus, morphine as

well as a majority of narcotic analgesicsare functionally basic compounds

bothpharmaceutically (dosage forms) and physiologically. Most of the other natural or

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semi synthetic narcotics are derived by peripheral modifications in morphine

pentacyclic ring system. Substitution of N-methyl moiety of thebaines with methyl

cyclopropyl group produce mixed agonist/antagonist narcotics like buprenorphine.

Structures of selected narcotics are depicted in figure 4.2.

Figure 4.2: Structures of selected narcotics: morphine (1), heroin (2), buprenorphine (3),

pethidine (4), and fentanyl (5)

Stimulants and narcotics in general were among the first analytes to be tested

insystematic doping controls. In the late 1950s, based on chemistry that provided

characteristic and more or less quantitative data by means of color reactions, the

capability of gas chromatography (GC) to separate compounds relevant for doping

controls was recognized and introduced into sports drug testing to measure various

classes of analyte.[7-11]

Analyzers such as flame ionization and nitrogen–phosphorus

detectors (FID and NPD, respectively) as well as ionization β-ray (strontium 90) or

electron capture detectors were used, and sample extraction and concentration

methodologies were mostly adapted from earlier purely „„chemical‟‟ procedures. The

enormous complexity of biologic matrices and the continuously increasing number of

drugs in the prohibited list, however, necessitated more specific and unequivocal

analyzers than forinstance NPD and FID alone.

This resulted in the frequent use of GC equipped with NPD plus mass spectrometry

(MS), a combination that allows the exploitation of advantages provided by both

analytical techniques simultaneously. MS is commonly operated using electron

ionization (EI), which frequently results in comprehensive fragmentation of analytes

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and thus hardly yields information on the molecular weight; however, the obtained EI

mass spectra contain diagnostic ions and provide detailed information that enables the

characterization and identification of target compounds. Moreover, various stimulants

and narcotics have been shown to produce stable molecular ions also under EI

conditions.

The considerable proton affinity of stimulants and narcotics enabled the use of robust

and sensitive instruments composed of liquid chromatography (LC) combined with

(tandem) mass spectrometers (LC-MS/MS) to detect and quantify stimulants and

narcotics in doping controls. [12,13]

The analytes are commonly ionized by means of

electrospray ionization (ESI) or atmospheric pressure chemical ionization that yields a

protonated molecule [M+H]+. Subsequent collision-induced dissociation (CID) of

[M+H] +

gives rise to product ion mass spectra that allow the sensitive and specific

analysis of analyte with the advantages that the intact molecular ion is recorded in

addition to diagnostic product ions and that no derivatization is required even in case

of heavy volatile orthermolabile analytes (eg, phase I or phase II metabolites). The

specificity of ion transitions (i.e., the direct correlation of precursor and product ions)

has been used to establish fast and sensitive detection assays that complement GC-

MS/NPD-based procedures. Though, stimulants and narcotics canbe detected using

LC-MS/MS still in the doping control laboratories, the most popular screening

methods for these drugsare on gas chromatography coupled with mass spectrometer or

NP detector.[13, 15]

This is because most of the stimulants and narcotics are volatile and

contains nitrogen in their structure hence are highly amenable for GC-MSD/NPD

analysis. In addition, the analysis on mass spectrometer is performed in full scan mode

which is a vital tool for retrospective analysis. Additionally, the use of technology in

various doping laboratories is based upon the availability of resources which makes it

a preferred method of choice.

Anti-doping analysis is conducted in two steps. Initially, screening of samples is

performed, in the case of a suspicious result; an additional selective confirmation is

carried out.[16]

As every sample has to be screened, the screening method has to be

highly sensitive and specific to ensure identification of suspected sample and in the

same time should minimize the probability of false suspects. Doping analysis requires

the use of several different chromatographic, mass spectrometric and immunological

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methods [17-19]

which makes it mandatory for all the doping control laboratories to

have a number of separate analytical procedures, thereby making screening of each

sample more complex, time-consuming and laborious. Therefore, the doping control

laboratories always try to have minimum possible number of screening procedure,

without the probability of false reporting.

In the current scenario, the use of stimulants and narcotics by the athletes is regulated

bytheWorld Anti-Doping Agency (WADA): they are indeedbanned, but only “in

competition” [2]

, as the activity ofthese drugs is effective over a relatively short period

of time andtherefore only if taken immediately prior the sportevent, they will cause an

performance enhancing effect. The WADA minimumrequired performance limit

(MRPL) for stimulants and narcotics (Figure 4.3) is indeed not a threshold value, nor

is it a limit ofdetection (LOD) or a limit of quantification (LOQ), but rathera

parameter to assess laboratory performance, and so an adverse analytical finding may

result from concentrationsbelow the MRPL, provided the identification criteria are

satisfied.[5,6]

Figure 4.3: Minimum required performance limit for stimulants and narcotics as per WADA TD

MRPL 2009

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Since the accreditation of National Dope Testing Laboratory (NDTL), India by World

Anti-Doping Agency (WADA) in 2008, the workflow at for screening of stimulants

and narcotics in urine sample comprised of sample preparation using alkaline liquid-

liquid extraction followed by injection on GC-NPD and further re-injection on GC-

MSD in scan mode for a suspicious sample. The analysis on GC-NPD is based only on

retention time but according to the criteria postulated by WADA, the

unequivocalidentification of suspicious substances should be achieved bythe

combination of both retention time and mass spectrometricdata.[6]

This is the reason

suspicious results obtained from GC-NPD were re-injected GC-MSD on data of more

specific method is required prior to proceed for confirmatory analysis. In addition to

this, most of the phenylalkylamines/amphetamine type stimulants (ATS) are close in

their chemical structure and produce overlapping retention times on GC-NPD, which

makes them difficult to differentiate. Hence, additional injection prior to proceeding

for confirmatory analysis has to be performed on GC-MSD for structural confirmation

based on mass spectrum.

During 2009, the laboratory was preparing for the testing of various major events

(XIX Commonwealth Games and I Singapore Youth Games) for which the doping

control tests were to be conducted in NDTL, India. The need was felt to reduce the

number of different screening procedures either by combining existing testing

protocols or by employing fast detection procedures which in turn will reduce turn-

around-time (TAT). In view of this, it was planned to employ the technique of GC-

NPD/MSD which is equipped with micro channel splitter (MCS) based dual detector

system. This would in turn aid in saving time of analysis, man power and would be

cost effective. This dual detector system will produce sensitive and specific detection

of nitrogen containing compounds on NPD and also full scan mass spectrum of all the

samples which may even be used in future retrospectively. The method would prove to

be highly beneficial during major games testing as it will aid in reducing TAT.

Hence, the setting up of the present method was targeted at minimizing the efforts,

time & resources for drug testing by deriving a single method which provides

simultaneous data of two detectors (NPD & MS) in a single run. The aim of present

work was to develop a fast & comprehensive method on GC-NPD/MSD for detection

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of free and volatile stimulants & narcoticsprohibited in sports in addition with few

analgesics, sedatives etc.which are relevant in drug of abuse testing.

Experimental

Chemicals and reagents

All chemicals and reagents wereanalytical or reagent grade. Tertiary butyl methyl

ether (TBME) and potassium hydroxide were purchased from Merck, Mumbai, India.

The certified reference standards of stimulants & narcotics and/or their metabolites

were obtained from established sources like Sigma-Aldrich, USA, National

Measurement Institute, Australia, Cerilliant, USA. Few standards were generously

provided by anti-doping laboratories of Cologne, Italy and Montreal. Water was

purified using a Milli-Q water purification system installed in the laboratory

(Millipore, Bedford, USA).

Sample Preparation

The sample preparation method used for extraction of stimulants and narcotics was the

classical method used in doping analysis for extraction of free & volatile stimulants &

narcotics which is termed as screening procedure-1 in our laboratory.[20]

The method

involved the described steps. To five ml of urine sample, 2 µg/ml of internal standard

(diphenylamine & N-methyl phenol thiazine) was added and samples pH was adjusted

to 14 by 500 µl of 5 normal KOH. Liquid-liquid extraction was performed with 2 ml

of TBME after adding 3 grams (approx.) of sodium sulfate to the samples for salting

out effect. After mixing for 20 minutes on horizontal shaker and centrifugation for 5

minutes at 3000 rpm, the ether layer was separated and directly transferred into

autosampler glass vials for analysis on GC-NPD/MSD. The scheme of sample

preparation is illustrated in the flow diagram in figure 4.4.

Preparation of standard solutions& quality control samples

The stock solutions of all the reference standards were prepared in ethanol at the

concentration of 1.0 mg/ml. For the standards available in salt form, appropriate

corrections factors were applied to convert in free base. Working standard mixtures

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were prepared at two different concentrations of (50 µg/ml &100 µg/ml) foreach

compound by mixing appropriate aliquotsof each stock solution and diluting with

ethanol. Urinary quality control (QC) samples were prepared with every batch at a

concentration levels of 500 ng/ml & 200 ng/ml for stimulants & narcotics respectively.

Thestock solution (1 mg/ml) of diphenylamine (DPA) & 10-N-methylphenothaizine

(NMPZ) was prepared and diluted at 2 µg/ml in ethanol to use as internal standard

(IS). All standard solutionsprepared were stored at 4ºC.

Figure 4.4: Sample extraction procedure for volatile and free stimulants and narcotics from urine

URINE SAMPLE 5ML

ADD 5N-KOH

0.5ML

VORTEX WELL

ADD 50 µL OF I.S. SOLUTION (DPA & NMPZ, 2 µg/ml)

ADD 2ML TBME

ADD APPROXI. 3GM NA2SO4

SHAKE FOR 20 MIN

CENTRIFUGE AT 3000 RPM FOR 5 MIN.

TRANSFER 2 ML TBME DIRECTLY IN

AUTOSAMPLER VIALS

INJECT ON GC-NPD/MSD

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Instrumentation

GC–MS analysis in scan mode was performed on an Agilent 7890A gas

chromatograph equipped with Agilent 7683B automatic liquid sampler and interfaced

to an Agilent 5975C inert mass-selective detector(70 eV, electron impact mode) and

installed with anUltra-2 (5% phenyl–95% methylpolysiloxane bonded

phase;12.5m×0.20mm I.D., 0.33µm film thickness) cross-linked capillarycolumn

(Agilent Technologies, Atlanta, GA, USA). Thetemperatures of injector, interface and

ion source were 280°C,300°C and 230°C respectively. Helium was used as carrier

gasat a flow rate of 1.2 ml/min (at 100°C) in constant pressure mode. Sample (4µl)

was introduced into the inlet in split injection mode (split ratio 5:1) and the column

temperature was set initially at 100°C (0 min) programmed to final 300°C at a rate of

20°C/min (4.5min). The mass range scanned was 40–450 amuat a rate of 1.53

scans/sec.

In the scan mode, at least three characteristicions including molecular ion (if detected)

for each analyte were used for peak-identification (Table 4.1). The electron multiplier

voltage (EMV) was 1750 V after applying a gain factor of 1.5. The mass spectrometer

was kept off for 1 minute for each injection to eliminate contamination due to solvent

and also increasing filament & EMV life. Each peak in the urine samples was

identified bymatching the area ratios of three ions with those of the direct standards.

The NPD was connected to the GC-MS using a dedicated micro channel splitter

(MCS). The device was installed in the oven compartmentand supplied with

continuous helium flow at constantpressure through auxiliary pneumatic control

device (Aux EPC). The column effluent was directly introduced in to MCS where it

was split in to two flows at calculated ratioentering the ion chamber (MS) and the

NPD detector.The NPD detector was operated at 320°C with constant flows of fuel

gas-Hydrogen (3 ml/min), reference gas-air (85 ml/min) and makeup-Helium (5

ml/min). The detector signal offset was kept at 40 for optimum results & enhanced

bead life.

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Method development and validation

The analytical method was developed and validated as per the WADA guidelines for

the anti-doping laboratories.[21]

For validation the parameters specificity, selectivity,

linearity, intra and inter-day precision, recovery, limit of detection (LOD), and

robustness were determined.

Recovery

The recoveries of analytes for which reference standards were available could be

estimated by spiking five replicates (for 3 days) of blank urine with each analyte at a

concentration of 500 & 200ng/ml for stimulants and narcotics respectively. Thepeak

area ratios between analyte and IS of extracted vsan unextracted sample were

calculated. Internal standard were added to the final ether extract in both spiked &

direct samples.

Specificity

Evaluation of specificity was carried out by analyzing six different spiked and six

different blank urine samples collected from healthy volunteers for significant

interfering peaks in the MSD & NPD output data at expected retention times of the

analytes.

Linearity & precision

The linearity of the method was determined by injecting non-extracted standards of

each analyte at five concentrations in the range of 25-1000 ng/ml (25, 50, 100, 500 &

1000) and the correlation coefficient was calculated by extrapolating the concentration

ratio against response ratio. Intra-day precision was determined at MRPL for each

compound using five replicates of spiked urine samples. The corresponding inter-

assay precision was calculated from samples prepared and analyzed at three different

days (n=5/day during 10 days). The precision of the method was determined by

calculation of the relative standard deviations (RSD %) of the mean of ion peak area

ratio of the analytes to internal standard. The precision of retention time was

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calculated as RSD% of relative retention time (RRT )of each analyte of IS for both

NPD & MS data (n=d/day for 3 days).

Limit of detection (LOD)

The LOD was defined as the lowest concentration of analyte that can be reliably

identified, measured with a signal-to-noise ratio (S/N>3) greater than 3. The S/N of

the least abundant diagnostic ion (preferably molecular ion) was calculated using ten

blank samples and ten fortified samples at concentration levels from 25 to 250 ng/ml.

Applicability to excretion study samples/routine doping control samples

A total of 11,400 doping control samples received in National Dope Testing

Laboratory (NDTL), India from 2008 to 2012 were analyzed by the developed method

for stimulants & narcotics, including samples of mega events vizI Youth Olympic

Games (2010), XIX Commonwealth Games (2010) & I Asian beach games (2011).

The method was also applied to excretion study samples after oral administration of

seligiline, pseudoephedrine, nicotine and tramadol to human volunteers. The study

was duly approved by the ethics committee of NDTL, India. The results of excretion

studies of these drugs are presented separately in the next chapters.

Results

Method development

The method allowed detection of more than 80 compounds of different chemistries

(stimulants and narcotics) listed by WADA and several other drugs of abuse like

sedatives, anti-histamines and analgesics. The physical and chemical properties of few

stimulants and narcotics tested by this method are enlisted in table 4.1. The method

was validated for all analytes except 22 compounds for which reference material was

not available; however these substances could be identified using respective positive

control samples. The short column allowed separation of most of the analytes in a run

of 14.5 minutes. The use of structural analogue as an internal standard is mandatory

only for quantitative confirmatory analysis [21]

; however its use in the developed

method was to monitor the extraction reproducibility. Hence, DPA and NMPZ were

the best suitable candidates. In the sample extraction procedure, the addition of

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anhydrous sodium sulphate increased the extraction into the organic phase of many of

the analytes.Due to the extraction at a high pH level the chromatographic biological

background was very low thus easing the correlation of data of both the detectors.

Table-4.1: Physical, chemical & pharmacological properties of few stimulants & narcotics

detected on GC-NPD/MSD

S.No Substance Formulae MW Human

metabolism

Target

compound

Pharmacologi

cal class

Chemic

al class

STIMULANTS

1. 3,4-

Methylenedioxyamphetamine (MDA)

C10H13NO2 179.22 Hepatic parent Monoaminergic, entactogenic

phenyl

ethylamine

2. 4-Methylamphetamine

C10H15N 149.23 Hepatic parent anorectic phenyl ethylam

ine

3. Amfepramone

C13H19NO 205.30 Hepatic parent Monoaminergic phenyl

ethylam

ine

/cathino

ne

4. Amphetamine

C9H13N 135.20 Hepatic,

deamination

dealkylationdem

ethylation

parent Monoaminergic phenyl

ethylam

ine

5. Benfluorex

C19H20F3NO2 351.36 Hepatic parent Anorectic/hypolipiodemic

fenflura

mine

6. Benzphetamine

C17H21N 239.35 Hepatic parent Monoaminergic/anorectic

phenyl

ethylam

ine

7. Benzylpiperazine

C11H16N2 176.25 Hepatic parent Monoaminergic Piperazi

ne

derivati

ve

8. Cathine

C9H13NO 151.20 Hepatic parent Monoaminergic phenylp

ropanol

amine

9. Clobenzorex

C16H18ClN 259.78 Hepatic

Parent

Amphetamine

Monoaminergic phenyl

ethylamine

10.

Dimethylamphetamine

C11H17N 163.25 Hepatic parent Monoaminergic phenyl ethylam

ine

11. Ephedrine

C10H15NO 165.23 Hepatic parent

Monoaminergic phenylp

ropanol

amine

12. Ethylamfetamine

C11H17N 163.25 Hepatic parent

norethylamph

etamine

Monoaminergic phenyl

ethylam

ine

13. Famprofazone

C24H31N 377.52 Hepatic

parent

Methampheta

mine

Analgesic phenyl

ethylam

ine-

pyrazol

one

derivati

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ve

14. Fencamfamine

C15H21N 215.33 Hepatic parent

Monoaminergic phenyl

ethylam

ine

15. Fencamine C20H28N6O2 384.48 Hepatic amphetamine Monoaminergic phenyl

ethylam

ine

16. Fenethylline

C18H23N5O2 341.40 Hepatic

Amphetamine Monoaminergic phenyl

ethylam

ine

17. Fenfluramine

C12H16F3N 231.26 Hepatic

Dealkylation

Norfenflurami

ne

Monoaminergic phenyl

ethylamine

18. Fenproporex

C12H16N2 188.26 Hepatic Converted to

amphetamine

Parent (5-9%) Amphetamine

(30 to 60%)

Monoaminergic phenyl ethylam

ine

19. Furfenorex

C15H19NO 229.31 Hepatic Methampheta

mine

Monoaminergic phenyl

ethylam

ine

20. Heptaminol C8H19NO 145.24 Hepatic Parent Monoaminergic alkanola

mine

21. Isometheptene

C9H19N 141.25 Hepatic Parent Monoaminergic alkylam

ine

22. MDMA

C11H15NO2 193.25 Hepatic,

O-

demethylenation

methylationgluc

uronide/sulfate

conjugation;

N-dealkylation,

Deamination,

Oxidation

Unchanged

(65%)

3,4-

methylenedio

xyamphetami

ne (7%),

Monoaminergic phenyl

ethylam

ine

23. Mefenorex C12H18ClN 211.73 Hepatic Amphetamine Monoaminergic phenyl

ethylam

ine

24. Mephentermine C11H17N 163.25 Hepatic Unchanged Monoaminergic phenyl

ethylamine

25. Methamphetamine C10H15N 149.23 Hepatic

Unchanged

(30-54%)

Amphetamine

(10-23%)

4-

hydroxymetha

mphetamine

4-

hydroxyamph

etamine

Monoaminergic phenyl

ethylam

ine

26. Nikethamide C10H14N2O 178.23 Hepatic Unchanged Resp. stim. Pyridine

carboxa

mide

27. Methylephedrine C11H17NO 179.26 Hepatic Unchanged Monoaminergic phenylp

ropanol

amine

28. Norfenfluramine C10H12F3N 203.20 Hepatic Unchanged Monoaminergic phenyl

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ethylam

ine

29. Ortetamine/ 2-

methylamphetamine

C10H15N 149.23 Hepatic Unchanged Monoaminergic phenyl

ethylam

ine

30. Pentylenetetrazol C6H10N4 138.17 Hepatic Unchanged Resp. stim./GABA

Azepine

derivati

ve

31. Phendimetrazine C12H17NO 191.27 Hepatic

Renal

phenmetrazin

e (~30% )

Monoaminergic Morpho

line

derivati

ve

32. Phenmetrazine C11H15NO 177.24 Hepatic

Unchanged

(19%)

Monoaminergic phenyl

ethylam

ine-

morpholine

derivati

ve

33. Phentermine C10H15N 149.23 Hepatic

Renal

Monoaminergic phenyl

ethylam

ine

34. Prenylamine C24H27N 329.48 Hepatic Renal

Amphetamine

Several others

phenyl

ethylam

ine

35. Cropropamide Hepatic Unchanged Resp. stim. Alkyla

mine

derivati

ve

36. Crotethamide Hepatic Unchanged Resp. stim. Alkyla

mine

derivati

ve

37. Prolintane C15H23N 217.35 Hepatic Renal

OH-

prolintane

conjugated

Monoaminergic Pyrrolid

ine

amine

38. Propylhexedrine C10H21N 155.29 Hepatic Unchanged Monoaminergic cycloalk

ylamine

39. Pseudoephedrine C10H15NO 165.23 Hepatic

(10–30%)

Renal (43–

96% )

Monoaminergic phenylp

ropanolamine

40. Selegiline C13H17N 187.28 Hepatic

Desmethylsel

egiline

l-

amphetamine

l-

methampheta

mine

MAOI

(monoamine oxidase inhibitor)

phenyl

ethylam

ine

41. Strychnine C21H22N2O

2

334.41 Hepatic

Unchanged

(10-20%)

Glycine antagonist

Terpene

indole

alkaloid

NARCOTICS

42. Codeine

C18H21NO3 299.36 Hepatic

Demethylation

Glucuronidation

Renal

Morphine

conjugated

Nor codeine

Narcotic analgesic/antitussive

morphin

e

alkaloid

43. Dextromoramide

C25H32N2O2 392.53 Oral Renal narcotic analgesic

Propion

anilide

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derivati

ve

44. Fentanyl

C22H28N2O 336.47 Hepatic

Unchanged

(60%)

<10%

Nor fentanyl

Narcotic analgesic

phenylp

iperadin

e

45. Methadone C21H27NO 309.44 Hepatic

Unchanged

Nor

methadone

EDDP

Narcotic analgesic

Propion

anilide

46. Oxycodone C18H21NO4 315.36 Hepatic

19%

unchanged

α and β

oxycodol, Oxymorphon

e

α and β

Oxymorphol

Noroxymorp

hone,

Noroxycodon

Narcotic analgesic

benzylis

oquinoli

ne

47. Pentazocine C19H27NO 285.42

Hepatic

Renal Narcotic analgesic

benzmo

rphans

48. Pethidine C15H21NO2 247.33 Hepatic

HydrolysisDemet

hylationGlucuro

nide Conjugation

Renal

Pethidinic

acid

Norpethidine

Narcotic analgesic

Phenylp

iperadin

e

49. Dextropropoxyphene C22H29NO2 339.4 Hepatic Renal

norpropoxyp

hene

Narcotic analgesic

Propion

anilide

The NPD remains one of the most sensitive detectors for stimulants even more

sensitive than MS in several cases; whilst the MSD in full scan mode provided the

structural information and differentiation of the analytes. Henceforth, the utility of

combining NPD & MS interfaced with GC seems to be promising. With the

advancements in pneumatic control & fast electronics, it became possible to use dual

detectors with GC with a single column using splitter. And so, the method described in

this chapter offered simultaneous detection of target & non-target analytes by RT

correlation of NPD & MS chromatographic peaks & instant full scan mass spectral

information.

Many stimulants are excreted in urine in the form of one or more metabolite apart

from parent. For examples, metabolites of few drugs like normethadone & EDDP

(metabolites of methadone), N-desmethylselegiline (metabolite of seleigiline),

norpethidine (pethidine metabolite), cotinine (nicotine by product), etc are excreted

unconjugated in human urine. The method could be successfully validated & applied

-116-

for detection of these metabolites. The method was validated and utilized in routine

after comparing with old method by parallel analysis for two months.

Optimization of GC-NPD/MSD parameters

Most of the CNS stimulants are derived from the basic phenylalkyamine structure.

Modifications involved are substitution at alkyl chain (e.g. Amfepramone by oxidation

of alkyl chain & bis substituted methylation of amino function; ephedrines by

hydroxylation of methylene moiety), amino functional group (e.g. N-ethyl

amphetamine after mono substituted ethylation of NH2), and rarely at aryl moiety (e.g.

fenfluramine by trifluoro methylation of aryl ring and methylation of amino group),

though backbone structure remains untouched in most derivatives.

Consequently, many analogues show an identical fragmentation pattern resulting in

similar base peak in positive electron impact (+EI) mass spectra. For instance

methamphetamine & phentermine showed identical base peak at m/z 58 (Figure-4.5),

likewise amphetamine & heptaminol had a base peak of m/z 44 (Figure 4.6). Chemical

modification in such cases may improve the quality & information of mass spectra,

however it does not eliminate the limitation of similar fragmentation pathways.

Nevertheless, these substances could be identified by RT based separation and

considering other more significant but less abundant ions. The molecular ion of

underivatized stimulants & narcotics are not always prominent in +EI ionization due

to very excessive fragmentation of molecular radical cation (M+·). However, most of

the phenylalkylamine & alkylamine stimulants generate molecular ion at detectable

abundance providing more reliability in MS analysis.

Many stimulants being volatile were co-eluted in the initial segment of the

chromatographicrun. It created problems of missing the retention time whenever

installing a new column and also consistency of retention times. To overcome this

drawback, identification was achieved using retention time locking and minor

diagnostic& differentiating ions of co-eluting molecules.As an example, figure 4.7

shows the ion chromatogram (m/z 72) forethyl amphetamine and fenfluramine which

are co-elutingand the ion chromatograms of the respective minor ionsm/z 159 and 220

which are separated at concentrations equal to500 ng/ml.

-117-

The RT reproducibility was determined for both NPD & MS peaks by calculating

relative retention times (RRT) to the IS (DPA). The RT & diagnostic ions (m/z)

along with molar mass & molecular ion are provided in table 4.2. For purpose of

identification, the relative ion intensities, RT & RRT of all compounds were compared

to a quality control sample fortified with authenticated standards at MRPL level in

accordance to TD2010IDCR. Although all substances successfully passed the criteria,

it was difficult to have diagnostic ions of relative intensity of >10% in early eluting

amphetamine type stimulants (ATS) due to structural factors. Most of these analytes

had a base peak of m/z 44 or m/z 58, besides other ions having abundances less than

5%. The mass ions with relative intensities below 10% are generally produced of

molecular radical cation or further loss of alkyl or hydroxyl moiety. Such ions are

fragmented instantly & intensively under high ionization energies and so the

intensities of intact ions are relatively uncertain & less reproducible.

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 3400

20000

40000

60000

80000

100000

120000

140000

160000

180000

200000

220000

240000

m /z -->

A bundance

Scan 155 (1.990 min): 112.D\ data.ms (-150) (-)58.0

91.0

134.1207.0184.2 266.9159.0 355.1

Methamphetamine

m/z 148

-118-

Figure 4.5: Methamphetamine and phentermine showing identical base peak but different

diagnostic ions

4 0 4 5 5 0 5 5 6 0 6 5 7 0 7 5 8 0 8 5 9 0 9 5 1 0 0 1 0 5 1 1 0 1 1 5 1 2 0 1 2 5 1 3 0 1 3 5 1 4 00

2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0

1 0 0 0 0

1 2 0 0 0

1 4 0 0 0

1 6 0 0 0

1 8 0 0 0

2 0 0 0 0

2 2 0 0 0

2 4 0 0 0

2 6 0 0 0

2 8 0 0 0

3 0 0 0 0

3 2 0 0 0

3 4 0 0 0

3 6 0 0 0

3 8 0 0 0

4 0 0 0 0

4 2 0 0 0

4 4 0 0 0

4 6 0 0 0

4 8 0 0 0

5 0 0 0 0

5 2 0 0 0

m / z - - >

A b u n d a n c e

S c a n 1 5 2 ( 1 . 8 1 1 m i n ) : 0 2 1 . D \ d a t a . m s ( - 1 3 9 ) ( - )5 8 . 0

9 1 . 1

4 2 . 0 1 3 4 . 11 1 7 . 1

7 7 . 05 0 . 0 6 6 . 0 1 0 4 . 0

4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 3 4 0 3 6 00

2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0

1 0 0 0 0

1 2 0 0 0

1 4 0 0 0

1 6 0 0 0

1 8 0 0 0

2 0 0 0 0

m / z - - >

A b u n d a n c e

Sc an 106 (1.705 min): 009.D \ data.ms (-101) (-)4 4 . 0

9 1 . 0

1 2 0 . 13 5 5 . 02 6 6 . 92 0 7 . 0

Phentermine

Amphetamine

m/z 134

-119-

Figure 4.6: Amphetamine and Heptaminol showing identical base peak but different diagnostic

ions

4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0

0

1 0 0 0

2 0 0 0

3 0 0 0

4 0 0 0

5 0 0 0

6 0 0 0

7 0 0 0

8 0 0 0

9 0 0 0

m / z - - >

A b u n d a n c e

S c a n 7 3 ( 1 . 5 1 2 m i n ) : 0 0 2 . D \ d a t a . m s ( - 7 1 ) ( - )

4 4 . 0

6 9 . 0 1 1 3 . 09 5 . 0

2 0 7 . 91 3 0 . 9 1 7 7 . 01 4 8 . 8 2 6 8 . 0

4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0

0

1 0 0 0

2 0 0 0

3 0 0 0

4 0 0 0

5 0 0 0

6 0 0 0

7 0 0 0

8 0 0 0

9 0 0 0

m / z - - >

A b u n d a n c e

# 2 1 1 4 2 : 2 - H e p t a n o l , 6 - a m i n o - 2 - m e t h y l -

4 4 . 0

6 9 . 01 1 3 . 09 5 . 0

4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0 2 6 0 2 8 00

1 0 0 0 0

2 0 0 0 0

3 0 0 0 0

4 0 0 0 0

5 0 0 0 0

6 0 0 0 0

7 0 0 0 0

8 0 0 0 0

9 0 0 0 0

1 0 0 0 0 0

1 1 0 0 0 0

1 2 0 0 0 0

1 3 0 0 0 0

m / z - - >

A b u n d a n c e

S c a n 2 0 3 (2 .2 7 0 m in ): 0 1 5 .D \ d a ta .m s (-1 9 9 ) (-)

7 2 . 1

4 4 . 09 1 . 0

1 1 7 . 0 1 4 8 . 12 8 0 . 82 0 8 . 0

Heptaminol

N-ethyl amphetamine

m/z 162

-120-

Figure 4.7: Co-eluting compounds ethyl amphetamine and fenfluramine showing common base

ion (m/z 72) and different diagnostic minor ions (m/z 159 and 220)

Table 4.2: Retention time (RT), base peak, molecular ion and other fragments of compounds

analyzed by GC-NPD/MSD

S.No. DRUG RT

(min)

CHARACTERSITIC MASS IONS (m/z)

Base Peak Mol. Ion Other Fragments

1. Diphenylamine(IS) 4.55 169 169 170

2. NMPZ(IS) 7.02 213 213 198

3. Acetophenone 1.9 109 151 43, 80, 53, 108

4. Amitryptylline 7.83 58 277 59, 30, 275, 217

5. Amphetamine 1.66 44 135 120,134

6. Benfluorex 1.72 105 350 192,159,149,216

7. Benzphetamine 5.91 91 239 65,56148

8. 1-Benzylpiperazine 3.78 91 176 134,176

9. Brompheneramine 7.36 247 * 248, 167, 180, 58, 194, 318

10. Bupropion 4.47 44 239 111,100,139

11. Caffeine 5.89 194 194 109,165

12. Cathine 2.76 44 151 77,132

13. Chlobenzorex 6.6 125 259 125,91,168

4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 3 4 00

5 0 0 0 0

1 0 0 0 0 0

1 5 0 0 0 0

2 0 0 0 0 0

2 5 0 0 0 0

3 0 0 0 0 0

3 5 0 0 0 0

4 0 0 0 0 0

4 5 0 0 0 0

5 0 0 0 0 0

m /z -->

A b u n d a n c e

Scan 202 (2.264 min): 018.D\ data.ms (-197) (-)7 2 .1

4 4 .0

1 5 9 .0

1 0 9 .0 2 1 6 .11 3 2 .9 1 8 7 .0 3 4 1 .0

Fenfluramine

-121-

14. Chlorpheneramine 7.0 203 274 58, 205, 168, 42, 167

15. Codeine 8.81 299 299 162,229,282,214

16. Cotinine 5.1 98 176 118, 119, 147, 99

17. Cropropamide 5.23 100 240 115,168,195

18. Crotethamide 4.94 86 226 69,154,181

19. Cyclobenzaprine 8.03 58 * 215, 202, 189, 275

20. 3,3 diphenpropylamine 5.69 194 211 165,116,179,152,211

21. Diazepam 9.14 256 284 283, 255, 221, 165

22. Diclofenac 7.6 214 277 242, 179, 178, 151

23. Desmethylsellegiline 2.9 82 * 91, 115, 172

24. Dextromoramide 11.0 100 * 56, 128, 265

25. Diethyl Propion 3.85 100 205 77,115,56

26. Dimethyl amphetamine 2.27 72 163 ,73,148,133

27. Diphenhydramine 6.08 58 * 165, 152, 227, 167, 255

28. Dextyromethorphen 7.55 59 271 150, 171, 214, 256

29. Ephedrine 3.07 58 165 105,117,132

30. EDDP Perchlorate 7.03 276 277 220, 262, 278, 56

31. Ethyl Amphetamine 2.21 72 163 162,148,103

32. Fluoxetine 6.14 44 * 104, 91, 78, 148

33. Fancamfamine 4.95 215 215 98,215,186

34. Fenetylline 10.57 250 341 250,70,181

35. Fenfluramine 2.25 72 231 109,44,159

36. Fenproporex 4.48 97 188 56,132,187

37. Fentanyl 10.11 245 336 245,189,146

38. Furfenorex 4.93 81 229 138,53

39. Heptaminol 1.6 44 145 113,128,59

40. Hydroxy cotinine 5.5 106 192 135, 119, 93, 78

41. Isometheptene 1.21 58 141 95,126,84,71

42. Ibuprofen 4.4 161 206 163, 119, 118, 164

43. Lamotrigene 9.06 185 255 187, 157, 114, 87

44. Lidocaine 6.28 86 234 58, 87, 56, 77

45. Mefenorex 4.36 120 211 120,122,84,196

46. Meperidine/Pethidine 5.39 71 247 172,247,218

47. Methamphetamine 1.92 58 149 91,134,

48. Mephentermine 2.34 72 163 148,117

49. MDA 3.69 44 179 77,105,179,136

50. MDMA 4.02 58 193 135,77,105

51. Methadone 7.54 72 309 294,165,309

-122-

*No molecular ion observed in the spectra

Nevertheless, a screening method is meant for preliminary identification & isolation of

suspicious samples for confirmatory analysis; the preliminary identification of

substances indeed refers to comparison of retention time relative to IS & mass spectral

fragments of more significance rather than abundance.

52. Methoxyphenamine 3.1 58 179 121,178,164

53. Methyl Ephedrine 3.33 72 179 77,105,

54. Nicotine 2.8 84 * 133, 161

55. Nikethamide 4.07 106 178 177,78,149

56. Nor nicotine 3.3 119 148 70, 147, 105, 120

57. Neonicotine 3.7 84 162 105, 133, 162

58. Norfluoxetine 6.03 30 * 134, 103, 191, 91

59. Nortryptylline 7.92 44 263 202, 203, 204, 191

60. Norfenfluramine 1.72 44 203 109,184,159

61. Ortetamine 2.22 44 149 105,115,148

62. Oxycodone 9.4 315 315 315,230,258

63. Paroxitine 9.5 44 329 192, 70, 41, 109

64. P-Methyl Amphetamine 2.17 44 149 105,134,117

65. Pentetrazole 4.28 55 138 82,138,41

66. Pentazocine 8.17 217 285 217,284,270

67. Phentermine 1.82 58 149 91,134,117

68. Phendimetrazine 3.63 57 191 70,191,85,191

69. Phenpromethamine 2.01 44 * 77,91,105,128

70. Pipradol 7.6 84 267 105,248,182

71. Prenylamine 9.44 58 * 238,167,152,91,115

72. Prolintane 4.6 126 217 174,91,70

73. Propylhexedrine 1.83 58 155 140,155

74. Propoxyphene 7.75 58 339 208,115

75. Pseudoephedrine 3.33 58 * 105,117,132

76. Ketamine 6.04 180 * 209, 152, 166, 194, 237

77. Selegiline 3.37 96 187 56,91

78. Strychnine 12.81 334 334 334,319,162

79. Tramadol 6.52 58 * 263, 135

80. N-desmethyl tramadol 6.7 44 * 188, 249

81. O-desmethyltramadol 6.9 58 249 46, 59, 55, 121

82. Tryptamine 5.2 130 160 131, 103, 51

-123-

The GC parameters were optimized to detect maximum possible substances of wide

molecular weights & volatility which were suitable for GC analysis and relevant in

drug of abuse analysis. The method was found capable of detecting highly volatile &

low molecular weight compounds (amphetamine &isomethepthne) on one hand; and

less volatile or higher molecular weight substances (strychnine) on the other hand. All

compounds were identified within 14.5 minutes of GC elution with solvent delay of

1.0 minute. The column was injected with 4 µl of sample volume through split liner at

the split ratio of 5:1 at 280°C to avoid saturation of line (leading to overloading of

column) and ensure vaporization of all the analytes of interest.

Usually a injection volume of 2 µl and inlet split ratio of 11:1 have been used in most

of the GC-MS methods employed in doing control. However, the relatively large

amount of injection volume & low split ratio have been set in the present method; so

that detectable amount of analyte molecules could reach to the two detectors used after

splitting through a micro channel splitter to consequently get the desired detection

levels. Moreover, the injection volume & split ratio were established by verifying the

liner type & the solvent vapor pressure to eliminate saturation of liner with solvent

which in turn could lead to loss of sample in injector. Good chromatographic

resolution was achieved for most of the compounds.The initial column temperature

was optimized at 100°C so as to avoid ghost peak of organic solvent & related volatile

impurities and also to retain &detect the low molar mass substances. A linear

increment of column temperature (100°C to 300°C @20°C) facilitated separation of

analytes of different volatility & molar mass in the column. The final column

temperature was held for 4.5 minutes to avoid retention of non-volatile or active

species on to the column.

The analysis of stimulants & other volatile substances on GC-MS can be more vital &

informative, if MS is operated in scan mode because it allows the identification of

many other related unknown species in addition to target substances. As the MRPLs of

stimulants & narcotic drugs are higher than other classes of substances [9]

, significant

detection levels were achieved while operating the mass spectrometer in full scan

mode in this method.

-124-

Method validation

Precision

Repeatability of retention time is mandatory & first step of method validation for both

qualitative & quantitative analysis. Précised RT exhibits the robustness of the

analytical instrument operated under certain parameters over a period of time.

Analysis of five replicates of QC samples for three days yielded stable retention times

(CV < 2%) for all of the compounds except amphetamine, p-methyl amphetamine &

isomethepthne which were eluted near to solvent front (within 0.7 minutes post

solvent delay of 1.0 minutes) and accounted with CV% in the range of 2.0 to 2.2. The

sample purification procedure involved in the measurement has a large impact of

stability of RT depending upon yield of extracts free of matrix. In the present method

no significant matrix effect on RT was observed because of very high pH used during

extraction which eliminated most of the endogenous amine interferences.

The method precision was estimated on QC samples spiked in negative urines of

different pH (5.5-8.5) and specific gravity (1.004-1.032 g/ml). The intermediate

precisions (intra-and inter-day) showed coefficients of variation less than 15% for all

analytes. The method was found to be repeatable with CV of < 10% over the entire

range of substances (Table 4.3).

Limit of detection (LOD)

The LOD of different compounds in the developed method is listed in table 4.3. All

stimulants were detected at concentrations far below WADA MRPL as well as LOD‟s

for narcotics were found to be at or below 50% of WADA MRPL with signal to noise

ratio above 3 using two diagnostic ions. The method allowed detection of many

analytes which are not prohibited at levels below 250 ng/ml.

Linearity

The linearity was evaluated for stimulants & narcotics from 25-1000 ng/ml (25, 50,

100, 500 & 1000). The correlation coefficients (R2) rangingfrom 0.986 to 0.999

showed the method linearity for all analytes over the specified concentrations.

-125-

Recovery (%)

The recovery percentage for all the analytes was found to be between 69-109%, (Table

4.3). The recoveries were sufficient to reliably identify the analytes at or below the

levels prescribed by WADA.

Specificity

An analytical method without any significant interference at retention times of

analytes of interest as well as absence of ions coming from interferences or

background is proposed as specific. No interferences were observed at the retention

time of analytes of IS in all the blank urines analyzed.

Table 4.3: Method validation results showing recovery percentage, precision and LOD of the

compounds analyzed by GC-NPD/MSD method

S.

No.

Compound WADA

MRPL

(ng/ml)

LOD

(ng/ml)

Recovery

(%)

RRT-

precision

(RSD%)

(n=5)

Inter-day

Precision

(RSD%)

(n=5X3)

Intra-day

precision

(RSD%)

(n=5)

1. Diphenylamine (ISTD) NA* NA - 1.1 6.2 3.2

2. NMPZ(ISTD) NA NA - 1.3 5.5 1.2

3. Amphetamine 500 50 91 2.1 8.8 5.1

4. Benfluorex 500 100 76 1.9 10.2 6.3

5. Benzphetamine. 500 50 98.8 1.8 5.4 4

6. 1-Benzylpiperazine 500 50 95.8 1.7 6.9 4.6

7. Bupropion 500 50 89.5 1 3.8 1.2

8. Caffeine 500 50 82 1.9 11.2 8.3

9. Cathine 500 100 94 1.5 2.4 1.1

10. Chlobenzorex 500 50 97 0.9 5.9 4.1

11. Codeine NA 50 88 1.0 8.1 7.6

12. Cotinine 50 20 92.5 1.1 7.6 4.9

13. Cropropamide 500 50 103 1.6 9.2 7.8

14. Crotethamide 500 50 97.3 1.6 10.2 5.6

15. 3,3 diphenpropylamine 500 50 98.5 1.2 8.6 6.4

-126-

16. Desmethylsellegiline 500 50 79 1.4 11.6 8.9

17. Dextromoramide 200 50 101 1.7 10.8 6.7

18. Diethylpropion 500 50 97.5 1 5.5 3.6

19. Dimethyl amphetamine 500 50 96.5 1.1 7.4 4.9

20. Ephedrine 500 100 99.5 1.5 3.6 2.2

21. EDDP Perchlorate 200 50 69 0.7 12.1 8.7

22. Ethyl Amphetamine 500 50 97.6 1.9 6.5 5.5

23. Fancamfamine 500 50 99.2 1.8 8.0 5.9

24. Fenetylline 500 100 89 1.9 4.7 3.1

25. Fenfluramine 500 50 91.6 1.8 8.3 6.9

26. Fenproporex 500 50 102 0.6 6.7 5.3

27. Fentanyl 10 50 88.8 0.9 10.0 8.8

28. Furfenorex 500 50 98.3 1.2 4.9 3.7

29. Heptaminol 500 100 94.6 1.6 11.1 8.4

30. Isometheptene 500 100 97.6 2.2 11.5 9.2

31. Mefenorex 500 50 107 1.9 6.1 4.8

32. Meperidine/Pethidine 200 50 87 1.1 6.6 3.8

33. Methamphetamine 500 50 84.8 1.9 9.7 6.6

34. Mephentermine 500 50 109 1.7 10.3 7.7

35. MDA 500 50 105.1 0.9 7.6 4.1

36. MDMA 500 50 104.7 0.8 8.2 6.4

37. Methadone 200 50 92.2 1.4 6.4 4.5

38. Methoxyphenamine 500 50 88.2 1.3 9.4 5.9

39. Methyl Ephedrine 500 100 102 1.2 3.1 2.5

40. Nicotine 50 20 88.9 1.2 7.2 5.1

41. Nikethamide 500 50 89.8 1.1 4.8 3.3

42. Nor nicotine 50 20 82.6 1.8 7.6 6.9

43. Norfenfluramine 500 50 95.8 2.1 10.9 7.1

44. Norfentanyl 200 100 88 0.8 11.8 9.4

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45. Ortetamine 500 50 81.2 2.0 11.6 6.8

46. Oxycodone 200 100 102.8 1.3 6.9 5.8

47. P-Methyl Amphetamine 500 50 92.6 2.1 10.7 8.4

48. Pentetrazole 500 50 79 1.5 8.8 4.1

49. Pentazocine 200 100 80.8 0.9 6.9 4.8

50. Phentermine 500 50 89.3 1.8 9.9 5

51. Phendimetrazine 500 50 74.2 1.3 7.2 5.6

52. Phenpromethamine 500 50 77.8 1.9 9.7 8

53. Pipradol 500 50 90.8 1.3 10.2 6.9

54. Prenylamine 500 50 93 1.6 11.1 9.9

55. Prolintane 500 50 100 1.1 7.6 6

56. Propylhexedrine 500 50 100 1.8 10.4 8.3

57. Propoxyphene NA 100 93.3 1.1 10.9 9.7

58. Pseudoephedrine 500 100 100.1 0.9 2.9 1.8

59. Selegiline 500 50 94.3 1.5 7.2 5.6

60. Strychnine 200 50 87.5 1.2 5.5 4.9

*NA: not applicable (indicates substances which are either not prohibited or included in WADA

monitoring program-2012; hence don’t have MRPL)

Applicability to routine analysis

The method was successfully applied to the analysis of 11,400in-competition routine

sample received in NDTL from 2008 to 2012.A total of 867 (7.6 %) adverse analytical

findings (AAFs) for various drugs of abuse were reported during the period(Figure

4.8). Out of the total adverse analytical findings, 18.6 % of AAFs were accounted for

stimulants & narcotics (Figure 4.9) detected by this method. The breakup of 7 major

analytes reported as AAF using this method is illustrated in figure 4.10.

-128-

Figure 4.8: Total number of in-competition samples tested and AAFs reported at NDTL,India

from 2008-2012

Figure 4.9: Year wise distribution of AAFs for stimulants & narcotics reported using GC-

NPD/MSD method in NDTL, India (2008-2012)

Total no. of IC samples-11400

AAFs- 867

Total no. of in-competition (IC) samples and total AAFs at NDTL india from 2008-2012

0.00

5.00

10.00

15.00

20.00

25.00

30.00

2008 2009 2010 2011 2012

10.81 % 7.87 %

26.98 %

21.36 %

14.55 %

Per

cen

t A

AF

Year

Stimulants & narcotics AAF % yearwise (2008-2012)

Average AAF% in 5 years= 18.6 %

-129-

Figure 4.10: Drug wise distribution of AAFs for stimulants in NDTL (2008-2012)

Discussion

Socially, stimulants & narcotics are referred to as substances of abuse rather than

substances of therapeutic importance. Stimulants & narcotics are banned in allsports

because they can produce alertness & analgesia, respectively. Both categories include

drugs with relatively short onset of action hence beneficial if ingested just prior to

event. As a result, both the classes (S6: stimulants & S7: narcotics) are forbidden in

sports only during competition.[21]

Stimulants are further classified in two section viz. „specified & non-specified‟ in

WADA prohibited list. Stimulants which are susceptible to unintentional doping

because of their presence in over the counter medicines, herbal/dietary supplements &

nutraceuticals or less likely to be abused as doping agents are termed as specified

stimulants. On the other hand, stimulants with potential of abuse but no therapeutic

use are regarded as non-specified. The sanctions for anti-doping rule violation may be

reduced if an athlete could establish that he has consumed a prohibited specified

stimulant inadvertently. Moreover, there are many specified stimulants which could

metabolize to non-specified substances and vice versa.

0

10

20

30

40

50

60

70

80

9088

55

6 6 4 1 1

54.7

34.2

3.7 3.7 2.5 0.6 0.6

Distribution of stimulants AAF reported using the proposed method

No. of AAF

% of total stimulants AAF

-130-

Consequently, several analytical challenges are imposed to correlate a parent

identified in a sample with its metabolic products as same metabolite could result from

one or more parent entities. It becomes more difficult in such cases where parent is

completely metabolized and only metabolites are excreted in urine. Apart from

pharmacological factors, various physical & chemical challenges like wider

chemistries, pKa, polarity & structural specificities limits use of a universal method

for detection of all stimulants & narcotics. For the detection of stimulants & narcotics

in urine in sports doping, minimum required performance levels (MRPL) of 500

ng/ml& 200 ng/ml have been fixed by WADA for accredited laboratories.[5]

Even

though the metabolismand elimination properties vary extensively and resultin

different urinary levels between the classes of stimulants & narcotics, the MRPLs are

sufficient to detect their abuse by athletes. As both the classes of drugs are abused just

before competition, lower dosages are less likely to produce the ergogenic effect;

hence a sample showing presence of a stimulant or narcotic at levels below 10 % of

MRPL (50 ng/ml & 20 ng/ml, respective) shall not be declared positive.[5]

Stimulants and narcotics are the oldest class of substances prohibited in sports. Both

the categories have been available through natural origin which has facilitated their

social & ergogenic abuse since ancient time. Due to this omnipresent knowledge of

potential of these substances, they were the first on the banned list. Several analytical

techniques have been proposed for the detection of doping agents; primarily among

them are Immunoassay, HPLC-UV-DAD, GC-NPD, GC-MS, andLC-MS/MS.[8-15]

Immediately after introduction of stimulants & narcotics as forbidden substances in

sports, attempts were made to invent a systemic detection method; the capability of

chromatography to separate components was utilized to detect sympathomimetic

agents on GC.[10, 11]

The detectors like FID, NPD or electron capture were used with

GC. Later during 1970s, inclusion of more number of substances as well as complex

interferences due to endogenous amines necessitated more selective & universal

analysers. Consequently, mass spectrometer interfaced with GC emerged with larger

role in doping control. Further, combination of GC with NPD & MS allowed

utilization of capacities of both the analyzers, simultaneously.[14, 15]

Although,

LCMS/MS based methods are now available for sensitive & trace level detection of

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polar, non-volatile & heat labile substances [13-15]

, the role of GC-NPD/MS remains

indispensible for comprehensive & sensitive analysis of volatile stimulants, narcotics

& other drugs of abuse. The GC methods promises fast and selective detection of non-

polar substances without any chemical modification and provide relatively robust &

repeatable data in terms of matrix interferences & detector background noise.

The mass spectrometer is normally used in electron impact (+) ionization in doping

control. The +EI mass spectra result from intensive fragmentation and thus provide

structural information through characteristic ions formed. Hence, GC-MS is still

considered as the method of choice for confirmatory analysis of volatile molecules,

specially stimulants & narcotics. The GC-NPD remained as a sole method for

screening identification of volatile and non-conjugated stimulants & narcotics for

many decades because of its simplicity at both sample preparation as well as

instrument operation. However, the combination of GC-NPD with mass spectrometry

provided confidence in screening analysis as it was possible to analyze the NPD peak

& its mass spectrum, simultaneously. The greatest advantage being exclusion of

suspicious samples due to interference of endogenous matrix during preliminary

analysis, hence less number of samples for confirmatory analysis. This resulted in

more comprehensive & cost effective analytical method for detection of volatile

substances.

The present method has been successfully used in screening & confirmation of various

drugs of abuse including stimulants & narcotics on GC-NPD/MSD. The current

method is capable of analysing 1 sample in 14.5 min. for 80 analytes against the two

separate run (each 15 min.) on the traditional GC-NPD & GC-MS method. The

method was developed on high end GC equipped with advanced electronic control

modules for fast temperature ramps & oven cooling, high capillary flows & signal

processing. The mass spectrometer was equipped with inert ionization source to

facilitate effective ionization of analytes minimizing noise ions coming from active

surfaces of ion chamber even at elevated temperatures. The triple axis mass detector

(TAD) was used to collect the amplified ions and convert them to analogue signal. The

TAD due to its structural physics & controlled voltages ensured capturing maximum

electrons coming from multiplier thus enhancing the sensitivity of analysis. This has

significantly improved the throughput where simultaneous analysis could be

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performed on NPD & MS data in comparison to two separate outputs in traditional

methods.

The separate injections on GC-NPD & GC-MS require splitting of sample extract,

availability of two equipment, additional human resource for instrument handling and

data analysis; which limits the feasibility of using these methods for preliminary

analysis during major events testing or even day to day analysis. Moreover, two

separate instrument & need of additional man power again limits the use in terms of

cost effectiveness, which in turn must be one of the most important points to a routine

analytical laboratory. The method developed has proven as high throughput &

comprehensive during the testing of major events viz. I Singapore Youth Olympic

Games and XIX Commonwealth games where a turnaround time of 24 hours was

required. The method has been found to be simple, robust and reliable with easy

operation & low maintenance. Since then, it has been used in the laboratory for in-

competition testing for more than 5 years and over 11,400 urine samples have been

analysed. The urine extracts are clear & without significant interferences or

contaminants, offering low maintenance & higher durability of consumables.

Conclusion

A rapid, comprehensive and sensitive method was developed utilizing dual detector

technology for the analysis of 80 stimulants &narcotics restricted for use in sports.

The experiments were carried out under standard mass spectrometric conditions for

+EI analysis on GC-NPD/MS. The method was validated according to the

International Standard for Laboratories [21]

as per World Anti-Doping Agency

enforcements. The analytical procedure enabled detection and identification of many

drugs and their metabolites, including most of the stimulants, 6-adrenergic agents and

narcotics (methadone, pentazocine and pethidine). In addition it is possible to detect

other nitrogen-containing drugs such as anti-histaminics, benzodiazepines, tricyclic

antidepressants and local anesthetics. The method was found to be simple, robust and

reliable with easy operation & low maintenance.

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