“rapid screening in doping analysis: separation and...
TRANSCRIPT
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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
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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.
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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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