Trends in Analytical Chemistry, Vol. 29, No.

3, 2010

Trends

Analytical procedures for

determination of cocaine and its
metabolites in biological samples
Monika Janicka, Agata Kot-Wasik, Jacek Namiesnik
The dangerous effects and increased usage of illicit drugs have inspired the
development of analytical methodologies. We based our review of analytical
procedures for determination of cocaine (COC) and its metabolites on literature from the past two decades. Our main aim was to compare the capabilities of different analytical techniques. The diversity of matrices from
which COC is determined (e.g., blood, hair, saliva, plasma, urine, inner
organs and meconium) has led to differences in sample preparation. To assess
drug exposure, it is essential to select the sample type and the instrumental
technique applied. We discuss appropriate sampling, extraction and purification techniques.
2010 Published by Elsevier Ltd.
Keywords: Analytical technique; Biological sample; Blood; Cocaine; Extraction; Hair;
Metabolite; Saliva; Sample preparation; Urine

1. Introduction
Monika Janicka*,
Agata Kot-Wasik,
Jacek Namiesnik,
Gdansk University of
Technology, Chemical Faculty,
Department of Analytical
Chemistry, ul. G. Narutowicza
11/12, 80-233 Gdansk, Poland

Corresponding author;
E-mail: mjanicka@hotmail.com

Cocaine (COC), an alkaloid of Erythroxylum coca plant, was used in ancient

civilizations for religious and ceremonial
reasons. After Spanish conquest of
South America, coca leaves were used
as a stimulant for native workers in
silver mines. Great change in the usage
of COC occurred in 1855 when a young
German chemist, Friedrich Gaedcke,
isolated it. It was used as a first anesthetic [1].
Combination with ethanol (cocaethylene, COE) gave it reinforcing potential, so
COC was a popular ingredient in wines
(e.g., Vin Mariani). In the USA in 1885,
John Pemberton developed Coca-Cola as
non-alcoholic drink in response to prohibition one glass of Coca-Cola contained
9 mg of COC. In 1903, the Coca-Cola
drink containing COC was removed from
the market [2]. Harrison Narcotic Tax in
1914 restricted the distribution, sale and
use of COC, although these practices were
still legal for registered companies and
individuals [3].

0165-9936/$ - see front matter 2010 Published by Elsevier Ltd. doi:10.1016/j.trac.2009.12.005

Two metabolic pathways of COC can be

distinguished: hydrolysis (non-enzymatic
or enzymatic with esterases) and oxidation
with oxidases. Hydrolysis occurs in blood
and tissues while oxidation occurs mainly
in liver. The first metabolic pathway
products are ecgonine methyl ester (EME)
and benzoylecgonine (BZE). Ecgonine
(ECG) is a product of non-enzymatic
hydrolysis of EME and/or enzymatic
hydrolysis of BZE. The oxidative pathway
leads to N-demethylation of COC, where
the main product is norcocaine (NOR) [4].
COC, in unchanged form, and its metabolites are excreted into urine.
The consequences of COC usage can
combine short-term and long-term effects.
Health effects of short-term use of COC are:
increased level of alertness; temperature
and euphoria; rapid breathing and rapid
pulse rate; irregular breathing; and,
irregular heart rhythm. Long term, COC
usage can cause cardiovascular complications (e.g., myocarditis, hypertension
and aortic dissection), loss of appetite,
restlessness, insomnia and many other
problems [5]. The toxicity of COC was
established to be greater than its metabolites. In cases of overdose of COC, administration of some specific compounds speed
up its change to less toxic metabolites. It is
necessary to measure the level of the drug
and its metabolites to achieve satisfactory
results in using COC [6].
The dangerous effects of this drug on
living organisms and current problems
associated with its wide use and availability have led to the development of
analytical procedures. Because of short
half-life of COC, its detection as a drug of
abuse is usually based on determination of

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H
N

H
N

CH3

CH3
O

O
O

NOR

COE

O
EtOH
H 3C
N

CH3
O
O

COC
O
H 3C

H 3C

CH3

OH
O
O
OH

BZE
EME

H 3C
N

O
OH
OH

ECG

Figure 1. Principal metabolites observed in cocaine metabolism.

its two principal metabolites, BZE and EME, or of its final

degradation products, ECG and NOR. Fig. 1 shows the
structures of metabolites of COC. Table 1 shows their
main characteristics.
Nowadays, there are various analytical techniques
applied for the drugs of abuse control in biological
samples. COC can be determined individually or simultaneously with other drugs in a broad range of matrices.
For identification and quantification of these drugs,
forensic-science analysis requires methodologies characterized by wide application and high specificity. Because of the difficulties in describing exposure (past or
current) to COC, various biological specimens have been
used for examination, the most common being blood,
urine, hair, perspiration, meconium, saliva, nails, teeth,
amniotic fluid and inner organs. Courts rely on results
from toxicological analyses and decide whether an
individual was under the influence of drugs during the
particular event (e.g., rape, robbery, or traffic and
workplace accidents [7]), so identification of the drug
and its metabolites in fluid or tissues should be objective
and reliable.

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In this article, we review analytical protocols proposed

within the past 10 years for verification of COC and its
metabolites in biological samples.

2. Sample preparation
In any analytical protocol for analysis of organic compounds, sample preparation is a critical step, which is
necessary in order to obtain reliable, accurate data [8].
The main aim of sample preparation is to separate and to
preconcentrate the target compound and, if possible, to
purify the extract. Apart from these main purposes, we
discuss other vital factors [e.g., cost effectiveness, high
sample throughput, enhanced analyte detectability and
identification potential, otherwise stated as speed, sensitivity, and selectivity (robustness)].
2.1. Problems and challenges
Before applying the analytical technique for qualitative
and quantitative measurement of drugs, it is important
to develop proper sample-preparation procedures.

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Table 1. Structures of cocaine and its major metabolites with their basic chemical characteristics
Chemical structure

Formula

H3C
N

Characteristic

C17H21NO4

molecular mass: 303.353 g/mol

solubility in water - 1800 mg/mL (20C)
stimulant of a central nervous system
stimulation lasts from 20 min to several hours
CAS Number [50-36-2]

C16H19NO4

molecular mass: 289.33 g/mol

primary metabolite of cocaine
detection period in trace amounts 24 days (chronic use up to 3 weeks)
CAS Number [519-09-5]

C10H17NO3

- molecular mass: 199.25 g/mol

- CAS Number [7143-09-1]

C9H15NO3

C16H19NO4

- molecular mass: 289.33 g/mol

- metabolite of the tropane alkaloid cocaine
- CAS Number [18717-72-1]

C18H23NO4

CH3
O
O

COCAINE
H3C
N

O
OH
O

BENZOYLECGONINE
H3C
N

CH3
O
OH

ECGONINE METHYL ESTER

H3C
N

O
OH

molecular mass: 185.22 g/mol

tropane alkaloid found naturally in coca leaves
density - 1.293 0.06 g/cm3
it is both metabolite and precursor of cocaine
CAS Number [481-37-8]

OH

ECGONINE
H
N

CH3
O
O

NORCOCAINE
H
N

CH3
O

molecular mass: 317.38 g/mol

formed in a body when cocaine and ethanol have been taken simultaneously
more euphoric stimulation
lasts a long time in the body
CAS Number [529-38-4]

COCAETHYLENE

Biological material is characterized by complicated

matrix composition, very low concentration of analytes
(e.g., pg/mg of hair, and ng/mL of blood and urine),
high possibility of interferences and extremely limited
access to reference materials. Such problems can be

overcome by selecting appropriate sample-preparation

procedures.
Matrix interferences can cause analytical problems,
resulting in analytical signal suppression or enhancement, or false-positive results coming from co-extraction

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(due to existence of substances having similar physicochemical properties). Matrix effects can be observed
when mass spectrometry (MS) is applied with both ionization types, but is more prevalent with electrospray
ionization (ESI) than atmospheric pressure chemical
ionization (APCI). ESI is clearly affected by components
with a wide polarity range, making this type of ionization highly susceptible to matrix effects. APCI proves less
susceptible to matrix effects than ESI, especially in the
presence of hydrophobic interferences. Suppression is
often observed in the beginning in chromatograms from
urine, oral fluids or plasma, probably due to the different
ionization processes, with APCI based on gas-phase
reactions and ESI mainly based on liquid-phase reactions.
Large differences in matrix effects are also observed
between sample-preparation techniques. In general,
acetonitrile protein precipitation in combination with
liquid chromatography with ESI and tandem MS (LCESI-MS2) has the greatest matrix effect, with major
suppression areas at the beginning and the end of the
chromatographic separation [9,10].
The presence of matrix effects also proves to be
dependent on the bio-fluid analyzed. Matrix components,
characteristic of each bio-fluid, can interfere at different
times. The major interferences in urine prove to be
hydrophilic residual components, most likely inorganic
salts. Oral fluid has more interferences than urine and
residual matrix components are of a hydrophilic and
hydrophobic nature, including proteins, amino acids,
and especially mucin. Finally, for plasma, residual
matrix components also have a wide polarity range, but
their concentrations are higher than those of oral fluid.
Physico-chemical properties of COC and its metabolites
(e.g., the non-polar character of COC or the hydrophilicity of BZE) require different approaches at the isolation
stage.
Careful optimization of sample-preparation procedures
is essential and crucial to obtain low limits of detection
(LODs) and reliable qualitative and quantitative data.
2.2. Sampling, storage and transport of biological
materials
The very first steps of any analytical procedure are
sampling, storage and transport of material. Defects in
sampling procedures can increase the time and the cost
of analysis and can also cause equivocal identification
and quantification. One of the main principles of analytical chemistry is the requirement for collection of
representative samples, which is especially difficult to
achieve when human material has to be analyzed. In
drug analysis, rules for sampling of biological material
are complicated, so sampling can be the weakest link in
the whole analytical procedure.
Taking into account the pharmacokinetic distribution
and the excretion of psychoactive substances from an
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organism, the most valuable material is urine because of

its wide window of detection. The problem of identifying if an individual was under the influence of a drug
at the site of an event can be solved by taking samples of
urine, on site and/or after a short time. Moreover, some
illicit drugs (just like COC itself) are metabolized too
rapidly to be measured in urine in routine screening.
When urine is screened for the presence of COC, it is
metabolite BZE that is measured. BZE has a biological
half-life of 58 h, which is much longer than that of COC
(0.51.5 h). The generally accepted time for BZE to be
cleared from urine is 35 days.
The length of time following drug use for which a
positive result may occur depends upon several factors,
including frequency of use and amount of drug, metabolic rate, excretion rate, drug half-life, and the drug
users age, weight, activity and diet.
There are several biological specimens that can be
obtained in non-invasive way saliva, hair and nails.
That kind of sample collection does not require special
facilities and close supervision of private functions of the
subject to eliminate adulteration. Research in 16 European countries showed that the most suitable matrix for
determining drugs is saliva, due to its availability, the
good correlation of results and low invasiveness. Compared to urine, drugs are found in saliva in parent form.
Saliva has a similar detection window to blood, the
advantage being strict affirmation of drug presence at
the time of examination. Unfortunately, economic circumstances limit wide use of toxicological analysis of
saliva at the sites of events, and that extends the time of
screening tests [11]. Also, another difficulty is that a cutoff level has not been precisely defined for analytical
methods in general use.
Body fluids (e.g., blood and urine) are usually collected
for laboratory examination. In plasma, the concentration of toxic drug is directly proportional to the individuals clinical state. When only blood is secured, its
complicated matrix composition can interrupt or even
make impossible toxicological assay. Also, screening
tests for plasma are relatively expensive. Collection of
only urine reduces the time of screening but lengthens
the detection window for drugs of abuse, making it
hard to define if the person examined was under the
influence of the drug at the moment of an event. It is
important to protect several specimens, from which
drugs can be determined, due to the need for precise
confirmation of whether the drugs abuse is occasional or
has a long history. An example of a procedure for sampling and analysis of saliva, urine and blood from living
people, and for sampling and analysis of urine, blood and
biological tissues from deceased is presented on Fig. 2.
One of the crucial points in non-conventional matrices
is the possibility to extend the time window of detection
from hours or days, as in blood and urine, to weeks or
months, as in nails and hair. In the assessing past

Living people

Substances similar-acting to alcohol

Screening analysis
in toxicological laboratory

End of examination, signing the

protocol
(negative screening result)

Sampling and analysis of blood, urine

and/or inner organs
(immunochemical methods)

Substances similar-acting to alcohol

On-site screening analysis

Saliva analysis
(immunochemical test)

YES
NO
Negative screening result

End of examination,
signing the protocol
(negative screening result)

Impossibility or refuse
to making examination, positive screening
result of saliva

NO
Analysis ofYES
blood, urine and/or
biological material
(immunochemical methods)
(GC-MS, LC-MS)

Confirmatory analysis
in toxicological laboratory

YES
Screening/confirmatory analysis
in toxicological laboratory

NO
End of examination and drawing up
a report
(negative result)

Confirmation of positive
result (qualitative
and quantitative analysis)

Sampling and analysis

of blood and/or urine
(ELISA, FPIA)

YES
End of examination
and drawing up a report
(negative screening result)

Negative screening result

YES
End of examination and drawing up
a report
(positive result)

Trends in Analytical Chemistry, Vol. 29, No. 3, 2010

Deceased

NO
Analysis
of blood and/or urine
(GC-MS, LC-MS)

Confirmatory analysis
in toxicological laboratory

NO

http://www.elsevier.com/locate/trac

End of examination
and drawing up a report
(negative result)

Confirmation of positive
result (qualitative
and quantitative analysis)

YES
End of examination and drawing up
a report
(positive result)

Figure 2. Algorithm of procedure for sampling and analysis of saliva, urine and blood from living people, and for sampling and analysis of urine, blood and biological tissues
from the deceased.

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Table 2. Estimates for detecting cocaine and/or its metabolites in saliva, blood, urine and hair
Matrix

Time for the detection

Urine

Up to 3 days

Saliva
Blood
Hair

Up to 10 h
Up to 10 h
Months

Comments

Ref.

The analysis of urine is most common, inexpensive method of testing for the presence of cocaine in
the human body. Urine testing is mostly preferred compared to other types of tests, mainly because it
is considered to be the most inexpensive and non-intrusive, and can be done at home.
This method is a short-term procedure, which can detect drugs that have been used recently.
This is an expensive test for detecting cocaine in the human body.
Hair testing is one of the effective ways of testing for cocaine content in the human body over a long
period of time. Whenever any substance enters the human body, it deposits a residue in the hair
shaft, which will show its presence.

[20]

chronic exposure to drugs of abuse, hair from both living

and dead humans is rightly considered the matrix of
choice. Recently, milk teeth have been proposed as a
matrix to measure drug content in monitoring cumulative exposure [12].
Table 2 sets out the estimated possibility of detection of
COC in different biological specimens as a function of
time, showing that hair samples are suitable for detecting illicit drugs in cases of long-term drug consumption
[13].
Many authors have been observed COC degrading to
BZE in standard solutions and urine samples [14].
Immediate freezing prevents degradation of COC in
urine samples and standard solutions. When the
investigated material can be stored only at +4C,
satisfactory COC stability for 90 days is ensured by
acidifying urine samples to pH = 5. Storage of urine at
pH = 5 and at +25C for longer than 60 days creates
a risk of significant degradation of the COC present in
the sample.
2.3. Extraction techniques
The diversity of samples of complex matrices, from which
COC is to be determined, leads to problems with sample
preparation. Nowadays, drugs are determined in various
specimens (e.g., meconium, amniotic fluid, teeth, sweat,
urine, saliva, hair, inner organs, blood and plasma).
Urine, hair and saliva are generally used for screening
assays. These specimens can contain proteins, lipids,
phospholipids, various enzymes, carbohydrates and
electrolytes, which interfere with determination of the
analytes of interest. The type of sample is one of a few
factors to take into account in choosing an analytical
technique.
Prior to final analysis, isolation and/or enrichment of
the biological specimens is usually needed. Use of proper
extraction and purification procedures maximizes
recovery of the analytes and eliminates negative effects
of impurities.
Biological material taken for illicit drug examination
can be liquid (e.g., urine, saliva, blood, plasma, or sweat)
or solid (e.g., hair, teeth, or fingernails).
Only screening methods applied for urine and saliva
samples require little or no sample preparation [15,16].

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[16]
[25]
[27]

Direct measurement is the biggest advantage of these

methods. Nevertheless, such simple, rapid analyses suffer from high LODs, which lead to false-negative results
or possible false-positive results caused by interferences.
In liquid samples, a preconcentration step is usually
made by liquid-liquid extraction (LLE) or solid-phase
extraction (SPE). Liquid-liquid extraction shows limited
extraction efficiency of very different analytes (polar,
basic and acid, relatively non-polar). Typically, the high
extraction efficiency of COC, NOR and COE at basic pH
and very low extraction efficiency of BZE and EME are
observed with organic solvents (several dichloromethane/isopropanol mixtures, or butyl acetate), the opposite
being true at acidic pH [18].
SPE is the most common technique for liquid-sample
preparation, due to the possibility of applying a wide
range of cartridges, which can separate analytes with
different properties, from acidic to basic. In most cases,
mixed-mode SPE cartridges are applied for COC and its
metabolites. Mixed mode (i.e. C18, C8 or C4 with cationexchange-functionalized silica) reduces matrix effects,
which cause ion suppression. In comparison with reversed-phase SPE cartridges, mixed mode is stable over a
wide range of pH, and gives high-purity drug extracts
and a better signal-to-noise ratio [1724].
Typically, an SPE procedure involves a number of
steps [e.g., conditioning of a sorbent, sample loading,
and washing (in which the analytes are retained on a
cartridge and interferences are washed out, or analytes
are eluted and interferences are retained)]. The conditions of each step must be carefully optimized for interactions between matrix, sorbent and analyte. Factors to
be optimized include: type of sorbent; solvent strength;
pH; ionic strength; solvent volume; and, flow rates.
Elution of interferences is selective removal of undesirable substances from the sorbent. Appropriate choices of
solvent and pH prevent analyte loss. SPE can separate
analytes from all kinds of biological samples.
To separate particulate matter from blood [18,25,26]
or urine [18], centrifugation is necessary, after which
the supernatant is transferred onto an SPE column.
Tissues have to be homogenized and centrifuged with
small amounts of solvent (e.g., NaF to prevent COC

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Table 3. Standard procedures for sample preparation for different specimens adopted according to the techniques of final determination
Biological specimen

Sample preparation

Adopted analytical technique

Ref.

Teeth

Pulverization of teeth, incubation with internal standard(IS) in 0.1 M HCl at

37C for 18 h, pH 6, extraction with chloroform/ isopropanol(9:1), centrifuging,
organic layer evaporation, derivatization with BSTFA/ 1%TMCS
Mixing with IS and methanol, centrifuging, organic layer evaporation to
dryness, dissolution in phosphate buffer (pH 6.0), extraction by Bond Elute
Certify SPE column, evaporation and dissolution in 1% acetic acid
Washing twice with solution of Tween 80, rinsing twice with distilled water,
enzymatic hydrolysis with solution of Pronase and dithiotreitol in Tris-HCl
buffer (pH 7.2), mixing with borax buffer (pH 8.5) and sodium chloride, SPME
(PDMS fiber) dipped in the mixture for 25 min, desorption time 5 min
Washing with methanol, incubation overnight in 0.1 M HCl at 45C, SPE
Washing with methylene chloride, sonication in phosphate buffer (pH 2.7) at
75C for 3 h, SPE
Decontamination by 5 min agitation in gas-tight vessel, washing with
petroleum benzene followed by methanol, add IS (MPPH), extraction for 4 h by
ultrasonication at 50C in methanol, spiked with IS mixture (COC-d3, BZE-d3),
evaporation and dissolution in acetonitrile and ammonium-acetate buffer
Washing with three solvents, extraction by ultrasonication with methanol for
2 h, evaporation and redissolving in phosphate buffer (pH 6.0), mixing mode
SPE cartridges and automated SPE device (Rapid Trace), evaporation and
dissolution in mobile phase
Washing with methylene chloride, aliquot incubated in 1 ml of methanol for
16 h at 40C, evaporation to dryness in the presence of 100 ll of MeOH/HCl
(99:1, v/v), evaporation in presence of deuterated analogs, redissolution of dry
extract in phosphate buffer (pH 8.4), extraction with methylene chloride/
isopropanol/n-heptane (50:17:33, v/v/v), agitated (95 cycles/min) and
centrifuged (3000 rpm), purification of organic layer by an additional acid
extraction (2 mL, 0.2 M HCl), neutralization by 1 M NaOH, addition of
phosphate buffer (pH 8.4), reextraction of aqueous layer with methylene
chloride, agitation, centrifugation, evaporation of organic layer to dryness,
silylation of drugs with BSTFA-TMCS at 70C for 20 min
Washing for 5 min with 5 ml of de-ionized water, 5 ml of petroleum benzene,
and 5 ml of dichloromethane, cutting hair into small pieces, ultrasonication in
methanol for 5 h at 50C, addition of deuterated standard, evaporation to
dryness, reconstitution in phosphate buffer (pH 6.0), SPE, derivatization of
extract with MSTFA, pyridine, isooctane for 15 min at 90C
Washing with isopropanol at 37C for 15 min, washing twice with 0.01 M
phosphate buffer with 0.1% BSA (pH 6) at 37C for 30 min, washing twice with
0.01 M phosphate buffer with 0.1% BSA (pH 6) at 37C for 60 min, enzymatic
digestion
Washing with 4 ml of methanol for 5 min, centrifugation for 5 min (2000 rpm),
filtration, cutting into 1-mm fragments, incubation with IS solution and 0.1 M
HCl, for 18 h at 45C, cooling, addition of phosphate buffer (pH 6.0, 0.1 M) and
2 M KOH, vortexing for 1 min, centrifuging for 5 min at 2000 rpm, SPE (Varian,
BondElut C2 cartridges), evaporation, redissolution with mobile phase
Incubation overnight in 0.25 M HCl at 45C, neutralization, liquid-liquid
extraction (Toxi Tubes A, Analytical Systems), evaporation, reconstitution with
mobile phase
Washing with aqueous solution 0.3% Tween 20 (2 20 ml), cutting into small
fragments, incubation overnight in 0.25 M HCl at 45C, neutralization with
NaOH, extraction into organic phase with Toxi-Tubes A, evaporation,
redissolution with mobile phase
Washing with aqueous solution 0.3% Tween 20 (2 20 ml), cutting into small
fragments, incubation overnight in 0.25 M HCl at 45C, neutralization with
NaOH, extraction into organic phase with Toxi-Tubes A, evaporation,
redissolution with bidistilled water
Washing with aqueous solution 0.3% Tween 20 (2 20 ml), rinsing with water,
drying at 37C, cutting into small fragments, incubation overnight in 0.12 M
HCl at 45C, neutralization with NaOH, extraction into organic phase with
Toxi-Tubes A, evaporation, redissolution with 0.05 M phosphate buffer (pH 5.0)

GC-MS

[12]

HPLC-MS

[22]

GC-MS

[33]

GC-MS
HPLC

[16]
[19]

HPLC-MS2

[43]

HPLC-MS2

[53]

GC-MS

[27]

GC-MS

[32]

HPLC-MS2

[45]

HPLC-FL

[50]

HPLC-ECAD

[55]

CZE-UV

[28]

CZE-MS

[29,30]

HPLC-FL CZE-UV

[39]

Meconium

Hair

(continued on next page)

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Table 3. (continued)
Biological specimen

Sample preparation

Adopted analytical technique

Ref.

Urine

Mixing with phosphate buffer (pH 6.0) SPE (Bond Elut Certify columns), eluate
evaporation, derivatization with BSTFA/1%TMS
SPE(LiChrolut TSC columns)
Filtration, mixing to pooled sample, cleaning on 96- well plate
Mixing with IS and Sorensen phosphate buffer (pH 7.4), SPE (Oasis MCX SPE
cartridges), evaporation at 50C, reconstitution in MeOH/H2O (5:95, v/v)
Mixing with IS, SPME (PDMS fiber) + derivatization reagent
buthylchloroformate, desorption time 15 min
Mixing sample with phosphate buffer (0.1 M, pH 6), methanolic IS solution
(200 ng/ml of butorphanol, MDMPA and 2-methylcocaine) and IS, SPE (Varian,
Bond Elut Certify), evaporation at 35C in the presence of hydrochloric acid
dissolved in methanol, dissolution in chromatographic solvent
Heavy atom and/or surfactants spotted on solid substrates, solutions spotted on
solid substrates, drying of solid substrates under infrared lamp, samples placed
in desiccators with CaSO4 chips
Mixing with sodium acetate buffer (pH 4.0), SPE (Clean Screen ZSDAU020
extraction column), derivatization with BSTFA/TMCS
Mixing with borax buffer (pH 9.0) and sodium chloride, SPME fiber (PDMS)
dipped in the mixture for 25 min, fiber placed in a injection port for 5 min
Mixing with water, HCl, centrifuging, supernatant transfer to SPE column,
evaporation to dryness and reconstitution in mobile phase
SPE (Bond Elut Certify column and Vac-Elut manifold), evaporation,
reconstitution in methylene chloride-2-propane, evaporation, one part mixed
with mobile phase for HPLC analysis, second part mixed with benzene,
pyridine and p-fluorococaine (for derivatization of EME)
SPE (BondElut Certify cartridge), evaporation at 65C, reconstitution with
mobile phase
Samples collected to tubes with sodium fluoride, stored in ice for 1530 min,
centrifuged for 5 min at 1315 rpm, alkalization with 0.1 M NaCO3, 0.1 M
NaHCO3 and carbonate buffer (pH 10.7), vortexing with lidocaine (IS), addition
of carbonate buffer (pH 10.7) and vortexing, addition of hexane and shaking on
an oscillating shaker for 3 min, centrifugation for 3 min at 1200 rpm, hexane
poured into a disposable borosilicate test tube, evaporation, reconstitution with
mobile phase
Samples drawn into heparinized syringes, centrifuged for 10 min at 3000 rpm,
plasma samples spiked with COC and BZE, centrifuged for 5 min at 3000 rpm
Blood collected to tubes with IS solution, addition of 0.64 M NaF, vortex-mixed
and centrifuged, SPE
Dialysate samples collected into glass vials (Chromacol Ltd., UK) containing 10
lL 0.03% acetic acid for an additional 240 min

GC-MS

[17]

HPLC-DAD
HPLC-MS2
UPLC-MS2

[20]
[42]
[24]

GC-MS

[35]

HPLC-TOF

[51]

RTP

[16]

GC-MS

[21]

GC-MS

[34]

HPLC-MS2

[18]

HPLC-UV

[46]

HPLC-DAD

[47]

HPLC-UV

[48]

HPLC-DAD

[26]

HPLC-MS2

[25]

HPLC-MS2

[44]

Saliva

Sweat
Blood, plasma

Brain dialysates

hydrolysis), then supernatant can be extracted with SPE

[25].
SPE is generally selected in sample-preparation procedures because of its simplicity, rapidity and high
selectivity. SPE provides the sample clean up, recovery,
and concentration necessary for accurate qualitative and
quantitative analysis.
Solid samples (e.g., teeth) need to be pulverized and
dissolved in HCl before analysis. In this case, LLE has
been proposed for isolation of analytes [12].
Hair is a complicated matrix. Drugs are typically extracted from it in two ways:
 digestion of the hair matrix and extraction of analytes
from the digest with LLE or SPE; or,
 extraction from the intact hair matrix.
The most popular extraction techniques in hair analysis are LLE [2731], SPE [15,19,32], solid-phase micro-

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extraction (SPME) [3335] and ultrasound extraction

[15,32]. SPME is mainly used when GC-MS is applied,
due to the simple sample-preparation procedure, without
need for derivatization [34,35]. Several authors have
described the fundamentals of hair extraction [36].
Table 3 shows examples of extraction procedures applied for different types of biological samples, as presented most recently by different authors working in the
field of drug analysis.

3. Identification and quantitative determination

A variety of analytical techniques is involved at the final
step, which enables identification and quantitative
determination of drugs of abuse. They can be divided in
two groups: screening and confirmatory techniques.

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Table 4. Limit of detection (LOD) values for methods of determination for cocaine from different biological specimens
Ref.

Specimen

Screening

Cut-off

Confirmation

[16]
[32]
[27]
[54]

Saliva
Hair
Hair
Urine

RTP
ELISA
ELISA
FPIA

0.1 ng/mg
0.5 ng/mg COC
50 ng/mL

[38]

Meconium

FPIA

[15]

EMIT

[35]
[34]
[21]
[33]

Hair
Urine
Saliva
Plasma
Sweat
Hair

0.1 ng/mg BZE

0.15 mg/L BZE
-

GC-MS
GC-MS
GC-MS
GC-MS

[12]
[23]

Teeth
Hair

GC-MS
GC-CI-MS2

[20]
[25]
[45]
[43]

Urine
Blood, amniotic fluid, placental and fetal tissues
Hair
Hair

HPTLC
-

HPLC-UV
HPLC-MS2
HPLC-MS2
HPLC-MS2

[44]
[18]

Brain dialysates
Blood
Urine

0.15 mg/L

HPLC-MS2
HPLC-MS2

[19]
[42]
[24]

Hair
Urine
Urine

HPLC-MS2
HPLC-MS2
UPLC-MS2

[22]

Meconium

0.061 lg/mL COC

0.058 lg/mL BZE
-

[51]
[55]
[46]
[48]
[47]
[26]

Oral fluid
Hair
Plasma
Canine serum
Plasma
Plasma

HPLC-TOF
HPLC-ECAD
HPLC-UV
HPLC-UV
HPLC-DAD
HPLC-DAD

[50]
[28]
[29]
[30]
[39]

Hair
Hair
Hair
Hair
Hair

RIA

0.1 ng/mg
0.1 ng/mg
0.1 ng/mg

HPLC-FL
CZE-DAD
CZE-MS
CZE-MS
HPLC-FL
CZE

Screening analyses commonly use either thin layer

chromatography (TLC) or immunochemical techniques
[e.g., enzyme-multiplied immunoassay technique (EMIT),
radioimmunoassay (RIA) and fluorescence-polarization
immunoassay (FPIA)] on various specimens (e.g., urine,
saliva or hair). Techniques that are more specific [e.g.,
gas chromatography with MS (GC-MS), LC with MS
(HPLC-MS), HPLC with UV (HPLC-UV) and high-performance TLC (HP-TLC)] are used for confirmation of positive results from immunological techniques.

GC-MS
GC-MS
CE-immunoassay
GC-MS
GC-MS

GC-MS

HPLC-MS

LOD
Sub-ng level
30 ng/mL
0.6 lg/g BZE
0.25 lg/g COC
0.5 lg/g BZE
5 ng/mL
19 ng/mL
2.5 ng/patch
0.08 ng/mg COC
0.02 ng/mg COE
2.5 ng/g
0.005 ng/mg COC, COE
0.025 ng/mg EME
0.5 lg /mL
<1 ppb
0.02 ng/mg
0.007 ng/mg COC
0.015 ng/mg BZE
0.009 ng/mg COE
1 pg/mL
0.001 ng/mg EME
0.002 ng/mg BZE
0.003 ng/mg COC
25 pg/mg
2.5 ng/mL
0.0014 lg/mL COC
0.0010 lg/mL BZE
0.9 ng/mg COC
1.2 ng/mg BZE
1.2 ng/mg COE
0.22 ng/mL
26.4 lg/L COC
3590 ng/mL
25 ng/mL
0.01 lg/mL COC, BZE, COE
0.03 lg/mL COC
0.10 lg/mL BZE
0.1 ng/mL
8 ng/mL COC
0.015 ng/mg COC
0.015 ng/mg COC
-

Table 4 gives an overview of screening and confirmatory techniques involved for the determination of COC
and metabolites in biological specimens.
3.1. Screening techniques
Perfect screening analysis should be fast, specific (i.e. no
false positive results obtained), sensitive (i.e. able to
determine very low concentrations of analytes in
samples), reliable (i.e. give identical results with different
laboratories and performers), require little or no sample

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preparation, easy to perform and cheap. A problematic

issue in screening is the need to avoid giving false-negative results, which ends the analysis, compared with
false positive results, which need to be confirmed by
more specific techniques (e.g., LC-MS or GC-MS). Most of
immunoassays are directed to detect BZE, the major
metabolite of COC.
3.1.1.
Enzyme-multiplied
immunoassay
technique
(EMIT). In EMIT, enzyme activity is measured spectrophotometrically. For determination of COC and its
metabolite, absorbance is monitored at kmax = 340 nm
[15]. EMIT assays are easy to automate and are cost
effective. Moreover, the contribution of interfering substances is minimized. However, intake of popular nonsteroidal anti-inflammatory drugs aspirin or tolmetin
can cause false-negative results due to urinary metabolites of these analgesics interfering with antibodies.
3.1.2.
Enzyme-linked
immunosorbent
assay
(ELISA). ELISA is easy to automate. With high sensitivity and a shelf life of the enzyme (label) of about one
year, it is suitable for drug testing in biological fluids and
hair. A disadvantage of ELISA is blocking the activity of
enzyme by antimicrobial agents added to preserve urine.
Cost of ELISA analysis is higher than that of other
immunoassays [27,32,37].
3.1.3. Fluorescence polarization immunoassay (FPIA).
FPIA is based on correlation of the intensity of polarized
light (emitted in the range k = 525550 nm) with concentration of the drug. Advantages of this technique are
low LOD, sensitivity, low matrix effects and long life of
reagents used.
FPIA is suitable for post mortem analysis of whole
blood, because generally it does not require any sample
preparation. FPIA can also be used successfully with
tissue homogenates, but sometimes it can produce falsepositive results.
FPIA was the technique of choice in determination
COC and BZE in meconium. The sensitivity for BZE was
0.6 lg/g meconium [38].
3.1.4. Radioimmunoassay (RIA). RIA is one of the most
efficient, convenient and accurate techniques for detection of small amounts of organic compounds in fluids
because radioactivity is fairly free of matrix interferences
and can be measured in small amounts [37].
A commercially available RIA kit (Coat-a-Count) can
be used to screen hair samples for COC and other drugs.
Qualitative and quantitative results obtained by RIA and
HPLC are similar [39].
Comparison between data obtained by RIA and GC-MS
for the determination of COC, BZE and EME in sweat
patches and urine proves the usefulness of RIA as a
screening method. Furthermore, RIA is cost-effective,
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specific, sensitive and, similarly to FPIA, does not require

sample preparation [40].
3.1.5. Thin-layer chromatography (TLC). TLC is used for
drug screening for forensic purposes, especially due to the
simplicity of performing it and the possibility of simultaneous determination of parent, metabolite and interfering drugs. Nevertheless, TLC is not sensitive and specific
enough to discriminate COC from substances naturally
found in biological samples (e.g., nicotine and caffeine).
Compared with TLC, HP-TLC provides better precision
and accuracy, takes less time and has lower cost of
analysis. Moreover, the use of an advanced type of
densitometer makes HP-TLC suitable for qualitative and
quantitative assay.
Nowadays, HP-TLC is used to detect COC, BZE and
COE simultaneously in urine samples. COE is an important marker for combined consumption of COC and
alcohol [11].
For the detection of BZE together with COC, the
metabolite should be converted into the parent compound, which gives a lower LOD and good separation
from interferences present in biological material [41].
3.1.6. Solid-surface room-temperature phosphorimetry
(SS-RTP). SS-RTP is a simple, sensitive and selective
technique, which can be used for screening analysis for
COC and BZE in saliva. However, it can be applied for a
wide variety of organic compounds. The assay is performed on filter paper, on which the saliva sample is
collected. Only 1 ll of sample is needed for complete
analysis. The LOD for SS-RTP is under the required value
established by the National Institute for Drug Abuse
(NIDA). COC and its metabolite were determined by the
phosphorescence bands with maximum excitation and
emission wavelengths at 258 nm and 413 nm [16]. The
diverse diets of volunteers from whose saliva samples
were obtained did not show interference from the complex composition of the matrix. Background emission of
non-spiked samples was visibly differentiated from
phosphorescence spectra of COC and its metabolite in
spiked samples.
3.2. Confirmatory techniques
Several analytical techniques for analyzing COC and its
metabolites have been reported in the literature. These
techniques range from TLC to HPLC and GC. However,
CE has also been successfully applied to the analysis of
illicit drugs.
3.2.1. HPLC. HPLC has been widely applied for the
main confirmatory analysis of drugs of abuse in forensic
science laboratories because it offers good selectivity and
LODs without need for derivatization. Little (inherent)
UV absorption of polar COC metabolites limits the usefulness of UV detection. Today, MS and MS2 are often

Trends in Analytical Chemistry, Vol. 29, No. 3, 2010

chosen for detection with HPLC, because of its versatility,

specificity, high sensitivity and simple sample preparation [37]. Many analytical protocols, based on HPLC,
have been reported for quantitation of COC.
COC, BZE, and EME are hydrophilic, basic compounds
with pKa values greater than 8. Consequently, buffer
salts or ion-pairing agents and a mobile phase with organic content are needed to ensure adequate retention
on a typical C18 stationary phase, the mobile phase
usually containing ammonium acetate or formic acid
with acetonitrile, water and/or methanol [19,25,42
45]. Methods mentioned were developed on 150-mm
columns, but the analytes can be also monitored using
30-mm columns [18]. A short column offers a short time
of analysis and narrow, symmetric peaks.
3.2.2. HPLC with ultraviolet detection (HPLC-UV). A
growing number of publications describe the use of GCMS, HPLC-MS and HPLC-MS2 for determination of COC
and its metabolites, showing that these are techniques of
choice. However, satisfactory results can also be obtained using HPLC-UV, which is cheaper and less complicated.
An isocratic HPLC procedure with UV detection at
235 nm determined COC and its metabolites in rat
plasma. For simultaneous determination of BZE, NOR
and EME with UV, it was necessary to derivatize EME
to p-fluorococaine [46]. Analytes were separated on a
C18 column with tropacocaine as internal standard.
Another gradient HPLC-diode array detection (DAD)
method was performed on a C8 column (250 mm)
with an acetonitrile-phosphate buffer (pH 6.53) as a
mobile phase to determine opioids, COC and their
metabolites [47]. A chromatogram of plasma spiked
with mixture of drugs is presented on Fig. 3. The
detector response was linear at concentrations in the
range 0.110 lg/ml.

Trends

A similar method was found where COC and COE were

determined in canine serum. A reversed-phase HPLC
method was performed on a 250-mm cyanoprolyl
column with detection at 230 nm. The mobile phase was
acetonitrile-phosphate buffer (pH 7.40) [48].
The level of concentration of COC and BZE in the
plasma of coca chewers was monitored at 230 nm with
bupivicaine hydrochloride as internal standard [49]. For
each compound, the UV spectrum was in the range 200
350 nm.
An HPLC-UV method for determination of COC and
BZE in human-blood-plasma samples was described. A
column switching method used an akyl-diol-silica ADSC18 extraction precolumn for separating the analytes
from proteins and endogenous compounds. For final
separation, an Alltech mixed-mode C18/cation-exchange analytical column was used with a UV-DAD
detector set at 235 nm. The advantage was overlap of
sample preparation. Typical chromatogram of drug-free
plasma and plasma spiked with COC and BZE is presented on Fig. 4. The sensitivity of these methods was
significantly lower than that of other methods reported
[26].
3.2.3. HPLC with fluorescence detection (HPLCFL). Low-cost FL instrumentation and native fluorescence of COC (excitation wavelength 230 nm and
emission wavelength 315 nm) means that this
technique does not require a derivatization procedure,
and that is the main advantage of procedures based on
HPLC-FL. FL achieves LODs of 1 ng/ml (0.2 ng injected) for COC and its metabolites [39,50].
3.2.4. HPLC with MS (HPLC-MS). MS is widely used
for confirmation because MS data provide identification
based on mass-spectral information, and can be used as
confirmatory evidence in courts of law.

Figure 3. Chromatogram of plasma spiked with morphine (MRP), codeine (CDN), benzoylecgonine (BZE), 6-acetylmorphine (6AM), cocaine
(COC), cocaethylene (COE), 2-ethylidene-1,5-dimethyldiphenylpyrrolidyne (EDDP) and methadone (MTD) [47].

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Figure 4. Chromatogram of cocaine (COC) and benzoylecgonine (BZE) after 50-ll direct injection of human blood plasma: A) drug-free blank
plasma; and, B) plasma spiked with 1 lg/mL of COC and BZE [26].

HPLC-MS working with selected-ion monitoring (SIM)

acquisition mode in positive electrospray ionization (ESI)
is nowadays preferred for detection of COC in biological
materials. The use of internal standards (e.g., cocaine-d3
or nalorfine) is common practice. Ions frequently monitored are: m/z 304, 212, 182. This technique with an
LOD of 5 ng/g and a few ng/ml is sensitive enough to
determine COC in meconium and urine, respectively
[22].
3.2.5. HPLC with tandem MS (HPLC-MS2). More reliable identification can be obtained by LC coupled to
tandem MS with a triple quadrupole. This is due to the
fact that the LC-MS2 product-ion spectra generated by
these techniques depend less on sample composition and
experimental settings. However, the product-ion-scan
approach requires the choice of a limited number of ions
to be monitored in the first quadrupole, which makes a
separate survey scan necessary to select the ions. The
present state of the art of MS2 detection results in
exceptionally high specificity and sensitivity, even when
complicated matrices are analyzed.
Quantitative analysis of the COC and metabolites from
whole blood and urine with minimal sample preparation
has been presented recently [18]. Multiple-reaction
monitoring (MRM) analysis uses two transitions set for
each analyte (one parent ion and two daughter ions is
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routine practice). Table 5 shows the parent and daughter ions monitored in most experiments.
There are several publications showing application of
LC-MS2 for the determination of COC in human hair
[19,43,45]. LC-MS2 data recorded in MRM mode give
excellent sensitivity and no need for complete separation
of analytes, as it is presented on Fig. 5.
To improve the efficiency and the quality of routinely
performed tests, an HPLC-MS2 method for illicit drugs is
converted to an ultra-performance LC tandem MS
(UPLC-MS2) method, which improved the chromatographic properties and decreased the overall run time.
COC and other commonly used and abused drugs were
separated on short columns with small particle size

Table 5. Parent and daughter ions used for quantification and as

qualifier
Compound
Cocaine (COC)
Benzoylecgonine (BZE)
Ecgonine methyl
ester (EME)
Ecgonine (ECG)
Norcocaine (NOR)
Cocaethylene (COE)

Transition 1 m/z

Transition 2 m/z

304.0 182.0
290.0 168.0
200.0 182.2

304.0 81.8
290.0 104.9
200.0 81.9

186.0 168.0
318.0 196.0
290.0 168.0

186.0 82.0
318.0 81.8
290.0 135.9

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Figure 5. Chromatogram of hair extract containing cocaine (COC), benzoylecgonine (BZE), cocaethylene (COE) and norcocaine (NOR) [19].

Table 6. Ions monitored in single-ion monitoring (SIM) for quantitative analysis of cocaine, ecgonine methyl ester-TMS, benzoylecgonine-TMS by GC-MS (underlined ions are those most frequently
selected for quantitation)
Compound

COC

EME-TMS

BE-TMS

m/z

82, 182, 303

82, 96, 271

82, 240, 360

(1.82.6 lm). Run times were reduced 23 times without negative effect on quality [24].
3.2.6. HPLC with time-of-flight MS (HPLC-TOFMS). Accurate-mass measurement provided by HPLCTOF-MS greatly increases confidence in identification,
because it inherently limits the possible number of candidate compounds the better the precision and the
accuracy of the mass measurement, the fewer the

number of compounds theoretically possible for a given

accurate mass. There were some publications presented
recently that discussed the application of HPLC-TOF-MS
for the determination of COC and its metabolites [51].
However, existing disadvantages (e.g., extremely high
cost of equipment and lack of personnel with high
qualifications) limit the use of HPLC-TOF-MS.
3.3. Gas chromatography (GC)
GC is a technique frequently used for determination of
drugs of abuse. COC, COE and EME can be assayed without
derivatization with MS or nitrogen-phosphorus detection
but some tend to tail in the analysis [37]. Determination of
low volatile compounds can be improved by derivatization. Reagent most often used for derivatization of COC is
N,O-bis(trimethylsilyl)trifluoroacetamide with 1% trimethyl-chlorosilane (BSTFA/TMCS). BSTFA obtains

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Figure 6. Chromatogram of a plasma sample, cocaine (COC) and cocaethylene (COE) [34].

Figure 7. Electropherogram of a mixture of drug standards, tetracaine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyethylamphetamine (MDE), cocaine (COC), morphine (MRP) and nalorphine (NRP) [28].

volatile, thermally stable derivates of COC and metabolites. The addition of TMCS enables derivatization of
amides, many secondary amines and hydroxyls that are
not derivatized by BSTFA alone. Other derivatization
reagents used for drugs of abuse include: N-methylN-(t-butyl-dimethylsilyl)trifluoroacetamide (MTBSTFA),
pentafluoropropionic anhydride (PFPA) and pentafluoropropionyl (PFP) [52].
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3.3.1. GC with MS (GC-MS). Different specimens may

be tested for illicit drugs with the opportunity to define a
profile of long-term usage. GC is generally employed for
confirmatory testing. Urine immunoassays are followed
by GC-MS with detection in single-ion-monitoring (SIM)
or scan-acquisition mode in the range 33450 amu
[31,32]. Table 6 presents ions routinely monitored in
GC-MS (SIM mode).

Trends in Analytical Chemistry, Vol. 29, No. 3, 2010

Monitoring of COC, COE and anhydroecgonine methyl

ester (AEME) shows not only the form but also different
ways of COC intake. AEME is a product of smoked COC
and existence of COE proves concurrent use of ethanol
and COC. Sensitivity and selectivity required by the law
can be obtained by the use of GC with an ion-trap
detector in positive chemical-ionization mode [23]. Quite
a lot of hair analyses are performed with GC with a mass
selective detector in SIM mode [15,33]. Sweat patches
after SPE and derivatization with BSTFA/TMCS can be
also analyzed by GC-MS in SIM mode [21].
Another outstanding application of GC-MS is determination of COC in the teeth of drug users, which were
pulverized and extracted with chloroform/ isopropanol
(9:1) [12].
When SPME is used to analyze the concentration of
COC in plasma, no derivatization step prior GC is needed.
The fiber containing analytes sorbed from plasma is directly placed in injection port of chromatograph.
Advantages of this method are solvent-free extraction,
simplicity and shorter time of analysis [34]. Fig. 6 shows
a typical chromatogram of plasma samples containing
COC and COE.
The use of SPME turned out to be substantially simpler
and faster than conventional sample processing, and GCMS was found to be specific, sensitive and selective
enough to determine the low drug concentrations to be
expected in plasma. This method therefore has the sensitivity and the selectivity required for clinical and
forensic toxicology.
3.4. Capillary electrophoresis (CE)
CE is an effective analytical tool for determination of illicit drugs in matrices with complicated composition. CE
provides good resolution, short separation time, high
mass selectivity, simultaneous determination of drugs
without need of derivatization, sustainability for coupling with MS and low consumption of reagents.
Changes in the composition of running buffer result in
different selectivity.
In recent years, capillary zone electrophoresis (CZE)
has gained popularity as an analytical tool for toxicological analysis of hair [28]. Fig. 7 shows an electropherogram of a mixture of drug standards. In most
applications, when CZE was coupled with DAD detector,
potassium phosphate at pH 2.5 was used as a running
buffer.
When CZE was combined with MS (ESI), the typical
running buffer was ammonium formate at pH 9.5
[29].

4. Summary
Ultimately, the determination of the appropriate specimen to analyze for detecting drug exposure will depend

Trends

on the specific needs of the monitoring program and

specimen availability. Each specimen has advantages
and limitations, including ease and invasiveness of collection, and different detection times, sensitivities and
specificities. Regardless of the specimen analyzed, confirmation of positive screening results is essential.
Development of an analytical protocol is focused on
multi-analyte determination for economic reasons. COC
and its metabolites are often determined in biological
specimens simultaneously with other drugs (e.g.,
opioids, cannabinoids, amphetamine and benzodiazepines) and metabolites.
Another issue to consider is the choice of screening
technique and its performance. It has to be sensitive
enough to determine low concentrations of analytes in
biological fluids (with low LODs of ng/ml or ng/g).
RIA is suitable for blood and tissue analysis as it
has no matrix effect. FPIA and EMIT can produce
false-negative results, because of high LODs, or falsepositive results, due to matrix interferences with antibodies.
Confirmation of positive results is not always possible
due to the high cost of equipment and the lack of personnel with high qualifications. However, when such
confirmation is needed, the most common technique is
HPLC with different modes of detection, as it can be
applied to multi-analyte samples due to its resolving
power. HPLC-TOF-MS and HPLC-ESI-MS2 are excellent
chromatographic confirmation methods for COC and its
metabolites.
Relative to GC-MS, HPLC reduces sample preparation
time by eliminating derivatization. The sensitivity is the
highest in HPLC-TOF-MS and HPLC-ESI-MS2, but accurate-mass determination, which is possible with LC-TOFMS, increases confidence in identification.
The next technique of choice is GC, but the need for
derivatization of non-volatile compounds is a serious
drawback, which can be avoided with SPME applied at
the sample-preparation stage.
CE is an interesting alternative to HPLC. With high
sensitivity, short analysis time and low running cost, CE
is finding more applications in drug testing.
The selection of an appropriate analytical procedure is
fundamental to achieving reliable, accurate results. The
growing number of illicit drugs on the market forces
scientists to develop new methodologies for simultaneously determining these hazardous substances.
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