Review

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Bioanalytical methods for the determination

of cocaine and metabolites in human
biological samples

Determination of cocaine and its metabolites in biological specimens is of great importance, not only in clinical and
forensic toxicology, but also in workplace drug testing. These compounds are normally screened for using sensitive
immunological methods. However, screening methods are unspecific and, therefore, the posterior confirmation of
presumably positive samples by a specific technique is mandatory. Although GCMS-based techniques are still the
most commonly used for confirmation purposes of cocaine and its metabolites in biological specimens, the advent
of LCMS and LCMS/MS has enabled the detection of even lower amounts of these drugs, which assumes particular
importance when sample volume available is small, as frequently occurs with oral fluid. This paper will review
recently-published papers that describe procedures for detection of cocaine and metabolites, not only in the most
commonly used specimens, such as blood and urine, but also in other alternative matrices (e.g., oral fluid and hair)
with a special focus on sample preparation and chromatographic analysis.

Cocaine is one of the most abused drugs
worldwide. Indeed, its use in the USA is only
exceeded by cannabis and nonmedical psycho-
therapeutics, including pain relievers, tranqui-
lizers, sedatives and stimulants [1] . According
to the 2008 Annual Report of the European
Monitoring Centre for Drugs and Drug
Addiction [301] , the use of stimulant drugs is
growing amongst Europeans, not only in pat-
terns of drug use ,which are usually seen within
the chronic and marginalized population of
problematic drug users, but also among the
better socially integrated groups of young peo-
ple who use drugs on a more recreational basis.
The same report concludes that cocaine is now
the most commonly used stimulant in many
countries in the south and west of Europe and
its use continues to grow.
As illicit drug consumption affects all mod-
ern societies, the assessment of situations of
drug abuse is of overwhelming importance
and therefore sensitive analytical methods
for the reliable determination of cocaine and
metabolites in biological samples are needed.
Bioanalytical methods for the determination
of cocaine in various biological samples will
be reviewed in this paper, with a special focus
on sample preparation and chromatographic
analysis. The studies were selected through the
references list of known published papers and
using the public MedLine database PubMed
with the following search strings: cocaine and
blood; cocaine and urine; cocaine and hair; M Barroso1, E Gallardo2,3
cocaine and oral fluid or saliva; and cocaine & JA Queiroz2,4
and meconium.
Author for correspondence
1
Instituto Nacional de Medicina
Disposition of cocaine Legal Delegao do Sul,
Cocaine is available for consumption under RuaManuel Bento de Sousa,
several different forms, including cocaine 3,1150219 Lisboa, Portugal
hydrochloride and free base alkaloid (also Tel.: +35 121 881 1800
known as crack) and its absorption depends Fax: +35 121 885 0078
on the route of administration. Indeed, cocaine E-mail: mbarroso@dlinml.mj.pt
can be absorbed through any mucus mem- 2
CICS: Centro de Investigao
brane, smoked or injected and, therefore, it can em Cincias da Sade,
be snorted, inhaled, or injected intravenously Universidade da Beira Interior,
or intramuscularly [2] . 6201001 Covilh, Portugal
Following absorption, the drug is rapidly 3
Tel.: +35 127 531 9700
distributed and approximately 90% is bound Fax: +351 275329099
to plasma proteins [3] . Cocaine has a short Email: egallardo@fcsaude.ubi.pt
half-life in plasma, which ranges from 0.5 to 4
E-mail: jqueiroz@ubi.pt
1.5h. The highest concentrations of cocaine
appear in the brain, spleen, kidney and lung.
Cocaine crosses the placenta and is found in Analytical methods
breast milk [4] . Procedures aiming to detect and
The majority of a cocaine dose (8090%) is quantitate single or multiple
metabolized to ecgonine methyl ester (EME) compounds in a sample.
Analytical methods discussed in
and benzoylecgonine (BE). While EME is this review involve analyte
produced via a rapid enzymatic hydrolysis by extraction from the matrix and
plasma and liver esterases, cocaine undergoes a instrumental analysis, usually by
spontaneous and pH-dependent nonenzymatic chromatographic methods
hydrolysis and, to a lesser extent, enzymatic
hydrolysis, to form BE. Indeed, even in water,
at pH values greater than neutrality, cocaine
is readily hydrolyzed to BE [5] . Less than 10%

10.4155/BIO.09.72 2009 Future Science Ltd Bioanalysis (2009) 1(5), 9771000 ISSN 1757-6180 977
Review | Barroso, Gallardo & Queiroz
Cocaine and metabolites
An alkaloid obtained from the
coca plant (Erythroxylon coca). It
is a highly addictive CNS
stimulant and one of the most
widely abused drugs nowadays.
Cocaine undergoes extensive
biotransformation reactions,
yielding several metabolites.
Benzoylecgonine and ecgonine
methyl ester are the two major
metabolites of cocaine
Biological specimens
In the context of this review a
biological specimen is any
specimen or sample of biological
origin, usually human, in which
abused drugs can be detected
and quantitated to document
drug exposure
of cocaine is metabolized in the liver to nor-
cocaine, which has significant pharmacologic
activity [3] .
Other related compounds include anhydro-
EME, a pyrolisis product that originates when
cocaine is smoked; and cocaethylene, which
forms when ethanol is concomitantly con-
sumed [6] . Several minor metabolites, such as
m- and p-hydroxycocaine, m- and p-hydroxy-
BE and nor-BE, have also been identified [1] .
The metabolic pathway of cocaine is shown in
Figure1.
Little cocaine is eliminated in urine as
unchanged drug (19%, depending on urine
pH) [5] and it can be detected up to 2436h[4] ,
while its major metabolites, which account for
8090% of urinary metabolites in humans, can
be detected in urine for 614days after chronic
cocaine use [2] . The elimination rate of cocaine
and metabolites in urine is affected by diuresis.
Therefore, any condition leading to an increase
in diuresis will enhance the excretion rate of
these compounds, as their fixation on tissues
will bedecreased.
Controlled administration studies were
extremely useful for a better understanding of
cocaine disposition and bioavailability [1,7,8] .
It should be noted, however, that pharmaco
kinetic parameters are usually evaluated in
plasma, while whole-blood samples are more
commonly used in forensic toxicology, espe-
cially in postmortem toxicology. Therefore,
care should be taken in results interpretation in
thesesituations.
Cocaine detection in
biologicalspecimens
Testing for cocaine and metabolites can be
performed in a variety of biological specimens,
including blood, urine, hair, oral fluid (saliva)
and meconium. Nonbiological samples, such as
currency notes, can also be tested for the presence
of cocaine [911] .
Blood, plasma & urine
These are by far the most-used specimens for the
detection of abused drugs, including cocaine, in
forensic toxicology. Blood and plasma samples

O O
OH HN O O
N OH
N
O O
OH
O O
OH Ecgonine
m-hydrobenzoylecgonine Norcocaine methyl ester

O O
OH O O
N N OH
N
O O

O Cocaine O Anydroecgonine
Benzoylecgonine methyl ester
CH
3 CH
2 OH

O O O O
OH O O O
N N N N

O O O O
OH OH
O O O O
OH
p-hydrobenzoylecgonine m-hydrococaine p-hydrococaine Cocaethylene

Figure1. Major metabolic pathway of cocaine.

978 Bioanalysis (2009) 1(5) future science group

 Bioanalytical methods for the determination of cocaine & metabolites
are relevant media in the assessment of short-
term use of drugs of abuse [12,13] and the ana
lysis of these specimens is mandatory in many
countries to identify impaired drivers in driving
under the influence of drugs situations [1416] .
The past use of cocaine can be assessed even
when it is no longer present in blood, by means
of its main metabolites, BE and EME, which
have longer plasma half-lives [2] .
On the other hand, urine is the most widely
used specimen to assess drug exposure and is
capable of providing evidence of drug use or
abstinence (for most drugs) for the preceding
2days. Indeed, this is the specimen of choice
for many laboratories and this is due to the often
higher concentrations at which the drugs (and
metabolites) appear in this specimen when com-
pared with blood or plasma, the ease of sampling
and the low invasiveness of the collection proce-
dure. In addition, urine is still the most efficient
test sample for monitoring recent exposure to
drugs presenting short detection periods [17] .
However, despite being a well-researched
technology, urinalysis presents some short
comings, including the possibility of sample
adulteration or substitution [18] . Moreover, urine
is not the specimen of choice in identifying drug
impaired driving situations. Indeed, as the detec-
tion windows are longer than those observed in
blood, an increase in the prevalence of positive
findings for some substances may be observed.
On the other hand, the urinary metabolites of
many drugs are not pharmacologically active
and, therefore, do not pose a particular problem
for traffic safety[16,19] .
These conventional biological specimens
present a number of drawbacks, especially when
dealing with living subjects. For instance, the
procedures used for their collection are often
invasive to the donor (e.g., blood collection)
and/or infringe on the individuals privacy
(e.g.,supervised urine collection). Furthermore,
urine samples can be adulterated or substituted,
invalidating analysis [20,21] .
To overcome these situations, a number of
additional samples may be analyzed and these
include the so-called alternative or unconven-
tional specimens, including hair, oral fluid,
sweat and meconium.
Hair
Hair is the most widely used alternative speci-
men in documenting drug exposure and presents
an overwhelming advantage over all the other
samples: its larger detection window (weeks to
future science group
| Review
months, depending on the length of the hair
shaft), which makes long-term histories acces-
sible through segmental analysis [22] . For this
reason, hair is often called a calendar of drug
exposure [23] . On the other hand, as drugs are
incorporated into the hair shaft from the blood-
stream during growth, they become entrapped
inside the hair and are not easily removed by
normal hygiene procedures. This allows, for
instance, the collection of an almost identical
hair sample several days after it was first col-
lected, which is important in the case of sus-
pected sample substitution or a break in the
chain of custody.
However, hair analysis presents some draw-
backs, from which the most important is the
possibility of reporting false-positive results
derived from environmental contamination[24] .
This issue assumes even more relevance for those
drugs which are more likely to be smoked, as
occurs with cannabis and cocaine, especially
if the consumption has legal implications. To
minimize this effect, the Society of Hair Testing
strongly recommends that hair analysis pro-
cedures include a washing step to remove the
drug and the use of parent drug-to-metabolite
ratios[25] . In addition, criteria for wash evalua-
tion and extensive washing procedures have been
proposed [2629] . This extensive washing is neces-
sary to demonstrate that the drug being recov-
ered by the extraction steps is not superficial or
surface drug, but internally deposited drug.
Oral fluid
Oral fluid testing presents two major advan-
tages. On one hand, the sample is collected in
a noninvasive and simple manner (under close
observation, if necessary), maintaining the
examinees privacy, being less prone to adultera-
tion or substitution. On the other hand, drug
concentrations in oral fluid reflect the nonpro-
tein-bound fraction, which is most relevant for
assessing degree of impairment because the free
fraction of the drug is pharmacologically active
[30,31] . Pharmacokinetics of illicit drugs, includ-
ing cocaine, in oral fluid have been reviewed by
Drummer [32] . In general, abused drugs are read-
ily detectable in oral fluid and can show similar
pharmacokinetics to plasma. This relationship is
important in the screening of suspected drug use
while driving, since in these situations invasive
collection procedures are not easily performed.
Concerning cocaine, its oral fluid-to-plasma
ratio is approximately three and this compound
and its metabolites can be detected for up to
www.future-science.com 979
Review | Barroso, Gallardo & Queiroz
17h after the last dose in self-reported users,
showing a longer terminal elimination half-
life than in plasma [32] . However, the amount
of sample available for analysis is usually small
(often <1ml) and therefore highly sensitive tech-
niques, namely using MS detection, are needed
to accomplish theanalysis.
Sweat, meconium & other specimens
The application of sweat in toxicological analysis
has been reviewed by Gallardo and Queiroz[22]
and Huestis etal. [33] . This type of analysis is
scarcely performed on a routine basis, since
the estimation of an accurate volume is diffi-
cult, impairing the evaluation of the obtained
concentrations. In addition, the collection of
adequate sample for analysis is not an easy task.
On the other hand, some of the patches used for
sample collection may be prone to environmen-
tal contamination, as studied by Kidwell and
Smith[34] .
A study has been conducted on the detec-
tion of several drugs in sweat after controlled
administration [33] and cocaine concentrations
in this specimen have been shown to depend
on the collection device and on the collection
site. Moreover, the concentration of the parent
compound in this specimen is higher than the
metabolites concentrations and this detection
pattern is distinctly different from that found
in urine specimens.
One interesting application of unconven-
tional biological samples is in the assessment
of prenatal drug exposure. Besides urine and
hair, other neonate specimens may be used for
this purpose, including meconium, umbili-
cal cord tissue and blood, vernix caseosa and
amniotic f luid [22,3546] . Cocaine is almost
never detected alone in meconium and the con-
centration of this compound and metabolites
in this specimen decreases rapidly 48h after
birth. The exception is BE, which remains
detectable for longer [38] . On the other hand,
the metabolic profile in newborns in meco-
nium differs from that of urine [40] . Indeed,
m-hydroxyBE is present in this specimen at
significant amounts and therefore meconium
testing should include this compound to avoid
false-negative results[40] .
As will be further discussed below, cocaine
and metabolites may also be detected in other
biological specimens, such as pericardial
fluid [47,48] , vitreous humor [4956] , nails [5765]
and several organs and tissues (brain, skeletal
muscle and bones, for example) [51,53,6674] .
980 Bioanalysis (2009) 1(5)
Bioanalytical procedures for cocaine
detection in biological specimens
In general, laboratorial analysis of drugs in
biofluids begins with a screening assay, usu-
ally by an immunoenzymatic approach, aim-
ing to detect or exclude a specific drug class.
These immunoassay techniques are extremely
important for laboratories with a high work-
load, requiring high sample throughput with
little or no sample pretreatment. Indeed, if
an adequate screening test is used, laborious
and expensive techniques such as LC or GC
coupled with MS will only be applied to the
confirmation of presumptive positive samples,
saving time and money. This is especially rel-
evant if one takes into account that most of
the samples routinely tested for drugs turn out
to be negative.
Immunoassay techniques are the most com-
monly-used methods for the screening of illicit
drugs in biological specimens, including not only
the most commonly used urine and blood samples
but also other matrices such as oral fluid and hair.
Several studies have been published on the use
of immunoassays for cocaine screening in various
matrices, including urine [7583] , blood[76,8488] ,
hair [8996] and oral fluid [97101] .
Immunoassays have evolved and the ultimate
stage of this evolution is presented by onsite
testing systems, which are important in various
situations, such as workplace drug testing or
assessment of impaired driving. These portable
devices have been developed for drug detection
in several biological fluids, including urine, oral
fluid and sweat [22,30,102109] .
These techniques represent highly sensitive
and cost-effective technology; however, due to
their lack of specificity, the confirmation of pre-
sumptive positive samples is mandatory. These
confirmation analyses are usually performed
using highly specific MS methods that allow
the identification of the present compound(s),
eluding false-positive results.
Biological specimens are very complex matri-
ces and therefore they cannot be directly ana-
lyzed. Indeed, proteins and lipids, as well as
other biomolecules are usually present at high
concentrations and this fact will affect ana-
lytical methods performance. Therefore and
to improve their detection, the analytes must
be extracted (isolated) from these matrices and
concentrated in a solvent that is compatible with
analytical instruments.
Several papers have been published on the
determination of cocaine and metabolites in
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 Bioanalytical methods for the determination of cocaine & metabolites | Review
biological specimens. These analytes are mostly These sample-cleanup procedures may be
extracted from biological samples by means of applied to different biological specimens and
SPE [23,33,71,110117] and liquidliquid extraction there is a growing tendency to use SPE for sam-
(LLE) [58,60,74,119123] . However, methods using ple cleanup, since lesser amounts of organic
solid-phase microextraction (SPME) [17,124129] solvents are required to accomplish the ana
and supercritical fluid extraction [130,131] are lysis. In addition, the extracts are often cleaner
also available. than those obtained using LLE and SPE is more

Table1. Bioanalytical procedures for determination of cocaine and metabolites in blood/plasma samples.
Compounds Sample Sample preparation Detection mode LOD (ng/ml); Ref.
volume (ml), LOQ (ng/ml)
matrix
AM, MA, MOR, MAM, MDA, MDEA, 0.15, plasma Protein precipitation LCMS/MS (ESI) 0.5; 0.8 (COC) [12]
MDMA, COC, BE, THC, THCCOOH, (methanol) 0.4; 0.6 (BE)
KET, PHEN
MA, MDA, MDEA, MDMA, MET, 0.5, blood LLE CZETOFMS (ESI) 2; 10 (COC) [136]
COC, BE, MOR, COD, MAM (Chloroform:IPA, 9:1) 5; 20 (BE)
COC, BE, EME, COET, ECG 0.5, blood Online SPE LCMS/MS (ESI) 3; 20 (COC) [137]
(Hysphere MM anion) 7; 8 (BE)
12; 36 (EME)
5; 15 (COET)
16; 47 (ECG)
COC, BE, EME, COET, MOR, COD, 2,blood SPE ELISA/GCMS 50; 50 (GCMS) [110]
MAM, HYCOD, OXCOD, HYMOR (ZSDAU020 Clean Screen)
COC, BE 1,blood SPE GCMS ; 50 [138]
(Bond Elut Certify)
COC, BE, COET, MOR, COD, MAM, 1,plasma MW-assisted LLE LCDAD 10; 50 [139]
MET, EDDP (Chloroform)
MOR, COD, MAM, PHOLC, COC, BE, 1,blood SPE EMIT/GCMS [111]
THC, THC-OH, THCCOOH, BUP, NBUP (Isolute HCX)
COC, BE, NCOC, EME, COET 0.2,blood SPE LCMS/MS (ESI) 13; 58 [140]
(SPEC MP1)
COC, COET 1, plasma SPME GCMS 19; 25 (COC) [141]
(100-m PDMS) 11 (COET)
MOR, MAM, AM, MA, MDA, 0.2, blood SPE LCMS (ESI) 0.5; 2 [112]
MDMA, MDEA, MBDB, COC, BE (Oasis HLB)
COC, BE, NBE, NCOC, ECG, EME, 3, blood SPE GCMS 2; 2 for all compounds [142]
mHBE, AEME, COET, NCOET, EEE (Bond Elut Certify) except:
25; 25 (NBE)
640; 800 (ECG)
50; 50 (mHBE)
13; 13 (AEME)
MOR, COD, MAM, COC, BE, COET, 1, plasma SPE LCDAD 10; 100 [143]
MET, EDDP (Bond Elut Certify)
AC: Acetylcodeine; ACE: Acetone; AEME: Anhydroecgonine methyl ester; AM: Amphetamine; APCI: Atmospheric pressure chemical ionization; API:Atmospheric pressure
ionization; BE: Benzoylecgonine; BUP: Buprenorphine; CBD: Cannabidiol; CBN: Cannabinol; CE: Capillary electrophoresis; CEC: Capillary electrochromatography;
CEDIA:Cloned enzyme donor immunoassay; CI: Chemical ionization; COC: Cocaine; COD: Codeine; COET: Cocaethylene; CSEI: Cation-selective exhaustive injection;
CZE:Capillary zone electrophoresis; DAD: Diode array detector; DI: Direct immersion; DMF: N,N-dimethyl formamide; ECG: Ecgonine; EDDP: 2-Ethylidene-1,5-dimethyl-
3,3-diphenylpyrrolidine; EEE: Ecgonine ethyl ester; EI: Electron ionization; EME: Ecgonine methyl ester; EMIT: Enzyme multiplied immunoassay technique;
EMOR:Ethylmorphine; EPH: Ephedrine; ESI: Electrospray ionization; FID: Flame ionization detector; FPIA: Fluorescence polarization immunoassay; GHB:g-hydroxybutyrate;
HER: Heroin; HMA: 3-hydroxy-4-methoxy-amphetamine; HMMA:3-hydroxy-4-methoxy- methamphetamine; HPTLC: High performance thin-layer chromatography;
HS:Headspace; HYCOD: Hydrocodone; HYMOR:Hydromorphone; IMS: Ion mobility spectrometry; IPA: Isopropyl alcohol; KET: Ketamine; LLE: Liquidliquid extraction;
LOD:Limit of detection; LOQ: Limit of quantitation; LSD: Lysergic acid diethylamide; MA: Metamphetamine; MAM: 6-acetylmorphine; MBDB: N-methyl-1-(3,4-
methylenedioxyphenyl)-2-butanamide; MDA:3,4-methylenedioxyamphetamine; MDB:3,4-methylenedioxybutanamine; MDEA:3,4-methylenedioxyethamphetamine;
MDMA:3,4-methylenedioxymethamphetamine; MECK: Micellar electrokinetic chromatography; MEK: Methyl ethyl ketone; MEPH: Methylephedrine; MEPS:
Microextraction by packed sorbent; MET: Metadone; mHBE: Meta-hydroxybenzoylecgonine; mHCOC: Meta-hydroxycocaine; MOR:Morphine; MOR-3-G: Morphine-3-
glucuronide; MOR-6-G: Morphine-6-glucuronide; MW: Microwave; NBE: Norbenzoylecgonine; NBUP: Norbuprenorphine; NCI: Negative chemical ionization;
NCOC:Norcocaine; NCOD: Norcodeine; NCOET:Norcocaethylene; NEPH: Norephedrine; NKET: Norketamine; NMF: N-methyl formamide; NMOR:Normorphine;
OXCOD:Oxycodone; OXMOR:Oxymorphone; PCI: Positive chemical ionization; PDMS: Polydimethylsiloxane; pHBE: Para-hydroxybenzoylecgonine; pHCOC: Para-
hydroxycocaine; PHEN: Phencyclidine; PHOLC: Pholcodine; pMETAM: para-Metoxyamphetamine; PSEPH: Pseudoephedrine; RIA:Radioimmunoassay; SACI: Surface
activated chemical ionization; SPME: Solid-phase microextraction; TFAA: Trifluoracetic acid; THC:D9-Tetrahydrocannabinol; THCCOOH: 11-nor-D9-tetrahydrocannabinol-9
carboxylic acid; THC-OH: 11-hydroxy- D9-tetrahydrocannabinol; TOF: Time-of-flight; UPLC:Ultra-performance liquid chromatography.

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Review | Barroso, Gallardo & Queiroz

Table2. Bioanalytical procedures for determination of cocaine and metabolites in urine samples.
Compounds Sample Sample Detection mode LOD (ng/ml); Ref.
amount (ml) preparation LOQ (ng/ml)
COC, BE, EME, mHBE, pHBE, NBE, ECG 3 SPE (Clean-Thru EMIT/GCMS (EI) ; 1 (COC) [7]
Clean-Thru ; 4 (BE)
ZCDAU020L) ; 1 (EME, mHBE, pHBE)
; 2 (NBE)
; 16 (ECG)
MOR-3-G, MOR, AM, MA, COD, MDA, 0.5 SPE (Oasis HLB) FPIA/LCMS/MS 0.0176; (COC) [151]
OXCOD, MDMA, MDEA, BE, NKET, KET, COC, (ESI) 0.211; (BE)
7- aminoflunitrazepam, PCP, N-desmethyl-
flunitrazepam, a -OH-alprazolam, a-OH-
triazolam, oxazepam, flunitrazepam,
nordiazepam, MET, diazepam, THCCOOH,
phenobarbital, butalbital, butalbarbital,
amobarbital, pentobarbital, secobarbital
MAM, COD, dihydrocodeine, MOR, NCOD, 1 Online extraction HPLC screening [152]
NMOR, noscapine, OXCOD, EDDP, tilidine, column with systems- toxicological
ephedrine, fencamfamine, AM, MDA, MDMA, loading buffer identification system
methoxyamphetamine, COC, BE and SPE (Bond and HPLC screening
Elut Certify) for systemsRemediHS
GCMS analysis GCMS (EI)
AM, MA, MOR, EMOR, MAM, HER, Immunochemical 2100; 5000 (COC) [153]
7-aminoflunitrazepam; BE, flunitrazepam, screening test 1600; 5000 (BE)
diazepam, COD, COC, 2-aminochlor- QuikScreen
benzophenone High performance
Thin layer
chromatography
CEUV/VIS detector
MA, AM. COC, MAM, nitrazepam, estazolam, 0.6 No sample LCMS (ESI) 5; 50 [154]
7-aminoflunitrazepam preparation
EME, BE, NCOC, COET 0.05 SPE (SPECMP1) LCMS/MS (ESI) 10; 5 ng/ml (EME) [140]
2; 8 ng/ml (BE)
10; 8 ng/ml (COC)
2; 8 ng/ml (NCOC)
2; 7 ng/ml (COET)
MOR, COD, MAM, COC, COET, BE, 1 MW-assisted LLE LCDAD 20; 100 (COC) [155]
MET, EDDP (chloroform) 40; 100 (BE)
BE 2 SPE (Cerex Fast GCMS 25; 25 [156]
Polychrom CLIN II)
COC, BE GCMS (EI) 1; [157]
Dihydrocodeine, EPH, MEPH, NEPH, 2 LLE (diethyl ether: GCMS 12.5; 50 (EME) [158]
pseudoephedrine, MA, MDA, MDMA, MDEA, chloroform, 4:1) 50; 100 (COC)
EME, COC, MOR, MAM
AM, MA, MDA, MDMA, MDEA, BE, 0.6 Inmunoassays ; 10 [159]
nordiazepam, desalkylflurazepam, oxazepam, (Instant-View
temazepam, OH-midazolam, lorazepam, TesTcup OnLine)
estazolam, aOHalprazolam, a-OH-triazolam, GCMS (EI)
diazepam, COD, MOR, HYCOD, HYMOR,
OXCOD, OXMOR, THC, phentermine, EPE,
pseudoephedrine, phenylpropanolamine
COC, COET 1 SPME (100m GCMS (EI) 5; 5 [17]
PDMS)
COC, COET 2 SPE (Bond Elut HPTLC 100; (COC) [160]
Certify)
COC, thebaine 9 Solvent CEDAD 2; 16 [161]
microextraction
COC, BE, EME, NBE, NCOC, ECG, mHBE, 3 SPE (Bond Elut GCMS (EI) 2l; 2 for all compounds, [142]
AEME, COET, NCOET, EEE Certify) except:
25; 25 (NBE)
640; 800 (ECG)
50; 50 (mHBE)
13; 13 (AEME)

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 Bioanalytical methods for the determination of cocaine & metabolites | Review

Table2. Bioanalytical procedures for determination of cocaine and metabolites in urine samples (cont.).
Compounds Sample Sample Detection mode LOD (ng/ml); Ref.
amount (ml) preparation LOQ (ng/ml)
COC, BE, EME, COET Few L MEPS (Clean TOF-MS (API) 22.9; 65 (EME) [144]
Screen DAU) 23.7; 75 (BE)
4.0; 95 (COC)
9.8; 75 (COET)
MOR, MAM, COD, EMOR, PHOLC, OXCOD, 0.5 SPE (Oasis MCX) EMIT/UPLCMS/MS 1; 2.9 (BE) [145]
COC, BE (ESI) 1.4; 3.5 (COC)
AM, MA, MDA, MDMA, MDEA, HER, COD, 0.5 SPE (SCX) CECDAD 10; 20 [146]
MAM, COC, MOR
BE, mHBE, pHBE, NBE 4.5 SPE online and LCMS/MS (ESI) 1. 2; 1.2 (BE, mHBE, [115]
manual (SPEWare) GCMS (EI) pHBE) using LCMS/MS
15; 15 (BE ) using GCMS
ECG, EME, BE, COC, COET 1 Online SPE LCMS/MS (ESI) 23; 69 (ECG) [147]
(Hysphere MM 11; 25 (EME)
Anion) 4; 13 (BE)
2; 7 (COC)
7; 21 (COET)
AM, MA, MDMA, MDA, MDEA, pMETAM, 1 SPE (GV-65) ELISA/GCMS ; 40 [116]
phentermine, amobarbital, butabarbital,
butalbital, pentobarbital, secobarbital,
alprazolam, -hydroxyalprazolam, clonazepam,
lorazepam, nordiazepam, oxazepam,
temazepam, THC, THCCOOH, carisoprodol,
meprobamate, BE, COC, COET, fentanyl,
norfentanyl, meperidine, normeperidine, MET,
EDDP, COD, dihydrocodeine, HYCOD, HYMOR,
MOR, OXCOD, OXMOR, norpropoxyphene,
propoxyphene
COC, BE, NCOC, COET 2 SPE (Oasis MCX) CECSEI-sweeping- 0.053; 200 (COET) [148]
MEKCDAD 0.040; 200 (NCOC)
and sweeping- 0.050; 200 (COC)
MEKCDAD 0.33; 200 (BE) using
CSEI-sweeping-MEKC
0.042; 0.2 (COET)
0.030; 0.2 (NCOC)
0.037; 0.2 (COC)
0.236; 1 (BE) using
sweeping-MEKC
COC, BE, MOR 1 SPE (Bond Elut Cozart rapid urine [149]
Certify) multipanel test
GCMS (EI)
EME, BE, MOR, COD, MAM, AM, MA, MDMA, 0.5 SPE (Oasis HLB) LCMS/MS (ESI) 0.5; 1 [150]
MDMA, MET, EDDP, LSD
Modafinil, selegiline, crotetamide, cropropamide, 1 LLE GCMS (EI) [119]
pentetrazol, EPH, NEPH, sibutramine, COC, BE, chloroform:IPA
EME, AM, MA, COD, dihydrocodeine (9:1)
AEME: Anhydroecgonine methyl ester; AM: Amphetamine; BE: Benzoylecgonine; CE: Capillary electrophoresis; COC: Cocaine; COD: Codeine; COET: Cocaethylene;
DAD: Diode array detector; ECG: Ecgonine; EDDP: 2-Ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine; EEE: Ecgonine ethyl ester; EI: Electron ionization; EME: Ecgonine
methyl ester; EMIT: Enzyme multiplied immunoassay technique; EMOR:Ethylmorphine; EPH: Ephedrine; ESI: Electrospray ionization; FPIA: Fluorescence polarization
immunoassay; HER: Heroin; HPTLC: High-performance thin-layer chromatography; HYCOD: Hydrocodone; HYMOR:Hydromorphone; KET: Ketamine; LLE:Liquid
liquid extraction; LOD: Limit of detection; LOQ: Limit of quantitation; MA: Metamphetamine; MAM: 6-acetylmorphine; MDA: 3,4-methylenedioxyamphetamine;
MDEA: 3,4-methylenedioxyethamphetamine; MDMA: 3,4-methylenedioxymethamphetamine; mHBE: Meta-hydroxybenzoylecgonine; mHCOC: Meta-hydroxycocaine;
MOR:Morphine; MOR-3-G: Morphine-3-glucuronide; NBUP: Norbuprenorphine; NCOET: Norcocaethylene; NCOC: Norcocaine; NEPH:Norephedrine;
NKET:Norketamine; NMOR:Normorphine; OXCOD: Oxycodone; OXMOR:Oxymorphone; PDMS: Polydimethylsiloxane; pHBE: Para-hydroxybenzoylecgonine;
SPME:Solid-phase microextraction; THC: D9-tetrahydrocannabinol; THCCOOH: 11-nor-D9-tetrahydrocannabinol-9 carboxylic acid.

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Table3. Bioanalytical procedures for determination of cocaine and metabolites in hair samples.
Compounds Sample Extraction Cleanup Detection LOD (pg/mg); Ref.
amount mode LOQ (pg/mg)
(mg)
COC, BE, MOR, COD, MAM 20 Methanol, 4h, No cleanup LCMS/MS 1; 10 [133]
40C, (ESI)
(ultrasonication)
COC, BE 20mg Methanol: SPE (Oasis MCX) GCMS (EI) 20; 50 (COC) [23]
hydrochloric acid, 15; 50 (BE)
0.1 M (2:1), 3 h,
65C
AM, MA, MDA, MDMA, MDEA, diazepam, 10 Methanol/ SPE (Bond Elut LCMS/MS 20100; 50340 [162]
nordiazepam, oxazepam, temazepam, COC, phosphate buffer, Certify and Clean (ESI)(Ion
BE, EME, COET, MOR, MAM, COD, 18 h, 45C Screen DAU) trap)
dihydrocodeine
COC, BE, COET 50 Methanol:TFAA SPE (Bond Elut MALDI [163]
(GCMS) (9:1), overnight, Certify) GCMS (EI)
1(MALDI) 45C for SPE methanol/TFAA
(for MALDI)
COC, BE, COET, HER, MOR, MAM, COD, AC 50 Methanol, 5 h, SPE (chromabond GCMS (EI) 10; 110 (COC) [134]
50C (sonication) drug) 30; 260 (BE)
50; 210 (COET)
Nicotine, cotinine, MOR, AM, MA, COD, 20 Acetonitrile: No cleanup LCMS/MS ; 12.5 (COC) [164]
MDMA, 7-aminonitrazepam, MAM, BE, COC, 25mM formic (ESI) ; 2.5 (BE)
zopiclone, 7-aminoclonazepam, acid (5:95), 18 h,
meprobamate, zolpidem, 37C
7-aminoflunitrazepam, BUP, oxazepam,
carisoprodol, MET, diazepam, alprazolam
COC 5 Hydrochloric acid, SPE (Bond Elut LC 100; 300 [165]
0.1M, 18 h, 45C C2cartridges) fluorescence
detection
COC, BE, CE, NCOC 1113 Enzymatic SPE (isolute SPE LCMS/MS 20; 20 [26]
hydrolysis columns) (ESI)
Methyl ecgonine, BE, COC, COD, MOR, Hydrochloric acid, SPE (Bond Elut GCMS (EI) 20; 90 [166]
MAM, scopolamine, nalorphine 0.1 M, overnight, CertifyLRC)
45C
AM, MDMA, MDA, benzodiazepines, COC, Methanol SPE (HCX ELISA/ [167]
BE, THC, THCCOOH, MAM, MOR, AC, HER, (sonication), 6 h, mixed mode) GCMS (EI
dihydrocodeine, COD NaOH and CI)
COC, BE, COD, MOR, MAM 50 Enzymatic SPE (Oasis HLB GCMS (EI) 40; 130 [168]
hydrolysis, 12 h, cartridges)
40C
COC, BE, NCOC COET 10 Phosphate buffer, SPE (Clin II, LCMS/MS 25; 50 [135]
0.025 M, 3 h, 6910353T) (APCI)
75C (sonication)
CBD, THC, CBN, AM, MA, MDMA, 50 Methanol, 16 h, LLE (dichloro- ELISA/ [89]
MDA, MDEA, EME, COC, COET, BE, COD, 40C methane: GCMS (EI)
MOR, MAM IPA:N-heptane,
50:17:33)
AM, MDMA, MDA, MDEA, MBDB, Methanol SPE (HCX ELISA/ [90]
diazepam, lorazepam, nordiazepam, (sonication), 6 h, SPEmixed mode) GCMS (EI
oxazepam, temazepam, COC, BE, AEME, NaOH and CI)
COET, THC, THCCOOH, THC-OH, CBD, CBN,
MAM, MOR, AC, HER, dihydrocodeine,
COD, MET, EDDP
AM, MA, MDMA, MDA, MOR, COD, COC, 100 Hydrochloric acid LLE (Toxi-Tubes A) CZE 15; 50 (COC) [169]
BE, MAM, EPH, PHOLC 0.1 M overnight MicroTOF 100; 330 (BE)
45C MS (ESI)

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Table3. Bioanalytical procedures for determination of cocaine and metabolites in hair samples (cont.).
Compounds Sample Extraction Cleanup Detection LOD (pg/mg); Ref.
amount mode LOQ (pg/mg)
(mg)
AM, BE, COC, COET, COD, MA, MDA, MOR, 20 Methanol, No cleanup LCMS/MS 50; 50 [170]
MAM, MDEA, MDMA overnight, 50C (APCI)
COC, BE 510 Methanol, No cleanup ELISA/ 100; [171]
overnight, 52C GCMS (EI)
Methyltestosterone, nandrolone, boldenone, 50 Methanol, No cleanup GCMS/MS 100; 100 [172]
fluoxymesterolone, COC, BE overnight, 56C (EI)
MAM, MOR, AM, MA, MDMA, MDA, BE, 100 Hydrochloric acid LLE (Toxi-Tubes A) CZEMS 15; 30 (COC) [121]
COC, EPH 0.1 M, overnight, (ESI; ion 100; (BE)
45C trap)
COC, BE, COET, NCOC 12 Enzymatic SPE (isolute SPE LCMS/MS [173]
hydrolysis columns) (ESI)
Ethyl glucuronide, COET, COC, EME, BE 50 Hydrochloric acid, SPE (Bond Elut GCMS (EI) ; 40 [174]
0.1 M, overnight Certify LRC)
COC, COET 50 Enzymatic SPME (100m GCMS (EI) 80; 400 (COC) [123]
hydrolysis, PDMS) 20; 400 (COET)
overnight, 37C
COC, BE 2050 Hydrochloric acid,No sample LCMS/MS 3; 10 (COC) [175]
0.1 M, 18 h, 45C cleanup (LCMS) (SACI) 20; 40 (BE)
SPE (C18; GCMS) GCMS (EI)
HER, MAM, MOR, COD, COC, BE,COET 50 Methanol, 5h, SPE (chromabond ELISA/ 10; 110 ( COC) [91]
50C (sonication) drug) GCMS (EI) 30; 260(BE)
50; 210 (COET)
AM, MA, MDMA, MDA, COC, COET, BE, 1050 Hydrochloric acid, SPE (Bakerbond GCMS (EI) ; 333 [176]
EME, COD, MAM, MOR, diazepam, 0.1 M, overnight, Narc-2 mixed-
nordiazepam, dihydrocodeine 50C mode)
AC: Acetylcodeine; AEME: Anhydroecgonine methyl ester; AM: Amphetamine; APCI: Atmospheric pressure chemical ionization; BE: Benzoylecgonine;
BUP:Buprenorphine; CBD: Cannabidiol; CBN: Cannabinol; COC: Cocaine; COD: Codeine; COET: Cocaethylene; CZE: Capillary zone electrophoresis;
EDDP:2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine; EI: Electron ionization; EME: Ecgonine methyl ester; EPH: Ephedrine; ESI: Electrospray ionization;
HER:Heroin; IPA: Isopropyl alcohol; LLE: Liquidliquid extraction; MA: Metamphetamine; MAM: 6-acetylmorphine; MDA: 3,4-methylenedioxyamphetamine;
MDEA:3,4-methylenedioxyethamphetamine; MDMA: 3,4-methylenedioxymethamphetamine; MET: Metadone; MOR:Morphine; NCOC:Norcocaine;
PHOLC:Pholcodine; SACI: Surface-activated chemical ionization; TFAA: Trifluoracetic acid; THC: D9 -tetrahydrocannabinol; THCCOOH: 11-nor-D9 -
tetrahydrocannabinol-9 carboxylic acid; THC-OH: 11-hydroxy- D9 -tetrahydrocannabinol; TOF: Time of flight.

Table4. Bioanalytical procedures for determination of cocaine and metabolites in oral fluid samples.
Compounds Sample Sample Sample Detection mode LOD (ng/ml); Ref.
amount (ml) collection preparation LOQ (ng/ml
AM, MA, MOR, MAM, MDA, MDEA, 0.15 Spitting No extraction LCMS/MS 0.1; ng/ml [12]
MDMA, COC, BE, THC, THC-COOH, KET, (protein
PHEN precipitation
with methanol)
AM, MA, MDA, MDMA, PHEN, COD, COC, BE, 0.5 QuantisalTM SPE (UCT LCMS/MS (Qtrap) 2; 2 [120]
MOR, HYCOD, HYDMOR, OXCOD, OXMOR Zsdau005)
AM, MDMA, THC, COC, MOR, COD, 0.5 Several LLE (ethyl GCMS (EI) [177]
diazepam,alprazolam devices acetate)
(comparison)
MOR, MAM, EDDP, MET, COC, BE, COET 1 Salivette MW-assisted LC-DAD 10; 50 [178]
LLE (chloroform)
LLE (Toxitubes A)
MOR, COD, MAM, MET, AM, MDA, 0.51 StatSure SPE (OASIS HLB) LCMS/MS (ESI) 500; 1000 [179]
MDMA, MDEA, MA, BE, COC, THC, Saliva
zolpidem, zopiclone, alprazolam, Sampler
clonazepam, oxazepam, nordiazepam,
lorazepam, flunitrazepam, diazepam,
diphenhydramine, amitriptyline

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Review | Barroso, Gallardo & Queiroz

Table4. Bioanalytical procedures for determination of cocaine and metabolites in oral fluid samples (cont.).
Compounds Sample Sample Sample Detection mode LOD (ng/ml); Ref.
amount (ml) collection preparation LOQ (ng/ml
Modafinil, selegiline, crotetamide, 1 LLE GCMS (EI) 5; 10 [119]
cropropamide, pentetrazol, EPH, NEPH, (chloroform/IPA)
sibutramine, COC, BE, EME, AM, MA,
COD,dihydrocodeine
AM, AM, MDA, MDEA, MDMA, MBDB, 0.3 Intercept SPE (HCX) ELISA/GCMS ;2 [180]
chlordiazepoxide, diazepam, lorazepam, (EI,NCI)/GCMS/MS
nordiazepam, oxazepam and temazepam, (EI)
BUP, NBUP, CBN, CBD, THC, THC-OH,
THCCOOH, AEME, BE, COET, COC, MET,
EDDP, AC, MAM, COD, dihydrocodeine,
HER, MOR
MOR, MAM, AM, MA, MDA, MDMA, 0.3 Spitting SPE (Oasis HLB) LCMS (ESI) 0.5; 1 [181]
MDEA, MBDB,COC, BE, THC
AM, MA, MDMA, MDA, MDEA, 1 Salivette SPE (Bond Elut GCMS (EI) 1.4; 4.1 (COC) [182]
phentermine, BE, COET, EME , THC, Certify) 2.6; 8.0 (BE)
THCCOOH, THC-OH, CBN, CBD, MAM, 4.2; 12.7 (EME)
MOR, COD, flurazepam, flunitrazepam, 2.4; 7.2 (COET)
chlorazepate, alprazolam, diazepam,
oxazepam, amitryptiline, paroxetine and
sertraline, haloperidol, chlorpromazine,
fluphenazine chlormethiazole, loratidine,
hydroxyzine, diphenhydramine,
valproicacid,gabapentin
MOR, MAM, COD, BUP, MET, AM, MA, 0.5 Intercept LLE LCMS/MS (ESI) ; < 0.78 (COC) [122]
MDA, MDMA, MDEA, COC, BE, THC, LSD, (ethylacetate: ; 7.2 (BE)
alprazolam, bromazepam, clonazepam, heptane, 4:1)
7-aminoclonazepam, diazepam,
N-desmethyldiazepam, 3OHdiazepam,
fenazepam, flunitrazepam,
7-aminoflunitrazepam, lorazepam,
nitrazepam, 7-aminonitrazepam, oxazepam,
zopiclone, zolpidem, carisoprodol,
meprobamate
AM, MA, MOR, MAM, COD, COC, BE, 1 QuantisalTM SPE (Oasis LCMS/MS (ESI) [183]
MET, oxazepam, THC MCXCOC)
(Bond Elut
CertifyBE)
COC, BE, COET, EME, EEE, ECG, pHBE, 1 Salivette No extraction LCMS/MS (APCI) 0.255; 110 [184]
mHBE, BE, pHCOC, mHCOC, NCOC, NCOET, (protein
MET, EDDP, AC, MAM, COD, NCOD, NMOR, precipitation
HER, noscapine, papaverine, MOR with acetonitrile)
COC, BE Stimulation GCMS (EI) 1; 8 [157]
citric acid
COC, AEME, EME, COET 0.5 Salivette Automated SPE GCMS/MS (PCI) 0.10.5; 25 [6]
(HCX Isolute)
THC, THC-OH, THCCOOH , AM, MA, 0.5 Drger Drug SPE (Bond Elut GCMS (EI) 20; (COC, [185]
MDMA, MDA, MDEA, MBDB, MDB, COC, Test oral fluid Certify HF) EME)
EME, BE, MOR, MAM, COD, collection 10; (BE)
dihydrocodeine, MET, EDDP device
49 illicit drugs including COC, BE, EME, 1 Omni-Sal SPE (Bond Elut GCMS (EI) 0.3; 0.9 (EME) [186]
COET Certify) LCMS/MS (ion trap) 3.4; 11.4 (BE)
0.9; 3.1 (COC)
1.0; 3.4 (COET)
Dihydrocodeine, COD, MOR, MAM, HER, 1 CRS opiates Dilution with Cozart RapiScan/ 15; (GCMS) [98]
COC, BE, COET and cocaine buffer and SPE GCMS
test system

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Table4. Bioanalytical procedures for determination of cocaine and metabolites in oral fluid samples (cont.).
Compounds Sample Sample Sample Detection mode LOD (ng/ml); Ref.
amount (ml) collection preparation LOQ (ng/ml
AM, MA, MDA, MDMA, COC, BE, MOR, 0.25 Intercept SPE LCMS/MS (ESI) 0.20.5; 2 [187]
MAM, COD (OasisMCX)
THC, MDMA, MA, MDA, opiates and Cozart SPE ELISA/GCMS/ [188]
cocaine and metabolites, benzodiazepines LCMS/MS
30 drugs of abuse including COC and BE 0.25 Spitting SPE (HCX) Fast GC/EIMS [117]
COC, BE, COET 0.1 Salivette SPE (IST LCMS-TOF (ESI) 1; 10 [189]
confirm HCX)
COC, BE, COET 1 Cozart SPE (Bond Elut ELISA/GCMS 15; 15 [100]
Rapiscan Certify)
oral fluid
collector
COC, BE, EME, COET 1 Salivette Dilution with Cozart RapiScan 2.5; 2.5 [101]
buffer and SPE Oral Fluid Drug
(Clean Screen Testing System/
ZSDAU020) ELISA/GCMS
COC, EME, BE 1 Salivette LLE GCMS (CI) 0.9 ng/ml; [190]
(ToxitubesA) 3.0ng/m (EME)
2.2 ng/ml;
7.4ng/ml (COC)
0.2 ng/ml;
0.8ng/ml (BE)
EDDP, COC, COET, AM, MA, MDMA, 0.2 Cozart DI-SPME and GCMS (EI) 20 and 100; 50 [191]
MDEA, MBDB, CBD, CBN, THC Rapiscan HS-SPME and 250 (COC)
System (30-m PDMS) 5 and 20; 10
and 100 (COET)
COC, AM, MA, THC, ethanol 2 Salivette SPME GC-FID 5; 5 [192]
(100-m PDMS)
COC, BE, COET, EME, AEME, EEE, ECG, p 0.2 No extraction LCMS/MS (APCI) 0.255; 510 [193]
and mHBE, p and mHCOC, NBE, NCOC, (protein
NCOET, MET, EDDP, AC, MAM, COD, precipitation
NCOD, NMOR, HER, MOR, propoxyphene, with acetonitrile)
noscapine, papaverine, methadol
COC, BE, EME, COET 1 Salivette Dilution with ELISA/GCMS (EI) 2.5; 2.5 [99]
buffer and SPE
(Clean Screen
ZSDAU020)
THC, BE, MOR, MAM, COD, PHEN, 1.2 Intercept Dilution with ELISA/GCMS/MS ;0.6 [194]
AM,MA DOA oral preservative
specimen solution/LLE
collection
device
THC, THC-OH, THCCOOH, AM, MA, 12 Spitting SPE GCMS (EI) 1; 2.5 (BE) [195]
MDMA, MDA, MDEA, MBDB, BE, COC, 3; 5 (COC,
EME, AEME, MOR, COD, MAM EME,AEME)
AM, MA, MDA, MDMA, MDEA, MOR, 0.2 Spitting SPE (Bond Elut LCMS-TOF (ESI) 0.29; 2 (BE) [196]
COD, COC, BE Certify) 0.22; 2 (COC)
COC, BE, EME, AEME, MOR, MAM, COD, 12 Spitting SPE (Bond Elut Drugwipe/GCMS [197]
AM, MDA, MDMA, MDEA, MBDB, EPH, Certify) (EI)
THC, CBN, CBD, THC-OH, THCCOOH
AC: Acetylcodeine; AEME: Anhydroecgonine methyl ester; AM: Amphetamine; APCI: Atmospheric pressure chemical ionization; BE: Benzoylecgonine; BUP: Buprenorphine;
CBD: Cannabidiol; CBN: Cannabinol; CI: Chemical ionization; COC: Cocaine; COD: Codeine; COET: Cocaethylene; DI: Direct immersion; ECG: Ecgonine; EDDP: 2-ethylidene-
1,5-dimethyl-3,3-diphenylpyrrolidine; EEE: Ecgonine ethyl ester; EI: Electron ionization; EME: Ecgonine methyl ester; ESI: Electrospray ionization; FID: Flame ionization
detector; HER: Heroin; IPA: Isopropyl alcohol; MA:Metamphetamine; MAM: 6-acetylmorphine; MBDB: N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamide;
MDEA:3,4-Methylenedioxyethamphetamine; MDMA: 3,4-Methylenedioxymethamphetamine; MET: Metadone; mHBE: Meta-hydroxybenzoylecgonine; mHCOC:
meta-Hydroxycocaine; MOR:Morphine; NBUP: Norbuprenorphine; NCOET: Norcocaethylene; NCOC: Norcocaine; NCOD:Norcodeine; NMOR:Normorphine; PCI: Positive
chemical ionization; pHBE: para-Hydroxybenzoylecgonine; pHCOC: para-Hydroxycocaine; SPME: Solid-phase microextraction; THC: D9-tetrahydrocannabinol;
THCCOOH:11-nor-D9-tetrahydrocannabinol-9 carboxylic acid; THC-OH: 11-hydroxy- D9-tetrahydrocannabinol; TOF:Time of flight.

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Table5. Bioanalytical procedures for determination of cocaine and metabolites in sweat samples.
Compounds Analyte extraction/cleanup Detection LOD; LOQ Ref.
mode
AEME, EME, MET, COC, BE, Sodium acetate buffer (pH 4.0)/SPE GCMS (EI) 2.5 ng/patch; 5 ng/patch [114]
COD, MOR, AC, MAM, HER (Clean Screen ZSDAU020)
Nicotine, cotinine, caffeine, 2ml HCl 0.1 M (2h at RT)/LLE GCMS (EI) 20 ng/patch; 50 ng/patch for all compunds [198]
MET, EDDP, COC, BE, EME, (chloroform:IPA, 9:1)
MOR, COD, MAM
Opiates and cocaine Sodium carbonate/sodium GCMS (EI) [199]
bicarbonate buffer 0.2 M pH 9/SPE IMS
(chromabond drug)
COC , BE, m and p HCOC, m Sodium acetate buffer (pH 4.0) GCMS (EI) 2.5 ng/patch (COC, BE, EME, EEE, COET) [200]
and pHBE, EME, COC, COET, 0.5M/SPE (Clean Screen DAU) 5.0 ng/patch for all other analytes
EEE, EME, NCOC
COC, BE, EME 0.2 M acetate/methanol (25:75) GCMS (EI) 4 ng/ml (COC) [201]
buffer (pH 5.0)/ 2 ng/ml (BE)
2 ng/ml (EME)
COC, COET 0.2 M acetate buffer (pH 5.0)/SPME GCMS (EI) 12.5 ng/patch [126]
(100-m PDMS)
COC, BE, EME Methanol/0.2 M acetate, pH5.0, GCMS 2 ng/patch [202]
(3:1)/SPE (Bond Elut Certify) (Ion Trap)
COC, BE, EME Methanol/0.2 M sodium acetate RIA/ 4 ng/patch [203]
pH5, (75:25)/SPE () GCMS (CI)
COC, BE, AEME, ecgonidine 0.2m acetate/methanol (25:75) GCMS (EI) [204]
buffer pH 5.0/SPE (Bond Elut
Certify)
COC, BE 0.1 M HCl/SPE (DAU SPE columns) ELISA/ 4 ng/ml; (COC) [205]
GCMS (CI) 2 ng/ml; (BE)
BE Methanol/0.2-M pH 5 sodium GCMS (EI) ; 4 ng/patch [206]
acetate, (75:25)/SPE ()
THC, THC-OH, THCCOOH; AM, Acetate buffer 0.1 M (pH 4)/SPE () GCMS (EI) 1 ng/wipe; 2.5 ng/wipe (BE) [195]
MA, MDMA, MDA, MDEA, 3 ng/wipe; 5 ng/wipe (COC, EME, AEME)
MBDB, BE, COC, EME, AEME,
MOR, COD, MAM
COC, BE, EME, HER, MAM, Methanol/0.2M pH 5 sodium RIA/ELISA 25 ng/patch;(COC, BE), 50 ng/patch; [207]
MOR acetate, 75:25 (EME)using ELISA
2.5 ng/patch; (COC), 50 ng/patch; (BE),
100 ng/patch;(EME) using RIA
COC, BE, HER, AM, MA, MDMA 0.1-M hydrochloric acid/SPE GCMS (EI) 2 ng/patch; [34]
(Zymark Rapid Trace)
COC, COD, BE, EME , MOR, Deionized water/SPE (Clean GCMS (EI) 1.25 ng/patch; 2.5 ng/patch (COC, BE, EME) [33]
NCOD, MAM, COET, NCOET, ScreenDAU) 1.255.0 ng/patch; for all other analytes
AEME, EEE
AC: Acetylcodeine; AEME: Anhydroecgonine methyl ester; AM: Amphetamine; BE: Benzoylecgonine; CI: Chemical ionization; COC: Cocaine; COD: Codeine;
COET:Cocaethylene; EDDP: 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine; EEE: Ecgonine ethyl ester; EI: Electron ionization; EME: Ecgonine methyl ester;
HER:Heroin; IMS: Ion mobility spectrometry; IPA: Isopropyl alcohol; LLE: Liquidliquid extraction; LOD: Limit of detection; LOQ: Limit of quantitation;
MA:Metamphetamine; MAM: 6-acetylmorphine; MBDB: N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamide; MDA: 3,4-methylenedioxyamphetamine;
MDEA:3,4-methylenedioxyethamphetamine; MDMA: 3,4-methylenedioxymethamphetamine; MET:Metadone; mHBE:Meta-hydroxybenzoylecgonine;
mHCOC:meta-Hydroxycocaine; MOR:Morphine; NCOC: Norcocaine; NCOET: Norcocaethylene; PDMS: Polydimethylsiloxane; pHBE: Para-hydroxybenzoylecgonine;
pHCOC:Para-hydroxycocaine; RIA: Radioimmunoassay; SPME: Solid-phase microextraction; THC: D9 -tetrahydrocannabinol; THCCOOH:11-nor-D9 -
tetrahydrocannabinol-9 carboxylic acid; THC-OH: 11-hydroxy- D9 -tetrahydrocannabinol.

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 Bioanalytical methods for the determination of cocaine & metabolites | Review

Table6. Bioanalytical procedures for determination of cocaine and metabolites in meconium samples.

Compounds Sample Sample preparation Detection mode LOD; LOQ Ref.

amount (ng/g)
(g)
AM, MA, pOHMAMP, COC, BE, COET, mHBE, 0.25 SPE (CleanScreen DAU) LCMS/MS (ESI) 0.5; 1 (COC, COET) [109]
nicotine, cotinine, hydroxycotinine, MAM, MOR, 1; 1 (BE)
COD, HYMOR, HYCOD, OXCOD, MET, EDDP, 2.5; (mHBE)
BUP, NBUP
COC, BE, COET, MOR, MAM, MOR-3-G, LCMS 3 (COC) [208]
MOR-6-G, COD, AM, MA, MDA, MDMA, GCMS 4 (BE, COET)
MDEA, THC, THC-OH, THCCOOH
COC, BE, mHBE, MOR, COD, HYCOD, 0.50 Buffer/SPE () GCMS 5 (COC, BE, mHBE) [209]
HYMOR, OXCOD, oxazepam, cotinine,
3-hydroxycotinine, norcotinine, nornicotine,
nicotine, BUP, NBUP, buprenorphine
glucuronide, norbuprenorphineglucuronide
COC, BE 0.20 Methanol and dilution RIA/GCMS 0.05 [171]
with phosphate-
buffered saline
COC, BE, COD, MOR, MAM 0.5 Methanol/SPE GCMS (EI) 30; 40 [210]
(OasisHLB)
COC, BE, COET, MOR, MAM, MOR-3-G, LCMS 3 (COC) [211]
MOR-6-G, COD, AM, MA, MDA, MDMA, 4 (BE, COET)
MDEA, THC, THC-OH, THCCOOH
mHBE, pHBE 1 Methanol/SPE (Bond LCMS (ESI) 1.5; 0.005 (pHBE) [212]
Elut Certify) 1.3; 5 (mHBE)
COC, BE, COET, mHB, amphetamines, FPIA/GCMS 25 [213]
cannabinoids, MOR, COD, benzodiazepines,
barbiturates
COC, BE, COET, MOR, MAM, MOR-3-G, 1 Methanol/SPE (Bond LCMS (ESI) 0.9; 3 (COC) [214]
MOR-6-G, COD Elut Certify) 1.2; 4 (BE, COET)
Opiates, cannabis, COC, amphetamines, LSD, FPIA/EMIT/GCMS [215]
benzodiazepines, MET
COC, BE, opiates, cannabis, benzodiazepines, 0.2 Methanol and dilution ELISA/GCMS 50 [216]
MET, barbiturates with phosphate-
buffered saline
COC, BE, COET, mHBE, MOR, COD, MAM, EMIT/GCMS 50 [217]
HYMOR, HYCOD, THC, AM, MA, PCP
AEME, ECG, EME, BE, COC, COET, NBE, 0.5 Methanol/SPE (Bond EMIT II/LCMS/MS 15 [218]
NCOET, NCOC, cocaine N-oxide, mHBE, pHBE, Elut Certify) (ESI)
mHCOC, pHCOC)
NCOC, BE, COC, NBE, EME, COET, mHBE, pHBE, 1 Methanol/SPE (Clean GCMS (EI) 1025 [219]
mHCOC, pHCOC Screen ZSDAU020)
HCCOOH, COC, BE, phencyclidine, MOR, 1 Methanol/Assay buffer EMIT/GCMS (EI) 50; 50 (BE) [220]
morphine, amphetamines (Imunnoassay)
40% potassium
phosphate/LLE
(chloroform:IPA, 9:1)
AEME: Anhydroecgonine methyl ester; AM: Amphetamine; BE: Benzoylecgonine; BUP: Buprenorphine; COC: Cocaine; COD: Codeine; COET: Cocaethylene;
ECG:Ecgonine; EDDP: 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine; EI: Electron ionization; EME: Ecgonine methyl ester; ESI: Electrospray ionization;
EMIT:Enzyme-multiplied immunoassay technique; FPIA: Fluorescence polarization immunoassay; HYCOD: Hydrocodone; HYMOR:Hydromorphone; IPA: Isopropyl
alcohol; MA: Metamphetamine; MAM:6-acetylmorphine; MDA: 3,4-methylenedioxyamphetamine; MDEA: 3,4-methylenedioxyethamphetamine;
MDMA:3,4-methylenedioxymethamphetamine; MET: Metadone; mHBE: Meta-hydroxybenzoylecgonine; mHCOC:Meta-hydroxycocaine; MOR:Morphine;
MOR-3-G:Morphine-3-glucuronide; MOR-6-G: Morphine-6-glucuronide; MS: Mass spectrometry; NBUP: Norbuprenorphine; NCOC: Norcocaine;
NCOET:Norcocaethylene; OXCOD: Oxycodone; pHBE: para-Hydroxybenzoylecgonine; pHCOC: Para-hydroxycocaine; RIA: Radioimmunoassay; THC: D9 -
tetrahydrocannabinol; THCCOOH: 11-nor-D9 -tetrahydrocannabinol-9 carboxylic acid; THC-OH: 11-hydroxy- D9 -tetrahydrocannabinol; TOF: Time of flight; UPLC:Ultra-
performance LC.

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Review | Barroso, Gallardo & Queiroz

Table7. Bioanalytical procedures for determination of cocaine and metabolites in other biological samples.
Compounds Specimen Sample Sample Detection LOD; LOQ Ref.
amount preparation mode
COC, BE, COET Pericardial fluid 1ml SPE (Bond Elut GCMS (EI) 15.9 ng/ml; 53.0 ng/ml (BE) [47]
Certify) 8.7 ng/ml; 29.0 ng/ml (COC)
3.9 ng/ml; 13.0 ng/ml (COET)
COC, MOR, COD, Pericardial fluid 1ml SPE (Bond Elut EMIT/ [48]
BE,COET Certify) GCMS (EI)
COC, BE, EME Bile 1ml SPE (Bond Elut GCFID 18 ng/ml; 60 ng/ml (COC) in bile [51]
Vitreous humor Certify) 25 ng/ml; 82 ng/ml (BE) in bile
and vitreous humour
27 ng/ml; 89 ng/ml (EME) in bile
24 ng/ml; 80 ng/ml (COC) in
vitreous humour
26 ng/ml; 85 ng/ml (EME) in
vitreous humour
COC, BE, MOR, COD, Vitreous humor LLE Cozart 5 ng/ml; 20 ng/ml [50]
dihydrocodeine, MET, Rapiscan/
EDDP, MAM, AM, MA GCMS (EI)
COC, BE Vitreous humor ml (vitreous SPE (Clean CEDIA [53]
Bile, humor) Screen (vitreous
Stomachcontents, 1ml (bile, ZSDAU020) (bile, humour)/
Solid-organ tissue bladder wash) bladder wash GCMS (EI)
MOR, MAM, COD, Vitreous humour 2ml SPE (ZSDAU020 ELISA/ 50 ng/ml; 50 ng/ml [110]
HYCOD, OXCOD, Clean Screen) GCMS (EI)
HYMOR, BE, COC, EME,
COET
COC, BE, MOR, MET, Nails 10mg SPE (Bond Elut GCMS (EI) 0.025 ng/mg; 0.025 ng/mg [57]
caffeine, nicotine, Certify LRC)
cotinine and nalorphine
COC, paracetamol Nails Raman [221]
spectroscopy
AEME, EME, COC, COET, Nails 100mg LLE (chloroform: GCMS (EI) 0.10 ng/mg (AEME) [58]
BE IPA:n-heptane, 0.50 ng/mg (EME)
50:17:33) 0.050 ng/mg (COC)
0.050 ng/mg (COET)
0.15 ng/mg (BE)
COC, BE, NCOC Nails 5mg SPE (Bond Elut GCMS (EI) 5 ng/mg (COC) [59]
Certify) 7 ng/mg (BE )
9 ng/mg (NCOC)
MOR, MAM, COC Nails 50mg LLE (chloroform/ GCMS (EI) 0.1 ng/mg [60]
IPA, 3:1)
SPE (Bond Elute
Certify)
COC, BE, NCOC, EME, Nails 2030mg LLE (chloroform/ GCMS (EI) 0.1 ng on column [61]
MAM, COET, nalorphine, IPA, 9:1; ethyl
HYCOD, COD, OXCOD, acetate/IPA, 9:1)
MOR
COC, BE Skin Skin, sebum SPE (Bond Elute GCMS (EI) [73]
Sebum and stratum Certify)
Interstitial fluid corneum ()
Stratum corneum 0.09ml of
interstitial fluid
AM, MA, COC, BE, COD, Skin 50mg SPE (ZSDAU020 GCMS (CI) 2.5 ng/biopsy; 2.5 ng/biopsy [72]
NCOC, COET, NCOET, Clean Screen) (COC, BE, COET)
AEME, MAM, MOR, LLE (hexane) 1.25 ng/biopsy; 2.5 ng/biopsy
EME, EEE (NCOC, NCOET)
5.0 ng/biopsy; 5.0 ng/biopsy
(EME, EEE)

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 Bioanalytical methods for the determination of cocaine & metabolites | Review

Table7. Bioanalytical procedures for determination of cocaine and metabolites in other biological samples (cont.).
Compounds Specimen Sample Sample Detection LOD; LOQ Ref.
amount preparation mode
MOR, BE, amitriptyline, Adipose tissue 13 g LLE (chloroform) GCMS (EI) [74]
nortriptyline, COC, Skin
OXCOD, tramadol,
diazepam, nordiazepam,
alprazolam, MET,
diphenhydramine, COET
COC, COET, BE, NBE, Muscle 3g SPE (Bond Elut GCMS (EI) 2 ng/ml; 2 ng/ml (COC, BE, [142]
NCOC, ECG, EME, mHBE, Certify) NCOC, EME, COET, NCOET, EEE)
AEME, NCOET, EEE 25 ng/ml; 25 ng/ml (NBE)
640 ng/ml; 800 ng/ml (ECG)
50 ng/ml; 50 ng/ml (mHBE)
13 ng/ml; 13 ng/ml (AEME)
Hydroxycotinine, nicotine, Brain 100mg SPE (ZSDAU020 LCMS/MS 2.5 pg/mg; 5 pg/mg (COC) [71]
COC, MAM, MOR, Clean Screen) (ESI) 12.5 pg/mg; 25 pg/mg (BE)
Cotinine, BE, EME, COD
MOR, BE, dihydrocodeine Brain 200mg SPE (IsoluteTM ) GCMS (EI) [222]
MOR, MAM, COC, Teeth 1g LLE (chloroform/ GCMS (EI) 2.5 ng/g; 7.5 ng/g (COC, BE, [223]
BE,COET IPA, 9:1) COET)
AEME: Anhydroecgonine methyl ester; AM: Amphetamine; BE: Benzoylecgonine; CEDIA: Cloned enzyme donor immunoassay; COC: Cocaine; COD: Codeine;
COET:Cocaethylene; ECG: Ecgonine; EDDP: 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine; EEE: Ecgonine ethyl ester; EI: Electron ionization; EME: Ecgonine
methyl ester; EMIT:Enzymemultiplied immunoassay technique; ESI: Electrospray ionization; FID: Flame ionization detector; HYMOR:Hydromorphone; IPA: Isopropyl
alcohol; LLE: Liquidliquid extraction; MA: Metamphetamine; MAM:6-acetylmorphine; MET:Metadone; mHBE: Meta-hydroxybenzoylecgonine;
HYCOD:Hydrocodone; MOR:Morphine; NCOC: Norcocaine; NCOET: Norcocaethylene; OXCOD:Oxycodone.

easily automated, providing greater sample
throughput.
Nevertheless, there are some biological speci-
mens for which additional sample treatment
procedures are required. This is the case with
hair, since sample decontamination and analyte
extraction from within the matrix are necessary
prior to sample cleanup and chromatographic
analysis. These decontamination and extraction
procedures, including those aiming to determine
cocaine and metabolites in hair samples, have
been reviewed [22,24,129,132,133] and involve rins-
ing the hair with different solvents and time-
consuming incubations at mild conditions.
However, methods for the analysis of cocaine
and metabolites in hair after short incubations
have been published [23,91,134136] . This is usually
the most critical and time-consuming step in hair
analysis. Indeed, in order to improve detection
and allow precise and accurate quantitation of
the drugs, these must be quantitatively extracted
from the hair shaft, that is, this extraction must
be complete. In fact, new analytical methods
should demonstrate their ability to extract the
entire drug that is present, but unfortunately
most published papers do not discuss in detail
this important issue concerning hair analysis.
MS methods are the state-of-the-art in the
determination of cocaine and metabolites in
biological specimens and this is mainly due to
the high sensitivity and specificity achieved.
Indeed, scientists are now able to detect drug
amounts that were unthinkable just a few years
ago. In addition, this type of technology allows
the unequivocal identification of the present
compound(s) and this is of outmost importance
from a forensic point of view, particularly in the
case of implications of the test result on the
examinees professional life or freedom.
Until a few years ago, GCMS was the main
analytical technique for quantitative determina-
tion of cocaine in biological specimens, as the
desired sensitivity could be met. However, the
introduction of alternative specimens, such as
oral fluid or hair, has brought new needs con-
cerning sensitivity. While cocaine levels in these
specimens can usually be detected using GC
MS, the fact that the sample is often limited is of
concern. Therefore, multi-analyte methods are
desired for the analysis of these specimens and
these are more efficiently developed using LC
MS. This does not mean that GCMS is inad-
equate for multi-analyte analysis, however; in
fact, multi-analyte methods have been published
for oral fluid [118] . Nonetheless, different drug
classes often require different derivatizing rea-
gents and this makes multi-analyte procedures
more laborious and time consuming. In addi-
tion, the background noise level is usually higher
in derivatized extracts and for some compounds

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Review | Barroso, Gallardo & Queiroz
the active sites in the molecules are not easily
accessed by the derivatizing reagent, originat-
ing inefficient or even incomplete reactions.
Furthermore, analysis of derivatized samples in
GCMS leads to a higher necessity for system
maintenance and this is important when a large
number of samples is expected to be analyzed.
On the contrary, in LCMS methods most
analytes do not need to be derivatized prior
to chromatography and this enables the use of
simpler sample-preparation procedures.
Electrospray ionization (ESI) and atmospheric
pressure chemical ionization are the most wide-
spread ionization techniques in LCMS meth-
ods. However, assessing which of these suits
better for developing a new procedure must be
made case by case. For example, atmospheric
pressure chemical ionization is more appropri-
ate for unionized analytes, while ESI interfaces
permit MS analyses of molecules in the molecu-
lar weight range of drugs of abuse. In spite of the
fact that it is more susceptible to matrix effects,
ESI is the most used ionization technique.
These matrix effects can cause ion suppression
or enhancement phenomena and it is manda-
tory that these are evaluated and documented
during method development and validation.
These effects are challenging the common per-
ception that the use of LCMS/MS practically
guarantees selectivity.
Cocaine and metabolites have been deter-
mined in biological samples by means of the
above mentioned techniques and analytical
methods published in the last decade (except
for hair, blood and urine analysis, for which a
3year window was used) are summarized in
Tables 17. Studies in which cocaine analysis is
described (not necessarily methodology papers)
were selected through the references list of
known published papers. Moreover, additional
papers were found by using the PubMed data-
base with the following search strings: cocaine
and blood; cocaine and urine; cocaine and
hair; cocaine and oral fluid or saliva; and
cocaine and meconium. Some of the reviewed
papers also aimed to detect other drug classes,
therefore presenting different limits and/or
sample preparation approaches. In these situa-
tions, only specific data related to cocaine and
metabolites is shown.
Future perspective
Cocaine is one of the most abused illicit drugs
worldwide and urine and blood samples have
been the most utilized biological specimens to
992 Bioanalysis (2009) 1(5)
document human exposure to it. These speci-
mens are widely used in most forensic toxicology
laboratories; however, valuable information can
be obtained by analysis of other samples, such as
hair, oral fluid or sweat. These unconventional
biological specimens should not be seen as substi-
tutes for traditional ones, but as complementary
samples. Indeed, there is a growing tendency to
use less invasive collection procedures to obtain
biological specimens for drug testing and this
can be easily assessed from the data in the tables,
in which the number of papers utilizing oral fluid
or hair surpassed the use of blood or urine.
Over the last few years a number of articles have
been published on cocaine determination in a vari-
ety of biological specimens and the application of
LCMS is increasing. Indeed, analytical instru-
ments are becoming more and more sensitive and
this allows the determination of smaller amounts
of the drugs and their metabolites. While the main
analytical problems concerning the determination
of cocaine and metabolites in urine, blood, hair
and oral fluid are in general adequately dealt with,
more data is needed concerning other specimens,
such as sweat and meconium.
Meconium is used to assess in utero drug expo-
sure. The metabolic profile of cocaine in newborns
based on analysis of meconium differs from that of
urine, as discussed above. Therefore, it is possible
that further cocaine metabolites will be detected
in meconium in the future, due to the increasing
sensitivity of analytical methods.
It is expected that analytical equipment will
become even more sensitive in the future and
the use of highly sensitive and accurate MS, such
as time-of-flight technology, will become more
widespread. In addition, there is also a trend for
reducing sample size and this may be accomplished
by the use of online systems combining sample
preparation and chromatography; these are already
being used for the analysis of cocaine in biological
samples. This reduction in sample size has obvious
advantages from the analytical point of view, since
matrix-borne compounds will affect analysis to a
lesser extent. Moreover, miniaturized instruments
(e.g., nano-LC systems) have been developed
and are being optimized, in spite of the fact that
their application to cocaine analysis in biological
specimens is not yet documented.
In addition, sample cleanup procedures are also
evolving: new techniques are being developed and
their applications to bioanalysis are being published
every year. Amongst these, the use of environmen-
tal friendly methods appears to be the guiding
principle for bioanalytical sciences in the future.
future science group
 Bioanalytical methods for the determination of cocaine & metabolites | Review
Financial & competing interests disclosure includes employment, consultancies, honoraria, stock
The authors have no relevant affiliations or financial ownership or options, expert testimony, grants or patents
involvement with any organization or entity with a received or pending, or royalties.
financial interest in or financial conflict with the subject No writing assistance was utilized in the production
matter or materials discussed in the manuscript. This of this manuscript.

Executive summary
Disposition of cocaine
Cocaine, one of the most abused drugs worldwide, can be administered in several forms, and can be snorted, inhaled or injected

intravenously or intramuscularly.
Cocaine has a short half-life in plasma, and the majority of a dose is metabolized to ecgonine methyl ester (EME) and
benzoylecgonine(BE).
Little cocaine is excreted unchanged, while its major metabolites, EME and BE, account for 8090% of urinary metabolites.
Cocaine detection in biological specimens
Blood plasma and urine

These are by far the most used specimens for the detection of cocaine in forensic toxicology.
Blood and plasma samples are relevant media in the assessment of short-term use of drugs of abuse, and the analysis of these
specimens is mandatory in many countries to identify impaired drivers in driving under the influence of drugs situations.
The past use of cocaine can be assessed even when it is no longer present in blood, by means of its main metabolites, BE and EME,
which have longer plasma half-lives.
Urine is the specimen of choice for many laboratories due to the often higher concentrations at which the drugs (and metabolites)
appear in this specimen when compared to blood or plasma, the ease of sampling and the low invasiveness of the
collectionprocedure.
One important drawback of urinalysis is the possibility of sample adulteration or substitution.
Hair

The most widely used alternative specimen in documenting drug exposure.

The most striking fact concerning hair analysis is the larger detection window, typically weeks to months (depending on the length
of the hair shaft).
The possibility of reporting a false-positive result derived from environmental contamination is the most important drawback of
hairanalysis.
Oral fluid
Abused drugs are readily detectable in oral fluid, and can show similar pharmacokinetics to plasma.
The oral fluid-to-plasma ratio of cocaine is approximately 3, and this compound and its metabolites can be detected for up to 17 h
after the last dose in self-reported users, showing a longer terminal elimination half-life than in plasma.
The possibility of correlation to plasma levels makes this specimen extremely valuable in determining impaired driving situations.
Sweat, meconium and other samples
Sweat analysis is scarcely performed on a routine basis, since the estimation of an accurate volume is difficult, impairing the
evaluation of the obtained concentrations.
Cocaine concentration in sweat depends on the collection device and the collection site.
Meconium, umbilical cord tissue and blood, vernix caseosa and amniotic fluid can be used to document prenatal drug exposure.
Meconium testing should include m-hydroxybenzoylecgonine to avoid false negative results, since this metabolite is present in this
specimen at significant amounts.
Other biological specimens for cocaine detection include pericardial fluid, vitreous humour, nails and several organs and tissues
(brain, skeletal muscle and bones).
Bioanalytical procedures for cocaine detection in biological specimens
Immunoassays

Extremely useful, since they allow the saving of time and money, which would be wasted in more expensive confirmatory methods
(usually mass spectrometric-based techniques), while most of the samples would be negative.
The presumptive positive samples must be confirmed by more specific techniques, namely mass spectrometric methods.
On-site portable devices can be used for the assessment of drug impaired driving situations and in workplace scenarios. The advantages
of on-site testing are rapid turn around times, reduced costs and the fact that the test can be carried out virtually anywhere.
Sample cleanup procedures
Biological specimens are complex samples, and therefore cannot be directly analysed
SPE, liquid-liquid extraction (LLE) and solid-phase microextraction are the most used sample cleanup procedures for cocaine
determination in biological samples.

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Review | Barroso, Gallardo & Queiroz
Executive summary (cont.)
Instrumental analysis
Mass spectrometric methods are the state of the art in the determination of cocaine and metabolites in biological specimens.
GCMS technology is available in most laboratories nowadays, and this is still the main technique for confirmation and quantitation
of drugs and their metabolites in biological specimens.
When the amount of sample available for analysis is limited, GCMS may not provide the desired sensitivity. Therefore more
sensitive techniques, such as LCMS(MS) may be a prerequisite.
LCMS permits the analysis of non-volatile, labile and/or high molecular weight compounds.
ESI and atmospheric pressure chemical ionization have become the most widespread ionizationtechniques.
The main drawback of LCMS procedures is their susceptibility to be affected by matrix constituents (e.g., ion suppression or
enhancement phenomena), and this assumes more relevance when ESI interfaces are used.
Matrix effects must be assessed and documented during method development and validation.

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