Anal Bioanal Chem (2007) 388:1437–1453

DOI 10.1007/s00216-007-1245-8

REVIEW

Bioanalytical procedures for determination of drugs of abuse

in oral fluid
Nele Samyn & Marleen Laloup & Gert De Boeck

Received: 23 January 2007 / Revised: 2 March 2007 / Accepted: 6 March 2007 / Published online: 3 April 2007
# Springer-Verlag 2007

Abstract Recent advances in analytical techniques have BSTFA N,O-Bis(trimethylsilyl)-trifluoroacetamide

enabled the detection of drugs and drug metabolites in oral DMSO dimethyl sulfoxide
fluid specimens. Although GC–MS is still commonly used DUID driving under the influence of drugs
in practice, many laboratories have developed and success- ECCI electron capture chemical ionization
fully validated methods for LC–MS(–MS) that can detect a ECD electron capture detection
large number of compounds in the limited sample volume EDDP 2-Ethylidene-1,5-dimethyl-3,3-
available. In addition, several enzyme immunoassays have diphenylpyrrolidine
been commercialized for the detection of drugs of abuse in EEE ecgonine ethyl ester
oral fluid samples, enabling the fast screening and selection EI electron impact
of presumably positive samples. A number of concerns are EIA enzyme immunoassay
discussed, such as the variability in the volume of sample EME ecgonine methyl ester
collected and its implications in terms of quantitative ESI electrospray ionization
measurements, and the drug recoveries of the many FA formic acid
different specimen collection systems on the market. GC-MS gas chromatography–mass spectrometry
Additional considerations that also receive attention are GC-GC- two-dimensional gas chromatography–
the importance of providing complete validation data with MS mass spectrometry
respect to analyte stability, matrix effect, and the choice of GC-MS- gas chromatography–tandem mass
collection method. MS spectrometry
HFBA heptafluorobutyric acid anhydride
Keywords Drugs of abuse . Oral fluid . Analytical methods . HFIP 1,1,1,3,3,3-Hexafluoro-2-propanol
Review HMA 3-Hydroxy-4-methoxyamphetamine
HMMA 4-Hydroxy-3-methoxymethamphetamine
Abbreviations HPLC high-pressure liquid chromatography
6-AM 6-Acetylmorphine LC-MS liquid chromatography–mass spectrometry
AGP α1-Acid glycoprotein LC-MS- liquid chromatography–tandem mass
AEME anhydroecgonine methyl ester MS spectrometry
APCI atmospheric pressure chemical ionization LLE liquid–liquid extraction
BDB 3,4-(Methylenedioxyphenyl)-2-butanamine LOD limit of detection
BE benzoylecgonine LOQ limit of quantification
LSD lysergic acid diethylamide
N. Samyn (*) : M. Laloup : G. De Boeck MBDB N-Methyl-1-(3,4-methylenedioxyphenyl)-2-
Laboratory of Toxicology, National Institute of Criminalistics butanamine
and Criminology (N.I.C.C.), Federal Public Service Justice,
Vilvoordsesteenweg 100,
MBTFA N-Methyl-bis(trifluoramide)
1120 Brussels, Belgium MDA 3,4-Methylenedioxyamphetamine
e-mail: nele.samyn@just.fgov.be MDEA 3,4-Methylenedioxyethylamphetamine
1438 Anal Bioanal Chem (2007) 388:1437–1453

MDMA 3,4-Methylenedioxymethamphetamine for passive diffusion [17, 18]. In addition, it has been
MSTFA N-Methyl-N-trimethylsilyl-trifluoroacetamide demonstrated that shortly after the abuse of a drug (by
MTBSTFA N-Methyl-N-tert-butyldimethylsilyl- smoking, nasal insufflation, and, to a lesser extent, oral
trifluoroacetamide ingestion), contamination of the oral cavity can lead to
NCI negative chemical ionization dramatically elevated concentrations of the parent drug in
NPD nitrogen–phosphorus detection oral fluid [17]. Several authors have investigated the
PCI positive chemical ionization pharmacokinetics of the most common drugs of abuse in
PFPA pentafluoropropionic anhydride oral fluid, thereby identifying the target analytes and typical
PFBPI N-Pentafluorobenzoyl-S-(−)-prolyl-1- concentration ranges for toxicological analysis [18].
imidazolide Oral fluid can be extracted and analyzed in the same
QTOF quadrupole-time-of-flight manner as other biological fluids, such as blood. However,
ROC receiver operating curves analytical sensitivity is a significant concern when con-
SPE solid-phase extraction ducting oral fluid drug testing, especially when the
SPME solid-phase microextraction screening of a large number of compounds is requested.
TBAH Tetrabutyl-ammonium hydroxide Decreased salivary secretion is a side effect of many drugs,
TBDMCS tert-Butyldimethylchlorosilane including stimulants, yielding small volumes of oral fluid
TFAA trifluoroacetic anhydride for analysis. In addition, the target range of concentrations
THC delta9-tetrahydrocannabinol is generally lower than for screening of a urine sample. The
THC- 11-Nor-delta9-tetrahydrocannabinol-9- use of a (commercial) collection device is often beneficial
COOH carboxylic acid since it provides a clean, more abundant specimen for
TMAH tetrabutylammonium hydroxide analysis, but a number of factors such as the actual volume
of oral fluid collected, the recovery of the analytes from the
device, and drug stability should be thoroughly evaluated.
Commercially available laboratory-based EIA kits for the
Introduction screening of different drug classes in oral fluid are designed
for use with specific collection devices. Chromatographic
Oral fluid has been used as an alternative to blood and urine methods used for quantification of different drugs in oral
in clinical and forensic toxicology [1–5]. The main benefit fluid include GC–MS [19, 20], which has long been the
of this body fluid is its noninvasive collection procedure standard technique, and LC–MS–MS [21–26], which is
compared with blood, and its minimal inconvenience increasingly used to analyze a large number of compounds
compared with urine sampling when observation of the in small sample volumes with suitable sensitivity. The use
donor is required to avoid adulteration, substitution, and of GC–MS–MS and GC–GC–MS has also been reported in
dilution of the sample. Moreover, oral fluid appears to be some specific studies where sensitivity is a major issue (e.g.,
superior to urine in correlating with serum analytical data for the detection of cannabinoids) [27, 28]. This paper will
and impairment symptoms of drivers under the influence of focus on validation data for and the benefits and pitfalls of
drugs [6, 7]. Furthermore, it may provide a cost-effective different analytical techniques used for drug testing in oral
approach for the screening of large populations. The use of fluid, as well as current limitations of on-site testing. It is
oral fluid testing for drugs of abuse in the workplace [8, 9] important to note that the absence of internationally accepted
and in treatment facilities has been reported [10–15]. guidelines hampers the actual use of oral fluid testing in a
Oral fluid originates from three pairs of major salivary legal context.
glands (parotis, submandibularis, and sublingualis), a great
number of minor salivary glands, the oral mucosa, and
gingival crevices. This mixture of fluids is referred to in the Sampling
literature as “saliva,” “whole saliva,” and “oral fluid,”
which is actually the preferred term for the testing fluid. Common methods of oral fluid collection are spitting,
The volume, composition, and viscosity is greatly influ- draining, suction, and collection on various types of
enced by a variety of factors [16]. The most common absorbent swabs. Spitting itself is usually a sufficient
mechanism of drug transport from the blood is passive stimulus to elicit a flow, but the sample volume is often
diffusion; it is limited to non-protein-bound, unionized insufficient and the intra- and intersubject variability is
molecules, with a molecular weight of less than 500 Da and large. The flow can be stimulated mechanically (via
a certain degree of lipophilicity. Generally, oral fluid Teflon™, chewing gum, etc.) or chemically (via lemon
predominantly contains the parent drug because of the drops or citric acid crystals), resulting in an oral fluid
higher lipid solubility and, therefore, the higher potential sample which differs in composition (e.g., has a higher pH)
Anal Bioanal Chem (2007) 388:1437–1453
from oral fluid collected without stimulation. A variety of
devices that allow the use of oral fluid testing for the drug
screening of large populations have been marketed over the
years. They promote easy, quick and reproducible collec-
tion as well as a cleaner specimen which is more suitable
for analysis. Commercial devices include Omni-Sal®
(Cozart Biosciences Ltd., Abingdon, UK), Salivette®
(Sarstedt AG, Rommelsdorf, Germany), Intercept® (Ora-
Sure Technologies, Bethlehem, PA, USA), Finger Collec-
tor® (Avitar Technologies, Inc., Canton, MA, USA),
ORALscreen™ (Avitar Technologies, Inc, Canton, MA,
USA), and Quantisal™ (Immunalysis Corporation,
Pomona, CA, USA). As a general rule, they consist of a
sorbent material that becomes saturated in the mouth of the
donor, and after removal the oral fluid is recovered by
centrifugation or by applying pressure. Often the device is
placed in a container that contains a stabilizing buffer
solution.
Significant differences in percent recovery from the
sorbent material for various drugs are reported, as well as a
large variability in the ultimately measured oral fluid
concentrations. A typical example is the observation that
THC, the major psychoactive constituent of cannabis,
remains bound to the sampling device and that an organic
solvent is needed to release it [20, 21, 29, 30]. A
modification of the sampling procedure for the Intercept®
collector, consisting of the addition of 2 mL of methanol to
the elution buffer, resulted in complete recovery of THC
over a large concentration range [29]. The recovery of other
drugs from the Intercept® device has also been investigated
[20, 21]. Losses of more than 30% for 7-aminoclonazepam
and LSD were noted, whereas variable recoveries were
observed for other compounds (e.g., buprenorphine, metha-
done, and zolpidem).
Quintela et al. performed an in vitro evaluation of the
Quantisal™ device for amphetamine, methamphetamine,
morphine, codeine, cocaine, BE, methadone, oxazepam,
and THC [31]. Recoveries for opioids, cocaine, amphet-
amines, and oxazepam exceeded 91%, and 82% was
obtained for BE. Especially noteworthy was the high
recovery of THC from the Quantisal™ collector (81.3–
91.4%). Previously published work with Salivette® showed
that significant amounts of cocaine, EME, morphine, 6-AM,
midazolam, diazepam, lorazepam, and tetrazepam remained
sequestered on the cotton roll [32–34]. An additional
problem when interpreting quantitative results is the diffi-
culty involved in estimating the actual volume of oral fluid
collected when a dilution buffer is used in the device. Kauert
et al. have shown that for the Intercept® device, reliable
quantitative results can be obtained when applying a
gravimetric determination of the amount of oral fluid [29].
Several authors have investigated the stability of specific
analytes like THC, morphine, 6-AM, BE, and designer
1439
amphetamines in the collected oral fluid sample [13, 17,
35–37]. For some compounds, e.g., flunitrazepam, poor
stability (even at +4 °C) was noted when no NaF was added
to oral fluid specimens collected by spitting [38]. The
stability of morphine, both on the collection pad and in the
preservative fluid of the Intercept® device, was assessed by
Niedbala et al. [13]. Morphine was stable at all temper-
atures tested under both conditions. In contrast, 6-AM
proved to be stable for just one week under the same
conditions. At −20 °C, the buffered oral fluid collected with
Intercept® showed only a small decline in THC concentra-
tion, whereas at 4 °C and 21 °C, significant decreases to
13% were noted after six weeks of storage [17]. In contrast,
Moore et al. showed that THC is stable in buffered oral
fluid collected with the Quantisal™ device, if the specimen
is stored in the refrigerator and if extended exposure to
fluorescent light is avoided [36]. However, in an evaluation
of the recovery and subsequent storage (ten days at room
temperature and +4 °C), Dickson et al. observed substantial
drug losses for this device and the Microgenics device [39].
Of the three systems studied, the Cozart product was the
only one acceptable for THC.
Sample pretreatment
The recovery of a target range of illicit drugs from oral fluid
has been performed using solid-phase extraction (SPE) and
liquid–liquid extraction (LLE) methods similar to those
already applied to blood samples. Since oral fluid contains
considerably less protein and lipid compared to blood or
plasma, it was suggested that this permits easier and faster
analysis. However, the development of fast and easy sample
preparation methods can be hampered by the higher mucin
content of oral fluid [40]. A sample collected by spitting
contains cell debris, food particles, and strings of mucus;
centrifugation is difficult because of the high viscosity. The
specimen has to be stored at +4 °C and measured as soon as
possible because of possible bacterial and fungal growth.
Freezing of the sample lowers the viscosity substantially so
that centrifugation can be easily performed after thawing.
Teixeira et al. [41] have reported much higher THC
concentrations in oral fluid obtained by spitting than those
measured in a previous report [30], which can be explained
by another pretreatment of the spitted samples before SPE.
The sample preparation technique can highly influence
the presence of matrix effects in the subsequent chromato-
graphic analysis [42]. In addition to the endogenous
substances present in oral fluid, specific collection devices
mostly include a preservation buffer containing other
compounds, such as stabilizing salts, nonionic surfactants
and antibacterial agents. This can result in long-term
problems in GC–MS analysis with respect to liner, column,
Table 1 GC-based procedures used for the quantification of drugs in oral fluid samples
1440

Compound Sample collection Sample Derivatization Stationary phase Chromatography Validation data Reference
clean-up

Amphetamine, methamphetamine, Spitting / S-(−)- HP-5MS GC–NCI–MS Selectivity, linearity, precision, and accuracy, [56]
MDMA, MDA, MDEA heptafluorobutyrylprolyl LOD, LOQ, recovery, stability (bench-top,
chloride processed sample, freeze/thaw), applicability
MDMA, MDA, MDEA, Spitting SPE HFBA HP-5MS GC–EI–MS Selectivity, linearity, precision, and accuracy, [54]
amphetamine, methamphetamine, LOD, LOQ, recovery, stability (bench-top,
HMMA, HMA processed sample), applicability
15 psychoactive amines Spitting / HFBA DB-5MS GC–EI–MS Selectivity, linearity, precision, [53]
and accuracy, LOD, LOQ, recovery,
applicability
Methamphetamine, amphetamine Spitting with citric acid SPE MTBSTFA+1% HP-1 or GC–EI–MS Linearity, precision, LOD, LOQ, [52]
candy stimulation and TBDMCS Phenomenex ZB1 applicability
Salivette® (neutral or
citric acid-treated)
MDMA, MDA, HMMA Spitting SPE MBTFA HP Ultra-2 GC–EI–MS Linearity, precision, and accuracy, LOQ, [51]
recovery, applicability
MBDB, BDB Spitting LLE HFBA HP-5MS GC–EI–MS Linearity, precision, LOD, recovery, [110]
applicability
D-amphetamine, L-amphetamine Spitting after LLE PFBPI 5% OV-275 coated GC–PCI–MS Linearity, accuracy, applicability [111]
mechanical on 100/120 mesh
stimulation (Teflon) Chromosorb W-AW
THC-COOH Intercept® device SPE HFIP/PFAA DB-5 GC–NCI–MS– Selectivity, linearity, precision, [27]
MS and accuracy, LOD, LOQ, recovery,
stability (bench-top, processed sample),
applicability
THC-COOH Quantisal™ device SPE HFIP/PFAA Primary column: GC–GC–ECCI– Selectivity, linearity, precision, LOQ, [28]
DB-35MS; MS recovery, applicability
secondary column:
DB-1
THC OraLine® IV LLE TMAH/DMSO and Optima5-MS GC–EI–MS Linearity, precision, LOQ, applicability [112]
s.a.t. device iodomethane
THC Intercept® device LLE TBAH/DMSO and HP5-MS GC–EI–MS Selectivity, linearity, precision, LOQ, [62]
iodomethane applicability
THC Intercept® device LLE BSTFA+1% TMCS 5% phenyl methyl GC–EI–MS–MS Linearity, precision, LOD, LOQ, [113]
silicone applicability
THC Salivette® LLE TBAH/DMSO and HP5-MS GC–EI–MS Linearity, precision, LOD, recovery, [61]
iodomethane applicability
THC Spitting? LLE PFPA OV-101 GC–ECD Selectivity, linearity, LOD, LOQ, [59]
applicability
Cocaine, AEME, EME, Salivette® Automated / DB-5MS GC–PCI–MS– Selectivity, linearity, precision, [19]
cocaethylene SPE MS and accuracy, LOD, LOQ, applicability
Cocaine, EME, BE Spitting, Salivette® ToxiTube® A BSTFA+1% TMCS 5% phenyl- GC–PCI–MS Linearity, precision, and accuracy, LOD, [32]
methylsiloxane LOQ, recovery, applicability
AEME Spitting? LLE BSTFA+1% TMCS HP-5MS GC–EI–MS Selectivity, linearity, precision, LOD, [68]
recovery, applicability
Anal Bioanal Chem (2007) 388:1437–1453
Table 1 (continued)

Compound Sample collection Sample Derivatization Stationary phase Chromatography Validation data Reference
clean-up

Cocaine, BE, norcocaine, Spitting with citric acid SPE vs. LLE BSTFA+1% TMCS HP-1 GC–EI–MS Selectivity, linearity, precision, and accuracy, [67]
benzoylnorecgonine, EME, candy stimulation LOD, recovery, applicability
cocaethylene, norcocaethylene,
AEME, EEE
Cocaine, BE Self-constructed SPE PFPA HP-1 GC–EI–MS Linearity, LOQ, applicability [66]
osmotic device
Cocaine, BE, EME Spitting with and SPE BSTFA+1% TMCS HP-1 GC–EI–MS Linearity, precision, applicability [65]
without citric acid
candy stimulation
Cocaine Spitting with citric acid LLE / 3% OV-17 on Gas GC–PCI–MS Linearity, precision, LOD, applicability [64]
candy stimulation Chrom Q (100/120 and GC–NPD
and 120/140 mesh)
Meperidine (1), tramadol (2), Quantisal™ device SPE (1) BSTFA+1% TMCS, DB-5MS GC–EI–MS Selectivity, linearity, precision, LOQ, [73]
oxycodone (3) (3 different (2) BSTFA+1% TMCS recovery, stability (bench-top,
chromatographic procedures, (3) MSFTA+1% TMCS processed sample), applicability
Anal Bioanal Chem (2007) 388:1437–1453

compounds not simultaneously

detected)
Codeine, morphine, 6-AM Spitting, Salivette® ToxiTube® A BSTFA+1% TMCS 5% phenyl- GC–PCI–MS Linearity, precision, and accuracy, LOD, [33]
methylsiloxane LOQ, recovery, applicability
Propoxyphene Quantisal™ device SPE / DB-5MS GC–EI–MS Selectivity, linearity, precision, LOD, LOQ, [72]
recovery, applicability
Codeine, morphine, 6-AM, Spitting?, artificial oral SPE (1) Methoxyamine, (2) DB-5 GC–EI–MS Linearity, precision, LOD, LOQ, [71]
hydrocodone, hydromorphone, fluid BSTFA+1% TMCS applicability
oxycodone
Codeine, norcodeine, morphine, Spitting with citric acid SPE (1) MTBSTFA+1% HP-1 or GC–EI–MS Linearity, precision, LOD, LOQ, [76]
normorphine candy stimulation and TBDMCS, (2) BSTFA Phenomenex ZB1 applicability
Salivette® (neutral or +1% TMCS
citric acid-treated)
Morphine, 6-AM, heroin Spitting? Filtration 1st aliquot: / 2nd aliquot: Rtx-5 GC–EI–MS Selectivity, linearity, precision, [70]
followed by MBTFA and accuracy, LOD, applicability
SPE
Methadone, EDDP Spitting? SPME vs. / HP-5 GC–EI–MS Linearity, precision, LOD, LOQ, [78]
LLE recovery, applicability
Methadone, EDDP Spitting? LLE / 3% SP-2250 on GC with Selectivity, linearity, precision, LOD, [114]
Supelcoport 100– thermionic recovery, applicability
120 mesh detection
Flunitrazepam, Spitting Centrifugation HFBA HP-1 GC–NCI–MS Linearity, precision, and accuracy, LOD, [38]
7-aminoflunitrazepam followed by LOQ, recovery, applicability
SPE
1441
1442
and ion source contamination [20]. If the clean-up of the
specimen is insufficient, subsequent LC–MS(–MS) mea-
surements will suffer from poor precision and accuracy
through a phenomenon called ion suppression or ion
enhancement [43–46], which will be discussed in the
following section on chromatographic methods.
Analytical methods
Part I: Chromatographic methods
Chromatographic methods should be fully validated accord-
ing to international recommendations, as critically reviewed
by Peters and Maurer [47]. For LC–MS–MS methods, the
presence of matrix effects should always be evaluated.
Mortier et al. reported that using a simple deproteinization
as sample clean-up can lead to important ion suppression
effects [48]. In experiments involving ESI of biological
extracts, it was shown that the main cause of ion suppression
is a change in the spray droplet solution properties caused by
the presence of non-volatile or less volatile solutes [49].
These non-volatile materials (e.g., salts, endogenous com-
pounds, drugs and metabolites) change the efficiency of
droplet formation or droplet evaporation, which in turn
affects the amount of charged ion in the gas phase that
ultimately reaches the detector. Dams et al. [42] performed
an in-depth evaluation of the effect of mobile phase,
ionization type, and sample preparation method on the
importance of the matrix effect in the LC–MS–MS analysis
of urine, plasma, and oral fluid. Neither protein precipitation
nor SPE provided sufficient clean-up of oral fluid samples
collected with Salivette® to eliminate a matrix effect
observed for morphine during LC–ESI–MS–MS analyses.
However, this did not seem to be the case for LC–APCI–
MS–MS. In general, oral fluid was shown to have more
interferences than urine, mainly in ESI, and residual matrix
components were both hydrophilic and hydrophobic.
We agree with the point of view of several authors that
any bioanalytical procedure using LC–MS–MS should only
be used routinely, and can only be accepted for publication
if ion suppression studies have been performed [43, 45, 46].
Tables 1 and 2 present an overview of validated GC and
HPLC methods used for the quantification of different
compounds in oral fluid samples. The table is limited to the
analysis of a single drug class; screenings for a larger
variety of compounds will be described in detail later in this
paper.
Amphetamines
Until 2001, only a few reports dealing with the quantifica-
tion of amphetamines in oral fluid were published.
Anal Bioanal Chem (2007) 388:1437–1453
Mancinelli et al. developed the first HPLC fluorimetric
procedure for MDMA, MDA, MDEA, and MBDB in
spitted oral fluid samples [50]. A sensitivity of 50 ng/mL
was achieved for all compounds. Unfortunately, the paper
lacks a detailed description of the dilution reagent and the
mobile phase used in the analytical procedure. Six years
later, the same technique was used for the same compounds
after LLE with Toxitube A® [35]. The analytical method
allowed good separation in only 10 min with a reported
LOQ of 10 ng/mL for all compounds. However, no internal
standard was used due to the difficulty involved in finding a
compound with native fluorescence and good chromato-
graphic properties under isocratic conditions. This could be
the reason for the higher %RSD observed during the
interassay precision experiments (ranging from 16.4 to
23.5% at 250 ng/mL).
In 2001, Navarro et al. applied a previously published
GC–MS method for plasma samples to the analysis of
MDMA and its metabolites MDA and HMMA in spitted
oral fluid samples obtained from a controlled study [51].
The LOQ were 5.7 ng/mL, 1 ng/mL, and 2.9 ng/mL for
MDMA, MDA, and HMMA respectively. Similarly,
Schepers et al. used an extended version of a method
published earlier for the analysis of sweat samples for the
quantification of methamphetamine and amphetamine in
oral fluid samples from a controlled study (LOD/LOQ:
2.5 ng/mL for both analytes) [52]. A rapid GC–EI–MS
assay for the simultaneous determination of 15 psychoac-
tive amines using only 100 μL of spitted sample was
developed and validated by Kankaanpää et al. [53].
Extraction and derivatization occurred in a single step
using a mixture of toluene, methylmexiletine (as internal
standard), and HFBA with direct injection of an aliquot of
the solvent phase into the GC–MS system. Different
columns were tested, with the bonded, crosslinked nonpolar
DB-5MS column yielding satisfactory separation of all
compounds. With a LOQ of 20 ng/mL for all compounds
tested, this method offers the rapidity of an immunoassay
combined with the versatility and accuracy of a confirma-
tion technique. Recently, Scheidweiler et al. described a
validated GC–EI–MS method for the quantification of
seven amphetamine compounds in oral fluid [54]. Using
400 μL of sample, a LOQ of 5 ng/mL was obtained for all
analytes, except for HMA and HMMA, where a LOQ of
25 ng/mL was observed.
Wood et al. developed a very sensitive LC–ESI–MS–MS
method for the quantification of the amphetamine class in
oral fluid samples [55]. Following a simple protein
precipitation step with methanol, a LOQ of 0.5–1.0 ng/mL
could be achieved using only 50 μL of sample. One
important item to note, however, is the lack of ion
suppression data, although only a simple sample clean-up
procedure was performed. The possible influence of the
Table 2 HPLC-based procedures used for the quantification of drugs in oral fluid samples

Compound Sample collection Sample clean-up Stationary Mobile phase Chromatography Validation data Reference
phase

MDMA, MDA, MDEA, MBDB Spitting LLE Kromasil 100 Isocratic with aqueous potassium HPLC with Selectivity, linearity, precision, LOD, LOQ, [35]
C8 dihydrogen phosphate and fluorescence recovery, stability (bench-top, long term)
acetonitrile detection
MDMA, MDA, MDEA, Spitting Deproteinization Hypersil Isocratic with aqueous LC–ESI+-MS- Selectivity, linearity, precision, and accuracy, [55]
amphetamine, methamphetamine, BDS C18 ammonium acetate and MS LOD, LOQ, applicability
ephedrine acetonitrile
MDMA, MDA, MDEA, MBDB Spitting Dilution LiChrocart- Isocratic with concentrated HPLC with Selectivity, linearity, precision, and accuracy, [50]
LiChrospher eluent, water and acetonitrile fluorescence LOD, LOQ
RP-18 detection
THC Spitting, Salivette® SPE XTerra MS Isocratic with aqueous ammonia LC–ESI+-MS Selectivity, linearity, precision, LOD, LOQ, [41]
Anal Bioanal Chem (2007) 388:1437–1453

C18 and acetonitrile recovery, applicability

THC Intercept® device LLE XTerra MS Isocratic with aqueous LC–ESI+-MS- Linearity, precision, and accuracy, LOQ, [24]
C18 ammonium formate and MS recovery, stability (bench-top, processed
methanol sample, freeze/thaw), matrix effects,
applicability
THC Spitting LLE XTerra MS Isocratic with aqueous formic LC–ESI+-MS Linearity, precision, and accuracy, LOQ, [63]
C18 acid and acetonitrile recovery, matrix effects, applicability
THC Spitting with citric Deproteinization μBondapak Isocratic with aqueous sodium HPLC with Linearity, precision, and accuracy, recovery, [60]
acid candy followed by C18 monochloroacetate and amperometric applicability
stimulation LLE monochloroacetic acid and detection
methanol
Cocaine, BE, Cocaethylene Salivette® SPE Hypersil Gradient with aqueous LC–ESI+-MS- Selectivity, linearity, precision, and accuracy, [69]
BDS C18 ammonium acetate, methanol MS LOD, LOQ, recovery, applicability
and acetonitrile
Morphine, 6-AM, codeine, heroin, Modified / Exsil BDS Gradient with aqueous LC–ESI+-MS- Linearity, precision, and accuracy, LOD, LOQ, [74]
acetylcodeine Intercept® device C8 ammonium acetate, formic acid, MS applicability
methanol and isopropanol
Dihydrocodeine, dihydrocodeine- Salivette® SPE Nucleosil Gradient with aqueous HPLC with Selectivity, linearity, precision, LOD, LOQ, [75]
6-glucuronide, dihydromorphine, 100 C18 triethylammonium phosphate fluorescence recovery, applicability
dihydromorphine-6-glucuronide, buffer and acetonitrile
dihydromorphine-3-glucuronide,
N-nordihydrocodeine
R-/S-methadone, R-/S-EDDP Salivette® Centrifugation α1-acid Isocratic with aqueous LC–ESI+-MS Linearity, precision, and accuracy, LOD, LOQ, [12]
after freezing glycoprotein ammonium acetate and recovery, stability (bench-top, processed
and thawing (AGP) acetonitrile sample, long-term), applicability
R-/S-Methadone Salivette® filtration α1-acid Isocratic with aqueous LC–ESI+-MS Linearity, precision, LOQ, recovery, [11]
glycoprotein ammonium acetate, N,N- applicability
(AGP) dimethyloctylamine and
isopropanol
Tetrazepam Spitting LLE XTerra MS Gradient with aqueous formic LC–ESI+-MS– Selectivity, linearity, precision, and accuracy, [79]
C18 acid and acetonitrile MS LOD, recovery, matrix effect, applicability
17 benzodiazepines and hypnotics Intercept® device LLE XTerra MS Gradient with aqueous formic LC–ESI+-MS– Selectivity, linearity, precision, and accuracy, [23]
C18 acid and acetonitrile MS LOQ, recovery, matrix effect, applicability
1443
1444 Anal Bioanal Chem (2007) 388:1437–1453

Reference
matrix effect on the precision and accuracy of this method
is, at least partially, reduced by the use of deuterated

[82]

[34]

Selectivity, linearity, precision, LOD, recovery, [81]

[80]
analogs as internal standards. In addition, due to the high
Selectivity, linearity, precision, and accuracy, sensitivity of the method and the expected concentrations of

Selectivity, linearity, precision, and accuracy,

amphetamines in oral fluid specimens, it is unlikely to yield
false negative results due to the suppression of the
LOD, LOQ, recovery, matrix effect,

LOD, LOQ, recovery, matrix effect,

Selectivity, linearity, precision, LOD,

response. Enantioselective analysis of amphetamines can
be helpful when interpreting toxicological results [56]. To
date, only one fast and sensitive assay has been presented
for the enantioselective determination of amphetamine
compounds in oral fluid samples collected by spitting.
Chromatography Validation data

After dilution with 0.2 mL of carbonate buffer (pH 9), oral

applicability

applicability

applicability

applicability
fluid samples (0.01–0.05 mL) were derivatized with S-(−)-
heptafluoro-butyrylprolyl chloride, and the resulting dia-
stereomers were extracted into 0.1 mL of cyclohexane and
directly analyzed by GC–NCI–MS. The method was linear
LC–ESI+-MS–

LC–ESI+-MS–

from 5 to 250 ng/mL per enantiomer of MDA and from 25

LC–ESI+-MS

LC–ESI+-MS

to 1250 ng/mL per enantiomer of amphetamine, metham-

phetamine, MDMA, and MDEA. For the determination of
MS

MS

MDEA enantiomers, the validation criteria applied were not

fulfilled. The method was fully validated and applied to
XTerra RP18 Gradient with aqueous formic

XTerra RP18 Gradient with aqueous formic

samples from a controlled study with MDMA and from

authentic DUID cases.
ammonium formate and

ammonium formate and

Novapak C18 Isocratic with aqueous

XTerra C18 Isocratic with aqueous

acid and acetonitrile

acid and acetonitrile

Cannabinoids
Mobile phase

acetonitrile

acetonitrile

Very little is known about the composition of cannabinoid

compounds in oral fluid samples. When cannabis is smoked,
THC is deposited in the oral cavity, and it appears that this
oral mucosal depot of active THC is the primary source of
Sample clean-up Stationary

the THC which is ultimately collected and measured during

phase

oral fluid testing [57]. In addition to THC, cannabidiol and

cannabinol are detected in oral fluid after cannabis is
smoked. Recently, Moore et al. and Day et al. demonstrated
the presence of the principal urinary metabolite THC-
COOH in the oral fluid matrix [27, 28]. In order to
Spitting with citric LLE

LLE

LLE

LLE

approach the required sensitivity, oral fluid samples were

neutral Salivette®

analyzed using GC–NCI–MS–MS [27] or GC–GC–MS

Sample collection

acid-treated and

[28]. The application of a prior separating column in this

assay allows the background associated with the extract to
Salivette®

Spitting

Spitting

be spread out over a longer time frame. A pressure switch

is then used to divert a narrow “window” of the effluent
from the first column that contains the analyte and
minimal background. To further focus the peak of interest,
the fraction from the first column is selectively transferred
to the analytical column, where a cryogenic trap allows the
analyte to be “cold-trapped.” The second analytical
Table 2 (continued)

9 benzodiazepines

column is of a different polarity to the first and provides

a further separation of the analyte from potential interfer-
Midazolam

Lorazepam
Compound

ences. In addition, the MS was operated in ECCI mode,

Zolpidem

which is a more selective ionization procedure, affecting

only compounds with sufficient electronegativity to
Anal Bioanal Chem (2007) 388:1437–1453
capture ionized electrons. Because less fragmentation
occurs at low source and quadrupole temperatures, fewer
ions are produced (in this case, two), but their selectivity is
critical. The collision gas used in this method was
ammonia, which improves the sensitivity of the assay.
Day et al. reported a LOQ of 10 pg/mL using GC–NCI–
MS–MS, whereas a five-fold higher sensitivity was
obtained with the single-quadrupole MS procedure of
Moore et al. As such, this paper represents a valuable
approach to improve the analytical limits of existing
techniques without the need for expensive investment in
more sophisticated instruments. Both methods used TFAA
and HFIP for derivatization, which, in the case of ECCI
detection, enhanced the overall electronegativity of the
molecule, contributing to the achieved sensitivity. The
addition of more electronegative groups was attempted
using PFPA and HFBA in place of TFAA, but neither
anhydride was particularly stable. In another report from
these authors, the same GC–GC–MS method was used to
show that the inclusion of this metabolite in the confir-
mation profile for cannabinoids in oral fluid increases the
confirmation rate of the immunoassay result by at least
9.7% [58].
By the mid 1980s, methods achieving a high sensitivity
for THC in oral fluid specimens (LOD: 1 ng/mL) using
GC–ECD [59] or HPLC with amperometric detection [60]
were already being reported. Later on, Kintz et al.
described the use of a GC–MS procedure to test for THC
in oral fluid samples collected with Salivette® [61].
Extraction of this compound from the cotton roll was
performed using a mixture of hexane/ethyl acetate (90:10,
v/v), followed by methylation with TBAH/DMSO and
iodomethane (LOQ = 1 ng/Salivette®). This method was
subsequently applied to specimens obtained from injured
drivers. Later on, these authors reported the detection of
THC in oral fluid samples collected with the Intercept®
device using the same analytical procedure [62].
Recently, several authors used LC–MS(–MS) for the
confirmation of THC positive samples using different
collection protocols [24, 41, 63]. An XTerra MS C18
column eluted with a mixture of acetonitrile/0.05% NH3
(70:30, v/v) [41], acetonitrile/0.1% FA (85:15, v/v) [63] or
methanol/1 mM ammonium formate (90:10) [24] was used
in these methods. Sample clean-up was performed using
either mixed-mode SPE and elution with hexane/ethyl
acetate (80:20, v/v) [41] or LLE with hexane [24, 63]. All
of these methods were successfully applied to the analysis
of specimens from roadside controls (Fig. 1), rock
festivals, and a controlled study with marijuana. The
LOQ obtained using tandem MS technology was markedly
lower than that obtained with single MS methods. Using
only 100 μL of sample, a LOQ of 0.5 ng/mL for THC
could be obtained [24].
1445
Cocaine and opioids
In 1987, a sensitive GC–NPD method using codeine as
internal standard was published for the detection of cocaine
in oral fluid (LOQ: 5 ng/mL) [64]. Later on, Kato et al.
determined cocaine and its metabolites using GC–EI–MS;
however, the sensitivity of the method was not reported
[65]. Simultaneously, Schramm et al. determined the
presence of cocaine and BE in oral fluid samples collected
with osmotic collection devices [66]. Cone et al. developed
a method for the simultaneous analysis of cocaine, coca-
ethylene, six of their metabolites, and AEME [67]. The
LODs were approximately 1 ng/mL for cocaine, EME, and
BE, and 3–6 ng/mL for the remaining analytes. Kintz et al.
used a complex LLE consisting of three steps, extraction
with a mixture of chloroform–isopropanol–n-heptane
(50:17:33, v/v/v), and then aqueous extraction with 0.2 M
HCl and back-extraction in a mixture of phosphate buffer–
NaOH–chloroform (25:12.5:62.5, v/v/v) for the detection of
cocaine and metabolites [68]. However, only validation
parameters for the pyrolysis product of cocaine, AEME,
were reported.
In the last five years, only three papers concerning the
quantification of cocaine and its metabolites in oral fluid
have been published [19, 32, 69]. Campora et al. used
samples collected by spitting for the validation, whereas
authentic samples were obtained with the Salivette® [32].
The drug recoveries from the Salivette® for EME, cocaine,
and BE ranged from 23 to 84%, hampering accurate
quantifications of these compounds. The same problem
was encountered when using a similar procedure for the
analysis of opiates [33]. Cognard et al. developed a semi-
automated SPE–GC–PCI–MS–MS method with isobutane
used as reagent gas for the simultaneous analysis of cocaine
and its metabolites in oral fluid samples [19]. Since BE was
not included in the method, a time-consuming derivatiza-
tion step could be eliminated. The LOQ was determined at
2 ng/mL for cocaine and cocaethylene, and 5 ng/mL for
EME. AEME could only be determined semi-quantitatively
due to the high %RSD observed during the inter- and intra-
assay validation experiments (ranging from 22.4 to 50.4%).
The exclusion of BE from the method, and the poor
precision data for AEME reduce the importance of this
publication.
Already in 1993, Goldberger et al. described an SPE
procedure for the isolation of heroin, 6-AM and morphine
from blood, plasma, oral fluid, and urine with subsequent
GC–MS detection [70]. A LOD of approximately 1 ng/mL
was observed for all compounds in all matrices. Several
methods for other opioids were developed [71–76]. Jones et
al. described the simultaneous determination of several
opiates in both hair and oral fluid samples using methox-
ime/BSTFA derivatives, enhancing the chromatographic
1446 Anal Bioanal Chem (2007) 388:1437–1453

4.96
100 a
318.2>196.1
% 2.27e4

2.50
0

4.99
100

315.2>193.1
% 2.83e4
3.28

4.96
100

315.2>259.3
%
1.26e4
3.28

0 Time (min)
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50

4.96
100
b
318.2>196.1
2.25e4
%

4.99
100

315.2>193.1
%
1.63e5

4.99
100

315.2>259.3
% 7.08e4

0 Time (min)
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50
Fig. 1 Typical MRM chromatograms obtained following the analysis qualifier; middle and bottom traces respectively). In addition, the
of two authentic oral fluid specimens (collected with Intercept®) presence of cannabidiol (at a retention time of 3.28), showing the same
obtained from a driver in a roadside setting. Concentrations were transitions as THC, was noted in a. Peak intensity is shown in the top
5.7 ng/mL (a) and 50.8 ng/mL (b). The figure shows the response for right-hand corner of each trace. Reproduced from [24] with the
THC-d3 (top trace) and for the two transitions of THC (quantifier and permission of Elsevier
Anal Bioanal Chem (2007) 388:1437–1453
separation of the prescription opiates [71]. The LOQ ranged
from 2 ng/mL for codeine, morphine, hydromorphone, 6-
AM, and oxycodone to 3 ng/mL for hydrocodone. This
method was also successfully applied to the analysis of oral
fluid samples collected after the ingestion of poppy seeds
[77]. Recently, the group of Moore published two reports
concerning the determination of propoxyphene (LOQ:
5 ng/mL) [72], meperidine, tramadol, and oxycodone
(10 ng/mL) [73] in oral fluid specimens. Also, the
succesful detection of acetylcodeine as a marker for illicit
heroin abuse in these specimens was reported [74].
Three reports dealing with the quantitative analysis of
methadone were published in the last five years [11, 12,
78], two of which were able to chromatographically
separate the R- and S-enantiomers of this compound [11,
12]. Both used a chiral AGP column, providing a good
chromatographic separation between the two enantiomers.
In addition, the report of Rosas et al. included the
simultaneous enantioselective determination of EDDP.
Benzodiazepines and neuroleptics
Because of their extensive protein binding, benzodiazepines
are only present in oral fluid in very low concentrations
(low-ng or pg range). Therefore, sensitive assays are
needed for the analysis of these samples.
Only one paper deals with the determination of fluni-
trazepam and its metabolite, 7-aminoflunitrazepam, in oral
fluid samples using GC–NCI–MS [38]. After the controlled
administration of 1 mg flunitrazepam, samples obtained by
spitting were derivatized using HFBA following extraction
using mixed-mode SPE cartridges. Due to the low pKa of
flunitrazepam, cartridges had to be washed with 1 N
phosphoric acid (pH 1) before washing with methanol to
retain this compound on the column. The reported LOQs
were 0.1 ng/mL and 0.15 ng/mL for flunitrazepam and 7-
aminoflunitrazepam, respectively.
In the last two years, several LC–ESI–MS(–MS)
methods dealing with the detection of one or more
benzodiazepines in oral fluid samples have been published
[23, 34, 79–82]. The group of Kintz has described the
detection of zolpidem, lorazepam, and tetrazepam in this
matrix after the controlled administration of these drugs
[79–81]. These authors published a validated procedure for
the screening of 17 benzodiazepines and hypnotics in oral
fluid after collection with the Intercept® device by LC–
MS–MS [23]. Extraction of 500 μL of this preserved oral
fluid was performed with diethyl ether/dichloromethane
(50:50, v/v). The LOQ ranged from 0.1 to 0.2 ng/mL. In
view of the large abuse potential of these prescription drugs
and the problems associated with their detection, this
method represents a valuable contribution to the field,
particularly for cases of driving under the influence.
1447
Chromatographic methods: target screening
Wang et al. described a GC method for the simultaneous
detection of cocaine, heroin, and 12 metabolites in hair,
plasma, oral fluid, and urine. Unfortunately, the method for
oral fluid was only validated for linearity and LOD (range:
1–5 ng/mL) and is therefore of limited importance. [83].
One of the first LC methods targeting different drugs of
abuse in one run used 200 μl of oral fluid and simulta-
neously quantified amphetamines, morphine, codeine,
cocaine, and BE by LC–MS–MS [25]. Separation was
performed with a Hypersil BDS phenyl column after
extraction with mixed-mode SPE cartridges, on a QTOF
instrument. Due to the limited linear range of the TOF
instrument used in this study, quadratic regression curves
were shown to generate the best fit for all compounds,
using in-house synthesized analogs as internal standards.
Excellent validation data were obtained using spiked oral
fluid samples collected by spitting; however, the developed
SPE method was not able to provide a suitable clean-up
when analyzing oral fluid collected with a specific device.
This is a typical example of the impact of the collection
method on the analytical validation. In a later report, Dams
et al. fully validated the use of LC–APCI–MS–MS in
combination with protein precipitation using acetonitrile for
the simultaneous quantification of 27 compounds, cocaine,
opioids, and their metabolites in 200 μL of spitted oral fluid
[22]. The analytes were separated on a Synergi Polar RP
column using a mixture of ammonium formate, FA, and
acetonitrile as mobile phase and a gradient elution.
Wood et al. developed a fully validated LC–ESI–MS–
MS method for the quantification of multiple basic drugs in
preserved oral fluid samples collected with the Intercept®
device [26]. Chromatographic separation was achieved
using a XTerra MS C18 analytical column and gradient
elution with 10 mM ammonium bicarbonate and methanol.
The optimized SPE (mixed-mode) procedure proved to be
highly effective for the elimination of matrix compounds
(Fig. 2). In addition, these authors showed that the pH of
the processed samples was of major concern for the
stability of cocaine and 6-AM, and that attention should
be paid to this factor when developing methods for the
simultaneous determination of drugs with important differ-
ences in chemical characteristics.
Recently, another LC–ESI–MS–MS method using positive
ionization mode was published for the screening of 32
compounds in preserved oral fluid (Intercept® device) [21].
In addition to the typical basic drugs of abuse, also THC,
several benzodiazepines and their metabolites, zolpidem,
zopiclone, LSD, carisoprodol, meprobamate, methadone,
and buprenorphine were included in the assay. Samples
were prepared by LLE with ethyl acetate/heptane (4:1, v/v).
LC-separation was achieved using an Atlantis dC18 column
1448
(2.1×50 mm, 3 μm particles), with a run time of 14 min. A
weighted (1/x) second-order regression line, which included
the origin, was applied for each compound. Correlation
coefficients (r2) were>0.981, except for benzoylecgonine
(BE), for which an r2 of 0.792 was observed. The between-
day %RSD ranged from 3.6% to 39.1%, and the within-day
%RSDs were 2.0–31.8%. A large and highly variable matrix
effect was observed for several compounds. Therefore, this
method should be considered as a screening method;
however, the presence of large differences in matrix effects
between various oral fluid extracts is a serious drawback of
this procedure due to the possibility of false negative results.
Recently, Smink et al. developed a LC–APCI+–MS(–
MS) method for the screening of 33 benzodiazepines in oral
fluid specimens [84]. The analytes were eluted from the
XTerra MS C18 analytical column using a gradient with
aqueous FA and methanol. The method was applied for the
confirmation of oral fluid specimens from drivers stopped
during a roadside survey (collected with Salivette®).
Unfortunately, validation data were not complete; only the
LODs, LOQs, and recoveries of those benzodiazepines
detected in the samples were reported.
Gunnar et al. described a well-validated GC–MS method
for the simultaneous quantification of 30 drugs of abuse
using 250 μL of oral fluid obtained by spitting [20]. The
Fig. 2 Evaluation of the matrix
on 6-AM response by post-col-
umn infusion following an in-
jection of a water-only control
(a), a blank sample prior to SPE
(b), and the same post SPE
clean-up (c). The dotted area
indicates the elution position for
6-AM. Peak intensity is shown
in the top right-hand corner of
each trace
Anal Bioanal Chem (2007) 388:1437–1453
drugs were eluted in two phases, the first one with a
mixture of toluene/ethyl acetate (80:20, v/v) eluting THC
and most of the benzodiazepines, and the second one with
acetonitrile/ammonia (100:4, v/v), containing amphet-
amines, opiates, cocaine, BE, midazolam, alprazolam, and
zolpidem. The lower vaporization volume of toluene in
comparison with acetonitrile allowed the use of higher
injection volumes without peak distortion or broadening.
The second eluate fraction was divided into two parts,
allowing the application of two distinct derivatization
procedures: HFBA was selected for the amphetamines,
and MSTFA for the other compounds in this fraction.
MTBSTFA was chosen for the derivatization of the
benzodiazepines and THC. Furthermore, various GC–MS
parameters were optimized, including fast temperature
programming and carrier gas flow-rate, pulsed flow split/
splitless injection, target tuning, and SIM time windows.
Thirty-meter GC columns with large internal diameters of
0.32 and 0.53 mm (DB-35MS and DB-5MS columns) were
selected due to their higher reproducibility and sensitivity
for certain molecules (e.g., benzodiazepines). Due to its
thorough validation, the large number of compounds
included, and the limited sample volume used, this report
represents a valuable contribution. The same method was
also applied to samples collected with the Intercept®
Anal Bioanal Chem (2007) 388:1437–1453
device. Due to the presence of nonvolatile macromolecules
in the blue buffer of this device, the selectivity of the
method was decreased in comparison with neat oral fluid.
A combination of GC–MS and LC–MS methods was
applied to oral fluid samples collected using a modified
Omni-Sal® device and extracted using a single mixed-mode
extraction cartridge [85]. Elution of 49 legal and illegal
drugs from the cartridge was performed using two different
elution mixtures: acetone/chloroform (50:50, v/v; fraction
1), and ethyl acetate/ammonia (98:2, v/v; fraction 2). GC–
MS of the underivatized and pentafluoropropionyl deriva-
tives and LC–ESI–MS–MS analyses were used in parallel:
fraction 1 contained diazepam and its three metabolites, and
was analyzed with LC–ESI–MS–MS; fraction 2 (including
the amphetamines, cocaine, opioids and their metabolites)
was divided into two portions to be analyzed by LC–ESI–
MS–MS and GC–MS. In order to acquire sufficient data
points across the peaks, extracts had to be injected twice
into the LC–MS–MS instrument.
In 2003, two different groups investigated the use of
SPME for the oral fluid testing of various drugs [86, 87].
The reported procedures had limitations related to the
nature of the drugs that could be determined.
Part II: Immunological procedures
Many toxicological laboratories use immunological tech-
niques to realize preliminary monitoring of a large number
of samples in a reduced period of time, which is then
followed by confirmation of the presumptive positive
samples with a chromatographic method that provides the
required specificity. Microtiter plate EIA offers a number
of distinct advantages for oral fluid testing: limited amount
of specimen needed (usually around 25 μl), the absence of
sample pretreatment, and the antibody’s sensitivity and
cross-reactivity for the parent drugs. In addition to
precision and cross-reactivity data, a correct validation of
the immunoassay includes a comparison with a well-
known and validated reference method, applied to the
same sample. The result of the reference analysis (positive
or negative) is interpreted using a pre-established cut-off
value. For the screening assay, the chosen cut-off value
should optimize the number of true positive and true
negative samples, and reduce the false positive and false
negative screening rate. A screening cut-off that is too low
will result in an excessive number of negative samples in
the confirmation analysis, which makes the time and cost
of analysis unnecessarily high. In contrast, cut-off values
that are too high will lower the number of presumptive
positive specimens but result in a low sensitivity with too
many false negative samples, which undermines the
deterrent effect of drug testing and the detection efficiency
in forensic and clinical applications. Cut-off concentrations
1449
for screening and confirmation in oral fluid that are
scientifically and legally defensible have been proposed
by a number of working groups in the US, UK, and
Australia [88–89; Clarke J, Altrix Healthcare, personal
communication, Jan 2007]. Only the Australian Standards®
are approved at this time; they are intended, but not limited
to, workplace, medico-legal or court-directed detection of
drugs of abuse in oral fluid [89]. Recommended cut-off
concentrations are set for undiluted oral fluid. To overcome
problems with the adsorption of certain drugs to the
collection devices, each manufacturer should be required
to document the recovery of all target analytes from the
devices. In addition, collection onto an absorbent swab with
subsequent transfer into a buffer causes difficulties when
interpreting quantitative results [90]. Some commercially
available EIA kits for oral fluid are clearly marketed with
specific collection devices, e.g., Orasure® micro-plate
Intercept kits and Cozart® micro-plate EIA. In the former
system, calibrators in oral fluid buffered matrix have been
used for quantification. However, the great variation in
collection volume for the Intercept® collector (0.05–0.8 ml)
[9, 90] makes correct interpretation of the analytical data
very difficult, leading to false negative results when the
amount of oral fluid collected is too small. In contrast, the
Cozart® collector consists of an indicator which turns blue
when 1 ml of oral fluid is collected. An arbitrary dilution
factor of 1:3 in buffer is used to calculate the concentrations
for neat oral fluid; this implies that calibration curves are
obtained with undiluted oral fluid. It should be pointed out
that the aim of the preliminary screening technique is to
reduce the amount of samples requiring confirmation with a
chromatographic method, and so the absolute quantitative
result is of no importance; results are expressed as
equivalents of the target analyte. When the calibrators
differ considerably from the tested specimens, the reported
equivalents should be interpreted with respect to one or
more positive controls with a concentration of target analyte
above or close to the chosen cut-off and a negative control
prepared in the same matrix as the samples and analyzed on
the same plate [91].
Table 3 gives an overview of the reports on immuno-
logical screening techniques for different compounds in
oral fluid. An external quality assessment scheme involving
the Intercept® collection device and the corresponding EIA
kits revealed that the major source of error observed was
the sensitivity, where the EIA failed to achieve the cut-off
values specified by the manufacturer [92]. An extensive
validation was performed for the Cozart® Microplate
Opiate EIA oral fluid kit, detecting different opiates in
preserved oral fluid samples from drug users in a treatment
program [93]. ROC analysis revealed an optimum screen-
ing cut-off of 30–50 ng/mL morphine equivalent in neat
oral fluid when using a confirmation cut-off of 30 ng/mL.
Table 3 Immunological screening procedures for the detection of drugs in oral fluid specimens
1450

Compound Sample collection Confirmation Screening (target analyte Validation data Recommended screening/ Reference
technique in calibrators) confirmation cut-off level
(ng/mL)

Morphine, codeine, Intercept® GC–MS–MS Orasure® opiate microplate Precision, LOD, cross- 10/10 [13]
heroin, 6-AM EIA (morphine) reactivity, applicability
Codeine Spitting with or without GC–MS Cozart® opiate Precision, cross-reactivity, 20/20 [94]
citric candy stimulation and EIA (codeine) specificity, sensitivity,
Salivette® (neutral or efficiency, applicability
citric acid-treated)
Morphine, codeine, Cozart® Rapiscan Saliva GC–MS Cozart® opiate EIA (morphine) Precision, interferences, 30–50/30 [93]
heroin, 6-AM, Collector specificity, sensitivity,
dihydrocodeine applicability
Methadone, EDDP Cozart® Rapiscan Saliva GC–MS Cozart® EIA (d-methadone) Cross-reactivity, specificity, 20–30/15–30 [115]
Collector sensitivity, applicability
Cocaine, BE Intercept® GC–MS STC cocaine metabolite EIA Precision, LOD, cross- 10/10 [37]
(BE) reactivity, interferences,
specificity, sensitivity,
accuracy, applicability
Cocaine, BE, EME, Spitting with or without GC–MS Cozart® cocaine EIA (BE) Precision, specificity, 20/8 30/15 [95]
cocaethylene citric candy stimulation sensitivity, efficiency,
and Salivette® applicability
(neutral or citric acid-treated)
Cocaine, BE, Cozart® Rapiscan Saliva Collector GC–MS Cozart® cocaine EIA (BE) Precision, interferences, 20–25/30 [97]
cocaethylene specificity, sensitivity,
applicability
MDMA, MDA Intercept® LC–MS–MS Cozart® amphetamine EIA Precision, cross- 51/10 [91]
(D-amphetamine) reactivity,
interferences, specificity,
sensitivity, applicability
Amphetamine, Cozart® Rapiscan Saliva Collector GC–MS Cozart® amphetamine Precision, LOD, cross- 40–45/30 [98]
methamphetamine, EIA (D-amphetamine) reactivity, interferences,
MDMA, MDA specificity, sensitivity,
applicability
Anal Bioanal Chem (2007) 388:1437–1453
Anal Bioanal Chem (2007) 388:1437–1453
Barnes et al. evaluated the same Opiate EIA oral fluid kit in
a controlled study with codeine, using different collection
methods [94]. Six opiate EIA/confirmation cut-off criteria
were applied. The best assay efficiency (95.6%) was
obtained with a 20/20 ng/mL cut-off for codeine, providing
a specificity of 98.7% and a sensitivity of 82.9%.
In a study with controlled administration of cocaine,
different screening cut-offs (10, 20, and 30 ng/mL), and
GC–MS cut-off levels (2.5, 8, 10, 15 ng/mL cocaine, BE,
and/or EME) were evaluated using the Cozart EIA oral
fluid kit [95]. The highest specificity (89.8%) was obtained
with a 30 ng/mL screening cut-off and a 15 ng/mL
confirmation cut-off, resulting in a sensitivity of 89.8%
and an overall best efficiency of 89.7%. When the same
EIA system was used with the Cozart® collection system,
Cooper et al. reported an optimum screening cut-off of 20–
25 ng/mL cocaine equivalents in the neat oral fluid, with a
very high sensitivity and specificity, using a GC–MS
confirmation cut-off of 30 ng/mL [96].
When using different collection methods with the
Cozart® EIA oral fluid kit for cocaine, Kim et al. [95]
reported that the generally lower pH produced in specimens
collected with the citric acid-treated Salivette® [mean pH
2.8] yielded a higher number of false positive results, and
thus a lower specificity, due to a decrease in absorbance.
Niedbala et al. reported that there was no significant effect
of oral fluid pH on the STC Cocaine Metabolite Micro-
Plate EIA when the sample pH was between 5.0 and 9.0
[37]. As for the confirmation analysis, it is of prime
importance to carefully evaluate the selected oral fluid
collection method for this type of analysis.
The effect of common foodstuffs and household chem-
icals was investigated for the Orasure® microplate Intercept
kits [37]. Potential interferences in the Cozart® micro-plate
EIA system were thoroughly evaluated, including the
sample adequacy indicator dye and everyday substances
such as alcohol, hard candy, chewing gum, coffee, tea,
orange juice, water, general food consumption, hemoglo-
bin, mouthwash, and smoking [93, 96–98].
Few papers describe the comparison of different immu-
noassays for the same application. Dickson et al. found that,
using the Cozart® collection system, a combination of three
different EIA systems provided the most sensitive and
discriminating assays for the drugs studied, namely THC,
methamphetamine, morphine, and benzodiazepines [39].
The state-of-the-art (commercialized or prototypes) on-
site drug tests for oral fluid were thoroughly evaluated in
the EC projects ROSITA and Rosita2 [99, 100]. Several
authors have compared the on-site test results of these
devices with laboratory results, including Cozart® Rapi-
Scan [96, 101–105], Drugwipe® [30, 62, 106], Toxiquick®
[107], Dräger® DrugTest [108], Salivette® [95], and
Oratect® [109]. Most of the newer generation on-site
1451
devices offer the ability to collect enough sample volume
(sometimes diluted with buffer) that can be used for
confirmation analysis in the laboratory. However, several
researchers also used separately collected oral fluid speci-
mens for the confirmation analysis, which complicates the
validation of the test device. Moreover, the absence of
internationally accepted cut-off values for oral fluid
hampers the comparison of on-site test results with
chromatographic results. At the end of the Rosita2 study,
based on the analytical evaluation (comparison to reference
methods in oral fluid and/or blood), no device was
considered reliable enough to be recommended for roadside
screening of drivers [99]. The sensitivity of the on-site
screening devices for cannabis and benzodiazepines needs
to be improved dramatically. For example, the performance
of the Dräger DrugTest® used to screen for THC in oral
fluid was evaluated using oral fluid and plasma as reference
samples. Whereas the specificity of the test was high (93–
100%), a sensitivity of only 50% was observed, indicating a
high number of false negative test results [91].
Conclusions
Sensitive, specific, and reliable analytical methods are now
available for the screening of and for confirmation analysis
of drugs of abuse in oral fluid specimens. For confirmation
analysis, a significant number of well-validated GC–MS
and LC–MS(–MS) methods have been published. For
screening purposes, each laboratory has to choose between
using rapid and commercially available EIA methods or a
chromatographic technique that permits the target screening
of a large number of compounds, such as LC–MS–MS.
Both require only a small amount of specimen for the
analysis. An efficient sample clean-up method for LC–MS
(–MS) procedures is required to obtain accurate and
reproducible quantitative data. On-line SPE methods
present a good alternative for high-throughput analysis; in
the authors’ laboratory, work is underway using an on-line
SPE–LC–ESI–MS–MS method for the detection of basic
drugs in oral fluid samples.
In any case, a thorough validation of the method needs to
be conducted according to international guidelines, includ-
ing data on selectivity, precision, accuracy, detection and
quantification limits, stability, evaluation of matrix effects,
and an in-depth evaluation of the collection method. It is
especially important to ensure that the same collection
system is used for validation experiments as for authentic
samples. External proficiency testing schemes are now
being developed and cut-off concentrations have been
proposed by scientific organizations. Appropriate certifica-
tion of laboratories worldwide and the introduction of legal
provisions in several countries will be the next step.
1452 Anal Bioanal Chem (2007) 388:1437–1453

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