The analysis of antipsychotic drugs in human matrices using LC-MS(/MS).

Abstract

Antipsychotic drugs (APs) are prescribed for a wide range of psychotic illnesses. With more than 35 APs currently available worldwide, this drug class has rapidly gained importance in both clinical and forensic settings. On account of their chemical properties, many APs are present in human specimens at very low concentrations, which complicate their detection using standard gas chromatography-mass spectrometry (GC-MS) procedures that often cannot provide the required sensitivity. Recent advances in liquid chromatography-(tandem) mass spectrometry LC-MS(/MS) technology have enabled accurate detection and quantification of these compounds in various human specimens, indicated by the increasing number of published methods. Method validation has been a particular focus of analytical chemistry in recent times. Recommendations set by several guidance documents are now widely accepted by the toxicology community, as reflected by the guidelines drafted by leading toxicological societies. This review provides a critical review of single-stage and tandem LC-MS procedures for the detection and quantification of APs, with a particular emphasis on appropriate method validation. The quality of published methods is inconsistent throughout the literature. While the majority of authors incorporate some validation experiments in their respective method development, a large number of published methods lack essential components of method validation, which are considered mandatory according to the guidelines. If adapting a method for the detection of APs for use in a laboratory, analysts should ensure successful validation experiments for appropriateness and completeness have been conducted, and perform additional experiments when indicated.

Full text

Page 1

The analysis of antipsychotic drugs in human
matrices using LC-MS(/MS)
Eva Saar,a* Jochen Beyer,a,bDimitri Gerostamoulosa,band
Olaf H. Drummera,b
Antipsychotic drugs (APs) are prescribed for a wide range of psychotic illnesses.With more than 35APs currently available world-
wide, this drug class has rapidly gained importance in both clinical and forensic settings. On account of their chemical properties,
many APs are present in human specimens at very low concentrations, which complicate their detection using standard gas chro-
matography–massspectrometry (GC-MS)proceduresthatoftencannot providetherequiredsensitivity.Recentadvancesinliquid
chromatography-(tandem) mass spectrometry LC-MS(/MS) technology have enabled accurate detection and quantification of
these compounds in various human specimens, indicated by the increasing number of published methods. Method validation
has been a particular focus of analytical chemistry in recent times. Recommendations set by several guidance documents are
now widely accepted by the toxicology community, as reflected by the guidelines drafted by leading toxicological societies. This
review provides a critical review of single-stage and tandem LC-MS procedures for the detection and quantification of APs, with a
particular emphasis on appropriate method validation.
The quality of published methods is inconsistent throughout the literature. While the majority of authors incorporate some
validation experiments in their respective method development, a large number of published methods lack essential compo-
nents of method validation, which are considered mandatory according to the guidelines.
If adapting a method for the detection of APs for use in a laboratory, analysts should ensure successful validation experi-
ments for appropriateness and completeness have been conducted, and perform additional experiments when indicated.
Copyright © 2012 John Wiley & Sons, Ltd.
Keywords: antipsychotic drugs; LC-MS(/MS); method validation
Introduction
In the 1950s, the phenothiazine derivative chlorpromazine was
the first drug introduced for the treatment of psychotic illnesses,
largely replacing electroconvulsive therapy and psychosurgery.
Subsequent to the success of chlorpromazine, a large number
of compounds were introduced for the treatment of patients
suffering from mental illnesses. The main category of neurolep-
tic drugs is the phenothiazine derivatives, butyrophenones,
and thioxanthenes, known as ‘typical’ antipsychotics (APs).
While these drugs show significant improvement in the symp-
toms of psychotic illness, they are also associated with
unwanted extrapyramidal side-effects resulting from their activ-
ity at dopamine receptors. A new generation of APs introduced
around 1995 largely overcame these side-effects via decreased
activity at dopamine receptors compared with their traditional
counterparts. These ‘second generation’ or ‘atypical’ APs now
account for the vast majority of AP prescriptions. Reports in
the USA indicate a steady increase from 1.0 M prescriptions in
1995 to 13.3 M in 2008, while typical agents decreased signifi-
cantly over the same timeframe.[1]However, studies in recent
years have shown that atypical APs are not free from side-
effects. An increased risk of mortality in addition to cardiovascu-
lar complications have been reported in patients suffering from
dementia when treated with atypical APs.[2]Furthermore,
second-generation APs do not only increase the risk of diabe-
tes[3]compared with typical agents, but also show a similar risk
of sudden cardiac death to their typical counterparts.[4]With
more than 35 APs currently available worldwide, this drug class
has rapidly gained importance in both a clinical and forensic
setting, which makes the ability to reliably detect APs in human
biological specimens a necessity.
In a clinical environment, the analysis of APs in blood is neces-
sary in order to monitor patient compliance and to maintain drug
concentrations within the recommended therapeutic range of
the respective drug. The absence of prescribed APs in a clinical
case may also indicate non-compliance, a common issue among
patients suffering from mental illness. In a forensic setting, the
detection of APs is crucial in determining whether these drugs
played a role in the cause of death. A sub-therapeutic concentra-
tion of an AP in forensic cases may be particularly relevant in
cases where mental disturbances have contributed to the death
of a person by another, for example, homicides. Analytically,
APs have been traditionally measured using gas chromatography
(GC) with mass spectrometry (MS).
Zhang et al.[5]presented an overview of bioanalytical methods
for the determination of APs up until 2007. The authors focused
primarily on GC and liquid-chromatography (LC) methods with
* Correspondence to: Ms Eva Saar, Victorian Institute of Forensic Medicine,
Department of Forensic Medicine, Monash University, 57–83 Kavanagh St,
Southbank, Australia. E-mail: eva.saar@monash.edu
a Department of Forensic Medicine, Monash University, Southbank, Victoria,
Australia
b Victorian Institute of Forensic Medicine, Southbank, Victoria, Australia
Drug Test. Analysis (2012)Copyright © 2012 John Wiley & Sons, Ltd.
Review
Drug Testing
and Analysis
Received: 24 January 2012 Revised: 17 February 2012 Accepted: 17 February 2012Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/dta.1337

Page 2

various detectors such as ultraviolet (UV), nitrogen phosphorus,
fluorescence, and electrochemical detection (EC), concluding that
LC was the most suitable separation technique for these mostly
involatile compounds. MS/MS in combination with LC now domi-
nates the analytical field, providing a particularly convenient tool in
the analysis of APs. The high sensitivity of LC-MS/MS methods often
allows analysis times to be substantially reduced compared with
traditionalUVandECmethods,whichisparticularlyusefulforalarge
sample throughput or when fast turn-around times are required.
Method validation has been a particular focus in recent times, in
order to ensure true performance of methods and provide an
objective tool to establish whether a method works as intended.
The reproducibility of an analytical method is mandatory in
preventing serious legal consequences that can result from discre-
pancies in forensic investigations. Specific guidelines for method
validation were published two decades ago[6,7]and have since been
revisitedbytheauthors[8,9]toproducecontemporaryguidelinesspec-
ifyingtheminimumrequirementsformethodvalidation.Theseguide-
linesarenowwidelyacceptedinthetoxicologycommunity,reflected
in guidelines drafted by leading toxicology societies such as The In-
ternational Association of Forensic Toxicologists (TIAFT), the Society
of Forensic Toxicologists (SOFT) and the Society of Toxicological
andForensicChemistry(GTFCh).However,alargenumberofmeth-
ods still exist that either lack crucial parts of validation, or that have
not adequately performed the obligatory validation experiments.
This paper provides a critical review of single-stage and tandem
LC-MS procedures for the detection and quantification of APs with
a particular emphasis on appropriate method validation.
Methods
Papers for this review were selected following a comprehensive
PubMed search for English articles using LC-MS or LC-MS/MS
methods for the detection of one or more APs in various human
specimens (blood, plasma, serum, urine, hair, saliva, and cerebro-
spinal fluid). Selected papers were reviewed for analytical details
and assessed with regard to the extent of validation studies
against current guidelines.[6–9]
Choice of biosamples
Blood is the preferred specimen for AP analysis as it provides the
most accurate representation of the relevant pharmacological
effects.Inaclinicalsetting,plasmaandserumarematricesofchoice
for drug analysis, as they are the most common specimens used in
diagnosticmedicine.Therapeuticdrugsmonitoring(TDM)methods
are common and are more likely to focus on one or very few
analytes. Whole blood is the most common specimen used in
forensic cases since lysis is common in death investigations, and
centrifugation shortly after collection is not always possible.[10]
Urine is a useful specimen for general unknown screening
(GUS) procedures, particularly when overdose is suspected and
qualitative results are required. APs are included in most pub-
lished non-targeted screening procedures as part of big libraries.
However, since these methods lack the ability to produce quanti-
tative results, they are less relevant for the detection of APs and
will not be discussed in this review.[11–13]Targeted published
methods for detection of APs in urine using LC-MS(/MS) are rare
and usually include an additional matrix.[14–17]
Hair has become an increasingly popular alternative specimen
to blood, as drugs and their metabolites are likely to remain in
hair samples long after the compounds have been eliminated
from the body. Segmental hair analysis in particular can provide
an indication of the long-term history of drug use in an individ-
ual. While hair analysis is frequently used as a tool in the analysis
of drugs of abuse, only a limited number of methods targeting
APs in hair using LC-(MS/)MS technology have been published
to date.[16,18–22]
Oral fluid is used as an alternative to blood, which has increas-
ingly gained importance due to the relatively short drug detection
windows inadditionto non-invasivecollectionofspecimens.These
factors make oral fluid a useful specimen in circumstances where
trained medical staff is not available, such as roadside and work-
place drug testing. APs are known to reduce salivary flow rate[23]
and may therefore not be ideal for detection in oral fluid. This is
reflected in the limited number of published methods for APs[24]
to date using this specimen.
Cerebrospinal fluid (CSF) is commonly analyzed in order to
help diagnose various diseases and conditions affecting the cen-
tral nervous system (CNS), such as meningitis and encephalitis. It
is also useful in diagnosing bleeding of the brain or tumours
within the CNS. CSF is most commonly obtained by lumbar punc-
ture, a complex and invasive procedure that requires specialized
medical staff. While it is likely that drug concentrations in CSF are
more closely related to pharmacological effects than blood con-
centrations, the complicated process of sample collection makes
it a less favourable specimen in drug analysis, with only one pub-
lished method for the detection of APs.[25]
General considerations
Sample volume and LLOQ
In published analytical methods, sample volumes below 0.1 ml
are rare,[24,26–28]whereas volumes closer to 1 ml are frequently
used. When selecting a sample volume for an analytical method
targeting APs, several factors must be considered. Using a small
sample volume in an analytical method provides several advan-
tages, including easier handling during sample extraction and
the ability to conduct analysis in cases where only limited speci-
mens are available – for example, post-mortem cases. However,
APs are mostly lipid-soluble weak bases, which are quickly
absorbed into body fat and organs following administration,
signifying a large volume of distribution (VD). Despite their high
VD, most common APs also significantly bind to plasma proteins
(Fb). Both the large VDand high Fb significantly reduce the
amount of unbound drug available in the blood for detection.
Analytical requirements dictate that the lowest therapeutic
blood concentration of a drug must be quantified. This equates
to determining the lower limit of quantification (LLOQ), usually
involving two different approaches: a signal-to-noise ratio (S/N)
of 10 is considered satisfactory[29]and so is a precision and accu-
racy of <20% at the desired LLOQ.[6,8]Huang et al.[30]reported
an S/N of 3 at the LLOQ, which is generally acceptable for a limit
of detection (LOD), but not for the LLOQ. However, they
conducted validation experiments which confirmed the preci-
sion and accuracy at the LLOQ to be within 20%, and therefore
meet acceptance criteria. It needs to be guaranteed that a
method is sufficiently sensitive to fulfill at least one of these
two criteria when selecting the sample volume. Table 1 shows
pharmacokinetic parameters of common APs.
E. Saar et al.
Drug Testing
and Analysis
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Single-analyte methods vs multi-analyte methods
Single-analyte methods are mostly used in a TDM-setting, where
only specific compounds are the target of drug monitoring. Meth-
ods targeting the atypical AP risperidone (RIS) should always in-
clude its major metabolite 9OH-risperidone (9OH RIS), also referred
to as paliperidone. 9OH RIS is formed by cytochrome (CYP) P450
enzymes, specifically CYP2D6, and is likely to contribute to the
in vivo effects of RIS.[31]Whilst plasma concentrations of RIS and
9OH RIS show a large variation between individuals,[32–34]RIS levels
are generally lower than 9OH RIS levels. In fact, a study measuring
plasma concentration of RIS and 9OH RIS after oral administration
of RIS in steady-state found RIS was not detectable at a LLOQ of
0.1 ng/ml in ~18% of all tested individuals, whereas 9OH RIS was
detected in all cases.[32]Measuring only the parent compound,
especiallyinTDMmethods,canthereforeleadtoinaccurateconclu-
sions regarding patient compliance.
While the same risk of interferences exists for single-analyte and
multi-analyte procedures, chances are higher that they will be
identified during method development when a greater number
of analytes are included in the method. Generally, multi-analyte
procedures are preferred over single-analyte approaches, as the
inclusion of a number of analytes in one method saves time
and resources.
Table 1. Pharmacokinetic parameters of common APs.
Drug Common daily oral dose
range in adults (mg)1
Blood concentrations
expected following therapeutic
use (ng/ml)2
t1/2(h)3
VD(L/Kg)4
9OH-Risperidone*
Amisulpride
Aripiprazole
Bromperidol
Buspirone
Chlorpromazine
Chlorprothixene
Clozapine
Flupentixol
Fluphenazine
Fluspirilene¥
Haloperidol
Levomepromazine
Loxapine
Melperone
Mesoridazine
Molindone
Olanzapine
Penfluridol
Perazine
Pericyazine
Perphenazine
Pimozide
Pipamperone
Prochlorperazine
Promazine
Quetiapine
Risperidone
Sertindole
Sulpiride
Thioridazine
Thiothixene
Trifluoperazine
Triflupromazine
Ziprasidone
Zotepine
Zuclopenthixol
3–12 10–100
50–400
50–350
1–20
1–10
30–300
20–200
200–800
1–15
2–20
N/A
5–50
15–60
10–100
5–40
15–100
~500
10–100
4–25
100–230
5–60
0.6–2.4
15–20
100–400
10–500
10–400
70–170
10–100
50–500
50–400
200–2000
N/A
1–50
30–100
50–120
5–300
5–100
23N/A
13–16
4.9
N/A
5–6
10–35
11–23
2–7
14.1
220
N/A
18–30
30
N/A
7–10
3–6
3–6
10–20
N/A
N/A
N/A
10–35
11–62
N/A
13–32
27–42
8–12
0.7–2.1
20–40
2.7
18
N/A
N/A
N/A
1.5–2.3
50–168
15–20
400–1200
10–30
1–15
20–30
200–600
40–80
300–450
3–6
1–5
2–5 (i.m)
1–15
25–50
20–100
100–400
100–400
50–100
5–20
11–27
60–90
15–35
3–12
7–119
8–12
6–17
19–39
13–58
21 days (decanoate)
18
15–30
3–4
2–4
2–9
1.2–2.8
21–54
70
8–15
N/A
8–12
28–214
12–30
14–27
7–17
6–7
3–20
N/A
4–11
26–36
12–36
7–18
N/A
2–8
12–30
12–28
20–60 (once per week)
50–600
15–60
12–24
7–10
80–120
15–40
200–800
300–450
2–6
12–20
400–600
150–300
6–30
15–20
165–375
40–160
75–300
20–50
1: Common daily oral dose data for the treatment of schizophrenia, psychoses or bipolar disorder from DrugdexWEvaluations in the MicromedexW
Internet database.[96]Where the drug is indicated for other disorders (e.g. depressive disorders obtained), dosages may vary.
2: Blood concentrations expected following therapeutic use obtained from TIAFT guidelines.[97]
3: Terminal elimination half–life and;4: Volume of distribution obtained from Baselt.[98]
* : Also referred to as ‘Paliperidone’.
¥: Only available as i.m. injection
Review: The analysis of antipsychotic drugs in human matrices
Drug Testing
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Sample preparation
Extraction of APs from blood, plasma, and serum
Table 2 shows an overview of currently published single-analyte
LC-MS(/MS) methods using blood, plasma, or serum. Table 3 con-
tains all published multi-analyte studies.
Due to the high specificity of LC-MS methods, it was initially
thought that the sample preparation step may not be as crucial
as with other analytical methods, particularly for MS/MS methods
since transitions greatly reduce the risk of interference from other
drugs. However, this view was soon revised. While endogenous
components might no longer be detected using LC-MS methods,
they can still significantly interfere with the quantification of a
drug.[35,36]
Therefore, liquid-liquid extraction (LLE)[25,30,37–51]and solid-
phase extraction (SPE)[15,16,52–59]are still most commonly used
as a sample treatment prior to injection into the LC-MS system,
as they provide the most thorough sample clean-up. Saar
et al.[60]systematically evaluated nine different combinations of
extraction solvents and buffers in order to find the most suitable
LLE method for the extraction of 19 APs.[60]The method showing
the best results overall for extraction recoveries and matrix
effects used trizma buffer and 1-chlorobutane (BuCl) and was
subsequently compared with a standard SPE method. While
extraction efficiencies were comparable between LLE and SPE
methods, blockages of SPE cartridges were a common problem,
especially when dealing with post-mortem samples. Nirogi
et al.[46]applied a similar approach when comparing six organic
solvents and their combinations in order to optimize extraction
recovery for their method targeting olanzapine (OLZ) in plasma.
A mixture of diethylether and dichloromethane (7:3, v/v) yielded
the highest recovery of OLZ and was therefore used in their
detection method. Gutteck et al.[48]stated that due to the differ-
ent ‘extraction coefficients... and different concentration ranges
in human serum’, four different extraction procedures had to be
applied for determination of thirteen antidepressants and five
APs. Minor variations in organic solvents used for the LLE, differ-
ences in the volumes of the mobiles phases and varying internal
standards mark the differences between the four methods. A
more practical approach would have been to have one extraction
method and chromatographic conditions that allowed the analy-
sis of all drugs in a single cost-effective method, especially since it
is not clear which factors resulted in the development of the four
different methods.
Simple protein-precipitation (PP) may be used for ‘cleaner’
matrices such as serum or plasma.[26,27,61,62]It needs to be noted,
however, that matrix effects must be investigated closely as PP
might fail to remove phospholipids from plasma or serum which
mightcauseinterferences.[63,64]Interestingly,KloseNielsenetal.[65]
compared LLE methods with different combinations of organic
solvents and SPE techniques prior to the development of their
method for the determination of OLZ in whole blood, and found
none of them to be functional. However, a simple PP appeared to
producesoundresults.Fewmethodsemployeddirect-injection,[14,28]
while one published method used direct injection in combination
with column switching[24]in order to decrease matrix influences.
One published approach uses solid-phase micro-extraction (SPME)
as a solvent-free and concentrating extraction technique.[17]While
traditionally combined with GC, employing heat assisted desorp-
tion from the fibre, a simple interface coupling SPME with LC
makes it functional for non-volatile substances. Online-SPE
has been applied in order to reduce human error and increase
time-efficiency.[57]Upscaling of the extraction is achieved by
work-up in the 96-well format.[27,53]
Extraction of APs from hair
Table 4 shows an overview of methods published for the detec-
tion of APs in hair, using LC-MS(/MS).
The Society of Hair Testing recommends that hair be washed
prior to analysis (e.g. in methanol (MeOH)) and the wash solution
be subsequently analyzed for drug content.[66]A high concentra-
tion of the drug of interest in the wash solution may indicate
external contamination of the hair sample. To date, however, a
conclusion has not been reached concerning the best deconta-
mination strategy.[67–73]
Among the most commonly used extraction procedures for
hair analysis are alkaline hydrolysis using NaOH followed by
SPE, or extraction with MeOH and aqueous buffer using an
ultrasonicator.[74]Whilst both techniques are used for analysis
of APs in hair, methods using NaOH appear to be preferable for
alkaline-stable drugs such as APs. Josefsson et al.
attempt a full validation of their LC-MS/MS method for the iden-
tification of 19 APs and their major metabolites in hair. Incubation
with NaOH was performed prior to extraction with BuCl and back
extraction into formic acid. Two SRM transitions were chosen per
AP (and where possible per metabolite) for identification of the
drugs of interest. The authors highlighted the importance of
including metabolites of drugs of interest in hair methods. In hair
analysis, the issue of incorporation of a drug into the hair from
external sources rather than ingestion is a frequent point of dis-
cussion, especially in court cases where an accused person denies
the use of a drug. For some drugs, the presence of metabolites in
a certain ratio to the parent drug can be an additional indication
that ingestion of the drug has occurred and facilitate interpreta-
tion of results of hair analyses.[75]
Nielsen et al.[20]tested different combinations and ratios of
organic and aqueous solvents prior to the development of their
detection and quantification method. This involved 52 common
pharmaceuticals and drugs of abuse in hair, including five APs.
This ‘mixed’ approach was fully validated in accordance with
international guidelines.[9]When extracting basic compounds
such as APs from hair, the use of a neutral or slightly acidic aque-
ous buffer is recommended in order to facilitate ionization of the
compounds prior to transition into the aqueous phase.[74]Mueller
et al.[19]and Weinmann et al.[22]performed ultrasonication with
MeOH prior to mixed-mode SPE. Thieme et al.[21]divided the
initial 50 mg segment of hair into individual hairs prior to analy-
sis; 30 fg on column was sufficient to detect clozapine in single
hairs. The authors, however, acknowledge the uncertainty associ-
ated with hair analysis, mainly resulting from the unknown recov-
ery of drug from hair combined with the uncertainty of the exact
length of single hair segments.
[16]did not
Extraction of APs from cerebrospinal fluid, oral fluid, and urine
Table 5 shows an overview of published methods for the detec-
tion of APs in CSF, oral fluid, and urine using LC-MS(/MS).
Several authors have attempted to validate previously devel-
oped methods for the detection of APs in plasma or blood for
urine[14,15,17]. Bogusz et al.[76]applied full-scan mode to urine
samples of patients treated with OLZ in order to find proposed
metabolites. A large number of OLZ metabolites in urine have
been confirmed by Kassahun et al. in their comprehensive study
E. Saar et al.
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Page 5

Table 2. Summary of single–analyte methods for the detection of APs in blood (a), plasma (b), and serum (c) using LC-MS/MS.
a)
Author
(Year)
Volume [ml]
Drugs
IS
Extraction
Column
Mobile Phase
Detection
mode
Validation data
Klose Nielsen
et al.[65](2009)
0.19
olanzapine
dibenzepine
acidic MeOH–induced PP Zorbax Extend C18
(50 x 2.1 mm,
5 mm)
gradient with 5 mM
ammonium hydroxide
in ACN and ACN
ESI, positive
mode, SRM,
MS/MS
linearity, selecticity,
matrix effects, reco-
very, LLOQ, precision,
accuracy, PS stability,
LT stability
Kollroser et al.[43]◊
(2001)
1
zuclopenthixol
flupentixol
LLE (ammonia solution
and ethylacetate)
Symmetry C18
Waters
(3.0 x 150 mm,
5 mm)
gradient with ACN and
0.1% formic acid
ESI, positive
mode, SRM,
MS/MS
linearity, accuracy,
precision, LOD
b)
Author
(Year)
Volume [ml]
Drugs
IS
Extraction
Column
Mobile Phase
Detection
mode
Validation data
Aravagiri et al.[37]
(2001)
0.5
clozapine,
norclozapine,
clozapine–N–
oxide
“a derivative of
risperidone”
LLE (ethyl acetate,
methylene chloride,
pentane)
Phenomenex C18
(50 x 4.6 mm,
5 mm)
isocratic with 60 mM
ammonium acetate
MeOH and ACN
ESI, positive
mode, SRM
precision, accuracy
Aravagiri et al.[38]
(2000)
0.5
risperidone, 9OH–
risperidone
R68808
LLE (0.5 ml sat solution of
sodium carbonate (pH =
10.5) 15% methylene
chloride in pentane
Phenomenex
phenyl hexyl
column (5 mm,
50 x 4.6 mm)
isocratic with 0.15 mM
ammonium acetate,
MeOH, and ACN
ESI, positive
mode, SRM
precision, accuracy,
Arinobu et al.[14] ##
(2002)
1
haloperidol, reduced
haloperidol, 4–(4–
chlorophenyl)–4–
hydroxypiperidine
4–[4–(4–
chlorophenyl)–4–
hydroxy–1–
piperidinyl]–(4–
chlorophenyl–1–
butanone
addition of 3 ml of dH2O
with 0.09% formic acid
and 20 mM ammonium
acetate, freezing,
thawing, centrifugation,
injection of 20mL of
supernatant
Mspak GF–310 4B
(50 x 4.6 mm)
gradient with formic acid
and 20 mM ammonium
acetate in dH2O and ACN
SSI, positive
mode, MS
linearity, LOD, precision,
accuracy
Barret et al.[52]
(2007)
0.5
quetiapine
clozapine
SPE
Atlantis dC18
(100 mm x
3 mm, 3 mm)
isocratic with ACN–MeOH–
0.01 M ammonium acetate
ESI, positive
mode, SRM,
MS/MS
selectivity, LOD,
LLOQ, recovery, ma-
trix effects, linearity,
precision, F/T and LT
stability, PS stability
Review: The analysis of antipsychotic drugs in human matrices
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Page 6

Author
(Year)
Volume [ml]
Drugs
IS
Extraction
Column
Mobile Phase
Detection
mode
Validation data
Bhatt et al.[62]
(2006)
0.1
risperidone, 9OH–
risperidone
methyl risperidone
PP (ACN)
Betasil C18column
(3 mm, 100 x
3 mm)
isocratic with ammonium
acetate and ACN
ESI, positive
mode, SRM
MS/MS
selectivity, linearity,
LLOQ, precision,
accuracy, recovery, F/
T and LT stability, PS
stability
De Meulder
et al.[15] ##(2006)
0.2
risperidone,
9OH–risperidone
2H2–13C2–risperidone
and2H2–13C2–
9OH–risperidone
SPE (mixed mode)
Chiralcel OJ
column
(50 mm x 4.6,
10 mm)
gradient with hexane,
0.01 mM ammonium
acetate in isopropanol,
0.01 mM ammonium
acetate in ethanol
ESI, positive
mode, SRM,
MS/MS
selectivity, precision,
accuracy, recovery, F/
T and LT stability, PS
stability
Flarakos et
al.[24] ##(2004)
0.025
risperidone,
9OH–risperidone
R068808
online cleanup, column
switching
Zorbax SB18(30 x
2.1 mm, 3.5 mm)
isocratic with
10 mM ammonium
acetate/ACN
SRM, MS/MS
linearity, selectivity,
precision, accuracy,
recovery, matrix
effects, F/T and
LT stability
Gschwend et al.[42]
(2006)
0.25
amisulpride
sulpiride
LLE (diisopropylether:
dichloromethane, 1:1)
Phenomenex
Synergi
Polar–RP analytical
column (75 mm
x
4.6 mm, 4 mm)
isocratic with 5 mM ammonium
formate/
ACN
ESI, positive
mode, SRM,
MS/MS
linearity, selectivity,
recovery, precision,
accuracy, F/T and LT
stability, PS stability
Kubo et al.[44]
(2005)
0.4
aripiprazole,
OPC–14857
OPC–14714
LLE (diethylether)
RP Chemcobond
ODS–W (150 x
2.1 mm, 5 mm)
isocratic with
dH2O /ACN
ESI, positive
mode, SRM,
MS/MS
selectivity, linearity,
accuracy, precision,
recovery, F/T and LT
stability, PS stability
Moody et al.[45]
(2004)
1
risperidone,
9OH–risperidone
RO68808
LLE (pentane/
methylene chloride)
Intersil 5 ODS3
(150 x 2.1 mm)
gradient with
dH2O and ACN
APCI, positive
mode, SRM,
MS/MS
selectivity, extraction
efficiency, accuracy,
precision, F/T stability,
LT stability
Nirogi et al.[46]
(2006)
0.5
olanzapine
loratadine
LLE (diethylether:
dichloromethane)
Inertsil ODS
column
(3 mm, 100 x 3 mm)
isocratic with 10 mM
ammonium acetate:ACN
ESI, positive
mode, SRM,
MS/MS
linearity, precision,
accuracy, LLOQ,
recovery, F/T and LT
stability, PS stability
Method A: R068809
Remmerie et al.[54]
(2003)
0.5
risperidone, 9OH-
risperidone
Method B:2H2-13C2-
risperidone and
SPE (10cc/130mg
Bond Elut Certify)
3–mm C18 BDS–
Hypersil
gradient with 0.01M
ammonium formate
and ACN
ESI, positive
mode, SRM,
MS/MS,
linearity, selectivity,
accuracy, precision,
recovery, PS stability,
Table 2. (Continued)
E. Saar et al.
Drug Testing
and Analysis
wileyonlinelibrary.com/journal/dta
Copyright © 2012 John Wiley & Sons, Ltd.Drug Test. Analysis (2012)

Page 7

2H2-13C2-9OH-
risperidone
column
(100?4.6mm)
LT stability, F/T
stability, matrix effects
Swart et al.[47]
(1998)
1
fluspirilene
?
LLE (4% isoamyl
alcohol in hexane)
Phenomenex Luna
C18
5mm,
150?2.1mm)
isocratic with MeOH
and dH2O
ESI, positive
mode,
scanning
product ion
spectrum
from m/z
130-500
selectivity, recovery,
LLOQ, accuracy,
precision, PS stability,
LT stability
c)
Author (Year)
Volume
[ml]
Drugs
IS
Extraction
Column
Mobile Phase
Detection
mode
Validation data
Huang et al.[30]
(2008)
0.3
risperidone
paroxetine
LLE (ACN)
Alltima–C18(2.1
mm?100 mm,
3 mm)
isocratic with
formic acid/ACN
ESI, positive
mode, SRM,
MS/MS
selectivity, linearity,
precision, accuracy,
recovery, PS stability,
F/T stability
Josefsson et al.
[25]##(2010)
0.2
olanzapine,
N–desmethylo-
lanzapine
olanzapine–d3
LLE (tert–butyl–
methyl–ether)
Synergi Hydro–
RP (50mm?
2mm, 2.5mm)
gradient with 10mM
ammonium formate
with formic acid
and MeOH with
formic acid
ESI, positive
mode, SRM,
MS/MS
linearity, LLOQ,
precision, accuracy,
recovery, matrix
effects, F/T stability,
LT stability
Nozaki et al.[26]
(2009)
0.03
zotepine
imipramine
PP (ACN)
Tosoh ODS–100 V
(50 mm?2mm,
5mm)
gradient with 10mM
ammonium formate
containing ACN and 10mM
ammonium formate
containing 90% ACN
ESI, positive
mode, EC–
MS/MS
linearity, LLOQ,
accuracy, precision,
recovery, matrix
effects
Abbreviations: ACN: acetonitrile, APCI: atmospheric pressure chemical ionization, dH2O: deionized water, EC: electrochemistry, ESI: electrospray ionization, F/T: freeze/thaw, LLOQ: lower limit of
quantification, LOD: limit of detection, LT: long term, m/z: mass over charge ratio, MeOH: methanol, SRM: selected reaction monitoring, MS/MS: tandem mass spectrometry, PP: protein
precipitation, PS: processed sample, SPE: solid phase extraction, SSI: sonic spray ionization
#: More drugs are included in this method but do not belong to the group of APs.
##: This method was applied to more than one matrix.
◊: Post–mortem specimens were analyzed in this method.
?: An IS appears to have been used but is not specified.
Review: The analysis of antipsychotic drugs in human matrices
Drug Testing
and Analysis
Drug Test. Analysis (2012) Copyright © 2012 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/dta

Page 8

Table 3. Summary of multi–analyte methods for the detection of APs in blood (a), plasma (b), and serum (c) using LC–MS/MS
a)
Author (Year) Sample [ml]]
Drugs
IS
Extraction
Column
Mobile Phase
Detection mode
Validation data
Josefsson
et al.[16] ◊ ##
(2003)
1
buspirone, chlorpromazine,
chlorprothixene, clozapine, dixyrazine,
flupentixol, fluphenazine, haloperidol,
hydroxyzine, levomepromazine,
melperone, olanzapine, perphenazine,
pimozide, prochlorperazine,
risperidone, thioridazine, ziprasidone,
zuclopenthixol#
N/A
SPE
Zorbax Stable Bond
Cyano column
(50 x 2.1 mm,
3.5 mm)
gradient with
different ratios
of MeOH:ACN:
20 mM
ammonium
formiate
ESI, positive
mode, SRM,
MS/MS
N/A
Kumazawa
et al.[17] ##
(2000)
1
perazine, thioridazine, prochlorperazine,
perphenazine, trifluoperazine,
flupentixol, fluphenazine,
thioproperazine#
propericiazine
SPME (polyacrylate–
coated fiber)
Capcell Pak C18
UG120, S–5 mm,
2.0 x 150
(Shiseido)
gradient with
10 mM
ammonium
acetate and ACN
ESI, full scan
m/z 50–500,
SRM, MS/MS
linearity, precision,
accuracy,
Roman
et al.[49] ◊
(2008)
1
buspirone, fluphenazine, flupentixol,
perphenazine, risperidone, 9OH–
risperidone, ziprasidone, zuclopenthixol
haloperidol–d4
LLE (trizma buffer,
methyl t–butyl ether)
Zorbax Stable
Bond Cyano
column
(50 x 2.1 mm,
3.5 mm)
gradient with
different ratios
MeOH, ACN, 20
mM ammonium
formate
ESI, positive
mode, SRM,
MS/MS
selectivity, linearity,
LLOQ, precision,
recovery, matrix
effects
Saar et al.[50]
(2010)
0.1
9OH-risperidone, amisulpride,
aripiprazole, bromperidol, buspirone,
chlorpromazine, chlorprothixene,
clozapine, droperidol, fluphenazine,
fluspirilene, haloperidol,
levomepromazine, loxapine,
melperone, mesoridazine, olanzapine,
perazine, pericyazine, perphenazine,
pimozide, pipamperone,
prochlorperazine, promazine,
promethazine, quetiapine, risperidone,
sulpiride, thioridazine, trifluoperazine,
triflupromazine, ziprasidone, zotepine,
zuclopenthixol
haloperidol–d4
LLE (trizma buffer,
1–chlororbutane)
Zorbax Eclipse
XCB–C18
(4.6 x 150, 5 mm)
gradient with
ammonium
formate and ACN
ESI, positive
mode, SRM,
MS/MS
selectivity, linearity,
accuracy,
precision,
PS stability, LT
stability,
LLOQ, extraction
efficiencies, matrix
effects, processefficiencies, F/T
stability
Seno et al.[95]
(1999)
1
flupentixol, perazine,prochlorperazine,
trifluoperazine,thioproperazine,perphenazine,
fluphenazine,propericiazine,thioridazine#
?
SPE
Capcell Pak C18UG80,
S–5 mm, 1.0 x 250
mm (Shiseido)
gradient with 10 mM
ammonium acetate
and ACN
ESI, positive mode,
SRM, MS/MS (for
Flupentixol)
linerarity, recovery,
Verweij
et al.[59]
(1994)
1
chlorprothixene, flupentixol, thiothixene,
zuclopenthixol
N/A
SPE (Bond Certify 3 cc
column (Varian)
HP 5 mm
Asahipak ODP–50,
4.0 x 125 mm
isocratic with ACN and
50 mM ammonium
acetate in dH2O
(85:15)
ESI, comparison of
fullscan and SRM
selectivity, linearity
E. Saar et al.
Drug Testing
and Analysis
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Copyright © 2012 John Wiley & Sons, Ltd. Drug Test. Analysis (2012)

Page 9

b)
Author (Year) Sample [ml]]
Drugs
IS
Extraction
Column
Mobile Phase
Detection mode
Validation data
Choong
et al.[55]
(2009)
0.5
aripiprazole, clozapine, olanzapine,
sertindole, dehydroaripiprazole,
norclozapine, dehydrosertindole#
remoxipride
SPE (mixed
mode support)
Xbridge C18column
(2.1 mm x 100,
3.5 mm)
gradient with
ammonium
acetate 20 mM
and ACN
ESI, positive mode,
MS, SIM
selectivity,
repeatability,
precision, trueness,
accuracy, matrix
effects, F/T and LT
stability, PS stability
Kollroser
et al.[28]
(2002)
0.05
clozapine, desmethylclozapine,
olanzapine
dibenzepine
direct injection
procedure, HPLC–
integrated sample
clean–up with OasisW
HLBextractioncolumn
(50 mm x 1.3, 5 mm)
Symmetry C18
Waters (3.0 x
150 mm, 5 mm)
isocratic with ACN/
formic acid
ESI, positive
mode, SRM,
MS/MS
selectivity, linearity,
recovery, LLOQ,
accuracy,
precision,
Kratzsch
et al.[56]
(2003)
0.5
amisulpride, bromperidol, clozapine,
droperidol, flupentixol, fluphenazine,
haloperidol, melperone, olanzapine,
perazine, pimozide, risperidone, sulpiride,
zotepine, zuclopenthixol, norclozapine,
clozapine–N–oxide, 9OH-risperidone
trimipramine–d3
SPE
Merck LiChroCART
column
(125 x 2 mm)
gradient with
5 mM aqueous
ammonium
formate and ACN
APCI, positive
mode, MS/MS,
SRM
selectivity, linearity,
accuracy, precision,
F/T stability, LT
stability, PS
stability, recovery
Remane et al.
[82](2011)
0.5
9OH-risperidone, amisulpride,
aripiprazole, benperidol, bromperidol,
chlorpromazine, clozapine, clozapine–
N–oxide, droperidol, flupentixol,
fluphenazine, fluspirilene, haloperidol,
levomepromazine, melperone,
norclozapine, perazine, perphenazine,
pimozide, pipamperone, promazine,
prothipendyl, quetiapine, risperidone,
sulpiride, thioridazine, ziprasidone,
zotepine#
citalopram–d6,
norclozapine–d8,
nordazepam–d5,
trimipramine–d3,
zolpidem–d6
LLE (butyl acetate/
ethyl acetate)
TF Hypersil GOLD
Phenyl column
(100 x 2.1 mm,
1.9 mm)
Gradient with
10 mM aqueous
ammonium
formate plus 0.1%
formic acid (pH =
3.4) and ACN plus
0.1% formic acid
APCI, positive
mode, MS/
MS, SRM
selectivity, linearity,
accuracy,
precision, ion
suppression/
enhancement of
co–elutinganalytes,
PS stability, LT
stability, LLOQ,
extraction
efficiencies, matrix
effects, process
efficiencies,
“crosstalk”, F/T
stability
Zhou et al.[51]
(2004)
0.5
clozapine, olanzapine, risperidone,
quetiapine
diazepam
LLE (ether)
Macherey–Nagel
C18 (2 mm x
125 mm, 3 mm)
isocratic with dH20
(formic acid: 2.7
mmol/l,
ammonium
acetate: 10 mmol
ESI, SRM,
accuracy, precision,
LT stability, F/T
stability
Review: The analysis of antipsychotic drugs in human matrices
Drug Testing
and Analysis
Drug Test. Analysis (2012)Copyright © 2012 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/dta

Page 10

Table 3. (Continued)
c)
Author (Year)
Sample [ml]]
Drugs
IS
Extraction
Column
Mobile Phase
Detection mode
Validation data
Gutteck et al.[48]
(2003)
1
flupentixol, fluphenazine,
pipamperone, thioridazine,
zuclopenthixol
imipramine–d3,
doxepine–d3,
chlorohaloperidol
LLE (n–hexane/
dichloromethane 4:1)
or dichloromethane
Silice Uptisphere
column RP C18
(12.5 cm x 2 mm,
5 mm)
isocratic with four
different
combinations of
50 mM acetate
buffer and ACN
ESI, positive mode,
MS, SIM
linearity,
selectivity,
precision,
accuracy,
recovery, LLOQ
Hasselstrom et
al.[27](2011)
0.06
clozapine, quetiapine,
ziprasidone
clozapine–d3,
quetiapine–d8,
ziprasidone–d8
Zinc sulphate, MeOH,
96–well plate
Zorbax SB–C8
(2.0 x 50 mm,
1.8 mm)
gradient with
formic acid
in dH2O and formic
acid in MeOH
ESI, positive mode,
SRM MS/MS,
selectivity,
recovery, matrix
effects, LLOQ,
precision,
trueness, LT
stability
Kirchherr et
al.[61](2006)
0.1
amisulpride, aripiprazole,
benperidol, chlorpromazine,
chlorprothixene, olanzapine,
flupentixol, fluphenazine,
haloperidol, 9OH-risperidone,
levomepromazine, olanzapine,
perazine, perphenazine,
pimozide, pipamperone,
quetiapine, risperidone,
sulpiride, thioridazine,
ziprasidone, zotepine,
zuclopenthixol
clonidine,
methylrisperidone,
MBHZ
PP (ACN:MeOH)
Chromolith Speed ROD
C18(50 mm x 4.6 mm,
5 mm)
gradient with
MeOH and
acetic acid
ESI, positive
mode, SRM,
MS/MS
linearity, accuracy,
precision, LLOQ,
recovery, matrix
effects
Niederlaender
et al.[57]
(2006)
N/A
clozapine, desmethylclozapine,
clozapine–N–oxide
mirtazapine
SPE (online)
Zorbax Eclipse XDB–C18
(4.6 x 150 mm, 5 mm)
isocratic with
MeOH–aqueous
ammonium acetate
buffer (25 mM)
ESI, positive
mode, MS, SIM
linearity,
recovery,
accuracy,
precision. LLOQ
Rittner et al.[58]
(2001)
1
clozapine, haloperidol,
levomepromazine, perazine,
pimozide, sulpiride#
flunitrazepam–d3
SPE
Symmetry WAT C18
(1.0x150mm,3.5mm)
gradient with
ACN, dH2O ,
MeOH
ESI, positive
mode, MS
fullscan mode
(m/z = 100–650)
N/A
Abbreviations: ACN: acetonitrile, APCI: atmospheric pressure chemical ionization, dH2O: deionized water, ESI: electrospray ionization, F/T: freeze/thaw, LLOQ: lower limit of quantification, LOD: limit of
detection, LT: long term, m/z: mass over charge ratio, MeOH: methanol, SRM: selected reaction monitoring, MS: single stage mass spectrometry, MS/MS: tandem mass spectrometry, PP: protein precipitation, PS: processed sample, SIM: single ion monitoring, SPE: solid phase ectraction, SPME: solid–phase micro–extraction
#: More drugs are included in this method but do not belong to the group of APs.
##: This method was applied to more than one matrix.
◊: Post–mortem specimens were analyzed in this method.
?: An IS appears to have been used but is not specified.
E. Saar et al.
Drug Testing
and Analysis
wileyonlinelibrary.com/journal/dta
Copyright © 2012 John Wiley & Sons, Ltd. Drug Test. Analysis (2012)

Page 11

Table 4. Summary of methods for the detection of APs in hair.
Author (Year)
Sample [g]
Drugs
IS
Extraction
Column
Mobile Phase
Detection mode
Validation data
Josefsson et al.[16] ##
(2003)
0.01–0.02
buspirone, chlorpromazine,
chlorprothixene, clozapine,
dixyrazine, flupentixol,
fluphenazine, haloperidol,
hydroxyzine, levomepromazine,
melperone, olanzapine,
perphenazine, pimozide,
prochlorperazine, risperidone,
thioridazine, ziprasidone,
zuclopenthixol#
N/A
Incubation for
15 min in 1 M
NaOH, 25 mM
trizma buffer,
extraction with
BuCl, back
extraction into
formic acid
Zorbax Stable Bond
Cyano column
(50 x 2.1 mm,
3.5 mm)
Gradient with
MeOH–ACN–20 mM
ammonium formate
and MeOH– ACN–20
mM ammonium
formate
ESI, positive mode,
MS/MS, SRM
N/A
McClean et al.[18]
(2000)
0.5
chlorpromazine, flupentixol,
trifluoperazine, risperidone
trimipramine
MeOH, NaOH, 4 M
hydrochloric acid,
final extraction
with hexane
Phenomenex
Luna C18
(150 x 4.6 mm)
Isocratic with 0.02
mol/L ammonium
acetate/0.1% acetic
acid in dH2O
and ACN
ESI, positive mode,
MS/MS, SRM
linearity, LOD,
recovery
Mueller et al.[19]
(2000)
0.05
pipamperone#
doxepine–d3
MeOH, SPE (mixed
mode)
RP–C8–select
B (2 mm x 125
mm, 5 mm)
Gradient with ACN 25%
aqueous ammonia
and formic acid
ESI/CID–MS, ProdI
scan, positive
mode, MS/MS, SRM
N/A
Nielsen et al.[20]
(2010)
0.01
chlorprothixene, clozapine,
levomepromazine,
promethazine, quetiapine#
mianserin– d3
Incubation with
MeOH:ACN:
ammonium
formate (2 mM,
8% ACN, pH = 5.3)
at 37?C for 18
hrs, Mini–Uniprep
vials (PTFE filter)
Waters
100mmx2.1mm
ACQUITY HSS T3
1.8 mm C18
Gradient with
0.05% formic
acid and MeOH
ESI, positive
mode, TOF–MS
LOD, LLOQ,
matrix effects,
selectivity,
carry–over,
linearity,
trueness,
precision
Thieme et al.[21]
(2007)
0.05 (divided into
single hairs for
segmentation)
clozapine, norclozapine
5–(4–methylphenyl)–
5–phenyl
hydantoine
Decontamination
with 5 ml
petroleumbenzene, ]
Ultrasonication
with 3 ml MeOH
for 3 hrs, reduce to
single hairs,
segmentation,
3hrsultrasonication
in 30uL dH2O /
MeOH (50/50)
Synergy Polar–RP
(Phenomenex,
75 mm x 2.0 mm,
4 mm)
Isocratic with
ammonium acetate
buffer in (50:50)
water and ACN
ESI, ProdI,
MS/MS, SRM
N/A
Review: The analysis of antipsychotic drugs in human matrices
Drug Testing
and Analysis
Drug Test. Analysis (2012)Copyright © 2012 John Wiley & Sons, Ltd.
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Page 12

Table 4. (Continued)
Author (Year)
Sample [g]
Drugs
IS
Extraction
Column
Mobile Phase
Detection mode
Validation data
Weinmann et al.[22]
(2002)
0.02–0.05
clozapine, norclozapine,
haloperidol, penfluridol,
thioridazine, northioridazine,
flupentixol, zuclopenthixol,
de–(hydroxyethyl)–
zuclopenthixol
doxepine–d3
Ultrasonication with
4 ml MeOH for
2 hrs, SPE
(mixed mode)
RP–C8–select
B (2 mm x
125 mm, 5 mm)
Gradient with 1 mM
ammonium formate/
0.1% formic acid,
and ACN/0.1%
formic acid
ESI, ProdI,
MS/MS, SRM
linearity, LOD,
LLOQ, recovery,
precision
Abbreviations: ACN: acetonitrile, BuCL: 1–chlorobutane, dH2O: deionized water, CID: collision induced dissociation ESI: electrospray ionization, LLOQ: lower limit of quantification, LOD: limit of detection,
MeOH: methanol, SRM: selected reaction monitoring, MS: single stage mass spectrometry, MS/MS: tandem mass spectrometry, NaOH: sodium hydroxide, ProdI: Product Ion Scan, SPE: solid phase
extraction, TOF: time of flight
#: More drugs are included in this method but do not belong to the group of APs.
##: This method was applied to more than one matrix.
Table 5. Summary of methods for the detection of APs in CSF, saliva, and urine using LC–MS/MS.
Author
(Year)
Matrix
Sample
[g]
Drugs
IS
Extraction
Stationary
Phase
Mobile Phase
Detection
mode
Validation data
Arinobu et
al.[14] ##
(2002)
urine
1
haloperidol, reduced haloperidol,
4–(4–chlorophenyl)–4–hydroxypiperidine
4–[4–(4–
chlorophenyl)–4–
hydroxy–1–
piperidinyl]–(4–
chlorophenyl–1–
butanone
addition of 3 ml of
dH2O with 0.09% formic
acid and 20 mM
ammonium acetate,
freezing, thawing,
centrifugation, injection
of 20mL of supernatant
Mspak GF–
310 4B (50 x
4.6 mm)
gradient with
formic acid and
20 mM ammonium
acetate in
dH2O (A) and ACN (B)
SSI,
positive
mode,
MS
LOD, precision,
accuracy
De
Meulder
et al.[15]
##(2008)
urine
0.2
risperidone, 9OH-risperidone
2H2–13C2–
risperidone and
2H2–13C2–9OH–
risperidone
SPE (mixed mode)
Chiralcel
OJ column
(50 mm x
4.6, 10 mm)
gradient with hexane,
0.01 mM ammonium
acetate in isopropanol,
0.01 mM ammonium
acetate in ethanol
ESI,positive
mode,
SRM,
MS/MS
selectivity,
precision,
accuracy, recovery,
F/TandLTstability,
PS stability
Flarakos et
al.[24] ##
(2004)
saliva
0.025
risperidone, 9OH-risperidone
R068808
online cleanup,
column switching
Zorbax
SB18(30 x 2.1
mm, 3.5 mm)
isocratic with
10 mM ammonium
acetate/ACN
N/A
linearity, selectivity,
precision,
accuracy,recovery,
matrix effects, F/T
and LT stability
E. Saar et al.
Drug Testing
and Analysis
wileyonlinelibrary.com/journal/dta
Copyright © 2012 John Wiley & Sons, Ltd.Drug Test. Analysis (2012)

Page 13

Table 5. (Continued)
Author
(Year)
Matrix
Sample
[g]
Drugs
IS
Extraction
Stationary
Phase
Mobile Phase
Detection
mode
Validation data
Kumazawa
et al.[17]
##(2000)
urine
1
perazine, thioridazine, prochlorperazine,
perphenazine, trifluoperazine, flupentixol,
fluphenazine, thioproperazine#
propericiazine
SPME (polyacrylate–
coated fiber)
Capcell Pak
C18UG120,
S–5 mm, 2.0
x 150
(Shiseido)
gradient with
10 mM ammonium
acetate and ACN
ESI, full
scan m/
z 50–
500,
SRM,
MS/MS
linearity,
precision,
accuracy,
Josefsson
et al.[25]
##(2010)
CSF
0.2
olanzapine, N–desmethylolanzapine
olanzapine–d3
LLE (tert–butyl–methyl–
ether)
Synergi
Hydro–RP
(50 mm x 2
mm, 2.5 mm)
gradient with
10 mM ammonium
formate with
formic acid
and MeOH with
formic acid
ESI,
positive
mode,
SRM,
MS/MS
linearity,
LLOQ, precision,
accuracy,
recovery, matrix
effects,
F/T stability,
LT stability
Bogusz et
al.[76] ##
(1999)
urine
1
olanzapine
LY170222
SPE
Super Spher
RP18(125 x
3 mm; 4 mm)
(Merck)
isocratic with
ACN/ammonium
formate, OLZ
metabolites
with Gradient
APCI,
positive
mode,
MS
recovery,
LLOQ, precision,
linearity,
selectivity,
F/T and LT
stability
Josefsson
et al.[16]
##(2003)
urine
0.5
buspirone, chlorpromazine, chlorprothixene,
clozapine, dixyrazine, flupentixol, fluphenazine,
haloperidol, hydroxyzine, levomepromazine,
melperone, olanzapine, perphenazine, pimozide,
prochlorperazine, risperidone, thioridazine,
ziprasidone, zuclopenthixol
N/A
SPE
Zorbax Stable
Bond Cyano
column (50
x 2.1 mm,
3.5 mm)
Gradient with
different ratios
of MeOH:ACN:20
mM ammonium
formiate
ESI,
positive
mode,
SRM,
MS/MS
N/A
Legend: ACN: acetonitrile, APCI: atmospheric pressure chemical ionization, dH2O: deionized water, ESI: electrospray ionization, F/T: freeze/thaw, LLOQ: lower limit of quantification, LOD: limit of detection,
LT: long term, m/z: mass over charge ratio, MeOH: methanol, SRM: selected reaction monitoring, MS: single stage mass spectrometry, MS/MS: tandem mass spectrometry, PS: processed sample, SIM:
single ion monitoring, SPE: solid phase extraction, SPME: solid–phase micro–extraction, SSI: sonic spray ionization
#: More drugs are included in this method but do not belong to the group of APs.
##: This method was applied to more than one matrix.
Review: The analysis of antipsychotic drugs in human matrices
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Page 14

of the metabolism of OLZ in humans.[77]It was hypothesized that
OLZ-10-N-glucuronide and N-desmethyl-OLZ would be present
in urine samples following OLZ ingestion. However, the com-
pounds were not unequivocally identified as a valid reference
standard was not available.
To the authors’ knowledge, the only method for the detection
of APs in oral fluid was published by Flarakos et al. in 2004.[24]
Their fully validated method applied online clean-up with column
switching for the detection of RIS and 9OH RIS in 25 ml saliva and
plasma, aiming to establish a salivary/plasma (S/P) ratio. A wide
range of S/P ratios obtained from 13 plasma and saliva samples
(seven adults and six children) confirmed that saliva analysis only
provided a qualitative tool for the presence of RIS and 9OH RIS
but did not allow a conclusion regarding plasma concentrations
at the time of sampling.
Josefsson et al. applied their detection method for OLZ and N-
desmethyl OLZ not only to serum but also to CSF.[25]The authors
postulated that the pharmacological effects of OLZ are likely to
be more closely related to its concentration in the CFS than in
serum. With a LLOQ of 0.2 ng/ml in plasma, the method showed
sufficient sensitivity for the expected low concentrations in CSF.
The authors postulated a linear correlation between serum and
CSF OLZ concentrations (r² = 0.77). While there were only six
individuals included in this study, the developed method was
successfully applied to a cohort of 37 individuals. The authors
also considered the influence of gender, age, smoking, and
pharmacogenetics, when investigating the ratio between OLZ
and metabolite concentrations in serum and CSF.[78]
LC separation
All APs possess hydrophobic properties and as such, all currently
published methods for the detection and quantification of APs in
biological matrices have employed reversed phase (RP) station-
ary phases, with mostly silica-based packings containing C8and
C18chains. Cabovska et al.[40]and de Meulder et al.[15]used chiral
columns in order to separate the (+) and (?) enantiomers of 9OH
RIS. 9OH RIS is the main metabolite of the atypical AP RIS and has
shown to be almost equipotent to risperidone in animal stud-
ies.[79]Due to its efficacy, racemic 9OH RIS (paliperidone) is also
marketed as a drug in its own right.[80]The separation of the
two enantiomers is useful for kinetic studies, as the formation
of the (+)-form appears to be catalyzed by CYP2D6, whereas
CYP3A4 and CYP3A5 are essential for the formation of the (?)-
form.[81]The separation of these enantiomers is usually not es-
sential in routine drug analysis.
Columns packed with <2 mm particles are referred to as ultra
high pressure LC (UHPLC) columns and are said to reduce analyt-
ical run times due to improved compound separation. This is de-
sirable in a TDM environment where a large number of samples
are tested for very few compounds. To the authors’ knowledge,
there are only two methods using UHPLC published to date. Has-
selstrom et al.[27]used a Zorbax SB-C8column with a particle size
of 1.8 mm, resulting in the detection and quantification of 13 anti-
depressants and APs, in addition to 13 deuterated IS over a total
analytical run time of 4 min. Remane et al.[82]covered a total of 62
compounds including 31 APs over a total run time of 26 min,
employing a TF Hypersil GOLD Phenyl column with a particle size
of 1.9 mm. A recent review, however, compared the separation
power of columns with particle sizes of 1.8 mm and 5 mm at a ‘fast’
(1 ml/l) and a ‘slow’ (0.3 ml/l) flow rate, and concluded that the
particle size was less significant than initially proposed. The col-
umn particle size appeared to make only a modest difference in
the peak height, peak width, or resolution, with the difference
for each parameter being less than a factor of 2. Higher flow rates
distinctively increased peak height by 6–7-fold and the peak
width decreased by about 3-fold when using the faster flow
rate.[64]In a post-mortem environment, larger particle sizes (3–5
mm) have proven to be favourable due to the higher robustness
which is required for more complex matrices such as whole
blood.[50]The presented methods show a wide range of isocratic
and gradient elutions, including various aqueous and organic elu-
tion solvents. Details are shown in the column ‘Mobile Phase’ in
Tables 3 and 4.
MS detection
Ionization of compounds in LC-MS technology is usually achieved
with either electrospray ionization (ESI) or atmospheric-pressure
chemical ionization (APCI). The reason ESI is used in the majority
of presented methods for the detection of APs is likely to be as-
sociated with the higher sensitivity achieved by ESI. Bhatt et al.
compared ESI with APCI, prior to development of their method
for the detection of RIS and 9OH RIS in plasma. They found APCI
to be less favourable when compared with ESI.[62]In a compre-
hensive study investigating the influence of anticoagulant and
lipemia on matrix effects when analyzing OLZ, Chin et al.
reported that the analyte response with APCI was five times less
than with ESI.[83]Therefore, the required LLOQ of 0.05 ng/ml for
OLZ was not achieved in APCI mode. The higher sensitivity
achieved by ESI, however, was at the expense of lower selectivity.
Many authors have found matrix effects to be more prominent
when applying ESI.[84,85]Ionization efficient neutral compounds
including matrix particles, co-eluting compounds, or additives
such as salts in biological samples, can compete with analytes
during the evaporation process. This is likely to lower the ioniza-
tion rate of the compounds of interest. It is further suggested that
during the evaporation process, the analyte of interest may pre-
cipitate from solution by itself or as a co-precipitate with non-vol-
atile sample components.[84]This highlights the need for thor-
ough sample clean-up prior to MS analysis and the assessment
of matrix-effects as a crucial part of method validation. This is dis-
cussed later in this paper.
Due to the predominantly basic properties of APs, ionization
takes place in the positive mode. The vast majority of published
methods apply selected reaction monitoring (SRM) as an easy
way for the detection and quantification of APs. International
guidelines[86–88]require a minimum of two SRM transitions for re-
liable identification of an analyte – unfortunately a large compo-
nent of SRM methods do not comply with this rule. The best ex-
ample of possible misidentification of a compound due to
monitoring a single SRM transition is the structurally similar O-
desmethyl metabolite of the antidepressant venlafaxine and the
synthetic opioid tramadol. Due to their almost identical chemical
structure, they do not only elute at the same time but also share
the most abundant transition (m/z 264.2 ! 58.2).[89]Less com-
mon examples in the field of APs include the structural isomers
promazine and promethazine (Figures 1a and 1b). These drugs
share the most abundant transition (m/z 285 ! 86), representing
the cleavage of the side chain[50]and also elute at the same time.
The isobaric compounds pipamperone and haloperidol
(Figures 2a and 2b) share the two most abundant transitions
E. Saar et al.
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and Analysis
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Copyright © 2012 John Wiley & Sons, Ltd. Drug Test. Analysis (2012)

Page 15

(m/z 376.2 ! 123 and m/z 376.0 ! 165).[50]If sensitivity can still
be maintained, it is recommended to pick a transition with a
smaller abundance for one of the two analytes or, alternatively,
add a third transition in order to guarantee reliable differentiation.
While MS in the SRM mode certainly provides an efficient tool
for compound identification, these examples highlight the need
to critically evaluate parameters (such as most abundant
transitions) provided by the instrument during compound
optimization. Few authors use screening procedures that allow
subsequent quantification of APs of interest.[17,47,58]
Validation issues
Tables 2 and 3 present an overview of single-analyte and multi-
analyte published methods, respectively, for the detection of APs in
blood, plasma and serum using LC-MS(/MS). It is generally accepted
that all methods must be validated using internationally accepted
guidelines. Specific validation criteria must be met to satisfy the
following minimum requirements:[7–9]selectivity, matrix effects, ex-
traction efficiency, process efficiency, processed sample stability, lin-
earity, accuracy, precision, and freeze-thaw stability. Although some
authors claim to have conducted all/specific components of the
method validation experiments, the quality and reputability of these
experimentsisnotconsistentacrossallpapers.Parameterswhichare
frequently associated with inconsistencies will be discussed below.
Internal standard
A variety of internal standards (IS) have been used in
the reviewed methods. Preferred internal standards are deuter-
ated compounds of the drug class of interest, such as
clozapine-d3,[27]haloperidol-d4,[49,50]olanzapine-d3,[25]quetia-
pine-d8,[27]and ziprasidone-d8.[27]If these IS are unavailable to
a laboratory, it is recommended to use a deuterated IS from a
different drug-class rather than an AP that is in therapeutic
use.[90]To the contrary, it has been suggested that high concen-
trations of a drug can influence the peak areas of their co-
injected deuterated analogues when using APCI mode with
isotope peaks (M + 1 to M + 3) of analytes contributing to the
peak area of the IS. This can lead to miscalculation of the IS
concentration and subsequently underestimation of the drugs
of interest. However, for masses (M + 5) and higher, no isotopic
contribution was observed.[91]
As co-medication and therapeutic use of a compound can
never be fully excluded, overestimation of an IS is likely to result
in underestimation of a drug concentration. Swart et al.[47]did
not achieve good results in their detection method for fluspiri-
lene in human plasma when using dimethothiazine as an IS. Their
decision not to use an IS at all defies the guidelines of acceptable
analytical practice. Particularly in cases where only few analytes
are included in a method, a suitable deuterated IS is preferred
in all instances. Unfortunately, this is not an isolated event. A
large number of analytical methods still use therapeutic drugs
as IS.[17,26,28,30,41,42,46,51,52,55,57,61,65,92]
Selectivity
In order to guarantee selectivity of an analytical method, it would
be ideal that all possible interferences arising from matrix com-
pounds, other drugs, and IS, are excluded. As this is impractical,
the analysis of six blank specimens from different sources is
widely considered acceptable[6]and is applied by most authors.
The testing of 10 blank specimens, however, has been employed
by some authors[50,56]and is encouraged for improved selectiv-
ity.[93]Josefsson et al.[25]performed method validation in
accordance with international guidelines in their method for
the detection of OLZ and N-desmethyl OLZ in CSF; however,
selectivity of the method was not investigated. This is surprising,
as despite the more invasive nature of sample collection
compared with taking blood, the authors obtained drug-free
CSF samples from six different patients. Several authors do not
state clearly how many different sources of blank specimens
were tested for interferences.[30,48]Klose Nielsen et al.[65]
examined the interferences from other possible drugs in forensic
samples by spiking blank blood samples with 66 common
drugs such as benzodiazepines, analgesics, antidepressants,
APs, b-blockers, narcotics and stimulants. Two ‘zero’ samples
(blank sample containing IS) should be included in validation-
experiments in order to exclude possible interferences of the IS
on the selectivity of the method.
Calibration
Linearity is an important part of method validation whenever
quantification of analytes via a standard curve is carried out,
Figure 1. Structures of promazine (a) and promethazine (b), their molec-
ular weights and the side-chain fragmen-tation resulting in the most
abundant fragment for both compounds (m/z=86).
Figure 2. Structures of pipamperone (a) and haloperidol (b), their molecular weights and the fragmentations resulting in the two most abundant frag-
ment for both compounds (m/z=123 and m/z=165).
Review: The analysis of antipsychotic drugs in human matrices
Drug Testing
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