Acta Psychiatr Scand 2016: 1–11 © 2016 John Wiley & Sons A/S.

© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
All rights reserved ACTA PSYCHIATRICA SCANDINAVICA
DOI: 10.1111/acps.12673

The significance of sampling time in

therapeutic drug monitoring of clozapine
Jakobsen MI, Larsen JR, Svensson CK, Johansen SS, Linnet K,
Nielsen J, Fink-Jensen A. The significance of sampling time in
therapeutic drug monitoring of clozapine.
Objective: Therapeutic drug monitoring (TDM) of clozapine is
standardized to 12-h postdose samplings. In clinical settings, sampling
time often deviates from this time point, although the importance of the
deviation is unknown. To this end, serum concentrations (s-) of
clozapine and its metabolite N-desmethyl-clozapine (norclozapine) were
measured at 12  1 and 2 h postdose.
Method: Forty-six patients with a diagnosis of schizophrenia, and on
stable clozapine treatment, were enrolled for hourly, venous blood
sampling at 10–14 h postdose.
Results: Minor changes in median percentage values were observed for
both s-clozapine ( 8.4%) and s-norclozapine (+1.2%) across the 4-h time
span. Maximum individual differences were 42.8% for s-clozapine and
38.4% for s-norclozapine. Compared to 12-h values, maximum median
differences were 8.4% for s-clozapine and 7.3% for s-norclozapine at
deviations of 2 h. Maximum individual differences were 52.6% for s-
clozapine and 105.0% for s-norclozapine. The magnitude of s-clozapine
differences was significantly associated with age, body mass index and the
presence of chronic basophilia or monocytosis.
Conclusion: The impact of deviations in clozapine TDM sampling time,
within the time span of 10–14 h postdose, seems of minor importance
when looking at median percentage differences. However, substantial
individual differences were observed, which implies a need to adhere to
a fixed sampling time.
M. I. Jakobsen1,2, J. R. Larsen1,2,
C. K. Svensson1,2,
S. S. Johansen3, K. Linnet3,
J. Nielsen4,5, A. Fink-Jensen1,2
1
Psychiatric Centre Copenhagen and Laboratory of
Neuropsychiatry, Department of Neuroscience and
Pharmacology, Rigshospitalet, Copenhagen O, 2Faculty
of Health and Medical Sciences, University of
Copenhagen, Copenhagen N, 3Section of Forensic
Chemistry, Department of Forensic Medicine, Faculty of
Health and Medical Sciences, University of Copenhagen,
Copenhagen O, 4Department of Clinical Medicine,
Aalborg University, Aalborg, and 5Psychiatry, Aalborg
University Hospital, Aalborg, Denmark
Key words: clozapine; norclozapine; sampling time;
serum concentration; therapeutic drug monitoring
Anders Fink-Jensen, Psychiatric Centre Copenhagen and
Department of Neuroscience and Pharmacology,
Rigshospitalet, Edel Sauntes Alle 10, DK-2100
Copenhagen O, Denmark.
E-mail: a.fink-jensen@dadlnet.dk
Accepted for publication November 2, 2016

Significant outcomes
• Both clozapine and norclozapine concentrations differed significantly within the 4-h time span,
although median percentage differences were minor
• Some individuals showed substantial percentage differences, indicating that deviations in sampling
time could influence the interpretation of individual TDM concentrations
• The magnitude of percentage clozapine differences was significantly associated with increasing age,
decreasing BMI and the presence of chronic monocytosis or basophilia

Limitations
• The sample size was small (n = 46), which limits the generalizability of the findings
• Multiple variables were tested for sensitive subgroup hypothesis generation, increasing the risk of
mass significance (type 1 errors)

1
Jakobsen et al.
Introduction
Clozapine is a highly effective, second-generation
antipsychotic, with a documented superiority to
other compounds, when treating patients with
treatment-resistant schizophrenia (1).
However, clozapine is also associated with sev-
eral potential life-threatening adverse effects, such
as an increased risk of myocarditis, ileus and
agranulocytosis (2), which is why treatment with
clozapine is restricted to patients with insufficient
response to at least two other antipsychotics (2).
The risk of developing a variety of haematological
side-effects, such as agranulocytosis (3), is also the
reason for the implementation of mandatory
haematological monitoring programmes world-
wide, for example in Europe, weekly white blood
cell (WBC) differential counts for the first
18 weeks of treatment, and monthly thereafter (2).
Most side-effects to clozapine treatment do
not have an established relationship with dose or
concentration levels (4), although the rate of
side-effects seems to double with concentrations
above 350 ng/ml (1071 nmol/l; conversion factor
3.06 (5)) (6). This is also the suggested lower
threshold for therapeutic response, as adequate
response is more likely to occur at concentra-
tions >350 ng/ml (1071 nmol/l) (7). Inefficiently,
low concentration levels will typically result in
treatment failure with relapse or withdrawal
symptoms and increased hospitalization (7, 8).
An upper limit for clozapine levels has not
reached consensus (9) as some patients may
require higher levels to show adequate response
(10); however, an alert level of 1000 ng/ml
(3060 nmol/l) has been suggested (5), addressing
a safety threshold, above which the risk of ful-
minant toxicity and CNS-related adverse reac-
tions, such as seizures, increases (9). Clozapine
levels thereby seem to be good predictors of
response, although the assessment of an ade-
quate response relies on a clinical evaluation as
well. Moreover, individual concentration-to-dose
(C/D) responses are somewhat unpredictable (7),
with an almost 50-fold reported variation in C/
D ratios for patients on fixed doses (11). This is
due to the complex nature of clozapine pharma-
cokinetics: clozapine is rapidly absorbed with a
mean 2.1 h time-to-peak plasma concentration
(range 0.4–4.2 h). Elimination is biphasic with
an average terminal half-life of 12 h (range 6–
26 h) (12). It is extensively metabolized in the
liver by various cytochrome P-450 (CYP)
enzymes, primarily by CYP1A2, with
N-desmethyl-clozapine (norclozapine) and cloza-
pine-N-oxide being the major metabolites (13).
2
Norclozapine is an active metabolite with sug-
gested antipsychotic and toxic effects on its own
(14, 15), and a reverse metabolism, back into the
parent compound, has been shown for the
metabolite clozapine-N-oxide (16). The turnover
through the various CYP systems is influenced
by several factors: smoking (17), caffeine con-
sumption (12, 18), genetics (19, 20), inflamma-
tion (21) and drug–drug interactions. Numerous
drugs have an effect on the activity of the CYP
systems; for example, some antibiotics, antide-
pressants, anticonvulsants, proton pump inhibi-
tors (PPI) and hormonal contraceptives (HC) (5,
12, 22) have been shown to markedly alter
clozapine levels. Other factors that have shown
to affect clozapine levels are gender, age and
bodyweight (4, 23).
Consequently, large inter- as well as intra-indivi-
dual variability in the C/D ratio has been reported
for clozapine-treated patients (7, 11). One study
suggests sampling time error as an important
source of the intra-individual variance in clozapine
concentrations (24). The authors of the mentioned
study argue that blood sampling for therapeutic
drug monitoring (TDM) of clozapine should fol-
low guidelines in order to be of relevance, as only
around 15% of the 773 investigated clozapine sam-
ples were collected at the standard 12-h time point
and that s-clozapine significantly correlated with
time from last administration (24).
TDM is a valuable and strongly recommended
clinical tool in the process of optimizing clozapine
treatment, while reducing the risk of concentra-
tion-dependent toxicity or relapse, especially sub-
sequent to changes in the concentration-affecting
parameters (4, 5). Although most hospitals have
standardized TDM of clozapine to 12-h samples,
accepted deviations in sampling time have not been
clearly defined. Consequently, sampling time can
deviate several hours from the 12-h time point
(24). The clinical impact of these deviations
remains to be elucidated.
Aims of the study
The aims of this study were to describe the impact
on serum (s-) clozapine and norclozapine levels, as
sampling time deviated 1 and 2 h from the stan-
dard 12-h time point, and to identify possible
impact-associated variables.
Material and methods
The study was approved by the local ethics com-
mittee of the Capital Region of Denmark and
the Danish health authorities. The study was
 Sampling time and clozapine concentrations

Table 1. Patient characteristics. Presented data are obtained from study interview, Table 1. (Continued)
medical records and trial assays
Patients characteristics (n = 46) Numbers
Patients characteristics (n = 46) Numbers
MR (NCLZ/CLZ), 10 h postdose 0.61 (0.33–1.17)
Demographics (n = 44)
Male gender* 26 (56.5) MR (NCLZ/CLZ), 12 h postdose 0.64 (0.36–1.39)
Age (years)† 42 (20–59) (n = 45)
Age ≥45 years* 21 (45.7)
Clinical assessments C/D, concentration-to-dose ratio; MR, metabolic ratio.
Clinical *Values are given as numbers and proportions (n (%)).
Smokers* 17 (37.0) †Values are given as median and range (min–max).
BMI (kg/cm2)† 30.9 (20.1–48.8) ‡Estimate based on daily cups (1.7 dl) of caffeinated beverages: coffee (73.6 mg/
Daily caffeine‡ (mg)† 744.9 (0–2224.2) cup), tea (42.5 mg/cup), cola (25.9 mg/cup), energy drinks (54.7 mg/cup) (26).
Baseline CRP ≥ 5 mg/l* 16 (34.8) §Chronic blood dyscrasia was defined as a minimum of 50% of the observations,
Observed chronic§ blood dyscrasias (n = 42)* for a given cell line, located either under or above the normal reference range. Only
Leukocytosis (>8.8 9 109/l) 16 (38.1) subjects with three or more observations within the retrospective period were
Basophilia (>0.1 9 109/l) 1 (2.4) included for chronic blood dyscrasia evaluation. The mean deviation from inclusion
Eosinopaenia (<0.04 9 109/l) 19 (45.2) day was 1.2  11.3 days. The mean period of observation was
Lymphocytosis (>3.5 9 109/l) 3 (7.1) 5.8  1.1 months, and the mean number of observations within the retrospective
Lymphopaenia (<1.0 9 109/l) 1 (2.4)
follow-up period was 6.9  3.2 observations.
Monocytosis (>7.6 9 109/l) 4 (9.6)
¶Medications of relevance defined as drug groups with known affecters of s-cloza-
Neutrophilia (>5.9 9 109/l) 13 (31.0)
pine.
Neutropaenia (<1.6 9 109/l) 1 (2.4)
Clozapine dosing
1 daily administration* 29 (63.0)
2 daily administrations* 12 (26.1)
registered at ClinicalTrials.gov, and the local
3 daily administrations* 4 (8.7) Good Clinical Practice (GCP) unit monitored
4 daily administrations* 1 (2.2) the study.
Dose, evening (mg)† 237.5 (50–500)
Dose, daily (mg)† 300.0 (50–800)
Co-medications of relevance*,¶ Participants
Antidepressants 21 (45.7)
Anticonvulsants 8 (17.4) Participants were recruited from out-patient ser-
Proton pump inhibitors 9 (19.6)
vices in the Capital Region of Denmark. Patients
Antibiotics 0 (0)
Hormonal contraceptives (n = 20) 6 (30.0) were included from September 2015 to December
Pharmacokinetic assessments 2015. All participants provided written informed
Clozapine (CLZ)† consent before entering the study, and all eligible
CLZ concentration, 10 h 1418.2 (144.8–6300.8)/463.4
postdose (nmol/l)/(ng/ml) (47.4–2059.2)
patients were screened by interview before final
(n = 44) inclusion.
CLZ concentration, 12 h 1258.6 (120.1–5870.4)/411.4 Inclusion criteria were as follows: (i) age 18–64,
postdose (nmol/l)/(ng/ml) (39.2–1918.3) (ii) diagnosis of schizophrenia according to the cri-
(n = 45)
C/D, 10 h postdose (ng/ml/mg) 1.56 (0.39–4.74)
teria of International Classification of Diseases,
(n = 44) 10th revision (ICD10) (25), or the Diagnostic and
C/D, 12 h postdose (ng/ml/mg) 1.42 (0.42–5.07) Statistical Manual of Mental Disorders, Fourth
(n = 45) Edition (DSM-IV) (26), (iii) unchanged dose and
Maximum percentage change, 16.2 (2.9–42.8)
across the 12-h time point (n = 45)
dosing of clozapine 30 days prior to trial, (iv) usual
Maximum percentage difference, 17.2 (6.1–52.6) night administration of clozapine between 9 and
related to 12-h values (n = 45) 12 p.m.
Norclozapine (NCLZ)† Exclusion criteria were as follows: (i) non- or
NCLZ concentration, 10 h 879.9 (163.7–2634.4)/287.6
postdose (nmol/l)/(ng/ml) (53.6–860.1) partial clozapine compliance the day before blood
(n = 44) sampling, (ii) clozapine ingestion in the morning of
NCLZ concentration, 12 h 786.1 (80.3–2738.9)/256.9 or during the trial, (iii) non-response to telephone
postdose (nmol/l)/(ng/ml) (26.1–895.1)
call at the scheduled time of evening clozapine
(n = 45)
Maximum percentage change, 14.2 (1.7–38.4) ingestion, (iv) significant change, defined as cessa-
across the 12-h time point (n = 45) tion, starting, doubling or halving in smoking or
Maximum percentage difference, 18.0 (5.2–105.0) caffeine habits within 30 days prior to trial, (v)
related to 12-h values (n = 45)
Metabolite and parent compound†
altered use of other antipsychotics or medications
Combined (CLZ+NCLZ) C/D, 10 h 2.55 (0.86–6.43) known to affect s-clozapine (Table 1), 30 days
postdose (ng/ml/mg) (n = 44) prior to trial (7 days for hormone-based contra-
Combined (CLZ+NCLZ) C/D, 12 h 2.38 (1.01–7.13) ceptives), (vi) pregnancy or breastfeeding, (vii)
postdose (ng/ml/mg) (n = 45)
alcohol or drug abuse that would affect participa-
tion of the trial (assessed per interview).

3
Jakobsen et al.
Study design
The study was conducted at the Psychiatric Centre
of Copenhagen, Rigshospitalet. Eligible patients
were scheduled for venous blood sampling at pre-
cisely 10, 11, 12, 13 and 14 h after their night
clozapine dosing. The drug was to be ingested at
the usual time of administration the evening before
blood sampling. For subjects with more than one
daily dosing of clozapine (i.e. dosing additional to
their night dosing between 9 and 12 p.m., see
Table 1), an eventual morning dosing of clozapine
was postponed until sampling time ended. Status
of in- and exclusion criteria, compliance and exact
time of drug administration was verified by tele-
phone contact with the patient at the time of last
drug administration. Sampling time was scheduled
based on the confirmed time of night clozapine
ingestion. Only blood samples drawn
within 5 min from schedule were accepted.
Primary endpoints were differences in clozapine
and norclozapine concentrations within the 4-h
time span.
Medical charts were retrospectively reviewed for
confirmation of in- and exclusion criteria and
extraction of secondary endpoint variables.
Secondary endpoints served for hypothesis gen-
eration in terms of subgroup analysis for correla-
tions between s-clozapine differences and selected
variables. Secondary endpoint variables included
the parameters gender, age (both as a continuous
and a binary variable: ≥45 years of age vs.
<45 years), body mass index (BMI), smoking
habits, daily caffeine consumption based on self-
reported amounts of caffeinated beverages (27),
C-reactive protein (CRP) levels at inclusion (≥5 mg/
l vs. <5 mg/l), relevant co-administrations (defined
as drug groups with known affecters of clozapine
concentration), clozapine doses (night and total
daily dose of clozapine), number of clozapine
administrations (one vs. multiple daily administra-
tions), clozapine concentration to daily dose ratio
(C/D), metabolic ratio for norclozapine:clozapine
(MR) and the presence of chronic blood dyscrasia.
WBC differential counts from the inclusion day, or
the observation nearest to the inclusion day, and
6 months retrospectively, were collected. Normal
WBC differential reference ranges (for usual clini-
cal evaluation) were set by the Department of Clin-
ical Biochemistry, Rigshospitalet (28). Chronic
blood dyscrasia was defined as a minimum of 50%
of the retrospective observations, for a given cell
line, located either under or above the normal ref-
erence range. Only, subjects with three or more cell
counts, within the retrospective period, were
included for chronic blood dyscrasia evaluation.
4
Laboratory analysis
Blood sampling was performed at fixed time points
(10, 11, 12, 13 and 14 h postdose), and 6 ml of
venous blood was collected at each time point for
the determination of drug concentrations. Blood
was collected in tubes with clot activator and left
to coagulate at room temperature, until the end of
sampling time. Collected blood was immediately
hereafter centrifuged at 4000 revolutions per min-
ute, and serum was extracted and stored at 20°C
until analysis. Serum from the first blood sample
was also used for measuring of baseline CRP.
S-clozapine and s-norclozapine assays were per-
formed at the Section of Forensic Chemistry,
University of Copenhagen. Serum concentrations
were measured from each blood sample by a
slightly modified solid-phase extraction and ultra-
performance liquid chromatography, combined
with tandem mass spectrometry (UPLC-MS-MS),
using isotope standards as internal controls for
each compound (29, 30).
A highly sensitive analysis for C-reactive protein
(hsCRP) was performed using a validated high-
sensitivity luminescence immunoassay (LIA) at the
Department of Clinical Biochemistry, Rigshospi-
talet.
Statistical analyses
SAS Enterprise Guide 7.1 was used for the analy-
ses, and only two-tailed P values <0.05 were con-
sidered statistically significant. In case of skewness
of values, natural logarithm (log) values were used
for statistical analyses.
For descriptive statistics, continuous outcomes
are presented as medians and ranges between mini-
mum and maximum values, or means and standard
deviations (SD), when applicable. Categorical
outcomes are reported as numbers and propor-
tions (n (%)).
For each participant, the percentage difference
in s-clozapine and s-norclozapine was calculated.
The 12-h time point was set as reference and com-
pared to concentrations measured at each of the
other time points (10, 11, 13 and 14 h postdose).
Likewise, the percentage changes of s-clozapine
and s-norclozapine concentrations between
11- and 13-h values and between 10- and 14-h
values (across the 12-h time point) were measured.
The student’s t-test for paired data was used to
analyse intra-individual differences in serum con-
centrations.
To identify potential subgroups with higher sen-
sitivity towards deviations in sampling time, we
identified both the maximum percentage difference
 Sampling time and clozapine concentrations

from the 12-h clozapine value, and the maximum
percentage clozapine change across the 12-h time
point (11- vs. 13- or 10- vs. 14-h differences), in
absolute numbers, for each subject. Pearson’s
correlation was then used to test for correlations
between maximum percentage clozapine differ-
ences, both compared to and across the 12-h time
point, and the continuous variables age, BMI,
daily caffeine consumption, clozapine dosage
(both daily and last dosage), reference clozapine
concentration (the 12-h or 10-h clozapine concen-
tration), reference norclozapine concentration
clozapine, C/D ratio, combined clozapine and
norclozapine C/D ratio, MR and maximum, per-
centage difference in norclozapine concentration.
The impact of the binary variables gender, age
≥45 years <, CRP status ≥5 mg/l <, smoking sta-
tus, co-administration of relevant medications
and number of daily clozapine administrations
(one vs. multiple) was also tested on maximum,
percentage clozapine differences, with the
student’s t-test for unpaired data.
Results
Fifty-four subjects were screened, and 48 subjects
(88.9%) were included in the study. Two subjects
were subsequently excluded: One patient suffered a
serious adverse reaction (SAR), that is agranulocy-
tosis and sepsis, during sampling time, and one
patient had had a clozapine dose adjustment
10 days prior to the trial and was excluded as a
screening failure.
Consequently, data from 46 (85.2%) subjects
were used for statistical analyses. Forty subjects
had all five of their blood samples drawn on time.
Five subjects had one missing value, and one sub-
ject had two missing values. Of the 46 included
subjects, 26 were males (56.5%). The participants
Table 2. Differences in s-clozapine and s-norclozapine values, compared intra-individually to both measured values at 12 h postdose and between values across the 12-h time
point, n = 46
had a median age of 42 years (range 20–59) and a
median BMI of 30.9 kg/m2 (range 20.1–48.8), and
they received a median daily clozapine dosage of
300.0 mg (range 50.0–800.0). A complete list of
patient characteristics is presented in Table 1.
Clozapine
Differences across the 12-h time point. The median
and range of 10-h concentrations are presented in
Table 1.
There was a general tendency towards a decrease
in s-clozapine over time with a median decrease of
8.4% and a mean absolute difference of 14.0%
(10.2) from 10 to 14 h postdose (Table 2). How-
ever, 11 patients (23.9%) showed increasing levels
during the 4-h period. S-clozapine time courses are
presented in Fig. S1.
S-clozapine values changed significantly
(P < 0.01) across the sampling period (between 11-
and 13-h values and between 10- and 14-h values),
with individual differences up to 39.1% and 42.8%
respectively (Table 2). Seven subjects (15.9%)
showed absolute differences above 25% (range
26.7–42.8) from 10 to 14 h postdose.
Differences compared to the 12-h values. The median
and range of 12-h s-clozapine concentrations are
presented in Table 1. The median and range of
percentage differences in measured levels of clozap-
ine, compared to 12-h values, are presented in
Table 2 and Fig. 1.
Measured levels before the 12-h time point were
significantly different from the 12-h reference con-
centration (P < 0.01) with individual differences
up to 50.8% and 52.6% at –1 and 2 h (Table 2).
Nine subjects (20.9%) showed absolute differences
above 25% (range 25.1 to 52.6) between 10- and
12-h values (Fig. 1).

s-clozapine s-norclozapine

Compared time points† n‡ % difference§ |%| difference¶ P value** % difference§ |%| difference¶ P value**

12 vs. 10 43 8.4 ( 32.2 to 52.6) 13.6  12.1 0.0001* 6.2 ( 28.1 to 105.0) 14.2  16.4 0.0418*
12 vs. 11 44 8.0 ( 37.5 to 50.8) 13.0  11.1 0.0034* 0.8 ( 24.8 to 53.0) 11.1  11.7 0.5462
12 vs. 13 42 3.4 ( 30.5 to 25.9) 8.7  7.4 0.1119 4.2 ( 16.3 to 31.3) 10.1  7.7 0.0283*
12 vs. 14 45 1.5 ( 31.1 to 44.0) 11.0  10.1 0.5041 7.3 ( 25.6 to 37.5) 13.0  8.7 0.0233*
11 vs. 13 42 8.6 ( 39.1 to 23.5) 11.9  9.3 0.0003* 2.7 ( 23.6 to 31.3) 11.1  7.6 0.3184
10 vs. 14 44 8.4 ( 42.8 to 20.4) 14.0  10.2 0.0001* 1.2 ( 38.4 to 35.5) 10.6  10.0 0.9767

*Significant P values (two-tailed).

†Concentrations measured at time points 10, 11, 12, 13 and 14 h postdose were compared.
‡Numbers of subjects with both values of concentration for comparison.
§Data presented as median and range (min–max) of percentage (%) difference between compared values.
¶Data presented as mean  SD of absolute percentage |%| difference.
**Difference in (log) serum concentration is tested by the Student’s t-test for paired data (P values).

5
Jakobsen et al.

(a) Median differences

10
8
6
Per cent

4 CLZ
2
NCLZ
0
–2
–4
10 11 12 13 14
Hours postdose

(b) Individual differences, CLZ

50

40

30

20
Per cent

10

–10

–20

–30

–40
10 11 12 13 14
Hours postdose

(c) Individual differences, NCLZ

110

90

70

50
Per cent

30

10

–10 Fig. 1. Median and individual

percentage differences in serum
clozapine (CLZ) and norclozapine
–30
(NCLZ) concentrations, compared
10 11 12 13 14
intra-individually to 12-h values (0%),
Hours postdose n = 45.

Differences in s-clozapine levels from the 12-h ref-
erence point and forth were insignificant, although
individuals showed differences up to 30.5% and
44.0% at +1 and 2 h respectively (Table 2). Four
subjects (8.9%) showed absolute differences above
25% (range 25.2–44.0) between 12- and 14-h val-
ues (Fig. 1).
Median s-clozapine differences reached a max-
imum of 8.0% and 8.4% at 1 and 2 h
(Table 2).

6

Norclozapine
Differences across the 12-h time point. The median
and range of 10-h concentrations are presented in
Table 1.
There was a general tendency towards an u-
shaped time course for s-norclozapine concentra-
tions over time with a median increase of 1.2%
and a mean absolute difference of 10.6%
(10.0) from 10 to 14 h postdose (Table 2).
S-norclozapine time courses are presented in
Fig. S1.
Individual changes across the 12-h time point
(between 11- and 13-h values and between 10- and
14-h values) went up to 31.3% and 38.4%, respec-
tively (Table 2), although the mean differences in
serum concentrations were insignificant (Table 2).
Three subjects (6.8%) showed absolute differences
above 25% (range 35.3–38.4%) from 10 to 14 h
postdose.
Differences compared to the 12-h values. Median
and range of 12-h concentrations are presented in
Table 1. Individual and median percentage differ-
ences in s-norclozapine, compared to 12-h values,
are presented in Table 2 and Fig. 1.
Within the 4-h time span, individual s-norcloza-
pine levels differed with a maximum of 31.3–
105.0% from the reference 12-h concentration
(Table 2). Four subjects (9.3%) showed absolute
differences above 25% (range 27.3–105.0%)
between 10- and 12-h values, and six subjects
(13.3%) exceeded 25% (range 25.4–37.5%) in dif-
ference between 12- and 14-h values (Fig. 1). The
differences in serum concentrations were signifi-
cant (P < 0.05), except from the difference between
11 and 12 h postdose values (Table 2).
Median s-norclozapine differences at 1 and 2 h
reached a maximum of 4.2% and 7.3% respec-
tively (Table 2).
Analyses for associated variables
Pearson’s correlation. A positive correlation was
found between maximum percentage s-clozapine
difference compared to the 12-h value and increas-
ing age (r = 0.30, P = 0.045) (Table 3). A negative
correlation was found between maximum per-
centage 12-h s-clozapine difference and increas-
ing BMI (r = 0.30, P = 0.046) (Table 3).
No other significant correlations were found
between the magnitude of clozapine differences
compared to 12-h values and the remaining contin-
uous variables. No significant correlations were
found between maximum percentage s-clozapine
change across the 12-h time point (11–13 or
Sampling time and clozapine concentrations
10–14 h postdose) and any of the continuous vari-
ables. The results of all tested correlations with
continuous data are presented in Table 3.
t-test. When age was submitted as a binary vari-
able, maximum percentage clozapine change
across the 12-h time point was significantly higher
for subjects aged ≥45 years (P = 0.047) (Table 4).
Subjects with chronically elevated basophiles or
monocytes also showed significantly higher clozap-
ine differences, and this was for both differences
compared to and across the 12-h time point
(Table 4).
No significant differences were observed for the
remaining binary variables. The results of all t-tests
with binary data are presented in Table 4.
Discussion
The main findings of this study were that median
and mean percentage differences in measured
clozapine and norclozapine concentrations, within
the 4-h time span, were minor. However, some
individuals experienced substantial variations in
clozapine and norclozapine levels. The maximum
intra-individual variation was larger for norclozap-
ine than for clozapine. A greater part of the change
in s-clozapine occurred before the 12-h time point,
whereas s-norclozapine changed significantly both
before and after the 12-h time point, although in
opposite directions (Table 2). Furthermore, we
found that age, BMI and the presence of chronic
monocytosis or basophilia seemed to be associated
with the magnitude of clozapine variation.
The mechanism behind the variation in mea-
sured plasma levels is thought to be multifactorial:
in general, some intra-individual variability in
measured concentrations over time should be
expected, naturally due to the elimination of drug
over time, but also due to analytical laboratory
variation. The coefficient of variation (CV%) for
our laboratory measurements was approximately
11%, in line with other studies (29), and would
have accounted for some of the varying clozapine
concentrations over time. MR is an index of meta-
bolic capacity. A high metabolic activity (a high
norclozapine:clozapine ratio) would imply a
greater variation in s-clozapine over time. Subjects
with high clozapine clearance are likely to require
a higher clozapine dosage to produce an effective
level of concentration, giving a low C/D. There-
fore, C/D is also a measure of clozapine clearance
and would be expected to inversely correlate with
the magnitude of clozapine variation (5, 31). The
fact that neither MR nor C/D seemed to correlate
with clozapine variation implies a noteworthy
7
Jakobsen et al.

Table 3. Test for correlations between maximum percentage difference in s-cloza- effects to clozapine treatment nor food consump-
pine and obtained continuous data
tion in relation to trial period were registered in
Maximum the present study though.
percentage Maximum Moreover, a reverse transformation into the par-
difference percentage ent compound has been shown for the metabolite
compared to 12-h change across the
values† 12-h time point
clozapine-N-oxide and some, less important, pro-
tein-reactive metabolites, which partially could
Continuous data n r‡ P value§ r‡ P value§ explain a rise in s-clozapine long after time to peak
Clinical
(16). Therefore, it seems plausible that deviations
Age (years) 45 0.30 0.0450* 0.25 0.1005 in sampling time (as a combination of drug excre-
BMI (kg/cm2) 45 0.30 0.0458* 0.05 0.7262 tion over time, analytical and metabolic variation)
Daily caffeine† (mg) 45 0.14 0.3780 0.01 0.9291 could contribute to a great deal of the reported
Dose, evening (mg) 45 0.14 0.3469 0.03 0.8243
Dose, daily (mg) 45 0.06 0.7117 0.13 0.3830 intra-individual differences in s-clozapine concen-
Clozapine (CLZ) trations.
S-CLZ, 10 h postdose† 44 0.11 0.4625 In a clinical perspective, our findings suggest
S-CLZ, 12 h postdose† 45 0.08 0.6111
that inconsistency in sampling time could give the
C/D¶ 10 h postdose† 44 0.30 0.1344
C/D¶ 12 h postdose† 45 0.20 0.1981 impression of a change in habitual clozapine levels
Norclozapine (NCLZ) for some patients, although no such change has
S-NCLZ, 10 h postdose† 44 0.08 0.6202 occurred. Moreover, one could speculate whether
S-NCLZ, 12 h postdose† 45 0.11 0.4607
Max. percentage difference, 45 0.16 0.2988
measured clozapine differences of this magnitude
10–14 h or 11–13 h could in fact disguise or even seriously amplify an
Max. percentage difference, 45 0.25 0.0969 actual change in s-clozapine, subsequent to alter-
related to 12-h values† ations in the previously described metabolism-
Metabolite and parent compound
Combined C/D¶, 10 h postdose† 44 0.23 0.1326
affecting parameters. These measurements could
Combined C/D¶, 12 h postdose† 45 -0.24 0.1068 make actual changes appear either insignificant or
MR**, 10 h postdose† 44 0.08 0.5913 more extreme as, for example, smoking cessation
MR**, 12 h postdose† 45 0.08 0.5984 has been reported to increase s-clozapine levels
*Significant P values (two-tailed). with a mean of around 72% (17), and cessation of
†Log-transformed data. caffeine intake may result in an approximately
‡Coefficient of correlation. 50% reduction in s-clozapine levels (12). This
§P value of correlation, tested with Pearson’s correlation analysis. could potentially have severe clinical implications.
¶C/D = concentration-to-dose ratio.
**MR = metabolic ratio (norclozapine : clozapine).
Furthermore, norclozapine showed a fluctuation
pattern as well, and even though its concentrations
were lower than those of clozapine, the relative dif-
contribution from analytical variation. However, ferences corresponded to those of clozapine, sug-
this could also be due to lack of statistical power. gesting that one should be cautious if interpreting
The complex individual pharmacokinetics of the combined concentrations of clozapine and nor-
clozapine is also expected to have contributed: clozapine (35).
for example, although there was a clear tendency The parameters age, gender, BMI, CRP ≥5 mg/
towards a decrease in clozapine levels over time, l, caffeine consumption, smoking status and co-
a rather large proportion of the subjects (23.9%) administrations such as oral contraceptives have
showed increasing serum levels during the 4-h been demonstrated to affect clozapine levels (4, 5,
period. Clozapine is rather rapidly absorbed, and 12, 17, 18, 21–23) and would therefore be expected
a delayed absorption seems unlikely. However, to correlate with the magnitude of clozapine varia-
we cannot rule out that the absorption rate has tion as a result of deviations in sampling time.
been erratic in some patients. Although hepatic However, in the present study, only two of these
metabolism is substantial, there is indirect evi- parameters (age and BMI) showed significance in
dence that presystemic metabolism in the gas- the correlation analyses. Previous studies have
trointestinal tract may account for a large shown significant differences in concentration to
contribution of the total clozapine clearance (32). dose ratios, when subjects aged ≥45 years were
Gastrointestinal side-effects are well known to compared to subjects aged < 45 years (36). When
clozapine treatment (33) and could be suspected we divided our participants into corresponding age
to affect absorption. Also, the co-administration groups, we also found a difference in the magni-
of food has, in one study, been demonstrated to tude of clozapine fluctuation. This was, however,
affect absorption rate (34), although product in the opposite direction than anticipated; that is,
summary informs otherwise (12). Neither adverse older subjects showed greater variance in their

8
 Sampling time and clozapine concentrations

Table 4. t-test for difference in means of maximum percentage clozapine differences

Maximum percentage difference compared to 12-h values Maximum percentage change across the 12-h time point

Group yes Group no Group yes Group no

Binary groups n Mean n Mean P† value n Mean n Mean P value

Male gender 26 21.8  10.8 19 17.5  8.7 0.1488 25 17.4  10.6 20 17.0  9.3 0.8913
Age ≥ 45 years 21 23.2  11.5 24 17.2  7.9 0.0548 21 20.3  10.9 24 14.5  8.3 0.0468*
Smoker 17 24.1  12.6 28 17.5  7.4 0.0806 16 16.8  10.5 29 17.5  9.8 0.8265
CRP ≥ 5 mg/l 16 20.2  11.9 29 19.8  9.2 0.9319 15 17.7  8.7 30 17.0  10.6 0.8383
Chronic leukocytosis 16 22.4  12.7 25 19.1  8.4 0.5106 15 18.7  9.6 26 17.0  10.1 0.6097
Chronic basophilia 1 53.0  N.A. 40 19.6  9.1 0.0314* 1 39.1  N.A. 40 17.1  9.3 0.0251*
Chronic eosinopaenia 18 18.2  8.8 23 22.1  11.3 0.2358 19 18.4  10.1 22 17.0  9.8 0.6549
Chronic lymphocytosis 3 28.0  21.3 38 19.8  9.2 0.3568 3 22.2  17.7 38 17.3  9.3 0.4154
Chronic lymphopaenia 1 30.2  N.A. 40 20.1  10.3 0.3134 1 21.8  N.A. 40 17.5  9.9 0.6760
Chronic monocytosis 4 34.5  15.8 37 18.9  8.6 0.0190* 3 28.9  8.8 38 16.8  9.4 0.0368*
Chronic neutrophilia 13 24.3  13.3 28 18.6  8.3 0.2113 12 16.9  11.4 29 17.9  9.3 0.7638
Chronic neutropaenia 1 12.6  N.A. 40 20.6  10.4 0.4890 1 17.5  N.A. 40 17.6  10.0 0.9890
1 daily dosing of clozapine 29 19.3  9.9 16 21.3  10.6 0.6403 29 17.8  9.4 16 16.2  11.0 0.6013
Co-medicated with ADs 20 18.9  8.5 25 20.9  11.3 0.6458 21 16.2  9.0 24 18.1  10.7 0.5217
Co-medicated with ACs 8 17.2  8.5 37 20.6  10.4 0.4134 8 12.4  6.9 37 18.3  10.2 0.1307
Co-medicated with PPIs 8 20.1  7.9 37 20.0  10.6 0.7775 9 18.5  5.0 36 16.9  10.8 0.6873
Co-medicated with HCs 6 13.3  5.0 13 19.4  9.5 0.1802 6 18.6  6.9 14 16.3  10.3 0.6205

ADs, antidepressants; ACs, anticonvulsants; PPIs, proton pump inhibitors; HCs, hormonal contraceptives.
*Significant P values (two-tailed).
†t-test based on log-transformed percentage differences.

clozapine concentrations, despite reports of an
age-dependent decrease in liver enzyme activity (4,
36). This highlights the associative, rather than
causal, character of the significant correlations.
Still, this indicates that patients ≥45 years of age
are more sensitive not only to dose, but also to
sampling time. Furthermore, our findings seem to
support the idea of high body fat composition as a
mean to an increased clozapine half-life (4) and
hence a more stable clozapine excretion. The fact
that we were unable to demonstrate correlations
for the other continuous variables, or found corre-
lations only for differences either related to or
across the 12-h time point, could be due to lack of
statistical power.
Due to the high availability of WBC differential
counts to clinicians, as a result of the treatment
guidelines for clozapine, and the puzzling interindi-
vidual variation in occurrence and type of clozap-
ine-induced blood dyscrasias (37), we included
chronic abnormal WBC counts as a secondary
endpoint variable. This was to test whether the
type of a chronic blood dyscrasia could be associ-
ated with the magnitude of s-clozapine variation in
time, despite the lack of direct causality. A possible
association between these parameters could help
identifying risk patients, without further tests and
costs, and hence be an important clinical tool in
patient-specific TDM optimizing. We found that
patients with chronic basophilia or monocytosis
had significantly higher maximum percentage dif-
ferences in s-clozapine, suggesting that patients
with these particular chronic blood dyscrasias are
more sensitive to deviations in sampling time.
However, only four patients showed blood dyscra-
sias of this nature, and one patient showed both
monocytosis and basophilia. Consequently, results
(especially that of basophilia) should be inter-
preted with caution.
Limitations of this study include the small num-
ber of subjects, which could be suspected to have
influenced statistical power, as in the case of the
only few significant correlations (potential type 2
errors). Another limitation regarding statistics is
the fact that an increasing number of tested vari-
ables also increase the risk of mass significance
(type 1 errors), and the need of a correction factor
was considered. However, our secondary endpoint
analyses served only as hypothesis generating tests,
showing associations (not necessarily causations)
and conclusions are merely suggestive. A correc-
tion factor at this point could have eliminated
potential important associations from future
studying. Further examination is needed to clarify
the association between clozapine pharmacokinet-
ics and the variables in question, to accurately
identify subgroups that are more susceptible to
deviations in sampling time.
Assessment of compliance relied on reports from
the patients. However, all included patients were
reached by telephone at the scheduled time of
clozapine administration for confirmation of their
clozapine consumption. Also, all patients were re-
interviewed regarding in- and exclusion criteria

9
Jakobsen et al.
(including time of last clozapine administration),
at the study facility, prior to final inclusion and
blood sampling.
Thus, strengths of this study are the high preci-
sion of sampling time and the documentation of
individual kinetics, addressing a relevant clinical
issue. This is, to our knowledge, the only study of
its kind.
In conclusion, the present study argues that the
general impact of deviations in clozapine TDM
sampling time, within the period of 10–14 h post-
dose, is minor. However, substantial individual
variations in serum clozapine and norclozapine
were observed, implying risk patients for whom the
deviations may be of clinical importance, thereby
implying a need to adhere to a fixed sampling time.
Acknowledgements
We thank patients for their participation in the study and the
health professionals who facilitated the contact. We also thank
biomedical laboratory scientist Bente Bennike for assistance
with blood sampling and serum extraction in multiple patient
settings.
Declaration of interest
Author Fink-Jensen has received an unrestricted grant form
Novo Nordisk for another clinical study. Author Larsen was
an investigator of the study that received the grant from Novo
Nordisk and is now working at Novo Nordisk A/S. The other
authors have nothing to declare.
The protocol can be obtained by contact to the correspond-
ing author.
Author Jakobsen performed the statistical analysis in con-
sultancy with the Statistical Advisory Service, Department of
Public Health, University of Copenhagen.
The study was registered at ClinicalTrials.gov at 13 Novem-
ber 2015, ID: NCT02625103.
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Supporting Information
Additional Supporting Information may be found in the online
version of this article:
Figure S1. Mean and individual time courses for s-clozapine
and s-norclozapine, 10–14 h postdose (n = 46).
11