Supplementary material
Supplementary Table 1. Schematic representation of one phase in a three-phase crossover study.
Washout time between phases was six weeks.
Day
Pretreatment with antimycotic at 07:00 a.m.
Tramadol at 08:00 a.m.
1
Placebo, terbinafine 250mg or itraconazole 200mg
2
Placebo, terbinafine 250mg or itraconazole 200mg
3
Placebo, terbinafine 250mg or itraconazole 200mg
4
Placebo, terbinafine 250mg or itraconazole 200mg
5
Placebo, terbinafine 250mg or itraconazole 200mg
Tramadol 50mg
Supplementary text - Bioanalytical methods
The concentrations of plasma tramadol and M1 were measured on an API 3000 liquid
chromatography–tandem mass spectrometry system (AB Sciex, Toronto, ON, Canada) as
previously described [1], with minor modifications. Prior to analysis a simple protein
precipitation of plasma samples with acetonitrile was performed, excluding the samples
containing very low plasma drug concentrations (48-h samples), which were prepared by
use of a MCX solid phase extraction (Waters, Milford, MA). Chromatography was
performed on an Atlantis T3 analytical column (2.1x100 mm; Waters) using 10mM formic
acid with 0.1% (v/v) trifluoroacetic acid (channel A) and methanol (channel B) as the
mobile phase. Tramadol-d6 and O-desmethyltramadol- d6 served as internal standards
and were added to plasma samples, plasma quality control samples and plasma reference
standards before other pre-analytical procedures. The recovery for both analytes were >
75%. The mass spectrometer was operated in positive multi-reaction monitoring (MRM+)
detection mode with electro spray ionization. The selected ion transitions used for
quantification were: m/z 264 to m/z 58 for tramadol, m/z 250 to m/z 58 for M1, and m/z 270
to m/z 64 and m/z 256 to m/z 64 for internal standards, respectively. The limit of
quantification for plasma tramadol and M1 was 0.02 ng/ml. The interday coefficients of
variation (CV%) were for tramadol 7.2% at 2 ng/ml, 7.8% at 10 ng/ml and 4.6% at 100
ng/ml, and for M1 4.9% at 1.75 ng/ml, 8.5% at 8.75 ng/ml, and 5.4% at 87.5 ng/ml. The
interday accuracy, expressed as the percentage relative error (RE%) from nominal
concentrations (2, 10, 100 mg/ml) was < 10%.
Plasma concentrations of terbinafine, itraconazole and hydroxyitraconazole were
determined as described before [2,3]. Their limit of quantification was 10 ng/ml. The CV for
terbinafine was 2.5% at 21 ng/ml, 3.7% at 92 ng/ml, and 2.0% at 699 ng/ml. The CV for
itraconazole was 8.0% at 19 ng/ml, 1.0% at 192 ng/ml, and 1.7% at 1200 ng/ml. The CV
for hydroxyitraconazole was 3.5% at 19 ng/ml, 0.9% at 192 ng/ml, and 2.2% at 1200
ng/ml. Similarly, concentrations of 5-HT and 5-HIAA in whole blood were analyzed as
described earlier [4]. Limits of quantification were 30 nmol/l for 5-HT and 3 nmol/l for 5HIAA. The CVs of both analytes were <10% at these levels and <5% in the actual
concentration ranges encountered in the study. Genotyping for CYP2D6 was performed
using a two-step multiplex primer extension method [5]. The concentrations of plasma
tramadol, O-desmethyltramadol (M1), terbinafine, itraconazole and hydroxyitraconazole
were determined in an in house lab, i.e. in the laboratory of the Department of Clinical
Pharmacology, University of Helsinki.
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