3 RESULTS
3.1 Classification of subjects
All 44 subjects were classified based on fasting, feeding, andCYP2B6 mutants. Among these subjects, 20 were placed in the
fasting group, while 24 were placed in the feeding group. The plasma
concentrations of BUP and HBUP were determined via HPLC-MS/MS. The
pharmacokinetic parameters were calculated in DAS 3.0 by applying a
non-compartmental method.CYP2B6*4/*6/*9genotypes and single-nucleotide polymorphism (SNP) were identified by
PCR-RFLP. The effects of the genetic polymorphism of CYP2B6 on
BUP were investigated in many studies, but the effects of a combination
of feeding and CYP2B6 genotype on the pharmacokinetics of BUP and
HBUP among Chinese subjects have never been examined. Therefore, this
study investigated the effects of high-fat diet and CYP2B6mutants on the pharmacokinetics of BUP and HBUP among Chinese subjects.
3.2 BUP and HBUP concentrations
The lower limits of quantification for BUP and HBUP were 0.500 ng/mL and
0.600 ng/mL, while the assay ranges used were 0.500 ng/mL–400 ng/mL and
0.600 ng/mL–480 ng/mL, respectively. The mean correlation coefficients
for BUP and HBUP were 0.9985 and 0.9960. The intraday and interday
precision and accuracy, which were measured by HPLC-MS/MS, were less
than ±15%. Our method satisfied the criteria of the Guidance for
Industry Bioanalytical Method Validation (FDA) and the Guideline for
Bioanalytical Method Validation (EMA).
Basing on the HPLC-MS/MS conditions in the mentioned concentration
assay, BUP, HBUP and venlafaxine are identified and quantified. The
structures and full-scan production spectra of the BUP, HBUP and
venlafaxine are shown in Fig. 2. The retention time of BUP, HBUP and
venlafaxine are 3.32 min, 2.89 min and 3.36 min, respectively. The BUP
and HBUP plasma concentrations of the 44 subjects were determined by
using the developed method. The concentration–time curve of the
subjects in the fasting and feeding groups is shown in Fig. 3. The
pharmacokinetic parameters were calculated by using DAS 3.0 and the
non-compartmental method, and the results can be seen in Tables 1 and 2.
3.3 Classification of CYP2B6
As shown in Table 3, the genotypes ofCYP2B6*4/*6/*9were categorized by 516G>T and785A>G mutation, and the numbers of different
genotypes are shown in Table 4. The subjects were grouped into the
following based on their wild and variant types: fastingCYP2B6*1/*1 (n=10), feedingCYP2B6*1/*1 (n=11), fastingCYP2B6 mutants (n=10), and feeding CYP2B6 mutants (n=13).
The fasting CYP2B6 mutants containedCYP2B6*1/*6 ,CYP2B6*1/*4 ,CYP2B6*4/*6 ,CYP2B6*6/*9 , andCYP2B6*1/*9 genotypes, while
the feeding CYP2B6 mutants containedCYP2B6*1/*6 ,CYP2B6*1/*4 ,CYP2B6*4/*6 , andCYP2B6*6/*6 genotypes.
3.4 Pharmacokinetic parameters of BUP and HBUP
The BUP and HBUP plasma concentrations were determined by using the
HPLC-MS/MS method. The concentration–time curves of BUP and HBUP
differentiated by fasting, feeding, and CYP2B6 mutants are shown
in Figs. 4 and 5. The pharmacokinetic parameters were calculated by
using DAS 3.0 and the non-compartmental method, and the results can be
seen in Tables 5 to 8. The AUC(0→96),
Cmax, and Tmax of BUP and HBUP in
fasting CYP2B6*1/*1 , fastingCYP2B6 mutants, feedingCYP2B6*1/*1 , and feedingCYP2B6 mutants can be seen in Figs. 4 and 5.