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.