Pregnant females with a diagnosis of bacterial vaginosis whose providers recommended treatment with metronidazole were eligible for enrollment in this study. To participate, they had to be 18 years of age or older, pregnant with singletons, and in a pregnancy window of 10–14 weeks, 22–26 weeks, or 34–38 weeks. Women were excluded from participation if there was anemia with a hematocrit of less than 28% and with a prior history or current medical examination consistent with the presence of clinically significant alterations in hepatic, renal, or gastrointestinal function. Decisions about diagnosis and treatment were made by the women’s own healthcare providers and were independent of participation in this study. Participants were considered eligible on medication use, comedication, smoking habits, intake of caffeine (eg, coffee, cola, tea), and daily use of prenatal vitamins.
All procedures involving human subjects were conducted according to the declaration of Helsinki and its actual amended version, the International Conference on Harmonization–Good Clinical Practices (ICH-GCP) guidelines. All women were enrolled with written informed consent under a protocol that was reviewed and approved by the University of Texas Medical Branch Institutional Review Board.
All subjects received metronidazole 500 mg orally twice daily for 3 consecutive days. Metronidazole was supplied as 250 mg tablets of Flagyl®. The pharmacokinetics of metronidazole were studied on each patient after a single oral dose on the morning of day 4. Blood samples were taken from the antecubital vein immediately prior to dosing and at 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, and 12 hours post ingestion. The plasma was centrifuged immediately and stored in polypropylene tubes at −70°C until analysis. Urine samples were collected over the same 12-hour interval at 0, 0–2, 2–4, 4–6, 6–8, 8–10, and 10–12 hours and stored at −70°C until analysis.
High performance liquid chromatography (HPLC) method
The HPLC system consisted of a 1525 binary HPLC pump, a 2489 dual λ absorbance detector, and a 717 autosampler controlled by a Millennium chromatography manager (Waters, Milford, MA, USA). An isocratic elution was performed on a reverse phase C18 column (Symmetry 4.6 mm×150 mm), 5 μm particle size, plus phenomenex guard cartridge. The mobile phase was a degassed and filtered (0.45 μm; Millipore) mixture of acetonitrile: water (7:93, v/v) adjusted to pH 6.5 using acetic acid. The flow-rate was 1 mL/min, and the UV detector wavelength was set at 317 nm. The retention time for metronidazole was 7.8 minutes under the described conditions.
Calibration curves were constructed by spiking known amounts of standards into blank plasma or urine. The plasma and urine standard curves for metronidazole were found to be linear in the range of 0.2–25 μg/mL and 2.5–200 μg/mL, respectively. The R-squared value was greater than 0.99.
Six replicates of the quality control (QC) samples at each concentration level were used to evaluate the intra-day precision and accuracy. Two replicates of the QC samples at each concentration level from 3 separate batches were used to evaluate the interday precision and accuracy. In plasma, the intra- and interassay mean precision was between 1.40% and 4.30%, and the mean accuracy was between -4.37% and 0.42% for metronidazole. In urine, the intra- and inter-assay mean precision ranged from 0.95% to 2.58%, and the mean accuracy was between −2.56% and −0.63%.
The recovery of metronidazole from the 3 different matrices was determined at 3 QC levels by comparing the peak area of samples that had been spiked with metronidazole prior to precipitation using perchloric acid with samples prepared using the standard solution in water. The average recovery of metronidazole from human plasma and urine was greater than 95%.
Plasma and urine sample preparation
Plasma samples were thawed at room temperature and vortexed, then centrifuged for 5 minutes at 6000g at approximately 4°C. To 250 μL of plasma, 7 μL of 70% perchloric acid were added. Samples were then vortexed (20 seconds) and centrifuged at 8000g for 10 minutes. Then the supernatant was collected and transferred to autosampler vials, and 25 μL was injected into the HPLC.
Urine samples were prepared by adding 5μL of 35% perchloric acid and 250 μL of urine (standard, quality control, or patient samples) to a 1.5-mL microcentrifuge tube. Each tube was vortex-mixed and then centrifuged at 8,000 × g for 5 minutes. An aliquot of 10 μL of supernatant was injected into HPLC system.
Pharmacokinetic analyses were conducted using Kinetica software version 5.0 (Thermo Scientific, Waltham, MA). All data were evaluated using a model independent approach. Plasma concentration-versus-time data were curve fit using a peeling algorithm to generate initial polyexponential parameter estimates. Final estimates of the terminal elimination rate constant (Ke) were determined from an iterative, nonlinear least squares regression algorithm. Individual maximum plasma drug concentration (Cmax) and time to Cmax (Tmax) were determined by visual inspection of the plasma concentration-versus-time profile. The area under the plasma concentration-versus-time curve at steady-state (AUCss) was determined using the log-linear trapezoidal rule. For participants whose plasma sampling was truncated prior to 12 hours, C12 was extrapolated from the best fit-curve, and the AUC to 12 hours (AUC0-12) was calculated by summation of AUC0-n +AUCn-12 where n represents the last measured time point. Apparent total plasma clearance (CL/F) and steady-state volume of distribution (Vdss/F) were calculated from the AUCss.
Metronidazole pharmacokinetic data for the study cohort were examined using standard descriptive statistics. One-way analysis of variance was used to compare calculated pharmacokinetic parameters between dosing cohorts. Univariable analysis of variance and nonlinear regression techniques were used to evaluate the relationship between demographic variables and pharmacokinetic parameter estimates. All analyses were performed in SPSS version 12.0 (SPSS, Chicago, IL), and P-values less than 0.05 were considered to indicate statistical significance.