Winnonlin v 5

Male Sprague Dawley (SD) rats (n = 5, weight 200–220 g), provided by Charles River Laboratories (Beijing, China), were used in the pharmacokinetics studies. The experimental protocol was approved by the Institutional Animal Care and Use Committee of Tsinghua University (Beijing, China). Rats were subcutaneously administered of DV1 (dissolved in physiological saline) at a single dose of 10 mg/kg. Blood samples were collected into heparin-coated polypropylene centrifuge tubes at 0, 5, 10, 15, 30 min and 1, 2, 4, 6, 8, 12, 24, 36 and 48 h after the dose. The plasma sample was obtained from blood collection by centrifugation at 3,500 × g for 10 min at 4 °C, and then frozen at −80 °C until analysis. The samples and QCs were analyzed by the LC-MS/MS described above. The pharmacokinetic parameters of DV1 were determined by using WinNonlin v. 5.2 (Pharsight, Cary, NC).

Naïve mice were administered a single 20 mg/kg dose of IP COC HCl, anesthetized with isoflurane at 10 and 30 min following COC administration and 50 μl of blood was collected by cardiac puncture, placed in a heparinized tube, centrifuged and the resulting plasma was removed and snap-frozen in liquid nitrogen. Whole brain was collected, rinsed, weighed and snap-frozen in liquid nitrogen. Plasma and brain samples were stored at −80°C until analyzed. Plasma and brain concentrations of COC, norcocaine (NOR) and benzoylecgonine (BZE) were quantified using liquid chromatography tandem mass spectrometry (Slawson et al. 2002 (link)).
Plasma and brain concentrations are expressed as ng/ml and ng/g, respectively. The area under the concentration vs. time curve (AUC) in plasma and brain was calculated for each animal using a linear-up log-down function (Winnonlin v5.2; Pharsight, St. Louis, MO, USA).

Details of the analytical method used in the study are reported in (Carter et al. 2012 (link)). Briefly, blood was collected over an 8-h period and immediately placed on ice. Plasma was isolated within 45 min of blood collection. Baseline and PK plasma samples were analyzed in the Vanderbilt Bioanalytical Mass Spectrometry Core Facility by nanoflow LC–MS/MS and HPLC–MS/MS repectively (Carter et al. 2012 (link)). Nanoflow LC was necessary to reliably quantitate the low (pg/ml) endogenous levels of melatonin and N-acetylserotonin (NAS). PK parameters [maximal concentration of melatonin (C_max), time to maximal concentration (T_max), AUC, half-life (t_1/2), clearance (CI), and volume of distribution (V_z)] were derived from plasma concentration versus time curves using the program Win-Nonlin v. 5.2 (Pharsight, Palo Alto, CA, USA). The data were analyzed using a noncompartment model as described in Carter et al. 2012 (link). The LC–MS/MS analytical method was validated on a small group of adult volunteers (two male, two female, weight 72–109 kg, age 28–64 years, height 1.65–1.83 m). For the adult population, calculated PK parameters were consistent with literature values (Table SI and S2). The AUC and C_max showed evidence of linear kinetics. The half-life (t_1/2) and T_max were not a function of dose.

All IVF outcome data were expressed as the mean ± SEM. Percentage of metaphase II oocytes and percentage of blastocyst-stage embryos were compared using a two-tailed paired Student’s t test. Total cell count, ICM cell count, TE cell count, and percentage of ICM to TE cells in blastocyst-stage embryos were assessed with Student’s t tests. Data were analyzed with Prism software (GraphPad Software). Differences were considered statistically significant at p< 0.05. Pharmacokinetic (PK) analysis of the serum ethanol concentrations was performed using WinNonlin v5.3 software (Pharsight Corp., Mountain View, CA, USA) to estimate time of peak (Tmax) ethanol concentrations using a one compartmental model and nonlinear regression analysis based on the Gauss-Newton (Levenberg and Hartly) method.