2.1 Materials and reagents
cPOA and tPOA were purchased from NU-CHEK (＞99%, Elysian, USA). HPLC-grade formic acid was obtained from Roe Scientific Inc. (Powell, OH, USA). HPLC-grade acetonitrile, methanol and MTBE（methyl tert-butyl ether）were obtained from Merck KGaA (Darm-stadt, Germany). Ultrapure water was from a Millipore Milli-Q system (Millipore Corp., Billerica, MA, USA). All other solvents or reagents were commercially available and reagent grade. Blank rat serum was collected from healthy male Sprague-Dawley rats weighing 280 ± 20 g (Laboratory Animal Center of Nanjing University of Chinese Medicine, Nanjing, China).
2.2 Chromatographic and mass spectrometric conditions
2.2.1 Liquid chromatography conditions
Analyte separations were performed on an Agilent UPLC-1290 system (Agilent Corp., Milford, MA, USA) using a BDS C18 column (2.1×100 mm, 2.1 μm, Thermo Fisher Scientific, USA). The mobile phase was included water (A) and acetonitrile (B) (A:B = 20:80, v/v) at a flow rate of 0.3 mL/min, and the injection volume was 1 μL.
2.2.2 Mass spectrometric conditions
Identification of cPOA and tPOA in serum samples was conducted using an AB 5500 Q-trap UPLC-MS/MS (ABSCIEX, Framingham, MA, USA) equipped with electro spray ionization (ESI). Quantitative analysis of cPOA and tPOA were also performed by UPLC-MS/MS. Detection was performed in negative ion mode under the following conditions: curtain gas at 35.0 L/h, and ion source gases at 50 L/h. AB Analyst 1.6.0 software (ABSCIEX, Framingham, MA, USA) was used for system control and data acquisition. ESI-MS/MS parameters are shown in Table 1. Detection was performed in the negative ion mode and conditions for cPOA and tPOA detection optimized using standards. Product ions obtained from deprotonated molecular ions of cPOA and tPOA included three main ions from each compound at m/z 235.2, 126.9, 111.0 and at m/z 234.7, 127, and 111.1, respectively.
2.3 Stock solutions and working solutions
Individual standard stock solutions of cPOA and tPOA (2.50 mg/mL, 2.24 mg/mL respectively) were prepared in acetonitrile. These stock solutions were serially diluted with acetonitrile to provide standard working solutions in the concentration range of 0.175 ~ 42.0 μg/mL for cPOA and tPOA. All solutions were stored at -20℃ and brought to room temperature before use.
2.4 Calibration standard curves and QC samples
Calibration standard (CS) curves were prepared by spiking 20 μL of the appropriate analyst working solution into 50 μL of blank rat serum. The effective concentrations were 0.1, 0.5, 2.5, 5, 10, 12 μg/mL for cPOA and tPOA. QC samples were prepared as compound samples for each concentration at 0.5 μg/mL for cPOA and tPOA, and stored at -20℃ until use. Rat serum samples, serving as QCs, were processed the following sample procedure as for unknown samples.
2.5 Serum sample preparation
50 μL of serum sample (blank, or pharmacokinetics serum sample) in a 2.0 mL centrifuge tube, 60 μL of aqueous solution with formic acid in 5%, 100 μL methanol, and 1250 μL MTBE were added and mixed by vortexing for 3 min. After centrifugation at 18,000 ×g for 10 min, the clear supernatant of 1 mL was extracted to a new centrifuge tube, blowed by flowing nitrogen, and redissolved by 200 μL acetonitrile solution. Centrifugation at 18,000 ×g for 10 min again, supernatant was injected into the UPLC-MS/MS system.
2.6 Method validation
Assay validation performed was based on the currently accepted FDA prescription and per guidelines of the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use . Each blank serum sample was processed through the extraction procedure and tested to ensure no rat serum interference with the analyte. While the serum sample preparation was 60 μL of aqueous solution with formic acid in 5%, 100 μL methanol, and 1250 μL MTBE were added and mixed by vortexing for 3 min. After centrifugation at 18,000 ×g for 10 min, the clear supernatant of 1 mL was extracted to a new centrifuge tube, blowed by flowing nitrogen, and redissolved by 200 μL acetonitrile solution.
The determination of the extraction recoveries of cPOA and tPOA was at three QC concentrations. And the calculation of the recoveries was by comparing analyte peak area ratios for each analyte in serum samples with those of analytes in the serum matrices by extracting analyte-free serum samples which were prior to chromatography. In extracted rat serum, matrix effects from endogenous substances were presented, which might have caused ion signal suppression or enhancement. Matrix effects at three QC concentrations (0.5, 2.5, and 10 ng/mL) were measured by comparing peak responses of samples post-extraction (A) with that of pure standard solution which contained equivalent amounts of the two compounds (B). The ratio (A/B × 100%) was used to evaluate the matrix effect and the extraction recovery and matrix effect of cPOA and tPOA were evaluated simultaneously by the same method.
During sample storage and processing procedures，the stability of cPOA and tPOA in rat serum was assessed by analyzing replicates (n=6) of three QC concentrations. The freeze–thaw stability was determined through three freeze–thaw cycles. All stability testing of QC samples were determined according to calibration curves of freshly prepared standards.
2.7 Pharmacokinetic studies of cPOA and tPOA
Male rats (ICR, 280±20 g) were obtained from the Laboratory Animal Center of Nanjing University of Chinese Medicine (Nanjing, China). Animal handling procedures followed standard operating procedure approved by the institutional animal care and use committee. All rats were dosed following overnight fasting except for water ad libitum. For pharmacokinetic studies, 18 male rats were randomly divided into three groups. In the first group, rats were administered intragastric gavage of normal saline with 75 mg/kg body weight. Blood samples were collected at the time points of 0, 10, 20, 30, 40, 60 min, and 2 h, 3 h, 6 h, 12 h, 24 h. Rats in the second group were administered i.g. of cPOA with 75 mg/kg body weight. Serial blood samples were collected in tubes via the orbital venous plexus before and at time points of 0, 10, 20, 30, 40, 60 min, 2 h, 3 h, 6 h, 12 h, 24 h, after administration. In the third group, rats were administered intragastric gavage of tPOA with 75 mg/kg body weight. Blood samples were collected at the time points of 0, 10, 20, 30, 40, 60 min, and 2 h, 3 h, 6 h, 12 h, 24 h. Serum was separated and stored frozen at -80℃ until analysis. The following main pharmacokinetic parameters were analyzed using the non-compartmental pharmacokinetics data analysis soft-ware of PK solution 2TM (Summit ResearchService, Montrose, CO, USA): area under curve from zero to the last measurable serum concentration point (AUC0–t, t=24h), maximum concentration (Cmax), time-to-maximum concen-tration (Tmax).