Chemicals and reagents
Acetonitrile (Fisher technologies Inc., USA) and methanol (Tianjin concord Science Co. Ltd., Tianjin, China) were of HPLC grade. Standard reference isoimperatorin and bergapten (purity >98 %) were purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Ethyl acetate and formic acid were of analytical grade. Deionized water was purified with a Milli-Q Academic ultra-pure water system (Millipore, Milford, MA, USA) and used for the HPLC mobile phase.
Apparatus and chromatographic conditions
HPLC analysis was performed on an Agilent 1100 HPLC (Agilent Technologies, USA) equipped with a quaternary pump, a degasser, an autosampler, a column thermostat and a fluorescence detector. An agilent fluorescence detector was coupled to the Agilent system. Separation was carried out with a Hedera™ ODS column (4.6 × 250 mm, 5 μm) by gradient elution at a temperature of 30 °C. Excitation and emission of the fluorescence detector was set to 288 and 478 nm, respectively. A constant flow rate of 1.0 mL min−1 and an injection volume of 30 μL were employed throughout the analysis. A mobile phase comprising of aqueous formic acid (0.1 %, v/v) (solvent system A) and acetonitrile (solvent system B) was employed with a gradient elution of 40–80 % B at 0 to 5 min, 80–85 % B at 5 to 10 min, 85–90 % B at 10 to 12 min, 90–95 % B at 12–15 min, 95 % B at 15–20 min. The re-equilibration time of gradient elution was 8 min.
Preparation of stock solution, calibration standards
In preparing the stock solution, appropriate amount of bergapten was weighed and dissolved in methanol to achieve a concentration of 1.0 mg mL−1. The chemical structures of bergapten and isoimperatorin are shown in Fig. 1. Working solutions of bergapten were then prepared by appropriate dilution with methanol for use. The stock solution of internal standard, isoimperatorin was also dissolved in methanol and diluted with methanol to a final concentration of 1 μg mL−1 and stored at 4 °C until analysis.
10 μL aliquots of bergapten working solutions were added to 100 μL drug-free rat plasma to obtain bergapten calibration standards (2, 4, 8, 20, 40, 100 and 100, 200, 500, 1000, 2500, 5000 ng mL−1) in plasma samples for two calibration curves.
Sample pretreatment and quality samples
To a 100 μL aliquot of plasma sample, 10 μL internal standard solutions were added. Samples were vortex-mixed for 2 min, extracted with 1000 μL ethyl acetate and then centrifuged for 10 min at 14,000 rpm. The supernatant was transferred into another centrifuge tube and evaporated to dryness using nitrogen gas. The dried residue was reconstituted by adding 100 μL methanol. The solution was shaken and ultrasonicated for 2 min. It was then centrifuged at 14,000 rpm for 10 min. A 30 μL of the solution was run with the HPLC and analysis was performed.
For the quality control (QC) samples (2, 6, 500 and 5000 ng mL−1), blank rat plasma was spiked with appropriate standard solutions of bergapten to the required plasma concentrations, followed by the same sample preparation and extraction method described above.
Method validation
Testing for specificity involved comparing the chromatograms of six different batches of blank rat plasma samples with that of their corresponding spiked plasma. The limit of detection (LOD) was defined as the amount of analyte that could be detected with a signal to noise ratio of 3. The lower limit of quantification (LLOQ), which is the lowest concentration in the standard curve at which the signal to noise ratio (S/N) was to be larger than 5, with relative standard deviation (RSD n = 6) within 20 % and accuracy in the range of 80 % to 120 according to the guidelines for industry (2001). In determining the linearity of the method, samples were prepared by spiking blank rat plasma with standard solutions (prepared in methanol) of bergapten to the concentrations: 2, 4, 8, 20, 40, 100 and 100, 200, 500, 1000, 2500 and 5000 ng mL−1 for the calibration curves. In determining the intra-day accuracy and precision, four quality control (QC) samples (n = 6) were assayed within the same day. This was in turn repeated once a day for 3 consecutive days to evaluate the inter-day precision along with the standard calibration curve. The determination of the extraction recoveries was performed by comparing the observed peak areas of bergapten in extracted plasma samples with those of the bergapten in non-processed plasma samples at the same theoretical concentrations. The tests for stability were investigated for bergapten in autosampler for 24 h, after 3 times freeze and thaw cycles and also after storing in a freezer at a temperature of −20 °C for 1 month.
Application to a pharmacokinetic study in rats
Male Sprague–Dawley rats (240–260 g) were fed with standard laboratory food and water and kept in an environmentally-controlled breeding room for at least 1 week before experimentation. The rats were fasted for 12 h and allowed free access to water prior to the experiments. The rats were randomly divided into 4 groups with eight rats in each group to diminish the individual variation. The first group was given bergapten intravenously at a dose of 5 mg kg−1 while the other three groups were given bergapten orally at doses of 5, 10 and 15 mg kg−1. Disposable sterilized syringes were used for intravenous administration and medical cotton ball was pressured on the wound until bloodless after injection. Blood samples (about 250 μL) were immediately collected in heparinized 1.5 mL polythene tubes from the suborbital vein at 0, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 12, 24 h after oral administration. For intravenous administration, time intervals were set at 0, 0.033, 0.083, 0.17, 0.25, 0.33, 0.5, 0.75, 1, 2, 3, 4, 6, 8, 12, 24 h for blood sampling. All blood samples were immediately centrifuged to separate plasma at 6000 rpm for 10 min. The plasma was transferred into clean tubes and stored at −20 °C until analysis. Animal welfare and experimental procedures were strictly in accordance with the guide for the care and use of laboratory animals and the related ethical regulations of Tianjin University of Traditional Chinese Medicine.
Excretion of bergapten in rat urine, feces and bile
Sixteen male Sprague–Dawley (SD) rats (250 ± 10 g) were divided into two groups (group1 were for collecting urine and fecal samples in metabolic cages while group 2 were for collecting bile samples from the bile duct using polyethylene tubes). For group 1, the rats were orally administrated with bergapten dissolved in 0.5 % CMC-Na at a dosage of 15 mg kg−1 and placed in metabolic cages enabling collection of urine and fecal samples separately. The urine and fecal samples were collected at time interval of 0–4, 4–8, 8–12, 12–24, 24–36, 36–48, 48–60, and 60–72 h. For Group 2, the rats were anaesthetised with chloral hydrate at a dose of 0.3 g kg−1 administered intraperitoneally. A polyethene tube was used in cannula ting the bile duct ensuring continuous flow of bile. Bile samples were then collected at different time intervals (0–1, 1–2, 2–3, 3–4, 4–5, 5–6, 6–7, 7–8, 8–9 and 9–10 h). After the volumes of urine and bile obtained were measured, these samples were stored at −20 °C until analysis. The preparation of the urine and bile samples were the same as the plasma sample preparation described above. The fecal samples, on the other hand, after collection were dried out in a drying oven at 40 °C. After measuring the weights of the fecal samples, they were crushed by a mortar to achieve a uniform powder. 0.1 g of powdered feces was measured and 1 mL methanol was added in 1.5 mL polythene tubes, mixed sufficiently for 3 min by vortexing and extracted ultrasonically for 30 min. Supernatant were transferred into vials for analysis using HPLC-FLD.
Data analysis
The DAS software (Drug and Statistics 1.0, Medical College of Wannan, China), a computer program was used in calculating the pharmacokinetic parameters after administering bergapten both intravenously and orally at dose of 5 and 5, 10, 15 mg kg−1, respectively. To choose the optimum compartment model for fitting the plasma concentration–time curve, the minimum Akaike’s information criterion (AIC) estimation was tested by calculating the lowest AIC value. The compartment model with minimum AIC is regarded as the best representation of the plasma concentration–time course data [10]. The bioavailability was calculated as follows: F = (AUCoral/AUCintravenous) × 100 %. Both AUC oral and AUC intravenous were estimated by one-compartment model.