The pharmacokinetics, bioavailability and excretion of bergapten after oral and intravenous administration in rats using high performance liquid chromatography with fluorescence detection

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 = (AUC_oral/AUC_intravenous) × 100 %. Both AUC oral and AUC intravenous were estimated by one-compartment model.

Optimization of the fluorescence spectra

The excitation and emission wavelengths of bergapten were optimized to obtain a suitable detection wavelength with an increased signal to noise (S/N). After several examinations, an excitation wavelength of 288 nm and emission wavelength of 478 nm was the most suitable fluorescence detection wavelength for bergapten and the IS isoimperatorin.

Method validation

Specificity

Figure 2 shows the representative chromatograms of blank plasma, blank plasma samples spiked with bergapten and plasma sample obtained from a rat following an injection of bergapten. The retention time of bergapten was 7.3 min and IS was 10.2 min. As described above, good resolution was achieved between analyte and IS and no substance from several different sources of rat plasma was observed interfering with the separation and quantitation of bergapten. In the real pharmacokinetic study samples, no metabolite or endogenous substance interfered with the determination of the analytes.

Calibration curve and lower limits of quantification

The model of calibration for the two calibration curves was selected based on the analysis of the data by linear regression and with weighting factor (1/x). The peak area ratio of bergapten to IS in rat plasma was linear in relation to the concentration of the analyte for the ranges, 2–100 ng mL⁻¹ and 100–5000 ng mL⁻¹. The regression equation for calibration one was Y = 0.006581X − 0.00793 (correlation coefficient, r = 0.9990), and that for the second calibration was Y = 0.007403X + 0.050226 (correlation coefficient, r = 0.9992) over the range 100-5000 ng mL⁻¹. The LOD for bergapten was found to be 1 ng mL⁻¹ (S/N ≥ 3) and LLOQ was 2 ng mL⁻¹(S/N ≥ 5).

Pharmacokinetic studies

Pharmacokinetics of bergapten in rats after intravenous administration

The pharmacokinetics of bergapten in rat plasma after administering bergapten both intravenously and orally was successfully studied using the developed method. After intravenous administration of bergapten at dose of 5 mg kg⁻¹ to rats, the mean plasma concentration–time profile of bergapten is shown in Fig. 3. Some important pharmacokinetic parameters have been listed in Table 4. The distribution of bergapten into the tissues was slow and this is indicated by the long distribution half-life, T_1/2α of 2 h. The plasma concentration of bergapten decreased gradually within the first 4 h after interavenous administration, and then slowly decreased to the LLOQ during the next 8 h. The average volume of distribution is 0.0027 ± 0.0006 L Kg⁻¹. The mean area for the plasma concentration–time curve from time zero to the last measurable plasma concentration point (AUC_0–tn) was 4391 ± 1363 ng (mL h)⁻¹ and the mean area under the plasma concentration–time curve from time zero to time infinity (AUC_0–∞) was 4474 ± 1322 ng (mL h)⁻¹.

Parameters	Low (5 mg kg−1)
Tmax (h)	0.0333
Cmax (ng mL−1)	2080 ± 484
AUC(0–tn) (ng mL−1 h−1)	4391 ± 1363
AUC(0–∞) (ng mL−1 h−1)	4474 ± 1323
V/F (L)	0.0027 ± 0.0006
T1/2α (h)	1.74 ± 0.21
MRT(0–tn) (h)	1.80 ± 0.10
MRT(0–∞) (h)	4.05 ± 3.81

Bioavailability of bergaten in rats after administration

The absolute oral bioavailability (F) were 80.1 ± 29.6 %, 94.0 ± 40.3 % and 69.5 ± 44.2 % for low, medium and high concentrations using the formula F = (AUC_oral/AUC_intravenous) × 100 %, based on the AUC_(0–∞) obtained after intravenous and oral administration. The AUC of the medium and high concentration were similar, it could be inferred that the absorption of bergapten reached its peak within the range of 10 to 15 mg kg⁻¹. It was demonstrated that bergapten might have a good absorption from the gastrointestinal tract in rat. It was also concluded that oral administration of bergapten may be the better route if it was developed the new drugs used in clinic.

Excretion study of bergapten in rat urine, feces and bile

The cumulative excretion of bergapten in urine, feces and bile were determined as shown in Fig. 4. After an oral administration of bergapten at a dose of 15 mg kg⁻¹ to the rats, bergapten could be detected in rat urine until 72 h. Bergapten increased rapidly in urine during a time period of 4–8 h. After 8 h however, there was a gradual increase of bergapten in urine. Bergapten exhibited an increased rise in fecal sample up until a period of 36 h, after which the detection of bergapten was stable with minimal increase or decrease until 72 h. The rise was rapid especially in the period of 4–8 h. Therefore, the time cumulative excretion percentage of bergapten in feces stabilized after 36 h. Owing to the rats’ physical conditions, bile samples were only collected for a period of 10 h. After a more gradual increase in bile for a period of 8 h, bergapten stabilized. As shown in Fig. 4, the time cumulative amounts of bergapten in feces were 27.99 ± 10.08 % of the total dose, demonstrating that bergapten was mainly excreted in the feces. Even though the time cumulative amount of bergapten in urine was 0.032 ± 0.019 %, it continued to increase in urine until 72 h.

Pharmacokinetic study of bergapten

The development of sensitive and specific assay of a drug is crucial to the study of drug pharmacokinetics. The HPLC-FLD was first developed to monitor the concentration of bergapten in solution to determine its suitability and sensitivity. The method was further optimized for the determination of bergapten in the rat plasma and has been validated to be sensitive to investigate the pharmacokinetics of bergapten in rats. Bergapten is an important furocoumarin because of its presence in many TCMs and the various therapeutic effects it possesses. Bergapten was rapidly absorbed by rats with the maximum plasma concentration achieved within 3 h after dosing (5 mg kg⁻¹) as seen in Table 1. The kinetic properties were fit to the one-compartment model after rats were given i.v. administration. The absorption T _1/2 after oral administration was 30 s, which shows that bergapten was also rapidly absorbed. The long distribution half-life could explain the reason for high bioavailability in rats after oral administration (T_1/2α (h) = 9.35 ± 3.06). The oral absolute bioavailability were 80.1 ± 29.6 %, 94.0 ± 40.3 % and 69.5 ± 44.2 % for low, medium and high concentration of bergapten, which showed that bergapten was provided with a higher degree of absorption from the gastrointestinal tract.

Method comparison with existing reports

A pharmacokinetic study of bergapten was performed in dog plasma using an LC–MS/MS method. Tmax and AUC_(0–∞) were 4.2 h and 3219.2 ± 211.4 ng (mL h)⁻¹ respectively which were comparable to that obtained from our experiment, giving a T_max and AUC_(0–∞) of 3–4.5 h and 3537 ± 1302 ng (mL h)⁻¹. The LLOQ as obtained from the LC–MS/MS experiment was 0.5 ng mL⁻¹ which differs from this study which was 2 ng mL⁻¹ [8]. Our experiment differs from the LC–MS/MS experiments done on bergapten in that we determined the oral bioavailability and excretion of bergapten in rats. HPLC-FLD offers a cheaper analytical tool option compared to the higher cost of LC–MS/MS and HPLC-FLD requires less technical know-how.