Validation of (S)-zaltoprofen assay in rat plasma by HPLC and its application in pharmacokinetics
study
C. V. Pham, S-H. Jung, C-W. Cho
ChungNam National University
Purpose
(S)-zaltoprofen ((S)-ZPF) is a non-steroidal anti-inflammatory drug, which has analgesics, antipyretics, and anti-inflammatory
activities. Some previous studies showed that (S)-ZPF exerted better anti-inflammatory and analgesic activities than racemic
zaltoprofen in rodents and also expressed a higher bioavailability compared to (R)-zaltoprofen. However, to our knowledge, no studies
have been reported on validation of quantification method of pure (S)-ZPF in rat plasma as well as an evaluation of bioavailability of
(S)-ZPF in rats. Hence, this study focused on establishing a validated method of (S)-ZPF in rat plasma using HPLC and also
evaluating the kinetic profiles of (S)-ZPF after oral and intravenous route administration.
Methods
The liquid-liquid extraction of (S)-ZPF and ketoprofen (IS) from rat plasma (0.125 mL) into the mixture of acetonitrile and
dichloromethane (1:6, v/v) was employed. The organic layer was separated and evaporated under a gentle stream of nitrogen at 40 °C.
The residue was reconstituted in the according mobile phase or MeOH and injected onto HPLC. Two different HPLC columns such as
the reversed column C18 (with mobile phase containing acetonitrile-distilled water (55:45, v/v) at a flow rate of 1.2 mL/min.) and the
chiral column Chiralcel OJ-H (with mobile phase containing n-hexane-2-propanol-trifluoroacetic acid (90:10:0.1, v/v/v) at a flow rate
of 0.8 mL/min) were used. The eluates were monitored using an ultraviolet (UV) detector set at 240 nm. To look into in vivo study of
(S)-ZPF in rats, six SD rats were employed according to the
Guiding Principles in the Use of Animals in Toxicology adopted by the
Society of Toxicology (USA) and the experimental protocols were approved by the Animal Care Committee of Chungnam National
University. The suspension of (S)-ZPF using in oral route was prepared in the mixture of 5% (v/v) DMSO containing 20% (w/v) PEG
400 in a 5% (w/v) glucose solution. To prepare of solution applied to intravenous administration, 5 mg of (S)-ZPF was dissolved
completely in ethanol and then given solution diluted 10 times with 20 % (w/v) PEG 400 solution. Blood samples (0.8 mL) were
collected from the orbital vein at desirable time points. Within 30 min following blood withdrawal, the samples were centrifuged at
15,000 rpm for 15 min at 4 °C. The plasma was collected, labeled and stored at -80 °C prior to application of above analytical method.
Results
The validation included assessment of linearity, recovery, accuracy, precision, and stability in rat plasma. Under mentioned reversed
HPLC conditions, (S)-ZPF and ketoprofen were detected separately at 4.16 and 2.95 minutes, respectively. Response was linear over
the calibration ranges of 0.1-75
g/mL with R
2
of 0.9998. The lower limit of quantification was 1.92 g/mL, and the percent
coefficient of variation for the QCs was within 3%. In addition, (S)-ZPF was stable under various store conditions such as three cycles
of freezing-thawing and 4 °C for 24 hr. The results also revealed that there was not any transformation of (S)-ZPF into (R)-zatoprofen
and absolute bioavailability of (S)-ZPF through oral administration was 71.89 ± 3.43%. In oral administration, (S)-zaltoprofen showed
the dramatic longer half-life (t
½
) of 9.34 ± 0.89 h compared to the half-life in intravenous administration at 6.04 ± 0.85 h.
Conclusion
The assay developed is linear, accurate, precise and reproducible for the analysis of (S)-zaltoprofen in rat plasma. The use of the
method can easily enable the characterization of (S)-ZPF pharmacokinetics after single oral and intravenous dose.