HIGH PERFORMANCE LIQUID CHROMATOGRAPHY FOR DETERMINING A NEW ANTICONVULSANT CANDIDATE, BENZALDEHYDE SEMICARBAZONE IN RAT PLASMA

Abstract

A simple, rapid, and sensitive high performance liquid chromatographic method using UV detection (HPLC-UV) for the determination of benzaldehyde semicarbazone (BS) in rat plasma was developed and validated using sodium diclofenac as an internal standard (IS). Analyses were performed on an HPLC system using a C₁₈ column and isocratic elution with mobile phase composed of water-methanol-acetonitrile (65:30:5, v/v/v) (pH 9.0), at a flow rate of 1.1 mL/min. The eluate was monitored using a UV detector at 282 nm. Plasma samples were deproteinized with methanol and 25 μL of the supernatant were injected into the system. The analysis was performed in 6 min and the retention times of BS and IS were approximately 4.9 and 2.6 min, respectively. The method was linear in the concentration range of 0.1–16.0 μg/mL (r² > 0.98). The lower limit of quantification (LLOQ) was 0.1 μg/mL. The accuracy of the method was within 15%. Intra and inter-day relative standard deviations were equal or less than 10.19% and 2.10%, respectively. The applicability of the HPLC-UV method for pharmacokinetic studies was tested using plasma samples obtained after oral administration of BS to male Wistar rats. No interference from endogenous substances was observed, showing the specificity of the method developed. The reported method can provide the necessary sensitivity, linearity, precision, accuracy, and specificity to allow the determination of BS in preclinical pharmacokinetic studies.

Keywords:

• benzaldehyde semicarbazone
• HPLC
• pharmacokinetic
• plasma
• validation

ACKNOWLEDGMENTS

The authors would like to thank IM – INOFAR/CNPq – Brazil (Process 420015/05-1) for financial support.

Notes

S.D., standard deviation; R.S.D., relative standard deviation.

Values are for n = 3 observations; S.D., standard deviation and R.S.D., relative standard deviation.

n = 6 observations.

Values are for n = 3 observations; S.D., standard deviation, R.S.D., relative standard deviation

^a At room temperature for 4 h;

^b Three freeze–thaw cycles within 3 days for 24 h and thawed at room temperature;

^c At −20°C for 70 days;

^d In an autosampler at 4°C for 12 h.