MICRO-SCALE METHOD FOR SEPARATION AND QUANTIFICATION OF ATENOLOL AND HYDROCHLOROTHIAZIDE BY SEQUENTIAL INJECTION CHROMATOGRAPHY

Abstract

A microscale stopped-flow separation manifold, which is termed sequential injection chromatograph (SIC), was exploited for separating and quantifying atenolol and hydrochlorothiazide. A short C18 separation column (4.6 × 25 mm) with a monolithic structure was used to offer a rapid and sufficient separation. Microscale fiber optic spectrometric devices were coupled with the SIC manifold for UV detection at 220 nm. The proposed SIC method was optimized, validated, and applied to both formulations single and binary mixture. The mobile phase composition (MPC) was optimized using the 2³ full-factorial design approach while other conditions were optimized using the univariate approach. The optimum MPC was 40 µmol/L phosphate:methanol:acetonitrile (85:7.5:7.5, v/v/v) at pH 3.0. Other optimum conditions were 40 µL/s for flow rate and 40 µL for sample volume. Satisfactory separation with a resolution of 1.6 and numbers of theoretical plates of more than 630 was achieved. The interesting features of the proposed SIC method are the microliter-scale of the consumed volume of mobile phase (3.50 mL) and high sample frequency (23 samples/hr).

Keywords:

• atenolol
• hydrochlorothiazide
• method validation
• multivariate analysis
• pharmaceutical analysis
• sequential injection chromatography

ACKNOWLEDGMENT

R.E.E. Elgorashe would like to thank the Sudan University of Science and Technology for allowing him to study for a PhD degree in chemistry. Dr. Idris also thanks King Khalid University for encouraging him to join the research team at the Sudan University of Science and Technology.

Notes

Fixed conditions: sample volume 40 µL, flow rate 40 µL/s, 50 µg/mL ATN, 25 µg/mL HCZ, and UV detection at 220 nm.

^a experiment number.

^b buffer concentration.

^c volume percentage of buffer.

^d volume percentages of acetonitrile and methanol.

^a phosphate:methanol:acetonitrile.

^b phosphate: acetonitrile.

^c consumed mobile phase volume.

^d peak height.

^e concentration (µg/mL).

^f plotted area under the peak.

^g limit of detection.

^h limit of quantification.

ⁱ relative standard deviation for 7 replicates in a day.

^j relative standard deviation for 5 replicates in 5 days within a week.