Salting-out induced liquid-liquid microextraction for alogliptin benzoate determination in human plasma by HPLC/UV

Abstract

Salting-out induced liquid-liquid microextraction method has been developed for plasma sample treatment before determination of alogliptin by high performance liquid chromatography with UV detection. Several parameters were optimized to achieve maximum enrichment, including type of extractant, volume of extractant, type of anion, type of cation, salt amount and pH. The optimum conditions were attained using 500 µL of acetonitrile, added to 1 mL of aqueous sample containing 250 mg of sodium chloride at pH 12. An RP-HPLC method was developed and validated according to the International Conference on Harmonization guidelines M10. The method was linear in the concentration range of 0.1 to 50 µg/mL (correlation coefficient = 0.997). The limit of detection was 0.019 µg/mL and limit of quantitation was 0.06 µg/mL. The method was accurate and precise with an average % recovery of 99.7% and a % relative standard deviation ranging between 1.5 and 2.5. These results showed that the salting-out induced liquid-liquid microextraction methods could be better than other sample preparation protocols in terms of sensitivity, easiness, solvent consumption and waste reduction.

Keywords: Alogliptin; Microextraction; Plasma; Salting-out; Sitagliptin.

Fig. 1

The chemical structure of the analyte (alogliptin benzoate) and the internal standard (sitagliptin phosphate monohydrate)

Fig. 2

Procedures of salting-out induced liquid–liquid microextraction for alogliptin benzoate from aqueous samples using sitagliptin phosphate monohydrate as an internal standard

Fig. 3

Effect of acetonitrile volume (µL) on the efficiency of SALLME of alogliptin benzoate

Fig. 4

Effect of extractant type on the efficiency of SALLME of alogliptin benzoate

Fig. 5

Effect of diluent pH on the efficiency of SALLME of alogliptin benzoate

Fig. 6

Effect of chloride amount (in mg) on the efficiency of SALLME of alogliptin benzoate

Fig. 7

Chromatographic separation of alogliptin benzoate and sitagliptin phosphate monohydrate using the proposed SALLME method (a) and the reported protein precipitation method (b)