Automated, continuous, and dynamic speciation of urinary arsenic in the bladder of living organisms using microdialysis sampling coupled on-line with high performance liquid chromatography and hydride generation atomic absorption spectrometry

Abstract

An on-line and fully automated method was developed for the continuous and dynamic in vivo monitoring of four arsenic species [arsenite (As^III), arsenate (As^V), monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA)] in urine of living organisms. In this method a microdialysis sampling technique was employed to couple on-line with high performance liquid chromatography (HPLC) and hydride generation atomic absorption spectrometry (HGAAS). Dialysates perfused through implanted microdialysis probes were collected with a sample loop of an on-line injector for direct and automated injection into HPLC system hyphenated with HGAAS. The saline (0.9% NaCl) solution was perfused at the rate of 1 μl min⁻¹ through the microdialysis probe and the dialysate was loaded into 50 μl of sample loop. The separation conditions were optimally selected to be in phosphate buffer solution at a pH 5.2 with a flow rate of 1.2 ml min⁻¹. The effluent from the HPLC was first mixed on-line at the exit of the column with HCl (1 M) solution and then mixed with a NaBH₄ (0.2% m/v) solution. Based on the optimal conditions obtained, linear ranges of 2.5–50 ng ml⁻¹ for As^III and 6.75–100 ng ml⁻¹ for the other three arsenic species were obtained. Detection limits of 1.00, 2.18, 1.03 and 2.17 ng ml⁻¹ were obtained for As^III, DMA, MMA and As^V, respectively. Typical precision values of 3.4% (As^III), 5.4% (DMA), 3.6% (MMA) and 7.5% (As^V) were obtained, respectively, at a 25 ng ml⁻¹ level. Recoveries close to 100%, relative to an aqueous standard, were observed for each species. The average in vivo recoveries of As^III, DMA, MMA and As^V in rat bladder urine were 56 ± 5%, 60 ± 9%, 49 ± 3% and 55 ± 7%, respectively. The use of an on-line microdialysis-HPLC-HGAAS system permitted the determination of four urinary arsenic species in the bladder of an anesthetized rat with a temporal resolution of 50 min sampling.