Total analysis system for the determination of uremic toxins in human plasma based on bead injection solid phase extraction hyphenated to mass spectrometry

Indoxyl sulfate (INDS) andp-cresol sulfate (pCS) are two of the most relevant uremic toxins that are recognized to have an essential role in chronic kidney disease (CKD) progression and associated cardiovascular risk. Thus, it is crucial to accurately assess their circulating levels in the body. Aiming at establishing an analytical strategy for quantification of INDS and pCS in human plasma, an automatic on-line micro-solid-phase extraction (& mu;SPE) procedure hyphenated to tandem mass spectrometry (MS/MS) detection without previous chromatographic separation was herein developed. The bead injection (BI) concept was used to implement the & mu;SPE procedure in the lab-on-valve (LOV) format. After studying the extraction conditions, the anion-exchange OASIS WAX sorbent beads (10 mg) and 99% ACN-H2O (15:85, v/v)-1% (v/v) NH4OH were chosen as sorbent and eluent, respectively, as they provided the highest analyte recoveries. Subsequently, the & mu;SPE-BI-LOV system was hyphenated on-line to a MS/MS detector and the full analytical cycle, comprising sample preparation and analytes detection, was completed in <20 min. The developed & mu;SPE-BI-LOV-MS methodology presented good linearity (r2 > 0.999) for quantification of the target analytes at concentrations ranging from 18 to 360 & mu;g mL-1 in plasma. LOQ values were 2 & mu;g mL-1 for INDS and 7 & mu;g mL-1 for pCS in plasma. Human plasma samples from healthy subjects and individuals with CKD were successfully analyzed using the developed approach. The proposed automatic methodology can be described as an eco-friendly strategy, with a favorable score of 0.64 after greenness evaluation using the AGREE metric.

Automated summary

Researchers have developed an automatic online analytical strategy based on micro-solid-phase extraction (μSPE) for quantification of the uremic toxins, indoxyl sulfate (INDS) and p-cresol sulfate (pCS), in human plasma without prior chromatographic separation. The μSPE-BI-LOV-MS methodology demonstrated good linearity and sensitivity for the target analytes, allowing for successful analysis of plasma samples from healthy subjects and individuals with chronic kidney disease (CKD).