Rapid and simple one-step membrane extraction for the determination of 8-hydroxy-2′-deoxyguanosine in human plasma by a combination of on-line solid phase extraction and LC–MS/MS

Abstract

A quantitative analytical method using automated on-line solid phase extraction (SPE) and liquid chromatography–electrospray tandem mass spectrometry (LC–ESI-MS/MS) for the determination of 8-OHdG (8-hydroxy-2′-deoxyguanosine) in human plasma was developed and validated. A one-step membrane extraction method for the plasma sample preparation and a C18 SPE column with simple extraction and purification were used for the on-line extraction. A C18 column was employed for LC separation and ESI-MS/MS was utilized for detection. 15N 5-8-OHdG ( 15N 5-8-hydroxy-2′-deoxyguanosine) was used as an internal standard for quantitative determination. The extraction, clean-up and analysis procedures were controlled by a fully automated six-port switch valve as one strategy to reduce the matrix effect and simultaneously improve detection sensitivity. Identification and quantification were based on the following transitions: m/ z 284 → 168 for 8-OHdG and m/ z 289 → 173 for 15N 5-8-OHdG. Satisfactory recovery was obtained, and the recovery ranged from 95.1 to 106.1% at trace levels in human plasma and urine, with a CV lower than 5.4%. Values for intraday and interday precision were between 2.3 and 6.8% for plasma and between 2.7 and 4.5% for urine, respectively. Values for the method accuracy of intraday and interday assays ranged from 93.0 and 100.5% for plasma and 110.2 and 119.4% for urine, respectively. The limits of detection (LOD) and LOQ were 0.008 ng/mL and 0.02 ng/mL, respectively.The applicability of this newly developed method was demonstrated by analysis of human plasma samples for an evaluation of the future risk of oxidative stress status in human exposure to nanoparticles and other diseases.