Simultaneous quantification of urinary 6‑sulfatoxymelatonin and 8‑hydroxy‑2′‑deoxyguanosine using liquid chromatography-tandem mass spectrometry

Abstract

Oxidative stress is involved in the pathophysiology of many diseases and natural aging. Urinary 8‑hydroxy‑2′‑deoxyguanosine (8‑OHdG), a stable product of DNA oxidative damage, has been widely used as an oxidative stress biomarker. However, only reporting 8‑OHdG level, as commonly done previously, does not take into account of damaged DNA molecules that have not been repaired and excreted in urine. Melatonin is known to help with DNA repairs and hence can modify 8‑OHdG levels. 6‑sulfatoxymelatonin (aMT6s) is a major metabolite of melatonin excreted in urine. Hence it is useful to measure both urinary 8‑OHdG and aMT6s together. Here we describe a newly developed and validated method to simultaneously measure these two compounds using high-performance liquid chromatography-tandem mass spectrometry. Using a liquid-liquid extraction (20% methanol) at a pH ~ 7, as opposed to a solid-phase extraction used previously, substantially expedited the sample pretreatment process. The calibration curves showed a linear range from 0.5 to 100 ng/mL for aMT6s with a R2 of 0.9999 and from 1.5 to 100 ng/mL for 8‑OHdG with R2 of 0.9985. The recovery for 8‑OHdG and aMT6s was 102 ± 2% and 88 ± 4%, respectively. The limits of detection were 0.1 ng/mL for aMT6s and 0.5 ng/mL for 8‑OHdG, while the lower limits of quantification were 0.3 ng/mL for aMT6s and 1.0 ng/mL for 8‑OHdG. For both compounds, the retention time standard deviation was 0.03 min, and inter-day and intra-day variability, expressed as relative standard deviation (RSD), ranged from 4.5%–15.8%. The new method was successfully applied to analyze both compounds in the first morning and midday urine voids from five volunteers. The results support that this method is capable of rapidly and accurately measuring urinary 8‑OHdG and aMT6s simultaneously. This new method is expected to have important applications in biomedical and environmental health studies involving the oxidative stress pathophysiological pathway.