A fully validated bioanalytical method using an UHPLC–MS/MS system for quantification of DNA and RNA oxidative stress biomarkers

Abstract

A new, rapid and effective ultra-high-performance liquid chromatography method with mass spectrometry detection is described for the separation and quantification of 8-hydroxy-2-deoxyguanosine, 8-hydroxyguanosine and creatinine in human urine. The present study uses an isotope-labelled internal standard ([N]-8-hydroxy-2-deoxyguanosine), a BIO core-shell stationary phase and an isocratic elution of methanol and water. Sample preparation of human urine was performed by solid-phase extraction (SPE) on Oasis HLB cartridges with methanol/water 50:50 (v/v) elution. Extraction recoveries ranged from 98.1% to 109.2%. Biological extracts showed high short-term stability. Several aspects of this procedure make it suitable for both clinical and research purposes: a short elution time of less than 3.2 min, an intra-day precision of 2.5-8.9%, an inter-day precision of 3.4-8.7% and low limits of quantification (27.7 nM for 8-hydroxyguanosine, 6.0 nM for 8-hydroxy-2-deoxyguanosine). Finally, simultaneous analysis of DNA and RNA oxidative stress biomarkers is a useful tool for monitoring disease progression in neurodegenerative disorders and cancer. Graphical abstract UHPLC-MS/MS analysis of DNA and RNA oxidative stress biomarkers.

Simultaneous analysis of multiple urinary biomarkers for the evaluation of oxidative stress by automated online in‐tube solid‐phase microextraction coupled with negative/positive ion‐switching mode liquid chromatography–tandem mass spectrometry

“…These MRM transitions and optimal MS/MS parameters are shown in Supporting Information Table S1. These findings are in agreement with previous reports . 8‐IP, 8‐OHdG, and 3‐NT were separated on LC using an Inertsil CX column.…”

“…The detection limits (S/N = 3) of 8‐IP, 8‐OHdG, and 3‐NT were 3.4, 12.6, and 21.5 pg/mL, respectively, and the in‐tube SPME method showed a 27 to 37‐fold higher sensitivity than the direct injection method (10 μL injection) (Table ). These sensitivities were higher than those of previous methods for 8‐IP (detection limit: 7–97 pg/mL) , 8‐OHdG (detection limit: 10–1019 pg/mL) , and 3‐NT (detection limit: 2–500 pg/mL) . The intra‐ and inter‐day precisions (RSD) obtained from the analysis of 0.2, 0.8, and 2.0 ng/mL standard mixtures containing 8‐IP, 8‐OHdG, and 3‐NT each were 1.1–5.0 and 2.3–6.5%, respectively ( n = 5; Table ).…”

“…In-tube SPME sensitivities were higher than those of previous methods for 8-IP (detection limit: 7-97 pg/mL) [37][38][39][40], 8-OHdG (detection limit: 10-1019 pg/mL) [40][41][42][43], and 3-NT (detection limit: 2-500 pg/mL) [44,45]. The intra-and inter-day precisions (RSD) obtained from the analysis of 0.2, 0.8, and 2.0 ng/mL standard mixtures containing 8-IP, 8-OHdG, and 3-NT each were 1.1-5.0 and 2.3-6.5%, respectively (n = 5; Table 1).…”

“…Methods used to measure the concentration of these biomarkers in human urine samples include ELISA [27][28][29], GC-MS/MS [30], CE [31,32], MEKC [33], HPLC [34][35][36], and LC-MS/MS [37][38][39][40][41][42][43][44][45]. Although commercially available ELISA kits are frequently used because of their benefits of reduced instrumentation, many kits must be used to determine the types of biological molecules damaged by oxidative stress.…”

“…In contrast, LC-MS/MS methods can sensitively and selectively measure concentrations of 8-IP [37][38][39][40], 8-OHdG [40][41][42][43], and 3-NT [44,45]. However, most LC-MS/MS methods require time consuming and troublesome sample preparation procedures, such as LLE or SPE, to remove other substances in urine samples before analysis.…”