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Abstract Ultraviolet (UV) radiation induces mutagenicity and cytotoxicity in human cells by the formation of DNA lesions, including cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs), mainly on thymine-thymine (TpT) dinucleotides. Here, we firstly synthesized the two TpT dimeric lesions with satisfactory yields using a unique UV-irradiated water droplet approach followed by HPLC purification. By the use of purified TpT lesions as standards, we further developed and optimized a quantitative UHPLC-Q-TOF/MS method for the detection of CPDs and 6-4PPs. After the optimization of the enzyme composition and the pH values of hydrolysis solution, a combination of snake venom phosphodiesterase, nuclease P1, and calf intestine alkaline phosphatase can be used for one-step enzymatic digestion to efficiently release the dimeric lesions (CPDs and 6-4PPs) from the genomic DNA. By the use of the one-step digestion and UHPLC-Q-TOF/MS assay for scanning all dimeric lesions, we demonstrate that only are TpT dimeric lesions detectable in genomic DNA of HCT116 cells upon UVC irradiation. The estimated frequency of the CPD of TpT increases from 28.7 to 409 per 106 bases with increasing UVC dosage from 40 J/m2 to 1200 J/m2, while the 6-4PP of TpT increases from 3.7 to 54 per 106 bases. The proposed UHPLC-Q-TOF/MS method is promising for accurate identification and quantitative detection of UV-induced dimeric lesions in cellular DNA. Highlights • Unique UV-irradiated water droplet approach followed by HPLC purification was developed to prepare TpT dimer standards. • A simplified one-step enzymatic process to release TpT dimeric lesions from genomic DNA. • TT CPD and T(6-4)T levels were detected by UHPLC-Q-TOF/MS in HCT116 cells upon UVC-irradiation. [ABSTRACT FROM AUTHOR]

8-Hydroxydeoxyguanosine (8-OHdG) is a widely-used biomarker of oxidative DNA damages. 8-OHdG in peripheral blood leukocyte is associated with mutation and cancer risk. The level of 8-OHdG in peripheral blood leukocytes can indicate a long-term response to oxidative stress rather than that in urine. Accurate identification and quantification of leukocyte 8-OHdG are essential for understanding its mechanism of formation, repair, and biological consequences. In this study, a fast and accurate ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC–MS/MS) method was developed and validated to detect 8-OHdG in human peripheral leukocyte. DNA in blood samples were extracted and digested, then subjected onto UHPLC–MS/MS using an isocratic elution on a Zorbax Eclipse Plus C18 column (2.1 × 100 mm, 1.8 μm). Multiple reaction monitoring (MRM) mode was adopted by using [ 15 N 5 ]-8-OHdG as an internal standard. The assay was linear over the concentration range of 1.0–100 nM with R 2 = 0.999. The accuracy for spiked samples was 90.9%–94.8%, and the intra-day precision was within 3.7%. The limit of detection (LOD) is 0.30 nM and limit of quantification (LOQ) is 1.0 nM with 5 μl of sample injection. By the analysis of human leukocyte 8-OHdG (n = 121) using the developed UHPLC–MS/MS method, it demonstrates that the level of leukocyte 8-OHdG of the cancer patients group (n = 46) is significantly higher than that of the health control (n = 75). [ABSTRACT FROM AUTHOR]

A sensitive and accurate stable isotope dilution UHPLC–MS/MS method was developed and validated for the detection and quantification of ProdG adducts in human urine, a surrogate for the ProdG adducts in genomic DNA of human. A specific solid phase extraction (SPE) approach was established for selective enrichment of urinary ProdG adducts and elimination of urinary matrix facilitating the coupled MS/MS detection. The recovery of the method is estimated about 84.8–107.2%, and the precision are about 0.8–3.6% for intraday and 2.8–10.0% for interday. Due to that the matrix effect is efficiently eliminated by SPE pretreatment, the limits of detection (LODs, S/N = 3) and quantification (LOQs, S/N = 10) are decreased to 100 and 300 amol for urinary ProdG adducts, respectively. By coupling the developed SPE pretreatment with the UHPLC–MS/MS analysis, ProdG adducts were accurately quantified in healthy human urine. [ABSTRACT FROM AUTHOR]