Patic DNA hydrolysates for 7-CEGua by liquid chromatographyelectrospray ionization-tandem mass spectrometry-selected reaction monitoring (LC-ESIMS/MS-SRM) DNA isolation and enzymatic hydrolysis had been carried out primarily as described [11]. In short, for hydrolysis, DNA (1 mg) was dissolved in 1 mL of 10 mM Tris-HCl/5 mM MgCl2 buffer (pH 7.0) containing [15N5]7-CEGua (1300 fmol). The DNA was enzymatically hydrolyzed for 70 min at 37 with 1060 units of DNase I (kind II, bovine pancreas), 0.05 units of phosphodiesterase I (sort II, Crotalus adamanteus venom), and 300 units of alkaline phosphatase (calf intestine). The hydrolysate, just after removal of a ten ?.. L aliquot for dGuo quantitation, was purified using a mixed mode cation exchange cartridge [MCX Vac RC, 60 mg (Waters Corp, Milford, MA)]. The cartridge was conditioned with 2 mL of CH3OH and 2 mL two H3PO4. The sample was acidified with ten ?.. L of 86 H3PO4. Just after the sample was applied, the cartridge was washed with 2 mL 0.1 H3PO4 and 2 mL of CH3OH, along with the analyte was eluted with 2 mL 3 NH4OH in CH3OH. This fraction was collected and concentrated to dryness. One mL of a freshly ready 10 CH3COCl solution in CH3OH was added towards the vial.102838-43-7 Chemical name The mixture was then heated for 1 h at 50 to convert 7-CEGua to its methyl ester, thenChem Biol Interact.Formula of 887310-61-4 Author manuscript; out there in PMC 2014 October 25.Wang et al.Pageconcentrated to dryness. The residue was dissolved in 1 mL 15 mM NH4OAc buffer (pH 6.6) and purified employing a Strata-X solid-phase extraction cartridge [33 ?.. m, 30 mg/1 mL (Phenomenex, Torrance, CA)]. The cartridge was conditioned with 1 mL CH3OH, 1 mL H2O and 1 mL 15 mM NH4OAc buffer (pH six.six). Following the sample was applied, the cartridge was washed with 1 mL 15 mM NH4OAc buffer (pH six.6), 1 mL H2O, and 1 mL 2 CH3OH. Finally the analyte was eluted with 1 mL of 80 CH3OH, this fraction was collected and evaporated to dryness.PMID:24275718 The residue was dissolved in 40 ?.. L of 15 mM NH4OAc buffer (pH six.6), and eight ?.. L aliquots were injected and analyzed by LC-ESI-MS/MS-SRM. Adduct analysis by LC-ESI-MS/MS-SRM was carried out using a TSQ Quantum Discovery Max triple quadrupole mass spectrometer (Thermo Scientific, Waltham, MA) interfaced with an Agilent 1100 capillary flow HPLC (Agilent Technologies, Palo Alto, CA) equipped with a 0.5 x 150 mm Hypersil Gold PFP column (Thermo). The column was operated at 30 as well as a flow rate of ten ?.. L/min. A ten min linear gradient from two to 35 CH3CN in 15 mM NH4OAc buffer (pH 6.six) was followed by a 35 CH3CN hold for 5 min, after which by a two min gradient from 35 to 80 CH3CN. The column was washed for 3 min with 80 CH3CN, then returned to two CH3CN in 2 min and ultimately re-equilibrated for 15 min. The MS parameters had been set as follows: spray voltage, four kV; sheath gas stress, 30; capillary temperature, 250 ; collision energy, 22 V; scan width, 0.1 amu; scan time, 0.4 s; Q1 peak width, 0.7; Q3 peak width, 0.7; Q2 stress, 1.0 mTorr; supply CID, 8V; and tube lens offset, 95V. Transitions monitored were as follows: m/z 238 [M + H]+! m/z 152 [BH]+ for 7-CEGua methyl ester; and m/z 243 [M + H]+! m/z 157 [BH]+ for [15N5]7-CEGua methyl ester. Calibration curves were constructed before each and every evaluation employing normal solutions of 7CEGua and [15N5]7-CEGua. A continual volume of [15N5]7-CEGua (1300 fmol) was mixed with differing amounts of 7-CEGua (ten, 20, 40, 60, 100, and 200 fmol), and these have been esterified with CH3COCl and CH3OH and analyzed by LC-ESI-MS/MS-SR.