Methanol (LC-grade), formic acid (for MS), ammonium formate (for MS) and thymidine (THY) were purchased from Sigma-Aldrich (Seelze, Germany). Zidovudine (ZDV) and zidovudine-d3 (ZDV-d3) were purchased from Toronto Research Chemicals (Toronto, Canada). Purified water was generated from a Merck Millipore Milli-Q-Integral 3-System (Darmstadt, Germany). Stock solutions of target analytes and surrogate standard solutions were prepared as listed in Table 1 and stored in the dark at −25 °C. Working solutions containing all analytes were prepared in methanol at 10, 1, 0.1 and 0.01 mg L−1, for surrogate standards at 1 mg L−1 and were stored at 4 °C.Table 1
Compounds, solvents and concentrations of stock solutions, retention times, MRM transitions (bold printed transitions are the most intense), declustering potential, collision energy, cell exit potential, dwell time, transition ratios and assignment of internal standards (IS).
| compound | stock solutions | LC/MS/MS-parameters | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| solvent | concentration [g L−1] | retention time [min] | [M+H]+ | MRM1 | MRM2 | DP | CE1 [eV] | CE2 [eV] | CXP1 [V] | CXP2 [V] | dwell time [ms] | Assignment of IS | |
| THY | H2O | 1.0 | 7.2 | 243 | 110 | 32 | 17 | 44 | 10 | 10 | 50 | ZDV-d3 | |
| ZDV | H2O | 1.0 | 11.2 | 268 | 110 | 34 | 17 | 45 | 10 | 10 | 50 | ZDV-d3 | |
| internal standard | |||||||||||||
| ZDV-d3 | MeOH | 0.1 | 11.1 | 271 | 113 | 31 | 15 | 43 | 10 | 10 | 50 | ||