Diazepam d5

Diazepam (C₁₆H₁₃ClN₂O, 284.74 g mol⁻¹) and norDiazepam (C₁₅H₁₁ClN₂O, 270.71 g mol⁻¹) (Fig. 1), Diazepam-d₅ (isotopic purity; d₀ 0.00%) and norDiazepam-d₅ (isotopic purity; d₀ 0.02%) were obtained as stock solutions in methanol (all with the chemical purity of 99%) from Cerilliant (Round Rock, TX, USA). Formic acid (> 99%, pro analysi) was purchased from Acros Organics (Morris Plains, NJ, USA). Ortho-phosphoric acid (85%, pro analysi) and sodium hydroxide (≥ 99%, pro analysi) came from Merck (Darmstadt, Germany). methanol (HPLC grade) and acetonitrile (HPLC grade) were from Fisher Scientific UK Limited (Loughborough, UK). A Milli-Q Academic water purification system (Billerica, MA, USA) with a Millipak-40 filter unit (0.22 μm, Billerica, MA, USA) was used to generate purified water.Fig. 1

The chemical structures of Diazepam and norDiazepam. Diazepam (R = CH₃) and norDiazepam (R = H)

The stock solutions of Diazepam, Diazepam-d₅, norDiazepam, and norDiazepam-d₅ were stored at − 28 °C in the dark and diluted to working solutions with a mixture of 10 mM Formic acid in purified water/methanol (1/1, v/v) or 5 mM Formic acid in purified water/acetonitrile (90/10, v/v). The working solutions were stored at 4 °C in the dark. Phosphate buffer (pH 7.0, 10 mM) was prepared from stock solutions of phosphoric acid (1 M) and sodium hydroxide (1 M).

Estazolam, and the internal standard (IS) diazepam-d₅ standard stock solutions (1 mg/ml) were purchased from Cerilliant (Round Rock, TX, United States). Methanol, and acetonitrile (≥99.9%, suitable for UPLC) were purchased from Sigma-Aldrich (St. Louis, MO, United States). Ammonium formate and formic acid were obtained from Fluka (Buchs, Switzerland). Dithiothreitol (DTT) powder (≥99%, HPLC-grade) was obtained from Sigma-Aldrich (St. Louis, MO, United States). Deionized water was purified using a Milli-Q system (Millipore, MA, United States). Custom-made transparent stationery tape ruled in 0.4 mm squares was purchased from Qian Jie Metal Label Factory (Shanghai, China).
Working solutions of estazolam (2000, 1000, 400, 100, 50, 20, and 10 pg/ml) were diluted in methanol from a stock solution and stored at -20°C. The hair extraction medium (EM) was prepared by dissolving DTT in a mixture of methanol/acetonitrile/2 mM ammonium formate (8% acetonitrile, pH 5.3) (25:25:50, v/v/v) at a concentration of 10 mg/ml. The diazepam-d₅ (internal standard, IS) working solution (10 pg/ml) was diluted with EM solution.