Diazepam

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).