Transl Clin Pharmacol.  2017 Dec;25(4):173-178. 10.12793/tcp.2017.25.4.173.

Development of a simple and sensitive HPLC-MS/MS method for determination of diazepam in human plasma and its application to a bioequivalence study

• Kim DH
• Cho JY
• Chae SI
• Kang BK
• An TG
• Shim WS
• Noh YS
• Hwang SJ
• Chung EK
• Lee KT

• KMID: 2406998
• DOI: http://doi.org/10.12793/tcp.2017.25.4.173

Abstract

We developed a simple, sensitive, and effective ultra-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) method with an electrospray ionization (ESI) interface in multiple reaction monitoring (MRM) and positive ion modes to determine diazepam concentrations in human plasma using voriconazole as an internal standard (IS). Diazepam and IS were detected at transition 285.2→193.1 and 350.2→127.1, respectively. After liquid-liquid extraction (LLE) using 1.2 ml of ethyl acetate:n-hexane (80:20, v/v), diazepam and IS were eluted on a Phenomenex Cadenza CD-C18 column (150 × 3.0 mm, 3 µm) with an isocratic mobile phase (10 mM ammonium acetate in water:methanol [5:95, v/v]) at a flow rate of 0.4 mL/min. The peak retention time was 2.32 min for diazepam and 2.01 min for IS, respectively. The lower limit of quantitation (LLOQ) was 0.5 ng/mL (S/N > 10) using 50 µL of plasma, and no interferences were observed in chromatograms. Our analytical method was fully validated and successfully applied to a bioequivalence study of two formulations of diazepam in healthy Korean volunteers.

Keyword

Liquid chromatography-tandem mass spectrometry; human plasma; method validation; pharmacokinetic study; diazepam

MeSH Terms

Ammonium Compounds
Diazepam*
Humans*
Liquid-Liquid Extraction
Mass Spectrometry
Methods*
Plasma*
Therapeutic Equivalency*
Volunteers
Voriconazole
Diazepam
Voriconazole

Figure

• 

  Figure 1 Product ion mass spectra and the pattern of fragmentation of (A) diazepam and (B) voriconazole (IS).

• 

  Figure 2 Multiple reaction monitoring chromatograms of (A) blank human plasma, (B) plasma spiked with IS only (voriconazole, 200 ng /mL), (C) plasma spiked with 0.5 ng/mL diazepam (LLOQ) and IS and (D) sample plasma from a volunteer after an oral administration of 2 mg diazepam (measured diazepam concentration: 75.728 ng/mL).

• 

  Figure 3 Mean plasma concentration-time profile of diazepam in the plasma after an oral dose of 2-mg diazepam Samjin tablet (●, reference formulation) and Daewon tablet (○, test formulation) administered to healthy volunteers (n = 40).

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