title: Characterization and evaluation of two-dimensional microfluidic chip-HPLC coupled to tandem mass spectrometry for quantitative analysis of 7-aminoflunitrazepam in human urine.
abstract: Microfluidic chip-based high-performance-liquid-chromatography coupled to mass spectrometry (chip-HPLC-MS) has been widely used in proteomic research due to its enhanced sensitivity. We employed a chip-HPLC-MS system for determining small molecules such as drug metabolites in biological fluids. This chip-HPLC-MS system integrates a microfluidic switch, a 2-dimensional column design including an enrichment column (160 nL) for sample pre-concentration and an analytical column for chromatographic separation, as well as a nanospray emitter on a single polyimide chip. In this study, a relatively large sample volume (500 nL) was injected into the enrichment column for pre-concentration and an additional 4 μL of the initial mobile phase was applied to remove un-retained components from the sample matrix prior to chromatographic separation. The 2-dimensional column design provides the advantages of online sample concentration and reducing matrix influence on MS detection. 7-Aminoflunitrazepam (7-aminoFM2), a major metabolite of flunitrazepam (FM2), was determined in urine samples using the integrated chip-HPLC-MS system. The linear range was 0.1-10 ng mL(-1) and the method detection limit (signal-to-noise ratio of 3) was 0.05 ng mL(-1) for 7-aminoFM2. After consecutive liquid-liquid extraction (LLE) and solid-phase extraction (SPE), the chip-HPLC-MS exhibited high correlation between 7-aminoFM2 spiked Milli-Q water and 7-aminoFM2 spiked urine samples. This system also showed good precision (n = 5) and recovery for spiked urine samples at the levels of 0.1, 1.0, and 10 ng mL(-1). Intra-day and inter-day precision were 2.0-7.1% and 4.3-6.0%, respectively. Clinical urine samples were also analyzed by this chip-HPLC-MS system and acceptable relative differences (-1.3 to -13.0%) compared with the results using a GC-MC method were determined. Due to its high sensitivity and ease of operation, the chip-HPLC-MS system can be utilized for the determination of small molecules such as drug metabolites and neurotransmitters in biological fluids for clinical diagnosis.
authors.affiliation: Department of Chemistry, Soochow University, Taipei, Taiwan.
authors.forename: Hsin-Yu
authors.initials: HY
authors.lastname: Bai
authors.forename: Shu-Ling
authors.initials: SL
authors.lastname: Lin
authors.forename: Shen-An
authors.initials: SA
authors.lastname: Chan
authors.forename: Ming-Ren
authors.initials: MR
authors.lastname: Fuh
mesh_terms: D014151:Anti-Anxiety Agents / Q000302:isolation & purification / Q000378:metabolism / Q000652:urine*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005445:Flunitrazepam / Q000031:analogs & derivatives* / Q000302:isolation & purification / Q000378:metabolism / Q000652:urine; D006801:Humans; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*; D052616:Solid Phase Extraction; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D014151:Anti-Anxiety Agents; D005445:Flunitrazepam; C036343:7-aminoflunitrazepam