Fast and sensitive determination of urinary 1-hydroxypyrene by packed capillary column switching liquid chromatography coupled to micro-electrospray time-of-flight mass spectrometry

The present work reports capillary liquid chromatographic column switching methodology tailored for fast, sensitive and selective determination of 1-hydroxypyrene (1-OHP) in human urine using micro-electrospray ionization time-of-flight mass spectrometric detection. Samples (100 μl) of deconjugated, water diluted and filtered urine samples were loaded onto a 150 μm I.D.×30 mm 10 μm Kromasil C18 pre-column, providing on-line sample clean-up and analyte enrichment, prior to back flushed elution onto a 150 μm I.D.×100 mm 3.5 μm Kromasil C18 analytical column. Loading flow rates up to 100 μl/min in addition to the use of isocratic elution by a mobile phase composition of acetonitrile/water (70/30, v/v) containing 5 mM ammonium acetate provided elution of 1-OHP within 5.5 min and a total analysis time of less than 15 min with manual operation. Ionization was performed in the negative mode and 1-OHP was observed as [M–H]− at m/z 217.08. The method was validated over the concentration range 0.2–40 ng/ml 1-OHP in pre-treated urine, yielding a coefficient of correlation of 0.997. The within-assay (n=6) and between-assay (n=6) precisions were in the range 6.4–7.3 and 7.0–8.1%, respectively, and the recoveries were in the range 96.2–97.5 within the investigated concentration range. The method mass limit of detection was 2 pg, corresponding to a 1-OHP concentration limit of detection of 20 pg/ml (0.09 nmol/l) diluted urine or 0.3 ng/ml (1.35 nmol/l) urine.