Journal of Pharmaceutical and Biomedical Analysis xxx (2005) xxx–xxx Short communication Investigating the human metabolism of acetaminophen using UPLC and exact mass oa-TOF MS Kelly A. Johnson, Robert Plumb ∗ Waters Corporation, 34 Maple Street, M/S GC, Milford, MA 01757, USA Received 7 March 2004; received in revised form 28 April 2005; accepted 29 April 2005 Abstract The ability to rapidly detect and characterize drug metabolites in biological fluids often relies on a combination of a high quality chromato- graphic separation and sensitive high resolution mass spectrometry. Here, the performance of two high throughput LC/MS approaches, namely monolith columns and sub-2 m particle Ultra Performance Liquid Chromatography (UPLC) columns is compared for the detection and identi- fication of the human metabolites of acetaminophen in urine. The UPLC system produced approximately three times the sensitivity and detected more metabolites than the monolithic column approach. The sharp peaks produced by UPLC seem to be particularly advantageous when cou- pled to electrospray mass spectrometry, apparently reducing ion suppression leading to superior sensitivity and hence lower limits of detection. © 2005 Elsevier B.V. All rights reserved. Keywords: HPLC; LC/MS; UPLC 1. Introduction The main reasons that compounds are eliminated from the drug discovery process are related to efficacy or toxicity [1,2]. Observed toxicity may be due to the dosed compound itself or a metabolite and, as part of any drug discovery activity, it is important to screen for the presence of such toxic metabo- lites [3]. Metabolite detection and identification is usually performed by LC/MS(MS) [4]. During such a screening pro- cess, speed and sensitivity are important factors, as there are typical tens to hundreds of compounds to be evaluated and the candidate compound may be dosed at very low levels to mice and rats. Both of these factor place significant strain on the analytical process. The rate of analysis depends on the ability of the chromatography system to reliably separate the components of interest in the sample. This ability is a mea- sure of the peak capacity per unit time of the chromatography system [5]. There have been several successful approaches to increasing analytical throughput in drug discovery including; the use of short columns with rapid gradients [6–8], cassette ∗ Corresponding author. E-mail address: Rob Plumb@waters.com (R. Plumb). dosing and analysis [9], monolithic column chromatography [10,11] and, more recently, Ultra Performance Liquid Chro- matography (UPLC) [12]. Of these approaches the monolithic columns and UPLC are the most promising as they offer high throughput with good chromatographic performance. The monolithic columns give a chromatographic performance equivalent to 4 m particle columns. They utilize the high permeability of the stationary phase allowing long columns to be employed with high linear velocity mobile phases, hence generating rapid separations with little loss in chromatographic per- formance. Ultra Performance Liquid Chromatography takes advantage of the flat nature of the van Demmter plot for sub 2 m stationary phases to generate higher chromatographic performance. These UPLC materials can be operated at very high mobile phase linear velocities with no loss in chromato- graphic performance [12]. In order to compare the relative performance of these two chromatographic approaches, the human metabolism of the common analgesic compound acetaminophen (paraceta- mol) was investigated. Acetaminophen is an over the counter medicine used for the treatment of headaches and fevers that has been available for many years; however, it is not 0731-7085/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jpba.2005.04.048 PBA-5337; No. of Pages 6