Measurement of phenol and p-cresol in urine and feces using vacuum microdistillation and high-performance liquid chromatography Roger A.King,Bruce L.May, Debbie A.Davies,Anthony R.Bird * Food Futures National Research Flagship, CSIRO Human Nutrition, Adelaide,SA 5000,Australia a r t i c l e i n f o Article history: Received 28 April 2008 Available online 26 September 2008 Keywords: Cresols Feces HPLC Human Phenol Urine Vacuum microdistillation a b s t r a c t In this article,we describe a simple, sensitive,accurate,and repeatable method for the measurement of phenol and p-cresol (4-methylphenol) in human urine and feces. We examined a number of parameters to identify an optimal extraction protocol. Purification of sample extracts was achieved by low-temper- ature vacuum microdistillation. Separation was achieved in approximately 15 min by high-performance liquid chromatography (HPLC) with quantification by fluorescence at 284/310 nm. Limits of detection for phenol were 2 ng/ml for urine and 20 ng/g for feces, and those for p-cresol were 10 ng/ml for urine and 100 ng/g for feces. For comparison, approximate mean values for urine are 3 l g/ml for phenol and 30 l g/ ml for p-cresol, and those for feces are 1 l g/g for phenol and 50 l g/g for p-cresol. An experienced analyst can process 60 samples each day using this method. Crown Copyright Ó 2008 Published by Elsevier Inc. All rights reserved. Measurement ofphenols and cresols in feces and urine is of interest for two main reasons. First, these substances serve as markers for monitoring environmental exposure to aromatic hydrocarbons such as benzene and toluene [1,2]. Second,phenol and p-cresol (4-methylphenol) are putative biomarkers of large bo- wel health both in humans [3–5] and in animal models [6]. Diets that promote the generation of these potentially toxic metabolites are associated with enhanced rates of colonic DNA damage and greater risk of large bowelcancer [6,7].Although environmental exposure can lead to the generation of a large number of phenols and cresols [1,8], only phenol and p-cresol, which arise from bacte- rial action on dietary aromatic amino acids [9], are present in nor- mal samples. A number of high-performance liquid chromatography (HPLC) 1 and gas chromatography (GC) methods for the measurement of phe- nols and cresols in urine have been published [1,8–14]. In contrast to urine, we are aware of only two methods for feces [9,11]. In urine, phenols and cresols are present largely as glucuronide and sulfate conjugates [15,16], and before chromatography these must be hydrolyzed using acid [2,9–13] or enzymatic [1,8,13,15] methods. Hydrolysis is not required for fecal samples [9] because, as is the case for bile acids [17], endogenous bacterial hydrolases generate the free forms of phenols and cresols. Initial extracts have most commonly been obtained by simple extraction from acidified samples using a variety of solvents,including dichloromethane [1], isopropyl ether [2,14,15], t-butyl ether [8], hexane [10], and diethyl ether [11]. Other methods have used distillation [9,13] and solid phase extraction [12]. Most methods [1,11–15,18,19], but not all methods [2,9],use an internal standard, and these have included 4-ethylphenol [14,19],4-chlorophenol [1,11],2,6-dimethylphenol [13,15],and o- cresol [18].For HPLC methods, analytes are detected and quantified with ultraviolet (UV) [1,2,11],fluorescence [9,14,15], electrochemi- cal [13],or chemiluminescence [12] methods. Measurement of phenol and p-cresol in urine and feces presents a challenge because phenol is usually present at concentrations at least one to two orders of magnitude less than p-cresol and be- tween-individual differences in concentrations can also be ofa similar magnitude. In the current study, and as expanded on below, we examined a number of elements of the assay of phenols and cresols in urine and feces with a view to optimizing and validating their measurement. With regard to choice of internal standards,we have exam- ined a number of substances to identify the best candidate. For sample cleanup, distillation is inherently superior to solvent extraction because it removes all nonvolatile substancesand provides a purer extract [9,13]. This is particularly important for fecal samples. However, previously reported distillation methods for phenols and cresols have involved collection of up to 50 ml of distillate [9,13,20] and process only one sample at a time, thereby limiting throughput.For the measurement of short-chain fatty acids in feces, our group has used a simple multisample low-temperature vacuum microdistillation appara- tus [21] based on a single-sample apparatus described by others 0003-2697/$ - see front matter Crown Copyright Ó 2008 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2008.09.034 * Corresponding author.Fax: +61 88303 8899. E-mail address: tony.bird@csiro.au (A.R. Bird). 1 Abbreviationsused: HPLC, high-performance liquid chromatography;GC, gas chromatography; UV, ultraviolet. Analytical Biochemistry 384 (2009) 27–33 Contents lists available at ScienceDirect Analytical Biochemistry j o u r n a l h om e pa ge : w w w . e l s e v i e r . c o m / l o c a t e / y a b i o