Journal of Chromatography A, 1303 (2013) 48–53 Contents lists available at SciVerse ScienceDirect Journal of Chromatography A j our nal homep age: www.elsevier.com/locate/chroma Analysis of organophosphate flame retardant diester metabolites in human urine by liquid chromatography electrospray ionisation tandem mass spectrometry Nele Van den Eede a , Hugo Neels a , Philippe G. Jorens b , Adrian Covaci a,∗ a Toxicological Center, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium b Clinical Pharmacology/Toxicology, University of Antwerp, Antwerp University Hospital, Wilrijkstraat 10, 2630 Edegem, Belgium a r t i c l e i n f o Article history: Received 4 April 2013 Received in revised form 7 June 2013 Accepted 19 June 2013 Available online 26 June 2013 Keywords: Dialkyl phosphates Diphenyl phosphate Phosphorus flame retardants PFR Metabolites Urine a b s t r a c t A new analytical method was developed for the determination of dialkyl and diaryl phosphates (DAPs), which are metabolites of organophosphate triesters (PFRs), in human urine. Target DAPs included dibutyl phosphate (DBP), diphenyl phosphate (DPHP), bis(2-butoxyethyl) phosphate (BBOEP), bis(2-chloroethyl) phosphate (BCEP), bis(1-chloro-2-propyl) phosphate (BCPP), and bis(1,3-dichloro-2-propyl) phosphate (BDCIPP). Sample preparation was based on solid phase extraction using a weak anion exchange sorbent (Oasis WAX). Although several instrumental techniques have been tested, best results were obtained with reversed phase liquid chromatography–negative electrospray ionisation tandem mass spectrome- try (LC–ESI-MS/MS) taking the total analysis time into account. Method accuracy at 3 ng/mL in pooled urine ranged between 69 and 119% (recovery), while inter-day imprecision (as relative standard devi- ation) was <31%. The performance of the LC–MS/MS method was compared to a method based on gas chromatography–electron impact tandem mass spectrometry (GC–MS/MS) and a good correlation (Pear- son r = 0.82, p < 0.01) between the results of these two methods was obtained for DPHP. LC–MS/MS analysis was more suitable for DPHP and BBOEP with respective method limits of quantification (mLOQ) of 0.3 and 0.15 ng/mL. In contrast, GC–MS/MS had a better sensitivity for BCEP, BCIPP, and BDCIPP, their respective mLOQs being 0.1, 0.06, 0.02 ng/mL, compared to 1.2, 3.7, and 0.5 ng/mL by LC–MS/MS. A set of urine sam- ples from volunteers was analysed, in which DPHP was the major DAP metabolite. A significant increase of DPHP levels was observed in the group of smokers (geometric mean of 1.55 ng/mL) compared to the non-smokers (geometric mean of 0.88 ng/mL). Metabolic transformation of triphenyl phosphate to DPHP by metabolic enzymes induced in smokers could be an explanation for this observation. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Organophosphate triesters are used as additive flame retar- dants (PFRs) and plasticisers in polymers, paints, synthetic rubber, varnishes, lacquers, and hydraulic fluids [1,2]. Since the restriction in the use of the flame-retarding polybrominated diphenyl ethers (PBDEs), the consumption of PFRs has increased resulting in house dust levels which exceed the levels of polybrominated diphenyl ethers in dust [3,4]. In Belgian dust, the dominating PFRs were tris(2-butoxyethyl) phosphate (TBOEP, 2.03 g/g), tris(1-chloro- 2-propyl) phosphate (TCIPP, 1.38 g/g), and triphenyl phosphate (TPHP, 0.5 g/g) [4]. As daily ingestion estimates are 20 mg of indoor dust on average, adults could be exposed to amounts as high as 260 ng PFRs per day [4]. Experiments in laboratory animals indicated that PFRs, such as tris(1,3-dichloro-2-propyl) phosphate, ∗ Corresponding author. Tel.: +32 3 265 2498; fax: +32 3 265 2722. E-mail address: adrian.covaci@ua.ac.be (A. Covaci). tris(2-chloroethyl) phosphate, and tri-n-butyl phosphate, undergo hydrolysis after uptake in the body to form dialkyl or diaryl phos- phates (DAPs) [5,6]. Indeed, recent publications demonstrated the presence of several DAPs in human urine, including dibutyl phosphate (DBP), bis(2-chloroethyl) phosphate (BCEP), bis(1- chloro-2-propyl) phosphate (BCIPP), bis(1,3-dichloro-2-propyl) phosphate (BDCIPP), diphenyl phosphate (DPHP), and bis(2- butoxyethyl) phosphate (BBOEP). However, not all these metabo- lites were combined in a single analytical protocol using urine [7–10]. In this study, we have developed a method based on solid-phase extraction (SPE) and liquid chromatography–tandem mass spec- trometry (LC–MS/MS) for the determination of BCEP, BCIPP, DBP, DPHP, BDCIPP, and BBOEP in human urine. Through the use of an important number of synthesised labelled internal standards, we could properly assess the method performance and the matrix effects for BDICPP and BBOEP. The final optimised method was com- pared to the GC–MS/MS method proposed by Schindler et al. [7]. Advantages and disadvantages of both methods were evaluated. 0021-9673/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2013.06.042