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Impact of Urine Preservation Methods and Duration of Storage on Measured Levels of Environmental Contaminants

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Impact of Urine Preservation Methods and Duration of Storage on Measured Levels of Environmental Contaminants Journal of Exposure Science and Environmental Epidemiology (2006) 16, 39–48 r 2006 Nature Publishing Group All rights reserved 1559-0631/06/$30.00 www.nature.com/jes Impact of urine preservation methods and duration of storage on measured levels of environmental contaminants JANE A. HOPPIN,a ROSS ULMER,b ANTONIA M. CALAFAT,c DANA B. BARR,c SUSAN V. BAKER,d HELLE M. MELTZERe AND KJERSTI S. RØNNINGENe aEpidemiology Branch, NIEHS, NIH, DHHS, Research Triangle Park, North Carolina, USA bWestat, Research Triangle Park, North Carolina, USA cNational Center for Environmental Health, Centers for Disease Control and Prevention, DHHS, Atlanta, Georgia, USA dCODA Research, Research Triangle Park, North Carolina, USA eNorwegian Institute of Public Health, Oslo, Norway Collection of urine samples in human studies involves choices regarding shipping, sample preservation, and storage that may ultimately influence future analysis. As more studies collect and archive urine samples to evaluate environmental exposures in the future, we were interested in assessing the impact of urine preservative, storage temperature, and time since collection on nonpersistent contaminants in urine samples. In spiked urine samples stored in three types of urine vacutainers (no preservative, boric acid, and chlorhexidine), we measured five groups of contaminants to assess the levels of these analytes at five time points (0, 24, 48, and 72 h, and 1 week) and at two temperatures (room temperature and 41C). The target chemicals were bisphenol A (BPA), metabolites of organophosphate (OP), carbamate, and pyrethroid insecticides, chlorinated phenols, and phthalate monoesters, and were measured using five different mass spectrometry-based methods. Three samples were analyzed at each time point, with the exception of BPA. Repeated measures analysis of variance was used to evaluate effects of storage time, temperature, and preservative. Stability was summarized with percent change in mean concentration from time 0. In general, most analytes were stable under all conditions with changes in mean concentration over time, temperature, and preservative being generally less than 20%, with the exception of the OP metabolites in the presence of boric acid. The effect of storage temperature was less important than time since collection. The precision of the laboratory measurements was high allowing us to observe small differences, which may not be important when categorizing individuals into broader exposure groups. Journal of Exposure Science and Environmental Epidemiology (2006) 16, 39–48. doi:10.1038/sj.jea.7500435; published online 6 July 2005 Keywords: sample storage and shipment, cohort studies, analytical chemistry, urine. Introduction (Gunter, 1997; Landi and Caporaso, 1997). Choices made regarding shipment and storage of samples may influence Urine specimens are being collected and stored as part of the ability to measure specific analytes in the future. To standard protocols in cohort studies designed to assess ensure the integrity of the specimens during sample shipment exposure to nonpersistent environmentalcontaminants and processing, efforts are taken to prevent bacterialgrowth and to ensure timely receipt by the laboratory for long-term 1. Abbreviations: ANOVA, analysis of variance; APCI, atmospheric storage. Methods to prevent bacterialgrowth include pressure chemicalionization; BPA, bisphenolA; CDC, Centers for Disease Controland Prevention; CV, coefficient of variation; DAP, shipment of samples on ice or use of preservatives. Ideally, dialkylphosphates; DEDTP, diethyldithiophosphate; DEP, diethylpho- timely sample shipment usually involves sample receipt sphate; DETP, diethylthiophosphate; DMDTP, dimethyldithiopho- within 24 h of collection, but due to unexpected delays in sphate; DMP, dimethylphosphate; GC-MS/MS, gas chromatography- shipping, samples may not be received until a few days tandem mass spectrometry; HPLC-MS/MS, high-performance liquid following sample collection. Few data are currently available chromatography-tandem mass spectrometry; LOD, limit of detection; NC, not calculated; MEP, monoethyl phthalate; MOP, monooctyl to evaluate any detrimental effects that urine preservatives or phthalate; OP, organophosphate; RT, room temperature; SD, standard delayed sample handling might have on the integrity of the deviation; SPE, solid phase extraction; TCPY, 3,5,6-trichloro-2- biological specimens to be used for measuring environmental pyridinol. analytes. 2. Address all correspondence to: Dr J.A. Hoppin, NIEHS, Epidemiology To assess urine transport options as part of the Norwegian Branch, MD A3-05, PO Box 12233, Research Triangle Park, NC 27709- Mother and Child Cohort Study, a prospective study of 2233, USA. Tel.: þ 1-919-541-7622. Fax: þ 1-919-541-2511. 100,000 pregnant women and their children (for more E-mail: [email protected] Received 22 December 2004; accepted 29 April 2005; published online information see www.fhi.no), we conducted a pilot study to 6 July 2005 evaluate the impact of different urine preservatives on levels Hoppinetal. Urine preservation effects of common environmentalcontaminants or their metabolites spiking each pool are listed in Table 2. In most instances, in urine. We were interested in addressing three issues related these concentrations are within the range of concentrations to selection of shipping container and shipping temperature: normally found in the general US population. In cases where (1) Does the sample preservative influence the ability to the normalUS concentration range is near or belowthe detect a wide range of environmentalcontaminants in urine? method limit of detection, higher concentrations were used to (2) Does the shipping temperature influence the ability to allow us to accurately detect differences among the preserva- accurately measure environmental contaminants in urine? tion methods tested. Standards used for spiking were and (3) Does the time from collection to long-term storage prepared according to the procedures outlined in the influence the urinary concentration of the analytes? analytical methods (Bravo et al., 2002, 2004; Kuklenyik et al., 2003; Silva et al., 2003; Barr et al., 2004; Olsson et al., 2004). Nine to 15 aliquots of each pool were dispensed into Materials and methods each of three urine storage tubes, two containing preserva- tives to prevent bacterialcontamination. We used pooled urine samples to evaluate the impact of Three types of commercially available urine vacutainers sample storage and shipping conditions on environmental (Becton Dickinson) were evaluated: no preservative (10 ml), analytes. We measured environmental contaminants at up to boric acid tube (6 ml) containing boric acid/sodium formate five time points and three different storage temperatures using (lyophilized at bottom of tube; no additive volume listed), mass spectrometry-based analytical methods. Table 1 outlines and chlorhexidine tube (8 ml) containing chlorhexidine, ethyl the elements of our study design. For each analyte, up to 45 paraben, and sodium propionate. The preservatives prevent conditions (3 replicates  3temperatures 5 storage times) bacterialgrowth. At the beginning of the study, 6–10 mlof were evaluated. Three different urine pools were created each pooled urine sample were aliquoted into a urine using urine obtained from severaladultanonymous donors. vacutainer. Urine was in the tube and in contact with the One poolwas spiked with bisphenolA (BPA); one poolwas preservative throughout the duration of the experiment. spiked with phthalate metabolites; and one pool was spiked Three replicate tubes were used for each time point over the with metabolites of nonpersistent pesticides (for the three course of the study. For the BPA analysis, two replicate different pesticide methods). The sample concentrations after tubes were used for each time point, because only a limited number of tubes were available. As sample shipment can occur at a variety of temperatures Ta bl e 1 . Preservative experiment details. (room temperature (RT), on ice, or frozen), we assessed the impact of temperature choice by storing the tubes containing Three urine storage tube types the pooled urine at RT (on the bench in the lab), in the No preservative refrigerator, and in the freezer (for phthalates and BPA Boric acid, sodium formate, sorbital only). Samples were stored at these temperatures throughout Chlorohexidine, ethyl paraben, sodium propionate the duration of the experiment. Three temperatures The time between sample collection and processing by the Room temperature laboratory for long-term storage can vary as a result of a Refrigerator (41C) number of conditions including weekend shipment of Freezera (À201C) samples, weather delays, and other unforeseen circumstances. To determine which analyses were influenced by time since Five time points 0 collection, we analyzed the concentrations of all analytes at 24 h four time points: time 0, 24, 48, and 72 h after collection. 48 h Samples for phthalates and BPA were also analyzed 1 week 72 h after collection. b 1week We used five analytical methods routinely performed at the Five analytical methods NationalCenters for EnvironmentalHealthlaboratory at the BisphenolA Centers for Disease Controland Prevention (CDC) to Phthalates measure organic contaminants in urine, including phthalate Organophosphorus pesticide metabolites metabolites, BPA, organophosphate, carbamate and pyre- Multiclass pesticide method throid insecticide metabolites, chlorinated phenols, herbicide Phenolic
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