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Technical Notes
Quantifying Phthalate Metabolites in Human Meconium and Semen Using Automated Off-Line Solid-Phase Extraction Coupled with On-Line SPE and Isotope-Dilution High-Performance Liquid Chromatography-Tandem Mass Spectrometry Kayoko Kato, Manori J. Silva, Larry L. Needham, and Antonia M. Calafat*
Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, Georgia 30341
We developed an analytical method using off-line solidphase extraction (SPE) coupled with on-line SPE and isotope-dilution high-performance liquid chromatographytandem mass spectrometry (HPLC-MS/MS) to determine the concentrations of phthalate metabolites in human meconium and in semen. First, we used off-line SPE to remove interfering proteins and other biomolecules from the samples. Then, we preconcentrated the phthalate metabolites in the extract using on-line SPE before measuring them by HPLC-MS/MS. For most of the analytes, the limits of detection ranged between 0.2 and 0.7 ng/g for meconium and between 0.3 and 0.7 ng/mL for semen. The recovery after off-line SPE varied for most analytes between 65 and 99% at concentrations ranging from 3.0 to 30.0 ng/mL in semen and between 67 and 103% at concentrations ranging from 2.0 to 10.0 ng/mL in meconium. Precision measured by the relative standard deviation ranged from 3.2 to 19.1% for intraday and from 3.9 to 18.6% for interday. We validated this novel approachswhich is applicable to other biological matrixes, including serum and breast milkson spiked samples and on five meconium samples and one pooled semen sample from people with no known occupational exposure to phthalates. Phthalate esters are a family of multifunctional compounds widely used as plasticizers, solvents, or additives in many diverse products such as poly(vinyl chloride) (PVC) materials, pharmaceuticals and medical devices, pesticides, lubricants, and personal care products. Humans have been exposed to phthalates through the manufacture, ubiquitous use, and disposal of PVC materials and other phthalate-containing products.1 Results from numerous animal studies suggest that some phthalates and their metabolites are reproductive and develop* Corresponding author. Phone: 770-488-7891. Fax: 770-488-4371. E-mail:
[email protected]. (1) Hauser, R.; Calafat, A. M. Occup. Environ. Med. 2005, 62, 806-818. 10.1021/ac0608220 Not subject to U.S. Copyright. Publ. 2006 Am. Chem. Soc.
Published on Web 08/03/2006
mental toxicants2-7 and that fetuses and peripubertal animals are more sensitive to these substances than are adults.8 Data on the relations between human health and phthalate exposure at all life stages are rather limited. Since the early 2000s, researchers have explored possible associations between phthalate exposure and several health outcomes, including altered semen quality, reduced anogenital distance in male infants, and allergic and asthma symptoms in children.9-12 Humans metabolize phthalates to the phthalate monoesters, which can be further metabolized to other oxidative metabolites before being excreted in urine and feces.13-16 Therefore, by measuring phthalate metabolites in biologic matrixes, researchers (2) Gray, L. E.; Ostby, J.; Furr, J.; Price, M.; Veeramachaneni, D. N. R.; Parks, L. Toxicol. Sci. 2000, 58, 350-365. (3) Gray, L. E.; Barlow, N. J.; Furr, J. R.; Brock, J.; Silva, M. J.; Barr, D. B.; Ostby, J. S. Toxicol. Sci. 2003, 72, 283. (4) Parks, L. G.; Ostby, J. S.; Lambright, C. R.; Abbott, B. D.; Klinefelter, G. R.; Barlow, N. J.; Gray, L. E. Toxicol. Sci. 2000, 58, 339-349. (5) Ema, M.; Miyawaki, E. Reprod. Toxicol. 2002, 16, 71-76. (6) Ema, M.; Miyawaki, E.; Hirose, A.; Kamata, E. Reprod. Toxicol. 2003, 17, 407-412. (7) Ema, M.; Miyawaki, E. Toxicol. Sci. 2003, 72, 274. (8) Sjoberg, P.; Lindqvist, N. G.; Ploen, L. Environ. Health Perspect. 1986, 65, 237-242. (9) Bornehag, C. G.; Sundell, J.; Weschler, C. J.; Sigsgaard, T.; Lundgren, B.; Hasselgren, M.; Hagerhed-Engman, L. Environ. Health Perspect. 2004, 112, 1393-1397. (10) Duty, S. M.; Silva, M. J.; Barr, D. B.; Brock, J. W.; Ryan, L.; Chen, Z. Y.; Herrick, R. F.; Christiani, D. C.; Hauser, R. Epidemiology 2003, 14, 269277. (11) Jonsson, B. A. G.; Richthoff, J.; Rylander, L.; Giwercman, A.; Hagmar, L. Epidemiology 2005, 16, 487-493. (12) Swan, S. H.; Main, K. M.; Liu, F.; Stewart, S. L.; Kruse, R. L.; Calafat, A. M.; Mao, C. S.; Redmon, J. B.; Ternand, C. L.; Sullivan, S.; Teague, J. L. Environ. Health Perspect. 2005, 113, 1056-1061. (13) ATSDR. Toxicological Profile for Diethyl Phthalate (DEP); Agency for Toxic Substances and Disease Registry: Atlanta, GA, 1995; (http://www.atsdr.cdc.gov/toxprofiles/tp73.html). (14) ATSDR. Toxicological Profile for Di-n-octyl Phthalate (DNOP); Agency for Toxic Substances and Disease Registry: Atlanta, GA, 1997; (http:// www.atsdr.cdc.gov/toxprofiles/tp95.html). (15) ATSDR. Toxicological Profile for Di-n-butyl Phthalate (DBP); Agency for Toxic Substances and Disease Registry: Atlanta, GA, 2001; (http:// www.atsdr.cdc.gov/toxprofiles/tp135.html).
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can obtain information on the bioavailability of phthalates for exposure assessment.17,18 Although urine is the most common matrix for assessing exposure to phthalates, other matrixes have also been used, including serum, breast milk, amniotic fluid, and saliva.19-25 As concerns arise about exposure to phthalates during critical periods of human development, assessing phthalate exposure using biologic matrixes other than urine may become increasingly desirable, especially for assessing exposure of potentially vulnerable populations or specific population groups. Meconium, the digested residue of swallowed amniotic fluid, begins to accumulate in the human fetus at approximately week 16 of gestation and is not excreted until after delivery. Meconium often is used to detect fetal exposure to common drugs of abuse and other xenobiotics, suggesting that meconium can be a valid dosimeter of prenatal exposure to these compounds.26-37 Similarly, concentrations of phthalate metabolites in meconium may represent the cumulative exposure to phthalates during gestation. Semen is a mixture of prostatic fluid, seminal vesicle fluid, and spermatozoa. Pharmaceuticals and some environmental chemicals have been measured in semen.38,39 Previous studies point to potential associations between the urinary concentrations of some phthalate metabolites and both semen quality and DNA damage (16) ATSDR. Toxicological Profile for Di(2-ethylhexyl) phthalate (DEHP); Agency for Toxic Substances and Disease Registry: Atlanta, GA, 2002; (http:// www.atsdr.cdc.gov/toxprofiles/tp9.html). (17) Koch, H. M.; Drexler, H.; Angerer, J. Int. J. Hyg. Environ. Health 2003, 206, 1-7. (18) Silva, M. J.; Barr, D. B.; Reidy, J. A.; Malek, N. A.; Hodge, C. C.; Caudill, S. P.; Brock, J. W.; Needham, L. L.; Calafat, A. M. Environ. Health Perspect. 2004, 112, 331-338. (19) Kato, K.; Silva, M. J.; Brock, J. W.; Reidy, J. A.; Malek, N. A.; Hodge, C. C.; Nakazawa, H.; Needham, L. L.; Barr, D. B. J. Anal. Toxicol. 2003, 27, 284289. (20) Koch, H. M.; Bolt, H. M.; Preuss, R.; Angerer, J. Arch. Toxicol. 2005, 79, 367-376. (21) Koch, H. M.; Angerer, J.; Drexler, H.; Eckstein, R.; Weisbach, V. Int. J. Hyg. Environ. Health 2005, 208, 489-498. (22) Silva, M. J.; Reidy, J. A.; Herbert, A. R.; Preau, J. L.; Needham, L. L.; Calafat, A. M. Bull. Environ. Contam. Toxicol. 2004, 72, 1226-1231. (23) Silva, M. J.; Samandar, E.; Preau, J. L.; Reidy, J. A.; Needham, L. L.; Calafat, A. M. J. Anal. Toxicol. 2005, 29, 819-824. (24) Silva, M. J.; Reidy, J. A.; Samandar, E.; Herbert, A. R.; Needham, L. L.; Calafat, A. M. Arch. Toxicol. 2005, 79, 647-652. (25) Calafat, A. M.; Slakman, A. R.; Silva, M. J.; Herbert, A. R.; Needham, L. L. J. Chromatogr., B 2004, 805, 49-56. (26) Bearer, C. F.; Santiago, L. M.; O’Riordan, M. A.; Buck, K.; Lei, S. C.; Singer, L. T. J. Pediatr. 2005, 146, 824-830. (27) Bielawski, D.; Ostrea, E.; Posecion, N.; Corrion, M.; Seagraves, J. Chromatographia 2005, 62, 623-629. (28) Coles, R.; Clements, T. T.; Nelson, G. J.; McMillin, G. A.; Urry, F. M. J. Anal. Toxicol. 2005, 29, 522-527. (29) Pichini, S.; Puig, C.; Zuccaro, P.; Marchei, E.; Pellegrini, M.; Murillo, J.; Vall, O.; Pacifici, R.; Garcia-Algar, S. Forensic Sci. Int. 2005, 153, 59-65. (30) Pichini, S.; Pacifici, R.; Pellegrini, M.; Marchei, E.; Perez-Alarcon, E.; Puig, C.; Vall, O.; Garcia-Algar, O. J. Chromatogr., B 2003, 794, 281-292. (31) Pichini, S.; Pacifici, R.; Pellegrini, M.; Marchei, E.; Lozano, J.; Murillo, J.; Vall, O.; Garcia-Algar, O. Anal. Chem. 2004, 76, 2124-2132. (32) Whyatt, R. M.; Barr, D. B. Environ. Health Perspect. 2001, 109, 417-420. (33) Moore, C.; Negrusz, A.; Lewis, D. J. Chromatogr., B 1998, 713, 137-146. (34) Moore, C. M.; Lewis, D. E.; Leikin, J. B. J. Forensic Sci. 1996, 41, 10571059. (35) Le, N. L.; Reiter, A.; Tomlinson, K.; Jones, J.; Moore, C. J. Anal. Toxicol. 2005, 29, 54-57. (36) Chan, D.; Caprara, D.; Blanchette, P.; Klein, J.; Koren, G. Clin. Biochem. 2004, 37, 429-438. (37) Gareri, J.; Klein, J.; Koren, G. Clin. Chim. Acta 2006, 366, 101-111. (38) Pichini, S.; Zuccaro, P.; Pacifici, R. Clin. Pharm. 1994, 26, 356-373. (39) Klemmt, L.; Scialli, A. R. Birth Defects Res. Part B-Dev. Reprod. Toxicol. 2005, 74, 119-131.
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in sperm.11,40,41 However, no data exist on the potential relation between an internal measure of phthalate exposure, estimated from the urinary concentrations of phthalate metabolites, and target organ dose, estimated from the concentrations of these same metabolites in semen. No published methods exist for measuring phthalate metabolites in meconium or semen. Here we present methods of measuring phthalate metabolites in these biologic matrixes. Our analytical approach, which also could be used for measuring phthalate metabolites in other biomatrixes, such as serum and breast milk, involves off-line solid-phase extraction (SPE), followed by on-line SPE and tandem mass spectrometric analysis. MATERIALS AND METHODS Reagents and Standards. We purchased the following from Cambridge Isotope Laboratories, Inc. (Andover, MA): monomethyl phthalate (MMP), monoethyl phthalate (MEP), mono-nbutyl phthalate (MBP), monocyclohexyl phthalate (MCHP), monobenzyl phthalate (MBzP), mono(2-ethylhexyl) phthalate (MEHP), mono(2-ethyl-5-oxohexyl) phthalate (MEOHP), mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono-n-octyl phthalate (MOP), mono-3-methyl-5-dimethylhexyl phthalate (monoisononyl phthalate, MNP), and mono-3-methyl-7-methyloctyl phthalate (monoisodecyl phthalate, MDP; >99.9%) and their 13Clabeled internal standards (>99.9%); 13C4-4-methylumbelliferone; D4-dimethyl phthalate (D4-DMP); D4-diethyl phthalate (D4-DEP); D4-dibutyl phthalate (D4-DBP); and D4-bis(2-ethylhexyl) phthalate (D4-DEHP). We obtained mono(3-carboxypropyl) phthalate (MCPP) and 13C4-MCPP from Los Alamos National Laboratory (Los Alamos, NM). We purchased 4-methylumbelliferone and its glucuronide, ammonium acetate (>98%), and acetic acid (glacial) from Sigma-Aldrich (St. Louis, MO). Monoisobutyl phthalate (MiBP), D4-MiBP, mono(2-ethyl-5-carboxypentyl) phthalate (MECPP), and D4-MECPP were gifts from Professor Ju¨rgen Angerer (University of Erlangen-Nuremberg, Germany). We purchased acetonitrile, methanol, and water (HPLC grade) from Tedia (Fairfield, OH); ammonium hydroxide (30%), sodium dihydrogen phosphate, phosphoric acid, and sodium hydroxide from J. T. Baker (Phillipsburg, NJ); β-glucuronidase (Escherichia coli-K12) from Roche Biomedical (Mannheim, Germany); and human pooled meconium collected in 2003 from Battelle (Columbus, OH) for method validation and preparation of quality control (QC) materials. Goat meconium, used for method development, was a donation from Ms. Joyce Rodriguez (CDC, Atlanta, GA). The semen we used for method development and validation was pooled from residual specimens collected for an ongoing study with Dr. Russ Hauser (Harvard School of Public Health, Boston, MA). We purchased bovine serum from Invitrogen Corp. (Carlsbad, CA). Sample Preparation. Meconium. We measured ∼0.5 g of meconium into a 10-mL centrifuge tube (Nalge Nunc International) and added 3 mL of H3PO4 (100 mM) to denature enzymes and to free adsorbed phthalate metabolites from the solid matrix. Using a VX2500 multitube vortexer (VWR International, West Chester, PA), we vortex mixed the contents of the centrifuge tube for 20 min. We then added NaOH (1 M, 0.3 mL) to adjust the pH (40) Hauser, R.; Williams, P.; Altshul, L.; Calafat, A. M. Environ. Health Perspect. 2005, 113, 425-430. (41) Duty, S. M.; Ackerman, R. M.; Calafat, A. M.; Hauser, R. Environ. Health Perspect. 2005, 113, 1530-1535.
to 6.5, followed by solutions of internal standard (0.1 mL) and β-glucuronidase (5 µL in 0.25 mL of 1 M NH4Ac buffer, pH 6.5). The spiked meconium was incubated at 37 °C for 90 min. We added formic acid (100 mM, 2 mL) to the samples after incubation. The meconium suspension was loaded into an Avanti J-25I centrifuge (Beckman Coulter, Fullerton, CA) and centrifuged for 60 min at 25 000 rpm. The supernatant was decanted into a borosilicate glass tube for off-line SPE. Semen. We acidified semen (0.3 mL) in a 10-mL glass tube by adding H3PO4 (1 M, 0.04 mL) to denature enzymes present in the matrix and vortex mixed it. To adjust the pH to 6.5, we added NaOH (1 M, 0.06 mL) followed by an internal standard solution containing isotope-labeled analogues of the analytes (0.1 mL) and β-glucuronidase solution (5 µL in 0.25 mL of 1 M NH4Ac buffer, pH 6.5). The sample was vortex mixed for 1 min in a VX2500 multitube vortexer and incubated at 37 °C for 90 min. We added formic acid (100 mM, 2 mL) to the samples after incubation and before off-line automated SPE. An analogous procedure could be used for breast milk (1 mL) and serum (0.5 mL). Off-Line Automated SPE. We extracted the phthalate metabolites on a Rapid Trace automated SPE system (Zymark Corp., Hopkinton, MA) using Oasis HLB 60 mg/3 mL SPE cartridges (Waters Corp., Milford, MA). The cartridges were conditioned with 2 mL of methanol and 2 mL of water. The sample solution was loaded onto the SPE cartridge at a rate of 1 mL/min. The cartridge was rinsed with 0.1 M formic acid (2 mL), 40% aqueous methanol (3 mL), and water (3 mL) at 1 mL/min and air-dried for 0.5 min. The analytes were eluted with acetonitrile (0.5 mL) at 1 mL/min, and the eluate was concentrated to dryness under a stream of dry nitrogen (UHP grade) in a Turbovap evaporator (Zymark Corp.) for 15 min at 55 °C. Subsequently, we suspended the residue in 0.5 mL of 0.1% acetic acid in water, transferred it to a 1.5-mL high-recovery screw-cap glass autosampler vial (Agilent, Palo Alto, CA), and placed it on the HPLC autosampler for analysis. Reagent blanks were prepared with water. Reagent blanks and QC materials were processed using the same procedure. On-Line SPE-HPLC-MS/MS. Our previously developed method of on-line sample preconcentration and cleanup for urine was adopted.42 The on-line SPE-HPLC-MS/MS system was built on a ThermoFinnigan Surveyor liquid chromatograph (ThermoFinnigan, San Jose, CA) coupled with a ThermoFinnigan TSQ Quantum triple-quadrupole mass spectrometer equipped with an electrospray ionization interface (ESI), a ThermoFinnigan Surveyor LC pump, and a 10-port switching valve (Rheodyne MX7960, Rehnert Park, CA). The ThermoFinnigan Surveyor liquid chromatograph, including the switching valve and Surveyor LC pump, was controlled by the ThermoFinnigan Xcalibur software. We used ESI in the negative ion mode to form negatively charged analyte ions at the interface under the following fixed instrument settings: spray ion voltage, -3800 V; sheath gas (N2) pressure, 25 arbitrary units; auxiliary gas (N2) pressure, 10 arbitrary units; capillary temperature, 280 °C; tube lens voltage, -140 V; collision gas (Ar) pressure, 1.5 mTorr. Ionization parameters and collision cell parameters were optimized for each analyte. The source collision-induced dissociation voltage was set to 10 V to breakdown acetate clusters. Unit resolution was used (42) Kato, K.; Silva, M. J.; Needham, L. L.; Calafat, A. M. Anal. Chem. 2005, 77, 2985-2991.
for both Q1 and Q3 quadrupoles. The mass spectrometer was set in selective reaction monitoring mode. Two precursor/product ion combinations (one for quantification and one for confirmation) were monitored for each analyte; one precursor/product ion combination for quantification was monitored for the isotopelabeled analytes (see Table S1, Supporting Information). Data Analysis. For data acquisition and analysis, we used a program created with the Xcalibur software on a PC-based data system. The data analysis program automatically selected and integrated each ion of interest in the chromatogram. The identity of the phthalate metabolites was confirmed by matching retention times with the isotope-labeled internal standards. We manually corrected the peak integrations, if necessary. A response factor (RF), calculated as the peak area of each analyte ion divided by the peak area of its isotope-labeled standard, was used for quantification. For increased selectivity, we determined the ratio of the peak area of the quantification ion to the confirmation ion for each analyte. We used 10 standard analyte concentrations encompassing the entire linear range of the method (0.1-800 ng/ mL, depending on the analyte) to construct daily calibration curves, weighted by the reciprocal of the standard amount (1/x), of RF versus the standard amount. To monitor sensitivity changes, we injected the full set of standards once before and once after all samples (e.g., QCs, blanks, unknowns). The calibration curves were linear over 3 orders of magnitude and had correlation coefficients exceeding 0.99. Because the slopes of the calibration curves were very similar regardless of the matrix used (water, semen, or meconium), we used calibration curves obtained from water for quantification. We saved the calibration data, along with the integrated peak areas for each analyte and retention times, in a Microsoft Excel file, exported the data to a Microsoft Access database, and statistically analyzed the data using SAS statistical software (SAS Institute, Cary, NC). Column Regeneration. Using a ThermoFinnigan Spectra system P1000 pump, we regenerated Chromolith Flash RP-18e (4.6 mm × 25 mm, Merck KGaA) SPE and Betasil Phenyl (3 µm, 2.1 × 150 mm; ThermoElectron Corp., Bellefonte, PA) HPLC columns by pumping 60 units/mL proteinase K solution (0.2 mg/ mL in water, Fermentas Life Science, Hanover, MD) at 1.0 mL/ min for 5 min (Chromolith Flash RP-18e) or at 0.3 mL/min for 10 min (Betasil Phenyl). Then, we incubated the columns in a water bath for 20 min at 60 °C. After incubation, the columns were washed with water (1.0 mL/min for 5 min for Chromolith Flash RP-18e and 0.3 mL/min for 10 min for Betasil Phenyl) and acetonitrile and capped until use. Analysis of Semen and Meconium for Endogenous Esterase Activity. We added a methanol solution containing D4-DMP, D4-DEP, D4-DBP, and D4-DEHP (0.1 mL, 4000 ng/mL each) to 1 mL of semen and 1 g of meconium. The spiked meconium or semen was incubated at 37 °C. Samples were processed as described previously and analyzed for D4-MMP, D4-MEP, D4-MBP, and D4-MEHP. Preparation of QC Materials. Because of the limited amount of semen available, we used bovine serum QCs for analyses of semen samples. Bovine serum was pooled to create QC low (QCL, 0.5-10.0 ng/mL) and QC high (QCH, 2.5-40 ng/mL) pools. Phosphoric acid (0.2 M) was added to the QC pools, and additional phthalate metabolite standards were added as needed to achieve Analytical Chemistry, Vol. 78, No. 18, September 15, 2006
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Figure 1. Time course of phthalate diester hydrolysis by endogenous esterases in meconium and semen.
the desired concentrations. We mixed the pools uniformly, dispensed them into prerinsed glass vials, and stored them at -70 °C until use. QC characterization involved 60 discrete measurements for each analyte on 10 separate days. To prepare the meconium QC pools, we made a paste by adding 1:6 water (by weight) to pooled human meconium. We added phosphoric acid (0.1 M) and mixed the acidified meconium paste in a commercial blender to obtain a homogeneous mixture. The paste was divided among two beakers to create two pools. Each pool was fortified with additional phthalate metabolites as needed, reblended, and stored in prerinsed glass vials at -70 °C until use. QC characterization involved 60 discrete measurements for each analyte on 10 separate days. RESULTS AND DISCUSSION We used concentrations of D4-MMP, D4-MBP, D4-MEP, and D4-MEHP to determine the endogenous esterase activity in semen and meconium that had been spiked with D4-DMP, D4-DBP, D4DEP, and D4-DEHP, respectively, and incubated for 90 min at 37 °C. In meconium, after 90 min the extent of hydrolysis was 20% for D4-DMP, 10% for D4-DEP and D4-DBP, and 5% for D4-DEHP (Figure 1). In semen, the percentage of hydrolyzed diesters was greater than 65% for D4-DMP, greater than 35% for D4-DEP, 30% for D4-DBP, and ∼10% for D4-DEHP. The differences observed in the extent of diester hydrolysis between the two matrixes suggest that the enzyme activity could be specific for the chemical composition of each matrix, although other unknown reasons cannot be ruled out. Furthermore, these data suggest that meconium and semen, like serum19 and milk,25 contain esterases capable of hydrolyzing phthalate diesters into monoesters. Therefore, to minimize the potential contamination with phthalate diesters during sampling, storage, or analysis, meconium or semen must be treated to deactivate the enzymes after collection, or specimens need to be collected in phthalate-free containers. Of interest, some phthalate metabolites, specifically the oxidative metabolites such as MEHHP, MEOHP, MECPP, and MCPP cannot be formed as a result of the hydrolysis of the diester phthalate by esterases. Oxidative metabolism involves additional 6654
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enzymes.14,16 Therefore, concentrations of these oxidative metabolites in meconium or semen are a more selective measure of phthalate diester exposure than the hydrolytic monoester metabolites (formed by the hydrolysis of the phthalate diesters).13-16 The biological matrixes used in this study are complex. Therefore, trace analysis of phthalate metabolites and other environmental chemicals is challenging because endogenous matrix biomolecules may affect the extraction and the chromatographic separation of the analytes of interest. Our automated off-line SPE procedure likely minimizes potential matrix effects for three reasons. First, because of the relatively large particle size (30 µm) of the OASIS HLB copolymer solvent, large molecules (including proteins) are not retained. Second, diluting the sample with formic acid disrupts the interactions of the phthalate metabolites with the matrix macromolecules; thus, the analytes of interest are retained by the SPE polymer whereas large molecules are lost in the breakthrough. Third, the thorough washing of the SPE cartridge with 2 mL of 0.1 M formic acid, 3 mL of 40% aqueous methanol, and 3 mL of water will minimize major interferences in the extract. We also devised a column cleanup protocol using a solution of proteinase that permitted reusing the on-line SPE columns and substantially increasing the life of the HPLC columns. With this treatment, one SPE column could be used for more than 200 injections of semen samples and for 40 injections of meconium samples. Similarly, one HPLC column could be used for the injection of 500 semen samples and 100 meconium samples. We found that the regeneration of the SPE and HPLC columns and daily maintenance of the ion-transfer tube of the mass spectrometer were instrumental for the optimal performance of the method. The analytical limits of detection (LODs) for each analyte were determined as 3S0, where S0 is the standard deviation as the concentration approaches zero.43 S0, determined from five to seven repeated measurements of the lowest five standards, was the y-intercept of the best-fit line of a plot of the standard deviation of these measurements versus the standard concentration. LODs (43) Taylor, J. K. Quality Assurance of Chemical Measurements; Lewis Publishers: Chelsea, MI, 1987.
Table 1. Off-Line Solid-Phase Extraction Recoveries, Their RSD, and Limits of Detection of Phthalate Metabolites in Semen and Meconiuma semen
Table 2. Interday and Intraday Precision (Expressed as RSD) of Measurements of Phthalate Metabolite Concentrations in Spiked QC Poolsa
meconium
meconium
% recovery/% RSD %
recoveryd/
metabolite
low concnb
high concnc
LOD
% RSD
LOD
MCPP MMP MEP MECPP MiBP MBP MEOHP MEHHP MCHP MBzP MEHP MOP MNP MDP
98.7/6.0 92.5/8.1 97.1/5.8 82.7/4.1 82.1/3.3 90.9/3.6 82.1/3.4 88.3/6.5 89.5/7.4 83.8/9.2 79.5/8.3 59.6/7.5 48.3/7.8 49.6/6.0
82.0/1.3 76.5/3.9 90.7/6.3 93.9/6.6 88.5/7.2 74.4/8.0 75.6/4.2 87.4/3.6 74.5/4.6 68.1/1.3 65.3/7.7 57.3/2.2 46.0/4.6 50.3/1.8
0.4 0.5 0.7 0.5 0.3 0.5 0.5 0.5 0.3 0.5 0.6 0.5 0.8 0.9
67.0/10.6 100.6/14.3 98.8/10.1 85.7/10.8 68.9/11.6 75.2/8.6 102.5/13.2 86.1/7.6 87.9/7.1 103.1/6.1 79.8/6.9 85.4/5.9 79.8/8.2 75.3/4.9
0.3 0.2 0.3 0.5 0.3 0.3 0.7 0.6 0.3 0.3 0.6 1.2 1.5 2.3
a Note: Off-line solid-phase extraction recoveries were obtained from quintuplicate measurements at each concentration. b Concentrations ranged from 3.0 to 7.0 ng/mL, depending on the analyte. c Concentrations ranged from 10.0 to 30.0 ng/mL, depending on the analyte. d Concentrations ranged from 2.0 to 10.0 ng/mL, depending on the analyte.
were less than 0.7 ng/mL for semen and less than 0.7 ng/g for meconium for most analytes (Table 1). The LODs for MINP and MIDP, the metabolites with the highest number of carbons in the alkyl chain, were somewhat higher than for the rest of analytes, especially in meconium (Table 1). We obtained off-line SPE recoveries for each analyte, expressed as a percentage of the expected value, from quintuplicate measurements at two concentrations. The value was calculated as RFb/RFa, where RF is the analyte response factor obtained from spiking the sample with phthalate metabolites (before the centrifugation step for meconium) before (RFb) and after (RFa) SPE. In semen, SPE recoveries (RSD) ranged from 80 to 99% (3.39.2%) for relatively low-level concentrations (3.0-7.0 ng/mL), and from 65 to 94% (1.3-8.0%) for relatively high-level concentrations (10.0-30.0 ng/mL). In meconium, SPE recoveries (RSD) ranged from 67 to 103% (4.9-14.3%) at concentrations of 2.0-10.0 ng/ mL (Table 1). We used pooled meconium and semen for these determinations, and variations in SPE recovery when applying the method to individual specimens cannot be ruled out. Interday precision, calculated as RSD of 50 repeated measurements over a 10-day period, ranged from 3.9 to 18.6%. Similarly, intraday precision, estimated from five repeated measurements, ranged from 3.2 to 19.1% (Table 2). These values indicate the good reproducibility of this trace analysis method. Although we previously developed analytical methods to measure phthalate metabolites in serum and breast milk,19,23,25 because the concentration of phthalate metabolites in these matrixes is relatively low, we needed new methods with increased sensitivity. Our new analytical approach also can be applied to serum and breast milk (Tables S2 and S3, Supporting Information), with resulting improved analytical sensitivity for most of the analytes. We measured the concentrations of phthalate monoesters in five meconium samples collected from five babies born during
serumb
metabolite
intraday QCL/QCH
interday QCL/QCH
intraday QCL/QCH
interday QCL/QCH
MCPP MMP MEP MiBP MBP MEOHP MEHHP MBzP MEHP
12.8/10.5 3.2/6.2 8.5/4.7 7.0/13.9 12.7/12.7 12.7/4.4 13.7/10.4 8.4/11.3 6.4/9.5
6.0/5.6 9.1/6.4 10.3/4.3 13.2/10.8 11.0/15.2 3.9/4.1 6.1/6.0 10.9/12.6 7.9/6.4
8.2/7.0 14.4/4.9 9.4/7.6 19.1/17.3 7.1/12.0 7.3/3.9 6.3/6.0 11.3/12.8 10.2/7.3
18.6/12.8 10.6/8.9 11.3/9.8 18.3/17.6 12.7/18.6 15.2/12.6 16.2/11.8 14.5/13.3 16.8/12.9
a Concentrations ranged from 0.5 to 10.0 ng/mL (QCL) and 2.5 to 40 ng/mL (QCH), depending on the analyte. N ) 5 (for intraday precision); N ) 50 (for interday precision) b Bovine serum was used for semen analysis.
2004 and 2005 in Atlanta, GA, and in one pooled semen sample. No personal identifiers were available for any of the samples analyzed. We detected MECPP, an oxidative metabolite of MEHP, in all meconium samples analyzed and in the pooled semen sample; other phthalate metabolites were also detected (Table S4, Figure S1, Supporting Information). These data confirm the validity of our method for assessing environmental exposure to phthalates using meconium and semen. ACKNOWLEDGMENT This research was supported in part by an appointment (K.K.) to the Research Participation Program at the Centers for Disease Control and Prevention (CDC), National Center for Environmental Health, Division of Laboratory Sciences. The appointment was administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and CDC. The use of trade names is for identification only and does not constitute endorsement by the U.S. Department of Health and Human Services or the Centers for Disease Control and Prevention. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of CDC. SUPPORTING INFORMATION AVAILABLE Table detailing the precursor and product ion transitions of the phthalate metabolites and their isotope-labeled analogues, collision energies, and retention times; table showing the off-line SPE recoveries, their RSD percent, and LODs of phthalate metabolites in breast milk and serum; table detailing interday and intraday precision (expressed as the RSD) of measurements of phthalate metabolite concentrations in spiked QC breast milk pools; table showing the concentrations of selected phthalate monoesters in meconium and semen; and figure including the chromatograms of samples of human meconium and semen. This material is available free of charge via the Internet at http:// pubs.acs.org. Received for review May 3, 2006. Accepted June 22, 2006. AC0608220 Analytical Chemistry, Vol. 78, No. 18, September 15, 2006
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