Determination of Seven Urinary Metabolites of Organophosphate Esters using Liquid Chromatography-Tandem Mass Spectrometry

CHINESE JOURNAL OF ANALYTICAL CHEMISTRY Volume 45, Issue 11, November 2017 Online English edition of the Chinese language journal

Cite this article as: Chin J Anal Chem, 2017, 45(11), 1648–1654.

RESEARCH PAPER

Determination of Seven Urinary Metabolites of Organophosphate Esters using Liquid ChromatographyTandem Mass Spectrometry LI Pei1,2, ZENG Xiang-Ying1, CUI Jun-Tao1,2, ZHAO Ling-Juan1, YU Zhi-Qiang1,* 1

State Key Laboratory of Organic Geochemistry, Guangdong Key Laboratory of Environment and Resources, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou 510640, China 2 University of Chinese Academy of Sciences, Beijing 100049, China

Abstract: A simple method was developed for simultaneous determination of seven urinary metabolites of organophosphate esters (OPs) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Based on different physical and chemical properties of these OP metabolites, their enrichment and clean-up were performed through solid phase extraction to obtain high-efficient solid phase extraction cartridges, and the washing and elution conditions were optimized. At the same time, the kinetic parameters and mass spectrometry parameters were investigated for getting the qualitative and quantitative characteristic ion pairs for analysis of each metabolites. The results showed that the Oasis WAX solid phase extraction cartridge was suitable for sample enrichment and clean-up, and the optimal elution solvents were 2 mL of 5% ammonia in methanol and 2 mL of methanol. The recoveries of six analytes ranged from 60.5 to 104.0%, whereas the recovery of diethyl phosphate ranged from 17.8% to 36.2%. The complete baseline separations of seven analytes were achieved under optimized chromatographic conditions. The limits of detection and limits of quantification of the seven analytes ranged from 0.005 to 0.2 μg L‒1 and 0.02 to 0.5 μg L‒1, respectively. The intra-day and inter-day precision results (RSD ≤ 15.4%) showed that this method had good stability and reproducibility. This method was subsequently used to determine OP metabolites in 10 urine samples from the general population in Guangzhou city. The concentrations of the seven OP metabolites in urine samples ranged from 0.5 to 6.7 μg L‒1. Key Words:

1

Liquid chromatography-tandem mass spectrometry; Organophosphate esters; Urine; Metabolites

Introduction

Due to the gradual prohibition of brominated flame retardants, organophosphate esters (OPs) have been widely used as substitutes, and the production and consumption of OPs have rapidly increased[1]. OPs are used as additives inflame retardants, plasticizers and anti-foaming agents in a variety of industries, including construction, textile, electronics, chemical and petroleum industry[2]. It has been reported that OPs have become a global environmental pollutant and are distributed in various environmental matrices,

such as air, water, dust, soil, sediment and biota samples[3]. Toxicological studies have verified that OPs could induce neurotoxicity, carcinogenicity, teratogenicity and endocrine disrupting effects. Given the wide distribution of OPs and their potential human health effects[4,5], it is necessary to monitor human exposure levels to OPs and provide basic data for human health risk assessment. According to the results of in vivo and in vitro studies, OPs are absorbed and rapidly metabolized into their dialkyl and diaryl phosphate analogs, and OP diesters are the major detected metabolites[6,7], which are mainly excreted through

________________________ Received 21 June 2017; accepted 19 July 2017 *Corresponding author. Email: zhiqiang@ gig.ac.cn This work was supported by the National Natural Science Funds for Distinguished Young Scholars of China (No. 41225013), and the Strategic Priority Research Program of the Chinese Academy of Sciences (No. XDB14010202). This is contribution No. IS-2448 from GIGCAS. Copyright © 2017, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(17)61048-X

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urine after metabolism in human body. Therefore, determination of OP diesters in human urine is an important method for assessing human exposure levels to OPs. In general, most studies have focused on the establishment and improvement of analytical methods for urinary OP metabolites, and few studies have investigated human exposure to OPs. To date, two major instrumental analytical methods have been used to determine OP diesters. One is gas chromatography coupled to tandem mass spectrometry (GC-MS/MS). Due to the polarity of OP diesters, these target compounds require derivatization. For example, Schindler et al[8,9] used solid phase extraction (SPE) for the initial enrichment and clean-up of urine sample, and then the target OP diesters were derivatized with pentafluorobenzylbromide, and further clean-up through SPE. The extracts were subsequently analyzed by GC-MS/MS. The pretreatment procedure of this method was complicated, and thus was unsuitable for large-scale epidemiological investigations. Another analytical method is liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Su et al[10] developed a highly sensitive method based on ultra-high pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) to determine OP diesters in human urine. Four non-chlorinated OP diesters were directly determined using UHPLC-electrospray (−)-MS/MS, while UHPLC-ESI (+)MS/MS was used to determine 3 chlorinated OP diesters following methylation using diazomethane. The sensitivity for determination of OPs metabolites was significantly increased. However, due to the complicated matrix effects of urine, the derivatization reaction requires a higher level of technology to ensure its repeatability. Cequier et al[11] reported a highthroughput method based on ultra-performance LC coupled with time-of-flight mass spectrometry. The urine samples did not require pretreatment and were directly injected into the instrument. The entire analysis took less than 3 min; however, the method suffered from severe matrix effects and their limits of detection were high. In addition, van den Eede et al[12] and Cooper et al[13]also reported a rapid method based on SPE and LC-MS/MS for simultaneous determination of six urinary OP diesters. Urine samples were enriched by SPE using a weak mixed anion exchange sorbent and directly analyzed by LC-MS/MS. To date, these aforementioned methods satisfied the basic requirements for OP diester analysis, but had some disadvantages, such as severe matrix effects, higher limit of detection and few OP diesters were detected. Therefore, in the present study, based on the environmental investigation of OPs in China[14,15], seven OP metabolites in human urine were selected as target compounds. A simple and rapid method was established by optimization of SPE cartridges, sample clean-up and instrumental parameters.

2 2.1

Experimental Instruments and reagents

An Agilent 1100 series liquid chromatograph (Agilent Technologies, Palo Alto, CA, USA) coupled to API4000 triple quadrupole mass spectrometer (Applied Biosystems, Foster City, CA, USA) and a Heraeus™ Labofuge™ 200 centrifuge (Thermo Fisher Scientific, Dreieich, Germany) were used in this study. A solid phase extraction device with 12 holes was obtained from Supeclo (Bethlehem, PA, USA). A Milli-Q Unique-R10 water purification system was obtained from Research Scientific Instruments Co. (Millipore, USA). Oasis WAX Extraction Cartridges (60 mg, 3 mL) were obtained from Waters company (Milford, MA, USA). For standards used in this study, diethyl phosphate (DEP) and dibutyl phosphate (DBP) were purchased from Chem Service Inc. (West Chester, PA, USA). Diphenyl phosphate (DPhP), bis(2-chloroethyl) phosphate (BCEP), bis(1-chloro-2propyl) phosphate (BCPP), D10-DPhP, D8-BCEP and D12-BCPP were purchased from Toronto Research Chemicals Inc. (Toronto, Ontario, Canada). Bis(1,3-dichloro-2-propyl) phosphate(BDCPP) was purchased from Wellington Laboratories (Guelph, Canada). Dibenzyl phosphate (DTP) was obtained from Tokyo Chemical Industry, Japan. Methanol (LC grade) was obtained from Merck (Darmstadt, Germany). Acetic acid (LC grade) was obtained from Tedia Company (Fairfield, OH, USA). Ammonia (28%‒30%) and anhydrous sodium acetate (analytical grade) were obtained from Anpel (Shanghai, China). 2.2

Urine sample collection

All urine samples were collected between October 2014 and July 2015 from the Center for Reproductive Medicine of Nanfang Hospital, Southern Medical University, China. All participants were informed about this study and provided a signed informed consent. In this study, ten urine samples were analyzed for method validation. Urine was collected in clean polyethylene plastic bottles and stored at -80oC until analysis. In addition, fifty volunteers from our laboratory were recruited and approximately 10 mL urine was collected from each volunteer. These urine samples from volunteers were blended to form a mixed urine matrix for method optimization. 2.3

Urine sample pretreatment

Urine samples were thawed and centrifuged at 3000 r/min for 10 min. Two milliliters of supernatant were placed in a glass tube and 10 ng of surrogates (D10-DPhP, D8-BCEP and D12-BCPP) were added. Urine pH was adjusted to 5.0 with 200 μL 0.1 M sodium acetate buffer. The urine was homogenized and placed in the dark for 6‒8 h. Before sample extraction, the SPE column was conditioned with 2 mL of methanol, 2 mL of 5% ammonia in methanol and 3 mL of sodium acetate buffer. After urine sample was loaded, the SPE column was washed with 2 mL of 30% methanol in water (pH

LI Pei et al. / Chinese Journal of Analytical Chemistry, 2017, 45(11): 1648–1654

was adjusted to 5.0 with buffer) and dried with nitrogen. The analytes were then eluted with 2 mL of 5% ammonia in methanol and 2 mL of methanol. The extracts were evaporated until dry, and re-dissolved in 200 μL of 50% methanol in water until instrumental analysis. 2.4

Instrumental analysis

An Agilent Zorbax SB-C18 (250 mm × 4.6 mm, 5 μm) was used as the separation column. 10 mM of ammonium formate buffer (pH = 9.2) (A), methanol (B) and acetonitrile (C) were used as mobile phases. Flow rate was 0.4 mL min‒1 and the column temperature was set to 35 oC and maintained during analysis. A gradient elution was used as shown in Table 1. An API 4000 triple quadrupole mass spectrometer (Applied Biosystems, Foster City, CA, USA) was used to determine OP diesters. Electrospray ionization negative ion mode with multiple reaction monitoring (MRM) was used. The mass parameters were as follows: ionization voltage ‒4500 V, source temperature 450 oC, nebulizer gas 50 psi, auxiliary gas 60 psi, and curtain gas 30 psi. The retention time and MS/MS parameters of all analytes are shown in Table 2.

3 3.1

Results and discussion Optimization of SPE

The SPE cartridge is an important factor in sample clean-up and enrichment of urinary OP diesters. In this study, we chose Oasis WAX, Oasis HLB and Bond Elut C18 as SPE cartridges for optimization. The results showed that Bond Elut C18 gave poor extraction recoveries which were lower than 50% for all OP diesters. This might be explained by higher polarity and lower pKa values of OP diesters, as they occurred in an anion state because pH of human urine is 5‒8[11]. Therefore, these OP diesters were not effectively adsorbed on the reversedphase non-polar sorbent Bond Elut C18. However, Oasis HLB was not suitable for enrichment of highly water-soluble compounds, such as DEP and BCEP, with extraction recoveries of 19.4% and 32.2%, respectively. Therefore, Oasis

WAX, a weak anion exchange sorbent cartridge, was chosen as the optimal sorbent. Although Oasis WAX had DBP background contamination, it exhibited good extraction efficiency[12]. This background contamination could be subtracted from the average background concentration. This phenomenon was also verified by van den Eede et al[12]. Several studies have optimized SPE cartridges. Cooper et al[13] used a Strata X-AW column and achieved higher recoveries, whereas van den Eede et al[12] observed severe matrix effects when using Strata X-AW. They also found that the Bond Elut NH3 column resulted in an 80% loss of analytes during the loading and washing step and the final recoveries of OP diesters were extremely low. We chose Oasis WAX as the SPE cartridge for OP diesters analysis. After choosing the SPE cartridge, effects of pH value, washing solvents and elution solvents on recovery of OP diesters were assessed. Due to their high polarity and lower pKa values (< 3.0)[13], OP diesters likely existed in the anion state as pH of humans urine usually ranges from 5 to 8. It is necessary to adjust the pH of urine samples to ensure satisfactory enrichment efficiency. On one hand, because Oasis WAX is a weak anion exchange sorbent, the most suitable pH value of the sample to be analyzed should be in the range of two units higher than pKa value of analyte and two units lower than that of sorbent (pKa, ~6.5 for Oasis WAX). On the other hand, dilution of samples with buffer could reduce the ionic strength of the sample and low ionic strength will facilitate exchange of target analytes with weak anions in the Oasis WAX sorbent. For these reasons, the effects of urine sample pH on extraction recoveries were determined in this study by adding different volumes of 0.1 M Table 1 Gradient elution program of the mobile phase Time 0 5 8 15 18 20 26 30 35 42

Water (%) pH = 9.2 65 65 50 20 0 0 0 50 65 65

Methanol (%) 35 35 45 75 95 95 95 50 35 35

Acetonitrile (%) 0 0 5 5 5 5 5 0 0 0

Table 2 Instrumental parameters for the seven OP metabolites and internal standards Retention time

Quantitative transition

Qualitative transition

Declustering potential

Collision energy

(min)

(m/z)

(m/z)

(V)

(eV)

Diethyl phosphate

5.4

152.9>78.7

152.9>125.0

-25

-25

Bis(2-chloroethyl) phosphate

6.7

221.1>35.0

221.1>37.0

-30

-21

10.2/11.2/12.5

249.0>35.0

249.0>37.0

-23

-25

Compound

Bis(2-chloropropyl) phosphate Diphenyl phosphate

15.7

249.1>93.1

249.1>155.0

-38

-32

Di-n-butyl phosphate

16.5

209.0>78.7

209.0>152.9

-25

-24

Dibenzyl phosphate

17.1

276.9>78.7

276.9>107.0

-45

-33

Bis(1,3-dichloro-2-propyl) phosphate

17.3

316.9>35.0

319.0>35.0

-25

-30

D8-BCEP

6.7

229.2>35.0

229.2>37.0

-30

-21

D12-BCPP

9.7/10.8/12.1

261.1>35.0

261.1>37.0

-25

-22

D10-DPhP

15.5

259.0>98.1

259.0>160.0

-38

-33

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NaAC-HAC buffer, and the results are shown in Fig.1. When the urine pH was adjusted to 5.0 with 200 μL NaAC-HAC buffer, good extraction recoveries of all compounds were obtained. Before final elution, sufficient washing is very important in order to remove the biological matrix. In this study, different ratios of methanol in water were tested as the washing solvent, and all solvents were adjusted to pH 5 with 0.1 M sodium acetate buffer before use. Figure 2 shows the effects of washing solvents on recoveries. As shown in Fig.2, when content of methanol in washing solution was in the range of 5%‒15%, the urine matrix was not sufficiently removed and severe matrix interferences was observed during instrumental analysis. When methanol ratio was increased to 40%, target analytes were washed out, resulting in lower extraction recovery. Therefore, 30% methanol in water (pH 5) was chosen as the washing solvent to achieve good recoveries and reduced matrix effects during instrumental analysis. When the sample was loaded onto the SPE sorbent, the analytes were adsorbed onto the sorbent by ionic interactions between the analytes and sorbent. Therefore, in order to elute the analytes, it was necessary to neutralize most of the sorbent’s positive charge, reduce ionic interactions between the sorbent and analytes and facilitate elution of the analyte from the cartridge. A buffer with high ionic strength or an organic solvent with a small amount of acid or base are usually used as elution solvents. In this study, 2 mL of 5% ammonia in methanol was used as the elution solvent which was consistent with the results of another study[12]. However, we found that after elution with 2 mL of 5% ammonia in methanol, an additional 2 mL of methanol as elution solvent increased the recovery of DTP. This might be caused by the high lipophilicity of DTP. Therefore, 2 mL of 5% ammonia in methanol and 2 mL of methanol were chosen as the final elution solution. 3.2

Optimization of instrumental parameters

Previous studies showed that 10 mM ammonium formate buffer at pH 9.2 instead of water as the mobile phase (A) significantly improved analysis sensitivity of OP diesters[11,12]. Methanol and buffer are widely used as mobile phases to separate OP diesters in LC. However, our study found that due to the stronger elution capacity of acetonitrile compared with methanol, the addition of a small amount of acetonitrile to the mobile phase contributed to the baseline separation of all seven targets and reduced retention time of the analytes. Baseline separation of all seven targets from the separation column avoided competitive ionization of compounds at the ion source and improved analysis sensitivity. Therefore, 10 mM of ammonium formate buffer at pH 9.2 (A), methanol (B) and acetonitrile (C) were used as mobile phases in this study.

Fig.1 Effect of urine pH value on recovery

Fig.2 Effect of washing solvents on recovery

The optimization of MS parameters for determination of OP diesters were as follows: (1) Precursor ions were determined by analyzing an authentic standard under MS1 full scan mode. (2) Product ions were further determined by analyzing a standard in product ion scan mode, and at least three m/z values were chosen for quantification and qualification. (3) The declustering potential (DP) and collision energy (CE) for each MRM transition were optimized by analysis of the standard at different DPs and CEs in MRM mode. The final quantitative and qualitative ion pairs, as well as the DPs and CEs in MRM mode for LC-MS/MS analysis of OP diesters are listed in Table 2. Typical total ion chromatograms of the standard solution and spiked urine samples (Fig.3) shows that all target compounds were baseline separated. 3.3

Method performance

The developed method was assessed by recovery, repeatability, linearity, limit of detection and the matrix effect. Recoveries were determined using a spiked mixed urine matrix at three levels (10, 50 and 100 μg L‒1) in sextuplicate. The recovery results of each compound at the three concentration levels are shown in Table 3. The results indicated that the recoveries of OP diesters ranged from 60.5% to 104.0%, with the exception of DEP, which only ranged from 17.8% to 36.2%. The low recoveries of DEP may be the result of severe matrix effects. Due to its high polarity and water solubility, DEP is easy to co-elute with urine interferences during the washing

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Fig.3 Total ion chromatograms of OP metabolites. (A) Chromatograms of all analytes in standard solution; (B) Chromatograms of BCEP and BCPP and their corresponding internal compounds in standard solution; (C) Chromatograms of all analytes in a spiked urine sample; (D) Chromatograms of BCEP and BCPP and their corresponding internal compounds in a spiked urine sample Table 3 Recovery, limit of detection, limit of quantification and repeatability for seven OP metabolites (n = 6) Compound ‒1

DEP BCEP BCPP DPhP DBP DTP BDCPP

10 μg L 36.2±2.0 63.0±10.4 72.7±14.6 96.3±11.3 104.0±1.7 91.7±10.8 91.7±12.2

Recovery (%) ± SD Spiking levels 50 μg L‒1 27.8±0.7 60.5±13.5 83.7±12.0 92.3±4.3 99.8±8.9 98.2±6.1 83.3±3.2

‒1

100 μg L 17.8±1.5 74.7±6.5 89.5±9.0 97.1±3.9 97.6±2.7 93.8±7.9 79.7±8.3

step and elute near the injection front in chromatography separation. This result was consistent with other studies which also reported extremely low extraction recovery of DEP[16]. However, Reemtsma et al[17] used a liquid-liquid extraction (LLE) method to analyze urinary OP metabolites and the recovery of DEP was 114%. Therefore, a LLE method may be an effective method for improving the extraction recovery of DEP. All compounds showed good linearity in the range of 0.5‒100.0 μg L‒1 with correlation coefficients ranging from 0.996 to 0.999. The LOD and LOQ were defined as signalto-noise ratio of 3 and 10, respectively. As shown in Table 3, LODs for all analytes ranged from 0.005 to 0.200 μg L‒1, and LOQs ranged from 0.020 to 0.500 μg L‒1. Under optimized conditions, the matrix effect was calculated by comparing the peak area of the analyte in the spiked urine matrix and the peak area of an equal amount of

Intra-day precision (%)

Inter-day precision (%)

LOD (μg L‒1)

LOQ (μg L‒1)

5.7 9.4 10.7 2.0 1.9 1.9 4.8

11.6 15.4 10.7 10.5 9.5 11.6 12.7

0.030 0.100 0.200 0.005 0.006 0.007 0.050

0.100 0.300 0.500 0.020 0.020 0.020 0.200

analyte in spiked water/methanol after subtracting the background level of the analyte in the non-spiked urine matrix. The detail formula for calculating the matrix effect is as follow: ME (%) = [(A1 ‒ A0 ‒ A2)/A2] × 100 (1) ME represents the matrix effect. A0 represents the peak area of background analyte in the non-spiked urine matrix. A1 represents the peak area of analyte in the spiked urine matrix. A2 represents the peak area of analyte in spiked water/methanol. Negative results represent signal suppression, positive results mean signal enhancement and zero represents no matrix effect. The results showed that matrix effects were significant from ‒56% (DEP) to 26% (DPhP) with most analytes showing signal suppression. However, the matrix effect in this study was much lower than that for the reported literature (‒40%‒166%)[12]. This confirmed that the developed method effectively removed interferences with the efficient pretreatment method and LC separation to achieve accurate

LI Pei et al. / Chinese Journal of Analytical Chemistry, 2017, 45(11): 1648–1654

important scientific data for the human-health risk assessment.

results. Compared with other methods used for the analysis of OP diesters in urine (Table 4), this method does not require derivatization and is simple, rapid and sensitive. Under optimal conditions, all analytes had good extraction recoveries ranging from 60.5% to 104.0%, except for DEP which had an extraction recovery ranging from 17.8% to 36.2%. The inter-day and intra-day precision results showed that the developed method had good repeatability with relative standard deviations less than 15.4%. 3.4

4

Conclusions

In this study, SPE combined with LC-MS/MS analysis was developed to determine seven OP diesters in human urine. The pretreatment was performed by SPE enrichment and clean-up. Two milliliters of urine were diluted with 200 μL of 0.1 M NaAC-HAC buffer, the diluted samples were enriched by a SPE column, and all analytes were eluted with 2 mL of 5% ammonia in methanol and 2 mL of methanol. This method had good recoveries and high reproducibility, and can be used for analyzing OP metabolites in urine sample with large sample size. The chromatographic separation of the analytes was carried out using an analytical column with a large capacity, which reduced the matrix interferences and improved the detection sensitivity of halogenated OP metabolites. However, compared with non-halogenated OP metabolites, the detection sensitivity of halogenated OP metabolites was much lower. Therefore, it is necessary to explore effective methods to improve the sensitivity of detection of urinary halogenated OP metabolites in the future.

Practical application

The developed SPE combined with LC-MS/MS was used to determine seven OP diesters in ten urine samples from women in Guangzhou, China. The concentrations of the seven OP diesters in urine samples are shown in Table 5. All compounds were detected in the samples. The detection rate for all compounds was higher than 50%, except for BCPP which was only detected in one urine sample. Total concentrations of the seven OP diesters ranged from 0.5 to 6.7 μg L‒1. DEP, BCEP and DBP were the major OP metabolites detected in human urine. Studies on human exposure to OPs can provide

Table 4 Comparison of parameters between the developed method and methods reported in the literature for analysis of OP metabolites

a

Sample preparation a

Instrumental analysis

Analyte

LOD (μg L‒1)

Ref.

SPE (Isolute ENV+ and Bond Elut PSA + Florisil)

GC-MS/MS

DBP, BCPP, BCEP, DPhP, DmCP, DpCP

0.1‒1

[8,9]

SPE (Strata X-AW)

LC-MS/MS

BDCPP, DPhP

0.008‒0.2

[13]

LLE SPE (Oasis WAX)

LC-MS/MS LC-MS/MS

DEP, DiBP, DnBP, DPhP, DBEP, DEHPetc. BCEP, BCPP, BDCPP, DBP, DPhP, BBOEP

0.3‒11 1.3‒25

[17] [12]

Direct injection

LC-TOF MS

BCEP, BCPP, DBP, DPhP, BDCPP, BBOEP

0.1‒25

[11] [10]

SPE (ISOLUTE aminopropyl)

UPLC-MS/MS

DBP, DPhP, BBOEP, DEHP, BCEP, BCPP, BDCPP

0.02‒0.2

SPE (Oasis WAX)

UPLC-MS/MS

BDCPP, DPhP, BBOEP, BCPP, BCEP, DpCP etc.

0.08‒0.25

[18]

SPE (Oasis WAX)

LC-MS/MS

DEP, BCEP, BCPP, DBP, DPhP, BDCPP, DTP

0.005‒0.2

This study

Type of SPE column in brackets. DmCP: di-m-cresylphosphate; DpCP: di-p-cresylphosphate; BBOEP: bis(2-butoxyethyl)phosphate; DEHP: di(2-ethylhexyl)phosphate.

Table 5 Concentrations of OP metabolites in 10 participants (μg L‒1) Sample No. S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 a

DEP 0.29 0.41 0.12 0.13 0.59 0.45 0.32 0.10 0.07 0.13

BCEP 0.35 3.88 ND ND ND ND ND 0.35 0.37 0.84

BCPP b

ND 0.91 ND ND ND ND ND ND ND ND

DPhP

DBP

DTP

BDCPP

Total DAPsa

0.06 0.11 0.06 0.08 0.63 0.03 0.08 0.02 0.03 0.05

0.67 0.81 2.53 0.93 ND ND ND 0.89 ND 0.19

0.05 0.03 0.06 0.04 0.01 0.02 0.02 0.04 0.03 0.02

ND 0.51 ND 0.08 0.07 0.07 0.08 0.13 0.07 0.20

1.41 6.66 2.77 1.26 1.31 0.58 0.50 1.52 0.57 1.44

Total DAPs: sum of seven OP diesters. b ND: not detected.

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