CHINESE JOURNAL OF ANALYTICAL CHEMISTRYVolume 37, Issue 4, April 2009 Online English edition of the Chinese language journal

Cite this article as: Chin J Anal Chem, 2009, 37(4), 585–588.

Received 12 September 2008; accepted 27 October 2008 * Corresponding author. Email: jinjun3799@yahoo.com.cn This work was supported by the National Natural Science Foundation of China (No. 20507023) and the 985 Engineering of China (No. CUN985-3-3). Copyright © 2009, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(08)60097-3

RESEARCH PAPER

Determination of Hexabromocyclododecane Diastereomers in Soil by Ultra Performance Liquid Chromatography-Electrospray Ion Source /Tandem Mass Spectrometry JIN Jun1,*, YANG Cong-Qiao1, WANG Ying1, LIU An-Ming2

1 Central University for Nationalities, College of Life and Environmental Science, Beijing 100081, China 2 Coastal People’s Hospital, Weifang 262737, China

Abstract: A new method has been developed to the trace level measurement of -, -, and -HBCD (hexabromocyclododecane) diastereomers (C12H18Br6) in soil samples by accelerated solvent extraction-ultra performance liquid chromatography-electrospray ion source-triple quadrupole mass spectrometry (ASE-UPLC-ESI-MS/MS). Soil samples were extracted with 3:1 hexane/acetone (V/V) and was further cleaned with a multilayer silica column (15 mm i.d.) filled from the bottom with 6 g of activated silica, 3 g of H2SO4/silica 44% (w/w), and 3 g dried Na2SO4. Quantification of -, -, and -HBCD diastereomers in samples was performed by the internal standard method. The recovery of triplicate blank soil samples was (104.6 ± 3.7)% for the spiked level of 54 ng HBCD. The concentrations of HBCD in soil samples collected near a HBCD manufactory ranged from 2.8–144.5 ng g–1 (dry weight). The diastereomer profile of HBCD in the soil samples was dominated by -HBCD (73.7 ± 4.7)% and followed by -HBCD (14.6% ± 3.4)% and -HBCD (11.7 ± 1.7)%. Key Words: HBCD diastereomers; Ultra performance liquid chromatography-electrospray ion source/tandem mass spectrometry; Soil; Accelerated solvent extraction

1 Introduction

Hexabromocyclododecane (HBCD) was the third most produced brominated flame retardants (BFRs) in the world. Hexabromocyclododecane was used primarily in Expanded polystyrene (EPS) and Upholstery textiles, and HBCD has no technically suitable alternative in Extruded polystyrene[1]. The annual world market consumption of HBCD was estimated to be greater than 22000 tons[2]. Hexabromocyclododecane was used as additive BFRs and easily released from the products into the environment. Due to its low degradability and high Octanol-Water partition coefficient (Kow), this substance has a relatively high bioaccumulation potential in the adipose tissue of living organisms[3]. Its long-range transportability has been testified in many studies[4–6], thus environment pollution caused by the production and usage of HBCD could possibly be spread

far away. Hexabromocyclododecane could influence the animal

endocrine and immune parameters, and HBCD may cause the same effect to human health as DDT and PCBs, in terms of inducing genetic recombination, which was known to provoke a number of diseases, including cancer[7]. As the environment level of HBCD and the possibility of acute toxic effect were very low, its adverse effects to environment and humans were not easily perceived. Substantial and irreversible harm induced by HBCD may occur due to its massive use, environmental persistence, and biological toxicity. Thus, HBCD has been testified as a potential contamination, and the analysis of HBCD level in environment has been a new hot research topic.

However, environmental data of HBCD diastereomers were currently still insufficient. In order to develop further research on environmental habit, fate, environmental effects, and

JIN Jun et al. / Chinese Journal of Analytical Chemistry, 2009, 37(4): 585–588

biological toxicity of HBCD, rapid and efficient analysis method dedicated to trace level measurement of HBCD diastereomers must be developed. Hexabromocyclododecane has traditionally been determined using gas chromatography coupled with mass spectrometric detection (GC-MS) or liquid chromatography coupled with mass spectrometric detection (LC-MS). However, because thermally induced rearrangements, as well as decomposition of the compound, occurred at temperatures > 240 °C[8], GC-MS, which required high temperature, does not allow the quantification of individual isomers. A new method dedicated to trace level measurement of

-, -, and -HBCD diastereomers in soil samples by accelerated solvent extraction-ultra performance liquid chromatography-electrospray ion source-triple quadrupole mass spectrometry (ASE-UPLC-ESI-MS/MS) has been developed in this study.

2 Experimental 2.1 Instruments and reagents

An ultra performance liquid chromatography-electrospray

ion source coupled to triple quadrupole mass spectrometry (Waters, USA) was used. The UPLC separation was obtained using Waters C18 reversed-phase column (2.1 mm × 50 mm, 1.7 m, Waters, USA). A Dionex accelerated solvent extraction 300 system was used for sample extraction (Dionex, USA). A Heidolph rotary evaporator (Heidolph, Germany) was used. Hexane, acetone, and dichloromethane were of pesticide grade (Tedia, USA); methanol and acetonitrile were of HPLC grade (Baker, USA). Water was purified using a Milli-Q system (Millipore, USA). Other chemicals were of analytical grade (Beihua, Beijing). High purity nitrogen gas and argon gas were purchased from Chengxin Company (Chengxin, Beijing). 13C12-labeled -HBCD and native 12C12- -, -, and -HBCD standards were purchased from Cambridge Isotope Laboratories (99%, USA).

2.2 Method 2.2.1 Preparation of standard solution

Quantification of HBCD diastereomers was performed

using internal standard method. A series of 12C12- -, -, and -HBCD mixture standard solutions were prepared, and

13C12-labeled -HBCD standard was added. The solutions with constant volume were prepared for use.

2.2.2 Sampling and pretreatment

The seven soil samples were collected manually at 0–5 cm

depth from seven sampling sites that are located near a HBCD product manufactory in China on July, 2007. The soil samples

were collected in stainless steel metal box and stored in Car Fridge, and then transported back to the lab and stored at –18 ºC until analysis.

Ten grams of dried soil samples were weighed and spiked with 13C12- -HBCD standard. The samples were extracted over 4 static cycles with 5 min per cycle. Samples were extracted with 3:1 hexane/acetone (V/V) at 120 ºC. After each extraction procedure, additional 4 times of manual system rinse progress was adopted to avoid possible contamination between samples. The extractions were concentrated to 2–3 ml by rotary evaporation and was further purified with one multilayer silica column (15 mm i.d.) filled from the bottom with 6 g of activated silica, 3 g of H2SO4/silica 44% (w/w), and 3 g dried Na2SO4. The samples were eluted with 30 ml hexane and 100 ml hexane/DCM (1:1). The second fraction, which contained the target HBCD, was concentrated to 2–3 ml, and the solvent was exchanged to methanol for LC-MS/MS analysis.

2.2.3 LC-MS parameters

Separation of -, -, and -HBCD diastereomers was

achieved using a Waters UPLC system. Injected volume was 3 l. Elution solvents were methanol (A) and 20% acetonitrile/water (B). Mobile phase composition (A:B, V/V) was 80:20, and the flow rate was set at 0.25 ml min–1. The column temperature was 35 ºC, and the sample temperature was 10 ºC. Mass spectrometric data were acquired in negative electrospray ionization (ESI–). Target compounds were determined by multiple reactions monitoring mode (MRM). The ion signals from m/z 640.6 m/z 78.9 and m/z 652.4 m/z 78.9 transitions were monitored for C12-HBCD and 13C12-labeled HBCD isomers, respectively. Capillary voltage was 2.5 kV, and cone voltage was 20 V. Source temperature was 120 ºC, and desolvation temperature was 400 ºC. Collision energy was 15. Desolvation gas flow rate was 550 l h–1, cone gas flow rate was 130 l h–1, and collision gas flow rate was 0.15 ml min-1.

3 Results and discussion

3.1 Identification and quantification

The chromatograms for the HBCD standard solutions and

the soil samples were acquired at MS scan mode. The relative retention times of the three chromatographic peaks in extract chromatogram for soil samples were consistent with that of -, -, and -HBCD in standard solutions (Fig.1).

Hexabromocyclododecane diastereomers in soil samples were further identified by comparing spectrum of HBCD in standard solution and that in soil and plant samples. The molecular formula for HBCD is C12H18Br6. The relative abundance of the molecular ions was 1:6:15:20:15:6:1 in

JIN Jun et al. / Chinese Journal of Analytical Chemistry, 2009, 37(4): 585–588

spectrum for soil samples, which was consistent with that for HBCD standard solutions (Fig.2). The m/z 641 was the peak for molecular ion, and the m/z 635, 637, 639, 643, 645 and 647 were the peaks for isotopic ions. Then, it could be confirmed that there was HBCD in the soil samples.

For the purpose of selecting characteristic ion, the daughter scan spectrum of HBCD standard solution was acquired. Only the ion with relative high abundance and better stability was suitable to be used as characteristic ion and then paired with the parent ion for quantification. As shown in Fig.3, the Br- ion has the highest relative abundance, and it has more stability than other fragment ions.

Fig.1 Chromatogram for HBCD standard solution (a) and extract chromatogram for soil sample (b)

Fig.2 Spectrum of HBCD standard solution (a) and spectrum of

HBCD in soil sample (b)

Fig.3 Daughter scan spectrum of HBCD standard solution

Mass spectrometric data were acquired in ESI– that was performed in multiple reactions monitoring mode (MRM). Quantification of -, -, and -HBCD was obtained using MassLynx V4.1 software (Waters) based on the ion signal from the m/z 640.6 m/z 78.9 and m/z 652.4 m/z 78.9. 3.2 Calibration curve and detection limits

The standard solutions were detected under the above

instrument conditions. The individual HBCD isomer amount was calculated using the calibration curve by the ratio of the peak area of HBCD isomer to that of 13C12-labeled -HBCD. The calibration curves and the corresponding correlation coefficients for -, - and -HBCD were y = 1.1525x + 0.0384, r = 0.9997; y = 1.267x + 0.0015, r = 0.9999; y = 1.5198x + 0.317, r = 0.9999. The linear range for -, - and -HBCD were 2–500 μg l–1, 2–500 g l–1, and 4–1000 g l–1, respectively. The limit of detection for -HBCD was 2.4 pg. The method detection limits was 20 pg g–1.

3.3 Recovery and quality accuracy

The average recovery of -, -, - and HBCD in triplicate

spiked soil samples (10 g) was (113.3 ± 1.7)%, (102.5 ± 3.8)%, (104.0 ± 3.4)%, and (104.6 ± 3.7)% for the spiked level of 54 ng HBCD. The method was sufficiently robust to accommodate a satisfying quantitative analysis.

3.4 HBCD in soil samples

The seven soil samples were determined with the

developed method. -, -, and -HBCD were detected in all samples, and the concentrations of HBCD ranged from 2.8 to 144.5 ng g–1 (dry weight). The HBCD in soil samples was dominated by -HBCD (73.7 ± 4.7)% and followed by

-HBCD (14.6 ± 3.4)% and -HBCD (11.7 ± 1.7)%. The developed method for the measurement of -, -, and

-HBCD diastereomers in soil samples by ASE-UPLC-ESI- MS/MS has some significant advantages: (1) In comparison with Soxhlet extraction, which usually need 24–48 h, accelerated solvent extraction could shorten the extraction time to about 35 min. Then, it could substantially reduce the analysis cycle time and guarantee the measurement accuracy; the automatic control of operating conditions could enhance the quality of parallel sample analysis; through the optimization of extraction parameters, satisfying recovery could also be obtained. (2) The adoption of UPLC system could shorten the separation time of -, -, and -HBCD diastereomers and ensure good separation efficiency. In addition, it could reduce the injection volume and was more suitable for trace level measurement of environment samples. (3) In comparison with atmospheric pressure chemical ionization (APCI), ESI could significantly enhance the signal

JIN Jun et al. / Chinese Journal of Analytical Chemistry, 2009, 37(4): 585–588

response, and it improved the instrument detection limits and method detection limits. (4) Through the monitor of [M–H]– Br– transition, other possible impurities could be excluded at the greatest extent. This may further guarantee the qualification and quantification of HBCD diastereomers. References [1] Liu W Z, Jin J, Wang Y. Chinese Journal of Environmental

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