Yokogawa Analytical Systems Inc., Nakacho, Musashino-shi, Tokyo , Japan.

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1 Determination of organochlorine pesticides in river water by gas chromatography-negative-ion chemical-ionization mass spectrometry using large volume injection Sadao Nakamura,* Takashi Yamagami and Shigeki Daishima Yokogawa Analytical Systems Inc., Nakacho, Musashino-shi, Tokyo , Japan. THE ANALYST FULL PAPER Received 22nd May 2001, Accepted 31st July 2001 First published as an Advance Article on the web 21st September 2001 A method for the determination of 24 organochlorine pesticides by gas chromatography-mass spectrometry (GC-MS) with negative-ion chemical-ionization (NICI) using programmable temperature vaporizer (PTV)-based large volume injection (LVI) is described. The ion source temperature was determined to be 150 C for the optimized NICI-selected ion monitoring (SIM) conditions. PTV inlet parameters were also optimized. The sensitivities of the pesticides by splitless-gc-nici-ms were approximately times higher than those of the pesticides by splitless-gc-ei-ms. The sensitivities of the pesticides by LVI-GC-NICI-MS were over 80 times higher than those of the pesticides by spiltless-gc-nici-ms. This method was applied to the determination of the pesticides in river water using micro liquid liquid extraction as sample preparation. The recoveries of the pesticides from a river water sample spiked with standards at 2 ng l 21 and 20 ng l 21 were % (RSD, %) and % (RSD, %), respectively. The methodical detection limits ranged from to 2.2 ng l 21 Introduction The human race has been synthesizing or isolating lots of chemicals. Many of them are toxic to many organisms and the environment. Organochlorine compounds, such as polychlorinated biphenyls (PCBs) and chlorinated pesticides, are also toxic contaminants. It is important to monitor organochlorine compounds at low levels in the environment. Therefore, we need a sensitive method for determining organochlorine compounds in the environment. To achieve lower detection limits, we generally need a method with high concentration sample preparation such as gas chromatography-electron ionization mass spectrometry (GC-EI-MS) using liquid liquid extraction or solid-phase extraction with ~ 1000-fold concentration for aqueous samples. 1,2 In GC, there are generally some approaches to improve detection limits: concentrate samples; increase the sensitivity of the detector; increase the selectivity of the detector; and increase the injection volume. For the determination of organochlorine pesticides, negative-ion chemical-ionization mass spectrometry (NICI-MS) has a big advantage for the sensitivity and selectivity of the detector. Electron capture NICI provides high sensitivity and selectivity for electrophilic compounds. Therefore, GC-NICI-MS has been applied for the trace level determinations of electrophilic compounds containing halogens, nitro groups, and highly conjugated systems in environmental samples. 3 5 On the other hand, large volume injection (LVI) is another approach to improve the detection limits. The typical injection volume for capillary column analysis is 1 to 2 ml. With the LVI technique, good chromatography can usually be obtained with injection volumes of up to several hundreds of microliters. The majority of the solvent is evaporated before transfer of the sample to the analytical column. LVI is a good technique for the trace analysis of semivolatile compounds. 6 8 Bagheri et al. reported the determination of PCBs in biological samples by GC-NICI-MS off-line combined with liquid chromatography (LC) as the separation. 9 Their method provides high sensitivity and selectivity for PCBs with four or more chlorine atoms. Louter and coworkers reported the determination of organochlorine pesticides in water samples by on-line solid-phase extraction (SPE)- GC-EI-MS with direct injection of ml of sample extract on GC via retention gap techniques using an on-column interface Their method allows the detection of the target compounds at levels below 0.1 mg l 21 using a sample volume of 10 ml. Hankemeier et al. developed a method for the determination of triazines and organophosphorus pesticides by automated on-line SPE-GC-flame-ionization detection (FID). 13 Their method allows the detection of the target compounds at mg l 21 levels in river water using an on-column interface with retention gap techniques for the injection of 50 ml of sample extract. In this study, the combination of programmable temperature vaporizer (PTV)-based LVI and NICI-MS was used for the determination of 24 organochlorine pesticides. Since the combination provided much lower detection limits, a micro liquid liquid extraction was used to simplify sample preparation. This technique reduces the extraction time and the total volume of sample and solvent required for the analysis, and eliminates the step of concentrating the solvent. Very few studies on the determination of organochlorine pesticides have been performed by GC-NICI-MS using LVI. We developed a GC-NICI-MS method using LVI for the determination of organochlorine pesticides. The method was optimized and then applied to the determination of organochlorine pesticides in river water. Experimental Chemicals The 24 organochlorine pesticides were obtained from Wako (Osaka, Japan). Acetone and hexane, both of reagent grade, were purchased from Merck (Darmstadt, Germany). Stock standard solutions of the individual pesticides were prepared by diluting each compound to a concentration of 1.0 mg ml 21 in acetone. Sodium chloride (NaCl), reagent grade, was purchased from Merck Analyst, 2001, 126, DOI: /b104501f This journal is The Royal Society of Chemistry 2001

2 Sample preparation (micro liquid liquid extraction) River water was collected from the Singapore River. 0.3 g of NaCl was added to the sample (10 ml), and then the vial was shaken until the NaCl had completely dissolved. Two ml of hexane was added to the sample, and then the vial was shaken for 3 min. Then, 100 ml of the organic layer was analyzed by GC-NICI-MS with PTV. Instrumentation All GC-MS analyses were performed using an Agilent 6890/5973 (Agilent Technologies, Palo Alto, CA, USA) equipped with PTV. The separation was carried out on a HP- 5MS capillary column (30 m mm id mm film thickness). Helium was used as the carrier gas with a column flow rate of 1.2 ml min 21 in the constant flow mode, and methane served as the CI reagent gas with a flow rate of 2.5 ml min 21. The column temperature was held at 90 C for 5.32 min, then programmed at 20 C min 21 to 170 C, then at 5 C min 21 to 250 C and finally at 15 C min 21 to 280 C, which was held for 2.5 min. The PTV inlet was equipped with a deactivated empty glass liner with multi baffles (1.5 mm id). Solvent vent mode was used for LVI, and twenty 5 ml injections were made for a total of 100 ml by an autosampler equipped with a 10 ml syringe. The PTV parameters of pause time between injections, initial temperature, final temperature, vent flow rate, sample transfer time, and injection volume were optimized. The pause time between injections was chosen as 0 s over the range between 0 and 4 s: the responses for the target compounds slightly decreased with increasing the time. The final temperature was chosen as 300 C over the range between 200 and 400 C: although the responses for the target compounds slightly increased with increasing the temperature, 300 C was selected for decreasing the transfer of sample matrix to the capillary column. The sample transfer time was selected as 1 min over the range between 0 and 2 min: the responses for the target compounds slightly increased with increasing the time until 1 min. The injection volume was chosen as 100 ml for practical repeatability; the responses for the target compounds were linear between 50 and 350 ml. The optimization of the initial temperature and vent flow rate is described in the Results and Discussion section. The vent flow rate was set at 50 ml min 21, and the vent pressure was set at 0 kpa until the injection sequence was done (3.22 min). The normal inlet pressure was restored and the vent flow was turned off at 3.22 min. The vent flow remained off until it was set to 100 ml min 21 at 5.32 min. The inlet temperature was held at 20 C for 3.32 min, then programmed at 280 C min 21 to 300 C, which was held for 1 min, then at 250 C min 21 to 350 C, which was held for 2.8 min, and finally at 20 C min 21 to 250 C. The transfer line temperature was kept at 280 C. The ion source temperature was chosen as 150 C after the optimization over the temperature range between 150 and 270 C. The mass spectrometer was operated in the NICI mode and with a scan range of m/z 10 to 570 at 1.36 scans s 21. In the selected ion monitoring (SIM) mode, monitoring ions are listed in Table 1 and the ions were monitored with a dwell time of 35 to 150 ms per single ion. Results and discussion Optimization of the PTV inlet parameters of initial temperature and vent flow rate PTV inlet parameters were optimized over the inlet initial temperature range between 0 and 60 C and the vent flow rate range between 25 and 200 ml min 21 (standard mixture concentration: 500 pg ml 21 each; injection volume: 40 ml). Fig. 1 shows the effect of the inlet temperature and the vent flow rate on peak areas for a-bhc, trans-nonachlor, and mirex. Most of the organochlorine pesticides showed similar results for the various combinations of the inlet temperature and the vent flow rate. At the inlet temperatures of 40, 50, and 60 C, most of the 24 pesticides showed smaller peak areas over the whole range of vent flow rates. The target compounds presumed to be vented in vapor together with the solvent because peak areas tended to be Table 1 Comparison of detection limits (DL, at S/N = 3) of EI-SIM using splitless, NICI-SIM using splitless, and NICI-SIM using LVI EI-SIM NICI-SIM Monitor ions Splitless a DL/pg ml 21 Monitor ions Splitless a DL/pg ml 21 LVI b DL/pg ml 21 1 a-bhc HCB b-bhc g-bhc d-bhc heptachlor aldrin heptachlor epoxide c 770 c 1.3 c 9 oxychlordane trans-chlordane o,p-dde a-endosulfan cis-chlordane trans-nonachlor dieldrin d 300 d 1.3 d 16 p,pa-dde e 1700 e 5.6 e 17 o,p-ddd endrin b-endosulfan p,pa-ddd endosulfan sulfate p,pa-ddt methoxychlor mirex a Injection volume: 1 ml. b Injection volume: 100 ml. c m/z 35 could not be monitored because this compound overlapped oxychlordane under this condition. d m/z 35 could not be monitored because this compound overlapped p,p -DDE under this condition. e m/z 35 could not be monitored because this compound overlapped dieldrin under this condition. Analyst, 2001, 126,

3 smaller with increasing inlet temperature and/or vent flow rate. At the inlet temperatures of 0 C, most of the pesticides showed maximum peak areas under the vent flow rate of 100 ml min 21. At 10 C, they showed maximum peak areas under 100 ml min 21. At 20 C, they showed maximum peak areas under 50 ml min 21. At 30 C, they showed maximum peak areas under 25 ml min 21. The repeatabilities of peak areas under the conditions provided maximum peak areas at each temperature (at 0 C with a vent flow rate of 100 ml min 21, at 10 C with a vent flow rate of 100 ml min 21, at 20 C with a vent flow rate of 50 ml min 21, and at 30 C with a vent flow rate of 25 ml min 21 ) were 3.7, 3.1, 3.2, and 5.5%, respectively, as average RSD value (n = 6) of all the pesticides. Therefore, the parameters of 10 C with a vent flow rate of 100 ml min 21 and 20 C with a vent flow rate of 50 ml min 21 showed better repeatabilities. For the sensitivity, the parameters of 20 C with a vent flow rate of 50 ml min 21 Fig. 1 Effect of inlet initial temperature and vent flow rate on peak areas for a-bhc,trans-nonachlor, and mirex. 5: 0 C, -: 10 C, $: 20 C, 3: 30 C, +: 40 C, 1 50 C, 8 60 C. Fig. 2 SIM chromatograms of organochlorine pesticides extracted from (upper) the river water spiked with the standards (20 pg) and (lower) the non-spiked river water by LVI-GC-NICI-MS. 1: a-bhc, 2: HCB, 3: b-bhc, 4: g-bhc, 5: d-bhc, 6: heptachlor, 7: aldrin, 8: heptachlor epoxide, 9: oxychlordane, 10: trans-chlordane, 11: o,p-dde, 12: a-endosulfan, 13: cis-chlordane, 14: trans-nonachlor, 15: dieldrin, 16: p,p -DDE, 17: o,p-ddd, 18: endrin, 19: b- endosulfan, 20: p,p -DDD, 21: endosulfan sulfate, 22 p,p DDT, 23: methoxychlor, 24: mirex Analyst, 2001, 126,

4 provided better results than those of 10 C with a vent flow rate of 100 ml min 21. The inlet initial temperature and vent flow rate were chosen as 20 C and 50 ml min 21, respectively. Determination of organochlorine pesticides in SIM mode A comparison of the detection limit levels (at a signal-to-noise ratio of 3) of the organochlorine pesticides by splitless-gc-ei- MS, splitless-gc-nici-ms, and LVI-GC-NICI-MS is shown in Table 1. The sensitivities of the target compounds by splitless- GC-NICI-MS were times higher than those of the target compounds by splitless-gc-ei-ms except for heptachlor epoxide, o,p-dde, p,p -DDE, o,p-ddd, p,p -DDD, p,p -DDT, and methoxychlor. For heptachlor epoxide, m/z 35 (the largest ion) could not be monitored because this compound overlapped oxychlordane under this condition. For o,p-dde, p,p -DDE, o,p-ddd, p,p -DDD, p,p -DDT, and methoxychlor, since the sensitivities by NICI-MS were times higher than those by EI-MS, these pesticides did not provide high sensitivities for NICI-MS. For HCB, heptachlor, aldrin, oxychlordane, transchlordane, a-endosulfan, cis-chlordane, trans-nonachlor, dieldrin, endrin, b-endosulfan, endosulfan sulfate, and mirex, these pesticides provided high sensitivities for NICI-MS because the sensitivity by NICI-MS were over 40 times higher than those by EI-MS. The sensitivities of the target compounds by LVI-GC-NICI- MS were over 80 times higher than those of the target compounds by splitless-gc-nici-ms. The detection limits using LVI-GC-NICI-MS ranged from to 5.6 pg ml 21. The linearity and repeatability of the NICI-MS method using LVI were tested and the results are listed in Table 2. The calibration curves for all the pesticides were linear at 12 levels of concentration ranging from 0.2 to 1000 pg ml 21 with correlation coefficients between and The repeatabilities, expressed as a RSD (n = 6), for peak areas of all the pesticides were between 1.7 and 17% at 1 pg ml 21 and between 2.4 and 14% at 10 pg ml 21. The repeatabilities of the splitless- GC-EI-SIM method and the splitless-gc-nici-sim method were also demonstrated and the results are listed in Table 2. The repeatabilities for peak areas of all the pesticides were between 0.9 and 12% at pg ml 21 by the splitless-gc-ei-sim method, and between 1.5 and 11% at 500 pg ml 21 and between 2.2 and 8.2% at pg ml 21 by the splitless-gc-nici-sim method. NICI-MS showed almost the same performance as EI- MS at lower levels as judged by the comparison of RSD values. Although RSD values of o,p-ddd, endrin, p,p -DDD, and methoxychlor showed over 10% due to worse signal-to-noise ratio, LVI-GC-NICI-MS showed almost the same performance as splitless-gc-nici-ms. For o,p-ddd, endrin, p,p -DDD, and methoxychlor, good repeatabilities were obtained at 100 pg ml 21 with RSD values between 3.3 and 7.9%. Application to river water Interference of river water matrix. Standards (2 and 20 pg: 0.2 and 2 ng l 21 as concentration in river water) were added to 10 ml of a river water sample, and then the river water was treated by the micro liquid liquid extraction method described in the Experimental section. The extract spiked with the standards and the non-spiked extract were analyzed by GC- NICI-MS using LVI. a-bhc, ß-BHC, g-bhc, endosulfan sulfate, and p,p -DDT were also detected in the non-spiked sample (a-bhc: 0.30, b-bhc: 0.38, g-bhc: 0.75, endosulfan sulfate: 0.59, and p,p -DDT: 0.61 ng l 21 ). All the pesticides could be determined without interference from the river matrix. Fig. 2 shows NICI-SIM chromatograms of the pesticides extracted from the river water spiked with the standards (20 pg) and from the non-spiked river water. The detection limits evaluated in the river water are listed in Table 3, and ranged from to 2.2 ng l 21. Recovery. The standards were added to 10 ml of a river water sample (20 and 200 pg as the spiked amounts; 2 and 20 ng l 21 as the concentration in the river water), and then the spiked Table 2 Repeatabilities (n = 6) and correlation coefficients of calibration curves NICI-SIM splitless a LVI b EI-SIM splitless a RSD (%) pg ml 21 RSD (%) 500 pg RSD (%) 1 pg Correlation c ml pg ml 21 ml pg ml 21 coefficient 1 a-bhc HCB b-bhc g-bhc d-bhc heptachlor aldrin heptachlor epoxide 2.1 nd 7.3 nd oxychlordane trans-chlordane o,p-dde a-endosulfan cis-chlordane trans-nonachlor dieldrin nd p,pa-dde 2.2 nd 8.2 nd o,p-ddd 2.4 nd 4.7 nd endrin nd b-endosulfan p,pa-ddd 3.0 nd 5.0 nd endosulfan sulfate p,pa-ddt 2.2 nd methoxychlor 2.4 nd 3.9 nd mirex a Injection volume: 1 ml. b Injection volume: 100 ml. c Concentration range: pg ml 21 nd: not detected. Analyst, 2001, 126,

5 Table 3 Recoveries of organochlorine pesticides from river water and reproducibilities (n = 6), and methodical detection limits Recovery, % (RSD, %) spiked 20 pg in 10 ml river water Recovery, % (RSD, %) spiked 200 pg in 10 ml river water Methodical detection limits/ng l 21 1 a-bhc 85 (4.7) 102 (3.9) HCB 75 (3.4) 93 (2.1) b-bhc 89 (5.5) 104 (2.4) g-bhc 86 (6.1) 103 (2.9) d-bhc 89 (4.9) 102 (2.7) heptachlor 107 (8.6) 97 (5.6) aldrin 82 (4.9) 92 (1.6) heptachlor epoxide 111 (15) 97 (3.0) oxychlordane 88 (3.2) 101 (1.7) trans-chlordane 83 (3.3) 98 (0.8) o,p-dde 93 (5.1) 100 (2.6) a-endosulfane 92 (3.9) 100 (1.4) cis-chlordane 85 (3.1) 97 (0.5) trans-nonachlor 85 (2.9) 95 (3.9) dieldrin 106 (15) 95 (1.9) p,pa-dde 104 (12) 95 (2.3) o,p-ddd 93 (6.9) 103 (2.8) endrin 105 (5.9) 113 (1.5) b-endosulfan 94 (3.7) 99 (1.2) p,pa-ddd 97 (11) 100 (2.3) endosulfan sulfate 95 (4.9) 102 (1.5) p,pa-ddt 98 (4.3) 103 (1.4) methoxychlor 11 (8.8) 105 (2.0) mirex 89 (3.8) 97 (2.0) sample was treated by the micro liquid liquid extraction method. Subsequently, the extract was analyzed by GC-NICI- MS using LVI. The recovery and reproducibility were tested and the results are listed in Table 3. Good recoveries were obtained for all the pesticides with values between 75 and 111% at 2 ng l 21 and between 92 and 105% at 20 ng l 21. Practical reproducibility was obtained for all the pesticides with RSD values (n = 6) between 2.9 and 15% at 2 ng l 21 and between 0.5 and 5.6% at 20 ng l 21 for the peak areas. Conclusions A rapid, sensitive, and selective GC-NICI-MS method for the determination of 24 organochlorine pesticides in river water was developed. Significant advantages of the method are that NICI-MS provided high sensitivity for the pesticides; PTVbased LVI improved the sensitivities by the 100 ml injection volume. Therefore, the combination of NICI-MS and LVI allows the detection of levels in the range ng l 21 level of the pesticides in river water using micro liquid liquid extraction (5-fold concentration). The method also provides a wide range of linearity, satisfactory recovery, and good reproducibility. References 1 H. M. Kuch and K. Ballschmiter, Fresenius J. Anal. Chem., 2000, 366, T. Ibaraki, C. Oguma, A. Tanabe, K. Kawata, M. Sakai and I. Kifune, Bunseki Kagaku, 1999, 48, M. Yasin, P. J. Baugh, P. Hancock, G. A. Bonwick, D. H. Davies and R. Armitage, Rapid Commun. Mass Spectrom., 1995, 9, G. A. Bonwick, C. Sun, P. Abdul-Latif, P. J. Baugh, C. J. Smith, R. Armitage and D. H. Davies, J. Chromatogr. A, 1995, 707, P. Haglund, T. Alsberg, Å. Bergman and B. Jansson, Chemosphere, 1987, 16, H.-J. Stan and M. Linkerhägner, J. Chromatogr. A, 1996, 727, J. C. Bosboom, H.-G. Janssen, H.-G. J. Mol and C. A. Cramers, J. Chromatogr. A, 1996, 724, H. G. J. Mol, M. Althuizen, H.-G. Janssen and C. A. Cramers, J. High Resolut. Chromatogr., 1996, 19, H. Bagheri, P. E. G. Leonards, R. T. Ghijsen and U. A. Th. Brinkman, Int. J. Environ. Anal. Chem., 1993, 50, A. J. H. Louter, C. A. van Beekvelt, P. Cid Montanes, J. Slobodnik, J. J. Vreuls and U. A. Th. Brinkman, J. Chromatogr. A, 1996, 725, J. Slobodnik, A. C. Hogenboom, A. J. H. Louter and U. A. Th. Brinkman, J. Chromatogr. A, 1996, 730, J. Slobodnik, A. J. H. Louter, J. J. Vreuls, I. Liska and U. A. Th. Brinkman, J. Chromatogr. A, 1997, 768, Th. Hankemeier, P. C. Steketee, J. J. Vreuls and U. A. Th. Brinkman, J. Chromatogr. A, 1996, 750, Analyst, 2001, 126,