Gel Permeation Chromatography Purification and Gas Chromatography-Mass Spectrometry Detection of Multi-Pesticide Residues in Traditional Chinese Medicine

Wan-E Zhuang, Zhen-Bin Gong

.

DOI: 10.4236/ajac.2012.31005                

Abstract

The measurement of 23 organochlorine, organophosphorus, and pyrethroid pesticides in typical traditional Chinese medicine (TCM), flos lonicerae, was made using gel permeation chromatography (GPC) purification and gas chroma- tography-mass spectrometry (GC-MS) detection. The pesticides were extracted with ultrasonic device and 5.0 mL mixture of ethyl acetate and cyclohexane (1:1, v/v). Coextractants from sample matrices which may have interfere to the qualitative and quantitative analysis, such as pigments, were removed using GPC purification. Simultaneous full scan and selective ion monitor (scan/SIM) mode for GC-MS was used for qualitative and quantitative analysis, which pro- vided retention time and characteristic fragments ratio for each pesticide so as to positively identify each analyte. Rela- tive standard deviations (RSDs) were within 7.7% (5.0 - 22.5 μg/kg, n = 3). The recoveries of pesticide standards at the spiked concentration of 5.0 - 22.5 μg/kg were between 87.1% and 110.9%. Limits of detection (LODs) for the analytes were 0.16 - 3.2 μg/kg, which could meet the demand of routine analysis and TCM quality control.

Keywords

Traditional Chinese Medicine; Multi-Pesticide Residue; Flos Lonicerae; Gel Permeation Chromatography; Gas Chromatography-Mass Spectrometry

1. Introduction

Traditional Chinese Medicine (TCM) usually means medicinal plants. Herb water, mixed tablet, and powder from the extracts of herbs are the general styles in clinical practice. The usage of TCM in China has a very long history. The influence of TCM on health care system has been profound in Asia as well as in the West in recent years [1-3]. The concerning of contaminations of heavy metals [4-6] and pesticides [2,6-9], which may be introduced during the cultivation, transportation, preparation and preservation, has been increased as the popularity of TCM enlarged.

Pesticides using to control various insect pests all over the world have advanced agriculture to gain great productivity [10,11]. In the mean time, they contaminate the environment [12] and endanger human health [13]. Some pesticides are neural destroyers [14], and some act as hormones, which may disturb human endocritic system [15]. Most of the pesticides are bio-accumulated and may be transferred along the food chain [16], similar to the environmental behavior of heavy metals. For these reasons, the contents of pesticide residues in Chinese herbs have been concerning by public in China and the other areas of the world. There have methods reported for the analysis of pesticide residues in TCM [9,17,18]. However, a rapid procedure or screening method to determine organochlorine, organophosphorus, and pyrethroid pesticides in TCM, especially for complex matrix medicinal herbs, such as flos lonicerae, is of great significance in quality control activities.

Typically, measurement of multi-residue in complex matrices comprises sample pre-treatment, separation and detection by gas chromatography-mass spectrometry (GCMS) [19,20]. The pre-treatment usually is time-consuming, which includes extraction, purification, and enhancement of the analytes to reduce or eliminate possible interference to the accurate detection. Soxhlet extraction [21], accelerated solvent extraction (ASE) [16], supercritical fluid extraction (SFE) [17], solid phase extraction (SPE) [12,13], microwave-assisted extraction (MAE) [22], matrix solid phase disperse extraction (MSPDE) [19,23], and disperse solid phase extraction (DSPE, QuEChERS) [24-26] have been investigated for pesticides analysis. Gel permeation chromatography (GPC), as reported in bibliography [10,16,27,28], may be one of the best techniques for the analysis of multi-residue of pesticides in TCM, which separates lower molecular weight target pesticides from higher molecular weight chemical matrices, such as pigments. Gas chromatography-mass spectrometry can do two-tier identification and confirmation with the retention time and the relative ratios of characteristic ions of the pesticide. The select ion monitor (SIM) can eliminate matrix influence and enhance selectivity and sensitivity effectively. The double mode of scan plus SIM mode (scan/SIM) can qualify and quantify target compounds simultaneously in a single injection.

The purpose of this study was to develop a novel method for accurately and simultaneously determination of organochlorine, organophosphorus, and pyrethroid pesticides in flos lonicerae. The advantage of GPC purification and good separation and high sensitivity of GC-MS was investigated in qualitative identification and quantitative detection of multi-pesticide in complex chemical matrices.

2. Experimental

2.1. Chemicals and Reagents

Acetone, acetonitrile, cyclohexane and ethyl acetate were all of HPLC grade and purchased from Thermal Fisher Co. (USA). The 23-pesticide standards were from Chem Service Co. (USA), which were of purity ≥ 98.1%.

1000 µg/mL stock solutions for each pesticide were prepared with acetone and stored in freezer at –18˚C. 10 µg/mL mixed standard solution for daily work were obtained by mixing and diluting stock solutions with acetone, and stored in a refrigerator at 4˚C.

2.2. Apparatus

Agilent 7890A gas chromatography, 5975C mass spectrometer, and 7683B auto sample injector was used (Agilent Technologies, USA). Data acquisition, data processing, and instrumental control were performed with Agilent Enhanced ChemStation (Agilent Technologies, USA). A DB-5 MS fused silica capillary column of 30 m × 0.25 mm with 0.25 µm film thickness from Agilent Technologies was used.

The GPC system (Vario, LC Tech, Germany) consisted of high pressure pump, auto-sampler with 24-sample vials (10 mL) and 5.0 mL sample loop, GPC column, and 24 fraction collected vials (100 mL). The clean-up GPC column was packed polystyrene-divinylbenzene (Bio-Beads S-X3, 400 mm × 25 mm I.D., 200 - 400 mesh).

MS2 mini-shaker (IKA, Germany), B5200S-OT ultrasonic extract device (Branson, USA), centrifuge (Shanghai, China), auto concentrating apparatus (EVA Ш, LC Tech, Germany), and laboratory-built nitrogen evaporator were used.

2.3. Sample Extraction and Purification

1.000 g of ~2 kg homogenized dry flos lonicerae sample was extracted with 5.0 mL mixed ethyl acetate and cyclohexane (1:1, v/v) for 10 min. The extraction was repeated for 3 times. Combine the extracts together, and then concentrated with a nitrogen stream to 10.0 mL. 5.0 mL of the extract solution was injected and separated on GPC with a mixed mobile phase of ethyl acetate and cyclohexane (1:1, v/v) at a flow rate of 5.0 mL/min. The fraction containing the analyzed pesticides was collected within the retention time of 17 to 36 min (totally 95.0 mL of eluant). The GPC fraction was evaporated and concentrated to 5.0 mL using EVA Ⅲ rotary evaporator at 40˚C, and further concentrated to near dry with nitrogen stream, re-dissolved with 0.5 mL mixture of ethyl acetate and cyclohexane (1:1, v/v) for GC-MS analysis.

2.4. GC-MS Conditions

Carrier gas was high purity of Helium (≥99.999%). The separation of all pesticides was performed with a constant pressure mode. The injection port of GC was 250˚C. 1.0 µL of pretreated sample solution was injected in splitless mode (split valve closed for 0.75 min). The retention time was locked by chlorpyrifos-methyl. Programmed temperature for GC oven was initially 50˚C for 1 min, increased to 125˚C at a rate of 25˚C/min, and then to 300˚C at 10˚C/min, and finally maintained at 300˚C for 10 min until all the analytes eluted.

Electron impact (EI) ionization source was used at 70 eV. The interface between the GC and mass detector was maintained at 280˚C. The temperature for EI source and quadrupole were set at 230˚C and 150˚C, respectively. The solvent delay time was set to 3.5 min. Full scan and selected ion mode (Scan/SIM) were used to qualitative identification and quantitative detection of multi-pesticide.

Figure 1 is the chromatograms of 23-pesticide standard and standard spiked flos lonicerae sample at optimized operating conditions. All pesticides in Figure 1 have been separated and eluted before the retention time 23.237 min. Additional 8 min at 300˚C was set for column cleaning-up and ready for next injection

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	K. Chan, “Progress in Traditional Chinese Medicine,” Trends in Pharmacological Sciences, Vol. 16, No. 6, 1995, pp. 182-187. doi:10.1016/S0165-6147(00)89019-7
[2]	K. Chan, “Some Aspects of Toxic Contaminants in Herbal Medicines,” Che-mosphere, Vol. 52, No. 9, 2003, pp. 1361-1371. doi:10.1016/S0045-6535(03)00471-5
[3]	P. Drasar and J. Moravcova, “Recent Advances in Analy- sis of Chinese Medical Plants and Traditional Medi- cines,” Journal of Chromatography B-Analytical Tech- nologies in the Biomedical and Life Sciences, Vol. 812, No. 1-2, 2004, pp. 3-21. doi:10.1016/S1570-0232(04)00741-X
[4]	E. Ernst, “Toxic Heavy Metals and Undeclared Drugs in Asian Herbal Medi-cines,” Trends in Pharmacological Sciences, Vol. 23, No. 3, 2002, pp. 136-139. doi:10.1016/S0165-6147(00)01972-6
[5]	R. J. Huang, Z. X. Zhuang, Y. Tai, R. F. Huang, X. R. Wang and F. S. C. Lee, “Direct Analysis of Mercury in Traditional Chinese Medicines Using Thermolysis Cou- pled with Online Atomic Absorption Spectrometry,” Ta- lanta, Vol. 68, No. 3, 2006, pp. 728-734. doi:10.1016/j.talanta.2005.05.014
[6]	F. M. Li, Z. L. Xiong, X. M. Lu, F. Qin and X. Q. Li, “Strategy and Chromatographic Technology of Quality Control for Traditional Chinese Medi-cines,” Chinese Jour- nal of Chromatography, Vol. 24, No. 6, 2006, pp. 537- 544. doi: 10.1016/S1872-2059(06)60022-9
[7]	J. F. Deng, “Clinical and Laboratory Investigations in Herbal Poisonings,” Toxicology, Vol. 181, 2002, pp. 571- 576. doi:10.1016/S0300-483X(02)00484-5
[8]	J. Jung, M. Her-manns-Clausen and W. Weinmann, “An- orectic Sibutramine Detected in a Chinese Herbal Drug for Weight Loss,” Forensic Science International, Vol. 161, No. 2-3, 2006, pp. 221-222. doi:10.1016/j.forsciint.2006.02.052
[9]	J. Xue, L. L. Hao and F. Peng, “Residues of 18 Or- ganochlorine Pesticides in 30 Traditional Chinese Medi- cines,” Chemosphere, Vol. 71, No. 6, 2008, pp. 1051- 1055. doi:10.1016/j.chemosphere.2007.11.014
[10]	A. G. Frenich, P. P. Bolanos and J. L. M. Vidal, “Mul- tiresidue Analysis of Pes-ticides in Animal Liver by Gas Chromatography Using Triple Quadrupole Tandem Mass Spectrometry,” Journal of Chroma-tography A, Vol. 1153, No. 1-2, 2007, pp. 194-202. doi:10.1016/j.chroma.2007.01.066
[11]	S. N. Sinha and M. Odetokun, “Liquid Chromatography Mass Spectrometer (LC-MS/MS) Study of Distribution Patterns of Base Peak Ions and Reaction Mechanism with Quantification of Pesticides in Drinking Water Using a Lyophilization Technique,” American Journal of Ana- lytical Chemistry, Vol. 2, No. 5, 2011, pp. 511-521. doi:10.4236/ajac.2011.25061
[12]	S. Wang, P. Zhao, G. Min and G. Z. Fang, “Multi-Resi- due Determination of Pesticides in Water Using Multi- Walled Carbon Nanotubes SolidPhase Extraction and Gas Chromatography-Mass Spectrometry,” Journal of Chro- matography A, Vol. 1165, No. 1-2, 2007, pp. 166-171. doi:10.1016/j.chroma.2007.07.061
[13]	P. Liu, Q. Liu, Y. Ma, J. Liu and X. Jia, “Analysis of Pesticide Multiresidues in Rice by Gas Chromatography- Mass Spectrometry Coupled with Solid Phase Extrac- tion,” Chinese Journal of Chromatography, Vol. 24, No. 3, 2006, pp. 228-234. doi:10.1016/S1872-2059(06)60011-4
[14]	C. S. Roegge, O. A. Timofeeva, F. J. Seidler, T. A. Slot- kin and E. D. Levin, “De-velopmental Diazinon Neuro- toxicity in Rats: Later Effects on Emotional Response,” Brain Research Bulletin, Vol. 75, No. 1, 2008, pp. 166- 172. doi:10.1016/j.brainresbull.2007.08.008
[15]	R. McKinlay, J. A. Plant, J. N. B. Bell and N. Voulvoulis, “Calculating Human Exposure to Endocrine Disrupting Pesticides Via Agricultural and Non-Agricultural Expo- sure Routes,” Science of the Total Environment, Vol. 398, No. 1-3, 2008, pp. 1-12. doi:10.1016/j.scitotenv.2008.02.056
[16]	A. G. Frenich, J. L. M. Vidal, A. D. C. Sicilia, M. J. G. Rodriguez, P. P. Bolanos and R. G. A. C. Contaminan, “Multiresidue Analysis of Organochlorine and Organo- phosphorus Pesticides in Muscle of Chicken, Pork and Lamb by Gas Chromatography-Triple Quadrupole Mass Spectrometry,” Analytica Chimica Acta, Vol. 558, No. 1-2, 2006, pp. 42-52. doi:10.1016/j.aca.2005.11.012
[17]	Y. C. Ling, H. C. Teng and C. Cartwright, “Supercritical Fluid Extraction and Clean-up of Organochlorine Pesti- cides in Chinese Herbal Medicine,” Journal of Chroma- tography A, Vol. 835, No. 1-2, 1999, pp. 145-157. doi:10.1016/S0021-9673(98)01077-2
[18]	M. Barriada-Pereira, E. Concha-Grana, M. J. Gonzalez- Castro, S. Muniate-gui-Lorenzo, P. Lopez-Mahia, D. Prada- Rodriguez and E. Fer-nandez-Fernandez, “Microwave- Assisted Extraction Versus Soxhlet Extraction in the Ana- lysis of 21 Organochlorine Pes-ticides in Plants,” Journal of Chromatography A, Vol. 1008, No. 1, 2003, pp. 115- 122. doi:10.1016/S0021-9673(03)01061-6
[19]	P. P. Bolanos, A. G. Frenich and J. L. M. Vidal, “Appli- cation of Gas Chromato-graphy-Triple Quadrupole Mass Spectrometry in the Quantifi-cation-Confirmation of Pes- ticides and Polychlorinated Bi-phenyls in Eggs at Trace Levels,” Journal of Chromatography A, Vol. 1167, No. 1, 2007, pp. 9-17. doi:10.1016/j.chroma.2007.08.019
[20]	Z. Q. Huang, Y. J. Li, B. Chen and S. Z. Yao, “Simulta- neous Determination of 102 Pesticide Residues in Chi- nese Teas by Gas Chromatogra-phy-Mass Spectrometry,” Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences, Vol. 853, No. 1-2, 2007, pp. 154-162. doi:10.1016/j.jchromb.2007.03.013
[21]	S. H. Hong, U. H. Yim, W. J. Shim, J. R. Oh, P. H. Viet and P. S. Park, “Persistent Organochlorine Residues in Estuarine and Marine Sediments from Ha Long Bay, Hai Phong Bay, and Ba Lat Estuary, Vietnam,” Chemosphere, Vol. 72, No. 8, 2008, pp. 1193-1202. doi:10.1016/j.chemosphere.2008.02.051
[22]	S. B. Singh, G. D. Foster and S. U. Khan, “Determination of Thiophanate Methyl and Carbendazim Residues in Vegetable Samples Using Mi-crowave-Assisted Extrac- tion,” Journal of Chromatography A, Vol. 1148, No. 2, 2007, pp. 152-157. doi:10.1016/j.chroma.2007.03.019
[23]	M. G. D. Silva, A. Aquino, H. S. Drea and S. Navickiene, “Simultaneous Deter-mination of Eight Pesticide Residues in Coconut Using Mspd and Gc/Ms,” Talanta, Vol. 76, No. 3, 2008, pp. 680-684. doi:10.1016/j.talanta.2008.04.018
[24]	C. Lesueur, P. Knittl, M. Gartner, A. Mentler and M. Fuerhacker, “Analysis of 140 Pesticides from Conven- tional Farming Foodstuff Samples after Extraction with the Modified Quechers Method,” Food Control, Vol. 19, No. 9, 2008, pp. 906-914. doi:10.1016/j.foodcont.2007.09.002
[25]	T. D. Nguyen, E. M. Han, M. S. Seo, S. R. Kim, M. Y. Yun, D. M. Lee and G. H. Lee, “A Multi-Residue Method for the Determination of 203 Pesticides in Rice Paddies Using Gas Chromatography/Mass Spectrometry,” Ana- lytica Chimica Acta, Vol. 619, No. 1, 2008, pp. 67-74. doi:10.1016/j.aca.2008.03.031
[26]	T. D. Nguyen, J. E. Yu, D. M. Lee and G. H. Lee, “A Multiresidue Method for the Deter-mination of 107 Pesti- cides in Cabbage and Radish Using Quechers Sample Preparation Method and Gas Chromatography Mass Spec- trometry,” Food Chemistry, Vol. 110, No. 1, 2008, pp. 207-213. doi:10.1016/j.foodchem.2008.01.036
[27]	B. Hu, W. Song, L. Xie and T. Shao, “Determination of 33 Pesticides in Tea Using Accelerated Solvent Extrac- tion/Gel Permeation Chromatography and Solid Phase Ex- traction/Gas Chromato-graphy-Mass Spectrometry,” Chinese Journal of Chromatogra-phy, Vol. 26, No. 1, 2008, pp. 22-28. doi:10.1016/S1872-2059(08)60009-7
[28]	F. Goni, R. Lopez, A. Etxeandia, E. Millan, A. Vives and P. Amiano, “Method for the Determination of Selected Organochlorine Pesticides and Polychlorinated Biphenyls in Human Serum Based on a Gel Permeation Chroma- tographic Clean-Up,” Chemosphere, Vol. 76, No. 11, 2009, pp. 1533-1539. doi:10.1016/j.chemosphere.2009.05.041
[29]	C. Ferrer, M. J. Gomez, J. F. Garcia-Reyes, I. Ferrer, E. M. Thurman and A. R. Fernandez-Alba, “Determination of Pesticide Residues in Olives and Olive Oil by Matrix Solid-Phase Dispersion Followed by Gas Chromatogra- phy/Mass Spectrometry and Liquid Chromatography/ Tandem Mass Spectrometry,” Journal of Chromatogra- phy, A, Vol. 1069, No. 2, 2005, pp. 183-194. doi:10.1016/j.chroma.2005.02.015