GAS CHROMATOGRAPHY-MASS SPECTROMETRY (GC/MS)

Journal Title: The analysis of common metabolites of Organophosphorus Pesticides in Urine by Gas Chromatography-Mass Spectrometry (GC/MS)

Gas Chromatography-Mass Spectrometry is a common type of chromatography used in analytic chemistry for separating, quantifying, identifying and analyzing

compounds that can be vaporized without decomposition. Besides, it is also used for determining of molecular weight and element compositions of unknown or ganic compounds in complex mixtures. However, a broad of samples that only can be analyzed as long as the compounds are sufficiently thermal stable and volatile for gas chromatographic.

Figure1: Gas Chromatography-Mass Spectrometry The general application is included quantitation of pollutants in drinking and wastewater using official U.S. Environmental Protection Agency (EPA) methods;

quantitation of drugs and their metabolites in blood and urine for both pharmacological and forensic applications; identification of unknown organic compounds in hazardous waste dumps; identification of reaction products by synthetic chemists; and analysis of industrial products for quality control.

The component of Gas Chromatography-Mass Spectrometry includes carries gas, sample injection port, columns, column temperature and detector.

Figure 2: schematic diagram of gas chromatograph

a) Carries gas: The carrier gas must be chemically inert. Commonly used gases include nitrogen, helium, argon, and carbon dioxide. The choice of carrier gas is often dependent upon the type of detector which is used. The carrier gas system also contains a molecular sieve to remove water and other impurities.

b) Sample injection port: in order to get optimum column efficiency, the sample should not be too large, and the most common injection method is where a micro syringe is used to inject sample through a rubber septum into a flash vapouriser port at the head of the column. The temperature of the sample port is usually about 50C higher than the boiling point of the least volatile component of the sample.

c) Columns: There are two general types of column, packed and capillary (also known as open tubular). Packed columns contain a finely divided, inert, solid support material (commonly based on diatomaceous earth) coated with liquid stationary phase. Most packed columns are 1.5 - 10m in length and have an internal diameter of 2 - 4mm. Capillary columns have an internal diameter of a few tenths of a millimeter. They can be one of two types; wall-coated open tubular (WCOT) or support-coated open tubular (SCOT). d) Columns temperature: The column temperature must be controlled to within tenths of a degree. e) Detector: There are six types of common detector which include Mass Spectrometer (GC/MS), Flame Ionization Detector (FID), Thermal Conductivity Detector (TCD) Electron Capture Detector (ECD), Nitrogen Phosphorous Detector (NPD), and Atomic Emission Detector (AED). Detector Mass Spectrometer (GC/MS) Flame Ionization Detector (FID) Thermal Conductivity Detector (TCD) Electron Capture Detector (ECD) Used for Identification of unknown compounds Compounds containing Carbon Universal detection for gases without Carbon Selective detection of Nitrogen and Phosphor containing compounds Nitrogen Phosphorous Detector (NPD) Atomic Emission Detector (AED) Selective detection of halogen containing compounds Selective detection of elemental composition

In this study, it was to study the common metabolites of organophosphorus pesticides in human urine by using gas Chromatography-Mass Spectrometry in order to know which metabolites may be used as the exposure biomarkers to pesticides. There were four common dialkyl phosphates include O,O-Dimethyl phosphate (DMP), O,O-Dimethyl phosphorodithioate (DMDTP), O,O-Diethyl phosphate (DEP), and O,O-Diethyl phophorothionate (DETP) will be anlaysed by using gas

chromatography. However, before running the metabolites by using GC/MS, solid phase extraction was used for extraction of organophosphate metabolites from urine and the extract was derivatized. The detector of this study, mass spectrometer (MS) is

better than used GC/Flame Photometric Detector (FPD) due to the ability of MS in indentifying of these metabolites. The instrument in this study include Hewlett-Packard Model 5890 Series II Plus gas Chromatograph directly interfaced to a HP 5972 mass selective detector was used. For the data analysis, a HPG 1034C MS chemstation was used. The extraction of biological sample was done by SPE-24G Extraction kit. A HP fused-silica capillary column (length 25m, i.d. 0.2mm, film thickness 0.33m) was coupled to the ion source. Helium at a flow rate of 0.8 ml/min was employed as a carrier gas. Samples were injected in split at a ratio of 10:1. The injector and the transfer line GC/MS were at 280C and 300C, respectiviely. The oven temperature was set at 70C initially, subsequently increased by 4C per min to 150C, held there for 1 min, subsequently increased 20C per min to 310C, and held there for 1 min. A 2 l aliquot of a derivatized sample was injected. The mass spectrometer was operated at 70 eV in the electron impact (EI) mode using SCAN or SIM (selected ion monitoring). The operating conditions of GS/MS and the selected ion groups for the identification of 4 metabolites in SIM mode were listed in table 1.

After that, all the standards of four metabolites were prepared in methanol at a concentration of 1 mg/ml and stored at 4C in the dark. Any analytical method mixed

standard of all four metabolites was prepared in acetone. After prepared, derivatization of standard was made. The dry residue (containing 100g of each metabolite) was dissolved in 100l of a mixture of HMDS-TMCS-pyridine (2:1:10, v/v/v). The mixture was stirred on vortex.

The extraction procedure for recovery test is the cychloexyl extraction cartridge was used to extract dialkyl phosphate metabolites from urine. A 3ml aliquot of urine was pipetted into a 15ml test tube. A 30l aliquot of the metabolites standard solution (10g/ml) was spiked in the urine sample (3ml). Sodium chloride, ammonium sulfates, ammonium acetate were added as a salts, and acetic acid, or hydrochloric acid was added as a pH modifier, and the mixture was vortexed for 1 min. then, the cyclohexyl extraction catridge was pre-washed with one column volume (3ml) of methanol, water, and salt-saturated water. The urine sample was applied to the cartridge. The cartridge was washed with 2ml 20% methanol in hexane. Under a negative pressure (30mmHg), the cartridge was then placed into a 15mL graduated centrifuge tube and eluted with 3 ml methanol. Triphenyphosphate (10g/ml, 25l) was added as an internal standard and 0.4g powered anhydrous sodium sulfate was added to the methanol eluate, and the solution was vortex-mixed. After removing sodium sulfate, methanol was evaporated under nitrogen stream. The centrifuge tube was dried in vacuum desiccators with P2O5/KOH for 15 min. An 100l portion of the derivatization solution (HMDS/TMCS/pyridine, 2:1:10, v/v/v) was added, and the mixture was vortex-mixed for 1 min. the extraction procedure of urinary metabolites was same as Scheme 1. Then, it was identifed by gas Chromatography-Mass Spectrometry. The phenthorate was orally administered to the two male Sprague-Dawley rats (200 mg/kg, in 0.5% Carboxymethyl-cellulose solution). Urine samples were collected for 24 hours in metabolic cages. A blank urine sample was collected before the admistration of phenthate to rats.

In this study, in can be concluded that, the calibration curve obtained from each metabolites standard using by GC/MS/SIM has shown good linearity and detection limits of metabolites were range of 0.05-0.1g/ml in urine. The DMP, DMDTP, monomethyl phosphate and dimethyl- thiophospate were detected in urine after oral administration of phenthoate of rats. So, the result of this study may be applied to development of exposure biomarker for monitoring of environmental pollutants.