Assignment GCMS.docx

7/29/2019 Assignment GCMS.docx

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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 ofchromatography 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 beanalyzed 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;
http://en.wikipedia.org/wiki/Chromatographyhttp://en.wikipedia.org/wiki/Analytic_chemistryhttp://en.wikipedia.org/wiki/Separation_processhttp://en.wikipedia.org/wiki/Vaporizedhttp://en.wikipedia.org/wiki/Chemical_decompositionhttp://en.wikipedia.org/wiki/Chemical_decompositionhttp://en.wikipedia.org/wiki/Vaporizedhttp://en.wikipedia.org/wiki/Separation_processhttp://en.wikipedia.org/wiki/Analytic_chemistryhttp://en.wikipedia.org/wiki/Chromatography


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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 usedgases 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, thesample 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.


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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) orsupport-coated open

tubular(SCOT).

d) Columns temperature: The column temperature must be controlled towithin tenths of a degree.

e) Detector: There are six types of common detector which include MassSpectrometer (GC/MS), Flame Ionization Detector (FID), Thermal

Conductivity Detector (TCD) Electron Capture Detector (ECD), Nitrogen

Phosphorous Detector (NPD), and Atomic Emission Detector (AED).

Detector Used for

Mass Spectrometer (GC/MS) Identification of unknown compounds

Flame Ionization Detector (FID) Compounds containing Carbon

Thermal Conductivity Detector (TCD) Universal detection for gases without Carbon

Electron Capture Detector (ECD) Selective detection of Nitrogen and Phosphor

containing compounds

Nitrogen Phosphorous Detector (NPD) Selective detection of halogen containing compounds

Atomic Emission Detector (AED) 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


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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


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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.


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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.

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