The instrument of GC/MS/MS is similar to GC-MS, with an exception where a second phase of mass fragmentation is added .Here a second quadrapole is used in an instrument which is known as MS/MS. Here first the sample is subjected to gas chromatography and then placed in mass spectrometer where each ion is fragmented1 MS/MS consist of two quadrupoles :Q1 and Q2 , Q1 is last filter and Q2 is precursor ion. Both the quadruples are connected by a collision cell. They may be used for scanning or static mode which depends on the type of MS-MS analysis performed. Analysis include different types they are product ion scan, single reaction monitoring (SRM), Precursor ion scan and multiple reaction monitoring2. Finally the product ions are selected by the third quadropole (Q3) Mass filter . The target compounds are divided into six steps in the development of an MS/MS method. They are (1) specifying segment breaks (2). Selection of precursor ion (3) adjustment of Q value (4) examination isolation efficiency (5).Optimization of collision induced dissociation (CID) Voltage (6) Defining the scan range of the product3.
This technique is very sensitive, effective. It provides great selectivity against matrix interference5.This technique has the properties like reproducibility, repeatability and specificity which are very advantageous. This technique improves the limit of detection and limit of quantification values6.This technique is rapid and sensitive. It requires less sample preparation by which different types of metabolites are accurately measured. It is having high through output7.By this technique the classical mass spectra can be obtained. In this low electric field were observed. Q2 acts as a good focusing device, it is useful for mixture screening. This technique is tolerated to high pressure ionization sources8.MS/MS allows improvement in signal to noise ratio(S/N) for detecting compounds which are present in mixtures. This technique improves the detection limit for complex mixtures9.
The main disadvantage of this technique is that it can analyse only the compounds having vapour pressure exceeding around 10-10 torr. Analysis can be done only for the compounds with low pressures. By this technique determination of the position on the aromatic ring is difficult. Some isomeric compounds cannot be differentiated by mass spectrometry; they can be separated only by chromatography. This technique shows incompatibility between two techniques. The compound used in the gas chromatograph is a trace component present in GC-carrier gas. It requires pressure about 760 torr. But the mass spectrometer uses vacuum about 10-6 to 10-5 torr for the operation. It is a major problem in this technique10. In this technique lower E-collisions and incomplete fragmentations were observed. It is not suitable for pulsed ionization methods. The mass range and resolution were limited. MS/MS spectra is mostly instrument dependent8.
This technique is used for the analysis of environmental samples, mainly pesticides. It is the powerful technique which is used for quantitation of low levels of target compounds by using high sample matrix background2.This technique is used in doping analysis , It is applied in forensic science , drug testing and this method is used for the identification of pesticide residues in food . This method is used in biomarker analysis for oil exploration. This applied for the trace analysis of dioxine11.It is applied for the analysis of acidic phytohormones and related regulators12.This technique is used for the identification of biological matrices like blood, hair and urine etc.They are used in clinical and forensic sciences6. This technique is used to identify and quantitate the volatile and semi volatile organic compounds in complex mixtures. It is used for the determining molecular weights and elemental compositions of unknown compounds. This technique is also used to determine the structure of unknown organic compounds both by matching their spectra with reference spectra. This method is used to quantitate pollutants presenting drinking and waste water. This technique is used to quantitate Drugs and their metabolites in blood and urine. It is used to identify unknown organic compounds in hazardous waste dumps. This technique is applied to identifying reaction products by synthetic organic chemists. This technique is employed to analyzed industrial products for quality control10.This technique is used in newborn screening7.This technique is used for the detection of aminoacidemia, organic acidemias,it is also used for the detection of disorders of fatty acid oxidation7.It is used for the metabolic disease screening in newborns. This method is used in detecting the sequence of different proteins13. This technique is used for the organic trace analysis14.
Presently there have been increased human estrogens in marine ecosystem which are showing their effects on marine habitat. The mot significant sources of human estrogens in the marine environment are the raw sewage discharge. For this, a technique has been developed called as GC-MS-MS for analyzing natural estrogens and estrone (E1) and 17β - estradiol (E2) and many other synthetic estrogens. This female sex hormone plays an important role in feminization and other reproducible abnormalities. These hormones are secreted by all vertebrates. All the natural estrogens are presented in free form like glucuronide or sulfate conjugates which are executed in urine. Mostly, the steroid estrogens are identified at low ng/L levels in the receiving waters. At these levels, it includes vitellogenin, a protein present in reproducing females.
Mussels are used as bio-indicators which are developed for investigation of the many organic concomitants like alkyl phenols and other estrogenic chemicals. For the analysis of volatile compounds in waste water effluents GC-MS is most frequently used method. Analysis of these estrogens can be done by not using any derivatization but leads to poor chromatography. The detection limits should be between 2 and 5 ng/L. For advanced detection limits GC-MS/MS is used. Analytical methods which are sensitive are based on this technique. For detecting estrogens, the detection was at least one order of magnitude. Hence GC-MS/MS method is used for steroid estrogens in mussels.
For the analysis, the chemical and reagents required are estrogenic steroids E1 , E2 EE2, DES and 5β- cholestan- 3β -ol, silicagel,TMS,derivatization reagent,1%tri methyl chlorosilane,stock solution of steroid standards. The analysis was performed by ion trap MS-MS system connected with temperature programmable injector.GC separation was done on DB-5-column by using injector. The temperature of injector must be 50 to 300̊ C at 100̊ C /min. The temperature of the column must be at 50̊ C to 3.5 mins. The MS was operated in electron impact mode (EI).Then it is placed for the recovery of target analytes from mussels (n=3), which was found above 60% with RSD ranging from 8% to 13%. For aqueous samples the recoveries were found above 80% ranges from 3% to 7%. The detection limits for the target analytes were found at 0.1 ng/g to 1.0 ng/g for mussel sample analysis. For analysis of water sample the range was found at 0.5 ng/l to 1.2 ng/l
The significant use of method can be seen by analysis of environmental samples from ST.John's and Halifax Canada. Here, the raw sewage is directly discharged into harbors. The detection range of estrone and 17β -estradiol were found to be 1.5 ng/l and 1.8 ng/l in sea water samples which are collected from ST.John's harbor. The trace quantities of estrone were measured in some mussels which are collected from Halifax harbor.
Discharging of raw sewage at Halifax and ST.John's harbors are the primary sources of estrogenic steroids in these locations. These are potent endocrine disruptic chemicals which are exposed to marine aquatic organisms to chemicals which are helpful to scientific community. This result indicates the presence of natural estrogens and fecal biomarker coprostarol in samples of sea water. Hence, due to fast metabolism, the steroid estrogens are detected in trace levels in mussels15.
GC-MS-MS technique is used for detecting drugs whose mass spectra is unavailable. In this technique the detection of drugs can be done by following mass frontier. Simple laws of organic chemistry and predictions can be made theoretically by choosing electron impact or chemical ionization. The mass frontier is similar to mass spectra interpreter at tips of finger which helps in the development of MS-MS method, target compounds can be isolated from the matrix and falls positives from the analysis can be eliminated. Hence this is an effective method.
Journal of mass spectrometry,vol,31,129-137(1996) by Edmond de Hoffmann.
Test book of gass chromatography ---mass spectroscopy chapter 31 by Ronald A. Hites
Proc.Natl.Acad.Sci USA vol.83,pp.6233-6237,September 1986 Chemistry,protein sequencing by Tandem Mass Spectroscopy by DONALD F.HUNT,JOHN R.YATES,JEFFREY SHBANOWITZ etc
16. Journal of Chemosphere volume 76,Issue8,Auguest 2009,pages 1156-1162 by Gursankar Saravanabhavan ,Robert Helleur and Jocelyne Hellou.