Metabolism of drug is that, the procedure by which drugs are transformed in the body to a form that is more readily evacuated, the metabolic fate of specific drug in terms of identification and quantification of the metabolites is request to know that, if its use it will not cause more problems than the medical condition it is product to cure the patient. In addition, it is very important to the drug companies to see how it works and help to provide drugs with more efficient.
Drug metabolism (or biotransformation) is the track which drug are made more water-soluble to subserve their removing from the body. There is a wide latitude of different drug structure, while there are only proportionally small number of chemical reaction lead to the production of metabolites.
What is Liquid chromatography/Mass spectrometry(LC/MS) ?
As it is named, it is an important method conjoins two separation procedures: Liquid chromatography(LC) and Mass spectrometry(MS). Both of these techniques are known for quite long time and they have been developed and used widely.
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LC is explained as that, it is a physical produce of separation in which the compound to be separated is apportioned between two phases, first one is stationary ( the stationary phase), while the other ( the mobile phase) travel in define direction. Any modern chromatographic system may be include four component parts which are:
Device for sample introduction.
A mobile phase.
A stationary phase.
These four components are used in different number of chromatographic techniques. For example, the injector in gas chromatography used to introduce the sample is of great importance and have to be selected in light of the specialties of the analyte under investigation (their stability and volatility) and the quantum of the analyte present, correct choice will give a successful analysis. In LC the injector is used to allow introduction of the analyte into the liquid stream. The mobile phase which are conjoint with the separation that occurs in a chromatographic system are the mobile phase and stationary phase.
It is known as an instrument which can forming, separating and detecting, either atomic or molecular, based on their mass-to-charge ratio. A mass-to-charge ratio of an ion is often abridge as m/z. A mass spectrometry can be contained four component parts which are:
A method of sample introduction.
A method of ion production.
A method of ion separation.
Facilities for ion detection and data manipulation.
Tandem Mass spectrometry:
It is a technique which can provide both the molecular weight of the analyte and the information concerning the structure of the molecule. However, the ionization technique is widely used for LC-MS, are designative 'soft ionization' in that make primarily molecular species with fragmentation.
Tandem mass spectrometry (MS-MS) is a method which cover a number of techniques in one stage of mass spectrometry. It is not necessarily that, the first stage is isolate an ion of interest and a second stage is used to probe the respect of this ion with others. The two steps of mass spectrometry are related in certain ways in order to provide the intended analytical information.
There are a considerable number of different MS-MS experiments that can be carried out but there are four which most widely used and these are:
The product ion scan.
The precursor-ion scan.
The constant-neutral-loss scan.
Selected decomposition monitoring.
-Instrument of MS-MS:
In this part there is one Instrument which will be mentioned and that is the Triple Quadrupole.
The Triple Quadrupole:
This one is most widely used in MS-MS instrument. As it is named, it contains three of sets of quadrupole rods in series. The second set of rods is not used as a mass separation device but as collision cell, while the fragmentation of ions transmitted by the first set of quadrupole rods is carried out and the third set is used as a device for focusing any produced ions. By controlling both sets of rods to allow the transition of ions of a single m/z ratio or a range of m/z values to give the intendment analytical information.
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Fig.1. Schematic of a triple quadrupole mass spectrometer. (Ardrey,2003).
Technique of MS-MS:
In this part also just one technique of MS-MS will be mentioned which is Selected Reaction Monitoring. In this technique, the fragmentation of a chosen precursor ion to a chosen product ion is monitored. This is carried out by revising each of the steps of mass spectrometry to send a single ion ( i.e. the precursor ion by MS1 and the product ion by MS3).
-Desorption electrospray ionization (DESI) and Direct analysis in real time (DART) techniques:
DESI and DART are atmospheric pressure desorption ionization techniques, which they can product ions of diverse chemical nature directly from solid surface for MS/MS analysis. These techniques are widely used and they give an ability to analyses samples by simple step, which is positioning a surface near the sampling inlet of an APIequipped.
Fig.2. (DART and DESI)( Elsevier,2008)
A prototype of API MS was evolved to used for analysis in nonlaboratory environments. The prototype API MS used a compact vacuum system and smaller pump to fit into a 0.1m-3 box and weight <45 Kg. In this technique the instrument can capable of tandem MS analyses because of the CIT (capillary isotachophoresis) mass analyzer and presents result which similar to those generated using benchtop API3D quadrupole ion trap instrument. DART and DESI are used for analysis molecules from over-thecounter drug and explosives.
Stanozolol (17_-hydroxy-17_-methyl-5_-androst-2-eno(3,2- c)-pyrazole( Fig. 3, compound 1) is one of the doping, which is an amphetamine stimulant a lot. The misuse of stanozolol is normally showed by the target exposing of the metabolite 3-hydroxy-stanozolol ( Fig. 3, metabolite 4). The exposing of this compound can be done by Gas Chromatography-Mass Spectrometry (GS-MS) or by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). Stanozolol metabolisms to 19 metabolites and all of them can be detected by using LC-MS/MS, but in this work just four metabolites will be mentioned.
In this application of LC-MS/MS the product of ion has been chosen to be at three position which are m/z 81,97 and 145. The product ion at m/z 81 is selected as a specific for stanozolol metabolites without a modification in A- or N-rings. Whereas, the product ion at m/z 97, 145 for metabolites hudroxylated in N-ring and 4-hydroxy-stanozolol metabolites respectively. With these condition the parent drug and up to 15 metabolites are found in positive doping test sample.
Fig.3. Proposed metabolic pathway for stanozolol. (Steroids, 2009).
Analysis of unconjugated component was done by changing the PH of the urine(5 ml) to 9.2 by increment of approximately 0.3 g sodium hydrogen carbonate: potassium carbonate (2:1, w:w). then, liquid- liquid extraction (LLE) was accomplished by adding 5 ml diethyl ether and mixing that about 20 min. After that, the sample was centrifuged and when the organic layer was appeared it separated and evaporated under oxygen free nitrogen (OFN) at 40c0.
In respect of conjugated and unconjugated component, there was 1 ml of phosphate buffer (PH7) added and 50 ml of the Î²-glucuronidase solution was added to 5 ml urine. Then the sample was hydrolysed about 2.5 h at 56c0. Next, the sample was cooled to room temperature, extraction was accomplished as described for the uncojugated component.
Regarding to LC-MS/MS analysis, the leftover was dissolved into 20 ml of methanol : water (50:50, v:v), then 20 ml of solution were directly injected into the system.
Precursor ion analysis (QqQ):
Selecting the making ion at m/z 81,97 and 145 was applied using the triple quadrupole instrument (QqQ) in precursor ion scan method. The collision energy for m/z 81 and 91 was 45 eV and for m/z 145 was 30 eV. The width of peak was setting at 0.7 Da and the scan rate at 0.4 s/scan. Column, mobile phase composition (LC-MS/MS studies) were used.
selected-reaction monitoring (SRM) analysis:
A SRM manner using the triple quadruple instrument was promoted for LC-LC/MS detection of stanozolol for each metabolite. The acquisition time was set at 50 ms for each motion.
Precursor ion scan and natural loss method were useful tools for detection of steroid metabolites. Because of the lower amount of interferences the more specific the approach the higher number of metabolites identified. Wherefore, specific ions were chosen for detection of stanzolol metabolite. The ion at m/z 81 has been noted for stanzolol derivatives without any modification in the N-ring, the ion at m/z 97 was reported for stanzolol derivatives with a hydroxyl group in the N-ring, while the ion at m/z 145 was noted to 4-hydrolysed-stanzolol derivatives. When human urine was applied no metabolites were found in non-hydrolysed urine, compatible with period reported results, which articulate that, less than 5% of stanzolol metabolites are found in the uncojugated component. Whereas, after hydrolysis 15 metabolites were detected. By using precursor ion scan of m/z 81 articulating an unaltered N-ring six compounds were detected. Five metabolites were detected using the precursor ion of m/z 97 and four appeared using the precursor ion scan of m/z 145. Some of these compounds were identify by using the analysis of available standards. As a result, compound 1 was assigned to stanozolol, metabolite 2 to 16Î²-hydroxy-stanozolol, metabolite 3 to 4Î²-hydroxy-stanozolol and metabolite 4 to 3-hydroxy-stanozolol.
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Fig. 4. Precursor ion chromatogram at m/z 81 (top), m/z 97 (medium) and m/z 145 (below) for (a) human urine declared negative and (b) human urine declared positive for stanozolol. Numbers within brackets refer to metabolites in Fig. 3. (Steroids, 2009).
LC-MS/MS QqQ in precursor ion scan is very useful method and allowed to detect 19 metabolites of stanozolol. As a result, LC-MS/MS can be used to exposition of drugs by using sample of urine and decide if that drug is beneficial or not. In addition, helps to appear the weakness of that drug and helps drug companies to provide and product efficient drug.