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Question:

What is Metabolomics. What is LC/MS and GC/MS also analysis GC/MS and LC/MS for analyzing steroids. Also Tell some  Advantages of GC/MS in steroid profiling in paediatrics.

 

 

Answer:

Introduction:

Metabolomics is the study of metabolites and since there is huge variety of metabolites present hence a comprehensive research of metabolites today requires both LC/MS (Liquid chromatography–mass spectrometry) and GC/MS (Gas chromatography–mass spectrometry). However not all the metabolomic studies in practice are comprehensive, the factors such as chemical characteristics of the metabolites, the cost and the sample matrix lead to make a choice between LC/MS and GC/MS (Lu, 2008). The present paper attempts to make a critical comparison between the two techniques in the detection of steroids. The paper will review some of the characteristics of LC/MS and GC/MS and present factors that have to be considered for choosing one of the techniques.

LC/MS is form of HPLC system having a mass spec detector attached to it. The chemicals are being separated by the HPLC by the column chromatography and this process is reverse phase chromatography. In the presence of hydrophilic solvent (such as water) the metabolite binds to the column due to hydrophobic interactions and then eluted by the hydrophobic solvent for example methanol. As the metabolites elute out from one end of the column they then enters the mass detector from where metabolites the solvent is removed and are ionized. The mass spectrometers are able to differentiate number of chemicals mixed together on the basis of their masses. Hence it is not necessary to bother to separate chemicals with HPLC first (Lu, 2008).

GC/MS is a technique consisting of gas chromatography with a mass spectrometer. A complex mixture of chemicals are separated, identified and quantified. With these characteristics the GC/MS is an ideal technique for analyzing number of low molecular weight compounds that are present in environment. The compound that is to be analyzed using GC/MS has to be volatile and thermally stable. Apart from this the functionalised compounds may require some modifications such as derivatization before analyzing it by GC/MS. This is done to avoid any unwanted adsorption effects that would otherwise result into inferior data quality.

 

The samples before subjected to GC/MS analysis have to be first dissolved in organic solvent and then the solvent has to be extracted and the extract is subjected for various analyses. The sample when injected into the GC is vaporised and moves into the chromatographic column with the help of carrier gas. As the sample flows through the column the compounds of interest from the mixture gets separated with respect to the interaction of the metabolite with the coating of the column as well as the carrier gas (Hübschmann, 2008).

Comparative analysis of GC/MS and LC/MS for analyzing steroids

The steroid profiling has always been most important part of diagnosis related to steroid hormone disorders and metabolism. Various techniques have been used from decades such as thin layer chromatography and gas chromatography-mass spectrometry. Apart from this many other immunoassays have been used extensively for plasma steroid hormones quantification in common diagnostic processes. These techniques have been central to the clinical disorder diagnosis but the data are not fully reliable as the problem of cross reactivity has never been solved completely for many analytes thus influencing the specificity. When the concentration of the hormones to be quantified is very less such as in the case of pediatric patients or in the case of postmenopausal women problem occurs. Further there is variability present in the interassay of common measurements such as estradiol, testosterone and the progesterone. These variabilities have been reduced significantly by using liquid chromatography-mass spectrometry. Nowadays the immunoassays are rarely used as the latest generation of LC/MS and GC/MS have been proved to be superior to such assays. Many diagnostic laboratories have adopted LC/MS due to its superior immunoassays, its specificity and its providing good linearity even at very low concentrations. Though LC/MS is steroid analysis has emerged as a mainstream diagnostic technology but it has many limitations with one major drawback that the analyte measurements are always targeted even though the panels of the steroids are being measured in a single run (Krone, 2010).

An alternative analytical technique to LC/MS available is known as GC/MS which is available for more than 40 years. The GC/MS is used for the analysis of metabolites of steroid hormones as well as their precursors. This technique can be sued both in targeted and comprehensive mode. A scanned GC/MS run consist of all the excreted steroids and the data can be looked for any required analyte even after long period of analysis (Vogeser, 2007).The advantage of LC/MS is the rapid and required analysis of a little amount of compounds at very high sensitivity and is easily automated. Derivatization and hydrolysis of any conjugates is not required. Hence this makes the LC/MS an attractive and ideal diagnostic technique. However structural characterization of some novel steroids cannot be carried out with this technique and the scanning will result into ill defined metabolites and the data obtained is difficult to interpret. Here the GC/MS has the advantage over the LC/MS of providing better resolution. Recently the resolution of the LC column has improved greatly with the use of small particle size but still the data is compromised due to the short run time. In GC/MS derivatization of the steroids is required with the use of methyloxime-trimethylsilyl ether. This is the disadvantage with this technique as this consumes time and need extra labour.

 

However this allows the characterization of the structures through comparatively easier determination of the number of C=O and C-OH groups. The MS fragmentation takes place due to derivatization hence huge information is available on the genesis of the particular ions that are generated from the analysis of GC/MS. This results into easier determination of the functional groups and their position on the steroid molecule. The GC/MS analysis have been proved to be superior in separating the epimeric steroids hence it is very useful specifically in the metabolism disorders such as 17β-hydroxysteroid dehydrogenase type 3 deficiency and others. When GC/MS is at scan mode non targeted steroid profiling and identification of novel compounds can be done along with identification of their synthesis and metabolism pathways and this data can be stored for longer period of time to be used in future (Bowden, 2009).When small amount of required analyte is to be measured with high background GC/MS is invaluable for this LC/MS as very small amount of sample ka be analysed easily (Rauh, 2009).

Advantages of GC/MS in steroid profiling in paediatrics

With the introduction of LC/MS the amount of the sample required for the analysis of steroid hormone has been greatly reduced in comparison to other immunoassays and the steroid profiling has also been established successfully as the diagnostic test method for the differentiation between the defects in the steroid disorders. This technique has been established as the confirmation tests in the screening of neonatal congenital adrenal hyperplasia relying only upon blood samples. Hence it has to be kept in mind that this technique relies only upon the blood samples which in a pediatric settings may not be easy to obtain as it is traumatizing both for the patient and their parents. Moreover the blood sampling only reflects single time point than the integrated picture of the steroid metabolome for a longer period of time which is possible in GS/MS steroid analysis technique. Another specific advantage that GC/MS has over LC/MS is that it is non-selective in nature that is a scan will run for all the excreted steroids which will give an oerall picture of single metabolome. One of the major drawbacks of using GC/MS technique in the clinical steroid analysis is the presenting data in the simple form which can be interpreted by the endocrinologists. The endocrinologists find it difficult to analyse the intricacies of the steroid metabolism. A more user friendly presentation of GC/MS data has to be established which can make the complex metabolomes easier to interpret and understandable by the clinicians and the scientists.

Conclusion:

With the advent in the technologies of LC/MS the most classical steroid analytical methodology can be replaced by measuring the hormones and the precursors specifically for the known analytes. With LC/MS appropriate values are obtained mostly for the first time specifically in pediatric and females where low concentrations of androgens and estrogens are present and the work is important and requires reproducibility (Rauh, 2009). However in spite of this the GC/MS still plays a major role in the analysis of the rare and unidentified conditions and is able to retain its place as the eminent discovery in defining new and unknown pathways of metabolism of the unknown steroids. Among the two the GC/MS appears to be the best technique for the study of metabolism and is a good option in the pediatric condition when a small amount of blood sampling is desired. This method still remains the core of the prenatal diagnosis of steroid disorders by analysing the maternal urine or the amniotic fluid. However from analysing both the technique it can be concluded that both the technique in steroid analysis if complementary more than competing.

References:

Bowden, J. A., Colosi, D. M., Mora-Montero, D. C., Garrett, T. J., & Yost, R. A. (2009). Enhancement of chemical derivatization of steroids by gas chromatography/mass spectrometry (GC/MS). Journal of Chromatography B,877(27), 3237-3242.

 

Hübschmann, H. J. (2008). Handbook of GC/MS: fundamentals and applications. John Wiley & Sons.

Krone, N., Hughes, B. A., Lavery, G. G., Stewart, P. M., Arlt, W., & Shackleton, C. H. (2010). Gas chromatography/mass spectrometry (GC/MS) remains a pre-eminent discovery tool in clinical steroid investigations even in the era of fast liquid chromatography tandem mass spectrometry (LC/MS/MS).The Journal of steroid biochemistry and molecular biology, 121(3), 496-504.

Lu, X., Zhao, X., Bai, C., Zhao, C., Lu, G., & Xu, G. (2008). LC–MS-based metabonomics analysis. Journal of Chromatography B, 866(1), 64-76.

Rauh, M. (2009). Steroid measurement with LC–MS/MS in pediatric endocrinology. Molecular and cellular endocrinology, 301(1), 272-281.

Vogeser, M., & Parhofer, K. G. (2007). Liquid chromatography tandem-mass spectrometry (LC-MS/MS)-technique and applications in endocrinology.Experimental and clinical endocrinology & diabetes, 115(09), 559-570.

 

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