Department of Clinical Pathology, Cleveland Clinic, Cleveland, OH 44195, USA
Received date: July 27, 2014; Accepted date: July 28, 2014; Published date: September 01, 2014
Citation: Bunch DR, Wang S (2014) Steroid Analysis by Liquid Chromatography- Mass Spectrometry: Derivatization Consideration. J Chromatograph Separat Techniq 5:e122. doi:10.4172/2157-7064.1000e122
Copyright: © 2014 Bunch DR, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Steroids are small non-polar cholesterol derived hormones that regulate metabolic processes through the endocrine systems , and are essential to proper physical development, sexual differentiation and maturation, and metabolic homeostasis. Abnormal steroid hormone production can indicate various human diseases such as congenital metabolic defects, hyperplasia, and cancer. Accurate steroid assessment in circulation is essential for determining pathophysiological conditions and forming treatment strategies.
Steroid measurement was first achieved through radioimmunoassay (RIA) and later by direct immunoassays (IA). RIA requires sample pretreatment to produce reliable results. Direct IAs are quick and require no pre-extraction steps and are currently the most commonly used steroid assays within clinical laboratories . Liquid chromatography tandem mass spectrometry (LC-MS/MS) leverages specificity for steroid analysis to eliminate the accuracy issues associated with IAs, which are commonly due to interfering substances such as heterophilic antibodies that tend to produce false positives  or negatives  and limited antibody specificity, which cross react with other steroids . LC-MS/MS is quickly becoming the preferred technology for steroid determination.
The major analytical issues with LC-MS/MS are ionization efficiency, matrix effects, and isobaric interferences . Some steroids such as pregnenolone, 17-hydroxypregnenolone, and dehydroepiandro stenedione are particularly difficult to ionize using normal LC-MS/MS ionization techniques, electrospray or atmospheric chemical ionization. One method for addressing this is chemical derivation. For steroids, these chemical derivations usually utilize the keto or hydroxyl groups to add chargeable moieties allowing for improved ionization . Keto group derivation is usually performed with a Schiff base type reaction to attach a new moiety. These moieties can carry charged groups (nitrogen-Girard P and T) or groups that are easily ionized such as hydroxyl (hydroxylamine), carboxylic acid (carboxymethoxylamine), or sulfur groups (p-toluenesulfonyl hydrazone). The compounds capable of derivatizing the hydroxyl groups (2-fluoro-1-methylpyridinium p-toluenesulfonate and dansyl chloride) have similar motifs . In addition to the improved ionization, these chemical additions can improve chromatographic separation of isobaric interferences and substances causing matrix effects. The additional moieties can also change the parent ion fragmentation pattern yielding high abundance product ions that distinguish between isobars. The disadvantages of the derivatization strategies are three folds: 1) extra labor time is needed to perform the assays; 2) precision may be compromised dues to extra steps introduced; 3) accuracy could be reduced due to the possible hydrolysis of conjugates under the derivatization conditions . One should consider the steroid panel components and the clinical needs to determine if derivatization is necessary.