The New Standard for Precise Quantitative Assays: Liquid Chromatography-tandem mass spectrometry (LC-MS/MS)

2009, Volume 20, Number 3


David F. Keren M.D.

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Articles about current advances in clinical laboratory procedures tend to focus on the remarkable ability of molecular amplification and sequencing procedures for nucleic acids.  But another technique has been moving into clinical laboratories over the past few years and is revolutionizing precision in quantitative analysis of a wide variety of molecules.  It is LC-MS/MS.

The combination of the physical separating properties of liquid chromatography with the powerful mass analysis provided by mass spectrometry results in a rapid and precise analysis of complex mixtures of molecules that can be performed in minutes.  Basically, the LC separates the molecules by properties such as charge and the MS determines the weight of the molecules electronically.  In tandem mass spectrometry, two mass spectrometers are connected so that while in the first mass spectrometer, molecules present are converted to an ionized form and the size of the molecules present is determined.  Then, these molecules are broken apart in a collision cell chamber.  The fragments are identified by being compared to a known standard.  Precise quantitation of the compounds of interest is achieved by comparing the ratio of the patient sample fragments/internal standard to the ratio of known standard fragments/internal standard.

In the present issue of the Warde Report, our Director of Toxicology, Dr. Ntei Abudu illustrates the use of LC-MS/MS to solve a long-term problem that enzyme-immunoassays (EIA) and high-pressure liquid chromatography (HPLC) had in distinguishing between cortisol and other structurally similar molecules, such as cortisone. These vexing cross-reactivities could produce erroneous results that confounded the diagnosis of Cushing’s syndrome.  As Dr. Abudu demonstrates in our current issue, with LC-MS/MS such structurally similar molecules readily can be separated and a precise measurement ensured.

But the utility of LC-MS/MS goes far beyond improving precision in endocrine steroid analysis.  It excels in detecting molecules at low concentrations in a small volume of fluid.  For toxicology, it can simultaneously measure a wide variety of drugs and metabolites.  Indeed, it is becoming the new standard for testing drugs of abuse in addition to disease markers. 

At Warde Medical Laboratory, we use LC-MS/MS to detect immunosuppressive drugs (Cyclosporine A, Tacrolimus and Sirolimus), Methyl Malonic Acid, Levetiracetam, Topiramate and Opiates.  Dr. Abudu is currently working on using LC-MS/MS to evaluate meconium for the presence of Opiates, Cocaine metabolites, THC, Amphetamine, PCP and Methadone. 

Further Reading: T. M. Annesley. Application of Commercial Calibrators for the Analysis of Immunosuppressant Drugs in Whole Blood. Clin. Chem., 2005; 51:457. J. C. Drees, J. A. Stone, K. R. Olson, K. H. Meier, A. M. Gelb, and A. H. B. Wu Clinical Utility of an LC-MS/MS Seizure Panel for Common Drugs Involved in Drug-Induced Seizures. Clin. Chem., 2009;55:126. S. O'Halloran and K. F. Ilett. Evaluation of a Deuterium-Labeled Internal Standard for the Measurement of Sirolimus by High-Throughput HPLC Electrospray Ionization Tandem Mass Spectrometry. Clin. Chem., 2008;54:1386. H.-A. Lakso, P. Appelblad, and J. Schneede. Quantification of Methylmalonic Acid in Human Plasma with Hydrophilic Interaction Liquid Chromatography Separation and Mass Spectrometric Detection. Clin. Chem.2008;54:2028. R. L. Taylor, S. K. Grebe, and R. J. Singh. Quantitative, Highly Sensitive Liquid Chromatography-Tandem Mass Spectrometry Method for Detection of Synthetic Corticosteroids. Clin. Chem., 2004; 50:2345.