Fig. 1
Schematic diagram of a mass spectrometry analysis
2.1 GC-MS
GC-MS has been used in clinical laboratories for several decades. A typical GC-MS uses helium gas, sample injection port, capillary column, mass spectrophotometer, vacuum system, detector, and a data analysis system. Typical analysis involves analyte extraction from a sample, concentration of the extract, sample derivatization if the compound of interest is not volatile or is heat-labile, and injection of sample in GC-MS. Once analyte ions are fragmented, either selected ions can be analyzed (selected ion monitoring) or all fragments can be analyzed (total ion monitoring) to produce a mass spectrum. Selected ion monitoring is more sensitive than total ion monitoring, and is generally used in quantitative analysis. Total ion monitoring is very useful in the identification of unknown compounds. It is like a fingerprint since a specific mass spectrum will be produced by the fragmentation of a specific analyte. GC-MS is suitable for analysis of small molecules that are volatile, non-polar, and thermally stable.
2.2 LC-MS
In recent years LC-MS, particularly LC-MS/MS , has gained popularity and has become the method of choice, particularly for the analysis of hormones and proteins . Wide array of analytes can be measured by LC-MS/MS as compared to GC-MS. Analytes that are heat labile and difficult to derivative are more suited for LC-MS analysis. Furthermore, sample preparation is generally less involved as compared to GC-MS. Disadvantages of LC-MS/MS are less reproducible mass spectra, higher maintenance and cost as compared to GC-MS.
2.3 Time-of-Flight (TOF)-MS
In TOF-MS, an ion’s mass-to-charge ratio is determined by calculating the time required by the ion to travel a fixed distance into a flight tube. Under a fixed electrical field, lighter ions travel faster than the heavier ions. The major advantages of TOF-MS analyzers are high mass resolution and exact mass measurements. For example, at m/z of 100 and resolution of 2 ppm, mass error is less than m/z 0.0002. This provides adequate information to assign initial molecular formulae to a compound for further identification and confirmation. High-resolution TOF-MS is being used in drug and metabolites screening and identification. Matrix-assisted laser desorption ionization (MALDI)-TOF is being increasingly used in the identification of proteins and bacteria.
3 Clinical Applications
MS is increasingly being used in clinical laboratory for the analysis of wide array of analytes. Most common applications of MS are in the fields of therapeutic drug monitoring, toxicology, endocrinology, and inborn error of metabolism . In recent years, emerging applications include pathogen identification, proteomics, and genomics.
3.1 Therapeutic Drug Monitoring and Toxicology
In clinical laboratory, immunoassays are the commonly used methods for therapeutic drug monitoring. Immunoassays are also commonly used for screening few drugs of abuse. Since immunoassays are not available for a number of drugs and they are non-specific, MS is used for the assay of these drugs and confirmation of immunoassay-positive results for drugs of abuse. Although GC-MS is still widely used in therapeutic drug monitoring and toxicology, LC-MS/MS is being increasingly used due to ease of sample preparation. Using GC-MS or LC-MS/MS, 100–1000s of drugs and toxins can be screened in a single analytical run.
3.2 Endocrinology
Although immunoassays remain mainstay for the determination of hormones, MS is increasingly used for the assay of hormones [3, 4]. Immunoassays have inherent degree of non-specificity and variability among different assays particularly for steroid hormones and catecholamines and their metabolites [5]. For example, steroid hormone immunoassays generally overestimate real concentrations, and there is a significant inter-laboratory variability [6, 7]. This makes difficult to follow-up a patient over long period of time due to assay changes within a laboratory or use of different immunoassays among different laboratories. Although both GC-MS and LC-MS/MS are used for the determination of hormone assay, the later is becoming a preferred technique due to ease of sample preparation. Table 1 lists commonly assayed hormones using mass spectrometry .
Table 1
Hormones assays by mass spectrometry
• Aldosterone |
• Androstenedione |
• Catecholamines |
• Coenzyme Q |
• Cortisol |
• Corticosterone |
• Cortisone |
• Deoxycortisol |
• Dehydroepiandrosterone |
• 1,25-Dihydroxyvitamin D |
• Estrone |
• Estradiol |
• 17-Hydroxyprogesterone |
• 17-Hydroxypregnenolone |
• 25-Hydroxyvitamin D |
• Insulin |
• Pancreatic polypeptide |
• Parathyroid hormone (PTH) |
• Metanephrines |
• Testosterone |
3.3 Inborn Error of Metabolism
Mass spectrometry has shown great promise in the screening and confirmation of inborn error of metabolism . LC-MS/MS is widely used in newborn screening to detect wide array of metabolic disorders including disorders of amino acids, organic acids, and fatty acids metabolism [8, 9]. GC-MS has been used for several decades and still remains the most commonly used technique for the analysis of urine organic acids . HPLC coupled with spectrophotometry had been the mainstay method for analysis of amino acids. This method is very time-consuming and is being replaced by LC-MS/MS . Other commonly measured analytes for the detection of metabolic disorders include acylcarnitines, bile acids, purines, and pyrimidines. Tables 2 and 3 list the disorders screened by tandem mass spectrometry in newborn screening , and other metabolic disorders and metabolites diagnosed/assayed by mass spectrometry .
Table 2
Metabolic disorders screened by tandem mass spectrometry through newborn screening
Organic acidurias • Isovaleric acidemia • Glutaric aciduria 1 and 2 • 3-Hydroxy-3-methylglutaric aciduria • Multiple carboxylase deficiency • Methylmalonic acid emia due to mutase deficiency • 3-Methylcrotonyl CoA carboxylase deficiency • Methylmalonic acid emia due to cobalamin A and B defects • Propionic acidemia • 3-Ketothiolase |
Fatty acids oxidation defects • Medium chain acyl-CoA dehydrogenase • Very long chain acyl-CoA dehydrogenase • Long chain 3-hydroxy-acyl-CoA dehydrogenase • Trifunctional protein deficiency • Carnitine uptake defect • Short chain acyl-CoA dehydrogenase |
Amino acids/urea cycle
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