Historically, mass spectrometry had been of limited use in clinical laboratories; however, the combination of atmospheric pressure ionization (API) methods with tandem mass spectrometry has opened up this technology to the clinical laboratory arena. The novelty of this technology seems to be similar to that of immunoassays in the 1970s and polymerase chain reaction, or PCR, in the 1980s.1 Tandem mass spectrometry found its way into clinical laboratories in the early 1990s, with the analysis of acylcarnitines and amino acids from neonatal blood spots. During the past decade, liquid chromatography tandem mass spectrometry (LC-MS/MS) has played an increasingly important role in clinical analysis. It is common now to find it in laboratories with applications for endocrinology, toxicology, pharmacology, and therapeutic drug monitoring. As the prices for basic LC-MS/MS systems decrease, more clinical labs will venture into this technology.
Overview of LC-MS/MS
Mass spectrometry measures the mass-to-charge ratio (m/z) of a molecule, which has been ionized, and its fragment ions. A mass spectrum is the plot of m/z versus the ion intensity or ion abundance.2 A tandem mass spectrometer has two mass selective devices (quadrupoles) arranged in a series that selects the precursor ion (of a particular m/z) of the respective analyte, which is then directed into a collision cell where a very low flow of a collision gas is responsible for the disintegration of the precursor ions into several product ions. The second quadrupole positioned behind the collision cell is used to scan for the product ions that are formed according to their respective m/z ratio.
The collision cell itself may represent a small quadrupole; hence, the term triple quadrupole mass spectrometer can be used synonymously. For quantification with LC-MS/MS, after LC separation of the respective analyte, the molecules are introduced into the mass spectrometer via API, which allows for ion formation. There are two primary ionization techniques, electrospray ionization (ESI) and atmospheric-pressure chemical ionization (APCI). The use of ESI is primarily for the analysis of charged species; whereas, APCI is used for uncharged or difficult to charge species. ESI is a “soft” ionization (very low energy) process, which usually yields the intact parent molecule with one or multiple charges.
Strengths of LC-MS/MS
Specificity: Selection of the precursor ion with the first quadrupole and the product fragment ion with the second quadrupole by the tandem mass spectrometer can exhibit highly specific detection for a given molecule.
Wide range of applications: In contrast to GC/MS, the application of LC-MS/MS is not limited to volatile molecules (usually with molecular weights below 500 Da). Since most biologically active molecules are polar, thermolabile, and non-volatile, LC-MS/MS is better geared towards the detection of such molecules. Furthermore, sample preparation is usually simple and does not require derivatization techniques. LC-MS/MS assays are generally optimized to shorter runtimes; therefore, far higher sample throughput can be realized when compared to GC/MS.
Flexibility: New assays can typically be developed in house (“home brew”) with a high degree of flexibility and within a short time, provided a comprehensive assay validation is carried out.
Abundance of information: A single analytical LC-MS/MS run, due to the fast ion-selection electronics, multi-parametric, quasi-parallel analyses with the mass spectrometer, can produce a large number of quantitative or qualitative results.
Areas of application
Newborn screening: The initial foray of LC-MS/MS into the clinical arena was its application to newborn screening of inborn errors of metabolism.3 Currently, in many industrialized countries, LC-MS/MS is used in the routine newborn screening for inborn errors of metabolism. At present, newborn screening in dried blood spots encompasses more than 30 diseases, which include organic acidemias, aminoacidopathies, and defects of fatty-acid oxidation. In addition, tandem mass spectrometry has been used to screen for other metabolites such as urine oligosaccharides,4 sulfatide,5 very long chain fatty acids,6 long chain bile acids,7 methylmalonic acid,8 and for the investigation of porphyrias9 and screening for patients at risk of inherited disorders of purine and pyrimidine metabolism.10
Therapeutic drug monitoring: The development of the immunosuppressant sirolimus (rapamycin) for the prevention of organ rejections after transplantation was the major stimulus behind the introduction of LC-MS/MS into the clinical laboratory. LC-MS/MS methods are available for the simultaneous determination of tacrolimus, sirolumus, and cyclosporine.11 LC-MS/MS methods are available for a variety of drug classes such as cytotoxic drugs,12 antiretroviral drugs,13 tricyclic antidepressants,14 anticonvulsants,15 anti-epileptics16 … . As the drive towards more individualized drug dosing continues, LC-MS/MS analysis of these drug classes will become more and more routine.
Drugs-of-abuse and pain-management testing: GC/MS is still the most widely used technique for toxicology testing; however, LC-MS/MS is gaining ground in that area due to simple sample preparation, no requirement for derivatization, and shorter run times. Direct drug screening from urine has been reported17 and methods are available for multidrug panels for drugs-of-abuse screening,18 and for the confirmation of benzodiazepines,19 the measurement of numerous drugs such as buprenorphine, and its metabolites,20 amphetamines,21 opioids.22
Endocrinology and steroid testing: Radioimmunoassay and enzyme immunoassays were the main techniques for the measurement of steroids in the clinical laboratories. Immunoassays suffer from many limitations such as the lack of specificity, limited dynamic range, and matrix effects. The advent of LC-MS/MS is viewed as an innovative analytical technology applicable to a wide number of analyses in the endocrinology laboratory, mainly the quantification of various steroids. The analysis of 25(OH) vitamin D3 and D2 levels by LC-MS/MS has been gaining momentum,23 and LC-MS/MS methods have been developed for a variety of steroids such as testosterone, aldosterone, cortisol, progesterone, estriol… .24
The LC-MS/MS technology has to overcome certain challenges before making the jump from a specialized, technically complex technique into mainstream clinical laboratory testing. Although the instrumentation has decreased in price and complexity, challenges such as developing assays, standardizing assays across the board, and training and maintaining highly qualified technical staff remain.
Method development and validation: The majority of LC-MS/MS methods used in the clinical lab can be placed under the lab-developed test category, since they were “home brewed.” The labs implementing this technology are expected to develop the test and undertake a thorough validation before putting these tests into routine use. The labs are expected to prepare in house or acquire commercially high-quality standards, deuterated internal standards, and quality-control materials. Efforts are underway from the manufacturing companies to present Food and Drug Administration- (FDA-) approved test kits for the use by the clinical laboratory. The first such kit was the Waters MassTrak Immunosuppressants kit (Waters, Milford, MA) which was FDA-cleared for the quantification of the immunosuppressant Tacrolimus (FK506; Prograf) in liver- and kidney-transplant patients’ whole blood samples as an aid in the management of tacrolimus therapy. As similar kits become FDA cleared, and as more commercial calibrators, quality-control materials, and standards become available, these will help alleviate some of the challenges widespread use of LC-MS/MS in the clinical labs.
Standardization: The advantage of highly specific measurements is offset by between lab differences among various LC-MS/MS methods since the majority are “home brewed.” The lack of standardization and harmonization in mass spectrometry is a major challenge that needs to be overcome for more widespread use of this technology in the clinical laboratories. Efforts are underway to standardize certain LC-MS/MS methods such as testosterone and vitamin D. The Endocrine Society and the Centers for Disease Control and Prevention came out early 2010 with a consensus statement to ensure highly accurate testosterone testing that will result in improved diagnosis, treatment, and prevention of disease through the use of standardized assays.25 This effort aims at achieving highly accurate and reliable testosterone measurement using both LC-MS/MS and immunoassays. In the U.K., an external proficiency-testing scheme for vitamin-D analysis — termed DEQAS, for vitamin D external quality-assurance scheme — is available. The overall aim of DEQAS is to ensure the analytical reliability of 25 hydroxyvitamin D (25OHD) and 1,25 dihydroxyvitamin D (1,25(OH)2D) assays.
Technical expertise: Training and maintaining highly qualified staff is needed not just for method development and validation but also for day-to-day operation as well. Doctoral-level talent might be needed for implementing the tests and for troubleshooting but not for the day-to-day operations of the mass spectrometer.
Laboratory technologists with mass-spectrometry expertise are more than capable of handling everyday operations; however, finding such expertise is challenging. In order to bring laboratory personnel up to speed with mass spectrometry, the American Association for Clinical Chemistry has established a new nine-course certificate program in using tandem mass spectrometry in the clinical laboratory.
Charbel Abou-Diwan, PhD
, is a clinical chemistry post-doctoral Fellow at the department of Pathology at Emory University School of Medicine, Atlanta GA.
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- Rashed MS, et al. Screening blood spots for inborn errors of metabolism by electrospray tandem mass spectrometry with a microplate batch process and a computer algorithm for automated flagging of abnormal profiles. Clin Chem. 1997; 43(7):1129-1141.
- Rozaklis T, et al. Determination of oligosaccharides in Pompe disease by electrospray ionization tandem mass spectrometry. Clin Chem. 2002;48(1):131-139.
- Whitfield PD, et al. Characterization of urinary sulfatides in metachromatic leukodystrophy using electrospray ionization-tandem mass spectrometry. Mol Genet Metab. 2001;73(1):30-37.
- Johnson DW. Alkyldimethylaminoethyl ester iodides for improved analysis of fatty acids by electrospray ionization tandem mass spectrometry. Rapid Commun Mass Spectrom. 2000;14(21):2019-2024.
- Johnson DW, et al. Rapid and quantitative analysis of unconjugated C(27) bile acids in plasma and blood samples by tandem mass spectrometry. J Lipid Res. 2001; 42(1):9-16.
- Magera MJ, et al. Methylmalonic acid measured in plasma and urine by stable-isotope dilution and electrospray tandem mass spectrometry. Clin Chem. 2000; 46(11):1804-1810.
- Ford RE, et al. Quantitative measurement of porphobilinogen in urine by stable-isotope dilution liquid chromatography-tandem mass spectrometry. Clin Chem. 2001;47(9):1627-1632.
- Ito T, et al. Rapid screening of high-risk patients for disorders of purine and pyrimidine metabolism using HPLC-electrospray tandem mass spectrometry of liquid urine or urine-soaked filter paper strips. Clin Chem. 2000;46(4):445-452.
- Volosov A, Napoli KL, Soldin SJ. Simultaneous simple and fast quantification of three major immunosuppressants by liquid chromatography—tandem mass-spectrometry. Clin Biochem. 2001;34(4):285-290.
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- Gu J, Soldin SJ. Modification of tandem mass spectrometric method to permit simultaneous quantification of 17 anti-HIV drugs which include atazanavir and tipranavir. Clin Chem Acta. 2007;378(1-2):222-224.
- Kollroser M, Schober C. Simultaneous determination of seven tricyclic antidepressant drugs in human plasma by direct-injection HPLC-APCI-MS-MS with an ion trap detector. Ther Drug Monit. 2002;24(4):537-544.
- Oertel R, et al. Simultaneous determination of three anticon-vulsants using hydrophilic interaction LC-MS. J Sep Sci. 2009;32(2):238-243.
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- Glover SJ, Allen KR. Measurement of benzodiazepines in urine by liquid chromatography-tandem mass spectrometry: confirmation of samples screened by immunoassay. Ann Clin Biochem. 2010;47(2):111-117.
- Polettini A, Huestis MA, Simultaneous determination of buprenorphine, norbuprenorphine, and buprenorphine-glucuronide in plasma by liquid chromatography-tandem mass spectrometry. J Chromatogr. B: Biomedical Sciences and Applications, 2001. 754(2): p. 447-459.
- Wu TY, Fuh MR. Determination of amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxyethylamphetamine, and 3,4-methylenedioxymethamphetamine in urine by online solid-phase extraction and ion-pairing liquid chromatography with detection by electrospray tandem mass spectrometry. Rapid Commun Mass Spectrom. 2005;19(6):775-780.
- Musshoff F, et al. An automated and fully validated LC-MS/MS procedure for the simultaneous determination of 11 opioids used in palliative care, with 5 of their metabolites. J Mass Spectrom. 2006;41(5):633-640.
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- Rosner W, Vesper H. Toward excellence in testosterone testing: a consensus statement. J Clin Endocrinol Metab. 2010;95(10):4542-4548.