LC-MS/MS in the Clinical Laboratory - Where to From Here?

Abstract

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has seen enormous growth in clinical laboratories during the last 10-15 years. It offers analytical specificity superior to that of immunoassays or conventional high performance/pressure liquid chromatography (HPLC) for low molecular weight analytes and has higher throughput than gas chromatography-mass spectrometry (GC-MS). Drug/Toxicology and Biochemical Genetics/Newborn Screening laboratories were at the vanguard of clinical LC-MS/MS use, but have been eclipsed by Endocrine laboratories. In USA reference/referral laboratories, most steroids and biogenic amines are now assayed by LC-MS/MS, and the technology has started to penetrate into smaller laboratories. Assays for mineralo- and gluco-corticoids and their precursors, sex steroids, metanephrines and 25-hydroxy vitamin D highlight the advantages of LC-MS/MS.However, several limitations of LC-MS/MS have become apparent, centring on the interacting triangle of sensitivity - specificity - throughput. While sample throughput is higher than for conventional HPLC or GC-MS, it lags behind automated immunoassays. Techniques which improve throughput include direct sample injection, LC-multiplexing and samplemultiplexing. Measures to improve specificity and sensitivity include sample clean-up and optimising chromatography to avoid interferences and ion suppression due to sample-matrix components. Next generation instrumentation may offer additional benefits.The next challenge for clinical LC-MS/MS is peptide/protein analysis. The quest for multi-biomarker profiles for various diseases has largely failed, but targeted peptide and protein testing by LC-MS/MS, directed at analytical and clinical questions that need to be answered, is proving highly successful. We anticipate that this will result in similar growth of clinical protein/peptide LC-MS/MS as has been seen for low molecular weight applications.

Figure 1.

Principal components of a tandem mass spectrometer. (A) The sample is ionised in the source, passes into the 1^st mass filter (Q1), then into the collision cell (Q2), followed by the 2^nd mass filter (Q3), and finally the detector. (B) and (C) depict schematically the two principal types of ionisation-sources that are in use in current clinical LC-MS/MS instruments, electrospray ionisation (ESI, B) and atmospheric pressure chemical ionisation (APCI, C). In ESI, the solvent-analyte flow from the LC passes into the source through a positively charged, very narrow capillary, and gets nebulised as microscopic, positively charged solvent-analyte droplets. These droplets fly towards the negatively-charged faceplate, with solvent evaporating on the way, until they disintegrate in a Coulomb explosion, when the repulsive charge of their ionised components exceeds their surface tension. The individual ionised analyte molecules then pass through the faceplate entry hole into the mass spectrometer. In APCI, the solvent-analyte stream from the LC is vaporised by heated nebuliser gas and the polar components of the solvent(s) vapour are ionised by a high-current discharge of a Corona needle. The solvent molecules subsequently transfer their charge to ionisable analyte molecules, which pass through the faceplate entry hole into the mass spectrometer.

Figure 2.

The five principal experiments that can be performed with mass filtering tandem mass spectrometers: Full Scan (A), Product Ion Scan (B), Precursor Ion Scan (C), Neutral Loss Scan (D) and Selective (Multiple) Reaction Monitoring (E). CID, collision-induced dissociation.

Figure 3.

Comparison of HPLC-UV (A) and LC-MS/MS (B) chromatograms of urinary free cortisol measurements of the same patient sample that contains potentially interfering substances (carbamazepine). (A) Several potentially interfering peaks are visible on HPLC-UV, with one of the carbamazepine metabolites co-eluting with cortisol and the cortisone peak being barely separated from the cortisol peak, despite a run time of 30 minutes. (B) No interferences are seen by LC-MS/MS with only 3 minutes run time and the cortisone peak shows baseline separation from the cortisol peak. Note the cortisol isotopic internal standard overlaying the cortisol peak. Reprinted from Taylor et al. (Clin Chem 2002;48:1511–9) with permission from Clinical Chemistry.

Figure 4.

Atmospheric pressure chemical ionisation precursor ion (top) and product ion (bottom) scan of oestradiol derivatised with dansyl chloride. Reprinted from Nelson et al. (Clin Chem 2004;50:373–84) with permission from Clinical Chemistry.

Figure 5.

Oestradiol (E2) measurement in cell culture medium shows relatively poor signal-to-noise for a sample containing approximately 2 pg/mL of E2 (left panel). After optimisation of chromatography, with a longer run time, the signal intensity is improved by a factor of ∼10 and the signal-to-noise ratio shows even greater improvement (right panel).

Figure 6.

Monthly testosterone test volumes at the Mayo Clinic Rochester Endocrine laboratory from January 2004 to August 2010. All three depicted testosterone assays require measurement of total testosterone by LC-MS/MS.

Figure 7.

Diagram depicting the principle of LC-multiplexing (courtesy Thermo). The total chromatographic run time is 4 minutes for each sample. However, the data-window of interest is only 1 minute wide. By staggering injections from four LC systems – a different injection every minute – and letting the LC flow before and after the window of interest go to waste, four injections can be ‘squeezed’ into a 4 minute usage of the tandem mass spectrometer.

Figure 8.

Derivatisation of 25-hydroxyvitamin D (25OHD) by triazoline-diones (TADs). 25OHD (A) reacts irreversibly with the reactive group of TADs, which can contain various different ‘R’ groups (examples in B), to form a derivatised 25OHD. The derivatised 25OHD fragments in Q2 through its molecular back bone (C), yielding analyte-specific product ions. Reprinted from Netzel et al. (Clin Chem 2011;57:431–40) with permission from Clinical Chemistry.

Figure 9.

Chromatograms of five differentially TAD-labelled patient samples containing 25OHD₂, 25OHD₃ and their respective internal standards (20 peaks overall). Reprinted from Netzel et al. (Clin Chem 2011;57:431–40) with permission from Clinical Chemistry.

Figure 10.

Gas-chromatography, selective reaction monitoring (SRM) tandem mass spectrometry (GC-MS/MS) of a sample containing 0.1 pg/mL of oestradiol. The long column (∼30 metres) and high temperatures used in GC minimise background, ion suppression, and interferences, and allow the MS/MS to show its full potential in SRM mode. The analytical sensitivity in this example is nearly 10-fold better than what can be achieved by LC-MS/MS. Courtesy of Agilent.

Figure 11.

Prednisolone created by acid-base step during extraction of free cortisol from urine. (A) An interference can be detected in extracted samples, eluting just before the cortisol peak (top of panel A), but not in unextracted samples (bottom panel of panel A). (B) Subjecting the cortisol peak and the interference peak to a high resolution MS/MS scan on a quadrupole time-of-flight tandem mass spectrometer shows a spectrum consistent with prednisolone for the interference and a typical cortisol spectrum for the cortisol peak.

Figure 12.

Urine albumin measurement by LC-MS/MS. (A) Spectra and chromatogram of human and bovine serum albumin urine subjected to LC-MS. There is in-source breakage of intact albumin and the 3+ and 4+ charge species of the N-terminal 24 amino acid fragment give a high intensity, proteotypic signal in both cases. (B) and (C) Method comparison of LC-MS with immunoturbidimetry (B) and HPLC-UV (C). Reprinted from Babic et al. (Clin Chem 2006;52:2155–7) with permission from Clinical Chemistry.

Figure 13.

Workflow of measurement of intact parathyroid hormone (1–84 PTH) by LC-MS/MS in serum and plasma. PTH from patient samples and 15N-labelled recombinant 1–84 PTH are captured with antibodies on polystyrene beads (1 and 2), washed (3) and trypsin digested (4). The digest is then subjected to LC-MS/MS with SRM settings to detect the amino acid 1–13 proteotypic trypsin fragment. Reprinted from Kumar et al. (Clin Chem 2010;56:306–13) with permission from Clinical Chemistry.

Figure 14.

Method comparison between LC-MS/MS and immunoassay (Roche Cobas) measurement of PTH. (A) Scatterplot with regression fit and confidence intervals for slope and fit. (B) Bland-Altman plot showing percentage difference between methods plotted against the mean PTH of both methods. There is evidence for non-linearity of the difference, with the difference between the two assays first declining from a high percentage low bias for the immunoassay to a lesser percentage bias, and then to increasingly higher percentage biases for the immunoassays at very high PTH concentrations. Reprinted from Kumar et al. (Clin Chem 2010;56:306–13) with permission from Clinical Chemistry.

Figure 15.

Example of a standard curve of LC-MS/MS measurement of angiotensin II in plasma.