Paul J. Taylor

The development of HPLC-atmospheric pressure ionization-mass spectrometry (HPLC-MS) has presented clinical laboratories with a powerful analytic tool. The aim of this paper is to provide an overview of the current status of HPLC-MS in the clinical laboratory; to discuss the challenges to mass spectrometry in this setting; and to present some of the latest developments in instrumentation and illustrate their potential application. Currently, the major clinical applications for HPLC-MS are neonatal screening for metabolic disorders, therapeutic drug monitoring of immunosuppressant and HIV/AIDS drugs, and toxicological investigations. The major barrier to the uptake of this technology in the clinical laboratory is the initial capital outlay for instrumentation. A secondary reason is the lack of suitably trained scientists. The challenges that clinical HPLC-MS face are (1) ease of use and automation, (2) interpatient variability in relation to matrix effects, (3) availability of suitable internal standards, and (4) harmonization of methods to meet regulatory requirements. The development of the triple quadrupole linear ion trap mass analyzer allows the quantification power of a triple quadrupole mass analyzer to be combined with the scanning ability of an ion trap. This hybrid instrument allows different permutations of scan combinations. The combination of selected reactant monitoring and MS3 is an attractive combination for quantification. The ion source, atmospheric pressure photoionization, has recently been developed and is well suited to nonpolar analytes, although its role is yet to be established. This ion source complements other interfaces used in HPLC-MS. Both of these advances in instrumentation add to the potential applications of HPLC-MS. How HPLC-MS goes forward into the clinical laboratory is dependent on clinical scientists, instrument manufacturers, and regulatory authorities.

The calcineurin inhibitor cyclosporin A (CsA) is used as a primary immunosuppressant in solid organ transplantation and for the treatment of many autoimmune diseases. Individualization of therapy is required because CsA has high inter- and intrapatient pharmacokinetic variability, the potential for drug interactions, a narrow therapeutic index, and important compliance issues (1). The use of the CsA formulation Neoral® has been associated with a shift in monitoring from trough (predose) to 2-h postdose (C2) whole blood samples (2)(3). To provide efficient CsA therapeutic drug-monitoring, a rapid turnaround of results, high selectivity, and high sensitivity are required (4). These requirements have led to the development of several high-throughput HPLC–mass spectrometry (HPLC-MS) methods (4)(5)(6)(7)(8)(9). For quantification purposes, internal standardization is used in all methods. Currently, there is no consensus on the preferred internal standard for CsA measurement. To date, the internal standards that have been used can be classified into 3 types: isotope-labeled CsA (10)(11), a structural analog (8)(12), or a structurally unrelated compound (4)(5). The aim of this study was to evaluate the analytical performance of these 3 classes of internal standards with deuterium-labeled CsA (CsA-d12), cyclosporin D (CsD), and ascomycin for the quantification of CsA by a high-throughput HPLC-MS method. The HPLC-MS method in this study is a modification based on the previously reported assay by Keevil et al. (5). Whole blood samples (50 μL) were treated with 0.1 mol/L zinc sulfate (200 μL) followed by acetonitrile (500 μL) containing internal standard (50 μg/L). Because of interference from CsA-d12 in the CsD mass transition, 2 extractions were performed and analyzed: 1 with …

The authors describe a reverse-phase high-performance liquid chromatography-electrospray-tandem mass spectrometry method for the measurement of nicotine in human plasma. Samples (500 μL) with added deuterium-labeled d3-nicotine as an internal standard (IS) were treated with a 2-step process of ether extraction (6 mL) followed by back-extraction into 0.1% formic acid (50 μL). Chromatography was performed on a phenyl Novapak column with a mobile phase consisting of 50% 10 mM ammonium formate (pH 3.3) and acetonitrile (50:50, vol/vol). A flow rate of 0.2 mL/min resulted in a total analysis time of 5 minutes per sample. Mass spectrometric detection was by selected reactant monitoring (nicotine m/z 163.2→130.2; IS m/z 166.2→87.2). The assay was linear from 0.5 to 100 μg/L (r > 0.993, n = 9). The accuracy and imprecision of the method for quality control samples were 87.5% to 113% and 75%. The method described has good process efficiency, stabilized nicotine, avoided concentration steps, and most importantly minimized potential contamination. Further, we have established that water-based standards and controls are interchangeable with plasma-based samples. This method was used successfully to measure the pharmacokinetic profiles of subjects involved in the development of an aerosol inhalation drug delivery system.

The currently expanding range of immunosuppressant agents has placed new challenges on therapeutic drug-monitoring (TDM) services. Many of these drugs require the measurement of concentrations with subsequent dosage adjustment to maximize efficacy while minimizing toxicity. HPLC-mass spectrometry (HPLC-MS) is a relatively new technique for drug quantification and thus TDM of immunosuppressant drugs. Although mass spectrometry relies on producing, differentiating, and detecting ions in the gas phase, the development of the atmospheric pressure ionization interface (electrospray and chemical ionization) has enabled the direct coupling of solution introduction of compounds, via HPLC, to the mass analyzer. The impetus for using HPLC-MS for immunosuppressant measurement has come from the highly potent low-dose immunosuppressant drugs tacrolimus and sirolimus, which have low nanogram per milliliter circulating concentrations. A number of strategies have been reported for sample preparation and ways to automate these processes with solid-phase extraction and 2-dimensional chromatography. The disadvantages of HPLC-MS are initial cost of equipment and availability of suitably skilled scientific staff. The advantages of HPLC-mass spectrometry are high sensitivity, specificity, small sample requirements, minimal sample preparation, rapid throughput, and simultaneous measurement. Further, scientists have the ability to develop methods to measure new immunosuppressant drugs by HPLC-MS before commercial assays become available. With potential applications increasing in immunosuppressive drug monitoring, it can be envisaged that HPLC-MS may become standard equipment in TDM laboratories of the future.

Conflicting conclusions have been drawn from comparisons of high-performance liquid chromatography (HPLC) and the Abbott Diagnostics monoclonal fluorescence polarization immunoassay (mFPIA) for cyclosporin. The aim of this study was to compare whole blood cyclosporin A (CsA) concentrations measured by both mFPIA and HPLC in liver and heart transplant patients. One hundred and twenty-four liver and 62 heart transplant patient samples were assayed by both methods. Assay imprecision for both methods during the studies was < 7% over the range 150-800 micrograms/L. At an HPLC-determined concentration of 100 micrograms/L, mFPIA overestimated CsA by 60% (liver) and 77% (heart). At 300 micrograms/L, the overestimation was 40% (liver) and 45% (heart). On this basis, the mFPIA is not interchangeable with HPLC.