Performance of the Roche Total Mycophenolic Acid® assay on the Cobas Integra 400®, Cobas 6000® and comparison to LC-MS/MS in liver transplant patients

Background: Mycophenolic acid (MPA) is an immunosuppressant for which therapeutic drug monitoring (TDM) is performed for optimal prophylaxis and avoidance of toxicity in transplant patients. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is ideally suited for TDM of MPA. There have been several method comparisons of the Roche Total MPA assay, but none have been performed with respect to liver transplant patients. Methods: We validated the Roche Total MPA assay on the Cobas Integra 400 and Cobas 6000 and compared it to a validated LC-MS/MS (API 2000 (TM)) method. Fifty-five EDTA plasma samples from liver transplant patients were measured with the Roche assay on these platforms and compared to the LC-MS/MS results. Results: Validation of the LC-MS/MS, Cobas Integra 400 and 6000 was performed with good results. The LC-MS/MS/Integra 400/Cobas 6000 were linear up to 30, 15 and 17 mg/L, respectively. Imprecision was <10% for LC-MS/MS and <7% for the Roche assay on both platforms. The samples showed good agreement with LC-MS/MS. Passing-Bablok regression analysis showed Cobas Integra (mg/L) = 1.02 x LC-MS/MS (mg/L)-0.50 and Cobas 6000 (mg/L) = 0.98 x LC-MS/MS-0.47. Conclusions: The Roche Total Mycophenolic Acid-assay is suitable for measuring total MPA in plasma from liver transplant patients and is a good alternative for LC-MS/MS

Liquid Chromatography Electron Capture Dissociation Tandem Mass Spectrometry (LC-ECD-MS/MS) versus Liquid Chromatography Collision-induced Dissociation Tandem Mass Spectrometry (LC-CID-MS/MS) for the Identification of Proteins

Electron capture dissociation (ECD) offers many advantages over the more traditional fragmentation techniques for the analysis of peptides and proteins, although the question remains: How suitable is ECD for incorporation within proteomic strategies for the identification of proteins? Here, we compare LC-ECD-MS/MS and LC-CID-MS/MS as techniques for the identification of proteins.Experiments were performed on a hybrid linear ion trap–Fourier transform ion cyclotron resonance mass spectrometer. Replicate analyses of a six-protein (bovine serum albumin, apo-transferrin,lysozyme, cytochrome c, alcohol dehydrogenase, and β-galactosidase) tryptic digest were performed and the results analyzed on the basis of overall protein sequence coverage and sequence tag lengths within individual peptides. The results show that although protein coverage was lower for LC-ECDMS/MS than for LC-CID-MS/MS, LC-ECD-MS/MS resulted in longer peptide sequence tags,providing greater confidence in protein assignment

MS-DIAL: data-independent MS/MS deconvolution for comprehensive metabolome analysis.

Data-independent acquisition (DIA) in liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS) provides comprehensive untargeted acquisition of molecular data. We provide an open-source software pipeline, which we call MS-DIAL, for DIA-based identification and quantification of small molecules by mass spectral deconvolution. For a reversed-phase LC-MS/MS analysis of nine algal strains, MS-DIAL using an enriched LipidBlast library identified 1,023 lipid compounds, highlighting the chemotaxonomic relationships between the algal strains

Matrix effect in bio-analysis of illicit drugs with LC-MS/MS: Influence of ionization type, sample preparation, and biofluid

AbstractThe purpose of the present work was to evaluate the synergistic effect of ionization type, sample preparation technique, and bio-fluid on the presence of matrix effect in quantitative liquid chromatography (LC)-MS/MS analysis of illicit drugs by post-column infusion experiments with morphine (10-μg/mL solution). Three bio-fluids (urine, oral fluid, and plasma) were pretreated with four sample preparation procedures [direct injection, dilution, protein precipitation, solid-phase extraction (SPE)] and analyzed by both LC-electrospray ionization (ESI)-MS/MS and LC-atmospheric pressure chemical ionization (APCI)-MS/MS. Our results indicated that both ionization types showed matrix effect, but ESI was more susceptible than APCI. Sample preparation could reduce (clean up) or magnify (pre-concentrate) matrix effect. Residual matrix components were specific to each bio-fluid and interfered at different time points in the chromatogram. We evaluated matrix effect in an early stage of method development and combined optimal ionization type and sample preparation technique for each bio-fluid. Simple dilution of urine was sufficient to allow for the analysis of the analytes of interest by LC-APCI-MS/MS. Acetonitrile protein precipitation provided both sample clean up and concentration for oral fluid analysis, while SPE was necessary for extensive clean up of plasma prior to LC-APCI-MS/MS

Impact of glucuronide interferences on therapeutic drug monitoring of posaconazole by tandem mass spectrometry

Background: Posaconazole is a novel antifungal drug for oral application intended especially for therapy of invasive mycoses. Due to variable gastrointestinal absorption, adverse side effects, and suspected drug-drug interactions, therapeutic drug monitoring (TDM) of posaconazole is recommended. Method: A fast ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for quantification of posaconazole with a run-time &lt;3 min was developed and compared to a LC-MS/MS method and HPLC method with fluorescence detection. Results: During evaluation of UPLC-MS/MS, two earlier eluting peaks were observed in the MRM trace of posaconazole. This was only seen in patient samples, but not in spiked calibrator samples. Comparison with LC-MS/MS disclosed a significant bias with higher concentrations measured by LC-MS/MS, while UPLC-MS/MS showed excellent agreement with the commercially available HPLC method. In the LC-MS/MS procedure, comparably wide and left side shifted peaks were noticed. This could be ascribed to in-source fragmentation of conjugate metabolites during electrospray ionisation. Precursor and product ion scans confirmed the assumption that the additional compounds are posaconazole glucuronides. Reducing the cone voltage led to disappearance of the glucuronide peaks. Slight modification of the LC-MS/MS method enabled separation of the main interference, leading to significantly reduced deviation. Conclusions: These results highlight the necessity to reliably eliminate interference from labile drug metabolites for correct TDM results, either by sufficient separation or selective MS conditions. The presented UPLC-MS/MS method provides a reliable and fast assay for TDM of posaconazole. Clin Chem Lab Med 2010; 48:1723-31

Current developments in LC-MS for pharmaceutical analysis

The current developments in liquid chromatography-mass spectrometry (LC-MS) and its applications to the analysis of pharmaceuticals are reviewed. Various mass spectrometric techniques, including electrospray and nanospray ionization, atmospheric pressure chemical ionization and photoionization and their interface with liquid chromatographic techniques are described. These include high performance liquid chromatography, capillary electrophoresis and capillary electrochromatography and the advantages and disadvantages of each technique are discussed. The applications of LC-MS to the studies of in vitro and in vivo drug metabolism, identification and characterization of impurities in pharmaceuticals, analysis of chiral impurities in drug substances and high-throughput LC-MS-MS systems for applications in the “accelerated drug discovery” process are described

Automated processing of whole blood samples for the determination of immunosuppressants by liquid chromatography tandem-mass spectrometry

Background: Liquid chromatography tandem-mass spectrometry (LC-MS/MS) is an efficient technology for routine determination of immunosuppressants in whole blood; however, time-consuming manual sample preparation remains a significant limitation of this technique. Methods: Using a commercially available robotic pipetting system (Tecan Freedom EVO), we developed an automated sample-preparation protocol for quantification of tacrolimus in whole blood by LC-MS/MS. Barcode reading, sample resuspension, transfer of whole blood aliquots into a deep-well plate, addition of internal standard solution, mixing, and protein precipitation by addition of an organic solvent is performed by the robotic system. After centrifugation of the plate, the deproteinized supernatants are submitted to on-line solid phase extraction, using column switching prior to LC-MS/MS analysis. The only manual actions within the entire process are decapping of the tubes, and transfer of the deep-well plate from the robotic system to a centrifuge and finally to the HPLC autosampler. Whole blood pools were used to assess the reproducibility of the entire analytical system for measuring tacrolimus concentrations. Results: A total coefficient of variation of 1.7% was found for the entire automated analytical process (n=40; mean tacrolimus concentration, 5.3 mu g/L). Close agreement between tacrolimus results obtained after manual and automated sample preparation was observed. Conclusions: The analytical system described here, comprising automated protein precipitation, on-line solid phase extraction and LC-MS/MS analysis, is convenient and precise, and minimizes hands-on time and the risk of mistakes in the quantification of whole blood immunosuppressant concentrations compared to conventional methods

An Optimized Data Structure for High Throughput 3D Proteomics Data: mzRTree

As an emerging field, MS-based proteomics still requires software tools for efficiently storing and accessing experimental data. In this work, we focus on the management of LC-MS data, which are typically made available in standard XML-based portable formats. The structures that are currently employed to manage these data can be highly inefficient, especially when dealing with high-throughput profile data. LC-MS datasets are usually accessed through 2D range queries. Optimizing this type of operation could dramatically reduce the complexity of data analysis. We propose a novel data structure for LC-MS datasets, called mzRTree, which embodies a scalable index based on the R-tree data structure. mzRTree can be efficiently created from the XML-based data formats and it is suitable for handling very large datasets. We experimentally show that, on all range queries, mzRTree outperforms other known structures used for LC-MS data, even on those queries these structures are optimized for. Besides, mzRTree is also more space efficient. As a result, mzRTree reduces data analysis computational costs for very large profile datasets.Comment: Paper details: 10 pages, 7 figures, 2 tables. To be published in Journal of Proteomics. Source code available at http://www.dei.unipd.it/mzrtre