Investigation and Resolution of Interference in the LC-QTOF-MS Detection of 4-MePPP.

An interference resulting in the false-positive detection of the synthetic cathinone 4-MePPP in urine was suspected following the recent addition of 4-MePPP spectral data to an LC-QTOF-MS drug library. Although positive detection criteria were achieved, it was noted that all urine samples suspected of containing 4-MePPP also concurrently contained high levels of tramadol and its associated metabolites. Using QTOF-MS software elucidation tools, candidate compounds for the suspected interference were proposed. To provide further confidence in the identity of the interference, in silico fragmentation tools were used to match product ions generated in the analysis with product ions predicted from the theoretical fragmentation of candidate compounds. The ability of the suspected interference to subsequently produce the required product ions for spectral library identification of 4-MePPP was also tested. This information was used to provide a high preliminary confidence in the compound identity prior to purchase and subsequent confirmation with certified reference material. A co-eluting isobaric interference was identified and confirmed as an in-source fragment of the tramadol metabolite, N,N-bisdesmethyltramadol. Proposed resolutions for this interference are also described and subsequently validated by retrospective interrogation of previous cases of suspected interference.

Effect of caffeine on adenosine-induced reversible perfusion defects assessed by automated analysis.

OBJECTIVES: This prospective study investigated the effects of caffeine ingestion on the extent of adenosine-induced perfusion abnormalities during myocardial perfusion imaging (MPI). METHODS: Thirty patients with inducible perfusion abnormalities on standard (caffeineabstinent) adenosine MPI underwent repeat testing with supplementary coffee intake. Baseline and test MPIs were assessed for stress percent defect, rest percent defect, and percent defect reversibility. Plasma levels of caffeine and metabolites were assessed on both occasions and correlated with MPI findings. RESULTS: Despite significant increases in caffeine [mean difference 3,106 µg/L (95% CI 2,460 to 3,752 µg/L; P < .001)] and metabolite concentrations over a wide range, there was no statistically significant change in stress percent defect and percent defect reversibility between the baseline and test scans. The increase in caffeine concentration between the baseline and the test phases did not affect percent defect reversibility (average change -0.003 for every 100 µg/L increase; 95% CI -0.17 to 0.16; P = .97). CONCLUSION: There was no significant relationship between the extent of adenosine-induced coronary flow heterogeneity and the serum concentration of caffeine or its principal metabolites. Hence, the stringent requirements for prolonged abstinence from caffeine before adenosine MPI - based on limited studies - appear ill-founded.

Measurement and stability of FTY720 in human whole blood by high-performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry.

We report here a validated method for the quantification of a new immunosuppressant drug FTY720, using HPLC-tandem mass spectrometry. Whole blood samples (500 microl) were subjected to liquid-liquid extraction, in the presence of an internal standard (Y-32919). Mass spectrometric detection was by selected reaction monitoring with an atmospheric pressure chemical ionization source in positive ionization mode (FTY720: m/z 308.3-->255.3). The assay was linear from 0.2 to 25 microg/l (r(2)>0.997, n=5). The inter- and intra-day analytical recovery and imprecision for quality control samples (0.5, 7 and 15 microg/l) were 95.8-103.2 and <5.5%, respectively. At the lower limit of quantification (0.2 microg/l) the inter- and intra-day analytical recovery was 99.0-102.8% with imprecision of <7.6% (n=5). The assay had a mean relative recovery of 100.5+/-5.8% (n=15). Extracted samples were stable for 16 h. FTY720 quality control samples were stable at room temperature for 16 h, at 4 degrees C for at least 8 days and when taken through at least three freeze-thaw cycles. In conclusion, the method described displays analytical performance characteristics that are suitable for pharmacokinetic studies in humans.

Evaluation of a fluorescent polarization immunoassay for whole blood everolimus determination using samples from renal transplant recipients.

OBJECTIVES: This study compared the performance of a fluorescent polarization immunoassay (FPIA) against HPLC-tandem mass spectrometry (HPLC-MS) for the measurement of everolimus in renal transplant recipients. DESIGN AND METHODS: A total of 333 pre-dose samples from 45 renal transplant patients were analyzed by FPIA and HPLC-MS. RESULTS: The inter-batch inaccuracy and precision of the FPIA for control samples were

A rapid HPLC-mass spectrometry cyclosporin method suitable for current monitoring practices.

OBJECTIVES: Cyclosporin is an immunosuppressant drug with a narrow therapeutic window. Trough and 2-h post-dose blood samples are currently used for therapeutic drug monitoring in solid organ transplant recipients. The aim of the current study was to develop a rapid HPLC-tandem mass spectrometry (HPLC-MS) method for the measurement of cyclosporin in whole blood that was not only suitable for the clinical setting but also considered a reference method. METHODS: Blood samples (50 muL) were prepared by protein precipitation followed by C(18) solid-phase extraction while using d(12) cyclosporin as the internal standard. Mass spectrometric detection was by selected reaction monitoring with an electrospray interface in positive ionization mode. RESULTS: The assay was linear from 10 to 2000 microg/L (r(2)>0.996, n=9). Inter-day analytical recovery and imprecision using whole blood quality control samples at 10, 30, 400, 1500, and 2000 microg/L were 94.9--103.5% and <7.7%, respectively (n=5). The assay had a mean relative recovery of 101.6%. Ion suppression was<8.0% of the total signal (n=15). Extracted samples were stable for 6 h. Patient samples, measured by this method and compared with a validated HPLC-UV assay, revealed a strong correlation (r=0.998) and excellent agreement with a mean percentage bias of 2.1% (n=60). CONCLUSION: This high-throughput method provides accurate, precise, and specific measurement of cyclosporin in blood over a wide analytical range, thus making it suitable for current clinical monitoring strategies.