Absorptive chemistry based extraction for LC-MS/MS analysis of small molecule analytes from biological fluids - an application for 25-hydroxyvitamin D.

BACKGROUND: Already available sample preparation technologies for liquid chromatography-tandem mass spectrometry have substantial shortcomings with respect to automation. A novel approach is based on gel-like polymeric material with defined absorption chemistry, which is immobilized in micro-plate wells. It is referred to as Tecan Immobilized Coating Extraction™ (TICE™) technology and it enables easy automation on liquid handling systems. We aimed to study the performance of Tecan AC Extraction Plate™ based on this principle by addressing 25-hydroxyvitamin D (25OHD) as an exemplary analyte. METHODS: A protocol for extraction of 25OHD from serum samples based on TICE™ technology was implemented on a robotic liquid handling system Freedom EVO® (Tecan). An isotope-dilution ultra-performance liquid chromatography-tandem mass spectrometry method was used for quantification. Performance was tested according to a comprehensive protocol. RESULTS: Linearity was found over a range from 4.3 to 65.8 ng/mL for 25OHD3. The coefficients of variation for the intra-day and inter-day precision were <6% and accuracy ranged between 96.9% and 99.8% for 25OHD3. Recovery was 84% and efficient control of matrix effects was verified. High sample throughput could be observed with 96 samples prepared in <60 min. Close agreement of results was found for clinical samples analyzed with a second tandem mass spectrometry method based on protein precipitation and two-dimensional ultra-performance liquid chromatography for sample preparation (r=0.988, n=73). CONCLUSIONS: The new TICE™ technology was found to be a useful process for sample preparation in clinical mass spectrometry. Full automation suited for routine analysis was achieved.

No endogenous ouabain is detectable in human plasma by ultra-sensitive UPLC-MS/MS.

BACKGROUND: The presence of a binding site for cardiac glycosides, such as digitoxin and digoxin, in the sodium-potassium-ATPase, stimulated attempts to isolate endogenous cardiotonic steroids. Using immunoassays, clinical studies found the cardenolide ouabain to be secreted endogenously in response to exercise and untreated hypertension and to be correlated with severity of clinical conditions such as kidney failure and dilated cardiomyopathy. The assays used were not standardized and the mean concentrations of endogenous ouabain reported for healthy controls ranged from 60 to 530 pmol/l. None of these immunoassays is available any more. Therefore, the aim of this study was to develop a highly specific and reliable method for measurement of ouabain in human plasma based on isotope dilution liquid chromatography tandem-mass spectrometry (ID-LC-MS/MS). METHOD: An ultra-sensitive and specific ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed which applied solid phase extraction of plasma for sample preparation. RESULTS: The method was comprehensively validated and had a lower limit of quantification of 1.7 pmol/l. However, despite this very low detection limit ouabain was not observed in plasma samples from patients with and without heart failure. CONCLUSION: Our results suggest that immunoassays previously used to quantify assumed endogenous ouabain detected compounds which are not structurally identical with ouabain. Cross reactivity of structurally related compounds of endogenous origin may cause these discrepancies between immunological and mass spectrometric analyses. Conclusive characterization of assumed endogenous counterparts of digoxin in a biomarker discovery approach seems to require distinct analytical techniques.

Simultaneous quantification of four vitamin D metabolites in human serum using high performance liquid chromatography tandem mass spectrometry for vitamin D profiling.

OBJECTIVES: For quantification of 25-hydroxyvitamin D(3) (25OH-D(3)), 25-hydroxyvitamin D(2) (25OH-D(2)), 3-epi-25-hydroxyvitamin D(3) (3-epi-25OH-D(3)) and 24R,25-dihydroxyvitamin D(3) (24R,25(OH)(2)-D(3)) in human serum a high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method was developed and validated. DESIGN AND METHODS: After protein precipitation further purification is achieved with on-line sample preparation using a reversed phase (RP) C-4 column. Chromatographic separation is realized by a RP-column with core shell material and pentafluorophenyl (PFP) selectivity. Atmospheric pressure chemical ionization in the positive ion mode with multi-reaction monitoring is used for analyte detection. RESULTS: Baseline separation of the analytes is achieved below 10 min. The method is linear over the range 4.0-265.3 nmol/L for 25OH-D(3), 3.9-183.6 nmol/L for 25OH-D(2), 2.0-133.8 nmol/L for 3-epi-25OH-D(3) and 2.8-129.9 nmol/L for 24R,25(OH)(2)-D(3) (r(2)>0.998). The limit of quantification is 4.0 nmol/L for 25OH-D(3), 3.9 nmol/L for 25OH-D(2), 2.0 nmol/L for 3-epi-25OH-D(3) and 2.8 nmol/L for 24R,25(OH)(2)-D(3). The CVs for the intra-day and inter-day precision are <5% and <4%, respectively. Metabolite levels for a set of 50 human serum samples have been determined and resulted in the detection of considerable amounts of 3-epi-25OH-D(3) and 24R,25(OH)(2)-D(3). CONCLUSIONS: This highly specific HPLC-MS/MS method is suitable for vitamin D profiling. There is a correlation between 25OH-D(3) and 24R,25(OH)(2)-D(3). Serum concentration of 24R,25(OH)(2)-D(3) increases disproportionally with increasing concentration of 25OH-D(3).