Hematocrit-independent recovery of immunosuppressants from DBS using heated flow-through desorption.

BACKGROUND: We applied our concept for automated flow-through desorption of DBS to investigate the effect of desorption temperature on recovery. For this purpose, a method has been developed for the determination of four immunosuppressants in DBS. RESULTS: We compared recoveries of four immunosuppressants for measurements with and without temperature-enhanced desorption at different hematocrit (Ht) levels. Temperature-enhanced desorption increased recovery substantially for tacrolimus, sirolimus and everolimus at all Ht values and for cyclosporine At high Ht. In addition, recovery became largely independent from Ht variations. Under the optimized conditions, a brief validation using spiked blood samples showed that the method complies with acceptance criteria for quantitative bioanalysis. CONCLUSION: This method enables a quantitative analysis of immunosuppressants in DBS independent from the Ht.

Steroidogenesis in the adrenal dysfunction of critical illness: impact of etomidate.

INTRODUCTION: This study was aimed at characterizing basal and adrenocorticotropic hormone (ACTH)-induced steroidogenesis in sepsis and nonsepsis patients with a suspicion of critical illness-related corticosteroid insufficiency (CIRCI), taking the use of etomidate-inhibiting 11ß-hydroxylase into account. METHOD: This was a prospective study in a mixed surgical/medical intensive care unit (ICU) of a university hospital. The patients were 62 critically ill patients with a clinical suspicion of CIRCI. The patients underwent a 250-µg ACTH test (n = 67). ACTH, adrenal steroids, substrates, and precursors (modified tandem mass spectrometry) also were measured. Clinical characteristics including use of etomidate to facilitate intubation (n = 14 within 72 hours of ACTH testing) were recorded. RESULTS: At the time of ACTH testing, patients had septic (n = 43) or nonseptic critical illness (n = 24). Baseline cortisol directly related to sepsis and endogenous ACTH, independent of etomidate use. Etomidate was associated with a lower baseline cortisol and cortisol/11ß-deoxycortisol ratio as well as higher 11ß-deoxycortisol, reflecting greater 11ß-hydroxylase inhibition in nonsepsis than in sepsis. Cortisol increases < 250 mM in exogenous ACTH were associated with relatively low baseline (HDL-) cholesterol, and high endogenous ACTH with low cortisol/ACTH ratio, independent of etomidate. Although cortisol increases with exogenous ACTH, levels were lower in sepsis than in nonsepsis patients, and etomidate was associated with diminished increases in cortisol with exogenous ACTH, so that its use increased, albeit nonsignificantly, low cortisol increases to exogenous ACTH from 38% to 57%, in both conditions. CONCLUSIONS: A single dose of etomidate may attenuate stimulated more than basal cortisol synthesis. However, it may only partly contribute, particularly in the stressed sepsis patient, to the adrenal dysfunction of CIRCI, in addition to substrate deficiency.

A fast liquid chromatographic tandem mass spectrometric method for the simultaneous determination of total homocysteine and methylmalonic acid.

A liquid chromatography(LC)/electrospray ionization tandem mass spectrometry (MS) method for the quantitative determination of total homocysteine and methylmalonic acid and the monitoring of methionine, homocystine and succinic acid in plasma has been developed. The analytes are determined under the presence of the deuterated internal standards methylmalonic acid-d (3) and homocystine-d (8). Although methylmalonic acid can be determined directly, a reduction step has to be carried out to ensure the measurement of total homocysteine. Ultrafiltration was applied afterwards to deproteinize the samples prior to LC/MS injection. LC/MS analysis is carried out isocratically using a mobile phase consisting of 5% methanol and 95% of a 0.06 M formic acid solution on a reversed-phase C18 column at a flow rate of 0.5 mL/min. The MS measurement was separated into several periods: homocysteine, homocystine and methionine were determined in the positive-ion mode, whereas the determinations of methylmalonic acid and succinic acid were carried out in the negative-ion mode. The intraday coefficients of variation (CVs) were 2.9% or less and 3.2% or less for homocysteine and methylmalonic acid, respectively. Interday CVs ranged from 3.8 to 5.9% for homocysteine and from 3.5 to 6.3% for methylmalonic acid. Analyte concentrations could reliably be determined, also far below the reference values. Furthermore, the linearity was determined and a correlation study with respect to the existing homocysteine and methylmalonic acid methods at Medisch Spectrum Twente Hospital was carried out.

Labeling strategies for bioassays.

Different labeling strategies for enzymatic assays and immunoassays are reviewed. Techniques which make use of direct detection of a label, e.g. radioimmunoassays, are discussed, as are techniques in which the label is associated with inherent signal amplification. Examples of the latter, e.g. enzyme-linked immunosorbent assays or nanoparticle-label based assays, are presented. Coupling of the bioassays to chromatographic separations adds selectivity but renders the assays more difficult to apply. The advantages and drawbacks of the different analytical principles, including future perspectives, are discussed and compared. Selected applications from clinical, pharmaceutical, and environmental analysis are provided as examples.

Analysis of microperoxidases using liquid chromatography, post-column substrate conversion and fluorescence detection.

A liquid chromatographic method with on-line activity determination for microperoxidases has been developed. After enzymatic digestion of a cytochrome, possibly under formation of microperoxidases, the product mixture is separated by reversed-phase liquid chromatography. The products first pass a diode-array detector, and are then subjected to a reaction with 4-(N-methylhydrazino)-7-nitro-2,1,3-benzooxadiazole (MNBDH) and hydrogen peroxide. In a reaction coil, microperoxidases catalyze the reaction under formation of the fluorescent 4-(N-methylamino)-7-nitro-2,1,3-benzooxadiazole (MNBDA). Quantification of the microperoxidases is performed using a fluorescence detector at an excitation wavelength of 470 nm and an emission wavelength of 545 nm, respectively. For this LC-based detection system, limits of detection are 3 x 10(-8) mol/L, limits of quantification are 9 x 10(-8) mol/L, and a linear range from 9 x 10(-8) mol/L to 3 x 10(-6) mol/L is obtained for the microperoxidases MP-9 and MP-11. A highly active microperoxidase MP-6 was found in the reaction of cytochrome c from bovine heart with protease from streptomyces griseus.

Liquid chromatographic/mass spectrometric investigation on the reaction products in the peroxidase-catalyzed oxidation of o-phenylenediamine by hydrogen peroxide.

Mass spectrometric evidence was obtained to confirm that the main reaction product of the horseradish peroxidase (POD)-catalyzed oxidation of o-phenylenediamine (OPD) by hydrogen peroxide is 2,3-diaminophenazine. Although this reaction is one of the most widespread detection schemes in enzyme-linked immunosorbent assays (ELISAs), the literature data on the identity of the reaction product(s) have been strongly contradictory throughout the last few decades. Liquid chromatography with UV/Vis and mass spectrometric detection as well as exact mass measurements after LC fraction collection have led to the unambiguous identification of 2,3-diaminophenazine as main reaction product. 2,2'-Diaminoazobenzene, which is frequently described in other publications to be the major reaction product, was not detected at all.