Improved capillary electrophoresis determination of carbohydrate-deficient transferrin including on-line immunosubtraction.

The instrumental analysis of carbohydrate-deficient transferrin (CDT), a recognized marker of chronic alcohol abuse, is most commonly carried out by high-performance liquid chromatography (HPLC) or capillary zone electrophoresis (CZE). Between these two techniques, CZE shows higher efficiency and productivity, but is often reported to be inferior to HPLC in terms of selectivity, because of a less specific ultraviolet detection wavelength than HPLC. On these grounds, the present work was aimed at the development of an improved CZE method for CDT determination, including an on-line immunosubtraction step specifically aimed at enhancing the analytical specificity of CZE determination. The analytical conditions were as follows: uncoated fused silica capillary, 30 microm x 60 cm (L = 50 cm to detector); running buffer, 100 mmol/L borate and 6 mmol/L DAB (1,4-diaminobutane), pH 8.3; voltage, 30 kV; temperature, 25 degrees C; detection, 200 nm. Under the described CZE conditions, a baseline separation between all the CDT related peaks was achieved with good analytical performances in terms of both precision and accuracy. In order to achieve unequivocal recognition of the CDT peaks, an in-capillary immunosubtraction step was included by loading a plug of anti-human transferrin antibody solution after the sample plug. This analytical approach was applied successfully to recognize CDT peaks in the presence of potential interferences.

High-throughput LC-MS/MS method for monitoring sirolimus and everolimus in the routine clinical laboratory.

BACKGROUND: Immunosuppressant therapeutic drug monitoring (TDM) is an important requirement in post-transplant patient care. In recent years, high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has become a valid alternative to antibody-based immunoassays in TDM due to its high specificity and sensitivity. Furthermore, this technology allows for the simultaneous measurement of several immunosuppressive drugs. The aim of the present study was to establish a straightforward, robust, and high-throughput LC-MS/MS method for the simultaneous determination of sirolimus and everolimus in whole blood in order to replace immunoassays in our routine practice. METHODS: Five-level blood calibrators were employed for assay development, while three materials at different concentrations were used for internal quality control. The proposed method uses protein precipitation for sample preparation. Analyses were performed using a triple quadrupole LC-MS/MS, with a C18 held at 60°C. Using an appropriate gradient elution profile and SPE on-line, elution times for all compounds analysed were 2.6 min with a total run-time of 3.5 min. RESULTS: Calibration curves were linear throughout the selected ranges. The intra- and inter-assay CVs (<7%), the limit of quantification (0.2 µg/L) and accuracy were highly satisfactory. On testing the results using the international proficiency testing scheme (UK-NEQAS), the performance of the proposed method was found to be highly reliable. CONCLUSIONS: The findings made by us indicate that the proposed method is of value, since it is speedy, straightforward, accurate, and applicable to different LC-MS/MS instruments for the routine TDM of organ transplant recipients.

HPLC determination of plasma dimethylarginines: method validation and preliminary clinical application.

BACKGROUND: Asymmetric dimethylarginine (ADMA) has been suggested as a possible marker of endothelial dysfunction, and interest in its use in clinical practice is increasing. However, the potential role of symmetric dimethylarginine (SDMA) as an endogenous marker of renal function, has been less widely investigated. The aims of the present study were therefore to determine reference values for dimethylarginines in plasma after method validation, and to ascertain ADMA plasma concentrations in patients with disorders characterized by endothelial dysfunction; a further end-point was to investigate the relationship between SDMA plasma concentrations and estimated GFR (eGFR) as well as plasmatic creatinine in patients with chronic kidney disease (CKD). MATERIALS AND METHODS: HPLC with fluorescence detection was used for the determination of plasma dimethylarginines. To verify the clinical usefulness of ADMA and SDMA, values from 4 groups of patients at a high risk of cardiovascular complications as well renal dysfunction (chronic heart failure n=126; type II diabetes n=43; pulmonary arterial hypertension n=17; chronic kidney disease n=42) were evaluated, and compared with the reference values, obtained from 225 blood donors. RESULTS: The intra- and inter-assay CVs (<5.2%), the absolute and relative recoveries (96-106%) were highly satisfactory. ADMA levels were significantly elevated in all groups of patients compared with controls (p<0.001) with the exception of samples from patients with type II diabetes. SDMA levels were significantly elevated both in the patients with chronic kidney disease and in the patients with type II diabetes complicated by renal insufficiency, the values being closely correlated with both eGFR (R=0.740) and plasmatic creatinine (R=0.700). CONCLUSIONS: The findings made in the present study shows that ADMA levels are significantly increased in patients with diseases associated with endothelial dysfunction This molecule might, therefore, be used as a biochemical marker for the evaluation of endothelial function. Furthermore, the preliminary results reported suggest that SDMA might be a reliable marker of renal function, especially in peadiatric populations, for which the use of eGFR is not recommended.

Determination of ethoxylated bisphenol A dimethacrylate monomers in dental composites by micellar electrokinetic chromatography.

Separation of monomeric constituents of ethoxylated bisphenol A (BIS-EMA) with between 2 and 15 ethoxy groups per phenol in the molecule was investigated with micellar electrokinetic chromatography at different volume percentages methanol in the background electrolyte (10 mM sodium dodecylsulfate, 100 mM borate-50 mM phosphate buffer, pH 7.0). The conditions allowed the differentiation of the lower from the higher BIS-EMA homologues and of isomers, and enabled the characterisation of commercial dental composite materials. The decay curve for the light induced radical polymerisation of BIS-EMA in composite specimens was determined. The content of leachable monomers after light curing was quantified, resulting in 6% of the initial value after the recommended curing time. The method is suited to determine monomer constituents in polymerised composite material in the ppm range.

Microemulsion electrokinetic chromatography applied for separation of levetiracetam from other antiepileptic drugs in polypharmacy.

Microemulsion electrokinetic chromatography was applied for the separation of levetiracetam from other antiepileptic drugs (primidone, phenobarbital, phenytoin, lamotrigine and carbamazepine) that are potentially coadministered in therapy of patients. The influence of the composition of the microemulsion system (with sodium dodecyl sulfate as charged surfactant) was investigated, modifying the kind of cosurfactant (lower alcohols from C3 to C5), the pH (and salinity) of the aqueous background electrolyte, and the ratio of aqueous phase to organic constituents forming the microdroplets of the oil-in-water emulsion. Separation selectivity was depending on all these parameters, resulting even in changes of the migration sequence of the analytes. Only moderate correlation was observed for the microemulsion system compared with a micellar system, both consisting of the aqueous borate buffer (pH 9.2) and SDS as micelle former (linear correlation coefficient for analyte mobilities is 0.974). The sample solvent plays an important role on the shape of the resulting chromatograms: methanol at concentrations higher than 35% impairs peak shape and separation efficiency. The microemulsion method (with 93.76% aqueous borate buffer (pH 9.2, 10 mM), 0.48% n-octane, 1.80% SDS, 3.96% 1-butanol, all w/w) is suitable for the determination of levetiracetam in human plasma (combined with a sample pretreatment based on solid-phase extraction).