Immune Monitoring of Mycophenolate Mofetil Activity in Healthy Volunteers Using Ex Vivo T Cell Function Assays.

Mycophenolate mofetil (MMF) is part of the standard immunosuppressive treatment after transplantation and usually given as "one-dose-fits-all" together with a calcineurin inhibitor (CNI). Although drug concentrations are frequently monitored, there is still a group of patients who experience side effects related to excessive or insufficient immune suppression. We therefore aimed to identify biomarkers that reflect the overall immune status of the patient and might support individualized dosing. We previously studied immune biomarkers for CNIs and aimed to investigate whether these are also suitable to monitor MMF activity. Healthy volunteers received a single dose of MMF or placebo, after which IMPDH enzymatic activity, T cell proliferation, and cytokine production were measured and compared to MPA (MMF's active metabolite) concentration in three different matrices (plasma, peripheral blood mononuclear cells, and T cells). MPA concentrations in T cells exceeded those in PBMCs, but all intracellular concentrations correlated strongly with plasma concentrations. At clinically relevant MPA concentrations, IL-2 and IFN-γ production was mildly suppressed, while MPA T cell proliferation was strongly inhibited. Based on these data, it is expected that monitoring of T cell proliferation in MMF-treated transplantation patients may be a valid strategy to avoid excessive immune suppression.

Application of microbore HPLC in combination with tandem MS for the quantification of rosuvastatin in human plasma.

The potential of microbore high-performance liquid chromatography (HPLC) in combination with tandem mass spectrometry (MS/MS) for the sensitive detection of rosuvastatin (Crestor) in human plasma was investigated. Three microbore HPLC columns with internal diameters (i.d.) of 0.5, 1.0 and 2.0 mm were evaluated for column efficiency and mass sensitivity, and compared to a conventional 4.6 mm i.d. column. The 2.0 and 1.0 mm i.d. columns performed very well while the 0.5 mm i.d. column was slightly less efficient, this is probably due to a lower packing density. Good results with respect to gains in mass sensitivity compared to the conventional analytical column were achieved with the 2.0 and 1.0 mm columns. Thus, the 2.0 mm i.d. column had an improved signal-to-noise (S/N) ratio of 16 whilst the 1.0 mm i.d. column had an improved S/N ratio of greater than 70. Experiments with the 1.0 mm i.d. HPLC column were performed to determine the robustness of the microbore method for human plasma extracts after sample preparation using solid-phase extraction (SPE). A number of problems were encountered with extracts including high backgrounds, the blocking of the column and a rapid deterioration in column performance. The blocking of the column by particulates was solved by off-line filtration of the sample extracts. Peak tailing of the analytes and high background, both of which were due to endogenous interferences in the extracts, were eliminated using gradient elution. Using these approaches over 500 injections of plasma extracts were achieved without significant deterioration in assay performance. Quantities of rosuvastatin of 0.3 pg on-column could be detected and cross-validation experiments demonstrated that the conventional and the microbore HPLC-MS/MS methods provided similar information on the concentration of rosuvastatin but with greatly reduced sample consumption using the microbore method.

Contactless conductivity detection of synthetic polymers in non-aqueous size-exclusion electrokinetic chromatography.

A capacitively coupled contactless conductivity detection (CCD) system has been applied for the detection of neutral synthetic polymers in capillary size-exclusion electrokinetic chromatography (SEEC). Polystyrene standards, that were used as a model compounds, were separated on a capillary column packed with porous 10 microm silica particles with an electrokinetically driven mobile phase, and detected by CCD and UV detection simultaneously. Mass-calibration curves for polystyrene were constructed. Satisfactory results were obtained for the linearity, the run-to-run repeatability (<0.2% for the relative retention and <4% for the peak area) and the robustness of the detector. One of the major issues in this preliminary study was to investigate the origin of the peaks observed for the polystyrene standards. The effect of the molar mass of the polystyrenes on the sensitivity was small. Therefore, the signals obtained could not be explained as the result of an increased viscosity and a decreased solution conductivity of the solute zone. An alternative hypothesis is suggested, and recommendations for further research are given.

Characterization of polyethylene glycols and polypropylene glycols by capillary zone electrophoresis and micellar electrokinetic chromatography.

Methods based on capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) have been developed and optimized for the separation of polyethylene glycols (PEGs) and polypropylene glycols (PPGs). To provide for charge and detectability both types of polymeric compounds were derivatized with phthalic anhydride (PhAH) or 1,2,4-benzenetricarboxylic anhydride (BTA) before the separation. Derivatization with BTA yielded more complex electropherograms, due to the occurrence of different isomeric reaction products for every PEG or PPG species. Effective mobilities of the PhAH derivatives were related to the number of monomer units in the polymers in a straightforward way. The CZE method could also be used to determine the monomer-number distribution of random and block PEG-PPG copolymers. For an MEKC analysis the PEGs and PPGs were derivatized with phenyl isocyanate. Oligomers of PEGs could be separated up to molecular masses of 5,000, while for the more hydrophobic PPGs oligomeric separation was only accomplished for molecular masses of up to 1,500. Due to a strongly different separation mechanism for the PEG and PPG derivatives in the MEKC system. a complete group separation of the two types of polymer molecules could be obtained.

Characterization of glycerin-based polyols by capillary electrophoresis.

Methods based on capillary electrophoresis (CE) have been developed to obtain the molar mass distribution (MMD) of glycerin-based polyols and details on the presence of mono- and difunctional byproducts in technical samples. Prior to the analyses the hydroxy end-groups of the trifunctional polyols were converted to chargeable and UV-active moieties with phthalic anhydride (PhAH) as the derivatization reagent. With a method of capillary zone electrophoresis (CZE) samples of glycerin-based polyols with average molar masses up to 6000 were separated according to their charge-to-size ratio. The separations were carried out with a buffer solution containing 50% (v/v) acetonitrile and 10 mM sodium tetraborate, and for detection UV absorption at 220 nm was measured. An approximately linear relation between the reciprocal of the effective mobilities and the degree of polymerisation of the glycerin-based polyols was found. Therefore, the proposed CZE system could be used to determine the degree of polymerisation and polydispersity of technical glycerin-based polyol samples. The effect of the presence of sodium dodecyl sulfate (SDS) in the buffer solution on the CE separation of linear polyethylene glycols (PEGs), polypropylene glycols (PPGs) and ethylene oxide-propylene oxide (EO-PO) copolymers with different molar masses was investigated. The interaction between the charged polymer derivatives and SDS ions in solution increased strongly with the degree of polymerisation and the amount PO in the chain of the polymeric compounds. This behaviour made it possible to invert the migration order of EO-PO containing polymers of different size. With a background electrolyte (BGE) composition of 10mM SDS and 25% (v/v) acetonitrile in borate buffer mono- and difunctional byproducts were separated from the main glycerin-based polyols based on their number of end-groups. Accurate quantities for the mono- and difunctional impurities in technical glycerin-based polyol products were determined.

Determination of the degree of substitution and its distribution of carboxymethylcelluloses by capillary zone electrophoresis.

A method based on capillary zone electrophoresis (CZE) has been developed to determine the degree of substitution (DS) of carboxymethylcellulose (CMC). Separations were performed with borate buffer (pH 9, ionic strength 20 mM) as background electrolyte in capillaries of 75 microm ID, with an applied voltage of 10 kV, and for detection UV absorption at 196 nm was measured. The use of an internal standard (phthalic acid) to correct for mobility variations resulted in a strong improvement of the precision of the DS determination. Experiments with indirect UV detection indicated that the peak widths obtained actually reflect the variation in mobility, and with that of the DS value, of CMC samples. With the proposed method not only the average DS value but also its dispersity could be established for technical CMC samples. A small but definite effect of the polymeric size on the mobilities was observed. Therefore, DS calibration curves will have to be determined for a specific MM range. Since the size effect is small, a classification of CMCs as low-, middle-, or high MM will be sufficient to obtain accurate data on the DS distribution.