Weak Anion-exchange Liquid Chromatography Research Articles

Characterization of mercury-containing protein in human plasma

Characterization of mercury binding protein in the human body is very important for understanding the metabolism and the mechanism of toxication of ingested mercuric compounds. In this study, mercury-containing protein in human plasma was separated by on-line heart-cutting two-dimensional high performance liquid chromatography (2D-HPLC). This 2D separation system used size exclusion liquid chromatography (SEC) followed by weak anion exchange liquid chromatography (WAX) and the two LC parts were coupled by a six-port valve equipped with a storage loop and controled by the computer. The WAX effluent was determined by both UV detection and inductively coupled plasma-mass spectrometry (ICP-MS) to locate the mercury-containing protein. A unique mercury-containing protein fraction was obtained by 2D-HPLC separation and subsequently identified by HPLC coupled with linear ion trap-Fourier transform ion cyclotron resonance mass spectrometry (HPLC-LTQ-FT). The database search confirmed that the mercury-containing protein in the human plasma is human serum albumin (HSA). The stoichiometry and thermodyamics interaction of inorganic mercury (Hg(2+)) with HSA was studied by isothermal titration calorimetry (ITC) and two binding types were observed. Mercury-containing protein in human plasma was separated and identified in the present study and it is important for understanding the metabolism of mercury in the human body.

Simultaneous quantification of emtricitabine and tenofovir nucleotides in peripheral blood mononuclear cells using weak anion-exchange liquid chromatography coupled with tandem mass spectrometry

Emtricitabine (FTC) and tenofovir (TFV) are widely used antiviral agents that require intracellular phosphorylation to become active. This article describes the development and validation of an assay for the simultaneous quantification of FTC mono-, di- and triphosphate (FTC-MP, -DP and -TP), TFV and TFV mono- and diphosphate (TFV-MP and -DP) in peripheral blood mononuclear cells. Reference compounds and internal standards were obtained by thermal degradation of FTC-TP, TFV-DP, stable isotope-labeled TFV-DP and stable isotope-labeled cytosine triphosphate. Cells were lysed in methanol:water (70:30, v/v) and the extracted nucleotides were analyzed using weak anion-exchange chromatography coupled with tandem mass spectrometry. Calibration ranges in PBMC lysate from 0.727 to 36.4, 1.33 to 66.4 and 1.29 to 64.6 nM for FTC-MP, FTC-DP and FTC-TP and from 1.51 to 75.6, 1.54 to 77.2 and 2.54 to 127 nM for TFV, TFV-MP and TFV-DP, respectively, were validated. Accuracies were within −10.3 and 16.7% deviation at the lower limit of quantification at which the coefficients of variation were less than 18.2%. At the other tested levels accuracies were within −14.3 and 9.81% deviation and the coefficients of variation lower than 14.7%. The stability of the compounds was assessed under various analytically relevant conditions. The method was successfully applied to clinical samples.

Development and validation of an assay for the quantitative determination of cladribine nucleotides in MDCKII cells and culture medium using weak anion‐exchange liquid chromatography coupled with tandem mass spectrometry

The development and validation of an assay for the quantitative analysis of cladribine mono-, di- and triphosphate (2-chloro, 2'-deoxyadenosine 5'-mono-, di- and triphosphate or 2CdAMP, 2CdADP and 2CdATP) in culture medium (Optimem) and cell lysate is described. Cladribine mono- and diphosphate reference compounds were obtained by thermal degradation of cladribine triphosphate. The reference compounds were characterized using ion-pairing reversed-phase high-performance liquid chromatography with ultraviolet detection. The bioanalytical assay for 2CdAMP, 2CdADP and 2CdATP is based on weak anion-exchange liquid chromatography coupled with tandem mass spectrometry in the positive ion mode (WAXLC/MS/MS). A fused-silica electrospray capillary was used instead of a stainless steel electrospray capillary to minimize adsorption of analytes and thus decrease variation in the analyte signals. Dynamic ranges of 1.11-27.7, 0.550-55.0 and 1.31-52.3 nM for 2CdAMP, 2CdADP and 2CdATP, respectively, were validated in culture medium and cell lysate. Optimem samples required stabilization with 30% methanol to prevent conversion of 2CdATP into 2CdAMP and 2CdADP. All intra- and interday accuracies and precisions were within +/-20%. The stability of the compounds was assessed under various analytically relevant conditions. The method was successfully used to investigate cladribine nucleotide transport in vitro in Madin-Darby canine kidney II (MDCKII) cells.

Quantitative analysis of gemcitabine triphosphate in human peripheral blood mononuclear cells using weak anion‐exchange liquid chromatography coupled with tandem mass spectrometry

Gemcitabine triphosphate (dFdCTP) is a highly active metabolite of gemcitabine. It is formed intra-cellularly via the phosphorylation of gemcitabine by deoxycytidine kinase. The monitoring of dFdCTP in human peripheral blood mononuclear cells (PBMCs), in addition to plasma concentrations of gemcitabine and its metabolite 2',2'-difluorodeoxyuridine, is considered very useful in determining pharmacokinetic-pharmacodynamic relationships. We describe a novel sensitive assay for the quantification of dFdCTP in human PBMCs. The method is based on weak anion-exchange liquid chromatography and detection with tandem mass spectrometry (LC-MS/MS). The assay has been validated from 1 ng/ml (lower limit of quantification, LLOQ) to 25 ng/ml (upper limit of quantification, ULOQ) using 180 microl aliquots of PBMC extracts containing approximately 0.648 mg protein or 3.8 x 10(6) lysed PBMCs. The LLOQ is equivalent to 94 fmol/10(6) cells (1 ng/ml = 0.18 ng/180 microl or 0.18 ng/0.648 mg protein = 0.047 ng/10(6) cells or 94 fmol/10(6) cells). This highly sensitive assay is capable of quantifying about 200-fold lower concentrations of dFdCTP in human PBMCs than currently available methods.

Novel direct detection method for quantitative determination of intracellular nucleoside triphosphates using weak anion exchange liquid chromatography/tandem mass spectrometry.

A novel analytical method has been developed for direct quantification of intracellular nucleoside triphosphates (NTPs). Lysates of human peripheral blood mononuclear cells (PBMCs) were extracted by protein precipitation, and the filtered extracts were analyzed by weak anion exchange liquid chromatography (WAX-LC) coupled to detection by mass spectrometry (MS). Compared with ion pairing (IP)-LC/MS/MS, the only MS-compatible direct detection method for NTPs currently available, the new method completely avoids the usage of ion-pairing reagents and has a shorter analytical time of only 2 min. The method was validated and is being used to determine the amount of the triphosphate metabolite of D-D4FC (DPC817), an investigational HIV nucleoside reverse transcriptase inhibitor (NRTI), in human PBMC samples from clinical studies. By using a PE Sciex API 4000 triple quadrupole instrument operating in positive ion MRM mode, the method was able to achieve a lower limit of quantitation (LLOQ) of 5 fmol/10(6) cells in samples containing 3 x 10(6) lysed cells (6 fmol on-column). With minor adaptation, the method described here may be suitable for analyzing other NTPs. This paper also provides a discussion of the unique retention characteristics of WAX-LC, the principles of which may prove to be valuable for designing other forms of directly coupled ion-exchange (IX)-LC/MS methods suited for high sensitivity quantitative analysis.