Determination of thalidomide by high performance liquid chromatography: plasma pharmacokinetic studies in the rat

A sensitive and simple high performance liquid chromatography (HPLC) method was developed and validated for the determination of thalidomide in rat plasma. Chromatography was accomplished with a reversed-phase Hypersil C18 column. Mobile phase consisted of acetonitrile-10 mM ammonium acetate buffer (pH 5.50) (28:72, v/v), at a flow rate of 0.8 ml/min. Thalidomide was monitored by ultraviolet detector at 220 nm and it gave a linear response as a function of concentration over 0.02–50 μM. The limit of quantitation in rat plasma was 0.50 ng (0.02 μM plasma concentration) with an aliquot of 20 μl. Results from a 3-day validation study indicated that this method allows for simple and rapid quantitation of thalidomide with excellent accuracy and reliability. Using this validated assay, the effect of coadministered irinotecan (CPT-11) on the plasma pharmacokinetics of thalidomide in rats was determined. Coadministration of CPT-11 (intravenously, 60 mg/kg) increased the maximum plasma concentration (C_max) and area under the plasma concentration–time curve (AUC_{0–10 h}) of thalidomide by 32.29 and 11.66%, respectively, as compared to the control, but none of the effect of CPT-11 was of statistical significance (P > 0.05). Concomitant CPT-11 also caused a 10.04% decrease in plasma clearance (CL) and 14.51% decrease in volume of distribution (V_d) (P > 0.05). These results suggest that coadministered CPT-11 did not significantly alter the plasma pharmacokinetics of thalidomide in rats. Further studies are warranted to explore the pharmacokinetic and pharmacodynamic interactions between CPT-11 and thalidomide.

Preliminary evaluation of monolithic column high-performance liquid chromatography with tris(2,2’-bipyridyl)ruthenium(II) chemiluminescence detection for the determination of quetiapine in human body fluids

High-performance liquid chromatography (HPLC) with tris(2,2-bipyridyl)ruthenium(II) chemiluminescence detection methodology is reported for the determination of the atypical antipsychotic drug quetiapine and the observation of its major active and inactive metabolites in human urine and serum. The method uses a monolithic chromatographic column allowing high flow rates of 3mL min−1 enabling rapid quantification. Flow injection analysis (FIA) with tris(2,2-bipyridyl)ruthenium(II) chemiluminescence detection and HPLC time of flight mass spectrometry (TOF-MS) were used for the determination of quetiapine in a pharmaceutical preparation to establish its suitability as a calibration standard. The limit of detection achieved with FIA was 2×10−11 mol L−1 in simple aqueous solution. The limits of detection achieved with HPLC were 7×10−8 and 2×10−10 mol L−1 in urine and serum, respectively. The calibration range for FIA was between 5×10−9 and 1×10−6 mol L−1. The calibration ranges for HPLC were between 1×10−7–1×10−4 and 1×10−8–1×10−4 mol L−1 in urine and serum, respectively. The quetiapine concentrations in patient samples were found to be 3×10−6 mol L−1 in urine and 7×10−7 mol L−1 in serum. Without the need for preconcentration, the HPLC detection limits compared favourably with those in previously published methodologies. The metabolites were identified using HPLC-TOF-MS.

Determination of resin acids in pulp mill effluent and receiving waters by high performance liquid chromatography

Resin acids are reported to be of major toxicological importance in pulp mill effluents for Rainbow Trout. Their determination, using a high performance liquid chromatographic method, is described.

The analysis of café espresso using two-dimensional reversed phase–reversed phase high performance liquid chromatography with UV-absorbance and chemiluminescence detection

In this study, an activity based screening technique combining two-dimensional liquid chromatography (2DHPLC) with UV-absorbance and chemiluminescence detection was applied to study “Ristretto”, "Decaffeinatto” and “Volluto” espresso coffees. This technique, which coupled the separation power of 2DHPLC with the sensitivity and selectivity of the chemiluminescence detection, offers great potential for screening complex samples for antioxidant compounds. Detailed information regarding the complexity of the sample, and the variation between these three coffees could be obtained using this multidimensional-hyphenated method of analysis.

High-performance liquid chromatography with post-column 2,2′-diphenyl-1-picrylhydrazyl radical scavenging assay : methodological considerations and application to complex samples

An improved post-column 2,2´-diphenyl-1-picrylhydrazyl (DPPH•) radical scavenging assay for the screening of antioxidants in complex matrices was developed. Experimental parameters believed to be influential to DPPH• response were studied in a univariate approach. Optimum conditions were found to be: 5×10⁻⁵M DPPH• reagent prepared in a 75% methanol: 25% 40mM citric acid–sodium citrate buffer (pH 6) solution, degassed with nitrogen; reaction coil of 2m×0.25mm i.d. PEEK tubing; detection at 521 nm; analysis at room temperature. The analytical utility of this protocol was evaluated by screening for antioxidants in thyme and green tea, in comparison with two commonly employed methodologies.

High performance liquid chromatography with two simultaneous on-line antioxidant assays : evaluation and comparison of espresso coffees

The antioxidant profiles of various espresso coffees were established using HPLC with UV-absorbance detection and two rapid, simultaneous, on-line chemical assays that enabled the relative reactivity of sample components to be screened. The assays were based on (i) the colour change associated with reduction of the 2,2´-diphenyl-1-picrylhydrazyl radical (DPPH•); and (ii) the emission of light (chemiluminescence) upon reaction with acidic potassium permanganate. Results from the two approaches were similar and reflected the complex array of antioxidant species present in the samples. However, some differences in selectivity were observed. Chromatograms generated with the chemiluminescence assay contained more peaks, which was ascribed to the greater sensitivity of the reagent towards minor, readily oxidisable sample components. The three coffee samples produced closely related profiles, signifying their fundamentally similar chemical compositions and origin. Nevertheless, the overall intensity and complexity of the samples in both UV absorption and antioxidant assay chromatograms were aligned with the manufacturers description of flavour intensity and character.

Determination of intracellular glutathione and glutathione disulfide using high performance liquid chromatography with acidic potassium permanganate chemiluminescence detection

Measurement of glutathione (GSH) and glutathione disulfide (GSSG) is a crucial tool to assess cellular redox state. Herein we report a direct approach to determine intracellular GSH based on a rapid chromatographic separation coupled with acidic potassium permanganate chemiluminescence detection, which was extended to GSSG by incorporating thiol blocking and disulfide bond reduction. Importantly, this simple procedure avoids derivatisation of GSH (thus minimising auto-oxidation) and overcomes problems encountered when deriving the concentration of GSSG from ‘total GSH’. The linear range and limit of detection for both analytes were 7.5 × 10−7 to 1 × 10−5 M, and 5 × 10−7 M, respectively. GSH and GSSG were determined in cultured muscle cells treated for 24 h with glucose oxidase (0, 15, 30, 100, 250 and 500 mU mL−1), which exposed them to a continuous source of reactive oxygen species (ROS). Both analyte concentrations were greater in myotubes treated with 100 or 250 mU mL−1 glucose oxidase (compared to untreated controls), but were significantly lower in myotubes treated with 500 mU mL−1 (p < 0.05), which was rationalised by considering measurements of H2O2 and cell viability. However, the GSH/GSSG ratio in myotubes treated with 100, 250 and 500 mU mL−1 glucose oxidase exhibited a dose-dependent decrease that reflected the increase in intracellular ROS.

Retention mechanism divergence of a mixed mode stationary phase for high performance liquid chromatography

Mixed mode stationary phases utilize secondary retention mechanisms to add a dimensionality to the surface of high performance liquid chromatography (HPLC) adsorbents. This approach was used by several authors to improve the separation performance of single dimension separations. We explored the magnitude of these secondary interactions by performing an off-line two-dimensional (2D)-HPLC separation with a Scherzo SM-C18 column of a β-lactoglobulin tryptic digest with a mobile phase pH of 7 in the first dimension and 2 in the second. Mechanism divergence was determined using the peak capacity and a geometric approach to factor analysis, to measure the correlation. This separation was repeated with a C18 stationary phase as a control. It was found that the C18 column had a correlation coefficient of 0.784, smaller than the mixed mode column, 0.884. This indicated that the retention mechanisms of the C18 column were more divergent under these two pH environments than the mixed mode column. However, the SM-C18 still provided alternative selectivity of the peptides to that of the C18 and could be considered as a good alternative for further 2D-HPLC separations.

π-Selective stationary phases: (I) Influence of the spacer chain length of phenyl type phases on the aromatic and methylene selectivity of aromatic compounds in reversed phase high performance liquid chromatography

Phenyl type stationary phases of increasing spacer chain length (phenyl, methyl phenyl, ethyl phenyl, propyl phenyl and butyl phenyl, with 0–4 carbon atoms in the spacer chain, respectively) were synthesised and packed in house to determine the impact that the spacer chain length has on the retention process. Two trends in the aromatic selectivity, q_aromatic, were observed, depending on whether the number of carbon atoms in the spacer chain is even or odd. Linear log k′ vs ϕ plots were obtained for each stationary phase and the S coefficient was determined from the gradient of these plots. For the phenyl type phases, the S vs n_c plots of the retention factors of linear polycyclic aromatic hydrocarbons vs the number of rings exhibit a distinct discontinuity that between 3 and 4 rings, which increases with increasing spacer chain length for even phases but decreases for odd phases. Accordingly, we suggest that the retention factors depend differently on the number of carbon atoms in the spacer chain depending on whether this number is even or odd and that this effect is caused by different orientations of the aromatic ring relative to the silica surface.