Automated solid-phase extraction method for the determination of atovaquone in capillary blood applied onto sampling paper by rapid high-performance liquid chromatography.

A bioanalytical method for the determination of atovaquone in 100 microl blood-spots by solid-phase extraction and high-performance liquid chromatography has been developed and validated. Atovaquone was extracted from the sampling paper in 0.2 M phosphoric acid and a structurally similar internal standard was added with acetonitrile before being loaded onto a C8 end-capped solid-phase extraction column. Atovaquone and internal standard were analysed by high-performance liquid chromatography on a C18 J'Sphere ODS-M80 (150 x 4.0 mm) column with mobile phase acetonitrile-phosphate buffer, 0.01 M, pH 7.0 (65:35, v/v) and UV detection at 277 nm. The intra-assay precision was 2.7% at 12.00 microM and 13.5% at 1.00 microM. The inter-assay precision was 3.3% at 12.00 microM and 15.6% at 1.00 microM. The lower limit of quantification was 1.00 microM. The limit of detection was 0.50 microM.

Improved method for the simultaneous determination of proguanil and its metabolites by high-performance liquid chromatography and solid-phase extraction of 100-microl capillary blood samples dried on sampling paper.

An improved method is presented for the determination of proguanil, cycloguanil and 4-chlorophenylbiguanide in 100-microl capillary blood samples applied to sampling paper. This method also utilises a solid-phase extraction technique and high-performance liquid chromatography. Different kinds of sampling paper, such as ion-exchange and cellulose sampling paper were tested. The best elution recovery (70-80%) was obtained after treatment of cellulose sampling paper with a quaternary ammonium compound. The limit of determination was 50 nmol/l for cycloguanil and 4-chlorophenylbiguanide and 125 nmol/l for proguanil using 100 microl capillary blood. The stability of the analytes and elution performance from sampling paper was validated at different temperature and storage time. Venous blood and capillary blood concentrations of proguanil and metabolites were found to be similar.

Improved validated assay for the determination of proguanil and its metabolites in plasma, whole blood, and urine using solid-phase extraction and high-performance liquid chromatography.

An improved and validated method is presented for the determination of proguanil, cycloguanil, and 4-chlorophenylbiguanide in plasma, whole blood, and urine using solid-phase extraction (SPE) technique and reversed-phase high-performance liquid chromatography (HPLC). The HPLC method uses isocratic elution with acetonitrile:phosphate buffer 0.1 mol/l, pH 2.6 (21.5:78.5 vol/vol) at a flow rate of 1.0 ml/min for the separation. The recovery of proguanil and metabolites ranged from 82% to 104%. The limit of determination was 20 nmol/l for proguanil and its metabolites in plasma and approximately 50 nmol/l for proguanil and metabolites in whole blood. Different stationary phases for HPLC and SPE were tested and the best chromatographic separation from endogenous constituents and other antimalarial drugs was achieved with cyanopropyl stationary phases.

Determination of chloroquine and its metabolites in urine: a field method based on ion-pair extraction.

A new straightforward photometric method for the assay of the antimalarial drug chloroquine and its metabolites in urine is described. The method involves an ion-pair extraction procedure with dichloromethane using the acid-base indicator bromthymol blue as counter-ion. The ion pair formed with chloroquine in the organic phase is yellow, and absorbance is measured at lambda = 410 nm using a filter photometer. The absorbance is a linear function of concentration up to 400 mumol/l (120 mg/l) chloroquine. The method is suitable for the determination of chloroquine and its metabolites in urine down to a limiting concentration of about 10 mumol/l (3 mg/l). Additionally, the method is suitable for semiquantitative visual estimation of the concentration of chloroquine in urine. A single dose of 5 mg/kg chloroquine base could be determined in urine from two volunteers for at least 8 days after administration of the drug. The results obtained for the analysis of chloroquine and its metabolites with the colorimetric method described here correlate well with those obtained using high performance liquid chromatography.

Effect of use of gel-barrier sampling tubes on determination of some antiepileptic drugs in serum.

We evaluated the effect on drug concentration of the duration of sample storage in three different tubes for blood collection: SST (Becton Dickinson), AutoSep (Terumo), and Microvette (Sarstedt), all of which contain an inert barrier (e.g., gel) for cleaner separation of serum from coagulum during centrifugation. With two of the tubes (SST and AutoSep) we examined samples taken from patients undergoing treatment with phenobarbital, phenytoin, and carbamazepine. We found significant decrease in the drug, concentration after 24 h with the serum standing over the gel barrier in the SST tubes for phenobarbital and both the SST and AutoSep tubes for carbamazepine. Adding aqueous drug solutions or serum samples supplemented with the above drugs in therapeutic concentrations to the three types of tubes resulted in a more pronounced decrease of concentration in the SST and Auto-Sep tubes than in the Microvette tubes.

Haemoglobin determination without cyanide on Hemalog-8.

The most common method for determination of haemoglobin is the cyanmet-haemoglobin method which is recommended by the International Committee for Standardization in Haematology (ICSH). Determination of haemoglobin is one of the most used methods in hospital laboratories. One disadvantage with the method is the use of cyanide in the reagent, which demands cautious preparation and use. The intention of our investigation was to replace the cyanide reagent for Hemalog-8 with a reagent not containing cyanide. It is shown that the change of reagent did not influence the results of the analysis.

Binding of bilirubin and long-chain fatty acids to human serum albumin with general remarks on displacement of firmly bound ligands.

Binding of bilirubin to human serum albumin was studied by estimation of the free bilirubin concentration from the rate of oxidation with hydrogen peroxide and peroxidase, and by spectrophotometry: nI = 1, kI = 7 x 10(7) l/mol; nII = 1, kII = 5 x 10)5) l/mol, at pH 7.4, 37 degrees C, ionic strength 0.1. Palmitate or oleate in excess of 4 mol per mol albumin, influences the high-affinity binding of bilirubin as described by an empirical equation. Theoretical consideration of competitive displacement of a biologically active substance, firmly bound in an inactive state to a macromolecular carrier, demonstrates that significant displacement may occur on addition of another ligand with a lower binding constant. Displacement of bilirubin from its high-infinity site by fatty acids and drugs is thermodynamically feasible and probably clinically important.

The effects of overloading in density-gradient centrifugation.

The effects of overloading of the sample zone in density gradient centrifugation have been studied by use of a three-component shelf-lavered sample in which the total protein concentration was increased by addition of different amounts of albumin. It is found that overloading of the gradient gives rise to particle movements which are not predictable from the Svedberg equation. The two typical effects of overloading are dislocation of the zone mass centres and changes in the zone shapes. It is found that the magnitude of the calculated sedimentation coefficients increases nearly linearly with increasing sample load. The changes in zone shapes are found to depend on the specific load and two different patterns may be distinguished. The zone of the sample component which causes the overloading is defined as primarily overloaded and the others as secondarily overloaded. In primarily overloaded zones the original Gaussian shape is lost, while in secondarily overloaded zones the Gaussian zone shape is maintained, although a zone broadening is seen. Extreme high loads are found to be able to divide single zones. As a whole these experiments show that evidence for a non-overloaded set of experimental conditions must be provided, when density gradient centrifugation is used for determination of sedimentation coefficients. For preparative gradient centrifugations the power of resolution will decrease with increasing sample load. A simple method to detect overloading in density gradient centrifugations is described.

Properties and immunochemical reactivities of carboxy-modified human serum albumin.

Seven carboxy modified and four amino modified derivatives of human serum albumin have been prepared and studied by optical rotatory dispersion measurements, gel filtration, immunoelectrophoresis, immunodiffusion and by use of an ammonium sulphate technique. It is found that carboxy groups are of major importance for the maintenance of the structure and function of human serum albumin, and that carboxy modification has a much more profound effect than amino modification has to the same relative extent.

Binding of bilirubin to low-affinity sites of human serum albumin in vitro followed by co-crystallization.

Human serum albumin has three hydrophobic binding sites for bilirubin, one high affinity and two subordinate sites. At pH 7.4 the two molecules of bilirubin bound at the latter sites are unstable, since the equilibrium concentration of free bilirubin is higher than the solubility. Co-crystallization takes place, resulting in a suspension of micro-crystals containing about 200 molecules of bilirubin for each albumin. The fresh suspension shows a high optical rotation with a negative Cotton effect, indicating that the bilirubin molecules are arranged in an optically active pattern, the asymmetry originating from the albumin.