Review: HPLC determination of fumonisin mycotoxins.

An overview of liquid chromatographic methods, mainly employing fluorescence detection together with sample pre-treatment methods, is presented for the determination of the toxic group of fumonisin mycotoxins in various matrices.

Quantitative determination of p-aminosalicylic acid and its degradation product m-aminophenol in pellets by ion-pair high-performance liquid chromatography applying the monolithic Chromolith Speedrod RP-18e column.

An ion-pair high performance liquid chromatographic method was developed for the simultaneous determination of p-aminosalicylic acid (PAS) and its degradation product m-aminophenol (MAP) in a newly developed multiparticular drug delivery system. Owing to the concentration differences of PAS and MAP, acetanilide and sulfanilic acid were used as internal standards, respectively. The separation was performed on a Chromolith SpeedROD RP-18e column, a new packing material consisting of monolithic rods of highly porous silica. The mobile phase composition was of 20 mm phosphate buffer, 20 mm tetrabutylammonium hydrogen sulphate and 16% (v/v) methanol adjusted to pH 6.8, at a flow-rate of 1.0 mL/min, resulting in a run-time of about 6 min. Detection was by UV at 233 nm. The method was validated and proved to be useful for stability testing of the new dosage form. Separation efficiency was compared between the new packing material Chromolith SpeedROD RP-18e and the conventional reversed-phase cartridge LiChroCART 125-4 (5 microm). A robustness test was carried out on both columns and different separation parameters (retention, resolution, run time, temperature) were determined.

Application of an alkyl-diol silica precolumn in a column-switching system for the determination of meloxicam in plasma.

The group of LiChrospher alkyl-diol silica (ADS) phases that make part of the unique family of restricted-access materials, have been developed as special packings used in the liquid chromatographic integrated sample processing of biofluids. The advantage of these phases lies in the possibility of direct injection of untreated plasma. An on-line elimination of the protein matrix is achieved with a quantitative recovery together with an on-column enrichment. The present method describes a hand-operated on-line switching high-performance liquid chromatographic system for the determination of meloxicam. Spiked plasma samples were introduced on the ADS precolumn using a 0.05 M phosphate buffer, pH 6.0. After washing with the buffer the ADS column was backflushed with the mobile phase 0.05 M phosphate buffer-30% (v/v) acetonitrile (ACN)-25 mM t-butylamine (TBA) at a pH of 7.0, thus transferring the analyte to the analytical column LiChrocart 125-4 LiChrospher RP-8. The eluent was monitored by a UV-detector set at 364 nm. The developed column-switching method is fully applicable to plasma injections.

Comparison of morphine and hydromorphone analysis on reversed phase columns with different diameters.

A comparison of a reversed phase high-performance liquid chromatographic (HPLC) method performed on columns with different internal diameters is reported for the quantitative routine determination of morphine.HCl and hydromorphone.HCl in solutions used for intramuscular injection. The method is based on the ion-pairing properties of sodium dodecyl sulphate (SDS) with alkaloids on a reversed phase LiChrospher RP-18 packing material and UV-detection at 230 nm. The mobile phase consisted of an acetonitrile: water mixture 35:65 (v/v) containing 0.5% (w/v) SDS and 0.4% (v/v) acetic acid. Precision, linearity and limit of detection were compared on the 2, 3 and 4 mm i.d. x 125 mm columns. A robustness test for the determination of hydromorphone.HCl was also evaluated.

Optimization of the chiral separation of some 2-arylpropionic acids on an avidin column by modeling a combined response.

The enantiomeric separation of some nonsteroidal antiinflammatory drugs was investigated on an avidin column. An experimental design approach (central composite design) was used to evaluate the effects of three method parameters (pH, concentration of organic modifier, and buffer concentration) on the analysis time and the resolution, as well as to model these responses. This revealed that the organic modifier concentration and sometimes the pH are significant parameters to control because of their influence on both analysis time and resolution. Furthermore, the central composite design results were combined in a multicriteria decision-making approach in order to obtain a set of optimal experimental conditions leading to the most desirable compromise between resolution and analysis time.

A validated HPLC method for the determination of thiazinamium methylsulphate in pharmaceutical preparations.

The phenothiazine derivative thiazinamium methylsulphate is a drug with antihistaminic and anticholinergic properties, often used in some types of obstructive lung diseases. Because there is a lack of chromatographic data available for its determination, the objective of the present investigation was to develop a sensitive and rapid HPLC method for the quantitative estimation of thiazinamium methylsulphate in a pharmaceutical dosage form, applicable to routine analysis. The drug was chromatographed on a C18-reversed phase system applying a Licrocart column (LiChrospher 100 RP 18, 125 x 4 mm) with a mobile phase consisting of acetonitrile-water (3:7, v/v), employing as ion-pairing agent octanesulphonic acid sodium salt (20 mM) together with N,N-dimethyloctylamine (20 mM), adjusted to pH 3. Detection occurred at 254 nm. Propylparaben was used as an internal standard. The method was applied to solutions for intramuscular injection containing thiazinamium methylsulphate (65 mg/2 ml). Since little sample preparation is required, most analyses can be carried out within 15 min. The optimized method was validated and provided acceptable results with respect to linearity (r = 0.9999), precision and accuracy in the concentration range of 26-78 microg/ml. The proposed method is presently employed to investigate the stability of thiazinamium methylsulphate in solutions for intramuscular injection in the presence of anti-oxidizing agents.

Detection in the liquid phase applying chemiluminescence.

Several chemiluminescence-based reactions are applicable to the determination of various bio-pharmaceutically important analytes, and they can be applied for monitoring chemiluminescence emission using flow injection, liquid chromatographic and capillary electrophoretic analysis, as well as for the development of chemiluminescence-based sensors or in immunoassays. As in general the emission intensity is linearly proportional to the concentration of any of the reagents, the technique allows the analysis of different species involved in the light-producing reaction, amongst which are the chemiluminescent reagent, oxidants, inhibitors, cofactors, catalysts, some fluorophore, etc. The present overview illustrates some important applications of the last decade on this rather unfamiliar luminescence technique to detectional challenges in the liquid phase. The required instrumentation is limited as no external light source is needed. Also, the technique opens perspectives for increasing detection sensitivity in miniaturized flowing streams. On the other hand, several drawbacks still limit full application, eg dependence of the emission signal upon a number of environmental factors forcing the analyst to make a compromise between separating and measuring conditions, a lack of selectivity in specific cases, the critical detection of the signal at strictly defined periods, especially in the case of sharp emission vs time profiles, and the development of detection devices in capillary electrophoresis.

Application of the restricted-access precolumn packing material alkyl-diol silica in a column-switching system for the determination of ketoprofen enantiomers in horse plasma.

The group of LiChrospher ADS (alkyl-diol silica) sorbents that make part of a unique family of restricted-access materials, have been developed as special packings for precolumns used in the LC-integrated sample processing of biofluids. The advantage of these sorbents lies in the direct injection of untreated biological fluids, that is without sample clean-up, the elimination of the protein matrix with a quantitative recovery together with an on-column enrichment. The present method is based on previous work applying UV detection at 260 nm for ketoprofen determinations. Plasma samples introduced to the ADS precolumn using a 0.1 M phosphate buffer, pH 7.0. After washing with the buffer the ADS column was backflushed with the mobile phase 0.01 M phosphate buffer-6% (v/v) 2-propanol-5 mM octanoic acid at a pH of 5.5, thus transporting the analytes to the chiral-HSA (human serum albumin) (100x4.0 mm) column where the separation of the ketoprofen enantiomers was achieved with a resolution factor of 1.4. The developed column-switching method was fully applicable to plasma injections.

Direct HPLC analysis of ketoprofen in horse plasma applying an ADS-restricted access-phase.

Making up part of the unique family of restricted access materials (RAM) the Lichrospher ADS (alkyl-diol silica) sorbents have been developed as special packing materials for precolumns used for LC-integrated sample processing of biofluids. The advantage of such phases consists of direct injection of untreated biological fluids without sample clean-up and elimination of the protein matrix together with an on-column enrichment. The plasma samples, with internal standard phenacetin added (not essential), were brought onto the precolumn (C-18 ADS, 25 micron, 25 x 4 mm i.d.) using a phosphate buffer, 0.1 M, pH 7.0. After washing with the buffer, the ADS column was backflushed with the mobile phase phosphate buffer 0. 05 M pH 7.0: acetonitrile (80:20), thus transporting the analytes onto a reversed-phase column Ecocart 125-3 HPLC cartridge with a LiChrocart 4-4 guard column, both packed with LiChrospher 5 micron 100 RP-18; after separation detection was performed in UV at 260 nm. Essential features of the method include the novel precolumn packing, the absence of sample pretreatment, a quantitave recovery, good precision and accuracy, as well as a considerable reduction of analysis time compared to conventional manual methods applied in bioavailability studies.

Bioavailability of racemic ketoprofen in healthy horses following rectal administration.

Ketoprofen (KTP) is a chiral non-steroidal anti-inflammatory drug (NSAID) of the propionic acid class, approved by the FDA for the allevation of pain associated with musculoskeletal disorders in horses. The present study was designed to examine the bioavailability of ketoprofen enantiomers after rectal administration of the racemate to healthy horses. One gram of racemic ketoprofen was injected intravenously and administered rectally as a fat based suppository in a cross-over design study (n = 4). Blood samples were analysed for KTP enantiomers using HPLC. After IV administration, the S(+) enantiomer concentrations in plasma were higher than the R(-) enantiomer concentrations and the AUC(0-12 h) for the S(+) enantiomer was significantly higher than for the R(-) enantiomer. Following rectal administration C(max) and AUC(0-12 h) were significantly higher for the S(+) than for the R(-) enantiomer. Bioavailability after rectal administration was low. Since there was no significant difference in bioavailability between the two enantiomers, it is assumed that no pre-systemic inversion from R(-) to S(+) occurred after rectal administration of racemic KTP to horses.

A first and straightforward approach to detection limits for narrow-bore HPLC and CE.

Two methodologies have been proposed for a simple statistical estimation of the detection limits in microbore-HPLC and CE techniques. Due to the impossibility to measure the blank, by using the extrapolated or approximated expressions it is possible to obtain a straightforward approach of the detection limits from the study of the residuals of the calibration data set, avoiding tedious treatment of the signals. The utility of these expressions has been checked on the data from an experimental microbore HPLC system.

Cerium (IV)-based chemiluminescence analysis of hydrochlorothiazide.

A flow-injection analytical method for the determination of hydrochlorothiazide is presented. The method is based on the chemiluminescence reaction of hydrochlorothiazide with cerium(IV) in sulphuric acid, sensitized by the fluorescent dye rhodamine 6G. The proposed procedure allows quantitation of hydrochlorothiazide in the concentration range of 0.33-130 mumol l(-1) with a detection limit of 0.15 mumol l(-1), an RSD of 2.4% at 10 mumol l(-1) and a sample measurement frequency of 200 h(-1). The method was successfully applied to the determination of hydrochlorothiazide in pharmaceutical preparations containing, amongst others, lactose, maize starch, calcium phosphate, magnesium stearate, potassium chloride and E 110 (disodium-6-hydroxy-5-(4-sulphonatophenylazo) naphthalene-2-sulphonate) as the concomitant species. Apart from the single formulation, hydrochlorothiazide was also determined in tablets combined with the antihypertensive lisinopril.