Packed-column supercritical fluid chromatography for the purity analysis of clevidipine, a new dihydropyridine drug.

In this paper we describe a packed column supercritical fluid chromatography method that can be used for the analysis of a new dihydropyridine substance. The method is based on methanol-modified carbon dioxide as the mobile phase and Hypersil bare silica as column support at a column temperature of 50 degrees C and 150 bar as back pressure. Using an adjusted methanol gradient the most likely by-products can be separated and detected (240 nm) within 13 min. Occasionally the column needed treatment with 4 mM citric acid in the methanol modifier in order to give a narrow peak of an acidic analogue. The present method can detect analogues at the 0.1% (w/w) level. The precision at this level for one of the analogues was 5.9% RSD. This method shows a higher selectivity than a corresponding reversed-phase liquid chromatographic method.

GLC determination of nicotinamide in multivitamin formulations after conversion to nicotinonitrile.

A method was developed for the quantitative GLC determination of nicotinamide. It is based on the conversion of nicotinamide to nicotinonitrile. This dehydration reaction is mediated with trifluoroacetic anhydride and catalyzed by base. The GLC properties of this nitrile are excellent. The reaction conditions, extraction, and stability properties of the derivative as well as the choice of internal standard are discussed.

Electron-capture GC determination of subnanogram amounts of emepronium bromide in serum.

Barium peroxide in an acidic medium was utilized to increase the sensitivity in the benzophenone method for the determination of the quaternary ammonium compound emepronium bromide. The method is comprised of ion-pair extraction, oxidation, and quantitative determination of benzophenone by electron-capture GC. By employing small extraction and reaction volumes, the method was used in the 0.2--8-ng/ml range with a relative standard deviation of 2.5% at the 1-ng/ml level. The application of the method to human serum samples after a single oral dose demonstrated that the elimination phase for emepronium in serum had a half-life of 7--11 hr.

Polar organic phase liquid chromatography with packed capillary columns using a vancomycin chiral stationary phase

Vancomycin immobilized on silica served as the chiral stationary phase (CSP) in this investigation with polar organic solvents as the mobile phase in liquid chromatography (LC). It was shown that trace amounts of water were beneficial for improving peak shape and efficiency. To regulate the retention and selectivity an acid and/or base were added to the mobile phase where an excess of acid was shown to be preferential for enantioseparation. An unusual increase in selectivity with increasing temperature was shown for the acidic drug, thalidomide. Additionally, nonlinear van't Hoff plots were obtained for metoprolol enantiomers that showed increased retention with increasing temperature. Metoprolol also showed unusual behavior in the polar organic phase when water was added to resemble reversed-phase chromatography, with minimum retention observed at high water or high methanol concentrations. In both instances a high degree of electrostatic interaction between metoprolol and vancomycin was concluded. Metoprolol and ten of its analogs were examined on this CSP to evaluate the enantiorecognition process. A comparison in enantioselectivity for a number of acidic and basic drugs using this CSP was also carried out using the polar organic phase, reversed phase, and normal phase LC which were all compared to the results obtained in supercritical fluid chromatography (SFC). Polar organic phase LC offered a better separation of basic molecules while reversed phase LC was preferred for the resolution of acids. SFC showed the broadest enantioselectivity overall and normal phase LC indicated similar properties, as expected, to SFC but with lower column efficiency. Copyright 2000 Wiley-Liss, Inc.

Dissolution test for felodipine tablets using chemical oxidation in situ to maintain 'sink conditions'.

A test model is described for the determination of the dissolution rate of the vasodilator, felodipine, a derivative of dihydropyridine that is practically insoluble in water. 'Sink conditions' are maintained by means of an oxidizing agent, ceric sulphate, which reacts rapidly with dissolved drug molecules in the dissolution fluid. A pyridine derivative is formed quantitatively in the oxidation reaction. The amount of dissolved felodipine is calculated from the concentration of the pyridine derivative, as determined by reversed-phase liquid chromatography. Dissolution rates depend on the concentration of the oxidizing agent so that high concentrations accelerate dissolution. The dissolution test suggested for 25-mg felodipine tablets is performed in 500 ml fluid that contains 5 mM ceric sulphate in 0.12 M sulphuric acid. The test is performed on single tablets with USP paddle equipment. Dissolution rates for nine different tablet compositions are correlated to such bioavailability parameters as maximum plasma concentration and total area under the plasma concentration-time curve. Interferences and limitations of the method are discussed.

Direct derivatization of drugs in untreated biological samples for gas chromatographic analysis.

The possibilities to derivatize an analyte directly in the biological sample are reviewed with examples from our own experiences and from the literature. Techniques, such as extractive acylation, alkylation and benzoylation, are frequently used. Improvement of the extractability of the drug from the matrix is a common feature, especially with hydrophilic compounds, where sometimes cyclizing reactions can be employed. Several analytes are reactive or labile in the sample and can be trapped in derivatization reactions in situ. In many cases, two-phase reactions lead to milder derivatization conditions (e.g. dealkylation of tertiary amines), which is favourable from a clean-up point of view.

Direct separation of captopril diastereoisomers including their rotational isomers by RP-LC using a teicoplanin column.

A direct reversed-phase liquid chromatography (LC) method has been developed for the separation and analysis of captopril and its 2R,2S diastereoisomer using a teicoplanin stationary phase. The proline containing diastereoisomers, which are known to form conformers in aqueous solution, were also separated from their rotational isomers. The influence of temperature, different organic modifiers and buffer type, concentration and pH were optimised to obtain a working resolution between the two diastereoisomers and their respective rotational isomers. The diastereoisomeric purity of several commercial captopril batches was subsequently evaluated using a 0.05% triethylammonium acetate (TEAA) buffer (pH 3.8) run at 1.0 ml/min with mobile phase reservoir and column temperature controlled at 0 degrees C. Throughout the study online UV diode array and mass spectrometry detection was carried out simultaneously to confirm that peaks eluting from the teicoplanin column were in fact captopril and not its readily converted disulphide dimer. Additionally, as a result of the greater detection sensitivity of mass spectrometry, it also facilitated a more accurate optimisation study where trace amounts of the rotational isomers were found to be present in the baseline at temperatures higher than optimum.

Identification of a new by-product detected in metoprolol tartrate.

A new impurity has been found in some batches of metoprolol tartrate. As the amount exceeded 0.1% it was of interest to deduce the structure. Techniques involved in solving the problem were LC, LC-MS and GC-MS. The LC systems showed that the impurity and metoprolol behaved differently to modifications of the mobile phase, indicating that there were differences in the functional groups. LC-MS was used to determine the molecular weight, which was 74 mass units higher than metoprolol. A hydrogen-deuterium shift technique using micro column LC-MS gave the information that three hydrogen atoms were bound to heteroatoms, i.e. one more than in metoprolol. This led to the conclusion that the impurity had three extra carbon and two extra oxygen atoms. It was supposedly a by-product in the synthesis. Knowledge of the synthesis steps for beta-receptor blocking drugs suggested three possible structures. Two were independently synthesized and one of these was found to be identical to the impurity.

Formation of mono(ethylhexyl)phthalate from di(ethylhexyl)phthalate in human plasma stored in PVC bags and its presence in fractionated plasma proteins.

The formation of mono(ethylhexyl)phthalate (MEHP) from di(ethylhexyl)phthalate in human plasma stored in bags of polyvinylchloride has been studied. Substantial amounts were formed and in ten bags from 4 to 56microgram/ml were found. After 2 weeks at room temperature the concentration of MEHP had increased to values between 27 and 79 microgram/ml. However, MEHP was also disappearing as shown in a recovery experiment. Of the fractionated proteins albumin contained MEHP in amounts from less than 3 to 290 microgram/g.

Capillary supercritical fluid chromatography of aliphatic amines. Studies on the selectivity and symmetry with three different columns using carbon dioxide or nitrous oxide as mobile phase.

The supercritical fluid chromatography of intact aliphatic amines with different columns is described. One group of amines was based on N,N-dimethyl-n-octylamine and related primary and secondary amines, and the other on the amino alcohol metoprolol and several of its analogues. Columns with three different phases were investigated, one non-polar coated with 5% phenyl methyl polysiloxane and two more polar with 25% cyanopropyl methylphenyl polysiloxane and Carbowax 20M. Generally, equal molar amounts were injected under splitless conditions and the peak symmetry was recorded. The system with the non-polar silicone phase was more inert, followed by the wax-phase column. The cyanopropyl column gave severe peak tailing although it was loaded with five times more of the amines than the other columns. The selectivity was investigated and was found higher with the two polar columns. Both showed a marked increase in the retention of amines with free hydrogens. With nitrous oxide the selectivity was almost the same as that with carbon dioxide as mobile phase. The nature of the flame ionization detector changed, however, giving a negative baseline drift on pressure programming. An interesting conclusion is that the amines are chromatographed as such with carbon dioxide as the mobile phase.

Phosgene as a derivatizing reagent prior to gas and liquid chromatography.

The use of phosgene as a derivatizing agent for bifunctional compounds prior to gas and liquid chromatographic analysis is reviewed. Applications include gas chromatographic determinations of metoprolol and its metabolites in biological fluids, enantiomeric separations of beta-blocking drugs and sympathomimetic agents on a chiral stationary phase and liquid chromatographic enantiomer separations.

Gas chromatography with nitrogen-selective detection of metoprolol from human plasma after reaction with phosgene.

A method for the determination of therapeutic levels of metoprolol in human plasma is presented. Metoprolol and the internal standard are extracted from the buffered plasma sample to an organic phase containing 4 X 10(-3) M phosgene. After 10 min the organic phase is taken to dryness. The residue is dissolved in ethyl acetate and the formed oxazolidine derivatives are analyzed by gas chromatography with nitrogen-selective detection. With packed columns, rectilinear standard curves through the origin were obtained down to 80 nmoles/l of plasma. The precision of the method at 200 nmoles/l was 1.5% (n = 8). The sensitivity of the method was improved by using capillary column gas chromatography. Linear standard curves were obtained down to 10 nmoles/l of metoprolol in plasma. The precision of the method at the 50 nmoles/l level was 2.2% (n = 7). With this simple and straightforward method using extractive derivatization 30 samples can be handled in a day.

Determination of tocainide in human plasma by gas chromatography with nitrogen-selective detection after Schiff base formation.

Tocainide is a primary amine with antiarrhythmic properties derived from lidocaine. For biopharmaceutical and pharmacokinetic purposes an assay was developed that made use of Schiff base formation with methyl isobutyl ketone and gas chromatography with nitrogen-selective detection. The derivatization procedure was performed at 85 C for 10 min, although a longer time at this temperature caused degradation of the product. Of several structural analogues the p-methyl one was the internal standard of choice. The amine was extracted from alkaline samples with dichloromethane and, after evaporation, reconstituted in methyl isobutyl ketone. From plasma the yields were lower than those from aqueous samples but the addition of hydroxylamine 30 min before the extraction process resulted in the same yields. Hydroxylamine probably acts as a competitor for carbonyl groups in the biological sample. In addition to the enhanced yields patients' samples extracted after hydroxylamine treatment were analysed with better precision. With nitrogen-selective detection 500 nmol/l in a 0.5-ml sample could be quantified, which is well below the therapeutic levels. The method compared favourably with a liquid chromatography assay.

Determination of hydrazine in hydralazine by capillary gas chromatography with nitrogen-selective detection after benzaldehyde derivatization.

A method for the determination of hydralazine substance is described. Hydrazine is derivatized in aqueous media with benzaldehyde to benzalazine. After extraction to an organic phase containing a homologue as marker, the sample is subjected to capillary column gas chromatography with nitrogen-selective detection. A prolonged reaction with 0.1 M benzaldehyde of 20 min or more led to an increased level of benzalazine when hydralazine was analysed. An increase was also observed if the aqueous hydralazine sample had been allowed to stand for some time before analysis. The final method involved the use of a 5-min reaction time, fresh solutions and the standard addition principle. The levels of hydrazine found in hydralazine hydrochloride were below 1 ppm (as bases, 1 ng/mg).

Pharmacokinetics of oxazepam in healthy volunteers.

The pharmacokinetics of oxazepam was studied in healthy volunteers. When oral doses of 15 mg were given to eight subjects on two occasions 2.5 years apart the individual areas under the plasma concentration time curves did not differ significantly. Plasma clearance ranged between 0.050 and 0.171 x kg-1 x h-1 and half-lives from 5.9 to 25 hours. The urinary recovery of oxazepam conjugates was 67 +/- 15 S.D.% of the dose given and the total recovery including faecal oxazepam was 70 +/- 15 S.D. %. On multiple dosing (5 mg t.i.d.) stable steady-state concentrations were established. There was a tendency for slightly lower steady-state concentrations than predicted from single oral doses. During steady-state 86 +/- 17 S.D. % of the dose was recovered in the urine over a 24 hour period.