Chiral supercritical fluid chromatography on porous graphitic carbon using commercial dimethyl beta-cyclodextrins as mobile phase additive.

Using dimethylated-beta-cyclodextrin mixtures (MeCD) as chiral selectors in CO2-polar modifier mobile phase and porous graphitic carbon as solid-phase, chiral supercritical (or subcritical) fluid chromatography was performed. The adsorbed quantity of MeCD onto the porous graphitic carbon (Hypercarb) was measured for various chiral selector concentrations using the breakthrough method with evaporative light scattering detector. The effects of MeCD concentration in the mobile phase, the nature of the polar modifier, the outlet pressure, the column temperature and the nature of the commercial MeCD mixture on the retention and the enantioselectivities were studied. For a given solute, the enantioselectivity is greatly dependent on the commercial MeCD mixture used. The retention mechanism was also studied. From the data, we find that the dominant mechanism for the chiral discrimination is the diastereoisomeric complexation in the mobile phase.

Fast enantioselective separation of clevidipine and a dihydropyridine substituted acid by SFC on Chiralpak AD.

Conditions for the fast separation of the enantiomers of a dihydropyridine substituted acid on a 50 x 4.6 mm ID short Chiralpak AD column with 2-propanol modified carbon dioxide as the mobile phase are presented. A high throughout of samples can be accomplished through the continuous sample loading of the loop of the injector. If a continuous data collection was used 10 separations could be performed in about 5 min with a precision of 0.6% RSD for the area ratio (n = 10). The parent drug clevidipine can also be analyzed and its enantiomeric composition determined after alkaline hydrolysis into its acid, either through hydrolysis followed by extraction to dichloromethane or by direct analysis of the hydrolysis media. About 1 min is required for each run. Using 0.1 M of methanolic sodium hydroxide 2 min are sufficient for the hydrolysis, and, including weighing, only 5 min are required to analyze clevidipine.

Packed-column supercritical fluid chromatography for the analysis of isosorbide-5-mononitrate and related compounds in bulk substance and tablets.

We describe a packed-column supercritical fluid chromatographic method that can be used for the analysis of isosorbide-5-mononitrate (5-ISMN) bulk substance and the 5-ISMN content of Imdur tablets. The method is based on methanol-modified carbon dioxide as the mobile phase and porous graphitized carbon (PGC, Hypercarb) as column support at 40 degrees C and 100 bar back pressure. The method makes it possible to simultaneously determine 5-ISMN and related compounds. In order to elute NO(3)(-) with acceptable retention time a quarternary ammonium hydrogen sulfate salt is added to the methanol modifier. An almost linear increase of the retention time with increasing carbon content of the counter ion was found. Tetramethyl ammonium hydrogen sulfate 5 mM in methanol was used in the final method as polar modifier for the simultaneous determination of possible degradation products within 12 min. The present method can separate and detect related compounds such as isosorbide-2, 5-dinitrate, isomannidemononitrate and isosorbide-2-mononitrate at the 0.1% (w/w) level as required by regulatory guidelines. Nitrate can be detected down to about 0.02% (w/w). Repeated analyses of ground tablet powder gave an assay precision for isosorbide-5-mononitrate of 1.4% (R.S.D., eight samples and two injections of each). For related substances at an area percent of 0. 1 the precision was less than 10%.

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.

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.

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).

Analysis and stereoselective metabolism after separate oral doses of tocainide enantiomers to healthy volunteers.

The potential of stereoselective metabolism of tocainide was studied in six healthy volunteers after separate oral administration of the pure enantiomers in solution. A method was developed to convert the N-carbamoylglucuronide of tocainide in plasma and urine by base treatment to a hydantoin derivative which after extraction and silation was analysed by selected ion monitoring using a deuterated internal standard. Analytical problems concerning side-reactions during derivatization of the conjugate are discussed. The peak plasma levels of the enantiomers, observed at less than or equal to 2 h after dosing, were similar but plasma clearances and terminal half-lives were different after oral administration of (R)-tocainide (195.5 +/- 20.1 ml min-1 and 9.7 +/- 0.8 h) and (S)-tocainide (110.2 +/- 10.5 ml min-1 and 14.5 +/- 1.7 h). Over 0-96 h the averaged urinary recovery of (R)-tocainide was 36 per cent and of (S)-tocainide 50 per cent. Stereoselective metabolism was a likely mechanism for the observed differences as the urinary recovery of the conjugate formed from (R)-tocainide differed substantially from that of (S)-tocainide (45 vs 1.2 per cent of given dose). Plasma t1/2 of the (R)- and (S)-conjugate were 9.9 and 18.7 h, respectively, indicating formation rate limited kinetics of the metabolite. The renal clearances of the conjugates were not significantly different (131 vs 97 ml min-1).

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.

Analysis of alpha-hydroxy metabolites of metoprolol in human urine after phosgene/trimethylsilyl derivatization.

Three metoprolol metabolites containing an alpha-hydroxy group were identified in human urine by capillary column gas chromatography/mass spectrometry. After aqueous phase cyclization with phosgene the neutral or acidic derivatives formed were isolated by solvent extraction at pH 10 or 3, respectively. Following silylation the electron impact mass spectra of the metabolites exhibited a characteristic ion at m/z 336 of high abundance which originated from cleavage of the bond adjacent to the alpha-OTMS group. Most probably the identified compounds were formed by further biotransformations of alpha-hydroxy metoprolol, which is a primary metabolite. The analytical method is applicable to detect the metoprolol metabolites reported so far. A quantitative assay for one of the metabolites (H 119/72) with nitrogen selective detection is described. The total amount of this metabolite excreted by one subject within 24 h after dosing was about 0.25% of the given dose.

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 derivatization of alprenolol and its 4-hydroxy metabolite in urine with phosgene and methanol prior to analysis by capillary column gas chromatography.

A method for the determination of alprenolol and its 4-hydroxy metabolite has been developed. The urine sample is made alkaline with buffer (pH 12) and derivatized with 60 microliter of 2 M phosgene in toluene with vigorous shaking. In the presence of 2.5% methanol, an oxazolidineone methyl carbonate is formed from 4-hydroxy alprenolol. The now neutral derivatives are extracted with an equal volume of dichloromethane. After evaporation of the organic phase, the residue is taken up in a small volume of ethyl acetate and subjected to capillary column gas chromatography with CP-Sil 8 as the stationary phase. The precision was 2.1% at the 3.3 micrograms/ml level of the metabolite in urine (n = 8). The isopentylamino analogue was used as the internal standard.

Simultaneous determination of metoprolol and metabolites in urine by capillary column gas chromatography as oxazolidineone and trimethylsilyl derivatives.

A method for the determination of metoprolol and its main metabolites in urine is presented. The method comprises derivatization of the aminopropanol side-chain with phosgene at alkaline pH and isolation in an organic phase at acidic pH. After trimethylsilylation, separation and quantification are performed by capillary column gas chromatography with flame ionization detection. The reaction is performed at pH 12 with 60 microliters of 2 M phosgene in toluene added in three portions. Diethyl ether--dichloromethane is used as extraction medium and bis(trimethylsilyl) acetamide as silylating agent. With spiked samples linear standard curves were obtained for metoprolol and three of its main metabolites with a detection limit varying between 4 and 20 mumol/l of urine. The method was applied to urine samples from a normal individual who had taken 292 mumol of metoprolol as tartrate.

Monitoring of S- and R-tocainide in human plasma after heptafluorobutyrylation, separation on Chirasil-Val and electron-capture detection.

The conditions for the heptafluorobutyrylation of tocainide have been studied. An almost instantaneous reaction was obtained with 0.01% of heptafluorobutyric anhydride in toluene at 40 degrees C. Higher anhydride concentration caused degradation of the initially formed derivative, mainly by the loss of water, as shown by mass spectral analysis. Tocainide was isolated from plasma by extraction into dichloromethane at alkaline pH. Gas chromatographic separation was performed with a fused-silica capillary column coated with a methyl silicone gum. The enantiomers were separated on a glass capillary column coated with Chirasil-Val. Upon analysing 0.1 ml of plasma eight times the precision was 4.7% at the 10 mumol/1 level for the S-form of tocainide.

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.

Electron-capture gas chromatography of plasma sulphonylureas after extractive methylation.

Conditions for the extractive alkylation of eight sulphonylurea hypoglycemic drugs have been evaluated. Extractive methylation of the compounds was achieved within 90 min using tetrabutylammonium as counter-ion (0.1 M at pH = 6.9) with 5% methyl iodide in dichloro-methane as organic phase. Mass spectral analysis showed derivatives methylated at the sulphonamide nitrogen. A higher pH or use of tetrapentylammonium as counter-ion caused hydrolysis of the sulphonylureas. The derivatives showed a high electron-capture response with minimum concentrations detectable in the range 1-4 x 10(-16) moles sec-1. Therapeutic plasma concentrations of glipzide and tolbutamide were determined by direct extractive methylation of the compounds from the plasma sample. The glipizide derivative was determined by electron-capture gas chromatography down to about 20 ng/ml in a 0.5-ml plasma sample. The relative standard deviation at the 0.2 microgram/ml level of glipizide was 6% (n = 6). The corresponding figure in the determination of tolbutamide at the 10 microgram/ml level was 3% (n = 10).

Determination of free fatty acids as pentafluorobenzyl esters by electron capture gas chromatography.

A method for the determination of free fatty acids in minute serum samples has been developed. The acids are esterified by extractive alkylation, using tetrabutylammonium as a counter ion and pentafluorobenzyl bromide as alkylating reagent. The derivatization of palmitic acid required a reaction time of 25 min. The excess of pentafluorobenzyl bromide is removed by coupling it with a phenoalkylamine and extraction of the product into an acidic aqueous phase. Quantitation is carried out by gas chromatography with electron capture detection. The precision achieved in the determination of 6.2 mug of palmitic acid in 50 mul of mouse serum was 6.1% (S.D.).