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A chromatographic method is described for separation of substituted dihydropyridines on Hypercarb, porous-graphitic carbon as support and methanol-modified carbon dioxide containing Tween 60® deactivation additive, as mobile phase. Enantiomers of a dihydropyridine substituted acid were resolved after addition of Z-(L)-arginine as ion-pairing counter ion. A 0.2 mM concentration was sufficient to achieve a resolution factor>2 or baseline resolution. The system was used to estimate the minor enantiomer below 0.1% level. [ABSTRACT FROM AUTHOR]

Enantiomeric separations of four 2-substituted propionic acid drugs and two related acids have been studied using normal phase liquid chromatography with amylose (tris 3,5-dimethylphenylcarbamate) coated on silica as support (Chiralpak AD). At standard conditions (i.e. flow-rate, 1.0 ml/min; column temperature, 30 degrees C) the elution order can be reversed when the polar alcohol modifier in isohexane, 2-propanol, is replaced by methanol/ethanol 2:1. This is the case for ibuprofen with 2.5% (v/v) alcohol and for mandelic acid with 10% (v/v) alcohol using synthetic mixtures with unequal proportions of the respective enantiomer. Thermodynamic studies in the range 10-45 degrees C on retention and selectivity of ibuprofen and mandelic acid gave both linear and curved plots. These results stress the importance of investigating enantiomer elution order during the development of enantioselective methods when both old and new CSPs are evaluated. One should also keep in mind that reversal can take place for rather common analytes in well established enantioselective chromatographic systems.

Metoprolol and a number of related amino alcohols and similar analytes have been chromatographed on aminopropyl (APS) and ethylpyridine (EPS) silica columns. The mobile phase was carbon dioxide with methanol as modifier and no amine additive was present. Optimal isocratic conditions for the selectivity were evaluated based on experiments using design of experiments. A central composite circumscribed model for each column was used. Factors were column temperature, back-pressure and % (v/v) of modifier. The responses were retention and selectivity versus metoprolol. The % of modifier mainly controlled the retention on both columns but pressure and temperature could also be important for optimizing the selectivity between the amino alcohols. The compounds could be divided into four and five groups on both columns, with respect to the selectivity. Furthermore, on the aminopropyl silica the analytes were more spread out whereas on the ethylpyridine silica, due to its aromaticity, retention and selectivity were closer. For optimal conditions the column temperature and back-pressure should be high and the modifier concentration low. A comparison of the selectivity using optimized conditions show a few switches of retention order between the two columns. On aminopropyl silica an aldehyde failed to be eluted owing to Schiff-base formation. Peak symmetry and column efficiency were briefly studied for some structurally close analogues. This revealed some activity from the columns that affected analytes that had less protected amino groups, a methyl group instead of isopropyl. The tailing was more marked with the ethylpyridine column even with the more bulky alkyl substituents. Plate number N was a better measure than the asymmetry factor since some analyte peaks broadened without serious deterioration of symmetry compared to homologues.

In this paper, a chromatographic system based on carbon dioxide with methanol as mobile phase, and diol silica as stationary phase has been investigated for metoprolol and related amino alcohols by addition of strong acids to systems with triethylamine base as primary additive. Standard conditions used were 10% of methanol, containing 24 mM of acid and 18 mM of triethylamine, in carbon dioxide with a flow rate of 1.5 ml min(-1). The column dimensions were 125 mm x 4 mm I.D. and kept at 40 degrees C with a back pressure of 150 bar. Effects on selectivity were stronger with trifluoroacetic acid than with ethanesulfonic acid. From a large set of related analytes, it was shown that selectivity changes were significant when the structure close to the nitrogen of the amino alcohol analyte differed. The stability of the column in the short time perspective was examined and it showed negligible changes. For a diastereoisomeric pair, not resolved in a basic system with triethylamine nor by addition of ethanesulfonic acid, resolution improved to about 2.1 with trifluoroacetic acid. The described approach offers a way to tune the selectivity of SFC systems when amines are analyzed without the need to change stationary phase for the chromatographic separation.

A method for the determination of sodium stearyl fumarate aqueous suspension is described. This straightforward method is based on homogenisation of the sample, dilution of a known aliquot with methanol to a suitable clear solution and mixing with an internal standard; (S)-naproxen. Separation and quantification is performed by packed column supercritical fluid chromatography on a commercial tartaric acid network polymeric column (tertbutylbenzoyl) with UV-detection at 214 nm. The precision of the presented method upon repeated analysis of a 20 mg/ml suspension is 0.5% (n = 8), and the yield is near 100%. Less than 5 min is required for the chromatographic separation with a resolution of about 3 to the internal standard. With some modification of the chromatographic conditions water samples can also be analysed.

Enantiomeric separations of four 2-substituted propionic acid drugs have been studied using packed-column supercritical fluid chromatography (SFC) with amylose tris(3,5-dimethylphenylcarbamate) coated on silica as support (Chiralpak AD). Under standard conditions (i.e., flow rate, 1.5 ml/min; column temperature, 30 degrees C; back-pressure, 150 bar), the order of elution could be reversed when the polar alcohol modifier methanol in carbon dioxide was replaced by 2-propanol for ibuprofen, ketoprofen, and naproxen. For flurbiprofen, with the highest selectivity factor, no reversal was observed, although selectivity was reduced significantly with higher alcohols. Naproxen and flurbiprofen were also investigated with 2-butanol and 2-pentanol. The former showed reversal of elution order but not the latter. For higher alcohol modifiers, including 2-propanol, the peak symmetry was poor but could be improved by addition of citric acid in the alcohol modifier. These results stress the importance to investigate enantiomer elution order during the development of enantioselective methods and when chromatographic conditions are optimized. Preliminary experiments with column temperatures over the range of -15 to 45 degrees C revealed that, in a few cases, reversal took place with a change in temperature only., (Copyright 2005 Wiley-Liss, Inc.)

This paper describes packed column supercritical fluid chromatography (SFC) for the analysis of a peroxysome proliferator-activating gamma-receptor agonist that is a carboxylic acid. Evaluation of conditions for the separation of this candidate drug and related compounds in bulk substance is described. A Chiralcel OD column was used for this purpose due to its high selective retention of related substances and relative inertness, though the enantioselectivity was negligible, with methanol as polar modifier. A high enantioselectivity was obtained on Chiralpak AD and it was possible to determine the enantiomeric purity within 10 min on a 5 cm short column. Both the achiral and the chiral systems were run without acid additive in the mobile phase and the level of detection of impurities by area was about 0.1%. For the analysis of samples dissolved in water, without any isolation step, 2-propanol was used as modifier. Due to the column surface activity, evidently generated by injected water, citric acid 1 mM was included as additive in the 2-propanol in order to maintain symmetric and undistorted peak shape. The detection limit for the assay was 21 microg mL(-1) (50 nmol mL(-1)) for 5 microL injected (R.S.D. 6.4%, n = 8). A 5 cm short Chiralcel OD column was used. Determination of enantiomeric purity of the drug in aqueous samples required increased sensitivity. The sample was acidified and extracted into a small volume of 1-pentanol, out of which 25 microl was analyzed by SFC. The minor enantiomer at the 3% (w/w) level added could be confirmed. Its ratio remained constant during the procedure as measured relative to a reference solution in organic media.

An enantioselective anion exchanger based on tert-butylcarbamoylquinine as chiral selector and thiol-modified silica as chromatographic support was applied for the enantiomer separation of the shortacting calcium antagonist clevidipine after its hydrolysis to methyl 4-(2',3'-dichlorophenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate. This hydrolyzed derivative of clevidipine is the primary metabolite and precursor of the parent drug. Method development included the steps of optimization of the composition of the mobile phase (pH, type and content of organic modifier, polar organic mode), screening of various structural analogs of above mentioned CSP, and evaluation of the effect of the flow rate. These detailed studies gave insight into the operational mode of the CSP, which is basically an enantioselective anion exchange mechanism. The polar organic mode turned out to be advantageous with regard to enantioselectivity and resolution. The optimized method makes use of an eluent composed of 0.125% acetic acid in acetonitrile with flow rate of 1 ml/min at a constant temperature of 25 degrees C, and allows the separation of the both enantiomers with enantioselectivity alpha of 1.25 and a resolution RS of 3.0 within 10 min. On the above mentioned quinine carbamate phase the (R)-enantiomer is stronger retained, while a change to the corresponding quinidine carbamate CSP allows the reversal of the elution order.

In the recent years, it has repeatedly been stated that amines react with CO2 and can therefore not be chromatographed under supercritical conditions with CO2. The aim of the present work is to elucidate the structural requirements and conditions that can lead to the reaction of an amine analyte with CO2 and, if this occurs, the structure of the formed product. The use of on-line nuclear magnetic resonance (NMR) spectroscopy with a flow probe for supercritical fluid chromatography (SFC) enables the investigation of these unstable analytes in supercritical mediums. Several alkyl-substituted secondary benzylamines and some primary aromatic amines were dissolved in supercritical CO2 and investigated by employing on-line SFC-1H NMR spectroscopy. It was found that the condition of carbamic acid formation depends on the steric properties of the substituents of the amine. A 2-isopropylamino alcohol compound, metoprolol, was also investigated with the setup. No carbamic acid could be detected with the present conditions.

The use of L-(+)-tartaric acid as a chiral mobile phase additive (CMPA) has been investigated in a packed-column SFC system. The CMPA, carbon dioxide, and methanol, containing a high concentration of aliphatic amine additive, were used as the mobile phase and Hypercarb as support [Gyllenhaal O., Karlsson A., SFC of metoprolol and other amino alcohols on Hypercarb (in preparation)]. Good enantioselectivities were obtained for tertiary amine homologues of 2-amino alcohols, used as beta-adrenoreceptor-blocking drugs. Moderate selectivities were observed for aromatic compounds having a second substituent in the ortho-position. The overall retention was influenced by the aromaticity of the analytes as well as the presence of free electron pairs in the molecule. Increased concentrations of CMPA gave higher retention and also increased the enantioselectivity. The practical utility of this present enantioselective system was demonstrated on one batch of (S)-metoprolol that was N-methylated with methyl iodide. The enantiomeric separation was accomplished within 10 min., (Copyright 2002 Elsevier Science B.V.)

A method for the analysis of aqueous samples of isosorbide-5-mononitrate (5-ISMN) is presented. It is based on packed column supercritical fluid chromatography (SFC) using 20% of 2-propanol in carbon dioxide as the mobile phase and a diol silica column as the stationary phase. Using the described conditions it is possible to quantitate 5-ISMN released from Imdur tablets in gastric media. The precision upon repeated injections was 2% (RSD) at the 20 microg/ml level (n=8), using peak height measurements, when the solution was circulated through the sample loop of the injector. Samples from drug release testing that had been analyzed with reversed phase LC were analyzed with the present method and the results agreed well. It is also possible to monitor the drug released in a dissolution-testing vessel through direct on-line continuous loading (recirculation) of the sample loop of the SFC instrument.

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.

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).

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).

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.

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).

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.

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.

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.).

The derivatization of benzenesulphonamide, N-ethylbenzenesulphonamide and N-phenylbenzenesulphonamide with trifluoroacetic and heptafluorobutyric anhydride and pentafluorobenzyl bromide has been studied. A rapid quantitative acylation is obtained in benzene in the presence of trimethylamine. Pentafluorobenzylation is performed by the extractive alkylation technique using tetrabutylammonium as counter ion and methylene chloride as solvent. Less than 20 min are required for a quantitative derivatization. The derivatized sulphonamides have a hydrophobic character, making them very suitable for gas chromatography. Trifluoroacetylation and heptafluorobenzylation decreases it. The derivatives have a high electron-capture detector response (minimum detectable quantity, 1-2 X 10-minus 16 moles/sec). A standard curve is given for the determination of N-phenylbenzenesulphonamide as trifluoroacetyl derivative in the range 1.8-90 ng/ml.

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.

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.

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.

A gas chromatographic method for the determination of phenobarbital in saliva has been developed. Phenobarbital is converted into its bispentafluorobenzyl derivative by extractive alkylation at pH 9 with 0.1 M tetrabutylammonium ion as extracting agent and 0.1 M pentafluorobenzyl bromide as alkylating reagent in methylene chloride. A reaction time of 20 min is required. Quantitation is effected by electorn-capture detection in a gas chromatograph equipped with a pre-column venting system for removal of methylene chloride and pentafluorobenzyl bromide. This procedure allows the direct introduction of the reaction mixture into the gas chromatograph. A 60-ng amount of phenobarbital in 100 mul of human saliva can be determined with a precision of 1.9% (S.D.) and a recovery of 93%.