The sensitivity of the cis/trans-isomerization of enalapril and enalaprilat to solvent conditions.

The cis- and trans-isomers of enalapril and enalaprilat can be resolved by HPLC and by capillary electrophoresis. The isomeric content of enalapril is perturbed by the ionization of both its carboxyl and amine groups, while the isomeric content of enalaprilat is only perturbed by the ionization of its amine group. Increasing the hydrophobicity of the analyte solvent, as reflected in its molar polarization, increases the Z (cis) content of enalapril and markedly decreases the kinetics for isomerization. Far UV circular dichroic measurements suggest that the increase in Z (cis) content of enalapril is due to protonation of its carboxylate group. Taken together, the in-vitro properties of enalapril and enalaprilat suggest that the in-vivo transformation of the prodrug enalapril to the inhibitor enalaprilat and its delivery to angiotensin-converting enzyme should not be significantly limited by cis/trans-isomerization.

Analysis of the isomeric composition of the proline peptide bond in an angiotensin-converting enzyme inhibitor using capillary electrophoresis.

The cis and trans isomeric composition of a proline peptide bond can be determined by routine free-solution capillary electrophoresis measurements provided that one isomeric form is preferentially stabilized by a dissociable ionic group. This capability is illustrated using the angiotensin converting enzyme (ACE) inhibitor (S)-1-N-[1-(ethoxycarbonyl)-3-phenylpropyl]-L-ala-L-pro, which has the trade name enalapril. Electropherograms indicate that the two isomeric forms of enalapril can be separated with baseline resolution at 15 degrees C using capillary buffers having pH values in the dissociation ranges of the enalapril carboxyl group, pK(cis) and pK(trans) of 2.6 and 3.1, and of the enalapril amine group, pK(cis) and pK(trans) of 5.9 and 5.6. Such baseline resolution indicates that the isomeric composition does not change during analysis, facilitating measurement of the isomer composition of a sample prior to its injection into the capillary. Thus the effect of pH, ionic strength, or an aprotic solvent on the isomeric composition of enalapril can be measured under uniform analytical conditions. The trans isomer composition changes from 68% in the cationic form, pH <2, to 50% in the isoelectric form, pH approximately 4.5, to 60% in the anionic form, pH >7. Addition of salt to the isoelectric form or addition of an aprotic solvent to any form prior to analysis increases the trans isomer composition. Similar analyses can be made using the alternative ACE inhibitors captopril and enalaprilat.

Nonaromatic hydroxylation of bupivacaine during continuous epidural infusion in man.

Less than 11% of the dose of bupivacaine could be accounted for in urine from 10 patients receiving continuous epidural infusions. HPLC analysis of metabolites confirmed (S)-bupivacaine was more extensively metabolised than (R)-bupivacaine, and dealkylation was the predominant metabolic pathway although co-elution of metabolites made quantitation difficult. The percentage of (S)-2',6'-pipecoloxylidide and co-eluting metabolites excreted relative to (R)-2',6'-pipecoloxylidide from three patients was 0.32+/-0.05, while for seven patients it was 1.28+/-0.09. Conversely, the percentage of (S)-3'-hydroxy bupivacaine and co-eluants excreted relative to (R)-2',6'-pipecoloxylidide from the three patients (1.76+/-0.48) was greater than the seven patients (0.19+/-0.09). Urinary metabolites were analysed for evidence of aliphatic hydroxylation of bupivacaine. Chiral liquid chromatography-mass spectrometry (LC-MS) on an alpha(1)-glycoprotein column at pH 7 used hydroxylamine acetate as the volatile mobile phase. Compounds tentatively identified as hydroxybupivacaines by MRM were verified by their product ion spectra in a subsequent MS-MS run. Eighteen oxygenated metabolites of bupivacaine were detected, half of which were hydroxylated on nonaromatic groups. Equal numbers of mono- and dihydroxybupivacaines were excreted. There was no evidence to suggest the presence of (S)-4'-hydroxybupivacaine, 2'-hydroxymethylbupivacaine, 3'-hydroxy-2',6'-pipecoloxylidide or a piperidone. The metabolite previously identified as (S)-4'-hydroxybupivacaine was not hydroxylated on the xylyl group.

Isocratic liquid chromatographic assay for monitoring the degradation of luteinizing hormone releasing hormone by extracts from the gastrointestinal tract of possums.

A simple HPLC method to separate human luteinizing hormone releasing hormone (LHRH) from its metabolites using an isocratic elution is described. Intact LHRH and five metabolites were separated in 11.4 min. The calibration curve (peak area versus concentration) was linear over the concentration range 1.25-35 microg/ml (r(2)=0.99) with the intercept not significantly different from zero (P>0.05). Intra-day and inter-day variability of the assay was less than 5% for repeat injections of 5, 14.5 and 29 microg/ml. The method was applied to evaluate the susceptibility of LHRH to enzymes present in the lumen and mucosal extracts of the gastrointestinal tract of possums. The major degradation products of LHRH were identified by HPLC separation, amino acid analysis and mass spectrometry as LHRH (1-5), LHRH (1-4), LHRH (1-3) and LHRH (3-4).

Quantitative capillary electrophoresis assay for the proteolytic stability of luteinizing hormone-releasing hormones.

A rapid and simple capillary electrophoresis (CE) assay for measuring the stability of luteinizing hormone-releasing hormone (LHRH) analogues in the presence of intestinal enzymes has been developed and validated. Buffer pH and sample stacking were important factors in controlling resolution and reproducibility. The CE assay for human (h) and salmon LHRH analogues between 0.05 and 0.25 mM was linear for peak height versus concentration (r2>0.99). Analysis of hLHRH at 0.1 mM had an intra-day relative standard deviation of 1.25% and an inter-day relative standard deviation of 5.0%. The method was applied to the stability of LHRH analogues in salmon intestinal digests.