Determination of circular dichroism and ultraviolet spectral parameters of norgestimate- and other Delta(4)-3-ketosteroid oxime isomers via normal phase HPLC method.

The oxime formation reaction of therapeutical progestogen (levonorgestrel, levonorgestrel acetate, norethisterone), androgen (methyltestosterone, testosterone phenylpropionate) and anabolic (nortestosterone phenylpropionate) Delta(4)-3-ketosteroids has been investigated. The ketosteroid-hydroxylamine reaction was monitored by reversed phase HPLC system. It was established, that under the experimental conditions applied the oxime formation was complete within 2 h. The reaction leads to the formation of Z and E oxime isomers. The isomers of norgestimate (levonorgestrel 17-acetate oxime) and other Delta(4)-3-ketosteroid oximes have been separated by a new normal phase HPLC method. The identification (elution order assignation) and determination of the formation ratio of the isomers have been performed by 1H NMR spectroscopy on the basis of the chemical shift differences of 4-H signals. The on-line CD and UV spectra of the pure oxime isomers were recorded and then molar ellipticities and absorbances of the isomers were calculated in the wavelength range of 200-300 nm via parameter estimation method.

Optical resolution of a series of potential cholecystokinin antagonist 4(3H)-quinazolone derivatives by chiral liquid chromatography on alpha1-acid glycoprotein stationary phase.

Optical resolution of the enantiomers of new 4(3H)-quinazolone derivatives is investigated using the alpha1-acid glycoprotein chiral stationary phase (Chiral-AGP). Stereoselective separation of the model compounds can be controlled by varying the pH and adding uncharged organic modifiers (acetonitrile and 2-propanol) to the mobile phase. For the majority of quinazolone derivatives, Chiral-AGP is proved to be an excellent enantioselector, because optimized chromatographic conditions allow for the baseline separation of the enantiomers. Separation factors between 1.19 and 1.85 are obtained. The effects of acetonitrile and 2-propanol on the chromatographic behavior of the model compounds are quite different because of their different hydrophobic- and hydrogen-bonding properties. The eluent pH and organic modifier concentration also contributes to the chiral recognition by altering the protein environment. The analysis of the experimental results leads to new information about the chromatographic mechanism on a Chiral-AGP surface.

Determination of delta4-3-ketosteroids based on oxime formation by difference circular dichroism spectroscopy.

delta4-3-Ketosteroids exhibit an intensive negative Cotton effect on the circular dichroism (CD) spectra in the wavelength range for the n-pi* electronic transition (270-350 nm). With hydroxylamine hydrochloride, delta4-3-ketosteroid compounds can be transformed into oxime derivatives. Following oxime formation, positive ellipticity with low intensity can be registered in this wavelength range. The quantitative determination of delta4-3-ketosteroids is based on the considerable difference between the ellipticities before and after oxime formation. The difference ellipticity for the six ketosteroids examined (norethisterone, levonorgestrel, levonorgestrel acetate, methyltestosterone, testosterone phenylpropionate, nortestosterone phenylpropionate) varies linearly with the concentration in the interval 6 x 10(-6)-3 x 10(-3) mol/L. The method can be well applied to determination of delta4-3-ketosteroid contamination of norgestimate [(+)-13-ethyl-17-hydroxy-18,19-dinor-17alpha-pregn4-en-20-yn-3-one oxime acetate]; 0.02-10% impurity can be measured.

Ion-pair partition of quarternary ammonium drugs: the influence of counter ions of different lipophilicity, size, and flexibility.

PURPOSE: The ion-pair partition of quaternary ammonium (QA) pharmacons with organic counter ions of different lipophilicity, size, shape and flexibility was studied to elucidate relationships between ion-pair formation and chemical structure. METHODS: The apparent partition coefficient (P') of 4 QAs was measured in octanol/pH 7.4 phosphate buffer system by the shake-flask method as a function of molar excess of ten counter ions (Y), namely: mesylate (MES), acetate (AC), pyruvate (PYRU), nicotinate (NIC), hydrogenfumarate (HFUM), hydrogenmaleate (HMAL), p-toluenesulfonate (PTS), caproate (CPR), deoxycholate (DOC) and prostaglandin E1 anion (PGE1). RESULTS: Based on 118 of highly precise logP' values (SD< 0.05), the intrinsic lipophilicity (without external counter ions) and the ion-pair partition of QAs (with different counter ions) were characterized. Linear correlation was found between the logP' of ion-pairs and the size of the counter ions described by the solvent accessible surface area (SASA). The lipophilicity increasing effect of the counter ions were quantified and the following order was established: DOC approximate to PGE1 >> CPR approximate to PTS >> NIC approximate to HMAL >> PYRU approximate to AC approximate to MES approximate to HFUM. Analyzing the lipophilicity/molar ratio (QA:Y) profile, the differences in the ion-pair formation were shown and attributed to the differences in the flexibility/rigidity and size both of QA and Y. CONCLUSIONS: Since the largest (in average, 300 X) lipophilicity enhancement was found by the influence of DOC and PGE1 and considerable (on average 40 X) increase was observed by CPR and PTS, it was concluded that bile acids and prostaglandin anions may play a significant role in the ion-pair transport of quaternary ammonium drugs and caproic acid and p-toluenesulfonic acid may be useful salt forming agents to improve the pharmacokinetics of hydrophilic drugs.

Determination of absolute configuration of ketamine enantiomers by HPLC-CD-UV technique.

Recognizing that the stereochemical structure of NMDA receptor antagonist ketamine provides valuable data about the relationship between its conformation and absolute configuration by CD-UV analysis, a method for the identification of ketamine enantiomers is proposed which avoids the need for authentic samples of the enantiomers. The ketamine enantiomers were separated by HPLC using Chiralcel OJ stationary phase. The in situ registration of CD and UV spectra, together with the application of the octant rule for cyclohexanone derivatives, makes possible the direct assignment of the eluted ketamine enantiomers.

[High pressure liquid chromatographic methods for compendium of purity tests]. / Gyógyszerkönyvi tisztaságvizsgálati feladatok megoldása nagyhatékonyságú folyadékkromatográfiával.

The paper is addressed to the institute of Pharmaceutical Chemistry, Budapest, on the 50th anniversary of its foundation, paying tribute to Prof. Antal Végh, the first director of the Institute and his first deputy dr. László Auber and all the members of the Staff having merit in developing the research activity at the Department. After surveying the early papers published from the fifties through the seventies, concerning the profile of drug purity control, the factors increasing HPLC selectivity are shortly reviewed. In the concluding part of the paper, some recent results, as HPLC purity control methods suitable for compendial purposes (methylxanthines, opium alkaloids, tropane-alkaloids, enantiomer purity control of different drug compounds on glycoprotein and chirelcel columns) are summarized.

[Novel chiroptical methods in pharmaceutical analysis]. / Ujabb kiroptikai módszerek a gyógyszeranalízisben.

Chiroptical spectroscopy, and its double and triple hyphenated combinations with UV-VIS spectroscopy and/or separation techniques constitute great progress in pharmaceutical analysis, of which five recently developed methods are surveyed here, as follows: The determination of enantiomeric purity by double, CD/UV detection without, and with the latter one provides enhanced sensitivity and selectivity. The determination of chromatographic peak homogeneity, by double detection, and peak slicing by recording. The separation, identification and four stereoisomers of doubly chiral compounds, by HPLC separation on chiral column, and optical-chiroptical detection.

[Competitive binding of tetrabutylammonium and cetylpyridinium cations on C18 surfaces]. / Tetrabutilammónium és cetilpiridinium kationok kompetitív kitõdése C18 felületen.

The adsorption behavior of tetrabutilammonium (TBA) and cetylpyridinium (CP) cations was studied on Hypersil 5-ODS sorbent. After establishing, that CP has about 5 fold stronger affinity to the C18 surface, than the structurally different, symmetric, spheric molecule of TBA, the adsorption from their common solution was also examined. The data of "competitive binding" seem to show, that TBA should have certain selective binding sites on the C18 surface, from which, it can not be displaced by the CP even in the case the latter is present in a 4 fold excess in the mobile phase. The maximal coverage was found 52.9% (CP) and 12% (TBA) i.e. every second of C18 groups is covered by CP cations and only 1/8th of C18 groups may interact with TBA. The accessible, proton releasing silanol content, related to the total C18 content of the column, was found very low.

[Equilibrium solubility of new potential drugs in water, 1-octanol and cyclohexane]. / Uj potenciális gyógyszervegyületek egyensúlyi oldhatósági vizsgálata vízben, 1-oktanolban és ciklohexánban.

A method was developed for the determinations of the exact value of isotherm solubility equilibrium of organic compounds in various solvents, that can be used as a standard operating procedure, too. It was used for the determination of equilibrium solubility data of twenty two new organic compounds in three solvents: water, 1-octanol and cyclohexane. The data of ten imidazo-quinazolone and twelve pyrido-quinazolone derivatives were compared with their chemical structures moreover within and between the structural and solvents series. It was proved, that these compounds are not ideal solutes, due to a specific interaction with the solvents which is constant for the imidazo-quinazolones, while it is randomly altering at the different pyrido-quinazolones. The octanol/water partitioning data were calculated using octanol and water solubility data, and those were compared with separately, accurately measured data obtained by the traditional shake-flask method. Our data support, that--as it can be found in most of the literature--at compounds which form special interaction with the solvent the partition coefficient cannot be calculated from the solubility data, and reversely the water solubility cannot be estimated using the determined partition coefficient and some correction factors. It was demonstrated, that the calculated partition coefficient can be biased randomly up to and order of magnitude, and in some cases even the tendency of those is false in a series.

Heterocondensed quinazolones: synthesis and protein-tyrosine kinase inhibitory activity of 3,4-dihydro-1H,6H-[1,4] oxazino-[3,4-b]quinazolin-6-one derivatives.

1-Benzylidene (2-14) and 1-phenylhydrazono derivatives (15-29) of 3,4-dihydro-1H,6H-[1,4]oxazino[3,4-b]quinazolin-6-one (1) were obtained from the condensation reactions of 1 with a series of aromatic aldehydes and by direct diazonium coupling with aryl-diazonium chlorides. The substances were tested for their ability to inhibit the tyrosine kinase activity of SW-620 (human colon carcinoma) cells. Compounds 8, 10, 12 and 13 showed remarkable inhibitory activity.

Acid-base properties of terbutaline in terms of protonation macro- and microconstants.

The acid/base chemistry of terbutaline was characterized at the molecular level in terms of protonation macroconstants and microconstants. The macroconstants were measured by potentiometry and calculated by standard evaluation methods. The stepwise macroconstant values were log K1 = 11.01, log K2 = 9.89, and log K3 = 8.57 at 25.0 degrees C and 0.2 M ionic strength. The microconstants were deduced using the relationships between macro- and microconstants and an appropriate data set of model compounds (resorcinol and phenylephrine). The molecule of terbutaline contains three ionizable functional groups. In the unprotonated form of the molecule, the two identical phenolate groups are slightly more basic than the secondary amino group, whereas the amino basicity significantly exceeds that of the phenolate site, when the other phenol is protonated. This is due to the large phenolate-phenolate intramolecular interaction. The phenolate-phenolate and the phenolate-amino interactivity parameters were found to be -1.21 and -0.41 log E units, respectively.

Determination of protonation macro- and microconstants and octanol/water partition coefficient of the antiinflammatory drug niflumic acid.

The drug niflumic acid is an amphoteric substance with overlapping pKa values. The acid-base chemistry of the molecule has been characterized in terms of protonation macroconstants (with reference to stoichiometric ionizations) and microconstants (with reference to ionizations of individual species). The proton-binding sites were assigned using 1H and 13C NMR spectroscopy. Due to the very poor water solubility of niflumic acid, the aqueous pKa values were determined from the apparent ionization constants in methanol-water solutions of various proportions by extrapolation to zero co-solvent using the Yasuda-Shedlovsky procedure. The kz tautomerization microconstant of the equilibrium unionized form<-->zwitterionic form was determined from mixtures of organic solvent (dioxane or methanol) with aqueous buffer (at the pH of isoelectric point) by UV spectroscopy, and used for calculation of the other protonation microconstants. The zwitterionic form of the molecule predominates over the uncharged form, the concentration being maximal at the isoelectric pH. The apparent partition coefficients (Papp) of niflumic acid were measured in octanol/water solution by the shake-flask method over a wide pH range. The lipophilicity profile (logPapp vs pH) shows a parabolic shape near its maximum at the isoelectric point. A relationship derived between Papp, PXH0(micropartition coefficient of the uncharged microspecies) and PX-(partition coefficient of the anion) is valid for amphoteric drugs, in cases where the partition of the unionized form and the ion-pair partition of anion can be confirmed. The logP values of microspecies indicate the high lipophilicity of niflumic acid, which is consistent with its good skin penetration and absorption.

[Correlation between the structures and physicochemical properties of chemotherapeutic fluoroquinolone agents]. / Osszefüggés kemoterápiás fluorokinolonok szerkezete és fiziko-kémiai tulajdonságai között.

The acid-base properties, the lipophilicity and the HPLC behaviour of nine antibacterial fluoroquinolone derivatives were studied to reveal relationship between these physico-chemical parameters and the chemical structure. Basicity of compounds with two proton-binding sites is depicted here in terms of protonation macro- and microconstants. The concentrations of microspecies (cation, zwitterion, neutral and anion) were calculated in the function of pH. The microspeciation of fluoroquinolones were used to derive relationship between the apparent and true octanol/water partition coefficients. An analysis of structure-chromatographic behaviour has been performed utilizing the retention values was determined in a chromatographic system methanol-aqueous phosphate buffer/ODS. Close correlation was found between the lipophilicity and chromatographic behaviour of the tested compounds when pH dependence and the influence of an ionpairing agent were investigated.

Partitioning behaviour of local anesthetic fomocaine derivatives.

The octanol/water partitioning behaviour of local anesthetic fomocaine derivatives has been investigated. The true partition coefficients obtained from log P(app) values reflect well the high lipophilicity of these aminoalkylaryl-ether type compounds. A consequence of this is that the partitioning of the protonated form is not negligible as was proved by the log P(i) values of the ionized molecule measured at pH 4.0. The effect of ionization on partitioning was studied and characterized by the pQ values. This ionization influence on lipophilicity was found quite constant (pQ approximately 4) among the hydroxypiperidine analogs. A parabolic relationship was obtained between the acute toxicity and the log P values of several of the examined compounds.

Relationship between partitioning properties and (calculated) molecular surface. SPR investigation of imidazoquinazolone derivatives.

Structure-property relationship (SPR) investigations have been carried out for a series of N-bridged compounds synthetized recently as potential antithrombotic agents. The octanol/water partition coefficient and reversed phase HPLC retention (log k') were determined experimentally for ten imidazoquinazolone derivatives. Calculations including total geometry optimization and surface area have been carried out. The isotropic surface area (ISA) of supermolecules were obtained. The good correlations were found between log P, log k' and ISA values, provide simple and reliable means by using a single structural parameter for the log P prediction of polycyclic N-bridged compounds.

The combined use of vibrostimulation and in vitro fertilization: successful pregnancy outcome from a retrograde specimen obtained from a spinal cord-injured male.

While pregnancies have been documented through the independent use of the vibrator method, from other methods of procuring ejaculate from spinal cord injured men, and from artificial insemination using a retrograde specimen, we believe that this is the first case report of a live birth resulting from a retrograde ejaculate obtained by vibration from a spinal cord-injured male whose partner underwent in vitro fertilization. Vibrostimulation may well be successful in the two-thirds of men whose spinal cord lesions are at the T10 neurological level and above, who have an intact bulbocavernosus reflex and anal tone but no pain or temperature sensation of the genitalia. Blood pressure monitoring, prevention of autonomic dysreflexia, alkalinization, dilution and infection control of urine, and retrograde specimen retrieval are all important techniques to ensure patient safety and optimal ejaculates. The timing of ovulation and insemination is the crucial factor for the partner of a SCI male whose sperm quality is poor. A complete gynecological workup, including studies of tubal patency, should be done before embarking on a series of artificial inseminations. Stimulation of ovulation and well-timed inseminations should optimize the chance of conception. Depending on semen analysis, female partner factors, and emotional and financial costs, IVF can appropriately be either an early or a final option.

Sex and disability are not mutally exclusive. Evaluation and management.

Managing sexual problems in persons with disabilities is a team effort. Physicians have three special roles; identifying problems that are sexual; assessing these problems; and providing psychological, pharmacologic, or surgical treatment methods in the context of the patients' rehabilitation programs.

[HPLC-investigations of imidazoquinazolone derivatives. Part 2]. / Imidazokinazolon-származékok folyadékkromatográfiás vizsgálata. II.

In a previous paper (2) authors, reported good correlation between the HPLC (log k') and TLC (RM) retention data and the 1-octanol/water partition coefficient (log l') of imidazoquinazolone derivatives synthetized in their laboratory. In the present work the behaviour of the compounds in HPLC systems containing ion-pairing agents (camphorsulfonic acid, Na-dodecyl sulfate) has been studied. While, in reversed phase systems no change of retention indicating the formation of ion-pairs or molecule complexes was observed, camphor-sulfonic acid caused significant increase of retention in the chromatographic systems with bare silica as stationary phase. This phenomenon is interpreted by the formation of camphorsulfonic acid double layer on the silica surface and as a consequence of mixed retention mechanism. The results of a detailed study on the latter subject will be published in a next paper. Double peak formation was observed in reversed phase system in case of C1-C3 disubstituted derivatives. This was interpreted with the formation of isomeric mixtures. Theoretical considerations as well as the identical spectra of the two peaks make plausible the appearance and separation of conformational isomers.

[HPC purity tests for caffeine, theophylline and theobromine]. / Koffein, teofillin, teobromin tisztaságvizsgálata HPLC módszerrel.

After giving a survey of the literature of the HPLC methods for the determination of caffeine, theophylline and theobromine two methods are presented for their purity test, i.e. their detection in each other at the trace level. In the reversed-phase system the stationary phase was C18-silica and the 2:3 mixture of methanol and water was used as the eluent at a detection wavelength of 254 nm. The k' values for theobromine, theophylline and caffeine are 0,25, 0,62 and 1,12, respectively. This system enables the detection of 0,2% of theobromine and 0,1% of theophylline in caffeine and 0,2% of caffeine in theobromine or theophylline. In the normal phase system silica was used as the stationary phase while the eluent was 85:15:0,05 mixture of chloroform, dioxane and formic acid. The wavelength of the detection was 273 nm. The k' values of caffeine, theobromine and theophylline are 1,5 4,0 and 5,6, respectively. This system also enables the detection of 0,1% of theophylline and theobromine in caffeine or 0,2% of caffeine in the former. Theophyline and theobromine can be detected in each other at the 0,1% level.

Protonation equilibria of quinolone antibacterials.

The acid-base properties of seven antibacterial 7-piperazinyl fluoroquinolone derivatives were studied by potentiometry and UV and NMR spectroscopy. These molecules contain two proton-binding sites of similar basicity, namely, the piperazine amino and the carboxylate groups, as proven by 1H NMR spectroscopy. The basicities are quantitated at the molecular level in terms of macroconstants, and also at the submolecular level in terms of microconstants. The microconstants are then used to calculate the concentration of the positive, zwitterionic, neutral, and negatively charged species (microspeciation). The zwitterionic forms always predominate over their neutral protonation isomers, but the zwitterionic:neutral concentration ratio is considerably different for the examined fluoroquinolone derivatives.