Metal ion coordination of macromolecular bioligands: formation of zinc(II) complex of hyaluronic acid.

The coordination of zinc(II) ion to hyaluronate (Hya), a natural copolymer, in aqueous solution at pH 6 was investigated by potentiometric and circular dichroism (CD) spectroscopic methods, and by monitoring the changes in macroscopic properties by high-precision measurements. The zinc(II)-selective electrode, and CD measurements proved the binding of zinc(II) by Hya. A number of Hya fragments (Mr approximately 3.3 x 10(3)-1.4 x 10(6)) were studied to estimate the contributions of the polyelectrolyte effect, the solvation and host-guest interactions to the extra stabilization of the macromolecular zinc(II) complexes as compared with the monomeric unit. The zinc(II) ion activity increase reflected a stability decrease for the fragments with Mr < 4 x 10(4). This molecular weight differs from that where cleavage of the Hya skeleton starts (approximately 5 x 10(5), according to the size-exclusion gel, and anion-exchange chromatographic behavior of the Hya fragments) and from that where the polyelectrolyte effect stops (approximately 6 x 10(3)). The excess volumes and Bingham shear yield values of the solutions revealed the transformation of the coherent random coil structure stabilized by intermolecular association in the NaHya to an intramolecular association producing the globular structure of the ZnHya molecule, with a smaller but more strongly bound solvate water sheet.

Enantiomeric separations and their application in pharmaceutical analysis using chiral eluents.

The most important problems of enantiomeric separations using chiral eluents are discussed. The methods have been compared with respect to enantiomeric purity of reagents, reagent selection and separation variables. The most important considerations for methods based on inclusion-complexation with different CDs and on using chiral counter ions are summarised and compared. A new possibility for the separation of enantiomeric compounds with the aid of a combination of ion-pair and inclusion-complex formation has been introduced. As a consequence of this investigation, the selectivity of the separation can be significantly improved; those ionic isomers and enantiomers which cannot be separated or are unsatisfactorily separated by ion-pair chromatography or by inclusion-complex formation, can be separated by the combined technique. Comparison of methods applicable for enantiomeric separations is also given with respect to solving specific analytical tasks in the field of pharmaceutical analysis.

Estimation of impurity profiles of drugs and related materials. Part VIII: Combined application of high-performance liquid chromatography and NMR spectroscopy in the impurity profiling of drugs.

The usefulness of the joint application of HPLC and NMR spectroscopy in drug impurity profiling is demonstrated by the following examples: (1) identification of Z and E isomers of 17 alpha-ethynyl-4-oestrene-3 beta, 17-diol-3-acetate-17-(3'-acetoxy-2'-butenoate) in ethynodiol diacetate; (2) identification of the p-tolyl analogue as the impurity of enalapril maleate; (3) identification and quantification of 2'-dehydro-pipecuronium bromide in pipecuronium bromide. The possibilities of utilizing NMR spectroscopy for the identification and quantification of the impurities with and without their isolation are discussed.

Estimation of impurity profiles of drugs and related materials: part XXI. HPLC/UV/MS study of the impurity profile of ethynodiol diacetate.

The impurity profile of ethynodiol diacetate was investigated using the HPLC/UV/MS method. Using the slightly modified HPLC method of USP 24 two impurities, earlier isolated by preparative HPLC and investigated by NMR spectroscopy were separated and characterised. The mass spectra amended by the diode-array UV spectra supported the earlier found structures (E and Z isomers of 17alpha-ethinyl-estr-4-ene-3beta,17-diol-3-acetate-17-(3'-acetoxy-2'-butenoate). Another, hitherto not described impurity, 17alpha-ethinyl-estr-4-ene-3beta,17-diol-3-acetate-17-(3-oxo-butanoate) has also been separated and characterised by means of its mass spectrum, NMR and UV spectra.

Analysis of steroids Part 50. Derivatization of ketosteroids for their separation and determination by capillary electrophoresis.

4-Ene-3-ketosteroids and 17-ketosteroids were quantitatively transformed into the corresponding hydrazones using Girard P and T reagents, respectively. The positively charged derivatives were separated by capillary electrophoresis. The spectrophotometric characteristics of the derivatives permitted their sensitive detection in the 230-280 nm range. The steroids investigated included nortestosterone and its phenylpropionate, norethisterone and its oenanthate, d,l-norgestrel, dehydroepiandrosterone, androstenedione and ethisterone.

Estimation of impurity profiles of drugs and related materials. Part 14: the role of HPLC/diode-array UV spectroscopy in the identification of minor components (impurities, degradation products, metabolites) in various matrices.

The possibility of the rapid identification of drug related minor components by HPLC/diode-array UV spectroscopy is demonstrated by three examples. Hydroxylated impurities (degradation products) of norgestrel (6 alpha and beta, 10 beta-hydroxy derivatives) were identified on the basis of their UV spectra and retention matching with the synthesized impurities. The position of the phenolic hydroxyl groups in the mono- and dihydroxylated metabolites of bisaramil was established by UV spectroscopy and retention matching with the synthesized metabolites. The discrimination between the isomeric 4-ene-3-ketone and 1-ene-3-ketone components in crude 19-nortestosterone, product of the Birch reduction of 3-methoxy-1,3,5(10)-oestratriene-17 beta-ol, was also based on the diode-array UV spectra.

Estimation of impurity profiles of drugs and related materials Part 18. Impurities and degradation products of mazipredone.

Reversed-phase HPLC methods using C-18 and C-8 columns as well as various isocratic and gradient systems with aqueous ammonium acetate, methanol and acetonitrile are described for the separation of the impurities of mazipredone (11beta,17-dihydroxy-21-(4-methyl-1-piperazinyl)-pregna-1,4-diene- 3,20-dione hydrochloride). These methods were used also for the estimation of the hydrolytic and oxidative degradation pathways of mazipredone in 0.1 M hydrochloric acid and sodium hydroxide at 80 degrees C. With the aid of HPLC-(APCI)-MS and HPLC-diode-array UV techniques 15 impurities and degradation products have been identified.

Estimation of impurity profiles in drugs and related materials. Part 11--The role of chromatographic and spectroscopic methods in the estimation of side-reactions in drug syntheses.

Impurities in drugs are classified on the basis of the types of side-reactions in drug syntheses resulting in their formation. This is shown by summarizing the authors' earlier results in the field of impurity profiling of 19-nor-steroids, ethynodiol diacetate, mazipredone, pipecuronium bromide, flumecinol, enalapril, pyridinol carbamate, phenylbutazone, thymotrinan and some new results related to danazol and famotidine.

Estimation of impurity profiles of drugs and related materials: part 19: theme with variations. Identification of impurities in 3-oxosteroids.

Due to the varied reactions leading to the 3-oxo group in steroids and the reactivity of its environment, a large number of impurities related to this group are formed during the reaction steps and the degradation studies. In this paper the experiences from the authors laboratory with the 3-oxo-related impurities in 19-nor-4-ene-3-oxosteroids (norgestrel, norethisterone, nandrolone, its esters and Nestorone) as well as corticosteroids (prednisolone, mazipredone, etc) are presented. The impurities include saturated 3-ones, 1-ene-3-ones, 5(10)-ene-3-ones, 3-deoxo and 3-ethinyl-3,5-diene derivatives, 6-ene, 8(14)-ene, 6,8(14)-diene, 6-hydroxy (alpha and beta), 10beta-hydroxy and 6-one derivatives in 4-ene-3-oxosteroids and 8(9)-ene, 9(11)-ene, 11alpha-hydroxy, 11-oxo and 4-ene-3-one derivatives in 11beta-hydroxy-1,4-diene-3-oxosteroids. The chromatographic, spectroscopic and hyphenated techniques used in this study include TLC, GC, HPLC with diode array UV detector, GC-MS, LC-MS and NMR methods.

Determination of ethinylestradiol in tablets by quantitative TLC method.

A new quantitative TLC method was developed for the determination of ethinylestradiol in tablets. The possible adsorption of ethinylestradiol on the starch can be avoided by enzymatic degradation, which enabled the easy extraction of the active ingredients from the tablet powder. The quantitative evaluation of the separated compounds was performed by densitometry at a wavelength of 280 nm. The relative standard deviation of the method was found to be better than +/- 2.6% in every case except Ambosex tablet where this value was +/- 4.7%. On the basis of the results obtained the method can be proposed for the routine analysis of tablets containing small amount of ethinylestradiol.

Investigation of the hydrolytic decomposition of 5-iodine-2'-desoxyuridine by high-performance liquid chromatography.

A new HPLC method was developed for the monitoring of the hydrolytic decomposition of 5-iodine-2'-desoxyuridine in aqueous solution and for the stability assay of this compound in formulation. The separation of 5-iodine-2'-desoxyuridine and its decomposition products (5-iodineuracil and uracil) was performed on a u Bondapack C18 column using a mixture of water and methanol as eluent. On the basis of the experimental data, the kinetic order and the rate-constants of both consecutive reactions were determined.

[High performance liquid chromatography and capillary electrophoresis investigation of the neww immunostimulant tetrapeptide drug, thymocartin]. / Az új immunstimuláló tetrapeptid, a thymocartin nagyhatékonyságú folyadékkromatográfiás és kapillár elektroforetikus vizsgálata.

High-performance liquid chromatographic (HPLC) and capillary electrophoretic (CE) methods have been developed for the separation of 22 related peptides (potential and real impurities) from a new immunostimulant tetrapeptide derivative (thymocartin, Arg-Lys-Asp-Val) being under clinical examination. The described methods are suitable for the purity control of the bulk drug material. In the course of the reversed-phase ion-pair HPLC separations C-18 columns (Hypersil BDS and Ultrasphere IP) were used. In the case of using sodium hexanesulphonate as the ion-pairing reagent, the eluent contained phosphate buffer (pH = 3) and 32% v/v methanol, while in another method a gradient system with a sodium perchlorate solution (pH = 2 set by perchloric acid) and acetonitrile was applied. The optimum separation in the CE investigations was achieved at pH = 3 using triethylammonium phosphate buffer and 10% v/v acetonitrile as the organic modifier.

[Are ultraviolet and visible spectroscopy and spectrophotometry obsolete methods in pharmaceutical analysis?]. / Elavult módszereknek tekinthetók-e az ultraibolya spektroszkópia és spektrofotometria a gyógyszeranalitikában?

It has been investigated if UV-VIS spectroscopy and spectrophotometry can be regarded to be obsolete methods in pharmaceutical analysis. The conclusions are as follows. As a consequence of the introduction and spreading of highly efficient spectroscopic methods in the structural analysis of organic compounds the importance of UV-VIS spectroscopy as a structure elucidation tool has greatly decreased. At the same time, however, diode-array UV spectrophotometers used as HPLC detectors have created very convenient possibilities for the identification of minor components (impurities, degradation products, etc.) in drugs. This statement is illustrated by several practical examples. On the basis of some data taken from the British Pharmacopoeia 1998 it is stated that UV spectrophotometry as a quantitative analytical method still belongs to the most frequently used analytical techniques in pharmaceutical analysis. At the same time, however, the authors are of negative opinion about the up-to-dateness and usefulness of colorimetric methods still very often published for the determination of drug substances.

[Analysis of steroids. Part 45: Analytical investigation of pipecuronium bromide (Arduan)]. / Adatok a szteránvázas vegyületek analitikájához 45. Pipecuronium bromid (Arduan) analitikai vizsgálata.

The following methods are described for the analytical investigation of pipecuronium bromide. 1. HPLC method. Of the several systems tried for the separation and quantification of impurities and degradation products the best results were obtained using silica as the stationary phase and 43:43:14 mixture of methanol, acetonitrile and concentrated aqueous ammonia containing 0.1 mole/l each of ammonium chloride and ammonium carbonate as the eluent. The validation of this method is presented. The above described aggressive eluent can be successfully replaced by an ion-pairing system using silica as the stationary phase and 96:4 mixture of acetonitrile and water containing 0.1 mole/l sodium perchlorate as the eluent. 2. Thin-layer chromatography. TLC systems are described for the separation and densitometric quantification of the impurities and degradation products of pipecuronium bromide. 3. Spectrophotometry. Two methods are described. The ester groups of the molecule can be determined by the iron(III)-hydroxamate method while for the ion-pair extraction of the quaternary ammonium steroid picric acid or bromthymol blue are used as the reagents. 4. Titrimetry. In addition to the titration with acetous perchloric acid for the assay of the bulk material a microtitration method is described for the determination of pipecuronium bromide in individual lyophylized ampoules (potentiometric titration with 0.1 M silver nitrate).

Selection of high-performance liquid chromatographic methods in pharmaceutical analysis. IV. Selection of most applicable separation system.

Different analytical tasks in the pharmaceutical analysis can be classified according to the separation problems into three main groups: trace analysis, assay methods and separation of closely related compounds including isomers. The most important requirements of high-performance liquid chromatographic (HPLC) methods with respect of the separation problems are summarized. Considerations and recommendations for the selection of the most applicable HPLC system to solve particular analytical problems are discussed. HPLC methods can be compared on the basis of the system resolution (SR) and system selectivity (SS). Criteria developed for the characterization of HPLC methods considering the difficulties created by the different analytical problems are established. The principles of the selection of the most applicable separation systems are demonstrated through some practical examples in pharmaceutical analysis.

Enantiomeric derivatization for biomedical chromatography.

Derivatization reactions aimed at creating the basis for the chromatographic resolution of biologically and pharmaceutically important enantiomers are reviewed, with emphasis on the literature published in the last 10 years. Three main aspects of chiral derivatization are discussed. (a) Enantiomers containing suitable functional groups (amino, carboxyl, hydroxyl, epoxy, etc.) are transformed into covalently bonded diastereomeric derivatives using homochiral derivatizing agents. The diastereomers formed (esters, amides, urethanes, urea and thiourea, etc., derivatives) can be separated on achiral stationary phases. The derivatization reactions often afford further advantages, such as the improvement of chromatographic properties and the detectability of the solutes using UV and fluorimetric detectors. (b) Covalent but achiral derivatization is often necessary even with the use of chiral stationary phases enabling in principle direct enantioseparations (Pirkle-type columns, cyclodextrin-bonded phases, glycoprotein column and functionalized cellulose columns). The main goals of these derivatization reactions (which are analogous to those discussed above), are to introduce functional groups into the molecule of the enantiomers that improve the possibilities for chiral interactions or block functional groups to avoid non-specific interactions. (c) In the broader sense, the dynamic formation of diastereomers using chiral mobile phase additives (cyclodextrins, various reagents to form diastereomeric ion pairs, adducts, mixed metal complexes) can also be considered to be chiral derivatization reactions and is therefore briefly discussed also.