Terpenoid Hydrazones as Biomembrane Penetration Enhancers: FT-IR Spectroscopy and Fluorescence Probe Studies.

Hydrazones based on mono- and bicyclic terpenoids (verbenone, menthone and carvone) have been investigated in vitro as potential biomembrane penetration enhancers. In this regard, liposomes composed of lecithin or cardiolipin as phospholipid phase components with incorporated fluorescence probes have been prepared using the thin-film ultrasonic dispersion method. The mean particle size of the obtained liposomes, established using laser diffraction, was found to be 583 ± 0.95 nm, allowing us to categorize them as multilamellar vesicles (MLVs) according to their morphology. Pursuant to fluorescence analysis, we may assume a reduction in microviscosity and, consequently, a decrease in the packing density of lecithin and cardiolipin lipids to be the major mechanism of action for terpenoid hydrazones 1-15. In order to determine the molecular organization of the lipid matrix, lipids were isolated from rat strata cornea (SCs) and their interaction with tested compounds was studied by means of Fourier transform infrared spectroscopy. FT-IR examination suggested that these hydrazones fluidized the SC lipids via the disruption of the hydrogen-bonded network formed by polar groups of SC constituents. The relationship between the structure of terpenoid hydrazones and their ability to enhance biomembrane penetration is discussed.

Development and Validation of HPLC-DAD and UHPLC-DAD Methods for the Simultaneous Determination of Guanylhydrazone Derivatives Employing a Factorial Design.

Guanylhydrazones are molecules with great pharmacological potential in various therapeutic areas, including antitumoral activity. Factorial design is an excellent tool in the optimization of a chromatographic method, because it is possible quickly change factors such as temperature, mobile phase composition, mobile phase pH, column length, among others to establish the optimal conditions of analysis. The aim of the present work was to develop and validate a HPLC and UHPLC methods for the simultaneous determination of guanylhydrazones with anticancer activity employing experimental design. Precise, exact, linear and robust HPLC and UHPLC methods were developed and validated for the simultaneous quantification of the guanylhydrazones LQM10, LQM14, and LQM17. The UHPLC method was more economic, with a four times less solvent consumption, and 20 times less injection volume, what allowed better column performance. Comparing the empirical approach employed in the HPLC method development to the DoE approach employed in the UHPLC method development, we can conclude that the factorial design made the method development faster, more practical and rational. This resulted in methods that can be employed in the analysis, evaluation and quality control of these new synthetic guanylhydrazones.

An optimized method for the measurement of acetaldehyde by high-performance liquid chromatography.

BACKGROUND: Acetaldehyde is produced during ethanol metabolism predominantly in the liver by alcohol dehydrogenase and rapidly eliminated by oxidation to acetate via aldehyde dehydrogenase. Assessment of circulating acetaldehyde levels in biological matrices is performed by headspace gas chromatography and reverse phase high-performance liquid chromatography (RP-HPLC). METHODS: We have developed an optimized method for the measurement of acetaldehyde by RP-HPLC in hepatoma cell culture medium, blood, and plasma. After sample deproteinization, acetaldehyde was derivatized with 2,4-dinitrophenylhydrazine (DNPH). The reaction was optimized for pH, amount of derivatization reagent, time, and temperature. Extraction methods of the acetaldehyde-hydrazone (AcH-DNP) stable derivative and product stability studies were carried out. Acetaldehyde was identified by its retention time in comparison with AcH-DNP standard, using a new chromatography gradient program, and quantitated based on external reference standards and standard addition calibration curves in the presence and absence of ethanol. RESULTS: Derivatization of acetaldehyde was performed at pH 4.0 with an 80-fold molar excess of DNPH. The reaction was completed in 40 minutes at ambient temperature, and the product was stable for 2 days. A clear separation of AcH-DNP from DNPH was obtained with a new 11-minute chromatography program. Acetaldehyde detection was linear up to 80 µM. The recovery of acetaldehyde was >88% in culture media and >78% in plasma. We quantitatively determined the ethanol-derived acetaldehyde in hepatoma cells, rat blood and plasma with a detection limit around 3 µM. The accuracy of the method was <9% for intraday and <15% for interday measurements, in small volume (70 µl) plasma sampling. CONCLUSIONS: An optimized method for the quantitative determination of acetaldehyde in biological systems was developed using derivatization with DNPH, followed by a short RP-HPLC separation of AcH-DNP. The method has an extended linear range, is reproducible and applicable to small-volume sampling of culture media and biological fluids.

A highly selective and sensitive in vivo fluorosensor for zinc(II) without cytotoxicity.

A highly selective and sensitive fluorescent Zn(2+) sensor, 2,6-bis(2-hydroxy-benzoic acid hydrazide)-4-methylphenol (1), was designed and synthesized. In aqueous THF (4 : 6 v/v) ligand 1 induces a 2 : 1 complex formation with respect to Zn(2+) at physiological pH. This probe features visible light excitation(390 nm) and emission (490 nm) profiles, excellent selectivity responses for Zn(2+)over other competing biological metal ions with K(d) < 1 pM(2), LOD < 1 ng L(-1) and about 680 fold enhancement in fluorescent intensity upon Zn(2+) binding. It also exhibits cell permeability and intracellular Zn(2+) sensing in A375 human melanoma cancer cell.

[Bronchial hyperresponsiveness and factors of bronchial inflammation under the exercise-induced asthma in schoolchildren].

88 schoolchildren with bronchial asthma were observed at the department of pulmonary disease and allergology of the Chernivtsi regional children clinical hospital. The study showed that the content of aldehyde- and ketoderivatives of 2,4-dinitrophenylhydrazones of neutral character in the expired air condensate (AKDNPH) was presumably higher in patients with the exercise-induced bronchial asthma thus indicating the more significant activity of the inflammatory process in bronchi under the studied phenotype of the disease. Non-specific bronchial hyperresponsiveness was presumably higher in children suffering exercise-induced bronchial asthma due to bronchial hyperresponsiveness as well as hyperreactivity when compared with the patients having activity independent asthma attacks. The factor of bronchial spasm was significantly associated with the content of AKDNPH of the basic character and nitric oxide metabolites in the expired air condensate in the patients with the exercise-induced bronchial asthma.

HPLC-DAD and MS/MS analysis of novel drug candidates from the group of aromatic hydrazones revealing the presence of geometric isomers.

Salicylaldehyde isonicotinoyl hydrazone (SIH) is an iron-chelating aromatic hydrazone with promising pharmacological properties. However, it suffers from relatively short biological half-life. Hence, two novel derivates of SIH, HAP-INH and HPP-INH were synthesized in order to overcome this pharmacokinetic drawback. The aim of the present study was to employ HPLC-DAD and HPLC-MS/MS methods to investigate the identity of the putative impurities of these newly prepared substances, which are being formed in aqueous environment. At first, it was shown that their retention times as well as UV spectra did not correspond to any expected synthetic precursor, by-product or degradation product. HPLC-DAD analysis confirmed purity of peaks and revealed close but not identical UV spectra of putative impurities and corresponding hydrazones. The subsequent HPLC-MS/MS analyses using ESI and the ion trap mass analyzer showed the identical molecular ions (in both modes) as well as their fragmentation, which implicated presence of geometric isomers. This suggestion was further supported by the NMR analyses. Since the Z/E isomers can have different biological activities, results of this study might be of great importance for further development of the aroylhydrazones as novel drug candidates as well as from the theoretical point of view.

Glyoxal sample preparation for high-performance liquid chromatographic detection of 2,4-dinitro-phenylhydrazone derivative: Suppression of polymerization and mono-derivative formation by using methanol medium.

Some difficulties on a sampling of gaseous glyoxal using DNPH-silica cartridge were discussed, and an alternative sampling procedure was proposed. When glyoxal was sampled using the cartridge, it partially formed mono-hydrazone with various degrees, whereas it was quantitatively converted into its bis-hydrazone when sample gas was directly bubbled into a DNPH acidic solution. Additionally, glyoxal polymerized on the inner wall of the system during trapping to the silica cartridge. We found that the polymerization of glyoxal was effectively suppressed by dissolving glyoxal into methanol under reduced pressure; glyoxal in methanol readily reacted with DNPH in a hydrochloric acid solution and gave bis-hydrazone derivative, without any interference of methanol and any formation of mono-hydrazone derivative. When glyoxal was sampled by the proposed method, 102% of recovery was obtained, whereas it was reduced to 92.7% when the trapped glyoxal was dissolved into methanol under atmosphere.

Kinetic spectrophotometric determination of ethamsylate in dosage forms.

A simple and sensitive kinetic method has been developed for the determination of ethamsylate (ESL) in its pharmaceutical preparations. The method is based upon oxidation of ESL with 3-methyl-2-benzothiazolinone hydrazone hydrochloride in presence of cerium (IV) ammonium sulfate at room temperature for 20 min. The absorbance of the reaction product is measured at 514 nm. The absorbance-concentration plot was rectilinear over the range of 4-30 microg/mL (r = 0.9999). The lower detection limit was 0.267 microl/mL (9.110 x 10(-6) M) and the lower quantitation limit was 0.808 microg/mL. The different experimental parameters affecting the development and stability of the reaction product were studied and optimized. The proposed method was applied to the determination of ESL in formulations, and the results obtained were in good agreement with those obtained using a reference method. The proposed method was also used for the in vitro detection of ESL in spiked human plasma at its therapeutic concentration level.

Analysis of oxysterols by electrospray tandem mass spectrometry.

Oxysterols are oxygenated derivatives of cholesterol. They are intermediates in cholesterol excretion pathways and may also be regarded as transport forms of cholesterol. The introduction of additional hydroxyl groups to the cholesterol skeleton facilitates the flux of oxysterols across the blood brain barrier, and oxysterols have been implicated in mediating a number of cholesterol-induced metabolic effects. Oxysterols are difficult to analyze by atmospheric pressure ionization mass spectrometry on account of the absence of basic or acidic functional groups in their structures. In this communication, we report a method for the derivatization and analysis of oxysterols by electrospray mass spectrometry. Oxysterols with a 3beta-hydroxy-Delta5 structure were converted by cholesterol oxidase to 3-oxo-Delta4 steroids and then derivatized with the Girard P reagent to give Girard P hydrazones, which were subsequently analyzed by tandem mass spectrometry. The improvement in sensitivity for the analysis of 25-hydroxycholesterol upon oxidation and derivatization was over 1000.

1H, 13C, NH, HH, CH COSY, HH NOESY NMR and UV-vis studies of Solophenyl red 3BL dye azo-hydrazone tautomerism in various solvents.

Azo-hydrazone tautomeric behavior of polyazo Solophenyl red 3BL (C.I. Direct 80) dye in different solvents (water, methanol and DMSO) was investigated using 1H, 13C, NH, HH, CH COSY, HH NOESY NMR techniques and UV-vis spectroscopy. Two-dimensional NMR experiments were used to assign 1H, 13C and 15N NMR lines unambiguously. Results showed that the hydrazone-form proton NMR signal appeared in the weakest field with respect to tetramethylsilane, in comparison with the amide and phenolic proton NMR signals. UV-vis absorption spectroscopic evidences showed that azo-hydrazone mixture exists in water and DMSO solvents, but in methanol, only azo tautomer was dominant, which was in a good agreement with NMR spectroscopic results.

Airborne aldehydes in cabin-air of commercial aircraft: Measurement by HPLC with UV absorbance detection of 2,4-dinitrophenylhydrazones.

This paper presents the strategy and results of in-flight measurements of airborne aldehydes during normal operation and reported "smell events" on commercial aircraft. The aldehyde-measurement is a part of a large-scale study on cabin-air quality. The aims of this study were to describe cabin-air quality in general and to detect chemical abnormalities during the so-called "smell-events". Adsorption and derivatization of airborne aldehydes on 2,4-dinitrophenylhydrazine coated silica gel (DNPH-cartridge) was applied using tailor-made sampling kits. Samples were collected with battery supplied personal air sampling pumps during different flight phases. Furthermore, the influence of ozone was investigated by simultaneous sampling with and without ozone absorption unit (ozone converter) assembled to the DNPH-cartridges and found to be negligible. The method was validated for 14 aldehydes and found to be precise (RSD, 5.5-10.6%) and accurate (recovery, 98-103 %), with LOD levels being 0.3-0.6 µg/m(3). According to occupational exposure limits (OEL) or indoor air guidelines no unusual or noticeable aldehyde pollution was observed. In total, 353 aldehyde samples were taken from two types of aircraft. Formaldehyde (overall average 5.7 µg/m(3), overall median 4.9 µg/m(3), range 0.4-44 µg/m(3)), acetaldehyde (overall average 6.5 µg/m(3), overall median 4.6, range 0.3-90 µg/m(3)) and mostly very low concentrations of other aldehydes were measured on 108 flights. Simultaneous adsorption and derivatization of airborne aldehydes on DNPH-cartridges to the Schiff bases and their HPLC analysis with UV absorbance detection is a useful method to measure aldehydes in cabin-air of commercial aircraft.

Influence of the labeling group on ionization and fragmentation of carbohydrates in mass spectrometry.

The ionization and fragmentation behaviors of carbohydrate derivatives prepared by reaction with 2-aminobenzamide (AB), 1-phenyl-3-methyl-5-pyrazolone (PMP), and phenylhydrazine (PHN) were compared under identical mass spectrometric conditions. It has been shown that the intensities of signals in MS spectra depend on the kind of saccharides investigated and reducing end labels used. PMP sialyllactose, when ionized by ESI/MALDI, produced a mixture of [M + H]+, [M + Na]+, [M - H + 2Na]+ ions in the positive mode and [M - H]-, [M + Na - 2H]- ions in the negative mode. The AB and PHN derivatives formed abundant [M + H]+ and [M - H]- ions in ESI, and by matrix-assisted laser desorption/ionization (MALDI) produced abundant [M + Na]+ ions. PMP- and reduced AB-sialyllactose produced only Y-type fragment ions under both MS/MS sources. In the electrospray ionization (ESI)-MS/MS spectrum of PHN-sialyllactose, abundant ions corresponded to B, Z cleavages and in its MALDI-MS/MS spectrum, the abundant ions were consistent with Y glycosidic cleavages with the concurrence of B, C, and cross-ring fragment ions. In the MALDI-MS spectra of oligosaccharides acquired immediately after derivatization, it was possible to detect only PHN derivatives. After purification, spectra of all three types of derivatives showed high signal-to-noise ratios with the most abundant ions observed for AB reduced saccharides. [M + Na]+ ions were the dominant products and their fragmentation patterns were influenced by the type of the labeling and the kind of oligosaccharide considered. In the MALDI-PSD and -MS/MS spectra of AB-derivatized glycans, higher m/z fragment ions corresponded to B and Y cleavages and the loss of bisecting GlcNAc appeared as a weak signal or was not detected at all. Fragmentation patterns observed in the spectra of hybrid/complex PHN and PMP glycans were more comparable-higher m/z fragments corresponded to B and C glycosidic cleavages. For PHN glycans, the abundance of ions resulting from the loss of bisecting GlcNAc depended on the number of residues linked to the 6-positioned mannose. Also, PHN and PMP derivatives produced cross-ring cleavages with abundances higher than observed in the spectra of AB derivatized oligosaccharides. For high-mannose glycans, the most informative cleavages were provided by AB and PHN type of labeling. Here, PMP produced dominant Y-cleavages from the chitobiose while other ions produced weak signals.

LC-UV/MS methods for the analysis of prochelator-boronyl salicylaldehyde isonicotinoyl hydrazone (BSIH) and its active chelator salicylaldehyde isonicotinoyl hydrazone (SIH).

Salicylaldehyde isonicotinoyl hydrazone (SIH) is an intracellular iron chelator with well documented potential to protect against oxidative injury both in vitro and in vivo. However, it suffers from short biological half-life caused by fast hydrolysis of the hydrazone bond. Recently, a concept of boronate prochelators has been introduced as a strategy that might overcome these limitations. This study presents two complementary analytical methods for detecting the prochelator-boronyl salicylaldehyde isonicotinoyl hydrazone-BSIH along with its active metal-binding chelator SIH in different solution matrices and concentration ranges. An LC-UV method for determination of BSIH and SIH in buffer and cell culture medium was validated over concentrations of 7-115 and 4-115 µM, respectively, and applied to BSIH activation experiments in vitro. An LC-MS assay was validated for quantification of BSIH and SIH in plasma over the concentration range of 0.06-23 and 0.24-23 µM, respectively, and applied to stability studies in plasma in vitro as well as analysis of plasma taken after i.v. administration of BSIH to rats. A Zorbax-RP bonus column and mobile phases containing either phosphate buffer with EDTA or ammonium formate and methanol/acetonitrile mixture provided suitable conditions for the LC-UV and LC-MS analysis, respectively. Samples were diluted or precipitated with methanol prior to analysis. These separative analytical techniques establish the first validated protocols to investigate BSIH activation by hydrogen peroxide in multiple matrices, directly compare the stabilities of the prochelator and its chelator in plasma, and provide the first basic pharmacokinetic data of this prochelator. Experiments reveal that BSIH is stable in all media tested and is partially converted to SIH by H2O2. The observed integrity of BSIH in plasma samples from the in vivo study suggests that the concept of prochelation might be a promising strategy for further development of aroylhydrazone cytoprotective agents.

Identification of cholesterol-bound aldehydes in copper-oxidized low density lipoprotein.

Lipid-soluble cholesteryl ester core aldehydes (aldehydes still bound to the cholesterol ring) were identified among the products of copper-catalyzed peroxidation of human low density lipoprotein (LDL). The LDL was exposed to oxygenated buffer and 5 microM CuSO4 for 24 h. The core aldehydes were isolated as the dinitrophenylhydrazones, and were identified by reverse-phase HPLC with mass spectrometry. The major components were the C4-C10 oxoalkanoyl esters of cholesterol and 7-ketocholesterol, and accounted for 1-2% of the cholesteryl linoleate and arachidonate consumed.

Detecting and identifying volatile aldehydes as dinitrophenylhydrazones using gas chromatography mass spectrometry.

The detection of aldehydes has become an important measure of lipid oxidation in biological milieu. Aldehyde 2,4-dinitrophenylhydrazones are easily prepared and readily purified by HPLC and/or TLC and have proven useful for the detection of aldehydes. The lower limit of detection for dinitrophenylhydrazones was significantly reduced by using gas chromatography-mass spectrometric (GC-MS) techniques. Individual dinitrophenylhydrazones were readily separated by GC and detected by both positive and negative ion MS. The two major ions in negative ion spectra were the 182 m/z fragment ion and the molecular ion. Positive ion spectra showed strong ions corresponding to the protonated molecular ion and a protonated iminium ion. The greatest sensitivity was obtained with negative ion detection (10 pg per injection). However, more structural information was obtained from analysis of the positive ion spectra. Dinitrophenylhydrazones of hydroxyaldehydes, like 4-hydroxynonenal, were analyzed after converting the dinitrophenylhydrazones into trimethylsiloxylethers. GC-MS with negative ion detection was used to identify and quantitate the release of 4-hydroxynonenal by alveolar macrophages exposed to nitrogen dioxide.

High-performance liquid chromatographic determination of plasma glycolic acid in healthy subjects and in cases of hyperoxaluria syndromes.

A liquid chromatographic procedure for the determination of glycolic acid in plasma is proposed. The system is based on pre-column derivatization of the alpha-keto acid by means of phenylhydrazine, coupled with the enzymatic oxidation of glycolate to glyoxylate. The phenylhydrazone formed is separated by reversed-phase liquid chromatography and detected by UV absorption. The measured within and between-batch CV imprecision was 2.6 and 11.3%, respectively, at 5.68 mumol/l glycolate concentration; the analytical recovery was 102.0 +/- 7.3% and the minimum detectable concentration of glycolate was 0.3 mumol/l. The reference interval for plasma glycolate was 4.51 to 12.20 mumol/l (n = 14). Results of determinations of plasma samples from uremic patients, patients with type I primary hyperoxaluria and patients with chronic renal failure secondary to systemic oxalosis are reported.

Qualitative and quantitative high-performance liquid chromatographic analysis of aldehydes in Brazilian sugar cane spirits and other distilled alcoholic beverages.

A study is presented on the high-performance liquid chromatographic analysis of eighteen aldehydes in Brazilian sugar cane spirits and other international brandies. The aldehydes were separated by reversed-phase high-performance liquid chromatography as 2,4-dinitrophenylhydrazones (DNPHs). A very good chromatographic separation was achieved for eighteen different aldehyde-DNPHs. The proposed methodology is quite simple and not very time-consuming. Ten aldehydes were identified in 75 beverages and quantified using the external standard method with UV detection at 365 nm. A detailed knowledge of the aldehyde content should significantly contribute to improving the quality control of distilled spirits.

Spectrophotometric determination of isoniazid in presence of its hydrazones.

A spectrophotometric determination of isoniazid in the presence of its hydrazones was developed. The method involves the reaction between isoniazid and 2,3-dichloro-1,4-naphthoquinone in the presence of ammonia in an ethanolic medium. The colored product has an absorbance maxium at 640 nm. The Lambert-Beer law is obeyed in the 1--14-microgram/ml range. The proposed method was applied to the analysis of isoniazid tablets. In commercial tablets, hydrazone formation due to the reaction between isoniazid and lactose was detected by TLC. The analysis of lactose-containing isoniazid tablets showed 10--22% lower recovery than that obtained by the official method. Hydrazone formation in tablets probably interferes with isoniazid bioavailability.

In vitro kinetic studies of the reaction of hydralazine and its acetone hydrazone with pyruvic acid.

To understand the reaction between hydralazine (HP) or its acetone hydrazone (HAH), a metabolite of HP and pyruvic acid, a new selective HPLC method for simultaneous determination of HP, HAH, and hydralazine pyruvic acid hydrazone (HPH) was developed. In vitro degradation of HAH and formation of HP and HPH were investigated at pH 7.4 and 37 degrees C in the presence or absence of pyruvic acid. Hydralazine degraded slowly according to an apparent first-order rate (7.46 x 10(-2)h-1). The degenerative reaction of HAH, accompanied by simultaneous hydrolysis to the parent drug HP, was also subject to apparent first-order loss (3.00 x 10(-1)h-1). In addition, HAH was partly converted to HP and HPH in the presence of pyruvic acid. For the formation pathway of HPH, a model that included the direct reaction of HAH with pyruvic acid and the secondary formation mediated by back-conversion to HP gave a better fit to the experimental data than the model consisting of the latter reaction only. About 10% of the HPH formed was generated by the direct reaction of HAH with pyruvic acid, based on the rate constants estimated. These results suggest that the formation of HPH is not all accomplished through back-conversion to HP.

Determination of lipid ester ozonides and core aldehydes by high-performance liquid chromatography with on-line mass spectrometry.

Unsaturated triacylglycerols (TG) and choline (PC) and ethanolamine (PE) phosphatides of known structure were subjected to ozonization and reduction with triphenylphosphine to yield the corresponding lipid ester core aldehydes. Mono- and di-C9 aldehyde palmitoylglycerols were prepared from oleoyldipalmitoyl and oleoyllinoleoylpalmitoyl glycerols, respectively, while egg yolk PC and PE provided the mono-C5 and mono-C9 aldehydes of palmitoyl-and stearoyl glycerophospholipids. The aldehydes were isolated in the free form and as the dinitrophenylhydrazone (DNPH) derivatives by thin-layer chromatography (TLC). The intermediate ozonides, free aldehydes and hydrazones were identified by reversed phase high performance liquid chromatography (HPLC) with on-line negative ion thermospray and normal phase HPLC with on-line positive ion electrospray mass spectrometry (LC-MS). The synthetic aldehydes were used as carriers during isolation from natural sources and as reference compounds in quantitative analyses.