Validation of Ultraviolet-visible and High-performance Liquid Chromatographic Methods for the Determination of Sodium p-Aminosalicylate and m-Aminophenol in a New Pharmaceutical Formulation.

Sodium p-aminosalycilate is an orphan drug used in patients affected with Multidrug-resistant Tuberculosis. Two methods, high-performance liquid chromatographic and ultraviolet spectrophotometric for the quantitative determination of sodium p-aminosalycilate and its degradation product m-aminophenol in a new pharmaceutical formulation, powder for extemporaneous reconstitution, were developed in the present work. The parameters linearity, precision, accuracy, specificity, robustness, limit of detection, and limit of quantification were also studied. Chromatography was carried out by reverse-phase technique on an RP-18 column with a mobile phase composed of 50 mM monobasic/dibasic phosphate buffer and methanol (42.5:42.5:15 v/v/v) with 1.9 g of hidroxytetrabutylammonium ionic pare adjusted to pH 7.0 with orthophosphoric acid. The ultraviolet spectrophotometric method was performed at 254 nm and 280 nm for quantification of sodium p-aminosalycilate and m-aminophenol, respectively. The proposed methods are highly sensitive, precise, and accurate and can be used for the reliable quantification of sodium p-aminosalycilate in the new alternative formulation. High-performance liquid chromatographic approach demonstrated to be a stability-indicating method, therefore suitable for the investigation of the chemical stability of sodium p-aminosalycilate.

Preparation and characterisation of novel spray-dried nano-structured para-aminosalicylic acid particulates for pulmonary delivery: impact of ammonium carbonate on morphology, chemical composition and solid state.

OBJECTIVES: The objective of this work was to spray dry p-aminosalicylic acid (PAS) and its ammonium salt and to investigate the impact of the pore-forming agent, ammonium carbonate (AC), on the morphological, aerodynamic and physicochemical properties of the resulting powders. METHODS: Microparticles were prepared by spray drying from ethanol/water solvent systems. Their solid-state properties were evaluated by scanning electron microscopy, powder X-ray diffraction, differential scanning calorimetry, thermogravimetric analysis and in-vitro deposition, using the twin impinger. KEY FINDINGS: The physicochemical properties of PAS were altered on spray drying with AC and a new solid state was produced. The solution composition impacted on the morphology of the resulting powders, which ranged from irregular crystal agglomerates to spherical crystal clusters and porous microparticles. The chemical composition, structure and morphology were dependent on process inlet temperature, low inlet temperatures resulting in a novel solid of stoichiometry; PAS : ammonia : water, 2 : 1 : 0.5. At higher temperatures pure PAS was obtained. In-vitro deposition studies showed an increase in emitted dose from spray dried drug, relative to the micronised PAS. CONCLUSIONS: Under appropriate process conditions AC interacts with the acidic PAS, resulting in the formation of a novel solid-state drug phase. Spray-dried PAS powders have potential for pulmonary delivery.

HPLC analysis of para-aminosalicylic acid and its metabolite in plasma, cerebrospinal fluid and brain tissues.

Para-aminosalicylic acid (PAS), an approved drug for treatment of tuberculosis, is a promising therapeutic agent for treatment of manganese (Mn)-induced parkinsonian syndromes. Lack of a quantifying method, however, has hindered the clinical evaluation of its efficacy and there upon new drug development. This study was aimed at developing a simple and effective method to quantify PAS and its major metabolite, N-acetyl-para-aminosalicylic acid (AcPAS), in plasma, cerebrospinal fluid (CSF) and tissues. Biological samples underwent one-step protein precipitation. The supernatant was fractionated on a reversed-phase C18 column with a gradient mobile system, followed by on-line fluorescence detection. The lower limits of quantification for both PAS and AcPAS were 50 ng/ml of plasma and 17 ng/g of tissues. The intra-day and inter-day precision values did not exceed 5% and 8%, respectively, in all three matrices. The method was used to quantify PAS and AcPAS in rat plasma and brain following a single iv injection of PAS. Data showed a greater amount of PAS than AcPAS in plasma, while a greater amount of AcPAS than PAS was found in brain tissues. The method has been proven to be sensitive, reproducible, and practically useful for laboratory and clinical investigations of PAS in treatment of Mn Parkinsonism.

Quantitative determination of p-aminosalicylic acid and its degradation product m-aminophenol in pellets by ion-pair high-performance liquid chromatography applying the monolithic Chromolith Speedrod RP-18e column.

An ion-pair high performance liquid chromatographic method was developed for the simultaneous determination of p-aminosalicylic acid (PAS) and its degradation product m-aminophenol (MAP) in a newly developed multiparticular drug delivery system. Owing to the concentration differences of PAS and MAP, acetanilide and sulfanilic acid were used as internal standards, respectively. The separation was performed on a Chromolith SpeedROD RP-18e column, a new packing material consisting of monolithic rods of highly porous silica. The mobile phase composition was of 20 mm phosphate buffer, 20 mm tetrabutylammonium hydrogen sulphate and 16% (v/v) methanol adjusted to pH 6.8, at a flow-rate of 1.0 mL/min, resulting in a run-time of about 6 min. Detection was by UV at 233 nm. The method was validated and proved to be useful for stability testing of the new dosage form. Separation efficiency was compared between the new packing material Chromolith SpeedROD RP-18e and the conventional reversed-phase cartridge LiChroCART 125-4 (5 microm). A robustness test was carried out on both columns and different separation parameters (retention, resolution, run time, temperature) were determined.

Genetic loci contributing to age-related hippocampal lesions in mice.

C57BL/6J mice develop genetically determined age-related hippocampal granular deposits that have some similarities to lesions seen in the brains of human patients with tau protein related neurodegenerative disorders ("tauopathies"). We sought to identify the genetic loci responsible for these in an F2 intercross of inbred mouse strains C57BL/6J and DBA/2J, using quantitative trait locus (QTL) analysis. Hippocampal lesions were shown to be PAS positive, H and E negative, and immunoreactive for tau protein and alpha synuclein, but not to Abeta 1-40 or Abeta 1-42, or for ubiquitin. These were quantitated by histomorphometry, and QTL analysis revealed a locus on chromosome 7 with a lod score of 6.5 as well as two suggestive loci on chromosome 10. The genomic data indicate that the genetic basis is complex, but with one locus playing a major role in lesion formation. These lesions may represent a useful model for investigating dysregulation of tau protein in the hippocampus.

Apparatus and method for interfacing capillary electrophoresis and chemiluminescence nitrogen detection for the analysis of nitrogen-containing compounds.

We have developed an interface that allows the specific detection of nitrogen-containing compounds by using a chemiluminescence nitrogen detector. The feasibility of using this interface was demonstrated by separating and detecting two nitrogen-containing compounds, p-aminosalicylic acid and L-phenylalanine. Although baseline separation was achieved, the theoretical plates were lower when compared to UV detection (25000 vs. approximately 85000). A sensitivity of 75 ng (approximately 500 pmol) per injection was achieved with this system which is adequate for pharmaceutical and biotech applications.

[Application of charge transfer reaction in the determination of aminosalicylate sodium].

A spectrophotometric method for the determination of aminosalicylate sodium has been developed based on the charge transfer reaction. The Beer's law is obeyed in the range of 40-160 microg/mL of aminosalicylate sodium. The apparent molar absorptivity of the complex at 660nm is 1.17 X 10(3)L x mol(-1). The average standard deviation of ten determination is 0.67%. The composition of aminosalicylate sodium 2, 6-Dichloroquinone chlorimide complex is 1:1. The proposed method has been applied to the determination of aminosalicylate sodium tablets with satisfactory results.

Controlled trial of 4-ASA in ulcerative colitis.

A six-week placebo-controlled trial of the efficacy and safety of 6 g per day of 4-aminosalicylic acid (4-ASA) was conducted in 30 subjects with mild to moderately severe ulcerative colitis. Subjects were stratified into groups having distal (< 60 cm) or more extensive (> 60 cm) disease. Diarrhea, bleeding, sigmoidoscopic and biopsy appearance, and physician global assessment were scored to judge efficacy. Safety was evaluated by monitoring untoward symptoms and laboratory values. Median percent improvement was significantly greater (P < 0.05) in the 4-ASA > 60-cm group (42.7%) than in the placebo > 60-cm group (21.2%), but 4-ASA was not better than placebo for the < 60-cm group or the total study group. Severe dyspepsia (one subject), abnormal AST (transient in five, persistent in one) and elevated lipase without pancreatitis (six subjects) were noted. Thus 6 g 4-ASA for six weeks was more effective than placebo in mild to moderate ulcerative colitis extending more than 60 cm above the anus, but not in distal disease, and the drug was generally well tolerated.

Simple spectrofluorometric determination of p-aminobenzoic and p-aminosalicylic acids in biological fluids by use of terbium-sensitized luminescence.

A novel, sensitive, and selective method has been developed for determination of p-aminobenzoic (PABA) and p-aminosalicylic (PAS) acids in the N-benzoyl-L-tyrosyl-PABA/ PAS test. PAS is measured as a ternary complex with terbium and EDTA (lambda(ex) = 324 nm, lambda(em) = 546 nm) in alkaline aqueous solution (pH approximately 12.6), whereas both compounds (PABA and PAS) are measured as ternary complexes with terbium and tri-n-octylphosphine oxide (lambda(ex) = 292 nm, lambda(em) = 546 nm) in weakly acidic aqueous solution (pH approximately 5.5). We inve stigated and implemented optimum conditions for formation of these complexes, yielding respective detection limits for PABA and PAS of 0.07 and 0.02 micromol/L and ranges of application of 0-10 and 0-40 micromol/L (final concentration). The method has been successfully applied to determinations of PABA and PAS in urine and, after alkaline hydrolysis, to determinations of PABA in serum that has been deproteinized with acetonitrile. Within-run imprecision of the PABA determination ranges from 0.8% to 4.2 % for urine samples and from 3.9% to 8.2% for serum samples; day-to-day imprecision varies from 3.2% to 10% for serum samples.

Determination of isoniazid in pharmaceutical preparations by reaction with radiochloramine-B.

A method has been developed for the determination of isoniazid (INH) at microgram level in pharmaceutical preparations based on the oxidation of isoniazid by radiochloramine-B. Interference of vitamin C has been overcome by precipitation as lead ascorbate and that of rifampicin and p-aminosalicylic acid by selective extraction. Amounts as low as 25 micrograms of INH can be determined.

Mechanism of decarboxylation of p-aminosalicylic acid.

The rate of decarboxylation of p-aminosalicylic acid (1) in aqueous solutions was studied at 25 degrees C (mu = 0.5) as a function of pH and buffer concentration. A pH-rate profile was generated by using the rate constants extrapolated to zero buffer concentration. The profile was bell-shaped, with the maximum rate of decarboxylation near the isoelectric pH. The rate constants obtained in buffered solutions indicated general acid catalysis. Bronsted behavior appeared to be adhered to. The two ionization constants of 1 were determined spectrophotometrically at 25 degrees C and at an ionic strength of 0.5. An HPLC method was used to characterize the degradation products of the reaction. Kinetic solvent deuterium isotope effects were studied to further confirm the mechanism of decarboxylation. Below pH 7.0, the mechanism of 1 decarboxylation is the rate controlling proton attack on the carbon-alpha to the carboxylic acid group of 1 anion and the ampholyte, followed by the rapid decarboxylation of the formed intermediate.

Age-related changes in the gastric submucosal arterioles and gastric acid secretion.

The purpose of this study was to determine whether alterations in the reactivity of gastric submucosal arterioles to norepinephrine and changes in acid secretion occur in the same time period in the rat. Experiments were performed on animals 6 weeks and 10, 18, and 25 months of age. Arteriolar diameter was measured by an image splitting in vivo microscopy technique before and after superfusion of the gastric submucosa with graded doses of norepinephrine. Acid secretion was measured in the pylorus-ligated rat. The percentage of arteriolar media stained with periodic acid-Schiff reagent (an age-related change) was determined for each age group. A dose-dependent response to norepinephrine was observed in 6-week and 10-, 18-, and 25-month age groups. Only in the 25-month-old rats were arterioles significantly more responsive to norepinephrine. Acid secretion at 10, 18, and 25 months was significantly less (about one-quarter) than that observed in the 6-week-old animals. Periodic acid-Schiff positive material deposited in the arteriolar media was significantly greater in the 18- and 25-month-old rats than in younger animals. We conclude that by 10 months of age in the rat, acid secretion is markedly reduced. The reactivity of the gastric submucosal arterioles is significantly increased at 25 months, but in the 18-month-old animal, when increased periodic acid-Schiff deposits are clearly evident, the reactivity to norepinephrine is not different from the 6-week-old rat.

Normal and abnormal development of the epididymis of the fetus and infant.

The fetal epididymis shows a weak periodic acid Schiff (PAS)-positive secretion by the 25th week of gestation which gradually increases during the following weeks and reaches its peak in the later weeks of pregnancy. It decreases again after the 3rd month of life. The organ continues to secrete throughout childhood, although in a much reduced degree. It can be assumed that androgens, produced by the fetus in response to stimulation by maternal and placental hormones, are responsible for the secretion of the organ during fetal life. The normal development and function of the epididymis in the fetus and infant can be impaired in the presence of various congenital malformations. Hormonal therapy to the mother during pregnancy may also affect the organ. However, it is possible that in the absence of any serious structural damage to the organ, the purely functional changes may only be of a temporary nature and not detrimental to fertility in later life.