Towards harmonization of solutions used for cystic fibrosis diagnosis by nasal potential difference measurements: A formulation approach with CHESS® software.

BACKGROUND: One diagnosis of cystic fibrosis involves measuring the nasal transepithelial potential difference (NPD) as a complementary technique in the forms of the disease, where the sweat test is non-discriminating. The NPD is measured using solutions with and without chlorides, containing a variety of substances whose activities on nasal mucus membranes are studied or assessed. Among the solutions described in the literature and used in specialized centers, none seems to be best adapted for industrial production for reasons of stability (formulas of the international consensus of Rowe et al. and formulas of Knowles et al.) and/or potential toxicity (formulas of Middleton et al.). OBJECTIVE(S): Defining new formulas, according to those of the international consensus, with greater physicochemical and microbiological stability. METHODS: The reformulation tests were conducted on the formulas of Rowe et al., using CHESS® (CHemical Equilibrium of Species and Surfaces) software for modeling aqueous systems that substantially reduced the number of experiments. CHESS® software was first validated using models of ideal and non-ideal solutions. Thereafter, experimentation was carried out for the sake of comparison with theoretical data. RESULTS: CHESS® software using models of ideal and non-ideal solutions were validated. The experimentation confirmed the theoretical data, and new formulas were assessed based on their physicochemical (pH, content, Osmolality) and microbiological stability. CONCLUSION: The new formulas defined here guarantee excellent physicochemical and microbiological stability of diagnostic solutions, indispensable criteria for harmonizing and comparing results from different specialized centers using NPD measurements. These new formulas apply to the harmonization approach of techniques for measuring the nasal transepithelial potential difference.

Optimising concentrations of antimicrobial agents in pharmaceutical preparations: Case of an oral solution of glycerol and an ophthalmic solution containing cysteamine.

INTRODUCTION: In the context of current distrust of antimicrobial preservatives, the quantities of these substances in two pharmaceutical formulas were studied: an ophthalmic solution of cysteamine preserved benzalkonium chloride at 1mg/5mL and Glycerotone(®) preserved with sorbic acid at 0.1g/100g. The purpose of this work was to verify that a reduction of the quantities of preservative continues to fulfil the requirements for antimicrobial preservation. MATERIAL AND METHODS: The Test of efficacy of antimicrobial preservation, section 5.1.3 of the 8th edition of the European Pharmacopoeia, was carried out on each formulation prepared with decreasing quantities of preservative. RESULTS: The results show that formulations whose preservative concentration was reduced by a factor of four remained compliant with standards. It is to be noted that in formulas without preservative, fungal growth was observed in both the solution of Glycerotone(®) and the ophthalmic solution containing cysteamine. DISCUSSION: Although there is no question that an antimicrobial preservative is necessary, the quantity of preservative can be reduced without deteriorating the quality of the pharmaceutical product but the minimal effective concentration remains to be determined. CONCLUSION: The formulations of many pharmaceutical products should therefore be examined in order to limit the quantities of preservative while continuing to guarantee patient's safety.

[Solutions for organ preservation and other cardioplegic liquid formulations. Role of the hospital pharmacist]. / Solutions pour conservation d'organes et pour cardioplégie. Le rôle du pharmacien hospitalier.

Solid organ transplantation is an increasing need and a well-established activity which requires maintaining the quality of the transplant from procurement through the entire, storage, transport and graft procedure. Solutions for organ preservation play a key role in this procedure, by minimizing the deleterious effects of both ischemia and reperfusion. As such, their qualitative and quantitative compositions have to be optimized and validated. The development strategy and formulations proposed for these solutions are analyzed in this review as well as the results of the clinical studies which have set up the relevant pharmacological and physicochemical criteria. The French regulatory status of these products is also discussed. A clear distinction has to be made between solutions for organ preservation which are classified as produits thérapeutiques annexes (therapeutic ancillary products) and cardioplegic liquid formulations which are considered as medicinal products and are subject to marketing approval. Finally, the roles of the hospital pharmacist in the evaluation, selection, purchase and proper use of these products are described.

Sensitive quantification of diphemanil methyl sulphate in human plasma by liquid chromatography-tandem mass spectrometry.

A simple detection system with a high-performance liquid chromatography (HPLC) with positive ionisation-tandem mass spectrometry (ESI-MS/MS) for determining diphemanil methylsulphate (DMS) levels in human plasma using 4-diphemanylmethylene,1-methylpiperidine as an internal standard (I.S.), is proposed. The acquisition was performed with the multiple reactional monitoring (MRM) mode, by monitoring the transitions: m/z 278>262 for DMS and m/z 263>247 for the I.S. The method involved a simple single-step deproteinisation with acetonitrile. The analyte was chromatographed on a Zorbax C18 reversed-phase chromatographic column by isocratic elution with 10(-3)M ammonium acetate and 10(-3)M hexafluorobutyric acid, adjusted to pH 7.0 with ammoniac/acetonitrile (40/60, v/v). The results were linear over the studied range (0.5-50.0 ng mL(-1)) and the total analysis time for each run was 10 min. The mean extraction apparent recoveries expressed at the 95% intervals of confidence were 94-104% for DMS and 92-106% for the I.S. The intra- and inter-assay precisions were 4.6-8.4% and 2.9-10.6%, respectively. The limit of quantification was 0.15 ng mL(-1). The devised assay was successfully applied to the residual concentrations monitoring in infant.

Stability studies of ionised and non-ionised 3,4-diaminopyridine: hypothesis of degradation pathways and chemical structure of degradation products.

3,4-Diaminopyridine is used to treat some symptoms met in Lambert-Eaton myasthenia syndrome. It was shown efficient to reduce a form of variable muscle weakness and fatigability typical of the disease and correlated to a block of acetylcholine release. In France, 3,4-diaminopyridine is nowadays given to patients under capsules form and the status of hospital preparation. Whatever the diluant used in the formulation, the stability period could not exceed 12 months. Preliminary studies were made on a salt form in order to test the influence of various stress factors and determine if there is interaction between them. From this study, the most influent stress condition, presence of hydrogen peroxide, was selected and a comparative study was performed to compare the stability of molecular and salt species. Solutions of each species were exposed to 5 or 15% of hydrogen peroxide and analyzed at 8, 24, 72 and 216 h of degradation by HPLC-UV. Fractions of detected impurities were purified and collected by semi-preparative HPLC-UV and analyzed by HPLC-UV-ESI-MS and IR spectroscopy in order to determine their structure hypotheses. Theses experiments demonstrate that the salt species were more stable under oxidative stress condition than molecular species. The two main degradation products were collected and identified as 4-amino, 3-nitropyridine and 3,4-diaminopyridine-N-oxide when the molecular form was degraded whereas only 4-amino, 3-nitropyridine was found in less quantity in the salt solutions. Nitrogen pyridine and pyridine amine could not easily be oxidized by hydrogen peroxide in salt comparatively to molecular species due to the lone pair of electron engaged in a bound with hydrogen in the first case and by resonance change of the pyridine in the second case. This modification of structure promoted different pathways of degradation for the salt form which are more dependent of energy. Owing to the better stability of the salt species, a new pharmaceutical form containing it was developed to assess its stability under ICH standard conditions allowing an industrial manufacture of this drug.

Validated, stability-indicated quantitative purity test for triethylenetetramine tetrachlorhydrate by automated multiple development.

There is a monography of Triethylenetetramine dichlorhydrate (Trientine) in the United States Pharmacopeia. But neither the base nor the salts di- or tetra-chlorhydrate are in the European Pharmacopeia. Triethylène tetramine tetrachlorhydrate, used by AGEPS now as matural, is more soluble then triethylene tetramine dichlorhydrate. It is administred to patients with Wilson's disease, which results from a congenital lack of the copper metabolism. A quantitative purity test of this drug by automated multiple development high-performance thin-layer chromatography is developed and validated. The validation parameters tested are specifically characterized by retention factor, linearity, limits of detection and quantitation of several nanograms, reliability, and accuracy. To determine impurities, the monography of triethylenetetramine dichlorhydrate in the American Pharmacopeia is tested. This method in classic developing tank requires two mobile phases and is not quantitative. Assays in high-performance liquid chromatography with a different column and mobile phase did not give good results for the separation of impurities. Thus, it is not possible to perform comparative validation of the separation of the impurities. Only the assay of triethylenetetramine with potentiometer detection has been validated.

[Migration of plastic material components into food]. / Migration dans les aliments de composants des matériaux plastiques.

Despite constant improvement in the control of interactions between packaging materials and food, the problem remains worrisome. The theoretical study of various type of reactions (absorption, migration, permeation) as well as influential factors are described. The analysis of the French regulation is detailed with a special focus on the January 2003 order. After the description of assay protocols using simulated fluids, the main issues are studied and the future of manufacturing processes and materials is presented.

Speciation of arsenic and selenium compounds by ion-pair reversed-phase chromatography with electrothermic atomic absorption spectrometry. Application of experimental design for chromatographic optimisation.

An off-line system is proposed consisting of ion-pair reversed-phase liquid chromatography, collections of fractions at the outflow of the column and furnace atomic absorption spectrometry. The so-called system allowed determination of both arsenic and selenium species mainly found in the environment and in mammals (arsenite, arsenate, monomethylarsonate, dimethylarsinate, selenite, selenate, selenocystamine, selenocystine, selenomethionine and selenoethionine). In order to study the retention behaviour of these compounds and to estimate the optimal conditions for the chromatographic separation, central composite designs were used to evaluate the influence of the eluent parameters such as pH, tetrabutylammonium phosphate (TBA) concentration and sodium hydrogenphosphate amounts. The retention factors of each species and the selectivity were established as response criteria. Response surfaces and isoresponse curves were drawn from the mathematical models and enabled one to determine the optimal conditions and to visualise the method robustness. The predicted optimal zone was situated at pH 5.5-6.5, 4.0 mM Na2HPO4 and 3.0-4.0 mM TBA. Regression models suggested linearity for the studied compounds in the range 25-200 microg selenium and arsenic per litre investigated.

Determination of A 3,4-diaminopyridine in plasma by liquid chromatography with electrochemical detection using solid-phase extraction.

In order to quantify a small amount of a drug, 3,4-diaminopyridine (3,4-DAP), in animal plasma samples, an analytical method was developed. It involved an extraction of 3,4-DAP and phenylephrine, used as internal standard (IS), from plasma with solid-phase extraction (SPE) on C18 cartridges. This analytical method is a hyphenated technique based on high-performance liquid chromatography with electrochemical detection (HPLC-EC) whose purpose is to obtain first a sensitive method and second a satisfying separation between 3,4-DAP and phenylephrine. The analytical method is accurate, specific, and linear between 10 and 500 g of 3,4-DAP per litre. The recovery of 3,4-DAP is estimated at 70.8% with a 95% confidence interval of (66.0 -75.6%). Intermediate precision was evaluated on three quality control samples; the intra-day precision was estimated at 13.5, 9.1, 7.8% and the inter-day precision at 17.9, 8.4, 9.3%. The limit of quantification of the method was evaluated at 10 g l-1. First toxicokinetic parameters determined on dogs plasma samples after one 3,4-DAP oral administration of 1 mg kg-1 were: Cmax=395.7 microg l-1; Tmax =15 min; t1/2=113.6 min; Clearance/F=16.8 ml kg-1 min-1 and Vd/F=2.7 l kg -1.

Simultaneous determination of water-soluble vitamins by over-pressure layer chromatography and photodensitometric detection.

An over-pressure layer chromatographic procedure with photodensitometric detection for the simultaneous determination of water-soluble vitamins in multivitamin pharmaceutical preparations was developed and evaluated. The method uses high-performance TLC (HPTLC) plates with silica gel as the thin-layer, and an n-butanol:pyridine:water mixture (50:35:15, v/v/v) as mobile phase at a rate of 0.25 mL/min for baseline separation. The quantitation was carried out without derivatization (vitamin B1, vitamin B2, vitamin B6, folic acid, nicotinamide, vitamin C) or after spraying ninhydrin reagent (calcium pantothenate) or 4-dimethylaminocinnamaldehyde (vitamin B12, biotin). This was applied to the analysis of multivitamin solutions. Satisfactory relative standard deviations and good recovery were obtained for all the vitamins examined. It was concluded that this method is fast, accurate, specific, and suitable for routine quality control use.

[Not Available]. / Etude du pouvoir oxydant du pentoxyde de vanadium en milieu non aqueux Application a l'oxydation de molecules organiques oxygenees monofonctionelles.

Oxidation with vanadium pentoxide in aqueous sulphuric acid has certain limitations, mainly because of the instability of vanadosulphate complexes in aqueous media. Hence the possibility of use of less strongly dissociative solvents than water has been examined with a view to enhancing the stability and oxidizing power of these complexes. Some simple organic oxygen compounds (alcohols, aldehydes, ketones and acids) have been examined as reductants, together with some others (acetals and esters) which are difficult to study in aqueous media because of the hydrophobic character. The results show that alcohols are more resistant to attack in non-aqueous medium than in water, and that the longer-chain alcohols are more easily oxidized. The aldehydes are more difficult to oxidize than ketones, as is also the case in water. The acids, also as in water, react only very feebly. The use of non-aqueous media extends the range of oxidation with vanadate to some substances insoluble in water (such as epoxides) which display sufficient reactivity.

Determination of n-acetylmuramoyl-l-alanyl-d-isoglutamine in liposomes by HPLC.

A method using reversed-phase high-pressure liquid chromatography (with spectrometric detection at 218nm) is described for the determination in new pharmaceutical preparations (liposomes) of a new immunostimulating agent (N-acetylmuramoyl-l-alanyl-d-isoglutamine). Separation was achieved with a mu-bondapak column and phosphate buffer (pH 2.5)-methanol mixture (93:7 v/v) as eluent, at a flow-rate of 2 ml min . Sodium acetate was used as an internal standard. The detector response at 218 nm was linear in the range 10-170 mug ml . The method is simple and accurate.

Determination of total cysteamine in human serum by a high-performance liquid chromatography with fluorescence detection.

A convenient, reliable and rapid method for determination of total cysteamine in human plasma by high-performance liquid chromatography with fluorescence detection is reported. This assay involves reduction of samples with dithiothreitol, derivatization of total cysteamine by addition of monobromobimane and protein precipitation by perchloric acid. The calibration curve was linear in the range 2-150 nmol ml-1 and the detection limit was 0.5 nmol ml-1. This method was successfully applied for a pharmacokinetic study of three cysteamine derivatives in healthy volunteers without any interference from coexisting substances.

Stability of stored methacholine solutions: study of hydrolysis kinetic by IP-LC.

Methacholine chloride is a powerful cholinergic bronchoconstrictor agent used during bronchial airway hyper-responsiveness diagnosis. Methacholine is susceptible to hydrolysis in aqueous solutions in acetic acid and beta-methylcholine. In the present work, kinetics of hydrolysis with different solvents (water and phosphate-buffered saline (PBS) pH 7.4) at different temperatures have been studied using a newly developed high-performance liquid chromatography. At 4 degrees C, kinetic determination of hydrolysis in methacholine chloride solutions (50 mg/ml) shows no hydrolysis in either aqueous or phosphate-buffered solutions over a 40-day period. At 30 degrees C, concentration of unbuffered methacholine chloride solutions remained unchanged, but buffered methacholine chloride solutions have degradation up to 5.5% over a 40-day period. At 40 degrees C, concentration of unbuffered methacholine chloride has degradation up to 5% and buffered methacholine chloride solutions have degradation up to 10% over a 40-day period. Methacholine chloride solutions are susceptibly to be used in hospital pharmacy at different concentrations. We have studied pH and osmolality for methacholine solutions prepared with different diluents potentially used in hospital pharmacies, i.e. deionized water, 0.9% NaCl and PBS pH 7.4. We have demonstrated that methacholine solutions prepared with deionized water at 50 mg/ml and diluted with PBS pH 7.4 from 5 to 40 mg/ml are isoosmotic and potentially available for inhalation tests to measure non-specific bronchial hyper-responsiveness.

LC determination of diphemanil methylsulfate: application to stability study of stored pharmaceutical formulations.

Diphemanil methylsulfate (DMS) is a synthetic antimuscarinic agent classically used in infants for vagal hypertonia-related symptoms. A normal-phase, isocratic liquid chromatographic method was developed for the quantitative determination of DMS in bulk drugs and in pharmaceutical forms. The method has been completely validated and robustness of this method has been studied. The limit of detection (LOD) for DMS impurities namely, impurity 1 and 2 were found to be 11 and 46 ng/ml. The limit of quantitation (LOQ) was found to be 49 and 139 ng/ml for impurity 1 and 2, respectively. The stability studies have been performed for 2 and 10 mg DMS tablets subjected at various temperatures: 25 degrees C (long term storage condition) and 40 degrees C (accelerated storage condition) for 18 and 6 months, respectively. At 25 degrees C, the samples were found to be stable for the study period. At 40 degrees C, 2 and 10 mg DMS tablets showed degradation up to 5 and 10% over a 6-month period.

Various techniques for the routine evaluation of the degradation of glucose in parenteral solutions--a critical study.

A practical approach is described for studying the influence of various physicochemical factors on the degradation of glucose in parenteral solutions sterilized by heating in an autoclave. Five routine analytical methods are discussed: pH determination; direct ultraviolet absorption measurement (BP) method; liquid chromatography of 5-HMF; thin-layer chromatography of sugars, carboxylic acids and carbonyl species; and enzymatic determination of glucose. The effects of various factors on the degradation of glucose were studied: glucose concentration (10%, 30%, 50%); pH of solution before sterilization (2-10); sterilization cycle (103 min at 110 degrees C, 20 min at 120 degrees C, 3 min at 134 degrees C; same Fo); time of heating at 120 degrees C (30, 40, 60 min); and the presence of salts (sodium acetate, sodium lactate, sodium chloride). The results demonstrate the importance of these factors in influencing the rate of glucose degradation during sterilization. In the presence of salts, 5-HMF is not the most important product of degradation and the BP assay is not suitable for evaluation of glucose breakdown. The authors propose two control procedures. For simple solutions of glucose, the BP method is suitable. In the presence of salts the glucose oxidase method should be used.

Cyanide assay: statistical comparison of a new gas chromatographic calibration method versus the classical spectrophotometric method.

This work compares two different methods for assaying hydrogen cyanide, a spectrophotometric method and a headspace gas chromatographic method, each with its own reference standard generation. In the first method, the reference standards are cyanide solutions. In the second method, the reference standards are based upon the in situ reduction of ethyl thiocyanate by dithiothreitol to produce hydrogen cyanide. Furthermore, hydrogen cyanide concentration in the blood of patients who were poisoned by smoke inhalation or who committed suicide by cyanide ingestion is determined using both methods. Results are discussed using statistical comparison.

New calibration method for gas chromatographic assay of carbon monoxide in blood.

The calibration method described is based on the in situ formation of carbon monoxide in a closed system by the reaction of hot concentrated sulfuric acid with formic acid. Carbon monoxide is released from hemoglobin by treatment with 85% phosphoric acid, diluted 1:2 (v/v). Carbon monoxide is analyzed by means of headspace gas chromatography on a Porapak Q 80-100 column, following catalytic reduction to methane, using a flame-ionization detector. The method was validated by comparing the results obtained for blood samples from patients suffering from carbon monoxide poisoning and previously analyzed by means of spectrophotometry.

Serum selenium deficiency in myocardial infarction and congestive cardiomyopathy.

Serum selenium concentration was prospectively measured using the method of thermoelectric atomic absorption spectrophotometry in 129 study participants distributed as follows in three groups: control group (n = 48) volunteers without known cardiac or lung disease; group I: patients with congestive cardiomyopathy (n = 48), group II: patients with myocardial infarction (n = 31). Serum selenium level was 84.73 +/- 1.79 micrograms.l-1 (m +/- sem) in the control group, 68.53 +/- 2.26 micrograms.l-1 in group I and 73.55 +/- 2.33 micrograms.l-1 in group II. The difference between group I and group II versus the control group was significant (p less than 0.01). There was no significant difference between group I and II.

[Artificial nutrition in hospitals: general principles of quality control]. / Prothèses nutritives en milieu hospitalier: principes généraux du contrôle de qualité.

Sterile pharmaceutical preparations must be supplied to clinical wards; their high degree of safety is given by hospital pharmacists who are responsible for quality control of these preparations. Various considerations on the choice of protocol are suggested. Good manufacturing practice is to carry out quality controls at every stage of production of total parenteral nutrition fluids. However, these controls are made difficult by the poor equipment of hospital pharmacies and by the absence of specific pharmaceutical monographs. The selected protocol involves: preliminary controls, which determine the practicability of the suggested formulation, as well as its expiration date: amino acid determinations using liquid chromatography, electrolyte analysis and concentration, pH, evolution of particle size in fat emulsion according to storage conditions, choice of plastic container; microbiological controls: every two months and every time the pharmaceutical staff changes; moreover, a search for bacterial endotoxins using a chromogenic substrate (LAL technique) with "microstrips" is suggested; a sample from each admixture is kept under refrigeration for 15 days; controls during fabrication to check, at the end of the process, if good manufacturing practices have been realized: microbiological sampling of the working area and elaboration of a written procedure; control after fabrication of each nutrient admixture: determination of various elements such as sodium, potassium and dextrose in a 5 ml sample. These assays enable the checking of high standards of fabrication. Hospital pharmacies, despite their reduced equipment, can answer by these procedures to the physicians' needs and supply sterile TPN meeting pharmaceutical standards.