Determination of l-cysteine origin on the basis of its δ15N values.

The majority of l-cysteine is obtained industrially by hydrolysis of animal materials, such as poultry feathers. Despite widespread belief, there is little evidence that human hair is used as a source material and its use is explicitly banned in the European Union (2000/63/EC decision). We developed an isotope ratio mass spectrometric (EA-IRMS) method to determine carbon and nitrogen isotopic ratio in cysteine preparations and related compounds, e.g. cystine and carbocysteine. A threshold relying on the 15N/14N was established to differentiate between hair and feathers; a value below 6.6‰ indicates a poultry feathers origin. Global uncertainty of measurement was found to be 0.1‰ for δ15N (sample size of 0.5-1.8 mg).

Development and validation of a stability indicating method for S-carboxymethyl-L-cysteine and related degradation products in oral syrup formulation.

A stability-indicating method for the determination of S-carboxymethyl-L-cysteine and related degradation impurities in Exputex® 250mg/5mL syrup was developed in anion-exchange liquid chromatography mode. A forced degradation study supported the method development to ensure stability indicating conditions. Aqueous solutions of the active pharmaceutical ingredient and syrup samples at different pH-values were stress-tested in different thermal, light exposure and headspace conditions. One degradation product was detected in thermal stress studies at 60°C and 80°C in the pH range 5.0-7.0 and was identified by mass spectrometry as 5-oxo-thiomorpholine-3-carboxylic acid (lactam of carbocysteine). A second degradation product was only generated in moderately strong oxidizing conditions (0.5% H2O2 aqueous solution) and was identified as S-carboxymethyl-L-cysteine-(R/S)-sulphoxide (carbocysteine sulphoxide). The method was developed on a Zorbax SAX column, in isocratic mode. The mobile phase consisted of 200mM phosphate solution at pH 4.0 and acetonitrile (50:50 v/v) and UV detection was performed at a wavelength of 205nm. The method was linear for carbocysteine (R>0.9982) over a concentration range of 2.5-50µg/mL and 0.4-0.6mg/mL. Linearity for the impurities was shown from the LOQ to 50µg/mL. Specificity was verified and accuracy demonstrated for the active ingredient and its degradation products in syrup samples at 3 levels around their respective specification limits. Repeatability, intermediate precision and inter-laboratory reproducibility were assessed on three commercial batches, analyzed in triplicate by two operators at both the transferring and the receiving site and demonstrated a successful method transfer to the manufacturing quality control laboratory.

Diagnostic capability of salivary biomarkers in the assessment of head and neck cancer: A systematic review and meta-analysis.

The purpose of this systematic review and meta-analysis was to evaluate the diagnostic value of salivary biological markers in the diagnosis of head and neck carcinoma. Studies were gathered by searching Cochrane, EMBASE, LILACS, MEDLINE, and PubMed. The references were also crosschecked and a partial grey literature search was undertaken using Google Scholar. The methodology of selected studies was evaluated using the 14-item Quality Assessment Tool for Diagnostic Accuracy Studies. After a two-step selection process, 15 articles were identified and subjected to qualitative and quantitative analyses. The studies were homogeneous, and all had high methodological quality. Combined biomarkers demonstrated better accuracy with higher sensitivity and specificity than those tested individually. Furthermore, the salivary biomarkers reviewed predicted the early stages of head and neck carcinoma better than the advanced stages. A restricted set of five single biomarkers (interleukin-8, choline, pipecolinic acid, l-phenylalanine, and S-carboxymethyl-l-cysteine) as well as combined biomarkers demonstrated excellent diagnostic test accuracy. The present systematic review confirms the potential value of a selected set of salivary biomarkers as diagnostic tools for head and neck carcinoma.

Hair sulfur amino acid analysis.

This overview emphasizes present aspects of sulfur-containing amino acids in hair. A selection of analytical procedures to determine cystine, cysteine, S-sulfocysteine, cystine oxide, cysteic acid, lanthionine and lysinoalanine are presented. The methods relate to intact hair or partial and total hydrolysates and comprise chromatography, titration, colorimetry, polarography and spectroscopy. For the analysis of cysteine, cystine and cystine oxides, polarography and spectroscopy are the methods of choice. Cysteic acid, lanthionine and lysinoalanine are analysed by means of ion-exchange chromatography (Spackman et al., 1958) after total hydrolysis.

Ion-chromatographic determination of carbocisteine in pharmaceuticals based on non-suppressed conductimetric detection.

A novel method for the determination of carbocisteine (S-CMC), a mucolytic and expectorant drug with an acidic amino acid structure, was developed and validated, using non-suppressed ion-chromatographic system with conductimetric detection, and anion or cation exchange columns. Among the various combinations of column type and eluent composition tested, a cation exchange column with a 0.25 mM tri-fluoroacetic acid (TFA) as eluent in isocratic mode at 1.2 ml/min gave the best results. S-CMC was very well separated from all common amino acids (resolution > 2.6). The retention time was 3.5 min and the asymmetry factor 1.1. A linear calibration curve from 17 to 400 microg/ml (r = 0.99994), with a detection limit of 0.14 microg (5.6 microg/ml-25 microl injection volume) and a precision of 1.5% R.S.D. (100 microg/ml, n = 3) was achieved. The proposed method was applied for the determination of S-CMC content in intensely colored commercial formulations (syrups). No interference from excipients was found and the only pretreatment step was the appropriate dilution with the mobile phase. Recovery from standard additions was ranged from 96.0 to 104.9% and precision (R.S.D., n = 3) 1.8-3.6%.

Direct determination of s-carboxymethyl-l-cysteine in syrups by reversed-phase high-performance liquid chromatography.

A simple reversed-phase high-performance liquid chromatography method was developed for the determination of s-carboxymethyl-l-cysteine in syrup preparations. The experiments were performed without specific sample pre-treatment. The LC conditions used were acetonitrile-10 mM sodium dihydrogenphosphate buffer, pH 2.0 (1:99, v/v) on a C(18) Inersil column with a flow rate of 1.5 ml/min. Ultraviolet detection was carried out at 240 nm. The method showed excellent linearity (r(2)>0.9998) over the concentration range tested (0.8-25.6 mg/ml) with good precision and accuracy (%R.S.D. 0.7%). Recoveries were good (>99%) with a limit of detection and limit of quantitation of 0.1 and 0.8 mg/ml. Other compositions in the syrup vehicle did not interfere the analysis of s-carboxymethyl-l-cysteine.

An investigation of the metabolism of S-carboxymethyl-L-cysteine in man using a novel HPLC-ECD method.

The metabolism of an oral dose of S-carboxymethyl-L-cysteine (SCMC) in man has been studied; the quantitative determination of SCMC, S-methyl-L-cysteine (SMC) and their sulphoxide metabolites (SCMCO and SMCO), in urine, was carried out using high performance liquid chromatography (HPLC) with electrochemical detection (ECD); the possibility of stereospecific sulphoxidation was investigated.

Impurity profiling of carbocisteine by HPLC-CAD, qNMR and UV/vis spectroscopy.

For the impurity profiling of the mucolytic and anti-inflammatory drug carbocisteine a high performance liquid chromatographic (HPLC) method using corona charged aerosol detection (CAD) was developed and fully validated following the ICH guideline Q2(R1). The response was linear (R²>0.995) over a small concentration range (0.05-0.25 or 0.10-0.60% respectively) and a detection limit of at least 0.03% was registered. The separation was achieved on a mixed mode column combining hydrophobic C18 and strong cation exchange retention mechanisms using a mass spectrometer compatible volatile mobile phase consisting of trifluoroacetic acid 10 mM and acetonitrile 12% (V/V). Impurities, not assessable by HPLC-CAD such as the volatile chloroacetic acid and the unstable cysteine, were determined by quantitative NMR (qNMR) with maleic acid as internal standard and UV/vis spectroscopy after reaction with Ellman's reagent, respectively. Six batches of three different manufacturers were tested by means of those methods. The purity varied from below 99.0 to higher than 99.8 per cent. The major impurities of all batches were the starting material cystine and N,S-dicarboxymethylcysteine being a synthesis by-product.

Fluorimetric determination of some sulfur containing compounds through complex formation with terbium (Tb+3) and uranium (U+3).

Two simple, sensitive and specific fluorimetric methods have been developed for the determination of some sulphur containing compounds namely, Acetylcysteine (Ac), Carbocisteine (Cc) and Thioctic acid (Th) using terbium Tb+3 and uranium U+3 ions as fluorescent probes. The proposed methods involve the formation of a ternary complex with Tb+3 in presence of Tris-buffer method (I) and a binary complex with aqueous uranyl acetate solution method (II). The fluorescence quenching of Tb+3 at 510, 488 and 540 nm (lambda(ex) 250, 241 and 268 nm) and of uranyl acetate at 512 nm (lambda(ex) 240 nm) due to the complex formation was quantitatively measured for Ac, Cc and Th, respectively. The reaction conditions and the fluorescence spectral properties of the complexes have been investigated. Under the described conditions, the proposed methods were applicable over the concentration range (0.2-2.5 microg ml(-1)), (1-4 microg ml(-1)) and (0.5-3.5 microg ml(-1)) with mean percentage recoveries 99.74+/-0.36, 99.70+/-0.52 and 99.43+/-0.23 for method (I) and (0.5-6 microg ml(-1)), (0.5-5 microg ml(-1)), and (1-6 microg ml(-1)) with mean percentage recoveries 99.38+/-0.20, 99.82+/-0.28 and 99.93+/-0.32 for method (II), for the three cited drugs, respectively. The proposed methods were successfully applied for the determination of the studied compounds in bulk powders and in pharmaceutical formulations, as well as in presence of their related substances. The results obtained were found to be in agree statistically with those obtained by official and reported ones. The two methods were validated according to USP guidelines and also assessed by applying the standard addition technique.

Degradação forçada e análise fatorial para caracterização da estabilidade de formulações líquidas de carbocisteína / Forced Degradation and Factorial Analysis of Stability of Liquid Formulations containing Carbocisteine

Este estudo teve como objetivo avaliar a estabilidade de xaropes contendo carbocisteína, submetidos à degradação forçada, utilizando Desenho Experimental Fatorial (DEF). Os fatores avaliados foram pH (5,0; 6,5; 8,0), presença ou ausência de EDTA dissódico e metabissulfito de sódio (0,1%). Para o estudo de degradação forçada, as formulações foram submetidas a estresse térmico (50 °C e 75% UR) e oxidação com peróxido de hidrogênio a 3%. Posteriormente, as formulações foram analisadas quanto ao pH, propriedades organolépticas e teor de fármaco por CLAE-UV, nos tempos 0, 15 e 35 dias. Os resultados mostraram que as formulações submetidas à degradação forçada sofreram uma diminuição no teor do fármaco, enquanto que o pH se manteve relativamente estável. Em relação a cor, apenas as formulações que não possuíam antioxidantes mostraram-se mais escuras. A análise dos resultados do DEF mostrou interação significativa (p<0,05) para os fatores pH/metabissulfito e EDTA/metabissulfito. As formulações contendo metabissulfito em pH 5,0 apresentaram maior degradação e as formulações com metabissulfito sem EDTA também não foram eficientes para impedir a degradação da carbocisteína.

The aim of this study was to use Factorial Design (FD) to assess the stability of carbocisteine syrups subjected to forced degradation. The factors assessed were pH (5.0; 6.5; 8.0), presence or absence of disodium EDTA and sodium metabisulfite (0.1%). For the study of forced degradation, the formulations were subjected to thermal stress (50°C and 75% RH) and oxidation with 3% hydrogen peroxide. The formulations were analyzed for pH, organoleptic properties and drug content by HPLC-UV, at 0, 15 and 35 days. The results showed that the formulations exposed to forced degradation suffered a fall in drug content, while the pH remained relatively stable. Regarding the color, only the formulations without antioxidant exhibited a darker coloration. The results of FD revealed significant interactions (p<0.05) for pH/metabisulfite and EDTA / metabisulfite. Formulations containing metabisulfite at pH 5.0 showed the greatest degradation and those with metabisulfite and without EDTA were also not effective in preventing the degradation of carbocisteine.

S-carboxymethylcysteine sulfone: instability to acid hydrolysis and unreactivity with N-terminal reagents.

An examination of the properties and reactivity of S-carboxymethylcysteine sulfone indicated that, unlike S-carboxymethylcysteine, the sulfone is not stable under acid hydrolysis conditions and decomposes to yield alanine. Unlike S-carboxymethylcysteine, the sulfone is resistant to N-derivatization by the dansyl reagent or by phenylisothiocyanate. Efforts were made to determine if spontaneous cyclization of the sulfone to the corresponding thiazane (lactam) accounts for lack of reactivity. These included i.r. spectroscopy, natural abundance 13C-n.m.r. spectroscopy and differential scanning calorimetry, but yielded equivocal results concerning the existence of the cyclic form in solution. Solubility behavior of the sulfone after lyophilization from strongly acid solutions was consistent with conversion of the open chain form to the cyclic form on addition of water.

Preparation of a two-disulfide bonded enzymically active derivative from hen egg lysozyme.

A method has been developed for preparation of an enzymically active two-disulfide bonded derivative from hen egg lysozyme. Lysozyme (0.15 mM) is incubated with 2 mM dithiothreitol at pH 7.8, 23 degrees for 40 min. The products are reacted with [1-14C]iodoacetic acid and then purified by gel filtration and ion-exchange chromatography. An enzymically active derivative containing 4 mol of [1-14C] carboxymethyl groups and no free sulfhydryl groups is obtained in approximately 18% yield. Examinations of hydrodynamic volume, tryptophan fluorescence, CD and tryptic peptides containing [1-14C] carboxymethyl cysteine indicate that this derivative contains two presumably native disulfide bonds and two open disulfide bonds between Cys 6 and Cys 127 and between Cys 76 and Cys 94. The rest of the species in the incubation mixture are intact lysozyme. Thus, the species containing two presumably native disulfide bonds and four free sulfhydryl groups at Cys 6, Cys 76, Cys 94 and Cys 127 appears to be only the intermediate accumulating during reduction of lysozyme with dithiothreitol.

High pressure liquid chromatographic determination of carbocisteine in human plasma and urine.

A method is described for the direct analysis of the amino acid carbocisteine in plasma and urine samples, following reaction with dabsyl chloride. Dabsylated carbocisteine is subjected to high pressure liquid chromatography with spectrophotometric detection at 425 nm. The usefulness of the method for bioavailability studies is discussed and compared with methods currently in use.

Instability of DTNB-treated globin or haemoglobin.

Human haemoglobin or globin in its native form reacts with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) with uptake of two 3-carboxylato-4-nitrothiophenol groups, one for each of the reactive thiols at the beta93 positions. Attempts to isolate the DTNB-treated globin by the acetone-HC1 method, which unfolds the protein chains, result in disulphide interchange and oxidation of almost all the uncoupled "masked" thiol groups. This modification is in marked contrast to the stability of haemoglobin or globin treated with reagents such as iodoacetic acid or N-ethylmaleimide that do not form disulphide bonds in blocking the thiol groups. The derivatized globin chains have been separated by urea-thiol buffer chromatography on carboxymethycellulose columns. Amino acid analysis and peptide mapping established the presence and location of disulphide bonds, whilst gel filtration in urea buffers and sodium dodecyl sulphate acrylamide gel electrophoresis defined the size of the products.

High-sulphur proteins from alpha-keratins. II. Isolation and partial characterization of purified components from mouse hair.

The present paper continues the study of the reduced and S-carboxymethylated high-sulphur proteins from mouse hair. Fractions have been obtained in a substantially purified form by fractional precipitation with ammonium sulphate at pH 6, followed by ion exchange chromatography on cellulose phosphate at pH 2.6. Approximately 80% by weight of the high-sulphur proteins fall into the ultra-high-sulphur category (carboxymethylcysteine content greater than 26 residues per 100 residues), and they cover a molecular weight range of 17000-28000. The components show a remarkable diversity in amino acid composition; for example the contents of arginine and glycine each vary by about 3:1. The remainder of the proteins contain 17-20 residues per 100 residues of carboxymethylcysteine, are smaller in size (molecular weight 11500), and also show great diversity in overall amino acid composition. Molecular weights were determined by chromatography on controlled-pore glass and confirmed by gel filtration on Sephadex G-100 and Sepharose 6B. A comparison of the results suggests that the values obtained should be reliable to within 10%.

Relative bioavailability of carbocysteine from three dosage forms, investigated in healthy volunteers.

The aim of the present study was to evaluate the bioavailability of a new tablet formulation of carbocysteine relative against two other oral carbocysteine containing dosage forms, viz. a syrup and capsules. Plasma levels and urine concentrations of carbocysteine were monitored, following oral administration of all three dosage forms to healthy human volunteers, by direct derivatization of carbocysteine using dabsylchloride and subsequent high performance liquid chromatography. There was no difference in bioavailability of carbocysteine from these dosage forms as expressed by the respective areas under the plasma concentration-time curves and total amounts of unchanged carbocysteine excreted in urine.