Applicability of capillary electrophoresis-frontal analysis for displacement studies: Effect of several drugs on l-tryptophan and lidocaine binding to human serum albumin.

The effective concentration of a drug in the blood, i.e. the concentration of a free drug in the blood, is influenced by the strength of drug binding onto plasma proteins. Besides its efficacy, these interactions subsequently influence the liberation, absorption, distribution, metabolism, excretion, and toxicological properties of the drug. It is important to not only determine the binding strength and stoichiometry, but also the binding site of a drug on the plasma protein molecule, because the co-administration of drugs with the same binding site can affect the above-mentioned concentration and as a result the pharmacological behavior of the drugs and lead to side effects caused by the change in free drug concentration, its toxicity. In this study, the binding characteristics of six drugs with human serum albumin, the most abundant protein in human plasma, were determined by capillary electrophoresis-frontal analysis, and the obtained values of binding parameters were compared with the literature data. The effect of several drugs and site markers on the binding of l-tryptophan and lidocaine to human serum albumin was investigated in subsequent displacement studies which thus demonstrated the usability of capillary electrophoresis as an automated high-throughput screening method for drug-protein binding studies.

Quantification and assessment of detection capability in imaging mass spectrometry using a revised mimetic tissue model.

Aim: A revised method of preparing the mimetic tissue model for quantitative imaging mass spectrometry (IMS) is evaluated. Concepts of assessing detection capability are adapted from other imaging or mass spectrometry (MS)-based technologies to improve upon the reliability of IMS quantification. Materials & methods: The mimetic tissue model is prepared by serially freezing spiked-tissue homogenates into a cylindrical mold to create a plug of tissue with a stepped concentration gradient of matrix-matched standards. Weighted least squares (WLS) linear regression is applied due to the heteroscedastisity (change in variance with intensity) of most MS data. Results & conclusions: Imaging poses several caveats for quantification which are unique compared with other MS-based methods. Aspects of the design, construction, application, and evaluation of the matrix-matched standard curve for the mimetic tissue model are discussed. In addition, the criticality of the ion distribution in the design of a purposeful liquid chromatography coupled to mass spectrometry (LC-MS) validation is reviewed.

Chromatographic studies of chlorpropamide interactions with normal and glycated human serum albumin based on affinity microcolumns.

Sulfonylurea drugs have significant binding to proteins in blood, with most of this binding believed to occur with human serum albumin (HSA). High performance affinity chromatography and affinity microcolumns containing immobilized HSA were used to investigate binding by the sulfonylurea drug chlorpropamide to normal HSA and glycated HSA, which is a modified form of HSA that has an increased serum concentration in diabetes. Experiments employing frontal analysis indicated that the binding by chlorpropamide gave a good fit to a two-site model for both normal HSA and glycated HSA samples that were representative of controlled or advanced diabetes. These interactions involved a set of moderate-to-high affinity sites and a set of lower affinity sites, with binding constants in the range of 6.2-9.9 × 104 M-1 and 0.18-0.57 × 104 M-1, respectively, at pH 7.4 and 37 °C. Competition studies utilizing a zonal elution format demonstrated that chlorpropamide could interact at both Sudlow sites I and II of HSA, with affinities in the range expected for the moderate-to-high affinity sites of this drug. The affinity of chlorpropamide at Sudlow site I had a small increase of up to 1.2-fold when comparing the normal HSA and glycated HSA samples. Chlorpropamide gave a larger 1.4- to over 1.5-fold increase at Sudlow site II when the affinity of this drug was compared between normal HSA and the same samples of glycated HSA. These results were compared to those obtained previously with other sulfonylurea drugs to help determine how glycation can change the overall and site-selective binding strength of these drugs with HSA at levels of protein modification that are seen in patients with diabetes.

NMR search for polymorphic phase transformations in chlorpropamide form-A at high pressures.

The high-pressure effects on chlorpropamide (C10H13ClN2O3S) form-A have been studied by 1H NMR spectroscopy at high pressures up to 800 MPa in the temperature range 90-300 K. A study of the NMR second moment and spin-lattice relaxation time has been completed by a calculation of the steric hindrances for molecular reorientations and simulations of the second moment of the NMR line by the Monte-Carlo method, which enabled a precise description of molecular dynamics in the compound studied. Reorientations of the methyl group, oscillations and reorientations of the chlorophenyl ring and reorientations of the propyl group have been revealed and respective activation parameters extracted. No phase transformation of the compound form-A has been detected.

Gingival crevicular fluid PGE2, IL-1beta, t-PA, PAI-2 levels in type 2 diabetes and relationship with periodontal disease.

OBJECTIVES: To evaluate if type 2 diabetes mellitus increase gingival crevicular fluid (GCF) levels of prostaglandin E(2) (PGE(2)), interleukin-1beta (IL-1beta), tissue-type plasminogen activator (t-PA), and plasminogen activator inhibitor-2 (PAI-2). DESIGN AND METHODS: Seventeen type 2 diabetic patients with periodontal disease (DM), 17 otherwise healthy periodontally diseased patients (PD) and 17 systemically and periodontally healthy control subjects (H) were enrolled. Clinical periodontal measurements were recorded at six sites/tooth. GCF samples were analyzed by ELISA. Data were tested by statistical tests. RESULTS: DM group revealed lower IL-1beta levels than PD group (p<0.01). PGE(2), t-PA and PAI-2 levels were similar in DM and PD groups (p>0.05). PGE(2), t-PA levels were higher in DM and PD groups than H group (p<0.05). PAI-2 level was higher in DM group than H group (p<0.05). GCF total amount of PGE(2) in DM group exhibited significant correlations with all clinical periodontal measurements (p<0.05). CONCLUSION: Type 2 diabetes in this study seems not to increase GCF levels of the evaluated inflammatory mediators.

Low level determination of p-toluenesulfonate and benzenesulfonate esters in drug substance by high performance liquid chromatography/mass spectrometry.

GC/FID and HPLC/MS single ion monitoring methods have been evaluated for the determination of trace levels of methyl, ethyl and isopropyl esters of p-toluenesulfonic acid and methyl, ethyl, isopropyl and n-butyl esters of benzenesulfonic acid in drug substances. These sulfonate esters have been highlighted as potential genotoxins. HPLC/MS was found to be more promising and limits of quantification were between 2.5 and 5 ng/mL, which enabled detection limits in drug substance at 0.01-0.1 ppm for a 50 mg/mL solution. For one drug substance excellent recoveries of 94-95% were obtained at the 1.0 ppm level, however, with a second drug substance, a besylate salt, recoveries ranged from 86% to 100% and were dependant on the sample preparation. Limited stability of the sulfonate esters in various potential sample solutions indicated that samples may need to be prepared immediately before injection.

LC/MS/MS method for the analysis of acrylamide and glycidamide hemoglobin adducts.

Hemoglobin adducts of acrylamide and its primary metabolite, glycidamide are used as biomarkers of acrylamide exposure. Several methods for analyzing these biomarkers in blood have been described previously. These methods were developed to analyze small numbers of samples, not the high sample throughput that is needed in population screening. Obtaining data on exposure of the US population to acrylamide through food and other sources is important to initiate appropriate public health activities. As part of the Centers for Disease Control and Prevention biomonitoring activities, we developed a high throughput liquid chromatography tandem mass spectrometry (LC/MS/MS) method for hemoglobin adducts of acrylamide. The LC/MS/MS method consists of using the Edman reaction and isolating the reaction products by protein precipitation and solid-phase extraction (SPE). Quantitation is achieved by using stable-isotope labeled peptides as internal standards. The method is performed on an automated liquid handling and SPE system. It provides good sensitivity in the low-exposure range as assessed in pooled samples and enables differentiation between smokers and non smokers.

Quantitative determination of polymorphic composition in intact compacts by parallel-beam X-ray powder diffractometry.

This paper details the development of a method using parallel-beam X-ray powder diffractometry as a novel means of determining polymorphic composition in intact compacts. Two polymorphic systems, chlorpropamide and glycine, were selected. The polymorphic components were weighed, mixed, and compressed using a Carver press with 3/8-in. concave tooling. The compacts were then analyzed using parallel-beam X-ray powder diffractometry in transmission geometry. The data were processed using the profile-fitting module in the Shimadzu XRD-6000 software V 4.1 (for NT 4.0/98). The integrated intensity ratio of a selected peak for each crystal form was used for quantitation of each polymorph. Excellent linear correlation was observed for both polymorphic systems. The convex shape of the compact surface had no effect on the XRD patterns. Since parallel-beam X-ray diffractometry is not sensitive to the shape of the sample surface, it provides a simple method for quantifying polymorphs in intact compacts. Further work to extend this to formulated tablets is ongoing. The relatively larger variation in one of the peaks in the chlorpropamide study was found to be consistent with the computational analysis of the slip behavior of the stable polymorph. This method provides the first reported non-invasive X-ray diffraction pattern quantitation of crystal forms in intact compacts.

The qualitative and quantitative analysis of chlorpropamide polymorphic mixtures by near-infrared Fourier transform Raman spectroscopy.

We analyzed binary mixtures of polymorphs A and B of chlorpropamide ((1-[4-chlorobenzenesulphonyl]-3-propyl urea)) by near-infrared Fourier transform Raman spectroscopy (FTRS). The individual polymorphs were prepared and characterized by differential scanning calorimetry (DSC), Fourier transform infrared (FT-IR) microscopy, and physical appearance. The FTR spectra of the two polymorphs showed distinct differences which result from "crystal splitting" effects. A series of 13 different mixtures of polymorph A and B was prepared by geometric mixing and their FTR spectra statistically analysed by factor analysis programming. Predictions of the A/B polymorphic composition of mixtures were made and compared with the theoretical values. The results demonstrate that FTRS combined with factor analysis programming may be successfully applied to the in situ monitoring of the A/B polymorphic nature of a chlorpropamide sample.

An infrared study of tautomerism in acetohexamide polymorphs.

Infrared data determined for known polymorphic forms and some new derivatives of acetohexamide and related compounds support the view that acetohexamide polymorphs exhibit keto-enol tautomerism. They indicate that type A polymorphs exist in the enol form, probably stabilized by intramolecular bonding between an O-H and S = O group to form a six-membered ring. Type B polymorphs exist in the keto form with the urea carbonyl group intermolecularly bonded to a sulphonamide N-H. The new evidence disputes previous interpretations of the data.

Determinaçäo de prometazina e clopromazina em soluçoes injetáveis por cromatografia líquida de alto desempenho / Determination of promethazine and chloropromazine in injections by high perfomace liquid chromatography

A cromatografia líquida de alto desempenho (CLAD) é uma técnica adequada para a análise de fenoriazínicos uma vez que é eficiente, rápida, precisa e sensível. Nesta pesquisa foi padronizado um método por cromatografia líquida para a análise quantitativa simultânea dos cloridratos de prometazina e clorpromazina en injetáveis. Um sistema em fase reversa foi empregado consistindo de uma coluna LiChrosorb RP-18 (5 micronn) fase estacionária e metanol-água-amônia (88:11:1) como fase móvel. Operou-se em temperatura ambiente e o fluxo foi de 1,0 mL/min. As amostras após preparaçäo e extraçäo foram injetadas na coluna (cloridrato de prometazina = 20,a microng/mL, cloridrato de clorpromazina = 10,0 microng/mL). Os compostos foram detectados a 254 nm. O método pode ser usado para controle de qualidade durante a produçäo e no produto terminado

Liquid chromatographic determination of chlorpropamide in tablet dosage forms: collaborative study.

A reverse-phase liquid chromatographic method was developed for determining chlorpropamide in tablet dosage forms. Linearity was established over the range 0.2-2.0 micrograms at a wavelength of 240 nm. The Associate Referee obtained a mean recovery for a synthetic tablet mixture of 99.2%, with a relative standard deviation (RSD) of 1.41. For an authentic tablet mixture, collaborators obtained a mean recovery of 99.6% with an RSD of 0.60%. RSDs were 1.24% for 250 mg/tablet commercial product and also for 100 mg/tablet commercial product. The method has been adopted official first action.

The assay and stability of chlorpropamide in solid dispersion with urea.

Thin layer chromatography followed by reflectance densitometry has been used to evaluate the stability of chlorpropamide-urea during the fusion process. Urea was found to decompose to biuret and chlorpropamide to p-chlorobenzenesulphonamide: several other unidentified decomposition products were detected. The energy for decomposition of chlorpropamide was 57.1 kJmol-1 for melts containing 15 and 30% chlorpropamide. Decomposition followed apparent first order kinetics.

Stability-indicating high-performance liquid chromatographic determination of chlorpropamide, tolbutamide, and their respective sulfonamide degradates.

A quantitative high-performance liquid chromatographic method for the determination of chlorpropamide, tolbutamide, and their respective hydrolysis products, p-chlorobenzenesulfonamide and p-toluenesulfonamide, in solid dosage forms was developed. The method is stability indicating and can be used to determine the sulfonamide hydrolysis product and the intact drug in the presence of minor degradates. Method reproducibility, demonstrated by repeated injections of a calibration standard, was 1.21%. The lower limit of quantitation of the hydrolysis products, p-chlorobenzenesulfonamide and p-toluenesulfonamide, was 0.2 microgram/5-microliter injection. The accuracy of the method for intact drugs was determined by comparison of the HPLC results to those obtained by the appropriate USP or BP assays. The mean of the results obtained by the two methods differed by 0.7% for chlorpropamide and 0.3% for tolbutamide. Pure drug samples were spiked with amounts of the hydrolysis products ranging from 20 to 120% of the intact content. The mean percent recovery for p-chlorobenzenesulfonamide was 98.6%; for p-toluenesulfonamide, it was 100.6%. A qualitative TLC procedure for the detection of chlorpropamide, p-chlorobenzenesulfonamide, dipropylurea, propylurea, n-propylamine, tolbutamide, p-toluenesulfonamide, dibutylurea, butylurea, and n-butylamine is also described.

The effect of composition and ageing on the dissolution rates of chlorpropamide-urea solid dispersions.

Discs of chlorpropamide and urea have been prepared by (a) melting and (b) compression. Intrinsic and relative dissolution rates of the discs have been measured and the dissolution process investigated microscopically. Higher dissolution rates were found from melts than from physical mixes. The optimum dissolution rate composition found was for a melt composed of 30% w/w chlorpropamide which possessed an intrinsic dissolution rate 930 times greater than for the pure drug. Sphere formation, during dissolution rate measurement has been observed and a likely mechanism proposed to account for its occurrence. Dissolution rates generally increased with age for most melt compositions.

Phase equilibria and stability characteristics of chlorpropamide-urea solid dispersions.

Physical mixtures and melts of various compositions of chlorpropamide and urea have been prepared. The phase diagrams and the effects of ageing of the systems have been measured by differential scanning calorimetry. The eutectic composition was found to contain 89% w/w chlorpropamide. Greater concentrations of chlorpropamide produced solid solutions of urea in chlorpropamide, whereas solid solution formation did not occur at compositions less than 89%. Melts in the range 50-100% chlorpropamide, which included the eutectic, existed as glass solids. The effect of ageing produced generally an increase in the liquidus peak temperature which was considered to be due to a gradual increase in crystal size.

Gas chromatographic and mass spectrometric analysis of the products of methylation of sulfonylurea drugs.

Gas chromatographic analysis of the products of reaction of diazomethane with tolbutamide and chlorpropamide indicates the formation of three compounds in both cases. As expected, N-methylation (at sulfonamide nitrogen) is the predominant reaction; minor amounts of O-methylated product are also observed. The third product in both cases is the N-methylsulfonamide formed by decomposition of the N-methylated sulfonylurea during gas chromatography. Electron impact and chemulfonylurea during gas chromatography. Electron impact and chemical ionization mass spectrometric analysis, as well as 1H nuclear magnetic resonance examination of samples collected from gas chromatography, confirm the structural assignments. Additionally, proton magnetic resonance analysis of the crude reaction products established that N-methylsulfonamides are not formed in the course of the diazomethane reaction and that the O-methylated derivatives are true products of the reaction. The use of a paramagnetic shift reagent allowed direct estimation of the ratios of N- to O-methylation, and the demonstration that these ratios are not vitiated during gas chromatographic analysis.