A Study of peptide-peptide interaction by matrix-assisted laser desorption/ionization

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry was used to study peptide-peptide interaction. The interaction was seen when 6-aza-2-thiothymine was used as a matrix (pH 5.4), but was disrupted with a more acidic matrix, alpha-cyano-4-hydroxycinnamic acid (pH 2.0). In the present study, we show that dynorphin, an opioid peptide, and five of its fragments that contain two adjacent basic residues (Arg6-Arg7), all interact noncovalently with peptides that contain two to five adjacent acidic residues (Asp or Glu). Two other nonrelated peptides containing two (Arg6-Arg7) or three (Arg1-Lys2-Arg3) adjacent basic amino acid residues were studied and exhibited the same behavior. However, peptides containing adjacent Lys or His did not form noncovalent complexes with acidic peptides. The noncovalent bonding was sufficiently stable that digestion with trypsin only cleaved Arg and Lys residues that were not involved in hydrogen bonding with the acidic residues. In an equimolar mixture of dynorphin, dynorphin fragments (containing the motif RR), and an acidic peptide (minigastrin), the acidic peptide preferentially complexed with dynorphin. If the concentration of minigastrin was increased 10 fold, noncovalent interaction was seen with dynorphin and all its fragments containing the motif RR. In the absence of dynorphin, minigastrin formed noncovalent complexes with all dynorphin fragments. These findings suggest that conformation, equilibrium, and concentration do play a role in the occurrence of peptide-peptide interaction. Observations from this study include: (1) ionic bonds were not disrupted by enzymatic digests, (2) conformation and concentration influenced complex formation, and (3) the complex did not form with fragments of dynorphin or unrelated peptides that did not contain the motifs RR or RKR, nor with a fragment of dynorphin where Arg7 was mutated to a phenylalanine residue. These findings strongly suggest that peptide-peptide interaction does occur, and can be studied by MALDI if near physiologic pH is maintained.     

Spectrophotometric determination of some pharmaceutically-important aminoquinoline antimalarials

A simple and rapid spectrophotometric method for the assay of amodiaquine hydrochloride, chloroquine phosphate and primequine phosphate is described. The method is based on the interaction of the drug with tetracyanoethylene to give a stable charge transfer complex. The spectra of the complex show maxima at 413, 415 and 415nm, respectively, with high apparent molar absorptivities. Beer's law is obeyed in the concentration ranges 2–12, 1–8 and 2–12 g ml^–1 of the three drugs studied. The proposed method is applied to the determination of these drugs in certain formulations and the results are favourably comparable to the official methods.     

Determination of 1,4-benzodiazepines in drug dosage forms by difference spectrophotometry

A spectrophotometric method has been developed for the determination of seven 1,4-benzodiazepines, namely: chlordiazepoxide, diazepam, nitrazepam, oxazepam, lorazepam, temazepam and cinolazepam. The method involves reduction of the target compounds using Zn/HCl and measuring the difference in the absorbance before and after reduction. The quantities of zinc and acid required for efficient reduction were carefully studied. 1–20 g ml^–1 of the compounds could be determined with < 1% error. The method was applied successfully to the determination of these compounds in pharmaceutical dosage forms. The results were sufficiently accurate and precise and comparable to those from the official methods.     

Direct current polarographic determination of chlorprothixene and thiothixene after bromination

A direct current (dc) polarographic method is described for the determination of chlorprothixene and thiothixene after reaction with bromine. The bromo derivatives of the two drugs show a well-defined, diffusion-controlled cathodic wave; the height of the latter increases with an increase in the concentration of the depolarizers. The different experimental conditions were carefully studied and incorporated into the procedure. The proposed method was further applied to the studied compounds in dosage forms and the results obtained were compared favorably to those given by compendial methods.     

First isolation of sesquiterpenes and flavonoids from Zingiber spectabile and identification of zerumbone as the major cell growth inhibitory component

Nine sesquiterpenes and eight flavonoids were isolated from Zingiber spectabile for the first time. Structures were determined by spectroscopic methods. The major compound zerumbone (1) was found to be the most active (IC(50) 13 microg mL(-1)) in cell growth inhibitory assay against colon carcinoma SW480 cells.     

Conventional and First Derivative Synchronous Fluorometric Determination of Ethamsylate in Pharmaceutical Preparations and Biological Fluids. Application to Stability Studies

Two simple, accurate and highly sensitive spectrofluorometric methods were developed for the determination of ethamsylate (ETM). Method I is based on measuring the native fluorescence of ethamsylate in water at 354 nm after excitation at 302 nm. The calibration plot was rectilinear over the range of 0.05–1 μg/mL for ETM with limits of detection and quantitation of 7.9 and 26 ng/mL, respectively. Method II involved synchronous and first derivative synchronous fluorometric methods for the simultaneous determination of ethamsylate (ETM) and hydroquinone (HQ) which is considered as an impurity and/or acidic degradation product. The synchronous fluorescence of both the drug and its impurity were measured in methanol at Δ λ of 40 nm. The peak amplitudes (¹D) were estimated at 293.85 or 334.17 nm for ETM and at 309.05 nm for HQ. Good linearity was obtained for ETM over the ranges 0.1–1.4 μg/mL and 0.1–1.0 μg/mL at 293.85 and 334.17 nm, respectively. For HQ, the calibration plot was rectilinear over the range of 0.01–0.14 μg/mL at 309.05 nm. Limits of detection were 20, 2.01 ng/mL and limits of quantitation were 60, 6.7 ng/mL for ETM and HQ by method II, respectively. Both methods were successfully applied to commercial ampoules and tablets. The results were in good agreement with those obtained by the reference method. Method I was utilized to study the stability of ETM and its degradation kinetics using peroxide. The apparent first-order rate constant, half-life times and activation energy of the degradation process were calculated. Method I was further extended to the in-vitro and in-vivo determination of ETM in spiked and real plasma samples. The mean% recoveries were 99.57 ± 3.85 and 89.39 ± 5.93 for spiked and real human plasma, respectively.     

Liquid chromatographic method for the simultaneous determination of eprosartan and hydrochlorothiazide in tablets and human plasma

A new, specific, and sensitive RP-HPLC method was developed for the simultaneous determination of eprosartan (EPR) and hydrochlorothiazide (HCT). Good chromatographic separation was achieved using a 250 x 4.6 mm id, 5 microm particle size Symmetry C18 column. The mobile phase acetonitrile-0.1 M phosphate buffer (35+65, v/v), pH 4.5, was pumped at a flow rate of 1 mL/min, with UV detection at 275 nm. The method showed good linearity in the ranges of 0.5-50 and 0.1-10 microg/mL, with LOD of 0.06 and 0.02 microg/mL and LOQ of 0.20 and 0.08 microg/mL for EPR and HCT, respectively. The proposed method was successfully applied for the analysis of the studied drugs in their synthetic mixture and co-formulated tablets. The method was further extended to the in vitro and in vivo determination of the two drugs in spiked and real human plasma. Interference likely to be encountered from the co-administered drugs was studied.     

Optimization of automated matrix deposition for biomolecular mapping using a spotter

Imaging mass spectrometry using matrix-assisted laser desorption/ionization allows the detailed mapping of biomolecules directly from tissue. Matrix deposition is the key step for successful imaging. The appropriate concentration and deposition of matrix is critical for extraction, desorption, and ionization of molecules from tissue without losing molecular localization. The main challenge to meet these criteria is to deposit matrix droplets homogeneously on the tissue section. This work shows how a chemical inkjet printer was used for this purpose resulting in the imaging of phosphatidylcholines and sulfatides. The intricacies involved in effective matrix deposition are discussed.