Determination of melatonin in rat pineal,plasma and retina by high-performance liquid chromatography with electrochemical detection

A sensitive method for the routine measurement of endogenous melatonin (MEL) in pineal, retina and plasma rat tissues has been developed using reversed-phase high-performance liquid chromatography with electrochemical detection. Quantification limit for MEL was 0.2 ng/mg protein in pineal, 15 pg/ml in plasma and 2.0 pg/mg protein in retina. To improve both MEL quantification and the reproducibility of the assay, an internal standard was used when an extraction in organic solvent was required, in contrast with other available chromatographic methods. MEL values and the circadian profile obtained in this study from both rat pineal and plasma agree with those reported previously. This method allows MEL detection in mammal retina, particularly in rat, where MEL levels are very low.     

Comparative studies on aldose reductase from bovine, rat and human lens

A purification scheme for aldose reductase (alditol: NADP+ 1-oxidoreductase, EC 1.1.1.21) developed using bovine lens tissue including an affinity chromatographic step is presented which is particularly suited for small quantities of lenses. Using the affinity chromatographic method as a key step also makes it possible to obtain preparations of rat lens aldose reductase which are homogeneous. The behavior of crude preparations of aldose reductase from human lens on both ion-exchange and affinity chromatography was similar to the chromatographic behavior of the enzyme from rat and bovine lens. Comparative studies of aldose reductase obtained from the lenses of the three species demonstrate the similarity of the enzymes. These comparisons were based on molecular weights, isoelectric points, chromatographic behavior and kinetic data. Homotropic cooperativity for both NADPH and glyceraldehyde, as evidenced by a downward curvature in the Lineweaver-Burk double-reciprocal plots, had been demonstrated for aldose reductase obtained from bovine lens (Sheaff, C.M. and Doughty, C.C. (1976) J. Biol. Chem. 251, 2696-2702). Similarly, cooperativity was observed with the enzyme from both rat and human lenses and the apparent Km values at both high and low concentrations of substrate are comparable for the lens aldose reductases from all three species for both substrates.     

LC–MS/MS method using unbonded silica column and aqueous/methanol mobile phase for the simultaneous quantification of a drug candidate and co-administered metformin in rat plasma

BMS-754807 and metformin were co-administered in drug discovery studies which required the quantitation of both compounds in plasma. Since the two compounds are chemically and structurally dissimilar, developing a single bioanalytical method presented a number of chromatographic challenges including the achievement of appropriate retention times and peak shapes on a single analytical column. To address this chromatographic challenge, we investigated different LC columns under different gradient elution schemes using aqueous/organic mobile phases. Using unbonded silica column and aqueous/methanol mobile phase, we were able to obtain robust and well-resolving chromatographic conditions to support the development and implementation of a single LC–MS/MS bioanalytical method. The use of sub-2 micron particle sizes and a high flow rate, which are attainable with UPLC systems, enhanced the method. The method performance evaluation showed that the method easily met the normally used acceptance criteria for bioanalytical methods, namely a deviation of ±15% from the nominal concentration except at lower limit of quantitation (LLOQ), where ±20% is accepted. The reported LLOQ of 7.8 ng/ml, for both BMS-754807 and metformin, was adequate to support the pharmacokinetic studies.     

Ginkgolides and bilobalide: their physical, chromatographic and spectroscopic properties

Ginkgolides A, B, C, J, K, L and M and bilobalide are rare terpene trilactones that have been isolated from leaves and root bark of the Chinese tree Ginkgo biloba. The structures of the highly oxidized ginkgolides were independently elucidated in the 1960s by the groups of Nakanishi and Sakabe. Later these compounds were found to be potent and selective antagonists of platelet activating factor, which fact triggered much new research. During the past 40 years, much physical, chromatographic and spectroscopic data have been published on these compounds in various, sometimes inaccessible, sources. The published melting points, solubility in different solvents, ionization constants, chromatographic behaviour, specific optical rotations, UV, IR, MS and NMR data, and X-ray studies are summarized and, where necessary, discussed. The literature until April 2005 has been reviewed.     

Drug level monitoring - analytical techniques,metabolism, and pharmacokinetics,by W. Sadée and G.C.M. Beelen,Wiley-Interscience,New York, 1980, XIII + 495 pp., price £22.20, ISBN 0-471-0488-1

A reversed-phase high-performance liquid chromatographic method for the determination of tetrabenazine and a hydroxy metabolite in plasma is described. Tetrabenazine and the hydroxy metabolite are quantified as their dehydro derivatives using fluorescence detection. This method has been applied to the analysis of plasma samples from patients with Huntington's chorea and has been found to be sensitive, reliable and specific for tetrabenazine and the hydroxy metabolite. The plasma concentrations of tetrabenazine found in patients were lower than could be detected using previously published methods.     

Determination of MK-287, a new platelet-activating factor antagonist, in plasma and serum by gas chromatography chemical ionization mass spectrometry

MK-287 is a novel platelet-activating factor antagonist. A sensitive and specific gas chromatographic/mass spectrometric assay has been developed for the determination of the drug in serum and plasma. The assay utilizes an extraction with methyl-t-butyl ether and subsequent trimethylsilylation of the hydroxyl function. The gas chromatographic/mass spectrometric determinations are carried out with temperature-programmed capillary gas chromatography and ammonia negative chemical ionization mass spectrometry. The method has sufficient sensitivity, precision, accuracy and selectivity for the analysis of drug concentrations in clinical samples.     

Dispersion of protein solutions in short, open capillary tubes as a method for rapid molecular weight determination

A method has been developed by which the molecular weight of proteins and other freely diffusing species can be estimated on the basis of chromatographic peak shapes developed by injection of a sample into an open capillary tube in a liquid chromatography system. In chromatographic peaks obtained from such a system, there are contributions from both convection and diffusion. Thus, peak shape is dependent upon the diffusion coefficient of the molecular species, the flow rate, and the length of the capillary tube. In the work reported here it has been found that for samples of different proteins ranging from 2000 to 14,000 molecular weight, each injected at the same mobile phase flow rate, the ratio (R) of h1, the height of the peak primarily due to convection, to h2, the height of the "makeup" peak, primarily due to diffusion from the capillary wall, is a direct measure of protein molecular weight. Linear plots of R vs molecular weight are obtained under certain conditions.     

Investigation, identification and dosage of local anesthetics and antihistaminics in cosmetic products

A rapid and accurate reversed-phase HPLC method for separation and simultaneous quantitation of some local anesthetics, antihistamines and preservatives in skin cosmetics is described. The investigated compounds (procaine, lidocaine, mepivacaine, bupivacaine, benzocaine, brompheniramine, benzoic acid, methyl, ethyl and propyl p-hydroxybenzoate, o-phenylphenol) are extracted by ultrasonic treatment in methanol from cosmetic form. Methanol-aqueous orthophosphoric acid (pH 2.8) containing 0.9% dibutylamine has been used as eluent. The influence of different percentages of methanol on chromatographic behaviour has been studied using both isocratic conditions and gradient elution program. The gradient program allows a rapid resolution and quantitation also for acidic preservatives.     

Simultaneous microdetermination of theophylline, caffeine and phenobarbital in blood collected on paper

A sensitive and selective gas chromatographic method has been developed for the simultaneous determination of theophylline, caffeine and phenobarbital. Blood collection is performed by dropping 30 μl of blood onto a disc of a special paper. Vinbarbital is used for quantitation by the internal standard method. The chromatographic separation is performed on a 3% OV-17 column, after pentylation of the methylxanthine and internal standard, and the compounds are detected with a nitrogen-sensitive detector.The sensitivity of the method allows the monitoring of theophylline therapy in premature newborns by the differential determination of caffeine and theophylline. The sampling method does not affect the accuracy and precision and is very suitable for the collection of small blood samples.     

A study of strontium binding to albumins, by a chromatographic method involving atomic emission spectrometric detection

A chromatographic method involving ICP-AES (inductively coupled plasma atomic emission spectrometry) detection has been successfully applied for the study of strontium-protein complexes. The chromatographic step involves the use of gel filtration-a large-zone Hummel and Dreyer method-which allows to dissociate the bound metallic ions and the free ones. This step is followed by an ICP-AES analysis of fractions collected throughout the chromatographic experiment: the concentration of ionic metallic species in solution can therefore be calculated. Two proteins have been tested: bovine serum albumin, which showed only weak interactions with Sr2+ ions, and bovine alpha-lactalbumin: this protein, well-known for its calcium binding capacity, proved to interact strongly with strontium. The influence of various parameters on the formation of strontium-lactalbumin complexes were determined, namely temperature, pH. Competition experiments between Sr2+ ions and, respectively Na+ and Ca2+ ions were also performed, by varying ionic strength of the medium, and by using both apo and native forms of bovine alpha-lactalbumin.     

Continuous preparative liquid chromatography in the downstrem processing of biotechnological products

Continuous counter‐current chromatographic processes have been successfully used in the petrochemical and sugar industry over the last 30 years. Only recently has simulated moving bed (SMB)‐technology attracted widespread interest in the pharmaceutical industry, mainly as a very efficient system for chromatographic enantioseparation. The application of this technique to the downstream processing of biotechnological products requires some specific changes to meet the special demands of bioproduct isolation. Production processes are set up on an multi‐ton scale, for example, for the purification of fructose with both yield and purity higher than 90%. Examples for other mono‐ and oligosaccharides are reported. In the purification of fatty acids or fat soluble vitamins, SMB technology under supercritical fluid conditions gives additional benefits and increases the productivity by a factor of four when a pressure gradient is applied. Another field of operation is the isolation of drug compounds from natural sources where different batch‐ and SMB‐chromatographic steps could be successfully combined. First examples are reported for cyclosporine A and paclitaxel isolation. Finally, step‐gradient elution modes can be used continuously, as demonstrated for the isolation of monoclonal antibodies.     

Preparative chromatographic separation: Simulated moving bed and modified chromatography methods

Chromatography has been the method of choice for the separation of complex biological mixtures for analytical purposes, particularly for the last fifty years. Its use has recently been extended to preparative separation where the productivity relative to the amount of resin and solvent used is a matter of concern. To overcome the inherent thermodynamic inefficiency of batch chromatography, as exemplified by the partial temporal usage of the resin and dilution of the product with the solvent, chromatography has been continually modified by separation engineers. Column switching and recycling represent some of the process modifications that have brought high productivity to chromatography. Recently, the simulated moving bed (SMB) method, which claims a high separation efficiency based on counter-current moving bed chromatography, has become the mainstay of preparative separation, especially in chiral separation. Accordingly, this paper reviews the current status of SMB, along with several chromatographic modification, which may be helpful in routine laboratory and industrial chromatographic practices.     

A combined screening and in silico strategy for the rapid design of integrated downstream processes for process and product-related impurity removal

Integrated designs of chromatographic processes for purification of biopharmaceuticals provides potential gains in operational efficiency and reductions of costs and material requirements. We describe a combined method using screening and in silico algorithms for ranking chromatographic steps to rapidly design orthogonally selective integrated processes for purifying protein therapeutics from both process- and product-related impurities. IFN-α2b produced in Pichia pastoris containing a significant product variant challenge was used as a case study. The product and product-related variants were screened on a set of 14 multimodal, ion exchange, and hydrophobic charge induction chromatography resins under various pH and salt linear gradient conditions. Data generated from reversed-phase chromatography of the fractions collected were used to generate a retention database for IFN-α2b and its variants. These data, in combination with a previously constructed process-related impurity database for P. pastoris, were input into an in silico process development tool that generated and ranked all possible integrated chromatographic sequences for their ability to remove both process and product-related impurities. Top-ranking outputs guided the experimental refinement of two successful three step purification processes, one comprising all bind-elute steps and the other having two bind-elute steps and a flowthrough operation. This approach suggests a new platform-like approach for rapidly designing purification processes for a range of proteins where separations of both process- and product-related impurities are needed.     

Thin-layer chromatographic methods to isolate 32P-labeled 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP): determination of cellular PRPP pools and assay of PRPP synthetase activity.

Conditions are described where 5-phosphoribosyl-α-1-pyrophosphate (PRPP) can be determined by thin-layer chromatographic methods commonly used for the determination of nucleoside triphosphate pools in ³²P-labeled bacteria. A two-dimensional chromatographic system is described where very small pools of PRPP (about 0.03 μmol per gram dry weight bacteria) can be determined. In a uni-dimensional chromatographic system the lower limit for detection of PRPP pools is about 0.3 μmol per gram dry weight bacteria. This uni-dimensional system offers an assay also for PRPP synthetase activity even in crude extracts using [γ-³²P]ATP as a substrate. The assay is highly specific due to the chromatographic isolation of PRPP and is very sensitive due to the use of ³²P labeling.The chromatographic methods for determination of PRPP pools and of activities of PRPP synthetase have been applied to the analysis of some mutants of Salmonella typhimurium and have provided results that agree well with the results obtained by conventional methods of PRPP analysis.     

A convenient and versatile method for the purification of CoA thiol esters

A simple and versatile chromatographic procedure has been described for the purification of fatty acyl-CoA thiol esters, ranging in chain length from C₂ to C₁₈. The method yields products that are over 90% pure, as judged by several criteria, in yields of 80% or greater. It may also be applicable to analytical procedures where the resolution and isolation of acylated CoA, CoASH, CoA-S-S-CoA and adenine nucleotide from a large number of reaction mixtures is required.     

Determination of d- and l-aspartate in cell culturing medium,within cells of MPT1 cell line and in rat blood by a column-switching high-performance liquid chromatographic method

HPLC fluorometric methods have been used to analyze trace amounts of d-amino acids in biological samples. In this study, we established an expedient column-switching fluorometric HPLC system that would improve the analysis of d-amino acids, in particular d-aspartate (Asp). Our system consists of the fluorogenic derivatization of amino acids with NBD-F and two chromatographic steps, one that separates individual amino acids in reverse phase mode and another that separates the chiral forms of each amino acid in normal-phase mode. The two separation steps are linked through a trapping column by an automated column-switching system. In addition, sample preparation is simplified and improved, where trichloroacetic acid is used for deproteinization, and borate buffer, pH 9.5 is employed for the fluorescent derivatization. The detection limit for d-Asp in culturing medium is 5 nM. The resulting peak heights correlated well with concentrations that ranged from 12.5 to 250 nM for both d- and l-Asp. The present method was applied to determine d- and l-Asp levels in cell culturing medium, and within cells of MPT1 cell line. The detected cellular levels of d- and l-Asp agree with those detected by our previous method. In addition, this method was used to measure d- and l-Asp levels in rat blood samples, and the results are consistent with the reported values.     

Rapid thin-layer chromatographic method for the simultaneous determination of carbamazepine,diphenylhydantoin, mephenytoin,phenobarbital and primidone in serum

A thin-layer chromatographic method for the simultaneous determination of five anticonvulsant drugs is presented. The serum is extracted with toluene and the dried extract is dissolved in chloroform and applied on to a thin-layer chromatographic plate. After development, the plate is scanned at 215 nm without staining. The drug peaks are well defined. Most of the interfering substances that occur naturally in serum are soluble in and eliminated by the liquid front.     

Self-interaction chromatography of proteins on a microfluidic monolith

A novel miniaturized system has been developed for measuring protein-protein interactions in solution with high efficiency and speed, and minimal use of protein. A chromatographic monolith synthesized in a capillary is used in the method to make interaction measurements by self-interaction chromatography (SIC) in a manner that, compared to column methods, is more efficient as well as more readily practicable even if only small amounts of protein are available. The microfluidic monolith requires much less protein for both column synthesis and the chromatographic measurements than a conventional SIC system, and in addition offers improved mass transfer and hence higher chromatographic efficiency than for previous SIC miniaturization systems. Protein self-interactions for catalase as a model protein, quantified by measurement of second virial coefficients, B(22), were determined by SIC and follow trends that are consistent with previously reported values. Different column derivatization conditions were studied in order to optimize the chromatographic behavior of the microfluidic system for SIC measurements. Chromatographic sensitivity can be further increased by using different column synthesis conditions.     

Fluorescence polarization assay of plasmin, plasminogen, and plasminogen activator

A chromatographic method involving medium-pressure liquid chromatography on alumina impregnated with silver nitrate is described for the separation of a series of closely related C₂₇ sterol precursors of cholesterol differing only in the number and location of olefinic double bonds. The features of the described system are compared with those of previously described thin-layer, gas-liquid, gravity column, and high-pressure liquid chromatographic methods.