Mostafa K. Mesbah

Precise Measurement of the G + C Content of Deoxyribonucleic Acid by High-Performance Liquid Chromatography

High-performance liquid chromatography is a promising alternative for determining the G+C content of bacterial deoxyribonucleic acid (DNA). The method which we evaluated involves enzymatic degradation of the DNA to nucleosides by P1 nuclease and bovine intestinal mucosa alkaline phosphatase, separation of the nucleosides by high-performance liquid chromatography, and calculation of the G+C content from the apparent ratios of deoxyguanosine and thymidine. Because the nucleosides are released from the DNA at different rates, incomplete degradation produces large errors in the apparent G+C content. For partially purified DNA, salts are a major source of interference in degradation. However, when the salts are carefully removed, the preparation and degradation of DNA contribute little error to the determination of G+C content. This method also requires careful selection of the chromatographic conditions to ensure separation of the major nucleosides from the nucleosides of modified bases and precise control of the flow rates. Both of these conditions are achievable with standard equipment and C18 reversed-phase columns. Then the method is precise, and the relative standard deviations of replicate measurements are close to 0.1%. It is also rapid, and a single measurement requires about 15 min. It requires small amounts of sample, and the G+C content can be determined from DNA isolated from a single bacterial colony. It is not affected by contamination with ribonucleic acid. Because this method yields a direct measurement, it may also be more accurate than indirect methods, such as the buoyant density and thermal denaturation methods. In addition, for highly purified DNA, the extent of modification can be determined.

Burkholdines 1097 and 1229, potent antifungal peptides from Burkholderia ambifaria 2.2N

Potent antifungal cyclic lipopeptides, burkholdines (Bk), were isolated from a culture of Burkholderia ambifaria 2.2N. Bk-1229 (1) and Bk-1097 (2) are octapeptides comprised of nonproteinogenic amino acids, including beta-hydroxytyrosine, beta-hydroxyasparagine, and a new fatty acyl amino acid. 1 and 2 are fungicidal against a panel of fungi with potencies 2-60-fold better than amphotericin B control.

Rapid and simultaneous determination of antioxidant markers and caffeine in commercial teas and dietary supplements by HPLC-DAD.

A simple and fast reverse-phase high-performance liquid chromatography procedure coupled with photodiode array detector (RP-HPLC-DAD) was developed and validated for the analysis of major catechins, proanthocyanidin (procyanidin B2) and caffeine in 25 different natural complex matrices containing Camellia sinensis L. and/or grape seed extracts, two popular plant extracts that have been widely used as natural antioxidants in various food and beverage applications. Using an isocratic elution system, separation of all compounds was achieved within 12 min. Excellent linearity was observed for all of the standard calibration curves, and the correlation coefficients were above 0.9997. Limits of detection for all of the analyzed compounds ranged between 2.80×10(-3) and 2.51×10(-2) μg mL(-1); limits of quantitation ranged between 9.30×10(-3) and 8.36×10(-2) μg mL(-1). The developed method was found to be accurate and sensitive and is ideally suited for rapid, routine analysis of principal components in these well-known natural antioxidants.

Determination of the G+C Content of Prokaryotes

Abstract The mol% G+C content of bacteria is a key in the complete description of novel genera and higher taxa, and also influences several physiological properties such as codon usage and DNA stability. Since differences in the G+C content can be used in the differentiation between different bacterial groups, the description of a standardized and uniform procedure for its determination is critical. This chapter provides an overview of many common methods as well as a detailed protocol for determination of the G+C content by reverse-phase high-performance liquid chromatography. Detailed protocols on DNA digestion and chromatographic nucleoside separation are presented, and methods employed for buffer and reagent preparation and storage are described. The method of G+C content calculation is also presented. High-performance liquid chromatography has proved to be one of the most accurate methods for determination of G+C content and will allow more precise comparison of values determined by different researchers and laboratories worldwide.

New Validated Methods for the Simultaneous Determination of Two Multicomponent Mixtures Containing Guaiphenesin in Syrup by HPLC and Chemometrics‐Assisted UV‐Spectroscopy

Abstract High pressure liquid chromatographic (HPLC) and spectrophotometric methods are developed for the determination of two multicomponent mixtures containing guaiphenesin, dextromethorphane hydrobromide, and sodium benzoate together with either phenylephrine hydrochloride, chlorpheniramine maleate, and butylparaben (mixture 1) or ephedrine hydrochloride and diphenhydramine hydrochloride (mixture 2). The HPLC method depended on using an ODS column with mobile phase consisting of acetonitrile −10 mM potassium dihydrogen phosphate, pH 2.7 (40∶60 v/v) containing 5 mM heptane sulfonic acid sodium salt (for mix 1) and a cyanopropyl column with mobile phase consisting of acetonitrile −12 mM ammonium acetate, pH 5 (40∶60 v/v) (for mix 2) and UV detection at 214 nm. The cyanopropyl column is much less hydrophobic, less sterically restricted to the penetration of bulky solute molecules into the stationary phase, and has weaker hydrogen‐bond acidity than the ODS column. So the cyanopropyl column is more suitable for separation of components of mix 2. The chemometric‐assisted spectrophotometric method with, principal component regression (PCR) and partial least squares (PLS‐1) was used. For the chemometric method a calibration set of the mixture consisting of each compound in each mixture was prepared in distilled water. The absorbance data in the UV spectra were measured in the spectral region (210–240 or 210–224 nm for mix 1 and mix 2, respectively, as this range provided the greatest amount of information about the two mixture components). The spectrophotometric method does not require a separation step. The proposed methods were successfully applied for the analysis of the two multicomponents combinations in laboratory‐prepared mixtures and in commercial syrups, and the results were compared with each other.

HPLC–DAD Determination of Seven Antioxidants and Caffeine in Different Phytopharmaceuticals

A high-performance liquid chromatography method employing diode array detection was developed to determine levels of the major catechins, proanthocyanidin (procyanidin B2), caffeine, thymoquinone and carvacrol and its isomer, thymol, which are present in different natural complex matrices found in commercial products of Camellia sinensis L. and/or Nigella sativa L. Reversed-phase separation was performed on a C18 column by using gradient elution by varying the proportions of solvent A (distilled water containing 0.05% orthophosphoric acid) and solvent B (acetonitrile), with a flow rate of 1.5 mL/min and duration of 31 min. Excellent linearity was observed for all standard calibration curves, and correlation coefficients were above 0.9996. The developed method is efficient, with high reproducibility and sensitivity, and is ideally suited for rapid and routine analysis of principal components in these promising medicinal plants.

Microbial production of 1α-hydroxyvitamin D3 from vitamin D3

The microbial transformation of vitamin D3 (1) by the fungi Candida maltosa R42 and Botrytis allii NRRL 2502 was investigated. Incubation of compound 1 with C. maltosa R42 and B. allii NRRL 2502 produced the same three more polar metabolites in small yields. The main metabolite was identified as 1α-hydroxyvitamin D3 (2). This biotransformation has utility as a possible tool for the production of 1α-hydroxyvitamin D3 from the readily available vitamin D3 for patients with compromised kidney function.

QUANTITATIVE DETERMINATION OF GLUTATHIONE IN PRESENCE OF ITS DEGRADANT IN A PHARMACEUTICAL PREPARATION USING HPLC-DAD AND IDENTIFICATION BY LC-ESI-MS

A simple and rapid method for the quantification of the well-known multifunctional antioxidant glutathione in presence of its degradant in a pharmaceutical preparation using HPLC with a photodiode array detector was developed and validated for linearity, repeatability, limit of detection, and limit of quantitation. In addition, a high performance liquid chromatography coupled with electrospray ionization mass spectrometry method was described for its identification. Chromatographic separation and quantitation were made on a 250 mm × 4.6 mm (i.d.) C 18 column using 0.1% formic acid in water–methanol, in the ratio of (90:10, v/v) as mobile phase for the HPLC–DAD method. For structural identification chromatographic separation was performed on an YMC TM ODS-AQ 150 mm × 2 mm (i.d.) C 18 column using 0.1% formic acid in deionized water–methanol in ratio of (90:10, v/v) as mobile phase for LC-ESI-MS method. This analytical method provides a sensitive, rapid, and simple method with minimal sample preparation suitable for the stability control and routine analysis of glutathione-based commercial preparations.