Cheryl L. Flurer

Analysis of antibiotics by capillary electrophoresis

The broad category of antibiotics encompasses some of the most widely prescribed pharmaceuticals in the world. As is the case with any pharmaceutical, an antibiotic must be characterized in terms of its potency or activity, and the presence and quantity of impurities. Additionally, any residue or metabolite that may be present as a result of its use must be monitored. Many capillary electrophoretic techniques have been utilized in the analysis of antibiotics, addressing the various aspects of their quantitation, profiling, and monitoring. Some of the more recent applications are summarized in this review article.

Determination of ephedrine compounds in nutritional supplements by cyclodextrin-modified capillary electrophoresis

Capillary electrophoresis was utilized for the separation, identification, and quantitation of ten stereoisomers in the ephedrine family. Chiral discrimination was accomplished through the use of hydroxypropyl-beta-cyclodextrin, and separation was enhanced at pH 2 in the presence of tetramethylammonium chloride and sodium dodecyl sulfate. Calibration plots of the ephedrines were linear over the range 4-100 micrograms/ml. This method was used in the analysis of nutritional supplements that contain Ma Huang, a Chinese herbal preparation that is made from plants in the genus Ephedra.

The analysis of aminoglycoside antibiotics by capillary electrophoresis

The analyses of aminoglycoside antibiotics by capillary electrophoresis utilizing borate complexation and direct UV detection are discussed. Twelve aminoglycosides were studied and separated to demonstrate identification capabilities, with migration time RSDs from 0.21 to 0.44% (n = 6) for individual components. This buffer system permitted the detection of minor impurities such as precursors or closely related fermentation products. Quantification of dihydrostreptomycin and streptomycin was accomplished in 160 mM sodium tetraborate decahydrate with linearity over the range 0.050-1.0 mg ml-1. Determination of the purity of bulk dihydrostreptomycin was possible by the addition of the cationic surfactant myristyltrimethylammonium bromide. This reversed the electroosmotic flow, thereby reversing the migration order, and causing the streptomycin impurity to migrate before the dihydrostreptomycin main peak. Quantification was also demonstrated with the closely related compounds amikacin, bekanamycin, kanamycin A, and tobramycin, using sisomicin as an internal standard. The reproducibility of the method was typically 2-3% over 1 day, and 2% day-to-day. These studies illustrate the use of capillary electrophoresis for the identification and quantification of selected aminoglycosides as potential alternative methods to the assays given by the US Pharmacopeia.

Quantitation of gentamicin sulfate in injectable solutions by capillary electrophoresis

The results presented in this communication establish the use of capillary electrophoresis as a tool in the analysis of injectable solutions containing the antibiotic complex gentamicin sulfate. The utilization of a borate buffer leads to the separation of the individual components and their visualization by direct UV detection. This rapid and straightforward procedure yields qualitative and quantitative information simultaneously, and could be utilized as an alternative to the multiple assays required by current US Pharmacopoeia protocols.

Chemical profiling of pharmaceuticals by capillary electrophoresis in the determination of drug origin

Capillary electrophoresis has been utilized to detect trace components in bulk pharmaceutical products, with emphasis on the identification of differences among manufacturers that can be used for source verification in suspect/counterfeit cases. Micellar electrokinetic capillary chromatography with sodium dodecyl sulfate was used in the analyses of beta-lactam antibiotics. The aminoglycoside clindamycin phosphate and the macrolide erythromycin stearate were analyzed using borate buffers with direct UV detection. Methyl-beta-cyclodextrin was used as a buffer additive in the erythromycin studies. Determination of product potency using peak area ratios has been demonstrated for ampicillin and clindamycin phosphate.

Structural characterization of sulfoaildenafil, an analog of sildenafil.

Phosphodiesterase type 5 (PDE-5) inhibitors represent a class of drugs used primarily in the treatment of erectile dysfunction. Currently, three PDE-5 inhibitors have been approved by the U.S. Food and Drug Administration (FDA) for use in the United States: sildenafil citrate, tadalafil, and vardenafil hydrochloride trihydrate. A bulk material, labeled as an ingredient for a dietary supplement, was analyzed for the presence of PDE-5 inhibitors. The compound that was detected displayed structural similarities to sildenafil, and was characterized further using LC-MS(n), FTICRMS, X-ray crystallography and NMR. The compound was given the name sulfoaildenafil. When compared to sildenafil, sulfoaildenafil contains a sulfur atom substitution for the oxygen atom in the pyrazolopyrimidine portion of the molecule, and a 3,5-dimethyl substitution on the piperazine ring, rather than the 4-methyl moiety. The X-ray crystallographic data indicate that the material in this sample is comprised of two polymorphs, which may affect the chemical and/or biological properties of any product formulated with this compound.

Detection of adulteration of locust bean gum with guar gum by capillary electrophoresis and polarized light microscopy.

Capillary electrophoresis (CE) and polarized light microscopy (PLM) were utilized in the detection of the adulteration of locust bean gum with guar gum. For CE analyses, standards of locust bean and guar gums were extracted with 30% CH3CN, removing the residual proteins from the gum matrix. A 8.75 mM NaH2PO4-20.6 mm Na2b4O7 buffer, pH 9, was used to separate these proteins and to identify marker proteins that were present in the guar gum. These markers did not co-migrate with components in the extracts of mechanically processed locust bean gum, and are used as indicators of adulteration. Using PLM with toluidine blue and iodine staining techniques, unadulterated locust bean gum samples were distinguished from mixed samples through the differential staining of components in locust bean versus guar and tara gums. These experiments in the use of CE and PLM provide orthogonal and complementary methods for the verification of ‘true’ positives and the elimination of ‘false’ positives.

Characterization of galactomannans by capillary electrophoresis.

Capillary electrophoresis (CE) was utilized in the characterization of various galactomannans. Standards of gums were extracted with 50% CH3CN to remove the residual proteins from the gum matrix. Separation buffers were optimized with respect to pH, buffer concentration and presence of sodium dodecyl sulphate, yielding protein profiles from which the desired information could be obtained. Examples are given of the profiles generated by various gums and gum blends to aid in the verification of component presence, and to demonstrate levels of adulteration detectable under the buffer conditions used.

Determination of Phosphodiesterase-5 Inhibitors and Analogs Using High-Performance Liquid Chromatography with Ultraviolet Detection

A considerable number of erectile dysfunction products, and dietary supplements suspected of containing phosphodiesterase-5 (PDE-5) inhibitors, have been analyzed by the US Food and Drug Administration. Often these samples are found to contain the approved active pharmaceutical ingredients (APIs) such as sildenafil, tadalafil or vardenafil. However, analogs of these APIs have also been identified in many samples and products containing multiple PDE-5 inhibitors have also been found. A single high-performance liquid chromatography with ultraviolet detection method has been developed for the determination of sildenafil, tadalafil, vardenafil and a number of commonly encountered analogs in pharmaceutical dosage forms and dietary supplement products, including tablets, capsules, bulk powders, troches and liquids. This method was designed as an alternative to methods developed for the determination of a single PDE-5 inhibitor. Using this protocol, 14 PDE-5 inhibitor compounds can be separated and determined in a single analysis.

Development and implementation of a pass/fail field-friendly method for detecting sildenafil in suspect pharmaceutical tablets using a handheld Raman spectrometer and silver colloids

HIGHLIGHTSHandheld Raman spectrometers and silver colloids are used to detect sildenafil in suspect tablets.The method is based on a pass/fail platform that can be taught to non‐experts in about 20 min.The method involves measuring sample solutions directly through glass vials (no drying required).The method proved to be 92.6% effective on average for 117 counterfeit and unapproved drugs.The method yielded detection limits in samples as low as 10 ppm. ABSTRACT A simple, fast, sensitive and effective pass/fail field‐friendly method has been developed for detecting sildenafil in suspect Viagra and unapproved tablets using handheld Raman spectrometers and silver colloids. The method involves dissolving a portion of a tablet in water followed by filtration and addition of silver colloids, resulting in a solution that can be measured directly through a glass vial. Over one hundred counterfeit Viagra and unapproved tablets were examined on three different devices during the method development phase of the study. While the pass/fail approach was found to be 92.6% effective on average, the efficacy increased to 97.4% on average when coupled with the softwares “Discover Mode” feature that allows the user to compare a suspect spectrum to that of a stored sildenafil spectrum. The lowest concentration of sildenafil in a water/colloid solution that yielded a “Pass” was found to be 7.6 &mgr;g/mL or 7.6 parts per million (ppm). For the analysis of suspect tablets, this value was found to be as low as 10 &mgr;g/mL and as high as 625 &mgr;g/mL. This variability was likely related to the tablet formulation, e.g., concentration of sildenafil, presence and concentration of water‐soluble and/or water‐insoluble ingredients. However, since most counterfeit Viagra and unapproved tablets contain >50 mg sildenafil per tablet, such low concentrations will not be encountered often. Limited in‐lab and in‐field validation studies were conducted in which analysts/field agents followed the procedure outlined in this study for small sample sets. These individuals were provided with written instructions, a ˜20 min demonstration regarding how to perform the procedure and use the instrument, and a kit with field‐friendly supplies (purified bottled water from a local grocery store, disposable plastic pipettes, eye‐dropper with a silver colloid solution, etc.). The method proved to be 98.3% and 91.7% effective for the in‐lab and in‐field validation studies, respectively, which demonstrated the ruggedness, simplicity and practicality of the method.