Highly sensitive ergotamine fluorescent determination in pharmaceuticals and biological samples prior cloud point extraction

WANG, CHIEN CHUN; FERNANDEZ, LILIANA; GOMEZ, ROXANA A.

Santa Fe

Congreso; Reunión Internacional de Ciencias Farmacéuticas RICiFa; 2012

Introduction Organized surfactant media have found increasing application in many areas of separation science, due to their singular properties and unquestionable advantages with respect to conventional phase separation techniques. The crucial factor in successful application of micellar systems is associated with the ability to selectively solubilizing and interacting with solute molecules. Cloud point extraction (CPE) has demonstrated the efficiency for concentrating several analytes from aqueous samples, including organic pollutants such as pesticides, aromatic hydrocarbons and to monitoring drug level in biological samples (1), with the advantages of needing small amount of non-toxic reagents and generating environmental friendly wastes. Ergotamine belongs to the ergot family of alkaloids, secondary metabolites of Claviceps ssp. It was first isolated in 1918 and has been used for therapeutic purposes since the 1950s to treat vascular headache. It has antiserotonin effects, -adrenergic blocking activity and a direct stimulating action on smooth muscle, especially of blood vessels and uterus (oxytocic effect) (2). This drug is highly toxic and in large, repeated doses can produce symptoms of ergot poisoning, known as ergotism, manifesting painful seizures, spasms, diarrhea, psychosis, hallucinations, nausea, vomiting and the marked vasoconstriction can cause gangrene, especially in distal structures such as fingers and toes. In this work, the high extraction efficiency of CPE procedure has been combined with the inherent sensitivity of molecular fluorescence for the analysis of ergotamine. Emission advantages of undiluted surfactant rich-phase in rigid gel state have been explored, differently to the well known CPE procedure, in which the surfactant-rich phase is diluted in order to diminish its high viscosity. Materials and methods A Shimadzu RF-5301PC spectrofluorimeter equipped with a Xenon discharge lamp and a micro-quartz cell of 1 cm pathlength (0.3 mL) were used for fluorescence measurements. Ergotamine tartrate used for standard solution was kindly provided by Andrómaco S.A. (Bs. As., Argentine). Extracting solution was prepared from an anionic surfactant PONPE 7.5 (polyoxyethylene(7.5)nonylphenylether) ethanol and water (10:40:50) (v/v). Pharmaceuticals sample solutions were obtained dissolving tablets of two trademarks labeled as containing 1 mg ergotamine in association with caffeine, methochlopramide and dipyrone (TetralginTM CRAVERI, Bs. As., Argentine) and with caffeine and ibuprofen (Ibupirac MigraTM PFIZER, Bs. As., Argentine). Aliquots of 10 mL of urine were centrifuged for 5 min; 3 mL of supernatants were collected from each tube and stored at 5 ºC. Aliquots of 5 mL of saliva were diluted at double in volume with water and were centrifuged for 10 min; 2 mL of supernatants were collected and stored at 5 ºC. For general procedure, aliquots of standard working/sample solution were mixed with 1 mL of borax solution (1 10-2 mol L-1, pH 8.5), 300 μL of extracting solution and made up to 10 mL in a set of centrifuge tubes. After 10 min of centrifugation, the systems were cooled in an ice bath for 5 min to facilitate the phase separation. The remaining viscous surfactant-rich phase was then transferred by micropipette into the 300 μL quartz cell and the fluorescent emissions were measured at λem = 425 nm using λex = 313 nm. Results One important characteristic of ergotamine is the alkalinity of N6 with pKa values of 6.5. Thus ergotamine is positively charged in acidic solutions and neutral at pH values above 7.0. Therefore, ergotamine initially present in aqueous solution, at pH 8.5 is solubilized into the hydrophobic micellar core of the extractant, where the molecule is found as neutral form. After cloud point (for PONPE 7.5 occurs at room temperature) the homogeneous aqueous/surfactant solution is separated in two marked phases and ergotamine is extracted into the small surfactant rich phase. After pouring off the upper aqueous phase, the fluorescence emission of ergotamine was recorded directly from the highly viscous surfactant rich phase. In contrast to the traditional CPE procedure where the surfactant rich phase is diluted in order to facilitate its transference to the measurement cell; in the present methodology the spectral advantages of the gel state medium were explored. The enhancement of the enrichment factor is due to the viscous microenvironment which favored emission pathway deactivation of ergotamine instead of non-radiation relaxation of its excited state. Under optimal conditions, the calibration curve for ergotamine CPE was performed according to the general procedure. A linear calibration curve was obtained from 3.81 10-7 to 0.11 μg mL-1 with detection and quantification limits of 0.11 and 0.38 pg mL-1, respectively. In order to validate the accuracy of the methodology, a recovery study was applied to determine ergotamine in two trademarks of tablets and the obtained results were statistically compared to those obtained from an official method (UV-vis spectrophotometry) and from capillary electrophoresis, giving satisfactory results. The versatility of this methodology has been demonstrated by analysis of ergotamine in complex matrix such as urine and saliva. Conclusions The developed methodology for Ergotamine determination combines all known advantages of CPE with the inherent sensitivity of spectrofluorimetry. In comparison to traditional separation techniques, it is a simple operating procedure using non-toxic and environmentally friendly reagents. The distinctive characteristic of this proposal is the use of undiluted surfactant-rich phase in the determinative step with an enrichment factor of 1325 respect to the ethanol solution of ergotamine. The present methodology was successfully applied to the quality control of ergotamine in commercial pharmaceutical formulations. This methodology shows potentiality and versatility for the quality control of ergotamine in pharmaceutical formulations as well as in biological fluids drug monitoring. Acknowledgments The authors wish to thank to CONICET (Consejo Nacional de Investigaciones Científicas y Tecnológicas) and National University of San Luis (Project 22/Q828) for the financial support.