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European Pharmaceutical Students’ Association
DESIGNING AND VALIDATING AN HPLCUV METHOD FOR CONCENTRATION DETERMINATION OF 4-FLUORAMPHETAMINE IN HUMAN BLOOD SERUM Author: F. de Vries, BSc Scientific Coordinator: Dr F. Flesch, Ir. W.J.M. van den Bogaard, Ir. G.A.H Korte-Bouws Institution: Utrecht University
INTRODUCTION: The rising use of 4-fluoroamphetamine (4-FMP) has caused more presentations of intoxications in Dutch hospitals. Yet, there is no universal method to determine 4-FMP concentration in patients’ blood. AIM: Therefore the purpose of this thesis was to design and validate an HPLC-UV method for concentration determination of 4-FMP in human blood serum. MATERIAL AND METHODS: The 4-FMP was purchased online. Identification was executed via a melting point test, chloride test and via IR-spectrum, ESI-MS, 1H NMR and 13C NMR spectrum determination. For the HPLC-UV analysis, human blood serum was spiked with various concentrations 4-fluoramphetamine (respectively; 40 (HQC), 10, 4, 1 (MQC), 0.4 and 0.1 (LQC) µg/mL). 50 µL internal standard (amphetamine) was added and the samples were prepared for HPLC-UV analysis. This preparation consisted of basifying the spiked serum with 50 µL 1.0M NaOH and adding 5 mL ether, to extract 4-FMP and amphetamine to the organic-phase. This was transferred to a new tube and acidified with 200 µL 0.01 M HCl, extracting 4-FMP and amphetamine back to the water-phase. The ether was removed and the remainder was blown dry with nitrogen. 25 µL was injected, in duplo, in the HPLC-UV. The eluent consisted of 17.5% acetonitril plus 10 mM perchloric acid. The flow was 0.05 mL/min measured at a wavelength of 257 nm. RESULTS:The identification tests validated that the purchased substance was 4-FMP·HCl. UV-spectrometry determined that the
purity of the 4-FMP was 94,53% and that the substance contained no interfering components. Based on 3 parallels in duplo, the preparation methods’ recovery was 93.74% for 4-flouramphetamine and 91.11% for amphetamine. Via a 1/X transformed calibration curve (Y = 0.01955x – 3.043·10-5, R2 0.996) the accuracy was determined. 100% of the samples fell within the requirements. The average peak height of the 0.1 µg/mL LQC sample fulfilled the requirements of minimally being 5 times larger than noise. Furthermore, the deviations of the samples fell within the requirements, confirming the precision of the method. The stability of the stock-solution during 11 months was acceptable and there were no interferences with commonly used medicines. CONCLUSION: Identification results display that the purity of the 4-FMP·HCl was well enough for usage during the method design phase. The self-designed preparation and analysis method was accurate, precise and robust enough to be validated. Therefore this method could be used to determine the 4-FMP concentration in human bloodserum.
