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Changes in the level of
DESIGNING AND VALIDATING AN HPLC
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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.
Questions & answers
Please, tell us a little bit more about yourself. II am Fenna, a 25 year old Dutch pharmacy student. I have completed my bachelor’s degree at Utrecht University and I am currently halfway through my master’s degree at Leiden University. After completing my master’s degree I would like to pursue a doctorate degree and specialise as a hospital pharmacist. My interests within the field of pharmacy are; toxicology, therapeutic drug monitoring, illegal substances and pharmaceutical analysis. amphetamine. This way I could identify the amphetamine structure, remaining only the fluor atom and its location to identify. A 1H NMR and 13C NMR analysis were used to validate the presence of the fluor atom on the fourth carbon atom of the benzene ring.
In your opinion, what is the benefit of joining ESSP and what advice do you have for students undertaking research in the future? Joining ESSP can result in a (first) publication. This publication can form an addition to your resume. Besides, the process of writing an article and getting it published is an educational experience. My advice: take into account that research will take more time than anticipated. You will have major setbacks and weeks without any progress. But in the end, you will get the desired results.
Tell us a bit more about your research and its significance. The Netherlands and illegal drug use go handin-hand. The constant development of new designer drugs form a problem for, amongst others, drug-users, medics and authorities. For my bachelor thesis I designed and validated a method to determine 4-fluoroamphetamine concentrations in human blood serum. The 4-fluoroamphetamine level could be an indicator for the severity of intoxication, and thus could be useful to optimise the treatment.
What was the biggest challenge while carrying out the research and how did you overcome that? The 4-fluoroamphetamine used during research was ordered online. Therefore Ineeded to identify the substance as 4-fluoroamphetamine. Due to the short existence of the drug, there was barely any referential information available. To overcome this problem, I used the small amount of available data about 4-fluoroamphetamine and combined this with data about
