NATURE OF THE MAIN CONTAMINANT IN THE DRUG PRIMAQUINE di-PHOSPHATE: COMPARISON of HPLC,

1. SPONSORS University of Oslo DEPARTMENT OF MOLECULAR BIOSCIENCE, UNIVERSITY OF OSLO, P.O.BOX 1041, BLINDERN, 0316 OSLO, NORWAY NATURE OF THE MAIN CONTAMINANT IN THE DRUG PRIMAQUINE di-PHOSPHATE: COMPARISON of HPLC, GC and SFC METHODS of ANALYSIS E-Mail: ilia.brondz@biokjemi.uio.no ILIA BRONDZ1*, UWE KLEIN1, LARYSA KARALIOVA2, VASO VLACHOS3, PAUL OAKLEY3, ROBERT LEIDEBORG4 andFELIX MIKHAILITSYN5. 1Department of Molecular Biosciences, University of Oslo, Oslo, Norway. E-mail: ilia.brondz@biokjemi.uio.no 2Jupiter AS, 1400 Ski, Norway. 3Berger SFC, Mettler-Toledo, Newark, DE, USA.. 4 Mettler-Toledo AutoChem, Stockholm, Sweden. 5Martsinovsky Institute of medical parasitology and tropical medicine, Moscow, Russia. The aim of this work was to compare the GC, HPLC and SFC methods to separate quinocide contaminant in the anti-malaria drug primaquine from the primaquine. INTRODUCTION Malaria is one of the most deadly diseases on earth with an estimated death rate of up to 2.7 million humans per year. The drug primaquine di-phosphate is used for causative treatment of infections. Using HPLC equipped with a chiral column and HPLC-MS equipped with a reverse-phase column, we were able to show that the main contaminant of primaquine is the positional isomer quinocide [1]. Using GC, GC-MS we supported previously results and were able to work out the detailed fragmentation map for primaquine and quinocide and to compare the fragmentation during the MS [2]. HPLC methods for analyzing primaquine are main methods in the Pharmacopeias and in pharmaceutical industries. It has previously been assumed that the main contaminant of primaquine is the enantiomer, and a possible separation of the racemate has been reported. In some publications, the separation of the positional isomer from primaquine was mistaken as separation of the stereoisomer. Separation of isomers should be supported by MS analysis. However, by HPLC on the columns used [1] the isomers of primaquine were not adequately separated Fig. 1 and therefore it is difficult to quantify the highly toxic contaminant, quinocide. A novel method for separation of quinocide from primaquine was developed on the HPLC Discovery HS-F5 column resulting in base-line resolution suitable for quantification Fig. 2. Packed-column SFC was also tested as an alternative to HPLC and revealed more realistic quantification Fig. 3 and 4. CONCLUSION 1. The most suitable HPLC column for separation of quinocide from primaquine is Discovery HS-F5 (SUPELCO). The column gives base-line separation of the important contaminant of primaquine. 2. The most suitable methodology to analyze contaminants in the drug primaquine is SFC. SFC gives thye possibility to analyze samples in the absence of water, and prolongs the life time of the contaminant to achieve the correct analytical results. MATERIALS AND METHODS The details about the HPLC analysis are described in [1], an Agilent 1100 HPLC chromatograph with diode array detector (DAD) fitted with the Discovery column was used. The Discovery HS-F5 column, 25cm x 4.6mm i.d. 5um particle size (Supelco, Bellefonte, PA, USA) was utilized to enhance the separation. Base-line separation was achieved isocratically by varying the buffer proportion in the acetonitrile mobile phase at a flow rate of 1.0 mL/min, Fig 2. The mobile phase composition finally chosen was acetonitrile/20 mM ammonium acetate in distilled water, pH 7.0, 50/50. The analytes were detected at 268 nm with reference at 300 nm. The details about GC analysis are described in [2]. For the SFC analysis a Berger SFC MiniGram system (Berger Instruments, Inc., DE, USA) equipped with 2-Ethyl-Pyridine or Discovery HS-F5 column was used. Mobile phase was liquid CO2 with gradient (EtOH + 0.4%DiEtAm), detection was done by a Knauer UV detector at 268 nm. Resolution on the 2-Ethyl-Pyridine column is shown in Fig. 3, and on Discovery HS-F5 is shown in Fig. 4. DISCUSSION The HPLC equipped with the Discovery HS-F5 column has unique selectivity for the positional isomers of primaquine and excellent separation characteristics in comparison with Adsorbosphere Nucleotide-Nucleoside, 7um, Chirex (S)-VAL and Chirex (R)-NEA as well as other stationary phases. We used SFC equipped with 2-Ethyl-Pyridine and Discovery HS-F5 columns to evaluate biological test results published in [1] which were in contradiction to analytical quantification. It was possible to show that the actual quantity of contaminant quinocide in drug primaquine di-phosphate used by industry is much higher than is shown in HPLC, because of instability of quinocide. SFC generally shows better separation power for isomers than HPLC and the Discovery HS-F5 phase shows stronger separation power than any other of the columns tested in experiments with HPLC and SFC Fig. 1 - 4. Fig. 1 Fig. 2 Fig. 3 1.. Ilia Brondz, Dimitris Mantzilas, Uwe Klein, Dag Ekeberg, Erlend Hvattum, Marina N. Lebedeva, Felix S. Mikhailitsyn, Gasan D. Souleimanov, Johan Røe, Nature of the main contaminant in the anti malaria drug primaquine di-phosphate: a qualitative isomer analysis, Journal of Chromatography B, 800 (2004) p. 211. 2. Ilia Brondz, Uwe Klein, Dag Ekeberg, Dimitris Mantzilas, Erlend Hvattum, Henrik Schultz, Felix S. Mikhailitsyn, Nature of the main contaminant in the anti malaria drug primaquine di-phosphate: GC-MS analysis, Asian Journal of Chemistry, 17 (3) (2005) in press. Fig. 4