1 CycloLab Cyclodextrin R&D Laboratory Ltd., Budapest, Hungary, e-mail : szeman .j @cyclolab.hu

1. Evaluation of European Pharmacopoeia Method for Analysis of Hydroxypropylbetadex: Proposal for Improvement Katalin Csabai,1 Julianna Szemán1, Gábor Varga2, Lajos Szente1 1CycloLabCyclodextrin R&D Laboratory Ltd., Budapest, Hungary, e-mail: szeman.j@cyclolab.hu 2ChiroQuest Chiral Technologies Development Ltd., Budapest, Hungary INTRODUCTION Pharmacopoeia method Description: European Pharmacopoeia 5.04 (Page1771-73) Related substances. Liquid chromatography Column: - size: l=0.30m, Ø=3.9mm - stationary phase: phenylsilyl silica gel for chromatography R, - temperature: 40C Mobile phase: water for chromatography R Flow rate: 1.5 ml/min Detection: differential refractometer, at 40C Run time: 3 times the retention time of BCD Relative retention (r):with reference to impurity B (PRG) (tR = about 2.5 min.);impurity A (BCD) about 4.2;Hydroxypropylbetadex about 6for the beginning of the elution System suitability: -resolution: minimum 4 between the peaks due to impurity BCDand impurity PRG Hydroxypropylbetadex elutes as a very wide peak or several peaks Hydroxypropylbetadex ((2-hydroxy)propyl b-cyclodextrin, HPBCD) a statistically substituted derivative of Betadex (b-cyclodextrin, BCD), has long been used successfully as additive in drug delivery to increase the aqueous solubility and stability of drugs - even in marketed drug products. For identification and characterisation of the statistically substituted cyclodextrin derivatives – like HPBCD – fingerprint chromatograms obtained on reversed phase (C8, C18, Phenyl) or normal phase (amino bonded silica) HPLC column are used [1-10]. European Pharmacopoeia (EP) prescribes phenylsilyl silica gel stationary phase with water as mobile phase for determination of the remnant un-substituted BCD and propylene glycol (PRG) in HPBCD. The separationis based oninclusion complex formation between of the phenyl groups on the stationary phases and the analyte cyclodextrins. The different separation potency of phenyl columns (surface coverage, free silanols) obtained from different manufacturers, however, has a strong influence on the separation of cyclodextrin derivatives [10]. In this work we have studied applicability of the Pharmacopoeia method considering the knowledge of the inclusion complex formation properties of the substituted cyclodextrin derivatives. An alternative analysis method is also given using a special phenyl column developed for cyclodextrin analysis [11]. RESULTS AND DISCUSSION The components of HPBCD form strong inclusion complexes with phenyl groups depending on the degree of substitution of the respective components. In all probability, some components of HPBCD can not be eluted from the column by water. Theoretical considerations Adaptation of EP method on different phenyl columns AlphaBond Phenyl mBondapackPhenyl YMC-Pack Phenyl l : 0.3m, Ø:4.6mm, particle size:10mm l : 0.30m, Ø:3.9mm, particle size: 10mm l : 0.25m, Ø:4.6mm, particle size: 5mm • Propylene glycol (PRG) has very low retention on phenyl columns (retention factor 0.1-1.1), therefore its evaluation is disturbed by the system peaks caused by the water content of samples • The resolution between BCD and PRG is better than the prescribed limit (Rs minimum 4), except AlphaBond column • Although the tested columns meets the requirement of the EP method, the relative retention of BCD is lower than the given value • The relative retention of the first peak of HPBCD is lower than the given value (r ~ 6) • The resolution between the BCD peak and the first HPBCD peak is very low on the AlphaBond column • The baseline is not stable at the prescribed run time (3 times the retention time of BCD), components of HPBCD remained on the column??? Chromatograms of BCD, PRG and HPBCD Mobile phase: water, RI detection RIU 220 Run time 200 PRG BCD 180 160 140 120 Characteristic data on three different phenyl columns 10 20 30 40 min Chromatograms of BCD, PRG and HPBCD Mobile phase: water, gradient with methanol,ELS detection ADC1 A, ADC1 (P:\ARCHIV~1\HPBCD-EP\HPB0322\HPBG2R2.D) PMP1, Solvent B Norm. 100 80 60 BCD 40 BCD 20 High amount of HPBCD was washed by the methanol gradient from the columns 0 0 5 10 15 20 25 min Effect of the non-eluted HPBCD on the separation Alternative method Consecutive injection of HPBCD(mBondapack column,run time: 3 times of BCD retention time) Washing procedure using RI detection Description:. Liquid chromatography Column: - size: l=0.25m, Ø=4.0 mm - stationary phase: CD-Screen,a special phenyl type column, developed and tested for separation of cyclodextrins - temperature:30C. Mobile phase: methanol and water 45 : 55 Flow rate: 0.7 ml/min. Detection: differential refractometer, at 40C Run time: 6 times the retention time of BCD(depends on the degree of substitution of HPBCD) Relative retention:with reference to impurity B (PRG) (tR = about 4.3 min.); BCD about 4.8; HPBCD about 6.2for the beginning of the elution System suitability:-resolution: minimum 2 between peaks due to BCD and the first peak of HPBCD • Washing with methanol starts from the run time, and takes minimum 15 min. • Wash-back to water mobile phase: to get stable base line takes about 80 min. • The baseline is not stable, and shifted to higher refraction values, therefore the evaluation of peak areas is doubtful • The retention time of BCD peak shows decreasing tendency Change of reference chromatograms Change of HPBCD chromatograms Necessary run + wash time time about 100 min. RIU RIU 240 No. of injection first peak No. of injection 350 220 6 BCD 6 PRG 4 200 300 HPBCD 4 2 PRG 180 ADC1 A, ADC1 CHANNEL A (S:\HPLC1\CDDERI~1\HPBE0311\HPBCD11.D) BCD 250 Pump 1, Solvent B: VIZ:MEOH=10:90 (11-Mar-05, 11:28:33) 2 1 mV 160 1 200 100 140 150 120 80 100 100 Reproducible chromatograms Six replicate injections of HPBCD Characteristic fingerprint chromatograms HPBCD samples with different degrees of substitution 0 2 4 6 8 10 12 14 16 18 min 0 2 4 6 8 10 12 14 16 18 min 60 Analysis Washing period BCD 40 CONCLUSIONS 20 0 5 10 15 20 25 min Pharmacopoeia method  Components of HPBCD remained on the column resulted in changed column performance after consecutive injectionsinstable chromatographic system  Washing HPBCD with methanol long analysis time  Resolution between BCD and the first peak of HPBCD is not prescribed, but can influence the evaluation of BCD peak  System peaks disturb the evaluation of propylene glycol (PRG) peak Alternative method  All components of HPBCD are eluted from the columnstable chromatographic system, reproducible chromatograms, acceptable run time  HPBCD elutes as a characteristic fingerprint (possibility of identification)  Evaluation of BCD peakis reproducible, resolution between BCD and the first peak of HPBCD is a system suitability factor  System peaks still disturb the evaluation of propylene glycol (PRG) peak  use of GC method is advisable REFERENCES [1]  G. 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Csabai, K. Kékesi, l. Szente, G. Varga; J. Chromatography A, 1116, 76-82 (2006) ACKNOWLEDGEMENT The authors are grateful to Ms. Zs. Zachár and Ms. E. Erdei to their valuable technical assistance. The work was supplied by the National Research Fund (NKFP-1A-041/2004 and NKFP1-012/2005). EXPERIMENTAL Apparatus: Agilent 1050 HPLC system with Evaporative Light Scattering Detector PL-ELS 1000, (Polymer Laboratories), or Refractive Index Detector ERC-7515B (Erkatech) ELS Detector parameters: Evaporation: 110°C, Nebulization: 90 °C, Gas flow: 1.2 l/min. RI Detector parameters: Fast mode, 40°C Columns:AlphaBond Phenyl (Alltech Chromatography, USA), mBondapack Phenyl (Waters Corp., USA), YMC-Pack Phenyl (YMC Europe GmbH), CD-Screen (ChiroQuest Ltd, Hungary) Samples:BCD and HPBCD were products of Cyclolab Ltd., Hungary and Wacker Chemie, Germany.