Transcript Document

Evaluation of European Pharmacopoeia Method for Analysis of
Hydroxypropylbetadex: Proposal for Improvement
Katalin Csabai,1 Julianna Szemán1, Gábor Varga2, Lajos Szente1
1 CycloLab
2
Cyclodextrin R&D Laboratory Ltd., Budapest, Hungary, e-mail: [email protected]
ChiroQuest Chiral Technologies Development Ltd., Budapest, Hungary
INTRODUCTION
Pharmacopoeia method
Description: European Pharmacopoeia 5.04 (Page 1771-73)
Related substances. Liquid chromatography
Column:
- size: l = 0.30 m, Ø=3.9 mm
- 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 6 for the beginning o f the elution
System suitability: - resolution: minimum 4 between the peaks due to impurity
BCD
and 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 separation is based
on inclusion 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.
AlphaBond Phenyl
mBondapack Phenyl
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
Adaptation of EP method on different phenyl columns
• 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???
Characteristic data on three different phenyl columns
Chromatograms of BCD, PRG and HPBCD
Mobile phase: water, RI detection
Retention time of
PRG
Prescribed: ~2.5 min
Relative ret. of
BCD
Prescribed: ~ 4
Relative ret. of
HPBCD
Prescribed: ~ 6
Resolution
BCD / PRG
Prescribed: minimum 4
ADC1 A, ADC1 (E:\HPB042\HPB0920\HPBCD1.D)
Run time
RIU
140
Run time
BCD
180
PRG
110
Run time
120
PRG
200
150
PRG
BCD
220
130
130
120
110
160
100
100
140
90
90
80
120
70
80
5
Phenyl
stationary
phase
RIU
ADC1 A, ADC1 (S:\HPLC2\2005RE~ 1\HPB05\HPBE0301\HPBCD2.D)
RIU
BCD
Theoretical considerations
Resolution
BCD / HPBCD
Not prescribed
10
15
20
25
30
35
40 min
10
20
30
40
10
min
20
30
40
50
60
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
ADC1 A, ADC1 CHANNEL A (S:\HPLC1\CDDERI~1\HPBE0311\HPBCD11.D)
Pump 1, Solvent B: VIZ:MEOH=10:90 (11-Mar-05, 11:28:33)
AD C1 A, AD C1 CHANN EL A (E:\C DPHDATA\YMC0921\H PBCD 1.D)
Pump 1, Solv ent B: MeOH-DV 9-1 v /v (21-Sep-04, 12:58:47)
Norm.
mV
mV
100
140
AlphaBond
2.25
2.7
3.8
2.46
100
1.3
120
80
80
2.62
2.4
3.7
9.81
100
4.2
60
60
2.6
4.8
13.1
8.9
40
40
60
BCD
3.11
80
BCD
YMC-Pack
BCD
BCD
mBondapack
20
40
20
20
High amount of HPBCD was washed by the methanol gradient from the columns
0
0
0
0
5
10
15
20
25
5
10
Washing procedure using RI detection
(mBondapack column, run time: 3 times of BCD retention time)
• 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 HPBCD chromatograms
Necessary run + wash time time about 100 min.
RIU
No. of injection
350
180
2
No. of injection
RIU
160
6
140
4
HPBCD
200
4
BCD
PRG
250
PRG
6
BCD
220
300
ADC1 A, ADC1 (G:\DATA\HPBE0823\HPBCDWU1.D)
ADC1 A, ADC1 (G:\DATA\HPBE0823\HPBCDWU2.D)
first peak
240
120
100
1
80
1
200
1. injection
2. injection after washing
2
160
25
min
10
15
20
25
min
Alternative method
Consecutive injection of HPBCD
RIU
20
5
Effect of the non-eluted HPBCD on the separation
Change of reference chromatograms
15
min
140
60
150
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 6.2 for the beginning of the elution
System suitability:- resolution: minimum 2 between peaks due to BCD and the
first peak of HPBCD
120
40
4
6
8
10
12
14
16
18
min
100
0
2
4
6
8
10
12
14
16
18
min
10
Analysis
20
30
40
50
60
70
80
90
min
Reproducible chromatograms
Six replicate injections of HPBCD
Washing period
RIU
CONCLUSIONS
Characteristic fingerprint chromatograms
HPBCD samples with different degrees of substitution
180
160
ADC1 A, ADC1 (E:\HPB042\HPB1025\HPBVAL1.D)
RIU
140
130
120
110
DS = 3.5
BCD
2
100
90
80
10
ADC1 A, ADC1 (E:\HPB042\HPB1025\74B008.D)
RIU
140
110
100
20
30
BCD
0
H PB C D 1. pe a k
100
40
min
DS = 4.3
90
Pharmacopoeia method
100
BCD
PR G
120
80
70
10
ADC1 A, ADC1 (E:\HPB042\HPB1025\74B004.D)
20
30
40
min
RIU
 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
REFERENCES
[1] G. Liu, D. M. Goodall, J.S. Loran; Chirality, 5, 220-223 (1993)
[2] N. Rabearimonjy, S. Ounnar, M. Righezza, M. Dreux; Proc. 9th Int. Symp. Cyclodextrins, 1999, p.
19-22, Ed. J.J.T. Labandeira, J.L. Vila-Jato; Kluwer Academic Publishers, Dordrecht. Netherlands
[3] K. Koizumi, Y. Kubota, T. Utamura, S. Horiyama; J. Chromatogr., 368, 329-337 (1986)
[4] Y. Kubota, T. Tanimoto, S. Horiyama, K. Koizumi; Carbohydr. Res., 192, 159-166 (1989)
[5] G. Schomburg, A. Deege, H. Hinrichs, E. Hübinger; J. High Resolut. Chromatogr., 15, 579-584 (1992)
[6] I. Caron, C. Elfakir, M. Dreux; J. Higy Resolut. Chromatogr. 21, 554-560, (1998)
[7] I. Caron, A. Salvador, C. Elafkir, B. Herbretau, M. Dreux; J. Chromatogr. A, 746, 103-108 (1996)
[8] I. Caron, C. Elafkir, M. Dreux; J. Liq. Chrom. & Rel. Technol., 20, 1015-1035 (1997)
[9] A. Salvador, B. Herbretau, M. Dreux; J. Chromatogr. A, 855, 645-656 (1999)
[10] I. Caron, C. Elafkir, M. Dreux; Chromatographia 47, 383-390 (1998)
[11] J. Szemán, K. Csabai, K. Kékesi, l. Szente, G. Varga; J. Chromatography A, 1116, 76-82 (2006)
90
87.5
85
DS = 6.2
BCD
 Components of HPBCD remained on the column resulted in
changed column performance after consecutive injections
 instable chromatographic system
92.5
80
82.5
80
77.5
60
0
10
20
30
40
min
10
20
30
40
min
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 peak is 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
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.