Evaluation of European Pharmacopoeia Method for Analysis of Evaluation of European Pharmacopoeia Method for Analysis of

Hydroxypropylbetadex: Proposal for ImprovementHydroxypropylbetadex: Proposal for Improvement

1 CycloLab Cyclodextrin R&D Laboratory Ltd., Budapest, Hungary, e-mail: szeman.j@cyclolab.hu 2 ChiroQuest Chiral Technologies Development Ltd., Budapest, Hungary

Katalin Csabai,1 Julianna Szemán1, Gábor Varga2, Lajos Szente1

Hydroxypropylbetadex ((2-hydroxy)propyl -cyclodextrin, HPBCD) a statistically substituted derivative of Betadex (-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].

INTRODUCTIONINTRODUCTION

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: 40CMobile phase: water for chromatography R

Flow rate: 1.5 ml/min

Detection: differential refractometer, at 40CRun 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

RESULTS RESULTS ANDAND DISCUSSION DISCUSSION

Theoretical considerations 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.

• 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???

Adaptation of EP method on different phenyl columns

Characteristic data on three different phenyl columns

AlphaBond Phenyl

Chromatograms of BCD, PRG and HPBCD Mobile phase: water, RI detection

l : 0.25m, Ø:4.6mm, particle size: 5m

YMC-Pack PhenylBondapack Phenyll : 0.3m, Ø:4.6mm, particle size:10m l : 0.30m, Ø:3.9mm, particle size: 10m

min5 10 15 20 25

mV

0

20

40

60

80

100

ADC1 A, ADC1 CHANNEL A (S:\HPLC1\CDDERI~1\HPBE0311\HPBCD11.D)

BC

D

Pump 1, Solvent B: VIZ:MEOH=10:90 (11-Mar-05, 11:28:33)

min5 10 15 20 25

mV

0

20

40

60

80

100

120

140

ADC1 A, ADC1 CHANNEL A (E:\CDPHDATA\YMC0921\HPBCD1.D)

BC

D

Pump 1, Solvent B: MeOH-DV 9-1 v/v (21-Sep-04, 12:58:47)

Chromatograms of BCD, PRG and HPBCD Mobile phase: water, gradient with methanol, ELS detection

High amount of HPBCD was washed by the methanol gradient from the columns

Effect of the non-eluted HPBCD on the separation

Consecutive injection of HPBCD (Bondapack column, run time: 3 times of BCD retention time)

Washing procedure using RI detection

• 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

min10 20 30 40 50 60 70 80 90

RIU

40

60

80

100

120

140

160

ADC1 A, ADC1 (G:\DATA\HPBE0823\HPBCDWU1.D)

1. injection

2. injection after washing

ADC1 A, ADC1 (G:\DATA\HPBE0823\HPBCDWU2.D)

CONCLUSIONSCONCLUSIONS

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

[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)

REFERENCES

Alternative methodAlternative method

Description:. Liquid chromatographyColumn: - 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: 30C.Mobile phase: methanol and water 45 : 55Flow rate: 0.7 ml/min.Detection: differential refractometer, at 40CRun 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 elutionSystem suitability:- resolution: minimum 2 between peaks due to BCD and the

first peak of HPBCD

min0 10 20 30 40

RIU

60

80

100

120

140

160

180

BC

DH

PB

CD

1.

peak

PR

G

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

Reproducible chromatograms Six replicate injections of HPBCD

Characteristic fingerprint chromatograms HPBCD samples with different degrees of substitution

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°CColumns: AlphaBond Phenyl (Alltech Chromatography, USA), Bondapack 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.

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

ACKNOWLEDGEMENT

• 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.

Necessary run + wash time time about 100 min.

Analysis Washing period

min10 20 30 40

RIU

80

90

100

110

120

130

140

ADC1 A, ADC1 (E:\HPB042\HPB1025\HPBVAL1.D)

DS = 3.5

BC

D

min10 20 30 40

RIU

70

80

90

100

110

ADC1 A, ADC1 (E:\HPB042\HPB1025\74B008.D)

DS = 4.3

BC

D

min10 20 30 40

RIU

77.5

80

82.5

85

87.5

90

92.5

ADC1 A, ADC1 (E:\HPB042\HPB1025\74B004.D)

DS = 6.2

BC

D

min5 10 15 20 25 30 35 40

RIU

80

90

100

110

120

130

ADC1 A, ADC1 (S:\HPLC2\2005RE~1\HPB05\HPBE0301\HPBCD2.D)

Ru

n t

ime

BC

DP

RG

min10 20 30 40 50 60

RIU

70

80

90

100

110

120

130

140

150

ADC1 A, ADC1 (E:\HPB042\HPB0920\HPBCD1.D)

PR

G BC

D

Ru

n t

ime

Pharmacopoeia method

min0 5 10 15 20 25

Norm.

0

20

40

60

80

100

ADC1 A, ADC1 (P:\ARCHIV~1\HPBCD-EP\HPB0322\HPBG2R2.D)

BC

D

PMP1, Solvent B

min10 20 30 40

RIU

120

140

160

180

200

220

PR

G

BC

D

Ru

n t

ime

min0 2 4 6 8 10 12 14 16 18

RIU

100

120

140

160

180

200

220

240

PR

G

BC

D

HP

BC

Dfi

rst p

eak

No. of injection

6

4

2

1

min0 2 4 6 8 10 12 14 16 18

RIU

100

150

200

250

300

350

PR

G

BC

D

No. of injection

6

4

2

1

BC

D

13.1

9.81

2.46

Resolution BCD / PRG

Prescribed: minimum 4

8.94.82.63.11YMC-Pack

4.23.72.42.62Bondapack

1.33.82.72.25AlphaBond

Resolution BCD / HPBCDNot prescribed

Relative ret. of HPBCD

Prescribed: ~ 6

Relative ret. of BCD

Prescribed: ~ 4

Retention time of PRG

Prescribed: ~2.5 min

Phenyl stationary

phase

13.1

9.81

2.46

Resolution BCD / PRG

Prescribed: minimum 4

8.94.82.63.11YMC-Pack

4.23.72.42.62Bondapack

1.33.82.72.25AlphaBond

Resolution BCD / HPBCDNot prescribed

Relative ret. of HPBCD

Prescribed: ~ 6

Relative ret. of BCD

Prescribed: ~ 4

Retention time of PRG

Prescribed: ~2.5 min

Phenyl stationary

phase