1. NITRO-PHENYL-CARBAMIDE HPLC STATIONARY PHASES;
CD-SCREEN COLUMN FOR ANALYSIS OF CYCLODEXTRIN-DERIVATIVES AND -SELECT COLUMN FOR GENERAL PURPOSE APPLICATIONS
Gábor Varga2, Katalin Csabai1, Lajos Szente1, Imre Klebovich3, Krisztina Ludányi3, Julianna Szemán1
1 CYCLOLAB Cyclodextrin R&D Laboratory Ltd., Illatos u. 7.,Budapest, H-1097, Hungary,
2 CHIROQUEST Chiral Technologies Development Ltd., Rumbach S. u. 7., Budapest, H-1075, Hungary,
3 SEMMELWEIS UNIVERSITY, Faculty of Pharmacy, Department of Pharmaceutics, Hőgyes E. u. 7., Budapest, H-1092, Hungary,
INTRODUCTION
In our previous work novel stationary phase was prepared by bonding N-(4-nitrophenyl)-carbamide group to the silica gel matrix [1,2]. The new phase has primarily been developed for analysis of cyclodextrins (CDs) and their derivatives.
Taking into account the structure of this selector it seemed to be a promising tool for the separation of various families of compounds. Based on theoretical considerations several types of interactions can be expected with different types of molecules. The main characteristic property of this stationary phase is the essential role of π-π interactions in the retention mechanism [3, 4]. Due to the electron-withdrawing nitro-group in para position, retention forces become stronger and shape selectivity can be significantly better compared to other commercially available phenyl-bonded silica phases [5].
In general, hydrogen bonding was regarded as one of the reasons of poor peak symmetry and low efficiency of chromatographic media. However, the rationally planned, well-defined, non silanol type hydrogen bonding capability can play an important role in the selectivity and retention. Due to the balanced ratio of several types of interactions, it can be supposed that this stationary phase would be suitable both for reversed and normal phase applications, as well.
Although the numerous types of p-p active HPLC phases have been already designed and investigated [3, 4], columns having diverse and multiple interacting sites for selectivity tuning can still expect great interest to solve separation problems e.g. in orthogonal chromatographic systems. The aim of this work was to study the separation potency of 4-nitrophenyl-carbamide stationary phases and to optimize its capability for different separation problems.
STRUCTURE OF THE SELECTOR
dipole
H-acceptor δ+
H-donors π-acceptor
hydrophylic-hydrophobic
Illustration of the interaction between the apolar cavity of cyclodextrin and the nitrophenyl-carbamide selector
Illustration of electrostatic potential surface difference of 4-nitrophenyl and phenyl-carbamide in complex with phenol and formation of hidrogen bonding
PRINCIPLE OF SEPARATION
The steric possibilities of the interactions were examined by molecular modelling methods. Both selector and solute molecular models were geometry-optimized using HyperChem MM+ molecular mechanics computational method. The energy minimization (relaxation) of the system consisting of these molecules together was the next step using the same method. The resulted complexes show clearly the presence of one or more hydrogen-bridges, depending on the chemical structure of analyte. Examining the electrostatic potential surfaces of the molecules in these complexes the role of the electron- withdrawing nitro-group in the retention mechanism can be easily understood.
RESULTS AND DISCUSSION
p-SELECT
CD-SCREEN
higher surface coverage compared to the CD-Screen stationary phase, to obtain strong p - p interaction
fully endcapped to eliminate the silanol interactions, to obtain well defined H-bonding
optimized surface coverage, fitting to the size of cyclodextrin molecules
secondary interactions with free silanols to increase the selectivity
Comparison of the results of hydroxypropyl-betadex analysis, obtained by European Pharmacopoeia method and CD-Screen column
Comparison to phenyl column
Polar analytes
Apolar, aromatic analytes
EP method
CD-Screen
π-Select
Conventional phenyl-column
k’=0.54
k’=0.25
Separation of phenol and caffeine
higher retention due to the -  interactions and well defined hydrogen-bonding
residual silanols are eliminated, good peak shape
in pH 2-8 interval the retention time of phenol is relatively high and does not depend on the pH (on conventional phenyl column the retention time of phenol is near to t0 at higher pH)
Separation of apolar, aromatic analytes
high retention on the -Select column, in spite of the fact that the carbon-content of this stationary phase is very low
the shape-selectivity and CH2-selectivity are higher, even diethyl-phthalate and biphenyl are well resolved.
π-Select
Conventional phenyl-column
C: 4.7%
C: 7.0%
BCD
Eluent: water
prescribed stop time
Number of
theoretical plates
for BCD:
1800 plates/m
Using mass-sensitive detector and gradient elution, it can be stated, that approx. 80% of hydroxypropyl-betadex is not eluted from the column before the prescribed stop time. Capacity factor of BCD peak is decreasing continuously due to the accumulation of the sample components on the stationary phase.
BCD
Eluent: 45% methanol
Number of
theoretical plates
for BCD:
32000 plates/m
During 20 min. HP-betadex components are eluted completely. Due to the unique retention mechanism and balanced hydrophilic/hydrophobic interactions, CD-Screen column makes it possible to perform fast, reproducible and high accuracy analysis, furthermore, it provides more detailed information on the sample.
Examination of the degradation products of HP-betadex on CD-Screen column
Eluent: MeOH-water 30:70
1.: Phenol 2.: Caffeine
Eluent: MeOH-water 60:40
1.: Dimethyl-phtalate; 2.: Diethyl-phtalate
3.: Biphenyl 4.: o-Terphenyl
CDs and CD derivatives are relatively stable substances, only a few articles can be found on their decomposition. However, to follow the hydrolytical, oxidative or enzymatic decomposition of CDs and their derivatives even in drug formulations can be interesting not only as research subject, but also from practical point of view.
Shape selectivity of p-Select column Polyaromatic hydrocarbons 1 2 3 6 7
1. Benzothiophene
4. Dibenzothiophene
7. Benzo[g,h,i]perylene
6. Benzo[a]pyrene
3. Fluoranthene
2. Fluorene
5. Pyrene
Averaged mass spectra of the small, substituted maltooligomers
Stressed HPBCD sample, ELS detection
Averaged mass spectra of the intact HPBCD 1 2 4 3 5 GL
1GL
1-2-3 HP
2GL HP
3GL
2-3-4 HP
4GL HP
5GL
Degradation products
HP-betadex
DS=4
DS=6
DS=8
Eluent: MeOH-water 85:15
Eluent: MeOH-water 75:25
Prostaglandine intermediate product
Diastereomer peptides
Quantity of the degradation products vs. the number of glucose and HP units
Extracted ion chromatograms of the degradation products
Averaged mass spectra of the linear, substituted maltoheptaoses
Z and E
R and S *
Plate number 16685
asymmetry: 1,189
Plate number: 5649
asymmetry: 1,261
π-Select 250x4 mm
LiChrosphere Si x4 mm *
Tyr-Pro-Phe-Atc-NH2
* racemic amino-acid
Atc-NH2: 2-amino-decaline-2-carboxylic acid
1 GL
2 GL
3 GL
5 GL
4 GL
6 GL
7 GL
Ring opening products,
linear, substituted maltoheptaoses
further fragmentation products, smaller substituted maltooligomers t0
DS=4
DS=6
DS=8
Normal phase application Eluent: n-hexane - t-buthyl-methyl-ether 97:3
Eluent: 0.1% TFA in water-0.1% TFA in MeOH 75:25
CONCLUSIONS
Cetirizine and related compounds C2 A* F* C1 B* C3 C* E*
Cetirizine
Separation of various families of compounds have been investigated on 4-nitrophenyl-carbamide bonded stationary phase in both reversed and normal phase systems.The new chromatographic media kept its retentive capability even in extremely polar or apolar conditions and proved to be suitable for the separation of different type of substances:
p-Select:
 separation of polyaromatic hydrocarbons
 separation of diastereomers in reversed and normal phase conditions, as well
 analysis of basic drugs in acidic conditions
CD-Screen:
 provides an alternative method for analysis of residual BCD content of HP-betadex
 due to the special retention mechanism, intact CD-derivatives and maltooligomers are well separated, hence, development of a stability indicating method is realizable
Column: Pi-Select 150x4 mm, 3 μm
Eluent: methanol-50 mM KH2PO4 pH :62
Flow: 0.8 ml/min.
Temperature: 40 C
* Determination of marked impurities is prescribed by Ph. Eur. 5
REFERENCES
EXPERIMENTAL
[1] PCT Application Number PCT/HU 05/00043, May 30, 2005
[2] J. Szemán, K. Csabai, K. Kékesi, L. Szente, G. Varga; J. Chromatogr. A, 1116, 76-82, (2005)
[3]   J. Horak, W. Lindner ; J. Chromatogr. A, 1043, (2004)
[4]  J. Horak, N. M. Maier, W. Lindner; J. Chromatogr. A, 1045, (2004)
[5]  I. Caron, C. Elafkir, M. Dreux; Chromatographia 47, (1998)
[6] A. Salvador, B. Herbretau, M. Dreux; J. Chromatogr. A, 855, (1999)
Apparatus: Agilent 1050 HPLC system with UV-VIS Detector at 205 or 254 nm. For detection of cyclodextrins Evaporative Light Scattering Detector PL-ELS 1000, (Polymer Laboratories) was used (Evaporation: 110°C, Nebulization: 90 °C, Gas flow: 1.2 l/min)
Columns: The stationary phases (Hungarian Patent Application Pending 2004) were prepared by ChiroQuest Ltd. Column size: 250 mm x 4.0 mm I.D; Mobile phases: methanol – water or acetonitrile – water; Column temperature: 30 °C; Flow rate: 1.0 ml/min.
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