nitro-phenyl-carbamide hplc stationary phases; cd-screen column for analysis of...
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NITRO-PHENYL-CARBAMIDE HPLC STATIONARY PHASES;CD-SCREEN COLUMN FOR ANALYSIS OF CYCLODEXTRIN-DERIVATIVES
AND -SELECT COLUMN FOR GENERAL PURPOSE APPLICATIONSGábor 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, e-mail: [email protected] 2 CHIROQUEST Chiral Technologies Development Ltd., Rumbach S. u. 7., Budapest, H-1075, Hungary, e-mail: [email protected] 3 SEMMELWEIS UNIVERSITY, Faculty of Pharmacy, Department of Pharmaceutics, Hőgyes E. u. 7., Budapest, H-1092, Hungary, e-mail: [email protected]
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 - 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.
INTRODUCTIONINTRODUCTION
N+
O-
O
NH
OO
NHCH2CH2CH2
O
O Si δ+
H-acceptor
H-donors π-acceptor
STRUCTURE OF THE SELECTORSTRUCTURE OF THE SELECTOR
PRINCIPLE OF SEPARATIONPRINCIPLE OF SEPARATION
RESULTS AND DISCUSSIONRESULTS AND DISCUSSION
Illustration of the interaction between the apolar cavity of cyclodextrin and the nitrophenyl-carbamide selector
• higher surface coverage compared to the CD-Screen stationary phase, to obtain strong 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
-SELECT-SELECT CD-SCREENCD-SCREEN
Illustration of electrostatic potential surface difference of 4-nitrophenyl and phenyl-carbamide in complex with
phenol and formation of hidrogen bonding
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.
min0 2 4 6 8 10 12 14 16 18
mAU
0
5
10
15
20 1
2
min0 2 4 6 8 10 12 14 16 18
mAU
0
5
10
15
20
25
12
π-Select
Conventional phenyl-column
k’=0.54
k’=0.25
Polar analytes
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.
min0 2 4 6 8
mAU
0
2.5
5
7.5
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20
2
3 4
1
min0 2 4 6 8
mAU
0
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1
2+3
4
π-Select
Conventional phenyl-column
C: 4.7%
C: 7.0%
Eluent: MeOH-water 30:701.: Phenol 2.: Caffeine
Eluent: MeOH-water 60:401.: Dimethyl-phtalate; 2.: Diethyl-phtalate3.: Biphenyl 4.: o-Terphenyl
Apolar, aromatic analytes
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.
Shape selectivity of -Select columnPolyaromatic hydrocarbons
[min.]Time
0 1 2 3 4 5 6 7
[mV]
Vol
tage
0
200
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600
800
TOH_MeOH_85_ism
1
2 3
6 7
[min.]Time
0 2 4 6 8
[mV]
Vol
tage
0
100
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ToL_MeOH_70
12
4
35
S
S
1. Benzothiophene
4. Dibenzothiophene
7. Benzo[g,h,i]perylene
6. Benzo[a]pyrene
3. Fluoranthene
2. Fluorene 5. Pyrene
min0 2.5 5 7.5 10 12.5 15 17.5 20
mAU
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min0 2.5 5 7.5 10 12.5 15 17.5 20
mAU
-10
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40
Plate number 16685
asymmetry: 1,189
Plate number: 5649
asymmetry: 1,261
π-Select 250x4 mm
LiChrosphere Si 60 250x4 mm
Prostaglandine intermediate product
Eluent: MeOH-water 85:15 Eluent: MeOH-water 75:25
O
O O Si
COOCH3
Z and E
R and S*
Diastereomer peptides
Cetirizine and related compounds
[min.]Time
0 10 20 30 40 50
[µV]
Vol
tage
0
100
200
300
400
piszelebmtpppn
*Tyr-Pro-Phe-Atc-NH2* racemic amino-acidAtc-NH2: 2-amino-decaline-2-carboxylic acid
Column: Pi-Select 150x4 mm, 3 μmEluent: methanol-50 mM KH2PO4 pH 2.2 38:62Flow: 0.8 ml/min.Temperature: 40 C
* Determination of marked impurities is prescribed by Ph. Eur. 5
CD-ScreenEP method
Examination of the degradation products of HP-betadex on CD-Screen column
Stressed HPBCD sample, ELS Stressed HPBCD sample, ELS detectiondetection
Averaged mass spectra of the Averaged mass spectra of the intact HPBCDintact HPBCD
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.
Quantity of the degradation products vs. Quantity of the degradation products vs. the number of glucose and HP unitsthe number of glucose and HP units
Extracted ion chromatograms of Extracted ion chromatograms of the degradation productsthe degradation products
CONCLUSIONSCONCLUSIONSNormal phase application Eluent: n-hexane - t-buthyl-methyl-ether 97:3
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.
EXPERIMENTAL
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:-Select:-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: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
[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, 177-194 (2004)[4] J. Horak, N. M. Maier, W. Lindner; J. Chromatogr. A, 1045, 43-58 (2004)[5] I. Caron, C. Elafkir, M. Dreux; Chromatographia 47, 383-390 (1998)[6] A. Salvador, B. Herbretau, M. Dreux; J. Chromatogr. A, 855, 645-656 (1999)
REFERENCES
Averaged mass spectra of the Averaged mass spectra of the linear, substituted maltoheptaoseslinear, substituted maltoheptaoses
Eluent: 0.1% TFA in water-0.1% TFA in MeOH 75:25
Comparison of the results of hydroxypropyl-betadex analysis, obtained by European Pharmacopoeia method and CD-Screen columnComparison to phenyl column
dipole
hydrophylic-hydrophobic
0
BCD
Eluent: water
prescribed stop time
Number of theoretical plates
for BCD: 1800 plates/m
m2 4 6 8 1 1 1 1 1
V
30
35
40
45
50
55
60
65
70
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.
[min.]Time
0 5 10 15 20
[V]
Vol
tage
0
100
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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
Averaged mass spectra of the small, Averaged mass spectra of the small, substituted maltooligomerssubstituted maltooligomers
1 2 3 4 5 6 7 8 9 10 11 12 13GL1
GL4
GL7
hydroxypropyl units
Glucose units
m/z200 400 600 800 1000 1200
0
20
40
60
80
*MSD1 SPC, time=4.948:13.468 of D:\DOC\MS\CD_HCL.D API-ES, Pos, Scan, Frag: 150
Max: 5368
GL
1GL1-2-3 HP
2GL1-2-3-4 HP
3GL2-3-4 HP
4GL2-3-4-5 HP
5GL2-3-4 HP
ClO
OH
ON
N
ClH2
[min.]Time
5 10 15 20
[mV]
Vo
ltage
0
2
4
6
8
10
C2A*
F*
C1
B*
C3C*
E*
Cetirizine
Malév Hungarian Airlines supports our participation at the conferenceMalév Hungarian Airlines supports our participation at the conference
m/z1350 1400 1450 1500 1550 1600 1650 1700
0
20
40
60
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100
*MSD1 SPC, time=13.168:19.401 of D:\DOC\MS\CD_HCL.D API-ES, Pos, Scan, Frag: 150
Max: 12081
DS=4
DS=6
DS=8
m/z1400 1450 1500 1550 1600 1650 1700
0
20
40
60
80
100
*MSD1 SPC, time=20.545:26.335 of D:\DOC\MS\CD_HCL.D API-ES, Pos, Scan, Frag: 150
Max: 5416
DS=4
DS=6
DS=8
min0 5 10 15 20 25 30
mV
0
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Degradation products
HP-betadex