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Electrophoresis 2005 26 1106 1113 CE separation of etodolac enantiomers 1107. them by mass spectrometry with electrospray ionization CD DS 3 carboxyethylated g cyclodextrin CE g CD. Vancomycin is a glycopeptide type macrocyclic antibiotic DS 4 acetylated b cyclodextrin Ac b CD DS 7. with a limited pH working range 4 pH 8 adsorbs to acetylated g cyclodextrin Ac g CD DS 7 carbox. the wall of untreated fused silica capillaries and has ymethylated g cyclodextrin CM g CD DS 3 g cyclo. strong absorption in the UV Vis region On the other hand dextrin g CD and HP b CD DS 3 were from Cyclolab. a permethylated g CD was used as chiral selector by Budapest Hungary In addition HP b CD DS 4 7 and. Mayer and Schurig 18 in capillary electrochromato RAMEB DS 10 5 14 7 were also obtained from Sigma. graphy They showed the usefulness of open tubular Madrid Spain Water used to prepare solutions was pu. columns for the enantioseparation of NSAIDs including rified with a Milli Q system from Millipore Bedford MA. etodolac However only with the permethylated g CD USA All solutions were filtered prior to use through. Chirasil Dex it was possible to achieve a resolution of 0 45 mm pore size disposable nylon filters from Sugelabor. 1 6 for etodolac enantiomers in 30 min Madrid Spain Etodolac was from Sigma The pharma. ceutical tablets analyzed were obtained in a pharmacy of. Our goal was to develop a chiral separation method by CE Ismira Turkey Table 1 shows the composition of these. using cyclodextrins CDs as chiral selectors due to their formulations. potential for chiral separations commercial availability. stability and easy use in comparison with other chiral. Table 1 Composition of the three different commercial. selectors as for example macrocyclic antibiotics or pro formulations analyzed. teins which present similar practical problems Then in. this work we have made a screening of different CD Commercial Composition. derivatives of neutral and anionic nature to select the formulation. most appropriate chiral selector for the enantioresolution. of etodolac by CE With the slected chiral selector effect Tablet A Etodolac 200 mg per tablet Fe2O3 and TiO2. Tablet B Etodolac 300 mg per tablet Fe2O3 FeO and TiO2. on the enantioresolution of the buffer concentration. Tablet C Etodolac 300 mg per tablet Fe2O3 and TiO2. degree of substitution commercial origin of the chiral. selector and concentration of the CD as well as instru. mental parameters such as temperature and the, separation voltage have been studied to achieve the best 2 2 Apparatus. resolution of etodolac enantiomers Finally the validation. All experiments were performed on a HP3D CE system. of the chiral method and its use to quantify the amount of. Hewlett Packard Waldbronn Germany equipped with, etodolac in different pharmaceutical preparations have. an on column diode array detector DAD Instrument, also been performed. control and data acquisition was performed with the HP. 3D CE ChemStation software Separations were per, formed on uncoated fused silica capillaries of 50 mm ID. 2 Materials and methods and 375 mm OD purchased from Composite Metal Ser. vices Worcester England Capillaries had a total length. 2 1 Reagents and samples of 58 5 cm and 50 cm to the detector The capillary tem. perature was 157C and UV detection was performed at. All reagents employed for the preparation of the separa 225 nm with a bandwidth of 10 nm using a reference. tion buffers were of analytical grade Disodium hydrogen wavelength of 360 nm with a bandwidth of 50 nm and a. phosphate hydrate and sodium acetate were supplied response time of 0 3 s To adjust the pH of the separation. by Panreac Barcelona Spain Dimethyl sulfoxide buffers a 744 pH meter from Metrohm Herisau Switzer. DMSO was from Scharlab Barcelona Spain Sodium land was used. hydroxide sodium dihydrogen phosphate dihydrate and. ortophosphoric acid were from Merck Darmstadt Ger, many b Cyclodextrin b CD randomly methylated 2 3 Procedures.
b cyclodextrin RAMEB degree of substitution DS aver. age number of substituents on one cyclodextrin ring Before its first use a new capillary was flushed 1 bar with. 12 14 2 hydroxypropylated b cyclodextrin HP b 1 M NaOH for 30 min followed by 30 min with water and. CD DS 4 2 carboxymethylated b cyclodextrin CM b 60 min with buffer Between introduction of samples the. CD DS 3 and succinylated b cyclodextrin Succ b capillary was conditioned with 0 1 M NaOH for 1 min fol. CD DS 3 were purchased from Fluka Buchs Switzer lowed by the separation buffer for 2 min Injections were. land 2 Hydroxy buten 3 yl b cyclodextrin HBen b made by pressure 50 mbar for 3 s of sample followed by. CD DS 5 carboxyethylated b cyclodextrin CE b 50 mbar for 3 s of buffer and the applied voltage was. 2005 WILEY VCH Verlag GmbH Co KGaA Weinheim, 1108 R R de Pablos et al Electrophoresis 2005 26 1106 1113. 20 kV Buffer separation solutions were prepared by dis Table 2 for screening their capability as chiral selectors for. solving the same amount of the acid and basic compo etodolac enantiomers All these CDs were used in 50 mM. nents in order to get the desired pH the pKa of the phosphate buffer at pH 7 0 This buffer was used because. selected acid The appropriate amount of each CD was it enabled the larger difference between the EOF and eto. weighed and dissolved into these buffer solutions at a dolac signals the pKa of etodolac is 4 65 In addition the. final concentration of 20 mM Stock solutions were pre buffer pH is similar to physiological conditions which is. pared by dissolving the appropriate amount of etodolac in desirable for pharmacokinetic studies where biological. DMSO to achieve a final concentration of 2000 mg L fluids at this pH e g blood may be analyzed. These solutions were diluted in 1 1 v v water DMSO to. get diluted solutions with concentrations of etodolac be As shown in Table 2 the anionic cyclodextrins CM g CD. tween 10 and 200 mg L 5 and 100 mg L of each enan CM b CD CE g CD CE b CD and Succ b CD and the. tiomer Sample solutions of the three commercial for neutral cyclodextrins b CD RAMEB from Sigma Ac b. mulations analyzed were prepared by crushing and mix CD and Ac g CD all of them at a concentration of 20 mM. ing homogeneously five tablets of each commercial did not show enantioresolution power for etodolac With. formulation and weighing about 0 1 g 0 11 0 13 g of the 20 mM RAMEB from Fluka or HBen b CD poor enantio. resultant powder which was dissolved in DMSO at a final resolutions were observed Rs 0 7 The highest enan. concentration of 5000 mg L These sample solutions tioresolutions were obtained for the neutral HP b CD and. were diluted 1 1 v v in water DMSO to get solution con g CD at a concentration of 20 mM in phosphate buffer at. centrations of 100 mg L All solutions buffers standards pH 7 0 see Table 2 As a consequence we selected HP. and samples were filtered through 0 45 mm pore size b CD as chiral selector for the separation of etodolac. nylon filter membranes and sonicated before use enantiomers because it is less expensive than g CD. Table 2 Enantioresolutions Rs of etodolac enantiomers. 2 4 Data treatment obtained with the different CD derivatives stud. ied at a 20 mM concentration, Resolution was obtained from the ChemStation software. using the equation Rs 1 18 t22t1 w1 2 11w1 2 2 where t1 CD commercial supplier Enantioresolution. and t2 are the migration times of etodolac enantiomers. and w1 2 1 w1 2 2 are their peak widths at half height Lim Neutral CDs b CD Fluka 2. RAMEB Sigma 2, its of detection LODs 3sa b and quantitation LOQs. RAMEB Fluka 1, 10sa b were determined from the standard error of the. HP b CD Cyclolab 11, intercept sa and the slope b of the calibration curve HBen b CD Cyclolab 1.
obtained by analysis of variance ANOVA 19 Control Ac b CD Cyclolab 2. graphs elaborated for stability studies were made plotting g CD Cyclolab 11. corrected peak areas as a function of time The central Ac g CD Cyclolab 2. values X were calculated as the averaged value of six. Anionic CDs Succ b CD Fluka 2, repeated runs and the control limits which enable to CM b CD Fluka 2. visualize the values in and out of control were calculated CE b CD Cyclolab 2. as X 6 3s where s is the standard deviation of the six CM g CD Cyclolab 2. replicate values 20 Experimental data analysis and pa CE g CD Cyclolab 2. rameters were calculated using Excel Microsoft XP and. Statgraphics Plus Version 5 0 softwares 2 Rs 0 1 Rs 0 7 11 0 7 Rs 1. 3 Results and discussion With the selected CD the influence on enantiomeric res. olution of the temperature and separation voltage was. 3 1 Method optimization studied to choose the most appropriate values of these. parameters The variation of the resolution for etodolac. The crucial step to achieve an enantiomeric separation is enantiomers as a function of the capillary temperature in. the choice of the chiral selector CDs are the favourite the range of 15 457C showed that the resolution gradu. chiral selectors in CE and they have shown to separate ally decreases as the temperature increases This effect. the enantiomers of a huge amount of chiral drugs Due to was observed previously with the same CD derivative by. their availability stability and potential we selected dif our research group 21 and could be attributed to the. ferent CD derivatives of neutral and anionic nature see increase in the stability of selector selectand complexes. 2005 WILEY VCH Verlag GmbH Co KGaA Weinheim, Electrophoresis 2005 26 1106 1113 CE separation of etodolac enantiomers 1109. that takes place when the temperature is decreased 16 HP b CD is a randomly substituted CD manufactured in a. 21 22 Therefore all subsequent separations were per different way by the three different suppliers which. formed at 157C However a low influence of the separa provide different DSs see Section 2 Thus depending on. tion voltage on the enantioresolution of drugs is generally the DS the substitution patterns can be different and. obtained For etodolac enantiomers a similar enantio even side chain substitution e g 2 2 hydroxy propoxy. resolution was obtained for 15 and 20 kV whereas the propyl substituents is possible In addition the DS. resolution decreased for 30 kV Since the migration times depends on the chemical reaction used base low high. decreased when increasing the applied voltage 20 kV of temperature and large differences between batches and. separation voltage was used in our experiments production sites may be obtained under some conditions. Due to these problems a trend in chiral analysis by CE is. the use of single isomer CDs which are able to achieve. The effect of the buffer concentration on the separation of. higher reproducibility and selectivity than randomly. etodolac enantiomers was investigated by using different. substituted CDs 23 However it is important to note that. concentrations 40 50 80 and 100 mM phosphate buffer. HP b CD which has been shown to be the best chiral. containing 20 mM HP b CD As it can be seen in Fig 2. selector for etodolac is not a single isomer Cyclolab. there is a linear increment of the enantioresolution with. produces the single isomer monoHp b CD but its aque. the phosphate buffer concentration from 40 to 100 mM. ous solutility is very poor 0 05 g 100 cm3 being not. This can be explained because when the buffer con, useful in aqueous CE For this reason we studied the. centration increases the solute wall interactions and the. effect of the concentration of this CD from three different. undesirable effects of electrodispersion are minimized. commercial suppliers on the resolution of etodolac enan. 16 Therefore 100 mM phosphate buffer at pH 7, tiomers This study was performed because the con. 140 mA was used for further experiments, centration of the chiral selector affects directly the chiral.
properties 21 24 As can be seen in Fig 3 when varying. The influence of the concentration and the degree of the concentration of HP b CD of the three different com. substitution of HP b CD commercial origin in the mercial origins from 10 to 60 mM the maximum enantio. selected buffer was also studied The commercial origin resolution for etodolac was observed at a 20 mM CD con. of the chiral selector in the separation buffer was found to centration In addition HP b CD from Fluka DS 4 2. be a very decisive factor influencing the enantiomeric gets always the best enantioresolution values for all the. resolution of the etodolac enantiomers This is because concentrations studied As a consequence this con. centration of the HP b CD DS 4 2 was chosen for the. enantiomeric separation of etodolac, Figure 2 Effect of the buffer concentration on the enan. tioresolution of etodolac for a phosphate buffer pH 7. with 20 mM HP b CD Cyclolab as chiral selector CE, conditions temperature 157C untreated fused silica Figure 3 Influence of the concentration and the DS. capillary 58 5 cm 50 cm to the detector window 650 mm commercial origin effect of the HP b CD in 100 mM. ID applied voltage 20 kV injection 50 mbar63 s UV phosphate buffer pH 7 on the enantioresolution of eto. detection at 230 nm dolac CE conditions as in Fig 2. 2005 WILEY VCH Verlag GmbH Co KGaA Weinheim, 1110 R R de Pablos et al Electrophoresis 2005 26 1106 1113. The detection conditions for etodolac enantiomers were 3 2 Method validation. also studied in this work As it is known the response time. of the detector affects the baseline noise and the enan Prior to the validation of the chiral method developed for. Separation of etodolac enantiomers by capillary electrophoresis Validation and application of the chiral method to the analysis of commercial formulations Separation of etodolac enantiomers which exhibit different biological activity and pharmacokinetic profiles has been achieved using the randomly substituted 2 hydroxy propyl b cyclodextrin HP b CD as chiral selector in capillary

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