Influence of Moile Phse Composition on the Preprtive Seprtion of Profens y Chirl Liquid Chromtogrphy António E. Rieiro, Luís S. Pis, Alírio E. Rodrigues Lortory of Seprtion nd Rection Engineering School of Technology nd Mngement, Brgnç Polytechnic Institute, Cmpus de Snt Apolóni, Aprtdo 34, 53-857 Brgnç, Portugl Fculty of Engineering, University of Porto, Ru Dr. Roerto Fris, 4-465 Porto, Portugl Keywords: Enntiomer seprtion, Preprtive chromtogrphy, Moile phse composition, Profens, Nonsteroidl nti-inflmtory drugs. Topic: Industril Processes - Phrmceuticl. Astrct Liquid chirl chromtogrphy of ketoprofen nd fluriprofen enntiomers is crried out using n mylose-sed sttionry phse. The moile phses used for profens chirl seprtions re usully hydrocron-lcohol comintion, with high hydrocron content. However, profens show poor soluilities in hydrocron solvents when compred to lcohols. When the finl ojective is high productivity preprtive seprtions, esides retention time, selectivity nd column efficiency, soluility of the rcemic drug is lwys mndtory spect to tke into ccount. This work shows tht n increse of the lcoholic content in the moile phse is possile without decrese in selectivity nd column efficiency. Considering the chirl seprtion of ketoprofen nd fluriprofen enntiomers, results show tht the moile phse needs only smll quntity of cidic modifier nd cn e composed y high or even pure lcoholic content. Additionlly, it is found tht the type of lcohol to e used cn differ, depending on the profen rcemic mixture to e seprted. Introduction The chirlity of drugs is n importnt issue for the phrmceuticl industry, since the different enntiomers of rcemic drug my hve distinct phrmcologicl ctivities, phrmcokinetic nd phrmcodynmic effects. Becuse of its chirl selectivity, humn ody rects with rcemic drug differently, nd metolise ech enntiomer on seprte pthwys producing different phrmcologicl ctivity. Thus, one isomer my produce the desired therpeutic ctivities, while the other my e inctive or even, in worst cses, produce unwnted effects. Fluriprofen [-(-fluoro-4- iphenyl)-propionic cid] nd ketoprofen [-(3-enzoylphenyl)-propionic cid] (Figure ), elong to fmily of chemicls nmed -rylpropionic cids, or profens, n importnt su-clss of the frequently prescried nd used drugs clled nonsteroidl nti-inflmmtory drugs (NSAIDs). The min primry indictions for NSAIDs therpy include rheumtoid rthritis, osteorthritis, cute gouty rthritis, nkylosing spondylitis nd dysmenorrhe (DeRuiter, ). The importnce of profens is supported y the fct tht, in the lst twenty yers, drugs like spirin, phenzone derivtives or cetminophen re eing supplemented y profens (Brune nd Hinz, 998). O COOH COOH () Ketoprofen () Fluriprofen Figure. Chemicl structures of ketoprofen () nd fluriprofen (). F Corresponding uthor. Tel + 35-73-3387. E-mil: pis@ip.pt
Some studies refer tht, while the nti-inflmmtory effect nd gstric toxicity is ssocited minly with the S-enntiomers (Wechter et l., ), R-enntiomers ply mjor role in nlgesi (Bertini nd Cselli, 999) eing less toxic thn the S-enntiomers or the rcemic form (Geisslinger nd Schile, 996). The phrmokinetics in terms of sorption, distriution, metolism, protein inding nd elimintion my e different for the two enntiomers, leding to inter-individul vriility in clinicl response nd drug toxicity. Therefore, there is need for the development of preprtivescle seprtion method for this clss of drugs. Due to its good sensitivity, reproduciility nd low chromtogrphic interferences, high performnce liquid chromtogrphy (HPLC) using chirl sttionry phses (CSPs) hs een the most employed enntioseprtion method of profens. The phenylcrmte derivtives of polyscchrides, prticulrly cellulose nd mylose, show high chirl recognition when used s CSPs for HPLC (Yshim, ). Among the mny derivtives, the mylose 3,5-dimethylphenylcrmte (e.g. Chirlpk AD, Dicel, Jpn) is the most used on the seprtion of profen rcemtes. Considering the preprtive seprtion of these clss of enntiomers, the choice of the moile phse composition is criticl issue, since directly ffects the system productivity y influencing retention time, selectivity, column efficiency nd soluility of the rcemte (Fncotte, ). The ojective of this work is to study how moile phse composition, in terms of cidic nd lcoholic modifiers, influences the profen enntioseprtion. Equipment nd Mterils All nlysis were performed on Jsco HPLC system with n UV-575 multiwvelength detector set t 6 nm, nd Rheodyne 775(i) injection vlve with µl loop. The column used ws n mylose tris(3,5-dimethylphenylcrmte) coted on µm silic-gel sustrte (Chirlpk AD, 5 mm L 4,6 mm ID) from Dicel Chemicl Industries (Jpn). Methnol, ethnol, isopropyl lcohol, cetonitrile nd n-hexne, ll HPLC grde, trifluorocetic cid (TFA) spectrophotometric grde,,3,5- tri-tert-utylenzene (s non-retined component), rcemic fluriprofen nd rcemic ketoprofen of nlyticl grde, were ll purchsed from Sigm (Mdrid, Spin). 3 Results nd Discussion 3. Soluility mesurements nd pressure drop Rcemic ketoprofen nd fluriprofen were used for soluility mesurements in different solvents. First, the influence of the composition of mixture contining ethnol/n-hexne/. ws studied. Lter, ketoprofen soluility ws mesured in five different pure solvents: n-hexne, cetonitrile, isopropyl lcohol, ethnol, nd methnol. All former mesurements were crried out t 5 ºC. Additionlly, the influence of temperture on ketoprofen soluility ws studied for pure ethnol nd methnol. Figure shows tht the lcoholic (ethnol) content in the moile phse drsticlly influences profen soluility: ketoprofen enntiomers re insolule in pure n-hexne solvent nd present high soluility in pure ethnol. Soluility (g ketoprofen / g solution) 5 4 3 P/L (tm/cm)..9.6.3 4 6 8 % EtO H 3 4 5 6 7 u (cm /m in) Figure. Effect of the lcoholic content on ketoprofen soluility (solvent: ethnol/n-hexne with.; T=5ºC). Figure 3. Pressure drop for different solvents: pure isopropnol (filled circles), pure ethnol (filled squres), pure methnol (open circles), nd n 8% n- hexne/% ethnol mixture (open squres). All solvents with.% TFA.
Soluility mesurements otined for different solvents shows tht, t 5 ºC, ketoprofen enntiomers hve incresing soluilities for pure cetonitrile, isopropyl lcohol, ethnol, nd methnol. These results confirm tht rcemic drugs hve considerly higher soluilities in lcoholic solvents thn in the trditionl moile phses used in nlyticl chirl seprtion, consisting in n lcohol-hydrocron comintion, with high hydrocron content (Miller et l., 999). Results lso shows the expected incresing soluilities with temperture. Additionl soluility mesurements were crried out for fluriprofen enntiomers. Although showing lower soluilities thn ketoprofen, the fluriprofen enntiomers present the sme increse in soluility with the increse of the lcoholic content (dt not shown). In preprtive scle perspective, it is of crucil importnce the pressure drop otined in the dsorent ed. Figure 3 shows tht lower pressure drops re otined for methnol; lower thn for ethnol nd even lower thn for isopropyl lcohol. 3. Effect of cidic modifier Concerning the seprtion of ketoprofen nd fluriprofen enntiomers, the effect of the cidic modifier content on the cpcity nd selectivity (Figures 4 nd 5) nd on column efficiency (Figures 6 nd 7) ws studied, using moile phse composition of 8% n-hexne/% ethnol with TFA (,.,.5,., nd.5% v/v). cpcity fctor, k i.5.4.3. cpcity fctor, k i.5.75.5.8.7.6.5..5.4.3.6.9..5.3.6.9..5.3.6.9..5.3.3.6.9..5 Figure 4. Effect of the cidic modifier content () on the seprtion prmeters for ketoprofen enntiomers: () cpcity fctors (circles for the less nd squres for the more retined enntiomer); () selectivity (moile phse: 8% n-hexne/% ethnol; T=5ºC). Figure 5. Effect of the cidic modifier content () on the seprtion prmeters for fluriprofen enntiomers: () cpcity fctors (circles for the less nd squres for the more retined enntiomer); () selectivity (moile phse: 8% n-hexne/% ethnol; T=5ºC). The results clerly show tht the introduction of the cidic modifier decrese retention of oth enntiomers nd increse selectivity. However, it ws found tht smll concentrtion of TFA (.%) is enough to ensure seprtion nd no etter performnces re otined with higher TFA contents. 5 % TFA 5 5 % TFA 5 % TFA 5.,.5,. nd.5% TFA 5 % TFA.,.5,. nd.5 % TFA 5 5.,.5,. nd.5 % TFA 5 5.,.5,. nd.5 % TFA 3 4 5 6 7 u (cm/min) 3 4 5 6 7 3 4 5 6 7 3 4 5 6 7 Figure 6. Effect of the cidic modifier content () on column efficiency for ketoprofen enntiomers: () less retined; () more retined enntiomer (moile phse: 8% n-hexne/% ethnol with,.,.5,., nd.5% TFA; T=5ºC). Figure 7. Effect of the cidic modifier content () on column efficiency for fluriprofen enntiomers: () less retined; () more retined enntiomer (moile phse: 8% n-hexne/% ethnol with,.,.5,., nd.5% TFA; T=5ºC).
This fct is clerly shown in Figures 6 nd 7: for oth ketoprofen nd fluriprofen enntiomers, the column efficiency is low ( HETP = 5 µm) if no TFA is dded. Introducing.% TFA content, the HETP for oth profens nd oth enntiomers vries etween 5 nd 3 µm. No etter results re otined for higher TFA contents. Additionl experiments were crried out for moile phse contining pure methnol nd TFA modifier, nd similr conclusion ws found:.% TFA content is enough to ensure seprtion of profen enntiomers on Chirlpk AD sttionry phse. 3.3 Effect of lcoholic modifier 3.3. Hydrocron lcohol mixtures Experiments were crried out using different n-hexne/ethnol rtios: 8/, 5/5, 35/65, /8, /9 (%v/v), nd pure ethnol; ll mixtures contining.% TFA. The results otined re presented in Figures 8 to. cpcity fctor, k i.5.45.4.35.3.5 cpcity fctor, k i.75.5.5.8.75.7.65.6 5 4 6 8. 4 6 8 4 6 8 4 6 8 Figure 8. Effect of the lcoholic modifier content () on the seprtion prmeters for ketoprofen enntiomers: () cpcity fctors (circles for the less nd squres for the more retined enntiomer); () selectivity (moile phse: n-hexne/ethnol mixtures, with.% TFA; T=5ºC). Figure 9. Effect of the lcoholic modifier content () on the seprtion prmeters for fluriprofen enntiomers: () cpcity fctors (circles for the less nd squres for the more retined enntiomer); () selectivity (moile phse: n-hexne/ethnol mixtures, with.% TFA; T=5ºC). 5 4 5 4 5 4 5 4 3 3 3 3 3 4 5 6 7 3 4 5 6 7 3 4 5 6 7 3 4 5 6 7 Figure. Effect of the lcoholic modifier content () on column efficiency for ketoprofen enntiomers: () less retined; () more retined enntiomer (moile phse: n-hexne/ethnol mixtures, with.% TFA ; T=5ºC). Ethnol content: % (open tringles); 5% (filled tringles); 65% (open circles); 8% (filled circles); 9% (open squres); % (filled squres). Figure. Effect of the lcoholic modifier content () on column efficiency for fluriprofen enntiomers: () less retined; () more retined enntiomer (moile phse: n-hexne/ethnol mixtures, with.% TFA; T=5ºC). Ethnol content: % (open tringles); 5% (filled tringles); 65% (open circles); 8% (filled circles); 9% (open squres); % (filled squres). Anlysing Figures 8 nd 9, we conclude tht retention (cpcity fctors) diminish with the increment of the lcoholic (ethnol) content. However, selectivity remins under reltively constnt vlues. The sme occurs in terms of column efficiency (Figures nd ). These results revel tht the use of pure lcoholic solvents is possile for chirl seprtions, nd eneficil t preprtive scle.
3.3. Alcohol Alcohol mixtures The effect of the content of ethnol/methnol moile phses on seprtion ws studied for oth ketoprofen nd fluriprofen systems. The study ws crried out using different ethnol/methnol composition rtios: /, /8, 4/6, 6/4, 8/ nd / (%v/v); ll with.. Figures nd 3 show distinct results respectively for ketoprofen nd fluriprofen enntiomers..5.8.5. cpcity fctor, k i.4.3.6.4 cpcity fctor, k i.4.3.8....6...4 4 6 8 4 6 8 4 6 8 4 6 8 Figure. Effect of the content of n ethnol/methnol moile phse on the seprtion prmeters for ketoprofen enntiomers: () cpcity fctors (circles for the less nd squres for the more retined enntiomer); () selectivity (moile phse: ethnol/methnol mixtures,.% TFA; T=5ºC). Figure 3. Effect of the content of n ethnol/methnol moile phse on the seprtion prmeters for fluriprofen enntiomers: () cpcity fctors (circles for the less nd squres for the more retined enntiomer); () selectivity (moile phse: ethnol/methnol mixtures,.% TFA; T=5ºC). For ketoprofen enntiomers, oth retention nd selectivity generlly increse with the increse of the ethnol content. Low selectivities nd resolutions re otined for high methnol content. For fluriprofen enntiomers, the retention of the first enntiomer is only slightly ffected y the composition of the ethnol/methnol moile phse, while the retention of the second enntiomer increses with the increse of the methnol content. Selectivity lso increses with the increment of the methnol content. We conclude tht the chirl seprtion of ketoprofen enntiomers cn e otined using pure ethnol moile phse, while seprtion of fluriprofen enntiomers cn e etter ccomplished with pure methnol solvent. Both solutions re otined with low retentions, which is n dvntge in preprtive scle perspective. Figure 4 shows the correspondent column efficiency ( HETP ) for ketoprofen nd fluriprofen enntiomers, using the selected pure lcohol moile phse. 35 35 3 3 5 5 5 5 3 4 5 6 7 3 4 5 6 7 u (cm/min) u (cm/min) Figure 4. vn Deemter curves (column efficiency versus superficil velocity) for: () ketoprofen chirl seprtion in pure ethnol; () fluriprofen chirl seprtion in pure methnol. Circles for the less nd squres for the more retined enntiomers. Both moile phses with.% TFA; T=5ºC.
4 Conclusions The moile phses used for profens chirl seprtions re usully hydrocron-lcohol comintion, with high hydrocron content. However, profens show poor soluilities in hydrocron solvents. When the finl ojective is the high productivity preprtive seprtions, soluility of the rcemic drug is of crucil importnce. The results presented show tht n increse of the lcoholic content in the moile phse is possile without decrese on selectivity. Considering the preprtive production of pure profen enntiomers using n mylose-sed chirl sttionry phse, results show tht the optimum moile phse needs only smll quntity of cidic modifier (.% TFA) nd cn e otined under pure lcohol content. The use of pure lcohol solvents increses soluility of the rcemte nd decreses retention time, oth dvntges in preprtive scle point of view. Besides, the use of pure solvents lso simplifies its reutiliztion in production seprtion process. Considering the chirl seprtion of profen rcemic mixtures, this work shows tht the choice of the etter moile phse is not strightforwrd tsk. Pure methnol (with low quntity of TFA cidic modifier) should e used to seprte fluriprofen enntiomers: esides higher soluility, the use of methnol presents higher selectivity nd lower pressure drop. However, considering the seprtion of ketoprofen enntiomers, pure methnol should e replced y pure ethnol, since the former moile phse presents low selectivities for this system. Acknowledgements Finncil support from the portuguese R&D foundtion, Fundção pr Ciênci e Tecnologi (project POCTI/388/EQU/), is grtefully cknowledged. References Bertini, R., Cselli, G. (999). Anlgesic Effect of Ketoprofen Is Minly Associted to its R- Enntiomer: Role of Cytokine Modultion. Anlgesi 4, 8-86. Brune, K., Hinz, B. (998). -Arylpropionsuren stellenwert in der schmerztherpie. Der Schermz 6, 383-388. DeRuiter, J. (). Principles of drug ction, Fll (ville t http://www.duc. uurn.edu/~deruij/nsids_.pdf) Frncotte, E. (). Enntioselective chromtogrphy s powerful lterntive for the preprtion of drug enntiomers. J. Chromtogr. A 96, 379-397. Geisslinger, G., Schile, H. (996). New insights into the site nd more of ntinoceptive ction of fluriprofen entiomers. J. Clin. Phrmcol. 36, 53-5. Miller, L.,Orihuel, C., Fronek, R., Murphy, J. (999). Preprtive chromtogrphic resolution of enntiomers using polr orgnic solvents with polyscchride chirl sttionry phses. J. Chromtogr. A 865, -6. Wechter, W., Leipold, D., Murry, E., Quiggle, D., McCrcken, J., Brrios, R., Greenerg, N. (). E-7869 (R-Fluriprofen) Inhiits Progression of Prostte Cncer in the TRAMP Mouse. Cncer Res. 6, 3-8. Yshim, E. (). Polyscchride-sed chirl sttionry phses for high-performnce liquid chromtogrphy enntioseprtion. J. Chromtogr. A 96, 5-5.