citalopram ( cit ) , chemically 1-(3-(dimethylamino ) propyl)-1-(4-fluorophenyl)-5-phthalancarbo - nitrile , is a chiral drug , widely used to treat depression and panic anxiety . citalopram possesses one asymmetric carbon , giving a pair of enantiomers , ( s)-citalopram and ( r)-citalopram . the s form citalopram has been proven to have much higher medicinal potency than the racemic form due to pharmacologically inactive and even counteracting nature of the r form citalopram . the s form citalopram , escitalopram , is commercially available around the world under the brand name lexapro or cipralex . food and drug administration ( fda ) in 1992 , all drugs with chiral centers are recommended to be marketed in the form of pure enantiomer , and other enantiomers without any medicinal effects are considered as impurities . in order to quantitatively determine the enantiomeric composition of citalopram , various analytical methods have been researched and developed in recent years [ 521 ] . most of them involve the employment of high performance liquid chromatography ( hplc ) or supercritical fluid chromatography ( sfc ) , equipped with chiral stationary phases [ 517 ] . as early as in 1995 [ 6 , 7 ] , the pioneer work on enantiomeric separation of citalopram by hplc was conducted by rochat et al . , using a chiralcel od column and an acetylated -cyclobond column . since then , many efforts were made to use a number of commercially available chiral stationary phases for analyzing enantiomeric purity of citalopram , including chiralcel od - r column , chirobiotic v column [ 911 ] , chiralcel od - h column , chiral - agp column , and chiralpak ad column . most recently , li et al .   explored the enantioseparation of cit using supercritical fluid chromatography equipped with a chiralpak ad column . the effect of organic modifiers in mobile phase , column temperature , and operation pressure was studied . additionally , some studies were focused on the development of more efficient chiral stationary phases . for example , zhang et al .   developed a copolymer chiral stationary phase derived from ( 1r , 2r)-(+)-1 , 2-diphenylethylenediamine . chen et al .   prepared a novel stationary phase containing two structurally similar chiral selectors synthesized from d - tartrates . these newly developed chiral stationary phases have demonstrated improved enantioselectivity and have been successfully applied on chiral separation of cit . contrary to using chiral stationary phases , another viable option is to add a chiral additive into the mobile phase to form a pair of transient diastereomeric complexes , followed by chromatographic separation on an achiral column . introducing chiral additives in mobile phase has a number of advantages over using chiral stationary phases [ 22 , 23 ] . first , the method is less expensive . as compared to conventional achiral columns , chiral columns , in general , are of high price , less stability , and low capacity and have a relatively short lifetime . , the approach based on chiral mobile phases can work in both a normal phase and a reversed phase operation mode . cyclodextrin and its derivatives [ 24 , 25 ] have been recognized as the most prevalent chiral mobile phase additives due to the nature of being nontoxic , nonflammable , nonvolatile , stable over a wide range of ph and negligible absorption in the uv ranges broadly used in chromatographic detection . they , therefore , have been successfully applied in the enantiomeric separation of a variety of racemates reported in a review article , and recently , of mandelic acid derivatives , pantoprazole , cathinone and amphetamine derivatives , phenyllactic acid , 2-arylpropionic acid , sertraline , beta - carboline alkaloids , and amlodipine . the goal of this research work is to explore the use of sulfobutylether--cyclodextrin ( sbe--cd ) , a newly developed cyclodextrin derivative as a chiral mobile phase additive to enantioseparation citalopram on a silica - based reversed phase c18 column . sbe--cd was considered as the chiral mobile phase additive in our work because of its favorable structure and modified physicochemical properties , relative to native cyclodextrin . as illustrated in figure 2 , the presence of sulfobutyl group results in stronger acidity , leading to an increased solubility in water , while the cavity structure is well preserved . this enables sbe--cd in mobile phase to readily form complexes with the guest molecule , citalopram . from the steric structure point of view , the sulfobutyl group may partially block the mouth of the cyclodextrin and consequently alter the complexation behavior , thus resulting in variation of chiral selectivity . our study indicated that sbe--cd was an effective chiral mobile phase additive , exhibiting high enantioselectivity ability . to the best of our knowledge , this work is the first attempt of employing sulfobutylether--cyclodextrin ( sbe--cd ) as a chiral mobile phase additive to separate citalopram on an achiral column instead of chiral stationary phases , offering a promising alternative for citalopram enantiomers quantitation . racemic citalopram hydrobromide and escitalopram oxalate were provided by huahai pharma ( taizhou , china ) with purity larger than 99.0% . ( suzhou , china ) and acetonitrile was from j&k ( beijing , china ) . sodium dihydrogen phosphate ( analytical grade ) was purchased from l.f . fine - chemical ( shanghai , china ) and phosphoric acid ( analytical grade ) from national medicine ( shanghai , china ) . standard buffers of ph was from national medicine ( shanghai , china ) and purity water from wahaha ( hangzhou , china ) . the hplc system ( hegong instrument , shanghai , china ) consisted of a vertex sti p5000 pump , a sti uv detector ( wavelength range of 190700  nm ) , 10  l sample loop ( rheodyne ) , and a 7725i sampler . a leici phs-25 ph ( shanghai , china ) meter equipped with an e-201-c combined glass electrode was used for ph measurements . it was calibrated using standard buffers of ph values of 4.01 , 6.86 , and 9.18 . the chromatographic and the integrated data were collected using a n3000 data - acquiring software based on a window xp computer system . before being delivered to the hplc system , the mobile phase was filtered through 0.45  m ptfe filters and was degassed . chromatographic separation was conducted on a hedera ods-2 silica gel c18 column ( 250  mm  4.6  mm  5.0  m , hanbang , china ) in the reversed phase operating mode , with a mobile phase consisting of aqueous buffer ( sodium dihydrogen phosphate and sbe--cd ) , methanol , and acetonitrile with the volumetric ratio of 21  :  3  :  1 . the measurements were carried out in an isocratic elution mode at ambient temperature with a flow rate of 1.0  ml / min , an injection volume of 10  l , and uv detection at 240  nm . the conversion of citalopram hydrobromide into racemic citalopram free base was carried out as reported in the literature [ 34 , 35 ] . 10  g of citalopram hydrobromide was suspended in 20  ml water and 20  ml dichloromethane . a solution of sodium hydroxide ( 1.3  m ) was slowly added until ph value of 10 was reached . the mixture was stirred for 30  min and settled for additional 30  min , and then the two resulting phases were separated . the organic phase was washed three times using 30  ml water each time and then concentrated on a rotary evaporator to obtain translucent oil , citalopram - free base in dichloromethane . the rest of the solvent in free base was further removed through a vacuum oven for 1  h at 80c , giving free base citalopram in white crystalline . the melting point of the final product was 9093c , determined by a melting point apparatus . the conversion of escitalopram oxalate into ( s)-cit free base was performed in the same way . typically , 0.0035  g racemic citalopram was weighed and dissolved in 50  ml mobile phase solution in a volumetric flask , with the aid of ultrasonication , giving cit concentration of 0.22  mm . an aliquot of racemic cit and ( s)-cit was dissolved in the mobile phase , and 10  l of the mixture was submitted to chromatographic analysis under the above - mentioned conditions . examining the two elution peak areas , the later peak had a larger area , corresponding to ( s)-cit . in chromatographic separation , the ph value of mobile phase is one of the most important factors influencing the resolution of enantiomers . in this work , the ph value was set in the range of 2.54.0 based on the following considerations . a very low ph value promotes the protonation of sbe--cd while it negatively impacts the packing materials in hplc column . however , under a neutral ( ph of 7 ) condition , dissociation of sulfobutyl group is not complete , leading to less diastereomeric complex formation . results are illustrated in figure 3 . here , k 
 r and k 
 s represent the retention factors of ( r)-cit and ( s)-cit , respectively , and r is the resolution . as seen from figure 3 , the ph of aqueous buffer strongly affected the resolution of cit enantiomers . the resolution of cit enantiomers decreased from 1.30 to 1.05 when the ph was increased from 2.5 to 4.0 , while the retention factors increased with the increase of ph . the ph range examined , cit was almost completely protonated in the aqueous buffer ; thus , cit mainly existed as cations . alternatively , sbe--cd possessed a sulfobutyl group , and the degree of dissociation was significantly impacted by the ph of aqueous buffer . the lower ph led to a high proportion of undissociated sulfobutyl group , which may alter the interactions between sbe--cd and cit enantiomers . however , too low ph of aqueous buffer may have adverse effects on column and chromatographic system employed . in this study the effect of sbe--cd concentration on enantioseparation was investigated under the ph of 2.5 , and the results are summarized in table 1 . a range of sbe--cd concentrations from 4  mm to 16  mm was tested . obviously , the concentration of sbe--cd has a slight impact on the resolution but a significant effect on the retention time . considering that the pressure of column would increase with the increase in sbe--cd concentration , the sbe--cd concentration of 12  mm was chosen as a compromise between a good resolution and a relatively short retention time . the sodium dihydrogen phosphate concentration affects the stability of inclusion complexes formed between the chiral additive and analyte to certain degree [ 25 , 36 ] ( hereby , ( r)-citsbe--cd and ( s)-citsbe--cd ) , further influencing the resolution and retention time . the effect of concentration of sodium dihydrogen phosphate on enantioseparation was examined by varying the concentration of sodium dihydrogen phosphate from 0 to 12  mm , and the results are shown in figure 4 . it is clearly shown that a concentration of 5  mm of sodium dihydrogen phosphate was the best , giving a high resolution and low retention factors under our experimental conditions . the mobile phase composition was found to influence the retention time and resolution of cit enantiomers . further investigation was conducted by varying the volumetric ratio of aqueous buffer , methanol , and acetonitrile under the following conditions : ph of 2.5 , sbe--cd concentration of 12  mm , and sodium dihydrogen phosphate concentration of 5  mm . as shown in table 2 , the variation of organic modifiers ' content has substantial impact on enantioseparation of cit . upon increasing the proportions of methanol or acetonitrile , both retention time and resolution the volume ratio of 21  :  3  :  1 was selected . the effect of column temperature on the resolution of cit enantiomers was studied in a range of 20c to 35c . when the column temperature was increased , the chromatographic parameters including retention factor and resolution decreased accordingly as seen in table 3 . to achieve a fair balance between higher resolution and lower retention time , 25c was chosen as the operating temperature . in summary , under the experimental scope in this study , the optimal operating conditions were determined to be as follows : mobile phase containing a mixture of aqueous buffer , methanol , and acetonitrile with a ratio of 21  :  3  :  1 ( v / v / v ) ; the concentrations of sodium dihydrogen phosphate and sbe--cd in aqueous buffer of 5  mm and 12  mm , respectively ; ph of 2.5 ; flow rate of 1.0  ml / min ; detection wavelength of 240  nm ; column temperature of 25c ; injection volume of 10  l ; and racemic cit concentration of 0.22  mm . the proposed method was validated through the examination of precision , linearity , limit of detection ( lod ) , and limit of quantitation ( loq ) . sequentially , six sample solutions were injected to the column under the same chromatographic conditions . the rsds of retention time were 1.9% for ( r)-cit and 2.3% for ( s)-cit , respectively ; the rsds of peak areas were 1.5% for ( r)-cit and 2.8% for ( s)-cit , respectively , and the rsd of resolution was 2.5% . the accuracy was evaluated by determining the percent recovery through standard additions . as shown in table 4 , the recovery of ( r)-cit and ( s)-cit is 93.8% and 93.7% , respectively , demonstrating that the reported method is accurate for analyzing ( r)-cit and ( s)-cit enantiomers . linearity was examined by conducing triplicate injections at six concentration levels of racemic cit ( ranging from 0.01 to 0.06 regression equations ( the peak area versus the concentration of each enantiomer in g / l ) showed fairly good linearity with regression coefficients ( r 
 ) of 0.9988 for ( r)-cit and 0.9995 for ( s)-cit , being presented as follows:(1)y=1.85760107cr+3774r2=0.9988,y=1.93443107cs1755r2=0.9995 . limit of detection ( lod ) is defined as the lowest analyte concentration that can be detected , giving accuracy within 15% ( bias ) of the nominal value , and 15% of interassay precision . limit of quantitation ( lod ) is defined as the lowest analyte concentration that can be detected to produce a significant response . , lod and loq were calculated using signal / noise ( s / n ) ratio method . lod was taken as the concentration of analyte in which the s / n was 3 while loq was taken as the concentration of analyte where s / n was 10 . the lods of ( r)-cit and ( s)-cit were found to be 0.070  g / ml and 0.076  g / ml , respectively . loqs were 0.235  g / ml for ( r)-cit and 0.254  g / ml for ( s)-cit , respectively . this paper describes a new and reliable method for cit enantioseparation by hplc , using sbe--cd as a chiral mobile phase additive . sulfobutylether--cyclodextrin was used for the first time as a chiral mobile phase additive for determining the enantiomeric composition of racemic citalopram , rather than involving costly chiral stationary phases . the chromatographic conditions were optimized , and a fairly good enantioseparation was obtained under the following conditions : aqueous buffer consisting of 5  mm sodium dihydrogen phosphate and 12  mm sbe--cd with the ph of 2.5 , the ratio of aqueous buffer , methanol , and acetonitrile of 21  :  3  :  1 ( v / v / v ) , and the column temperature of 25c . however , it is noteworthy that the retention time was 75 minutes , relatively long , which might undermine the efficiency and robustness of this newly developed method . more research is needed to improve retention time , possibly through changing organic modifiers and/or adding a second chiral selector .
enantiomeric separation of citalopram ( cit ) was developed using a reversed phase hplc ( rp - hplc ) with sulfobutylether--cyclodextrin ( sbe--cd ) as a chiral mobile phase additive . 
 the effects of the ph value of aqueous buffer , concentration of chiral additive , composition of mobile phase , and column temperature on the enantioseparation of cit were investigated on the hedera ods-2 c18 column ( 250  mm  4.6  mm  5.0  um ) . 
 a satisfactory resolution was achieved at 25c using a mobile phase consisting of a mixture of aqueous buffer ( ph of 2.5 , 5  mm sodium dihydrogen phosphate , and 12  mm sbe--cd ) , methanol , and acetonitrile with a volumetric ratio of 21  :  3  :  1 and flow rate of 1.0  ml / min . 
 this analytical method was evaluated by examining the precision ( lower than 3.0% ) , linearity ( regression coefficients close to 1 ) , limit of detection ( 0.070  g / ml for ( r)-cit and 0.076  g / ml for ( s)-cit ) , and limit of quantitation ( 0.235  g / ml for ( r)-cit and 0.254  g / ml for ( s)-cit ) .