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Use of vancomycin silica stationary phase in packed Part IV: Enantiomer separation of fluoxetine and norfluoxetine employing UV high sensitivity Chimiche, Consiglio Nazionaledelle Ricerche, Area dellaRicerca di Roma, P.O. Box 10, The enantiomeric separation of the antidepressant drug fluoxetine and its main metabolite (norfluoxetine) was achieved by using capillary electrochromatography employing packed capillaries with a vancomycin stationary phase. The capillary (21, 23, or 25 cm675 lm ID), with frits at both ends, was connected to a commercially available high sensitivity UV detection cell to improve the detection limit. With the aim of optimizing the separation of the two pairs of enantiomers in the same run, several parameters were studied: these included the mobile phase composition (buffer pH, organic solvent ratio), the capillary temperature, the length of the packed capillary, and the packed stationary phase type. Experiments were performed using a mobile phase containing aqueous ammonium acetate pH 6 (5 mM final concentration) Bologna, Via Belmeloro 6,40126 Bologna, Italy dissolved in 90% acetonitrile/methanol (MeCN/MeOH) with various organic modifierratios. Increasing MeOH concentration improved the enantioresolution for both analy-tes. However, the two compounds were not separated under these conditions. Theaqueous electrolyte of pH 6 was dissolved at the same concentration as describedabove in organic solvent (MeCN/MeOH, 70/20 v/v) and used for further studies inwhich the length of the stationary phase and its composition were changed. Capilla-ries of 21, 23, and 25 cm length were packed bed length with vancomycin-diol mixedwith silica (3:1) or with vancomycin-diol only and tested for the separation of Flx andNFlx enantiomers. Limits of detection (LOD) and limits of quantification (LOQ) as lowas 25 and 50 ng/mL, respectively, were observed for each racemic analyte.
Key Words: Electrochromatography; Enantiomers; Chiral; Packed capillaries; Drugs;High sensitivity UV detection cell; Received: May 23, 2002; revised: June 14, 2002; accepted: June 14, 2002 chiral compounds are administered as racemic mixtures.
Therefore, with the help of the regulatory authorities, con- siderable effort is being invested in the development of In the last decade great attention has been paid by several chiral separation methods to control the enantiomeric pur- research groups to the separation and quantitation of ity of drugs as well as for pharmacokinetic/pharmacody- chiral compounds in biomedical, pharmaceutical, environ- namic (PK/PD) and/or clinical studies. The analytical mental, and other sciences. Since it has been shown that methods developed should offer good performance such the pharmacological activity and toxicological properties as high efficiency and resolution allowing rapid analysis at of two enantiomers of a certain drug can be different the demand for chiral separation methods by research andapplication groups is continuously increasing [1]. Some Several separation techniques possess the above men-tioned characteristics and consequently are currently Correspondence: Salvatore Fanali, CNR-Istituto di Metodo- employed in the field of chiral analysis; they include high logie Chimiche, Area della Ricerca di Roma, P.O. Box 10, performance liquid chromatography (HPLC), gas chroma- 00016 Monterotondo Scalo (Roma), Italy.
E-mail: tography (GC), supercritical fluid chromatography (SFC), and capillary electrophoresis (CE) [2 – 11].
Abbreviations: acetonitrile, MeCN; enantioresolution factor,Rs; fluoxetine, Flx; methanol, MeOH; norfluoxetine, NFlx; re- In CE several separation modes have been studied in tention time, tR; retention factor, k; supercritical fluid chroma- order to improve the selectivity of the separation and among them capillary electrochromatography (CEC) i 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim seems to be very promising according to the results evaluated in order to find the optimum experimental condi- tions. Finally biological samples spiked with Flx and NFlxracemic mixtures were analyzed.
In CEC improvement of peak efficiency and peak resolu-tion can be easily obtained because the mobile phase iselectrodriven through the stationary phase, thus avoidingthe peak dispersion observed in pressure driven system such as HPLC. The above mentioned properties makeCEC a very attractive separation technique also for the analysis of enantiomers. A wide number of chiral selec-tors, most of them previously used in HPLC, have been Electrochromatographic experiments were carried out applied in CEC for enantiomer separations employing using an automatic HP 3DCE capillary electrophoresis either packed, or open or monolithic columns [11, 16 – 17].
instrument (Agilent, Waldbronn, Germany) equipped with Several racemic analytes belonging to different classes a diode-array UV detector operating at 195 nm. A constant such as derivatized amino acids, herbicides, pharmaceuti- voltage of 25 kV (unless otherwise stated) was applied and cals, etc. have been resolved into their enantiomers the temperature of the capillary cartridge was set in the employing stationary phases containing cyclodextrins or range 15 – 308C. The inlet and the outlet ends of the capil- their derivatives, proteins, cellulose derivatives, Pirkle lary were pressurized at 500 kPa during runs in order to type phases, molecularly imprinted polymers (MIP), weak avoid bubble formation. Fused silica capillaries (75 lm ID, anion exchange type materials, polymethacrylate, glyco- 375 lm OD) were purchased from Composite Metal Ser- peptide antibiotics (GAs), etc. [18 – 32].
vices (Hallow, UK). The packed capillary was connectedto a high sensitivity detection cell with a path length of GAs were first used by Armstrong et al. for the enantio- 1,200 lm (Agilent Technologies, Waldbronn, Germany).
meric separation of a wide number of analytes in HPLC The fused silica capillaries were packed by using a LC se- [33] and later vancomycin and teicoplanin were success- ries 10 pump (Perkin Elmer, Palo Alto, CA, USA). Samples fully applied as chiral selectors in CEC using both packed were injected at the anodic end of the capillary by pressure and monolithic stationary phases in reversed-phase or (unless otherwise stated) at 500 kPa60.4 min followed by polar organic modes [18, 19, 34 – 38]. Recently we a buffer plug injection at 500 kPa60.2 min.
showed the practical applicability of CEC to biomedicalanalysis employing a packed capillary with a silica-vanco-mycin stationary phase for the separation, in the same run, of the two enantiomer pairs of venlafaxine and O-des- Vancomycin hydrochloride was purchased from Sigma methylvenlafaxine in human plasma [36].
(St. Louis, MO, USA). S-venlafaxine, racemic fluoxetine dl-N-Methyl-3-phenyl-3[(a,a,a-trifluoro-p-tolyl)oxy]propyl- (Flx), norfluoxetine (N-Flx), R-nor-fluoxetine, and S-nor- amine, fluoxetine, a selective serotonin reuptake inhibitor fluoxetine were gift from C.B. Eap (UnitØ de Biochimie et drug, is widely used worldwide as an antidepressant due Psychopharmacologie adulte, site de Cery, Switzerland).
to its efficacy and reduced side effects [39]. It was also For the chemical structure of Flx and NFlx, see Figure 1.
used in the therapy of alimentary disorders, bulimia andsevere obesity [40, 41]. Demethylation of fluoxetine, as aconsequence of hepatic metabolism, produces a mainactive metabolite, the norfluoxetine (NFlx). The pharma-cological activity of norfluoxetine has also been documen-ted; however, only the S-isomer exhibited similar activityto the parent drug [42], The aim of this work was to develop a rapid and sensitivemethod for the simultaneous enantiomeric separation of Figure 1. Chemical structures of fluoxetine (Flx) and nor- fluoxetine and its metabolite norfluoxetine by using a fused silica capillary packed with vancomycin-derivatizedsilica stationary phase. In order to increase the detectionsensitivity the packed capillary was connected to an LiChrospher diol silica phase and LiChrospher Si-60, both extended path length UV detection cell. The effects of the with 5 lm particle diameter, sodium cyanoborohydride mobile phase composition (organic modifier type and con- and sodium periodate were from Merck (Darmstadt, Ger- centration), capillary effective length, and chiral stationary many). Ammonium acetate, methanol (MeOH), acetoni- trile (MeCN), all of HPLC analytical grade, were pur- Use of vancomycin silica stationary phase Figure 2. Scheme of the CEC set-up used for the experi-ments.
2.3 Vancomycin stationary phase and CEC packed The chiral stationary phase (CSP) used in this work wassynthesized in our laboratory modifying a publishedmethod [19]; vancomycin was chemically bound toLiChrospher diol silica as described in our previous work[20]. In order to prepare the packed capillaries (75 lm) infollowing procedure was adopted: first a porous temporary Figure 3. Effect of organic modifier ratio present in the frit was prepared by dipping the end of the capillary in a mobile phase on a) retention factor (k) and b) enantioresolu-tion factor (Rs) of fluoxetine and norfluoxetine enantiomers.
slurry of 2 lm silica particles in water and burning with an The capillary (23 cm effective length), was completely electric heater. Secondly, a short zone (about 5 cm) was packed with a stationary phase mixture of vancomycin-diol/ packed with a diol-silica slurry in water and then the van- silica (3:1, w/w), 21 cm; the outlet end was 8.5 cm; 75 lm comycin stationary phase or vancomycin stationary was the ID. Mobile phase 100 mM ammonium acetate pH 6,5% (v/v)/5% water and 90% (v/v) of MeOH-MeCN at different phase/silica (3 : 1, w/w) was packed for the desired length ratios. Applied voltage and capillary temperature were 25 kV (21 or 25 cm) and finally diol-silica slurry was again packed for 5 cm. Frits were prepared as close as possible 500 kPa60.4 min of 50 lg/mL of each racemic analyte. For to the vancomycin packed zone; the capillary was cut, other experimental conditions see text.
equilibrated for 30 min with the mobile phase using the LCpump, connected to the high sensitivity detection cell, and MeOH, which changed in the range 0 – 90 and 90 – 0% positioned in the cartridge for the CEC experiments. An respectively). As expected, modification of the ratio of the untreated fused silica capillary (8.5 cm675 lm ID) was organic solvents (concentration ratio) present in the connected to the detection cell by fitting the appropriate mobile phase caused alterations of the flow velocity due screw (outlet), as shown in Figure 2.
to change of i) the medium viscosity, ii) the dielectric con-stant, and iii) the double layer and zeta potential on boththe stationary phase and the capillary wall. In fact the EOFincreased on decreasing the MeCN concentration from 90% (tEOF, 3.9 min) to 60% (tEOF, 3.4 min), where the vis-cosity of the methanol-acetonitrile mixture reaches a mini- Based on our previous results achieved in CEC [36] by mum, thus altering the magnitude of the EOF in the oppo- using a packed capillary with vancomycin-modified silica site manner. It then decreased, reaching a minimum at particles for the enantioresolution of basic compounds, an aqueous-polar organic solvent mixture (5 mM ammoniumacetate pH 6/acetonitrile-methanol) was selected as Figure 3.a shows the effect of organic modifier ratio on mobile phase in order to study the influence of the organic the retention factor (k) of the two pairs of enantiomers. As modifier on the enantioresolution of fluoxetine and nor- shown in the Figure, when the mobile phase contained fluoxetine. The overall concentration of organic modifier in 0% MeOH (90% MeCN) the retention factors of the two the mobile phase was always 90% (v/v) (x + y = 90%, enantiomers of NFlx were higher than those of Flx enan- where x and y were the concentrations of MeCN and tiomers. On increasing the concentration of MeOH to 10 and 20%, a decrease of k was recorded for the two pairsof enantiomers. Further increase of MeOH concentrationin the mobile phase resulted in an increase of k for the fourstudied compounds. However, the highest values of theretention factor were achieved when the mobile phasecontained 90% of MeOH (0% MeCN). From the abovementioned data it is possible to conclude that i) at 50%MeOH the two pairs of enantiomers were moving very Figure 4. Electrochromatogram of the separation of the two close to each other, exhibiting very similar retention fac- couples of fluoxetine and norfluoxetine enantiomers usingvancomycin CSP packed capillary and extended path length tors, ii) at MeOH concentrations higher than 50% the elu- UV detection cell. Experimental conditions: capillary 25 cm tion order of Flx and NFlx was reversed, with Flx being packed with vancomycin stationary phase; mobile phase, eluted behind NFlx. Therefore, the composition of the 5% of 100 mM ammonium acetate pH 6/5% water/MeCN mobile phase (organic modifier ratio) plays a very impor- (55%, v/v)- MeOH (35%, v/v); applied voltage, 27 kV; capil- tant role in determining both the chiral and the achiral lary temperature, 208C; vials pressurized at 400 kPa duringthe CEC run. Electrokinetic injection 15 kV610 s of 0.5 lg/ separation. This change in selectivity could be related to mL of each racemic compounds dissolved in water; the con- the difference in dissociation constants and solvation of centration of S-venlafaxine (S-Vx) was 0.25 lg/mL. For other the analytes in the solvents of various compositions.
Furthermore, changes in pKa values of the studied com-pounds could not be excluded on modification of mixture the two enantiomers of Flx from those of its metabolite we composition, e. g., increase of MeCN concentration investigated the effect of a different stationary phase com- increased the separation capability of the stationary phase position and length of capillary. Capillaries of 21, 23, and of Flx and NFlx; on the other hand, raising the alcohol con- 25 cm length were packed with stationary phases contain- centration, while increasing the enantioresolution of both ing vancomycin-modified diol/silica (3 : 1) or with vanco- racemic analytes, caused a decrease of their achiral reso- Increasing the length of the packed bed did not improve Resolution of the two pairs of fluoxetine and nor-fluoxetine the chiral resolution of either racemic analyte; however, enantiomers was achieved at any ratio of organic modifier longer elution times were recorded. Satisfactory results studied. Figure 3.b shows the effect of organic solvent were obtained on packing the capillaries with vancomycin ratio on the enantioresolution factor.
stationary phase only. However, longer retention timeswere observed due either to the greater amount of vanco- Rs of both Flx and NFlx enantiomers increased on raising mycin or to the slight reduction of the electroosmotic flow.
the MeOH concentration in the mobile phase up to 45%.
Rs of Flx first decreased at 50 and 60% MeOH and then The best results (optimum chiral and achiral resolution of rose, reaching a maximum at 90%. In the case of NFlx, the Flx and NFlx analytes) were obtained employing the capil- enantioresolution decreased on increasing the MeOH lary fully packed with vancomycin-modified diol particles concentration up to 80%, showing a higher Rs value at of 25 cm length and using a mobile phase containing 5% 90% MeOH. Therefore the highest enantioresolution was of 100 mM aqueous ammonium acetate pH 6/5% water achieved for both analytes in the absence of MeCN. The and 90% of organic modifier (55% MeCN and 35% higher enantioresolution achieved in the presence of only MeOH). Figure 4 shows an electrochromatogram of the MeOH for both racemic compounds is probably due to the CEC separation of Flx and NFlx enantiomers using the longer time spent by the enantiomers in contact with the above described experimental conditions.
chiral stationary phase. Although the mobile phase with The same standard mixture was analyzed ten times under lower content of MeOH did not allow baseline resolution of the same conditions, recording the electrochromato- NFlx enantiomers, higher efficiency and shorter elution grams, measuring the retention times, peak areas, and times (data not shown) were observed.
calculating the enantioresolution factors (Rs). The stan-dard deviation (STD%) of retention time was found to be Further experiments were performed in order to improve 1.2 and 1.1 for Flx and NFlx enantiomers, respectively.
the selectivity of the separation of the two pairs of enantio- The enantioresolution factor STD% was 4.1 and 6.7% for mers, changing the capillary temperature in the range Flx and NFlx, respectively. The limit of detection (LOD, 15 – 308C. A temperature of 208C was selected for enan- signal to noise ratio 3 : 1) was 25 ng/mL for both racemic tiomeric separation because no substantial differences Flx and NFlx while the limit of quantitation (LOQ, signal to were noticed in comparison with the data obtained at low noise ratio 10:1) was 50 ng/mL for each racemic analytes.
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