Journal of Biochemical and Biophysical Methods, 25 (1992) 273-284
273
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-022X/92/$05.00
JBBM 00970
Separation of (R)- and (S)-naproxen using rnicellar chromatography and an a~-acid-glycoprotein column: application for chiral monitoring in human liver rnicrosomes by coupled-column chromatography D. Haupt, C. Pettersson and D. Westerlund Deparl'ment of Analytical Pharmacetaical Chemistry, Uposala UniversityBiomedical Centre, Uppsala (Sweden) (Received 3 July 1992) (Revised version received 14 August 1992) (Accepted 14 August 1992)
Summary A column-switching system for fast determination of (R)- and (S)-naproxen in liver microsomes has been developed. The centrifuged sample was injected directly onto a pre-column with octadecyl~ated silica. The retained analytes were then directed to an at-AGP column using a mobile phase composed of phosphate buffer (pH 6.5), dimethyloetylamine (30 mM) and the nonionic surfactant, Tween ® 20 (40 g/i). The method gave high absolute recoveries and good repeatabilities: 99.6% (1.7% relative standard deviation) and 94.9% (2.4% R.S.D.) for the (R)- and (S)-naproxen, respectively. The use of a surfactant in combination with an aliphatic amine in the mobile phase invo~Tes reduced retention times with retained tmantioselectivity. Furthermore, the presence of the surfactant makes it possible to inject biological samples directly into the chromatographic s~stem. Key words: (R,S)-Naproxen; Quantitation; Direct injection; Micellar chromatography: (Human liver microsome)
Introduction The enantiomers of pharmaceutical drugs often induce disparate biological effects. Differences in metabolic, pharmacokinetic, toxicological as well as in Correspondence address: C. Petters.~,on, Dept. of Analytical Pharmaceutical Chemistry, Uppsala University Biomedical Centre, Box 574, S-751 23 Uppsala, Sweden.
274
pharmacodynamic properties have been observed for several drugs [1]. Furthermore, stereoselective bioinversion, i.e., a transformation of the enantiomer that was administered into its antipode, has been observed in several cases, for instance the non-steroidal anti-inflammatory drugs ibuprofen and naproxen [2]; the inactive (R) form was transformed into the pharmacologically active (S) form. Wechter et al. [3] have proposed an enzymatic pathway for this chiral inversion process and postulated the existence of an R-(-)-arylpropionic acid isomerase system. Studies of stereoselective mechanisms in biological systems require efficient analytical techniques fer qua~ttitation of the separate enantiomers in serum, urine, liver microsomes and other complex biological materials. Chiral chromatography is currently the most suitable technique for separation and determination of enantiomers in biological samples. Chromatographic systems that allow direct injection of sample solutions or methods that require a minimum of pre-treatment (e.g., dilution or centrifug?.t,.'on)of the samples are preferred as they increase the sample throu,~hput arid improve the method performance. Coupled-column chromatogra. phy with aqueous mobile phases enable,,; i~ljections of biological samples after a minimum of pre-treatment and has been applied in bioanalysis for quantitation of, for instance, enantiomeric terbutalin [4], warfarin [5] and metoprolol [6]. One or more pre-columns were used to concentrate and isolate the enantiomers from proteins and other interfering endogenous molecules. In addition, the pre-column protected the chiral column from impairment due to contamination from the biological matrix. In this study, an immobilized protein phase, a t-acid glycoprotein (Enantiopac ®), was used for the separation of (R,S)-naproxen in a column-switching system. The mobile-phase composition was optimized with respect to resolution (Rs), time of analysis and applicability in coupled column chromatography. Separation of hydrophobic enantiomers on protein stationary phases may be problematic as the protein phases only can be used with a limited amount of organic modifiers present in the mobile phase. Micelles improve the solubility of hydrophobic compounds and were therefore used in this study to control the retention of naproxen enantiomers. Surfactants in concentrations above CMC (critical miceile concentration) are attractive mobile-phase additives in bioanalysis of enantiomers as they allow direct injection of serum and urine samples onto the separation column [7]. The studies in this paper have mainly been performed with a first-generation Enantiopac column. Recent improvements of commercially available columns with immobilized at-AOP, i.e., Chirai-AGP ®, involves improved stability and lifetime of the material [8]. The retention characteristics of chirai compounds are also in many cases different using the ChiraI-AGP compared to Enantiopac. Naproxen, for example, is much less retained c n Chiral-AOP than on Enantiopac [8]. Some experiments were performed with the new-generation Chiral-AGP in order to see whether the addition of micellar agents (Tween ® 20) also offered ~dvanlages in this case, Le., decreased capacity factors and improved separation. The bebaviour was quite similar. Adding a mixture of Tween 20 and N,N-di. methyloctylamine to the mobile phase decreased the retention time and increased the resolution of naproxen when using Enantiopac as well as Chiral AGP.
275
Thus, the principles presented in this paper, i.e., a decrease of retention times by applying micellar phases in combination with retained stereoselectivity, seem to be essential features that should be applicable to other analytes giving high retentions on present-day chiral-protein columns.
Materials and Methods Apparatus The HPLC system used was a Beckman 114 M pump (Beckman Instruments, Berkeley, CA, USA), a Merk-Hitachi 655A-22 UV detector measuring at 254 nm and a LDC FluoroMonitor III Model 1311 (LDC, Riviera Beach, FL, USA), operating at an excitation wavelength of 254 nm and emission cut-off at 360 nm. The irjector was a Rheodyne 7120 syringe-loading injector (Rheodyne, Berkeley, CA, USA) equipped with a 20-~tl loop. A Valco CV-3-HPAX three-port valve (Valco Instruments, Houston, Texas, USA) was used as a venting valve. The pre-column was a LiChroma tube 21 × 6.6 mm I.D. of stainless steel (Handy and Harman Tube, Norristown, PA, lISA) eq!J~pp~d with modified Swagelok connectors and a 2-~m screen at the outlet' and a spreader at the inlet. The pressure regulator used to control the back-pressure when applying the pre-column venting technique was constructed according to the method of Lindner et al. [9]. The bulk material for one of the Enantiopac colum,~ was kindly supplied by LKB (Bromma, Sweden). The columns, injector and reservoir were thermostated using a HETO Type 02 PT 923 TC (Birkercd, Denmark). The pH measurements were made with an AG 9100 Metrohm 632 pH meter (Herisau, Switzerland) equipped with a Type 1014 glass pH electrode.
Chemicals (-)-(R)- and (+)-(S)2-(6-methoxy-2-naphthyl)propionic acid (naproxen) were gifts from Astra AB (S6dert~lje, Sweden). N,N-dimethyloctylamin (DMOA) was purchased from ICN Pharmaceuticals (Plainview, NY, USA). Tween 20 (zur synthese), (2)-propanol (p.a.), sodium laurylsulphate, sodium dihydrogenphosphate, disodium hydrogensulphate and Lichrosorb RP-18 (7 and 10 ~M) were from E. Merck (Darmstadt, Germany). The human liver microsome suspensions (approx. 10 mg/ml) were a gift from the Dept. of Clinical Pharmacology, Karolinska Instituter, Huddinge Hospital (Huddinge, Sweden).
Column preparation and chromatographic technique Three a~-acid glycoprotein columns (Nos. 1, 2 and 3) were used in this study. The first one, En~mtiopac No. 1 (100 × 4.0 ram), was a gift from LKB (Bromma, Sweden). The secoad column was slurry-packed according to the packing procedure given by LKB. Disopyramide enantiomers were used to test the chiral column. The asyml~,etry factor (Asf) of the later eluted enantiomer was calculated and the column wa,~ accepted if 0.7 < Asf < 1.4. The al-~acid glycoprotein column No. 2 was used fox the final test, i.e., determination of (R,S).naproxen in liver microsome suspension. The third column was Chiral-AGP (ChromTech AB, Nots-
276 from pum~) 1
2
3
4
5
6
to detector
to waste
Fig. 1. Coupled-column system. (1) 2-ml loop for plug fluid; (2) sample injector; (3) pre-column; (4) three-port venting valve; (5) laboratory-built pressure regulator; (6) separation column.
borg, Sweden). The analytical column and pre-column of LiChrosorb RP-18 were slurry-packed at high pressure. The mobile phases were prepared by dissolving the additives in a phosphate buffer with an ionic strength of 0.1. The column temperature was 25.0°C and the flow-rate 0.3 ml/min unless otherwise stated.
Sample preparation The spiked samples were l:repared by mixing 400 /zi o/ a ~aproxen stock solution (0.218-0.239 raM), 100 ~1 of a liver microsome suspensi ~ and 1500/~1 of mobile phase. The samples ~ere centrifuged at 3200 × g f~ 10 rain before injection. A small precipitate was observed. The absolute recovery of naproxen from human liver microsomes was determined by comparing peak areas, measured by triangulation, obtained from injection of standard solutions and from spiked microsome samples, processed according to the complete analytical procedure.
Column-switchingprocedure The column-switching system used is shown in Fig. 1. Initially, a pre-column plug-venting technique developed by Daoud et al. [10] was applied to avoid any accidental precipitation of proteins due to mixing with incompatible mobile phases. A 2.0-ml plug (e.g., phosphate buffer, pH 3] was introduced by the firat injector and the sample was then injected in the middle part of the plug stream by the second injector. Thus, the sample solution was transferred to the p~'e-column surrounded by a neat buffer solution. The naproxen was retained on the short pre-column whereas the proteins were unretained as they are mainly excluded from the pores of the solid phase. When the plug containing the proteins had passed the pre-column and been eluted to waste, the three-port valve was switched and the eluted naproxen enantiomers were introduced onto the Enantiopac column. In studies of recoveries from the pre-column, the chiral column was exchanged for an achiral column (LiChrosorb RP-8). The composition of the plug fluid was chosen in order to give a high capacity factor for the naproxen enantiomers on the pre-column (LiChrosorb RP 18 or Nucleosil C18). However, most of the studies were performed without the plug, i.e., direct injection of centrifuged biological samples onto the pre-column. Resu|ts and Discussion
Recoveries Initially the pre-column plug-venting technique was studied. Selecting a plug of pH 3.0, where the enantiomers are mainly present in uncharged form and strongly
277 TABLE 1 Recovery of naproxen when using pre-column venting-plug technique Precolumn Separation column Mobile phase Solute Flow-rate Temperature Detector
Nucleosil C18 (10 x 4.6 ram) Lichrosorb RP-8 (7/tM, 100 x 4.6 mm) 30 mM DMOA and 40 g/I Tween 20 in phosphate buffer (pH 7.5, I - 0.1) R- and S-naproxen, 2.2-2.4 nmol 1.0 ml/min 25.0°C Excitation 254 nm emission, cut-off 360 nm
Sample
Plug
Recovery (n = 4) %
S-( + )-naproxen R-( - ~naproxen R-( - )-naproxen
phosphate buffer, pH 3.0 phosphate buffer pH 3.0 30 mM DMOA in phosphate buffer, pH 7.5 30 mM DMOA in phosphate buffer, pH 7.5 30 mM DMOA in phosphate buffer, pH 7.5
93.3 97.0 98.7
R-(-)-naproxen in spiked liver microsome solution S-(+)-naproxen in spiked liver mierosome solution
97.0 93.3
retained on the pre-column, gave 93.3 and 97.0% recovery for the (S)-naproxen and (R)-naproxen, respectively (Table 1). High recoveries (> 93%) were also obtained when the plug was a phosphate buffer solution of pH 7.5 containing a counter-ion DMOA. The enantiomers are then ionized and retained as ion-pairs with the hydrophobic cation DMOA. No significant differences in recoveries were
TABLE 2 Recovery of S-(+)-naproxen with and without plug Precolumn Separation column Mobile phase Solute Flow-rate Temperature Detector
Lichrosorb RP-18 (21 x 4.6 mm) Lichrosorb RP-8 (7 ~tM, 100 x 4.6 mm) 30 mM DMOA and 40 g/! Tween 20 in phosphate buffer (pH 7.5, ! = 0.1) S-(+)-naproxen (0.48 nmol) in liver microsome suspension 1.0 ml/min 32.5°C Excitation 254 nm emission, cut-off 360 nm
Plug
Sample work-up
30 mM DMOA in phosphate buffer, pH 7.5 m
centrifuged
Recovery
n
Between-day repeatability (R.S.D. %)
97.8
4
99.2
10
10|
52
drifting b~seline increasing back pressure |.9
278
_/ A, I
TiME
I
10.4 8.1
I
0
(rnin) Fig. 2 Determination of (R,S).naproxen in spiked liver microsome suspension. Pre-column: Lichrosorb RP-18 10 ttm (21 x4.6 mm I.D.); column: Enantiopac, 100×4.0 mm (No. 2); mobile phase, 30 mM DMOA and 40 g/I Tween 20 in phosphate buffer (pH 6.5, I - 0.1); solute: 44 ~M (R)-naproxen, 48 ~tM (S)-naproxen; flow-rate, 0.3 ml/min; temperature, 35.0°C.
observed from standard solutions and the liver microsomes solutions. However, transferring the naproxen to the analytical column gave rise to a drifting baseline that made it difficult to accurately quantify the enantiomeric composition. Direct injection of microsome solution without a plug gave high recoveries (Table 2), but the back pressure increased after six injections and made any accurate quantitation impossible. The remedy for these disadvantageous properties was a centrifugation (3200 ×g for 10 min) of the sample before the injection, which eliminated microparticles responsible for blockage of the column and causing high back-pressure and unstable baseline. The stability and recovery study showed a more than 99% recovery of (S)-naproxen with good day-to-day reproducibility (R.S.D. 1.9%, n = 10). No significant change in back pressure or column efficiency was observed after 52 injections of 20 ~! liver microsome suspension. A typical chromatogram (Fig. 2), with an Enantiopac column coupled on-line with the pre-column, demonstrates an almost complete separation ( R s - 1.43) of the enantiomers in spiked liver-microsome suspension. The fluorometric detection provided a very high selectivity towards endogenous compounds - no interfering peaks were observed. The recoveries of (R)- and (S)-naproxen were found to be 99.6% (R.S.D. 1.7%, n --4) and 94.9% (R.S.D. 2.4%, n = 4), respectively.
Effects on retention and selectivity by charged modifiers Long retention times and insufficient resolution of naproxen enantiomers were obtained on Enantiopac when using phosphate buffer (pH 6.4) as the eluent (Table 3). As previously reported [11] the addition of dimethyloctylamine (DMOA) to the mobile phase improved the resolution. The regulation of retention and stereoselectivity on Enantiopac by charged modifiel,s in the mobile phase has been studied extensively [12] and it has been suggested that the modifiers improve the separation by competing for achirai and chiral-binding sites on the aracid glycoprotein or by conformational changes of the immobilized protein. The presence of DMOA in the mobile phase, however, also increased the capacity factors for the naproxen enantiomers. In an attempt to decrease retention times, isopropanol was added to the DMOA containing mobile phase. However, in order to obtain k' < 10 it was necessary to add at least 8% (v/v) of 2-propanol, which led to a complete loss of
279 TABLE 3 Influence of 2-propanol and charged modifiers on chiral separation Column Mobile phase Solute Flow-rate Temperature
Enantiopac, 100× 4.0 mm (No. 1) Additives in phosphate buffer (pH 6.4, I = 0~l) R- and S-naproxen, 1.0-1.1 nmol 0.3 ml/min 25.0°C
Additives
k~
N
as/R
Rs
0.5 mM DMOA 1.0 mM DMOA 0.6 mM DMOA + 8% 2-pmpanol (v/v) 1.0 mM DMOA + 1.0 mM laurylsulphate 1.1 mM DMOA + 5.0 mM laurylsulphate
19 21 25
700 1100 1000
1.30 1.60 1.77
1.4 2.6 3.1
1200
1.00
-
600
1.49
2.0
600
1.00
-
9.7 11 6.8
the enantioselectivity. An alternative way to reduce the retention could be by addition of a co-ion, i.e., an ion of the same charge as the solute, to the mobile phase. The co-ion competes with naproxen for the limited binding capacity of the immcbilized at-acid glycoprotein (Enantiopac) [12]. Adding 1.0 mM of laurylsulfate (critical micelle concentration (CMC) approx. 8.3 mM [13]) to a mobile phase containing 1.0 mM DMOA decreased the k' for (R)-naproxer~ by a factor of two ( k ~ - 11), but the loss of enantioselectivity at 5.0 mM of the anion, giving a k~ = 6, 8, revealed the limitations in controlling retention by laurylsulphate. In conclusion, isocratic elution, using charged modifiers (DMOA and laurylsulphate) in a phosphate buffer as the mobile phase, promoted a complete resolution of (R,S)-naproxen on Enantiopac. However, this mobile-phase composition was less suitable for routine analysis of biological samples as it gave high capacity factors and low column efficiency.
Effects of uncharged micellar agents Surfactants in concentrations above CMC have been used in liquid chromatography for regulation of retention and selectivity [14]. Furthermore, the powerful solvating properties of nonionic and ionic detergents (micellar chromatography) have allowed drug analysis by direct injection of untreated serum and urine in chromatographic systems [7]. The addition of laurylsulphate in high concentrations to the mobile phase seems to impair the stereoselectivity for naproxen (Table 3). As an alternative a nonionic surfactant, Tween 20, was investigated as mobile-phase additive. The change in k' for (R)-naproxen when adding Tween 20 (structure in Fig. 3) in concentrations above CMC is shown in Fig. 4. The CMC for Tween 20 is 0.14 g/l [13]. Observe the presence of chiral carbons in the structure (Fig. 3); however, the surfactant used is probably a racemic mixture. Distribution of the
280 ~f H(OCHz.CHa)aO- -
.0.~ O(CH=.CH~.O)nH ~ I - C H . CH.aO• CO, C,THaa - - O ( C H = • el'l=O)nH
n
Tween 20 =Sorbimacrogolilauras
(ca) 6
Fig. 3. Structure of the suffactant Tween 20.
tR rain) 80
TWEEN 20 glL
Fig. 4. Influence of Tween 20 on retention and stereoselectivity. Column: Ewiantiopac (No. 1); mobile phase: TWeen 20 in phosphate buffer (pH 6.5, 1 = 0.1); solute: (o) 1.0-2.0 nmol (R)-naproxen, (e) 1.0-2.2,1tool ($)-naproxen; flow-rate, 0.3 ml/min; temperature, 25.0°(3.
enantiomers to the miceiles in the mobile phase will accelerate their elution from the column. However, the drastic decrease in enantioselectivity (Fig. 4) when the surfactant was present in the mobile phase indicated a change in the binding
..j (rain)
TiME
2.8
1.3
0
Fig. 5 Resolution of the enantiomers of naproxen. Column: Chiral-AGP 100x 4.0 ram; mobile phase, 30 mM DMOA and 40 g/! Tween® 20 in phosphate buffer (pH 6.5, /=0.1); solute" 0.15 nmol (R)-naproxen, 0.18 nmol (S)-naproxen; flow-rate, 0.9 mi/min; temperature, 25.0°C.
281 TABLE 4 Influence of N,N-dimethyioctylamine (DMOA) on chiral resolution with micellar mobile-phase Column Mobile phase Solute Flow-rate Temperature
Enantiopac 100 × 4.0 mm (No. 1) 40 g/! Tween 20 in phosphate buffer (pH 7.5, I -- 0.1) R- and S-naproxen, 2.2-2.4 nmol 0.3 ml/min 35.0°C DMOA (mM)
k~ N
aS/R Rs
5.0
10
20
4.3 1500 1.19 1.3
3.3 1300 1.31 1.6
2.6 760 1.44 1.8
properties of the protein ,phase. No chiral separation was obtained using Tween 20 as mobile-phase additive and LiChrosorb RP-18 as achiral phase. Therefore the change in enantioselectivity is not due to the solvation of enantiomers in the mobile-phase as these equilibria are non-stereoselective. As discussed above, the addition of DMOA to the mobile-phase increased the enantioselectivity and resolution of naproxen enantiomers on Enantiopac. Similarly, DMOA improved the selectivity and resolution with micellar mobile-phases (Table 4). Furthermore, increasing the amine concentration gave rise to a decrease in the capacity factors for the naproxen enantiomers (Table 4). An almost complete resolution for (R)and (S)-naproxen in liver microsomes solution could be obtained within less than 14 min by using 30 mM of DMOA and 40 g/l Tween 20 in phosphate buffer (pH 6.5) as the mobile-phase (Fig. 2). The optimal mobile-phase pH differed for the two Enantiopac columns; No. 1 gave the highest resolution at pH 7..5, while No. 2 had its best performance at pH 6.5. Otherwise the columns showed the same general behaviour but exhibited some individual differences. Disopyramide was, for example, better resolved on Enantiopac No. 1 than on column No. 2 (Rs ffi 4.0 and 3.2, respectively). The influence of pH and column temperature on the chiral resolution with Tween 20 present in the mobile-phase was also investigated (Tables 5 and 6). The selectivity and the efficiency were highest at high pH, where the acid is mainly present as anion; pK a of naproxen is 4.2 [15]. Under these conditions naproxen enantiomers were distributed as ion-pairs with DMOA to the protein stationary phase as well as to the micelles. However, the net charge of the protein stationary phase and the protein conformation will depend on pH, making the effects of a change in pH difficult to interpret and predict. Interestingly, increasing the column temperature from 16.5°C to 45°C had no significant effect on the stereosekectivity when Tween 20 was present in the mobile-phase, whereas the efficiency and in consequence the resolution of (R,S)-naproxen improved. Thus, Enantiopac used at an elevated temperature with a mobile-phase containing Tween 20 and DMOA
282 TABLE 5
Influence of pH on chiral resolution in presence of Tween 20 and N,N-dimeth?loctylamine Enantiopae 100 x 4.0 (No. 1) 5.0 mM DMOA and 40 g/l Tween 20 in phosphate buffer (I = 0.1) R- and S-naproxen, 2.2-2.4 nmol 0.3 ml/min 35.00C
Column Mobile phase Solute Flow-rate Temperature pH k~ N
aR/s Rs
7.5
6.5
5.6
4.5
4.3 1500 1.19 1.3
8.2 1300 1.15 1.1
4.2 800 1.20 1.0
1.5 370 1.09 0.3
promotes complete separation of naproxen enantiomers within a reasonable time of analysis.
Chromatographic characteristics of the second generation of AGP columns (Chiral. AGP) Since all experiments described so far have been performed with the first-generation AGP column, it was of interest to study whether the second generation column, Chiral-AGP, had similar properties. The retentions of the naproxen enantiomers are less strong on the new column, k ~t being 5 compared to 19 with neat buffer solutions (pH 6.4-6.5) as the mobile-phase (see Tables 7 and 3, respectively). However, applying a micellar mobile-phase containing DMOA resulted in similar effects, i.e., decreased retentions and retained selecti, 6' and resolutioa (see Tables 4, 5 and 7).
TABLE 6
Influence of temperature on chiral resolution with a micellar mobile phase Column Mobile phase Solute Flow-rate
Enantiopac 100 × 4.0 (No. 1) 5.0 mM DMOA and 40 g/i Tween 20 in phosphate buffer (pH 6.5, !-- 0.1) R. and S-naproxen, 2.2-2.4 nmol 0.3 ml/min Temperature (°C)
k~ N aR/S Rs
16.5
25.0
35.0
45.0
8.0 680 1,16 0.8
11 830 1.13 0.8
8.2 1300 1.15 1.1
6.6 1700 1.16 1.2
283 TABLE 7 Influence of N,N-dimethyloctylamine (DMOA) on chiral resolution with micellar mobile-phase and the second generation a~-AGP column Chiral-AGP, 100 × 4.0 mm (R)- and (S)-naproxen, 0.29-0.35 nmol 0.9 ml/min 25.0°C
Column Solute Flow-rate Temperature Mobile phase phosphate buffer (pH 6.5, I - 0.1) 30 mM DMOA and 40 g/! Tween 20 in phosphate buffer (pH 6.5, i m 0.1)
k~
N
as/•
Rs
5.0
680
1.12
1.5
0.68
300
3.59
2.7
It is therefore highly probable that a similar approach to that described in this paper for direct injection and chiral separation with the first-generation AGP columns is also applicable for the new generation of the protein c¢~umn.
Simplified description of the methed and ~¢s application Micellar chromatography using Tween 20 and DMOA as additives in phosphate buffer as mobilephase and a~-AGP as the chiral stationary phase decreased the retention times for R/S-naproxen with retained stereoselectivity. A coupled-column system with a pre-column connected in series with the chiral column could be used for fast analysis of the enantiomeric composition of naproxen in liver microsomes after injection of the centrifuged biological fluid.
Acknowledgements This project was supported by the Swedish Natural Science Research Council (project No. K-Ku 6248-301). We are grateful for research grants provided by the Swedish Academy of Pharmaceutical Sciences and the I.F. Foundation for Pharmaceutical Research.
References 1 Ariens, E.J. (1989) Raccmates - an impediment in the use of drugs and agrochemicals in Chiral separations by HPLC. Krstulovic, A.M. (Ed.), Ellis Horwood, Chichester, pp. 31-68. 2 Hurt, A.J. and Caldwell, J. (1984) The importance of stereochemistry in the clinical pharnlacokinetics of the 2~arylpropionic acid non-steroidal antiinflammatory drugs. Clin. Pharm~,cokinet. 9, 371-373.
284 3 Wechter, W.J., Loughhead, D.G, Reiser, R.J., Vangiessen, G.J. and Kaiser, D.G. (1974). Enzymatic inversion at saturated carbon: nature and mechanism of the inversion of R-(-)-p-iso-butyi hydratropic acid. Biochem. Biophys. Res. Commun. 61, 833-837. 4 Edholm, L.-E., Lindberg, C., Pauisson, J. and Walhagen, A. (1988). Determination of drug enantiomers in biological samples by coupled column liquid chromatography and liquid chromatography-mass spectrometry. J. Chromatogr. 424, 61-72. 5 Chu, Y.-Q. and Wainer, I.W. (1988). The measurement of warfarin enantiomers in serum using coupled achiral/chiral high-performance liquid chromatography. Pharm. Res. 5, 680-683. 6 Walhagen, A., Edholm, L.-E., Heeremans, C.E.M., van der Hoeven, R.A.M., Niessen, W.M.A., Tjaden, U.R. and van der Greta,f, J. (1989) Coupled column chromatography-mass spectrometry, Thermospray liquid chromatographic-mass spectrometric and liquid chromatographic-tandem mass spectrometric analysis of metoprolol enantiomers in plasma using phase-system switching. J. Chromagogr. 474, 257-263. 7 Cline Love, L.J., Zibas, S., Noroski, J. and Arunyanan, M. (1985) Direct injection of untreated serum using non-ionic and ionic micellar liquid chromatography for determination of drugs. J. Pharm. Biomed. AnaL, 3, 511-521. 8 Keta, J.R. (1991) ('!Jromatographic separation of the optical isomers of naproxen. J. Chromatogr. 543, 355-366. 9 Lindner, W. and R~tckendorfer~ H. (1983) HPLC - residue analysis of the herbicide pyridate in cereals. Int. J. Environ. Anal. Chem. 16, 205-218. 10 Daoud, N., Arvids~on, T. and Wahlund, K.G. (1987) Precolumn-venting plug technique for direct injection of untreated blood plasma samples into reversed-phase liquid chromatographic systems. J. Chromatogr. 385, 311-322. 11 Hermansson, J. and Eriksson, M. (1986) Direct liquid chromatographic resolution of acidic drugs using a chiral at-acid glycoprotein column (Enantiopac ®) J. Liq. Chromatogr. 9, 621-639. 12 Hermansson, J. and Schill, G. (1989) Separation of chiral compounds with at-acid 81ycoprotein as selector in High-performance liquid chromatography. Brown, P.R. and Hartwick, R.A. (Eds.), Wiley, pp. 337-3'74. 13 Mukerjee, P. and Mysels, K.J. (1971) Critical Mice'd Concentrations of aqueous Suffactant Systems, U.S. Govern~nent Printing Office, Washington, PC. 14 Cline Love, L.$. and Fett., Joseph J. (1991) Optimization of selectivity in micellar chromatographic procedures for the determinatit)n of drugs in urine by direct injection. J. Pharm. Biomed. Anal. 9, 323~333, 15 Martindale, The extra Pharmacopoeia, Twenty-eighth edition, The Pharmaceutical Press, London, XXVI.
273
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-022X/92/$05.00
JBBM 00970
Separation of (R)- and (S)-naproxen using rnicellar chromatography and an a~-acid-glycoprotein column: application for chiral monitoring in human liver rnicrosomes by coupled-column chromatography D. Haupt, C. Pettersson and D. Westerlund Deparl'ment of Analytical Pharmacetaical Chemistry, Uposala UniversityBiomedical Centre, Uppsala (Sweden) (Received 3 July 1992) (Revised version received 14 August 1992) (Accepted 14 August 1992)
Summary A column-switching system for fast determination of (R)- and (S)-naproxen in liver microsomes has been developed. The centrifuged sample was injected directly onto a pre-column with octadecyl~ated silica. The retained analytes were then directed to an at-AGP column using a mobile phase composed of phosphate buffer (pH 6.5), dimethyloetylamine (30 mM) and the nonionic surfactant, Tween ® 20 (40 g/i). The method gave high absolute recoveries and good repeatabilities: 99.6% (1.7% relative standard deviation) and 94.9% (2.4% R.S.D.) for the (R)- and (S)-naproxen, respectively. The use of a surfactant in combination with an aliphatic amine in the mobile phase invo~Tes reduced retention times with retained tmantioselectivity. Furthermore, the presence of the surfactant makes it possible to inject biological samples directly into the chromatographic s~stem. Key words: (R,S)-Naproxen; Quantitation; Direct injection; Micellar chromatography: (Human liver microsome)
Introduction The enantiomers of pharmaceutical drugs often induce disparate biological effects. Differences in metabolic, pharmacokinetic, toxicological as well as in Correspondence address: C. Petters.~,on, Dept. of Analytical Pharmaceutical Chemistry, Uppsala University Biomedical Centre, Box 574, S-751 23 Uppsala, Sweden.
274
pharmacodynamic properties have been observed for several drugs [1]. Furthermore, stereoselective bioinversion, i.e., a transformation of the enantiomer that was administered into its antipode, has been observed in several cases, for instance the non-steroidal anti-inflammatory drugs ibuprofen and naproxen [2]; the inactive (R) form was transformed into the pharmacologically active (S) form. Wechter et al. [3] have proposed an enzymatic pathway for this chiral inversion process and postulated the existence of an R-(-)-arylpropionic acid isomerase system. Studies of stereoselective mechanisms in biological systems require efficient analytical techniques fer qua~ttitation of the separate enantiomers in serum, urine, liver microsomes and other complex biological materials. Chiral chromatography is currently the most suitable technique for separation and determination of enantiomers in biological samples. Chromatographic systems that allow direct injection of sample solutions or methods that require a minimum of pre-treatment (e.g., dilution or centrifug?.t,.'on)of the samples are preferred as they increase the sample throu,~hput arid improve the method performance. Coupled-column chromatogra. phy with aqueous mobile phases enable,,; i~ljections of biological samples after a minimum of pre-treatment and has been applied in bioanalysis for quantitation of, for instance, enantiomeric terbutalin [4], warfarin [5] and metoprolol [6]. One or more pre-columns were used to concentrate and isolate the enantiomers from proteins and other interfering endogenous molecules. In addition, the pre-column protected the chiral column from impairment due to contamination from the biological matrix. In this study, an immobilized protein phase, a t-acid glycoprotein (Enantiopac ®), was used for the separation of (R,S)-naproxen in a column-switching system. The mobile-phase composition was optimized with respect to resolution (Rs), time of analysis and applicability in coupled column chromatography. Separation of hydrophobic enantiomers on protein stationary phases may be problematic as the protein phases only can be used with a limited amount of organic modifiers present in the mobile phase. Micelles improve the solubility of hydrophobic compounds and were therefore used in this study to control the retention of naproxen enantiomers. Surfactants in concentrations above CMC (critical miceile concentration) are attractive mobile-phase additives in bioanalysis of enantiomers as they allow direct injection of serum and urine samples onto the separation column [7]. The studies in this paper have mainly been performed with a first-generation Enantiopac column. Recent improvements of commercially available columns with immobilized at-AOP, i.e., Chirai-AGP ®, involves improved stability and lifetime of the material [8]. The retention characteristics of chirai compounds are also in many cases different using the ChiraI-AGP compared to Enantiopac. Naproxen, for example, is much less retained c n Chiral-AOP than on Enantiopac [8]. Some experiments were performed with the new-generation Chiral-AGP in order to see whether the addition of micellar agents (Tween ® 20) also offered ~dvanlages in this case, Le., decreased capacity factors and improved separation. The bebaviour was quite similar. Adding a mixture of Tween 20 and N,N-di. methyloctylamine to the mobile phase decreased the retention time and increased the resolution of naproxen when using Enantiopac as well as Chiral AGP.
275
Thus, the principles presented in this paper, i.e., a decrease of retention times by applying micellar phases in combination with retained stereoselectivity, seem to be essential features that should be applicable to other analytes giving high retentions on present-day chiral-protein columns.
Materials and Methods Apparatus The HPLC system used was a Beckman 114 M pump (Beckman Instruments, Berkeley, CA, USA), a Merk-Hitachi 655A-22 UV detector measuring at 254 nm and a LDC FluoroMonitor III Model 1311 (LDC, Riviera Beach, FL, USA), operating at an excitation wavelength of 254 nm and emission cut-off at 360 nm. The irjector was a Rheodyne 7120 syringe-loading injector (Rheodyne, Berkeley, CA, USA) equipped with a 20-~tl loop. A Valco CV-3-HPAX three-port valve (Valco Instruments, Houston, Texas, USA) was used as a venting valve. The pre-column was a LiChroma tube 21 × 6.6 mm I.D. of stainless steel (Handy and Harman Tube, Norristown, PA, lISA) eq!J~pp~d with modified Swagelok connectors and a 2-~m screen at the outlet' and a spreader at the inlet. The pressure regulator used to control the back-pressure when applying the pre-column venting technique was constructed according to the method of Lindner et al. [9]. The bulk material for one of the Enantiopac colum,~ was kindly supplied by LKB (Bromma, Sweden). The columns, injector and reservoir were thermostated using a HETO Type 02 PT 923 TC (Birkercd, Denmark). The pH measurements were made with an AG 9100 Metrohm 632 pH meter (Herisau, Switzerland) equipped with a Type 1014 glass pH electrode.
Chemicals (-)-(R)- and (+)-(S)2-(6-methoxy-2-naphthyl)propionic acid (naproxen) were gifts from Astra AB (S6dert~lje, Sweden). N,N-dimethyloctylamin (DMOA) was purchased from ICN Pharmaceuticals (Plainview, NY, USA). Tween 20 (zur synthese), (2)-propanol (p.a.), sodium laurylsulphate, sodium dihydrogenphosphate, disodium hydrogensulphate and Lichrosorb RP-18 (7 and 10 ~M) were from E. Merck (Darmstadt, Germany). The human liver microsome suspensions (approx. 10 mg/ml) were a gift from the Dept. of Clinical Pharmacology, Karolinska Instituter, Huddinge Hospital (Huddinge, Sweden).
Column preparation and chromatographic technique Three a~-acid glycoprotein columns (Nos. 1, 2 and 3) were used in this study. The first one, En~mtiopac No. 1 (100 × 4.0 ram), was a gift from LKB (Bromma, Sweden). The secoad column was slurry-packed according to the packing procedure given by LKB. Disopyramide enantiomers were used to test the chiral column. The asyml~,etry factor (Asf) of the later eluted enantiomer was calculated and the column wa,~ accepted if 0.7 < Asf < 1.4. The al-~acid glycoprotein column No. 2 was used fox the final test, i.e., determination of (R,S).naproxen in liver microsome suspension. The third column was Chiral-AGP (ChromTech AB, Nots-
276 from pum~) 1
2
3
4
5
6
to detector
to waste
Fig. 1. Coupled-column system. (1) 2-ml loop for plug fluid; (2) sample injector; (3) pre-column; (4) three-port venting valve; (5) laboratory-built pressure regulator; (6) separation column.
borg, Sweden). The analytical column and pre-column of LiChrosorb RP-18 were slurry-packed at high pressure. The mobile phases were prepared by dissolving the additives in a phosphate buffer with an ionic strength of 0.1. The column temperature was 25.0°C and the flow-rate 0.3 ml/min unless otherwise stated.
Sample preparation The spiked samples were l:repared by mixing 400 /zi o/ a ~aproxen stock solution (0.218-0.239 raM), 100 ~1 of a liver microsome suspensi ~ and 1500/~1 of mobile phase. The samples ~ere centrifuged at 3200 × g f~ 10 rain before injection. A small precipitate was observed. The absolute recovery of naproxen from human liver microsomes was determined by comparing peak areas, measured by triangulation, obtained from injection of standard solutions and from spiked microsome samples, processed according to the complete analytical procedure.
Column-switchingprocedure The column-switching system used is shown in Fig. 1. Initially, a pre-column plug-venting technique developed by Daoud et al. [10] was applied to avoid any accidental precipitation of proteins due to mixing with incompatible mobile phases. A 2.0-ml plug (e.g., phosphate buffer, pH 3] was introduced by the firat injector and the sample was then injected in the middle part of the plug stream by the second injector. Thus, the sample solution was transferred to the p~'e-column surrounded by a neat buffer solution. The naproxen was retained on the short pre-column whereas the proteins were unretained as they are mainly excluded from the pores of the solid phase. When the plug containing the proteins had passed the pre-column and been eluted to waste, the three-port valve was switched and the eluted naproxen enantiomers were introduced onto the Enantiopac column. In studies of recoveries from the pre-column, the chiral column was exchanged for an achiral column (LiChrosorb RP-8). The composition of the plug fluid was chosen in order to give a high capacity factor for the naproxen enantiomers on the pre-column (LiChrosorb RP 18 or Nucleosil C18). However, most of the studies were performed without the plug, i.e., direct injection of centrifuged biological samples onto the pre-column. Resu|ts and Discussion
Recoveries Initially the pre-column plug-venting technique was studied. Selecting a plug of pH 3.0, where the enantiomers are mainly present in uncharged form and strongly
277 TABLE 1 Recovery of naproxen when using pre-column venting-plug technique Precolumn Separation column Mobile phase Solute Flow-rate Temperature Detector
Nucleosil C18 (10 x 4.6 ram) Lichrosorb RP-8 (7/tM, 100 x 4.6 mm) 30 mM DMOA and 40 g/I Tween 20 in phosphate buffer (pH 7.5, I - 0.1) R- and S-naproxen, 2.2-2.4 nmol 1.0 ml/min 25.0°C Excitation 254 nm emission, cut-off 360 nm
Sample
Plug
Recovery (n = 4) %
S-( + )-naproxen R-( - ~naproxen R-( - )-naproxen
phosphate buffer, pH 3.0 phosphate buffer pH 3.0 30 mM DMOA in phosphate buffer, pH 7.5 30 mM DMOA in phosphate buffer, pH 7.5 30 mM DMOA in phosphate buffer, pH 7.5
93.3 97.0 98.7
R-(-)-naproxen in spiked liver microsome solution S-(+)-naproxen in spiked liver mierosome solution
97.0 93.3
retained on the pre-column, gave 93.3 and 97.0% recovery for the (S)-naproxen and (R)-naproxen, respectively (Table 1). High recoveries (> 93%) were also obtained when the plug was a phosphate buffer solution of pH 7.5 containing a counter-ion DMOA. The enantiomers are then ionized and retained as ion-pairs with the hydrophobic cation DMOA. No significant differences in recoveries were
TABLE 2 Recovery of S-(+)-naproxen with and without plug Precolumn Separation column Mobile phase Solute Flow-rate Temperature Detector
Lichrosorb RP-18 (21 x 4.6 mm) Lichrosorb RP-8 (7 ~tM, 100 x 4.6 mm) 30 mM DMOA and 40 g/! Tween 20 in phosphate buffer (pH 7.5, ! = 0.1) S-(+)-naproxen (0.48 nmol) in liver microsome suspension 1.0 ml/min 32.5°C Excitation 254 nm emission, cut-off 360 nm
Plug
Sample work-up
30 mM DMOA in phosphate buffer, pH 7.5 m
centrifuged
Recovery
n
Between-day repeatability (R.S.D. %)
97.8
4
99.2
10
10|
52
drifting b~seline increasing back pressure |.9
278
_/ A, I
TiME
I
10.4 8.1
I
0
(rnin) Fig. 2 Determination of (R,S).naproxen in spiked liver microsome suspension. Pre-column: Lichrosorb RP-18 10 ttm (21 x4.6 mm I.D.); column: Enantiopac, 100×4.0 mm (No. 2); mobile phase, 30 mM DMOA and 40 g/I Tween 20 in phosphate buffer (pH 6.5, I - 0.1); solute: 44 ~M (R)-naproxen, 48 ~tM (S)-naproxen; flow-rate, 0.3 ml/min; temperature, 35.0°C.
observed from standard solutions and the liver microsomes solutions. However, transferring the naproxen to the analytical column gave rise to a drifting baseline that made it difficult to accurately quantify the enantiomeric composition. Direct injection of microsome solution without a plug gave high recoveries (Table 2), but the back pressure increased after six injections and made any accurate quantitation impossible. The remedy for these disadvantageous properties was a centrifugation (3200 ×g for 10 min) of the sample before the injection, which eliminated microparticles responsible for blockage of the column and causing high back-pressure and unstable baseline. The stability and recovery study showed a more than 99% recovery of (S)-naproxen with good day-to-day reproducibility (R.S.D. 1.9%, n = 10). No significant change in back pressure or column efficiency was observed after 52 injections of 20 ~! liver microsome suspension. A typical chromatogram (Fig. 2), with an Enantiopac column coupled on-line with the pre-column, demonstrates an almost complete separation ( R s - 1.43) of the enantiomers in spiked liver-microsome suspension. The fluorometric detection provided a very high selectivity towards endogenous compounds - no interfering peaks were observed. The recoveries of (R)- and (S)-naproxen were found to be 99.6% (R.S.D. 1.7%, n --4) and 94.9% (R.S.D. 2.4%, n = 4), respectively.
Effects on retention and selectivity by charged modifiers Long retention times and insufficient resolution of naproxen enantiomers were obtained on Enantiopac when using phosphate buffer (pH 6.4) as the eluent (Table 3). As previously reported [11] the addition of dimethyloctylamine (DMOA) to the mobile phase improved the resolution. The regulation of retention and stereoselectivity on Enantiopac by charged modifiel,s in the mobile phase has been studied extensively [12] and it has been suggested that the modifiers improve the separation by competing for achirai and chiral-binding sites on the aracid glycoprotein or by conformational changes of the immobilized protein. The presence of DMOA in the mobile phase, however, also increased the capacity factors for the naproxen enantiomers. In an attempt to decrease retention times, isopropanol was added to the DMOA containing mobile phase. However, in order to obtain k' < 10 it was necessary to add at least 8% (v/v) of 2-propanol, which led to a complete loss of
279 TABLE 3 Influence of 2-propanol and charged modifiers on chiral separation Column Mobile phase Solute Flow-rate Temperature
Enantiopac, 100× 4.0 mm (No. 1) Additives in phosphate buffer (pH 6.4, I = 0~l) R- and S-naproxen, 1.0-1.1 nmol 0.3 ml/min 25.0°C
Additives
k~
N
as/R
Rs
0.5 mM DMOA 1.0 mM DMOA 0.6 mM DMOA + 8% 2-pmpanol (v/v) 1.0 mM DMOA + 1.0 mM laurylsulphate 1.1 mM DMOA + 5.0 mM laurylsulphate
19 21 25
700 1100 1000
1.30 1.60 1.77
1.4 2.6 3.1
1200
1.00
-
600
1.49
2.0
600
1.00
-
9.7 11 6.8
the enantioselectivity. An alternative way to reduce the retention could be by addition of a co-ion, i.e., an ion of the same charge as the solute, to the mobile phase. The co-ion competes with naproxen for the limited binding capacity of the immcbilized at-acid glycoprotein (Enantiopac) [12]. Adding 1.0 mM of laurylsulfate (critical micelle concentration (CMC) approx. 8.3 mM [13]) to a mobile phase containing 1.0 mM DMOA decreased the k' for (R)-naproxer~ by a factor of two ( k ~ - 11), but the loss of enantioselectivity at 5.0 mM of the anion, giving a k~ = 6, 8, revealed the limitations in controlling retention by laurylsulphate. In conclusion, isocratic elution, using charged modifiers (DMOA and laurylsulphate) in a phosphate buffer as the mobile phase, promoted a complete resolution of (R,S)-naproxen on Enantiopac. However, this mobile-phase composition was less suitable for routine analysis of biological samples as it gave high capacity factors and low column efficiency.
Effects of uncharged micellar agents Surfactants in concentrations above CMC have been used in liquid chromatography for regulation of retention and selectivity [14]. Furthermore, the powerful solvating properties of nonionic and ionic detergents (micellar chromatography) have allowed drug analysis by direct injection of untreated serum and urine in chromatographic systems [7]. The addition of laurylsulphate in high concentrations to the mobile phase seems to impair the stereoselectivity for naproxen (Table 3). As an alternative a nonionic surfactant, Tween 20, was investigated as mobile-phase additive. The change in k' for (R)-naproxen when adding Tween 20 (structure in Fig. 3) in concentrations above CMC is shown in Fig. 4. The CMC for Tween 20 is 0.14 g/l [13]. Observe the presence of chiral carbons in the structure (Fig. 3); however, the surfactant used is probably a racemic mixture. Distribution of the
280 ~f H(OCHz.CHa)aO- -
.0.~ O(CH=.CH~.O)nH ~ I - C H . CH.aO• CO, C,THaa - - O ( C H = • el'l=O)nH
n
Tween 20 =Sorbimacrogolilauras
(ca) 6
Fig. 3. Structure of the suffactant Tween 20.
tR rain) 80
TWEEN 20 glL
Fig. 4. Influence of Tween 20 on retention and stereoselectivity. Column: Ewiantiopac (No. 1); mobile phase: TWeen 20 in phosphate buffer (pH 6.5, 1 = 0.1); solute: (o) 1.0-2.0 nmol (R)-naproxen, (e) 1.0-2.2,1tool ($)-naproxen; flow-rate, 0.3 ml/min; temperature, 25.0°(3.
enantiomers to the miceiles in the mobile phase will accelerate their elution from the column. However, the drastic decrease in enantioselectivity (Fig. 4) when the surfactant was present in the mobile phase indicated a change in the binding
..j (rain)
TiME
2.8
1.3
0
Fig. 5 Resolution of the enantiomers of naproxen. Column: Chiral-AGP 100x 4.0 ram; mobile phase, 30 mM DMOA and 40 g/! Tween® 20 in phosphate buffer (pH 6.5, /=0.1); solute" 0.15 nmol (R)-naproxen, 0.18 nmol (S)-naproxen; flow-rate, 0.9 mi/min; temperature, 25.0°C.
281 TABLE 4 Influence of N,N-dimethyioctylamine (DMOA) on chiral resolution with micellar mobile-phase Column Mobile phase Solute Flow-rate Temperature
Enantiopac 100 × 4.0 mm (No. 1) 40 g/! Tween 20 in phosphate buffer (pH 7.5, I -- 0.1) R- and S-naproxen, 2.2-2.4 nmol 0.3 ml/min 35.0°C DMOA (mM)
k~ N
aS/R Rs
5.0
10
20
4.3 1500 1.19 1.3
3.3 1300 1.31 1.6
2.6 760 1.44 1.8
properties of the protein ,phase. No chiral separation was obtained using Tween 20 as mobile-phase additive and LiChrosorb RP-18 as achiral phase. Therefore the change in enantioselectivity is not due to the solvation of enantiomers in the mobile-phase as these equilibria are non-stereoselective. As discussed above, the addition of DMOA to the mobile-phase increased the enantioselectivity and resolution of naproxen enantiomers on Enantiopac. Similarly, DMOA improved the selectivity and resolution with micellar mobile-phases (Table 4). Furthermore, increasing the amine concentration gave rise to a decrease in the capacity factors for the naproxen enantiomers (Table 4). An almost complete resolution for (R)and (S)-naproxen in liver microsomes solution could be obtained within less than 14 min by using 30 mM of DMOA and 40 g/l Tween 20 in phosphate buffer (pH 6.5) as the mobile-phase (Fig. 2). The optimal mobile-phase pH differed for the two Enantiopac columns; No. 1 gave the highest resolution at pH 7..5, while No. 2 had its best performance at pH 6.5. Otherwise the columns showed the same general behaviour but exhibited some individual differences. Disopyramide was, for example, better resolved on Enantiopac No. 1 than on column No. 2 (Rs ffi 4.0 and 3.2, respectively). The influence of pH and column temperature on the chiral resolution with Tween 20 present in the mobile-phase was also investigated (Tables 5 and 6). The selectivity and the efficiency were highest at high pH, where the acid is mainly present as anion; pK a of naproxen is 4.2 [15]. Under these conditions naproxen enantiomers were distributed as ion-pairs with DMOA to the protein stationary phase as well as to the micelles. However, the net charge of the protein stationary phase and the protein conformation will depend on pH, making the effects of a change in pH difficult to interpret and predict. Interestingly, increasing the column temperature from 16.5°C to 45°C had no significant effect on the stereosekectivity when Tween 20 was present in the mobile-phase, whereas the efficiency and in consequence the resolution of (R,S)-naproxen improved. Thus, Enantiopac used at an elevated temperature with a mobile-phase containing Tween 20 and DMOA
282 TABLE 5
Influence of pH on chiral resolution in presence of Tween 20 and N,N-dimeth?loctylamine Enantiopae 100 x 4.0 (No. 1) 5.0 mM DMOA and 40 g/l Tween 20 in phosphate buffer (I = 0.1) R- and S-naproxen, 2.2-2.4 nmol 0.3 ml/min 35.00C
Column Mobile phase Solute Flow-rate Temperature pH k~ N
aR/s Rs
7.5
6.5
5.6
4.5
4.3 1500 1.19 1.3
8.2 1300 1.15 1.1
4.2 800 1.20 1.0
1.5 370 1.09 0.3
promotes complete separation of naproxen enantiomers within a reasonable time of analysis.
Chromatographic characteristics of the second generation of AGP columns (Chiral. AGP) Since all experiments described so far have been performed with the first-generation AGP column, it was of interest to study whether the second generation column, Chiral-AGP, had similar properties. The retentions of the naproxen enantiomers are less strong on the new column, k ~t being 5 compared to 19 with neat buffer solutions (pH 6.4-6.5) as the mobile-phase (see Tables 7 and 3, respectively). However, applying a micellar mobile-phase containing DMOA resulted in similar effects, i.e., decreased retentions and retained selecti, 6' and resolutioa (see Tables 4, 5 and 7).
TABLE 6
Influence of temperature on chiral resolution with a micellar mobile phase Column Mobile phase Solute Flow-rate
Enantiopac 100 × 4.0 (No. 1) 5.0 mM DMOA and 40 g/i Tween 20 in phosphate buffer (pH 6.5, !-- 0.1) R. and S-naproxen, 2.2-2.4 nmol 0.3 ml/min Temperature (°C)
k~ N aR/S Rs
16.5
25.0
35.0
45.0
8.0 680 1,16 0.8
11 830 1.13 0.8
8.2 1300 1.15 1.1
6.6 1700 1.16 1.2
283 TABLE 7 Influence of N,N-dimethyloctylamine (DMOA) on chiral resolution with micellar mobile-phase and the second generation a~-AGP column Chiral-AGP, 100 × 4.0 mm (R)- and (S)-naproxen, 0.29-0.35 nmol 0.9 ml/min 25.0°C
Column Solute Flow-rate Temperature Mobile phase phosphate buffer (pH 6.5, I - 0.1) 30 mM DMOA and 40 g/! Tween 20 in phosphate buffer (pH 6.5, i m 0.1)
k~
N
as/•
Rs
5.0
680
1.12
1.5
0.68
300
3.59
2.7
It is therefore highly probable that a similar approach to that described in this paper for direct injection and chiral separation with the first-generation AGP columns is also applicable for the new generation of the protein c¢~umn.
Simplified description of the methed and ~¢s application Micellar chromatography using Tween 20 and DMOA as additives in phosphate buffer as mobilephase and a~-AGP as the chiral stationary phase decreased the retention times for R/S-naproxen with retained stereoselectivity. A coupled-column system with a pre-column connected in series with the chiral column could be used for fast analysis of the enantiomeric composition of naproxen in liver microsomes after injection of the centrifuged biological fluid.
Acknowledgements This project was supported by the Swedish Natural Science Research Council (project No. K-Ku 6248-301). We are grateful for research grants provided by the Swedish Academy of Pharmaceutical Sciences and the I.F. Foundation for Pharmaceutical Research.
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