(49g) Competitive Equilibrium Adsorption Behavior of Anesthetic Bupivacaine Enantiomers and Triethylamine Onto Kromasil® CHI-TBB

Competitive equilibrium adsorption behavior of anesthetic bupivacaine enantiomers and triethylamine onto Kromasil^® CHI-TBB.

Introduction:

Today the regulatory authorities require that new drugs have been tested with respect to each enantiomer already at the initial stage of drug development. This requires rapid methods for purification of mg-g amounts of the individual enantiomers of the chiral drugs. Such a method is preparative chiral chromatography, performed with a battery of stationary phases with selectivity for different types of compounds and with high chiral capacities. Non-chromatographic methods, such as crystallisation using diasteromeric salt formation or asymmetric synthesis, are too time-consuming and tedious for this short-term purpose.

Bupivacaine, (±)-1-butyl-N-(2,6-dimethylphenyl)-2-piperidinecarboxamide (Figure 1), an amide type local anesthetic widely used since the 1960s for its long duration of effects in surgery and obstetrics for sustained peripheral and central nerve blockade, is synthesized and used as the racemate (equimolar mixture 1:1).14-16 Bupivacaine enantiomers differ pharmacologically. For example, R-(+)-bupivacaine is more toxic to the central nervous and the cardiovascular systems than S-(-)-bupivacaine. Bupivacaine enantiomers also differ pharmacokinetically. For example, it has been found in sheep and humans that the clearance of S-(-)-bupivacaine is 20 ? 40% greater than R-(+)-bupivacaine and it has been found that the tissue-blood distribution coefficient for many tissues is greater for S-(-)-bupivacaine that for R-(+)-bupivacaine. Hence it is clear that an in depth understanding of the pharmacology of bupivacaine is not possible without a thorough study of its component enantiomers.

Figure 1. Chemical structure of bupivacaine. * indicates the chiral center

In preparative chiral liquid chromatography loading capacity is one of the critical factors and indicates the maximum amount of racemic mixture the column can tolerate without compromising resolution. Therefore, it has a determining impact on throughput. The optimization of enantiomer separations at the preparative scale and the scale-up of separation processes from batch chromatography to continuous separations like simulated moving bed (SMB) require a proper understanding of the adsorption equilibria under nonlinear conditions.

In this paper the competitive adsorption isotherms of bupivacaine enantiomers and trethylamine onto Kromasil CHI-TBB was evaluated. The elution profiles under nonlinear conditions (overloaded conditions) were also performed by pulse experiments and related to the isotherm model. These results will allow us to design and operate experiments in large scale chromatographic systems.

Experimental Section:

 Materials:

The racemic mixture, as well as pure enantiomers of anesthetic bupivacaine, was furnished by Cristália Pharmaceutical Company (Itapira, SP ? Brazil). The mobile phase used in this work was n-hexane/2-propanol/acetic acid/triethylamine (98/2/0.3/0.05 v/v). n-Hexane and 2-propanol was purchased from Mallinckrodt Baker, Inc. (USA). Acetic acid and triethylamine was purchased from Sigma-Aldrich (USA). The inert tracer 1,3,5-tri-tert-butylbenzene (TTBB) were obtained from Sigma-Aldrich (USA).

 Equipment

The semi-preparative chiral column (25 cm x 1 cm I.D.) used in experiments was Kromasil® CHI-TBB furnished by Eka-Nobel (Sweden). Kromasil® CHI-TBB column is packed with 16 µm of particle diameter and 100 Å of internal pore diameter of Kromasil silica which is covalent bonded with O,O'-bis[4-tert-butyl-benzoyl]-N,N'-diallyl-L-tartardiamide.

The experiments were carried out using a single chromatographic column in a high-performance liquid chromatographic (HPLC) system consisted of a dual pump Waters 1252 equipped with a Waters 2487 UV detector and digital data acquisition system (Breeze software) and temperature controller. TTTB, pure enantiomers and the racemic mixture of bupivacaine were dissolved separately in the mobile phase in order to prepare the solutions. These solutions and the mobile phase were previously filtered in Millipore filter system (0.45 µm Millipore filter) and degasified in a Cole Parmer 8892 ultrasonic bath.

Experimental procedure

All the experiment was measured at room temperature (25 ºC ±1 ºC) and 2.0 mL/min of mobile phase flow-rate. The signal was monitored by the UV detector with a wavelength (l) of 270 nm. The competitive isotherm data were measured inverse method following the procedure described by Forssén et al¹. Several experiments were accomplished varying the feed concentration of racemic mixture from 1.0 to 10.0 g/L.

Results and discussion

The overloaded elution experiments of racemic mixture of bupivacaine reported by Silva Jr. et al.² were here compared to the competitive isotherms. Figure 5 shows the concentration overload profiles at fixed volume injection (20 µL). The decrease of retention times with increase concentration were observed for both enantiomers. In spite of the increase of the peak asymmetry with increasing injection concentration the separation still happens for base line. Figure 6 shows the volume overload profiles at 10.0 g/L. A similar behavior can be drawn as for the concentration overload profiles. However, the decrease in the retention times and peak asymmetry was more remarked. This result was expected, because with the increase of the injection volume there is a larger amount of molecule competing for the available sites of the adsorbent and the saturation of the column happens more quickly.

At overloaded conditions the elution profiles is controlled by thermodynamic of adsorption and the mass transfer effects are negligible.

The elution profiles of bupivacaine enantiomers under overloaded conditions are in agreement with the fitted adsorption isotherms. The nonlinear behavior with decrease in the slope of the equilibrium isotherms leads to a decrease in the retention times of the overload elution profiles. According to the Langmuir model the adsorbent has a finite number of sites which in overload conditions should be filled out more quickly. Once the sites completely filled out the bupivacaine enantiomers are eluted earlier.

Figure 2. Concentration overloaded profiles (1.5, 2.0, 10.0 g/L) of the bupivacaine enantiomers at fixed volume injection (20 µL) and flow-rate at 3.0 mL/min.

Figure 3. Volume overload profiles (20, 50 and 200 µL) of bupivacaine enantiomers at fixed feed concentration injection (10.0 g/L) and flow-rate at 3.0 mL/min.

The nonlinear behavior of adsorption isotherms was observed and the experimental data were well fitted to competitive Langmuir isotherm model. Overloaded elution profiles were performed at different injection conditions and the results were correlated with the fitted model of competitive adsorption.

References:

1.    P. Forssén, R. Arnell,  M. Kaspereitc, A. Seidel-Morgenstern, T. Fornstedt, Journal of Chromatography A, 1212 (2008) 89?97.

2.    Silva Junior, I. J.; Veredas, V.; Carpes, M. J. S.; Santana, C. C.; Adsorption 2005, 11, 123.