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Enantiomeric Purities of Pharmaceuticals Using Carbohydrate-based Isothiocyanates

By: Manfred P. Schneider, FB C – Bergische Universität Wuppertal,D-42097 Wuppertal, Germany, AnalytiX Issue 3

Introduction

In the first two papers of this series (Analytix Nr. 1/2010 and 2/2010), the efficient enantiomeric analysis of (a) alkyl oxiranes, and (b) amino acids was described using inexpensive reversed phase columns (as an alternative to high cost so-called “chiral columns”) in combination with monosaccharide based isothiocyanates as derivatization reagents (Figure 1). While alkyloxiranes have to be converted fi rst into the corresponding ß-amino alcohols by reaction with isopropyl amine, all amino acids (proteinogenic, non-proteinogenic and nonnatural) can be converted directly (straight out of the bottle or the body fl uid, respectively) into the corresponding diastereomeric thioureas, which can be injected directly into the HPLC. Base-line separations were observed in almost all cases, establishing a highly efficient and generally applicable method for the analysis of these classes of molecules.

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Pharmaceuticals

The carbohydrate-based isothiocyanates have also been shown to be highly suitable for the enantiomeric analysis of neurotransmitters (e.g. adrenaline and related molecules), numerous pharmaceuticals carrying functional amino groups, such as ß-adrenergic blockers, various pharmaceuticals such as penicillamine and mexiletine, and fine chemicals such as 1-phenyl-2-aminoethanol. Representative examples of these classes of molecules are shown in Figure 1.

 

Figure 1.Pharmacologically active molecules with functional amino groups

Figure 1. Pharmacologically active molecules with functional amino groups

The close relationship between the biological (physiological) activities of these molecules and their absolute configurations is well established. Frequently, only one enantiomer (Eutomer) has the desired pharmacological activity, while its antipode is inactive (Distomer), shows undesired side-eff ects, or is even toxic. Inactive enantiomers are also frequently referred to as Xenobiotics in the sense of pollutants. Several of these compounds are known for their illicit use in doping, as narcotics or psychotropic agents, and for their illegal use in food and feed. It is well established that in so-called ß-adrenergic blockers, the pharmacological activity resides in the (S)-enantiomers, while the (R)-enantiomer of penicillamine is highly toxic. On the other hand, the neurotransmitter activity of adrenaline resides largely in the (R)-enantiomer. Many more similar examples can be found in the literature. Clearly, in view of the fact that novel pharmaceuticals of this kind are increasingly used in the form of single enantiomers, the determination of enantiomeric ratios/purities is of increasing importance. Examples include the monitor of (a) enantioselective syntheses, (b) quality control in manufacturing, (c) stability and metabolic rate in biological systems (e.g. serum), and (d) analysis of illicit drugs and narcotics in body fl uids (e.g. doping/body building/athletic sports). Advantageously, all of these compounds can be analyzed without any prior manipulation (“straight out of the bottle” or the reaction medium, e.g. biological fl uids). They react under mild conditions and at a rapid rate (at room temperature) with the mono-saccharide isothiocyanates, leading to the corresponding diastereomeric thioureas, as exemplified in Figure 2. These, in turn, can be injected – without the need for further purifi cation – directly into the HPLC.

 

Figure 2. Pharmaceuticals (here a beta-blocker) with functionalized amino groups: formation of diastereomeric thioureas (schematic) [* denotes center of chirality]

Figure 2. Pharmaceuticals (here a beta-blocker) with functionalized amino groups: formation of diastereomeric thioureas (schematic) [* denotes center of chirality]

As derivatives of natural mono-saccharides (Figure 1), all of the employed reagents are enantiomerically pure by defi nition, and the ratios of thus produced diastereomers directly refl ect the enantiomeric composition of the chiral amino compound in question. This requires, of course, that both enantiomers of a racemic mixture react rapidly and quantitatively, and with the same rate in order to avoid a diastereoselectivity during the derivatization process. For new target molecules, this must be ascertained in every case by calibration with the corresponding racemate. The described strategy frequently has distinct advantages over the so-called direct method employing chiral stationary phases in that (a) the separation of diastereomers is usually more simple to perform and often provides better resolutions, (b) the choice of chromatographic conditions is much greater and thus can be more easily optimized, and (c) the reagents contain chromophores (fluorophores) for convenient UV- or fluorescence detection. In view of the range of novel derivatization reagents which recently became available (PGITC, PGalITC, NGalITC) [5], the method is an interesting alternative to so-called chiral columns.

In principle, all of the above reagents can be employed for the above pharmaceuticals. Thus, Nimura et al.1 achieved base-line separations in the analysis of adrenaline (epinephrine) and noradrenaline (norepinephrine) using GITC (Aldrich T5783) and AITC (90245). Adrenaline is only present in minute quantities in lidocain local anesthetics; nevertheless this method allowed the quantitative determination of the enantiomeric ratio in more than 250 commercially available anesthetics2. Using the same reagents, a series of diff erently substituted amphetamines were analyzed3. The method also works well for the analysis of ephedrine, pseudoephedrine and norephedrine4. Further examples include the enantiomeric analysis of the antiarrhythmic agent mexiletine in human plasma using GITC5 and the corresponding analysis of a whole series of beta-adrenergic antagonists (ß-blockers) such as propranolol, atenolol and sotalol (Figure 2)6. The introduction of benzoyl groups (BGITC Aldrich 335622)7 and naphthoyl groups (NGaIITC 04669)8 considerably enhanced the UV- and fl uorescence detection of the corresponding thioureas by factors of 6 (BIGTC) to 40 (NGalITC). Furthermore, the introduction of these residues frequently resulted in a considerable improvement in the separation of these diastereomers, as did the incorporation of extremely bulky pivaloyl groups such as in PGITC (44891) and PGaIITC (88102). It should also be noted that the described methodology is not limited to the referenced pharmaceuticals, but can be potentially extended to all chiral compounds carrying functional amino groups such as a wide variety of amino alcohols.

 

Table 1. Mobile phase: acetonitrile: 0.1% TFA/H20 = 70:30

Table 1.Mobile phase: acetonitrile: 0.1% TFA/H20 = 70:30

 

Table 2. Mobile phase: acetonitrile : 0.1% TFA/H2O = 60:40

Table 2. Mobile phase: acetonitrile : 0.1% TFA/H2O = 60:40

 

Table 3.Mobile phase: MeOH: H2O = 80:20 to 90:10

Table 3.Mobile phase: MeOH: H2O = 80:20 to 90:10

While simple RP-18 columns are generally employed, the separation conditions can be varied widely in order to achieve the best separating conditions. Various different mobile phases have been used ranging from MeOH : phosphate buff er (pH 2.8)1 over MeOH : H2O : phosphate buffer (pH 7) to acetonitrile : water : 0.1% trifluoroacetic acid in order to optimize the separation conditions. In certain cases the reagent may interfere with the separation, having the same or similar retention time. The addition of small amounts of ethanolamine or hydrazine is sufficient to destroy excess reagent by formation of the corresponding thioureas, which elute at different retention times.

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Summary

The method described above allows the rapid, efficient and inexpensive determination of enantiomeric purities in a wide variety of structurally varied pharmaceuticals and fine chemicals. By using the suitable derivatization reagent, base-line separations are observed in nearly all cases. The procedure is quite general and applicable to (a) detecting enantiomeric ratios of pharmaceuticals, in addition to biological samples; (b) determining racemizations and diff erences in metabolic degradation; (c) monitoring asymmetric syntheses; and (d) detecting molecules in illicit drug abuse and doping. The method is clearly adaptable to automation using reaction batteries and auto-samplers. The technique is applicable both on a laboratory scale and in on-line quality control. It is thus highly suitable for monitoring asymmetric syntheses including enzyme-catalyzed transformations.

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Experimental

5 mg of the corresponding pharmaceutical is dissolved in 50% (v/v) aqueous acetonitrile (or dimethylformamide) containing 0.55% (v/v) triethyl amine (in this case, hydrochlorides are employed) to give a final volume of 10 mL. To 50 μL of this stock solution 50 μL of 066% (w/v) BGITC in acetonitrile is added. The resulting solution is shaken on a laboratory shaker for 30 min, after which 10 μL of 0.26% (v/v) ethanolamine (or hydrazine) in acetonitrile is added and shaking is continued for another 10 min. Ethanolamine (hydrazine) reacts with any excess of BGITC and the resulting thiourea derivative is eluted well behind any of the amino acid derivatives. The mixture is then diluted to a final volume of 1 mL and a 10 μL aliquot is injected into the HPLC. (RP-18, mobile phase MeOH : H2O [67 mM phosphate buffer (pH 7) = 65:27:8 up to 70:25:5 and 80:15:5], depending on the case, flow rate 0.5 mL/min, compare Tables).

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Materials

     

References

  1. Nimura, N., Kasahara, Y., Kinoshita, T., resolution of enantiomers of norepinephrine and epinephrine by reversedphase high-performance liquidchromatography J. Chromatogr. 213 (1981) 327–330.
  2. Allgire, J.F., Juenge, E.C., Adamo, C.P., Sullivan, G.M., Kirchhoefer, R.D. High-performance liquid chromatographic determination of d-/l-epinephrine enantiomer ratio in lidocain-epinephrine local anesthetics; J. Chromatogr. 325 (1985) 249–254.
  3. Miller, K.J., Gal, J.,Ames, M.M. High-performance liquid chromatographic resolution of enantiomers of 1-Phenyl-2-1minopropanes (amphetamines) with four chiral reagents, J. Chromatogr. 307 (1984) 335 –342.
  4. Gal, J. Resolution of the enantiomers of ephedrine, norephedrine and pseudoephedrine by high-performance liquid chromatography, J. Chromatogr. 307 (1984) 220 –233.
  5. Grech-Belanger, O., Turgeon, J. High-performance liquid chromatographic assay for mexiletine enantiomers in human plasma, J. Chromatogr. 337 (1985) 172-177.
  6. Sedman, A.J., Gal, J. resolution of the enantiomers of propranolol and other beta-adrenergic antagonists by highperformance liquid chromatography, J. Chromatogr. Biomedical Appl. 278(1983) 199 –203.
  7. Lobell, M., Schneider, M., 2,3,4,6-Tetra-O-benzoyl-ß-Dglucopyranosyl isothiocyanante: an efficient reagent for the determination of enantiomeric purities of amino acids, ß-adrenergic blockers and alkyloxiranes by high-performance liquid chromatography using standard reversed phase columns, J. Chromatogr. 633 (1993) 287–294.
  8. Schneider, M., recent results, unpublished.

 

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