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Technical Note Rapid Chiral Separation of Dextro and Levo- Methorphan using Capillary ABSTRACT: The chiral differentiation of the Dextro- and Levo- Methorphan is obtained in under 4 minutes with excellent peak shapes using capillary electrophoresis with dynamically coated capillaries. Dynamic coating of the capillary surface is accomplished by rapid flushes of 0.1 N sodium hydroxide, water, a buffer containing a polycation coating reagent, and a reagent containing methanol and a polyanionic coating reagent containing hydroxypropyl-beta-cyclodextrin. KEYWORDS: Dextromethorphan, Levomethorphan, Chiral Analysis, Capillary Electrophoresis, Dynamically Coated Capillaries, Forensic Chemistry
Dextromethorphan is an antitussive agent commonly found in Over-the-Counter (OTC) cough and cold pharmaceuticals (and more recently in Ecstasy (MDMA) mimic or combination tablets). Levomethorphan is a narcotic analgesic that is not commercially available, and therefore is not commonly submitted to forensic laboratories. Nonetheless, the differentiation and identification of these enantiomers is important in the United States, since Dextromethorphan is not controlled while Levomethorphan is a Schedule II controlled substance. However, the differentiation of Dextro- and Levo- Methorphan is challenging. Methorphan is a tertiary amine which is not amenable to derivatization; therefore, the use of chiral derivatizing reagents (to form diastereomers for analysis on an achiral gas chromatography (GC) or high performance liquid chromatography (HPLC) column) is not a viable approach. Instead, relatively expensive chiral columns are required to resolve the two isomers using either GC or HPLC [1]. Capillary electrophoresis (CE) allows for the separation of enantiomers on conventional capillaries by utilizing run buffers containing chiral additives. Micellar electrokinetic chromatography (MEKC) [2], and electrokinetic chromatography (ECC) [3], both with single wavelength UV detection, and free zone capillary electrophoresis (CZE) with secondary equilibria and PDA-UV detection [4], have all been previously used to resolve the enantiomers of methorphan. However, run times in excess of 15 minutes were required. For the separation of basic drugs at low pH, faster, more precise migration times and higher plate counts are obtained using dynamically coated versus uncoated capillaries. The use of a chiral additive (such as a cyclodextrin) imparts the additional selectivity needed for the analysis of enantiomers [5]. Using the procedure developed by Chevigne and Janssens [6], the capillary, after base hydrolysis, is sequentially coated with a polycation and then with a polyanion. The run buffer (with or without an added cyclodextrin) is the final coating reagent. This process produces coated capillaries with a higher and more robust electroosmotic flow (EOF) at lower pH values versus uncoated capillaries. In addition, the coated capillary surface has more favorable kinetics. In the present study, the rapid chiral analysis of methorphan using a dynamically coated capillary approach is reported.
Chemicals Instrumentation
and Procedures Standard and Sample Preparation For standard solutions, an appropriate amount of standard Dextro- and Levo- Methorphan was weighed into an appropriate volumetric flask and diluted to volume with injection solvent, in order to obtain a final concentration of approximately 0.05 mg/mL of each component. These were sonicated for 15 minutes, then filtered. For sample solutions, an appropriate amount of powder was weighed into a volumetric flask and diluted to volume with injection solvent, in order to obtain a final concentration approximately equal to that of standard. These were also sonicated for 15 minutes, then filtered. All standard and sample solutions were filtered with 0.45 µm Nylon syringe filters (MicroSolv). Capillary Electrophoresis Conditions
Dahlen and Lenz used CZE with an uncoated capillary and with added HP-ß-CD to resolve Dextro- and Levo- Methorphan in under 16 minutes [4]. In the present study, using the same run buffer but on a dynamically coated capillary, identical results were obtained in under 4 minutes (see Figure 1). Highly precise separations were obtained, as demonstrated by excellent run-to-run migration time precision (% RSD = 0.1, n = 7). Because the peaks are so narrow, identification can be difficult based on migration time alone; however, co-injection of sample and either standard eliminates any ambiguities. Relative migration time data (relative to Dextromethorphan) of solutes commonly found with Dextromethorphan is given in Table 1. The non-controlled substances are included because they and Dextromethorphan are commonly combined in various OTC pharmaceuticals. The controlled substances are included since they and Dextromethorphan are occasionally identified in Ecstasy (MDMA) mimic and combination tablets. If needed, n-butylamphetamine can be used as an internal standard in this method for the quantitation of Methorphan. The present procedure is compatible with previously reported methodology for the CE analysis of a wide variety of seized drugs using the same capillary with dynamic coatings [5]. Classes of compounds that can be analyzed using this methodology (using higher CD concentrations than used in this study) include the phenethylamines and the methylenedioxyphenethylamines. Specific compounds that can be analyzed using this methodology include propoxyphene, cocaine, oxycodone, heroin, lysergic acid diethylamide (LSD), opium, psilocybe mushrooms, gamma-hydroxybutyrate (GHB), and gamma-butyrolactone (GBL).
1. Pihlainen K, Kostiainen R. Effect of the eluent on enanatiomer separation of controlled drugs by liquid chromatography ultraviolet absorbance detection electrospray ionization tandem mass spectrometry using vancomycin and native beta cyclodextrin chiral stationary phases. Journal of Chromatography A 2004;1033:91-99. 2. Aumatell A, Wells RJ. Chiral differentiation of the optical isomers of racemethorphan and racemorphan in urine by capillary zone electrophoresis. Journal of Chromatographic Science 1993;31:502-508. 3. Lurie IS. Application of capillary electrophoresis to the analysis of seized drugs. American Laboratory 1996;28(2):26-34. 4. Dahlen J, Lenz M. Chiral analysis of substances of forensic interest using cyclodextrin modified capillary electrophoresis. Presented at ENSFI working group, Riva del Garda, Italy, May 2004. 5.
Lurie IS, Hays PA, Parker K. Capillary electrophoresis analysis of
a wide variety of seized drugs using the same capillary with dynamic
coatings. Electrophoresis 2004;25:1580-1591. [Note: Substrates analyzed
in the study include the enantiomers of norpseudoephedrine, pseudoephedrine,
ephedrine, amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine
(MDA), 6. Chevigne R, Janssens J. US Patent #5,611,903, 3/18/97. Table 1. Relative Migration Times of Solutes Commonly Found with Dextromethorphan.
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