Tropane analysis

Mateus, L., Cherkaoui, S., Christen, P., and Veuthey, J. L. (1998). Capillary electrophoresis for the analysis of tropane alkaloids pharmaceutical and phytochemical applications. ]. Pharm. Biomed. Anal. 18, 815-825. 

One of the earliest reports of SCILs in LC concerned the analysis of ephed-rines [54] and tropane alkaloids [55]. In these studies, the N,N-dialkylimid-azolium motif was covalently attached through a 3-mercaptopropylsilane linker. Retention of the analytes seemed to be governed predominantly through reversed-phase interactions. In comparison with conventional reversed-phase media, the IL-based stationary phase provided better peak shape and resolution while simultaneously allowing for reduction in the organic content of the mobile phase. 

A new method for the rapid separation of alkaloids, inter alia atropine, has been elaborated, using chromatography on paper that is impregnated with zirconium oxide.57 Cation-exchange h.p.l.c. analysis of tropane alkaloids has been developed,58 followed by post-column derivatization using the fluorimetric ion-pair technique.58,59 Gas chromatography of tropanes has been reviewed.60... 

Proton61 and carbon-1362 n.m.r. spectroscopy have recently been used extensively for the analysis of tropane alkaloids. The non-equivalence of H-6 and H-7 on the one hand and of H-2 and H-4 on the other in atropine and scopolamine is due to the non-symmetrical shielding by the tropic acid moiety. The configuration of A-substituents, e.g. N-oxides, has been determined by studying the deshielding of the 6- and 7/3-hydrogens. All of these results should be useful for the identification of new tropane alkaloids and other related systems in the future. 

Liquid Chromatography-Mass Spectrometric Analysis of Tropane Alkaloids in Mammalian Samples Techniques and Applications... 

The present book chapter presents an actual summary on the use of LC-MS-based procedures applied to mammalian samples for qualitative and quantitative analysis of tropane alkaloids (TA). Relevant literature obtained from PubMed database search and references cited therein is summarized and commented to provide an overview on various natural TA structures and their related pharmaceutical derivatives. A comprehensive unique set of TA was compiled that is categorized by diverse fields of analytical applications. 

Table 1 Log P and SMILES code of tropane alkaloids used in LC-MS-based analysis...

Table 5 Tropane alkaloid analysis in plasma or serum by LC-MS and LC-MS/MS for pharmacokinetic studies... 

Fig. 2 Simultaneous LC-MS/MS analysis of diverse tropane alkaloids. Analysis of plasma samples was performed according to John et al. [50] using an Atlantis T3 C18 column (150 mmx4.6 mm I.D., 5 pm) in gradient mode (as indicated) with solvent A (0.1 %v/v FA) and solvent B (ACN/water 80 20 v/v 0.1% v/v FA) at 1 ml/min and 30 °C. Detection was done by positive ESI MS/MS in MRM mode...

Fig. 7 Selected biotransformation products of diverse tropane alkaloids identified by LC-MS/MS approaches. Depicted metabolites are only an exert of those presented by Chen et al. for anisodamine (Ada) [6], atropine (Atr) [51], and scopolamine (Sep) [7] as well as by He et al. for benztropine (Benz) [59]. Structures were identified from in vivo and in vitro samples following diverse modes of MS/MS analysis as discussed in the section Biotransformation studies...

Incidental and accidental intake of atropine and scopolamine, which are the main tropane alkaloids in plants of the solanecae family, may provoke poisoning of man and livestock [11,13-15, 55, 57,119-122] causing agitation, aggression, hallucinations, dry mouth and skin, mydriasis, loss of consciousness followed by coma combined with tachycardia, hypotension, and hyperthermia [57, 121], A detailed statistical analysis of paediatric plant exposures in Germany within the years 1998-2004 has been provided by Pietsch et al. [123], They found that most prevalent victims of accidental plant exposures are children in the age of 1-6 years presumably being misled by the attractive plump berries. 

Auriola S, Martinsen A, Oksman-Caldentey KM, Naaranlahti T (1991) Analysis of tropane alkaloids with thermospray high-performance liquid chromatography-mass spectrometry. J Chromatogr 562 737-744... 

Aehle E, Drager B (2010) Tropane alkaloid analysis by chromatographic and electrophoretic techniques an update. J Chromatogr B 878 1391-1406... 

Current methods for tropane alkaloids analysis have been well covered in the literature. An excellent comprehensive review written by B. Drager [45] appeared in 2002, describing the analysis of tropane and related alkaloids in plant material. Sample preparation procedures were reviewed, as well as the analytical methods used for performing the separation and detection of tropane alkaloids, such as gas chromatography (GC), liquid chromatography (LC), and capillary electrophoresis (CE). Therefore, this chapter will not describe in detail these well-known analytical methods but discuss some recently developed applications for the analysis of tropane alkaloids in plant material and biological matrices. 

The isolation of atropine, scopolamine, and cocaine occurred long before the development of modern analytical techniques. Gas chromatography was the first instrumental technique available in the field of separation science and thus it is not surprising that these alkaloids were firstly analyzed by GC despite their low volatility. With the advent of capillary columns and the proliferation of various sample introduction and detection methods, GC has evolved as the dominant analytical technique for screening, identification, and quantitation of tropane alkaloids of plant origin as well as in biological fluids. The state-of-the-art of GC analysis of tropane alkaloids has been the subject of two comprehensive reviews [45,58]. We shall therefore mainly focus on publications which have appeared since 2002. 

The usefulness of GC-MS analysis for biosynthetic studies was demonstrated by Patterson and O Hagan [74] in their investigation of the conversion of littorine to hyoscyamine after feeding transformed root cultures of Datura stramonium with deuterium-labeled phenyllactic adds. This study complements previous investigations on the biosynthesis of the tropate ester moiety of hyoscyamine and scopolamine [75], where GC-MS played a key role. It also has general relevance in the biosynthetic pathway of tropane alkaloids in the entire plant kingdom [76]. 

Gas chromatography of cocaine of plant origin has mainly involved the analysis of the coca plant [77-79]. Identification and quantitation GC methods of minor naturally occurring tropane alkaloids in illicit cocaine samples have also been reviewed [80]. Moore et al. presented an in-depth methodology for the analysis of the coca plant by GC-FID, GC-ECD, and GC-MS for the identification of alkaloids of unknown structure [81]. Recently, Casale et al. [82] have analyzed the seeds from Erythroxylum coca for their alkaloidal content. Several tropane alkaloids were detected and characterized and it appeared that methylecgonidine (MEG) was the primary constituent and not an analytical artifact. 

Fig. 12.2 Separation of four isomeric tropane alkaloids by GC (a) Analysis in linear programmed temperature CC-MS using a conventional 30 m x 0,25 mm i.d. X 0.25 pm film thickness HP5-IVIS column, (b) Fast GC...

The analysis of tropane alkaloids by liquid chromatography has been performed for over 30 years. The first high-performance liquid chromatography (HPLC) analysis of tropane alkaloids was published in 1973 and concerned the separation of atropine and scopolamine as well as homatropine and apoatropine [102] the first analysis of these compounds in plant material appeared 12 years later [103]. 

Analysis of tropane alkaloids in biological fluids has been developed mostly for cocaine and metabolites. Indeed, it is well recognized that cocaine remains one of the most widely consumed drugs of abuse worldwide. Generally, reversed phase liquid chromatography coupled with UV-VIS detection is employed for these analyses. 

The on-line coupling of CE with electrospray ionization mass spectrometry (CE-ESI-MS) allows high separation efficiency together with high sensitivity and selectivity as well as molecular structural information. A CE-UV-ESI-MS method was developed for the analysis of hoscyamine, scopolamine, and other tropane derivatives [131]. The differentiation of hyoscyamine from littorine, commonly encountered in plant material, was demonstrated using in-source collision-induced dissociation. The developed method was applied to the analysis of these alkaloids in Belladonna leaf extract and in Datura Candida x D. awreahairy root extract. Recently, CE coupled with electrochemiluminescence detection has been used for the determination of atropine and scopolamine in Flos daturae [132]. 

Tropane alkaloids are an important class of natural products possessing different and interesting pharmacological activities. Hyoscyamine (atropine in the racemate form), scopolamine, and cocaine are the major representatives of this class. They are commonly found in plant materials, mainly in genera belonging to three families Solanaceae, Erythroxylaceae, and Convolvulaceae. The importance of these compounds requires that there are accurate analytical methods for their determination in plants and in biological matrices. This chapter describes the state-of-the-art of analytical procedures (extraction and analysis) for analyzing tropane alkaloids. 

Analyses of tropane alkaloids are mainly carried out by GC and HPLC and to a lesser extent by CE. This review describes recent applications developed for the analysis of this class of compounds in plant materials and biological matrices. Of course, mass spectrometry is generally used as the detection technique because of its high sensitivity and selectivity, but other techniques such as UV, fluorescence, flame ionization detection, nuclear magnetic resonance, among others have also been investigated. Finally, desorption electrospray ionization mass spectrometry is reported as a new interesting detection technique for the rapid analysis of samples without any sample preparation. 

To a solution of nor-3-/3-(4-fluorophenyl)tropane-2-/3-carboxylic acid methyl ester (0.2 mmol) dissolved in 10 ml acetonitrile was added in succession the product from Step 1 (0.2 mmol), KI (0.2 mmol), and K2CO3 (2.0 mmol), and the slurry refluxed overnight. The solution was cooled, 2 g silica gel added, and concentrated. The residue was purified by chromatography on a silica gel column with NH40H/EtOAc/hexanes, 0.5%1 1, and the product isolated in 72% yield. The product was then converted into the dihydrogen chloride salt which had a mp = 166-168 °C. H-NMR, IR, elemental analysis data supplied. 

---