Quantitation of Alkaloids

A variety of methods for quantitative determination of major opium alkaloids have been reported by different workers, such as gravimetric/ volumetric methods, colorimetric methods, spectrofluorimetric methods, etc. However, separation is usually achieved by thin layer (TLC) or paper chromatography (PC). Today, advanced analytical methods, like gas (GLC)/high performance liquid chromatography (HPLC) are more frequently employed for this purpose. In the following sections, we describe these methods briefly, with greater emphasis on GLC and HPLC. 

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Whilst gas chromatography has been used for the analysis of many of the lycoctonine-based alkaloids [52], the larger, less volatile, and more thermally labile MSAL compounds require analytical procedures such as TLC and HPLC for separation and detection. For example, both normal phase liquid chromatography [53] and reversed phase liquid chromatography [54] with UV detection have been used for separation, detection, and quantitation of alkaloids from Delphinium species associated with livestock poisonings in the western US and Canada. The introduction of API techniques has allowed the analysis of all types of diterpene alkaloids by direct MS methods and with MS methods coupled to liquid chromatography. 

This is also a reversed-phase system which has been applied for separation and quantitation of alkaloids from cell suspension cultures of Catharanthus roseus including vinblastine (123). p Bondapak Cig. 

The catalytic hydrogenation of enamide 34 in the presence of 0.5-1 mol% of (/ )-BINAP complex in a 5 1 mixture of ethanol and dichloromethane under 1-4 atm of hydrogen affords 35 with quantitative yield and higher than 99.5% ee. The (fi)-isomer is not reduced under similar reduction conditions. This approach provides a route to a number of alkaloid compounds (Scheme 6-19).47... 

Methylated phenols very often form part of the molecule of natural products, in particular, of alkaloids. In the elucidation of their constitution the quantitative determination of the methoxyl groups is of great importance. This determination is carried out by the excellent method of Zeisel, in which the methyl group is removed as methyl iodide by the action of concentrated hydriodic acid. This method (directions, p. 80) should be practised with the anisole already prepared. 

Callaway JC, Raymon LP, Fleam WL, McKenna DJ, Grob CS, Brito GS, Mash DC. (1996). Quantitation of N,N-dimethyltryptamine and harmala alkaloids... 

The concurrent development of analytical techniques for the isolation, detection, and quantitation of indole alkaloids in small amounts of cellular material has been addressed by Hdfle and co-workers 134), who developed a reversed-phase system using either methanol-water-triethylamine or acetonitrile-triethylammonium formate buffer as the eluept. Two prepacked cartridge systems were found to be very effective, for the rapid preparation of the alkaloid mixture from the cell contents. ... 

The stationary phase may be a solid or liquid on a solid support. The mechanisms responsible for distribution between phases include surface absorption, ion exchange, relative solubilities and steric affects . High performance liquid chromatography is a useful method for quinolizidine alkaloid analysis, especially when pure standards are available". This method was recently used for alkaloid metabolite extraction and analysis . A simple reversed-phase liquid chromatographic method has been developed for the simultaneous quantitation of four anticancerous alkaloids vincristine, vinblastine, and their precursors catharanthine and vindoline using a specific HPLC column . 

Gas-liquid chromatography is a qualitative, but also quantitative, method of alkaloid analysis. It is very sensitive. The only problem concerns the distribution of the alkaloid mixture in the chromatographic process and the identification of alkaloids, which must be achieved by a different technique . A very positive characteristic is the possibility of totally computerizing this method of alkaloid detection. 

Meeker, J. E. and Kilgore, W. W. 1987. Identification and quantitation of the Alkaloids of Lupinus latifolius. Journal of Agriculture and Food Chemistry, 35 431 33. 

It has been demonstrated that ILMs are suitable for qualitative and quantitative analyses of low-molecular weight compounds of biological interest, for example, carbohydrates, vitamins and amino acids [38], and glycolipids [40]. ILMs were further used for fhe direct analysis of alkaloids, anesthetics and antibiotics, separated by thin-layer chromatography (TLC) [46]. For this purpose, the ILM was spotted onto the fractions on the TLC-plates and the complete plate was measured in MALDI MS without the need for additional pretreatment of the TLC-samples. The mass deviation inherently caused by the inhomogeneous surface of fhe TLC-plafe was balanced by using the... 

Method. To 40 nmoles of alkaloid in a small centrifuge tube are added 200 /il of a 0.1% solution of DNS-C1 in acetone followed by 200 jul of 0.1 M sodium carbonate. (A 6-7 fold molar excess of DNS-C1 is usually required for quantitative conversion of the alkaloid into the DNS derivative.) The reaction mixture is warmed at 45 °C for 20 min. The tube is then cooled and 2 ml of benzene are added. The centrifuge tube is stoppered and shaken. An aliquot portion of the resulting solution is used for chromatography. The derivatives obtained for several alkaloids are listed in Table 4.21, together with their emission wavelengths (excitation, ca. 365 nm) and the relative fluorescence intensities of 10 nmoles of the derivatives in 5 ml of benzene (corrected for background) [121]. 

McCooeye, M., Ding, L., Gardner, G. J., Fraser, C. A., Lam, J., Sturgeon, R. E., and Mester, Z. (2003). Separation and quantitation of the stereoisomers of ephedra alkaloids in natural health products using flow injection-electrospray ionization-high field asymmetric waveform ion mobility spectrometry-mass spectrometry. Anal. Chem. 75 2538-2542. 

Quantitation of Napelline.—The pharmacological activity of napelline (35)38 has prompted the development of a method for the quantitation of this alkaloid in raw plant materials.39 This method consists of the extraction of the total alkaloids, chromatographic separation, and a micro-scale acid-base titration of the napelline eluate in a non-aqueous medium. The total alkaloids were exhaustively extracted from a sodium carbonate suspension with chloroform. This extract was concentrated, dissolved in acetone, and chromatographed on silica-gel plates, with a standard reference of napelline as a marker. The appropriate bands were quantitatively removed and extracted, and the extracts were concentrated to dryness. These residues were dissolved in glacial acetic acid and titrated with 0.01N perchloric acid. The standard deviation of this method was 3.39 x 10 3. No limits of detection are reported. 

Even though much is already known about the toxicity of diterpene alkaloids that contribute to the toxicity of Consolida, Delphinium, and Aconitium species, no antiviral study has been so far reported on this type of alkaloids. Therefore, no SAR studies have been encountered by us on the antiviral or antimicrobial activities of these alkaloids. However, a quantitative SAR analysis performed on a number of diterpene alkaloids isolated from an Aconitum sp. indicated that biological activity of these alkaloids may be related to their toxicity rather than to a specific pharmacological action [40]. In a current study on 43 norditerpenoid alkaloids from Consolida, Delphinium, and Aconitum species against several tumor cell lines, lycoctonine and browniine were... 

The filter disc is transferred to a stopperred flask containing ethanol and propan-2-ol, and shaken to extract the water present which is then determined by gas-liquid chromatography the quantitity of water present is calculated from the ratio of the areas of the peaks for water (unknown) and ethanol (internal standard). The alkaloids are extracted from the filter disc using sulphuric acid and determined by a specially developed autoanalyser procedure based on the Koenig reaction this is shown schematically in Fig. 21. Since the procedure was developed specially for this application, results obtained from it were compared closely with those produced by the traditional manual method, a steam distillation technique, before it was adopted as a standard method. 

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. 

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. 

LC-MS of Alkaloids Qualitative Profiling, Quantitative Analysis, and Structural Identification... 

Gas chromatography coupled to mass spectrometry is a widely used analytical method [31] but does require prior reduction of the involatile N-oxides. Additionally, there are problems associated with some alkaloids that require prederivatization to enhance the GC characteristics. These requirements for prederivatization can adversely affect the GG-MS interpretation, especially at trace levels of alkaloids [32]. Gombined SPE-LC-MS approaches have provided methods for qualitative profiling and quantitative analysis of pyrrolizidine alkaloids and their N-oxides. 

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