Alkaloids, HPLC systems

Ipecac contains many isoquinoline alkaloids as mentioned above and the value of the regenerated plants through tissue culture greatly depends on their phytochemical similarities to the crude drug. Therefore, improved HPLC system for the analysis of the isoquinoline alkaloids, not only emetine and cephaeline but also minor constituents, was investigated [19]. 

Many other HPLC systems have been reported for identification and quantitation of hyoscyamine and other tropane alkaloids. Several of these are presented in the following table 8. 

A number of papers have been published on the HPLC analysis of alkaloids in order to study the possibilities for such analyses in general. The papers comprise a wide variety of alkaloids and HPLC systems for their separation. The data published in these papers may be useful in connection with many separation and other HPLC problems in the analysis of alkaloids. Therefore some of the data of general interest are summarized in the present chapter. However, in other chapters of this book, HPLC systems that may be useful for alkaloid analysis in general, have also been dealt with (see Table 2.1). 

Because atropine and 1-hyoscyamine behave similar in all HPLC systems, the term atropine will be used in the text and in all tables and figures to describe both alkaloids. 

Using an affinity probe technique it has become possible to identify natural products that bond to DNA (79). Extracts derived from Albizia amara (Leguminosae) were found to demonstrate activity in a recently developed HPLC system designed to detect compounds capable of interacting with DNA. Further investigation led to the procurement of four sets of alkaloid isolates X1-X4 that were found to be macrocyclic pithecolobine alkaloids. Isolate Xi found to inhibit the catalytic activity of DNA polymerase, RNA polymerase, and HIV-1 reverse transcriptase. It has been identified as a mixture of budmunchiamines A (89), B (90), and C (91), in the ratio 4 1 1 80), whose structures were determined by spectroscopic methods 81). In further detailed bioassay-guided isolation, six more spermine macrocyclic alkaloids, budmunchiamines D-I (92-97) were obtained 82). The structures of these substances were confirmed by spectral analysis and comparison with the related alkaloids budmunchiamines A (89)-C (91). (See Fig. 10.)... 

Methods for Production of Alkaloids in Root Cultures and Analysis of Products 211 Table 4. Examples of HPLC systems for selected alkaloids (see references in text)... 

Unlike a UHPLC system, in the HPLC system, both the reversed-phase [46,47] and the normal-phase [29,48-50] mode have reportedly been used to determine procyanidins and alkaloids in cocoa samples. This is explained because, until now, no UHPLC columns with polar stationary phase (normal-phase mode) with sub-2 pm have been developed. Initial reports for the determination of procyanidins and alkaloids in cocoa samples by HPLC technique were based on the normal-phase mode, and monomer procyanidins through decamer procyanidins were resolved. In this normal phase, two different stationary phases were reported, an unmodified silica column [29,50] and a dihydroxypropyl-bonded silica [48,49]. 

A hybrid FIA/HPLC system incorporating monolithic column chromatography was developed for the determination of six opiate alkaloids (morphine, pseudomorphine, codeine, oripavine, ethylmorphine, and thebaine) and four biogenic amines (vanilman-delic acid, serotonin, 5-hydroxyindole-3-acetic acid, and homovanillic acid) in human urine, using tris(2,2 -bipyridyl)ruthenium(III) and acidic potassium permanganate CL detection (Adcock et al., 2007). This hybrid system approaches the automation and separation efficiency of HPLC while maintaining the positive attributes of FIA, such as manifold versatility, speed of analysis, and portability. 

Acceptable analytical criteria for (-f)-tubocurarine have been established (402, 450). Information on TLC and HPLC systems that will allow both the detection and quantitation of the alkaloid and its congeners in curare and biological materials, as well as in pharmaceutical preparations, has been compiled (263, 403-405, 450, 560). ... 

Both normal phase [53] and reversed phase [54] HPLC methods have been used for the separation of diterpene alkaloids. Reversed phase HPLC coupled to APCI mass spectrometry has been used for the analysis of diterpene alkaloids of Aconitum spp. [64,65] and normal phase HPLC conditions [53] have been successfully used with APCI-MS for the detection of diterpene alkaloids in Delphinium species [56]. However, caution should be observed in the use of APCI sources with some normal phase HPLC solvents such as hexane, to ensure no oxygen is introduced into the system producing a possible explosive mixture in the API source. 

System V This system is a combination of ion-pair and column switching HPLC to determine tropane alkaloids, mainly hyoscyamine, in complex preparations of gastrointestinal drugs using three pumps in the system (141). 

However, under basic conditions the stability of silica gel is poor18. Depending on the nature of the organic solvents used 1n combination with the basic modifier, the silica gel is dissolved at various rates. This limits the applicability of silica gel as stationary phase in the HPLC analysis of alkaloids. For certain solvent systems the column life can... 

In a series of papers, Segal 1 and co-workers described various HPLC methods for the analysis of pyrrolizidine alkaloids in plant material. A cyano-type column in combination with tetrahydrofuran - 0.01 M ammonium carbonate was used (Fig.3.3) for the isolation and identification of some senecio alkaloids11,13 13,3 . A gradient of 13% tetrahydrofuran, increasing to 26%, gave good separations. Similar results could be obtained with an isocratic system containing 16% tetrahydrofuran. 

Fig.4.9). HPLC was prefered over GLC because of the decomposition of some of the alkaloids during GLC. Some other straight-phase systems for drugs of abuse are dealt with in Chapter 74,12. 

A similar solvent system was also used by Rasmussen et al.51 for the analysis of morphine in organic poppy extracts (Fig. 7.13). The extracts could be analyzed without any purification prior to HPLC analysis. Gimet and Filloux performed analyses on alkaloids -including opium alkaloids - in pharmaceutical preparations, and used a microparticulate silica gel column and diethyl ether or diethyl ether saturated with water as mobile phase. In both cases 0.4% diethylamine was added to the mobile phase (Fig. 7.14). An increase... 

HPLC has been extensively applied in the analysis of indole alkaloids - for quite different purposes. A series of papers on the analysis of drugs of abuse includes the analysis of one or more indole alkaloids, particularly strychnine, a common adulterant of heroin (Table 8.16). Moreover strychnine and brucine have often been used as test compounds for a number of separation systems 2, 1 2 32,4 43 as well as internal standards1 33. 

Jarvie et al. used a microparticulate silica gel column for the determination of colchicine in plasma. Iorio et al. isolated and identified some impurities of colchicine by means of HPLC, TLC and MS. Alkaloid separation was performed on silica gel with a gradient system of chloroform - methanol. 

An HPLC method was devised for the separation of the alkaloids from other components of extracts of Schumanniophyton species [42]. A reverse-phase system was employed consisting of a C-18 stationary phase... 

A very detailed review of the HPLC methods has been carried out [7]. The author has described the stationary phase, the mobile phase, flow rate, and detector system used by researchers since 1973. We would like to describe the other analytical methods such as the capillary electrophoresis, flow injection analysis and two-dimensional fluorescence spectroscopy which have foimd applications in ergot alkaloid research. 

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