Automated multiple development thin-layer chromatography for separation of opiate alkaloids and derivatives

doi:10.1016/j.chroma.2005.05.023

Journal of Chromatography A

Volume 1080, Issue 2, 8 July 2005, Pages 186-191

https://doi.org/10.1016/j.chroma.2005.05.023 Get rights and content

Abstract

There are three types of opiate alkaloids. First, the poppy alkaloids: morphine, codeine, thebaine, noscapine and papaverine; then, the semi-synthetic and synthetic derivatives used in therapy as antitussives and analgesics, such as pholcodine, ethylmorphine and dextromethorphan; at last narcotic compounds, diacetylmorphine (heroin) and opiates employed as substitutes in treatment of addiction: buprenorphine and methadone. For classical thin-layer chromatography (TLC) of opium alkaloids, it is necessary to use complex eluents with strong alkaline substances to obtain a clean separation between morphinan and isoquinoline compounds. This study purposes the planar chromatographic analysis of these substances by the automated multiple development (AMD) compared with results obtained by classical TLC method. The aim of this work was to achieve the best separation of these opiate alkaloids and derivatives by this modern technique of planar chromatography. The AMD system provided a clean separation for each of three opiates groups studied and the best results have been obtained with universal gradient: methanol 100, methanol–dichloromethane 50/50, dichloromethane 100, dichloromethane 100, hexane 100 for opium alkaloids and with gradient A: 5% of 28% ammonia in methanol 100, acetone 100, acetone 100, ethyl acetate–dichloromethane 50/50, dichloromethane 100 for antitussives and substitutes. Two reagents were used for the detection of alkaloids by spraying: Dragendorff and iodoplatinate reagents [17]. The detection limits with these two reagents were 1 μg for ethylmorphine, thebaine, papaverine, codeine, and 2 μg for morphine and noscapine and other alkaloids.

Introduction

Opiates are the most powerful known pain relievers, they are derivatives of opium and can produce euphoria but also they are used as analgesics. Opiates can be classified according to three series. The first one is constituted by the poppy alkaloids: morphine, codeine, thebaine, noscapine and papaverine (Fig. 1); the second category included mainly semi-synthetic derivatives of morphine or synthetic compounds are used in therapy as antitussives and analgesics: pholcodine, ethylmorphine (codethyline), dextromethorphan (Fig. 2); at last, the third class is composed of narcotic compounds: diacetylmorphine (heroin), and other opiates; they are employed as substitutes in treatment of addiction: buprenorphine, and methadone (Fig. 3).

Automated multiple development (AMD) is an instrumental technique of planar chromatography [1], [2], [3], [4] which uses an eluent gradient starting from the most polar to the least polar [5]; the migration is performed by successive steps (15–25) and at each new development the proportions of the eluent constituents change; so the polarity is decreasing when the distance increases (Fig. 5).

Gradient development with linear eluotropic profile [6] leads to a band re-concentration improving the separation [1]. A successful separation depends mainly on the choice of the solvent components, optimisation of the shape of the gradient, the stepwise movement of the elution front [3], [7] and the repeated developments increase the resolution [8].

This chromatographic technique is available for separation of plant extracts [9], [10], [11], [12], [13], [14], [15], [16], mainly herbal drugs which present an interest with therapy activity and chemical substances belonging to various classes as: essential oils, alkaloids, resins, phenolic compounds. Besides, this method has permitted to analyse various chemical classes of alkaloids and phenolic compounds [10], [11], [12] with similar chemical properties and also isomeric position compounds [11]. In the field of detection of abuses, AMD was used with success for the chromatographic analysis of cannabinoids [13].

The aim of this work was to apply the performances of AMD to the separation of opium alkaloids, antitussives and narcotic compounds and to obtain a clean separation for each group studied.

Section snippets

Apparatus and materials

Chromatographic separations were performed using the AMD system (Camag, Muttenz, Switzerland).

Plates used were HPTLC silica gel F₂₅₄ 10 cm × 20 cm on glass, layer thickness 0.1 mm, Article 11764, and HPTLC silica gel F₂₅₄ 10 cm × 20 cm on glass, layer thickness 0.2 mm, Article 5642 (Merck, Darmstadt, Germany).

Samples were applied with a Linomat IV system (Camag, Muttenz, Switzerland).

All the solvents were analytical grade and purchased from Carlo Erba Reactifs (Val de Reuil, France). Before use, the

Separation of opiate alkaloids by classical TLC

The classical thin-layer chromatography analysis of opium alkaloids is not easy because these alkaloids belong to two different groups: morphinan (morphine, codeine and thebaine) and benzylisoquinoline (noscapine, papaverine) and their chromatographic behaviour is very different; the morphinan group presents low hR_F and isoquinoline group very high hR_F. For these reasons the authors use generally complex eluents, for example: diisopropyl ether–ethanol–diethylamine (97:2:1) [18],

Conclusion

Planar chromatography is always a choice method for the analysis of natural products and medicinal plants. It has become a modern technique with the arrival of automated apparatus for the application of samples and automated development chambers (OPLC and AMD) which are available techniques particularly to separate constituents in crude extracts.

OPLC and AMD are more appropriate in many instances because these techniques give better reproducible hR_F values in well-defined experimental

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Citation Excerpt :
They have been used directly and indirectly (via semi-synthesis) as valuable drugs due to potent pharmacological activities, for example analgesic properties of codeine and morphine, the coronary vasodilator function of papaverine, the potentially anti-cancer drug and cough suppressant features of noscapine [1–3]. An overview on the reported separation approaches indicated that there are numerous appropriate techniques available for qualification and quantitative analysis of opium alkaloids such as thin-layer chromatography [4], capillary electrophoresis [5,6], gas chromatography [7] and high-performance liquid chromatography (HPLC), with a vast diversity of detection methods [8–10], particularly mass spectrometry (MS) [11–14], UV absorbance and/or fluorescence detections [8], and chemiluminescence [9]. Analysis of opium alkaloids is typically performed by applying reversed-phase high-performance liquid chromatography (RP-HPLC), accompanied by ion pairing reagents.
The separation of a mixture containing five major opium alkaloids, namely morphine, codeine, thebaine, noscapine and papaverine has been investigated in hydrophilic interaction liquid chromatography (HILIC) mode using five different stationary phases: bare silica, zwitterion, aminopropyl, diol and cyanopropyl. In order to propose the appropriate column for separation and purification, retention behaviors of the five natural opioids have been studied on mentioned HILIC stationary phases. The mechanism of separation in diverse HILIC media, based on the formation of water-rich layer on surface of the HILIC stationary phases and the physicochemical properties of opium alkaloids, such as pKa (acidic pK) and the octanol-water distribution coefficient (log Do/w) are discussed. Chromatographic responses including modified limit of detection LOD_m, signal to noise ratio (S/N)_m, and defined modified R_Sm have considered for suggestion of the suitable column for quantitative/qualitative and preparative purposes. According to the obtained results, diol stationary phase is best suited for analytical chromatography, whereas bare silica and zwitterionic stationary phases are appropriate for preparative applications.

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Automated multiple development thin-layer chromatography for separation of opiate alkaloids and derivatives

Abstract

Introduction

Section snippets

Apparatus and materials

Separation of opiate alkaloids by classical TLC

Conclusion

J. Pharm. Biomed. Anal.

Farmaco

J. Chromatogr. A

J. Chromatogr.

Fresenius Z. Anal. Chem.

J. Planar Chromatogr.

Thin-Layer Chromatography, Techniques and Applications

J. Liq. Chromatogr.

J. Planar Chromatogr.

J. Planar Chromatogr. Mod. TLC