Rapid determination of total solanesol in tobacco leaf by ultrasound-assisted extraction with RP-HPLC and ESI-TOF/MS

Abstract

A reliable and rapid method based on high-performance liquid chromatography (HPLC-UV) and positive ion electrospray-time of flight mass spectrometry (ESI-TOF/MS) has been developed for the characterization and quantification of solanesol in extracts of tobacco leaves from different sources. The solanesol was extracted from tobacco leaf via saponification and ultrasonic-assist extraction, and the extraction conditions were optimized. The HPLC conditions are as following: Hypersil C₄ (4.6 mm × 150 mm, 5 μm) column, acetonitrile and water as mobile phase, flow-rate is 0.8 ml/min, detection length of UV is 202 nm, injection volume is 10 μl. The results indicated that the developed HPLC method is simple, sensitive and reliable for the determination of solanesol in tobacco leaves with a linear dynamic range of 3.65–4672 ng, a detection limit of 1.83 ng, and an average recovery of 98.7%. The method has been applied to analyze and compare different tobacco samples. The results show that the solanesol content in samples of different geographic locations varies widely from 0.20 to 1.50%. When different parts of the tobacco plant are compared, the top parts of the leaves are more abundant in solanesol content than those of lower parts.

Introduction

Solanesol (C₄₅H₇₄O) is a trisesquiterpenoid alcohol (polyisoprenoid alcohols) which was first isolated from flue-cured tobacco by Rowland et al [1]. The chemical structure of solanesol is illustrated in Fig. 1. Solanesol has diverse applications as cardiac stimulant, lipid antioxidant and antibiotic, etc. [2]. Besides being used as a medicine itself, the chemical is also used widely as an intermediate for the synthesis of coenzyme Q₁₀ or vitamin K_2. In these processes, the solanesol molecule is incorporated as a side-chain component in the product molecules. Solanesol as a chemical is generally isolated from natural plant species rather than synthesized chemically. Among these plant species, the notable ones include tobacco leaves, mulberry leaves and silkworm feces. In both mulberry leaves and silkworm feces, the contents of solanesol are very low. As was reported by Rowland et al. [1], solanesol is present in comparatively larger quantities only in tobacco leaves, making it the major commercial source of solanesol. The solanesol content of tobacco leaves depends upon a number of factors including the type of tobacco, the stalk position, and the growing time of the plant. Several studies have been reported pertaining to these subjects [3], [4], [5], [6]. The reported content of solanesol in tobacco leaves range from 0.3 to 3% of the dry weight of tobacco leaves [7], [8], [9]. A substantial portion of the solanesol in tobacco is as esters of fatty acids rather than free molecules. To extract the solanesol component, these “bound”(esterified) solanesol must first be liberated by alkaline hydrolysis [3].

Many analytical techniques including, thin-layer chromatography [10], [11], gas chromatography [4], [6], [12], high-performance liquid chromatography [13], [14], [15], [16], [17], [18], etc. have been developed to assay solanesol in tobacco leaves and tobacco smoke. The thin-layer chromatographic densitometric method as reported by woolen and Jones is problematic because of considerable solanesol decomposition on the plates [11]. Gas chromatography method is also considered undesirable because it involves lengthy extraction and derivatization procedures. HPLC is a popular method, and various detectors including UV, RID, ELSD have been used to determine solanesol [13], [14], [15], [16], [17], [18]. Reports on mass spectrometric analysis of solanesol has been scarce although one paper has been published recently on the determination of solanesol in tobacco smoke by HPLC-ES–MS [16].

In the present paper, a rapid and sensitive RP-HPLC-UV method has been developed for the quantitative determination of solanesol in the crude extract of tobacco leaf. Consideration has been given especially on the selection of HPLC conditions with optimum separation efficiency and better detection sensitivity. ESI-TOF/MS coupled with HPLC has also been applied to the analysis of tobacco leaf extracts and the identification of solanesol. In addition, a new extraction process involving saponification followed by ultrasound-assisted extraction was optimized to maximize solanesol extraction yield. The developed method has been applied to compare solanesol yield from tobacco leaves of different breeds.