Analysis of 32 toxic natural substances in herbal products by liquid chromatography quadrupole linear ion trap mass spectrometry

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Highlights

  • LC–MS/MS EPI method was developed for analysis of toxic natural substances.

  • QuEChERS procedure enabled extraction of target analytes from herbal supplements.

  • MRM-IDA-EPI enable both qualification and quantification in one run.

  • EPI spectrum library enable screening without standard.

Abstract

In this study, an LC–MS/MS EPI method was developed for simultaneous determination of 32 toxic natural substances in herbal products. The analytes include aconite alkaloids, lobelia alkaloids, solanaceous alkaloids, digitalis steroid glycosides, strychnine, tetrahydropalmatine etc. They werecommonly used in herbal products. The target analytes were extracted from the samples using theQuEChERS method and analysed using AB SCIEX QTRAP 5500 coupled with Agilent HPLC 1260. Thecolumn used was biphenyl reversed phase analytical column. Mobile phase A and B were deionizedwater and methanol respectively, both containing 5 mM ammonium formate and 0.1% formic acid. TheMRM-IDA-EPI method enabled quantification and confirmation of the analytes in a single run. The EPIwas used for the qualitative analysis while the MRM was used for the quantitative analysis. Limits ofdetection were determined to be below 10 μg/kg for the majority of the analytes. The recoveries forthose commonly detected natural substances were in the acceptable range of 70–120%.

Introduction

The use of herbal products has significantly increased during the last few decades. There were more than 10,000 registered Chinese Proprietary Medicines in Singapore up to June 2014 [1]. In US, sales of herbal products had increased steadily over the last decade [2]. In year 2000, the sales were 4.2 billion, and it exceeded 6 billion in 2013. Consumers often assume that the use of herbal medicines is safer because they occur in nature or come from plants. Although herbal products and supplements are advertised as ‘natural’, they are not necessarily safe to the human body and free of potential health risks [3], [4]. A large number of active ingredients in herbal plants are toxic if overdose or without proper preparation, such as aconite alkaloids, colchicine alkaloids, digitalis glycosides etc., which may cause serious illness, injury or even death [5], [6].

Most of the previous reports were focused on analysis of certain species of plants [9], [10], [11], [12], [13], [14], [15], [16], [17], [18] and can only detect limited number of toxic natural substances. Thus, a specific method that can detect the presence of a wide range of toxic natural substances commonly occurring in herbal products is needed for the safeguarding of public health.

In this study, aconite alkaloids, aristolochic acids, atropine, colchine alkaloids, ephedrine alkaloids, lobelia alkaloids, solanaceous alkaloids, digitalis glycosides and some other toxic substances that are commonly used in herbal products in Singapore market were analysed. The aim of this study was to develop a method using liquid chromatography-coupled quadrupole tandem mass spectrometry (LC–MS/MS) for simultaneous quantification and detection of a wide range of toxic natural substances in herbal products in a single run. An EPI (Enhanced Product Ion) spectrum library was built up and can be used for the screening of these compounds.

Section snippets

Chemicals and reagents

Deionized water (18  cm−1) was obtained from a Millipore Milli-Q Integral 5 purification system (EMD Millipore, Merck KGaA, Darmstadt, Germany). HPLC grade acetonitrile and methanol were supplied by RCI Labscan (Bangkok, Thailand). Formic acid was supplied by Merck (Darmstadt, Germany). Ammonium formate was supplied by Alfa Aesar (Ward Hill, MA, USA), anhydrous magnesium sulphate was supplied by Sigma–Aldrich (St. Louis, MO, USA) and sodium chloride was supplied by RCI Labscan (Bangkok,

Optimization of the extraction procedure

There were many reported methods on the analysis of certain classes of toxic substances (e.g. aconite alkaloids [11], aristolochic acids [12], [13], berberine [14], lobelia alkaloids [15], methoxsalen [16], solanaceous alkaloids [17] and tetrahydropalmatine [18] etc.) in herbal medicines. Most of them use generic sample preparation, extraction with methanol or acetonitrile followed by centrifugation. These methods were simple and rapid, but the disadvantage of such procedures is the

Conclusion

A method based on QuECHERS extraction and LC–MS/MS was successfully developed for detection and quantification of a wide range of toxic natural substances in herbal products. The study demonstrated the application of an EPI library for multi-analyte screening and identification without the need of standards for each analysis. No significant differences for the library matching were observed between samples containing the multi-analyte mixture and those containing only single analyte.

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