Elsevier

Talanta

Volume 138, 1 June 2015, Pages 240-246
Talanta

Development of a Stationary Phase Optimised Selectivity Liquid Chromatography based screening method for adulterations of food supplements for the treatment of pain

https://doi.org/10.1016/j.talanta.2015.03.010Get rights and content

Highlights

  • Development of a POP-LC screening method for illegal dietary supplement to treat pain.

  • Selection of optimised stationary phase combination>gradient optimisation.

  • Validation of the screening method.

  • Application to real samples.

Abstract

Illegally adulterated dietary supplements are an increasing problem worldwide. One of the important groups of often adulterated products are the dietary supplements, sold for the treatment of pain. These often contain analgesics, a heterogeneous group of molecules, containing both hydrophilic and hydrophobic compounds. The development of a screening method for these components, especially when mass spectrometric detection is not available, necessitates chromatographic separation, difficult to achieve with traditional chromatographic columns.

In this paper Stationary Phase Optimised Selectivity Liquid Chromatography was used for the development of a screening method for nine analgesics, codeine and caffeine, often present in this type of dietary supplements. The method shows a good separation of all the compounds, allowing the screening to be performed with diode array detection and is fully compatible with mass spectrometry. The method was validated for its selectivity following the guidelines as described for the screening of pesticide residues and residues of veterinary medicines in food.

Introduction

The distribution of counterfeited and substandard medicines is an increasing problem worldwide. In developing countries it is estimated that about 30% of the medicine market is covered by counterfeit medicines. Here not only essentially lifesaving medicines like antibiotics, anti-malarial products, HIV inhibitors, etc. are counterfeited, resulting in higher mortalities, but also in resistance of certain viruses and bacteria, due to inadequate treatment. In industrialised countries less than 1% of the medicine market is affected. In these countries mostly counterfeited life style drugs are encountered. Sometimes some counterfeited antibiotics or other essential drugs were detected in both the United States as in the European Union [1], [2], [3], [4]. Another increasing problem is the availability of adulterated dietary supplements and traditional medicines [1]. Dietary supplements and traditional medicines are now freely available through internet or in some special shops. Care should be advised, since these products, especially when they are sold by internet sites, disclosing their identity or other illegal suppliers, are often adulterated with pharmaceutical ingredients. Essentially four important indication groups of adulterated dietary supplements can be distinguished: slimming, potency enhancement, muscle building and treatment of pain [1], [5]. This paper will focus on the latter group. Dietary supplements for the treatment of pain, sold through an illegal supply chain, often claim to be of herbal nature or to contain 100% natural compounds, but are in fact adulterated with analgesics. The group of analgesic medicines is constituted of a heterogeneous group of molecules with a wide variety of molecular structures and log P values. This makes it difficult to develop a screening approach with both good separations for the more hydrophilic compounds and good separation and detection of the more hydrophobic molecules. Even if detection with mass spectrometry does not necessitate separation, it is recommended for a screening method to have a certain separation of the molecules. The more, not all laboratories confronted with the analysis of adulterated dietary supplements are equipped with mass spectrometry and therefore a screening method allowing good separation and identification based on retention time and diode array data is advantageous.

Stationary Phase Optimised Selectivity Liquid Chromatography or SOS-LC is a relatively unexplored technique to solve separations in which both groups of hydrophilic as well as groups of hydrophobic molecules have to be separated and detected in the same run. In SOS-LC the stationary phase becomes a tunable parameter by serial connexion of column segments, with variable lengths, of different stationary phases [6]. The basic development kit sold by Bischoff chromatography (Bischoff Analysentechnik u. geraete GmbH, Leonburg, Germany) consists of segments of 1, 2, 4, 6 and 8 cm of five different stationary phases, i.e. ProntoSIL C18 SH-2, ProntoSIL C18 EPS-2, ProntoSIL Phenyl-2, ProntoSIL CN-2 and ProntoSIL C30 [7]. The basic idea is to run samples, containing the molecules of interest on the different stationary phases separately and based on the retention times obtained, calculate the optimal combination of segments for the separation. In the literature only a few applications using SOS-LC are described. Zedda et al. [8] proposed a liquid chromatographic screening method with mass spectrometric detection, based on SOS-LC, for polymer electrolyte membrane degradation products. Keuhnle et al. [9] and De Beer et al. [6] both presented SOS-LC methods for the separation and the analysis of different steroids and Chen et al. [10] presented the potential of SOS-LC in the domain of green chemistry. All these applications are focussed on the targeted screening of a series of molecules and this in an as short as possible run. The situation of laboratories, charged with the screening of suspected dietary supplements is however different. Often a set of molecules belonging to a therapeutic class is selected for the development of the screening method. The separation is optimised, but the run is kept quite long in order to ensure the detection of other (possibly unknown) molecules during the run. This is necessary since a large variety of molecules, possibly of a total different therapeutic class as the one suggested by the indication, might be present as well as designer molecules. The advantage of SOS-LC in the development of screening methods is the fact that both the stationary as well as the mobile phase can be adapted to obtain the best separation/detection of the compounds of interest. The basic idea of SOS-LC is that you keep the mobile phase composition constant and the stationary phase is adapted by changing type and length of the column segments in order to achieve the optimal separation. The idea in the presented approach is that both stationary phase and mobile phase can be adapted in order to obtain good separation of a set of molecules that are hard to separate using a classic chromatographic column.

In this paper a screening method using SOS-LC for dietary supplements with the treatment of pain as indication was developed. Therefore retention times for a set of 10 often found adulterants were measured isocratically on the five stationary phases of the Bischoff development kit. Caffeine was used as the 11th component since it is present in the majority of the dietary supplements for the treatment of pain analysed in our laboratory. Including caffeine in the development/validation set ensure that the presence of caffeine will not interfere with the screening. Based on isocratically measured retention times the optimal stationary phase was calculated and used for gradient optimisation. After validation the method was applied for the screening of five samples seized by the Belgian authorities.

Section snippets

Chemicals and reagents

The reference standards for paracetamol, acetyl salicylic acid, aminopyrine, caffeine, phenylbutazone, and ibuprofen were purchased from Fagron (Waregem, Belgium). Ketoprofen, naproxen, aceclofenac, diclofenac sodium salt and indomethacin were purchased from Sigma-Aldrich (St. Louis, USA) and Codein Phosphate Hemihydrate from Conforma (Destelbergen, Belgium). Uracil for determination of death volumes was purchased from Across (Geel, Belgium).

For the preparation of the mobile phases ammonium

Selection of the optimal column

For the development of the screening method 11 molecules often encountered in dietary supplements sold for the treatment of pain were selected. Nine of these molecules are non-steroidal anti-inflammatory drugs (NSAID). Codeine and caffeine were added to the development set, since they have a synergetic effect with NSAIDs and are, especially caffeine, frequently encountered in these types of dietary supplements. Table 2 shows the 11 molecules chosen, together with their calculated log P values.

Conclusion

A screening method for the detection of analgesics, codeine and caffeine in counterfeit medicines and potentially adulterated dietary supplements was developed, based on the principles of SOS-LC. The development performed, shows clearly the potential of SOS-LC to solve difficult separation problems, especially when a mixture of strongly hydrophilic and hydrophobic compounds is targeted. Indeed it would be very hard to detect all molecules of the development set in one run and have them all

References (14)

  • M. Johansson et al.

    J. Pharm. Biomed. Anal.

    (2014)
  • M. Zedda et al.

    J. Chromatogr. A

    (2009)
  • K. Chen et al.

    J. Chromatogr. A

    (2010)
  • E. Deconinck et al.

    J. Chromatogr. Sci.

    (2013)
  • 〈http://www.prlog.org/10124036-global-pharmaceutical-market-forcast-to-2012.html〉 (accessed...
  • A. Deisingh

    Analyst

    (2005)
  • 〈http://www.who.int/impact/FinalBrochureWHA2008a.pdf〉 (accessed...
There are more references available in the full text version of this article.

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