Determination of Caffeine in Dietary Supplements by Miniaturized Portable Liquid Chromatography

Nowadays a wide variety of dietary supplements (capsules, herbal preparations, gels, plant extracts) are easily available in local markets and e-commerce platforms with a broad spectrum of claimed properties such as anti-aging, stimulating, fat-burning, anti-inflammatory, anti-carcinogenic and many others, and new formulations are constantly being introduced [1]. Among them, products derived from botanical species are of special concern. This is because they are perceived as safe for many consumers due to their natural origin, with the resulting risk of excessive and uncontrolled use to obtain rapid results [2]. Dietary supplements derived from plants are usually categorized as foods; thus, the regulations setup for their preparation, distribution and labeling are not as strict as those established for pharmaceutical products [3]. Unlike pharmaceuticals, the amount of the active ingredients in these products may not be specified. This is the case of supplements containing caffeine, a central nervous stimulant with many effects on the body metabolism. Caffeine is naturally present in different plants, mainly in coffee and tea, which are ingredients in most dietary supplements aimed at enhancing the physical performance; caffeine is also a common ingredient of slimming products. Caffeine levels in raw stuff such as coffee beans and tea leaves, are widely variable depending on the source [4]. Therefore, even if the amount of the botanical species used to prepare these products is specified by the producer, the accurate amount of caffeine in the final products is rarely known [4,5]. In addition, it is a common practice to add caffeine to increase the stimulating and thermogenic effects of the final product. There are different risks associated with a high intake of caffeine, which could be aggravated by synergistic effects of combined substances [2,6]. For these reasons there is an increasing demand of analytical methods that can be used to estimate the quality, effectiveness and safety of dietary supplements, regarding their caffeine content [7], [8], [9].

To date, conventional liquid chromatography (LC) is the technique of choice for the determination of caffeine in dietary supplements as well as in beverages obtained from plants [2,4,[8], [9], [10], [11], [12]]. However, in many instances the methods reported involve the employment of sophisticated instrumentation; thus, they may be unsuitable for routine tests. A greater availability of simple and rapid methods would facilitate a better control for this kind of products [2,8].

Recent progresses in LC technology have led to an increasing interest in miniaturized separations, and miniaturized LC is now considered reliable alternative to conventional LC in many areas of application. The reduction of column diameters, particles sizes and flows results in diminished band-broadening, which in turn results in better resolution and enhanced mass sensitivity over conventional LC [13,14]. However, in the analysis of dietary supplements miniaturized LC has only been applied to a few compounds, mainly amino acids and peptides, fatty acids, flavonoids, and terpenes [15,16]. In other words, no methods based on miniaturized LC have been developed for the quantification of caffeine in this kind of products.

During the past years, the advances in miniaturization have led to the development of portable LC systems, most of them fabricated in-lab [17], [18], [19]. Because of their inherent simplicity and reduced dimensions, portable instruments are becoming increasingly used in different fields. Even in well-equipped laboratories, portable miniaturized LC offers important advantages such as reduced time of analysis (thus, high sample throughput), low energy use and low generation of wastes [20]. These kinds of systems can be also beneficial in cases where the reliability of a product is a priority or when the samples present limited stability [17]. A labmade capLC system was developed by Coates et al. for the on-site analysis of different compounds, including caffeine, but this compound was only analyzed in drinks (a cola drink, an energy drink and brewed coffee) [21]. We have recently developed a method using a commercially available portable chromatograph for the analysis of caffeine and other methylxantines in environmental waters [22]. In the past few years, different alternatives have also been proposed for the rapid analysis of caffeine with non-chromatographic miniaturized portable devices. For example, a fluorimetric device with a smartphone was developed for the determination of caffeine in different drinks and medicines [23].Very recently, Wang et al. reported an assay for the determination of caffeine and other active ingredients of tea by using a pocket-sized near-infrared spectrometer [24]. To the best of our knowledge, none of these methods has been applied to the analysis of complex matrices such as dietary botanical supplements.

Taking into account the current state of the art, the main goals of the present study were: (i) to optimize conditions for the analysis of caffeine using benchtop capLC and benchtop nanoLC, and starting from their respective analytical parameters, to establish the possibilities of a portable LC instrument for (ii) the determination of caffeine in dietary supplements of botanical origin. The analyte was previously extracted from the samples in methanol by ultrasound assisted extraction (UAE). For processing the extracts into the benchtop nanoLC system an in-tube solid-phase microextraction (IT-SPME) arrangement (Fig. 1) was used for a further on-line purification of the analyte [25]. On the basis of the results obtained, a new method for the rapid measurement of caffeine in dietary supplements by miniaturized portable LC with a commercially available instrument is presented. The method has been applied to the analysis of different products, namely two green coffee extract-based products, one of them derived from decaffeinated coffee, and a fat burning formulation. To the best of our knowledge, this is the first study that reports the employment of miniaturized LC for the analysis of caffeine in dietary supplements. This is also the first application of miniaturized portable LC to the analysis of samples of botanical origin.