Analytical Methods A rapid HPLC column switching method for sample preparation and determination of b-carotene in food supplements Ivana Brabcová, Markéta Hlavác ˇková, Dalibor Šatínsky ´ ⇑ , Petr Solich Department of Analytical Chemistry, Faculty of Pharmacy, Charles University, Heyrovského 1203, Hradec Králové 500 05, Czech Republic article info Article history: Received 20 December 2011 Received in revised form 8 November 2012 Accepted 19 April 2013 Available online 30 April 2013 Keywords: b-carotene On-line sample preparation Column-switching HPLC Food supplements abstract A simple and automated HPLC column-switching method with rapid sample pretreatment has been developed for quantitative determination of b-carotene in food supplements. Commercially samples of food supplements were dissolved in chloroform with help of saponification with 1 M solution of sodium hydroxide in ultrasound bath. A 20-min sample dissolution/extraction step was necessary before chro- matography analysis to transfer b-carotene from solid state of food supplements preparations (cap- sules, tablets) to chloroform solution. Sample volume – 3 lL of chloroform phase was directly injected into the HPLC system. Next on-line sample clean-up was achieved on the pretreatment precolumn Chro- molith Guard Cartridge RP-18e (Merck), 10 Â 4.6 mm, with a washing mobile phase (methanol:water, 92:8, (v/v)) at a flow rate of 1.5 mL/min. Valve switch to analytical column was set at 2.5 min in a back-flush mode. After column switching to the analytical column Ascentis Express C-18, 30 Â 4.6 mm, particle size 2.7 lm (Sigma Aldrich), the separation and determination of b-carotene in food supplements was performed using a mobile phase consisting of 100% methanol, column temperature at 60 °C and flow rate 1.5 mL/min. The detector was set at 450 nm. Under the optimum chromatographic conditions stan- dard calibration curve was measured with good linearity – correlation coefficient for b-carotene (r 2 = 0.999014; n = 6) between the peak areas and concentration of b-carotene 20–200 lg/mL. Accuracy of the method defined as a mean recovery was in the range 96.66–102.40%. The intraday method preci- sion was satisfactory at three concentration levels 20, 125 and 200 lg/mL and relative standard devia- tions were in the range 0.90–1.02%. The chromatography method has shown high sample throughput during column-switching pretreatment process and analysis in one step in short time (6 min) of the whole chromatographic analysis. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction b-carotene is an important biological compound that is widely distributed in fruits and vegetables. It is converted in the human body to retinol (vitamin A) that is essential for proper function of the retina, skin and mucous membranes. Biological studies have shown that the dietary intake of b-carotene may reduce the risk of cancer and infectious diseases. b-carotene protects plant, human and animal cells (most mucous membranes and skin) from the destructive effects of UV radiation. As an antioxidant deactivates harmful free radicals, thus slows down the ageing process. Traditionally, the separation and determination of carotenoids (including b-carotene) was often performed using high perfor- mance liquid chromatography (HPLC) with a reversed phase C-18 columns and detection at visible part of spectra (Iwase, 2002; Kar- ppi, Nurmi, Alonso, Lorencio, & Nyyssönen, 2008; Pupin, Dennis, & Toledo, 1999). However, most HPLC methods employ time con- suming and laborious sample preparation steps such as pressur- ized liquid extraction (PLE) (Plaza et al., 2011; Sanagi, See, Ibrahim, & Naim, 2005), liquid–liquid extraction (LLE) (Burgos et al., 2009; Jackson, Lien, White, Bruns, & Kuhlman, 1998; Karppi et al., 2008; Pupin et al., 1999), supercritical fluid extraction (SFE) (Birtigh, Johannsen, Brunner, & Nair, 1995; Chuang & Brunner, 2006), solid phase extraction (SPE) (Iwase, 2002; Shen et al., 2009; Sánchez, Granados, Serrana, & Martínez, 2010), ultrasound- assisted extraction (UAE) (Plaza et al., 2011), or matrix solid-phase dispersion (MSPD) (Gentili & Caretti, 2011; Putzbach et al., 2005), which are carried out in off-line mode before sample injection into the chromatography system. In addition, using above mentioned sample pretreatment methods could be accompanied with risk of loss of analytes during sample manipulation and greater biohazard. Nowadays the application of on-line extraction on special SPE sorbents is the growing trend in sample preparation procedures (Sadílek, Šatínsky ´ , & Solich, 2007). The introduction of special extraction sorbents, such as restricted access materials (RAM), al- lows the direct and repetitive injection of complex biological 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.04.063 ⇑ Corresponding author. Tel.: +420 495067228; fax: +420 495518718. E-mail address: satinsky@faf.cuni.cz (D. Šatínsky ´ ). Food Chemistry 141 (2013) 1433–1437 Contents lists available at SciVerse ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem