Automated interface for hyphenation of planar chromatography with mass spectrometry Heinrich Luftmann 1 , Mario Aranda 2 y and Gertrud E. Morlock 2 * 1 Institute of Organic Chemistry, University of Mu ¨nster, Mu ¨nster, Germany 2 Institute of Food Chemistry, University of Hohenheim, Stuttgart, Germany Received 20 September 2007; Revised 26 September 2007; Accepted 26 September 2007 A fully automated interface to couple high-performance thin-layer chromatography (HPTLC) with mass spectrometry (MS) is described. This universal hands-free interface connects intact normal- phase plates to any liquid chromatography/mass spectrometry (LC/MS) system without any adjust- ments or modifications to the mass spectrometer. The interface extracts the complete substance band with its depth profile and thus allows detections in the pg/band range. The high performance of the automated interface was evaluated through caffeine quantification in real samples, viz., energy drinks and pharmaceutical tablets, without internal standard. Following chromatographic separation on silica gel 60 F 254 HPTLC plates, caffeine bands were eluted from the plate by means of the automated interface to the electrospray ionization (ESI) source of a triple-quadrupole mass spec- trometer. Since in full scan mode only the protonated molecule [MRH] R was observed, caffeine quantification was performed using the selected-ion monitoring (SIM) mode at m/z 195. The validation showed highly reliable results for the linear range (R 2 ¼ 0.9973), repeatability (RSD ¼ 5.6%, n ¼ 6) and intermediate precision (RSD ¼ 1.5%, n ¼ 3). Regarding accuracy the results obtained by HPTLC/MS were not statistically different (F-test, t-test) from those obtained by validated HPTLC/ UV methods. Hence, this interface proved to be one of the most reliable and universal interfaces for HPTLC/MS. Copyright # 2007 John Wiley & Sons, Ltd. Planar chromatography on high-performance thin-layer chromatography (HPTLC) plates is a well-known cost- effective offline separation technique. In recent years the entire system reproducibility, from application to detection, has been enhanced through automatization of the equipment making it useful and reliable for quantitative analysis. Extraordinary helpful – and at the same time an under- estimated feature – is the fact that planar chromatography shows also what is left at the start, which would never be seen in column chromatography, and gives a more comprehensive sample image when unknowns have to be analyzed. The system versatility, i.e. parallel chromatog- raphy of up to 36 runs, wide range of application volumes (nL to mL), automated-single or multiple-development 1 and multiple detection by fluorescence (FLD), UV and Vis, 2,3 together with less sample preparation than other chromato- graphic techniques 4,5 and the simplicity of carrying out in situ pre- and post-chromatographic derivatization, 2,6 provide planar chromatography with a still-treasured position amongst the analytical tools for high-throughput analysis. However, the coupling with mass spectrometry (MS) has been a weakness of this technique in comparison with the advances achieved for high-performance liquid chromatography (HPLC), gas chromatography (GC) and capillary electrophoresis (CE). Due to the selective detection offered by MS, several approaches for HPTLC/MS hyphena- tion have been proposed during the extensive research done in the last decade, e.g. fast atom bombardment (FAB), 7 secondary ion mass spectrometry (SIMS), 8,9 matrix-assisted laser desorption/ionization (MALDI), 10,11 continuous wave diode LD/atmospheric pressure chemical ionization (APCI), 12 extraction using an automated surface sampling probe via liquid microjunction with electrospray ionization (ESI), 13 automated sampling with desorption ESI (DESI), 14 direct analysis in real time (DART), 15 and plunger-based extractors. 16,17 Considering the advantages of ultra thin-layer chromatography (UTLC), 18 i.e. decreased detection limits, reduced solvent consumption and increased speed, other authors have developed UTLC/MALDI, 19 UTLC/DESI, 20 and UTLC/SIMS. 8 All these approaches have the great advantage that the planar chromatographic separation can be performed regardless of MS-suitable solvents. All HPTLC/MS systems were first assayed as proof- of-concept, then, after successful qualitative results, several of these systems were also evaluated for quantitative determinations with promising results. However, besides that many of these approaches needed an internal standard (IS) to carry out the quantification, each system has its own RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom. 2007; 21: 3772–3776 Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/rcm.3276 *Correspondence to: G. E. Morlock, Institute of Food Chemistry, University of Hohenheim, Garbenstrasse 28, 70599 Stuttgart, Germany. E-mail: gmorlock@uni-hohenheim.de y On leave from the Department of Food Science, Nutrition and Dietetic, Faculty of Pharmacy, University of Concepcion, Con- cepcion, Chile. Copyright # 2007 John Wiley & Sons, Ltd.