of electrospray ionization, HPLC was coupled to MS. In contrast with GC, HPLC has lower chromatographic resolution, but requires no derivatization for polar molecules, and separates molecules in the liquid phase. Additionally HPLC has the advantage that a much wider range of analytes can be measured with a higher sensitivity than GC methods. Capillary electrophoresis (CE) has a higher theoretical separation efficiency than HPLC (although requiring much more time per separation), and is suitable for use with a wider range of metabolite classes than is GC. As for all electrophoretic techniques, it is most appropriate for charged analytes. Detection methods Mass spectrometry (MS) is used to identify and quantify metabolites after optional separation by GC, HPLC, or CE. GC-MS was the first hyphenated technique to be developed. Identification leverages the distinct patterns in which analytes fragment. These patterns can be thought of as a mass spectral fingerprint. Libraries exist that allow identification of a metabolite according to this fragmentation pattern . MS is both sensitive and can be very specific. There are also a number of techniques which use MS as a stand-alone technology: the sample is infused directly into the mass spectrometer with no prior separation, and the MS provides sufficient selectivity to both separate and to detect metabolites. For analysis by mass spectrometry the analytes must be imparted with a charge and transferred to the gas phase. Electron ionization (EI) is the most common ionization technique applies to GC separations as it is amenable to low pressures. EI also produces fragmentation of the analyte, both providing structural information while increasing the complexity of the data and possibly obscuring the molecular ion. Atmospheric-pressure chemical ionization (APCI) is an atmospheric pressure technique that can be applied to all the above separation techniques. APCI is a gas phase ionization method, which provides slightly more aggressive ionization than ESI which is suitable for less polar compounds. Electrospray ionization (ESI) is the most common ionization technique applied in LC/MS. This soft ionization is most successful for polar molecules with ionizable functional groups. Another commonly used soft ionization technique is secondary electrospray ionization (SESI). Surface-based mass analysis has seen a resurgence in the past decade, with new MS technologies focused on increasing sensitivity, minimizing background, and reducing sample preparation. The ability to analyze metabolites directly from biofluids and tissues continues to challenge current MS technology, largely because of the limits imposed by the complexity