1 3 DOI 10.1007/s10337-017-3337-8 Chromatographia ORIGINAL A 14 Parameter Study of UHPLC’s for Method Development Transfer and Troubleshooting Imad A. Haidar Ahmad 1 · Frank Hrovat 1 · Arianne Soliven 1 · Adrian Clarke 2 · Paul Boswell 3 · Thomas Tarara 1 · Andrei Blasko 1 Received: 10 January 2017 / Revised: 24 May 2017 / Accepted: 31 May 2017 © Springer-Verlag GmbH Germany 2017 of such issues are: retention time irreproducibility, low sen- sitivity, method transfer failure, etc. Keywords UHPLC · UPLC · Ultra-high pressure liquid chromatography · Mixing accuracy · Mixing precision · Temperature accuracy and precision · Baseline noise · Baseline drift · Detector linearity · Injector linearity · Wavelength accuracy · Dwell volume, and extracolumn volume · Sample temperature · Pressure ripple · Flow rate accuracy · Flow rate precision Introduction High performance liquid chromatography (HPLC) is the most used analytical technique in industry after weighing and pH measurement. HPLC is utilized in industries such as pharmaceuticals, food, forensic, environmental, cos- metic, etc. Analytical results acquired from chromatogra- phy are fundamental in making reliable decisions during drug development. Hence, the accuracy, precision, reli- ability and robustness of a chromatography system must be evaluated to gauge the best operating system, and condi- tions to deliver high quality products. The empirical determination of a UHPLC’s practical performance is of high importance when developing and transferring methods, to create a comprehensive under- standing and realistic tolerances in terms of method perfor- mance and accuracy and precision of the desired analytical result. It also assists us with rapid troubleshooting when deviations occur. Therefore, only after such a compre- hensive analysis of each system’s capabilities and modus operandi, can rational tolerances be set. The acceptance cri- teria may either be wider to account for vendor-to-vendor deviations, or it may be restricted for an instrument from Abstract Five ultra-high pressure liquid chromatography (UHPLC) instruments were compared to one another by examining the overall system performance and key func- tions of the system parts including: pump, auto-sampler, thermo-stated column compartment, and the detector. The five UHPLC systems used in this study were: ThermoFisher Vanquish, Agilent 1290 Infinity I, Agilent 1290 Infinity II, Waters Acquity I-Class, and Shimadzu Nexera X2. The identities of the systems were blinded in the results and discussion section to use this study for scientific purposes only rather than for competition and marketing. The follow- ing tests were performed to evaluate and compare the five UHPLC systems: injector linearity and precision, sample carryover, sample (autosampler) temperature accuracy, col- umn temperature accuracy and precision, pressure ripple, pump mixing accuracy, flow rate accuracy, detector drift and noise, detector linearity, wavelength accuracy, extra- column volume, and dwell volume determination. This study presents an approach on how to test the performance of UHPLC systems along with potential problems that ana- lysts may face when using the UHPLC systems, examples Imad A. Haidar Ahmad and Frank Hrovat are first authors. * Imad A. Haidar Ahmad iahmad.umn@gmail.com * Andrei Blasko Andrei.Blasko@Novartis.com 1 Novartis Pharmaceuticals Corporation, Technical Research and Development, PHAD, San Carlos, CA, USA 2 Novartis Pharma AG, Chemical and Analytical Development, CHAD, Basel, Switzerland 3 Department of Horticultural Science, University of Minnesota, St. Paul, MN, USA