Chromatography focus on


Application of ChromSword Software for Automatic HPLC Method Development and Robustness Studies.


Separation of Terbinafine and Impurities by François Vogel (Novartis AG, Basel, Switzerland) Sergey Galushko (ChromSword, Muehltal, Germany) Corresponding author. Tel: +49 6151 136777; Email: galushko@chromsword.de


This presentation describes the application of automated HPLC method development for separation of a mixture of terbinafine and impurities. In our approach the system was specified to start with rapid optimisation steps automatically exploring the entire design space through software intelligence to find the best analysis conditions. Screening of different columns, solvents and buffers, instrument parameters as well as rapid optimisation, fine-tuning, robustness studies, and documentation have been implemented automatically in one software platform.


The system was used effectively for optimisation of separation of terbinafine and impurities in the reversed-phase mode with the Quality by Design (QbD) principles. ChromSwordAuto®


and AutoRobust software can significantly reduce method development time.


Terbinafine hydrochloride, (E)-N-(6,6-dimethyl-2-hepten-4-ynyl)-N-methyl-1-naphtalene methanamine hydrochloride (Lamisil) is an active pharmaceutical ingredient with antifungal activity used in different drug formulations. The remaining patent or exclusivity for Lamisil expired on 30 June, 2007 and the US Food and Drug Administration has approved the first generic versions of prescription Lamisil (terbinafine hydrochloride) tablets. There are only a limited number of methods described for the determination of terbinafine, its impurities and degradation products by reversed-phase HPLC. Published methods are either long-term or require ion-pair additives or aggressive conditions that reduce lifetime of reversed-phase columns [1-3].


HPLC method development is frequently a time consuming process which requires tests of different columns, mobile phases and other conditions to provide a reliable and robust method to be used for impurities profiling or quality control analyses. In this article we describe how time for method development can be reduced substantially utilising specialised HPLC (UHPLC) method development system controlled by automated method development and robustness tests software.


Instruments Agilent Technologies 1290 Method Development Systems with


Method Development Software ChromSwordAuto®


Results and Discussion Automated method development process included the following steps:


1. Columns set selection 2. Rapid optimisation 3. Fine optimisation


4. Retention models building, fine tuning 5. Robustness studies


Step 1. Columns set selection.


The columns set was selected form ChromSword column characteristics database. The column database software module builds selectivity maps of more than 100 commercially available reversed-phase columns. These maps allow selection of columns with different selectivity for a different concentration of acetonitrile and methanol in mobile phases. The columns set described in the experimental part of the article is one of many method development column combinations which can be chosen from the column selectivity maps.


DAD, two thermostated column compartments, internal 8 pos. column selector and external 12 pos. solvent selector Step 2. Rapid Optimisation. software for computer-assisted and automatic HPLC method development


(ChromSword). AutoRobust software for automatic robustness studies of HPLC methods (ChromSword).


Both software are compatible with Empower (Waters), ChemStation, OpenLab (Agilent Technologies), Chromeleon (Thermo Fisher) chromatography data systems.


Columns Set(100 x 4.6cm): 1. SynergiFusionRP, 4µ (Phenomenex) 2. X-TerraRP18.3.5µ (Waters) 3. Kinetex C18, 2.6µ (Phenomenex) 4. HyperSelectHiPurityC18, 5µ (ES Industries) 5. Gemini NXC18, 5µ (Phenomenex) 6. Lichrospher100RP18, 5µ (Merck KGaA)


Mobile phases: 0.1%TEA+HAc pH 7.3 with Acetonitrile and Methanol


The rapid optimisation automated algorithm performs only few runs to find optimal conditions for every column/solvent/buffer combination. The rapid optimisation algorithm starts without any preliminary information about a sample or initial retention data. The first gradient run is a scouting and the second and following runs are results of optimisation. As a results good isocratic, linear or multi-step gradient conditions can be found. In this step 6 columns and 2 organic solvents (ACN and MeOH) were automatically tested with totally 36 runs during 18 hours.


Kinetex C18, 2.6µ column with methanol as an organic solvent provided the best separation with a minimal time. In Figure 1 a chromatogram and a gradient profile that the system found automatically are shown for the critical pair of isomers.


Step 3. Fine optimisation.


The fine optimisation step was done to find optimal and alternative methods for separation of the mixture with the best combination of a column\solvent which was found after the Step 2. During the fine optimisation process the system performs detailed study of a sample for impurities profiling, peak tracking, building retention models and searches for isocratic and gradient conditions to separate all components of a mixture. In this step the 4 alternative gradient methods were proposed and retention models constructed for all sample components.


Step 4. Retention models building and off-line optimisation (optional)


This step is optional because the software finds conditions automatically. However it was interesting to build retention models of retention behaviour of all components to simulate chromatograms and to find alternative conditions for separation of a mixture.


INTERNATIONAL LABMATE - MARCH 2013


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