11


Table 1. Time contributions for automated method development Step


Analyst (hours)


1 2 3 4 5


Columns set selection Rapid optimisation Fine Optimisation Fine Tuning


Robustness studies


0.5 1 1 1 1


ChromSword (hours)


--------------------- 18 11 3 4


Figure 2. Separation of terbinafine and impurities. Gradient. 0 min. – 50% B, 1.0 min. - 80%, 11.0 – 80%. T = 30°C; flow rate 1.0 ml/min, WL 280nm.


Automated method development.


How long does it take? Time contributions for automated method development are shown in the Table 1.


It is necessary to note that the experiments were performed with UHPLC system and it takes 30-50% more time for automated work with HPLC instrument.


Figure 1. Separation of a critical pair of terbinafine impurities after rapid optimisation.


A useful option is prediction of retention from chemical structures and column properties. For terbinafine the software predicted retention factor of 1.7 at 90% and 11 at 76% of methanol in a mobile phase for the Kinetex C18 column. Those values were experimentally obtained at 91% and 78% of methanol in a mobile phase. Such predictability only from structural formulae and column properties can save time to find practically good separation conditions. Fine-tuning procedure applied after input data of only one run allows prediction of retention for practically acceptable level.


ChromSword can automatically optimise gradient profiles with more than three variables. Typically method development software can build resolution maps for 2D or 3D dimensions -


gradient time, temperature, pH or other variable. In this case initial (C0) and final concentration (Ct) of a solvent in a mobile phase are constant and specified by a user and only gradient time (Gt) of a linear gradient and temperature (T) can be optimised. ChromSword uses the Monte- Carlo procedure of optimisation and optimises simultaneously C0, Ct, Gt and T for a linear gradients (4D) and also position of every gradient node (concentration and time) and


temperature for the whole gradient profile for multistep gradients with up to 100 nodes. Automated optimisation proposed a method with full separation of all compounds and run time less than 10 min (Figure 2). This method was selected for the next step to test robustness.


Step 4. Automatic robustness tests


Robustness studies are important stage of method development process to build design space and determine operating range of method variables according the quality by design (QbD) approach.


The following design for automatic robustness studies with AutoRobust module was used:


1. Temperature, gradient slope, flow rate – the full factorial design when effect of all possible combinations of method variables are studied


2. Temperature, gradient slope, flow rate, wave length, injection volume, equilibration time, three column batches – Plackett–Burman design when a statistical design of experiments is created automatically for 12 runs and 7 two-level factors.


The design plans are created fully automatically that allows to avoid human factor for possible errors. Results of studies confirmed robustness of the method and possibility to transfer it to other laboratories.


An important point for method development in regulated laboratories are detailed reporting of all steps. ChromSwordAuto and AutoRobust generate reports automatically and this enables an analyst save time for preparation of necessary project documentation.


Interested in publishing a Technical Article?


Contact Gwyneth on +44 (0)1727 855574 or email:


gwyneth@intlabmate.com Conclusion


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


The robust rapid method with analysis time less than 10 min. has been developed


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 use of automatic procedures implemented in ChromSwordAuto® and AutoRobust software can significantly reduce method development time.


The technology allows to test automatically many different conditions in a short period of time. It allows to obtain comprehensive and important information about a sample, critical parameters for peak shape and resolution and to find the best separation conditions.


References 1. Marinela Florea, Corina-Cristina Arama, Crina-Maria Monciu. Farmacia 2009, Vol LVII, p. 82


2. Vimal Domadiya*, Rambir Singh, Rakesh kumar Jat, Rakshit Chokshi. Pharm Analysis & Quality Assurance . Vol. 2012 , Article ID- "Inventi:ppaqa/307/12 " , 2012


3. Matysová L, Solich P, Marek P, Havlíková L, Nováková L, Sícha J. Talanta. 2006 Jan 15;68(3):713-20.


INTERNATIONAL LABMATE - MARCH 2013


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