19


mixture without the doxepin isomers) of the chosen chromatographic conditions can be maintained using the following chromatographic operating parameter ranges of tG


: 45 ± 1 [min], T: 50 ± 1 [°C], ternary eluent composition: 80 ± 1 [%B1:B2], flow rate: 1.0 ± 0.1 mL/min, dwell volume: 1.18 ± 0.05 mL, Bstart: 5±1%B. A comparison of the predicted and experimental retention times for this chromatographic condition within the 3-D resolution map is shown in Fig. 10. The correlation was observed to be excellent, the average difference between predicted and experimental retention times is approximately 0.4 min and the largest difference is 0.7 min.


The tG –T-ternary resolution model also allows Figure 8. 3-D Resolution space showing the tG -T-AN- plane with eluent B: B2. Colour code as shown in Fig. 6.


the chromatographer to rapidly establish that the use of a binary eluent containing MeOH is also feasible for the separation of the sample mixture (without the doxepin isomers) which, for economic reasons and ease of method transfer, would be very attractive. Examination of the tG


–T-ternary resolution


model (Fig. 6, eluent B: 100% B1)(“MeOH plane”) indicates that the binary LC method using MeOH would be sufficiently robust (i.e. Rs, crit, >1.6) if operated within the following operating parameters – tG


: 22 ± 1 [min], T: 55


± 1 [°C], ternary eluent composition: 100% B1 (MeOH modifier), flow rate: 1.0 ± 0.1 mL/min, dwell volume: 1.18 ± 0.05 mL, Bstart: 5±1%B. Fig. 11 once again highlights that excellent correlation was observed, the average difference between predicted and experimental retention times is approximately 0.1 min and the largest difference is 0.2 min.


Summary Figure 9. 3-D Resolution space showing the tG -T-plane for (B1:B2)(80:20 V/V) as eluent B. Colour code as shown in Fig. 6. 3.6 Selection and verification of optimum


robust chromatographic conditions Given the fact that there was excellent correlation between the three predicted and experimental validation runs, it was expected that the accuracy of the simulated chromatograms within the resolution cube would also be good. This was confirmed by comparison of the predicted and experimental chromatograms using the chromatographic conditions: tG


: 45 min,


T: 50 °C and a ternary eluent composition: (B1:B2 80:20 V/V) (i.e. the dark blue circle represented in Fig. 7).


The experimental chromatogram was obtained using a different batch of stationary phase material, differing buffer batch and a different mobile phase preparation and the chromatography performed on a different day which highlighted that, even with a worst case scenario, excellent correlation between experimental and modelled data is obtained and that the chosen chromatographic conditions are robust (see Fig 10).


Evaluation of the tG –T-ternary resolution


model (80% MeOH plane – see Fig. 9) permits the chromatographer to rapidly establish that the robustness (i.e. Rs,crit > 1.5 for the sample


A method for the separation of all 20 basic drug molecules and two neutral components was rapidly achieved and was proven to be robust with respect to the three critical operating parameters – gradient time, temperature and ternary mobile phase composition. The methodology presented in this work is in accordance with Quality by Design (QbD) principles and results in the definition and visualization of the Design Space and the identification of robust working regions for the chromatographic conditions.


The HPLC modelling software has been shown to increase efficiency and productivity in routine method development, optimization and method transfer. The new 3-D modelling technology locates the global optimum of highly influential chromatographic operating parameters with respect to separation, analysis time and robustness.


The graphical presentation of the critical parameters with an optimization Design Space greatly assists in assessing the robustness of the chromatographic separation.


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