14


December 2010


AutoChrom (Advanced Chemistry Development, Toronto, Canada), ChromSword (Merck KGaA, Darmstadt, Germany) and Fusion (S-Matrix, Eureka, CA, USA) also support the principles of QbD in that the software enables the systematic design of experiments leading to an increased comprehension and understanding of the influences of the chromatographic operating parameters on the separation.


In the RP-LC arena, retention modelling has been mostly employed in the separation of small molecular pharmaceuticals including synthesis impurities and degradation products of widely differing polarities [4-11]


approach has been also successfully used for peptides and proteins [12,13] [13], metabolites [10,14]


, oligonucleotides [15-20], environmental pollutants [21-23]


, complex plant mixtures .


A major reason for the extensive use of these retention modelling packages within modern method development laboratories resides in their excellent prediction accuracy for analyte retention and resolution [4,24-26]


and the


flexibility of the software, which can be used to model isocratic or gradient separations as a function of variables such as percentage organic, gradient time, gradient steps, pH, temperature, ion pairing reagent concentration or ionic strength in a continuous way.


The use of computer modelling is extremely attractive, as only limited input data is required in order to rapidly obtain accurate optimum separation conditions.


In addition to the 1-dimensional modelling (or one factor at-a-time, OFAT) described above, which help to understand peak movements, some software packages can now accurately perform 2-dimensional modelling, i.e. simultaneous variation of any two-separation variables for a chromatographic procedure. The 2-dimensional (2-D) approaches have a much more pronounced effect on the separation selectivity than the additive effect of the two individual variables [27]


. Examples


include gradient time (tG) vs pH, percentage organic in the mobile phase (%B) vs pH, tG


vs


temperature (T), ionic strength vs temperature among many others possible combinations and show excellent results with U(H)PLC and sub-2-µm columns in industrial analysis by the


. The 2-D technology has recently been extended to 3-D modelling [28]


where it has


been elegantly shown that, based on only twelve input experiments and after building the Resolution Cube, the chromatographer can investigate in excess of 106


(=1 million) virtual chromatograms with extremely high


group Fekete and Fekete at Richter Pharma [3, 31,32]


precision and can evaluate the interactions of tG tG


, temperature (T) and ternary composition or


-T-pH, or other combinations and find the best separation in seconds. A resolution space can be presented in a virtual mode which allows the establishment of robust HPLC methods, and their visualization, in an extremely efficient way.


This present paper investigates the application of the DryLab®


2010, 3-D multi-


factorial optimization modelling software of three critical HPLC method parameters, i.e. gradient time (tG composition (B1:B2


. However, the


), temperature (T) and ternary ), (where the ratio of the


two organic modifiers is varied) based on 3x4 experiments in the separation of 20 pharmaceutically relevant basic molecules and 2 neutral compounds as controls. Examining the effect of the experimental operating variables on critical resolution and selectivity was carried out in such a way as to systematically vary all three factors simultaneously. The basic element was a gradient time–temperature (tG


–T) plane, which


was repeated at three different ternary compositions of eluent B between methanol and acetonitrile. The so-defined volume enables the investigation of the critical resolution (Rs, crit


) for a part of the Design


Space of this complex pharmaceutically relevant sample mixture. 3-D modelling offers visual support of the Design Space which generates more flexibility and establishes more robust HPLC regions for utilisation. Multi-dimensional robust regions can be successfully defined and graphically depicted. The use of multi-factorial approaches to HPLC method development will undoubtedly result in a reduction in development costs associated with trial and error, generate highly robust methods and enable smoother method transfer between different laboratories in a global economy.


2. Experimental 2.1 Chemicals, compounds and reagents Acetonitrile (AN) and methanol (MeOH) (both HPLC grade) were supplied by Lab-Scan Analytical Sciences (Gliwice, Poland). HPLC grade water was provided by Romil Ltd. (Cambridgeshire, UK). Amiloride hydrochloride, benzylalcohol, benzylamine hydrochloride, (S)-(+)-chlorpheniramine maleate, desipramine hydrochloride, diphenhydramine hydrochloride, (±)-nicotine, nortriptyline hydrochloride, oxprenolol hydrochloride, phenol, pindolol, procainamide hydrochloride, quinine, salbutamol hemisulphate,


benzyltrimethylammonium chloride, doxepin hydrochloride (85:15 E:Z-isomer distribution)


and terbutaline hemisulfate were purchased from Sigma-Aldrich Company Ltd. (Dorset, UK). Quinoxaline was supplied by Acros Organics (Geel, Belgium). ARC 68397, ARD 12495 and remacemide hydrochloride were a generous gift from Astra Zeneca R & D Charnwood (Loughborough, UK). Individual stock solutions of pindolol, benzylalcohol and quinoxaline were prepared at a concentration of 0.5 mg/mL in (AN:H2


O) (1:1)(V:V), Quinine


was prepared at a concentration of 0.5 mg/mL in 20 mM potassium dihydrogen-phosphate pH 2.7 in (AN:H2


O)(20:80)(V:V), ARC 68397 was


prepared at a concentration of 0.5 mg/mL in 20 mM potassium dihydrogen - phosphate pH 2.7 in H2


O and all other compounds were


prepared at a concentration of 0.5 mg/mL in H2


O. A mixture of the 22 compounds was


prepared by mixing equal volumes (50 µL) of the individual solutions.


2.2 Instrumentation HPLC separations were performed on an Agilent Technologies 1100 LC with ChemStation v. 9.03 LC software (Agilent Technologies, Cheadle, Cheshire, UK) equipped with a binary pump, a vacuum degasser, cooled autosampler, temperature controlled column compartment and a diode array detector. Data acquisition was performed using the Agilent ChemStation.


2.3 High Performance Liquid Chromatography (HPLC)


Eluent A: 20 mM KH2 PO4 PO4 pH 2.7 in H2 O.


Eluent B: consisted of 3 different eluents B1: 20 mM KH2


(65 : 35 V/V)


B2: 20 mM KH2PO4 (65 : 35 V/V)


B3: 20 mM KH2PO4 pH 2.7 in AN : water pH 2.7 in MeOH : AN : water (32.5 : 32.5 : 35 V/V)


Gradient range: 5->100% eluent B in all experiments


At least 20 column volumes (ca. 30 mL) of the appropriate mobile phase were flushed through the column prior to commencing the testing. ACE 3 C18, 3 µm, 150 × 4.6 mm columns were supplied by Hichrom Ltd. (Reading, UK). All analyses comprised of duplicate 5 µL injections. Other conditions included: flow rate of 1.0 mL/min and detection at 214 and 254 nm. The system dwell volume was experimentally determined as 1.18 mL. Gradients of 15, 30 and 45 min (5%B to 100%B which equates to 3.3 to 65 % total organic) were performed using the different mobile phase compositions as described above. Each gradient run was performed at 40 and 60°C. A typical solvent-


pH 2.7 in MeOH : water


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