doi:10.1016/j.chroma.2006.04.065 
Copyright © 2006 Elsevier B.V. All rights reserved.
Application of a column selection system and DryLab software for high-performance liquid chromatography method development
Ryan M. Kriskoa, Kieran McLaughlinb, 
, 
, Michael J. Koenigbauerb and Craig E. Luntea
aDepartment of Chemistry, University of Kansas, Mallott Hall, Lawrence, KS 66045, USA
bPharmaceutical and Analytical Research & Development, AstraZeneca, Wilmington, DE 19805, USA
Received 23 March 2005;
revised 14 April 2006;
accepted 25 April 2006.
Available online 18 May 2006.
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Abstract
This paper describes a strategy for the development of chromatographic methods for drug candidates based upon the use of simple MS compatible mobile phases and optimization of the chromatographic selectivity through variations of the stationary phase and mobile phase pH. The strategy employs an automated column selection system and a series of HPLC columns, varying in hydrophobicity and silanol activity, in combination with DryLab software to develop chromatographic methods for the separation of mixtures of bupivacaine and its metabolites; acidic, basic, and neutral compounds; and atenolol, nitrendipine, and their degradation products.
Keywords: HPLC; DryLab; Automated method development
Fig. 1. Structures of desbutylbupivacaine (1), 4′-hydroxybupivacaine (2), 3′-hydroxybupivacaine (3) and bupivacaine (4).
Fig. 2. Structures of lidocaine (1), mepivacaine (2), prilocaine (3), bupivacaine (4), amitriptyline (5), prednisolone (6), naproxen (7) and ibuprofen (8).
Fig. 3. Structures of p-hydroxyphenylacetamide (1), atenolol (2), p-hydroxyphenyl acetic acid (3), atenolol acid (4), nitropyridine (5) and nitrendipine (6).
Fig. 4. Simulated DryLab chromatograms of bupivacaine and it's desmethyl, 3′-hydroxybupivacaine and 4′-hydroxybupivacaine metabolites on the Prodigy (a), Symmetry (b), Inertsil C8-3 (c), Symmetry Shield (d), Ace C8 (e), Zorbax Eclipse XDB (f), Hypersil BDS (g) and XTerra MS C8 (h) columns.
Fig. 5. Experimentally verified isocratic separation of bupivacaine and it is desmethyl, 3′-hydroxybupivacaine and 4′-hydroxybupivacaine metabolites using the chromatographic conditions as predicted by DryLab on the Prodigy (a), Symmetry (b), Inertsil C8-3 (c), Symmetry Shield (d), Ace C8 (e), Zorbax Eclipse XDB (f), Hypersil BDS (g) and XTerra MS C8 (h) columns. Peak identities: desbutylbupivacaine (1), 4′-hydroxybupivacaine (2), 3′-hydroxybupivacaine (3) and bupivacaine (4). The USP tailing factor of bupivacaine is indicated on the left hand side of each chromatogram. Peak order is the same for all chromatograms.
Fig. 6. Gradient chromatograms at pH 2.7 of lidocaine (1), mepivacaine (2), prilocaine (3), bupivacaine (4), amitriptyline (5), prednisolone (6), naproxen (7) and ibuprofen (8) on the XTerra MS C8 (a), Prodigy (b), Inertsil C8-3 (c) and Symmetry (d) columns. Peak order is the same for all chromatograms.
Fig. 7. Gradient chromatograms at pH 10.5 of lidocaine (1), mepivacaine (2), prilocaine (3), bupivacaine (4), amitriptyline (5), prednisolone (6), naproxen (7) and ibuprofen (8) on the Zorbax Extend C18 (a), XTerra MS C8 (b) and XTterra Phenyl (c) columns. Peak order is the same for all chromatograms.
Fig. 8. Gradient chromatograms of atenolol, nitrendipine and their degradation products on a Hypersil BDS (a) and Ace C8 (b) columns at a mobile phase of pH 2.7 and on a Zorbax Extend C18 column at a mobile phase of pH 10.5 (c). Peak identities: p-hydroxyphenylacetamide (1), atenolol (2), p-hydroxyphenyl acetic acid (3), atenolol acid (4), nitropyridine (5) and nitrendipine (6).
Fig. 9. Gradient chromatograms of atenolol (1 mg/ml), nitrendipine and their degradation products on a Hypersil BDS (a) and Ace C8 (b) columns at a mobile phase of pH 2.7. Peak identities: p-hydroxyphenylacetamide (1), atenolol (2), p-hydroxyphenyl acetic acid (3), atenolol acid (4), nitropyridine (5) and nitrendipine (6).
Table 1.
Column selectivity parameters [15]
a H, column hydrophobicity.
b S, accessibility of the stationary phase to insertion of “bulky” molecules (“steric interaction,” similar to “shape selectivity”).
c A, column hydrogen-bond acidity due to non-ionized silanols.
d B, column hydrogen-bond basicity due to basic groups in the stationary-phase (e.g., embedded polar groups).
e C (2.8), column cation-exchange activity due to ionized silanols (at pH 2.8).
f C (7.0), column cation-exchange activity due to ionized silanols (at pH 7.0).
g Information not currently available for this column.
Table 2.
Retention time comparison [r = texp/tsim] for simulated, tsim, DryLab (Fig. 4) and experimentally, texp, verified (Fig. 5) separations of (1) desbutylbupivacaine, (2) 4′-hydroxybupivacaine, (3) 3′-hydroxybupivacaine and (4) bupivacaine. Column identifications (a–h) are as described in Fig. 4
Table 3.
Gradient program for Fig. 8a–c
Mobile phase for Fig. 8a and b: A = water containing 0.1% formic acid (pH 2.7) and B = acetonitrile containing 0.1% formic acid. Mobile phase for Fig. 8c: A = water containing 0.1% ammonium hydroxide (pH 10.5) and B = acetonitrile containing 0.1% ammonium hydroxide.
a Fig. 8a.
b Fig. 8b.
c Fig. 8c.
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