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CHROMATOGRAPHY
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Figure 2. Challenges in pharmaceutical analysis illustrated by a simulated chromatogram
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Impurities with no UV-chromophores are easily missed by commonly-used HPLC-UV method.
Phosphate buff ers are frequently used for better chroma- tographic performance, but they are not MS compatible.
• Multiple HPLC methods are needed for chiral compounds, especially those with multiple chiral centers.
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Some strongly-retained components may not elute in the method run time.
For these challenges, while the overly-retained components are manageable by holding the organic solvent at higher percentage for a longer time, others are diffi cult to overcome by a conventional, one- dimensional separation.
Figure 1. (a) 2DLC peak capacity illustrated by retention plane in a perfect case of two dimensions are totally orthogonal (b) The correlation of the retention factors k’ of method A and method B (c) The correlation of the retention factors k’ of method X and method Y
Challenges in HPLC Analysis of Pharmaceuticals
Bringing well-characterized, high quality drug to patients quickly is a major task in pharmaceutical analysis. 2DLC has been used to solve the separation challenges that cannot be achieved by one-dimensional HPLC (1DLC), and to increase the separation speed of 1D separation. Figure 2 illustrates some key challenges of using conventional HPLC in pharmaceutical analysis. These are listed below.
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Peak co-elution is a major concern. Peaks could co-elute with API and other impurities or components.
Poorly retained peaks that eluted in the solvent front are frequently overlooked.
40 | | November/December 2013
It is important to note that impurity levels are typically low in pharmaceutical materials since the safety bar is high and the regulatory requirements are strict. This is diff erent from proteomics and natural products, where multiple components may present at relatively higher levels. The diff erent sensitivity requirement and number of components make a diff erence when choosing the best 2DLC mode for analysis. Pharmaceutical materials are typically made from high purity starting materials and strictly-controlled manufacturing process are used to ensure the impurities are controlled at low levels. Multiple factors must be considered when setting the criteria for impurity control, but generally ICH Q3A [5] guidance recommends setting 0.05% as the reporting limit, 0.10% as the identifi cation limit and 0.15% as the quantifi cation limit for drug substance, and ICH Q3B [6] is referred to for drug product analysis. In the case of genotoxic impurities, ppm level control may be required.
2DLC Separation Modes
Heartcutting 2DLC Heartcutting 2DLC is an eff ective way to resolve pharmaceutical challenges [7-11]. It typically transfers one or two fractions of interest
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