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CHROMATOGRAPHY
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2DLC has been used to solve the separation challenges that
cannot be achieved by one-dimensional HPLC (1DLC).
Figure 4. The 2D (top) and 3D (bottom) profi les of a
specifi city of an established 1DLC method. In this case, the established method is used in the 1st D, and orthogonal methods are used in the 2nd D to screen the appropriate sample set to check if there is any peak that might be missed by the established method. The 1DLC method may need to be updated based on the fi ndings from 2DLC. The other approach is to use 2DLC in the situation that a 1DLC method is in lack of specifi city, and then a 2nd D separation is used to compensate the method specifi city.
3. Impurity and Degradation Profi ling
Forced degradation samples are typically more complicated than the clinical materials. Rapid comprehensive impurity profi ling is highly desirable in evaluating the stability of drug candidates, as well as developing a stability-indicating HPLC method. Using UHPLC in the 2nd D is highly eff ective to facilitate the comprehensive impurity profi le screening [19].
Figure 4 shows the separation of a pharmaceutical material with structure-related impurities by using on-line comprehensive mode 2DLC. A commercial software was used to generate the profi le plots. Diff erent columns and mobile phases were used in each dimension to maximize the method orthogonality and consequently peak capacity in the 2D space.
Hyphenated detection is highly desirable for complete sample profi ling. As indicated earlier, some of the components or impurities may not have UV-chromophores. Multi-dimensional separation coupled with hyphenated detection is a powerful way to get a complete profi le of a sample [18, 20]. Our lab uses UV-CAD-MS detection in 2DLC work for impurity and degradation profi ling [9].
42 | | November/December 2013
pharmaceutical material with structure-related impurities using comprehensive 2DLC
4. Quantifi cation by 2DLC
Figure 5 shows the 2DLC separation and quantifi cation of a low- level impurity that co-eluted with the main peak in the fi rst dimension. Single complete heart-cutting of the whole peak in the 1st dimension was employed. The single complete heart-cutting is a preferred separation mode for quantifi cation because there is no sample loss and data analysis is straightforward. The impurity spiked at levels from 0.05% to 10% was sensitively quantifi ed with good linearity and accuracy. Comprehensive 2D mode quantifi cation using advanced software was reported by Reichenbach et al. and Mondello et al. [21-23].
5. 2DLC-MS
Low-level impurities that co-elute with or elute close to the major component or stronger ionization species could be signifi cantly suppressed, and thus undetected. 2DLC-MS is an eff ective way to reduce signal suppression caused by matrix interference from strongly ionized components, as well as non-volatile buff ers, such as phosphate buff ers. 2DLC-MS enables using phosphate buff er in the 1st dimension to achieve the desired chromatographic performance, while using MS compatible mobile phases in the 2nd D to obtain the MS structural information [9, 24]. Additionally, 2DLC-MS gives unambiguous peak tracking. Mass spectrometry sometimes is treated as a “third added dimension” to 2DLC to generate more information of compounds that may not be fully resolved chromatographically [11, 25].
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