« CHROMATOGRAPHY
from the fi rst dimension (1st D) to the second dimension (2nd D). The instrumentation setup is simple and data analysis is straightforward. A distinct advantage of heartcutting is the 2nd D method run time is independent of the sampling rate from the 1st D; therefore a relatively longer method can be used to increase resolution and sensitivity. These factors are particularly important for trace level analysis. The tradeoff is longer separation time especially when multiple peaks are of interest. To mitigate this issue, we developed an on-line multi- heartcutting 2DLC system [9]. In pharmaceutical analysis, normally only certain peaks or certain regions in the 1st D are of interest and it is not necessary to transfer the entire 1st D eluent to the 2nd D. The on-line multi-heartcutting mode allows greater fl exibility.
Comprehensive 2DLC
Comprehensive-mode 2DLC typically transfers the entire 1st D eluent in fractions into the 2nd D. A comprehensive profi le of the sample can be obtained in a short time period by using this mode. It is popular for samples containing a large number of relatively high-level components, such as proteomics, natural products and polymers. Stoll gave an excellent review of recent progress in online comprehensive 2DLC for non-proteomic applications [12]. The applications of on-line comprehensive 2DLC in pharmaceutical analysis remain limited in the literature although this has increased in recent years [13-15] .
In comprehensive 2DLC mode, the 2nd D separation must be very fast to accommodate the 1st D sampling rate and avoid sample loss. For a fast method, for instance with a 24-second cycle time, the chromatographic performance such as resolution and sensitivity can be signifi cantly reduced, especially for low level impurities. The introduction of UHPLC and new column technologies such as sub-2 µm and core-shell (or fused-core) particles greatly improved this issue. The selective comprehensive separation mode reported by Stoll and co-workers is an eff ective way to resolve this issue [16-17]. Elevated temperature is also a popular approach to increase the separation speed, but it is not preferred for pharmaceutical tests due to the uncertainty of compounds stability under high temperature.
On-line comprehensive 2DLC can generate a large amount of data in a short time, but the data analysis is challenging. The background noise from the valve switching can signifi cantly interfere with the low- level peak signal. Improved hardware as well as advanced software is critical to fully utilize the separation power of on-line comprehensive 2DLC in pharmaceutical analysis for complex samples, such as forced degradation studies.
Offl ine 2DLC
Offl ine 2DLC transfers the fractions from the 1st dimension to the 2nd dimension not in real time. It can be carried out on a conventional 1D HPLC. The fractions are collected manually or by a fraction collector, and then re-injected to the same or diff erent HPLC for further analysis, typically with a diff erent column or mobile phase. It can be operated in either heart-cutting or comprehensive modes. Because of its simplicity and fl exibility of the sample fraction treatment, such as evaporating the solvent to concentrate the sample, it is still popular in research [18- 19], as well as in practical use. Offl ine 2DLC is popular in pharmaceutical
labs for qualitative work, e.g. separate matrix or buff er interference in the 1st D and do MS structure elucidation in the 2nd D. Apparently, it is more time- and labor-consuming.
Applications of 2DLC in Pharmaceutical Analysis
1. Peak Co-Elution and Peak Purity
A major application of 2DLC is to separate peaks that co-eluted in the conventional HPLC methods [9, 10, 15]. This is the basis for peak purity assessment, method specifi city, stability-indicating method development and many other applications in the below sections. Figure 3 shows the 2DLC separation of a stability sample of an active pharmaceutical ingredient (API) spiked with three process- related impurities. The 1st D used a typical reversed-phase column and phosphate buff er/acetonitrile gradient separation. The API and three impurities were separated. Each of the four peaks were further evaluated in the 2nd D by using on-line multi-heartcutting 2DLC technology to evaluate if there are any hidden impurities. The 2nd D method used a diff erent type of reversed-phase column and formic acid/ACN gradient separation. Each of the fractions were found to have small peaks that were hidden by co-elution in the 1st D.
2. Method Specifi city and Stability-indicating Method Development
Method specifi city is a component of method development and validation. Specifi city is the ability to assess the analyte unequivocally in the presence of impurities, degradants, the matrix, etc. There are two ways to use 2DLC for method specifi city and similarly for stability- indicating method development.
One approach is to check the
Figure 3. Peak co-elution analysis using 2DLC
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