By Jerry “Junior” Mizell
Associate Director, Analytical Services
Diode-Array Detection (DAD) or Photodiode-Array Detection (PDA) is an analytical technique that can be used to determine the purity of an analyte or related impurity peak eluting during an HPLC separation.
The use of DAD to demonstrate method specificity is allowed by the International Council for Harmonisation (ICH) guideline Q2(R1).
The diode array detector uses the same principles of operation as a variable wavelength detector (VWD). However, the array of diodes enables simultaneous acquisition across a range of wavelengths, rather than just a single one. Spectral acquisition used in conjunction with a chromatographic separation is a technique that allows the analyst to collect multiple spectra across a chromatographic peak. Once these spectra have been collected from an HPLC analysis, they can be used to perform an assessment of spectral peak purity mathematically and possible identification.
During a routine sample analysis, an impurity peak eluting at approximately 52 minutes was observed in chromatography for a sample preparation. This peak had a similar retention time of a suspected degradant and positive identification analysis using DAD was begun. In order to determine what the unknown impurity was, sample and impurity marker solutions were analyzed using the validated stability indicating method employing Diode Array Detection. From spectral data collected, an attempt was going to be made to identify the impurity observed in the sample preparation.
A chromatogram of the sample preparation and extracted UV/VIS spectra are shown in Figures 1 and 2, respectively. A chromatogram of the impurity preparation and extracted UV/VIS spectra are shown in Figures 3 and 4, respectively.
Figure 1. Chromatogram of sample preparation containing impurity at 52 minutes
Figure 2. Extracted UV spectra for chromatographic peaks shown in Figure 1
Figure 3. Impurity marker preparation of suspected unknown impurity
Figure 4. Extracted UV/VIS spectrum of impurity marker preparation
At this point, the extracted UV/VIS spectrum for the unknown impurity shown in Figure 2 was compared to the UV/VIS spectrum shown in Figure 4 for the known impurity. Both the unknown impurity and the known impurity exhibited a similar UV/VIS profile with both spectra containing primary UV maxima in the 261 nm to 263 nm range.
It is noted that the secondary UV maximum in Figure 2 is due to non-baseline resolution between the impurity and the active. Along with retention time, this confirms the identity of the impurity in the sample preparation.
This is a very simple and straightforward example of how Diode-Array Detection (DAD) can be used to identify unknown peaks when observed in chromatography.
Junior Mizell manages a team of group leaders responsible for conducting analytical laboratory services for Metrics Contract Services clients. Among other duties, he reviews and approves regulatory submissions; reviews specifications, methodology validation, stability testing, analytical investigations and release reports for raw and in-process materials, active pharmaceutical ingredients and finished products; and reviews and approves method validation protocols and reports. A member of the American Chemical Society and AAPS, Mr. Mizell joined Metrics in 1995 after working at Burroughs Wellcome, now part of GSK. He holds a bachelor’s of science degree in chemistry from East Carolina University.