Daniel N. Hayes is associate principal scientist at Cambrex High Point Analytical Research and Development.

Daniel N. Hayes

Analytical scientists in the pharmaceutical industry are tasked with ensuring the highâperformance liquid chromatography (HPLC) purity methods that they develop and validate provide complete separation of compounds, and this work can yield unexpected findings that lead to improvements in methods and manufacturing processes.

	In the following case study, peak spectral analysis was used as an analytical tool to determine the cause of peak purity failure during HPLC method validation. This analysis resulted in the separation and identification of a previously unknown impurity, and the development and validation of a new HPLC method that allowed this impurity to be separated and independently determined.

	Early phase HPLC purity/assay validations typically include the following components, based on International Council for Harmonization (ICH) Q2(R1) guidelines (1): 

		Specificity-the ability of a method to separate the analyte, or compound of interest, from other components expected to be present in the sample 

		Linearity-the ability of a method to provide results within a specified concentration range that are directly proportional to concentration of the analyte 

		Accuracy-the ability of a method to produce assay results near the true value 

		Precision-the ability of a method to give consistently reproducible results 

		Detection limit-the lowest concentration of analyte that can be detected 

		Quantitation limit-the lowest concentration of analyte that can be consistently measured 

		Forced degradation-exposing the drug substance to severe environmental and chemical stresses to rapidly evaluate material stability. 

	This final component is performed according to the ICH guideline Q1A(R2), Section 2.1.2, and will demonstrate that a method is stability-indicating (2).

	During a recent validation on an API that was being manufactured for a client, forced degradation studies were performed on the API using the client-supplied method. During the study, the HPLC-diode array detector (DAD) peak purity analysis of the analyte peak failed for all conditions, including the control. Failure was not observed during pre-validation exercises, which were performed on a different, previous batch of API. This indicated that a new, unknown impurity could be co-eluting with the analyte peak.

	Using Chemstation software (Agilent), the ultraviolet (UV) spectra of the analyte peak collected by the diode array are analysed for spectral purity, giving the purity factor. A purity factor of ≥990 generally indicates a peak is pure and suggests no other peaks co-elute.

Peak purity analysis results for an older lot are shown in 

, where the upper-left quadrant shows the chromatogram, with its baseline enlarged for detail. The green bar under the peak indicates the peak has a purity factor above the threshold of 990, with an observed result of 999.433, which is shown in the upper-right quadrant. The lower-right quadrant shows the analysis of spectral purity within the peak, as indicated by the wide line that remains within the green bar, and the lower-left quadrant includes the peak spectral results taken from five points across the peak. 

 show the new, failing, lot of API, which has a purity factor of 835.895. The wide line beneath the peak in the lower-right quadrant indicates the presence of a spectral impurity on the tail of the peak. A careful review of the spectra in the lowerâleft quadrant indicates that one of the spectral plots differs from the others, as it is shifted to the right, and this corresponds to the co-eluting impurity. This shift pattern would be useful in developing a method for the new impurity, as it implied that the impurity responded better than the analyte at a wavelength of about 290 nm.

	The three-dimensional spectral results of the analyte peak were also modelled, and the area around the baseline enlarged for detail. The spectral map of the pure analyte peak obtained from the older lot is shown in 

, which plots wavelengths between 240 and 310 nm against time (11.6 minutes to 10.9 minutes, right to left). Note that the peak is reversed from the normal chromatogram to allow the spectral details to be observed. The spectral map of analyte peak from the impure lot is shown in 

 (using the same axis scale), and the impurity shows up as a ‘bulge’ on the main peak.

	The method that was being validated was performed at a wavelength of 260 nm, but when the impure analyte peak was evaluated at 290 nm, it became clear that there were two distinct compounds. Overlays of the impure analyte peak at 260 nm and 290 nm wavelengths were created, as shown in 

. The later peak (right, red, 290 nm) shows the location of the bulge in the three-dimensional model in 

 indicated that it might be possible to separate the later-eluting new compound from the analyte, based on the differences in properties of the two molecules. As the identity of the new impurity was unknown, data from the manufacturing campaign of the impure batch were reviewed to check for information that might point to the source of the impurity. HPLC data from the manufacturing batch record indicated that the impurity was introduced early in the manufacturing. The data also revealed that the method used for in-process checks was able to separate out the impurity from an intermediate material with the use of a different column and mobile phase.

	Isolated intermediate material from the manufacturing campaign that produced the impurityâcontaining API was analysed using the in-process HPLC method, column, and mobile phase. Although the method was not optimized for this separation, the impurity appeared as a peak before the main intermediate peak, and was resolved from the intermediate peak. Additional analysis showed a spectral profile similar to the impurity in the final compound. The HPLC results are shown in 

. Spectral results are shown in an overlay in 

, where the impurity has a spectral maximum of approximately 274 nm, compared to the intermediate, which has a spectral maximum of 260 nm. 

	Additional method development was performed on the API, leading to a final set of conditions that successfully separated out the impurity. Although the intermediate and the API have a similar structure (the intermediate molecule contributes a significant structural fragment to the API), the API is less polar, and elutes much later than the intermediate shown in the previous figures. Using the final method conditions, the impurity elutes first, as shown in the example chromatogram in 

, and the API peak was determined to be spectrally pure. 

	Spectral results from the peaks in the example chromatogram are shown in 

, where the impurity peak has a spectral maximum at 274 nm. Because the impurity’s spectral maximum was different from the API’s maximum at 260 nm, a response factor correction for the impurity was empirically determined and added to the new method. This new method was subsequently validated and implemented prior to release of the next batch (a clean-up of the impure batch of API), demonstrating the versatility and utility of spectral analysis as an analytical tool. 

Validation of Analytical Procedures: Text and Methodology

, (ICH, November 2005).
	2. ICH, Q1A (R2) 

Stability Testing of New Drug Substances and Products

	When referring to this article, please cite it as D. Hayes, " Utilizing Spectral Analysis in HPLC Diode Array to Discover Impurities,"

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Articles

Investigation of peak purity failure during HPLC method validation led to discovery of a co-eluting impurity under the main peak. Spectral analysis, including three-dimensional modelling, was used to characterize the peak, leading to the development of a new HPLC method for analysing impurity content.