Toward Single-Calibrant Quantification in HPLC. A Comparison of Three Detection Strategies:  Evaporative Light Scattering, Chemiluminescent Nitrogen, and Proton NMR

Abstract

There is an urgent need for detection technologies that enable accurate and precise quantification of solutions containing small organic molecules in a manner that is rapid, cheap, non-labor-intensive, readily automated, and without a requirement for specific analyte standards. We provide a theoretical analysis that predicts that the logarithmic nature of the working domain of the evaporative light-scattering detector (ELSD) will normally bias toward underestimation of chromatographically resolved impurities, resulting in an overestimation of analyte purity. This analysis is confirmed by experiments with flow injection analysis (FIA) and gradient reversed-phase high performance liquid chromatography (RP-HPLC). Quantification is further compromised by the dependence of response parameters on the matrix composition and hence on the retention time of the analyte. Attempts were made to ameliorate these problems by using the response surface of a single compound to calibrate throughout the HPLC gradient. A chemiluminescent nitrogen detector (CLND) was also used in a similar manner, and the performance of the two techniques were compared against those of each other and that of a reference standard technique. A protocol for this purpose was developed using proton nuclear magnetic resonance (1H NMR) and the ERETIC method to enable quantification by integrating proton signals. The double-blind comparison exercise confirmed molar nitrogen CLND response to be sufficiently stable and robust across a methanol gradient to be used with a single external nitrogenous calibrant to quantify nitrogen-containing compounds of known molecular formula. The performance of HPLC-CLND was very similar to that of NMR, while that of HPLC-ELSD was seen to be significantly worse, showing it to be unsuitable for the purpose of single-calibrant quantification. We report details and experience of our use of RP-HPLC-CLND-MS to characterize and quantify small amounts of solutions of novel compounds at nominal levels of 10mM in microtiter plate (MTP) format.

Streamlined approach to high‐quality purification and identification of compound series using high‐resolution MS and NMR

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Accurate quantification of modified cyclic peptides without the need for authentic standards

“…[41][42] Proton NMR quantification (qNMR) by ERETIC is a non-destructive and rapid way of providing accurate analyte concentrations 43 by using an indirect internal reference signal that represents a known concentration. This averts the need to determine a compoundspecific response factor, 44 making qNMR an accurate and straightforward technique for quantification. The drawbacks to this method are that it requires relatively pure samples of large size that would allow sufficient signal to noise ratio (>150:1) 9 and an internal certified reference material.…”

Development of a Reversed-Phase HPLC Impurity Method for a UV Variable Isomeric Mixture of a CRF Drug Substance Intermediate with the Assistance of Corona CAD

“…A recently developed detection technology, Corona charged aerosol detection (CAD), has been used as an analytical tool for determining relative response factors for unknown impurities with divergent UV responses because it can provide constant response for different compounds, especially those of similar structure [2][3][4]. As an aerosol-based "universal" detector, Corona CAD has become increasingly useful in the analytical chemistry field [5][6][7][8][9][10].…”