Review and Optimization of Linearity and Precision in Quantitative HPLC–ELSD with Chemometrics

Sep 30, 2008



High performance liquid chromatography (HPLC) with evaporative light scattering detection (ELSD) continues to grow in popularity as a "quasi-universal" detector as shown in Table I (1–88 please see the complete table in the online version of this article at http://chromatographyonline.findpharma.com/lcgc/issue/issueDetail.jsp?id=15886/). Improvements in ELSD instrument design, including low-temperature evaporation, have been commercialized recently (40). Potential improvements that are currently patent applications (39,41,52) or laboratory experiments (33), including condensation nucleation light-scattering prototypes (42), along with techniques such as volatile mobile phase additives (9,28,32) and higher molecular weight perfluorinated ion-pairing reagents (25,61–68), can further enhance the sensitivity of ELSD. Two overviews of ELSD have been published in LCGC (59,60) in 2003 and 2004, and an excellent review (38) of 20 years of progress in ELSD up to mid-2005 has been published. A more recent review, which includes a graph of the increase in ELSD publications versus time, also has been published (97), so our review of selected publications covering quantitative HPLC–ELSD begins in late 2004.

HPLC–ELSD is a simple, robust technique. It is especially useful for analytes without significant chromophores, or with mobile phases that have UV chromophores (43,51,79). It also has been used as a surrogate mass spectrometry (MS) detector during method development, albeit with lower sensitivity. It is a mass flow sensitive detection method — it responds to larger amounts (quantities) of analytes, and it gives a larger relative response the larger the amount or mass of the analyte passing through the detector — rather than a concentration detection method, as is UV detection. Quantitative HPLC–ELSD has been used recently to study analytes from dried leeches (44), fungi (13), and medicinal plants (7,14–16,18,22,26,27,29,45,48,49,88–92), human intestinal (28) and bronchoalveolar (2) fluids and human breast milk (72), as well as blood (12,57), bile acids (24), underivatized amino acids (44,62), sugars (46,75), lipids (4,9,12,19,24,28,50,55,56,72,76,79), foods (3,4,50,55–56,76,79–87), antibiotics (17,25,54,63–68), pharmaceutical excipients (9,11,20,21,47,58,70), surfactants (73), detergents (74), nutritional supplements (19,30,61), polymers (6,69), and many "traditional" pharmaceutical compounds (1,53,57,77). However, it has been unsuccessful with some combinatorial chemical libraries (5,8,36) to date. As a vote of confidence in quantitative ELSD, in 2006, NIST (30) published two cross-validated quantitative HPLC methods (LC–MS and HPLC–ELSD with isocratic elution) for the analysis of its Standard Reference Material 3280, Biotin. Although the LC–MS method was far superior in terms of quantitative sensitivity (LOD, LOQ), both methods produced similar quantitative results. NIST concluded that either method alone could be used potentially as a reference method for the quantification of biotin in multivitamin tablets.