Publication InformationLi, B.; Calvet, A.; Casamayou-Boucau, Y.; Morris, C.; Ryder, A.G. (2015) 'Low-content quantification in powders using Raman spectroscopy: a facile chemometric approach to sub 0.1% limits of detection'. Analytical Chemistry, 87 (6):3419-3428. PublisherAmerican Chemical Society Low-content quantification in powders using Raman spectroscopy: a facile chemometric approach to sub 0.1% limits of detection.Boyan Li, Amandine Calvet, Yannick Casamayou-Boucau, Cheryl Morris, and Alan G. Ryder* Nanoscale Biophotonics Laboratory, School of Chemistry, National University of Ireland, Galway, Galway, Ireland.* To whom correspondence should be addressed.Tel: +3539149 2943 Email: alan.ryder@nuigalway.ie ABSTRACT A robust and accurate analytical methodology for low-content (<0.1%) quantification in the solid-state using Raman spectroscopy, sub-sampling, and chemometrics was demonstrated using a piracetam-proline model. The method involved a 5-step process: collection of relatively large number of spectra (8410) from each sample by Raman mapping, meticulous data pretreatment to remove spectral artefacts, use of a 0-100% concentration range partial least squares (PLS) regression model to estimate concentration at each pixel, use of a more-accurate, reduced concentration range PLS model to accurately calculate analyte concentration at each pixel, and finally statistical analysis of all 8000+ concentration predictions to produce an accurate overall sample concentration. The relative prediction accuracy was ~2.4% for a 0.05~1.0% concentration range and the limit of detection was comparable to high performance liquid chromatography (0.03% versus 0.041%). For data pretreatment, we developed a unique cosmic ray removal method and used an automated baseline correction method, neither of which required subjective user intervention and thus were fully automatable. The method is applicable to systems, which cannot be easily analyzed chromatographically such as hydrate, polymorph, or solvate contamination.

Anisotropy resolved multidimensional emission spectroscopy (ARMES) provides valuable insights into multi-fluorophore proteins (Groza et al 2015 Anal. Chim. Acta 886 133-42). Fluorescence anisotropy adds to the multidimensional fluorescence dataset information about the physical size of the fluorophores and/or the rigidity of the surrounding micro-environment. The first ARMES studies used standard thin film polarizers (TFP) that had negligible transmission between 250 and 290 nm, preventing accurate measurement of intrinsic protein fluorescence from tyrosine and tryptophan. Replacing TFP with pairs of broadband wire grid polarizers enabled standard fluorescence spectrometers to accurately measure anisotropies between 250 and 300 nm, which was validated with solutions of perylene in the UV and Erythrosin B and Phloxine B in the visible. In all cases, anisotropies were accurate to better than ±1% when compared to literature measurements made with Glan Thompson or TFP polarizers. Better dual wire grid polarizer UV transmittance and the use of excitation-emission matrix measurements for ARMES required complete Rayleigh scatter elimination. This was achieved by chemometric modelling rather than classical interpolation, which enabled the acquisition of pure anisotropy patterns over wider spectral ranges. In combination, these three improvements permit the accurate implementation of ARMES for studying intrinsic protein fluorescence.

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Intrinsic fluorescence spectroscopy (IFS) measurements for protein structural analysis can be enhanced by the use of anisotropy resolved multidimensional emission spectroscopy (ARMES). ARMES attempts to overcome the tryptophan (Trp) and tyrosine (Tyr) spectral overlap problem and resolve emitting components by combining anisotropy measurements with chemometric analysis. Here we investigate for the first time the application of polarized excitation-emission matrix (pEEM) measurements and Parallel Factor (PARAFAC) analysis to study IFS from an Immunoglobulin G (IgG) type protein, rabbit IgG (rIgG), in its native state. Protein IFS is a non-trilinear system primarily because of Förster resonance energy transfer (FRET). Non-trilinearity is also caused by inner filter effects, and Rayleigh/Raman scattering, both of which can be corrected by data pre-processing. However, IFS FRET cannot be corrected for, and thus here we carefully evaluated the impact of various different data preprocessing methods on IFS data which used for PARAFAC. Care must be taken with data preprocessing and interpolation, as both had an impact on PARAFAC modelling and the recovered anisotropy values, with residual shot noise from the Rayleigh scatter which overlapped the emission blue edge being the root cause.pEEM spectra from thawed rIgG solutions (15-35 °C temperature range) were collected with an expectation being that this temperature range should cause sufficient emission variation to facilitate component resolution but without major structural changes. However, the only significant changes observed were of the overall intensity due to thermal motion induced quenching and this was confirmed by the PARAFAC scores. PARAFAC resolved one major component (>99%) for the emission data (polarized and unpolarized) which mostly represented the large Tyr-to-Trp hetero-FRET process, with a second, very weak component (<1%) apparently a contribution from directly excited Trp emission. PARAFAC scores recovered from normalized pEEM data showed minimal change which was further proof for negligible structural change. The results of this study serves as the starting point for the use of PARAFAC analysis of IFS from IgG type proteins and important processes such as denaturation and aggregation.

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