At-Line Monitoring of the Extraction Process of Rosmarini Folium via Wet Chemical Assays, UHPLC Analysis, and Newly Developed Near-Infrared Spectroscopic Analysis Methods

Abstract

The present study demonstrates the applicability of at-line monitoring of the extraction process of Rosmarinus officinalis L. leaves (Rosmarini folium) and the development of near-infrared (NIR) spectroscopic analysis methods. Therefore, whole dried Rosmarini folium samples were extracted by maceration with 70% (v/v) ethanol. For the experimental design three different specimen-taking plans were chosen. At first, monitoring was carried out using three common analytical methods: (a) total hydroxycinnamic derivatives according to the European Pharmacopoeia, (b) total phenolic content according to Folin-Ciocalteu, and (c) rosmarinic acid content measured by UHPLC-UV analysis. Precision validation of the wet chemical assays revealed a repeatability of (a) 0.12% relative standard deviation (RSD), (b) 1.1% RSD, and (c) 0.28% RSD, as well as an intermediate precision of (a) 4.1% RSD, (b) 1.3% RSD, and (c) 0.55% RSD. The collected extracts were analyzed with a NIR spectrometer using a temperature-controlled liquid attachment. Samples were measured in transmission mode with an optical path length of 1 mm. The combination of the recorded spectra and the previously obtained analytical reference values in conjunction with multivariate data analysis enabled the successful establishment of partial least squares regression (PLSR) models. Coefficients of determination (R²) were: (a) 0.94, (b) 0.96, and (c) 0.93 (obtained by test-set validation). Since Pearson correlation analysis revealed that the reference analyses correlated with each other just one of the PSLR models is required. Therefore, it is suggested that PLSR model (b) be used for monitoring the extraction process of Rosmarini folium. The application of NIR spectroscopy provides a fast and non-invasive alternative analysis method, which can subsequently be implemented for on- or in-line process control.

Keywords: Folin–Ciocalteu; near-infrared spectroscopy; phytoextraction; total hydroxycinnamic derivatives; ultra-high performance liquid chromatography.

Figure 1

Chromatograms of (a) rosemary extract (red line) in 70% v/v ethanol (50 g/L) after 3 h continued stirred extraction, and (b) rosmarinic acid reference solution (black line), measured at 330 nm.

Figure 2

(a) Raw near-infrared (NIR) spectra of all 90 samples; (b) section from the raw NIR spectra showing the wavenumber region used for PLSR model calculation; (c) first derivate (13 smoothing points) and standard normal variate (SNV)-transformed NIR spectra region used for total hydroxycinnamic derivatives (THCD) in mg/kg and gallic acid equivalents (GAE)% PLSR model calculation; and (d) second derivate (23 smoothing points) and SNV-transformed NIR spectra region used for rosmarinic acid (RA)% PLSR model calculation.

Figure 3

Predicted versus reference plots (left column) and regression coefficient plots (right column) for the best test-set validated PLSR models for: (a) and (b) THCD in mg/kg, (c) and (d) GAE%, and (e) and (f) RA%.

Figure 4

Monitoring of the extraction process of Rosmarini folium via (a) Folin–Ciocalteu reference analysis and (b) NIR spectroscopy.