## 2 Chromatographic Techniques for Carotenoids Analysis
### High-Performance Liquid Chromatography
 & 0.9 & UV-Vis & 45 & 51–300 & 155–909 & 83–107 & [13] \\ \hline Sweet red peppers (_Capsicum autumn_ & Latetin, Zexanthin, &
\begin{tabular}{c} C30 YMC column \\ \(\mathrm{\beta}\)-Crytoplasmin, \\ \(\mathrm{\beta}\)-Crytoplasmin, \\ \(\mathrm{\alpha}\)-Cornetne, \\ \(\mathrm{\beta}\)-Crytoplasmin, \\ \(\mathrm{\alpha  
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HPLC is often coupled with detection systems such as ultraviolet-visible (UV-Vis) spectroscopy and mass spectrometry (MS). UV-Vis detection provides a cost-effective method for quantification based on absorbance spectra, which typically exhibit two to three maxima between 400-550 nm [12; 13; 14; 15; 16; 17; 18; 19; 20; 21; 22; 23; 24; 25]. In contrast, HPLC-MS and tandem mass spectrometry (HPLC-MS/MS) allow for precise identification and structural characterization of carotenoids with parent ions typically ranging between 536-600 Da [13; 22; 23; 24; 25]. Most applications usually employ atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) as ionization sources for MS. APCI is widely used for nonpolar carotenoids (e.g., \(\beta\)-carotene, \(\alpha\)-carotene, and lycopene) due to its ability to efficiently ionize lipophilic compounds [22; 23; 24]. This technique operates at higher temperatures, facilitating the ionization of volatile compounds while minimizing fragmentation, often producing a dominant [M+H]+ molecular ion [30]. In contrast, ESI is preferred for oxygenated carotenoids (xanthophylls), such as lutein, zeaxanthin, and violaxanthin, as it provides softer ionization, reducing fragmentation and yielding intact molecular ions in both positive ([M+H]+) and negative ([M-H]-) modes [25]. ESI is particularly advantageous for carotenoids with hydroxyl or epoxy groups, improving detection sensitivity in complex food matrices [25; 30]. While both techniques offer valuable insights into carotenoid composition, APCI is better suited for nonpolar species, whereas ESI excels in analyzing more polar carotenoids. The selection of ionization mode should be carefully considered based on the target analytes' structural characteristics and the food matrix's complexity [30]. Advanced MS techniques, such as quadrupole time-of-flight (QTOF) and Orbitrap MS, significantly enhance carotenoid determination by enabling high-resolution separation and structural elucidation [31; 32]. QTOF-MS with ion mobility spectrometry allows rapid differentiation of isomeric carotenoids based on collision cross-section values, distinguishing cis- and all-trans forms within milliseconds while minimizing ionization-induced isomerization [32]. Meanwhile, Orbitrap MS provides exact mass measurements and detailed fragmentation pathways, offering insights into functional groups, polyene backbones, and double-bond locations [31]. Although some isomeric carotenoids remain challenging to differentiate solely by fragmentation, combining these MS techniques with optimized chromatography, diode array detection (DAD), and nuclear magnetic resonance (NMR) enhances accuracy and structural confirmation. Further, it improves the accuracy of detecting trace levels of carotenoids.