Ultra-high-performance liquid chromatography–tandem triple quadrupole mass spectrometry (UHPLC-MS/MS)
-
UHPLC, an advanced form of HPLC known for its enhanced capabilities in terms of resolution, analysis speed, and sensitivity
[71][38], has paved the way for more sophisticated analytical methodologies. One such prominent application is the utilization of UHPLC-MS/MS for the precise determination of vitamin A levels in biological tissues, particularly in blood samples
[72][39]. The approach demonstrates rapidity, accuracy, and heightened sensitivity, coupled with straightforward preprocessing procedures. Mass spectrometric analysis is carried out in the positive ion mode using the multiple reaction monitoring mode, while quantification is facilitated through the utilization of the internal standard method
[72][39].
Generally, chromatographic techniques offer a multitude of benefits in the assessment of vitamin A, including superior sensitivity and specificity, adaptability across various sample types, and reduced sample preparation requirements
[73,74][40][41]. Despite these advantages, chromatography methodologies also exhibit certain drawbacks, such as prolonged analysis time, significant financial investment, and susceptibility to matrix effects
[45,73][11][40].
5. Nuclear Magnetic Resonance (NMR) Spectroscopy
NMR spectroscopy is a powerful analytical technique employed in the determination of retinol, leveraging the magnetic properties of hydrogen nuclei
[75][42]. Grounded in the principle of resonance, it involves the matching of electromagnetic radiation frequencies to specific energy transitions of the nuclei, thereby facilitating the comprehensive analysis of the chemical structure and environment of retinol
[76][43]. The phenomenon of chemical shift, denoting the displacement of the resonance frequency relative to a standard reference, plays a critical role in discerning the functional groups and molecular constituents of retinol
[75][42]. Additionally, the integration of NMR signals allows for quantitative insights into the relative abundance of distinct types of hydrogen atoms within various retinol molecules
[77][44].
NMR spectroscopy offers several advantages for the precise quantification of vitamin A in samples, owing to its high sensitivity, versatility, and dependable analytical capabilities
[78][45]. However, it comes with certain drawbacks, including significant financial costs and the requirement for specialized equipment and expertise
[79][46].
6. Near-Infrared Spectroscopy (NIRS)
NIRS is a non-invasive analytical technique widely used for studying the molecular structure, composition, and concentration of various substances
[80][47]. It functions on the basis of the unique light absorption properties of different molecules at specific wavelengths, enabling the identification and quantification of compounds of interest. Near-infrared light, falling within the 800- to 2500-nanometer wavelength range, is particularly favored for its ability to penetrate biological samples effectively, making it a suitable tool for analyzing complex biological materials
[81][48].
In practical applications, NIRS involves the transmission of near-infrared light through a sample, followed by the measurement of the absorbed or reflected light
[82][49]. Through the analysis of absorption and reflection patterns, researchers can discern the presence and concentration of specific compounds or functional groups within the sample. Notably, NIR spectroscopy has also found utility in the examination of retinol within vitamin compositions
[38][50].
NIRS is recognized as a non-destructive, expedient, and cost-effective technique for determining retinol in diverse samples
[83,84][51][52]. Nevertheless, its sensitivity and accuracy are relatively restricted compared to standard reference methods such as HPLC, necessitating calibration with these methods
[85][53]. It is imperative to note that NIRS might not be universally applicable across all sample types, as certain samples could potentially introduce interference during the analysis process
[83][51].
7. Enzyme-Linked Immunosorbent Assays (ELISAs) for Biological Tissues
ELISA kits are used to measure the amount of a specific substance, such as vitamin A, in a sample. The kits use antibodies to detect and measure the substance, and there are different types of ELISA kits available depending on the specific needs of the experiment
[86][54]. For example, the competitive EIA ELISA kit uses a competitive inhibition method to measure the amount of vitamin A in a sample
[87,88][55][56], while the sandwich ELISA kit uses a sandwich method
[89][57]. ELISA kits can be used to measure vitamin A in a variety of biological tissues, including plasma, serum, tissue homogenates, cell lysates, and cell culture supernatants
[90][58].
ELISA kits for measuring vitamin A in biological tissues offer the advantages of high sensitivity and user-friendliness for processing multiple samples simultaneously, but they can be time-consuming and may lack sensitivity for detecting low levels of vitamin A
[91][59]. Cross-reactivity with other substances in the sample can also lead to occasional false positives.