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Shastak, Y.; Pelletier, W.; Kuntz, A. Quantitative Vitamin A Analysis Approaches. Encyclopedia. Available online: https://encyclopedia.pub/entry/54936 (accessed on 04 July 2024).
Shastak Y, Pelletier W, Kuntz A. Quantitative Vitamin A Analysis Approaches. Encyclopedia. Available at: https://encyclopedia.pub/entry/54936. Accessed July 04, 2024.
Shastak, Yauheni, Wolf Pelletier, Andrea Kuntz. "Quantitative Vitamin A Analysis Approaches" Encyclopedia, https://encyclopedia.pub/entry/54936 (accessed July 04, 2024).
Shastak, Y., Pelletier, W., & Kuntz, A. (2024, February 08). Quantitative Vitamin A Analysis Approaches. In Encyclopedia. https://encyclopedia.pub/entry/54936
Shastak, Yauheni, et al. "Quantitative Vitamin A Analysis Approaches." Encyclopedia. Web. 08 February, 2024.
Quantitative Vitamin A Analysis Approaches
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This entry is adapted from the peer-reviewed paper 10.3390/analytica5010004

Ensuring that animals receive a well-balanced diet and appropriate feed formulation can significantly enhance animal productivity, improve the quality of their products, and promote better animal welfare. Vitamin A, also known as retinol, is a vital micronutrient crucial for maintaining optimal health and well-being in livestock. It plays a central role in various physiological functions, including immune response regulation, vision maintenance, and cellular differentiation. Consequently, the accurate determination of vitamin A levels in animal feed and premixes is essential for ensuring animal health and production. Beyond preventing deficiencies and associated health issues, it forms the foundation for their overall growth and performance.

vitamin A analysis colorimetric assays HPLC GLC LLC UPC NMR NIRS

1. Introduction

In the realm of animal nutrition, ensuring the precise measurement of vitamin A content is of utmost importance. It is not merely a matter of scientific accuracy, but a critical factor in guaranteeing the safety and quality of the end products consumed by livestock [1]. With the increasing emphasis on food safety standards, the precise determination of vitamin A underscores the commitment to providing nutritious and safe feed for animals, safeguarding their well-being, and subsequently ensuring the quality of animal-derived products [2]. Likewise, evaluating the retinol levels in animal tissues is crucial for assessing nutritional status, preventing illnesses, and improving the general health and productivity of livestock [3].
However, the pursuit of accurate results in vitamin A determination faces several challenges. Researchers and analysts encounter various obstacles that impede the attainment of reliable outcomes, presenting a significant predicament in the analysis of animal feed and other samples [2][4]. Challenges range from intricate sample preparation and extraction procedures to the complexities associated with analytical methodologies and the absence of standardized regulatory guidelines [5]. Achieving precision in vitamin A determination necessitates a comprehensive understanding of these challenges and their implications.
In the field of nutritional science, the precise evaluation of vitamin A plays a pivotal role in understanding dietary sufficiency and identifying potential deficiencies. Similarly, the assessment of retinol levels in animal tissues is indispensable for gauging nutritional well-being, preventing diseases, and enhancing overall animal health and productivity. The following chapters explore the intricate landscape of analytical techniques employed in the quantification of vitamin A, offering a succinct explanation of the fundamental principles that underlie these methodologies.

2. Colorimetric Assays

Colorimetric assays have long been employed as a dependable means of quantifying vitamin A or retinol within various samples [6]. The methodology involves introducing a chromogenic reagent into a soluble fortified food or feed sample, resulting in a reaction with retinol and the development of a distinctive color complex [7]. The intensity of the resultant color is directly proportional to the concentration of vitamin A in the sample. The fundamental principles of colorimetric assays used for measuring vitamin A are represented by the following methods:
  • The Carr and Price assay: This method involves the quantitative evaluation of retinol utilizing antimony trichloride (SbCl3) as a crucial component [8].
  • The Sobel and Werbin assay: This assay employs activated 1,3-dichloro-2-propanol to react with vitamin A, as initially proposed by Sobel and Werbin [9] and later expounded upon by Blake and Moran [10].
  • Trifluoroacetic acid-based colorimetric determination: This technique relies on the interaction of a vitamin A solution in food or feed materials with several Lewis acids, resulting in the transient manifestation of a blue color [6].
Colorimetric assays offer several advantages. They are a cost-effective and straightforward alternative to complex methodologies such as high-performance liquid chromatography (HPLC) [11]. Additionally, their versatility enables the measurement of vitamin A in diverse samples, ranging from foods and feeds to biological fluids [12][13]. Nevertheless, utilizing colorimetric assays for measuring vitamin A or retinol does have its drawbacks. They tend to be less sensitive when compared to more sophisticated techniques like HPLC [14]. Moreover, the presence of interfering substances within the sample can significantly impact the accuracy of the results [15]. Lastly, the lack of specificity for vitamin A or retinol means that these assays can inadvertently identify other compounds forming stable color complexes with the reagent, leading to potential inaccuracies [16].

3. Spectrophotometric Analyses

Spectrophotometric analysis represents a widely utilized approach for the determination of retinol levels in food and feed samples [8][17]. This technique operates on the fundamental principle that all-trans-retinol in isopropanol exhibits maximal absorption at 325 nm [8][17]. The analytical methods for quantifying vitamin A rely on the effective dispersion of the fortified food, premix, or feed matrix to liberate vitamin A, including its various esters [7]. To facilitate this process, a UV-light irradiation system is employed, which serves to degrade the retinol. The setup for this system may include a simple configuration, such as employing a UV lamp along with a protective curtain to safeguard operators from potential exposure to light [18]. Key to the success of this approach is the meticulous determination of the optimal duration of irradiation and the precise positioning of the UV light in relation to the solutions under examination [7].
Spectrophotometric analysis for assessing retinol offers both advantages and challenges. It is lauded for its simplicity, cost-effectiveness, and high sensitivity to trace amounts of retinol [8]. Additionally, its non-destructive nature allows for sample reusability, contributing to resource conservation [11]. Extensive validation across various food and feed matrices underscores its reliability, yet the method is susceptible to yielding inaccurate results in the presence of interfering substances [8]. Ensuring accurate analysis requires meticulous sample preparation and the effective extraction of vitamin A from complex food/feed matrices [19]. The lack of specificity in differentiating retinol from other compounds with similar light absorption characteristics presents a challenge [19]. Notably, it is less suitable for the simultaneous determination of retinol and its esters, necessitating alternative analytical approaches in such cases [20].

4. Chromatographic Techniques

Chromatographic techniques for the analysis of vitamin A are grounded in the fundamental principle of differentiating components based on their distinct interactions with a stationary phase and a mobile phase [21]. These methodologies capitalize on the varying affinities exhibited by the different forms of vitamin A towards the stationary phase, facilitating their separation and eventual quantification [22][23]. Several types of chromatography have been employed for the purpose of vitamin A analysis.
a.
High-performance liquid chromatography (HPLC)
HPLC is a widely used analytical technique that employs high-pressure pumps to propel the mobile phase, comprising the sample, through a column containing the stationary phase [24]. The methodology for determining retinol in food, premix, feed, and biological samples using HPLC commonly involves an isocratic, aqueous solvent system consisting of a mixture of methanol and water as the mobile phase, enabling the isolation of retinol from the extracted sample [25][26]. The column is a crucial element in the HPLC setup, serving as the site for the separation of sample components [27]. The fundamental principle behind HPLC separation relies on the distribution of the analyte (sample) between the mobile phase (eluent) and the stationary phase (packing material) [28]. In the case of retinol, a reverse-phase column is commonly employed as the stationary phase, facilitating the segregation of retinol from other sample constituents based on variations in their hydrophobicity [29]. Prior to HPLC analysis, the retinol must be extracted from the sample and prepared for injection into the HPLC system. This typically involves a lipid extraction step aimed at eliminating interfering lipids and other components [30]. Subsequent to the separation of retinol by the HPLC system, identification and quantification become imperative. This task is typically accomplished using a UV detector, which gauges the absorbance of retinol at a specific wavelength [31].
b.
Gas–liquid chromatography (GLC)
GLC is an established analytical technique employed for the separation and analysis of volatile compounds within a given sample [32]. Prior to the chromatographic process, the sample undergoes an extraction and purification procedure to eliminate any potentially confounding substances [32]. The utilization of a glass column, packed with meticulously chosen glass beads, is imperative to enable the effective separation of retinol from other compounds within the sample [33].
A crucial aspect of GLC revolves around the selection of an inert carrier gas that does not interact with the sample constituents. Helium, known for its inert nature, has emerged as a preferred choice for the carrier gas in GLC [34]. Furthermore, precise regulation of the column temperature is paramount to ensure optimal separation of the compounds within the sample. For instance, the introduction of elevated column temperatures and extended retention times can facilitate the generation of anhydro-retinol during gas chromatography, thereby serving as an effective assay for retinol analysis [33].
To accurately detect the effluent from the column, the implementation of an appropriate detector, such as a mass spectrometer, is fundamental in the GLC process. This detector aids in the identification and quantification of the separated compounds, thereby facilitating comprehensive analysis and interpretation [32].
c.
Liquid–liquid chromatography (LLC)
LLC is a separation technique in which the stationary phase comprises a liquid supported on a solid, while the mobile phase is also a liquid. LLC amalgamates the principles of liquid–liquid extraction and chromatography [35]. Similar to chromatography, one of the phases involved remains stationary during the separation process. However, several significant distinctions differentiate LLC from traditional chromatography utilizing solid stationary phases, such as HPLC [35].
In the extraction of retinol from the sample, a suitable solvent is employed. The selection of the solvent is contingent upon the characteristics of the sample and the specific type of liquid chromatography utilized. Within this method, the compounds are segregated based on their distribution between the two immiscible liquid phases [29][36].
d.
Waters UltraPerformance Convergence Chromatography (UPC)
UPC represents a modern separation technique that harnesses compressed carbon dioxide as the primary mobile phase [37]. This method capitalizes on the utilization of sub-2 μm particle chromatography columns, taking advantage of the low-viscosity properties of CO2 and a sophisticated chromatography system. This approach distinguishes itself from conventional HPLC and notably enhances the sensitivity of the assay. Moreover, UPC demonstrates a significant reduction in solvent waste generation in comparison to traditional liquid chromatography [37].
e.
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 [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 [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 [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 [40][41]. Despite these advantages, chromatography methodologies also exhibit certain drawbacks, such as prolonged analysis time, significant financial investment, and susceptibility to matrix effects [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 [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 [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 [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 [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 [45]. However, it comes with certain drawbacks, including significant financial costs and the requirement for specialized equipment and expertise [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 [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 [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 [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 [50].
NIRS is recognized as a non-destructive, expedient, and cost-effective technique for determining retinol in diverse samples [51][52]. Nevertheless, its sensitivity and accuracy are relatively restricted compared to standard reference methods such as HPLC, necessitating calibration with these methods [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 [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 [54]. For example, the competitive EIA ELISA kit uses a competitive inhibition method to measure the amount of vitamin A in a sample [55][56], while the sandwich ELISA kit uses a sandwich method [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 [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 [59]. Cross-reactivity with other substances in the sample can also lead to occasional false positives.

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Subjects: Agriculture, Dairy & Animal Science
Contributors MDPI registered users' name will be linked to their SciProfiles pages. To register with us, please refer to https://encyclopedia.pub/register :
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