A Comprehensive Review of Analytical Approaches for Carotenoids Assessment in Plant-Based Foods: Advances, Applications, and Future Directions

Featured Application

The advancements in carotenoid analysis discussed in this review have significant applications in food quality assessment, nutritional research, and functional ingredient development. Integrating green chromatography, miniaturized detection systems, and artificial intelligence can revolutionize analytical workflows, reducing environmental impact and enhancing real-time food monitoring. These innovations hold promise for industries focused on food fortification, dietary supplements, and plant-based functional foods, ensuring improved bioavailability and regulatory compliance.

Abstract

Carotenoids are essential bioactive compounds in plant-based foods, valued for their antioxidant properties and role in human health. Accurate quantification of these pigments is critical for food science, nutrition, and health research, yet their analysis remains challenging due to structural complexity, susceptibility to degradation, and matrix interferences. This review comprehensively evaluates analytical techniques for carotenoid assessment, focusing on chromatographic advancements, emerging detection strategies, and sustainability considerations. High-performance liquid chromatography remains the gold standard due to its precision, while novel approaches such as supercritical fluid chromatography and core–shell particle technology enhance efficiency and environmental sustainability. Machine learning and lab-on-a-chip technologies are also emerging as promising tools for rapid, cost-effective, and miniaturized analysis. Challenges in standardization, regulatory gaps, and the limited availability of certified reference materials persist, emphasizing the need for fully validated analytical methodologies. Future research should prioritize green analytical techniques and interdisciplinary strategies to improve sensitivity, reproducibility, and environmental impact. This review provides a critical resource for researchers and industry professionals willing to refine carotenoid analysis for food science, nutrition, and biotechnology applications.

1. Introduction

Carotenoids, a class of naturally occurring pigments, play a critical role in plant biology and human health [1,2,3]. These compounds contribute to the vibrant red, orange, and yellow hues of fruits and vegetables and are essential for photosynthesis and plant photoprotection [1,2]. In humans, carotenoids such as β-carotene and lycopene are known for their antioxidant properties and their role as precursors to vitamin A, contributing to immune function, vision, and cellular health [2]. Given their significant role in human health and nutrition, precise quantification of carotenoids in plant-based foods is essential [1,2,3]. This analysis is pivotal for improving food quality, optimizing bioavailability, and informing nutritional guidelines.

Understanding carotenoid composition in plant foods is particularly important due to their health benefits and role in dietary interventions for chronic diseases, including cardiovascular conditions and certain cancers [4]. Although advancements in analytical methods have improved carotenoid quantification, their structural diversity, susceptibility to degradation, and interactions with other compounds within food matrices present persistent challenges. For instance, carotenoids exhibit various molecular structures, including differences in chain length, functional groups, and conjugated double bonds, all of which influence their physical, chemical, and biological properties. Figure 1 illustrates the molecular structures of major carotenoids, highlighting their structural variability and the functional groups that contribute to their unique properties. Understanding these structures is essential for addressing analytical challenges and optimizing quantification methods.

The accurate analysis of carotenoids also aids in understanding their broader health implications. These compounds have modulated oxidative stress and inflammation, reducing the risk of chronic diseases such as macular degeneration, cardiovascular diseases, and certain types of cancer [5]. Furthermore, as precursors to vitamin A, carotenoids are crucial in preventing deficiency-related conditions like xerophthalmia. Recent studies, such as those utilizing spectroscopic methods, have linked carotenoid biomarkers to dietary intake, highlighting their potential to enhance immune responses and promote skin health [6].

Chromatographic methods have become the cornerstone of carotenoid analysis due to their precision and reliability in quantifying individual compounds [7]. Techniques such as high-performance liquid chromatography (HPLC) and ultra-high-performance liquid chromatography (UHPLC) offer robust approaches for achieving rapid separation and quantification, even in complex plant matrices [8]. Emerging methods like supercritical fluid chromatography (SFC) offer eco-friendly alternatives with high separation efficiency, especially for nonpolar carotenoids, while advancements in sample preparation, including enzymatic treatments and green solvents, are enhancing recovery rates and reducing analytical errors, driving the field toward more sustainable practices. [9,10]. Despite these advancements, challenges like carotenoid degradation during analysis, matrix interferences, and the lack of standardized reference materials persist. Overcoming these hurdles requires interdisciplinary approaches integrating chemistry, biochemistry, and food science [11]. Additionally, regulatory frameworks must evolve to accommodate novel analytical techniques and ensure global standardization.

Despite significant advancements in carotenoid analysis, there remains a need for a comprehensive evaluation of how different methodologies compare in terms of accuracy, efficiency, and applicability. Traditional techniques, such as HPLC, have provided reliable quantification for decades, but newer technologies, including UHPLC and SFC, are demonstrating enhanced precision and sustainability. However, many studies focus on isolated aspects of carotenoid analysis rather than presenting an integrated perspective that incorporates developments in sample preparation, detection technologies, and regulatory considerations. This review seeks to bridge this gap by providing a holistic assessment of contemporary and emerging analytical strategies, contributing to the ongoing effort to refine and standardize carotenoid quantification in plant-based foods.

2. Chromatographic Techniques for Carotenoids Analysis

The analysis of carotenoids in food matrices relies on various chromatographic techniques, each offering unique separation, identification, and quantification advantages. Figure 2 presents the number of methods published for each of these techniques since 2015.

These methods address carotenoids’ structural complexity and diversity by including a separation step based on the interaction between the stationary, analyte, and mobile phases, which depends on specific properties such as polarity in liquid chromatography. Analyte determination is then performed using different detectors selected according to the goals of the analysis, facilitating accurate and reliable analysis in different applications. HPLC is the most widely used method for carotenoid determination due to its reliability, sensitivity, and ability to separate a wide range of compounds with high resolution. Since 2021, there has been a slight increase in the use of UHPLC, likely driven by its advantages in speed, resolution, and solvent efficiency, though its adoption remains limited due to higher costs and specialized column requirements [7]. Other techniques, such as SFC and GC, are rarely used, as SFC is still emerging as an alternative for nonpolar carotenoids [9]. Despite its lower frequency of use, TLC has remained stable over the years and continues to serve as a valuable complementary technique, particularly for preliminary screening and qualitative analysis. In some studies, TLC has been used alongside HPLC, highlighting its role as a supporting method for quick assessments before further chromatographic quantification [11]. These trends suggest that while HPLC remains the dominant technique, combining different analytical methods can improve accuracy and robustness in carotenoid research. A detailed discussion of the specific features of these techniques for carotenoid analysis, along with their advantages and limitations, will be presented in the following sections.

2.1. High-Performance Liquid Chromatography

HPLC is the most widely employed method for carotenoid analysis due to its precision and ability to separate individual carotenoids effectively.

Table 1. HPLC methods for carotenoid analysis in food matrices since 2014 to date.

Sample	Analytes	Stationary Phase	Mobile Phase	Flow Rate (mL/min)	Detector System	Analysis Time (min)	LOD (ng/g)	LOQ (ng/g)	Recovery (%)	Ref.
Red chili peppers (9 varieties)	Capsanthin, Zeaxanthin, Lutein, β-Cryptoxanthin, β-Carotene	Spherisorb ODS-2 C18 (250 mm × 4.6 mm, 5 μm)	MeOH/THF/ACN/Acetone	0.5/1.0/1.5	DAD	25	20–63	67–209	88–107	[12]
Legumes (7 species, including beans, peas, and lentils)	Lutein, Zeaxanthin, β-Carotene, α-Carotene, β-Cryptoxanthin	C30 (250 × 4.6 mm I.D., S-5 μm)	MTBE, MeOH-20 mM ammonium acetate, and water	0.9	UV-Vis	45	51–300	155–909	83–107	[13]
Sweet red peppers (Capsicum annuum L.)	Lutein, Zeaxanthin, α-Carotene, β-Carotene, β-Cryptoxanthin, Violaxanthin, Capsanthin, Phytoene, Phytofluene	C30 YMC column (5 μm, 250 × 4.6 mm i.d.)	0.1% TMA in MeOH and MTBE	NR	DAD	21	NR	NR	NR	[14]
Tomatoes (Cherry and Pear varieties; Solanum lycopersicum)	Lutein, β-Carotene, Lycopene	C30 column (3.0 mm × 150 mm, 2.6 μm)	MeOH/MTBE/H₂O	0.4	DAD	20	3–46	8–1530	86–116	[15]
Brown algal extracts	Fucoxanthin and β-Carotene	Nova-Pak C18 (3.9 × 150 mm, 4 µm)	AAc/MeOH/EtOAc	0.5	DAD	40	500–560	NR	93–103	[16]
Starchy staples (potato, cassava, sweet potato, yam, taro)	Lutein, Zeaxanthin, β-Cryptoxanthin, α-Carotene, β-Carotene	Prontosil 200–3-C30 (150 × 4.6 mm; 3 μm)	MeOH/H₂O (95:5) and MTBE/MeOH/H₂O (85:10:5)	2	DAD	15	65.5 –113	241–410	NR	[17]
Wild cherry tomatoes	Lutein, Zeaxanthin, β-carotene, Lycopene	YMC-C30 (250 × 4.6 mm, 5 µm)	MeOH/MTBE/H₂O	0.8	DAD-MS	33	3.66–8.11	17.2–244	NR	[18]
Fruit juices (commercial and fresh samples from citrus, berries, and tropical fruits)	β-Carotene, (all-E)-Lutein, β-Cryptoxanthin, (all-E)-Zeaxanthin, Phytoene, (all-E)-Violaxanthin, (9′Z)-Neoxanthin, (all-E)-Antheraxanthin	BEH C18 (100 mm × 2.1 mm; 1.7 μm)	ACN–MeOH and H₂O	0.5	DAD	16.6	22–157	70–52	75–104	[19]
Microalgae	30 identified carotenoids	Poroshell 120 EC-C18 (150 mm × 3.0 mm; 2.7 μm)	0.1% FA in H2O-MeOH (1:1 v/v) and in MTBT-MeOH (8:2 v/v)	0.2	DAD-QTOF-MS	55	NR	NR	NR	[20]
Citrus fruits (mandarin, lemon, sweet orange, bergamot)	Violaxanthin, Neoxanthin, Lutein Epoxide, Antheraxanthin, Luteoxanthin, Lutein, Zeaxanthin, β-Cryptoxanthin, α-Carotene, β-Carotene	C30 (250 × 2.1 mm; 3 μm)	ACN/MBTE/MeOH and water	0.4	DAD	NR	NR	NR	NR	[21]
Dried red peppers (Capsicum frutescens var.)	23 identified carotenoids	ORBAX Eclipse XDB C18 column (4.6 mm × 150 mm, 5 μm)	H₂O and Acetone	0.6	DAD-APCI/MS/MS	37	NR	NR	NR	[22]
Soybean seeds	Lutein, Zeaxanthin, β-Carotene, α-Carotene, β-Cryptoxanthin	C30 (250 × 4.6 mm, 5 µm)	EtOH/ACO	0.9	UV-Vis	77–84	5.1–30	15.5–90.9	83–106	[13]
Coffee berries	Violaxanthin, Neoxanthin, Chlorophyll b, Lutein, Chlorophyll a, α-Carotene, β-Carotene	C30 (150 × 3 mm; 3 μm)	MeOH/MTBE/water	0.42	DAD-APCI/MS	90	0.5 –3.8	0.48 –12	NR	[23]
Apricots	27 identified carotenoids	C30 (250 × 4.6 mm; 5 μm)	MeOH/MTBE/water	1	DAD-APCI/MS	140	NR	NR	NR	[24]
Sea Buckthorn (Hippophae rhamnoides L.)	19 identified carotenoids	BEH C18 (150 mm × 2.1 mm; 1.7 μm)	EtOAc/ACN/water	0.4	DAD-ESI/MS	27	NR	NR	NR	[25]

Ref., references; DAD, diode array detector; UV-Vis, ultraviolet-visible detector; HPLC, high-performance liquid chromatography; MeOH, methanol; THF, tetrahydrofuran; AAc, ammonium acetate; ACN, acetonitrile; MTBE, methyl tert-butyl ether; TMA, trimethylamine; NR, not reported; DAD-APCI/MS, diode array detector with atmospheric pressure chemical ionization mass spectrometry; DAD-ESI/MS, diode array detector with electrospray ionization mass spectrometry; EtOAc, ethyl acetate.

compares the determination of carotenoids in different foods using HPLC coupled with different detection systems [12,13,14,15,16,17,18,19,20,21,22,23,24,25].

Reversed-phase liquid chromatography (RP-LC) is particularly popular, using C18 and C30 columns with non-polar stationary phases to achieve excellent resolution [12,13,14,15,16,17,18,19,20,21,22,23,24,25]. C18 phases, such as Spherisorb ODS-2 and BEH C18, are suitable for hydrophobic carotenoids [12,13,18,19,20,22,25], while C30 phases, like YMC C30 and Prontosil C30, offer superior separation of structurally similar compounds and geometric isomers [13,14,15,16,17,21,23,24]. As seen in Kinetex C18, core–shell technology enhances separation efficiency, whereas sub-2 μm particles in BEH C18 facilitate UHPLC applications, reducing analysis time while maintaining high sensitivity [13,18,19,20,25].

The choice of mobile phases in HPLC depends on the polarity and solubility of carotenoids. Commonly used solvents include methanol (MeOH), acetonitrile (ACN), methyl tert-butyl ether (MTBE), tetrahydrofuran (THF), acetone, and water. Some methods incorporate EtOAc for improved solubility, while ammonium acetate (NH₄OAc) is used as a buffer modifier [13,16]. Environmental concerns arise from solvents such as ACN, MeOH, and MTBE, which pose toxicity and waste disposal issues [26]. MTBE, in particular, is environmentally persistent and a potential groundwater contaminant [26]. Sustainable alternatives, such as ethyl acetate, are being explored to reduce environmental impact [25,26,27]. Modifiers like trifluoroacetic acid and trimethylamine improve peak shape but require careful handling due to their corrosiveness and volatility [14,20,28,29].

Column dimensions in HPLC vary depending on the analytical needs. Standard 4.6 mm I.D. columns with lengths between 150 and 250 mm provide high-resolution separations. In comparison, narrow-bore (2.1 mm I.D.) columns, such as BEH C18, are used in UHPLC for increased sensitivity and lower solvent consumption [18,20]. Flow rates range from 0.3 to 2.0 mL/min, with higher rates for larger-particle columns and lower for sub-2 μm particle columns. The analysis time spans from 6 to 140 min, with shorter columns and smaller particle sizes leading to faster analyses, while longer columns ensure better separation for complex matrices.

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., β-carotene, α-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.

2.2. Gas Chromatography (GC)

Gas chromatography (GC) is not commonly used for carotenoid analysis due to these compounds’ volatility and thermal stability limitations [33]. Unlike liquid chromatography (LC), which is well-suited for carotenoids in their native forms, GC is primarily used to analyze volatile carotenoid-derived compounds (CPs), particularly apocarotenoids and short-chain degradation products [23,33]. The efficiency of GC for these compounds depends on extraction, pretreatment (derivatization), and instrumental conditions.

Extraction methods for volatile CPs often involve solvent extraction, steam distillation, or solid phase microextraction (SPME), depending on the sample matrix [33]. Derivatization is often necessary to enhance volatility and thermal stability; for instance, N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide and the Sweeley reagent (10% v/v hexamethyldisilazane–trimethylchlorosilane, 2:1 in pyridine, heated at 45 °C for 30 min) are frequently used in CP analysis [23]. However, some compounds, such as β-ionone (β-IO), β-cyclocitral (β-CC), dihydroactinidiolide, and epoxy-β-ionone, can be analyzed without derivatization [23] GC analysis is performed using capillary columns such as BPX5 from SGE Analytical Science, Zebron ZB-1 from Phenomenex, DB-5MS and CP Wax 58CB from Agilent Thecnologies, and STABILWAX-DA from Restek Corporation, with temperature gradients ranging from 40 °C to 310 °C, depending on the sample composition [23,33,34]. These optimized extractions, pretreatment, and instrumental techniques allow GC to be a viable method for analyzing volatile CPs despite its limitations for intact carotenoids.

2.3. Thin-Layer Chromatography

Thin-layer chromatography (TLC) is a simple and cost-effective technique often used for the preliminary screening of carotenoids. It involves the separation of carotenoids on a stationary phase, such as silica gel, using solvent systems optimized for their polarity. While TLC lacks the precision and quantification capabilities of HPLC or GC, it remains valid for rapid qualitative analysis and initial profiling of carotenoid mixtures.

Table 2. TLC methods for carotenoid analysis in food matrices since 2015 to date.

Sample	Analyte	Stationary Phase	Developing Solvent	Determination	Ref.
Tomato leaf extracts	β-carotene, and lutein	TLC silica gel 60 F254 plates (5 cm × 10 cm)	PE:CE:EtOAc:ACO: tOH (60:16:10:10:6 v/v)	Raman Spectroscopy (Handheld Metrohm MiraDS, 785 nm laser)	[35]
Dietary supplements	β-carotene, canthaxanthin, astaxanthin, lutein and zeaxanthin	HPTLC Silica Gel 60 F254 Plates 20 × 10 cm	PE:CH:EtOAc:Ac:EtOH (60:16:10:10:6, v/v/v/v/v)	UV (440 nm) using a TLC Scanner	[36]
Aesculus hippocastanum leaves	Alloxanthin, β,β-Carotene, 9′-cis-Neoxanthin, Diadinoxanthin, Diatoxanthin, Fucoxanthin, Lutein, Myxoxanthophyll, Peridinin, Violaxanthin	TLC Silica gel 60; 20 × 20 cm aluminum sheets	0.8% n-PA in light PE (v/v)	UV-Vis (350–750 nm, 390–710 nm, 400–700 nm); Handheld UV lamp; JENWAY 7315 spectrophotometer	[37]
Dietary supplements	Lutein, zeaxanthin	TLC Si60 F254s glass plates	n-hept:EtOAc (9:1, v/v) and n-hept:Ac: EtOAc (55:25:20, v/v/v) sequentially	UV detection (450 nm) with densitometry; BMD-TLC combined with HPLC–DAD–ESI–MS	[38]
Dietary supplements and fruit juices	β-Carotene	TLC Aluminiumoxid 60 F254 neutral	CH3Cl:MeOH:Ac:NH4OH (10:22:53:0.2, v/v/v/v)	Densitometric detection at 450 nm using TLC Scanner 3 (CAMAG, Muttenz, Swirzerland) with Cats 1.3.4 software	[39]
Phoenix sylvestris fruit epicarp	β-carotene, lutein	HPTLC Silica Gel 60 F254, 10 × 20 cm plates	PE (60–80 °C): Ac (70:30, v/v)	UV detection at 450 nm, densitometry	[40]
various spices, pastes, sauces, and palm oils	Carotenoids (unspecified), for authentication	HPTLC Silica Gel 60 Nano-SIL-PAH caffeine-impregnated plates	Isohex–EMK (5:1, v/v)	UV-Vis detection, post-chromatographic UV irradiation, HPTLC-vis-HPLC-DAD-ESI-MS	[41]
Haloferax larsenii NCIM 5678 (isolated from Pachpadra Salt Lake, Rajasthan)	Bacterioruberin and its derivatives	Silica gel F254 TLC plate	ACO:n-hept (50:50, v/v)	UV-Vis detection (460, 490, 520 nm)	[42]
Orange peel waste	ζ-carotene, β-cryptoxanthin, other carotenoids	HPTLC Silica Gel 60 F254 plates	Cyclohex–MTBE (various ratios, v/v)	UV-Vis detection (200–500 nm); Densitometry at 450 nm	[43]
Shrimp (Penaeus semisulcatus, Fenneropenaeus indicus, Metapenaeus ensis, Penaeus monodon)	Astaxanthin, β-carotene, zeaxanthin	Silica Gel G TLC plates	EtOAc:hex (7:7, v/v)	UV-Vis detection (461 nm); TLC analysis (Rf values 0.65, 0.85)	[44]

Ref., reference; PE, petroleum ether; cyclohex, cyclohexane; EtOAc, ethyl acetate; ACO, acetone; EtOH, ethanol; n-PA, n-propanol; n-hept, n-heptane; Isohex, iso-hexane; EMK, ethyl methyl ketone; MTBE, methyl tert-butyl ether; TLC, thin-layer chromatography; HPTLC, high-performance thin-layer chromatography; F254, fluorescent indicator 254 nm; UV, ultraviolet; Vis, visible; DAD, diode array detector; ESI, electrospray ionization source; MS, mass spectrometry; Rf, retention factor.

summarizes different TLC-based methods for carotenoid determination used since 2009.

These studies employed various stationary phases, developing solvents, and detection techniques to identify carotenoids in different food matrices [35,36,37,38,39,40,41,42,43,44]. Most studies used silica gel-based TLC plates, either standard (TLC Silica Gel 60) or high-performance variants (HPTLC Silica Gel 60 F254) [35,36,37,40,41,43], with some employing aluminum-backed silica gel or nano-silica gel plates for better separation [39,41]. Developing solvents varied significantly, with petroleum ether-based systems being common, while more polar solvents like THF/methylene chloride/n-hexane and chloroform/methanol/acetone/ammonium hydroxide were also used [36,40]. Some solvents, such as methylene chloride and chloroform, pose environmental concerns due to their toxicity and disposal challenges, making greener alternatives a critical consideration [45]. Detection methods mainly relied on UV-Vis, with 425 and 461 nm detection wavelengths, sometimes supplemented by densitometry or Raman spectroscopy [35,38]. More advanced approaches, such as TLC-HPLC-DAD-ESI-MS, were employed in certain studies to improve identification accuracy [38,41].

The number of carotenoids analyzed varied among studies. Simple food matrices, such as dietary supplements [36,38], typically contained three to five carotenoids, whereas complex samples, like Aesculus hippocastanum leaves, exhibited up to ten different carotenoids [37]. Advanced detection techniques were employed for more complex mixtures, while single carotenoids, such as β-carotene in fruit juices, were identified using more straightforward UV-Vis detection [39]. The foods analyzed included pollen, dietary supplements, fruit juices, leaves, spices, pastes, sauces, palm oils, shrimp, and orange peel waste. The main carotenoids identified were β-carotene, lutein, zeaxanthin, astaxanthin, cryptoxanthin, bacterioruberin, and several xanthophyll derivatives.

A recent study by Saini et al. presents an innovative approach using preparative TLC for purifying xanthophyll carotenoids from lettuce [46]. This method utilizes Hyflo-Super-Cel–MgO (Heavy)–calcium sulfate hemihydrate (9:9:2 w/w) as an adsorbent and acetone– hexane (1:1) as the mobile phase, achieving 95–96% purity in a single step without interference from chlorophylls or minor carotenoids. This approach simplifies the purification process while maintaining high purity levels, making it suitable for analytical and preparative applications. Additionally, the study explored the anticancer potential of the purified carotenoids, emphasizing their health benefits. This analysis highlights the versatility of TLC in carotenoid research, from preliminary screening to purification and bioactivity studies. While TLC cannot replace more advanced chromatographic techniques, it remains a valuable tool for qualitative analysis and initial compound separation.

2.4. Supercritical Fluid Chromatography

Supercritical fluid chromatography (SFC) is gaining attention as an environmentally friendly alternative to traditional methods. It employs supercritical carbon dioxide as the mobile phase, reducing the use of organic solvents and offering efficient separation, particularly for non-polar carotenoids. Coupled with detectors like DAD and MS, SFC provides reliable quantification and identification while supporting sustainable analytical practices.

Table 3. SFC methods for carotenoid analysis in food matrices since 2016 to date.

Sample	Analyte	Stationary phase	BPR Bar	T °C	MP	Detector	Ref.
Hemp Seed Oil and Waste Fish Oil	α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, ergocalciferol, cholecalciferol	HSS C18 SB (3.0 × 100 mm; 1.8 μm).	124.1	35	MeOH + CO2	DAD	[47]
Microalgae, rosehip.	α-carotene, β-carotene, lycopene, canthaxanthin, lutein, zeaxanthin, neoxanthin, β-cryptoxanthin, astaxanthin, and violaxanthin.	1-AA (3.0 × 100 mm, 1.7 μm).	160	35	MeOH + CO2	PDA–QTOF MS	[48]
Dietary supplements	α-tocopherol, α-tocotrienol, β-tocopherol, β-tocotrienol, γ-tocopherol, γ-tocotrienol, δ-tocopherol, δ-tocotrienol, tocopherol acetate.	BEH-2-EP (3.0 × 100 mm, 1.7 μm).	130	50	MeOH + CO2	DAD	[49]
Capsicum chinense (Habanero pepper)	Apocarotenoids (10)	Ascentis Express C30 (150 × 4.6 mm, 2.7 µm)	150	35	MeOH + CO2	APCI-QqQ/MS	[50]
Egg yolk (from laying hens supplemented with β-carotene)	β-Carotene, lutein, zeaxanthin	Venusil XBP C30 (250 × 4.6 mm, 5 μm)	160	60	IPA + CO2	DAD-MS/MS	[51]

Ref., reference; HSS, high-strength silica; C18, octadecylsilane (18-carbon chain); SB, surface-bonded; MeOH, methanol; CO₂, carbon dioxide; PDA, photodiode array; QTOF MS, quadrupole time-of-flight mass spectrometry; BEH, ethylene-bridged hybrid; 2-EP, 2-ethylpyridine; DAD, diode array detection; DAD-MS/MS, diode array detection with tandem mass spectrometry; APCI-QqQ/MS, atmospheric pressure chemical ionization quadrupole–QqQ mass spectrometry; IPA, isopropanol.

outlines different SFC-based carotenoid and vitamin determination methods over the past decade [44,45,46,47,48,49,50,51].

The stationary phases include HSS C18 SB, 1-AA, BEH-2-EP, Ascentis Express C30, and Venusil XBP C30. These phases offer varied selectivity, with C30-based columns often preferred for carotenoid separation due to their affinity for non-polar compounds. The back pressure regulators used in these studies ranged from 124.1 to 160 bar, ensuring stable CO₂ conditions for efficient separation. The temperature settings varied from 35 °C to 60 °C, with higher temperatures generally improving analyte solubility and reducing retention times. The mobile phase typically consisted of MeOH combined with CO₂, though one study utilized isopropanol (IPA) as a modifier to enhance the solubility of specific carotenoids [51].

The number of analytes detected varied significantly across studies. For example, tocopherols and tocotrienols in dietary supplements and oils [47,49] included up to nine compounds. In contrast, carotenoid profiling in microalgae and rosehip [48] involved up to ten analytes, such as α-carotene, β-carotene, lycopene, canthaxanthin, lutein, zeaxanthin, neoxanthin, β-cryptoxanthin, astaxanthin, and violaxanthin. Detection systems ranged from DAD to advanced QTOF-MS and APCI-QqQ/MS, which allowed for precise mass-based identification. The study on egg yolk from hens supplemented with β-carotene utilized DAD-MS/MS, enabling spectral and structural confirmation of β-carotene, lutein, and zeaxanthin [51]. The food matrices analyzed included hemp seed oil, waste fish oil, dietary supplements, microalgae, rosehip, habanero peppers, and egg yolk. These diverse sources highlight the broad applicability of SFC for lipid-soluble vitamins and carotenoids. Capsicum chinense [50] was particularly interesting as it contained ten apocarotenoids, demonstrating SFC’s ability to separate structurally similar compounds effectively. A recent study by Donato et al. developed a two-dimensional separation method (SFC × RP-UHPLC) to improve the analysis of carotenoids in red chili pepper (Capsicum annuum L.) [52]. This approach combined SFC using an Ascentis ES Cyano column (250 × 1.0 mm, 5 µm) from Supelco (Darmstadt, Germany) in the first dimension, followed by reversed-phase ultra-high performance liquid chromatography (RP-UHPLC) on an Acquity UPLC BEH C18 column (50 × 2.1 mm, 1.7 µm) from Waters (MA, USA) in the second dimension. This method achieved a more efficient separation of 50 carotenoids, including β-carotene, capsanthin, lutein, zeaxanthin, phytofluene, neurosporene, β-cryptoxanthin, and capsorubin. This approach improved analytical accuracy by classifying carotenoids into 15 structural groups and resolving co-elution challenges. Additionally, combining DAD, Q-ToF MS, and ion mobility spectrometry (IMS) allowed for detailed cis/trans isomer differentiation [52,53]. Compared to conventional NP-LC × RP-LC, this method reduced analysis time from 120 to 60 min. It decreased solvent consumption by 90%, demonstrating the potential of SFC × RP-UHPLC for advanced carotenoid profiling [52,53]. This highlights the evolving role of SFC in food analysis, offering both sustainability and enhanced separation efficiency.

3. Sample Preparation for Carotenoids Determination

Carotenoid isolation includes several essential steps: sample pretreatment (which involves drying, grinding, and storing to stabilize carotenoids), extraction, clean-up (using solid-phase extraction (SPE) or liquid–liquid extraction (LLE) to remove any interfering compounds), chemical modification (such as saponification and derivatization), and pre-concentration (in some instances).

The compact structure of plant cell walls and the association of carotenoids with various molecules, such as proteins and fatty acids, require preliminary steps to mitigate the influence of the matrix before extraction. These steps aim to break down barriers, facilitate solvent penetration into the tissue, and improve mass transfer. The type of pre-treatment used before extraction is directly related to the cells’ processing characteristics. Several cell disruption methods are available, including physical methods (e.g., cryogenic grinding, bead milling, cooking, high-pressure homogenization, ultrasonication, etc.), chemical methods (acids and surfactants), biological methods (germination), and enzymatic lysis. Enhancing the effectiveness of cell wall damage can increase carotenoid yields by up to tenfold [54]. In the case of samples rich in water, it may be advisable to freeze-dry prior to extraction, while matrices rich in starch should be heat-treated or enzymatically hydrolyzed to enhance the extraction process of carotenoids. In turn, matrices rich in proteins require protein precipitation with acids, while samples rich in fat or pigment require often saponification before further analysis. Though sample preparation varies significantly between different laboratories, it should be of great importance to standardize sample preparation procedures to compare obtained data. Additionally, despite the variety of methods, several limitations need to be addressed, including energy consumption, toxicity, and the stability of metabolites observed during the extraction of carotenoids [55].

3.1. Sample Extraction Techniques

The extraction process aims to isolate the targeted compound from the surrounding matrix using a solvent with a similar polarity or using the affinity of that compound to the absorbent phase. The most commonly used extraction method for carotenoids relies on organic solvents, which can pose significant risks to the environment and human health [45]. In response to these concerns, there has been a recent evolution in carotenoid extraction techniques aimed at reducing these risks while improving process efficiency and cost-effectiveness. As a result, new extraction technologies are being explored, such as ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), supercritical fluid extraction (SFE), enzyme-assisted extraction (EAE), pulsed electric field (PEF) extraction, subcritical water extraction (SWE), high-hydrostatic-pressure extraction (HHPE) and ohmic heating (OH). Additionally, ongoing efforts are to develop new green or biobased solvents that can be successfully used along with the abovementioned methods.

3.1.1. Solvent Extraction

Methods used for solvent extraction of carotenoids include refluxing in a boiling water bath (Soxhlet), mechanical stirring, and maceration [56]. Carotenoids are overall nonpolar compounds. The most used solvents for carotenoid extraction are chloroform, dichloromethane, and tetrahydrofuran, with solubility ranging from 1000 to 10,000 mg·L⁻¹ [57]. However, there are also more polar carotenoids, such as xanthophylls, which require the extraction of polar solvents or mixtures containing polar solvents. It has been observed that acetone successfully dissolves both xanthophylls and carotenes [58]. The choice of solvent for carotenoid extraction is dictated not only by the compound’s polarity, chemical stability, or solubility but also by the toxicity of the solvent. Recently, especially for products designed for human consumption, alcohol has been predominantly used as a solvent. Hydrophilic solvents (EtOH, MeOH, acetone, etc.) are also preferably used to enhance the extractability of carotenoids from high-moisture samples. The Soxhlet method is characterized by a simple design, continuous process, and easy visual monitoring while requiring a low solvent flow. However, there is a higher possibility of carotenoid degradation due to longer process times and higher temperatures (varying from 40 up to 100 °C depending on the solvent type used). Thus, lower temperatures not exceeding 60 °C as well as limited exposure to light, oxygen and acidic conditions is also highly recommended for this method. In contrast, maceration decreases the degradation of thermolabile carotenoids, reduces organic solvent usage, and increases yields but is less effective for samples containing water [59].

3.1.2. Biobased Solvent Extraction

Bio-based solvents are more environmentally friendly than conventional solvents derived from petrochemicals [60]. These solvents, such as plant materials, are produced from renewable biomass and are known for their lower toxicity, making them safer for human health [60]. They can be classified into four categories based on the biomass used for production: (I) lignocellulosic, (II) starch and sugar, (III) oil and protein-based, and (IV) other food and forestry waste [61]. One extensively studied green solvent for extracting carotenoids is ethyl lactate [62].

Ethyl lactate is produced industrially through the continuous esterification of lactic acid and alcohol with an acid catalyst. Research indicates that the extraction efficiency of carotenoids using ethyl lactate is significantly higher, yielding 243 mg/kg, compared to conventional solvents like acetone (51.90 mg/kg), hexane (34.45 mg/kg), and ethanol (17.57 mg/kg) [63]. When combined with UAE, its efficiency increases up to 8.7 times compared to acetone, 7 times to ethanol, and 1.6 times to sunflower oil [64].

Another effective bio-based solvent is 2-methyltetrahydrofuran (2-MeTHF), obtained by catalytic furfural hydrogenation derived from plant polysaccharides. It has no reported mutagenic or genotoxic effects and has been shown to extract up to 12.5 times more carotenoids from olive pomace than n-hexane [65]. Ionic liquids (ILs), composed of organic cations and inorganic anions, are non-flammable but vary in biodegradability and toxicity. While concerns exist about their environmental impact, their ability to be reused makes them attractive for green extraction processes. ILs have demonstrated greater efficiency than acetone for carotenoid extraction and outperform MeOH in lutein extraction [66,67].

Deep eutectic solvents (DESs), often called “solvents of the 21st century”, are formed by hydrogen bonds between two or three components [68]. Natural deep eutectic solvents (NADESs), prepared from plant-based ingredients, are considered safer alternatives. Studies confirm that DES and NADES outperform conventional solvents in carotenoid extraction [69,70]. However, some DES formulations pose toxicity risks to aquatic and terrestrial ecosystems, making NADES with edible components the safest choice [70]. Vegetable oils have emerged as a sustainable alternative for carotenoid extraction, eliminating the need for conventional solvents while also protecting carotenoids from oxidation. Extracted carotenoids can be directly incorporated into food without additional purification. Commonly used oils include flaxseed, sunflower, corn, soy, olive, and peanut [70]. Sharma and Bhat (2021) found that corn oil extracted twice as many carotenoids from pumpkin peels as hexane/isopropyl alcohol, yielding 33.70 µg/g versus 16.20 µg/g [71]. Similarly, Elik et al. (2020) reported that while n-hexane, ethanol, and acetone provided higher recovery (~87%) than flaxseed oil (77.48%), the ability to avoid conventional solvents remains a key advantage [72].

3.1.3. Green Extraction Technologies

Various green extraction methods have been developed to improve carotenoid recovery while reducing environmental impact. Figure 3 plots the number of methods published for each extraction method since 2015 to date.

Among these, solvent extraction, UAE, and supercritical fluid extraction (SFE) are the most widely studied [62].

• UAE utilizes sound waves above 20 kHz to generate acoustic cavitation, which disrupts plant cell walls, enhancing solvent penetration and accelerating extraction. Studies have shown significant efficiency gains using UAE. Bhimjiyani et al. (2021) reported a 50% increase in carotenoid yield from sea buckthorn, while Stupar et al. (2021) observed that UAE extracted 151.41 µg/mL of β-carotene, compared to 96.74 µg/mL with NADEs [73,74]. Similarly, Sharma et al. (2021) demonstrated that UAE combined with corn oil extracted 38.03 µg/g of carotenoids from pumpkin, nearly twice the amount obtained using hexane–isopropyl alcohol (19.21 µg/g) [71]. Extraction efficiency in the UAE depends on several parameters. Increasing ultrasound intensity produces a proportional rise in carotenoid yield [75]. Other influencing factors include extraction time, solvent-to-solid ratio, and temperature, with extraction time having the most significant effect, while temperature has minimal impact [76].
• SFE utilizes fluids in a supercritical state, characterized by solvent power as liquids and mass transfer properties as gas which accelerates extraction. Carbon dioxide is the most commonly used solvent due to its low critical temperature (31.1 °C) and pressure (7.38 MPa), making it suitable for heat-sensitive carotenoids [77]. SFE has demonstrated high extraction efficiencies. Lima et al. (2019) optimized SFE for carotenoids in 15 vegetable and fruit waste matrices, achieving up to 96.2% recovery, while Sanzo et al. (2018) reported a 98.6% recovery of astaxanthin from H. pluvialis [78,79]. Despite its advantages, slow extraction kinetics limit SFE’s efficiency. To address this, EAE and UAE can be combined with SFE to improve cell wall disruption and enhance carotenoid release [80].
• MAE applies microwave radiation to heat solvents through dipole rotation and ionic conduction, increasing cell permeability and accelerating extraction. It requires less solvent and shorter processing times but is unsuitable for thermolabile carotenoids. Comparisons between MAE and UAE show that while UAE achieved a higher yield (267 mg/100 g DW) at 200 W for 80 min, MAE still provided a notable improvement over conventional methods, yielding 262 mg/100 g DW at 120 W for 25 min. Additionally, UAE extracts exhibited higher antioxidant capacity and preserved more bioactive compounds, though they required significantly more energy consumption (229 kcal vs. 43 kcal for MAE). These findings highlight UAE’s effectiveness for carotenoid recovery while underscoring the need for energy optimization in industrial applications. [81].
• EAE utilizes cell-wall-degrading enzymes (e.g., cellulases, pectinases, proteases) to enhance carotenoid release [82,83]. Strati et al. (2015) observed a 10-fold increase in lycopene and a 6-fold increase in total carotenoids from tomato paste following enzyme pretreatment [82]. Similarly, Lavecchia and Zuorro (2008) reported a 20-fold rise in lycopene extraction from tomato waste using cellulase and pectinase [84]. EAE is particularly beneficial for wet samples, eliminating the need for drying before extraction [82,83].
• PEF extraction involves short-duration electric pulses (nanoseconds to milliseconds) to increase cell membrane permeability, facilitating carotenoid extraction. Moderate electric fields (up to 10 kV/cm) with low energy input have enhanced carotenoid recovery without degradation or isomerization [85].
• HHPE applies pressures between 100 MPa and 1000 MPa at moderate temperatures (<60 °C) to disrupt cell membranes, improving carotenoid release. Strati et al. (2015) found that 700 MPa treatment increased carotenoid yield by up to 64% [82].
• SWE exploits water’s unique properties under high temperatures (100–320 °C) and pressures (20–150 bar), allowing it to behave similarly to organic solvents. Studies have shown that SWE can achieve comparable efficiency to solvent extraction [80].
• OH extraction applies alternating electrical currents, generating uniform heating and inducing electroporation, which improves carotenoid release while minimizing oxidation and degradation [86].

3.2. Saponification and Chemical Modifications

After extraction, alkaline saponification is commonly used to hydrolyze carotenoid esters into their free forms while removing interfering lipids and chlorophylls, improving chromatographic resolution [87]. This step typically involves potassium hydroxide or sodium hydroxide, with concentrations adjusted based on the sample matrix and carotenoid composition [87,88]. However, alkaline-sensitive carotenoids such as lutein, violaxanthin, and astaxanthin can degrade, leading to byproducts like astacin [87,89]. For these compounds, enzymatic hydrolysis is recommended as a milder alternative.

Studies have examined the impact of temperature and reaction time on carotenoid stability during saponification. While earlier research suggested that prolonged exposure to high temperatures promotes degradation and isomerization, Hong et al. (2023) found no significant loss of carotenoids across various temperatures (25–65 °C) and reaction times (10–70 min) when using a 15% KOH solution [90]. These findings indicate that controlled saponification conditions can effectively process carotenoid-rich samples without excessive degradation.

In some cases, chemical modifications are applied to improve the stability, detection, and ionization efficiency of carotenoids in HPLC and MS-based analyses. These modifications enhance chromatographic behavior and refine identification and quantification. Protonation strategies, such as the use of 1% formic acid in MeOH, have been shown to stabilize diapocarotenoids and improve ionization efficiency during LC-MS analysis, facilitating the detection of low-abundance carotenoid metabolites [20]. Additionally, ammonium acetate and ammonium formate buffers are commonly used to enhance ionization stability in high-resolution mass spectrometry [18]. These approaches significantly contribute to the accurate profiling of carotenoid-derived compounds, supporting further studies on their biological roles and analytical characterization.

4. Carotenoids Analysis in Plant-Based Foods

As presented in

Table 4. Analytical methods and food sources of carotenoids.

Carotenoid	Food Source	Determination	Refs.
Lycopene	Tomatoes, Microalgae, rosehip	HPLC-DAD UHPLC-DAD SFC-PDA-QTOF MS	[8,45]
β-Carotene	Tomatoes, Sweet Pepper, Soybean, Peach Palm, Maize (Zea mays L.), Microalgae, rosehip, orange peel, Phoenix sylvestris fruit epicarp	HPLC-DAD HPLC-PAD-MS/MS HPLC-UV-VIS SFC-PDA-QTOF MSHPTLC-UV	[8,21,37,40,45,92,93,94]
Lutein	Tomatoes, Soybean, Goji Berry, Durum Wheat Pasta, Microalgae, rosehip, Phoenix sylvestris fruit epicarp	HPLC-DAD HPLC-UV-VIS SFC-PDA-QTOF MS HPTLC-UV	[12,21,37,40,45,95,96]
Zeaxanthin	Sweet Pepper, Soybean, Goji Berry, Microalgae, rosehip	HPLC-PAD-MS/MS HPLC-UV-VIS SFC-PDA-QTOF MS	[21,45,92,95]
Capsanthin	Sweet Pepper, Chili Peppers	HPLC-PAD-MS/MS HPLC-UV/Vis	[92,97]
Astaxanthin	Microalgae, rosehip	HPLC-MS SFC-PDA-QTOF MS	[45,95]
γ-Carotene	Peach Palm	HPLC-DAD	[93]

Ref., reference; HPLC, high-performance liquid chromatography; UHPLC, ultra-high-performance liquid chromatography; DAD, diode array detector; PAD, photodiode array detector; MS/MS, tandem mass spectrometry; HPLC-MS, high-performance liquid chromatography-mass spectrometry; UV-VIS, ultraviolet-visible spectroscopy; SFC, supercritical fluid chromatography.

, HPLC coupled with different detection systems remains the most commonly used method for food carotenoid determination. However, other food matrices present unique analytical challenges, requiring tailored chromatographic conditions [91]. This section explores carotenoid analysis in key plant-based food sources.

4.1. Vegetable Oils and Fats

Vegetable oils and fats, especially from avocado, palm, and olive, are rich sources of non-polar carotenoids such as lutein, lycopene, and β-carotene. However, their lipophilic nature complicates carotenoid extraction and quantification, requiring efficient solvent extraction methods as those discussed in the previous section followed by saponification to remove interfering lipids before chromatographic separation [98]. Advances in detection methods, such as DAD and MS, have improved the identification of carotenoids in complex oil matrices with greater specificity and sensitivity [99,100].

Moreover, the structural instability of carotenoids due to oxidation and thermal processing poses additional challenges, as degradation products may interfere with quantification. In this context, C30 columns are particularly useful for enhancing the separation of geometrical isomers, which frequently occur due to oil refining and storage conditions [101]. Studies focusing on olive oil analysis have demonstrated how optimizing chromatographic conditions is essential to accurately measure carotenoid content throughout processing and storage [102].

4.2. Nuts, Seeds, and Legumes

The high lipid content and complex structure of nuts, seeds, and legumes introduce additional difficulties in carotenoid extraction. These matrices require careful sample preparation to prevent lipid oxidation and improve analyte recovery. These matrices contain significant amounts of lutein, β-carotene, and zeaxanthin, especially in their raw form. To address these limitations HPLC is the primary analytical technique, often in combination with DAD or tandem MS, to enhance the simultaneous detection of multiple carotenoids in a single run [103].

In soybeans and flaxseeds, carotenoid profiling has relied on HPLC-DAD, while sunflower seeds have been investigated for both carotenoid and lipid-soluble antioxidant content. The fiber and protein matrix of legumes, such as lentils and chickpeas, can bind carotenoids, reducing their extractability [104]. To address this, researchers have adopted UPLC for improved resolution and faster analysis times, while HPLC-UV-Vis techniques have been used to assess the impact of thermal processing on carotenoid degradation. [105].

4.3. Cereal Grains and Related Products

Cereal grains, including maize, wheat, and barley, contain lower carotenoid concentrations compared to lipid-rich matrices, making high-sensitivity detection techniques essential. Yellow maize, in particular, has high lutein and zeaxanthin levels, making it a key focus in carotenoid research. Due to the presence of esterified carotenoids, saponification is commonly used before chromatographic separation, ensuring complete extraction [106,107].

Various chromatographic techniques have been developed to study carotenoids in cereal grains, particularly in biofortification research. For example, HPLC with C18 or C30 columns has been used to examine the increased carotenoid content in biofortified maize cultivars. In wheat and barley, where carotenoid levels are lower, more sensitive detection methods such as tandem MS combined with HPLC are required [108].

4.4. Emerging Plant-Based Protein Sources

New plant-based protein sources, such as microalgae, duckweed, and protein isolates from peas and soy, have gained interest due to their carotenoid-rich composition and potential nutritional benefits. However, analyzing these compounds is challenging due to the complex protein and pigment composition, which can interfere with chromatographic separation [109,110]. HPLC is widely used for carotenoid analysis in microalgae, particularly in combination with MS [111].

SFC has also been explored as an alternative for carotenoid extraction from plant-based protein sources due to its shorter run times and lower solvent usage. Advances in chromatographic techniques have enhanced research on carotenoid bioaccessibility, providing insights into the potential health benefits of including these foods in plant-based diets [112].

Carotenoids are in smaller amounts in plant-based protein isolates such as peas and soy, requiring highly sensitive detection methods. Techniques such as UPLC-MS and HPLC-MS have been beneficial for quantifying trace carotenoid levels in these matrices, supporting the development of fortified plant-based protein products [113,114].

Carotenoid quantification in different food matrices requires matrix-specific extraction and analytical strategies to account for variations in lipid content, fiber interactions, and processing effects. While HPLC remains the gold standard, techniques such as UPLC, tandem MS, and SFC are increasingly being used to enhance sensitivity and efficiency. The specific challenges posed by oils, seeds, cereals, and plant-based proteins highlight the importance of adapting chromatographic conditions to improve analytical accuracy. As plant-based diets become more popular, accurate carotenoid measurement and evaluation are increasingly crucial for understanding their functional and nutritional significance. The following sections will discuss the advantages and limitations of these techniques in greater detail, providing a comprehensive understanding of their applications in carotenoid analysis.

5. Challenges and Limitations in Carotenoids Analysis

Carotenoid analysis presents various challenges, including extraction efficiency, stability, analytical sensitivity, and regulatory compliance. While these limitations impact reproducibility and comparability across different studies, advancements in extraction and detection techniques offer solutions to mitigate these issues. The following sections discuss key challenges and how recent innovations contribute to overcoming them.

5.1. Challenges in Sample Preparation

Sample preparation is critical for ensuring efficient carotenoid recovery while minimizing degradation, environmental impact, and overall analytical performance. However, several limitations exist:

• Exhaustive extraction requirements: Carotenoids are strongly bounded to plant cell matrices and lipophilic compounds, requiring high solvent volumes and prolonged processing times in techniques such as maceration, Soxhlet extraction, and solvent-assisted extraction. This increases the risk of degradation and variability in extraction efficiency [53,54,55,56,57,58,115]. To improve extraction selectivity and reduce solvent usage, techniques such as SWE and SFE have gained attention, as they offer cleaner, eco-friendly alternatives with reduced processing times [77,78,79,80,85].
• Extensive use of organic solvents and chemicals: Traditional extraction relies on solvents like hexane and acetone, posing environmental and safety concerns [48,49,50,51,52,53,54,55,56,57,58]. While DES and ionic liquids (ILs) provide promising green alternatives, their validation for food applications remains incomplete. Further research is needed to standardize low-toxicity, biodegradable solvents for regulatory approval [42,59].
• Stability and degradation issues: Carotenoids are susceptible isomerization, and thermal degradation, affecting quantification accuracy [116]. Enzymatic-assisted extraction (EAE) and pulsed electric field (PEF) technologies have demonstrated the potential to reduce degradation by operating under mild conditions [81,110]. Additionally, nitrogen flushing and low-temperature extractions help preserve carotenoid integrity during sample preparation [117,118].
• Lack of universally applicable extraction methods: Due to matrix-dependent variations in carotenoid interactions with proteins, fibers, and lipids, extraction conditions must be tailored. [119]. A single standardized method applicable to all matrices is currently lacking, making interlaboratory comparisons and method validation even more complex. A promising solution lies in emerging hybrid approaches, such as combining UAE with enzymatic treatments, which have improved recovery in complex food matrices [75,86]. Further optimization of multi-step protocols is necessary for interlaboratory standardization.

Table 5. Advantages and limitations of carotenoid extraction technologies.

Extraction	Advantages	Limitations	Extracted Carotenoids	Refs.
SE	Simple, widely used, effective for lipophilic carotenoids	High solvent consumption, environmental concerns, potential for oxidation and carotenoid degradation	β-carotene, lycopene, lutein, zeaxanthin, phytoene, phytofluene, violaxanthin, neoxanthin	[57,58]
UAE	Reduced time, lower solvent use, enhanced mass transfer	Ultrasound intensity and frequency must be optimized, risk of oxidation due to cavitation effects	β-carotene, lutein, zeaxanthin, astaxanthin, lycopene, canthaxanthin, neoxanthin	[5,73,74,75,76]
MAE	Highly selective, fast, efficient, improved extraction yields	Heat-sensitive carotenoids may degrade, potential formation of cis-isomers, solvent selection is critical	β-carotene, lutein, lycopene, violaxanthin, zeaxanthin, canthaxanthin, capsanthin	[8,70,71,81]
SFE	Use of sostainable solvent (CO2), high purity, solvent-free extraction	Expensive equipment, requires specific pressure and temperature control for optimal yield	Lycopene, β-carotene, phytoene, phytofluene, lutein, zeaxanthin, violaxanthin	[77,78,79,80]
EAE	Mild conditions, environmentally friendly, preserves carotenoid bioactivity	Long extraction times, enzyme cost, enzyme specificity affects efficiency, limited applications	Lutein, β-carotene, zeaxanthin, violaxanthin, astaxanthin, neoxanthin, antheraxanthin	[80,82,83]
PEF	Enhances cell permeability, facilitates solvent penetration, low energy input	Requires optimization for each matrix, may not be effective for all carotenoid-rich tissues	Lutein, β-carotene, zeaxanthin, capsanthin, astaxanthin, lycopene, neoxanthin	[84,110,120]
HHPE	Effective for cell disruption, moderate temperatures prevent degradation, retains bioactivity	High-pressure equipment required, limited scalability, requires precise control of pressure conditions	Lutein, β-carotene, astaxanthin, zeaxanthin, lycopene, phytoene, phytofluene	[81,82]
SWE	Comparable to solvent efficiency, avoids organic solvents, effective for polar carotenoids	High temperatures can degrade carotenoids, may favor cis-isomerization, risk of oxidation if not properly controlled	Lutein, β-carotene, violaxanthin, neoxanthin, zeaxanthin, canthaxanthin, astaxanthin	[85,121]
OH	Uniform heating reduces oxidation risk, enhances mass transfer	Requires specialized equipment, temperature control is critical to prevent carotenoid isomerization	β-carotene, lycopene, astaxanthin, lutein, zeaxanthin, violaxanthin, capsanthin	[86,122]

Ref., reference; SE, solvent extraction; UAE, ultrasound-assisted extraction; MAE, microwave-assisted extraction; SFE, supercritical fluid extraction, EAE, enzyme-assisted extraction; PEF, pulsed electric field extraction; HHPE, high-hydrostatic-pressure extraction; SWE, subcritical water extraction; OH, ohmic heating.

summarizes the key advantages and limitations of commonly used extraction techniques and the carotenoids most frequently extracted using each method.

5.2. Challenges in Detection Systems

Carotenoid detection relies on various chromatographic and spectroscopic techniques, but each approach presents specific limitations:

• Low resolution of TLC: While TLC remains a cost-effective screening method, its low resolution restricts its ability to separate and quantify individual carotenoids, especially those with similar structures [23,33,34,35,36,37,38,39,40,41]. This limitation makes TLC unsuitable for complex food matrices where multiple carotenoids coexist. To enhance separation efficiency, high-performance thin-layer chromatography (HPTLC) with densitometric detection has been proposed, allowing for semi-quantitative analysis of carotenoids [23,33,34,35,36,37,38,39,40,41].
• High cost and environmental concerns of HPLC, UHPLC, and SFC: While HPLC and UHPLC remain the gold standards for carotenoid analysis, their high solvent consumption and operational costs limit accessibility particularly in low-resource settings [123]. Supercritical fluid chromatography (SFC) has been introduced as a solvent-reducing alternative, particularly for nonpolar carotenoids. However, wider adoption requires greater standardization of SFC methodologies to improve reproducibility and regulatory acceptance [124].
• Detection sensitivity and matrix interferences: DAD and UV-Vis spectroscopy are commonly used for carotenoid detection, but they face interference challenges in complex food matrices [125]. The integration of tandem MS (HPLC-MS/MS, UPLC-MS/MS) has significantly improved specificity and sensitivity, particularly for trace carotenoids in low-concentration samples [125,126]. These techniques are now widely used for food authentication and quality control.

5.3. Challenges in Regulatory and Standardization

Standardizing carotenoid analysis is essential for ensuring data comparability and method reliability. However, several regulatory gaps remain:

• Lack of official standardized methods for all carotenoids: While regulatory agencies such as AOAC International and the European Food Safety Authority (EFSA) provide validated methods for β-carotene analysis, official methods covering a broader spectrum of carotenoids are still lacking [127]. This gap affects the accuracy and reproducibility of carotenoid quantification across different laboratories. Standardization efforts should focus on harmonizing protocols for food safety testing and nutritional labeling.
• Limited availability of analytical standards: Certified reference materials and analytical standards are not available for all carotenoids, especially for minor and newly identified carotenoids, limiting the ability to perform accurate quantification [128]. This challenge is particularly relevant for minor carotenoids and newly discovered derivatives, where standard synthesis and commercial availability remain constrained. Expanding the availability of commercially synthesized carotenoids could support regulatory compliance and method validation.
• High determination limits (low sensitivity) and incomplete validation: Many existing methods exhibit high limits of detection (LODs) and limits of quantification (LOQs), restricting their applicability to trace-level carotenoid analysis in specific food matrices [129]. The adoption of high-resolution MS and isotope dilution methods has enhanced precision, particularly in regulatory food testing laboratories. Furthermore, incomplete analytical validation of carotenoid determination methods, including interlaboratory reproducibility studies, hinders regulatory approval and method harmonization.
• Alignment with food safety regulations: Chromatographic techniques must align with food industry regulations, particularly in regions with strict labeling requirements. For example, HPLC and MS-based methods are widely accepted in FDA and EFSA regulations for carotenoid content verification in fortified foods [130,131]. Further regulatory updates may be needed to accommodate emerging techniques, such as SFC and novel solvent-free extractions.

6. Advances in Analytical Strategies and Future Directions

Carotenoid analysis continues to evolve, driven by advancements in chromatographic techniques, detection systems, and sustainability considerations Figure 4. While HPLC and UHPLC remain the gold standard, new approaches aim to enhance separation efficiency, increase detection sensitivity, and reduce environmental impact. This section explores key innovations in separation techniques, future trends in analytical methodologies, and efforts toward sustainability in carotenoid analysis.

6.1. Innovations in Separation Techniques for Carotenoids Analysis

One of the main areas of advancement in carotenoid analysis is the development of novel packing materials for chromatographic columns. Core–shell technology, which employs superficially porous particles, has significantly improved separation efficiency by reducing band broadening and enhancing resolution compared to fully porous particles [132]. Additionally, C30 stationary phases have been optimized to improve isomer separation, which is critical for accurately distinguishing structurally similar carotenoids [13,14]. These materials allow for faster analysis times while maintaining high sensitivity, making them particularly useful in UHPLC applications [18,19,20].

Another key innovation is the adoption of sustainable mobile phases. SFC has gained attention as an eco-friendly alternative to HPLC, utilizing supercritical CO₂ as the mobile phase, thereby reducing organic solvent consumption [9]. Similarly, DES and ILs have been investigated as greener alternatives, though further validation is required before they can be widely adopted in carotenoid research [68,69,70,71,72].

Advancements in hyphenated techniques, such as UHPLC-QTOF-MS, SFC-MS, and bidimensional chromatography coupled with MS, offer enhanced structural characterization and quantification by integrating advanced chromatographic separation with high-resolution MS [51,52,53]. These approaches enable detailed profiling of carotenoids, including identifying novel derivatives and isomeric forms. Integrating IMS with MS has further enhanced the differentiation of closely related carotenoid compounds based on their collision cross-section values [32,52,53].

6.2. Future Trends in Carotenoids Analysis

Recent machine learning (ML) innovations have significantly impacted carotenoid analysis, particularly in non-invasive detection and data processing. Traditional techniques such as UPLC-MS/MS, while highly sensitive, are time-consuming and require complex sample preparation. ML-based approaches offer an alternative by improving predictive modeling, classification, and real-time quantification [133].

One of the most promising applications is deep learning-based spectral analysis. Machine learning algorithms process chromaticity measurements (L, a, b* values) to estimate carotenoid concentrations with high accuracy**, eliminating the need for destructive chemical extractions. For example, long short-term memory (LSTM) networks have been trained to predict carotenoid levels in aquaculture species, demonstrating accuracy improvements of over 450 times compared to conventional methods [133].

Additionally, ML has been applied in human plasma studies, correlating beta-carotene levels with lifestyle factors such as diet, smoking, and alcohol consumption. Rough set theory and decision trees have successfully classified plasma carotenoid levels with >90% accuracy, providing insights into how carotenoid bioavailability is affected by external variables [134]. These applications suggest that ML can be integrated into large-scale nutritional and clinical studies, making carotenoid quantification more accessible and data-driven.

Alongside AI-driven techniques, lab-on-a-chip (LOC) technologies have emerged as a revolutionary approach to miniaturizing and automating carotenoid analysis. LOC systems integrate sample preparation, separation, and detection into a single microfluidic platform, reducing reagent consumption and analysis time while enabling on-site and portable applications [135].

Recent optofluidic LOC devices have been optimized for carotenoid detection in biological samples, leveraging microchannel-based light absorption measurements. These systems have been used to analyze microalgae pigments, demonstrating high sensitivity and the ability to handle small sample volumes [135].

Additionally, miniaturized Raman spectroscopy has been integrated into LOC platforms, enhancing non-destructive, high-throughput screening of carotenoids. Portable Raman instruments have successfully detected carotenoids in complex matrices under extreme conditions, such as remote desert environments, showcasing the potential for field applications in food safety, agriculture, and biomedical research [136].

By integrating Raman spectroscopy with LOC systems, researchers aim to enhance detection capabilities while maintaining low-cost, high-throughput analysis [137]. Future developments in laser-based miniaturized systems and on-chip spectrometric detection could lead to highly portable platforms for carotenoid analysis, improving accessibility and applicability in diverse fields such as food science, agriculture, and biomedical research.

While LOC and ML approaches offer transformative improvements, challenges remain in standardizing these technologies for regulatory acceptance and ensuring compatibility with existing chromatographic protocols. Continued research is needed to validate these emerging methods against conventional techniques before they can be fully integrated into industrial and regulatory settings.

6.3. Sustainability in Carotenoid Analysis

Sustainability has become a core focus in carotenoid analysis, with researchers aiming to minimize solvent use, waste generation, and energy consumption [138]. The adoption of solvent-free extractions and micro-scale chromatography systems has been a major step forward in reducing environmental impact [64]. SWE has been successfully used to extract carotenoids from fruit and vegetable by-products, aligning with green chemistry principles [139].

Additionally, the valorization of fruit and vegetable agro-wastes has gained attention as an eco-friendly approach to carotenoid production. By utilizing peels, seeds, and other by-products, researchers are developing sustainable extraction processes that align with circular economy principles [140]. These approaches reduce food waste and enhance carotenoid bioavailability through innovative nanotechnology and encapsulation strategies.

Another promising approach is the development of biodegradable and recyclable chromatographic materials. Bio-based sorbents and membranes derived from plant-based polymers are being explored as sustainable alternatives to synthetic stationary phases, offering renewable options for column packing [64].

Finally, the development of automated and high-throughput methods improves efficiency while reducing the environmental footprint of carotenoid analysis. Automation minimizes solvent use, decreases energy consumption, and enhances reproducibility, aligning with modern green laboratory practices [139]. Additionally, life cycle assessments of extraction techniques provide valuable insights into their sustainability, helping to identify the most environmentally friendly and scalable methods for carotenoid analysis.

7. Methodology for Bibliographic Search

A structured bibliographic search was conducted using Web of Science, Scopus, PubMed, and Google Scholar to ensure a comprehensive and systematic review of the literature. The search focused on carotenoid extraction methods and analytical techniques, using predefined keywords and Boolean operators to refine the results. The keywords included terms related to extraction techniques such as solvent extraction, ultrasound-assisted extraction, supercritical fluid extraction, microwave-assisted extraction, enzymatic-assisted extraction, pulsed electric field, high-hydrostatic-pressure extraction, subcritical water extraction, and ohmic heating extraction, as well as analytical techniques such as HPLC, UHPLC, GC, TLC, and SFC.

To ensure the relevance and quality of the selected studies, only peer-reviewed articles published between 2014 and 2024 in English were considered. Studies had to focus on specific analytical techniques or their combinations, with a particular emphasis on food matrices and edible plants. Conference papers, book chapters, and preprints were excluded from the review. The selection process followed a multi-stage screening approach, beginning with a title and abstract review to filter relevant studies, followed by a full-text review for final inclusion. Five researchers conducted the screening process, starting with an independent evaluation before reaching a consensus-based selection to ensure accuracy and consistency in study inclusion. This methodology was designed to improve the transparency, reproducibility, and reliability of the literature review.

8. Final Remarks

This comprehensive review highlights the pivotal role of advanced analytical techniques in accurately assessing carotenoids in plant-based foods. HPLC remains the gold standard due to its precision and adaptability across diverse food matrices. Emerging methods such as supercritical fluid chromatography and core–shell particle technology further improve separation efficiency while minimizing environmental impact. Moreover, innovations like machine learning and lab-on-a-chip systems are promising tools for real-time, sustainable carotenoid analysis. Despite significant progress, persistent challenges remain, including carotenoid degradation, matrix interferences, and a lack of standardized reference materials. The review underscores the importance of interdisciplinary collaboration among chemistry, biochemistry, and food science to develop more robust and eco-friendly analytical methods. Furthermore, nuclear magnetic resonance (NMR) spectroscopy and omics-based approaches are identified as critical tools for advancing structural elucidation and metabolic profiling of carotenoids.

These advancements support food quality and nutritional research and have far-reaching implications for developing functional ingredients, dietary supplements, and fortified foods, ultimately contributing to public health and sustainable food systems.