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Article

A Modified Extraction and Saponification Method for the Determination of Carotenoids in the Fruit of Capsicum annuum

by
Hung Trieu Hong
1,2,*,
Rimjhim Agarwal
1,
Tatsuyoshi Takagi
1,
Michael E. Netzel
1,
Stephen M. Harper
2 and
Tim J. O’Hare
1
1
Centre for Nutrition and Food Sciences, Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Coopers Plains, Brisbane, QLD 4108, Australia
2
School of Agriculture and Food Sustainability, The University of Queensland, Gatton, QLD 4343, Australia
*
Author to whom correspondence should be addressed.
Agriculture 2025, 15(6), 646; https://doi.org/10.3390/agriculture15060646
Submission received: 6 February 2025 / Revised: 9 March 2025 / Accepted: 14 March 2025 / Published: 18 March 2025
(This article belongs to the Section Agricultural Product Quality and Safety)
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Abstract

Quantification of free and bound carotenoids in pigmented fruit and vegetable matrices has previously been challenging due to carotenoid instability, degradation during extraction, and the prevalence of predominant carotenoid esters. The aim of the present study was to develop an optimized extraction procedure that minimises the loss of free and bound carotenoids by utilising a combination of extraction solutions, followed by an improved saponification process. A mixture of hexane, dichloromethane, ethanol and water achieved the highest extraction efficiency (>97%) from the chili/capsicum matrix. The study also addressed the previously unexplained loss of carotenoids during saponification by adding phosphate buffer to the sample–extract mixture, which prevented soap micelle formation. Additionally, the duration and temperature of the saponification procedure and pH of the final extraction solution were further optimised to achieve a higher total carotenoid recovery. A total of 48 free and bound carotenoids were identified in the capsicum fruit samples using UHPLC-DAD-MS/MS. The total carotenoid content within six bell pepper and chili fruits ranged between 1.63 (green bell capsicum) and 32.08 mg/100 g fresh weight (sweet red baby capsicum). The current methodology potentially could be used in a broad range of different carotenoid-containing matrices and commodities.

1. Introduction

Capsicum annuum fruits, such as bell peppers and some chilies, are known to be rich in carotenoids, consisting of a combination of free- and esterified-carotenoids [1,2]. Although several carotenoids have been reported to exist in a free form (violaxanthin, luteoxanthin, antheraxanthin, capsanthin, capsorubin, lutein, zeaxanthin, β-cryptoxanthin, α- and β-carotene) [1,3], the hydroxyl groups at either end of xanthophyll carotenoids can undergo esterification with different fatty acids [4]. Consequently, a high number of carotenoid mono- and diesters exist naturally in both bell pepper and chili fruits [1]. Esterified xanthophylls are formed during fruit ripening [5] and are both more stable and more lipophilic than their unesterified counterparts, resulting in an increased carotenoid storage capacity in ripe capsicum fruits [6,7].
Although analytical procedures for determining carotenoids in bell pepper and chili fruit have been published [1,7,8], there is a significant issue with underestimation of carotenoid concentration due to the susceptibility of pigments to oxidants, degradation and cis-trans photoisomerization when exposed to heat, light, acids, and prolonged extraction procedures [9]. Carotenoids are often extracted along with other unwanted compounds such as chlorophylls, lipids, and fatty acids that are co-embedded in the plant cell matrix [10,11]. These additional compounds can further interfere with accurate carotenoid quantification in detection systems such as UV/VIS or photodiode array, resulting in an underestimation of carotenoid concentration. Xanthophyll esters also have similar polarity and elution times [3,4], leading to overlapping peaks and incorrect identification and quantification [12].
These challenges are due to the strong tendency of xanthophylls to remain linked to fatty acids and/or the matrix of plant cells [12], as well as their instability when exposed to prolonged extraction procedures at high temperatures [9,10]. Saponification is necessary to release the free carotenoids from their ester forms with fatty acids. The reaction releases not only carotenoids but also degrades chlorophylls [13], which eliminate any interference of these compounds with the carotenoid signals in the chromatographic system [14], a crucial issue in capsicum fruit, which gradually reduce in chlorophyll content during ripening. Although saponification enhances the recovery of zeaxanthin and β-carotene [15], it has also been reported to be the main cause for significant losses of β-carotene (20–30%) and xanthophylls (up to 50%) [16]. While saponification temperature is a key factor affecting the stability of carotenoids and the reaction rate of saponification, no published studies could be found that identified an optimum temperature to minimise carotenoid losses during saponification of bell peppers or chilies. Information about saponification temperature is very limited, and it appears that temperatures used in previous studies have been selected on a random rather than from a systematic investigation [17,18,19,20]. Furthermore, the KOH concentrations used in the saponification reaction vary considerably, from 1.1% KOH in methanol to 40% [8,18,21], as does the reaction time, ranging from a few minutes [22] to 16 h [10,12]. In addition, treating samples with potassium hydroxide during the saponification procedure may cause Cannizzaro reactions, aldol condensations, ring-fission of carotenoid non-enolizable aldehydes, carbonyl carotenoids, and carotenoid epoxides [23]. As a result, artifact organic molecules are generated during the saponification process that may further negatively affect the recovery of carotenoids.
The aim of the present study was to develop and validate comprehensive extraction and saponification procedure for the accurate quantification of carotenoids in Capsicum annuum fruits. The study also identified key steps in the extraction and saponification procedure that contribute to carotenoid underestimation. The proposed method was then applied to identify and quantify carotenoids in six different examples of bell pepper and chili fruit.

2. Materials and Methods

2.1. Plant Materials

Green, red bell capsicum and sweet baby capsicums with orange, yellow and red colour (Figure S1) [24] were purchased from Queensland growers selling at ALDI supermarket, Coopers Plains, Brisbane, Queensland, Australia. White chili fruit were purchased at local supermarkets in Brisbane, QLD, Australia. Six fruits from each variety of orange bell pepper and orange ‘Bulgarian’ were harvested randomly at the Gatton Research Facility, QLD, Australia and immediately transported to the Health and Food Sciences Precinct at Coopers Plains (QLD).

2.2. Chemicals

Zeaxanthin, capsanthin, β-carotene, and lutein standards were obtained from Sapphire Bioscience (Redfern, NSW, Australia). Other solvents and chemicals were purchased from Merck (Darmstadt, Germany) or Sigma-Aldrich (Castle Hill, NSW, Australia) at HPLC or analytical grade. Milli-Q water (Millipore Australia Pty Ltd., Kilsyth, VIC, Australia) was utilized throughout the study unless otherwise stated.

2.3. Solutions

The study used an extraction solution made of hexane and dichloromethane (7:3, v/v) containing 0.1% butylated hydroxytoluene (BHT). The solution was employed to isolate carotenoids from aqueous ethanol solution. Potassium hydroxide (KOH) pellets were dissolved in neat methanol to prepare solutions with concentrations of 3%, 5%, 10%, 15%, and 30% of KOH in methanol to investigate the impact of varying KOH concentrations on saponification efficiency. About 1 mg of lutein and zeaxanthin each was dissolved in 10 mL ethanol. Additionally, 1 mg each of β-carotene, capsanthin (external standards), and trans-β-apo-8′-carotenal (internal standard) were mixed with acetone solvent to create master stock solutions at a concentration of 100 mg/L for each carotenoid. The exact concentrations of these stock solutions were verified using a UV-Vis spectrophotometer. The real concentrations of master stock solutions were calculated from their extinction coefficient and the absorptivity reported previously [14,25] before it was used as a ‘known’ standard concentration for carotenoid analysis. Working solutions at nine concentrations from 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 20, to 50 mg·L−1 were prepared daily from the stock solutions with methanol: methyl tert-butyl ether (MTBE) (1:1, v/v) with 0.1% BHT), matrix-free solution.

2.4. Sample Preparation

A composite of 60 freeze-dried ‘Bulgarian’ fruits was selected as a representative of the chili/capsicum matrix. This variety was chosen because it contains a higher concentration of carotenoid as well as more carotenoid components compared to other chili or bell pepper varieties (Table 1) [26]. The predominant carotenoid compounds in ’Bulgarian’ chili include violaxanthin, lutein, zeaxanthin, antheraxanthin, and their esters (Table 1). In contrast, capsanthin and its esters are predominant in red chili/capsicum [27], while zeaxanthin, lutein, and their esters are more common in orange bell peppers and chili fruit [26].
Six fruits per variety were collected and stored in −20 °C. Freeze dried ‘Bulgarian’ fruits followed by frozen capsicum samples were placed into milling vessels, with liquid nitrogen added and allowed to evaporate. The vessels were then positioned in a MM400 Retsch Mixer Mill (Haan, Germany) and operated at 30 Hz for 60 s. Approximately 0.5 g of each sample was placed to a 50 mL Falcon tube for carotenoid extraction. ‘Bulgarian’ power was used throughout the optimization procedure, following five proposed methods and carotenoid analysis.

2.5. Optimisation of the Extraction Solution

In order to optimise extraction solvents for bell pepper and chili matrices, four common previous extraction solvents (M1, M2, M3, M4, and M5) were used to extract carotenoids from ‘Bulgarian’ chili fruit. Solvents tested included ethanol (M1) [11], acetone (M2) [28], a combination of hexane and acetone (M3) [4], three consecutive aliquots of a petroleum ether/methanol/ethyl acetate (1:1:1, v:v:v) mixture (M4) [29], and a combination of ethanol, 10% sodium chloride (7:3, v:v), and re-extraction with hexane and dichloromethane (DCM) (8:2, v:v) (M5) [14]. Solvents were compared and utilised under the same extraction conditions.

2.6. Optimisation of Saponification Temperature and Reaction Time

The effect of temperature on the saponification reaction was investigated using an aliquot of non-saponified extract from the ‘Bulgarian’ chili fruit. A range of different temperatures (25 °C, 35 °C, 45 °C, 55 °C, and 65 °C) was assessed, using 15% KOH in methanol as the saponifying agent for 60 min. Following the optimisation of temperature, the reaction time was optimised at 35 °C by performing the saponification reaction for 4 min, 10 min, 20 min, 40 min, 50 min, 60 min, and 70 min on an orbital shaker (RP1812, Paton Scientific, Victor Harbor, SA, Australia) at 100 rpm.

2.7. Optimisation of Carotenoid Recovery After Saponification

The recovery of carotenoids following saponification was investigated using aliquots of ‘Bulgarian’ fruit extracts saponified with 1 mL KOH (15%) in methanol under dim light and nitrogen atmosphere at 35 °C on an orbital shaker at 100 rpm for 10 min. Increasing volumes (0.5 mL, 1.0 mL, 1.5 mL, 2.0 mL, 2.5 mL, 3.0 mL, 3.5 mL, and 4.0 mL) of 2 M phosphate buffer (pH 2) with 5 mL Mili-Q water were introduced to the reaction mixture. The mixture was shaken for 10 s prior to the addition of 10 mL of the extraction solution (hexane and DCM (8:2, v:v)). The mixture was vortexed for 30 s, then centrifuged at 4000 rpm for 5 min at 25 °C. The extract (upper layer) was collected and subsequently washed with 3 mL of 0.1 M phosphate buffer (pH 7) to deacidify the extract, and then dried under nitrogen. The result samples were dissolved in 1 mL of matrix-free solution and then filtered through a 0.22 µm syringe filter into HPLC vials for carotenoid analysis.

2.8. Method Validation

The analytical procedure was validated in accordance with NATA guidelines (National Association of Testing Authorities, Australia) [30].

2.8.1. Principles of Analytical Calibration and Carotenoid Recovery

Standard solutions of each carotenoid (lutein, zeaxanthin, β-cryptoxanthin, β-carotene, and capsanthin) were prepared by spiking appropriate volumes of carotenoid stock solutions (100 µg/mL) with matrix-free solution. Five calibration curves were created for each, covering the concentration range of 0.01–40 µg/mL for the above listed external carotenoid standards. These calibration curves were employed to quantify carotenoids in the chili/capsicum samples.
Recoveries of carotenoids (β-carotene, β-cryptoxanthin and lutein) during the extraction, and saponification procedure were determined by spiking appropriate volumes of lutein, β-cryptoxanthin, and β-carotene stock solutions with the matrix solution (the extract of white chili fruit) to generate low (3 µg/mL), medium (10 µg/mL), and high (30 µg/mL) concentrations. The spiked mixtures were extracted and saponified following the above procedure with seven (n = 7) technical replicates. The spiked extracts were analysed, and recovery rates were determined by comparing the detected concentrations with the spiked concentrations of lutein, β-cryptoxanthin, and β-carotene.

2.8.2. Limit of Detection (LOD) and Quantification (LOQ)

The LOD and LOQ were established based on a signal-to-noise ratio of 3:1 and 10:1, respectively. The white chili extract (the matrix solution) was spiked with lutein, capsanthin, β-cryptoxanthin, and β-carotene to determine the LOD and LOQ of the instrumental method.

2.8.3. Matrix Effect

Lutein, capsanthin, β-cryptoxanthin, and β-carotene were spiked into the matrix solution and matrix-free solution at a concentration range of 0.01–40 µg/mL. The matrix effect (ME) was calculated by the differences of the slopes of the calibration curves (Ss/Sm) in the matrix-free solution and in the matrix solution. The relative ratio of the two slopes indicates the effect of matrix components on the signal strength of carotenoids [30].
% ME = ( S m S s − 1 ) ∗ 100 %

2.8.4. Precision and Accuracy

β-carotene, lutein, and capsanthin were selected to analyse accuracy and precision of the current method due to their predominance in chili/capsicum fruit. In addition, lutein (yellow) and capsanthin (red) represent xanthophylls, whilst β-carotene represents carotenes. β-carotene, lutein, and capsanthin were spiked into the white chili sample at three different concentrations (low-3 µg/mL, medium-10 µg/mL and high-30 µg/mL) over three consecutive days. Two UPLC-PDA instruments were employed, operating under the same instrumental conditions.

2.9. Carotenoid Analysis

Samples (approximately 0.5 g of flesh tissues) were mixed with ethanol (7 mL) containing 0.1% BHT (w/v). The mixture was vortexed for 20 s at room temperature. Sodium chloride (3 mL, 10% (w/v)) was introduced to assist with the layer separation. Two immiscible liquids (layers) were generated by adding 10 mL of hexane/dichloromethane (DCM) (8:2, v:v), and carotenoids were extracted from the top-layer after centrifugation at 4000 rpm for 5 min at 25 °C (Eppendorf 5804R and 5810R centrifuge, Eppendorf, Hamburg, Germany). The upper layer was collected to a 50 mL falcon tube, while the pellet was extracted two more times until the pellet was discoloured. The combined upper layers were then evaporated using a centrifugal evaporator (mi-Vac Duo concentrator Genevac, model DUP-23050-H00, Ipswich, UK) at 25 °C, over 45 min. A nitrogen stream was utilised to dry samples before being reconstituted in 2 mL of matrix-free solution. While one aliquot of non-saponified extract (1 mL) was filtered through a 0.22 µm syringe filter, then transferred into HPLC vials for carotenoid analysis, and another aliquot (1 mL) was saponified with 1 mL of KOH 15% (w/v) in methanol for 10 min under nitrogen atmosphere. The mixture was placed on an orbital shaker (RP1812, Paton Scientific, Victor Harbor, SA, Australia) at 100 rpm for 60 min at 35 °C. The saponification reaction was stopped by adding 3 mL of buffer (2 M, pH 2) so that the final mixture had a pH-value of 3. This mixture was then subjected to a liquid–liquid extraction using 10 mL of the extraction solution, and centrifuged at 3000 rpm for 2 min. The carotenoid layer was extracted and washed with 3 mL phosphate buffer (0.1 M, pH 7). The solvent was evaporated using a nitrogen stream, and the pellet was dissolved in 1 mL of matrix-free solution and filtered via a 0.22 µm syringe filter into HPLC vials for carotenoid analysis.

2.10. Carotenoid Identification and Quantification

2.10.1. Carotenoid Identification

Carotenoids were identified using a Shimadzu UHPLC-DAD-APCI-MS (Shimadzu, Kyoto, Japan) equipped with a Nexera X2 UHPLC system consisting of two degassers (DGU-20A3R and DGU-20A5R), a system controller (CBM-30A), an autosampler (SIL-30AC), three pumps (LC-30AD), column heater (CTO-20AC) and diode-array detectors (DAD) detector (SPD-M30A). The Nexera X2 UHPLC system was coupled to a MS-8060 triple quadrupole mass spectrometer (Shimadzu) and the APCI source was operated with a nebulizer gas flow of 3 L/min, drying gas flow of 5 L/min, desolvation line (DL) temperature of 200 °C, interface temperature of 350 °C, and heat block temperature of 200 °C. Full MS scans in the positive mode were operated in the range of m/z 100–1200. Labsolutions LCMS software Ver.5.85 (Shimadzu) was used for instrument control and data-processing. The absorption spectra of carotenoids exhibit three maxima in the visible range of the spectrum, between 400 and 500 nm [25]. Therefore, the current study monitored carotenoids by a DAD detector at 450 nm and additional spectra in the range of 190–800 nm [12,31]. External standards of lutein, violaxanthin, capsanthin, zeaxanthin, β-carotene and β-cryptoxanthin were spiked into the extract samples to determine their specific elution times.

2.10.2. Carotenoid Quantification

Carotenoids were quantified by a Waters AcquityTM UPLC-PDA system (Waters, Milford, MA, USA). UV/Vis spectra were captured in the range of 190–600 nm and carotenoids were analysed at 450 nm using EmpowerTM software (Empower 3.0, Waters Corporation, Milford, MA, USA). Chromatographic separation was carried out on a YMC C30 Carotenoid Column (250 mm × 4.6 mm ID, 3 μm; Waters) at a flowrate of 0.6 mL/min and a column oven temperature of 25 °C. The 40 min gradient elution started isocratic at 20% mobile phase B (MTBE, 0.1% formic acid) and 80% mobile phase A (methanol, 0.1% formic acid) for 1 min, increasing to 25% B in 18 min and 30% within 9 min, before a sharp increase to 70% in 4 min, then holding for 2 min, conditioning for 1.2 min, and finally re-equilibrating for 4.8 min. The concentrations of individual carotenoids were determined by external calibration curves of lutein, zeaxanthin, capsanthin, and β-carotene. Minor carotenoid compounds and carotenoid esters were calculated as “equivalents” using the calibration curve of one of the main carotenoids resembling the chemical structure best. The total carotenoid content was calculated as the sum of all detectable (≥LOQ) carotenoid compounds in chili/capsicum fruit.

2.11. Statistical Analysis

A one-way analysis of variance (ANOVA) was used to access variances in the carotenoid content in chilies/capsicum fruit during method validation and optimization, including temperature, time, extraction solutions, and potassium hydroxide concentration. Statistical analyses were performed using Minitab 17 software for Windows (Minitab Inc., State College, PA, USA). Between three and seven technical replicates (n = 7) were used during the optimization and validation process. Differences between means were regarded as significant if the p-value was equal or less than 0.05.

3. Results

3.1. Optimisation of the Extraction Solution and pH Levels for the Recovery of Carotenoids During the Saponification Procedure

Carotenoid extraction efficiency was found to vary significantly (p < 0.05) between the five extraction solutions tested (Figure 1A). M4 and M5, in which a combination of methanol/ethyl acetate/petroleum ether or a combination of ethanol, 10% sodium chloride (7:3, v:v) and re-extraction with hexane and dichloromethane (DCM) (8:2, v:v) were utilised, exhibited the best extraction efficiency of carotenoids from the ‘Bulgarian’ fruit, with the extraction capacity up to 99.6% (133.1 mg/100 g DW) and 100% of carotenoid (133.6 mg/100 g DW), respectively. Pure (100%) ethanol alone (M2) was the least efficient extraction solution, with only 45.4% (60.6 mg/100 g DW) of carotenoids extracted. The results indicate the important functionality of a combination of different solvents in liquid–liquid extraction. Aqueous ethanol penetrates better to the cell walls than pure organic solvent, and efficiently extracts more carotenoids from the sample matrix, showing a similar extraction efficiency of aqueous ethanol to what was found in a previous study of anthocyanin in purple sweetcorn [32]. Liquid–liquid extraction also accelerates the extraction efficiency, excludes contaminants, and simplifies quantification steps. In contrast, pure organic solvents exhibit a lower degree of extraction due to instantly coagulating proteins in plant cells, which prevents further penetration of extraction solvents [33].

3.2. Optimised Saponification Temperature and Saponification Duration

The present trial indicated that the range of saponification temperatures and saponification times tested did not significantly differ (p < 0.05) in their effect on carotenoid recovery (Figure 2A,B). Although a saponification temperature of 35 °C and a saponification time of 10 min yielded the greatest carotenoid concentration, temperature between 25 to 65 °C, and times between 4 and 70 min were not significantly different. These findings indicate that saponification of the chili tissue extract was a fast reaction. Therefore, prolonging the reaction time or increasing the reaction temperature had no effect on carotenoid recoveries, despite both factors being previously reported to accelerate the degradation rate, cis-trans isomerization, and rearrangement of carotenoids [9,10].

3.3. Method Validation

3.3.1. Recovery of Carotenoids During Saponification

The optimisation of the temperature and the length of reaction for the saponification procedure was carried out to assess the recovery of the main carotenoids in ‘Bulgarian’ chili fruit. External standards (β-cryptoxanthin, β-carotene, and lutein) were spiked into the sample matrix (white chili extract) at three concentrations (low, 3 µg/mL; medium, 10 µg/mL; and high, 30 µg/mL) that cover the common concentration range of carotenoid observed in chili/capsicum fruit [8,27]. The results show that the new extraction and saponification procedure improved the recovery of carotenoids in the chili matrix (Table S1) [24]. More than 97% recovery of carotenoid was found after saponification, compared to previous studies that reported much lower recoveries of zeaxanthin and lutein (63.7% and 43.8, respectively) [20]. Similarly, Divya et al [16] reported a loss of 50% lutein occurring during extraction and saponification when water was used to remove the remaining KOH (alkali) after saponification.

3.3.2. LOD, LOQ, and Matrix Effect

The current LODs and LOQs of β-cryptoxanthin (0.05 and 0.15 mg·L−1), β-carotene (0.03 and 0.09 mg·L−1), lutein (0.06 and 0.18 mg·L−1), and zeaxanthin (0.05 and 0.15 mg·L−1) are shown in Table S1 [24]. The LOD and LOQ values are consistent with previous values reported with other fruits and vegetables [34]. The matrix effect was assessed by comparing the standard curves of the matrix-free extract solution with those of the white chili matrix, in which no carotenoids were present. A high coefficient of determination (R2 > 0.99) was obtained from the calibration curves of β-carotene, zeaxanthin, and lutein with the matrix effect being less than 7.5%.

3.3.3. Precision and Accuracy

The relative variation of the current analytical method was determined by assessing the relative changes of β-carotene, lutein and zeaxanthin at three different concentration levels that were spiked to samples over three days, with seven replicates (n = 7). The observed percentage of the relative standard deviation (RSD%) from 1.3 to 5.4% for intra-day variation, and from 1.7 to 8.7% for inter-day variation, indicated a reliable method for carotenoid quantification. The analysis of spiked samples across two different instruments resulted in the RSD% ranging from 3.7 to 7.7%. Overall, the obtained validation results indicated that the current UHPLC-PDA method is reproducible, accurate and reliable (Table S2) [24].

3.4. Identification of Carotenoids in Chili/Capsicum Fruit

External standards of β-carotene, β-cryptoxanthin, zeaxanthin, lutein, and violaxanthin were spiked at different concentrations to identify their specific retention times, molecular masses, and product ions to aid in the identification of the main carotenoids in ‘Bulgarian’ chili and other pigmented capsicums. Other carotenoids were determined based on their parent masses, specific fragmentation patterns and comparison with literature reports (Table 1 and Figure S2) [24]. For example, zeaxanthin dipalmitate (m/z 1045.8, Figure S2) was identified by the molecular ion at m/z 1045.8 and the fragment ion at m/z 789.7 [M + H − 256]+ corresponding to the loss of a palmitic acid. Likewise, zeaxanthin myristate-palmitate (m/z 1017.8) was confirmed by the detection of its fragment ion at m/z 789.6 [M + H − 228]+, representing the loss of myristic acid. The fragment ion at m/z 761.6 [M + H − 256]+ corresponded to zeaxanthin myristate-palmitate after elimination of palmitic acid and m/z 533.4 [M + H − 256–228]+ representing the loss of myristic acid and palmitic acid (Figure S2) [24].

3.5. Quantification of Carotenoids in Pigmented Chili/Capsicums

Red sweet baby capsicum (RSC) had the highest carotenoid concentration (32.08 mg/100 g FW (fresh weight)), followed by red bell capsicum (RC, 27.10 mg/100 g FW). Orange sweet baby capsicum (OSC) contained a significant higher carotenoid, 25.29 mg/100 g FW, than orange bell capsicum, 18.09 mg lutein equivalents/100 g FW. Yellow sweet baby capsicum (YSC) and green bell capsicum (GC) had the least carotenoid content with 3.92 and 1.63 mg/100 g FW, respectively. ‘Bulgarian’ chili fruit displayed a similar carotenoid content to RSC, 30.17 mg lutein equivalents/100 g FW. The findings are in agreement with previous studies reporting the carotenoid concentration in pigmented capsicums [35,36].

4. Discussion

Saponification is a widely used procedure for analysis of carotenoids in vegetables and fruits. It helps to remove unwanted lipids and chlorophyll while hydrolysing carotenoid-esters to release free carotenoids [9]. During saponification, the reaction of fatty acids and lipids with potassium hydroxide (KOH) produces soap, which acts as a detergent on lipophilic carotenoids in the aqueous phase within the aqueous extraction layer to create micelles. This forms micelles, creating a physical barrier that inhibits free carotenoids from transferring into the non-aqueous extraction layer.
As a result, low carotenoid recovery during saponification has been reported in numerous studies, and was previously thought to be due to a high degradation/oxidisation rate of carotenoids within alkaline environments [9,10,37]. However, a recent study suggest that micelle formation, rather than degradation alone, is the primary cause of reduced carotenoid recovery reported by Hong et al [14]. Our results support this hypothesis. As shown in Figure 1B, acidifying the solution after saponification significantly improved carotenoid recovery from ‘Bulgarian’ chili fruit (p < 0.05). While decreasing the pH from 7.0 to 3.0 showed only a slight trend (not significant), neutralizing the alkaline conditions (pH from 12.5 to 7.0) led to a significant increase in carotenoid recovery from 89.2 mg/100 g FW to 125.5 mg/100 g FW, respectively. This suggests that neutralizing the solution and inhibiting soap formation is key to improving carotenoid extraction.
In this study, we replaced the water (typically used in previous studies to facilitate phase separation) [20,38] with the addition of a 2 M aqueous phosphate buffer (pH 2), which neutralized the pH to 7.0 or below. The presence of the highly ionized sodium phosphate in the buffer helped to prevent soap formation from the carotenoid esters, further enhancing carotenoid recovery.
The HPLC-PDA chromatogram of the carotenoid profile at 450 nm of “Bulgarian” chili before saponification (A) revealed that the extraction solution had 48 different carotenoids, eluted from 6 min to 39 min. These findings were similar to previous research on carotenoid profiles in red pepper pods, where 42 non-esterified carotenoids and carotenoid esters were found [4]. The presence of 23 carotenoids, eluted within a duration of 31–39 min indicates their similar chemical forms and polarities. These last 23 compounds were also the predominant carotenoids in the “Bulgarian” chili fruit. The differences in absorption maximum λmax (Table 1) serve as a key classification component for different xanthophylls, such as λmax 444 nm indicating lutein’s esters, λmax 450 nm -zeaxanthin’s esters, λmax 427 nm -mutatoxanthin’s esters, or λmax 422 nm- luteoxanthin’s esters. Selected ion monitoring (SIM) in positive and negative modes of the molecular ion indicate that they are mono or diesters of xanthophylls with fatty acids, such as lauric acid, myristic acid, butyric acid, and palmitate acid (Table 1). The overlay of mass spectra in Figure 2B indicates many co-eluted xanthophyll’s esters, which is a major issue for quantification of individual carotenoids. In contrast, Figure 3C shows a simpler carotenoid profile of “Bulgarian” chili after saponification. Consequently, quantifying carotenoids in “Bulgarian” chili after saponification was achieved using external standards.
Carotenoid esters are the predominant carotenoid compounds in ‘Bulgarian’ chili and other pigmented capsicums before saponification. These esters are formed by the attachment of fatty acids, such as lauric acid, myristic acid, and palmitic acid, to the carotenoid molecule (Table 1). Carotenoid esters are highly diverse in terms of isomerization and often coelute with other carotenoids or carotenoid esters, making them difficult to quantify due to overlapping retention times and high background noise in chromatographic analysis (Figure 3A,B). This coelution was a primary reason for optimizing the saponification procedure in this study.
After saponification, the carotenoid profile of “Bulgarian” chili became simpler, with 16 different carotenoids being identified. This carotenoid profile is in agreement with previous findings on carotenoids in chili and capsicum fruits [4,26]. Individual carotenoids were identified and quantified based on externally available standards, molecular mass, masses of fragment ions, and their unique λmax, as shown in Table 2 [4].
The carotenoid profiles of red, orange, yellow, and green capsicums observed in this study are in agreement with previous reports [1,2]. Interestingly, despite the similar orange pigmentation of orange bell capsicum, sweet baby capsicums, and ‘Bulgarian’ chili, lutein (a yellow carotenoid) was the predominant carotenoid in ‘Bulgarian’ chili, while zeaxanthin (an orange carotenoid) dominated in the orange capsicums (Table 3). This finding highlights the diversity of carotenoid composition in pigmented capsicums, even among varieties with similar coloration.
Overall, carotenoid concentrations varied significantly (p < 0.05) among red, orange, and yellow capsicum cultivars. In addition to differences in total carotenoid content, the predominant carotenoids also differed between varieties. For example, red capsicums contained approximately 60% capsanthin (a red carotenoid), while orange mini sweet capsicums had 52% zeaxanthin (an orange carotenoid). Zeaxanthin also dominated in orange bell capsicum (OC), making up 64% of the total carotenoids. In contrast, ‘Bulgarian’ chili and yellow sweet baby capsicum (YSC) had more than 50% lutein (a yellow carotenoid), which was the predominant carotenoid in both ‘Bulgarian’ chili (about 50%) and green bell capsicum (GC) (approximately 31%).

5. Conclusions

The present study optimised the carotenoid extraction and saponification procedure to more accurately determine carotenoid concentration in chili/capsicum fruit using UHPLC-DAD-MS/MS. A combination of four solvents (hexane, dichloromethane, ethanol, and water) efficiently extracted the carotenoids from these complex plant matrices. A fully validated method with both low LOQ and high accuracy to quantify carotenoids following saponification was developed. The method is applicable to a broad range of pigmented chilies and capsicums and potentially to other carotenoid-containing commodities. A total of 48 carotenoid compounds, including free carotenoids and esters, could be identified in ‘Bulgarian’ chili using UHPLC-PDA-MS/MS. Importantly, the main reason for poor carotenoid recovery during the saponification procedure was identified and remedied by using phosphate buffer to stop the detrimental effect of soap formation. As a consequence, a considerably higher recovery of carotenoids (>97%) was achieved after saponification.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agriculture15060646/s1, Figure S1: Orange (A), green and red bell paper capsicum (B); ‘Bulgarian’ chili (C); orange, yellow and red sweet baby capsicums (D); Figure S2: Fragmentation and mass spectrum of zeaxanthin dipalmitate (A) and zeaxanthin myristate-palmitate (B) in positive ion mode conducted by a Shimadzu UHPLC-MS/MS; diagrams of fragmentation locations within zeaxanthin dipalmitate (A) and zeaxanthin myristate-palmitate (B) have been indicated; Table S1: LOD and LOQ of lutein, β-carotene, zeaxanthin, and β-cryptoxanthin in sample-free matrix solution, and recovery of lutein and β-carotene at three different concentrations. Data are means of technical replicates (n = 7); Table S2: Precision and accuracy of the optimised extraction and saponification method using the sample matrix.

Author Contributions

Conceptualization, H.T.H.; methodology, H.T.H.; software, H.T.H.; validation, H.T.H., T.T. and R.A.; formal analysis, H.T.H.; investigation, H.T.H.; and T.J.O.; resources, T.J.O.; writing—original draft preparation, H.T.H.; writing—review and editing, H.T.H., T.T., M.E.N., S.M.H., T.J.O. and R.A.; supervision, T.J.O.; project administration, T.J.O.; funding acquisition, T.J.O. All authors have read and agreed to the published version of the manuscript.

Funding

This project was partially funded by Hort innovation, Australia as part of ‘Naturally Nutritious’ project (HN15001).

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The original data presented in the study are openly available in FigShare at [https://doi.org/10.6084/m9.figshare.28464680, accessed on 13 March 2025].

Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations

The following abbreviations are used in this manuscript:
UV/VISultraviolet–visible spectroscopy
KOHpotassium hydroxide
HPLCHigh-performance liquid chromatography
QLDQueensland
BHTbutylated hydroxytoluene
MTBEmethyl tert-butyl ether
DCMdichloromethane
NATANational Association of Testing Authorities, Australia
LODlimit of detection
LOQlimit of quantification
MEmatrix effect
UHPLCUltra high performance liquid chromatography
DADDiode array detector
APCIAtmospheric pressure chemical ionization
MSMass spectrometer

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Agriculture 15 00646 g001
Figure 1. Optimised extraction solution for ‘Bulgarian’ chili (A), M1: acetone, M2: ethanol, M3: acetone/hexane (1:1 v/v), M4: a petroleum ether/ethyl acetate/methanol (1:1:1, v/v/v) mixture, M5: ethanol followed by sodium chloride 10% and DCM/hexane (2:8, v/v). Optimised pH levels for the best recovery of carotenoid after saponification of ‘Bulgarian’ chili fruit (B). Values within columns followed by different letters indicate significant differences (p < 0.05).
Figure 1. Optimised extraction solution for ‘Bulgarian’ chili (A), M1: acetone, M2: ethanol, M3: acetone/hexane (1:1 v/v), M4: a petroleum ether/ethyl acetate/methanol (1:1:1, v/v/v) mixture, M5: ethanol followed by sodium chloride 10% and DCM/hexane (2:8, v/v). Optimised pH levels for the best recovery of carotenoid after saponification of ‘Bulgarian’ chili fruit (B). Values within columns followed by different letters indicate significant differences (p < 0.05).
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Figure 2. Optimised saponification time (A) and saponification temperature (B) for the determination of carotenoids in ‘Bulgarian’ chili fruit. Values within columns followed by different letters indicate significant differences (p < 0.05).
Figure 2. Optimised saponification time (A) and saponification temperature (B) for the determination of carotenoids in ‘Bulgarian’ chili fruit. Values within columns followed by different letters indicate significant differences (p < 0.05).
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Agriculture 15 00646 g003
Figure 3. The carotenoid profile of ‘Bulgarian’ chili before saponification (A) and after saponification (C) by UHPLC-PDA; profiling carotenoid ester in ‘Bulgarian’ chili fruit by mass spectrometer operated by selected ion monitoring (SIM) in positive mode (B) indicated m/z values in SIM chromatograms (peak 15–48 in Table 1, with chromatogram 1 represented for m/z 779.6; chromatogram 2 for m/z 811.6; chromatogram 3 for m/z 735.5; chromatogram 4 for m/z 795.6; chromatogram 5 for m/z 783.6; chromatogram 6 for m/z 933.7; chromatogram 7 for m/z 107.8; chromatogram 8 for m/z 763.6; chromatogram 9 for m/z 961.8; chromatogram 10 for m/z. 965.6; chromatogram 11 for m/z 989.8; chromatogram 12 for m/z 1045.9; chromatogram 13 for m/z 1049.8; chromatogram 14 for m/z 993.6; chromatogram 15 for m/z 1005.6).
Figure 3. The carotenoid profile of ‘Bulgarian’ chili before saponification (A) and after saponification (C) by UHPLC-PDA; profiling carotenoid ester in ‘Bulgarian’ chili fruit by mass spectrometer operated by selected ion monitoring (SIM) in positive mode (B) indicated m/z values in SIM chromatograms (peak 15–48 in Table 1, with chromatogram 1 represented for m/z 779.6; chromatogram 2 for m/z 811.6; chromatogram 3 for m/z 735.5; chromatogram 4 for m/z 795.6; chromatogram 5 for m/z 783.6; chromatogram 6 for m/z 933.7; chromatogram 7 for m/z 107.8; chromatogram 8 for m/z 763.6; chromatogram 9 for m/z 961.8; chromatogram 10 for m/z. 965.6; chromatogram 11 for m/z 989.8; chromatogram 12 for m/z 1045.9; chromatogram 13 for m/z 1049.8; chromatogram 14 for m/z 993.6; chromatogram 15 for m/z 1005.6).
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Table 1. Identification of carotenoids detected in ‘Bulgarian’ chili using a Shimadzu UHPLC-DAD-APCI-MS/MS system in positive ion (+) and negative ion (−) mode.
Table 1. Identification of carotenoids detected in ‘Bulgarian’ chili using a Shimadzu UHPLC-DAD-APCI-MS/MS system in positive ion (+) and negative ion (−) mode.
Peak NoCarotenoidsrt (min)λ (nm)m/z
(−)		Fragment Ion (m/z) (−)m/z (+)Fragment Ion (m/z) (+)
1Unknown6.27379398420618.4582.3; 277.2; 219.1585.4391.3; 270.3; 256.3
2Violaxanthin6.72399439469600.4582.3; 448.9; 407.7601.3583.4; 565.5; 509.3
3Violaxanthin isomer6.91399439469600.4582.3; 448.9; 407.7601.3583.4; 565.5; 509.3
4(9-Z)-Luteoxanthin7.41399421448600.4448.8; 407.4601.3554.5; 536.4
5(all-E)-Violaxanthin7.81420441468600.4582.3; 448.9; 407.7601.3583.4; 565.5; 509.3
6Unknown8.09400420440600.4575.3; 407.9; 575.3601.3575.4; 397.3
7Unknown8.8400422448600.4491.3; 448.9601.3573.4; 335.3
8Unknown9.7380401426600.4582.4; 465.2; 448.4601.3583.4
9(all-E)-Antheraxanthin10401426471584.3566.3; 281.3585.4577.4, 339.3, 313.3
10(9-Z)-Antheraxanthin10.6401426471584.3566.3; 281.3585.4577.4, 339.3, 313.3
11Unknown10.9382401426600.4255.2601.3583.4; 551.4; 509.4
12(all-E)-Lutein11.7422444472568.4455.4; 450.0569.4551.4; 459.3; 335.3
13(all-E)-Mutatoxanthin12.2400427452584.4568.4; 464.5; 449.7585.4567.4; 409.4; 575.5
14(all-E)-Zeaxanthin13.6425450475568.4465.5; 449.6; 430.1569.4551.4; 177.6
15(all-E)-Cryptoxanthin *18.5423445472552.4784.6; 654.6; 466.6553.4767.5; 533.2, 453
16Luteoxanthin laurate *19.2400427448782.5465.9; 277.2783.6765.5; 597.4; 335
17Luteoxanthin myristate *19.6399427448810.5782.5811.5811.5
18β-Cryptoxanthin22.6422446470552.4536.4; 464.9553.4595.4
19Luteoxanthin myristate *23.8400422448810.5684.8; 464.9811.6793.6; 599.5
20Luteoxanthin myristate isomer *24.1400422448810.5536.4811.6537.3; 793.6
21(all-E)-carotene *27.1379399423536.4794.5;537.4851.6; 449.6, 383.3
22Antheraxanthin myristate *27.5425443471794.6810.5; 552.4795.5851.6; 553.4
23β-Carotene *29423446473536.4810.6; 766.5; 838.5537.4853.6; 839.6; 811.6
24Violaxanthin butyrate-laurate *30400441468853.7750.5; 766.5; 810.5855.6733.5; 767.5; 811.6
25Antheraxanthin myristate *31.1425444470794.5932.6795933.6
26Violaxanthin caprate-myristate *31.4425448475964.6838.6; 794.6; 750.5965.6839.6; 795.6; 751.5
27β-Carotene isomer *31.7425451476536.4750.5; 964.7; 855.6537.4965.7; 857.7; 795.6; 751.5
28Antheraxanthin myristate *32.1425446470794.6838.5795.6839.6; 601.5; 533.4
29Lutein laurate-myristate *32.6415444470959.7992.6; 750.5; 734.5961.8993.7; 751.5; 735.6
30Unknown *32.9422444470881.7992.7; 959.7883.7993.7; 961.8
31Zeaxanthin myristate *33.1422448475778.5536.4779.6577.5; 551.4; 537.4
32Lutein laurate-laurate *34.2422444471992.61020.7; 883.6; 734.5993.61021.8; 885.7; 735.6
33Lutein myristate-myristate *34.5400423448987.7948.6; 992.6989.8993.7; 965.6; 949.6
34Antheraxanthin laurate-laurate *34.7423448472948.71048.7; 1021.7, 992.7949.71049.7, 1021.7, 993.7
35Unknown *35.04044264461020.7992.61021.7965.7; 909.7
36Cryptoxanthin laurate *35.4425449475734.51048.7; 1021.7735.51049.7, 1021.7
37Violaxanthin myristate-palmitate *35.54264464721048.71020.7; 762.5; 734.51049.81021.8; 735.6; 765.5
38Cryptoxanthin myristate *35.6425447473762.61020.7; 734.5763.61021.8; 735.5
39Violaxanthin myristate-myristate *35.84274444691020.7762.6; 734.51021.8763.6; 735.7
40Antheraxanthin laurate-myristate *36.14254464721004.6948.7; 734.51005.6949.8; 735.6
41Violaxanthin palmitate-palmitate *36.54254484751076.71004.7; 1048.71077.61049.8; 965.7; 1005.8; 763.6
42Violaxanthin myristate-myristate *36.94254464721020.61004.7; 976.71021.71005.8, 977.7; 733.5
43Zeaxanthin laurate-laurate *37.1425450477932.61076.8, 761.5933.71076.8; 762.5
44Lutein myristate-palmitate *37.74254484721048.71032.7; 1004.7; 986.71049.81033.8; 1005.8; 987.7
45Zeaxanthin laurate- myristate *37.9425450477960.71004.7; 760.6961.71005.8, 761.6
46Zeaxanthin myristate-myristate *38.5425450476988.71032.7; 1016.8989.81033.8; 1016.8
47Zeaxanthin myristate-palmitate *39.34254514771016.71060.6; 449.71017.81061.9; 789.6
48Zeaxanthin palmitate-palmitate *39.94254514781044.81016.7; 963.51045.91017.8; 789.6; 761.6; 533.4
* Carotenoids coeluted with others carotenoid esters; C12: lauric acid; C14 myristic acid; C16 palmitic acid.
Table 2. Identification of the main carotenoids detected in pigmented capsicums after saponification.
Table 2. Identification of the main carotenoids detected in pigmented capsicums after saponification.
CarotenoidsRT (min)λmax (nm)m/zMS/MS
Luteoxanthin7.4400, 424, 450601.5583.4; 554.5; 536.4
Violaxanthin6.72; 6.91; 7.8420, 439, 468601.5583.4; 565.5; 509.3
Capsorubin9.2480601.5583.5; 430.4; 176.9
Antheraxanthin9.8420, 444, 471585.5561.5; 545.5; 401.3
Cis-capsanthin10.6468585.5567.4
(all-E)-Lutein11.5422, 444, 472569.4552.5; 431.2; 176.8
(all-E)-Mutatoxanthin12.0400, 427, 452585.5567.4; 409.4; 575.5
(all-E)-Capsanthin12.7473585.5567.5; 493.4; 479.4
(all-E)-Zeaxanthin13.3425, 450, 478568.4552.4; 431.2; 176.8
Cis-Capsanthin14.8468585.5567.4
α-Cryptoxanthin18.1425, 450, 484533.4177.8
β-Cryptoxanthin22.2425, 451, 484533.4178.0; 120.7
α-Carotene28.1420, 447, 474537.5177.6
Cis-α-Carotene29.7420, 447, 474537.5177.6
β-Carotene31.4425, 451, 478537.5177.6
Cis-β-Carotene32.7423, 447, 472537.5178.3
Table 3. Individual carotenoid and total carotenoid concentration in pigmented capsicums (RSC: red sweet capsicum, RC: red capsicum, OSC: orange sweet capsicum, OC: orange capsicum, GC: green capsicum, YSC yellow sweet capsicum, and BgC: ‘Bulgarian’ chili) determined by UHPLC-DAD at 450 nm.
Table 3. Individual carotenoid and total carotenoid concentration in pigmented capsicums (RSC: red sweet capsicum, RC: red capsicum, OSC: orange sweet capsicum, OC: orange capsicum, GC: green capsicum, YSC yellow sweet capsicum, and BgC: ‘Bulgarian’ chili) determined by UHPLC-DAD at 450 nm.
SamplesRSCRCOSCOCGCYSCBgC
mg/100 g FW
Violaxanthin1.79 ± 0.111.89 ± 0.080.12 ± 0.010.05 ± 0.000.29 ± 0.020.21 ± 0.012.53 ± 0.41
Luteoxanthin1.07 ± 0.060.97 ± 0.020.71 ± 0.06ndnd0.22 ± 0.021.37 ± 0.33
Antheraxanthin1.82 ± 0.141.53 ± 0.050.04 ± 0.010.26 ± 0.030.04 ± 0.000.02 ± 0.001.51 ± 0.24
Capsorubin1.68 ± 0.081.88 ± 0.02ndndndndnd
Cis-Capsanthin1.31 ± 0.071.63 ± 0.020.03 ± 0.00ndnd0.01 ± 0.00nd
Lutein0.43 ± 0.070.39 ± 0.033.20 ± 0.202.76 ± 0.170.50 ± 0.021.78 ± 0.1015.55 ± 0.48
Capsanthin17.93 ± 0.8815.92 ± 0.2ndnd0.33 ± 0.01ndnd
Mutatoxanthin0.61 ± 0.050.51 ± 0.043.85 ± 0.180.69 ± 0.110.11 ± 0.010.36 ± 0.102.75 ± 0.05
Zeaxanthin1.97 ± 0.240.54 ± 0.0213.15 ± 1.0111.51 ± 1.090.21 ± 0.030.47 ± 0.013.99 ± 0.18
α-Cryptoxanthin0.07 ± 0.000.06 ± 0.000.24 ± 0.020.79 ± 0.11nd0.14 ± 0.010.66 ± 0.19
β-Cryptoxanthin0.78 ± 0.090.17 ± 0.021.01 ± 0.090.76 ± 0.09nd0.05 ± 0.010.56 ± 0.06
α-Carotene0.71 ± 0.150.46 ± 0.061.31 ± 0.210.53 ± 0.080.03 ± 0.000.41 ± 0.050.24 ± 0.12
β-Carotene1.91 ± 0.391.15 ± 0.221.61 ± 0.140.76 ± 0.080.10 ± 0.020.25 ± 0.031.01 ± 0.05
Total carotenoid concentration32.08 ± 2.2527.10 ± 0.4225.29 ± 1.5018.09 ± 1.761.63 ± 0.113.92 ± 0.3230.17 ± 2.11
nd: carotenoids were not detected, or the signals were lower than the LOQ; data are means ± SD, n = 3.
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Hong, H.T.; Agarwal, R.; Takagi, T.; Netzel, M.E.; Harper, S.M.; O’Hare, T.J. A Modified Extraction and Saponification Method for the Determination of Carotenoids in the Fruit of Capsicum annuum. Agriculture 2025, 15, 646. https://doi.org/10.3390/agriculture15060646

AMA Style

Hong HT, Agarwal R, Takagi T, Netzel ME, Harper SM, O’Hare TJ. A Modified Extraction and Saponification Method for the Determination of Carotenoids in the Fruit of Capsicum annuum. Agriculture. 2025; 15(6):646. https://doi.org/10.3390/agriculture15060646

Chicago/Turabian Style

Hong, Hung Trieu, Rimjhim Agarwal, Tatsuyoshi Takagi, Michael E. Netzel, Stephen M. Harper, and Tim J. O’Hare. 2025. "A Modified Extraction and Saponification Method for the Determination of Carotenoids in the Fruit of Capsicum annuum" Agriculture 15, no. 6: 646. https://doi.org/10.3390/agriculture15060646

APA Style

Hong, H. T., Agarwal, R., Takagi, T., Netzel, M. E., Harper, S. M., & O’Hare, T. J. (2025). A Modified Extraction and Saponification Method for the Determination of Carotenoids in the Fruit of Capsicum annuum. Agriculture, 15(6), 646. https://doi.org/10.3390/agriculture15060646

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