Abstract
This study presents the development and validation of a robust HPLC-UV method for the simultaneous detection and quantification of vitamin D2, vitamin D3, ergosterol, and 7-dehydrocholesterol in a single analytical run, adhering to ICH Q2 (R1) guidelines. Application of the HPLC-UV method to six vegetable oils demonstrated inherent vitamin D content and its enhancement under different radiation conditions. Oils were exposed to direct sunlight, UVA, and UVB radiation, with UVB showing the most significant impact on vitamin D levels. Notably, UVB exposure increased vitamin D3 in castor oil from 2.82 μg mL−1 to 4.29 μg mL−1 and vitamin D2 in mustard oil from 0.73 μg mL−1 to 1.89 μg mL−1. The findings highlight the potential of UVB radiation to convert vitamin D precursors to active forms more effectively than other radiation sources. This study provides a novel approach for naturally enriching vitamin D in vegetable oils, offering a promising strategy to mitigate vitamin D deficiencies in populations with limited sun exposure or dietary restrictions. The developed HPLC-UV method and resultant insights into the radiation-induced enhancement of vitamin D content in oils contribute valuable knowledge to nutritional science.
1 Introduction
Vitamin D, an essential fat-soluble secosteroid, plays a crucial role in human health, encompassing calcium homeostasis, bone health, immune modulation, cellular proliferation, and gene expression regulation [1–6]. The primary forms of vitamin D are vitamin D2 (ergocalciferol) and D3 (cholecalciferol), which are derived from their precursors ergosterol and 7-dehydrocholesterol [7, 8]. Endogenous synthesis, driven by UVB radiation, remains a principal source of vitamin D. UVB catalyzes the conversion of 7-dehydrocholesterol to pre vitamin D3, which then isomerizes to vitamin D3 [9–11]. Similarly, ergosterol in fungi and yeast converts to vitamin D2 upon UVB exposure [12–16]. Natural sources of vitamin D include sunlight, fatty fish, cod liver oil, egg yolks, and fortified foods [17, 18]. However, limited sun exposure, dietary restrictions, and cultural practices necessitate finding additional vitamin D sources [19–21]. Vegetable oils, rich in vitamin D precursors, offer a promising source of vitamin D through photo mediated biotransformation upon exposure to sunlight or artificial UV radiation. This potential enhancement of dietary vitamin D intake is particularly valuable in regions with limited sunlight [22, 23]. The efficiency of this conversion is influenced by the oil matrix composition and specific radiation wavelengths, making it essential to understand these interactions for optimizing the use of vegetable oils as dietary vitamin D sources.
The conversion of ergosterol to vitamin D2 and 7-dehydrocholesterol to vitamin D3 is a remarkable process catalyzed by ultraviolet (UV) radiation, primarily from sunlight [11, 14]. UV light, with wavelengths shorter than visible light (100–400 nm), is divided into UVC (100–280 nm), UVB (280–320 nm), and UVA (320–400 nm). Only about 1% of solar UVB radiation reaches the Earth's surface, even during peak sunlight hours in summer, due to the efficient absorption by the stratospheric ozone layer, which absorbs all UVC and nearly 99% of UVB radiation [24–27].
Building upon the pioneering work of Jasinghe and Perera, this investigation explores the conversion of ergosterol into vitamin D2 across diverse mushroom varieties under UVA irradiation. The experimental setup involves a precisely calibrated 2 h exposure to UVA light facilitated by a specialized UVA lamp. This meticulous approach reveals significant conversion of ergosterol into vitamin D2 during this specific irradiation window [28]. In a complementary study, Keflie et al., explore the relationship between natural UVB exposure and vitamin D2 levels in oyster mushrooms under subtropical conditions. Their study reveals a notable increase in vitamin D2 content due to UVB exposure, supported by rigorous statistical analysis of sun exposure durations on vitamin D2 levels. Interestingly, the research shows no significant discrepancy in vitamin D2 content among different size groups after 8 h of sun exposure, but a significant shift is observed after prolonged 16-h exposure, highlighting the nuanced dynamics of vitamin D2 levels during extended sun exposure [29]. Mau et al. further investigate the impact of UV exposure on vitamin D2 content in fresh common and high-temperature mushrooms. Their meticulous experimentation exposes these mushrooms to UV radiation durations ranging from 0 to 2 h. Consistently, the study reveals an increase in vitamin D2 content alongside a decrease in ergosterol content. The peak transformation occurs after a 2-h UV exposure, providing detailed insights into the relationship between UV radiation and vitamin D2 levels [30].
Developing robust analytical methods for detecting and quantifying vitamin D is crucial, given the importance of this vitamin in human health. Various techniques, including GC, HPLC-UV, GC-MS, LC-MS, and LC-MS/MS, have been utilized for this purpose. Each technique has its merits and demerits, such as the high costs of instruments and analyses, as well as sensitivity to temperature fluctuations [31–34]. LC-MS/MS, in particular, is a powerful and highly sensitive method that combines the separation efficiency of liquid chromatography with the selectivity of mass spectrometry, allowing precise identification and quantification of vitamin D compounds even at trace levels. However, the high cost and need for specialized expertise limit its accessibility for many laboratories [34–43]. Alternatively, HPLC-UV is widely used for vitamin D analysis due to its excellent separation, selectivity, and cost-effectiveness, despite lacking sensitivity for trace-level detection [44–51]. Given the significance of this analysis, our current work aims to detect and quantify vitamin D2, D3, ergosterol, and 7-dehydrocholesterol in a single run. This task is particularly challenging due to the structural similarities of vitamin D2 with ergosterol and vitamin D3 with 7-dehydrocholesterol. To the best of our knowledge, although existing HPLC methods can separate vitamin D2 and D3, no documented studies have achieved the simultaneous separation of all four compounds in a single analytical run.
In this study, our primary objective is to develop an HPLC-UV method capable of concurrently detecting and quantifying all four vitamin D compounds in a single run, followed by method validation according to established ICH Q2 (R1) guidelines [52–54]. Subsequently, this newly developed methodology will be applied to comprehensively analyze vitamin D2, vitamin D3 ergosterol, and 7-dehydrocholesterol, in various vegetable oil samples. The accuracy of peak detection will be further confirmed using LC–MS, thereby ensuring the robustness of our analytical approach.
In the broader context of this research, vegetable oils will be subjected to various radiation conditions, including direct sunlight exposure and controlled artificial UVA and UVB radiation facilitated by specialized UV lamps. Following radiation exposure, HPLC analysis will be performed to investigate the effects of radiation on vitamin D precursors in vegetable oils. This research aims to provide valuable insights into optimizing nutritional enrichment in dietary oils and understanding the impact of different radiation sources on vitamin D synthetic pathways.
2 Materials and methods
2.1 Materials
Standard compounds of vitamin D2, vitamin D3, ergosterol, and 7-dehydrocholesterol were procured from Sigma-Aldrich. HPLC grade solvents, including n-hexane, methanol, ethanol, acetonitrile, and isopropyl alcohol, were obtained from Merck and Sigma-Aldrich. Additional reagents, such as potassium hydroxide (KOH), butylated hydroxytoluene (BHT), sodium ascorbate, and other chemicals, were also purchased from Merck and Sigma-Aldrich. All solvents and reagents were of analytical grade and used as received without additional purification.
2.2 Samples
Six distinct vegetable oil samples mustard oil, sesame oil, coconut oil, castor oil, almond oil, and groundnut (peanut) oil were subjected to analysis. These samples were sourced directly from farmers located in the southern region of Tamil Nadu, India. The oils were extracted using traditional cold-pressing methods, ensuring that no external additives of vitamin D or related compounds were introduced during production. In this traditional process, dried oil seeds are crushed using a heavy wooden pestle inside a large wooden mortar. The ground seeds undergo pressing, allowing the oil to naturally drain out. Subsequently, the extracted oils are collected and filtered through fine cloths to eliminate impurities before analysis. This approach maintains the oils' natural integrity, providing a pure sample suitable for the precise detection and quantification of vitamin D2, vitamin D3, ergosterol, and 7-dehydrocholesterol.
2.3 Sun exposure, UVA and UVB irradiation of vegetable oils
To comprehensively assess the impact of different radiation conditions on vegetable oil samples, a systematic division into four distinct sets was carried out. The determination of exposure sources and durations was guided by references in the literature [27–30, 55]. Notably, the study did not aim to ascertain whether the chosen exposure times and radiation doses were optimal for the conversion process; rather, it focused on exploring the varied responses of the vegetable oil samples under these conditions.
2.3.1 Control sample
In the first set, designated as the untreated control group, samples of mustard oil, groundnut oil, sesame oil, castor oil, coconut oil and almond oil (each weighing 50 mL) were individually placed in transparent glass bottles. These bottles were sealed with tight screw caps and shielded with aluminium foil to prevent exposure to light. The control samples were stored in a freezer at −18 °C to maintain their integrity throughout the duration of the experiment.
2.3.2 Sun exposed sample
The second batch of samples underwent controlled direct sunlight exposure. 50 mL samples of mustard oil, groundnut oil, sesame oil, castor oil, coconut oil, and almond oil were placed in transparent glass bottles with secure screw caps, following the procedure used for the control group. Each sample was positioned horizontally and exposed to sunlight from 9:00 am to 5:00 pm over two consecutive cloudless days in April 2023 in Chennai, Tamil Nadu, India (12° 59′ N, 80° 10′ E, 15 m above sea level). The ambient temperatures during sunlight exposure ranged from 27 to 37 °C. After exposure, each sample was covered with aluminium foil and stored in a freezer, similar to the control group, for subsequent analysis.
2.3.3 UVA irradiated sample
In the third set of experiments, a 50 mL portion of each oil sample (mustard oil, groundnut oil, sesame oil, castor oil, coconut oil, and almond oil) was placed in separate transparent glass bottles and exposed to UVA radiation. The UVA radiation exposure was performed using a Luzchem Photoreactor Model LZC-4V (Luzchem Research Inc., Canada), which emits UV radiation at 350 nm. The exposure duration was 2 h. After the exposure, the samples were shielded with aluminium foil to prevent further light exposure and stored in a freezer until further analysis.
2.3.4 UVB irradiated sample
In the fourth set of experiments, 50 mL samples of each oil were placed in transparent glass bottles and exposed to UVB radiation. The UVB radiation exposure was conducted using two UV-B lamps (15W, G15T8E; Sankyo Denki, Tokyo, Japan), each emitting ultraviolet rays in the range of 280–360 nm with a peak at 306 nm. The exposure duration was 2 h. After the exposure period, the samples were carefully covered with aluminium foil to prevent further light exposure and stored in a freezer until further analysis.
This rigorous experimental setup allowed for a comprehensive investigation into the potential effects of different environmental conditions on the studied vegetable oils, with precise storage and analytical protocols adhered to throughout the study. All the samples were meticulously collected for HPLC analysis, with the aim of precisely determining and quantifying the levels of vitamin D2, vitamin D3, ergosterol, and 7-dehydrocholesterol, employing the developed HPLC method. Simultaneously, within each set of these samples, one oil sample was selected for a thorough physicochemical analysis. This analysis encompassed key parameters such as moisture content, refractive index, specific gravity, acid value, saponification value, and unsaponifiable matters. The objective was to assess whether radiation exposure had any discernible impact on these standard quality parameters of the oils, aligning with the stipulated guidelines set forth by the Bureau of Indian Standards (BIS) [56–60].
2.4 Standard preparation
The standard solutions required for analysis were prepared following established protocols outlined in the literature [61]. Initially, stock solutions of each standard compound vitamin D2, vitamin D3, ergosterol, and 7-dehydrocholesterol were prepared in methanol at a concentration of 1.0 mg mL−1. From these stock solutions, working standard solutions were subsequently prepared at a concentration of 20 μg mL−1, along with an internal standard solution at 1.30 μg mL−1 specifically used for the analysis. Calibration mix standard solutions were prepared by diluting appropriate volumes of the initial stock solution to ensure accurate quantification during analysis.
2.5 Sample preparation
In the process of sample preparation, saponification and liquid-liquid extraction are the necessary stages. Our procedure was refined with careful consideration of the EN standard, EN 12821:2009 and supplemented by insights from various relevant literature sources, which were adapted through multiple modifications [46, 48, 62, 63] and the procedure as described. Weighted accurately 8.0 g of sample into a 500 mL round bottom flask and added 1.0 g of sodium ascorbate, 50 mL of ethanol, 1.0 mL of internal standard solution (1.30 μg mL−1) and 50 mL of 20% KOH solution. Fitted a reflux condenser and placed in a water bath, hydrolysed at 85 °C for 45 min. Once the reaction was complete, the resulting solution was allowed to cool and then the mixture was transferred to a 500 mL separating funnel. Into the separating funnel, 50 mL of an ethanol solution was added, followed by extraction with 150 mL of hexane. The organic layers were then combined and washed with water until neutral. To the organic layer was added 0.01 g of BHT and the solvent was evaporated under reduced pressure using rotary evaporator. The dried sample reconstituted with 5.0 mL methanol and filtered using 0.45 μm syringe filter and the sample obtained was taken for HPLC-UV and LC- MS analysis.
2.6 Instrumentation
2.6.1 High-performance liquid chromatography (HPLC)
The HPLC system used was a Shimadzu Prominence iSeries (model 2030C 3D, Shimadzu Corporation, Kyoto, Japan), featuring an auto-injector valve and a photodiode array (PDA) detector set to 265 nm. A Shim-pack Scepter HD-C18 column (250 mm × 4.6 mm; 5 µm, Shimadzu, Japan) maintained at 30 °C was employed for separation. An isocratic elution method with a mobile phase consisting of acetonitrile and isopropyl alcohol (90:10, v/v) at a flow rate of 1.0 mL min−1 was utilized. A sample volume of 20 µL was injected for each analysis. Identification of compounds was based on the retention times of standard compounds, with further confirmation by matching the UV spectra. Data processing and calculations were carried out using Lab Solutions software, Version 6.115.
2.6.2 LC-APCI-MS analysis
2.6.2.1 Separation
Liquid chromatographic analysis was conducted using a Shimadzu Nexera UHPLC system (Shimadzu Corporation, Kyoto, Japan) connected with a thermostated column compartment. Chromatographic separation utilized a Shim-pack Scepter C18-120 column (150 mm × 2.1 mm, 1.9 µm particle size) (Shimadzu, Japan). The mobile phase comprised an isocratic elution with 0.05% formic acid in acetonitrile (90:10, v/v) at 1.0 mL min−1 flow rate, with the column temperature set at 30 °C. A 50 µL aliquot of the sample was injected after flushing the needle with 2-propanol for 10 s to ensure cleanliness. Moreover, a cleaning routine for the injection valve was initiated post-equilibration to mitigate potential carry-over from prior samples.
2.6.2.2 Mass spectrum Analysis
Mass spectrum analysis was conducted using a Shimadzu LCMS 8050 Triple Quadrupole system (Shimadzu Corporation, Kyoto, Japan) featuring an atmospheric pressure chemical ionization (APCI) source, with nitrogen utilized as the collision gas. Mass detection was performed in both positive and negative ion modes. The source parameters were optimized by injecting compounds through an auto-sampler to maximize peak intensity. Optimal operating conditions were determined to be an ESI voltage of 3.5 eV, heat block temperature of 200 °C, DL temperature of 200 °C, and a nebulizing gas flow of 3.0 L min−1. Instrument control and data acquisition were managed using Lab Solutions software (version 5.99 SP2, Shimadzu Corporation).
2.7 Method validation
The developed HPLC method underwent validation according to the ICH guidelines (ICH Q2R1) to confirm the reliability of results for parameters such as linearity, accuracy, precision, robustness, limit of quantification (LOQ), and limit of detection (LOD) [53–55]. To determine linearity, serial dilutions ranging from 0.2 to 1.5 μg mL−1 were prepared for Vitamin D2, Vitamin D3, ergosterol, and 7-dehydrocholesterol in methanol, with each dilution done in triplicate, validating the range between 0.2 and 1.5 μg mL−1. Accuracy was evaluated using a recovery method by spiking pre-analyzed samples with standard solutions at various concentrations, including LOQ (0.26 μg mL−1 for Vitamin D2, 0.18 μg mL−1 for Vitamin D3, 0.30 μg mL−1 for ergosterol, and 0.36 μg mL−1 for 7-dehydrocholesterol), 100% (0.60 μg mL−1), and 250% (1.50 μg mL−1). This procedure was repeated six times, and the percentage recovery of each analyte was calculated by comparing the peak areas of spiked samples to those of standard solutions at corresponding concentration levels. Precision was examined through repeatability and intermediate precision, with six replicates of 0.6 μg mL−1 analyzed consecutively to assess repeatability, and variations between different days and analysts checked within the linearity range to evaluate intermediate precision. The LOD and LOQ were calculated using signal-to-noise (S/N) ratios of 3:1 and 10:1, respectively, by injecting known concentrations of serial dilutions of the standard solutions under consistent chromatographic conditions. These parameters were confirmed using the standard deviation (σ) method and derived from the slope (S) of the calibration curve. Method robustness was tested by altering the temperature (29, 30, and 31 °C) and adjusting the mobile phase flow rate by ±1%.
3 Results and discussion
3.1 Optimization of the chromatographic separation
The principal aim of this study was to establish and validate a HPLC method capable of concurrently detecting and quantifying the four primary vitamin D compounds: vitamin D2, vitamin D3, ergosterol, and 7-dehydrocholesterol in a single analytical run. Given the inherent hydrophobic nature of vitamin D and their derivatives, the method of choice for chromatography has been reversed-phase (RP) HPLC, a widely accepted and employed technique in this field [64]. In line with established practices, it prompted us to employ the RP-HPLC methods for our study. RP-HPLC was predicated on the use of a non-polar stationary phase, typically realized through the employment of an octadecyl column, paired with a polar mobile phase [65–67].
In our quest to attain optimal resolution for all four compounds within a single chromatogram, we systematically explored various C18 columns, employing distinct combinations of acetonitrile: methanol and acetonitrile: isopropyl alcohol as mobile phases in an isocratic elution mode [65]. Our initial experimentation commenced with the Phenomenex Kromasil 5u 100A C18 column [52] (a), employing a mobile phase mixture of acetonitrile: methanol and acetonitrile: isopropyl alcohol, as delineated in supplementary Table S1. Despite the well-known reputation of octadecyl silane columns for facilitating excellent separation, our efforts, unfortunately, yielded suboptimal resolution, as exemplified in supplementary Fig. S1 (entries 1). Undeterred by this initial setback, we embarked on a comprehensive exploration of alternative columns (b-f) featuring diverse stationary phases. Simultaneously, we pursued chromatographic separation using three distinct combinations of mobile phases (entries 2–6) [65, 68]. Regrettably, regardless of our endeavours, the stationary phase's pivotal role in achieving satisfactory resolution became evident, and none of our attempts yielded the desired separation efficiency.
In light of these challenges, an extensive review of pertinent literature directed us toward adopting a column characterized by a stationary phase composed of fully porous organosilica hybrid – octadecyl material ((g), Shim-pack Scepter HD-C18 column). This strategic decision proved to be pivotal, as it resulted in the successful attainment of a high level of separation for vitamin D2, vitamin D3, ergosterol, and 7-dehydrocholesterol. Notably, we conducted experiments with various mobile phase combinations and identified the optimal condition as a mixture of acetonitrile and isopropyl alcohol at a 90:10, v/v ratio, coupled with a flow rate of 1.0 mL min−1. It's important to highlight that all four compounds were calculated using a single λmax of 265 nm, simplifying the analytical process. Retention times for standard vitamin D2, vitamin D3, ergosterol, and 7-dehydrocholesterol were visually determined to be 20.91, 22.32, 33.48, and 35.80 min, respectively, as illustrated in Fig. 1. This streamlined approach ensured consistency and efficiency in our analysis.
Simultaneous determination of vitamin D2, vitamin D3, ergosterol, and 7-dehydrocholesterol by HPLC-UV method. Chromatogram is a standard mixture of 0.60 μg mL−1 of 1) vitamin D2, 2) vitamin D3, 3) ergosterol, and 4) 7-dehydrocholesterol
Citation: Acta Chromatographica 2025; 10.1556/1326.2024.01275
3.2 System suitability parameters
System suitability parameters were analyzed to assess the consistency of system performance. Six replicates of a standard mixture solution containing a known concentration (0.60 μg mL−1) of each standard compound—Vitamin D2, Vitamin D3, ergosterol, and 7-dehydrocholesterol—were injected, and column performance metrics such as tailing factor, retention time, and number of theoretical plates were recorded (Table S2). The HPLC method demonstrated excellent peak separation and symmetry under optimized experimental conditions, as evidenced by the system suitability parameters. The tailing factors were 1.01 for Vitamin D2, 1.03 for Vitamin D3, 1.01 for ergosterol, and 1.00 for 7-dehydrocholesterol, all well within the acceptance criterion of ≤ 2.0. These low tailing factors indicate good peak symmetry, reflecting the efficiency of the column and the effectiveness of the chromatographic conditions in maintaining peak shape. Additionally, the number of theoretical plates was 93,236 for Vitamin D2, 96,301 for Vitamin D3, 98,992 for ergosterol, and 98,925 for 7-dehydrocholesterol, significantly surpassing the acceptance criterion of ≥ 2,000. These high theoretical plate numbers indicate a high degree of column efficiency, resulting in sharp and well-defined peaks. Moreover, the resolution between vitamin D2 and vitamin D3 was 2.01, between vitamin D3 and ergosterol was 12.16 between ergosterol and 7-dehydrocholesterol was 2.08. Both values are well above the commonly accepted threshold of 1.5 for adequate separation, demonstrating that the method provides excellent peak separation. This high resolution ensures that peaks are well-separated with minimal overlap, allowing for accurate and precise quantification of each analyte.
3.2.1 Linearity and range
Linearity was evaluated by analyzing various concentrations of standard solutions for Vitamin D2, Vitamin D3, ergosterol, and 7-dehydrocholesterol. The response was linear across the concentration range of 0.20–1.50 μg mL−1, which was established as the working range of the method. The peak area and concentration data were subjected to linear least-squares regression analysis to derive the calibration equation and correlation coefficient (Table 1). The linearity of the calibration curves was confirmed by high correlation coefficient values (R2) of 0.9979 for Vitamin D2, 0.9989 for Vitamin D3, 0.9972 for ergosterol, and 0.9960 for 7-dehydrocholesterol. Additionally, the %RSD for the correlation coefficients was less than 2%, indicating excellent linearity and consistency in the method.
Validation parameters of RP-HPLC method for vitamin D2, vitamin D3, ergosterol, and 7-dehydrocholesterol
| S.No | Parameters | Vitamin D2 | Vitamin D3 | Ergosterol | 7-dehydrocholesterol |
| 1 | Retention Time (min)±%RSD | 20.91 ± 0.024 | 22.32 ± 0.025 | 33.48 ± 0.055 | 35.80 ± 0.095 |
| 2 | Regression equation | y = 190,46x + 406.68 | y = 424,27x + 558.22 | y = 102,80x + 566.28 | y = 9165.9x + 112.87 |
| 3 | Correlation coefficient (R2) | 0.9979 | 0.9989 | 0.9972 | 0.9960 |
| 4 | Limit of detection (LOD) (μg mL−1) | 0.08 | 0.06 | 0.10 | 0.11 |
| 5 | Limit of quantification (LOQ) (μg mL−1) | 0.26 | 0.18 | 0.30 | 0.36 |
| 6 | S/N ration of LOD | 17.37 | 15.14 | 8.58 | 6.78 |
| 7 | S/N ration of LOQ | 50.93 | 46.50 | 21.18 | 15.35 |
| Accuracy Calculation: Recovery in % | |||||
| 8 | Recovery % at LOQ±%RSD | 97.56 ± 1.27 | 98.41 ± 1.38 | 97.15 ± 2.01 | 98.30 ± 1.18 |
| 9 | Recovery % at 100%±%RSD | 99.02 ± 1.06 | 99.18 ± 1.04 | 97.30 ± 2.12 | 97.94 ± 1.62 |
| 10 | Recovery % at 250%±%RSD | 99.36 ± 1.19 | 98.51 ± 0.99 | 98.83 ± 2.09 | 99.30 ± 1.45 |
| Precision Calculation: Intraday analysis RSD % | |||||
| 11 | Analyst 1 | 1.30 | 0.68 | 0.69 | 0.70 |
| 12 | Analyst 2 | 0.71 | 1.27 | 0.86 | 0.80 |
| Precision Calculation: Interday analysis RSD % | |||||
| 13 | Day 1 | 1.30 | 0.68 | 0.69 | 0.70 |
| 14 | Day 2 | 1.17 | 1.16 | 1.74 | 1.81 |
3.2.2 Accuracy
The accuracy of the method was evaluated using the recovery method. The method involved extracting and analyzing a coconut oil sample spiked with LOQ, 100%, and 150% of each of the four standard substances. The recovery rates ranged from 97% to 99% (Table 1), demonstrating the method's accuracy.
3.2.3 Precision
Precision was assessed by evaluating the repeatability of sample injections and peak area measurements, expressed as %RSD (Table 1). Repeatability and intermediate precision were determined at three different concentrations (LOQ, 0.6, and 1.50 μg mL−1) for both within-day and day-to-day analyses. The %RSD values were consistently below 2%, indicating that the method's repeatability and intermediate precision were within acceptable limits.
3.2.4 LOQ and LOD
The method demonstrated high sensitivity for detecting and quantifying the target compounds. The LOD, based on a signal-to-noise ratio of 3:1, was 0.08 μg mL−1 for Vitamin D2, 0.06 μg mL−1 for Vitamin D3, 0.10 μg mL−1 for ergosterol, and 0.11 μg mL−1 for 7-dehydrocholesterol. The LOQ, based on a signal-to-noise ratio of 10:1, was 0.26 μg mL−1 for Vitamin D2, 0.18 μg mL−1 for Vitamin D3, 0.30 μg mL−1 for ergosterol, and 0.36 μg mL−1 for 7-dehydrocholesterol (Table 1).
3.2.5 Robustness
Robustness was tested by introducing small, deliberate changes in temperature (±1 °C) and the mobile phase flow rate (±1%). There were no significant changes in the results of the developed method under these modified conditions. The standard deviation of peak areas for each set of conditions was calculated and found to be less than 2% (Table 2). These low %RSD values indicate that the method is robust.
Robustness of the RP HPLC method
| Parameters | Conditions | Change in RSD% | |||
| Vitamin D2 | Vitamin D3 | Ergosterol | 7-dehydrocholesterol | ||
| Flow rate | 1.01 mL min−1 | 0.29 | 0.04 | 1.65 | 1.30 |
| 1.0 mL min−1 | 1.06 | 1.04 | 1.56 | 1.62 | |
| 0.99 mL min−1 | 0.40 | 0.29 | 1.90 | 1.26 | |
| Oven Temperature | 31 °C | 1.81 | 0.64 | 0.33 | 0.75 |
| 30 °C | 1.06 | 1.04 | 1.56 | 1.62 | |
| 29 °C | 1.77 | 0.86 | 0.68 | 0.77 | |
3.3 Quantification of vitamin D compounds in vegetable oils
The application of developed HPLC method was extended to six commercially available vegetable oils, specifically mustard oil, groundnut oil, sesame oil, castor oil, coconut oil, and almond oil, intended for human nutrition, underwent comprehensive characterization to assess the vitamin D2, vitamin D3, ergosterol, and 7-dehydrocholesterol in these oils. Samples were subjected to the saponification process followed by the extraction with hexane as per the procedure mentioned in the literature. The final samples were analysed using HPLC under the validated conditions with a acetonitrile: isopropyl alcohol (90:10, v/v ratio) as the mobile phase. The separation of the four compounds was achieved on the specific Shim-pack Scepter HD-C18 column (250 mm × 4.6 mm; 5 µm). The four compounds present in the sample were confirmed by comparing the retention time of UV absorption spectrum of the corresponding standard compound. Notably, λmax 265 was used to detect and quantify both the vitamin D2 and vitamin D3, ergosterol, and 7-dehydrocholesterol. Notably, castor oil exhibited the highest amount of vitamin D3 at 2.82 μg mL−1 Fig. 2, while other oils, including mustard oil, groundnut oil, sesame oil, and almond oil, featured detectable levels of Vitamin D3.
Chromatogram of Untreated Castor Oil Sample: The chromatogram illustrates the separation and detection of the following compounds: 1) Vitamin D2 (RT-20.90), 2) Vitamin D3 (RT-22.16), 3) Ergosterol (RT-33.37), and 4) 7-dehydrocholesterol (RT-35.72)
Citation: Acta Chromatographica 2025; 10.1556/1326.2024.01275
Vitamin D2 was identified exclusively in Mustard oil, quantified at 0.73 μg mL−1 Fig. 3, with no other oils exhibiting quantifiable amounts of vitamin D2. Interestingly, Coconut oil, among the six oils, did not reveal the presence of either vitamin D2 or D3, Importantly, our results unequivocally corroborated the existence of both ergosterol and 7-dehydrocholesterol in the examined vegetable oil samples and described in the Table 3. These findings are consistent with prior research, highlighting the natural occurrence of these compounds within various plant oils and underscoring their potential as precursors for vitamin D biosynthesis [69].
Chromatogram of Untreated Mustard Oil Sample: The chromatogram illustrates the separation and detection of the following compounds: 1) Vitamin D2 (RT-21.17), 2) Vitamin D3 (RT-22.19), 3) Ergosterol (RT-34.52), and 4) 7-dehydrocholesterol (RT-35.93)
Citation: Acta Chromatographica 2025; 10.1556/1326.2024.01275
Quantities of vitamin D2, vitamin D3, ergosterol, and 7-dehydrocholesterol in various vegetable oils using RP-HPLC- UV method
| Oil Samples | Vegetable oil control samples (qty in mg mL−1) | |||
| Vitamin D2 | Vitamin D3 | Ergosterol | 7-dehydrocholesterol | |
| Mustard oil | 0.73 | 0.48 | 3.06 | 0.83 |
| Ground nut oil | <0.26 | 0.45 | 3.35 | 1.38 |
| Sesame oil | <0.26 | 0.78 | 3.08 | 1.19 |
| Castor oil | <0.26 | 2.82 | 7.54 | 2.52 |
| Coconut oil | <0.26 | <0.18 | 1.07 | 1.40 |
| Almond oil | <0.26 | <0.18 | 0.48 | 1.64 |
3.3.1 Reconfirmation of HPLC detected peaks by LC- MS method in vegetable oils
Vegetable oils are known to contain a variety of aliphatic constituents apart from the anticipated molecules viz., vitamin D2, D3, ergosterol and 7-dehydrocholesterol. Hence, it is very difficult to confirm the correctness of the HPLC peaks corresponding to the vitamin D and its precursors even if the samples are spiked with the authentic molecules. To overcome this ambiguity, the corresponding mass values are usually analysed. Among the various methods available for the mass analysis, LC-MS method is highly recommended. Given the significance, HPLC online coupled with atmospheric pressure chemical ionization (APCI) tandem mass spectrometry (LC–MS) method was used to reconfirm the presence of vitamin D2, vitamin D3, ergosterol, and 7-dehydrocholesterol in all vegetable oil samples in order to check the correctness of the developed HPLC method. Interestingly, this technique integrates the identification capabilities of high-performance liquid chromatography with the sensitivity and specificity offered by mass spectrometry, offering a robust method to confirm the presence of identified molecules in the oil samples.
The strategy involved correlating the retention times and the mass spectrometry spectral information of vitamin D2, vitamin D3, ergosterol, and 7-dehydrocholesterol in the vegetable oil samples with those of pure standards. This approach serves as a valuable method for accurate identification and confirmation of these compounds in the oil samples, ensuring the precision and validity of the analytical results.
The positive ion full-scan APCI-MS spectrum in Fig. 4 illustrates the characteristic signatures of vitamin D2, vitamin D3, ergosterol, and 7-dehydrocholesterol standards. Specifically, the mass spectra of vitamin D2 (A) and vitamin D3 (B) show the presence of the parent ion [M + H]+ at mass-to-charge ratios (m/z) of 397 and 385, respectively. Similarly, the mass spectra of ergosterol (C) and 7-dehydrocholesterol (D) exhibit the parent ion [M + H–H2O]+ with m/z values of 379 and 367, as detailed in Table S3.
APCI mass spectra showing the parent ions and their corresponding m/z values of standards: The mass spectra of Vitamin D2 (A) and Vitamin D3 (B) show the parent ion [M+H]+ at m/z values of 397 and 385, respectively. Similarly, the mass spectra of Ergosterol (C) and 7-dehydrocholesterol (D) exhibit the parent ion [M + H–H2O]+ with m/z values of 379 and 367, respectively
Citation: Acta Chromatographica 2025; 10.1556/1326.2024.01275
The mass spectra obtained for both mustard oil and groundnut oil samples are shown in Figs S2 and S3. These spectra were characterized by the presence of protonated molecules [M + H]+ at m/z 397 and m/z 385 for vitamin D2 (A)and vitamin D3 (B), and [M + H–H2O)]+ at m/z 379 and 367 for ergosterol (C) and 7-dehydrocholesterol (D), respectively. These mass spectral data offer concrete evidence for accurate identification of these compounds in the specific oil samples under investigation.
Due to the complexity of the organic matrix present in the oil samples, it is important to prove its correctness of the vitamin D compounds in the vegetable oils. Therefore, oil samples were rechecked using LC-MS. Initially, retention times of standard compounds with mass spectral information were analyzed and confirmed. Subsequently, analysis of vegetable oil samples was subjected under the standard conditions. The mass analysis of each sample by the LC-MS method indicated the presence of distinct parent ions. Pleasingly, LC-MS method is in complete agreement with the current HPLC method for its correctness for all the identified compounds in oil samples.
3.4 Effect of sunlight, UVA and UVB exposure on vitamin D content in vegetable oils
Identifying new sources of vitamin D or enhancing its content in regular foodstuffs is crucial for maintaining adequate vitamin D levels. In this study, we found that some vegetable oils contain both vitamin D2 and D3, along with their precursors, ergosterol, and 7-dehydrocholesterol. This discovery provides novel insights into dietary vitamin D enrichment. Previous studies have reported the impact of UV radiation on the conversion of vitamin D precursors. To further investigate this phenomenon in vegetable oils, we examined the effects of sun exposure, UVA, and UVB radiation on vitamin D content. We conducted a series of experiments where various vegetable oils were exposed to these radiation sources and monitored the resulting changes in the concentrations of vitamin D compounds.
3.4.1 Sunlight exposure
Six vegetable oil samples were exposed to direct sunlight from 9:00 am to 5:00 pm over two days. Minor variations in vitamin D content were observed in most oils. Notably, the vitamin D2 content in mustard oil showed an increase, while vitamin D3 levels in mustard, sesame, and almond oils were also enhanced due to sunlight exposure. However, these changes were not substantial. The comparison of vitamin D2 and D3 levels before and after exposure is detailed in Table 4.
Comparison of the effect of radiation in the vitamin D2 and D3 content in vegetable oils
| Vegetable oil samples | Vitamin D2 | Vitamin D3 | ||||||
| Control | After sun exposure | After UVA | After UVB | Control | After sun exposure | After UVA | After UVB | |
| Mustard oil | 0.73 | 0.81 | 1.44 | 1.89 | 0.48 | 0.53 | 0.80 | 1.24 |
| Ground nut oil | <0.26 | <0.26 | <0.26 | <0.26 | 0.45 | 0.47 | 0.62 | 0.95 |
| Sesame oil | <0.26 | <0.26 | <0.26 | <0.26 | 0.78 | 0.93 | 0.79 | 0.81 |
| Castor oil | <0.26 | <0.26 | <0.26 | <0.26 | 2.82 | 2.99 | 3.27 | 4.29 |
| Coconut oil | <0.26 | <0.26 | <0.26 | <0.26 | <0.18 | <0.18 | <0.18 | <0.18 |
| Almond oil | <0.26 | <0.26 | <0.26 | <0.26 | <0.18 | 0.35 | 0.51 | 0.66 |
3.4.2 UVA and UVB radiation exposure
Two additional sets of oil samples were subjected to UVA and UVB radiation, respectively, to assess the impact of these specific wavelengths on the vitamin D content of vegetable oils. Compared to sunlight exposure, both UVA and UVB radiation markedly enhanced the vitamin D levels in the oils. UVB radiation, in particular, demonstrated the greatest enhancement in vitamin D content, corroborating previous studies that highlight UVB's superior efficacy in converting precursors to active vitamin D forms. Among the six oils studied, UVB radiation significantly increased the vitamin D3 content in castor oil, with levels rising from 2.82 μg mL−1 to 4.29 μg mL−1. Mustard oil also exhibited a notable increase in vitamin D2 content under UVB exposure, with levels increasing from 0.73 μg mL−1 to 1.89 μg mL−1. These results align with the established understanding that UVB radiation, due to its specific wavelength, effectively catalyzes the conversion of corresponding vitamin D forms. The comparative results of vitamin D2 and D3 levels before and after exposure to UVA and UVB radiation are summarized in Table 4.
Our study revealed notable differences in the conversion rates of precursor compounds to vitamin D under varying radiation conditions. Oils subjected to artificial UVB radiation exhibited the highest conversion rates, followed by those exposed to artificial UVA radiation. Oils exposed to direct sunlight demonstrated the lowest conversion rates. The consistent transformation of 7-dehydrocholesterol into vitamin D3 was observed across all oils and conditions, with conversion rates proportional to the initial 7-dehydrocholesterol concentration.
Castor oil, rich in 7-dehydrocholesterol, showed the highest conversion levels under UVB radiation, with a significant increase in vitamin D3 after 2 hs of exposure. Mustard oil also displayed a substantial rise in vitamin D3, while groundnut oil showed a moderate increase. Interestingly, mustard oil exhibited a significant transformation in vitamin D2 content under UVB radiation, as detailed in Table 4. Despite ergosterol being more prevalent than 7-dehydrocholesterol in some oils, no significant conversion to vitamin D2 was noted under any condition, except in mustard oil samples. Coconut oil consistently showed low or negligible conversion rates across all conditions.
The results highlight distinct transformation patterns, with UVB radiation emerging as the most effective catalyst for precursor conversion. Oils exposed to UVB radiation showed significantly higher conversion rates compared to other sources, as illustrated in Figs 5 and 6. This finding aligns with previous research emphasizing the critical role of UVB in initiating vitamin D synthesis [22, 29, 55, 70]. The efficiency of UVB-induced biotransformation underscores its potential for enhancing the vitamin D content of vegetable oils, offering a valuable strategy for natural fortification.
Comparison of the effect of sunlight, UVA and UVB radiation on vitamin D3 in vegetable oils
Citation: Acta Chromatographica 2025; 10.1556/1326.2024.01275
Comparison of the effect of sunlight, UVA and UVB radiation on vitamin D2 in Mustard oil
Citation: Acta Chromatographica 2025; 10.1556/1326.2024.01275
While UVA radiation had a lesser but still significant influence on precursor conversion, it was less effective than UVB. The increased vitamin D2 and D3 content in UVA-exposed oils suggests that this radiation source can contribute to vitamin D synthesis, albeit to a lesser extent. Surprisingly, direct sunlight exposure had the least effect on precursor conversion. This may be related to geographical or climatic factors that require further exploration. These findings suggest that, in this specific context, natural sunlight may not be as efficient as UVB radiation in driving the conversion process.
In parallel, within each batch of these samples, a single oil sample is meticulously selected and subjected to a comprehensive analysis of physicochemical parameters. These included crucial metrics such as moisture content, refractive index, specific gravity, acid value, saponification value, and unsaponifiable matter, all rigorously evaluated in accordance with the IS standards stipulated by the Bureau of Indian Standards (BIS) for their respective oils. Our exhaustive analysis, documented in Table 5, unequivocally demonstrates that these parameters remained virtually unchanged. Notably, all measurements consistently fell well within the specified limits. These findings provide robust confirmation that diverse radiation exposures had no discernible impact on the physicochemical attributes of the oils, reinforcing their safety and suitability for human applications.
Physicochemical parameters of vegetable oils as per IS standard specification
| Sample details | Condition | Requirement as per IS standard | |||||
| Moisture % | Specific gravity @ 25 °C | Unsaponifiable matter % | Refractive @ 40 °C | Saponification value | Acid value | ||
| Mustard oil | Control (untreated) | 0.08 | 0.9108 | 1 | 1.4658 | 175.4 | 1.12 |
| After direct Sun exposure | 0.06 | 0.9108 | 1.05 | 1.4651 | 172.5 | 1.13 | |
| After UVA exposure | 0.07 | 0.9111 | 1.05 | 1.4655 | 175.1 | 1.13 | |
| After UVB exposure | 0.07 | 0.9121 | 1.04 | 1.4653 | 173.4 | 1.12 | |
| Groundnut oil | Control (untreated) | 0.07 | 0.905 | 0.65 | 1.462 | 190 | 0.32 |
| After direct Sun exposure | 0.07 | 0.904 | 0.76 | 1.4623 | 188 | 0.4 | |
| After UVA exposure | 0.08 | 0.906 | 0.75 | 1.4632 | 187 | 0.3 | |
| After UVB exposure | 0.08 | 0.91 | 0.72 | 1.4632 | 188 | 0.36 | |
| Sesame oil | Control (untreated) | 0.08 | 0.9154 | 1.44 | 1.4651 | 190.67 | 2.48 |
| After direct Sun exposure | 0.09 | 0.9152 | 1.4 | 1.4651 | 192.04 | 2.49 | |
| After UVA exposure | 0.07 | 0.915 | 1.5 | 1.4656 | 192.56 | 2.39 | |
| After UVB exposure | 0.08 | 0.9152 | 1.46 | 1.4653 | 192.43 | 2.42 | |
| Castor oil | Control (untreated) | 0.09 | 0.9574 | 0.61 | 1.4721 | 184.29 | 0.54 |
| After direct Sun exposure | 0.07 | 0.9571 | 0.48 | 1.4722 | 183.92 | 0.55 | |
| After UVA exposure | 0.09 | 0.9574 | 0.57 | 1.4721 | 184.29 | 0.56 | |
| After UVB exposure | 0.08 | 0.9573 | 0.51 | 1.4721 | 184.92 | 0.55 | |
| Coconut oil | Control (untreated) | 0.08 | 0.9176 | 0.25 | 1.4489 | 267.39 | 0.42 |
| After direct Sun exposure | 0.07 | 0.9166 | 0.4 | 1.4489 | 260.23 | 0.42 | |
| After UVA exposure | 0.08 | 0.9169 | 0.34 | 1.4488 | 264 | 0.42 | |
| After UVB exposure | 0.07 | 0.9167 | 0.3 | 1.4489 | 265.5 | 0.41 | |
| Almond oil | Control (untreated) | 0.07 | 0.9166 | 0.6 | 1.468 | 165 | 2.4 |
| After direct Sun exposure | 0.08 | 0.916 | 0.54 | 1.469 | 168 | 2.6 | |
| After UVA exposure | 0.09 | 0.9163 | 0.55 | 1.469 | 167 | 2.2 | |
| After UVB exposure | 0.07 | 0.9161 | 0.55 | 1.471 | 165 | 2.4 | |
4 Conclusion
This study successfully developed and validated an HPLC-UV method for the simultaneous detection and quantification of Vitamin D2, Vitamin D3, ergosterol, and 7-dehydrocholesterol in a single analytical run, adhering to ICH Q2 (R1) guidelines. The method's robustness was confirmed through LC-MS, underscoring its reliability for comprehensive analysis. Application of the validated HPLC-UV method to various vegetable oils revealed their Vitamin D content, and the impact of different radiation types—sunlight, UVA, and UVB—on these levels was investigated. Both UVA and UVB radiation enhanced Vitamin D content in vegetable oils, with UVB radiation showing the most pronounced effect. Specifically, UVB exposure increased Vitamin D3 content in castor oil from 2.82 μg mL−1 to 4.29 μg mL−1 and Vitamin D2 content in mustard oil from 0.73 μg mL−1 to 1.89 μg mL−1. These findings underscore the superior efficacy of UVB radiation in converting Vitamin D precursors to their active forms compared to other radiation sources. The developed method and resultant insights offer a valuable approach for naturally enhancing Vitamin D content in vegetable oils, providing a promising strategy for addressing Vitamin D deficiencies in populations with limited sun exposure or dietary restrictions.
Acknowledgment
This study was supported by Selective Excellence Research Initiative -2021- SRM SIT (SERI – 2021).
Supplementary material
Supplementary data to this article can be found online at https://doi.org/10.1556/1326.2024.01275.
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