A Validated High-Performance Thin-Layer Chromatography Method for Analyzing Fat-Soluble Vitamins in Commercial Pharmaceutical Preparations

Featured Application

The developed HPTLC method was able to detect and quantify four targeted fat-soluble vitamins in commercial pharmaceutical preparations.

Abstract

A simple, cost-effective, and efficient novel high-performance thin-layer chromatography (HPTLC) tool was developed and validated in accordance with International Conference on Harmonisation (ICH) guidelines for the detection and quantification of four fat-soluble vitamins, D₂, D₃, E, and K₁, using chloroform: cyclohexane (55:45, v/v) as mobile phase. The detection and quantification limits were found to be 30.86 and 93.52 ng/band for vitamin D₂, 19.44 and 58.92 ng/band for vitamin D₃, 14.17 and 42.95 ng/band for vitamin E, and 0.86 and 2.61 ng/band for vitamin K₁, which were similar or lower than those reported in previous methods. The advantage of the developed method is that it uses a simple mobile phase in a single development step and has low detection and quantification limits. The application of the developed HPTLC method was successfully demonstrated with the quantitative analysis of these vitamins in some commercially available pharmaceutical preparations.

1. Introduction

Vitamins can be classified as fat-soluble and water-soluble vitamins based on their respective solubility profiles. Fat-soluble vitamins include vitamins A, D, E, and K whereas water-soluble vitamins include members of the vitamin B-complex (vitamins B₁, B₂, B₃, B₅, B₆, B₇, B₉, and B₁₂) as well as vitamin C. Vitamins are essential for cellular development and growth and also play a crucial role in metabolic processes and cellular regulation, thus they are important for maintaining health [1,2]. When vitamin levels are insufficient to meet physiological requirements, vitamin intake can be increased either through diet, nutraceutical supplements, or pharmaceutical preparations [2]. Commonly fat-soluble vitamins are incorporated in these formulations as acetate in the case of vitamin A, as α-tocopherol or tocopheryl acetate for vitamin E, in the form of either cholecalciferol (vitamin D₃) or ergocalciferol (vitamin D₂) and as phylloquinone (vitamin K₁) or menaquinones (vitamin K₂) [3,4]. As both deficiency and excess intake of vitamins can cause health problems [5], it is crucial to establish analytical methods that enable the evaluation of the quality of the nutraceutical and pharmaceutical preparations in terms of meeting their stated vitamin content.

In earlier studies, a wide range of analytical methods has been employed, including spectrophotometry, high-performance liquid chromatography (HPLC), reverse phase high-performance liquid chromatography (RP-HPLC), liquid-chromatography/mass spectrometry (LC-MS), liquid-chromatography/UV-spectrometry (LC-UV), liquid chromatography coupled with diode array/mass spectrometry (LC-DAD-MC) and gas-liquid chromatography (GLC), for the analysis of fat-soluble vitamins in complex matrices like human plasma and serum, pharmaceutical preparations and food items such as fruits and vegetables [2,6,7,8,9,10,11]. In recent years, High-performance thin-layer chromatography (HPTLC) has also been developed into a popular analytical tool based on its simple, multifunctional, highly adaptable and economically efficient analysis process [5]. Moreover, high sample throughput coupled with minimal sample preparation steps facilitate a highly efficient and rapid analysis using HPTLC [4].

In light of the above, HPTLC has become widely popular for the analysis of targeted molecules in various complex matrices like food items (e.g., honey), pharmaceutical formulations, and also nutraceutical preparations [12,13,14]. Specifically in the context of fat-soluble vitamins, previous studies found that HPTLC analysis is a “green” technique to analyze vitamin D₃ in commercial pharmaceutical products [15]. Another study revealed that using a two-step development coupled with densitometric detection, vitamins A and E as well as some water-soluble vitamins can be determined simultaneously by HPTLC [16] whereas other studies found that fat-soluble vitamins can be effectively analyzed in human plasma and food items using HPTLC [4,17,18].

Table 1. Published studies using HPTLC for the analysis of fat-soluble vitamins.

Vitamins	Mobile Phases	Solvent	Stationary Phase	Number of Development Steps	Detection	LOD and LOQ (ng/band)	Type of Sample	References
D3	Chloroform: Diethyl ether (90:10, v/v)	Methanol	60F254S (20 × 10 cm)	1	Densitometry	17.54 and 52.62	Pharmaceutical products	[15]
D3	Ethanol: Water (70:30, v/v)	Methanol	RP-60F254S (20 × 10 cm)	1	Densitometry	8.47 and 25.41	Pharmaceutical products	[15]
A and E	Benzene and a 0.02 M aqueous micellar solution of sodium dodecyl sulfate (SDS)	Methanol	High-performance Sorbfil plates	2	Optical densitometer and densitometry	-	Standards	[16]
A and E	Chloroform: Cyclohexane (55:45, v/v)	Methanol and n-heptane	Silica gel 60F254 (20 × 10 cm and 10 × 10 cm)	1	Densitometry	0.16 and 1.2; 0.89 and 5.71	Human plasma	[18]
α-, β-, γ-, and δ-Tocopherols	Ethanol-water (10:0; 9.5:0.5; 9.0:1, v/v)	Chloroform	RP-18-HPTLC (10 × 10 cm)	1	Dipyridyl iron reagent	-	Various biological samples	[17]
K1	Methanol: Ethanol: Isopropanol: Water (75:5:5:15, v/v/v/v)	Ethanol	Silica gel 60F254 (20 × 10 cm)	1	Densitometry	0.19–0.85 and 0.57–2.50	Pharmaceutical products, vegetables, and fruits	[4]

LOD = Limit of detection, LOQ = Limit of quantification.

provides an overview of previously published HPTLC-based methods for the analysis of fat-soluble vitamins. Reviewing these studies, it can be concluded that there are still some limitations to the quantitative analysis of fat-soluble vitamins either as pure substances or in biological, pharmaceuticals, or food items using existing HPTLC-based methods. For example, they were found to be unable to adequately separate more than two fat-soluble vitamins simultaneously using a single development step.

Addressing those limitations, the aim of the current study was to develop and validate a HPTLC-based method for the simultaneous quantification of four fat-soluble vitamins including D₂, D₃, E, and K₁ (Figure 1) in pharmaceutical preparations using a simple mobile phase and a single development step.

2. Materials and Methods

2.1. Chemicals and Reagents

All chemicals and reagents used in this study were of analytical grade. The vitamin standards (Ergocalciferol—vitamin D₂, cholecalciferol—vitamin D₃, d-alpha-tocopherol—vitamin E, and phytomenadione—vitamin K₁) as well as chloroform were sourced from Sigma-Aldrich (Bayswater, VIC, Australia). Cyclohexane and ethanol were bought from Chem-Supply (Gillman, Adelaide, South Australia) and methanol from Scharlau (Barcelona, Spain). Silica gel 60 F254 HPTLC glass plates (20 cm × 10 cm) were purchased from Merck KGaA (Darmstadt, Germany).

2.2. Commercial Samples

Three different dosage forms (i.e., hard gelatine capsules, soft gelatine capsules, IV solution) of four commercially available pharmaceutical preparations were sourced from Chemist Warehouse (Australia). The samples were labelled S1–S4 (

Table 2. Analyzed pharmaceutical preparations.

Sample ID	Details of Products	Content Declared
S1	Hard Gelatine Capsules	Vitamin D2 (75 µg), Protein (47 mg), Fat (3 mg), Carbohydrates (40 mg), Sodium (0.009 mg), Sugars (0.15 mg)
S2	Soft Gelatine Capsules	Vitamin D3 (175 µg), Soya oil, Gelatine, Glycerol, Fractionated coconut oil, Opacode monogramming ink S1-1-17823 Black and Purified water
S3	Soft Gelatine Capsules	Vitamin E (335.6 mg), Gelatine glycerol, Purified water, Sorbitol solution (70%), Soya oil
S4	IV Solution	Vitamin K1 (2 mg), Glycocholic acid (10.92 mg), Sodium hydroxide (0.92 mg), Lecithin (15.12 mg), Hydrochloric acid 25% (0.02 mg) and Water for injection (0.2 mL)

IV = Intravenous.

) and stored at 4 °C before analysis.

2.3. Sample Preparation

2.3.1. Preparation of Stock Solutions and Mobile Phase

Ethanolic solutions were prepared for the four fat-soluble vitamin standards at different concentration ranges: 100–300 µg/mL for vitamin D₂, D₃, and E and 20–60 µg/mL for vitamin K₁. Due to the vitamins’ inherent instability, the stock solutions were prepared freshly before every analysis. Different mobile phases were trialled (

Table 3. Trial experiments with different mobile phase compositions.

MP No.	Mobile Phases	Solvent Composition (v/v)	Remarks	References
1	Chloroform: Diethyl ether	90:10	Vitamins were not adequately separated	[15]
2	Ethanol: Water	70:30	Vitamins were not adequately separated	[15]
3	Toluene: Ethyl Acetate: Formic Acid	6:5:1	Vitamins were not adequately separated	[19]
4	Ethanol: Water	19:1	Vitamin D2 could be separated from the other vitamins but the band was not sharp	[17]
5	Chloroform: Cyclohexane	55:45	Vitamins were separated but D2 and D3 had an identical Rf value	[18]

) and a mobile phase comprising chloroform and cyclohexane at a ratio of 55:45 (v/v) was found to be best for the analysis of the four fat-soluble vitamins.

2.3.2. Preparation of Pharmaceutical Samples for HPTLC Analysis

Ten capsules were randomly sampled to determine the percentage of the stated content of the vitamins in commercial hard gelatine capsule dosage form (S1). Each hard gelatine capsule was carefully opened, and the powder content was collected and weighed. For analysis, 1200 mg powder, equivalent to four capsules, was weighed into a 25-mL volumetric flask. Absolute ethanol was added, and extraction was aided with vortexing (MX-S, China) for 5 min before the flasks were made up to volume. The suspension was filtered and immediately used for HPTLC analysis. For products S2 and S3, which were soft gelatine capsule formulations, five capsules were randomly selected, the content of the capsules was carefully squeezed out, and a total of 750 mg (S2) and 30 mg (S3) equivalent to a single capsule were sampled respectively into separate 100 mL volumetric flasks before the content was made up to volume with ethanol. For product S4, 0.2 mL of the injection solution was collected and mixed in 100 mL ethanol. The prepared solutions were immediately used for HPTLC analysis. All analyses were carried out in three replications.

2.4. HPTLC Method and Instruments

2.4.1. Application of Standards and Samples

A semi-automated HPTLC applicator (Linomat 5, CAMAG, Muttenz, Switzerland) was employed to apply the standard solutions onto the HPTLC plates. Application volumes ranged from 1–3 μL for vitamins D₂, D₃, and E and 2–6 μL for vitamin K₁. The standard solutions were applied at a rate of 30 nL/s with the application points located 20 mm from the side edges and 8.0 mm from the lower edges of the HPTLC plates, resulting in 8.0 mm long bands spaced 11.4 mm apart. Calibration curves were constructed over a concentration range of 100–300 ng/μL for vitamins D₂, D₃, and E and 20–60 ng/μL for vitamin K₁.

2.4.2. Sample Development

The chromatographic separation was carried out using silica gel 60 F254 HPTLC plates (glass plates 20 × 10 cm) as stationary phase. Chloroform: cyclohexane (55:45, v/v) was used as a mobile phase at ambient temperature in an activated (33% relative humidity) and saturated development chamber (ADC2, CAMAG). The plates were pre-conditioned with the mobile phase for 5 min and the development chamber was saturated for 45 min before the plates were automatically developed with 10 mL of the mobile phase to a migration distance of 75 mm prior to being dried for 5 min. A specialized HPTLC software (visionCATS v3.1, CAMAG) was used to operate the HPTLC instrument modules. The documentation of the chromatographic results at 254 nm was carried out by an HPTLC imaging device (TLC Visualiser 2, CAMAG). After imaging, a TLC Scanner 4 (CAMAG) was used to analyze the developed plates with a scanning speed of 20 mm/s at 263 nm (vitamin D₂), 270 nm (vitamin D₃), 272 nm (vitamin E) and 255 nm (vitamin K₁), which correspond to the respective absorbance maxima (λ_max) of the investigated fat-soluble vitamins.

2.5. Method Validation

The developed HPTLC method for the quantification of fat-soluble vitamins D₂, D₃, E and K₁, was validated for specificity, linearity, sensitivity, precision, accuracy, repeatability, and robustness in accordance with the International Conference on Harmonisation (ICH) guidelines Q2 (R1) [20,21].

2.5.1. Specificity

Specificity assesses the capability of the chosen method to detect the analyte of interest in the presence of other substances or closely related compounds. In HPTLC analysis specificity is ensured through the complete separation of the peak(s) of the analyte(s) from other peaks as well as by their distinct retardation factors (R_F). In this study, specificity focused on the adequate separation of the four investigated fat-soluble vitamins, D₂, D₃, E, and K₁.

2.5.2. Linearity

The linearity of an analytical procedure defines the ability to generate data that are directly proportional to the analyte’s concentration over a specified range. To evaluate the linearity of the proposed method, three replicate calibration curves with five data points for each fat-soluble vitamin were constructed in a concentration range of 100–300 ng/band for the standard solution of vitamins D₂, D₃, and E and 20–60 ng/band for vitamin K₁ standard. Peak areas versus the respective concentrations of vitamin D₂ and D₃ standards and peak height versus the respective concentrations of vitamin E and K₁ standards were evaluated by linear regression analysis and the coefficient of determination (r²), slope (m), y-intercept (c), and standard deviation (SD) of the calibration curves calculated. Calculations were done by using Microsoft Excel.

2.5.3. Sensitivity

The ability to detect and quantify the analyte in a sample matrix is defined as the sensitivity of an analytical method. It is commonly expressed as limit of detection (LOD) and the limit of quantification (LOQ), which refer in the case of LOD to the minimum amount of the sample that can be detected but not necessarily quantified and in the case of LOQ to the lowest concentration of the analyte that can be quantified with acceptable accuracy, precision, and reproducibility under the specific experimental conditions. In this study, based on the evaluation of three calibration curves, the LOD and LOQ values for each fat-soluble vitamin standard were determined using the formulas stipulated in the ICH guidelines [20]:

LOD = 3.3 σ/S and

LOQ = 10 σ/S

where σ is the regression line’s standard deviation (SD) and S is the average slope value of triplicate calibration curves.

2.5.4. Precision

The degree of consistency among individual test results when the method is repeatedly applied to multiple samples under the same prescribed conditions but at different times, on different instruments, and/or by different laboratories is defined as its precision. In this study intra-day and inter-day precision were assessed to validate the method. For this, triplicate analyses of three different concentrations of the respective fat-soluble vitamin samples were carried out either on the same day or over three days using 150, 200, and 250 ng/band for vitamin D₂, D₃, and E and 30, 40, and 50 ng/band for vitamin K₁. Precision was expressed as the percent relative standard deviation (%RSD) of the results obtained for peak areas for vitamin D₂ and D₃ as well as peak heights for vitamin E and K₁.

2.5.5. Accuracy

The accuracy of the method expresses the closeness of agreement between the value, which is accepted either as a conventional true value or an accepted reference value, and the value found. The accuracy of the method, which reflects the closeness of agreement between experimental and theoretical analysis results, was tested by calculating the percentage recovery of each vitamin at three different concentration levels, 150, 200, and 250 ng/band for vitamins D₂, D₃ and E, and 30, 40, and 50 ng/band for vitamin K₁. Each experiment was performed in triplicate, and the % recovery and %RSD of the vitamin standards were used to determine the accuracy of the method.

2.5.6. Repeatability (System Precision)

Obtaining precise results under the same operating conditions over a short time interval reflects the repeatability of an analytical method. In this study, repeatability was assured by analyzing standard solutions five times at a concentration of 200 ng/band for vitamin D₂, D₃, and E, and 40 ng/band for vitamin K₁. Results were expressed as %RSD of standards.

2.5.7. Robustness

The robustness of a method is the ability to produce reliable and reproducible results following some small changes to the experimental conditions. In this study, robustness was measured by analysing three different concentrations of vitamin standards: 200 ng/band for vitamin D₂, D₃, and E, and 40 ng/band for vitamin K₁ after a slight change in the mobile phase volume (±2 mL), mobile phase composition (chloroform: cyclohexane, 55:47 and 57:45, v/v), and saturation time of the development chamber (±5 min). The results were tested for robustness regarding % recovery and R_F values for the individual vitamins.

3. Results and Discussion

3.1. Mobile Phase Selection

The objective of the study was to develop a simple and suitable HPTLC method for quantifying four fat-soluble vitamins in complex matrices like pharmaceutical preparations in a single development step. During the method development stage of this study, various mobile phases were trialled to accomplish this goal (Table 3). Mobile phase (MP) 5 was found to be the optimum mobile phase, which ensured adequate separation of the fat-soluble vitamins D, K, and E based on their respective R_F values (Figure 2). However, given their closely related structures (Figure 1), the optimised mobile phase could not separate the two forms of vitamin D, D₁ and D₂, as they produced the same R_F value (0.20). However, since vitamin D₂ is only sourced from plants and fungi and vitamin D₃ is derived from animals, these two vitamins are rarely combined in a single nutraceutical preparation as the former is mainly found in vegan friendly preparations [3,22]. The developed method is thus suitable for the quantitative analysis of fat-soluble vitamins as long as only one form of vitamin D is present. The optimised mobile phase was used previously to quantify retinol (vitamin A) and a-tocopherol (vitamin E) in human serum [18], however, in this study, satisfactory analysis results for vitamin A could not be achieved.

3.2. Evaluation of Visual Images and Selection of Quantification Method

A set of images at 254 nm were obtained and visionCATS (CAMAG) software was used to interpret each of the images. Percent (%) recovery and correlation coefficient (r²) values were found to be consistent for all four fat-soluble vitamins at 254 nm over the entire concentration ranges of their respective calibration curves. For the quantification of vitamins D₂ and D₃, absorbance peak area versus concentration of their calibration curves were found to produce the best results with high levels of reproducibility, whereas standard curves using peak height versus concentration were found to be most suited to quantify vitamins E and K₁ (Figure S1).

3.3. Chromatographic Results and UV–Vis Spectra

Vitamins D₂, D₃, E and K₁ were well separated using the optimized chromatographic system and produced distinct R_F values: R_F 0.20 for vitamins D₂ and D₃, 0.46 for vitamin E, and 0.68 for vitamin K₁. To determine their absorbance maxima (λ_max) for optimal detection, the UV–Vis spectrum of each fat-soluble vitamin was recorded. The λ_max values for vitamin D₂, D₃, E and K₁ were found to be 263 nm, 270 nm, 272 nm and 255 nm, respectively (Figure S2). Although their R_F values were identical, the λ_max values of vitamin D₂ and D₃ differed slightly with 263 nm for vitamin D₂ and 270 nm for vitamin D₃, however, this difference was not sufficient to quantify them accurately individually at their respective absorbance maxima.

3.4. Method Validation

3.4.1. Specificity

Respective volumes of the four fat-soluble vitamin standards (2 µL for vitamin D₂, D₃, and E; 4 µL for vitamin K₁) were applied and the HPTLC plates developed with the optimized mobile phase chloroform: cyclohexane (55:45, v/v). The specificity of the developed method was confirmed by distinctly different R_F values for the four vitamins (Figure 2), 0.20 for vitamins D₂ and D₃, 0.46 for vitamin E, and 0.68 for vitamin K₁.

3.4.2. Linearity

The linearity of the method was validated rigorously through linear regression analysis, with the correlation coefficients (r²) derived from the five-point calibration curves for the respective fat-soluble vitamins. The resulting linearity ranges were 100–300 ng/band for vitamin D₂, D₃ and E and 20–60 ng/band for vitamin K₁. The r² values of three replicate experiments for all analyzed fat-soluble vitamins were found to be more than 0.98, confirming adequate linearity of the method in the defined concentration range (

Table 4. Linear regression analysis for the calibration curves of vitamins D₂, D₃, E, and K₁.

Vitamins	RF	Run	Linearity Range (ng/band)	Regression Equation	Correlation Coefficient (r2)	Slope (Average)	y-Intercept (SD)	LOD (ng)	LOQ (ng)
D2	0.20	Run 1	100–300	y = 5 × 10−6x − 6 × 10−5	0.9944	6.53 × 10−6	6.11 × 10−5	30.86	93.52
		Run 2	100–300	y = 8 × 10−6x − 2 × 10−5	0.9928
		Run 3	100–300	y = 7 × 10−6x − 1 × 10−4	0.9969
D3	0.20	Run 1	100–300	y = 8 × 10−6x + 1 × 10−4	0.9977	7.07 × 10−6	4.16 × 10−5	19.44	58.92
		Run 2	100–300	y = 5 × 10−6x + 8 × 10−5	0.9959
		Run 3	100–300	y = 7 × 10−6x + 2 × 10−4	0.9978
E	0.46	Run 1	100–300	y = 3 × 10−4x + 3.5 × 10−3	0.9984	3.07 × 10−4	1.32 × 10−3	14.17	42.95
		Run 2	100–300	y = 3 × 10−4x + 9 × 10−4	0.9929
		Run 3	100–300	y = 3 × 10−4x + 2.7 × 10−3	0.9888
K1	0.68	Run 1	20–60	y = 1.1 × 10−3x + 2.9 × 10−2	0.9931	1.37 × 10−3	3.58 × 10−4	0.86	2.61
		Run 2	20–60	y = 1.6 × 10−3x + 3 × 10−3	0.9978
		Run 3	20–60	y = 1.4 × 10−3x + 3 × 10−3	0.9973

).

3.4.3. Sensitivity (LOD and LOQ)

The average slope values and y-intercepts obtained from linear regression analysis from the respective fat-soluble vitamins’ standard curves were used to calculate the LOD and LOQ of each vitamin (Table 4). The LOD for vitamin D₂, D₃, E, and K₁ was 30.86 ng/band, 19.44 ng/band, 14.17 ng/band, and 0.86 ng/band, respectively, and the LOQ was 93.52 ng/band, 58.92 ng/band, 42.95 ng/band and 2.61 ng/band, respectively (Table 4). In comparison with previously developed HPTLC methods, with a LOD of 17.54 and 0.19 ng/band and a LOQ of 52.62 and 2.50 ng/band respectively we were able to determine and quantify vitamin D₃ and K₁ with similar limits [4,15]. Moreover, the newly developed method can also detect and quantify two other fat-soluble vitamins, D₂ and E, with a low LOD and LOQ as seen in Table 4 whereas a previous study was only able to determine and quantify vitamin E (LOD and LOQ 0.89 and 5.71 ng/band respectively [18]). However, no other HPTLC-based study was found that allows for the quantification of vitamin D₂ in complex matrices.

3.4.4. Accuracy

Based on sample recovery, the method’s accuracy and level of conformity were assessed. The standard addition method was used to calculate sample recovery by taking the percentage mean recovery of each vitamin sample. The findings of this analysis demonstrate that the accuracy level of the developed method was high with percent mean recovery ranging from 99.37% to 101.53% for vitamin D₂, 100.89% to 101.51% for vitamin D₃, 99.67% to 102.51% for vitamin E, and 97.64% to 102.18% for vitamin K₁. According to ICH guidelines, all results fall within the acceptable limit (

Table 5. Recovery of vitamins D₂, D₃, E, and K₁.

Theoretical Amount (ng/band)	Run 1			Run 2			Run 3
	Amount Recovered (ng/band)	% Recovery	% Mean Recovery	Amount Recovered (ng/band)	% Recovery	% Mean Recovery	Amount Recovered (ng/band)	% Recovery	% Mean Recovery
Vitamin D2
150	146.52	97.68		157.30	104.87		155.70	103.80
200	198.60	99.30	99.37	193.00	96.50	99.75	203.60	101.80	101.53
250	252.86	101.14		244.70	97.88		247.50	99.00
Vitamin D3
150	157.70	105.13		150.80	100.53		153.40	102.27
200	198.30	99.15	101.51	201.30	100.65	101.15	201.60	100.80	100.89
250	250.60	100.24		255.70	102.28		249.00	99.60
Vitamin E
150	152.10	101.40		148.50	99.00		158.30	105.53
200	200.57	100.28	99.67	202.40	101.20	100.51	198.40	99.20	102.51
250	243.30	97.32		253.30	101.32		257.00	102.80
Vitamin K1
30	29.9	99.67		29.24	97.47		30.40	101.33
40	41.04	102.60	102.18	39.53	98.83	97.64	38.98	97.45	100.64
50	52.14	104.28		48.32	96.64		51.57	103.14

).

3.4.5. Precision

The study’s intra-day and intermediate (inter-day) precision were calculated based on %RSD values, which ranged from 0.92% to 4.11%, and thus were found to be within acceptable limits as per ICH guidelines (

Table 6. Precision study of vitamins D₂, D₃, E, and K₁ (Intra-Day).

Theoretical Amount (ng/band)	Precision of the Method (Intra-Day)
	Run 1 Measured Amount (ng/band)	Run 2 Measured Amount (ng/band)	Run 3 Measured Amount (ng/band)	Mean (ng/band)	SD	%RSD
Vitamin D2
150	156.80	153.60	150.70	153.70	3.05	1.99
200	203.30	195.53	205.47	201.43	5.22	2.59
250	245.43	248.43	252.53	248.80	3.56	1.43
Vitamin D3
150	156.57	150.90	160.35	155.94	4.76	3.05
200	204.90	197.40	209.00	203.77	5.88	2.89
250	253.30	255.05	258.95	255.77	2.89	1.13
Vitamin E
150	154.10	148.40	147.33	149.94	3.64	2.43
200	193.40	196.00	204.10	197.83	5.58	2.82
250	248.00	250.20	255.80	251.33	4.02	1.60
Vitamin K1
30	30.78	30.12	29.45	30.12	0.66	2.20
40	41.00	39.85	41.17	40.67	0.72	1.77
50	52.70	49.78	49.47	50.65	1.78	3.52

and

Table 7. Precision study of vitamin D₂, D₃, E, and K₁ (Inter-Day).

Theoretical Amount (ng/band)	Precision of the Method (Inter-Day)
	Run 1 Measured Amount (ng/band)	Run 2 Measured Amount (ng/band)	Run 3 Measured Amount (ng/band)	Mean (ng/band)	SD	%RSD
Vitamin D2
150	154.30	152.30	144.30	150.30	5.29	3.52
200	196.90	199.97	201.80	199.56	2.48	1.24
250	257.96	247.00	253.20	252.72	5.50	2.17
Vitamin D3
150	156.20	152.00	145.35	151.18	5.47	3.62
200	202.50	198.80	200.43	200.58	1.85	0.92
250	260.70	249.50	253.75	254.65	5.65	2.22
Vitamin E
150	156.37	153.70	158.90	156.32	3.68	2.35
200	191.63	206.90	204.80	201.11	8.27	4.11
250	248.00	243.30	251.10	247.47	3.93	1.59
Vitamin K1
30	31.00	30.07	31.30	30.79	0.64	2.09
40	42.13	39.63	39.33	40.36	1.54	3.81
50	49.42	48.80	50.57	49.60	0.90	1.81

), indicating a high degree of precision of the developed method.

3.4.6. Repeatability

The method’s repeatability was expressed as SD and %RSD. The findings demonstrate that with 1.58-2.76%, the %RSD was within the acceptable limits of the ICH guidelines (

Table 8. Repeatability (System precision).

D2 Theoretical Amount (ng/band)	D2 Measured Amount (ng/band)	D3 Theoretical Amount (ng/band)	D3 Measured Amount (ng/band)	E Theoretical Amount (ng/band)	E Measured Amount (ng/band)	K1 Theoretical Amount (ng/band)	K1 Measured Amount (ng/band)
200	203.20	200	199.20	200	195.40	40	40.96
200	195.50	200	201.50	200	198.30	40	41.84
200	197.80	200	205.80	200	197.40	40	40.37
200	208.30	200	197.80	200	205.90	40	39.36
200	197.40	200	199.03	200	197.90	40	39.17
Average	200.44	Average	200.67	Average	198.98	Average	40.34
SD	5.24	SD	3.17	SD	4.03	SD	1.11
%RSD	2.62	%RSD	1.58	%RSD	2.02	%RSD	2.76

), which confirmed the high confidence level in the methods’ repeatability.

3.4.7. Robustness

Small changes in mobile phase volume, development chamber saturation time, and composition of the mobile phase were intentionally made to assess the method’s robustness. The findings of the robustness study are shown in

Table 9. Robustness: Change in mobile phase volume.

Mobile Phase Volume	D2			D3			E			K1
	Theoretical Amount (ng/band)	% Recovery	RF (Mean ± SD)	Theoretical Amount (ng/band)	% Recovery	RF (Mean ± SD)	Theoretical Amount (ng/band)	% Recovery	RF (Mean ± SD)	Theoretical Amount (ng/band)	% Recovery	RF (Mean ± SD)
8 mL	150	101.33	0.20 ± 0.01	150	102.00	0.20 ± 0.03	150	96.93	0.46 ± 0.04	30	101.03	0.68 ± 0.03
	200	97.15		200	102.90		200	102.75		40	103.43
	250	98.72		250	104.72		250	100.68		50	101.82
12 mL	150	102.73	0.20 ± 0.03	150	98.33	0.20 ± 0.03	150	100.07	0.46 ± 0.05	30	99.37	0.68 ± 0.02
	200	99.10		200	102.35		200	100.40		40	104.40
	250	98.40		250	100.92		250	99.24		50	98.60

,

Table 10. Robustness: Change in saturation time.

Saturation Time (Min)	D2			D3			E			K1
	Theoretical Amount (ng/band)	% Recovery	RF (Mean ± SD)	Theoretical Amount (ng/band)	% Recovery	RF (Mean ± SD)	Theoretical Amount (ng/band)	% Recovery	RF (Mean ± SD)	Theoretical Amount (ng/band)	% Recovery	RF (Mean ± SD)
15	150	105.60	0.20 ± 0.02	150	100.87	0.20 ± 0.05	150	100.07	0.46 ± 0.04	30	101.67	0.68 ± 0.03
	200	102.25		200	102.45		200	98.05		40	103.08
	250	104.80		250	102.92		250	99.24		50	99.50
25	150	101.93	0.20 ± 0.03	150	103.07	0.20 ± 0.03	150	103.93	0.46 ± 0.05	30	105.33	0.68 ± 0.04
	200	104.25		200	98.55		200	102.55		40	103.75
	250	102.92		250	103.58		250	102.36		50	100.42

and

Table 11. Robustness: Change in mobile phase composition.

Mobile Phase Composition	D2			D3			E			K1
	Theoretical Amount (ng/band)	% Recovery	RF (Mean ± SD)	Theoretical Amount (ng/band)	% Recovery	RF (Mean ± SD)	Theoretical Amount (ng/band)	% Recovery	RF (Mean ± SD)	Theoretical Amount (ng/band)	% Recovery	RF (Mean ± SD)
Chloroform: Cyclohexane (55:47)	150	97.53	0.20 ± 0.02	150	103.27	0.20 ± 0.02	150	98.67	0.46 ± 0.04	30	104.87	0.69 ± 0.04
	200	96.35		200	100.70		200	102.45		40	104.65
	250	98.24		250	102.56		250	100.32		50	98.44
Chloroform: Cyclohexane (57:45)	150	101.80	0.20 ± 0.03	150	100.60	0.20 ± 0.03	150	101.40	0.46 ± 0.02	30	103.30	0.69 ± 0.03
	200	99.15		200	99.35		200	102.85		40	102.45
	250	100.16		250	103.48		250	100.36		50	100.20

. As can be seen from the data presented in these tables, small changes in the respective parameters had minimal effects on the R_F values of the investigated vitamins and did not affect their accurate quantification based on mean % recovery calculations.

4. Application of the Method

This study aimed to develop and validate a HPTLC method suitable for the analysis of fat-soluble vitamins in various pharmaceutical preparations. Three different types of pharmaceutical dosage forms (hard gelatine capsules, soft gelatine capsules, and injection-Table 2) were selected to demonstrate the applicability of the method. The results of the quantification study are shown in

Table 12. Determined the stated content of vitamins D₂, D₃, E and K₁ in commercial samples.

Commercial Samples	Vitamins	Stated Content (mg)	Average Sample Mass (mg)	Calculated Content (mg)			Mean ± SD	% Stated Content
				Run 1	Run 2	Run 3
S1	D2	0.075	1200.00	0.069	0.076	0.073	0.073 ± 0.003	97.24
S2	D3	0.175	750.00	0.164	0.181	0.177	0.174 ± 0.009	99.42
S3	E	335.60	30.00	344.97	348.66	348.94	347.52 ± 2.21	103.55
S4	K1	2.00	200	1.96	1.92	1.78	1.89 ± 0.03	94.42

. The findings revealed that 97.24% of the label claimed for vitamin D₂ in the S1 sample could be recovered based on three replicate analyses. For samples S2, S3, and S4, the vitamin content analyzed expressed as a percentage of the labelled content was 99.42% for vitamin D₃, 103.55% for vitamin E, and 94.42% for vitamin K₁, respectively (Table 12). All the samples contained different excipients, as listed in Table 2, but none was found to interfere with the quantification, demonstrating the broad applicability of the method.

The developed method appears more convenient than previous HPTLC-based methods developed for the analysis of fat-soluble vitamins as it only used a simple mobile phase in a single development step with satisfactory accuracy, precision and robustness. Moreover, its sensitivity is also good with LOD and LOQ levels that were found to be similar or lower compared to previously published methods [4,15,16,17,18]. Moreover, the newly developed method can analyze four fat-soluble vitamins, vitamin D₂, D₃, E and K₁ whereas previously developed HPTLC methods can only identify and quantify a single vitamin, like vitamin D₃ in pharmaceutical preparations, vitamin A and E in human plasma and vitamin K₁ in pharmaceutical preparations, fruits and vegetables [4,15,16,17,18]. This illustrates the novelty of the newly developed HPTLC method in comparison of previous methods.

Despite its advantages over previous studies, a limitation of the method is its inability to separate vitamin D₂ and D₃. However, this does not seem to have practical implications as formulations always tend to contain either one or the other but not both types of vitamin D at the same time [3].

5. Conclusions

Fat-soluble vitamins play an important role in various physiological processes like bone and vision health, immunity and blood coagulation. To meet the required intake levels of these vitamins, dietary supplements like nutraceuticals or pharmaceutical formulations might need to be taken to complement dietary intake. To ensure the quality of these preparations, a simple and efficient HPTLC tool was developed and validated which can assist in the simultaneous quantitative analysis of four fat-soluble vitamins, D₂, D₃, E and K₁. The developed method appeared more convenient than previous HPTLC-based methods developed for the analysis of fat-soluble vitamins as it only used a simple mobile phase in a single development step with satisfactory accuracy, precision and robustness. Thus, the method has been demonstrated to successfully quantify a range of fat-soluble vitamins in complex pharmaceutical preparations.