Process Development and Validation of Reverse-Phase High-Performance Liquid Chromatography Method for Simultaneous Quantification of Quercetin, Thymoquinone, and Pterostilbene

Abstract

The simultaneous HPLC method for quantifying Quercetin (Que), Thymoquinone (Thy), and Pterostilbene (Pte) aims at the precise measurement of these polyphenols alone or in complex mixtures, targeting their therapeutic potential in disorders such as diabetes and epilepsy. The method focuses on quantifying Que, Thy, and Pte, utilizing optimized reversed-phase HPLC conditions as per ICH Q2(R1) standards. Key validation aspects include linearity, specificity, precision, and accuracy, ensuring compliance for quality control in nanomedicine and nutraceuticals, and the method’s applications support pharmacokinetic studies and stability testing, contributing to personalized medicine and addressing pharmaco-resistance. The HPLC method development and validation were performed on a phenyl column using the mobile phase consisting of solvent A (0.1% orthophosphoric acid in HPLC water) and solvent B (acetonitrile) at a ratio of 55:45 in an isocratic elution mode at a flow rate of 1 mL/min and at a column temperature of 35 °C. Ultraviolet detection was measured at 254 nm. Moreover, the method was validated for accuracy, precision, linearity, specificity, and sensitivity. The retention time for tested Que, Thy, and Pte was observed at 4.15 min, 8.70 min, and 10.75 min, respectively. Limits of detection for Que, Thy, and Pte were 1.55 μg/mL, 2.40 μg/mL, and 70.79 µg/mL, whereas limits of quantification were 4.69 μg/mL, 7.28 μg/mL, and 214.52 µg/mL, respectively. Linearity and correlation coefficients for Que, Thy, and Pte were found in the range of 50–250 μg/mL (0.9999), 50–250 μg/mL (0.9999), and 620–3100 μg/mL (0.9996), respectively. A reasonable level of accuracy was indicated by the tested method suggesting extremely high recovery levels (98–102%). The separation of tested compounds was achieved within 11 min. The developed and validated RP-HPLC–UV method was successfully applied for the identification and quantification of Que, Thy, and Pte for their combined estimation in complex formulations. From the validation study, it was found that the tested method is specific, accurate, precise, reliable, and reproducible.

1. Introduction

The quantification of bioactive molecules from intricate matrices, especially in pharmaceutical products, presents substantial problems due to their varied chemical characteristics and concentration variances. Plant extracts and biological fluids are examples of biological matrices that include several different components whose quantities might be affected by processing techniques and geographic origin, making qualitative and quantitative studies more challenging. Isolating, identifying, and measuring bioactive compounds that are present at trace quantities among many interfering molecules is a major difficulty. Low-concentration molecules with significant biological activity are frequently overlooked by traditional fractionation procedures, which can be ineffective [1,2]. Additionally, the complex interactions between several components may be the source of this mix-specific bioactivity, requiring analytical techniques that appropriately capture these dynamics. Advanced approaches are essential for addressing these analytical challenges [3]. Because it is easy to use and versatile for a variety of chemical characteristics, high-performance liquid chromatography (HPLC), or HPLC combined with mass spectrometry (HPLC–MS/MS) [4,5], high-performance thin-layer chromatography (HPTLC) [6] has become a common method for assessing bioactive compounds. Improved techniques, such as thorough two-dimensional liquid chromatography (LC × LC) [7] and sophisticated dereplication techniques, greatly increase the chemical identification and separation efficiency in complicated samples. In pharmaceutical development, these exact analytical methods are crucial for stability assessments, quality control, and verifying the therapeutic effectiveness of bioactive formulations [8].

Considering the earliest times, herbal remedies have been utilized as dietary supplements or medications, due to the presence of abundances of physiologically active substances with curative advantages. Medicinal plants are increasingly valued worldwide due to their limited negative and beneficial effects on human health. Plant-based metabolites and nutritional supplements have become more popular in recent years [9,10]. The bioactive substances present in medicinal plants provide a wide range of therapeutic benefits, such as antidiabetic [11], analgesic [12], antiviral [13], anticancer [14], antimalarial [15], and anti-inflammatory [16] activities. These treatments are common among urban impoverished individuals and in rural communities with little accessibility to contemporary medications. The pharmacological attributes of plants that are abundant in bioactive phytocompounds such as alkaloids [17], flavonoids [18], tannins [19], stilbenoids [20], and polyphenols [21] have been utilized to treat diseases [22,23]. In the identification and characterization of substances, separation becomes difficult since plant extracts are combinations of different phytocompounds. The chromatography techniques are strong and secure analytical techniques that can be used to determine their structure and biological activity, including plant-based products, and estimate the amounts and chemical constituents of those components. Although HPLC techniques have several drawbacks, as they have evolved, they have become more cost-effective and appropriate for addressing enormous quantities of substances [24].

A bioactive flavonoid discovered in plant-based products, Que (Figure 1), is well-known for its anti-cancer [25], anti-inflammatory [26], antidiabetic [27], cardiovascular [28], and neurodegenerative [29] properties with antioxidant benefits [30]. The literature indicates that it may slow the progression of degenerative conditions such as atopic dermatitis, renal fibrosis, myocarditis, and neurological disorders by preventing metastasis and triggering apoptosis [31]. Research has demonstrated that it is effective in preventing human metastatic osteosarcoma cells from expressing PTHR1 [32]. Numerous plants, fruits, and vegetables, such as onions, apples, tea, brassicas, grapes, almonds, and more, contain Que [23]. Estimating Que in novel formulations, calculating out the right dosage for treatment, and enhancing our understanding of plant characteristics all depend on detecting Que levels in natural resources [33].

A naturally generated bioactive substance, Thy (Figure 1) is extracted from the black seed oil of the Nigella sativa plant. Several studies have shown its potential for use in anticancer treatment and prevention [34,35]. It causes apoptosis by inhibiting the activity of the extracellular signal-related kinase, Akt, and NF-kB signaling pathways [36]. It has also been demonstrated that Thy promotes bone repair [37]. Thy, on the other hand, has a sluggish rate of absorption, strong hydrophobicity, rapid metabolism, limited bioavailability, and poor water solubility [38]. With proof of efficacy in addressing metabolic disorders such as hyperglycemia, hypertension, and hypercholesterolemia, black seed oil has been utilized for both preventative and therapeutic purposes [39]. While the thymol isomers such as carvacrol, t-anethole, and 4-terpineol contribute to the overall antioxidative activity, Thy and its derivatives are responsible for the antioxidative impact of black seed oils [40,41]. The traditional cold press extraction process delivers poor yields and is more prone to oxidation. Innovative pretreatment methods have been developed to enhance oil yields and quality features while lowering extraction time, solvent usage, and energy consumption [42,43]. These methods include microwaving, enzymatic digestion, pulsed electric fields, and ultrasonication [44].

Similarly, it has been demonstrated that the Pte stilbenoid compound, natural sources of which include blueberries, peanuts, grapes, and traditional medicinal herbs such as the tree wood of Pterocarpus marsupium, which is the 3,5-dimethoxy motif at the A-phenyl ring of resveratrol (Figure 1), has promising chemo-preventive and therapeutic effects [45]. Due to its structural properties, it is more physiologically active, lipophilic, and accessible than resveratrol. Additionally, Pte can be discovered in traditional herbal medicines used to treat diabetes [46].

By employing a thorough approach that includes both qualitative and quantitative evaluations of markers unique to each plant, any polyherbal compositions or any herbal formulations can be standardized. A comprehensive and simultaneous determining approach is required since it is difficult to design separate strategies to estimate each marker [47]. The precision, accuracy, and cost-effectiveness of this approach should enable the more effective qualitative and quantitative evaluations of many markers. In an individual quantitative analysis, the novelty is the capacity to estimate three markers simultaneously using just one methodology. HPLC procedures to detect Que, Thy, and Pte have been verified in earlier research (

Table 1. Selected publications on HPLC analysis of Que, Thy, and Pte.

Sample	Method	Chromatographic Conditions				Column Used	Ref.
		Mobile Phase	Flow Rate	Detection	Inj. Volume
Thymoquinone
Thy content in black cumin seeds (Nigella sativa) oil	HPLC diode array UV Vis	40% H2O and 60% of acetonitrile	1 mL/min	254 nm	20 μL	Inertsil ODS-3: (5 µm, 4.6 mm × 250 mm)	[48]
Accelerated solvent extraction (ASE) of Thy	UHPLC–DAD	Acetonitrile 50%: 2 mM ammonium formate 50%	0.2 mL/min	256 nm	10 µL	Accucore™ Vanquish™ C18 (1.5 µm; 100 mm × 2.1 mm)	[49]
Thy in seed powder of Nigella sativa (NS)	HPLC–UV	Methanol/acetonitrile/buffer (2.2 mM ammonium formate): 35:50:15	0.9 mL/min	249 nm	15 μL	Symmetry® LC-18 (150 mm × 3.9 mm × 5 μm)	[50]
Thy	UHPLC–PDA	Methanol/water (80:20)	0.3 mL/min	260 nm	5 µL	Pinnacle DB Cyanom (1.9 µm; 30 cm ×  2.1 cm)	[51]
Extract of Thy	RP-HPLC–UV	Water and methanol (40:60)	1.5  mL/min	254 nm	10 μL	Inertsil ODS-3v: (5 µm, 4.6 mm × 150 mm)	[52]
Thy	RP-HPLC–UV	Water/methanol (25:75)	1 mL/min	253 nm	20 μL	Luna C18 (250 mm × 4.60 mm, 5 µm)	[53]
Thy	HPLC–florescence detector	20 mM KH2PO4 buffer (pH adjusted to 2.7 ± 0.05 using 50% OPA (60:40)	1 mL/min	274 excitations and 340 emissions	25 µL	Symmetry LC 18 (150 mm × 3.9 mm, 5 µm)	[54]
Quercetin
Que in the extract of Eruca Sativa	HPLC–UV	Methanol/water with β-cyclodextrin (5 mM) with 0.1% orthophosphoric acid (70:30)	1 mL/min	370 nm	10 μL	Lichrosphere-100, C 18 (25 cm × 4.6 mm, 5 μm)	[55]
Que in C. reflexa	HPLC–UV	0.025 M NaH2PO4 buffer—ACN (pH—2.6) (72:28)	1.2 mL/min	378 nm	20 μL	Cosmosil C18-column (150 mm × 4.6 mm, 5 μm)	[56]
Que in three plant drugs from genus Sedum	HPLC–DAD	Methanol and 0.40% phosphoric acid (49:51)	1 mL/min	-	20 μL	Agilent Eclips XDB- C18 (4.6 × 250 mm, 5 µm)	[57]
Que in Ocimum sanctum and Tinosporacordifolia	HPLC–UV	Acetonitrile and methanol (50:50)	1 mL/min	256 nm	20 μL	C-18 column with 250 mm × 4.60 mm	[58]
Pterostilbene
Pte and Pte capsules	HPLC–UV	Acetonitrile and water (90:10)	1 mL/min	254 nm	20 μL	Nucleodur (150 mm × 4.6 mm, 5 μm)	[59]
Pte	HPLC–UV	Water/ACN (35:65)	1 mL/min	306 nm	20 μL	ACE C-18 Column (150 × 4.6 mm, 3 um)	[60]
Pte	HPLC–PDA	0.1% Trifluoroacetic acid in water/acetonitrile (10:90)	0.6 mL/min	219 nm	20 μL	Phenomenex C8 (250 mm × 4.6 mm, 5 µm)	[61]
Pte-loaded nanoemulsion	HPLC–PDA	Methanol and distilled water (60:40)	1 mL/min	319 nm	10 μL	Phenomenex LunaC18 (250 ×  4.6 mm, 5 μm)	[62]

); however, there is currently no approved analytical method for the simultaneous detection of these phytocompounds together. A specific herb-based HPLC approach might not be enough since pharmaceutical compositions contain both active and inactive chemicals. It is essential to establish a validated analytical approach for quality control and treatment efficacy monitoring. In order to ascertain the quantities of active phytocompounds in herbal formulations, this study attempts to implement a validated HPLC technique.

2. Materials and Methods

Quercetin (purity > 98%), Thymoquinone (purity 99%), and Pterostilbene (purity > 99%) were purchased from Yucca Enterprises, Mumbai, India. HPLC-grade methanol, acetonitrile, and orthophosphoric acid were procured from Fisher Scientific; Avantor, Maharashtra; and Finar, Gujarat, respectively. HPLC-grade water used was ultra-pure with specific resistance of 18 MΩ·cm obtained after filtration through Millipak@Express20 (0.22 μm; Sigma Aldrich, Singapore). Milli-Q by Merck was used throughout the investigation. The solvents were filtered through a 0.45 μm filter (Millipore) and degassed in an ultrasonic bath (Remi Instruments) before use. All the data of Perkin Elmer Flexar HPLC system were acquired and processed using PerkinElmer Total Chrome Workstation 6.3.2 software.

2.1. Preparation of Stock Solutions and Working Mixture Solution

A comprehensive stock solution, with a concentration of 1000 μg/mL, was prepared for bioactive markers, namely Que (Quecertin), Thy (Thymoquinone), and Pte (Ptrostilbene), utilizing HPLC-grade methanol. In order to ensure consistency and accuracy in the assessment of these essential bioactive substances, the standardized solution forms the fundamental elements for further investigation. Stock solutions of Que, Thy, and Pte were diluted with methanol, resulting in the preparation of 5 mL solutions, each attaining a concentration of 50 μg/mL for Que and Thy and 620 μg/mL for Pte for their respective bioactive markers. A higher working concentration was used for Pte to compensate for its comparatively lower UV absorbance at the selected detection wavelength (254 nm), thereby ensuring adequate detector response and reliable quantification within the linear range of the method. A stable and precise working solution combination preceding quantitative analysis is ensured by the intricate methodology for their combined estimation in intricate formulations. All standard and sample solutions were prepared and stored in amber-colored volumetric flasks and vials, and exposure to direct light was avoided throughout handling and analysis. Solutions were freshly prepared or stored at 4 °C and analyzed within the validated stability period.

2.2. Selection of Detection Wavelength

The sensitivity of HPLC method that uses UV detection depends upon proper selection of wavelength detection for tested phytoconstituents. Thus, before development of initial method, an appropriate detection wavelength of the targeted phytoconstituents is required to be measured to have an idea about the λ_max of the components. At the initial stage of the method development, the λ_max of the concerned phytoconstituents was measured. For this purpose, working standards of Que, Thy, and Pte were dissolved in and diluted with HPLC-grade methanol separately to prepare final working solutions of these compounds containing 50 μg/mL for Que and Thy and 620 μg/mL for Pte. Each standard working solution was scanned over the entire UV range of 199 nm to 400 nm using methanol as blank in a double beam UV–vis spectrophotometer (DS-1800, Shimadzu, Kyoto, Japan).

2.3. Selection of Chromatographic Conditions

Proper selection of the HPLC method depends upon the nature of the sample (non-ionizable or ionizable or neutral molecule), its molecular weight, and solubility. RP-HPLC was selected for the initial separation due to its simplicity and suitability. To optimize the chromatographic conditions, the effect of chromatographic variables such as mobile phase, pH, flow rate, and solvent ratio was studied. The resulting chromatograms were recorded, and the chromatographic parameters such as tailing factor and column efficiency were calculated as reported in this article. Finally, the condition that gives the best symmetry and capacity factor was selected for the estimation of Que, Thy, and Pte.

Simultaneous quantification of tested three phytocompounds such as Que, Thy, and Pte was estimated using RP-HPLC and was carried out on PerkinElmer HPLC system (model: Flexar) having analytical/FX 10 UPLC Pump, Flexar LC autosampler fitted with 250 μL syringe and 100 μL sample loop, as well as UV/Visible HPLC detector using following chromatographic conditions: stationary phase: Perkin Elmer Phenyl Column (250 mm × 4.6 mm, 5 μm) and mobile phase: 0.1% orthophosphoric acid in HPLC water/acetonitrile. Temperature of the column was 35 °C at 1 mL/min flow rate and detected at 254 nm. The chromatogram of separated compounds was recorded at 254 nm.

2.4. Method Validation

Validation is crucial in the pharmaceutical industry, ensuring that the performance of analytical procedures is accurate and reproducible across different laboratories and equipment. The validity of the RP-HPLC methodology was thoroughly evaluated in accordance with the standards established by the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH). Essential attributes including linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, robustness, and specificity were all elements of the thorough validation procedure. The result guarantees the strategy’s resilience, precision, and authenticity, which qualifies it for sophisticated investigations [63,64].

For tested phytocompounds, linearity was demonstrated by injecting five standard mixture solutions at concentrations ranging from 50 to 250 μg/mL for Que and Thy and 620–3100 μg/mL for Pte. Using the standard deviation (SD) of the calibration curve’s slope (s) and intercept (σ), the LOD and LOQ were calculated using the formulas LOD = 3.3 σ/s and LOQ = 10 σ/s. Three spiked solutions of known concentrations (80%, 100%, and 120%) of the targeted phytochemicals were analyzed, and the recovery percentage was estimated, in order to assess the accuracy of the method. Three injections of the standard solutions were made on the same day (intra-day) at two different times and two consecutive days (inter-day) to evaluate precision. By altering the chromatographic parameters, such as the temperature of the column, the flow rate, and the detection wavelength, robustness was assessed.

3. Results

3.1. Selection of Detection Wavelength

The UV overlay spectrum of Que, Thy, and Pte spectra (Figure 2) was recorded for the determination of the λ_max of all the phytoconstituents. The λ_max of Que, Thy, and Pte was detected as 255.5 nm, 252.5 nm, and 306 nm, respectively. The merged overlay of the UV absorption spectrum for the tested three phytoconstituents was obtained to determine the isosbestic wavelength with maximum absorption of all the phytoconstituents.

The HPLC analysis was performed using UV detection at 254 nm to enable the simultaneous quantification of all analytes within a single chromatographic run. Although Pte exhibits a higher absorption maximum around 306–310 nm in acetonitrile, its conjugated aromatic stilbene structure provides sufficient absorbance at 254 nm due to π–π* electronic transitions. UV spectral scanning of Pte in the mobile phase (acetonitrile–0.1% orthophosphoric acid in HPLC water) confirmed the appreciable absorbance at 254 nm with a stable baseline. During method development, Pte showed a linear detector response at 254 nm over the tested concentration range, with acceptable sensitivity, precision, and accuracy. Validation results demonstrated satisfactory linearity (R² = 0.9996), and the limits of detection and quantification met the analytical requirements. Therefore, the selected wavelength was considered suitable for the reliable quantification of Pte while maintaining method simplicity and robustness for simultaneous analysis [59]. A single detection wavelength of 254 nm was selected as a compromise wavelength at which all analytes exhibited adequate absorbance and acceptable LOD/LOQ values in accordance with ICH Q2(R1), enabling robust simultaneous quantification without wavelength switching.

3.2. Selection and Optimization of Chromatographic Conditions

The optimization of HPLC methods involves systematic adjustments of various parameters such as the column, mobile phase, and operating conditions to improve the resolution, sensitivity, and robustness while minimizing runtime. Unfortunately, all three compounds, Que, Thy, and Pte, exhibit poor water solubility but differ in their solubility in organic solvents [20,65]. Pte stands out as the most lipophilic. Que has extremely limited water solubility; Thy shows sparing solubility in water. There is no validated RP-HPLC method for simultaneously quantifying Que, Thy, and Pte, though individual methods exist, as mentioned earlier. A chromatographic protocol for analyzing these compounds using various trials has been executed, initiated with different ratios of a mobile phase constituting water as an aqueous phase and methanol (MeOH) as an organic phase and using a C-18 column as a stationary phase in an isocratic and gradient elution mode at different flow rates. Initial attempts using a C18 column (254 mm) failed to achieve adequate separation of the three compounds. Therefore, the stationary phase was replaced with a phenyl column, which provided desirable separation of all analytes. However, changing the stationary phase itself is not adequate to achieve suitable separations. With methanol replaced by acetonitrile (ACN), the aqueous phase is replaced by an acidified aqueous phase (0.1% orthophosphoric acid). To achieve adequate resolution and minimize co-elution, fine-tune the organic phase ratio and the pH of the aqueous phase (acidic) maintained to suppress the ionization of Que and enhance peak shapes for all analytes involved. Adjustments to the ACN percentage of ±5–10% can be made based on the performance observed during the run, particularly to address any overlaps or early elution of Thy. The effective chromatographic separation conditions include an acidified aqueous phase containing 0.1% orthophosphoric acid with ACN, with a typical ratio of 55:45 with flow rate 1 mL/min, and a wavelength of 254 nm is optimized and achieves the desired resolution. HPLC determination of the drugs was carried out by considering the solubility, stability, column performance, detection sensitivity, and flow rate; a mixture of 0.1% orthophosphoric acid in HPLC water and acetonitrile in the ratio of 55:45 v/v was selected as a mobile phase for method development (

Table 2. Optimized chromatographic conditions for the method of quantification of three phytoconstituents, Que, Thy, and Pte.

Parameters	Description
Column	Perkin Elmer Phenyl Column (250 mm × 4.6 mm, 5 μm)
Detection wavelength	254 nm
Flow rate	1.0 mL/min
Temperature	35 °C
Injection volume	5 μL
Mobile phase	0.1% orthophosphoric acid in HPLC water/acetonitrile (55:45 v/v)

). The chromatogram of separated compounds was recorded and the chromatogram is shown in Figure 3.

3.3. System Suitability Parameters

The system suitability test for chromatographic method standardization was performed before the validation of the HPLC method development. Six replicate injections of the prepared solutions of Que, Thy, and Pte were injected and peak asymmetry factor or tailing factor, number of theoretical plates, resolution, and RSD of peak areas were determined. The values of these parameters were found to be satisfactory in accordance with the requirements of USP. The results are summarized in Table 3.

The RSD (%) peak response for major peaks for the standard solution should not be more than 2.0% [66]. The number of theoretical plates should not be less than 2000 and the tailing factor should be less than 2.0. The RSD (%), number of theoretical plates, and tailing factors for Que, Thy, and Pte were within the acceptance criteria. The system suitability parameters on six replicate injections for the developed method were tested and the RSD, tailing factor, and number of theoretical plates were found to be in the acceptable limits. The capacity factor and resolution of the said compounds were greater than 2.0.

Table 3. System suitability data of Que, Thy, and Pte.

System Suitability Parameters	Quercetin	Thymoquinone	Pterostilbene
Retention time (min)	4.15 ± 0.02	8.70 ± 0.03	10.75 ± 0.04
Number of theoretical plates	38,625.188	68,421.531	64,878.88
Peak height	227,553.31	322,070.97	378,706.53
Tailing factor	1.2908	1.0095	1.0168
Signal to noise ratio	5688.83	1894.53	2103.92
Mean peak area	716,517.66	1,658,838.67	2,482,514.55
SD	1363.110	6063.106	9108.190
Relative SD	0.190	0.365	0.366
Capacity factor (k′) (t0: 1 min)	3.15	7.70	9.75
Resolution	-	3.03	2.98
Separation factor (α)	-	3.24	1.31

Table 3. System suitability data of Que, Thy, and Pte.

System Suitability Parameters	Quercetin	Thymoquinone	Pterostilbene
Retention time (min)	4.15 ± 0.02	8.70 ± 0.03	10.75 ± 0.04
Number of theoretical plates	38,625.188	68,421.531	64,878.88
Peak height	227,553.31	322,070.97	378,706.53
Tailing factor	1.2908	1.0095	1.0168
Signal to noise ratio	5688.83	1894.53	2103.92
Mean peak area	716,517.66	1,658,838.67	2,482,514.55
SD	1363.110	6063.106	9108.190
Relative SD	0.190	0.365	0.366
Capacity factor (k′) (t0: 1 min)	3.15	7.70	9.75
Resolution	-	3.03	2.98
Separation factor (α)	-	3.24	1.31

3.4. Method Validation

The developed method was validated and the results with respect to the various validation parameters are discussed thoroughly.

3.4.1. Linearity

For the estimation of Que, Thy, and Pte, a series of mixed standards were prepared in the concentration range of linearity. In this experiment, five concentrations of three standard working solutions of Que, Thy, and Pte were prepared in a constant range of 50 to 250 µg/mL, 50 to 250 µg/mL, and 620 to 3100 µg/mL to examine their linearity. This was evaluated as per the slope and regression values obtained from the standards tested. The calibration curves were plotted between the concentration and AUC observed at the selected wavelength. The results of linearity analysis indicate that the drug components are linear with respect to the concentration range applied in the method (Figure 4). LOD and LOQ were calculated and all the results related to linearity are shown in Table 4.

Table 4. Linearity data for Que, Thy, and Pte.

Linearity Parameter	Quercetin	Thymoquinone	Pterostilbene
Correlation coefficient (r2)	0.9999	0.9999	0.9996
Regression equation (y)	14049x + 20650	32355x + 62879	3877x + 14572
Linearity range (μg/mL)	50–250 μg/mL	50–250 μg/mL	620–3100 μg/mL
Residual standard deviation	6588.217509	23,558.51111	83,187.36082
Limit of detection	1.55 μg/mL	2.40 μg/mL	70.79 μg/mL
Limit of quantification	4.69 μg/mL	7.28 μg/mL	214.52 μg/mL

Note: Results obtained from three replicates.

Table 4. Linearity data for Que, Thy, and Pte.

Linearity Parameter	Quercetin	Thymoquinone	Pterostilbene
Correlation coefficient (r2)	0.9999	0.9999	0.9996
Regression equation (y)	14049x + 20650	32355x + 62879	3877x + 14572
Linearity range (μg/mL)	50–250 μg/mL	50–250 μg/mL	620–3100 μg/mL
Residual standard deviation	6588.217509	23,558.51111	83,187.36082
Limit of detection	1.55 μg/mL	2.40 μg/mL	70.79 μg/mL
Limit of quantification	4.69 μg/mL	7.28 μg/mL	214.52 μg/mL

Note: Results obtained from three replicates.

Figure 4. Linearity curve for the phytocompounds Que, Thy, and Pte.

Figure 4. Linearity curve for the phytocompounds Que, Thy, and Pte.

3.4.2. Accuracy

Accuracy, which was assessed against three repetitions at three spiked concentrations (80%, 100%, and 120%) of the substance being tested, is the degree of agreement between analytical techniques and traditional true or reference values. The accuracy for the developed method studied at different spike levels and the results show that the recovery (%) was found within the limit (

Table 5. Recovery study data of the phytocompounds Que, Thy, and Pte.

Recovery Study	Quercetin			Thymoquinone			Pterostilbene
Spike level	80%	100%	120%	80%	100%	120%	80%	100%	120%
Concentration taken (μg/mL)	50	50	50	50	50	50	620	620	620
Concentration added (μg/mL)	40	50	60	40	50	60	496	620	744
Recovered content	90.37	99.99	109.97	89.49	99.48	109.35	1120.02	1242.15	1364.92
Mean percentage of recovery ± SD	100.41 ± 0.483	99.99 ± 0.174	99.97 ± 0.179	99.44 ± 0.414	99.48 ± 0.312	99.41 ± 0.190	100.36 ± 0.666	100.17 ± 0.627	100.06 ± 0.540
RSD (%)	0.350	0.228	0.083	0.230	0.128	0.106	0.375	0.058	0.112

).

3.4.3. Precision

The precision of this method can be ascertained by calculating the relative standard deviation of each test response. Inter-day precision assesses differences in analyses when performed on multiple days inside the same laboratory, whereas intra-day precision describes the constant use of the same analytical processes inside the same laboratory over a brief period of time. RSD (%), which is the measure of instrument accuracy, is obtained by applying the same concentration repeatedly under similar circumstances. To assess inter-day precision across a two consecutive day period, the study employed standard solutions of Que, Thy, and Pte at fixed concentrations, each with three repetitions (

Table 6. Precision data of Que, Thy, and Pte.

Parameters	Quercetin	Thymoquinone	Pterostilbene
Inter-Day (Day 1)
Mean peak area	620,400.15	1,738,496.22	2,521,265.64
SD	610.4464	5916.723	2956.208
Relative SD	0.098396	0.340336	0.117251
Inter-Day (Day 2)
Mean peak area	617,057.16	1,744,775.29	2,539,296.00
SD	995.2106	2759.759972	2035.197078
Relative SD	0.161283	0.158172	0.080148
Intra-Day
Mean peak area	650,664.28	1,731,903.2	2,508,017.9
SD	850.2117	9344.877	4722.844
Relative SD	0.130668	0.539573	0.18831

).

3.4.4. Robustness

To assess the acceptability of the method under the slight variations in the chromatographic conditions, robustness analysis was carried out. The analysis was carried out on the changes in different flow rates, different column temperatures, and different detection wavelengths (

Table 7. Robustness data of Que, Thy, and Pte by altering the detection wavelength, flow rates, and column temperature.

Parameters	Quercetin	Thymoquinone	Pterostilbene
By altering the detection wavelength of 252 nm
Retention time (min)	4.099	8.894	11.07
Mean peak area	92,616.35	153,644.3	218,083.9
Standard deviation	489.8528	480.2017	1500.679
Relative standard deviation	0.528905	0.312541	0.68812
Tailing factor	1.3	1.1	1.1
Number of theoretical plate count	34,575.51	65,568.74	61,055.44
By altering the detection wavelength of 254 nm
Retention time (min)	4.00	8.658	10.705
Mean peak area	96,142.17	166,323	190,213
Standard deviation	103.1943	225.3557	239.7754
Relative standard deviation	0.107335	0.135493	0.126056
Tailing factor	1.2	1.0	1.0
Number of theoretical plate count	38,625.188	68,421.531	64,878.88
By altering the detection wavelength of 256 nm
Retention time (min)	4.077	8.859	11.104
Mean peak area	95,669.25	162,180	154,565.5
Standard deviation	185.0442	326.4393	796.7615
Relative standard deviation	0.193421	0.201282	0.515485
Tailing factor	1.2	1.0	1.0
Number of theoretical plate count	40,752.2	70,021.1	65,333.11
By altering the flow rate: 0.9 mL/min
Retention time (min)	4.52	9.82	12.28
Mean peak area	90,553.53	159,284.73	180,551.96
Standard deviation	165.959	295.0118	156.476
Relative standard deviation	0.1832	0.18521	0.086665
Tailing factor	1.4	1.1	1.1
Number of theoretical plate count	32,876.7	63,891.5	61,222.09
By altering the flow rate: 1.0 mL/min
Retention time (min)	4.019	8.733	10.868
Mean peak area	95,478.68	161,276.28	182,396.79
Standard deviation	154.7664	255.6494	454.5533
Relative standard deviation	0.162095	0.158516	0.249211
Tailing factor	1.2	1.0	1.0
Number of theoretical plate count	38,625.188	68,421.531	64,878.88
By altering the flow rate: 1.1 mL/min
Retention time (min)	3.710	8.069	10.126
Mean peak area	92,716.04	160,504.90	179,568.54
Standard deviation	156.2852	467.7465	267.2273
Relative standard deviation	0.168563	0.291422	0.148816
Tailing factor	1.2	1.0	1.0
Number of theoretical plate count	40,025.2	70,120.5	65,333.11
By altering the temperature: 30 °C
Retention time (min)	4.031	8.731	10.854
Mean peak area	93,372.6	164,516.2	189,005
Standard deviation	511.0089	1023.95	659.9987
Relative standard deviation	0.547279	0.6224	0.349197
Tailing factor	1.2	1.1	1.1
Number of theoretical plate count	36,600.349	66,995.135	62,967.16
By altering the temperature: 35 °C
Retention time (min)	4.018	8.709	10.814
Mean peak area	97,229.11	164,724.2	183,317.3
Standard deviation	73.09304	173.5275	235.8738
Relative standard deviation	0.075176	0.105344	0.12867
Tailing factor	1.2	1.0	1.0
Number of theoretical plate count	39,325.194	69,271.015	65,105.995
By altering the temperature: 40 °C
Retention time (min)	4.018	8.708	10.815
Mean peak area	96,657.06	164,565.1	186,573.503
Standard deviation	203.744	383.1356	1239.142
Relative standard deviation	0.2107	0.232817	0.664
Tailing factor	1.2	1.0	1.0
Number of theoretical plate count	39,388.7	69,070.8	65,333.11

). The statistical parameters like retention time, tailing factor, number of theoretical plate count, and RSD (%) were calculated. The results demonstrated that regardless of all variation in the system conditions, the method was robust as the RSD (%) was found to be well below the acceptable limit of 2.0%.

4. Discussion

On the background of the findings from the literature survey, the present study was designed to undertake experimental study for the development of a highly sensitive, new simultaneous HPLC method for the rapid separation and accurate quantification of Que, Thy, and Pte when present in combination in pharmaceutical preparation. The present study was also focused on evaluating the precision, accuracy, and robustness of the developed method. For this purpose, the validation of said developed method was carried out as per ICH guidelines. Under optimized conditions, Que, Thy, and Pte showed good selectivity and resolution; all the results of the validation data for the developed method are given in

Table 8. Summarized validation parameters of Que, Thy, and Pte.

Parameters		Acceptable Criteria	Obtained Result
			Quercetin	Thymoquinone	Pterostilbene
Linearity		Correlation coefficient ≥ 0.999	0.9999	0.9999	0.9996
Range		80–120%	50–250 μg/mL	50–250 μg/mL	620–3100 μg/mL
LOD		-	1.55 μg/mL	2.40 μg/mL	70.79 µg/mL
LOQ		-	4.69 μg/mL	7.28 μg/mL	214.52 µg/mL
Precision Repeatability		RSD < 2%	0.130668	0.539573	0.18831
Inter-day precision		RSD < 2%	0.098396	0.340336	0.117251
			0.161283	0.158172	0.080148
Accuracy	80% Spike	Recovery (98–102%)	100.41 ± 0.483	99.44 ± 0.414	100.36 ± 0.666
	100% Spike		99.99 ± 0.174	99.48 ± 0.312	100.17 ± 0.627
	120% Spike		99.97 ± 0.179	99.41 ± 0.190	100.06 ± 0.540

.

For the satisfactory separation of all the compounds, various mobile phase systems with different compositions and pH were tried on reverse-phase columns at the isocratic elution mode of the solvent delivery system. For this purpose, several trials were carried out for the development and optimization of ideal chromatographic conditions. A satisfactory separation of Que, Thy, and Pte was achieved on a phenyl column using the mobile phase consisting of solvent A (0.1% orthophosphoric acid in HPLC water) and solvent B (acetonitrile) with an isocratic elution mode using a 55:45 ratio at a flow rate of 1 mL/min and at a column temperature 35 °C with ultraviolet detection at 254 nm (Table 2). Methanol concentration (>80%) led to overlapping the peak of Pte with Thy as well as increased RT and reduced sensitivity for all the compounds, while at methanol concentration (<50%) separation occurred but the sequence change with prolonged RT was observed (Figure 5). The desired separation with reasonable RT for all the phytoconstituents was obtained when methanol was replaced with acetonitrile with a concentration range between 50 and 25%; however, 45% of acetonitrile concentration was found to produce the best resolution between Que, Thy, and Pte with optimum peak height, minimum tailing, and maximum HETP.

In comparison to phosphate buffer, the acidic solution produces sharper and more resolved peaks of all three compounds, which clearly indicate that 0.1% orthophosphoric acid is more suitable in the development of the ideal chromatographic conditions for the satisfactory separation of said compounds. The identification of Que, Thy, and Pte was confirmed by comparing the RT of the peaks obtained with the sample solution to that the RT of the peaks obtained by the standard solutions of Que, Thy, and Pte. The specificity of the developed LC method has been elucidated in Figure 3, where complete the separation of Quercetin, Thymoquinone, and Pterostilbene was observed. The mean RT ± SD values for Que, Thy, and Pte were 4.15 ± 0.02, 8.70 ± 0.03, and 10.75 ± 0.04, respectively.

The linearity of the LC method for Que, Thy, and Pte was evaluated over 50–250 μg/mL (Que, Thy) and 620–3100 μg/mL (Pte), with each concentration analyzed in triplicate (Table 4). Calibration curves showed high correlation coefficients and intercepts not significantly different from zero, meeting ICH guidelines. This confirms the method’s reliability across the tested concentration ranges. Additionally, capacity factors (k′), selectivity (α), tailing factors (T), and peak areas were assessed (Table 4). From the characteristic parameters of the regression equation, it was revealed that the obtained value of the correlation coefficient of Que, Thy, and Pte was within the acceptable criteria of the ICH guidelines. Precision, including repeatability and inter-day variation, was evaluated via the relative standard deviations (RSD%) of repeated injections, demonstrating consistent performance.

In the case of the repeatability study, six different injections of the same standard concentrations of Que and Thy (50 μg/mL) and Pte (620 μg/mL) were performed and the RSD (%) of the mean peak area of those six different injections of Que, Thy, and Pte was calculated statistically (Table 3), which was found to be 0.190%, 0.365%, and 0.0366% for Que, Thy, and Pte, respectively. The obtained RSD (%) of the drugs was found to be with the acceptable limit.

LOD and LOQ were calculated based on the standard deviation of the response (SD) and the slope of the calibration curve(s) at levels approximate to the LOD according to the formulae. In this study, the LOD was determined to be 1.55 μg/mL (Que), 2.40 μg/mL (Thy), and 70.79 μg/mL (Pte). The RSDs (%) for all the phytocompounds were found to be 0.130%, 0.539%, and 0.188%, respectively. LOQ was determined to be 4.69 μg/mL (Que), 7.28 μg/mL (Thy), and 214.52 μg/mL (Pte).

The calibration range for Que, Thy, and Pte was established as 50–250 μg/mL, 50–250 μg/mL, and 620–3100 μg/mL, respectively, reflecting practical concentrations in pharmaceutical products. Accuracy was verified via recovery studies at 80%, 100%, and 120% spiking levels, with recoveries between 98% and 102% (Table 5), demonstrating good accuracy. Inter-day precision, assessed by triplicate injections over multiple days at specified concentrations, showed RSDs of 0.161%, 0.158%, and 0.080% for the three compounds, all within ICH guidelines (Table 6). Variation in mean peak areas remained below 1.5%, confirming method precision and reproducibility.

The different results obtained with the different tested precision studies clearly reveal the reliability of the method for the quantitative determination of Que, Thy, and Pte within the established range. Robustness measures the capacity of the analytical method by the deliberate variations introduced into the method parameters. Several experiments such as detections of wavelength alteration (252 nm to 256 nm), flow rates (0.9–1.1 mL/min), and different temperatures (35 °C ± 5 °C) in the developed LC method reflect the changes likely to arise in different test environments. Analysis was performed in triplicate using the prepared solution containing Que, Thy 50 μg/mL, and Pte 620 μg/mL. The calculated RSD (%) values of each of the above-mentioned parameters were compared with the respective values obtained under optimum chromatographic conditions (Table 7).

5. Conclusions

The research work embodied in this article for the simultaneous method of the development and validation of Que, Thy, and Pte was carried out. The research work aimed to develop a new, highly sensitive, precise, and robust method for the simultaneous determination of Que, Thy, and Pte in bulk as well as in other dosage forms. The RP-HPLC–UV method developed allows for the reliable simultaneous quantification of Que, Thy, and Pte, satisfying ICH guidelines for system suitability, linearity, precision, accuracy, robustness, and sensitivity. Chromatographic conditions, including a phenyl column and optimized mobile phase for isocratic elution, resulted in well-resolved peaks for each analyte without interference from other components, confirming specificity. Calibration curves showed high linearity with correlation coefficients over 0.999, ensuring proportionality between the peak area and concentration. Validation studies demonstrated intra- and inter-day precision within %RSD limits of ≤2%, and recovery rates ranged from 98 to 102%, indicating robust accuracy across different concentrations. The method’s sensitivity was supported by the low LOD and LOQ values, suitable for pre-formulation and release studies. Additionally, small variations in the chromatographic parameters did not compromise performance, indicating robustness for routine quality control. A significant finding is the simultaneous resolution and quantification of the three distinct phytoconstituents at a single optimized UV wavelength under a simple isocratic method, a novel approach not previously reported together.