Chili pepper (Capsicum annuum L.), a perennial plant in the Solanaceae family, is widely consumed as a vegetable and pungent spice, particularly in Asia. Peppers include varieties such as sweet and hot peppers and are cultivated worldwide. They are considered to have exceptional nutritional value due to their high palatability and broad range of culinary applications (Ding et al., 2010; Padilha et al., 2015). Moreover, peppers are an extensively utilized spice frequently incorporated into a large diversity of dish, including kimchi, a traditional Korean delicacy. The growing interest in spices is attributed to the discovery of their beneficial effects on human health (Cho et al., 2020). Among various peppers, C. annuum L. cultivar Dangjo (DJ) was, developed in Korea through pedigree selection, induced mutation, and crossbreeding. It is notable for its potent α-glucosidase inhibitors, whose content is five times more than that in other Capsicum species (Kim et al., 2022; Kim et al., 2023).

Quercitrin, also known as 2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-chromen-3-yl 6-deoxy-α-L-mannopyranoside or quercetin 3-rhamnoside, is a natural flavonoid found in various plant parts, including flowers, leaves, and fruits. A glycoside of quercetin and rhamnose, quercitrin belongs to the flavanol glycosides subclass and is widely distributed in nature. Quercitrin has potent biological activities, including anti-inflammatory, antioxidant, anti-carcinogenic, and anti-leishmanial; it also helps prevent diarrhea and protect against UV-induced cell death and apoptosis (Jadczak et al., 2010; Tang et al., 2019; Thetsana et al., 2019). Additionally, quercitrin’s anti-cancer properties, particularly against non-small cell lung cancer, and its ability to inhibit pro-inflammatory mediators highlight its potential in diverse therapeutic areas (Truong et al., 2016).

Standardizing quercitrin measurement in DJ requires an analytical method to be developed and validated to quantify quercitrin. The validation of an analytical procedure is essential for demonstrating its suitability and ensuring that it generates reproducible, consistent, and effective outcomes. Validation involves defining the performance characteristics of a developed chromatographic system through validation parameters, including selectivity, linearity and linear range, sensitivity, accuracy, and precision. It is essential to establish a method’s reliability, particularly for new or updated medications, to ensure its effectiveness and consistency (Perwitasari et al., 2014; Jayawardhane et al., 2021; Varma et al., 2021). By validating the method, researchers can establish confidence in the precision of quercitrin quantification and are enabled accurately assess its content in DJE samples.

Quercitrin, a flavonoid found in plants like peppers, can be effectively quantified using reverse-phase high-performance liquid chromatography (HPLC). HPLC offers faster equilibration and better reproducibility for retention time. Validating the HPLC method ensures accuracy and reliability, which are crucial for many applications (Hu et al., 2009). This way, researchers are enabled to make meaningful comparisons and study applications in areas such as food quality control, pharmaceutical development, and nutritional assessments (Hu et al., 2009).

This study aimed to highlight the importance of validating the method for standardized quantification quercitrin in DJ and outline the key parameters, such as selectivity, accuracy, precision, and sensitivity, that are critical for ensuring the reliability and consistency of the analytical method.

Materials

Quercitrin was obtained from ChemFaces (catalog number: CFN98850); methanol and water were procured from Fisher Scientific; formic acid was obtained from Sigma-Aldrich.

Chemicals and reagents

This study focused on analyzing quercitrin, also known as quercetin 3-rhamnoside, with a purity ≥98%. DJ was the plant material under investigation. Methanol and water were utilized as solvents for the both mobile phase preparation and the sample solutions, while formic acid was incorporated to enhance the chromatographic performance.

Sample solution preparation and extraction

Quercitrin was extracted from freeze-dried DJ (DJE) by first weighing 1 g of the sample, which corresponded to approximately 0.315 mg/g of quercitrin, the indicator ingredient quercitrin. Then, the sample was placed into a 50 mL volumetric flask.

Then, 40 mL of methanol was added to the flask, which was subsequently sealed with a lid. The sealed flask was subjected to ultrasonic extraction using an ultrasonicator at 500 W and 65°C for 60 min. Following the extraction process, the flask was allowed to cool to room temperature. Afterward, 50 mL of methanol was added to the flask, and the mixture was stirred to achieve homogeneity. Next, the resulting solution was filtered through a 0.45-μm membrane filter (Minisart^TM RC Hydrophilic, 15 mm) to obtain the test solution for further analysis.

Instruments

Chromatographic analysis was conducted using an Agilent HPLC 1200 system and an Agilent HPLC 1100 system (Agilent Technologies) equipped with a diode array detector (DAD). Chromatographic separation was achieved using a CAPCELL PAK C18 UG120 column with dimensions of 4.6×250 mm and a particle size of 5 μm (Osaka Soda Co., Ltd.). Additionally, an ultrasonicator (JAC-5020, Kodo Technical Research Co., Ltd.) was used in the experimental setup.

Chromatographic conditions

The analysis was performed on an Agilent HPLC 1200 Series system equipped with a DAD set at 360 nm for recording chromatograms. Chromatographic separation was conducted on a C18 column at 40°C. The mobile phase comprised a mixture of 0.1% formic acid solution (solvent A) and 100% methanol (solvent B). The solvents were applied as follows: 0 to 40 min, 30% B; 40 to 41 min, 50% B; 41 to 43 min, 100% B; 43 to 43.1 min, 30% B; 43.1 to 49 min, 30% (B). The injection volume for each sample was set at 10 μL.

Standard solution preparation

The quercitrin standard solution was prepared by weighing precisely 5 mg of quercitrin and transferring it to a 100-mL volumetric flask. Quercitrin was dissolved in methanol, and ultrasonication was applied at 500 W for 10 min to ensure complete dissolution. Afterward, the solution was diluted with methanol to produce a standard stock solution of 50 mg/L. The stock solution was further diluted with methanol to prepare standard solutions with concentrations of 2.5, 5.0, 7.5, 10.0, 12.5, and 15.0 mg/L for calibration purposes. Then, these standard solutions were used in subsequent analyses as references for quantification.

Method validation

The HPLC method was validated according to the Association of Official Agricultural Chemists International guidelines (AOAC International, 2002). Specificity was assessed by analyzing quercitrin standard solutions at 10 μg/mL to confirm that the other components in DJ samples did not interfere with the peak of quercitrin peak. Linearity was evaluated by preparing quercitrin standard solutions at concentrations of 2.5, 5.0, 7.5, 10.0, 12.5, and 15.0 mg/L and analyzing each concentration in triplicates (n=3) constructing calibration curves, and determining linearity with the correlation coefficient (R²). The accuracy was tested by spiking DJE samples with quercitrin at low (3.15 μg/mL), medium (6.30 μg/mL), or high (9.45 μg/mL) levels, analyzing each level in triplicate (n=3) comparing the measured concentrations with the expected values, calculating the percentages of recovery. Precision was assessed by evaluating repeatability and analyzing a quercitrin standard solution five times within one day at three different concentrations [80%, 100%, and 120% of a target amount of 1,000 mg (100%)]. Precision was also assessed by confirming reproducibility and performing analyses on different days and with different operators or equipment, or both, with all tests conducted in five replicates. The relative standard deviation (RSD) was calculated to confirm the method precision, with an acceptable RSD typically less than 8%.

Statistical analysis

All analyses were conducted in three or five repetitions. The mean values and standard deviations were calculated using Microsoft Excel, while the statistical significance between groups was assessed using the SPSS software. Duncan’s multiple range test was applied to compare the mean values across the different groups, with the significance level set at P>0.05.

Validation methods

The HPLC method for measuring quercitrin in the C. annuum L. cultivar DJ was validated for specificity, linearity, accuracy, and precision (Table 1). Specificity was confirmed with a retention time of approximately 21 min and no peak interference. Linearity was demonstrated across concentrations of 2.5 to 15.0 μg/mL with an average R² of 0.99975. The accuracy, assessed by calculating the recovery rates of the spiked samples, ranged from 89.02% to 99.30% with %RSD between 0.50% and 5.95%. Precision was confirmed by demonstrating reproducibility with samples with quercitrin content ranging from 0.316 to 0.344 mg/g (%RSD of 0.835% to 1.379%) and repeatability with samples with quercitrin content ranging from 0.290 to 0.326 mg/g (%RSD of 0.94% to 3.21%).

Table 1 . Validation methods.

Parameter	Evaluation method	Result
Specificity	Obtained the HPLC analysis retention time	∙Retention time for both: approximately 21 min ∙No peak interference phenomena were observed ∙Quercitrin peak spectrum in the standard solution and sample (DJE) matches
Linearity	Confirmed with six concentrations of the standard substance	∙A range of 2.5－15.0 μg/mL was established to assess the accuracy and precision ∙Average coefficient of determination (R2): 0.99975
Accuracy	Evaluated the recovery rate by spiking the samples with the standard substance at three different concentrations	Recovery rate: 89.02%－99.30% %RSD range: 0.50%－5.95%
Precision	Evaluated reproducibility by having multiple analysts in each laboratory analyze the same sample using the same instrumentation. Confirmed repeatability by having an analyst assess the content of three or more samples	∙ReproducibilityQuercitrin content: 0.316－0.344 mg/g% RSD range: 0.835%－1.379% ∙RepeatabilityQuercitrin content: 0.290－0.326 mg/g%RSD range: 0.94%－3.21%

HPLC, high-performance liquid chromatography; DJE, Dangjo extract; RSD, relative standard deviation..

Specificity

Retention time: Applying the analytical method to both the quercitrin standards and DJE samples yielded consistent results, with a uniform display of detected peaks. Notably, peaks were observed in both the standard and sample solutions at 21.734 and 21.775 min, respectively; thus, the peaks occurred at approximately 21 min (Fig. 1). This similarity in retention times strongly suggests that these peaks corresponded to the same substance, likely quercitrin; therefore, the reliability of the analytical method was confirmed. Additionally, the clear resolution of the test solution from the surrounding peaks underscored the effectiveness of chromatographic separation. Robust separation ensures minimal interference from other components, enhancing the accuracy and specificity of the analysis. Overall, the consistent peak profiles for the standard and sample solutions validate the method’s capability to accurately identify and quantify quercitrin in DJE samples.

Figure 1. Chromatogram of quercitrin. (A) A standard solution. (B) A sample solution.

Peak purity: Peak purity was evaluated by gathering supplementary information essential to select suitable analytical conditions that ensure specific determination. A peak was deemed pure if its peak purity index exceeded the single point threshold, yielding a positive value for the minimum peak purity index (Jain et al., 2014). The purity of the quercitrin peak detected in the standard and DJE sample solutions was assessed by conducting a detailed examination of the UV peak spectrum (Fig. 2). The comparison of the UV spectra between the standard solutions and the DJE sample solutions revealed consistent quercitrin peak characteristics, confirming that the detected peaks represented quercitrin. This consistency underscores the reliability of the HPLC method.

Figure 2. The peak spectrum of quercitrin in standard and sample solution. (A) Quercitrin standard solution (10 mg/L). (B) Dangjo extract mineral quercitrin.

In addition, a purity factor analysis confirmed these findings. The standard quercitrin solutions had a purity factor of 998.90 (9 out of 63 spectra exceeding the threshold) and the DJE sample exhibited a purity factor of 998.01 (14 out of 60 spectra exceeding the threshold). Both solutions demonstrated a high degree of purity with minimal impurities; these results validated the HPLC method. Furthermore, this confirmation of peak purity enhances confidence in the accuracy of quantifying the quercitrin content of the DJE sample, again validating the HPLC method.

Linearity

The linearity of a calibration curve is typically evaluated using the coefficient of correlation, r, or the coefficient of determination, R². A correlation coefficient close to unity (r=1) strongly suggests linearity (Moosavi and Ghassabian, 2018). The evaluation of the linearity of the HPLC method was based on the outcomes obtained from analyzing the standard quercitrin solution at various concentrations. Through repeated assessments, the HPLC method was found to exhibit a linear response within the concentration range of 2.5 to 15.0 μg/mL. This finding indicated that as the concentration of quercitrin increased, the response of the analytical method also increased in a predictable and consistent manner. The observed slope values ranged from 19.71 to 21.25, further supporting the linear relationship between the concentration and the response (Table 2). Additionally, the high R² values (0.99972, 0.99973, and 0.99981) signified strong correlation coefficients, reinforcing the robustness of the linear regression model used to assess linearity. The detection limit for quercitrin in our analytical procedure was established at 0.30 μg/mL, representing the lowest concentration at which quercitrin could be reliably detected under standard operating conditions. The quantitation limit, defined as the lowest concentration at which quercitrin could be quantitatively measured with acceptable precision and accuracy, was determined to be 0.92 μg/mL. These values demonstrated the method’s reliability and suitability for accurately quantifying quercitrin within the specified concentration range, thereby ensuring the precision and accuracy of the results.

Table 2 . Standard linearity of quercitrin.

Parameter	Slope	R2	r
Quercitrin 1	21.25	0.99973	0.99986
Quercitrin 2	19.71	0.99981	0.99990
Quercitrin 3	20.00	0.99972	0.99986

R²: Coefficient of determination, which indicates the proportion of variance explained by the model. Values closer to 1 suggest a stronger linear relationship. r: Correlation coefficient, representing the strength and direction of the linear relationship. Values closer to 1 or —1 indicate a strong relationship..

Accuracy and recovery

The accuracy, a critical parameter in analytical chemistry, is assessed using the recovery calculation following a standard addition procedure. This approach enables researchers to determine the extent to which the measured results align with the true value of the parameter under investigation. Additionally, the accuracy can be confirmed by comparing the results obtained from the proposed analytical method with those derived from a reference method or another established technique known for its accuracy and precision. This comprehensive assessment of accuracy ensures the reliability and trustworthiness of an analytical method, enhancing confidence in the accuracy of the results obtained for the parameter of interest (Chandran and Singh, 2007; Wang et al., 2014).

The accuracy of the HPLC method was assessed (Table 3). The recovery tests yielded a recovery rate within the range of 89.02% to 99.30% for all samples. The RSD was within the range of 0.50% to 5.95%. Testing at three quercitrin concentrations (3.15, 6.30, and 9.45 μg/mL), as guided by the AOAC recommendation of measuring three concentrations based on 100%, helped us confirm that the method was accurate and reliable across the entire working range of quercitrin and that it was suitable for the intended use. Additionally, there was no significant difference between the groups spiked with quercitrin at 3.15, 6.30, and 9.45 μg/mL based on Duncan’s test, as the means were nearly identical across different subsets at a significance level of 0.05. Overall, the average recovery rate of 94.08% suggested that the analytical method was reliable and robust. It met the AOAC standard of 0.01% (0.1 mg/g) or higher and fell within the acceptable range of 85－110 (AOAC International, 2002). A high recovery rate indicates that a method effectively extracts and quantifies the analyte from the sample matrix, contributing to the accuracy of the results. Conversely, a low recovery may lead to inaccuracies in the measurement, affecting the overall accuracy of the method. Therefore, achieving satisfactory recovery rates is essential for ensuring the accuracy and reliability of the analytical method, as per the AOAC standard. These findings provide confidence in the accuracy and reliability of the HPLC method for determining the quercitrin content in DJEs.

Table 3 . Accuracy of the results.

Sample (mg/g)	Spiking concentration (μg/mL)	Detected standard concentration (mg/g)	Recovery rate (%)	Overall average of recovery rate (%)	%RSD
0.315	−	−	−	94.08	−
0.473	3.15	0.1551)	98.38		5.95
		0.1561)	99.30
		0.1401)	89.02
0.630	6.30	0.2871)	91.13		1.26
		0.2901)	92.03
		0.2941)	93.44
0.788	9.45	0.4461)	94.30		0.50
		0.4451)	94.13
		0.4491)	95.02

¹⁾All groups display no significant differences among themselves based on Duncan’s test..

RSD, relative standard deviation..

Precision

An analytical method’s precision refers to the level of agreement between a series of measurements obtained from multiple samplings of the same standardized sample under prescribed conditions. It assesses the degree of reproducibility or repeatability of a method, which is often expressed as the standard deviation (SD) or RSD (coefficient of variation). Precision, a critical parameter in analytical chemistry, evaluating the consistency of individual test results when a method is repeatedly applied to homogenous samples under normal operating conditions (Gupta, 2020; Chavan and Desai, 2022).

Reproducibility (inter-assay precision): Reproducibility measures the variation in a measurement system caused by different operators and environmental factors. An error may occur when different inspectors measure the same product under identical conditions, often due to insufficient training or non-standard measurement methods (Burdick et al., 2003; Pan, 2006; Kazemi et al., 2010). The reproducibility of the HPLC method was evaluated by testing pretreated samples, having two different analysts analyze the samples with different machine series, and comparing the results. The analysis generated an average quercitrin content of 0.323 to 0.341 mg/g, with an SD of 0.004 to 0.003 mg/g (Table 4). Our results demonstrated a satisfactory level of quercitrin content, which aligned well with the expectations for this type of analysis.

Table 4 . Reproducibility of the results.

Analyst	Machine	Quercitrin (mg/g)	Average (mg/g)	SD (mg/g)	%RSD
Person 1	Agilent 1200	0.323	0.3231)	0.004	1.379
		0.326
		0.322
		0.316
		0.327
Person 2	Agilent 1100	0.340	0.3411)	0.003	0.835
		0.340
		0.344
		0.337
		0.343

¹⁾All groups display no significant differences among themselves based on Duncan’s test..

SD, standard deviation; RSD, relative standard deviation..

Meanwhile, other research on quercitrin content has been conducted. For example, Kim et al. (2023) reported a quercitrin content of 0.7 mg/g in a similar cultivar. Similarly, Kim et al. (2022) reported a quercitrin concentration of 0.4097 mg/g. The variation in quercitrin content among these studies is attributable to differences in the extraction methods, cultivation conditions, and genetic variability of the analyzed cultivars. Here, the calculated RSD were 1.379% and 0.835%, indicating that the reproducibility of the analysis was within the acceptable range of 8% as specified by the AOAC standard of 0.01% (0.1 mg/g) (AOAC International, 2002). There was no significant difference between the two different analysts and machine series based on Duncan’s test, as the means were nearly identical across different subsets at a significance level of 0.05. The observed RSD value suggested that the variations among the measurements performed by the different analysts and machine series were relatively low, indicating the HPLC method’s satisfactory reproducibility. This finding underscores the reliability of the HPLC method for quantifying quercitrin in DJEs.

Repeatability (intra-assay precision): Repeatability refers to the variability in repeated measurements under consistent conditions. It involves determining the repeatability of a single instrument’s repeated measurements, assuming that factors, such as environment and operator remain constant and the time between measurements is minimized (McAlinden et al., 2015). Synonyms for repeatability include reliability, stability, consistency, and predictability. A measuring device is considered reliable if it consistently produces the same value under the same conditions for its intended application (Mazumder et al., 2011). In analytical chemistry, repeatability refers to the consistency of result under consistent conditions involving the same analyst, reagents, and equipment, usually within a short period. It is evaluated by determining the SD of the simultaneous duplicates or replicates. Replicates should be performed at different times within the same day, on different samples, and at varied concentrations to ensure a comprehensive assessment. This approach provides a more accurate representation of repeatability, considering the potential variations in the experimental conditions and sample characteristics (AOAC International, 2002).

In this study, we confirm the repeatability of the HPLC method for analyzing quercitrin content of DJEs by collecting samples in three different concentrations [80%, 100%, and 120% of the target sample amount of 1,000 mg (100%)], with five replicates each, and measuring the quercitrin content was measured according to each pre-treatment and analysis procedure. The average quercitrin content fell within the range of 0.290 to 0.326 mg/g while the %RSD range was analyzed at each concentration from 0.94 to 3.21% (Table 5). Based on Duncan’s test, there was no significant difference between the groups; the means for the 80, 100, and 120 groups were similar, appearing in different subsets at a significance level of 0.05. Hence, the %RSD observed across five repetitions using varied sample quantities remained within 4% of the established standard of 0.01% as per the AOAC guidelines (AOAC International, 2002). This finding confirms the satisfactory repeatability of the HPLC method.

Table 5 . Repeatability of the results.

Sample (%)	Area	Quercitrin (mg/g)	Average (mg/g)	SD (mg/g)	%RSD
80	97.265	0.313	0.3061)	0.010	3.21
	95.298	0.307
	89.555	0.290
	94.689	0.306
	97.715	0.315
100	126.089	0.320	0.3181)	0.003	0.94
	123.282	0.313
	125.425	0.319
	124.092	0.315
	125.966	0.320
120	150.524	0.316	0.3171)	0.005	1.62
	148.345	0.312
	155.310	0.326
	150.425	0.316
	150.919	0.317

¹⁾All groups display no significant differences among themselves based on Duncan’s test..

SD, standard deviation; RSD, relative standard deviation..

In this study, we have developed and validated an HPLC method for quantifying quercitrin in DJEs. The method has demonstrated high specificity, as evidenced by the consistent retention times and peak spectra observed in both the standard and sample solutions. The method’s linearity was confirmed within the concentration range of 2.5 to 15.0 μg/mL, with strong correlation coefficients indicating a predictable relationship between the concentration and the response. The accuracy assessment revealed satisfactory recovery rates (89.02%－99.30%) and low RSD values, suggesting the reliability and robustness of the HPLC method. The result from the precision analysis further supported the method’s reliability, with the result from both intra-assay and inter-assay precision analysis falling within acceptable limits, as per the AOAC standard.

In summary, these findings underscore the suitability of the developed HPLC method for the accurate and precise quantification of quercitrin in DJE samples. The standardization of quercitrin measurement and contributes to quality control efforts in the food and pharmaceutical industries. Moreover, the method’s reliability and reproducibility render it valuable for further investigation into the pharmacological properties of quercitrin and its potential applications in various therapeutic areas. The use of response surface methodology to optimize the extraction and HPLC analysis processes is recommended to improve the efficiency and precision of quercitrin extraction and quantification from DJ extract.

This research was supported by the “2021 Discovering Functional Crops Project” of The Food Industry Promotional Agency of Korea.

The authors declare no conflict of interest.

Concept and design: JES, SKJ. Analysis and interpretation: BRS. Data collection: BRS. Writing the article: ANF. Critical revision of the article: MJK. Final approval of the article: all authors. Obtained funding: SKJ. Overall responsibility: SKJ.