pharmaceuticals

Article
Validation of an HPLC-DAD Method for Quercetin Quantification
in Nanoparticles
Daniel Carvalho 1 , Ângelo Jesus 1,2 , Cláudia Pinho 1,2 , Rita Ferraz Oliveira 1,2 , Fernando Moreira 1,2, *
and Ana Isabel Oliveira 1,2

1 Escola Superior de Saúde, Instituto Politécnico do Porto, Rua Dr. António Bernardino de Almeida,
4200-072 Porto, Portugal; [email protected] (D.C.); [email protected] (Â.J.); [email protected] (C.P.);
[email protected] (R.F.O.); [email protected] (A.I.O.)
2 REQUIMTE-LAQV, Escola Superior de Saúde, Instituto Politécnico do Porto, Rua Dr. António Bernardino de
Almeida, 4200-072 Porto, Portugal
* Correspondence: [email protected]

Abstract: The evaluation of the efficacy of incorporation of quercetin in nanoparticles is crucial,

both for the development and quality control of pharmaceutical formulations. The validation of
analytical methods for the precise quantification of quercetin is useful for the evaluation of various
potential quercetin delivery systems and quercetin pharmacokinetics. This work aimed to validate
a high-performance liquid chromatography with diode array detection (HPLC-DAD) method for
quercetin detection and quantification in nanoparticles. Different mobile phase conditions and detec-
tion wavelengths (254 and 368 nm) were tested, and the major validation parameters were assessed
(precision, accuracy, linearity, sensitivity, stability, and selectivity). The best peak resolution was
obtained when quercetin was analyzed at 368 nm with a mobile phase of 1.5% acetic acid and a
water/acetonitrile/methanol ratio of 55:40:5. Under these conditions, quercetin also eluted rapidly
(retention time of 3.6 min). The method proved to be linear (R2 > 0.995), specific, and repeatable
(variation coefficient between 2.4% and 6.7%) and presented intermediate precision (variation co-
efficient between 7.2% and 9.4%). The accuracy of the analysis ranged between 88.6% and 110.7%,
and detection and quantification limits were 0.046 and 0.14 µg/mL, respectively. Quercetin solutions
Citation: Carvalho, D.; Jesus, Â.;
were more stable when stored at 4 ◦ C than at room temperature or −20 ◦ C. This validated method
Pinho, C.; Oliveira, R.F.; Moreira, F.; satisfied more parameters of bias assessment than most recent methods for quercetin determination
Oliveira, A.I. Validation of an and presented itself as more sensitive and efficient than general spectrophotometric methods. The
HPLC-DAD Method for Quercetin method was successfully used for the analysis of quercetin incorporation in nanoparticles and will be
Quantification in Nanoparticles. evaluated in the future for its adequacy for the determination of quercetin in more complex matrices.
Pharmaceuticals 2023, 16, 1736.
https://doi.org/10.3390/ Keywords: analysis; chromatography; HPLC; quercetin; validation
ph16121736

Academic Editor: Roberta Rocca

Received: 17 November 2023 1. Introduction

Revised: 13 December 2023
High-performance liquid chromatography (HPLC) is one of the most used chromatog-
Accepted: 14 December 2023
Published: 17 December 2023
raphy techniques given its versatility, easy operation, and minimal solution preparation [1].
Additionally, unlike gas chromatography (GC), HPLC can be used for thermolabile and
nonvolatile compounds that may be present in different matrices. Allowing both qualitative
and quantitative approaches, HPLC enables the detection of several analytes from the same
Copyright: © 2023 by the authors. sample and the convenient adaptation of several chromatographic conditions [1,2].
Licensee MDPI, Basel, Switzerland. UV/visible detectors are amongst the most used detectors in HPLC analysis, often in
This article is an open access article the form of a diode array detector (DAD). DAD enables simultaneous acquisition of the
distributed under the terms and spectra of the peaks within a range of wavelengths. This feature enables the selection of the
conditions of the Creative Commons best-fitting wavelength, resulting in increased sensitivity [2,3].
Attribution (CC BY) license (https://
All the analytical methods, whether newly developed or optimized, must go through
creativecommons.org/licenses/by/
a validation process for the compounds in the particular study to guarantee the reliability
4.0/).

Pharmaceuticals 2023, 16, 1736. https://doi.org/10.3390/ph16121736 https://www.mdpi.com/journal/pharmaceuticals

Pharmaceuticals 2023, 16, 1736 2 of 14

of results and ensure the scientific value of the developed method, assuring its reproducibil-
ity [4,5]. Several organizations have established guidelines indicating the validation process
to be followed. The Food and Drug Administration (FDA), International Conference on
Harmonization (ICH), and World Health Organization (WHO) are some of those examples.
According to ICH, linearity, specificity/selectivity, limit of detection (LOD), limit of quan-
tification (LOQ), precision, accuracy, robustness, and range of analysis are parameters that
must be evaluated [6].
Quercetin, a flavonol, has received great attention in recent years due to its strong
antioxidant [7], anti-inflammatory [8], cardiovascular [9], anticancer [10], and gastric protec-
tion [11] properties. It can be found in various fruits, vegetables, and plants [12,13] and has
been shown to be effective in preventing and treating several cardiovascular diseases [9],
neurodegenerative diseases [14], and cancer [10]. Quercetin is soluble in organic solvents,
such as methanol (4.0 mg/mL, being moderately soluble at 37 ◦ C) and dimethyl sulfoxide
(150 mg/mL at 25 ◦ C, presenting high solubility). On the other hand, it has low aqueous
solubility (approximately 0.01 mg/mL at 25 ◦ C) [15]. Despite its multiple potentialities
for human health, its physicochemical properties, such as low bioavailability, easiest en-
zymatic degradation, and rapid metabolism, limit its application in the pharmaceutical
industry [10]. Different attempts have been made to improve these properties using various
methodologies [16,17].
Recently published reviews have depicted the sample preparation [18] and develop-
ment and validation of analytical methods for the determination of quercetin from different
matrices [19,20]. It was concluded that chromatography methods constitute the group of
techniques most widely used for that purpose [19], while HPLC coupled with spectropho-
tometry detectors was the most popular method (surpassing mass spectrometry detectors).
These findings suggest that most laboratories have conditions to analyze quercetin by
HPLC-DAD and can benefit from the publication of optimized HPLC-DAD methods [20].
In one of the reviews, a risk-of-bias assessment with emphasis on method comparison was
performed. The assessment was based on a previously published study that considered the
evaluation of eight variables [21]. It was concluded that although all the included studies
mentioned/presented totally or partially at least four of the eight parameters, only one of
the studies fully satisfied more than four of the parameters (the study fully satisfied six of
the parameters) [20]. These results further demonstrate the need for more complete and
rigorous validation plans for quercetin determination.
To demonstrate improvements in biological effects, the efficient inclusion of quercetin
in different distribution systems, and even the presence of quercetin in natural products, the
validation of analytical methods that enable the detection and quantification of quercetin
in the most varied matrices is crucial. Some of the important issues to be addressed include
the full validation of methods that can be adopted in commonly existing equipment in most
laboratories, such as HPLC-DAD; the characterization of parameters such as stability that
has been scarcely depicted; and overcoming difficulties in distinguishing quercetin from
structurally similar compounds. The main objective of the present study was to optimize
and validate an HPLC-DAD method for quercetin detection and quantification for the
analysis of quercetin in synthetic nanoparticles.

2. Results
A higher chromatographic signal intensity was observed when the analysis took
place at 368 nm than when the analysis took place at 254 nm. As for the mobile phase
configuration, the ratio of 55:40:5 of water/acetonitrile/methanol, acidified with 1.5% acetic
acid and with a variable flow rate between 1.0 and 1.3 mL/min proved to be the best option
for quercetin quantification (Figure 1).
Pharmaceuticals 2023, 16, x FOR PEER REVIEW 3 of 15
Pharmaceuticals 2023, 16, 1736 3 of 14

Figure 1. Chromatogram of quercetin (150 µg/mL) exhibiting the retention time at 3.6 min.

Figure 1. Chromatogram
Regarding of quercetin
linearity, (150 µg/mL)
a calibration curve exhibiting
comprising thenine
retention time at
standard 3.6 min.
concentrations was
constructed. Additionally, two adjusted calibration curves comprising the five lowest
Regarding linearity,
concentrations a calibration
of the linear curve comprising
range (adjusted calibration nine
curvestandard concentrations
1) and the was
five highest concen-
constructed. Additionally, two adjusted calibration curves comprising the five
trations of the linear range (adjusted calibration curve 2) were also generated. The linearity lowest
concentrations of the linear
of all three calibration range
curves was (adjusted
evaluatedcalibration
regarding thecurve 1) and the coefficient,
determination five highestthe
concentrations of the linear range (adjusted calibration curve 2) were also generated.
correlation coefficient, and their concordance after backcalculation. These results are Thede-
linearity
picted inofTables
all three
1 andcalibration
2. Figures S1curves
and S2was evaluated
provided in theregarding the determination
Supplementary File exhibit the
coefficient, the correlation
obtained calibration coefficient,
curves, and S3
and Figures their
andconcordance after
S4, also in the backcalculation.
Supplementary File,These
present
results are depicted
the difference plots.in Tables 1 and 2. Figures S1 and S2 provided in the Supplementary
File exhibit the obtained calibration curves, and Figures S3 and S4, also in the
Supplementary File,
Table 1. Linearity present
range, the difference
regression equation,plots.
determination coefficient, and correlation coefficient
of quercetin.
Table 1. Linearity range, regression equation, determination coefficient, and correlation coefficient
of quercetin.Linearity Range (µg/mL)
Compound—Calibration Curve Calibration Equation r2 r
QUE—nonadjusted Compound— 0.14–245 Linearity Range y = 15,485x + 11,659 0.9976 0.9988
QUE—adjusted [1] 0.14–5 y Calibration Equation
= 15,262x − 271.48 r2
0.9994 r
0.9997
Calibration Curve (µg/mL)
QUE—adjusted [2] 5–245 y = 15,317x + 42,292 0.9954 0.9977
QUE—nonadjusted 0.14–245 y = 15,485x + 11,659 0.9976 0.9988
QUE: quercetin; r: correlation coefficient; r2 : determination coefficient.
QUE—adjusted [1] 0.14–5 y = 15,262x − 271.48 0.9994 0.9997
QUE—adjusted
Table 2. Backcalculation of quercetin standards= with
[2] 5–245 y 15,317x + 42,292
deviations. 0.9954 0.9977
QUE: quercetin; r: correlation coefficient; r2: determination coefficient.
QUE QUE—Nonadjusted QUE—Adjusted [1] QUE—Adjusted [2]
Concentration Accordance Accordance Accordance
(µg/mL) [Obtained] [Obtained] [Obtained]
(%) (%) (%)
0.14 −0.62 −442 0.15 107 - -
0.35 −0.37 −106 0.41 117 - -
0.57 −0.29 −51 0.49 86 - -
2.8 1.98 71 2.8 100 - -
5 4.16 83 5 100 2.2 44
65 64.25 100 - - 63 97
125 130.06 104 - - 129 103
185 193.26 104 - - 193 104
245 236.40 96 - - 234 96
[Obtained]: obtained concentration; QUE: quercetin.
Pharmaceuticals 2023, 16, 1736 4 of 14

The linearity of all three calibration curves and their respective equations was ad-
ditionally determined with the intercept of the ordinate at origin and relative standard
deviation (RSD) of the slope criteria, as shown in Table 3.

Table 3. Data from the analysis of intercept of the ordinate at origin and relative standard deviation
of the slope criteria for linearity evaluation.

95% Confidence Interval

Compound—Calibration Curve m sm Slope RSD (%)
Minimum Maximum
QUE—nonadjusted 15,485 289.47 1.87 −65,423 88,741
QUE—adjusted [1] 15,262 211.13 1.38 −2006 1463
QUE—adjusted [2] 15,317 601.69 3.93 −247,002 331,585
QUE: quercetin; m: slope; sm : slope standard deviation; RSD: relative standard deviation.

As for sensitivity determination, an LOD of 0.046 µg/mL and a LOQ of 0.14 µg/mL
were obtained.
Intra- and interday precision results are presented in Table 4. Regarding repeatability,
RSDs were equal to/lower than 6.74%, whereas intermediate precision presented a variation
equal to/lower than 9.42%.

Table 4. Analytical data for precision (intra- and interday) of quercetin.

Intraday Precision—RSD Interday Precision—RSD

QUE Concentration (µg/mL)
(%) (%)
0.35 5.66 9.42
0.57 5.47 8.19
5 6.74 6.87
125 2.41 7.38
185 2.64 7.18
Values were obtained from five independent samples (n = 5) in triplicate; QUE: quercetin; RSD: relative standard
deviation.

The accuracy of the method was determined relative to the adjusted equations. The
concentrations of 0.35, 0.49, and 0.57 µg/mL, relative to the adjusted Equation (1), resulted
in accuracies between 88.6% and 104.1%. The concentrations of 49, 125, and 196 µg/mL,
relative to the adjusted Equation (2), resulted in accuracies between 96.8% and 110.7%
(Table 5).

Table 5. Analytical data for accuracy of quercetin.

QUE Concentration (µg/mL) Obtained Concentration Accuracy (%)

0.35 0.31 88.6
0.49 0.51 104.1
0.57 0.52 91.2
49 52 106.1
125 121 96.8
196 217 110.7
QUE: quercetin.

To estimate the specificity of the method, besides quercetin, mixed solutions containing
rutin and kaempferol were also analyzed. Rutin presented a retention time of 2.5 min and
kaempferol a retention time of 5.4 min. Both chromatographic peaks were visually distinct
from the quercetin chromatographic peak, enabling their identification and quantification.
After promoting a slight variation in optimized chromatographic parameters, namely,
pH of the mobile phase (+0.11) and flow rate (+0.2 mL/min), it was possible to observe a
diminishing of the quercetin peak areas (Table 6).
Pharmaceuticals 2023, 16, 1736 5 of 14

Table 6. Analytical data for robustness evaluation regarding pH and flow rate parameters.

Difference of
% of Peak Area in
Retention Time
Parameter Relation to Retention
Method Parameter Peak Area in Relation to
Value Optimized Time (min)
Optimized
Method
Method (min)
Optimized 3.32 7,304,837 N/A 3.8 N/A
variation for
pH
robustness 3.43 6,741,931 92.29 3.8 0.0 min
evaluation
Optimized 1.3 mL/min 6,111,868 N/A 3.7 N/A
variation for
Flow rate
robustness 1.5 mL/min 4,299,546 70.35 2.4 −1.3 min
evaluation
N/A: Not applicable.

The results of stability studies of quercetin after 5 and 7 days of storage under different
conditions (−20 ◦ C, 4 ◦ C, and room temperature) are depicted in Table 7.

Table 7. Analytical data for stability of quercetin after 5 and 7 days in three storage conditions
(−20 ◦ C, 4 ◦ C, and room temperature) for three levels of concentration.

Concentration Day 0 Day 5 Day 7

Condition
(µg/mL) Mean (n = 3) Mean (n = 3) Stability (%) Mean (n = 3) Stability (%)
0.57 7129 7929 111.22 8737 122.57
−20 ◦ C 5 71,284 78,463 110.07 77,477 108.69
125 2,170,741 2,347,331 108.14 2,155,391 99.29
0.57 8048 8758 108.82 8697 111.41
4 ◦C 5 74,799 83,905 112.17 78,945 105.54
125 2,123,647 2,318,601 109.18 2,255,046 106.19
0.57 7799 6674 85.58 5336 68.42
Room
5 73,338 75,772 103.32 67,525 92.07
temperature
125 2,246,811 2,474,717 110.14 2,378,043 105.84

3. Discussion
3.1. Chromatographic Conditions
DAD detectors enable the evaluation of a wide range of wavelengths in the UV and
visible region (190–900 nm). Accordingly, the most suitable wavelength can be chosen for
the analyte under study [2]. The two most commonly studied wavelengths for quercetin
detection and quantification (254 [22,23] and 368 [24] nm) were evaluated. The higher
chromatographic signal intensity at 368 nm is related to the quercetin molecular structure,
more precisely to its B and C rings and hydroxyl groups, which demonstrate an intense ab-
sorption in a wavelength range between 300 and 400 nm due to electron transitions [25,26].
Previously published methods developed by Aguilar-Sánchez et al. [27], Ang et al. [28],
Antal et al. [29], Sree et al. [30], and Sousa et al. [31] used more distinct columns and detec-
tors to those available in our laboratory to perform the analysis. Therefore, by choosing the
method developed by Zu et al. [24] to test initial conditions, there was a higher probability
of more efficient implementation. After verifying a pronounced tailing phenomenon with
the adoption of the mobile phase composition described in the study by Zu et al. [24]
(consisting of a mixture of water, acetonitrile, and methanol (45:15:40) in an isocratic elution
and at a flow rate of 1.0 mL/min), different conditions were tested. Accordingly, the
percentage of water was tested between 45% and 70% (increases of 5%), the percentage
of acetonitrile was tested between 15% and 40% (increases of 5%), and the percentage of
methanol was tested between 0% and 40% (increases of 5%). Acetic acid was included at
Pharmaceuticals 2023, 16, 1736 6 of 14

concentrations of 0%, 0.5%, 1.0%, and 1.5%. The tested flow rate conditions varied between
1.0 and 1.5 mL/min, and both isocratic and gradient conditions were tested. The chromato-
graphic condition that ensured the biggest reduction in peak tailing was considered as
the primary criterion for the choice of mobile phase. The secondary criteria was the least
consumption of organic solvents (for economic and ecological purposes). As previously
mentioned, a ratio of 55:40:5 of water/acetonitrile/methanol, acidified with 1.5% acetic
acid and with a variable flow rate between 1.0 and 1.3 mL/min proved to be the best option
for quercetin quantification.

3.2. Method Validation

3.2.1. Linearity
This method was tested for linearity using linear regression and correlation coefficient.
Guidelines define that a minimum of five concentration levels should be analyzed to
construct a calibration curve, along with three analyses. This was accomplished in this case
because nine standard concentrations were used in triplicate [6].
A linear regression was observed between the peak area and analyte concentrations
after visual inspection, ranging between 0.14 and 245 µg/mL with a correlation coefficient
of 0.999, thus proving the method’s linearity. It is generally assumed that the method is
more linear the closer the correlation coefficient is to 1 [4,32]. Although linearity was proven
for the referred concentration range, a subdivision of the calibration curve was performed.
When the curve standards were backcalculated and the respective deviations were analyzed,
there was a reduction in the deviations of the adjusted equation in comparison to the
unadjusted equation (Table 2). Thus, to achieve the most accurate quantification as possible
and cover a broad linearity range of concentrations, it was necessary to divide the calibration
curve into two, with each one containing five points, resulting in a calibration curve
with a linearity range for lower concentrations (0.14 to 5 µg/mL) and another for higher
concentrations (5 to 245 µg/mL). This demonstrated the suitability of the method for
estimating a concentration of 5 µg/mL and encompassing a larger range than the studies of
Abdelkawy et al. [22] (linearity range: 0.10–25 µg/mL), Olszewska [33] (0.1–77.92 µg/mL),
Ang et al. [28] (1.25–200 µg/mL), Antal et al. [29] (6.0–192 µg/mL), Careri et al. [34]
(0.45–57.60 µg/mL), Chen et al. [35] (1.11–22.10 µg/mL), Zu et al. [24] (19–280 µg/mL),
Zhang et al. [36] (10,000–350,000 µg/mL), and He et al. [37] (14–70 µg/mL). This broad
range of concentrations is particularly useful in cases in which there is no estimated
concentration of quercetin.
However, some authors consider that the correlation coefficient is not the most appro-
priate parameter for linearity determination [38]. Therefore, linearity was also determined
using the intercept of the ordinate at origin and RSD of the slope criteria [6,38].
After analysis of the results (Table 3), it can be concluded that both criteria were satis-
fied. According to the intercept of the ordinate at origin criterion, zero should be contained
in the ordinate at the origin, and this was verified in all the curves [6,38]. According to the
slope RSD criterion, the standard error of the slope should be less than 5%, which was also
checked, and the errors were between 1.38% and 3.93% [38]. Thus, it can be stated that a
strong linearity relationship was observed in all the calibration curves.

3.2.2. Detection and Quantification Limits

The obtained LOD and LOQ values (0.046 and 0.14 µg/mL, respectively), along with
the calibration curve slope, showed the high sensitivity of the analytical method in question
for concentration values in the µg/mL range. In addition, the value found for LOQ
was concurrent with the concentration of the lowest standard in linearity. Accordingly,
it may be affirmed that it is possible to analyze quercetin accurately and precisely in
samples with analyte concentrations within the determined linearity range. The evidenced
sensitivity may be considered noninferior to the previously published methods on quercetin
detection by Zu et al. [24] (LOD = 3.0 µg/mL), Choudhari et al. [39] (LOD = 0.66 µg/mL,
LOQ = 2.0 µg/mL), and Abdelkawy et al. [22] (LOD = 0.05 µg/mL, LOQ = 0.1 µg/mL).
Pharmaceuticals 2023, 16, 1736 7 of 14

3.2.3. Precision
Regarding precision, the guidelines recommend that at least three concentrations and
a minimum of nine measurements covering the linearity range should be assessed and
determined by the RSD [6,32]. In this case, precision was evaluated for five concentration
standards with 15 measurements each (five samples/independent tests in triplicate), cover-
ing the entire range and including the orders of magnitude. Concentrations of 0.35 and
0.57 µg/mL were deemed relevant for the adjusted calibration curve 1, whereas concentra-
tions of 125 and 185 µg/mL were relevant for the adjusted calibration curve 2. Because the
5 µg/mL concentration was included in both the linear ranges of calibration curves 1 and 2,
its evaluation of precision was considered adequate for both ranges. The RSD of repeatabil-
ity showed values ranging from 2.71% (125 µg/mL) to 6.74% (5 µg/mL), while the RSD of
intermediate precision varied between 6.87% (5 µg/mL) and 9.42% (0.35 µg/mL), revealing
a slight positive tendency (increased precision came with an increased standard concen-
tration) [40]. According to the guidelines, RSD should be less than 15% for each standard
concentration tested, which was verified in the optimized method, confirming the method’s
repeatability and intermediate precision and showing that although small variations in
analytical method conditions may occur, precise quantification is achievable [32,40].

3.2.4. Accuracy
The closeness of the obtained value after chromatogram analysis with the real value
determines the accuracy, and it should be established throughout the linearity range as
it is one of the most important parameters to be evaluated [6,32]. This parameter could
be studied by the application of different techniques, in which case, a reference standard
is used. Guidelines define that the determination of accuracy should follow a minimum
number of analyses and tested concentrations equivalent to that defined for precision,
as previously mentioned [4,6]. For accuracy, six levels of concentrations were tested
in triplicate. The three lower concentrations (0.35, 0.49 µg/mL, and 0.57 µg/mL) were
considered to study the accuracy of the adjusted calibration curve 1, whereas the three
higher concentrations (49, 125, and 196 µg/mL) were included to study the accuracy of the
adjusted calibration curve 2. Accuracy should be equal to or lower than 15% variation for
every concentration level tested, that is, it should be between 85% and 115% [32].
Conveniently applying the most adequate curve equation according to the expected
concentration, the accuracy ranged between 88.6% (0.35 µg/mL) and 110.7% (196 µg/mL),
with the results compiled in Table 5.

3.2.5. Specificity/Selectivity
To ensure that it would be possible to distinguish quercetin from similar compounds,
two other polyphenolic compounds [6] whose chromatographic separation from quercetin
is considered to be challenging were selected (rutin and kaempferol) [41]. Both rutin
and kaempferol exhibit the same backbone as quercetin; however, kaempferol does not
present a hydroxyl group in the C3’ position [42], and in the case of rutin, a sugar group is
present in the C3 position [43]. From the results, it is possible to infer that the optimized
method demonstrates good selectivity as a clear separation of the three compounds was
verified with good resolutions, which also suggests the method’s accuracy, precision, and
linearity [6].
The results demonstrate an improvement in the simultaneous detection of these three
flavonoids as previous studies described the need for an optimization process or large
variations in chromatographic conditions for their convenient separation [41].

3.2.6. Robustness
According to the ICH, robustness should be evaluated as part of the development
process before execution of the analytical procedure validation study, and it fundamentally
represents the reliability of an analytical procedure after intentional variations in parame-
ters. Regarding HPLC analysis, modifications in the mobile phase are considered adequate
Pharmaceuticals 2023, 16, 1736 8 of 14

to evaluate the method’s capacity to meet the expected performance requirements during
normal use [6]. In this study, a slight modification in pH (from 3.32 to 3.43) resulted in a
7.71% decrease in the peak area, although not contributing to a modification in retention
time. On the other hand, a slight increase in flow rate (from 1.3 to 1.5 mL/min) resulted
in variations in both the peak area (−29.65%) and retention time (−1.3 min). Despite its
fast elution of quercetin, this method results in a tailing increase, further compromising
peak integration.

3.2.7. Stability
Solution stability is crucial to obtain reliable and reproducible results because a long-
run analysis may occur or a repetition of sample analysis may be needed, thus ensuring its
integrity throughout time if stored correctly [40].
In this study, three distinct concentration levels were evaluated and submitted to
different storage conditions, for different time lengths. The obtained values were compared
to the tested concentrations used to determine the accuracy [32].
Concentration variations were calculated for the fifth and seventh day of storage in
comparison to the solution preparation day, and it was used to establish their stability.
These values were between 68.42% and 122.57% (Table 7).
Following storage at −20 ◦ C, stability ranged between 108.14% and 111.22% after
5 days and 99.29% and 122.57% after 7 days. After storage at 4 ◦ C, the variation was
between 108.82% and 112.17% after 5 days and between 105.54% and 111.41% after 7 days.
At room temperature, stability was in the range of 85.58% to 110.14% and 68.42% to 105.84%
after 5 and 7 days, respectively. Therefore, the best storage condition was at 4 ◦ C, with the
solutions remaining stable for 7 days when correctly stored as there was less discrepancy
in the stability values compared to the original concentration and amongst themselves. It
was also observed that there was greater stability at higher concentrations compared to
lower concentrations. Stability results above 100% suggest that solvent evaporation may
occur with a consequent increase in quercetin concentration, whereas the observed values
below 100% may be justified by the degradation of the analyte.

3.3. Overall Assessment of the Method Performance and Quercetin Quantification in Nanoparticles
A comparison of the analytical methods to determine the one that is most fit for pur-
pose may be very challenging owing to the different features that need to be considered.
Additionally, consistent bias assessments specifically developed for validation of analytical
methods are scarce. Nevertheless, a recently published review considering studies pub-
lished between 2018 and 2022, performed a bias assessment and concluded about the most
fit-for-purpose methods, for quercetin determination. The factors that need to be considered
to determine the most suitable method were found to be the retention time, the sensitivity
of the methods, and the results of a bias assessment [20]. Regarding retention time, in the
present study, a 3.6 min retention time was recorded. Only one of the studies included in
the review could provide a more efficient analysis for quercetin, presenting a retention time
of 2.8 min [44]. In terms of sensitivity, the method described herein may be considered more
sensitive than any of the spectrophotometric methods included in the mentioned review,
presenting an LOD of 0.046 µg/mL. In the review, the spectrophotometric method of Srivas-
tava et al. [45], displaying an LOD of 0.33 µg/mL, was considered the most sensitive. With
regard to the bias assessment, this study fully satisfied six of the eight parameters: (i) the
criteria for acceptable performance was established according to the guidelines, (ii) the
reference method using reference material was performed, (iii) the x–y plot of the data was
presented, (iv) the difference plot was provided, (v) the regression analysis was considered,
and (vi) the linearity test was performed and interpreted; only one of the methods included
in the review fully satisfied a maximum of six of the parameters. Accordingly, the present
spectrophotometric method developed and validated for the determination of quercetin
may be considered superior to the most recent spectrophotometric methods.
Pharmaceuticals 2023, 16, 1736 9 of 14

The fact that the present method enabled the simultaneous determination of rutin and
kaempferol alongside quercetin suggests that it may be used, at least for qualitative pur-
poses, for preparations containing a mixture of the three compounds. The fact that quercetin
remained satisfactorily stable at 4 ◦ C for at least seven days after sample preparation is also
an important finding that may influence, for example, reanalysis procedures.
The method described herein is currently employed in our laboratory for quercetin
detection in nanoparticles.
Polycaprolactone nanoparticles have been used for the improvement of quercetin
properties. These nanoparticles are important delivery systems for the treatment of several
pathologies once they enable the delivery of compounds in target-specific sites or enable
their applications. Therefore, quercetin quantification in this matrix has become useful
and imperative.
Quercetin can be detected in a supernatant by evaluating the free compound or after
extraction from the nanoparticles. When quercetin is free in a supernatant (water), the
preparation of samples consists of the dilution of quercetin in methanol and acetonitrile to
obtain a solvent ratio of water/acetonitrile/methanol of 45:15:40. For the extraction from
nanoparticles, the nanoparticles are first broken down by dissolving them in methanol
assisted by ultrasound and then centrifugated to obtain a quercetin solution, which is then
diluted in water and acetonitrile.
Thus, the method’s reproducibility is noteworthy as it can detect and quantify quercetin
after using different extraction methods depending on the type of analysis (direct or indi-
rect) and the method used to prepare the standards.
Its selectivity is also notable as no interferents were detected during the analysis.
Interferents could eventually appear as a result of the nanoparticle coating or products
derived from quercetin that might arise during the production process.

3.4. Limitations
Despite different attempts to suppress the existence of a tailing phenomenon in the
chromatographic profile of quercetin, it could only be diminished and not fully eliminated.
Previous studies have described similar chromatograms when determining quercetin by
spectrophotometric detectors and after reviewing analytical methods for this purpose.
Mansour et al. [19] concluded that tailing is a frequent incident when using reverse-phase
chromatography. Tailing was depicted in quercetin chromatograms of methods developed
for the analysis of Bauhinia variegata Linn flower extract [46], German grape wines [47],
Ginkgo biloba leaf extract [48], Costus igneus leafs [49], Hibiscus sabdariffa L. calyces [50], and
Brassica oleracea L. var. capitata plant material [51]. Particularly regarding reverse-phase
chromatography methods like the one described herein, tailing is known to be due to
“mixed-mode” retention, where the hydrophobic monolayer comprises one type of site and
the “active” silanols comprise a second type of site [52]. Although commonly described, it
is important to acknowledge that the tailing phenomenon is undesirable, may compromise
the quantification accuracy, and constitutes an important limitation to the present study.
Although the presently described HPLC-DAD method has proved to be fit for the
analysis of quercetin in pharmaceutical preparations, the adequacy for determination of
quercetin in complex matrices—both from biological matrices and from natural sources—is
unknown. It is important to acknowledge that the complexity of those matrices may in-
crease noise in the chromatogram and result in coeluting peaks. For more complex matrices
and especially if low analyte levels are to be expected, mass spectrometric methods may be
more appropriate given their increased sensitivity. Recently, mass spectrometric methods
for the detection of quercetin in complex matrices have been used for the analysis of sam-
ples of Ziziphus jujuba and Ziziphus nummularia plants [53], berries [54], and Polygonatum
verticillatum rhizomes [55].
Pharmaceuticals 2023, 16, 1736 10 of 14

4. Materials and Methods

4.1. Reagents
Methanol (≥99.8%; 32.04 g/mol) and acetonitrile (≥99.9%; 41.05 g/mol) were pur-
chased from Fisher Scientific (Loughborough, UK). Acetic acid (60.05 g/mol) was purchased
from VWR® (Fontenay-sous-Bois, France). Quercetin (≥95.0%; 302.24 g/mol) and rutin
(≥94.0%; 610.52 g/mol) were purchased from Sigma-Aldrich® Co. (St. Louis, MO, USA).
Kaempferol (≥98.0%; 286.24 g/mol) was purchased from Santa Cruz Biotechnology, Inc.
(Dallas, TA, USA).

4.2. Chromatographic Conditions

HPLC analysis was conducted on a JASCO® equipment with a reverse-phase C-18
column (LiChroCART® 250-4, LiChrosorb® , RP-18, 250 mm × 4 mm, 5 µm), a DAD (MD-
4010, Jasco® , Oklahoma City, OK, USA), an autosampler (AS-4050, Jasco® , Oklahoma
City, OK, USA), and a pump (PU-4180, Jasco® , Oklahoma City, OK, USA). ChromNav®
2.0 was the software employed for data acquisition and analysis, which was set at room
temperature with an injection volume of 10 µL. Detection was performed at 254 and 368 nm.
Different mobile phase mixtures were evaluated in an isocratic elution. Previously purified
water was filtered with a 0.22 µm nylon filter, and all solvents were degassed by ultrasound.
A ratio of 55:40:5 of water/acetonitrile/methanol, acidified with 1.5% acetic acid, was used
as the mobile phase. The flow rate was as follows: 1.000 (0–3.5 min), 1.300 (3.5–8 min), and
1.000 (8 min until analysis finale) mL/min. The chromatographic run was set for 10 min.

4.3. Standard Solution Preparation

Quercetin stock solution (0.49 mg/mL) was prepared in a water/acetonitrile/methanol
ratio of 45:15:40. Then, successive dilutions were made to obtain the standard solutions.
Rutin and kaempferol solutions were obtained following the same procedure.

4.4. Method Validation

4.4.1. Linearity and Range
The linearity of the analytical method optimized for quercetin quantification was
determined using nine quercetin standard solutions (0.14, 0.35, 0.57, 2.8, 5, 65, 125, 185,
and 245 µg/mL) [6,32]. Analysis was performed over different days in triplicate, and a
calibration line comprising all analyses was constructed.
One of the strategies to evaluate linearity is the RSD of the slope [6,38]. Accordingly,
RSD was calculated using Equation (1):

RSD (%) = sm /m × 100 (1)

where “sm ” represents the slope standard deviation and “m” represents the slope.

4.4.2. Limit of Detection and Limit of Quantification

As recommended, LOD and LOQ were determined based on the calibration curve
(specifically, using the calibration curve slope) and standard deviation of the response
obtained through the analysis of 10 blank samples analyzed in triplicate [6,56]. These
parameters were determined employing Equations (2) and (3), respectively [6]:

LOD = 3.3 × δ/s (2)

LOQ = 10 × δ/s (3)

where “δ” represents the standard deviation of the response, and “s” represents the calibra-
tion curve slope.
Pharmaceuticals 2023, 16, 1736 11 of 14

4.4.3. Precision
Intra- and interday precisions were calculated by analyzing quercetin standard solu-
tions in five distinct concentrations (0.35, 0.57, 5, 125, and 185 µg/mL) in triplicate and
subsequently expressed under RSD [6] (Equation (4)):

RSD (%) = s/X × 100 (4)

where s represents the standard deviation of a series of measurements, and X represents

the mean value from the independent variable.

4.4.4. Accuracy
Regarding the method’s accuracy determination, six quercetin standard solutions with
known concentrations (0.35, 0.49, 0.57, 49, 125, and 196 µg/mL) were injected in triplicate
for subsequent backcalculation of the area values obtained in the calibration equation,
and further comparison of the concentration was carried out with the values of the actual
concentrations [6] through Equation (5) (n = 5):

Accuracy = Experimental value/Real value × 100 (5)

4.4.5. Specificity/Selectivity
Different solutions were prepared with two other compounds whose chemical struc-
ture was similar to quercetin and which can be found in several matrices, together with
quercetin, such as medicinal plants: rutin and kaempferol [57]. Solutions containing 0.245
µg/mL of each of the three compounds were prepared and analyzed in triplicate to verify
the ability to separate the peaks of the different compounds under analysis [6].

4.4.6. Robustness
Robustness was assessed in relation to slight variations in two parameters of the
mobile phase, as suggested by the ICH guidelines: pH and flow rate [6]. Thus, a 1.1
increase in pH and an increase of 0.2 mL/min in flow rate were both individually studied
and compared to the optimized method. The differences regarding peak area and retention
time were determined.

4.4.7. Stability
Three concentrations of standard solutions (0.57, 5, and 125 µg/mL) were analyzed
in three different storage conditions (−20 ◦ C, 4 ◦ C, and room temperature) at different
times (5 and 7 days postpreparation) to determine the stability of the solutions prepared
for analysis [5]. Stability was calculated by comparison with the day of its preparation, and
variations in the results were determined using Equation (6):

Stability (%) = Concentration (day x)/Concentration (day 0) × 100 (6)

5. Conclusions
A simple, rapid, accurate, and precise method was successfully optimized and vali-
dated following existing guidelines for quercetin detection and quantification. The method
comprised a wide concentration range with the application of adjusted equations for
quantification depending on the concentration of quercetin after first analyzing using a
nonadjusted equation. Quercetin was rapidly eluted, enabling a short analysis time and,
consequently, a more ecologic and economic approach than most previously reported meth-
ods. This may be a promising method for analysis of quercetin contained in nanoparticles
for new, effective, and safe biological and therapeutic approaches. Given the sensitivity of
the method, its efficiency, and the fulfilment of most bias assessment parameters, it stands
as a particularly convenient method compared to most recently published studies using the
HPLC-DAD technology, which is available widespread, for quercetin determination. This
Pharmaceuticals 2023, 16, 1736 12 of 14

method will be evaluated in the future for its suitability for the determination of quercetin
in more complex matrices and the possible inclusion of additional analytes.

Supplementary Materials: The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/ph16121736/s1, Figure S1: Calibration curve of quercetin—nonadjusted
for a linear range of 0.14–245 µg/mL; Figure S2: Calibration curve of quercetin. a: Quercetin—
adjusted [1] for a linear range of 0.14–5 µg/mL. b: Quercetin—adjusted [2] for a linear range of
5–245 µg/mL; Figure S3: Difference plot of concentrations between 0.14 and 5 µg/mL, exhibiting
the variance between the linear theoretical range (discontinuous line), backcalculation of the con-
centration given by the nonadjusted calibration curve (orange dots), and backcalculation of the
concentration given by the adjusted calibration curve (gray dots); Figure S4: Difference plot of
concentrations between 5 and 245 µg/mL, exhibiting the variance between the linear theoretical
range (discontinuous line), backcalculation of the concentration given by the nonadjusted calibration
curve (orange dots), and backcalculation of the concentration given by the adjusted calibration curve
(gray dots).
Author Contributions: Conceptualization, D.C., F.M. and A.I.O.; methodology, D.C., F.M. and C.P.;
software, D.C. and Â.J.; validation, D.C., F.M. and R.F.O.; formal analysis, A.I.O., C.P. and R.F.O.;
investigation, D.C. and Â.J.; resources, A.I.O. and Â.J.; data curation, D.C.; writing—original draft
preparation, D.C., F.M. and A.I.O.; writing—review and editing, R.F.O., Â.J. and C.P.; supervision,
A.I.O. and F.M. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is contained within the article and Supplementary Material.
Conflicts of Interest: The authors declare no conflict of interest.

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