agronomy

Article

A Sustainable Reversed-Phase HPTLC Method for theQuantitative Estimation of Hesperidin in Traditional andUltrasound-Assisted Extracts of Different Varieties of CitrusFruit Peels and Commercial Tablets

Ahmed I. Foudah 1 , Faiyaz Shakeel 2 , Prawez Alam 1, Mohammed H. Alqarni 1, Maged S. Abdel-Kader 1,3

and Sultan Alshehri 2,*

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Citation: Foudah, A.I.; Shakeel, F.;

Alam, P.; Alqarni, M.H.; Abdel-Kader,

M.S.; Alshehri, S. A Sustainable

Reversed-Phase HPTLC Method for

the Quantitative Estimation of

Hesperidin in Traditional and

Ultrasound-Assisted Extracts of

Different Varieties of Citrus Fruit

Peels and Commercial Tablets.

Agronomy 2021, 11, 1744.

https://doi.org/10.3390/

agronomy11091744

Academic Editors: Mercedes

Vazquez Espinosa and Gerardo

Fernández Barbero

Received: 9 August 2021

Accepted: 28 August 2021

Published: 30 August 2021

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4.0/).

1 Department of Pharmacognosy, College of Pharmacy, Prince Sattam Bin Abdulaziz University, P.O. Box 173,Al-Kharj 11942, Saudi Arabia; a.foudah@psau.edu.sa (A.I.F.); prawez_pharma@yahoo.com (P.A.);m.alqarni@psau.edu.sa (M.H.A.); mpharm101@hotmail.com (M.S.A.-K.)

2 Department of Pharmaceutics, College of Pharmacy, King Saud University, P.O. Box 2457,Riyadh 11451, Saudi Arabia; faiyazs@fastmail.fm

3 Department of Pharmacognosy, Faculty of Pharmacy, Alexandria University, Alexandria 21215, Egypt* Correspondence: salshehri1@ksu.edu.sa

Abstract: Hesperidin (HSP) is a bioactive flavanone glycoside, present abundantly in the variety ofcitrus fruits. The environmental safety and sustainability of the reported analytical assays of HSPanalysis have not been considered in the literature. Hence, a sensitive and sustainable “reversed-phase high-performance thin-layer chromatography (RP-HPTLC)” method has been developed andvalidated for HSP analysis in traditional (TE) and ultrasound-based (UBE) extracts of four differentvarieties of citrus fruit peels and its commercial tablet dosage forms. The binary combination ofgreen solvents such as ethanol-water (50:50, v v−1) was used as the mobile phase. The detection ofHSP was performed at 287 nm. The sustainable RP-HPTLC method was linear in 20–2000 ng band−1

range. The studied validation parameters, including accuracy, precision, robustness, sensitivity wereacceptable for HSP analysis. The content of HSP in TE of four different varieties of citrus fruitsincluding grapefruit peels (Citrus paradisi), mosambi peels (Citrus limetta), lemon peels (Citrus lemon),and orange peels (Citrus sinensis) was detected as 8.26, 6.94, 5.90, and 6.81% w w−1, respectively. Thecontent of HSP in TE of commercial formulations A and B was detected as 5.31 and 5.55% w w−1,respectively. However, the content of HSP in UBE of grapefruit peels, mosambi peels, lemon peels,and orange peels was detected as 11.41, 8.86, 7.98, and 8.64% w w−1, respectively. The content ofHSP in UBE of commercial formulations A and B was detected as 6.72 and 6.92% w w−1, respectively.The greenness score of the sustainable RP-HPTLC method was predicted as 0.83 using analyticalGREEnness (AGREE) metric approach, indicated the excellent greenness profile of the RP-HPTLCmethod. UBE procedure for HSP was superior over its TE procedure. These observations andresults suggested that the present RP-HPTLC method can be successfully used for the quantitativeestimation of HSP in the variety of citrus fruit peels and its commercial formulations. In addition,this method is simple, rapid, precise, accurate, and economical compared to the reported analyticalmethods of HSP analysis. It is also safe and sustainable method due to the use of ethanol-watersolvents systems, as both the solvents are green solvents compared to the solvents used in reportedanalytical methods of HSP analysis.

Keywords: AGREE; hesperidin; sustainable RP-HPTLC; citrus fruit peels; validation

1. Introduction

Citrus fruits are important crops, which produced worldwide [1,2]. Traditionally,the by-products of citrus fruits such as pulps, seeds, and peels represent a problem intheir disposal because these wastes were burned, which produced carbon dioxide and

Agronomy 2021, 11, 1744. https://doi.org/10.3390/agronomy11091744 https://www.mdpi.com/journal/agronomy

Agronomy 2021, 11, 1744 2 of 14

other harmful gases [1]. However, new ecofriendly approcahes have been developed nowto obtain new high-value products [2]. Interestingly, citrus peels have natural bioactivecompounds such as flavonoids, which have several therapeutic effects [2,3]. Hesperidin(HSP) is a flavanone glycoside, which is present in large amounts in the peels of variouscitrus fruits [4,5]. It possesses various therapeutic activities such as antioxidant [4,5],analgesic [6], anti-inflammatory [6–8], antiallergic [9], cardioprotective [10], and anticanceractivities [11] in literature.

Various analytical techniques have been reported for the quantitative estimation ofHSP either alone or in combination with other bioflavonoids in various food products,commercial formulations, and plant-based materials. An ultra-violet (UV) spectrometrymethod was applied for the simultaneous analysis of HSP and diosmin in tablet dosageform [12]. UV-spectrometry-based assay has also been reported for the quantitative estima-tion of HSP in dry matter of citrus tissues [13]. The main disadvantage of UV-spectrometrymethods is their sensitivity [12,13]. The variety of “high-performance liquid chromatogra-phy (HPLC)” assays have been established for the quantitative estimation of HSP eitheralone or in combination with other bioflavonoids in commercial formulations, citus fruitpeels, and citrus juices [14–19]. In most of the reported HPLC methods of HSP analysis,the mobile phase used was the binary combination of methanol and water (W). Methanolis a toxic solvent and hence these methods are not safe and sustainable for HSP analy-sis [14–19]. “Liquid chromatography tandem mass spectrometry (LC-MS)” assay has alsobeen established for the determination of HSP in combination with other bioflavonoidsin its plant-based materials [20]. Various LC-MS-based assays were also applied for thedetermination of HSP in combination with other bioflavonoids in the biological fluids ofrats and humans [21–25]. An “ultra-performance liquid chromatography tandem massspectrometry (UPLC-MS)” method has also been established for the simultaneous analysisof HSP, neo-HSP, naringin, naringenin, meranzin hydrate, and hesperitin in rat plasmasamples and plant extract [26]. In reported LC-MS and UPLC-MS methods of HSP analysis,the mobile phase used was the binary combination of acetonitrile-W or acetonitrile-buffers.Acetonitrile is a highly toxic solvent and hence reported LC-MS and UPLC-MS methodsare not safe and sustainable for HSP analysis [20–26]. A fluorimetry method has also beenestablished for the simultaneous estimation of HSP and diosmin in combined dosage formand human plasma samples via complex formation using terbium [27]. Reported fluorime-try method was not sensitive enough for HSP analysis in human plasma samples [27]. Asingle normal-phase “high-performance thin-layer chromatography (HPTLC)” assay isavailable for the quantitative estimation of HSP in four different varieties of citrus fruitpeels [28]. The ternary combination of ethyl acetate-methanol-W was used as the mobilephase for HSP analysis using reported HPTLC method. Although, ethyl acetate and W aregreen solvents, but methanol is a toxic solvent and hence this method was not completelysafe and sustainable for HSP analysis [28]. After detailed evaluation of published reportson quantitative estimation of HSP in dosage forms, plant extracts, and biological fluids,we have found that the safety and sustainability of reported analytical assays were notassessed. Recently, a sustainable reversed-phase HPTLC (RP-HPTLC) assay has beenreported for the determination of pterostilbene in comparison to the normal-phase HPTLCin its capsule dosage form. The combination of ethanol (EtOH)-W was used as the mobilephase in reported RP-HPTLC method of pterostilbene [29]. However, the composition ofmobile phase and the nature of analytes of present study were different from reportedmethod of pterostilbene analysis. In addition, the sustainable RP-HPTLC assay has not yetutilized for the quantitative analysis of HSP in its different varieties of citrus fruit peelsand its commercial formulations. Sustainable HPTLC assays offer various advantages overHPLC and other analytical assays [30–33]. Therefore, the sustainable RP-HPTLC assay forthe quantitative analysis of HSP was chosen in this research. Various metric approacheshave been documented for the greenness assessment of the analytical methods [32–37].However, only “analytical GREEnness (AGREE)” metric approach utilizes all 12 differentprinciples of the “green analytical chemistry (GAC)” for this purpose [36]. Hence, the

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AGREE metric approach was used for the greenness evaluation of the sustainable RP-HPTLC assay [36]. Based on all these assumptions, the present research was aimed toestablish a sensitive and sustainable RP-HPTLC assay for the quantitative analysis of HSPin four different varieties of citrus fruit peels including grapefruit peels (Citrus paradisi),mosambi peels (Citrus limetta), lemon peels (Citrus lemon), and orange peels (Citrus sinensis)and two different commercial formulations. The sustainable RP-HPTLC assay for thequantitative analysis of HSP was validated using “International Council for Harmonization(ICH)” Q2 (R1) recommendations [38].

2. Materials and Methods2.1. Plant Materials

Four different variety of citrus fruits including, grape fruits, mosambi fruits, lemonfruits, and orange fruits were purchased from the hyper market of Al-Kharj, Saudi Arabia.All four plant materials were authenticated by a taxonomist at “Medicinal, Aromatic andPoisonous Plants Research Center (MAPPRC), College of Pharmacy, King Saud Univer-sity, Riyadh, Saudi Arabia”. A voucher specimen (number 13998) was deposited in the“Herbarium of College of Pharmacy, King Saud University, Riyadh, Saudi Arabia”.

2.2. Chemicals and Reagents

The reference standard of HSP (purity: 99%) was procured from “Sigma Aldrich(St. Louis, MO, USA)”. HPLC-grade EtOH was procured from “E-Merck (Darmstadt,Germany)”. Deionized/HPLC-grade W was obtained from a “Milli-Q water purifier unit(E-Merck, Darmstadt, Germany)”. Other solvents/reagents were of analytical grades. Thecommercial tablet formulations of HSP i.e., formulation A and formulation B were procuredfrom a pharmaceutical market in “Alexandria, Egypt”.

2.3. Chromatography

The quantitative analysis of HSP in its working standard, four different varieties ofcitrus fruit peels, and commercial tablet dosage forms was performed by “HPTLC CAMAGTLC system (CAMAG, Muttenz, Switzerland)”. The RP-HPTLC estimation of HSP wascarried out via “10 × 20 cm2 aluminum plates pre-coated with RP silica gel 60 F254S plates(E-Merck, Darmstadt, Germany)”. The samples were spotted as the 6 mm bands withthe help of a “CAMAG Automatic TLC Sampler 4 (ATS4) Sample Applicator (CAMAG,Geneva, Switzerland)”. The applicator for sampling was connected to the “CAMAGmicroliter Syringe (Hamilton, Bonaduz, Switzerland)”. The rate of applicationfor thequantification of HSP was fixed constant at 150 nL s−1. The RP silica gel TLC plates weredeveloped in a “CAMAG automatic developing chamber 2 (ADC2) (CAMAG, Muttenz,Switzerland)” with a distance of 80 mm under linear ascending mode. The mobile phasewas the binary combination green solvents such as EtOH-W (50:50, v v−1). The chamber forthe development was saturated previously with the vapors of EtOH-W for 30 min at 22 ◦C.The detection of HSP was carried out at 287 nm. The slit dimensions (band length × width)and scanning rate were fixed at 4 × 0.45 mm2 and 20 mm s−1, respectively. Each analysiswas performed in triplicates. The software utilized for the data processing and analysiswas “WinCAT’s (version 1.4.3.6336, CAMAG, Muttenz, Switzerland)”.

2.4. HSP Calibration Plot and Quality Control (QC) Samples

The weighed amount of HSP (10 mg) was dissolved in 100 mL of EtOH-W (50:50, v v−1)binary solvent mixtures in order to obtain the stock solution of 100 µg mL−1. The variablevolumes of this stock solution were diluted again with EtOH-W (50:50, v v−1) binary solventmixtures to obtain the HSP concentrations in 20–2000 ng band−1 range. The preparedsolutions of HSP with variable concentrations were applied to the RP silica gel TLC plates.The chromatographic response for HSP was recorded for each HSP concentration using thesustainable RP-HPTLC method. The HSP calibration plot was constructed by plotting theHSP concentrations against its recorded TLC response. Additionally, three different QC

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samples, including low QC (LQC; 60 ng band−1), middle QC (MQC; 800 ng band−1), andhigh QC (HQC; 2000 ng band−1) samples were prepared separately for validation studiesof the sustainable RP-HPTLC method.

2.5. Sample Preparation for the Quantitative Analysis of HSP in TE of Citrus Fruit Peels

The air-dried peels (5 g) of four different varieties of citrus fruits including grapefruits(Citrus paradisi), mosambi fruits (Citrus limetta), lemon fruits (Citrus lemon), and orangefruits (Citrus sinensis) were coarsely powdered, defatted with petroleum ether and thenexhaustively extracted a Soxhlet apparatus with hot methanol for 72 h. The solvent wasevaporated and the residue was separately dissolved in methanol in 50 mL volumetricflask. The obtained samples were used for the quantitative analysis of HSP in TE of citrusfruit peels using the sustainable RP-HPTLC assay at 287 nm.

2.6. Sample Preparation for the Quantitative Analysis of HSP in UBE of Citrus Fruit Peels

The UBE of the air-dried peels (5 g) of four different varieties of citrus fruits wasperformed using ultrasonic vibrations using the “Bransonic series (Model CPX5800H-E;Princeton, NJ, USA)”. The methanol was evaporated using a rotary vacuum evaporator andthe residue was dissolved in 50 mL of methanol in a volumetric flask. It was ultrasonicatedat 50 ◦C for 1 h. The obtained samples were used for the quantitative analysis of HSP inUBE of four different varieties of citrus fruit peels using the sustainable RP-HPTLC assayat 287 nm.

2.7. Sample Processing for the Quantitative Analysis of HSP in TE of Commercial TabletDosage Forms

Two different tablet dosage forms (one brand containing 500 mg of HSP and anotherbrand containing 50 mg of HSP) were weighed and an average weight for each brand wasrecorded. The tablets were crushed to obtain uniform powder. A powder containing 50 mgof HSP was extracted using hot methanol. The obtained mixture was filtered to removeany insoluble materials of the tablet and sonicated for 30 min. Approximately 1.0 mL ofthis stock was diluted again with methanol to obtain 50 mL solution. The obtained samplewas used for the quantitative estimation of HSP in TE of tablet dosage forms using thesustainable RP-HPTLC assay at 287 nm.

2.8. Sample Preparation for the Quantitative Analysis of HSP in UBE of Commercial TabletDosage Forms

The UBE extraction of the HSP from commercial tablets was performed by ultrasonicvibrations using the “Bransonic series (Model CPX5800H-E; Princeton, NJ, USA)”. Bothbrands of HSP containing tablet dosage forms were crushed to obtain fine powder. A pow-der containing 50 mg of HSP was dissolved in hot methanol. The solvent was evaporatedusing a rotary vacuum evaporator and the residue was dissolved in 50 mL of methanol.The obtained solution was ultrasonicated at 70 ◦C for 1 h. The obtained samples were usedfor the quantitative estimation of HSP in UBE of tablet dosage forms using the sustainableRP-HPTLC assay at 287 nm.

2.9. Validation Studies

The sustainable RP-HPTLC assay for the quantitative estimation of HSP was validatedfor various validation parameters according to the ICH-Q2 (R1) recommendations [38]. TheHSP linearity was evaluated by plotting HSP concentrations against its measured HPTLCarea. The HSP linearity was determined in 20–2000 ng band−1 range for the sustainableRP-HPTLC method. The system suitability parameters for the sustainable RP-HPTLC assaywere determined in terms of “retardation factor (Rf), asymmetry factor (As), and number oftheoretical plates per meter (N m−1)”. The “Rf, As, and N m−1” values were determined,by adopting their formulae reported in literature [39].

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The accuracy for the sustainable RP-HPTLC assay was evaluated as the % recov-ery, which was obtained at LQC (60 ng band−1), MQC (800 ng band−1), and HQC(2000 ng band−1) for the sustainable RP-HPTLC assay.

The precision for the sustainable RP-HPTLC assay was measured as intra/interdayprecision. Intraday precision was evaluated by the determination of HSP at LQC, MQC,and HQC on the same day. Interday precision was evaluated by the determination of HSPat LQC, MQC and HQC on three different days [38]. Each precision was evaluated in sixdifferent replicates (n = 6).

The robustness was estimated by making some minor modifications in the mobilephase for the sustainable RP-HPTLC assay. For this, the original EtOH-W (50:50, v v−1)mobile phase was modified to EtOH-W (52:48, v v−1) and EtOH-W (48:52, v v−1) mobilephases, and the required analytical responses were noted [38].

The sensitivity for the sustainable RP-HPTLC assay was determined as “detection(LOD) and quantification (LOQ) limits” by standard deviation technique. The “LODand LOQ” of HSP for the sustainable RP-HPTLC assay was calculated by adopting theirstandard formulae reported in literature [38,39].

The peak purity/specificity for the sustainable RP-HPTLC assay was determined bycomparing the Rf values and spectrodensitograms of HSP in four different varieties ofcitrus fruits and tablet dosage forms with those of pure HSP.

2.10. Quantitative Estimation of HSP in TE and UBE of Citrus Fruits and Commercial Formulations

The processed samples of TE and UBE of four different varieties of citrus fruit peelsand commercial formulations were spotted to the RP silica gel TLC plates and their HPTLCresponses were recorded. The HSP contents in all processed samples were determinedfrom the calibration plot of HSP for the sustainable RP-HPTLC assay.

2.11. Greenness Evaluation

The greenness profile for the sustainable RP-HPTLC assay was assessed using “AGREEmetric approach” [36]. The AGREE scores (0.0–1.0) of the sustainable RP-HPTLC assay werepredicted using the “AGREE: The Analytical Greenness Calculator (version 0.5, GdanskUniversity of Technology, Gdansk, Poland, 2020)”.

3. Results and Discussion3.1. Method Development

Upon exhaustive analysis of reported analytical methods of HSP quantification, wefound that the environmental safety and sustainability/greenness of the reported analyticalmethods have not been taken into consideration. Accordingly, the current study was per-formed to establish a rapid, sensitive, and sustainable RP-HPTLC assay for the quantitativeestimation of HSP in TE and UBE of four different varieties of citrus fruit peels and twodifferent tablet dosage forms.

For the determination of HSP using the sustainable RP-HPTLC assay, differentamounts of EtOH and W such as EtOH-W (50:50, v v−1), EtOH-W (60:40, v v−1), EtOH-W(70:30, v v−1), EtOH-W (80:20, v v−1), and EtOH-W (90:10, v v−1) were studied as thegreen solvent mixtures/mobile phases for the development of an acceptable band for thequantitative analysis of HSP in TE and UBE of four different varieties of citrus fruit peelsand two different commercial formulations. The green solvent system was developedunder chamber saturation conditions and its pictogram is summarized in Figure 1.

From the results, it was observed that EtOH-W (60:40, v v−1), EtOH-W (70:30, v v−1),EtOH-W (80:20, v v−1), and EtOH-W (90:10, v v−1) green solvent mixtures presented a poorspectrodensitogram of HSP with a high As value (As = 1.19). While, the EtOH-W (50:50,v v−1) green solvent mixtures had shown to present a well-separated spectrodensitogramof HSP at Rf = 0.73 ± 0.03 with an acceptable As value (As = 1.05) as shown in Figure 2.

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Figure 1. Thin-layer chromatography (TLC)-chromoplate of standard hesperidin (HSP), citrus peel extracts, and commercial tablets developed using EtOH-W (50:50, v v−1) as the mobile phase for a sustainable “reversed-phase high-performance thin-layer chromatography (RP-HPTLC)” assay.

From the results, it was observed that EtOH-W (60:40, v v−1), EtOH-W (70:30, v v−1), EtOH-W (80:20, v v−1), and EtOH-W (90:10, v v−1) green solvent mixtures presented a poor spectrodensitogram of HSP with a high As value (As = 1.19). While, the EtOH-W (50:50, v v−1) green solvent mixtures had shown to present a well-separated spectrodensitogram of HSP at Rf = 0.73 ± 0.03 with an acceptable As value (As = 1.05) as shown in Figure 2.

Figure 2. Representative spectrodensitogram of standard HSP for the sustainable RP-HPTLC method.

Accordingly, the EtOH-W (50:50, v v−1) combination was chosen as the final mobile phase for the quantitative estimation of HSP in TE and UBE of four different varieties of citrus fruit peels and two different commercial formulations. The spectrodensitograms for the sustainable RP-HPTLC assay were recorded densitometrically and the maximum

Figure 1. Thin-layer chromatography (TLC)-chromoplate of standard hesperidin (HSP), citrus peel extracts, and commercialtablets developed using EtOH-W (50:50, v v−1) as the mobile phase for a sustainable “reversed-phase high-performancethin-layer chromatography (RP-HPTLC)” assay.

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Figure 1. Thin-layer chromatography (TLC)-chromoplate of standard hesperidin (HSP), citrus peel extracts, and commercial tablets developed using EtOH-W (50:50, v v−1) as the mobile phase for a sustainable “reversed-phase high-performance thin-layer chromatography (RP-HPTLC)” assay.

From the results, it was observed that EtOH-W (60:40, v v−1), EtOH-W (70:30, v v−1), EtOH-W (80:20, v v−1), and EtOH-W (90:10, v v−1) green solvent mixtures presented a poor spectrodensitogram of HSP with a high As value (As = 1.19). While, the EtOH-W (50:50, v v−1) green solvent mixtures had shown to present a well-separated spectrodensitogram of HSP at Rf = 0.73 ± 0.03 with an acceptable As value (As = 1.05) as shown in Figure 2.

Figure 2. Representative spectrodensitogram of standard HSP for the sustainable RP-HPTLC method.

Accordingly, the EtOH-W (50:50, v v−1) combination was chosen as the final mobile phase for the quantitative estimation of HSP in TE and UBE of four different varieties of citrus fruit peels and two different commercial formulations. The spectrodensitograms for the sustainable RP-HPTLC assay were recorded densitometrically and the maximum

Figure 2. Representative spectrodensitogram of standard HSP for the sustainable RP-HPTLC method.

Accordingly, the EtOH-W (50:50, v v−1) combination was chosen as the final mobilephase for the quantitative estimation of HSP in TE and UBE of four different varieties ofcitrus fruit peels and two different commercial formulations. The spectrodensitogramsfor the sustainable RP-HPTLC assay were recorded densitometrically and the maximumchromatography response was recorded at 287 nm for the sustainable RP-HPTLC assay.Accordingly, the entire analyses of HSP were carried out at 287 nm.

3.2. Validation Studies

The sustainable RP-HPTLC assay for the quantitative estimation of HSP was validatedfor “linearity range, system suitability parameters, accuracy, precision, robustness, sensi-tivity, and peak purity/specificity” according to the ICH guidelines [38]. The results forthe linear regression analysis of the calibration plot of HSP for the sustainable RP-HPTLC

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assay are listed in Table 1. The HSP calibration plot was linear in 20–2000 ng band−1 rangefor the sustainable RP-HPTLC assay. The value of “determination coefficient (R2)” for HSPwas determined as 0.9997 for the sustainable RP-HPTLC assay. These results indicatedgood linearity between the HSP concentration and its HPTLC area.

Table 1. Results for the regression analysis for the quantitative analysis of hesperidin (HSP) using asustainable “reversed-phase high-performance thin-layer chromatography (RP-HPTLC)” method(mean ± SD; n = 6).

Parameters Values

Linearity range (ng band−1) 20–2000Regression equation y = 34.463x − 112.940

R2 0.9995Slope ± SD 34.463 ± 1.870

Intercept ± SD 112.940 ± 4.420Standard error of slope 0.76

Standard error of intercept 1.8095% confidence interval of slope 31.17–37.74

95% confidence interval of intercept 105.17–120.70LOD ± SD (ng band−1) 7.02 ± 0.28LOQ ± SD (ng band−1) 21.06 ± 0.84

The parameters for the system suitability of the sustainable RP-HPTLC assay werestudied and results are listed in Table 2. The “Rf, As, and N m−1” for the sustainableRP-HPTLC assay were determined as 0.73 ± 0.03, 1.05 ± 0.04, and 5154 ± 3.11, respectively.These data suggested that the sustainable RP-HPTLC assay was suitable for the quantitativeestimation of HSP in TE and UBE of four different varieties of citrus fruit peels and twodifferent commercial formulations.

Table 2. System suitability parameters, including retardation factor (Rf), asymmetry factor (As),and number of theoretical plates per meter (N m−1) of HSP for a sustainable RP-HPTLC method(mean ± SD; n = 3).

Parameters Value

Rf 0.73 ± 0.03As 1.05 ± 0.04

N m−1 5154 ± 3.11

The results for the accuracy determination for the sustainable RP-HPTLC assay areshown in Table 3. The % recovery of HSP for the sustainable RP-HPTLC assay wasdetermined as 101.73%, 101.30%, and 99.21% at LQC, MQC, and HQC, respectively. Thehigh values of % recoveries suggested the accuracy of the sustainable RP-HPTLC assay forthe quantitative estimation of HSP in TE and UBE of four different varieties of citrus fruitpeels and two different commercial formulations.

Table 3. Evaluation of accuracy of HSP for a sustainable RP-HPTLC method (mean ± SD; n = 6).

Conc. (ng band−1) Conc. Found (ng band−1) ± SD Recovery (%) CV (%)

60 61.04 ± 0.51 101.73 0.83800 810.42 ± 4.13 101.30 0.502000 1984.31 ± 6.62 99.21 0.33

The precision for the sustainable RP-HPTLC assay was evaluated in terms of the per-cent of the coefficient of variation (% CV) and results listed in Table 4. The % CVs of HSPfor the sustainable RP-HPTLC assay were determined as 0.73%, 0.47%, and 0.32% at LQC,

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MQC, and HQC, respectively for the intraday precision. The % CVs of HSP for the sustain-able RP-HPTLC assay were estimated as 0.80%, 0.49%, and 0.36% at LQC, MQC and HQC,respectively for the interday precision. The low values of % CV suggested the precision ofthe sustainable RP-HPTLC assay for the quantitative estimation of HSP in TE and UBE offour different varieties of citrus fruit peels and two different commercial formulations.

Table 4. Evaluation of intra/inter-day precision of HSP for a sustainable RP-HPTLC assay (mean ± SD; n = 6).

Conc.(ng band−1)

Intraday Precision Interday Precision

Conc. (ng band−1) ± SD Standard Error CV (%) Conc. (ng band−1) ± SD Standard Error CV (%)

60 58.84 ± 0.43 0.17 0.73 59.54 ± 0.48 0.19 0.80800 794.81 ± 3.81 1.55 0.47 812.41 ± 4.02 1.64 0.49

2000 2016.15 ± 6.61 2.69 0.32 1983.54 ± 7.17 2.92 0.36

The resulting data of the robustness evaluation for the sustainable RP-HPTLC assayare listed in Table 5. The % CVs for the robustness evaluation were recorded as 0.57–0.62%for the sustainable RP-HPTLC assay. The Rf values of HSP were recorded in 0.72–0.74 rangefor the sustainable RP-HPTLC assay. The narrow variations in the Rf values of HSP and low% CVs suggested the robustness of the sustainable RP-HPTLC assay for the quantitativeanalysis of HSP in TE and UBE of four different varieties of citrus fruit peels and twodifferent commercial formulations.

Table 5. Results of robustness evaluation for HSP for a sustainable RP-HPTLC assay (mean ± SD; n = 6).

Conc.(ng band−1)

Mobile Phase Composition (Ethanol/Water) Results

Original Used Level Conc. (ng band−1) ± SD % CV Rf

52:48 +2.0 784.18 ± 4.51 0.57 0.72800 50:50 50:50 0.0 804.54 ± 4.73 0.58 0.73

48:52 −2.0 821.34 ± 5.10 0.62 0.74

The sensitivity for the sustainable RP-HPTLC assay was determined as “LOD andLOQ” and their data are listed in Table 1. The “LOD and LOQ” for the sustainable RP-HPTLC assay were estimated as 7.02 ± 0.28 and 21.06 ± 0.84 ng band−1, respectivelyfor the quantitative analysis of HSP. These data of “LOD and LOQ” for the sustainableRP-HPTLC assay suggested the sensitivity of the sustainable RP-HPTLC assay for thequantitative analysis of HSP in TE and UBE of four different varieties of citrus fruit peelsand two different commercial formulations.

The peak purity/specificity for the sustainable RP-HPTLC assay was determined bycomparing the overlaid spectrodensitograms of HSP in four different varieties of citrusfruits and two different commercial formulations with those of pure HSP. The overlaidspectrodensitograms of pure HSP and HSP in four different varieties of citrus fruits andtwo different commercial formulations are summarized in Figure 3. The maximum chro-matography response for HSP in pure HSP and four different varieties of citrus fruitsand two different commercial formulations was recorded at 287 nm for the sustainableRP-HPTLC assay. The identical spectrodensitograms, Rf values, and wavelength of HSP inpure HSP, four different varieties of citrus fruits and two different commercial formulationsindicated the peak purity/specificity for the sustainable RP-HPTLC assay.

3.3. Application of Sustainable RP-HPTLC Assay for Quantitative Estimation of HSP in VariousTE and UBE of Different Varieties of Citrus Fruit Peels and Commercial Formulations

The sustainable RP-HPTLC assay could be an alternative of conventional analyticalmethods for the quantitative analysis of HSP in TE and UBE of four different varieties ofcitrus fruit peels and two different commercial formulations. The spectrodensitometrypeaks of HSP from TE and UBE of four different varieties of citrus fruit peels and two

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different commercial formulations were identified by comparing their single TLC spotat Rf = 0.73 ± 0.03 with those of a pure HSP for the sustainable RP-HPTLC assay. Therepresentative spectrodensitogram of HSP in TE of grapefruit peels and lemon peels ispresented in Figure 4. The spectrodensitogram for HSP in grapefruit peels (Figure 4A) andlemon peels (Figure 4B) presented the identical peaks of HSP with those of pure HSP forthe sustainable RP-HPTLC assay.

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RP-HPTLC assay. The identical spectrodensitograms, Rf values, and wavelength of HSP in pure HSP, four different varieties of citrus fruits and two different commercial formulations indicated the peak purity/specificity for the sustainable RP-HPTLC assay.

Figure 3. Overlaid spectrodensitograms of (A) standard HSP, (B) grapefruit peels, (C) lemon peels, (D) mosambi peels, (E) orange peels, (F) commercial formulations A, and (G) commercial formulations B.

3.3. Application of Sustainable RP-HPTLC Assay for Quantitative Estimation of HSP in Various TE and UBE of Different Varieties of Citrus Fruit Peels and Commercial Formulations

The sustainable RP-HPTLC assay could be an alternative of conventional analytical methods for the quantitative analysis of HSP in TE and UBE of four different varieties of citrus fruit peels and two different commercial formulations. The spectrodensitometry peaks of HSP from TE and UBE of four different varieties of citrus fruit peels and two different commercial formulations were identified by comparing their single TLC spot at Rf = 0.73 ± 0.03 with those of a pure HSP for the sustainable RP-HPTLC assay. The representative spectrodensitogram of HSP in TE of grapefruit peels and lemon peels is presented in Figure 4. The spectrodensitogram for HSP in grapefruit peels (Figure 4A) and lemon peels (Figure 4B) presented the identical peaks of HSP with those of pure HSP for the sustainable RP-HPTLC assay.

Figure 3. Overlaid spectrodensitograms of (A) standard HSP, (B) grapefruit peels, (C) lemon peels, (D) mosambi peels, (E)orange peels, (F) commercial formulations A, and (G) commercial formulations B.

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RP-HPTLC assay. The identical spectrodensitograms, Rf values, and wavelength of HSP in pure HSP, four different varieties of citrus fruits and two different commercial formulations indicated the peak purity/specificity for the sustainable RP-HPTLC assay.

Figure 3. Overlaid spectrodensitograms of (A) standard HSP, (B) grapefruit peels, (C) lemon peels, (D) mosambi peels, (E) orange peels, (F) commercial formulations A, and (G) commercial formulations B.

3.3. Application of Sustainable RP-HPTLC Assay for Quantitative Estimation of HSP in Various TE and UBE of Different Varieties of Citrus Fruit Peels and Commercial Formulations

The sustainable RP-HPTLC assay could be an alternative of conventional analytical methods for the quantitative analysis of HSP in TE and UBE of four different varieties of citrus fruit peels and two different commercial formulations. The spectrodensitometry peaks of HSP from TE and UBE of four different varieties of citrus fruit peels and two different commercial formulations were identified by comparing their single TLC spot at Rf = 0.73 ± 0.03 with those of a pure HSP for the sustainable RP-HPTLC assay. The representative spectrodensitogram of HSP in TE of grapefruit peels and lemon peels is presented in Figure 4. The spectrodensitogram for HSP in grapefruit peels (Figure 4A) and lemon peels (Figure 4B) presented the identical peaks of HSP with those of pure HSP for the sustainable RP-HPTLC assay.

Figure 4. Spectrodensitograms of HSP in methanolic extract of (A) grapefruit peels and (B) lemon feels obtained usingRP-HPTLC method.

The representative spectrodensitogram of HSP in TE of mosambi peels and orangepeels is presented in Figure 5. The spectrodensitogram for HSP in mosambi peels (Figure 5A)and orange peels (Figure 5B) also presented the identical peaks of HSP with those of pureHSP for the sustainable RP-HPTLC assay.

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Figure 4. Spectrodensitograms of HSP in methanolic extract of (A) grapefruit peels and (B) lemon feels obtained using RP-HPTLC method.

The representative spectrodensitogram of HSP in TE of mosambi peels and orange peels is presented in Figure 5. The spectrodensitogram for HSP in mosambi peels (Figure 5A) and orange peels (Figure 5B) also presented the identical peaks of HSP with those of pure HSP for the sustainable RP-HPTLC assay.

Figure 5. Spectrodensitograms of HSP in methanolic extract of (A) mosambi peels and (B) orange peels obtained using RP-HPTLC method.

The representative spectrodensitogram of HSP in TE of commercial formulation A and commercial formulation B is presented in Figure 6. The spectrodensitogram for HSP in commercial formulation A (Figure 6A) and commercial formulation B (Figure 6B) also showed the identical peaks of HSP with those of pure HSP for the sustainable RP-HPTLC assay. In addition, some additional peaks were also recorded in spectrodensitograms of TE of four different varieties of citrus fruit peels and two different commercial formulations, suggesting the specificity of the sustainable RP-HPTLC assay for the quantitative estimation of HSP in the presence of excipients/impurities. Due to this observation, it is expected that the sustainable RP-HPTLC assay could be able to separate the potential degradation products of HSP.

The content (% w w−1) of HSP in TE and UBE of four different varieties of citrus fruit peels and two different tablet dosage forms was obtained from the calibration plot of HSP and resulting data are shown in Table 6. The content of HSP in TE of grapefruit peels, mosambi peels, lemon peels, and orange peels was detected as 8.26 ± 0.42, 6.94 ± 0.33, 5.90 ± 0.31, and 6.81 ± 0.28% w w−1, respectively. The content of HSP in TE of commercial formulations A and B was detected as 5.31 ± 0.25 and 5.55 ± 0.27% w w−1, respectively. However, the content of HSP in UBE of grapefruit peels, mosambi peels, lemon peels, and orange peels was detected as 11.41 ± 0.51, 8.86 ± 0.48, 7.98 ± 0.45, and 8.64 ± 0.50% w w−1, respectively. The content of HSP in UBE of commercial formulations A and B was detected as 6.72 ± 0.35 and 6.92 ± 0.38% w w−1, respectively. The content of HSP was recorded maximum in TE and UBE of grapefruit peels compared to the other citrus fruit peels and commercial formulations. Generally, the content of HSP was significantly higher in UBE of all four different varieties of citrus fruit peels and two different commercial formulations compared to their TE (p < 0.05). Based on these data and results, the UBE procedure for the extraction of HSP in four different varieties of citrus fruit peels and two different commercial formulations has been proposed as superior over its TE procedure.

Figure 5. Spectrodensitograms of HSP in methanolic extract of (A) mosambi peels and (B) orange peels obtained usingRP-HPTLC method.

The representative spectrodensitogram of HSP in TE of commercial formulation Aand commercial formulation B is presented in Figure 6. The spectrodensitogram for HSPin commercial formulation A (Figure 6A) and commercial formulation B (Figure 6B) alsoshowed the identical peaks of HSP with those of pure HSP for the sustainable RP-HPTLCassay. In addition, some additional peaks were also recorded in spectrodensitograms of TEof four different varieties of citrus fruit peels and two different commercial formulations,suggesting the specificity of the sustainable RP-HPTLC assay for the quantitative estimationof HSP in the presence of excipients/impurities. Due to this observation, it is expectedthat the sustainable RP-HPTLC assay could be able to separate the potential degradationproducts of HSP.

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Figure 6. Spectrodensitograms of HSP in methanolic extract of (A) commercial formulation A and (B) commercial formulation B obtained using RP-HPTLC method.

Table 6. Application of a sustainable RP-HPTLC method for the quantitative estimation of HSP in TE and UBE of four different varieties of citrus fruit peels and commercial formulations (mean ± SD; n = 3).

Samples Traditional Extraction Ultrasound-Based Extraction Amount of HSP (% w w−1)

Grapefruit peels (Citrus paradise) 8.26 ± 0.42 11.41 ± 0.51 Mosambi peels (Citrus limetta) 6.94 ± 0.33 8.86 ± 0.48

Lemon peels (Citrus lemon) 5.90 ± 0.31 7.98 ± 0.45 Orange peels (Citrus sinensis) 6.81 ± 0.28 8.64 ± 0.50

Formulation A 5.31 ± 0.25 6.72 ± 0.35 Formulation A 5.55 ± 0.27 6.92 ± 0.38

Overall, these results suggested that the sustainable RP-HPTLC assay can be efficiently used for the quantitative estimation of HSP in the wide variety of food and pharmaceutical products having HSP as one of the phytoconstituents.

3.4. Greenness Assessment Various metric approaches are used for the greenness assessment of the analytical

techniques [32–37]. Nevertheless, only AGREE metric approach uses all 12 principles of GAC for this purpose [36]. Accordingly, the greenness profile of the sustainable RP-HPTLC assay was assessed using “AGREE: The Analytical Greenness Calculator (version 0.5, Gdansk University of Technology, Gdansk, Poland, 2020)”. The predicted AGREE score with respect to 12 different principles of GAC for the sustainable RP-HPTLC assay is summarized in Figure 7. The AGREE score for the sustainable RP-HPTLC assay was predicted as 0.83, suggesting the excellent green analytical assay for the quantitative analysis of HSP.

Figure 6. Spectrodensitograms of HSP in methanolic extract of (A) commercial formulation A and (B) commercial formula-tion B obtained using RP-HPTLC method.

The content (% w w−1) of HSP in TE and UBE of four different varieties of citrus fruitpeels and two different tablet dosage forms was obtained from the calibration plot of HSPand resulting data are shown in Table 6. The content of HSP in TE of grapefruit peels,mosambi peels, lemon peels, and orange peels was detected as 8.26 ± 0.42, 6.94 ± 0.33,5.90 ± 0.31, and 6.81 ± 0.28% w w−1, respectively. The content of HSP in TE of commercialformulations A and B was detected as 5.31 ± 0.25 and 5.55 ± 0.27% w w−1, respectively.However, the content of HSP in UBE of grapefruit peels, mosambi peels, lemon peels, andorange peels was detected as 11.41 ± 0.51, 8.86 ± 0.48, 7.98 ± 0.45, and 8.64 ± 0.50% ww−1, respectively. The content of HSP in UBE of commercial formulations A and B wasdetected as 6.72 ± 0.35 and 6.92 ± 0.38% w w−1, respectively. The content of HSP wasrecorded maximum in TE and UBE of grapefruit peels compared to the other citrus fruitpeels and commercial formulations. Generally, the content of HSP was significantly higherin UBE of all four different varieties of citrus fruit peels and two different commercial

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formulations compared to their TE (p < 0.05). Based on these data and results, the UBEprocedure for the extraction of HSP in four different varieties of citrus fruit peels and twodifferent commercial formulations has been proposed as superior over its TE procedure.

Table 6. Application of a sustainable RP-HPTLC method for the quantitative estimation of HSP in TE and UBE of fourdifferent varieties of citrus fruit peels and commercial formulations (mean ± SD; n = 3).

Samples Traditional Extraction Ultrasound-Based Extraction

Amount of HSP (% w w−1)

Grapefruit peels (Citrus paradise) 8.26 ± 0.42 11.41 ± 0.51Mosambi peels (Citrus limetta) 6.94 ± 0.33 8.86 ± 0.48

Lemon peels (Citrus lemon) 5.90 ± 0.31 7.98 ± 0.45Orange peels (Citrus sinensis) 6.81 ± 0.28 8.64 ± 0.50

Formulation A 5.31 ± 0.25 6.72 ± 0.35Formulation A 5.55 ± 0.27 6.92 ± 0.38

Overall, these results suggested that the sustainable RP-HPTLC assay can be efficientlyused for the quantitative estimation of HSP in the wide variety of food and pharmaceuticalproducts having HSP as one of the phytoconstituents.

3.4. Greenness Assessment

Various metric approaches are used for the greenness assessment of the analyticaltechniques [32–37]. Nevertheless, only AGREE metric approach uses all 12 principlesof GAC for this purpose [36]. Accordingly, the greenness profile of the sustainable RP-HPTLC assay was assessed using “AGREE: The Analytical Greenness Calculator (version0.5, Gdansk University of Technology, Gdansk, Poland, 2020)”. The predicted AGREEscore with respect to 12 different principles of GAC for the sustainable RP-HPTLC assayis summarized in Figure 7. The AGREE score for the sustainable RP-HPTLC assay waspredicted as 0.83, suggesting the excellent green analytical assay for the quantitativeanalysis of HSP.

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Figure 7. Analytical GREEnness (AGREE) score for the sustainable RP-HPTLC method.

3.5. Comparison with Reported Analytical Methods The sustainable RP-HPTLC assay for the quantitative estimation of HSP was

compared with reported analytical assays. The results for comparison are listed in Table 7. Various parameters like as “linearity, accuracy, and precision” of the sustainable RP-HPTLC assay were compared with reported analytical assays. The linearity range and accuracy of a literature HPLC assay have been reported as 20–100 μg mL−1 and 89.70–107.00%, respectively, which were much inferior to sustainable RP-HPTLC assay (linearity range = 20–2000 ng band−1 and accuracy = 99.21–101.73%) [16]. However, the precision of reported HPLC assay was comparable to the sustainable RP-HPTLC assay [16].

Table 7. Comparative evaluation of the present RP-HPTLC assay with reported analytical assays for the quantitative analysis of HSP.

Analytical Method Linearity Range Accuracy (% Recovery) Precision (% CV) Ref. HPLC 20–100 (μg mL−1) 89.70–107.00 0.44–1.80 [16] LC-MS 0.25–2.50 (μg mL−1) 89.10–114.90 4.40–8.70 [20]

Spectrophotometry 5.00–50.00 (μg mL−1) 98.66–101.00 0.05–0.21 [12] Fluorimetry 3.86 × 10−6–1.64 × 10−5 (mol) 97.04–98.34 0.48–0.71 [27]

Normal-phase HPTLC 100–800 (ng band−1) 98.55–99.38 0.76–1.20 [28] RP-HPTLC 20–2000 (ng band−1) 99.21–101.73 0.32–0.80 Present work

The linearity range, accuracy, and precision of a literature LC-MS assay have been reported as 0.25–2.50 μg mL−1, 89.10–114.90%, and 4.40–8.70%, respectively, which were also much inferior to sustainable RP-HPTLC assay [20]. The linearity range of a reported spectrophotometry method has been reported as 5.00–50.00 μg mL−1, which was inferior to the sustainable RP-HPTLC assay [12]. However, the accuracy and precision of this method were within the limit of ICH guidelines and hence similar to the sustainable RP-HPTLC method [12]. The linearity range and accuracy of a reported fluorimetry method were also inferior to the sustainable RP-HPTLC assay, but its precision was within the limit of ICH guidelines [27]. The linearity range of a reported normal-phase HPTLC assay has been reported as 100–800 ng band−1, which was also inferior to the sustainable RP-HPTLC assay [28]. However, the accuracy and precision of this method were within the

Figure 7. Analytical GREEnness (AGREE) score for the sustainable RP-HPTLC method.

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3.5. Comparison with Reported Analytical Methods

The sustainable RP-HPTLC assay for the quantitative estimation of HSP was com-pared with reported analytical assays. The results for comparison are listed in Table 7.Various parameters like as “linearity, accuracy, and precision” of the sustainable RP-HPTLCassay were compared with reported analytical assays. The linearity range and accuracyof a literature HPLC assay have been reported as 20–100 µg mL−1 and 89.70–107.00%,respectively, which were much inferior to sustainable RP-HPTLC assay (linearity range =20–2000 ng band−1 and accuracy = 99.21–101.73%) [16]. However, the precision of reportedHPLC assay was comparable to the sustainable RP-HPTLC assay [16].

Table 7. Comparative evaluation of the present RP-HPTLC assay with reported analytical assays for the quantitativeanalysis of HSP.

Analytical Method Linearity Range Accuracy (% Recovery) Precision (% CV) Ref.

HPLC 20–100 (µg mL−1) 89.70–107.00 0.44–1.80 [16]LC-MS 0.25–2.50 (µg mL−1) 89.10–114.90 4.40–8.70 [20]

Spectrophotometry 5.00–50.00 (µg mL−1) 98.66–101.00 0.05–0.21 [12]Fluorimetry 3.86 × 10−6–1.64 × 10−5 (mol) 97.04–98.34 0.48–0.71 [27]

Normal-phase HPTLC 100–800 (ng band−1) 98.55–99.38 0.76–1.20 [28]RP-HPTLC 20–2000 (ng band−1) 99.21–101.73 0.32–0.80 Present work

The linearity range, accuracy, and precision of a literature LC-MS assay have beenreported as 0.25–2.50 µg mL−1, 89.10–114.90%, and 4.40–8.70%, respectively, which werealso much inferior to sustainable RP-HPTLC assay [20]. The linearity range of a reportedspectrophotometry method has been reported as 5.00–50.00 µg mL−1, which was inferior tothe sustainable RP-HPTLC assay [12]. However, the accuracy and precision of this methodwere within the limit of ICH guidelines and hence similar to the sustainable RP-HPTLCmethod [12]. The linearity range and accuracy of a reported fluorimetry method werealso inferior to the sustainable RP-HPTLC assay, but its precision was within the limit ofICH guidelines [27]. The linearity range of a reported normal-phase HPTLC assay hasbeen reported as 100–800 ng band−1, which was also inferior to the sustainable RP-HPTLCassay [28]. However, the accuracy and precision of this method were within the limit ofICH guidelines [28]. Overall, the sustainable RP-HPTLC assay for HSP analysis was foundto be superior over all reported analytical assays.

4. Conclusions

The sustainable RP-HPTLC assay has been established for the quantitative estimationof HSP in TE and UBE of four different varieties of citrus fruit peels and two differenttablet dosage forms. The sustainable RP-HPTLC assay was found to be highly-sensitive,rapid, accurate, precise, robust, and sustainable for the quantification of HSP. The contentsof HSP were found significantly higher in UBE of four different varieties of citrus fruitpeels and two different commercial formulations compared to their TE. Therefore, UBE-based procedure has been considered as the preferred procedure for the extraction of HSPfrom citrus fruit peels and commercial formulations. The sustainable RP-HPTLC assaywas found to be superior over all reported analytical assays for the quantitative analysisof HSP. The recorded AGREE score for the sustainable RP- HPTLC assay indicated theexcellent greenness profile for pharmaceutical analysis of HSP. These observation andresults suggested that the sustainable RP-HPTLC assay can be used for the quantitativeestimation of HSP in the wide range of food, pharmaceutical, and plant-based products.

Author Contributions: Conceptualization, A.I.F. and S.A.; methodology, P.A., M.H.A., A.I.F. andF.S.; software, F.S.; validation, S.A. and M.S.A.-K.; formal analysis, M.H.A.; investigation, P.A. andA.I.F.; resources, S.A.; data curation, M.H.A.; writing—original draft preparation, F.S.; writing—review and editing, S.A., P.A. and A.I.F.; visualization, M.S.A.-K.; supervision, S.A. and A.I.F.; project

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administration, S.A.; funding acquisition, S.A. All authors have read and agreed to the publishedversion of the manuscript.

Funding: This research was funded by the Researchers Supporting Project (number RSP-2021/146)at King Saud University, Riyadh, Saudi Arabia. The APC was funded by RSP.

Data Availability Statement: This study did not report any data.

Acknowledgments: Authors are thankful to Researchers Supporting Project (number RSP-2021/146)at King Saud University, Riyadh, Saudi Arabia.

Conflicts of Interest: The authors declare no conflict of interest.

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