Development and Validation of a HPLC–PDA Method for the Simultaneous Determination of Berberine, Palmatine, Geniposide, and Paeoniﬂorin in Haedoksamul-Tang

applied sciences

Article Development and Validation of a HPLC–PDA Method for the Simultaneous Determination of Berberine, Palmatine, Geniposide, and Paeoniﬂorin in Haedoksamul-tang

Beom-Geun Jo 1, Kyung-Hwa Kang 2,* and Min Hye Yang 1,*

1 College of Pharmacy, Pusan National University, Busan 46241, Korea; [email protected] 2 Department of Physiology, College of Korean Medicine, Dongeui University, Busan 47227, Korea * Correspondence: [email protected] (K.-H.K.); [email protected] (M.H.Y.); Tel.: +82-51-890-3309 (K.-H.K.); +82-51-510-2811 (M.H.Y.); Fax: +82-51-853-4036 (K.-H.K.); +82-51-513-6754 (M.H.Y.) Received: 11 July 2020; Accepted: 6 August 2020; Published: 7 August 2020

Abstract: Haedoksamul-tang (HST) is a traditional medical prescription comprising eight medicinal herbs: Angelica gigas, Cnidium officinale, Coptis japonica, Gardenia jasminoides, Paeonia lactiflora, Phellodendron amurense, Rehmannia glutinosa, and Scutellaria baicalensis. HST is used to treat blood circulation disorders and has anti-inflammatory, hemostatic, and anticonvulsant effects. In this study, a high-performance liquid chromatography/photodiode array detector (HPLC–PDA) method was developed and validated for the simultaneous determination of four marker compounds in HST, namely, berberine, palmatine, geniposide, and paeoniflorin. Four standard solutions and HST sample solutions were analyzed using a reverse-phase SunFire®C column (4.6 250 mm, 18 × 5 µm) using a 0.05% aqueous formic acid/methanol gradient. The column temperature, flow rate, injection volume, and wavelengths used were 28 2 °C, 1.0 mL/min, 10.0 µL, and 230 nm and 240 nm, ± respectively. Calibration curves of the four marker compounds showed good linearity (r2 0.9994), ≥ and limits of detection (LODs) and quantification (LOQs) were in the ranges 0.131–0.296 µg/mL and 0.398–0.898 µg/mL, respectively. Ranges of intra- and inter-day precisions and accuracies values were 96.74–102.53% and 97.95–100.83%, respectively, and relative standard deviation (RSD) values were all <4%. Recoveries averaged 92.33–116.72% with RSD values <5%. Quantitative analysis for the four marker compounds showed geniposide (10.77 mg/g) was most abundant in HST.

Keywords: Haedoksamul-tang; traditional medical prescription; HPLC-PDA; geniposide

1. Introduction Haedoksamul-tang (HST), also known as Onchungeum in Korea, Wen-Qing-Yin in China, and Unsei-in in Japan, is a traditional medical prescription comprising Hwangryunhaedok-tang (HHT) and Samul-tang (SMT). According to the Donguibogam (principles and practice of eastern medicine), HST has been traditionally used to treat abnormal uterine bleeding and abdominal pain in women or to treat flooding accompanied by a yellowish complexion, stomach aches, and alternating chills [1]. According to several studies, HST exhibits diverse therapeutic effects on inflammation [2–7], atopic dermatitis [8–11], blood stasis [12,13], Behcet’s syndrome [14,15], hyperlipidemia [16], and oxidative stress [17–20]. HST consists of eight medicinal herbs: Angelica gigas (Apiaceae), Cnidium oﬃcinale (Umbelliferae), Coptis japonica (Ranunculaceae), Gardenia jasminoides (Rubiaceae), Paeonia lactiﬂora (Paeoniaceae), Phellodendron amurense (Rutaceae), Rehmannia glutinosa (Orobanchaceae), and Scutellaria baicalensis

Appl. Sci. 2020, 10, 5482; doi:10.3390/app10165482 www.mdpi.com/journal/applsci Appl. Sci. 2020, 10, 5482 2 of 9

(Lamiaceae). The major components of the eight medicinal herbs in HST are: flavones (e.g., baicalin, wogonin, and baicalein) from Scutellaria baicalensis [21], alkaloids (e.g., berberine, palmatine, coptisine, jatrorrhizine, and epiberberine) from Coptis japonica [22], alkaloids (e.g., palmatine, phellodendrine, magnoflorine, and berberine) from Phellodendron amurense [23], iridoids (e.g., geniposide, genipin, genipin-1-β gentiobioside, and geniposidic acid) from Gardenia jasminoides [24], iridoids (e.g., catalpol) from Rehmannia glutinosa [25], coumarins (e.g., decursin, decursinol, decursinol angelate, and nodakenin) from Angelica gigas [26], monoterpenoids (e.g., paeoniflorin, albiflorin, benzoylpaeoniflorin, and oxypaeoniflorin) and phenols (e.g., paeonol and (+)-catechin) from Paeonia lactiflora [27], and phthalides (e.g., senkyunolideA, Z-ligustilide, and Z-butylidenephthalide) and ferulic acid from Cnidium officinale [28]. Qualitative and quantitative analyses of HHT and SMT by high-performance liquid chromatography (HPLC) using photodiode array (PDA) detectors have been previously reported [29–32]. Recently, a method for simultaneous quantification of six biomarkers (berberine, baicalin, ferulic acid, geniposide, hydorxymethoxylfurfural, and paeoniflorin) in HST by high-performance liquid chromatography/diode array detector (HPLC-DAD) was developed [33]. However, analysis results depended on geographic sources and cultivation environments, and the analysis data was inadequate for standardizing HST quality. In the present study, we devised and validated a HPLC-PDA method for the simultaneous determination of four biomarkers in HST (berberine from Coptis japonica, palmatine from Phellodendron amurense, geniposide from Gardenia jasminoides, paeoniflorin from Paeonia lactiflora).

2. Materials and Methods

2.1. Plant Materials and Chemicals Scutellaria baicalensis was purchased from Hyunjin pharmaceutical Corp., Coptis japonica from Miryung herb medicine Ltd., Gardenia jasminoides, Phellodendron amurense, and Rehmannia glutinosa from Taechang pharmaceutical Corp., Angelica gigas, Cnidium officinale, and Paeonia lactiflora from Jechen traditional herbal market. All herbal materials were authenticated by Professor Eun Ju Jeong of the Department of Agronomy and Medicinal Plant Resources at Gyeongnam National University of Science and Technology (Jinju, South Korea). Voucher specimens (PNU-0028~PNU-0035) were deposited at the College of Pharmacy, Pusan National University. Geniposide was isolated from G. jasminoides by column chromatography in our laboratory and used as a reference standard. The structure of geniposide was determined by nuclear magnetic resonance (NMR) [34], and their purities by HPLC ( 98.0%). Other reference standards: berberine ≥ ( 98.0%), palmatine ( 98.0%), and paeoniflorin ( 98.0%) were purchased from ChemFaces Biochemical ≥ ≥ ≥ Co., Ltd. (Wuhan, China). Water, acetonitrile, methanol (Honeywell Burdick and Jackson, Muskegon, MI, USA), and formic acid (DAEJUNG Chemicals & Metals Co., Ltd., Siheung-si, Korea) were of HPLC grade.

2.2. Preparation of Haedoksamul-Tang (HST) Extract The eight herbal materials (100 g of each) were mixed and crushed for HST extraction, and then extracted for 3 h in 8 L of distilled water using an ultrasonic extractor. The extract was passed through ﬁlter paper (Advantec, Tokyo, Japan), concentrated under vacuum at 50 ◦C, and freeze-dried. The lyophilized HST extract was sieved to a particle size of <0.425 mm with a standard sieve and 117.6 g of HST (hereafter referred to as HST) was obtained (yield, 14.7%).

2.3. Preparation of Samples and Standard Solutions Sample solutions contained HST (40.0 mg) dissolved in distilled water at a concentration of 20.0 mg/mL, and then sonicated for 10 min at room temperature and filtered through a 0.5 µm polytetrafluoroethylene (PTFE) syringe filter (13JP050AN, Advantec, Tokyo, Japan) prior to injection. Appl. Sci. 2020, 10, 5482 3 of 9 Appl. Sci. 2020, 10, x 3 of 9

StandardStandard stockstock solutionssolutions of thethe fourfour referencereference standardsstandards (Figure(Figure1 ),1), berberine, berberine, palmatine,palmatine, geniposide,geniposide, and and paeoniflorin paeoniﬂorin,, were were prepared prepared at concentrations at concentrations of 500.0 of 500.0μg/mL,µg 500.0/mL, μg/mL, 500.0 µ 1000.0g/mL, 1000.0μg/mL,µ gand/mL, 1000.0 and 1000.0 μg/mLµ,g respectively,/mL, respectively, in methanol. in methanol. All solutions All solutions were were stored stored in a refrigerator in a refrigerator at 4 at°C 4 until◦C until required required for analysis. for analysis.

Figure 1.1. Chemical structures of standard compounds.compounds. 2.4. High-Performance Liquid Chromatography–Photodiode Array (HPLC–PDA) Equipment and Chromatographic2.4. High-Performance Conditions Liquid Chromatography–Photodiode Array (HPLC–PDA) Equipment and Chromatographic Conditions Simultaneous analysis of the berberine, palmatine, geniposide, and paeoniflorin in HST was Simultaneous analysis of the berberine, palmatine, geniposide, and paeoniflorin in HST was performed using a Waters Alliance system (Waters Corporation, Milford, MA 01757, USA) consisting performed using a Waters Alliance system (Waters Corporation, Milford, MA 01757, USA) consisting of an e 2695 separation module and a 2998 photodiode array (PDA) detector. All chromatographic of an e 2695 separation module and a 2998 photodiode array (PDA) detector. All chromatographic data were recorded and analyzed using Empower three chromatography data software. The four data were recorded and analyzed using Empower three chromatography data software. The four components were separated using a reverse-phase SunFire® C18 column (100 Å, 4.6 250 mm ID, components were separated using a reverse-phase SunFire® C18 column (100 Å, 4.6 × ×250 mm ID, 5 5 µm particle size, Waters), which was maintained at 28 2 °C. The mobile phase consisted of 0.05% μm particle size, Waters), which was maintained at 28 ± 2 ℃. The mobile phase consisted of 0.05% (v/v) formic acid in water (A) and methanol (B); solvents were degassed prior to analysis. The optimum (v/v) formic acid in water (A) and methanol (B); solvents were degassed prior to analysis. The gradient elution system used was as follows: 0–30 min, 10–50% (B); 30–35 min, 50–90% (B); 35–45 min, optimum gradient elution system used was as follows: 0–30 min, 10–50% (B); 30–35 min, 50–90% (B); 90–90% (B); and 45–46 min, 90–10% (B). The flow rate and injection volume used were 1.0 mL/min and 35–45 min, 90–90% (B); and 45–46 min, 90–10% (B). The flow rate and injection volume used were 1.0 10.0 µL, and detection wavelengths for quantification and analysis were set at 230 nm and 240 nm. mL/min and 10.0 μL, and detection wavelengths for quantification and analysis were set at 230 nm 2.5.and Method240 nm. Validation

2.5. MethodThe HPLC Validation analytical method devised was evaluated by determining its specificity, linearity, limit of detection (LOD), limit of quantitation (LOQ), precision, and recovery performance as detailed by the InternationalThe HPLC analytical Conference method on Harmonisation devised was (ICH) evaluated guidelines by determining [35]. its specificity, linearity, limit of detection (LOD), limit of quantitation (LOQ), precision, and recovery performance as detailed 2.5.1.by the Specificity International Conference on Harmonisation (ICH) guidelines [35]. The specificity was determined by comparing chromatograms and PDA spectral patterns 2.5.1. Specificity (λ = 200–400 nm) obtained from standard mixtures and HST samples. The specificity was determined by comparing chromatograms and PDA spectral patterns (λ = 2.5.2.200–400 Linearity, nm) obtained Limit of from Detection standard (LOD), mixtures and Limitand HST of Quantitation samples. (LOQ) Calibration curves of berberine, palmatine, geniposide, and paeoniflorin stock solutions were plotted2.5.2. Linearity, over the Limit concentration of Detection ranges (LOD), 2.50–25.00 and Limit ofµg Q/mL,uantitation 2.50–25.00 (LOQµg) /mL, 2.50–25.00 µg/mL, and 2.50–25.00Calibrationµ gcurves/mL, respectively. of berberine, Solutions palmatine, were geniposide, analyzed inand quintuplicate, paeoniflorin and stock calibration solutions curves were wereplotted obtained over the by concentration plotting peak ranges areas (Y)2.50versus–25.00compound μg/mL, 2.50 concentrations–25.00 μg/mL, (x,2.50µg–/25.00mL). μg/mL, Correlation and coe2.50ffi–25.00cients μg/mL (r2) obtained, respectively from calibration. Solutions curveswere analyzed were used in toquintuplicate evaluate linearity,, and calibration and all r2 valuescurves exceededwere obtained 0.999. by LOD plotting and LOQ peak were area calculateds (Y) versus using compound the standard concentration deviationss (x of, μg/mL). y-intercepts Correlation (σ) and calibrationcoefficients curve (r2) obtained slopes (S), from as follows:calibration curves were used to evaluate linearity, and all r2 values exceeded 0.999. LOD and LOQ were calculated using theσ standard deviations of y-intercepts (σ) and calibration curve slopes (S), as follows: LOD = 3.3 (1) × S LOD = 3.3 × (1) S σ Appl. Sci. 2020, 10, 5482 4 of 9

σ LOQ = 10 (2) × S

2.5.3. Precision The precision of the developed method was determined by inter-day and intra-day testing at three diﬀerent concentrations. Intra-day precision was measured by performing analyses three times per day, and inter-day precision was measured by repeating analyses three times per day over three consecutive days. Precisions were determined by calculating relative standard deviation (RSD) using:

standard deviation (SD) RSD (%) = 100 mean measured amount ×

2.5.4. Recovery Recovery testing was performed to verify the accuracy of the optimized HPLC–PDA method. Recovery was evaluated by spiking standard solution and HST samples with diﬀerent amounts of berberine, palmatine, geniposide, and paeoniﬂorin. All analyses were performed in triplicate. Recovery was calculated as follows:

(found amount original amount) Recovery (%) = − 100 spiked amount ×

3. Results and Discussion

3.1. Optimization of HPLC–PDA Conditions Optimal HPLC–PDA conditions were obtained by optimizing column type, column temperature, ® and mobile phase variables. For efficient separation of peaks, a SunFire C18 column (5 µm, 100 Å, 4.6 mm 250 mm, Waters) was used at a column temperature of 28 2 °C with a water (0.05% formic × ± acid)/methanol gradient elution system. The maximum ultraviolet (UV) absorption wavelengths of the four components were confirmed using PDA UV spectra (λ = 200–400 nm), and wavelengths of 230, Appl. Sci. 2020, 10, x 230, 240, and 230 nm were selected for berberine, palmatine, geniposide, and paeoniflorin, respectively 5 of 9 (Figure3).

Figure 2. Cont.

Figure 2. High-performance liquid chromatography (HPLC) chromatograms of standard mixtures (A) and a Haedoksamul-tang (HST) sample (B). (1), berberine; (2), palmatine; (3), geniposide; (4), paeoniflorin.

3.2. Method Validation

3.2.1. Specificity Method specificity was evaluated by comparing retention time and UV spectra of standard mixtures and HST samples. Comparisons showed retention times and UV absorbances of HST samples and the four reference standards were well matched (Figure 3). The wavelengths used for analysis were as follows; berberine (λmax = 228.3, 263.8, and 346.2 nm), palmatine (λmax = 226.0, 273.3, and 343.8 nm), geniposide (λmax = 239.0 nm), and paeoniflorin (λmax = 231.9 and 274.5 nm).

Figure 3. Photodiode array (PDA) detector spectra of the 4 reference standards (A–D) and of a HST extract (A′–D′). A, berberine; B, palmatine; C, geniposide; D, paeoniflorin.

3.2.2. Linearity, LOD and LOQ HPLC–PDA linearity results for berberine, palmatine, geniposide and paeoniflorin showed excellent linearity (r2 ≥ 0.9994) in the concentration range 2.50–25.00 μg/mL for berberine and Appl. Sci. 2020, 10, x 5 of 9

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Figure 2. Cont. Figure 2. High-performanceFigureFigure 2. 3. High-performanceHigh-performance liquid chromatography liquid liquid chromatography chromatography (HPLC) (HPLC)(HPLC) chromatograms chromatograms chromatograms of of standard standard mixtures mixtures of standard mixtures ((AA)) and and a a Haedoksamul-tang Haedoksamul-tang (HST) (HST) sample sample (B ().B ).(1), (1), berberine; berberine; (2), (2),palmatine; palmatine; (3), geniposide; (3), geniposide; (4), (A) and a Haedoksamul-tangpaeoniflorin.(4), paeoniﬂorin. (HST) sample (B). (1), berberine; (2), palmatine; (3), geniposide; (4), paeoniflorin. 3.2.3.2. Method Method Validation Validation

3.2.1.3.2.1. Specificity Specificity 3.2. Method Validation MethodMethod specificity specificity was was evaluated evaluated by by comparing comparing retention retention time time and and UV UV spectra spectra of of standard standard mixturesmixtures andand HSTHST samples. samples. Comparisons Comparisons showed showed retention retention times times and UVand absorbances UV absorbances of HST samplesof HST samplesand the fourand referencethe four reference standards standards were well matchedwere we (Figurell matched4). The (Figure wavelengths 3). The wavelengths used for analysis used were for 3.2.1. Specificity λ λ analysisas follows; were berberine as follows; ( max berberine= 228.3, 263.8,(λmax = and 228.3, 346.2 263.8, nm), and palmatine 346.2 nm), ( max palmatine= 226.0,273.3, (λmax = and 226.0, 343.8 273.3, nm), λ λ andgeniposide 343.8 nm), ( max geniposide= 239.0 nm), (λmax and = 239.0 paeoniflorin nm), and ( paeoniflorinmax = 231.9 and(λmax 274.5 = 231.9 nm). and 274.5 nm). Method specificity was evaluated by comparing retention time and UV spectra of standard mixtures and HST samples. Comparisons showed retention times and UV absorbances of HST samples and the four reference standards were well matched (Figure 3). The wavelengths used for analysis were as follows; berberine (λmax = 228.3, 263.8, and 346.2 nm), palmatine (λmax = 226.0, 273.3, and 343.8 nm), geniposide (λmax = 239.0 nm), and paeoniflorin (λmax = 231.9 and 274.5 nm).

FigureFigure 3. 4. PhotodiodePhotodiode array array (PDA) (PDA) detector detector spec spectratra of of the the 4 4 reference reference standards standards ( (AA––DD)) and and of of a a HST HST extractextract ( (AA′0––DD′).0). A (,A) berberine;, berberine; B, palmatine; (B), palmatine; C, geniposide; (C), geniposide; D, paeoniflorin. (D), paeoniflorin. 3.2.2. Linearity, LOD and LOQ 3.2.2. Linearity, LOD and LOQ HPLC–PDA linearity results for berberine, palmatine, geniposide and paeoniflorin showed HPLC–PDA linearity results for berberine, palmatine, geniposide and paeoniflorin showed excellent linearity (r2 0.9994) in the concentration range 2.50–25.00 µg/mL for berberine and palmatine excellent linearity (r2≥ ≥ 0.9994) in the concentration range 2.50–25.00 μg/mL for berberine and and 5.00–50.00 µg/mL of geniposide and paeoniflorin. The LOD and LOQ values of the four compounds ranged from 0.131–0.296 µg/mL and 0.398–0.898 µg/mL, respectively (Table1).

Figure 3. Photodiode array (PDA) detector spectra of the 4 reference standards (A–D) and of a HST extract (A′–D′). A, berberine; B, palmatine; C, geniposide; D, paeoniflorin.

Table 1. Retention times, linear range, calibration curves, limits of detection (LODs), and limits of quantitation (LOQs) of the four compounds (n = 5).

λa t b RSDc Linear Range Regression LOD LOQ Compound R r2 e (nm) (min) (%) (µg/mL) Equationd (µg/mL) (µg/mL) Y = 59,041.58 x + berberine 230 20.20 0.26 2.50–25.00 0.9998 0.131 0.398 7174.76 Y = 44,562.64 x + palmatine 230 21.07 0.25 2.50–25.00 0.9999 0.179 0.543 7204.29 Y = 23,807.44 x + geniposide 240 22.26 0.39 5.00–50.00 0.9998 0.217 0.659 6159.4 Y = 19,555.98 x paeoniﬂorin 230 24.11 0.38 5.00–50.00 − 0.9994 0.296 0.898 2874.87 a Detection wavelength; b Retention times; c Relative standard deviation; d Y: peak area, x: concentration (µg/mL).; e Coeﬃcient of determination.

3.2.3. Precision Precision was determined by intra-day and inter-day analysis. The intra-day and inter-day RSDs of the four compounds ranged from 0.111% to 2.857% and from 0.946% to 3.593%, respectively. Accuracy values ranged from 96.74% to 102.53% and from 97.95% to 100.83%, respectively (Table2). The intra-day and inter-day RSDs of berberine, palmatine, geniposide, and paeoniﬂorin were all <4%. Thus, the developed method was found to be both reliable and reproducible.

Table 2. Precision and accuracy of the HPLC–PDA method for the four compounds.

Intra-Day (n = 3) Inter-Day (n = 3) Conc. Measured Measured Compound RSD Accuracy RSD Accuracy (µg/mL) Conc. Conc. (%) (%) (%) (%) (µg/mL) (µg/mL) 3.91 3.92 2.348 102.53 3.85 2.281 100.45 berberine 5.21 5.02 0.697 98.28 5.04 1.068 98.70 7.81 7.68 0.111 100.36 7.68 0.946 100.38 3.91 3.90 2.127 101.79 3.86 2.840 100.83 palmatine 5.21 4.99 0.826 97.71 5.01 1.774 98.22 7.81 7.69 1.230 100.48 7.69 1.109 100.49 7.81 7.78 1.135 101.63 7.68 1.467 100.34 geniposide 10.42 9.88 0.280 96.74 10.00 1.061 97.95 15.63 15.50 1.526 101.22 15.41 1.118 100.64 7.81 7.81 1.693 101.96 7.64 3.593 99.76 paeoniﬂorin 10.42 10.03 1.731 98.23 10.09 1.274 98.85 15.63 15.30 2.857 99.91 15.38 2.444 100.42

3.2.4. Recovery Recovery testing was performed in triplicate using the method developed by spiking HST samples with reference standards. The average recoveries of berberine, palmatine, geniposide, and paeoniﬂorin ranged from 92.33% to 116.72% and RSD values from 0.731% to 4.705% (Table3). Therefore, the developed assay method was found to be suitable for analyzing levels of the four compounds in HST samples. Appl. Sci. 2020, 10, 5482 7 of 9

Table 3. Recoveries of the four compounds in Haedoksamul-tang (HST) samples (n = 3).

Spiked Amount Found Amount Compound Recovery (%) SD RSD (%) (µg/mL) (µg/mL) 2.60 58.33 92.97 0.032 3.486 Berberine 5.21 59.88 90.52 0.013 1.404 7.81 72.74 108.46 0.040 3.693 2.60 31.06 98.97 0.033 3.365 palmatine 5.21 31.94 92.33 0.043 4.705 7.81 40.25 113.13 0.033 2.881 5.21 319.60 104.35 0.009 0.893 geniposide 10.42 326.28 104.71 0.012 1.099 15.63 367.28 116.72 0.009 0.731 5.21 141.21 95.42 0.040 4.148 paeoniﬂorin 10.42 151.04 98.05 0.015 1.571 15.63 178.61 114.41 0.034 2.955

3.3. Quantitative Analysis of Four Marker Compounds in HST The HPLC–PDA based method showed that HST contained 2.11 mg/g of berberine, 1.54 mg/g of palmatine, 10.77 mg/g of geniposide, and 7.04 mg/g paeoniflorin, and all RSD values were <3% (Table4). In other words, geniposide, the major compound in Gardenia jasminoides, was the most abundant, followed by paeoniflorin, the major compound in Paeonia lactiflora.

Table 4. Amounts of the four compounds in HST (n = 4).

Compound Mean (mg/g) SD RSD (%) berberine 2.11 0.018 0.854 palmatine 1.54 0.033 2.141 geniposide 10.77 0.249 2.313 paeoniﬂorin 7.04 0.117 1.667

4. Conclusions Summarizing, we developed and verified a HPLC–PDA-based analytical method for the simultaneous determination of berberine, palmatine, geniposide, and paeoniflorin in HST. The optimized method utilized a 0.05% formic acid in water/methanol gradient elution system and a reverse-phase C18 column. Validation of the optimized analytical method was performed by determining specificities, linearities, limits of detection (LODs), and limits of quantification (LOQs) and using precision and recovery tests. In addition, we confirmed that geniposide (the marker compound of Gardenia jasminoides) is abundant in HST. We hope that the method developed and data acquired during the course of this study will be found useful for efficient quality control and standardization of HST.

Author Contributions: Conceptualization, K.-H.K. and M.H.Y.; performing experiments and analyzing data, B.-G.J.; Writing—Original draft preparation, B.-G.J. and M.H.Y.; funding acquisition, K.-H.K. and M.H.Y.All authors read and agreed to the published version of the manuscript. Funding: This research was supported by the National Research Foundation of Korea (NRF) funded by the Korean government (Grant no. NRF-2018R1A2A3075248). Conﬂicts of Interest: The authors have no conﬂict of interest to declare.

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