# Analytical Method Validation of Gamijakyakgamchobuja-Tang (KCHO-1, Mecasin) Preparation

Authors: Tingting Wang; Seongjin Lee; Muhack Yang; Eunhye Cha; Jongwon Jang; Sungchul Kim
Journal: Evidence-Based Complementary and Alternative Medicine
 (2019)

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## Abstract

Previous studies have confirmed that KCHO-1 (Mecasin) was developed to alleviate the symptoms of Amyotrophic Lateral Sclerosis (ALS). And its toxicity test has also been carried out. The aim of this study is confirming the validation and stability of concentration analysis method of the Mecasin preparations using HPLC. As a conclusion, we found that the preparations at the concentrations of 50mg/ml and 200mg/ml in sterilized distilled water were homogeneous and it was stable for 4 hours at room temperature and 7 days refrigerated condition (2~8°C). And this method for analyzing the concentration of the Mecasin preparations has been found to be suitable. This study is helpful to promote development of reliable manufacturing medicine and good researches through definitive quality control of Mecasin as complex herbal medicine, aiming to provide help for the treatment of ALS.


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## Body

## 1. Introduction

Gamijakyakgamchobuja-tang (KCHO-1, Mecasin) is a new prescription reported to have anti-inflammatory and antioxidant properties [1]. The constituents of Mecasin are Radix Paeoniae Alba, Radix Glycyrrhizae, Radix Aconiti Lateralis Preparata, Radix Salviae Miltiorrhizae, Rhizoma Gastrodiae, Radix Polygalae, Curcuma Root, Fructus Chaenomelis, and Rhizoma Atractylodis Japonicae (Table 1) [2]. This medicine has been used mainly for alleviating pain, muscle spasms, and cold syndrome due to blood deficiency for centuries in traditional oriental medicine [3]. In recent years of medical research, we have found that it has a role in reducing pain, GABA neuron regeneration and NO reduction in neuropathic pain rats [1], antiseizure, analgesic, antipyretic, anti-inflammatory, and antiulcer effects, suppressing the progress of osteoarthritis, neuroprotective, and antineuroinflammatory effects and safety in both in vitro and in vivo trials [4–12]. More concretely, in the first mechanism of action of Mecasin, KCHO-1 increases cellular resistance to glutamate or H2O2-induced oxidative injury in HT22 cells, presumably through ERK and p38 pathways and Nrf2/ARE-dependent HO-1 expression (Figure 1) [2]. And in the second mechanism of action of Mecasin, KCHO-1 upregulated HO-1 expression by promoting the nuclear translocation of Nrf2 in mouse BV2 microglia, and it suppressed the production of proinflammatory mediators and cytokines through suppression of IκB-α phosphorylation and degradation and NF-κB nuclear translocation in LPS-stimulated microglia (Figure 2) [2, 7].Table 1
The constituents of Mecasin.
Scientific Names Parts 1 Curcuma longa Radix  2 Salvia miltiorrhiza Radix  3 Gastrodia elata Rhizoma  4 Pseudocydonia sinensis Fructus  5 Paeonia lactiflora Radix  6 Polygala tenuifolia Radix  7 Glycyrrhiza uralensis Radix  8 Atractylodes japonica Rhizoma  9 Aconitum carmichaeli Radix PreparataFigure 1
1st mechanism of action of Mecasin.Figure 2
2nd mechanism of action of Mecasin.In addition, several studies have been conducted on the compositions of KCHO-1 and its toxicity. Now it is time to advance the study of KCHO-1, and the ultimate goal is to apply it to the clinic to help the treatment of ALS. And for quality control of Mecasin, confirming the validation and stability of concentration analysis method of the Mecasin preparations has become a major issue. We could promote development of reliable manufacturing medicine and good researches through definitive quality control of Mecasin as complex herbal medicine by the validation and stability studies.The experiments for this research were conducted at the Korea Testing & Research Institute (KTR), an institution authorized to perform nonclinical studies under the GLP regulations.


## 2. Materials and Methods

### 2.1. Analytical Method Validation

(1) Test Article and Vehicle Control. The test article was KCHO-1, which was provided by the Nervous & Muscular System Disease Clinical Research Center of Wonkwang University Gwang-ju Korean Medicine Hospital and stored at room temperature (1~30°C). The vehicle control was sterilized distilled water manufactured by Korean Sci. Standard substances were Curcumin, Glycyrrhizic Acid, and Salvianolic Acid B. All were provided by ChromaDex, with assay of 97.7, 93.3, and 96.7%, respectively.(2) Reagents and Equipment. The reagents were acetonitrile, purified water, methanol, ethanol (Burdick and Jackson, USA). Balance (KG-EQM-042) and micropipette (KG-EQM-359) were used as equipment. Shimadzu HPLC (KG-EQM-352(8)) was used as the analytical instrument and the analysis conditions were as follows (Table 2).Table 2
Analytical conditions of KCHO-1 using HPLC.
Mobile phase Time (min) Water (%) Acetonitrile (%) 0 90 10 30 90 10 70 13 87 72 13 87 73 90 10 85 90 10  Flow rate 1.0 mL/min  Injection volume 20μL  Column CAPCELLPAK 5 C18, 250× 2.6 mm, 5 μm  Column oven temperature 40°C  Detector wavelength (PDA) 254 (Glycyrrhizic acid), 280 (Salvianolic acid B), 420 (Curcumin) nm  Run time 85 min(3) Preparation of Solvents and Methods(i)  Mobile Phase A line: water B line: acetonitrileThe mobile phase was used within 7 days.(ii)  Diluent. Ethanol: methanol (50:50, v/v) was used as diluent solvent.(iii)  Standard Solution(a) Curcumin. 20mg of the standard substance was weighed and placed in a 10mL volumetric flask, and diluted solvent was added to the line. This was used as a stock solution. The stock solution was diluted to 1, 5, 10, 50, and 100μg/mL with dilution solvent and used as a standard solution.(b) Glycyrrhizic Acid. 20mg of the standard substance was weighed and placed in a 10mL volumetric flask, and diluted solvent was added to the line. This was used as a stock solution. The stock solution was diluted to 5, 25, 50, 250, and 500μg/mL with dilution solvent and used as a standard solution.(c) Salvianolic Acid B. 20mg of the standard substance was weighed and placed in a 10mL volumetric flask, and diluted solvent was added to the line. This was used as a stock solution. The stock solution was diluted to 5, 25, 50, 250, and 500μg/mL with dilution solvent and used as a standard solution.(4) QC Sample(a) Curcumin. A 50μg/mL concentration of the standard solution was used.(b) Glycyrrhizic Acid. A 250μg/mL concentration of the standard solution was used.(c) Salvianolic Acid B. A 250μg/mL concentration of the standard solution was used.(5) Preparation of Test Article and Treatment of Preparation. 1500 mg and 6000 mg of the test article were weighed and added with a vehicle (sterilized distilled water), shaken, and adjusted to 30mL. Concentrations of preparations for homogeneity and stability tests were 50mg/mL and 200mg/mL. 1mL of the preparation at a concentration of 50mg/mL and 200mg/mL was diluted with a diluting solvent and injected in 20 mL each into HPLC within the calibration curve range.(6) Quantitation. The quantitative value of the preparation was calculated by the following equation after substituting the peak area of the measured value into the calibration curve (y = ax + b).(i)
The quantitative value of the preparation: the measured value × dilution factorThe coefficient of variation, accuracy, and rate of variation were calculated as follows.(i)
The coefficient of variation: (Standard deviation of quantitative values ÷ Average of quantitative values) × 100(ii)
The accuracy: (Average of quantitative values ÷ Theoretical concentration) × 100(iii)
The rate of variation: [(Average of Quantitative value after storage - Quantitative value average immediately after sample preparation) ÷ Quantitative value average immediately after sample preparation]) × 100(4) Preparation Analysis(i)  System Suitability. The coefficient of variation was calculated by repeatedly measuring QC samples 5 times. The criterion was that the coefficient of variation of peak area and retention time was less than 3%.(ii)  Linearity. The concentration of the standard solution was measured once each time, and the correlation coefficient between the concentration of the standard solution and the peak area was calculated. The criterion was that the correlation coefficient r was 0.9950 or more. The results used for linearity validation were used as calibration curves for stability analysis.(iii)  Specificity. Blank samples were measured and the presence or absence of interference peaks was confirmed at the same position as the retention time of the test substance. The criterion is that the peaks of the test substance exhibit sufficient shape for quantification and that there is no interference peak at the same retention time as the test substance.(iv)  Intraday. The preparation was sampled three times in the middle layer and measured once per sample. The criterion was that the variation coefficient of the quantitative value was 15% or less and the accuracy was 75 to 125%.(v)  Stability in Autosampler. In order to confirm the stable time in the autosampler, the samples used in the intraday were left in the autosampler for a certain time and then remeasured. The criterion was that the variation coefficient of the quantitative value was 15% or less and the variation rate with respect to the initial concentration was within ±25%.(vi)  Homogeneity. The preparation was sampled each three times in the upper, middle, and lower layers and measured once per sample. The results of the middle layer were used as a result of intraday. The criterion was that the variation coefficient of the quantitative value was 15% or less and the accuracy was 75 to 125%.(vii)  Stability(a) Room Temperature for 4 Hours. The preparation for each dose was left at room temperature for 4 hours, sampled three times in the middle layer, and measured once per sample; the stability was confirmed. The criterion was that the variation coefficient of the quantitative value was 15% or less and the variation rate with respect to the initial concentration was within ±25%.(b) Refrigeration for 7 Days. The preparation for each dose was left at refrigerated condition (2~8°C) for 7 days, sampled three times in the middle layer, and measured once per sample; the stability was confirmed. The criterion was that the variation coefficient of the quantitative value was 15% or less and the variation rate with respect to the initial concentration was within ±25%.(8) QC (Quality Control). The qc samples were measured three times at the end of the analysis. The criterion was that the coefficient of variation of the analysis result was less than 10% and the accuracy was 80~120%.


## 2.1. Analytical Method Validation

(1) Test Article and Vehicle Control. The test article was KCHO-1, which was provided by the Nervous & Muscular System Disease Clinical Research Center of Wonkwang University Gwang-ju Korean Medicine Hospital and stored at room temperature (1~30°C). The vehicle control was sterilized distilled water manufactured by Korean Sci. Standard substances were Curcumin, Glycyrrhizic Acid, and Salvianolic Acid B. All were provided by ChromaDex, with assay of 97.7, 93.3, and 96.7%, respectively.(2) Reagents and Equipment. The reagents were acetonitrile, purified water, methanol, ethanol (Burdick and Jackson, USA). Balance (KG-EQM-042) and micropipette (KG-EQM-359) were used as equipment. Shimadzu HPLC (KG-EQM-352(8)) was used as the analytical instrument and the analysis conditions were as follows (Table 2).Table 2
Analytical conditions of KCHO-1 using HPLC.
Mobile phase Time (min) Water (%) Acetonitrile (%) 0 90 10 30 90 10 70 13 87 72 13 87 73 90 10 85 90 10  Flow rate 1.0 mL/min  Injection volume 20μL  Column CAPCELLPAK 5 C18, 250× 2.6 mm, 5 μm  Column oven temperature 40°C  Detector wavelength (PDA) 254 (Glycyrrhizic acid), 280 (Salvianolic acid B), 420 (Curcumin) nm  Run time 85 min(3) Preparation of Solvents and Methods(i)  Mobile Phase A line: water B line: acetonitrileThe mobile phase was used within 7 days.(ii)  Diluent. Ethanol: methanol (50:50, v/v) was used as diluent solvent.(iii)  Standard Solution(a) Curcumin. 20mg of the standard substance was weighed and placed in a 10mL volumetric flask, and diluted solvent was added to the line. This was used as a stock solution. The stock solution was diluted to 1, 5, 10, 50, and 100μg/mL with dilution solvent and used as a standard solution.(b) Glycyrrhizic Acid. 20mg of the standard substance was weighed and placed in a 10mL volumetric flask, and diluted solvent was added to the line. This was used as a stock solution. The stock solution was diluted to 5, 25, 50, 250, and 500μg/mL with dilution solvent and used as a standard solution.(c) Salvianolic Acid B. 20mg of the standard substance was weighed and placed in a 10mL volumetric flask, and diluted solvent was added to the line. This was used as a stock solution. The stock solution was diluted to 5, 25, 50, 250, and 500μg/mL with dilution solvent and used as a standard solution.(4) QC Sample(a) Curcumin. A 50μg/mL concentration of the standard solution was used.(b) Glycyrrhizic Acid. A 250μg/mL concentration of the standard solution was used.(c) Salvianolic Acid B. A 250μg/mL concentration of the standard solution was used.(5) Preparation of Test Article and Treatment of Preparation. 1500 mg and 6000 mg of the test article were weighed and added with a vehicle (sterilized distilled water), shaken, and adjusted to 30mL. Concentrations of preparations for homogeneity and stability tests were 50mg/mL and 200mg/mL. 1mL of the preparation at a concentration of 50mg/mL and 200mg/mL was diluted with a diluting solvent and injected in 20 mL each into HPLC within the calibration curve range.(6) Quantitation. The quantitative value of the preparation was calculated by the following equation after substituting the peak area of the measured value into the calibration curve (y = ax + b).(i)
The quantitative value of the preparation: the measured value × dilution factorThe coefficient of variation, accuracy, and rate of variation were calculated as follows.(i)
The coefficient of variation: (Standard deviation of quantitative values ÷ Average of quantitative values) × 100(ii)
The accuracy: (Average of quantitative values ÷ Theoretical concentration) × 100(iii)
The rate of variation: [(Average of Quantitative value after storage - Quantitative value average immediately after sample preparation) ÷ Quantitative value average immediately after sample preparation]) × 100(4) Preparation Analysis(i)  System Suitability. The coefficient of variation was calculated by repeatedly measuring QC samples 5 times. The criterion was that the coefficient of variation of peak area and retention time was less than 3%.(ii)  Linearity. The concentration of the standard solution was measured once each time, and the correlation coefficient between the concentration of the standard solution and the peak area was calculated. The criterion was that the correlation coefficient r was 0.9950 or more. The results used for linearity validation were used as calibration curves for stability analysis.(iii)  Specificity. Blank samples were measured and the presence or absence of interference peaks was confirmed at the same position as the retention time of the test substance. The criterion is that the peaks of the test substance exhibit sufficient shape for quantification and that there is no interference peak at the same retention time as the test substance.(iv)  Intraday. The preparation was sampled three times in the middle layer and measured once per sample. The criterion was that the variation coefficient of the quantitative value was 15% or less and the accuracy was 75 to 125%.(v)  Stability in Autosampler. In order to confirm the stable time in the autosampler, the samples used in the intraday were left in the autosampler for a certain time and then remeasured. The criterion was that the variation coefficient of the quantitative value was 15% or less and the variation rate with respect to the initial concentration was within ±25%.(vi)  Homogeneity. The preparation was sampled each three times in the upper, middle, and lower layers and measured once per sample. The results of the middle layer were used as a result of intraday. The criterion was that the variation coefficient of the quantitative value was 15% or less and the accuracy was 75 to 125%.(vii)  Stability(a) Room Temperature for 4 Hours. The preparation for each dose was left at room temperature for 4 hours, sampled three times in the middle layer, and measured once per sample; the stability was confirmed. The criterion was that the variation coefficient of the quantitative value was 15% or less and the variation rate with respect to the initial concentration was within ±25%.(b) Refrigeration for 7 Days. The preparation for each dose was left at refrigerated condition (2~8°C) for 7 days, sampled three times in the middle layer, and measured once per sample; the stability was confirmed. The criterion was that the variation coefficient of the quantitative value was 15% or less and the variation rate with respect to the initial concentration was within ±25%.(8) QC (Quality Control). The qc samples were measured three times at the end of the analysis. The criterion was that the coefficient of variation of the analysis result was less than 10% and the accuracy was 80~120%.


## 3. Results

### 3.1. Analytical Method Validation

(1) System Suitability. The coefficient of variation of the peak area and retention time measured five times repeatedly at a concentration of 50μg/ mL of the QC sample of the Curcumin was 0.20% and 0.03%.The coefficient of variation of the peak area and retention time measured five times repeatedly at a concentration of 250μg/ mL of the QC sample of the Glycyrrhizic Acid was 0.32% and 0.04%.The coefficient of variation of the peak area and retention time measured five times repeatedly at a concentration of 250μg/ mL of the QC sample of the Salvianolic Acid B was 0.37% and 0.02%. The results are shown in Table 3.Table 3
System suitability.
(a)
Curcumin
Concentration of standard solution (μg/mL) Classification No.1 No.2 No.3 No.4 No.5 Mean SD CV (%) 50 Peak area 4524661 4514684 4516341 4517396 4499824 4514581 9089 0.20 R/Time 58.43 58.42 58.41 58.45 58.41 58.43 0.02 0.03  (b)
Glycyrrhizic Acid
Concentration of standard solution (μg/mL) Classification No.1 No.2 No.3 No.4 No.5 Mean SD CV (%) 50 Peak area 3923859 3913992 3910725 3923579 3893235 3913078 12516 0.32 R/Time 55.37 55.36 55.35 55.39 55.35 55.36 0.02 0.04  (c)
Salvianolic Acid B
Concentration of standard solution (μg/mL) Classification No.1 No.2 No.3 No.4 No.5 Mean SD CV (%) 50 Peak area 2079298 2079339 2082767 2064939 2068935 2075056 7675 0.37 R/Time 46.47 46.46 46.45 46.48 46.45 46.46 0.01 0.02(2) Linearity. The correlation coefficient r of the calibration curve measured at the concentration range of 1 to 100 μg /mL of the standard solution of the Curcumin was 1.0000 on day 0 and 0.9999 on day 7.The correlation coefficientr of the calibration curve measured at the concentration range of 5 to 500 μg /mL of the standard solution of the Glycyrrhizic Acid was 1.0000 on day 0 and 1.0000 on day 7.The correlation coefficientr of the calibration curve measured at the concentration range of 5 to 500 μg /mL of the standard solution of the Salvianolic Acid B was 0.9998 on day 0 and 1.0000 on day 7. The results are shown in Tables 4 and 5.Table 4
Accuracy of calibration curves (Day 0).
(a)
Curcumin
No. Concentration of standard solution (μg/mL) Peak Area Measured concentration (μg/mL) 1 1 85263.00 1.15  2 5 433268.00 4.97  3 10 888555.00 9.98  4 50 4511765.00 49.81  5 100 9086517.00 100.10 y=90965.1551x − 18969.5488, r=1.0000  (b)
Glycyrrhizic Acid
No. Concentration of standard solution (μg/mL) Peak Area Measured concentration (μg/mL) 1 5 78714.00 5.30  2 25 382923.00 24.61  3 50 788267.00 50.33  4 250 3928115.00 249.56  5 500 7878090.00 500.20 y=15759.4853x − 4852.7604, r=1.0000  (c)
Salvianolic Acid B
No. Concentration of standard solution (μg/mL) Peak Area Measured concentration (μg/mL) 1 5 32793.00 8.51  2 25 177058.00 25.17  3 50 384874.00 49.17  4 250 2073814.00 244.24  5 500 4313601.00 502.92 y=8658.3059x − 40850.7837, r=0.9998Table 5
Accuracy of calibration curves (Day 7).
(a)
Curcumin
No. Concentration of standard solution Peak Area Measured concentration (μg/mL) (μg/mL) 1 1 99129.00 0.66  2 5 573255.00 4.97  3 10 1110862.00 9.85  4 50 5642990.00 51.01  5 100 10983038.00 99.51 y=110099.9926x + 26535.0464, r=0.9999  (b)
Glycyrrhizic Acid
No. Concentration of standard solution Peak Area Measured concentration (μg/mL) (μg/mL) 1 5 70795.00 5.45  2 25 361460.00 25.03  3 50 736343.00 50.29  4 250 3679238.00 248.53  5 500 7422854.00 500.70 y=14845.4655x– 10159.4707, r=1.0000  (c)
Salvianolic Acid B
No. Concentration of standard solution Peak Area Measured concentration (μg/mL) (μg/mL) 1 5 36633.00 6.23  2 25 197146.00 25.21  3 50 409263.00 50.30  4 250 2069324.00 246.63  5 500 4225420.00 501.63 y=8455.2959x– 16021.9262, r=1.0000(3) Specificity. The preparation exhibited sufficient shape for analysis and no component that affected the peak of the test substance during the analysis was detected. The results are shown in Figures 3, 4, and 5.Figure 3
Chromatogram of specificity (Curcumin).Figure 4
Chromatogram of specificity (Glycyrrhizic Acid).Figure 5
Chromatogram of specificity (Salvianolic Acid B).(4) Intraday. The coefficient of variation of the test substance was 0.82% and 1.66% and the accuracy was 116.99% and 116.32% at a concentration of Curcumin of 50mg/mL and 200mg/mL of the preparation by each dose.The coefficient of variation of the test substance was 2.07% and 0.64% and the accuracy was 78.64% and 79.02% at a concentration of Glycyrrhizic Acid of 50mg/mL and 200mg/mL of the preparation by each dose.The coefficient of variation of the test substance was 0.86% and 1.30% and the accuracy was 95.61% and 94.70% at a concentration of Salvianolic Acid B of 50mg/mL and 200mg/mL of the preparation by each dose. The results are shown in Table6.Table 6
Accuracy and precision of intraday variation.
(a)
Curcumin
Concentration of dosing formulation (μg/mL) Measured concentration (μg/mL) Mean (μg/mL) SD CV (%) Accuracy (%) No.1 No.2 No.3 39.5 46.52 45.79 46.33 46.21 0.38 0.82 116.99 158.0 186.52 180.49 184.37 183.79 3.06 1.66 116.32  (b)
Glycyrrhizic Acid
Concentration of dosing formulation (μg/mL) Measured concentration (μg/mL) Mean (μg/mL) SD CV (%) Accuracy (%) No.1 No.2 No.3 305.0 234.75 240.17 244.67 239.86 4.97 2.07 78.64 1220.0 961.40 959.78 971.12 964.10 6.13 0.64 79.02  (c)
Salvianolic Acid B
Concentration of dosing formulation (μg/mL) Measured concentration (μg/mL) Mean (μg/mL) SD CV (%) Accuracy (%) No.1 No.2 No.3 985.0 932.67 944.18 948.37 941.74 8.13 0.86 95.61 3940.0 3762.21 3675.21 3755.96 3731.13 48.53 1.30 94.70(5) Stability in Autosampler. As a result of confirming the stability of the Curcumin concentration in the autosampler at 50mg/mL and 200mg/mL for each dose, the variation rates with respect to the initial concentration were 1.15% and 0.85%, and the coefficient of variation was 0.59% and 0.88%.As a result of confirming the stability of the Glycyrrhizic Acid concentration in the autosampler at 50mg/mL and 200mg/mL for each dose, the variation rates with respect to the initial concentration were 0.99% and 1.85%, and the coefficient of variation was 0.42% and 0.95%.As a result of confirming the stability of the Salvianolic Acid B concentration in the autosampler at 50mg/mL and 200mg/mL for each dose, the variation rates with respect to the initial concentration were 1.31% and 0.22%, and the coefficient of variation was 1.08% and 0.45%. The results are shown in Table7.Table 7
Stability of treated sample in the autosampler.
(a)
Curcumin
Title Concentration of the dosing formulation (μg/mL) Measured concentration Mean (μg/mL) SD CV (%) Accuracy (%) Variation (%) (μg/mL) No.1 No.2 No.3 Start 39.5 46.52 45.79 46.33 46.21 0.38 0.82 116.99 - 158.0 186.52 180.49 184.37 183.79 3.06 1.66 116.32 -  End 39.5 47.06 46.57 46.59 46.74 0.28 0.59 118.33 1.15 158.0 186.74 183.55 185.76 185.35 1.63 0.88 117.31 0.85  (b)
Glycyrrhizic Acid
Title Concentration of the dosing formulation (μg/mL) Measured concentration Mean (μg/mL) SD CV (%) Accuracy (%) Variation (%) (μg/mL) No.1 No.2 No.3 Start 305.0 234.75 240.17 244.67 239.86 4.97 2.07 78.64 - 1220.0 961.40 959.78 971.12 964.10 6.13 0.64 79.02 -  End 305.0 241.22 242.24 243.27 242.24 1.03 0.42 79.42 0.99 1220.0 943.30 956.29 939.06 946.22 8.98 0.95 77.56 -1.85  (c)
Salvianolic Acid B
Title Concentration of the dosing formulation (μg/mL) Measured concentration Mean (μg/mL) SD CV (%) Accuracy (%) Variation (%) (μg/mL) No.1 No.2 No.3 Start 985.0 932.67 944.18 948.37 941.74 8.13 0.86 95.61 - 3940.0 3762.21 3675.21 3755.96 3731.13 48.53 1.30 94.70 -  End 985.0 945.25 965.36 951.74 954.12 10.26 1.08 96.86 1.31 3940.0 3758.11 3726.34 3733.48 3739.31 16.67 0.45 94.91 0.22(6) Homogeneity. The homogeneity of the upper, middle, and lower layers at the concentrations of Curcumin at 50mg/mL and 200mg/mL of the preparations was confirmed. The coefficient of variation was 2.52% and 1.73%, and the accuracy was 117.65% and 115.91%.The homogeneity of the upper, middle, and lower layers at the concentrations of Glycyrrhizic Acid at 50mg/mL and 200mg/mL of the preparations was confirmed. The coefficient of variation was 3.35% and 2.08%, and the accuracy was 81.53% and 78.97%.The homogeneity of the upper, middle, and lower layers at the concentrations of Salvianolic Acid B at 50mg/mL and 200mg/mL of the preparations was confirmed. The coefficient of variation was 2.39% and 1.40%, and the accuracy was 97.49% and 95.17%. The results are shown in Table8.Table 8
Homogeneity of dosing formulation.
(a)
Curcumin
Concentration of dosing formulation (μg/mL) Measured concentration (μg/mL) Mean (μg/mL) SD CV (%) Accuracy (%) No.1 No.2 No.3 39.5 Upper 46.67 47.97 46.87 46.47 1.17 2.52 117.65 Middle 46.66 46.08 46.08 Lower 43.99 47.82 46.06  158.0 Upper 177.77 182.59 187.16 183.13 3.17 1.73 115.91 Middle 186.58 182.00 184.79 Lower 183.97 179.00 184.33  (b)
Glycyrrhizic Acid
Concentration of dosing formulation (μg/mL) Measured concentration (μg/mL) Mean (μg/mL) SD CV (%) Accuracy (%) No.1 No.2 No.3 305.0 Upper 252.52 260.77 261.73 248.68 8.32 3.35 81.53 Middle 238.85 248.52 245.37 Lower 243.74 247.37 239.27  1220.0 Upper 975.53 947.51 962.39 963.45 20.06 2.08 78.97 Middle 938.64 955.67 954.80 Lower 951.83 980.93 1003.77  (c)
Salvianolic Acid B
Concentration of dosing formulation (μg/mL) Measured concentration (μg/mL) Mean (μg/mL) SD CV (%) Accuracy (%) No.1 No.2 No.3 985.0 Upper 966.20 1001.76 988.87 960.32 22.91 2.39 97.49 Middle 934.32 958.07 945.25 Lower 938.01 964.54 945.84  3940.0 Upper 3739.49 3721.20 3813.87 3749.74 52.53 1.40 95.17 Middle 3759.39 3704.46 3731.31 Lower 3694.78 3728.22 3854.97(7) Stability(i)  Room Temperature for 4 Hours. As a result of confirming the stability of the concentration of Curcumin at 50mg/mL and 200mg/mL of the preparation at room temperature for 4 hours, the variation rates with respect to the initial concentration immediately after preparation was -1.04% and -0.39%, and the coefficient of variation was 1.66% and 2.78%.As a result of confirming the stability of the concentration of Glycyrrhizic Acid at 50mg/mL and 200mg/mL of the preparation at room temperature for 4 hours, the variation rates with respect to the initial concentration immediately after preparation were 1.19% and -0.76%, and the coefficient of variation was 4.36% and 1.49%.As a result of confirming the stability of the concentration of Salvianolic Acid B at 50mg/mL and 200mg/mL of the preparation at room temperature for 4 hours, the variation rates with respect to the initial concentration immediately after preparation were -0.30% and 0.52%, and the coefficient of variation was 2.69% and 1.63%. The results are shown in Table9.Table 9
Stability of the dosing formulations for 4 hours at room temperature.
(a)
Curcumin
Time (hr) Concentration of the dosing formulation (μg/mL) Measured concentration Mean (μg/mL) SD CV (%) Accuracy (%) Variation (%) (μg/mL) No.1 No.2 No.3 0 39.5 46.52 45.79 46.33 46.21 0.38 0.82 116.99 - 158.0 186.52 180.49 184.37 183.79 3.06 1.66 116.32 -  4 39.5 46.37 45.93 44.89 45.73 0.76 1.66 115.77 -1.04 158.0 178.89 181.59 188.74 183.07 5.09 2.78 115.87 -0.39  (b)
Glycyrrhizic Acid
Time (hr) Concentration of the dosing formulation (μg/mL) Measured concentration Mean (μg/mL) SD CV (%) Accuracy (%) Variation (%) (μg/mL) No.1 No.2 No.3 0 305.0 234.75 240.17 244.67 239.86 4.97 2.07 78.64 - 1220.0 961.40 959.78 971.12 964.10 6.13 0.64 79.02 -  4 305.0 254.16 240.70 233.31 242.72 10.57 4.36 79.58 1.19 1220.0 947.84 973.20 949.28 956.77 14.24 1.49 78.42 -0.76  (c)
Salvianolic Acid B
Time (hr) Concentration of the dosing formulation (μg/mL) Measured concentration Mean (μg/mL) SD CV (%) Accuracy (%) Variation (%) (μg/mL) No.1 No.2 No.3 0 985.0 932.67 944.18 948.37 941.74 8.13 0.86 95.61 - 3940.0 3762.21 3675.21 3755.96 3731.13 48.53 1.30 94.70 -  4 985.0 964.42 938.51 913.89 938.94 25.27 2.69 95.32 -0.30 3940.0 3680.07 3779.39 3791.90 3750.45 61.27 1.63 95.19 0.52(ii)  Refrigeration for 7 Days. As a result of confirming the stability of the concentration of Curcumin at 50mg/mL and 200mg/mL of the preparation at refrigerated condition (2~8°C) for 7 days, the variation rates with respect to the initial concentration immediately after preparation were -18.39% and -18.53%, and the coefficient of variation was 2.80% and 3.37%.As a result of confirming the stability of the concentration of Glycyrrhizic Acid at 50mg/mL and 200mg/mL of the preparation at refrigerated condition (2~8°C) for 7 days, the variation rates with respect to the initial concentration immediately after preparation were 13.18% and 17.02%, and the coefficient of variation was 2.48% and 2.99%.As a result of confirming the stability of the concentration of Salvianolic Acid B at 50mg/mL and 200mg/mL of the preparation at refrigerated condition (2~8°C) for 7 days, the variation rates with respect to the initial concentration immediately after preparation were 4.18% and 7.01%, and the coefficient of variation was 2.26% and 2.91%. The results are shown in Table10.Table 10
Stability of the dosing formulations for 7 days under refrigeration.
(a)
Curcumin
Day Concentration of the dosing formulation (μg/mL) Measured concentration Mean (μg/mL) SD CV (%) Accuracy (%) Variation (%) (μg/mL) No.1 No.2 No.3 0 39.5 46.52 45.79 46.33 46.21 0.38 0.82 116.99 - 158.0 186.52 180.49 184.37 183.79 3.06 1.66 116.32 -  7 39.5 37.92 38.65 36.57 37.71 1.06 2.80 95.48 -18.39 158.0 143.93 152.77 152.53 149.74 5.04 3.37 94.77 -18.53  (b)
Glycyrrhizic Acid
Day Concentration of the dosing formulation (μg/mL) Measured concentration Mean (μg/mL) SD CV (%) Accuracy (%) Variation (%) (μg/mL) No.1 No.2 No.3 0 305.0 234.75 240.17 244.67 239.86 4.97 2.07 78.64 - 1220.0 961.40 959.78 971.12 964.10 6.13 0.64 79.02 -  7 305.0 277.24 273.06 264.09 271.46 6.72 2.48 89.00 13.18 1220.0 1099.67 1165.43 1119.48 1128.19 33.73 2.99 92.47 17.02  (c)
Salvianolic Acid B
Day Concentration of the dosing formulation (μg/mL) Measured concentration Mean (μg/mL) SD CV (%) Accuracy (%) Variation (%) (μg/mL) No.1 No.2 No.3 0 985.0 932.67 944.18 948.37 941.74 8.13 0.86 95.61 - 3940.0 3762.21 3675.21 3755.96 3731.13 48.53 1.30 94.70 -  7 985.0 996.37 991.24 955.72 981.11 22.14 2.26 99.61 4.18 3940.0 3879.51 4111.82 3986.46 3992.60 116.28 2.91 101.34 7.01(8) QC (Quality Control). When the concentration of 50μg/mL of the QC sample of Curcumin was measured three times at the end of the analysis, the coefficient of variation was 0.41% and the accuracy was 101.78%.When the concentration of 250μg/mL of the QC sample of Glycyrrhizic Acid was measured three times at the end of the analysis, the coefficient of variation was 0.46% and the accuracy was 101.21%.When the concentration of 250μg/mL of the QC sample of Salvianolic Acid B was measured three times at the end of the analysis, the coefficient of variation was 0.39% and the accuracy was 96.92%. The results are shown in Table 11.Table 11
Accuracy and precision of QC sample.
(a)
Curcumin
Concentration of total catechins (μg/mL) Measured concentration (μg/mL) Mean (μg/mL) SD CV (%) Accuracy (%) No.1 No.2 No.3 50 50.66 51.07 50.93 50.89 0.21 0.41 101.78  (b)
Glycyrrhizic Acid
Concentration of total catechins (μg/mL) Measured concentration (μg/mL) Mean (μg/mL) SD CV (%) Accuracy (%) No.1 No.2 No.3 250 251.68 253.84 253.55 253.02 1.17 0.46 101.21  (c)
Salvianolic Acid B
Concentration of total catechins (μg/mL) Measured concentration (μg/mL) Mean (μg/mL) SD CV (%) Accuracy (%) No.1 No.2 No.3 250 242.70 242.95 241.21 242.29 0.94 0.39 96.92


## 3.1. Analytical Method Validation

(1) System Suitability. The coefficient of variation of the peak area and retention time measured five times repeatedly at a concentration of 50μg/ mL of the QC sample of the Curcumin was 0.20% and 0.03%.The coefficient of variation of the peak area and retention time measured five times repeatedly at a concentration of 250μg/ mL of the QC sample of the Glycyrrhizic Acid was 0.32% and 0.04%.The coefficient of variation of the peak area and retention time measured five times repeatedly at a concentration of 250μg/ mL of the QC sample of the Salvianolic Acid B was 0.37% and 0.02%. The results are shown in Table 3.Table 3
System suitability.
(a)
Curcumin
Concentration of standard solution (μg/mL) Classification No.1 No.2 No.3 No.4 No.5 Mean SD CV (%) 50 Peak area 4524661 4514684 4516341 4517396 4499824 4514581 9089 0.20 R/Time 58.43 58.42 58.41 58.45 58.41 58.43 0.02 0.03  (b)
Glycyrrhizic Acid
Concentration of standard solution (μg/mL) Classification No.1 No.2 No.3 No.4 No.5 Mean SD CV (%) 50 Peak area 3923859 3913992 3910725 3923579 3893235 3913078 12516 0.32 R/Time 55.37 55.36 55.35 55.39 55.35 55.36 0.02 0.04  (c)
Salvianolic Acid B
Concentration of standard solution (μg/mL) Classification No.1 No.2 No.3 No.4 No.5 Mean SD CV (%) 50 Peak area 2079298 2079339 2082767 2064939 2068935 2075056 7675 0.37 R/Time 46.47 46.46 46.45 46.48 46.45 46.46 0.01 0.02(2) Linearity. The correlation coefficient r of the calibration curve measured at the concentration range of 1 to 100 μg /mL of the standard solution of the Curcumin was 1.0000 on day 0 and 0.9999 on day 7.The correlation coefficientr of the calibration curve measured at the concentration range of 5 to 500 μg /mL of the standard solution of the Glycyrrhizic Acid was 1.0000 on day 0 and 1.0000 on day 7.The correlation coefficientr of the calibration curve measured at the concentration range of 5 to 500 μg /mL of the standard solution of the Salvianolic Acid B was 0.9998 on day 0 and 1.0000 on day 7. The results are shown in Tables 4 and 5.Table 4
Accuracy of calibration curves (Day 0).
(a)
Curcumin
No. Concentration of standard solution (μg/mL) Peak Area Measured concentration (μg/mL) 1 1 85263.00 1.15  2 5 433268.00 4.97  3 10 888555.00 9.98  4 50 4511765.00 49.81  5 100 9086517.00 100.10 y=90965.1551x − 18969.5488, r=1.0000  (b)
Glycyrrhizic Acid
No. Concentration of standard solution (μg/mL) Peak Area Measured concentration (μg/mL) 1 5 78714.00 5.30  2 25 382923.00 24.61  3 50 788267.00 50.33  4 250 3928115.00 249.56  5 500 7878090.00 500.20 y=15759.4853x − 4852.7604, r=1.0000  (c)
Salvianolic Acid B
No. Concentration of standard solution (μg/mL) Peak Area Measured concentration (μg/mL) 1 5 32793.00 8.51  2 25 177058.00 25.17  3 50 384874.00 49.17  4 250 2073814.00 244.24  5 500 4313601.00 502.92 y=8658.3059x − 40850.7837, r=0.9998Table 5
Accuracy of calibration curves (Day 7).
(a)
Curcumin
No. Concentration of standard solution Peak Area Measured concentration (μg/mL) (μg/mL) 1 1 99129.00 0.66  2 5 573255.00 4.97  3 10 1110862.00 9.85  4 50 5642990.00 51.01  5 100 10983038.00 99.51 y=110099.9926x + 26535.0464, r=0.9999  (b)
Glycyrrhizic Acid
No. Concentration of standard solution Peak Area Measured concentration (μg/mL) (μg/mL) 1 5 70795.00 5.45  2 25 361460.00 25.03  3 50 736343.00 50.29  4 250 3679238.00 248.53  5 500 7422854.00 500.70 y=14845.4655x– 10159.4707, r=1.0000  (c)
Salvianolic Acid B
No. Concentration of standard solution Peak Area Measured concentration (μg/mL) (μg/mL) 1 5 36633.00 6.23  2 25 197146.00 25.21  3 50 409263.00 50.30  4 250 2069324.00 246.63  5 500 4225420.00 501.63 y=8455.2959x– 16021.9262, r=1.0000(3) Specificity. The preparation exhibited sufficient shape for analysis and no component that affected the peak of the test substance during the analysis was detected. The results are shown in Figures 3, 4, and 5.Figure 3
Chromatogram of specificity (Curcumin).Figure 4
Chromatogram of specificity (Glycyrrhizic Acid).Figure 5
Chromatogram of specificity (Salvianolic Acid B).(4) Intraday. The coefficient of variation of the test substance was 0.82% and 1.66% and the accuracy was 116.99% and 116.32% at a concentration of Curcumin of 50mg/mL and 200mg/mL of the preparation by each dose.The coefficient of variation of the test substance was 2.07% and 0.64% and the accuracy was 78.64% and 79.02% at a concentration of Glycyrrhizic Acid of 50mg/mL and 200mg/mL of the preparation by each dose.The coefficient of variation of the test substance was 0.86% and 1.30% and the accuracy was 95.61% and 94.70% at a concentration of Salvianolic Acid B of 50mg/mL and 200mg/mL of the preparation by each dose. The results are shown in Table6.Table 6
Accuracy and precision of intraday variation.
(a)
Curcumin
Concentration of dosing formulation (μg/mL) Measured concentration (μg/mL) Mean (μg/mL) SD CV (%) Accuracy (%) No.1 No.2 No.3 39.5 46.52 45.79 46.33 46.21 0.38 0.82 116.99 158.0 186.52 180.49 184.37 183.79 3.06 1.66 116.32  (b)
Glycyrrhizic Acid
Concentration of dosing formulation (μg/mL) Measured concentration (μg/mL) Mean (μg/mL) SD CV (%) Accuracy (%) No.1 No.2 No.3 305.0 234.75 240.17 244.67 239.86 4.97 2.07 78.64 1220.0 961.40 959.78 971.12 964.10 6.13 0.64 79.02  (c)
Salvianolic Acid B
Concentration of dosing formulation (μg/mL) Measured concentration (μg/mL) Mean (μg/mL) SD CV (%) Accuracy (%) No.1 No.2 No.3 985.0 932.67 944.18 948.37 941.74 8.13 0.86 95.61 3940.0 3762.21 3675.21 3755.96 3731.13 48.53 1.30 94.70(5) Stability in Autosampler. As a result of confirming the stability of the Curcumin concentration in the autosampler at 50mg/mL and 200mg/mL for each dose, the variation rates with respect to the initial concentration were 1.15% and 0.85%, and the coefficient of variation was 0.59% and 0.88%.As a result of confirming the stability of the Glycyrrhizic Acid concentration in the autosampler at 50mg/mL and 200mg/mL for each dose, the variation rates with respect to the initial concentration were 0.99% and 1.85%, and the coefficient of variation was 0.42% and 0.95%.As a result of confirming the stability of the Salvianolic Acid B concentration in the autosampler at 50mg/mL and 200mg/mL for each dose, the variation rates with respect to the initial concentration were 1.31% and 0.22%, and the coefficient of variation was 1.08% and 0.45%. The results are shown in Table7.Table 7
Stability of treated sample in the autosampler.
(a)
Curcumin
Title Concentration of the dosing formulation (μg/mL) Measured concentration Mean (μg/mL) SD CV (%) Accuracy (%) Variation (%) (μg/mL) No.1 No.2 No.3 Start 39.5 46.52 45.79 46.33 46.21 0.38 0.82 116.99 - 158.0 186.52 180.49 184.37 183.79 3.06 1.66 116.32 -  End 39.5 47.06 46.57 46.59 46.74 0.28 0.59 118.33 1.15 158.0 186.74 183.55 185.76 185.35 1.63 0.88 117.31 0.85  (b)
Glycyrrhizic Acid
Title Concentration of the dosing formulation (μg/mL) Measured concentration Mean (μg/mL) SD CV (%) Accuracy (%) Variation (%) (μg/mL) No.1 No.2 No.3 Start 305.0 234.75 240.17 244.67 239.86 4.97 2.07 78.64 - 1220.0 961.40 959.78 971.12 964.10 6.13 0.64 79.02 -  End 305.0 241.22 242.24 243.27 242.24 1.03 0.42 79.42 0.99 1220.0 943.30 956.29 939.06 946.22 8.98 0.95 77.56 -1.85  (c)
Salvianolic Acid B
Title Concentration of the dosing formulation (μg/mL) Measured concentration Mean (μg/mL) SD CV (%) Accuracy (%) Variation (%) (μg/mL) No.1 No.2 No.3 Start 985.0 932.67 944.18 948.37 941.74 8.13 0.86 95.61 - 3940.0 3762.21 3675.21 3755.96 3731.13 48.53 1.30 94.70 -  End 985.0 945.25 965.36 951.74 954.12 10.26 1.08 96.86 1.31 3940.0 3758.11 3726.34 3733.48 3739.31 16.67 0.45 94.91 0.22(6) Homogeneity. The homogeneity of the upper, middle, and lower layers at the concentrations of Curcumin at 50mg/mL and 200mg/mL of the preparations was confirmed. The coefficient of variation was 2.52% and 1.73%, and the accuracy was 117.65% and 115.91%.The homogeneity of the upper, middle, and lower layers at the concentrations of Glycyrrhizic Acid at 50mg/mL and 200mg/mL of the preparations was confirmed. The coefficient of variation was 3.35% and 2.08%, and the accuracy was 81.53% and 78.97%.The homogeneity of the upper, middle, and lower layers at the concentrations of Salvianolic Acid B at 50mg/mL and 200mg/mL of the preparations was confirmed. The coefficient of variation was 2.39% and 1.40%, and the accuracy was 97.49% and 95.17%. The results are shown in Table8.Table 8
Homogeneity of dosing formulation.
(a)
Curcumin
Concentration of dosing formulation (μg/mL) Measured concentration (μg/mL) Mean (μg/mL) SD CV (%) Accuracy (%) No.1 No.2 No.3 39.5 Upper 46.67 47.97 46.87 46.47 1.17 2.52 117.65 Middle 46.66 46.08 46.08 Lower 43.99 47.82 46.06  158.0 Upper 177.77 182.59 187.16 183.13 3.17 1.73 115.91 Middle 186.58 182.00 184.79 Lower 183.97 179.00 184.33  (b)
Glycyrrhizic Acid
Concentration of dosing formulation (μg/mL) Measured concentration (μg/mL) Mean (μg/mL) SD CV (%) Accuracy (%) No.1 No.2 No.3 305.0 Upper 252.52 260.77 261.73 248.68 8.32 3.35 81.53 Middle 238.85 248.52 245.37 Lower 243.74 247.37 239.27  1220.0 Upper 975.53 947.51 962.39 963.45 20.06 2.08 78.97 Middle 938.64 955.67 954.80 Lower 951.83 980.93 1003.77  (c)
Salvianolic Acid B
Concentration of dosing formulation (μg/mL) Measured concentration (μg/mL) Mean (μg/mL) SD CV (%) Accuracy (%) No.1 No.2 No.3 985.0 Upper 966.20 1001.76 988.87 960.32 22.91 2.39 97.49 Middle 934.32 958.07 945.25 Lower 938.01 964.54 945.84  3940.0 Upper 3739.49 3721.20 3813.87 3749.74 52.53 1.40 95.17 Middle 3759.39 3704.46 3731.31 Lower 3694.78 3728.22 3854.97(7) Stability(i)  Room Temperature for 4 Hours. As a result of confirming the stability of the concentration of Curcumin at 50mg/mL and 200mg/mL of the preparation at room temperature for 4 hours, the variation rates with respect to the initial concentration immediately after preparation was -1.04% and -0.39%, and the coefficient of variation was 1.66% and 2.78%.As a result of confirming the stability of the concentration of Glycyrrhizic Acid at 50mg/mL and 200mg/mL of the preparation at room temperature for 4 hours, the variation rates with respect to the initial concentration immediately after preparation were 1.19% and -0.76%, and the coefficient of variation was 4.36% and 1.49%.As a result of confirming the stability of the concentration of Salvianolic Acid B at 50mg/mL and 200mg/mL of the preparation at room temperature for 4 hours, the variation rates with respect to the initial concentration immediately after preparation were -0.30% and 0.52%, and the coefficient of variation was 2.69% and 1.63%. The results are shown in Table9.Table 9
Stability of the dosing formulations for 4 hours at room temperature.
(a)
Curcumin
Time (hr) Concentration of the dosing formulation (μg/mL) Measured concentration Mean (μg/mL) SD CV (%) Accuracy (%) Variation (%) (μg/mL) No.1 No.2 No.3 0 39.5 46.52 45.79 46.33 46.21 0.38 0.82 116.99 - 158.0 186.52 180.49 184.37 183.79 3.06 1.66 116.32 -  4 39.5 46.37 45.93 44.89 45.73 0.76 1.66 115.77 -1.04 158.0 178.89 181.59 188.74 183.07 5.09 2.78 115.87 -0.39  (b)
Glycyrrhizic Acid
Time (hr) Concentration of the dosing formulation (μg/mL) Measured concentration Mean (μg/mL) SD CV (%) Accuracy (%) Variation (%) (μg/mL) No.1 No.2 No.3 0 305.0 234.75 240.17 244.67 239.86 4.97 2.07 78.64 - 1220.0 961.40 959.78 971.12 964.10 6.13 0.64 79.02 -  4 305.0 254.16 240.70 233.31 242.72 10.57 4.36 79.58 1.19 1220.0 947.84 973.20 949.28 956.77 14.24 1.49 78.42 -0.76  (c)
Salvianolic Acid B
Time (hr) Concentration of the dosing formulation (μg/mL) Measured concentration Mean (μg/mL) SD CV (%) Accuracy (%) Variation (%) (μg/mL) No.1 No.2 No.3 0 985.0 932.67 944.18 948.37 941.74 8.13 0.86 95.61 - 3940.0 3762.21 3675.21 3755.96 3731.13 48.53 1.30 94.70 -  4 985.0 964.42 938.51 913.89 938.94 25.27 2.69 95.32 -0.30 3940.0 3680.07 3779.39 3791.90 3750.45 61.27 1.63 95.19 0.52(ii)  Refrigeration for 7 Days. As a result of confirming the stability of the concentration of Curcumin at 50mg/mL and 200mg/mL of the preparation at refrigerated condition (2~8°C) for 7 days, the variation rates with respect to the initial concentration immediately after preparation were -18.39% and -18.53%, and the coefficient of variation was 2.80% and 3.37%.As a result of confirming the stability of the concentration of Glycyrrhizic Acid at 50mg/mL and 200mg/mL of the preparation at refrigerated condition (2~8°C) for 7 days, the variation rates with respect to the initial concentration immediately after preparation were 13.18% and 17.02%, and the coefficient of variation was 2.48% and 2.99%.As a result of confirming the stability of the concentration of Salvianolic Acid B at 50mg/mL and 200mg/mL of the preparation at refrigerated condition (2~8°C) for 7 days, the variation rates with respect to the initial concentration immediately after preparation were 4.18% and 7.01%, and the coefficient of variation was 2.26% and 2.91%. The results are shown in Table10.Table 10
Stability of the dosing formulations for 7 days under refrigeration.
(a)
Curcumin
Day Concentration of the dosing formulation (μg/mL) Measured concentration Mean (μg/mL) SD CV (%) Accuracy (%) Variation (%) (μg/mL) No.1 No.2 No.3 0 39.5 46.52 45.79 46.33 46.21 0.38 0.82 116.99 - 158.0 186.52 180.49 184.37 183.79 3.06 1.66 116.32 -  7 39.5 37.92 38.65 36.57 37.71 1.06 2.80 95.48 -18.39 158.0 143.93 152.77 152.53 149.74 5.04 3.37 94.77 -18.53  (b)
Glycyrrhizic Acid
Day Concentration of the dosing formulation (μg/mL) Measured concentration Mean (μg/mL) SD CV (%) Accuracy (%) Variation (%) (μg/mL) No.1 No.2 No.3 0 305.0 234.75 240.17 244.67 239.86 4.97 2.07 78.64 - 1220.0 961.40 959.78 971.12 964.10 6.13 0.64 79.02 -  7 305.0 277.24 273.06 264.09 271.46 6.72 2.48 89.00 13.18 1220.0 1099.67 1165.43 1119.48 1128.19 33.73 2.99 92.47 17.02  (c)
Salvianolic Acid B
Day Concentration of the dosing formulation (μg/mL) Measured concentration Mean (μg/mL) SD CV (%) Accuracy (%) Variation (%) (μg/mL) No.1 No.2 No.3 0 985.0 932.67 944.18 948.37 941.74 8.13 0.86 95.61 - 3940.0 3762.21 3675.21 3755.96 3731.13 48.53 1.30 94.70 -  7 985.0 996.37 991.24 955.72 981.11 22.14 2.26 99.61 4.18 3940.0 3879.51 4111.82 3986.46 3992.60 116.28 2.91 101.34 7.01(8) QC (Quality Control). When the concentration of 50μg/mL of the QC sample of Curcumin was measured three times at the end of the analysis, the coefficient of variation was 0.41% and the accuracy was 101.78%.When the concentration of 250μg/mL of the QC sample of Glycyrrhizic Acid was measured three times at the end of the analysis, the coefficient of variation was 0.46% and the accuracy was 101.21%.When the concentration of 250μg/mL of the QC sample of Salvianolic Acid B was measured three times at the end of the analysis, the coefficient of variation was 0.39% and the accuracy was 96.92%. The results are shown in Table 11.Table 11
Accuracy and precision of QC sample.
(a)
Curcumin
Concentration of total catechins (μg/mL) Measured concentration (μg/mL) Mean (μg/mL) SD CV (%) Accuracy (%) No.1 No.2 No.3 50 50.66 51.07 50.93 50.89 0.21 0.41 101.78  (b)
Glycyrrhizic Acid
Concentration of total catechins (μg/mL) Measured concentration (μg/mL) Mean (μg/mL) SD CV (%) Accuracy (%) No.1 No.2 No.3 250 251.68 253.84 253.55 253.02 1.17 0.46 101.21  (c)
Salvianolic Acid B
Concentration of total catechins (μg/mL) Measured concentration (μg/mL) Mean (μg/mL) SD CV (%) Accuracy (%) No.1 No.2 No.3 250 242.70 242.95 241.21 242.29 0.94 0.39 96.92


## 4. Discussion

Validation was performed to quantitate the concentration of the preparation to be used in the efficiency and toxicity test. As a result of the validation analysis, the peak area and the coefficient of variation of the retention time, which were measured QC samples 5 times repeatedly, satisfied all of the criteria. The linearity measured in the concentration range of the standard solution also satisfied criteria of both the correlation coefficient and the accuracy. The peak of the preparation showed a sufficient shape for analysis, and no ingredient that affected the peak of the test article was detected in the blank sample. As a result of intraday, the coefficient of variation and accuracy of the test articles in the preparations at concentrations of 50mg/mL and 200mg/mL satisfied all the criteria. 50mg/mL and 200mg/mL of the preparation were allowed to left in autosampler for a certain time and then their stability was confirmed. As a result, the variation rate and the coefficient of variation for the initial concentration of the test article for 5 hours satisfied all the criteria. The homogeneity of the upper, middle, and lower layers in the preparations at concentrations of 50mg/mL and 200mg/mL was checked. The coefficient of variation and accuracy were all satisfied the criteria. To confirm the stability, the preparation at 50mg/mL and 200mg/mL was maintained at room temperature for 4 hours and at refrigerated condition (2~8°C) for 7 days. The variation rate of the initial concentration immediately after preparation and the coefficient of variation were all satisfied with the criterion. In addition, the coefficient of variation and accuracy at the QC sample concentration satisfied all the criteria.


## 5. Conclusion

This method for analyzing the concentration of KCHO-1 preparations has been found to be suitable. The preparations at the concentrations of 50mg/ml and 200mg/ml in sterilized distilled water were homogeneous and it was stable for 4 hours at room temperature and 7-day refrigerated condition (2~8°C).



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*Source: 7824146-2019-05-22.xml*