Chemical and Pharmaceutical Bulletin Advance Publication by J-STAGE Advance Publication DOI:10.1248/cpb.c21-00160 March 30, 2021

1 Chem Pharm Bull 2 Regular Article

3

4 quantification in mirifica crude drugs and products by

5 single-reference UPLC/PDA/MS using relative molar sensitivities to kwakhurin

6

7 Sayaka MASADA, Junko HOSOE, Ryoko ARAI, Yosuke DEMIZU,

8 Takashi HAKAMATSUKA, Yukihiro GODA, Nahoko UCHIYAMA*

9

10 National Institute of Health Sciences, 3-25-26, Tonomachi, Kawasaki, Kanagawa

11 210-9501, Japan

12

13 *Corresponding author

14 Institution: National Institute of Health Sciences, Division of Pharmacognosy,

15 Phytochemistry and Narcotics

16 Address: 3-25-26, Tonomachi, Kawasaki, Kanagawa 210-9501, Japan

17 E-mail: [email protected]

18

Ⓒ 2021 The Pharmaceutical Society of Japan 19 Summary

20 Owing to occasional health damages caused by health food products derived from

21 (PM), the Japanese government has designated PM as an “ingredient

22 calling for special attention”. Miroestrol is a specific isolated from PM and

23 possesses very strong estrogenic activity enough to induces side effects in small

24 amount. Therefore, routine analyses for miroestrol quantification is recommended to

25 control the safety and quality of PM products. However, miroestrol content in PM is

26 quite low, and commercial reagent for its detection is rarely available. In this study, we

27 developed a quantitative analysis method for miroestrol in PM without using its

28 analytical standard by using the relative molar sensitivity (RMS) of miroestrol to

29 kwakhurin, another PM-specific isoflavone, as a reference standard. The RMS value

30 was obtained by an offline combination of 1H-quantitative NMR spectroscopy and a

31 LC/PDA and miroestrol content was determined by single-reference LC/PDA using

32 RMS. Furthermore, we investigated miroestrol content in commercially available PM

33 crude drugs and products, and the RMS method was compared with the conventional

34 calibration curve method in terms of performance. The rate of concordance of

35 miroestrol contents determined by two method was 89 – 101%. The results revealed

36 that our developed LC/PDA/MS method with RMS using kwakhurin as a reference

Chemical and Pharmaceutical Bulletin Advance Publication 37 standard was accurate for routine monitoring of miroestrol content in PM crude drugs

38 and products to control their quality.

39

40 Keywords:

41 Pueraria mirifica; quality control; miroestrol; kwakhurin; relative molar sensitivity;

42 quantitative NMR

43

44

Chemical and Pharmaceutical Bulletin Advance Publication 45 Introduction

46 Pueraria candollei Wall. ex Benth var. mirifica (Airy Shaw and Suvat.) Niyomdham

47 (commonly termed P. mirifica, PM) is a popular -rich belonging to

48 the family. Its tuberous roots, named White Kwao Keur, have been used in

49 Thai traditional medicine for rejuvenation and for the treatment of menopausal

50 symptoms. Notably, PM contains the unique such as miroestrol (MIR),

51 deoxymiroestrol (dMIR), and kwakhurin (KWA), as well as popular estrogenic

52 such as , , and [1, 2]. Although its content in PM

53 is low (< 0.005%), MIR was found to have the considerably highest estrogenic activity

54 (i.e. approximately 1,000-fold stronger than that of genistein and daidzein).

55 Subsequently, Ishikawa et al. isolated dMIR as an alternative and more active

56 compound (approximately 10-fold stronger) than MIR and suggested that MIR was an

57 artifact easily converted from dMIR by aerial oxidation [3]. They also reported a

58 quantitative analysis focusing on the more stable MIR and KWA as a marker

59 compound for the standardization of PM [4]. Although the total synthesis of MIR was

60 previously reported by two research groups [5, 6], a mass-producible method has not

61 been realized because of its complicated structure requiring manipulation for

62 stereoselective reaction and separation of structural isomers.

Chemical and Pharmaceutical Bulletin Advance Publication 63 At present, there are many food products derived from PM in the global healthcare

64 market claiming to have rejuvenating and antiaging effects, as well as potential to

65 improve skin appearance, infertility, and menopausal disorders. However, more than

66 200 adverse events possibly caused by such products had been reported from 2012 to

67 2017 in Japan, and the Ministry of Health, Labour and Welfare (MHLW) has released a

68 cautionary notice [7]. Subsequently, MHLW has amended the Enforcement Regulation

69 of the Food Sanitation Act to designate PM as one of four “designated ingredients

70 calling for special attention” and required manufacturers to appropriately produce PM

71 products and to properly manage its quality [8, 9]. To control the quality of PM

72 products, identification of the characteristic marker compounds and quantification of

73 the active compounds are essential. Several studies have reported conventional

74 quantification methods for MIR and its derivatives in PM using HPLC [4, 10]

75 and in PM dietary supplements using LC/MS/MS [11]. However, detection of MIR in

76 PM may be difficult owing to its low content and the multiple interfering compounds

77 in crude drugs or other ingredients in products. Additionally, not only analytical

78 standards but also commercial reagent-grade MIR is hardly available for routine

79 analysis. The lack of commercial reagents results in a bottleneck effect in the

Chemical and Pharmaceutical Bulletin Advance Publication 80 standardization of the quantification method for routine monitoring of MIR content in

81 PM crude drugs and products.

82 Recently, Sugimoto et al., have developed a single-reference HPLC method with

83 relative molar sensitivity (RMS) determined by off-line combination of 1H-quantitaive

84 NMR (1H-qNMR) and LC/PDA for quantitative analysis of analytes in natural

85 products [12-16]. A single reference standard is used as an internal standard in the

86 1H-qNMR and HPLC methods to calculate the RMS values of each targeted analyte in

87 various samples. These values can be determined as analyte-specific factors from the

88 response ratios obtained by HPLC and the molar ratios obtained by 1H-qNMR. Once

89 the RMS value of analytes to the reference standard was determined by the reagent

90 manufactures, the concentrations of the targeted analytes in natural products are able to

91 be calculated using the external reference standard without a reliable standard and

92 calibration curve. In fact, Masumoto et al. developed single-reference analysis of

93 perillaldehyde in perilla herbs based on an indirect standard

1 94 (3-(trimethylsilyl)-a-propanesulfonic acid-d6 sodium salt) for H-qNMR and a external

95 reference standard (diphenyl sulfone) for RMS [15] and the quantitative assay defined

96 in the Japanese Pharmacopoeia is planning to be revised based on this method [17].

97 Using this approach with RMS, we can propose a standard method for evaluating PM

Chemical and Pharmaceutical Bulletin Advance Publication 98 without using a MIR standard (Fig. 1). In this study, we employed KWA as a reference

99 standard instead of an external reference standard for simultaneous confirmation of the

100 characteristic peaks and quantification of MIR in PM crude drugs and products. We

101 developed a single-reference LC/PDA/MS method with RMS for PM identification and

102 for MIR quantification to evaluate the quality of PM crude drugs and products. MIR

103 content in PM crude drugs and commercial products was determined by the RMS

104 method using KWA as an internal reference standard, and the performance of the

105 developed method was validated by comparison with the conventional absolute

106 calibration curve method.

107

108 Results and discussion

109 First, we established a repeatable LC method to identify the MIR peak using the

110 relative retention time to KWA peak and determine the RMS. Isocratic LC conditions

111 are usually developed for the RMS method to minimize the variance between LC

112 instruments, but it was impossible to separate MIR, KWA, and numerous other

113 components in PM rapidly and simultaneously under isocratic conditions because of

114 their various physicality. Therefore, we adopted a stepwise UPLC system for the RMS

115 method. Under the finalized condition within a 25-min analysis, MIR was detected at

Chemical and Pharmaceutical Bulletin Advance Publication 116 6.9 min with absorption maxima at 214 and 285 nm, and KWA was detected at 16.0

117 min with absorption maxima at 218 and 291 nm (Fig. 2a-c). Thus, the relative retention

118 time of MIR peak was determined as 0.43 to KWA peak. For reliable quantitative

119 determination of an analyte using the single-reference method with RMS, it has been

120 reported to be ideal to select the reference that has the same absorption maximum as

121 the analyte for reducing the influence of differences in the absorption spectral

122 resolution on the intensity of response from the detectors [18]. Because the absorption

123 curve of KWA was relatively shallow from 280 to 300 nm, the detection wavelength

124 for the RMS method was set at 285 nm, the absorption maximum of MIR. The peaks

125 of MIR and KWA were confirmed in the negative mass with the SIM mode as

126 deprotonated molecules [M-H]- at m/z 357 and 367 (Fig. 2d, e). The purities of MIR

127 and KWA used in this study was 98.71±0.031% and 86.08±0.122% by 1H-qNMR,

128 respectively.

129 To confirm the linear range of the LC/PDA method, 10 points of calibration

130 standards for MIR were analyzed, and a calibration curve was constructed based on

131 the chromatographic peak areas. Clear linear trends with a correlation coefficient (R2)

132 > 0.999 was achieved over a concentration range of 0.2–100 μg/mL. The limits of

Chemical and Pharmaceutical Bulletin Advance Publication 133 detection and quantification by the absolute calibration method were determined to be

134 0.004 and 0.0103 μg/mL, respectively.

135 To determine the exact RMS value of MIR to KWA, NMR mix std. was prepared at

136 a concentration of 1 mg/mL and subjected to 1H-qNMR analysis (Fig. 3). As the

137 quantitative signal used for 1H-qNMR, the objective signal should be well separated

138 from other signals derived from the analyte, reference compound, and any impurities

139 that may be present. The 1H-qNMR spectrum showed that position 2 signal from MIR

140 (approximately 6.25 ppm) and position 2 signal from KWA (approximately 7.56 ppm)

141 were well separated from other peaks. Therefore, they were applied as the objective

142 peaks for the calculation of molecular ratios (Rn). The molecular ratio of MIR to KWA

143 was determined to be 1.02737 with high accuracy (RSD = 0.121%, Table S1).

144 To determine the applicable concentration range of RMS, LC mix std. solutions

145 were prepared by diluting NMR mix std. at each concentration of MIR and KWA to

146 0.6, 3, and 15 μg/mL, and then analyzed. Although the RMS values of LC mix std. at

147 0.6 μg/mL slightly varied (RSD > 3.5%), the invariance of the RMS values obtained

148 from LC mix std. at the other concentrations was confirmed with enough accuracy

149 (RSD < 1.0%, Table 1). Therefore, the applicable concentration range of LC mix std.

150 containing MIR and KWA was determined to be 3 to 15 μg/mL. For system suitability

Chemical and Pharmaceutical Bulletin Advance Publication 151 assay, LC mix std. solution at 3 μg/mL was injected six times, and the RMS value of

152 MIR to KWA was determined as 0.2553. This RMS value was used for the subsequent

153 quantitative analyses.

154 Before the quantitative analysis of real samples, detection of MIR and KWA peaks

155 from PM crude drugs (PMC) and commercial products (PMP) were confirmed by

156 LC/PDA/MS (Fig. 4). Among the crude drug samples, PMC-001, 004, and 005

157 contained both peaks, and these crude drug samples were considered to be derived

158 from the correct species. In contrast, PMC-002 showed totally different chromatogram

159 from the other samples’ pattern, and neither MIR nor KWA peaks were detected. This

160 result indicated that PMC-002 was derived from wrong species. Because the

161 chromatogram pattern of PMC-002 was similar to that obtained from the Red Kwao

162 Keur derived from , this species could be used for PMC-002 (data not

163 shown). Thus, it was considered that MIR and KWA could be useful as

164 species-specific marker compounds to discriminate PM (White Kwao Keur) from Red

165 Kwao Keur. In the case of PMC-003, the KWA peak was not detected even under the

166 SIM mode, but MIR peaks were identified on the UV and MS chromatograms. This

167 sample was estimated to be derived from PM owing to the presence of MIR, but

168 further investigation is needed to verify its original species.

Chemical and Pharmaceutical Bulletin Advance Publication 169 Among the PM commercial products, PMP-001, 003, 004, and 005 showed both

170 peaks at least under the SIM mode, and it was indicated that the correct species were

171 used for these samples. On the contrary, PMP-002 showed totally different

172 chromatogram from the other samples’ patterns, and only KWA peak was detected. It

173 was considered that MIR and KWA content was quite low in this product, as PMP-002

174 was composed of three plant extracts, including PM extract, in a tablet form. Taken

175 together, it was confirmed that the developed LC/PDA/MS method could apply to

176 identify MIR and KWA in PM crude drugs and commercial products. Once the sample

177 was analyzed by LC/PDA/MS, MIR peak was able to be detected by using relative

178 retention time to KWA on LC/PDA for quantitative analysis. These results also

179 indicated that the developed LC/PDA/MS method was useful to confirm the botanical

180 origin of PM crude drugs and commercial products.

181 Furthermore, KWA content in PM crude drugs (PMC) and commercial products

182 (PMP) were determined by the absolute calibration curve method using the LC/PDA

183 data. The calibration curve for KWA was linear (R2>0.999) in the range of 0.1–100

184 μg/mL. The limit of quantification (LOQ) was 0.013 μg/mL, with S/N ≥10. The KWA

185 content varied with a range of 0.35–5.36 μg/g in the crude drug samples (PMC-001

186 and PMC-004, Table S2). These values were in well accordance with the previously

Chemical and Pharmaceutical Bulletin Advance Publication 187 reported isolation yield (0.00076% [2] and 0.0064–0.0067% [10]). KWA content in the

188 product samples was calculated to be in a range of 0.68–7.25 μg/g (Table S2). The

189 peak of KWA obtained from PMP-003 was at the trace level. Because this is the first

190 report of the quantification of KWA content in PMC and PMP, further investigations

191 are needed to elucidate the effect of geographic and climatic factors on the biosynthesis

192 and accumulation of KWA.

193 To validate the RMS method for MIR quantification, MIR content in PM crude

194 drugs and commercial products was calculated by a single-reference LC/PDA method

195 with RMS using KWA as a reference standard, and the value was then compared with

196 that obtained by the conventional absolute calibration method (Table 2). First, MIR

197 content was determined using the absolute calibration curve. The values were within a

198 range of 15.12–31.83 μg/g in the four crude drugs. MIR content in the product samples

199 was calculated to be in a range of 4.04–13.58 μg/g in PMP-001, 004, and 005.

200 Subsequently, MIR content was determined by the RMS method using reference

201 standards at three different concentrations. When 5 μg/mL KWA solution was used as

202 a standard for the RMS method, MIR content were calculated to be in a range of

203 14.96–32.08 μg/g in crude drugs, and in a range of 3.61–25.19 μg/g in product samples

204 (Table 2). These values correspond to 89–101% of the content values obtained by the

Chemical and Pharmaceutical Bulletin Advance Publication 205 calibration curve method. In fact, the difference in MIR content between these two

206 methods was within up to 2% in the cases of PMC. Because the absolute calibration

207 curve has y-axis intercept, errors on the quantitative values tend to be large at low

208 concentrations. It is a possible reason why the difference between these two methods

209 was larger in PMP than in PMC. The obtained quantification values were in well

210 agreement with the previous results from PM raw materials (0.002% [2] and 0.004%

211 [10]) and with the concentration in PM dietary supplements previously determined by

212 LC/MS/MS (7.6 to 27.7 μg/g [11]). These results revealed that the single-reference

213 method with RMS was suitable for quantitative assays of MIR in PM samples without

214 using a MIR standard reagent.

215

216 Conclusion

217 In this study, we developed a LC/PDA/MS method for the qualification and

218 quantification of PM crude drugs and products using RMS of MIR to KWA. The

219 applicable concentration range of LC mix std containing MIR and KWA for RMS was

220 determined from 3 to 15 μg/mL by off-line combination of 1H-qNMR and LC/PDA,

221 and the RMS was determined as 0.2553. Next, we assayed five PM crude drugs and

222 five PM products by LC/PDA/MS at qualitative and quantitative levels. We confirmed

Chemical and Pharmaceutical Bulletin Advance Publication 223 the botanical origin of samples by detecting MIR and KWA peaks on SIM

224 chromatograms, and found that at least one sample from crude drugs was probably not

225 derived from PM. MIR content in 10 samples was determined by the single-reference

226 method with RMS using KWA as a reference standard and compared with that

227 determined by the conventional absolute calibration method. The differences between

228 the quantitative values obtained by the two methods were less than 2% in the crude

229 drug samples and less than 11% in the product samples. The significant advantages of

230 the developed single-reference method with RMS include cost-effectiveness and

231 simplicity. Under the same analytical conditions as those used for the first RMS

232 determination, the RMS can be considered the same value without re-analyses.

233 Considering its sensitivity and reactivity to MIR, the LC/PDA/MS method was

234 considered sufficiently accurate for routine monitoring of MIR content in PM crude

235 drugs and products to control their quality. Notably, simultaneous identification and

236 quantification was achieved in this study by using a species-specific internal

237 compound as a reference standard without using a standard for analyte or a certified

238 analytical standard. This approach can be applied to other plant materials and products

239 to overcome the unavailability of reagent-grade analytes for routine analysis.

240

Chemical and Pharmaceutical Bulletin Advance Publication 241 Materials and methods

242 Materials and reagents

243 P. mirifica crude drugs were purchased from raw material suppliers in Japan and on

244 a local market in Thailand. Commercial food products containing PM were purchased

245 online from their Japanese manufactures in 2019. Details are shown in Table S3.

246 Authentic MIR (C20H22O6, Mw = 358.39) was kindly provided by Dr. Tsutomu

247 Ishikawa, emeritus professor at Chiba University. KWA (C21H20O6, Mw = 368.39) was

248 synthesized as previously reported [19]. 1,4-Bis(trimethylsilyl)benzene-d4

249 (1,4-BTMSB-d4; C12H18D4Si2; Mw = 226.50; Code No. 024-17031; Lot. TWN2900;

250 purity 99.9%), a certified reference material, was purchased from Fujifilm Wako Pure

251 Chemical Industries, Ltd. and used as a reference standard for both chemical shift and

252 qNMR measurements. Methanol-d4 (Lot. A0373436; deuteration rate 100.0 atom % D)

253 was purchased from Acros Organics. All other solvents of HPLC or LC/MS grade was

254 purchased from Kanto Chemical Co., Inc. An Ultra-Microbalance XPR2UV (Mettler

255 Toledo) with a minimum reading of 0.0001 mg and a Multipette Xstream electric

256 auto-pipette (Eppendorf) were used for accurate measurements of weight and volume.

257

258 Instruments and parameters

Chemical and Pharmaceutical Bulletin Advance Publication 259 1H-qNMR analysis was performed for determination of the molar ratios of

260 MIR/KWA using JNM-ECA600 (600 MHz; JEOL Ltd.). The observed spectrum

261 width was 20 ppm. A digital filter was used. The center of the spectrum was set at 5

262 ppm. The pulse width was set to the time at which a 90-degree pulse was obtained.

263 Acquisition time, 4 s; digital resolution, 0.25 Hz; and delay time, 60 s. An auto FG

264 shim was used for shim adjustment. The determination temperature was set at room

265 temperature (20–30°C). We performed 13C decoupling with MPF8. The dummy scan

266 was performed twice, and the scan was performed 64 times. The determination was

267 performed thrice in accordance with the internal standard method (AQARI: Accurate

268 quantitative NMR with internal reference substance) to ensure that a signal to noise

269 ratio (S/N) of the quantitative signal was 200 or higher. Purity Pro, manufactured by

270 JEOL Ltd., was used for NMR data processing. The trimethylsilyl peak of the

271 reference standard for qNMR (1,4-BTMSB-d4) was set at 0 ppm. Phase correction and

272 baseline correction were performed manually. The integration range for each peak was

273 determined using a manual method.

274 LC/PDA/MS analyses were performed using Acquity UPLC H-class coupled with

275 Xevo TQD (Waters Co.). An ACQUITY UPLC HSS C18 column (2.1 mm i.d. × 100

276 mm, 1.8 μm; Waters Co.) was used for chromatography at a flow rate of 0.3 mL/min

Chemical and Pharmaceutical Bulletin Advance Publication 277 and a column temperature of 55°C. The mobile phase was composed of A (0.1%

278 formic acid in water) and B (0.1% formic acid in acetonitrile) with a stepwise elution:

279 0–8 min, 10% B; 8–10 min, 15%–27% B; 10–18 min, 27% B; 18.5–20 min, 98% B. A

280 triple quadrupole mass spectrometer was operated with an electrospray ionization

281 (ESI) source in the negative mode with a capillary voltage of 2.5 kV, and the cone

282 voltage was set to 40 V. The desolvation and cone gas flow were set to 1000 and 50

283 L/h, respectively, and were obtained using a nitrogen source. The source temperature

284 was set to 150°C, and the desolvation temperature was set to 550°C. Full-scan mass

285 spectra were collected over the range m/z 100–1200, and single-ion monitoring (SIM)

286 mass was collected using the target ions at [M-H]- m/z 357 for MIR and [M-H]- m/z

287 367 for KWA, respectively. All analyses and acquisitions were performed using the

288 MassLynx 4.1 and TargetLynx 4.1 software (Waters Co.).

289

290 Determination of RMS of MIR to KWA

291 Briefly, 1.5 mg of MIR, 1.5 mg of KWA, and 1 mg of 1,4-BTMSB-d4 (reference

292 standard for both chemical shift and qNMR) were accurately weighed in a same vial,

293 and then dissolved in 1 mL of methanol-d4 to obtain a mixed standard solution (NMR

Chemical and Pharmaceutical Bulletin Advance Publication 1 294 mix std). Using H-qNMR, the molar ratio (Rn) of MIR to KWA in NMR mix std was

295 calculated using the following equation:

푛푀퐼푅 푆푀퐼푅 퐻퐾푊퐴 푅푛 = = × 푛퐾푊퐴 푆퐾푊퐴 퐻푀퐼푅

296 where n is the mole number (mol), S is the average of 1H signal areas obtained by three

297 measurements, and H is the number of 1H nuclei in one molecule contributing to S.

298 Next, the NMR mix std was diluted 2500-, 500-, and 100-fold with methanol to

299 obtain mixed standard solutions (LC mix std) at low (each concentration of MIR and

300 KWA was 0.6 μg/mL), medium (each concentration was 3 μg/mL), and high (each

301 concentration was 15 μg/mL) concentrations, respectively, for LC/PDA/MS analysis.

302 LC mix std at medium concentration was injected six times to confirm the

303 reproducibility of the peak areas of MIR and KWA under the measurement conditions

304 shown above. The response ratio (Rr) of MIR to KWA in LC mix std was determined

305 as follows:

퐴푀퐼푅 푅푟 = 퐴퐾푊퐴

306 where A is the average of chromatographic peak areas obtained by six measurements at

307 285 nm.

308 The RMS of MIR to KWA is expressed by the following equation:

푅 푅푀푆 = 푟 푅푛

Chemical and Pharmaceutical Bulletin Advance Publication 309

310 Determination of the purity of KWA and MIR

311 To determine the purities of KWA and MIR, the NMR spectrum of NMR mix std

312 was analyzed and the trimethylsilyl peak of 1,4-BTMSB-d4 was set at 0 ppm. The

313 purity of KWA was calculated using the following equation:

푆퐾푊퐴 퐻퐵푇푀푆퐵 푀퐾푊퐴 푊퐵푇푀푆퐵 푃퐾푊퐴 = × × × × 푃퐵푇푀푆퐵 푆퐵푇푀푆퐵 퐻퐾푊퐴 푀퐵푇푀푆퐵 푊퐾푊퐴

314 where S is the average of 1H signal areas obtained by three measurements, H is the

315 number of 1H nuclei in one molecule contributing to the 1H signal area, M represents

316 the molar mass (g/mol), W is the amount (mg) of compound weighed for NMR mix std,

317 and PBTMSB is the certified purity of 1,4-BTMSB-d4 (%, w/w).

318 The purity of MIR was determined using the same method.

319

320 Preparation of sample and standard solutions

321 In brief, 0.4 g of each powdered sample was accurately weighed in a precipitation

322 tube for centrifugal separation, dissolved in 2 mL of 80% methanol, and then sonicated

323 for 15 min. After centrifugation, the supernatant was transferred to a volumetric flask,

324 and 80% methanol was added to a final volume of exactly 2 mL. The resultant solution

325 was filtered through a 0.45-μm Ultrefree-MC filter (Merk KGaA) and applied to the

Chemical and Pharmaceutical Bulletin Advance Publication 326 LC/PDA/MS system. Triplicate solutions for each sample were prepared, and each

327 solution was injected three times. In addition, 1 mg of KWA was precisely weighed

328 and dissolved in exactly 1 mL of methanol. This solution was then diluted 200-fold

329 with methanol to obtain KWA standard solutions at a final concentration of 5 μg/mL.

330

331 Quantitative determination of MIR by the single-reference method with RMS

332 Sample and standard solutions were subjected to LC/PDA/MS. Using the peak areas

333 of MIR in the sample solutions and those of KWA in the standard solutions at 285 nm,

334 MIR content in each sample solution was determined as follows:

퐴푀퐼푅 푚푀퐼푅 푊푆푇퐷 푃퐾푊퐴 1 푉푠푎푚푝푙푒 퐶표푛푡푀퐼푅 = × × × × × 퐴푆푇퐷 푚퐾푊퐴 푉푆푇퐷 100 푅푀푆 푊푠푎푚푝푙푒

335 where A is the average of chromatographic peak areas obtained by three measurements,

336 m is the mole weight, W is the amount (mg), V is the volume (mL), and PKWA is the

337 determined purity of KWA (%, w/w).

338

339 Quantitative determination of MIR by the absolute calibration method

340 Next, 1 mg of MIR was precisely weighed and dissolved in exactly 1 mL of

341 methanol and was diluted to 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50 and 100 μg/mL as MIR

342 calibration standards. These concentrations did not reflect the purity of MIR.

Chemical and Pharmaceutical Bulletin Advance Publication 343 Calibration standards were subjected to LC/PDA/MS, and the calibration curve was

344 constructed based on the chromatographic peak areas. Subsequently, MIR content in

345 each sample solution analyzed above was determined using the following equation:

(퐴푀퐼푅 − 퐼푛푡) 푃푀퐼푅 푉푠푎푚푝푙푒 퐶표푛푡푀퐼푅 = × × 푆푙표푝푒 100 푊푠푎푚푝푙푒

346 where A is the average of chromatographic peak areas obtained by three measurements,

347 Int and Slope are the intercept and slope of the calibration curve, respectively, W is the

348 amount (mg), V is the volume (mL), and PMIR is the determined purity of MIR (%,

349 w/w).

350 With the same procedure, the calibration curve of KWA was constructed, and KWA

351 content in each sample solution analyzed above was determined.

352

353 Acknowledgements

354 We are grateful to Dr. Tsutomu Ishikawa for kindly providing authentic MIR. We also

355 thank Dr. Naoko Sato-Masumoto for providing useful advice. This study was

356 supported by a grant from the Ministry of Health, Labour, and Welfare of Japan.

357

358 Conflict of interest

359 The authors declare no conflict of interest.

Chemical and Pharmaceutical Bulletin Advance Publication 360 Supplementary Materials

361 The online version of this article contains supplementary materials.

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383 9. Ministry of Health, Labour and Welfare, Japan.Designated ingredients stipulated

384 by MHLW based on the provisions of Article 8, Paragraph 1 of the Food

385 Sanitation Act. (No. 119 of 2020 Ordinance of MHLW, in Japanese). <

386 https://www.mhlw.go.jp/content/000614389.pdf.> cited 5, February, 2021.

387 10. Yusakul G, Putalun W, Udomsin O, Juengwatanatrkul T, Chiachantipyuth C.

388 Fitoterapia 82, 203–207 (2011).

389 11. Lee JH, Kim JY, Cho SH, Jeong JH, Cho S, Park HJ, Baek SY. J Chromatogr. Sci.,

390 55, 214–221 (2017).

391 12. Kitamaki Y, Saito N, Yamazaki T, Otsuka S, Nakamura S, Nishizaki Y, Sugimoto

392 N, Numata M, Ihara T. Anal. Chem., 89, 6963–6968 (2017).

393 13. Takahashi M, Nishizaki Y, Sugimoto N, Sato K, Inoue K. J Chromatogr. A, 1555,

394 45–52 (2018).

Chemical and Pharmaceutical Bulletin Advance Publication 395 14. Nishizaki Y, Sato-Masumoto N, Nakanishi A, Hashizume Y, Tandia M, Yamazaki

396 T, Kuroe M, Numata M, Ihara T, Sugimoto N, Sato K. J Food Hyg. Soc. Jpn., 59,

397 1–10 (2018).

398 15. Masumoto N, Nishizaki Y, Maruyama T, Igarashi Y, Nakajima K, Yamazaki T,

399 Kuroe M, Numata M, Ihara T, Sugimoto N, Sato K. J Nat. Med., 73, 566–576

400 (2019).

401 16. Ohtsuki T, Matsuoka K, Fuji Y, Nishizaki Y, Masumoto N, Sugimoto N, Sato K,

402 Matsufuji H. Plos one, 15, e0243175 (2020).

403 17. Pharmaceuticals and Medical Devices Agency (PMDA). “JP drafts for public

404 comments. (3 June, 2019)”, cited 5

405 February, 2021.

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407 Numata M, Ihara T, Ito Y, Sugimoto N, Sato K. Food Addit. Contam. Part A, 35,

408 838–847 (2018).

409 19. Tsuji G, Yusa M, Masada S, Yokoo H, Hosoe J, Hakamatsuka T, Demizu Y,

410 Uchiyama N. Chem. Pharm. Bull., 68, 797-801 (2020).

Chemical and Pharmaceutical Bulletin Advance Publication 411 412 Table 1 Response ratios and relative molar sensitivities of MIR to KWA under different concentrations of LC mix std. Concentration* Measurement Measurement Measurement Average RSD

(μg/mL) 1 2 3 (%)

0.6 Rr 0.22212 0.22474 0.23738 0.22808

RMS 0.21620 0.21875 0.23106 0.22200 3.6

3 Rr 0.25936 0.26198 0.26052 0.26062

RMS 0.25245 0.25500 0.25358 0.25368 0.51

15 Rr 0.26363 0.26244 0.26311 0.26306

RMS 0.25660 0.25545 0.25610 0.25605 0.23

413 * Each concentration of MIR and KWA in LC mix std 414 415

Chemical and Pharmaceutical Bulletin Advance Publication 416 Table 2 MIR content in Pueraria mirifica crude drugs and products determined by the single-reference method with relative molar 417 sensitivity using KWA standard solution at 5 μg/mL and that determined by the absolute calibration curve method Sample ID Labeled name MIR content (μg/g)a Rate of

RMS Calibration curve concordance

method method [%]

PMC-001 Powdered PM 32.08 31.83 100.78%

PMC-002 Powdered PM ND ND

PMC-003 White Kwao Keur 18.21 18.29 99.55%

PMC-004 White Kwao Keur 23.22 23.18 100.16%

PMC-005 White Kwao Keur 14.96 15.12 98.95%

PMP-001 Powdered PM 13.38 13.58 98.55%

PMP-002 Powdered PM extract ND ND and two other plant extracts PMP-003 Powdered Pueraria spp. traceb traceb

PMP-004 Powdered PM 25.19 25.11 100.34%

PMP-005 Powdered PM 3.61 4.04 89.38%

418 a Each value of the content (μg/g) is the average of three measurements for triplicate sample solutions.

419 b Detected only by SIM mass

420

Chemical and Pharmaceutical Bulletin Advance Publication 1.0e-2

AU 5.0e-3 6.87 135

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 421

Relative molar Reference RMS mix1.0e-2 sensitivity standard standard KWA

1

H-qNMRAU 5.0e-3 6.85 (μg/mL) KWA 141 MIR 2.00 4.00 6.00Sample 8.00 Peak 10.00detection 12.00 14.00 16.00 18.00 20.00 Molecular ratio, by relative retention time S /H ÷ S /H W (mg) Peak (%) MIR MIR KWA KWA identification

1.0e-2 PPM 6.8 6.6 6.4 6.2 6.0 MIR5.8 Chemical shift (ppm) V (mL) KWA LC-PDA

AU 5.0e-3 MIR 6.87 66 KWA Time 2.00 4.00 6.00 8.00 Retention10.00 time12.00 (min) 14.00 16.00 18.00 20.00 Response ratio, (mg/g) AMIR/AKWA

Retention time (min) 422 423

Chemical and Pharmaceutical Bulletin Advance Publication 424 Fig. 1 Schematic representation of LC quantification method with RMS for determination of MIR using KWA as a reference standard. 425 P, purity (%); S, 1H signal area; H, ;1H nuclei number; A, chromatographic peak area; Conc, concentration (μg/mL); W, amount (mg); 426 V, volume (mL); Cont, content (mg/g); m, mole weight 427 (Color figure can be accessed in the online version.) 428

Chemical and Pharmaceutical Bulletin Advance Publication 429

430

431

[M-H]- [M-H]-

432

433

Chemical and Pharmaceutical Bulletin Advance Publication 434 Fig. 2 LC/PDA/MS spectrum and spectra of LC mix std at high concentration. (a) LC 435 chromatogram of MIR and KWA. Absorption spectra of MIR (b) and KWA (c). Mass 436 scan spectra of MIR (d) and KWA (e).

Chemical and Pharmaceutical Bulletin Advance Publication 437

4 6 1,4-BTMSB-d 3 5 7 Water Solvent 4

8 2 9 15 10 16 1 14 5” 4” 11 13 17 20 3” 12 18 8 21 2” 2 1” 19 7 3 6’ Miroestrol (MIR) 6 M-2 5 4 1’ 5’ (6.25ppm) 2’ 4’ 3’ K-2 (7.56 ppm) Kwakhurin (KWA)

PPM 6.30 6.25 6.20 6.15 6.10 6.05 M-21 M-20

PPM K-5’-OMe 7.60 7.55 7.50 7.45 7.40 7.35 K-5’”-Me

M-7 K-4’”-Me M-4 M-18 M-16a M-16b K-3’ K-6 K-2 CH3OH M-12 M-1 K-8 M-19 K-1” M-2 K-2” K-5 M-9 M-13

PPM 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0

Chemical and Pharmaceutical Bulletin Advance Publication 438 439 Fig. 3 1H-qNMR spectrum of NMR mix std. with structures of MIR and KWA. Atom numbering was determined according to 440 references [4, 19]. Quantitative signals are enlarged. 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457

Chemical and Pharmaceutical Bulletin Advance Publication 458

459

Chemical and Pharmaceutical Bulletin Advance Publication 460 461 Fig. 4 Representative chromatogram of Pueraria mirifica crude drug sample, PMC-001. (a) LC chromatograms at 285 nm. (b) SIM 462 chromatogram at m/z 357. (c) SIM chromatogram at m/z 3

Chemical and Pharmaceutical Bulletin Advance Publication