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 Miroestrol quantification in Pueraria 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 Pueraria mirifica (PM), the Japanese government has designated PM as an “ingredient
22 calling for special attention”. Miroestrol is a specific isoflavone 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 phytoestrogen-rich plant belonging to
48 the Fabaceae 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 phytoestrogens such as miroestrol (MIR),
51 deoxymiroestrol (dMIR), and kwakhurin (KWA), as well as popular estrogenic
52 isoflavones such as genistein, daidzein, and puerarin [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 plants 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 Butea superba, 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.
Chemical and Pharmaceutical Bulletin Advance Publication 362 References
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377 378 aria-letter_4.pdf.>, cited 5, February, 2021. Chemical and Pharmaceutical Bulletin Advance Publication 379 8. Ministry of Health, Labour and Welfare, Japan. Amendment of the Food Sanitation 380 Act -Obligation to notify health damage incident caused by intake of their food 381 products containing the designated ingredients or components. < 382 https://www.mhlw.go.jp/content/000530354.pdf.> cited 5, February, 2021. 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)” 405 February, 2021. 406 18. Nishizaki Y, Sato-Masumoto N, Mikawa T, Nakashima K, Yamazaki T, Kuroe M, 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