Chem Pharm Bull Regular Article Miroestrol Quantification in Pueraria

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.

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