Analytical Sciences Advance Publication by J-STAGE Received October 5, 2018; Accepted November 30, 2018; Published online on December 14, 2018 DOI: 10.2116/analsci.18P441

Separation of Synephrine enantiomers in Fruits by a Reversed Phase

HPLC After Chiral Precolumn Derivatization

Sohei Tanaka,*1 Misaki Sekiguchi,*1 Atsushi Yamamoto,*2 Sen-ichi Aizawa,*3

Kanta Sato,*4 Atsushi Taga,*4 Hiroyuki Terashima,*5 Yoshimi Ishihara,*1

Shuji Kodama,∗1†

*1 School of Science, Tokai University, 4-1-1 Kitakaname, Hiratsuka, Kanagawa

259-1292, Japan

*2 Department of Biological Chemistry, College of Bioscience and Biotechnology,

Chubu University, 1200 Matsumoto-cho, Kasugai-shi, Aichi 487-8501, Japan

*3 Graduate School of Science and Engineering, University of Toyama, 3190 Gofuku,

Toyama 930-8555, Japan

*4 School of Pharmacy, Kinki University, 3-4-1 Kowakae, Higashi-Osaka 577-8502,

Japan

*5 GL Sciences Inc., 30F, Tokyo Square Tower, 22-1 Nishishinjuku 6-chome,

Shinjuku-ku, Tokyo 163-1130, Japan

†To whom correspondence should be addressed.

Shuji Kodama, School of Science, Tokai University, 4-1-1 Kitakaname, Hiratsuka,

Kanagawa 259-129, Japan

E-mail: [email protected]

Hiroyuki Terashima: [email protected]

1 Analytical Sciences Advance Publication by J-STAGE Received October 5, 2018; Accepted November 30, 2018; Published online on December 14, 2018 DOI: 10.2116/analsci.18P441

1 ABSTRACT

2 Racemic synephrine, which was transformed into diastereomers by derivatization with

3 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosil isothiocyanate, was resolved by a reversed

4 phase HPLC with UV detection at 254 nm. The total contents of synephrine

5 enantiomers in citrus fruit samples were exocarp > mesocarp > endocarp > sarcocarp,

6 suggesting that synephrine content of outer side of citrus fruits was higher than that of

7 the inner side. (R)-Synephrine was detected in exocarp of eleven fresh citrus fruits

8 except for , , and samples. (S)-Synephrine was determined in

9 exocarp of four citrus fruits (mikan, , , and samples) and

10 the ratio of (S)-synephrine to total synephrine was 0.5−0.9%. The racemization of

11 (R)-synephrine in aqueous solution during heating at 100 °C was also examined. An

12 increase in the heating time brought about an increase in the (S)-synephrine content in

13 a linear fashion. The racemization was found to be significantly reduced by addition of

14 D-fructose, D-maltose, D-glucose, D-mannose or D-galactose, but not D-sucrose or

15 D-mannitol. It is suggested that the reducibility of sugars may result in the inhibition of

16 racemization.

17

18 Keywords: Enantioseparation; synephrine; diastereomer; citrus; HPLC.

19

20

21

2 Analytical Sciences Advance Publication by J-STAGE Received October 5, 2018; Accepted November 30, 2018; Published online on December 14, 2018 DOI: 10.2116/analsci.18P441

22 Introduction

23 Synephrine (Fig. 1), a phenethylamine alkaloid, is known to be present in the peel

24 and the edible part of Citrus fruits.1-5 Since synephrine is structurally similar to

25 adrenergic agonist such as adrenaline, noradrenaline, and ephedrine, effects of

26 synephrine on the cardiovascular system are attributable to adrenergic stimulation. In

27 general, vasoconstriction occurs when ligands bind to α-adrenergic receptors, while

28 binding to β-1 adrenergic receptors result in cardiovascular contractility and increased

29 heart rate. Ligand binding to β-2 adrenergic receptors is associated with

30 bronchodilation.6 However, synephrine binds much more poorly to α-1, α-2, β-1, and

31 β-2 adrenergic receptors than other ligands such as adrenaline.7 Various studies have

32 shown that synephrine binds to β-3 adrenergic receptors, resulting in an increase in the

33 body’s ability to breakdown fats. Binding to β-3 adrenergic receptors dose not

34 influence heart rate or blood pressure, but regulate lipid and carbohydrate

35 metabolism.5 According to Stohs,5 synephrine exhibits greater adrenergic receptor

36 binding in rodents than in humans, while data from animals cannot be directly

37 compared to those of humans. Bitter orange extracts, which contain synephrine, are

38 widely used for weight loss/weight management, energy production, and sports

39 performance. Synephrine is a chiral compound and its enantiomers have been shown to

40 exert different pharmacological activities on α- and β-adrenergic receptors. That is,

41 (R)-synephrine is from 1 to 2 orders of magnitude more active than its

42 (S)-enantiomer.2,8

43 Synephrine has been analyzed by HPLC with ultraviolet detector,9-15 HPLC with

44 mass spectrometry,11,16-18 and Raman spectrometry.19 It was reported that the amount

45 of synephrine in citrus fruits was varied according to the citrus species, parts of fruits,

46 and the maturation period.9-13,16 Arbo et al. revealed a variation on the content of

3 Analytical Sciences Advance Publication by J-STAGE Received October 5, 2018; Accepted November 30, 2018; Published online on December 14, 2018 DOI: 10.2116/analsci.18P441

47 synephrine in Citrus. sinensis according to the maturation period and found that its

48 content was inversely proportional to the size of the fruit.10 Synephrine enantiomers in

49 citrus fruit samples have been also separated by HPLC with chiral columns20-23.

50 Direct and indirect methods have evolved as the main strategies for the

51 enantioseparation.24-26 A direct method, which does not require chemical derivatization,

52 is based on a chiral stationary phase or with a chiral selector in a mobile phase on an

53 achiral stationary phase. An indirect method is based on the formation of

54 diastereomers by derivatization of analyte enantiomers with a chiral reagent. Gal and

55 Brown27 reported an indirect HPLC method for the chiral separation of adrenergic

56 agent including synephrine based in derivatizing with 2,3,4,6-tetra-O-acetyl-β-D-

57 glucopyranosil isothiocyanate (TAG-ITC) under basic condition. But this indirect

58 method was not applied for real samples.

59 It was reported that (S)-synephrine in peels of six citrus fruits including mikan and

60 orange was not detected (less than 1 mg/100g) and that (R)-synephrine was the only

61 enantiomer isolated from citrus fruits.22 The same results were obtained using mikan23

62 and bitter orange,20 but Pellati et al.21 determined (S)-synephrine in fresh fruits pulp of

63 bitter orange, in which the ratio of (S)- and (R)-synephrines was 7.6:92.4. They also

64 reported that the ratio of (S)-synephrine to total synephrine extended over 4.8−14.4 %

65 in Evodia fruits.28 Kusu et al.23 found both (S)- and (R)-synephrines were detected in

66 two orange juices and a marmalade. They suggested that (S)-synephrine may possibly

67 be formed during production steps. Thus, it remains unclear whether (S)-synephrine is

68 contained in citrus fruits, or not. In order to clarify the question, we developed an

69 HPLC method for analysis of synephrine enantiomers in citrus fruits, citrus juices, and

70 citrus products after synephrine was derivatized with TAG-ITC at neutral pH. We also

71 studied the effect of sugars on the racemization of (R)-synephrine.

4 Analytical Sciences Advance Publication by J-STAGE Received October 5, 2018; Accepted November 30, 2018; Published online on December 14, 2018 DOI: 10.2116/analsci.18P441

72 Experimental

73 Reagents and chemicals

74 Racemic synephrine and acetonitrile were obtained from Sigma (St. Louis, MO,

75 USA). Methanol was from Kanto chemicals (Tokyo, Japan). (R)-(−)-Synephrine,

76 racemic octopamine, tyramine, and 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosil

77 isothiocyanate (TAG-ITC) were from Tokyo Kasei (Tokyo, Japan). Sodium

78 dihydrogenphosphate dihydrate and other chemicals (analytical grade) were obtained

79 from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan).

80

81 Apparatus for HPLC and Mobile Phase Conditions.

82 The HPLC system consisted of a Jasco (Hachioji, Japan) model PU-2080 pump, a

83 Jasco Model UV-2075 detector, a Rheodyne (Cotati, CA, USA) manual injector, a

84 Shimadzu (Kyoto, Japan) column oven Model CTO-10Avp, and a Shimadzu degasser

85 Model DGU-14A. InertSustain C18 column (5 µm, 4.6 mm i.d. x 150 mm, GL

86 Sciences, Tokyo, Japan) was used. A mobile phase consisted of 20% acetonitrile, 20%

87 methanol and 30 mM phosphate buffer (pH 7.0). Elution was carried out at a flow rate

88 of 1.0 mL/min at 40 °C. Analytes were detected at 254 nm. Data acquisition and

89 processing were conducted with a Chromato-PRO (Runtime Instrument, Kanagawa,

90 Japan). For LC/MS analysis, an LC 7400 series (Hitachi) equipped with an Agilent

91 6140 quadrupole mass spectrometer was used. LC/MS separation was performed on a

92 InertSustain C18 column (3 µm, 2.1 mm x 250 mm, GL Sciences) with a mobile phase

93 consisting of acetonitrile/water/formic acid (40/60/0.1, v/v/v) at a flow rate of 0.2

94 mL/min at 40 °C. ESI conditions (positive ion mode) were as follows: drying gas

95 temperature, 250 °C; drying gas flow, 10 L/min; capillary voltage, 4 kV. 1H nuclear

96 magnetic resonance (NMR) spectra were recorded on a JEOL ECS400 (400 MHz)

5 Analytical Sciences Advance Publication by J-STAGE Received October 5, 2018; Accepted November 30, 2018; Published online on December 14, 2018 DOI: 10.2116/analsci.18P441

97 spectrometer in CD3OD. Chemical shifts have been reported in δ ppm units with

98 reference to the internal standard tetramethylsilane (Si(CH3)4, 0.00 ppm).

99

100 Preparation of Racemic Synephrine Derivatized with TAG-ITC.

101 Stock solution of racemic synephrine (10 mM) was prepared with water, and stored

102 at −15 °C. TAG-ITC (20 mM) solution was prepared with acetonitrile. Solutions of

103 synephrine (100 µL), 200 mM phosphate buffer (pH 7.0) (200 µL), and acetonitrile

104 (150 µL) and TAG-ITC (50 µL) were mixed. The mixture was incubated at 40 °C for

105 20 min and then synephrine derivative was analyzed by HPLC.

106

107 Sample Extraction and Preparation.

108 Thirteen brands of citrus fruits, four brands of dried citrus fruits, four brands of

109 citrus juices, two brands of orange marmalade, and a brand of canned mikan (Table 1)

110 were purchased from local markets. A bitter orange (Citrus aurantium) was kindly

111 gifted by JA Aira Izu. Mikan (), orange (Citrus sinensis), and bitter

112 orange (Citrus aurantium) were divided into exocarp, mesocarp, endocarp, and

113 sarcocarp. For other citrus fruits, exocarp was used. Citrus products were not devided,

114 whole products were used. Synephrine in each sample was extracted with a Microtec

115 (Funabashi, Japan) Physcotron homogenizer model NS-52 in 10 mL of 30%

116 acetonitrile. Then, the mixture was centrifuged at 1,200 xg for 5 min. The supernatant

117 was filtered with a 0.2 μm filter and the filtrate was applied to a Sep-Pak Plus Short

118 tC18 cartridge (Waters, Milford, USA). The first nonbinding fraction (0−2 mL) was

119 discarded and the next nonbinding fraction (2−3mL) was collected. The nonbinding

120 fraction was used as sample solution for derivatization with TAG-ITC.

121

122 6 Analytical Sciences Advance Publication by J-STAGE Received October 5, 2018; Accepted November 30, 2018; Published online on December 14, 2018 DOI: 10.2116/analsci.18P441

123 Results and discussion

124 Factors Affecting Chiral Separation.

125 Generally, amino group could be derivatized with isothiocyanate under basic

126 conditions. Gal and Brown reported synephrine was derivatized with

127 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosil isothiocyanate (TAG-ITC) in the presence of

128 sodium bicarbonate. We also derivatized synephrine with TAG-ITC at 40 °C for 20

129 min in the presence of 0.4% (v/v) triethylamine. The derivatized synephrine was

130 enantioseparated by reversed phase HPLC with an InertSustain C18 column using a

131 mobile phase containing 40% (v/v) acetonitrile and 0.1% (v/v) phosphoric acid.

132 However, many minor peaks were observed around peaks of synephrine enantiomers.

133 This result was the same as that obtained using 10 mM tetraborate buffer (pH 9.2)

134 instead of 0.4% (v/v) triethylamine. We tried to examine the derivatization under lower

135 pH conditions using 80 mM phosphate buffer. The minor peaks were decreased with

136 decreasing pH of derivatized media. Native synephrine peak was not observed above

137 pH 7, and peak area of synephrine enantiomers at pH 7 or 8 was larger than that at pH

138 6. Therefore, pH 7 was selected for the derivatization of synephrine with TAG-ITC.

139 The derivatized racemic mixture was applied to LC(ESI+)-MS. The correct mass of

140 both ingredients gave peaks with protonation at m/z = 557, suggesting that (R)- and

141 (S)-synephrines reacted with TAG-ITC at a ratio of 1:1. 1H NMR spectra of synephrine

142 TAG-ITC adduct was compared with that of synephrine. Although most of the aryl

143 protons of synephrine were not shifted, the N-methyl and N-methylene signals were

144 shifted upfield by derivatization. It indicated that amino group of synephrine was

145 reacted with TAG-ITC.

146 The effect of reaction time (0−30 min) on the derivatization of synephrine with

147 TAG-ITC was examined using a reaction mixture containing 1 mM racemic

7 Analytical Sciences Advance Publication by J-STAGE Received October 5, 2018; Accepted November 30, 2018; Published online on December 14, 2018 DOI: 10.2116/analsci.18P441

148 synephrine, 4 mM TAG-ITC, and 80 mM phosphate buffer (pH 7.0) at 40 °C. Peak

149 area of derivatized synephrine increased with an increase in incubation time up to 20

150 min, and then kept constant up to 30 min. Therefore, the incubation time was

151 determined to be 20 min.

152 In our preliminary study, a mobile phase containing 40% (v/v) acetonitrile and 0.1%

153 (v/v) phosphoric acid was used and two peaks of synephrine enantiomers were well

154 separated (Fig. 2-A). However, when citrus samples were analyzed, matrix peaks were

155 overlapped with synephrine peaks. Thus, we investigated the effect of mobile phase

156 mixed with acetonitrile and methanol on the separation and the retention time of

157 derivatized synephrine (Fig. 2). Derivatized synephrine was enantioseparated by HPLC

158 at any ratios of acetonitrile and methanol concentrations. A mobile phase containing

159 20% (v/v) acetonitrile, 20% (v/v) methanol and 0.1% (v/v) phosphoric acid was used

160 for further experiments considering the higher separation, a moderately short retention

161 time of synephrine, and the better separation of contaminants contained in citrus fruit

162 samples. It has been reported that octopamine and tyramine were minor components in

163 citrus peels.9,12-14,21 Using the above HPLC and derivatization conditions, three

164 phenethylamines (racemic octopamine, racemic synephrine and tyramine, each 0.3

165 mM) were analyzed (Fig. 3A). These phenethylamine derivatives were found to be

166 well separated with the elution order of octopamine, synephrine, and tyramine, which

167 order was the same as that of native phenethylamines reported by using reversed phase

168 HPLC.9,12,13,21 Racemic octopamine was not enantioseparated by the above HPLC

169 method. Each peak area of racemic octopamine and tyramine was about half of the

170 total peak area of synephrine. Derivatization rates of octopamine and tyramine were

171 slower than that of synephrine, and this may be attributed to difference between

172 primary and secondary amines. The (R)-synephrine (0.5 mM), which is commercially

8 Analytical Sciences Advance Publication by J-STAGE Received October 5, 2018; Accepted November 30, 2018; Published online on December 14, 2018 DOI: 10.2116/analsci.18P441

173 available, was derivatized with TAG-ITC, and the derivative was analyzed by the

174 proposed method (Fig. 3B). Since the peak corresponding to the (S)-synephrine

175 derivative was not detected, it was found that any racemization of (R)-synephrine did

176 not occur during derivatization process.

177 Synephrine was subjected to the proposed HPLC method using the above optimum

178 conditions. The limit of detection (LOD) of each synephrine enantiomer defined as a

179 signal-to-noise ratio of 3 was 0.0005 mM (0.0836 mg/100g) and the limit of

180 quantification (LOQ) of each enantiomer defined as a signal-to-noise ratio of 10 was

181 0.0015 mM (0.251 mg/100g). Linearity (r2 > 0.999) was demonstrated in the

182 concentration range of 0.0015−1 mM by each standard curve (9 points) for (R)- and

183 (S)-synephrines. The repeatability of five consecutive determinations was evaluated at

184 0.005 mM, 0.05 mM, and 0.5 mM for (R)- and (S)-synephrine. Good repeatabilities of

185 peak areas (RSD < 1.2%) and retention times (RSD < 0.2%) were obtained for both

186 enantiomers. Interday repeatabilities of peak areas and retention time in three days

187 (n=5, each day) were less than 0.4% and 0.3% RSD, respectively, at 0.5 mM for each

188 enantiomer. When a standard solution (0.3 mL) containing 83.6 mg/ L each enantiomer

189 of synephrine (final concentration: 50.2 mg each enantiomer per g samples) was added

190 to mikan, orange, and bitter orange peel samples (0.5 g), recoveries of (R)- and

191 (S)-synephrines were between 93 and 107%.

192

193 Separation of synephrine enantiomers in citrus fruit samples

194 Kusu et al.23 studied an HPLC method using a chiral column for synephrine in

195 Citrus unshiu and reported that synephrine content was mesocarp > endocarp >

196 exocarp > sarcocarp. On the other hand, Arbo et al.10 reported that its content was peel

197 > albedo > pulp. The (S)- and (R)-synephrine contents in Citrus fruit samples were

9 Analytical Sciences Advance Publication by J-STAGE Received October 5, 2018; Accepted November 30, 2018; Published online on December 14, 2018 DOI: 10.2116/analsci.18P441

198 analyzed by the proposed HPLC method (Table 1). The representative chromatograms

199 were shown in Fig. 4. The total contents of synephrine enantiomers in mikan were

200 exocarp > mesocarp > endocarp > sarcocarp, and the same results were also obtained

201 with orange and bitter orange samples, suggesting that synephrine content of outer side

202 of citrus fruits was higher than that of the inner side. Synephrine was detected in

203 exocarp of eleven citrus fruits except for lemon, lime, and grapefruit (Tables 1 and 2).

204 (S)-Synephrine was detected in exocarp of a mikan (Citrus unshiu), two

205 (Citrus unshiu × Citrus sinensis) and a ponkan (Citrus reticulate) and the ratio of

206 (S)-synephrine to total synephrine was 0.5−0.9%. (S)-Synephrine was detected in

207 exocarps of bitter orange, , , and hassaku samples, but the contents of

208 their samples were significantly low (0.08−0.25 mg/100g). Synephrine enantiomers in

209 products of citrus fruits were also analyzed (Table 3). (R)-Synephrine was detected in

210 three dried citrus fruits, four citrus juices, two orange marmalades, and a canned mikan,

211 but (S)-synephrine was not detected.

212

213 The effect of sugars on the racemization of (R)-synephrine

214 Pellati et al.29 studied the racemization of synephrine enantiomer. They found that

215 the racemizaton hardly occurred in buffer solutions at pH values between 3 and 8 at 80

216 °C after 48 h (less than 10% isomerization) and showed a possible mechanism of

217 racemization of (R)-synephrine in acidic and basic media. We also examined the

218 racemization of (R)-synephrine (0.5 mM) in aqueous solution at 100 °C (Fig. 5). pH of

219 the solution was 9.6. An increase in the heating time (2 h) brought about an increase in

220 the ratio of (S)-synephrine to total synephrine enantiomers in a linear fashion. The total

221 peak areas of (S)- and (R)-synephrines were not affected during heating, showing no

222 decomposition. Eroglu et al. reported the effect of thermal treatment on synephrine

10 Analytical Sciences Advance Publication by J-STAGE Received October 5, 2018; Accepted November 30, 2018; Published online on December 14, 2018 DOI: 10.2116/analsci.18P441

223 content of bitter along with ascorbic acid and sugar contents, where no

224 significant effect on the total synephrine content was obserbed.30 It has been reported

225 that orange juices contain 4-6% (w/v) sucrose, 2-3% (w/v) glucose, and 2-3% (w/v)

226 fructose.31-33 When 0.5 mM (R)-synephrine was heated at 100 °C for 2 h in the

227 presence of 5% (w/v) D-sucrose, the production of (S)-synephrine by the racemization

228 slightly decreased. However, the racemization was found to be significantly reduced by

229 addition of D-glucose or D-fructose instead of sucrose. Since both D-glucose and

230 D-fructose are reducing sugars, the reducibility of sugars may result in the inhibition of

231 the racemization. In order to confirm the suggestion, effect of concentrations of several

232 sugars (3−100 mM) on the racemization of (R)-synephrine was studied (Fig. 6). Five

233 reducing sugars (D-fructose, D-maltose, D-glucose, D-mannose, and D-galactose)

234 suppressed the racemization of (R)-synephrine, but two non-reducing sugars

235 (D-sucrose and D-mannitol) did not affect the racemization. Among the five sugars,

236 D-fructose and D-maltose showed the strong suppression at lower concentration less

237 than 30 mM compared to other three sugars. Further studies are necessary to clarify the

238 mechanism.

239

240 In conclusion, using a derivatization with TAG-ITC, an HPLC method for the

241 separation of the synephrine enantiomers was developed. Generally, primary amino

242 group could be derivatized with isothiocyanate under basic conditions. However, the

243 optimum pH for derivatization of synephrine, having secondary amino group, with

244 TAG-ITC was found to be 7. Using the proposed method, synephrine enantiomers in

245 14 citrus fruits and 11 citrus fruit products were analyzed. A small amount of

246 (S)-synephrine was found to be detected in exocarp of a mikan (Citrus unshiu), two

247 tangors (Citrus unshiu × Citrus sinensis) and a ponkan (Citrus reticulate), where the

11 Analytical Sciences Advance Publication by J-STAGE Received October 5, 2018; Accepted November 30, 2018; Published online on December 14, 2018 DOI: 10.2116/analsci.18P441

248 ratio of (S)-synephrine to total synephrine was less than 1%. Effect of sugars on the

249 racemization of (R)-synephrine during heating at 100 °C was also studied. D-Sucrose

250 and D-mannitol did not affect the racemization. However, an increase in the

251 concentration of D-fructose, D-maltose, D-glucose, D-mannose or D-galactose caused

252 a decrease in the racemization. Therefore, it is suggested that the reducibility of sugars

253 may result in the inhibition of racemization.

254

12 Analytical Sciences Advance Publication by J-STAGE Received October 5, 2018; Accepted November 30, 2018; Published online on December 14, 2018 DOI: 10.2116/analsci.18P441

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15 Analytical Sciences Advance Publication by J-STAGE Received October 5, 2018; Accepted November 30, 2018; Published online on December 14, 2018 DOI: 10.2116/analsci.18P441

308 Legends for Figures

309 Fig. 1. Reaction scheme of synephrine derivatization with TAG-ITC.

310

311 Fig. 2. Effect of mixing ratios of acetonitrile and methanol in mobile phase on the

312 enantioseparation of synephrine. HPLC was conducted by using an InertSustain C18

313 column with a mobile phase consisting of 30 mM phosphate buffer (pH 7.0) and

314 several ratios of acetonitrile and methanol at 40 °C. R and S represent (R)-synephrine

315 and (S)-synephrine, respectively.

316

317 Fig. 3. Chromatograms of racemic octopamine, racemic synephrine, and tyramine

318 (A), and (R)-synephrine (B) derivatized with TAG-ITC. HPLC was conducted by using

319 an InertSustain C18 column with a mobile phase consisting of 20% acetonitrile, 20%

320 methanol and 30 mM phosphate buffer (pH 7.0) at 40 °C. R and S represent

321 (R)-synephrine and (S)-synephrine, respectively.

322

323 Fig. 4. Chromatograms of standard solution (A) and derivatized synephrine extracted

324 from exocarp (B), mesocarp (C), endocarp (D), and sarcocarp (E) of mikan sample A

325 in Table 1. HPLC was conducted by using an InertSustain C18 column with a mobile

326 phase consisting of 20% acetonitrile, 20% methanol and 30 mM phosphate buffer (pH

327 7.0) at 40 °C. R and S represent (R)-synephrine and (S)-synephrine, respectively.

328

329 Fig. 5. Effect of heating on the racemization of (R)-synephrine. (R)-synephrine (0.5

330 mM) (closed circles) or that with 5% of D-sucrose (open square), D-glucose (open

331 triangle) or D-fructose (open circle) were heated at 100 °C, and then, derivatized with

332 TAG-ITC. HPLC was conducted by using an InertSustain C18 column with a mobile

16 Analytical Sciences Advance Publication by J-STAGE Received October 5, 2018; Accepted November 30, 2018; Published online on December 14, 2018 DOI: 10.2116/analsci.18P441

333 phase consisting of 20% acetonitrile, 20% methanol and 30 mM phosphate buffer (pH

334 7.0) at 40 °C.

335

336 Fig. 6. Effect of concentrations of sugars on the racemization of (R)-synephrine by

337 heating. (R)-synephrine (0.5 mM) with 3−100 mM sugars were heated at 100 °C, and

338 then, derivatized with TAG-ITC. HPLC was conducted by using an InertSustain C18

339 column with a mobile phase consisting of 20% acetonitrile, 20% methanol and 30 mM

340 phosphate buffer (pH 7.0) at 40 °C. D-Mannitol (closed squares); D-sucrose (open

341 squares); D-galactose (open rhombs); D-mannose (closed rhombs); D-glucose (open

342 circles); D-maltose (closed triangles); D-fructose (open triangles).

17 Analytical Sciences Advance Publication by J-STAGE Received October 5, 2018; Accepted November 30, 2018; Published online on December 14, 2018 DOI: 10.2116/analsci.18P441

343 Table 1. Concentrations of (S)- and (R)-synephrines in each organ of fresh citrus fruit samples (R)-Synephrine (S)-Synephrine Name Species Organ (mg/100g) (mg/100g) A mikan Citrus unshiu exocarp 84.6 ± 0.90a 0.75 ± 0.46 mesocarp 72.7 ± 0.70 < LOQb endocarp 33.4 ± 0.72 < LOQ sarcocarp 6.5 ± 1.10 NDc B orange Citrus sinensis exocarp 24.1 ± 4.45 ND mesocarp 15.1 ± 1.01 ND endocarp 8.3 ± 0.22 ND sarcocarp 1.9 ± 0.96 ND

C bitter orange () Citrus aurantium exocarp 26.4 ± 0.87 < LOQ mesocarp 17.4 ± 0.96 ND endocarp 9.4 ± 0.96 ND sarcocarp 2.1 ± 1.03 ND 344 a mean ± standard deviation, n=3. 345 b less than 0.25 mg/100g. 346 c less than 0.08 mg/100g. 347

18 Analytical Sciences Advance Publication by J-STAGE Received October 5, 2018; Accepted November 30, 2018; Published online on December 14, 2018 DOI: 10.2116/analsci.18P441

348 Table 2. Concentrations of (S)- and (R)-synephrines in exocarp of fresh citrus fruit samples (R)-Synephrine (S)-Synephrine Name Species (mg/100g) (mg/100g) D citrus fruit hybrid (kanpei) Citrus unshiu × Citrus sinensis 64.7 ± 0.92a 0.31 ± 2.73 E citrus fruit hybrid () Citrus unshiu × Citrus sinensis 92.6 ± 1.28 0.64 ± 0.97 F ponkan Citrus reticulate var poonensis 64.5 ± 0.60 0.47 ± 2.03 G iyokan Citrus iyo 50.4 ± 0.76 < LOQb H dekopon Citrus dekopon 25.3 ± 1.04 < LOQ I hassaku Citrus hassaku 12.2 ± 0.36 < LOQ J amanatsu Citrus natsudaidai 8.8 ± 1.30 ND K Citrus junos 12.1 ± 1.03 ND L lemon Citrus limon ND ND M lime Citrus aurantifolia ND ND N grapefruit Citrus paradisi ND ND 349 a mean ± standard deviation, n=3. 350 b less than 0.25 mg/100g. 351 c less than 0.08 mg/100g. 352

19 Analytical Sciences Advance Publication by J-STAGE Received October 5, 2018; Accepted November 30, 2018; Published online on December 14, 2018 DOI: 10.2116/analsci.18P441

353 Table 3 Concentrations of (S)- and (R)-synephrines in citrus fruit products (R)-synephrine (S)-synephrine samples (mg/100g) (mg/100g) O dried citrus fruits round slice of orange 1.6 ± 1.02a NDb P round slice of citrus fruit hybrid () 3.5 ± 1.20 ND Q orange peel 2.1 ± 1.31 ND R segment of mikan 3.3 ± 0.98 ND S juices orange 2.5 ± 1.03 ND T orange 0.5 ± 0.60 ND U mikan 4.7 ± 1.77 ND V mikan + orange 2.2 ± 0.56 ND W orange marmalade 3.6 ± 0.56 ND X 0.7 ± 0.20 ND Y canned mikan 1.4 ± 0.66 ND 354 a mean ± standard deviation, n=3. 355 b less than 0.08 mg/100g. 356

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