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 Citrus 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 lemon, lime, and grapefruit samples. (S)-Synephrine was determined in 9 exocarp of four citrus fruits (mikan, orange, bitter orange, and ponkan 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.