ANALYTICAL SCIENCES APRIL 2019, VOL. 35 407 2019 © The Japan Society for Analytical Chemistry 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 and 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, Aichi 487–8501, Japan *3 Graduate School of Science and Engineering, University of Toyama, 3190 Gofuku, Toyama 930–8555, Japan *4 Department of Pharmacy, Kindai University, 3-4-1 Kowakae, Higashi-Osaka 577–8502, Japan *5 GL Sciences Inc., 30F, Tokyo Square Tower, 6-22-1 Nishishinjuku, Shinjuku, Tokyo 163–1130, Japan Racemic synephrine, which was transformed into diastereomers by derivatization with 2,3,4,6-tetra-O-acetyl-β-D- glucopyranosil isothiocyanate, was resolved by a reversed phase HPLC with UV detection at 254 nm. The total contents of synephrine enantiomers in citrus fruit samples were exocarp > mesocarp > endocarp > sarcocarp, suggesting that synephrine content of outer side of citrus fruits was higher than that of the inner side. (R)-Synephrine was detected in exocarp of eleven fresh citrus fruits, except for lemon, lime, and grapefruit samples. (S)-Synephrine was determined in the exocarp of four citrus fruits (mikan, orange, bitter orange, and ponkan samples) and the ratio of (S)-synephrine to total synephrine was 0.5 – 0.9%. The racemization of (R)-synephrine in aqueous solution during heating at 100°C was also examined. An increase in the heating time brought about an increase in the (S)-synephrine content in a linear fashion. The racemization was found to be significantly reduced by the addition of D-fructose, D-maltose, D-glucose, D-mannose or D-galactose, but not D-sucrose or D-mannitol. It is suggested that the reducibility of sugars may result in the inhibition of racemization. Keywords Enantioseparation, synephrine, diastereomer, citrus, HPLC (Received October 5, 2018; Accepted November 30, 2018; Advance Publication Released Online by J-STAGE December 14, 2018) to those of humans. Bitter orange extracts, which contain Introduction synephrine, are widely used for weight loss/weight management, energy production, and sports performance. Synephrine is a Synephrine (Fig. 1), a phenethylamine alkaloid, is known to be chiral compound and its enantiomers have been shown to exert present in the peel and edible parts of Citrus fruits.1–5 Since different pharmacological activities on α- and β-adrenergic synephrine is structurally similar to adrenergic agonist, such as receptors. That is, (R)-synephrine is from 1 to 2 orders of adrenaline, noradrenaline, and ephedrine, the effects of magnitude more active than its (S)-enantiomer.2,8 synephrine on the cardiovascular system are attributable to Synephrine has been analyzed by HPLC with an ultraviolet adrenergic stimulation. In general, vasoconstriction occurs detector,9–15 HPLC with mass spectrometry,11,16–18 and Raman when ligands bind to α-adrenergic receptors, while binding to spectrometry.19 It was reported that the amount of synephrine in β-1 adrenergic receptors result in cardiovascular contractility citrus fruits was varied according to the citrus species, parts of and increased heart rate. Ligand binding to β-2 adrenergic fruits, and the maturation period.9–13,16 Arbo et al. revealed a receptors is associated with bronchodilation.6 However, variation on the content of synephrine in Citrus. sinensis synephrine binds much more poorly to α-1, α-2, β-1, and β-2 according to the maturation period and found that its content adrenergic receptors than other ligands, such as adrenaline.7 was inversely proportional to the size of the fruit.10 Synephrine Various studies have shown that synephrine binds to β-3 enantiomers in citrus fruit samples have also been separated by adrenergic receptors, resulting in an increase in the body’s HPLC with chiral columns.20–23 ability to breakdown fats. Binding to β-3 adrenergic receptors Direct and indirect methods have evolved as the main dose not influence heart rate or blood pressure, but regulate lipid strategies for enantioseparation.24–26 A direct method, which and carbohydrate metabolism.5 According to Stohs,5 synephrine does not require chemical derivatization, is based on a chiral exhibits greater adrenergic receptor binding in rodents than in stationary phase or with a chiral selector in a mobile phase on humans, while data from animals cannot be directly compared an achiral stationary phase. An indirect method is based on the formation of diastereomers by derivatization of analyte † To whom correspondence should be addressed. enantiomers with a chiral reagent. Gal and Brown27 reported an E-mail: [email protected] indirect HPLC method for the chiral separation of an adrenergic 408 ANALYTICAL SCIENCES APRIL 2019, VOL. 35 Fig. 1 Reaction scheme of synephrine derivatization with TAG-ITC. agent including synephrine based in derivatizing with spectrometer was used. LC/MS separation was performed on a 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosil isothiocyanate (TAG- InertSustain C18 column (3 μm, 2.1 mm × 250 mm, GL ITC) under basic condition. But this indirect method was not Sciences) with a mobile phase consisting of acetonitrile/water/ applied for real samples. formic acid (40/60/0.1, v/v/v) at a flow rate of 0.2 mL/min at It was reported that (S)-synephrine in the peels of six citrus 40°C. ESI conditions (positive ion mode) were as follows: fruits, including mikan and orange, was not detected (less than drying gas temperature, 250°C; drying gas flow, 10 L/min; 1 mg/100 g) and that (R)-synephrine was the only enantiomer capillary voltage, 4 kV. 1H nuclear magnetic resonance (NMR) isolated from citrus fruits.22 The same results were obtained spectra were recorded on a JEOL ECS400 (400 MHz) 23 20 21 using mikan and bitter orange, but Pellati et al. determined spectrometer in CD3OD. Chemical shifts have been reported in (S)-synephrine in fresh fruits pulp of bitter orange, in which the δ ppm units with reference to the internal standard ratio of (S)- and (R)-synephrines was 7.6:92.4. They also tetramethylsilane (Si(CH3)4, 0.00 ppm). reported that the ratio of (S)-synephrine to total synephrine extended over 4.8 – 14.4% in Evodia fruits.28 Kusu et al.23 Preparation of racemic synephrine derivatized with TAG-ITC found both (S)- and (R)-synephrines were detected in two orange A stock solution of racemic synephrine (10 mM) was prepared juices and a marmalade. They suggested that (S)-synephrine with water, and stored at –15°C. TAG-ITC (20 mM) solution may possibly be formed during production steps. Thus, it was prepared with acetonitrile. Solutions of synephrine remains unclear whether (S)-synephrine is contained in citrus (100 μL), 200 mM phosphate buffer (pH 7.0) (200 μL), and fruits, or not. In order to clarify the question, we developed an acetonitrile (150 μL) and TAG-ITC (50 μL) were mixed. The HPLC method for the analysis of synephrine enantiomers in mixture was incubated at 40°C for 20 min and then the citrus fruits, citrus juices, and citrus products after synephrine synephrine derivative was analyzed by HPLC. was derivatized with TAG-ITC at neutral pH. We also studied the effect of sugars on the racemization of (R)-synephrine. Sample extraction and preparation Thirteen brands of citrus fruits, four brands of dried citrus fruits, four brands of citrus juices, two brands of orange Experimental marmalade, and a brand of canned mikan (Table 1) were purchased from local markets. A bitter orange (Citrus aurantium) Reagents and chemicals was kindly gifted by JA Aira Izu. Mikan (Citrus unshiu), orange Racemic synephrine and acetonitrile were obtained from (Citrus sinensis), and bitter orange (Citrus aurantium) were Sigma (St. Louis, MO, USA). Methanol was from Kanto divided into exocarp, mesocarp, endocarp, and sarcocarp. For Chemicals (Tokyo, Japan). (R)-(–)-Synephrine, racemic octo- other citrus fruits, exocarp was used. The citrus products were pamine, tyramine, and 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosil not divided, whole products were used. Synephrine in each isothiocyanate (TAG-ITC) were from Tokyo Kasei (Tokyo, sample was extracted with a Microtec (Funabashi, Japan) Japan). Sodium dihydrogenphosphate dihydrate and other Physcotron homogenizer Model NS-52 in 10 mL of 30% chemicals (analytical grade) were obtained from FUJIFILM acetonitrile. Then, the mixture was centrifuged at 1200g for Wako Pure Chemical Corporation (Osaka, Japan). 5 min. The supernatant was filtered with a 0.2-μm filter and the filtrate was applied to a Sep-Pak Plus Short tC18 cartridge Apparatus for HPLC and mobile phase conditions (Waters, Milford, USA). The first nonbinding fraction The HPLC system consisted of a Jasco (Hachioji, Japan) (0 – 2 mL) was discarded and the next nonbinding fraction Model PU-2080 pump, a Jasco Model UV-2075 detector, a (2 – 3 mL) was collected. The nonbinding fraction was used as Rheodyne (Cotati, CA, USA) manual injector, a Shimadzu a sample solution for derivatization with TAG-ITC. (Kyoto, Japan) column oven Model CTO-10Avp, and a Shimadzu degasser Model DGU-14A. InertSustain C18 column (5 μm, 4.6 mm i.d. × 150 mm, GL Sciences, Tokyo, Japan) was Results and Discussion used. A mobile phase consisted of 20% acetonitrile, 20% methanol and 30 mM phosphate buffer (pH 7.0). Elution was Factors affecting chiral separation carried out at a flow rate of 1.0 mL/min at 40°C. Analytes were Generally, the amino group could be derivatized with detected at 254 nm.