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www.rsc.org/methods Page 1 of 28 Analytical Methods
1 2 3 Simultaneous Quantification of seventeen Bioactive Components in Rhizome and 4 5 Aerial Parts of Alpinia officinarum Hance Sampled at Different Growing Periods 6 7 Using Liquid Chromatography/Quadrupole Tandem Mass Spectrometry 8 9 10 Jun-Qing Zhang †,# , Yong Wang †,# , Hai-Long Li †, Wen Qi †, Hang Yin †, Nian-Kai Zeng †, Wei-Yong Lai †, 11 12 Na Wei †, Shou-Qian Cheng †, ‡, Sheng-Li Kang †, Feng Chen †,* , You-Bin Li †, ‡,* 13 14 † Hainan Provincial Key Laboratory of R&D of Tropical Herbs, School of Pharmacy, Hainan Medical 15 16 University, Haikou 571101, China 17 ‡ 18 Nanjing University of Chinese Medicine, Nanjing 210046, China 19 20 21 Manuscript 22 23
24 25 26 27 28 29 30
31 Accepted 32 33 34 35 36 37 38
39
40 Methods
41 42 43 44 45 46 # These authors contributed equally to this work. 47 48 * Corresponding authors at: School of Pharmacy, Hainan Medical University, Haikou 571101, 49 50 China. Tel.: +86 898 66895337; Fax: +86 898 66893460. 51 Analytical 52 E-mail addresses: [email protected] (Feng Chen) and [email protected] (Y.-B. Li). 53 54 55 56 57 58 59 1 60 Analytical Methods Page 2 of 28
1 2 3 4 1 ABSTRACT 5 6 2 The rhizomes of Alpinia officinarum Hance (Zingiberaceae family) have been used as 7 8 9 3 antiemetics, stomachics and analgesics in Asia for centuries. Unfortunately, the aerial 10 11 4 parts were thrown away as wastes whilst harvesting the rhizomes of A. officinarum . 12 13 14 5 Recently, scientists reported that the ethanol extract of aerial parts displayed 15 16 6 anti proliferation activity through mitochondrial pathway induced cell apoptosis. 17 18 19 7 However, the chemical composition information of this extract remained largely 20 21 8 unknown. We have identified sixteen chemicals including twelve flavonoids and four Manuscript 22 23 24 9 diarylheptanoids from the methanol extraction of A. officinarum leaves using liquid 25 26 10 chromatography/tandem mass spectrometry (LC MS/MS). In order to better explore 27 28 29 11 the potential value of the aerial parts, we need to know what the main constituents 30
31 12 occurring in the aerial parts and how the contents of these chemicals are influenced by Accepted 32 33 34 13 the growing periods. In the present study, a LC MS/MS method was developed and 35 36 14 validated for determination of seventeen compounds both occurring in the aerial parts 37 38 39 15 and rhizomes sampled at different growing periods. Validation indices evaluated were
40 Methods 41 16 satisfactory and the method was successfully employed to analyze the 42 43 44 17 above mentioned plant samples. Notably, we found that the contents of these 45 46 18 compounds except for quercetin were higher in rhizomes than those of compounds in 47 48 49 19 aerial parts. The six major constituents both in aerial parts and rhizomes were 50 51 Analytical 20 galangin, kaempferide, hexahydrocurcumin, pinocembrin, chrysin and isorhamnetin. 52 53 54 21 Moreover, the content changes’ trends of most of the monitored phytochemicals along 55 56 22 with sampled periods were almost similar between the aerial parts and the rhizomes. 57 58 59 2 60 Page 3 of 28 Analytical Methods
1 2 3 4 23 Our study should be of value in arousing everyone’s interest to make the best use of 5 6 24 aerial parts of A. officinarum in the future. 7 8 9 25 Keywords: Alpinia officinarum Hance; rhizomes and aerial parts; harvest times; 10 11 12 26 LC MS/MS 13 14 15 16 17 18 19 20 21 Manuscript 22 23 24 25 26 27 28 29 30
31 Accepted 32 33 34 35 36 37 38 39
40 Methods 41 42 43 44 45 46 47 48 49 50 51 Analytical 52 53 54 55 56 57 58 59 3 60 Analytical Methods Page 4 of 28
1 2 3 4 27 Introduction 5 6 28 Plant secondary metabolites are critical for not only the function and value of the 7 8 9 29 compounds within the plants themselves, but also the relationship involves biological 10 11 30 interaction of plants with other organisms and with their environment 1. A plant that is 12 13 14 31 able to synthesize some compounds that can disturb the physiological functions of an 15 16 32 herbivore may have a selective advantage over one that does not. The secondary 17 18 19 33 metabolites may be utilized to establish interlocking relationship within the contexts 20 21 34 of pollination, seed dispersal or protection of the plant by another organism. Generally Manuscript 22 23 24 35 speaking, the secondary chemicals are biosynthesized, accumulated under certain 25 26 36 conditions and then transported within the plant to a site of storage or are deposited on 27 28 2 29 37 the surface . From an evolving point of view, it appears that these products are often 30
31 38 concentrated in the most vulnerable tissues. Accepted 32 33 34 39 Alpinia officinarum Hance (Zingiberaceae family), known as lesser galangal, is a 35 36 40 famous traditional herb and mainly distributed in the Southern China such as 37 38 39 41 Guangdong province and Hainan Island. A. officinarum rhizomes that used as
40 Methods 41 42 medicinal parts have been used as antiemetics, stomachics and analgesics in Asia for 42 43 44 43 centuries 3. Recently, a review article of our group has summarized the advances in 45 46 44 studies on chemical constituents in A. officinarum rhizomes and their pharmacological 47 48 49 45 activities 4. Some bioactive components of rhizomes have been listed as essential oil, 50 51 Analytical 46 flavonoids, diarylheptanoids, phenylpropanoids, glycosides and other constituents in 52 53 54 47 this article. Especially, the flavonoids such as galangin and diarylheptanoids are the 55 56 48 main constituents and have exhibited various pharmacological activities including 57 58 59 4 60 Page 5 of 28 Analytical Methods
1 2 3 4 49 antimicrobial, antiviral, antitumor, antioxidant roles, gastric ulcer protective activities 5 6 50 and usually used as a hemostat for treating gastrointestinal hemorrhages. Therefore, 7 8 9 51 flavonoids and diarylheptanoids are always used as marker compounds for quality 10 11 52 control of A. officinarum rhizomes and its extracts and some Chinese traditional 12 13 14 53 patent medicine. 15 16 54 Unlike the rhizome, the aerial parts of A. officinarum are not widely concerned. 17 18 19 55 Zhang et al. identified five flavonoids including galangin, 3 O methylgalangin, 20 21 5 56 pinocembrin, pinobaksin and kaempferide from the ethanol extract of the aerial parts . Manuscript 22 23 24 57 A Chinese patent (CN104138368A) provided a process for producing a purified 25 26 58 extract (AO 95) from the aerial parts by ethanol extraction and subsequent 27 28 29 59 purification via macro porous adsorptive resins. This AO 95 extract displayed 30
31 60 anti proliferation activity through mitochondrial pathway induced cell apoptosis. Accepted 32 33 34 61 However, the chemical composition information of this extract was not provided in 35 36 62 this patent. Liquid chromatography/tandem mass spectrometry (LC MS/MS) 37 38 6 9 39 63 technique has been used to biological molecules structure determination . Recently,
40 Methods 41 64 we identified sixteen chemicals including twelve flavonoids and four diarylheptanoids 42 43 44 65 from the methanol extraction of A. officinarum leaves using LC MS/MS ) with 45 46 10 66 selected reaction monitoring mode . Twelve flavonoids included chrysin, 47 48 49 67 pinocembrin, tectochrysin, apigenin, galangin, 3 O methylgalangin, acacetin, 50 51 Analytical 68 kaempferol, kaempferide, quercetin, isorhamnetin and rutin. Four diarylheptanoids 52 53 54 69 were yakuchinone A, oxyphyllacinol, hexahydrocurcumin and hannokinol. These 55 56 70 secondary metabolites may contribute to the above mentioned anti cancer activity. 57 58 59 5 60 Analytical Methods Page 6 of 28
1 2 3 4 71 Therefore, the aerial part of A. officinarum has potential to be used as the medicinal 5 6 72 part in the future. 7 8 9 73 In order to better explore the potential value of the aerial parts, we need to know 10 11 74 what the main constituents occurring in the aerial parts and how the contents of these 12 13 14 75 chemicals are influenced by the growing periods. Furthermore, as time goes on, the 15 16 76 transportation of these secondary metabolites from aerial parts to rhizomes is also 17 18 19 77 need to be characterized. In the present study, seventeen compounds both occurring in 20 21 78 the aerial parts and rhizomes sampled at different growing periods were monitored Manuscript 22 23 24 79 and quantified using LC MS/MS. Notably, we found that the content changes’ trends 25 26 80 of most of the target phytochemicals along with sampled periods were almost similar 27 28 29 81 between aerial parts and rhizomes. 30
31 Accepted 32 33 34 35 36 37 38 39
40 Methods 41 42 43 44 45 46 47 48 49 50 51 Analytical 52 53 54 55 56 57 58 59 6 60 Page 7 of 28 Analytical Methods
1 2 3 4 82 Material and Methods 5 6 7 83 Chemical and Reagents 8 9 84 Reference standard of nootkatone was obtained from Sigma Aldrich (St Louis, MO, 10 11 12 85 USA). Yakuchinone A was purchased from Chenfun Medical Technology (Shanghai) 13 14 86 Co., Ltd. (Shanghai, China). Hexahydrocurcumin and Hannokinol were purchased 15 16 17 87 from BioBioPha Co., Ltd (Kunming, China). Apigenin and pinocembrin were 18 19 88 obtained from Shanghai YuanYe Bio Technology Co., Ltd (Shanghai, China). 20 21 Manuscript 22 89 Acacetin was bought from Nanjing Zelang Pha Co. Ltd (Nanjing, China). Galangin, 23 24 90 rutin, quercetin, kaempferol, luteolin and isorhamnetin were purchased from National 25 26 27 91 Institutes for Food and Drug Control (Beijing, China). Tectochrysin, izalpinin, chrysin 28 29 92 and kaempferide were separated from A. oxyphylla fruits. Diarylheptanoid was 30
31 Accepted 32 93 separated and prepared from the rhizomes of A. officinarum . The purities of these 33 34 94 reference standards were over 98.0%. HPLC grade methanol and acetonitrile were 35 36 37 95 products of Merck (Darmstadt, Germany). HPLC grade formic acid was purchased 38 39 96 from Aladdin Industrial Inc. (Shanghai, China). HPLC grade water was prepared with
40 Methods 41 42 97 a Millipore Milli Q Integral 3 cabinet water purifying system (Bedford, MA, USA). 43 44 45 98 The other chemical reagents, analytical grade or better, were obtained from Hainan 46 47 99 YiGao Instrument Co., Ltd (Haikou, China). The chemical structures of 18 plant 48 49 50 100 secondary metabolites are shown in Figure 1. 51 Analytical 52 53 101 (Insert Figure 1 here) 54 55 56 102 The aerial parts and rhizomes of 3 year old A. officinarum were collected from 57 58 59 7 60 Analytical Methods Page 8 of 28
1 2 3 4 103 the planting base of Hainan Lin Feng Yuan Industrial Co., Ltd. The sampling periods 5 6 104 and corresponding serial numbers are shown in Figure 2. 7 8 9 105 (Insert Figure 2 here) 10 11 12 106 13 Sample Preparation for Analysis 14 15 107 In order to perform the determinations on these plant secondary metabolites, we 16 17 108 18 developed an extraction protocol specific for these plant samples. All the rhizomes 19 20 109 and aerial parts of A. officinarum were dried in an electric drying oven (6CH 18, 21 Manuscript 22 110 23 Xingmin Tea Machinery Co., Ltd., Anxi, China) at 40°C until dry. The freshly dried 24 25 111 plant samples were manually shucked into little segments, which were smashed using 26 27 28 112 a smashing machine (FW100, Taisite Instrument, Tianjin, China) and then sieved 29 30 113 manually by a 60 mesh. The resulting fine powders and residue were mixed evenly.
31 Accepted 32 33 114 An aliquot (0.5 g) was weighed precisely and macerated with 50 fold methanol and 34 35 115 then ultrasonicated three times for 30 min each. For each extraction, the resulting 36 37 38 116 extract solutions were centrifuged at 13000 rpm for 10 min (Kubota 5922, Kubota 39
40 117 Corporation, Tokyo, Japan). 1 mL of supernatant was sampled and the remaining was Methods 41 42 43 118 discarded. The residue was extracted with methanol for another 2 times. The sampled 44 45 119 methanol extracts (3 mL) were combined and centrifuged at 13000 rpm for 10 min to 46 47 48 120 obtain the supernatant fractions that were frozen at –20ºC until analysis. The extract 49 50
121 solution was appropriately diluted with methanol before analysis. Finally, a 10 L Analytical 51 52 53 122 aliquot was injected into the LC MS/MS system for quantitative analysis. 54 55 56 123 Instrumentation and LC-MS/MS Conditions 57 58 59 8 60 Page 9 of 28 Analytical Methods
1 2 3 4 124 A component LC MS/MS system consisted of an API 4000 plus mass spectrometer 5 6 125 (AB SCIEX, Toronto, Canada) interfaced via a Turbo V ion source with a Shimadzu 7 8 9 126 Prominence UFLC chromatographic system (Shimadzu Corporation, Kyoto, Japan) 10 11 127 and operated with AB SCIEX Analyst software. The UFLC system is equipped with 12 13 14 128 two LC 20AD pumps, a model DGU 20A 3R degasser unit, a SIL 20A HT 15 16 129 auto sampler and a CTO 20A column oven. 17 18 19 130 Chromatographic separation was achieved using a Phenomenex Kinetex 2.6 20 21 11 131 XB C18 (2.10 mm i.d. × 50 mm) . The LC mobile phase at a flow rate of 0.3 Manuscript 22 23 1 24 132 mL ∙min consisted of 0.1‰ formic acid in water (A) and methanol (B) using a 25 26 133 gradient elution as follows: from 0% B to 2% B in 0.01 min, hold for 1 min; from 2% 27 28 29 134 B to 35% B in 0.01 min, hold for 3 min; from 35% B to 90% B in 11 min; back to 2% 30
31 135 B in 0.01 min; maintain 4.99 min. The temperature of the column was controlled at Accepted 32 33 34 136 40ºC. A 0.5 m biocompatible inline filter (Upchurch Scientific, Oak Harbor, WA, 35 36 137 USA) was used before the chromatographic column. 37 38 39 138 The parameters of the electrospray ionization (ESI) source operating in positive
40 Methods 41 139 ionization mode were as follows: collision gas flow (N ): level 4; curtain gas (N2): 25 42 2 43 44 140 psi; nebulizer gas (N2, Gas I): 55 psi; heated dry gas (N2, Gas II): 55 psi; IonSpray 45 46 141 voltage (v): +5,500 v and dry temperature (TEM): 550 ºC. MS/MS operating 47 48 49 142 conditions were optimized by infusion of the standard solution (1 g∙mL 1) of each 50 51 Analytical 143 analytes into the ESI source via a syringe pump (11plus, Harvard Apparatus, Holliston, 52 53 54 144 MA, USA). Quantification was performed using multiple reaction monitoring (MRM) 55 56 145 modes for the following transitions. The MRMs of nootkatone, yakuchinone A, 57 58 59 9 60 Analytical Methods Page 10 of 28
1 2 3 4 146 tectochrysin, izalpinin, chrysin, kaempferide, apigenin 4’,7 dimethylther, kaempferol, 5 6 147 galangin, hannokinol, hexahydrocurcumin, pinocembrin, isorhamnetin, luteolin, rutin, 7 8 9 148 apigenin, acacetin and quercetin were m/z 219.2 →163.0 (the optimal collision ene rgy, 10 11 149 22 V), 313.2→136.9 (13 V), 269.1→226.0 (43.5 V), 285.0→242.0 (43 V), 12 13 14 150 255.1 →152.9 (42 V), 301.1→286.0 (37 V) , 299.2 →256.0 (45 V) , 287.2→153.0 (45 15 16 151 V), 271.2→153.0 (43.8 V), 313.8→256.3 (31.5 V), 375.2→357.2 (9 V), 17 18 19 152 257.1→153.0 (30.5 V), 317.2→302.2 (35.2 V), 287.1→153.0 (45.1 V), 611.2→303.0 20 21 153 (25.4 V), 271.1→153.0 (42.8 V), 285.1→242.0 (46.5 V) and 303.1→153.0 (47 V), Manuscript 22 23 24 154 respectively, with a scan time of 20 ms for each ion pair. The product ion spectra of 25 26 155 protonated molecules are shown in Figure 3. 27 28 29 156 (Insert Figure 3 here) 30
31 Accepted 32 33 157 Validation of Analytical Method 34 35 158 The quantitative LC MS/MS method was validated with respect to linearity, recovery, 36 37 1 38 159 precision and sensitivity. Twelve stock solutions containing about 1 mg ∙mL of 39
40 160 nootkatone, yakuchinone A, chrysin, kaempferol, galangin, hannokinol, Methods 41 42 43 161 hexahydrocurcumin, pinocembrin, luteolin, rutin, quercetin and diarylheptanoid were 44 45 162 prepared independently in methanol excluding hexahydrocurcumin, which was 46 47 48 163 dissolved in acetonitrile. Kaempferide, tectochrysin, izalpinin, apigenin, isorhamnetin 49 50
164 and acacetin were prepared in methanol and the concentration of standards was 1.41, Analytical 51 52 1 53 165 1.035, 0.5, 0.5, 0.3 and 0.332 mg ∙mL , respectively. 54 55 166 For calibration aims, working solutions were freshly prepared via diluting each 56 57 58 167 stock solution with methanol or acetonitrile. Calibration curves were established on 59 10 60 Page 11 of 28 Analytical Methods
1 2 3 1 4 168 six data points covering the designed concentration such as 2 2000 ng mL . Each 5 6 169 calibration curve was obtained by plotting the peak area vs the concentration of the 7 8 9 170 standard. The linearity was assessed by calculation of a regression line using the least 10 11 171 squares method. The limits of detection (LOD) and quantification (LOQ) were 12 13 14 172 evaluated by analysis of the peak height vs the baseline noise at a signal to noise ratio 15 16 173 of 3:1 and 10:1, respectively. Reliabilities of the extraction method were evaluated by 17 18 19 174 adding reference standards (25 ng for C-02 , C-12 and 50 ng for the other compounds) 20 21 175 into the powdered plant material of the two selected rhizomes and aerial parts samples, Manuscript 22 23 24 176 and the recovery of the added standards was measured. Precision was assessed by the 25 26 177 evaluation of the repeatability (intra day precision) and by intermediate precision 27 28 29 178 (inter day precision). The intra day precision was determined by analyzing six 30
31 179 replicate plant samples in a day, which was performed by the same technician. The Accepted 32 33 34 180 inter day precision was obtained through measuring the above mentioned six plant 35 36 181 samples in three consecutive days and by two different analysts. 37 38 39
40 Methods 41 42 43 44 45 46 47 48 49 50 51 Analytical 52 53 54 55 56 57 58 59 11 60 Analytical Methods Page 12 of 28
1 2 3 4 182 Results and Discussion 5 6 7 183 Selection of the Extraction Method 8 9 184 Plant samples were extracted using published method 11 13 by our group with a slight 10 11 12 185 modification. In this study, we chose methanol as the extraction solvent and 13 14 186 sonication procedure for the extraction of the target constituents. Sonication combined 15 16 17 187 with methanol extraction provides mechanical disruption and thereby aiding in 18 19 188 extraction. In order to control the uniformity of sonication procedure, fresh running 20 21 Manuscript 22 189 water was added into the bath of the ultrasonic extraction device (40 KHz, 80 W, 23 24 190 Kunshan Ultrasonic Instruments, Kunshan, China) for each extraction. After having 25 26 27 191 completed the third sonication extraction, the ultrasonic device was kept on standby 28 29 192 for 1 hour in case of overheats. Our results revealed that the recoveries of the 17 plant 30
31 Accepted 32 193 secondary metabolites were more than 85% by single extraction and almost 100% 33 34 194 through triple extraction for aerial parts and rhizomes samples. Therefore, the sample 35 36 37 195 was macerated with 50 fold methanol and then ultrasonicated three times for 30 min 38 39 196 each.
40 Methods 41 42 43 197 Method Validation 44 45 198 Representative chromatograms for 17 reference standards and aerial parts samples, as 46 47 48 199 well as rhizomes samples of A. officinarum , are shown in Figure 4. No interfering 49 50
200 peaks were noted and good resolution was achieved among the monitored chemicals, Analytical 51 52 53 201 which were mainly eluted within 7.5 14 min except for hannokinol (retention time 54 55 202 7.07 min, data not shown) during our 20 min gradient program. Exogenous chemical 56 57 58 203 coming from plastic consumables such as inserts tubes, pipette tips interfered with 59 12 60 Page 13 of 28 Analytical Methods
1 2 3 4 204 hannokinol analysis. We failed to overcome this interference because we had to apply 5 6 205 various plastic products during our assay procedure. 7 8 9 206 (Insert Figure 4 here) 10 11 12 207 13 Standard curves were linear over the dynamic ranges with correlation 14 15 208 coefficients greater than 0.99 (see Table 1 ). The LODs and LOQs were in the range of 16 17 209 ∙ 1 ∙ 1 18 0.0141–2 ng mL and 0.1–10 ng mL , respectively. Mean recovery (standard 19 20 210 addition approach) of each target compound from aerial parts samples and rhizomes 21 Manuscript 22 211 23 samples exceeded 85.5% (range = 85.5 112%, Table 1 ), indicating that various plant 24 25 212 background matrices had little effect on the quantification analysis. For this standard 26 27 28 213 addition approach, the accurate amounts of the standards are added to real samples, in 29 30 214 which the concentration of target analyte has been determined, and then extracted and
31 Accepted 32 33 215 analyzed. The average percentage recoveries are evaluated by calculating the ratio of 34 35 216 detected amount versus added amount. Actually, standard addition approach was used 36 37 14 38 217 to analyze pharmaceutical residues in environmental samples , diarrheic shellfish 39 15 16 40 218 poisoning toxins and pharmaceutical residues in drinking water . Methods 41 42 43 219 As shown in Table 1 , the RSD values of intra and inter day variations of the 44 45 220 seventeen secondary metabolites occurring in aerial parts and rhizomes were almost 46 47 48 221 less than 10%. Overall, these results indicated that the established method was 49 50
222 accurate and reliable. Analytical 51 52 53 223 (Insert Table 1 here) 54 55 56 224 57 Quantitative Assay of 17 Constituents occurring in the Aerial parts and 58 59 13 60 Analytical Methods Page 14 of 28
1 2 3 4 225 Rhizomes of A. officinarum 5 6 226 The content levels of the 17 constituents are summarized in Figure 5a and 5b . Besides 7 8 9 227 quercetin ( C-12 ), the content levels of the sixteen monitored compounds in rhizomes 10 11 228 were 1.26 – 14.0 times higher than those of in aerial parts. The rank order of the best 12 13 14 229 six compound for their content differences was as follows: kaempferide ( C-18 , 14 15 16 230 times) > izalpinin ( C-16, 10.9 times) > diarylheptanoid (C-03 , 7.47 times) > 17 18 19 231 hexahydrocurcumin ( C-05 , 7.17 times) > nootkatone ( C-01 , 6.57 times) > galangin 20 21 232 (C-06 , 6.08 times). For quercetin, its concentration in aerial parts was 1.42 fold higher Manuscript 22 23 24 233 than that of in rhizomes. The six major constituents both in aerial parts and rhizomes 25 26 234 were the same, i.e. , galangin ( C-06 ) > kaempferide ( C-18 ) > hexahydrocurcumin 27 28 29 235 (C-05 ) > pinocembrin ( C-07 ) > chrysin ( C-14 ) > isorhamnetin ( C-08 ). Their highest 30
31 236 content levels were larger than 100 g∙g 1. Of particular, the highest content levels of Accepted 32 33 1 1 34 237 galangin ( C-06 ) were 17.7 mg ∙g (1.77%) in rhizomes and 2.92 mg ∙g (0.29%) in 35 36 238 aerial parts, respectively. Similarly, kaempferide and hexahydrocurcumin in rhizomes 37 38 1 1 39 239 were 5.11 mg ∙g (0.51%) and 1.29 mg ∙g (0.13%), respectively. Zhai et al. measured
40 Methods 41 240 the active flavonoids including galangin, kaempferide, quercetin and isorhamnetin of 42 43 44 241 A. officinarum rhizomes from 19 different localities of Hainan province 17. The 45 46 242 content of galangin and kaempferide was at 0.07 1.56% and 0.06 0.64%, respectively. 47 48 49 243 Therefore, galangin was the predominant constituent both in aerial parts and rhizomes 50 51 Analytical 244 of A. officinarum . 52 53 54 55 245 (Insert Figure 5a and 5b here) 56 57 58 246 As shown in Figure 5a and 5b , the contents of the seventeen constituents 59 14 60 Page 15 of 28 Analytical Methods
1 2 3 4 247 occurring in aerial parts and rhizomes changed obviously with different growing 5 6 248 periods (from 19 February to 31 August). The differences of these phytochemicals 7 8 9 249 covered 1.96 61.5 and 1.93 116 times for rhizomes and aerial parts, respectively. The 10 11 250 content of apigenin ( C-11 ) in rhizomes sampled at 19 February (S-01 ) was 61.5 times 12 13 14 251 higher than that at 30 May (S-07). Similarly, the content of chrysin ( C-14) in aerial 15 16 252 parts sampled at August 16 ( S-11) was 116 fold higher than that at 15 June ( S-08). 17 18 19 253 The content variability of the best six phytochemicals along with different growth 20 21 254 time both in rhizomes and aerial parts samples was as follows: galangin ( C-06 , Manuscript 22 23 24 255 6.84/3.74), kaempferide ( C-18 , 3.37/2.71), hexahydrocurcumin ( C-05 , 14.2/37.4), 25 26 256 pinocembrin ( C-07 , 5.01/2.45), chrysin ( C-14 , 58.9/166) and isorhamnetin ( C-08 , 27 28 29 257 3.22/2.43). Of particular, the contents of these five major flavonoids except for 30
31 258 chrysin in rhizomes maintained at a relatively higher level during the period of 15 Accepted 32 33 34 259 June –15 July and amounted to highest levels at 31 August. As for galangin, Deng et 35 36 260 al . determined its content in rhizomes of A. officinarum harvested in different months 37 38 18 39 261 using a reversed phase HPLC method . The content levels ranged from 0.74% (May)
40 Methods 41 262 to 1.39% (October). During the period of July to October, the content was relatively 42 43 44 263 stable ranging from 1.06% to 1.39%. Our results revealed that the content variability 45 46 264 of galangin in rhizomes covered 0.26 1.77%. On the other hand, the content levels of 47 48 49 265 galangin in aerial parts changed from 0.08% to 0.29%. Therefore, one must fully 50 51 Analytical 266 consider the impact of harvest time on the contents of phytochemicals. The Chinese 52 53 54 267 Pharmacopeia recommends that the best harvest period for rhizomes is in late summer 55 56 268 and early autumn (i.e. , August and September) 19. This recommendation looks like 57 58 59 15 60 Analytical Methods Page 16 of 28
1 2 3 4 269 reasonable based on our results. 5 6 270 As shown in Figure 5a and 5b , the content changes of the seventeen 7 8 9 271 phytochemicals with growth time were almost similar between aerial parts and 10 11 272 rhizomes. The content time curves presented two types, “dumbbell” or “parallel bars”. 12 13 14 273 For the “dumbbell” type curves, such as yakuchinone a ( C-02 ), hexahydrocurcumin 15 16 274 (C-05 ), luteolin ( C-09 ), apigenin ( C-11 ), acacetin ( C-13 ), chrysin ( C-14 ) and 17 18 19 275 kaempferol ( C-17 ), the contents during the period of 14 April 15 July retained at 20 21 276 relatively low levels. This period looked like a handle of the dumbbell and the handle Manuscript 22 23 24 277 thickness was different for this type phytochemicals indicating the content variability 25 26 278 among aerial parts or rhizomes samples. On the other hand, for the “parallel bars” 27 28 29 279 type chemicals, including nootkatone ( C-01 ), diarylheptanoid ( C-03 ), galangin ( C-06 ), 30
31 280 pinocembrin (C-07 ), isorhamnetin ( C-08 ), and so on, the contents both in aerial parts Accepted 32 33 34 281 and rhizomes had smaller fluctuation from 19 February ( S-01 ) to 31 August ( S-12 ). 35 36 282 Currently, it is still unclear how to explain the content variations. 37 38 39 283 The rhizomes of A. officinarum are utilized as medical parts for traditional
40 Methods 41 284 medicine, such as stimulant and carminative. It is especially useful in flatulence, 42 43 44 285 dyspepsia, vomiting and sickness at stomach, being recommended as a remedy for 45 46 286 sea sickness, sometimes for fever or as a stimulant. In addition, galangal is used in 47 48 49 287 cattle medicine 20 . Unfortunately, the aerial parts were thrown away as wastes whilst 50 51 Analytical 288 harvesting the rhizomes of A. officinarum . Like rhizome, the aerial parts contain 52 53 54 289 multiple phytochemicals such as flavonoids (e.g. galangin), diarylheptanoids (e.g. 55 56 290 hexahydrocurcumin) and sesquiterpenes (e.g. nootkatone) although their contents are 57 58 59 16 60 Page 17 of 28 Analytical Methods
1 2 3 4 291 relatively lower than those of rhizomes based on our results. In addition, the yield of 5 6 292 aerial parts of A. officinarum was comparable or less than that of rhizomes. Therefore, 7 8 9 293 additional work is required to assess the bioactive properties of the aerial parts and 10 11 294 make the best use of these valuable medicinal plant resources. 12 13 14 295 In summary, in this study we reported the development and validation of a 15 16 296 LC MS/MS and successfully employed this method to analyze the seventeen 17 18 19 297 phytochemicals present in aerial parts and rhizomes of A. officinarum . Our results 20 21 298 revealed that (1) validation indices evaluated were satisfactory; (2) the target Manuscript 22 23 24 299 seventeen phytochemicals were measurable in aerial parts and rhizomes sample; (3) 25 26 300 the contents of these compounds except for quercetin were higher in rhizomes than 27 28 29 301 those of compounds in aerial parts; (4) the six major constituents both in aerial parts 30
31 302 and rhizomes were galangin, kaempferide, hexahydrocurcumin, pinocembrin, chrysin Accepted 32 33 34 303 and isorhamnetin; (5) the content changes’ trends of most of the monitored 35 36 304 phytochemicals along with sampled periods were almost similar between aerial parts 37 38 39 305 and rhizomes; and (6) the amplitude of variation along with growth period for each
40 Methods 41 306 phytochemical was different. Our study should be of value in arousing everyone’s 42 43 44 307 interest in the future to make the best use of the aerial parts, rather than just rhizomes, 45 46 308 of A. officinarum , valuable medicinal plant resources. 47 48 49 50 51 Analytical 52 53 54 55 56 57 58 59 17 60 Analytical Methods Page 18 of 28
1 2 3 4 309 Acknowledgements 5 6 310 This work was supported by Grant ZDZX2013008 3 and ZDXM 2015078 from the 7 8 9 311 Hainan Science and Technology Major Project, Grant 2015ZY06 from Hainan Special 10 11 312 Plan for the Modernization of Chinese Medicines, Grant HNKY2014 50 from the 12 13 14 313 Hainan provincial project on higher education & teaching reform. 15 16 314 The authors wish express their thanks to Xian Yue Lin (Hainan LinFengYuan 17 18 19 315 industrial Co., Ltd, Haikou, China) for his help in collecting A. officinarum Hance 20 21 316 from the planting base of Hainan Lin Feng Yuan Industrial Co., Ltd. Manuscript 22 23 24 317 25 26 318 Conflicts of Interest 27 28 29 319 There are no competing interests to declare. 30
31 Accepted 32 33 34 35 36 37 38 39
40 Methods 41 42 43 44 45 46 47 48 49 50 51 Analytical 52 53 54 55 56 57 58 59 18 60 Page 19 of 28 Analytical Methods
1 2 3 4 320 References 5 6
7 321 1. J.B. Harborne, Plant Ecology, Second Edition, 1997; pp 132-155. 8 9 322 2. F. Chen, H.L. Li, Y.F. Tan, W.W. Guan, J.Q. Zhang, Y.H. Li, Y.S. Zhao, Z.M. Qin, Molecules . 2014, 10 11 323 19 , 4510-4523. 12 13 14 324 3. T. Kakegawa, S. Takase, E. Masubuchi, K. Yasukawa, Evid Based Complement Alternat Med. 15 16 325 2014, 2014 , 204797. 17 18
19 326 4. H.F. Li, Y.H. Li, Y. Wang, N. Wei, Y.F. Tan, J.Q. Zhang, Chin J Experiment Tradi Med Formula , 20 21 327 2014, 20 , 236-244. Manuscript 22 23 328 5. H. Zhang, L.W. Xu, P. Wu, X.Y. Wei, J Trop Subtrop Botany , 2014 , 22, 89-92. 24 25 26 329 6. Gao Y, McLuckey SA, J Mass Spectrom, 2012 , 47, 364-369. 27 28 330 7. Gao Y, McLuckey SA, Rapid Commun Mass Sp , 2013 , 27, 249-257. 29 30 331 8. Gao Y, Yang J, Cancilla MT, Meng F, McLuckey SA, Anal Chem , 2013 , 85, 4713-4720. 31 Accepted 32 33 332 9. Webb IK, Gao Y, Londry FA, McLuckey SA, J Mass Spectrom , 2013 , 48, 1059-1065. 34 35
36 333 10. Tan YF, Li HL, Li YB, Li YH, Lai WY, Wang Y, Chen F, Kang SL, Chin J Experi Tradi Med Formula , 37 38 334 2015, 21, 37-40. 39
40 335 11. F. Chen, H.L. Li, Y.H Li, Y.F. Tan, J.Q. Zhang, Chem Cent J . 2013 , 7, 131. Methods 41 42 43 336 12. Y.H. Li, F. Chen, J.F. Wang, Y. Wang, J.Q. Zhang, T. Guo, Chem Cent J . 2013 , 7, 134. 44 45 337 13. F. Chen,; H.L. Li, Y.F. Tan, W.W. Guan, J.Q. Zhang, Y.H. Li, Y.S. Zhao, Z.M. Qin, Molecules . 2014 , 46 47 48 338 19, 4510-4523 49 50 339 14. M. Petrović,; M.D. Hernando, M.S. Díaz-Cruz, D. Barceló, J Chromatogr A . 2005 , 1067, 1-14. 51 Analytical 52 340 15. S. Ito, K. Tsukada, J Chromatogr A . 2002 , 943, 39-46. 53 54 55 341 16. N. Cimetiere, I. Soutrel, M. Lemasle, A. Laplanche, A. Crocq, Environ Technol . 2013 , 34, 56 57 342 3031-3041. 58 59 19 60 Analytical Methods Page 20 of 28
1 2 3 4 343 17. H.L. Zhai, Q. Li, H. Wang, D.J. Liang, Y.B. Zeng, C.H. Cai, W.L. Mei, H.F. Dai, J Trop Biology , 5 6 344 2014 , 5, 188-193 7 8 9 345 18. Y.F. Deng, L.N. Feng, H. Luo, Chin Pharm J . 2010 , 45, 1593-1596. 10 11 346 19. Chinese Pharmacopoeia Commission. Pharmacopoeia of the People’s Republic of China , 12 13 14 347 People’s Medical Publishing House, Beijing, 2005 ; vol I, pp 270–271, 15 16 348 20. http://www.botanical.com/botanical/mgmh/g/galang01.html 17 18 19 20 21 Manuscript 22 23 24 25 26 27 28 29 30
31 Accepted 32 33 34 35 36 37 38 39
40 Methods 41 42 43 44 45 46 47 48 49 50 51 Analytical 52 53 54 55 56 57 58 59 20 60 Page 21 of 28 Analytical Methods
1 2 3 4 349 Figure legends 5 6 350 Figure 1 Chemical structures of interest occurring in A. officinarum aerial parts and 7 8 9 351 rhizomes. These chemicals are given ID numbers in parentheses. 10 11 12 352 Figure 2 Cartoon for different sampling periods for A. officinarum aerial parts and 13 14 353 rhizomes. 15 16 17 354 18 Figure 3 Product ion spectra of protonated phytochemicals of interest. 19 20 21 355 Figure 4 Representative chromatograms for seventeen reference standards (upper Manuscript 22 23 356 panel, the concentration level was 2000 ng/mL) and aerial parts samples (bottom 24 25 26 357 panel, harvested at August 16, 2014 with sample no. S_11 ), as well as rhizomes 27 28 358 samples (middle panel, harvested at March 29, 2014 with sample no. S_03 ) of A. 29 30 359 officinarum . 31 Accepted 32 33 34 360 Figure 5 Content sampling period curves for seventeen phytochemicals. 35 36 37 38 39
40 Methods 41 42 43 44 45 46 47 48 49 50 51 Analytical 52 53 54 55 56 57 58 59 21 60 Analytical Methods Page 22 of 28
1 2 3 Figure 1 4 5 O O 6 O O
7 HO 8 9 Nootkatone (C-01) Yakuchinone A (C-02) Diarylheptanoid (C-03) 10 11 OH OH O OH HO O H CO OCH 12 3 3 OH 13 HO OH HO OH OH O 14 Hannokinol (C-04) Hexahydrocurcumin (C-05) Galangin (C-06) OCH 15 3 OH OH 16 OH HO O HO O HO O 17 18 OH OH O OH O OH O 19 Pinocembrin (C-07) Isorhamnetin (C-08) Luteolin (C-09) 20 OH OH 21 OH OH OH Manuscript 22 HO O HO O HO O 23 O-rutinose OH 24 OH O OH O OH O 25 Rutin (C-10) Apigenin (C-11) Quercetin(C-12) 26 OCH 3 27 HO O HO O O O 28 OH O OH O OH O 29 Acacetin (C-13) Chrysin (C-14) Tectochrysin (C-15)
30 OH O
31 O O HO O HO O Accepted 32 OH OH OH 33 OH O OH O OH O 34 Izalpinin (C-16) Kaempferol (C-17) Kaempferide (C-18) 35 36 37 38 39
40 Methods 41 42 43 44 45 46 47 48 49 50 51 Analytical 52 53 54 55 56 57 58 59 22 60 Page 23 of 28 Analytical Methods
1 2 3 Figure 2 4 5 6 7 8 9 10 11 12 S 01 S 02 S 03 S 04 S 05 13 _20140219 _20140311 _20140329 _20140414 _20140429 14 15 16 17 18 19 20 21 Manuscript 22 23 S 06 S 07 S 08 S 09 24 _20140515 _20140530 _20140615 _20140630 25 26 27 28 29 30
31 Accepted 32 33 34 35 S 10 S 11 S 12 _20140715 _20140816 _20140831 36 37 38 39
40 Methods 41 42 43 44 45 46 47 48 49 50 51 Analytical 52 53 54 55 56 57 58 59 23 60 Analytical Methods Page 24 of 28
1 2 3 Figure 3 4 5 100 100 81.1 121.1 149.1 Max. 3.2e7 cps. Max. 5.3e6 cps. 6 111.1 163.0 303.0 465.1 135.1 93.1 [M+H] + [M+H] + 7 50 69.1 50 177.0 219.2 611.2 8 C-01 449.0 C-10 9 0 0 100 100 10 137.0 Max. 1.1e8 cps. 153.0 [M+H] +Max. 1.5e7 cps. 119.1 11 [M+H] + 271.1 50 50 145.0 313.2 12 163.0 295.1 C-02 243.1 C-11 13 0 0 14 100 100 105.0 Max. 7.8e6 cps. Max. 1.7e6 cps. 303.1 15 + + 50 91.0 133.0 [M+H] 50 [M+H] 16 263.0 137.1 153.0 229.1 257.0 117.0 159.0 C-03 285.0 C-12 17 0 245.0 0 18 100 100 256.3 Max. 4.9e6 cps. 242.0 Max. 3.4e6 cps. 19 [M+H] + 50 318.3 50 20 133.1 270.0 [M+H] + 102.1 285.0 21 300.3 152.9 212.2 C-04 C-13 Manuscript 22 0 0 100 100 23 357.2 Max. 5.6e5 cps. 152.9 Max. 8.0e7 cps. [M+H] + 103.0 [M+H] + 24 50 50 69.1 129.0 255.1 25 177.0 375.2 C-05 C-14 26 0 0 100 100 27 Relative intensity (cps, %) 153.0 Max. 3.7e5 cps. 226.1 Max. 6.3e7 cps. 105.1 + 28 [M+H] + [M+H] 50 141.0 50 269.1 69.1 197.0 271.2 29 167.0 254.1 C-06 129.0 197.0 C-15 30 0 0
31 100 100 Accepted Max. 1.2e6 cps. Max. 1.5e8 cps. 131.0 152.9 242.0 32 [M+H] + [M+H] + 105.0 50 257.1 50 33 166.9 285.1 196.0 270.0 173.0 C-07 91.1 124.0 C-16 34 0 0 35 100 100 Max. 7.8e4 cps. Max. 1.1e7 cps. 36 152.9 302.2 121.1 153.0 + 229.0 [M+H] + [M+H] 37 50 285.0 317.2 50 93.1 287.2 217.1 137.1 69.1 165.0 213.0 38 C-08 258.0 C-17 39 0 0 100 100 Max. 2.6e6 cps. 40 153.0 Methods + 152.9 230.0 Max. 1.5e7 cps. [M+H] 258.0 135.1 + 41 50 287.1 50 286.0 [M+H] 135.0 301.0 42 117.0 161.1 164.9 201.0 269.0 240.9 C-09 C-18 43 0 0 44 m/z, Da 45 46 47 48 49 50 51 Analytical 52 53 54 55 56 57 58 59 24 60 Page 25 of 28 Analytical Methods
1 2 3 Figure 4 4 5 C-02 100 C-15 6 Max. 5.4e6 cps. Standards C-13 C-01 7 C-07 C-03 8 C-16 9 50 C-09 C-06 10 C-11 C-14 C-05 C-08 11 C-10 C-12 C-17 C-18 12 0
13 100 C-14 Max. 2.0e6 cps. 14 Rhizomes-S_03 15 C-07 16 C-06 C-13 50 C-05 17 C-08 C-17 C-18 18 C-12 C-10 C-11 C-02 C-03 19 C-09 C-16 20 0 C-06
21 intensity (cps, Relative %) 100 Max. 4.2e6 cps. Manuscript Aerial parts-S_11 C-07 22 C-18 23 C-13
24 50 C-05 C-14 25 26 C-11 C-08 C-02 C-03 C-10 C-17 C-16 27 0 28 29 0 5 10 15 20 30 Time (min)
31 Accepted 32 33 34 35 36 37 38 39
40 Methods 41 42 43 44 45 46 47 48 49 50 51 Analytical 52 53 54 55 56 57 58 59 25 60 Analytical Methods Page 26 of 28
1 2 3 Figure 5a 4 5 0.60 3500 6 Contents ( g∙∙∙g 1) Aerial parts Contents ( g∙∙∙g 1) Aerial parts 7 8 0.30 1750
9 0 S-01 S-02 S-03 S-04 S-05 S-06 S-07 S-08 S-09 S-10 S-11 S-12 0 S-01 S-02 S-03 S-04 S-05 S-06 S-07 S-08 S-09 S-10 S-11 S-12 10 11 2.00 6000
C 01 Rhizomes C 06 Rhizomes 12 4.00 20000 2.50 140 13 Contents ( g∙∙∙g 1) Aerial parts Contents ( g∙∙∙g 1) Aerial parts 14 1.25 70 15
16 0 0
17 18 5.00 400
C 02 Rhizomes C 07 Rhizomes 19 10.0 800 8.00 40.0 20 Contents ( g∙∙∙g 1) Aerial parts Contents ( g∙∙∙g 1) Aerial parts 21 4.00 20.0 Manuscript 22 23 0 0 24 25 35.0 70
C 03 Rhizomes C 08 Rhizomes 26 70.0 140 250 2.50 27 Contents ( g∙∙∙g 1) Aerial parts Contents ( g∙∙∙g 1) Aerial parts
28 125 1.25 29 30 0
31 Accepted 32 800 2.50 C 05 Rhizomes C 09 Rhizomes 33 1600 5.00
34 35 36 37 38 39
40 Methods 41 42 43 44 45 46 47 48 49 50 51 Analytical 52 53 54 55 56 57 58 59 26 60 Page 27 of 28 Analytical Methods
1 2 3 4 5 6 7 Figure 5b
8 14.0 12.0 70.0 9 Contents ( g∙∙∙g 1) Aerial parts Contents ( g∙∙∙g 1) Aerial parts Contents ( g∙∙∙g 1) Aerial parts
10 7.0 6.0 35.0 11
12 0 S-01 S-02 S-03 S-04 S-05 S-06 S-07 S-08 S-09 S-10 S-11 S-12 0 S-01 S-02 S-03 S-04 S-05 S-06 S-07 S-08 S-09 S-10 S-11 S-12 0 S-01 S-02 S-03 S-04 S-05 S-06 S-07 S-08 S-09 S-10 S-11 S-12 13 14 20.0 25.0 22.5 15 C 10 Rhizomes C 11 Rhizomes C 12 Rhizomes 16 40.0 50.0 45.0 Manuscript 18.0 400 0.20 17 Contents ( g∙∙∙g 1) Aerial parts Contents ( g∙∙∙g 1) Aerial parts Contents ( g∙∙∙g 1) Aerial parts 18 9.0 200 0.10 19
20 0 0 0 21
22 20.0 300 0.15
23 Accepted C 13 Rhizomes C 14 Rhizomes C 15 Rhizomes 24 40.0 600 0.30 25 0.90 2.50 450 Contents ( g∙∙∙g 1) Aerial parts Contents ( g∙∙∙g 1) Aerial parts Contents ( g∙∙∙g 1) Aerial parts 26 27 0.45 1.25 225 28 29 0 0 0
30 Methods 31 4.50 3.00 3000
32 C 16 Rhizomes C 17 Rhizomes C 18 Rhizomes 33 9.00 6.00 6000 34 35 36 37 38 Analytical 39 40 41 27 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Analytical Methods Page 28 of 28
1 2 3 4 5 6 7 Table 1 Validation parameters 8 LOD LOQ Precision (Intra-/inter-day; RSD, %) Recovery (mean %; RSD %) 9 Analytes Linearity (range, r, weighting factor) 10 (ng∙mL -1) (ng∙mL -1) Aerial parts Rhizomes Aerial parts Rhizomes 11 C-01 0.05 1 1-100, r=0.9963, 1/x 2 2.37%; 9.96% 2.28%; 3.41% 105 (2.61%) 102 (2.90%) 12 2 13 C-02 0.05 1 1-100, r=0.9933, 1/x 4.53%; 7.51% 4.81%; 5.57% 85.9 (0.46%) 91.5 (1.15%) 2 14 C-03 0.01 0.1 1-100, r=0.9943, 1/x 4.83%; 4.45% 5.04%; 4.08% 86.7 (2.54%) 85.9 (4.28%) 15 C-05 1 2 2-2000, r=0.9975, 1/x 5.21%; 6.20% 9.28%; 8.76% 91.6 (1.98%) 88.1 (9.66%) 16 C-06 0.01 2 2-1000, r=0.9991, 1/x 8.38%; 8.01% 8.40%; 5.42% 98.1 (5.77%) 90.8 (9.32%) Manuscript 17 2 18 C-07 0.05 0.5 1-100, r=0.9936, 1/x 3.27%; 4.04% 3.24%; 4.38% 88.9 (1.88%) 87.6 (2.32%) 19 C-08 1 2 2-1000, r=0.9993, 1/x 2.44%; 3.22% 7.86%; 6.21% 102 (3.68%) 94.2 (5.03%) 20 C-09 1 2 2-2000, r=0.9985, 1/x 7.11%; 5.89% 5.64%; 5.80% 110 (4.31%) 109 (2.98%) 21 C-10 1 2 2-2000, r=0.9992, 1/x 3.85%; 4.12% 4.04%; 4.32% 106 (6.56%) 103 (10.6%) 22 C-11 0.5 1 1-100, r=0.9971, 1/x 2 4.36%; 3.36% 2.07%; 2.90% 112 (1.98%) 95.2 (4.58%)
23 Accepted 24 C-12 5 10 10-2000, r=0.9989, 1/x 7.23%; 8.12% 5.03%; 6.63% 97.6 (7.85%) 93.4 (5.48%) 25 C-13 0.05 0.1 1-100, r=0.9939, 1/x 2 4.15%; 2.94% 7.78%; 2.27% 111 (3.19%) 95.2 (3.96%) 26 C-14 0.5 1 1-100, r=0.9947, 1/x 2 2.76%; 4.44% 6.30%; 14.8% 94.5 (2.98%) 91.6 (2.78%) 27 2 28 C-15 0.05 0.1 1-100, r=0.9904, 1/x 2.02%; 2.31% 3.98%; 3.29% 86.0 (0.84%) 85.5 (2.67%) 29 C-16 0.05 0.1 1-100, r=0.9943, 1/x 2 6.98%; 6.38% 5.28%; 5.13% 89.0 (3.86%) 87.0 (1.18%)
30 C-17 2 5 2-2000, r=0.9980, 1/x 5.39%; 6.82% 8.41%; 6.82% 106 (7.57%) 102 (4.47%) Methods 31 C-18 0.0141 1.41 1.41-1410, r=0.9987, 1/x 2.30%; 9.30% 7.27%; 7.55% 103 (5.47%) 112 (3.57%) 32 33 34 35 36 37 38 Analytical 39 40 41 28 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60