Pharmacokinetics of Daikenchuto, a Traditional Japanese Medicine (Kampo) After Single Oral Administration to Healthy Japanese Volunteers

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Pharmacokinetics of daikenchuto, a traditional Japanese medicine (Kampo) after single oral administration to healthy Japanese volunteers

Masaya Munekage, Hiroyuki Kitagawa, Kengo Ichikawa, Junko Watanabe, Katsuyuki Aoki,

Toru Kono, Kazuhiro Hanazaki

Department of Surgery, Kochi Medical School, Nankoku, Kochi, Japan (M.M., H.K., K.I., K.H); Downloaded from

Tsumura Laboratories, TSUMURA & CO., Ami, Ibaraki, Japan (J.W.); Pharmaceutical & Quality

Research Department, TSUMURA & CO., Ami, Ibaraki , Japan (K.A.); Division of dmd.aspetjournals.org Gastroenterologic and General Surgery, Department of Surgery, Asahikawa Medical University,

Hokkaido, Japan (T.K.).

at ASPET Journals on September 26, 2021

Copyright 2011 by the American Society for Pharmacology and Experimental Therapeutics. DMD Fast Forward. Published on July 1, 2011 as DOI: 10.1124/dmd.111.040097 This article has not been copyedited and formatted. The final version may differ from this version.

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Running title: Pharmacokinetics study of daikenchuto

Address correspondence to: Kazuhiro Hanazaki, M.D., Ph.D. Department of Surgery, Kochi

Medical School, Oko-cho kohasu, Nankoku-shi, Kochi 783-8505, Japan. E-mail: [email protected] , Phone: 81-88-880-2370, Fax: 81-88-880-2371

Number of text pages: 17 Downloaded from

Number of Tables: 1

Number of Figures: 2 dmd.aspetjournals.org Number of References: 17

Number of Words:

Abstract: 199 at ASPET Journals on September 26, 2021

Introduction: 377

Results and Discussion: 855

ABBREVIATIONS: TJ-100, daikenchuto; HAS, hydroxy-α-sanshool; HBS, hydroxy-β-sanshool; 6S, [6]-shogaol; 10S, [10]-shogaol; GRB1, ginsenoside Rb1; GRG1, ginsenoside Rg1; HPLC, high-performance liquid chromatography; LC, liquid chromatography;

MS, mass spectrometry; MS/MS, tandem mass spectrometry

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ABSTRACT

The pharmacokinetics of Daikenchuto (TJ-100), a pharmaceutical grade traditional Japanese medicine, were investigated in healthy Japanese volunteers after single oral administration of 2.5,

5 and 10g doses. Six ingredients [hydroxy-α-sanshool (HAS), hydroxy-β-sanshool (HBS),

[6]-shogaol (6S), [10]-shogaol (10S), ginsenoside Rb1(GRB1) and ginsenoside Rg1(GRG1)] of

TJ-100 were determined by using LC-MS/MS. The results indicated that HAS an i ngredient derived from Zanthoxylum fruit, exhibited the highest plasma concentration among the 6 Downloaded from ingredients investigated. The plasma concentrations of HA S, HBS, 6S an d 10S reached maximum concentration (approximately 400, 80, 0.14 and 0.6 ng/mL, respectively after 5 g dmd.aspetjournals.org administration of T J-100) within 30 min after administration and the mean half-life was approximately 2 hr. Thus, these compounds were rapidly absorbed and eliminated. The plasma concentration of GRB1 reached maximum concentration (2 ng/mL after 5 g administration of at ASPET Journals on September 26, 2021

TJ-100) at approximately 4 hr after administration and the half life of GRB1 was approximately

40hr. The plasma concentration of GRG1 was extremely low (< 0.023 ng/mL). The pharmacokinetics of HAS, HBS, 6S, and 10S, were linear within the range of 2.5 g to 10 g/day of TJ-100. On the other hand, the kinetics of GRB1 and GRG1 were not proportional to dosage, and plateauing was observed.

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INTRODUCTION

Daikenchuto (TJ-100) is a pharmaceutical grade traditional Japanese medicine consisting of Japanese pepper

(zanthoxylum fruit), processed ginger, and ginseng with maltose as an additive. In Japan, TJ-100 is routinely used in the modern medical care system as a prescription drug for the treatment of various gastrointestinal disorders, including postoperative ileus, postoperative intestinal paralysis and adhesive bowel obstructions [Itoh et al., 2002;

Kono et al., 2009]. Results from a recent clinical pharmacological study have shown that TJ-100 accelerates intestinal transit in healthy humans [Manabe et al., 2010]. Furthermore, basic pharmacological studies have Downloaded from suggested that TJ-100 and its ingredients have a wide variety of biological effects, including prokinetic effect largely attributed to the activity of zantoxylum fruit [Tokita et al., 2007; Kawasaki et al., 2007; Jin et al., 2001], dmd.aspetjournals.org contraction and relaxation of intestinal smooth muscle [Kito et al., 2006], increase of intestinal blood flow due to the activity of ginger ingredients [Kono et al., 2008; Murata et al., 2002], anti-inflammatory effect [Kono et al., 2010], and suppression of bacterial translocation possibly mediated by ginseng ingredients [Yoshikawa et al., 2008]. at ASPET Journals on September 26, 2021

Estimating the factors involved in the absorption, distribution, metabolism, and excretion (ADME) of the multiple constituents in a Kampo medicine is an ongoing challenge, and obtaining a consistent picture of its mechanism of action is difficult as well. To solve these problems, we have conducted preliminary clinical trials to obtain the profiles of compounds absorbed in the plasma and urine after a single oral administration of 15 g of TJ-100 in 4 healthy volunteers [Iwabu et al., 2010]. In that study, we constructed an LC-MS/MS analysis to detect as many compounds as possible, and successfully identified 44 ingredients of TJ-100. Twenty-three ingredients and their metabolites were detected in the plasma, indicating that a large number of TJ-100 ingredients were actually absorbed and metabolized.

Based on the results, we have developed assay methods for six compounds, hydroxy-α-sanshool, hydroxyl-β-sanshool, [6]-shogaol, [10]-shogaol, ginsenoside Rb1 and ginsenoside Rg1 using the following criteria: 1) Plasma concentration of the compounds have enough strength to establish

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DMD #040097 the validated determination methods, 2) Enables the measurement at least one compound per botanical raw material, 3) Pharmacological activities of the selected compounds have been previously reported.

In this study, we focused on the pharmacokinetics of the ingredients of TJ-100.

MATERIALS AND METHODS

Chemicals and Reagents. TSUMURA Daikenchuto Extract Granules was manufactured by

TSUMURA & CO. Fifteen grams of TSUMURA Daikenchuto Extract Granules contains 1.25 g Downloaded from of a dried extract prepared from a mixture of three herbs (5.0 g of processed ginger, 3.0 g of ginseng and 2.0 g of Japanese pepper), and 10.0 g of maltose. Extra pure grade acetonitrile and dmd.aspetjournals.org HPLC grade acetic acid were purchased from Wako Pure Chemical Industries, Ltd. (Osaka,

Japan). Asiaticoside was purchased from Extrasynthese (Genay, France) and nonivamide from

Enzo Life Sciences, Inc. (Farmingdale, NY). Water was purified using a pure water supply at ASPET Journals on September 26, 2021 system (MILLI-Q, Nihon Millipore Ltd., Japan). Oasis HLB μElution Plate (Nihon Waters K.K,

Japan) was used as the solid phase extraction (SPE) plate. The isolation, synthesis and identification of the 6 authentic standards of TJ-100 ingredients were described in the previous paper [Iwabu et al., 2010]. The structural formulas of the authentic standards are provided in

Supplemental Figure 1.

Clinical Trial Design, Administration, and Sample Collection. This was a randomized, open label, three-arm, three-period study (Japic CTI-101114) conducted at the Kochi Medical School.

Nineteen subjects participated in this study but three subjects were excluded from the pharmacokinetic analysis because they did not meet the eligibility criteria. Safety endpoint was evaluated in 19 subjects. Participants were fasted from 12 hours before and 4 hours after administration. All foods and drinks (including spices) containing ginseng, Japanese pepper, and

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DMD #040097 ginger were strictly prohibited from 3 days prior to dispensing the study medication until completion of each treatment phase. Blood samples (7 mL each) were collected from the medial cubital vein into evacuated tubes containing heparin just before and at 0.25, 0.5, 1, 2, 3, 4, 8, 12,

24 and 48 hours after administration and were immediately centrifuged (1700×g, 10 min).

Plasma fractions were stored at -20°C until analysis. All study procedures were conducted in accordance with the ethical principles of the Declaration of Helsinki, consistent with the Good

Clinical Practice guidelines, and approved by the Institutional Review Board at Kochi Medical Downloaded from

School Hospital. Subjects gave their written informed consent before participating in the study.

Determination of plasma concentration of TJ-100 ingredients. The concentrations of six dmd.aspetjournals.org TJ-100 ingredients were determined according to the method reported by Iwabu et al. [Iwabu et al., 2010] with modification. In brief, 200 µL of the plasma samples were mixed with 200 µL of

0.1 mol/L hydrochloric acid, 10 µL of IS solution (schizandrin: 10 ng/mL for HAS and HBS, at ASPET Journals on September 26, 2021 nonivamide: 10 ng/mL for 6S and 10S, and asiaticoside: 100 ng/mL for GRB1 and GRG1) and

10 µL of water/acetonitrile (7:3, v/v). The resulting mixture was loaded onto an Oasis HLB

µElution 96-well plate (30 µm), which was washed with 200 µL of methanol and 200 µL of water. The plate was then washed with 200 µL of water and eluted with 50 µL of acetonitrile. A

40-µL portion of the eluate was mixed with 60 µL of water and a 20-µL portion was injected into the Agilent 1200 series HPLC system (Agilent Technologies, Inc.) with Sciex API 5000 (AB

Sciex Pte. Ltd.). YMC-Pack ODA-AQ(3 μm, 150 × 2.0 mm I.D., YMC Co., Ltd) was used for analyzing HAS, HBS, GRB1 and GRG1; YMC-Pack ODA-AQ(3 μm, 50 × 2.0 mm I.D., YMC

Co., Ltd) was used for analyzing 6S and 10S. The analytical methods were validated according to the FDA Guidance for Industry Bioanalytical Method Validation. These analytical methods and their validation data are summarized in Supplemental data (Supplemental Table1, 2, and 3.)

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Pharmacokinetic Analysis. Pharmacokinetic parameters were estimated using the WinNonlin version 5.2 (Pharsight, Corporation, Mountain View, CA). Experimentally observed values of maximum concentration (Cmax) and time to maximum concentration (tmax) after TJ-100 administration were used for the analysis . The area under plasma concentration-time curve from zero to time t (AUC0-last) was calculated from time zero to last detected time. Apparent elimination half-life (t1/2) was calculated divided by loge2/ke where ke means the terminal Downloaded from elimination rate constant. Cmax and AUC0-last are presented as mean ± SD. Apparent elimination half-life and tmax are presented as median with range.

Evaluation of Linearity of Dosage-Exposure Relations. The dose-proportionality was analyzed dmd.aspetjournals.org via a power model fitted as a linear mixed effect model which included the period effect and random subject effect (equation 1). () += ββ ( ) +++ επ

ln PK ijk 10 ln Doseij akj ijk . (1) at ASPET Journals on September 26, 2021

The ln(PK)ijk means natural logarithm of the AUC0-last or Cmax in group i (i=A,B,C) during period j (j=1,2,3) in the subject (k=1,2…ni). The Doseij is the administrated dose (g) of the test drug in group i during period j. The πj is the term to express the fixed period effect, and the ak is the term to express the subject specific effect which is assumed to be normally distributed around mean 0 with inter-subject standard deviation of σa. The εijk is the error term with mean 0 and standard deviation σ. The β1 is the parameter to be used for dose-proportionality evaluation and β0 is the intercept term. When the value of β1 significantly differs from 1, the hypothesis of dose-proportionality is rejected. Evaluation of linearity of dosage-exposure relations analysis were conducted using SAS9.1.3 (SAS Institute Inc., 2004).

RESULTS AND DISCUSSION

Study subjects and adverse effects.

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Sixteenth healthy subjects [age: 20-37 years, height: 150.7-189.7 cm, weight: 44.5-72.5 kg, and body mass index (BMI): 18.5-24.1 kg/m2] were included in the pharmacokinetic analysis in this study. There was an uneven gender ratio in the study (13 men and 3 women). Six adverse experiences were observed in four patients, all of which were deviations from normal reference laboratory values. However, these deviations were judged by the principal investigator as physiological changes, thus causal relationship with TJ-100 was denied.

Pharmacokinetics in human subjects. Downloaded from

The plasma concentration–time profiles in human subject for each dose (2.5, 5 and 10 g) are shown in Figure 1 and the pharmacokinetic parameters of the 6 main TJ-100 ingredients are dmd.aspetjournals.org summarized in Table 1.

HAS, which is a constituent of zanthoxylum fruit, exhibited the highest plasma concentration among the six compounds measured. The plasma concentration of HAS reached maximum at ASPET Journals on September 26, 2021 concentration within 30 min after administration and its median half-life was 1.6-1.7 h, indicating its rapid absorption and elimination. The plasma concentration of HBS showed the parallel pattern to the plasma concentration of the HAS. It has been reported that both HAS and

HBS have unique biological activities and play an important role in the efficacy of TJ-100

[Hayakawa et al., 1999; Tokita et al., 2007 a; Tokita et al., 2007 b]. Various in vitro experimental systems have demonstrated that HAS exerts its pharmacological effects in the range of 0.1 to 100

μM [Koo et al., 2007; Jin et al., 2001]. In this study, the maximum concentration of HAS in plasma was 0.76 to 2.66 μM. Taken together, these result suggest that HAS absorption into the blood circulation may contribute to the pharmacological effects of orally administered TJ-100.

These findings are intriguing in light of the fact that several studies strongly suggest the localized effect of TJ-100, that is, TJ-100 ingredients mainly affect the mucosal, nervous, and smooth

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DMD #040097 muscle cells in the intestinal lumen. For example, intraduodenal and intrajejunal TJ-100 administration induced phasic contractions in the duodenum and proximal jejunum, respectively, and those contractions migrated distally [Shibata et al., 1999; Jin et al., 2001]. No motor response was obtained proximal to the site of administration, suggesting that direct contact with the intestinal mucosa is essential for TJ-100 to exert its effects. The results of this pharmacokinetic study underscores the importance of clarifying whether HAS has systemic biological effects. Downloaded from

The plasma concentrations of 6S and 10S, the constituents of processed ginger, were significantly lower than those of zanthoxylum fruit constituents. The values of Cmax of 6S were dmd.aspetjournals.org observed within 15 min after administration and its elimination half-life was 0.3-0.6 hr.

Meanwhile, 10S reached maximum plasma concentrations within 30 min after administration and its elimination rate was slightly lower than that of 6S at 0.8-1.5 hr. Although the plasma at ASPET Journals on September 26, 2021 concentrations of 6S and 10S were extremely low, it is possible that their intestinal concentrations were sufficient to exert an effect in the intestinal lumen.

Conversely, the plasma concentration of GRB1, which is one of the ginseng constituents, gradually increased with a tmax of approximately 4 hr followed by maximum plasma concentrations. Thereafter, GRB1 was gradually eliminated with a half-life of approximately 40 hr. The tmax and the half-life of GRB1 indicate a relatively slow increase and gradual disappearance. The plasma concentrations of GRB1 are therefore expected to increase to levels higher than the concentrations observed in this study when TJ-100 is administered repeatedly.

From the present data, we anticipate that five half-lives can be measured once a steady-state blood concentration of GRB1 is achieved after about 1-2 weeks of repeated administration.

Although there are a few adverse reports related to the long term use of TJ-100 in Japan,

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DMD #040097 repeated-dose pharmacokinetic trials will be required to clarify this point. In addition, the information on plasma concentration of GRB1 after repeated TJ-100 is useful to discuss the possible involvement of the ingredient in TJ-100’s clinical effect.

The calculation of elimination half-life of GRG1 was possible in only a few subjects because the plasma concentration of GRG1 was low and less than the lower limit of quantitation (0.01 ng/mL) in many of the subjects.

Analysis of Linearity in Dosage-Exposure Relations. Downloaded from

The dose-proportionality of AUC0-last is graphically displayed in Figure 2. The β1 of HAS, HBS,

6S, and 10S for AUC0-last was close to one for doses ranging between 2.5 g and 10 g of TJ-100. dmd.aspetjournals.org These result suggested that the pharmacokinetics of HAS, HBS, 6S, and 10S were linear within the dose range of 2.5-10 g/day of TJ-100. However, a 90% confidence interval of the β1 value for

GRB1 did not include 1. While the AUC0-last value of GRB1 was very limited because the last at ASPET Journals on September 26, 2021 observation time points were only 50% of the estimated half-life, the result inferred a plateauing phenomenon of GRB1. The analysis for GRG1 has not been conducted because only a few subjects had detectable plasma concentrations. The result of dose-proportionality for Cmax was similar to that of AUC0-last.

The present study provides a strong basis for future ADME studies to help create a consistent kinetic profile and to generate robust evidence on the pharmacological activity of TJ-100.

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ACKNOWLEDGEMENT

We thank Yuka Takezaki (Department of Surgery, Kochi Medical School) for her help in the clinical trial and technical assistance, Kenichiro Hayashi (Medi-Chem Business Segment,

Mitsubishi Chemical Medience Corporation, Kumamoto, Japan) for the analysis of TJ-100 constituents, and Hideaki Uehara (Kampo Research Planning Department, Sales and Marketing

Division., TSUMURA & CO., Tokyo, Japan) for the evaluation of the linearity and sensitivity analysis on this study. Downloaded from

dmd.aspetjournals.org at ASPET Journals on September 26, 2021

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AUTHORSHIP CONTRIBUTIONS

Participated in research design: Munekage, Kitagawa, Ichikawa, Kono, Hanazaki

Conducted experiments: Munekage, Kitagawa, Ichikawa, Kono, Hanazaki

Performed data analysis: Munekage, Kitagawa, Ichikawa, Watanabe, Kono, Hanazaki

Contributed to the writing of the manuscript: Munekage, Kitagawa, Ichikawa, Watanabe, Aoki,

Kono, Hanazaki

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REFERENCES

Hayakawa T, Kase Y, Saito K, Hashimoto K, Ishige A, Komatsu Y, and Sasaki H (1999)

Pharmacological studies of the effect of Dai-kenchu-to on spontaneous contraction of

isolated rabbit jejunum. J Smooth Muscle Res 35(2):55-62.

Itoh T, Yamakawa J, Mai M, Yamaguchi N, and Kanda T (2002) The effect of the herbal

medicine dai-kenchu-to on post-operative ileus. J Int Med Res 30:428–432.

Jin XL, Shibata C, Naito H, Ueno T, Funayama Y, Fukushima K, Matsuno S, and Sasaki I Downloaded from

(2001) Intraduodenal and intrajejunal administration of the herbal medicine,

dai-kenchu-tou, stimulates small intestinal motility via cholinergic receptors in conscious dmd.aspetjournals.org dogs. Dig Dis Sci 46(6):1171-6.

Iwabu J, Watanabe J, Hirakura K, Ozaki Y, and Hanazaki K (2010) Profiling of the Compounds

Absorbed in Human Plasma and Urine after Oral Administration of a Traditional at ASPET Journals on September 26, 2021

Japanese (Kampo) Medicine, Daikenchuto. Drug Metab Dispos, 38 (1):2040-2048.

Kawasaki N, Nakada K, Nakayoshi T, Furukawa Y, Suzuki Y, Hanyu N, and Yanaga K (2007)

Effect of Dai-kenchu-to on gastrointestinal motility based on differences in the site and

timing of administration. Dig Dis Sci 52(10):2684-94.

Kito Y and Suzuki H (2006) Effects of Dai-kenchu-to on spontaneous activity in the mouse

small intestine. J Smooth Muscle Res 42(6):189-201.

Koo JY, Jang Y, Cho H, Lee CH, Jang KH, Chang YH, Shin J, and Oh U (2007)

Hydroxy-alpha-sanshool activates TRPV1 and TRPA1 in sensory neurons. Eur J

Neurosci 26(5):1139-47.

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Kono T, Koseki T, Chiba S, Ebisawa Y, Chisato N, Iwamoto J, and Kasai S. (2008) Colonic

vascular conductance increased by Daikenchuto via calcitonin gene-related peptide and

receptor-activity modifying protein 1. J Surg Res 150(1):78-84.

Kono T, Kanematsu T, and Kitajima M (2009) Exodus of Kampo, traditional Japanese medicine,

from the complementary and alternative medicines: is it time yet? Surgery 146:837–840.

Kono T, Kaneko A, Hira Y, Suzuki T, Chisato N, Ohtake N, Miura N, and Watanabe T. (2010)

Anti-colitis and -adhesion effects of daikenchuto via endogenous adrenomedullin Downloaded from

enhancement in Crohn's disease mouse model. J Crohns Colitis 4(2):161-70.

Manabe N, Camilleri M, Rao A, Wong BS, Burton D, Busciglio I, Zinsmeister AR, and Haruma dmd.aspetjournals.org K (2010) Effect of daikenchuto (TU-100) on gastrointestinal and colonic transit in

humans. Am J Physiol Gastrointest Liver Physiol 298:G970–G975.

Murata P, Kase Y, Ishige A, Sasaki H, Kurosawa S, and Nakamura T (2002) The herbal at ASPET Journals on September 26, 2021

medicine Dai-kenchu-to and one of its active components [6]-shogaol increase intestinal

blood flow in rats. Life Sci 70:2061–2070.

SAS Institute Inc. 2004. SAS OnlineDoc(r) 9.1.3. Cary, NC: SAS Institute

Shibata C, Sasaki I, Naito H, Ueno T, and Matsuno S (1999) The herbal medicine

Dai-Kenchu-Tou stimulates upper gut motility through cholinergic and

5-hydroxytryptamine 3 receptors in conscious dogs. Surgery126(5):918-24.

Tokita Y, Yuzurihara M, Sakaguchi M, Satoh K, and Kase Y. (2007 a) The pharmacological

effects of Daikenchuto, a traditional herbal medicine, on delayed gastrointestinal transit

in rat postoperative ileus. J Pharmacol Sci 104(4):303-10.

DMD Fast Forward. Published on July 1, 2011 as DOI: 10.1124/dmd.111.040097 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #040097

Tokita Y, Satoh K, Sakaguchi M, Endoh Y, Mori I, Yuzurihara M, Sakakibara I, Kase Y, Takeda

and S, Sasaki H (2007 b) The preventive effect of Daikenchuto on postoperative

adhesion-induced intestinal obstruction in rats. Inflammopharmacology 15(2):65-6.

Yoshikawa K, Kurita N, Higashijima J, Miyatani T, Miyamoto H, Nishioka M, and Shimada M

(2008) Kampo medicine "Dai-kenchu-to" prevents bacterial translocation in rats. Dig Dis

Sci 53(7):1824-1831.

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 26, 2021

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FOOTNOTES

This study was supported by a grant from TSUMURA & CO., Tokyo, Japan.

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LEGENDS FOR FIGURES

Figure 1 A semi-logarithmic plot of plasma concentrations of main TJ-100 ingredients in human healthy volunteers. The results of quantification of main TJ-100 ingredients in human plasma are shown. A: HAS, B: HBS, C: 6S, D: 10S, E: GRB1, F: GRG1. 2.5 g (closed circle), 5 g (closed square), 10 g (closed triangle). Values are means of the results ± S.D. (n = 15 or 16).

Downloaded from Figure 2 Relations between dosage and AUC0-last of HAS, HSB, 6S, 10S and GRB1 after oral administration of TJ-100 in healthy volunteers (n = 16). Each data represents geometric mean of AUC0-last. Solid line represents predicted curve given by power model fitting. HAS dmd.aspetjournals.org (closed circle), HBS(open circle), 6S (closed square), 10S (open square),GRB1(closed triangle). at ASPET Journals on September 26, 2021

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Table 1. Pharmacokinetic Parameters for Components of TJ-100

Dose AUC(0-last)* C * t † t † N max 1/2 max (g/day) (ng・hr/mL) (ng/mL) (hr) (hr) 2.5 15 349±136 209±100 1.65(1.01-2.48) 0.250(0.229-1.00) HAS 5 16 658±223 391±136 1.71(1.04-3.26) 0.258(0.233-0.633) 10 16 1290±329 664±165 1.62(1.46-3.22) 0.475(0.233-0.550) 2.5 15 66.3±35.0 42.2±25.5 1.14(0.478-1.71) 0.267(0.229-1.00) HBS 5 16 130±81.4 80.4±45.9 1.17(0.681-1.76) 0.258(0.233-0.633) 10 16 234±88.4 131±52.6 1.09(0.925-1.70) 0.475(0.233-0.550) 2.5 15 0.0306±0.0211 0.0762±0.0542 0.618(0.280-0.956) 0.233(0.229-0.550) a 6S 5 16 0.0751±0.0571 0.142±0.109 0.312(0.286-0.793) 0.242(0.233-0.500) Downloaded from 10 16 0.173±0.0907 0.262±0.125 0.429(0.302-0.526) 0.250(0.233-0.533) 2.5 15 0.397±0.372 0.290±0.157 1.46(0.463-4.66) 0.500(0.229-0.983) 10Sb 5 16 0.821±0.522 0.636±0.341 0.851(0.559-3.45) 0.317(0.233-3.00) 10 16 1.48±0.760 1.20±0.742 0.812(0.460-5.42) 0.483(0.233-1.90) 2.5 15 1.33±0.653 0.0504±0.0189 37.8(12.2-101) 4.00(2.98-8.00) dmd.aspetjournals.org GRB1 5 16 2.27±0.839 0.0744±0.0229 41.0(21.3-330) 4.02(1.98-12.0) 10 16 3.45±1.39 0.123±0.0466 40.1(20.3-5398)) 3.98(2.92-7.98) 2.5 15 0.191±0.241 0.0198±0.0320 27.8(2.07-53.5) 2.48(0.500-8.00) GRG1c 5 16 0.209±0.236 0.0209±0.00910 4.40(2.06-1390) 1.00(0.233-3.93) 10 16 0.113±0.0802 0.0230±0.0117 14.3(2.69-171) 1.96(0.983-23.7) at ASPET Journals on September 26, 2021 *：Data represent the means ± S.D. †：Data represent the median (range) a：The elimination half-life was calculated from 2 subjects at the dose of 2.5g, 10 subjects at the dose of 5 g, and 12 subjects at the dose of 10 g. b：The elimination half-life was calculated from 10 subjects at the dose of 2.5g, 15 subjects at the dose of 5 g, and 16 subjects at the dose of 10 g. c：The elimination half-life was calculated from 2 subjects at the dose of 2.5g, 3 subjects at the dose of 5 g, and 7 subjects at the dose of 10 g.

DMD Fast Forward. Published on July 1, 2011 as DOI: 10.1124/dmd.111.040097 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from dmd.aspetjournals.org at ASPET Journals on September 26, 2021 DMD Fast Forward. Published on July 1, 2011 as DOI: 10.1124/dmd.111.040097 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from dmd.aspetjournals.org at ASPET Journals on September 26, 2021 DMD #040097 Supplemental data

Title: Pharmacokinetics of daikenchuto, a traditional Japanese medicine (Kampo) after single oral administration to healthy Japanese volunteers

Authors: Masaya Munekage, Hiroyuki Kitagawa, Kengo Ichikawa, Junko Watanabe,

Katsuyuki Aoki, Toru Kono, Kazuhiro Hanazaki

Journal: Drug Metabolism Disposition DMD #040097 Supplemental data

Supplemental data

Supplemental Figure 1. Structural formulas of TJ-100 ingredients and ISs DMD #040097 Supplemental data

Supplemental Table 1. Summary of assay method （Zanthoxylum Fuit）

Analyte Hydroxy-α-sanshool（HAS）, Hydroxy-β-sanshool（HBS） Internal standard Schizandrin LC-MS/MS system API5000 (AB SCIEX) Biological sample and volume Human plasma 0.5 mL Calibration curve range 0.01 to 10 ng/mL

LC-MS/MS conditions Analytical column YMC-Pack ODA-AQ, 3 μm, 150 × 2.0 mm I.D. (YMC Co., Ltd.) Guard column YMC-Pack ODA-AQ, 3 μm, 10 × 2.0 mm I.D. (YMC Co., Ltd.) Mobile phase 0.2 vol% Acetic acid in water/acetonitrile (55:45, v/v) Flow rate 0.2 mL/min Column temp 40°C Autosampler temp 7°C Injection volume 20 µL Analytical time 15 min Ionization mode ESI (Electrospray ionization, Turbo ion spray probe), positive Scan mode MRM (multiple reaction monitoring) Monitoring ion hydroxy-α-sanshool m/z 264.3 → m/z 106.9 hydroxy-β-sanshool m/z 264.3 → m/z 106.9 Schizandrin m/z 433.4 → m/z 384.0

Optimized mass spectrometer acquisition parameters used for analyte quantitation CUR (psi) 40 GS1 (psi) 50 GS2 (psi) 60 TEM (℃) 600 CAD (psi) 8 CE (V) 23 CUR, Curtain Gas; GS, Ion source gas; TEM, Temperature; CAD, Collision Gas; CE, Collision energy.

DMD #040097 Supplemental data

Validation results Validation Items Results Hydroxy-α-sanshool No interfering Selectivity Hydroxy-β-sanshool No interfering Schizandrin (IS) No interfering 0.05 ng/mL 114.2 ± 7.4 % HAS Recovery 8 ng/mL 81.3 ± 6.7 % (Nominal concentration) 0.05 ng/mL 93.2 ± 8.4 % HBS 0.05, 8 ng/mL 8 ng/mL 85.2 ± 3.5 % Schizandrin (IS) 8 ng/mL 91.0 ± 2.1 % Correlation coefficient (r) 0.9955 to 0.9995 Linearity HAS LLOQ RE: -2.6 to 8.0 % Accuracy (Nominal concentration) Others RE: -14.0 to 15.0 % 0.01, 0.02, 0.05, 0.1, Correlation coefficient (r) 0.9965 to 0.9995 0.5, 1, 5, 10 ng/mL HBS LLOQ RE: -2.7 to 7.0 % Accuracy Others RE: -12.0 to 14.0 % LLOQ RE: -1.1 % Accuracy ULOQ RE: 9.0 % HAS LLOQ CV: 12.0 % Limits of quantification Precision ULOQ CV: 3.7 % (Nominal concentration) LLOQ RE: -5.2 % 0.01, 10 ng/mL Accuracy ULOQ RE: 2.0 % HBS LLOQ CV: 11.6 % Precision ULOQ CV: 2.0 % Continue DMD #040097 Supplemental data

Validation Items Results 0.02 ng/mL RE: 2.5 % Accuracy 0.5 ng/mL RE: -12.0 % 8 ng/mL RE: 2.8 % HAS 0.02 ng/mL CV: 8.3 % Intra-batch Precision 0.5 ng/mL CV: 2.5 % (Nominal 8 ng/mL CV: 1.3 % concentration) 0.02 ng/mL RE: -0.5 % 0.02, 0.5, 8 ng/mL Accuracy 0.5 ng/mL RE: -6.8 % 8 ng/mL RE: -0.5 % HBS 0.02 ng/mL CV: 7.0 % Precision 0.5 ng/mL CV: 1.9 % 8 ng/mL CV: 2.3 % 0.02 ng/mL RE: -1.5 % Accuracy 0.5 ng/mL RE: -1.2 % 8 ng/mL RE: 6.9 % HAS 0.02 ng/mL CV: 8.1 % Inter-batch Precision 0.5 ng/mL CV: 9.5 % (Nominal 8 ng/mL CV: 4.2 % concentration) 0.02 ng/mL RE: -3.0 % 0.02, 0.5, 8 ng/mL Accuracy 0.5 ng/mL RE: 1.4 % 8 ng/mL RE: 6.9 % HBS 0.02 ng/mL CV: 5.7 % Precision 0.5 ng/mL CV: 6.3 % 8 ng/mL CV: 5.6 % 0.02 ng/mL RR: 105.4 % Stability in Plasma 1h 8 ng/mL RR: 100.4 % (Short-term Stability) HAS 0.02 ng/mL RR: 104.9 % (Nominal 4h 8 ng/mL RR: 98.7 % concentration) 0.02 ng/mL RR: 94.5 % 0.02, 8 ng/mL 1h 8 ng/mL RR: 102.9 % Room temperature, HBS 0.02 ng/mL RR: 105.0 % for 1h, 4h 4h 8 ng/mL RR: 99.0 % Continue DMD #040097 Supplemental data

Validation Items Results 0.02 ng/mL RR: 100.5 % 1 week 8 ng/mL RR: 103.3 % 0.02 ng/mL RR: 104.9 % 2 weeks 8 ng/mL RR: 104.6 % 0.02 ng/mL RR: 95.1 % HAS 1 month 8 ng/mL RR: 91.1 % Stability in Plasma 0.02 ng/mL RR: 110.8 % (long-term Stability) 2 months 8 ng/mL RR: 98.5 % (Nominal 0.02 ng/mL RR: 103.0 % concentration) 6 months 8 ng/mL RR: 90.9 % 0.02, 8 ng/mL 0.02 ng/mL RR: 108.0 % at -20 ℃, 1 week 8 ng/mL RR: 112.8 % for 1 week, 2 weeks, 0.02 ng/mL RR: 102.5 % 1 month, 2 months, and 2 weeks 8 ng/mL RR: 109.5 % 6 months 0.02 ng/mL RR: 106.0 % HBS 1 month 8 ng/mL RR: 104.6 % 0.02 ng/mL RR: 103.5 % 2 months 8 ng/mL RR: 102.9 % 0.02 ng/mL RR: 96.5 % 6 months 8 ng/mL RR: 90.6 % Post-preparative 0.02 ng/mL RR: 90.6 % stability HAS 24h (Stability in Extract) 8 ng/mL RR: 92.9 % (Nominal concentration) 0.02 ng/mL RR: 95.0 %

0.02, 8 ng/mL HBS 24h in auto-sampler set at 8 ng/mL RR: 98.5 % 7 ℃, for 24h 0.02 ng/mL RR:98.5 % HAS 3 cycle Freeze and Thaw 8 ng/mL RR: 86.8 % Stability 0.02 ng/mL RR:107.0 % HBS 3 cycle 8 ng/mL RR: 105.5 % Continue DMD #040097 Supplemental data

Validation Items Results Accuracy RE: 4.6 % Appropriateness of 10-fold Precision CV: 3.6 % Dilution Method HAS Accuracy RE: 9.0 % (using blank plasma) 100-fold Precision CV: 3.9 % (Nominal Accuracy RE: 10.8 % concentration) 10-fold Precision CV: 3.2 % 50 ng/mL HBS Accuracy RE: 5.4 % 100-fold Precision CV: 1.7 %

DMD #040097 Supplemental data

Supplemental Table 2. Summary of assay method （Processed ginger）

Analyte [6]-shogaol（6S）, [10]-shogaol（10S） Internal standard Nonivamide LC/MS/MS system API5000 (AB SCIEX) Biological sample and volume Human plasma 0.5 mL Calibration curve range 0.02 to 10 ng/mL

LC-MS/MS conditions Analytical column YMC-Pack ODA-AQ, 3 μm, 50 × 2.0 mm I.D. (YMC Co., Ltd.) Guard column YMC-Pack ODA-AQ, 3 μm, 10 × 2.0 mm I.D. Mobile phase (A) 0.2 vol% Acetic acid in water (B) 0.2 vol% Acetic acid in acetonitrile gradient of 65-70-65% (B) over 15 min Flow rate 0.2 mL/min (0 to 12 min), 0.3 mL/min (12.1 to 15.0 min) Column temp 40°C Autosampler temp 7°C Injection volume 20 µL Analytical time 15 min Ionization mode ESI (Electrospray ionization, Turbo ion spray probe), positive Scan mode MRM (multiple reaction monitoring) Monitoring ion [6]-shogaol m/z 277.1 → m/z 137.0 [10]-shogaol m/z 333.3 → m/z 137.0 Nonivamide m/z 294.1 → m/z 137.0

Optimized mass spectrometer acquisition parameters used for analyte quantitation CUR (psi) 40 GS1 (psi) 60 GS2 (psi) 40 TEM (℃) 700 CAD (psi) 4 CE (V) 17（[6]-shogaol）, 25([10]-shogaol) CUR, Curtain Gas; GS, Ion source gas; TEM, Temperature; CAD, Collision Gas; CE, Collision energy.

DMD #040097 Supplemental data

Validation results Validation Items Results 6S No interfering Selectivity 10S No interfering Nonivamide (IS) No interfering 0.05 ng/mL 79.7 ± 1.2 % 6S Recovery 8 ng/mL 79.3 ± 0.8 % (Nominal concentration) 0.05 ng/mL 15.5 ± 0.1 % 10S 0.05, 8 ng/mL 8 ng/mL 15.3 ± 0.3 % Nonivamide (IS) 8 ng/mL 93.6 ± 0.9 % Correlation coefficient (r) 0.9970 to 0.9999 Linearity 6S LLOQ RE: -2.0 to 6.0 % Accuracy (Nominal concentration) Others RE: -11.6 to 9.0 % 0.02, 0.05, 0.1, Correlation coefficient (r) 0.9920 to 0.9991 0.5, 1, 5, 10 ng/mL 10S LLOQ RE: -5.5 to 5.0 % Accuracy Others RE: -14.6 to 14.8 % LLOQ RE: -2.5 % Accuracy ULOQ RE: 1.0 % 6S LLOQ CV: 1.5 % Limits of quantification Precision ULOQ CV: 1.0 % (Nominal concentration) LLOQ RE: 9.0 % 0.01, 10 ng/mL Accuracy ULOQ RE: -14.6 % 10S LLOQ CV: 4.1 % Precision ULOQ CV: 1.5 % Continue DMD #040097 Supplemental data

Validation Items Results 0.05 ng/mL RE: 1.4 % Accuracy 0.5 ng/mL RE: 5.2 % 8 ng/mL RE: 0.8 % 6S 0.05 ng/mL CV: 1.4 % Intra-batch Precision 0.5 ng/mL CV: 3.0 % (Nominal 8 ng/mL CV: 2.9 % concentration) 0.05 ng/mL RE: 6.6 % 0.05, 0.5, 8 ng/mL Accuracy 0.5 ng/mL RE: -5.8 % 8 ng/mL RE: 4.4 % 10S 0.05 ng/mL CV: 3.8 % Precision 0.5 ng/mL CV: 10.8 % 8 ng/mL CV: 6.5 % 0.05 ng/mL RE: -1.4 % Accuracy 0.5 ng/mL RE: 2.4 % 8 ng/mL RE: 0.5 % 6S 0.05 ng/mL CV: 3.2 % Inter-batch Precision 0.5 ng/mL CV: 3.1 % (Nominal 8 ng/mL CV: 4.7 % concentration) 0.05 ng/mL RE: 0.4 % 0.05, 0.5, 8 ng/mL Accuracy 0.5 ng/mL RE: -1.8 % 8 ng/mL RE: 5.5 % 10S 0.05 ng/mL CV: 11.8 % Precision 0.5 ng/mL CV: 10.6 % 8 ng/mL CV: 8.1 % 0.05 ng/mL RR: 99.6 % Stability in Plasma 1h 8 ng/mL RR: 98.7 % (Short-term Stability) 6S 0.05 ng/mL RR: 94.6 % (Nominal 4h 8 ng/mL RR: 95.3 % concentration) 0.05 ng/mL RR: 104.3 % 0.05, 8 ng/mL 1h 8 ng/mL RR: 99.8 % Room temperature, 10S 0.05 ng/mL RR: 105.2 % for 1h, 4h 4h 8 ng/mL RR: 108.6 % Continue DMD #040097 Supplemental data

Validation Items Results 0.05 ng/mL RR: 96.7 % 1 week 8 ng/mL RR: 103.4 % 0.05 ng/mL RR: 99.8 % 2 week 8 ng/mL RR: 109.7 % 0.05 ng/mL RR: 97.5 % 6S 1 month 8 ng/mL RR: 106.5 % Stability in Plasma 0.05ng/mL RR: 96.9 % (long-term Stability) 2 month 8 ng/mL RR: 107.1 % (Nominal 0.05 ng/mL RR: 99.0 % concentration) 6 month 8 ng/mL RR: 108.6 % 0.05, 8 ng/mL 0.05 ng/mL RR: 98.5 % at -20 ℃, 1 week 8 ng/mL RR: 91.1 % for 1 week, 2 weeks, 0.05 ng/mL RR: 85.4 % 1 month, 2 months, and 2 week 8 ng/mL RR: 95.7 % 6 months 0.05 ng/mL RR: 93.3 % 10S 1 month 8 ng/mL RR: 108.3 % 0.05ng/mL RR: 97.2 % 2 month 8 ng/mL RR: 96.8 % 0.05 ng/mL RR: 85.6 % 6 month 8 ng/mL RR: 88.4 % Post-preparative 0.05 ng/mL RR: 114.5 % stability 6S 24h (Stability in Extract) 8 ng/mL RR: 110.7 % (Nominal concentration) 0.05 ng/mL RR: 105.4 %

0.05, 8 ng/mL 10S 24h in auto-sampler set at 8 ng/mL RR: 106.4 % 7 ℃, for 24h 0.05 ng/mL RR:86.9 % 6S 3 cycle Freeze and Thaw 8 ng/mL RR: 101.1 % Stability 0.05 ng/mL RR:88.0 % 10S 3 cycle 8 ng/mL RR: 106.3 % Continue DMD #040097 Supplemental data

Validation Items Results Accuracy RE: 8.4 % Appropriateness of 10-fold Precision CV: 1.7 % Dilution Method 6S Accuracy RE: 1.4 % (using blank plasma) 100-fold Precision CV: 1.0 % (Nominal Accuracy RE: 1.6 % concentration) 10-fold Precision CV: 1.8 % 50 ng/mL 10S Accuracy RE: 2.0 % 100-fold Precision CV: 1.2 %

DMD #040097 Supplemental data

Supplemental Table 3. Summary of assay method （Ginseng）

Analyte Ginsenoside Rb1（GRB1）,

Ginsenoside Rg1（GRG1） Internal standard Asiaticoside LC/MS/MS system API5000 (AB SCIEX) Biological sample and volume Human plasma 0.5 mL Calibration curve range 0.01 to 10 ng/mL

LC-MS/MS conditions Analytical column YMC-Pack ODA-AQ, 3 μm, 150 × 2.0 mm I.D. Guard column YMC-Pack ODA-AQ, 3 μm, 10 × 2.0 mm I.D. Mobile phase (A) 0.2 vol% Acetic acid in water (B) 0.2 vol% Acetic acid in acetonitrile gradient of 10-55-10% (B) over 15 min Flow rate 0.2 mL/min (0 to 12 min), 0.3 mL/min (12.1 to 15.0 min) Column temp 40°C Autosampler temp 7°C Injection volume 20 µL Analytical time 15 min Ionization mode ESI (Electrospray ionization, Turbo ion spray probe), positive Scan mode MRM (multiple reaction monitoring)

Monitoring ion ginsenoside Rb1 m/z 1109.7 → m/z 325.1

ginsenoside Rg1 m/z 801.6 → m/z 423.4 Asiaticoside m/z 959.6 → m/z 453.5

Optimized mass spectrometer acquisition parameters used for analyte quantitation CUR (psi) 20 GS1 (psi) 80 GS2 (psi) 10 TEM (℃) 450 CAD (psi) 4

CE (V) 30 (ginsenoside Rb1), 13 (ginsenoside Rg1) CUR, Curtain Gas; GS, Ion source gas; TEM, Temperature; CAD, Collision Gas; CE, Collision energy.

DMD #040097 Supplemental data

Validation results Validation Items Results GRB1 No interfering Selectivity GRG1 No interfering Asiaticoside (IS) No interfering 0.05 ng/mL 57.1 ± 4.0 % GRB1 Recovery 8 ng/mL 57.7 ± 1.9 % (Nominal concentration) 0.05 ng/mL 81.1 ± 1.8 % GRG1 0.05, 8 ng/mL 8 ng/mL 84.5 ± 5.2% Asiaticoside (IS) 8 ng/mL 72.4 ± 0.8 % Correlation coefficient (r) 0.9959 to 0.9989 Linearity GRB1 LLOQ RE: -4.8 to 7.0 % Accuracy (Nominal concentration) Others RE: -15.0 to 13.0 % 0.01, 0.02, 0.05, 0.1, Correlation coefficient (r) 0.9954 to 0.9992 0.5, 1, 5, 10 ng/mL GRG1 LLOQ RE: -3.8 to 7.0 % Accuracy Others RE: -14.3 to 14.0 % LLOQ RE: 4.0 % Accuracy ULOQ RE: 3.0 % GRB1 LLOQ CV: 12.5 % Limits of quantification Precision ULOQ CV: 1.9 % (Nominal concentration) LLOQ RE: 5.0 % 0.01, 10 ng/mL Accuracy ULOQ RE: 6.0 % GRG1 LLOQ CV: 14.3 % Precision ULOQ CV: 3.8 % Continue DMD #040097 Supplemental data

Validation Items Results 0.02 ng/mL RE: 2.0 % Accuracy 0.5 ng/mL RE: 9.4 % 8 ng/mL RE: 6.6 % GRB1 0.02 ng/mL CV: 9.3 % Intra-batch Precision 0.5 ng/mL CV: 2.0 % (Nominal 8 ng/mL CV: 8.6 % concentration) 0.02 ng/mL RE: -0.5 % 0.02, 0.5, 8 ng/mL Accuracy 0.5 ng/mL RE: 0.2 % 8 ng/mL RE: -1.9 % GRG1 0.02 ng/mL CV: 14.6 % Precision 0.5 ng/mL CV: 4.0 % 8 ng/mL CV: 3.8 % 0.02 ng/mL RE: 3.5 % Accuracy 0.5 ng/mL RE: 4.8 % 8 ng/mL RE: 7.4 % GRB1 0.02 ng/mL CV: 11.6 % Inter-batch Precision 0.5 ng/mL CV: 6.7 % (Nominal 8 ng/mL CV: 5.7 % concentration) 0.02 ng/mL RE: 4.0 % 0.02, 0.5, 8 ng/mL Accuracy 0.5 ng/mL RE: 1.8 % 8 ng/mL RE: 5.5 % GRG1 0.02 ng/mL CV: 12.5 % Precision 0.5 ng/mL CV: 6.1 % 8 ng/mL CV: 6.6 % 0.02 ng/mL RR: 110.4 % Stability in Plasma 1h 8 ng/mL RR: 107.6 % (Short-term Stability) GRB1 0.02 ng/mL RR: 100.5 % (Nominal 4h 8 ng/mL RR: 110.2 % concentration) 0.02 ng/mL RR: 100.5 % 0.02, 8 ng/mL 1h 8 ng/mL RR: 90.9 % Room temperature, GRG1 0.02 ng/mL RR: 90.9 % for 1h, 4h 4h 8 ng/mL RR: 89.4 % Continue DMD #040097 Supplemental data

Validation Items Results 0.02 ng/mL RR: 102.5 % 1 week 8 ng/mL RR: 98.0 % 0.02 ng/mL RR: 107.0 % 2 weeks 8 ng/mL RR: 102.7 % 0.02 ng/mL RR: 87.6 % GRB1 1 month 8 ng/mL RR: 96.9 % Stability in Plasma 0.02 ng/mL RR: 101.5 % (long-term Stability) 2 months 8 ng/mL RR: 100.4 % (Nominal 0.02 ng/mL RR: 93.5 % concentration) 6 months 8 ng/mL RR: 85.7 % 0.02, 8 ng/mL 0.02 ng/mL RR: 91.9 % at -20 ℃, 1 week 8 ng/mL RR: 97.3 % for 1 week, 2 weeks, 0.02 ng/mL RR: 109.1 % 1 month, 2 months, and 2 weeks 8 ng/mL RR: 105.7 % 6 months 0.02 ng/mL RR: 101.0 % GRG1 1 month 8 ng/mL RR: 102.2 % 0.02 ng/mL RR: 100.5 % 2 months 8 ng/mL RR: 98.3 % 0.02 ng/mL RR: 113.6 % 6 months 8 ng/mL RR: 85.2 % Post-preparative 0.02 ng/mL RR: 104.0 % stability GRB1 24h (Stability in Extract) 8 ng/mL RR: 102.0 % (Nominal concentration) 0.02 ng/mL RR: 99.5 %

0.02, 8 ng/mL GRG1 24h in auto-sampler set at 8 ng/mL RR: 105.5 % 7 ℃, for 24h 0.02 ng/mL RR:108.0 % GRB1 3 cycle Freeze and Thaw 8 ng/mL RR: 96.4 % Stability 0.02 ng/mL RR: 96.0 % GRG1 3 cycle 8 ng/mL RR: 92.8 % Continue DMD #040097 Supplemental data

Validation Items Results Accuracy RE: 2.6 % Appropriateness of 10-fold Precision CV: 2.1 % Dilution Method GRB1 Accuracy RE: -3.6 % (using blank plasma) 100-fold Precision CV: 1.9 % (Nominal Accuracy RE: 1.4 % concentration) 10-fold Precision CV: 2.8 % 50 ng/mL GRG1 Accuracy RE: 4.6 % 100-fold Precision CV: 4.6 %