Pharmacokinetics of Rhodamine 110 and Its Organ Distribution in

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Pharmacokinetics of Rhodamine 110 and its Organ Distribution in Rats Shiau-Han Jiang, Yung-Yi Cheng, Teh-Ia Huo, and Tung-Hu Tsai J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02685 • Publication Date (Web): 10 Aug 2017 Downloaded from http://pubs.acs.org on August 13, 2017

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Journal of Agricultural and Food Chemistry

Pharmacokinetics of Rhodamine 110 and its Organ Distribution in Rats

Shiau-Han Jiang a,1, Yung-Yi Cheng b,1, Teh-Ia Huo a, Tung-Hu Tsai b, c, d, e*

a

Institute of Pharmacology, National Yang-Ming University, Taipei, Taiwan

b

Institute of Traditional Medicine, National Yang-Ming University, Taipei, Taiwan

c

Graduate Institute of Acupuncture Science, China Medical University, Taichung,

Taiwan d

School of Pharmacy, College of Pharmacy, Kaohsiung Medical University,

Kaohsiung, Taiwan e

Department of Chemical Engineering, National United University, Miaoli, Taiwan

AUTHOR INFORMATION 1

contributed equally

*Corresponding Author: Tung-Hu Tsai, Ph.D. Professor  Fax: (886-2) 2822 5044. Phone: (886-2) 2826 7115. E-mail: [email protected]

1

ACS Paragon Plus Environment


2


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1


ABSTRACT

2

Rhodamine dyes have been banned as food additives due to their potential

3

tumorigenicity. Rhodamine 110 is illegal as a food additive, although its

4

pharmacokinetics have not been characterized, and no accurate bioanalytical methods

5

are available to quantify rhodamine 110. The aim of this study was to develop and

6

validate a fast, stable, sensitive method to quantify rhodamine 110 using

7

high-performance liquid chromatography coupled to tandem mass spectrometry

8

(HPLC-MS/MS) to assess its pharmacokinetics and organ distribution in awake rats.

9

Rhodamine 110 exhibited linear pharmacokinetics and slow elimination after oral

10

administration. Furthermore, its oral bioavailability was approximately 34-35%. The

11

distribution in the liver and kidney suggests that these organs are primarily

12

responsible for rhodamine 110 metabolism and elimination. Our investigation

13

describes the pharmacokinetics and a quantification method for rhodamine 110,

14

improving our understanding of the food safety of rhodamine dyes.

15 16 17

Keywords: Rhodamine 110; rhodamine dyes; food additives; pharmacokinetics;

18

toxicokinetics; HPLC-MS/MS.

19 20 21 22 23

3



24 25

INTRODUCTION

26 27

Rhodamine derivatives, except for erythrosine, have recently been banned for use as

28

food additives to enhance the durability of coloring.1 A notorious rhodamine

29

derivative, rhodamine B, has been illegally used in foodstuffs according to documents

30

from the US Food and Drug Administration (U.S. FDA).1,2 Based on animal studies,

31

rhodamine B metabolites are produced via de-ethylation in the liver.1,3 The final

32

metabolite, rhodamine 110 (3,6-diaminofluoran) is a xanthene dye similar to

33

fluorescein and eosin.3,5 Webb et al. suggested that rhodamine 110 is less toxic than

34

the parent molecule based on the intravenous LD50 acute toxicity values of 89.5

35

mg/kg and 140.0 mg/kg for rhodamine B and rhodamine 110, respectively.3 Both

36

molecules induced liver and kidney enlargement after ingestion, and male rats showed

37

more significant increases than female rats after rhodamine 110 exposure.3 In addition,

38

testis weight increased in male rats dosed with rhodamine 110.3 Furthermore, in vitro

39

studies have shown that acidic rhodamine 110 accumulates in mitochondria in a

40

cationic form, which alters the pH in this cellular compartment.4 Jeannot's and

41

Lampidis's research demonstrated that rhodamine 110 accumulates in human

42

lymphoblastoid cells and Friend leukemia cells. No cytotoxicity to human

43

lymphoblastoid cells was observed below 10 μM, but rhodamine 110 caused Friend

44

leukemia cells to die at concentrations above 100 μM.6,7

45 46

The persistent and strong fluorescence properties of rhodamine 110 arise from its

47

abundant conjugated π bonds. It is slightly soluble in water but highly soluble in

48

methanol and dimethyl sulfoxide. Rhodamine 110 is widely applied in the biomedical

49

and industrial fields as a fluorescent probe and stain and is potentially useful for in 4


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vivo cancer diagnoses due to its excellent photostability and photophysical

51

properties.8 Fluorescent rhodamine 110 has been conjugated to amino acids, peptides

52

and small molecules to probe cancer cells in bio-imaging applications.9-11 However,

53

there are unavoidable limitations to determining the fluorescence level at target sites,

54

which can be affected by factors involved in auto-oxidation,12 temperature,

55

fixation or subsequent processing.13 Accurately quantifying drug concentrations is

56

also influenced by these factors. Thus, high-performance liquid chromatography

57

tandem mass spectrometry (HPLC-MS/MS) is a useful, precise tool for analyte

58

quantification. Several analytical methods to detect rhodamine derivatives in

59

foodstuffs,14-17,19 fish,16 soft drinks,17 waste water,17 ink,18 and cosmetics17 have been

60

developed, but few reports have quantified rhodamine dyes in biological samples or

61

assessed their pharmacokinetics.1,20,21 Two articles were identified in searching the

62

PubMed database for the keywords “rhodamine 110 or 3,6-diaminofluoran” and

63

“LC-MS”.

64

chromatography–tandem mass spectrometry” and “pharmacokinetic or toxicokinetic”

65

as keywords, no articles regarding the pharmacokinetics or toxicokinetics of

66

rhodamine 110 were retrieved. In previous studies, food products containing

67

rhodamine 110 were analyzed using LCIT-TOF-MS and HPLC-MS/MS methods with

68

reverse C18 columns,19,22 indicating that LC-MS/MS can be used to quantify analytes

69

with high sensitivity and high resolution compared to other analytical methods.

However,

when

using

“rhodamine

110”

13

and

tissue

“liquid

70 71

Rhodamine 110 is a potentially mutagenic dye according to the aforementioned

72

studies. Because of its bright colors and properties that allow for easy dyeing, it might

73

be deliberately used as a food colorant. However, rhodamine 110 is not listed as a

74

food colorant that is accepted by the FDA of Taiwan or that of United States.23,24

75

Owing to safety considerations, we tested the hypothesis that there is a potential risk 5



76

of ingesting rhodamine 110 if it were illegally added to foodstuffs. Although there are

77

limited toxicity reports for rhodamine 110, human exposure to this chemical would be

78

a potential food safety problem. Finding clinical records that prove the damaging

79

effects on health of rhodamine 110 is another important issue. We take a

80

forward-looking attitude to analyze the possibility of the damaging effects on health

81

of rhodamine 110 because its parent molecule (rhodamine B) has been illegally used

82

in food. Thus, it is necessary to investigate the pharmacokinetics and organ

83

distribution of rhodamine 110 to support food safety evaluations. To date, there have

84

been no reports describing analytical methods or sample preparation methods to

85

detect rhodamine 110 in biological samples. Therefore, the aim of this study was to

86

develop a validated bioanalytical method to investigate the pharmacokinetics, organ

87

distribution, and oral bioavailability of rhodamine 110 in rats.

88 89

MATERIAL AND METHODS

90 91

Reagents and Materials

92 93

Rhodamine 110 chloride, carvedilol, pentobarbital sodium, urethane, α-chloralose,

94

polyethylene glycol 400 and heparin sodium were purchased from Sigma-Aldrich (St.

95

Louis, MO, USA). LC-MS grade methanol and formic acid were purchased from E.

96

Merck (Darmstadt, Germany). Triple-de-ionized water obtained from Millipore

97

(Bedford, MA, USA) was used for all of the aqueous solutions.

98 99

Sample Preparation

100 6


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Plasma and organ samples (50 μL) were mixed with 150 μL methanol containing (10

102

ng/mL) for protein precipitation. The samples were centrifuged at 16,000 × g for 10

103

min at 4 C. The supernatant was filtered into an Eppendorf tube through a 0.22-μm

104

filter and transferred to an auto-sampler for UHPLC-MS/MS analysis.

105 106

Preparation of Stock and Calibration Standards

107 108

Rhodamine 110 chloride was dissolved in methanol at a concentration of 1 mg/mL as

109

a stock solution. Working solutions were prepared by diluting the stock solution in

110

50% methanol. Carvedilol was dissolved in methanol for the stock solution (10 μg

111

/mL) and diluted to a concentration of 10 ng/mL, with methanol as an internal

112

standard (IS). All of the stock solutions were stored at -20 C until analysis.

113 114

UHPLC-MS/MS Conditions

115 116

The UHPLC-MS/MS system consisted of an LC-MS-8030 triple-quadrupole mass

117

spectrometer (Shimadzu, Kyoto, Japan) coupled to a CBM-20A HPLC system

118

(Shimadzu, Kyoto, Japan). The CBM-20A HPLC system controller comprised an

119

SIL-20AC XR auto-sampler, a DGU-20A degasser, and two LC-20AD pumps. Before

120

quantitation, the mass spectrometer parameters were optimized as follows: the

121

nebulizing gas (nitrogen) flow and drying gas (nitrogen) flow were set at 3 L/min and

122

15 L/min, respectively; the collision gas (argon) pressure was set at 230 kPa; the

123

interface voltage was 3.8 kV; the desolvation line temperature was 250 C; the oven

124

temperature was 40 C; and the heat block temperature was 400 C. Ions were

125

detected in selective reaction monitoring (SRM) mode. The quantification of

126

rhodamine 110 was performed using the SRM mode with collision-induced 7



127

dissociation. The precursor ion of rhodamine 110 was found in full-scan positive-ion

128

mode and was observed at m/z 331.1. After impact with a collision energy of -38 eV,

129

the product ion of rhodamine 110 was at m/z 287.2, which was selected for

130

quantification (Figure S1). The fragmentation pattern of rhodamine 110 in our study

131

was the same as that reported in a previous study19. The mass transition of carvedilol

132

(IS) was determined using the same procedure. The transition of m/z 407.1 → 100.1

133

was chosen (Figure S1); the collision energy was -35 eV for carvedilol.

134 135

The analytical column was a C18 reverse-phase column (100 × 2.1 mm, 2.6 μm;

136

Phenomenex, Kinetex C18, Torrance, California, USA) coupled to a C18 guard

137

column (0.5 μm depth × 0.004 i.d., Phenomenex, AF0-8497, Torrance, California,

138

USA) for chromatographic separation. The mobile phase consisted of 0.1% formic

139

acid as the aqueous phase (A) and 100% methanol as the organic phase (B), with a

140

gradient elution system. The gradient program was set as follows: 0.01-1 min, 30% B;

141

1-1.5 min, 30-75% B; 1.5-4.5 min, 75% B; 4.5-5 min, 75-30% B; 5-6 min, 30% B.

142

The flow rate was set at 0.2 mL/min. The temperature of the auto-sampler was 8 C.

143

The injection volume was five μL.

144 145

Validation Method

146 147

To demonstrate that the developed method is reliable and reproducible, the

148

bioanalytical method was validated according to FDA guidelines.25 The method

149

validation included accuracy, precision, matrix effect, recovery and stability tests.

150 151

Linearity, Accuracy, and Precision 8


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A calibration curve was generated for the peak area ratio of rhodamine 110 and the IS

153

versus concentration. Linearity was defined as a coefficient correlation (r2) of at least

154

0.995 for the calibration curve. To evaluate precision and accuracy, blank plasma

155

samples spiked with rhodamine 110 were prepared at low, medium and high

156

concentrations and tested on the same day (intra-day), with five replicates then tested

157

on five consecutive days (inter-day). Precision (relative standard deviation, R.S.D., %)

158

refers to the closeness of individual analyte measurements to each other and was

159

calculated from the standard deviation and observed concentration (Cobs) as follows:

160

precision (R.S.D., %) = [standard deviation (S.D.)/Cobs] × 100%. Accuracy (bias, %)

161

describes the closeness of a measured value to the true value. The accuracy was

162

calculated using the nominal concentration (Cnom), and the mean value for the

163

observed concentration (Cobs) was calculated as follows: accuracy (bias, %) = [(Cnom

164

-Cobs)/Cnom] × 100%. Precision and accuracy should be within ± 15% except for at the

165

lower limit of quantification (LLOQ). The precision and accuracy of the LLOQ

166

should not deviate by more than 20%, which is defined as a S/N (signal-to-noise ratio)

167

less than 10.

168 169

Matrix Effect and Recovery

170

The matrix effect and recovery were studied using three sets processed at low,

171

medium and high concentrations within the linear range, whereas carvedilol (IS) was

172

measured at 10 ng/mL. Set 1 was a neat standard solution without a biological matrix.

173

Sets 2 and 3 refer to standard solutions spiked with biological matrix after and before

174

extraction, respectively. The sample preparation of the three sets was as follows:

175 176

Set 1: Standard solution. The 5-μL standard solution aliquot was spiked with 195 μL 0.1% formic acid/methanol (50:50, v/v). 9



177

Set 2: Standard solution spiked with post-extraction matrix. A blank plasma or

178

organ tissue sample was processed in the same manner described for sample

179

preparation (section 2.6). The standard solutions (5 μL) were added to the obtained

180

blank matrix (195 μL) and filtered through a 0.22-μm filter before analysis.

181

Set 3: Standard solution spiked with pre-extraction matrix. The standard solutions

182

(5 μL) were mixed with blank a plasma or organ tissue sample (195 μL) and then

183

prepared as in section 2.6.

184 185

The percentage of the matrix effect and recovery were calculated according to the

186

peak area of set 2/set 1 and set 3/set 2, respectively. The results are presented as the

187

means ± standard deviation (S.D.) of triplicate experiments.

188 189

Stability

190

Experiments to test rhodamine 110 stability in various biological samples were

191

performed under different conditions at a low, medium and high concentration within

192

the linear range. Stability was evaluated after being exposed to short-term storage,

193

auto-sampler storage, freeze-thaw cycle and long-term storage conditions. Stability

194

was measured using the standard solution spiked with various biological matrices

195

prepared as described in section 2.6. The short-term stability test was performed at

196

room temperature for 4 h. Stability under auto-sampler storage was determined after

197

storage in an auto-sampler for 8 h. Freeze-thaw stability was assessed three times after

198

storing samples at -20 C for 24 h and then thawing them at room temperature for 1 h.

199

Long-term stability was measured by keeping samples at -20 C for 28 days. The

200

stock solution stability procedure also included an evaluation of rhodamine 110 at

201

room temperature.

202 10


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Experimental Animals

204 205

Adult male Sprague-Dawley rats (230 ± 20 g) were obtained from the Laboratory

206

Animal Center at National Yang-Ming University, Taipei, Taiwan. The animal

207

experimental protocol (IUCAC no. 1040902) was reviewed and approved by the

208

Institutional Animal Care and Use Committee of National Yang-Ming University and

209

was in compliance with the guidelines of the National Research Council, USA26. All

210

of the experimental animals were housed at a temperature of 24 ± 1 C with a 12 h

211

light/dark cycle. Food and water were freely accessible to experimental animals at all

212

times. The rats were randomly divided into groups (n = 6 for each group) and were

213

fasted for 8 h before drug administration.

214 215

Freely Moving Rat Model

216

After the rats were anesthetized with pentobarbital sodium (50 mg/kg, i.p.), the

217

surgical sites were shaved and cleaned with a 75% ethanol solution. All of the

218

surgical instruments were sterilized with 75% ethanol. The right jugular vein was

219

cumulated toward the right atrium with polyethylene tubing (PE-50), and the catheter

220

was transferred to the dorsal neck region and fixed for blood sampling. After surgery,

221

each rat was placed in a clean cage and allowed to recover for at least 12 h.

222 223

After the rats recovered, rhodamine 110 in polyethylene glycol 400 (0.3 mg/mL and 1

224

mg/mL) was administered at 3 mg/kg and 10 mg/kg by oral gavage. Blood samples

225

(150 μL) were collected from the right jugular vein 5, 15, 30, 60, 120, 180, 240, 300,

226

360, 480 and 720 min after drug administration. After each sampling, 100 μL normal

227

saline was administered via catheter to compensate for the loss of body fluid, and a 50

228

μL heparin solution (20 IU heparin/mL normal saline) was provided to prevent 11



229

coagulation. Blood samples were centrifuged at 16,000 × g for 10 min at 4 C to

230

obtain plasma, which was stored at −20C until analysis.

231 232

Intravenous Administration of Rhodamine 110

233

A mixture of urethane (1 g/kg) and α-chloralose (100 mg/kg) was administered

234

intraperitoneally (i.p.) to anesthetize rats. Before surgery, the surgical sites were

235

shaved and cleaned with 75% ethanol. The right jugular vein and left femoral vein

236

were catheterized with polyethylene tubing (PE-50) for blood collection and drug

237

administration, respectively.

238 239

After surgery, rhodamine 110 in polyethylene glycol 400 (1 mg/mL) was

240

administered intravenously to rats at 3 mg/kg (n = 6). A 150 μL blood sample was

241

collected from the right jugular vein 5, 15, 30, 60, 120, 180, 240, 300, 360, 480, 720

242

min after drug administration. Then, 100 μL of normal saline was administered via

243

the right jugular vein to compensate for body fluid loss, and 50 μL of a heparin

244

solution (20 IU heparin/mL normal saline) was provided to prevent blood clotting.

245

After blood collection, the sample preparation was the same as in section 2.5.1.

246 247

Organ Distribution

248

Rhodamine 110 dissolved in polyethylene glycol 400 (0.3 mg/mL) was administered

249

orally at a single dose of 3 mg/kg (n = 6). After drug administration, a mixture of

250

urethane (1 g/kg) and α-chloralose (0.1 g/kg) administered i.p. was used to anesthetize

251

rats. After ingesting the drug for 180 min, 150 μL of blood was collected via cardiac

252

puncture, and the rats were sacrificed by exsanguination while unconsciousness. The

253

liver, heart, spleen, lungs, kidneys, and brain were collected and weighed. The organs

254

were homogenized with 50% methanol (5x volume for heart, spleen, lungs, and brain; 12


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10x volume for liver and kidneys). After centrifugation at 16,000 × g for 10 min, the

256

supernatants were stored at -20 C until sample preparation.

257 258

Data and Statistical Analysis

259

Profiling Solution Software Ver. 1.1 (Shimadzu, Kyoto, Japan) was used to analyze

260

the chromatogram data. Pharmacokinetic parameters were calculated using

261

WinNonlin

262

non-compartmental model for groups administered intravenous and oral treatments.

263

All data are expressed as the means ± S.D.

Standard

Edition

1.0

(Pharsight

Corp.,

CA,

USA)

with

a

264 265

RESULTS AND DISCUSSION

266 267

For superior separation and qualification, rhodamine 110 and carvedilol were

268

analyzed in the optimized mobile phase, which contained 0.1% formic acid and

269

methanol. Formic acid was selected as the aqueous phase to reduce peak tailing.

270

Gradient elution was used to obtain well-resolved peak shapes and proper retention

271

times (3.8 and 4.1 min) for rhodamine 110 and carvedilol in various biological

272

matrices with a C18 reverse-phase column (100 × 2.1 mm, 2.6 μm). Representative

273

UHPLC-MS/MS chromatograms shown in Figure 1 are for the following samples:

274

blank plasma, rhodamine 110 spiked with matrix and biological samples after the oral

275

administration of 10 mg/kg rhodamine 110. The analytes were not observed in the

276

blank biological matrix, demonstrating that they were not endogenous. Additionally,

277

the smooth baseline and lack of an interfering signal in these chromatograms

278

demonstrates acceptable selectivity to identify rhodamine 110 in biological samples.

279

This analytical method had the advantages of specificity and a high sensitivity for

280

detecting rhodamine 110. 13



281 282

Excluding the liver matrix, the validation calibration curve for rhodamine 110 in rat

283

plasma and organ samples ranged from 50 to 500 ng/mL. The calibration curve was

284

linear over a range of 50-1000 ng/mL for the rat liver matrix. The linearity of the

285

calibration curves was assessed using coefficients of determination (r2) obtained from

286

the regression lines. All correlation coefficients (r2) for rhodamine 110 were greater

287

than 0.995, thus meeting the FDA guidelines. The LLOQ for plasma and organ

288

samples was 50 ng/mL.

289 290

The accuracy (as % bias) and precision (as % R.S.D) were all acceptable within one

291

day and over five consecutive days. The accuracy and precision for analyzing plasma

292

samples ranged from -2.34 to 17.88% and 3.67 to 8.71, respectively, at low (50

293

ng/mL), medium (100 ng/mL) and high (500 ng/mL) concentrations of rhodamine 110.

294

The accuracy for analyzing various organ tissue samples ranged from -13.68 to

295

13.48%, -10.26 to 7.73, -5.66 to 5.00, -5.48 to 12.30, -15.73 to 6.44, and -1.35 to 9.90

296

for the liver, heart, spleen, lungs, kidneys and brain, respectively. The precision of

297

analyzing various organ samples ranged from 1.61 to 19.32, 1.93 to 7.99, 0.55 to 3.31,

298

3.23 to 19.07, 1.05 to 9.28 and 1.05 and 11.70 for the liver, heart, spleen, lungs,

299

kidneys and brain, respectively. The summarized results are shown in Table 1. The

300

variation in accuracy and precision were irregular but met the FDA guidelines. The

301

method validation results imply that all duplicated processes were precise, accurate,

302

reproducible and appropriate for the intended purpose.

303 304

The values for the matrix effect and recovery of rhodamine 110 for plasma at low,

305

medium and high concentrations were within 107-117% and 101-103%, respectively.

306

Moreover, the matrix effects of rhodamine 110 for organ samples showed high ion 14


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307

enhancement, especially in lung samples. The mean recovery values were 101, 110,

308

115, 118, 106, 107, and 108% for plasma, liver, heart, spleen, lungs, kidneys, and

309

brain, respectively. Detailed data are summarized in Supplementary Table S1.

310

According to the FDA guidelines, the recovery value for each organ sample was

311

acceptable. The removal of protein by denaturation or precipitation is an effective

312

sample preparation method that is often applied to plasma samples prior to analysis.27

313

Reagents commonly used to precipitate proteins include acids, organic solvents,

314

organic salts or metallic ion solutions. The efficiency of protein precipitation with 15

315

mL methanol added to 5 mL plasma was evaluated and showed a high efficiency

316

(above 95%).28 Our method used three volumes of methanol to reduce protein

317

solubility, leading to precipitation. The advantages of the deproteinization method in

318

our study were a reduced preparation time and a simple clean-up procedure, as well as

319

the ability to perform a direct analysis by LC-MS/MS.

320 321

The stability of the analytes in various matrices after being exposed to short-term,

322

auto-sampler, freeze-thaw and long-term storage conditions was examined in

323

triplicate. Acceptable outcomes were obtained for samples stored in these four

324

different conditions according to FDA guidelines. No significant degradation of

325

rhodamine 110 in the plasma, liver, heart, spleen, lungs, or kidneys was observed

326

under these four different conditions. The results demonstrate that rhodamine 110 can

327

be stably maintained under various storage conditions; the results are shown in Table

328

2.

329 330

The pharmacokinetics of rhodamine 110 were assessed following oral administration

331

at two dosages (3 and 10 mg/kg) and intravenous administration at one dosage (3

332

mg/kg). The mean plasma concentration–time profile of rhodamine 110 in rats after 15



333

exposure is shown in Figure 2. Pharmacokinetic parameters were calculated using an

334

extravascular input and IV-bolus input, non-compartmental model analysis conducted

335

with WinNonlin Standard Edition. The pharmacokinetic parameters of rhodamine 110

336

are shown in Table 3 and indicate that the maximum plasma concentrations (Cmax) of

337

the two oral dosages were 283.4 and 657.0 ng/mL, which were reached at 140 and

338

210 min, respectively. This indicates that rhodamine 110 absorption was not rapid

339

after ingestion, as it took over two h to be absorbed from the intestines into the blood.

340

The areas under the concentration–time curves (AUCs) for the two dosages were

341

138.1 ± 20.3 and 444.0 ± 170.8 h ng/mL. The pharmacokinetic data demonstrate that

342

the AUC was proportional to the administered oral dose of rhodamine 110 (3 mg/kg

343

and 10 mg/kg). Furthermore, the clearance (Cl) of the two orally administered doses

344

was 7.94 and 8.61 mL/min/kg, respectively, which was not significantly different

345

from Cl following intravenous administration. The low clearance rate, long half-life,

346

and long mean residence time of rhodamine 110 suggest that the elimination process

347

might be slow. In addition, comparing the AUC and Cl values of the two dosages,

348

rhodamine 110 exhibited linear pharmacokinetics after oral administration. The oral

349

bioavailability was derived from the AUC after intravenous and oral administration

350

using the formula F (%) = 100 × (AUCoral/doseoral)/(AUCiv/doseiv). Thus, the oral

351

bioavailability of rhodamine 110 at two oral dosages was approximately 34-36%.

352 353

Three hours after ingesting rhodamine 110, transfer was apparent, maintaining a

354

dynamic equilibrium between the first and second compartments according to the

355

pharmacokinetic profile following oral administration. To clarify the major

356

distribution sites of rhodamine 110 three h after oral administration at a dosage of 3

357

mg/kg, various organ samples were collected. The rhodamine 110 levels in the liver,

358

heart, spleen, lungs, kidneys, brain and plasma were 1853.3 ± 233.9, 199.9 ± 75.6, 16


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205.9 ± 44.6, 212.0 ± 38.5, 1175.5 ± 150.7, 15.0 ± 7.4 ng/g and 190.2 ± 9.5 ng/mL,

360

respectively. As Figure 3 shows, the rhodamine 110 concentration in various organs

361

exhibited the following order: liver > kidneys > lungs > spleen > heart > brain.

362

Comparing organ and plasma samples, the distribution in the liver was 9.7 times

363

higher than in plasma and 6.1 times higher in the kidneys than in plasma. This

364

suggests that the liver and kidneys were the organs primarily metabolizing rhodamine

365

110, leading to its accumulation at these sites, and that these are the primary organs

366

for rhodamine 110 clearance. The lowest level of rhodamine 110 was detected in the

367

brain, which was less than 10% of the plasma level; this finding indicates that

368

rhodamine 110 might permeate the blood-brain barrier. In summary, the distribution

369

of rhodamine 110 in organs was highest in the liver, followed by that in the kidneys

370

and then in the brain.

371 372

We investigated the pharmacokinetics of rhodamine B, another rhodamine derivative,

373

in an article published.1 Tmax values were 0.46 and 3.5 h for orally administered (10

374

mg/kg) rhodamine B and rhodamine 110, respectively, indicating that the absorption

375

of rhodamine B is faster than of rhodamine 110. The oral bioavailabilities of

376

rhodamine B and rhodamine 110 were 9.84 and 34.65%, respectively, which shows

377

that the more hydrophilic dye, rhodamine 110, has better water solubility but a lower

378

permeability than rhodamine B as demonstrated by the longer the absorption time of

379

rhodamine 110. The clearance rates of rhodamine B and rhodamine 110 were 78.2

380

and 516.6 mL/h/kg, respectively, suggesting the rhodamine 110 is eliminated faster

381

than rhodamine B, which could explain why the toxicity of rhodamine 110 is much

382

lower than that of its parent molecule. Overall, we conclude that rhodamine 110

383

probably does not accumulate in animal bodies if the oral dosage is less than 10

384

mg/kg because rhodamine 110 has linear pharmacokinetics. 17



Page 18 of 35

385 386

A fast and simple UHPLC-MS/MS method was established for analyzing rhodamine

387

110 in various biological samples, including rat liver, heart, spleen, lungs, kidneys,

388

brain and plasma. The values for the matrix effect, recovery, and stability in rat

389

plasma and organ tissue samples were acceptable according to the FDA biological

390

method validation guidelines (2007). The pharmacokinetic results suggest that

391

rhodamine 110 has linear pharmacokinetics and a slow elimination, and the oral

392

bioavailability was approximately 36% in rats. Based on the organ distribution

393

assessment data, rhodamine 110 mostly accumulated in the liver and kidneys and, to a

394

lesser degree, in the brain. Our constructive findings offer sufficient information to

395

evaluate the food safety and pharmacokinetics of rhodamine 110.

396 397

ABBREVIATIONS USED

398

AUC, area under the concentration−time curve; Cmax, maximum plasma concentration;

399

Cl,

400

high-performance liquid chromatography tandem mass spectrometry; IS, internal

401

standard; LCIT-TOF-MS, ultrafast liquid chromatography-ion trap time of flight mass

402

spectrometry; LD50, median lethal dose; LLOQ, lower limit of quantification; R.S.D.,

403

relative standard deviation; SRM, selective reaction monitoring mode; S/N,

404

signal-to-noise.

clearance;

FDA,

Food

and

Drug

Administration;

HPLC-MS/MS,

405 406

ACKNOWLEDGMENTS

407

We appreciate the PK Lab members who assisted and supported this study.

408 409

SUPPORTING INFORMATION 18


Page 19 of 35


410

Typical HPLC-MS/MS chromatograms for organs and data on the matrix effects and

411

recovery of rhodamine 110 in plasma and organ samples are available free of charge

412

at http://pubs.acs.org.

413 414

19



415

20


Page 20 of 35

Page 21 of 35


REFERENCES 1.

Cheng, Y. Y.; Tsai, T. H. Pharmacokinetics and biodistribution of the illegal food colorant rhodamine B in rats. J. Agric. Food Chem. 2017, 65, 1078-1085.

2.

U.S. FDA. Detention without Physical Examination and Guidance of Foods Containing Illegal and/or Undeclared Colors; Import Alert 4502; U.S. Department of Health and Human Services, Food and Drug Administration Center for Drug Evaluation and Research (CDER): Washington, DC, USA, 2016. 

3.

Webb, J. M.; Hansen, W. H.; Desmond, A.; Fitzhugh, O. G. Biochemical and toxicologic studies of rhodamine B and 3,6-diaminofluoran. Toxicol. Appl. Pharmacol. 1961, 3, 696−706

4.

Jeannot, V.; Salmon, J.-M.; Deumié, M.; Viallet, P. Intracellular accumulation of rhodamine 110 in single living cells, J. Histochem. Cytochem. 45 (1997) 403-412.

5.

Webb, J. M.; Hansen, W. H. Studies of the metabolism of rhodamine B. Toxicol. Appl. Pharmacol. 1961, 3, 86–95.

6.

Alford, R.; Simpson, H.M.; Duberman, J.; Hill, G.C.; Ogawa, M.; Regino, C.; Kobayashi, H.; Choyke, P.L.; Toxicity of organic fluorophores used in molecular imaging: literature review, Mol. Imag. 2009, 8, 341-354.

7.

Nestmann, E.R.; Douglas, G.R.; Matula, T.I.; Grant, C.E.; Kowbel, D.J. Mutagenic activity of rhodamine dyes and their impurities as detected by mutation induction in Salmonella and DNA damage in Chinese hamster ovary cells, Cancer Res., 1979, 39, 4412−4417.

8.

Beija, M.; Afonso, C.A.; Martinho, J.M. Synthesis and applications of Rhodamine derivatives as fluorescent probes, Chem. Soc. Rev., 2009, 38, 2410-2433. 21



9.

Fei, X.; Gu, Y. Progress in modifications and applications of fluorescent dye probe, Prog. Nat. Sci., 2009, 19, 1-7.

10. Kobayashi, H.; Ogawa, M.; Alford, R.; Choyke, P. L. and Urano, Y. New Strategies for Fluorescent Probe Design in Medical Diagnostic Imaging, Chem Rev. 2010, 110, 2620–2640. 11. Longmire, M. R.; Ogawa, M.; Hama, Y.; Kosaka, N.; Regino, C. A.S.; Choyke, P. L. and Kobayashi, H. Determination of Optimal Rhodamine Fluorophore for in Vivo Optical Imaging. Bioconjug. Chem. 2008, 19, 1735-1742. 12. Yazdani, M. Concerns in the application of fluorescent probes DCDHF-DA, DHR 123 and DHE to measure reactive oxygen species in vitro, Toxicol. In Vitro, 30 (2015) 578–582. 13. Jensen, E. C. Use of Fluorescent Probes: Their Effect on Cell Biology and Limitations. AR Insights, 2012, 12, 2031−2036. 14. Sun, H.; Wang, L.; Ai, L. Determination of banned 10 azo-dyes in hot chili products by gel permeation chromatography liquid chromatography electrospray ionization tandem mass spectrometry, J. Chromatogr. A., 2007,1164, 120–128. 15. Tatebe, C.; Zhong, X.; Ohtsuki, T.; Kubota, H.; Sato, K.; Akiyama, H.; A simple and rapid chromatographic method to determine unauthorized basic colorants (rhodamine B, auramine O, and pararosaniline) in processed foods. Food Sci. Nutr. 2014, 2, 547–556. 16. Reyns, T.; Belpaire, C.; Geeraerts, C.; Van Loco, J.; Multi-dye residue analysis of triarylmethane, xanthene, phenothiazine and phenoxazine dyes in fish tissues by ultra-performance liquid chromatography–tandem mass spectrometry, J. Chromatogr. B. 2014, 953–954, 92–101.

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17. Soylak, M.; Unsal, Y. E.; Yilmaz, E.; Tuzen, M. Determination of rhodamine B in soft drink, waste water and lipstick samples after solid phase extraction. Food Chem. Toxicol. 2011, 49, 1796−1799. 18. Chen, H.; Meng, H.-H.; Lee, H.-C.; Cho, L.-L.; Tsai, S.-C.; Huang, M.-T.; Hsiao, C.-T.; Lin, A.C.-Y.; Chen, S.-J.; Lee, J.C.-I. Identification of Rhodamine 6G and Rhodamine B dyes present in ballpoint pen inks using high-performance liquid chromatography and UV-Vis spectrometry, Forensic Sci. J, 2007, 6, 21−37. 19. Hu, X.; Xiao, G.; Pan, W.; Mao, X.; Li, P. Simultaneous determination of 7 rhodamine

dyes

in

hot

chili

products

by high

performance

liquid

chromatography-tandem mass spectrometry, Chin. J. Chromatogr., 2010, 28, 590−595. 20. Cheng, Y.Y.; Tsai, T.H. A validated LC-MS/MS determination method for the illegal food additive rhodamine B: Applications of a pharmacokinetic study in rats, J. Pharm. Biomed. Anal., 2016, 125, 394−399. 21. Mason, R. W.; Edwards, I. R. High-performance liquid chromatographic determination of rhodamine B in rabbit and human plasma. J. Chromatogr. 1989, 491, 468–472. 22. Zhang, D.; Wang, L.; Chen, X.; Wang, J.; Cao, H.; Huang, L. Determination of ten basic dyes in meat products by ultra fast liquid chromatography-ion trap time of flight mass spectrometry, Chin. J. Chromatogr., 2012, 30, 770−776. 23. The Food and Drug Administration of Taiwan, Act Governing Food Safety and Sanitation,

Ministry

of

Health

and

Welfare

of

Taiwan,

http://law.moj.gov.tw/Eng/LawClass/LawContent.aspx?PCODE=L0040001. 24. U.S. Food and Drug Administration, Summary of Color Additives for Use in the United States in Foods, Drugs, Cosmetics, and Medical Devices. 2015, 23



https://www.fda.gov/forindustry/coloradditives/coloradditiveinventories/ucm115 641.htm 25. U.S. FDA. Guidance for Industry, Bioanalytical Method Validation; U.S. Department of Health and Human Services, Food and Drug Administration Center for Drug Evaluation and Research (CDER): Washington, DC, USA, 2001.  26. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research, in: N.R. Council (Ed.), http://www.nap.edu/catalog/10732.html, 2003. 27. McDowall, R.D. Sample preparation for biomedical analysis, J. Chromatogr., 1989, 492 , 3-58. 28. Blanchard, J. Evaluation of the relative efficacy of various techniques for deproteinizing plasma samples prior to high-performance liquid chromatographic analysis, J. Chromatogr.1981, 226, 455−460.

24


Page 24 of 35

Page 25 of 35


FUNDING Funding for this study was provided in part by research grants from the Ministry of Science and Technology of Taiwan (MOST 105-2113-M-010-004).

25



FIGURE CAPTIONS

Figure 1. Representative UHPLC-MS/MS chromatograms of (A) blank plasma; (B) blank plasma spiked with rhodamine 110 (250 ng/mL) and carvedilol (10 ng/mL); (C) plasma sample collected 30 min after the oral administration of rhodamine 110 (10 mg/kg). Peak 1: rhodamine 110, peak 2: carvedilol.

Figure 2. Mean plasma concentration–time profile in rat plasma after the administration of rhodamine 110: (○) rhodamine 110 (3 mg/kg, per os, p.o.); (▼) rhodamine 110 (10 mg/kg, p.o.); (●) rhodamine 110 (3 mg/kg, i.v.). Data are expressed as the means ± S.D. (n = 6).

Figure 3. Biodistribution of rhodamine 110 in the liver, heart, spleen, lungs, kidneys, brain, and plasma after oral administration of rhodamine 110 (3 mg/kg). Data are expressed as the means ± S.D. (n = 6); units of concentration for plasma and organ samples are ng/mL and ng/g, respectively.

26


Page 26 of 35

Page 27 of 35


Figure 1.

27



Rhodamine 110 concentration (ng/mL)

10000

Page 28 of 35

Rhodamine 110, 3 mg/kg, i.v. Rhodamine 110, 3 mg/kg, p.o. Rhodamine 110, 10 mg/kg, p.o.

1000

100

10

1 0

2

4

6

8

Time (hours) Figure 2.

28


10

12

14

Page 29 of 35


Figure 3

29



Page 30 of 35

Table 1. Inter-day and Intra-day Assay Precision (% R.S.D.) and Accuracy (% bias) Using the UHPLC-MS/MS Method to Quantify Rhodamine 110 in Various Biological Samples

Nominal

Intra-day

Inter-day

Conc.

Observe conc. Accuracy

Precision

Observed

Accuracy

Precision

(ng/mL)

(ng/mL)

Bias (%)

R.S.D (%)

Conc. (ng/mL)

Bias (%)

R.S.D (%)

50

51.16 ± 3.07

2.32

6.00

58.94 ± 4.25

17.88

7.21

100

105.4 ± 4.3

5.40

4.07

113.6 ± 9.9

13.60

8.71

500

488.3 ± 17.9

-2.34

3.67

526.7 ± 38.4

5.34

7.29

50

43.16 ± 1.43

-13.68

3.31

48.65 ± 9.40

-2.70

19.32

500

542.3 ± 19.8

8.46

3.65

567.4 ± 44.9

13.48

7.91

1000

976. 9 ± 15.7

-2.31

1.61

1006 ± 54

0.60

5.37

50

50.70 ± 2.94

1.40

5.80

47.68 ± 3.81

-4.64

7.99

100

107.7 ± 4.2

7.70

3.90

100.4 ± 7.0

0.40

6.97

500

502.3 ± 9.7

0.46

1.93

448.7 ± 32.1

-10.26

7.15

50

47.99 ± 1.50

-4.02

3.13

47.17 ± 1.56

-5.66

3.31

100

101.5 ± 2.0

1.50

1.93

105.0 ± 1.6

5.00

1.52

500

496.0 ± 13.7

-0.80

2.75

494.7 ± 2.7

-1.06

0.55

Plasma

Liver

Heart

Spleen

Lungs 30


Page 31 of 35


50

47.26 ± 3.80

-5.48

7.92

49.93 ± 9.52

-0.14

19.07

100

99.25 ± 4.10

-0.75

4.13

112.3 ± 15.1

12.30

13.45

500

498.4 ± 16.1

-0.32

3.23

513.7 ± 49.3

2.74

9.60

50

49.99 ± 4.64

-0.02

9.28

46.06 ± 2.76

-15.73

5.99

100

100.6 ± 5.8

0.60

5.77

103.2 ± 2.3

6.44

2.23

500

491.2 ± 13.5

-1.76

2.75

491.6 ± 5.0

-3.36

1.02

50

50.76 ± 1.45

1.52

2.86

49.59 ± 5.80

-0.80

11.70

100

109.9 ± 1.3

9.90

1.18

103.8 ± 4.4

3.78

4.24

500

496.8 ± 7.0

-0.64

1.41

493.2 ± 5.2

-1.35

1.05

Kidneys

Brain

Data are expressed as the means ± S.D. (n = 5); precision, R.S.D (%) = S.D. / Cobs × 100; accuracy, bias (%) = [(Cobs - Cnom) / Cnom] × 100.

31



Page 32 of 35

Table 2. Stability of Rhodamine 110 in Various Biological Samples

Nominal

Stock Solution Short-term

Auto-sampler

Freeze-thaw

Long-term

Stability (%)

Stability (%)

Stability (%)

Stability (%)

Concentration

Stability

(ng/mL)

(%) -0.67 ± 1.07 2.49 ± 1.16 -1.24 ± 0.78

Plasma 50

-8.80 ± 6.88

0.84 ± 3.95

-13.35 ± 1.26

0.20 ± 1.23

100

-5.23 ± 4.90

-1.96 ± 1.56

-6.12 ± 5.71

-3.15 ± 1.15

500

-4.64 ± 2.98

1.64 ± 1.71

-6.68 ± 4.58

-3.20 ± 0.47

50

2.23 ± 6.09

6.79 ± 10.7

5.09 ± 4.01

-7.51 ± 7.82

500

4.67 ± 3.35

7.37 ± 4.29

1.32 ± 4.06

0.30 ± 6.75

1000

4.71 ± 4.06

2.99 ± 2.36

0.58 ± 1.82

8.18 ± 3.49

50

-0.69 ± 4.66

3.43 ± 4.98

2.44 ± 3.83

-8.00 ± 6.54

100

-2.65 ± 5.71

-2.54 ± 2.09

-5.69 ± 6.36

-5.91 ± 4.17

500

-6.32 ± 2.34

-1.58 ± 1.00

-3.91 ± 3.34

2.81 ± 2.10

50

-2.68 ± 2.59

-2.40 ± 0.51

-1.12 ± 9.34

0.96 ± 5.74

100

-1.51 ± 3.26

1.50 ± 1.39

0.30 ± 0.87

1.57 ± 5.67

500

-1.55 ± 1.88

-1.57 ± 2.07

-1.60 ± 1.51

3.14 ± 2.56

Liver

Heart

Spleen

32


Page 33 of 35


Lungs 50

-0.56 ± 1.53

-3.42 ± 2.81

-4.13 ± 2.57

-1.39 ± 1.67

100

-2.35 ± 1.51

0.40 ± 1.73

-5.94 ± 4.58

-3.07 ± 1.58

500

0.19 ± 1.49

-1.87 ± 1.79

-3.92 ± 1.28

0.12 ± 1.73

50

-6.68 ± 6.27

-3.93 ± 5.34

6.79 ± 5.31

3.35 ± 3.27

100

-5.13 ± 2.51

-4.45 ± 1.91

0.33 ± 4.23

2.84 ± 5.96

500

-0.05 ± 1.91

-3.42 ± 0.48

-0.78 ± 1.86

5.09 ± 6.62

50

4.22 ± 1.05

6.79 ± 3.90

12.42 ± 2.22

-4.56 ± 2.67

100

4.91 ± 2.14

4.92 ± 1.19

8.62 ± 3.37

-4.77 ± 1.42

500

-0.99 ± 2.37

3.92 ± 0.78

3.31 ± 1.95

0.02 ± 2.12

Kidneys

Brain

Data are expressed as the means ± S.D. (n = 3).

33



Page 34 of 35

Table 3. Pharmacokinetic Parameters of Rhodamine 110 in Rats

Pharmacokinetic

Rhodamine 110

Parameters

3 mg/kg, i.v.

C0 (ng/mL)

1715 ± 585

3 mg/kg, p.o.

10 mg/kg, p.o.

Cmax (ng/mL)

283.4 ± 60.9

657.0 ± 126.7

Tmax (min)

140.0 ± 62.0

210.0 ± 32.9

t½ (min)

428.1 ± 142.2

182.0 ± 13.8

298.5 ± 173.8

AUC (min μg/mL)

384.3 ± 196.2

138.1 ± 20.3

444.0 ± 170.8

Cl (mL/min/kg)

9.63 ± 4.69

7.94 ± 1.15

8.61 ± 2.67

Vss (mL/kg)

5002 ± 1223 2096 ± 415

3332 ± 1081

365.8 ± 31.2

541.8 ± 274.3

35.94 ± 5.29

34.65 ± 13.33

Vd (mL/kg) MRT (min)

592.6 ± 202.2

F (%) a

Rhodamine 110 Rhodamine 110

Abbreviations: AUC, area under concentration-time curve; t1/2, half-life; Tmax, time

of peak plasma level; C0, initial drug concentration; Cmax, maximum concentration; Cl, clearance; MRT, mean residence time; Vss, apparent volume of distribution at steady state; Vd, volume of distribution; F, bioavailability. Data are expressed as the means ± S.D. (n = 6).

34


Page 35 of 35


Graphic for Table of Contents

35


Pharmacokinetics of Rhodamine 110 and Its Organ Distribution in

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