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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: thtsai@ym.edu.tw
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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
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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
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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
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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
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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
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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
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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
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Set 2: Standard solution spiked with post-extraction matrix. A blank plasma or
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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.
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Experimental Animals
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Adult male Sprague-Dawley rats (230 ± 20 g) were obtained from the Laboratory
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Animal Center at National Yang-Ming University, Taipei, Taiwan. The animal
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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
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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
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coagulation. Blood samples were centrifuged at 16,000 × g for 10 min at 4 C to
230
obtain plasma, which was stored at −20C 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
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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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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
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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
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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
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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
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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
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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
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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.
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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).
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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.
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Figure 1.
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Rhodamine 110 concentration (ng/mL)
10000
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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.
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12
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Figure 3
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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
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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.
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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
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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).
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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).
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