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Fluorescence derivatization method for sensitive chromatographic determination of zidovudine based on the Huisgen reaction
Accepted Manuscript Title: Fluorescence derivatization method for sensitive chromatographic determination of zidovudine based on the Huisgen reaction Author: Yuki Maeda Naoya Kishikawa Kaname Ohyama Mitsuhiro Wada Rie Ikeda Naotaka Kuroda PII: DOI: Reference:
S0021-9673(14)00913-3 http://dx.doi.org/doi:10.1016/j.chroma.2014.06.017 CHROMA 355493
To appear in:
Journal of Chromatography A
Received date: Revised date: Accepted date:
1-5-2014 3-6-2014 3-6-2014
Please cite this article as: Y. Maeda, N. Kishikawa, K. Ohyama, M. Wada, R. Ikeda, N. Kuroda, Fluorescence derivatization method for sensitive chromatographic determination of zidovudine based on the Huisgen reaction, Journal of Chromatography A (2014), http://dx.doi.org/10.1016/j.chroma.2014.06.017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Fluorescence derivatization method for sensitive chromatographic determination
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of zidovudine based on the Huisgen reaction
3 Yuki Maeda, Naoya Kishikawa, Kaname Ohyama, Mitsuhiro Wada, Rie Ikeda and
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Naotaka Kuroda
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Graduate School of Biomedical Sciences, Course of Pharmaceutical Sciences,
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Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
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an
10 *Corresponding author: Naotaka Kuroda
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Graduate School of Biomedical Sciences, Course of Pharmaceutical Sciences,
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Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
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Phone: +81-95-819-2894
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E-mail: [email protected]
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Ac ce p
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Fax: +81-95-819-2444
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Abstract
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A novel pre-column fluorescence derivatization method for chromatographic analysis of azide compounds was developed based on the Huisgen reaction, which is a
20
specific cycloaddition reaction between an alkyne and an azide.
21
synthesized a fluorescent alkyne, 2-(4-ethynylphenyl)-4,5-diphenyl-1H-imidazole
22
(DIB-ET) as a reagent.
23
fluorophore and reactive center, respectively.
24
of DIB-ET, a high-performance liquid chromatography with fluorescence detection
25
method was developed for the determination of zidovudine as a model of azide
26
compound.
27
sulfate and L-ascorbic acid as catalysts, and the formed derivative was detected
28
fluorometrically.
29
of zidovudine in plasma samples with the detection limit of 0.28 ng mL-1 at a S/N = 3.
30
Finally, the proposed method could be applied to determine the zidovudine
31
concentration in rat plasma after administration of zidovudine without interference
33 34 35 36
cr
We designed and
DIB-ET has a lophine skeleton carrying an alkyne acting as
an
us
In order to evaluate the practicality
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Zidovudine could be reacted with DIB-ET in the presence of copper (II)
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The proposed method allows sensitive and selective determination
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from biological components.
Keywords: Huisgen reaction, Fluorescence derivatization, Azide group, Zidovudine, Fluorescent alkyne
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1. Introduction
37 38
It is important to understand the pharmacokinetics of drugs that can cause beneficial or adverse side effects even in small amounts.
40
analytical method is necessary to determine small amounts of drugs in biological
41
samples.
42
detection is a powerful analytical technique to determine drugs owing to its high
43
sensitivity and selectivity [1, 2].
44
fluorescence, derivatization reagents have been frequently adopted in order to convert
45
non-fluorescent drugs to strongly fluorescent derivatives.
46
of fluorescence derivatization reagents have been developed and applied for the
47
determination of small amounts of drugs in biological samples [3-7].
48
fluorescence derivatization reagents can easily react with co-existing biological
49
components and the resultant products can interfere with the detection of target drugs.
50
In order to overcome this problem, we have been attempted to develop a novel
51
fluorescence reagent that can react with target drug selectively even in the presence of
53 54 55 56
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High-performance liquid chromatography (HPLC) with fluorescence
us
But, since most drugs do not possess native
However,
d
M
an
Up to now, various types
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52
Therefore, a sensitive
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39
biological components, based on specific chemical reactions.
We so far developed a
fluorescent aryl boronic acid, 4-(4,5-diphenyl-1H-imidazol-2-yl)phenylboronic acid (DPA, a lophine derivative) as a fluorescence derivatization reagent for aryl halides based on the Suzuki coupling reaction, which is a cross-coupling reaction between
aryl halides and aryl boronic acids [8-11].
It was found that DPA could react
57
selectively with aryl halide drugs such as haloperidol in the presence of biological
58
components.
59
4-(4,5-diphenyl-1H-imidazol-2-yl)iodobenzene, could be used as a specific
60
fluorescence derivatization reagent for alprenolol that has a terminal double bond
61
moiety based on the Mizoroki-Heck coupling reaction, which is a reaction between
Also, we recently reported that a lophine based fluorescent aryl iodide,
3
Page 3 of 23
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aryl halides and terminal double bonds [12].
63
strategy, we attempted to develop a new type of specific fluorescence derivatization
64
reagent for azide group based on the Huisgen reaction. The Huisgen reaction [13-16] is a specific cycloaddition reaction between an
ip t
65
As an expansion of the analytical
66
alkyne and an azide in the presence of copper (I) as a catalyst to form stable
67
five-membered triazole ring [17].
68
representative reaction of click chemistry and is frequently used to build functional
69
molecules in various research fields.
70
reaction, we considered that fluorescent alkyne and fluorescent azide could be used as
71
specific derivatization reagent for azide group and alkyne moiety, respectively.
72
Huisgen reaction proceeds under mild conditions even in the presence of water, while
73
many general derivatization reactions often require hard conditions such as high
74
temperature and long reaction time, and are likely inhibited by water.
75
fluorescence derivatization reaction based on the Huisgen reaction should be suitable
76
for biomedical analysis.
77
fluorescent alkyne, 2-(4-ethynylphenyl)-4,5-diphenyl-1H-imidazole (DIB-ET) as a
79 80 81 82
cr
The
Therefore,
d
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an
us
Owing to the high specificity of the Huisgen
te
From these aspects, we designed and synthesized a
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The Huisgen reaction is known as the most
specific fluorescence derivatization reagent for azide group (Fig. 1).
DIB-ET has a
lophine skeleton carrying an alkyne acting as fluorophore and reactive center, respectively.
In the present study, we applied DIB-ET to the development of
determination method for zidovudine as a model of azide compound.
Zidovudine is
an antiretroviral drug used for the treatment of human immunodeficiency virus (HIV)
83
infection [18, 19].
Since the adverse side effects of zidovudine, such as lactic
84
acidosis or myelosuppression were reported [20], the sensitive and selective
85
determination method for zidovudine should be useful to facilitate the safe dosing of
86
zidovudine.
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derivative (DIB-zidovudine) after reaction with DIB-ET (Fig. 1), and DIB-zidovudine
We confirmed that zidovudine could be converted to fluorescent
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could be sensitively determined by HPLC with fluorescence detection.
Furthermore,
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the developed method was successfully applied to the determination of zidovudine in
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rat plasma after administration without any interference from biological components.
92
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91 2. Experimental
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2.1. Materials and reagents
Zidovudine was purchased from Tokyo Chemical Industries (Tokyo, Japan).
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Copper (II) sulfate, L-ascorbic acid, benzil and ammonium acetate were obtained from
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Nacalai Tesque (Osaka, Japan).
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Louis, MO, USA).
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(Tokyo).
an
4-Ethynylbenzaldehyde was from Sigma (St.
Cellulose acetate membrane filter (0.45 µM) was from Advantec
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Wistar male rats were obtained from Kyudo Experimental Animal
Laboratory (Saga, Japan).
DIB-ET and DIB-zidovudine were synthesized as
101
described in later sections.
Water was distilled and passed through a Pure Line
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WL21P system (Yamato, Tokyo, Japan).
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purity and quality available.
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te
All other chemicals were of the highest
Stock solution of zidovudine was prepared in methanol
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and stored at 4 ºC.
DIB-ET, copper (II) sulfate and L-ascorbic acid dissolved in
methanol just before use.
2.2. Equipment
The HPLC system consisted of two Shimadzu LC-10AT pumps (Kyoto,
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Japan), a Shimadzu RF-20Axs fluorescence detector, a Rheodyne (Cotati, CA, USA)
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7125 injector with a 20-µL loop and Chromato-Pro chromatography data acquisition
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system (Run Time Corporation, Kanagawa, Japan).
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Fluorescence spectra were measured with a Shimadzu RF-1500 spectrofluorophotometer.
Mass spectral data were obtained with a JEOL
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JMS-700N spectrometer (Tokyo, Japan).
Elemental analyses were performed on a
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Perkin Elmer 2400II (Norwalk, CT, USA).
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Yanagimoto MP-53 melting point apparatus (Kyoto, Japan).
Melting points were measured with a
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Chromatographic separation was performed on a Cosmosil 5C18-AR-II
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(250×4.6 mm i.d., Nacalai Tesque, Osaka, Japan) column with a gradient elution
121
program using solvent A (acetonitrile-5 mM Tris-HCl buffer (pH 7.4) (50:50, v/v%))
122
and solvent B (acetonitrile).
123
0% B (0–9.5 min), 0% B to 100% B linearly (9.5–10.0 min), and 100% B (10.0–19.0
124
min).
125
excitation and emission wavelength were set at 310 nm and 400 nm, respectively.
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an
The gradient program was programmed as follows:
130 131 132 133 134 135
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DIB-ET was synthesized according to the previous papers [12, 21].
Benzil
(157.5 mg, 0.75 mmol), ammonium acetate (500 mg, 6.5 mmol) and
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2.4. Synthesis of DIB-ET
d
126 127
The
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The flow-rate was set at 1.0 mL min-1 at ambient temperature.
4-ethynylbenzaldehyde (97.5 mg, 0.81 mmol) were dissolved in 1.5 mL of acetic acid. This mixture was heated at 90 °C for 8 h. mixture was poured into cold water.
After cooling to room temperature, the
The resultant precipitate was obtained as
yellow crystals; yield: 191 mg, 78%, mp: 264 °C.
Elemental analysis; calculated for
C23H16N2: C, 86.22%; H, 5.03%, N, 8.74%, found: C, 86.17%, H, 5.00%, N, 8.82%.
FAB-MS (m/z) calculated: 321 [M+H]+, found 321.
136 137 138 139
2.5. Synthesis of authentic DIB-zidovudine DIB-ET (30.0 mg, 94 µmol), zidovudine (24.9 mg, 94 µmol), copper (II) sulfate (224.7 mg, 900 µmol) and L-ascorbic acid (159 mg, 900 µmol) were dissolved
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in 10 mL of tetrahydrofuran-water (50:50, v/v%).
After heating at 50 °C for 2 h, the
141
solution was evaporated and filtered.
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mL of acetonitrile and water to give DIB-zidovudine as yellow crystals; yield: 28 mg,
143
51%, mp: 248-250 °C.
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H, 4.97%, N, 16.68%, found: C, 67.57%, H, 5.22%, N, 16.89%. FAB-MS (m/z)
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calculated: 588 [M+H]+, found 588.
The resulting precipitate was washed with 10
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Elemental analysis; calculated for C33H29N7 O4: C, 67.45%;
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2.6. Fluorescent derivatization procedure for standard zidovudine
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To 25 µL 0.32-1336 ng mL-1 zidovudine in methanol, 50 µL 4 mM DIB-ET in
148
methanol, 25 µL 40 mM copper (II) sulfate and 180 mM L-ascorbic acid in methanol
150
were successively added.
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reduction of copper (II) sulfate with L-ascorbic acid.
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reaction mixture was heated at 40 ºC for 5 min.
153
filter, a 20 µL of aliquot was injected into HPLC system.
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After filtration through a membrane
2.7. Assay procedure for zidovudine in rat plasma Wistar male rats (240 ± 2 g) were anesthetized with ethyl carbamate (1.5 mg
kg-1).
Zidovudine administration was performed via right inguinal vein at a dose of
10 mg kg-1 [22].
Blood samples were collected through indwelling arterial catheters,
transferred to heparinized collection tubes and centrifuged at 2000×g for 10 min.
After centrifugation, the plasma was separated and stored at -80 °C in the dark until
161
analysis.
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University Animal Care and Use Committee.
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After vortex-mixing, the
Ac ce p
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In this procedure, copper (I) was generated by the
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an
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The experiment was performed with an approval of the Nagasaki
Fifty microliters of methanol was added into 50 µL of plasma samples.
164
Then, samples were centrifuged at 5000×g for 10 min.
165
supernatant was collected and was subjected to the derivatization reaction as
7
Twenty-five microliters of
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previously described.
Pharmacokinetic parameters were determined by moment
167
analysis using a single-dose i.v. bolus drug administration model.
168
parameters were area under the curve (AUC), mean residence time (MRT), terminal
169
phase half life (T1/2), volume of distribution (Vd) and clearance (CL).
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3.1. Fluorescence characteristics of DIB-ET and DIB-zidovudine
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The studied
The fluorescence spectra of DIB-ET were measured in methanol and the maximum excitation and emission wavelengths of DIB-ET were found to be 310 and
176
400 nm, respectively.
177
wavelengths of DIB-zidovudine were almost identical to those of DIB-ET.
178
addition, the fluorescence intensity of DIB-zidovudine was almost identical to that of
179
same concentration of DIB-ET.
180
that the fluorescence characteristics of DIB-ET were not changed after the reaction
181
with zidovudine although the extension of the conjugation could be occurred by the
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On the other hand, the maximum excitation and emission In
te
d
In view of results described above, it was suggested
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an
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formation of triazole ring.
3.2. Optimization of fluorescent derivatization conditions Figure 2 (A) and (B) show typical chromatograms obtained from the reagent
blank and the reaction mixture of zidovudine with DIB-ET, respectively.
The peak
187
of DIB-zidovudine was detected at 8 min, while the excess DIB-ET was eluted later
188
than DIB-zidovudine and detected at approximately 15 min.
189
To obtain higher reactivity, the conditions used for the derivatization reaction
190
were optimized using a standard solution of zidovudine.
191
solvent on the reactivity was examined with methanol, tetrahydrofuran,
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The effect of reaction
Page 8 of 23
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N,N-dimethylformamide, 1-buthanol, ethanol and acetonitrile.
193
solvents, the optimal result was obtained with methanol (Fig. S1, supplementary
194
data).
195
methanol was selected as the reaction solvent.
196
investigated over a range of 0.5-6 mM and the maximum peak area of
197
DIB-zidovudine was obtained using 4 mM DIB-ET (Fig. 3A).
198
copper (II) sulfate was examined over a range of 10–120 mM, and the maximum and
199
constant peak area was obtained at a concentration over 30 mM (Fig. S2,
200
supplementary data); 40 mM copper (II) sulfate was selected.
201
concentration of L-ascorbic acid was examined over a range of 100-600 mM and 180
202
mM was selected because it gave maximum peak areas (Fig. S3, supplementary data).
203
The effects of reaction temperature and time were investigated (Fig. 3B).
204
almost same reactivity was obtained at temperature higher than 40 ºC, and the
205
maximum and constant reactivity was obtained for more than 5 min.
206
hand at room temperature, the reactivity was slightly reduced as compared with
207
heating.
209 210 211 212 213
In addition, since methanol can be used for the deproteinization of plasma,
ip t
The concentration of DIB-ET was
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The concentration of
The
On the other
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an
The effect of
As a result, 40 °C and 5 min were chosen for the reaction temperature and
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208
Among these
time, respectively, because these conditions gave high and constant reactivity. Under the optimized conditions, the reaction yield, which was estimated by comparing the peak areas of DIB-zidovudine in the reaction mixture with that of authentic DIB-zidovudine, was 78%.
3.3.
Calibration curve, detection limit and repeatability Validation is carried out according to PDA guideline for bioanalytical method
214 215
[23].
A calibration curve was prepared by spiking blank plasma with varying
216
concentration of zidovudine.
217
peak area and zidovudine concentration over the range from 0.32 to 1336 ng mL-1.
A linear relationship (r = 0.999) was obtained between
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The slope and intercept of regression equation (mean ± standard error, n = 3) were
219
4.03×105 ± 1.5×104 and 1.1×102 ± 8.8×10, respectively.
220
zidovudine in rat plasma was 0.28 ng mL-1 (20 fmol/injection) at a signal-to-noise
221
ratio (S/N) of 3.
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that of HPLC with UV detection [24], 3 times higher than that of HPLC with
223
electrochemical detection [25] and 3 times higher than that of HPLC with
224
electrospray ionization tandem mass spectrometry [26].
225
method was 4 times less sensitive than radio-immunoassay [27], the proposed method
226
does not require special and hazardous materials unlike radio-immunoassay.
227
Accuracy and precision of the proposed method were examined using three levels
228
(2.67, 53.4 and 802 ng mL-1) of zidovudine in rat plasma.
229
within- and between-day accuracy ranged from 91.1% to 105% with %RSD of
230
precision less than 5.2%.
231
sufficient accuracy and precision.
235 236 237 238
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Although the proposed
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As shown in Table 1, the
d
Therefore, it was proved that the proposed method showed
3.4.
Determination of zidovudine in rat plasma after administration
Ac ce p
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The sensitivity of the proposed method was 26 times higher than
te
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The detection limit of
To demonstrate the practicality of the proposed method, the zidovudine
concentration in rat plasma after a single dose administration was determined. Typical chromatograms of blank rat plasma and rat plasma collected after 15 min after the administration of zidovudine (10 mg kg-1) are shown in Fig. 4 (A) and (B), respectively.
The peak of DIB-zidovudine could be detected clearly and there were
239
no interfering peaks derived from plasma components.
These results mean that
240
DIB-ET reacted selectively with zidovudine and did not react with most of biological
241
components.
242
zidovudine.
243
administration.
Figure 5 shows mean plasma concentration vs. time profiles of The zidovudine concentration in rat plasma decreased with time after The pharmacokinetic parameters (mean ± standard error, n = 3)
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were summarized as follows; AUC0→∞ 6190 ± 2290 min mg L-1; MRT 180.89 ±
245
65.71 min; T1/2 121.58 ± 47.16 min; Vd 68.02 ± 10.70 mL; CL 0.39 ± 0.15 L h-1 kg-1.
246
The results are consistent with reported results in previous study [28].
247
practicality of the proposed method was confirmed by real sample analysis.
248
consideration of high sensitivity and selectivity, the proposed method should be
249
useful for therapeutic drug monitoring of zidovudine in routine clinical practice with
250
simple procedure for sample preparation.
cr
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In
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251 4. Conclusion
an
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The
253
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In this study, we developed a sensitive and selective HPLC determination
254 255
method for zidovudine by fluorescent alkyne, DIB-ET as a specific fluorescence
256
derivatization reagent.
257
based on the Huisgen reaction and the DIB-zidovudine could be detected clearly on
258
the chromatogram without interference from biological components.
259
derivatization method was successfully applied to the determination of zidovudine in
261 262 263 264
d
te
Ac ce p
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DIB-ET reacted selectively with azide group of zidovudine
rat plasma after administration.
The proposed
In the present study, the Huisgen reaction could be
subjected to fluorescent derivatization reaction for chromatographic analysis for the first time.
In further investigation, we will attempt to develop a selective
derivatization method for alkyne compounds by using fluorescent azide compound as a reagent on the basis of the Huisgen reaction.
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[18] G.J. Veal, D.J. Back, Metabolism of zidovudine, Gen. Pharmacol. 26 (1995)
331
1469-1475.
333 334 335 336 337
te
Ac ce p
332
d
329
[19] M.A. Raviolo, J.M. Sanchez, M.C. Briñón, M.A. Perillo, Determination of liposome permeability of ionizable carbamates of zidovudine by steady state fluorescence spectroscopy, Colloids Surf. B 61 (2008) 188-198.
[20] M. Gerschenson, K. Brinkman, Mitochondrial dysfunction in AIDS and its treatment, Mitochondrion 4 (2004) 763-777.
[21] K. Nakashima, H. Yamasaki, N. Kuroda, S. Akiyama, Evaluation of lophine
338
derivatives as chemiluminogens by a flow-injection method, Anal. Chim. Acta 303
339
(1995) 103-107.
340 341
[22] L. Zhang, Y. Deng, Y. Li, H. Wu, S. Xu, Investigation of the pharmacokinetics and determination of cholesteryl carbonate zidovudine in rat plasma by
14
Page 14 of 23
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non-aqueous reversed-phase high performance liquid chromatography with UV
343
detection, J. Chromatogr. B 853 (2007) 163-167.
344
[23] U.S. Department of Health and Human Services Food and Drug Administration, Center for Drug Evaluation and Research (CDER), and Center for Veterinary
346
Medicine (CVM), Guidance for Industry, Bioanalytical Method Validation,
347
http://www.fda.gov/downloads/prugs/Guisdances/ucm070107.pdf (accessed 18
348
Jan., 2014).
cr
[24] G. Ramachandran, A.K. Hemanthkumar, V. Kumaraswami, S. Swaminathan, A
us
349
ip t
345
simple and rapid liquid chromatography method for simultaneous determination of
351
zidovudine and nevirapine in plasma, J. Chromatogr. B 843 (2006) 339-344.
an
350
[25] Y. Chen, Y.X. Liu, Z.D. Chen, M.L. Chen, Y. Zhu, Determination of zidovudine
353
using anion exchange chromatography with integrated pulsed amperometric
354
detection, Chin. Chem. Lett. 23 (2012) 715-718. [26] K.B. Kenney, S.A. Wring, R.M. Carr, G.N. Wells, J.A. Dunn, Simultaneous
d
355
M
352
determination of zidovudine and lamivudine in human serum using HPLC with
357
tandem mass spectrometry, J. Pharm. Biomed. Anal. 22 (2000) 967-983.
359 360 361 362
Ac ce p
358
te
356
[27] K. Peter, J.P. Lalezari, J.G. Gambertoglio, Quantification of zidovudine and individual zidovudine phosphates in peripheral blood mononuclear cells by a combined isocratic high performance liquid chromatography radioimmunoassay method, J. Pharm. Biomed. Anal. 14 (1996) 491-499.
[28] S.D. Brown, M.G. Bartlett, C.A. White, Pharmacokinetics of intravenous
363
acyclovir, zidovudine, and acyclovir-zidovudine in pregnant rats, Antimicrob.
364
Agents Chemother. 47 (2003) 991-996.
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Figure captions
366
368
Fig. 1. Fluorescence derivatization reaction of zidovudine with DIB-ET based on the Huisgen reaction.
ip t
367
369
371
Fig. 2. Chromatograms of (A) reagent blank and (B) standard solution of 5 µM
cr
370
zidovudine.
373
us
372
Fig. 3. (A) Effect of DIB-ET concentration on the peak area of zidovudine. Derivatization conditions: concentrations of zidovudine, copper (II) sulfate
375
and L-ascorbic acid were 2 µM, 120 mM and 600 mM, respectively.
376
Reaction temperature and time were 50 ˚C and 60 min, respectively.
377
(B) Effects of reaction temperature and reaction time on the peak area of
378
zidovudine. Derivatization conditions: concentrations of zidovudine, DIB-ET,
379
copper (II) sulfate and L-ascorbic acid were 2 µM, 4 mM, 40 mM and 180
380
mM, respectively.
382 383 384 385
M
d
te
Ac ce p
381
an
374
Fig. 4. Chromatograms of (A) rat plasma sample and (B) that obtained at 15 min after i.v. administration of zidovudine (10 mg kg-1).
Fig. 5. Mean plasma concentration-time profiles of zidovudine in rat plasma
386
following administration via right inguinal vein at a dose of 10 mg kg-1 to
387
Wistar male rats (data are means ± standard error, n = 3).
388
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388
Table1 Within- and between-day accuracy and precision of the proposed method
389
for determination of zidovudine in rat plasma.
Zidovudine -1
(ng mL )
a
Between-day (n=5)
Accuracy
Precision
Accuracy
(%)
(RSD a, %)
(%)
2.67
91.1
4.8
92.3
53.4
103
3.2
105
1.9
802
99.1
3.2
103
1.6
Relative standard deviation
ip t (RSD a, %)
cr
5.2
an
391
Precision
us
390
Within-day (n=5)
Ac ce p
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d
M
392
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Page 17 of 23
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an
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Highlights A fluorescence derivatization method for determination of zidovudine is described. Zidovudine can be reacted with fluorescent alkyne based on the Huisgen reaction. The method allows sensitive and selective determination of zidovudine in plasma.
Ac ce p
392 393 394 395 396
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Figure 2
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Figure 3
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Figure 4
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S0021-9673(14)00913-3 http://dx.doi.org/doi:10.1016/j.chroma.2014.06.017 CHROMA 355493
To appear in:
Journal of Chromatography A
Received date: Revised date: Accepted date:
1-5-2014 3-6-2014 3-6-2014
Please cite this article as: Y. Maeda, N. Kishikawa, K. Ohyama, M. Wada, R. Ikeda, N. Kuroda, Fluorescence derivatization method for sensitive chromatographic determination of zidovudine based on the Huisgen reaction, Journal of Chromatography A (2014), http://dx.doi.org/10.1016/j.chroma.2014.06.017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Fluorescence derivatization method for sensitive chromatographic determination
2
of zidovudine based on the Huisgen reaction
3 Yuki Maeda, Naoya Kishikawa, Kaname Ohyama, Mitsuhiro Wada, Rie Ikeda and
5
Naotaka Kuroda
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4
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6
Graduate School of Biomedical Sciences, Course of Pharmaceutical Sciences,
8
Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
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7
9
an
10 *Corresponding author: Naotaka Kuroda
12
Graduate School of Biomedical Sciences, Course of Pharmaceutical Sciences,
13
Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
14
Phone: +81-95-819-2894
15
E-mail: [email protected]
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11
Ac ce p
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Fax: +81-95-819-2444
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Page 1 of 23
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Abstract
17 18
A novel pre-column fluorescence derivatization method for chromatographic analysis of azide compounds was developed based on the Huisgen reaction, which is a
20
specific cycloaddition reaction between an alkyne and an azide.
21
synthesized a fluorescent alkyne, 2-(4-ethynylphenyl)-4,5-diphenyl-1H-imidazole
22
(DIB-ET) as a reagent.
23
fluorophore and reactive center, respectively.
24
of DIB-ET, a high-performance liquid chromatography with fluorescence detection
25
method was developed for the determination of zidovudine as a model of azide
26
compound.
27
sulfate and L-ascorbic acid as catalysts, and the formed derivative was detected
28
fluorometrically.
29
of zidovudine in plasma samples with the detection limit of 0.28 ng mL-1 at a S/N = 3.
30
Finally, the proposed method could be applied to determine the zidovudine
31
concentration in rat plasma after administration of zidovudine without interference
33 34 35 36
cr
We designed and
DIB-ET has a lophine skeleton carrying an alkyne acting as
an
us
In order to evaluate the practicality
M
Zidovudine could be reacted with DIB-ET in the presence of copper (II)
te
d
The proposed method allows sensitive and selective determination
Ac ce p
32
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19
from biological components.
Keywords: Huisgen reaction, Fluorescence derivatization, Azide group, Zidovudine, Fluorescent alkyne
2
Page 2 of 23
36
1. Introduction
37 38
It is important to understand the pharmacokinetics of drugs that can cause beneficial or adverse side effects even in small amounts.
40
analytical method is necessary to determine small amounts of drugs in biological
41
samples.
42
detection is a powerful analytical technique to determine drugs owing to its high
43
sensitivity and selectivity [1, 2].
44
fluorescence, derivatization reagents have been frequently adopted in order to convert
45
non-fluorescent drugs to strongly fluorescent derivatives.
46
of fluorescence derivatization reagents have been developed and applied for the
47
determination of small amounts of drugs in biological samples [3-7].
48
fluorescence derivatization reagents can easily react with co-existing biological
49
components and the resultant products can interfere with the detection of target drugs.
50
In order to overcome this problem, we have been attempted to develop a novel
51
fluorescence reagent that can react with target drug selectively even in the presence of
53 54 55 56
cr
High-performance liquid chromatography (HPLC) with fluorescence
us
But, since most drugs do not possess native
However,
d
M
an
Up to now, various types
te
Ac ce p
52
Therefore, a sensitive
ip t
39
biological components, based on specific chemical reactions.
We so far developed a
fluorescent aryl boronic acid, 4-(4,5-diphenyl-1H-imidazol-2-yl)phenylboronic acid (DPA, a lophine derivative) as a fluorescence derivatization reagent for aryl halides based on the Suzuki coupling reaction, which is a cross-coupling reaction between
aryl halides and aryl boronic acids [8-11].
It was found that DPA could react
57
selectively with aryl halide drugs such as haloperidol in the presence of biological
58
components.
59
4-(4,5-diphenyl-1H-imidazol-2-yl)iodobenzene, could be used as a specific
60
fluorescence derivatization reagent for alprenolol that has a terminal double bond
61
moiety based on the Mizoroki-Heck coupling reaction, which is a reaction between
Also, we recently reported that a lophine based fluorescent aryl iodide,
3
Page 3 of 23
62
aryl halides and terminal double bonds [12].
63
strategy, we attempted to develop a new type of specific fluorescence derivatization
64
reagent for azide group based on the Huisgen reaction. The Huisgen reaction [13-16] is a specific cycloaddition reaction between an
ip t
65
As an expansion of the analytical
66
alkyne and an azide in the presence of copper (I) as a catalyst to form stable
67
five-membered triazole ring [17].
68
representative reaction of click chemistry and is frequently used to build functional
69
molecules in various research fields.
70
reaction, we considered that fluorescent alkyne and fluorescent azide could be used as
71
specific derivatization reagent for azide group and alkyne moiety, respectively.
72
Huisgen reaction proceeds under mild conditions even in the presence of water, while
73
many general derivatization reactions often require hard conditions such as high
74
temperature and long reaction time, and are likely inhibited by water.
75
fluorescence derivatization reaction based on the Huisgen reaction should be suitable
76
for biomedical analysis.
77
fluorescent alkyne, 2-(4-ethynylphenyl)-4,5-diphenyl-1H-imidazole (DIB-ET) as a
79 80 81 82
cr
The
Therefore,
d
M
an
us
Owing to the high specificity of the Huisgen
te
From these aspects, we designed and synthesized a
Ac ce p
78
The Huisgen reaction is known as the most
specific fluorescence derivatization reagent for azide group (Fig. 1).
DIB-ET has a
lophine skeleton carrying an alkyne acting as fluorophore and reactive center, respectively.
In the present study, we applied DIB-ET to the development of
determination method for zidovudine as a model of azide compound.
Zidovudine is
an antiretroviral drug used for the treatment of human immunodeficiency virus (HIV)
83
infection [18, 19].
Since the adverse side effects of zidovudine, such as lactic
84
acidosis or myelosuppression were reported [20], the sensitive and selective
85
determination method for zidovudine should be useful to facilitate the safe dosing of
86
zidovudine.
87
derivative (DIB-zidovudine) after reaction with DIB-ET (Fig. 1), and DIB-zidovudine
We confirmed that zidovudine could be converted to fluorescent
4
Page 4 of 23
88
could be sensitively determined by HPLC with fluorescence detection.
Furthermore,
89
the developed method was successfully applied to the determination of zidovudine in
90
rat plasma after administration without any interference from biological components.
92
ip t
91 2. Experimental
95
2.1. Materials and reagents
Zidovudine was purchased from Tokyo Chemical Industries (Tokyo, Japan).
us
94
cr
93
96
Copper (II) sulfate, L-ascorbic acid, benzil and ammonium acetate were obtained from
97
Nacalai Tesque (Osaka, Japan).
98
Louis, MO, USA).
99
(Tokyo).
an
4-Ethynylbenzaldehyde was from Sigma (St.
Cellulose acetate membrane filter (0.45 µM) was from Advantec
M
Wistar male rats were obtained from Kyudo Experimental Animal
Laboratory (Saga, Japan).
DIB-ET and DIB-zidovudine were synthesized as
101
described in later sections.
Water was distilled and passed through a Pure Line
102
WL21P system (Yamato, Tokyo, Japan).
103
purity and quality available.
105 106 107 108
te
All other chemicals were of the highest
Stock solution of zidovudine was prepared in methanol
Ac ce p
104
d
100
and stored at 4 ºC.
DIB-ET, copper (II) sulfate and L-ascorbic acid dissolved in
methanol just before use.
2.2. Equipment
The HPLC system consisted of two Shimadzu LC-10AT pumps (Kyoto,
109
Japan), a Shimadzu RF-20Axs fluorescence detector, a Rheodyne (Cotati, CA, USA)
110
7125 injector with a 20-µL loop and Chromato-Pro chromatography data acquisition
111
system (Run Time Corporation, Kanagawa, Japan).
112 113
Fluorescence spectra were measured with a Shimadzu RF-1500 spectrofluorophotometer.
Mass spectral data were obtained with a JEOL
5
Page 5 of 23
114
JMS-700N spectrometer (Tokyo, Japan).
Elemental analyses were performed on a
115
Perkin Elmer 2400II (Norwalk, CT, USA).
116
Yanagimoto MP-53 melting point apparatus (Kyoto, Japan).
Melting points were measured with a
118
ip t
117 2.3. HPLC conditions
Chromatographic separation was performed on a Cosmosil 5C18-AR-II
cr
119
(250×4.6 mm i.d., Nacalai Tesque, Osaka, Japan) column with a gradient elution
121
program using solvent A (acetonitrile-5 mM Tris-HCl buffer (pH 7.4) (50:50, v/v%))
122
and solvent B (acetonitrile).
123
0% B (0–9.5 min), 0% B to 100% B linearly (9.5–10.0 min), and 100% B (10.0–19.0
124
min).
125
excitation and emission wavelength were set at 310 nm and 400 nm, respectively.
us
120
an
The gradient program was programmed as follows:
130 131 132 133 134 135
te
129
DIB-ET was synthesized according to the previous papers [12, 21].
Benzil
(157.5 mg, 0.75 mmol), ammonium acetate (500 mg, 6.5 mmol) and
Ac ce p
128
2.4. Synthesis of DIB-ET
d
126 127
The
M
The flow-rate was set at 1.0 mL min-1 at ambient temperature.
4-ethynylbenzaldehyde (97.5 mg, 0.81 mmol) were dissolved in 1.5 mL of acetic acid. This mixture was heated at 90 °C for 8 h. mixture was poured into cold water.
After cooling to room temperature, the
The resultant precipitate was obtained as
yellow crystals; yield: 191 mg, 78%, mp: 264 °C.
Elemental analysis; calculated for
C23H16N2: C, 86.22%; H, 5.03%, N, 8.74%, found: C, 86.17%, H, 5.00%, N, 8.82%.
FAB-MS (m/z) calculated: 321 [M+H]+, found 321.
136 137 138 139
2.5. Synthesis of authentic DIB-zidovudine DIB-ET (30.0 mg, 94 µmol), zidovudine (24.9 mg, 94 µmol), copper (II) sulfate (224.7 mg, 900 µmol) and L-ascorbic acid (159 mg, 900 µmol) were dissolved
6
Page 6 of 23
140
in 10 mL of tetrahydrofuran-water (50:50, v/v%).
After heating at 50 °C for 2 h, the
141
solution was evaporated and filtered.
142
mL of acetonitrile and water to give DIB-zidovudine as yellow crystals; yield: 28 mg,
143
51%, mp: 248-250 °C.
144
H, 4.97%, N, 16.68%, found: C, 67.57%, H, 5.22%, N, 16.89%. FAB-MS (m/z)
145
calculated: 588 [M+H]+, found 588.
The resulting precipitate was washed with 10
cr
ip t
Elemental analysis; calculated for C33H29N7 O4: C, 67.45%;
146
2.6. Fluorescent derivatization procedure for standard zidovudine
us
147
To 25 µL 0.32-1336 ng mL-1 zidovudine in methanol, 50 µL 4 mM DIB-ET in
148
methanol, 25 µL 40 mM copper (II) sulfate and 180 mM L-ascorbic acid in methanol
150
were successively added.
151
reduction of copper (II) sulfate with L-ascorbic acid.
152
reaction mixture was heated at 40 ºC for 5 min.
153
filter, a 20 µL of aliquot was injected into HPLC system.
157 158 159 160
M
d
After filtration through a membrane
2.7. Assay procedure for zidovudine in rat plasma Wistar male rats (240 ± 2 g) were anesthetized with ethyl carbamate (1.5 mg
kg-1).
Zidovudine administration was performed via right inguinal vein at a dose of
10 mg kg-1 [22].
Blood samples were collected through indwelling arterial catheters,
transferred to heparinized collection tubes and centrifuged at 2000×g for 10 min.
After centrifugation, the plasma was separated and stored at -80 °C in the dark until
161
analysis.
162
University Animal Care and Use Committee.
163
After vortex-mixing, the
Ac ce p
156
In this procedure, copper (I) was generated by the
te
154 155
an
149
The experiment was performed with an approval of the Nagasaki
Fifty microliters of methanol was added into 50 µL of plasma samples.
164
Then, samples were centrifuged at 5000×g for 10 min.
165
supernatant was collected and was subjected to the derivatization reaction as
7
Twenty-five microliters of
Page 7 of 23
166
previously described.
Pharmacokinetic parameters were determined by moment
167
analysis using a single-dose i.v. bolus drug administration model.
168
parameters were area under the curve (AUC), mean residence time (MRT), terminal
169
phase half life (T1/2), volume of distribution (Vd) and clearance (CL).
ip t
170 3. Result and discussion
cr
171 172
174
3.1. Fluorescence characteristics of DIB-ET and DIB-zidovudine
us
173
The studied
The fluorescence spectra of DIB-ET were measured in methanol and the maximum excitation and emission wavelengths of DIB-ET were found to be 310 and
176
400 nm, respectively.
177
wavelengths of DIB-zidovudine were almost identical to those of DIB-ET.
178
addition, the fluorescence intensity of DIB-zidovudine was almost identical to that of
179
same concentration of DIB-ET.
180
that the fluorescence characteristics of DIB-ET were not changed after the reaction
181
with zidovudine although the extension of the conjugation could be occurred by the
183 184 185 186
M
On the other hand, the maximum excitation and emission In
te
d
In view of results described above, it was suggested
Ac ce p
182
an
175
formation of triazole ring.
3.2. Optimization of fluorescent derivatization conditions Figure 2 (A) and (B) show typical chromatograms obtained from the reagent
blank and the reaction mixture of zidovudine with DIB-ET, respectively.
The peak
187
of DIB-zidovudine was detected at 8 min, while the excess DIB-ET was eluted later
188
than DIB-zidovudine and detected at approximately 15 min.
189
To obtain higher reactivity, the conditions used for the derivatization reaction
190
were optimized using a standard solution of zidovudine.
191
solvent on the reactivity was examined with methanol, tetrahydrofuran,
8
The effect of reaction
Page 8 of 23
192
N,N-dimethylformamide, 1-buthanol, ethanol and acetonitrile.
193
solvents, the optimal result was obtained with methanol (Fig. S1, supplementary
194
data).
195
methanol was selected as the reaction solvent.
196
investigated over a range of 0.5-6 mM and the maximum peak area of
197
DIB-zidovudine was obtained using 4 mM DIB-ET (Fig. 3A).
198
copper (II) sulfate was examined over a range of 10–120 mM, and the maximum and
199
constant peak area was obtained at a concentration over 30 mM (Fig. S2,
200
supplementary data); 40 mM copper (II) sulfate was selected.
201
concentration of L-ascorbic acid was examined over a range of 100-600 mM and 180
202
mM was selected because it gave maximum peak areas (Fig. S3, supplementary data).
203
The effects of reaction temperature and time were investigated (Fig. 3B).
204
almost same reactivity was obtained at temperature higher than 40 ºC, and the
205
maximum and constant reactivity was obtained for more than 5 min.
206
hand at room temperature, the reactivity was slightly reduced as compared with
207
heating.
209 210 211 212 213
In addition, since methanol can be used for the deproteinization of plasma,
ip t
The concentration of DIB-ET was
us
cr
The concentration of
The
On the other
te
d
M
an
The effect of
As a result, 40 °C and 5 min were chosen for the reaction temperature and
Ac ce p
208
Among these
time, respectively, because these conditions gave high and constant reactivity. Under the optimized conditions, the reaction yield, which was estimated by comparing the peak areas of DIB-zidovudine in the reaction mixture with that of authentic DIB-zidovudine, was 78%.
3.3.
Calibration curve, detection limit and repeatability Validation is carried out according to PDA guideline for bioanalytical method
214 215
[23].
A calibration curve was prepared by spiking blank plasma with varying
216
concentration of zidovudine.
217
peak area and zidovudine concentration over the range from 0.32 to 1336 ng mL-1.
A linear relationship (r = 0.999) was obtained between
9
Page 9 of 23
218
The slope and intercept of regression equation (mean ± standard error, n = 3) were
219
4.03×105 ± 1.5×104 and 1.1×102 ± 8.8×10, respectively.
220
zidovudine in rat plasma was 0.28 ng mL-1 (20 fmol/injection) at a signal-to-noise
221
ratio (S/N) of 3.
222
that of HPLC with UV detection [24], 3 times higher than that of HPLC with
223
electrochemical detection [25] and 3 times higher than that of HPLC with
224
electrospray ionization tandem mass spectrometry [26].
225
method was 4 times less sensitive than radio-immunoassay [27], the proposed method
226
does not require special and hazardous materials unlike radio-immunoassay.
227
Accuracy and precision of the proposed method were examined using three levels
228
(2.67, 53.4 and 802 ng mL-1) of zidovudine in rat plasma.
229
within- and between-day accuracy ranged from 91.1% to 105% with %RSD of
230
precision less than 5.2%.
231
sufficient accuracy and precision.
235 236 237 238
ip t
cr
an
us
Although the proposed
M
As shown in Table 1, the
d
Therefore, it was proved that the proposed method showed
3.4.
Determination of zidovudine in rat plasma after administration
Ac ce p
234
The sensitivity of the proposed method was 26 times higher than
te
232 233
The detection limit of
To demonstrate the practicality of the proposed method, the zidovudine
concentration in rat plasma after a single dose administration was determined. Typical chromatograms of blank rat plasma and rat plasma collected after 15 min after the administration of zidovudine (10 mg kg-1) are shown in Fig. 4 (A) and (B), respectively.
The peak of DIB-zidovudine could be detected clearly and there were
239
no interfering peaks derived from plasma components.
These results mean that
240
DIB-ET reacted selectively with zidovudine and did not react with most of biological
241
components.
242
zidovudine.
243
administration.
Figure 5 shows mean plasma concentration vs. time profiles of The zidovudine concentration in rat plasma decreased with time after The pharmacokinetic parameters (mean ± standard error, n = 3)
10
Page 10 of 23
244
were summarized as follows; AUC0→∞ 6190 ± 2290 min mg L-1; MRT 180.89 ±
245
65.71 min; T1/2 121.58 ± 47.16 min; Vd 68.02 ± 10.70 mL; CL 0.39 ± 0.15 L h-1 kg-1.
246
The results are consistent with reported results in previous study [28].
247
practicality of the proposed method was confirmed by real sample analysis.
248
consideration of high sensitivity and selectivity, the proposed method should be
249
useful for therapeutic drug monitoring of zidovudine in routine clinical practice with
250
simple procedure for sample preparation.
cr
ip t
In
us
251 4. Conclusion
an
252
The
253
M
In this study, we developed a sensitive and selective HPLC determination
254 255
method for zidovudine by fluorescent alkyne, DIB-ET as a specific fluorescence
256
derivatization reagent.
257
based on the Huisgen reaction and the DIB-zidovudine could be detected clearly on
258
the chromatogram without interference from biological components.
259
derivatization method was successfully applied to the determination of zidovudine in
261 262 263 264
d
te
Ac ce p
260
DIB-ET reacted selectively with azide group of zidovudine
rat plasma after administration.
The proposed
In the present study, the Huisgen reaction could be
subjected to fluorescent derivatization reaction for chromatographic analysis for the first time.
In further investigation, we will attempt to develop a selective
derivatization method for alkyne compounds by using fluorescent azide compound as a reagent on the basis of the Huisgen reaction.
11
Page 11 of 23
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References
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Figure captions
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Fig. 1. Fluorescence derivatization reaction of zidovudine with DIB-ET based on the Huisgen reaction.
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Fig. 2. Chromatograms of (A) reagent blank and (B) standard solution of 5 µM
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zidovudine.
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Fig. 3. (A) Effect of DIB-ET concentration on the peak area of zidovudine. Derivatization conditions: concentrations of zidovudine, copper (II) sulfate
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and L-ascorbic acid were 2 µM, 120 mM and 600 mM, respectively.
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Reaction temperature and time were 50 ˚C and 60 min, respectively.
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(B) Effects of reaction temperature and reaction time on the peak area of
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zidovudine. Derivatization conditions: concentrations of zidovudine, DIB-ET,
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copper (II) sulfate and L-ascorbic acid were 2 µM, 4 mM, 40 mM and 180
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mM, respectively.
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Fig. 4. Chromatograms of (A) rat plasma sample and (B) that obtained at 15 min after i.v. administration of zidovudine (10 mg kg-1).
Fig. 5. Mean plasma concentration-time profiles of zidovudine in rat plasma
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following administration via right inguinal vein at a dose of 10 mg kg-1 to
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Wistar male rats (data are means ± standard error, n = 3).
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Table1 Within- and between-day accuracy and precision of the proposed method
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for determination of zidovudine in rat plasma.
Zidovudine -1
(ng mL )
a
Between-day (n=5)
Accuracy
Precision
Accuracy
(%)
(RSD a, %)
(%)
2.67
91.1
4.8
92.3
53.4
103
3.2
105
1.9
802
99.1
3.2
103
1.6
Relative standard deviation
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Precision
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Within-day (n=5)
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Highlights A fluorescence derivatization method for determination of zidovudine is described. Zidovudine can be reacted with fluorescent alkyne based on the Huisgen reaction. The method allows sensitive and selective determination of zidovudine in plasma.
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Figure 5
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