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(the nal concentration of KI is 40 mg mLꢀ1), shake well, and
react in a 60 ꢂC water bath for 30 min. Then, 1 mL of the
derivatization reagent solution were added to the same 10 mL
vial. Aer being vortexed for 10 s and diluted to 10 mL with
acetonitrile, the mixture was allowed to react at 60 ꢂC water bath
for 90 min. The reaction solution was analyzed by HPLC-UV
directly and the injection volume was 20 mL. For application,
4 mL of oroxylin A (5 mg mLꢀ1) or antipyrine (12.5 mg mLꢀ1) in
acetonitrile replaced the 4 mL of mixed standard working
solutions and the other step was the same. The derivatization
scheme of benzyl halides with 4-NPP is shown in Fig. 2.
2. Experimental
2.1 Chemicals and reagents
1-(4-Nitrophenyl) piperazine (4-NPP, 98%), methyl 4-(bromo-
methyl) benzoate (4-BMB, 98%), 2-chlorobenzyl chloride (2-
CBC, 98%), 4-chlorobenzyl chloride (4-CBC, 98%) and 2,6-
dichlorobenzyl bromide (2,6-DCB, 98%) were obtained from
Energy Chemical (Shanghai, China). 1-(2-Nitrophenyl)pipera-
zine (2-NPP, 98%) and benzyl chloride (BC, 99%) were
purchased from the TCI Development Co. Ltd (Shanghai,
China). Benzyl bromide (BB, 98%) was obtained from the
Macklin Biochemical Co. Ltd (Shanghai, China). 2-Nitro-
phenylhydrazine (2-NPH, 97%) and potassium iodide (KI, 99%)
were purchased from Aladdin (Shanghai, China). 2-Nitroaniline
(2-NA, 98.5%) and 4-nitroaniline (4-NA, 98%) were obtained
from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China).
Oroxylin A was synthesized as the active pharmaceutical ingre-
dient in the Department of Medicinal Chemistry, China Phar-
maceutical University (Nanjing, China).14 Antipyrine was
purchased from Sinopharm group Co. Ltd (Shanghai, China) as
the active pharmaceutical ingredient. The structures of these
compounds are shown in Fig. 1. Water was puried using
a Millipore Milli-Q system (Bedford, MA, USA). HPLC grade
acetonitrile was obtained from TEDIA (Faireld, USA). All other
reagents were of analytical grade and obtained from conven-
tional commercial sources.
2.5 Method validation
To demonstrate the feasibility of the newly developed method,
validation was performed under the optimal conditions in
relation to specicity, linearity, limit of quantitation (LOQ),
limit of detection (LOD), precision, accuracy and stability. These
validation parameters were validated according to the ICH
guidelines15 and Chinese Pharmacopoeia.16
Oroxylin A and antipyrine were used as the representative
drug substances. Since the analytical method implications were
that the limits could be 10 times greater than the default life-
time TTC (threshold of toxicological concern) limit (1.5 mg per
day),17 15 mg per day was selected as the TTC of the study.
Calculation of the limit applied for benzyl halides is as follows:
the TTC value divided by the maximum daily dose (g per day).18
Based on the maximum daily dose for both antipyrine and
2.2 Instrumentation and chromatographic conditions
oroxylin A of 600 mg per day, their GTIs are required to be
controlled at a concentration limit of 25 mg gꢀ1
.
19,20
The experiments were performed on a Shimadzu LC-20AT series
HPLC system (Kyoto, Japan) with a SPD-20A UV detector. A LC-
solution workstation (Shimadzu, Kyoto, Japan) was used to
control the system and date acquisition. An InertSustain® C18
column (250 mm ꢁ 4.6 mm, 5 mm) was used for the analysis.
The mobile phase was composed of acetonitrile (solvent A) and
5 mM ammonium acetate solution (solvent B). The chromato-
graphic separation was achieved using the following gradient
elution: 0–10 min, 70% A; 10–15 min, ramping from 70% to
85% A; 15–20 min, 85% A, with a constant ow rate of 1.0
mL minꢀ1. The column temperature was kept at 30 ꢂC. The
typical injection volume was 20 mL.
3. Results and discussion
3.1 Selection of derivatization reagents
Since 2-NPH has been reported as a suitable derivatization
reagent for detecting acyl chlorides in the near visible range
(395 nm),11 the UV spectrum of benzyl halides aer derivatiza-
tion with 2-NPH was studied rstly. Fig. 3A showed that the
maximum absorption wavelength of derivatization product was
just 261 nm, which was not suitable for benzyl halides analysis.
Hence, a new derivatization reagent was urgently needed.
Nitroaniline (NAs) and nitrophenylhydrazine (NPPs) are two
other kinds of potential derivatization reagents with the
maximum absorbance wavelength in the near visible range.13
The results showed that the derivatization reaction between
benzyl bromide and NAs (2-NA, 4-NA) hardly proceeded. It may
be attributed to the strong conjugation of nitrobenzene group,
leading to the weaker nucleophilic ability of amine (–NH2) in
the NAs structures. On the contrary, because the aliphatic
amine (–NH–) was far away from the nitrobenzene ring in the
NPPs structures, the reaction rates between NPPs (2-NPP, 4-
NPP) and benzyl bromide were much higher. Furthermore, the
derivatization product of 4-NPP stood out given that it exhibited
strong absorption at 392 nm (Fig. 3B, the red line) compared
with that of 2-NPP (Fig. 3B, the black line). Other selected benzyl
2.3 Preparation of solutions
Separate solutions of 2-NPH, 2-NA, 4-NA, 2-NPP and 4-NPP at
the concentration of 3 mg mLꢀ1 were freshly prepared in
acetonitrile for the derivatization experiments. A standard stock
solution of KI was freshly prepared each day by dissolved KI (2.0
g) in water (10 mL). Separate stock solutions containing 1 mg
mLꢀ1 of each benzyl halides were prepared in acetonitrile.
Before use, a series of mixed standard working solutions (0.1–1
mg mLꢀ1) were prepared by diluting each stock solution with the
same solvent.
2.4 Derivatization
Accurately remove 4 mL of mixed standard working solutions, halides showed similar spectral characteristics (Fig. S1†). In
place it in a 10 mL vial, add 1 mL of 200 mg mLꢀ1 KI solution order to further verify that the red shi effect of the reaction of
25798 | RSC Adv., 2019, 9, 25797–25804
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