Dexamethasone (DEX) : Ondansetron (OND) Granisetron (GRA) Tropisetron (TRO) Azasetron (AZA)

1.
Introduction
Dexamethasone (DEX) (Figure 1(a)), a synthetic corticosteroid, has long been used as
antiemetic agent in patients undergoing cancer chemotherapy [1, 2], being effective for both
acute and delayed nausea and vomiting. Selective 5-hydroxytryptamine type 3 (5-HT3)
receptor antagonists, including ondansetron (OND) (Figure 1(b)), granisetron (GRA) (Figure
1(c)), tropisetron (TRO) (Figure 1(d)), and azasetron (AZA) (Figure 1(e)), are effective and
potent antiemetic drugs which are recommended by clinical practice guidelines for patients
undergoing surgery and cancer chemotherapy [3–6]. Literature survey has revealed that
coadministration of antiemetics from different classes could be a more effective antiemetic
treatment modality, and DEX is a standard component of antiemetic combination regimens
with 5-HT3 antagonists [3, 7–10]. Hence, mixtures of 5-HT3 receptor antagonists, alone or in
combination with DEX, are often used in clinical practice to relieve chemotherapy induced
nausea and vomiting. However, there are no commercially available such drug mixtures, and
they must be prepared in the hospital pharmacy departments for clinical use. From the
literature survey it is evident that various analytical methods were available for the
determination of DEX, OND, GRA, TRO, and AZA with other combinations by using high-
performance liquid chromatography (HPLC) [11–31]. No method has been reported in the
literature for simultaneous estimation of DEX, OND, GRA, TRO, and AZA in infusion
samples. Hence, the objective of the current study was to develop and validate a simple,
rapid, accurate, and precise HPLC method for simultaneous estimation of DEX, OND, GRA,
TRO, and AZA in infusion samples.
2. Experimental
2.1. Chemicals and Reagents. The working standards of DEX sodium phosphate, OND
hydrochloride, GRA hydrochloride, TRO hydrochloride, and AZA hydrochloride were
purchased from the National Institutes for Food and Drug Control (Beijing, China) and stored
at 4∘C. The pharmaceutical formulations used in this study were DEX sodium phosphate
injection 5mg/1mL (Cisen Pharmaceutical Co., Ltd., Shandong, China), OND hydrochloride
injection 4mg/2mL (Qilu Pharmaceutical Co., Ltd., Shandong, China), GRA hydrochloride
injection 3mg/3mL (Cinkate Pharmaceutical Corporation, Suzhou, China), TRO
hydrochloride injection 5mg/1mL (Qilu PharmaceuticalCo., Ltd., Shandong, China), and
AZA hydrochloride injection 10 mg/2mL (Wanma Pharmaceutical Co., Ltd., Zhejiang,
China). The solution of 0.9% NaCl used to prepare the sample mixtures was from Kelun
Pharmaceutical Co., Ltd. (Sichuan, China). Potassium dihydrogen phosphate KH2PO4,
triethylamine, and phosphoric acid of AR Grade were obtained from Xilong Chemical Ltd.
(Guangdong, China). HPLC grade acetonitrile was purchased from Fisher Scientific
International (St Louis,MO, USA). Ultrapure water was purified using a Milli-Q system
(Millipore, Bedford, MA, USA).
2.2. HPLC Instrumentation and Chromatographic Conditions. An UltiMate_ 3000 standard

high pressure liquid chromatographic instrument (Dionex,Germany) composed of an
UltiMate 3000 quaternary gradient pump, an ASI-100 autosampler, a TCC-100 thermostat
column oven, and an ultraviolet detector (DAD) was employed in the study. Data acquisition
was carried out using Chromeleon_ software. Chromatographic analyses were performed on a
Phenomenex C18 column (4.6mm × 150 mm, 5 𝜇m) and using the mobile phase of
acetonitrile-50mM KH2PO4 buffer-triethylamine (25 : 74 : 1; pH adjusted to 4.0 using
diluted phosphoric acid). The mobile phase was prepared daily and filtered through a 0.22𝜇m
membrane filter (Millipore Corp., USA). The flow rate of the mobile phase was kept at 1.0
mL/min. The selected detection wavelengths for DEX, OND, GRA, TRO, and AZA were 241
nm, 310 nm, 302 nm, 285 nm, and 302 nm, respectively. The assay was performed at 30∘C
and injection volume was 20 𝜇L.
2.3. Preparation of Stock and Working Solutions. We accurately weighed and transferred
20mg of DEX sodium phosphate, 16 mg of OND hydrochloride, 12mg of GRA
hydrochloride, 10 mg of TRO hydrochloride, and 20 mg of AZA hydrochloride working
standard into a 100mL volumetric flask.We added about 70mL of deionised water and used
sonication for dissolving completely and made volume up to the mark with the same solvent
to obtain the final concentration of 0.2mg/mL of DEX sodium phosphate, 0.16mg/mL of
OND hydrochloride, 0.12mg/mL of GRA hydrochloride, 0.1mg/mL of TRO hydrochloride,
and 0.2mg/mL of AZA hydrochloride, respectively. The solutions were kept at –20∘C until
use. Fresh working standard solutions were prepared by diluting the stock solution with
deionised water to the required concentrations before use.
2.4. Preparation of Infusion Samples. In order to mimic a concentration range relevant to

clinical practice and the conditions commonly occurring in hospitals, four sample infusion
solutions were prepared under aseptic conditions in laminar flow hoods. Solution 1. 2mL
(10mg) DEX sodium phosphate injectable solution and 4mL (8mg) OND hydrochloride
injectable solution were transferred to a 100mL polyolefin bags and filled with 0.9% sodium
chloride injection. solution and 3mL (3 mg) GRA hydrochloride injectable solution were
transferred to a 100mL polyolefin bags and filled with 0.9% sodium chloride injection.
Solution 3. 2mL (10mg) DEX sodium phosphate injectable solution and 1mL (5 mg) TRO
hydrochloride injectable solution were transferred to a 100mL polyolefin bags and filled with
0.9% sodium chloride injection. Solution 4. 2mL (10mg) AZA hydrochloride injectable
solution was transferred to a 100mL polyolefin bags and filled with 0.9% sodium chloride
injection.
2.5. Validation of the Method. The developed analytical method was subjected to validation
with respect to various parameters such as linearity, intra- and interday precision, accuracy,
limit of quantification (LOQ), limit of detection (LOD), and reproducibility for each analyte.
2.6. Physicochemical Stability Study of the Infusion Samples. The compatibility and stability
studies of the infusion samples were performed at 25±0.5∘C; all the solutions were protected
from light exposure and checked at predetermined times: 0, 2, 4, 8, 24, and 48 hours. At the
specified times, the infusion samples were examined for the changes in appearance and the
pH value of the mixture was also determined in a digital Crison phs-3c pH meter. Moreover,
the concentrations of the drugs were determined at each analysis by the above described
HPLC-DAD method. In the concentrations’ analysis, 2mL samples were collected from each
polyolefin bag and were diluted 1 : 5 in deionised water before injection into HPLC system
so as to be within the range covered by the calibration curves.

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1.

2.2. HPLC Instrumentation and Chromatographic Conditions. An UltiMate_ 3000 standard

2.4. Preparation of Infusion Samples. In order to mimic a concentration range relevant to

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