154
FARMACIA, 2008, Vol.LVI, 2
THE LIQUID CHROMATOGRAPHIC ASSAY
OF ONDANSETRON HYDROCHLORIDE AND
ITS IMPURITIES USING A NEW
STATIONARY PHASE
ANDREEA VARVARA1*, CRINA-MARIA MONCIU2, CORINA ARAMĂ2,
C. POPESCU1
1
Raw Materials and Finished Products Control Department, National
Medicines Agency, Av. Sanatescu Street no. 48, Bucharest, Romania
2
Analytical Chemistry Department, Faculty of Pharmacy, UMF Carol
Davila, Traian Vuia Street no. 6, Bucharest, Romania
* corresponding author: andreea.varvara@anm.ro
Abstract
A high performance liquid chromatography method for separation and assay of ondansetron
hydrochloride and some of its possible impurities in bulk and dosage forms was developed.
Taking into account that ondansetron hydrochloride and its impurities have
polarizable structures, the objective of this work was to identify a new suitable stationary
phase for an optimal separation of these polar compounds. In such cases, the use of porous
graphitic carbon (PCG), a stationary phase with good retention properties of very polar
analytes was elected. PCG is different from classical stationary phases and has special
ability to retain these analytes types and to separate closely related compounds, due to a
combination of two mechanisms: adsorption and charge induced interactions of a polar
analyte with the polarizable surface of graphite.
We studied the behavior of ondansetron hydrochloride and its possible impurities
in a chromatographic system using a Hypercarb column and mixtures of acetonitrile and
polar solvents with acidic modifiers (trifluoroacetic acid) as mobile phase. The research
resulted in a new method for separation and quantitative estimation of ondansetron
hydrochloride and its impurities in bulk and dosage forms.
Rezumat
Lucrarea prezintă o nouă metodă lichid cromatografică de înaltă performanţă
pentru identificarea şi separarea clorhidratului de ondansetron şi a posibilelor sale impuritaţi.
Având în vedere faptul că atât clorhidratul de ondansetron, cât şi impuritaţile sale
au structuri polarizabile, scopul studiului efectuat a fost identificarea unei noi faze
staţionare pentru separare optimă a acestor compuşi polari. În acest caz, s-a optat pentru
folosirea cărbunelui grafitic poros, o fază staţionară diferită de cele clasice, cu bune
proprietaţi de retenţie şi separare a compuşilor foarte polari, cu structuri chimice
asemănătoare. Separarea se explică prin asocierea a două mecanisme: adsorbţia şi
interacţiile induse electric ale analitului polar pe suprafaţa polarizabilă a grafitului.
S-a studiat comportarea clorhidratului deondansetron şi a posibilelor sale
impuritaţi într-un sistem cromatografic folosind o coloană Hypercarb şi amestecuri de
acetonitril şi solvenţi polari cu modificatori acizi (acid trifluoracetic) ca fază mobilă.
Rezultatele obţinute au condus la elaborarea unei noi metode pentru separarea şi
determinarea cantitativă a clorhidratului de ondansteron şi a impurităţilor sale din substanţa
activă şi din forme farmaceutice.
155
FARMACIA, 2008, Vol.LVI, 2


Ondansetron hydrochloride
Liquid chromatographic assay
INTRODUCTION
Ondansetron
((3RS)-9-methyl-3-[(2-methyl-1H-imidazol-1yl)methyl], 1,2,3,9-tetrahydro-4H-carbazol-4-one, as hydrochloride dihydrate)
(figure 1) is a selective 5-HT3 receptor antagonist used in the treatment of
nausea and vomiting related to cancer chemotherapy or radiotherapy [1].
A monograph for quantitative determination of ondansetron
hydrochloride dihydrate was first introduced in the European Pharmacopoeia
5th edition [2]. In the United States Pharmacopoeia USP 28 is presented a
monograph of the active substance and the dosage form (injectable solution)
[3]. The RP-HPLC assay methods by UV detection at 216 nm, with a
spherical nitrile silica gel column as stationary phase and buffered aqueous
acetonitrile as mobile phase are mentioned in both monographs.
The same method is applied for quantification of two major related
substances of ondansetron hydrochloride dihydrate described in both
monographs: (3RS)-3-[(dimethylamino)methyl]-9-methyl-1,2,3,9-tetrahydro4H-carbazol-4-one (impurity A) and 9-methyl-1,2,3,9-tetrahydro-4Hcarbazol-4-one (impurity C) (figure 1), with admissibility limits of 0.2%.
(a)
(b)
(c)
Figure 1
Structures of ondansetron hydrochloride dihydrate (a), impurity A (b) and impurity C (c)
Several authors reported various similar methods using nitrile,
octylsilyl or phenyl silica gel as stationary phase and mixtures of polar
solvents as mobile phases [4-9]. A HPLC assay of ondansetron enantiomers
in human serum using a reverse phase cellulose-based chiral stationary phase
is also published [10]. Recently, a separation of ondansetron and its
pharmacopoeial impurities on a zirconium oxide-based stationary phase as an
alternative to clasical silica-based stationary phases was accomplished [11].
The specific retention mechanisms of Hypercarb column (with
100% porous graphitic carbon structure) consisting of dispersive
interactions between analyte – graphite surface (higher retention as the
156
FARMACIA, 2008, Vol.LVI, 2
hydrophobicity of the molecule increases) and on the other side chargeinduced interactions of polar analytes with the polarizable surface of
graphite, recommend it for the separation of very polar analytes [12-16].
On this basis we have established chromatographic conditions for
the separation and assay of ondansetron hydrochloride dihydrate using
Hypercarb column and a mobile phase consisting of acetonitrile/2-propanol
(50:50) and 0.05% (w/V) trifluoroacetic acid [17]. Unfortunately, these
conditions are not convenient for the separation of ondansetron
hydrochloride dihydrate and its related compounds. That is why we
developed optimized chromatographic conditions suitable for the separation
of ondansetron hydrochloride dihydrate and two of its potential impurities
(impurity A and impurity C).
MATERIALS AND METHODS
Liquid chromatography system and chromatographic condition
A Waters 600 quaternary pump system, equipped with a Waters 996
DAD and an autosampler was used. The Hypercarb column (batch number
PGC315), 100 x 4.6 mm, 5 μm was offered by Thermo Electron Corporation.
After the preliminary tests made in order to identify the critical
parameters, the chromatographic conditions were optimized as it follows:
mobile phase was acetonitrile containing 0.1% (w/V) trifluoroacetic acid
(TFA) / water (90:10). Frequently, TFA is reported to act as a competitive
electronic modifier which reduces polar retention in order to obtain elution
in a reasonable run time [18]. The Hypercarb column was set at 50°C and
the flow rate was 1.5 mL/minute.
According to UV spectra of each compound, the wavelength of 216
nm was chosen for quantitative determination (figure 2).
(a)
(b)
Figure 2
The overlaid UV spectra of impurity A and impurity C (a)
versus ondansetron hydrochloride (b)
FARMACIA, 2008, Vol.LVI, 2
157
Reagents
Drug standards
Ondansetron hydrochloride dihydrate working standards (10.1% water
content), impurity A and impurity C were provided by Dr. Reddy’s (India).
Active substance and dosage forms
Ondansetron hydrochloride dihydrate active substance was also
provided by Dr. Reddy’s (India). A value of 9.8% water content was
determinated by Karl Fischer titration.
Dosage forms: Osetron 4 mg, injectable solution (iv) (Dr. Reddy’s,
India), containing 4 mg ondansetron/2 mL and Emeset 8 mg/4 mL,
injectable / infusion solution (iv), (Cipla Ltd., India), containing 2 mg
ondansetron/mL.
Solvents and chemicals
All solvents were LiChrosolv® HPLC grade, obtained from Merck
(Germany). Ultrapure water obtained with a Milli-Q UF Plus water
purification system was used throughout the study.
Standard and sample solutions
Standard ondansetron hydrochloride dihydrate solution (a): an
accurately weighted amount of 20 mg ondansetron hydrochloride dihydrate
working standard, corresponding to 16 mg ondansetron, is dissolved in a 10
mL volumetric flask with methanol. After a brief sonication, bring to
volume with the same solvent. A dilution of 1 mL in a 10 mL volumetric
flask with methanol is prepared (0.2 mg/mL ondansetron hydrochloride
dihydrate, corresponding to 0.16 mg/mL ondansetron).
Standard ondansetron hydrochloride dihydrate solution (b): a
dilution of 1 mL standard solution (a) in a 50 mL volumetric flask with
methanol is prepared (4 μg/mL ondansetron hydrochloride dihydrate).
Standard impurity A solution: an accurately weighted amount of 1
mg impurity A is dissolved in a 10 mL volumetric flask with methanol. After
a brief sonication, bring to volume with the same solvent. A dilution of 1 mL
in a 25 mL volumetric flask with methanol is prepared (4 μg/mL impurity A).
Standard impurity C solution: an accurately weighted amount of 1
mg impurity C is dissolved in a 10 mL volumetric flask with methanol. After
a brief sonication, bring to volume with the same solvent. A dilution of 1 mL
in a 25 mL volumetric flask with methanol is prepared (4 μg/mL impurity C).
Standard impurity A and impurity C mixture solution: 1 mL of
Standard impurity A solution and 1 mL of Standard impurity C solution are
diluted in a 10 mL volumetric flask with methanol (0.4 μg/mL ondansetron
impurity A and 0.4 μg/mL ondansetron impurity C).
158
FARMACIA, 2008, Vol.LVI, 2
System suitability solution: 1 mL of Standard ondansetron
hydrochloride dihydrate solution (b), 1 mL of Standard impurity A solution
and 1 mL of Standard impurity C solution are diluted in a 10 mL volumetric
flask with methanol (0.4 μg/mL ondansetron hydrochloride dihydrate, 0.4
μg/mL impurity A and 0.4 μg/mL impurity C).
Sample solutions of active substance: were preparated in the same
way as the Standard ondansetron hydrochloride dihydrate solution (a).
Sample solutions of dosage forms: 0.8 mL of dosage form
(injectable solution) is diluted in a 10 mL volumetric flask with methanol.
RESULTS AND DISCUSSION
A volume of 100 μL from the system suitability solution is
injected. The resolution between the peaks due to impurity A and
ondansetron should not be less 2.0.
Assay of the active substance: 10 μL of 6 replicates Standard
ondansetron hydrochloride dihydrate solution (a) and 2 replicates of 6
independent sample solutions were injected.
(a)
(b)
Figure 3
The chromatogram of ondansetron as active substance (a) and dosage forms (b)
The results obtained for both preparations types (active substance
and dosage forms) are reported in table I.
159
FARMACIA, 2008, Vol.LVI, 2
Table I
Results reported for assay of ondansetron
hydrochloride active substance and dosage forms
Preparation
type
Assay results (%)
Avera
ge
RSD
(%)
Active
substance
99.04
100.44
101.77
100.42
101.56
100.60
100.64
0.97
Osetron
101.69
100.98
101.21
102.17
102.07
101.48
101.60
0.46
102.90
104.42
103.20
103.52
103.33
105.02
103.73
0.78
Emeset
Confidence
interval
(n=6, P=95%)
100.22 – 101.06 %
(as ondansetron
hydrochloride)
101.40 – 101.80 %
(2.03– 2.04
mg/mL)
(as ondansetron)
103.38 – 104.08 %
(2.07– 2.08
mg/mL)
(as ondansetron)
Assay of the related compounds: 100 μL of 6 replicates Standard
impurity A and impurity C mixture solution and 6 independent sample
solutions were injected. The relative retention time with reference to the
retention time of ondansetron (around 3 minutes) are: impurity A – about
0.7, impurity C – about 6.6. Figure 4 shows the chromatographic separation
in the System suitability solution (a) and in the Standard impurity A and
impurity C mixture solution (b).
(a)
(b)
Figure 4
The separation of ondansetron hydrochloride and its impurities
160
FARMACIA, 2008, Vol.LVI, 2
In figure 5 is presented the chromatographic separation of the
active substance.
Figure 5
The chromatogram of ondansetron hydrochloride active substance
Impurity A and impurity C were below the limit of detection for
both sample types (active substance and dosage forms).
Validation of chromatographic procedure
The analytical procedure was validated according to ICH
requirements [19].
Assay of the active substance
Linearity: an accurately weighted amount of 20 mg of ondansetron
hydrochloride dihydrate working standard, corresponding to 16 mg
ondansetron, is dissolved in a 10 mL volumetric flask with methanol
(ondansetron hydrochloride dihydrate stock solution). Three independent
injections of 5 diluted standard solutions of 0.16–0.24 mg/mL ondansetron
hydrochloride dihydrate, corresponding to 80–120% range are made. The
graphical representation of regression line and the detector response factor
are presented in figure 6 and 7.
Detector response factor
Peak area /
concentration
Peak area / 1000
Linearity
10000
8000
6000
4000
2000
0
y = 32.132x - 65.069
R2 = 0.9996
0
100
200
Concentration (μg/mL)
Figure 6
Assay linearity – regression line
300
40000
20000
0
0
100
200
300
Concentration (μg/mL)
Figure 7
Assay linearity - detector response
161
FARMACIA, 2008, Vol.LVI, 2
Repeatability: RSD% (6 injections) for 0.2 mg/mL ondansetron
hydrochloride dihydrate was 0.27 and for diluted dosage form sample was 0.22.
Specificity: no interference with solvent and placebo were detected.
Accuracy: the recovery of ondansetron hydrochloride dihydrate working
standard in 3 diluted samples with known concentration of 95, 100 and 105% from
the target value of 0.2 mg/mL ondansetron hydrochloride dihydrate was
determinated. The confidence interval was 98.39 – 101.56% (average 99.97%).
Synthetic mixtures with known quantities of active substance (95,
100 and 105% from the target value of 2.5 mg ondansetron hydrochloride
dihydrate corresponding to 2 mg/mL ondansteron) in placebo mixtures were
prepared and diluted as prescribed. The confidence interval for the recovery
of ondansetron hydrochloride dihydrate in “Osetron synthetic mixtures” was
98.35– 102.97% (average 100.66%) and “Emeset synthetic mixtures was
98.84– 102.19% (average 100.52%).
LOD: 1.21 μg/mL ondansetron hydrochloride dihydrate (S/N = 3),
LOQ: 4.0 μg/mL ondansetron hydrochloride dihydrate (S/N=10).
Stability of solution was studied both for standards and diluted
samples (of active substance and medicinal products). The stability of solution
was preserved for 24 hours, at room temperature or in refrigerator (2-8ºC).
Assay of ondansetron impurity A and ondansetron impurity C
Linearity: an accurately weighted amount of 1 mg impurity A and 1
mg impurity C are separately dissolved in a 10 mL volumetric flask with
methanol. A dilution of 1 mL in a 25 mL volumetric flask with methanol is
made (impurity A and impurity C stock solution). 1 mL of ondansetron
hydrochloride dihydrate stock solution and successive dilution of each
impurity stock solution with methanol were prepared (0.32 – 0.48 μg/mL
impurity A and 0.32–0.48 μg/mL impurity C corresponding to 80–120%
from the target value of 0.4 μg/mL, equivalent to 0.2% from the standard
solution of 0.2 mg/mL ondansetron hydrochloride dihydrate). Injections of
each 5 solutions are made. The results are presented in figures 8- 11.
Linearity - Impurity A
Linearity - Impurity C
200000
200000
Peak Area
Peak area
250000
150000
100000
y = 333,8x + 27473
R2 = 0,9992
50000
0
0
100
200
300
400
500
600
Concentration (x 1000 μg/mL)
Figure 8
Linearity of impurity A – regression line
150000
100000
y = 350,85x + 2041
R2 = 0,9992
50000
0
0
100
200
300
400
500
600
Concentration (x 1000 μg/mL)
Figure 9
Linearity of impurity C – regression line
162
FARMACIA, 2008, Vol.LVI, 2
Detector response factor - Impurity C
600
400
200
0
0
100
200
300
400
500
Concentration (x 1000 μg/mL)
Figure 10
Linearity of impurity A - detector
response factor
600
Peak area /
conc
Peak area /
conc
Detector response factor - Impurity A
400
200
0
0
100
200
300
400
500
600
Concentration (x 1000 μg/mL)
Figure 11
Linearity of impurity C - detector
response factor
Repeatability: RSD% (6 injections) for ondansetron hydrochloride
dihydrate was 0.27, for impurity A was 0.64 and for impurity C was1.24.
Specificity: no interference with solvent and placebo were detected.
Accuracy: the recovery of impurity A and impurity C in 3 diluted
samples with known concentration of 90, 100 and 110% from the target
value of 0.4 μg/mL impurity A and 0.4 μg/mL impurity C in the presence of
0.2 mg/mL ondansetron hydrochloride dihydrate was determinated. The
confidence interval was 100.69 – 101.41% (average 101.05%) for impurity
A and 98.61 – 101.69% (average 100.15%) for impurity C.
Spiked samples with known quantities of impurity A and impurity
C (90, 100 and 110% of the target value of 0.4 μg/mL in the presence of 0.2
mg/mL ondansetron hydrochloride dihydrate (prepared as a synthetic
mixture in placebo) were injected and the recovery results were examinated.
The confidence interval for the recovery of impurity A in “Osetron
synthetic mixtures” was 100.58 – 101.74% (average 101.16%) “Emeset
synthetic mixtures” was 100.38 – 101.21% (average 100.79%).
The confidence interval for the recovery of impurity C in “Osetron
synthetic mixtures” was 99.00 – 101.40% (average 100.20%) and in
“Emeset synthetic mixtures” was 98.63 – 101.72% (average 100.18%).
Impurity A - LOD: 0.05 μg/mL (S/N = 3), LOQ: 0.21 μg/mL (S/N = 10),
Impurity C - LOD: 0.04 μg/mL (S/N = 3), LOQ: 0.15 μg/mL (S/N = 10).
Stability of ondansteron and its impurities solution was studied
both for mixture standard and diluted samples (of active substance and
dosage forms). The stability of impurities solutions were preserved for 24
hours, at room temperature or in refrigerator (2-8ºC).
A comparative study between the new established method for assay and
purity of ondansteron hydrochloride dihydate and the USP method* was made.
(* - L10 column - Waters Spherisorb S5 CN RP (batch number 011181411),
150 x 4.6 mm, 5μm, mobile phase: acetonitrile : phosphate buffer pH=5.4
(monobasic sodium phosphate 0.02M for active substance / monobasic
163
FARMACIA, 2008, Vol.LVI, 2
potassium phosphate 0.02M for injectable solution) (50 : 50), UV detection
at 216 nm, flow rate: 1.5 mL/minute).
The assay results are presented in table II. The F value (ANOVA)
proved that there is no significant difference between the two sets of
measurements.
Table II
Comparative results reported for assay of ondansetron hydrochloride (n=6, P=95%)
Porous graphitic carbon
Spherisorb CN
Preparation Average
F value
Confidence
Average
Confidence
type
(%)
interval (%)
(%)
interval (%)
Active
100.64
100.22 –
99.73
99.20 –
1.63
substance
101.06
100.27
Osetron
2mg/mL
101.60
101.40 –
101.80
100.60
100.46 –
100.75
1.91
Emeset
2mg/mL
103.73
103.38 –
104.08
101.31
101.07 –
101.55
2.09
Impurity A and impurity C were below LOD for both sample types.
CONCLUSIONS
A new and different from classical stationary phases, porous
graphitic carbon, was used in a validated HPLC method for separation and
assay of ondansetron hydrochloride and two of its potential impurities in
bulk and dosage forms. The results were similar to those obtained using the
current USP monographs.
REFERENCES
1. Dollery C, editor., Ondansetron (hydrochloride), Therapeutic Drugs,
Churcill Livingstone 1999, 2nd Edition: O21-O24
2. XXX, the European Pharmacopoeia 5th edition, Strasbourg, 2005
3. XXX, the United States Pharmacopoeia USP 28, Rockville, 2005
4. Bauer, S.; Stormer, E.; Kaiser, R.; Tremblay, P.B.; Brockmoller, J.;
Roots, I., Simultaneous determination of ondansetron and tropisetron
in human plasma using HPLC with UV detection, Biomedical
Chromatography, 2002, 16, 187-190
5. Depot, M.; Leroux, S.; Caille, G., High resolution liquid
chromatographic method using ultraviolet detection for
164
FARMACIA, 2008, Vol.LVI, 2
determination of ondansetron in human plasma, Journal of
Chromatography B, 1997, 693, 399-406
6. Salem, I. I.; Lopez, J. M. R.; Galan, A. C., Ondansetron
hydrochloride, Anal. Profiles Drug Subst. Excipients, 2001, 27, 301338
7. Lahuerta Zamora, L; Martinez Calatayud, J., Martinez Tamayo E.,
Spectral and thermal properties of ondansetron, Revista Brasileira de
Ciencias Farmaceuticas, 1999, 35(1), 119-126
8. Zheng, H.; Pan, Wei; Wang, Yan, Determination of ondansetron
hydrochloride in human plasma by HPLC, Zhongguo Yiyao Gongye
Zazhi 2002, 33(12), 603-605, CA2004140:121955 h
9. Dotsikas, Y.; Kousoulos, C.; Tsatsou, G.; Loukas, L.Y.,
Development and validation of a rapid 96-well format based liquidliquid extraction and liquid chromatograph -tandem mass
spectrometry analysis method for ondansetron in human plasma,
Journal of Chromatography B, 2006, 836, 79-82
10. Liu, J.; Stewart James, T., High-performance liquid chromatographic
analysis of ondansetron enantiomers in human serum using a
reversed-phase cellulose-based chiral stationary phase and solidphase extraction, Journal of Chromatography B, 1997, 694, 179-184
11. Zizkovsky, V.; Kucera, R.; Klimes, J., Potential employment of nonsilica-based stationary phases in pharmaceutical analysis, Journal of
Pharmaceuticla and Biomedical Analysis, 2007, 44, 5, 52-55
12. Ayrton. J.; Evans.M.B.; Harris. A.J.; Plumb, R.S., Porous graphitic
carbon promise for the rapid chromatographic analysis of polar
drugs metabolites, Journal of Chromatography B, 1995, 667(1),
173-178
13. Forgacs, E.; Cserhati. T., Relationship between retention
characterisation and physicochemical parameters of solutes on
porous graphitized carbon column, Journal of Pharmaceutical and
Biomedical Analysis, 1998, 18(4-5), 505-510
14. XXX, Method Development Guide for Hypercarb Columns, Thermo
Electron Corporation, 2004
15. XXX, Chromatography Columns and Consumables, 2006-2007,
Thermo Electron Corporation, 2005
16. Ross, P., LC - GC Europe, 2000, Vol. 13, no. 5
17. Varvara, A.; Monciu, C-M.; Aramă, C.; Popescu, C., A new
stationary phase in the liquid chromatographic assay of ondansetron
hydrochloride, Farmacia, 2007, Vol. LV, No. 2, 121-131
FARMACIA, 2008, Vol.LVI, 2
165
18. Balcan, M.; Cserhati. T.; Forgacs, E., Influence of organic modifiers
on the retention characterisation of graphitized carbon column,
Analytical Letters, 1997, 30(4), 883-893
19. XXX, Validation of Analytical Procedures: Text and Methodology
Q2(R1), ICH Harmonised Tripartite Guideline, International
Conference on Harmonisation of Technical Requirements for
Registration of Pharmaceuticals for Human Use, November 2005