development of a validated rp-hplc method for separation - farmacia

534
FARMACIA, 2009, Vol. 57, 5
DEVELOPMENT OF A VALIDATED RP-HPLC
METHOD FOR SEPARATION AND
DETERMINATION OF PROCESS-RELATED
IMPURITIES OF OMEPRAZOLE
IN BULK DRUGS
CRISTINA IUGA 1* , MARIUS BOJIŢĂ 1 , SORIN E. LEUCUŢA 2
University of Medicine and Pharmacy „Iuliu Haţieganu”, Faculty of
Pharmacy, 400349, str. Louis Pasteur 6, Cluj Napoca, Romania
1 Department of Drugs Analysis
2 Department of Pharmaceutical Technology and Biopharmacy
*corresponding author: iugac@umfcluj.ro
Abstract
A gradient reversed phase liquid chromatographic (RP-LC) method has been developed
and subsequently validated for the determination of omeprazole and its process-related impurities
(noted as: impurity A, B, C, D, G, H).. Separation was achieved with a Zorbax Extend C 18 column
and acetonitrile: water: triethylamine1% (pH adjusted to 9.5) as eluent, at a flow rate of 0.8 mL/min.
UV detection was performed at 280 nm. The described method was linear over a range of 40.6-
203µg/mL for omeprazol, 0.9556-14.334µg/mL for impurity A, 1.1568-17.352µg/mL for impurity
B, 1.0772-16.158µg/mL for impurity C, 1.289-19.344 µg/mL for impurity D and 0.7968-
11.952µg/mL for impurity H. The accuracy of the method has been demonstrated at 5 concentration
levels in the range of 60–140% of the specification limit and the recovery of impurities was found to
be in the range of 90–109%. The method is simple, rapid, selective, accurate and useful for indicating
the stability of omeprazole from dosage forms. The method can be useful in the quality control of
bulk manufacturing and pharmaceutical formulations.
Rezumat
A fost elaborată şi validată o metodă RP-HPLC de separare şi determinare a
omeprazolului şi impurităţilor sale de sinteză (notate astfel: impuritatea A, B, C, D, G, H).
Separarea s-a realizat pe o coloană ZorbaxExtend C 18 cu o fază mobilă formată din
acetonitril: apa: trietilamină 1% (pH=9,5), cu un debit de 0,8 mL/min. Detecţia a fost UV la
280nm. Metoda descrisă a fost lineară pe domeniul de concentraţii de 40,6-203µg/mL
pentru omeprazol, 0,9556-14,334µg/mL pentru impuritatea A, 1,1568-17,352µg/mL pentru
impuritatea B, 1,0772-16,158µg/mL pentru impuritatea C, 1,289-19,344 µg/mL pentru
impuritatea D and 0,7968-11,952µg/mL pentru impuritatea H. Acurateţea metodei a fost
demonstrată pentru 5 nivele de concentraţie în domeniul de 60–140% făţă de limitele
specificate, gradul de regăsire pentru impurităţi a fost cuprins în domeniul 90–109%.
Metoda este simplă, rapidă, selectivă, are acurateţe şi precizie, şi poate fi folosită şi pentru
evaluarea stabilităţii omeprazolului în forme farmaceutice. Este o metodă utilă de control al
purităţii omeprazolului din materia primă şi din formele farmaceutice.
Keywords: omeprazole; process-related impurities; RP-HPLC; validation

FARMACIA, 2009, Vol. 57, 5 535
Introduction
Omeprazole (OPZ), 5-methoxy-2-[[(4-methoxy-3,5-dimethyl-2-
pyridinyl)methyl]sulphinyl]-1H-benzimidazole is a substituted benzimidazole
compound and a prototype anti-secretory agent, being the first “proton pump
inhibitor” widely used for the prophylaxis and treatment of gastro-duodenal
ulcers and for the treatment of symptomatic gastro-oesophageal reflux. It acts
by interacting with H + /K + ATPase in the secretory membranes of the parietal
cells and is very effective in the treatment of Zollinger–Ellison syndrome.
It is a lipophilic, weak base with pKa 1 = 4.2 and pKa 2 = 9 and can be
degraded unless it is protected against acid conditions. OPZ contains a
tricoordinated sulphur atom in a pyramidal structure and therefore can exist in two
different optically active forms, (S)- and (R)-omeprazole. OPZ was first approved
as a racemic mixture, but the (S) isomer was recently introduced on the market [1].
Few methods for the determination of the impurities either in bulk
drugs or pharmaceuticals have been reported. In the last few years, it can be
observed an increased interest for identification and quantification of
impurities in bulk drugs using new methodologies [2, 3].
There are only a few analytical methods available in literature for
the determination of omeprazole and its related impurities (Fig. 1) in bulk
drugs and pharmaceuticals [1,4,5].
Thus, there is a need for the development of analytical methods,
which will be useful for monitoring the levels of impurities in the finished
products with omeprazole during process development.
Experimental
Materials
Samples of omeprazole (batch nr. 90807/BA22347) and its related
impurities - impurity A (batch nr. 17998/ BA20692), impurity B (batch nr.
Rx935/BA20697), impurity C (batch nr. 310790/BA20693), impurity D
(batch nr. 210997/BA22424), impurity G (batch nr. 321_E/BA20694) and
impurity H (batch nr. AOP-324-1/BA22425) - were obtained from Union
Quimica Farmaceutica, Barcelona, Spain. HPLC grade acetonitrile,
triethylamine and orthophosforic acid 85% were obtained from Merck,
Darmstadt, Germany. LC grade water was deionized with Milli-Q and then
filtered using Milli-Q Academic, Millipore water purification system
(Milford, MA, USA). All other reagents were of analytical grade purity.
Equipment
The LC system, HPLC Agilent Series 1100, consisted of binary
gradient pump, autosampler, column oven and a UV detector. The output
signal was monitored and integrated using HP Chemstation software.

536
FARMACIA, 2009, Vol. 57, 5
Figure 1
Chemical structures of omeprazole and its process-related impurities
Solutions
Mobile phase.
A. Water: triethylamine 1% pH adjusted to 9.5 with 85%, w/v
H 3 PO 4 and acetonitrile in ratio (90:10v/v). It was filtered through a 0.45 µm
nylon membrane filter prior to use and degassed for 15 min.
B. Acetonitrile
Dilution Solution. A mixture of sodium tetraborat 0.01M and
acetonitrile in ratio (3:1 v/v)

FARMACIA, 2009, Vol. 57, 5 537
Impurities standard solutions. 0.5mg/mL impurities stock
standard solutions were prepared in methanol. These solutions were kept at
+4°C, protected from light. In these conditions the solutions were stable for
two months. Working solutions were prepared by dilution to a concentration
of 1 µg/mL and injected into the system.
Omeprazole standard solution. 1.015mg/mL omeprazole stock
standard solution was prepared in methanol and was kept at +4°C, protected
from light. In these conditions it was stable for two months. Omeprazole
working solutions were prepared by dilution using previously prepared
impurities solutions at concentrations between 40-200µg/mL and injected
into the system.
Sample solution. 2mg/mL solution of omeprazole sample was
prepared in methanol and diluted to a concentration of 0.200µg/mL and
injected into the system.
Forced degradation samples for the specificity study.
Omeprazole was kept in a climatic chamber at 40±5°C, 75±5%
humidity and was exposed to artificial light for 6 months to study the
formation of the degradation products. Every two weeks, an omeprazole
sample was withdrawn from the climatic chamber to perform the analyse.
Chromatographic conditions. A Zorbax Extend C 18 analytical
column (150mm×4.6 mm, 5µm packing) (Agilent) was used for analysis at
25°C. The mobile phase was pumped through the column at a flow rate of
0.8 mL/min. Gradient program was: 0min(18%B), 7.2min(18%B),
10.5min(50%B), 12min(50%B), 16min(18%B) and 18min(18%B). The
sample injection volume was 20µL. The UV detector was set to a
wavelength of 280 nm for the detection.
Results and discussion
Method development
In order to develop a suitable and robust LC method for the
determination of omeprazole and its process-related impurities, different
mobile phases and columns were employed to achieve the best separation
and resolution. Finally, the mobile phase consisting of water:triethylamine
1% pH adjusted to 9.5 with 85% H 3 PO 4 and acetonitrile at a flow rate of 0.8
mL/min, using a ZorbaxExtend C 18 (150mm×4.6mm, 5µm) column was
found to be appropriate, allowing good separation of omeprazole and its
process-related impurities. Because, the impurity D and the impurity G were
not separated in the described chromatographic conditions, we choose to use
only impurity D along with others impurities in future analysis. The reason

538
FARMACIA, 2009, Vol. 57, 5
for this choice is because it is a higher probability to find impurity D than
impurity G as degradation product in time.
Omeprazole and its process-related impurities show significant UV
absorbance at the wavelength of 280nm. Hence, this wavelength has been
chosen for detection.
Method validation
Specificity. In the above conditions, impurity A eluted first
followed by impurities D, B, omeprazole, H and C. The chromatogram
showing the separation of omeprazole and all its process-related impurities
is shown in Fig. 2.
Figure 2
The chromatogram of omeprazole and its impurities (standard solutions)
Linearity. Calibration curves were performed in the following
concentration ranges: for omeprazol (40.6-203 µg/mL), impurity A (0.9556-
14.334 µg/mL), impurity B (1.1568-17.352 µg/mL), impurity C (1.0772-
16.158 µg/mL), impurity D (1.289-19.344 µg/mL) and impurity H (0.7968-
11.952 µg/mL). The parameters of linearity curves are shown in table I.
Table I
Parameters of linearity curves of omeprazole and its impurities
Slope
(b)
Intercept
(a)
r Limit of detection (LOD )
(µg/mL)
Omeprazole 39.210 -29.414 0.9997 0.70
Impurity A 30.397 -12.095 0.9998 0.40
Impurity B 57.505 -14.112 0.9992 0.20
Impurity C 36.713 -18.166 0.9998 0.50
Impurity D 39.920 -1.554 0.9999 0.03
Impurity H 46.287 -10.037 0.9989 0.20

FARMACIA, 2009, Vol. 57, 5 539
Accuracy. The accuracy of the analytical method was evaluated by
analysing standard solutions of omeprazole and its related impurities at five
concentration levels. The obtained values are reported in Table II.
Compound
Omeprazole
Impurity A
Impurity B
Impurity C
Impurity D
Impurity H
Theoretical
concentration
(µg/mL)
Calculated
concentration
µg/mL ±SD
Table II
Accuracy of omeprazole and its impurities
Recovery % Accuracy
±SD
%
40.60 41.28 ± 0.03 101.67 ± 0.06 1.67
81.20 83.69 ± 0.56 103.07 ± 0.69 3.07
121.80 122.35 ± 0.53 100.45 ± 0.43 0.45
162.40 165.96 ± 0.61 102.19 ± 0.38 2.19
203.00 206.50 ± 0.58 101.72 ± 0.28 1.72
0.96 1.0 ± 0.03 109.18 ± 3.02 9.18
1.91 1.89 ± 0.15 99.13 ± 7.86 -0.87
3.82 3.90 ± 0.17 102.09 ± 4.37 2.09
7.64 7.11 ± 0.03 93.02 ± 0.38 -6.98
14.33 14.27 ± 0.05 99.58 ± 0.36 -0.42
1.15 1.15 ± 0.01 100.29 ± 1.00 0.29
2.13 2.13 ± 0.02 100.16 ± 0.98 0.16
4.62 4.52 ± 0.01 97.84 ± 0.22 -2.16
9.25 9.20 ± 0.05 99.46 ± 0.56 -0.54
17.35 17.33 ± 0.02 99.90 ± 0.09 -0.10
1.07 1.05 ± 0.03 98.13 ± 2.80 -1.87
2.15 2.15 ± 0.04 100.16 ± 1.76 0.16
4.30 4.33 ± 0.02 100.62 ± 0.36 0.62
8.61 8.51 ± 0.08 98.80 ± 0.93 -1.20
16.15 16.22 ± 0.02 100.45 ± 0.09 0.45
1.28 1.17 ± 0.02 91.41 ± 1.35 -8.59
2.52 2.46 ± 0.02 97.62 ± 0.69 -2.38
5.15 5.12 ± 0.02 99.42 ± 0.34 -0.58
10.31 10.32 ± 0.03 100.06 ± 0.31 0.06
19.34 19.30 ± 0.11 99.79 ± 0.58 -0.21
0.79 0.86 ± 0.07 108.86 ± 8.77 8.86
1.59 1.62 ± 0.04 101.68 ± 2.38 1.68
3.18 2.93 ± 0.03 92.03 ± 0.79 -7.97
6.37 6.27 ± 0.02 98.38 ± 0.33 -1.62
11.95 12.24 ± 0.69 102.45 ± 5.77 2.45
Precision. The precision of the analytical method was evaluated by
analysing standard solutions of omeprazole and its related impurities at
100% level. The obtained values for intra-day precision are reported in
Table III and for inter-day precision are reported in Table IV.

540
FARMACIA, 2009, Vol. 57, 5
Compound
Table III
Intra-day precision of the RP-HPLC method,
evaluated by analysing standard solutions of omeprazole and its impurities
Theoretical
concentration
(µg/mL)
Calculated
concentration
µg/mL ±SD
CV %
Omeprazole 121.80 121.74 ± 0.105 0.12
Impurity A 3.82 4.07 ± 0.007 0.18
Impurity B 4.62 4.53 ± 0.008 0.29
Impurity C 4.30 3.74 ± 0.010 0.26
Impurity D 5.15 5.07 ± 0.005 0.13
Impurity H 3.18 2.70 ± 0.009 0.34
Compound
Table IV
Inter-day precision of the RP-HPLC method,
evaluated by analysing standard solutions of omeprazole and its impurities
Theoretical
concentration
(µg/mL)
Calculated
concentration
µg/mL ±SD
CV %
Omeprazole 121.80 121.905 ± 0.479 0.39
Impurity A 3.82 4.131 ± 0.049 1.20
Impurity B 4.62 4.539 ± 0.016 0.35
Impurity C 4.30 3.828 ± 0.101 2.66
Impurity D 5.15 5.109 ± 0.028 0.55
Impurity H 3.18 2.715 ± 0.038 1.42
Limit of detection (LOD). Limits of detection for omeprazole and
its impurities were calculated based on the signal to noise ratio (S/N=10σ),
and were between 0.7µg/mL and 0.03 µg/mL, being presented in table I.
Limit of quantification (LOQ). Limits of quantitation for
omeprazole and its impurities were calculated based on the signal to noise
ratio (S/N=10σ), and were the following: omeprazole (2.331µg/mL),
impurity A (3.331µg/mL), impurity B (0.666µg/mL), impurity C
(1.665µg/mL), impurity D (0.099µg/mL), impurity H (0.666µg/mL).
Conclusion
A gradient reversed phase HPLC method has been developed and
validated for the determination of omeprazole and its process-related impurities
from bulk drug. This chromatographic assay fulfilled all the requirements for
being a reliable and feasible method, including accuracy, linearity, recovery
and precision. It is a highly specific and precise analytical procedure.
Therefore, this HPLC method can be used as a routine sample
analysis.

FARMACIA, 2009, Vol. 57, 5 541
Acknowledgements
The authors would like to acknowledge the help of Union Quimica
Farmaceutica, Barcelona, for providing process-related impurities of
omeprazole.
References
1. Espinosa Bosh M, Ruiz Sanchez A.J., Sanchez Rojas F., Bosch Ojeda C.
Analytical methodologies for the determination of omeprazole: An overview.
Journal of Pharmaceutical and Biomedical Analysis 2007; 44: 831–844.
2. Varvara A., Monciu C.M, Arama C., Popescu C. The HPLC assay of
ondansetron hydrochloride and its impurities using a new stationary phase.
Farmacia 2008; 56 (2):154-165.
3. Constantinescu D., Curea E. Reversed phase high performance liquid
chromatography (RP-HPLC) determination of lisinopril and its degradation
products in stability and compatibility studies. Farmacia 2008; 56 (1) :50-56.
4. Iuga C., Moldovan M., Popa A., Leucuţa S.E. Validation of HPLC-UV
method for analysis of omeprazole in presence of its metabolites in human
plasma. Farmacia, 2008, 56 (3): 254-260.
5. Ribani M, Collins CH, Bottoli CB. Validation of chromatographic methods:
evaluation of detection and quantification limits in the determination of
impurities in omeprazole. J Chromatogr A. 2007; (1156) 1-2:201-205.
___________________________
Manuscript received: 25.03.2009