Chapter-7 Determination of Assay and Related Substances...
-
Upload
hoangkhanh -
Category
Documents
-
view
235 -
download
3
Transcript of Chapter-7 Determination of Assay and Related Substances...
208
Overview:
Method development and validation of rapid stability indicating method
for determination of omeprazole (OME) and its related substances in
solid oral dosage form is presented in this chapter.
Chapter-7
Determination of Assay and
Related Substances of Omeprazole in
Pharmaceutical Dosage Form
209
Determination of Assay and Related Substances of
Omeprazole in Pharmaceutical Dosage Form
7.1 LITERATURE REVIEW
Impurity profiling of active pharmaceutical ingredients (API) in both bulk material and finalized
formulations is one of the most challenging tasks of pharmaceutical analytical chemists under
industrial environment [1]. The presence of unwanted or in certain cases unknown chemicals,
even in small amounts, may influence not only the therapeutic efficacy but also the safety of the
pharmaceutical products [2]. For these reasons, all major international pharmacopoeias have
established maximum allowed limits for related compounds for both bulk and formulated APIs.
As per the requirements of various regulatory authorities, the impurity profile study of drug
substances and drug products has to be carried out using a suitable analytical method in the final
product [3, 4].
Literature search revealed that, papers on degradation of omeprazole [5], determination by UV
spectrophotometry method [6], omeprazole (OME) in human plasma and urine by LC-MS-MS
[7], colorimetric method [8], determination of S-omeprazole, R-omeprazole and racemic
omeprazole [9] are available, but as such there is no validated method available, which reports
more known and unknown impurities precisely and significantly for omeprazole (OME), as such
and in drug product. It’s validated analytical performance in terms of major parameters such as
selectivity, accuracy, precision and sensitivity is adequate for the routine quality control of the
purity of omeprazole containing pharmaceutical formulations. The important part of method is
with help of single injection, quantification of omeprazole and its degradable impurities and
process impurities. Common method for assay and related substances, as well as analytical
methods, are validated to ensure they are suitable for their intended use and give accurate and
reliable data.
Chapter-7
210
7.2 THE SCOPE AND OBJECTIVES OF PRESENT STUDY
There is no stability-indicating HPLC method reported in the literature that can adequately
separate all impurities and accurately quantify omeprazole API and dosage forms. It is, therefore,
felt necessary to develop a new rapid, stability-indicating method for the related substance
determination and quantitative determination of omeprazole. An attempt is made to determine
whether HPLC can reduce analysis times without compromising the resolution and sensitivity.
The objectives of the present work are as follow:
To developed a rapid, stability indicating RP-HPLC method for determination of
omeprazole and its related substances in solid oral dosage form.
Forced degradation study.
To separates omeprazole from its all impurities (Benzamithazone, N-Oxide, Sulphone,
Des-Methoxy and Sulphide) known impurities/ degradation products and any unknown
degradation product generated during forced degradation study.
Perform analytical method validation for the proposed method as per ICH guideline.
7.3 OMEPRAZOLE
Omeprazole is highly effective inhibitor of gastric acid secretion used in the therapy of stomach
ulcers and zollinger-ellison syndrome. The drug inhibits the H(+)-K(+)-ATPase (H(+)-K(+)-
exchanging ATPase) in the proton pump of gastric parietal cells [10, 11]. The chemical IUPAC
name of omeprazole is 6-methoxy-2-[(4-methoxy-3,5-dimethylpyridin-2-yl)methylsulfinyl]-1H-
benzimidazole. Its empirical formula is C17H19N3O3S, and its structural formula is shown in
Figure 7.1 [12]; omeprazole Mg is a white to off-white free-flowing crystalline powder with a
molecular weight of 713.1. The route of synthesis of omeprazole Mg resulted five known
impurities, Benzamidazole, N-Oxide, Sulphone, Des-Methoxy and Sulphide. The presented
method is capable for quantification of all known and unknown impurities of omeprazole Mg
accurately.
Determination of assay and related substances of omeprazole in pharmaceutical dosage form
211
Figure 7.1 3D and 2D Structure of omeprazole
Indications
For the treatment of acid-reflux disorders (GERD), peptic ulcer disease, H. pylori eradication,
and prevention of gastroinetestinal bleeds with NSAID use.
Pharmacodynamics
Omeprazole is a compound that inhibits gastric acid secretion and is indicated in the treatment of
gastroesophageal reflux disease (GERD), the healing of erosive esophagitis, and H.
pylori eradication to reduce the risk of duodenal ulcer recurrence. Omeprazole belongs to a new
class of antisecretory compounds, the substituted benzimidazoles, that do not exhibit
anticholinergic or H2 histamine antagonistic properties, but that suppress gastric acid secretion
by specific inhibition of the H+/K
+ ATPase at the secretory surface of the gastric parietal cell. As
a result, it inhibits acid secretion into the gastric lumen. This effect is dose-related and leads to
inhibition of both basal and stimulated acid secretion irrespective of the stimulus.
Mechanism of action
Omeprazole is a proton pump inhibitor that suppresses gastric acid secretion by specific
inhibition of the H+/K
+-ATPase in the gastric parietal cell. By acting specifically on the proton
pump, omeprazole blocks the final step in acid production, thus reducing gastric acidity.
Chapter-7
212
Absorption
Absorption is rapid, absolute bioavailability (compared to intravenous administration) is about
30-40% at doses of 20-40 mg.
Protein binding
95%
Metabolism
Hepatic
Route of elimination
Urinary excretion is a primary route of excretion of omeprazole metabolites.
Half life
0.5-1 hour
Toxicity
Symptoms of overdose include confusion, drowsiness, blurred vision, tachycardia, nausea,
diaphoresis, flushing, headache, and dry mouth.
7.4 EXPERIMENTAL
7.4.1 Materials and reagents
Omeprazole tablets, placebo, omeprazole working standard (90.2% w/w), benzamidazole
working standard (99.7% w/w), N-oxide working standard (98.4% w/w), sulphone working
standard (98.7% w/w), des-methoxy working standard (95.1% w/w) and sulphide working
standard (99.8% w/w) are provided by Cadila Pharmaceutical Ltd. Dholka, Ahmedabad, India.
HPLC grade methanol and acetonitrile are obtained from J.T. Baker (NJ., USA). GR grade
glycine and sodium hydroxide are obtained from Merck Ltd (Mumbai, India). Nylon membrane
Determination of assay and related substances of omeprazole in pharmaceutical dosage form
213
filters (0.45 µ), nylon syringe filters and PVDF syringe filters are purchased from Pall Life
Science Limited (India). High purity water is generated with Milli-Q Plus water purification
system (Millipore, Milford, MA, USA).
7.4.2 Equipments
Cintex digital water bath (Mumbai, India) is used for specificity study. Photo stability studies are
carried out in a photo-stability chamber (SUNTEST XLS+, ATLAS, Germany). Thermal
stability studies are performed in a dry air oven (Cintex, Mumbai, India).
7.4.3 Preparation of mobile phase
Mobile phase-A:
0.04 M glycine (adjusted pH 8.8 with diluted sodium hydroxide solution) buffer solution is used
as a MP-A.
Mobile Phase-B:
Mixture of acetonitrile and methanol in ratio of (83:17) is used as MP-B.
Table 7.1 Gradient Program
Time in min Mobile-A Mobile-B
0 88 12
20 40 60
21 88 12
25 88 12
7.4.4 Diluent preparation
Dissolve 4.47 gm of Na2HPO4 and 0.73 gm of NaH2PO4 in water. Make the volume up to 1000
mL with water. Dilute 250mL of this solution to 1000 mL with water (pH 8.0, adjusted with
dilute NaOH solution). Mix buffer (pH 8.0) and acetonitrile at a ratio of 90:10.
Chapter-7
214
7.4.5 Standard solution (RS)
An accurately weight and transfer about 5 mg of each impurity and 5 mg of OME standard into
100mL volumetric flask dissolve and dilute with diluent and mix. Dilute 5.0mL of this solution
to 50mL with diluent.
7.4.6 Resolution solution
An accurately weight and transfer about 1 mg of each impurity and 50 mg OME standard into
100mL volumetric flask, dissolve and dilute up to the mark with diluent, mix well.
7.4.7 Standard solution (Assay)
An accurately weight and transfer about 25 mg of OME working standard in to 50mL volumetric
flask add 10mL of dimethylformamide to dissolve it, dilute up to the mark with diluent (500
µg/mL), mix well.
7.4.8 Sample solution preparation
Transfer 5 tablets in to 200mL volumetric flask add about 10mL of dimethylformamide and
sonicate for five minutes and add about 150mL of diluent and sonicate for 20 minutes, dilute to
volume with diluent. Filter this solution with 0.45µ nylon filter.
7.4.9 Placebo solution preparation
Transfer 5 placebo tablets in to 200 mL volumetric flask add about 10mL of dimethylformamide
and sonicate for five minutes and add about 150mL of diluent and sonicate for 20 minutes, dilute
to volume with diluent. Filter this solution with 0.45µ nylon filter.
7.4.10 Chromatographic conditions
The liquid chromatography consisted of an Agilent and waters system, equipped with automatic
sample injector and PDA detector. For data collection and calculation chemstation Software and
Waters EmpowerTM
-2 software is used. The chromatographic condition is optimized using a
Zorbax XDB C8 (150 x 4.6mm, 5µ) column. 0.04M glycine buffer (pH adjusted 8.8) as a MP-A
Determination of assay and related substances of omeprazole in pharmaceutical dosage form
215
and mixture of acetonitrile: methanol (83:17) as a MP-B. The mobile phase is filtered through a
0.45µ Nylon membrane filter and degassed under vacuum prior to use. The flow rate is 1.2
mL/min with gradient program [Table 7.1]. The monitoring wavelength is 302 nm and the
injection volume is 10 µL with maintaining column oven temperature at 25ºC and sample cooler
temperature is 10˚C.
7.5 METHOD VALIDATION
The method described herein has been validated for simultaneous determination of assay and its
related substances by RP-HPLC.
7.5.1 Specificity
Forced degradation studies are performed to demonstrate selectivity and stability-indicating
capability of the proposed method. The sample are exposed to acid hydrolysis [0.1 N HCl (5
mL), 25°C, 15 min], alkaline hydrolysis [0.1N NaOH (5 mL), 60°C, 3h], oxidative [1 % H2O2 (5
mL), 60°C, 3h], water hydrolysis [at 60°C for 4h], thermal [104°C, 4h], and photolytic
degradation [1.2 million Lux hours]. All exposed samples are than analysed by the proposed
method.
7.5.2 System suitability
Inject diluent, one injection of resolution solution, six replicate of standard solution (RS), five
replicate injection of standard solution (Assay) and check the system suitability as follows.
1) In resolution solution, the resolution between OME and des-methoxy should not less than 2.0.
2) The % RSD of area due to all impurities peaks and OME peaks in six replicate injections of
standard solution (RS) should not more than 5.0.
3) The % RSD of the area due to OME in five replicate injection of standard preparation (Assay)
should not more than 2.0.
Chapter-7
216
7.5.3 Precision
The precision of the system is determined using the sample preparation procedure described
above for six real samples of formulation and analysis using the same proposed method.
Intermediate precision is studied by other scientist, using different columns, different HPLC, and
is performed on different day.
7.5.4 Accuracy
To confirm the accuracy of the proposed method, recovery experiments are carried out by the
standard addition technique for assay and related substances. The accuracy of the assay method
for OME is evaluated in triplicate (n=3) at the three concentrations of 250, 500 and 750 µg/mL
(50, 100 and 150 %) of drug product, and the recovery is calculated for each added (externally
spiked) concentration. The mean of percentage recoveries (n=9) and the relative standard
deviation are calculated. For all impurities, the recovery is determined in triplicate (n=3) for
LOQ, 0.5, 1.0 and 1.5 µg/mL (LOQ, 50, 100, and 150 %) and the recovery of the impurities are
calculated. The mean of percentage recoveries (n=12) and the relative standard deviation are also
calculated for related substances.
7.5.5 Linearity
For related substances test, linearity is demonstrated from 0.1 to 1.5µg/mL of standard
concentration of impurities using a minimum of ten calibration levels (0.10, 0.15, 0.20, 0.25,
0.30, 0.50, 0.75, 1.0, 1.25 and 1.50µg/mL) for benzamidazole, N-oxide, suphone, des-methoxy,
and sulphide impurities. For assay test, linearity is demonstrated from 50% to 150% of standard
concentration using a minimum of five calibration levels (250, 375, 500, 625 and 750µg/mL) for
OME. The method of linear regression is used for data evaluation. The peak areas of the standard
compounds are plotted against the respective OME, benzamidazole, N-oxide, sulphone, des-
methoxy and sulphide concentrations. Linearity is described by the linearity equation, correlation
coefficient and Y-intercept bias is also determined.
Determination of assay and related substances of omeprazole in pharmaceutical dosage form
217
7.5.6 Limit of detection (LOD) and limit of quantification (LOQ)
Limit of detection and quantification concentrations of omeprazole determined based on standard
deviation of response and slope method. The linearity study is performed in the range of 10% to
150.0% of the limit concentration of OME, benzamedazole, N-Oxide, sulphone, des-methoxy
and sulphide, considering 0.2% limit for all known impurities and 0.10% for any unknown
impurities. Duplicate injection of linearity solutions are injected performed. Linearity graph of
concentration in µg/mL (X-axis) versus peak area response (Y-axis) is plotted. LOD and LOQ
concentrations of omeprazole and its impurities are determined on the basis of equation given
below.
Limit of Detection = (3.3 X σ) / S
Limit of Quantification = (10 X σ) / S
Where, σ = Residual standard deviation of regression line
S = Slope of calibration curve
Injected six replicate injections of these LOD and LOQ concentrations and ensured the peak is
detected and responses are measured.
7.5.7 Robustness
The robustness is a measure of the capacity of a method to remain unaffected by small but
deliberate changes, change in wavelength (± 2.0nm), change in column oven temperature (±
5°C), changes in flow rate (± 0.1mL/min), and change in buffer pH (± 0.2). All important
characteristic including resolution between des-methoxy and OME, tailing factor, theoretical
plate number and the retention behaviour of interested compound are evaluated.
7.5.8 Solution stability
The stability of the sample solution (for related substances) is established by storage of the
sample solution at 10°C temperature for 25h. The sample solution is re-analyzed after 7h, 16h,
Chapter-7
218
21h, 25h and the results of the analysis are compared with the results of the fresh sample. The
stability of standard solution and sample solution for assay are established by the storage of them
at ambient temperature for 54h. The standard and sample solution are re-injected after 7h, 16h,
21h, 25h, 32h, 44h and 54h, % difference is calculated for it.
7.5.9 Filter compatibility
Filter compatibility is performed for 0.45 μ nylon syringe filter and 0.45 μ PVDF syringe filter.
To confirm the filter compatibility in proposed analytical method, filtration recovery experiment
is carried out by sample filtration technique. Sample is filtered through both syringe filters and
percentage assay and impurities are calculated and compared against centrifuged sample.
7.6 RESULTS AND DISCUSSION
7.6.1 Method Development and Optimization
The main objective of the RP-HPLC method development is to rapid assay and related
substances determination of omeprazole in pharmaceutical formulation. Developed method
should be able to determine assay (AS) and related substances (RS) in single run and should be
accurate, reproducible, robust, stability indicating, filter compatible, linear, free of interference
from blank/ placebo/ impurities / degradation products and straightforward enough for routine
use in quality control laboratory.
The spiked solution of omeprazole (500 µg/mL), benzamidazole (10 µg/mL), N-oxide (10
µg/mL), sulphone (10 µg/mL), des-methoxy (10 µg/mL) and sulphide (10 µg/mL) is subjected to
separation by RP-HPLC. Initially the separation of all compounds is studied using water as a
mobile phase-A (MP-A) and acetonitrile (ACN) as a mobile phase-B (MP-B) on a HPLC column
Hypersil BDS (C8, 150 x 4.6mm, 5µ) using a Waters (HPLC) system with the linear gradient
program. The flow rate of 1.0 mL/min is selected with regards to the backpressure and analysis
Determination of assay and related substances of omeprazole in pharmaceutical dosage form
219
time as well. Various types of MP-A and MP-B are studied to optimize the method; some of
them are summarized in Table 7.2 with the associated observations.
Table 7.2 Summary of method optimization
Experimental condition Observation
Water (MP-A) and ACN (MP-B), linear gradient;
Hypersil BDS-C8 (150 × 4.6 mm, 5µ); 25°C
No resolution found between
omeprazole and impurities
0.01 M KH2PO4, pH-7.0 (MP-A) and ACN (MP-B),
linear gradient; Hypersil BDS-C8 (150 × 4.6 mm, 5µ);
25°C
Peak shape is not good
0.04 M Glycine pH-9.0 (MP-A) and ACN (MP-B), linear
gradient; Zorbax Eclipse XDB C8 (4.6 x 150 mm) 5 µm;
25°C
Peak separation observed but
peak shape is not good
0.04 M Glycine pH-8.8(MP-A) and ACN : methanol,
(83:17)(MP-B), linear gradient; Zorbax Eclipse XDB-C8
(150 x 4.6 mm, 5µ); 25°C
Satisfactory peak separation
and sharp peak shape
ACN … Acetonitrile
Based on above experimental study, the optimized HPLC parameters are; flow rate 1.2 mL/min;
column oven temperature 25°C; 0.04 M glycine buffer pH-8.8 as MP-A and, a mixture of ACN,
and MeOH in the ratio of 83:17 v/v as MP-B. In order to achieve symmetrical peak shape of all
substances and more resolution between OME and des-Methoxy, different stationary phases are
explored. Finally the desired separation with symmetrical peaks is obtained using Zorbax Eclipse
XDB C8 (150 x 4.6 mm, 5 µ) column. Column oven temperature is also studied and found that
25°C is more appropriate with respect to separation and peak shape. Based on compounds UV
response, 302nm is found to be more appropriate for determination of OME and its impurities
from single run. OME, benzamidazole, N-oxide, sulphone, des-methoxy and sulphide are well
resolved from each other and there is no chromatographic interference observed due to blank and
placebo in a reasonable time of 25.0 minutes, which are presented in Figure 7.2.
Chapter-7
220
Figure 7.2 Expanded overlaid specimen chromatograms of blank, placebo and sample
7.6.2 Analytical Parameters and Validation
After satisfactory development of the method it is subjected to method validation as per ICH
guideline [13]. The method is validated to demonstrate that it is suitable for its intended purpose
by the standard procedure to evaluate adequate validation characteristics (specificity, system
suitability, precision, accuracy, linearity, LOD, LOQ, robustness, solution stability and filter
compatibility).
7.6.2.1 Specificity
Specificity is the ability of the method to measure the analyte response in the presence of its
potential impurities. Forced degradation studies are performed to demonstrate selectivity and
stability indicating capability of the proposed RP-HPLC method. Figure 7.2 is show that there is
no any interferences at the RT (retention time) of OME due to blank, placebo and impurities.
Stress studies are performed at concentration of 500 µg/mL of OME to provide the stability
indicating property and specificity of the proposed method.
The sample is exposed to acid hydrolysis, alkaline hydrolysis, oxidative, water hydrolysis,
thermal, and photolytic degradation to evaluate the ability of the proposed method to separate
OME from its degradation products. Minor degradation products are observed when OME is
Determination of assay and related substances of omeprazole in pharmaceutical dosage form
221
subjected to base, heat, photolytic and hydrolytic conditions. Significant degradation is observed
when the drug product is subjected to acid hydrolysis, and oxidation. The purity of the peaks
obtained from the stressed sample is verified using the PDA detector. The results of forced
degradation study are summarized in Table 7.3. Specimen chromatogram of specificity study
presented in Figure 7.3 to 7.11.
Table 7.3 Summary of forced degradation results
Degradation
condition
Mass
balance#
Purity Observation
Control sample 99.8 Pass NA
Acidic hydrolysis 100.1 Pass Significant degradation observed
Alkaline hydrolysis 98.8 Pass No significant degradation observed
Oxidation 97.5 Pass Significant degradation observed
Water hydrolysis 98.4 Pass No significant degradation observed
Thermal (solid) 98.3 Pass No significant degradation observed
Photolytic 99.1 Pass No significant degradation observed
NA= Not applicable; # = % assay + % known impurities + area % unknown impurities
Figure 7.3 Specimen chromatogram of diluent
Chapter-7
222
Figure 7.4 Specimen chromatogram of placebo
Figure 7.5 Specimen chromatogram of OME
Figure 7.6 Specimen chromatogram of benzamedazole
Determination of assay and related substances of omeprazole in pharmaceutical dosage form
223
Figure 7.7 Specimen chromatogram of N-oxide
Figure 7.8 Specimen chromatogram of sulphone
Figure 7.9 Specimen chromatogram of des-methoxy
Chapter-7
224
Figure 7.10 Specimen chromatogram of sulphide
Figure 7.11 Specimen chromatogram of impurities spiked in OME sample
7.6.2.2 System suitability
The % RSD of area counts of five replicate injections is below 1.0 % in OME standard solution
(Assay). The % RSD of all impurities area counts for six replicate injections are below 1.0% in
standard solution (RS). The resolution between des-methoxy and OME is more than 3.0 in
resolution solution. The all parameters complied with the acceptance criteria and system
suitability is established. The system suitability result for system precision, method precision and
intermediate precision are summarized in Table 7.4.
Determination of assay and related substances of omeprazole in pharmaceutical dosage form
225
Table 7.4 System suitability results (system precision, method precision and intermediate
precision)
Test Parameters % RSD Resolution between OME
and Des-Methoxy
System precision Omeprazole (assay) 0.1 3.1
Benzamedazole 0.4
N-Oxide 0.5
Sulphone 0.3
Des-Methoxy 0.2
Sulphide 0.2
Omeprazole (RS) 0.9
Precision Omeprazole (assay) 0.1 3.0
Benzamedazole 0.3
N-Oxide 0.4
Sulphone 0.2
Des-Methoxy 0.1
Sulphide 0.4
Omeprazole (RS) 0.7
Intermediate
precision
Omeprazole (assay) 0.2 3.1
Benzamedazole 0.1
N-Oxide 0.4
Sulphone 0.3
Des-Methoxy 0.5
Sulphide 0.2
Omeprazole (RS) 0.2
RS … Related substance
7.6.2.3 Precision
In this study, % RSD of sample is observed, by injecting six sets of sample solution. %RSD for
assay and related substances results of six samples are calculated. The result should not deviate
more than 1.0% for assay value and for related substances it’s not deviate more than 0.01% for
all impurities. It is well within the acceptances criteria for all the sets and method found
repeatable and precise for intended purpose. Method precision and intermediate precision results
are summarized in Table 7.5.
Chapter-7
226
Table 7.5 Precision (n=6) and intermediate precision (n=6) results
Substance Precision Intermediate precision
% Difference Mean % % RSD Mean % % RSD
OME (1000µg/mL) 99.1 1.0 98.8 0.8 0.3
Benzamedazole 0.006 7.7 0.008 6.8 0.002
N-Oxide 0.086 0.5 0.086 1.7 0.000
Sulphone 0.227 1.9 0.226 2.6 0.001
Des-Methoxy 0.031 2.7 0.032 3.1 0.001
Sulphide 0.041 1.8 0.040 4.3 0.001
Unknown impurity 0.050 1.0 0.051 2.1 0.001
Total impurities 0.441 3.2 0.443 2.5 0.002
7.6.2.4 Accuracy
Accuracy of a method is defined as the closeness of the measured value to the true value for the
sample. Accuracy has been performed for RS in the range of LOQ to 150.0% (LOQ, 50.0%,
100.0%, and 150.0%) of target concentration of OME considering limit 0.1% (limit of individual
unknown impurity) and 0.2% of (limit of all known impurities). Accuracy has been performed
for assay in the range of 50 to 150 % of target concentration (500 µg/mL).
The % recovery for the range 50.0% to 150.0% of target concentration has found within the
acceptance criteria with acceptable % RSD of NMT 2.0 at each level. The recovery at each level
is within 98.0% to 102.0%. The recovery at each level of each impurity is within 80% to 120%
of target concentration. This indicates that the method is accurate for the analysis of OME assay
and related substances method. Accuracy study results are summarized in Table 7.6. Overlaid
specimen chromatograms of accuracy study (related substance) are presented in Figure 7.12.
Determination of assay and related substances of omeprazole in pharmaceutical dosage form
227
Figure 7.12 Overlaid specimen chromatograms of accuracy study
Table 7.6 Accuracy results
Substance
LOQ (n=3) At 50% (n=3) At 100% (n=3) At 150% (n=3)
%
Rec.$
%
RSD
%
Rec.$
%
RSD
%
Rec.$
%
RSD
%
Rec.$
%
RSD
OME (Assay) NA NA 99.6 0.8 99.2 0.5 101.2 0.7
#Benzamedazole 85.1 2.3 82.8 1.3 85.4 0.3 89.3 4.7
#N-Oxide 87.1 1.2 91.3 1.5 94.7 2.1 96.0 1.7
#Sulphone 98.4 0.8 103.5 2.1 98.9 1.7 93.3 1.0
#Des-Methoxy 81.3 1.1 85.9 2.0 87.4 1.0 85.1 0.3
#Sulphide 81.3 1.6 82.9 0.1 97.3 0.6 92.2 0.2
Unknown impurity 99.9 2.4 100.1 0.2 100.0 0.2 99.7 0.1
$... Recovery, #... For Related Substances, NA… Not applicable
7.6.2.5 Linearity
Concentration levels are 50, 75, 100, 125 and 150% of the claimed analyte concentration of
assay, corresponding to the range of about 250-750 µg/mL. For related substances each
impurities concentration levels are LOQ, 15%, 20%, 25%, 30%, 50%, 75%, 100%, 125% and
Chapter-7
228
150% of the claimed analyte concentration of impurities, corresponding to the range of about
LOQ to1.5 mcg/mL. The calibration curve obtained by plotting the peak area versus the
concentration is linear in the mentioned concentration range of 50% to 150% for assay and LOQ
to 150% for related substances. For assay correlation coefficient of linearity curve is less than
0.999 and Y-intercept bias is within ± 2.0% of 100% linearity response. For RS correlation
coefficient of linearity curve is less than 0.998 and Y-intercept bias is within ± 5.0% of 100%
linearity response.
Linearity regression statistic results are summarized in the Table 7.7. Linear curve of OME
(assay), OME (RS) and all known impurities are presented in Figure 7.13 to 7.19. Obtained
results are indicated that the method is linear up to the specified range of concentrations.
Overlaid specimen chromatograms of linearity (RS) study are presented in Figure 7.20.
Table 7.7 Regression statistics
Compound Linearity
(µg/mL)
Correlation
coefficient (r2)
Linearity (Equation) Y- intercept
bias at 100%
Omeprazole (Assay) 250 to 750 0.999 Y= 21034x + 5521.8 0.6
Benzamidazole 0.1 to 1.5 0.999 y= 47630x-372.9 0.8
N-Oxide 0.1 to 1.5 0.999 y= 13718x + 99.4 0.7
Sulphone 0.1 to 1.5 0.999 y= 14419x + 40.9 0.3
Des-Methoxy 0.1 to 1.5 0.999 y= 18966x + 1374.9 2.7
Sulphide 0.1 to 1.5 0.999 y= 17722x + 595.5 3.2
Omeprazole (RS) 0.1 to 1.5 0.999 y= 59502x-372.9 2.2
Determination of assay and related substances of omeprazole in pharmaceutical dosage form
229
Figure 7.13 Linearity curve of omeprazole (assay)
Figure 7.14 Linearity curve of benzamedazole
Figure 7.15 Linearity curve of N-Oxide
y = 21034x + 5521.8
R² = 0.9997
0
2000000
4000000
6000000
8000000
10000000
12000000
14000000
16000000
0 100 200 300 400 500 600 700 800
Are
a
Conc. In ppm
Linearity of Omeprazole (Assay)
y = 47630x - 372.9
R² = 0.9998
0.000
10000.000
20000.000
30000.000
40000.000
50000.000
60000.000
70000.000
80000.000
0.000 0.200 0.400 0.600 0.800 1.000 1.200 1.400 1.600
Are
a
Conc. in ppm
Linearity of Benzamedazone
y = 13718x + 99.4
R² = 0.9998
0.000
5000.000
10000.000
15000.000
20000.000
25000.000
0.000 0.200 0.400 0.600 0.800 1.000 1.200 1.400 1.600 1.800
Are
a
Conc. in ppm
Linearity of N-Oxide
Chapter-7
230
Figure 7.16 Linearity curve of sulphone
Figure 7.17 Linearity curve of Des-Methoxy
Figure 7.18 Linearity curve of sulphide
y = 14419x + 40.9
R² = 0.9999
0.000
5000.000
10000.000
15000.000
20000.000
25000.000
0.000 0.200 0.400 0.600 0.800 1.000 1.200 1.400 1.600 1.800
Are
a
Conc. In ppm
Linearity of Sulphone
y = 18966x + 1374.9
R² = 0.9999
0.000
5000.000
10000.000
15000.000
20000.000
25000.000
30000.000
35000.000
0.000 0.200 0.400 0.600 0.800 1.000 1.200 1.400 1.600
Are
a
Conc. In ppm
Linearity of Des-Methoxy
y = 17722x + 595.5
R² = 0.9998
0.000
5000.000
10000.000
15000.000
20000.000
25000.000
30000.000
0.000 0.200 0.400 0.600 0.800 1.000 1.200 1.400 1.600 1.800
Are
a
Conc. In ppm
Linearity of Sulphide
Determination of assay and related substances of omeprazole in pharmaceutical dosage form
231
Figure 7.19 Linearity curve of omeprazole (RS)
Figure 7.20 Overlaid specimen chromatograms of linearity study (RS)
7.6.2.6 Limit of detection (LOD) and limit of quantification (LOQ)
The detection limit of individual analytical procedure is the lowest amount of analyte in a sample
which can be detected but not necessarily quantified as an exact value and quantification limit is
the lowest amount of analyte in a sample which can be quantitatively determined with suitable
precision and accuracy. Limit of detection and quantification concentrations of omeprazole
determined based on standard deviation of response and slope method.
y = 59502x - 372.92
R² = 0.9998
0.00
10000.00
20000.00
30000.00
40000.00
50000.00
60000.00
70000.00
80000.00
0.000 0.200 0.400 0.600 0.800 1.000 1.200 1.400
Are
a
Conc. In ppm
Omeprazole (Related Substance)
Chapter-7
232
Duplicate injection of linearity solutions are injected into chromatography system. Linearity
graph of concentration in µg/mL (X-axis) versus peak area response (Y-axis) is plotted. The
correlation coefficient, slope of regression line and RSD of regression line are calculated.
Injected six replicate injections of these LOD and LOQ concentrations and ensured the peak is
detected and responses are measured. Chromatogram of LOD and LOQ are presented in Figure
7.21 and Figure 7.22 respectively. Results for LOD, LOQ and LOQ precision are summarized in
Table 7.8.
Figure 7.21 Specimen chromatogram of LOD
Figure 7.22 Specimen chromatogram of LOQ
Determination of assay and related substances of omeprazole in pharmaceutical dosage form
233
Table 7.8 Results of LOD, LOQ and LOQ precision (n=6)
7.6.2.7 Robustness
The robustness of an analytical procedure is a measure of its capacity to remain unaffected by
small but deliberate variations in method parameters and provides an indication of its reliability
during normal usage. No significant effect is observed on system suitability parameters such as
RSD, and resolution, when small but deliberate changes are made to chromatographic conditions.
The results are summarized in Table 7.9, along with the system suitability parameters of normal
conditions. Thus, the method is found to be robust with respect to variability in applied
conditions.
Table 7.9 Robustness study results
Condition Name of component Resolution between Des-
Methoxy and OME
% RSD of five
injections
Normal
methodology
Omeprazole (Assay) 3.0 0.13
Benzamidazone 0.34
N-Oxide 0.42
Sulphone 0.23
Des-Methoxy 0.11
Sulphide 0.40
Omeprazole (RS) 0.72
Name of
compound LOD (µg/mL)
LOQ
(µg/mL)
LOQ precision
(% RSD)
OME 0.060 0.180 2.2
Benzamedazole 0.020 0.060 2.4
N-Oxide 0.016 0.048 4.2
Sulphone 0.010 0.030 2.5
Des-Methoxy 0.015 0.045 1.7
Sulphide 0.017 0.051 2.5
Chapter-7
234
At 300 nm
Wavelength
Omeprazole (Assay) 3.0 0.36
Benzamidazone 0.40
N-Oxide 0.33
Sulphone 0.32
Des-Methoxy 0.57
Sulphide 0.29
Omeprazole (RS) 0.43
At 304 nm
Wavelength
Omeprazole (Assay) 3.0 0.18
Benzamidazone 0.32
N-Oxide 0.19
Sulphone 0.11
Des-Methoxy 0.95
Sulphide 0.05
Omeprazole (RS) 0.31
Column temperature
35˚C
Omeprazole (Assay) 3.1 0.05
Benzamidazone 0.50
N-Oxide 0.31
Sulphone 0.03
Des-Methoxy 1.50
Sulphide 0.04
Omeprazole (RS) 0.56
Column temperature
25˚C
Omeprazole (Assay) 3.2 0.07
Benzamidazone 0.38
N-Oxide 0.11
Sulphone 0.08
Des-Methoxy 0.23
Sulphide 0.04
Omeprazole (RS) 0.78
At 1.3 mL/min flow Omeprazole (Assay) 2.9 0.11
Benzamidazone 0.28
N-Oxide 0.16
Sulphone 0.37
Des-Methoxy 0.11
Determination of assay and related substances of omeprazole in pharmaceutical dosage form
235
Sulphide 0.10
Omeprazole (RS) 1.21
At 1.1 mL/min flow Omeprazole (Assay) 3.1 0.19
Benzamidazone 1.58
N-Oxide 0.14
Sulphone 0.37
Des-Methoxy 0.37
Sulphide 0.21
Omeprazole (RS) 0.23
Buffer pH 8.6 Omeprazole (Assay) 2.9 0.16
Benzamidazone 0.47
N-Oxide 0.21
Sulphone 0.16
Des-Methoxy 0.59
Sulphide 0.16
Omeprazole (RS) 0.79
Buffer pH 9.0 Omeprazole (Assay) 3.1 0.36
Benzamidazone 0.90
N-Oxide 0.36
Sulphone 0.41
Des-Methoxy 0.58
Sulphide 0.31
Omeprazole (RS) 0.47
7.6.2.8 Solution stability
The solution stability is checked for sample preparation from initial to 25h for related substances
and initial to 54h for assay. For related substances % difference is not more than 0.1. For assay %
area difference is not more than 2.0. The results obtained are well within the acceptance criteria
up to 25h at 10˚C temperature. Solution stability result of sample, standard for assay and related
substance summarized in Table 7.10 and 7.11.
Chapter-7
236
Table 7.10 Solution stability of sample preparation for related substances
Time in
Hours Benzamidazole Sulphone N-Oxide
Uk-
(RRT-
0.36)
Uk-
(RRT-
0.85)
Total
impurity
%
Difference
Initial 0.02 0.25 0.08 0.03 0.05 0.43 NA
7 h 0.01 0.27 0.08 0.03 0.06 0.45 -0.02
16 h 0.01 0.29 0.08 0.03 0.07 0.48 -0.05
21 h 0.01 0.30 0.08 0.03 0.08 0.50 -0.07
25 h 0.02 0.30 0.08 0.03 0.08 0.52 -0.09
NA … Not Applicable; Uk … un known; RRT … related retention time
Table 7.11 Solution stability of standard and sample preparation for assay
Time in
Hours
Area of
standard
solution
% Difference
Area of
sample
solution
% Difference
Initial 10071745 NA 10696650 NA
7 h 10049421 0.22 10715280 0.17
16 h 10045327 0.26 10677310 0.18
21 h 10070728 0.01 10691846 0.04
25 h 10073984 0.02 10702234 0.05
32 h 10059240 0.12 10698153 0.01
44 h 10074238 0.02 10729678 0.31
54 h 10043342 0.28 10677789 0.18
NA… Not Applicable
7.6.2.9 Filter compatibility
The filter compatibility is studied for two different filters 0.45 µm PVDF filter and 0.45 µm
nylon filters. A single set of sample solution is prepared and some of the portion of this solution
is centrifuged, filtered 10mL of sample solution through 0.45μm nylon filter and 0.45µm PVDF
filter. Centrifuged sample solution and filtered solutions are injected in the chromatography
system. For assay, the % difference in assay value is less than 1.0, from centrifuge solution
Determination of assay and related substances of omeprazole in pharmaceutical dosage form
237
value. For RS, the % difference in the value is less than 0.05, from centrifuge solution value.
Filter compatibility study results for assay and RS are summarized in Table 7.12 and 7.13,
respectively. Based on obtained results PVDF and Nylon both the filters are compatible for the
developed method.
Table 7.12 Filter compatibility study result for Assay
Component Name Centrifuge sample Sample filter with
Nylon filter
Sample filter with
PVDF filter
Omeprazole 100.0% 99.8% 99.5%
% difference Not applicable 0.2% 0.5%
Table 7.13 Filter compatibility study result for related substance
Name of
Impurity
Centrifuged
sample
Sample filter
with Nylon
%Difference
against
centrifuge
Sample
filter with
PVDF
%Difference
against
centrifuge
Un Single Max 0.04% 0.03% 0.01% 0.01% 0.01%
Benzamidazole 0.16% 0.15% 0.01% 0.16% 0.00%
N-Oxide 0.14% 0.14% 0.00% 0.15% 0.01%
Sulphone 0.15% 0.15% 0.00% 0.14% 0.01%
Des-methoxy 0.15% 0.14% 0.01% 0.15% 0.00%
Sulphide 0.16% 0.15% 0.01% 0.15% 0.01%
Total impurities 0.80% 0.76% 0.04% 0.76% 0.04%
Un … unknown; Max … Maximum
7.7 CALCULATION FORMULA
7.7.1 Assay (% w/w)
Calculated the quantity, in mg, of OME in the portion of solid oral pharmaceutical formulation
using the following formula:
Chapter-7
238
Where,
Cstd = Concentration of standard solution in mg/mL
Cs = Concentration of sample solution in mg/mL
Rs = Compound peak response obtained from the sample preparation
Rstd = Compound peak response (mean peak area) obtained from the standard preparation
7.7.2 Impurity (% w/w)
Calculate % impurity present in the finished product formulation using the following formula:
Where,
Cstd = Concentration of standard solution in mg/mL
Cs = Concentration of sample solution in mg/mL
Rs = Compound peak response obtained from the sample preparation
Rstd = Compound peak response (mean peak area) obtained from the standard preparation
7.7.3 Relative standard deviation (% RSD)
It is expressed by the following formula and calculated using Microsoft excel program in a
computer.
Where,
SD= Standard deviation of measurements
= Mean value of measurements
Determination of assay and related substances of omeprazole in pharmaceutical dosage form
239
7.7.4 Accuracy (% Recovery)
It is calculated using the following equation:
7.8 CONCLUSION
A precise and accurate method is successfully developed and validated for simultaneous
determination of omeprazole and its related substances in omeprazole dosage form, to meet
global regulatory requirements. The methodology is evaluated for specificity, linearity, precision,
accuracy and range in order to establish the suitability of the analytical method. Stability of
analytical solution, filter compatibility, LOD and LOQ is also studied. The total run time is 25.0
min, within which the drug and their degradation products are eventually separated. This method
can be successfully applied for the routine analysis as well as stability study.
Chapter-7
240
7.9 REFERENCES
[1] Jigar M and Vyas NG, “Development and validation of stability indicating method for
quantification of nebivolol and their related substance by HPLC-UV-PDA detection in
its pharmaceutical drug product” Int J of Pharm Tech Resea, 2009; 1(4): 1139-1147.
[2] Van de WA, Xhonneux R, Reneman R and Janssen P, “Cardiovascular effects of dl-
nebivolol and its enantiomers-a comparison with those of atenolol” Eur J Pharma,
1988; 156(1): 95-103.
[3] ICH Guideline Q3A (R2), Impurities in New Drug Substances, October 25, 2006.
[4] ICH Guideline Q7A, Good Manufacturing Practice Guide for Active pharmaceutical
Ingredients, November 2005.
[5] Della GM, Iesce MR, Previtera L, Rubino M, Temussi F and Brigante M, “Degradation
of lansoprazole and omeprazole in the aquatic environment” Chemosphere, 2006; 63(7):
1087-1093.
[6] Abdel-Aziz MW, Omayma AR, Azza AG, Hoda M and Marwa SM,
“Spectrophotometric determination of omeprazole, lansoprazole and pantoprazole in
pharmaceutical formulations” J of Pharma and Biom Anal, 2002; 30(4): 1133-1142.
[7] Espinosa BM, Ruiz SAJ, Sánchez RF and Bosch OC, “Analytical methodologies for the
determination of omeprazole: An overview” J of Pharma and Biom Anal, 2007; 44(4):
831-844.
[8] el-Kousy NM and Bebawy LI, “Stability-indicating methods for determining
omeprazole and octylonium bromide in the presence of their degradation products”
J AOAC Int, 1999; 82(3): 599-606.
[9] Hassan-Alin M, Andersson T, Niazi M, Röhss K, “A pharmacokinetic study comparing
single and repeated oral doses of 20 mg and 40 mg omeprazole and its two optical
isomers, S-omeprazole (esomeprazole) and R-omeprazole, in healthy subjects” Eur J
Clin Pharmacol, 2005; 60(11): 779-784.
[10] Tommy A, Johan H, Kerstin R, Anders W, “Pharmacokinetics and effect on caffeine
metabolism of the proton pump inhibitors, omeprazole, lansoprazole, and pantoprazole”
British J of Clinical Pharmac, 1998; 45(4): 369-375.
Determination of assay and related substances of omeprazole in pharmaceutical dosage form
241
[11] Oosterhuis B, “Omeprazole: Pharmacology, Pharmacokinetics and Interactions”
J Pharma Bio Research Int, 1989; 44(1): 9-17.
[12] www.wikipedia.org
[13] ICH, Validation of Analytical Procedure, Text and Methodology Q2(R1), International
conference on Harmonization, IFPMA, Geneva, Switzerland, 2005.