INTRODUCTION:

Lansoprazole, an acid proton-pump inhibitor, used as an antiulcer drug in the treatment and maintenance of healing of duodenal or gastric ulcers, erosive and reflux esophagitis, NSAID-induced ulcer, Zollinger-Ellison syndrome, and Barrett's esophagus^[1,2]. Lansoprazole is active against Helicobacter pylori ^[3].

It belongs to a class of anti secretory compounds, the substituted benzimidazoles, that do not exhibit anticholinergic or histamine H₂-receptor antagonist properties, but rather suppress gastric acid secretion by specific inhibition of the (H⁺,K⁺)-ATP ase enzyme system at the secretory surface of the gastric parietal cell, because this enzyme system is regarded as the acid (proton) pump within the parietal cell, Lansoprazole has been characterized as a gastric acid-pump inhibitor, in that it blocks the final step of acid production. This effect is dose-related and leads to inhibition of both basal and stimulated gastric acid secretion irrespective of the stimulus ^[4-6].

There are UV ^[7-11], Electrochemical ^[12-16], HPLC ^[17-29] and HPTLC ^[30-31] methods reported for estimation Lansoprazole in single, in presence of other components in dosage forms and in biological fluids.

There is no method reported for the estimation of related substances present in bulk drug, it is thought worthwhile to develop a method for the separation and quantification of related compounds present with Lansoprazole.

Specifications of Impurities present with Lansoprazole

Name of the impurity	Specification
Sulphone(Related compound-A)	Not more than 0.4%
Sulphide(Related compound-C)	Not more than 0.1%
N-Oxide(Related compound-B)	Not more than 0.1%
Any single maximum unknown impurity	Not more than 0.1%
Total impurities	Not more than 0.7%

MATERIALS AND METHODS:

An Agilent Ultra High Pressure Liquid Chromatographic system (Agilent 1290 series) with diode array detector equipped with Chemostation soft ware, Hypersil Gold C18, (2.1 x 100 mm, 1.8 µ) column were used. Millipore water, Methanol of HPLC grade, Acetonitrile of HPLC grade (Merck), tryethylamine (Merck), orthophoric acid (Merck), Lansoprazole working standard, Lansoprazole sample, sulphide, sulphone and N-oxide impurities were used.

Preparation of Standard:

Accurately weighed about 12.5mg working standard (RLAN 2) and transferred into a 50ml volumetric flask, added 5 ml of methanol dissolved and made up the volume with diluent and sonicated to degas the solution.

Preparation of Sample:

Accurately weighed about 12.5mg sample (RLAN 2) and transferred into a 50ml volumetric flask, added 5 ml of methanol dissolved and make up the volume with diluent and sonicated to degas the solution.

Preparation of impurity blends stock solution:

Accurately weighed about 12.5 mg of each impurity and transferred into a 50ml volumetric flask separately, added 5 ml of methanol dissolved and make up the volume with ml diluent and sonicated to degas the solution

RESULTS AND DISCUSSION:

METHOD DEVELOPMENT

The LC method described here for the determination of Lansoprazole and its related substances was developed based on methods reported in the literature. Although there was no method originally developed for the analysis of related substances, two methods were selected depending upon the mobile phase used. They include: a combination of disodium hydrogen phosphate buffer and acetonitrile (S. Muthu Kumar et al.), a combination of phosphate buffer and acetonitrile (B. Prasanna Kumar Reddy et al), and a combination of phosphate buffer, tryethylamine (TEA) and acetonitrile (Idrees F. Al-Momani et al), all used isocratic elution.

Some parameters were kept unchanged when testing the different methods. Each method was tested on the same column, namely Hypersil Gold (2.1X100mm, 1.8 µ). This was chosen for its beneficial price-quality ratio. Column temperature was set at minimum 25^oC. The flow rate was set at 0.3 mL/min. Since Lansoprazole is sparingly soluble in water, water and acetonitrile solution was selected as the diluent.

Using a combination of water and acetonitrile as mobile phase with gradient elution, no good separation between the peaks could be achieved, even with various gradients. It was not possible to separate properly the impurity eluted immediately after the principal peak using this mobile phase system.

Using a combination of water and organic buffer (acetonitrile: water: triethylamine, 80: 20: 0.005, pH adjusted to 6.0 with ortho phosphoric acid) as mobile phase gave better separation. Initially two Solvents, A consisting of 100% of water and B, 80: 20: 0.005 ACN: water; triethylamie water were used. The gradient program used was: 0–6 min, 80% A–20% B; 6–10 min, linear increase of mobile phase B from 20 to 40%; 10-15min, 60% B; 15–17 min, linear return to the initial conditions; re equilibration with 80% A–20% B.

Since this method gave the best separation, it was further optimized by adapting the percentages of organic buffer and water in the mobile phase and by changing the gradient program in order to achieve a good separation of all peaks noticed in the chromatogram within a short period of time. Next, different column temperatures and buffer pH were examined in order to find the best conditions.

In the final method, Solvent A consisted of 100 volumes of milli Q water and Solvent B of 70 volumes of ACN, 20 volumes of milli Q water, and 0.005 volumes of triethylamine (Solvent’s pH was adjusted to 7.0 with ortho phosphoric acid). The gradient program used is given below. UV detection was performed at 285 nm and the column was kept at 25^oC temperature and the injection volume 3 µl.

Optimized Chromatographic Conditions

Column	: Hypersil Gold (2.1X100mm, 1.8 µ)
Detector wavelength	: 285 nm
Flow rate	: 0.3 ml/min
Injection volume	: 3 µl
Column oven temperature	: 250C
Run time	: 20 minutes.
Diluent	: Transferred 100ml of Solvent B and 900 ml of Solvent A into a 1000ml beaker and mixed. Filtered the solution through 0.45µ membrane filter and sonicated to degas the solution.

Gradient program:

Time	0.0	6	10	12	14	17	20
Solvent A	70	55	35	20	55	70	70
Solvent-B	30	45	65	80	45	30	30

Solvent-A: Milli q water 1000ml

Solvent-B: Transferred 1600ml of Acetonitrile, 400 ml of water into a 2000 ml beaker and then 10 ml of triethylamine added then the pH to 7 adjusted with dilute orthophosphoric acid, solution was filtered the solution through 0.45µ membrane filter and sonicated to degas

Solvent-A: Milli q water 1000ml

Solvent-B: Transferred 1600ml of Acetonitrile, 400 ml of water into a 2000ml beaker and then 10 ml of triethylamine added then the pH to 7 adjusted with dilute orthophosphoric acid, solution was filtered the solution through 0.45µ membrane filter and sonicated to degas.

METHOD VALIDATION

System Suitability

Standard solution spiked with impurities injected six times into the system and chromatograms were recorded. The system suitability tests were done before each parameter throughout the validation. Results were shown in table no 1.

Specificity

The blank solution, standard solution, sample solution and impurity blend solutions injected separately into the system, chromatograms were recorded and results were evaluated.

The % RSD of retention time and peak area was less than 2, tailing factor is less 2, theoretical plates were more than 4000 for lansoprazole, resolution between lansoprazole and impurities was more than 6.0. The specificity shows that there is no interference and the method detects the impurities without any interference

Limit of Detection and Quantification

Prepared series of dilutions of impurities and analyte in different concentrations and injected them into the chromatographic system till the signal to noise ratio is between 2 and 3 for limit of detection and signal to noise ration ratio is between 9.5-10.4 for limit of quantification.

The detection limit was found to be 0.0025 mg/ml, 0.0066 mg/ml and 0.0023 mg/ml for N-Oxide, Sulphone and Sulphide respectively.

The quantification limit was found to be as 0.0075 mg/ml, 0.0100 mg/ml and 0.0023 mg/ml for N-Oxide, Sulphone and Sulphide respectively.

Table No 2 Results of Limit of Detection

Impurity/ Analyte	Conc. in mg /ml	S/N Ratio	Acceptance criteria
N-Oxide(Impurity-B)	0.0025	2.1	The signal to noise ratio should be between 2.0 and 3.0
Sulphone(Impurity-A)	0.0066	2.2
Sulphide(Impurity-C)	0.0023	2.5

Table No 3 Results of Limit of Quantification

Impurity/ Analyte	Conc. in mg/ml	S/N Ratio	Acceptance criteria
N-Oxide(Impurity-B)	0.0075	9.6	The signal to noise ratio should be between 9.5 and 10.6
Sulphone(Impurity-A)	0.0100	9.6
Sulphide(Impurity-C)	0.0070	9.6

Precision at Limit of Quantification Level

Prepared six individual solutions containing impurities concentration at limit of quantification level Injected each solution once and calculated the % RSD for the area of impurities. % RSD of peak area for sulphone, sulphide and N-oxide were found to be as 9.7, 6.9 and 6.5 respectively.

Table No 4 Results of Precision of Limit of Quantification Level

Preparation	Sulphone	Sulphide	N-Oxide
1	4.38314	1.27134	1.37207
2	4.28931	1.28931	1.32641
3	4.41325	1.31152	1.26541
4	4.65861	1.25561	1.29901
5	4.86411	1.36521	1.38946
6	4.38925	1.39531	1.29841
Average	4.499612	1.31471	1.325128
STDEV	0.198146	0.050126	0.043409
%RSD	4.403615	3.812687	3.275833
Acceptance criteria: % RSD of individual impurities area should not be more than 10.0%

Accuracy at Limit of Quantification Level

Prepared in triplicate standard solutions containing impurities at limit of quantification level, and to this impurities were added in small amounts (spiked) and amount in added was calculated in terms of % recovery. The mean recovery for sulphone, sulphide and N-oxide was found to 97.4%, 101.5% and 94.7% respectively.

Table No 5 Results of Accuracy at Limit Quantification Level

Preparation	Sulphone	Sulphide	N-Oxide
1	94.3	113.5	95.4
2	97.1	97.1	89.8
3	100.9	94.0	98.9
Average	97.4	101.5	94.7
Acceptance criteria: The % recovery should be in between 80.0 and 120.0

Table No 6 Linearity result data and for Sulphone

% Concentration	Concentration in mg/ml	Area of Sulphone
25%	0.0100	4.38094
50%	0.0200	8.61214
75%	0.0300	13.45249
100%	0.0400	17.58836
125%	0.0500	21.03921
150%	0.0600	25.96074
Correlation		0.999073
Slope		42.66174
Y-Intercept		0.240705
%Y-Intercept		1.4

Fig No 3 Graph Linearity of Sulphone

Fig No 4 Graph Linearity of N-Oxide

Linearity

Linearity was demonstrated by injecting impurities at limit of quantification level, 25%, 50%, 75%, 100%, 125% and 150% with respect to the specification level. Plotted the calibration curve by taking concentration on X-axis and peak area on Y-axis, calculated the correlation coefficient and % y-intercept at 100% specification level. The linearity study reveals that the method is linear from LOQ to 150%.

Method Precision

Six different sample were prepared at two levels, specification (100%) and 150% level prepared and assayed using the method and %RSD calculated. % RSD for individual impurities at specification level (100%) and 150% level was found to be well under limit.

Accuracy

Accuracy was determined by adding the known amounts of analyte and impurities at 50%, 100% and 150% of specification level.. The accuracy is calculated in terms of % recovery of analyte and impurities. The mean % recovery at three different concentration levels was found be very well under limit.

Table No 7 Linearity result data and for N-Oxide

% Concentration	Concentration in mg /ml	Area of N-Oxide
25%	0.0075	1.37207
50%	0.0150	2.82444
75%	0.0225	4.52201
100%	0.0300	5.93650
125%	0.0375	7.46001
150%	0.0450	8.84465
Correlation		0.999643
Slope		60.2104
Y-Intercept		-0.10846
%Y-Intercept		-1.8

Table No 8 Linearity result data and for Sulphide

% Conc.	Concentration in mg/ml	Area of Sulphide
25%	0.0070	1.28334
50%	0.0140	2.49188
75%	0.0210	3.78600
100%	0.0280	5.16863
125%	0.0350	6.33379
150%	0.0420	7.61440
Correlation		0.999806
Slope		50.9299
Y-Intercept		-0.01003
%Y-Intercept		-0.2

Ruggedness

Ruggedness was demonstrated by determining the precision of method by analyzing same sample on different system, by different analyst and on different column.

System Suitability by 2^nd analyst similarly as first analyst standard solution spiked with impurities injected five times into the system and chromatograms were recorded

Method precision studies were performed in similar manner as first analyst by second analyst

The system suitability parameters and method precision was not changed much when different analyst performed analysis on different system.

Fig No 5 Graph Linearity of Sulphide

Table No 9 Results of Method Precision

Preparation	Lansoprazole		Sulphone		Sulphide		N-Oxide
	100%	150%	100%	150%	100%	150%	100%	150%
1	99.32	99.56	0.25	0.27	0.091	0.096	0.10	0.11
2	99.46	99.36	0.26	0.29	0.10	0.096	0.10	0.12
3	99.12	100.03	0.26	0.27	0.09	0.095	0.09	0.11
4	100.16	100.41	0.26	0.26	0.10	0.095	0.09	0.10
5	99.81	99.89	0.25	0.29	0.10	0.096	0.09	0.10
6	99.61	99.94	0.24	0.26	0.10	0.099	0.10	0.10
Avg	99.58	99.865	0.253	0.273	0.096	0.097	0.095	0.106
Std Dev	0.369	0.368	0.0085	0.013	0.004	0.003	0.005	0.008
%RSD	0.371	0.368	3.2233	4.998	5.076	3.847	5.765	7.654
Acceptance criteria: The % RSD for main analyte and impurity content should not be more than 2.0 and 10.0 respectively

Table No 10 Results of Accuracy

	Lansoprazole			Sulphone			Sulphide			N-Oxide
	50%	100%	150%	50%	100%	150%	50%	100%	150%	50%	100%	150%
1	99.85	99.69	100.04	113.6	111.8	111.5	86	93.5	105.3	106.2	104.4	106.1
2	99.32	99.34	99.85	114.4	114	114.4	86.7	95.5	86.7	105.2	103.6	105.2
3	99.65	99.81	99.41	113.5	114.2	113.5	86	92.8	86	102.9	103.6	102.9
Avg	99.6	99.61	99.76	113.8	113.3	113.1	86.2	93.9	92.7	104.8	103.9	104.7
Acceptance criteria: The % recovery of analyte and impurities should be between 85.0 -115.0 and 98%-102% respectively

Table No 11 Results of System Suitability by second analyst

Parameter	Result	Acceptance Criteria
Theoretical plates factor for Lansoprazole	98563	Not less than 2000
Tailing Factor for Lansoprazole	1.10	Not more than 2.0
The % of RSD of area for five injections of Lansoprazole	0.61	Not more than 1.0
The % of RSD of area for five injections of Sulphone	0.51	Not more than 10.0
The % of RSD of area for five injections of N-Oxide	0.81	Not more than 10.0
The % of RSD of area for five injections of Sulphide	0.69	Not more than10.0
The resolution between Lansoprazole and N-Oxide	19.36	Not less than 2.0
The resolution between Lansoprazole and Sulphone	6.31	Not less than 2.0
The resolution between Sulphone and Sulphide	10.42	Not less than 2.0

Table No 12 Results of precision by second analyst

Preparation	Lansoprazole		Sulphone		Sulphide		N-Oxide
Preparation	100%	150%	100%	150%	100%	150%	100%	150%
1	99.46	99.86	0.35	0.37	0.09	0.11	0.10	0.09
2	99.32	100.09	0.36	0.39	0.10	0.11	0.10	0.11
3	98.85	100.16	0.36	0.37	0.09	0.10	0.09	0.11
4	99.12	99.76	0.36	0.36	0.10	0.12	0.09	0.12
5	99.73	99.94	0.35	0.39	0.10	0.12	0.09	0.11
6	99.86	99.67	0.34	0.36	0.10	0.10	0.10	0.09
Avg	99.390	99.913	0.353	0.373	0.096	0.110	0.095	0.105
Std Dev	0.377	0.188	0.008	0.013	0.005	0.008	0.005	0.012
%RSD	0.379	0.189	2.310	3.659	5.342	8.131	5.765	11.664
Acceptance criteria: The % RSD for main analyte and impurity content should not be more than 2.0 and 10.0 respectively

Table No 13 Results of Flow rate Variation

Parameter	0.2 ml/min.	0.3 ml/min.	0.4ml/min.	Acceptance Criteria
Theoretical plates for Lansoprazole	95642	98600	91185	Not less than 2000
Tailing factor for Lansoprazole	1.49	1.14	1.194	Not more than 2.0
The % of RSD of area for five injections of Lansoprazole	1.12	0.39	0.61	Not less than 1.0
The % of RSD of area for five injections of Sulphone	0.89	0.56	0.76	Not more than 10.0
The % of RSD of area for five injections of N-Oxide	1.01	0.76	0.99	Not more than 10.0
The % of RSD of area for five injections of Sulphide	0.96	0.99	0.89	Not more than 10.0
The resolution between Lansoprazole and N-Oxide	19.01	19.11	19.09	Not Less than 2.0
The resolution between Lansoprazole and Sulphone	6.09	6.17	6.14	Not Less than 2.0
The resolution between Sulphone and Sulphide	10.16	10.59	10.29	Not less than 2.0

Table No 14 Results of Column Oven Temperature Variation

Parameter	20^oC	25^oC	30^oC	Acceptance Criteria
Theoretical plates for Lansoprazole	95642	97600	91185	Not less than 2000
Tailing factor for Lansoprazole	1.130	1.19	1.294	Not more than 2.0
The % of RSD of area for five injections of Lansoprazole	1.49	1.14	1.194	Not less than 1.0
The % of RSD of area for five injections of Sulphone	1.32	0.45	0.84	Not more than 10.0
The % of RSD of area for five injections of N-Oxide	0.95	0.61	0.87	Not more than 10.0
The % of RSD of area for five injections of Sulphide	1.17	0.66	1.07	Not more than 10.0
The resolution between Lansoprazole and N-Oxide	6.01	6.19	6.12	Not more than 2.0
The resolution between Lansoprazole and Sulphone	18.93	19.29	18.97	Not more than 2.0
The resolution between Sulphone and Sulphide	10.16	10.35	10.08	Not less than 2.0

Injected blank once, standard solution spiked with impurity solution five times into chromatograph with the above mentioned conditions, chromatograms were recorded and evaluated for system suitability parameters. There was no change in system suitability parameters.

Variation in pH:

Adjusted the pH as per the following two conditions separately while other parameters remain unchanged. Injected blank once, standard solution spiked with impurity solution five times into chromatograph with the above mentioned conditions, chromatograms were recorded and evaluated for system suitability parameters. There was no change in system suitability parameters.

CONCLUSION:

From the above experimental data on the various method validation parameters, it is proved that this method which was designed to determine for related substances present in Lansoprazole bulk by UHPLC is precise, accurate, linear, rugged, and robust throughout range from LOQ to 150% of the specification limit. Hence, the method can be used for routine application.

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Received on 30.05.2012 Modified on 08.06.2012

Asian J. Research Chem. 5(7): July, 2012; Page 859-865

Parameter	pH 6.9	pH 7.0	pH 7.1	Acceptance Criteria
Theoretical plates for Lansoprazole	94326	98312	90462	Not less than 2000
Tailing factor for Lansoprazole	1.41	1.14	1.32	Not more than 2.0
The % of RSD of area for five injections of Lansoprazole	1.23	0.64	1.09	Not less than 1.0
The % of RSD of area for five injections of Sulphone	1.01	0.55	0.94	Not more than 10.0
The % of RSD of area for five injections of N-Oxide	0.75	0.53	0.96	Not more than 10.0
The % of RSD of area for five injections of Sulphide	0.96	0.61	1.11	Not more than 10.0
The resolution between Lansoprazole and N-Oxide	6.21	6.27	6.02	Not less than 2.0
The resolution between Lansoprazole and Sulphone	18.87	19.19	18.98	Not less than 2.0
The resolution between Sulphone and Sulphide	10.16	10.49	10.08	Not less than 2.0