Determination of Pantoprazole Sodium and Lansoprazole in Individual Tablet Dosage Forms by RP-HPLC Using Single Mobile Phase

A simple, sensitive and precise high performance liquid chromatographic method for the analysis of pantoprazole sodium and lansoprazole has been developed, validated and used for the determination of compounds in commercial pharmaceutical products. The compounds were well separated an isocratically on a C18 column [Inertsil C18, 5 μ, 150 mm x 4.6 mm] utilizing a mobile phase consisting of acetonitrile: phosphate buffer (60:40, v/v, pH 7.0) at a flow rate of 1.0 mL/min with UV detection at 230 nm. The retention time of pantoprazole sodium and lansoprazole was found to be 2.017 min and 2.538. The procedure was validated for linearity (Correlation coefficient=0.999). The study showed that reversed-phase liquid chromatography is sensitive and selective for the determination of pantoprazole sodium and lansoprazole using single mobile phase. Keywards: Pantoprazole sodium, Lansoprazole, RP-HPLC, Method validation and Tablets


Experimental
A High Performance Liquid Chromatograph system, with LC solutions data handling system (Shimadzu-LC 2010) with an auto sampler was used for the analysis.The data was recorded using LC 2010 solutions software.The purity determination performed on a stainless steel column 150 mm long, 4.6 mm internal diameter filled with octadecyl silane chemically bonded to porous silica particles of 5 µm diameter (Inertsil C 18 , 5µ , 150 mm x 4.6 mm, make: Shimadzu ltd, Japan) with the mobile phase containing acetonitrile and phosphate buffer in the ratio of 60:40 (v/v pH 7.0) at ambient temperature.Flow rate was kept at 0.8 mL/min, and the elution was monitored at 260 nm.

Reagents and chemicals
Pantoprazole sodium and lansoprazole working standard were used from Smilax Laboratories Limited.For the estimation of pantoprazole sodium and lansoprazole in bulk and commercial formulations of lansoprazole brand (Pantin-20, Genex laboratories LAN, Intas laboratories), 20 tablets were obtained from retail pharmacies.Each tablet was labeled contain 20 mg of pantoprazole sodium and 30 mg of lansoprazole and had an expiry of not less than 365 days at the time of study.HPLC grade sodium dihydrogen phosphate (NaH 2 PO 4 ), disodium hydrogen phosphate (Na 2 HPO 4 ) and acetonitrile procured from Merck, India.High pure water was prepared by using Millipore Milli Q plus purification system.

Preparation of mobile phase
Mobile phase was prepared by mixing 700 mL of acetonitrile with 300 mL of phosphate buffer and its pH adjusted to 7.0.The mobile phase was sonicated for 15 min and then it was filtered through a 0.45 µ membrane filter paper.

Preparation of stock and standard solutions
Accurately weighed 25 mg of test sample taken into a clean dry 50 mL volumetric flask, dissolved and diluted to the mark with mobile phase.This solution was marked as sample solution.This solution contains 0.5 mg/mL of sample.Qualified working standard of pantoprazole sodium and lansoprazole was used to carry out validation exercise.The potency of working standard was 99.78% and 99.85%.With the optimized chromatographic conditions, a steady baseline was recorded, the standard solution was injected and the chromatogram was recorded.This procedure was repeated to sample preparation.

Assay in formulations
In case of marketed formulations, five accurately weighed tablets were crushed to a fine powder and an amount equivalent to 10 mg of pantoprazole sodium and lansoprazole was added into different 100 mL volumetric flasks and volume was made up with acetonitrile and methanol mixture.The samples were filtered through a 0.45 µm membrane filter, different serial dilutions (3.10, 6.20, 12.40, 25 µg/mL) were made from this solution in 25 mL volumetric flask and were injected for HPLC analysis.The retention time of pantoprazole sodium and lansoprazole was found to be 2.017 min and 2.538 (Figure 1 and Figure 2).

Method validation
The method was validated for the parameters like specificity, range and linearity, limit of detection (LOD), limit of quantitation (LOQ), accuracy and precision.In addition, system suitability parameters were also calculated.To demonstrate specificity in the presence of excipients used in formulation, pantoprazole sodium was spiked (at approximately 25 µg/mL) in drug product, chromatogram was observed and compared with that of raw material.To evaluate the linearity, the LOD and LOQ of the method in reference drug, different serial dilutions (0.0980, 0.180, 0.80, 1.50, 3.10, 6.30, 12.50 and 25 µg/mL) were prepared from the standard stock solutions of pantoprazole sodium and lansoprazole in 25 mL volumetric flasks and volume made up with diluent which is mixture of 60:40 acetonitrile & methanol.The samples were injected (10 µL) and signals from the samples were recorded which were compared with those of blank.LOD and LOQ values were calculated as signal-to-noise ratio of 3:1 and 10:1 respectively.To determine accuracy of the method, working standard of pantoprazole sodium was prepared in triplicate at three concentration levels (10, 20 and 25 µg/mL) and analyzed.Repeatability of the method was checked by analyzing six replicate samples of pantoprazole sodium (at the 100% concentration level) and calculating relative standard deviation (%RSD).To determine intermediate precision, standard solutions of pantoprazole sodium and lansoprazole at eight concentration levels were analyzed three times within the same day (intra-day variation) and three other days (inter-day variation).

System suitability
The HPLC system was equilibrated with the initial mobile phase composition followed by 10 injections of the same standard.These 10 consecutive injections were used to evaluate the system suitability on each day of method validation.The system suitability parameters including capacity factor >2, resolution>3 and asymmetric factor <2.All parameters were satisfactory with good specificity for the stability assessment of pantoprazole sodium and lansoprazole.Theoretical plates of the column were >3000.

Accuracy
The accuracy of an analytical method is the closeness of test results obtained by that method to true value.In case of the assay of a drug in a formulated product, accuracy may be determined by application of the analytical method to synthetic mixtures of the drug product components to which known amount of analyte has been added within the range of method.If it is not possible to obtain samples of all drug product components, it may be acceptable to add known quantities of the analyte to the drug product (i.e."to spike").In our studies, the later technique was adopted and pantoprazole sodium was spiked in drug product.The result of accuracy (Table 1) revealed that the method was found accurate for all above purposes.

Precision
Precision is the degree of reproducibility or repeatability of the analytical method under normal operating conditions.The method passed the test for repeatability as determined by %RSD of the area of the peaks of six replicate injections at 100% test concentration.The results of intraand inter-day variation of pantoprazole sodium and lansoprazole were shown in Table 2.

Table 1 .
Accuracy/recovery of pantoprazole sodium and lansoprazole.

Table 2 .
Intermediate precision of the method.