Development and Validation of RP-HPLC Method for The Estimation of Proton Pump Inhibitor in Bulk and Pharmaceutical Dosage Form

Drugs are physiological compounds used for disease treatment, alleviation, or prevention. Originating from botanical substances, they have evolved through scientific advancements, enhancing the quality of pharmaceutical compounds and synthetic processes. [1-2]

Significance of specificity of analytical methods in drug analysis

The pharmaceutical sector uses liquid chromatography technology, notably LC-UV, to assess drug material purity, identifying UV-active analytes with accuracy and precision.

Importance of impurity quantification in pharmaceutical industry

The pharmaceutical sector preserves purity and health advantages by limiting impurities depending on nature, toxicity, and dosage, with limited commercialization necessary to manage contaminants.

Pharmaceutical Analysis

Pharmaceutical analysis is a vital part of analytical chemistry, involving the separation, identification, and determination of components in matter samples. It ensures drug quality, quality control, and uses physical and physicochemical methods. [3-5]

High Performance Liquid Chromatography (HPLC)

High performance liquid chromatography (HPLC) is an advanced method with high resolution, small diameter columns, and controlled flow of the mobile phase. It is highly automated and sensitive, and is often used in the pharmaceutical sector to qualify medications before release. Reversed phase HPLC (RP-HPLC) is also used.

Normal phase HPLC

Although it is classified as "normal", it is not the most widely utilised kind of HPLC. The column is filled with microscopic silica particles, and the solvent is non-polar. A typical column has an internal diameter of 4.6 mm (and may be less than that), and a length of 150 to 250 mm. Polar chemicals in the mixture being transported through the column will adhere longer to the polar silica than non-polar ones. The non-polar chemicals will therefore travel more swiftly through the column.

Reverse phase HPLC

Reverse phase HPLC involves altering silica to non-polar by attaching hydrocarbon chains, using a polar solvent, and observing the attraction between the solvent and polar molecules. Non-polar chemicals are less soluble and slow down passage through the column.

Functional description of the instrument

The HPLC comprises of the following component (Figure 1): Mobile phase reservoir Pump Injector Column Detector and Data system.

Figure 1: HPLC Instrumentation

Glass bottles serve as prevalent solvent reservoirs, equipped with lids, Teflon tubing, and filters for interfacing with the pump inlet and purge gas (helium) for the elimination of air.

Pump

High-pressure pumps propel solvents through stationary phase beds, ensuring flow rate stability and incorporating cost-effective attributes such as electronic feedback and multi-headed combinations. Their flow rate ranges from 0.01 to 5 mL/min, with a maximum pressure of 300 MPa, and includes integrated degassing systems.

Isocratic flow and gradient elution

Isocratic separation preserves a stable mobile phase composition, whereas gradient elution alters it. Reverse-phase chromatography employs solvent A as water and solvent B as an organic solvent.

Figure-02 : High pressure Gradient Flow Chart

Injector

The introduction of LC systems includes injection valves, auto samplers, microprocessors, liquid chromatography, and solvent dissolution for liquid samples, utilizing straightforward techniques such as injection valves.

Column

HPLC columns, typically 5-25 cm long, are packed with tiny diameter particles for sample capacity, mobile phase consumption, speed, and resolution. Larger diameter columns may be needed for pure substances. Packing involves experience and specialized equipment; hence prepacked columns are suggested for most chromatographers.

Column Efficiency

Column efficiency refers to the performance of the stationary phase to accomplish specified separations. This comprises how well the column is packed and its kinetic performance. The efficiency of a column can be tested by numerous ways which may or may not be affected by chromatographic abnormalities, such as "tailing" or appearance of a "front." For this reason, efficiency might be an intriguing value since manufacturers may utilize different approaches in determining the efficiency of their columns.

Column-packing materials

Silica (SiO2? XH2O) is a commonly used material for packing materials, offering a wide range of commercial products with surface areas ranging from 100 to 800 m2/g and particle sizes from 3 to 50 µm. The silanol groups on the surface provide polar character, which is used in adsorption chromatography utilizing non-polar chemical eluents. The binding of hydrocarbon chains to silica generates a non-polar surface ideal for reversed phase chromatography employing combinations of water and organic solvents. Intermediate surface polarities occur from bonding to silica of other organic compounds. Separation of chemicals in mixtures takes place slowly via differential adsorption on a stationary silica phase, with partition playing an essential role.

Detector

Optical detectors are often employed in liquid chromatographic systems to monitor light intensity differences produced by UV absorption, fluorescence emission, or refractive index changes. These voltage variations are captured on a strip chart recorder and transmitted into a computer for retention time and peak area data. Other frequent detectors include UV, refractive index, fluorescence, and electrochemical detectors, with UV being the most sensitive.

Data system

Electronic data systems improve signal analysis by boosting accuracy and precision while lowering operator focus. Preprogrammed computing integrators are suited for regular analysis, while intelligent devices like data stations or minicomputers give higher control levels. Intelligent processors in chromatographs enable automated possibilities, complicated data analysis, and software safe controls to reduce inadvertent usage. [6-12]

HPLC Method Development and Valiation

HPLC method development involves selecting instruments, defining analyte parameters, and optimizing criteria. Results are reviewed for improvement, with optimization focusing on efficiency and minimizing experimental input.

The column and flow rate

To ensure irreproducible sample retention during technique development, sturdy and repeatable columns are necessary. A C8 or C18 column made from less acidic silica is recommended for all samples. For low pH and low temperatures, sterically protected bonded phase column packing is preferred. A column with a flow rate of 2 mL/min is suited for diverse mobile phases.

Figure - 03: Separation of components in column

The mobile phase

Acetonitrile is the chosen organic solvent for mobile phase mixes due to UV transmittance and low viscosity. Methanol is a reasonable option. Amine modifiers like tetrahydrofuran are less desirable due to lengthier column equilibration durations and other difficulties. The mobile phase's pH should be determined depending on stability and retention of column silanols. A pH of 2 to 2.5 is recommended for stable columns.

Injection volume

For optimal detection sensitivity, use 25-50 μL injection. For smaller column diameters or smaller particles, use smaller volumes. Dissolve samples in water or acetonitrile, then utilize mobile phase for technique development. Dissolve samples in acetonitrile or methanol.

Equilibration of the column with the mobile phase

The analytical column is equilibrated with the mobile phase before analysis, ensuring precise retention data. Equilibration is important for changes in column, mobile phase, or temperature during technique development. Reliability can be checked by cleaning the column with new mobile phase and reinjecting the sample. Retention times should not change by more than 0.02 minutes between runs.

Performance calculations

The overall system performance was accessed by calculating the following values:

1. Relative retention
2. Theoretical plates
3. Capacity factor
4. Resolution
5. Peak asymmetry
6. Plates per meter [13-18]

Validation of Analytical Data

Method validation seeks to guarantee a method fulfills its intended function, considering aspects including sampling procedure, sample preparation, chromatographic separation, and detection, with consistency in analytical parameters.

Validation Parameters:

Method validation will be evaluated with different validation parameters are evaluated. Those are listed as below.

• System Suitability
• Specificity
• Precision
• Linearity
• Limit of Detection and Quantitation
• Accuracy
• Stability in Analytical Solution
• Robustness

Recovery

Absolute recovery of a technique is assessed by comparing the response of a processed spiked matrix standard to the pure standard without sample pretreatment. It determines if the method delivers a response for the total amount of analyte in the sample. If an internal standard is utilized, its recovery should be established independently. [19-20]

Response function

Chromatographic analysis employs peak area to construct a calibration model, proving its accuracy by assessing differences between observed and fitted values for six specific concentrations.

Sensitivity

Sensitivity in analytical procedures is determined by calibration line slope and limits of quantitation (LOQ). Over-range concentrations should be diluted and re-assayed.

Precision

Precision in analytical methods evaluates random error and agreement between replicate measurements. It contains intra-assay precision, which allows repeating the same methodology with the same analyst, equipment, and chemicals, and inter-assay precision, which allows repeating under various conditions. Validation should test precision at three unique concentrations.

Accuracy

The analytical method's accuracy is determined by the percentage bias, which is calculated by comparing the measured value to the true value. Validation assures accuracy within ± 15% at all concentrations.

Table- 01: Details of Instruments and their applications

Figure - 04: Method Validation Protocol [30]

Drug Profile

Dexlansoprazole

Structure

Chemical Name: (2-([3-methyl-4-(2,2,2-trifluoroethoxy) pyridin-2-yl] methyl sulfinyl) -1H-benzo[d]imidazole

Description: White to off-white powder.

Molecular formula: C16H14F3N3O2 S

Molecular weight: 369.36

Solubility: Insoluble in water, soluble in DMSO, ethanol and free soluble in methanol & organic solvent

Category: Dexlansoprazole is in a class of medications called proton pump inhibitors. It works by decreasing the amount of acid made in the stomach

Mechanism of Action: Dexlansoprazole blocks the H/K ATPase at the gastric parietal cell's secretory surface, preventing the discharge of hydrochloric acid into the gastric lumen.

Pharmacodynamics: Dexlansoprazole, a proton pump inhibitor, lowers gastric acid secretion, but may increase susceptibility to infections, vitamin deficiencies, hypomagnesemia, and false positive results in tests.

Absorption: Dexlansoprazole's dual delayed-release formulation includes two unique peaks, with plasma concentration-time profiles. Its mean Cmax and AUC values increase dose-proportionally after oral administration, with meals raising Cmax and AUC.

Metabolism: Dexlansoprazole is extensively metabolized in the liver, undergoing oxidation, reduction, sulfation, glucuronidation, and glutathione conjugation to create inactive metabolites, largely derived from CYP2C19-mediated hydroxylation.

Route of elimination: Dexlansoprazole, provided to six healthy male participants, indicated that around 50.7% of the radioactivity was eliminated in urine and 47.6% in feces.

Adverse effects: Clinical trials revealed substantial adverse effects such as diarrhea, stomach pain, bloating, nausea, upper respiratory tract infection, vomiting, and flatulence (≥2%).

Uses: Dexlansoprazole is used for erosive esophagitis and GRD heartburn, lasting longer than lansoprazole and requiring less frequent usage, but lacks clear evidence of improved efficacy.

MATERIALS & METHODS

Materials

The drug used for present investigation was obtained from Swapnroop Drugs and Pharmaceuticals Maharashtra.

Details of Pure Drug

Table No.02: Details of API

Marketed Preparation

Table No.03: Details of Marketed preparation

Reagents and Chemicals

All reagents and chemicals used were of AR grade and HPLC grade.

Table No.04: List of Reagent and Chemicals used.

Instruments

Table No.05: List of Instruments used.

METHODS & PROCEDURE

Identification and characterization of drug

Before starting experimental work, it's necessary to examine the drug's physical and chemical properties, enabling the selection of solvents and the development of a rigorous analytical approach.

Selection and procurement of drug

Dexlansoprazole (DEXL) was chosen as a model drug candidate for method development and validation, gifted by the pharmaceutical sector India, and studied for physical attributes.

Physico-chemical characterization

The physico-chemical characterisation of drug molecule is crucial with reference to its purity, identification in development and validation of analytical method. The numerous tools utilized for characterisation of medicinal compounds include melting point, UV spectroscopy, solubility research, etc.

Solubility Studies

The solubility of both drugs was evaluated in several solvents to discover a common solvent for simultaneous drug quantification in a combination.

Melting point range determination

The melting point of Dexlansoprazole (DEXL) was evaluated by inserting a small sample in a capillary tube and noting the findings in Table 06.

FT-IR analysis

The FTIR 8400S spectrometer (Shimadzu) was used to record the IR absorbance spectrum of DEXL, which helps comprehend its chemical structure by evaluating the exact frequencies of light absorbed by molecules, based on their molecular surface shape, vibronic coupling, and atom mass.

UV Spectroscopy Analysis

The Shimadzu1800-UV visible spectrophotometer and 1cm quartz cells were used to produce the ultraviolet absorption spectrum of DEXL, examining wavelength maxima as indicated in table no. 07.

Selection of mobile phase

Preparation of standard solutions

DEXL standard solution

A exact quantity of DEXL was dissolved in methanol, resulting in a final concentration of approximately 30 μg/ml, after further dilution with 20 ml methanol.

Procedure

Methanol equilibrated, DEXL combination was evaluated, and several solvents were utilized to produce stable separation. Mobile phase compositions were assessed for adequate separation under specific chromatographic settings.

1. 1. 1. Methanol: Water (90:10)
      2. Methanol: Water (80:20)
      3. Methanol: Water (70:30)
      4. Acetonitrile: Water (90:10)
      5. Acetonitrile: Water (80:20)
      6. Acetonitrile: Phosphate Buffer (90:10) pH 5.5
      7. Acetonitrile: Phosphate Buffer (80:20) pH 5.5
      8. Acetonitrile: Phosphate Buffer (70:30) pH 5

The mobile phase comprising Acetonitrile: Phosphate Buffer (70:30) pH 5 was chosen because to its sharp repeatable retention time for DEXL.

Chromatographic conditions

The following chromatographic conditions were established by trial and error and were kept constant throughout method.

Column: Intersil 4.6 (id) x 250 mm

Particle size packing : 5 μm

Stationary phases      : C18 Intersil

Mobile phase : Acetonitrile: Phosphate Buffer (70:30) pH 5

Detection wavelength : 275 nm

Flow rate    : 1 ml/min.

Temperature  : Ambient

Sample size : 20 μL

Preparation of calibration curve:

Preparation of standard solutions:

DEXL standard stock solution

10 mg of DEXL was dissolved in methanol to 100 ml mark, then diluted with mobile phase to get varied concentrations.

Procedure

The mobile phase equilibrated with the stationary phase, and DEXL concentrations from 3-30 μg/ml were injected, with peak area recorded and graph plotted in Fig. No.12.

System suitability test

System suitability is a pharmacopoeial requirement ensuring adequate resolution and reproducibility of chromatographic systems for analysis, tested using data from five replicate injections of standard solutions.

Preparation of standard drug solution.

 DEXL standard solution

A precise weighed quantity of 10 mg of DEXL was dissolved in mobile phase, diluted to a final concentration of 30 μg/ml.

Procedure

The mobile phase was equilibrated with the stationary phase until a steady baseline was achieved, and a standard drug solution was injected in five replicates.

Application of proposed method for estimation of DEXL Laboratory Sample Preparation of laboratory mixture (standard)

A 10 mg DEXL was weighed, shaken, and diluted to obtain a 30 μg/ml laboratory sample.

Preparation of laboratory mixture (sample)

Five DEXL laboratory mixtures were prepared by weighing drug samples, comparing peak area, and estimating drug amounts using specific methods.

Where-

Application of proposed method for estimation of DEXL in formulation

Standard stock solution

A 10 mg DEXL solution was weighed, shaken, and mixed with mobile phase to create laboratory mixtures with a concentration of 30 μg/ml.

Sample solution preparation

The tablets were weighed, averaged, ground, and powdered to 10 mg of DEXL. The powder was diluted, filtered, and sonicated before being added to a flask.

Procedure

Standard and sample solution were injected separately, chromatograms recorded, and DEXL content calculated by comparing sample peak with standard. Tablet drug amount calculated using formula.

Assay mgmi=AtAs+DsDt+WsWt+p100×wtmgmlof test sample

% Label claim =    Assay (mg/ml) × 100

                              Label claim in mg/ml

Where -

At = Area count for sample solution.

As = Area count for standard solution.

Ds = Dilution factor for standard.

Dt = Dilution factor for sample

P = Potency of drug.

Validation parameters

Accuracy

It was ascertained on the basis of recovery studies performed by standard addition method. The results of recovery studies and statistical data are recorded in Table No. 13

Preparation of standard solution

Pre-analysed formulation was mixed with standard DEXL solution, adjusted, and filtered. Drug contribution was determined from total drugs, and drug content calculated using marketed formula.

The % Recovery was then calculated by using formula

Where,

%Recovery=AB+C×100

Results are shown in the Table No. 13

Precision

The precision of an analytical method, expressed as S.D or R.S.D of series of measurements, was confirmed through replicate estimation of drugs using the proposed method.

Ruggedness

Ruggedness refers to an analytical method's ability to withstand minor changes in experimental conditions, studied under two conditions: days and the analyzer.

Interday (Different days)

as under marketed formulation analysis on different days. The % label claim was calculated. Data obtained for day 1, day 2, and day 3 is shown in Table No. 15

Intraday

It was performed by using same procedure as under marketed formulation analysis and absorbance recorded at 3 hrs. interval within a day. The percent label claim was calculated using formula, Result and statistical data are shown in Table No. 16

Different analyst

The sample solution was prepared by two different analysts and same procedure was followed as described earlier. The % label claim was calculated as done in marketed formulation estimation.

Specificity

Specificity was measured as ability of the proposed method to obtain well separated peak for DEXL without any interference from component of matrix.

Mean retention time for –

DEXL – 4.573

The values obtained were very close to that in standard laboratory mixture indicates no interference from the component of matrix.

Typical chromatogram is shown in the Fig. No. 14

Linearity and range

USP tablet powder was diluted to 80%-120% of test concentration, revealing DEXL's linearity within ± 20% of the test concentration, as shown in Fig. No.15.

Robustness

The robustness study confirmed that the method's reliability remains unaffected by small variations in parameters and environmental factors, indicating its suitability for normal usage.

Limit of Detection (LOD) and Limit of Quantitation (LOQ):

Limit of detection (LOD) and limit of quantitation (LOQ) are the lowest analyte in a sample that can be detected but not quantitated accurately.

LOD=3.3 σS

LOQ=10 σS

Where,

σ = The standard deviation of the response S = The slope of the calibration curve

The results of LOD and LOQ are shown in table 20

RESULT & DISCUSSION

Dexlansoprazole (DEXL) is a commonly used clinical drug, with a purity of 99.8% reported by the supplier. The study analyzed its physico-chemical characterization using melting point, UV spectroscopy, and solubility studies.

Melting point range determination

The drug's melting point was determined by placing a small sample in a capillary tube and holding it on a melting point apparatus, as shown in Table 06.

Table No 06: Melting point range analysis result

FT-IR analysis

FT-IR of Dexlansoprazole

The FTIR 8400S spectrometer (Shimadzu) was used to record the IR absorbance spectrum of Dexlansoprazole (DEXL) within the 4000 to 400 cm-1 range.

Fig No 05: FT-IR Spectra of DEXL

IR spectroscopy theory explains how molecules absorb specific light frequencies, providing information about their structure and functional groups. The FTIR spectra of DEXL confirmed its drug's properties.

UV Spectroscopy Analysis

The Shimadzu1800-UV visible spectrophotometer and 1cm quartz cells were used to obtain the ultraviolet absorption spectrum of DEXL, analyzing wavelength maxima as shown in table no. 07

Fig. No. 06: - UV Spectra of DEXL

Table no 07: Drug wavelength maxima (λ max)

From the spectra the wavelengths selected for estimation of drug was 275 nm.

Selection of mobile phase

Preparation of standard solutions

DEXL standard solution

DEXL was dissolved in acetonitrile, diluted to 30 μg/ml, and filtered through Whatman filter paper. Different mobile phase compositions were evaluated for acceptable separation.

Table no 08: List of mobile phase tried

From various mobile phases tried, mobile phase containing Acetonitrile: Phosphate Buffer (70:30) pH 5 was selected, since it gives sharp reproducible retention time for DEXL.

Fig. No.07: Trial Chromatogram obtained by using Methanol: Water (90:10) as mobile phase.

Fig. No.08: Trial Chromatogram obtained by using Acetonitrile: water (80:20) as mobile phase

Fig. No.09: Trial Chromatogram obtained by using Acetonitrile: Phosphate Buffer (90:10) pH 5.5 as mobile phase.

Fig. No.10: Final Chromatogram obtained by using Acetonitrile: Phosphate Buffer (70:30) pH 5 as mobile phase of DEXL.

Fig. No.11: Blank Chromatogram obtained by using Acetonitrile: Phosphate Buffer (70:30) pH 5 as mobile phase.

Chromatographic conditions:

The following chromatographic conditions were established by trial and error and were kept constant throughout method.

Column: Intersil 4.6 (id) x 250 mm

Particle size packing : 5 μm

Stationary phases      : C18 Intersil

Mobile phase : Acetonitrile: Phosphate Buffer (70:30) pH 5

Detection wavelength            : 275 nm

Flow rate    : 1 ml/min.

Temperature  : Ambient

Sample size : 20 μL

Preparation of calibration curve

The mobile phase equilibrated with the stationary phase, and DEXL drug solutions were injected at concentrations ranging from 3-30 μg/ml, with a graph plotted.

Table No. 09: Observation of standard curve of DEXL

Fig. No. 12: Standard calibration curve for DEXL

System suitability test

System suitability is a pharmacopoeial requirement ensuring adequate resolution and reproducibility of chromatographic systems for analysis, tested using data from five replicate injections of standard solutions.

Table No. 10: Result of System Suitability Study

Application of proposed method for estimation of DEXL Laboratory Sample

The standard and Sample solution of DEXL was prepared and inject. The peak area of standard and sample laboratory was compared to obtain the concentration.

Table No 11.: Results and statistical data for estimation of DEXL in lab. Sample

Application of proposed method for estimation of DEXL in formulation

Standard and sample solutions were injected separately, chromatograms recorded, and major peaks measured. DEXL content was calculated by comparing sample peak with standard.

Fig. No.13: Chromatogram obtained by formulation of DEXL

Validation parameters

Accuracy

It was ascertained on the basis of recovery studies performed by standard addition method. The results of recovery studies and statistical data are recorded in Table No. 13

Table No.13: Results and statistical data for Recovery study of DEXL

Precision

The precision of an analytical method, expressed as S.D or R.S.D, was determined through replicate estimation of drugs using the proposed method, as shown in the table.

Table No.14: Results and statistical data of Precision Study

Brand Name: Dexlanzol 30

Ruggedness

The studies of ruggedness were carried out under two different conditions-

1. Days
2. Analyst.

Interday (Different days)

The same procedure was performed on different days for marketed formulation analysis, and the % label claim was calculated, as shown in Table No. 15.

Table No.15: Results and statistical data of Interday Study

Intraday

The study used the same procedure as the marketed formulation analysis, recording absorbance at 3 hours intervals within a day, and calculated the percent label claim using a formula.

Table No.16: Results and statistical data of Intraday Study

Brand Name: Dexlanzol 30

Different analyst

Two different analysts prepared the sample solution using the same procedure as before, and the % label claim was calculated as per the marketed formulation estimation.

Table No.17: Result and statistical data of Different analyst study

Specificity
The proposed method demonstrated specificity by obtaining a well-separated peak for DEXL without matrix interference, with a mean retention time of 4.573, closely resembling standard laboratory mixture.

Fig. No.14: Chromatogram obtained by formulation of DEXL

USP tablet powder was diluted to 80%-120% of test concentration, revealing DEXL's linearity within ± 20% of the test concentration, as shown in Fig. No. 15.

Table No.18: Observations of Linearity and range study for DEXL.

Fig. No.15: -Plot of linearity and range study for DEXL

The robustness study found that factors selected remained unaffected by small variations in mobile phase, wavelength, and flow rate, indicating the method's suitability remains within the limit.

Table No.19: Result of Robustness study of DEXL

Limit of detection refers to the minimum amount of analyte in a sample that can be detected but not quantitated accurately.

Table 20: LOD & LOQ of DEXL