Development And Validation of RP-HPLC Method for Estimation of Esmolol HCL from Semisolid Dosage Form

The chemical name of Esmolol Hydrochloride is methyl 3-(4-{2 -hydroxy-3-[(propan-2-yl) amino] propoxy} phenyl) propanoate hydrochloride.

Esmolol Hydrochloride is the salt form of Esmolol, which belongs to the class II anti-arrhythmic drugs. Esmolol hydrochloride blocks β-1 receptors in cardiac muscle and at higher doses, it will occlude β-2 receptors and vascular smooth muscle thus directing to its muscle relaxation 2, 3. It has a molecular formula of C₁₆H₂₅NO₄•HCl with molecular weight 331.8 g/mol. The purpose of this study is to develop a simple, precise, and accurate RP-HPLC method for the estimation of Esmolol Hydrochloride from a semi-solid dosage form and to validate the developed method with study of different parameters as per ICH guidelines as No reported RP-HPLC methods for estimation of Esmolol Hydrochloride in Semi solid dosage forms were found.

Esmolol Hydrochloride

MATERIALS AND METHODS:

Materials

Drug Substance: Working standards Esmolol Hydrochloride (99.9%), were procured from Mehta API.

Esmolol hydrochloride finished product:

Brand name: Diulcus Gel

Product name: Esmolol HCL 14% w/w

Manufacturer: IPCA Lab Ltd.

Instrumentation:

HPLC instrument with UV-visible detector – Agilent Infinity II

Software: Open lab

Column - Intsil C18 column (300 mm x 3.9 mm, 5 µm)

UV-visible Spectrophotometer - SHIMADZU 1601 Software - UV probe, version- 2.34

Digital Analytical Balance - OHSUS

Ultra Sonicator -PS 21

Digital pH meter – Lab India

Controlled temperature water bath

Hot air Oven – Kesar

FTIR – SHIMADZU

Melting Point Apparatus – ANALAB   Model: ThermoCal50

Chemicals and Reagents: Esmolol Hydrochloride API, Acetonitrile HPLC Grade, Phosphate Buffer, Tetrahydrofuran (THF), Ammonium acetate buffer, Hydrochloric Acid, Methanol HPLC Grade, Sodium Hydroxide pellet, Water: Distill water(Milli-Q), HPLC grade water.

Preparation of Mobile Phase: Acetonitrile:6.8g/L Ammonium acetate buffer (15:85) (pH 5)

Preparation of Standard Solution: Weigh accurately 70 mg of Esmolol hydrochloride WS and transfer into 100 ml volumetric flask. Add water about 60 ml then dissolved it then mark up to 100 ml with water. (Concentration of Esmolol hydrochloride is 700 µg /ml)

Preparation of Sample solution: Weigh 500 mg (Equivalent to 70 mg) of topical gel and transfer into 100 ml volumetric flask. Add water about 60 ml then dissolved it then mark up to 100 ml with Mobile Phase.

(Concentration of Esmolol hydrochloride is 700 µg /ml)

Preparation of system suitability solution: Take standard solution for system suitability.

System suitability requirement:

Tailing factor: NMT 2.0 for Esmolol peak

Relative standard deviation: NMT 2.0%   

Chromatographic Conditions:

Column: Intsil C₁₈ column (300 mm x 3.9 mm, 5 µm)

Flow rate: 1.5ml/min

Total run time (min): 20 min.

Injection volume (µl) : 20

Column Temperature(°C): Ambient

Detection Wavelength(nm) : UV 221 nm

IDENTIFICATION AND CHARACTERIZATION

The identification of taken standard API for experimental work had done for confirmation of its identity, standard quality and purity. The identification had done by taking IR and UV spectra, solubility study and melting point determination.

Solubility Study

The solubility of Esmolol Hydrochloride was practically determined by taking 100 mg of the drugs in 100 ml volumetric flasks, adding required quantity of solvent at room temperature and shaken for few minutes. Solubility data for each study was observed and recorded in Table 2.0

Table 1.0 Solubility table as per IP’2014 specification

Table 2.0 Solubility Data of Esmolol Hydrochloride

Melting point of Esmolol Hydrochloride has been determined. The melting points of the compounds were taken by open capillary method.

Table 3.0 Melting Point of Drugs

The IR Spectra of Esmolol Hydrochloride with its functional group identification, were shown in the following graph. IR spectra scanning of sample: Esmolol Hydrochloride

Figure 1.0 IR Spectra of Esmolol Hydrochloride

Table 4.0 IR Spectral interpretation of Esmolol Hydrochloride

SELECTION OF WAVELENGTH:

The RP-HPLC Method used UV detection which is depends on proper selection of detection wavelength. An ideal wavelength is important to get good response for the drugs that can be detected. To determine wavelength for measurement, standard spectra of Esmolol Hydrochloride was scanned between 200- 400nm against diluents. Peak is obtained at 221nm.

Figure 2.0 Overlay UV Spectra of Esmolol Hydrochloride

SELECTION OF COLUMN:

For RP-HPLC method, various columns are available but our main aim is to resolve drug in the presence of excipients. So the C18 column was selected for estimation of Esmolol Hydrochloride. Intsil C18 column (300 mm x 3.9 mm, 5 µm) column was chosen to give good peak shape and high resolution, which also provides high peak symmetry, good retention to drug and facilitates the separation of the drug without the interference of excipients within short run time.

Snapshot of Intsil C₁₈ column (300 mm x 3.9 mm, 5 µm):

SELECTION OF MOBILE PHASE

TRIAL 1:

Column: Intsil C18 column (300 mm x 3.9 mm, 5 µm)

Mobile Phase:  Methanol (MeOH) : 6.8gm/L Phosphate buffer (5:95) (pH 3)

Detection:  221 nm

Flow rate:   1.5 ml/min

Run Time:  32 minutes

Observation: No peak detected.

Figure 3.0 Trial 1: Chromatogram of Esmolol Hydrochloride

TRIAL 2:

Column: Intsil C18 column (300 mm x 3.9 mm, 5 µm)

Mobile Phase: Methanol (MeOH):  6.8gm/L Phosphate buffer (5:95) (pH 5)

Detection:  221 nm

Flow rate:   1.5 ml/min

Run Time:  32 minutes

Observation: No peak detected.

Figure 4.0 Trial 2: Chromatogram of Esmolol Hydrochloride

TRIAL 3:

Column: Intsil C18 column (300 mm x 3.9 mm, 5 µm)

Mobile Phase: Methanol (MeOH): 6.8gm/L Phosphate buffer (10:90) (pH 5)

Detection:  221 nm

Flow rate:   1.5 ml/min

Run Time:  20 minutes

Observation: Two Peak Identified.

Figure 5.0 Trial 3: Chromatogram of Esmolol Hydrochloride

TRIAL 4:

Column: Intsil C18 column (300 mm x 3.9 mm, 5 µm)

Mobile Phase: Tetrahydrofuran (THF) : 6.8gm/L Phosphate buffer (5:95) (pH 4)

Detection:  221 nm

Flow rate:   1.5 ml/min

Run Time:  20 minutes

Observation: No peak detected.

Figure 6.0 Trial 4: Chromatogram of Esmolol Hydrochloride

TRIAL 5

Column: Intsil C18 column (300 mm x 3.9 mm, 5 µm)

Mobile Phase: Tetrahydrofuran (THF) : 6.8gm/L Phosphate buffer (10:90) (pH 4)

Detection:  221 nm

Flow rate:   1.5 ml/min

Run Time:  10 minutes

Observation: Peak Shape not good.

Figure 7.0 Trial 5: Chromatogram of Esmolol Hydrochloride

TRIAL 6

Column: Intsil C18 column (300 mm x 3.9 mm, 5 µm)

Mobile Phase: Tetrahydrofuran (THF) : 6.8gm/L Phosphate buffer (10:90) (pH 5)

Detection:  221 nm

Flow rate:   1.5 ml/min

Run Time:  10 minutes

Observation: Peak not identified.

Figure 8.0 Trial 6: Chromatogram of Esmolol Hydrochloride

Column: Intsil C18 column (300 mm x 3.9 mm, 5 µm)

Mobile Phase: Acetonitrile (ACN) : 6.8 g/L Ammonium acetate buffer  (5:95) (pH 3)

Detection:  221 nm

Flow rate: 1.5 ml/min

Run Time:  10 minutes

Observation: No peak detected.

Figure 9.0 Trial  7: Chromatogram of Esmolol Hydrochloride

TRIAL 8  

Column: Intsil C18 column (300 mm x 3.9 mm, 5 µm)

Mobile Phase: Acetonitrile (ACN) : 6.8 g/L Ammonium acetate buffer(5:95) (pH 5)

Detection:  221 nm

Flow rate: 1.5 ml/min

Run Time:  10 minutes

Observation: Peak not identified.

Figure 10.0 Trial 8: Chromatogram of Esmolol Hydrochloride

TRIAL 9

Column: Intsil C18 column (300 mm x 3.9 mm, 5 µm)

Mobile Phase: Acetonitrile (ACN) : 6.8 g/L Ammonium acetate buffer   (6:94) (pH 5)

Detection:  221 nm

Flow rate:  1.5 ml/min

Run Time:  10 minutes

Observation: Negative peak observed

Figure 11.0 Trial 9: Chromatogram of Esmolol Hydrochloride

TRIAL 10

Column: Intsil C18 column (300 mm x 3.9 mm, 5 µm)

Mobile Phase:  Acetonitrile (ACN) : 6.8 g/L Ammonium acetate buffer  (8:92) (pH 5)

Detection:  221 nm

Flow rate: 1.5 ml/min

Run Time:  20 minutes

Observation: Broadening observed.

Figure 12.0 Trial 10: Chromatogram of Esmolol Hydrochloride

TRIAL 11

Column: Intsil C18 column (300 mm x 3.9 mm, 5 µm)

Mobile Phase: Acetonitrile (ACN) : 6.8 g/L Ammonium acetate buffer  (10:90) (pH 5)

Detection:  221 nm

Flow rate: 1.5 ml/min

Run Time:  7 minutes

Observation: Tailing observed.

Figure 13.0 Trial 11: Chromatogram of Esmolol Hydrochloride

TRIAL 12-1

Column: Intsil C18 column (300 mm x 3.9 mm, 5 µm)

Mobile Phase: Acetonitrile (ACN) : 6.8 g/L Ammonium acetate buffer (15:85) (pH 5)

Detection:  221 nm

Flow rate: 1.5 ml/min

Run Time:  20 minutes

Observation: Peak shape was observed good and sharp. Tailing factor below 1.5.

Figure 14.0 Trial 12-1: Chromatogram of Esmolol Hydrochloride

TRIAL 12-2

Column: Intsil C18 column (300 mm x 3.9 mm, 5 µm)

Mobile Phase: Acetonitrile (ACN) :  6.8 g/L Ammonium acetate buffer (15:85) (pH 5)

Detection:  221 nm

Flow rate: 1.5 ml/min

Run Time:  20 minutes

Observation: Peak shape was observed good and sharp. Tailing factor below 1.5.

Figure 15.0 Trial 12-2: Chromatogram of Esmolol Hydrochloride

TRIAL 12-3

Column: Intsil C18 column (300 mm x 3.9 mm, 5 µm)

Mobile Phase: Acetonitrile (ACN) :  6.8 g/L Ammonium acetate buffer(15:85) (pH 5)

Detection:  221 nm

Flow rate: 1.5 ml/min

Run Time:  20 minutes

Observation: Peak shape was observed good and sharp. Tailing factor below 1.5.

Figure 16.0 Trial 12-3: Chromatogram of Esmolol Hydrochloride

Optimized Chromatographic conditions :

Table 5.0 Optimized Chromatographic conditions

Method validation is the process of documenting or proving that an analytical method provides analytical data acceptable for the intended use. The need to validate a method and the procedure to be followed are matters of professional judgement, although well-prescribed procedures and guidelines are now available that aid in decision making. According to that the various validation parameters to validate each and every above stated method are:

• Accuracy
• Precision (Repeatability and Reproducibility)
• Linearity
• Range
• Selectivity/ Specificity
• Robustness/ Ruggedness
• Limit of Detection (LOD)/ Limit of Quantification (LOQ)

Accuracy

The accuracy of an analytical method is the closeness of test results obtained by that method to the 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 quantities of the analyte have been added (i.e. “to spike”).

Accuracy is calculated as the percentage of recovery by the assay of the known added amount of analyte in the sample or as the difference between the mean and the accepted true value, to gather with confidence intervals. Dosage form assays commonly provide accuracy within 3-5 % of the true value.

The ICH documents recommend that accuracy should be assessed using a minimum of nine determinations over a minimum of three concentration levels, covering the specified range (i.e. three concentrations and three replicated of each concentration).

It may often be expressed as the recovery by the assay of known added amounts

of analyte. Samples are prepared &analyzed and the recoveries of each are calculated.

% Recovery = Spiked sample result - Unspiked sample result) / Spiked sample result) × 100%

Acceptance criteria:

• The percentage recovery should be within 100 ± 2.0% for the average of each set of three weights.
• Each individual sample recovery should lie within the range of 98%-102%.
• Coefficient of determination (r2) should be greater than 0.998.
• There should be no curvature in the residuals plot.

Precision

The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. Precision may be considered at three levels: repeatability, intermediate precision and reproducibility. Precision should be investigated using homogeneous, authentic samples. However, if it is not possible to obtain a homogeneous sample it may be investigated using artificially prepared samples or a sample solution. The precision of an analytical procedure is usually expressed as the variance, standard deviation or coefficient of variation of a series of measurements.

Repeatability

Repeatability expresses the precision under the same operating conditions over a short interval of time. Repeatability is also termed intra-assay precision.

Intermediate precision

The extent to which intermediate precision should be established depends on the circumstances under which the procedure is intended to be used. The applicant should establish the effects of random events on the precision of the analytical procedure. Typical variations to be studied include days, analysts, equipment, etc. It is not considered necessary to study these effects individually. The use of an experimental design (matrix) is encouraged.

% RSD or coefficient of variance (CV) is expressed as,

             RSD = CV = SD/X  × 100

             Standard Error = SD/√n

Where, n = Number of Replicates taken in the set.

Reproducibility

Reproducibility expresses the precision between laboratories (collaborative studies, usually applied to standardization of methodology). The ICH documents recommends that repeatability should be assessed using a minimum of nine determinations covering the specified range for the procedure (i.e. three concentrations and three replicates of each concentration).

Linearity

A linear relationship should be evaluated across the range (see section 3) of the analytical procedure. It may be demonstrated directly on the active substance (by dilution of a standard stock solution) and/or on separate weighings of synthetic mixtures of the product components, using the proposed procedure. The latter aspect can be studied during investigation of the range.

Linearity should be evaluated by visual inspection of a plot of signals as a function of analyte concentration or content. If there is a linear relationship, test results should be evaluated by appropriate statistical methods, for example, by calculation of a regression line by the method of least squares. In some cases, to obtain linearity between assays and sample concentrations, the test data may need to be subjected to a mathematical transformation prior to the regression analysis. Data from the regression line itself may be helpful to provide mathematical estimates of the degree of linearity. The correlation coefficient, y-intercept, slope of the regression line and residual sum of squares should be submitted. A plot of the data should be included. In addition, an analysis of the deviation of the actual data points from the regression line may also be helpful for evaluating linearity. Some analytical procedures, such as immunoassays, do not demonstrate linearity after any transformation. In this case, the analytical response should be described by an appropriate function of the concentration (amount) of an analyte in a sample.

For the establishment of linearity, a minimum of 5 concentrations is recommended. Other

approaches should be justified.

Acceptance criteria:

• Coefficient of determination (r2) should be greater than 0.998.
• There should be no curvature in the residuals plot.
• The Y-intercept should not significantly depart from zero.

Range

The range of an analytical method is the interval between the upper and lower levels of analyte (including these levels) that has been demonstrated to be determining with a suitable level of precision, accuracy and linearity using the method as written. The range is expressed in the same units as test results (eg. percent, parts per million) obtained by analytical method.

Acceptance criteria:

• Coefficient of determination (r2) should be greater than 0.998.
• There should be no curvature in the residuals plot.
• The Y-intercept should not significantly depart from zero.

Specificity

Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present. Typically these might include impurities, degradants, matrix etc.

Lack of specificity of an individual analytical procedure may be compensated by other supporting analytical procedure(s). This definition has the following implications:

Identification: To ensure the identity of an analyte.

Purity Tests: To ensure that all the analytical procedures performed allow an accurate statement of the content of impurities of an analyte, i.e. related substances test, heavy metals, residual solvents content etc.

Assay (content or potency): To provide an exact result which allows an accurate statement on content or potency of the analyte in a sample.

Robustness

The robustness of an analytical method is a measure of its capacity to remain unaffected by small but deliberate variation in method parameters and provides an indication of its reliability during normal usage. The determination of robustness requires that methods characteristic are assessed when one or more operating parameter varied.

Rogudness

The ruggedness of an analytical method is the degree of reproducibility of test results obtained by the analysis of the same samples under a variety of normal test conditions such as different laboratories, different analysts, using operational and environmental conditions that may differ but are still within the specified parameters of the assay.

The testing of ruggedness is normally suggested when the method is to be used in more than one laboratory. Ruggedness is normally expressed as the lack of the influence on the test results of operational and environmental variables of the analytical method.

For the determination of ruggedness, the degree of reproducibility of test result is determined as function of the assay variable. This reproducibility may be compared to the precision of the assay under normal condition to obtain a measure of the ruggedness of the analytical method.

Limit of Detection

The limit of detection (LOD) is the lowest amount of analyte in a sample that can be detected, but not necessarily quantified, under the stated experimental conditions. It is usually expressed as the concentration of analyte in the sample. The determination of the limit of detection of instrumental procedures is carried out by determining the signal-to-noise ratio by comparing test results from the samples with known concentration of analyte with those of blank samples and establishing the minimum level at which the analyte can be reliably detected. A signal-to-noise ratio of 2:1 or 3:1 is generally accepted.

The signal-to-noise ratio is determined by dividing the base peak by the standard deviation of all data points below a set threshold. Limit of detection is calculated by taking the concentration of the peak of interest divided by three times the signal-to noise ratio. Other approaches depend on the determination of the slope of the calibration curve and the standard deviation of responses as shown below:

LOD = 3.3 × SD / Slope of calibration curve

Where, SD = Standard deviation of intercepts of calibration curves.

Limit of Quantification

The limit of quantification (LOQ) is the lowest amount of analyte in a sample that can be determined  with  acceptable precision and  accuracy. It is  usually expressed as the concentration of analyte in the sample. It can be determined by comparing measured signals of samples with known low concentrations of analyte with those of blank samples. The minimum concentration at which the analyte can reliably be quantified is established.

A typical acceptable signal- to- noise ratio is 10:1. Other approaches depend on the determination of the slope of the calibration curve and the standard deviation of responses.

LOQ = 10 × SD/Slope of calibration curve

Where, Standard Deviation of intercepts of calibration curves.

RESULT AND DISCUSSION

Analytical Method Validation

Specificity

The Chromatogram of Esmolol Hydrochloride Standard (Figure 18.0) and Esmolol Hydrochloride Sample (Figure 19.0) Show no interference with the Chromatogram of Esmolol Hydrochloride Blank (Figure 17.0), So the developed method is Specific.

Figure 17.0 Chromatogram of Esmolol Hydrochloride Blank

Figure 18.0 Chromatogram of Esmolol Hydrochloride Standard

Figure 19.0 Chromatogram of Esmolol Hydrochloride Sample

Linearity and Range

The linearity range for Esmolol Hydrochloride was found to be in the range of 350 to 1050 µg/ml. Correlation coefficient (r2) for calibration curve of Esmolol hydrochloride should be found 0.998.

Linearity stock solution: Transfer an accurately weighed quantity of about 140 mg of Esmolol hydrochloride in 100 ml volumetric flask. Add about 60 ml of mobile phase and sonicate to dissolve it. Make volume up to the mark with mobile phase.

50% (350 PPM): Pipette out 2.5 from Stock solution and dilute up to 10 ml with diluent.

80% (560 PPM): Pipette out 4.0 from stock solution and dilute up to 10 ml with diluent.

100%(700 PPM): Pipette out 5.0 from stock solution and dilute up to 10 ml with diluent.

120% (840 PPM): Pipette out 6.0 from stock solution and dilute up to 10 ml with diluent.

150%(1050 PPM): Pipette out 7.5 from stock solution and dilute up to 10 ml with diluent.

Figure 20.0 Overlay Chromatogram of five different concentrations of Esmolol Hydrochloride

Table 6.0 Calibration data for Esmolol Hydrochloride (n=5)

Figure 21.0: Calibration curve of Esmolol Hydrochloride

DISCUSSION

Linearity range for Esmolol Hydrochloride and Misoprostol was found to be in the range of 350 to 1050 µg/ml.

Range:

Range is the interval between upper and lower concentration of analyte in sample for which it has been demonstrated that the analytical method has suitable level of precision, accuracy and linearity. The linear response was observed over range of 350-1050 µg/ml for Esmolol Hydrochloride.

Table 7.0 Range Data

Precision

Precision of analytical procedure express the closeness of agreement between the value, which is accepted either as a conventional true value or as an accepted reference value and the value found.

System Precision/Repeatability

Prepared standard solution as per method.

Table 8.0 Area of 5 replicate standard injections

Method Precision/Intermediate precision Day 1

Repeatability of the method for the estimation of assay for Esmolol hydrochloride Repeatability expresses the precision under the same operating conditions over a short interval of time.

Blank, standard solution and sample solution prepared as per method.

Table 9.0 Observation of method precision

Discussion and Conclusion: %RSD of five replicate standard injections is within acceptance criteria (NMT 2.0%) and %RSD of test solution is within acceptance criteria (NMT 2.0%)

Intermediate Method Precision (Day 2)

For day 2- same instrument, different day and different analyst

Table 10.0 Observation for Day 2 precision results

Mean of Day 1 and Day 2 precision

Table 11.0 Observation mean results of Day 1 and Day 2

Discussion and Conclusion: % RSD between day 2 with Method precision or day 1 standard solution area is within acceptance criteria (NMT 2.0%) % RSD between day 2 and Method precision or day 1 test solution area is within acceptance criteria (NMT 2.0%).

Accuracy

Accuracy of an analytical procedure express the closeness to the agreement between the value, which is accept either as a conventional true value and the test result.

Demonstrate the accuracy of the test method by preparing recovery samples at the level 80%,100% and 120% if target concentration. Prepare the recovery samples in triplicate at each level and as per sample solution.

% Recovery = (Experimental value/Actual value) × 100

Table 12.0 % Recovery result

Discussion: Result obtain reveals that % recovery was Esmolol Hydrochloride by this method was found within the range of acceptance criteria in ICH i.e 98.35 – 99.86%

LOD and LOQ:

Calibration curve was repeated for five times and the standard deviation  of the intercept was calculated. Then LOD and LOQ were calculated as  follows.

Limit of Detection:

LOD=3.3σS

            = 1.462 µg/ml

Limit of Quantification:

LOQ=10σS

         = 10*2.977/6.7204

         = 4.430 µg/ml

Discussion:

The propose method can detect Esmolol Hydrochloride at low level and it also quantified small amount of drug precisely. So, it was concluded that the proposed method is very sensitive in nature.

Robustness

Variation in Flow rate : Variated flow rate ± 0.1 ml/min. (1.4 ml/min and 1.6 ml/min)

Mobile phase, blank, standard solution and Sample solution prepared as per method.

Table 13.0   Robustness result for Flow rate

Conclusion:

% RSD of Assay Esmolol hydrochloride is within acceptance criteria (NMT 2.0%)

Variation in Wavelength: Variated in wavelength ± 1 nm (220 nm and 222 nm)

Mobile phase, blank, standard solution and Sample solution prepared as per method.

Table 14.0   Robustness result for wavelength

Conclusion:

% RSD of Assay of Esmolol hydrochloride is within acceptance criteria (NMT 2.0%)

Stability of solution (Standard solution)

Mobile phase, blank, standard solution and Sample solution prepared as per method.

Conclusion: % Difference between the initial area and respective time interval value is within Acceptance criteria (NMT 2.0%)

Stability of test solution

Conclusion: % Difference between the initial %assay and respective time interval value is within Acceptance criteria (NMT 2.0%)

These %RSD value was found to be less than ± 2.0 indicated that the method is precise. No significant changes in the Peak area were observed, proving that the developed method is rugged and robust.

Application of the proposed method for analysis of Esmolol Hydrochloride in formulation.

Chromatogram of the Test solution containing 700µg/ml of Esmolol Hydrochloride was recorded and peak areas were noted for estimation of Esmolol Hydrochloride The concentration of Esmolol Hydrochloride in formulation was determined against the standard Esmolol Hydrochloride.

Figure 22.0   Chromatogram of Esmolol Hydrochloride

SUMMARY OF VALIDATION PARAMETER ON HPLC

Summary of validation parameter are shown in below table. All the parameters for substance met the criteria of ICH guideline for the method validation and found to be suitable for routine quantitative analysis in pharmaceutical dosage forms. The result of linearity, accuracy, precision proved to be within limits with lower limits of detection and quantification. Robustness of method was confirmed as no significant in the were observed on analysis by subjecting the method to slight change in the method condition. Assay results obtained by proposed method are fair agreement.