Estimation of Tranexamic Acid Andethamsylate Using RP-HPLC

Chapter-3 Experimental work

3. EXPERIMENTAL WORK

3.1 MATERIALS AND METHODS

Table 2. List of Chemical and standers used

Table 3. List of instruments used

3.2. Method development for the simultaneous estimation of Tranexamic acid andethamsylate by using RP-HPLC.

1. Selection of mobile phase
2. Selection of detectionwavelength
3. Selection of column
4. Selection of solvent delivery system
5. Selection of flow rate
6. Selection of column temperature
7. Selection of diluent
8. Selection of test concentration and injection volume

3.2.1. Selection of mobile phase

Phosphate Buffer: Methanol (30:70)

3.2.2. Selection of wavelength

10mg Tranexamic acid and Ethamsylate were dissolved in mobile phase. The overlay spectrum was used for selection of wavelength for Tranexamic acid and Ethamsylate The iso-bestic point was taken as detection wavelength 286nm.

3.2.3. Selection of column

• Heart of HPLC made of 316 grade stainless steel packed with stationary phase.

Silica based columns with different cross linking’s in the increasing order of polarity are as follows:

——- Non-polar———-moderately polar——–Polar———-

C₁₈< C₈< C₆< Phenyl < Amino

Reasons: Better separation,

Good tailing factor.

3.2.4. Selection of solvent delivery system

• Always preferable solvent delivery system.
• More chance of getting reproducible result on retention time of analytes.
• More economic than gradient technique.

3.2.5. Selection of flow rate

Acceptable limit: – Not more than 2.5 ml/min

• Flow rate selected was 1.0ml/min
• Flow rate is selected based on

1. Retention time

2. Column back pressure

3. Peak symmetry.

4. Separation of impurities.

Reasons:

• For earlier elution of analyte and elution of all impurities within 10 min
• Information from the reference method in literature.

3.2.6. Selection of diluent

• Selection of diluents is based on the solubility of the analyte
• Diluent selected: Phosphate Buffer: Methanol (30:70 % v/v)

Reason:

• Analyte is soluble in acetonitrile and water.

3.2.7. Selection of column temperature

• Preferable temperature is ambient or room temperature.

Reasons:

• To elute all impurities along with analyte with in 10 min of run time.
• Less retention time
• Good peak shape
• Higher theoretical plates.
• Good resolution.

3.2.8. Selection of test concentration and injection volume

Test concentration is finalized after it is proved that API is completely extractable at the selected test concentration.

• Test concentration is fixed based upon the response of API peak at selected detector wavelength.
• Tranexamic Acid and Ethamsylate label claimed 25mg and 50 mg
• And the test concentration selected is 100ppm
• Injection volume selected is 20µL.

Reason: good peak area, retention time, peak symmetry

Chromatographic trails for simultaneous estimation Tranexamic acid Ethamsylate

TRIAL 1

Fig. 4: Trial 1

Observation: 100% Methanol used for this trial, flow rate was 1ml/min at ambient temperature. Faster elution of the analyte takes place .

TRIAL 2

Fig. 5: Trial 2

Observation: Methanol and water was used in the ratio of 70:30. The flow rate was 1ml/min at ambient temperature.Couldn’t get consistent retention time

TRIAL 3

Fig. 6: Trial 3

Observation: Methanol and Phosphate Buffer used in the ratio of (30:70 ) Couldn’t get consistent retention time

Discussion: The above trials indicating that RT for the drug was not constant and elution time was faster which not prefered for the analysis.

TRAIL 4

Optimizing method

Fig. 7: Developed Chromatogram

Discussion: All the experiments were complete by the higher than developed method and the consequences were acceptable.

Optimized chromatographic conditions for simultaneous estimation of Tranexamic Acid and Ethamsylate

Trail 4: (Optimized Chromatographic Conditions)

Assay procedure

Preparation of 0.2M phosphate buffer:

Buffer solution prepares by dissolving 2.72g of Potassium dihydrogen ortho phosphate (KH₂PO₄) in 1L of water and the degassing of the solution.

Diluents Preparation:

1L of diluents was prepared by mixing 300 ml of 0.02 M Phosphate Buffer and 700 ml of Methanol.

Preparation of stock solution:

Accurately weighed 10 mg of the both Tranexamic Acid and Ethamsylate is transferred to 10 ml fresh and dry volumetric flask. The amount was making up to the mark among the Methanol and mixed well. This yielded a stock solution with concentration 1000 ppm of Tranexamic Acid and Ethamsylate mixture.

Preparation of standard solution:

Accurately amount of 0.25 and 0.25 ml of the Tranexamic Acid and Ethamsylate stock solution transferred to 10 ml clean and dried volumetric flask. Then compose up the amount up to the mark among the diluents and mix well. Finally the standard stock solution with concentrations of 25 ppm and 25 ppm of Tranexamic Acid and Ethamsylate respectively.

Procedure

20Lof the standard and sample was injected into the chromatographic system and areas for the Tranexamic acid and Ethamsylate from the peaks were used for calculating the % assay by using the formulae.

Assay calculation

AT WS DT P Avg. Wt

Assay % =————– x ———-x ——— x ———-x—————— 100

AS DS WT 100 Label Claim

Where:

AT = Average area counts of sample preparation.

AS = Average area counts of standard preparation.

WS=Weight of working standard taken in mg.

P= Percentage purity of working standard

LC = Label Claim of Tranexamic acid , Ethamsylate mg/ml.

3.4 METHOD VALIDATION

3.4.1 ANALYTICAL METHOD VALIDATION

Validation parameters

• Specificity
• Linearity
• Range

• Accuracy
• Precision
  • System precision
  • Repeatability
  • Intermediate Precision

• Detection Limit
• Quantitation Limit
• Robustness

1. Specificity

The system suitability for specificity was carried out to determine whether there are any interference of any impurities in retention time of analytical peak. The study was performed by injecting blank.

2. Linearity

The linearity is a systematic method its ability (within a given range) to get assessment results, which are directly relative to the absorption (amount) of analyte in sample.

Preparation of standard stock solution:

Accurately weighed 10 mg of the both tranexamic acid and Ethamsylate was transferred in to 10 ml fresh and dry volumetric flask.

After that the amount was made up to the mark with the Methanol and mix well. This yielded a stock solution amid attention 1000 ppm of tranexamic acid with Ethamsylate mixture.

Preparation of standard solution:

Accurately amount of 0.25 and 0.25 ml of the tranexamic acid and Ethamsylate stock was transferred to 10 ml clean and dry volumetric flask. Then the volume was made up to the mark with the diluent and mixed well. This yielded a standard stock solution with concentrations of 25ppm and 25ppm of tranexamic acid and Ethamsylate respectively¹⁰

Procedure: Prepared a series of standard solutions not less than five during the particular concentration range along with investigate them like for each method.

Acceptance criteria: The correlation coefficient should be not less than 0.9990

3. Range

The range of a systematic process is the gap between the superior and lower concentration of analyte in sample for which it has been established to the investigative practice was a suitable level of accuracy, precision and linearity.

Acceptance criteria: Linearity, Precision and Recovery should be shown.

The logic behind this parameter was – typical concentration range was essential between which the actual concentration should fall when performing real sample analysis.¹⁰

4. Accuracy

Preparation of standard stock solution:

Accurately amount of 0.25 and 0.25 ml of the tranexamic acid and Ethamsylate stock solution transfer to 10 ml fresh and dried volumetric flask. Make up the volume up to mark with the diluents and mix well. The standard stock solution with concentrations of 25 ppm and 25 ppm of tranexamic acid and Ethamsylate respectively.

Method procedure: Prepared solutions in triplicate at levels 80%, 100% and 120s% of test concentrations using for tranexamic acid and Ethamsylate working Standards as per the test method and injected each solution in triplicate.

Sample Are 100

% Recovery = ———————– x —————— x 100

Standared Area conc. in %

Accuracy normally refers to the difference between the mean of the set of results and the true or correct value for the quantity measured. According to IUPAC accuracy relates to the difference between results (or mean) and the true value. For analytical methods, there are two possible ways of determining the accuracy, absolute method and comparative method. Accuracy is best reported as percentage bias, which is calculated from the expression

Procedure: Known amount of drug substance spiked with known amount of standard drug- minimum of three levels (80%, 100% & 120% of test concentration), each level was triplicate.

Acceptance criteria: Assay recovery should be between 97%-103%.¹⁰

5. Precision

Preparation of standard solution:

Accurately amount of 0.25 and 0.25 ml of the tranexamic acid and Ethamsylate stock solution transferred to 10 ml clean and dried volumetric flask.

Subsequently make up the volume up to the mark among the diluent and well mixed. Finally the standard stock solution with concentrations of 25 ppm and 25 ppm of tranexamic acid and Ethamsylate respectively.

Method precision: Six individual preparations were prepared using single batch of tranexamic acid and Ethamsylate functioning standard as for each test process and injected each one solutions.

Injection precision:

Solo preparation was prepared using single batch of Tranexamic acid and Ethamsylate effective standard as for each urbanized process in addition to injected six injections^10.

Acceptance Criteria:

1. RSD should not be more than 2.0% for five replicate injections of standard.

6. Ruggedness

Preparation of standard solution:

Accurately amount of 0.25 and 0.25 ml of the tranexamic acid and Ethamsylate stock solution transferred to 10 ml clean and dried volumetric flask. Subsequently make up the quantity up to the mark among the diluents and well mix. Finally the standard stock solution with concentrations of 25 ppm and 25 ppm of tranexamic acid and Ethamsylate respectively.

Method Procedure: The standard solution was individually prepared as per the test method and injected each solution in six times using different system, analyst, and date.

Acceptance Criteria: Overall RSD should not be more than 2.0 %.

7. Limit detection and limit of quantitation

LOD: Lowest amount of analyte in a sample that can be detected but not necessarily quanities, under the stated experimental conditions.

Preparation of standard solution:

Accurately amount of 0.25 and 0.25 ml of the tranexamic acid and Ethamsylate stock solution transferred to 10 ml clean and dried volumetric flask. Then build up the quantity up to the mark with the diluents and mix well. Finally the standard stock solution with concentrations of 25 ppm and 25 ppm of Tranexamic acid and Ethamsylate respectively.

Method Procedure:

The mobile phase was permissible to run equilibrate with stationary phase up to good baseline was obtained. The different concentration ranging from 0.01 to 0.1ppm of tranexamic acid and 0.01 to 0.1ppm Ethamsylate was injected and peaks were recorded. 0.03 and 0.03ppm for tranexamic acid and Ethamsylate concentrations were detected respectively.

LOD can be calculated based on signal-noise ratio,by using following formula

LOD = S/N

Where,

S = Signal Obtained From LOD Solution.

N = Average Baseline Noise Obtained from Blank

Acceptance criteria for LOD and LOQ

8. Robustness

Preparation of standard solution:

Accurately amount of 0.25 and 0.25 ml of the tranexamic acid and Ethamsylate stock solution transferred to 10 ml clean and dried volumetric flask. After that make up the quantity up to the mark with diluent and well mixed. Finally the standard stock solution with concentrations of 25 ppm and 25 ppm of tranexamic acid and Ethamsylate respectively.

Method procedure:

1. Flow: The standard solution was prepared and injected for the two times with (+1) flow rate.

2. Mobile Phase: The standard solution was prepared and injected for the two times with (+5) Mobile Phase composition.

Appraise of its capability to remain unchanged by minute, but conscious variations in process parameters and provides signal of its reliability during its normal usage.

Procedure: samples were analyzed under the following conditions.¹⁰

3. Stability studies

In the rational design and evaluation of dosage forms for the drugs, the stability of the activity components must be a major criterion in determining their stability. The medicine has to reach the patient in an active and acceptable form maintaining the criteria for acceptable equality.

The quality of the product has to be retained as long as the product is offered for sale or for administration to the patient. ¹⁰

Acceptance Criteria: Overall RSD should not be more than 2.0 %.

9. System suitability

Preparation of standard solution:

Accurately amount of 0.25 and 0.25 ml of the tranexamic acid and Ethamsylate stock solution transferred to 10 ml clean and dried volumetric flask. Subsequently make up the amount up to the mark with diluent and well mixed.

Finally the standard stock solution with concentrations of 25 ppm and 25 ppm of tranexamic acid and Ethamsylate respectively.

Procedure: Standard solution was prepared and injected six times to test the performance of the chromatographic instrument.

Acceptance Criteria:

1. RSD should not be more than 2.0% for five replicate injections of standard

2. USP Tailing for tranexamic acid and Ethamsylate peak in not more than 2.0

3. The column efficiency as determined for tranexamic acid and ethamsylate

Plate Count should not be more than 2000.

Dept.of Pharmaceutical Analysis JNTUA-OTRI, Ananthapuramu Page 1