Analytical Method Development of Dissolution by using HPLC

1.0  OBJECTIVE

To lay down the Procedure for Analytical Method development of Dissolution by using HPLC.

2.0 SCOPE

This SOP is applicable for Procedure for Analytical Method development of Dissolution by using HPLC in company Name

3.0 BACKGROUND

NIL

4.0 RESPONSIBILITY

4.1 All Analytical Research Personnel is responsible to follow the SOP. 

4.2 Group Leader Analytical Research to ensure the Procedures to be followed.      

4.3 Head Analytical Research or his designee to ensure overall compliance.

5.0  PROCEDURE

5.1 Literature survey

5.1.1 Conduct literature survey and collect information about solubility of the drug substance, suggested dissolution media, dissolution parameters and suggested detection methods.

5.2 Solubility profile:

5.2.1Conduct solubility studies of the drug substance in dissolution media having pH 1 to 7.5, Stimulated gastric or intestinal fluid (with or without enzymes).The following are generally suggested media to check the solubility of the drug against pH.

1) 0.1N HCI & / 0.01NHCI.

2) pH 2.1 Simulated gastric fluid, fasted state (SGF).

3) pH 4.0 Acetate buffer.

4) pH 5.0 Phosphate buffer & pH 5.0 Simulated intestinal fluid, fed state(SDF).

5) pH 6.0 phosphate buffer .

6) pH 7.2 Phosphate buffer and pH 6.8 simulated intestinal fluid fasted state(SIF).

7) pH 7.5 Phosphate buffer.             

5.2.2 In case. the drug substance does not have any solubility in the above mentioned media, then media having surfactants with different concentrations can be chosen.

Eg.: SLS (Sodium Lauryl Sulphate), Polysorbate, etc.,

5.2.3 It is important to control the grade of surfactants, because use of different grades of Surfactants could affect the solubility of the drug.

5.2.4 Conduct solubility study by following the SOP " Determination of solubility of a drug substance" and establish the maximum solubility of the drug substance in mg/ML.

5.3 Selection of flow rate:

Flow rate shall be selected based on the following data.

(i) Retention times.

(ii) Column back pressures.

(iii)Separation of impurities.

(iv) Peak symmetries.  

(v) Theoretical plate count.   

Check the ruggedness of the method by varying the flow rate by ±0.2 ml from the selected flow rate.

Select the flow rate which gives least retention times, good peak symmetries, least Back pressures and better separation of blank, Placebo and impurity peaks from Analyte peak.

5.4 Selection of column temperature :

Always it is preferable to optimize the chromatographic conditions with column temperature as 27°C. However, if the peak symmetry could not be achieved by any combination of column and mobile phase, then the column temperatures above ambient can be adopted.

The Increase in column temperature generally will result in reduction in peak asymmetry and peak retentions. When found necessary, the column temperatures between 30°C to 60°C shall be adopted. If a column temperature of above 60°C is found to be necessary, Instrument which can support and column packing materials which can withstand to that temperature will be chosen.

5.5 Selection of Diluent for Test preparation :

Diluent for test preparation is selected initially based on solubility of the drug substance. However, finalization of diluent is based on its extraction efficiency, peak symmetries and resolution of impurities from Analyte peak and diluent blank injection interference. Select a diluent in which drug substance is soluble, in which the extraction is complete,due to which there is no blank Interference, in which the peak symmetry and resolution between impurities and analyte peak Is found to be satisfactory.

Inject the diluent blank, placebo and test solutions spiked with known impurities into the chromatographic system and establish the non-interference of blank and placebo in estimation of analyte and the effect of diluent on resolution of impurities from Analyte peak and peak symmetry.

5.6 Optimization of Extraction procedure :

In general, methods followed for extraction are sonication, rotary shaking etc. In some cases where the analyte is not extracted by the above procedures heating can be adapted if the substance is stable and should not precipitate upon cooling to room temperature.

Conduct experiments to optimize the extraction of analyte in presence of excipients at different test concentrations using the diluent chosen at different time intervals of sonication or rotary shaking or both and select the test concentration at which the extraction is most efficient.

Experiments shall be conducted by spiking API into placebo, using the final blend or tablets. Dropping and crushing methods to be studied for tablets or pellets.     

5.7 Selection of test concentration and Injection volume :

The test concentration is generally chosen based upon the response of analyte peak at the selected detector wavelength.

However, the test concentration shall be finalized after it is proved that drug substance is completely extractable at the selected test concentration.

Generally an injection volume of 5 to 50 µl is recommended for estimation of drug substance.

However, if the extractions are found to be difficult then the test concentrations can be kept low and the injection volume can be increased up to 200 µl. But it is to be ensured that at the selected Injection volume the column is not overloaded, Separation of impurities and placebo peaks from analyte peak and the peak symmetry are not compromised.

After the test concentration and the diluent are finalized, prepare a test solution and keep the filtered solution in closed condition in a stoppered flask on bench top and observe for any precipitation or turbidity after 24 hours. The solution should not show any turbidity/precipitation.

5.8 Establishment of stability of Standard and test preparations :

Prepare the standard and test solutions and establish the stability of solutions for at least 24 hours. As far as possible select a diluent in which test solution is stable for at least 24 hours. If the solution is found to be unstable by its nature, then conduct the solution stability on hourly basis and incorporate the stability of solution in test method.

Stability of standard and test solutions in the Auto sampler shall be established (on hourly basis) for 24 hours. For light sensitive materials, solution stability in the presence of light shall be established.

5.9 Establishment of System suitability :

System suitability parameter is to be selected based on the criticality of separation between impurities and analyte. In general, resolution factor for the closely eluting compounds is selected as a system suitability requirement. If the separation of impurities and degradants from analyte peak is found to be

satisfactory, there is no need keep a resolution factor as a system suitability parameter. In Such a case, only standard reproducibility and asymmetry of standard peak can be adopted as a system suitability requirement. 

5.10 Placebo interference:

5.10.1 Establishment of non-interference of placebo in the estimation of % dissolution of a particular drug product needs to be done prior to finalizing a detection method i.e HPLC method.

5.10.2 Placebo interference, If the placebo solution exhibits considerable absorbance (more than 2%) In such a case only HPLC detection method will be employed.

5.10.3 In case of capsule dosage form, perform the placebo interference along with the Capsule shell.

5.10.4 Prior to finalizing HPLC detection method, placebo solution shall be injected and ensured that it shows no peak at the retention time of analyte peak.

5.11 Preparation of standard solution and Optimization of standard concentration:

5.11.1 It is generally optimized by preparing the standard in following two different ways

a) Stock in dissolution medium followed by further dilution (if any) in dissolution medium.

b) Stock in suitable solvent (methanol. etc.,) followed by further dilution (if any) in dissolution medium.

5.11.2 If any drug substance is easily soluble in the dissolution medium under consideration then the standard can be prepared as per point (a) of 5.5.1.

5.11.3If the drug substance is not soluble in the dissolution medium under consideration. then the stock can be prepared in suitable solvent  (methanol, etc.,) followed by further dilution in dissolution medium i.e. as per point (b) of 5.5.1, provided it is not precipitating out. This is generally followed for medium such as pH 6.8 SIF and pH 5.0 SDF.

5.11.4 The concentration of standard is optimized to the level of the expected test concentration. However if there are more than one dosage forms for a particular

drug product then a stock standard concentration is fixed, which can be further diluted accordingly so as to get the concentration equivalent to the respective test

concentration for the respective dosage forms.

5.12 Establishment of sink conditions:

5.12.1 Note: Sink condition need not be performed for compendial methods.

5.12.2 Establishment of sink condition is useful in selecting the dissolution medium. A medium in which not less than 1.5 times the highest dose of active ingredient is soluble in the selected amount of dissolution medium (generally the volume is taken as 900 ml) can be considered as passing the requirement of sink condition.

5.12.3 For Sink condition establishment, the dissolution will be done at room temperature(~25°C) and at an RPM of 250.

5.12.4 However, if the acceptance criteria for selecting a dissolution medium based on the sink condition is found to be not passing at room temperature then in such a case the dissolution can be performed at 37.5°C.

5.13 Establishment of Linearity of Detector response:

5.13.1 Establishment of linearity of detector response is essential especially in case of drug substance with very low response. In such a case, it is essential that the peak maxima or the absorption maxima is easily detected by UV detection method at the fixed threshold.

5.13.2 The linearity of detector response is generally established in the range of 25% to150% of the target concentration.

5.13.3 For multiple dosage strengths, the linearity is conducted for 25% of lowest strength to 150% of highest strength.

5.14 Selection of dissolution medium:

5.14.1 Select the medium which meets the following acceptance criteria for sink conditions.  5.14.2   Criteria: Not less than 1.5 times the highest dose of active ingredient will be soluble in selected amount of dissolution medium (Normally the dissolution volume is considered as 900 mL).

5.14.3 Perform dissolution profiles of innovator product in the selected media based on solubility of the drug substance in that medium. Initially obtain profile data on at least 4 media having pH ranging from 1-8 (select 2 media on acidic side and 2 media towards alkaline side with paddle or basket apparatus and at preferably 50 RPM. he media which gives a better discriminating release (about 50% or less at 10 minutes and 100% at 60 minutes or 75 minutes) shall be selected as a media for release and stability testing).

5.14.4 In case, no single medium is giving a discriminating release, then the medium which gives a slightly faster release can also be selected (Eg. About 85% at 10 min, 95% at 20min and 100% at 30 minutes).

5.14.5 In case, no medium is giving 100% release at 60 or 90 minutes, then based on the data obtained, select an interval between 30 - 60 min and fix the specification accordingly.                                                                                                                

5.14.6 Generally media such as pH 2.1 simulated gastric fluid (fasted state), pH 6.8 simulated intestinal fluid (fasted state) and pH 5.0 simulated intestinal fluid (fed state) are selected to simulate the conditions of gastro intestinal tract

5.15 Selection of Apparatus RPM and volume of dissolution media:

5.15.1 Select either paddles/baskets/or other apparatus, initially based on any literature reference (Eg. USP, USPF, SBOA,. Etc.).

5.15.2 If there is no literature reference available, for immediate release products, select paddles apparatus, RPM as 50 and obtain profile data.

5.15.3 For USP apparatus Type I i.e baskets, the initial experiments shall be done at 100RPM. Depending on the release nature, the RPM can be lowered to 50 or

increased to 150. In any case the aim should be to obtain discriminating release.

5.15.4 For USP apparatus Type II i.e paddles, the initial experiments shall be done at 50RPM however if the release is faster, i,e more than 85% at 10 min and 100% at 20 min, then the RPM can be lowered to 25 or if the release is very slow (not releasing 100% even at 120 min) then RPM can be increased to 75.

Note: An RPM of 25 is generally not prescribed for a release and stability testing purpose. But can be used for profile comparison.

5.15.5 Increasing RPM more than 75 using paddles is generally not encouraged unless 100 RPM is proved to be very essential with justification and supporting data. In any case, an RPM greater than 100 is highly discouraged.

5.15.6 Depending upon the nature of profiles, variation in apparatus (baskets, paddles or others) can also be performed to check for a better discriminating profile.

5.15.7 Generally the volume of dissolution medium used is 900 mL however it can be either 750 mL or 1000 mL so as to get a discriminating profiles.

6.0 REFERENCES

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7.0 VERSION HISTORY