Pre-Formula Research Review

Specificity - selectivity of the method

Compare the chromatograms of 6 blank plasma samples of 6 individuals with blank plasma samples mixed with MES standards at LLOQ concentration (50 ng/ml) and IS. On the chromatogram of the standard sample (with MES concentration corresponding to the lowest concentration of the standard curve), the peaks of MES and IS must be clearly identified, completely separated from the impurity peaks. The response of the blank sample at the time coinciding with the retention time of MES must not exceed 20% of the response of MES at LLOQ concentration. The response of the blank sample at the time coinciding with the retention time of IS must not exceed 5% of the response of IS.

Standard curve and linear range

Prepare 8 MES standard samples with concentrations ranging from 50 ng/ml to 30000 ng/ml in plasma. Analyze according to the established procedure. Analyze each concentration 5 times. Determine the correlation between the MES concentration (x) in the sample and the MES/IS peak area ratio.

(y) by linear regression method, using the density coefficient (1/x 2 ). The standard curve must have a correlation coefficient r ≥ 0.95, the accuracy compared to the actual value of the concentration must reach from 85 - 115 %, the point with the lowest concentration of the standard curve (LLOQ) allows the accuracy to be in the range of 80 - 120 % and at least 75% of the points of the standard curve meet the above standard, including the sample with the lowest concentration and the highest concentration.

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Lower limit of quantitation (LLOQ)

Analyze blank plasma samples, plasma samples containing internal standard IS and MES standards with a concentration of about 50 ng/ml (LLOQ samples). Determine the concentration of mesalamine in LLOQ samples from the standard curve conducted in parallel under the same conditions. The standard sample with the lowest MES concentration of the standard curve is considered LLOQ when the MES peak is clearly identified, completely separated from the impurity peaks; the response of the LLOQ sample must be at least 5 times higher than the response of the blank sample at the same retention time; the accuracy must be 80 - 120% compared to the actual concentration and the precision when analyzing at least 5 independent LLOQ samples must be ≤ 20%.

Accuracy, intra-day and inter-day precision

Accuracy and precision were performed on 4 different concentration levels (LLOQ, LQC, MQC, HQC), with concentrations of 50; 150; 2000; 2400 ng/ml respectively. Determination of MES content in the sample using the standard curve method and % ratio

between the concentration determined from the standard curve and the theoretical concentration. Determine the accuracy of the method by comparing the quantified value with the actual value in the sample. Determine the intra-day repeatability by calculating the CV % deviation between the values of the quantification times of each concentration analyzed on the same day. Determine the inter-day repeatability by calculating the CV % deviation between the values of the quantification times of each concentration analyzed on at least 3 different days. The method achieves intra-day and inter-day accuracy and precision when the accuracy at each concentration is in the range of 85 - 115 %, except at the LLOQ point where 80 - 120 % is accepted; intra-day repeatability has a CV % value ≤ 15 %; inter-day repeatability has a CV value ≤ 15 %. Particularly, the LLOQ concentration allows a CV value ≤ 20 %.

Accuracy, precision of the method when diluted

Prepare samples AC1, AC2, AC3 with concentrations of approximately 300, 4000 and 48000 ng/ml respectively. Dilute 2 times, process and analyze the chromatography of those samples. Based on the standard curve conducted under the same conditions, determine the concentration of MES in the diluted samples and then calculate the concentration of MES in the original sample. Determine the accuracy of the method by comparing the quantified value with the actual value in the sample. Determine the precision by calculating the CV % deviation between the values of the quantification times. The method meets the requirements when the accuracy is in the range of 85

- 115% of actual value and CV value ≤ 15 %.

Recovery Rate

IS recovery rate: Process 6 blank plasma samples containing internal standard (concentration 700ng/mL) according to the selected procedure. Analyze and determine the IS peak area. In parallel, determine the IS peak area of 6 internal standard samples mixed in methanol - H2O (50:50) with corresponding concentrations. Conduct chromatography and determine the IS recovery rate by comparing the response of IS in the extracted QC samples - with the response of IS in the standard sample mixed in the sample diluent (not extracted).

MES recovery rate: Prepare 6 samples at each concentration of LQC, MQC, HQC mixed in plasma and mixed in methanol - water (50:50). Process the samples according to the established method. Conduct chromatography, determine the MES recovery rate by comparing the results.

MES response in QC samples with extraction – with MES response in standards diluted in sample diluent (without extraction).

The method of extraction and sample treatment is suitable when the recovery rate of active ingredient is not more than 110% and not less than 30%; the CV % value between the responses of MES, IS in QC samples through extraction at each concentration is ≤ 15%; the CV % value between the responses of MES, IS in QC samples through extraction at each concentration must be ≤ 10%; the average recovery rate at MES concentrations does not differ by more than  15%.

Influence of sample background

Prepare 6 blank plasma samples of different origins, process according to the procedure to obtain the sample matrix solution. Prepare standard samples at LQC and HQC concentrations in the corresponding sample matrix solutions. At the same time, prepare standard samples at LQC and HQC concentrations in methanol - water solvent (50:50). Analyze the chromatography of the above samples. Record the chromatograms and peak responses. The matrix influence factor MF of MES (or IS) is determined by the peak response ratio of MES (or IS) in the sample matrix solution compared to that in methanol - water solvent (50:50). The method meets the requirements when the MF values of MES and IS must be in the range of 0.85 - 1.15; the CV value of the ratio MF MES /MF IS ≤ 15 %

Cross contamination

Prepare 06 blank plasma samples, 06 standard samples mixed in plasma at LLOQ concentration, 06 standard samples mixed in plasma at ULOQ concentration. Inject 06 LLOQ samples and inject blank samples alternately after ULOQ samples. Record the chromatograms and peak responses. The method achieves cross-contamination when the response of the blank sample at the time coinciding with the retention time of MES must not exceed 20% of the response of the LLOQ sample; the response of the blank sample at the time coinciding with the retention time of IS must not exceed 5% of the response of the LLOQ sample.

Stability of the active substance in plasma

Stability of plasma sample after 3 freeze-thaw cycles: Stability study after 3 freeze-thaw cycles was performed on 2 concentrations LQC and HQC. Store the sample at -25º C and thaw at room temperature. After 3 freeze-thaw cycles, analyze to determine the concentration of MESA in the sample. Compare with the results of determining the concentration of MES

present in the samples analyzed immediately after preparation (initial concentration). The MES concentration in the sample after 3 freeze-thaw cycles must differ from the initial concentration by no more than 15% and the CV % value between the quantitative results at each concentration must be less than or equal to 15%.

Short-term stability of plasma samples at room temperature: Analyze plasma samples at 2 concentrations LQC and HQC after thawing and storing at room temperature for a certain period of time, compared with the concentration of the sample processed immediately after thawing. The MES concentration in the sample processed after storing at room temperature for a certain period of time must differ from the concentration of the sample processed immediately by no more than 15% and the CV% value between the quantitative results at each concentration must be less than or equal to 15%.

Stability of sample after processing (in auto-sampler): Compare the concentration of sample at LQC and HQC concentrations stored in auto-sampler after a certain period of time and the concentration of the injected sample immediately after processing. The concentration of sample after a certain period of storage in auto-sampler differs from the concentration of injected sample immediately after processing by no more than 15% and the CV% value between the quantitative results at each concentration must be less than or equal to 15%.

Long-term plasma sample stability: The minimum long-term stability test period must be sufficient to collect blood samples and analyze all plasma samples from the test dogs. Store plasma samples at 2 concentrations LQC and HQC at -25 ± 5ºC, analyze samples at baseline and after each specified storage period. Compare the concentration of the plasma sample after storage with the concentration of the sample at baseline. The MES concentration in the sample after a certain period of storage must differ from the initial concentration by no more than 15% and the CV % value between the quantitative results at each concentration must be less than or equal to 15%.

2.2.4.2. Evaluation of pre-formulation research

Nature

Observe the color of the solution or powder with the naked eye.

Mesalamine content

Conducted similarly to the method for quantifying mesalamine in kernel pellets.

2.2.4.3. Evaluation of pellets

Appearance Evaluation

Observe with the naked eye that the pellets must be intact, uniform in color, spherical in shape and not stuck together.

Pellet preparation efficiency

Pellet preparation efficiency is calculated according to the following formula:

In there:

H% =

x 100%

- m: Mass of pellets obtained with size 0.8 – 1 mm

- M: Initial solid material mass (excluding dissolving solvent)

Flowability

Determination on the machine (ERWEKA GWF) measuring the flow rate of granules and powders with a hopper diameter of 9 mm. The pellet mass used for each measurement is 100 g. The flow rate is calculated according to the formula: v = tgα. In which, v is the flow rate (g/s); α is the angle between the straight line representing the dependence of the mass of the pellet flowing on time and the x-axis (time axis).

Loss on drying

Loss on drying of pellets is determined by the loss on drying method on a Sartorius MA 30 rapid moisture balance. Weigh about 5 g of pellets, grind finely, put 1 g on the balance pan, set the temperature to 105 o C in automatic mode, monitor and read the result.

Determination of mesalamine in kernel pellets

Preparation of test sample: weigh an amount of preparation equivalent to about 400 mg of mesalamine, dissolve in 50 ml of 1 N hydrochloric acid and dilute with water to a concentration of 0.2 mg/ml. Filter through a 0.45 mm pore membrane to obtain the test sample.

Quantification was performed by HPLC under validated conditions (section 2.2.4.1).

Calculate the percentage (g/100g) of mesalamine (C 7 H 7 NO 3 ) content in the sample according to the formula:

𝑅 𝑢 𝑥 𝑚 𝑠 𝑥 𝑃 𝑠 % 𝑥 (1 − 𝐴 𝑠 %)𝑥 𝑉 𝑢 𝑥 𝑀 𝑣

𝑅 𝑠 𝑥 𝑚 𝑢 𝑥 𝑉 𝑠 𝑥 500

- R u /R s : Mesalamine peak area of test solution, standard solution

- P s /A s : Standard content and moisture

- m u /m s : Test mass, standard (mg)

- V u /V s : Dilution factor of test solution, standard

- M v : Average mass of pellet containing 500 mg mesalamine (mg)

In vitro dissolution evaluation of human pellets

The solubility of mesalamine from kernel pellets was assessed using an Erweka dissolution tester with the following parameters:

- Stirring device, stirring speed 50 rpm

- Dissolving environment temperature: 37 ± 0.5 o C

- Dissolution medium: 900 ml of phosphate buffer solution pH 6.8

- Weigh an amount of preparation equivalent to 500 mg of mesalamine into a solubility test cup.

- Sampling time: Take 10ml sample at times 1; 2; 3; 4 and 5 hours, filter and dilute with dissolution medium if necessary. Add 10ml of dissolution medium after sampling.

Prepare a standard solution of 0.025 mg/ml concentration in the dissolution medium. Calculate the amount of dissolved mesalamine by comparing the UV absorbance of the test solution and the standard in pH 6.8 medium at 330 nm wavelength according to the formula



n  1 

n  Vm Vl C i 

In there:

X  i  1   100 + X

n P

X n : % of active ingredient released at time n

C n : Concentration of active ingredient quantified at the time of sampling n (mg/ml)

Ci : Quantitative concentration of active ingredient of sample taken at time points before time point n

Vm: Volume of dissolution medium (ml)

Vl: Sampling volume (ml)

P: Active ingredient content according to label

X: % of active ingredient released into the environment in the previous stage

2.2.4.4. Evaluation of pellet bags

Nature

Observe with the naked eye that the pellets after film coating must be intact, have uniform color, be spherical and not stick together.

Film thickness

Indirectly determined based on the “Crust Volume Ratio” (TLVB), calculated by the formula:

In there:

𝑇𝐿𝑉𝐵 = (

𝑚 2 − 𝑚 1

𝑚 1

) 𝑥100%

- m 1 : Pellet weight before packaging (g)

- m 2 : Pellet weight after wrapping (g)

Determination of mesalamine in pellets

The method for determining mesalamine in film-coated pellets is carried out in the same way as for determining mesalamine in kernel pellets.

In vitro dissolution evaluation of coated pellets

Because the pellet coating film using Eudragit S100 has pH-dependent solubility (soluble at pH

> 7), therefore in vitro dissolution was assessed under 2 conditions using the Erweka dissolution tester:

- Condition 1 [86]

+ Stirring device, speed 50 rpm.

+ Dissolving environment temperature: 37 ± 0.5 o C

+Dissolution test medium: 900 ml of 0.1 N hydrochloric acid solution (pH 1.2) and phosphate buffer pH 6.8.

+ Weigh an amount of preparation equivalent to 500 mg of mesalamine into a solubility test cup, test at pH 1.2 for 2 hours, then replace the solution.

Liquid in beaker with pH 6.8 buffer solution and test in the following hours.

+ Sampling time: Take 10 ml after 1; 2; 3; 4; 5; 6; 7 and 8 hours, filter and dilute with dissolution medium if necessary. Add 10 ml of dissolution medium after sampling

- Condition 2 [104], [89]

+ Stirring device, speed 50 rpm.

+ Dissolving environment temperature: 37 ± 0.5 o C

+ Dissolving medium: 900 ml of 0.1 N hydrochloric acid solution (pH 1.2), phosphate buffer pH 7.4 and phosphate buffer pH 6.8.

+ Weigh an amount of preparation equivalent to 500 mg of mesalamine into a solubility test cup, test in a pH 1.2 environment for 2 hours, then replace the liquid in the cup with a pH 7.4 phosphate buffer solution, after 3 hours of testing in this environment, replace the liquid in the cup with a pH 6.8 buffer solution and test in the following hours.

+ Sampling time: Take 10 ml after 2; 3; 4; 5; 7; 9 and 11 hours, filter and dilute with dissolution medium if necessary. Add 10 ml of dissolution medium after sampling.

Prepare a standard solution of 0.025 mg/ml concentration in dissolution medium. Calculate the amount of mesalamine released by comparing the UV absorbance of the test solution and the standard in pH 1.2 medium at 301 nm; pH 6.8 and pH 7.4 at 330 nm according to the formula



n  1 

n  Vm Vl C i 

In there:

X  i  1   100 + X

n P

X n : % of active ingredient released at time n

C n : Concentration of active ingredient quantified at the time of sampling n (mg/ml)

Ci : Quantitative concentration of active ingredient of sample taken at time points before time point n