Method Development, Validation, And Degradation Studies Of Fosdenopterin

Studying anti-cancer medications in this laboratory over the last five years, the author has worked on developing new techniques to monitor their stability. An overview of pharmaceutical manufacturing in terms of quality maintenance, the requirement for stability-indicating test techniques, and validation will be covered thoroughly before the experimental procedures used and the findings obtained are described. Pemetrexed, erlotinib hydrochloride, nilotinib hydrochloride, and dasatinib hydrochloride are among the anticancer medications chosen for stability suggesting studies.

The quality of medications on the market has been the subject of significant scrutiny in recent years. Both the pharmaceutical and the bulk drug businesses face the problem of producing high-quality goods. Each industry's production must be subjected to stringent quality control tests if it is to remain of high quality and pure. Raw ingredients, manufacturing methods, and the kind of crystallisation and purifying procedure all have a role in determining the active medicinal ingredient's purity. Analytical chemistry progress is inextricably linked to modifications in the purity concept. The pharmacopoeias establish purity as well as impurity restrictions, some of which may be quite strict. Because they separate and measure components concurrently, modern separation technologies are obviously dominating in scientific research today. This makes it simpler to separate and characterise contaminants. Unwanted compounds that persist in pharmaceuticals or emerge during formulation or develop as a result of ageing of both the active pharmaceutical ingredients (APIs) and formed APIs to medications (1-4). Even at minute concentrations, these unwelcome substances may impair the effectiveness and safety of pharmaceuticals. Impurity levels in APIs and drug formulations are gradually being regulated by many pharmacopoeias, including the British Pharmacopoeia (BP) and the United States Pharmacopoeia (USP). Impurities in novel medicinal compounds, products, and residual solvents have been outlined by the International Conference on Harmonization (ICH) (5-7). One way to describe the contaminants in an API batch is to describe the recognised and undiscovered impurities present in that batch

2. MATERIALS AND METHODS

1. Equipment:

Table No.1: List of Apparatus used in HPLC

(b) Reagents & Chemicals

Table No.2: List of chemicals used in HPLC Method

Determination of Working Wavelength (λ_max):

In estimation of the drug isobestic wavelength was used. Isobestic point is the wavelength where the molar absorptivity is the same for the substances that are inter convertible. So this wavelength was used in estimation of drug accurately.

The wavelength of maximum absorption of the solution of the drug in mixture of Acetonitrile and 0.1% OPA (30:70) were scanned using PDA Detector within the wavelength region of 200–400 nm against Acetonitrile and 0.1% OPA (30:70) as blank. The absorption curve shows isobestic point at 220 nm. Thus 220 nm was selected as detector wavelength for the HPLC chromatographic method.

Chromatographic conditions:

During the selection of chromatographic conditions, numbers of trails were carried out and the best trail was selected for optimized method.

Preparation of standard solution

Accurately weigh and transfer 50 mg of Fosdenopterin working standard into a 100 ml clean dry volumetric flask add diluent and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution)

Further pipette 5 ml of the above stock solutions into a 50 ml volumetric flask and dilute up to the mark with diluent. (50ppm of Fosdenopterin)

Preparation of Sample solution

Accurately weigh and transfer 50 mg of Fosdenopterin sample into a 100 ml clean dry volumetric flask add diluent and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution)

Further pipette 5 ml of the above stock solutions into a 50 ml volumetric flask and dilute up to the mark with diluent. (50ppm of Fosdenopterin)

Trails in optimization of chromatographic condition

Table No.3: TRIAL-1 Chromatographic conditions

Table No.4: TRIAL-2 Chromatographic conditions

Table No.5: TRIAL-3 Chromatographic conditions

Table No.6: TRIAL-4 Chromatographic conditions

Table No.7: TRIAL-5 Chromatographic conditions

Table No.8: TRIAL-6 Chromatographic conditions (Optimized)

The Fosdenopterin peak was observed at 4.358 min with peak area 3239455, tailing factor 1.05. This trial was optimized.

General preparations

Preparation of Mobile Phase: Mobile phase was prepared by mixing 0.1% OPA and ACN taken in the ratio 70:30. It was filtered through 0.45μ membrane filter to remove the impurities which may interfere in the final chromatogram.

Chromatographic condition:

Use suitable High Performance Liquid Chromatographic equipped with PDA detector.

Column                                   :    X-Bridge Phenyl (250x4.6mm, 5µ)

Mobile phase ratio                  :   Acetonitrile and 0.1% OPA   (30:70)

Detection wavelength             :   220 nm

Flow rate                                  :   1ml/min

Injection volume                     :    10µl

Run time                                 :    6min

Preparation of Diluent: Mobile phase was used as a diluent.

Preparation of standard solution

Accurately weigh and transfer 50 mg of Fosdenopterin working standard into a 100 ml clean dry volumetric flask add diluent and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution)Further pipette 5 ml of the above stock solutions into a 50 ml volumetric flask and dilute up to the mark with diluent. (50ppm of Fosdenopterin)

Preparation of Sample solution

Accurately weigh and transfer 50 mg of Fosdenopterin sample into a 100 ml clean dry volumetric flask add diluent and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution)Further pipette 5 ml of the above stock solutions into a 50 ml volumetric flask and dilute up to the mark with diluent. (50ppm of Fosdenopterin)

Procedure:

Inject 10 mL of the standard, sample into the chromatographic system and measure the areas for Fosdenopterin peak and calculate the %Assay by using the formulae.

SYSTEM SUITABILITY:

Tailing factor for the peak due to Fosdenopterin in Standard solution should not be more than 2.0 Theoretical plates for the Fosdenopterin peak in Standard solution should not be less than 2000.

Formula for Assay:  
% Assay=ATAS*WSDS*DTWT*Average weightLabel Claim*P100*100

Where:            AT       = average area counts of test (sample) preparation.

AS       = average area counts of standard preparation.

WS      = Weight of working standard taken in mg.

DS       = Dilution of working standard in ml.

DT       = Dilution of test (sample) in ml.

WT      = Weight of test (sample) taken in mg.

P          = Percentage purity of working standard

LC       = Label Claim mg/ml.

METHOD VALIDATION SUMMARY:

Specificity:

Specificity of an analytical method is ability to measure specifically the analyte of interest without interference from blank and known impurities. For this purpose blank chromatogram, standard chromatogram and sample chromatogram were recorded. The chromatogram of blank shows no response at the retention times of drugs which confirms the response of drug was specific.

LINEARITY:

Preparation of stock solution:

Accurately weigh and transfer 50mg of Fosdenopterin working standard into a 100 ml clean dry volumetric flask add diluent and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution)

Preparation of Level – I (12.5ppm of Fosdenopterin):

1.25 ml of above stock solutions has taken in different 50 ml of volumetric flasks, dilute up to the mark with diluent.

Preparation of Level – II (25ppm of Fosdenopterin):

2.5 ml of above stock solutions has taken in different 50 ml of volumetric flasks, dilute up to the mark with diluent.

Preparation of Level – III (37.5ppm of Fosdenopterin):

3.75 ml of above stock solutions has taken in different 50 ml of volumetric flasks, dilute up to the mark with diluent.

Preparation of Level – IV (50ppm of Fosdenopterin):

5 ml of above stock solutions has taken in different 50 ml of volumetric flasks, dilute up to the mark with diluent.

Preparation of Level –V (62.5ppm of Fosdenopterin ):

6.25 ml of above stock solutions has taken in different 50 ml of volumetric flasks, dilute up to the mark with diluents.

Preparation of Level – VI (75ppm of Fosdenopterin)

7.5 ml of above stock solutions has taken in different 50 ml of volumetric flasks, dilute up to the mark with diluent.

Procedure:

Inject each level into the chromatographic system and measure the peak area.

Plot a graph of peak area versus concentration (on X-axis concentration and on Y-axis Peak area) and calculate the correlation coefficient.

Range:

The Range of an analytical method is the interval between the upper and lower levels of analyte (including these levels) that have been demonstrated with precision, accuracy and linearity

Acceptance Criteria:

Correlation coefficient should be not less than 0.999.

Preparation Accuracy Sample solutions:

For preparation of 50% solution (With respect to target Assay concentration):

Accurately weigh and transfer 25mg of Fosdenopterin sample into a 100 ml clean dry volumetric flask add diluent and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution)

Further pipette 5 ml of the above stock solutions into a 50ml volumetric flask and dilute up to the mark with diluent. (25ppm of Fosdenopterin)

For preparation of 100% solution (With respect to target assay concentration):

Accurately weigh and transfer 50 mg of Fosdenopterin sample into a 100 ml clean dry volumetric flask add diluent and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution)

Further pipette 5ml of the above stock solutions into a 50ml volumetric flask and dilute up to the mark with diluent. (50ppm of Fosdenopterin)

For preparation of 150% solution (With respect to target assay concentration):

Accurately weigh and transfer 75mg of Fosdenopterin sample into a 100 ml clean dry volumetric flask add diluent and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution)

Further pipette 5 ml of the above stock solutions into a 50ml volumetric flask and dilute up to the mark with diluent. (75ppm of Fosdenopterin)

Procedure:

Inject the standard solution, Accuracy -50%, Accuracy -100% and Accuracy -150% solutions.

Acceptance Criteria:

The % Recovery for each level should be between 98.0 to 102.0%

Precision

Precision is the degree of repeatability of an analytical method under normal operation conditions. Precision is of 3 types

1. System precision

2. Method precision

3. Intermediate precision (a. Intra-day precision, b. Inter-day precision)

System precision is checked by using standard chemical substance to ensure that analytical system is working properly. In this peak area and % of drug of six determinations measured and % RSD should be calculated.

In method precision, a homogenous sample of single batch should be analyzed 6 times. This indicates whether a method is giving constant results for a single batch. In this analyze the sample six times and calculate the % RSD.

The precision of the instrument was checked by repeatedly injecting (n=6) solutions of 50ppm of Fosdenopterin).

Acceptance Criteria:

The % RSD for the absorbance of six replicate injections results should not be more than 2%.

ROBUSTNESS:

As part of the Robustness, deliberate change in the Flow rate, Mobile Phase composition, Temperature Variation was made to evaluate the impact on the method.

A. The flow rate was varied at 0.8 ml/min to 1.2ml/min.

Standard solution 50ppm of Fosdenopterin was prepared and analysed using the varied flow rates along with method flow rate. On evaluation of the above results, it can be concluded that the variation in flow rate affected the method significantly. Hence it indicates that the method is robust even by change in the flow rate ±20%.

B. The variation of Organic Phase ratio.

Standard solution of 50ppm of Fosdenopterin was prepared and analysed using the varied in mobile phase ratio.

Limit of detection (LOD) and limit of quantification (LOQ):

The limit of detection (LOD) limit of quantification (LOQ) of the drug carry was calculated using the following equation as per international conference harmonization (ICH) guidelines.

LOD = 3.3 X σ /S

LOQ = 10 X σ /S

LOD for Fosdenopterin was found to be 1.5µg/mL and LOQ for Fosdenopterin was found to be 5µg/ml.

DEGRADATION STUDIES:

Preparation of stock:

Accurately weigh and transfer 50mg of Fosdenopterin working standard into a 100 ml clean dry volumetric flask add diluent and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution)

Acid degradation:

Pipette 5 ml of above solution into a 50ml volumetric flask and 3 ml of 1N HCl was added. Then, the volumetric flask was kept at 60ºC for 6 hours and then neutralized with 1 N NaOH and make up to 50ml with diluent. Filter the solution with 0.22 microns syringe filters and place in vials.

Alkali degradation:

Pipette 5 ml of above solution into a 50ml volumetric flask and add 3ml of 1N NaOH was added. Then, the volumetric flask was kept at 60ºC for 6 hours and then neutralized with 1N HCl and make up to 50ml with diluent. Filter the solution with 0.22 microns syringe filters and place in vials.

Peroxide degradation

Pipette 5 ml above stock solution  into a 50ml volumetric flask, 1 ml of 3% w/v of hydrogen peroxide added in 50 ml of volumetric flask and the volume was made up to the mark with diluent. The volumetric flask was then kept at room temperature for 15 min. Filter the solution with 0.45 microns syringe filters and place in vials.

Reduction degradation

Pipette 5ml of Stock solution transferred into 50ml volumetric flask to this add 1ml of 10% Sodium Bisulphate and kept on bench top for 10min then the remaining procedure is same as the test preparation.

Hydrolysis degradation

Pipette 5ml above stock solution  into a 50ml volumetric flask, 1 ml of water added in 50 ml of volumetric flask and the volume was made up to the mark with diluent. The volumetric flask was then kept at room temperature for 15 min. Filter the solution with 0.45 microns syringe filters and place in vials.

3. RESULTS AND DISCUSSION

3.1 RP-HPLC METHOD

Determination of Working Wavelength (λ_max):

Fig No.:1PDA - Spectrum of Fosdenopterin

3.2.      Optimization of chromatographic conditions

Fig No-2: chromatogram of Trial-1

Fig No-3: chromatogram of Trial-2

Fig No-4: chromatogram of Trial-3

Fig No-5: chromatogram of Trial-4

Fig No-6: chromatogram of Trial-5

Fig No-7: chromatogram of Trial-6

Table 11: Optimized chromatographic conditions

Specificity:

Fig No.10: Chromatogram of blank

Fig No.11: Chromatogram of placebo.

Fig No.12: Chromatogram of standard

Fig No.13: Chromatogram of sample

ANALYTICAL METHOD VALIDATION (HPLC)

The method was validated for its linearity range, accuracy, precision, and specificity. Mevalidation was carried out as per ICH guidelines.

Linearity:

Fig No.14: Chromatogram of Linearity-1

Fig No.15: Chromatogram of Linearity-2

Fig No.16: Chromatogram of Linearity-3

Fig No.17: Chromatogram of Linearity-4

Fig No.18: Chromatogram of Linearity-5

Fig No19: Chromatogram of Linearity-6

Table No.12: Results of linearity for Fosdenopterin

Fig No.20: Calibration curve for Fosdenopterin at 220 nm

Accuracy:

Table No.13: Accuracy results of Fosdenopterin by RP-HPLC method

Fig No.21: Chromatogram for Accuracy 50%

Fig No.22: Chromatogram for Accuracy 100%

Fig No.23: Chromatogram for Accuracy 150%

Precision:

Table No.14: Standard results for Fosdenopterin by RP-HPLC method

Table No.15: Method Precision for Fosdenopterin by RP-HPLC method

Fig No.24: Chromatogram of Method Precision

Acceptance Criteria: The % RSD for the area of six standard injections results should not be more than 2%.

Table No.16: Intermediate Precision for Fosdenopterin by RP-HPLC method

Fig No.25: Chromatogram of Intermediate Precision

Acceptance Criteria: The % RSD for the area of six standard injections results should not be more than 2%.

Robustness:

Table No.17: Robustness results of Fosdenopterin by RP-HPLC

Fig No.26: chromatogram for less flow rate (0.8 ml)

Fig No.27: chromatogram for more flow rate (1.2mL)

Fig No.28: chromatogram for less Organic Phase (27:73)

Fig No.29: chromatogram for more Organic Phase (33:67)

LOD and LOQ:

Table No.18: Sensitivity parameters (LOD & LOQ) by RP-HPLC

Fig No.30: chromatogram of LOD

Fig No.31: chromatogram of LOQ

DEGRADATION STUDIES:

Fig No.32: Chromatogram of Control degradation

Fig No.33: Chromatogram of Acid degradation

Fig No.34: Chromatogram of Alkali degradation

      Fig No.35: Chromatogram of Peroxide degradation

Fig No.36: Chromatogram of Reduction degradation

Fig No.37: Chromatogram of Hydrolysis degradation

Table No.19: Forced Degradation results for Fosdenopterin