VALIDATED ANALYTICAL METHOD DEVELOPMENT AND CHARACTERIZATION OF FORCED DEGRADATIVE PRODUCTS OF MITAPIVAT AND THEIR TOXICITY PREDICTION BY IN-SILICO STUDIES

VALIDATED ANALYTICAL METHOD DEVELOPMENT AND CHARACTERIZATION OF FORCED DEGRADATIVE PRODUCTS OF MITAPIVAT AND THEIR TOXICITY PREDICTION BY IN-SILICO STUDIES

S. K. Mastanamma *, Sunitha Chitturi, MD. Naseemunnisa and V. Anitha Kumari

Department of Pharmaceutical Analysis, Anu College of Pharmaceutical Sciences, Acharya Nagarjuna University, Guntur, Andhra Pradesh, India.

ABSTRACT: The current study reports to validate a stability indicating RP-HPLC method for estimation of mitapivat in bulk, pharmaceutical formulations and FD characterization by using MS method. The chromatographic separation was accomplished on Waters X-Bridge phenyl column (150× 4.6mm, 3.5µm). With the mobile phase consisting of acetonitrile and 0.1% OPA (80ː20v/v) at a flow rate of 1ml/min. The eluents were monitored by UV detector at 234nm. The developed liquid chromatographic method was validated with respect to accuracy, precision, linearity, robustness, range, limit of detection (LOD), limit of quantification (LOQ). The drug was subjected to various stress conditions such as acid, alkali, peroxide, reduction, photolytic, hydrolysis and thermal degradation. Among all the stress condition 5degradation products were obtained they were DP-1, Dp-2, Dp-3, Dp-4, Dp-5. They were subjected to mass characterization. The obtained structures were subjected to in-silico studies using Swiss ADME, pk CSM web server, protox2. The SWISS ADME web server and pk CSM web server were used for the prediction of pharmacokinetic properties and toxicity of the drug and its degradation products. The drug and the DP-1 don’t show any toxicity. Whereas DP-2, DP-3, DP-4, DP-5 shows immune toxicity. Hence the method was used for impurity profiling for mitapivat.

Keywords: RP-HPLC, Mitapivat, Mass characterization, Degradation products, In-silico studies

INTRODUCTION: Mitapivat N-(4-{[4-(Cyclo-propylmethyl) - 1-piperazinyl] carbonyl} phenyl)-8-quinoline sulfonamide. Mitapivat   belongs to a group of drugs known as pyruvate kinase activators. In patients with pyruvate kinase deficiency, mitapivat is used to treat hemolytic anemia, a blood condition which happens when red blood cells are destroyed more rapidly than they can be generated within the body.

The FDA has approved it   under the Pyrukynd (India) brand in a range of strengths starting at 5 mg ¹.  Literature survey revealed that few analytical techniques have been published individually or in combination with other drugs ^2-6. However, no method was reported for estimation of drug by using   HPLC method. The development of an established stability indicating RP-HPLC has been attempted. LC-MS/MS method for estimating Mitapivat and FD Characterization; in-silico investigations using soft ware’s to determine their toxicity ¹.

MATERIALS AND METHODS:

Instrument: A Waters Alliance e-2695 type HPLC with a column oven, auto sampler, and degasser was utilized for the analysis. The HPLC system was linked to the SCIEX QTRAP 5500 mass spectrometer, which features an electro spray ionization interface. The SCIEX program was utilized to the interpretation of the chromatogram's data.

The interface mode of the mass spectrometer has been set to positive ion electrospray ionization. To measure the Mitapivat, a multiple reactions monitoring mode has been implemented. Working Parameters have been set as follows:

• Collision energy: 13V
• Ion spray voltage: 5500V
• Source temperature: 550^oC
• Drying gas temperature: 120-250°C
• Collision gas: nitrogen
• Drying gas flow stream: 5 mL/min
• Declustering potential: 40V
• Entrance potential: 10V
• Exit Potential: 7V
• Dwell time: 1sec

Drug Samples: Working standards of Mitapivat were obtained as a gift samples from Shree icon laboratories, Vijayawada, India. Commercially available Mitapivat tablets were purchased from local pharmacy

Determination of Working Wavelength (λmax): The PDA Detector was used to scan the wavelength between 200–400 nm for the highest absorption of the drug solution in the mixture of acetonitrile and 0.1% OPA (80:20) versus acetonitrile and 0.1% OPA (80:20) as a blank. Maximum absorption was demonstrated by the absorption curve at 234 nm. Therefore, the HPLC chromatographic process used 234 nm was the detection wavelength.

Standard Solution Preparation: Weigh and transfer 8 mg of the working standard Mitapivat accurately into a 10 ml dry volumetric flask. Then, add the diluent and sonicate to completely dissolve it. Use the same solvent to bring the volume up to the desired level. (Stock solution) Moreover pipette out 1ml of the above stock solutions into a10ml volumetric flask and diluting to the appropriate mark with diluent (80ppm of Mitapivat).

Sample Solution Preparation: Take 20 commercially available marketed tablets, accurately weighed and further transferred into mortor and pestle. Titurated it to fine powder. And take 50mg equivalent  of powder in to a volumetric flask, add diluent and sonicate it up to 30min to dissolve, and centrifuge for 30 min and finally make up to the mark with the same diluent. Then it was filtered through 0.45µ injection filter. Further pipette 0.8 ml of the above stock solutions into a10mlvolumetric flask and dilute up to the mark with diluent. (80ppm of Mitapivat).

The Mitapivat peak was observed at 2.855 min with peak area 3161273, tailing factor 0.90. This trial was optimized.

Preparation of 0.1% OPA Buffer Solution: 1ml of Ortho Phosphoric acid is dissolved in1litre of HPLC grade water and filter through 0.45µ nylon filter.

Preparation of Mobile Phase: The mobile phase was developed by combining 0.1% OPA with 20:80 ACN. A 0.45μ membrane filter was used to filter it out of any impurities that would have affected the final chromatogram.

Chromatographic Condition:

Use Waters Acquity HPLC:

Column: Waters X- Bridge phenyl (150x4.6mm, 3.5µm) Mobile phase ratio: Acetonitrile and 0.1% OPA (80:20)

Detection wavelength: 234nm

Flow rate:  1ml/min

Injection volume: 10µl

Runtime: 5min

Assay Procedure: Measure the area of the Mitapivat peak after injecting 10 uL of the standard into the chromatographic apparatus. Then, use the formulas to get the assay percentage.

Formula for Assay:

%Assay = AT/AS*WS/DS* DT/WT* Average weight /Label claim*P/100*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

System Suitability: The theoretical plates for the Mitapivat peak in Standard solution should not be less than 2000. The tailing factor for the peak due to Mitapivat in Standard solution should not be more than 2.0.

Specificity: The ability of an analytical method to quantify an analyte of interest precisely without affect from known and blank contaminants is known as specificity. Blank, standard, sample chromatograms were recorded for this purpose. The fact that the blank chromatogram exhibits no reaction during the drug retention periods indicates that APA response was specific.

Linearity: A series of aliquots were prepared ranging from 20µg/ml to 120µg/ml were prepared using mobile phase from the stock solution. The peak area should be measured after six injections of each concentration into the HPLC apparatus. Graph the relationship between peak area and concentration (on X-axis concentration and on Y-axis Average Peak area) and measure the correlation coefficient.

Range: The interval between the highest and lower levels of analyte (including these levels) that have been proven with precision, accuracy, and linearity is known as the analytical method's range.

Accuracy: For recovery studies 50% 100% 150% solution concentrations were prepared by adding standard solution to pre analysed concentration using standard addition method. The prepared concentrations were then injected into the HPLC system 3 times each and their peak areas and %RSD were calculated.

Precision: The degree of repeatability that an analytical method exhibits under typical operating conditions is known as precision. There are three different types of precision.

1. System precision
2. Method precision
3. Intermediate precision (a. Intra-day precision, b. Inter day precision)

To make sure the analytical system is operating correctly, system precision is verified using a standard chemical substance. The percentage RSD should be calculated in this peak area, where the drug percentage of the six determinations is measured. A homogeneous sample from a single batch should be examined six times for method precision. This shows how a procedure is producing consistent outcomes for a particular batch. This involves six analyses of the sample to determine the percentage RSD. Six solutions of 80 ppm Mitapivat were injected repeatedly to test the instrument's precision.

Robustness: Deliberate changes were made to the Flow rate, Mobile Phase composition, and Temperature Variation as part of the Robustness to assess the effect on the procedure. A range of 0.98 ml/min to 1.02 ml/min was observed for the flow rate. With the use of method flow rate and a variety of flow rates, a standard solution containing 80 ppm of mitapivat was created and examined. It is clear from analyzing the previous information that the approach was greatly impacted by the flow rate change. That means that even with a ±2% deviation from the flow rate, the method remains stable. The change in the ratio of the Organic Phase. Using various kinds of mobile phase ratios, a standard solution containing 80 ppm of mitapivat was produced and analyzed.

Limit of Detection (LOD) and Limit of Quantification (LOQ): The following formula was used to determine the drug performs limit of detection (LOD) and limit of quantification (LOQ) in accordance with international conference harmonization (ICH) standards.

LOD =3.3 × σ / S LOQ = 10 X σ / S

Mitapivat's LOD was determined to be 0.48µg/mL and its LOQ to be 1.6µg/ml.

Degradation Studies:

Acid Degradation: Pipette 1 ml of above solution into a 10ml volumetric flask and1 ml of 1N HCl was added. Then, the volumetric flask was kept at 60ºC for 1 hour in water bath and then neutralized with 1 N NaOH  was added and make up to 10ml with diluents.

Alkali Degradation: Pipette 1ml of above solution into a 10ml volumetric flask and add 1ml of 1NNaOH was added. Then, the volumetric flask was kept at 60ºC for1 hour in water bath and then neutralized with 1N HCl and make up to 10ml with diluent.

Peroxide Degradation: Pipette 1 ml above stock solution was added to a 10 ml volumetric flask, 1 ml of 3 percent w/v hydrogen peroxide was added to the flask and the volume was built up to the mark using diluent. The volumetric flask was then maintained at 60^oC for 1 hour in water bath. After that, the volumetric flask was left at room temperature for 15 minutes.

Reduction Degradation: Pipette 1 ml above stock solution was added to a10 ml volumetric flask, 1 ml of 10 percent w/v Sodium bi sulphate was added to the flask and the volume was built up to the mark using diluent. The volumetric flask was then maintained at 60^oC for 1 hour in water bath. After that, the volumetric flask was left at room temperature for 15 minutes.

Hydrolysis Degradation: Pipette 1ml of above-stock solution was added to a 10ml volumetric flask, 1ml of HPLC grade water was added to a flask and the volume was built up to the required volume with diluent. The volumetric flask was then maintained at 60^oC for1 hour in water bath. After that, the volumetric flask was left at room temperature for 15 minutes.

Photolytic Degradation: Mitapivat sample and control wrapped with aluminum foil was placed in photostability chamber for 3 hours. Subsequently, the material was extracted, diluted using diluents, and introduced into an HPLC for analysis.

Thermal Induced Degradation: A mitapivat sample was collected in a petridish and heated to 105°C in a hot air oven for three hours. Subsequently, the material was extracted, diluted using diluents, and introduced into an HPLC for analysis. A 0.22u syringe was used to filter the fluid through the strained samples.

In-silico screening of Forced Degradation Products of Mitapivat ²:

 Software used in In-silico Study of Mitapivat:

1. SwissADME
2. Protox2
3. pKcsm

All the degradation products obtained from the degradation of Mitapivat were screened for toxicity and ADME properties using Swiss ADME, Protox 2, pKcsm².

RESULTS AND DISCUSSION:

Collision-Induced Dissociation of Mitapivat:

Mitapivat: Fig 16(B) shows the fragmentation mechanism of Mitapivat and the ESI spectrum showed the most intense [M+H]+ ion of m/z-450.1726. The MS/MS spectrum of Mitapivat displayed abundant product ions at m/z-355.0991 (loss of C6H13N from m/z-450.1726), m/z- 228.0569 (loss of C9H7N from m/z 355.0991), m/z-149.0841 (loss of H3NO2S from m/z 228.0569) and m/z-54.0470 (loss of C5H9NO from m/z 149.0841). The MS/MS experiments combined with accurate mass measurements have confirmed the proposed

DP1: The fragmentation mechanism of DP1 has been shown in Fig. 17(B), and the most intense [M+H]+ ion of m/z-486.1492 was seen in the ESI spectrum under conditions of acid degradation. Abundant product ions were observed in the DP1 MS/MS spectra at m/z-348.0335 (loss of C8H16N2 from m/z-486.1492), m/z-193.0197 (loss of C7H5ClNO- from m/z 348.0335) and m/z-129.0578 (loss of SO2 from m/z 193.0197). The suggested scheme has been validated by the MS/MS tests in conjunction with precise mass measurements.

DP2: The fragmentation mechanism of DP2 has been shown in Fig. 18(B), and the most intense [M+H]+ ion of m/z-472.1545 was seen in the ESI spectrum under conditions of alkali degradation. Abundant product ions were observed in the DP2 MS/MS spectra at m/z-334.0388 (loss of C8H16N2 from m/z-472.1545), m/z-193.0197 (loss of C7H6NNaO from m/z 334.0388) and m/z-129.0578 (loss of SO2 from m/z 193.0197). The suggested scheme has been validated by the MS/MS tests in conjunction with precise mass measurements.

DP3: The fragmentation mechanism of DP3 has been shown in Fig. 19(B) and the most intense [M+H]+ ion of m/z-466.1675 was seen in the ESI spectrum under conditions of peroxide degradation. A lot of product ions were seen in the DP3 MS/MS spectra at m/z-328.0518 (where C8H16N2 was lost from m/z-466.1675), m/z-193.0197 (where C7H7NO2 was lost from m/z 328.0518), and m/z-129.0578 (where SO2 was lost from m/z 193.0197). The suggested scheme has been validated by the MS/MS tests in conjunction with precise mass measurements.

DP4: The fragmentation mechanism of DP4 has been shown in Fig. 20(B) and the most intense [M+H]+ ion of m/z-400.2263 was seen in the ESI spectrum under conditions of thermal deterioration. A lot of product ions were seen in the DP4 MS/MS spectra at m/z-262.1106 (where C8H16N2 was lost from m/z-400.2263), m/z-135.0684 (where C9H7N was lost from m/z 262.1106), and m/z-56.0262 (where C5H9N was lost from m/z 135.0684). The suggested scheme has been validated by the MS/MS tests in conjunction with precise mass measurements.

DP5: The fragmentation mechanism of DP5 has been shown in Fig. 21(B), and the most intense [M+H]+ ion of m/z-466.1675 was seen in the ESI spectrum under conditions of hydrolysis degradation.

A lot of product ions were seen in the DP5 MS/MS spectra at m/z-328.0518 (where C8H16N2 was lost from m/z-466.1675), m/z-193.0197 (where C7H7NO2 was lost from m/z 328.0518) and m/z-129.0578 (where SO2 was lost from m/z 193.0197). The suggested scheme has been validated by the MS/MS tests in conjunction with precise mass measurements.

TABLE 1: OPTIMIZED CHROMATOGRAPHIC CONDITIONS

TABLE 2: SYSTEM SUITABILITY PARAMETERS FOR MITAPIVAT

TABLE 3: ASSAY OF MITAPIVAT

TABLE 4: RESULTS OF LINEARITY FOR MITAPIVAT

TABLE 5: SYSTEM PRECISION TABLE OF MITAPIVAT

TABLE 6: METHOD PRECISION FOR MITAPIVAT

TABLE 7: INTERMEDIATE PRECISION (DAY VARIATION) FOR MITAPIVAT  

TABLE 8: ACCURACY RESULTS OF MITAPIVAT

TABLE 9: ROBUSTNESS RESULTS OF MITAPIVAT

TABLE 10: SENSITIVITY PARAMETERS (LOD & LOQ)

TABLE 11: FORCED DEGRADATION RESULTS OF MITAPIVAT

TABLE 12: LC-MS/MS DATA OF MITPIVAT AND ITS DEGRADATION PRODUCTS AND SOME MAJOR FRAGMENTS

TABLE 13: ADME PREDICTION (SWISS ADME)

TABLE 14: PROTOX2 PREDICTION

TABLE 15: PKCSM PREDICTION

FIG. 1: MOLECULAR STRUCTURE OF MITAPIVAT

FIG. 2: PDA- SPECTRUM OFMITAPIVAT

FIG. 3: OPTIMIZED CHROMATOGRAM

FIG. 4: CHROMATOGRAM OF FORMULATION

FIG. 5: CHROMATOGRAM OF BLANK

FIG. 6: CHROMATOGRAM OF PLACEBO

FIG. 7: CALIBRATION CURVE FOR MITAPIVAT AT 259NM

FIG. 8(A): CHROMATOGRAM OF ACID DEGRADATION

Degradation Studies:

FIG. 8(B): PURITY PLOT OF ACID DEGRADATION

FIG. 9 (A): CHROMATOGRAM OF ALKALI DEGRADATION

FIG. 9(B): PURITY PLOT OF ALKALI DEGRADATION

FIG. 10(A): CHROMATOGRAM OF PEROXIDE DEGRADATION

FIG. 10(B): PURITY PLOT OF PEROXIDE DEGRADATION

FIG. 11(A): CHROMATOGRAM OF REDUCTION DEGRADATION

FIG. 11(B): PURITY PLOT OF REDUCTION DEGRADATION

FIG. 12(A): CHROMATOGRAM OF HYDROLYSIS DEGRADATION

FIG. 12(B): PURITY PLOT OF HYDROLYSIS DEGRADATION

FIG. 13(A): CHROMATOGRAM OF THERMAL DEGRADATION

FIG. 13(B): PURITY PLOT OF THERMAL DEGRADATION

FIG. 14(A): CHROMATOGRAM OF PHOTOLYTIC DEGRADATION

FIG. 14 (B): PURITY PLOT OF PHOTOLYTIC DEGRADATION

FIG. 15(A): MASS SPECTRA OF MITAPIVAT

FIG. 15(B): FRAGMENTATION PATHWAY OF MITAPIVAT

FIG. 16 (A): MASS SPECTRA OF ACID IMPURITY (DP-1)

FIG. 16(B): FRAGMENTATION PATH WAY OF ACID IMPURITY (DP-1)

FIG. 17 (A): MASS SPECTRA OF ALKALI IMPURITY (DP-2)

FIG. 17(B): FRAGMENTATION PATHWAY OF ALKALI IMPURITY (DP-2)

FIG. 18 (A): MASS SPECTRA OF PEROXIDE IMPURITY (DP-3)

FIG. 18(B): FRAGMENTATION PATHWAY OF PEROXIDE IMPURITY (DP-3)

FIG. 19(A): MASS SPECTRA OF THERMAL IMPURITY (DP-4)

FIG. 19(B): FRAGMENTATION PATHWAY OF THERMAL IMPURITY (DP-4)

FIG. 20(A): MASS SPECTRA OF HYDROLYSIS IMPURITY (DP-5)

FIG. 20(B): FRAGMENTATION PATH WAY OF HYDROLYSIS IMPURITY (DP-5)

FIG. 21: REACTION OF MITAPIVAT WITH HCL

FIG. 22: REACTION OF MITAPIVAT WITH NAOH

FIG. 23: REACTION OF MITAPIVAT WITH H2O2

FIG. 24: REACTION OF MITAPIVAT ON HEATING

FIG. 25: REACTION OF MITAPIVAT WITH WATER

CONCLUSION: The current study found that the stability indicating assay method combining RP-HPLC and FD Characterization with LC-MS was simple, reliable and non-interfering with degradation products or placebo. Thus, this can be applied to regular Mitapivat analysis.

Further investigation of degradation products was done by using in-silico techniques. All the degradation products obtained from the degradation of Mitapivat were screened for toxicity and ADME properties using Swiss ADME, pkCSM web server, protox 2. The SWISS ADME web server and pkCSM web server were used for the prediction of pharmacokinetic properties and toxicity of the drug and its degradation products. The intestinal absorption of the drug was 94.859% where as the degradation products DP-1, DP-3, DP-5 shows drastic decrease in the absorption. Only DP-4 shows the property of BBB permeation. Along with the drug, Mitapivat DP-2, DP-3, DP-4, DP-5 are CYP 3A4 inhibitors while DP-1 does not show CYP 3A4 inhibition. The predicted LD-50 of DP-4 is very much less than the drug and the degradation products which shows that the margin of safety of DP-4 is very less. The drug and theDP-1 doesn’t show any toxicity while DP-2, DP-3, DP-4, DP-5 shows immune toxicity. The volume of distribution is more for DP-4 while DP-2 has low volume of distribution.

ACKNOWLEDGEMENT: Authors are thankful to shree icon pharmaceutical laboratories for providing mitapivat drug as a gift sample. Authors are thankful to University College of pharmaceutical sciences for providing library and laboratory facilities.

CONFLICT OF INTEREST: No conflicts of interest.

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5. van Dijk MJ, Rab MAE and van Oirschot BA: One year safety and efficacy of mitapivat in sickle cell disease: follow -up results of a phase 2, open label study 2023; 7(24).
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   How to cite this article:

   Mastanamma SK, Sunitha C, Naseemunnisa MD and Kumari VA: Validated analytical method development and characterization of forced degradative products of mitapivat and their toxicity prediction by in-silico studies. Int J Pharm Sci & Res 2025; 16(1): 184-00. doi: 10.13040/IJPSR.0975-8232.16(1).184-00.

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