Quantification of Asciminib and its impurities: A RP-UPLC study

 

Siriki Pallavi, Gummadi Sowjanya*

Department of Pharmaceutical Analysis, GITAM School of Pharmacy,

GITAM (Deemed to be University), Visakhapatnam, Andhra Pradesh, India.

*Corresponding Author E-mail: statinen@gitam.edu

 

ABSTRACT:

A simple isocratic RP-UPLC-PDA detection method was developed for Asciminib and its process-related impurities. The chromatographic separation of Asciminib and its related impurities was achieved on the Agilent Eclipse C18 (150 × 4.6 mm, 3.5 µ) column with 0.1 percent v/v formic acid and acetonitrile (50:50 v/v) as a mobile phase at a flow rate of 1.0 mL/min. over a 5 min. run time analysed at 232 nm. The proposed method was validated according to the International Council for Harmonization (ICH) guidelines. The linearity was established from 75.00 - 450.00 µg/mL (Asciminib), 2.50 - 15.00 µg/mL (ASC Imp-1, ASC Imp-2) and 1.25 -7.50 µg/mL (ASC Imp-3) with regression coefficient ˃ 0.999. The respective computed LOD and LOQ values were 3.0, 10.0 µg/mL for Asciminib, 1.0, 3.0 µg/mL for both Imp-1 & Imp-2 and 0.5, 1.5 µg/mL for Imp-3. The method was further studied for forced degradation.

 

KEYWORDS: RP-UPLC, PDA, Asciminib, process-related impurities, validation, degradation.

 

 

INTRODUCTION: 

Asciminib is chemically N-4-[chloro(difluoro) methoxy] phenyl]-6-[(3R)-3-hydroxypyrrolidin-1-yl]-5-(1H-pyrazol-5-yl) pyridine-3-carboxamide. Pharmacologically Asciminib is a protein kinase inhibitor and was marketed as Scemblix. It is used for the treatment of Philadelphia chromosome-positive chronic myeloid leukemia (Ph+CML). Asciminib, is a substrate of the CYP3A4 enzyme and an inhibitor of CYP3A4, CYP2C9 and P-glycoprotein.

 

Imp-1, chemically 6-[(R)-3-hydroxypyrrolidin-1-yl]-5-(1H-pyrazol-5-yl) pyridine-3-carboxamide is a white powder bearing molecular weight 273.1226 g/mol., and molecular formula C13H15N5O2.  Imp-2, chemically 5-amino-N-(4-hydroxy phenyl) pyridine-3-carboxamide with molecular weight 229.0851 g/mol., molecular formula C12H11N3O2 is a white to off white powder. Imp-3, chemically 3-(1H-pyrazol-5-yl) pyridine-2-amine bearing molecular weight 160.0749 g/mol. and molecular formula C8H8N4 is in the form of white crystals.  All the three impurities have their solubility in methanol.

 

Impurity quantification (ICH Q3A R2 and Q3B R21,2) in method development and validation  is essential for ensuring product safety, quality, and regulatory compliance. It also plays a crucial role in process optimization and batch-to-batch consistency, ultimately contributing to the overall success of manufacturing operations.

 

Literature survey reported only a few analytical (UPLC, HPLC) 3-5 and bio-analytical (LC-MS/MS) 6 methods for Asciminib so far. Therefore an attempt was made to quantify Ascminib and its impurities 1, 2 & 3 (Figure 1) using ultra-performance liquid chromatography.

 

 

 

Asciminib

Asciminib Imp-1

 

 

 

 

Asciminib Imp-2

Asciminib Imp-3

Figure 1: Chemical structure of Asciminib and its related impurities

 

MATERIALS AND METHODS:

Chemicals and reagents

Acetonitrile, HPLC water of HPLC-grade; formic acid and ortho phosphoric acid of analytical grade were purchased from Merck (India) Ltd., Mumbai.  Pure drug Asciminib and its impurities 1, 2 & 3 were procured from Cadila health care Ltd, Ahmedabad, India.

 

Equipment and chromatographic conditions

Agilent 1290 Infinity II liquid chromatography monitored with Empower 2.0 data handling system were used to separate Asciminib and its impurities 1, 2 & 3.The analysis was performed on an Agilent Eclipse C18 (150x4.6 mm, 3.5 μ) column with isocratic elution using acetonitrile and 0.1% v/v formic acid (50:50 v/v) at a flow rate of 1.0 mL/min. and 232 nm PDA detection. The separation was carried out within 5 min. run time.

 

Preparation of  reagents and solutions

Preparation of 0.1 % v/v formic acid

1 mL of formic acid was dissolved in 1 litre of HPLC grade water and filtered through vacuum (0.45 µ membrane filter).

 

Preparation of mobile phase and diluent

Acetonitrile and formic acid mixed in 50:50 v/v  proportion was used as mobile phase and diluent.

 

Asciminib standard stock

Accurately about 30 mg of Asciminib was weighed, transferred into a 10 mL volumetric flask. Then a 7 mL diluent was added, sonicated for 10 min. to dissolve the contents and finally made upto the mark with diluent (3000 µg/mL). The standard aliquots were prepared from this stock.

 

Asciminib sample stock

Asciminib tablet powder equivalent to 30 mg (each tablet contains 40 mg of Asciminib) was transferred into a 10 mL volumetric flask and 7 mL diluent was added. This was sonicated for 20 min. and made upto the mark with diluent and filtered through a 0.45µ syringe filter. Further dilutions were made from this stock.

 

Impurity stock solution

10 mg each of Imp-1, Imp-2 and 5 mg of Imp-3 were accurately weighed into a 100 mL volumetric flask, and 70 mL diluent was added. This was sonicated for 20 min. and made upto the mark with diluent (100 µg/mL (for each Imp-1 and 2) and 50 µg/mL (for Imp-3).

 

Spiked standard solution

5 mL of Asciminib standard stock was transferred into a 50 mL volumetric flask, 5 mL of impurity standard stock solution was spiked and made upto the mark with diluent.

Spiked sample solution

5 mL Asciminib sample stock was transferred into a 50 mL volumetric flask. Then,  a 5 mL impurity stock was added to it and made upto the mark with diluent.

 

Method development

Trials were performed using X-Bridge Phenyl (250 × 4.6mm, 5µm) column and ACN: 0.1 % v/v TFA mobile phase in 80:20; 70:30; 65:35 v/v proportions respectively but symmetrical peaks were not achieved. Later trials were done on Agilent eclipse C18 (150 × 4.6 mm, 3.5 µ) using mobile phase of ACN: 0.1 % v/v formic acid in 60:40; 55:45 proportions respectively but the peak responses were high. Ultimately an optimized condition was attained on Agilent Eclipse C18 (150, 4.6 mm, 3.5 µ) column and ACN: 0.1 % v/v formic acid (50:50 v/v).

 

Method validation

The optimized method was validated for  system suitability, linearity, specificity, LOD & LOQ, precision, robustness, and  accuracy according to ICH Q2 (R1)7 guidelines.

 

System suitability

System suitability was performed by injecting spiked standard solution containing 300 µg/mL of Asciminib, 10 µg/mL each of Imp-1, Imp-2 and 5 µg/mL of Imp-3 in six replicates. From the values attained, the % RSD was computed and reproducibility was assessed.

 

Linearity

Six linearity aliquots each of Asciminib, Imp-1, Imp-2 and Imp-3 1.25, 2.50, 3.75, 5.00, 6.25, 7.50 mL  were were respectively pipetted from separate standard stock, impurity stocks of Imp-1, Imp-2 and Imp-3 into labeled volumetric flasks. Each solution was injected, and analyzed. From the calibration curve plotted, the slope Y-intercept and R2 for ASC, ASC Imp-1, ASC Imp-2, ASC Imp-3 were recorded.

 

Limit of detection and Limit of quantification

The LOD, LOQ for Asciminib and its impurities were estimated using signal/noise ratio. So, a series of solutions diluted with known concentrations were injected and analyzed.

 

Specificity

Specificity is the ability to measure the response of Asciminib in the presence of degradants, solvents etc. So, a blank, a standard spiked with the respective impurities and a sample spiked with impurities were prepared, injected and analyzed.

 

Precision, Robustness and Accuracy

The precision of the analytical technique is the degree of proximity of the sequence of measurements obtained from multiple homogeneous samples. The precision of the method was calculated by injecting six freshly prepared homogenous spiked sample  aliquots containing concentrations of 300 µg/mL (ASC), 10 µg/mL (ASC Imp-1 & ASC Imp-2) and 5 µg/mL (ASC Imp-3). The solutions were analyzed on the same day and other day respectively. The percent RSD was assessed.

 

Intentionally, flow rate (± 0.1 mL/min.) and mobile phase composition (± 5 parts) were varied from the optimized condition achieved. The effect of the deliberate modifications on the system suitability parameters and the % RSD were verified.

 

The accuracy of the method was achieved by measuring the recovery at three levels of concentrations (50%, 100% and 150%). The amount recovered and % RSD for Asciminib including its impurities was computed from the data obtained.

 

Forced degradation studies 8,9

A 5 mL sample stock was transferred to three different labeled volumetric flasks. Each 1 mL of 1N HCl, 1N NaOH, 10 % v/v H2O2 were added to three flasks respectively and the solutions were heated in a water bath for over 15 min. at 60 oC. Later the respective acid and alkali sotutions were cooled and neutralized. In a separate volumetric flask, a 5 mL sample stock was taken and exposed for 1.2 million lux hours in a photolytic chamber. For thermal study, 10 mg sample was kept in a hot air oven for15 min. at 105 oC. Later from this, 3  mg was transferred to a 10 mL flask. Then all the above degradation solutions were spiked with the impurities stock of 5 mL and made upto volume with the diluent. These solutions were injected and analyzed.

 

RESULTS AND DISCUSSION:

Method optimization

After a series of trials, an optimized condition (Table 1) with symmetrical peaks (Figure 2) and satisfactory values of system suitability parameters was achieved.

Table 1: Optimized chromatographic conditions for Asciminib and its impurities

Parameter

Value

Mobile phase

ACN: 0.1 % v/v formic acid (50:50 v/v)

Column

Agilent Eclipse C18

(150 × 4.6 mm, 3.5 µ)

Flow rate (mL/min.)

1.0

Mode of Elution

Isocratic

Detection wavlength (nm)

232

İnjection volume (µL)

10

 

Retention time (min.)

ASC

3.389

ASC Imp-1

1.425

ASC Imp-2

1.932

ASC Imp-3

2.455

Run time (min.)

5.0

ASC: Asciminib; Imp: impurity

 

 

Figure 2: Optimized chromatogram for Asciminib and its impurities

 

System suitability

The system suitability parameters were reproducible (Table 2) and the computed % RSD was less than 2.0 for Asciminib and its impurities.

 

Linearity

A six point calibration curve was plotted with peak areas against its respective concentrations (Figure 3). From this calibration curve, it was noticed that linearity was satisfied from 75-450 µg/mL, 2.5-15 µg/mL, 2.5-15 µg/mL and 1.25-7.5 µg/mL for ASC, ASC Imp-1, ASC Imp-2 and ASC Imp-3 (Table 3) respectively. The least possible amounts detected and quantified calculated by signal to noise ratio were recorded in Table 4.

 

 

Table 2: Results of system suitability for Asciminib and its impurities

Compound name

RT (min.)

(n=6)

RRT (min.)

(n=6)

Area count

(n=6)

USP plate count

(n=6)

USP tailing

(n=6)

USP resolution

(n=6)

% RSD

(n=6)

ASC

3.389

-

11591162

8067

1.06

6.55

1.68

ASC Imp-1

1.425

0.42

904072

2209

1.05

-

0.19

ASC Imp-2

1.932

1.35

935614

4739

1.28

4.33

0.24

ASC Imp-3

2.455

1.27

354862

5792

1.37

4.29

0.79

n =  mean of six determinations

 

 

 

ASC

ASC Imp-1

 

 

 

ASC Imp-2

ASC Imp-3

Figure 3: Calibration curves of Asciminib and its related impurities

 

Table 3: Linearity results of Asciminib and its impurities

Linearity

ASC

ASC Imp-1

ASC Imp-2

ASC Imp-3

Conc.

(µg/mL)

Area count

Conc.

(µg/mL)

Area count

  Conc.

  (µg/mL)

Area count

   Conc.

  (µg/mL)

Area count

Linearity-1

75.00

3393309

2.50

251457

2.50

250585

1.25

99478

Linearity-2

150.00

6013221

5.00

468532

5.00

442156

2.50

185469

Linearity-3

225.00

9425746

7.50

659837

7.50

678546

3.75

282145

Linearity-4

300.00

11638823

10.00

900850

10.00

938896

5.00

357027

Linearity-5

375.00

14865287

12.50

1114574

12.50

1185463

6.25

463259

Linearity-6

450.00

17842654

15.00

1315646

15.00

1366594

7.50

555698

Slope (m)

39094.06

87221.29

92375.40

73320.40

Intercept (C)

229414.29

18825.50

1790.21

2630.79

R2

0.99922

0.99956

0.99917

0.99953

R2 regression coefficient, Conc. Concentration

 

Table 4: Results of LOD and LOQ

Compund name

LOD (µg/mL)

LOQ (µg/mL)

ASC

3.0

10.0

ASC Imp-1

1.0

3.0

ASC Imp-2

1.0

3.0

ASC Imp-3

0.5

1.5

 

 

Specificity

On analyzing a blank, a standard aliquot spiked with impurities and a sample spiked with impurities, it was inferred that any kind of interference at the RTs of  Asciminib and its respective impurities (Figure 4) was not detected due to solvents and other unwanted degradants etc.

 

 

 

Blank

Standard spiked with impurities

Sample aliquot spiked with impurities

Figure 4: Chromatograms for specificity

 

Precision, Accuracy and Robustness

Six replicates of a sample solution containing Asciminib and its impurities were analysed on the same day and on a different day. From the peak areas achieved, % RSD values were calculated (Table 5). The % RSD was 1.28 (ASC), 0.06 (ASC Imp-1), 0.23 (ASC Imp-2), 0.41 (ASC Imp-3) for method precision and 2.38 (ASC), 0.19 (ASC Imp-1), 0.24 (ASC Imp-2), 0.79 (ASC Imp-3) for intermediate precision. The recovery values computed at the three levels for Asciminib and its impurities were recorded in Table 7. From the results achieved for varied conditions of flow rate and mobile phase composition,  the system suitability parameters and computed % RSD results were presented in Table 6. The RTs, peak areas,  plate count tailing and resolution of ASC and its three impurities were affected to a little extent confirming the robustness of the method.

 

Table 5: Results of Precision

Compound name

RT (min.)

(n=6)

RRT (min.) (n=6)

Peak area ± % RSD

(n=6)

Mass recovery (% w/w)

(n=6)

Method Precision

ASC

3.39

-

11300290 ± 1.28

-

ASC Imp-1

1.47

0.43

902644 ± 0.06

7.90

ASC Imp-2

1.94

0.58

935395 ± 0.23

8.27

ASC Imp-3

2.49

0.73

352699 ± 0.41

3.12

Intermediate Precision

ASC

3.38

-

11242495 ± 2.38

-

ASC Imp-1

1.45

0.43

903586 ± 0.19

7.91

ASC Imp-2

1.93

0.57

933737 ± 0.24

8.28

ASC Imp-3

2.49

0.74

354701 ± 0.79

3.14

 

Table 6: Robustness results of Asciminib and its impurities

Compound name

Parameter variation

Retention time (min.)

(n=3)

Area count, % RSD

(n=3)

USP plate count

(n=3)

USP tailing

(n=3)

USP resolution

(n=3)

 

 

 

ASC

Flow rate (mL/min.)

0.9

2.840

11850838, 0.09

7559

1.65

6.46

1.0

3.389

11654278, 1.68

8067

1.06

6.55

1.1

2.840

9545114, 0.23

7559

1.65

6.46

Mobile phase (v/v)

55:45

2.820

11682572, 0.12

6935

1.60

5.51

50:50

3.389

11654278, 1.68

8067

1.06

6.55

45:55

4.140

11954430, 0.20

2741

0.47

9.42

 

 

ASC Imp-1

Flow rate (mL/min.)

0.9

1.220

746928, 0.19

2056

1.23

-

1.0

1.425

907451, 0.19

2209

1.05

-

1.1

1.220

746928, 0.19

2056

1.23

-

Mobile phase (v/v)

55:45

1.330

894339, 0.15

2132

1.41

-

50:50

1.425

907451, 0.19

2209

1.05

-

45:55

1.560

974149, 0.16

2322

1.30

-

 

 

ASC Imp-2

Flow rate (mL/min.)

0.9

1.630

775429, 0.23

4152

1.35

4.21

1.0

1.932

936521 ,0.24

4739

1.28

4.33

1.1

1.630

775429, 0.23

4152

1.35

4.21

Mobile phase (v/v)

55:45

1.770

935092, 0.33

4340

1.59

4.25

50:50

1.932

936521 ,0.24

4739

1.28

4.33

45:55

2.181

1004048, 0.19

5450

1.50

4.32

 

 

ASC Imp-3

Flow rate (mL/min.)

0.9

2.040

305864, 0.63

5257

1.62

4.46

1.0

2.455

356241 ,0.79

5792

1.37

4.29

1.1

2.040

383460, 0.79

6748

1.62

4.46

Mobile phase (v/v)

55:45

2.128

332268, 0.53

5358

1.57

3.70

50:50

2.455

356241 ,0.79

5792

1.37

4.29

45:55

2.966

402116, 0.09

6958

1.33

6.28

n = mean of three determinations

Table 7: Accuracy results of Asciminib and its impurities

Level

(%)

ASC

ASC Imp-1

ASC Imp-2

ASC Imp-3

Amount recovered (µg/mL)

(n=3)

Recovery (% w/w), % RSD

(n=3)

Amount recovered (µg/mL)

(n=3)

Recovery (% w/w), % RSD

(n=3)

Amount recovered (µg/mL)

(n=3)

Recovery (% w/w), % RSD

(n=3)

Amount recovered (µg/mL)

(n=3)

Recovery (% w/w), % RSD

(n=3)

50

6.613

100.2, 0.36

0.050

100.7, 0.65

0.050

99.2,0.56

0.013

99.7, 1.36

100

13.218

100.2, 1.26

0.010

99.8,0.10

0.100

100.0,0.26

0.025

100.3, 0.61

150

19.684

99.4,1.19

0.149

99.5,1.13

0.149

100.4,0.45

0.038

100.5, 0.32

n mean of three determinations

 

Forced degradation studies

The impurities were clearly separated (Figure 5) and the % degradation obtained (Table 8) was recorded along with the system suitability parameters. The purity angle  was less than the purity threshold. Major  % degradation was achieved in case of acid, alkali, peroxide, thermal treatments and minor in photolytic condition. The retentions times were specific and the chromatographic peak of ASC was not hindered in any manner due to the respective impurities spiked.

 

 

 

Control degradation

Acid degradation

 

 

Alkali degradation

Peroxide degradation

 

 

Photolytic degradation

Thermal degradation

Figure 5: Chromatograms for forced degradation studies

 

 

Table 8: Results of forced degradation studies of Asciminib

Condition

RT (min.)

Area count

USP plate count

USP resolution

USP tailing

% Degradation (% w/w)

Purity angle

Purity threshold

Control

3.360

11545132

8032

3.86

0.89

-

0.519

5.826

Acid

3.382

10275478

8054

3.97

1.54

11.0

0.537

5.234

Alkali

3.367

10223654

7563

3.86

0.98

11.4

0.538

5.244

Oxidation

3.384

10084832

7892

3.86

0.91

12.6

0.546

5.368

Thermal

3.375

10493756

8034

3.02

1.13

9.1

0.554

5.548

Photolysis

3.346

11219984

8077

3.85

0.89

2.8

0.592

5.374

 

CONCLUSION:

A  simultaneuos RP-UPLC separation method was established for Asciminib and its impurities. Further the method underwent validation. The method was linear, specifc, reproducible, and accurate. This was the first quantification method reported for ASC along with its impurities. So, the proposed separation process can be undoubtedly utilized for future purpose in quality control applications.

 

ACKNOWLEDGEMENT:

I express my heartfelt gratitude to M/s GITAM School of pharmacy, Visakhapatnam and Shree Icon Laboratories, Vijayawada for supporting and facilitating the research.

 

CONFLICT OF INTEREST:

There were no conflicts in any manner among the authors.

 

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Received on 08.05.2024            Modified on 05.06.2024

Accepted on 14.07.2024           © RJPT All right reserved

Research J. Pharm. and Tech 2024; 17(7):3430-3436.

DOI: 10.52711/0974-360X.2024.00537