DEVELOPMENT, VALIDATION AND CHARACTERIZATION OF …

www.wjpps.com │ Vol 9, Issue 11, 2020. │ ISO 9001:2015 Certified Journal │

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Vikrant et al. World Journal of Pharmacy and Pharmaceutical Sciences

DEVELOPMENT, VALIDATION AND CHARACTERIZATION OF

DEGRADANTS BY STABILITY INDICATING HPLC METHOD FOR

ESTIMATION OF ELIGLUSTAT

Vikrant Salode*1, Madhuri Game

1 and Gouri Salode

2

1Vidyabharti College of Pharmacy, Amravati Camp, Amravati (MS), India-444602.

2P. R. Pote Patil College of Pharmacy, Amravati (MS), India 444602.

ABSTRACT

Objective: The aim of this study was to develop a validated stability

indicating Reverse Phase High-performance liquid chromatography

(RP-HPLC) method for estimation of Eliglustat and characterization of

degradants by mass spectroscopy. Method: The RP-HPLC method

was developed using the HiQSil C18 column (250 x 4.6 mm, 5µm) and

the mobile phase was selected Methanol: Ammonium Acetate Buffer

(80:20 v/v) with a flow rate of 1.0 ml / min with detection wavelength

selected at 284 nm. Drug retention time (RT) was at 3.560 min ± 0.034

min. Eliglustat has subjected to various stress testing conditions, such

as hydrolysis of acid and base, thermal degradation, oxidation and

photolysis. Result: The method has been validated with regard to linearity, accuracy,

precision and robustness. Linear regression analysis data indicated a good linear relationship

with a correlation coefficient (R2) of 0.987 over the 5-30 μg/ml concentration range. Stress

results showed that there was a significant degradation of drug in acidic condition, the

degradation product of which characterized using LC-MS technique Conclusion: The

method developed was found to be quick, sensitive, selective, precise, accurate, and robust

for Eliglustat analysis which can be adopted for routine drug analysis.

KEYWORDS: High performance liquid chromatography (HPLC), Mass spectroscopy (MS)

Eliglustat, Stress degradation, Validation.

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

SJIF Impact Factor 7.632

Volume 9, Issue 11, 1198-1210 Research Article ISSN 2278 – 4357

*Corresponding Author

Vikrant Salode

Vidyabharti College of

Pharmacy, Amravati Camp,

Amravati (MS), India-

444602.

Article Received on

10 September 2020,

Revised on 30 Sept. 2020,

Accepted on 20 Oct. 2020

DOI: 10.20959/wjpps202011-17706


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INTRODUCTION

Eliglustat chemically N-[(1R,2R)-1-(2,3-dihydro-1,4-benzodioxin-6-yl)-1-hydroxy-3-

pyrrolidin-1-ylpropan-2-yl]octanamide (Figure1). Eliglustat is an oral inhibitor of

glucosylceramide synthase which is used in the therapy for the long-term treatment of type 1

Gaucher disease.[1]

Literature survey reveals that few reports have been documented using

high-performance liquid chromatography (HPLC) for analytical method development and

validation to estimate eliglustat,[2]

Quantification of Eliglustat in rat plasma by HPLC-UV

method,[3]

LC-MS method on Quantification of Eliglustat in rat plasma,[4]

Isolation and

characterization of degradation products of Eliglustat tartarate using 2D-NMR and HRMS

using RP-UPLC.[5]

This work describes a simple, reliable HPLC system and validation

according to ICH guidelines,[6,7]

for the determination of Eliglustat in bulk form.

o

o

NH

NO

OH

Figure 1: Chemical structure of Eliglustat.

MATERIALS AND METHODS

Reagents and chemicals

Methanol (HPLC grade), Methanol (AR Grade) were purchased from LobaChemie Pvt. Ltd.,

Mumbai. Ammonium Acetate (HPLC), Hydrochloric acid (HCl), 30%Hydrogen Peroxide

(H2O2), and sodium hydroxide (NaOH); all were purchased from LobaChemie Pvt. Ltd.,

Mumbai India. HPLC Grade water conductivity of 0.05 μs/cm from extra pure Lab link water

purifier system.

Chromatographic conditions

HPLC system used was JASCO system equipped with model PU 2080 Plus pump, Rheodyne

sample injection port (20μl), JASCO MD2010 Plus detector and Borwin chromatography

software (version 1.5). A chromatographic column used was HiQSil C18 (250 x 4.6 mm,

5µm) at flow rate of 1.0 ml/min using Methanol: Ammonium acetate Buffer (80:20 v/v) as

mobile phase and wavelength detection at 284 nm.


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Selection of detection wavelength

From the standard stock solution (1000 μg/ml) further dilutions were made using methanol

and solution was scanned over the range of 200-400 nm and the spectra was obtained. It was

observed that the drug showed maximum absorbance at 284 nm. Representative UV spectrum

of Eliglustat (Figure 2).

Figure 2: UV spectra of Eliglustat (10 µg/ml).

Preparation of Standard stock solution

Standard stock solution of drug was prepared by dissolving 10 mg of the drug in 10 ml of

methanol to procure concentration of 1000 μg/ml. From the standard stock solution, working

standard solution was prepared by dissolving 1 ml into 10 ml methanol to procure 100 μg/ml

of eliglustat. Representative chromatograph is shown in Figure 3. The retention time (RT) of

drug was 3.560 min ± 0.034 min.

Figure 3: Standard solution of Eliglustat (100 μg/ml).


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Preparation of sample solution (Capsule formulation)

Contents of 20 capsules each containing 84 mg of Eliglustat (CERDELGA by Genzyme)

were weighed and powdered. Powder equivalent to 10 mg of drug was transferred to 10 ml

volumetric flask and volume was made up with methanol to get concentration (1000µg/ml)

and was sonicated for 10 min. Solution was filtered, from this solution 1 ml of drug was

taken in 10 ml volumetric flask and volume was made up with methanol. Further dilution in

mobile phase was made to get concentration 10 µg/ml.

Stress Degradation Studies of Bulk Drug

Stress degradation studies of bulk drug have been performed to demonstrate how drug quality

varies under the influence of a number of environmental conditions such as acidic, alkaline

hydrolysis, oxidation, dry heat and photolytic degradation. At concentration of 100 µg/ml all

stability tests are performed.

Degradation under alkali hydrolysis condition

1 ml of standard stock solution of Eliglustat (1000 µg/ml) was mixed with 1 ml of 1N NaOH

and kept aside for one hour. The solution was neutralized and final volume made to 10 ml

with mobile phase to get 100 µg/ml solution, the resultant solution was then injected and

chromatogram was recorded. Alkali degradation blank is prepared in the same way without

using analyte. On alkali hydrolysis Eliglustat was degraded 2.311 % with no degradation

peaks.

Figure 4: Alkali Hydrolysis of Eliglustat (100 μg/ml).

Degradation under acid hydrolysis condition

1 ml of standard stock solution of Eliglustat (1000 µg/ml) was mixed with 1 ml of 1N HCl

and kept aside for one hour. The solution was neutralized and final volume made to 10 ml


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with mobile phase to get 100 µg/ml solution, the resultant solution was then injected and

chromatogram was recorded. Acid degradation blank is prepared in the same way without

using analyte. On acid Hydrolysis Eliglustat was degraded 22.333 % with two degradation

peaks.

Figure 5: Acid Hydrolysis of Eliglustat (100 μg/ml).

Degradation under oxidative condition

1 ml of standard stock solution of Eliglustat (1000 µg/ml) was mixed with 1 ml of 30% H2O2

and kept aside for one hour. The volume made to 10 ml with mobile phase to get 100 µg/ml

solution, the resultant solution was then injected and chromatogram was recorded. On

Oxidative Hydrolysis Eliglustat was degraded 3.498 % with no degradation peaks.

Figure 6: Oxidation of Eliglustat (100μg/ml).


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Degradation under thermal condition

Eliglustat powder was heated at 800 C for 1 hour in hot air oven. After exposure drug was

dissolved in methanol to get 1000 µg/ml solution. From this solution 1 ml was diluted to 10

ml with mobile phase to get 100 µg/ml. The resultant solution was then injected and

chromatogram was recorded. No degradation was observed.

Figure 7: Thermal Degradation of Eliglustat (100 μg/ml).

Photo-degradation studies: The photo degradation stability study of the drug was studied by

exposing the drug to UV light providing illumination of NLT 200 watt hr/m2

followed by

exposure to cool white fluorescence light of NLT 1.2 million Lux-Hr. After exposure

accurately weighed 10 mg of drug was transferred to 10 ml of volumetric flask; the volume

was made up with methanol. Further dilution made with mobile phase to get 100 µg/ml as

final concentration and was injected to get chromatogram. No degradation was observed.

Figure 8: Photolytic Degradation of Eliglustat.


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Summary of Degradation Parameters

Table 1: Summary of Degradation.

Stress condition/Duration % Recovery R.T. of degraded

products

Alkaline/ 1 N NaOH / at room temperature 01 hour 97.689 -

Acidic/ 1 N HCl/ at room temperature 01 hour 77.667 EG1 – 2.693 min

EG2 – 2.973 min

Oxidative/ 30 % H2O2 / at room temperature 01 hour 96.502 -

Dry heat / 80ºC/ 01 hour 98.232 -

UV illumination NLT 200 watt hours/square meter

followed by Fluroscent light NLT 1.2 X 106 Lux hr

99.305 -

Identification, Characterization And Prediction Of Degradation Products By Lc–MS

Forced degradation studies were carried out as per ICH guidelines Q1A (R2).[6]

All stressed

solid samples and solutions were well protected, covered with aluminum foil, and kept in a

refrigerator at -4 °C until analysis. LC-MS study was carried out for standard drug as well as

for forced degradation samples of drug for acid, alkali and oxidation under which degradation

was observed during development of stability indicating assay method.

Successful separation of two of the major degradation products under acidic condition was

observed by high performance liquid chromatography (HPLC) using ZorbaxEclipise XDB-

C18 (150 mm× 3.0 mm, 3.55 μm) analytical column. It was identified and characterized by

liquid chromatography–electrospray ionization mass spectrometry (LC–ESI/MS) with

accurate mass measurements up to four decimals.

Mass Spectroscopy Conditions

Mass Spec parameters were optimized for Eliglustat to get highest intensity while tuning for

scan to identify the [M + H]. Quantification was achieved with MS detection in positive ion

mode for the drugs and its degrading products. Agilent LC-MS Q – TOF (6200 series

TOF/6500 series) (5301 Stevens Creek Blvd, Santa Clara, CA 95051, United States)

equipped with a dual AJS ESI with improved sensitivity [AJS – ESI: Agilent Jet Stream

Electro spray Ionizer] and Q-TOF B.05.01software version. The Min Range and Max Range

in acquisition mode was set to 60 and 1000, respectively with scan rate of 2.00 spectra/sec.


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Figure 9: Chromatograph of Eliglustat subjected to Acidic stress.

Figure 10: LC-MS Spectrum of Eliglustat from acidic stress sample (Retention Time –

7.56 min).

Figure 11: LC -MS Spectrum of DP1 of Eliglustat stress sample (Retention Time – 8.01

min).


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Figure 12: LC -MS Spectrum of DP2 of Eliglustat stress sample (Retention Time – 8.65

min).

Figure 13: Probable degradation pathway of Eliglustat acid stress sample.

Validation of Analytical Method

The method validation was performed as per ICH Q2 (R1) guidelines.[7]

Specificity

The specificity of the method was ascertained by peak purity profiling studies. The peak

purity values were found to be more than 992, indicating the no interference of any other

peak of degradation product, impurity or matrix.


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Linearity

From the standard stock solution (1000 µg/ml) of Eliglustat, solution was prepared containing

100 µg/ml of Eliglustat with methanol. This solution was further used to prepare range of

solutions containing six different concentrations. The linearity (relationship between peak

area and concentration) was determined by analyzing six solutions over the concentration

range of5-30 µg/ml. Linearity curve is represented in Figure9.

Figure 14: Calibration curve of Eliglustat.

Precision

The precision of the method was demonstrated by intra-day and inter-day variation studies. In

the Intra-day studies, 3 replicates of 3different concentrations were analyzed in a day and

percentage RSD was calculated. For the inter day variation studies, 3 different concentrations

were analyzed on 3 consecutive days and percentage RSD were calculated. The results

obtained for Intraday and Inter day variations are shown in Table 2 and Table 3, respectively.

Table 1: Intra-day precision study of Eliglustat.

Theo. Conc (mcg/ml) Area Practical

Conc (mcg/ml) % assay Avg* SD RSD

10 1413430.190 9.988 99.881

10 1429582.890 10.132 101.315 101.315 0.717 0.708

10 1421184.070 10.057 100.570

20 2563761.89 20.203 101.013

20 2583351.84 20.377 101.883 101.522 0.453 0.447

20 2578556.865 20.334 101.670

25 3025154.67 24.300 97.199

25 3036488.15 24.400 97.601 98.584 2.061 2.090

25 3130821.41 25.238 100.952


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Table 3: Inter-day precision of Eliglustat.

Theo. Conc (mcg/ml) Area Practical

Conc (mcg/ml) % assay Avg* SD RSD

10 1422445.786 10.068 100.682

10 1422096.290 10.065 100.651 100.581 0.149 0.148

10 1419381.038 10.041 100.410

20 2594307.620 20.474 102.369

20 2585701.830 20.397 101.987 101.960 0.424 0.416

20 2575234.725 20.305 101.523

25 3164242.980 25.535 102.139

25 3102893.600 24.990 99.960 101.168 1.109 1.096

25 3143578.290 25.351 101.405

Limit of Detection (LOD) and Limit of Quantification (LOQ)

LOD and LOQ were calculated from the formula 3.3 σ / Sand 10 σ / S, respectively, Whereσ

= standard deviation of y- intercept, S = slope of the calibration curve. The LOD and LOQ

were found to be 0.652 and 1.977.

Assay

CERDELGA 84 mg capsule formulation analysis was carried out as mentioned under section

preparation of sample solution. Procedure was repeated for six times. Sample solution was

injected and area was recorded. Concentration and % recovery was determined from linear

equation. (Table 4)

Table 4: Assay of marketed formulation.

Sr. No. Peak area Amount Recovered (µg/ml) % Recovery

1 1417947.89 10.028 100.282

2 1419274.84 10.040 100.400

3 1405828.15 9.920 99.206

4 1413450.02 9.988 99.882

5 1401947.09 9.886 98.861

6 1406390.29 9.925 99.255

Mean 1410806.38 9.964 99.648

SD 7106.003 0.063 0.630

%RSD 0.503 0.633 0.633

Accuracy

To check accuracy of the method, recovery studies were carried by spiking the standard drug

to the sample solution, at three different levels around 50, 100 and 150 %. Basic

concentration of sample solution chosen was 10 µg/ml. % recovery was determined from

linearity equation. The results obtained are shown in (Table 5).


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Table 5: Recovery studies of Eliglustat.

Level

Conc. of

Sample

solution

(µg/ml)

Conc. of

Standard

solution spiked

(µg/ml)

Area

Amount

recovered

(µg/ml)

% recovery

50% 10 5

1976827.83 14.99

99.90 1980105.24 15.01

1972265.63 14.94

100% 10 10

2562436.23 20.19

100.49 2520212.26 19.81

2573761.89 20.29

150% 10 15

3085114.64 99.32

98.43 3055154.67 98.26

3040172.55 97.72

Robustness

Robustness of the method was checked by carrying out the analysis under conditions during

which mobile phase composition, detection wavelength (± 1 nm), flow rate (± 0.05 ml/min)

were altered and the effect on the area were noted. Robustness of the method checked after

deliberate alterations of the analytical parameters showed that areas of peaks of interest

remained unaffected by small changes of the operational parameters indicating that the

method is robust.

RESULTS AND DISCUSSION

The developed method was found to be simple, sensitive, specific, accurate, and repeatable

for analysis of Eliglustat in bulk and pharmaceutical dosage form without any interference

from the excipients. Separation was carried on HiQ SilC18 column, (250 x 4.6 mm, 5µm).

Methanol: Ammonium acetate Buffer in the ratio of 80:20 v/v was used as mobile phase at

flow rate of 1 ml/min and detection at 284 nm. The retention time (RT) of drug was 3.560

min ± 0.034 min. The method was validated with respect to linearity, range, method precision

(intraday and inter day), accuracy and robustness. The data of linear regression analysis

indicated a good linear relationship over the range of 5-30 μg/ml concentrations with a

correlation coefficient (R2) of 0.994. The % assay was found to be 99.648 ± 0.633. The

results indicated the suitability of the method to study stability of Eliglustat under various

forced degradation conditions and characterization of degradation product using LC-MS

technique. Two degradation products were identified under acidic stress studies and

characterized using LC–MS supported by accurate mass measurements. Major degradant EG-


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1 and EG-2 were isolated and characterized. In-silico probable profile predicted and

demonstrated.

CONCLUSION

A simple, accurate, precise and sensitive high-performance liquid chromatographic (HPLC)

method has been developed and validated for the analysis of Eliglustat in bulk and in tablet

dosage form. The proposed method successfully separated the compound with degradants and

estimate the active content.

ACKNOWLEDGEMENT

Authors are thankful to the Management and Principal of Vidyabharti College of Pharmacy,

Amravati for providing required facilities for research work.

REFERENCES

1. https://pubchem.ncbi.nlm.nih.gov/.

2. Amena Tazeen, Dr. R. Vani, Dr. M. Sunitha. method development and validation for the

estimation of eliglustat using RP-HPLC method in bulk and pharmaceutical dosage form.

Analytical Indo American Journal of Pharmaceutical Research, 2017; 7(11): 920-927.

3. Reddy VK, Swamy N, Rathod R, Senugupta P. A Bioanalytical Method for Eliglustat

Quantification in Rat Plasma, Journal of chromatographic Science, 2019; 57(7): 600-605.

4. Jingjing Chen, Yuanyuan Shao, Huidan Zhu, Xiufang Chen, Xuemei Ye. Liquid

chromatography-tandem mass spectrometric method for the quantification of eliglustat in

rat plasma and the application in a pre-clinical study. J Pharm Biomed Anal, 177: 112858

https://doi.org/10.1016/j.jpba.2019.112858.

5. Umamaheshwar Puppala, Koduri S V Srinivas, K.V. Reddy, Muralidharan

Kaliyaperumal, R. Doddipalla, B.R. Jogi. Isolation and Characterization of Novel

Degradation Products of Eliglustat Tartrate Using 2D-NMR and HRMS: Development

and Validation of Stability Indicating RP-UPLC Method for Quantification of Assay and

Characterized Impurities. Analytical Chemistry Letters, 2020; 10(1): 1 – 20.

6. ICH guidelines for stability testing of new drug substances and products Q1A (R2),

Geneva, Switzerland, 2004.

7. ICH guidelines for validation of analytical procedures: text and methodology Q2 (R1),

Geneva, Switzerland, 2005.

https://pubchem.ncbi.nlm.nih.gov/

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