Arun

Kumar Kuna et.al., Int. J. Res. Pharm. Sci., 10(2), 865-873

ORIGINAL ARTICLE

INTERNATIONAL JOURNAL OF RESEARCH IN

PHARMACEUTICAL SCIENCES
Published by Pharmascope Publications Journal Home Page: www.pharmascope.org/ijrps

A novel RP- HPLC method development and forced degradation studies for
semaglutide in active pharmaceutical ingredients and pharmaceutical
dosage forms
Arun Kumar Kuna*, Ganapaty S, Radha GV
Department of Pharmaceutical Analysis, Gitam Institute of Pharmacy, Gitam University (Deemed to be
University) Rushikonda, Visakhapatnam- 530 045, Andhra Pradesh, India

Article History: ABSTRACT

Received on: 23.11.2018 This research objective is for the development of a specific and simple
Revised on: 12.02.2019 method to trace Semaglutide presence in active pharmaceutical ingredient
Accepted on: 15.02.2019 and pharmaceutical dosages. As part of a study on Semaglutide drug, solvents
of HPLC grade waters HPLC instrument (Empower software) with PDA de-
Keywords: tector, ultrasonicator (Make: Labman) and pH meter (Make: Adwa) are used.
The Method was optimized with mobile phase with a composition of buffer
and solvent were of 60:40%v/v, flow maintained was 1.0ml/min, the injec-
Semaglutide,
tion volume of 10µl, run time was 5min. All separations were performed with
RP-HPLC,
PDA detector and column used was Discovery C18 150 x 4.6mm, 5µ. Results
Validation,
for the developed method are accurate and specific. The detection
Method Development,
wavelength was 292 nm, the retention time for Semaglutide was 2.689min,
Detection
linearity resulted with r2= 0.9998, % RSD for precision was 1.0; %mean
recovery for accuracy was in the range of 99.73 to 100.29. This study report
is for industrial application for determining Semaglutide presence in phar-

maceutical ingredient and dosages.

* Corresponding Author 2017). Researchers at the University of Leeds and

Novo Nordisk reported in 2017 that it could also be
Name: Arun Kumar Kuna
used for the treatment of obesity (Blundell et al.,
Phone: +91-9949782803
2017).
Email: kunaarun@yahoo.co.in
Semaglutide Figure 1 is chemically known as 17-
ISSN: 0975-7538 {[(1R)-3-[(2-{2-[({2-[2-({[(5S)-5-[(2S)-2-[(2S)-2-
DOI: https://doi.org/10.26452/ijrps.v10i2.263 [(2S)-2-{2-[(2S)-2-[(2S)-2-[(2S)-2-[(2S)-2-[(2S)-
2-[(2S)-2-[(2S)-2-[(2S)-2-[(2S,3R)-2-[(2S)-2-
Production and Hosted by [(2S,3R)-2-{2-[(2S)-2-{2-[(2S)-2-amino-3-(1H-
imidazol-4-yl)propanamido]-2-
Pharmascope.org methylpropanamido}-4-
© 2018 Pharmascope Publications. All rights reserved.
carboxybutanamido]acetamido}-3-
INTRODUCTION hydroxybutanamido]-3-phenyl propanamido]-3-
hydroxybutanamido]-3-hydroxypropanamido]-3-
In type-2 diabetes (Novo Nordisk, 2016) treatment carboxypropanamido]-3-methylbutanamido]-3-
Semaglutide is used. It is a glucagon-like peptide-1 hydroxypropanamido]-3-hydroxypropanamido]-
receptor agonist (Marso et al., 1834). It lowers the 3-(4-hydroxyphenyl) propanamido]-4-
blood sugar level by increasing the production of methylpentanamido]-4-
insulin. Glucagon-like peptide-1 has 2- carboxybutanamido]acetamido}-4-carbamoyl
aminoisobutyric acid and arginine whereas butanamido] propan amido]propanamido]-5-
Semaglutide has two amino acid substitutions at {[(1S)-1-{[(1S)-1-{[(1S,2S)-1-{[(1S)-1-{[(1S)-1-
positions 8 and 34 (Lau et al., 2015). Semaglutide {[(1S)-1-{[(1S)-1-{[(1S)-4-carbamimidamido-1-
was approved by US FDA in 2017. It can be used as [({[(1S)-4-carbamimidamido-1-[(carboxy methyl)
an injection-type or oral-type drug (Davies et al., carbamoyl]
© Pharmascope Publications | International Journal of Research in Pharmaceutical Sciences 865
 Arun Kumar Kuna et al., Int. J. Res. Pharm. Sci., 10(2), 865-873

butyl]carbamoyl}methyl)carbamoyl]butyl]carbam Sample preparation

oyl}-2-methylpropyl] carbamoyl}-3-
1.5ml injection volume (drug) equivalent for 2.0
methylbutyl]carbamoyl}-2-(1H-indol-3-
mg of Semaglutide taken to a 50ml analytical glass
yl)ethyl]carbamoyl} ethyl]carbamoyl} -2-
flask, 10ml diluent added and mixing performed
methylbutyl]carbamoyl}-2-
for 25 minutes. Derived the required quantity of
phenylethyl]carbamoyl}-3-carboxypropyl]
volume with diluents HPLC filter used for the
carbamoyl}pentyl]
filtration process. 0.5ml of above-prepared
carbamoyl}methoxy)ethoxy]ethyl}carbamoyl)met
solution transferred to the 10ml analytical flask for
hoxy]ethoxy}ethyl)carbamoyl]-1-carboxy propyl]
volume make with diluents (10µg/ml
carbamoyl}heptadecanoic acid (Drug bank, 2017).
Semaglutide).
From the literature survey, it was known that there
Details of Validation
were no methods developed and validated for the
determination of Semaglutide with RP-HPLC. So Specificity: Drug quantities were determined with
this study on RP-HPLC was developed with an aim this study by the support of dilutions in
to keep this research method simple, sensitive and appropriate quantities for determining the
accurate. This method was developed and chromatographs. The drug was pre-weighed and
validated with accuracy, linearity, Limit of used for spiking during the study.
Detection, Limit of Quantification, precision,
Suitability of System: Method developed with this
specificity, other validations and degradation
parameter to observe the performance-related
related studies. factor. It is used to ensure adequate performance
MATERIAL AND METHODS of the chromatographic system.
Materials: The drug Semaglutide was kindly gifted Linearity and Range: In the chromatographic
by Spectrum Laboratories, Hyderabad. HPLC method, linearity generates data which helps to
water, acetonitrile and OPA used are of Merck evaluate the correlation coefficients. These are
grade. Instruments like Waters HPLC (Empower related to the analyte concentration and
software), ultrasonicator (Make: Labman) and pH proportionality with the given range. Analyte
meter (Make: Adwa) are used for developing this extents with respect to upper and lower intervals
method. were determined in this range system. The
linearity of Semaglutide was 2.5 μg/ml to 15
Instrument and its conditions
μg/ml. The correlation was derived as 0.9998.
A study performed on HPLC (Make: Waters) which
Precision: This study has two parts; they are
has detector PDA. Empower software was used for
interring day (between days) and intraday (same
analysis of data derived as a part of the study.
day). The study considered by estimating the
Chromatographic separation was performed on
equivalent response five times for interday and
column Discovery C18 150 x 4.6mm, 5µ. Gradient
intraday. These results are linked to the RSD
binary pump was used with ambient column
(Relative Standard Deviation). Studies related to
temperature. The components of the mobile phase repeatability done by response estimation of
used for this gradient elution are Buffer (0.1% concentrations of drug analysed. The generated
OPA): Acetonitrile (60:40 %v/v). Volume
results are reported in terms of percentage,
considered for injection was 10 µl. Detection
termed as percentage relative standard deviation.
performed at 292nm by the help of a detector.
Accuracy: Recovery calculated for Semaglutide by
Selection of method and Mobile phase the method of Spiking. Amount of known quantity
Solvent and buffer composition were 40:60%v/v drug was added for a solution sample which is pre-
for this study. 0.1% Ortho Phosphoric Acid was qualified. Estimations were done by measuring the
prepared by considering 1ml makeup to 1000ml curve. Curve derived by calibration with the help of
with the help of Milli-Q Water in-order to make-up peak area determination and applying the peak
the mobile phase. areas for deriving the straight line equation.
Standard preparation Detection/ Quantification limits
2.5mg Semaglutide working Standards was The level of quantification (LOQ) and detection
measured and transferred into 25 ml clean and (LOD) were conducted with the application of a
dried glass flask, diluents 10ml was added, signal to noise methodology.
sonication done for 10 min. And volume was made
Degradation studies
with diluents (100µg/ml Semaglutide).
Oxidation: 1 ml of Semaglutide stock solution, 1
ml hydrogen peroxide with a concentration of 20
866 © Pharmascope Publications | International Journal of Research in Pharmaceutical Sciences

 Arun Kumar Kuna et.al., Int. J. Res. Pharm. Sci., 10(2), 865-873

Figure 1: Specificity Chromatogram for blank

Table 5: Results for accuracy
Spiked Concentration Peak area Added qty. Qty. Found Recovery % Mean
(μg/ml) (n=3) (μg/ml) (μg/ml) (Z) (Z)
50 1040884 5 5.03 100.53 99.73
1035390 4.95 98.94
1038000 4.98 99.70
100 1382618 10 9.97 99.74 100.12
1382650 9.97 99.74
1390486 10.09 100.87
150 1725059 15 14.93 99.54 100.29
1732539 15.04 100.26
1740956 15.16 101.07
Table 7: Forced degradation study
S. No Study Retention Area Plate count Tailing
1 Acid degradation 2.663 646394 3326 1.1
2 Base degradation 2.648 641859 3535 1.0
3 Peroxide degradation 2.638 632885 3071 1.2
4 Thermal degradation 2.673 674101 3691 1.1
5 UV degradation 2.634 675204 2714 1.2
6 Water degradation 2.657 682198 3081 1.2

percent added. For 30 minutes’ solution was kept was diluted in order so that 10ppm solution can be
at 60°C. During this study, the solution was diluted obtained. 10 µl of the obtained solution was
in order so that 10ppm solution can be obtained. considered for injection into HPLC, and recorded
10 µl of the obtained solution was considered for graphs were used for the analysis of samples
injection into HPLC, and recorded graphs were stability.
used for the analysis of samples stability. It is the
Alkali Studies
stability of the prepared sample used for the study.
To 1ml of 2N Sodium hydroxide, 1ml of a prepared
Degradation Study of Acid stock solution of Semaglutide was added, and the
1 ml of Semaglutide stock solution, 1 ml hydrogen obtained solution was diluted to 10ppm. Injection
peroxide with a concentration of 2N added. This of 10 µl was taken into the HPLC system and
preparation was refluxed for 30 minutes by recorded the chromatographs. Activities
considering 60°C. During this study, the solution performed for 30min by considering 60°C.

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 Arun Kumar Kuna et al., Int. J. Res. Pharm. Sci., 10(2), 865-873

Degradation Studies of dry heat Table 2: System Suitability data

Factors Result Acceptance
Maintained conditions are for 6 hours with 105°C.
Limit
During this study, the solution was in over with
Rt 2.689 --
stated conditions. Solution diluted to 10ppm.
Resolution factor NA --
Injection of 10 µl was taken into the HPLC system
Number of theoretical 3145 More than
and recorded the chromatographs.
plates (N) 2000
Photo Stability studies Tailing factor (T) 1.2 Less than 2
This study performed by considering 10ppm Linearity and Range
solution and it is exposed to UV light with the help
of UV chamber for 7 days with photostability Linearity was determined for a concentration of
chamber. The derived solution was diluted for the 2.5 to 15 µg/ml. The correlation coefficient was
0.9998. Linearity results were represented in
study of 10ppm. Injected 10 µl into the HPLC
system, recorded the chromatographs to Table 3 and co-relation graph in Figure 3.
Table 3: Results of Linearity and range
determine the sample stability.
Serial Concentration (µg/ml) Peak Area
Degradation Study with Water 1 2.5 185200
Stress testing at neutral condition was studied by 2 5 347963
refluxing Semaglutide in water for 6hrs by 3 7.5 513667
maintaining a temperature of 60ºc. In this study, 4 10 688904
the resultant solution was diluted to the 10ppm 5 12.5 867268
solution, and 10 µl was injected. This determines 6 15 1043533
the stability of the sample applied for the
degradation study.
RESULTS AND DISCUSSION

Figure 3: Correlation graph

Precision
Figure 2: Structure of Semaglutide
In method validation, intraday and interday
Specificity precision determined. The %RSD for interday
This RP-HPLC method was specific. The retention precision is 0.5 and intraday is 1.0. Precision
time for Semaglutide was 2.689 min. Specificity results were represented in Table 4.
results were represented in Table 1, Figure 2a for Table 4: Results for intraday and interday pre-
blank and Figure 2b for the drug. cision
Table 1: Specificity Data for RP-HPLC S. No Intraday Interday
S. Peak Observation precision Area precision Area
No Name 1 682866 671865
1 Blank Nil 2 679715 676730
2 Placebo Nil 3 685659 670652
3 Standard Rt = 2.689min λ max = 292nm 4 684941 673626
5 698604 679618
System suitability 6 691844 673981
Tailing factor was 1.2 (T), and a number of Mean 687272 674412
theoretical plates were found to be 3145 (N). Std Dev 6839.2 3283.4
System Suitability results were represented in %RSD 1.0 0.5
Table 2.

868 © Pharmascope Publications | International Journal of Research in Pharmaceutical Sciences

 Arun Kumar Kuna et.al., Int. J. Res. Pharm. Sci., 10(2), 865-873

Figure 4: LOD Chromatograph

Figure 5: LOQ Chromatograph

Figure 6: Chromatograph of Acid degradation

Figure 7: Degradation purity plot of Acid

© Pharmascope Publications | International Journal of Research in Pharmaceutical Sciences 869

 Arun Kumar Kuna et al., Int. J. Res. Pharm. Sci., 10(2), 865-873

Figure 8: Degradation chromatograph of Base

Figure 9: Degradation purity plot of Base

Figure 10: Degradation chromatograph of Peroxide

Accuracy determined to be 0.26µg/ml for Semaglutide. The

level of quantification (LOQ) and detection (LOD)
Percentage Mean recovery of accuracy was from
were represented in Table 6 and Figure 4, Figure 5.
99.73 to 100.29. Accuracy results were
Detection sensitivity was determined through
represented in Table 5.
LOD, and the concentration that can be quantified
Table 6: LOD & LOQ at lowest is derived from LOQ.
Results
Parameter Degradation studies
LOD LOQ
RT 2.624 2.634 Degradation studies related to acid, base, peroxide,
Area 23464 85711 thermal, UV and water were represented in Table
Plate count 2571 2550 7. Figure 6 represents acid degradation
USP Tailing 1.5 1.0 chromatograph, and Figure 7 represents the
degradation of acid purity plot. Figure 8 represents
Detection and quantification limits: The LOD degradation chromatograph of base and Figure 9
was found to be 0.08µg/ml, and the LOQ was represents degradation purity plot of a base.
870 © Pharmascope Publications | International Journal of Research in Pharmaceutical Sciences

 Arun Kumar Kuna et.al., Int. J. Res. Pharm. Sci., 10(2), 865-873

Figure 11: Degradation purity plot of Peroxide

Figure 12: Thermal degradation chromatograph

Figure 13: Thermal degradation purity plot

Figure 14: UV degradation Chromatograph

© Pharmascope Publications | International Journal of Research in Pharmaceutical Sciences 871
 Arun Kumar Kuna et al., Int. J. Res. Pharm. Sci., 10(2), 865-873

Figure 15: UV degradation purity plot

Figure 16: Water degradation chromatograph

Figure 17: Water degradation purity plot

Figure 10 represents peroxide degradation methods have contributed for more accurate
chromatograph, and Figure 11 represents the determination of drug by HPLC study.
peroxide degradation purity plot. Figure 12
CONCLUSION
represents thermal degradation chromatograph,
and Figure 13 represents the thermal degradation The method developed was simple, applicable and
purity plot. Figure 13 represents UV degradation sensitive with a validated approach. This analytical
chromatograph, and Figure 14 represents the UV method for determination and elution of
degradation purity plot. Figure 15 represents Semaglutide can be applied for day to day analysis,
water degradation chromatograph, and Figure 16 routine analysis and experimental analysis.
represents water degradation purity plot. Results generated through this study indicate that
Degradation studies performed on Semaglutide this method is accurate.
drug in order to prove that drug considered for this
CONFLICT OF INTEREST
study has stability and the same property is being
exhibited during an experiment with the help of No conflicts of interest.
this study performed. Prepared and executed

872 © Pharmascope Publications | International Journal of Research in Pharmaceutical Sciences

 Arun Kumar Kuna et.al., Int. J. Res. Pharm. Sci., 10(2), 865-873

Acknowledgment
We thank Dr Jagadeesh Kumar Kuna, RMC
Kakinada for his encouragement and support in
providing necessary support for the work. We
greatly acknowledge the receipt of material and
research support from Spectrum Labs, Hyderabad.
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