INTRODUCTION:^1-8

Semaglutide is a glucagon-like peptide 1 (GLP-1) analog used to manage type 2 diabetes along with lifestyle changes. This drug reduces glycosylated hemoglobin (HbA1c) levels and reduces body weight, proving to be effective for patients with type 2 diabetes.¹IUPACname of Semaglutide is 17-{[(1R)-3-[(2-{2-[({2-[2-({[(5S)-5-[(2S)-2-[(2S)-2-[(2S)-2-{2- [(2S)-2-[(2S)-2-[(2S)-2-[(2S)-2- [(2S)-2-[(2S)-2- [(2S)-2-[(2S)-2-[(2S,3R)-2-[(2S)-2- [(2S,3R)-2-{2-[(2S)-2-{2-[(2S)-2-amino-3- (1H-imidazol-4-yl) propanamido]-2-methylpropanamido} -4-carboxybutanamido] acetamido} -3-hydroxybutanamido] -3-phenylpropanamido] -3-hydroxy butanamido] -3-hydroxypropanamido]-3-carboxypropanamido] -3-methylbutanamido] -3-hydroxypropanamido]-3-hydroxypropanamido]-3- (4-hydroxyphenyl) propanamido]-4- methylpentanamido]-4-carboxybutanamido] acetamido} -4-carbamoylbutanamido] propanamido] propanamido]-5-{[(1S)-1-{[(1S)-1- {[(1S,2S)-1-{[(1S)-1-{[(1S)-1-{[(1S)-1-{[(1S)-1-{[(1S)-4-carbamimidamido-1-[({[(1S)-4-carbamimidamido-1-[(carboxymethyl) carbamoyl]butyl] carbamoyl} methyl) carbamoyl]butyl] carbamoyl}-2-methylpropyl] carbamoyl}-3-methylbutyl] carbamoyl}-2-(1H-indol-3-yl)ethyl] carbamoyl}ethyl] carbamoyl}-2-methylbutyl] carbamoyl}-2-phenylethyl] carbamoyl}-3 carboxypropyl] carbamoyl} pentyl] carbamoyl} methoxy) ethoxy] ethyl} carbamoyl) methoxy] ethoxy}ethyl) carbamoyl]-1-carboxypropyl] carbamoyl} heptadecanoic acid. The molecular weight and chemical structures were 720.196g/mol and C₁₈₇H₂₉₁N₄₅O_59. Structure of Semaglutide shown in Fig No: 1

There is only one procedure is reported for quantification of Semaglutide. Updating of analytical methods is necessary based on requirements. This method developed with the aim of stable, rapid, economic, accurate, precise and robust method. This is suitable for determination of drug substance and drug product as well as per ICH guidelines.

Fig No 1 Structure of Semaglutide.

MATERIALS AND METHODS:

Materials:

Semagluitde API is received as gift sample from Sprectrum Labs. grade Acetonitrile, Methanol, Millipore MilliQ water and Glacial acetic acid were purchased from Merck, Germany, Regis Technologies Inc, USA.

Equipment:

The HPLC system is used for method development and method validation. Detection was carried by Waters with a diode array detector (model: 2996 detector 2487 separation module). The output signal was supervised and processed using Waters Empower 2 Software. LC GC Ragward Dual Range balance was used to perform weighing. Photostability studies were carried out in a photo stability chamber. Thermal stability studies were performed in at thermostat dry air.

Liquid Chromatographic Conditions:

Preparation of 28µg/ml of Semaglutide Standard Solution:

Accurately weighed 14mg of Samaglutide pharmaceutical substance and transferred into 10ml dry volumetric flask. Add 3/4th volume of diluent(0.01N Potassium dihyrogen ortho phosphate: Acetonitrile (50:50)), sonicated for 5 minutes and make up to the final volume with diluent.

1ml from the above two stock solutions was taken into a 10ml volumetric flask and made up to 10ml. (28µg/ml of Semaglutide)

Preparation of 28µg/ml of Semaglutide Sample Preparation:

5 tablets (Rybelsus) were weighed and the average weight of each tablet was calculated, then the weight equivalent to 1 tablet was transferred into a 50mL volumetric flask, 25mL of diluent added and sonicated for 25 min, further the volume made up with diluent and filtered. (280µg/ml of Semaglutide)

1ml was pipette out into a 10ml volumetric flask and made up to 10ml with diluent. (28µg/ml of Semaglutide).

Validation of Semaglutide by HPLC:

The RP-HPLC method for stability indicating method for estimation of Semaglutide was validated for the parameters like linearity, accuracy, reproducibility, specificity, robustness, LOD, LOQ and stability as per the ICH guidelines.

System Suitability:

System suitability studies used to verify the system performance. The system suitability was evaluated by six replicated standards of Semaglutide at a concentration of 28µg/ml of Semaglutide. All the parameters like relative standard deviation, peak tailing and theoretical plate number were measured.

Specificity/stability:

Evaluation of specificity of HPLC method was used to ensure that there was no interference of excipients and degradants in the formulations. Stress studies were formed by using concentration of 28µg/ml of Semaglutide active pharmaceutical ingredient to provide stability indicating property and specificity of proposed method. Degradation studies were evaluated under accelerated conditions. Sample solutions were exposed to photolytic stress (1.2 million lux hours followed by 200 Watthours), heat (exposed at 105°C for 15h), acid (1N HCl for 2 hours at 60°C), base (1N NaOH for 2 hours at 60°C), oxidation (10% peroxide for 30 min at 60°C), and humidity (exposedto85% RH for 72 hours).

Linearity:

If the obtained test results are directly proportional to the concentration of analyte that indicates the linearity of method. Linearity studies were performed by injecting three replicates of six different concentrations of Semaglutide (7, 14, 21, 28, 35, 42μg/ml). Calibration curve was plotted for average areas and concentration. Correlation coefficient, slope and intercept values were calculated from the calibration curve. In general, correlation coefficient value (r²) should be 0.999 is considered as acceptable fit for the data to regression line.

Accuracy:

Closeness between true value and measured value is considered as accuracy. Accuracy is determined by calculating the percentage recovery of analyte present in the three replicate samples of three different concentrations (14, 28, 42μg/ml) of standard solutions of Semaglutide. The mean percentage recovery of sample should be within 99-101% W/V to be accepted.

Precision:

Degree of agreement among the individual tests is considered as precision. Precision also considered as reproducibility of results of the developed method. Precision was checked by injecting six replicate standard preparations at a concentration of 28μg/ml and percentage relative standard deviation was calculated. Intermediated precision also assessed by using different analyst and a different instrument in the same laboratory. As per ICH guidelines the %RSD should be less than 2%.

Limits of detection (LOD) and quantification (LOQ):

LOD is the lowest concentration in a sample that can be detectable, but not necessary to quantify under given experimental specification. LOQ is the lowest concentration of analyte that can be determined with acceptable precision and accuracy. LOD and LOQ were calculated by sung the formulas LOD = 3.3SD/S and LOQ = 10SD/S, where S= slope of calibration curve and SD = standard deviation of peak area. LOD and LOQ also determined at signal-to-noise ratio of 3:1 and 10:1 respectively.

Robustness:

The developed method was remain unaffected by small, but deliberate variations in method parameters is considered as robustness. Evaluation of robustness indicated the reliability of method during normal usage. Under deliberate variations in parameters peak resolution, tailing and number of theoretical plates were evaluated. To study the outcome of the flow rate on the developed method, it was changed ± 0.2mL/minute. The effect of column temperature on the developed method was studied at ±5°C (instead of 30°C). The mobile phase composition was changed ±10% from the initial composition of organic phase. In all the above varied conditions, the aqueous component of the mobile phase was held constant.

Assay:

Assay is a method used to determine the amount of Semaglutide present in pharmaceutical product. Rybelsus conventional tablet bearing the label claim 14mg of Semaglutide used as sample to conduct assay. Six replicate sample solutions of 28μg/ml concentration were injected and percentage purity was calculated.

RESULTS AND DISCUSSION:

Development of HPLC method and Optimization:

C18 column (Azilent C18 150x 4.6, 5mm) is used as stationary phase and 0.01N Potassium dihyrogen ortho phosphate: Acetonitrile (50:50) used as a mobile phase at a flow rate of 1.0mL/min . The run time was 5 min and the injection volume was 10μl. Detection was done at the wavelength of 230nm. The operating temperature of HPLC system was 30°C. Retention time was found to be 2.222min. Chromatogram was shown in Fig No 2.

Fig 2: Optimized Chromatogram

System Suitability:

The system suitability was assessed by six replicated standards of Semaglutide at a concentration of 28µg/ml of Semaglutide. All the parameters like relative standard deviation, peak tailing and theoretical plate number were measured. The acceptance criterion was less than 2% percentage relative standard deviation. Results were shown in Table no 1.

Table No.1 Results for System Suitability

Injection	Rt	Area	USP plate count	USP tailing
Injection-1	2.220	1064993	8645	1.25
Injection-2	2.221	1052653	8005	1.25
Injection-3	2.221	1065689	8394	1.27
Injection-4	2.221	1045413	8170	1.26
Injection-5	2.222	1061636	8292	1.23
Injection-6	2.223	1050121	7922	1.29
Avg		1056751
SD		8496.9
%RSD		0.8

Specificity/stability:

Blank, placebo and degradation samples were analyzed with the above mentioned HPLC conditions using a PDA detector to monitor the homogeneity and purity of the Semaglutide. Blank, Placebo, Individual impurities of Semaglutide verified and proved to be non-interfering with each other thus proving the specificity of the method, retention time of Semaglutide. Degradation was not observed in photolytic stress, humidity and thermal stress studies. Degradant peaks were observed in oxidative, acidic, alkaline conditions. It was interesting to note that all the peaks due to degradation were well resolved from the peaks of Semaglutide. The verification of peak purity indicates that there is no interference from degradants, facilitating error-free quantification of Semaglutide. Hence the method is considered to be “stability-indicating.” The specificity results were shown in Table 2.

Table no.2 Forced degradation Results

S. No	Condition	Semaglutide Area	Semaglutide % Rec	% Degradation Semaglutide
1	acid	998165	94.45	5.55
2	Base	1012607	95.81	4.19
3	Peroxide	1012891	95.84	4.16
4	Thermal	1034078	97.85	2.15
5	UV	1046158	98.99	1.01
6	Humid	1051469	99.49	0.51

Linearity:

Linear calibration plots are tested at different concentration levels. The correlation coefficient obtained was 0.999 for Semaglutide. The slope and y-intercept values were also in satisfactory limits, which confirmed good linearity between peak areas and concentration. Results were mentioned in table 3. The linearity graph was shown in image 3.

Table No.3: Linearity Observation of Semaglutide

S. No.	Linearity Level	Areas	AVG Area	Concentration
1	I	275217	275039.5	7ppm
		274862
2	II	536441	536387.5	14ppm
		536334
3	III	792340	797934.5	21ppm
		803529
4	IV	1053972	1059238	28ppm
		1064503
5	V	1329321	1321876	35ppm
		1314431
6	VI	1573723	1581478	42ppm
		1589233
Correlation Coefficient				0.999

Fig 3: Linearity of Semaglutide

Accuracy:

Recovery of Semaglutide was found to be 99.0% to 101.0%. The summary of % recovery for individual data was mentioned in Table 4.

Precision:

The percentage RSD for the area of six standard injections results should not be more than 2%. From the results of precision, the % RSD data of Semaglutide was 0.8%. From the results obtained for the intermediated precision study % RSD data of Semaglutide was 0.2% for Semaglutide high precision of the method. Results were within the limit as per ICH guidelines. The results are shown in Tables 5 and 6.

Table No 4: Accuracy results

%	TRIAL	AREA(y)	m	C	y-c	Total Conc	Added Conc	Std Conc	Amt Rec	% Rec	AVG %Rec
50%	1	1579887	37556	7319	1572568	41.87262	14	28	13.87	99.09	100.3
	2	1592068	37556	7319	1584749	42.19696	14	28	14.20	101.41
	3	1587738	37556	7319	1580419	42.08166	14	28	14.08	100.58
100%	1	2097964	37556	7319	2090645	55.6674	28	28	27.67	98.81	99.17
	2	2100350	37556	7319	2093031	55.73094	28	28	27.73	99.04
	3	2107126	37556	7319	2099807	55.91136	28	28	27.91	99.68
150%	1	2630139	37556	7319	2622820	69.83758	42	28	41.84	99.61	100.68
	2	2651341	37556	7319	2644022	70.40212	42	28	42.40	100.96
	3	2659506	37556	7319	2652187	70.61953	42	28	42.62	101.48

Table no.5 Results for Precision

Injection	Area
Injection-1	1064993
Injection-2	1052653
Injection-3	1065689
Injection-4	1045413
Injection-5	1061636
Injection-6	1050121
Avg	1056751
SD	8496.9
%RSD	0.8

Table no.6 Results for Intermediate Precision

INJECTION	AREA
Injection-1	1054867
Injection-2	1057759
Injection-3	1058435
Injection-4	1061281
Injection-5	1058625
Injection-6	1061861
Avg	1058805
SD	2539.3
%RSD	0.2

Limits of detection (LOD) and quantification (LOQ):

LOD and LOQ values were found to be 0.007µg/ml and 0.022µg/ml respectively. It was considered that results were within the limits as per ICH guidelines.

Robustness:

Robustness studies were studied by deliberate change in the flow rate (+ 2ml/min), column temperature (+5^oC) and mobile phase ratio. No significant effect was observed on system suitability parameters deliberate change such as resolution, RSD, tailing factor, or the theoretical plates of Semaglutide. Thus, the method was found to be robust with respect to variability in applied conditions.

Assay:

The assay result was found to be 99.99 % w/w for Semaglutide. The results were within the limits as per the ICH guidelines and standard and sample results were shown in table 7.

Table no.7 Assay Results

S. No	Standard Area	Sample Area	% Assay
1	1067527	1064993	100.77
2	1052591	1052653	99.60
3	1062423	1065689	100.84
4	1063984	1045413	98.92
5	1035821	1061636	100.45
6	1052388	1050121	99.36
AVG	1055789	1056751	99.99

CONCLUSION:

The proposed HPLC method was found to be stable, simple, specific, precise, accurate, rapid and economical for estimation of Semaglutide pharmaceutical substance and pharmaceutical product. Chromatographic separation of semaglutide was achieved on Azilent C18 150x 4.6, 5mm. Potassium dihyrogen ortho phosphate Buffer: Acetonitrile: taken in the ratio 60:40 was used as mobile phase, at the flow rate of 1.0ml/min with PDA detectors at 230nm. The retention times were 2.222min for Semaglutide. The developed method was validated in terms of accuracy, precision, linearity, robustness and ruggedness, and results will be validated statistically and performed stability studies according to ICH guidelines. The system suitability parameters were within limit; hence it was concluded that the system was suitable to perform the assay. The developed method was stable under accelerated conditions like acid, basic, oxidation, thermal and humid conditions. Therefore, it was concluded that the performed method can be used for determination Semaglutide in pharmaceutical formulations.

ACKNOWLEDGEMENT:

The authors wish to thank the management of Vels Institute of Science, Technology and Advanced Studies (VISTAS), for supporting this work. The authors wish to thank Spectrum labs, for providing facilities to conduct this work. The authors wish to acknowledge the Hetero labs for providing the samples for their research. They would also like to thank colleagues for their support to complete research work.

CONFLICT OF INTEREST:

The authors have no conflict of interests.

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Received on 07.02.2020 Modified on 17.04.2020

Research J. Pharm. and Tech 2021; 14(3):1385-1389.

DOI: 10.5958/0974-360X.2021.00247.X