INTRODUCTION:

A reagent that is being used more and more is 1,2-Naphthoquinone-4-sulfonate (NQS), which is used in ultraviolet-visible (UV-Vis) spectrophotometric detection to analyze amines and amino acids. Both primary and secondary amino groups can react with NQS in a basic solution at moderate temperatures to create derivatives that can be detected using spectrophotometry.

Sitagliptin is a medication used for the treatment of type 2 diabetes. It belongs to the class of drugs called dipeptidyl peptidase 4- (DPP-4) inhibitors. Sitagliptin works by increasing the body's production of incretin hormones, a type of hormone which encourage insulin release and prevent glucagon synthesis, so decreasing blood sugar levels.

Sitagliptin is used to improve glycaemic control in people with diabetes type 2 mellitus. It can be taken alone or in combination with other antidiabetic medications such metformin, which sulfonylureas, or insulin.^1,2

Sitagliptin is a medication to prevent diabetes, the chemical formula⁵ of sitagliptin drug-C16H15F6N5O as shown in Figure 1.

Fig. 1: Structure of Sitagliptin

MATERIALS AND METHODS:

Chemicals required:

Sitagliptin drug, 1,2-Napthoquinone, 4-Sulphonate reagent, Methanol, 14 pH Buffer solution, Distilled water.

Instruments required:

Double beam UV-Visible Spectrophotometer, Weighing balance.

Preparation of reagents:

Buffer solution of pH 14: 4 gm of Sodium Hydroxide (salt of strong base) was taken and dissolve 2 gr of Potassium Chloride (salt of strong acid) in 100ml Volumetric flask with distilled water.^9,10,11

Preparation of 1,2-Napthoquinone 4-Sulphonate Reagent: Take 0.05gm of NQS reagent in 50 ml Volumetric flask and make up the volume with Distilled water.

Preparation of standard Stock solution: Take 0.01g of Sitagliptin pure drug(Gift sample from Laurel pharma labs) in 10ml Volumetric flask, dissolve and make up with methanol.^6,7,8

Preparation of Sitagliptin Sample solution:

Sitagliptin dose form assay:

The tablets' uniform weight: Taken ten tablets, each of which was weighed individually and then combined. Ten tablets were ground into a fine powder after being weighed. Ten milliliter volumetric flask, 10 mg of the corresponding Sitagliptin tablets were weighed, dissolved with methanol using sonication, and then filled up with methanol. After preparing a further dilution to 50µg/ml, 1ml of the pH14 buffer solution and 1 ml of NQS reagent were added. By utilizing the formula "y = mx + c," the percentage of the assay was determined to be 98.78%.

The percentage test was determined to be between 98 to 102%, which is within limits.

Method Development:

1ml of standard solution (SGT) was taken to that 1ml of 14 pH Buffer solution was added next to that add 1ml of 1,2-Napthoquinone, 4- Sulphonate was added formation of Reddish orange coloured complexas shown in Figure 2.

Chemical Reaction:

Fig. 2: Chemical reaction

The optimized wavelength is 454nm and the absorbance is 1.2545 as shown in Figure 3. The optimized temperature is 24 C for 1 hours, the colour is optimized and the colour is Reddish orange colour.

Fig. 3: Wavelength of sitagliptin standard solution

The test was performed by using different pH levels to optimize the colour. Finally, the method was optimized for pH14 value and the results of three different ranges as shown in the Fig 4.

Fig. 4: Colour stability with respect to pH

RESULTS AND DISCUSSION:

Method Validation Parameters:

Guidelines: This technique was verified in accordance with ICH specifications Q2R1, The validation parameters were set in compliance to the International Conference on Harmonization's (ICH) specifications^3,4

Linearity:

Fig. 5: Linearity data

Result: The correlation coefficient (r2) has been determined to be 0.9975, being within the acceptable range according to ICH criteria as shown in Figure 5

Accuracy:

50%: 20 ppm (standard) spiked to 50 ppm (sample); 1 ml of the spiked standard solution and 1 ml of the sample were pipetted into a 10-ml volumetric flask. 100%: 40ppm (standard) spiked to 50ppm (sample); 1 ml of the spiked standard solution and 1 ml of the sample were pipetted into a 10-ml volumetric flask. 150%: 60 ppm (standard) spiked to 50 ppm (sample); 1 ml of the spiked standard solution and 1 ml of the sample were pipetted into a 10-ml volumetric flask. Three degrees of additions of chemicals and reagents (50%, 100%, and 150%) were made. 1 ml of pH 14 buffer and 1 ml of NQS reagent. Add water to make up the remaining volume, and the colour appears to be a complicated reddish-orange.

THE RESULTS:

The accuracy recovery range was determined to be 98.5–100.1%, which is within the ICH (98–102%) criteria as shown in Table 1.

Precision:

Results of the preparation of six times 6 solutions are listed below. It was discovered that the Sitagliptin's %RSD of repeatability was 0.0126. For intra-day precision, the percentage of the standard deviation (RSD) was found to be 0.0192, in the morning and 0.016 in the eveningshown in Table 2,3,4. Similarly, the percentage RSD for inter-day precision was determined to be 0.0165 on day 1 and 0.0156 on day 2. All of those results, as per ICH criteria, were within the acceptable range^12-15

Repeatability of Sitagliptin:

Table 2: Repeatability precisionof sitagliptin

Concentration(µg/ml)	Repeatability Absorbance
50 µg/ml	0.6626
50 µg/ml	0.6623
50 µg/ml	0.6624
50 µg/ml	0.6625
50 µg/ml	0.6627
50 µg/ml	0.6622
Mean	0.66245
Standard Deviation	0.0000836
%RSD	0.0126

Table 3: Intra-day precision data

Concentration(µg/ml)	Absorbance
50 µg/ml	Morning	Evening
50 µg/ml	0.6632	0.6623
50 µg/ml	0.6634	0.6624
50 µg/ml	0.6638	0.6628
50 µg/ml	0.6639	0.6622
50 µg/ml	0.6637	0.6625
50 µg/ml	0.6633	0.6628
Mean	0.66355	0.6625
Standard Deviation	0.000128	0.00011
%RSD	0.0192	0.016

Table 4: Inter-day precision data

Concentration(µg/ml)	Absorbance
50 µg/ml	Day 1	Day 2
50 µg/ml	0.6642	0.6652
50 µg/ml	0.6648	0.6653
50 µg/ml	0.6643	0.6658
50 µg/ml	0.6647	0.6657
50 µg/ml	0.6642	0.6654
50 µg/ml	0.6644	0.6656
Mean	0.6644	0.6655
Standard Deviation	0.00011	0.000104
%RSD	0.0165	0.0156

The Limits of Quantification (LOQ) and Limits of Detection (LOD):

It was determined that the limits of detection (LOD) and limits of quantification (LOQ) were, respectively, 0.02376µg/ml and 0.072µg/ml.

Table 5: Robustness data of sitagliptin.

Concentration (µg/ml)	Absorbance at 453nm	Absorbance at 454nm	Absorbance at 455nm
50 µg/ml	0.6723	0.6626	0.6692
50 µg/ml	0.6748	0.6623	0.6694
50 µg/ml	0.6742	0.6624	0.6695
50 µg/ml	0.6726	0.6625	0.6694
50 µg/ml	0.6782	0.6627	0.6698
50 µg/ml	0.6794	0.6622	0.6694
Mean	0.67525	0.66245	0.66945
SD	0.00130	0.0000836	0.0000883
%RSD	0.192	0.0126	0.0131

Two separate analysts conducted the ruggedness analysis; the results were Analyst1%RSD 0.0192 and Analyst2%RSD 0.016shown in Table 5. We examined the robustness using two wavelengths, +1(%RSD 0.0131) along with -1(%RSD 0.192), and the findings were within the acceptable ranges according to ICH guidance.^16-25

Bioanalytical Method Development:

After adding 1 ml of plasma sample to 1 ml of 50 ppm sitagliptin standard drug, 2 ml of diethyl ether and 4 ml of isopropanol, the mixture was centrifuged at 1000 rpm for 15 minutes. The supernatant was then transferred into a 10 ml volumetric flask, where it was combined with 1 ml of NaoH and 1 ml of NQS reagent. The final amount was adjusted to 10 ml using diluent. The absorbance of the solution was measured at a particular wavelength of 454 nm in comparison to the blank methanol. % The bioanalytical method's recovery was found to be 98.58%.

DISCUSSION:

The developed UV-visible spectrophotometric method for Sitagliptin using NQS reagent proved to be simple, accurate, and sensitive. The drug formed a stable reddish-orange complex with NQS at pH 14, showing maximum absorbance at 454 nm. The method showed good linearity (5–120 μg/ml, r² = 0.9975), with accuracy (98.5–100.1%) and precision (%RSD < 2%) within ICH limits. Sensitivity was confirmed by low LOD and LOQ values. The method was robust, rugged, and successfully applied to plasma samples with 98.58% recovery. These results confirm its suitability for routine quality control and bioanalytical applications.

CONCLUSION:

The above developed method is simple, economical and precise for analytical validation of Sitagliptin. The results obtained are within the limits hence the developed method is useful for routine analysis. It demonstrates excellent linearity, precision, and accuracy within ICH guidelines and forms a stable complex with significant absorbance at 454 nm. The method is robust and effectively applicable to plasma samples, confirming its reliability for routine quality control and bioanalytical use.

ACKNOWLEDGEMENT:

The Department of Pharmaceutical Analysis team and our beloved Principal, Prof. M. Sumakanth Mam, deserve special recognition for providing this opportunity.

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Received on 18.10.2024 Revised on 11.02.2025

Accepted on 07.05.2025 Published on 13.01.2026

Available online from January 17, 2026

Research J. Pharmacy and Technology. 2026;19(1):296-300.

DOI: 10.52711/0974-360X.2026.00042

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Percentage Level	Sample Absorbance	Spiking Absorbance	Total Absorbance	% Recovery	Mean % Recovery
50% (50ppm +20ppm	0.6620	0.3159	0.9770	99.7%	99.1%
			0.9745	98.92%
			0.9740	98.76%
100% (50ppm +40ppm)	0.6620	0.5768	1.2355	99.42%	99.41%
			1.2359	99.49%
			1.2350	99.34%
150% (50ppm + 60ppm)	0.6620	0.7865	1.4485	100.0%	100.3%
			1.4501	100.2%
			1.4562	100.9%