Analytical Method Development and
Validation of UV-Visible Spectrophotometric
Method for the Estimation of Saxagliptin in
Deepika Joshi1*, Bhavana Singh1, Archana Rautela2 and Nidhi Semwal1
1School of Pharmaceutical Sciences, India
2Gyani Inder Singh Institute of Professional Studies, India
Submission: March 24, 2021; Published: April 22, 2021
*Corresponding author: Deepika Joshi, School of Pharmaceutical Sciences, SGRRU, India
How to cite this article:Deepika J, Bhavana, Archana R, Nidhi S. Analytical Method Development and Validation of UV-Visible Spectrophotometric Method
for the Estimation of Saxagliptin in Gastric Medium. Glob J Pharmaceu Sci. 2020; 8(2): 555735. DOI: 10.19080/GJPPS.2021.08.555735.
Aim: A simple, accurate, precise, cost effective, rapid and sensitive UV/visible spectrophotometric method was developed for the determination of Saxagliptin in active pharmaceutical dosage form. The developed method was validated as per ICH guidelines.
Method: The purity of saxagliptin was characterized by solubility profile, melting point, Fourier Transform Infra-Red. The drug was analyzed using UV/visible spectrophotometric method was validated in terms of linearity, accuracy, precision, specificity, Limit of detection and Limit of quantitation. The solvent used was methanol: water (15:85, v/v) and the wavelength corresponding to maximum absorbance of the drug were found at 204 nm.
Result: Melting point of drug was found 101°C nearly corresponds to its actual melting range. The linear response for concentration range of 2-10 μg/ml of saxagliptin was recorded with y = 0.1126x - 0.0103, regression coefficient r2 = 0.99068. The accuracy was found between 93.75- 104.16%. Precision for intra-day and inter-day was found to be within the limits. To establish the sensitivity of the method, limit of detection (LOD) and limit of quantification (LOQ) were determined which were found to be 6.77 μg /mL and 20.33 μg /mL respectively.
Conclusion: The drug was confirmed by interpretation of UV spectra. Hence proposed method stands out validated and thus may be used for routine analysis of Saxagliptin in pharmaceutical dosage forms.
Keywords: Spectrophotometric method; Saxagliptin; Methanol; Water; Melting point
Abbreviations: UV spectroscopy: Ultraviolet Spectroscopy; ICH: International Conference on Harmonisation; DPP: Dipeptidyl Peptidase; FTIR: Fourier Transform Infra-Red; LOD: Limit of Detection; LOQ: Limit of Quantification
Saxagliptin (SXG) is chemically (1S, 3S, 5S)-2- [(2S)-2-Amino-2-(3 hydroxytricyclo [22.214.171.124,7] dec- 1-yl) acetyl]-2-azabicyclo [3.1.0] hexane-3-carbonitrile previously identified as BMS-477118 as shown in figure 1. The empirical formula is C18H25N3O2.H2O, and the molecular weight is 333.43 [1-4]. Saxagliptin is an oral hypoglycemic or anti-diabetic drug of the dipeptidyl peptidase-4 (DDP-4) inhibitor class. The inhibition of DPP-4 increases levels active of glucagon like peptide 1 (GLP-1), which inhibits glucagon production from pancreatic alpha cells and increases production of insulin from pancreatic beta cells. In 2009, U.S. Food and Drug Administration (FDA) approved
saxagliptin and sold under the brand name Onglyza. In adults with type-2 diabetes mellitus, saxagliptin is suggested as an add-on to diet and exercise to improve glycemic control [5-11].
Literature survey reveals that the drug can be estimated only by LC-MS/MS, spectrophotometric method have been reported [12,13]. The aim of the present work was to develop a simple, sensitive, precise and accurate UV/Visible spectrophotometric method for the determination of saxagliptin in its pure form and pharmaceutical formulations further, to validate the developed method.
All the chemicals used were of analytical grade. All the
solutions were freshly prepared in Methanol and 0.1N HCl (15:85,
v/v). Authentic of saxagliptin were obtained as gift samples from
Mylan Laboratories Limited, Hyderabad
Saxagliptin was estimated using UV-Visible spectrophotometer
(Shimadzu model 1800 double beam) at the wavelength of
maximum absorption (204 nm) in acidic medium containing
0.1N HCl. The drug was characterized by solubility, melting
point, and Fourier Transform Infra-Red (FTIR) techniques. The
analysis of the drug was carried out by UV-Visible method which
was validated analytical parameters like linearity, precision, and
accuracy as per guidelines laid down by International Conference
on Harmonization (ICH).
Excess amount of saxagliptin was dissolved in 10 ml distilled
water till a saturated solution was obtained. The saturated
solution of saxagliptin was stirred for 48 hrs on magnetic stirrer
at 100rpm and room temperature (at 25 ± 1ºC). Then the sample
was centrifuged for 10 min at 10,000 rpm. Clear supernant was
collected using 0.22 μm syringe filter and analysed using UV
spectrophotometer. The results were analysed and noted .
Melting point of saxagliptin was determined using capillary
melting point apparatus. In this method a small quantity of drug
was filled in a capillary tube open both the ends and it was placed
along with thermometer in melting point apparatus.
FTIR spectrum was used as an analytical technique for
identification of pure drug sample by KBr method using FTIR
spectrometer (Agilent Technologies, USA). Peaks of individual
pure drug were compared with reference FTIR peaks. Samples
were previously prepared with KBr at 1: 5 (sample: KBr, w/w). KBr
disks were prepared by compressing the powders at a pressure of
5 tons for 3 min in a hydraulic press and scanned against a blank
KBr disk at wave numbers ranging from 500 to 4000 cm-1 with a
resolution of 1.0 cm-1 .
Saxagliptin was weighed equivalent to 100mg and transferred
into 100ml volumetric flask then 15ml of methanol was added and
shaked well to dissolve it after that the volume was made up to
100ml with 0.1N HCl. From that 2ml of solution was withdrawn
and taken in 100ml volumetric flask. The volume was adjusted
with diluent up to up to 100ml with 0.1N HCl [16,17] as shown
in table 1.
Prepare a series of dilute solution from the above stock
solution according to table 2 in 10 ml volumetric flask. Measure the absorbance of the solutions at 204 nm using distilled water
as a blank. Plot a graph by taking concentration (μg/ml) on X-axis
and absorbance on Y-axis. This plot gives a straight line and the
linearity can be determined using y = mx + C formula. From the
calibration curve calculate the Coefficient of determination R2
value, slope m and intercept C using the following formula, OR by
Pipette out 1,2,3,4,5,6,7, and 8 ml of working solution and
transfer into separate 10 ml volumetric flasks. Dilute all of them
to 10 ml with water to get solution of concentrations to 2, 4,
6,8,10,12,14,16 μg/ml respectively
Appropriate aliquots of saxagliptin working standard solutions
were taken in different 10 ml volumetric asks and diluted up to
the mark with distilled water to obtain final concentrations of 2,
4, 6, 8, 10 μg/ml. Calibration curves were constructed by plotting
absorbance versus concentrations and regression equations were
calculated for both the drugs.
Intraday precision was determined by analyzing the drugs
at concentrations (4μg/mL) and each concentration for three
times, on the same day. Inter-day precision was determined
similarly, but the analysis is carried out daily, for two consecutive
days. Repeatability (intraday) of the method was determined
by analyzing six samples of the same concentrations of the drug
(4μg/mL). The absorbance of each was measured and reported in
terms of relative standard deviation to obtain the variation.
The accuracy of the method was determined by calculating
recoveries of saxagliptin by method of standard additions at three
different levels 60, 100 and 140 %. Mean percentage recovery was
determined. Recovery values were calculated and shown in table
The Detection Limit of an individual analytical procedure is
the lowest amount of analyte in a sample which can be detected
but not necessarily quantitated as an exact value. The detection
limit (LOD) may be expressed as. LOD= 3.3σ/S
Where σ = Relative standard deviation of the response. S = the
slope of the calibration curve (of the analyte).
FTIR spectrum for identification of pure drug sample was
done by FTIR spectrometer. Major absorption peaks of saxagliptin
was found at 3450.12 cm-1
(N-H stretching), 2912.61 cm-1
(C-H stretching), 3301 cm-1
(OH stretching), 1614.47 cm-1
stretching), 1255.70 cm-1
(C-O stretching) as shown in figure 2
which corresponds to the literature peaks confirming the purity
of drug sample.
Selection of detection wavelength: The wavelength of
saxagliptin was determined to be 204 nm as shown in figure 3.
Preparation of standard plot for saxagliptin: Absorbance
of the resultant solution was measured at 204 nm using blank.
A graph was plotted between the concentrations and their
respective absorbance. The response of the drug was found linear
in the entire investigational range of 2 to 10μg/ml as shown in
table 1. The calibration curve showed the linear equation as, y
= 0.1126x - 0.0103, with a correlation coefficient, r2 = 0.99068,
where y represents absorbance (optical density) and x represents
the concentration (μg /ml) as shown in figure 4.
Method validation: The developed method was validated as
per ICH guidelines for the following parameters:
Linearity: The linearity for Saxagliptin was found to be linear
in the range of 2-10 μg/ml. The regression equation was found to
be y = 0.1126x - 0.0103, r2 = 0.99068.
Range: The observed range of saxagliptin in test solution was
observed from 0.097± 0.0005 to 0.871± 0.001.
Accuracy: The accuracy of the analytical method for
Saxagliptin was determined at 60%, 100% and 140% levels of
standard solution. Absorbance was measured at 204 nm and
results were expressed in terms of % recoveries in table 2.
Precision: The Intra-day and Inter-day precision were
carried out using same optimized conditions. The precision
(measurement of inter-day, intra-day repeatability) results
showed good reproducibility with the relative standard deviation
(% RSD) below 2.0 % as shown in table 3 & 4 respectively. This
indicated that the method was highly precise.
Limit of detection (LOD) and limit of quantification (LOQ):
LOD and LOQ of method were determined to be 6.77 μg /mL and
20.33 μg /mL respectively. LOD and LOQ indicate that method was
highly sensitive and fast.
The method was validated and found to be simple, sensitive,
accurate and precise as per ICH guidelines . The % RSD for
the validation parameters was found to be less than 2%. Hence
proposed method may be used for routine analysis of these drugs
in pharmaceutical dosage forms. Accuracy of proposed method
was confirmed by performing accuracy studies that showed the
results within the range. Precision of proposed UV method was
confirmed by performing intra-day and inter-day precision.
Results were well within acceptance criteria that indicate
excellent scope of the method for the determination of Saxagliptin
in pharmaceutical dosage forms and bulk.
All authors have contributed significantly in the preparation
of the manuscript and are in agreement with the content of
the manuscript and agree to submission to Global Journal of
Pharmacy & Pharmaceutical Sciences, Juniper Publishers.
Authors are grateful to Mylan Laboratories Limited, Hyderabad for providing the gift sample. The authors express gratitude to
School of Pharmaceutical Sciences, SGRRU, Dehradun.
The authors declare that there is no conflict of interest that
could be prescribed as prejudging the impartiality of the review.
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
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