Int J App Pharm, Vol 16, Issue 5, 2024, 349-357Original Article
DEVELOPMENT AND VALIDATION OF A SPECIFIC AND UNIQUE DUAL POLARITY ESI-LC-MS/MS METHOD FOR SIMULTANEOUS DETERMINATION OF SEMAGLUTIDE AND DAPAGLIFLOZIN IN HUMAN PLASMA
JAGAPATHI RAJU VATSAVAYI
, NALANDA BABY REVU*
GITAM School of Pharmacy, GITAM (Deemed to be University), Visakhapatnam-530045, Andhra Pradesh (State), India
*Corresponding author: Nalanda Baby Revu; Email: nrevu@gitam.edu
Received: 09 Apr 2024, Revised and Accepted: 24 Jun 2024
ABSTRACT
Objective: To develop a method capable of simultaneous quantification and estimation of semaglutide and dapagliflozin which are being studied as a prospective combination therapy for treating Diabetes.
Methods: An intricate protein precipitation extraction technique was employed using verapamil and tolbutamide as internal standards for semaglutide and dapagliflozin, respectively. The two compounds were separated on a Kinetex C18 (50 mm x 2.1 mm, 5 µ Particle size) column, with a dual polarity ionization Electro Spray Ionization (ESI) on a Liquid chromatograph Tandem Mass Spectrometry (LC-MS/MS) instrument. The detection was carried out with a Multiple Reaction Monitoring (MRM) method, and a gradient program utilizing Acetonitrile and water as mobile phases to achieve a separation in 3 min.
Results: The method established was proved linear over a working range of 1.00 ng/ml to 1000 ng/ml and 2.00 ng/ml to 2000 ng/ml for semaglutide (r2>0.98) and dapagliflozin (r2>0.98) respectively in human plasma. The accuracy, recovery, and matrix effects were within acceptable limits. The stability was also established under various conditions as necessitated by the International Council for Harmonisation of Technical Requirements of Pharmaceuticals for Human Use (ICH) M10 guideline on Bioanalytical method validation.
Conclusion: This highly selective and sensitive method where 1.00 ng/ml for semaglutide and 2.00 ng/ml for dapagliflozin as the Lower Limit of Quantification (LLOQ) can be utilized for estimation in human plasma will facilitate the further application to pharmacokinetic and bioequivalence studies for combination of these two drugs in pharmaceutical dosage forms.
Keywords: Semaglutide, Dapagliflozin, Electro spray ionization, Method validation, Mass spectrometer
© 2024 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/)
DOI: https://dx.doi.org/10.22159/ijap.2024v16i5.51095 Journal homepage: https://innovareacademics.in/journals/index.php/ijap
INTRODUCTION
Semaglutide is an Incretin mimetics class of anti-diabetes medication used in type-2 diabetes. It is a Glucagon-like peptide-1 receptor agonist. It helps in the release of insulin from the pancreas in instances where there are high blood glucose levels [1]. It is also used as an obesity medication for weight management as it reduces hunger and body fat. Semaglutide injection is not an alternative to Insulin and cannot be used in Type 1 diabetes or Diabetic Ketoacidosis. It also appears to enhance the growth of pancreatic β cells which are mainly responsible for Insulin production and release [2, 3]. The subcutaneous once-a-week administration was approved by the United States Food and Drug Administration (USFDA) in 2017 and the European Medicines Agency (EMA) in 2018 for Type 2 diabetes treatment [4-10]. Lee et al. have developed a method for estimating semaglutide in rat plasma and brain [11]. Dapagliflozin is a Gliflozin class drug, an oral glucose-lowering drug used for the treatment of Type 2 diabetes. It is categorized as a Sodium/glucose co-transporter 2 (SGLT2) inhibitor [12-16]. Inhibition of SGLT2 transporter results in the increased excretion of both Sodium and Glucose in the urine thereby also exerting its diuretic effect which results in body weight and blood pressure reduction. There are some reported Liquid chromatograph Tandem Mass Spectrometry (LC-MS/MS) methods for determining dapagliflozin in rat plasma [17-18]. Though quite a few bioanalytical methods are reported for semaglutide and dapagliflozin individually, there is no bioanalytical method reported so far for simultaneous estimation of semaglutide and dapagliflozin in human plasma employing LC-MS/MS.
As there is an unmet need for estimating semaglutide and dapagliflozin in human plasma, this combined bioanalytical method is developed and validated for the first time in human plasma and can be directly applied in clinical studies. This method was validated as per The USFDA Guidance on Bioanalytical method validation of 2018 [19] and the International Council for Harmonisation of Technical Requirements of Pharmaceuticals for Human Use (ICH) M10 guidelines on Bioanalytical method validation and study sample analysis [20].
MATERIALS AND METHODS
Reagents and chemicals
The Methanol and Acetonitrile were procured from J. T. Baker. Type 1 Milli-Q Water procured internally from the water purification system Millipore. Human Blood and Plasma lots were procured from Delta Laboratories. Anticoagulant Dipotassium Ethylene Diamine Tetraacetic acid (K2EDTA) was procured from Merck, semaglutide, dapagliflozin, verapamil, and tolbutamide Analytical Standards were obtained as gift samples.
Instrumentation
The chemicals were weighed using a Mettler Toledo microbalance MX5. The solutions were degassed with an ultrasonicator. Ultra performance Liquid chromatography equipment (Shimadzu Nexera) coupled to a Sciex 5500 LC-MS/MS was used for Chromatography and Centrifuge Sorvall, and a Vortex mixer were used for extraction. This study employed a novel dual polarity Multiple Reaction Monitoring (MRM) method in mass spectrometry, which was not explored previously.
Preparation of stock and working solutions for analyte and internal standard
Stock solutions of semaglutide and dapagliflozin (2 mg/ml) were prepared by dissolving 10 mg in a 5 ml volumetric flask. These stocks were prepared separately in Methanol. The calibration standards were prepared from the first stock of both analytes and quality control samples were prepared from the respective second stocks. The Internal standards stocks of verapamil and tolbutamide were prepared by dissolving 5 mg in a 5 ml volumetric flask with Methanol to prepare a 1 mg/ml stock. The intermediate stocks were followed by spiking solutions containing semaglutide and dapagliflozin (20 to 20000 and 40 to 40000 ng/ml respectively) prepared using a serial dilution approach using 50% methanol in water. 125 µl** of internal standard stock solution was added to a 50 ml measuring flask and made up to volume with 50:50 v/v Methanol: Water to obtain 2.5 µg/ml working solution.
Sample preparation
Protein Precipitation (PPT) extraction method was employed for sample preparation. To prepare calibration samples of semaglutide and dapagliflozin in the plasma, 95 μl of the blank human plasma was mixed with 5 μl of the standard working solution, 10 μl of the IS working solution and precipitated using 750 μl of methanol in a 1.5 ml microcentrifuge tube. After mixing on a vortex mixer for 10 min, the mixture was centrifuged at 4500 rpm for 15 min at 8 °C. After centrifugation approximately 500 μl was aliquoted into a 1 ml autosampler vial. An aliquot of 3 μl of the supernatant was injected into the LC-MS/MS.
Chromatographic condition
In gradient mode, a reversed-phase chromatographic separation was achieved on Kinetex C18 (50 mm x 2.1 mm, 5 µ Particle size) at 35 °C using mobile phase (A: Milli-Q water and B: Acetonitrile). The autosampler temperature was sustained at 8 °C. The Liquid Chromatograph (LC) binary gradient program was employed with a run time of 3.0 min. The flow rate employed was 0.5 ml/min for the run. All the mobile phases were filtered using a 0.22 µM membrane filter to prevent clogging of mobile phase lines.
Mass spectrometric conditions
An Atmospheric Pressure Ionization (API) 5500 with an Electro Spray Ionization (ESI) interface operated in MRM (alternate between negative and positive scans). The semaglutide is fragmented in positive mode and dapagliflozin in negative mode. The instrument was optimized for semaglutide, dapagliflozin, and internal standards verapamil and tolbutamide during tuning at a concentration of 100 ng/ml prepared in Acetonitrile and water solution (50:50) and infused at a flow rate of 15 µl**/min through a Hamilton pump infused into the mass spectrometer. The MRM transitions chosen were m/z 1029.2→1302.6 for semaglutide, 407.2→329.1 for dapagliflozin and 455.2→165.2 for verapamil (Internal Standard for semaglutide) and 269.1→106.0 for tolbutamide (Internal standard for dapagliflozin). The optimized mass spectrometric conditions for semaglutide and dapagliflozin for quantification were: ESI probe with a source temperature of 500 °C; ion spray voltage 5500; curtain gas, 20 psi, nebulizing gas (GS1) 45 psi, heater gas (GS2) 45 psi, Declustering Potential (100eV)and Collision energy (35eV) for semaglutide and verapamil. The dapagliflozin and tolbutamide were analysed with a Declustering Potential (-80eV), and Collision energy of (-17 and-10 eV). The entrance potential and cell exit potential were maintained at 10 eV for positive mode analytes and-10 eV for negative mode analytes. Ultra-high pure nitrogen gas was employed as collision gas. The optimized parameters were sufficient to attain linearity for the selected calibration curve range.
Data analysis
Data processing was carried out using Analyst 1.6.3 software. The calibration curves were constructed using the response factor (area ratio of analyte peak area and IS peak area) to the analyte concentration using linear regression model y = mx+c where y denotes the observed area ratio, m is for slope and c is for intercept respectively, with weighting factor 1/x2. The acceptance criteria were established to be>0.98 for the coefficient of determination (r2) with a minimum of 6 non-zero calibration curve standards i. e, at least 75% of the standards should be acceptable (±20% bias for Lower Limit of Quantification (LLOQ) and ±15% bias for other standards) for a calibration curve.
Bioanalytical method validation
Calibration and quality control samples
Calibration curves were made by spiking 5 µl** of spiking solution to 95 µl** of blank human plasma. The final concentrations in the plasma samples were 1.00, 3.00, 10.6, 106, 303, 485, 775, 1000 and 2.00, 6.00, 21.2, 212, 605, 970, 1550, 2000ng/ml. The Quality control (QC) samples were set at concentrations of 1.01, 2.82, 469, 750, and 2.02, 5.65, 940, and 1500 ng/ml (Lower Limit of quantification QC, Low QC, Mid QC, High QC) for semaglutide and dapagliflozin respectively.
Preparation of plasma calibration standards and quality control samples
5 µl** of the cocktail working solutions prepared above are spiked in 95 µl** of blank human plasma. This was the standard solution.
Calibration curve
The calibration curve was prepared using 8 standards excluding standard zero encompassing the range (1.00–1000 ng/ml) and (2.00–2000 ng/ml) for semaglutide and dapagliflozin respectively. The linearity was assessed by a weighted (1/x2) least squares regression analysis.
Linearity was assessed by plotting calibration curves in human plasma in each quantitative run.
Table 1: Acceptance criteria for calibration standards
| Level | Accuracy |
| LLOQ | ±20% |
| All standards except LLOQ | ±15% |
Accuracy and precision
To evaluate the Accuracy and Precision of this method, Quality Control samples at four concentrations (Lower Limit of Quantification Quality control (LLOQQC), Low Quality Control (LQC), Mid Quality Control (MQC), and High Quality Control (HQC) were analyzed in six replicates for each analyte on three different days from extracted plasma. The acceptance criteria are as follows
Table 2: Acceptance criteria for quality control samples
| QC level | Accuracy | Precision |
| LLOQQC | ±20% | ≤20% |
| LQC, MQC and HQC | ±15% | ≤15% |
Specificity and selectivity
The specificity of the method was gauged by studying six different plasma lots to investigate the likely interferences at the retention time for both analytes and internal standards. The six blank lots were processed without analytes or internal standards. The acceptance criteria were that at least five out of the tested six blanks must have<20% area interference to that of the LLOQ level response at the corresponding retention time. Selectivity of the assay was established by analyzing six individual blank plasma lots with individual analyte spiking and checking for interference at other analyte and Internal Standard retention time.
Recovery
Recoveries were evaluated at three QC levels LQC, MQC and HQC (2.82, 469, and 750 ng/ml for semaglutide and 5.65, 940, and 1500 ng/ml for dapagliflozin) by comparing the peak area in spiked pooled human plasma samples with those of analyte spiked in neat solutions.
Matrix effect
The matrix effect assessment was optimally suggested by Matuszewski et al. [21]. However, the latest approach suggested by ICH M10 guidelines was adopted wherein the back-calculated concentrations were obtained from 6 different interference-free lots and the mean concentration across the 6 lots was calculated. In this methodology, the matrix effects were assessed to check the impact of co-eluting matrix components which might trigger potential ion suppression or enhancement.
The matrix effect was evaluated at the LQC and HQC levels. Each of the selected blanks including one hemolyzed plasma lot were spiked with LQC and HQC and were quantified under a calibration curve. The acceptable criteria for the matrix factor should be a mean accuracy of 85% to 115%.
Stability experiments
The stability of semaglutide and dapagliflozin in human plasma was assessed by analyzing six replicates of QC samples at low and high levels in four different exposure conditions as follows; (1) The spiked samples at room temperature (ambient temperature for>18 h; (2) The processed samples at autosampler temperature i. e. 8 °C for 24 h, (3) four freeze and thaw cycles (24 h per cycle), and (4) For 14 d in the deep freezer at-80 °C. Samples were considered stable if the relative error was within 85–115% of the nominal concentration.
RESULTS AND DISCUSSION
Method optimization
The aim was to develop a robust bioanalytical method best suited for the simultaneous estimation of semaglutide and dapagliflozin in a single bioanalytical run on an LC-MS/MS platform. During the development of the extraction method due to the difference in physiochemical parameters between semaglutide and dapagliflozin, we adopted a protein precipitation method to maximize the recovery of both analytes. The existing methods for semaglutide in human plasma and related pharmacokinetic studies employed an LLOQ of 3 ng/ml [16-24]. During tuning of semaglutide in positive mode, the most prominent precursor ion 1029.2 was selected as a multiply charged [M+4] considering the large molecular weight of 4114 daltons. The product ion of 1302.6 was the chosen fragment which yielded the best chromatographic response. The main difficulty was overcoming dapagliflozin ionization, forming an adduct in formic acid. Therefore formic acid and its salts like Ammonium formate were not used in the mobile phase. Dapagliflozin was tuned in negative mode with a precursor ion of 407.2. The product of 329.1 along with the precursor of 407.2 yielded the best response. As semaglutide was ionisable in positive mode and dapagliflozin in negative mode we adopted a dual polarity ESI LC-MS/MS method for their simultaneous estimation. The MRM parameters for MS/MS determination were optimized to maximize the response for both analytes. The currently available method can quantify plasma concentrations of semaglutide in human plasma from 3 to 250 ng/ml by Kapitza et al. [22]. The validated method in this article has a wider linear dynamic range from 1 to 1000 ng/ml helpful even in cases of dose reduction due to other comorbidities. The currently available method for dapagliflozin utilizes an Acetate adduct for the parent ion which was 467.1 as reported by Shah PA et al. [23]. However, we have chosen a mobile phase to yield a parent ion 407.2 devoid of adduct and an enhanced selectivity at the chosen LLOQ of 2 ng/ml. Van der Aart-van der Beek AB et al. [24] reported an upper limit of quantification as 500 ng/ml for dapagliflozin utilizing a 200 µl** processing volume. However, we have achieved an upper limit of 2000 ng/ml with a 100 µl** sample processing volume.
Fig. 1: Structure of semaglutide
Fig. 2: Structure of dapagliflozin
Fig. 3: Full scan mass spectrum of semaglutide
Fig. 4: Product ion mass spectrum of semaglutide
Fig. 5: Full scan mass spectrum of dapagliflozin
Table 3: Precision and accuracy data of calibration curve standards for semaglutide and dapagliflozin in human plasma
| Matrix | Analyte | Nominal concentration (ng/ml) | Back calculated conc. (ng/ml) | n | CV (%) | RE (%) |
| Human Plasma | Semaglutide | 1.00 | 0.97 | 3 | 13.52 | 97.0 |
| 3.00 | 2.88 | 3 | 3.76 | 96.0 | ||
| 10.6 | 9.57 | 3 | 3.19 | 90.3 | ||
| 106 | 97.3 | 3 | 3.89 | 91.8 | ||
| 303 | 292 | 3 | 3.14 | 96.4 | ||
| 485 | 476 | 3 | 2.44 | 98.1 | ||
| 775 | 788 | 3 | 2.04 | 101.7 | ||
| 1000 | 1030 | 3 | 1.60 | 103.2 | ||
| Dapagliflozin | 2.00 | 1.98 | 3 | 13.88 | 99.2 | |
| 6.00 | 5.55 | 3 | 9.14 | 92.5 | ||
| 21.2 | 20.8 | 3 | 3.47 | 98.3 | ||
| 212 | 206 | 3 | 6.04 | 97.0 | ||
| 605 | 628 | 3 | 7.13 | 103.9 | ||
| 970 | 943 | 3 | 4.28 | 97.3 | ||
| 1550 | 1460 | 3 | 3.57 | 94.1 | ||
| 2000 | 1930 | 3 | 3.84 | 96.6 | ||
n=3 replicates at each concentration, Data presented in mean, %CV, and %RE
Calibration curves
The calibration standards were analyzed at specified standard concentrations for semaglutide and dapagliflozin from 1.00 to 1000 ng/ml and 2.00 to 2000 ng/ml respectively. The calibration curve for both analytes displayed a good linear relationship across the concentration range. The eight-point calibration curves were plotted for both analytes with linear fit 1/x2 weighting regression factor. The mean correlation coefficient for both analytes in human plasma was 0.998. The calibration curve results for both analytes in human plasma are summarized in table 3.
Fig. 6: Linearity plot of semaglutide
Fig. 7: Linearity plot of dapagliflozin
Specificity and selectivity
Specificity and selectivity employing this method in plasma were studied in six different lots along with a hyperlipidaemic and a hemolyzed lot. This is done to determine the degree of endogenous interferences that might contribute to the chromatographic area count of the analyte or internal standard. Each blank lot was processed as 2 replicates (1 devoid of any spiking and 1 spiked with the spiking solution of LLOQ and Internal Standard). The interference in the blank sample corresponding to each lot was compared against the average peak response at the LLOQ level and respective internal standard. As shown in fig. 8 and 11, no interference was detected at the retention time of semaglutide and dapagliflozin and its internal standards, verapamil and tolbutamide in any of the blank samples.
Fig. 8: Extracted blank of semaglutide
Fig. 9: Extracted LLOQ of semaglutide
Fig. 10: Extracted ULOQ of semaglutide
Fig. 11: Extracted blank of dapagliflozin
Fig. 12: Extracted LLOQ of dapagliflozin
Fig. 13: Extracted ULOQ of dapagliflozin
Accuracy and precision
The intra-day and the inter-day accuracy and precision outcomes for semaglutide in plasma are accessible in tables 4 and 5 and dapagliflozin is presented in tables 6 and 7 respectively. The accuracy and precision were evaluated at the selected four levels of QCs by using six replicates at each QC level. Quality Control levels employed were (1.01, 2.82, 469, and 750ng/ml) for semaglutide and (2.02, 5.65, 940, and 1500ng/ml) for dapagliflozin.
For semaglutide at each QC level, the intra-day precision was between 3.19% and 11.28 %, and accuracy was between 95.2% and 103.0%. In the case of inter-day, the precision was between 3.31% and 7.68%and accuracy was between 97.6 % and 100.6% for human plasma.
For dapagliflozin at each QC level, the intra-day precision was between 3.55% and 7.10% and accuracy was between 95.0% and 102.0%. In the case of inter-day, the precision was between 3.83% and 10.47 % and the accuracy was between 98.4 % and 101.0% for human plasma.
Extraction recovery and matrix effect
As shown in table 8 and table 9, the extraction recovery for semaglutide was 75.4±10.71% to 73.4±4.39 and 75.5±5.29 in human plasma at LQC, MQC, and HQC respectively. Similarly, the extraction recovery for dapagliflozin in human plasma was 80.1±4.12 to 82.9±5.02 and 81.8±3.88 in human plasma at LQC, MQC, and HQC respectively. The mean Matrix effect calculated from the six independent lots for human plasma at Low QC and High QC levels were within the acceptance range of 85% to 115% as shown in tables 10 and 11.
Table 4: Intra-day precision and accuracy for the estimation of semaglutide
| QC level | Nominal conc. (ng/ml) | Back calculated conc. (ng/ml) | Precision (%) | Accuracy (%) |
| LLOQC | 1.01 | 1.00±0.11 | 11.28 | 98.5 |
| LQC | 2.82 | 2.69± 0.21 | 7.99 | 95.2 |
| MQC | 469 | 460±43.12 | 9.37 | 98.2 |
| HQC | 750 | 772±24.64 | 3.19 | 103.0 |
n=6 replicates in each concentration, Data presented in (mean±SD)
Table 5: Inter-day precision and accuracy for the estimation of semaglutide
| QC level | Nominal conc. (ng/ml) | Back calculated conc. (ng/ml) | Precision (%) | Accuracy (%) |
| LLOQC | 1.01 | 1.00±0.06 | 6.32 | 98.9 |
| LQC | 2.82 | 2.75±0.21 | 7.68 | 97.6 |
| MQC | 469 | 469±28.80 | 6.15 | 99.9 |
| HQC | 750 | 755±24.94 | 3.31 | 100.6 |
n=6 replicates in each concentration, Data presented in (mean±SD)
Table 6: Intra-day precision and accuracy for the estimation of dapagliflozin
| QC Level | Nominal conc. (ng/ml) | Back calculated conc. (ng/ml) | Precision (%) | Accuracy (%) |
| LLOQC | 2.02 | 1.92±0.14 | 7.10 | 95.0 |
| LQC | 5.65 | 5.64± 0.37 | 6.61 | 99.7 |
| MQC | 940 | 959±34.08 | 3.55 | 102.0 |
| HQC | 1500 | 1480±71.53 | 4.84 | 98.5 |
n=6 replicates in each concentration, Data presented in (mean±SD)
Table 7: Intra-day precision and accuracy for the estimation of dapagliflozin
| QC level | Nominal conc. (ng/ml) | Back calculated conc. (ng/ml) | Precision (%) | Accuracy (%) |
| LLOQC | 2.02 | 2.00±0.21 | 10.47 | 98.8 |
| LQC | 5.65 | 5.56±0.25 | 4.53 | 98.4 |
| MQC | 940 | 950±36.38 | 3.83 | 101.0 |
| HQC | 1500 | 1490±60.99 | 4.09 | 99.3 |
n=6 replicates in each concentration, Data presented in (mean±SD)
Table 8: Extraction recovery of semaglutide in human plasma
| Concentration (ng/ml) | Mean recovery (%) | % CV |
| 2.82 | 75.4 | 10.71 |
| 469 | 73.4 | 4.39 |
| 750 | 75.5 | 5.29 |
n=6 replicates in each concentration, Data presented in mean and %CV
Table 9: Extraction recovery of dapagliflozin in human plasma
| Concentration (ng/ml) | Mean recovery (%) | %CV |
| 5.65 | 80.1 | 4.12 |
| 940 | 82.9 | 5.02 |
| 1500 | 81.8 | 3.88 |
n=6 replicates in each concentration, Data presented in mean and % CV
Table 10: Matrix effect of semaglutide in human plasma
| Concentration (ng/ml) | Mean matrix effect (%) | %CV |
| 2.82 | 100.5 | 9.21 |
| 750 | 98.7 | 3.45 |
n=6 replicates in each concentration, Data presented in mean and % CV
Table 11: Matrix effect of dapagliflozin in human plasma
| Concentration (ng/ml) | Mean matrix effect (%) | % CV |
| 5.65 | 97.9 | 2.34 |
| 1500 | 100.0 | 4.85 |
n=6 replicates in each concentration, Data presented in mean and %CV
Stability
The stability of semaglutide and dapagliflozin was evaluated in human plasma and the results are shown in tables 12 and 13, representing that no significant degradation happened at ambient temperature, or for the processed samples in autosampler, after four freeze and thaw cycles or on long storage at-80 °C in freezers.
Table 12: Stability of semaglutide in human plasma
| Experimental condition | Sample conc. (ng/ml) | Measured conc. (ng/ml) | CV (%) | Stability (%) |
| Human plasma | ||||
| 18 h at room temperature 24 °C | 2.82 | 2.69±0.21 | 7.99 | 95.2 |
| 750 | 772±24.6 | 3.19 | 103.0 | |
| 24 h in the autosampler 8 °C | 2.82 | 2.72±0.25 | 9.06 | 96.5 |
| 469 | 459±43.5 | 9.50 | 97.8 | |
| 750 | 768±22.3 | 2.90 | 102.4 | |
| 24 h at-80 °C then exposed to 4 freeze and thaw cycles | 2.82 | 2.74±0.15 | 5.64 | 97.0 |
| 750 | 750±14.8 | 1.97 | 100.1 | |
| 14 d at-80 °C | 2.82 | 2.78 ±0.20 | 7.08 | 98.6 |
| 750 | 740±22.8 | 3.07 | 98.8 |
Measured concentration provided in mean±SD, All stability measurements performed with n=6 replicates
Table 13: Stability of dapagliflozin in human plasma
| Experimental condition | Sample conc. (ng/ml) | Measured conc. (ng/ml) | CV (%) | Stability (%) |
| Human plasma | ||||
| 18 h at room temperature 24 °C | 5.65 | 5.64±0.37 | 6.61 | 99.7 |
| 1500 | 1480±71.5 | 4.83 | 98.7 | |
| 24 h in the autosampler 8 °C | 5.65 | 4.92±0.37 | 7.49 | 87.0 |
| 940 | 846±45.3 | 5.36 | 90.0 | |
| 1500 | 1550±47.0 | 3.03 | 103.3 | |
| 24 h at-80 °C then exposed to 4 freeze and thaw cycles | 5.65 | 5.52±0.23 | 4.08 | 97.7 |
| 1500 | 1510±39.1 | 2.59 | 100.7 | |
| 14 d at-80 °C | 5.65 | 5.54±0.35 | 6.25 | 98.0 |
| 1500 | 1480±74.4 | 5.03 | 98.7 |
Measured concentration provided in mean±SD, All stability measurements performed with n=6 replicates
CONCLUSION
In the current study, we have developed and thoroughly validated a robust, intricate, selective, and cost-effective LC-MS/MS method to quantify two antidiabetic drugs semaglutide and dapagliflozin simultaneously in human plasma. This method will facilitate the further application of pharmacokinetic and bioequivalence studies for these two drugs in pharmaceutical dosage forms or their new pharmaceutical formulation. This simple extraction technique and the optimized chromatographic conditions gave the best outcomes. This detailed procedure is user-friendly and will allow fast analysis procedures for further application to clinical studies.
ACKNOWLEDGEMENT
The authors are thankful to the management of GITAM University, Visakhapatnam, Andhra Pradesh, India, for providing the necessary support to carry out the research work
FUNDING
Nil
AUTHORS CONTRIBUTIONS
All the authors have contributed equally towards this article.
CONFLICT OF INTERESTS
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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