A SIMPLE, SENSITIVE AND FAST SINGLE STEP EXTRACTION METHOD FOR DETERMINATION OF EMPAGLIFLOZIN IN HUMAN PLASMA USING LC-MS/MSHTML Full Text
A SIMPLE, SENSITIVE AND FAST SINGLE STEP EXTRACTION METHOD FOR DETERMINATION OF EMPAGLIFLOZIN IN HUMAN PLASMA USING LC-MS/MS
Amit Raval 1, Cheepurupalli Prasad 2, Rajaram Shivaji Rao Patil 3 and Atmakuri Chaitanya Krishna * 1
College of Pharmacy 1, Pacific Academy of Higher Education and Research University, Pratap Hills, Pratap Nagar Ext., Airport Road, Udaipur - 313024, Rajasthan, India.
Vignan Institute of Pharmaceutical Technology 2, Besides Visakhapatnam Special Economic Zone, Kapujaggraju Peta, Duvvada, Visakhapatnam - 530049, Andhra Pradesh, India.
Omacon Analytical Solutions LLP 3, 119-120, 1st Floor, Building 5, Swastik Regalia, Ghodbunder Rd, Waghbil, Thane West - 400615, Maharashtra, India.
ABSTRACT: A simple, sensitive, and fast single-step extraction method for estimating empagliflozin in human plasma using LC-MS/MS was developed and validated for pharmacokinetics evaluations. Plasma samples were basified before solid-phase extraction on SOLA (30 mg / 1 mL cartridges). Separations were carried out on a normal reverse phase C18 column (Hypersil BDS 100 × 4.6, 5µ mm column) for 3.5 minutes at a flow rate of 0.6 mL/min. Ten µL of the SPE eluent is directly injected onto LC-MS/MS to quantify the analyte from 1.563-800.000 ng/mL using a single SRM transition (m/z: 449.140à371.100) in negative ion mode. During method validation, selectivity, matrix effect, recovery, carry-over effect, stability studies, inter-day, and intra-day precision and accuracy experiments were conducted per USFDA guidelines. Method validation data has successfully met the acceptance criteria making it suitable for use in routine bio-analytical laboratories. The scope of this assay can be extended to cover the requirement of preclinical, toxicology, and PK/PD studies.
Liquid chromatography-mass spectrometry, Empagliflozin, Bio-analytical method, Human Plasma
INTRODUCTION: Empagliflozin is indicated as an adjunct to diet and exercise to improve glycemic control, assist in weight loss and reduce blood pressure in adult patients with type 2 diabetes. Empagliflozin inhibits the sodium-glucose co-transporter 2 which is responsible for the reabsorption of glucose from the glomerular filtrate in the kidneys resulting in glucuretic effect 1-5. Based on the pharmacokinetic study data, the analytical method required for analysis of empagliflozin in human plasma must be sensitive to detect concentrations as low as 1.5 ng/mL.
Moreover, the linear response relationship till the upper limit of quantification determines the applicability of the analytical method for multiple doses of the drug and makes the method preferable.
Few liquid chromatography-tandem mass spectro-metry (LC-MS/MS) 6-13 and diode array detectors (DAD/PDA) 14, 15 based bio-analytical methods were reportedly using ultra-performance liquid chromatography (UPLC) systems for estimation of empagliflozin in human plasma. In this method, we present a simple, sensitive, high-throughput, and robust method for the determination of empagliflozin in human plasma using the HPLC-MS/MS method in negative ion mode. The current study employs a simple and single-step extraction procedure with sample volumes as low as 200 µL, and the solid phase eluent is directly injected onto LC-MS/MS.
Run time was shorter under normal HPLC conditions with the ability to analyze over 400 samples per day.
MATERIALS: Methanol (gradient grade), acetonitrile (gradient grade), ammonium bicarbonate (LR grade), and ammonium acetate (LR grade) were purchased from Merck. Water (LCMS grade) was used in-house from Milli Q system. Empagliflozin and empagliflozin D4 were procured from Vivan life sciences. Blank plasma was purchased from the blood bank. The chemical structure of empagliflozin is presented below in Fig. 1.
FIG. 1: STRUCTURE OF EMPAGLIFLOZIN
Instrumentation and Analytical Conditions: Ultimate 3000 HPLC system interfaced with a TSQ Endura Ultra triple quadrupole mass spectrometer (Thermo Fisher Scientific Inc) was used for analysis. As empagliflozin is basic in nature, a heated electro-spray-ionization source was operated in the negative ion mode. Moreover, empagliflozin is moderately polar and lipophilic. Therefore, a reverse-phase LC column (Hypersil BDS C18 column-Dimensions: 100 × 4.6 mm, 5 µm) was used employed to resolve the analyte at oven temperature of 40 ºC. Acetonitrile: methanol: 10 mM ammonium bicarbonate in water (85:10:5 v/v/v) was used as a mobile phase. The retention time of both empagliflozin and empagliflozin D4 was found to be the same at 1.8 min with a total run time of 3.5 minutes.
Mass spectrometry analysis was performed with the following optimized parameters: Sheath gas 50 (arb), auxiliary gas pressure 30 (arb), capillary temperature 300 °C, Q2 gas pressure 1.2 m Torr, ion spray voltage 3000 V, and vaporizer temperature 30 ºC. SRM (Selected reaction monitoring) transitions for quantification were m/z 449.140→371.100 for empagliflozin and 453.170 → 375.15 for empagliflozin D4.
Preparation of Standard Solutions and Quality Control Samples: Standard solutions of empagliflozin (100 µg/mL) and empagliflozin D4 (100 µg/mL) were prepared in methanol. Intermediate stock solutions of both analyte and internal standard (20µg/mL) were prepared in diluent (50% methanol in water) along with internal standard dilution (1µg/mL). Ten level calibrators and four-level controls were prepared in human plasma containing sodium heparin as anticoagulant from 1.563-800.000 ng/mL and 1.000-200.000 ng/mL, respectively.
Sample Preparation: Two hundred microliters of pre-spiked plasma samples were dispensed into microcentrifuge tubes. 30 µL of internal standard dilution (1000.000 pg/mL of empagliflozin D4) was dispended into all non-zero samples. Thirty microliters of diluent (50% methanol) were added to blank samples. Five hundred microliters of 2 mM ammonium acetate in water were used for pre-treatment to unionize the analyte and facilitate the reverse phase interactions during solid-phase extraction. Samples were vortexed to mix, and solid-phase extraction cartridges (SOLA-30 mg, 1mL) were conditioned and equilibrated with 1 mL methanol followed by 1 mL water (to improve water wettability and easily allow plasma samples to diffuse through the sorbent). Seven hundred and thirty microliters of pre-treated plasma sample were dispensed into the cartridges. Empagliflozin and internal standard were retained on the sorbent while the plasma components pass through the cartridges to waste due to gravity. To evaluate the retention mechanism of the analyte with the sorbent, solvents of different elution strength were verified in comparison with the physicochemical properties of empagliflozin, mainly solubility, polarity, and pH. This study has helped in eliminating other interfering compounds and separate the cleaner extracts of empagliflozin. Optimal clean-up of the samples was achieved with two wash solutions. Cartridges were initially cleaned with 900 µL water (2 times) followed by 900 µL 30% methanol in water. After drying the cartridges with nitrogen gas for 2 min, the analyte was extracted with 350 µL of acetonitrile: methanol: 2 mM ammonium acetate in water (70:20:10 v/v/v). The eluent was transferred to the HPLC vials, and 10µL was injected for LC-MS/MS analysis.
Method Validation: Selectivity, linearity, precision, accuracy, recovery, and stability experiments were performed as per USFDA guidelines.
Representative chromatograms and a calibration curve of empagliflozin are presented in Fig. 2 and 3.
FIG. 2: REPRESENTATIVE CHROMATOGRAMS OF EMPAGLIFLOZIN
FIG. 3: CALIBRATION CURVE OF EMPAGLIFLOZIN IN HUMAN PLASMA FROM 1.563-800.000 ng/ml
Precision and accuracy (P&A) batches (includes ruggedness and stability PA batch) were analysed with the calibration cure ranging from 1.563-800.000 ng/mL. A straight-line equation (y=mx+c) with 1/x2 weighing factor has been used to quantify the back-calculated concentration of the calibrators and the coefficient of determination (r2) was greater than 0.994 in all 5 PA batches. Summary of back-calculated concentrations and calibration curve parameters were presented below in Table 1-2.
TABLE 1: PRECISION AND ACCURACY
|Standard name||Nominal concentration (ng/ml)||P&A-01||P&A-
|% CV||% Nominal|
Mean statistical data are expressed as mean ± SD [n=5].
TABLE 2: CALIBRATION CURVE PARAMETERS SUMMARY
|Result Table ID||Slope||Y-Intercept||Regression Coefficient
Specificity and selectivity of the method was assessed in 6 different lots of human plasma containing sodium heparin as an anticoagulant. Haemolyzed and lipidemic (each lot) were also used for the evaluation of selectivity of empagliflozin. % interference in blank was found to be less than 9% in all lots when compared against LLOQ area of empagliflozin. Results were presented below in Table 3.
TABLE 3: SELECTIVITY
|Matrix Lot no||Area in Blank Matrix at analyte RT||Area of LLOQ||%Interference at retention time of Empagliflozin||Area in Blank Matrix at IS RT||Area of LLOQ||% Interference at retention time of Empagliflozin D4|
|LOT 7 (HEMOLYZED)||111||1532||7.25||300||43639||0.69|
|LOT 8 (LIPIDEMIC)||130||1472||8.83||252||44337||0.57|
Intra-day precision and accuracy was evaluated in 6 replicates of control samples at LLOQ QC, LQC, MQC and HQC over one PA batch was found to be between 1.14-3.39% and 101.18-103.79 respectively. Intra-day precision and accuracy results were presented in Table 4.
TABLE 4: INTRA-DAY PRECISION AND ACCURACY
|QC concentration||LLOQ QC||LQC||MQC||HQC|
|Mean ± SD (n=6)||1.62±0.05||6.40±0.21||50.59±0.98||204.47±2.34|
Mean statistical data are expressed as mean ± SD [n=6]
TABLE 5: INTER-DAY PRECISION AND ACCURACY
|QC concentration||LLOQ QC||LQC||MQC||HQC|
|Mean ± SD (n=30)||1.58±0.14||6.34±0.30||52.79±2.17||207.34±2.97|
Mean statistical data are expressed as mean ± SD [n=30]
Inter-day precision and accuracy experiments were evaluated in 5 batches at the same levels mentioned above and the results were found to be between 1.43-8.72% and 100.77-105.57, respectively. Results of Inter-day precision and accuracy were tabulated in Table 5.
Matrix effect was studied for both empagliflozin and empagliflozin D4 in eight lots of plasma (6 normal, 1 haemolyzed, and 1 lipidemic plasma). IS normalized matrix factor was calculated as a ratio of response ratio of post extracted spiked sample upon aqueous sample at both HQC and LQC concentration levels, and mean IS normalized matrix factor was found to be 1.00 and 0.95, respectively. Results of IS normalized matrix effect experiment were provided below in Table 6.
TABLE 6: MATRIX EFFECT EXPERIMENT
|QC||Response ratio of matrix effect sample||Response ratio of aqueous standard||Matrix factor||QC||Response ratio of matrix effect sample||Response ratio of aqueous standard||Matrix factor|
|Mean IS normalized matrix factor of LQC [mean ± SD (n=8)]||1.00±0.09||Mean IS normalized matrix factor of HQC [mean ± SD (n=8)]||0.95±0.08|
|% CV||8.86||% CV||8.22|
The average recovery of empagliflozin was obtained by calculating the response ratio of extracted and aqueous samples at LQC, MQC, HQC levels and was found to be 93.73% and 102.16% for empagliflozin and empagliflozin D4, respectively. Results of the recovery experiment were presented in Table 7.
TABLE 7: RECOVERY OF EMPAGLIFLOZIN
|QC||Response of extracted sample||Response of Unextracted sample||% Recovery|
|Mean recovery (mean ± SD)||93.73±1.91|
Mean statistical data are expressed as mean ± SD [n=6x3]
TABLE 8: STABILITY EXPERIMENTS IN BIOLOGICAL MATRIX
|Comparison QC details||Mean ± SD (n=6)||% CV||% Nominal|
|Freshly spiked LQC||6.40±0.0.21||3.28||102.46|
|Freshly spiked HQC||211.14±4.07||1.93||105.57|
|Stability QC details||Mean ± SD (n=6)||% CV||% Nominal|
|Autosampler stability-LQC (24 hrs)||6.44±0..28||4.28||103.05|
|Autosampler stability-HQC (24 hrs)||205.24±1.72||0.84||102.62|
|FT 5th Cycle LQC (-50 °C)||7.02±0.09||1.21||112.3|
|FT 5th Cycle HQC (-50 °C)||209.68±1.75||0.84||104.84|
|FT 5th Cycle LQC (-20 °C)||6.90±0.17||2.42||110.39|
|FT 5th Cycle HQC (-20 °C)||207.09±2.31||1.11||103.54|
|Wet extract stability LQC (40 hrs)||6.19±0.19||3.01||99.07|
|Wet extract stability HQC (40 hrs)||210.27±0.78||0.37||105.13|
|Bench top stability (LQC) (8 hrs)||6.49±0.20||3.13||103.91|
|Bench top stability (HQC) (8 hrs)||208.26±3.71||1.78||104.13|
|Long term matrix stability (LQC) (50 days)||6.18±0.15||2.51||98.96|
|Long term matrix stability (HQC) (50 days)||209.88±0.92||0.44||104.94|
Stability experiments in matrix were conducted for bench-top, freeze-thaw (at -50 °C and at -20 °C), autosampler, wet extract, and long-term storage (at -50°C). Stock solution stability, reinjection reproducibility, ruggedness and dilution integrity were also performed during method validation. Results of these experiments met the acceptance criteria and were provided in Table 8.
DISCUSSION: Validation parameters and acceptance criteria of the results are mentioned below in Table 9.
TABLE 9: VALIDATION PARAMETERS AND THEIR ACCEPTANCE CRITERIA
|S. no.||Parameter||Acceptance criteria|
|1||Linearity (includes ruggedness and stability)||Minimum 5-point standards are required for building a calibration curve|
|Two consecutive standards should not fail|
|First and last calibration standard should not fail|
|At least 75 % of the calibration curve standards should be with the acceptable limits for accuracy and precision|
|% Accuracy and precision should be within 85-115% for all standards except LLOQ.|
|% Accuracy and precision should be within 80-120% for LLOQ|
|2||PA [(inter-day and intra-day) and (stability studies)]||% Accuracy and precision should be within 85-115% for all QCs except LOQ QC.|
|% Accuracy and precision should be within 80-120% for LOQ QC.|
|At least 67% of the quality control samples should be within specified criteria for precision and accuracy|
|At least 50% quality control samples should meet the criteria specified for accuracy and precision|
|3||Specificity and selectivity||% interference at the retention time of the analyte in the blank sample should not be more than 20% of the peak area of analyte|
|% interference at the retention time of the internal standard in the blank sample should not be more than 5% of the peak area of the internal standard|
|4||Matrix effect||Mean matrix factor and is normalized matrix factor should be between 0.85-1.15|
|5||Recovery||No as such criteria defined for % recovery. The precision obtained for mean and global recovery should be with in±15%|
|6||Dilution integrity (DI)||Precision and accuracy of the DI QCs should be within 85-115 %|
|7||Limit of detection (LOD)||Precision and accuracy of the sensitivity samples should be within 85-115%|
Validation data was assessed as per the criteria mentioned above, and the results of all these parameters have met the acceptance criteria.
CONCLUSION: This newly developed bio-analytical method employs a relatively simple and fast extraction method as additional processing steps like centrifugation and evaporation are not required. The developed method was successfully validated as per USFDA guidelines, and it is suitable for the determination of empagliflozin in human plasma using LC-MS/MS for pharmaco-kinetic evaluations.
ACKNOWLEDGEMENT: The authors are thankful to the management of Pacific Academy of Higher Education and Research University, Udaipur, Rajasthan, India, for providing the necessary facilities to carry out the research work.
CONFLICTS OF INTEREST: There is no conflict of interest to declare.
- Kalra and Sanjay: Sodium-Glucose Co-Transporter-2 (SGLT2) Inhibitors: A Review of Their Basic and Clinical Pharmacology. Diabetes therapy: research, treatment and education of diabetes and related disorders 2014; 5(2): 355-66.
- Heise T, Seman L, Macha S, Jones P, Marquart A, Pinnetti S, Woerle HJ and Dugi K: Safety, tolerability, pharmacokinetics, and pharmacodynamics of multiple rising doses of empagliflozin in patients with type 2 diabetes mellitus. Diabetes therapy: research, treatment and education of diabetes and related disorders 2013; 4(2): 331-45.
- Heise T, Seewaldt‐Becker E, Seman L, Macha S, Pinnetti S and Woerle HJ: Safety, tolerability, pharmacokinetics and pharmacodynamics following 4 weeks' treatment with empagliflozin once daily in patients with type 2 diabetes. Diabetes, Obesity and Metabolism 2013; 15(7): 613-21.
- Kanada S, Koiwai K, Taniguchi A, Sarashina A, Seman L and Woerle HJ: Pharmacokinetics, pharmacodynamics, safety and tolerability of 4 weeks’ treatment with empagliflozin in Japanese patients with type 2 diabetes mellitus. J of Diabetes Investigation 2013; 4: 613-17.
- Empagliflozin product information. U.S. Food and Drug Administration 2016.
- Ayoub BM, Mowaka S, Elzanfaly ES, Ashoush N, Elmazar MM and Mousa SA: Pharmacokinetic evaluation of empagliflozin in healthy egyptian volunteers using LC-MS/MS and comparison with other ethnic populations. Scientific reports 2017; 7(1): 2583.
- Ayoub BM and Mowaka S: LC–MS/MS Determination of Empagliflozin and Metformin. Journal of Chromato-graphic Science 2017; 55(7): 742-47.
- Ayoub BM: UPLC simultaneous determination of empagliflozin, linagliptin and metformin. Royal Society of Chemistry advances 2015; 5: 95703-96709.
- Ayoub BM: Enhancement of plasma extraction recovery of empagliflozin and metformin combination using liquid-liquid extraction and vacuum evaporation techniques. Der Pharma Chemica 2016; 8(10): 163-66.
- Shah PA, Shrivastav PS and George A: Mixed-mode solid phase extraction combined with LC-MS/MS for determination of empagliflozin and linagliptin in human plasma. Microchemical Journal 2019; 145: 523-31.
- Rajesh PMN and Wagh P: Simultaneous extraction of metformin, linagliptin and empagliflozin from human plasma using mixed-mode SPE with oasis (WCX) and analysis by LC-MS/MS. Waters Corporation 2018; 1-2.
- Wattamwar T, Mungantiwar A, Halde S and Pandita N: Development of simultaneous determination of empagliflozin and metformin in human plasma using liquid chromatography–mass spectrometry and application to pharmacokinetics. European Journal of Mass Spectrometry 2019; 0(00): 1-14.
- Chen L, Mao Y, Sharp DE, Schadt S, Pagels S, Press R, Cheng T3, Potchoiba MJ and Collins W: Pharmaco-kinetics, Biotransformation, Distribution and Excretion of Empagliflozin, a Sodium-Glucose Co-Transporter (SGLT 2) Inhibitor, in Mice, Rats, and Dogs. Journal of Pharmaceutics & Drug Development 2015; 3(3): 1-12.
- Mabrouk MM, Soliman SM, El‑Agizy HM and Mansou FR: A UPLC/DAD method for simultaneous determination of empaglifozin and three related substances in spiked human plasma. Biomed Central Chem 2019; 13(83): 1-9.
- Padmaja N, Desalegn T, Sharathbabu M and Veerabhadram G: New validated RP-HPLC method for the estimation of empagliflozin in human plasma. Int J Pharm Sci & Res 2018; 49: 4885-89.
How to cite this article:
Raval A, Prasad C, Patil RSR and Krishna AC: A simple, sensitive and fast single step extraction method for determination of empagliflozin in human plasma using LC-MS/MS. Int J Pharm Sci & Res 2021; 12(6): 3457-63. doi: 10.13040/IJPSR.0975-8232.12(6). 3457-63.
All © 2013 are reserved by the International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.
A. Raval, C. Prasad, R. S. R. Patil and A. C. Krishna *
College of Pharmacy, Pacific Academy of Higher Education and Research University, Udaipur, Rajasthan, India.
22 June 2020
15 October 2020
04 May 2021
01 June 2021