• INTRODUCTION
  • MATERIALS AND METHODS
  • RESULTS AND DISCUSSION
  • CONCLUSION
  • FUNDING
  • AUTHORS CONTRIBUTIONS
  • CONFLICT OF INTERESTS
  • REFERENCES

    • Int J App Pharm, Vol 16, Issue 6, 2024, 176-181Original Article

    BIOANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR THE DETERMINATION OF ASCIMINIB ANTICANCER DRUG IN BIOLOGICAL MATRICES BY LC–ESI-MS/MS

    MANTRAVADI ANUSHA, KUMAR SHIVA GUBBIYAPPA*

    Department of Pharmaceutical Analysis, GITAM School of Pharmacy, GITAM Deemed to be Univeristy, Rudraram, Patancheru, Telangana, India
    *Corresponding author: Kumar Shiva Gubbiyappa; *Email: sgubbiya@gitam.edu

    Received: 25 Jul 2024, Revised and Accepted: 02 Sep 2024

    ABSTRACT

    Objective: A unique liquid chromatography-mass spectrometry technique is essential for determining the concentration of asciminib in biological matrices, and its development is of the utmost importance.

    Methods: The samples that were processed were separated using a Reversed Phase-Phenomenex (100 mm x 4.6 mm, 5 µm) C18 analytical column. The column was equipped with an isocratic moveable phase that consisted of 0.1% (v/v) HCOOH and acetonitrile at a ratio of 18:82% (v/v). The flow rate of the phase was 0.70 ml/min. For asciminib, the multiple reaction monitoring mode was used at m/z 450.23/257.3, while for canagliflozin, it was used at m/z 445.13/267.31.

    Results: With a correlation coefficient of 0.9998, the method was linear for asciminib throughout the concentration range of 1.0-2100.00 ng/ml. Each day's accuracy percentage relative standard deviation was within 5.74%. For analytes at the low-quality control level, the mean matrix factors ranged from 96.34 to 104.85% with a % Coefficient of Variance (CV) of 4.21; at the high-quality control level, the range was from 94.62 to 103.88% with a %CV of 3.67.

    Conclusion: The method that has been developed has the potential to be used to examine the pharmacokinetics and toxicokinetics of asciminib in various biological samples for both forensic and clinical purposes.

    Keywords: Asciminib, Cancer, FDA, Sensitivity, Precision, Accuracy

    © 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.2024v16i6.52154 Journal homepage: https://innovareacademics.in/journals/index.php/ijap=

    INTRODUCTION

    asciminib is a tyrosine kinase inhibitor used to treat chronic-phase Ph+Chronic Myeloid Leukemia (CML). More precisely, it hinders the activity of the Abelson Kinase (ABL1) in the Breakpoint Cluster Region (BCR)-ABL1 fusion protein, which is responsible for promoting the growth of CML in most affected individuals. It has shown effectiveness in treating Ph+CML with a T315I mutation, which causes resistance to therapy with the mutant BCR-ABL1 compared to the normal BCR-ABL1. The development CML is mostly caused by the Philadelphia chromosomal translocation, which creates a fusion oncogene called BCR ABL1 by combining the ABL and BCR genes. The interaction of this gene leads to the synthesis of a fusion protein, BCR-ABL1, which exhibits transformative properties and elevated tyrosine kinases that promote the growth of CML [1-3].

    Fig. 1: Chemical structure of asciminib

    Asciminib is an allosteric inhibitor that targets the BCR ABL1 tyrosine kinase. By binding to the myristoyl pockets of a specific component of the fusion proteins called ABL1, it has the potential to inhibit the oncogenic activity that would otherwise be triggered by the fusion proteins. This is achieved by immobilizing the fusion proteins in an inactive conformation [4]. The Area Under the concentration-time Curve (AUCtau) and maximum concentration (Cmax) reached steady state levels at a dosage of 40 mg administered twice daily were 5,262 ng. h/ml and 793 ng/ml, respectively. The chemical is formally known as N-4-[chloro (difluoro) methoxy] phenyl. The chemical compound is-6-[(3R)-3-hydroxy pyrrolidin-1-yl]. The compound is-5-(1H-pyrazol-5-yl). The compound pyridine-3-carboxamide hydrochloride has a chemical formula of C20H18ClF2N5O3 and a molar mass of 449.84 g•mol−1.

    The literature on asciminib indicates that two analytical methods were used to measure the amount of asciminib: Ultra-Performance Liquid Chromatography (UPLC) [7] and Liquid Chromatography-Mass Spectrometry (LC-MS/MS) [8]. However, there was a need for an analytical technique to accurately measure the amount of asciminib in the biosamples, with a high level of sensitivity and a wider range of concentrations. The present work focuses on developing an LC-MS/MS technique to accurately measure asciminib levels in plasma samples.

    MATERIALS AND METHODS

    Reagent and chemicals

    We acquired canagliflozin from Hetero pharmaceuticals in Hyderabad, India, which has a purity level of 99.52%. In Mumbai, India, Novartis Ltd. supplied the asciminib. The Vivekananda Blood Bank in Hyderabad, India, supplied the drug-free human plasma K2-Ethylenediaminetetraacetic Acid (EDTA) anticoagulant. The study effort used a High-Performance Liquid Chromatography (HPLC)-water purification system developed by Milli-pore in the United States of America, known as MilliQ. We sourced our high-grade ethyl acetate, ammonium acetate, HCOOH, Acetonitrile (ACN), and liquid chromatographic grade methanol from local vendors.

    Mass instrument

    In order to accomplish Multiple Reaction Monitoring (MRM), the+ve ionization method made use of the electro spray ionization technique. A diluted medication stock solution was injected to improve the operating parameters. The fluxes of nebulizer gas and auxiliary gas were 35 and 50 psi, respectively. An initial temperature of 350 °C was established. Unit resolution was used to monitor Q3 and Q1. Excellent peak intensities were achieved by improvising protonation of analytes when HCOOH was added to the mobile phase. For canagliflozin, the MRM mode was seen at m/z 445.13/267.31, while for asciminib, it was m/z 450.23/257.3. Using the analyst software 1.5.1 regression line, we were able to estimate the sample concentrations. Here, the peak response ratio technique was used.

    Quality control (QC) and calibration standards preparation

    The analyte stock solutions were processed in acetonitrile at a concentration of 1000 μg/ml. In order to acquire a concentration range of 1.0-2100.0 ng/ml using acetonitrile, the resultant solution was diluted serially. A mixture of 960 μl of pooled plasma with K2 EDTA and 20 μl of diluted asciminib was used to prepare the calibration standards [9-12]. Twenty microliters of Internal Standard (IS) dilution were added to the final solution after it was produced. A freezer was used to hold solutions with concentrations ranging from 1.0 to 2100.0 ng/ml.

    Three different concentrations of quality control standards High-Quality Control (HQC), Median Quality Control (MQC), and Low-Quality Control (LQC) were handled. Following the same procedures as the calibration standard solutions, these QC solutions were adjusted to 1575.00, 1050.00, and 2.8 ng/ml for HQC, MQC, and LQC, respectively. One conical flask was used to process 1 mg/ml stock quantities of canagliflozin in acetonitrile, while the other flask served as an IS. A 1.0 µg/ml solution was obtained by diluting the resultant stock with ACN.

    Chromatography

    An isocratic mobile phase containing 0.1% v/v HCOOH and ACN at a ratio of 18:82, (% v/v) was used at a flowrate of 0.70 ml/min, and 10 µl** of the sample was injected onto a Reversed Phase-Phenomenex (100 mm × 4.6 mm, 5 µm) C18 analytical column. The whole chromatographic period was 3.0 min, and the analytical column was kept at 45 °C. The chromatographic system used a combination of a Shimadzu HPLC and an Applied Biosystems API-5000 mass spectrometer.

    Protocol for sample preparation

    Moving 150 µl** of plasma and 100 µl** of canagliflozin (1 µg/ml) into a prelabeled tube and subjecting it to sonication for 10 min was the procedure for executing the drug solution. Centrifugation at 3500 rpm/min for 20 min separated asciminib and canagliflozin from a solution containing 5.0 ml of a solvent system consisting of acetonitrile and ethyl acetate in a 4:3 ratio. The organic component was separated and then dried using a lyophilizer. The dried residue was dissolved in 250 µl** of a mobile solvent and thereafter transferred to LC-vials [13-15]. The LC-MS/MS apparatus was charged with the contents of these vials.

    Validation of analytical method

    Based on United States Food and Drug Administration (USFDA)-guidelines for sensitivity, specificity, linearity, stability, accuracy, and inter-and intraday precision, the developed LC-MS/MS work was validated [12–14].

    RESULTS AND DISCUSSION

    Method development

    The selective and sensitive LC-MS/MS method needed precise sample cleaning procedures to quantify very low quantities of pharmaceutical formulations in biological sample solutions. The processing of biological materials typically included one of three methods: Solid-Phase Extraction (SPE), Protein Precipitation (PPT), or Liquid-Liquid Extraction (LLE). There is a possibility of matrix component interference, however the PPT technique using organic segments proved straightforward. After that, we gave LLE a go using ethyl acetate and ACN as solvents. As a conclusion, the sample extraction solvent combination of ACN and ethyl acetate (4:3 ratio) produced satisfactory recovery.

    Analytical method validation

    Selectivity

    Eight separate lots of human plasma, including 1-lipemic and 1-hemolytic lots, were used to test the method's selectivity [16]. At the drug and IS retention times, no interference components were found when the blanks' peak areas were compared to the spiked LOQ standard's regions. In fig. 2, the corresponding peaks were shown.

    Fig. 2: Chromatograms of asciminib blank plasma (A), spiked LLOQ (B) samples

    Recovery

    Asciminib and IS recovery values were found to be good and repeatable using this process, demonstrating that it is a robust analytical method [17, 18]. Analyte extraction recoveries were evaluated by comparing peak responses from spiked plasma samples (n = 6) prior to extraction with aqueous sample solution. All QC standards had an average recovery rate of 94.36% (within the specified margin of error). Table 1 (fig. 3) shows the results, which indicate that the IS recovery was 97.69%.

    Table 1: Extraction recovery rates of analytes

    Concentration level X Y % Recovery % mean recoveryn %RSD
    IS 1013246 989837 97.69 94.36 3.14
    HQC 1979779 1887523 95.34
    MQC 1319854 1226670 92.94
    LQC 3518 3314.195 94.17

    n=3; X, mean recoveries of unextracted samples; Y, mean recoveries of extracted samples; IS, Internal Standard; HQC, High-Quality Control; RSD, Relative Standard Deviation.

    Fig. 3: Chromatograms of asciminib LQC (A), MQC (B) and HQC (C) samples

    Linearity and sensitivity

    Analyte concentrations between 1.0 and 2100.00 ng/ml were used to produce an eight-point calibration curve. To examine the linear response of the calibration curve, the ratios of the drug's peak responses (y) to IS were plotted against the drug's concentration (x). Equation for linearity was determined to be y = 0.0012x-0.0002, and the correlation coefficient was more than 0.999 (0.9998). In order to get precise results for each calibration standard, the concentration measurements were tested again. The analyte's calibration point’s % RSD value ranges were 2.17-4.84, as shown in table 2.

    Table 2: Calibration standards for asciminib

    CS-ID Concentration (ng/ml) Meann (ng/ml) %RSD %RE
    CS-1 1 0.988 3.62 1.1
    CS-2 2.8 2.747 2.97 1.856
    CS-3 60 57.588 4.28 4.017
    CS-4 260 264.864 3.25 -1.871
    CS-5 650 678.443 4.86 -4.376
    CS-6 1100 1126.083 2.17 -2.372
    CS-7 1575 1524.206 4.84 3.224889
    CS-8 2100 2193.893 3.83 -4.473

    n=3, CS, Calibration Standards; RSD, Relative Standard Deviation; RE, Relative Error

    Accuracy and precision

    For all analytical concentrations, the accuracy and precision were tested for both intra-and inter-day groups using six replicate solutions at QC levels (n = 6). The standards used were a HQC control at 1575.00 ng/ml, a middle quality control at 1050.00 ng/ml, a LQC at 2.8 ng/ml, and a Lower Limit of Quantification (LLOQ) at 1.0 ng/ml. Table 3 displays the results for accuracy and precision. The percentage relative error ranged from-1.9 to 4.85% of the nominal values, while the intraday and interday precision %RSDs were within 5.74%.

    Table 3: Intra-batch and inter-batch precision and accuracy

    Concentration
    level    		 Nominal concentration
    (ng/ml)    		 Intra-batch Inter-batch
    Amount found
    (ng/ml)    		 %RSD %RE Amount found
    (ng/ml)    		 %RSD %RE
    HQC 1575 1647.221 4.91 4.58 1651.327 5.74 4.85
    MQC 1050 1091.412 2.85 3.94 1089.435 3.82 3.76
    LQC 2.8 2.903 4.31 3.68 2.921 2.17 4.32
    LLOQ 1 0.981 3.56 -1.9 0.971 3.64 -2.9

    HQC, High-Quality Control; MQC, Median Quality Control, LQC, Low-Quality Control; LLOQ, Lower Limit of Quantification; RSD, Relative Standard Deviation; RE, Relative Error

    Matrix effect

    Eight different lots, including lipemic-1 and hemolytic-1, were used to collect blank plasma samples in order to evaluate this parameter. Each batch was processed according to the sample preparation technique after 100 microliters of blank plasma was collected. Processing was carried out at either the LQC or HQC levels for both the aqueous and post-extracted solutions [19-22]. A formula was used to determine the matrix effect, which is defined as the percentage of the aqueous sample's peak response divided by the peak response of the postextraction samples, multiplied by 100. Table 4 shows that for analytes at the LQC level, the mean matrix factor ranged from 96.31% to 104.72% with a %CV of 4.57, whereas for analytes at the HQC level, it was 94.63% to 103.87% with a %CV of 3.71.

    Table 4: Matrix factor for asciminib

    S. No. HQC LQC
    Concentration in absence of matrix Concentration in presence of matrix Matrix factor Concentration in absence of matrix Concentration in presence of matrix Matrix factor
    1 1581.61 1506.33 95.24 2.678 2.542 94.89
    2 1563.57 1495.87 95.67 2.640 2.574 97.52
    3 1557.78 1594.26 102.34 2.750 2.648 96.31
    4 1465.49 1522.34 103.87 3.004 2.833 94.25
    5 1453.24 1416.62 97.48 2.67 2.795 104.72
    6 1610.87 1524.23 94.63 2.771 2.873 103.65
    Mean 98.20 98.58
    ± SD 3.60 4.15
    % CV 3.71 4.57

    HQC, High-Quality Control; LQC, Low-Quality Control; SD, Standard Deviation; CV, Coefficient of Variation

    Dilution integrity

    We made six separate copies of the sample solutions and compared them to a set of newly spiked calibration solutions to get an approximation. Researchers found that diluting the medication with plasma blank increased the upper concentration limit to 6,000 ng/ml [20]. With a coefficient of variation for the analyte below 4.09, the average back-calculated concentrations for 2-fold and 4-fold diluted sample solutions ranged from 95.78 to 103.52 percent.

    Carryover effect

    Use of sample solutions in the following order: lower limit of quantification quality control LLOQ QC of drug, plasma blank, and Upper Limit of Quantitation (ULOQ) of drug and plasma blank allowed for the processing of this parameter. During the analysis, no drug or IS carryover was found [21].

    Stability studies

    Both aqueous and matrix-based samples were subjected to stability testing. Both the long-term and short-term stability assessments of aqueous samples were assessed in the following ways:

    Aqueous solution stability

    The MQC standard solutions were processed and kept at 25.0 °C for 24 h to ensure short-term stability. Using a newly processed MQC standard sample solution and an injection of six duplicates of these standards at 25.0 °C, we evaluated any variations. At 94.95%, stability was the average. The MQC standard solutions were processed to ensure long-term stability, and the product was kept at 2-8 °C for 40 days. For the purpose of estimating any discrepancies with the newly processed MQC standard sample solution, six sets of these standards were injected. At 96.74%, stability was the average.

    Plasma stability in humans

    Using LQC and MQC standards processed in plasma solution, which were collected at-20.0 °C and maintained at 25.0 °C (room temperature) for seven hours, benchtop stability was achieved. For the purpose of estimating discrepancies with newly processed LQC and MQC standard sample solutions, six copies of these standards were inserted [17]. Between 96.34% and 95.31% of the time, stability was evident. After 22 h of processing in an auto sampler at 10.0 °C, the in-injector stability was determined to be 95.34-103.85% using LQC and MQC standards in plasma solution. Plasma solutions were tested for freeze-thaw stability using LQC and MQC standards. The expected percentage of recovery, after four freeze-thaw cycles, was determined to be between 98% and 103.85%. Results showed a wet extract stability of 94.87–93.71% when LQC and MQC standards were processed in a plasma solution at 25.0 °C for 7 h (table 5).

    Table 5: Stability results of asciminib

    Parameter QC level N M %RSD %Stability
    Wet extract stability LQC 2.8 2.763 4.63 98.69
    HQC 1575 1615.005 3.87 102.54
    Freeze–thaw stability LQC 2.8 2.656 3.94 94.87
    HQC 1575 1475.933 4.07 93.71
    Benchtop stability LQC 2.8 2.698 3.81 96.34
    HQC 1575 1498.928 2.93 95.17
    In-injector stability LQC 2.8 2.669 2.93 95.34
    HQC 1575 1635.638 2.51 103.85

    N, nominal concentration (ng/ml); M, mean concentrations (ng/ml) of analytes, HQC, High-Quality Control; LQC, Low-Quality Control; QC, Quality Control; RSD, Relative Standard Deviation

    Extended accuracy and precision run

    Forty sets of HQC and LQC, together with one batch of calibration curve standards, were prepared and estimated before the extended accuracy and precision run was conducted [19]. Table 6 displays the results pertaining to the precision and accuracy that were expanded. In terms of accuracy and stability, asciminib performed well at both the LQC and HQC levels, with scores of 3.89 and 95.34%, respectively.

    Table 6: Extended precision and accuracy analysis

    Analyte Conc. level A B %RSD %Stability
    asciminib HQC 1575 1635.282 4.12 103.74
    LQC 2.8 2.671 3.89 95.41

    A, Original concentration (ng/ml); B, mean of 6 concentrations (ng/ml), HQC, High-Quality Control; LQC, Low-Quality Control; RSD, Relative Standard Deviation

    CONCLUSION

    An LC–MS/MS method that is specific, accurate, and sensitive was developed and validated in a short amount of time for the purpose of determining the amount of asciminib present in human plasma. For the purpose of achieving consistent recovery results for both the drug and the IS, the LLE extraction technique was used in the method that was designed. The approach was validated in compliance with the criteria provided by the FDA across the concentration range of 1.0–2100.00 ng/ml for asciminib, and the correlation coefficient value was found to be 0.9998. Both the intraday and interday(RSDs were within 5.74% of the nominal values, and the rate of relative error ranged from-1.9 to 4.85 percent. At the LQC level, the mean matrix factor was found to be between 96.34 and 104.85%, with a % CV of 4.21. At the HQC level, the matrix factor range was 94.62 to 103.88%, with a %CV of 3.67. Because of this, the method that was created has the potential to be used for the purpose of effectively studying pharmacokinetics and toxicokinetics in forensic and clinical studies of asciminib in a variety of biological materials.

    FUNDING

    Nil

    AUTHORS CONTRIBUTIONS

    Both the authors have contributed in design, experiment and execution of the work.

    CONFLICT OF INTERESTS

    Declared none

    REFERENCES

    1. Breccia M, Colafigli G, Scalzulli E, Martelli M. Asciminib: an investigational agent for the treatment of chronic myeloid leukemia. Expert Opin Investig Drugs. 2021;30(8):803-11. doi: 10.1080/13543784.2021.1941863, PMID 34130563.

    2. Jones JK, Thompson EM. Allosteric inhibition of ABL kinases: therapeutic potential in cancer. Mol Cancer Ther. 2020;19(9):1763-9. doi: 10.1158/1535-7163.MCT-20-0069, PMID 32606014.

    3. Schoepfer J, Jahnke W, Berellini G, Buonamici S, Cotesta S, Cowan Jacob SW. Discovery of asciminib (ABL001) an allosteric inhibitor of the tyrosine kinase activity of BCR-ABL1. J Med Chem. 2018;61(18):8120-35. doi: 10.1021/acs.jmedchem.8b01040, PMID 30137981.

    4. Tran P, Hanna I, Eggimann FK, Schoepfer J, Ray T, Zhu B. Disposition of asciminib a potent BCR-ABL1 tyrosine kinase inhibitor in healthy male subjects. Xenobiotica. 2020;50(2):150-69. doi: 10.1080/00498254.2019.1594449, PMID 31006307.

    5. Hou JZ, YE JC, PU JJ, Liu H, Ding W, Zheng H. Novel agents and regimens for hematological malignancies: recent updates from 2020 ASH annual meeting. J Hematol Oncol. 2021;14(1):66. doi: 10.1186/s13045-021-01077-3, PMID 33879198.

    6. Khatri A, Wang J, Pendergast AM. Multifunctional ABL kinases in health and disease. J Cell Sci. 2016;129(1):9-16. doi: 10.1242/jcs.175521, PMID 26729027.

    7. Priya DS, Gubbiyappa, Rangapuram VA. A novel stability indicating reversed phase ultra performance liquid chromatography method for estimation of asciminib its bulk and tablet formulation. Int J Pharm Qual Assur. 2022;13(4):490-5. doi: 10.25258/ijpqa.13.4.23.

    8. Govindarao Y, Ganesh Kumar Y. Drug development and validation of a sensitive bio-analytical LCMS/MS method for quantification of asciminib a chronic myeloid leukemia drug in human plasma. J Drug Alc Res. 2023;12(5):1-15. doi: 10.4303/JDAR/236242.

    9. Lolla S, Gubbiyappa KS, Cheruku S, Bhikshapathi DV. Validation of an LC-MS/MS method for quantitation of fostemsavir in plasma. J Pharmacol Toxicol Methods. 2023 Mar-Apr;120:107254. doi: 10.1016/j.vascn.2023.107254, PMID 36863666.

    10. Bandaru AK, Bomma R, Shankar C, Bhikshapathi DV. Method development validation and application of liquid chromatography electrospray ionization mass spectrometry mass spectrometry method for the quantification of amprenavir in plasma samples. J Appl Pharm Sci. 2022;12(7):115-21. doi: 10.7324/JAPS.2022.120712.

    11. Ravi Y, Bhikshapathi D, Cheruku S, Rajkamal B. Development of fast and simple LC-ESI-MS/MS technique for the quantification of regorafenib; application to pharmacokinetics in healthy rabbits. Curr Pharm Anal. 2021;17(4):554-63. doi: 10.2174/1573412916666191111144707.

    12. FDA guidance for industry. Cent Drug Eval Res Biopharm Bioanal Method Validation; 2001. Available from: http://www.fda.gov/cder/guidance/index.htm.

    13. Sellappan M, Devakumar D. Development and validation of RP-HPLC method for the estimation of escitalopram oxalate and flupentixol dihydrochloride in combined dosage form and plasma. Int J Pharm Pharm Sci. 2021 Feb 1;13(2):61-6. doi: 10.22159/ijpps.2021v13i2.30158.

    14. Gurav P, DamLe M. Bioanalytical method for estimation of teriflunomidein human plasma. Int J Pharm Pharm Sci. 2022 Sep;14(9)19-23. doi: 10.22159/ijpps.2022v14i9.45151.

    15. Janiszewski J, Schneider P, Hoffmaster K, Swyden M, Wells D, Fouda H. Automated sample preparation using membrane microtiter extraction for bioanalytical mass spectrometry. Rapid Commun Mass Spectrom. 1997;11(9):1033-7. doi: 10.1002/(SICI)1097-0231(19970615)11:9<1033::AID-RCM951>3.0.CO;2-5, PMID 9204578.

    16. Puttagunta SB, Shaik RP, Bannoth CK, Challa BS, Awen BZ. Bioanalytical method for quantification of solifenacin in rat plasma by LC-MS/MS and its application to pharmacokinetic study. J Anal Sci Technol. 2014;5(1):35. doi: 10.1186/s40543-014-0035-0.

    17. Cheruku S, Darna B, Medipalli V, Nekkalapudi AR, Sadasivam RK. Bioanalytical method development and validation for the quantitation of larotrectinib in human plasma: application to pharmacokinetics in healthy rabbits. J Appl Pharm Sci. 2023;13(11):111-8. doi: 10.7324/JAPS.2023.127799.

    18. Dadhaniya T, Chaudhary K, Mehta P. Development of LC-MS/MS method for determination of iloperidone in rabbit plasma: application to a pharmacokinetic study. Int J Pharm Pharm Sci. 2013;7(4):294-7.

    19. Zhou J, Gao S, Zhang F, Jiang B, Zhan Q, Cai F. Liquid chromatography tandem mass spectrometry method for simultaneous determination of seven commonly used anticancer drugs in human plasma. J Chromatogr B Analyt Technol Biomed Life Sci. 2012;906:1-8. doi: 10.1016/j.jchromb.2012.07.033, PMID 22959676.

    20. Buszewski B, Noga S. Hydrophilic interaction liquid chromatography (HILIC)-a powerful separation technique. Anal Bioanal Chem. 2012;402(1):231-47. doi: 10.1007/s00216-011-5308-5, PMID 21879300.

    21. Ponnuri RN, Pragallapati P, NR, Mandava VB. A rapid and sensitive liquid chromatography mass spectrometry mass spectrometry method for estimation of pioglitazone keto pioglitazone and hydroxy pioglitazone in human plasma. Asian J Pharm Clin Res. 2017;10(12)120-8. doi: 10.22159/ajpcr.2017.v10i12.20284.

    22. Sura RS, Cvs S, Rachamalla SS. Cvs S, Rachamalla SS. Bioanalytical RP-HPLC method development and validation of clopidogrel bisulfate in wistar rat plasma and its application to pharmacokinetic study. Int J App Pharm. 2022;14(1):106-11. doi: 10.22159/ijap.2022v14i1.43328.