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IJCRR - 14(4), February, 2022

Pages: 50-55

Date of Publication: 15-Feb-2022


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A Novel Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (LC/ESI/MS/ MS) Method Development and Validation for the Quantification of Erdafitinib in Human Plasma

Author: Yarra Raviteja, G. Suresh

Category: Healthcare

Abstract:Introduction: A new specific, accurate and selective liquid chromatography/tandem mass spectrometry (LC-MS/MS) technique was desirable for the assessment of erdafitinib in human plasma. Aim: To develop an LC\?ESI-MS/MS method for the quantification of anticancer agent erdafitinib in human plasma and its validation as per the USFDA guidelines. Methodology: Propranolol (internal standard) reference drug utilized for the quantification of erdafitinib. Extraction of 250\?L plasma with liquid extraction technique, analyte components were eluted on symmetry (50 × 4.6mm; 5\?m) C18 analytical column. Mass detection system was executed by utilizing MRM (multiple reaction monitoring) with +ve ionization mode and electrospray ionization. Developed procedure was subjected for validation according to FDA guidelines in concentration levels between 69.95\?2798.00ng/mL for erdafitinib with r2 value of 0.9992. Results: The intraday and interday precision were within 4.11% and the assay accuracy was 97.95\?104.91 % of the nominal values. Matrix factor ranges from 95.61\? 103.84 % with a %CV of 3.63 for analyte at HQC and MQC levels, matrix factor range was 94.32\?104.01% with a %CV of 3.89. Conclusion: The developed process can be successfully applied for routine analysis in quality control, pharmacokinetic and forensic studies of biological samples.

Keywords: FGFR tyrosine kinase inhibitor, Erdafitinib, FDA, Accuracy, Linearity, Nominal values

Full Text:

INTRODUCTION

Erdafitinib is an inhibitor of FGFR (pan-fibroblast growth factor receptor) tyrosine kinases that is directed to treat patients with metastatic urothelial or locally advanced cancer which has: i) vulnerable FGFR 3 or FGFR 2 genetic alterations and ii) progressed during or following at least one line of prior platinum having chemotherapy with twelve months of adjuvant or neoadjuvant platinum comprising chemical therapy. Erdafitinib designated as N′-(3,5-Dimethoxy phenyl) -N′- [3- (1-methyl pyrazol -4-yl) quinoxalin -6-yl] -N-propan -2- ylethane-1,2- diamine with molecular formula and weight of C25H30N6O2 and 446.555 g·mol−1 respectively.1-5

FGFR is a transmembrane protein which express universally in normal tissue and also involved in numerous endogenous bio physiological procedures containing homeostasis of phosphate and cell proliferation, cell migration, vitamin D and cell anti-apoptotic signaling in different type of cells. Genetic changes or mutations like FGFR abnormalities like point mutations, gene amplification, and deregulation and chromosomal translocations of FGFR paths have been associated in pathogenesis of urothelial cancer, containing possibilities of such variations to all 4 FGFR-genes (FGFR-1, FGFR-2, FGFR-3, and FGFR-4).3,6,7 Deviations to the FGFRgenes are subsequently thought to stimulate cell propagation, relocation, anti-apoptosis and angiogenesis in many malignances comprising urothelial cancer. Drug is an oral specific pan-FGFR kinase inhibitor that bound and obstructs enzymatic action of expressed FGFR-1, FGFR-2, FGFR-3, and FGFR-4 based on in vitro data. In particular, erdafitinib demonstrates inhibition of FGFR phosphorylation and signaling as well as decreased cell viability in cell lines expressing FGFR genetic alterations, including point mutations, amplifications, and fusions.2,4,7

Literature on erdafitinib revealed that few analytical methods were reported on liquid chromatography8 and LC-MS/MS.9 The development of specific method alike LC-MS/MS is very vital for the quantification of erdafitinib in biological matrix.

MATERIALS AND METHODS

Chemicals and reagents

Erdafitinib (98.9 % purity) and propranolol (98.8 % purity) were acquired from Alembic Pharmaceuticals, Hyderabad, India. HPLC grade acetonitrile and methanol, ammonium acetate, ethyl acetate and HCOOH of the high grade quality were procured locally. Purified milli-Q water (Milford, MA) was utilized for entire research work.

Standards preparation

Processing of individual analyte stock solution(1000μg/ml) was done in methyl alcohol. The resulting solution was subjected for serial dilutions to get concentration range of 69.95–2798.00ng/mL with methanol.

Calibration standard solutions

For the processing of calibration standards, 20μl of erdafitinib diluted sample was mixed with 960μl of K2EDTA pooled plasma. To the resultant solution, 20μl of IS dilution was transferred to get final solution. The concentration range, 69.95–2798.00 ng/ml solutions were prepared and stored below −20 °C in a freezer.

Preparation of quality control samples

Quality control standard were processed at 3 dissimilar concentrations of high quality control(HQC) standards, median quality control(MQC)  standards and low quality control(LQC) standards. These QC (quality control) solutions were processed as per the calibration standard solutions to get 2098.5, 1399.00 and 195.86, ng/mL for HQC, MQC and LQC correspondingly.

Preparation of solution of internal standards

Propranolol was used as internal standard and 1mg/mL stock concentrations in methyl alcohol were processed in a separate conical flask. Resulting stock was made dilution with methyl alcohol to get 1µg/mL solution.

Sample preparation

Drug solution was executed by relocating 250µl of plasma and 50µL of propranolol (1µg/mL) in to a pre-labeled tube and sonicated for 2.0 min. Erdafitinib and propranolol were isolated with 5.0mL of ethyl acetate solvent system and the solution was subjected for centrifugation at 5000rpm/min for 25min. Organic layer was isolated and dried with lyophiliser. Residue after the drying was made solubilize in 250µl of movable solvent and then translocated to LC-vials. These vials were placed and injected into LC-MS/MS instrument.

Chromatography

10µL of sample was injected on reversed phase symmetry (50 × 4.6mm) 5 μm C18 analytical column with an isocratic movable phase comprising of methyl alcohol and 5mM ammonium acetate in 0.1% HCOOH in Proportion of 80:20, (%v/v) was utilized at a flow rate of 0.80ml/min. Analytical column was retained at 45°C and total chromatographic time was 3.5min. Chromatographic system was equipped with HPLC-Shimadzu combined with API-5000 Mass instrument of Applied Bio systems, America.

Mass instrument

Electro spray ionization technique was utilized and functioned in +ve ionization method for MRM. By injecting dilute stock solution of drug, the operating parameters were improvised as mentioned in Table 1. Auxiliary gas (GS2) and nebulizer gas (GS1) flows were 40 and 45 psi, respectively. Source temperature was set at 250 °C. Q3 and Q1 were monitored under unit resolution.  Upon addition of HCOOH to the mobile phase, protonation of analytes were improvised and excellent peak intensities were obtained. MRM mode monitored at:  m/z 447.25 → 388.17 for erdafitinib and   m/z 260.16 → 72.08 for propranolol. Concentrations of samples were estimated by regression line with the help of analyst software1.5.1. In this peak response ratio method was utilized.

Validation of analytical method

Developed LC–MS/MS work was subjected for validation according to USFDA-guidelines for sensitivity, inter and intraday precision, specificity,  linearity, stability and accuracy.10,11

RESULTS AND DISCUSSION

Method validation

Selectivity

Method selectivity was executed with 8 distinct human K2 EDTA human plasma samples including 1-hemolytic and 1-lipemic lot. Interferences observed were negligible during the analysis of analytes at respective retention times over the spiked response of LLOQ with IS (Fig.2).12-14

Recovery

Analyte recovery from extraction sample was evaluated by equating peak responses from six plasma sample solutions spiked prior to extraction against aqueous sample solutions.15, 16 Overall average recoveries of all three quality control standards was 93.15%. The recovery of IS was found to be 96.56 % and the findings were shown in Table 2 (Fig. 3 to 5).

Sensitivity and linearity

Eight points calibration curve was processed with analyte samples from 69.95 ng/ml to 2798.00 ng/ml. Peak response ratio(y) of analyte to IS was taken against nominal concentration(x) of analyte to evaluate linearity. Regression line has r2 value of 0.9992 with equation y= 0.005412 x+ 0.00128.16,17 Erdafitinib LLOQ was 69.95 ng/ml, which indicates the developed method sensitivity.    

Accuracy and precision

Inter and intraday accuracy and precision was evaluated by considering 6 replicates for  LLOQ, LQC, MQC and HQC standard solutions18, 19. The corresponding erdafitinib concentrations were 69.95, 195.86, 1399.00 and 2098.50 ng/ml. Accuracy and precision findings were represented in Table 3. Inter and intraday precision findings were ≤4.11% and the assay accuracy was 97.95–104.91 %.

Matrix effect

This parameter was evaluated by extracting 8 variable blank plasma samples including 1-lepemic and 1-hemolytic lot. From each blank plasma lot 100 microliters of sample was subjected for processing as per the sample preparation. Individual aqueous sample at HQC or LQC level was mixed to final solution(post-extraction matrix samples).16, 20 In a similar way individual aqueous sample solution at HQC or LQC level was executed with moveable solvent(aqueous sample without matrix).

Above processed solutions were injected six times at HQC and LQC levels and analyte and IS peak responses were compared. This parameter was evaluated by: Matrix effect (%) = A2/A1 × 100(%), where A1 = response of aqueous concentrations and A2 is the response of post-extracted concentrations. Average   (n = 6)   matrix factor ranges from 95.61– 103.84 % with a %CV of 3.63 for analyte at low and at high QC levels, the matrix factor range was 94.32–104.01%  with a  %CV  of  3.89 (Table 4).

Carryover effect

In this cleaning capability of washing solution utilized for infusion port and needle was evaluated.21 It was evaluated by injecting the samples in order of: LLOQQC, blank and ULOQQC and again blank solution. There was no effect of carryover observed throughout the study.

Dilution integrity

Developed process was subjected for dilution integrity at fourfold and twofold dilution. Six duplicate sample solutions were executed and subjected for evaluation over the fresh spiked standards. Upper limit was expandable upto 2798.00.ng/mL. Average back-calculation concentration values were present in between 94.46–104.52 % with a % CV of ≤2.83 for the analyte.15

Stability

Bench-top, freeze-thaw, in-injector and wet extract stability studies were executed for the evaluation human plasma sample stability. All these studies were evaluated at HQC and LQC levels. To process benchtop stability, standard QC samples were collected from freezer(−20°C) and thawed(25°C) upto 6 hours.  The percentage of stabilities were evaluated against the fresh sample solutions and found in between 95.97–98.11 %. To process the freeze-thaw stability, standard QC samples were subjected for freeze and thaw rotations for four times. The percentage of stabilities were evaluated against the fresh sample solutions and found in between 93.85 to 102.06 % .

To process the in-injector stability, standard QC samples were kept in an autosampler at 10°C for 24 hours.  The percentage of stabilities were evaluated against the fresh sample solutions and found in between 98.35–103.61 %. Wet extract stability was processed at 8 hours at 25°C and  The percentage of stabilities were evaluated against the fresh sample solutions and found in between 99.49–102.71 % (Table 5).

Extended accuracy and precision

To process the extended accuracy and precision, 1-set of calibration standards and forty sets of HQC and LQC standards, total ninety samples, were injected and subjected for the evaluation. The resultant findings were shown in Table 6. The precision for erdafitinib was <3.2 and stability at HQC and LQC levels were found to be 98.35% and 94.86% correspondingly.

CONCLUSION

A simple, specific and sensitive LC–MS/MS process for quantification of Erdafitinib in plasma samples was developed successfully and subjected for the validation as per the USFDA guidelines. Linearity was processed in between 69.95–2798.00ng/mL for erdafitinib with regression coefficient value of 0.9992. The intraday and interday precision were within 4.11% and the assay accuracy was 97.95–104.91 % of the nominal values. Matrix factor   ranges from 95.61– 103.84 % with a %CV of 3.63 for analyte at HQC level and at LQC level, the matrix factor range was 94.32–104.01%  with a  %CV  of  3.89. Stabilities revealed that the method has high degree of stability. The developed process can be utilized in bioequivalence and bioavailability studies for the quantification of erdafitinib in biological matrices.

ACKNOWLEDGEMENT

Authors acknowledge immense help received from the scholars whose articles are cited and included in reference to the manuscript. The authors are grateful to all the authors/ editors/publishers of those articles, journals, and books from where the literature of this article has been reviewed and discussed.

Source(s) of funding: No funding is involved.

Conflicts of interest: The authors declare no conflicting interest.

Author’s contribution: All the authors contributed equally.

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A Study by Jabbar Desai et al. entitled "Prevalence of Obstructive Airway Disease in Patients with Ischemic Heart Disease and Hypertension" is awarded Best Article for Vol 12 issue 17
A Study by Juna Byun et al. entitled "Study on Difference in Coronavirus-19 Related Anxiety between Face-to-face and Non-face-to-face Classes among University Students in South Korea" is awarded Best Article for Vol 12 issue 16
A Study by Sudha Ramachandra & Vinay Chavan entitled "Enhanced-Hybrid-Age Layered Population Structure (E-Hybrid-ALPS): A Genetic Algorithm with Adaptive Crossover for Molecular Docking Studies of Drug Discovery Process" is awarded Best article for Vol 12 issue 15
A Study by Varsha M. Shindhe et al. entitled "A Study on Effect of Smokeless Tobacco on Pulmonary Function Tests in Class IV Workers of USM-KLE (Universiti Sains Malaysia-Karnataka Lingayat Education Society) International Medical Programme, Belagavi" is awarded Best article of Vol 12 issue 14, July 2020
A study by Amruta Choudhary et al. entitled "Family Planning Knowledge, Attitude and Practice Among Women of Reproductive Age from Rural Area of Central India" is awarded Best Article for special issue "Modern Therapeutics Applications"
A study by Raunak Das entitled "Study of Cardiovascular Dysfunctions in Interstitial Lung Diseas epatients by Correlating the Levels of Serum NT PRO BNP and Microalbuminuria (Biomarkers of Cardiovascular Dysfunction) with Echocardiographic, Bronchoscopic and HighResolution Computed Tomography Findings of These ILD Patients" is awarded Best Article of Vol 12 issue 13 
A Study by Kannamani Ramasamy et al. entitled "COVID-19 Situation at Chennai City – Forecasting for the Better Pandemic Management" is awarded best article for  Vol 12 issue 12
A Study by Muhammet Lutfi SELCUK and Fatma entitled "Distinction of Gray and White Matter for Some Histological Staining Methods in New Zealand Rabbit's Brain" is awarded best article for  Vol 12 issue 11
A Study by Anamul Haq et al. entitled "Etiology of Abnormal Uterine Bleeding in Adolescents – Emphasis Upon Polycystic Ovarian Syndrome" is awarded best article for  Vol 12 issue 10
A Study by entitled "Estimation of Reference Interval of Serum Progesterone During Three Trimesters of Normal Pregnancy in a Tertiary Care Hospital of Kolkata" is awarded best article for  Vol 12 issue 09
A Study by Ilona Gracie De Souza & Pavan Kumar G. entitled "Effect of Releasing Myofascial Chain in Patients with Patellofemoral Pain Syndrome - A Randomized Clinical Trial" is awarded best article for  Vol 12 issue 08
A Study by Virendra Atam et. al. entitled "Clinical Profile and Short - Term Mortality Predictors in Acute Stroke with Emphasis on Stress Hyperglycemia and THRIVE Score : An Observational Study" is awarded best article for  Vol 12 issue 07
A Study by K. Krupashree et. al. entitled "Protective Effects of Picrorhizakurroa Against Fumonisin B1 Induced Hepatotoxicity in Mice" is awarded best article for issue Vol 10 issue 20
A study by Mithun K.P. et al "Larvicidal Activity of Crude Solanum Nigrum Leaf and Berries Extract Against Dengue Vector-Aedesaegypti" is awarded Best Article for Vol 10 issue 14 of IJCRR
A study by Asha Menon "Women in Child Care and Early Education: Truly Nontraditional Work" is awarded Best Article for Vol 10 issue 13
A study by Deep J. M. "Prevalence of Molar-Incisor Hypomineralization in 7-13 Years Old Children of Biratnagar, Nepal: A Cross Sectional Study" is awarded Best Article for Vol 10 issue 11 of IJCRR
A review by Chitra et al to analyse relation between Obesity and Type 2 diabetes is awarded 'Best Article' for Vol 10 issue 10 by IJCRR. 
A study by Karanpreet et al "Pregnancy Induced Hypertension: A Study on Its Multisystem Involvement" is given Best Paper Award for Vol 10 issue 09

List of Awardees

A Study by Ese Anibor et al. "Evaluation of Temporomandibular Joint Disorders Among Delta State University Students in Abraka, Nigeria" from Vol 13 issue 16 received Emerging Researcher Award


A Study by Alkhansa Mahmoud et al. entitled "mRNA Expression of Somatostatin Receptors (1-5) in MCF7 and MDA-MB231 Breast Cancer Cells" from Vol 13 issue 06 received Emerging Researcher Award


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International Journal of Current Research and Review (IJCRR) provides platform for researchers to publish and discuss their original research and review work. IJCRR can not be held responsible for views, opinions and written statements of researchers published in this journal

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