Validated HPLC-UV method for simultaneous quantification of PI3K inhibitors, copanlisib, duvelisib and idelalisib in rat plasma: Application to a pharmacokinetic study in rats

Anup Siddesh, Dhurvu Sriram, Ashok Zakkula*, Rajnish Kumar, Sreekanth Dittakavi, Mohd Zainuddin, Ravi Kumar Trivedi and Ramesh Mullangi

Abstract

Phosphatidylinositol 3-kinase (PI3K) inhibitors are novel class of anticancer drugs, which are approved to treat various malignancies. We report the development and validation of a highperformance liquid chromatography (HPLC) method for the simultaneous quantitation of three PI3K inhibitors namely copanlisib, duvelisib and idelalisib in rat plasma as per the FDA regulatory guidelines. The method involves extraction of copanlisib, duvelisib and idelalisib along with internal standard (IS; filgotinib) from rat plasma (100 µL) using a liquid-liquid extraction process. The chromatographic separation of the analytes was achieved using stepwise gradient elution on a Hypersil Gold C18 column. The UV detection wave length was set at max 280 nm. Copanlisib, duvelisib, idelalisib and the IS eluted at 7.16, 12.6, 11.9 and 9.86 min, respectively with a total run time of 15 min. The calibration curve ranged from 50 to 5000 ng/mL for all the analytes. Inter- and intraday precision and accuracy, stability studies, dilution integrity and incurred sample reanalysis were investigated for all three analytes, and the results met the acceptance criteria. The validated HPLC method was successfully applied to a pharmacokinetic study in rats.

KEY WORDS: Copanlisib, duvelisib, idelalisib, HPLC, method validation, rat plasma, pharmacokinetics

1. INTRODUCTION

Phosphatidylinositol 3-kinases (PI3Ks) play a fundamental role in cellular metabolism, motility, apoptosis, proliferation etc. (Liu, Cheng, Roberts & Zhao, 2009). Dysregulation of PI3K signaling pathway is reported frequently in human cancers, result in resistance to conventional therapies (Engelman, Luo & Cantley, 2006). Among three classes (I, II and III) of PI3Ks, class I exists in four isoforms namely , ,  and . PI3K- is known to play an important role in tumorogenesis, PI3K- showed evidence in thrombotic diseases and PI3K- and  are involved in inflammation and in the immune system (Greenwell, Flowers, Blum & Cohen, 2017). The need for alternative options for the treatment of cancer, inhibition of PI3K has become an attractive and viable target for novel anti-cancer therapy. In the last decade three PI3K inhibitors were approved. Idelalisib is the first-in-class highly selective oral PI3K- inhibitor approved for the treatment of relapsed chronic lymphocytic leukemia (CLL), relapsed follicular B-cell non-Hodgkin lymphoma (NHL) and relapsed small lymphocytic leukemia (SLL). Subsequently, copanlisib, which is a pan-PI3K inhibitor (predominant activity against PI3K- and PI3K- isoforms) was approved for the treatment of relapsed follicular lymphoma (FL) as an intravenous infusion. The third, PI3K inhibitor is duvelisib, approved for the treatment of relapsed or refractory CLL/SLL and FL. Duvelisib is also a dual PI3K inhibitor showing selectivity against PI3K- and PI3K- isoforms. Like idelalisib, duvelisib is also administered orally. Both idelalisib and duvelisib carry a boxed warning on their label. Post oral administration of duvelisib and idelalisib the maximum plasma concentrations in plasma (Cmax) attained between 1.5-2.0 h (Tmax). Duvelisib has shown dose-proportional increase in plasma exposure, however idelalisib exposure in plasma increase in less dose-proportional manner. The plasma protein binding of copanlisib and idelalisib was ~84% and it was >98% for idelalisib. All the three PI3K inhibitors are metabolized by CYP3A4 isoenzyme. Apart from CYP3A4, idelalisib was majorly metabolized by aldehyde oxidase. The major route of elimination for these drugs is through feces. The absolute oral bioavailability of duvelisib and idelalisib was found to be 42 and 34%, respectively. A brief overview on the activity, mechanism of action, dosage regimen and pharmacokinetics on these three PI3K inhibitors are presented in Supplementary Table 1.
To date, one mass spectrometry (LC-MS/MS) and one high-performance liquid chromatography (HPLC) bioanalytical methods were reported for the quantification of copanlisib (Dittakavi & Mullangi, 2019; Zakkula et al., 2020). For the quantification of idelalisib, few LC-MS/MS (Veeraraghavan et al., 2014; Huynh et al., 2018; Wang, Jia & Zhang, 2019; Arumugam, Mani & Chirinos, 2020) and HPLC (Suneetha & Sharmila, 2016) methods were reported in biological matrices. Very recently, Shao et al. (2020) reported an
UPLC-MS/MS method for the quantification of duvelisib in dog plasma. Supplementary Table 2 enlists the reported bioanalytical methods details for the quantitation of copanlisib, duvelisib and idelalisib. LC-MS/MS is often not available in most hospitals and many research laboratories as it is an expensive instrument and incurs huge amount for maintenance. Hence, alternative analytical methods like HPLC-UV method as a standard equipment as setting is simple, cheaper and easier to set up. Besides, there is no bioanalytical method reported for the simultaneous quantification of copanlisib, duvelisib and idelalisib in any biological matrix. Hence, we felt there is a need for an HPLC method for quantification of PI3K inhibitors, which can be used in hospitals (for routine therapeutic drug monitoring) and research laboratories (to support pharmacokinetic and/or toxicokinetic studies samples analysis). In clinic, the recommended doses for copanlisib, duvelisib and idelalisib was 60 mg, 25 mg (twice daily) and 150 mg (twice daily), respectively. At clinical doses, duvelisib showed ~70 ng/mL concentration in plasma (Flinn et al., 2018) at 24 h and the plasma concentration for idelalisib at 24 h was ~90 ng/mL (Arumugam, Mani & Chirinos, 2014). The plasma concentrations of these drugs were measured using a validated LC-MS/MS method. The aim of this work was to develop and validate a HPLC method for simultaneous quantitation of copanlisib, duvelisib and idelalisib in rat plasma and application to a pharmacokinetic study in rats. With the attained LLOQ (lower limit of quantitation) of 50 ng/mL in the present method, we believe the present validated HPLC can be used in place of LC-MS/MS to monitor these drugs concentration in plasma.

2. EXPERIMENTAL

2.1. Chemicals and reagents

Copanlisib (purity: 98.7%; Supplementary Fig. 1) was obtained from Aaron (Shanghai, China). Duvelisib (purity: 98%; Supplementary Fig. 1) and idelalisib (purity: 98%; Supplementary Fig. 1) were purchased from Angene International Limited (England, UK). Filgotinib [purity: >99%; internal standard (IS)] Tween-80, methyl cellulose and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). HPLC grade acetonitrile and methanol were purchased from J. T. Baker (Pittsburgh, PA, USA). Analytical grade ammonium formate was purchased from S.D Fine Chemicals (Mumbai, India). All other chemicals and reagents were of analytical grade and used without further purification. The control rat K2.EDTA plasma was procured from Animal House, Jubilant Biosys (Bangalore, India).

2.2. HPLC operating conditions

Waters 2695 Alliance HPLC system (Waters, Milford, USA) equipped with performance PLUS inline degasser along with an auto-sampler, column oven and photo diode array (PDA) detector set at max 280 nm was used for the analysis of copanlisib, duvelisib and idelalisib along with IS in the present study. Base line separation of copanlisib, duvelisib and idelalisib and the IS in the processed samples was achieved on an Hypersil Gold C18 column (250  4.0 mm, 5 µ; Thermo Scientific, USA) maintained at 40 ± 1°C using a binary mobile phase system consisted of 10 mM ammonium formate, pH: 4.2 (adjusted with formic acid) and acetonitrile run as per the gradient program given in Table 1. The flow-rate and injection volume were 1.00 mL/min and 50 µL, respectively.

2.3. Preparation of stock solutions for copanlisib, duvelisib, idelalisib and the IS Two separate primary stock solutions of copanlisib, duvelisib and idelalisib were prepared to facilitate the preparation of calibration curve (CC) and quality control (QC) samples.

Individual primary stock solution of all the analytes at 200 µg/mL was prepared in 0.1 N HCl: DMSO: methanol (0.2:0.2:99.8, v/v). Similarly, the primary stock solution of the IS (1000 µg/mL) was prepared in methanol. The primary stock solutions of copanlisib, duvelisib and idelalisib and the IS were stored at -20 ± 5°C, which were found to be stable for 50 days. The primary stock solution of IS was appropriately diluted with methanol to prepare the working IS solution (500 ng/mL).

2.4. Preparation of calibration curve standards and quality control samples

The first set of primary stock solutions of copanlisib, duvelisib and idelalisib were diluted appropriately using water:methanol (20:80, v/v) and composite stock solutions were made by successively dilution, which were subsequently used to prepare calibration curve (CC) standards. Calibration samples were prepared by spiking 90 µL of blank rat plasma with the composite working solution of analytes (10 µL) on the day of analysis. Calibration curve standard consists of a set of eight non-zero concentrations for all the analytes was prepared.
The calibrators were 50, 100, 375, 875, 1500, 3100, 4100 and 5000 ng/mL. Samples for the determination of precision and accuracy were prepared by spiking blank rat plasma in bulk with the second composite working stock solution of analytes at appropriate concentrations and 100 L aliquots were distributed into different tubes. The QCs were prepared at 50 ng/mL (lower limit of quantification quality control; LLOQ QC), 150 μg/mL (low quality control; LQC), 2600 μg/mL (medium quality control; MQC) and 3950 μg/mL (high quality control; HQC). All the QCs were stored together at -80 ± 10°C until analysis.

2.5. Sample preparation

To an aliquot of 90 µL of blank rat plasma, 10 μL of stock solution (CCs and QCs) was added. To this 1 mL of extraction solvent dichloromethane:ethyl acetate (30:70; v/v) enriched with IS was added and vortex mixed for 3.0 min, followed by centrifugation for 5 min at 14000 rpm in a refrigerated centrifuge (Eppendorf 5424R) maintained at 5°C. The organic layer (800 µL) was separated and evaporated to dryness at 50ºC using a gentle stream of nitrogen (Turbovap®, Zymark®, Kopkinton, MA, USA). The residue was reconstituted in 100 µL of 80% methanol, transferred into HPLC vial for sample analysis on HPLC.

2.6. Validation procedures

A full validation according to the US FDA guidance was performed for the quantitation of copanlisib, duvelisib and idelalisib in rat plasma (DHHS, FDA, CDER, & CVM, 2018). The selectivity of the proposed method was assessed by evaluating the presence of interfering the peaks at the retention times of copanlisib, duvelisib, idelalisib and the IS in six different batches of blank rat plasma samples. The auto-inject carry over was determined by injecting the highest calibration standard (5000 ng/mL) followed by injection of rat plasma blank samples. Recovery of copanlisib, duvelisib and idelalisib was determined by comparing their respective response from QCs (LQC and HQC) after the extraction process against their nonextracted samples above aqueous solutions. Intra- and inter-day accuracy and precision were determined at four QC levels [LLOQ QC (50 ng/mL), LQC (150 ng/mL), MQC (2600 ng/mL) and HQC (3950 ng/mL)] along with calibration curve (50-5000 ng/mL). Stability (auto-sampler, bench-top, freeze-thaw and long-term) studies, dilution effect and incurred sample reanalysis (ISR) were also evaluated as per guidelines requirement (DHHS, FDA, CDER, & CVM, 2018).

2.7. Pharmacokinetic study in mice

Four male Sprague Dawley rats (weigh range: 218-221 g) were procured from Vivo Biotech, Hyderabad, India and housed at Jubilant Animal House facility (having 12/12 h light/dark cycles with controlled humidity and temperature) for a period of seven days (during this period rats had free access to feed and water) before performing pharmacokinetic study [approved by Institutional Animal Ethics Committee (IAEC/JDC/2020/223)]. Following 12 h overnight fast (during the fasting period animals had free access to water) rats received duvelisib (50 mg/kg) and idelalisib (50 mg/kg) were co-administered orally [suspension formulation prepared using 0.1% Tween-80 with methyl cellulose (0.5% in water); strength: 5.0 mg/mL; dose volume: 10 mL/kg]. Serail blood samples were collected at pre-determined time points (0.25, 0.5, 1, 2, 4, 8, 10, 24, 30 and 36 h) through tail vein of each rat (using Micropipettes, Drummond Scientific, PA, USA; catalogue number: 1-000-0500) into polypropylene tubes (having K2.EDTA as an anti-coagulant). Plasma was harvested by centrifuging the blood using Biofuge (Hereaus, Germany) at 1760 g for 5 min and stored frozen at -80 ± 10°C until analysis. Rats were allowed to access feed 2 h post-dosing.
Along with plasma samples, LQC, MQC and HQC samples (made in blank plasma) were assayed in duplicate and were distributed among unknown samples in the analytical run. The criteria for acceptance of the analytical runs encompassed the following: (i) 67% of the QC samples accuracy must be within 85-115% of the nominal concentration (ii) not less than 50% at each QC concentration level must meet the acceptance criteria (DHHS, FDA, CDER, & CVM, 2018). The pharmacokinetic parameters were calculated by using Phoenix WinNonlin software (version 8.1; Pharsight Corporation, Mountain View, CA).

3. RESULTS AND DISCUSSION

3.1. Chromatographic conditions

To ensure good peak shapes for copanlisib, duvelisib and idelalisib, maintain their retention time and minimize the total run time, great effort was spent on column selection, gradient profile development and organic solvent selection. As a starting point, the earlier reported HPLC-UV method (Zakkula et al., 2020) for copanlisib was explored but in this method there was no resolution between duvelisib and idelalisib. As duvelisib and idelalisib are structurally very close and having a pKa value of 3.99 and 4.27, respectively it posed a challenge to get a resolution between these two drugs. In the mobile phase having phosphoric acid/formic acid/trifluoro acetic acid buffer in combination with either methanol or acetonitrile there was no resolution between duvelisib and idelalisib. Subsequently, by adjusting the pH of the 10 mM ammonium formate and acetonitrile mobile phase to pH 4.2 (with formic acid), which is close the pKa value of duvelisib and idelalisib and by using binary gradient elution (Table 1) with a total run time to 15 min, we have achieved the resolution between duvelisib and idelalisib (Fig. 1). Among the various columns (Hypersil Gold, Symmetry Shield, Hypersil, Atlantis etc.) evaluated only Hypersil Gold C18 column (250  4.0 mm, 5 µ) provided good resolution and peak shape for all the analytes along with the IS. Tofacitinib, which was used as an IS in the determination of copanlisib in mice plasma using HPLC-UV method (Zakkula et al., 2020) eluted very close to idelalisib in the present method optimized conditions. Hence, we tried the readily available kinase inhibitors (enasidenib, filgotinib, larotrectinib etc.) in our lab and found that filgotinib was found to be suitable for the present study. Selection of a proper wavelength plays a crucial role in determination of method sensitivity. Maximum absorbance for copanlisib was found at 310 nm, however due to similar structural features the maximum absorbance for duvelisib and idelalisib was found at ~270 nm. However, in order to facilitate the detection of all three analytes simultaneously with good sensitivity the UV detector was set at max 280 nm.

3.2. Recovery

In the previously reported methods, the recovery of copanlisib from mice plasma was achieved with liquid-liquid extraction (Dittakavi & Mullangi, 2019; Zakkula et al., 2020) and the recovery ranged between 60-83%. For idelalisib, the recovery ranged between 81-92% from different pre-clinical species (rat/rabbit/dog) and patient plasma, which was achieved either by simple protein precipitation (Suneetha & Sharmila 2016; Huynh et al., 2018) or liquid-liquid extraction (Veeraraghavan et al., 2014; Wang, Jia & Zhang, 2019). For duvelisib, Shao et al. (2020) reported simple protein precipitation technique for the effective recovery from dog plasma. In order to get maximum extraction efficiency, several organic solvents (dichloromethane, ethyl acetate, tert-butyl methyl ether; alone or in different proportions) were explored and finally we have chosen dichloromethane:ethyl acetate (30:70, v/v). The recovery was 88% for copanlisib, duvelisib and idelalisib from rat plasma. We did not explore solid-phase extraction technique as the liquid-liquid extraction method gave a satisfactory recovery for copanlisib, duvelisib and idelalisib. The recovery data (mean ± S.D) for copanlisib, duvelisib and idelalisib at LQC and HQC and for the IS (at 500 ng/mL) is shown in Table 2.

3.3. Selectivity

Fig. 1a, 1b and 1c show an overlay of blank rat plasma, rat plasma spiked with three analytes at their LLOQ (50 ng/mL) along with the IS and a 2.0 h time point oral pharmacokinetic study sample showing the peaks of duvelisib and idelalisib, respectively. It is evident from this overlay that endogenous components of rat plasma did not show no interference at the retention times of copanlisib, duvelisib, idelalisib and the IS indicating that the method is selective. The retention time of copanlisib, duvelisib, idelalisib and the IS was 7.16, 12.6, 11.9 and 9.86 min, respectively.

3.4. Sensitivity and carry over

The lowest limit of reliable quantification for each analyte was set at the concentration of the LLOQ (n=6 for each analyte). The precision (%RSD) and accuracy (%RE) for copanlisib at LLOQ (50 ng/mL) were found to be 8.86 and 97.4%. Similarly, for duvelisib and idelalisib the precision (%RSD) and accuracy (%RE) were 8.98 and 102%; 7.33 and 105%, respectively. We did not observe any carry-over produced by the highest calibration sample on the following injected rat blank plasma extracted sample for all the three analytes.

3.5. Calibration curve

The plasma calibration curve was constructed using eight calibration standards (viz., 50, 100, 375, 875, 1500, 3100, 4100 and 5000 ng/mL). The calibration standard curve had a reliable reproducibility over the standard concentrations across the calibration range. Calibration curve was prepared by determining the best fit of peak-area ratios (peak area analyte/peak area of the IS) versus concentration, and fitted to the y = mx + c using two weighting models, 1/X and 1/X2. A regression equation with a weighting factor of 1/X2 of each drug to the IS concentration was found to produce the best fit for the concentrationdetector response relationship. The mean ± SD slope and intercept values for copanlisib, duvelisib and idelalisib were 0.0003 ± 0.00005 and 0.009 ± 0.002; 0.0003 ± 0.00004 and = 4) was found to be >0.998. The lowest concentration with the RSD <20% was taken as LLOQ and was found to be 50 ng/mL for all the analytes. The accuracy observed for the mean of back-calculated concentrations for four calibration curves was within 89.9-103%; while the precision (%CV) values ranged from 1.99-4.95% for all the analytes.

3.6. Accuracy and precision

Accuracy and precision data for intra- and inter-day rat plasma samples are presented in Table 2. The assay values on both the occasions (intra- and inter-day) were found to be within the accepted variable limits. The data show that the method possesses adequate accuracy and repeatability for copanlisib, duvelisib and idelalisib in rat plasma samples.

3.7. Stability

Table 3 summarizes the results of stability studies conducted for copanlisib, duvelisib, idelalisib in rat plasma. The measured concentrations for these analytes at their respective LQC and HQC deviated within ±15% of the nominal concentrations in a battery of stability tests viz., in-injector (24 h), bench-top (6 h), repeated three freeze/thaw cycles and freezer stability at -80  10°C for at least for 30 days (Table 3) supported the stability of copanlisib, duvelisib and idelalisib at various stability conditions.

3.8. Dilution Effect

The dilution integrity was confirmed for QC samples that exceeded the upper limit of standard calibration curve. The mean accuracy and precision for copanlisib, duvelisib, idelalisib for the 10x diluted (with rat blank plasma) test samples was found to be less than 1.03, 1.06 and 1.09% (accuracy) and 3.95, 4.93 and 5.25% (precision), respectively, which show the ability to dilute samples up to a dilution factor of ten in a linear fashion.

3.9. Incurred samples reanalysis

For ISR analysis, a total of 10 samples each for duvelisib (2 samples each from 4 h and 12 h and 3 samples each from 10 h and 24 h) and idelalisib (2 samples each from 2 h and 4 h and 3 samples each from 12 h and 24 h) were chosen from rat pharmacokinetic study. All the samples selected for ISR met the acceptance criteria and the data is presented in Supplementary Fig 2a and 2b for duvelisib and idelalisib, respectively. The back calculated accuracy values ranged between 93.5 to 111.3% for duvelisib and 94.3 to 109.6% for idelalisib from the initial assay results.

4. Pharmacokinetic Study

The oral pharmacokinetic profile (time versus plasma concentrations) for duvelisib and idelalisib following oral co-administration to rats is presented in Fig. 2. Both duvelisib and idelalisib were quantifiable up to 36 h post oral administration to rats. The pharmacokinetic parameters are presented in Table 4. Post oral administration both the drugs attained maximum plasma concentration (Cmax) at 2.00 h (Tmax). At 50 mg/kg dose, the Cmax (7.10 µg/mL) of duvelisib was around 2-fold more over the Cmax (3.57 µg/mL) of idelalisib.
Similarly, the AUC0- (area under curve from time zero to infinity) of duvelisib was slightly
(1.4-fold) higher over the AUC0- of idelalisib (65.8 µg  h/mL vs. 47.6 µg  h/mL). However, both the drugs half-life (T1/2) was found to be ~6.00 h as both the drugs elimination profile was more or less similar (Fig. 4). In summary the validated method was sensitive enough to calculate the oral pharmacokinetic parameters for duvelisib and idelalisib. Earlier, we have reported the application of a validated HPLC-UV method for quantification of copanlisib in mice plasma and its applicability (Zakkula et al., 2020) hence in the current study we did not dose copanlisib to show the applicability of the present method.
Flinn et al. (2018) have reported the plasma concentrations in patients with advanced hematological malignancies. In this dose escalation study duvelisib was administered twice daily. The doses were 8, 15, 25, 35, 50, 60, 75 and 100 mg. At the effective clinical dose, the plasma concentration at 24 h was ~70 ng/mL. At 35 mg dose the 24 h concentration was ~100 ng/mL, however beyond 35 mg dose the plasma concentration at 24 h was ~220 ng/mL (Finn et al., 2018). In a bioequivalence study, Arumugam, Mani & Chirinos (2014) tested 150 mg tablets of reference (Zydelig) and generic (Abbott) formulation in healthy adult humans. In this study at 24 h the plasma concentration for idelalisib was ~90 ng/mL (Arumugam, Mani & Chirinos, 2014). We strongly believe that the current method with little or no modifications can be extended for therapeutic drug monitoring of duvelisib and idelalisib in clinic post oral administration. Besides, our method finds application in the routine pharmacokinetic and/or toxicokinetic studies in pre-clinical species post administration of marketed PI3K inhibitors. Though our method offers potential utility in therapeutic drug monitoring the main draw backs, which we foresee are longer run time, less sensitive and low throughput when compared to LC-MS/MS methods.

5. CONCLUSION

A simple reversed-phase HPLC-UV method for determination Filgotinib of copanlisib, duvelisib, idelalisib in rat plasma has been developed and validated. The proposed method is highly specific, accurate, precise and reproducible. All the validation parameters were within the acceptable limits for a bioanalytical method as per regulatory guidelines. This method has been successfully applied to a pharmacokinetic study in rats.

References

Aliqopa (Copanlisib). (2017). Highlights of prescribing information.
Arumugam, A., Mani, A., & Chirinos, J. (2020). A full replicate in vivo bioequivalence study of two idelalisib 150 mg tablets in fasted healthy human subjects. Journal of Bioequivalence & Bioavailability, 12, 391.
Copiktra (Duvelisib). (2018). Highlights of prescribing information. https://www.accessdata.fda.gov/drugsatfda_docs/label/2018/211155s000lbl.pdf.
Dittakavi, S. & Mullangi, R. (2019). LC-ESI-MS/MS determination of copanlisib, a novel PI3K inhibitor in mouse plasma and its application to a pharmacokinetic study in mice. Biomedical Chromatography, 33, e4460.
Engelman, J.A., Luo, J., & Cantley, L.C. (2006). The evolution of phosphatidylinositol 3 kinases as regulators of growth and metabolism. Nature Reviews. Genetics, 7,
Flinn, I.W., O’Brien, S., Kahl, B., …Horwitz, S. (2018). Duvelisib, a novel oral dual inhibitor of PI3K-δ,γ, is clinically active in advanced hematologic malignancies. Blood, 131, 877-887.
Greenwell, I.B., Flowers, C.R., Blum, K.A., & Cohen, J.B. (2017). Clinical use of PI3K inhibitors in B-cell lymphoid malignancies: today and tomorrow. Expert Review of Anticancer Therapy, 17, 271-279.
Huynh, H.H., Roessle, C., Sauvageon, H., … Goldwirt, L. (2018). Quantification of idelalisib in human plasma by ultra-performance liquid chromatography coupled to mass spectrometry in negative ionization mode. Therapeutic Drug Monitoring, 40, 237244.
Liu, P., Cheng, H., Roberts, T.M., & Zhao, J.J. (2009). Targeting the phosphoinositide 3 kinase pathway in cancer. Nature Reviews. Drug Discovery, 8, 627-644.
Shao, Y., Xie, S., Zhu, H., Du, X., & Xu, R. (2020). Development of a novel and quick LCMS/MS method for the pharmacokinetic analysis of duvelisib in beagle dogs. Journal of Pharmaceutical and Biomedical Analysis, 187, 113355.
Suneetha, D., & Sharmila, D. (2016). Method development and validation for the estimation of idelalisib in rabbit plasma by HPLC. International Journal of Pharmaceutical Sciences and Research, 7, 4998-5005.
US DHHS, FDA, CDER, CVM, Guidance for Industry: Bioanalytical Method Validation, U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Center for Veterinary Medicine (CV), (2018), Rockville, MD, USA.
Veeraraghavan, S., Thappali, S., Viswanadha, S., … Rangasamy, M. (2014). Simultaneous quantification of idelalisib, fludarabine and lenalidomide in rat plasma by using high-performance liquid chromatography coupled with heated electrospray ionization tandem mass spectrometry. Journal of Chromatography B, 949-950, 63-69.
Wang, C., Jia, F., & Zhang, Y. (2019). Simultaneous determination of idelalisib and its metabolite GS-563117 in dog plasma by LC-MS/MS: Application to a pharmacokinetic study. Biomedical Chromatography, 33, e4511.
Zakkula, A., Kurakula, P.K., Dittakavi, S., … Mullangi, R. (2020). Validated HPLC method for quantification of copanlisib in mice plasma: application to a pharmacokinetic study. ADMET & DMPK, 8, 113-121.
Zydelig (Idelalisib). (2014). Highlights of prescribing information.