Suresh P. Sulochana

Review of the validated HPLC and LC‐MS/MS methods for determination of drugs used in clinical practice for Alzheimer's disease

Alzheimers disease (AD) is a neurological disorder and is the most frequent type of dementia among elderly people. Donepezil, rivastigmine, galantamine, tacrine and memantine are the US Food and Drug Administration approved oral drugs used in the treatment of AD. Quantitation of these drugs in various biological matrices and monitoring them in long-term treatment is essential to titer the dose of these drugs and ensure patient compliance. This review provides a comprehensive account of various HPLC and LC-MS/MS assays, which have been successfully employed to measure the drug levels in various biological matrices arising from preclinical and clinical studies. In addition, this review collates various considerations such as internal standard selection, extraction schemes, matrix effect, selectivity evaluation and optimization of mass spectrometric conditions to enable the development of sound bioanalytical methods for quantitation of Alzheimers drugs. Overall LC-MS/MS methods have proven to be the choice of bioanalytical method for the quantification of Alzheimers drugs in both preclinical and clinical studies. In conclusion, important features of LC-MS/MS methodology for Alzheimers drugs include shortened analysis time, increased throughput, selectivity and lower cost of analysis.

LC–MS/MS-ESI method for simultaneous quantification of darolutamide and its active metabolite, ORM-15341 in mice plasma and its application to a pharmacokinetic study

Graphical abstract Figure. No Caption available. HighlightsFirst LC–MS/MS method being reported for the simultaneous quantification of darolutamide and ORM‐15341 in mice plasma.Simple sample processing method and shorter run time (2.5 min) enables this method as a high throughput assay.Method was validated as per regulatory guidelines.The method is specific, precise, accurate and no matrix effect was observed.Method was used in a mice pharmacokinetic study to derive the pharmacokinetic parameters for analytes. Abstract A sensitive and rapid LC–MS/MS method was developed and validated for the simultaneous quantitation of darolutamide and its active metabolite i.e. ORM‐15341 in 50 &mgr;L mice plasma using bicalutamide as an internal standard (I.S.) as per regulatory guidelines. Sample processing was accomplished through liquid‐liquid extraction. Chromatographic separation was achieved using an Atlantis C18 column with an isocratic mobile phase comprising 0.2% formic acid:acetonitrile (35:65, v/v) at a flow rate of 0.8 mL/min within 2.5 min. Detection and quantitation were done by multiple reaction monitoring on a triple quadrupole mass spectrometer following the transitions: m/z 397 → 202, 395 → 202 and 429 → 255 for darolutamide, ORM‐15341 and I.S, respectively in the negative ionization mode. The calibration curve was linear from 0.61–1097 ng/mL for both darolutamide and ORM‐15341. The intra‐ and inter‐day precisions were in the range of 1.34–13.8 and 4.85–12.9 and 3.91–13.7 and 6.54–14.2%, for darolutamide and ORM‐15341, respectively. Darolutamide and ORM‐15341 were found to be stable under different stability conditions. The validated method was applied to a pharmacokinetic study in mice.

Validation of an LC–MS/MS method for simultaneous quantitation of enzalutamide, N-desmethylenzalutamide, apalutamide, darolutamide and ORM-15341 in mice plasma and its application to a mice pharmacokinetic study

HighlightsFirst LC–MS/MS method for the quantification of second generation non‐steroidal anti‐androgens in mice plasma.Simple sample processing method and 2.5 min run time enables this method as a high throughput assay.Method was validated as per regulatory guidelines.Method is specific, precise, accurate and linear from 1.07–2000 ng/mL for the analytes.Method application was shown in a mice pharmacokinetic study to derive the pharmacokinetic parameters. ABSTRACT A sensitive and rapid LC–MS/MS method was developed and validated for the simultaneous quantitation of enzalutamide, N‐desmethylenzalutamide (active metabolite of enzalutamide), apalutamide, darolutamide and ORM‐15341 (active metabolite of darolutamide) in mice plasma as per regulatory guidelines. The analytes and the internal standard (I.S.: apalutamide‐d3) were extracted from 50 &mgr;L mice plasma by simple protein precipitation using acetonitrile, followed by chromatographic separation using an Atlantis C18 column with an isocratic mobile (0.2% formic acid:acetonitrile; 30:70, v/v) at a flow rate of 0.8 mL/min within 2.5 min. Detection and quantitation was done by multiple reaction monitoring on a triple quadrupole mass spectrometer following the transitions: m/z 465 → 209, 451 → 195, 478 → 450, 399 → 178, 397 → 194 and 481 → 453 for enzalutamide, N‐desmethylenzalutamide, apalutamide, darolutamide, ORM‐15341 and the I.S. respectively. The calibration curves were linear from 1.07 to 2000 ng/mL with r2 ≥0.99 for all the analytes. The intra‐ and inter‐batch accuracy and precision (% CV) across quality controls varied from 88.5–111% and 1.13–13.1, 85.4–106% and 3.15–14.3, respectively for all the analytes. All the analytes were found to be stable under different conditions. The method was applied to an intravenous pharmacokinetic study in mice.

LC-ESI-MS/MS determination of defactinib, a novel FAK inhibitor in mice plasma and its application to a pharmacokinetic study in mice

Graphical abstract Figure. No caption available. HighlightsFirst bioanalytical method being reported for quantification of defactinib in mice plasma on LC–MS/MS.Method was validated as per regulatory guidelines.The method is specific, precise, accurate and no matrix effect was observed and linear from 0.13 to 106 ng/mL.The method was successfully used in a mice pharmacokinetic study and for the first time pharmacokinetic parameters for defactinib in mice. ABSTRACT A sensitive, specific, selective and rapid LC‐ESI–MS/MS method has been developed and validated for the quantification of defactinib in mice plasma using 13C3,15N‐tofacitinib as an internal standard (I.S.). Sample preparation was accomplished through a liquid–liquid extraction process. Baseline chromatographic resolution of defactinib and the I.S. was achieved on an Atlantis dC18 column using an isocratic mobile phase comprising 0.2% formic acid in water and acetonitrile (25:75, v/v) delivered at a flow rate of 0.5 mL/min. Defactinib and the I.S. eluted at ˜1.59 and 0.99 min, respectively. The total chromatographic run time was 2.50 min. A linear response function was established in the concentration range of 0.13–106 ng/mL. Method validation was performed as per regulatory guidelines and the results met the acceptance criteria. The intra‐ and inter‐day accuracy and precision were in the range of 5.57–13.3 and 8.63–12.1%, respectively. Defactinib was found to be stable under various stability conditions. This novel method has been applied to a pharmacokinetic study in mice.

Validation of an LC-ESI–MS/MS method for the determination of apalutamide, a novel non-steroidal anti-androgen in mice plasma and its application to a pharmacokinetic study in mice

Graphical abstract Figure. No Caption available. HighlightsFirst LC–MS/MS method on quantification of apalutamide in mice plasma.Simple sample processing method and short run time enables this method as high throughput assay.Method was validated as per regulatory guidelines and linear from 1.02–2030 ng/mL.The method is specific, precise, accurate and no matrix effect was observed.The method was successfully used in a mice PK study. Abstract A sensitive, specific, selective and rapid LC‐ESI–MS/MS method has been developed and validated for the quantification of apalutamide in mice plasma using apalutamide‐d3 as an internal standard (I.S.). Sample preparation was accomplished through a simple protein precipitation process. Chromatography of apalutamide and the I.S. was achieved on an Atlantis dC18 column using an isocratic mobile phase comprising 0.2% formic acid in water and acetonitrile (20:80, v/v) delivered at a flow rate of 0.8 mL/min. LC–MS/MS was operated under the multiple reaction‐monitoring mode (MRM) using the electrospray ionization technique in positive ion mode and the transitions of m/z 478 → 450 and m/z 481 → 453 were used to measure the derivative of apalutamide and the I.S, respectively. The total chromatographic run time was 2.5 min and the elution of apalutamide and I.S. occurred at 1.10 and 1.09 min, respectively. Method validation was performed as per regulatory guidelines and the results met the acceptance criteria. Linearity was established in the concentration range of 1.02–2030 ng/mL (r> 0.995) for apalutamide. The intra‐ and inter‐day accuracy and precision for apalutamide in mice plasma were in the range of 2.11–8.44 and 2.51–6.09%, respectively. Apalutamide was found to be stable under various stability conditions. This novel method has been applied to a pharmacokinetic study in mice.

Validated Chiral LC-ESI-MS/MS Method for the Simultaneous Quantification of Darolutamide Diastereomers and Its Active Metabolite in Mice Plasma: Application to a Pharmacokinetic Study

A simple, selective and reliable LC-MS/MS method was developed and validated for the simultaneous quantitation of darolutamide diastereomers (diastereomer-1 and diastereomer-2) and its active metabolite i. e. ORM-15341 in mice plasma using warfarin as an internal standard (IS) as per the regulatory guidelines. Plasma samples were extracted by liquid-liquid extraction and the chromatographic separation was achieved on a Chiralpak IA column with an isocratic mobile phase 5 mM ammonium acetate:absolute alcohol (20:80, v/v) at a flow rate of 1.0 mL/min. Detection and quantitation was done by multiple reaction monitoring on a triple quadrupole mass spectrometer following the transitions: m/z 397→202, 395→202 and 307→250 for darolutamide diastereomers, ORM-15341 and the IS, respectively in the negative ionization mode. The calibration curves were linear (r>0.992) in the range of 100-2400 ng/mL for all the analytes. The intra- and inter-day precisions were in the range of 1.25-10.2 and 1.58-12.3; 2.85-5.68 and 1.85-9.58; 2.34-12.1 and 2.58-7.38 for diastereomer-1, diastereomer-2 and ORM-15341, respectively. Both diastereomers and ORM-15341 were found to be stable under different stability conditions. The validated method was applied to a pharmacokinetic study in mice.

Validation of a chiral LC-MS/MS-ESI method for the simultaneous quantification of darolutamide diastereomers in mouse plasma and its application to a stereoselective pharmacokinetic study in mice

A simple, selective and reliable LC-MS/MS method was validated for simultaneous quantitation of darolutamide diastereomers in 50 μL mouse plasma using warfarin as an internal standard (IS) as per regulatory guidelines. Plasma samples were extracted by liquid-liquid extraction and the chromatographic separation was achieved on a Chiralpak IA column with an isocratic mobile phase 5 mm ammonium acetate-absolute alcohol (20:80, v/v) at a flow rate of 1.0 mL/min. Detection and quantitation was done in multiple reaction monitoring mode following the transitions m/z 397 → 202 and 307 → 250 for darolutamide diastereomers and the IS, respectively, in the negative ionization mode. The linearity range was 100-2400 ng/mL for each diastereomer. The intra- and inter-day precisions were in the ranges of 1.78-4.20 and 4.34-14.6, and 3.63-4.74 and 4.78-5.15 for diastereomer-1 and diastereomer-2, respectively. Both diastereomers were found to be stable under different stability conditions. The validated method was applied to a pharmacokinetic study in mice. Following oral administration of darolutamide at 10 mg/kg, maximum concentration in plasma was 4189 and 726 ng/mL for diastereomer-1 and diastereomer-2, respectively. The terminal half-life was found to be ~0.50 h for both the diastereomers. The AUC(0-t) was found to be 18,961 ng*h/mL for diastereomer-1 and 1340 ng*h/mL diastereomer-2.

Highly Sensitive LC–MS-MS Method for the Determination of Tacrine in Rat Plasma: Application to Pharmacokinetic Studies in Rats

A rapid and highly sensitive assay method has been developed and validated for the estimation of tacrine in rat plasma using liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive-ion mode. The assay procedure involves a simple liquid-liquid extraction of tacrine and phenacetin (internal standard, IS) from rat plasma using ethyl acetate. Chromatographic separation was achieved with 0.2% formic acid : acetonitrile (30 : 70, v/v) at a flow rate of 0.50 mL/min on an Atlantis dC18 column with a total run time of 3.0 min. The MS-MS ion transitions monitored were 199.10 → 171.20 for tacrine and 180.10 → 110.10 for IS. Method validation was performed as per United States Food and Drug Administration (US FDA) guidelines and the results met the acceptance criteria. The lower limit of quantification achieved was 0.008 ng/mL and linearity was observed from 0.008 to 53.4 ng/mL. The intra- and inter-day precision was in the range of 2.76-12.5 and 5.15-12.8%, respectively. This novel method has been applied to a pharmacokinetic study in rats.

Prediction of Human Pharmacokinetics of Bendamustine from Preclinical Species Pharmacokinetics Based on Normalizing Time Course Profiles

Bendamustine, an alkylating anticancer agent, is used to treat chronic lymphocytic leukemia by intravenous infusion alone or in combination. The work aimed to develop a method to predict time vs. concentration profile for humans based on preclinical pharmacokinetics using the assumption of superimposability of normalized time course profiles of animals and humans. Standard allometric equations with/without correction factors (CF) were also used in prediction. The Vss was predicted by simple allometry of 0.312W0.871 (r2=0.987), where W is body weight; predicted Vss (19.71 L) was similar to the reported value (20.10 L). However, CL prediction involved both simple and CF allometry. Best proximity CL (543 vs. 598 mL/min) was obtained with maximum life span correction (MLP) [2.46W1.215 (r2=0.988)]. Normalized curves were obtained by normalizing the time (with mean residence time) vs. concentration (with dose/Vss) in animal species. The concentration vs. time profile in humans after intravenous infusion was then simulated using normalized curve for each animal species and the values of CL and Vss were predicted for humans. In summary the findings indicate that normalized time course approach could predict the bendamustine human pharmacokinetics and such an approach could be prospectively applied for analog drugs of this class.

Determination of Tofacitinib in Mice Whole Blood on Dried Blood Spots Using LC-ESI-MS/MS: Application to Pharmacokinetic Study in Mice.

A simple, sensitive and rapid assay method has been developed and validated as per regulatory guideline for the estimation of tofacitinib on mice dried blood spots (DBS) using liquid chromatography coupled to tandem mass spectrometry with electro spray ionization in the positive-ion mode. The method employs liquid extraction of tofacitinib from DBS disk of mice whole blood followed by chromatographic separation using 5 mM ammonium acetate (pH 6.5):acetonitrile (20:80, v/v) at a flow rate of 0.60 mL/min on an X-Terra Phenyl column with a total run time 2.5 min. The MS/MS ion transitions monitored were m/z 313→149 for tofacitinib and m/z 316→149 for the internal standard (13C3, 15N-tofacitinib). The assay was linear in the range of 0.99-1980 ng/mL. The intra- and inter-day precision was in the range of 1.17-10.3 and 3.37-10.9%, respectively. Stability studies showed that tofacitinib was stable on DBS cards for one month. This novel method has been applied to analyze the DBS samples of tofacitinib obtained from a pharmacokinetic study in mice.