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An overview of published dried blood spot (DBS) methods for the quantitation of various classes of anticancer drugs from clinical and preclinical studies is presented. The increased reporting of DBS methods in the literature for quantitation of various classes of drugs is a testimony to their utility in bioanalytical applications. While DBS offers several advantages as compared with conventional wet sampling techniques, there remain a number of nuances that may impede the assay adaptability of DBS method in routine quantitative bioanalysis. This review covers several case studies of DBS application in the quantitation of anticancer drugs. Some perspectives are provided on the optimization of the DBS method with respect to the selection of DBS card, spot volume, hematocrit effect and other regular validation parameters, which are essential in quantitative bioanalysis. Some thoughts are provided on the existing gaps in the DBS method and possible remedial measure(s) to address such gaps.Although DBS methods have great potential, there is the need for a global consensus including regulatory support on the type of validation experiments to be performed to support quantitative data.

A simple, specific and reproducible high-performance liquid chromatography (HPLC) assay method has been developed and validated for the quantitation of second generation antiandrogens and their active metabolites namely apalutamide, enzalutamide, N-desmethylenzalutamide (active metabolite of enzalutamide), darolutamide and ORM-15341 (active metabolite of darolutamide) in mice plasma. The method involves extraction of apalutamide, enzalutamide, N-desmethylenzalutamide, darolutamide and ORM-15341 along with internal standard (IS) from 100 µL mice plasma through a simple protein precipitation process. The chromatographic analysis was performed on a Waters Alliance HPLC system using a gradient mobile phase (comprising 10 mM ammonium acetate and acetonitrile in a flow-gradient) and X-Terra Phenyl column. The UV detection wave length was set at λmax 250 nm. Apalutamide, enzalutamide, N-desmethylenzalutamide, darolutamide and ORM-15341 and the IS eluted at 13.6, 11.4, 9.68, 6.11, 6.93 and 4.69 min, respectively with a total run time of 15 min. Method validation was performed as per regulatory guidelines and the results met the acceptance criteria. The calibration curve was linear over a concentration range of 209 – 5215 ng/mL (r 2=0.998). The intra- and inter-day precisions were in the range of 0.56–13.5 and 1.04–13.9%, respectively. The validated HPLC method was successfully applied to a pharmacokinetic study in mice.

<p>A simple, sensitive and rapid assay method has been developed and validated for the estimation of enzalutamide, N-desmethylenzalutamide (active metabolite of enzalutamide), darolutamide and ORM-15341 (active metabolite of darolutamide) 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 utilizes liquid extraction of enzalutamide, N-desmethylenzalutamide, darolutamide and ORM-15341 from 3 mm punched disks from DBS cards (spiked or study samples). The extracted sample was chromatographed using an isocratic mobile phase (0.2 % formic acid : acetonitrile; 30:70, v/v) on an Atlantis dC18 column. The total run time was 2.5 min. The MS/MS ion transitions monitored were m/z 465 → m/z 209, m/z 451 →  m/z 195, m/z 399 → m/z 178, m/z 397 →  m/z 194 and m/z 481 → m/z 453 for enzalutamide, N-desmethyl­enzalutamide, darolutamide, ORM-15341 and the IS (apalutamide-d₃), respectively. Method validation was performed as per regulatory guideline. The assay had a good linearity over the range of 0.93-2000 ng/mL. The intra- and inter-batch accuracy and precision (%RE &amp; RSD) across quality controls met the acceptance criteria for all the analytes. Stability studies showed that all the analytes were stable on DBS cards for one month. This novel method has been applied to analyze the DBS samples of enzalutamide, N-desmethylenzalutamide, darolutamide and ORM-15341 obtained from a pharmacokinetic study in mice.</p>

Immuno-oncology (IO) is an emerging option to treat cancer malignancies. In the last two years, IO has accounted for more than 90% of the new active drugs in various therapeutic indications of oncology drug development. Bioanalytical methods used for the quantitation of various IO small molecule drugs have been summarized in this review. The most commonly used are HPLC and LC-MS/MS methods. Determination of IO drugs from biological matrices involves drug extraction from the biological matrix, which is mostly achieved by simple protein precipitation, liquid-liquid extraction and solid-phase extraction. Subsequently, quantitation is usually achieved by LC-MS/MS, but HPLC-UV has also been employed. The bioanalytical methods reported for each drug are briefly discussed and tabulated for easy access. Our review indicates that LC-MS/MS is a versatile and reliable tool for the sensitive, rapid and robust quantitation of IO drugs.

Alzheimer's 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 Alzheimer's drugs. Overall LC-MS/MS methods have proven to be the choice of bioanalytical method for the quantification of Alzheimer's drugs in both preclinical and clinical studies. In conclusion, important features of LC-MS/MS methodology for Alzheimer's drugs include shortened analysis time, increased throughput, selectivity and lower cost of analysis.

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 313→149 for tofacitinib and 316→149 for the internal standard (CN-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.