Total Run Time Research Articles

Related Substances Method Development and Validation of Axitinib, Zanubrutinib and Upadacitinib Using RP-HPLC and its Degradation Products were Characterized by Using LC-MS/MS

Background: Axitinib was used to treat carcinoma of renal cells and Upadacitinib was used to treat rheumatoid arthritis and Zanubrutinib used as an anti cancer medication for mantle cell lymphoma. In the current application, Zanubrutinib, Upadacitinib and Axitinib and their associated substances will be developed and validated. Materials and Methods: The optimised method includes the Zanubrutinib, Upadacitinib and Axitinib gradient elution and associated flow rates with dimensions of 1 mL/min and phenyl column X-bridge (150 x 4.6 mm, 3.5 μ). A mobile phase was employed with 1.2 g of Hexane sulphonic acid of pH-2.5 adjusted with ortho-phosphoric acid (buffer) and acetonitrile. Zanubrutinib, Upadacitinib and Axitinib were separated from their associated substances for a total run time of 60 min. The approach developed has been validated in accordance with the ICH guidelines. Results: The test concentration LOD and LOQ were established for both drugs and for their impurities. The diagrams that were drawn were straightforward with an R2 regression coefficient > 0.999. As part of the method validation, recovery, specificity, linearity, accuracy, robustness was determined and the results were found to be within an acceptable range. Conclusion: HPLC was used to validate the method in terms of accuracy, linearity, method precision, accuracy, limit of detection, limit of quantification, robustness, and degradation, and LC-MS/MS was used to characterise the degradation products. The system was validated in all of these aspects.

Profiling the BLAST bioinformatics application for load balancing on high-performance computing clusters

BackgroundThe Basic Local Alignment Search Tool (BLAST) is a suite of commonly used algorithms for identifying matches between biological sequences. The user supplies a database file and query file of sequences for BLAST to find identical sequences between the two. The typical millions of database and query sequences make BLAST computationally challenging but also well suited for parallelization on high-performance computing clusters. The efficacy of parallelization depends on the data partitioning, where the optimal data partitioning relies on an accurate performance model. In previous studies, a BLAST job was sped up by 27 times by partitioning the database and query among thousands of processor nodes. However, the optimality of the partitioning method was not studied. Unlike BLAST performance models proposed in the literature that usually have problem size and hardware configuration as the only variables, the execution time of a BLAST job is a function of database size, query size, and hardware capability. In this work, the nucleotide BLAST application BLASTN was profiled using three methods: shell-level profiling with the Unix “time” command, code-level profiling with the built-in “profiler” module, and system-level profiling with the Unix “gprof” program. The runtimes were measured for six node types, using six different database files and 15 query files, on a heterogeneous HPC cluster with 500+ nodes. The empirical measurement data were fitted with quadratic functions to develop performance models that were used to guide the data parallelization for BLASTN jobs.ResultsProfiling results showed that BLASTN contains more than 34,500 different functions, but a single function, RunMTBySplitDB, takes 99.12% of the total runtime. Among its 53 child functions, five core functions were identified to make up 92.12% of the overall BLASTN runtime. Based on the performance models, static load balancing algorithms can be applied to the BLASTN input data to minimize the runtime of the longest job on an HPC cluster. Four test cases being run on homogeneous and heterogeneous clusters were tested. Experiment results showed that the runtime can be reduced by 81% on a homogeneous cluster and by 20% on a heterogeneous cluster by re-distributing the workload.DiscussionOptimal data partitioning can improve BLASTN’s overall runtime 5.4-fold in comparison with dividing the database and query into the same number of fragments. The proposed methodology can be used in the other applications in the BLAST+ suite or any other application as long as source code is available.

Validation of Colchicine Assay Method for Therapeutic Drug Monitoring in Human Plasma

Colchicine (COL) reduces the frequency of attacks in Familial Mediterranean Fever (FMF) patients and is effective in preventing and arresting renal amyloidosis in most patients. COL has a narrow therapeutic window. The blood concentration to achieve therapeutic effects can be determined by Therapeutic Drug Monitoring (TDM). However, the use of selective and sensitive analytical methods is necessary for achieving success with TDM. The purpose of this study is to develop and validate a new method for quantitative assay of COL in human plasma samples by liquid chromatograph- tandem mass spectrometry (LC-MS/MS). In our study, to 1ml plasma sample, 0.25 ml internal standard solution was added. The solution was extracted by liquid-liquid extraction (LLE). The method was validated according to the European Medicines Agency (EMEA). The total run time was 8 min in LC-MS/MS. The method has been validated over the 0.25 - 8.0 ng/mL calibration range for COL. It was seen that the method has been validated since the results of the analysis meet the EMEA criteria. In our study, COL plasma levels were found to be approximately 1.097±0.42 ng/ml in 40 FMF patients using an oral dose of 1.5-2 mg/day. A validated, rapid, simple, cost-effective, and sensitive LC-MS/MS method was developed and optimized for quantitation of COL in plasma. It has been thought that pharmacokinetic studies of COL in plasma can be performed easily using this validated method

Development and Validation of RP-HPLC method for the analysis of Moxifloxacin and its degradation products

In the present work, RP-HPLC method was developed for the analysis of Moxifloxacin in the marketed ophthalmic formulation. This eye solution is used to treat infections of the eye including bacterial conjunctivitis. The chromatographic separation was achieved with UV detection at 292 nm. The stationary phase used is Sunfire C18 column (250 mm × 4.6 mm, 5μm particle size column). The mobile phase contains a mixture of methanol: ACN: water with 0.1% TEA in isocratic elution mode. The total run time was 10min. The proposed method is found having linearity in the concentration range of 2-12μg/ml with co-relation coefficient of 0.999. This method was validated in accordance with International Conference on Harmonization (ICH) guidelines. The developed method is found simple and accurate. Due to its simplicity, rapidness, precision and accuracy, it may be used for laboratory analysis.

Accelerating LHC event generation with simplified pilot runs and fast PDFs

Poor computing efficiency of precision event generators for LHC physics has become a bottleneck for Monte-Carlo event simulation campaigns. We provide solutions to this problem by focusing on two major components of general-purpose event generators: The PDF evaluator and the matrix-element generator. For a typical production setup in the ATLAS experiment, we show that the two can consume about 80% of the total runtime. Using NLO simulations of pprightarrow ell ^+ell ^-+text {jets} and pprightarrow tbar{t}+text {jets} as an example, we demonstrate that the computing footprint of Lhapdf and Sherpa can be reduced by factors of order 10, while maintaining the formal accuracy of the event sample. The improved codes are made publicly available.

Application of Box-Behnken experimental design and response surface methodology for selecting the optimum RP-HPLC conditions for the simultaneous determination of methocarbamol, indomethacin and betamethasone in their pharmaceutical dosage form

An isocratic RP-HPLC method has been developed for the separation and determination of methocarbamol (MTL), indomethacin (IND), and betamethasone (BET) in combined dosage form using an Inertsil ODS-3v C18 (250 × 4.6 mm, 5 μm) column with UV- detection at 235 nm. Experimental design using Box-Behnken design (BBD) was applied to study the response surface during method optimization and to achieve a good separation with a minimum number of experimental runs. The three independent parameters were pH of buffer, % of acetonitrile and flow rate of the mobile phase while the peak resolution of IND from MTL and the peak resolution of BET from IND (R2) were taken as responses to obtain mathematical models. The composite desirability was employed to optimize a set of responses overall (peak resolutions). The predicted optimum assay conditions include a mobile phase composition of acetonitrile and phosphate buffer (pH 5.95) in a ratio of 79:21, v/v, pumped at a flow rate of 1.4 mL min−1. With this ideal condition, the optimized method was able to achieve baseline separation of the three drugs with good resolution and a total run time of less than 7 min. The linearity of MTL, IND, and BET was determined in the concentration ranges of 5–600 µg mL− 1, 5–300 µg mL− 1, and 5–300 µg mL− 1 and the regression coefficients were 0.9994, 0.9998, and 0.9998, respectively. The average percent recoveries for the accuracy were determined to be 100.41 ± 0.60%, 100.86 ± 0.86%, and 100.99 ± 0.65% for MTL, IND, and BET, respectively. The R.S.D.% of the intra-day precision was found to be less than 1%, while the R.S.D.% of the inter-day precision was found to be less than 2%. The RP-HPLC method was fully validated with regard to linearity, accuracy, precision, specificity, and robustness as per ICH recommendations. The proposed method has various applications in quality control and routine analysis of the investigated drugs in their pharmaceutical dosage forms and laboratory-prepared mixtures with the goal of reducing laboratory waste, analysis time, and effort.

Bioanalytical Method Development And Validation Of Darunavir In Biological Matrices Using Uplc-Ms-Ms

Background: To develop a simple and accurate method for the determination of Darunavir in human plasma by liquid-liquid extraction method with Verapamil as an internal standard using UPLC-MS/MS. Methods: The separation was achieved on Agilent, Zorbax XDB C18 (2.1 x 50 mm ID, 5 μm) column. The samples were chromatographed using the mobile phase consisting of acetonitrile and 2mM ammonium formate with 0.1% formic acid in water (70:30 v/v) at a flow rate of 0.12 ml/min in a mass spectrometer ESI chamber. The Darunavir and Verapamil were eluted at 1.35 and 1.13 min respectively with a total run time of 3 min. Results: A linear response was established at 10- 2,000 ng/ml with a correlation coefficient of 0.9919. The % accuracy was found to be 98% and 102.40%. A rapid method was developed for the determination of Darunavir in human plasma. The method was strictly validated according to the ICH guidelines.

Validated LC-MS/MS method for the determination of copanlisib in mouse dried blood spots.

Copanlisib is a dual PI3K-δ inhibitor, used in follicular lymphoma treatment. In this research, we report a validated LC-MS/MS method for quantifying copanlisib from a mouse dried blood spot (DBS). We validated the method in line with the guidelines of the US Food and Drug Administration. The liquid-liquid extraction technique was used to extract copanlisib from the DBS discs. We used an Atlantis dC18 column and isocratic mobile phase for the chromatographic separation of copanlisib and the internal standard (idelalisib). The flow was 0.90 ml/min. Under the optimized chromatographic conditions, the retentions of copanlisib and the internal standard were 0.98 and 0.93 min, respectively. Each injection total run time was 2.50 min. The MS/MS ion transitions monitored were m/z 481.31→128.00 and 416.10→176.10 for copanlisib and the internal standard (IS) idelalisib, respectively. We have used a broad calibration range (1.01-4,797 ng/ml) with a determination coefficient (r2 ) of 0.997. All of the evaluated parameters met the acceptance criteria. Hematocrit did not influence the DBS copanlisib concentrations. We have used the validated method to derive the intravenous pharmacokinetic parameters by quantifying copanlisib in mouse plasma.

Development of normal phase HPLC based method for the determination of retinyl palmitate in fortified edible oils

ABSTRACT Fortification of edible oil with retinyl palmitate (vitamin A) is a significant advancement to fulfill the nutritional deficiency of major population. The growing interest in the enrichment of foods warrants a simple and accurate method to analyze fortified products. This study developed an easy and reliable strategy based on normal-phase HPLC analysis to determine the edible oil’s retinyl palmitate. We also validated the method according to the International Conference on Harmonization (ICH) guidelines. The analysis was performed with a DB silica column (250 cm × 4.6 mm; 5 μm), with a mobile phase composed of n-heptane and isopropyl alcohol at a ratio of 75:25 v/v and a flow rate of 1.0 ml/min. The detection was performed at a wavelength of 326 nm, and the retention time of vitamin A was around 8 min, with a total run time of 10 min. The calibration plot gave a linear relationship (R2 = 0.9998) over the concentration range of 3.93–63 μg/ml. The LOD and LOQ were 0.029 and 0.096 μg/ml, respectively. The accuracy of the proposed method was determined by recovery studies and was found with a mean recovery of >95%. RSD% of the determination of precision was <2%. The results of robustness and solutions stability studies were within acceptable limits. The proposed method showed excellent linearity, accuracy, precision, specificity, robustness, LOD, LOQ, and system suitability results within the acceptable criteria.

Simple and Rapid LC-MS/MS Method for Determination of Perampanel in Human Plasma and Application to Bioequivalence Study

Background: Perampanel (PER) is a third-generation antiepileptic drug (AED). Several methods have been developed for the quantification of perampanel in plasma. The pharmacokinetic characteristics of perampanel in healthy Chinese ubjects have not been comprehensively reported. Objective: A simple, fast and sensitive LC-MS/MS method was established and validated for the quantification of perampanel in human plasma and its application to a bioequivalence study. Methods: Chromatographic separation was accomplished on a ZORBAX Eclipse XDB-Phenyl column (4.6 mm × 75 mm, 3.5 μm) using a binary gradient with mobile phase (A) (water containing 5 mmol/L ammonium acetate and 0.1% formic acid and (B) acetonitrile-water (95:5, v/v) at a flow rate of 0.9 mL/min and sample preparation was by one-step protein precipitation via acetonitrile. Results: The total run time in this study was 4.5 min and the retention time of perampanel and perampanel-d5 (internal standard) were 2.30 min and 2.32 min, respectively. The method was developed and validated over the concentration range of 2.00-500 ng/mL for perampanel, with a correlation coefficient greater than 0.9992. The inter-day precision was 3.1%-3.8% and accuracy was 98.9%-103.5%. The intra-day precision was 2.4%-6.8% and the accuracy was 97.6%- 104.9%. The extraction recovery ranged from 99.23%-103.84% and the matrix effect was not significant. Perampanel was proved to be stable in solution and human plasma under different tested conditions. The validated method was successfully applied to a randomized, open-label, 2- period, crossover bioequivalence study in healthy Chinese subjects, and the results indicated that bioequivalence was achieved for 2 formulations of the 4-mg perampanel tablet under both fasting and fed conditions, and both treatments were safe and well-tolerated by all study subjects. Conclusion: The validated method was successfully applied to a bioequivalence study of perampanel in human plasma and has achieved satisfactory results.

Simultaneous separation of naproxen and 6-O-desmethylnaproxen metabolite in saliva samples by liquid chromatography-tandem mass spectrometry: Pharmacokinetic study of naproxen alone and associated with esomeprazol-Results.

After performing liquid-liquid extraction with ethyl acetate and HCl, samples from 12 volunteers who performed sequential collections after taking a tablet of naproxen alone (n = 6) or associated with esomeprazole (n = 6) were analyzed in a triple quadrupole mass spectrometer 8040 LC MS/MS Shimadzu. Separation of naproxen and its main metabolite 6-O-desmethylnaproxen was performed in a Shim-Pack XR-ODS 75Lx2.0 column and C18 pre-column at 40°C using a mixture of methanol and ammonium acetate 10 mM (70:30, v/v) with an injection rate of 0.3 ml/min. The total analytical run time for each sample was 5 min. The association of naproxen with esomeprazole take considerably longer time to reach the maximum concentration [Tmax 0.17 h (interquartile range, 0.13-1.95) for naproxen alone and 13.18*h (interquartile range, 10.12-27.15) for naproxen with esomeprazole, p = 0.002], also to be eliminated [T1/2 0.12 h (interquartile range, 0.09-1.35) for naproxen alone and 9.16*h (interquartile range, 7.16-41.40) for naproxen with esomeprazole, p = 0.002] and lower maximum concentrations (Cmax 4.6 ± 2.5 ug/mL for naproxen alone and 2.04 ± 0.78* μg/mL, p = 0.038). The association of naproxen with esomeprazole showed increased values of AUC0-t [82.06* h*μg/mL (interquartile range, 51.90-157.00) with esomeprazole and 2.97 h*μg/mL (interquartile range, 1.82-7.84) naproxen alone, p = 0.002] in drug concentrations in relation to the naproxen tablet alone, probably, such differences are due to the delay in the absorption of naproxen when it is associated with the drug proton pump inhibitor, esomeprazole. As well as reduced values of full clearance when naproxen is combined with esomeprazole (0.07* μg/h (interquartile range, 0.005-0.01) with esomeprazole and 7.29 μg/h (interquartile range, 3.17-16.23) in naproxen alone, p = 0.002). Both naproxen and 6-O-desmethylnaproxen in saliva samples can be effectively quantified using LC-MS/MS, this methodology proved to be rapid, sensitive, accurate and selective for each drug and allows for the analysis of their pharmacokinetic parameters, in both situations.

Optimization of a GC-MS method for the detection of bioactive compounds from the green peel of pomelo cultivar ‘PO52’

Pomelo is the largest and most popular citrus fruit known to possess diverse health benefits. Sabah’s fruit basket, Tenom, is famous for pomelo production. Among the pomelo cultivars being planted in Tenom, the PO52 cultivar is the most abundant one. To date, the bioactive compounds of the PO52 cultivar are not well studied. Although numerous aroma-active compounds from the peels of Malaysian pomelo have been reported, there are still many potential non-aromatic bioactive compounds that are yet to be discovered. In this study, we analysed the peel extract of pomelo PO52 using gas chromatography coupled to a single quadrupole mass spectrometry (GC-MS) approach. We first analysed the pomelo peel extracts using two different GC-MS methods, one can detect 45 organic compounds with good separation, while the other can detect phytosterols and pentacyclic triterpenoids in Clinacanthus nutans roots. In our study, these two methods were able to detect bioactive compounds from the pomelo peels but with a longer running time and the generated total ion chromatograms (TIC) were also not smooth. Therefore, we further optimized the two methods by changing the temperature ramping and holding times of the GC-MS. Compared to the first two methods, the optimized method had the shortest total running time (38 min) and the total ion chromatogram (TIC) produced was smooth throughout the run time. Furthermore, bioactive compounds that have not been reported for pomelo peel before, such as osthole, α-tocopherol, gamma-sitosterol, friedelan-3-one and others were also detected using this optimized method. In conclusion, the optimized method is suitable for the detection of non-aromatic compounds present in the PO52 peels that can be subsequently applied in the next stage for large-scale analysis.

DLA-H: A Deep Learning Accelerator for Histopathologic Image Classification.

It is more than a decade since machine learning and especially its leading subtype deep learning have become one of the most interesting topics in almost all areas of science and industry. In numerous contexts, at least one of the applications of deep learning is utilized or is going to be utilized. Using deep learning for image classification is now very popular and widely used in various use cases. Many types of research in medical sciences have been focused on the advantages of deep learning for image classification problems. Some recent researches show more than 90% accuracy for breast tissue classification which is a breakthrough. A huge number of computations in deep neural networks are considered a big challenge both from software and hardware point of view. From the architectural perspective, this big amount of computing operations will result in high power consumption and computation runtime. This led to the emersion of deep learning accelerators which are designed mainly for improving performance and energy efficiency. Data reuse and localization are two great opportunities for achieving energy-efficient computations with lower runtime. Data flows are mainly designed based on these important parameters. In this paper, DLA-H and BJS, a deep learning accelerator, and its data flow for histopathologic image classification are proposed. The simulation results with the MAESTRO tool showed 756 cycles for total runtime and [Formula: see text] GFLOPS roofline throughput that is an extreme performance improvement in comparison to current general-purpose deep learning accelerators and data flows.

A Simulation of Facility Layout Improvement at Local Food Industry

As Salaiport become well known recently, demand for their product keep increasing. However, no proper layout of the workstation and the manual production process tend to reduce the production process and cause ergonomics problems. Simulation is a problem solving methodology for the solution of many real world problems, including to determine an effective layout for the workers in order to improve the productivity and efficiency of the production. The main aims of this study is to propose a layout for Erul Food Industry that optimize processing time and minimize the cost in view of ergonomics. The Sketchup software is used to design the production line layout and the FlexSim software is used to identify the best design based on time and cost of processing. Based on the simulation results, Design 2 is the best design as it has minimum processing time and cost. It can be concluded that the shorter total running time of production, the lower cost of production.

Comprehensive Online Reversed-Phase × Chiral Two-Dimensional Liquid Chromatography-Mass Spectrometry with Data-Independent Sequential Window Acquisition of All Theoretical Fragment-Ion Spectra-Acquisition for Untargeted Enantioselective Amino Acid Analysis.

This work presents an advanced analytical platform for untargeted enantioselective amino acid analysis (eAAA) by comprehensive achiral × chiral 2D-LC hyphenated to ESI-QTOF-MS/MS utilizing data-independent SWATH (sequential window acquisition of all theoretical fragment-ion spectra) technology. The methodology involves N-terminal pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC; AccQ) as retention, selectivity, and MS tag, supporting retention and UV detection in RPLC (1D), chiral recognition, and thus enantioselectivity by the core-shell tandem column composed of a quinine carbamate weak anion exchanger (QN-AX) and a zwitterionic chiral ion-exchanger (ZWIX(+)) (2D) as well as the ionization efficiency during positive electrospray ionization due to a high proton affinity of the AQC label. Furthermore, the urea-type MS tag gives rise to the generation of AQC-tag characteristic signature fragments in MS2. The latter allows the chemoselective mass spectrometric filtering of targeted and untargeted N-derivatized amino acids or related labeled species. The chiral core-shell tandem column provides a complete enantioselective amino acid profile of all proteinogenic amino acids within 1 min, with full baseline separation of all enantiomers, but without resolution of isomeric Ile/allo-Ile (aIle)/Leu, which can be resolved by RPLC. The entire LC × LC separation occurs within a total run time of 60 min (1D), with the chiral 2D operated in gradient elution mode and a cycle time of 60 s. A strategy to mine the 2D-LC-SWATH data is presented and demonstrated for the qualitative eAAA of two peptide hydrolysate samples of therapeutic peptides containing common and uncommon as well as primary and secondary amino acids. Absolute configuration assignment of amino acids using template matching for all proteinogenic amino acids was made feasible due to method robustness and the inclusion of an isotopically labeled L-[U-13C15N]-AA standard. The quantification performance of this LC × LC-MS/MS assay was also evaluated. Accuracies were acceptable for the majority of AAs enabling AA composition determination in peptide hydrolysates simultaneously with configuration assignment, as exemplified by oxytocin. This methodology represents a step toward truly untargeted 2D enantioselective amino acid analysis and metabolomics.

A liquid chromatography-tandem mass spectrometry method for simultaneous quantification of thirty-nine tyrosine kinase inhibitors in human plasma

Currently, the use of targeted drugs such as tyrosine kinase inhibitors (TKIs) plays an important role in clinical therapy. As the number of approved TKIs continues to increase, existing analysis methods will not be able to meet the growing needs, and will hamper the development of therapeutic drug monitoring (TDM) of TKIs. Based on LC-MS/MS technology, this study tends to develop and validate a multi-component analysis method for simultaneous determination of the concentrations of 39 TKIs in plasma. Spiked plasma was blended with isotope labelled internal standards, and injected into the LC-MS/MS system after protein precipitation by acetonitrile. Chromatographic separation was achieved using an ODS-4 column with gradient elution of formic acid/water (1:1000; v/v) and acetonitrile. Analytes detection was conducted in positive ionisation mode using MRM. The total run time was 8 min. The method validation was conducted by assessing the following parameters: selectivity, linearity and the lower limit of qualification, accuracy and precision, stability, matrix effect and recovery. The concentrations of 39 TKIs showed good linearity within the range of their respective standard curves in plasma, the accuracy of all quality control samples ranged from 85.9% to 114.1%, and the precision was lower than 13.3%. The extraction recovery ranged from 92.6% to 114.7%, and the matrix effect of plasma was lower than 11.3%. This new method was successfully developed, can be used for the determination of drug concentrations in multiple patients with different kinds of TKIs, and will therefore be suitable for TDM of 39 TKIs.

Determination of steroid hormones in grey seal (Halichoerus grypus) blood plasma using convergence chromatography tandem mass spectrometry

A hybrid solid phase extraction (HybridSPE) protocol tailored to ultra-performance convergence chromatography tandem mass spectrometry (UPC2-MS/MS) was developed for the determination of 19 steroid hormones in grey seal (Halichoerus grypus) blood plasma. In this study, the protocol demonstrated acceptable absolute recoveries ranging from 33 to 90%. The chromatographic separation was carried out using a gradient elution program with a total run time of 5 min. For most target analytes, the method repeatability ranged from 1.9 to 24% and the method limits of quantification (mLOQs) ranged from 0.03 to 1.67 ng/mL. A total of 9 plasma samples were analysed to demonstrate the applicability of the developed method, and 13 steroid hormones were quantified in grey seal pup plasma. The most prevalent steroids: cortisol, cortisone, corticosterone, 11-deoxycortisol, progesterone and 17α-hydroxyprogesterone were detected at concentrations in the range of 12.6–40.1, 7.10–24.2, 0.74–10.7, 1.06–5.72, 0.38–4.38 and <mLOQ - 1.01 ng/mL, respectively. To our knowledge, this is the first study to determine steroid hormones in the plasma of pinnipeds using convergence chromatography.

DDEL-07. DETERMINATION OF TOTAL AND UNBOUND LEVELS OF A POTENT INHIBITOR OF POLY ADP-RIBOSE POLYMERASE, NIRAPARIB, IN HUMAN PLASMA, CEREBROSPINAL FLUID AND BRAIN TUMOR BY A VALIDATED LC-MS/MS ASSAY

Abstract BACKGROUND Niraparib is an orally available inhibitor of poly ADP-ribose polymerase with high selectivity for PARP1 and PARP2. It is currently approved in the United States for the treatment of ovarian, fallopian tube, and primary peritoneal cancer. Herein we present the development and validation of an LC-MS/MS method for determination of total and unbound levels of niraparib in human plasma, cerebrospinal fluid (CSF) and glioblastoma tissue. METHODS Niraparib was extracted from human plasma, CSF and glioblastoma homogenate samples by protein precipitation with methanol. Unbound fractions of niraparib in plasma and brain tissues were determined by equilibrium dialysis. Validation was performed using Sciex ExionLC UHPLC system coupled with Sciex QTRAP 6500+ MS/MS detector using positive electrospray ionization mode. The method was validated according to FDA guidelines and CAP/CLIA regulations. RESULTS The method was validated for 1-10,000 nmol/L concentration range using plasma as matrix for all biospecimens. The chromatographic separation was performed on Phenomenex Kinetex™ PS C18 column with total run time of 3.5 min with gradient elution. Maximum coefficient of variation for intra- and inter-day precision was 10.6% and the accuracy was within 92.8-118.5% in all matrices. The analyte is stable in plasma and brain homogenate for at least 6 hours at room temperature (RT) and at least 32 days at -20°C. Stability of stock and working solutions was demonstrated for at least 21 hours (RT) and 41 days (4°C). The fraction unbound of niraparib in human plasma and brain were determined to be 0.15 and 0.05, respectively. CONCLUSIONS A sensitive and rapid bioanalytical method is developed and validated to quantify total and unbound niraparib levels in human plasma, CSF and glioblastoma tissue. This method is currently employed to assess plasma pharmacokinetics and CNS penetration of niraparib in newly-diagnosed glioblastoma and recurrent IDH1/2(+) ATRX mutant glioma patients (NCT05076513).

BIOM-25. DETERMINATION OF LEVELS OF SMALL MOLECULAR WEIGHT THIOLS AND DISULFIDES IN MOUSE AND HUMAN NORMAL BRAIN AND GLIOBLASTOMA TISSUE

Abstract BACKGROUND Small molecular weight (SMW) thiols play an essential role in many biochemical processes, particularly in maintaining redox homeostasis. Herein, we report on the development and qualification of a sensitive and rapid LC-MS/MS method for the determination of oxidative stress biomarkers glutathione (GSH), cysteine (Cys), glutathione disulfide (GSSG), cystine (CySS), as well as the interrelated precursors and products methionine (Met), homocysteine (Hcy), cystathionine (Cysth), gamma glutamyl-cysteine (Glu-Cys), and cysteinylglycine (Cys-Gly) in both mouse and human normal brain and glioblastoma tissue. METHODS Mouse and human tissue samples were homogenized with N-ethylmaleimide solution to prevent thiol oxidation. Analytes were then extracted from homogenate samples by protein precipitation with 2% formic acid in acetonitrile. Separate calibration curves were prepared for thiols and disulfides in analytical solutions using stable isotope-labeled internal standards (IS). Three levels of quality control samples were prepared in mouse and human brain homogenates. The detection was performed on Sciex QTRAP 6500+ mass spectrometer in positive electrospray ionization mode. RESULTS The concentration ranges of 0.04-40 µmol/L for Glu-Cys/Hcy, 4-400 µmol/L for Met/GSH/Cys/Cys-Gly and 0.05-20 µmol/L for GSSG/CySS/Cysth were established using a linear regression model. A gradient elution was applied on Intrada Amino Acid column to achieve the optimal chromatographic separation with a 5 minute total run time. At least three precision and accuracy runs were conducted on different days. The stability of analytes in biological matrix and in analytical solutions has been tested at room temperature, 4 °C, and -80 °C for appropriate sample preparation, analysis, and storage. CONCLUSIONS A bioanalytical method to quantify a panel of SMW thiols and disulfides has been successfully developed, qualified, and utilized to assess the levels of these biomolecules in mouse normal brain and glioblastoma tissue as well as in glioblastoma tissue of patients enrolled in the sonodynamic therapy clinical trial (NCT04559685).

CYP2C9 Polymorphism Influence in PK/PD Model of Naproxen and 6-O-Desmethylnaproxen in Oral Fluid.

Polymorphisms in CYP2C9 can significantly interfere with the pharmacokinetic (PK) and pharmacodynamic (PD) parameters of nonsteroidal anti-inflammatory drugs (NSAIDs), including naproxen. The present research aimed to study the PK/PD parameters of naproxen and its metabolite, 6-O-desmethylnaproxen, associated with allelic variations of CYP2C9. In our study, a rapid, selective, and sensitive Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) method was developed and validated for the determination of naproxen and its main metabolite, 6-O-desmethylnaproxen, in oral fluid. Naproxen and its main metabolite were separated using a Shim-Pack XR-ODS 75L × 2.0 column and C18 pre-column at 40 °C using a mixture of methanol and 10 mM ammonium acetate (70:30, v/v), with an injection flow of 0.3 mL/min. The total analytical run time was 3 min. The volunteers, previously genotyped for CYP2C9 (16 ancestral—CYP2C9 *1 and 12 with the presence of polymorphism—CYP2C9 *2 or *3), had their oral fluids collected sequentially before and after taking a naproxen tablet (500 mg) at the following times: 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6 8, 11, 24, 48, 72 and 96 h. Significant differences in the PK parameters (* p < 0.05) of naproxen in the oral fluid were: Vd/F (L): 98.86 (55.58−322.07) and 380.22 (261.84−1097.99); Kel (1/h): 0.84 (0.69−1.34) and 1.86 (1.09−4.06), in ancestral and mutated CYP2C9 *2 and/or *3, respectively. For 6-O-desmethylnaproxen, no PK parameters were significantly different between groups. The analysis of prostaglandin E2 (PGE2) proved to be effective and sensitive for PD parameters analysis and showed higher levels in the mutated group (p < 0.05). Both naproxen and its main metabolite, 6-O-desmethylnaproxen, and PGE2 in oral fluid can be effectively quantified using LC-MS/MS after a 500 mg oral dose of naproxen. Our method proved to be effective and sensitive to determine the lower limit of quantification of naproxen and its metabolite, 6-O-desmethylnaproxen, in oral fluid (2.4 ng/mL). All validation data, such as accuracy, precision, and repeatability intra- and inter-assay, were less than 15%. Allelic variations of CYP2C9 may be considered relevant in the PK of naproxen and its main metabolite, 6-O-desmethylnaproxen.