Development, validation and application of mass spectrometry methods for clinical pharmacokinetics, neurotransmitter and human habitat studies

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Farmacología y Terapéutica. Fecha de lectura: 19-12-201

Improvement and validation of a high-performance liquid chromatography in tandem mass spectrometry method for monitoring of omeprazole in Plasma

Omeprazole (OME) is a proton pump inhibitor (PPI) with 58% bioavailability after single oral dose, presenting large inter-individual variations and significant drug-drug interactions. A simple and rapid liquid chromatography in tandem with mass spectrometry (LC/MS-MS) with solid phase extraction (SPE) and isotope-labelled internal standard (IS) method was developed to monitor the plasma levels of OME for application in pharmacokinetics and drug-drug interactions studies. OME and its IS (OME-D3), were eluted with Zorbax extend C-18 rapid resolution (4.6 mm x 50 mm, 3.5 μm) at 25ºC, under isocratic conditions through a mobile phase consisting of 1 mM ammonium acetate, pH 8.5 (55%), and acetonitrile (ACN, 45%). The flow rate was 0.8 mL/min and the run time of chromatogram was 1.2 min. OME was detected and quantified by LC-MS/MS with positive electrospray ionization (ESI) that operates in multiple-reaction monitoring (MRM) mode. The method was linear in the range of 1.5- 2000 ng/mL for OME. The validation assays of accuracy and precision, matrix effect, extraction recovery and stability of the samples for OME did not deviate more than 20% for the lower limit of quantification (LLOQ) and no more than 15% for other quality controls (QCs), according to regulatory agencies.This work was also supported by FIS No. PI052124 and CA12/00122 to ARN and FPU12/02220 to AW

The adrenal medulla modulates mechanical allodynia in a rat model of neuropathic pain

We have investigated whether the stress response mediated by the adrenal medulla in rats subjected to chronic constriction injury of the sciatic nerve (CCI) modulates their nocifensive behavior. Treatment with SK29661 (300 mg/kg; intraperitoneal (I.P.)), a selective inhibitor of phenylethanolamine N-methyltransferase (PNMT) that converts noradrenaline (NA) into adrenaline (A), fully reverted mechanical allodynia in the injured hind paw without affecting mechanical sensitivity in the contralateral paw. The effect was fast and reversible and was associated with a decrease in the A to NA ratio (A/NA) in the adrenal gland and circulating blood, an A/NA that was elevated by CCI. 1,2,3,4-tetrahydroisoquinoline-7-sulfonamide (SKF29661) did not a ect exocytosis evoked by Ca2+ entry as well as major ionic conductances (voltage-gated Na+, Ca2+, and K+ channels, nicotinic acetylcholine receptors) involved in stimulus-secretion coupling in chroma n cells, suggesting that it acted by changing the relative content of the two adrenal catecholamines. Denervation of the adrenal medulla by surgical splanchnectomy attenuated mechanical allodynia in neuropathic animals, hence confirming the involvement of the adrenal medulla in the pathophysiology of the CCI model. Inhibition of PNMT appears to be an effective and probably safe way to modulate adrenal medulla activity and, in turn, to alleviate pain secondary to the injury of a peripheral nerveThis research was funded by the SPANISH MINISTER OF SCIENCE AND INNOVATION, grants BFU2011-26253, BFU2015-70067-REDC to A.R.A., and SAF2016-78892 to A.G.G, and by UNIVERSIDAD COMPLUTENSE DE MADRID, grant PR75/18-21593 to A.R.A

The effects of aripiprazole and olanzapine on pupillary light reflex and its relationship with pharmacogenetics in a randomized multiple-dose trial

Aims: Pupillography is a noninvasive and cost-effective method to determine autonomic nerve activity. Genetic variants in cytochrome P450 (CYP), dopamine receptor (DRD2, DRD3), serotonin receptor (HTR2A, HTR2C) and ATP-binding cassette subfamily B (ABCB1) genes, among others, were previously associated with the pharmacokinetics and pharmacodynamics of antipsychotic drugs. Our aim was to evaluate the effects of aripiprazole and olanzapine on pupillary light reflex related to pharmacogenetics. Methods: Twenty-four healthy volunteers receiving 5 oral doses of 10 mg aripiprazole and 5 mg olanzapine tablets were genotyped for 46 polymorphisms by quantitative polymerase chain reaction. Pupil examination was performed by automated pupillometry. Aripiprazole, dehydro-aripiprazole and olanzapine plasma concentrations were measured by high-performance liquid chromatography–tandem mass spectrometry. Results: Aripiprazole affected pupil contraction: it caused dilatation after the administration of the first dose, then caused constriction after each dosing. It induced changes in all pupillometric parameters (P '.05). Olanzapine only altered minimum pupil size (P =.046). Polymorphisms in CYP3A, HTR2A, UGT1A1, DRD2 and ABCB1 affected pupil size, the time of onset of constriction, pupil recovery and constriction velocity. Aripiprazole, dehydro-aripiprazole and olanzapine pharmacokinetics were significantly affected by polymorphisms in CYP2D6, CYP3A, CYP1A2, ABCB1 and UGT1A1 genes. Conclusions: In conclusion, aripiprazole and its main metabolite, dehydro-aripiprazole altered pupil contraction, but olanzapine did not have such an effect. Many polymorphisms may influence pupillometric parameters and several polymorphisms had an effect on aripiprazole, dehydro-aripiprazole and olanzapine pharmacokinetics. Pupillography could be a useful tool for the determination of autonomic nerve activity during antipsychotic treatment.Consejería de Educación, Juventud y Deporte, Comunidad de Madrid, Grant/Award Number: PEJD-2017-PRE/BMD-4164; H2020 Marie Skłodowska-Curie Actions, Grant/Award Number: 72123

The Neuroprotector Benzothiazepine CGP37157 Extends Lifespan in C. elegans Worms

The benzothiazepine CGP37157 has shown neuroprotective effects in several in vitro models of excitotoxicity involving dysregulation of intracellular Ca2+ homeostasis. Although its mechanism of neuroprotection is unclear, it is probably related with some of its effects on Ca2+ homeostasis. CGP37157 is a well-known inhibitor of the mitochondrial Na+/Ca2+ exchanger (mNCX). However, it is not very specific and also blocks several other Ca2+ channels and transporters, including voltage-gated Ca2+ channels, plasma membrane Na+/Ca2+ exchanger and the Ca2+ homeostasis modulator 1 channel (CALHM1). In the present work, we have studied if CGP37157 could also induce changes in life expectancy. We now report that CGP37157 extends C. elegans lifespan by 10%–15% with a bell-shaped concentration-response, with high concentrations producing no effect. The effect was even larger (25% increase in life expectancy) in worms fed with heat-inactivated bacteria. The worm CGP37157 concentration producing maximum effect was measured by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and was close to the IC50 for inhibition of the Na+/Ca2+ exchanger. CGP37157 also extended the lifespan in eat-2 mutants (a model for caloric restriction), suggesting that caloric restriction is not involved in the mechanism of lifespan extension. Actually, CGP37157 produced no effect in mutants of the TOR pathway (daf15/unc24) or the insulin/insulin-like growth factor-1 (IGF-1) pathway (daf-2), indicating that the effect involves these pathways. Moreover, CGP37157 was also ineffective in nuo-6 mutants, which have a defect in the mitochondrial respiratory chain complex I. Since it has been described that neuroprotection by this compound in cell cultures is abolished by mitochondrial inhibitors, this suggests that life extension in C. elegans and neuroprotection in cell cultures may share a similar mechanism involving mitochondria

Safety and cardiovascular effects of multiple-dose administration of aripiprazole and olanzapine in a randomised clinical trial

[Objective] To assess adverse events (AEs) and safety of aripiprazole (ARI) and olanzapine (OLA) treatment. [Methods] Twenty-four healthy volunteers receiving five daily oral doses of 10 mg ARI and 5 mg OLA in a crossover clinical trial were genotyped for 46 polymorphisms in 14 genes by qPCR. Drug plasma concentrations were measured by high-performance liquid chromatography tandem mass spectrometry. Blood pressure (BP) and 12-lead electrocardiogram were measured in supine position. AEs were also recorded. [Results] ARI decreased diastolic BP on the first day and decreased QTc on the third and fifth day. OLA had a systolic and diastolic BP, heart rate and QTc lowering effect on the first day. Polymorphisms in ADRA2A, COMT, DRD3 and HTR2A genes were significantly associated to these changes. The most frequent adverse drug reactions (ADRs) to ARI were somnolence, headache, insomnia, dizziness, restlessness, palpitations, akathisia and nausea while were somnolence, dizziness, asthenia, constipation, dry mouth, headache and nausea to OLA. Additionally, HTR2A, HTR2C, DRD2, DRD3, OPRM1, UGT1A1 and CYP1A2 polymorphisms had a role in the development of ADRs. [Conclusions] OLA induced more cardiovascular changes; however, more ADRs were registered to ARI. In addition, some polymorphisms may explain the difference in the incidence of these effects among subjects.D. Koller is co-financed by the H2020 Marie Sklodowska-Curie Innovative Training Network 721236 grant. M. Navares is co-financed by the “Consejería de Educación, Juventud y Deporte” PEJ-2018-TL/BMD-11080 grant from “Comunidad de Madrid” and “Fondo Social Europeo”.Peer reviewe

Simultaneous determination of six antipsychotics, two of their metabolites and caffeine in human plasma by LC-MS/MS using a phospholipid-removal microelution-solid phase extraction method for sample preparation

A simple and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated in human plasma for the simultaneous determination of aripiprazole (ARI) and its metabolite dehydro-aripiprazole (DARI); olanzapine (OLA), risperidone (RIS), paliperidone (PAL), quetiapine (QUE), clozapine (CLO) and caffeine (CAF). CAF is included to the method because it can have an influence on drug metabolism due to competitive inhibition. The above mentioned compounds and their isotope-labeled internal standards were extracted from 200 µL human plasma samples by both, effective phospholipids-eliminating three-step microelution-solid-phase extraction (µ-SPE) and protein precipitation (PPT) for comparison. A combination of formic acid (0.2%)-acetonitrile (pH 3.0; 65:35, v/v) was used as mobile phase and the chromatogram was run under gradient conditions at a flow rate of 0.6 mL/min. Run time lasted 6 min, followed by a re-equilibration time of 3 min. All analytes were monitored by mass spectrometric detection operating in multiple reaction monitoring mode and the method was validated covering the corresponding therapeutic ranges: 0.18–120 ng/mL for ARI, 0.25–80 ng/mL for DARI, 1.00–100 ng/mL for OLA, 0.70–60 ng/mL for RIS, 0.20–30 ng/mL for PAL, 0.50–160 ng/mL for QUE, 0.50–1000 ng/mL for CLO, and finally 1200–3700 ng/mL for CAF. The method was validated based on the recommendations of regulatory agencies through tests of precision, accuracy, extraction recovery, identity confirmation, trueness, matrix effect, process efficiency, stability, selectivity, linearity and carry-over effect fulfilling the guideline requirements. Our µ-SPE method results in the elimination of more than 99% of early eluting and more than 92% of late-eluting phospholipids compared to PPT. Additionally, the method was successfully applied for quantifying ARI and OLA plasma concentrations from healthy volunteers.This work and D. Koller were supported by the H2020 Marie Skłodowska-Curie Actions Innovative Training Network 721236 grant. P. Zubiaur is co-financed by “Consejería de Educación, Juventud y Deporte” from “Comunidad de Madrid” and “Fondo Social Europeo”.Peer reviewe

Dataset related to article: Simultaneous determination of six antipsychotics, two of their metabolites and caffeine in human plasma by LC-MS/MS using a phospholipid-removal microelution-solid phase extraction method for sample preparation

File 1. This xls files shows the data related to the main charateristics used for the development and validation of the new method as long as the measures for letting assure the repeatability and intermediate precision and accuracy values. The results obtained for the pharmacokinetic parameters are also included in the file File 2 This jpg file shows the product ion spectra and chemical structures of the antipsychotics and their metabolites obtained by collision-induced dissociation (CID) of the indicated precursor ions [M+H]+. The fragmentation patterns of all analytes are indicated by an arrow on their chemical structure of each analyte. The results are presented as the percentage of counts versus Mass-to-Charge (m/z). All mass peaks have been normalized to the most abundantA simple and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated in human plasma for the simultaneous determination of aripiprazole (ARI) and its metabolite dehydro-aripiprazole (DARI); olanzapine (OLA), risperidone (RIS), paliperidone (PAL), quetiapine (QUE), clozapine (CLO) and caffeine (CAF). CAF is included to the method because it can have an influence on drug metabolism due to competitive inhibition. The above mentioned compounds and their isotope-labeled internal standards were extracted from 200 µL human plasma samples by both, effective phospholipids-eliminating three-step microelution-solid-phase extraction (µ-SPE) and protein precipitation (PPT) for comparison. A combination of formic acid (0.2%)-acetonitrile (pH 3.0; 65:35, v/v) was used as mobile phase and the chromatogram was run under gradient conditions at a flow rate of 0.6 mL/min. Run time lasted 6 min, followed by a re-equilibration time of 3 min. All analytes were monitored by mass spectrometric detection operating in multiple reaction monitoring mode and the method was validated covering the corresponding therapeutic ranges: 0.18-120 ng/mL for ARI, 0.25-80 ng/mL for DARI, 1.00-100 ng/mL for OLA, 0.70-60 ng/mL for RIS, 0.20-30 ng/mL for PAL, 0.50-160 ng/mL for QUE, 0.50-1000 ng/mL for CLO, and finally 1200-3700 ng/mL for CAF. The method was validated based on the recommendations of regulatory agencies through tests of precision, accuracy, extraction recovery, identity confirmation, trueness, matrix effect, process efficiency, stability, selectivity, linearity and carry-over effect fulfilling the guideline requirements. Our µ-SPE method results in the elimination of more than 99% of early eluting and more than 92% of late-eluting phospholipids compared to PPT. Additionally, the method was successfully applied for quantifying ARI and OLA plasma concentrations from healthy volunteers.European Comission ITN Treatment H2020-MSCA-ITN-721236Peer reviewe

Effective phospholipids removing microelution-solid phase extraction LC-MS/MS method for simultaneous plasma quantification of aripiprazole and dehydro-aripiprazole: Application to human pharmacokinetic studies

A simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for simultaneous quantification of aripiprazole and its active metabolite, dehydro-aripiprazole, in human plasma. Stable isotopically labeled aripiprazole, aripiprazole-D8, has been used as the internal standard (IS) for both analytes. Only 200 μl of human plasma was needed for analyte extraction, using effective phospholipids-eliminating three-step microelution-solid-phase extraction (SPE, Oasis PRiME HLB 96-well μElution Plate). An ACE C18-PFP column was applied for chromatographic separation at 25 °C, protected by a 0.2-μm on-line filter. A combination of ammonium formate (5 mM)-acetonitrile (pH 4.0; 65:35, v/v) was used as mobile phase and the chromatogram was run under gradient conditions at a flow rate of 0.6 ml/min. Run time lasted 5 min, followed by a re-equilibration time of 3 min, to give a total run time of 8 min. Five μl of the sample was injected into the chromatographic system. Aripiprazole, dehydro-aripiprazole and IS were detected using the mode multiple reaction monitoring in the positive ionization mode. The method was linear in the concentration range of 0.18–110 ng/ml and 0.35–100 ng/ml for aripiprazole and dehydro-aripiprazole, respectively. Our method has been validated according to the recommendations of regulatory agencies through tests of precision, accuracy, recovery, matrix effect, stability, sensitivity, selectivity and carry-over. Our microelution-SPE method removes more than 99% of main plasma phospholipids compared to protein precipitation and was successfully applied to several bioequivalence studies.The authors wish to thank Instituto-Fundación Teófilo Hernando for its continued financial support. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 721236. This work was also supported by FPU12/02220 from MECD to AW.Peer reviewe