A LC-MS/MS method for the simultaneous determination of 6-cyanodopamine, 6-nitrodopamine, 6-nitrodopa, 6-nitroadrenaline and 6-bromodopamine in human plasma and its clinical application in patients with chronic kidney disease.

The aim of this study was to develop a high-performance liquid chromatography-tandem mass spectrometry method for the determination of 6-cyanodopamine, 6-nitrodopamine, 6-nitrodopa, 6-nitroadrenaline and 6-bromodopamine in human plasma samples. Strata-X 33 µm solid-phase extraction cartridges were used for the extraction of the catecholamines from human plasma samples. The catecholamines were separated in a 150 × 3 mm Shim-pack GIST C18-AQ column with 3 µm particle size, placed in an oven at 40°C and perfused with 82% mobile phase A (acetonitrile-H2O; 90:10, v/v) + 0.4% acetic acid and 18% mobile phase B (deionized H2O) + 0.2% formic acid at a flow rate of 340 µl/min in isocratic mode. The injected volume was 4 µl and the run lasted 4 min. The method was linear from 0.1 to 20 ng/ml and the lower limit of quantification was 0.1 ng/ml for all analytes. The method was applied to evaluate the plasma levels of catecholamines in plasma of patients with chronic kidney disease and allowed the detection for the first time of circulating levels of the novel catecholamines 6-bromodopamine and 6-cyanodopamine.

Mass Spectrometry-Based Characterization of Protein Aggregates in Tissues and Biofluids.

Protein aggregation is a common mechanism in multiple neurodegenerative and heart diseases and the accumulation of proteins in aggregates is toxic to cells, causing injury and death. The degree of protein aggregation directly correlates with the severity of the disease. Misfolded proteins present thermodynamic barriers that culminate in the loss of structure and function and the exposure of hydrophobic residues. The exposure of hydrophobic residues is the driving force behind protein aggregation, as it reduces surface free energy and increases the propensity for the formation of large insoluble aggregates. Exploring the protein content of aggregates is fundamental to understanding their formation mechanism and pathophysiological effects. We demonstrate here a method for isolating aggregated protein content in human plasma and mouse brain samples. The samples were characterized by mass spectrometry analysis, transmission electron microscopy, and western blotting. We report the identification of proteins associated with neurodegenerative diseases in the isolated pellets. The western blotting analyses of the isolated pellet showed the positivity for CD89 and CD63, consolidated markers of exosomes, confirming the presence of exosomes within the pellet but not in the supernatant in human plasma. Notably, the concomitant isolation of exosomes together with the protein aggregates was feasible starting from 200 µL of human plasma. Moreover, the presented methodology separated albumin from the aggregated pellet, allowing identification of larger diversity of proteins through mass spectrometry analysis.

Extracellular vesicles from human plasma dampen inflammation and promote tissue repair functions in macrophages.

Although inflammation is a vital defence response to infection, if left uncontrolled, it can lead to pathology. Macrophages are critical players both in driving the inflammatory response and in the subsequent events required for restoring tissue homeostasis. Extracellular vesicles (EVs) are membrane-enclosed structures released by cells that mediate intercellular communication and are present in all biological fluids, including blood. Herein, we show that extracellular vesicles from plasma (pEVs) play a relevant role in the control of inflammation by counteracting PAMP-induced macrophage activation. Indeed, pEV-treatment of macrophages simultaneously with or prior to PAMP exposure reduced the secretion of pro-inflammatory IL-6 and TNF-α and increased IL-10 response. This anti-inflammatory activity was associated with the promotion of tissue-repair functions in macrophages, characterized by augmented efferocytosis and pro-angiogenic capacity, and increased expression of VEGFa, CD300e, RGS2 and CD93, genes involved in cell growth and tissue remodelling. We also show that simultaneous stimulation of macrophages with a PAMP and pEVs promoted COX2 expression and CREB phosphorylation as well as the accumulation of higher concentrations of PGE2 in cell culture supernatants. Remarkably, the anti-inflammatory activity of pEVs was abolished if cells were treated with a pharmacological inhibitor of COX2, indicating that pEV-mediated induction of COX2 is critical for the pEV-mediated inhibition of inflammation. Finally, we show that pEVs added to monocytes prior to their M-CSF-induced differentiation to macrophages increased efferocytosis and diminished pro-inflammatory cytokine responses to PAMP stimulation. In conclusion, our results suggest that pEVs are endogenous homeostatic modulators of macrophages, activating the PGE2/CREB pathway, decreasing the production of inflammatory cytokines and promoting tissue repair functions.

Novel Multipotent Amantadine-M30D Hybrids with Highly Selective Butyrylcholinesterase Inhibition and Neuroprotective Effects as Effective Anti-Alzheimer's Agents.

As a contribution to the development of new dual/multifunctional drugs, a novel therapeutical scaffold merging key structural features from memantine and M30D was designed, synthesized, and explored for its AChE/BuChE inhibitory activity and neuroprotective effects. All synthetized hybrids were not able to inhibit AChE, but most of them exhibit inhibition with high selectivity toward butyrylcholinesterase (BuChE). Notably, among the tested compounds, amantadine/M30D hybrids with six, seven, nine, and twelve methylene groups in the spacer (5d, 5e, 5f, and 5g) not only highlighted having the best potency and selective butyrylcholinesterase inhibition greater than 83% but also, particularly 5e and 5d, elicited considerable neuroprotection when evaluated in pretreatment conditions, by reducing injury effects caused by glutamate with maximum protection reached about 47.82 ± 0.81% (5e) and 42 ± 2.20% (5d) in comparison with memantine (37.27 ± 2.69%). Likewise, we chose 5e as the hit compound, which in a glutamate excitotoxity coculture model prevented astroglia reactivity and neuronal death, as well as a 91% restoration of calcium levels and an increasing ATP level in both pre-/post-treatments of 61.48 ± 4.60 and 45.16 ± 10.55%, respectively. Regarding docking studies, a blockade of the NMDA channel pore by 5e would explain its neuroprotective response. Finally, the hit compound 5e exhibited in vitro blood-brain barrier (BBB) permeability and human plasma stability, as well as an optimal in silico neuropharmacokinetic profile. From a therapeutic perspective, merging key pharmacophoric features from memantine and M30D provides a new medicinal scaffold with dual-/multifunctional properties and human plasma stability for the future development of potential drugs for treating AD.

Alginate-based hydrogel fiber as a restricted access material for microextraction of drugs in biological samples.

A novel lab-made alginate-based hydrogel device was successfully prepared and applied as a sorption material for the solid-phase microextraction of drugs (fluoxetine and its metabolite, norfluoxetine) in human plasma, with subsequent determination by high performance liquid chromatography-fluorescence detection (HPLC-FD). When supported in a polypropylene hollow fiber, the alginate was able to extract the analytes and functioned as a restricted access material, excluding >95 % of proteins from the biological matrix. The results indicate the potential use of this phase/device for quantitative drugs extraction from biological matrices at concentrations compatible with those typical in the literature (0.5 µg mL-1), and with satisfactory precision (13.4 % for fluoxetine and 6.2 % for norfluoxetine). Such outcomes, promoted by a simple and inexpensive material, open a new perspective of exploration of hydrogels as the sorption phase in biological matrices, a concept previously unexplored in the literature.

Chemometric optimization of salting-out assisted liquid-liquid extraction (SALLE) combined with LC-MS/MS for the analysis of carvedilol enantiomers in human plasma: Application to clinical pharmacokinetics.

Carvedilol is a commonly used antihypertensive whose oral absorption is limited by low solubility and significant first-pass metabolism. This work aimed to apply chemometrics for the optimization of a salting-out assisted liquid-liquid extraction (SALLE) combined with LC-MS/MS to analyze carvedilol enantiomers in plasma samples. Method development and validation were driven for application in pharmacokinetic studies. Parameters that influence the efficiency of SALLE were evaluated using a fractional factorial 24-1 design with 4 factors and a central composite design was used to evaluate the optimal extraction condition. Carvedilol enantiomers and the internal standard lidocaine were separated on an Astec® Chirobiotic® V column and a mixture of methanol:ethanol (90:10, v/v) with 0.02% diethylamine and 0.18% acetic acid as mobile phase. The positive ion mode on electrospray ionization was used to monitor the transitions of m/z 407 > 100 and 235 > 86 for carvedilol enantiomers and lidocaine, respectively. Acetonitrile and ammonium acetate solution were selected for sample preparation by SALLE. Surface graphs and the desirability test were used to define the optimized SALLE conditions which resulted in 93% recovery for both carvedilol enantiomers. The method was linear in the range of 0.5 to 100 ng/mL in plasma, with a lower limit of quantification of 0.5 ng/mL. Within-run and between-run precision (as the relative standard deviation) were all < 9.74% and accuracy (as relative error) did not exceed ± 10.30%. Residual effect and matrix effect were not observed. Carvedilol enantiomers were stable in plasma under the storage, preparation, and analysis conditions. The validated method was successfully applied to analyze carvedilol in plasma samples from patients previously submitted to a Roux-en-Y gastric bypass surgery treated with a single oral dose of 25 mg racemic-carvedilol. Higher plasma concentrations were observed for (R)-(+)-carvedilol when compared to (S)-(-)-carvedilol in two patients post-bariatric surgery.

Visual Assessment of Blood Plasma versus Optical Transmittance and Refractive Index Measurements for Quantifying Lipemia.

Today, visual classification of the degree of lipemia in blood samples is frequently performed in clinical laboratories. However, achieving standardization of this classification at low cost and with fewer resources is an objective that is still under development. In this work, a comparison is made between the visual inspection and optical measurements of blood plasma for quantifying lipemia. The plasma refractive index's real part was measured using an Abbe refractometer and transmittance measurements were made at a 589 nm wavelength and wavelengths ranging from 320 to 1100 nm in the spectral region, respectively. Taking the slope of the transmittance spectrum at two specific wavelengths, it is possible to establish a more standardized selection criterion and implement it quickly using low-cost optical devices. Furthermore, using the proposed transmittance-spectrum-slope method, statistically significant differences (p < 0.05) were found between healthy blood samples and lipemia 1, 2, 3, and 4. However, there were no statistical differences between lipemia 1 and 2.

Isolation and Identification of Dengue Virus Interactome with Human Plasma Proteins by Affinity Purification-Mass Spectrometry.

Viral proteins evolve to benefit the interaction with host proteins during the infection and replication processes. A comprehensive understanding of virus interactome with host proteins may thus lead to the identification of molecular targets for infection inhibition. We present a procedure for isolating and identifying the dengue virus interactome with human plasma proteins. It comprises the fractionation of human plasma by anion exchange chromatography, followed by affinity purification and mass spectrometry identification of the captured proteins. This procedure was applied to the characterization of the interactions of the four serotypes of dengue virus with human plasma proteins, mediated by the domain III of the envelope protein of the virus. The resulting interactome comprises 62 proteins, six of which were validated as new direct interactions of the virus with its human host.

Systemic Effects of Hemorrhagic Snake Venom Metalloproteinases: Untargeted Peptidomics to Explore the Pathodegradome of Plasma Proteins.

Hemorrhage induced by snake venom metalloproteinases (SVMPs) is a complex phenomenon that involves capillary disruption and blood extravasation. HF3 (hemorrhagic factor 3) is an extremely hemorrhagic SVMP of Bothrops jararaca venom. Studies using proteomic approaches revealed targets of HF3 among intracellular and extracellular proteins. However, the role of the cleavage of plasma proteins in the context of the hemorrhage remains not fully understood. The main goal of this study was to analyze the degradome of HF3 in human plasma. For this purpose, approaches for the depletion of the most abundant proteins, and for the enrichment of low abundant proteins of human plasma, were used to minimize the dynamic range of protein concentration, in order to assess the proteolytic activity of HF3 on a wide spectrum of proteins, and to detect the degradation products using mass spectrometry-based untargeted peptidomics. The results revealed the hydrolysis products generated by HF3 and allowed the identification of cleavage sites. A total of 61 plasma proteins were identified as cleaved by HF3. Some of these proteins corroborate previous studies, and others are new HF3 targets, including proteins of the coagulation cascade, of the complement system, proteins acting on the modulation of inflammation, and plasma proteinase inhibitors. Overall, the data indicate that HF3 escapes inhibition and sculpts the plasma proteome by degrading key proteins and generating peptides that may act synergistically in the hemorrhagic process.

Characterization of a new SARS-CoV-2 variant that emerged in Brazil.

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a key role in viral infectivity. It is also the major antigen stimulating the host's protective immune response, specifically, the production of neutralizing antibodies. Recently, a new variant of SARS-CoV-2 possessing multiple mutations in the S protein, designated P.1, emerged in Brazil. Here, we characterized a P.1 variant isolated in Japan by using Syrian hamsters, a well-established small animal model for the study of SARS-CoV-2 disease (COVID-19). In hamsters, the variant showed replicative abilities and pathogenicity similar to those of early and contemporary strains (i.e., SARS-CoV-2 bearing aspartic acid [D] or glycine [G] at position 614 of the S protein). Sera and/or plasma from convalescent patients and BNT162b2 messenger RNA vaccinees showed comparable neutralization titers across the P.1 variant, S-614D, and S-614G strains. In contrast, the S-614D and S-614G strains were less well recognized than the P.1 variant by serum from a P.1-infected patient. Prior infection with S-614D or S-614G strains efficiently prevented the replication of the P.1 variant in the lower respiratory tract of hamsters upon reinfection. In addition, passive transfer of neutralizing antibodies to hamsters infected with the P.1 variant or the S-614G strain led to reduced virus replication in the lower respiratory tract. However, the effect was less pronounced against the P.1 variant than the S-614G strain. These findings suggest that the P.1 variant may be somewhat antigenically different from the early and contemporary strains of SARS-CoV-2.

Evaluation of the influence of temperature on the protein-tannic acid complex.

Precipitation of blood products from plasma fractionation has played a fundamental role in the industrial purification of important therapeutic products. Only a few studies have been reported by using tannins as proteins precipitant agent from whole plasma while, several conditions have been analyzed. Here, we decided to verify the effect of the temperature on the precipitation process of plasma proteins using tannic acid (TA). Plasma proteins were precipitated with tannic acid by using different temperature incubations. Subsequently, the protein-TA complex was analyzed by SDS-PAGE and quantified. In addition, the protein activity of the complex was measured after heating, as well as the structural changes of the complexes were accompanied by thermogravimetric analysis, differential scanning calorimetry and circular dichroism. In all conditions tested, tannic acid was able to precipitate without selectively separating the proteins in the mixture by using different temperatures during the precipitation process. Furthermore, the protein concentration from the plasma precipitate was not affected by different temperatures and the plasma precipitate was able to dissolve fibrin clots in vitro.

Systemic effects of hemorrhagic snake venom metalloproteinases: untargeted peptidomics to explore the pathodegradome of plasma proteins

Hemorrhage induced by snake venom metalloproteinases (SVMPs) is a complex phenomenon that involves capillary disruption and blood extravasation. HF3 (hemorrhagic factor 3) is an extremely hemorrhagic SVMP of Bothrops jararaca venom. Studies using proteomic approaches revealed targets of HF3 among intracellular and extracellular proteins. However, the role of the cleavage of plasma proteins in the context of the hemorrhage remains not fully understood. The main goal of this study was to analyze the degradome of HF3 in human plasma. For this purpose, approaches for the depletion of the most abundant proteins, and for the enrichment of low abundant proteins of human plasma, were used to minimize the dynamic range of protein concentration, in order to assess the proteolytic activity of HF3 on a wide spectrum of proteins, and to detect the degradation products using mass spectrometry-based untargeted peptidomics. The results revealed the hydrolysis products generated by HF3 and allowed the identification of cleavage sites. A total of 61 plasma proteins were identified as cleaved by HF3. Some of these proteins corroborate previous studies, and others are new HF3 targets, including proteins of the coagulation cascade, of the complement system, proteins acting on the modulation of inflammation, and plasma proteinase inhibitors. Overall, the data indicate that HF3 escapes inhibition and sculpts the plasma proteome by degrading key proteins and generating peptides that may act synergistically in the hemorrhagic process.

Plasma Tau Variants Detected by a Novel Anti-Tau Monoclonal Antibody: A Potential Biomarker for Alzheimer's Disease.

BACKGROUND: A major drawback in Alzheimer's disease (AD) is the lack of validated biomarkers for routine clinical diagnostic. We have reported earlier a novel blood biomarker, named Alz-tau®, based on variants of platelet tau. This marker evaluates the ratio of high molecular weight tau (HMWtau) and the low molecular weight (LMWtau) tau. OBJECTIVE: To analyze a potential novel source of antigen for Alz-tau®, plasma tau, detected by immunoreactivity with the novel monoclonal antibody, tau51. METHODS: We evaluated tau variants in plasma precipitated with ammonium sulfate from 36 AD patients and 15 control subjects by western blot with this novel monoclonal antibody. RESULTS: The HMW/LMWtau ratio was statistically different between AD patients and controls. CONCLUSIONS: Plasma tau variants are suitable to be considered as a novel antigen source for the Alz-tau® biomarker for AD.

Microextraction by packed sorbent and high performance liquid chromatography for simultaneous determination of lumefantrine and desbutyl-lumefantrine in plasma samples.

A bioanalytical method for the determination of lumefantrine and its metabolite desbutyl-lumefantrine in plasma samples using microextraction by packed sorbent (MEPS) and high-performance liquid chromatography was developed and validated. A complete factorial planning and surface response approach were employed to optimize the extraction parameters sample volume, dilution, aspirated sample volume and extraction cycles. The method employed C18 MEPS sorbent and diazepam as internal standard (IS). Separation was performed on a Luna C18 column (250 mm × 4.6 mm, 5 µm) at 35 °C, with mobile phase composed of acetonitrile and 0.05 % trifluoroacetic acid (68:32, v/v), detection at 305 nm and injection volume of 25 µL. The developed method showed to be selective, precise, accurate and linear in the range of 50-5000 ng/mL for lumefantrine and desbutyl-lumefantrine. Using the optimized MEPS procedure, high recovery rates were obtained for both analytes and IS (92.2 %-99.0 %). The method was successfully applied for the determination of lumefantrine and its metabolite in human plasma samples after oral administration of lumefantrine tablets in healthy volunteers.

Direct chiral LC-MS/MS analysis of fexofenadine enantiomers in plasma and urine with application in a maternal-fetal pharmacokinetic study.

This study shows the development and validation of two enantioselective LC-MS/MS methods for the determination of fexofenadine in biological matrices including the elution order determination. Plasma (200 µL) or urine (50 µL) aliquots were added to the internal standard solution [(S)-(-)-metoprolol] and extracted in the acid medium with chloroform. Resolution of the (R)-(+)- and (S)-(-)-fexofenadine enantiomers was performed in a Chirobiotic V column. The methods showed linearity at the range of 0.025-100 ng/mL plasma and 0.02-10 µg/mL urine for each fexofenadine enantiomer. These methods were applied to the maternal-fetal pharmacokinetics of fexofenadine enantiomers in plasma and urine of parturient women (n = 8) treated with a single oral 60 mg dose of racemic fexofenadine. Enantiomeric ratio in plasma (AUC0-∞(R)-(+)/(S)-(-)) was close to 1.5, nevertheless in urine was closed to unity. The transplacental transfer was approximately 18% for both fexofenadine enantiomers. The enantioselective methods can also be useful in future clinical studies of chiral discrimination of drug transporters.

LC-QToF-MS method for quantification of ethambutol, isoniazid, pyrazinamide and rifampicin in human plasma and its application.

In this research, we developed and validated a liquid chromatography coupled to mass spectrometry (LC-QToF-MS) method for simultaneous quantification of the anti-tuberculosis drugs ethambutol, isoniazid, pyrazinamide and rifampicin in human plasma. Plasma samples spiked with cimetidine (internal standard) were extracted using protein precipitation with acetonitrile containing 1% formic acid. Separation was performed using a C18 column under flow gradient conditions with water and acetonitrile, both containing 5 mm ammonium formate and 0.1% formic acid. The method was validated according to the ANVISA and US Food and Drug Administration guidelines for bioanalytical method validation. The calibration curve was linear over a concentration range of 0.2-5 µg ml-1 for ethambutol, 0.2-7.5 µg ml-1 for isoniazid, 1-40 µg ml-1 for pyrazinamide and 0.25-2 µg ml-1 for rifampicin, all with adequate precision and accuracy. The method was reproducible, selective and free of carryover and matrix effects. The validated LC-QToF-MS method was successfully applied to real samples and shown to be applicable to future therapeutic and pharmacokinetic monitoring studies.

Tannic acid as a precipitating agent of human plasma proteins.

The search for plasma proteins precipitation methods has been increasing due to the plasma protein therapeutic needs in world-wide. Thus, this work evaluates the tannic acid (TA) ability to precipitate proteins from human plasma. In this study, TA-plasma protein complexes were studied at different pH conditions, tannin/plasma ratio and reaction mixing time. The complexes formed from combinations of TA and plasma proteins were analyzed by gel electrophoresis, protein quantification, particle size, charge, mass spectrometry, microscopic image, and circular dichroism. It was possible to verify the precipitate formation in all tested pH values, with high precipitation at pH 5. The native PAGE analysis showed three mainly bands corresponding independent of the pHs used. It was possible to observe a gradual growing of precipitate protein in the first precipitation process (P1) when increased the TA/plasma ratio. 15 min of incubation was enough to precipitate 72.3% of proteins. Spectroscopic analyzes showed albumin signals and the electron microscopy analysis of IgG-TA confirmed the compact form of a precipitate. According to CD, formation of the IgG-TA complexes does not cause a major structural change of the protein. From the results obtained, it was possible to establish some parameters for plasma proteins precipitation using TA.

Biological sample preparation by using restricted-access nanoparticles prepared from bovine serum albumin: application to liquid chromatographic determination of ß-blockers.

Restricted-access nanoparticles (RANPs) were prepared from bovine serum albumin by coacervation. They have an average sized of 311 nm. They were characterized and used to capture the ß-blockers atenolol, metoprolol and propranolol from untreated biological samples. It is shown that both high protein affinity drugs (propranolol) and low protein affinity drugs (atenolol) could be rapidly extracted from plasma. This is revealed by kinetic and isothermal adsorption studies. On the other hand, almost all proteins from the sample were excluded. This demonstrates the efficiency of RANPs as restricted-access material. Sample preparation was carried out by solid phase microextraction using a probe obtained by the fixation of the RANPs at the end of a glass capillary. Atenolol (in concentrations from 100 to 1200 µg L-1), metoprolol (from 80 to 1000 µg L-1) and propranolol (from 15 to 200 µg L-1) were extracted from spiked plasma samples and analyzed by LC MS/MS without using a separation column. Correlation coefficients >0.99, good precision, accuracy, robustness, and lack of memory effects were observed for all of the analytes. The detection limits (at an S/N of 3) are 25.6, 14.6, and 3.8 µg L-1 for atenolol, metoprolol and propranolol, respectively. Ten samples can be simultaneously extracted within ∼15 min. Plasma samples of patients undergoing medical treatment were successfully analyzed with the method. Graphical abstract Schematic representation of a bovine serum albumin-based restricted access nanoparticle that exclude proteins from a human plasma sample but capture the small analytes.

UPLC-MS/MS method for gemfibrozil determination in plasma with application to a pharmacokinetic study in healthy Brazilian volunteers.

Gemfibrozil (GFZ) is a derivative of fibric acid and is used in the treatment of dyslipidemia. GFZ may affect the metabolism of various drugs, including statins, by inhibiting the sinusoidal influx transporter OATP1B1 and also CYP2C9 and CYP2C8 enzymes. This study presents the development and validation of a rapid, simple, sensitive and reproducible method of GFZ analysis in human plasma using UPLC-MS/MS. The method was applied in a pharmacokinetic study following administration of multiple doses of 600 mg GFZ every 12 h in healthy volunteers (n = 15). GFZ was separated on a C18 column using a mixture of 0.01% formic acid and acetonitrile (40:60, v/v) as the mobile phase at a flow rate of 0.4 mL/min. The method showed linearity in the range from 0.01 µg/mL to 100 µg/mL plasma. The coefficients of variation and the relative standard errors of the accuracy and precision analyses were <15%. The method allowed quantification of plasma concentrations of GFZ in the dose interval of the sixth day of administration of multiple oral doses of GFZ every 12 h. The pharmacokinetic parameters are presented as mean (95% CI): area under the plasma concentration versus time curve 88.84 (72.72-104.96) µg·h/mL, steady state mean plasma concentration 7.40 (6.06-8.75) µg/mL, minimum plasma concentration 1.24 (0.87-1.61) µg/mL, maximum plasma concentration 26.73 (21.31-32.15) µg/mL, time to reach maximum plasma concentration 2.28 (1.42-3.13) h, elimination half-life 2.81 (2.22-3.40) h, apparent total clearance 7.72 (5.85-9.58) L/h, apparent distribution volume 33.97 (18.41-49.53) L. In conclusion, the method for analysis of GFZ in human plasma showed sensitivity, linearity, precision and accuracy compatible with application in pharmacokinetic studies of multiple oral dose of 600 mg GFZ every 12 h.

A new HPLC-MS/MS method for the simultaneous quantification of SGLT2 inhibitors and metformin in plasma and its application to a pharmacokinetic study in healthy volunteers.

Monitoring the plasma concentrations of metformin and sodium-glucose cotransporter-2 inhibitors (canagliflozin, dapagliflozin and empagliflozin) is essential for pharmacokinetic and bioequivalence studies and therapeutic monitoring. The present work therefore aimed to develop and validate a high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) method for the simultaneous quantification of these drugs in human plasma. The analyses were performed using an Agilent 1200 HPLC system coupled to an Applied Biosystems API 3200 triple quadrupole MS/MS with electrospray ionization in positive ion mode. After one-step protein precipitation of plasma with acetonitrile containing 0.1% formic acid, chromatographic separation was achieved on an Xbridge C18 column, with a mobile phase consisting of a gradient of water and acetonitrile, both containing 1 mm ammonium formate and 0.1% formic acid. Quantification was performed in multiple reaction monitoring mode using m/z 130.1 → 71.1 for metformin, m/z 462.0 → 191.2 for canagliflozin, m/z 426.1 → 167.1 for dapagliflozin and m/z 468.0 → 354.9 for empagliflozin. The proposed method was validated and demonstrated to be adequate for the quantification of metformin, canagliflozin, dapagliflozin and empagliflozin for clinical monitoring, pharmacokinetics and bioequivalence studies.