New Validated Method for Quantification of Glycated Hemoglobin by LC-QToF-MS: Is HRMS Able to Quantify Clinical Samples?

High-resolution mass spectrometry is a powerful tool in clinical analysis but remains less explored due to its lower dynamic range and sensitivity compared to triple quadrupoles. Glycated hemoglobin (HbA1c) is the current gold standard biomarker to monitor the control of diabetes, representing long-term plasma glycemic levels. Due to its clinical importance, several methods have been developed for HbA1c quantification, using different principles; however, the results obtained with these techniques may differ according to the method adopted. Hence, there is a great need to standardize the current methods to quantify glycated hemoglobin. A new UPLC-QToF-MS method was fully validated and tested to quantify HbA1c in human samples. The peptides VHLTPE m/z 695.373 and gly-VHLTPE m/z 857.426, obtained via Glu-C digestion, were the selected peptides for quantification of HbA1c (mmol/mol). Chromatographic separation was obtained in a C18 column, maintained at 40 °C. The mobile phase was composed of water and acetonitrile, both containing 0.02% TFA and 0.1% acetic acid, and eluted in gradient mode. The method was fully validated, being considered linear in the range of 25-107 mmol/mol of HbA1c, and was sensitive, selective, precise, accurate, and free of matrix and carryover effects. The method was successfully applied to real samples, reaching about 90% agreement with reference method results, providing accurate and precise information on peptide mass, without laborious sample preparation. These results support the use of HRMS to improve the quality of quantitative results of HbA1c in health services and demonstrate a possible application of peptide investigation for clinical analysis in the near future.

Neuroprotective Effect of Caryocar brasiliense Camb. Leaves Is Associated with Anticholinesterase and Antioxidant Properties.

Pequi (Caryocar brasiliense) is an endemic species from Brazilian Cerrado, and their fruits are widely used in regional cuisine. In this work, a crude hydroalcoholic extract (CHE) of C. brasiliense leaves and its resulting fractions in hexane (HF), chloroform (CF), ethyl acetate (EAF), and butanol (BF) were investigated for their antioxidant properties and anticholinesterase activities. The antioxidant properties were evaluated by free radical scavenging and electroanalytical assays, which were further correlated with the total phenolic content and LC-MS results. The acetylcholinesterase and butyrylcholinesterase inhibitory activities were examined using Ellman's colorimetric method. The LC-MS analysis of EAF revealed the presence of gallic acid and quercetin. CHE and its fractions, EAF and BF, showed anticholinesterase and antioxidant activities, suggesting the association of both effects with the phenolic content. In addition, behavioral tests performed with CHE (10, 100, and 300 mg/kg) showed that it prevented mice memory impairment which resulted from aluminium intake. Moreover, CHE inhibited brain lipid peroxidation and acetyl and butyryl-cholinesterase activities and the extract's neuroprotective effect was reflected at the microscopic level. Therefore, the leaves of pequi are a potential source of phenolic antioxidants and can be potentially used in treatments of memory dysfunctions, such as those associated with neurodegenerative disorders.

New Pioglitazone Metabolites and Absence of Opened-Ring Metabolites in New N-Substituted Thiazolidinedione.

Thiazolidinediones (TZDs) are drugs used to treat type 2 diabetes mellitus; however, several safety concerns remain regarding the available drugs in this class. Therefore, the search for new TZD candidates is ongoing; metabolism studies play a crucial step in the development of new candidates. Pioglitazone, one of the most commonly used TZDs, and GQ-11, a new N-substituted TZD, were investigated in terms of their metabolic activity in rat and human liver microsomes to assess their metabolic stability and investigate their metabolites. Methods for preparation of samples were based on liquid-liquid extraction and protein precipitation. Quantitation was performed using liquid chromatography (LC)-tandem mass spectrometry, and the metabolite investigation was performed using ultraperformance LC coupled to a hybrid quadrupole-time of flight mass spectrometer. The predicted intrinsic clearance of GQ-11 was 70.3 and 46.1 ml/kg per minute for rats and humans, respectively. The predicted intrinsic clearance of pioglitazone was 24.1 and 15.9 ml/kg per minute for rats and humans, respectively. The pioglitazone metabolite investigation revealed two unpublished metabolites (M-D and M-A). M-A is a hydration product and may be related to the mechanism of ring opening and the toxicity of pioglitazone. The metabolites of GQ-11 are products of oxidation; no ring-opening metabolite was observed for GQ-11. In conclusion, under the same experimental conditions, a ring-opening metabolite was observed only for pioglitazone. The resistance of GQ-11 to the ring opening is probably related to N-substitution in the TZD ring.

Bioanalytical methods for the detection of antidiabetic drugs: a review.

Type 2 diabetes mellitus, a disease which prevalence has been progressively increasing worldwide, is characterized by chronic hyperglycemia resulting from the combination of inappropriate insulin secretion and/or resistance to insulin action. If left uncontrolled, diabetes is associated with complications such as dysfunction and failure of various organs, and even premature death. Along with lifestyle-modification strategies, several classes of oral antidiabetic agents can be employed for glycemic control. Thus, therapeutic drug monitoring of these drugs is essential to maintain appropriate treatment. This review discusses the most frequently employed analytical techniques and sample preparation systems to obtain a reliable and trustworthy method to quantify antidiabetic drugs in biological matrices. An adequate choice of internal standard, ideal chromatography conditions and most suitable analytical detector are reported.

New Metabolites of Coumarin Detected in Human Urine Using Ultra Performance Liquid Chromatography/Quadrupole-Time-of-Flight Tandem Mass Spectrometry.

Coumarin (1,2-benzopyrone) is a natural compound whose metabolism in humans was established in the 1970s. However, a new metabolite was recently identified in human plasma, indicating that the metabolism of coumarin has not been completely elucidated. To complement the knowledge of its metabolism, a rapid and sensitive method using UPLC-QTOF-MS was developed. A total of 12 metabolites was identified using MetaboLynxTM software, including eight metabolites not previously reported in human urine. The identified biotransformation included hydroxylation, glucuronidation, sulfation, methylation, and conjugation with N-acetylcysteine. The present work demonstrates that the metabolism study of coumarin was incomplete, possibly due to limitations of old techniques. The identification of eight inedited metabolites of such a simple molecule suggests that the information regarding the metabolism of other drugs may also be incomplete, and therefore, new investigations are necessary.

A systematic and critical review on bioanalytical method validation using the example of simultaneous quantitation of antidiabetic agents in blood.

A systematic and critical review was conducted on bioanalytical methods validated to quantify combinations of antidiabetic agents in human blood. The aim of this article was to verify how the validation process of bioanalytical methods is performed and the quality of the published records. The validation assays were evaluated according to international guidelines. The main problems in the validation process are pointed out and discussed to help researchers to choose methods that are truly reliable and can be successfully applied for their intended use. The combination of oral antidiabetic agents was chosen as these are some of the most studied drugs and several methods are present in the literature. Moreover, this article may be applied to the validation process of all bioanalytical.

Simultaneous Quantification of Antidiabetic Agents in Human Plasma by a UPLC-QToF-MS Method.

An ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry method for the simultaneous quantification of chlorpropamide, glibenclamide, gliclazide, glimepiride, metformin, nateglinide, pioglitazone, rosiglitazone, and vildagliptin in human plasma was developed and validated, using isoniazid and sulfaquinoxaline as internal standards. Following plasma protein precipitation using acetonitrile with 1% formic acid, chromatographic separation was performed on a cyano column using gradient elution with water and acetonitrile, both containing 0.1% formic acid. Detection was performed in a quadrupole time-of-flight analyzer, using electrospray ionization operated in the positive mode. Data from validation studies demonstrated that the new method is highly sensitive, selective, precise (RSD < 10%), accurate (RE < 12%), linear (r > 0.99), free of matrix and has no residual effects. The developed method was successfully applied to volunteers' plasma samples. Hence, this method was demonstrated to be appropriate for clinical monitoring of antidiabetic agents.

A kinetic study of the main guaco metabolites using syrup formulation and the identification of an alternative route of coumarin metabolism in humans.

For decades guaco species have been empirically used for the treatment of respiratory diseases. However, studies have shown that the toxic and therapeutic effects of the main guaco metabolites are dose-dependent, and none clinical study was done to evaluate the behavior of these substances in humans. In this work, a pilot study measuring the kinetic profile of the main guaco metabolites was performed leading to the knowledge of an alternative route of coumarin metabolism in humans. Initial screenings demonstrated that the administration of 60 mL of guaco syrup (single dose) did not provide sufficient levels of coumarin (COU), 7-hydroxycoumarin (7-HCOU), o-coumaric acid (OCA) and kaurenoic acid (KAU). The pharmacokinetic parameters were calculated by orally administering 60 mL of guaco syrup spiked with 1500 mg of COU. The kinetic study demonstrated that the plasmatic levels of 7-HCOU (considered the main metabolite of COU) were 10 times lower than the levels of COU, and the kinetic profile of 7-HCOU suggests sequential metabolism in the liver with low access of 7-HCOU to the systemic circulation. The study also demonstrated that OCA is one of the main bioavailable metabolites of COU. Therefore, the hydrolysis of the lactone ring forming a carboxylated compound is one of the possible routes of COU metabolism in humans. The half-lives of COU, 7-HCOU and OCA were approximately 4.0, 1.0 and 3.0 h, respectively and there was evidence that the recommended dosage of guaco syrup did not provide sufficient levels of COU, 7-HCOU or OCA to obtain a bronchodilation effect. Clinical studies are necessary to prove the efficacy and safety of products based on guaco.

Endothelium-Dependent Vasorelaxant Effect of Butanolic Fraction from Caryocar brasiliense Camb. Leaves in Rat Thoracic Aorta.

Caryocar brasiliense Camb. "pequi" is a native plant from the Cerrado region of Brazil that contains bioactive components reported to be antioxidant agents. Previous work has demonstrated that dietary supplementation with pequi decreased the arterial pressure of volunteer athletes. We found that the crude hydroalcoholic extract (CHE) of C. brasiliense leaves relaxed, in a concentration-dependent manner, rat aortic rings precontracted with phenylephrine, and that the butanolic fraction (BF) produced an effect similar to that of the CHE. Aortic relaxation induced by BF was abolished by endothelium removal, by incubation of the nitric oxide synthase inhibitor L-NAME, or the soluble guanylatecyclase inhibitor ODQ. However, incubation with atropine and pyrilamine had no effect on the BF-induced vasorelaxation. Moreover, this effect was not inhibited by indomethacin and tetraethylammonium. The concentration-response curve to calcium in denuded-endothelium rings was not modified after incubation with BF, and the vasorelaxation by BF in endothelium-intact rings precontracted with KCl was abolished after incubation with L-NAME. In addition, administration of BF in anesthetized rats resulted in a reversible hypotension. The results reveal that C. brasiliense possesses both in vivo and in vitro activities and that the vascular effect of BF involves stimulation of the nitric oxide/cyclic GMP pathway.