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An UHPLC-MS/MS Method for Target Profiling of Stress-Related Phytohormones.
Novák, O, Floková, K
Methods in molecular biology (Clifton, N.J.). 2018;:183-192
Abstract
The methodology described here represents an improved strategy for analysis of a broad range of stress-related plant hormones including jasmonates, salicylic acid, abscisic acid, and auxin metabolites. The method conditions are optimized in order to reduce the background effect of complicated plant matrix, allow effective preconcentration and thus perform highly sensitive profiling of multiple plant hormones by ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS).
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Propafenone quantification in human plasma by high-performance liquid chromatography coupled with electrospray tandem mass spectrometry in a bioequivalence study
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Severino, B, Luisi, G, Donomae Iwamoto, R, Moreno, RA, Perez Teixeira, V, Di Vaio, P, Saccone, I, Magli, E, Santagada, V, Caliendo, G, et al
International journal of clinical pharmacology and therapeutics. 2018;(6):280-291
Abstract
Propafenone is an antiarrhythmic drug applied to ventricular arrhythmias, initially recognized as a sodium channel blocker. This study aims to evaluate the bioequivalence of two propafenone formulations (300 mg tablet) in healthy subjects under non-fasting conditions. The study was conducted as an open, randomized, 2-period design with a 2-sequence (RT, TR) with a 1-week washout interval. The subjects were selected for the study after having their health status previously assessed by a clinical evaluation and laboratory tests (biochemical and hematological parameters, and urinalysis). Debrisoquine phenotype of healthy subjects was determined by analysis of urinary excretion of debrisoquine and its major metabolite, 4-hydroxydebrisoquine. A single propafenone tablet (300 mg) was given in each occasion. Plasma propafenone concentrations were analyzed by liquid chromatography coupled to tandem mass spectrometry (HPLC/MS/MS) with positive ion electrospray ionization using multiple reactions monitoring (MRM). The geometric mean and 90% confidence intervals (CI) of propafenone/Ritmonorm® (T/R) percent ratio were 100.44% (88.39 - 114.13%) for AUClast, 99.84% (90.31 - 110.36%) for AUCinf, and 99.30% (90.08 - 109.47%) for Cmax. Since the 90% CI for Cmax, AUClast, and AUCinf ratios were all inside the 80 - 125% interval proposed by the US Food and Drug Administration Agency, it was concluded that the propafenone formulation elaborated by Biolab Sanus Farmacêutica Ltda. is bioequivalent to Ritmonorm® formulation for both the rate and the extent of absorption. The drug was well tolerated by the subjects, indicating that it is safe to perform propafenone bioequivalence studies in healthy subjects with intermediate/extensive metabolism.
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UHPLC-MS/MS method for determination of atorvastatin calcium in human plasma: Application to a pharmacokinetic study based on healthy volunteers with specific genotype.
Xia, B, Li, Y, Zhang, Y, Xue, M, Li, X, Xu, P, Xia, T, Chen, S
Journal of pharmaceutical and biomedical analysis. 2018;:428-435
Abstract
A rapid, selective and sensitive ultra high performance liquid chromatography coupled with tandem triple quaternary mass spectrometry (UHPLC-MS/MS) method was developed and validated for the quantitative determination of atorvastatin calcium (AC) in human plasma. Separation of AC and rosuvastatin calcium (internal standard, IS) were achieved on a Dikma Leapsil C18 reversed phase column (100 × 2.1 mm, 2.7 μm) with gradient elution using 0.2% (v/v) formic acid in water and acetonitrile as mobile phases, at the flow rate of 0.3 mL/min. AC and IS were detected using MS/MS with turbo ion pray source in negative mode by monitoring the precursor-to-product ion transitions m/z 557.0→453.0 for AC and m/z 480.0→418.0 for IS. The calibration curves were linear from 0.05 to 50 ng/mL with a correlation coefficient ( r2) of 0.9992 or better. Thereafter, 187 healthy candidates were checked to the genetic polymorphism analysis of SLCO1B1 521T>C(rs4149056), SLCO1B1 388A>G(rs2306283), CYP3A4 1*B(rs2740574), CYP3A4 1*G(rs2242480) and CYP3A5*3(rs776746) using fluorescence in situ hybridization technology. The genotype frequencies of wild-type homozygote, mutant heterozygote and mutant homozygote were 62.57%(TT), 34.22%(TC) and 3.21%(CC) for SLCO1B1 521T>C, and 8.56%(AA), 33.69%(AG) and 57.75%(GG) for SLCO1B1 388A>G, and 62.57%(CC), 34.22%(CT) and 3.21%(TT) for CYP3A4 1 G, and 58.29%(GG), 34.76%(GA) and 6.95%(AA) for CYP3A5*3, respectively. Furthermore, each tested genotype of CYP3A4 1B was wild type. Finally, 5 candidates with specific genotype described above were recruited to carry out the clinical pharmacokinetics of AC (n = 5). The validated UHPLC-MS/MS method was implemented in a high-throughput setting, capable of analyzing up to 288 samples per day, and was successfully applied to the pharmacokinetic study of AC based on healthy volunteers with specific genotype. The Cmax of AC in human volunteers with the specific genotype was nearly 10 times higher than that previous reported, indicating that genetic polymorphisms of these specific genotypes have significant influence on pharmacokinetics of atorvastatin.
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Simultaneous quantification of fentanyl, sufentanil, cefazolin, doxapram and keto-doxapram in plasma using liquid chromatography-tandem mass spectrometry.
Flint, RB, Bahmany, S, van der Nagel, BCH, Koch, BCP
Biomedical chromatography : BMC. 2018;(10):e4290
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Abstract
A simple and specific UPLC-MS/MS method was developed and validated for simultaneous quantification of fentanyl, sufentanil, cefazolin, doxapram and its active metabolite keto-doxapram. The internal standard was fentanyl-d5 for all analytes. Chromatographic separation was achieved with a reversed-phase Acquity UPLC HSS T3 column with a run-time of only 5.0 min per injected sample. Gradient elution was performed with a mobile phase consisting of ammonium acetate or formic acid in Milli-Q ultrapure water or in methanol with a total flow rate of 0.4 mL min-1 . A plasma volume of only 50 μL was required to achieve adequate accuracy and precision. Calibration curves of all five analytes were linear. All analytes were stable for at least 48 h in the autosampler. The method was validated according to US Food and Drug Administration guidelines. This method allows quantification of fentanyl, sufentanil, cefazolin, doxapram and keto-doxapram, which is useful for research as well as therapeutic drug monitoring, if applicable. The strength of this method is the combination of a small sample volume, a short run-time, a deuterated internal standard, an easy sample preparation method and the ability to simultaneously quantify all analytes in one run.
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Novel Application of the Two-Period Microtracer Approach to Determine Absolute Oral Bioavailability and Fraction Absorbed of Ertugliflozin.
Raje, S, Callegari, E, Sahasrabudhe, V, Vaz, A, Shi, H, Fluhler, E, Woolf, EJ, Schildknegt, K, Matschke, K, Alvey, C, et al
Clinical and translational science. 2018;(4):405-411
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Abstract
Ertugliflozin, a sodium glucose cotransporter-2 inhibitor, is approved in the United States for treatment of type 2 diabetes mellitus. A novel two-period study design with 14 C microtracer dosing in each period was used to determine absolute oral bioavailability (F) and fraction absorbed (Fa ) of ertugliflozin. Eight healthy adult men received 100-μg i.v. 14 C-ertugliflozin (400 nCi) dose 1 h after a 15-mg oral unlabeled ertugliflozin dose (period 1), followed by 100 μg 14 C-ertugliflozin orally along with 15 mg oral unlabeled ertugliflozin (period 2). Unlabeled ertugliflozin plasma concentrations were determined using high-performance liquid-chromatography tandem mass spectrometry (HPLC-MS/MS). 14 C-ertugliflozin plasma concentrations were determined using HPLC-accelerator mass spectrometry (AMS) and 14 C urine concentrations were determined using AMS. F ((area under the curve (AUC)p.o. /14 C-AUCi.v. )*(14 C-Dosei.v. /Dosep.o. )) and Fa ((14 C_Total_Urinep.o. /14 C_Total_Urinei.v. )* (14 C-Dosei.v. /14 C-Dosep.o. )) were estimated. Estimates of F and Fa were 105% and 111%, respectively. Oral absorption of ertugliflozin was complete under fasted conditions and F was ∼100%. Ertugliflozin was well tolerated.
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In Vivo Quantitative Monitoring of Subunit Stoichiometry for Metabolic Complexes.
Wilson, RS, Thelen, JJ
Journal of proteome research. 2018;(5):1773-1783
Abstract
Metabolic pathways often employ assemblies of individual enzymes to facilitate substrate channeling to improve thermodynamic efficiency and confer pathway directionality. It is often assumed that subunits to multienzyme complexes are coregulated and accumulate at fixed levels in vivo, reflecting complex stoichiometry. Such assumptions can be experimentally tested using modern tandem mass spectrometry, and herein we describe such an approach applied toward an important metabolic complex. The committed step of de novo fatty acid synthesis in the plastids of most plants is catalyzed by the multienzyme, heteromeric acetyl-CoA carboxylase (hetACCase). This complex is composed of four catalytic subunits and a recently discovered regulatory subunit resembling the biotin carboxyl carrier protein but lacking the biotinylation motif necessary for activity. To better understand this novel form of regulation, a targeted tandem mass-spectrometry-based assay was developed to absolutely quantify all subunits to the Arabidopsis thaliana hetACCase. After validation against pure, recombinant protein, this multiplexed assay was used to quantify hetACCase subunits in siliques in various stages of development. Quantitation provided a developmental profile of hetACCase and BADC protein expression that supports a recently proposed regulatory mechanism for hetACCase and demonstrates a promising application of targeted mass spectrometry for in vivo analysis of protein complexes.
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25-Hydroxyvitamin D Testing: Immunoassays Versus Tandem Mass Spectrometry.
Garg, U
Clinics in laboratory medicine. 2018;(3):439-453
Abstract
Vitamin D has been associated with many health conditions. Because of widespread deficiency in the general population, laboratory testing of vitamin D has increased exponentially in recent years. Currently, 25-hydroxyvitamin D (25[OH]D) is considered the best marker of vitamin D status. Automated immunoassays and tandem mass spectrometry are the most widely used assays for the measurement of 25(OH)D. Because a medical decision of vitamin D deficiency and treatment are made based on specific levels, it is important that different 25(OH)D assays are harmonized. Despite standardization efforts, significant differences remain among various methods and laboratories for the measurement of 25(OH)D.
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Evaluation of top-down mass spectral identification with homologous protein sequences.
Li, Z, He, B, Kou, Q, Wang, Z, Wu, S, Liu, Y, Feng, W, Liu, X
BMC bioinformatics. 2018;(Suppl 17):494
Abstract
BACKGROUND Top-down mass spectrometry has unique advantages in identifying proteoforms with multiple post-translational modifications and/or unknown alterations. Most software tools in this area search top-down mass spectra against a protein sequence database for proteoform identification. When the species studied in a mass spectrometry experiment lacks its proteome sequence database, a homologous protein sequence database can be used for proteoform identification. The accuracy of homologous protein sequences affects the sensitivity of proteoform identification and the accuracy of mass shift localization. RESULTS We tested TopPIC, a commonly used software tool for top-down mass spectral identification, on a top-down mass spectrometry data set of Escherichia coli K12 MG1655, and evaluated its performance using an Escherichia coli K12 MG1655 proteome database and a homologous protein database. The number of identified spectra with the homologous database was about half of that with the Escherichia coli K12 MG1655 database. We also tested TopPIC on a top-down mass spectrometry data set of human MCF-7 cells and obtained similar results. CONCLUSIONS Experimental results demonstrated that TopPIC is capable of identifying many proteoform spectrum matches and localizing unknown alterations using homologous protein sequences containing no more than 2 mutations.
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Vitamin D Assays.
Bikle, DD
Frontiers of hormone research. 2018;:14-30
Abstract
The number of requests for vitamin D metabolite measurements has increased dramatically over the past decade leading commercial laboratories to develop rapid high throughput assays. The measurement of 25-hydroxyvitamin D (25[OH]D) and to a lesser extent 1,25-dihydroxyvitamin D (1,25[OH]2D) dominates these requests, but requests for multiple metabolite measurements in the same sample are also increasing. The most commonly used methods include immunoassays and liquid chromatography/mass spectrometry (LC-MS). Each method has its advantages and disadvantages, but with improvements in technology, especially in LC-MS, this method is gaining ascendance due to its greater precision and flexibility. The use of standards from the National Institutes of Standards and Technology has substantially reduced the variability from laboratory to laboratory, thereby improving the reliability of these measurements. Although the current demand is for measurement of total vitamin D metabolite levels, these metabolites circulate in blood tightly bound to vitamin D binding protein (DBP) and albumin with less than 1% free. The free concentration may be a more accurate indicator of vitamin D status especially in individuals with DBP levels that deviate from the normal population. Thus, methods to measure the free concentration at least of 25(OH)D are becoming available and may supplement if not replace measurements of total levels.
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Direct quantitation of endogenous steroid sulfates in human urine by liquid chromatography-electrospray tandem mass spectrometry.
Esquivel, A, Alechaga, É, Monfort, N, Ventura, R
Drug testing and analysis. 2018;(11-12):1734-1743
Abstract
A method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the direct quantitation of endogenous steroid sulfates has been developed to be able to evaluate these metabolites as biomarkers to detect the misuse of endogenous androgenic anabolic steroids in sports. For sample preparation, a mixed-mode solid-phase extraction was optimized to eliminate the glucuronide fraction in the washing step thus obtaining only the sulfate fraction. Chromatographic separation was optimized to achieve adequate resolution between isomers. The electrospray ionization and the product ion mass spectra of the sulfates were studied in order to obtain the most specific and selective transitions. The method was validated for quantitative purposes for 11 steroid sulfates obtaining satisfactory values for linearity, accuracy, and intra- and inter-day precision (relative standard deviation better than 16.2%). Limits of quantitation ranged between 0.5 and 2 ng/mL. Extraction recoveries for sulfate metabolites were between 90 and 94%. Matrix effect ranged from 90 to 110% showing the absence of significant ion suppression/enhancement. Samples were found to be stable after 2 freeze/thaw cycles. The applicability of the method was checked by the analysis of 75 urine samples from healthy volunteers (54 males, 37 Caucasian and 17 Asian, and 21 Caucasian females) to evaluate the concentration levels of endogenous sulfate metabolites in basal conditions.