scholarly journals Optimized Protocol for the In Situ Derivatization of Glutathione with N-Ethylmaleimide in Cultured Cells and the Simultaneous Determination of Glutathione/Glutathione Disulfide Ratio by HPLC-UV-QTOF-MS

Metabolites ◽  
2020 ◽  
Vol 10 (7) ◽  
pp. 292
Author(s):  
Xueni Sun ◽  
Raffaela S. Berger ◽  
Paul Heinrich ◽  
Ibtissam Marchiq ◽  
Jacques Pouyssegur ◽  
...  
Keyword(s):  
Dynamic Range ◽  
Limit Of Detection ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Internal Standard ◽  
Glutathione Disulfide ◽  
Optimized Protocol ◽  
In Situ Derivatization ◽  
In Cells

Glutathione (GSH) and glutathione disulfide (GSSG) are commonly used to assess the oxidative status of a biological system. Various protocols are available for the analysis of GSH and GSSG in biomedical specimens. In this study, we present an optimized protocol for the in situ derivatization of GSH with N-ethylmaleimide (NEM) to prevent GSH autooxidation, and thus to preserve the GSH/GSSG ratio during sample preparation. The protocol comprises the incubation of cells in NEM containing phosphate buffered saline (PBS), followed by metabolite extraction with 80% methanol. Further, to preserve the use of QTOF-MS, which may lack the linear dynamic range required for the simultaneous quantification of GSH and GSSG in non-targeted metabolomics, we combined liquid chromatographic separation with the online monitoring of UV absorbance of GS-NEM at 210 nm and the detection of GSSG and its corresponding stable isotope-labeled internal standard by QTOF-MS operated with a 10 Da Q1 window. The limit of detection (LOD) for GS-NEM was 7.81 µM and the linear range extended from 15.63 µM to 1000 µM with a squared correlation coefficient R2 of 0.9997. The LOD for GSSG was 0.001 µM, and the lower limit of quantification (LLOQ) was 0.01 µM, with the linear (R2 = 0.9994) range extending up to 10 µM. The method showed high repeatability with intra-run and inter-run coefficients of variation of 3.48% and 2.51% for GS-NEM, and 3.11% and 3.66% for GSSG, respectively. Mean recoveries of three different spike-in levels (low, medium, high) of GSSG and GS-NEM were above 92%. Finally, the method was applied to the determination of changes in the GSH/GSSG ratio either in response to oxidative stress in cells lacking one or both monocarboxylate transporters MCT1 and MCT4, or in adaptation to the NADPH (nicotinamide adenine dinucleotide phosphate) consuming production of D-2-hydroxyglutarate in cells carrying mutations in the isocitrate dehydrogenase genes IDH1 and IDH2.

scholarly journals A new selective, and sensitive method for the determination of lixivaptan, a vasopressin 2 (V2)-receptor antagonist, in mouse plasma and its application in a pharmacokinetic study

2018 ◽  
Vol 16 (1) ◽  
pp. 614-620
Author(s):  
Haitham Alrabiah ◽  
Mohammed Abunassif ◽  
Sabry Attia ◽  
Gamal Abdel-Hafiz Mostafa
Keyword(s):  
Receptor Antagonist ◽  
Pharmacokinetic Study ◽  
Limit Of Detection ◽  
Internal Standard ◽  
Hplc Method ◽  
Maximum Plasma ◽  
Mouse Plasma ◽  
V2 Receptor ◽  
V2 Receptor Antagonist

AbstractA new, selective and sensitive HPLC method for the determination of lixivaptan, an oral selective vasopressin 2 (V2)-receptor antagonist, was investigated and validated. A Waters symmetry C18 column was used as a stationary phase in isocratic elution mode using a mobile phase composed of KH2PO4 (100 mM)-acetonitrile (40: 60, v/v) at a flow rate of 1.5 mL min-1. Diclofenac was used as the internal standard (IS). Lixivaptan and the IS were extracted from plasma by protein precipitation and were detected at 260 nm. Lixivaptan and diclofenac were eluted at 3.6 and 6.2 min, respectively. The developed method showed good linearity over the calibration range of 50 -1000 ng mL-1 with a lower limit of detection of 16.5 ng mL-1. The extraction percentage of lixivaptan in the mouse plasma was in the range of 88.88 - 114.43%, which indicates acceptable extraction. The aforementioned method was validated according to guidelines of the International Council on Harmonization (ICH). The intra- and inter-day coefficients of variation did not exceed 5.5%. This method was presented to be simple, sensitive, and accurate and was successfully adapted in a pharmacokinetic study of the profile of lixivaptan in mouse plasma. A mean maximum plasma concentration of lixivaptan of 113.82 ng mL-1 was achieved in 0.5 h after oral administration of a 10 mg kg-1 dose in mouse as determined using the developed method.

Determination of tigecycline in turkey plasma by LC-MS/MS: validation and application in a pharmacokinetic study

2017 ◽  
Vol 20 (2) ◽  
pp. 241-249 ◽  
Author(s):  
A. Jasiecka-Mikołajczyk ◽  
J.J. Jaroszewski
Keyword(s):  
Pharmacokinetic Study ◽  
High Performance ◽  
Limit Of Detection ◽  
Internal Standard ◽  
Reversed Phase ◽  
Selected Reaction Monitoring ◽  
Limit Of Quantification ◽  
Complicated Skin ◽  
Intravenous And Oral Administration

Abstract Tigecycline (TIG), a novel glycylcycline antibiotic, plays an important role in the management of complicated skin and intra-abdominal infections. The available data lack any description of a method for determination of TIG in avian plasma. In our study, a selective, accurate and reversed-phase high performance liquid chromatography-tandem mass spectrometry method was developed for the determination of TIG in turkey plasma. Sample preparation was based on protein precipitation and liquid-liquid extraction using 1,2-dichloroethane. Chromatographic separation of TIG and minocycline (internal standard, IS) was achieved on an Atlantis T3 column (150 mm × 3.0 mm, 3.0 μm) using gradient elution. The selected reaction monitoring transitions were performed at 293.60 m/z → 257.10 m/z for TIG and 458.00 m/z → 441.20 m/z for IS. The developed method was validated in terms of specificity, selectivity, linearity, lowest limit of quantification, limit of detection, precision, accuracy, matrix effect, carry-over effect, extraction recovery and stability. All parameters of the method submitted to validation met the acceptance criteria. The assay was linear over the concentration range of 0.01-100 μg/ml. This validated method was successfully applied to a TIG pharmacokinetic study in turkey after intravenous and oral administration at a dose of 10 mg/kg at various time-points.

scholarly journals A novel online coupling of ion selective electrode with the flow injection system for the determination of vitamin B1

2016 ◽  
Vol 13 (2) ◽  
pp. 458-469
Author(s):  
Baghdad Science Journal
Keyword(s):  
Flow Injection ◽  
Dynamic Range ◽  
Limit Of Detection ◽  
Vitamin B1 ◽  
Pharmaceutical Formulations ◽  
Ion Selective Electrode ◽  
Injection System ◽  
Selective Electrode ◽  
Flow Injection System

A simple, fast, selective of a new flow injection analysis method coupled with potentiometric detection was used to determine vitamin B1 in pharmaceutical formulations via the prepared new selective membranes. Two electrodes were constructed for the determination of vitamin B1 based on the ion-pair vitamin B1-phosphotungestic acid (B1-PTA) in a poly (vinyl chloride) supported with a plasticized di-butyl phthalate (DBPH) and di-butyl phosphate (DBP). Applications of these ion selective electrodes for the determination of vitamin B1 in the pharmaceutical preparations for batch and flow injection systems were described. The ion selective membrane exhibited a near-Nernstian slope values 56.88 and 58.53 mV / decade, with the linear dynamic range of vitamin B1 was 5 x 10-5- 1 x 10-2 and 1 x 10-4-1 x 10-2 mol.L-1, in batch and FIA, respectively. The limit of detection was 3.5 x 10-5 and 9.5 x 10-5 mol.L-1, with the percentage linearity 98.85 and 95.22 in batch and FIA, respectively. The suggested ion selective electrode has been utilized perfection in the determination of vitamin B1 in pharmaceutical formulations using batch and flow injection system, respectively.

scholarly journals Simultaneous Determination of Trace Mefenamic Acid in Pharmaceutical Samples via Flow Injection Fluorometry

2020 ◽  
Vol 10 (03) ◽  
pp. 395-401
Author(s):  
Mohammad K. Hammood ◽  
Maryam Hamed
Keyword(s):  
Flow Injection ◽  
Mefenamic Acid ◽  
Dynamic Range ◽  
Limit Of Detection ◽  
Basic Medium ◽  
Minimum Concentration ◽  
Pharmaceutical Samples ◽  
Fluorescence Measurements ◽  
532 Nm

Mefenamic acid belongs to non-steroidal anti-inflammatory drugs that are used widely for the treatment of analgesia. Our aim from this study is to establish a new assay for the quantitative determination of mefenamic acid (MFA) in the pharmaceutical sample by two sensitive and rapid flow injection-fluorometric methods. A homemade fluorometer was used in fluorescence measurements, which using solid-state laser diode 405 and 532 nm as a source, combined with a continuous flow injection technique. The first method depends on the effect of MFA on calcein blue (CLB) fluorescence at 405 nm. Another method is a study of rhodamine-6G (Rh-6G) fluorescence after adding MFA, and recording at 532 nm. Optimum parameters as fluorescent dye concentration, basic medium, flow rate, sample volume, purge time, and delay coil have been investigated. The dynamic range of MFA was 0.2 to 2 mmol.L-1; 0.5 to 2.3 mmol.L-1 with linearity percentage (% r2) 98.92 and 99.83%, for Rh-6G and CLB, respectively. Limit of detection at a minimum concentration in calibration curve 189.34 and 199.89 ng/sample, for Rh-6G and CLB, respectively. The comparison of developed methods with the classical method (UV-vis spectrophotometry) was achieved. The proposed methods were successfully applied for the determination of MFA in the pharmaceutical samples and can be used as an alternative method.

Gas-Liquid Chromatographic Determination of T-2 Toxin in Plasma

1983 ◽  
Vol 66 (4) ◽  
pp. 909-912 ◽  
Author(s):  
Steven P Swanson ◽  
Venkatachalam Ramaswamy ◽  
Val R Beasley ◽  
William B Buck ◽  
Harold H Burmeister
Keyword(s):  
Chromatographic Method ◽  
Limit Of Detection ◽  
Internal Standard ◽  
Aqueous Sodium Hydroxide ◽  
Chromatographic Determination ◽  
Florisil Column ◽  
Liquid Chromatographic Method ◽  
Liquid Chromatographic Determination ◽  
Liquid Chromatographic

Abstract The gas-liquid chromatographic method for the determination of T-2 toxin in plasma is described. The toxin is extracted with benzene, washed with aqueous sodium hydroxide, and chromatographed on a small Florisil column; the heptafluorobutyryl derivative is prepared by reaction with heptafluorobutyrylimidazole. The T-2 HFB derivative is chromatographed onOV-1 at 230°C and measured with an electron capture detector. Iso-T-2, an isomer of T-2 toxin, is added to samples as an internal standard before extraction. Recoveries averaged 98.0 ± 5.5% at levels ranging from 50 to 1000 ng/m L. The limit of detection is 25 ng/mL.

scholarly journals Determination of Tianeptine in Tablets by High-Performance Liquid Chromatography with Fluorescence Detection

2007 ◽  
Vol 90 (3) ◽  
pp. 720-724
Author(s):  
Sevgi Tatar Ulu
Keyword(s):  
Lower Limit ◽  
High Performance ◽  
Limit Of Detection ◽  
Internal Standard ◽  
Linear Calibration ◽  
Fluorescence Detector ◽  
Relative Standard ◽  
Validation Parameters ◽  
Limit Of Quantitation

Abstract A sensitive and selective high-performance liquid chromatographic method has been developed for the determination of tianeptine (Tia) in tablets. The method is based on derivatization of Tia with 4-chloro-7-nitrobenzofurazan (NBD-Cl). A mobile phase consisting of acetonitrile10 mM orthophosphoric acid (pH 2.5; 77 + 23) was used at a flow rate of 1 mL/min on a C18 column. The Tia-NBD derivative was monitored using a fluorescence detector, with emission set at 520 nm and excitation at 458 nm. Gabapentin was selected as an internal standard. Linear calibration graphs were obtained in the concentration range of 45300 ng/mL. The lower limit of detection (LOD) was 10 ng/mL at a signal-to-noise ratio of 4. The lower limit of quantitation (LOQ) was 45 ng/mL. The relative standard values for intra- and interday precision were <0.46 and <0.57%, respectively. The recovery of the drug samples ranged between 98.89 and 99.85%. No chromatographic interference from the tablet excipients was found. The proposed method was validated in terms of precision, robustness, recovery, LOD, and LOQ. All the validation parameters were within the acceptance range. The proposed method was applied for the determination of Tia in commercially available tablets. The results were compared with those obtained by an ultraviolet spectrophotometric method using t- and F-tests.

Gas-chromatographic determination of 5-fluorocytosine in human serum.

1976 ◽  
Vol 22 (6) ◽  
pp. 772-776 ◽  
Author(s):  
S A Harding ◽  
G F Johnson ◽  
H M Solomon
Keyword(s):  
Chromatographic Method ◽  
Limit Of Detection ◽  
Internal Standard ◽  
Chromatographic Determination ◽  
Mean Value ◽  
Normal Serum ◽  
Trimethylsilyl Derivative ◽  
Serum Samples ◽  
Gas Chromatographic Method

Abstract We describe a sensitive and precise gas-chromatographic method, in which cytosine is used as the internal standard, for determination of an antifungal agent, 5-fluorocytosine, in serum. The trimethylsilyl derivative of this drug is well separated from the internal standard and from normal serum constituents. Amphotericin B does not interfere with the determination of 5-fluorocytosine. The lower limit of detection for 5-fluorocytosine is 1 mg/liter when 200 mul of serum is analyzed. Within-run precision (CV), established by analysis of 10 replicates, was 4.5% at a concentration of 19.9 mg/liter. Twenty-five serum samples were analyzed for 5-fluorocytosine by a microbiological assay and by the gas-chromatographic method. Mean value observed with the bioassay was 78.5 mg/liter and with our procedure was 69.4 mg/liter. When values for our assay were regressed against values for the bioassay, slope of the least-squares line was 0.85, intercept was 2.7 mg/liter, and r was 0.93.

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