Determination of Eupatilin in Human Plasma by High-Performance Liquid Chromatography–Tandem Mass Spectrometry

A rapid, sensitive and selective liquid chromatography-tandem mass spectrometric method (LC-MS-MS) for the determination of eupatilin in human plasma has been developed. Eupatilin and an internal standard; (S)-N-(3-{3-fluoro-4-[6-(1-methyl-1H-tetrazol-5-yl)-pyridine-3-yl]-phenyl}-2-oxo-oxazolidin-5-ylmethyl)-acetamide (DA-7867) were extracted from human plasma by liquid-liquid extractionand analyzed on a phenyl-hexyl column using the mobile phase: acetonitrile-ammonium formate (10 mM, pH 3.0) (60:40, /). Analytes were detected using electrospray ionization-tandem mass spectrometry in multiple-reaction monitoring mode. The calibration curve was linear (r = 0.999) over the concentration range: 1.00–500 ng mL^–1 with a lower limit of quantification of 1.0 ng mL^–1 using a 100 L plasma sample. The precision (CV%) of this assay ranged: 2.4–7.0%, relative error: –7.0 to +2.0%. Recoveries of eupatilin ranged: 64.3–65.0%, with that of DA-7867 (internal standard) being 87.0 ± 5.3%.     

Determination of Methylphosphonic Acid in Human Blood Plasma by High-Performance Liquid Chromatography–Tandem Mass Spectrometry

Using high-performance liquid chromatography combined with tandem mass spectrometric detection, an approach has been developed for the determination of the most stable nerve agent biomarker, methylphosphonic acid, in human blood plasma. The proposed method is based on the derivatization of methylphosphonic acid with p-bromophenacyl bromide. The optimization of conditions for human plasma sample preparation, mass spectrometric detection conditions, and gradient elution program has been performed. The proposed approach has demonstrated satisfactory reproducibility and selectivity of the determination; the limit of detection for methylphosphonic acid in human plasma was 3 ng mL^–1.     

Determination of Fluoxetine in Human Plasma by Liquid Chromatography–Mass Spectrometry and Its Application

Abstract

A rapid, simple, and specific liquid chromatography–electrospray ionization–mass spectrometric method has been developed and validated for the determination of fluoxetine in human plasma. The method was validated with a linear range of 0.5–100 ng mL^?1, and the lowest limits of quantification were 0.5 ng mL^?1 for fluoxetine. The extraction efficiencies were about 65% and recoveries of method were in the range of 94.0–97.5%. The intraday relative standard deviation (RSD) was less than 11% and interday RSD was within 12%. The method has been successfully applied to the evaluation of pharmacokinetics and bioequivalence of fluoxetine.     

Trace Determination of Manganese in Urine by Graphite Furnace Atomic Absorption Spectrometry and Inductively Coupled Plasma–Mass Spectrometry

This paper describes a simple and sensitive method for the determination of manganese in human urine by graphite furnace atomic absorption spectroscopy (GFAAS), which includes sample preparation by microwave digestion. Matrix modifier combinations, the digestion power, pyrolysis, and atomization temperatures were optimized. A mixture of 5.0 µg Pd(NO₃)₂ and 3.2 µg Mg(NO₃)₂ modifier presented the best performance. The optimal temperatures for pyrolysis and atomization were 1500°C and 1950°C, respectively. The GFAAS method was compared to inductively coupled plasma–mass spectrometry (ICP–MS) for the determination of manganese in urine. Analytical figures of merit for GFAAS and ICP–MS were: accuracy (3.46%, 2.19%), precision (3.61%, 5.84%), LOD (0.109 µg · L^?1, 0.015 µg · L^?1), LOQ (0.327 µg · L^?1, 0.045 µg · L^?1), and recovery (80–100%, 74–89%). Both methods were employed for the determination of Mn in urine and the results were compared statistically.     

Rapid Determination of Coumatetralyl in Human Serum by High‐Performance Liquid Chromatography Coupled with Electrospray Ionization Tandem Mass Spectrometry

Abstract

A rapid, sensitive, and selective high‐performance liquid chromatography‐tandem mass spectrometric method (HPLC‐MS‐MS) for the determination of coumatetralyl in human serum using warfarin as an internal standard has been developed and validated. Coumatetralyl and the internal standard were extracted from the human serum samples by liquid‐liquid extraction with ethyl acetate, followed by separation on a XDB C₁₈ reversed‐phase column (150 mm×2.1 mm i.d., 5 µm) using a mobile phase consisting of acetic acid‐ammonium acetate (5 mmol/L, pH=4.5)/methanol (20:80, v/v) at a constant flow rate of 0.40 mL/min. Coumatetralyl and the internal standard were ionized by negative ion pneumatically assisted electrospray and detected in the multiple‐reaction monitoring mode using precursor→product ion combinations at m/z 291→247 and 307→161, respectively. The calibration curve was linear (r²=0.9945) in the concentration range of 0.5～100.0 ng/mL, with a lower limit of quantification of 0.5 ng/mL in human serum. Intra‐ and inter‐day relative standard deviations were less than 6.3 and 11.0%, respectively. The mean extraction recovery was 87.9% for coumatetralyl and 90.1% for the internal standard. This method is found to be able to determine trace coumatetralyl in human serum and can be used for the diagnosis of poisoned human beings.     

Elucidation of O-Phosphoryl and N-Phosphoryl Amino Acids by Electrospray Ionization Tandem Mass Spectrometry

Mass spectroscopic characteristics of phosphoryl amino acids were studied in detail by positive and negative electrospray ionization mass spectrometry (ESI-MS) in conjunction with tandem mass spectrometry (MS/MS). Besides N-diisopropyloxyphosphoryl amino acids (N-DIPP-AA), O-phospho- and O-diisopropyloxyphosphoryl amino acids (O-DIPP-AA) were studied and compared to N-DIPP-AA. The fragmentation pathways of [M H]^ ,[M Na]^ and [M-H]^- ions of phosphoryl amino acids were summarized. In addition to several similar patterns, each of them showed its characteristic fragmention.     

Determination of Phytate in Urine by High-Performance Liquid Chromatography–Mass Spectrometry

An indirect method is described for determination of phytate in human urine. The method is based on hydrolysis of the phytate and determination of myo-inositol, one of the hydrolysis products. Chromatographic separations were performed on an Aminex HPX-87C column with Milli-Q water as mobile phase; 5 mM ammonium acetate was added post-column. The detector counted positive ions by monitoring m/z=198, which corresponds to the adduct of myo-inositol with the ammonium cation. The relative standard deviations obtained for standards containing 0.5, 1, and 1.5 mg L^–1 phytate were 4.1, 3.0, and 2.7% respectively (n=5). The limit of detection was 60 g L^–1. Different urine samples were analyzed both by this method and by an alternative analytical method based on GC–MS. The results from both methods were comparable.     

Novel Rearrangement of Small Peptides in Electrospray Ionization Tandem Mass Spectrometry

Electrospray ionization mass spectrometry (ESI-MS) is a powerful method for sequencing peptides. A novel fragmentation pattern with the loss of a neutral fragment of 45 Da was observed with the dipeptides, tripeptides,tetrapeptides and pentapeptides containing phenylalanine or histidine residues. A novel rearrangement reaction with the extrusion of a formamide piece was studied and the rearrangement mechanism was proposed and confirmed by deuterium labeling experiments with ESI-MS^n and high-resolution mass spectrometry. These findings are potentially helpful in identifying the specific sequence pattern in the peptide sequencing.     

Analysis of the Aconitine Alkaloids in Chuanwu by Electrospray Ionization ／ Tandem Mass Spectrometry

An electrospray ionization / tandem mass spectrometric (ESUMS/MS) method was developed for the simultaneous identification and analysis of three aconitine alkaloids [mesacontine (MA), hypaconitine (HA), and aconitine (A)] as intact molecules at low nanogram level in Chinese traditional medicine Chuanwu decoction as well as in human whole blood extract without chromatographic separation.     

Determination of Toluene and Ethanol in Urine by Headspace and Cryotrapping Gas Chromatography–Mass Spectrometry

Toluene is the major volatile organic compound found in glue and is often used as a hallucinogenic for abusers. Use with alcohol increases the risk of adverse effects from toluene exposure. In this study, a headspace and cryotrapping gas chromatography–mass spectrometry method was developed and validated for the determination of toluene and ethanol in urine. Experimental and instrumental variables were investigated to optimize the method for sensitivity. Excess sodium sulfate was used as the salting-out reagent before the headspace protocol. Linear least squares regression with a 1/x weighting factor was used to construct calibration curves from 0.002 to 0.4?µg?mL^?1 for toluene and 10 to 2000?µg?mL^?1 for ethanol. The correlation coefficients exceeded 0.9993. The limits of detection were 0.0005?µg?mL^?1 for toluene and 0.21?µg?mL^?1 for ethanol. Intraday and interday precisions were within 5.4 and 11.5%, while intraday and interday accuracies were between ?11.3 to ?4.0% and ?11.0 to 1.2%, respectively. The method validation results for selectivity and stability were satisfactory. The validation results were used to estimate the expanded uncertainty and the contribution of individual steps in the method for the quantification of toluene and ethanol. The relative expanded uncertainties were 14.1% for toluene and 4.6% for ethanol.     

Determination of Flavonoids in Stellaria by High-Performance Liquid Chromatography–Tandem Mass Spectrometry

The genus Stellaria (Caryophyllaceae) presents widely distributed plants often used in traditional medicine. Flavonoids are highly active plant secondary metabolites that may be involved in some of the effects of Stellaria plants. In this study, two new high-performance liquid chromatography–electrospray ionization tandem mass spectrometry (HPLC–ESI-MS-MS) methods were developed for the determination of flavonoids and isoflavonoids in plant material. The separations were performed on a reverse-phase C₁₈ column with gradient elution using methanol and water with 0.5% acetic acid as the mobile phase. Multiple reaction monitoring was used for the tandem mass spectrometry detection and the two most intensive transitions were chosen for the identification of each analyte. The limits of detection and the limits of quantification were between 0.2 and 15.0 ng/mL and 0.6 and 50.0 ng/mL. The developed methods were successfully used for the analyses of four representatives of the genus Stellaria. All the studied herbs contained luteolin and its 7-O-glycosides, naringenin, kaempferol, quercetin, its glycoside rutin, apigenin, and its 7-O-glycoside. One coumarin, scopoletin, was also found. Isoflavones were primarily represented by genistein, genistin, and ononin. Some of the analytes were detected for the first time in Stellaria sp. The findings support that these methods are suitable for analyses of plant material.     

Determination of Phenylethanoid Glycosides in Lagotis brevituba Maxim. by High-Performance Liquid Chromatography–Electrospray Ionization Tandem Mass Spectrometry

High performance liquid chromatography coupled with electrospray ionization quadrupole-time-of-flight tandem mass spectrometry was used to profile the phenylethanoid glycosides in Lagotis brevituba. A total of twenty-three phenylethanoid glycosides were characterized by comparing the retention time and fragmentation with standards, accurate mass measurements, and fragmentation at low and high collision energies. Most phenylethanoid glycosides were reported in L. brevituba for the first time. This established method may be employed for comprehensive quality control of L. brevituba.     

Liquid Chromatography‐Electrospray Ionization Mass Spectrometric Method for Determination of Gliclazide in Human Plasma

Abstract

A rapid, sensitive, and specific liquid chromatography‐electrospray ionization mass spectrometric (LC‐ESI‐MS) method has been developed for quantification of gliclazide in human plasma. The analyte and tolbutamide (internal standard, I.S.) were extracted from plasma samples with n‐hexane–dichloromethane (1:1, v/v) and analyzed on a C₁₈ column. The chromatographic separation was achieved within 4.0 min by using methanol–0.5% formic acid (80:20, v/v) as mobile phase and the flow rate was 1.0 mL/min. Ion signals m/z 324.0 and 271.0 for gliclazide and internal standard were measured in the positive mode, respectively. The method was linear within the range of 2.5–2000 ng/mL. The lower limit of quantification (LLOQ) was 2.5 ng/mL. The intra‐ and inter‐day precisions were lower than 2.8% in terms of relative standard deviation (RSD). The inter‐day relative error (RE) as determined from quality control samples (QCs) ranged from ?1.93% to 1.85%. This validated method was successfully applied for the evaluation of pharmacokinetic profiles of gliclazide modified‐release tablets in 20 healthy volunteers.     

Determination of Floridoside and Isofloridoside in Red Algae by High-Performance Liquid Chromatography–Tandem Mass Spectrometry

A facile method based on liquid chromatography coupled with triple quadrupole mass spectrometry was established to determine floridoside and isofloridoside in red algae. Correlation coefficients of the calibration curves were larger than 0.9989, indicated good linearity. Detection limits of floridoside and isofloridoside were 0.05 and 0.20 ng/mL, respectively, and the limits of quantification were 0.1 and 0.4 ng/mL. The recoveries varied from 75.7% to 76.8%, and relative standard deviations of inter-day and intra-day precision were lower than 8.5% (n = 5). The effects of sea level variations in the intertidal zone on the osmotic role of floridoside and isofloridoside concentrations in seven red algae were investigated. It was shown that algae that inhabit higher levels in the intertidal zone contained higher concentrations of floridoside and isofloridoside. The results suggest that the presence of direct sun, exposure time, and temperature influenced to the concentrations of floridoside and isofloridoside due to the osmotic pressure adjustments.     

Gas Chromatography–Mass Spectrometry Determination of Pregabalin in Human Plasma Using Derivatization Method

A gas chromatography–mass spectrometry method for the determination of pregabalin in human plasma is described. The procedure involves precipitation of protein, liquid–liquid extraction with ethylene glycol monomethyl ether, and derivatization with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide in the presence of N-hydroxysuccinimide as additive. Separation was attained on HP column (30 m × 0. 25 mm ID, 0.25 μm) coupled with mass spectrometric detector using electron impact selected ion monitoring. The assay showed an excellent linearity in the concentration range of 0.36–10 μg mL^?1 with correlation coefficient (r²) values of 0.999. The intra- and inter-day assay variations for three different concentration levels were less than 10%. The limit of quantification was detected at 0.36 μg mL^?1. The method is highly specific, precise, accurate, and reproducible and could also be applied for the determination of pregabalin in human plasma.     

Determination and Characterization of the Uptake of Voriconazole in Human Lung Epithelial Cells by High-Performance Liquid Chromatography–Tandem Mass Spectrometry

Voriconazole is used to prevent invasive pulmonary aspergillosis. However, little is known about the concentrations of voriconazole in human lung epithelial cells (A549), which is the target for preventing invasive pulmonary aspergillosis. The goal of this study was to develop a high-performance liquid chromatography–tandem mass spectrometry method to quantify voriconazole in A549 cells. A triple-quadrupole mass spectrometer in selected reaction monitoring mode was used with positive electrospray ionization. The total duration of each run was 5?min. The calibration curves fit a least squares model for the voriconazole concentration ranging from 0.625 to 160?ng/mL. Intraday and interday coefficients of variation were less than 10%. Recoveries at the concentrations of the quality control samples where greater than 85%, and the matrix effects showed that the ratios of the peak response exhibited a 15% suppression of the signal in the matrix compared to water. Voriconazole may penetrate A549 cells. However, the voriconazole uptake was slow in A549 cells, reaching a plateau at 2?h, where the dose-dependent intracellular voriconazole concentrations were 1.98?±?0.38, 4.43?±?0.54, and 8.14?±?0.52?ng/mg protein for extracellular voriconazole concentrations of 5, 10, and 20?µg/mL, respectively. The uptake of voriconazole by A549 cells was linear at extracellular concentrations from 0 to 20?µg/mL. This study established a rapid and sensitive method suitable for determining voriconazole in A549 cells and described the kinetic properties of the absorption of voriconazole by A549 cells.     

Determination of Metformin in Human Plasma by Liquid Chromatography-tandem Mass Spectrometric Assay

Metformin (1,1-dimethylbiguanide) (Fig. 1) i san oral anti-hyperglycemic agent used in the treatment of non-insulin-dependent diabetes mellitus(type Ⅱ ). Owing to its weight-decreasing and serum lipid-normalizing effects, it is especially recommended for obese patients. Various analytical methods have been described for the measurement of metformin in biological fluids,     

Determination of Microcystins in Cyanobacteria by Supercritical Fluid Extraction and Liquid Chromatography‐Tandem Mass Spectrometry

Abstract

A method for the detection of microcystins (microcystin LR, RR, and YR) in cyanobacteria by supercritical fluid extraction (SFE) and liquid chromatography‐mass spectrometry (LC/MS) has been developed. Supercritical fluids for the analytical extraction of nonvolatile, higher molecular weight compound, and microcystins from cyanobacteria were investigated. The microcystins included in this study are sparsely soluble in neat supercritical fluid CO₂. However, the microcystins was successfully extracted with a ternary mixture (90% CO₂, 9.5% methanol, 0.5% water) at 40°C and 250 atm. The polar carbon dioxide‐aqueous methanol fluid system gave high extraction efficiency for the extraction of the polar microcystins from cyanobacteria. The microcystins were determined by liquid chromatography‐tandem mass spectrometry (LC/MS/MS).     

Simultaneous Determination of Flonicamid and its Metabolites in Tea by Liquid Chromatography–Tandem Mass Spectrometry

An analytical method was established to simultaneously quantify flonicamid and its metabolites 4-trifluoromethylnicotinic acid (TFNA), N-(4-trifluoromethylnicotinoyl) glycine (TFNG), and 4-trifluoromethylnicotinamide (TFNA-AM) in tea using orthogonal experimental design and liquid chromatography–tandem mass spectrometry (LC–MS/MS). Residues were extracted from the samples with acetonitrile containing 1% acetic acid and were purified with graphitized carbon black. The linearity of the method was excellent in the concentration range of 0.01–10?µg/mL, producing correlation coefficients greater than 0.996 for the target compounds. The limits of detection and quantification of all analytes in tea were 0.0013–0.013?mg/kg and 0.004–0.040?mg/kg, respectively. The average recoveries of flonicamid, TFNA, TFNG, and TFNA-AM ranged from 75.14 to 92.72%, with intra- and interday relative standard deviations of 1.07–9.75%. The proposed method was successfully applied to the terminal residue determination of flonicamid and its metabolites in dry tea processed from three field trials’ fresh samples. The determined total terminal residue concentrations of flonicamid 10?days after the last application at all three sites were below the maximum residue limit (MRL) set by the European Union (0.1?mg/kg) and the residues in all samples were lower than the MRL established by the United States Environmental Protection Agency (EPA) (8?mg/kg). This method may be used to meet the requirements for the determination of flonicamid and its metabolites that could provide guidance for establishing a MRL for flonicamid in tea in China.     

Simultaneous Determination of Free Cortisol,Cortisone and their Tetrahydrometabolites in Urine by Single Solvent Extraction and Liquid Chromatography–Tandem Mass Spectrometry

Abstract

A fast, efficient and low-cost high performance liquid chromatography–tandem mass spectrometry methodology was developed and validated for the simultaneous determination of free urinary cortisone, cortisol and their tetrahydro-metabolites. The developed method comprises a simple liquid-liquid extraction with CH₂Cl_2, followed by reversed-phase liquid chromatography–tandem mass spectrometry (LC–MS/MS) with electrospray ionization (ESI) in positive mode. The baseline chromatographic separation of the analytes, including the stereoisomers tetrahydrocortisol (THF) and allo-THF, was achieved on a Hypersil Gold C₁₈ column with a mobile phase consisting of 0.05%v/v formic acid in water—acetonitrile, using a gradient elution program. The influence of the mobile phase composition and the ESI parameters on the sensitivity of the method was extensively studied. Sample preparation was also optimized, testing two techniques: solid phase extraction (SPE) and liquid-liquid extraction (LLE). Recoveries ranged from 74.7% (a-THF) to 93.5% (cortisol) and the method limits of detection (MLD) ranged from 0.34?ng mL^?1 (cortisol) to 1.37?ng mL^?1 (THF). Intra- and inter-day coefficient of variation of the assay varied from1.5% (allo-THF) to 13% (tetrahydrocortisone) and from 3.6% (allo-THF) to 14.9% (tetrahydrocortisone), respectively. The method was applied for the analysis of urine samples from 53 healthy individuals with a mean age of 13.96?years in order to estimate the concentration of the five corticosteroids and the ratio of the metabolites. Associations between urinary cortisol/cortisone and serum cortisol/cortisone values were also characterized.