LC–MS–MS Separation and Simultaneous Determination of Tolterodine and its Active Metabolite, 5-Hydroxymethyl Tolterodine in Human Plasma

A selective, sensitive and high throughput LC–MS–MS method has been developed and validated for the chromatographic separation and quantitation of tolterodine (TOL) and its metabolite 5-hydroxymethyl TOL in human plasma. Sample clean-up concerned liquid–liquid extraction of the drug, metabolite and their respective labelled internal standards from 300 μL human plasma. Both the analytes were chromatographically separated on a Symmetry C18 (100 mm × 4.6 mm, 5 μm particle size) analytical column using 10 mM ammonium formate (pH 5.0 ± 0.1, adjusted with formic acid) and acetonitrile (35:65, v/v) as the mobile phase with a resolution factor of 2.72. The method was validated over the concentration range of 0.025–10.0 ng mL^?1 for both analytes. The process efficiency found for TOL and its metabolite was 98.3 and 99.5%, respectively. The method was successfully applied to a pivotal bioequivalence study in 41 healthy human subjects after oral administration of a 2 mg tablet formulation under fasting conditions.     

LC–MS–MS Determination of Stabilizers and Explosives Residues in Hand-Swabs

The contribution of explosive trace detection in samples from the hands of suspects has been fundamental in several forensic cases involving terrorists. This paper describes a method for the rapid extraction and unequivocal confirmation of some high potential explosives (trinitrotoluene, cyclotrimethylenetrinitramine, pentaerythritol tetranitrate, nitroglycerin) and two stabilizer (diphenylamine and ethylcentralite) residues in hand-swabs using liquid chromatography–tandem mass spectrometry. The extraction procedure of the analytes from the swabs is realized by solvent elution and the extracts are directly analyzed. Recoveries from spiked swabs range from 78 to 96%; the limits of quantification are between 0.04 and 1.8 ng injected and the inter-day method precision is less than 15%. The developed procedure was applied to the detection of explosives traces in samples after handling tests.     

Detection and Confirmation of Ginsenosides in Horse Urine by GC–MS and LC–MS

Ginseng has been used by the Chinese as a traditional herbal medicine for thousands of years. In view of the growing popularity in the use of ginseng preparations as natural remedies and food supplements worldwide, there is an increasing concern for their abuse in both human and animal sports. Ginsenosides are considered the major constituents of ginseng responsible for its pharmacological properties. In this study, a method was developed for the detection and confirmation of a number of ginsenosides in horse urine. The intact ginsenosides were detected and confirmed at 5–100 ng mL^?1 by LC–MS², and two deglycosylation metabolites, namely protopanaxadiol and protopanaxatriol, could both be detected and confirmed at 2 ng mL^?1 by GC–MS² after trimethylsilylation. The above GC–MS and LC–MS methods were then applied to study the in vitro metabolism of ginsenosides Rg1 and Rb1 and the in vivo urinary metabolites after oral administration of Rg1 to horses. Results obtained reveal the very first evidence for the existence of the metabolites, Rg1 and protopanaxatriol, as glucuronides in urine.     

Establishment of fingerprint of Gegen Qinlian decoction and its formula compatibility groups using UHPLC–MS/MS and its study to spectrum–effect relationship

Based on the establishment of analytical method of ultra-high performance liquid chromatography-tandem mass spectrometry fingerprint and obtaining pharmacodynamic information of the effects on IL-4, IL-10, TNF-α, and IFN-γ of serum and interstitial fluid of the damp-type ulcerative colitis mice of Gegen Qinlian decoction and its formula compatibility groups, the spectrum–effect relationship study was performed by using the method of principal component analysis. The study built the common pattern of UHPLC–MS/MS fingerprint of Gegen Qinlian decoction and formula compatibility groups and identified eight components which were rooted in monarch and subject drugs and represented the whole pharmacodynamic information of Gegen Qinlian decoction. The research on formula compatibility and spectrum–effect relationship can provide scientific basis for revealing pharmacodynamic material basis and compatibility regularity of Gegen Qinlian decoction.     

Determination of Dothiepin in Human Plasma by LC–ESI–MS and its Application to Bioequivalence Studies

An LC–MS method for the determination of dothiepin in human plasma was developed and validated. Sample preparation involved extraction with n-hexane:2-propanol (95:5). Separation was on an Ultimate XB C18 column (2.1 × 150 mm, 5 μm). A single-quadrupole mass spectrometer with an electrospray interface was operated in the selected-ion monitoring mode to detect the [M+H]⁺ ions at m/z 296 for dothiepin and at m/z 278 for the internal standard (amitriptylene). The method demonstrated good linearity from 0.78 ng mL^?1 (the LOQ) to100 ng mL^?1. The mean extraction recovery was 82.4% for dothiepin and and 84.2% for the internal standard. The intra-day and inter-day precision ranged from 8.5 to 11.4% and 9.7 to 12.1% (RSD), respectively. The method was successfully applied to bioequivalence studies of dothiepin hydrochloride tablets to obtain the pharmacokinetic parameters.     

Development and validation of novel LC–MS/MS method for determination of Lusutrombopag in rat plasma and its application to pharmacokinetic studies

A novel, simple and sensitive method for the determination of Lusutrombopag in rat plasma using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was developed and validated. The determination was performed on an API4000 triple quadrupole mass spectrometry in the multiple reaction monitoring mode using the respective [M+H]⁺ ions m/z 593.1 → 272.3 for Lusutrombopag. The limit of detection was 0.5 ng/mL, and the lower limit of quantification was 2.0 ng/mL in rat plasma. Good linearity was obtained over the range of 2.0–150.0 ng/mL and the correlation coefficient was found to be 0.9998. The intra and inter-day precisions were found to be 3.8–6.9% and 6.8–10.5%, respectively. The intra and inter-day accuracy derived from QC samples was found to be 2.5–4.9% and 5.5–7.2%, respectively. The analyte was stable under various conditions (at room temperature, during freeze-thaw, in the autosampler and under deep-freeze conditions). The F-test and t-test at 95% confidence level were subjected on data for statistical analysis. The developed method was successfully applied to the pharmacokinetic study in rats.     

Radiodegradation of nadolol in the solid state and identification of its radiolysis products by UHPLC–MS method

Nadolol ((2R*,3S*)-5-{[(2R*)-3-(tert-butylamino)-2-hydroxypropyl]oxy}-1,2,3,4 tetrahydronaphthalene-2,3-diol) in substantia was exposed to ionizing radiation generated by a beam of high-energy electrons in an accelerator, in the standard sterilisation dose of 25 kGy and in higher doses of 50 ? 400 kGy. The irradiated and non-irradiated (control) samples were analysed by the infrared spectrophotometry, electron paramagnetic resonance, differential scanning calorimetry (DSC) and ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC–MS). The irradiated samples were found to contain free radicals in concentrations much higher than that observed for the other irradiated β-blockers. On the basis of UHPLC–MS results, it was possible to establish structures of 11 compounds of the impurities and/or products of nadolol decomposition. The main product of radiodegradation was concluded to be formed as a result of abstraction of the hydroxyl group and aromatization of the tetrahydronaphthalene ring. The results of DSC measurements confirmed the presence of radiolysis products in the irradiated samples of nadolol. A shift of the endothermic peak corresponding to melting towards lower temperatures (by 4.4 °C at the dose of 400 kGy) was directly proportional to the doses of radiation used, which permits concluding that this method is sensitive and suitable for evaluation of radiodegradation of nadolol in solid phase.     

Assessment of robustness for an LC–MS–MS multi-method by response-surface methodology,and its sensitivity

Sensitive, fast, and robust multi-methods are required for the surveillance of the contamination of the drinking water resources by organic trace contaminants. In the present work an alternative strategy using response surface methodology (RSM) was applied for assessment of the robustness of a LC–MS–MS multi-method. The analytical method was optimised by means of a central composite design including six design variables. The main object was to evaluate the significance of the RSM results with regard to robustness and to the sensitivity to the mass transitions used in the multi-method. The robustness of the multi-method was represented by the curvature of the calculated response surfaces for the response value R. Furthermore, it could be demonstrated that the RSM was sensitive to changes made to the investigated data set and was able to clearly indicate the fraction of substances, which met the defined criterion for signal-to-noise-ratio.     

Simultaneous Determination of Tamsulosin and Dutasteride in Human Plasma by LC–MS–MS

A sensitive and selective liquid chromatographic tandem mass spectrometric (LC–MS–MS) method was developed for simultaneous identification and quantification of tamsulosin and dutasteride in human plasma, which was well applied to clinical study. The method was based on liquid–liquid extraction, followed by an LC procedure with a Gemini C-18, 50 mm × 2.0 mm (3 μm) column and using methanol:ammonium formate (97:3, v/v) as the mobile phase. Protonated ions formed by a turbo ionspray in positive mode were used to detect analytes and internal standard. MS–MS detection was by monitoring the fragmentation of 409.1 → 228.1 (m/z) for tamsulosin, 529.3 → 461.3 (m/z) for dutasteride and 373.2 → 305.3 (m/z) for finasteride (IS) on a triple quadrupole mass spectrometer. The lower limit of quantification for both tamsulosin and dutasteride was 1 ng mL^?1. The proposed method enables the unambiguous identification and quantification of tamsulosin and dutasteride for clinical drug monitoring.     

Multiresidue Analysis of Pesticides in Vegetables and Citrus Fruits by LC–MS–MS

In this work, an analytical multiresidue method using liquid chromatography tandem-mass spectrometry (LC–MS–MS) with triple quadrupole in selected reaction monitoring (SRM) mode for the simultaneous determination of 54 pesticides in vegetables (pepper and tomato) and citrus fruits (orange and lemon) has been developed. The procedure involves initial single phase extraction of sample with acetonitrile by agitation, followed by liquid–liquid partition aided by “salting out” process using NaCl. The average recovery by the LC–MS–MS method obtained for these compounds varied from 65.5 to 114.5% with a relative standard deviation between 2.3 and 8.3%. The method presents good linearity over the range assayed 10–500 μg L^?1 (except famoxadone 50–1,000 μg L^?1) and the detection limits for the pesticides studied varied from 0.03 to 14.9 μg kg^?1. The proposed method was used to determine pesticide levels in vegetables and citrus fruit samples from different experimental orchards and greenhouses from the Region of Murcia.     

Simultaneous Determination of Atenolol and Chlorthalidone by LC–MS–MS in Human Plasma

A simple, sensitive, selective and rapid liquid chromatography–tandem mass spectrometry method was developed and validated for the simultaneous separation and quantitation of atenolol and chlorthalidone in human plasma using metoprolol and hydrochlorothiazide as internal standard. Following solid phase extraction, the analytes were separated by an isocratic mobile phase on a reversed-phase C₁₈ column and analyzed by MS in the multiple reaction-monitoring mode (atenolol in positive and chlorthalidone in the negative ion mode). The limit of quantitation for this method was 10 and 15 ng mL^?1 and the linear dynamic range was generally 10–2,050 ng mL^?1 and 15–3,035 ng mL^?1 for atenolol and chlorthalidone, respectively.     

Py–GC–MS examination of intermediates in the vapor from rapid pyrolysis of larch wood and its model components

Py–GC–MS was used to examine the components of vapor from rapid pyrolysis of larch wood and its model components, i.e. example cellulose, xylan, and lignin, and their mixture in accordance with the proportion of the components in larch wood. In this study, a total of 97 compounds in 12 categories were identified in the pyrolysis vapor and were compared. It was found that the most abundant chemical species in these five types of pyrolysis vapor were different. Saccharides and ketones were the major compounds in the pyrolysis vapor from microcrystalline cellulose and xylan, respectively, whereas the most abundant compounds in the pyrolysis vapor from alkaline lignin were sulfur compounds and phenols. Saccharides and ketones were major components of the pyrolysis vapor from MMC, whereas the main compounds in the pyrolysis vapor from larch wood were ketones, phenols, aldehydes, and saccharides. The different composition of the pyrolysis vapor from larch wood and its model mixture was explained on the basis of their different structural frameworks and the non-structural components of larch wood. It was also concluded that the presence of non-structural components, including extractives and ash, affect the pyrolysis reaction of larch wood. Nevertheless, the detailed patterns of this process must be further studied.     

Sensitive Determination of Felodipine in Human and Dog Plasma by Use of Liquid–Liquid Extraction and LC–ESI–MS–MS

Summary A sensitive and selective liquid chromatographic method coupled with electrospray ionization tandem mass spectrometry (LC–ESI–MS–MS) has been developed for quantification of felodipine in human and dog plasma. Compounds were separated on a 2.0 mm × 150 mm, 5.0 m particle, C₈ column with 1 m m ammonium acetate–acetonitrile, 20:80, pH 6.0, as mobile phase at a flow rate of 200 L min^–1. Nifedipine was used as internal standard. Plasma samples were extracted with diethyl ether, the centrifuged upper layer was evaporated, the residue was reconstituted with mobile phase, and the reconstituted samples were injected. The analytical column lasted for at least 1000 injections. By use of multiple reaction monitoring (MRM) mode in MS–MS felodipine and nifedipine were detected without severe interference from the human or dog plasma matrix. Felodipine produced a protonated precursor ion ([M + H]⁺) at m/z 384 and a corresponding product ion at m/z 338. And internal standard (nifedipine) produced a protonated precursor ion ([M + H]⁺) at m/z 347 and a corresponding product ion at m/z 315. Detection of felodipine in human and dog plasma was accurate and precise, with a limit of quantification of 0.05 ng mL^–1. The method has been successfully applied to preliminary pharmacokinetic study of felodipine in human and dog plasma.     

Determination of Pentachloronitrobenzene and Its Metabolites in Ginseng by Matrix Solid-Phase Dispersion and GC–MS–MS

A rapid method was developed for the determination of pentachloronitrobenzene (PCNB) and its metabolites pentachloroaniline, pentachlorothioanisole residues in ginseng. Extraction and clean-up were carried out in a single step and analysis was accomplished by gas chromatography–mass spectrometry with multiple reaction monitoring. The main parameters affecting extraction yield and selectivity, such as type and amount of dispersant material, clean-up co-sorbent and extraction solvent were evaluated. The best results were obtained using 1 g ginseng, 2 g florisil as dispersant sorbent, 0.5 g neutral alumina as clean-up co-sorbent, and subsequent extraction with 10 mL acetone–n-hexane (5:5, v/v) with assisted sonication and repeated with another 5 mL of the same solvent mixture. The method was validated by analysis of ginseng samples fortified at different concentration levels (0.01–0.10 mg kg^?1). Average recoveries (n = 5) ranged from 85 to 95% with relative standard deviation between 2.5 and 11.2%. Spiked blank samples were used as standards to counteract the matrix effect observed in the chromatographic determination. The detection limits ranged from 0.2 to 0.9 µg kg^?1 in ginseng. The method was applied to the analysis of PCNB and its metabolite residues in commercial ginseng samples.     

LC–MS–MS Analysis of Mirtazapine in Plasma, and Determination of Pharmacokinetic Data for Rats

A sensitive LC–MS–MS method with electrospray ionization has been developed for analysis of mirtazapine in rat plasma. After addition of diazepam as internal standard, liquid–liquid extraction was used to produce a protein-free extract. Chromatographic separation was achieved on a 150 × 4.6 mm, 5 μm particle, ODS column with 84:16 (v/v) methanol–water containing 0.1% ammonium acetate and 0.01% glacial acetic acid as mobile phase. LC–MS–MS was performed in selected-ion-monitoring (SIM) mode using target fragment ions m/z 195.09 for mirtazapine and m/z 192.80 for the IS. Calibration plots were linear over the range of 0.516–618.8 ng mL^?1. The lower limit of quantification was 0.516 ng mL^?1. Intra-day and inter-day precision were better than 12.6 and 8.8%, respectively. Mean recovery of mirtazapine from plasma was in the range 87.41–90.06%; average recovery was 88.40% (RSD 3.95%). Significant gender differences between mirtazapine pharmacokinetic data were observed in this study.     

LC–MS–MS Determination of Nikethamide in Human Plasma

A sensitive LC–MS–MS method with electrospray ionization has been developed for determination of nikethamide in human plasma. After addition of atropine as internal standard, liquid–liquid extraction was used to produce a protein-free extract. Chromatographic separation was achieved on a 150 mm × 2.1 mm, 5 μm particle, Agilent Zorbax SB-C₁₈ column, with 45:55 (v/v) methanol–water containing 0.1% formic acid as mobile phase. LC–MS–MS was performed in multiple reaction monitoring mode using target fragment ions m/z 178.8 → 107.8 for nikethamide and m/z 289.9 → 123.8 for the internal standard. Calibration plots were linear over the range of 20.0–2,000 ng mL^?1. The lower limit of quantification was 20.0 ng mL^?1. Intra-day and inter-day precisions were better than 4.2 and 6.1%, respectively. Mean recovery of nikethamide from human plasma was in the range 65.3–71.1%.     

Application of CE–ICP–MS and CE–ESI–MS in metalloproteomics: challenges,developments, and limitations

Application of capillary electrophoresis (CE) as a high-resolution separation technique in metalloproteomics research is critically reviewed. The focus is on the requirements and challenges involved in coupling CE to sensitive element and molecule-specific detection techniques such as inductively coupled plasma mass spectrometry (ICP–MS) or electrospray ionisation mass spectrometry (ESI–MS). The complementary application of both detection techniques to the structural and functional characterisation of metal-binding proteins and their structural metal-binding moieties is emphasised. Beneficial aspects and limitations of mass spectrometry hyphenated to CE are discussed, on the basis of the literature published in this field over the last decade. Recent metalloproteomics applications of CE are reviewed to demonstrate its potential and limitations in modern biochemical speciation analysis and to indicate future directions of this technique.     

Speciation of aluminium in tea infusions by use of SEC and FPLC with ICP–OES and ES–MS–MS detection

Speciation of Al in tea infusions was studied by size exclusion chromatography (SEC) and anion-exchange fast protein liquid chromatography (FPLC). Fractions were collected throughout the chromatographic separations and Al was determined “off line” by inductively coupled plasma optical emission spectroscopy (ICP–OES). Black, green, and red tea samples were investigated. The total concentration of Al in tea infusions was determined by ICP–OES and ranged between 0.5 and 4 mg dm⁻³. The pH of tea infusions ranged between 5.3 and 5.5. Data from SEC–ICP–OES analysis indicated that 10–35% of total Al in tea infusions was eluted at a retention volume corresponding to a molecular mass of approximately 3800 Da. The remaining Al was adsorbed on the column resin. The same tea infusions were also analysed by anion-exchange FPLC–ICP–OES. It was found experimentally that the same percentage of total Al as from the SEC column was eluted at a retention volume that corresponded to negatively charged Al-citrate. The remaining Al was adsorbed on the column resin. Identification of Al-binding ligands eluting under the chromatographic peak was performed by electrospray ionisation tandem mass spectrometry (ES–MS–MS) analysis. It was proven that ionic Al species in tea infusions (10–35% of the total Al) corresponded to negatively charged Al-citrate. The remaining species that was adsorbed on the SEC or FPLC columns was most probably bound to phenolic compounds. Speciation of Al in tea with milk or lemon was also studied. Results for tea with milk indicated that Al-citrate was not transformed and that approximately 60% of total Al was transformed into high-molecular-mass Al species. This fraction was subjected to sodium dodecyl sulfonate polyacryl gel electrophoresis (SDS–PAGE). The results indicated that Al was occluded by milk proteins (mostly caseins). When citric acid was added to tea infusions the percentage of negatively charged Al-citrate remained either the same or increased to 40% of total Al.