Pressure Ionization Tandem Mass Spectrometry Research Articles

Ultra-high-performance liquid chromatography-atmospheric pressure ionization-tandem mass spectrometry method for the migration studies of primary aromatic amines from food contact materials

This work describes the development of an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the determination of 23 primary aromatic amines (PAAs) that can potentially migrate from food contact materials. The chromatographic separation was performed in a pentafluorophenylpropyl (PFPP) column achieving the separation of all PAAs in less than 6.5 min using water to acetonitrile (0.1% acetic acid in both solvents) as mobile phase and a gradient elution. The feasibility of atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) was evaluated as alternative to electrospray ionization (ESI) for the analysis of PAAs. Results showed that for most of the compounds, better responses were obtained with APCI, which shows the advantage of being less susceptible to matrix effects. Tandem mass spectrometry (MS/MS) fragmentation studies of [M + H]+ allowed for the selection of the two most characteristic and abundant product ions of the 23 PAAs which led to the development of a selective and sensitive UHPLC-APCI-MS/MS method with limits of detection ranging from 0.2 to 2 μg kg−1. Moreover, intra-day and inter-day precisions of the method in terms of relative standard deviation (RSD%) were lower than 10% and 15%, while trueness as relative error was <15% for most of the compounds. The UHPLC-APCI-MS/MS method was applied to the analysis of twenty black Nylon kitchenware samples that were submitted to migration tests using food simulant B (3% acetic acid, w/v), and the presence of PAAs were detected in eighteen samples at concentrations above the legislated limit (2 μg kg−1 of food or food simulants).Graphical abstract

Determination of banned dyes in red spices by ultra-high-performance liquid chromatography-atmospheric pressure ionization-tandem mass spectrometry

This paper reports the development of an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC‒MS/MS) method to determine eight banned dyes (Sudan I-IV, Sudan Orange, Sudan Red 7B, Para Red, Rhodamine B) in turmeric, curry, and chili products. For this purpose, the feasibility of electrospray (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) for the ionization of these compounds was evaluated. The tandem mass spectrometry (MS/MS) fragmentation of all targeted compounds was studied and both multistage mass spectrometry and high-resolution mass spectrometry were used to establish the fragmentation pathways and identify common fragmentation behaviors. Among the most significant ions, the most characteristic and abundant product ions observed on the triple quadrupole were selected to propose a selective and sensitive UHPLC‒MS/MS method (multiple reaction monitoring mode, MRM) of these target compounds in spices samples after a quick and easy extraction with acetonitrile. Matrix effect (ME) studies carried out in the three atmospheric pressure ionization sources have demonstrated that APCI showed the best performance with ME values ranging from 2 to 25%. Furthermore, the estimated quality parameters indicated the good performance of the proposed method, providing low method limits of detection (MLODs) (1–48 μg kg−1), good intra-day precision (RSD % < 15%), and accurate quantitation (relative error % < 15%). Finally, the applicability of the developed method was demonstrated by the analysis of turmeric, curry, and chili products. In total, 36 diverse samples coming from different countries were analyzed and although none of these compounds were detected above the MLODs, the analysis of spiked samples showed that the method was able to detect this family of compounds at low μg kg−1.

Determination of veterinary antibiotics in dairy manure slurry by LC-MS/MS

Veterinary antibiotics are used in the dairy animal for therapeutic purposes to better absorb nutrients and treat different diseases. However, developments of resistance against veterinary antibiotics which is generally disposed into the agricultural lands have been reported as a considerable concern. Therefore, this study was conducted to determine the veterinary antibiotics in dairy manure slurry obtained from three selected farms in various districts in the Marmara Region of Turkey. This study examined the quantification of monensin, lincomycin, amprolium and salinomycin in dairy manure samples by a liquid chromatography triple quadrupole mass spectrometry (LC-MS/MS). Atmospheric pressure ionization-tandem mass spectrometry was utilized on a benchtop triple quadrupole mass spectrometer operated in electrospray ionization (ESI) mode to analyze manure slurry samples. The procedures of the extraction and recovery studies were also stated.

Quantitative Analysis of Mixed Halogen Dioxins and Furans in Fire Debris Utilizing Atmospheric Pressure Ionization Gas Chromatography-Triple Quadrupole Mass Spectrometry.

Residential and commercial fires generate a complex mixture of volatile, semivolatile, and nonvolatile compounds. This study focused on the semi/nonvolatile components of fire debris to better understand firefighter exposure risks. Using the enhanced sensitivity of gas chromatography coupled to atmospheric pressure ionization-tandem mass spectrometry (APGC-MS/MS), complex fire debris samples collected from simulation fires were analyzed for the presence of potentially toxic polyhalogenated dibenzo-p-dioxins and dibenzofurans (PXDD/Fs and PBDD/Fs). Extensive method development was performed to create multiple reaction monitoring (MRM) methods for a wide range of PXDD/Fs from dihalogenated through hexa-halogenated homologue groups. Higher halogenated compounds were not observed due to difficulty eluting them off the long column used for analysis. This methodology was able to identify both polyhalogenated (mixed bromo-/chloro- and polybromo-) dibenzo-p-dioxins and dibenzofurans in the simulated burn study samples collected, with the dibenzofuran species being the dominant compounds in the samples. Levels of these compounds were quantified as total homologue groups due to the limitations of commercial congener availability. Concentration ranges in household simulation debris were observed at 0.01-5.32 ppb (PXDFs) and 0.18-82.11 ppb (PBDFs). Concentration ranges in electronics simulation debris were observed at 0.10-175.26 ppb (PXDFs) and 0.33-9254.41 ppb (PBDFs). Samples taken from the particulate matter coating the firefighters' helmets contained some of the highest levels of dibenzofurans, ranging from 4.10 ppb to 2.35 ppm. The data suggest that firefighters and first responders at fire scenes are exposed to a complex mixture of potentially hundreds to thousands of different polyhalogenated dibenzo-p-dioxins and dibenzofurans that could negatively impact their health.

Application of atmospheric pressure ionization techniques and tandem mass spectrometry for the characterization of petroleum components

Atmospheric solid analysis probe (ASAP) and atmospheric pressure gas chromatography (APGC) are relatively new ionization techniques. In this study, we applied these two techniques to characterize complex petroleum fraction samples under atmospheric pressure. The ion formation of petroleum standards (paraffins, isoparaffins, naphthenes and aromatic hydrocarbons) in ASAP and APGC was investigated. Two primary ionization pathways were observed, nitrogen fixation (addition) and charge transfer. Paraffins and isoparaffins standards are primarily ionized by nitrogen addition. Cyclic paraffins and aromatic compounds are primarily ionized by charge transfer. ASAP was applied to analyze a saturate fraction of petroleum vacuum resid under atmospheric pressure. An expanded composition coverage was demonstrated. APGC coupled with tandem mass spectrometry was demonstrated to characterize biomarkers in complex petroleum samples with high specificity, providing similar information as compared to the traditional gas chromatography electron ionization (GC–EI) under vacuum.

Products of the OH Radical-Initiated Reactions of Furan, 2- and 3-Methylfuran, and 2,3- and 2,5-Dimethylfuran in the Presence of NO

Products of the gas-phase reactions of OH radicals with furan, furan-d4, 2- and 3-methylfuran, and 2,3- and 2,5-dimethylfuran have been investigated in the presence of NO using direct air sampling atmospheric pressure ionization tandem mass spectrometry (API-MS and API-MS/MS), and gas chromatography with flame ionization and mass spectrometric detectors (GC-FID and GC-MS) to analyze samples collected onto annular denuders coated with XAD solid adsorbent and further coated with O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine for derivatization of carbonyl-containing compounds to their oximes. The products observed were unsaturated 1,4-dicarbonyls, unsaturated carbonyl-acids and/or hydroxy-furanones, and from 2,5-dimethylfuran, an unsaturated carbonyl-ester. Quantification of the unsaturated 1,4-dicarbonyls was carried out by GC-FID using 2,5-hexanedione as an internal standard, and the measured molar formation yields were: HC(O)CH═CHCHO (dominantly the E-isomer) from OH + furan, 75 ± 5%; CH3C(O)CH═CHCHO (dominantly the E-isomer) from OH + 2-methylfuran, 31 ± 5%; HC(O)C(CH3)═CHCHO (a E-/Z-mixture) from OH + 3-methylfuran, 38 ± 2%; and CH3C(O)C(CH3)═CHCHO from OH + 2,3-dimethylfuran, 8 ± 2%. In addition, a formation yield of 3-hexene-2,5-dione from OH + 2,5-dimethylfuran of 27% was obtained from a single experiment, in good agreement with a previous value of 24 ± 3% from GC-FID analyses of samples collected onto Tenax solid adsorbent without derivatization.

Atmospheric pressure ionization-tandem mass spectrometry of the phenicol drug family

In this work, the mass spectrometry behaviour of the veterinary drug family of phenicols, including chloramphenicol (CAP) and its related compounds thiamphenicol (TAP), florfenicol (FF) and FF amine (FFA), was studied. Several atmospheric pressure ionization sources, electrospray (ESI), atmospheric pressure chemical ionization and atmospheric pressure photoionization were compared. In all atmospheric pressure ionization sources, CAP, TAP and FF were ionized in both positive and negative modes; while for the metabolite FFA, only positive ionization was possible. In general, in positive mode, [M + H](+) dominated the mass spectrum for FFA, while the other compounds, CAP, TAP and FF, with lower proton affinity showed intense adducts with species present in the mobile phase. In negative mode, ESI and atmospheric pressure photoionization showed the deprotonated molecule [M-H](-), while atmospheric pressure chemical ionization provided the radical molecular ion by electron capture. All these ions were characterized by tandem mass spectrometry using the combined information obtained by multistage mass spectrometry and high-resolution mass spectrometry in a quadrupole-Orbitrap instrument. In general, the fragmentation occurred via cyclization and losses or fragmentation of the N-(alkyl)acetamide group, and common fragmentation pathways were established for this family of compounds. A new chemical structure for the product ion at m/z 257 for CAP, on the basis of the MS(3) and MS(4) spectra is proposed. Thermally assisted ESI and selected reaction monitoring are proposed for the determination of these compounds by ultra high-performance liquid chromatography coupled to tandem mass spectrometry, achieving instrumental detection limits down to 0.1 pg.

Quantitative analysis of five tobacco‐specific N‐nitrosamines in urine by liquid chromatography–atmospheric pressure ionization tandem mass spectrometry

A liquid chromatography tandem mass spectrometry (LC/MS/MS) method was developed and validated for the determination of five total tobacco-specific N-nitrosamines (TSNA), including free and conjugated forms in urine. The limits of detection for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol, N'-nitrosonornicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, N'-nitrosoanatabine and N'-nitrosoanabasine were 0.6, 0.6, 10.0, 0.4 and 0.4 pg/mL, respectively, with a linear calibration range of up to 20,000 pg/mL. Intra- and inter-day precision for TSNA measurements ranged from 0.82 to 3.67% and from 2.04 to 7.73% respectively. For total TSNAs, the β-glucuronidase amount was optimized for hydrolysis time and yield. Different liquid chromatography columns and mobile phases with different pH conditions were evaluated. The validated method was then applied to 50 smoker and 30 nonsmoker urine samples. Our results suggest that this sensitive and relatively simple analytical method is suitable for application to epidemiological investigations of health risks associated with the exposure to tobacco smoke or secondhand smoke in both smokers and nonsmokers.

Simultaneous Determination of Cyclosporine A, Tacrolimus, Sirolimus, and Everolimus in Whole-Blood Samples by LC-MS/MS

Objectives. Cyclosporine A (CyA), tacrolimus (TRL), sirolimus (SIR), and everolimus (RAD) are immunosuppressive drugs frequently used in organ transplantation. Our aim was to confirm a robust sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determination of CyA, TRL, SIR, and RAD in whole-blood samples. Materials and Methods. We used an integrated online solid-phase extraction-LC-MS/MS system and atmospheric pressure ionization tandem mass spectrometry (API-MS/MS) in the multiple reaction monitoring (MRM) detection mode. CyA, TRL, SIR, and RAD were simultaneously analyzed in whole blood treated with precipitation reagent taken from transplant patients. Results. System performance parameters were suitable for using this method as a high-throughput technique in clinical practice. The high concentration of one analyte in the sample did not affect the concentration of other analytes. Total analytical time was 2.5 min, and retention times of all analytes were shorter than 2 minutes. Conclusion. This LC-MS/MS method can be preferable for therapeutic drug monitoring of these immunosuppressive drugs (CyA, TRL, SRL, and RAD) in whole blood. Sample preparation was too short and simple in this method, and it permits robust, rapid, sensitive, selective, and simultaneous determination of these drugs.

Characterization of model compounds of processed lignin and the lignome by using atmospheric pressure ionization tandem mass spectrometry

In the search for alternatives to fossil fuel and the valuable chemicals now derived from crude oil, lignocellulosic biomass has attracted wide interest. Degradation of the cellulose and lignin components of the biomass results in complex mixtures. Hence, the ability to identify the products of processed cellulose and lignin is important. Unfortunately, mass spectrometric analysis of processed lignin (and the lignome in general) is hindered by poor ion generation and/or extensive fragmentation upon ionization. After examination of several different ionization approaches, we demonstrate here that ionization of model compounds of lignin degradation products and lignome by using positive ion mode electrospray ionization (ESI) doped with sodium chloride results in the formation of abundant adduct ions, [M+Na]+, with no accompanying fragmentation. Hence, this approach allows for the determination of the molecular weights of lignin degradation products (and lignome) directly in mixtures. However, no structural information can be obtained by examining collision-activated dissociation of the sodiated molecules. Hence, a different ionization method was identified. Doping negative ion mode ESI with hydroxide ions (from NaOH) yields only one type of an ion per analyte, the deprotonated analyte, without fragmentation. Valuable structural information can be obtained for these anions by subjecting them to multiple consecutive ion isolation and collision-activated dissociation (CAD) steps (up to MS7). This methodology significantly improves the information that can be obtained by mass spectrometric analysis for lignin degradation products and lignome.

Evaluation of a Modified QuEChERS Extraction of Multiple Classes of Pesticides from a Rice Paddy Soil by LC-APCI-MS/MS

A new method for the determination of clomazone, fipronil, tebuconazole, propiconazole, and azoxystrobin in samples of rice paddy soil is presented. The extraction of the pesticides from soil samples was performed by using a modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) method. Some extraction conditions such as salt addition, sample acidification, use of buffer, and cleanup step were evaluated. The optimized method dealt with a single extraction of the compounds under study with acidified acetonitrile, followed by the addition of MgSO(4) and NaCl prior to the final determination by liquid chromatography-atmospheric chemical pressure ionization-tandem mass spectrometry. Validation studies were carried out in soil samples. Recoveries of the spiked samples ranged between 70.3 and 120% with relative standard deviation lower than 18.2%. The limits of quantification were between 10 and 50 μg kg(-1). The method was applied to the analysis of real samples of soils where rice is cultivated.

Products of the OH radical-initiated reactions of 2- and 3-hexyl nitrate

Abstract Products of the gas-phase reaction of OH radicals with 2-hexyl nitrate (containing 13% 3-hexyl nitrate) have been investigated by gas chromatography with mass spectral and flame ionization detection, and by direct air sampling atmospheric pressure ionization tandem mass spectrometry. The products identified and quantified from 2-hexyl nitrate were: 2-hexanone (8.6 ± 1.3%), propanal (3.4 ± 0.8%), butanal (2.3 ± 0.6%) and 5-hydroxy-2-hexanone (25%), where the molar yields are given in parentheses. 3-Hexanone was observed from the 3-hexyl nitrate reaction, with a molar formation yield of 9.5 ± 2.1%. Organic nitrates of molecular weight 133, 161, 177 and 208 were also observed, and these are attributed to C 4 -carbonyl nitrate(s), C 6 -carbonyl-nitrates, C 6 -hydroxycarbonyl-nitrates and C 6 -dinitrates, respectively, and are expected to account for all or most of the non-quantified reaction products from OH + 2- and 3-hexyl nitrate. 5-Hydroxy-2-hexanone formation indicates that the CH 3 CH(ONO 2 )CH 2 CH 2 CH(O )CH 3 alkoxy radical dominantly reacts by isomerization.

Abstract 1291: A phase 1 open-label single-radiolabeled dose study of PF299804 in healthy male volunteers

Abstract Objectives: To characterize the primary routes of elimination of the second-generation, oral, irreversible, pan-HER tyrosine kinase inhibitor, PF299804; to evaluate the pharmacokinetics (PK) of total radioactivity and of PF299804; and to identify the metabolites of PF299804 in plasma, urine, and feces in healthy volunteers (HVs). Methods: This Phase 1 study evaluated the mass balance and PK of PF299804 in HVs (18-55 yrs), following a single 45-mg oral dose containing ∼100 µCi [14C] PF299804. Serial samples were collected over an 18-day period. Radioactivity measurements of blood, plasma, urine, and feces were conducted by liquid scintillation spectroscopy. Plasma samples were analyzed for PF299804 and the metabolite PF5199265 using high-performance liquid chromatography with atmospheric pressure ionization tandem mass spectrometry. Plasma, urine, and fecal samples were analyzed for metabolic profiling of PF299804; major metabolites of PF299804 were identified where possible. Results: 6 HVs (male; median age 31.5 yrs) received treatment and were evaluated for PK and safety. 78.8% of the radiolabeled material was excreted in feces, and a further 3.2% was recovered in urine. Peak concentrations of PF299804 in plasma generally occurred 12 hrs after oral dosing, and 6 hrs post-dose for PF5199265. Plasma exposure for the metabolite was approximately one-third of that for the parent compound. Terminal plasma half-life (t1/2) averaged about 55 hrs for PF299804 and 73 hrs for PF5199265. Peak concentrations of total radioactivity in plasma occurred at 12 hrs. Geometric mean Cmax was approximately 2-fold higher and total exposure (AUCinf) was almost 6-fold higher for total radioactivity than for PF299804 in plasma. t1/2 for plasma radioactivity was 182 hrs. PF299804 and its O-desmethyl metabolite, PF5199265, were the major drug-related components in plasma. Other trace components were observed in the profile. The most abundant drug-related components identified in excreta were proposed to be PF299804, PF5199265, a cysteine conjugate (M2), and a mono-oxygenated metabolite (M7). There were no serious adverse events (AEs), severe AEs, or deaths during the study. Mild AEs were reported in 2 HVs: dermatitis acneiform, dizziness, insomnia, and photophobia (all considered related to study drug). Conclusions: Following single-dose administration of [14C] PF299804 the radiolabeled material was primarily eliminated in feces (78.8%) and, to a lesser degree, in urine (3.2%). Estimates of exposure for total radioactivity in plasma (Cmax and AUCinf) were 2- and 6-fold higher, respectively, than exposure for plasma PF299804, indicating the presence of circulating metabolic product(s). The metabolic profiles indicated that [14C] PF299804 underwent oxidative and conjugative metabolism in HVs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1291. doi:10.1158/1538-7445.AM2011-1291

Pharmacokinetic Comparisons of Bromfenac in DuraSite and Xibrom

The purpose of this study was to compare the ocular pharmacokinetics of experimental solutions of bromfenac in DuraSite(®) to Xibrom™ (bromfenac ophthalmic solution) 0.09%. The bromfenac content was measured in the aqueous humor of 84 Dutch Belted rabbits after a single dose of either 0.045% or 0.09% bromfenac in DuraSite in the left eye and the commercial preparation in the right eye. The drug content in the aqueous humor was measured at 0.5, 1, 2, 4, 8, 12, and 24 h after instillation. In a separate multi-dose study, rabbits received one drop of the 0.09% experimental or commercial preparation, 3 times daily for 14 days. For both experiments the drug content in ocular tissues was analyzed using liquid chromatography atmospheric pressure ionization tandem mass spectrometry. In single-dose experiments, the concentration of bromfenac in the aqueous humor was higher with the experimental preparations than with the commercial solution. The area under the concentration-time curve of 0.045% and 0.09% bromfenac in DuraSite was ∼2 and 4-fold higher than that of commercial bromfenac ophthalmic solution, 0.09%. After multi-dose experiments, ocular tissue concentrations of bromfenac were ∼3 times higher for the experimental than for the commercial formulation. The study demonstrates that the DuraSite topical drug delivery system can deliver bromfenac to various ocular tissues and attain considerably higher concentrations than the commercially available eye drop formulation. The higher aqueous concentration sustained with these experimental formulations could broaden the utility of bromfenac and/or reduce the currently approved dosing frequency of this drug.

Products and Mechanisms of the Gas-Phase Reactions of OH Radicals with 1-Octene and 7-Tetradecene in the Presence of NO

Products of the gas-phase reactions of OH radicals with 1-octene and 7-tetradecene have been investigated at 296 +/- 2 K and atmospheric pressure of air, using gas chromatography, direct air sampling atmospheric pressure ionization tandem mass spectrometry, and in situ Fourier transform infrared spectroscopy. We observe the hydroxynitrate(s) formed from reaction of the hydroxyalkylperoxy radicals with NO; heptanal and 4-hydroxyhexanal from decomposition of the 1,2-hydroxyoctoxy and 7-hydroxy-8-tetradecoxy radicals; and dihydroxynitrates and dihydroxycarbonyls formed after isomerization of the intermediate 1,2-hydroxyalkoxy radicals. Formic acid formation was also observed from the 1-octene reaction, in approximately 3% yield. In the presence of approximately 1.5 x 10(14) molecules cm(-3) of NO, the respective molar formation yields of heptanal and 4-hydroxyhexanal were 28 +/- 3% and 4% from 1-octene, and 86 +/- 11% and 6% from 7-tetradecene. The 4-hydroxyhexanal yield increased with increasing NO concentration, and we attribute 4-hydroxyhexanal + HC(O)OH formation to a minor pathway of the RC(*)HOH + O(2) reaction in the presence of NO. The reaction pathways occurring are discussed.

Determination of metformin in mouse, rat, dog and human plasma samples by laser diode thermal desorption/atmospheric pressure chemical ionization tandem mass spectrometry

A simple, rapid and robust high-throughput assay for the quantitative analysis of metformin in plasma from different species using laser diode thermal desorption interfaced with atmospheric chemical pressure ionization tandem mass spectrometry (LDTD-APCI-MSMS) was developed for use in a pharmaceutical discovery environment. In order to minimize sample preparation a generic protein precipitation method was used to extract metformin from the plasma. Laser diode thermal desorption is a relatively new sample introduction method, the optimization of the instrumental parameters are presented. The method was successfully applied to spiked mouse, rat, dog and human plasma samples and was subsequently used to determine the oral pharmacokinetics of metformin after dosing to male rats in order to support drug discovery projects. The deviations for intra-assay accuracy and precision across the four species were less than 30% at all calibration and quality control levels.

In-vitro metabolism of isotetrandrine, a bisbenzylisoquinoline alkaloid, in rat hepatic S9 fraction by high-performance liquid chromatography-atmospheric pressure ionization mass spectrometry.

The objective of this study was to investigate the in-vitro metabolism of isotetrandrine, a bisbenzylisoquinoline alkaloid, using rat hepatic S9 fraction and to profile and identify its metabolites using high-performance liquid chromatography-atmospheric pressure ionization mass spectrometry (HPLC-MS) and tandem mass spectrometry (MS/MS). Isotetrandrine was incubated at a concentration of 100 microg mL(-1) with male rat hepatic S9 fraction in the presence of an NADPH generating system (Tris buffer, pH 7.4, 37 degrees C). Samples were removed at 60 min after reaction initiation. Unchanged isotetrandrine (approximately 63% of the sample) and four metabolites were profiled, characterized and tentatively identified using solvent extraction, methyl derivatization, and HPLC-MS and MS/MS techniques. Isotetrandrine metabolites were mainly formed via two main pathways, N-demethylation and isoquinoline ring oxidation. The first pathway produced a major metabolite, N-desmethyl isotetrandrine (approximately 16% of the sample). The second pathway produced three minor oxidized metabolites, hydroxy-isotetrandrine (approximately 6% of the sample), oxo-isotetrandrine (approximately 7% of the sample), and oxohydroxy-isotetrandrine (approximately 7% of the sample). Diazomethane treatment of these metabolites did not produce any methyl derivatives and therefore the hydroxylated sites of the metabolites were tentatively assigned at the heterocyclic moieties of the isoquinoline rings. In conclusion, isotetrandrine is substantially metabolized in this in-vitro rat hepatic system.

Fragmentation Energy Index for Universalization of Fragmentation Energy in Ion Trap Mass Spectrometers for the Analysis of Chemical Weapon Convention Related Chemicals by Atmospheric Pressure Ionization-Tandem Mass Spectrometry Analysis

The use of mass spectra generated at 70 eV in electron ionization (EI) as a universal standard for EI has helped in the generation of searchable library databases and had a profound influence on the analytical applications of gas chromatography/mass spectrometry (GC/MS), similarly for liquid chromatography tandem mass spectrometry (LC-MS/MS), suggesting a novel method to normalize the collisional energy for the universalization of fragmentation energy for the analysis of Chemical Weapon Convention (CWC)-related chemicals by atmospheric pressure ionization-tandem mass spectrometry (API-MS(n)) using three-dimensional (3D) ion trap instruments. For normalizing fragmentation energy a "fragmentation energy index" (FEI) is proposed which is an arbitrary scale based on the fact of specific MS/MS fragmentation obtained at different collisional energies for the reference chemicals which are not CWC scheduled compounds. FEI 6 for the generation of an MS(n) library-searchable mass spectral database is recommended.

Determination of duloxetine in human plasma by liquid chromatography with atmospheric pressure ionization–tandem mass spectrometry and its application to pharmacokinetic study

A rapid, sensitive and accurate liquid chromatographic–tandem mass spectrometry (LC–MS–MS) method is described for the determination of duloxetine in human plasma. Duloxetine was extracted from plasma using methanol and separated on a C18 column. The mobile phase consisting of a mixture of acetonitrile and 5 mM ammonium acetate (45:55, v/v, pH 3.5) was delivered at a flow rate of 0.3 ml/min. Atmospheric pressure ionization (API) source was operated in positive ion mode. Multiple reaction monitoring (MRM) mode using the transitions of m/ z 298.1 → m/ z 44.0 and m/ z 376.2 → m/ z 123.2 were used to quantify duloxetine and internal standard (I.S.), respectively. The linearity was obtained over the concentration range of 0.1–50.0 ng/ml and the lower limit of quantitation (LLOQ) was 0.1 ng/ml. This method was successfully applied to pharmacokinetic study of a duloxetine formulation product after oral administration to healthy human subjects.

A liquid chromatography/atmospheric pressure ionization tandem mass spectrometry quantitation method for nevirapine and its two oxidative metabolites, 2‐hydroxynevirapine and nevirapine 4‐carboxylic acid, and pharmacokinetics in baboons

A rapid highly sensitive and specific electrospray ionization (ESI) liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for quantification of nevirapine (NVP) and its two metabolites, 2-hydroxynevirapine (2-OHNVP) and nevirapine 4-carboxylic acid (4-CANVP), in baboon serum was developed and validated. Nevirapine, 2-OHNVP, 4-CANVP, and the internal standard, hesperetin, were extracted from baboon serum with ethyl acetate. Components in the extract were separated on a 50 x 2.1 mm Aquasil C(18) 5 microm stainless steel column by isocratic elution with 40% acetonitrile/0.1% formic acid at a flow rate of 0.2 mL/min. The liquid flow was passed through a pre-source splitter and 5% of the eluant was introduced into the atmospheric pressure ionization (API) source. The components were analyzed in the multiple-reaction monitoring (MRM) mode as the precursor/product ion pair of m/z 267.2/226.2 for NVP, 283.0/161.2 for 2-OHNVP, 297.2/279.2 for 4-CANVP, and 303.2/177.2 for hesperetin. Linear calibration curves were obtained in the range of 1-1000 ng/mL for NVP and 2-OHNVP and 5-1000 ng/mL for 4-CANVP, using 0.2 mL baboon serum, respectively. The within-day and between-day precisions were <10% for NVP and 2-OHNVP, and <11.5% for 4-CANVP. Due to the similar structures and fragmentation patterns of 2-OHNVP and 3-OHNVP, it is not expected that the LC/MS/MS can differentiate 2-OHNVP and 3-OHNVP and they were assayed as a composite. The method was applied to a single-dose escalation study of NVP in non-pregnant baboons (Papio anubis) to characterize the pharmacokinetics of NVP, 2-OHNVP plus 3-OHNVP, and 4-CANVP, and to determine the appropriate dose necessary to achieve comparable peak serum concentration of NVP as reported in healthy human adults.