Monitoring Mass Spectrometry Research Articles

O25: HPLC-HRMS vs UPLC-MS/MS for a systematic toxicological screening in hair

Aims The aim of the present work was to compare the performances of HPLC-High Resolution Mass Spectrometry (HPLC-HRMS) in full scan and UPLC- Multiple mass spectrometry (UPLC-MS/MS) in multiple reaction monitoring (MRM) mode for the systematic, general unknown screening of drugs and pharmaceuticals in hair samples collected for clinical toxicology purposes at the local University Hospital. Methods 10 mg hair were washed and extracted by a rapid, micropulverized method already published. Extracts were analyzed in two different instruments, the former consisting of an Orbitrap coupled to a Finnigan Surveyor HPLC, the latter of a Xevo TQD triple quadrupole coupled to an Acquity UPLC. For HPLC, an Atlantis T3 (150 × 2,1 mm, 3 μm) was used while the UPLC employed a HSS C18 (150 × 2.1 × 1,8 μm), with a gradient of ammonium acetate and acetonitrile (formic acid 0.1%) at 300 μL/min and 400 μL/min respectively. The Orbitrap was operated in positive ion electrospray mode at a resolution of 60000 with mass locking, in full scan mode, with a in-house developed library of 600 compounds; the identificatiion criteria were 2 ppm accuracy of the exact mass of the protonated molecule, ± 0.1 min of the retention time, a minimum area count of 10000 arbitrary units; the Xevo TQD worked in switching ion polarity electrospray mode, and in MRM using a Waters toxicology library of 800 (untargeted screening) or 180 compounds (targeted screening), a recognition window of ±0.2 min and a minimum area count of 600 arbitraty units. Results Compounds belonging to drugs of abuse and/or to pharmaceutical drugs could be identified with both analytical approaches in the same hair samples. In more than 100 hair samples collected in the clinical settings in a year, drugs belonging to the following therapeutic categories were identified: anaesthetics, anti-epileptics, antidepressants, benzodiazepines, sedatives, hypnotics, opioids, anti-psychotics, neuroleptics, barbiturates, stimulants, non steroidal analgesics, steroids, beta-blockers, diuretics, antibiotics, antiarrhythmic agents, antihypertensive drugs, anti-coagulants, hypoglycaemic agents. The screening results were in every case confirmed by injection of the tentatively identified analyte(s) in methanol standard mixtures. When possible, the compliance with prescribed therapies or alleged use/abuse was verified. Conclusions The UPLC-MRM method turned to be more rapid than the HPLC-HRMS (14 min vs 30 min total run time); the possibility of polarity switching could be applied in routine only in the Xevo TQD allowing to easily identify acidic drugs such as non steroidal analgesics. The HPLC-Orbitrap, working in full scan, resulted superior for implementing the library with new molecules, since no adjustment of the acquisition method was needed, and for the postacquisition, retrospective screening of the raw files. In the UPLCMRM method, limits of identification with targeted analytes were in the order 0.1 – 0.2 ng/mg for basic drugs, with 10 mg hair samples, whereas in the HPLC-HRMS limits were generally lower.

Correlation of high levels of HER2 measured by multiplex mass spectrometry with increased overall survival in patients treated with anti-HER2-based therapy.

649 Background: There is evidence that increased absolute levels of the HER2 receptor in breast cancer positively correlates with a clinical benefit from anti-HER2 therapies. The current method of HER2 evaluation, immunohistochemistry (IHC), does not allow for absolute HER2 quantification and is prone to false-positives. Thus, a significant portion of patients classified as HER2-positive actually express low levels of the receptor. We quantitated absolute levels of HER2 using a mass spectrometry-based assay and correlated expression levels with the anti-HER2 therapy benefit. Methods: Formalin-fixed, paraffin-embedded (FFPE) tumor sections were microdissected and proteins were solubilized and digested by trypsin in Liquid Tissue buffer. After digestion, internal standards were added and absolute quantitation for multiple proteins was performed using selected reaction monitoring (SRM) mass spectrometry. Results: 26 HER2-positive (IHC 3+) breast cancer samples from patients treated with anti-HER2 therapy wer...

High‐throughput quantification of stabilizers in polymeric materials by flow injection tandem mass spectrometry

High-throughput methods for identification and quantification of stabilizers in plastic materials are of significant importance in order to evaluate the suitability of materials of unknown origin for specific application areas, to clarify reasons for failure of materials, or for comparison of materials from different sources. In the present study, a highly sensitive and rapid flow injection method coupled to selected reaction monitoring mass spectrometry (MS) for comprehensive analysis of 21 polymer stabilizers in polyolefins is demonstrated. A critical factor for this approach is the choice of ionization mode, as no separation was performed prior to MS detection. Differences between several ionization techniques regarding matrix effects are reported. Atmospheric pressure chemical ionization was found to be the most suitable ionization technique, with no significant matrix effects observed. The developed method has a linear dynamic range over two to three orders of magnitude with correlation coefficients better than 0.99 for all studied analytes. Following a multistep sample preparation protocol, the method allowed quantification down to minimum values of between 0.0001 and 0.04 wt% depending on the type of stabilizer. Results were compared to an established chromatographic approach and showed very good correlation (bias below 7.5%). The applicability of the optimized method could be demonstrated for both the qualitative and quantitative determination of polymer stabilizers in polyolefins. Furthermore, the described approach yields a complete analysis in a much shorter time than can be achieved with commonly applied chromatographic methods.

Determination of 39 polychlorinated biphenyls in indoor dust using ultrasonic extraction and gas chromatography-tandem mass spectrometry

A method for the determination of 39 polychlorinated biphenyls (PCBs) in indoor dust was developed. A vacuum cleaner was used for gathering the house dust. N-Hexane-dichloromethane (1: 1, v/v) was added and the extraction was performed in an ultrasonic bath. The supernatant was concentrated and then 0.1 mL n-hexane-dichloromethane (1: 1, v/v) was added. Gas chromatography-tandem mass spectrometry (GC-MS/MS) in selected-reaction monitoring (SRM) mode has been investigated for the determination of the 39 PCBs congeners in indoor dust. The 39 PCBs had highly efficient separation within 30 min and showed good linearity in the range of 0.1 - 100 microg/L with the correlation coefficients of 0.991 0 - 0.999 9. The spiked recoveries were 57.2% - 120.3%. The intra-day relative standard deviations (RSDs) were between 0.3% and 24.7%, while the inter-day RSDs were between 0.6% and 29.9%. This method has good linearity, high sensitivity, high accuracy and precision. Also, it is simple, rapid and low solvent consumption. The low chemical background interference allowed it to be used in more complex matrices.

Determination of antibiotics in oral hygiene products by high performance liquid chromatography-tandem mass spectrometry

A high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/ MS) method was developed for simultaneous determination of 13 antibiotics in oral hygiene products, including five tetracyclines, three macrolides, two quinolones, one beta-lactam and two lincosamides. The sample was extracted with 0.1% (volume percentage, same hereinafter) formic acid-acetonitrile (95:5, v/v), then centrifuged, filtered and diluted. The target compounds were separated on a C18 column (150 mm x 2.1 mm, 5 microm) with a gradient elution of 0. 1% formic acid and acetonitrile as the mobile phases, and detected by tandem mass spectrometry in positive electrospray ionization and multiple reaction monitoring (MRM) mode. The quantification of 13 antibiotics was performed by the external standard method. The calibration curves showed good linearity in the range of 5.0-50.0 microg/L with detection limits of 10.0 mg/kg. The recoveries of antibiotics in mouthwash and toothpaste samples at the three spiked levels of 10, 20 and 100 mg/kg were in the range of 80.1%-115% with the relative standard deviations in the range of 0.94%-8.69%. This method is accurate, reliable, simple, and suitable for the analysis of antibiotics in oral hygiene products.

Determination of seven toxaphene congeners in Ginseng and Milkvetch Root by gas chromatography-tandem mass spectrometry

A novel method for the determination of representative toxaphene congeners in traditional Chinese herbal medicines was developed. Ginseng and Milkvetch Root were selected as the samples and seven toxaphene congeners were selected as the monitoring objects. The samples were extracted by accelerated solvent extraction with cyclohexane-acetone (9:1, v/v), then cleaned-up by Florisil solid phase extraction with hexane as the eluent and the residues were detected by gas chromatography-electron ionization tandem mass spectrometry (GC-EI-MS/MS) in multiple reaction monitoring (MRM) mode. The performance was demonstrated by the analysis of Ginseng and Milkvetch Root samples spiked with toxaphene congeners at three concentration levels of 0.005, 0.01 and 0.1 mg/kg. The recoveries ranged from 72.4% to 105% with the relative standard deviations (RSDs) of 0.96%-10.4%. The limits of detection (LODs) were 0.2-1.7 microg/kg. This method is sensitive and efficient in the aspect of extraction, and can be applied to monitor the residue of toxaphene congeners in Ginseng and Milkvetch Root.

Simultaneous Determination of Amantadine and Rimantadine Residue in Animal Tissues by LC-MS/MS

A rapid determination method for amantadine and rimantadine in animal tissues was established using liquid chromatography-electrospray tandem mass spectrometry(LC-ESI-MS/MS) in the multiple reaction monitoring(MRM) mode.The samples were extracted with acetonitrile-1.0% trichloroacetic acid(50∶50,V/V) solution in ultrasonic,followed by a cleanup procedure with mixed cation exchange SPE.The eluate was dried under nitrogen blow,then dissolved in 1 mL methanol-0.2% formic acid(10∶90,V/V) solution and the aliquot was detected by LC-MS/MS.The chromatographic retention time and mass fragment ion abundance ratio were taken for qualitative confirmation,and external standard method was employed for quantification.The limit of detection(LOD) is 0.4 μg/kg,and the limit of quantification(LOQ) is 1.0 μg/kg in chicken,chicken liver,pork and pork liver.The correlation coefficent is greater than 0.999(r20.999) of the four animal tissues are obtained within 0.5—100.0 μg/L linear ranges.Recoveries are in a range of 70.7%—92.3% with the relative standard deviations(RSDs) varied from 1.7% to 11.7% at spiked levels of 5.0—100μg/kg,respectively.

Abstract 2505: Expression profiling of cofilin-1 in breast cancer cell lines and biopsies.

Abstract Introduction. Despite oestrogen and human epidermal growth factor receptor (HER2) targeted therapies improving patient survival, breast cancer continues to be a significant cause of premature death since many tumors do not respond to treatment or acquire resistance. Hence there remains the need to identify novel targets for therapy and biomarkers for prediction of response to treatment. A quantitative proteomics study of matched normal and tumor biopsies identified 63 proteins to be significantly increased or decreased in stage-specific tumors. Some have previously been associated with breast cancer, while others such as cofilin-1 represent new targets for investigation. Cofilin-1 was subject to a range of analyses to determine its association with breast cancer. Materials and Methods. Western blotting (WB) was performed on protein extracts from breast cancer cell lines and matched normal and tumor biopsies from Cypriot patients with different stages of the disease. Immunohistochemistry (IHC) was carried out on core biopsies from the Leeds Breast Tissue Bank. Multiple reaction monitoring (MRM) mass spectrometry was performed on trypsin-digested protein extracts from biopsies. Data from protein (Human Protein Atlas) and genomics (BioGPS, TiGER, UniGene) databases were assimilated with the experimental results. Results. WB indicated that cofilin-1 was ubiquitously expressed in tumor cell lines, representative of Luminal A, Luminal B, basal-like, claudin-low and HER2 phenotypes, at higher levels than normal breast cell lines. WB also indicated increased expression of cofilin-1 in invasive carcinoma tissues, compared to matched normal. Patients with ductal carcinoma in situ or fibroadenoma exhibited less clear results, either increasing slightly or remaining unchanged. MRM analysis of three cofilin-1 peptides in tissue extracts of invasive carcinoma patients indicated expression only in tumor. IHC of core biopsies exhibited strong staining for cofilin-1 in ductal invasive carcinoma tissues with no staining in normal breast cells. Genomics and proteomics databases indicate that cofilin-1 is expressed in a diverse range of normal tissues including major organs, gastro-intestinal tract, skin and tonsils. However, mRNA and protein levels are observed to be increased in skin, lung, liver, pancreatic and breast tumors. Discussion. The original quantitative proteomics data indicated only relatively small changes in expression (less than 2-fold) due to dynamic range limitations of the technique. Additional analytical approaches substantiated that cofilin-1 is significantly up-regulated in advanced breast cancer patients. Further patient sets are required to confirm the importance of cofilin-1 levels in early stages of breast cancer compared to benign tissues. Bioinformatics provided further confidence in our findings, highlighting the value of utilising databases for evidence of disease-specific proteins. Citation Format: Chris Sutton, Sadr ul-Shaheed, Paul Loadman, Valerie Speirs, Andrew Hanby, Andreas Hadjisavvas, Kyriacos Kyriacou. Expression profiling of cofilin-1 in breast cancer cell lines and biopsies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2505. doi:10.1158/1538-7445.AM2013-2505

Identification of the metabolites of lesogaberan using linear trap quadrupole orbitrap mass spectrometry and hydrophilic interaction liquid chromatography

1. In this study, hydrophilic interaction liquid chromatography (HILIC), radiochemical activity monitoring and linear trap quadrupole orbitrap mass spectrometry (MS) and tandem mass spectrometry (MS/MS) were used to identify the metabolites of a highly polar novel γ-aminobutyric acid type-B receptor agonist, lesogaberan, in rats.2. Urine was collected from three male Wistar rats for 24 h after dosing with 14C-labelled lesogaberan (170 mg/kg, 10 MBq/kg); plasma samples were taken 2 and 24 h after dosing. Pooled samples were separated by HILIC and eluents were analysed by radiochemical activity monitoring, MS and MS/MS.3. Only the parent compound was detected in plasma, but six metabolites (M1–M6) were detected in urine. Analysis of MS and MS/MS data and comparison with synthetic reference standards enabled the identification of the structure of each metabolite. M1 was identified as the N-acetylated species [(2R)-3-acetamido-2-fluoropropyl]-phosphinic acid, and M6 as [(2R)-3-amino-2-fluoropropyl]-phosphonic acid. Metabolites M2 and M5 were the alcohol and carboxylic acid species 3-hydroxypropyl-phosphinic acid and 3-hydroxyphosphonoyl-propanoic acid, respectively, both of which had lost the fluorine atom present in the parent compound. M3 was the corresponding carboxylic acid species retaining the fluorine atom, (2R)-2-fluoro-3-hydroxyphosphonoyl-propanoic acid. Finally M4 was identified as [(2R)-2-fluoro-3-guanidino-propyl]-phosphinic acid.

Determination of the residues of 3-methyl-quinoxaline-2-carboxylic acid and quinoxaline-2-carboxylic acid in animal origin foods by high performance liquid chromatography-tandem mass spectrometry

A high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/ MS) method was developed for the simultaneous quantitative determination of 3-methyl-quinoxaline-2-carboxylic acid (MQCA) and quinoxaline-2-carboxylic acid (QCA) as the marker residues for carbadox (CBX) and olaquindox (OLA), respectively, in the muscles and livers of porcine and chicken and in the muscles of fish and shrimp. The MQCA and QCA were deproteinated with 5% metaphosphoric acid in 10% methanol followed by liquid-liquid extraction. Further clean-up was performed by solid phase extraction (SPE) through mixed mode anion-exchange columns (Oasis MAX SPE). The separation of the compounds was carried on a Waters Xterra MS C18 column (150 mm x 2.1 mm, 5 microm) by a gradient elution using methanol and 0.2% formic acid as mobile phases. The analytes were detected by tandem mass spectrometry in multiple reaction monitoring (MRM) mode with positive electrospray ionization. The MQCA and QCA were quantified by internal standard method. The linear ranges were 1.0-20.0 microg/L and the correlation coefficients were not less than 0.9996. The average recoveries and relative standard deviations ranged from 62.4%-118% and 1.48%-28.1% respectively at the spiked levels of 0.1, 0.2 and 1.0 microg/kg for the both markers. The limit of quantitation (LOQ) was 0.1 microg/kg. The method is sensitive, accurate and suitable for the determination and confirmation of MQCA and QCA in animal origin foods.

LC–ESI-MS/MS analysis and pharmacokinetics of 6′-hydroxy justicidin A, a potential antitumor active component isolated from Justicia procumbens, in rats

A sensitive and accurate LC-ESI-MS/MS method was developed and validated of for the determination of 6'-hydroxy justicidin A (HJA), a potential antitumor active component isolated from Justicia procumbens in rat plasma using a simple liquid-liquid extraction (LLE) method for sample preparation. Chromatographic separation was achieved on an Agilent Zorbax-C(18) column (2.1 mm × 50 mm, 3.5 μm) using a step gradient program with the mobile phase of 0.1% formic acid aqueous solution and acetonitrile with 0.1% formic acid. HJA and IS (buspirone) were detected using electrospray positive ionization mass spectrometry in the multiple reaction monitoring (MRM) mode. This method demonstrated good linearity and did not show any endogenous interference with the active compound and IS peaks. The lower limit of quantification (LLOQ) of HJA was 0.50 ng/ml in 50 μl rat plasma. The developed and validated method has been successfully applied to the quantification and pharmacokinetic study of HJA in rats after intravenous and oral administration of 0.25 mg/kg HJA. The oral bioavailability (F) of HJA was estimated to be 36.0±13.4% with an elimination half-life (t(1/2)) value of 1.04±0.20 h.

Abstract 3933: ERα phosphorylation patterns at hinge domain Ser294 and Ser305 differentiate ligand-dependent from ligand-independent receptor activation

Abstract Domain-specific phosphorylation of estrogen receptor alpha (ERα) is critical for its transcription factor activity and may determine its predictive and therapeutic biomarker value. Recent attention has turned to the poorly understood ERα hinge domain in light of preliminary evidence that phosphorylation at serine 305 (pSer305) associates with poor clinical outcome and endocrine resistance. Using multiple reaction monitoring (MRM) mass spectrometry (MS), we have studied a number of ERα overexpressing human breast cancer cells lines (MCF-7, BT474, T47D) and found that robust ERα phosphorylation occurs on a neighboring hinge domain site at Ser294, but only upon ligand (estradiol, tamoxifen) stimulation of the receptor since growth factor cell stimulation (EGF, insulin, heregulin-α, forskolin) fails to induce pSer294. As well, pSer294 formation appears to be essential for full ligand induction of ERα gene transcription in experimental models. Selective inhibition of cyclin-dependent kinases (CDKs) by roscovitine or SNS-032 in the breast cancer cell lines blocks ligand induced pSer294 formation, indicating that this hinge domain phosphorylation is mediated by CDKs; likewise, cell-free studies with recombinant ERα and specific cyclin-CDK complexes reveal that Ser294 phosphorylation is most effectively induced by the transcription-regulating and cell cycle-independent kinases, CDK7 and CDK9. In contrast to this ligand specific induction of pSer294, western analysis of immunopurified ERα from MCF-7 probed with a newly available pSer305 monoclonal demonstrates that pSer305 formation occurs in response to growth factor stimulation (EGF, insulin, forskolin) but not in response to cell stimulation by ligand alone. Interestingly, co-treatment with ligand and growth factor reduces pSer294 formation relative to ligand stimulation alone while co-treatment reduces pSer305 formation relative to growth factor stimulation alone, suggesting that these two hinge phosphorylation sites not only respond reciprocally to specify different aspects of ERα function but are also antagonistically responsive to crosstalk. Unlike the better studied N-terminal domain phosphorylation sites at Ser118 and Ser167, which are phosphorylated jointly and additively upon combined ligand-dependent and ligand-independent ERα activation, the two hinge domain sites at Ser294 and Ser305 appear to respond in a reciprocal fashion differentiating ligand-dependent from ligand-independent ERα activation. Pending current development of a pSer294 monoclonal, future immunohistochemical interrogation of the ERα hinge domain phosphorylation status at Ser294 and Ser305 is expected to improve the biomarker value of this receptor by better predicting ERα overexpressing breast cancers most likely to respond to endocrine agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3933. doi:1538-7445.AM2012-3933

Simultaneous quantification of selective serotonin reuptake inhibitors and metabolitesin human plasma by liquid chromatography-electrospray mass spectrometry for therapeutic drug monitoring

Simultaneous quantification of selective serotonin reuptake inhibitors and metabolitesin human plasma by liquid chromatography-electrospray mass spectrometry for therapeutic drug monitoring

Dietary patterns and ethnicity are associated with distinct plasma proteomic groups

High-abundance plasma proteins are involved in disease-associated pathways and are useful in the diagnosis of nutritional and disease states. However, little is known about how concentrations of many plasma proteins vary between individuals from different ethnocultural groups with different dietary habits. We explored the association between plasma proteomic groups, dietary patterns, and ethnicity in the Toronto Nutrigenomics and Health Study, an ethnically diverse population of healthy young adults. Concentrations of 54 high-abundance plasma proteins were measured simultaneously by liquid chromatography/multiple-reaction monitoring-mass spectrometry in 1090 individuals. Principal components analysis was used to identify plasma proteomic groups. Linear regression was used to investigate relations between proteomic groups and previously identified dietary patterns (Western, prudent, Eastern). Differences in individual protein concentrations between ethnocultural groups were tested by using general linear models. Four independent principal components representative of proteomic groups were identified. Principal components 1 and 2 included proteins from multiple pathways. Component 3 was inflammatory, and component 4 included coagulation cascade proteins. East Asians and South Asians had lower component 1 scores, and East Asians had higher component 2 scores. South Asians had higher average scores for component 3. Individual protein concentrations also varied across ethnocultural groups. Principal component 1 was positively associated with the Western dietary pattern and inversely associated with the Eastern pattern. Component 3 was positively associated with the Eastern pattern. Plasma proteomic groups differ between young adults of diverse ethnocultural groups with different dietary habits. These differences may partly account for different rates of cardiometabolic disease later in life.

Rapid determination of ten polyphenols in Kudiezi injection using ultra-performance liquid chromatography-tandem mass spectrometry in multiple reaction monitoring mode

A simple and rapid method for simultaneous determination of ten polyphenols in Kudiezi injection has been developed and validated using ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) in multiple reaction monitoring (MRM) mode. The chromatographic separation was performed on an ACQUITY UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm) with a gradient of acetonitrile and 0.5% formic acid (v/v). Ten polyphenols were rapidly analyzed within 10 minutes. Very low limits of detection (LODs) of 0.002–0.630 μg L−1 and limits of quantitation (LOQs) of 0.005–2.930 μg L−1 of the ten components were achieved. The linear calibration range extended from 2.73 to 660.40 μg L−1. The developed method was validated in terms of good linearity (R2 > 0.9990), precision (less than 3.71%) and accuracy (from 97.35 to 102.02%) for ten components. The developed UPLC-ESI-MS/MS method was simple and useful for rapid determination of the constituents in Kudiezi injection.

Degradation of food dyes by zero-valent metals exposed to ultrasonic irradiation in water medium: optimization and electrospray ionization mass spectrometry monitoring

The reductive degradation of four prototype dyes largely employed by the food industry (Brilliant Blue, Amaranth, Sunset Yellow and Red 40), by zero-valent metals (Fe and Sn) under ultrasonic irradiation in acidic aqueous solution (pH 3.0), was studied. The reactions were monitored by direct infusion ESI(-)-MS (electrospray ionization mass spectrometry in the negative ion mode) as well as by UV-Vis and total organic carbon (TOC). The influence of major parameters (time of exposure to ultrasonic irradiation and mass of metal) on the degradation rates was firmly established by means of factorial design and surface response approaches. Hence, it was noticed that, under the optimized experimental conditions, metallic iron (Fe) was able to efficiently degrade all the dyes (with rates higher than 90% after 30 min reaction time), whereas metallic tin (Sn) exhibited a noticeably lower performance. Low mineralization rates were achieved probably due to the essentially reductive character of such process. Based on the results from the ESI(-)-MS monitoring, a plausible degradation route for the Brilliant Blue dye in aqueous medium by the Fe/US system was proposed. Hence, this route involves, as initial pathways, the hydrogenation of the exocyclic carbon-carbon double bond of the dye followed by the insertion of hydride at its imino moiety.

Real-time reaction monitoring using ion mobility-mass spectrometry

The potential of ion mobility (IM) spectrometry in combination with mass spectrometry (MS) for real-time reaction monitoring is reported. The combined IM-MS approach using electrospray ionization affords gas-phase analyte characterization based on both mass-to-charge (m/z) ratio and gas-phase ion mobility (drift time). The use of IM-MS analysis is demonstrated for the monitoring of the reaction products formed when 7-fluoro-6-hydroxy-2-methylindole is deprotonated by aqueous sodium hydroxide. Real-time reaction monitoring was carried out over a period of several hours, with the reaction mixture sampled and analysed at intervals of several minutes. Product ion relative intensity is enhanced selectively in the ion mobility-selected mass spectrum, compared to mass spectrometry alone. The combined IM-MS approach has potential as a rapid and selective technique to aid pharmaceutical process control and for the elucidation of reaction mechanism.

Rapid simultaneous determination of 42 psychoactive drugs and their metabolites in human plasma and urine by ultra performance liquid chromatography-tandem mass spectrometry

A rapid method for the simultaneous determination of 42 psychoactive drugs and their metabolites (barbitals, benzodiazepines, tricyclic antidepressants, phenothiazines, etc. ) in human plasma and urine was developed using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). After a simple protein precipitation step, the analysis of the drugs and the metabolites was achieved on an Acquity UPLC BEH C18 column by using the gradient elution with ammonium acetate and methanol-acetonitrile (1: 1, v/v) as the mobile phases, and detected by electrospray ionization-tandem mass spectrometry in the multiple reaction monitoring (MRM) mode operated simultaneously in both positive and negative modes by rapid switching, and quantified by matrix-match standard solution. The average recoveries were 60.2% - 125% and 64.5% - 126% for the drugs in plasma and urine except those of perphenazine, thioridazine and chloropromazine in plasma were 37.6% - 57.5%, 36.3% - 48.3%, 52.4% -67.4%, respectively; trazodone and diazepam in urine were 100% -142% and 108% - 177%, respectively. The relative standard deviations (RSDs) of all the drugs in plasma and urine were within 0.8% - 26% and 2.6%18% (n = 6), respectively. The detection limits of the 4 barbitals ranged from 20 to 100 mg/L and those of the other drugs ranged from 0.05 to 2.0 mg/L. The method is simple, selective and sensitive to detect drugs for both clinical and forensic purposes.

Ozonation of ethinylestradiol in aqueous-methanolic solution: direct monitoring by electrospray ionization mass spectrometry

The ozonolysis of ethinylestradiol (1), a synthetic steroidal estrogen, in an aqueous-methanolic solution was investigated. HPLC-UV analyses revealed that 1 was completely consumed after a 20 min reaction time either at pH 5 or 8. ESI(-)-MS (electrospray ionization mass spectrometry in the negative ion mode) monitoring also revealed the continuous consumption of 1 (detected as [1 – H]- of m/z 295) concomitantly with the emergence of oxidation products. Chemical structures were proposed for these products based on the data of MS and MS/MS (the m/z values and fragmentation profiles of the anionic specie, respectively). These data, in conjunction with the well-established knowledge about the reactivity of organic molecules toward ozone in aqueous solution, were evaluated and an unprecedented reaction route for the ozonation of 1 could thus be suggested. Hence, the first step in this reaction sequence was ascribed to involve a 1,3-dipolar cycloaddition of ozone at the phenolic ring of 1 yielding the di-hydroxylated product 2 (detected as [2 – H]- of m/z 311). The loss of acetylene as the unique dissociation channel for [2 – H]- thus confirmed that such hydroxylation occurred at the phenolic ring rather than the acetylenic moiety of 1. Subsequent oxidations were proposed to be the origin of a number of other products, all of them bearing the COOH functionally (this was verified by the characteristic loss of CO2 during the dissociation of the related deprotonated molecules). The ESI(-)-MS records also revealed notable differences between the reaction conducted at pH 5 and 8, i.e. at slightly acid or basic media, respectively.

Pesticide Residue Analysis of a Dietary Ingredient by Gas Chromatography/Selected-Ion Monitoring Mass Spectrometry Using Neutral Alumina Solid-Phase Extraction Cleanup

A sample cleanup procedure has been developed to remove coextractives that interfere with pesticide residue analysis of a dietary ingredient (Product B), an extract consisting of Scutellaria baicalensis and Acacia catechu. Samples were extracted using 1% acetic acid in acetonitrile, followed by solid-phase extraction and analysis by capillary gas chromatography with mass spectrometry in the selective-ion monitoring mode. Neutral alumina (alumina N) was found to be the most effective sorbent to remove coextractives from Product B; other materials that were tested but failed to remove interference were graphitized carbon black/primary-secondary amine (PSA), octadecylsilane (C18), Florisil, Oasis MCX, and strong anion exchange-PSA. The method was specifically developed for Product B, which was spiked with 41 organochlorine and organophosphorus pesticides, and resulted in the recovery of 80 to 120% at U.S. Pharmacopeia limits (0.06 to 4 microg/g) for the majority of the pesticides.