Rapid Communications in Mass Spectrometry

Draper, William M.; Behniwal, Paramjit; Wijekoon, Donald

doi: 10.1002/rcm.3653pmid: 18666199

The direct determination of dialkyl phosphates (DAPs) in water by strong anion‐exchange (SAX) liquid chromatography/atmospheric pressure chemical ionization (APCI) mass spectrometry was investigated. The SAX high‐performance liquid chromatography (HPLC) column was eluted with methanol/water gradients containing ammonium formate (AF) separating the DAPs which included six dimethyl‐ and diethyl‐substituted phosphates, thiophosphates, and dithiophosphates. The high buffer concentrations required for separation were compatible with −ve APCI, but in +ve APCI the DAPs were unstable giving anomalous ions such as [M+15]+ and [M+29]+. These ions are believed to result from ion molecule reactions with CH3OH 2+ in the plasma. DAPs are very stable in −ve APCI being detected as abundant [M–H]− ions, even with 200 mM AF. At higher AF concentration formate clusters ([M+45]− and [M+91]−) were seen. Fragmentation by collision‐activated dissociation (CAD) was more efficient for deprotonated ethyl‐substituted DAPs which lost ethylene followed by ethanol. APCI instrument detection limits were in the low ng/mL range and the response was highly linear. Isotope dilution quantitation using d10‐diethyl dithiophosphate (DEDTP) as an internal standard produced an instrument detection limit of 2 ng DEDTP/mL and method detection limit (MDL) of 9.3 ng/mL with accuracy of 99% (spike concentration, 25 ng/mL). DAP mixtures required storage in cold, dry conditions and alcohol solvents should be avoided because of solvolysis reactions. Copyright © 2008 John Wiley & Sons, Ltd.

Well, Reinhard; Flessa, Heinz

doi: 10.1002/rcm.3656pmid: 18666201

Isotopic signatures of N2O are increasingly used to constrain the total global flux and the relative contribution of nitrification and denitrification to N2O emissions. Interpretation of isotopic signatures of soil‐emitted N2O can be complicated by the isotopic effects of gas diffusion. The aim of our study was to measure the isotopic fractionation factors of diffusion for the isotopologues of N2O and to estimate the potential effect of diffusive fractionation during N2O fluxes from soils using simple simulations. Diffusion experiments were conducted to monitor isotopic signatures of N2O in reservoirs that lost N2O by defined diffusive fluxes. Two different mathematical approaches were used to derive diffusive isotope fractionation factors for 18O (ε18O), average 15N (εbulk) and 15N of the central (α‐) and peripheral (β‐) position within the linear N2O molecule (ε15Nα, ε15Nβ). The measured ε18O was −7.79 ± 0.27‰ and thus higher than the theoretical value of −8.7‰. Conversely, the measured εbulk (−5.23 ± 0.27‰) was lower than the theoretical value (−4.4‰). The measured site‐specific 15N fractionation factors were not equal, giving a difference between ε15Nα and ε15Nβ (εSP) of 1.55 ± 0.28‰. Diffusive fluxes of the N2O isotopologues from the soil pore space to the atmosphere were simulated, showing that isotopic signatures of N2O source pools and emitted N2O can be substantially different during periods of non‐steady state fluxes. Our results show that diffusive isotope fractionation should be taken into account when interpreting natural abundance isotopic signatures of N2O fluxes from soils. Copyright © 2008 John Wiley & Sons, Ltd.

Schettgen, Thomas; Musiol, Anita; Kraus, Thomas

doi: 10.1002/rcm.3659pmid: 18666198

Mercapturic acids are highly important and specific biomarkers of exposure to carcinogenic substances in occupational and environmental medicine. We have developed and validated a reliable, specific and very sensitive method for the simultaneous determination of five mercapturic acids derived from several high‐production chemicals used in industry, namely ethylene oxide, propylene oxide, acrylamide, acrolein and N,N‐dimethylformamide. Analytes are enriched and cleaned up from urinary matrix by offline solid‐phase extraction. The mercapturic acids are subsequently separated by means of high‐performance liquid chromatography on a Luna C8 (2) column and specifically quantified by tandem mass spectrometric detection using isotopically labelled analytes as internal standards. The limits of detection (LODs) for N‐acetyl‐S‐2‐carbamoylethylcysteine (AAMA) and N‐acetyl‐S‐2‐hydroxyethylcysteine (HEMA) were 2.5 µg/L and 0.5 µg/L urine, while for N‐acetyl‐S‐3‐hydroxypropylcysteine (3‐HPMA), N‐acetyl‐S‐2‐hydroxypropylcysteine (2‐HPMA) and N‐acetyl‐S‐(N‐methylcarbamoyl)cysteine (AMCC) it was 5 µg/L. These LODs were sufficient to detect the background exposure of the general population. We applied the method on spot urine samples of 28 subjects of the general population with no known occupational exposure to these substances. Median levels for AAMA, HEMA, 3‐HPMA, 2‐HPMA and AMCC in non‐smokers (n = 14) were 52.6, 2.0, 155, 7.1 and 113.6 µg/L, respectively. In smokers (n = 14), median levels for AAMA, HEMA, 3‐HPMA, 2‐HPMA and AMCC were 243, 5.3, 1681, 41.7 and 822 µg/L, respectively. Due to the simultaneous quantification of these mercapturic acids, our method is well suited for the screening of workers with multiple chemical exposures as well as the determination of the background excretion of the general population. Copyright © 2008 John Wiley & Sons, Ltd.

Kertesz, Vilmos; Van Berkel, Gary J.

doi: 10.1002/rcm.3662pmid: 18666197

The imaging resolution of desorption electrospray ionization mass spectrometry (DESI‐MS) was investigated using printed patterns on paper and thin‐layer chromatography (TLC) plate surfaces. Resolution approaching 40 µm was achieved with a typical DESI‐MS setup, which is approximately 5 times better than the best resolution reported previously. This improvement was accomplished with careful control of operational parameters (particularly spray tip‐to‐surface distance, solvent flow rate, and spacing of lane scans). In addition, an appropriately strong analyte/surface interaction and uniform surface texture on the size scale no larger than the desired imaging resolution were required to achieve this resolution. Overall, conditions providing the smallest possible effective desorption/ionization area in the DESI impact plume region and minimizing the analyte redistribution on the surface during analysis led to improved DESI‐MS imaging resolution. Published in 2008 by John Wiley & Sons, Ltd.

Sottani, Cristina; Rinaldi, Paola; Leoni, Emanuela; Poggi, Guido; Teragni, Cristina; Delmonte, Angelo; Minoia, Claudio

doi: 10.1002/rcm.3657pmid: 18666202

A reversed‐phase high‐performance liquid chromatography (rp‐HPLC) system interfaced with an electrospray ionization (ESI) source coupled to tandem mass spectrometry (MS/MS) was developed and validated for the determination of cyclophosphamide (CP), ifosfamide (IF), daunorubicin (DNR), doxorubicin (DXR), and epirubicin (EPI) in human urine. The analysis of samples containing multiple analytes with a dissimilar range of polarities was carried out using a conventional reversed‐phase chromatographic BDS Hypersil C8 column. The analytical run was 15 min. The triple quadrupole mass spectrometer was operated in positive ion mode and multiple reaction monitoring (MRM) was used for drug quantification. The method was validated over a concentration range of 0.2 to 4.0 µg · L−1 for CP, IF, DXR, EPI and 0.15–2.0 µg · L−1 for DNR in human urine. The lower limit of quantification (LLOQ) was 0.2 µg · L−1 for CP, IF, EPI and was set at 0.3 and 0.15 µg · L−1 for DXR and DNR, respectively. The relative standard deviations (RSD%) were <11.2% for inter‐ and intra‐day precisions. The overall accuracy was also within 114.7% for all analytes at the concentrations of the quality control samples. The potential of ionization suppression resulting from the endogenous biological material on the rp‐HPLC/MS/MS method was evaluated and measured. The feasibility of the proposed HPLC/ESI‐MS/MS procedure was demonstrated by analyzing urine samples from pharmacy technicians and nurses working in hospitals or personnel employed in drug‐manufacturing plants. Copyright © 2008 John Wiley & Sons, Ltd.

Gordin, Alexander; Fialkov, Alexander B.; Amirav, Aviv

doi: 10.1002/rcm.3654pmid: 18666200

A major benefit of gas chromatography/mass spectrometry (GC/MS) with a supersonic molecular beam (SMB) interface and its fly‐through ion source is the ability to obtain electron ionization of vibrationally cold molecules (cold EI), which show enhanced molecular ions. However, GC/MS with an SMB also has the flexibility to perform ‘classical EI’ mode of operation which provides mass spectra to mimic those in commercial 70 eV electron ionization MS libraries. Classical EI in SMB is obtained through simple reduction of the helium make‐up gas flow rate, which reduces the SMB cooling efficiency; hence the vibrational temperatures of the molecules are similar to those in traditional EI ion sources. In classical EI‐SMB mode, the relative abundance of the molecular ion can be tuned and, as a result, excellent identification probabilities and very good matching factors to the NIST MS library are obtained. Classical EI‐SMB with the fly‐through dual cage ion source has analyte sensitivity similar to that of the standard EI ion source of a basic GC/MS system. The fly‐through EI ion source in combination with the SMB interface can serve for cold EI, classical EI‐SMB, and cluster chemical ionization (CCI) modes of operation, all easily exchangeable through a simple and quick change (not involving hardware). Furthermore, the fly‐through ion source eliminates sample scattering from the walls of the ion source, and thus it offers full sample inertness, tailing‐free operation, and no ion‐molecule reaction interferences. It is also robust and enables increased column flow rate capability without affecting the sensitivity. Copyright © 2008 John Wiley & Sons, Ltd.

Vaezzadeh, Ali R.; Simicevic, Jovan; Chauvet, Alexis; François, Patrice; Zimmermann‐Ivol, Catherine G.; Lescuyer, Pierre; Deshusses, Jacques P. M.; Hochstrasser, Denis F.

doi: 10.1002/rcm.3658pmid: 18677718

Imaging Mass Spectrometry (IMS) has emerged as a powerful technique in the field of proteomics. The use of Immobilized pH Gradient–IsoElectric Focusing (IPG‐IEF) is also a new trend, as the first dimension of separation, in shotgun proteomics. We report a combination of these two outstanding technologies. This approach is based on the separation of shotgun‐produced peptides by IPG‐IEF. The peptides are then transferred by capillarity to a capture membrane, which is then scanned by the mass spectrometer to generate MS images. This high‐throughput methodology allows a preview of the sample to be obtained in a single day. We report the application of this new pipeline for differential comparison of the membrane proteome of two different strains of Staphylococcus aureus bacteria in a proof‐of‐principle experiment. Copyright © 2008 John Wiley & Sons, Ltd.

Zhang, Wei‐Dong; Qi, Lian‐Wen; Yang, Xiao‐Lin; Huang, Wen‐Zhe; Li, Ping; Yang, Zhong‐Lin

doi: 10.1002/rcm.3660pmid: 18668634

A novel methodology for the identification of tetorigenin and its metabolites in rat bile has been created using liquid chromatography (LC) combined with time‐of‐flight (TOF) and ion trap multiple mass spectrometry (IT‐MSn). As a means to discriminate amongst unknown organic compounds in complex biological matrices, the proposed methodology relies upon the combination of LC/TOF‐MS to provide accurate mass measurements in generating a molecular formula while benefiting from the complementary structural information provided by the LC/IT‐MSn. In this study, the combined approach has been applied to the metabolic fingerprinting chromatograms of rat bile samples before and after tectorigenin administration. All possible metabolites are investigated based on accurate mass data and isotope function using LC/TOF‐MS and structural confirmation using LC/IT‐MSn. Seven phase II metabolities of tectorigenin in rat bile have been successfully elucidated using this novel LC approach and are being reported for the first time. Copyright © 2008 John Wiley & Sons, Ltd.

Salomonsson, Matilda Lampinen; Bondesson, Ulf; Hedeland, Mikael

doi: 10.1002/rcm.3667pmid: 18677706

For the first time chemical derivatization of isomeric drug glucuronides with 1,2‐dimethylimidazole‐4‐sulfonyl chloride (DMISC) has been successfully applied as a tool for determining the site of conjugation. This provides a way to differentiate between glucuronide isomers containing aliphatic and phenolic hydroxyl groups. The analyses were performed with liquid chromatography/electrospray ion trap mass spectrometry (LC/ESI‐MSn). DMISC has previously been shown to react selectively with phenols in estrogens, thus improving sensitivity in ESI‐MS. The model compounds selected for this study were commercially available standards of formoterol, morphine, morphine‐3‐glucuronide (M3G), and morphine‐6‐glucuronide (M6G). Formoterol glucuronides were produced with an enzymatic method in house. Both formoterol and morphine possess one phenolic and one aliphatic hydroxyl group where glucuronidation could take place. The product ion mass spectra of the native morphine glucuronides were indistinguishable due to the initial neutral loss of monodehydrated glucuronic acid (176 u). However, a significant difference between the isomers was observed with DMISC derivatization, as only the form with a free phenol, M6G, gave a detectable reaction product. Formoterol formed two detectable glucuronide isomers in the enzymatic reaction. Their respective sites of conjugation could not be directly determined from the product ion spectra. Reaction with DMISC, however, gave a detectable product with only one of the isomers. Based on previous experience of the preferred DMISC reactions with phenols, and interpretation of the fragmentation pattern of the derivative, it was concluded that the reactive isomer had a free phenol, and was thus conjugated on the aliphatic chain. Copyright © 2008 John Wiley & Sons, Ltd.

Fiori, Jessica; Gotti, Roberto; Albini, Angelo; Cavrini, Vanni

doi: 10.1002/rcm.3661pmid: 18680092

The photostability of guaiazulene (1,4‐dimethyl‐7‐isopropylazulene; GA), a natural azulenic compound used in cosmetic and health‐care products, as well as in pharmaceutical preparations, was investigated in solution (methanol, ethanol, acetonitrile), by different techniques: gas chromatography/mass spectrometry (GC/MS) and high‐performance liquid chromatography combined with atmospheric pressure chemical ionization mass spectrometry and UV detection (LC/APCI‐MS and HPLC/UV). A solar simulator (xenon‐arc lamp) was used as UV‐A radiation source. The study involved: monitoring compound decomposition, identifying products of photodegradation (PPs), assessing the role of oxygen and evaluating the kinetics of the process. Minor PPs are volatile compounds and were characterized by GC/MS, while oligomeric polyoxygenated compounds, tentatively characterized on the basis of MS and MS/MS spectra, were found to be the main photoproducts. The photodegradation was found to be enhanced by the presence of oxygen; nevertheless, determination of the singlet oxygen quantum yield for GA gave a lower value than that for the reference standard Rose Bengal. The obtained results and the developed stability‐indicating methods (GC/MS and LC/MS) are of interest for stability studies and/or quality control purposes of GA as raw material or cosmetic products. Copyright © 2008 John Wiley & Sons, Ltd.