Preparation of doubly 13C-labelled benzylpenicillin as internal standard for residue analytical use with GC/MS and LC/MS

A simple and rapid method is described for the preparation of doubly ¹³C-labelled benzylpenicillin for use in isotope dilution analyses of benzylpenicillin residues. The product is characterized by IR, NMR, GC/MS and LC/MS. Tissue analyses demonstrate its advantage with residue analytical methods using mass spectrometric techniques for measurement. Different fragmentation patterns are observed when using GC/EI-MS or LC/electrospray-MS. The consequences for residue analysis are discussed. Received: 28 May 1997 / Revised: 23 July 1997 / Accepted: 25 July 1997     

Ion trap MS/MS of polychlorinated dibenzo-p-dioxins and dibenzofurans: confirming the concept of the molar response

It has been shown that the concept of molar response, which is applicable to single ion monitoring of M⁺, can also be employed for ion trap MS/MS measurements, simplifying the determination of PCDD/PCDF by an extended IDA approach.     

GC/MS on an LC/MS instrument using atmospheric pressure photoionization

Atmospheric pressure (AP) GC/MS was first introduced by Horning et al. [E.C. Horning, M.G. Horning, D.I. Carroll, I. Dzidic, R.N. Stillwell, Anal. Chem. 45 (1973) 936] using ⁶³Ni as a beta-emitter for ionization. Because, at the time special instrumentation was required, the technique was only applied with consistency to negative ion environmental studies where high sensitivity was required [T. Kinouchi, A.T.L. Miranda, L.G. Rushing, F.A. Beland, W.A. Korfmacher, J. High Resolut. Chromatogr., Chromatogr. Commun. 13 (1990) 281]. Currently, AP ion sources are commonly available on LC/MS instruments and recently a method was reported for converting an AP-LC/MS ion source to a combination AP-LC/MS:GC/MS source [C.N. McEwen, R.G. McKay, J. Am. Soc. Mass Spectrom. 16 (2005) 1730]. Here, we report the use of atmospheric pressure photoionization (APPI) with GC/MS and compare this to AP chemical ionization (APCI) GC/MS and electron ionization (EI) GC/MS. Using a nitrogen purge gas, we observe excellent chromatographic resolution and abundant molecular M⁺ and MH⁺ ions as well as structurally significant fragment ions. Comparison of a 9.8 eV UV lamp with a 10.6 eV lamp, as expected, shows that the higher energy lamp gives more universal ionization and more fragment ions than the lower energy lamp. While there are clear differences in the fragment ions observed by APPI-MS versus EI-MS, there are also similarities. As might be expected from the ionization mechanism, APPI ionization is similar to low energy EI. These odd electron fragment ions are useful in identifying unknown compounds by comparison to mass spectra in computer libraries.     

Calibration Point for Electron Ionization MS / MS spectra measured with multiquadrupole mass spectrometers

A protocol for establishing standard instrument conditions for measurement of product ion MS/MS spectra from parent ions produced by electron ionization is presented. Within this protocol, the ion at m/z 231 (C₅F₉ ⁺) from perfluorokerosene or perfluorotributylamine is selected as the parent ion and subjected to collision-induced dissociation. The relative intensities of product ions at m/z 69, 131, and 181 are monitored as a function of collision energy while keeping the target gas pressure constant within the range of 10^?4–10^?6 torr (measured), or a beam attenuation of approximately 30-70%. The collision energy at which the ion intensities for product ions at m/z 69 and 181 are equal is defined as the calibration point at that collision gas pressure; the intensity of the ion at m/z 131 is very close to this value as well. Electron ionization MS/MS spectra taken at the calibration point using two different multiquadrupole instruments show good reproducibility for several test compounds. The high degree of similarity may aid in the establishment of a MS/MS spectral library.     

Selected ion storage versus tandem MS/MS for organochlorine pesticides determination in drinking waters with SPME and GC-MS

The use of two modes for mass spectrometry (MS) detection with an ion trap instrument, selected ion storage (SIS) and tandem mass spectrometry (MS/MS), are compared for the solid-phase microextraction (SPME)–gas chromatography (GC) coupled to mass spectrometry (GC-MS) determination of 16 priority organochlorine pesticides (OCPs) in drinking water samples at the ultratrace levels (ng?L^?1) required by official guidelines in the European legislation. Experimental parameters investigated for the SPME sample preparation were: the type of coating (100?µm polydimethylsiloxane, PDMS, and 65?µm poly(dimethylsiloxane)–divinylbenzene, PDMS/DVB), SPME modality, extraction and desorption times and desorption temperature and the methanol percentage in the SPME working solution. Under the calculated optimal conditions two methodologies were developed, one for SIS and the other for MS/MS modes. The detection limits, precision and accuracy were evaluated for both alternatives and were appropriate to the official guidelines requirements. The SPME–GC-MS(SIS) methodology offered LODs from 0.2–6.6?ng?L^?1, precision below 13% and recoveries between 83 and 110%. The SPME–GC–MS/MS methodology provided limits of detection (LODs) ranging from 0.3 to 7.6 ng?L^?1, % RSD were ≤14% and recoveries of 79–108% were achieved. After the results observed within an Interlaboratory Exercise, the latest MS methodology was selected for the pursued analysis in real drinking water samples. Also, the good results in this round-robin exercise validate the proposed SPME–GC–MS/MS methodology.     

Pharmacokinetic studies of a novel tubulin inhibitor SK1326 in rat plasma by UPLC–MS/MS

A sensitive method for quantitation of SK1326 in rat plasma has been established using ultra-performance liquid chromatography–electrospray ionization tandem mass spectrometry (UPLC–ESI/MS/MS). SK1326 and the internal standard (tramadol) in plasma sample were extracted using acetonitrile. A centrifuged upper layer was then evaporated and reconstituted with a mobile phase of 0.5% formic acid–acetonitrile (35:65, v/v). The reconstituted samples were injected into a C₁₈ reversed-phase column. Using MS/MS in the multiple reaction monitoring mode, SK1326 and tramadol were detected without severe interference from the rat plasma matrix. SK1326 produced a protonated precursor ion ([M + H]⁺) at m/z 432.3 and a corresponding product ion at m/z 114.4. The internal standard produced a protonated precursor ion ([M + H]⁺) at m/z 264.4 and a corresponding product ion at m/z 58.1. Detection of SK1326 in rat plasma by the UPLC–ESI/MS/MS method was accurate and precise with a quantitation limit of 1.0 ng/mL. The validation, reproducibility, stability and recovery of the method were evaluated. The method has been successfully applied to pharmacokinetic studies of SK1326 in rat plasma. The pharmacokinetic parameters of SK1326 were evaluated after intravenous (at a dose of 10 mg/kg) and oral (at a dose of 20 mg/kg) administration of SK1326 in rats. After oral administration (20 mg/kg) of SK1326, the F (fraction absorbed) value was ~77.1%.     

LC/MS and LC/MS/MS based protocol for identification of dyes in historic textiles

Identification of dyes in historic textiles was until recently only based on reversed phase liquid chromatography and diode-array detection (RPLC–DAD). Although in the last years mass spectrometry (MS) is increasingly used as a detection system for liquid chromatography, most applications in the field are directed to identification of the molecular ions or in studies dedicated to degradation products which may be used as markers in RPLC–DAD. In the present work, an analytical protocol for the identification of dyes using RPLC/ESI/MS is presented. Atmospheric pressure electrospray ionization (ESI) was applied, in the negative ion monitoring mode. Both single stage and tandem MS (MS/MS) approaches were considered. An ion trap was used as mass analyzer. Experiments are based on the characterization of standards (natural dyes and/or dyed fibers) with the mass spectrometer sequentially working in the following modes: single MS/full scan, followed by plotting chromatograms through ion extraction (IEC) according to mass/charge ratios corresponding to molecular ions; single MS/selected ion monitoring (SIM) mode; tandem MS/single reaction monitoring (SRM) mode; tandem MS/multiple reactions monitoring (MRM) or product ion scanning modes. A faster chromatographic separation could be applied as MS detection readily balanced the selectivity of the analytical process. In a case study, 11 dyes from 3 biological sources were detected in a 0.5 mg historic sample.     

HPLC-MS/MS identification of tryptophan-derived tetrahydro-β-carboline derivatives in food

Based on electrospray ionization – tandem mass spectrometry coupled to liquid chromatography (HPLC-ESI-MS/MS) a method for separation and selective detection of 1,2,3,4-tetrahydro-β-carboline derivatives (THC) was developed. Retro-Diels Alder (RDA) fragmentation of the tetrahydropyrido moiety resulted in the characteristic neutral loss of 73 amu for tryptophan-derived THC-3-carboxylates. Accordingly, Pictet-Spengler condensation products of tryptamin exhibited product ions formed by loss of 29 amu. However, THC-1-carboxylates as obtained by reaction of tryptamin with α-oxo acids also yielded product ions [M+H-73]⁺, apparently originating from the combination of RDA-cleavage plus subsequent decarboxylation. As result, one had to consider the possibility of false-positive identification of THC-3-carboxylates in presence of isomeric THC-1-carboxylates. In order to overcome these analytical pitfalls, the unequivocal identification of trace amounts of THC-3-carboxylates by HPLC-MS/MS required the chromatographic separation of isomeric THC prior to selected reaction monitoring (SRM). Utilizing SRM, limits of detection for various THC were established in the 10 ng mL^–1 range. Subsequent analysis of food samples like seasoning sauce and yeast extract by HPLC-ESI-MS/MS revealed the presence of tryptamin-derived 1,2,3,4-tetrahydro-β-carboline-1-carboxylic acid, 1-methyl-tetrahydro-β-carboline-1-carboxylic acid, 1-carboxyethyl-tetrahydro-β-carboline and 1-carboxyethyl-tetrahydro-β-carboline-1-carboxylic acid beside established THC-3-carboxylates and -1,3-dicarboxylates. Received: 31 July 1997 / Revised: 23 October 1997 / Accepted: 30 October 1997     

LC/ESI‐MS/MS characterisation of lipopeptide biosurfactants produced by the Bacillus licheniformis V9T14 strain

Lipopeptide biosurfactants produced by the Bacillus licheniformis V9T14 strain showed an interesting anti‐adhesion activity against biofilm formation of human pathogenic bacterial strains. The chemical characterisation of the crude extract of V9T14 strain was first developed through electrospray ionisation mass spectrometry (ESI‐MS) and ESI‐MS/MS direct infusions: two sets of molecular ion species belonging to the fengycin and surfactin families were revealed and their structures defined, interpreting their product ion spectra. The LC/ESI‐MS analysis of the crude extract allowed to separate in different chromatogram ranges the homologues and the isoforms of the two lipopeptide families. The extract was then fractionated by silica gel chromatography in two main fractions, I and II. The purified biosurfactants were analysed through a new, rapid and suitable LC/ESI‐MS/MS method, which allowed characterising the composition and the structures of the produced lipopeptides. LC/ESI‐MS/MS analysis of fraction I showed the presence of C₁₃, C₁₄ and C₁₅ surfactin homologues, whose structures were confirmed by the product ion spectra of the sodiated molecules [M + Na]⁺ at m/z 1030, 1044 and 1058. LC/ESI‐MS/MS analysis of fraction II confirmed the presence of two main fengycin isoforms, with the protonated molecules [M + H]⁺ at m/z 1478 and 1506 corresponding to C₁₇ fengycin A and C₁₇ fengycin B, respectively. Other homologues (C₁₄ to C₁₆) were revealed and confirmed as belonging to fengycin A or B according to the retention times and the product ions generated, although with the same nominal mass. Finally, a relative percentage content of each homologue for both lipopeptides families in the whole extract was proposed. Copyright © 2010 John Wiley & Sons, Ltd.     

Factors that affect ion trap data-dependent MS/MS in proteomics

Quadrupole ion trap scanning parameters for performing bottom-up proteomics in a data-dependent fashion were evaluated on a Finnigan LCQ Deca mass spectrometer. Evaluation of parameters such as the number of averaged full scans, the number of averaged MS/MS scans, and ion injection times were necessary for acquiring high quality MS/MS spectra that yield favorable b and y ion coverage and high correlation to proteins using database searching algorithms. In this study, we demonstrated how the duty cycle of the mass spectrometer affects the number of peptides that can be successfully identified by SEQUEST using a model system of tryptic BSA peptides to mimic a typical complex mixture associated with bottom-up proteomics. The number of averaged scans and the duration of ion accumulation in the trap had a significant effect on the quality of acquired MS/MS spectra. For example, by increasing the ion injection time from 500 ms to 600 ms, peptide HLVDEPQNLIK improved from being improperly identified to being correctly identified with a SEQUEST cross-correlation score of 3.60. As a result of these experiments, we have devised the following set of ion trap parameters for performing bottom-up proteomics analysis in our laboratory: Three averaged full scans, five averaged MS/MS scans, and a maximum ion injection time of 600 ms.     

High‐speed MALDI MS/MS imaging mass spectrometry using continuous raster sampling

A matrix‐assisted laser desorption/ionization time of flight/time of flight tandem mass spectrometer (MALDI TOF/TOF) has been used for high‐speed precursor/fragment ion transition image acquisition. High‐throughput analysis is facilitated by an Nd:YLF solid state laser capable of pulse repetition rates up to 5 kHz, a high digitizer acquisition rate (up to 50 pixels/s), and continuous laser raster sampling. MS/MS experiments are enabled through the use of a precision timed ion selector, second source acceleration, and a dedicated collision cell. Continuous raster sampling is shown here to facilitate rapid MS/MS ion image acquisition from thin tissue sections for the drug rifampicin and for a common kidney lipid, SM4s(d18:1/24:1). The ability to confirm the structural identity of an analyte as part of the MS/MS imaging experiment is an essential part of the analysis. Additionally, the increase in sensitivity and specificity afforded by an MS/MS approach is highly advantageous, especially when interrogating complex chemical environments such as those in biological tissues. Herein, we report continuous laser raster sampling TOF/TOF imaging methodologies which demonstrate 8 to 14‐fold increases in throughput compared with existing MS/MS instrumentation, an important advantage when imaging large areas on tissues. Copyright © 2015 John Wiley & Sons, Ltd.     

Analysis of Chlormequat residues in grain using liquid chromatography-mass spectrometry (LC-MS/MS)

A fast, sensitive and specific method for routine determination of residues from Chlormequat (CAS no. 7003-89-6) is described. The method is based on a simple clean-up using an SPE-C₁₈ cartridge, high-performance liquid chromatography on a standard C₁₈ column (Spherisorb S5 ODS1) and specific detection and quantification by electrospray mass spectrometry (LC-MS/MS). ¹³C-Chlormequat was synthesised for use as internal standard. Samples were extracted with methanol – water – acetic acid. Internal standard and ammonium acetate were added before C₁₈-cartridge clean up and residues eluted with methanol – water – acetic acid, containing 50 mM ammonium acetate. Chromatographic separation was achieved using a solvent composed of acetonitrile – methanol – water – acetic acid (53:21:25:1 by volume), containing 50 mM ammonium acetate. Electrospray ionisation mass spectrometry was employed using m/z 58 (daughter ion of the Chlormequat quaternary ammonium ion, m/z 122) and m/z 61 (daughter ion of the ¹³C-Chlormequat quaternary ammonium ion, m/z 125) for quantification. The LC analysis time was 15 min and the limit of detection of the analytical method was 9 μg/kg. The performance of the method was demonstrated analysing grain material from an inter-comparison study. In Denmark the primary use of Chlormequat chloride (CCC, cycocel, or chlorocholin chloride, CAS no. 999-81-5) is for winter cereals and 11 such winter wheat samples from the Danish National Pesticide Survey were analysed. Residue contents were from below 0.01 up to 0.45 mg/kg, and thus below the EU maximum residue level of 2.0 mg/kg for wheat. Received: 22 December 1997 / Revised: 29 January 1998 / Accepted: 31 January 1998     

Mannose7 Glycan Isomer Characterization by IMS-MS/MS Analysis

The isomers of the Man₇GlcNAc₂ glycan obtained from bovine ribonuclease B have been characterized by ion mobility spectrometry-tandem mass spectrometry (IMS-MS/MS). In these experiments, [Man7 + 2Na]²⁺ precursors having different mobilities are selected by ion mobility spectrometry and analyzed by MS/MS techniques in an ion trap. The fragmentation spectra obtained for various precursor ions are specific, suggesting the isolation or enrichment of different glycan isomers. One fragment ion with a mass-to-charge ratio (m/z) of 903.8 is found to correspond to the loss of an internal mannose residue of a specific isomer. Extracted fragment ion drift time distributions (XFIDTDs) yield distinctive precursor ion drift time profiles indicating the existence of four separate isomers as proposed previously.     

Biopolymer sequencing using a triple quadrupole mass spectrometer in the ESI nozzle-skimmer/precursor ion MS/MS mode

A variety of model biopolymers, including oligonucleotides, oligosaccharides and a synthetic pharmaceutical agent, were sequenced using a triple quadrupole mass spectrometer equipped with an electrospray source and operated in a scan mode referred to as pseudo-MS3. This scan mode consists of three steps: (1) in-source collision-induced dissociation (CID) in the nozzle-skimmer (NS) region, (2) scanning of the fragment ions into the collision cell for further CID, and (3) passing of the secondary fragment ions through the final mass filter at a preselected mass, generally corresponding to the mass of a terminal sequence ion for the biopolymer. The mass spectra are recorded in the precursor ion MS/MS mode where ion selection and detection occur at the third stage of the triple quadrupole but the scan function is determined by the first stage. The advantages and limitations in using this pseudo-MS3 NS/precursor ion MS/MS scan mode for biopolymer sequencing are discussed.     

Particle beam LC/MS screening of polar compounds of contaminated ground water samples from a former gas plant

A commercial particle beam interface coupled to a single quadrupole mass spectrometer has been used for a nontarget screening of highly contaminated water samples from a former gas plant. A liquid chromatographic system has been used to separate hydrophilic compounds with a high percentage water gradient without any loss of efficiency in the MS interface. The ground water showed a high pollution burden in the total ion current chromatogram. Full scan EI mass spectra have been obtained for identification of unknown compounds which could be assigned to oxidation products of heterocyclic aromatic compounds, some known constituents of coal tar. The identification of 6(5H)-phenanthridinone has been confirmed by comparison with a corresponding standard. The difficulties of an unequivocal identification of isoquinolinone and methyl derivatives of quinolinone based on their EI mass spectra are discussed. Received: 7 March 1997 / Revised: 15 May 1997 / Accepted: 22 May 1997     

Particle beam LC/MS screening of polar compounds of contaminated ground water samples from a former gas plant

A commercial particle beam interface coupled to a single quadrupole mass spectrometer has been used for a nontarget screening of highly contaminated water samples from a former gas plant. A liquid chromatographic system has been used to separate hydrophilic compounds with a high percentage water gradient without any loss of efficiency in the MS interface. The ground water showed a high pollution burden in the total ion current chromatogram. Full scan EI mass spectra have been obtained for identification of unknown compounds which could be assigned to oxidation products of heterocyclic aromatic compounds, some known constituents of coal tar. The identification of 6(5H)-phenanthridinone has been confirmed by comparison with a corresponding standard. The difficulties of an unequivocal identification of isoquinolinone and methyl derivatives of quinolinone based on their EI mass spectra are discussed. Received: 7 March 1997 / Revised: 15 May 1997 / Accepted: 22 May 1997     

Determination of N-n-alkyl-dimethylmorpholines and their metabolites by high resolution GC/MS

An analytical method is described to determine simultaneously cis/trans N-n-alkyl-dimethylmorpholines and their metabolites, the 4-(ω-carboxy-n-alkyl) cis/trans-2,6-dimethylmorpholines by pyrolytic alkylation and high resolution GC/MS in aqueous systems and sediments. The substances in the sediment phase were analyzed by GC/MS after extraction with methanol and water, substances in the water phase could be directly determined by GC/MS. The analytical procedure also allows the determination of further carboxylic acids in complex aqueous systems. Received: 9 April 1996 / Revised: 10 June 1996 / Accepted: 14 June 1996     

HPLC‐DAD and HPLC‐ESI‐MS separation,determination and identification of the spin‐labeled diastereoisomers of podophyllotoxin

Spin‐labeled nitroxide derivatives of podophyllotoxin had better antitumor activity and less toxicity than that of the parent compounds. However, the 2‐H configurations of these spin‐labeled derivatives cannot be determined by nuclear magnetic resonance (NMR) methods. In the present paper, a high‐performance liquid chromatography‐diode array detection (HPLC‐DAD) and a high‐performance liquid chromatography‐electrospray ionization tandem mass spectrometry (HPLC‐ESI/MS/MS) method were developed and validated for the separation, identification of four pairs of diastereoisomers of spin‐labeled derivatives of podophyllotoxin at C‐2 position. In the HPLC‐ESI/MS spectra, each pair of diastereoisomers of the spin‐labeled derivatives in the mixture was directly confirmed and identified by [M+H]⁺ ions and ion ratios of relative abundance of [M‐ROH+H]⁺ (ion 397) to [M+H]⁺. When the [M‐ROH+H]⁺ ions (at m/z 397) were selected as the precursor ions to perform the MS/MS product ion scan. The product ions at m/z 313, 282, and 229 were the common diagnostic ions. The ion ratios of relative abundance of the [M‐ROH+H]⁺ (ion 397) to [M+H]⁺, [A+H]⁺ (ion 313) to [M‐ROH+H]⁺, [A+H‐OCH₃]⁺ (ion 282) to [M‐ROH+H]⁺ and [M‐ROH‐ArH+H]⁺ (ion 229) to [M‐ROH+H]⁺ of each pair of diastereoisomers of the derivatives specifically exhibited a stereochemical effect. Thus, by using identical chromatographic conditions, the combination of DAD and MS/MS data permitted the separation and identification of the four pairs of diastereoisomers of spin‐labeled derivatives of podophyllotoxin at C‐2 in the mixture.     

Dual parallel electrospray ionization and atmospheric pressure chemical ionization mass spectrometry (MS), MS/MS and MS/MS/MS for the analysis of triacylglycerols and triacylglycerol oxidation products

Two mass spectrometers, in parallel, were employed simultaneously for analysis of triacylglycerols in canola oil, for analysis of triolein oxidation products, and for analysis of triacylglycerol positional isomers separated using reversed-phase high-performance liquid chromatography. A triple quadrupole mass spectrometer was interfaced via an atmospheric pressure chemical ionization (APCI) interface to two reversed-phase liquid chromatographic columns in series. An ion trap mass spectrometer was coupled to the same two columns using an electrospray ionization (ESI) interface, with ammonium formate added as electrolyte. Electrospray ionization mass spectrometry (ESI-MS) under these conditions produced abundant ammonium adduct ions from triacylglycerols, which were then fragmented to produce MS/MS spectra and then fragmented further to produce MS/MS/MS spectra. ESI-MS/MS of the ammoniated adduct ions gave product ion mass spectra which were similar to mass spectra obtained by APCI-MS. ESI-MS/MS produced diacylglycerol fragment ions, and additional fragmentation (MS/MS/MS) produced [RCO](+) (acylium) ions, [RCOO+58](+) ions, and other related ions which allowed assignment of individual acyl chain identities. APCI-MS of triacylglycerol oxidation products produced spectra like those reported previously using APCI-MS. APCI-MS/MS produced ions related to individual fatty acid chains. ESI-MS of triacylglycerol oxidation products produced abundant ammonium adduct ions, even for those molecules which previously produced little or no intact molecular ions under APCI-MS conditions. Fragmentation (MS/MS) of the [M+NH(4)](+) ions produced results similar to those obtained by APCI-MS. Further fragmentation (MS/MS/MS) of the diacylglycerol fragments of oxidation products provided information on the oxidized individual fatty acyl chains. ESI-MS and APCI-MS were found to be complementary techniques, which together contributed to a better understanding of the identities of the products formed by oxidation of triacylglycerols.