Interactions of Pb2+ with fulvic acid by electrophoretically mediated on-capillary microanalysis

Studies of yessotoxin involving confirmation of fragmentation processes using a high-resolution orthogonal hybrid quadrupole time-of-flight (QqTOF) mass spectrometer and nanoLC hybrid quadrupole TOF MS have been undertaken. The fragmentation of YTX was studied in negative mode using nano electrospray (nanoESI) QqTOF mass spectrometry. Three major molecule-related ions were observed, [M - 2Na + H]-, [M - Na]- and [M - 2Na]2-, and fragmentation of the latter was studied in detail. This showed that product ions were formed as a consequence of charge-remote fragmentation processes that included a strong directional cleavage of the polyether rings of YTX. NanoLC coupled with QqTOF MS was used to determine YTX in small samples of the phytoplankton, Protoceratium reticulatum, by monitoring the [M - 2Na]2- ion at m/z 570. A PepMap C18 nanoLC column (75 microm x 10 cm, 100 A, 3 microm, LC Packings) was used and the solvent was acetonitrile/water (90:10 (v/v)) containing 1 mM ammonium acetate, at a flow rate of 400 nl/min, for 30 min. Calibrations obtained with YTX standard solutions were linear over four orders of magnitude, 0.75-250 ng/ml; r2 = 0.9947-0.9998. Phytoplankton cells (ca. 100-300) were picked, extracted with methanol/water (40:60), and the YTX concentration was determined over the range 0.011-0.020 ng/cell. The detection limit (3 x S/N) of this method was ca. 0.5 pg YTX on-column.     

Liquid chromatography with electrospray ion-trap mass spectrometry for the determination of anatoxins in cyanobacteria and drinking water

Anatoxin-a (AN) and homoanatoxin-a (HMAN) are potent neurotoxins produced by a number of cyanobacterial species. A new, sensitive liquid chromatography/multiple tandem mass spectrometry (LC/MS(n)) method has been developed for the determination of these neurotoxins. The LC system was coupled, via an electrospray ionisation (ESI) source, to an ion-trap mass spectrometer in positive ion mode. The [M+H](+) ions at m/z 166 (anatoxin-a) and m/z 180 (homoanatoxin-a) were used as the precursor ions for multiple MS experiments. MS(2)bond;MS(4) spectra displayed major fragment ions at m/z 149 (AN), 163 (HMAN), assigned to [Mbond;NH(3)+H](+); m/z 131 (AN), 145 (HMAN), assigned to [Mbond;NH(3)bond;H(2)O+H](+), and m/z 91 [C(7)H(7)](+). Although the chromatographic separation of these neurotoxins is problematic, reversed-phase LC, using a C(18) Luna column, proved successful. Calibration data for anatoxin-a using spiked water samples (10 mL) in LC/MS(n) modes were: LC/MS (25-1000 microg/L), r(2) = 0.998; LC/MS(2) (5-1000(microg/L), r(2) = 0.9993; LC/MS(3) (2.5-1000 microg/L), r(2) = 0.9997. Reproducibility data (% RSD, N = 3) for each LC/MS(n) mode ranged between 2.0 at 500 microg/L and 7.0 at 10 microg/L. The detection limit (S/N = 3) for AN was better than 0.03 ng (on-column) for LC/MS(3) which corresponded to 0.6 microg/L.     

Determination of the metabolites of gestrinone in human urine by high performance liquid chromatography, liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry

Gestrinone was studied by high performance liquid chromatography (HPLC) for screening and by gas chromatography/mass spectrometry (GC/MS) for confirmation. When the chromatograms of blank, spiked urine and dosed urine were compared by HPLC, two unknown metabolites were found and these were excreted as the conjugated forms. Metabolites 1 and 2 were tested by LC/MS and LC/MS/MS and both had parent ions at m/z 325. The fragment ion of metabolite 1 was at m/z 263 and ions for metabolite 2 were m/z 307 [MH - H(2)O](+), 289, 279 and 241. LC/MS/MS of m/z 263 as the parent ion of metabolite 1 gave fragment ions at m/z 245 and 217, which were assumed to be [263 - H(2)O](+) and [235 - H(2)O](+), respectively. The trimethylsilyl (TMS)-enol-TMS ether derivative of gestrinone displayed three peaks in its GC/MS chromatogram, formed by tautomerism.     

Isotope edited product ion assignment by α-N labeling of peptides with [2H3(50%)]2,4-Dinitrofluorobenzene

An isotopic modification of Sanger's method for identifying peptide N-termini has been developed to assist peptide sequencing by tandem mass spectrometry. Tryptic peptides, such as Val-His-Leu-Thr-Pro-Val-Glu-Lys, are derivatized with an equimolar mixture of 2,4-dinitrofluorobenzene and [2H3]2,4-dinitrofluorobenzene. Under optimized derivatization conditions, the alpha-amino group could be derivatized while the epsilon-amine of the lysine side chain and the imidazole of histidine remained underivatized. The alpha-dinitrophenyl modified peptides were characterized by electrospray ionization-tandem mass spectrometry (ESI-MS/MS) and liquid chromatography (LC)-ESI-MS. The [M + H]+ ions showed a doublet pattern with a delta m/z of 3 and the [M + 2H]2+ ions were recognized as doublets with a delta m/z of 1.5. MS/MS was employed where both isotopic [M + 2H]2+ ions were alternately subjected to collision-induced dissociation in the second quadrupole. Fragmentation in the ionization source generated identical product ion patterns that were observed during fragmentation in the second quadrupole. In the product ion mass spectra, the N-terminal a and b ions (no c ion observed) are doublets with a delta m/z of 3 or 1.5, while the C-terminal y and z ions (no x ion observed) are singlets appearing at identical masses. Thus, the product ions containing the N-terminus derivatized with a dinitrophenyl group are unequivocally distinguished from the product ions containing the C-terminus. The dinitrophenyl modification generally enhanced the production of a and b ions without diminishing y and z ion yields.     

Multiresidue method for determination of algal toxins in shellfish: single-laboratory validation and interlaboratory study

A method that uses liquid chromatography with tandem mass spectrometry (LC/MS/MS) has been developed for the highly sensitive and specific determination of amnesic shellfish poisoning toxins, diarrhetic shellfish poisoning toxins, and other lipophilic algal toxins and metabolites in shellfish. The method was subjected to a full single-laboratory validation and a limited interlaboratory study. Tissue homogenates are blended with methanol-water (9 + 1), and the centrifuged extract is cleaned up with a hexane wash. LC/MS/MS (triple quadrupole) is used for quantitative analysis with reversed-phase gradient elution (acidic buffer), electrospray ionization (positive and negative ion switching), and multiple-reaction monitoring. Ester forms of dinophysis toxins are detected as the parent toxins after hydrolysis of the methanolic extract. The method is quantitative for 6 key toxins when reference standards are available: azaspiracid-1 (AZA1), domoic acid (DA), gymnodimine (GYM), okadaic acid (OA), pectenotoxin-2 (PTX2), and yessotoxin (YTX). Relative response factors are used to estimate the concentrations of other toxins: azaspiracid-2 and -3 (AZA2 and AZA3), dinophysis toxin-1 and -2 (DTX1 and DTX2), other pectenotoxins (PTX1, PTX6, and PTX11), pectenotoxin secoacid metabolites (PTX2-SA and PTX11-SA) and their 7-epimers, spirolides, and homoYTX and YTX metabolites (45-OHYTX and carboxyYTX). Validation data have been gathered for Greenshell mussel, Pacific oyster, cockle, and scallop roe via fortification and natural contamination. For the 6 key toxins at fortification levels of 0.05-0.20 mg/kg, recoveries were 71-99% and single laboratory reproducibilities, relative standard deviations (RSDs), were 10-24%. Limits of detection were <0.02 mg/kg. Extractability data were also obtained for several toxins by using successive extractions of naturally contaminated mussel samples. A preliminary interlaboratory study was conducted with a set of toxin standards and 4 mussel extracts. The data sets from 8 laboratories for the 6 key toxins plus DTX1 and DTX2 gave within-laboratories repeatability (RSD(R)) of 8-12%, except for PTX-2. Between-laboratories reproducibility (RSDR) values were compared with the Horwitz criterion and ranged from good to adequate for 7 key toxins (HorRat values of 0.8-2.0).     

Negative ion electrospray and tandem mass spectrometric analysis of platelet activating factor (PAF) (1-hexadecyl-2-acetyl-glycerophosphocholine)

The analysis of 1-hexadecyl-2-acetyl-glycerophosphocholine (platelet activating factor, PAF) by negative ion and normal-phase liquid chromatography/tandem mass spectrometry (LC/MS/MS) was investigated as an alternative technique to the currently used gas chromatography/MS and positive ion LC/MS/MS procedures. The positive ion [M + H]+ derived from PAF and generated by electrospray ionization is abundant, but the potential presence of isobaric 1-octadecanoyl-2-lyso-glycerophosphocholine (stearoyl-lyso-GPC) and 1-hexadecanoyl-2-formyl-glycerophosphocholine (PFPC) in biological samples limits the use of the most abundant collision-induced decomposition (CID) transition (formation of the phosphocholine ion, m/z 524-->184) if chromatographic separation is not achieved. Less abundant CID product ions, such as loss of the neutral ketene molecule derived from the respective fatty acyl groups, provide the requisite specificity, but the intensity of these transitions yields a signal-to-noise ratio that greatly diminishes the analytical sensitivity. With negative ion LC/MS/MS, however, the molecular anions [M - 15]- derived from PAF, stearoyl-lyso-GPC and PFPC decompose to the carboxylate anions at m/z 59, 283 and 255, respectively, permitting discrimination of these isobaric molecules even without chromatographic separation. In addition, the CID of [M - 15]- was favorable, yielding ion currents of sufficient intensity to permit the measurement of PAF when isolated from small quantities of biological material. With the use of a stable isotopically labeled variant of PAF and isotope dilution, negative ion LC/MS/MS was found to measure PAF reliably even in the presence of the isobaric stearoyl-lyso-GPC and permitted the use of non-chlorinated mobile phases for normal-phase high-performance LC.     

Mass spectrometry for the characterization of unsulfated chondroitin oligosaccharides from 2-mers to 16-mers. Comparison with hyaluronic acid oligomers

This study reports for the first time the complete liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) and tandem mass spectrometry (MS/MS) analyses performed in negative ion mode of saturated unsulfated chondroitin oligosaccharides up to 16-mers and comparison with hyaluronic acid (HA) oligomers differing only in the nature of the hexosamine residue. MS/MS of the chondroitin disaccharide on the singly charged precursor at m/z 396.1 afforded a glycosidic cleavage C1 product ion at m/z 192.9. In the tetrasaccharide, C2 (m/z 396.0) and C3 (m/z 572.0) product anions were generated by glycosidic cleavage. A C5 [M-2H]2- product ion at m/z 475.1 was generated by the glycosidic cleavage of the hexasaccharide, and a C7 ion (m/z 664.6, charge state of -2) was produced from the octasaccharide. The same fragmentation pattern of deprotonated oligomers was observed for the largest oligosaccharides, from 10- to 16-mers. There has been no previous report of MS/MS spectra for unsulfated chondroitin oligomers of these sizes. Unsulfated saturated chondroitin oligosaccharides with x-mer units and larger than a tetrasaccharide dissociate to almost exclusively form CX-1-type ions. Saturated HA oligomers also afforded the same fragmentation pattern as deprotonated oligomers by ESI-MS and MS/MS analyses. Thus, under the experimental conditions used in the current study, we were unable to distinguish between unsulfated chondroitin and HA.     

Analysis of N7-(benzo[a]pyrene-6-yl)guanine in urine using two-step solid-phase extraction and isotope dilution with liquid chromatography/tandem mass spectrometry

Analysis of urinary N7-(benzo[a]pyren-6-yl)guanine (BP-6-N7Gua), a DNA adduct induced by benzo[a]pyrene, may serve as a risk-associated biomarker for exposure to polyaromatic hydrocarbons (PAHs). In this study a highly sensitive and specific analytical method, incorporating on-line sample preparation coupled with isotope-dilution liquid chromatography and tandem mass spectrometry (LC/MS/MS), was developed to quantitate this adduct in human urine. In order to achieve accurate quantitation, 15N5-labeled BP-6-N7Gua was synthesized to serve as the internal standard, and a two-step solid-phase extraction (SPE) procedure using C8 and SCX cartridges was used for sample cleanup. BP-6-7-N7Gua was analyzed using positive ion LC/MS/MS operated in multiple reaction monitoring (MRM) mode. The [M+H]+ ions at m/z 402 and 407 and the common fragment ion of [M+H]+ at m/z 252 were monitored for quantification. The recovery of this analyte after two-step SPE was 90%, and the limit of detection was 2.5 fmol/mL in 10 mL of urine. This highly specific and sensitive method for BP-6-N7Gua in urine may be applied to assess exposure to PAHs in coke-oven workers for future molecular epidemiology studies on health effects of PAHs.     

Thermochemistry of N-N containing ions: a threshold photoelectron photoion coincidence spectroscopy study of ionized methyl- and tetramethylhydrazine

The unimolecular dissociation reactions of the methylhydrazine (MH) and tetramethylhydrazine (TMH) radical cations have been investigated using tandem mass spectrometry and threshold photoelectron photoion coincidence spectroscopy in the photon energy ranges 9.60-31.95 eV (for the MH ion) and 7.74-29.94 eV (for the TMH ion). Methylhydrazine ions (CH3NHNH2(+*)) have three low-energy dissociation channels: hydrogen atom loss to form CH2NHNH2(+) (m/z 45), loss of a methyl radical to form NHNH2(+) (m/z 31), and loss of methane to form the fragment ion m/z 30, N2H2(+*). Tetramethylhydrazine ions only exhibit two dissociation reactions near threshold: that of methyl radical loss to form (CH3)2NNCH3(+) (m/z 73) and of methane loss to form the fragment ion m/z 72 with the empirical formula C3H8N2(+*). The experimental breakdown curves were modeled with Rice-Ramsperger-Kassel-Marcus theory, and it was found that, particularly for methyl radical loss, variational transition state theory was needed to obtain satisfactory fits to the data. The 0 K enthalpies of formation (delta(f)H0) for all fragment ions (m/z 73, m/z 72, m/z 45, m/z 31, and m/z 30) have been determined from the 0 K activation energies (E0) obtained from the fitting procedure: delta(f)H0[(CH3)2NNCH3(+)] = 833 +/- 5 kJ mol(-1), delta(f)H0 [C3H8N2(+*)] = 1064 +/- 5 kJ mol(-1), delta(f)H0[CH2NHNH2(+)] = 862 +/- 5 kJ mol(-1), delta(f)H0[NHNH2(+)] = 959 +/- 5 kJ mol(-1), and delta(f)H0[N2H2(+*)] = 1155 +/- 5 kJ mol(-1). The breakdown curves have been measured from threshold up to h nu approximately 32 eV for both hydrazine ions. As the photon energy increases, other dissociation products are observed and their appearance energies are reported.     

Determination and excretion study of gestrinone in human urine by high performance liquid chromatography and gas chromatography/mass spectrometry

Gestrinone was studied by HPLC for screening and by GC/MS for confirmation. Three unknown peaks were found by HPLC which are probably the metabolites of gestrinone, and conjugated gestrinone in dosed human urine. The metabolites and gestrinone were excreted as the conjugated forms. The total amounts of metabolite 1 and conjugated gestrinone, recovered after 48 h, were 0.20 and 0.32 mg, respectively. When metabolite 1 was tested by LC/MS and LC/MS/MS, the parent ion was m/z 327, [MH](+), and fragment ions were seen at m/z 309 [MH - H(2)O](+), 291 [MH - 2H(2)O](+), 283, 263 and 239. The TMS-enol-TMS ether derivative of gestrinone has three peaks in the GC/MS chromatogram formed by tautomerism. The reproducibility of the derivatization method was stable and recoveries were over 87% when spiked into blank urine.     

Discovering metabolites of post-harvest fungicides in citrus with liquid chromatography/time-of-flight mass spectrometry and ion trap tandem mass spectrometry

In this study, we benefit from the combination of liquid chromatography (LC)/time-of-flight (TOF) MS accurate mass measurements to generate elemental compositions of ions and LC/ion trap multiple MS (MSn) providing complementary structural information, which is useful for the elucidation of unknown organic compounds at trace levels in complex food extracts. We have applied this approach to investigate different citrus fruits extracts, and we have identified two post-harvest fungicides (imazalil and prochloraz), the main degradation product of imazalil ([M + H]+, m/z 257) and a non-previously reported prochloraz degradation product ([M + H]+, m/z 282). The database-mediated identification of the parent compounds was based on the generated elemental composition obtained from accurate mass measurements and additional qualitative information from the high resolution chlorine isotopic clusters of both the protonated molecules (imazalil, [M + H]+ 297.0556, <0.1 ppm error, 2-Cl; prochloraz, [M + H]+ 376.0381, 1.9 ppm error, 3-Cl) and their characteristic fragments ions (imazalil: m/z 255 and 159; prochloraz: m/z 308 and 266). The correlation between the structural information provided by ion trap MS/MS fragmentation pathways of the parent species and the TOF accurate mass elemental composition data of the degradation products were the key to elucidate the structures of the degradation products of both post-harvest fungicides. Finally, where standards were not available (prochloraz), further confirmation was obtained by synthesizing the proposed degradation product by acid hydrolysis of the parent standard and confirmation by LC/TOF-MS.     

An improved liquid chromatography/tandem mass spectrometry method for the determination of 8-oxo-7,8-dihydro-2'-deoxyguanosine in DNA samples using immunoaffinity column purification

The analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) represents an important biomarker of oxidative stress. A sensitive method for the detection of 8-oxodG in DNA samples has been developed that utilizes immunoaffinity column purification of 8-oxodG followed by liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) multiple reaction monitoring (MRM) mode analysis. An internal standard of stable-isotopically labelled 8-oxodG containing [(15)N(5)] was added prior to the enzymatic digestion of DNA to deoxynucleosides, which was then subjected to immunoaffinity column purification followed by microbore positive ion LC/MS/MS MRM. The 8-oxo-7,8-dihydroguanine (8-oxoG) base product ion at m/z 168 was monitored following cleavage of the glycosidic bond of the 8-oxodG [M+H](+) ion at m/z 284. Similar determinations were made for [(15)N(5)]8-oxodG by monitoring the [(15)N(5)]8-oxoG base product ion at m/z 173 formed from the [M+H](+) ion at m/z 289. The introduction of the immunoaffinity column purification step into the method represents a significant improvement for the accurate determination of 8-oxodG since all artefactual peaks that are observed following the direct injection of digested DNA onto the LC/MS/MS system are removed. The identity of these artefactual peaks has been confirmed to be 2'-deoxyguanosine (dG), thymidine (dT) and 2'-deoxyadenosine (dA). The presence of these artefactual peaks in MRM mode analysis can be explained as a consequence of a concentration effect due to their considerably higher relative abundance in DNA compared to 8-oxodG. The highest signal intensity was observed for the artefactual peak for dA due to the fact that the adenine base formed an adduct with methanol, which is a constituent of the mobile phase. The resulting [M+H](+) ion at m/z 284 (dA m/z 252 + CH(3)OH m/z 32) gave rise to a product ion at m/z 168 following the loss of deoxyribose in MRM mode analysis. Control calf thymus DNA was digested to deoxynucleosides and unmodfied deoxynucleosides were removed by immunoaffinity column purification; the enriched 8-oxodG was determined by LC/MS/MS MRM. The level of 8-oxodG in control calf thymus DNA was determined to be 28.8 +/- 1.2 8-oxodG per 10(6) unmodified nucleotides (n = 5) using 5 microg of digested DNA. The limit of detection of the microbore LC/MS/MS MRM for 8-oxodG was determined to be 25 fmol on-column with a signal-to-noise ratio of 3.5.     

Determination of azaspiracids in shellfish using liquid chromatography/tandem electrospray mass spectrometry.

Azaspiracid (AZA1), a recently discovered marine toxin, is responsible for the new human toxic syndrome, azaspiracid poisoning (AZP), which is caused by the consumption of contaminated shellfish. A new, sensitive liquid chromatography/mass spectrometry (LC/MS) method has been developed for the determination of AZA1 and its analogues, 8-methylazaspiracid (AZA2) and 22-demethylazaspiracid (AZA3). Separation of these toxins was achieved using reversed-phase LC and coupled, via an electrospray ionisation (ESI) source, to an ion-trap mass spectrometer. Spectra showed the protonated molecules, [M + H]+, and their major product ions, due to the sequential loss of two water molecules, [M + H - H2O]+, [M + H - 2H2O]+, in addition to fragment ions that are characteristic of these cyclic polyethers. A highly specific and sensitive LC/MS(3) analytical method was developed and, using shellfish extracts containing AZA1, the detection limit (S/N = 3) was 4 pg on-column, corresponding to 0.8 ng/mL. Using the protocol presented here, this is equivalent to 0.37 ng/g shellfish tissue and good linear calibrations were obtained for AZA1 in shellfish extracts (average r2 = 0.9988). Good reproducibility was achieved with % RSD values (N = 5) ranging from 1.5% (0.75 microg/mL) to 4.2% (0.05 microg/mL). An efficient procedure for the extraction of toxins from shellfish aided the development of a rapid protocol for the determination of the three predominant azaspiracids.     

Electrospray and chemical ionization mass spectrometry of di-n-butyl sulfate. Unimolecular chemistry of its protonated form and quantification method by liquid chromatography/electrospray ionization tandem mass spectrometry

Di-n-butyl sulfate (DNBS) has been studied by electrospray (ESI) and chemical (CI) ionization mass spectrometry. The use of methanol as solvent in electrospray ionization allows observation of relatively abundant [DNBS + CH(3)OH + H](+) ions (m/z 243) which upon collision dissociate to [DNBS + H](+) ions (m/z 211). In both ESI and CI experiments, it is found that [DNBS + H](+) ions lead to m/z 113 daughter ions. The composition of this m/z 113 fragment ion and its mechanism of formation have been established by high resolution measurements and CID-MIKE experiments. An 'internal substitution' reaction involving an ion-neutral intermediate is proposed to explain the formation of a [C(8)H(17)](+) ion (m/z 113) by loss of a H(2)SO(4) molecule. Finally, a LC/ESI-MS/MS quantification method is proposed in which a detection limit of di-n-butyl sulfate in the ppm range is obtained. It is suggested that the quantification method might be extended to higher dialkyl sulfates. Copyright 2000 John Wiley & Sons, Ltd.     

Development of a method for the identification of azaspiracid in shellfish by liquid chromatography-tandem mass spectrometry

Azaspiracid is the main toxin responsible for a number of recent human intoxications in Europe resulting from shellfish consumption. The first micro liquid chromatography-tandem mass spectrometry (micro-LC-MS-MS) method was developed for the determination of this novel shellfish poisoning toxin in mussels. The analyte was extracted from whole mussel meat with acetone and chromatographed on a C18 reversed-phase column (1.0 mm I.D.) by isocratic elution at 30 microl/min with acetonitrile-water (85:15, v/v), containing 0.03% trifluoroacetic acid. The toxin was ionised in an ionspray interface operating in the positive ion mode, where only the intact protonated molecule, [M+H]+, was generated at m/z 842. This served as precursor ion for collision-induced dissociation and three product ions, [M+H-nH2O]- with n=1-3, were identified for the unambiguous toxin confirmation by selected reaction monitoring LC-MS-MS analysis. A detection limit of 20 pg, based on a 3:1 signal-to-noise ratio, was achieved for the analyte. This LC-MS-MS method was successfully applied to determine azaspiracid in toxic cultivated shellfish from two regions of Ireland.     

Enhancement of atmospheric pressure chemical ionization for the determination of free and glycine-conjugated bile acids in human serum

A highly sensitive and accurate method based on the precolumn derivatization of bile acids (BA) with a high ionization efficiency labeling reagent 1,2-benzo-3,4-dihydrocarbazole-9-ethyl-benzenesulfonate (BDEBS) coupled with LC/MS has been developed. After derivatization, BA molecules introduced a weak basic nitrogen atom into the molecular core structure that was readily ionized in commonly used acidic HPLC mobile phases. Derivatives were sufficiently stable to be efficiently analyzed by atmospheric pressure chemical ionization (APCI)-MS/MS in positive-ion mode. The MS/MS spectra of BA derivatives showed an intense protonated molecular ion at m/z [M + H]+. The collision-induced dissociation of the molecular ion produced fragment ions at [MH-H2O]+, [MH-2H2O]+, [MH-3H2O]+. The characteristic fragment ions were at m/z 320.8, 262.8, and 243.7 corresponding to a cleavage of N-CO, O-CO, and C-OCO, respectively, and bonds of derivatized molecules. The selected reaction monitoring, based on the m/z [M+H]+ --> [MH-H2O]+, [MH-2H2O]+, [MH-3H2O]+, 320.8, 262.8, and 243.7 transitions, was highly specific for the BA derivatives. The LODs for APCI in a positive-ion mode, at an S/N of 5, were 44.36-153.6 fmol. The validation results showed high accuracy in the range of 93-107% and the mean interday precision for all standards was <15% at broad linear dynamic ranges (0.0244-25 nmol/mL). Good linear responses were observed with coefficients of > 0.9935 in APCI/MS detection. Therefore, the facile BDEBS derivatization coupled with mass spectrometric analysis allowed the development of a highly sensitive and specific method for the quantitation of trace levels of the free and glycine-conjugated BA from human serum samples.     

Liquid chromatography with mass spectrometry--detection of lipophilic shellfish toxins

A rapid multiple toxin method based on liquid chromatography with mass spectrometry (LC/MS) was developed for the detection of okadaic acid (OA), dinophysistoxin-1 (DTX-1), DTX-2, yessotoxin (YTX), homoYTX, 45-hydroxy-YTX, 45-hydroxyhomo-YTX, pectenotoxin-1 (PTX-1), PTX-2, azaspiracid-1 (AZA-1), AZA-2, and AZA-3. Toxins were extracted from shellfish using methanol-water (80%, v/v) and were analyzed using a C8 reversed-phase column with a 5 mM ammonium acetate-acetonitrile mobile phase under gradient conditions. The method was validated for the quantitative detection of OA, YTX, PTX-2, and AZA-1 in 4 species (mussels, Mytilus edulis; cockles, Cerastoderma edule; oysters, Crassostrea gigas; king scallop, Pecten maximus) of shellfish obtained from United Kingdom (UK) waters. Matrix interferences in the determination of the toxins in these species were investigated. The validated linear range of the method was 13-250 microg/kg for OA, PTX-2, and AZA-1 and 100-400 microg/kg for YTX. Recovery and precision ranged between 72-120 and 1-22%, respectively, over a fortification range of 40-160 microg/kg for OA, PTX-2, and AZA-1 and 100-400 microg/kg for YTX. The limit of detection, reproducibility, and repeatability of analysis showed acceptable performance characteristics. A further LC/MS method using an alkaline hydrolysis step was assessed for the detection of OA, DTX-1, and DTX-2 in their esterified forms. In combination with the LC/MS multiple toxin method, this allows detection of all toxin groups described in Commission Decision 2002/225/EC.     

Electrospray collision-induced dissociation of testosterone and testosterone hydroxy analogs

Complications with the gas chromatographic analysis of steroids prompted the use of alternative techniques for their identification. High-performance liquid chromatography/mass spectrometry with atmospheric pressure ionization allowed the collection of data for structural identification of these compounds. The objective of this study was to investigate the up-front collision-induced dissociation (UFCID) electrospray ionization (ESI) mass spectra of testosterone and monohydroxylated testosterones. The positive ion UFCID ESI mass spectrum of testosterone showed three significant ions at m/z 97, 109 and 123. The relative abundance of these ions in the UFCID ESI mass spectra of monohydroxylated testosterones varied with the position of the hydroxy group. Statistical data allowed the prediction of hydroxy group position on testosterone by evaluation of the relative abundance of the m/z 97, 109, 121 and 123 ions. Data from the ESI mass spectral analysis of testosterone in a deuterated solvent and from the analysis of cholestenone and 4-androstene-3 beta, 17 beta-diol indicated that the initial ionization of testosterone occurred at the 3-one position. CID parent ion monitoring analyses of the m/z 97, 109 and 123 ions indicated that each resulted from different fragmentation mechanisms and originated directly from the [M + H]+ parent ion. The elemental composition of these fragment ions is proposed based on evidence gathered from the CID analysis of the pseudo-molecular ions of [1,2-2H2]-, [2,2,4,6,6-2H5]-, [6,7-2H2]-, [7-2H]-, [19,19,19-2H3]- and [3,4-13C2]testosterone. The structure and a possible mechanism of formation of the m/z 109 and 123 ions is presented. The results of this study advance the understanding of the mechanisms of collision-induced fragmentation of ions.     

Simultaneous measurement of metabolic stability and metabolite identification of 7-methoxymethylthiazolo[3,2-a]pyrimidin-5-one derivatives in human liver microsomes using liquid chromatography/ion-trap mass spectrometry

A liquid chromatography/mass spectrometry (LC/MS) method using an atmospheric pressure chemical ionisation source was used to measure the metabolic stability and metabolite identification of 7-methoxymethylthiazolo[3,2-a]pyrimidin-5-one derivative (1) in human liver microsomes. After 15 min incubation with human liver microsomes, compound 1 exhibited metabolic turnover of 44%. Data-dependent tandem mass spectrometry (MS/MS) scanning was used to generate product ion spectra from the protonated ions of the compound and its metabolites. An unusual metabolite at m/z 407 corresponding to the [M-24+H]+ ion was identified for compound 1. Interestingly, the formation of the [M-24+H]+ ion was not observed in the analogues wherein the fused thieno double bond was substituted (2) and the thieno group replaced by a fused benzo derivative (3). Compounds 2 and 3 exhibited metabolic turnovers of 24 and 30%, yielding oxidative metabolites corresponding to [M+16] and [M+32]+, respectively. Based on these facts the mechanism for [M-24]+ formation in compound 1 through an initial epoxide formation on the double bond of the fused thieno ring followed by hydrolytic ring opening and deacylation is envisaged.     

Analysis of testosterone and dihydrotestosterone from biological fluids as the oxime derivatives using high-performance liquid chromatography/tandem mass spectrometry

A sensitive liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method for the simultaneous quantitative analysis of dihydrotestosterone (DHT) and testosterone (T) from biological fluids has been developed. Commercially available deuterated analogues were used as internal standards. Steroids were extracted from serum or testicular fluid with hexane/ethyl acetate, evaporated to dryness, and treated with hydroxylamine to form their oxime derivatives. Upon chromatographic separation, the compounds were quantified using selected reaction monitoring (SRM). For T, the [M+H](+) ion at m/z 304 and the fragment ion at m/z 124 were used as the precursor and product ions. For DHT the ion cluster [M+H+ACN](+) at m/z 347 and the dissociated ion [M+H](+) at m/z 306 were used as the precursor and product ions, respectively. The limits of detectability on-column were in the sub-femtomole range for both compounds and the intra-day coefficient of variation (CV) for analysis from serum was less than 7% for both compounds. Given its high reproducibility, sensitivity, and relative simplicity, this assay should be of use in determining androgen levels in biospecimens, particularly in settings where sample quantity or steroid concentration are low.