Determination of the Fusarium mycotoxins, fusaproliferin and beauvericin by high-performance liquid chromatography-electrospray ionization mass spectrometry.

A method is described using LC-MS for the detection of the mycotoxins fusaproliferin (FUS) and beauvericin (BEA) in cultures of Fusarium subglutinans and in naturally contaminated maize. Protonated molecular ion signals for FUS and BEA were observed at m/z 445 and m/z 784, respectively. Collision induced dissociation of the readily dehydrated protonated molecular ion of the sesterterpene FUS (m/z 427) led to the loss of another water molecule (m/z 409) and acetic acid (m/z 385), while the cyclic lactone trimer BEA fragmented to yield the protonated dimer (m/z 523) and monomer (m/z 262), respectively. Detection of FUS was best performed in the MS-MS mode while BEA displayed a stronger signal in the MS mode. The on-column instrumental detection limits for pure FUS and BEA were found to be 2 ng and 20 pg (S/N=2) while those in naturally contaminated maize were 1 microg/kg and 0.5 microg/kg, respectively. Five South African strains of F. subglutinans were analyzed following methanol extraction of which four produced FUS at levels between 330 mg/kg and 2630 mg/kg while only three produced BEA at levels between 140 mg/kg and 700 mg/kg. Application of this method to naturally contaminated maize samples from the Transkei region of South Africa showed FUS at levels of 8.8-39.6 microg/kg and BEA at 7.6-238.8 microg/kg.     

Electrospray ionization mass spectrometry monitoring of indigo carmine degradation by advanced oxidative processes

The degradation of the dye indigo carmine in aqueous solution induced by two oxidative processes (H(2)O(2)/iodide and O(3)) was investigated. The reactions were monitored by electrospray ionization mass spectrometry in the negative ion mode, ESI(-)-MS, and the intermediates and oxidation products characterized by ESI(-)-MS/MS. Both oxidative systems showed to be highly efficient in removing the color of the dye aqueous solutions. In the ESI(-)-MS of the indigo carmine solution treated with H(2)O(2) and H(2)O(2)/iodide, the presence of the ions of m/z 210 (indigo carmine in its anionic form, 1), 216, 226, 235, and 244 was noticeable. The anion of m/z 235 was proposed to be the unprecedented hydroperoxide intermediate 2 formed in solution via an electrophilic attack by hydroxyl and hydroperoxyl radicals of the exocyclic C=C bond of 1. This intermediate was suggested to be rapidly converted into the anionic forms of 2,3-dioxo-1H-indole-5-sulfonic acid (3, m/z 226), 2-amino-alpha-oxo-5-sulfo-benzeneacetic acid (4, m/z 244), and 2-amino-5-sulfo-benzoic acid (5, m/z 216). In the ESI(-)-MS of the indigo carmine solution treated with O(3), two main anions were detected: m/z 216 (5) and 244 (4). Both products were proposed to be produced via an unstable ozonide intermediate. Other anions in this ESI(-) mass spectrum were attributed to be [4 - H + Na](-) of m/z 266, [4 - H](2-) of m/z 121.5, and [5 - H](2-) of m/z 107.5. ESI-MS/MS data were consistent with the proposed structures for the anionic products 2-5.     

用Fourier变换质谱研究苯胺衍生物的自身化学电离

报导了3-苯甲酰氧基苯胺和P-溴代苯胺在FT质谱中的自身化学电离, 采用多共振离子消除技术研究离子-分子反应机理. 着重探讨系列离子m/z 200, 290, 380和470的生成机理.     

Analysis of triacetone triperoxide (TATP) and TATP synthetic intermediates by electrospray ionization mass spectrometry

The explosive triacetone triperoxide (TATP) has been analyzed by electrospray ionization mass spectrometry (ESI-MS) on a linear quadrupole instrument, giving a 62.5 ng limit of detection in full scan positive ion mode. In the ESI interface with no applied fragmentor voltage the m/z 245 [TATP + Na](+) ion was observed along with m/z 215 [TATP + Na - C(2)H(6)](+) and 81 [(CH(3))(2)CO + Na](+). When TATP was ionized by ESI with an applied fragmentor voltage of 75 V, ions at m/z 141 [C(4)H(6)O(4) + Na](+) and 172 [C(5)H(9)O(5) + Na](+) were also observed. When the precipitates formed in the synthesis of TATP were analyzed before the reaction was complete, a new series of ions was observed in which the ions were separated by 74 m/z units, with ions occurring at m/z 205, 279, 353, 427, 501, 575, 649 and 723. The series of evenly spaced ions is accounted for as oligomeric acetone carbonyl oxides terminated as hydroperoxides, [HOOC(CH(3))(2){OOC(CH(3))(2)}(n)OOH + Na](+) (n = 1, 2 ... 8). The ESI-MS spectra for this homologous series of oligoperoxides have previously been observed from the ozonolysis of tetramethylethylene at low temperatures. Precipitates from the incomplete reaction mixture, under an applied fragmentor voltage of 100 V in ESI, produced an additional ion observed at m/z 99 [C(2)H(4)O(3) + Na](+), and a set of ions separated by 74 m/z units occurring at m/z 173, 247, 321, 395, 469 and 543, proposed to correspond to [CH(3)CO{OOC(CH(3))(2)}(n)OOH + Na](+) (n = 1,2 ... 5). Support for the assigned structures was obtained through the analysis of both protiated and perdeuterated TATP samples.     

Arsenic speciation in chinese seaweeds using HPLC-ICP-MS and HPLC-ES-MS

Three common Chinese edible seaweeds, one brown (Laminaria japonica) and two red (Porphyra crispata and Eucheuma denticulatum), were examined for their total arsenic content. The As species were extracted with yields of 76.4, 69.8 and 25.0%, respectively. Anion-exchange and cation-exchange high-performance liquid chromatography (HPLC) in combination with inductively coupled plasma mass spectrometry (ICP-MS) were used for the separation of the different arsenic species in two of the three seaweed extracts (Laminaria and Porphyra). The main arsenic species in the algal extracts are arseno sugars, although it has been shown that the Laminaria seaweed contains significant amounts of dimethylarsinic acid (DMA). HPLC was coupled with electrospray mass spectrometry (ES-MS) for structural confirmation of the arsenic species. The mass spectrometer settings for the arseno sugars were optimised using standards. The conclusions drawn on the basis of HPLC-ICP-MS were confirmed by the HPLC-ES-MS data. The HPLC-ES-MS method is capable of determining both arseno sugars and DMA in the seaweeds. The unknown compounds seen in the HPLC-ICP-MS chromatogram of Laminaria could not be ascribed to trimethylarsenic oxide or tetramethylarsonium ion.     

Another designer steroid: discovery, synthesis, and detection of 'madol' in urine

Madol (17alpha-methyl-5alpha-androst-2-en-17beta-ol) was identified in an oily product received by our laboratory in the context of our investigations of designer steroids. The product allegedly contained an anabolic steroid not screened for in routine sport doping control urine tests. Madol was synthesized by Grignard methylation of 5alpha-androst-2-en-17-one and characterized by mass spectrometry and NMR spectroscopy. We developed a method for rapid screening of urine samples by gas chromatography/mass spectrometry (GC/MS) of trimethylsilylated madol (monitoring m/z 143, 270, and 345). A baboon administration study showed that madol and a metabolite are excreted in urine. In vitro incubation with human liver microsomes yielded the same metabolite. Madol is only the third steroid never commercially marketed to be found in the context of performance-enhancing drugs in sports.     

Preliminary investigation of the simultaneous detection of sugars,ascorbic acid,citric acid,and sodium benzoate in non-alcoholic beverages by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was used for the simultaneous detection of sugars, ascorbic acid, citric acid, sodium and/or potassium benzoate in non-alcoholic beverages, with meso-tetrakis(pentafluorophenyl)porphyrin (MW 974) as a matrix. Using potassium hydroxide as dopant, fructose/glucose was detected as the potassiated molecule at m/z 219, whereas potassiated sucrose, [Sucrose. K](+), was detected at m/z 381. Using sodium hydroxide as dopant, the fructose and sucrose ions were detected at m/z 203 and 365, respectively. Citric acid generated multiple ions at m/z 269, 307, and 345, which were assigned to [Citricbond;H+2K](+), [Citricbond;2H+3K](+), and [Citricbond;3H+4K](+), respectively. However, a stored methanolic solution of citric acid produced additional ions at m/z 283, 297, and 321, which were attributed to [Citricbond;2H+CH(3)+2K](+), [Citricbond;3H+2CH(3)+2K](+), and [Citricbond;3H+CH(3)+3K](+), respectively, due to esterification that took place during storage. The limits of detection in water were: ascorbic acid, 0.30 wt%; citric acid, 0.5 wt%; and sodium benzoate, 0.001 wt%. In the beverage formulations, the limits of detection were: ascorbic acid 0.3 wt%, citric acid 0.3 wt%, and sodium benzoate 0.02 wt%. Spiking a water or beverage solution that contained ascorbic and/or citric acid with less than 0.6 wt% of tartaric acid lowered the detection limits of ascorbic and citric acids to 0.2 wt%. This study demonstrates the potential for using MALDI-TOFMS in the quality control analyses of non-alcoholic beverages, particularly with regard to the detection of low molecular weight organic acids in commercial beverage formulations.     

A novel online approach to the determination of isotopic ratios for organically bound chlorine, bromine and sulphur

A novel approach for the measurement of (37)Cl, (81)Br and (34)S in organic compounds containing chlorine, bromine, and sulphur is presented to overcome some of the major drawbacks of existing methods. Contemporary methods either require reference materials with the exact molecular compositions of the substances to be tested, or necessitate several laborious offline procedures prior to isotope analysis. In our online setup, organic compounds are separated by gas chromatography (GC) coupled to a high-temperature reactor. Using hydrogen as a makeup gas, the reactor achieves quantitative conversion of chlorinated, brominated and sulphurated organic compounds into gaseous hydrogen chloride (HCl), hydrogen bromide (HBr), and hydrogen sulphide (H(2)S), respectively. In this study, the GC interface was coupled to a quadrupole mass spectrometer operated in single-ion mode. The ion traces of either H(35)Cl (m/z 36) and H(37)Cl (m/z 38), H(79)Br (m/z 80) and H(81)Br (m/z 82), or H(2)(32)S (m/z 34) and H(2)(34)S (m/z 36), were recorded to determine the isotopic ratios of chlorine, bromine, and sulphur isotopes. The conversion interface presented here provides a basis for a novel method for compound-specific isotope analysis of halogenated and sulphur-containing compounds. Rapid online measurements of organic chlorine-, bromine- and sulphur-containing mixtures will facilitate the isotopic analysis of compounds containing these elements, and broaden their usage in fields of environmental forensics employing isotopic concepts.     

Monitoring the degradation of tetracycline by ozone in aqueous medium via atmospheric pressure ionization mass spectrometry

The degradation of tetracycline (1) by ozone in aqueous solution was investigated. High performance liquid chromatography (HPLC), UV-visible spectroscopy (UV-Vis), and total organic carbon (TOC) analyses revealed that although tetracycline was quickly consumed under this oxidative condition, it did not mineralize at all. Continuous monitoring by electrospray ionization mass spectrometry in the positive ion mode, ESI(+)-MS, revealed that tetracycline (1), detected in its protonated form ([1 + H]+) of m/z 445, reacted to yield almost exclusively two unprecedented oxidation products (2 and 3) via a net insertion of one and two oxygen atoms, respectively. Compound 2, suggested to be formed via an initial 1,3-dipolar cycloaddition of ozone at the C11a-C12 double-bond of 1, and Compound 3, proposed to be produced via a subsequent ozone attack at the C2-C3 double-bond of 2, were detected in their protonated forms in the ESI(+)-MS, i.e., [2 + H]+ of m/z 461 and [3 + H]+ of m/z 477, and were further characterized by ESI(+)-MS(n). LC-APCI(+)-MS (liquid chromatography coupled with atmospheric pressure chemical ionization mass spectrometry in the positive ion mode) experiments corroborated the results.     

气相色谱-质谱联用法测定动物组织中氯霉素、氟甲砜霉素和甲砜霉素的残留量

建立了气相色谱-负离子化学电离源质谱同时测定动物组织中氯霉素(CAP)、甲砜霉素(TAP)和氟甲砜霉素(FF)残留量的方法。样品用乙酸乙酯提取,正己烷分配去脂肪,再用Florisil柱进一步净化,甲苯作为反应介质,用N,O-双(三甲基硅基)三氟乙酰胺(BSTFA)-三甲基氯硅烷(TMCS)(体积比为99∶1)进行硅烷化处理,用间硝基氯霉素(m-CAP)作为内标进行测定。CAP的检测限可达到0.03 μg/kg,TAP和FF的检测限可达到0.2 μg/kg;上述3种药物的标准曲线的线性相关系数均大于0.99。CAP,FF和TAP的批内测定的精密度(以相对标准偏差表示)依次为5.5%,10.4%和8.8%;批间测定的精密度依次为7.4%,20.7%和19.1%。回收率为80.0%~111.5%,相对标准偏差为1.2%~15.4%。该方法前处理步骤简单,处理后杂质干扰少,灵敏度高,适用性强,可用于猪肉及禽类、水产品等多种动物组织中氯霉素类药物残留的检测。     

Confirmatory method for macrolide residues in bovine tissues by micro-liquid chromatography-tandem mass spectrometry

A new confirmatory method for three macrolides (tylosin, tilmicosin and erythromycin) in bovine muscle, liver and kidney by micro-LC-MS-MS using an atmospheric pressure ionisation source and an ionspray interface has been developed. Roxithromycin was used as internal standard. The molecular related ions, [M+2H]2+, at m/z 435 for tilmicosin, and [M+H]+, at m/z 734 and 916 for erythromycin and tylosin, respectively, were the precursor ions for collision-induced-dissociation and two diagnostic product ions for each macrolide were identified for the unambiguous confirmation by selected reaction monitoring LC-MS-MS. Precision values (relative standard deviations) were all below 14.9%, whereas the overall accuracy (relative error) ranged from -17.7 to -9.8% for tylosin, from -17.5 to -10.7% for tilmicosin and from -19.6 to -13.7% for erythromycin, in all the investigated bovine tissues. The limits of quantification were 30 (muscle) or 40 (liver, kidney) microg kg(-1), 20 (muscle) or 150 (liver, kidney) microg kg(-1), 50 (muscle, liver) or 80 (kidney) microg kg(-1), 20 (muscle, liver) or 50 (kidney) microg kg(-1) for tylosin, tilmicosin, erytromycin and roxithromycin, respectively.     

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 of three major triterpenoids in epicuticular wax of cabbage (Brassica oleracea L.) by high-performance liquid chromatography with UV and mass spectrometric detection

Lupeol, together with alpha- and beta-amyrins in smaller quantities, has been found for the first time in the epicuticular wax of white cabbage (Brassica oleracea L. convar. capitata (L.) Alef. var. alba DC) leaf surface extract. The three triterpenoids were identified by a new high-performance liquid chromatographic (HPLC) method with UV and mass spectrometric (MS) detection using atmospheric pressure chemical ionization (APCI). All three isomeric compounds gave a parent ion peak at m/z 409 [M+H-18](+) and the relative intensities of some characteristic fragment ion peaks in tandem mass spectrometric (MS-MS) spectra of this parent ion enabled differentiation between the isomers. An additional peak at m/z 439 [M+H](+), which could be oleanonic or ursonic aldehyde, was detected by HPLC-APCI-MS. Saponification of cabbage leaf surface extract with 20% NaOH in methanol at 65 degrees C for 2h had no influence on lupeol, or alpha- or beta-amyrins, but lead to the formation of three additional compounds, which were not identified.     

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.     

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.     

Quantification of zolpidem in human plasma by liquid chromatography-electrospray ionization tandem mass spectrometry

A simple and robust method for quantification of zolpidem in human plasma has been established using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI MS/MS). Es-citalopram was used as an internal standard. Zolpidem and internal standard in plasma sample were extracted using solid-phase extraction cartridges (Oasis HLB, 1 cm3/30 mg). The samples were injected into a C8 reversed-phase column and the mobile phase used was acetonitrile-ammonium acetate (pH 4.6; 10 mm) (80:20, v/v) at a flow rate of 0.7 mL/min. Using MS/MS in the selected reaction-monitoring (SRM) mode, zolpidem and Es-citalopram were detected without any interference from human plasma matrix. Zolpidem produced a protonated precursor ion ([M+H]+) at m/z 308.1 and a corresponding product ion at m/z 235.1. The internal standard produced a protonated precursor ion ([M+H]+) at m/z 325.1 and a corresponding product ion at m/z 262.1. Detection of zolpidem in human plasma by the LC-ESI MS/MS method was accurate and precise with a quantification limit of 2.5 ng/mL. The proposed method was validated in the linear range 2.5-300 ng/mL. Reproducibility, recovery and stability of the method were evaluated. The method has been successfully applied to bioequivalence studies of zolpidem.     

Study of transaldolase deficiency in urine samples by capillary LC-MS/MS

Transaldolase (TAL) is a key enzyme of the pentose phosphate pathway (PPP). TAL deficiency is a newly recognized cause of liver cirrhosis. We have developed an ion-pair LC separation combined with negative ion electrospray MS/MS detection method to assess PPP metabolites in urine samples from TAL-deficient mice. Sedoheptulose 7-phosphate (S7P), C5-polyols D-arabitol and D-ribitol, and 6-phosphogluconate (6PG) levels were markedly increased in urine of TAL-deficient mice with respect to those of wild-type and heterozygote littermates. The detection limits of S7P, D-arabitol, and 6PG were 0.15 +/- 0.015 pmol, 3.5 +/- 0.41 pmol, and 0.61 +/- 0.055 pmol, respectively. The limit of quantitation was 0.4 +/- 0.024 nmol/ml for S7P, 1.6 +/- 0.11 nmol/ml for 6PG and 10 +/- 0.7 nmol/ml for D-arabitol. Additional metabolites, hexose 6-phosphates (m/z 259), D-ribose 5-phosphate and D-xylulose 5-phosphate (m/z 229), D-fructose 1,6-diphosphate (m/z 339), C6-polyols (m/z 181) and GSSG (m/z 611), that have been positively identified in mouse urine, showed similar levels in control and TAL-deficient mice.     

Unusual collision-induced dissociation of fluorated and non-fluorated alpha-nitrotoluene analogs in a gas chromatograph triple-stage quadrupole mass spectrometer under electron-capturing negative-ion chemical ionization conditions

Unusual collision-induced dissociation (CID) of perfluorated and non-perfluorated alpha-nitrotoluene analogs in a gas chromatograph triple-stage quadrupole (TSQ) mass spectrometer (GC-QqQ-MS) under electron-capturing negative-ion chemical ionization conditions is reported. CID of [M - 1]- of alpha-nitro-2,3,4,5,6-pentafluorotoluene (C6F5CH2-NO2) and alpha-nitro-2,5-difluorotoluene (C6H3F2CH2-NO2) produced an intense ion with m/z 66. By using 15N- or 18O-labelled C6F5CH2-NO2 analogs, we found that this anion has the formula C3NO. By contrast, CID of [M - 1]- of alpha-nitrotoluene (C6H5CH2-NO2) and alpha-nitro-3,5-difluorotoluene (C6H3F2CH2-NO2) produced an anion with m/z 86 with the formula C3H4NO2. The expected CID of the C-N-bond of all alpha-nitrotoluene analogs to form the nitrite anion (NO2-, m/z 46) did not occur. We propose mechanisms for the formation of the anions C3NO and C3H4NO2 in the collision chamber of the TSQ mass spectrometer. The most likely structures for the anion C3NO are :C=C=C=N--O and N triple bond C-C triple bond C--O-. The unique CID behavior of C6F5CH2--NO2 can be utilized to unequivocally identify and accurately quantify nitrite in biological fluids by GC-tandem MS.     

The use of high field/frequency EPR in studies of radical and metal sites in proteins and small inorganic models

Low temperature electron paramagnetic resonance (EPR) spectroscopy with frequencies between 95 and 345 GHz and magnetic fields up to 12 T have been used to study radicals and metal sites in proteins and small inorganic model complexes. We have studied radicals, Fe, Cu and Mn containing proteins. For S = 1/2 systems, the high frequency method can resolve the g-value anisotropy. It was used in mouse ribonucleotide reductase (RNR) to show the presence of a hydrogen bond to the tyrosyl radical oxygen. At 285 GHz the type 2 Cu(II) signal in the complex enzyme laccase is clearly resolved from the Hg(II) containing laccase peroxide adduct. For simple metal sites, the systems over S = 1/2 can be described by the spin Hamiltonian: H(S) = BgS + D[Sz2 - S(S + 1)/3 + E/D (Sx2 - Sy2)]. From the high frequency EPR the D-value can be determined directly by, (I) shifts of g(eff) for half-integer spin systems with large D-values as observed at 345 GHz on an Fe(II)-NO-EDTA complex, which is best described as S = 3/2 system with D = 11.5 cm(-1), E = 0.1 cm(-1) and gx = gy = gz = 2.0; (II) measuring the outermost signal, for systems with small D values, distant of (2S - 1) x absolute value(D) from the center of the spectrum as observed in S= 5/2 Fe(III)-EDTA. In Mn(II) substituted mouse RNR R2 protein the weakly interacting Mn(II) at X-band could be observed as decoupled Mn(II) at 285 GHz.     

Sensitive sulphur-specific detection of omeprazole metabolites in rat urine by high-performance liquid chromatography/inductively coupled plasma mass spectrometry

The use of high-performance liquid chromatography/inductively coupled plasma mass spectrometry (HPLC/ICPMS) with sulphur-specific detection was investigated as a method for obtaining metabolite profiles for the drug omeprazole administered as a 1:1 mixture of (32)S- and (34)S-labelled material. Analysis based on the monitoring of the chromatographic eluent at either m/z 32 or 34 was not successful due to insufficient sensitivity caused by interferences from polyatomic ions. However, reaction of sulphur with oxygen in the hexapole collision cell, combined with monitoring at m/z 48 (for (32)S) or m/z 50 (for (34)S), provided a facile method for metabolite profiling. Detection of m/z 48 was superior in sensitivity to detection of m/z 50.