Candidate reference method: Determination of valproic acid in serum by isotope-dilution mass spectrometry

Summary A method has been developed for the determination of valproic acid, without derivatization, in human serum by isotope-dilution mass spectrometry using labelled 2-propyl[3,3,3-d₃] valeric[5,5,5-d₃] acid as internal standard for accurate quantification of the concentration of valproic acid in the sample. After acidification, the analyte and internal standard are extracted withn-hexane. The amounts of valproic acid in the serum are calculated from the isotope ratio of valproic acid to labelled valproic acid, which is measured by electron impact (EI) and chemical ionization (CI) selected ion monitoring (SIM). The concentrations of valproic acid in sera measured using isotope-dilution mass spectrometry are compared with results from gas-liquid chromatography (GLC) and fluorescence polarization immunoassay (FPIA). The accuracy, precision and recovery of the GC-MS methods are discussed. The coefficient of variation determined from duplicate samples was less than 1.5%. The detection limit was 10 ng mL⁻¹ at a signal-to-noise ratio of 3:1. Part of this work was presented at the Kongre? der Deutschen Gesellschaft für Laboratoriumsmedizin, Berlin, 1994.     

Determination of theophylline in serum by high performance liquid chromatography/mass spectrometry with atmospheric pressure chemical-ionization (APCI HPLC/MS)

 A method for the determination of theophylline (TH), without derivatization, in serum by isotope dilution mass spectrometry using labelled [1, 3-¹⁵N₂-2-¹³C]theophylline (LTH) as internal standard is described. After deproteinization, the analyte is directly injected into a high performance liquid chromatography – mass spectrometer operating with atmospheric-pressure chemical-ionization (APCI HPLC/MS). The concentrations of TH in sera measured by APCI HPLC/MS are compared with results from gas chromatography – isotope dilution mass spectrometry (GC-ID/MS), high performance liquid chromatography (HPLC) and fluorescence polarization immunoassay (FPIA). The accuracy, precision and recovery of the APCI HPLC/MS and GC-ID/MS methods are discussed. The coefficient of variation (CV) determined from duplicate samples was less than 2%. The detection limit was 10 ng/ml at a signal-to-noise ratio of 3:1. Received: 17 January 1996/Revised: 26 March 1996/Accepted: 5 April 1996     

Determination of histamine in port wines and grape juices by ion-pair extraction and stable isotope dilution GC-MS

Summary An isotope dilution, GC-MS method for the quantitation of histamine in wines and grape juices was assayed. This method includes isolation of histamine and its, deuterated analogue [α,α,β,β-²H₄]histamine, used as the labelled internal standard, by ion-pair extraction with bis-2-ethylhexylphosphate, conversion to the volatile derivative: tris-pentafluorobenzyl-histamine, capillary GC separation and measurement of the abundance ratio of the (M-181)⁺ ions from labelled and unlabelled derivatives. The method was successfully applied to the determination of histamine in several samples of Port wine, red and white table wine and grape juice. Data are given on detectability, linearity, repeatability and recovery.     

Determination of the food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in human hair by solid-phase extraction and gas chromatography-mass spectrometry

Summary A method for the determination of the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in human hair has been developed and validated. Hair samples (200 mg) were dissolved in NaOH (1 M) and PhIP was isolated by successive solid-phase extraction on a polystyrene-divinylbenzene column and on a silica-based mixed-mode column with C₈ and-SO₃ ⁻ functional groups. Quantification was performed by gas chromatography-electron-impact ionization high-resolution mass spectrometry in selected-ion-monitoring mode. The method was validated for determination of PhIP in the concentration range 0.5–25 ng g⁻¹ hair with [²H₃]PhIP as internal standard. The limit of quantification was 0.26 ng g⁻¹ hair. Within-day and between-day precision were in the ranges 1–27% and 2–15% relative standard deviation, respectively. The hair sample used for method validation was found to contain 0.26 ng PhIP g⁻¹ hair.     

Analysis of 19-norandrosterone in human urine by gas chromatography–isotope-dilution mass spectrometry: method adopted by LGC for participation in the Comité Consultatif pour la Quantité de Matière (CCQM) Pilot Study P68

The method used at LGC for analysis of “total” 19-norandrosterone (19-norandrosterone glucuronide plus “free” 19-norandrosterone) in urine for the Comité Consultatif pour la Quantité de Matière Pilot Study (CCQM-P68) is described. The analytical method used was a modified version of the method developed at the National Measurement Institute of Australia, which used a hydrolysis and derivatisation procedure first described by the German Sports University. This method is routinely used by World Anti-Doping Agency-accredited laboratories for sports drug testing. The main modifications made to the method were the use of 19-norandrosterone glucuronide as a calibration standard and 19-norandrosterone glucuronide-d4 as an isotopically labelled internal standard, and the use of a bench-top quadrupole gas chromatograph–mass spectrometer. The results produced by LGC (2.14 ± 0.15 ng g⁻¹ expanded uncertainty, coverage factor k = 2) were in excellent agreement with those from other participating national metrology institutes and thus further validates the exact-matching isotope-dilution mass spectrometric procedures used at LGC for a wide range of reference measurement applications, including measurement of ng g⁻¹ levels of steroids in a biological matrix.     

A method for the determination of theophylline in serum by isotope dilution mass spectrometry

Summary A method for the determination of theophylline in human serum by the isotope dilution/mass spectrometric technique is described. As an internal standard labelled (1,3-¹⁵N₂-2-¹³C)theophylline is added to the serum sample. The analyte and internal standard are extracted with chloroform/2-propanol (90:10) and converted to the trimethylsilyl derivatives. The extraction and silylation procedures are checked by adding theophylline and internal standard in various concentrations to blank serum and determining the recovery. The trimethylsilyl derivatives of labelled and non-labelled theophylline are separated and detected by GC-MS with the mass spectrometer set to m/z 252 and 255. The amounts of theophylline in the serum are calculated from the isotope ratios measured by selected ion monitoring. The accuracy, precision and recovery of this GC-MS method are presented and discussed. The coefficient of variation determined from duplicate samples was less than 2.5%. The detection limit was 10 ng/ml at a signal-to-noise ratio of 3:1.Part of the work was presented at the 32th Kongreß der Deutschen Gesellschaft für Laboratoriumsmedizin, Frankfurt 1991     

Double exchange in polynuclear mixed-valence clusters. 2. Iron-sulfur proteins [Fe4S4]+ and [Fe4S4]3+

The energy states of tetrameric iron clusters with d⁶-d⁶-d⁶-d⁵ and d⁵-d⁵-d⁵-d⁶ electronic configurations, which form the metal frameworks of the [Fe₄S₄]⁺ and [Fe₄S₄]³⁺ iron-sulfur proteins, are calculated using a general approach. It is revealed that the structural distortions of these systems significantly affect the composition of the magnetic states. The effect of the isotropic Heisenberg exchange is considered. Valencia University, Spain. Bordeaux University, France. Moldova State University. Institute of Chemistry, Academy of Sciences, Moldova Republic. Translated fromZhurnal Strukturnoi Khimii, Vol. 37, No. 5, pp. 816–825, September–October, 1996. Translated by I. Izvekova     

Identification and determination of fumonisin FB1 and FB2 in corn and corn products by high-performance liquid chromatography-electrospray-ionization tandem mass spectrometry (HPLC-ESI-MS-MS)

Summary A new method has been developed for qualitative and quantitative analysis of fumonisin FB₁ and FB₂ in corn and corn products. After extractive sample preparation followed by solid phase extraction using a strong anionexchanger, highest selectivity and sensitivity was achieved by high-performance liquid chromatography-electrospray-ionization, tandem mass spectrometry (HPLC-ESI-MS-MS) analysis combined with selected reaction monitoring (SRM). With deuterated fumonisin FB₁-d₆ as internal standard the routine quantitation limit for FB₁ was set at 400 pg (0.8 ng g^–1 sample) with a signal-to-noise ratio of 101. Almost all corn grit and corn meal samples under study were found to be contaminated with fumonisin FB₁ and FB₂ in concentrations ranging from 1 to 200 ng g^–1. Lower fumonisin levels were found in cornflakes, popcorn and baby food, while no fumonisins were detected in canned sweet corn.     

Determination of steryl sulphates in invertebrate tissue by liquid chromatography–tandem mass spectrometry

A method for the identification and quantification of underivatised steryl sulphates in invertebrates by liquid chromatography (LC) coupled with tandem mass spectrometry (MS) involving a single cleanup step has been developed. Negative electrospray ionisation and positive and negative atmospheric-pressure chemical ionisation (APCI) spectra of steryl sulphate showed pseudomolecular ions ([M+H–H₂SO₄]⁺or [M–H]⁻). Collision-induced dissociation (CID) was efficient only in positive APCI. LC-MS in negative APCI was least susceptible to interference and possible differences in response factors. The detection limits (signal-to-noise ratio of 3) based on cholest-5-enyl-3-sulphate in positive and negative APCI modes are 3.66 and 0.73 pmol μL⁻¹, respectively. Calibration plots and response factors for cholest-5-enyl-3-sulphate relative to the internal standard, cholecalciferyl-3-sulphate, in both positive and negative polarities, were linear in the concentration range from 1.22 to 16.4 pmol μL⁻¹ with good coefficients of determination (R ²>0.98). It is suggested that the structure elucidation of steryl sulphates is best achieved in CID positive APCI mode, whereas their quantification should be carried out using negative APCI.     

Characterization of (<Superscript>13</Superscript>C<Subscript>24</Subscript>) T-2 toxin and its use as an internal standard for the quantification of T-2 toxin in cereals with HPLC–MS/MS

In this paper, the structure and the identity of fully ¹³C-substituted T-2 toxin were confirmed using high-resolution mass spectrometry, ¹H-NMR, ¹³C-NMR, tandem mass spectrometry and HPLC–DAD. The purity of this compound was estimated to be at least 98.8% according to UV data. The isotopic distribution of (¹³C₂₄) T-2 toxin indicated a total isotopic enrichment of 98.2 ± 1.0 atom% ¹³C, and the application of different MS measurement modes revealed the MS/MS fragmentation pattern of T-2 toxin. Furthermore, a stable isotope dilution mass spectrometry method for the quantification of T-2 toxin was developed using (¹³C₂₄) T-2 toxin as internal standard. The method was evaluated with and without conventional clean-up and validated for maize and oats. Both cereals showed strong matrix enhancement effects, which could be compensated for through the application of the isotope-substituted internal standard.     

Photodissociation of multilayered trimethylaluminum adsorbed on a cryogenic substrate: A time-of-flight mass-spectrometric study

Photodissociation of trimethylaluminum [Al(CH₃)₃] adsorbed on a silica (SiO₂) substrate at 110 K has been studied by multiphoton ionization time-of-flight mass spectrometry. Translational energy distributions of aluminum and AlCH₃ fragments can be fitted with a composition of two Maxwell–Boltzmann distributions. The two components are attributed to different environments of photodissociating parent molecules in the multilayer.     

Synthesis of [13C6]3,4-diaminobenzoic acid as a precursor for stable isotope labeled benzimidazoles

The synthesis of [¹³C₆]3,4-diaminobenzoic acid from commercially available [¹³C₆]aniline is described in six steps. Salient features of this route include the preparation of a differentially protected 3,4-diaminobenzonitrile, hydrogen gas free aromatic nitro group reduction with ammonium formate and facile benzimidazole ring closure of the ortho-arylenediamine with triethylorthoformate. This stable-labeled 3,4-diaminobenzoic acid is an ideal [M+6]isotopomer to synthesize complex benzimidazole fragments for mass spectrometry internal assays.     

Revisiting type-A uncertainties relating to the measurement of mass fraction of lead using isotope-dilution inductively coupled plasma mass spectrometry: a way of improving measurement precision and expanded uncertainty

A quadrupole inductively coupled plasma mass spectrometer (Q-ICP-MS) has been used for determination of lead in plant materials using isotope-dilution inductively coupled plasma mass spectrometry. The accuracy of the method was demonstrated by analysis of a matrix certified reference material, NIST SRM 1547 Peach Leaves. Specific instrumental parameters of Q-ICP-MS, including isotope analysis mode, integration time per point, number of points per mass, and number of measurements, were optimized to obtain the best measurement precision. The precision (expressed as relative standard deviation) associated with replicate measurement of the ²⁰⁸Pb/²⁰⁶Pb isotope ratio and its mass-bias correction factor was <0.2%. Following “Example A7” of the Eurachem/CITAC Guide, the relative expanded uncertainty, U _rel, (coverage factor k = 2) was found to be ±1.1%, which fulfilled the target value of ±2% maximum and was lower than the uncertainty of ±3.4% reported by NIST based on isotope-dilution thermal ionization mass spectrometry. Sample recovery of 99% was obtained.     

Electron impact ionization mass spectra of lithocholyl amides: Evidence for a C(20) to C(23) rearrangement involving the loss of a C4H9 fragment

Amides of lithocholic acid (3α-hydroxy-5β-cholan-24-oic acid) with 6-aminocaproic acid and 4-aminobutyric acid were prepared and examined by electron impact ionization mass spectrometry. Both these compounds gave an unusual [M ? 57]⁺ fragment. Since the product-ion analysis of [M ? 57]⁺ revealed the presence of fragments corresponding to the intact steroid nucleus in addition to that of the original amino acid (6-aminocaproic acid or 4-aminobutyric acid), we concluded that the integrity of the steroid amide had been retained in this fragment. The absence of this fragment from the product-ion spectrum of [M ? CH₃]⁺ rules out the sequential loss from the molecular ion of 15 + 42 u as the origin of this signal. Mass spectrometry of the 24-¹³C-labelled lithocholylcaproylamide showed the retention of the label in the [M ? 57]⁺ fragment. In contrast, the corresponding compound labelled with deuterium at C(23) showed a significant loss of the label during the formation of this product ion at [M ? 58]⁺. In addition, through a combination of derivatization and tandem mass spectrometry, it was demonstrated that this loss of 57 u represented a rearrangement with the expulsion of a C₄H₉ radical from the side-chain spanning C(20) to C(23) resulting in a truncated steroid-amide fragment. This fragmentation pattern has not been observed in bile acid conjugates with N-α-amino acids.     

Thermal decomposition of the [Pt(NH3)4]2+ complex in nax zeolite: effect of calcination procedure

The effect of the calcination procedure on the decomposition of the [Pt(NH₃)₄]²⁺ complex in a NaX zeolite was studied by mass spectrometry (MS-TPDE) and diffuse reflectance spectroscopy (DRS). The decomposition of the complex took place in two steps. In the first step, under oxygen, the [Pt(NH₃)₄]²⁺ complex was first converted to [Pt(NH₃)₂]²⁺ complex, accompanied by nitrogen release. In the second step, corresponding to the decomposition of the remaining two amine ligands, NO formation was also observed. Under He, the decomposition also occurred in two steps with H₂ liberation. A reaction scheme was proposed for these results.     

Determination of 30 Free Fatty Acids in Two Famous Tibetan Medicines by HPLC with Fluorescence Detection and Mass Spectrometric Identification

A simple and sensitive high-performance liquid chromatographic (HPLC) method with fluorescence detection and mass spectrometric identification has been developed for analysis of 30 long-chain and short-chain free fatty acids (FFAs). The fatty acids were derivatized to their esters with 1-[2-(p-toluenesulfonate)ethyl]-2-phenylimidazole-[4,5-f]-9,10-phenanthrene (TSPP) in N,N-dimethylformamide (DMF) at 90 °C with anhydrous K₂CO₃ as catalyst. A mixture of C₁–C₃₀ fatty acids was completely separated within 60 min by gradient elution on a reversed-phase C₈ column. Qualitative identification of the acids was performed by atmospheric-pressure chemical ionization mass spectrometry (APCI–MS) in positive-ion mode. The fluorescence excitation and emission wavelengths were 260 and 380 nm, respectively. Quantitative determination of the 30 acids in two Tibetan medicines Gentiana straminea and G. dahurica was performed. The results indicated that the medicines contained many FFAs. Linear correlation coefficients for the FFA derivatives were >0.9991. Relative standard deviations (RSDs, n = 6) for the fatty acid derivatives were <3%. Detection limits (at a signal-to-noise ratio of 3:1) were 3.1–38 fmol. When the fatty acid derivatives were determined in the two real samples results were satisfactory and the sensitivity and reproducibility of the method were good.     

Resonance capture of electrons by substituted pyrazoline molecules

The negative ion mass spectra and photoelectron spectra of substituted pyrazolines were studied. A correlation between the ionization energy of the highest occupied molecular orbital and the yield of [MeNHNH₂ ⁻] ions was found. Isomerization of molecular negative ions was studied by resonance electron capture mass spectrometry. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 4, pp. 701–704, April, 2000.     

Mass spectrometry of coordination compounds of transition metals with tetradentate ligands. 11. isomeric quasi-macrocyclic Ni(II) complexes based on S-substituted isothiocarbohydrazides and the structure of “small” ions derived from them

Two series of bonding isomers of Ni(II) coordination compounds with tetradentate quasimacrocyclic ligands based on S-substituted isothiocarbohydrazides were characterized by electron impact (EI) mass spectrometry and by tandem mass spectrometry methods. Conventional EI mass spectra were more isomer specific than metastable ion (MI) and collision induced dissociation (CID) mass spectra of the molecular ions. The MI (and CID) mass spectra of the isomers were very similar. This effect resulted from a facile randomization of Ni–N bonds in the ions possessing low internal energies, prior to their dissociation. The compounds were found to be convenient precursors for coordinatively unsaturated metal-containing ions, [NiL_n]⁺ and [RNiL_n]⁺ (n = 1, 2; L = NCCH₃, NCSCH₃; R = OH, NO). Most of these species had a structure of mono- or disolvated nickel ion. The dissociation of [HONiNCCH₃]⁺ ions was consistent with the formation of two isomers: one corresponding to the [HONi]⁺ ion solvated by acetonitrile and the other is a complex of H₂O with [NiNCCH₂]⁺. A structure of [HO,Ni,(NCCH₃)₂]⁺ ions was best represented by a five-membered cycle formed by two acetonitrile units and the metal atom with the OH group attached to one of the nitrogen atoms.     

Determination of trace levels of pyrethroid metabolites in human urine by capillary gas chromatography-high resolution mass spectrometry with negative chemical ionization

Summary Applications of high-resolution gas chromatography and high-resolution mass spectrometry (GC-MS) for identification and quantitation of trace amounts of pyrethroid metabolites in human urine samples are demonstrated. The method covers the pyrethroid metabolitescis- andtrans-3-(2,2-dichlorovinyl)-2,2-dimethyl-cyclopropane carboxylic acid (cis- andtrans-DCCA),cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane carboxylic acid (cis-DBCA), 4-fluoro-3-phenoxybenzoic acid (FPBA), and 3-phenoxybenzoic acid (3-PBA). After acid-induced hydrolysis of urine samples and exhaustive solvent extraction, a carbodiimide-coupled esterification of the free carboxylic acids with hexafluoroisopropanol (HFIP) is applied. Identification of the derivatives formed is achieved by low-resolution electron-impact mass spectrometry (EIMS) using an ion-trap detector. Quantitation was by capillary gas chromatography—high-resolution mass spectrometry using negative chemical ionization (GC-NCIMS). 2-Phenoxybenzoic acid (2-PBA) served as internal standard. The limits of detection forcis- andtrans-DCCA,cis-DBCA, FPBA and 3-PBA were 0.03 μg L⁻¹ or below. The applicability of the presented method was tested on urine samples of persons exposed to low levels of pyrethroids.