Giuseppe Centineo

Solid-phase microextraction as a clean-up and preconcentration procedure for organochlorine pesticides determination in fish tissue by gas chromatography with electron capture detection.

The feasibility of developing a single-step clean-up, enrichment procedure for organochlorine pesticides (OCPs) in fish tissue samples based on solid-phase microextraction (SPME) was investigated. The general analytical methodology developed combines conventional solid-liquid extraction of the OCPs from the sample using an organic solvent with SPME over the organic extract followed by gas chromatography-electron-capture detection (GC-ECD) analysis. Experimental SPME conditions such as extraction time, temperature and matrix effects were optimised. Under optimised conditions, precision, linearity range, detection limits and accuracy were evaluated. Detection limits obtained for fish tissue samples were in the range of 0.1-0.7 ng g(-1). Good recoveries (over 70% in all cases) were achieved from samples spiked at a concentration level of 10 ng g(-1). The accuracy of the developed SPME-GC-ECD procedure in real samples has been verified by analysing, using the standard addition method, a certified reference material (CRM 430, OCPs in pork fat) with satisfactory results.

Overcoming matrix effects in electrospray: Quantitation of β-agonists in complex matrices by isotope dilution liquid chromatography–mass spectrometry using singly 13C-labeled analogues

In this work, the implementation of isotope dilution mass spectrometry (IDMS) using minimal labeling and isotope pattern deconvolution (IPD) is evaluated as a strategy for the minimization of matrix effects during trace determination of β2-agonists in complex matrices by liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS). First, the parameters affecting the measurement of isotopic composition of organic compounds by liquid chromatography electrospray ionization high resolution mass spectrometry with a time-of-flight analyzer were evaluated using as a case of study three different β2-agonists: clenbuterol, clenproperol and brombuterol. Then, a calibration graph-free IDMS methodology was evaluated in order to overcome matrix effects in LC-ESI-MS in complex samples. In this procedure singly (13)C-labeled analogues of clenbuterol, clenproperol and brombuterol were employed in combination with IPD. Using this approach accurate and precise results were obtained in the simultaneous quantification of β2-agonists in human urine and bovine liver, even at the sub ngg(-1) and particularly in spite of the previously reported matrix effects. Recovery rates in the range of 97-114% in fortified human urine and from 95% to 111% in fortified bovine liver were obtained with RSD (%) of independent recovery experiments always lower than 6%. These results demonstrate that the proposed methodology based on the use of (13)C1-labeled standards and IPD is a reliable approach for accurate LC-MS quantitation of small molecules and compatible with full-scan high-resolution mass spectrometry.

Multiple spiking species-specific isotope dilution analysis by molecular mass spectrometry: simultaneous determination of inorganic mercury and methylmercury in fish tissues.

This work demonstrates, for the first time, the applicability of multiple spiking isotope dilution analysis to molecular mass spectrometry exemplified by the speciation analysis of mercury using GC(EI)MS instrumentation. A double spike isotope dilution approach using isotopically enriched mercury isotopes has been applied for the determination of inorganic mercury Hg(II) and methylmercury (MeHg) in fish reference materials. The method is based on the application of isotope pattern deconvolution for the simultaneous determination of degradation-corrected concentrations of methylmercury and inorganic mercury. Mass isotopomer distributions are employed instead of isotope ratios to calculate the corrected concentrations of the Hg species as well as the extent of species degradation reactions. The isotope pattern deconvolution equations developed here allow the calculation of the different molar fractions directly from the GC(EI)MS mass isotopomer distribution pattern and take into account possible impurities present in the spike solutions employed. The procedure has been successfully validated with the analysis of two different certified reference materials (BCR-464 and DOLT-4) and with the comparison of the results obtained by GC(ICP)MS. For the tuna fish matrix (BCR-464), no interconversion reactions were observed at the optimized conditions of open focused microwave extraction at 70 degrees C during 8 min. However, significant demethylation was found under the same conditions in the case of the certified dogfish liver DOLT-4. Methylation and demethylation factors were confirmed by GC(ICP)MS. Transformation reactions have been found to depend on the sample matrix and on the derivatization reagent employed. Thus, it is not possible to recommend optimum extraction conditions suitable for all types of matrices demonstrating the need to apply multiple spiking methodologies for the determination of MeHg and Hg(II) in biological samples. Double spike isotope dilution analysis methodologies using widespread GC(EI)MS instrumentation are proposed here for the routine analysis of inorganic mercury and methylmercury in fish samples. The estimated method detection limits were below 10 ng g(-1) for both mercury species. Precision was evaluated for the concentrations present in the certified reference materials (CRMs) which vary from 0.1 to 5 microg g(-1), achieving values of coefficients of variation ranging from 7% to 2%. The concentrations obtained in both CRMs analyzed were in excellent agreement with the certified values, demonstrating the accuracy of the method at these concentration levels.

Evaluation of minimal 13C-labelling for stable isotope dilution in organic analysis

A procedure for Stable Isotope Dilution Analysis in molecular Mass Spectrometry which does not require a methodological calibration graph and can be applied in combination with minimal labelling has been evaluated. This alternative approach is based on the determination of the molar fractions for each pure isotope pattern (natural abundance or labelled) contributing to the isotope pattern observed in the mixture of natural abundance and labelled molecules by multiple linear regression. Two labelled compounds, (13)C(1)-labelled or (13)C(6)-labelled phenol, were compared to study the influence of the number of (13)C atoms in the labelled molecule. The procedure was evaluated by comparing the results obtained for the determination of phenol in NIST 1584 CRM by GC-EI-MS using the classical isotope dilution calibration procedure and the new procedure based on multiple linear regression of isotope patterns without a calibration graph. The results obtained using the proposed procedure agreed well with both the certified values and those obtained using the classical Isotope Dilution Mass Spectrometry (IDMS) calibration procedures. For the evaluated procedure, a full uncertainty budget determination has been developed taking into account all uncertainty sources, including those derived from the uncertainties in the isotope patterns of the natural and labelled compounds. The measurements with the (13)C(1)-labelled phenol provided lower propagated uncertainties in comparison to the use of (13)C(6)-labelled phenol.

Comparison of three different ICP-MS instruments in the study of cadmium speciation in rabbit liver metallothionein-1 using reversed-phase HPLC and post-column isotope dilution analysisPresented at the 2002 Winter Conference on Plasma Spectrometry, Scottsdale, AZ, USA, January 6???12, 2002.

The coupling of reversed-phase high-performance liquid chromatography (RP-HPLC) with inductively coupled plasma mass spectrometry (ICP-MS) using post-column isotope dilution analysis (ID) was applied to the study of cadmium binding to standard rabbit liver metallothionein isoform 1 (MT-1). The analytical performance of three different ICP mass spectrometers, time of flight (TOF), double focussing (DF) and quadrupole (Q), was compared according to their performance for cadmium detection during chromatographic separation. Chromatographic conditions were optimised on a Vydac C8 VHP 259 RP column using a water–methanol gradient. Seven different sub-isoforms were distinguished by using this column coupled to a UV–Vis spectrophotometer, which greatly improved the resolution power exhibited by conventional anion-exchange chromatography. The ICP-DF-MS instrument was seriously affected by the methanol gradient and, thereafter, showed the worst performance in all cases. In contrast, the ICP-TOF-MS was able to resist up to 50% methanol with no serious changes in sensitivity or plasma stability. This instrument exhibited the lowest detection limits. The ICP-Q-MS provided results in between the other two instruments. In all cases, the use of post-column isotope dilution analysis was able to correct for matrix effects and signal drift, allowing for accurate determination of total MT concentration and the actual distribution of Cd-binding sub-isoforms.

Simultaneous determination of seven β2-agonists in human and bovine urine by isotope dilution liquid chromatography–tandem mass spectrometry using compound-specific minimally 13C-labelled analogues

Seven β2-agonist (clenproperol, clenbuterol, salbutamol, bronbuterol, ractopamine, clenpenterol and clencyclohexerol) were determined simultaneously in human and bovine urine by isotope dilution LC-ESI-MS/MS in a triple quadrupole instrument. The method is based on the application of multiple linear regression in combination with compound-specific minimally 13C-labelled analogues. Additionally, the increase of the bandpass of the first quadrupole during the selected reaction monitoring (SRM) measurement procedure allowed the simultaneous quantification of the seven compounds at sub ngg-1 levels in a single chromatogram without resorting to a methodological calibration graph. Recovery values at concentration levels between 5.0 and 0.05ngg-1 ranged from 95 to 110% in fortified bovine urine and from 91 to 108% in human urine, with relative standard deviations lower than 5% except for salbutamol and ractopamine. The proposed methodology was validated by analyzing the certified reference material BCR-503 (lyophilized bovine urine) certified for clenbuterol and salbutamol. The limits of detection (LOD) for a sample volume of 10mL of both human and bovine urine was found to be lower than 0.012ngg-1 for all compounds, except to salbutamol in bovine urine which was of 0.029ngg-1. The use of compound-specific isotopically labelled analogues minimally labelled in 13C minimized the occurrence of isotope effects and corrected for matrix effects during ESI ionization and can be efficiently applied for the quantification of ultra-trace concentrations of β2-agonists in human and bovine urine.