Peter Zöllner

Determination of the metabolites of nitrofuran antibiotics in animal tissue by high-performance liquid chromatography–tandem mass spectrometry

A LC-MS-MS method is presented to analyse simultaneously the metabolites of four nitrofuran antibacterial agents, furazolidone, furaltadone, nitrofurazone and nitrofurantoin in animal muscle tissue. Sample clean-up and analyte enrichment was performed by solid-phase extraction (SPE) with a polystyrene sorbent following combined hydrolysis of the protein-bound drug metabolites and derivatisation of the homogenised tissue with 2-nitrobenzaldehyde. Limits of detection of 0.5-5 ng g(-1) tissue and limits of determination of 2.5-10 ng g(-1) tissue were achieved using electrospray ionisation in positive mode. Analyte identification and quantification was performed according to EU guidelines, using multiple reaction monitoring (MRM) with one precursor ion and two product ions as identifiers. The use of an internal standard in combination with the simplified sample preparation led to a sensitive and reliable analysis method. The yield of the derivatisation reaction was between 66 and 74% and the recovery of SPE reached 92-105% for all values between 10 and 500 ng g(-1). The developed analytical protocol has been applied to contaminated tissue samples of furazolidone- and furaltadone-treated pigs and allowed unequivocal identification and quantification of the metabolites.

Comparison of methods for the determination of ochratoxin A in wine

Abstract Different analytical methods for the determination of ochratoxin A (OTA) in wine have been compared. Sample clean-up was based on solid-phase extraction (SPE) with (i) immunoaffinity or (ii) RP-18 sorbent materials applying different experimental protocols. The detection of OTA was accomplished with high-performance liquid chromatography (HPLC) combined either with electrospray ionisation (ESI) tandem mass spectrometry (MS–MS) or fluorescence detection (FL). Comparative method evaluation was based on the investigation of 18 naturally contaminated red wine samples originating from different European countries. The analytical results are discussed in view of the respective method validation data and the corresponding experimental protocols. In general, analytical data obtained with RP-18 SPE combined with LC–MS–MS detection and immunoaffinity extraction combined with FL offered comparable good results in the sub-ppb concentration level indicating that high selectivity of either the sample clean-up or, alternatively the detection system are equally well-suited to guarantee an accurate OTA analysis in wine.

Determination of zearalenone in grains by high-performance liquid chromatography–tandem mass spectrometry after solid-phase extraction with RP-18 columns or immunoaffinity columns

In this paper a robust, sensitive and selective LC-MS-MS method for the determination of zearalenone (ZON) in several cereals is described. Sample preparation was performed by extraction of the commodities with a mixture of acetonitrile and water followed by solid-phase extraction with RP-18 columns or immunoaffinity columns. The selective determination of ZON was achieved with an atmospheric pressure chemical ionization interface. Using the negative ion mode a detection limit of 0.5 microg/kg and a determination limit of 1 microg/kg grain was achieved, which is by a factor of 100 more sensitive than the positive ion mode. Zearalanone (ZAN), which does not occur in nature, was used as internal standard for quantification. A linear working range from 1.0 microg/kg to 1000 microg/kg could be achieved in grains with a standard deviation of 4% and recovery rates around 100%. All these results were independent from the grain matrices (maize, barley, oats, wheat) when ZAN was used as internal standard. Sample preparation with RP-18 and immunoaffinity materials gave comparable results. In addition, the method was successfully used for the investigation of naturally contaminated maize samples in the course of an interlaboratory comparison test.

Determination of zeranol, taleranol, zearalenone, α- and β-zearalenol in urine and tissue by high-performance liquid chromatography-tandem mass spectrometry

SummaryAn LC-MS/MS method has been developed for the sensitive determination of the anabolic compound zeranol, its epimer taleranol and the mycotoxins zearalenone, α-zearalenol and β-zearalenol in animal urine and meat samples. Sample preparation included extraction of meat samples and enzymatic digest of urine samples followed by solid phase extraction with RP-18 columns for sample clean-up. Mass spectrometric determination was carried out with an atmospheric pressure chemical ionisation interface (APCI) in the multi-reaction monitoring mode (MRM). Using the negative ion mode, a limit of detection between 0.1 and 0.5 ppb and a limit of determination between 0.5 and 1 ppb could be achieved. With zearalanone (ZAN) as internal standard, a linear range between 0.5 (1.0) and 100 ppb in urine samples (cow, pig) and between 1 and 100 ppb in meat samples (cow, calf, pig) could be established. Depending on the biological matrix and analyte, standard deviations were below 8.5%, with average recovery rates around 86% to 102% in spiked samples. The usefulness of the method developed was demonstrated via several contaminated cow and pig urine samples.

Determination of zearalenone and its metabolites α- and β-zearalenol in beer samples by high-performance liquid chromatography–tandem mass spectrometry

Abstract A fast, robust and sensitive LC–MS–MS method for the determination of zearalenone (ZON) and its metabolites α-zearalenol (α-ZOL) and β-zearalenol (β-ZOL) in beer samples is described. Sample preparation was performed by direct RP-18 solid-phase extraction of undiluted beer samples followed by selective determination of analytes by LC–MS–MS applying an atmospheric pressure chemical ionization (APCI) interface. Using the negative ion mode limits of determination of 0.03–0.06 μg l −1 beer and limits of quantification of 0.07–0.15 μg l −1 beer were achieved, which was distinctly more sensitive than in the positive ion mode. Twenty-three beer samples from different countries, produced from different grains and under different brewing conditions, were investigated by this method, but only in one sample could β-ZOL and ZON be detected. Independently of the type of beer, relative standard deviations between 2.1% and 3.3%, a linear working range of 0.15 μg l −1 to 500 μg l −1 beer and recovery rates around 100% could be achieved when zearalanone (ZAN) was used as internal standard.

Characterization of stationary phases for gas chromatography by 29Si NMR spectroscopy

Abstract Commercially available polysiloxanes containing methyl, phenyl, and cyanoalkyl groups and most frequently used as stationary phases in gas chromatography, were characterized by 1 H and 29 Si nuclear magnetic resonance spectroscopy. Microstructure, molecular weight, end group characteristics, branching, and cyclic by-products were determined for all polymers. The microstructure of an alternating, symmetrically substituted dimethyl, diphenylpolysiloxane (SOP-50) determined by 29 Si nuclear magnetic resonance spectroscopy demonstrated few rearrangements after thermal treatment under gas chromatographic conditions at 420°C.

A new 75% diphenyl, 25% dimethyl‐polysiloxane coated on fused silica capillary columns for high temperature gas chromatography

The three 75% phenyl, 25% methyl-polysiloxanes OV-25, PS162, and a new laboratory-made polymer called SOP-75 were coated on fused silica capillary columns and tested in high temperature gas chromatography. The synthesis of SOP-75 as a methoxy-terminated, symmetrically substituted 75% diphenyl, 25% dimethyl-polysiloxane is reported. For better comparison the polymers were characterized by 1 H and 29 Si nuclear magnetic resonance spectroscopy. The coated fused silica capillary columns were evaluated in respect of their selectivity, inertness, separation efficiency, and bleeding. The three stationary phases exhibited similar selectivity and high inertness up to 400 or 410 C. A minimum allowable operating temperature was established, since the capillaries offered insufficient separation efficiency at low temperatures. SOP-75 offered the best chromatographic properties with superior separation efficiency compared to PS126 and OV-25. A commercially available capillary column with similar polarity was used for comparison, where increased separation efficiency and inertness but also increased bleeding of SOP-75 were found. Improved separation of triacylglycerol mixtures was obtained due to the increased polarity and thermal stability of SOP-75. So far, SOP-75 is the polysiloxane with the highest phenyl content that has been successfully coated onto fused silica capillary columns for high temperature gas chromatography.

MALDI mass spectrometry of biomolecules and synthetic polymers using alkali hexacyanoferrate (II) complexes and glycerol as matrix

K4[Fe(CN)6]/glycerol and Na4[Fe(CN)6]/glycerol have been investigated as liquid matrix systems for UV-MALDI MS applying a N2 laser. Analyte molecules were detected as sodium or potassium adduct ions and, in the case of proteins, as well as protonated molecular ions. Mass accuracies were comparable to those found with standard solid matrix systems with −0.06 to +0.05% deviation in the reflectron mode and with −0.24 to +0.13% in the linear mode. Useful results could be obtained within a mass range of 15 000 Da for single-charged proteins and 8000 Da for potassium cationized polyethylene glycols. Detection limits were found for hydrophilic compounds in the low picomol range and for lipophilic compounds as triacylglycerols or peracetylated and partially benzylated carbohydrates in the low femtomol range. As shown by scanning electron microscopic investigations, the generation of a thin homogenous matrix layer was essential for a successful mass spectrometric experiment. A very careful cleaning of the target surface with glacial acid prior to matrix deposition improved the formation of such a matrix film that maximum sensitivity as well as good reproducibility of the experiments could be achieved.

Characterization of stationary phases for gas chromatography by 29Si NMR spectroscopy. II. Silphenylene-siloxane copolymers.

A series of tetramethyl-p-silphenylene-siloxane copolymers with dimethyl, 1H,1H,2H,2H-perfluorodecylmethyl and diphenyl siloxy groups was prepared. 1H and 29Si nuclear magnetic resonance spectroscopy showed that the chosen reaction conditions provided polymers with diphenyl content up to 85%. The theoretical content of the monomer units correlated well with the measured content. Signal assignments of the copolymers and their corresponding chemical shifts are summarized. Information about alternating, randomized or block sequences was obtained by 29Si nuclear magnetic resonance spectroscopy. Limitations of the method for the determination of microstructure parameters are discussed.

Determination of mercapturic acids using 1,4-dihydroxynaphthalene, a new matrix for matrix-assisted UV laser desorption/ionization mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF-MS) was applied for the characterization of six N-acetylcysteine conjugates (mercapturic acids) of synthetic origin. The measurement of mercapturic acids can provide important information for the biomonitoring of exposure to electrophilic compounds. Five isomers of dihydroxynaphthalene were evaluated as a MALDI matrix. 1,4-dihydroxynaphthalene was found to be very effective and the most suitable and was therefore used for further measurements. It has efficient absorbance at 337 nm, low background matrix ion signals and low matrix adduction. In the positive-ion mode only very weak signals were detected. In the negative-ion mode, the spectra exhibited deprotonated molecular ions [M – H]− of high abundance and no adduct peaks or fragment ions were observed. This enabled reliable molecular mass determination of all six mercapturic acids. A detection limit of 1 pmol (signal-to-noise ratio = 3) applied to the target was achieved, which is distinctly more sensitive than other detection methods. In addition, the data obtained for intensities of the deprotonated molecular ion [M – H]− enabled the recording of the degradation kinetics of solutions with two distinct pH values during five days. Among six investigated compounds, only one, 1,2-dihydroxy-4-(N-acetylcysteinyl)butane (DIOL), was found not to be stable in aqueous solution.