Claudia Oellig

Miniaturized Planar Chromatography Using Office Peripherals

High-performance thin-layer chromatography is a separation technique commonly used to identify and quantify components in chemical mixtures. Sophisticated analytical tools are required to extract the full analytical power from this technique and especially for miniaturized planar chromatography its utility has not been harnessed. A new approach uses an elegant, simplified system assembled from ordinary consumer printers and scanners to perform separations on monolithic and nanostructured ultrathin-layer phases. This system is shown to outperform existing planar chromatographic tools for analysis on miniaturized plates. Analysis can be completed in a manner of minutes, running numerous samples in parallel at a reduced cost, with very low sample and reagent volumes, all using a familiar computer interface with common office peripherals.

Planar solid phase extraction clean-up for pesticide residue analysis in tea by liquid chromatography-mass spectrometry.

Efficient clean-up is indispensable for preventing matrix effects in multi-residue analysis of pesticides in food by liquid and gas chromatography (LC and GC) coupled to mass spectrometry (MS). High-throughput planar solid phase extraction (HTpSPE) was recently introduced as a new clean-up concept in residue analysis of pesticides in fruit and vegetables (C. Oellig, W. Schwack, 2011 [45]). Thin-layer chromatography (TLC) was used to completely separate pesticides from matrix compounds and to focus them into a sharp zone, followed by extraction of the target zone by the TLC-MS interface. As rather challenging matrices, tea samples were chosen in this study. Besides chlorophylls and polyphenols, high amount of caffeine is co-extracted resulting in strong matrix effects both in LC-MS and GC-MS. The former HTpSPE procedure was adapted to initial extracts of green and black tea resulting in colorless extracts nearly free of matrix effects and interferences, as shown for seven chemically representative pesticides (acetamiprid, penconazole, azoxystrobin, chlorpyrifos, pirimicarb, fenarimol, and mepanipyrim). LC-MS/MS calibration curves obtained in the range of 0.002-0.5 mg/kg from matrix-matched standards and solvent standards were nearly identical and demonstrated the effectiveness of clean-up by HTpSPE. Mean recoveries determined by LC-MS/MS against solvent standards at spiking levels of 0.01 and 0.1 mg/kg ranged between 72 and 114% with relative standard deviations (RSDs) of 0.7-4.7% (n=4), while LC-MS measurements of tea samples spiked at 1 mg/kg provided recoveries of 81-104% with RSDs of 1.2-4.9% (n=6). Using LC-MS/MS, the method showed high sensitivity with signal-to-noise ratios>10 for concentrations below 0.002 mg/kg. HTpSPE of one sample was done in a few minutes, while numerous samples were cleaned in parallel at minimal costs with very low sample and solvent consumption.

Planar solid phase extraction—A new clean-up concept in multi-residue analysis of pesticides by liquid chromatography–mass spectrometry

Efficient clean-up is indispensable for preventing matrix effects in multi-residue analysis of pesticides in food by liquid and gas chromatography coupled to mass spectrometry. As a completely new approach, highly automated planar chromatographic tools were applied for powerful clean-up, called high-throughput planar solid phase extraction (HTpSPE). Thin-layer chromatography (TLC) was used to completely separate pesticides from matrix compounds and to focus them into a sharp zone, followed by extraction of the target zone by the TLC-MS interface. HTpSPE resulted in extracts nearly free of interference and free of matrix effects, as shown for seven chemically representative pesticides in four different matrices (apples, cucumbers, red grapes, tomatoes). Regarding the clean-up step, quantification by LC-MS provided mean recovery (against solvent standards) of 90-104% with relative standard deviations of 0.3-4.1% (n=5) for two spiking levels of 0.1 and 0.5 mg/kg. Clean-up of one sample was completed in a manner of minutes, while running numerous samples in parallel at reduced costs, with very low sample and solvent volumes.

Planar solid phase extraction clean-up and microliter-flow injection analysis-time-of-flight mass spectrometry for multi-residue screening of pesticides in food.

For multi-residue analysis of pesticides in food, a sufficient clean-up is essential for avoiding matrix effects in liquid and gas chromatography (LC and GC) analysis coupled to mass spectrometry (MS). In the last two years, high-throughput planar solid phase extraction (HTpSPE) was established as a new clean-up concept for pesticide residue analysis in fruits and vegetables (C. Oellig, W. Schwack, 2011) and tea (C. Oellig, W. Schwack, 2012). HTpSPE results in matrix-free extracts almost free of interferences and matrix effects. In this study, a time-of-flight mass spectrometer (TOFMS) was applied to directly analyze HTpSPE extracts for pesticide residues. This HTpSPE-microliter-flow injection analysis (μL-FIA)-TOFMS approach detects all pesticides at once in a single mass spectrum, without a liquid chromatographic separation step. Complete sample information was obtained after the injection of the cleaned extract within a single peak. Recovery studies for seven representative pesticides in four different matrices (apples, red grapes, cucumbers, tomatoes) provided mean recoveries of 86-116% with relative standard deviations of 1.3-10% (n=5) using the mass signal intensities under the entire sample peak. Comparing the mass spectra of sample peaks from spiked extracts and solvent standards indicated the efficiency of HTpSPE clean-up. A pesticide database search detected all spiked pesticides with a low incidence of false-positives. HTpSPE of one sample required a few minutes, and numerous samples could be cleaned in parallel at minimal cost with low sample and solvent consumption. The μL-FIA-TOFMS screening then needed an additional 6min per sample. The novel screening approach was successfully applied to QuEChERS extracts of several real samples, and the pesticides identified by HTpSPE-μL-FIA-TOFMS were identical to the pesticides detected by common target LC-MS/MS analyses. The high degree of concordantly identified pesticides by the new developed HTpSPE-μL-FIA-TOFMS approach and target LC-MS/MS demonstrates the applicability as a routine screening method.

High-performance thin-layer chromatography (HPTLC) for the simultaneous quantification of the cyclic lipopeptides Surfactin, Iturin A and Fengycin in culture samples of Bacillus species

A high-performance thin-layer chromatography method has been established for the identification and simultaneous quantification of the cyclic lipopeptides Surfactin, Iturin A and Fengycin in Bacillus culture samples. B. subtilis DSM 10T, B. amyloliquefaciens DSM 7T and B. methylotrophicus DSM 23117 were used as model strains. Culture samples indicated that a sample pretreatment is necessary in order to run HPTLC analyses. A threefold extraction of the cell-free broth with the solvent chloroform/methanol (2:1, v/v) gave best results, when all three lipopeptides were included in the analysis. For the mobile phase, a two-step development was considered most suitable. The first development is conducted with chloroform/methanol/water (65:25:4, v/v/v) over a migration distance of 60mm and the second development using butanol/ethanol/0.1% acetic acid (1:4:1, v/v/v) over a migration distance of 60mm, as well. The method was validated according to Validation of Analytical Procedures: Methodology (FDA Guidance) with respect to the parameters linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy and recovery rate. A linear range with R2>0.99 was obtained for all samples from 30ng/zone up to 600ng/zone. The results indicated that quantification of Surfactin has to be performed after the first development (hRF=44), while Fengycin is quantified after the second development (hRF=36, hRF range=20-40). For Iturin A, the results demonstrated that quantification is in favor after the first (hRF=19) development, but also possible after the second (hRF=59) development. LOD and LOQ for Surfactin and Iturin A after the first development, and Fengycin after the second development were determined to be 16ng/zone and 47ng/zone, 13ng/zone and 39ng/zone, and 27ng/zone and 82ng/zone, respectively. Results further revealed the highly accurate and precise character of the developed method with a good inter- and intraday reproducibility. For the precision and accuracy, expressed as % recovery and relative standard deviation, respectively, the determined values did not exceed ±15% as specified by the FDA Guidance. The recovery assay conducted for samples obtained from two strains with the solvent chloroform/methanol (2:1, v/v), which was determined to be most suitable if all three lipopeptides are of interest, gave recoveries of 96.5% and 99.6%, 68.6% and 71.6%, and 102.5% and 95.2% for Surfactin, Iturin A and Fengycin, respectively. Overall, a suitable and reliable method for the simultaneous quantification of the lipopeptides Surfactin, Iturin A and Fengycin in biological samples using HPTLC was successfully developed and validated.

Screening for total ergot alkaloids in rye flour by planar solid phase extraction-fluorescence detection and mass spectrometry.

The analysis of ergot alkaloids is generally performed by high-performance liquid chromatography (HPLC) coupled to fluorescence detection (FLD) or mass selective detection. As for monitoring only the sum of ergot alkaloids is relevant, a fast and easy screening method for the determination of the total alkaloid content was developed using planar solid phase extraction (pSPE). Applying pSPE, recently introduced for pesticide residue analysis in fruits and vegetables (Oellig and Schwack, 2011) and tea (Oellig and Schwack, 2012), all ergot alkaloids are concentrated in a target zone followed by detection as the sum. The herein presented method includes an ammonium acetate buffered extraction step, followed by a fast liquid-liquid partitioning pre-cleaning before pSPE is performed on high-performance thin-layer chromatography (HPTLC) amino plates with a single methanol development to separate the ergot alkaloids from the remaining matrix and to collect them in a single zone. For quantitation, the native fluorescence was used after dipping the plate in n-hexane/paraffin solution for fluorescence enhancement. Limits of detection and quantitation of 0.07 and 0.24 mg/kg rye, respectively, expressed as ergocristine, were well below the currently applied quality criterion limit for rye. Near-100% recoveries were obtained at relevant spiking levels for different rye flour samples. Hence, the fast pSPE-FLD is an efficient and reliable method to screen for the total ergot alkaloid content in rye and a rapid alternative to the HPLC determination of individual alkaloids and to summing them up. HPTLC-MS additionally enables the identification of the ergot alkaloid composition by a single mass spectrum, when utilized as a fingerprint, offering an easy differentiation of Secale cornutum from different origins.

Acetonitrile extraction and dual-layer solid phase extraction clean-up for pesticide residue analysis in propolis

Propolis is a very complex mixture of substances that is produced by honey bees and is known to be a rather challenging matrix for residue analysis. Besides resins, flavonoids and phenols, high amount of wax is co-extracted resulting in immense matrix effects. Therefore a suitable clean-up is crucial and indispensable. In this study, a reliable solid phase extraction (SPE) clean-up was developed for pesticide residue analysis in propolis. The clean-up success was quickly and easily monitored by high-performance thin-layer chromatography with different detection possibilities. The final method consists of the extraction of propolis with acetonitrile according to the QuEChERS method followed by an effective extract purification on dual-layer SPE cartridges with spherical hydrophobic polystyrene-divinylbenzene resin/primary secondary amine as sorbent and a mixture of toluene/acetone (95:5, v/v) for elution. Besides fat-soluble components like waxes, flavonoids, and terpenoids, more polar compounds like organic acids, fatty acids, sugars and anthocyanins were also removed to large extent. Method performance was assessed by recovery experiments at spiking levels of 0.5 and 1mg/kg (n=5) for fourteen pesticides that are relevant for propolis. Mean recoveries determined by HPLC-MS against solvent standards were between 40 and 101%, while calculation against matrix-matched standards provided recoveries of 79-104%. Precision of recovery, assessed by relative standard deviations, were below 9%. Thus, the developed dual-layer SPE clean-up enables the reliable pesticide residue analysis in propolis and provides a suitable alternative to time-consuming clean-up procedures proposed in literature.

Determination of caffeine, theobromine and theophylline in Mate beer and Mate soft drinks by high-performance thin-layer chromatography

Mate beer and Mate soft drinks are beverages produced from the dried leaves of Ilex paraguariensis (Yerba Mate). In Yerba Mate, the xanthine derivatives caffeine, theobromine and theophylline, also known as methylxanthines, are important active components. The presented method for the determination of caffeine, theobromine and theophylline in Mate beer and Mate soft drinks by high-performance thin-layer chromatography with ultraviolet detection (HPTLC-UV) offers a fully automated and sensitive determination of the three methylxanthines. Filtration of the samples was followed by degassing, dilution with acetonitrile in the case of Mate beers for protein precipitation, and centrifugation before the extracts were analyzed by HPTLC-UV on LiChrospher silica gel plates with fluorescence indicator and acetone/toluene/chloroform (4:3:3, v/v/v) as the mobile phase. For quantitation, the absorbance was scanned at 274nm. Limits of detection and quantitation were 1 and 4ng/zone, respectively, for caffeine, theobromine and theophylline. With recoveries close to 100% and low standard deviations reliable results were guaranteed. Experimental Mate beers as well as Mate beers and Mate soft drinks from the market were analyzed for their concentrations of methylxanthines.

Lysergic acid amide as chemical marker for the total ergot alkaloids in rye flour – Determination by high-performance thin-layer chromatography–fluorescence detection

Ergot alkaloids are generally determined by high-performance liquid chromatography (HPLC) coupled to fluorescence detection (FLD) or mass selective detection, analyzing the individual compounds. However, fast and easy screening methods for the determination of the total ergot alkaloid content are more suitable, since for monitoring only the sum of the alkaloids is relevant. The herein presented screening uses lysergic acid amide (LSA) as chemical marker, formed from ergopeptine alkaloids, and ergometrine for the determination of the total ergot alkaloids in rye with high-performance thin-layer chromatography-fluorescence detection (HPTLC-FLD). An ammonium acetate buffered extraction step was followed by liquid-liquid partition for clean-up before the ergopeptine alkaloids were selectively transformed to LSA and analyzed by HPTLC-FLD on silica gel with isopropyl acetate/methanol/water/25% ammonium hydroxide solution (80:10:3.8:1.1, v/v/v/v) as the mobile phase. The enhanced native fluorescence of LSA and unaffected ergometrine was used for quantitation without any interfering matrix. Limits of detection and quantitation were 8 and 26μg LSA/kg rye, which enables the determination of the total ergot alkaloids far below the applied quality criterion limit for rye. Close to 100% recoveries for different rye flours at relevant spiking levels were obtained. Thus, reliable results were guaranteed, and the fast and efficient screening for the total ergot alkaloids in rye offers a rapid alternative to the HPLC analysis of the individual compounds.

Screening for 16-O-methylcafestol in roasted coffee by high-performance thin-layer chromatography–fluorescence detection – Determination of Coffea canephora admixtures to Coffea arabica

16-O-Methylcafestol (16-OMC), the characteristic diterpene exclusively present in Coffea canephora, is an excellent marker for Coffea canephora admixtures to Coffea arabica. Here we show a straightforward, selective and sensitive screening method for the determination of 16-OMC in roasted coffee by high-performance thin-layer chromatography with fluorescence detection (HPTLC-FLD). As internal standard, Sudan IV was used, and a direct saponification with 10% ethanolic potassium hydroxide solution was followed by solid supported liquid extraction with petroleum ether. 16-OMC was selectively derivatized with 2-naphthoyl chloride and analyzed by HPTLC-FLD on silica gel plates with cyclohexane/tert-butyl methyl ether/formic acid (86:14:2, v/v/v) as the mobile phase. The enhanced fluorescence was scanned at UV 244/>320nm. Limits of detection and quantitation of 5 and 14mg 16-OMC/kg coffee allowed the determination of Coffea canephora admixtures to Coffea arabica below 1%. Recoveries for blends of Coffea arabica with Coffea canephora were close to 100%.