Tim T. Häbe

Tracking and identification of antibacterial components in the essential oil of Tanacetum vulgare L. by the combination of high-performance thin-layer chromatography with direct bioautography and mass spectrometry

Two tansy (Tanacetum vulgare L.) essential oils were obtained by steam distillation of the capitula with subsequent liquid-liquid extraction (oil 1) or with use of an auxiliary phase for the trapping of the steam components (oil 2). These oils were investigated against Bacillus subtilis F1276, B. subtilis spizizenii (DSM 618), Xanthomonas euvesicatoria, Pseudomonas syringae pv. maculicola, Ralstonia solanacearum strain GMI1000 and Aliivibrio fischeri, using the coupling of high-performance thin-layer chromatography to direct bioautography (HPTLC-DB). Using this method with the potato and tomato pathogen R. solanacearum is shown for the first time. Due to the advanced extraction process, oil 2 was richer in components and provided more inhibition zones. The main bioactive components were identified by scanning HPTLC-Direct Analysis in Real Time mass spectrometry (HPTLC-DART-MS) and solid-phase microextraction gas chromatography electron impact MS (SPME-GC-EI-MS) as cis- and trans-chrysanthenol as well as trans-chrysanthenyl acetate. cis-Chrysanthenol exhibited antibacterial effects against all tested bacteria, whereas trans-chrysanthenol inhibited B. subtilis, R. solanacearum and A. fischeri. trans-Chrysanthenyl acetate was an inhibitor for X. euvesicatoria, R. solanacearum and A. fischeri. Although HPTLC-DART-MS resulted in a comparable fragmentation, the ionization characteristics and the recorded mass spectra clearly showed that DART is a softer ionization technique than EI. It is also more affected by ambient conditions and thus prone to additional oxidation products.

Quantitative surface scanning by Direct Analysis in Real Time mass spectrometry.

RATIONALE Only a few ambient ionization sources have been demonstrated to work quantitatively for surface scanning. A modification of the Direct Analysis in Real Time mass spectrometry (DART-MS) interface is needed to improve the precision during the scanning of a high-performance thin-layer chromatography (HPTLC) plate or any other surface or planar substrate, especially for quantitation without an internal standard correction. METHODS The substrate movement relative to the ion source outlet and the mass spectrometer inlet was optimized to improve the desorption, ionization, and capture of analytes. The substrate carrier was mounted at an angled position, thus reducing collisions between the deflected gas stream and the inner transfer tube wall. A special transfer tube, whose edge was angled towards the substrate and allowed a narrow set-up of the ambient air gap, captured the deflected DART gas stream. RESULTS For the repeated DART-MS scanning along five identical deposited bands of butyl-4-hydroxybenzoate a mean precision of 2.7% was obtained. A signal decay of 62% was observed after five scans. After HPTLC of methyl-4-hydroxybenzoate and butyl-4-hydroxybenzoate, mean determination coefficients of 0.9937 and 0.9906 were obtained for five calibrations on five plates, respectively. The mean recovery of two control standards was 94% with a mean repeatability of 9% (%RSD, n = 5) obtained on five different plates. CONCLUSIONS The DART SVPA-3DS system remained compact and the access to the substrate was kept wide open despite the optimized scan lane (spatial resolution at full width at half maximum 0.8 mm, height 3 mm). The performance data showed that the quantitative surface scanning was improved as well as the desorption efficacy and detectability using this modified DART-MS interface.

Microfabrication, separations, and detection by mass spectrometry on ultrathin-layer chromatography plates prepared via the low-pressure chemical vapor deposition of silicon nitride onto carbon nanotube templates

Microfabrication of ultrathin-layer chromatography (UTLC) plates via conformal deposition of silicon nitride by low-pressure chemical vapor deposition onto patterned carbon nanotube (CNT) scaffolds was demonstrated. After removal of the CNTs and hydroxylation, the resulting UTLC phase showed no expansion or distortion of their microfeatures and the absence/reduction of remaining nitrogenic species. Developing time of a mixture of lipophilic dyes on this UTLC plates was 86% shorter than on high-performance thin-layer chromatography (HPTLC) plates. A water-soluble food dye mixture was also separated resulting in low band broadening and reduced developing time compared to HPTLC. For the latter example, mobile phase optimization on a single UTLC plate consisted of 14 developments with different mobile phases, each preceded by a plate prewashing step. The same plate was again reused for additional 11 separations under varying conditions resulting in a development procedure with a mean separation efficiency of 233,000theoretical plates/m and a reduced mobile phase consumption of only 400μL. This repeated use proved the physical robustness of the ultrathin layer and its resistance to damage. The layer was highly suited for hyphenation to ambient mass spectrometry, including desorption electrospray ionization (DESI) mass spectrometry imaging and direct analysis in real time (DART) mass spectrometry.

Improved desorption/ionization and ion transmission in surface scanning by direct analysis in real time mass spectrometry

RATIONALE Modifications to the Direct Analysis in Real Time mass spectrometry (DART-MS) interface, its source cap and transfer tube were necessary to obtain highest efficiency in desorption and ionization from the sampling surface and in ion transmission into the MS system. These issues are crucial for the trace analysis of any surface and the hyphenation of high-performance thin-layer chromatography (HPTLC) with DART-MS. METHODS The ion source mounting was modified to enable short source caps to be utilized in combination with a short transfer tube. The grid voltage contact section was readjusted to increase the intensity of the metastable gas stream towards the substrate. Eighteen different cap and two transfer tube geometries (including gas-stream focusing), along with the influence of their distance from the mass spectrometer glass capillary, were investigated for best signal intensity. RESULTS Using shortened source caps with staged inner bore, a transfer tube with gas-stream focusing and an optimized mounting geometry for DART-MS scanning along five identical deposited bands (600 ng each) of butyl 4-hydroxybenzoate, an average signal precision of 3.6% was obtained and the signal intensity was increased by a factor of 34. The width of the gas impact area did not exceed 1.5 mm and the smallest FWHM was determined to be 0.9 mm. CONCLUSIONS The desorption strength, ionization efficacy and ion transmission were improved significantly giving increased detectability using this further modified DART-MS interface with reduced cap length and optimum transfer tube geometry. The resolution was comparable with state-of-the-art densitometry. With this setup, reliable HPTLC surface scanning is possible, even for substance amounts in the low-nanogram range.

Effect-Directed Discovery of Bioactive Compounds Followed by Highly Targeted Characterization, Isolation and Identification, Exemplarily Shown for Solidago virgaurea

A nontargeted, effect-directed screening (bioprofiling) and a subsequent highly targeted characterization of antibacterial compounds from plant matrices is demonstrated on the example of Solidago virgaurea root extracts. The procedure comprises high-performance thin-layer chromatography (HPTLC) coupled with six bacterial bioassays including two plant pathogens, a radical scavenging assay, an acetylcholinesterase assay as well as in situ and ex situ mass spectrometric analyses. In situ mass spectra were directly recorded from the adsorbent using the Direct Analysis in Real Time interface (HPTLC-DART-MS), whereas ex situ mass spectra were recorded using an elution head-based interface (HPTLC-ESI-MS). For further bioassay-guided isolation of the main antimicrobial compounds, flash chromatographic fractionation and semipreparative high-performance liquid chromatographic purification were used and nuclear magnetic resonance data allowed the identification of the unknown antimicrobial compounds as 2Z,8Z- and 2E,8Z-matricaria esters. The discovered antibacterial activity was confirmed and specified by a luminometric assay and as minimal inhibitory concentration in the liquid phase.

Profiling and classification of French propolis by combined multivariate data analysis of planar chromatograms and scanning direct analysis in real time mass spectra

Quality control of propolis is challenging, as it is a complex natural mixture of compounds, and thus, very difficult to analyze and standardize. Shown on the example of 30 French propolis samples, a strategy for an improved quality control was demonstrated in which high-performance thin-layer chromatography (HPTLC) fingerprints were evaluated in combination with selected mass signals obtained by desorption-based scanning mass spectrometry (MS). The French propolis sample extracts were separated by a newly developed reversed phase (RP)-HPTLC method. The fingerprints obtained by two different detection modes, i.e. after (1) derivatization and fluorescence detection (FLD) at UV 366nm and (2) scanning direct analysis in real time (DART)-MS, were analyzed by multivariate data analysis. Thus, RP-HPTLC-FLD and RP-HPTLC-DART-MS fingerprints were explored and the best classification was obtained using both methods in combination with pattern recognition techniques, such as principal component analysis. All investigated French propolis samples were divided in two types and characteristic patterns were observed. Phenolic compounds such as caffeic acid, p-coumaric acid, chrysin, pinobanksin, pinobanksin-3-acetate, galangin, kaempferol, tectochrysin and pinocembrin were identified as characteristic marker compounds of French propolis samples. This study expanded the research on the European poplar type of propolis and confirmed the presence of two botanically different types of propolis, known as the blue and orange types.

Office Chromatography: Precise printing of sample solutions on miniaturized thin-layer phases and utilization for scanning Direct Analysis in Real Time mass spectrometry

Office Chromatography combines achievements in office technologies with miniaturized planar chromatography. In the life sciences, printing of materials became an accepted technique, whereas in separation science, the use of printers for chromatography is at its infancy. A bubble-jet printer was modified for exact application on miniaturized plates. Technical modifications included the removal of all unnecessary parts and the improvement of the positioning system, purge unit and sample supply system. Evaluation was performed via a slide scanner and image evaluation software. Printing of a food dye mixture solution (n=5) led to a calculated mean deposition volume of 13±1nL/mm(2) per print-cycle. A mean determination coefficient (R(2); n=5) of 0.9990 was obtained for application of increasing volumes, executed via increasing band widths of 50-200μm (corresponding to 2-8nL). Using larger band widths and multiple print jobs, deposition volumes of up to the microliter scale represented an alternative to cost-intensive standard equipment. After print, separation, detection and digital evaluation of five food dyes, mean R(2) (n=5) were obtained between 0.9977 and 0.9995. The accuracy of printing was proven by mean recovery rates of 101-105% with repeatabilities of 3-7% (%RSD, n=5). The transfer to nanostructured ultrathin-layer plates proved the synergetic potential of these fields of research. First, this modified printer was suited for printing of finely graduated scales of three preservatives for determination of the spatial resolution of scanning Direct Analysis in Real Time mass spectrometry.

Miniaturization of Instrumental Planar Chromatography with Focus on Mass Spectrometry

The universal trends to miniaturization and to automation of technologies affect the further development of analytical instrumentation and methodologies. In planar chromatography, these trends are characterized by decreasing dimensions and thickness of the stationary phase and the development of respectively tailored equipment. The miniaturization of instrumental planar chromatography is reflected in the interdisciplinary office chromatography concept, in which achievements in office technologies (print and media technologies) and miniaturized layer developments are integrated. The office chromatography concept stimulates research towards miniaturization and will at last implement a fully online miniaturized system. The use of print techniques is still at its infancy in separation science, whereas printing of materials already became an accepted tool in the life sciences. Commercially, available printers were used for sample application on miniaturized plates and were stepwise modified for a precise and quantitative performance. Further miniaturization attempts led to miniaturized layers with different physical and chromatographic properties. The method transfer to such ultrathin-layer chromatography (UTLC) plates demonstrated the synergetic potential of this instrumental concept. Detection and evaluation tools have to be adjusted and integrated, too. Since the beginning of this millennium, several ionization techniques have been introduced or improved, and paved the way for mass spectrometry (MS) directly from miniaturized surfaces. UTLC was already coupled to MS via several ion source interfaces, such as secondary ionization, matrix-assisted laser desorption and ionization, electrospray ionization, desorption electrospray ionization and direct analysis in real time. However, the hyphenation to targeted, scanning or imaging MS still requires adaptations and methodic efforts to utilize UTLC layers.

Challenges in quantitative high-performance thin-layer chromatography — Part 1: Influence of densitometric settings on the result

There is no doubt that high-performance thin-layer chromatography (HPTLC) can be applied as a quantitative method if the technique is properly used. Densitometry is a commonly used detection mode for quantitation in HPTLC. The influence of instrumental settings on signal intensity, peak resolution, and peak positioning was rarely described in literature. Especially, quantitation of adjacent substance zones was critical when improper combinations of these settings merge. Future trends regarding ultrathin-layer chromatography and hyphenation to scanning or imaging mass spectrometry required the consideration of these delicate points. The influence of different instrumental settings on the obtained signal intensities was demonstrated for four separated parabens (each 150 ng band−1). The maximum mean signal deviations of all four compounds were 6.9% by the optical system, 16.8% by the scan slit dimension, 7.5% by the scan speed, and 1.5% by the data resolution. The influence of these settings on the quantitation of three parabens in two skin protection creams was investigated. Depending on the selected settings, deviations of the calculated substance amount of up to 5.6% were yielded, whereby determination coefficients of the polynomial calibration curves (60–300 ng band−1) varied between 0.9985 and 0.9999. The setting of integration markers between two adjacent peaks was demonstrated to be deficient if low spatial data resolution is applied; however, this challenging task will rise in interest due to the trend towards miniaturization.

Direct bioautography hyphenated to direct analysis in real time mass spectrometry: Chromatographic separation, bioassay and mass spectra, all in the same sample run

Mass spectra were recorded directly in situ the bioautogram, i.e., in the presence of microorganisms, bioassay medium and substrate reagent. The desorption-based direct analysis in real time mass spectrometry (DART-MS) was applied immediately after direct bioautography (DB). It turned out to be an advantageous combination, as it offered the possibility of a straightforward mass spectrometric detection of bioactive analytes within the bioautogram, and at the same time, it was discriminating microorganism cells and highly polar bioassay medium ingredients which could otherwise stress the MS system. DB-DART-MS was investigated for bioactive compounds in cosmetics using the Bacillus subtilis and Aliivibrio fischeri bioassays for detection of Gram-positive and Gram-negative antimicrobials, respectively, and the planar yeast estrogen screen for detection of estrogen-effective compounds. The influences of the three different bioassay matrices on the analyte response and DB-DART-MS performance on different layers were studied on the example of parabens in hand creams. It was shown that with increasing culture medium complexity, the ion suppression increased. As proof-of-principle, the mass spectrometric quantification at the nanogram level in situ the bioautogram was verified by comparison to HPTLC-DART-MS. The total paraben contents of hand creams 1 and 2 were 0.17-0.20% and 0.30-0.34%, respectively, depending on the method used (DB-DART-MS with two different bioassays or HPTLC-DART-MS as well as on RPW or NP plate). In contrast to the current practice of applying the sample twice and subjecting one track to the bioassay and another to MS, the introduced hyphenation DB-DART-MS is straightforward and highly efficient.