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  Karlovsky Lab  

ANALYTICAL CHEMISTRY LABORATORY

of Molecular Phytopathology and Mycotoxin Research Group

HPLC-IT and HPLC-QQQ Flash chromatography Preparative HPLC HPLC-QQQ

Our chemical analysis: a synopsis

We use chemical analysis in our research and provide it to partners at and outside the university. Our major technique is HPLD coupled with optical (DAD, fluorometry, ELSD) and mass spectrometric (ion trap, Q-TOF and triple quadrupole) detectors. Usage policies for our HPLC-Q-TOF and 500-MS ion trap have been formalized in a policy document. GC-FAIMS, spectrofluorometry, TLC and different kinds of electrophoresis (flat-bed, capillary, PAGE, denaturing gradient) are available, too. Flash chromatography, prep. HPLC and Clevenger apparatus are used for the purification of fungal and plant metabolites. The chemical lab operates several rotary evaporators and vacuum concentrators and possesses four fume hoods.

Contact

Prof. Dr. Petr Karlovsky (phone 0551-3912918), PD Dr. Franz Hadacek (phone 0551-3914418) and Dr. Anna Rathgeb (phone 0551-3913230).

Techniques

Mycotoxin analysis

HPLC-MS/MS in MRM mode is our standard multi-mycotoxin method. Because we work with diverse matrices such as grains, plants stems, single spikelets and bodies of insects, we calibrate our analysis with matrix-matched standards. Isotopically labelled internal standards are used to estimate in checks of method performance. Analytical standards not available commercially are prepared by purification from fungal cultures. We have been using a triple quadrupole (1200L) and an ion trap (500-MS) for mycotoxin analysis, as shown in this example from 2005. Since 2005 then we were continuously extending protocols to further mycotoxins and matrices. The acquisition of a two HPLC-MS/MS systems scheduled for July 2017 will substancially increase the sensitivity and throughput of our mycotoxin analysis.

LC1200

Our first MS was triple quadrupole LC1200 from Varian, which served us from 2004 till 2015.

500-MS opened 1200L opened

On the left you see our second MS, which is ion trap MS-500 from Varian, with an open housing. We purchased it in 2006 and still use it (October 2017). On the right you see triple quadrupole LC1200 with open housing.

Disassembled ion trap

This is an ion trap disassembled into smallest parts. Users are not expected to try this if they intend to use the machine again but Katharina does it regularly. The skillful and highly motivated team of our workshop supports her when troubleshooting is needed.

Although HPLC-MS/MS in MRM is a golden standard for multi-mycotoxin analysis today, HPLC-FD is well suitable for certain mycotoxins; in certain situations it is preferable to HPLC-MS/MS. For instance, we use HPLC-FD for the analysis of zearalenone in our own projects on detoxification as well as in collaborations. Ochratoxin A is another mycotoxin suitable for HPLC-FD; we published a solid bar microextraction protocol with HPLC-FD detection for ochratoxin A in wheat and maize (Toxins (2016) 7:3000-3011) and used HPLC-FD in projects on zearalenone and aflatoxins.

Ochratoxin A

Detection of ochratoxin A by HPLC-FD

HPLC-DAD proved useful in some mycotoxin projects, too, for instance in a recent work of Dr. Rosine Suchfort on enniatins. Another application of HPLC-DAD was a study of transformation products of fusaric acid, which for narrow peaks requires mobile phase additives that are not compatible with MS:

Fusaric acid

Transformation products of fusaric acid resolved by HPLC-DAD (Rasoul Abousaeedi)

Examples of publications relying on our mycotoxin analysis by HPLC-MS/MS:

1. Guo Z, Pfohl K, Karlovsky P, Dehne HW, Altincicek B (2016) Fumonisin B1 and beauvericin accumulation in wheat kernels after seed-borne infection with Fusarium proliferatum. Agric Food Sci 25: 138-145. OPEN ACCESS

3. Trümper C, Paffenholz K, Smit I, Kössler P, Karlovsky P, Braun HP, Pawelzik E (2016) Identification of regulated proteins in naked barley grains (Hordeum vulgare nudum) after Fusarium graminearum infection at different grain ripening stages. J Proteomics 133: 86-92.

3. Amato B, Pfohl K, Tonti S, Nipoti P, Dastjerdi R, Pisi A, Karlovsky P and Prodi A (2015) Fusarium proliferatum and fumonisin B1 co-occur with Fusarium species causing Fusarium Head Blight in durum wheat in Italy. Journal of Applied Botany and Food Quality 88: 228-233. OPEN ACCESS

Plant secondary metabolites

HPLC-DAD is well suitable for most plant metabolites. With the help of pure standards every metabolite absorbing UV light can easily be quantified. HPLC-DAD is however useful for nontargeted analysis, too. UV absorption spectra combined with relative retention time are offten sufficient to point at the chemical identity of such metabolite even when standards are not available, as shown on the following figure. Dr. Franz Hadacek maintains a library of UV spectra that allows us to identify many plant metabolites without mass spectra. Using related compounds with similar chromatographic behavior and known absorption coefficients allows rough concentration estimates for metabolites without standards; when we need accurate quantification of metabolites for which standards that are not commercially available, we make them by purification.

Arabidopsis metabolites

Metabolites identified in root exudates of Arabidopsis thaliana (Dr. Pervin Akter and Dr. Franz Hadacek)

HPLC-DAD can often reveal differences among complex metabolites mixtures, such as in the following example:

HPLC-DAD chromatograms of root exudates of maize under normal conditions (left) and under stress (right) (Dr. Pervin Akter and Dr. Franz Hadacek)

Phytohormone analysis

We use a modified ether extraction protocol, MRM and internal isotopically labelled standards. Analysis of phytohormones is also available in Prof. Feußner's lab.

Examples of recent papers with our phytohormone analysis:

1. Ulferts S, Delventhal R, Splivallo R, Karlovsky P, Schaffrath U (2015) Abscisic acid negatively interferes with basal defence of barley against Magnaporthe oryzae.BMC Plant Biology 15/7.OPEN ACCESS

2. Häffner E, Karlovsky P, Splivallo R, Traczewska A, Diederichsen E (2014): ERECTA, salicylic acid, abscisic acid and jasmonic acid modulate quantitative disease resistance of Arabidopsis thaliana to Verticillium longisporum. BMC Plant Biology 14/85. OPEN ACCESS

3. Ratzinger A, Riediger N, von Tiedemann A., Karlovsky P (2009): Salicylic acid and salicylic acid glucoside in xylem sap of Brassica napus infected with Verticillium longisporum. Journal of Plant Research 122:571-579. Download OPEN ACCESS.

Pesticide analysis

So far our lab provided pesticide analysis for two divisions of the Faculty of Agriculture and one division of the Faculty of Forest Sciences and Forest Ecology. The demand on pesticide analysis is likely to increase in future and may include identification of products of enzymatic transformations underlying resistance.

Metabolic profiling: nontargeted comparative analysis of metabolic profiles

The analytes are separated by HPLC on RP and detected in full-scan mode after electrospray ionization. The approach is similar to metabolomics but we use a different data processing strategy because the purpose is to identify MS signals intensified by a treatment rather than to record and identify all metabolites in a sample. Our data processing pipeline relies on a combination of commercial software and custom-made Perl scripts:

1. Noise reduction by CODA and peak detection
We use CODA algorithm as implemented ACD/Labs software, raw data are imported after conversion to NetCDF.

2. Chromatogram alignment
A custom Perl script is used to compensate for shifts in the retention time

3. Normalization of peak intensities and comparative analysis
We use a normalization algorithm that eliminates bias caused by strongly induced and/or suppressed metabolites on the normalization factor. Normalized intensities of sample groups (controls vs. treatment) are compared using criteria such as intensitives, variance, maximum no. of controls and treatments with/without the signal etc. The outcome of the analysis is the set of Rf-m/z pairs for candidate metabolites. These ideas were integrated into a MATLAB-based tool for MS data developed in collaboration with bioinformaticians and plant biochemists.

Metacolomics platform XCMS was used in some of our projects. XCMS is the best OA metabolomics platform we have tested but familiarization with the system takes time. For most PhD students in the past, our ACD/Labs & Perl pipeline was easier to learn. In small collaborative projects we may carry out the entire data analysis; for larger efforts, however, we recommend students to learn the system and process their data themselves. Comparing different parameter setting often helps to extract maximum information from the data.

If the metabolite has already been described, its identity can often be discerned based on m/z value of its molecular ion and fragments in MSn spectrum and UV absorption. Dr. Franz Hadacek maintains a database of UV spectra of metabolites that we have been studying, which allows us to tentatively identify these metabolite when we encounter them again based solely on their retention time and UV spectrum. Below you see an example of the analysis of maize root exudates by HPLC-DAD:

HPLC-DAD chromatograms of root exudates of maize under normal conditions (left) and under stress (right) with UV spectra recorded at major peaks (Dr. Pervin Akter and Dr. Franz Hadacek)

Information about biosynthetic capacity of the strain/species used as a source is helpful. Our Perl scripts search available databases automatically but unfortunately most metabolites relevant for the projects that we have been supporting are underrepresented in databases.

Often purification is necessary for the identification of compounds responding to a treatment (see below). Because the efficiency of ionization and thus intensity of the MS signal varies widely with structure of the analyte, wrong targets for purification were often selected in the past, resulting in purified metabolites in amounts insufficient for structure elucidation and biological assays. The evaporative light scattering detector (ELSD) will solve this problem.

Examples of papers in which we used nontargeted metabolic profiling:

1. Chatterjee S, Kuang Y, Splivallo R, Chatterjee P, Karlovsky P (2016) Interactions among filamentous fungi Aspergillus niger, Fusarium verticillioides and Clonostachys rosea: fungal biomass, diversity of secreted metabolites and fumonisin production. BMC Microbiology 16/83 (13 pp).OPEN ACCESS

2. Döll K, Chatterjee S, Scheu S, Karlovsky P, Rohlfs M (2013): Fungal metabolic plasticity and sexual development mediate induced resistance to arthropod fungivory. Proceedings of the Royal Society B 280:20131219. FREE ACCESS

3. Khorassani R, Hettwer Z, Ratzinger R, Steingrobe S, Karlovsky P, Claassen N (2011): Citramalic acid and salicylic acid in sugar beet root exudates solubilize soil phosphorus. BMC Plant Biology 11/111. OPEN ACCESS.

Analysis of transformation products

This service task gradually developed to support projects in which mycotoxins or other chemicals are transformed by microorganisms or purified enzymes into other compounds. We know the core structure of the products and can often identify MS signals of transformation products using a technique called neutral loss scan (NLS) on a triple quadrupole or by its substitution by the analysis of MS2 data generated in automatic, data-dependent acquisition. The new HPLC-MS system that we expect to arrive in the lab this year will greatly improve our ability to estimate structures of transformation products based on their MS spectra. Analysis of UV spectra of transformation products provides useful information for some mycotoxins and transformations.

MS2 usually provides sufficient information to generate hypotheses (hydroxylation, glycosylation etc.). MS3 and higher fragmentation levels are available on the ion trap when needed. Untypical modifications such as amidation with GABA recently discovered in our laboratory require purification.

Enniatin degradation

Detoxification of enniatins: Dr. Rosine Suchfort carried out the transformation and Dr. Kirstin Feußner from Prof. Ivo Feußner's lab generated HR-MS spectra

Purification of standards

For many metabolites commercial standards for HPLC analysis are not available. We purify such standards from plant and fungal extracts using flash chromatography with gradient elution, preparative HPLC, Clevenger apparatus and preparative TLC. The progress of purification is monitored by HPLC. The protocols used vary depending on the source of material and on the purpose of the purification. A sequence of several chromatographic steps is usually used. Typically flash chromatography on silica gel with a cyclohexane - ethyl acetate - methanol elution gradient is the first step, followed by chromatography on Sephadex LH20 and/or preparative HPLC on C18. Analytical as well as preparative TLC on normal and reverse phase is often used, too. Upon the initiative of Zana Jamal Kareem we recently established a Clevenger apparatus as a simple yet powerful system for gentle distillation of essential oils from plant material. Zana currently uses Clavenger in his work with henbane (Hyoscyamus niger) and sesame (Sesamum indicum).

Apart from the preparation of standards for analytical methods, purified metabolites are often needed for biological experiments, for instance for studies of toxicity and in enzymatic transformations. Below you see purification of enniatins as an example:

Purification of enniatins

Enniatins from Fusarium tricinctum as a raw extract (left) and after purification (right) (Dr. Rosine Suchfort)

Purification of enniatins

Preparative separation of individual enniatins from Fusarium tricinctum (Dr. Rosine Suchfort)

Flash chromatography is the fundamental method for natural product purification. We usually start with normal phase eluted with a gradient of cyclohexane, ethyl acetate and methanol. Reverse phase and other materials are also available; we use ready-made single-use cartridges in the first step but to achieve a better separation we often fill longer columns ourselves.

Flash chromatography

Flash chromatography: in a fume hood you see pumps on the bottom (up to 5 MPa), fraction collector on the top and a column on the right. The signal of the detector is displayed on a laptop screen.

Preparative HPLC is another fundamental method for the purification of natural products. It is often the final step of the purification. We used RP columns in preparative (25 mm diameter) and semipreparative (14 mm diameter) scales.

Flash chromatography

Flash chromatography: in a fume hood you see pumps on the bottom (up to 5 MPa), fraction collector on the top and a column on the right. The signal of the detector is displayed on a laptop screen.

Preparative TLC is a simple yet very useful method, especially in its preparative version. Preparative TLC is often used in our lab in combination with flash chromatography and/or preparative HPLC:

Flash chromatography

TLC box in a foome hood, with Dr. Yi Kuang preparing samples for loading

Analysis of volatiles compounds by GC

Some plant and fungal metabolites are volatile. These compounds are sampled from the gaseous phase and analyzed by GC. Directly in the lab we use a GC-FAIMS system which allows us to distinguish among isomers. GC-FAIMS is a young method; the choice of software for data processing is therefore limited. We developed our own software that works with raw data from the detector, allowing us to optimize the parameters for specific tasks. On the following figures you see raw FAIMS data collected by Johannes Ott for fungal VOCs and chromatograms extracted from these data by our software:

GC-FAIMS in Karlovsky lab

Raw histogram of GC-FAIMS with positive ionization (Johannes Ott)

GC-FAIMS in Karlovsky lab

Raw histogram of GC-FAIMS with negative ionization (Johannes Ott)

GC-FAIMS in Karlovsky lab

Normalized GC-FAIM chromatograms for positive (red) and negative (green) ionization; notice signals in neg. ionization hardly visible in raw histogram (Johannes Ott and Petr Karlovsky)

In many projects with VOCs we however rely on state of the art GC-MS equipment in the laboratory of Prof. Ivo Feußner. Since a suspension bridge connecting our labs was constructed as you see below, moving forth and back between our labs is comfortable even on rainy days.

Bridge between Feussner and Karlovsky labs

Bridge connecting our analytical chemistry lab (left) with the lab of Prof. Ivo Feußner (right), photo by Petr

Bridge from the other side, photo by Franz

Bridge from the other side, photo by Franz

Selection of targets

The selection of target metabolites for purification was tricky in the past because it was based on the results of comparative metabolic profiling by HPLC-MS. Strongly ionizable metabolites with nice MS signals might be present in very low concentrations, leading to insufficient amounts after purification. A new evaporative light scattering detector solved this problem.

 
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