For sample preparation of field collected material for an environmental metabolic fingerprinting approach a robust method was developed. The criteria for the choice of the most suitable extraction procedure of field-collected material were first of all reproducibility (the conservation of abundance/number of peaks from the same sample), and furthermore efficiency (number and abundance of peaks, which were assumed to be compounds or metabolites) and handling comfort in the field. Reproducibility of extraction method and resulting chromatograms is a necessary prerequisite for comparison of plant samples grown under different conditions or at different sites, which is displayed in changes of metabolic fingerprints. Sample collection and storing conditions were adapted to the typical situation of field trips, where liquid nitrogen and a freezer may not be available and it may take another week before a laboratory is reached for further adequate sample processing.
The pre-treatment of plant material had a high influence on the metabolic fingerprint. In the lab shock-freezing of leaves in liquid nitrogen is the preferred method for metabolic fingerprinting, because the metabolism can effectively be quenched, the frozen material can easily be ground and further extraction and processing is possible [10–12]. However, for field-trips most scientists might encounter difficulties to comply with legal requirements for transportation of liquid nitrogen for longer distances and the nitrogen will quickly evaporate. Storage on dry ice might be an alternative. However, samples freeze only slowly on dry ice compared to shock-freezing in liquid nitrogen, and dry ice can also only be used for short field-trips but will usually not last for one week. Collection of fresh material directly in solvent or air-drying of plant material are therefore potentially suitable alternatives for field sampling as a compromise. Samples of freshly processed leaves clumped most in a PCA biplot (Figure 1), indicating highest reproducibility. Extracts of freshly processed leaf material had low variance - and thus high similarity - of scores in the first and second principal components (PC 1 and PC 2). All other methods showed high variances on at least one principal component's scores (Figure 1, Table 1). Therefore, collection of fresh material directly in solvent is the preferred method of pre-treatment for samples collected in the field, where neither nitrogen nor dry ice is available. Lyophilisation of samples is a method often used for studies of target metabolites , as sample dry mass can be exactly determined and leaf material can be stored easily for later grinding and extraction. However, drying of samples may change the metabolite pattern to a large extent especially due to irreversible adsorption of metabolites on cell walls and membranes . Also, a lyophiliser cannot easily be brought to the field sampling site. When extracting fresh material, masses of sampled material can be approximately determined by weighing the dried pellet after extraction, as was done in this study.
Quenching of the metabolism can be reached by cutting the leaf material with a hand-held, electric disperser directly in the solvent mixture. The saw-teeth homogenise the leaf material to very small pieces and destroy the cell walls mechanically. But a preceding manual cutting of the leaves in pieces of about 5× 5 mm with scissors is important, because the dispenser cannot process whole leaves of P. lanceolata. The cutting process takes only a few seconds but still changes of metabolites with high turnover rates and hence of metabolic fingerprints could occur . The necessary manual pre-cutting of leaf material definitely is a drawback of this approach. The impact of time needed for cutting on metabolic fingerprints remains to be tested. From a practical point of view, the disperser can be plugged to an electrical generator or an electrical inverter converting DC electricity from sources like (car) batteries, solar panels or fuel cells to AC electricity at the sampling location. Grinding material with ball mills is not a reasonable option in the field, as these devices are big and difficult to carry along. Moreover grinding of fresh leaf pieces in a ball mill resulted in squeezed plant material cleaved to the bottom and the top of the Eppendorf tubes and thus insufficient homogenisation and quenching.
In general, the extraction procedure should be quantitative for any metabolite in the final sample mixture. In many metabolic fingerprinting or metabolite profiling studies, only one extraction is carried out (see, for example, [14, 32, 33]). However, one extraction resulted in less than 50% of metabolites (peak integration) in P. lanceolata samples, which is not sufficient. About 90% of metabolites could only be extracted after the fifth extraction (Table 2). Three extraction steps, which resulted in extraction of about three fourth of the total metabolite number, seem a useful compromise between handling time (which is rather high for five or more extractions) and extraction efficiency. Furthermore, the highest number of metabolites was gained from P. lanceolata, when all three extraction steps were done immediately in a row (Table 3). However, when plant material is sampled outdoors, it is usually impossible to accomplish several extraction steps in a row. Therefore, the effects of time between first and subsequent extractions, i.e., how long the plant material was kept in the initial solvent mixture, were tested at 4°C. Storing samples for one week in the first solvent is likely the most suitable method from a practical point of view, despite loss of a high peak number (Figure 2). Possibly the most reactive metabolites have undergone transformations resulting in a relatively inert extract after one week and thus more reproducible analysis results with the drawback of "loosing" some metabolites that may be of major importance.
The choice of the solvent for extraction is a crucial step in metabolite profiling and metabolic fingerprinting studies and might highly depend on the biological material and the metabolites of interest. Often, cold methanol is used for the extraction of polar compounds , but also various solvent mixtures were tested and evaluated for extraction qualities in metabolomics studies (see, for example, [11, 19, 34]). Initial extraction mixtures of methanol and dichloromethane or methanol and chloroform provide high metabolism quenching capability . This also allows later phase separation by addition of a small amount of water to partition the majority of non-polar metabolites such as lipids.
The shaking with water is essential for the extraction process of P. lanceolata leaves as could be shown by higher Ξ values (demonstrating higher similarity between replicate sample extractions), than samples processed without phase separation (Table 3). Ξ values of samples with phase separation were comparable to analytical replicate measurements indicating very high reproducibility of the method. With respect to peak numbers no significant differences could be found between mixtures containing different parts of dichloromethane (Figure 3, Table 3). Furthermore, extractions in mixtures of methanol with dichloromethane resulted in more reproducible results in comparison to those with chloroform in P. lanceolata. For analysis of polar compounds phase separation in methanol:dichloromethane is especially advantageous since the polar metabolites are then in the upper phase, which is accessible without contamination of the lower phase with a pipette while transferring this phase for further processing. After centrifugation precipitates will rest in the organic phase together with non-polar compounds. In general, mixtures of a higher proportion of methanol than chlorinated organic solvent result in better phase separation ratios (more aqueous phase compared to organic phase). With respect to both sample handling and reproducibility of results, the mixture of methanol:dichloromethane 2:1 and addition of 1 part water for phase separation is overall the most suitable extraction process for samples that have to stay in the initial solvent for one week, as usually necessary when field-sampling.
The temperature during storage of first extraction had no influence on the chemical pattern of the samples, at least when comparing storage at cool temperatures (4°C) and room temperature. However, cooling might be necessary if temperatures increase above 22°C. In any case, samples should be stored at a dark place to avoid degradation of light-sensitive metabolites .
The first extraction mixture, in which the sample is stored for one week, should preferably be a neutral solvent to prevent possible matrix or metabolite degradation, that can occur at acid or basic pH [18, 35]. Subsequent extraction steps may have a different pH to protonate or deprotonate metabolites, which are not well soluble at neutral pH in the aqueous phase, to enhance extraction efficiency. The differences in reproducibility, when extracting in different order of pH, were generally of minor values (Table 3). High Ξ values were obtained for initial extraction with pH 6. With regard to number of extractable metabolites, most could be gained in the extraction order neutral-acid-basic.
Often samples need to be stored after the complete extraction before they can be analysed in the available analytical platform. Storage at 4°C reduces physical changes within the samples (e.g., adsorption, aggregation) to a minimum, but at these temperature conditions chemical reactions may occur [34, 36]. In contrast, at -80°C chemical reactions can be avoided, but physical changes can take place more readily. The comparative analysis of samples stored after the final extraction at 4°C or -80°C showed that storage at 4°C led to a higher reproducibility. This is thus the preferred option for metabolic fingerprinting studies with P. lanceolata, but long-term effects of storage at these temperatures (for more than one week) need to be elucidated. In both cases the number of metabolites was significantly reduced in comparison to immediate processing of samples.
This protocol was optimised for extraction of P. lanceolata leaves and the described amounts and ratios of leaf material and solvents. Smaller or larger sample amounts might result in poor precision for several processing steps, and different ratios of sample amount to extraction solvent might influence extraction efficiency. For other biological material conditions might differ, depending on the given metabolite composition and their physico-chemical properties. Compromises with regard to metabolite number must be taken into account to gain highest reproducibility for analysis of field-collected samples. The protocol was established on standardised material grown in the laboratory. First experiments with field-collected material showed that the method is indeed highly applicable. In spite of all necessary compromises the method is sensitive enough to discriminate metabolic fingerprints of plant samples from different sites having different environmental conditions (e.g., soil, temperature etc.) from samples of the same site but with different treatments (land-use) (Figure 4). Samples from different sites could be discriminated in a PCA at PC 1, whereas different treatments significantly differed at PC 2. This clearly demonstrates the applicability of the proposed protocol for field sampling.