The current European Pharmacopoeia (Ph. Eur.) texts for Interferon (IFN)-alfa-2 include a nonspecific photometric protein assay using albumin as calibrator and a highly variable cell-based assay for the potency determination of the protective effects. A request was expressed by the Official Medicines Control Laboratories (OMCLs) for improved methods for the batch control of recombinant interferon alfa-2 bulk and market surveillance testing of finished products, including those formulated with Human Serum Albumin (HSA). A HPLC method was developed at the Medical Products Agency (MPA, Sweden) for the testing of IFN-alfa-2 products. An initial collaborative study run under the Biological Standardisation Programme (BSP; study code BSP039) revealed the need for minor changes to improve linearity of the calibration curves, assay reproducibility and robustness. The goal of the collaborative study, coded BSP071, was to transfer and further validate this improved HPLC method. Ten laboratories participated in the study. Four marketed IFN-alfa-2 preparations (one containing HSA) together with the Ph. Eur. Chemical Reference Substance (CRS) for IFN-alfa-2a and IFN-alfa-2b, and in-house reference standards from two manufacturers were used for the quantitative assay. The modified method was successfully transferred to all laboratories despite local variation in equipment. The resolution between the main and the oxidised forms of IFN-alfa-2 was improved compared to the results from the BSP039 study. The improved method even allowed partial resolution of an extra peak after the principal peak. Symmetry of the main IFN peak was acceptable for all samples in all laboratories. Calibration curves established with the Ph. Eur. IFN-alfa-2a and IFN-alfa-2b CRSs showed excellent linearity with intercepts close to the origin and coefficients of determination greater than 0.9995. Assay repeatability, intermediate precision and reproducibility varied with the tested sample within acceptable ranges. Test accuracy estimated by comparing the values obtained by the participants to the declared contents determined by the manufacturers was good despite the absence of a common reference preparation. In conclusion, the present study showed that the new method is suitable, reproducible and transferable. Proposals for the revision of Ph. Eur. texts are presented.

This study investigates the occurrence of the flavonoid phloridzin (phloretin-2'-O-β-D-glucoside) in root exudates of apple seedlings showing growth reduction related to apple replant disease (ARD). The disease is most likely caused by a complex of soil-borne fungi and bacteria, but the etiology remains to be elucidated. Information on specific exudation processes in the rhizosphere of apple seedlings could contribute to our understanding of the conditions triggering ARD development. To procure ARD symptoms, apple seedlings (Malus x domestica Borkh.) were grown in ARD-conductive soil. Root exudates were collected by submerging the roots in a solution of 0.05 mM CaCl2 for a period of 4 h. The fraction of phenolic root exudates was analyzed using HPLC/DAD (high performance liquid chromatography/diode array detector). Results suggest that (i) phloridzin is a constant root exudate of apple seedlings. It was the most abundant phenol in the collected exudates from replant-diseased as well as healthy seedlings. (ii) Phloridzin exudation, related to root dry matter, was the most intensive at the onset of ARD symptom development and lower during the period when symptoms were most severe or outgrown. (iii) In comparison to healthy seedlings, the phloridzin exudation of apple replantdiseased seedlings was significantly higher only at the onset of ARD symptom development, suggesting a response of the plants to infection. The finding of phloridzin in the root exudates of Malus x domestica Borkh. might have consequences for research on the etiology of ARD. Specialized pathogenic microorganisms could be attracted by this distinct compound. Since it is very characteristic of apple plants, phloridzin might be the compound that ARD-causing microorganisms utilize to recognize their host. For practical applications, phloridzin root exudation could therefore be a parameter in evaluating ARD-susceptibility of different rootstocks.

To date there is no standard procedure for the determination of proanthocyanidins (PAC). There is thus no common base for comparisons of declared contents of various preparations. This problem was made obvious by a comparative analysis of proanthocyanidins of lingonberry (Vaccinium vitis-idaea) and cranberry (Vaccinium macrocarpon). In five commercial preparations each with lingonberry and with cranberry in comparable galenical formulation the fraction of PAC was quantified by photometric determination and by RP-HPLC with UV detection. The respective mono-, di and trimeric compounds were identified by fluorescence detection and by mass-spectrometric determination. As expected, the photometric analysis led in all cases to considerably higher PAC contents than the specific HPLC method with UV detection. Lingonberry was shown to be a rich source of proanthocyanidins: On comparison of similar galenical formulations of lingonberry and cranberry higher contents were found in favour of lingonberry preparations in almost all cases with respect to the quantitative and the qualitative composition (differentiation between A- and B-type PAC).

The dynamics of soil organic matter are a key factor in controlling the terrestrial carbon cycle. Compound specific stable carbon isotope analysis has given new insight in to the stability of individual organic molecules in soil. For lignin, one of the major plant compounds, available data suggest the existence of both a labile (turnover time <1year) and a relatively stable (turnover time in the range of decades) pool. However, these data derive almost exclusively from agricultural soils in temperate climates. In order to extend the range both in ecosystem type and observed time span, we analysed a pasture soil in subtropical Australia that had experienced a land use change from rainforest 90years earlier. We determined the concentration and isotopic signature of lignin biomarkers and compared them to those in an existing rainforest soil nearby and to samples of the respective vegetation. The land use change shifted both the relative abundance of lignin biomarkers and their isotopic signatures. In particular, the isotope data indicate that the pasture soil contains only small proportions of inherited rainforest-derived lignin biomarkers, which are mostly close to or below detection limit. These drastic changes in biomarker composition indicate that the original lignin structure had little chance to persist in this soil over a century. Thus, the stable soil organic carbon identified an earlier study of this soil is probably highly altered material and lignin biomarkers are not a suitable proxy of this stable carbon.

Nitrogen fertilization did not affect decay of old lignin and SOC in a C-labeled arable soil over 36 years A. Hofmann, A. Heim, P. Gioacchini, A. Miltner, M. Gehre, and M. W. I. Schmidt Department of Geography, University of Zurich, Zurich, Switzerland Institute of Agricultural Chemistry, University of Bologna, Bologna, Italy UFZ – Helmholtz Centre for Environmental Research, Leipzig, Germany Received: 22 December 2008 – Accepted: 15 January 2009 – Published: 4 February 2009 Correspondence to: A. Heim (alexander.heim@geo.uzh.ch) Published by Copernicus Publications on behalf of the European Geosciences Union.

Retardation of soil organic carbon (SOC) decay after nitrogen addition to litter or soil has been suggested in several recent studies and has been attributed to a retardation in lignin decay. With our study we tested the long-term effect of mineral fertilization (N+P) on the decay of the SOC component lignin in arable soil. To achieve this, we tracked 13C-labeled lignin and SOC in an arable soil that is part of a 36-year field experiment (conversion from C3 to C4 crops) with two mineral fertilization levels. We could show that fertilization neither retarded nor enhanced the decay of old SOC or lignin over a period of 36 years, proposing that decay of lignin was less sensitive to fertilization than previously suggested. However, for new, C4-derived lignin there were indications that decay might have been enhanced by the fertilization treatment, whereas decay of new SOC was unaffected.

SummaryLignin has long been considered a relatively stable component of soil organic matter. However, recent studies suggest that lignin may turn over within years to decades in arable soil. Here we analysed lignin concentrations in an 18‐year field experiment under continuous silage maize, where two soils were sampled at six points in time. Our objectives were to examine the long‐term dynamics of (i) lignin derived from a previous C3‐vegetation and (ii) lignin derived from maize, as influenced by two levels of maize biomass input. Total lignin concentrations in soil were quantified by gas chromatography of lignin cupric oxide oxidation products. Compound‐specific 13C isotope analysis allowed discrimination between C3‐derived lignin and maize‐derived lignin. Degradation dynamics of C3‐derived lignin were independent of biomass input level, suggesting that priming did not affect soil lignin concentrations. After 18 years, approximately two‐thirds of the initial C3‐derived lignin remained in the soils, whereas, on average, 10% of the recent maize‐derived lignin input was retained. We suggest that lignin is effectively stabilized in these arable soils, although the mechanisms involved remain unclear.