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Hydrilla verticillata (L.f.) Royle exhibits an inducible C4- type photosynthetic cycle, but lacks Kranz anatomy. Leaves in the C4-type state (but not C3-type) contained up to 5-fold higher internal dissolved inorganic carbon (DIC) concentrationsthan the medium, indicating that they possessed a CO2-concentrating mechanism (CCM). Several lines of evidence indicated that the chloroplast was the likely site of CO2generation. From C4-type [DIC] measurements, the estimated chloroplastic free [CO2] was 400 mmol m-3 This gave a calculated 2% O2 inhibition of photosynthesis, which was identical to the measured value, and provided independent evidence that the estimated [CO2] was close to the true value. A homogeneous distribution of DIC in the C4- type leaf could not account for such a high [CO2], or the resultant low O2 inhibition. For C3-type leaves the estimated chloroplastic [CO2] was only 7 mmol m-3, which gave high, and similar, calculated and measured O2 inhibition values of 22 and 26%, respectively. The CCM did not appear to be located at the plasma membrane, as it operated at low and high pH, indicating that it was independent of use of HCO3- from the medium. Also, both C3- and C4-type Hydrilla leaves showed pH polarity in the light, with abaxial and adaxial boundary layer values of about pH 4.0 and 10.5, respectively. Thus, pH polarity was not a direct component of the CCM, though it probably improved access to HCO3. Additionally, iodoacetamide and methyl viologen greatly reduced abaxial acidification, but not the steady-state CCM. Inhibitor studies suggested that the CCM required photosynthetically generated ATP, but Calvin cycle activity was not essential. Both leaf types accumulated DIC in the dark by an ATP-requiring pro... [ABSTRACT FROM AUTHOR]

The conventional treatment for distal radius fractures typically involves immobilization of the injured extremity using a conventional forearm cast. These casts do cause all sorts of discomfort during wear and impose life-style restrictions on the wearer. Personalized 3D printed splints, designed using three-dimensional (3D) imaging systems, might overcome these problems. To obtain a patient specific splint, commercially available 3D camera systems are utilized to capture patient extremities, generating 3D models for splint design. This study investigates the feasibility of utilizing a new camera system (SPENTYS) to capture 3D surface scans of the forearm for the design of 3D printed splints. In a prospective observational cohort study involving 17 healthy participants, we conducted repeated 3D imaging using both the new (SPENTYS) and a reference system (3dMD) to assess intersystem accuracy and repeatability. The intersystem accuracy of the SPENTYS system was determined by comparison of the 3D surface scans with the reference system (3dMD). Comparison of consecutive images acquired per device determined the repeatability. Feasibility was measured with system usability score questionnaires distributed among professionals. The mean absolute difference between the two systems was 0.44 mm (SD:0.25). The mean absolute difference of the repeatability of the reference -and the SPENTYS system was respectively 0.40 mm (SD: 0.30) and 0.53 mm (SD: 0.25). Both repeatability and intersystem differences were within the self-reported 1 mm. The workflow was considered easy and effective, emphasizing the potential of this approach within a workflow to obtain patient specific splint., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 van Ginkel et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Nowadays routine residue monitoring involves the analysis of many compounds from different classes, mainly in urine. In the past two decades, developments heavily focused on the use of mass spectrometers (MS) and faster and more sensitive MS detectors have reached the market. However, chromatographic separation (CS) was rather ignored and the cognate developments in CS were not in line. As a result, residue analysis did not improve to the extent anticipated. CS by LC x LC is a promising technique and will enable a further increase in the range of compounds and compound classes that can be detected in a single run. In the present study, a self-built LC x LC system, using a 10 port valve, was connected to a single quadrupole MS with electrospray interface. Standards containing a mixture of sulphonamides, β-agonists and (steroid) hormones, 53 compounds, in total, were analysed. Results demonstrated that these compounds were well separated and could be detected at low levels in urine, i.e. limit of detection (LOD) from 1 µg L -1 for most β-agonists to 10 µg L -1 for some sulphonamides and most hormones. To enhance the sensitivity, optimisation was performed on an advanced commercial LC x LC system connected to a full scan accurate MS. This ultimately resulted in a fast high throughput untargeted method, including a simple sample clean-up in a 96-well format, for the analysis of urine samples.

A robust LC-MS/MS method was developed to quantify a large number of phase I and phase II steroids in urine. The decision limit is for most compounds lower than 1ngml -1 with a measurement uncertainty smaller than 30%. The method is fully validated and was applied to assess the influence of administered synthetic steroids and beta-agonists on the steroidogenesis. From three animal experiments, clenbuterol, diethylstilbestrol and stanozolol, the steroid profiles in urine of bovine animals were compared before and after treatment. It was demonstrated that the steroid profiles were altered due to these treatments. A predictive multivariate model was built to identify deviations from normal population steroid profiles. The abuse of synthetic steroids can be detected in urine samples from bovine animals using this model. The samples from the animal experiments were randomly analysed using this method and predictive model. It was shown that these samples were predicted correctly in the exogenous steroids group., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

According to EU legislation a confirmatory method used for residue analysis should be able to confirm the identity of a compound beyond reasonable doubt. To provide an adequate instrumental set-up, Commission Decision 2002/657/EC introduced the concept of "identification points". A second aspect to assure unequivocal confirmation, is the establishment of ion ratio and retention time criteria. Currently, the gold standard for confirmatory analysis of most veterinary drug residues is liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS) in selected reaction monitoring (SRM) acquisition mode, isolating one precursor ion and monitoring two a priori selected product ions, yielding 4 identification points. We comprehensively evaluated the use of different low and high resolution LC-MS(/MS) techniques and acquisition modes with respect to the selectivity of 100 veterinary drugs in liver, muscle and urine extracts aiming to critically review the currently established identification points system. A comparison among MS/MS in SRM mode with high resolution mass spectrometry (HRMS) in full scan, all ion fragmentation and targeted MS/MS was made based on a unique inter-laboratory study, which comprises 21 laboratories from four different continents and equipment from all major vendors. In total 186 samples were analysed yielding results for 9282 analyte/matrix combinations. It was observed that the false positive rate approximately doubles if no ion ratio criterion is applied indicating that this criterion is important to prevent false positive results. Full scan HRMS analysis, only monitoring the molecular ion and allowing a ±5 ppm mass tolerance is, in general, less selective than low resolution MS/MS using SRM, and thus full scan alone is considered not sufficient for confirmatory analysis. Furthermore, even though the number of data on all ion fragmentation and targeted MS/MS at high resolution was limited, based on the data obtained, it was observed that the acquisition mode as well as the mass resolution needed, very much depend on the matrix and the compound itself. For complex matrix extracts and non-selective compounds (worst-case situation), only targeted MS/MS, monitoring the precursor ion and a single product ion in HR-MS using a maximum of ±5 ppm mass deviation, leads to comparable selectivity and false positive and negative rate as SRM monitoring two product ions in LR-MS. We conclude that the currently applied identification point system as established in commission decision 2002/657/EC should be revised with respect to the allocation of identification points., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Besides the identification point system to assure adequate set-up of instrumentation, European Commission Decision 2002/657/EC includes performance criteria regarding relative ion abundances in mass spectrometry and chromatographic retention time. In confirmatory analysis, the relative abundance of two product ions, acquired in selected reaction monitoring mode, the ion ratio should be within certain ranges for confirmation of the identity of a substance. The acceptable tolerance of the ion ratio varies with the relative abundance of the two product ions and for retention time, CD 2002/657/EC allows a tolerance of 5%. Because of rapid technical advances in analytical instruments and new approaches applied in the field of contaminant testing in food products (multi-compound and multi-class methods) a critical assessment of these criteria is justified. In this study a large number of representative, though challenging sample extracts were prepared, including muscle, urine, milk and liver, spiked with 100 registered and banned veterinary drugs at levels ranging from 0.5 to 100 µg/kg. These extracts were analysed using SRM mode using different chromatographic conditions and mass spectrometers from different vendors. In the initial study, robust data was collected using four different instrumental set-ups. Based on a unique and highly relevant data set, consisting of over 39 000 data points, the ion ratio and retention time criteria for applicability in confirmatory analysis were assessed. The outcomes were verified based on a collaborative trial including laboratories from all over the world. It was concluded that the ion ratio deviation is not related to the value of the ion ratio, but rather to the intensity of the lowest product ion. Therefore a fixed ion ratio deviation tolerance of 50% (relative) is proposed, which also is applicable for compounds present at sub-ppb levels or having poor ionisation efficiency. Furthermore, it was observed that retention time shifts, when using gradient elution, as is common practice nowadays, are mainly observed for early eluting compounds. Therefore a maximum retention time deviation of 0.2 min (absolute) is proposed. These findings should serve as input for discussions on the revision of currently applied criteria and the establishment of a new, globally accepted, criterion document for confirmatory analysis. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

In this paper, the past, present, and (possible) future of the European analytical criteria for residues are described. The elaboration of the revision of Commission Decision 9312561EC was a long process starting in 1996 and ending with the formation of a European Commission (EC) working group in 1998. This working group took account of developments in scientific and technical knowledge at that time and produced a draft version of the revision within 6 months. The revision, finally published in 2002 (2002/657/EC), includes new ideas on the identification of analytes and the criteria for performance assessment as well as validation procedures. Currently (2009), the evolution in analytical equipment and progress in scientific research, accompanied by recent European regulatory changes, demands an update or revision of the 2002/657/EC.

The use of accurate mass measurement as a confirmation tool is examined on a TOF-MS and compared with confirmation using a triple quadrupole mass spectrometer (QqQ-MS). Confirmation of the identity of a substance using mass-spectrometric detection has been described. However, the use of accurate mass measurement for confirmatory analysis has not been taken into account. In this study, criteria for confirmation with accurate mass are proposed and feasibility is demonstrated. Mass accuracy better than 3ppm of the quasi-molecular ion and a fragment and their relative ratios determined with LC/TOF-MS are compared to the criteria of two transition ions and their ratio of LC/QqQ-MS. The results show that these criteria can be met for Trenbolone in samples of bovine urine and that single MS accurate mass measurement is comparable to nominal mass MS/MS for confirmation. The increase in popularity and availability of LC/TOF-MS instruments and the ease, of which exact masses can be measured, make it important to formulate criteria for this type of instrumentation. It is shown in this study that accurate mass measurement can be used for confirmatory analysis. However, more experiments need to be conducted to demonstrate the applicability of accurate mass measurement in general for residue analysis.

One potential explanation for the presence of beta-boldenone in calf urine is contamination of the sample with feces containing beta-boldenone. It has been demonstrated that after oral and intramuscular administration of beta-boldenone esters, several metabolites are formed and excreted in urine. One of the (minor) metabolites is 6beta-hydroxy-17alpha-boldenone. This paper describes an analytical method that can discriminate between unconjugated boldenone, its glucuronide- and sulphate-conjugates, 6beta-hydroxy-17alpha/beta-boldenone and coprostanol, a marker for fecal contamination. The method was applied to all samples suspected to contain boldenone within the Dutch National Residue Control Plan. Approximately 10,000 samples of urine were screened (LC-MS) in 2004-2005 by VWA-East, one of the official Dutch control laboratories, from which 261 samples were suspected to contain boldenone. These samples were all analyzed for their conjugation state, 6beta-hydroxy-17alpha/beta-boldenone and for the presence of coprostanol. Alfa-boldenone, the major metabolite in bovine urine after boldenone-ester administration, was found in a large number of these samples. The presence of alpha-boldenone was proven also to be a result of fecal contamination. None of the samples tested contained residues of the metabolite 6beta-hydroxy-17alpha/beta-boldenone. Not finding this metabolite indicates that the origin of alpha-boldenone-conjugates is endogenous. The results confirm that the presence of unconjugated beta-boldenone and alpha-boldenone conjugates next to alpha-boldenone are no indicators for illegal administration of boldenone-esters. No indications were obtained that conjugated beta-boldenone can be of endogenous origin.

The certification by inter-laboratory testing of two candidate reference materials (RMs) for the mass concentration of the anabolic agent clenbuterol in bovine eye material is described: RM 674 with ca 10 microg clenbuterol per kg of eye matrix and RM 673 clenbuterol-free eye matrix as the negative control (<0.50 microg kg(-1)). Both candidate RMs were certified by eleven EU laboratories, and sixty-six accepted replicate measurements were included in the "Certification Study". The precision of the measurement process was assessed by calculation of the standard variation determined within each laboratory during the certification step. The study was performed according to the "Guidelines for the production and certification of BCR reference materials" and to "ISO guide 31, 33, and 35". The certified clenbuterol mass concentration for clenbuterol-free eye material CRM 673 (calculated on the basis of clenbuterol as the free base) was <0.50 microg kg(-1). The corresponding concentration for clenbuterol-containing eye material CRM 674 was 9.42 +/- 0.88 microg kg(-1). These certified values are very close to the desired target concentration of <0.5 microg kg(-1) and ca 10 microg kg(-1). This study has demonstrated that successful certification of clenbuterol-containing and clenbuterol-free bovine eye materials is possible.

Within the EU Standards, Measurement and Testing Program (SMT) two clenbuterol reference materials (RMs) were developed. Since clenbuterol readily accumulates and is slowly depleted from pigmented tissues such as the retina, homogenized eye liquid content is the most sensitive tissue for the detection of clenbuterol misuse. Therefore, both of the RMs were produced from bovine eye matrix: a negative control--RM 673 eye reference material, clenbuterol free (<0.50 microg/kg eye matrix) and a positive--RM 674 eye reference material containing clenbuterol (approximately 10 microg/kg eye matrix). Eyes were sampled from 103 German Simmental cattle and the inner liquid content was homogenized to a wet homogenized liquid content (HLC). This clenbuterol negative pool was divided into two sub-pools, one of which was spiked with clenbuterol to a final concentration of 10 microg clenbuterol/kg HLC. Of each pool exactly 2.0 +/- 0.01 g (+/- 0.5%) portions were weighed into 790 containers. Lyophilization of the 1,580 containers was performed in one batch. Parameters for the filling of containers, dry matter content, and residual moisture were in accordance with EU requirements. A three-year stability study and two homogeneity studies at various storage temperatures (-60 degrees C, -20 degrees C, +4 degrees C, +20 degrees C, and +37 degrees C) were performed. Low variation was observed within all of the homogeneity studies, proving that each of the RMs were homogeneous and that this was independent of storage temperature and storage time. In the stability studies, measured clenbuterol concentrations remained constant for RM 673 under the detection limit at 0.15 +/- 0.01 microg clenbuterol equivalent/kg HLC (n = 110) and were also constant for RM 674 at 11.21 +/- 0.15 microg clenbuterol/kg HLC (n=150; measured as duplicates). These studies demonstrate that clenbuterol-containing and clenbuterol-free RMs in bovine eye matrix can be successfully produced. Based on the results described above, it is concluded that both RMs may be suitable as candidates for certification.