Evidence is presented that the performance of the rationally designed MALDI matrix 4-chloro-α-cyanocinnamic acid (ClCCA) in comparison to its well-established predecessor α-cyano-4-hydroxycinnamic acid (CHCA) is significantly dependent on the sample preparation, such as the choice of the target plate. In this context, it becomes clear that any rational designs of MALDI matrices and their successful employment have to consider a larger set of physicochemical parameters, including sample crystallization and morphology/topology, in addition to parameters of basic (solution and/or gas-phase) chemistry.

Under the auspices of the Organic Analysis Working Group (OAWG) of the Comité Consultatif pour la Quantité de Matière (CCQM) a key comparison, CCQM K55.b, was coordinated by the Bureau International des Poids et Mesures (BIPM) in 2010/2011. Nineteen national measurement institutes and the BIPM participated. Participants were required to assign the mass fraction of aldrin present as the main component in the comparison sample for CCQM-K55.b which consisted of technical grade aldrin obtained from the National Measurement Institute Australia that had been subject to serial recrystallization and drying prior to sub-division into the units supplied for the comparison.Aldrin was selected to be representative of the performance of a laboratory's measurement capability for the purity assignment of organic compounds of medium structural complexity [molar mass range 300 Da to 500 Da] and low polarity (pKOW < −2) for which related structure impurities can be quantified by capillary gas phase chromatography (GC).The key comparison reference value (KCRV) for the aldrin content of the material was 950.8 mg/g with a combined standard uncertainty of 0.85 mg/g. The KCRV was assigned by combination of KCRVs assigned by consensus from participant results for each orthogonal impurity class. The relative expanded uncertainties reported by laboratories having results consistent with the KCRV ranged from 0.3% to 0.6% using a mass balance approach and 0.5% to 1% using a qNMR method.The major analytical challenge posed by the material proved to be the detection and quantification of a significant amount of oligomeric organic material within the sample and most participants relying on a mass balance approach displayed a positive bias relative to the KCRV (overestimation of aldrin content) in excess of 10 mg/g due to not having adequate procedures in place to detect and quantify the non-volatile content—specifically the non-volatile organics content—of the comparison sample.There was in general excellent agreement between participants in the identification and the quantification of the total and individual related structure impurities, water content and the residual solvent content of the sample.The comparison demonstrated the utility of 1H NMR as an independent method for quantitative analysis of high purity compounds. In discussion of the participant results it was noted that while several had access to qNMR estimates for the aldrin content that were inconsistent with their mass balance determination they decided to accept the mass balance result and assumed a hidden bias in their NMR data. By contrast, laboratories that placed greater confidence in their qNMR result were able to resolve the discrepancy through additional studies that provided evidence of the presence of non-volatile organic impurity at the requisite level to bring their mass balance and qNMR estimates into agreement.Main text. To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison ...

Aphanothece cells could take up Na(+) and this uptake was strongly inhibited by the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP). Cells preloaded with Na(+) exhibited Na(+) extrusion ability upon energizing with glucose. Na(+) was also taken up by the plasma membranes supplied with ATP and the uptake was abolished by gramicidin D, monensin or Na(+)-ionophore. Orthovanadate and CCCP strongly inhibited Na(+) uptake, whereas N, N'-dicyclohexylcarbodiimide (DCCD) slightly inhibited the uptake. Plasma membranes could hydrolyse ATP in the presence of Na(+) but not with K(+), Ca(2+) and Li(+). The K(m) values for ATP and Na(+) were 1.66+/-0.12 and 25.0+/-1.8 mM, respectively, whereas the V(max) value was 0.66+/-0.05 mumol min(-1) mg(-1). Mg(2+) was required for ATPase activity whose optimal pH was 7.5. The ATPase was insensitive to N-ethylmaleimide, nitrate, thiocyanate, azide and ouabain, but was substantially inhibited by orthovanadate and DCCD. Amiloride, a Na(+)/H(+) antiporter inhibitor, and CCCP showed little or no effect. Gramicidin D and monensin stimulated ATPase activity. All these results suggest the existence of a P-type Na(+)-stimulated ATPase in Aphanothece halophytica. Plasma membranes from cells grown under salt stress condition showed higher ATPase activity than those from cells grown under nonstress condition.

Under the auspices of the Organic Analysis Working Group (OAWG) of the Comité Consultatif pour la Quantité de Matière (CCQM) a laboratory comparison, CCQM-P20.f, was coordinated by the Bureau International des Poids et Mesures (BIPM) in 2007/2008. Nine national measurement institutes, four expert laboratories and the BIPM participated in the comparison. Participants were required to assign the mass fraction of digoxin present as the main component in the comparison sample (CCQM-P20.f) which consisted of digoxin material obtained from a commercial supplier stated to comply with USP requirements.In addition to assigning the mass fraction content of digoxin for the material, participants were requested, but not obliged, to provide mass fraction estimates for the minor components they identified in each sample.In contrast with the previous round of the CCQM-P20 series, in which the mass fraction content of theophylline in two comparison samples (CCQM-P20.e.1 and CCQM-P20.e.2) was determined, a wider range of results were reported for the mass fraction content of digoxin in the CCQM-P20.f comparison.A minority of participants did not appear to use conditions capable of fully resolving and/or quantifying the major related structure impurities present in the comparison sample. Among those that did achieve suitable separations, there was further variation in their reported quantifications of the individual and total related substance content which reflected in part the limited availability of reference standards for these materials and the resulting assumptions that had to be made regarding the structure and response factors relative to digoxin for each individual impurity. This was particularly relevant because of the span of molecular masses of the impurities present in the sample, which ranged from aglycones to glycones with tetrameric carbohydrate chains, relative to that of digoxin.A significant additional factor also contributed to the observed variation of results. Unlike the CCQM-P20.e samples, in which the major impurities were solely related structure organic compounds, the CCQM-P20.f study material contained significant levels of residual organic solvents (ethanol, dichloromethane and to a lesser extent toluene). The majority of participants failed to detect and allow for the presence of this class of impurity, introducing a bias towards overestimation of digoxin content in most of the individual results.However, the uncertainty budgets produced by several participants were sufficiently conservative such that their reported results were nevertheless consistent with the reference value for digoxin content assigned using a consensus mass balance approach. The results of the comparison reinforce the conclusion from previous rounds of the CCQM-P20 study that care in developing and validating the suitability of the chromatographic separation method used to resolve the main component from the related structure impurities present is essential to obtaining reliable, comparable results when using the mass balance approa...

Under the auspices of the Organic Analysis Working Group (OAWG) of the Comité Consultatif pour la Quantité de Matière (CCQM) a laboratory comparison, CCQM-P20.e, was coordinated by the Bureau International de Poids et Mesures (BIPM) in 2006/2007. Nine national measurement institutes, two expert laboratories and the BIPM participated in the comparison. Participants were required to assign the mass fraction of theophylline present as the main component in two separate study samples (CCQM-P20.e.1 and CCQM-P20.e.2).CCQM-P20.e.1 consisted of a high-purity theophylline material obtained from a commercial supplier. CCQM-P20.e.2 consisted of theophylline to which known amounts of the related structure compounds theobromine and caffeine were added in a homogenous, gravimetrically controlled fashion. For the CCQM-P20.e.2 sample it was possible to estimate gravimetric reference values both for the main component and for the two spiked impurities.In addition to assigning the mass fraction content of theophylline for both materials, participants were requested but not obliged to provide mass fraction estimates for the minor components they identified in each sample.The results reported by the study participants for the mass fraction content of theophylline in both materials showed good levels of agreement both with each other and with the gravimetric reference value assigned to the CCQM-P20.e.2 material. There was also satisfactory agreement overall, albeit at higher levels of uncertainty, in the quantification data reported for the minor components present in both samples. In the few cases where a significant deviation was observed from the consensus values reported by the comparison participants or gravimetric reference values where these where available, they appeared to arise from the use of non-optimal chromatographic separation conditions.The results demonstrate the feasibility for laboratories to assign mass fraction content with associated absolute expanded uncertainties in the range 0.05% to 0.5% for solid organic compounds of high purity (mass fraction of main component >995 mg/g) and in the range 0.1% to 1% for compounds of lower purity (mass fraction of main component >980 mg/g).Main text. To reach the main text of this paper, click on Final Report.The final report has been peer-reviewed and approved for publication by the CCQM Working Group on Organic Analysis.

Key comparison CCQM-K86 was performed to demonstrate and document the capacity of interested national metrology institutes (NMIs) and designated institutes (DIs) in the determination of the relative quantity of two specific genomic DNA fragments present in a biological tissue. The study provides the support for the following measurement claim: "Quantification of the ratio of the number of copies of specified intact sequence fragments of a length in the range of 70 to 100 nucleotides in a single genomic DNA extract from ground maize seed materials".The study was carried out under the auspices of the Bioanalysis Working Group (BAWG) of the Comité Consultatif pour la Quantité de Matière (CCQM) and was piloted by the Institute for Reference Materials and Methods (IRMM) in Geel (Belgium).The following laboratories (in alphabetical order) participated in this key comparison: AIST (Japan), CENAM (Mexico), DMSc (Thailand), GLHK (Hong Kong), IRMM (European Union), KRISS (Republic of Korea), LGC (United Kingdom), MIRS/NIB (Slovenia), NIM (PR China), NIST (USA), NMIA (Australia), TÜBITAK UME (Turkey) and VNIIM (Russian Federation).The following laboratories (in alphabetical order) participated in a pilot study that was organized in parallel: LGC (United Kingdom), PKU (PR China), NFRI (Japan) and NIMT (Thailand).Good agreement was observed between the reported results of eleven participants.Main text. To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/.The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (MRA).

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) is well-known to be a powerful technique for the analysis of biological samples. By using glycerol-based liquid support matrices (LSMs) instead of conventional MALDI matrices the power of this technique can be extended further. In this study, we exploited LSMs for the identification of complex samples, that is, the Lactobacillus proteome and a bovine serum albumin (BSA) digest. Liquid and solid MALDI samples were manually and robotically prepared by coupling a nanoflow high-performance liquid chromatography (nanoHPLC) system to an automated MALDI sample spotting device. MS and MS/MS data were successfully acquired at the femtomole level using TOF/TOF as well as Q-TOF instrumentation and used for protein identification searching sequence databases. For the BSA digest analysis, liquid MALDI samples resulted in peptide mass fingerprints, which led to a higher confidence in protein identification compared with solid (crystalline) MALDI samples; however, postsource decay (PSD) MS/MS analysis of both the proteome of Lactobacillus plantarum WCFS1 cells and BSA digest showed that further optimization of the formation and detection of peptide fragment ions is still needed for liquid MALDI samples, as the MS/MS ion search score was lower than that for the solid MALDI samples, reflecting the poorer quality of the liquid MALDI-PSD spectra, which can be attributed to the differences in PSD parameters and their optimization that is currently achievable.

Key comparison CCQM-K86.c was performed to demonstrate the capacity of National Metrology Institutes (NMIs) and Designated Institutes (DIs) in the determination of the relative quantity of two specific genomic DNA fragments present in a canola powder. The study provides direct support for the following measurement claim: "Quantification of the ratio of the number of copies of specified intact sequence fragments of a length up to 150 nucleotides following extraction from an unprocessed, high fat/oil ground seed matrix, with a copy number ratio from 0.001 to 1". The study was carried out under the auspices of the Nucleic Acids Working Group (NAWG) of the Consultative Committee for Amount of Substance: Metrology in Chemistry and Biology (CCQM) and was jointly coordinated by the National Research Council of Canada (NRC) and the EU Joint Research Centre, Geel (JRC). The following laboratories (in alphabetical order) submitted measurement results in this key comparison study: Centro Nacional de Metrología, Mexico ("CENAM"); D.I. Mendeleyev Institute of Metrology, Russia ("VNIIM"); EU Joint Research Centre, Geel (JRC); Hong Kong Government Laboratory ("GLHK"); Instituto Nacional de Metrología de Colombia ("INM"); LGC (United Kingdom); National Institute of Biology, Slovenia ("NIB"); National Institute of Metrology, P.R. of China ("NIM China"/"NIMC" [figures]); National Institute of Metrology, Thailand (NIMT); National Measurement Institute, Australia ("NMIA"); National Metrology Institute of Japan, AIST, Japan ("NMIJ"); National Metrology Institute of Turkey ("TÜBITAK"). Main text To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).