APMP-APLAC joint proficiency testing programs for elemental analysis in food with metrological reference values: Assessment of participants' performance considering measurement uncertainties.

The Asia Pacific Metrology Program (APMP) and the Asia Pacific Laboratory Accreditation Cooperation (APLAC) joint Proficiency Testing (PT) programs for toxic elements such as cadmium (Cd) and lead (Pb) or nutritional elements such as iron (Fe) and zinc (Zn) in food were organized by the Korea Research Institute of Standards and Science (KRISS) with the aim of enhancing the quality of measurement and metrological traceability in various economies of the Asia Pacific region by evaluating the performance with rigorous evaluation. Three APMP-APLAC joint PT programs for elemental analyses were carried out by KRISS sequentially, where candidate certified reference materials (CRMs) were used as the PT materials and metrologically traceable certified reference values (RVs) were used as the PT assigned values for the evaluation of participants' results, which allows reliable evaluation of participant performance. This article describes the operation of the PTs and the overall performance of the participating laboratories. The effectiveness of these joint PT programs and trends in PT performance assessment are also discussed. These PT programs confirm the significant importance of using the metrologically traceable RVs instead of the consensus values from participants as the PT assigned value for reliable assessment. The lack of understanding of the concept of coverage factor, degree of freedom, standard uncertainty, and expanded uncertainty was revealed by some participants in these PT programs. Interpreting the zeta-scores or En scores, which are derived by using measurement uncertainties, in conjunction with the z-scores is highly meaningful for assessing participants' ability in measurement capabilities and measurement uncertainty evaluation. Assessment of participants' performance considering measurement uncertainties helps the participants to check how reasonable their measurement uncertainty estimation was. The results of PTs also demonstrated that these PT programs are useful for improving the measurement capability of the laboratories, whereas more capability-building in uncertainty evaluation is required for further improvement.

Ionic strength effect on molecular structure of hyaluronic acid investigated by flow field-flow fractionation and multiangle light scattering.

This study describes the effect of ionic strength on the molecular structure of hyaluronic acid (HA) in an aqueous solution using flow field-flow fractionation and multiangle light scattering (FlFFF-MALS). Sodium salts of HA (NaHA) raw materials (∼2 × 10(6) Da) dispersed in different concentrations of NaCl prepared by repeated dilution/ultrafiltration procedures were examined in order to study conformational changes in terms of the relationship between the radius of gyration and molecular weight (MW) and molecular weight distribution (MWD) of NaHA in solution. This was achieved by varying the ionic strength of the carrier solution used in a frit-inlet asymmetrical FlFFF (FIAF4) channel. Experiments showed that the average MW of NaHA increased as the ionic strength of the NaHA solution decreased due to enhanced entanglement or aggregation of HA molecules. Relatively large molecules (greater than ∼5 MDa) did not show a large increase in RMS radius value as the NaCl concentration decreased. Conversely, smaller species showed larger changes, suggesting molecular expansion at lower ionic strengths. When the ionic strength of the FlFFF carrier solution was decreased, the HA species in a salt-rich solution (0.2 M NaCl) underwent rapid molecular aggregation during FlFFF separation. However, when salt-depleted HA samples (I = 4.66∼0.38 mM) were analyzed with FFF carrier solutions of a high ionic strength, the changes in both molecular structure and size were somewhat reversible, although there was a delay in correction of the molecular structure.

Characterization of sodium hyaluronate blends using frit inlet asymmetrical flow field-flow fractionation and multiangle light scattering.

We characterized ultrahigh molecular weight sodium hyaluronate (NaHA) and blended pharmaceutical products containing NaHA using flow field-flow fractionation and multiangle light scattering-differential refractive index (FlFFF-MALS-DRI). NaHA is a water-soluble polysaccharide with a range of molecular weights (MW; 10(5)~10(8) Da) that is found in body fluids and tissues. NaHA is also used commercially in pharmaceutical and cosmetic applications. We used a frit inlet asymmetrical FlFFF channel to separate aqueous polymers according to their hydrodynamic size, and we used on-line measurements of light scattering to obtain the MW distribution (MWD) as well as structural information about NaHA in aqueous solution. In this study, we investigated NaHA and anti-adhesive blend mixtures of NaHA (a commercial NaHA blend mixture containing sodium carboxymethyl cellulose and a new blend with hydroxyethyl starch (HES)) to determine the molecular weight distribution MWD of NaHA and the blend mixtures and to obtain structural information about these compounds in aqueous solution. We also examined the characteristics of NaHA-HES-polylactic-co-glycolic acid film products exposed to gamma radiation for sterilization purposes.

Development of a certified reference material for the determination of acrylamide in potato chips.

A certified reference material (CRM), KRISS CRM 108-10-003, has been developed for analysis of acrylamide in potato chips, as a representative of carbohydrate-rich food cooked in high-temperature oil. The material was prepared by grinding commercially available potato chips to a paste which was then homogenized, bottled in 15-g units, and stored at -70 °C. Certification, homogeneity and stability testing, were carried out by liquid chromatography-isotope-dilution mass spectrometry (ID-LC-MS). A single ID-LC-MS measurement was performed for each of 10 selected units for certification and homogeneity assessment. The mean measurement result for the 10 bottles, 0.455 ± 0.012 mg kg(-1), was assigned as the certified value of the CRM. The between-bottle homogeneity was 0.8% of the certified value. The within-bottle homogeneity, tested by measuring three replicate sub-samples from each of three randomly selected bottles, was similar to the between-bottle homogeneity. The stability of the CRM under storage conditions (-70 °C) was tested for 21 months and no change in the acrylamide content was observed within the measurement uncertainty. Stability of the CRM at -20 °C (storage at user's site) and room temperature (for regular use and transportation) was also tested. Also presented is the newly designed procedure for evaluating the uncertainty of the certified value for the characterization scheme used in this study.

Depolymerization study of sodium hyaluronate by flow field-flow fractionation/multiangle light scattering.

Thermal depolymerization of ultrahigh-molecular-weight (UHMW) sodium hyaluronate (NaHA) was studied systematically by using frit-inlet asymmetrical flow field-flow fractionation/multiangle light scattering/differential refractive index (FI-AFlFFF/MALS/DRI). FI-AFlFFF was utilized for the size separation of NaHA samples which had been thermally degraded for varied treatment times, followed by light-scattering detection to determine MW and structural information of degraded NaHA products. Analysis of NaHA products showed time-dependent depolymerization of raw molecules into smaller-MW components, as well as unfolding of compact structures of UHMW NaHA. To determine whether the observed decrease in MW of sodium hyaluronate originated from the chain degradation of UHMW molecules or from dissociation of entangled complex particles that may have been formed by intermolecular association, narrow size fractions (1 x 10(7)-6 x 10(7) and >6 x 10(7) MW) of NaHA molecules were collected during FlFFF separation and followed by thermal treatment. Subsequent FI-AFlFFF/MALS analysis of collected fractions after thermal treatment suggested that the ultrahigh-MW region (>10(7) Da) of NaHA is likely to result from supermolecular structures formed by aggregation of large molecules.

Flow field-flow fractionation/multiangle light scattering of sodium hyaluronate from various degradation processes.

Sodium hyaluronate (NaHA) is an ultrahigh molecular weight polysaccharide that is found in body tissues, synovial fluid, the vitreous humor, and the umbilical cord, and the size characterization of NaHA is important in pharmaceutical applications. On-line field-flow fractionation/multiangle light scattering/differential refractive index (FlFFF/MALS/DRI) has been applied for the study of degradation efficiency of sodium hyaluronate (NaHA). A NaHA raw sample was degraded by different chemical or physical methods and the degraded NaHA samples were separated using field-programming FlFFF, in which separation is achieved by differences in diffusion coefficients or hydrodynamic diameters. Separation was followed by serial detection using MALS and DRI. Molecular weight distribution (MWD) and information relating to the radius of gyration of the NaHA samples were examined for the raw and degraded NaHA samples. Samples studied include: two different products of ultrasonic degradation, two products of alkaline degradation, and four different products of enzymatic degradation. While alkaline degradation showed a moderate degradation compared to ultrasonic and enzymatic methods in reducing average MW, the latter two degradation methods showed significant changes in average molecular weight and in conformation of NaHA.

Molecular weight and structure characterization of sodium hyaluronate and its gamma radiation degradation products by flow field-flow fractionation and on-line multiangle light scattering.

A combined flow field-flow fractionation (FlFFF)/multiangle light scattering (MALS)/differential refractive index (DRI) detection method has been utilized for the size fractionation and characterization of ultrahigh molecular weight (MW) sodium hyaluronate (NaHA) samples. Separation of broad MW NaHA polymers was carried out by a frit inlet asymmetrical FlFFF channel employed with a linear field programming method followed by the on-line monitoring of light scattering at multiple angles for the calculation of MW and for the study of the conformation of NaHA samples. NaHA samples examined were: (1) two different viscosity fractions of NaHA obtained by a refinement process and (2) NaHA products of gamma radiation degradation. While the NaHA samples of two different viscosity fractions exhibited clearly different MW distributions and similar conformation, the radiation degraded NaHA samples showed a clear difference in both MW distribution and polymer structure.

A strategy for establishing accurate quantitation standards of oligonucleotides: quantitation of phosphorus of DNA phosphodiester bonds using inductively coupled plasma-optical emission spectroscopy.

A novel approach for quantitation of DNA (oligonucleotides) with an unprecedented accuracy of approximately 1% is reported. Quantitation of DNA is commonly performed by measuring UV absorption or fluorescence from dyes intercalated into DNA. Both methods need accurate quantitation standards to yield more comparable results between laboratories. For establishing technically authentic standards for DNA quantitation, a new measurement approach carrying an inherent capability of absolute quantitation is demanded. The proposed approach is based on the stoichiometric existence of phosphorus (P) in DNA. The quantity of P from the phosphodiester backbone of a purified oligonucleotide was accurately determined using inductively coupled plasma-optical emission spectroscopy (ICP-OES) with yttrium internal standard via acid digestion. The number of moles of oligonucleotides was then calculated from that of P using the stoichiometry. The major issues regarding the validity of the suggested approach were (i) effective removal of extra P sources, (ii) quantitative recovery of P through the digestion process, and (iii) oligomeric purity of the target oligonucleotide. These issues were investigated experimentally using various analytical techniques such as matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), capillary electrophoresis, electrical conductometry, UV spectrometry, and gravimetry. In conclusion, it is feasible to certify pure oligonucleotide reference materials with uncertainties less than 1% using the proposed approach.