Characterization of Low-Molecular-Weight Heparins by Strong Anion-Exchange Chromatography.

Currently, detailed structural characterization of low-molecular-weight heparin (LMWH) products is an analytical subject of great interest. In this work, we carried out a comprehensive structural analysis of LMWHs and applied a modified pharmacopeial method, as well as methods developed by other researchers, to the analysis of novel biosimilar LMWH products; and, for the first time, compared the qualitative and quantitative composition of commercially available drugs (enoxaparin, nadroparin, and dalteparin). For this purpose, we used strong anion-exchange (SAX) chromatography with spectrophotometric detection because this method is more helpful, easier, and faster than other separation techniques for the detailed disaccharide analysis of new LMWH drugs. In addition, we subjected the obtained results to statistical analysis (factor analysis, t-test, and Newman-Keuls post hoc test).

Development and single-laboratory validation of a method for the determination of total fructans in infant formula.

A number of methods have previously been developed for the determination of fructans in food products, resulting in the frequently used AOAC Official Methods 997.08 and 999.03. Method 997.08 has a lower LOQ, but the accuracy may suffer if significant quantities of sucrose are present. Method 999.03 is less affected by sucrose, but the LOQ is not as low as that of the other method. In this work we have combined these two methods, using the sample preparation steps of AOAC Method 999.03 to remove sucrose and other free sugars, and the detection procedure of AOAC Method 997.08 to achieve a low LOQ. The resulting new method achieved an LOQ of 0.13 g/100 g with powdered products (using 1 g sample weight) and recoveries in the range of 83-103% in the presence of sucrose at 12 g/100 g. Expanded measurement uncertainties were calculated and ranged from +/-17.4% at oligofructose and fructooligosaccharide (FOS) concentrations of around 0.2 g/100 g to -/+21.0% at FOS concentrations of 0.6 g/100 g.

Ion chromatographic determination of phosphorus soluble in different extracting media in fertilizers.

A new method based on ion chromatography (IC) was developed for the determination of phosphorus in fertilizers. Fertilizers were extracted with water, mineral acids, and 2% formic acid, 2% citric acid, and neutral ammonium citrate solutions according to European Regulation No. 2003/2003 of the European Parliament and the Council of October 13, 2003, or the Decree of the Italian Agriculture Minister of June 17, 2002; the extracts were analyzed by direct injection, after simple filtration, by IC on an IonPac AS19 (250 x 4 mm id) column, using a KOH (21-50-21 mM) gradient and suppressed conductivity detection. The calibration plot was linear over the range of 5-50 mg/L (r(2) of >0.999). The method was evaluated by comparison with a gravimetric method according to established norms. Associated uncertainty at the 95% confidence level was established as 0.47% for the determination of 3-46% P2O5 by IC. A good chromatographic separation of phosphorus forms such as phosphates and phosphites, and some other important anions like nitrates, chlorides, and sulfates present in many commercial fertilizers was also possible, with a linear response over the range of 5-50 mg/L. After a more complete validation, this IC determination of phosphorus could replace more tedious methods such as those using gravimetric determinations.

Phosphopeptides enrichment using on-line two-dimensional strong cation exchange followed by reversed-phase liquid chromatography/mass spectrometry.

We have developed a method to isolate and enhance the detection of phosphopeptides using liquid chromatography (LC)/mass spectrometry on a tryptic-digested protein sample. The method uses an on-line two-dimensional chromatography approach that consists of strong cation exchange (SCX) followed by reversed-phase (RP) chromatography with mass spectrometric detection. At pH 2.6 or lower, tryptic phosphopeptides are not retained during the first-dimension SCX chromatography step. Thus the capture of these peptides in the flow-through by the second-dimension RP trap can dramatically reduce the complexity of the phosphopeptide chromatography, resulting in little or no suppression of the signal often caused by the coeluting nonphosphorylated peptides. The method provides higher phosphopeptide recovery and less nonspecific biding of acidic peptides than the commonly used enrichment methods, such as immobilized metal affinity chromatography. Since the widely adopted multidimensional LC strategy in shotgun proteomics uses a similar SCX-RP approach, the method can be adapted to detect and characterize phosphopeptides from a complex mixture in a single experiment. Limitations of the method are also discussed.

Spectrophotometric determination of enrofloxacin and pefloxacin through ion-pair complex formation.

Two simple, quick and sensitive spectrophotometric methods are described for the determination of enrofloxacin and Pefloxacin. The methods are based on the reaction of these drugs with bromophenol blue (BPB) and methyl orange (MO) in buffered aqueous solution at pH 2.3-2.5 in case of bromophenol blue and at pH 3.6 with MO to give highly coloured complex species, extractable with chloroform. The coloured products are quantitated spectrophotometrically at 420 and 424 nm for BPB and MO, respectively. Optimisation of the different experimental conditions is described. Beer's law is obeyed in the concentration ranges 2-12 and 2-18 microg ml(-1) with BPB and in the ranges 1-12 and 4-40 microg ml(-1)with MO for enrofloxacin and pefloxacin, respectively. The proposed methods are applied for determination of Enroxil oral solution, Peflacine tablets and Peflacine ampoules with mean percentage accuracies 99.5+/-0.99, 99.39+/-1.05 and 100.02+/-0.895, respectively, with BPB and 100.30+/-0.89, 100.25+/-0.98 and 100.20+/-0.72, respectively, with MO.

Development of a new method for the quantitative analysis of the extracellular polysaccharide of Neisseria meningitidis serogroup A by use of high-performance anion-exchange chromatography with pulsed-amperometric detection.

A new method for the quantitative determination of Neisseria meningitidis group A (MenA) capsular polysaccharide (CPS) has been developed. The method is based on trifluoracetic acid (TFA) hydrolysis of the CPS (2 M at 80 degrees C for 3 h), followed by chromatographic separation and quantification of the liberated mannosamine-6-phosphate from the area of the peak obtained using an IonPac AS11 column coupled to the sensitive pulsed amperometric detector ED40. The highly selective nature of this method circumvents the interference problems associated with the classical method based on a colorimetric assay for phosphorus. Provided that suitable hydrolysis conditions can be found, this chromatographic approach might be applicable to the quantification of other bacterial antigens containing phosphorylated sugars such as meningococcal groups H, L, X and Z, and pneumococcal serotypes 6, 10A and 19.

Liquid chromatographic determination of lysine, methionine, and threonine in pure amino acids (feed grade) and premixes: collaborative study.

A total of 17 laboratories (including one author's laboratory) participated in a collaborative study for determination of lysine, methionine, and threonine in trade products or concentrated amino acid premixes. Thirteen samples, 4 pure amino acids and 6 premixes, including 3 Youden matched pairs, were analyzed. The applied liquid chromatographic (LC) method using cation-exchange resin and post-column derivatization with ninhydrin or o-phthaldialdehyde was shown to be accurate and specific for the analytes. Titration procedures, normally used for the assay of pure amino acids, are unspecific and the accuracy of the results can be affected by impurities. Repeatability relative standard deviations, RSDr, ranged from 0.84 to 1.17% for pure amino acids and from 0.50 to 1.68% for premixes; reproducibility relative standard deviations RSDR, ranged from 1.52 to 2.31% for pure amino acids and from 1.48 to 2.59% for premixes. Recoveries were between 97.5 and 102.8% of the expected amino acid assays. The method has been adopted Official First Action status by AOAC INTERNATIONAL.

[The biosynthesis of 2H-labelled inosine by Bacillus subtilis bacteria in a heavy-hydrogen medium]. / Biosintez 2H-mechenogo inozina bakteriei Bacillus subtilis v tiazhelovodorodnoi srede.

We studied biosynthesis of the purine ribonucleoside inosine by the strain of Bacillus subtilis, which was adapted to deuterium through plating to individual colonies on 2% agar with 2H2O and subsequent selection. For growing the strain, a special heavy hydrogen medium with a high level of deuteration (89-90 at. % 2H) based on 2% hydrolyzate of the biomass of the methylotrophic Brevibacterium methylicum as a source of 2H-labeled growth substrates was obtained on 98 vol % 2H2O and 2 vol % [U-2H] methanol. We provide experimental data on the strain growth and accumulation of inosine in the culture medium. A study of the level of inosine deuteration using mass-spectroscopy of fast atoms has shown a polymorphism of deuterium incorporation in the molecule (isotope composition of inosine: 4 at. 2H, 20%; 5 at. 2H, 38%; 6 at. 2H, 28%; 7 at. 2H, 14%), deuterium being incorporated in the ribose and hypoxanthine fragments of the molecules.

A novel high-performance anion-exchange chromatographic method for the analysis of carrageenans and agars containing 3,6-anhydrogalactose.

A novel method has been developed to determine the sugar composition of 3,6-anhydrogalactose-containing polysaccharides, such as carrageenan and agar. The method is based on reductive hydrolysis with a methylmorpholine-borane complex in the presence of acid and subsequent high-performance anion-exchange chromatography analysis of the alditols without any derivatization. The method was validated by 13C NMR analysis of six carrageenans and three agars and by a previously used method based on derivatization to alditol acetates and gas-liquid chromatography analysis. The new method was found to be superior to the gas-liquid chromatography method as the analysis time was less than half. Also it was found to be more accurate and reproducible and no derivatization was required. The analysis of the six different carrageenan samples revealed that homogeneous mu- and nu-carrageenan, theoretically without 3,6-anhydrogalactose residues, cannot be isolated from red seaweeds. Consequently, the question arose if mu- and nu-carrageenans at all are present in seaweeds and if the current hypotheses regarding biosynthesis of carrageenans in the seaweeds are correct. The data demonstrated that carrageenans are highly complex natural polysaccharides, which are more irregular than assumed hitherto. The new analytical technique will permit elucidation of the detailed structure of seaweed polysaccharides and determination of their structure-property relationships.

Facile purification of fibrinogen fragments using a computer-based model with general applicability to the generation of salt gradients.

We and others have recently shown that specific fragments of cross-linked fibrin affect cell behavior. In order to develop a facile method for the preparative scale purification of fibrin fragment D dimer, a simple gradient generating system for conventional chromatography was developed and validated, and methods of fibrin fragment D dimer purification were compared. The experimentally measured salt concentration/time relationship fell directly on the model-predicted line. Model-predicted changes in the reservoir volume and/or salt concentration in the limit buffer affected both the initial slope and the shape of the concentration/time relationship. This gradient generation method was used to separate the D domains of fibrin(ogen) from the amino terminal region E domain using anion-exchange chromatography. While the predicted salt gradient was achieved, a salt-dependent separation was found to be less optimal than that of a pH-dependent separation, as validated by Coomassie-stained SDS-PAGE and by immunoblotting. In conclusion, a facile, user-friendly, computer-based method to predict and generate salt gradients was written and validated by direct experimentation. While fibrinogen fragment purification was acceptable using this system, both separation and yields of fibrinogen and fibrin fragments were superior using a pH-based separation technique.

Analysis of the N-acetylneuraminic acid and N-glycolylneuraminic acid contents of glycoproteins by high-pH anion-exchange chromatography with pulsed amperometric detection.

Presence or absence of N-acetylneuraminic acid (Neu5Ac) can change a sialylated glycoprotein's serum half-life and possibly its function. We evaluated the linearity, sensitivity, reproducibility, and accuracy of a HPAEC/PAD method to determine its suitability for routine simultaneous analysis of Neu5Ac and N-glycolylneuraminic acid (Neu5Gc). An effective internal standard for this analysis is 3-deoxy-d-glycero-d-galacto-2-nonulosonic acid (KDN). We investigated the effect of the Au working electrode recession and determined that linear range and sensitivity were dependent on electrode recession. Using an electrode that was 350 microm recessed from the electrode block, the minimum detection limits of Neu5Ac, KDN, and Neu5Gc were 2, 5, and 2 pmol, respectively, and were reduced to 1, 2, and 0.5 pmol using a new electrode. The response of standards was linear from 10 to 500 pmol (r2>0.99) regardless of electrode recession. When Neu5Ac, KDN, and Neu5Gc (200 pmol each) were analyzed repetitively for 48 h, area RSDs were <3%. Reproducibility was unaffected when injections of glycoprotein neuraminidase and acid digestions were interspersed with standard injections. Area RSDs of Neu5Ac and Neu5Gc improved when the internal standard was used. We determined the precision and accuracy of this method for both a recessed and a new working electrode by analyzing Neu5Ac and Neu5Gc contents of bovine fetuin and bovine and human transferrins. Results were consistent with published values and independent of the working electrode. The sensitivity, reproducibility, and accuracy of this method make it suitable for direct routine analysis of glycoprotein Neu5Ac and Neu5Gc contents.

Simultaneous determination of oxalate, citrate and sulfate in children's plasma with ion chromatography.

To improve our understanding of both diagnosis and treatment of diseases of oxalate metabolism, we first set out to establish a new ion-chromatographic method to determine normal plasma levels of oxalate, citrate and sulfate from single plasma samples. In 50 infants and children (23 girls, 27 boys, aged 0.2 to 17 years) with normal renal function, blood was drawn in Li-heparin tubes, placed on ice and preserved immediately with 40 microliters M HCl/ml plasma in two ultracentrifugation steps. For measurement, plasma was injected onto an ion chromatography system with NaOH as the mobile phase, and then run as a linear gradient from 5 mM to 52.5 mM over 21 minutes. Analysis yielded measurable and reproducible oxalate (6.43 +/- 1.06 microM/liter), citrate (79.3 +/- 27.4 microM/liter) and sulfate (235.0 +/- 85.3 microM/liter) levels, without any age and gender specific differences. The least detectable plasma oxalate level was < 0.3 microM with a high reliability and reproducibility (coefficient of variance 1.95 to 4.75%). In conclusion, we established a reproducible, precise method to determine the relevant plasma anions involved in mineral metabolism, which heretofore have not been easily measurable. Studies of diseases of oxalate and citrate metabolism are ongoing on the basis of the normal plasma values achieved in this study.

State-of-the-art of serum total calcium measurement as investigated by split-sample measurement with an ion chromatography candidate reference method.

We evaluated bias and inaccuracy of four frequently used routine test systems for serum total calcium, using a candidate reference method based on ion chromatography. The mean biases and 95% confidence intervals that we observed were 0.0 +/- 0.59% for Johnson & Johnson arsenazo(III), 1.3 +/- 0.62% for Beckman arsenazo(III), -0.4 +/- 0.44% for Beckman applying ion selective electrode measurement after sample dilution, and -1.9% +/- 0.42% for Boehringer o-cresolphthalein. The inaccuracy of all test systems was usually < 4.7% (calculated as deviation of singlicates from ion chromatography). Both bias and inaccuracy are discussed in the light of specifications set by expert groups or derived from the biological variation of serum total calcium. The study revealed that the intrinsic quality of commonly used test systems for serum total calcium satisfies even some of the more stringent criteria for method bias and inaccuracy.

The direct determination of urinary oxalate by non-suppressed ion chromatography.

In this paper we describe a simple ion chromatographic method for determination of oxalate in urine. Acidified urine was diluted 1:2 with 0.03 mol/l benzidine hydrochloride in 0.3 mol/l boric acid to precipitate sulphate. The supernatant was passed through a C18-cartridge and 100 microliters of eluant were injected into an ion chromatographic system. Oxalate was measured by nonsuppressed conductivity detection. The detection limit for urinary oxalate was 0.05 mmol/l. The recovery for spiked urine samples was 101.5% with a CV of 4.5%. The within- and between-assay coefficients of variation were less than 4.5% and 2.5%, respectively. We found the results obtained by this method to be statistically equivalent to an enzymatic assay and a different ion-chromatographic method.

Salivary and plaque acids in caries active and caries free subjects.

The levels of organic acids in the plaque and saliva of 30 subjects aged between 12 and 20 years were investigated. Fifteen of the subjects were caries free and 15 had active carious lesions. The variables examined were the DMFT, plaque index, bleeding index, the flow rate of saliva, daily intake of fibre and sucrose and the level of acids in resting plaque and saliva exposed to a glucose rinse. Posterior bitewing radiographs were taken to confirm the presence of carious lesions. The Kruskal Wallis one-way analysis of variance was used to compare caries free and caries active subjects. Caries active subjects consumed more sucrose (p < 0.001) and had lower levels of acetic acid in their saliva (p = 0.05) than caries free subjects. The acetic acid-acetate buffer system can act as a buffer which suggests that acetic acid in the saliva of caries free subjects may diffuse into plaque to act as an effective buffer to counteract the destructive effects of lactic acid.

Fractals for multicyclic synthesis conditions of biopolymers. Examples of oligonucleotide synthesis measured by high-performance capillary electrophoresis and ion-exchange high-performance liquid chromatography.

We have developed models of patterns for nucleotide chain growth. These patterns are measurable by high-performance capillary electrophoresis and ion-exchange high-performance liquid chromatography in crude products of solid-phase synthesized 30mer and 65mer oligodeoxyribonucleotide target sequences N. We introduce mathematical methods for finding characteristic values d(o) and p(o) for constant chemical modes of growth as well as d and p for non-constant chemical modes of growth (d = probability of propagation, p = probability of termination). These methods are employed by presenting the accompanying computer software developed by us in C code, Mathematica R languages, and Fortran. Characteristic values of the parameters d, p, and the target nucleotide length N describe the complete composition of the crude product. From this we have developed the relation 2 - [N/(N - 1)]/Da, measurable(N,d) as a universal quantitative measure for multicyclic synthesis conditions (D, fractal dimension and similarity exponent, respectively). We use this mathematical treatment to compare the efficiency of oligodeoxyribonucleotide syntheses of different target length N on polymer support materials. Further, we analyze selected syntheses of short and long oligodeoxyribonucleotides as well as single-stranded DNA sequences by well-known empirical autocorrelation, fast Fourier transformation, and embedding dimension techniques.

Nonlinear filtering for the estimation of glycated hemoglobin in the presence of fetal hemoglobin using microcolumn ion-exchange chromatography.

In this paper, an improved method based on nonlinear least-squares to estimate the percentage concentration of glycated hemoglobin levels HbA1ab, HbA1c, HbF, and other Hb variants is presented. If the existing method is enhanced with the improved method, the use of microcolumn ion-exchange chromatography for estimating glycated hemoglobin levels even for patients with elevated fetal hemoglobin (HbF) would provide a more realistic estimate.

Determination of short-chain aliphatic, oxo- and hydroxy-acids in drinking water at low microgram per liter concentrations.

A fast and reliable ion chromatography method has been developed and applied to study the formation and consumption of organic acid ozonation by-products in a drinking water treatment plant. Water samples are injected directly into the ion chromatograph using a large sample loop (740 mu l) without any sample preparation step other than possibly filtration. Organic and inorganic anions are determined by separation on a high-capacity anion-exchange column followed by conductivity detection. The average recovery for the organic acids investigated (beta-hydroxybutyric, acetic, glycolic, butyric, formic, alpha-ketobutyric and pyruvic acid) ranged from 96 to 105%, and their method detection limits ranged from 1 to 5 mu g/l. When applied to samples taken from a drinking water treatment plant, the method proved to be reliable.

Glycohemoglobin assays evaluated in a large-scale quality-control survey.

We report the results of a national quality-control survey on glycohemoglobin (GHb), monitored in France by the Société Française de Biologie Clinique on behalf of the authority of the "Agence du Médicament." A sample of lyophilized hemolysate was sent to 3109 laboratories. Results were obtained from 2770 laboratories. HbA1C, HbA1, and total GHb were measured by 50%, 24%, and 26% of the participants, respectively. Of these measurements, 79% of the HbA1C results and 76% of the total GHb results, but only 48% of the HbA1 results, were within the +/- 20% limits of the indicated target values. Mean values for the hemolysate ranged from 8% to 11% for HbA1C, from 7% to 12% for HbA1, and from 11% to 13% for total GHb. The interlaboratory CVs ranged from 3% to 20%, according to method used. So, methods used for GHb assay, which are based on various principles, exhibit very different analytical performances. Nonetheless, this large-scale study indicates that some techniques can support transferability of results from laboratory to laboratory.

Three assays for glycohemoglobin compared.

Using 123 specimens, we compared the concordance of three different methods for determining glycohemoglobin (GHb): the Diamat (Bio-Rad Laboratories), an automated analyzer measuring HbA1c by cation-exchange chromatography; an assay with the IMx analyzer (Abbott Laboratories), based on boronate affinity binding; and an HPLC method measuring HbA1c by cation-exchange chromatography on a PolyCAT A column (PolyLC Inc.). The Pearson's correlation coefficient between PolyCAT A and Diamat was 0.900 +/- 0.038 (mean +/- 2 SD) and between PolyCAT A and IMx, 0.857 +/- 0.042. However, up to twofold differences were seen in some samples. The proportion of GHb was consistently lower with the PolyCAT A method than with the other two assays, apparently because of better separation of HbA1c from nonglycated coeluting forms of Hb. The difference in glycation percentage between the PolyCAT A and Diamat methods is 2-3% over the whole concentration range. These results point to the limitations of Diamat as a reference method to be used to calibrate other methods for determining HbA1c. Further, a switch from one method to another is likely to cause considerable problems in the clinical follow-up of certain patients.