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We describe a procedure for quantification of vitamin K(1) in human plasma by HPLC. Samples, enriched with a vitamin K derivative as internal standard, were deproteinized, purified on polymeric RP-SPE cartridges and injected into HPLC equipped with a post-column on-line zinc metal reactor and a fluorometric detector. Median level in blood donors (n=87) was 1.967 nmol/L (0.93-4.01, 5th-95th percentiles), with a significant correlation between plasma levels and age (r=0.276, p=0.00958) and a lower (not significant) value in women than in men. This method, easy-to-handle and with a high throughput, can be used to identify covert states of vitamin K intake deficiency in patients thus at risk of alterations in blood clotting or bone mineralization.

Background and Objectives: Hereditary hyperferritinemia cataract syndrome is caused by mutations of the iron responsive elements (IREs) of L-ferritin mRNA. These alter the IRE structure and determine L-ferritin upregulation. IREs are located in 5'untranslated regions (5'UTR) of ferritin mRNAs. L-ferritin 5'UTR has been extensively studied and up to 21 different mutations have been identified. Only one mutation has been reported for H-ferritin 5'UTR; this mutation modified IRE structure and was apparently associated with high serum ferritin levels and iron overload., Design and Methods: To identify other mutations in H ferritin 5'UTR we developed a fast DNA scanning method based on denaturing high performance liquid chromatography (HPLC). Five artificial DNA mutants were produced in order to validate the analytical conditions of the system for the identification of all mutations by single runs at 68 degrees C. The system was used to screen 660 DNA samples from subjects with high serum ferritin levels., Results: Two abnormal patterns were identified carrying the mutations C20G and G34T. Structural data and the analysis of ferritin levels in red blood cells suggest that these mutations do not affect the functionality of the IRE., Interpretation and Conclusions: This large and first population analysis indicates that mutations in the H-ferritin 5'UTR are rare and do not seem to contribute to hyperferritinemia or iron overload.

Denaturing HPLC (DHPLC) is a useful technique for the fast screening of known and unknown heterozygous gene mutations. Most DNA mutations causing genetic disorders consist of nucleotide substitutions, but insertions and deletions occur, albeit less frequently. The heteroduplexes with insertions/deletions have gaps that may affect molecular stability differently from the mismatches caused by substitutions. Therefore, gaps and mismatches may be distinguished by DHPLC analysis, which is based on the differential thermal stability of amplicons with different characteristics. To verify this hypothesis, we examined 12 DNA samples containing insertions and deletions of different sizes (one to 29 residues) from four different genes (ABCA4, CFTR, FTL, and SLC11A3). We found that all of them were detected by DHPLC runs at 50 degrees C, which is considered a non-denaturing temperature, as well as by runs at the temperature optimized for mismatch recognition. The finding confirms that gaps reduce heteroduplex stability more than mismatches, and indicates that DHPLC analysis at low temperature may be applied to distinguish DNA deletions/insertions from substitutions., (Copyright 2003 Wiley-Liss, Inc.)

A novel high-performance liquid chromatographic (HPLC) method for the quantification of diclofenac in human plasma was set up. Samples, added with ibuprofen (used as internal standard) were purified by solid-phase extraction using Abselut Nexus cartridges (Varian) not requiring pre-conditioning. Drugs of interest were eluted directly into the autosampler vials and injected. The recovery of diclofenac was 92%, the analysis lasted 7 min with a sensitivity of 5 ng/ml and intra- and inter-day RSDs of 3 and 8%, respectively. The pharmacokinetics of diclofenac after oral and rectal administration in 10 healthy volunteers are reported.

The aim of this study was to optimise a method for gentamicin determination in an agar matrix and to investigate if and how agar composition can affect the gentamicin diffusion kinetics during the agar diffusion tests for antibiotics sensitivity. Gentamicin was separated by RP-HPLC and detected at 365 nm after pre-column derivatization with 1-fluoro-2,4-dinitrobenzene. Recovery (> or = 79%), linearity (r2 > or = 0.997) and sensitivity (1 microg/ml) were assessed using four different agar matrices. The kinetics of gentamicin diffusion tested on BioMerieux and DID manufacturers' products showed in uninoculated agar plates significant differences that were even more pronounced in the presence of Pseudomonas aeruginosa metabolism.

Chemokines are members of the chemotactic cytokines family implicated in various immunoregulatory functions. The CXC-chemokine stromal cell derived factor-1 (SDF-1alpha) was purified from the culture medium of murine bone marrow stromal cell line (MS-5) by affinity and reversed-phase liquid chromatography. Yield and purity were assessed by capillary electrophoresis (CE) with reference to the human SDF-1alpha from recombinant DNA technology. CE technique was useful to evaluate the purity of human SDF-1alpha from chemical synthesis and to resolve murine and human SDF-1alpha, differing by only one amino acid. Chemotactic activity of the murine SDF-1alpha was tested on the basis of CE quantification.

We have modified a high-performance liquid chromatographic (HPLC) procedure based on SBD-F (ammonium-7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate) pre-column derivatization to obtain an assay that is useful for routine clinical total plasma homocysteine (tHcy) analysis. The introduction of easily handled sodium borohydride instead of the traditional tri-n-butylphosphine in dimethylformamide as a reductant and a 14-min run-time using basic isocratic HPLC equipment are the more notable advantages. The addition of mercaptopropionylglycine as an internal standard contributed to improvements in the reproducibility of the assay, yielding within- and between-run precisions of 1.9 and 4% (C.V.), respectively. Reference values for fasting tHcy were 7.65+/-2.3 and 8.9+/-2.4 micromol/l, while post-methionine load gave tHcy levels of 19.9+/-5.5 and 26.8+/-5.5 micromol/l, for women and men, respectively (n=40).

The aim of this study was to set up a method for quantification of plasma mitomycin C (MMC) concentrations during intravesical chemotherapy delivered in the presence of local bladder hyperthermia (HT). In comparison with existing methods, this assay, characterized by relative simplicity and efficiency, resulted in the facilitation of performance with nondedicated instrumentation or nonspecialized staff. Purification from plasma matrix was carried out by solid-phase extraction under vaccuum. The purified drug was then collected directly into the vials of the HPLC autosampler. Chromatographic analysis was performed on a reversed-phase C18 column with water:acetonitrile (85:15 by vol) as the mobile phase and the UV detector set at 365 nm. The use of porfiromycin as internal standard provided a method with good within-day precision (CV 6.0% at 5 micrograms/L, n = 6), linearity (0.5-50 micrograms/L), and specificity. The lower limit of detection (< or = 0.5 microgram/L) proved to be suitable for plasma pharmacokinetics monitoring in two tested patients treated with MMC + HT for superficial bladder cancer.

This HPLC assay with o-phthalaldehyde precolumn derivatization is used to measure the total, oxidized, and protein-bound forms of glutathione in human blood, plasma, and rat tissue. Total glutathione (i.e., sum of reduced, oxidized, and protein-bound fractions) was determined after reduction with dithiothreitol and protein precipitation with perchloric acid (PCA). A preliminary selective blockage of free sulfhydryl groups with N-ethylmaleimide was necessary to evaluate the different oxidized forms. The assay showed high sensitivity (< 0.05 pmol injected) and good precision (within-day CVs of 5.5% to 6.4%), recovery (101% +/- 4%), and linearity (r > 0.999). Samples, after PCA acidification, were stable at room temperature and 4 degrees C for 3 days, and at -20 degrees C and -80 degrees C for > 1 month. The method (involving automated derivatization) not only is very rapid and simple but also allows immediate processing of many different biological samples.

A precolumn derivatization method was optimized for rapid and specific analysis of total urinary hydroxyproline by HPLC. After an overnight hydrolysis, urine samples dried and reconstituted with the internal standard cysteic acid (in sodium hydrogen carbonate, pH 9.3) were derivatized with N,N-diethyl-2,4-dinitro-5-fluoroaniline (FDNDEA) at 100 degrees C for 20 min. The DNDEA-hydroxyproline adduct was separated on an Ultrasphere ODS column with a mobile phase of acetate buffer (containing triethylamine, 6 mL/L, pH 4.3) and acetonitrile (80/20, by vol), and was detected at 360 nm. A single run took 18 min with a hydroxyproline retention time of 7.3 min. The assay showed a linear response to hydroxyproline concentrations from 5 to 100 mg/L with a detection limit of 0.8 ng injected, corresponding to 2 mg/L in urine. Mean (SD) analytical recovery was 94.2 (13)% and 104 (9)% at 10 and 50 mg/L, respectively. Within-run and between-run CVs (n = 10) were 3.74% and 4.33%, respectively, for 25 mg/L. Results for samples (n = 50) analyzed by HPLC (y) vs ion-exchange chromatography with postcolumn ninhydrin reaction (x) correlated well: y = 0.98x + 1.02 (r = 0.985, Sxy = 3.13). In another comparison, involving 173 samples, a colorimetric procedure (Hypronosticon, x) gave slightly higher values than the HPLC method (y): y = 0.83x + 2.21 (r = 0.937, Sxy = 4.6).

We compared two pre-column derivatization methods, o-phthaldialdehyde (OPA) and N,N-diethyl-2,4-dinitro-5-fluoroaniline (FDNDEA), for analysis of serum amino acids by reversed-phase high-performance liquid chromatography. Separations took 102 and 106 min for FDNDEA and OPA (reconditioning included), respectively, allowing a very good resolution of 30 amino acids by the former process and 38 by the latter. Linearity, within- and between-day variability and advantages in terms of accuracy and speed were studied for both methods. Twenty serum samples from healthy volunteers were assayed with OPA, FDNDEA and with the reference method of ion-exchange and post-column ninhydrin reaction (amino acid analyser), which took 170 min. The correlation between OPA and ninhydrin was good for all the amino acids (r = 0.959) except for the last-eluting lysine. Good agreement was found for FDNDEA (r = 0.987), which appeared in general to be a highly reproducible technique. Both pre-column methods were more sensitive than the post-column ninhydrin method.