Development and validation of an HPLC method for the determination of spironolactone and its metabolites in paediatric plasma samples

Abstract An HPLC method has been developed and validated for the determination of spironolactone, 7a-thiomethylspirolactone and canrenone in paediatric plasma samples. The method utilises 200 µl of plasma and sample preparation involves protein precipitation followed by Solid Phase Extraction (SPE). Determination of standard curves of peak height ratio (PHR) against concentration was performed by weighted least squares linear regression using a weighting factor of 1/concentration2. The developed method was found to be linear over concentration ranges of 30–1000 ng/ml for spironolactone and 25–1000 ng/ml for 7a-thiomethylspirolactone and canrenone. The lower limit of quantification for spironolactone, 7a-thiomethylspirolactone and canrenone were calculated as 28, 20 and 25 ng/ml, respectively. The method was shown to be applicable to the determination of spironolactone, 7a-thiomethylspirolactone and canrenone in paediatric plasma samples and also plasma from healthy human volunteers.

Quantitative analysis of MRP-8 in gingival crevicular fluid in periodontal health and disease using microbore HPLC.

BACKGROUND:

The protein components of GCF can be separated by reverse-phase microbore HPLC on a C18 column with detection on the basis of 214 nm absorbance. A single major symmetrical protein peak eluting with a retention time of 26 min (50% acetonitrile) was evident in gingival crevicular fluid (GCF) from periodontitis patients but not in healthy GCF. This protein was identified as human MRP-8 by N-terminal amino acid sequencing and liquid chromatography quadropole mass spectrometry.

AIMS:

To quantify the amount of MRP-8 detectable in GCF from individual healthy, gingivitis and periodontitis affected sites and to study the relationship, if any, between the levels of this responsive protein and periodontal health and disease.

METHODS:

GCF was sampled (30 s) from healthy, gingivitis, and periodontitis sites in peridontitis subjects (n=15) and from controls (n=5) with clinically healthy gingiva and no periodontitis. Purified MRP-8 was sequenced by Edmann degradation and the phenylthiohydantoin (PTH) amino acid yield determined (by comparison of peak area with external PTH amino acid standards). This value was subsequently used to calculate the relative amount of protein in the peak eluting with a retention time of 26.0 min (MRP-8) in individual GCF chromatograms.

RESULTS:

Higher levels of MRP-8 were detected in inflammatory sites: periodontitis 457.0 (281.0) ng; gingivitis 413.5 (394.5) ng compared with periodontally healthy sites in diseased subjects 14.6 (14.3) ng and in controls 18.6 (18.5) ng, p=0.003. There was at least 20-fold more MRP-8 in the inflammatory compared with the healthy sites studied.

CONCLUSIONS:

The preliminary data indicate that MRP-8 is present in GCF, with significantly greater amounts present at diseased than healthy sites. A systematic study of the relationship of this protein to periodontal disease could prove useful in further clarifying whether MRP-8 could be a reliable GCF biomarker of gingivitis and periodontitis.

Development and validation of an HPLC method for the rapid and simultaneous determination of 6-mercaptopurine and four of its metabolites in plasma and red blood cells

An HPLC method has been developed and validated for the rapid determination of mercaptopurine and four of its metabolites; thioguanine, thiouric acid, thioxanthine and methylmercaptopurine in plasma and red blood cells. The method involves a simple treatment procedure based on deproteinisation by perchloric acid followed by acid hydrolysis and heating for 45 min at 100 degrees C. The developed method was linear over the concentration range studied with a correlation coefficient >0.994 for all compounds in both plasma and erythrocytes. The lower limits of quantification were 13, 14, 3, 2, 95 pmol/8 x 101 RBCs and 2, 5, 2, 3, 20 ng/ml plasma for thioguanine, thiouric acid, mercaptopurine, thioxanthine and methylmercaptopurine, respectively. The method described is selective and sensitive enough to analyse the different metabolites in a single run under isocratic conditions. Furthermore, it has been shown to be applicable for monitoring these metabolites in paediatric patients due to the low volume requirement (200 mu l of plasma or erythrocytes) and has been successfully applied for investigating population pharmacokinetics, pharmacogenetics and non-adherence to therapy in these patients. (C) 2008 Elsevier B.V. All rights reserved.

Complete Sudoku - extra rules for more symmetry

We propose some extra rules to add to the well-known Sudoku puzzle and present an argument to justify their inclusion. The rules mean that puzzles can be created with fewer cells completed initially yet which still have only one solution. We have created a Web-based program which can be used to generate and solve both standard and extended (Complete) puzzles.

Estimating the masses of extra-solar planets

All extra-solar planet masses that have been derived spectroscopically are lower limits since the inclination of the orbit to our line-of-sight is unknown except for transiting systems. In theory, however, it is possible to determine the inclination angle, i, between the rotation axis of a star and an observer's line-of-sight from measurements of the projected equatorial velocity (v sin i), the stellar rotation period (P(rot)) and the stellar radius (R(*)). For stars which host planetary systems this allows the removal of the sin i dependency of extra-solar planet masses derived from spectroscopic observations under the assumption that the planetary orbits lie perpendicular to the stellar rotation axis.
We have carried out an extensive literature search and present a catalogue of v sin i, P(rot) and R(*) estimates for stars hosting extra-solar planets. In addition, we have used Hipparcos parallaxes and the Barnes-Evans relationship to further supplement the R(*) estimates obtained from the literature. Using this catalogue, we have obtained sin i estimates using a Markov-chain Monte Carlo analysis. This technique allows proper 1 Sigma two-tailed confidence limits to be placed on the derived sin i's along with the transit probability for each planet to be determined.
While we find that a small proportion of systems yield sin i's significantly greater than 1, most likely due to poor P(rot) estimations, the large majority are acceptable. We are further encouraged by the cases where we have data on transiting systems, as the technique indicates inclinations of similar to 90 degrees and high transit probabilities. In total, we are able to estimate the true masses of 133 extra-solar planets. Of these 133 extra-solar planets, only six have revised masses that place them above the 13M(J) deuterium burning limit; four of those six extra-solar planet candidates were already suspected to lie above the deuterium burning limit before correcting their masses for the sin i dependency. Our work reveals a population of high-mass extra-solar planets with low eccentricities, and we speculate that these extra-solar planets may represent the signature of different planetary formation mechanisms at work. Finally, we discuss future observations that should improve the robustness of this technique.