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 45min 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, 95pmol/8 x 10(8) RBCs and 2, 5, 2, 3, 20ng/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 (200microl of plasma or erythrocytes) and has been successfully applied for investigating population pharmacokinetics, pharmacogenetics and non-adherence to therapy in these patients.

One- and two-step spin-crossover behavior of [Fe(II)(isoxazole)6]2+ and the structure and magnetic properties of triangular [Fe(III)3O(OAc)6(isoxazole)3][ClO4)]

The structure and spin-crossover magnetic behavior of [FeII16][BF4]2 (1 = isoxazole) and [FeII16][ClO4]2 have been studied. [FeII16][BF4]2 undergoes two reversible spin-crossover transitions at 91 and 192 K, and is the first two-step spin transition to undergo a simultaneous crystallographic phase transition, but does not exhibit thermal hysteresis. The single-crystal structure determinations at 260 [space group P3̄, a = 17.4387(4) Å, c = 7.6847(2) Å] and at 130 K [space group P1̄, a = 17.0901(2) Å, b = 16.7481(2) Å, c = 7.5413(1) Å, α = 90.5309(6)°, β = 91.5231(6)°, γ = 117.8195(8)°] reveal two different iron sites, Fe1 and Fe2, in a 1:2 ratio. The room-temperature magnetic moment of 5.0 μB is consistent with high-spin Fe(II). A plateau in μ(T) having a moment of 3.3 μB centered at 130 K suggests a mixed spin system of some high-spin and some low-spin Fe(II) molecules. On the basis of the Fe−N bond distances at the two temperatures, and the molar fraction of high-spin molecules at the transition plateau, Fe1 and Fe2 can be assigned to the 91 and 192 K transitions, respectively. [FeII16][ClO4]2 [space group P3̄, a = 17.5829(3) Å, c = 7.8043(2) Å, β = 109.820 (3)°, T = 295 K] also possesses Fe1:Fe2 in a 1:2 ratio, and magnetic measurements show a single spin transition at 213 K, indicating that both Fe1 and Fe2 undergo a simultaneous spin transition. [FeII16][ClO4]2 slowly decomposes in solutions containing acetic anhydride to form [FeIII3O(OAc)613][ClO4] [space group I2, a = 10.1547(7) Å, b = 16.5497(11) Å, c = 10.3205(9) Å, β = 109.820 (3)°, T = 200 K]. The isosceles Fe3 unit contains two Fe···Fe distances of 3.2844(1) Å and a third Fe···Fe distance of 3.2857(1) Å. The magnetic data can be fit to a trinuclear model with ℋ = −2J(S1·S2 + S2·S3) − 2J13(S1·S3), where J = −27.1 and J13 = −32.5 cm-1.

Peptide binding to HLA-DP proteins at pH 5.0 and pH 7.0:a quantitative molecular docking study

Background: HLA-DPs are class II MHC proteins mediating immune responses to many diseases. Peptides bind MHC class II proteins in the acidic environment within endosomes. Acidic pH markedly elevates association rate constants but dissociation rates are almost unchanged in the pH range 5.0 - 7.0. This pH-driven effect can be explained by the protonation/deprotonation states of Histidine, whose imidazole has a pKa of 6.0. At pH 5.0, imidazole ring is protonated, making Histidine positively charged and very hydrophilic, while at pH 7.0 imidazole is unprotonated, making Histidine less hydrophilic. We develop here a method to predict peptide binding to the four most frequent HLA-DP proteins: DP1, DP41, DP42 and DP5, using a molecular docking protocol. Dockings to virtual combinatorial peptide libraries were performed at pH 5.0 and pH 7.0. Results: The X-ray structure of the peptide - HLA-DP2 protein complex was used as a starting template to model by homology the structure of the four DP proteins. The resulting models were used to produce virtual combinatorial peptide libraries constructed using the single amino acid substitution (SAAS) principle. Peptides were docked into the DP binding site using AutoDock at pH 5.0 and pH 7.0. The resulting scores were normalized and used to generate Docking Score-based Quantitative Matrices (DS-QMs). The predictive ability of these QMs was tested using an external test set of 484 known DP binders. They were also compared to existing servers for DP binding prediction. The models derived at pH 5.0 predict better than those derived at pH 7.0 and showed significantly improved predictions for three of the four DP proteins, when compared to the existing servers. They are able to recognize 50% of the known binders in the top 5% of predicted peptides. Conclusions: The higher predictive ability of DS-QMs derived at pH 5.0 may be rationalised by the additional hydrogen bond formed between the backbone carbonyl oxygen belonging to the peptide position before p1 (p-1) and the protonated ε-nitrogen of His 79β. Additionally, protonated His residues are well accepted at most of the peptide binding core positions which is in a good agreement with the overall negatively charged peptide binding site of most MHC proteins. © 2012 Patronov et al.; licensee BioMed Central Ltd.