Development of a multianalyte method for the determination of anabolic hormones in bovine urine by isotope-dilution GC–MS/MS

A sensitive, specific and selective multianalyte GC–MS/MS method has been developed for the determination of 11 anabolic hormones in bovine urine. After adjusting the urine pH to 4.8, the samples were spiked with deuterated internal standards and submitted to enzymatic hydrolysis with β-glucuronidase/arylsulfatase. Hormones were eluted with methanol through a C18 solid phase cartridge and submitted to a liquid–liquid extraction. Analytes were derivatized by adding N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane and GC–MS data were obtained in the positive electron impact tandem mass mode. Under these conditions, no matrix effects were observed and limit of detection values were in the range of 0.005 ng/mL (diethylstilbestrol) to 0.38 ng/mL (17α-methyltestosterone and 17α-ethynylestradiol). Recoveries from 81% (α-zeranol) to 149% (17α-methyltestosterone) were found under the selected conditions. These results were better than those found using heptafluorobutyric anhydride (HFBA) as derivative reagent and those measured in full scan and selective ion monitoring modes.     

Determination of anabolic steroids in muscle tissue by liquid chromatography–tandem mass spectrometry

A specific and sensitive multi-method based on liquid chromatography–tandem mass spectrometry using atmospheric pressure chemical ionization (LC–APCI–MS/MS) has been developed for the determination of 20 anabolic steroids in muscle tissue (diethylstilbestrol, β-estradiol, ethynylestradiol, α/β-boldenone, α/β-nortestosterone, methyltestosterone, β-trenbolone, triamcinolone acetonide, dexamethasone, flumethasone, α/β-zearalenol, α/β-zearalanol, zearalenone, melengestrol acetate, megestrol acetate and medroxyprogesterone acetate). The procedure involved hydrolysis, extraction with tert-butyl methyl ether, defattening and final clean-up with solid phase extraction (SPE) on Oasis HLB and Amino cartridges. The analytes were analyzed by reversed-phase LC–MS/MS, in positive and negative multiple reaction monitoring (MRM) mode, acquiring two diagnostic product ions from each of the chosen precursor ions for the unambiguous confirmation of the hormones. The method was validated at the validation level of 0.5 ng/g. The accuracy and precision of the method were satisfactory. The decision limits CCα ranged from 0.03 to 0.14 ng/g while the detection capabilities CCβ ranged from 0.05 to 0.24 ng/g. The developed method is sensitive and useful for detection, quantification and confirmation of these anabolic steroids in muscle tissue and can be used for residue control programs.     

IBX in an ionic liquid: eco-friendly oxidation of 17α-methylandrostan-3β,17β-diol, an intermediate in the synthesis of anabolic oxandrolone

An easily available hypervalent iodine(V) reagent, 2-iodoxybenzoic acid (IBX) immobilized in the ionic liquid [bmim][Br] was found to be an efficient and eco-friendly protocol for the oxidation of 17α-methylandrostan-3β,17β-diol (1). At ambient temperature oxidation of 1 with IBX gave mestanolone (2) in good yield and with an increased stoichiometric amount of IBX, oxidation adjacent to the carbonyl functionality (α,β-unsaturation) occurred to give dehydrogenated 17β-hydroxy-17α-methyl-Δ¹-androsten-3-one (3) as the major product in a one-pot reaction. The product is easily obtained by extraction with diethyl ether and evaporation of the solvent.     

Construction of the Skeleton of Phthalascidin, Mechanism of the Formation of the Key Tricyclic Lactam Intermediate

Phthalascidin is a structurally simplified version of Et-743, which is a potent anti-tumor marine natural product isolated from Ecteinascidia turbinata. Its antiproliferative activity is greater than that of the agents taxol, camptothecin, adriamycin, mitomycin C, cisplatin, bleomycin, and etoposide by 1-3 orders of magnitude. An elegant synthesis of Et-743 and phthalascidin has been reported by E. J. Corey and co-workers1,2. As part of our continuing program, we have also engaged in dev…     

Studies of the dissociation and the protonation of TB-chlorosulphophenol

The dissociation constant of each step for TB-chlorosulphophenol has been determined by potentiometric method, and the thermodynamic constants, △G°, △H° and △S°, of the dissociation process have been calculated. The protonation constants were measured by the spectrophotometric method. The pH values of various forms of anions of the chromogenic reagent at their concentrations were also calculated.     

Synthesis of two peptides of α-bungarotoxin and the participation of the amino acid residue Trp-28 of the neurotoxin in the antigenicity of the molecule

Using the method of solid-phase peptide synthesis, two peptides have been synthesized, one of which corresponds to the central ring structure of -bungarotoxin (-BTX), while the second has in position 28 a Gly residue in place of the Trp in the first peptide, and their interrelationship with antitoxin antibodies has been investigated. It has been shown that the amino acid residue Trp-28 of -BTX, which is the contact residue in binding with the acetylcholine receptor, also participates directly in binding with the active centers of antibodies to -BTX.Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 628–631, July–August, 1997.     

Determination of the equilibrium melting point of the β-form of polypropylene

The melting behavior of the -form of isotactic polypropylene (-iPP) was investigated as a function of crystallization time and temperature. Calcium suberate, a selective -nucleating agent was used to produce samples that consist entirely of -form i-PP. The experimental melting points were recorded at different crystallization times and were extrapolated to the start of the crystallization process in order to eliminate the effect of lamellar thickening. Using the non-linear Hoffman—Weeks approach to correlate these extrapolated experimental melting temperatures with the corresponding crystallization temperatures, an equilibrium melting point of 209°C was obtained for -iPP. The equilibrium melting point estimated through the non-linear Hoffman—Weeks analysis is about 30°C higher than that (T _m ⁰=177°C) obtained on the basis of the linear extrapolation. These results are consistent with earlier claims that a linear extrapolation of T _m–T _c data leads to an underestimation of the equilibrium melting point. The results obtained for -iPP exemplify the importance of accounting for both the isothermal lamellar thickening effects and the non-linearity in the T _m–T _c correlation, when the determination of an equilibrium melting point is carried out using a procedure based on the predictions of the Lauritzen—Hoffman secondary nucleation theory.This revised version was published online in November 2005 with corrections to the Cover Date.     

The applicability of the “straight-line method” of asmus in the determination of the composition of polynuclear complexes

The straight-line method of Asmus was originally developed for the determination of n in mononuclear complexes of the general form AB_n (n≧ 1). In the present investigation it is demonstrated that the method also can be used for determining the value of n in polynuclear complexes of the form A[_mB_n (m > 1). The method as suggested by Asmus, is, however, not capable of distinguishing between mono- and polynuclear species. It is further shown, that the straight-line method can be applied for the determination of the value of m.     

The effect of the primary structure of the polypeptide catalyst on the enantioselectivity of the Juliá-Colonna asymmetric epoxidation of enones

Epoxidation of chalcone (1), using basic hydrogen peroxide, catalysed by polypeptides with defined primary structures demonstrates that the residues in the chain near to the N-terminus determine the stereochemical outcome of the reaction.     

Addition of γ-silyloxyallyltins on ethyl glyoxylate: evaluation of the influence of the experimental conditions on the stereochemical course of the reaction

Ethyl glyoxylate was reacted with α-substituted γ-(t-butyldimethylsilyloxy)-allyltributyltin in order to obtain selectively each diastereomer of ethyl 3-(t-butyldimethylsilyloxy)-2-hydroxyhex-4-enoate and subsequently the corresponding diols. Diastereomers syn-E, anti-E and anti-Z were obtained in good yields with good to high selectivities and the obtained results were rationalized by consideration of cyclic or open transition states in agreement with the experimental conditions and with the structure of the starting reagents.     

The kinetics of the polymorphic transition of the α-form of 2,4,6,8,10,12-hexanitrohexaazaisowurtzitane

The α → γ polymorphic transition in hexanitrohexaazaisowurtzitane was studied by optical microscopy, calorimetry, IR spectroscopy, thermogravimetry, and X-ray diffraction. The thermal effect of the transition was determined. The kinetics of the process is complex because of the relation between structural rearrangement and transition with the removal of water stabilizing the structure of the α polymorph. Depending on the morphological characteristics of the initial sample, the polymorphic transition can follow a frontal-heterogeneous mechanism (single crystal → polycrystalline aggregate) or a quasi-homogeneous mechanism (single crystal → single crystal).     

Application of the Mössbauer spectroscopy to the study of the oxidation state of azaferrocene derivatives

Azaferrocene has two active sites of iron and nitrogen atoms. Drastic change of the oxidation state in iodine oxidation of azaferrocene is observed by introducing the methyl substituents into the pyrrole ring, while all the N-methylates show a similar electronic state. It was revealed that an introduction of methyl substituent to the pyrrole ring promotes the oxidation of nitrogen atom in pyrrole ring more than the central iron atom.     

Effect of the Composition of the Water–Dimethyl Sulfoxide Solvent on the Thermodynamics of the Formation of the [Ag(18-Crown-6)]+Complex

The effect of a water–dimethyl sulfoxide solvent (X _DMSO= 0–0.97, where X _DMSOis the mole fraction of DMSO) on the thermodynamics of complexation between Ag⁺and 18-crown-6 and the solvation of all reagents involved in this equilibrium were studied. In aqueous solutions, the complex is stable mainly because of the enthalpy contribution to _r G°. For X _DMSO> 0.3, the contributions from entropy and enthalpy become comparable in magnitude, but they are opposite in sign. In the binary solvent, the complex is most stable at X _DMSO= 0.2 to 0.3. Analysis of the enthalpy characteristics of reagent solvation showed that this solvent effect was due to the superposition of two opposite solvation contributions occurring with an increase in the DMSO concentration in the binary solvent, namely, the destabilization of the ligand solvate sphere and the formation of stable Ag⁺complexes with DMSO.     

Development of a molecularly imprinted polymer-matrix solid-phase dispersion method for selective determination of β-estradiol as anabolic growth promoter in goat milk

A simple, fast, and sensitive method for determination of 17 β-estradiol (E2) in goat milk samples has been developed by combining selective molecularly imprinted matrix solid-phase dispersion (MIP–MSPD) and liquid chromatography with diode-array detection (DAD). The molecularly imprinted polymer was synthesized by use of 17β-estradiol as template molecule, methacrylic acid as functional monomer, ethylene glycol dimethacrylate as crosslinker monomer, azobisisobutyronitrile as initiator, and acetonitrile as porogen, and was used as selective solid support for matrix solid-phase dispersion. The selected dispersant had high affinity for E2 in the goat milk matrix and the extract obtained was sufficiently clean for direct injection for HPLC analysis without any interferences from the matrix. The proposed MIP–MSPD method was validated for linearity, precision, accuracy, decision limit (CCα) and detection capability (CCβ), in accordance with European Commission Decision 2002/657/EC criteria. Linearity ranged from 0.3–10 μg g^?1 (correlation coefficient r ²?>?0.999). Mean recovery of E2 from goat milk samples at different spiked levels was between 89.5 and 92.2%, with RSD values within 1.3–2%. CCα and CCβ values were 0.36 and 0.39 μg g^?1, respectively. The developed MIP–MSPD method was successfully applied to direct determination of E2 in goat milk samples.