Rapid multi-residue method for the quantitative determination and confirmation of glucocorticosteroids in bovine milk using liquid chromatography-electrospray ionization-tandem mass spectrometry

Dexamethasone, betamethasone and prednisolone are synthetic glucocorticosteroids authorised for therapeutic use in bovine animals within the European Union. Dexamethasone and betamethasone are used mainly for the treatment of metabolic and inflammatory diseases. Prednisolone is used to treat bovine mastitis. Maximum residue limits (MRLs) of 0.3 μg kg⁻¹ for both dexamethasone and betamethasone and 6.0 μg kg⁻¹ for prednisolone in bovine milk have been established. 6α-Methylprednisolone and flumethasone are not authorised for use in bovine animals and are completely banned in bovine milk. The proposed method is based on deprotenisation of milk using 20% (w/v) trichloroacetic acid. Samples are filtered using glass microfibre filters and subject to clean-up using OASIS HLB solid phase extraction. Separation was achieved on a Hypercarb 100 mm × 2.1 mm × 5 μm column. Mobile phase was: 90/10 acetonitrile/0.1% formic acid in water; flow rate was 600 μL min⁻¹. The method allowed the rapid identification and confirmation of the five glucocorticosteroids according to the criteria laid down in Commission Decision 2002/657/EC. Matrix calibration curves for all compounds were linear in the interval 0.0 MRL to 2.0 MRL with a correlation coefficient (r²) higher than 0.96. Relative recoveries ranged from 97% for betamethasone to 111% for prednisolone. Precision at the MRL ranged from 3.8% for prednisolone to 13.8% for betamethasone. Decision limits, CCα, and detection capability, CCβ have been calculated for all compounds.     

Microbial hydrolysis of 17 β-acetoxy steroidal compounds during aromatization by Arthrobacter simplex

Steroidal compounds containing 17β-acetoxy group after aromatization by Arthrobacter simplex afforded 17β-hydroxy-, 17-keto- or unhydrolyzed 17β-acetoxy-ring A-aromatic compounds. 17-Keto-compounds were probably formed through the oxidation of the corresponding 17 β-hydroxyl group during fermentation with this microorganism. Hydrolysis of 17 β-acetoxy group is dependent upon other substituents present in the substrates.     

Rapid simultaneous determination of dexamethasone and betamethasone in milk by liquid chromatography tandem mass spectrometry with isotope dilution

A simple, sensitive and reliable analytical method for the rapid simultaneous determination of dexamethasone and betamethasone in milk by high performance liquid chromatography–negative electrospray ionization tandem mass spectrometry (HPLC–NESI-MS/MS) with isotope dilution was developed. Samples were directly purified through C₁₈ cartridge. Then the eluate was dried under nitrogen and residues were dissolved in mobile phase. Samples were analyzed by HPLC–MS/MS on a Hypercarb graphite column with a mixture of acetonitrile–water–formic acid as mobile phase. The samples were quantified using dexamethasone-D₄ as an internal standard. The procedure was validated according to the European Union regulation 2002/657/EC determining specificity, decision limit (CCα), detection capability (CCβ), trueness, precision, linearity and stability. The method is demonstrated to be suitable for the determination of dexamethasone and betamethasone in milk. The total time required for the analysis of one sample was about 35 min.     

Stereoselectivity in the electrophilic addition reactions of stigmast-22(23)-ene derivatives

Oxidation of 5α-stigmast-22-en-3- one (1) with m-chloroperbenzoic acid afforded [22R,23R]-epoxide 3 and [22S, 23S]-epoxide 2, in a 5:3 ratio. Reaction of 1 with iodine/silver acetate gave a mixture of iodoacetates 8 and 9, which on treatment with base yielded the single epoxide 2. Those results suggest that electrophiles may preferentially approach the Δ²²-bond from the side of the 21-Me group, in accordance with observations with the ergosterol-like side chain.     

Colorimetric Microdetermination of some Corticosteroid Drugs Using Indophenol as Chromophoric Reagent

Abstract

A simple, rapid and sensitive method for the determination of betamethasone (I), dexamethasone (II) and hydrocortisone (III), either in the pure form or in pharmaceutical formulations is described. The method is based on the development of a brown product with indophenoi in basic aqueous-ethanolic (50% v/v) medium. The optimum reaction conditions for the charge transfer complex formed were assessed. The absorbance measurements were made at 820, 816 and 822 nm for I, II and III respectively. The calibration graph was linear in the range 1-26, 1-32 and 1-35 μg/ml of I, II and III with slopes of 0.028, 0.021 and 0.024, respectively. For more accurate analysis, Ringbom optimum concentration ranges were 2.5-23.0, 3.0-28.5 and 3.0-33.0 μg/ml for I, II and III, respectively. The precision of the procedure was checked by calculating the relative standard deviation of ten replicate determinations on a sample containing 20 μg/ml for each drug and was found to be 1.67, 1.39 and 1.85% for I, II and III, respectively. Many common excepience and common drugs present in their dosage forms do not interfere, and the tolerable levels were evaluated. Results of analysis of pure drugs and their dosage forms by the proposed method are in good agreement with those of the British Pharmacopoeia 1993 procedure.     

Autooxidation of Δ17(20)-20-hydroxy derivatives of steroids. Synthesis of 3β-acetoxy-17α-hydroperoxy-16α-methylpregn-5-en-20-one and its reduction to 17α-hydroxy derivative

An efficient procedure was proposed for the synthesis of 3β-acetoxy-17α-hydroperoxy-16α-methylpregn-5-en-20-one. Optimal conditions were found for the combined process including 1,4-addition of methylmagnesium bromide at the Δ¹⁶-20-oxo fragment of dehydropregnenolone acetate and autooxidation of resulting bromomagnesium 3β-acetoxy-16α-methylpregna-5,17(20)-dien-20-olate. The subsequent reduction of the 17α-hydroperoxy group and hydrolysis of the 3β-acetoxy group afforded 17α-hydroxy-16α-methyl-substituted dehydropregnenolone acetate and its 3-hydroxy analog in high yield.     

Characteristic neutral loss of CH3CHO from Thr‐containing sodium‐associated peptides

A characteristic neutral loss of 44 Da is observed in the MS/MS spectra of Thr‐containing sodiated peptides. A combination of tandem mass spectrometry and quantum chemical calculations calculated at the B3LYP/6‐311G (d, p) level of ab initio theory is used to elucidate this fragmentation pathway. The high resolution mass spectrometry data indicate this neutral loss is acetaldehyde lost from the side chain of Thr rather than CO₂. The intensity of this neutral loss can be enhanced when Thr residue is far from the C‐terminus and when the C‐terminus is esterified as well. The mechanism of the acetaldehyde loss is proposed to adopt a McLafferty‐type rearrangement reaction, which involves a proton transfer from the hydroxyl of Thr side chain to its C‐terminal neighboring carbonyl oxygen inducing the cleavage of the C_a–C_β bond. This mechanism is further supported by examining the fragmentation of a [GT(tBu)G + Na]⁺ peptide derivative and by comparing the product ion spectra of [M + Na‐44]⁺ of [GTGA + Na]⁺ with [M + Na]⁺ of [GGGA + Na]⁺. A similar neutral loss of HCHO can also be detected in Ser‐containing peptides. Our computational results reveal that the most stable [GTG + Na]⁺ ion is present as a tridentate charge‐solvated structure and the dissociation leading to the 44 loss is dynamically and energetically favorable. Copyright © 2015 John Wiley & Sons, Ltd.     

Metabolic Transformation of (3R, 4R)-Δ1(7)-Tetrahydrocannabinol by a Rat Liver Microsomal Preparation

The metabolism of the non-psychotropic cannabinoid (3R, 4R)Δ¹⁽⁷⁾-tetrahydrocannabinol ( 1 ) (=Δ ¹⁽⁷⁾-THC) was investigated in a rat liver microsomal preparation. The metabolites obtained from the incubation mixture were separated, purified and identified by ¹H-NMR. spectroscopy and combined gas-liquid chromatography/mass spectrometry. Metabolites 3–10 are derived from Δ¹⁽⁷⁾-THC ( 1 ) by mono-hydroxylation in the isoprenoid moiety or the side chain of the molecule. Metabolites 11–16 are hydroxylated in the isoprenoid ring and the side chain simultaneously. The third group, metabolites 18–22 , is derived from the 1,7-epoxide 17 by hydrolysis of the oxirane ring, three of these metabolites bearing additional hydroxyl-groups in the isoprenoid part or the side chain. The mass spectra of the metabolites are discussed in detail and a new rule for the fragmentations of tetrahydrocannabinols is presented.     

Synthesis of 3β-Hydroxy[21-14C]-5β-pregn-8(14)-en-20-one from Chenodeoxycholic Acid

3β-Hydroxy[21-¹⁴C]5β-pregn-8(14)-en-20-one ( 17 ) was prepared from chenodeoxycholic acid ( 1a ). The synthetic sequence involved: (i) degradation of the bile-acid side chain to an etianic acid; (ii) formation of the 8(14)-double bond; (iii) inversion of the configuration at C(3); (iv) construction of the acetyl side chain at C(17) with the required isotopic label at C(21). Structures of all described products were confirmed by chemical and spectroscopic (IR, ¹H-NMR, ¹³C-NMR, MS) methods.     

The rearrangement of a steroid C20, C22-epoxide catalyzed by BF3 · Et2O

The rearrangement of the C₂₀, C₂₂-epoxide ( 3a ) catalyzed by BF₃ · Et₂O was investigated. The structures of 10 products obtained in this reaction ( 1a, 1b, 10, 11, 12 or 13, 14, 15, 16, 17 and 18 ) were determined and the mechanism discussed.     

A variation of Mattox rearrangement mechanism under alkaline condition

A variation of the Mattox rearrangement, a key degradation pathway under acidic conditions for corticosteroids possessing the 1,3-dihydroxyacetone side chain, has been found to occur for the 17,21-diesters of these corticosteroids but under the alkaline condition. The mechanism of this variation of the original Mattox rearrangement is proposed.     

Selective Hydrolysis of Terminal Glycosidic Bond in α-1-Acid Glycoprotein Promoted by Keggin and Wells–Dawson Type Heteropolyacids

The reactivity of a range of Keggin and Wells–Dawson type heteropolyacids (HPAs): H₃PW₁₂O₄₀ H₄SiW₁₂O₄₀, H₃PMo₁₂O₄₀, K₆P₂W₁₈O₆₂, and NaH₂W₁₂O₄, towards the heavily glycosylated α-1-acid glycoprotein (AGP) is reported. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) show that after incubation of the protein with HPAs at 80 °C and pH 2.8 complete hydrolysis of terminal glycosidic bond has been achieved, resulting in the removal of sialic acids with no observed destruction of the protein core or the residual glycan chains. The ¹H NMR spectroscopy confirmed that the released sialic acids preserve intact structure upon their excision from the protein, which makes the reported method suitable for the analysis of sialic acid modifications which play an important role in numerous biological processes. The presence of other sugars was not detected by ¹H NMR and HPAEC-PAD, suggesting that HPAs hydrolyze only the terminal glycosidic bond in the glycoprotein, resulting in the selective release of sialic acid from AGP. The kinetic results have shown that under equal temperature and pH conditions, the hydrolysis of the terminal glucosidic bond occurred faster in the presence of HPAs compared to conventional mineral acids. The observed rate constants were in the range 6,7×10⁻² −11,9×10⁻² min⁻¹ and the complete and selective excision of sialic acids could be achieved within 60 min of incubation. The Trp fluorescence and CD spectroscopy show that non-covalent interaction between HPA and protein takes place in solution which could lead to stabilization of the sialosyl cation that is formed during the glycosidic bond hydrolysis by anionic HPA cluster.     

Liquid-phase autooxidation of retinyl polyenes

The autooxidation of retinyl acetate and methyl retinoate was investigated in chlorobenzene at 45°C. The rates of thermal initiation in the retinyl acetate solutions were measured, and a value was determined of the rate constant for the reaction of oxygen with retinyl acetate (RH + O₂ → R· + HO₂·): k_io = (1.3 ± 0.2) × 10^?5 L/mol · s. The number of moles of oxygen absorbed per mole of polyene depends on the substrate concentration. A kinetic scheme for the methyl retinoate autooxidation was proposed which takes into account the isomerization of primary peroxy radicals, and the rate constants for different elementary reactions were estimated. The partial rate constant for “allylic” hydrogen abstraction from retinyl acetate was estimated to be ≥ 1.65 × 10³ L/mol · s. A probable propagation sequence was proposed for the autooxidation of retinyl acetate.     

Peptide models. XIV. Ab initio study on the role of side-chain backbone interaction stabilizing the building unit of right- and left-handed helices in peptides and proteins

Previous ab initio computations revealed that the conformational building unit of the right-handed helix (ϕ ≈ −54°, ψ ≈ −45°) is not an energy minimum on two-dimensional-type Ramachandran potential energy surfaces (E = E{ϕ, ψ}). Theoretical investigations were performed on several single-amino-acid diamides such as For-Gly-NH₂, For-L-Ala-NH₂, Ac-L-Ala-NHMe, and For-L-Val-NH₂ containing amino acid residues (e.g., Ala) which can often be found in helices as shown by X-ray data analysis of globular proteins. The current ab initio [self-consistent field (SCF)] results (based on four different basis sets [3-21G, 4-21G, 4-21G^*, and 6-31G^*]) presented point toward an intrinsic (i.e., non-environmental-assisted) stability of the right-handed helical subconformation of a simple amino acid diamide if the residue contains a polar side chain. Such is the case for a serine derivative when its (SINGLE BOND)CH₂OH side chain is favorably oriented. For the For-L-Ser-NH₂ model compound two slightly different right-handed helical backbone conformations were determined. Depending on the relative orientation of the side chain, the conformational monomer of the 3₁₀ helix (a sharper helical structure with an [i, i + 3]-type H-bond network) as well as the building block of the “standard” α-helix (the regular helical structure with an [i, i + 4]-type H-bond network) were determined computationally by geometry optimization. © 1997 John Wiley & Sons, Inc.     

On the synthesis of ecdysones

A synthesis of ecdysone is described by which the insect moulting hormone can be readily prepared. Oxidation of ergosterol gave the 6-keto-Δ⁷-function, and preparation of the Δ²-olefine followed by stereospecific hydroxylation led to the 2β,3β-glycol system. Ozonization furnished (20 S)-2β,3β-diacetoxy-20-formyl-5α-pregn-7-en-6-one into which the side chain was introduced by a Grignard reaction with 2-methyl-3-butyn-2-ol tetrahydropyran-2-yl ether and a subsequent reduction of the triple bond. Hydroxylation at C-14 and isomerization at C-5 gave ecdysone. By an interchange of the sequence of the reactions C-22 isoecdysone was obtained stereospecifically.     

Multi-morphological self-assembled structures of a rod-coil organometallic oligomer

A rod-coil amphiphilic organometallic oligomer based on o-ferrocenylcarbonyl benzoic acid (FcBA) as hydrophobic short rod and poly(ethylene glycol)₁₇ (PEG₁₇) as hydrophilic coil chain was synthesized via Friedel–Crafts acylation and esterification reaction and characterized by ¹H-NMR and FT-IR analyses. The rod-coil oligomer FcBA-PEG₁₇ with short-rod insoluble FcBA segment and long-coil PEG₁₇ segment could self-assemble into multi-morphological aggregates in water such as sphere, rod, strip and vesicle at different initial concentrations, which were characterized by transmission electron microscopy. Interestingly, two different kinds of vesicles were observed, where disfigured vesicles formed at low initial concentration and ideal vesicles at high initial concentration. And the disfigured vesicles can be transferred into ideal vesicles at lower initial concentration through the threading of rigid α-CDs onto the coil PEG₁₇ chains. A possible mechanism for the formation of multi-morphological self-assembled micelles was also proposed.     

Ta(CNDipp)6: An Isocyanide Analogue of Hexacarbonyltantalum(0)

Hexakis(2,6‐diisopropylphenylisocyanide)tantalum is the first isocyanide analogue of the highly unstable Ta(CO)₆ and represents the only well‐defined zerovalent tantalum complex to be prepared by conventional laboratory methods. Two prior examples of homoleptic Ta⁰ complexes are known, Ta(benzene)₂ and Ta(dmpe)₃, dmpe=1,2‐bis(dimethylphosphano)ethane, but these have only been accessed via ligand co‐condensation with tantalum vapor in a sophisticated metal‐atom reactor. Consistent with its 17‐electron nature, Ta(CNDipp)₆ undergoes facile one‐electron oxidation, reduction, or disproportionation reactions. In this sense, it qualitatively resembles V(CO)₆, the only paramagnetic homoleptic metal carbonyl isolable under ambient conditions.     

Synthesis of insulin fragments with disulfide bridges between intact chains A20-B19

The synthesis of six insulin fragments is described, in which various sequences of the two chains are linked by the disulfide bridge between A²⁰ and B¹⁹. The fragments in question are: A^20–21–B^19–21, A^20–21–B^18–21, A^20–21–B^17–21, A^19–21–B^19–21, A^16–21–B^18–21 and A^20–21–B^12–21. In order to build up the simpler fragments the disulfide bridge was established by oxidation with iodine of two S-trityl cysteine peptides in which the carboxyl and amino groups were protected by the t-butyl and t-butyloxycarbonyl residue. From the mixture obtained the unsymmetrical cystine peptide was separated in all cases from the two symmetrical ones by counter-current distribution. In the synthesis of the more complex fragments advantageous use was made of smaller unsymmetrical fragments prepared as above but having one amino group protected by the N-trityl residue. After selective elimination of this group it was possible to lengthen the peptide chain at this position. The free peptides were obtained by removal of the protecting groups with strong acids, in particular concentrated hydrochloric acid. While in this deprotecting step the disulfide bond was stable, conditions are discussed under which disproportionation was observed. None of the six synthetic insulin fragments showed activity in stimulating rat adipose tissue to convert ¹⁴C-labelled glucose to CO₂ in vitro.     

Interpretation of13C NMR spectra of 7-substituted 9,11-dideoxy-PGF 1 analogues and their synthons

A full interpretation of¹³C NMR spectra of the methyl esters of 7-hydroxy-and 7-oxo-9,11-dideoxy-PGF ₁ analogues and their synthons is reported.

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Interpretation von¹³C-NMR-Spektren von 7-substituierten 9,11-Dideoxy-PGF₁-Analogen und deren Synthonen

Zusammenfassung Es wird eine vollständige Zuordnung der¹³C-NMR-Resonanzen von den Methylestern der 7-Hydroxy- und 7-Oxo-9,11-dideoxy-PGF ₁-Analogen und ihrer Synthone beschrieben.