Stability of 7,8‐Dihydropterins in Air‐Equilibrated Aqueous Solutions

6‐Substituted 7,8‐dihydropterins (=2‐amino‐7,8‐dihydropteridin‐4(1H)‐ones) are heterocyclic compounds that occur in a wide range of living systems and participate in relevant biological functions. In air‐equilibrated aqueous solutions, these compounds react with dissolved O₂ (autooxidation). The rates of these reactions as well as the products formed strongly depend on the chemical structure of the substituents. 7,8‐Dihydro‐6‐methylpterin and 7,8‐dihydro‐6,7‐dimethylpterin that bear electron‐donor groups as substituents are the most reactive derivatives and undergo oxidation of the pterin moiety to yield the corresponding oxidized derivatives (6‐methylpterin and 6,7‐dimethylpterin, resp.). The oxidations of 7,8‐dihydrobiopterin, 7,8‐dihydroneopterin, and 7,8‐dihydrofolic acid are slower, and they yield 7,8‐dihydroxanthopterin as the main product. 7,8‐Dihydroxanthopterin, 6‐formyl‐7,8‐dihydropterin, and sepiapterin are rather stable, and their consumption in air‐equilibrated solutions is negligible for several days. The pseudo‐first‐order rate constants of the reactions between these compounds and O₂ at 25° and 40° are reported. The biological implications of the results obtained are also discussed.     

Bioanalysis of acetylcarnitine in cerebrospinal fluid by HILIC–mass spectrometry

Acetyl‐l ‐carnitine (ALCAR) is a potential biomarker for the modulation of brain neurotransmitter activity, but is also present in cerebrospinal fluid (CSF). Recent studies have utilized hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC‐MS/MS) based assays to detect and quantify ALCAR within biofluids such as urine, plasma and serum, using various sample pretreatment procedures. In order to address the need to quantify ALCAR in CSF on a high‐throughput scale, a new and simple HILIC‐MS/MS assay has been successfully developed and validated. For rapid analysis, CSF sample pretreatment was performed via ‘dilute and shoot’ directly onto an advanced HILIC column prior to MS/MS detection. This newly developed HILIC‐MS/MS assay shows good recoveries of ALCAR without the need for chemical derivatization and multistep sample extraction procedures. The employment of this assay is suitable for the high‐throughput bioanalysis and quantification of ALCAR within the CSF of various animal models and human clinical studies. Copyright © 2015 John Wiley & Sons, Ltd.     

Simple and validated UHPLC method coupled to UV detection for determination of daptomycin in human plasma and urine

Daptomycin, a lipopeptide antibiotic with excellent activity against Gram‐positive bacteria, is excreted primarily by the kidneys. Development of effective chromatographic methodologies for the determination of daptomycin in human specimens is necessary for clinical use. This study developed a simple and validated ultra‐high‐performance liquid chromatography method coupled to ultraviolet detection for determination of daptomycin in human plasma and urine. After the pretreatments involving protein precipitation, the supernatants were separated using a 2.3 µm particle size octadecylsilyl column, and the run time was 1 min. The calibration curves were linear over the concentration ranges of 2–200 mg/L for plasma and 25–300 mg/L for urine. Intra‐ and inter‐assay precision and accuracy values of plasma were within 13.5 and 92–100% and within 10.7 and 100–107%, respectively. Those of urine were within 5.0 and 101–104% and within 3.7 and 100–101%, respectively. The validated method was applied to the determination of plasma and urine samples in patients receiving 4–6 mg/kg of intravenous daptomycin, resulting in sufficient sensitivity for evaluating the plasma exposure and urinary excretion. In conclusion, the present method with acceptable analytical performance can be helpful for evaluating the pharmacokinetic disposition of daptomycin in clinical settings. Copyright © 2013 John Wiley & Sons, Ltd.     

Polymer monolith microextraction online coupled to hydrophilic interaction chromatography/mass spectrometry for analysis of β2‐agonist in human urine

An analytical method based on online combination of polymer monolith microextraction (PMME) technique with hydrophilic interaction LC (HILIC)/MS is presented. The extraction was performed with a poly(methacrylic acid‐co‐ethylene glycol dimethacrylate) monolithic column while the subsequent separation was carried out on a Luna silica column by HILIC. After 1:1 v/v dilution with 20 mM phosphate solution at pH 7.0 and centrifugation, urine sample was directly used for extraction. After optimization, 85% ACN (containing 0.3% formic acid v/v) was used for rapid online elution, which was also the mobile phase in HILIC to avoid band broadening during separation or carry‐over that was usually observed in PMME‐RP LC system. Online automation of extraction and separation procedures was realized under the control of a program in this study. The developed method was applied to rapid and sensitive monitoring of three β₂‐agonist traces in human urine. The LODs (S/N = 3) of the method were found to be 0.05–0.09 ng/mL of β₂‐agonists in urine. The recoveries of three β₂‐agonists spiked in five different urine samples ranged from 79.8 to 119.8%, with RSDs less than 18.0%.     

Analytical method for urinary metabolites as biomarkers for monitoring exposure to phthalates by gas chromatography/mass spectrometry

Phthalates, widely used as plasticizers, have been detected in indoor air, but there have been few reports on methods of analyzing urinary metabolites as biomarkers to monitor exposure to di‐n ‐pentyl phthalate or di‐n ‐hexyl phthalate. Presented here is a cost‐effective and sensitive analytical method for the determination of urinary metabolites of phthalates containing these two compounds. Nine urinary phthalate metabolites were enzymatically hydrolyzed and extracted with toluene: monomethyl phthalate, monoethyl phthalate, monoisobutyl phthalate, mono‐n‐ butyl phthalate, mono‐n‐ pentyl phthalate, mono‐n‐ hexyl phthalate, monocyclohexyl phthalate, monobenzyl phthalate and mono(2‐ethyl‐5‐carboxypentyl) phthalate. After transformation to their tert‐ butyldimethylsilyl derivatives, they were analyzed by gas chromatography/mass spectrometry in the electron impact ionization mode. The calibration curves for the metabolites were linear at urinary concentrations of up to 30 μg/L, showing that they could be determined accurately and precisely (detection limits 0.1–0.4 μg/L, quantification limits 0.3–1.3 μg/L). The urine samples collected could be stored for up to 1 month at −20°C. The proposed analytical method was used to examine urine samples from seven healthy volunteers. This method should be useful for monitoring phthalate exposure in the general population.     

A New Synthesis of Pteridines Substituted with Branched and Linear Alkenyl Groups at C(6). The Nitroso‐Ene Reaction of 4‐(Alkenoylamino)‐5‐nitrosopyrimidines

Pteridines substituted with a 1,1‐, 1,2‐, or 1,1,3‐substituted alkenyl group (mostly (E)‐configured) at C(6) were synthesized in high yields by the intramolecular nitroso‐ene reaction of 4‐(alkenoylamino)‐2‐amino‐6‐benzyloxy‐5‐nitroso‐ and 4‐(alkenoylamino)‐2,6‐diamino‐5‐nitrosopyrimidines. Thus, the N‐alkenoyl nitrosopyrimidines 4 and 5 provided the pteridines 6 and 7 , respectively, characterized by a 1,2‐disubstituted (E)‐alkenyl substituent, the C(4)‐(E)‐geranoyl amide 13 led regio‐ and stereoselectively to the (E)‐1,1,2‐trisubstituted alkenyl‐pteridine 16 , and the C(4)‐(Z) isomer 14 led to 17 possessing a 1,1‐disubstituted alkenyl group. The trifluoromethylated butenoyl amide 15 possessing a less highly nucleophilic alkenoyl group reacted more slowly to give the trifluoromethylated vinylpteridine 18 . Also the 4‐(alkenoylamino)‐2,6‐diamino‐5‐nitrosopyrimidine 20 reacted more slowly than 4 and 5 , and provided the pteridines 23 ; introduction of additional N‐acyl groups as in 21 and 22 led to a considerably faster ene reaction. The X‐ray crystal structure analysis of the nitroso amide 15 shows eight symmetrically independent molecules in the unit cell. In the crystalline state, the N,N‐dimethylformamidine derivative 9 of 6 forms a centrosymmetric dimer with the 7,8‐lactam group connected by intermolecular hydrogen bonds.     

Simultaneous determination of three anticon‐vulsants using hydrophilic interaction LC‐MS

A specific and automated method was developed to quantify the anticonvulsants gabapentin, pregabalin and vigabatrin simultaneously in human serum. Samples were prepared with a protein precipitation. The hydrophilic interaction chromatography (HILIC) with a mobile phase gradient was used to divide off ions of the matrix and for separation of the analytes. Four different HILIC‐columns and two different column temperatures were tested. The Tosoh‐Amid column gave the best results: single small peaks. The anticonvulsants were detected in the multiple reaction monitoring mode (MRM) with ESI‐MS‐MS. Using a volume of 100 μL biological sample the lowest point of the standard curve, i.e. the lower LOQs were 312 ng/mL. The described HILIC‐MS‐MS method is suitable for therapeutic drug monitoring and for clinical and pharmcokinetical investigations of the anticonvulsives.     

Studien in der Flavin-Reihe. XIX. Mitteilung. Anomale Reduktion des Flavinkerns: Pteridine aus (Iso)alloxazinen

The catalytic hydrogenation of isoalloxazines in aqueous solutions proceeds non stoichiometrically. This is explained by a reaction side path, which is competitive with the normal reversible 1,5-dihydrogenation and leads to the irreversible reduction of the ‘o-xyloquinoid’ (rather than benzenoid) subnucleus in the (iso)alloxazine system. In trifluoroacetic acid, this irreversible side reaction is found to be complete within several hours, while 8 H-equivalents are absorbed per flavin molecule. The products are shown to be cyclohexano-[g]-pteridine-2,4-diones in the normal 5,6,7,8-tetrahydro state, which are oxidized to yield, subsequently, 7,8-dihydro- and ‘fully oxidized’ pteridines (= 6,7,8,9-tetrahydrofavins). The spectral characteristics, tautomeric structures and pK-values are discussed in context with recent data on 6,7,8-trialkylpteridine-2,4-diones [3].     

Pteridines,Part CXI,Pteridine‐Based Photoaffinity Probes for Nitric Oxide Synthase and Aromatic Amino Acid Hydroxylases

Various 6‐substituted pteridines and 5,6,7,8‐tetrahydropterins carrying photolabile functions at the side chain (see 7 , 20 – 22 , 34 – 36 , 38 , and 39 ) as well as at the 5‐position (see 27 – 29 ) were synthesized from pterin and from 6‐phenylpterin ( 1 ) and 6‐(hydroxymethyl)pterin ( 10 ). Attachment of the photoaffinity labels via ester bonds required a special protecting‐group strategy based upon acid‐labile (see 30 – 33 ) and β‐eliminating blocking groups (see 17 – 19 ). The 6‐(4‐azidophenyl)pterin ( 7 ) was obtained from 6‐phenylpterin ( 1 ) via intermediates 2 and 4 – 6 , due to the low solubility of simple pterins in general. The pteridine derivatives 21 , 22 , 25 , 26 , 28 , 29 , 32 , 33 , 35 , 36 , 38 , and 39 were screened as inhibitors of neuronal (type I) NO synthase (see Table) from porcine cerebellum, of which 22 , 35 , 36 , and 38 showed interesting inhibitory activity with similar potency and effectiveness.     

Enhanced MCR-ALS modeling of HPLC with fast scan fluorimetric detection second-order data for quantitation of metabolic disorder marker pteridines in urine

This work presents the development of a liquid chromatographic method based on modeling entire fast scan fluorimetric detection second-order data with the multivariate curve resolution alternating least squares algorithm, for the simultaneous determination of five marker pteridines in urine samples.The modeling strategy involves the building of a single MCR-ALS model composed of matrices augmented in the spectral mode, i.e. time profiles remain invariant while spectra may change from sample to sample. This approach allowed us to separate and determine the whole analytes at once.The developed approach enabled us to determine five of the most important metabolic disorder marker pteridines: biopterin, neopterin, isoxanthopterin, pterin and xanthopterin, three of them presenting emission spectra with the same emission wavelength maxima. In addition, some of these analytes present overlapped time profiles. As a consequence of using the entire data sets, a considerable reduction of the data processing experimental time can be achieved. Results are compared with a previous strategy in which data were split in five different regions, and information about the figures of merit of the new strategy compared with the previously reported strategy is reported.     

Rapid identification of gallotannins from Chinese galls by matrix‐assisted laser desorption/ionization time‐of‐flight quadrupole ion trap mass spectrometry

Chinese gall, a conventional traditional Chinese medicine, contains high levels of gallotannins. A rapid method for direct analysis of the gallotannins without using any troublesome sample pretreatments was developed using matrix‐assisted laser desorption/ionization time‐of‐flight quadrupole ion trap mass spectrometry (MALDI‐QIT‐TOF MS) to successfully identify the gallotannin components in the crude extract of Chinese galls within several minutes. The high quality of the MS and MS² spectra acquired clearly showed that hydrolysable tannins in Chinese galls were identified as a series of the gallotannins with degrees of polymerization (DP) of 4–11 galloyl units. The MS² data indicated that the identified gallotannins with DP of 4–7 galloyl units had clear fragmentation with loss of 1–5 galloyl units which were further deprived of 1–3 water moieties. This technique may be used for rapid evaluation and screening of hydrolysable tannins in medicinal plants. Copyright © 2009 John Wiley & Sons, Ltd.     

Hydrophilic interaction liquid chromatography/tandem mass spectrometry for the simultaneous determination of dasatinib,imatinib and nilotinib in mouse plasma

Hydrophilic interaction liquid chromatography (HILIC) interfaced with atmospheric pressure ionization (API) sources and a tandem mass spectrometer (MS/MS) was developed for the simultaneous determination of dasatinib, imatinib and nilotinib in mouse plasma samples. The retention profiles of all analytes on several silica stationary phases under HILIC conditions were explored. The influences of experimental factors such as the compositions of mobile phases on the chromatographic performance and the ionization efficiency of all analytes in positive ion mode were investigated. The applicability of the proposed HILIC/MS/MS approach following a protein precipitation procedure for the quantitative determination of dasatinib, imatinib and nilotinib at low nano‐mole levels was examined with respect to assay specificity and linearity. The analytical results obtained by various HILIC/MS/MS approaches were found to be in good agreement with those obtained by reversed‐phase liquid chromatography/tandem mass spectrometry (RPLC/MS/MS) methods in terms of assay sample throughputs, sensitivity and accuracy. Furthermore, the potential of matrix ionization suppression on the proposed HILIC/MS/MS systems was investigated using the post‐column infusion technique. Copyright © 2009 John Wiley & Sons, Ltd.     

Surfactant‐assisted dispersive liquid–liquid microextraction combined with field‐amplified sample stacking in capillary electrophoresis for the determination of mexiletine and lidocaine

A sensitive method for the determination of mexiletine and lidocaine using surfactant‐assisted dispersive liquid–liquid microextraction coupled with capillary electrophoresis was developed. Triton X‐100 and dichloromethane were used as the dispersive agent and extraction solvent, respectively. After the extraction, mexiletine and lidocaine were analyzed using capillary electrophoresis with ultraviolet detection. The detection sensitivity was further enhanced through the use of field‐amplified sample stacking. Under optimal extraction and stacking conditions, the calibration curves were linear over a concentration range of 0.05–1.00 μM for mexiletine and 0.03–1.00 μM for lidocaine. The limits of detection (signal‐to‐noise ratio of 3) were 0.01 and 0.01 μM for mexiletine and lidocaine, respectively. An approximately 1141‐ to 1250‐fold improvement in sensitivity was observed for the two analytes compared with the injection of a standard solution without the surfactant‐assisted dispersive liquid–liquid microextraction and field‐amplified sample stacking procedures. This developed method was successfully applied to the determination of mexiletine and lidocaine in human urine and serum samples. Both precision and accuracy for urine and serum samples were less than 8.7 and 6.7%, respectively. The recoveries of the two analytes from urine and serum samples were 54.7–64.9% and 16.1–56.5%, respectively.     

Separation of complex sugar mixtures on a hydrolytically stable bidentate urea‐type stationary phase for hydrophilic interaction near ultra high performance liquid chromatography

A stationary phase bearing both bridged bis‐ureido and free amino groups (USP‐HILIC‐NH₂–2.5SP) for high‐speed hydrophilic interaction liquid chromatography separations was prepared using a one‐pot two‐step procedure starting from 2.5 μm totally porous silica particles. Highly polar compounds, such as polyols, hydroxybenzoic acids, and sugars, were successfully analyzed in shorter times and with higher peak efficiency, when compared to results obtained with a bidentate urea‐type column packed with 5 μm particles. Increased sugarophilicity and better peak shape were attested for the USP‐HILIC‐NH₂–2.5SP column (100 × 3.2 mm id) when compared with two commercially available UHPLC columns, namely an acquity BEH amide packed with totally porous 1.7 μm microparticles and a HILIC Kinetex column packed with core–shell 2.6 μm particles. Finally, the new column was employed in the separation of complex mixture of sugars (mono‐, di‐, and oligosaccharides) and in the analysis of beer samples. The resulting chromatograms showed good selectivity and overall resolution, while the catalyzing effect of the free amino moieties resulted in excellent peak shapes and in the absence of split peaks due to sugar anomerization phenomena.     

SO2‐Extrusion of an 8‐thiabicylo[3.2.1]octa‐2,6‐diene 8,8‐dioxide and rearrangement of the resulting cycloheptatriene

The reaction of 3,4‐di‐tert‐butyl‐thio‐phene 1‐oxide ( 8 ) with tetrachlorocyclopropene provided 6,7‐di‐tert‐butyl‐2,3,4,4‐tetrachloro‐8‐thia‐bicylo[3.2.1]octa‐2,6‐diene 8‐oxide ( 10 ), which was oxidized to the corresponding 8,8‐dioxide 16 by m‐chloroperbenzoic acid. The thermolysis of 16 in refluxing chlorobenzene, xylene, or octane gave 5‐tert‐ butyl‐1,2‐dichloro‐3‐[(1,1‐dich‐loro‐2,2‐dimethyl)‐pro‐ pyl]‐benzene ( 18 ) with extrusion of SO₂ and 2‐tert‐butyl‐4,5,6‐trichloro‐9,9‐dimethylbicyclo[5.2.0]nona‐1,3,5‐triene ( 19 ) with extrusion of SO₂ and HCl in 73–78% combined yields. On the other hand, the thermolysis of 16 in the presence of triethylamine gave 19 as the sole product in 98% yield. A mechanism that involves the initial formation of 4,5‐di‐tert‐butyl‐1,2,7,7‐tetrachlorocycloheptatriene ( 17 ) is proposed to ex‐ plain the observed products. © 2005 Wiley Periodicals, Inc. Heteroatom Chem 16:132–222, 2005; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/hc.20079     

Alkane Chain‐extended Pterin Through a Pendent Carboxylic Acid Acts as Triple Functioning Fluorophore, 1O2 Sensitizer and Membrane Binder

In order to develop a new long alkane chain pterin that leaves the pterin core largely unperturbed, we synthesized and photochemically characterized decyl pterin‐6‐carboxyl ester (CapC) that preserves the pterin amide group. CapC contains a decyl‐chain at the carboxylic acid position and a condensed DMF molecule at the N2 position. Occupation of the long alkane chain on the pendent carboxylic acid group retains the acid–base equilibrium of the pterin headgroup due to its somewhat remote location. This new CapC compound has relatively high fluorescence emission and singlet oxygen quantum yields attributed to the lack of through‐bond interaction between the long alkane chain and the pterin headgroup. The calculated lipophilicity is higher for CapC compared to parent pterin and pterin‐6‐carboxylic acid (Cap) and comparable to previously reported O‐ and N‐decyl‐pterin derivatives. CapC's binding constant K_b (8000 M^?1 in L‐α‐phosphatidylcholine from egg yolk) and Φ_F:Φ_? ratio (0.26:0.40) point to a unique triple function compound, although the hydrolytic stability of CapC is modest due to its ester conjugation. CapC is capable of the general triple action not only as a membrane intercalator, but also fluorophore and ¹O₂ sensitizer, leading to a “self‐monitoring” membrane fluorescent probe and a membrane photodamaging agent.     

New access to pyrazole,oxa(thia)diazole and oxadiazine derivatives

1,4‐Disubstituted thiosemicarbazides 1b–f reacted with ethenetetracarbonitrile ( 5 ) in di‐ methylformamide with formation of 2‐substituted 5‐phenyl‐1,3,4‐thiadiazoles 2a–f and 2‐substituted 5‐phenyl‐1,3,4‐oxadiazoles 4a–f . Upon addition of 5 to 1c–e in chlorobenzene, 3‐amino‐2‐benzoyl‐4,5,5‐tri‐ cyano‐2,5‐dihydro‐1H‐pyrazole‐1‐[N‐(4‐tricyanovi‐nyl)phenyl]carbothioamide ( 12 ), 5‐benzylamino‐, and 5‐allylamino‐4‐benzoyl‐2,3‐dihydro‐[1,3,4]thiadiazol‐ 2,2‐dicarbonitrile ( 13a,b ) and 5‐amino‐1‐benzoylpyrazole‐3,4‐dicarbonitrile ( 14 ) as well as 2‐phenyl‐ 4H‐[1,3,4]‐oxadiazine‐5,6‐dicarbonitrile ( 15 ) were formed. Rationales for the role of the solvent and the conversions observed are presented. © 2005 Wiley Periodicals, Inc. Heteroatom Chem 16:12–19, 2005; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/hc.20071     

LC-MS/MS method for the quantification of myo- and chiro-inositol as the urinary biomarkers of insulin resistance in human urine

A simple LC‐MS/MS method has been developed and validated for the quantification of endogenous myo‐ and chiro‐inositol in human urine. myo‐ and chiro‐Inositol were completely resolved from other carbohydrates and there were no interference peaks in human urine. The correlation coefficient (n = 3) was greater than 0.9991 over the range 0.05–25.0 µg/mL with the weighted (1/C²) least square method. Precision (%RSD) and accuracy (%RE) were 0–10.0% and 0–6.0% for the intra‐day assay (n = 5) and 0–14.3% and 0–10.0% for the inter‐day assay (n = 5). myo‐ and chiro‐Inositol have been shown to be stable in human urine stored at room temperature and for three freeze–thaw cycles. Copyright © 2011 John Wiley & Sons, Ltd.     

Determination of marker pteridines in urine by HPLC with fluorimetric detection and second-order multivariate calibration using MCR-ALS

A liquid chromatographic method has been developed, in combination with the multivariate curve resolution-alternating least squares algorithm (MCR-ALS), for the simultaneous determination of marker pteridines in urine samples. A central composite design has been applied to optimize the factors influencing the separation (buffer concentration, buffer pH, flow rate, oven temperature, mobile-phase composition). A set of 15 calibration samples were randomly prepared, in a concentration range of 0.5–10.5 ng mL⁻¹ for neopterin, biopterin, and pterin; 4.0–8.0 ng mL⁻¹ for xanthopterin; and 0.5–4.5 ng mL⁻¹ for isoxanthopterin. The validation was carried out with fortified urine samples from healthy adults. The optimized conditions were a mobile-phase composition of 10 mM citric buffer at pH 5.44 and acetonitrile (94.5/5.5, v/v), a flow rate of 1.0 mL min⁻¹, and an oven temperature of 25 °C. The detection system consisted of a fast-scanning spectrofluorimeter, which allows obtaining of second-order data matrices containing the fluorescence intensity as a function of retention time and emission wavelength. In this work, MCR-ALS was used to cope with coeluting interferences, on account of the second-order advantage inherent to this algorithm which, in addition, is able to handle data sets deviating from trilinearity, like the high-performance liquid chromatography data analyzed in the present report. The developed approach enabled us to determine five pteridines, some of them with overlapped profiles, reducing the experimental time and reagent consumption. Ratio values for pteridines/creatinine in urine, for infected children with different pathologies, are reported in this work.     

Synthesis and biological study of 6‐polyhydroxyalkylpteridines

6‐Polyhydroxyalkylpteridines are synthesised by oxidation of the corresponding pyrano[3,2‐g]pteridines, the latter ones having been obtained by condensation between 5,6‐diaminopyrimidines 1a,b and phenylhydra‐zones 2a‐e . The relative configuration at the chiral centers of the pyrano[3,2‐g]pteridines has been determined by nmr study and X‐ray analysis. The anti‐AIDS activity of several of these compounds has been tested.