Determination of (R)-xanthoanthrafil, a phosphodiesterase-5 inhibitor, in a dietary supplement promoted for sexual enhancement

We describe here the first case of the finding of xanthoanthrafil, a phosphodiesterase-5 inhibitor, in a dietary supplement. A methanol extract of the supplement product was first analyzed by TLC and HPLC. The results indicated that the extract contained an unknown compound. The molecular weight of the compound was 389 and the accurate mass showed its elemental composition to be C(19)H(23)N(3)O(6). Combined with this data, NMR analysis revealed the planar structure of the unknown compound to be N-(3,4-dimethoxybenzyl)-2-(1-hydroxypropan-2-ylamino)-5-nitrobenzamide. The R-configuration of this compound had been synthesized as a phosphodiesterase-5 inhibitor, formerly reported as FR226807 by Fujisawa Pharmaceutical Co., Ltd. The absolute configuration of the isolated compound was estimated to have R-configuration by its optical rotation. Considering its general properties, this compound is renamed as (R)-xanthoanthrafil with the agreement of Astellas Pharma Inc. which is the successor of Fujisawa Pharmaceutical Co., Ltd. Quantitative analysis revealed that the content of (R)-xanthoanthrafil in the product was about 31 mg/capsule.     

Simultaneous determination of synthetic phosphodiesterase-5 inhibitors found in a dietary supplement and pre-mixed bulk powders for dietary supplements using high-performance liquid chromatography with diode array detection and liquid chromatography-electrospray ionization tandem mass spectrometry

A high-performance liquid chromatography-diode array detection (HPLC-DAD) method and a liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method were developed to screen for the presence of synthetic phosphodiesterase-5 (PDE-5) inhibitors and their analogues, namely sildenafil, vardenafil, tadalafil, homosildenafil, acetildenafil and hydroxyhomosildenafil. The methods were applied to pre-market samples submitted to the Health Sciences Authority of Singapore (HSA) for testing. One sample was in the form of capsules while six other samples were pre-mixed bulk powder samples for dietary supplements to be repackaged or formulated into the final dosage forms (usually capsules). Identification of PDE-5 inhibitors and their analogues was achieved by comparing individual peak retention times, UV spectra and mass spectra with those of reference standards. The seven samples were found to contain at least one of the following compounds: sildenafil, vardenafil, hydroxyhomosildenafil, homosildenafil and acetildenafil. The five compounds were simultaneously determined by LC-ESI-MS/MS in multiple reactions monitoring (MRM) scan mode. The method has been validated for accuracy, precision, linearity and sensitivity.     

Simultaneous determination of a new phosphodiesterase-5 inhibitor DA-8159 and its active metabolite in human plasma by high performance liquid chromatography with tandem mass spectrometry

Summary An LC-MS-MS method for the simultaneous determination of DA-8159 and its active metabolite, DA-8164 in human plasma was developed. DA-8159, DA-8164 and the internal standard, sildenafil were extracted from human plasma with dichloromethane at basic pH. A reversephase HPLC separation was performed on Luna phenylhexyl column with the mixture of acetonitrile-ammonium formate (10 mM, pH 6.0) (60:40,v/v) as mobile phase. The detection of analytes was performed using an electrospray ionization tandem mass spectrometry in the multiple reaction monitoring mode. The method showed a satisfactory sensitivity (lower limits of quantification, 2.0 ng mL¹), precision, accuracy, recovery and selectivity. The successful determination of DA-8159 and DA-8164 in the plasma of a volunteer who ingested a single dose of 100 mg DA-8159 confirms that the present method can be used for plasma analysis for clinical trial.     

Identification of mutaprodenafil in a dietary supplement and its subsequent synthesis

We isolated a new illegal sildenafil analogue named mutaprodenafil from a dietary supplement for erectile dysfunction (ED) and proposed that it is an aildenafil derivative containing an imidazole moiety. We subsequently synthesized mutaprodenafil from a thioaildenafil and authenticated its structure.     

Determination of arsenic in food and dietary supplement standard reference materials by neutron activation analysis

Arsenic was measured in food and dietary supplement standard reference materials by neutron activation analysis for the purpose of assigning certified or reference As mass fractions and to assess material homogeneity. Instrumental neutron activation analysis was used to value assign As in candidate SRM 3532 Calcium Dietary Supplement and candidate SRM 3262 Hypericum perforatum (St. John’s Wort) Aerial Parts down to about 100 μg/kg. Values were also determined for two additional candidate St. John’s Wort SRMs with As mass fractions <100 μg/kg. The presence of significant amounts of ²⁴Na and ⁸²Br limited the reproducibility of the method below 100 μg/kg. For measurement of lower As mass fractions, a radiochemical neutron activation analysis method with extraction of As³⁺ into diethyl-dithiocarbamate in chloroform and detection limits down to 0.1 μg/kg. As was used to value-assign As mass fractions for SRM 3280 Multivitamin/Multielement Tablets and for candidate SRM 3233 Fortified Breakfast Cereal, and at <10 μg/kg in candidate SRM 1845a Whole Egg Powder.     

In vitro metabolism of a novel phosphodiesterase-5 inhibitor DA-8159 in rat liver preparations using liquid chromatography/electrospray mass spectrometry

The in vitro metabolism of a new erectogenic, DA-8159, has been studied by LC with UV detection and on-line LC-electrospray mass spectrometry using rat hepatic microsomal incubation and rat liver perfusion. Both rat liver microsomal incubation of DA-8159 in the presence of NADPH and single-pass liver perfusion of DA-8159 resulted in the formation of three metabolites (M1-3). M1 was tentatively identified as hydroxy-DA-8159. M2 and M3 were identified as N-demethyl-DA-8159 and 5-(2-propyloxy-5-aminosulphonylphenyl)-1-methyl-3-propyl-1,6-dihydro-7H-pyrazolo(4,3-d)pyrimidin-7-one (DA-8164), respectively, on the basis of LC-MS/MS analysis with authentic standards. Rat CYP2D1 was a major isozyme for the formation of hydroxy-DA-8159 and N-demethyl-DA-8159. CYP2C12 and CYP1A1 catalysed the oxidation of DA-8159 to DA-8164.     

Liquid chromatographic determination of S-adenosyl-L-methionine in dietary supplement tablets

A simple and reliable liquid chromatographic method was developed for the determination of S-adenosyl-L-methionine (SAM) in dietary supplement tablets. SAM in products was extracted with a phosphate buffer and separated from the mixture on a reversed-phase C8 column by ion-pair chromatography. A gradient mobile phase containing phosphate buffer, sodium octanesulfonate as the ion-pair reagent, and acetonitrile at a flow rate of 1.2 mL/min was used in the analysis. The UV detection wavelength was set at 257 nm. The calibration curve was linear over a range of 75-375 microg/mL for the SAM active ion with R2 = 0.9999. Replicate tests indicated good reproducibility of the method with a relative standard deviation of 0.9% (n = 8). The multiple extractions and recoveries from fortified products showed the high accuracy of the analysis. The use of the acidic buffer for SAM extraction and elution and the use of a fresh standard for each calibration to counteract the instability of the SAM compound significantly improved the accuracy of the method.     

One-hydrogen polarization in hydroformylation promoted by platinum-tin and iridium carbonyl complexes: a new type of parahydrogen-induced effect

The first use of parahydrogen-induced polarization (PHIP) in hydroformylation is described including a novel one-hydrogen polarization (oneH-PHIP) in the product RCHO proton. Observed in propanal formed in the reaction of trans-PtCl(COEt)(PPh3)2 + SnCl2 under parahydrogen, oneH-PHIP was examined using the model hydroformylation catalyst Ir(COEt)(CO)2(dppe) (dppe = bis(diphenylphosphino)ethane) that yields the characterized acyl dihydride IrH2(COEt)(CO)(dppe). It is found that oneH-PHIP occurs as a consequence of second-order effects in the acyl dihydride species coupled with stereospecific reductive elimination of aldehyde. Thus, even though hydrogen transfer to substrate in hydroformylation is nonpairwise, parahydrogen effects prove useful mechanistically.     

Synthesis of nucleoside 3',5'-cyclic boranophosphorothioate,a new type of cyclic nucleotide

The first examples of a borane-containing doubly P-modified chiral cyclic nucleoside monophosphate (cNMP), e.g., thymidine and 5-fluoro-2'-deoxyuridine 3',5'-cyclic boranophosphorothioates, have been synthesized; these cNMP analogues with increased lipophilicity could be potential anticancer prodrugs and useful probes for mechanistic studies.     

Study of distribution and absorption of a new type calcium supplement in rabbit by47Ca radioactive tracer technique

Ca-47 radioactive tracer was used to determine the phamocodynamics, distribution pattern, absorption and bioavailability of a new type calcium supplement in rabbit prepared by plasma technology. Our results indicate that calcium from the new nutrient is mainly deposited in bone 6 or 7 days after oral administration. The absorption rate of this new calcium preparation is estimated to be 30–40 %.     

Determination of total soy isoflavones in dietary supplements, supplement ingredients, and soy foods by high-performance liquid chromatography with ultraviolet detection: collaborative study

An interlaboratory study was conducted to evaluate a method for determining total soy isoflavones in dietary supplements, dietary supplement ingredients, and soy foods. Isoflavones were extracted using aqueous acetonitrile containing a small amount of dimethylsulfoxide (DMSO) and all 12 of the naturally occuring isoflavones in soy were determined by high-performance liquid chromatography (HPLC) with UV detection using apigenin as an internal standard. Fifteen samples (6 pairs of blind duplicates plus 3 additional samples) of soy isoflavone ingredients, soy isoflavone dietary supplements, soy flour, and soy protein products were successfully analyzed by 13 collaborating laboratories in 6 countries. For repeatability, the relative standard deviations (RSDr) ranged from 1.07 for samples containing over 400 mglg total isoflavones to 3.31 for samples containing 0.87 mg/g total isoflavones, and for reproducibility the RSDR values ranged from 2.29 for samples containing over 400 mg/g total isoflavones to 9.36 for samples containing 0.87 mg/g total isoflavones. HorRat values ranged from 1.00 to 1.62 for all samples containing at least 0.8 mg/g total isoflavones. One sample, containing very low total isoflavones (< 0.05 mg/g), gave RSDR values of 175 and a HorRat value of 17.6. This sample was deemed to be below the usable range of the method. The method provides accurate and precise results for analysis of soy isoflavones in dietary supplements and soy foods.     

Comparison of virtual high-throughput screening methods for the identification of phosphodiesterase-5 inhibitors

Reliable and effective virtual high-throughput screening (vHTS) methods are desperately needed to minimize the expenses involved in drug discovery projects. Here, we present an improvement to the negative image-based (NIB) screening: the shape, the electrostatics, and the solvation state of the target protein's ligand-binding site are included into the vHTS. Additionally, the initial vHTS results are postprocessed with molecular mechanics/generalized Born surface area (MMGBSA) calculations to estimate the favorability of ligand-protein interactions. The results show that docking produces very good early enrichment for phosphodiesterase-5 (PDE-5); however, in general, the NIB and the ligand-based screening performed better with or without the added electrostatics. Furthermore, the postprocessing of the NIB screening results using MMGBSA calculations improved the early enrichment for the PDE-5 considerably, thus, making hit discovery affordable.     

Validation of a high-performance thin-layer chromatography/densitometry method for the quantitative determination of glucosamine in a herbal dietary supplement

Two-dimensional gas chromatography coupled to triple quadrupole mass spectrometry (GC-GC-MS-MS) is presented as a robust, selective and sensitive method for the accurate quantification of enantiomeric fractions (EFs) of atropisomeric polychlorinated biphenyls (PCBs) in a range of environmental matrices. The method was subjected to a set of quality control and validation experiments. EF precision of the atropisomers 95, 149, 132, and 174 ranged from 0.7 to 1.4% in soil samples. Quantified amounts of these congeners in soil and compost samples (59相似文献   

Evaluation of new pregnane derivatives as 5alpha-reductase inhibitor

The objective of this study was to synthesize several new pregnane derivatives and evaluate them as antiandrogens. From the commercially available 16-dehydropregnenolone acetate (7), two new steroidal compounds were synthesized: 17alpha-hydroxy-17beta-methyl-16beta-phenyl-D-homoandrosta-1,4,6-triene-3,20-dione (18) and 17alpha-acetoxy-17beta-methyl-16beta-phenyl-D-homoandrosta-1,4,6-triene-3,20-dione (19). The 5alpha-reductase inhibitory effect of the new compounds 18 and 19 together with the previously synthesized intermediates 7, 8, 13, 16, and 17 was determined in three different models: gonadectomized hamster flank organs diameter size, incorporation of [1,2-(14)C]sodium acetate into lipids in flank organs and conversion of [3H]testosterone (T) to [3H]dihydrotestosterone (DHT) by Penicillium crustosum. The evaluation of these steroids was carried out with three different controls: one group was treated with vehicle, the second with T and the third group with T plus finasteride. The pharmacological results from this work demonstrated that T significantly increases the diameter of the pigmented spot on the flank organs (p<0.05) as well as the incorporation of labeled sodium acetate into lipids in gonadectomized hamster flank organs (from 0.125 to 0.255 nmol per gland). In this study we also observed that broth of Penicillium crustosum converted [3H]T to [3H]DHT in a manner comparable to that of the flank organs. All experiments indicated that finasteride as well as steroids 7, 8, 13, 16-19 reduced significantly the conversion of T to DHT in P. crustosum. These compounds also decrease the size of the pigmented spot in the flank organs as well as reducing the incorporation of radiolabeled sodium acetate into lipids; T and the control sample (treated with vehicle only) were used for comparison. Apparently the presence of the 4,6-diene-3,20-dione moiety and also the C-17 ester group produce a higher inhibitory effect on the parameters used. PPThe data from this study indicated also that the three models used for the pharmacological evaluation exhibited comparable results.     

Determination of bitter orange alkaloids in dietary supplement Standard Reference Materials by liquid chromatography with atmospheric-pressure ionization mass spectrometry

A liquid chromatographic atmospheric-pressure ionization electrospray mass spectrometry (LC–API–ES–MS) method has been developed for the determination of five bitter orange alkaloids (synephrine, octopamine, n-methyltyramine, tyramine, and hordenine) in bitter orange-containing dietary supplement standard reference materials (SRMs). The materials represent a variety of natural, extracted, and processed sample matrices. Two extraction techniques were evaluated: pressurized-fluid extraction (PFE) and sonication extraction. The influence of different solvents, extraction temperatures, and pH were investigated for a plant material and a processed sample. The LC method uses a new approach for the separation of highly polar alkaloids. A fluorinated, silica-based stationary phase separated the five alkaloids and the internal standard terbutaline in less than 20 min. This method enabled the determination of the dominant alkaloid synephrine and other minor alkaloids in a variety of dietary supplement SRMs.     

Discovery of kibdelomycin, a potent new class of bacterial type II topoisomerase inhibitor by chemical-genetic profiling in Staphylococcus aureus

Bacterial resistance to known therapeutics has led to an urgent need for new chemical classes of antibacterial agents. To address this we have applied?a Staphylococcus aureus fitness test strategy to natural products screening. Here we report the discovery of kibdelomycin, a novel class of antibiotics produced by a new member of the genus Kibdelosporangium. Kibdelomycin exhibits broad-spectrum, gram-positive antibacterial activity and is a potent inhibitor of DNA synthesis. We demonstrate through chemical genetic fitness test profiling and biochemical enzyme assays that kibdelomycin is a structurally new class of bacterial type II topoisomerase inhibitor preferentially inhibiting the ATPase activity of DNA gyrase and topoisomerase IV. Kibdelomycin is thus the first truly novel bacterial type II topoisomerase inhibitor with potent antibacterial activity discovered from natural product sources in more than six decades.     

Interspecies in vitro metabolism of the phosphodiesterase-4 (PDE4) inhibitor L-454,560

L-454,560 is a potent phosphodiesterase 4 (PDE4) inhibitor which was identified as a development candidate for the treatment of asthma and chronic obstructive pulmonary disease (COPD). As part of the discovery of this compound, interspecies in vitro metabolism data was generated using liver microsomes and hepatocytes in order to understand the metabolic fate of the compound. In microsomes, metabolism of the 3-methyl-1,2,4-oxadiazole ring was the predominant pathway observed, including ring cleavage. In rat hepatocytes, hydroxylation of the methyl group on the oxadiazole ring and double-bond isomerization were the most abundant metabolites observed. No major species differences were found in terms of microsomal metabolite profiles. The use of LC with UV and MS detection is highlighted, as well as information from tandem mass spectrometry and NMR.