Sildenafil and analogous phosphodiesterase type 5 (PDE-5) inhibitors in herbal food supplements sampled on the Dutch market.

Herbal food supplements, claiming to enhance sexual potency, may contain deliberately added active pharmacological ingredients (APIs) that can be used for the treatment of erectile dysfunction (ED). The aim of this study was to determine whether herbal food supplements on the Dutch market indeed contain APIs that inhibit phosphodiesterase type 5 (PDE-5) inhibitors, such as sildenafil and analogous PDE-5 inhibitors. Herbal food supplements intended to enhance sexual potency (n = 71), and two soft drinks, were sampled from 2003 up to and including 2012. In 23 herbal supplements, nine different PDE-5 inhibitors were identified; in a few cases (n = 3), more than one inhibitor was indentified. The presence of these APIs was however not stated on the label. The concentrations of PDE-5 inhibitors per dose unit were analysed. Furthermore, the potential pharmacologically active properties of the detected PDE-5 inhibitors were estimated by using data from the scientific and patent literature regarding (1) in vitro PDE-5 activity, (2) reported effective doses of registered drugs with PDE-5 inhibitor activity and (3) similarity to other structural analogues. It was concluded that 18 of the 23 herbal food supplements, when used as recommended, would have significant pharmacological effects due to added APIs. Adequate use of existing regulation and control measures seems necessary to protect consumers against the adverse effects of these products.

Monitoring by LC-MS/MS of 48 compounds of sildenafil, tadalafil, vardenafil and their analogues in illicit health food products in the Korean market advertised as enhancing male sexual performance.

More than 46 phosphodiesterase type 5 (PDE5) inhibitor analogues have been found to be present as illegal adulterants in various forms of health food products (powder, tablet, capsule, etc.), thereby placing the health of consumers at risk through product intake. In this study, 164 samples advertised to be effective at enhancing male sexual performance were collected over a 4-year period (2009-2012) from the Korean on-line or off-line market and screened. An LC-MS/MS method was employed to screen for the presence of 48 compounds including sildenafil, tadalafil, vardenafil and their analogues. Method validation established LOQs (0.30-10.00 ng ml(-1) or ng g(-1)) and recoveries (spiked in liquid sample, 84-112%; spiked in solid sample, 83-110%). Most of the illicit products screened were adulterated with 14 of the PDE5 derivatives under examination, including considerable amounts of sildenafil and tadalafil; of the 48 compounds, tadalafil was the most frequent adulterant (42.6%), followed by sildenafil (27.9%). Specifically, tadalafil concentration ranges (mg g(-1)) in the samples collected over the 4-year period were determined as follows: 2.91-52.20 (2009), 4.50-108.10 (2010), 0.37-101.40 (2011), and 0.08-138.69 mg g(-1) (2012). The concentration ranges (mg g(-1)) of sildenafil were also at high levels: 4.90-117.96 (2009), 1.30-369.93 (2010), 0.03-241.77 (2011), and 18.34-297.91 mg g(-1) (2012). The results of screening for PDE5 inhibitor pharmaceuticals as adulterants in illicit health food products are of great significance with respect to the protection of public health and consumer safety.

Internet-ordered viagra (sildenafil citrate) is rarely genuine.

INTRODUCTION: Counterfeit medication is a growing problem. This study assessed the requirement for prescription, cost, origin, and content of medications sold via the Internet and purporting to be the phosphodiesterase type 5 inhibitor Viagra (sildenafil citrate). METHODS: Pfizer monitored top search results for the query "buy Viagra" on the two leading Internet search engines in March 2011. Orders were placed from 22 unique Web sites claiming to sell Viagra manufactured by Pfizer. Tablets received were assessed for chemical composition. RESULTS: No Web site examined required a prescription for purchase or a health screening survey; 90% offered illegal "generic Viagra." Cost per tablet ranged from $3.28-$33.00. Shipment origins of purchases were Hong Kong (N = 11), the United States (N = 6), and the United Kingdom (N = 2) as well as Canada, China, and India (N = 1 each). Notably, the four Internet pharmacies claiming to be Canadian did not ship medication from a Canadian address. Of 22 sample tablets examined, 17 (77%) were counterfeit, 4 (18%) were authentic, and 1 (5%) was an illegal generic. Counterfeit tablets were analyzed for sildenafil citrate, the active pharmaceutical ingredient (API) of Viagra, and contents varied between 30% and 50% of the label claim. Counterfeits lacked product information leaflets, including appropriate safety warnings, and genuine Viagra formulations. CONCLUSION: Internet sites claiming to sell authentic Viagra shipped counterfeit medication 77% of the time; counterfeits usually came from non-U.S. addresses and had 30% to 50% of the labeled API claim. Caution is warranted when purchasing Viagra via the Internet.

Simultaneous quantification of methylated purines in DNA by isotope dilution LC-MS/MS coupled with automated solid-phase extraction.

Since methylation at the N-7 and O(6) positions of guanine and the N-3 position of adenine in DNA are the predominant reaction sites, N(7)-methylguanine (N(7)-MeG), O(6)-methylguanine (O(6)-MeG), and N(3)-methyladenine (N(3)-MeA) have been suggested as good biomarkers for assessing exposure to methylating agents. Here, we report the development of a sensitive and selective assay based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) to simultaneously measure N(7)-MeG, O(6)-MeG, and N(3)-MeA in DNA hydrolysates. With the use of isotope internal standards ((15)N(5)-N(7)-MeG, d(3)-O(6)-MeG, and d(3)-N(3)-MeA) and online solid-phase extraction, DNA hydrolysates can be directly analyzed within 12 min without prior sample purification. The limits of detection were 0.02, 0.002, and 0.01 ng/mL on-column (6.1, 0.6, and 3.4 fmol) for N(7)-MeG, O(6)-MeG, and N(3)-MeA, respectively. Inter- and intraday imprecision (CV) were 3.6-9.6% and 2.7-13.6%, respectively. Mean recoveries were 96-109%. This method was then applied to quantitate the amounts of methylated purines in calf thymus DNA treated with methyl methanesulfonate (MMS). The levels of N(7)-MeG, O(6)-MeG, and N(3)-MeA in calf thymus DNA increase with MMS concentration and incubation time. The ratio of relative yields of N(7)-MeG, O(6)-MeG, and N(3)-MeA in MMS-treated DNA was found to be 1.00:0.0032:0.119, respectively. This LC-MS/MS assay provides the sensitivity and high throughput required to evaluate the extent of methylated lesions in DNA induced by methylating agents.

Sequence signatures of nucleosome positioning in Caenorhabditis elegans.

Our recent investigation in the protist Trichomonas vaginalis suggested a DNA sequence periodicity with a unit length of 120.9 nt, which represents a sequence signature for nucleosome positioning. We now extended our observation in higher eukaryotes and identified a similar periodicity of 175 nt in length in Caenorhabditis elegans. In the process of defining the sequence compositional characteristics, we found that the 10.5-nt periodicity, the sequence signature of DNA double helix, may not be sufficient for cross-nucleosome positioning but provides essential guiding rails to facilitate positioning. We further dissected nucleosome-protected sequences and identified a strong positive purine (AG) gradient from the 5'-end to the 3'-end, and also learnt that the nucleosome-enriched regions are GC-rich as compared to the nucleosome-free sequences as purine content is positively correlated with GC content. Sequence characterization allowed us to develop a hidden Markov model (HMM) algorithm for decoding nucleosome positioning computationally, and based on a set of training data from the fifth chromosome of C. elegans, our algorithm predicted 60%-70% of the well-positioned nucleosomes, which is 15%-20% higher than random positioning. We concluded that nucleosomes are not randomly positioned on DNA sequences and yet bind to different genome regions with variable stability, well-positioned nucleosomes leave sequence signatures on DNA, and statistical positioning of nucleosomes across genome can be decoded computationally based on these sequence signatures.

[Determination of benzylaminopurine in agricultural products].

A simplified method for the determination of benzylaminopurine in agricultural products by LC/MS was investigated. Benzylaminopurine in agricultural products was extracted with acetone and the extract was concentrated to below 30 mL. Buffer solution of pH 9.0 and ethyl acetate were added to the residue, and the solution was shaken. The ethyl acetate layer was evaporated to dryness, and the residue was dissolved in acetone-n-hexane (1 : 1). The solution was applied to a SAX/PSA mini-column, which was then rinsed with acetone-n-hexane (1 : 1). Benzylaminopurine was eluted with acetone-n-hexane (1 : 1) containing 1% water. The eluates were evaporated to dryness, and the residue was dissolved in 10 mmol/L ammonium acetate-methanol (1 : 1). Benzylaminopurine was analyzed by LC/MS. The MS detection was performed in the selected ion monitoring (SIM) mode, with detection of the M+H(+) ion of benzylaminopurine (m/z 226) generated by electrospray ionization (ESI). Recoveries of benzylaminopurine from 15 agricultural products were in the range of 83.1-97.2%. The limits of detection (S/N=3) of benzylaminopurine in all samples except green tea and in green tea were 0.0003 and 0.0012 microg/g, respectively.

[Drug counterfeiting--the risk to the public health]. / Podrabianie leków--zagroienie dia zdrowia publicznego.

UNLABELLED: Pharmaceutical counterfeiting and purchasing medicines from illegal distribution channels have become more and more common problem in our country. Different medicines, especially erectile dysfunction drugs are involved. The aim of this study was the qualitative analysis of fake Levitra tablets and the estimation of the risk they bear to potential users. Tablets were secured by the police and delivered to Bayer office in 2006. RESULTS: Trace amount of sildenafil (the active ingredient of Viagra) and not vardenafil (the active ingredient of Levitra) was found in tablets described as "Levitra" (vardenafil). The presence of this substance was discovered by NIR--and Raman spectroscopy. The appearance of tablets and blisters corresponded to the original product. There were no paper boxes and patient information leaflets attached. As prescription medicines erectile dysfunction drugs should be purchased from a pharmacy only. They need to be used under strict medical control.

Verification of statistical-overlap theory in micellar electrokinetic chromatography.

The limited peak capacity of neutral compounds in micellar electrokinetic chromatography (MEKC) causes peak overlap in a simple 38-compound sample that is predicted by statistical-overlap theory (SOT). The low-concentration sample was prepared in-house from several compound classes to span the entire migration-time range and was resolved partially in a pH=7 phosphate buffer containing 50 mM sodium dodecyl sulfate. Peaks, singlets, doublets, and other multiplets were identified on the basis of known migration times and were counted at 13 voltages spanning 4 - 26 kV. These numbers agreed well with predictions of a simple SOT based on the assumption of an inhomogeneous Poisson distribution of migration times. Because the dispersion theory of MEKC is simple, the standard deviations of single-component peaks were modeled theoretically. As part of a new way to implement SOT, probability distributions of the numbers of peaks, singlets, and so on, were computed by Monte Carlo simulation. These distributions contain all theoretical information on peak multiplicity predictable by SOT and were used to evaluate the agreement between experiment and theory. The peak capacity of MEKC was calculated numerically and substituted into the simplest equations in SOT, affirming that peak overlap arises from limited peak capacity.

Uses of field desorption mass spectrometry in the pharmaceutical industry.

Field desorption (FD) is a gentle method of ionizing molecules from the solid state. The method is still in its infancy and remains more of an art than a science. Purines, pyrimidines, pteridines, and nucleosides represent some of the compound classes which are studied repeatedly in our laboratory. Often the primary information sought is the molecular weight. Many of these compounds are thermally unstable or nonvolatile. In these instances, FD mass spectrometry has proven to be quite helpful. Molecular weights are obtained in a short time, eliminating the need for more time-consuming derivatization or chemical degradation methods. FD has been applied to the study of drug metabolism and has shown great promise in determining polar metabolites, including direct analysis of glucuronide, sulfate, and amino acid conjugates. Some problems have been encountered in handling samples of biological origins. Salt impurities introduced by the separation procedure may interfere with the desorption process. Cluster ions and background noise make it difficult to assign molecular ions, and some compounds do not yield molecular ions with FD.