Development and establishment of a QuEChERS-based extraction method for determining tembotrione and its metabolite AE 1417268 in corn, corn oil and certain animal-origin foods by HPLC-MS/MS.

In this study, we established a rapid and sensitive HPLC-MS/MS method for quantitative determination of tembotrione and its metabolite AE 1417268 (M5) in corn, corn oil, beef, pork, chicken, and eggs. Both analytes eluted from a C18 column within 6 min. The electrospray ionisation-positive mode was used for tembotrione, whereas the negative mode was used for M5. Acetonitrile was the extractant for all samples, and the addition of 2 g NaCl to each homogenised sample facilitated the extraction of the analytes into acetonitrile. The extracted analytes were further purified by the addition of 50 mg of C18 sorbent (25 mg GCB was also added to corn samples) for subsequent HPLC-MS/MS analysis. Satisfactory calibration-curve linearities (R2 ≥ 0.997) were obtained for the two analytes at concentrations of 1-500 µg/L. Mean recoveries in different matrices ranged from 73.7% to 110.4%, whereas intra-day and inter-day relative standard deviations were <15% for all concentrations of spiked analytes. The limit of quantification was 2 µg/kg for each analyte in all matrices. In food samples obtained from markets, residual tembotrione and M5 were all below the limits of quantification. These results confirm that the method is suitable for the routine monitoring of residual tembotrione and M5 in a variety of food matrices.

Detection of adulterated drugs in traditional Chinese medicine and dietary supplements using hydrogen as a carrier gas.

Helium, a minor component of natural gas and radioactive minerals, is most commonly used as a carrier in gas chromatography-mass spectrometry (GC-MS). Its scarcity leads to limited availability and higher costs. In this experiment, hydrogen from a safe source of a hydrogen generator was tested as a substitutive carrier gas for the detection of adulterant in traditional Chinese medicine (TCM) and food supplements by GC-MS analysis. We found that the limits of detection (LODs) of using hydrogen were from 10 to 1000 µg/g. The levels of LODs tested among 170 drugs remain the same whether hydrogen or helium was used as a carrier gas with the exception of 7 drugs-benzbromarone, estradiol benzoate, bezafibrate, mefenamic acid, oxymetholone, piperidenafil and cetilistat. The real sample analysis results using hydrogen were as satisfactory as those using helium. In addition, the retention time was shortened after the chromatographic performance was optimized. In summary, it is worth considering hydrogen as a carrier gas due to its affordable costs, energy efficiency, carbon reduction and chromatographic advantages to detect adulterated drugs in TCM and dietary supplement using GC-MS.

Dispersive micro solid phase extraction (DMSPE) using polymer anion exchange (PAX) as the sorbent followed by UPLC-MS/MS for the rapid determination of four bisphenols in commercial edible oils.

The present work presents a novel and rapid analytical method for the simultaneous analysis of bisphenol A (BPA), bisphenol B (BPB), bisphenol F (BPF) and bisphenol S (BPS) in edible oil based on dispersive micro solid phase extraction (DMSPE) for the first time followed by isotope dilution-ultra high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The edible oil sample was dispersed by n-hexane and extracted with ammoniated methanol-water solution. Then the target analytes were dispersedly absorbed using the polymer anion exchange (PAX) as the sorbent and eluted by acidic methanol. After that, four bisphenols were separated on a C18 column by gradient elution with methanol and 0.05% ammonium hydroxide in water as mobile phase, detected by MS/MS under multiple reactions monitoring (MRM) mode and quantified by internal standard method. The PAX amounts, adsorption time, concentrations of formic acid in the elution solvent and volume of elution solvent for the DMSPE technique were optimized. The limit of detection and quantitation (LOD and LOQ), matrix effect, recovery and precision of the developed method were investigated. Results indicated that BPS and the rest three bisphenols displayed excellent linearity in the concentration ranges of 0.1-50µg/L and 0.5-250µg/L, respectively, with correlation coefficients (R2) all larger than 0.998. Achieved MLODs (S/N=3) varied between 0.1-0.4µg/kg for all bisphenols. The mean recoveries at three spiked levels in edible oil were in the range of 87.3-108%. Intra-day precision (n=6) and inter-day precision (n=5) were <9% and <11%, respectively. This method is of rapid-and-simple pretreatment, accurate and sensitive, and suitable for the simultaneous determination of bisphenols in edible oil.

A liquid chromatography-atmospheric pressure photoionization tandem mass spectrometric method for the determination of organosulfur compounds in petroleum asphalt cements.

We present a sensitive liquid chromatography-atmospheric pressure photoionization tandem mass spectrometric (UHPLC-APPI-MS/MS) method for the determination of selected organosulfur compounds in Brazilian asphalt cements. It was possible to detect 14 organosulfur compounds of different classes where sulfoxides and sulfones presented higher sensibility in ionization than thiophenes and aromatic sulfides. A dopant-assisted APPI method was also tested, however, when chromatographic flow rate was optimized a decrease in signal was observed for all compounds. PAHs were tested and ruled out as possible interfering compounds and the matrix effect of asphalt cements was within an acceptable range for the quantification of organosulfur compounds. The proposed method was found to have satisfactory linearity and accuracy with recoveries between 83.85 and 110.28% for thianaphthene and 3-methylbenzothiophene, respectively. Therefore, the method allowed the characterization of organosulfur compounds in Brazilian asphalt cements and demonstrated changes in the amount quantified in asphaltenic and maltenic fractions after the RTFOT+SUNTEST aging process.

Characterization of Sildenafil analogs by MS/MS and NMR: a guidance for detection and structure elucidation of phosphodiesterase-5 inhibitors.

Novel synthetic analogs of Sildenafil are constantly detected as adulterants in counterfeit drugs and dietary supplements. Their intake constitutes a serious health hazard as side effects are unknown. In this paper an investigation is carried out on NMR and MS/MS spectra of Sildenafil, Thiosildenafil, Acetildenafil and thirteen of their analogs: a list of key signals is reported and discussed with the intent to provide a tool that can help in detecting adulteration and in elucidating the structure of novel analogs. In this view extensive spectral data were reported, discussed and summarized in tables. A discussion on mass fragmentation and NMR chemical shifts is also provided to rationalize assignation. Moreover, a comprehensive information on the route of synthesis is provided for the benefit of those medicines control laboratories that need to synthesize analogs reference standards in-house.

[Analysis and identification of illegal constituents in health food products implicitly advertizing tonic or slimming effect in the National Institute of Health Sciences in Japan].

With the prefectural governments' aid of the purchase, the Division of Pharmacognosy, Phytochemistry and Narcotics, National Institute of Health Sciences (NIHS) successively has surveyed illegal constituents in health food products implicitly advertizing tonic or slimming effect since the fiscal year of 2002 (slimming type) or 2003 (tonic type). The average numbers of the analyzed products per year are about 100 (slimming type) and 150 (tonic type), respectively. We also continuously distribute standards of authentic samples of several illegal components such as N-nitrosofenfluramine (NFF) and sildenafil (SIL) to prefectural institutes and the average gross number per year is about 140. In the case of slimming type, the fact that the products containing NFF were widely sold in Japanese markets in 2002 is well known. In addition, phenolphthalein, fenfluramine, sibtramine, desdimethylsibtramine, orlistat, mazindol, Rhubarb, Senna Leaf, etc. have been found as illegal constituents. In the tonic type products, we have identified more than 20 synthetic compounds relating to the erectile dysfunction (ED) treatment drugs, SIL, vardenafil and tadalafil (TDF). Since 2005, their synthetic intermediates and the patented but non-approved PDE5 inhibitors also have been found. It should be noted that TDF was found in the shells of capsule in 2009 and that mutaprodenafil was found as pro-drug type illegal component in 2010. In this report identification method of these illegal constituents is briefly described and then analytical trend in this decade is reviewed.

Application of a high resolution benchtop quadrupole-Orbitrap mass spectrometry for the rapid screening, confirmation and quantification of illegal adulterated phosphodiesterase-5 inhibitors in herbal medicines and dietary supplements.

In this study, the applicability of high resolution quadrupole-Orbitrap (Q-Orbitrap) mass spectrometry for the simultaneous qualitative and quantitative analysis of illegal adulterated phosphodiesterase-5 inhibitors (PDE-5 inhibitors) in herbal medicines and dietary supplements was investigated. The mass spectrometer was operated in full MS scan/dd-MS(2) (data-dependent MS(2)) mode. The use of 70,000 FWHM mass resolution and narrow mass windows (5 ppm) could effectively improve the selectivity of the method, increasing the signal-to-noise ratio for the analytes. The response showed good linear relationship with the analytes' concentrations over wide ranges (e.g., 0.05-10 µg/g for sildenafil) with all the coefficient of determinations (r(2)) >0.9996. The detection limits (LODs) were in the range of 1.0-5.0 ng/g for different analytes. The recoveries ranged from 85.4% to 96.7%. The intra- and inter-day accuracies were in the range of -6.6 to 10.1%, while the intra- and inter-day precision ranged from 0.0039% to 13.2%. Among 68 batches of herbal medicines and 20 batches of dietary supplements (including 83 capsules, 3 pellets and 2 liquid) samples, sildenafil was detected in 8 dietary supplements, while noracetildenafil was detected in only one dietary supplement. The novel Q-Orbitrap mass spectrometry has been proved to be a very promising and powerful tool for routine screening of illegal adulterate in herbal medicines and dietary supplements, ensuring food safety and public health.

Screening of synthetic PDE-5 inhibitors and their analogues as adulterants: analytical techniques and challenges.

The popularity of phosphodiesterase type 5 (PDE-5) enzyme inhibitors for the treatment of erectile dysfunction has led to the increase in prevalence of illicit sexual performance enhancement products. PDE-5 inhibitors, namely sildenafil, tadalafil and vardenafil, and their unapproved designer analogues are being increasingly used as adulterants in the herbal products and health supplements marketed for sexual performance enhancement. To date, more than 50 unapproved analogues of prescription PDE-5 inhibitors were found as adulterants in the literature. To avoid detection of such adulteration by standard screening protocols, the perpetrators of such illegal products are investing time and resources to synthesize exotic analogues and devise novel means for adulteration. A comprehensive review of conventional and advance analytical techniques to detect and characterize the adulterants is presented. The rapid identification and structural elucidation of unknown analogues as adulterants is greatly enhanced by the wide myriad of analytical techniques employed, including high performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), liquid chromatography mass-spectrometry (LC-MS), nuclear magnetic resonance (NMR) spectroscopy, vibrational spectroscopy, liquid chromatography-Fourier transform ion cyclotron resonance-mass spectrometry (LC-FT-ICR-MS), liquid chromatograph-hybrid triple quadrupole linear ion trap mass spectrometer with information dependent acquisition, ultra high performance liquid chromatography-time of flight-mass spectrometry (UHPLC-TOF-MS), ion mobility spectroscopy (IMS) and immunoassay methods. The many challenges in detecting and characterizing such adulterants, and the need for concerted effort to curb adulteration in order to safe guard public safety and interest are discussed.

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.

Adulteration of purported herbal and natural sexual performance enhancement dietary supplements with synthetic phosphodiesterase type 5 inhibitors.

INTRODUCTION: Many products labeled "herbal" or "all natural" (herbal/natural) that claim to enhance sexual performance and imply use for the treatment of erectile dysfunction (ED) are marketed as over-the-counter (OTC) dietary supplements. However, adulteration with undeclared phosphodiesterase type 5 (PDE5) inhibitors appears widespread. AIM: To assess the availability, cost, origin, categorical content, and adulteration with PDE5 inhibitors of purported herbal/natural OTC dietary supplements claiming to naturally enhance sexual performance. METHODS: Pfizer Global Security coordinated sample collection (all from convenience stores and filling stations in two U.S. metropolitan areas except for seven from U.S. Customs seizures) and liquid chromatography/mass spectrometry examination. MAIN OUTCOME MEASURE: Adulteration with synthetic PDE5 inhibitors. RESULTS: Ninety-one samples labeled as 58 distinct products and priced from $2.99 to $17.99 were evaluated. Origin/manufacture was claimed as United States (n = 62), apparently Asian (n = 15), and not clearly identified (n = 14). Although no sample claimed to include synthetic substances, 74 (81%) contained PDE5-inhibitor pharmaceutical ingredients, including tadalafil and/or sildenafil (n = 40, of which 18 contained >110% of the highest approved drug product strength) or PDE5-inhibitor analogs (n = 34). Pronounced heterogeneity of contents between samples within individual products indicated minimal quality control during manufacture. Labeling was inadequate (e.g., lacking lot number and/or expiry date) for 17 products (23 samples) and inconsistent between samples within a given product (e.g., in manufacturer, lot number, and/or expiry date) for seven of 17 products having multiple samples. Only 14 samples warned against concomitant nitrate use. CONCLUSIONS: Ethical pharmaceutical companies are concerned for an unsuspecting public when their products are counterfeited, mislabeled, and illegally offered for sale in an unsafe manner. Because of the dangers of adulteration with synthetic PDE5 inhibitors, absent safety warnings, and lack of quality or consistent manufacture, men with ED unknowingly risk their health by using OTC herbal/natural products that claim to enhance sexual performance.

Rapid-screening detection of acetildenafils, sildenafils and avanafil by ion mobility spectrometry.

Ion mobility spectrometry was used as a rapid screening tool for the detection of acetildenafils, sildenafils and avanafil within adulterated herbal supplement matrices. Acetildenafils show a tendency for partial fragmentation during the desorption/ionization process affording two peaks in the ion mobility spectrum in addition to the intact compound. The fragmentation appears to occur α to the carbonyl group along the CN bond attaching the piperazine moiety, producing a common fragment (K0=1.0280 cm²V⁻¹s⁻¹) along with the respective piperazine fragment. The sildenafils and avanafil afford one molecular ion peak per compound.

Detection and structure elucidation of hydroxythiovardenafil as an adulterant in a herbal dietary supplement.

A new derivative of vardenafil was detected in an alleged herbal dietary supplement and identified as 2-(2-ethoxy-5-(4-(2-hydroxyethyl)piperazin-1-ylsulfonyl)phenyl)-5-methyl-7-propyl-imidazo[1,5-f][1,2,4]triazin-4(3H)-thione. Structure elucidation was carried out by LC-UV-MS/MS and NMR. Results obtained with high resolution MS and IR spectroscopy confirmed the proposed chemical structure. The compound was distinguished from hydroxyvardenafil, a second active substance identified in the same product, by the conversion of the oxo group to a thio group on the imidazo-triazin moiety. Hydroxythiovardenafil was therefore suggested as a proprietary name for the new molecule. This is the first paper to describe a thio-analog of vardenafil in a commercially available product.

Structure elucidation of a novel analog of sildenafil detected as an adulterant in a dietary supplement using LC-UV and LC/MS.

A sildenafil-related compound was detected in a dietary supplement marketed as an aphrodisiac. The compound was detected during analysis of the dietary supplement using LC-UV and LC/electrospray ionization-MS. The structure of the compound was established using high resolution MS, NMR spectrometry, and X-ray crystal structure analysis. The compound was identified as 5-(5-((3,5-dimethylpiperazin-1-yl)sulfonyl)-2-ethoxyphenyl)-l-methyl-7-((1-methyl-4-nitro-1H-imidazol-5-yl)thio)-3-propyl-1H-pyrazolo[4,3-d] pyrimidine. Based on this structure, the compound was named nitroprodenafil. The dietary supplement was found to contain 90 mg nitroprodenafil/capsule. This article describes the structural characterization of a new sildenafil-related compound. The compound was detected during analysis of a dietary supplement using LC-UV and LC/electrospray ionization (ESI)-MS. The structure was established using high resolution MS (HRMS), NMR spectrometry, and X-ray crystal structure analysis. The structures of methisosildenafil, thiomethisosildenafil, and this new analog, named nitroprodenafil (21), are shown in Figure 1. In the Demizu et al. report, the compound is named mutaprodenafil instead ofnitroprodenafil. Considering the naming right, the authors of this paper think the use of mutaprodenafil is appropriate as the compound name, although nitroprodenafil is used.

Screening for multiple phosphodiesterase type 5 inhibitor drugs in dietary supplement materials by flow injection mass spectrometry and their quantification by liquid chromatography tandem mass spectrometry.

A flow injection tandem mass spectrometry method (FI-MS/MS) has been developed to detect enzyme phosphodiesterase type 5 inhibitors, including tadalafil, sildenafil, and vardenafil. Multiple reaction monitoring (MRM) was used to detect the drugs and product ion ratios were used for identification. FI-MS/MS was used for semi-quantification and liquid chromatography tandem mass spectrometry (LC-MS/MS) was used for further confirmation and quantification. One of 13 samples has been found to be adulterated with prescription levels of tadalafil and also low level of sildenafil. The method can be used for screening large numbers of herbal products for adulteration since it takes less than 1 min without chromatographic separation on a column.

Analysis of herbal dietary supplements for sexual performance enhancement: first characterization of propoxyphenyl-thiohydroxyhomosildenafil and identification of sildenafil, thiosildenafil, phentolamine and tetrahydropalmatine as adulterants.

Nine herbal dietary supplements intended to be beverages for enhancing sexual performance were analyzed before their possible launch on the market. Four of them contained a sildenafil analog reported for the first time as an adulterant. After isolation and characterization using NMR, MS, IR and UV, this analog was named propoxyphenyl-thiohydroxyhomosildenafil as the ethoxy chain on the phenyl ring of the already known analog thiohydroxyhomosildenafil was replaced by a propoxy moiety. One formulation was tainted with thiosildenafil, another unapproved PDE-5 inhibitor. Sildenafil along with the natural alkaloid tetrahydropalmatine that has no documented effect for enhancing erectile dysfunction were identified in two formulations. Another formulation was adulterated with phentolamine, a drug that is not approved for boosting male sexual performance when taken orally. The last formulation containing osthole, a bioactive natural coumarine improving sexual dysfunction, is most probably truly natural.

Combining 2D and 3D in silico methods for rapid selection of potential PDE5 inhibitors from multimillion compounds' repositories: biological evaluation.

Rapid in silico selection of target focused libraries from commercial repositories is an attractive and cost-effective approach when starting new drug discovery projects. If structures of active compounds are available rapid 2D similarity search can be performed on multimillion compounds' databases. This in silico approach can be combined with physico-chemical parameter filtering based on the property space of the active compounds and 3D virtual screening if the structure of the target protein is available. A multi-step virtual screening procedure was developed and applied to select potential phosphodiesterase 5 (PDE5) inhibitors in real time. The combined 2D/3D in silico method resulted in the identification of 14 novel PDE5 inhibitors with <1 µMIC(50) values and the hit rate in the second in silico selection and in vitro screening round exceeded the 20%.

Identification of novel sildenafil-analogues in an adulterated herbal food supplement.

A new herbal product advertised as potency pill was sent for analysis by the local authority. The product was tested for the presence of potential derivatives of PDE-5 inhibitors, such as sildenafil, vardenafil, and tadalafil. Sildenafil analogues were identified, in which the piperazine ring and the sulfonyl group were replaced by a piperazinone and a hydroxyethyl structure, respectively. The chemical structures were established by LC-MS in ESI negative mode, UV and NMR spectroscopy (including DEPT, HSQC, HMBC, H,H-COSY, H,H-TOCSY and H,H-NOESY experiments). This is the first report of piperazinonafil and isopiperazinonafil as adulterant in an herbal food supplement.

Identification of thiosildenafil in a health supplement.

The presence of a sildenafil derivative, the thiosildenafil, in an herbal product has been evidenced first by HPTLC and later determined by isolation and analysis of spectroscopic data. The analyzed product is nowadays marketed as dietary supplement containing herbal extracts and claimed for male and female sexual improvement. This report is noteworthy since it is clear that adulterated materials can cause serious health problems if they are consumed as herbal "natural" products, generally considered deprived of toxicity by the consumers. The use of a simple and reliable method, based on HPTLC, to determine synthetic adulterations is reported in this paper.

Synthesis of core-shell magnetic molecularly imprinted polymers and detection of sildenafil and vardenafil in herbal dietary supplements.

An analytical procedure for selective extraction of sildenafil and vardenafil in herbal dietary supplements (HDSs) has been set up by using the magnetic molecularly imprinted polymers (MMIPs) as the extraction and clean-up materials, followed by high performance liquid chromatography-ultraviolet (HPLC-UV). The MMIPs were prepared by a surface molecular imprinting technique, using Fe(3)O(4) magnetite as a magnetically susceptible component, sildenafil as template molecule, 2-(trifluoromethyl) acrylic acid (TFMAA) as functional monomer, ethylene glycol dimethacrylate (EGDMA) as polymeric matrix components. The MMIPs were characterized by transmission electron microscope (TEM), Fourier transform infrared spectrometer (FT-IR), X-ray diffraction (XRD) and vibrating sample magnetometer (VSM), respectively. The heterogeneity of the MMIPs was modeled with the Freundlich isotherm equation. The resulting MMIPs had high recognition ability and fast binding kinetics for sildenafil. The MMIPs were used as dispersive solid-phase extraction (DSPE) materials to selectively extract sildenafil and vardenafil from HDSs, the contents of sildenafil and vardenafil were found to be 8.05 and 3.86 µg g(-1), respectively, and the average recoveries in spiked HDSs were 70.91-91.75% with a relative standard deviation (R.S.D.) below 7%. The MMIPs were successfully used to selectively enrich and determine sildenafil and vardenafil from HDSs.