A Novel UHPLC-MS/MS Method for the Quantification of Seven Opioids in Different Human Tissues.

BACKGROUND: Opioids are considered the cornerstone of pain management: they show good efficacy as a first-line therapy for moderate to severe cancer pain. Since pharmacokinetic/pharmacodynamic information about the tissue-specific effect and toxicity of opioids is still scarce, their quantification in post-mortem autoptic specimens could give interesting insights. METHODS: We describe an ultra-high-performance liquid chromatography coupled with tandem mass spectrometry method for the simultaneous quantification of methadone, morphine, oxycodone, hydrocodone, oxymorphone, hydromorphone and fentanyl in several tissues: liver, brain, kidney, abdominal adipose tissue, lung and blood plasma. The presented method has been applied on 28 autoptic samples from different organs obtained from four deceased PLWH who used opioids for palliative care during terminal disease. RESULTS: Sample preparation was based on tissue weighing, disruption, sonication with drug extraction medium and a protein precipitation protocol. The extracts were then dried, reconstituted and injected onto the LX50 QSight 220 (Perkin Elmer, Milan, Italy) system. Separation was obtained by a 7 min gradient run at 40 °C with a Kinetex Biphenyl 2.6 µm, 2.1 × 100 mm. Concerning the analyzed samples, higher opioids concentrations were observed in tissues than in plasma. Particularly, O-MOR and O-COD showed higher concentrations in kidney and liver than other tissues (>15-20 times greater) and blood plasma (>100 times greater). CONCLUSIONS: Results in terms of linearity, accuracy, precision, recovery and matrix effect fitted the recommendations of FDA and EMA guidelines, and the sensitivity was high enough to allow successful application on human autoptic specimens from an ethically approved clinical study, confirming its eligibility for post-mortem pharmacological/toxicological studies.

Switchable resolution in soft x-ray tomography of single cells.

The diversity of living cells, in both size and internal complexity, calls for imaging methods with adaptable spatial resolution. Soft x-ray tomography (SXT) is a three-dimensional imaging technique ideally suited to visualizing and quantifying the internal organization of single cells of varying sizes in a near-native state. The achievable resolution of the soft x-ray microscope is largely determined by the objective lens, but switching between objectives is extremely time-consuming and typically undertaken only during microscope maintenance procedures. Since the resolution of the optic is inversely proportional to the depth of focus, an optic capable of imaging the thickest cells is routinely selected. This unnecessarily limits the achievable resolution in smaller cells and eliminates the ability to obtain high-resolution images of regions of interest in larger cells. Here, we describe developments to overcome this shortfall and allow selection of microscope optics best suited to the specimen characteristics and data requirements. We demonstrate that switchable objective capability advances the flexibility of SXT to enable imaging cells ranging in size from bacteria to yeast and mammalian cells without physically modifying the microscope, and we demonstrate the use of this technology to image the same specimen with both optics.

An ultrahigh-resolution soft x-ray microscope for quantitative analysis of chemically heterogeneous nanomaterials.

The analysis of chemical states and morphology in nanomaterials is central to many areas of science. We address this need with an ultrahigh-resolution scanning transmission soft x-ray microscope. Our instrument provides multiple analysis tools in a compact assembly and can achieve few-nanometer spatial resolution and high chemical sensitivity via x-ray ptychography and conventional scanning microscopy. A novel scanning mechanism, coupled to advanced x-ray detectors, a high-brightness x-ray source, and high-performance computing for analysis provide a revolutionary step forward in terms of imaging speed and resolution. We present x-ray microscopy with 8-nm full-period spatial resolution and use this capability in conjunction with operando sample environments and cryogenic imaging, which are now routinely available. Our multimodal approach will find wide use across many fields of science and facilitate correlative analysis of materials with other types of probes.

Identification of benzamidenafil, a new class of phosphodiesterase-5 inhibitor, as an adulterant in a dietary supplement.

A sample labeled to be a natural herbal supplement for the enhancement of sexual function, was sent to Health Sciences Authority (HSA) of Singapore for testing. An unknown compound was detected and isolated from the product. The structure of the unknown compound was identified using LC-UV, high-resolution MS, ESI-MS/MS, IR, and NMR. The compound was characterized as a phosphodiesterase-5 (PDE-5) inhibitor, benzamidenafil. This is the first report of benzamidenafil, representing a new class of PDE-5 inhibitors, as an adulterant of a dietary supplement.

Isolation and identification of thiohomosildenafil and thiosildenafil in health supplements.

Two unknown compounds are detected and isolated from health supplements for the enhancement of sexual function. The structures of the unknown compounds are elucidated using high-resolution MS, ESI-MS/MS, NMR, UV and IR. One compound is identified as an analogue of sildenafil in which the oxygen atom is substituted with a sulfur atom in the pyrazolopyrimidine moiety, and an ethyl group instead of a methyl group is attached to the piperazinyl nitrogen. Hence, this compound is named thiohomosildenafil. Another compound is also a sildenafil analogue in which the oxygen atom is substituted with a sulfur atom in the pyrazolopyrimidine moiety. This compound is named thiosildenafil. Both the two compounds are first detected in health supplements. The UV, IR and completely assigned NMR data of thiohomosildenafil and thiosildenafil are first reported.

Ultra fast miniaturized real-time PCR: 40 cycles in less than six minutes.

We have designed, fabricated and tested a real-time PCR chip capable of conducting one thermal cycle in 8.5 s. This corresponds to 40 cycles of PCR in 5 min and 40 s. The PCR system was made of silicon micromachined into the shape of a cantilever terminated with a disc. The thin film heater and a temperature sensor were placed on the disc perimeter. Due to the system's thermal constant of 0.27 s, we have achieved a heating rate of 175 degrees C s(-1) and a cooling rate of -125 degrees C s(-1). A PCR sample encapsulated with mineral oil was dispensed onto a glass cover slip placed on the silicon disc. The PCR cycle time was then determined by heat transfer through the glass, which took only 0.5 s. A real-time PCR sample with a volume of 100 nl was tested using a FAM probe. As the single PCR device occupied an area of only a few square millimeters, devices could be combined into a parallel system to increase throughput.

Liquid chromatography ion trap/time-of-flight mass spectrometric study on the fragmentation of an acetildenafil analogue.

Liquid chromatography ion-trap time-of-flight mass spectrometry was employed to elucidate the fragmentation pathways of an analogue of acetildenafil. Based on the accurate masses of the parent ion, product ions and neutral losses of acetildenafil analogue, its fragmentation pathways were proposed. The information is useful for the on-line structural identification of unknown analogues of acetildenafil found as adulterants in herbal products.

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.

Detection of sildenafil analogues in herbal products for erectile dysfunction.

Sildenafil, the active ingredient in Viagra (Pfizer), is a prescription medicine used for erectile dysfunction. Compounds with chemical structures similar to that of sildenafil were isolated and purified during the analysis of some herbal products marketed for treatment of erectile dysfunction. Structural elucidation using liquid chromatography-diode array detection, infrared spectroscopy, liquid chromatography-tandem mass spectrometry, and nuclear magnetic resonance spectroscopy confirmed that the compounds were homosildenafil, hydroxyhomosildenafil, and acetildenafil. The implications of adulteration by compounds structurally related to prescription drugs are discussed. Unlike established drugs, the efficacy and safety of such analogues are largely unknown. This poses a great challenge for safety and health administrators to detect these modified structures and to regulate them. Consumers who use such adulterated products are at risk of developing serious adverse reactions, potentially leading to death. Greater collaboration and exchange of information between various health authorities, health professionals, academics, researchers, and industry, as well as public education, are key steps in the efforts to stem the growing trend of adulteration of herbal products by analogues of prescription drugs.

Piezoelectric polymer multilayer on flexible substrate for energy harvesting.

A piezoelectric polymer multilayer structure formed on a flexible substrate is investigated for mechanical energy harvesting under bending mode. Analytical and numerical models are developed to clarify the effect of material parameters critical to the energy harvesting performance of the bending multilayer structure. It is shown that the maximum power is proportional to the square of the piezoelectric stress coefficient and the inverse of dielectric permittivity of the piezoelectric polymer. It is further found that a piezoelectric multilayer with thinner electrodes can generate more electric energy in bending mode. The effect of improved impedance matching in the multilayer polymer on energy output is remarkable. Comparisons between piezoelectric ceramic multilayers and polymer multilayers on flexible substrate are discussed. The fabrication of a P(VDF-TrFE) multilayer structure with a thin Al electrode layer is experimentally demonstrated by a scalable dip-coating process on a flexible aluminum substrate. The results indicate that it is feasible to produce a piezoelectric polymer multilayer structure on flexible substrate for harvesting mechanical energy applicable for many low-power electronics.

Miniaturized acceleration sensors with in-plane polarized piezoelectric thin films produced by micromachining.

Miniaturized acceleration sensors employing piezoelectric thin films were fabricated through batch micromachining with silicon and silicon-on-insulator (SOI) wafers. The acceleration sensors comprised multiple suspension beams supporting a central seismic mass. Ferroelectric (Pb,La)(Zr,Ti) O(3) (PLZT) thin films were coated and in-plane polarized on the surfaces of the suspension beams for realizing electromechanical conversion through the piezoelectric effect. Interdigital electrodes were formed on the PLZT films and connected in parallel. Finite element analyses were conducted for the stress and strain distributions, providing guidance to the structural design, including optimizing electrode positioning for collecting the electrical output constructively. Uniformity of the beam thickness and sample consistency were significantly improved by using SOI wafers instead of silicon wafers. The measurement results showed that all the sensor samples had fundamental resonances of symmetric out-of-plane vibration mode at frequencies in the range of 8 to 35 kHz, depending on the sample dimensions. These sensors exhibited stable electrical outputs in response to acceleration input, achieving a high signal-to-noise ratio without any external amplifier or signal conditioning.

Detection of dehydroepiandrosterone and androsterone in a traditional Chinese herbal product.

Dehydroepiandrosterone (DHEA) and androsterone (ADT) were detected in a traditional Chinese herbal product by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography tandem mass spectrometry (LC-MS/MS). DHEA and ADT were tentatively identified by comparing their electron ionization (EI) mass spectra with those in the GC-MS Wiley database. A multiple reaction monitoring (MRM) scan was performed in LC-MS/MS to confirm the presence of the DHEA and ADT in the herbal product extract. Both the [M + H]+ and the [M + NH4]+ of DHEA and ADT were selected as the precursor ions. DHEA was detected with ion transitions m/z 306.4 --> 271.2, 306.4 --> 253.3, 289.2 --> 270.9, 289.3 --> 253.1 while ADT was detected with ion transitions m/z 308.5 --> 273.6, 308.5 --> 255.3, 291.5 --> 273.5, 291.5 --> 255.2, which confirmed the presence of the two steroid hormones in the herbal product. Limits of detection (LODs) of 0.2 microg ml(-1) for DHEA and 0.3 microg ml(-1) for ADT were found in methanolic standard solutions when [M +NH4]+ of DHEA and ADT were selected as the precursor ions, which allowed the detection of DHEA and ADT at trace level without time-consuming derivatization.

The nature of the gecko lizard adhesive force.

The extraordinary climbing skills of gecko lizards have been under investigation for a long time. Here we report results of direct measurement of single spatula forces in air with varying relative humidities and in water, by the force-distance method using an atomic force microscope. We have found that the presence of water strongly affects the adhesion force and from analysis of our results, we have demonstrated that the dominant force involved is the capillary force.