Gregory Doran

Screening of whey protein isolate hydrolysates for their dual functionality: Influence of heat pre-treatment and enzyme specificity

Heat pre-treated and non heat pre-treated whey protein isolate (WPI) were hydrolysed using α-chymotrypsin (chymotrypsin), pepsin and trypsin. The in vitro antioxidant activity, ACE-inhibition activity and surface hydrophobicities of the hydrolysates were measured in order to determine if peptides with dual functionalities were present. Dual functional peptides have both biological (e.g. antioxidant, ACE-inhibition, opioid activities) and technological (e.g. nanoemulsification abilities) functions in food systems. Heat pre-treatment marginally enhanced the hydrolysis of WPI by pepsin and trypsin but had no effect on WPI hydrolysis with chymotrypsin. With the exception of the hydrolysis by trypsin, heat pre-treatment did not affect the peptide profile of the hydrolysates as analysed using size exclusion chromatography, or the antioxidant activity (P>0.05). Heat pre-treatment significantly affected the ACE-inhibition activities and the surface hydrophobicities of the hydrolysates (P<0.05), which was a function of the specificity of the hydrolysing enzyme. Extended hydrolysis (up to 24 h) had no significant effect on the DH and the molecular weight profiles (P>0.05) but in some instances caused a reduction in the antioxidant activity of WPI hydrolysates. The chymotrypsin hydrolysate showed a broad MW size range, and was followed by pepsin and then trypsin. The bioactivities of the hydrolysates generally decreased in the order; chymotrypsin>trypsin>pepsin. This study showed that by manipulating protein conformation with pre-hydrolysis heat treatment, combined with careful enzyme selection, peptides with dual functionalities can be produced from WPI for use as functional ingredients in the manufacture of functional foods.

A rapid method for the simultaneous quantification of the major tocopherols, carotenoids, free and esterified sterols in canola (Brassica napus) oil using normal phase liquid chromatography

A normal phase high performance liquid chromatography (HPLC) method was developed to simultaneously quantify several prominent bioactive compounds in canola oil vis. α-tocopherol, γ-tocopherol, δ-tocopherol, β-carotene, lutein, β-sitosterol, campesterol and brassicasterol. The use of sequential diode array detection (DAD) and tandem mass spectrometry (MS/MS) allowed direct injection of oils, diluted in hexane without derivatisation or saponification, greatly reducing sample preparation time, and permitting the quantification of both free sterols and intact sterol esters. Further advantages over existing methods included increased analytical selectivity, and a chromatographic run time substantially less than other reported normal phase methods. The HPLC-DAD-MS/MS method was applied to freshly extracted canola oil samples as well as commercially available canola, palm fruit, sunflower and olive oils.

Sorption and degradation of fipronil in flooded anaerobic rice soils.

The fate of fipronil in flooded, reductive rice soils was modeled using a conceptual model. Rate constants for the various sorption and degradation processes were calculated from experimental studies involving intact soil cores, and the reductive degradation constant was used to calculate half-lives for fipronil on each soil. The data predicted that fipronil was subject to rapid, reductive degradation or immediate sorption to the soil and any sorbed fipronil desorbed was reductively degraded. The reductive metabolite, fipronil sulfide, accumulated over the 184 day duration of the experiment and sorbed rapidly to the soil, where it accumulated and did not appear to degrade. Neither fipronil nor fipronil sulfide was found beyond the top 1 cm of soil in Yanco soil, while a small amount of each chemical was found up to 4 cm deep in the Coleambally soil profile.

The acute toxicity of fipronil to two non-target invertebrates associated with mosquito breeding sites in Australia

Mosquito-borne arboviruses are a significant health issue in the irrigation areas of south-eastern Australia. Fipronil, a pyrazole insecticide with strong activity against larval Culex species, was tested for its acute effects on Simocephalus elizabethae (Daphniidae) and Polypedilum nubiferum (Chironomidae), two non-target invertebrates associated with Australian rice field mosquito habitats. Technical and formulated fipronil were assessed in the presence or absence of particulate artificial diets in 48 h static bioassays. LC(50) values for neonate S. elizabethae ranged from 11.13 to 19.12 μgl(-1) whilst those for final instar P. nubiferum ranged from 0.89 to 2.18 μgl(-1). Feeding during exposure significantly reduced the susceptibility of P. nubiferum to both technical and formulated fipronil. The effect of feeding was less consistent in Simocephalus bioassays, where much less food was present. We investigated whether adsorption to unconsumed food particles may have limited fipronil bioavailability by using solid-phase microextraction and gas chromatography to measure the available fipronil from fed and unfed systems 24h after establishment. Differences between the systems were not significant (P>0.05). The significantly higher LC(50) values in the fed Polypedilum bioassays do not appear to be a consequence of reduced fipronil bioavailability. Observed differences in toxicity probably reflect increased stresses associated with food deprivation in the unfed bioassays. Our results support published data on the toxicity of fipronil to aquatic invertebrates which suggest that the use of this material as a mosquito larvicide may cause disruption to aquatic ecosystems.

The sorption and degradation of the rice pesticides fipronil and thiobencarb on two Australian rice soils

The sorption and degradation of the rice pesticides fipronil and thiobencarb on 2 Australian rice-growing soils were investigated. Greater sorption of both pesticides occurred on the soil containing less organic carbon, possibly as a result of the type of organic carbon present, rather than the absolute amount. While sorption tended to appear greater in the 0–10 mm layer than the 10–20 mm layer, analysis showed the difference was not significant (P > 0.05). Under aerobic conditions, a lag period of 20 days in the degradation of thiobencarb occurred on the Yanco soil, but rapid degradation occurred on the Coleambally soil, and, while unlikely, may have been a consequence of preconditioning of the Coleambally soil microbial population. Degradation of thiobencarb under both non-flooded anaerobic and flooded anaerobic conditions differed significantly (P < 0.05) compared to aerobic conditions. Conversely, fipronil degraded rapidly over the first few days and then slowed, and was attributed to the co-metabolism of fipronil by soil microbes. While fipronil sulfide was produced under all oxic/anoxic conditions, its concentration was greatest under flooded anaerobic conditions, possibly as a result of greater exclusion of oxygen from the soil by the floodwater.

The presence of licit and illicit drugs in police stations and their implications for workplace drug testing

The presence of licit and illicit drug residues on surfaces was studied in 10 police stations and a central drug evidence store in New South Wales, Australia, with the results compared to similar surfaces in four public buildings (to establish a community baseline). The results of almost 850 workplace surface swabs were also compared to the outcome of drug analysis in urine and hair samples volunteered by police officers. Surfaces were swabbed with alcohol and the swabs were extracted and analysed by LC-MS/MS. Low level concentrations of the more commonly used drugs were detected at four public sites and one restricted access police office facility. Surface swabs taken in 10 city and country police stations yielded positive results for a broader suite of drugs than at background sites however 75-93% of the positive drug results detected in police stations were below 40ng, which is only slightly greater than the largest background result measured in the current study. This study indicates that contamination issues are more likely to be focussed in higher risk areas in police stations, such as counters and balances in charge areas, and surfaces within drug safes although front reception counters also returned surface contamination. All 64 urine samples collected in this study were negative, while only 2 of the 11 hair samples collected from donors resulted in trace concentrations for cocaine, but not its metabolite benzoylecgonine. Positive hair samples were only obtained from police donors in very high risk jobs, indicating that the exposure risk is low. Minor changes to the materials used as work surfaces, and some procedural changes in police stations and large evidence stores are suggested to decrease the likelihood of drugs contaminating work surfaces, thereby reducing the potential exposure of police officers to drugs in the workplace.

Quantification of licit and illicit drugs on typical police station work surfaces using LC-MS/MS

Licit and illicit drug use is widespread in the community and as a result, drug residues can be transferred onto handles and work surfaces in shared places. Police officers are more likely than members of the public to encounter drug residues while performing their work duties. As a result, sampling and analysis methodology must be developed to assess their work environments to determine which drug residues are present, at what concentration, and how long they may persist on the work surface to attempt to determine whether the residues pose a risk. The following reports a method for determining residues on work surfaces using cotton swabs, solvent extraction and analysis with LC-MS/MS. LC column type, swab extraction time, solvent composition, and analyte suppression were investigated. The reported method is simple, allows high throughput at low cost, simultaneously analyses for 23 licit and illicit drugs and metabolites, and has the scope for inclusion of additional analytes. Additionally, the method could be adapted easily to suit other organic chemicals, such as pesticides. The optimised method was used to investigate the persistence of 23 drugs and metabolites on five different surface types commonly found in police stations, under both dark and illuminated incubation conditions. The results demonstrated that different drugs within a given class can have dramatically different rates of loss, and general predictions cannot be made for other drugs in the same class. Illuminated incubation conditions generally accelerated the loss of drugs on surfaces, either by enhanced volatilisation, photocatalysis, or a combination of both. Only drugs such as amphetamine, methamphetamine and ketamine deviated from this trend because their disappearance from all surfaces under both incubation conditions was so rapid that no real difference was observed.

Simultaneous determination of niclosamide and its degradates in water by LC-MS/MS

A new method for the analysis of niclosamide (NIC) and its primary degradates 2-chloro-4-nitroaniline (2C4NA), aminoniclosamide (AN), hydroxyniclosamide (HN) and 5-chlorosalicylic acid (5CSA) in water was developed using direct injection LC-MS/MS. Methanol and acetonitrile mobile phases were compared. Methanol was superior for both separation and sensitivity for all chemicals. LLoQs for all chemicals were 3–50 times better in methanol than in acetonitrile, and baseline separation was observed for HN and 5CSA in methanol but not acetonitrile. The LLoQ for NIC in the current study was approximately 20 times lower than that previously reported using LC-MS/MS methodology, and 10–250 times lower for all chemicals than obtained by HPLC-UV/visible detection. The method reported in the current study relies upon smaller injection volumes and direct injection of water, eliminating time consuming clean-up steps and increasing sample throughput. The current method is also more selective for all four chemicals than existing HPLC-UV/visible methods, and is not susceptible to spectral interferences from DOM. The use of a shorter LC column with core shell particles resulted in shorter run times and lower mobile phase consumption than previously reported methods for NIC, which rely on traditional porous particle columns.

Work place drug testing of police officers after THC exposure during large volume cannabis seizures

Police officers responsible for the seizure and removal of illegally grown cannabis plants from indoor and outdoor growing operations face the prospect of THC exposure while performing their work duties. As a result, a study investigating the amount of THC on hands and uniforms of officers during raids on cannabis growing houses (CGHs) and forest cannabis plantations (FCPs) and in the air at these sites was conducted. Swabs of gloves/hands, chests, and heads/necks were collected and analysed for THC. Results of hand swabs indicated that officers removing plants from FCPs were exposed to THC concentrations up to 20 times those involved in raids at CGHs, which was mainly associated with the number and size of plants seized. Air samples collected inside cannabis houses showed no detectable THC. Air samples collected inside the cargo area of the storage trucks used during FCP raids indicated that THC can be volatilised when lush plants are compressed by other seized plants loaded on top of them in the truck over a period of several days, allowing composting of plants at the bottom of the load to commence. The elevated temperature and humidity inside the truck may assist the decarboxylation of THCA to THC, as well as increasing the rate of volatilisation of THC. More than 100 urine samples were collected from officers in raids on both CGHs and FCPs and all tested negative for THC. Removal of cannabis plants by officers often resulted in cuts, abrasions and ruptured blisters on exposed skin surfaces, particularly at FCPs. The results in this study suggest that even when small areas of damaged skin are directly exposed to THC by contact transfer, the likelihood of showing a positive THC urine test is low.

Pharmacokinetics of pergolide after intravenous administration to horses

OBJECTIVE To determine the pharmacokinetics of pergolide after IV administration to horses. ANIMALS 8 healthy adult horses. PROCEDURES Pergolide mesylate was administered IV at a dose of 20 μg/kg (equivalent to 15.2 μg of pergolide/kg) to each horse, and blood samples were collected over 48 hours. Pergolide concentrations in plasma were determined by means of high-performance liquid chromatography-tandem mass spectrometry, and pharmacokinetic parameters were determined on the basis of noncompartmental methods. RESULTS After IV administration of pergolide, mean ± SD clearance, elimination half-life, and initial volume of distribution were 959 ± 492 mL/h/kg, 5.64 ± 2.36 hours, and 0.79 ± 0.32 L/kg, respectively. CONCLUSIONS AND CLINICAL RELEVANCE With an elimination half-life of approximately 6 hours, twice-daily dosing may be more appropriate than once-daily dosing to reduce peak-trough fluctuation in pergolide concentrations. Further pharmacodynamic and pharmacokinetic studies of pergolide and its metabolites will be necessary to determine plasma concentrations that correlate with clinical effectiveness to determine the therapeutic range for the treatment of pituitary pars intermedia dysfunction.