The effect of the introduction of livestock on the erosion of alpine soils: a comparison of five dating techniques applied to sediments of the Australian alpine Blue Lake.

210Pb and 137Cs dating of bulk sediments obtained from the alpine Blue Lake, located in the Snowy Mountains of southeastern Australia, was applied here to date recent lacustrine sediments. In addition, the presence of Pinus pollen (a taxon introduced in Australia about 150 years ago) down to a sediment depth of 56 cm in the core is used to obtain a chronology for the upper part of the core. Accelerated Mass Spectrometry radiocarbon dates obtained from organic muds from the same core do not agree with the chronology constructed using the three other dating techniques. In addition, optically stimulated luminescence (OSL) dating of single quartz grains, from sediment-core samples collected from the same lake, was applied to date recent lacustrine sediments. The optical age of 185 ± 20 years for a sample at 60-62 cm depth, and 470 ± 50 years at 116-118 cm depth are well over 1000 years younger than the ages inferred from radiocarbon dates. We therefore infer that the 'old' radiocarbon ages result from carbon stored for considerable time within the catchment prior to its transport and deposition on the lake floor. As plant decomposition occurs at much slower rates in high altitude environments, these results bring into question the veracity of previously published radiocarbon dates from Blue Lake and alpine lake sediments in general. The deposition ages inferred from the 210Pb-137Cs and OSL dating, and the first appearance of Pinus pollen, indicate that for the 100-year period after European settlement (from the mid 1800s to early 1900s) the sediment-accumulation rate increased by a factor of about 2, from 0.19 ± 0.01 cm yr-1 to 0.35 ± 0.02 cm yr-1. In the 1900s the accumulation rate increased further to 0.60 cm yr-1. The accumulation rate was particularly rapid in the 20-year period from 1940-1960, reaching a rate 18 times higher than the pre-European rate in the mid-1950s. The increase in sedimentation rate is attributed to changes in land use resulting from European activities in the lake catchment, primarily through sheep and cattle grazing in the Blue Lake catchment.

Intelligence benefit of the 3-methoxytyramine to tyramine ratio in equine urine.

Equine urine analysis has evolved over time to detect thousands of urinary compounds for doping control in the horse racing industry. The longitudinal assessment of 3-methoxytyramine to tyramine ratio (3-MT/T) values in equine urine by GC-MS profiling was investigated to support the Racing NSW Equine Biological Passport (EBP) for detection of dopaminergic manipulation in racehorses. This involved comparison of routine urine samples to administration studies of Sinemet, a common Parkinson's disease medication containing levodopa. Using an endogenous reference compound (ERC) in a urinary ratio enabled greater confidence to provide intelligence of pharmaceutical manipulation as distinct from physiological variation. Population reference limits (PRLs) of 776 ng/ml for urinary 3-MT and 5.3 for 3-MT/T, together with the use of individual reference limits (IRLs), are proposed.

The equine metabolism of the catechol-O-methyltransferase enzyme inhibitor nitecapone.

The abuse of performance-enhancing catecholamine-based stimulants, such as levodopa, is controlled in horse racing through the application of a regulatory threshold for the common major metabolite. However, catechol-O-methyltransferase (COMT) enzyme inhibitors can be used to restrict the catalysis of the stimulant, and so the concurrent administration of both substances would be a viable strategy to enhance racing performance while removing the risk of exceeding the threshold. A 200 mg dose of nitecapone, a COMT inhibitor, was administered to a Thoroughbred horse, and we have analysed the blood (≤24 h) and urine (≤48 h) samples that were collected. The extracts, analysed by UHPLC coupled to a high-resolution accurate mass spectrometer, were consistent with the presence of nitecapone glucuronide in all the samples collected. An in-depth examination of the samples was then carried out using targeted accurate mass extracted ion chromatograms to identify whether the metabolites that have been found in other species were also present in the extracts. Once these were tentatively identified, MS/MS experiments were conducted on some of the metabolites (M1-M5), as well as decomposition products (DP1 and DP2), to verify that spectrum included MS fragments were consistent with their proposed structures. The accumulated data provided evidence that is consistent with this drug having been converted into many metabolites and a few decomposition products. An unexpected finding was that O-methylation was a very minor pathway until after the reduction of the 2,4-pentanedione side chain had occurred.

Metabolism and elimination of the catechol-o-methyltransferase inhibitor tolcapone in the horse.

The metabolism of the masking agent tolcapone in the horse has been investigated. This substance was found to have undergone various chemical transformations that produced a large variety of phase I metabolites, as well as glucuronide and sulfate conjugation. Confirmation of the presence of tolcapone and the 3-O-methylated metabolite in the blood samples collected up to 240 minutes and in urine obtained up to 24 hours, was successfully conducted using both gas chromatography- and liquid chromatography-tandem mass spectrometry techniques. The 3-O-methyl tolcapone is the better marker to use in a screening method because, in comparison to tolcapone, we have found that this substance offers superior chromatographic performance that should potentially give a lower limit of detection.

Fabrication of an on-line enzyme micro-reactor coupled to liquid chromatography-tandem mass spectrometry for the digestion of recombinant human erythropoietin.

Our aim was to develop a fast and efficient on-line method using micro-reactors for the digestion and deglycosylation of recombinant human erythropoietin extracted from equine plasma. The trypsin digestion micro reactors were fabricated using fused silica capillaries with either a dextran-modified coating or a porous monolith that was able to immobilise the enzyme. These were both found to be reasonably robust and durable, with the trypsin immobilised on dextran-modified fused silica capillaries offering better reproducibility than the micro-reactor based upon covalent attachment of this enzyme to the polymer. It is also evident that the enzyme attached micro reactors produced some tryptic peptides in a greater yield than in-solution digestion. A peptide-N-glycosidase F reactor was also fabricated and, when coupled with the trypsin reactor, the deaminated peptides T5 DAM and T9 DAM from recombinant human erythropoietin could also be detected by LC-ESI-MS/MS analysis. These results were better than those achieved using off-line digestion plus deglycosylation reactions and the analysis required far less time and effort to complete. The use of this on-line approach improved the sensitivity, efficiency and speed of our confirmation methodology that is based upon detecting the unique peptide segments of recombinant human erythropoietin that has been affinity extracted from positive equine plasma samples.

Monolithic poly (SPE-co-BVPE) capillary columns as a novel hydrophilic interaction liquid chromatography stationary phase for the separation of polar analytes.

A novel hydrophilic interaction liquid chromatography (HILIC) stationary phase was prepared by the co-polymerisation of zwitterionic N,N'-dimethyl-N-methacryloxyethyl-N-(3-sulfopropyl) ammonium betaine (SPE) and the crosslinker 1,2-bis(p-vinylphenyl) ethane (BVPE) in the presence of the porogens, toluene and methanol. Monolithic columns were produced by carrying out the α,α'-azoisobutyronitrile (AIBN) initiated reaction for 1, 2, 4, 8 and 12 h inside a 200 µm i.d. fused silica capillary at 75°C (water bath). The optimum polymerisation time was shown to be 2 h, as this resulted in good porosity, due to enlarged flow-channels and the presence of a higher proportion of mesopores provided a relatively larger surface area than the other columns. The chromatographic properties of the optimised poly (SPE-co-BVPE) monolithic column were evaluated with test mixtures containing both basic and neutral compounds in the HILIC gradient separation mode. This produced relatively sharp peaks (average peak width at half height=0.1 min) with average asymmetry factors of 1.4 and baseline resolution was obtained for all the compounds. Using the isocratic separation of the test mixture, the number of theoretical plates (N) per metre calculated was between 26,888 and 35,930 by using average values obtained for triplicate injections of the compounds thiourea, toluene and acrylamide.

Direct-injection screening for acidic drugs in plasma and neutral drugs in equine urine by differential-gradient LC-LC coupled MS/MS.

Direct-injection LC-LC hybrid tandem MS methods have been developed for undertaking broad-based screening for acidic drugs in protein-precipitated plasma and neutral doping agents in equine urine. In both analyses, analytes present in the matrix were trapped using a HLB extraction column before being refocused and separated on a Chromolith RP-18e monolithic analytical column using a controlled differential gradient generated by proportional dilution of the first column's eluent with water. Each method has been optimised by the adoption of a mobile phase and gradient that was tailored to enhance ionisation in the MS source while maintaining good chromatographic behaviour for the majority of the target drugs. The analytical column eluent was fed into the heated nebulizer (HN) part of the Duospray interface attached to a 4000 QTRAP mass spectrometer. Information dependent acquisition (IDA) with dynamic background subtraction (DBS) was configured to trigger a sensitive enhanced product ion (EPI) scan when a multiple reaction monitoring (MRM) survey scan signal exceeded the defined criteria. Ninety-one percent of acidic drugs in protein-precipitated plasma and 80% of the neutral compounds in equine urine were detected when spiked at 10 ng/ml.

Screening for basic drugs in equine urine using direct-injection differential-gradient LC-LC coupled to hybrid tandem MS/MS.

A rapid, selective and robust direct-injection LC/hybrid tandem MS method has been developed for simultaneous screening of more than 250 basic drugs in the supernatant of enzyme hydrolysed equine urine. Analytes, trapped using a short HLB extraction column, are refocused and separated on a Sunfire C(18) analytical column using a controlled differential gradient generated by proportional dilution of the first column's eluent with water. Independent data acquisition (IDA) was configured to trigger a sensitive enhanced product ion (EPI) scan when a multiple reaction monitoring (MRM) survey scan signal exceeded the defined criteria. The decision on whether or not to report a sample as a positive result was based upon both the presence of a MRM response within the correct retention time range and a qualitative match between the EPI spectrum obtained and the corresponding reference standard. Ninety seven percent of the drugs targeted by this method met our detection criteria when spiked into urine at 100 ng/ml; 199 were found at 10 ng/ml, 83 at 1 ng/ml and 4 at 0.1 ng/ml.