Improved method robustness and ruggedness in liquid chromatography-mass spectrometry by increasing the acid content of the mobile phase.

A routine multiresidue method developed for the detection and quantification of veterinary drug residues in animal-based food was used to analyze sheep (ovine) liver. Unlike when working with previously validated matrices (e.g., bovine liver), some of the analytes of interest chromatographed in the form of split- or even fully baseline separated peaks. In other cases a significantly longer retention times (tR) was observed. A detailed investigation led to the elucidation of taurocholic acid as the causative agent. This compound is present in sheep liver at significantly higher concentrations than in most other animal tissues. Taurocholic acid is a zwitterionic compound and likely acts as an ion pairing agent, which modifies the selectivity of the stationary phase in a highly spatial and dynamic way. Injecting smaller volumes of matrix extract or the use of a significantly higher formic acid concentration in the mobile phase reduced or even completely eliminated the peak splitting. A more detailed examination led to the observation that the problem is not restricted to this particular matrix and extraction procedure or the used stationary phase. In fact, a higher formic acid concentration (e.g., 1.0 % versus 0.1 %) significantly improves the peak shape of many analytes present in fortified matrix samples as well as in pure standard solutions. In addition, analytical column aging was observed as being slower with a higher formic acid concentration. Finally the peak shape of analytes interacting with the metallic parts along the flow path of the liquid chromatograph was also significantly improved. Use of 0.1 % acid in mobile phases is often taken for granted in LC-MS. Regardless of the stationary phase, a higher ionic strength better stabilizes the pH and reduces unwanted interactions, which ultimately improves the method robustness. Flow injection experiments often show that 0.1 % acid concentrations produce the highest analyte signals. Yet, the use of 1 % acid in the mobile phase often leads to narrower and therefore taller chromatographic peaks, which may lead to lower detection limits for many analytes and to an improved separation efficiency.

Optimized multimatrix calibration concept for liquid chromatography mass spectrometry-based bioanalysis methods.

In this paper, a calibration procedure for LC/MS-based bioanalysis methods, termed "A/B fortification", is proposed. The concept relies on the post-extraction fortification (B-spike) of an aliquot of the injection-ready sample extract for the determination and compensation of specific signal suppression or enhancement effects compared to matrix-free extract prepared in buffer or mobile phase. Conventional analyte recovery, observed due to the incomplete extraction of analytes from the sample or losses during a cleanup, is determined by the conventional pre-extraction fortification (A-spike) of a blank sample that belongs to the same type of matrix as the sample with the unknown analyte concentration. This approach permits a higher throughput than conventional sample fortification strategies. The results obtained by utilizing the A/B fortification concept were extensively compared against conventional methods (representative bank matrix fortification, sample fortification and internal standard). The proposed concept (based on the pre-fortification of a reference matrix and post-fortification of the sample) was found to be significantly less biased than internal standard-based techniques. The A/B fortification indicated a better accuracy than the sample fortification or representative blank matrix fortification approach and, most importantly, produced significantly fewer outliers. This was linked to the fact that in the case of the A/B fortification, the uncertainty of the subtraction of two peak areas (fortified minus unfortified sample) is reduced, because fortifications are not made prior to the extraction step but are made into the final injection-ready sample extract. Fortification into an injection-ready aliquot eliminates all sample processing-related differences (procedural errors), which can affect conventional sample fortification-based quantifications.

Does the ion mobility resolving power as provided by commercially available ion mobility quadrupole time-of-flight mass spectrometry instruments permit the unambiguous identification of small molecules in complex matrices?

Quadrupole based mass spectrometry based detection has experienced enormous improvements in terms of sensitivity over the last centuries. This development has not been equally matched with improvements in selectivity. Hence, the use of unit mass based MS/MS transitions or high resolution (HRMS) based extracted ion chromatograms is gradually becoming insufficient in the field of high sensitivity multi-residue analysis (e.g. pesticides in food). As a consequence, commercial instruments hyphenating ion mobility (IMS) with low or high resolution mass spectrometry based detection have appeared. The use of such an additional (frequently claimed to be orthogonal) dimension is intended to increase selectivity. In addition, IMS derived collision cross section (CCS) has been proposed to be used as an additional identification point for the unambiguous identification of trace compounds in complex matrices. It is the topic of this paper to investigate the benefit of using such a hyphenated technique for trace analysis of small molecules in complex matrices. The potential of CCS to serve as additional identification point has been critically evaluated. Discussed are the effect of CCS data on false detects and missing detects of analytes present at trace levels. This involves the investigation of the physical resolving power provided by HRMS, IMS and chromatography as well as the correlation among these parameters (orthogonality). It is the conclusion that currently commercially available travelling wave and linear drift tube based IMS devices with a resolving power of up to 50 permit a reduction of false detects, yet this comes at the price of a higher likelihood of missing detects. The reduction of missing detects and the use of CCS as potential confirmatory information would require IMS resolving powers above 100.

Corrigendum to: The acute physiological status of white sharks (Carcharodon carcharias) exhibits minimal variation after capture on SMART drumlines.

[This corrects the article DOI: 10.1093/conphys/coz042.].

The acute physiological status of white sharks (Carcharodon carcharias) exhibits minimal variation after capture on SMART drumlines.

Drumlines incorporating SMART (Shark-Management-Alert-in-Real-Time) technology are a new tool used in several bather protection programmes globally. In New South Wales (NSW), Australia, the white shark (Carcharodon carcharias) is a target species for SMART drumlines because they are often involved in attacks on humans. To understand white shark sensitivity to capture and to establish protocols around acceptable timeframes for responding to alerts, 47 juvenile and subadult white sharks were caught on SMART drumlines at five locations off the east coast of Australia. There was no at-vessel mortality during the sampling period. After capture, blood was sampled from each shark to assess its acute physiological status. Of the 18 metabolites investigated, only lactate and aspartate aminotransferase exhibited significant positive relationships with the capture duration on SMART drumlines. These results indicate that the capture process is relatively benign and that the current response times used here are appropriate to minimize long-term negative impacts on released white sharks. Where white sharks are likely to interact negatively with beachgoers, SMART drumlines can therefore be a useful addition to bather protection programmes that also aim to minimize harm to captured animals. Other shark species captured on SMART drumlines should also be investigated to gain broader understanding of potential physiological consequences of using this new technology.

High-resolution mass spectrometry-based multi-residue method covering relevant steroids, stilbenes and resorcylic acid lactones in a variety of animal-based matrices.

A new, relatively simple sample processing and detection workflow has been developed for the quantification and confirmation of banned growth-promoting substances in a wide variety of animal-based food products. The method covers all required compounds (belonging to the so-called A1, A3, A4 and B2f groups as termed by the relevant EU legislation) which are currently monitored by the official European community surveillance programs. The sample processing includes a thermal sample denaturation step, intended to prevent undesirable side-reactions during the following enzymatic deconjugation of covalently bound analytes. A pH-adjusted dual liquid/liquid-extraction produces sufficient clean extracts for a wide range of matrices (urine, muscle, liver, serum, full blood). The method has been validated using two hybrid quadrupole high-resolution mass spectrometers (Orbitrap and time-of-flight technology-based instruments). Full-scan data acquisition, interlaced with targeted modes (unit mass isolation of the precursors, followed by collision-induced fragmentation), produces sufficiently sensitive and selective detection of the analytes within all the validated matrices. The proposed method is an alternative to currently used methods that are restricted to a limited set of analytes and matrices.

Diagnostic accuracy of a prototype rapid chlamydia and gonorrhoea recombinase polymerase amplification assay: a multicentre cross-sectional preclinical evaluation.

OBJECTIVES: Rapid and accurate sexually transmitted infection diagnosis can reduce onward transmission and improve treatment efficacy. We evaluated the accuracy of a 15-minute run-time recombinase polymerase amplification-based prototype point-of-care test (TwistDx) for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG). METHODS: Prospective, multicentre study of symptomatic and asymptomatic patients attending three English sexual health clinics. Research samples provided were additional self-collected vulvovaginal swab (SCVS) (female participants) and first-catch urine (FCU) aliquot (female and male participants). Samples were processed blind to the comparator (routine clinic CT/NG nucleic acid amplification test (NAAT)) results. Discrepancies were resolved using Cepheid CT/NG GeneXpert. RESULTS: Both recombinase polymerase amplification and routine clinic NAAT results were available for 392 male and 395 female participants. CT positivity was 8.9% (35/392) (male FCU), 7.3% (29/395) (female FCU) and 7.1% (28/395) (SCVS). Corresponding NG positivity was 3.1% (12/392), 0.8% (3/395) and 0.8% (3/395). Specificity and positive predictive values were 100% for all sample types and both organisms, except male CT FCU (99.7% specificity (95% confidence interval (CI) 98.4-100.0; 356/357), 97.1% positive predictive value (95% CI 84.7-99.9; 33/34)). For CT, sensitivity was ≥94.3% for FCU and SCVS. CT sensitivity for female FCU was higher (100%; 95% CI, 88.1-100; 29/29) than for SCVS (96.4%; 95% CI, 81.7-99.9; 27/28). NG sensitivity and negative predictive values were 100% in FCU (male and female). CONCLUSIONS: This prototype test has excellent performance characteristics, comparable to currently used NAATs, and fulfils several World Health Organization ASSURED criteria. Its rapidity without loss of performance suggests that once further developed and commercialized, this test could positively affect clinical practice and public health.

Sublethal effects of angling and release on golden perch Macquaria ambigua: implications for reproduction and fish health.

The present study tested the hypothesis of no delayed sublethal effects of mild angling and release on the feeding, growth, somatic condition and gonadal development of golden perch Macquaria ambigua during gametogenesis. Subsamples of adult M. ambigua (n = 17-21 of 207), originally captured from the wild and stocked into ten 0·1 ha earthen ponds, were angled and released during early and late gametogenesis. Wild samples that were concurrently collected throughout the experiment underwent rapid and synchronous gonadal development and many spawned. While no spawning occurred in the ponds, most M. ambigua underwent normal gonadal development to maturity, including the angled fish. Angled fish also fed, maintained condition and actually grew faster than non-angled captive controls. Although females that were angled during late gametogenesis more readily ingested and retained baited hooks, neither their subsequent condition nor gonadal development was significantly affected. The predominance of null results was attributed to the combined effects of the flexible reproductive strategy of M. ambigua, the benignness of mouth hooking and immediate release, and possible methodological issues arising from differential hooking success of more aggressive and resilient individuals. The findings support earlier catch-and-release research, but contrast with reports of acute reproductive effects following capture and handling for aquaculture broodstock. This discrepancy highlights the need for research to specifically address welfare questions relevant to recreational fisheries across various species and angling scenarios.

Age and growth of mangrove red snapper Lutjanus argentimaculatus at its cool-water-range limits.

This study investigates the age and growth of Lutjanus argentimaculatus at its southern (cooler) range limits in eastern Australia. Specimens were collected from New South Wales and southern Queensland between November 2011 and December 2013. Fork lengths (LF ) ranged from 190 to 1019 mm, and ages ranged from 2+ to 57+ years. Growth was described by the von Bertalanffy growth function with coefficients L∞ = 874·92 mm, K = 0·087 year(-1) and t0 = -2·76 years. Estimates of the instantaneous natural mortality rate (M) ranged from 0·072 to 0·25. The LF (mm) and mass (W; g) relationship was represented by the equation: W=2·647×10-5LF2·92. The maximum age of 57+ years is the oldest reported for any lutjanid and comparisons with tropical studies suggest that the age-based demography of L. argentimaculatus follows a latitudinal gradient. High maximum ages and low natural mortality rates indicate considerable vulnerability to overexploitation at the species' cool-water-range limits. These results demonstrate the need to identify underlying processes driving latitudinal gradients in fish demography.

Determination of nitrofuran and chloramphenicol residues by high resolution mass spectrometry versus tandem quadrupole mass spectrometry.

An ultra-high performance liquid chromatography based method, coupled to high resolution mass spectrometry (UHPLC-HRMS), was developed to permit the detection and quantification of various nitrofuran and chloramphenicol residues in a number of animal based food products. This method is based on the hydrolysis of covalently bound metabolites and derivatization with 2-nitrobenzaldehyde. Clean-up is achieved by a liquid/liquid and a reversed phase/solid phase extraction. Not only are the four conventional nitrofurans (nitrofurantoin, furazolidone, nitrofurazone and furaltadone) detected, but also nifursol, nitrovin and nifuroxazide. Furthermore, an underivatizable nitrofuran (nifurpirinol) and another banned drug (chloramphenicol) can be quantified as well. The compounds are detected in the form of their precursor ions, [M+H](+) and [M-H](-), respectively. The mass resolving power of 70,000 FWHM, and the applied mass window ensure sufficient selectivity and sensitivity. Confirmation is obtained by monitoring the HRMS resolved product ions which were derived from the unit-mass resolved precursor ions. The multiplexing capability of the utilized Orbitrap instrument provides not only highly selective, but also sensitive confirmatory signals. This method has been validated according to the CD 2002/657/EC for the following matrices: muscle, liver, kidney, fish, honey, eggs and milk.

Reliability of veterinary drug residue confirmation: high resolution mass spectrometry versus tandem mass spectrometry.

Confirmation of suspected residues has been a long time domain of tandem triple quadrupole mass spectrometry (QqQ). The currently most widely used confirmation strategy relies on the use of two selected reaction monitoring signals (SRM). The details of this confirmation procedure are described in detail in the Commission Decision 93/256/EC (CD). On the other hand, high resolution mass spectrometry (HRMS) is nowadays increasingly used for trace analysis. Yet its utility for confirmatory purposes has not been well explored and utilized, since established confirmation strategies like the CD do not yet include rules for modern HRMS technologies. It is the focus of this paper to evaluate the likelihood of false positive and false negative confirmation results, when using a variety of HRMS based measurement modes as compared to conventional QqQ mass spectrometry. The experimental strategy relies on the chromatographic separation of a complex blank sample (bovine liver extract) and the subsequent monitoring of a number of dummy transitions respectively dummy accurate masses. The term "dummy" refers to precursor and derived product ions (based on a realistic neutral loss) whose elemental compositions (CxHyNzOdCle) were produced by a random number generator. Monitoring a large number of such hypothetical SRM's, or accurate masses inevitably produces a number of mass traces containing chromatographic peaks (false detects) which are caused by eluting matrix compounds. The number and intensity of these peaks were recorded and standardized to permit a comparison among the two employed MS technologies. QqQ performance (compounds which happen to produce a response in two SRM traces at identical retention time) was compared with a number of different HRMS(1) and HRMS(2) detection based modes. A HRMS confirmation criterion based on two full scans (an unfragmented and an all ion fragmented) was proposed. Compared to the CD criteria, a significantly lower probability of false positive and false negative findings is obtained by utilizing this criterion.

Multi-residue quantification of veterinary drugs in milk with a novel extraction and cleanup technique: salting out supported liquid extraction (SOSLE).

A quantitative liquid chromatography coupled with high-resolution mass spectrometry method was developed for the determination of more than one hundred compounds belonging to a variety of veterinary drug classes in bovine milk. Salting out supported liquid extraction (SOSLE), a novel extraction and cleanup technique, was introduced to ensure high extraction efficiency and good sample cleanup. The high salt (ammonium sulfate) concentration in the aqueous donor phase permits supported liquid/liquid extraction (SLE) with a relative polar organic acceptor phase (acetonitrile). This is different from traditional SLE, in which the need for phase separation results in the selection of organic solvents with intermediate polarities (e.g., ethyl acetate or dichloromethane). Hence, SOSLE is more efficient in recovering polar analytes than conventional SLE. SOSLE was also compared to classical approaches like solid phase extraction, QuEChERS and ultra-filtration. The proposed technique resulted in extracts of equal or superior cleanliness and with higher average recoveries than those obtained with QuEChERS or SPE. The recovery (median for all compounds) was 73% for QuEChERS, 83% for SPE and 91% for SOSLE. The most significant improvements were observed for polar analytes (penicillines, quinolones and tetracyclines) which are hardly recovered by QuEChERS. The chromatographic separation and detection was based on an ultra-high-performance liquid chromatography Q-Orbitrap system (Q-Exactive plus). The developed analytical method has been validated (based on the commission decision 2002/957/EC) as required for quantitative veterinary drug methods.

Genome sequencing and characterization of an extensively drug-resistant sequence type 111 serotype O12 hospital outbreak strain of Pseudomonas aeruginosa.

A series of extensively drug-resistant isolates of Pseudomonas aeruginosa from two outbreaks in UK hospitals were characterized by whole genome sequencing (WGS). Although these isolates were resistant to antibiotics other than colistin, we confirmed that they are still sensitive to disinfectants. The sequencing confirmed that isolates in the larger outbreak were serotype O12, and also revealed that they belonged to sequence type ST111, which is a major epidemic strain of P. aeruginosa throughout Europe. As this is the first reported sequence of an ST111 strain, the genome was examined in depth, focusing particularly on antibiotic resistance and potential virulence genes, and on the reported regions of genome plasticity. High degrees of sequence similarity were discovered between outbreak isolates collected from recently infected patients, isolates from sinks, an isolate from the sewer, and a historical isolate, suggesting that the ST111 strain has been endemic in the hospital for many years. The ability to translate easily from outbreak investigation to detailed genome biology by use of the same data demonstrates the flexibility of WGS application in a clinical setting.

Clinical signs of barotrauma in golden perch, Macquaria ambigua (Richardson), and associated effects on post-release mortality and health.

This study assessed the effects of different retrieval depths (2, 10 or 20 m), surface intervals (none or 15 min) and release methods (untreated, vented or recompressed) on the incidence of external and internal clinical signs of barotrauma (ECSB and ICSB) and post-release mortality in golden perch, Macquaria ambigua (Richardson). Fish were assessed for ECSB before and after surface intervals and either monitored for mortality over 3 days in two deep cages or killed for internal examination. When all fish were left untreated, short-term mortality increased with retrieval depth from 0% and 4.2% among 2 and 10-m fish, respectively, to 19.2% among 20-m fish; while surface interval only affected the incidence of two ECSB (excess buoyancy and a prolapsed cloaca). Mortality was also greater among 20-m fish that were subjected to a 15-min surface interval and left untreated (22.2%) or vented (22.2%) than those that were recompressed (5.6%). Of the ECSB, only exophthalmia was associated with increased mortality, with half of the affected fish dying. However, many fish retrieved from 10 and 20 m also sustained numerous ICSB, including compressed gonads or vital organs and ruptured or collapsed, haemorrhaging swimbladders that remained deflated for up to 3 days after release.

Resilience of inshore, juvenile snapper Pagrus auratus to angling and release.

This study assessed the mortality of 157 snapper Pagrus auratus (9-29 cm, total length, L(T) ) after being conventionally angled and then released into cages (along with 48 controls) for 4 days off south-eastern Australia. Fatalities were restricted to 12 angled fish (7·6%) and mostly attributed to the ingestion of hooks and especially their subsequent removal, which caused substantial blood loss and immediate death. Hook ingestion was significantly biased towards smaller fish (<21 cm L(T)) and attributed to a lower chance of anglers initially detecting these individuals on the line (allowing them to consume more of the baits). While mortalities might be reduced in future via (1) choosing terminal rigs that promote mouth hooking and (2) cutting the line on any-hook ingested fish, the results nevertheless validate releasing unwanted angled inshore juvenile P. auratus as a means for managing their exploitation.

Determination of aminoglycoside residues by liquid chromatography and tandem mass spectrometry in a variety of matrices.

A quantitative LC-MS/MS method was developed for the determination of 13 commonly used aminoglycoside antibiotics in meat (pork muscle, fish, and veal livers and kidneys). The proposed method is sufficiently sensitive and highly selective. Unlike other previously reported methods, it uses a simple clean-up procedure based on a strong cation-exchange solid-phase cartridge that permits high sample extract loading volumes. A unique elution regime based on a volatile buffer at intermediately high pH value in combination with an organic solvent provides quantitative elution of the various aminoglycosides. This methodology ensured that neither a breakthrough of weakly retained aminoglycosides (e.g. spectinomycin) nor the incomplete elution of strongly retained analytes (e.g. neo- and gentamycin) is observed. The single-step clean-up is fast and produces clean extracts that minimize matrix-related signal suppression in the electrospray interface.

Quantification of anthelmintic drug residues in milk and muscle tissues by liquid chromatography coupled to Orbitrap and liquid chromatography coupled to tandem mass spectrometry.

A simple method for the determination of some anthelmintic drugs and phenylbutazone residues in milk and muscle was developed. Following a fast and easy extraction and evaporation procedure, the extract was injected into an ultra performance liquid chromatography system coupled to a single stage Orbitrap detector. The high mass resolution of 50,000 full width at half maximum and corresponding narrow mass windows permitted a very selective and sensitive detection of analytes without requiring fragmentation of the observed [M+H](+) or [M+Na](+) ions. This eliminated some difficulties which have plagued the analysis of compounds belonging to the group of avermectins. The analytical method was validated according to the EU commission decision for Orbitrap based, but also for more traditional tandem mass spectrometry based detection and quantification. Equal repeatability but significantly higher sensitivity for critical compounds (avermectins) was obtained for the Orbitrap based detection. A result of this study was the conclusion that analytes with poor fragmentation properties (e.g. sodium-cationized molecules) can be more easily quantified by single stage high resolution mass spectrometry than by tandem mass spectrometry.

Development of an improved high resolution mass spectrometry based multi-residue method for veterinary drugs in various food matrices.

Multi-residue methods for veterinary drugs or pesticides in food are increasingly often based on ultra performance liquid chromatography (UPLC) coupled to high resolution mass spectrometry (HRMS). Previous available time of flight (TOF) technologies, showing resolutions up to 15,000 full width at half maximum (FWHM), were not sufficiently selective for monitoring low residue concentrations in difficult matrices (e.g. hormones in tissue or antibiotics in honey). The approach proposed in this paper is based on a single stage Orbitrap mass spectrometer operated at 50,000 FWHM. Extracts (liver and kidney) which were produced according to a validated multi-residue method (time of flight detection based) could not be analyzed by Orbitrap because of extensive signal suppression. This required the improvement of established extraction and clean-up procedures. The introduced, more extensive deproteinzation steps and dedicated instrumental settings successfully eliminated these detrimental suppression effects. The reported method, covering more than 100 different veterinary dugs, was validated according to the EU Commission Decision 2002/657/EEC. Validated matrices include muscle, kidney, liver, fish and honey. Significantly better performance parameters (e.g. linearity, reproducibility and detection limits) were obtained when comparing the new method with the older, TOF based method. These improvements are attributed to the higher resolution (50,000 versus 12,000 FWHM) and the superior mass stability of the of the Orbitrap over the previously utilized TOF instrument.

Moxifloxacin and pyrazinamide susceptibility testing in a complex case of multidrug-resistant tuberculosis.

Multidrug-resistant tuberculosis (MDR-TB) is a public health problem of global concern. It is critical that drug susceptibility testing (DST) methods accurately predict clinical response. We present a patient with a challenging case of MDR-TB with additional resistance to quinolones and pyrazinamide. Treatment with a regimen including high-dosage moxifloxacin, based on additional genotypic and phenotypic DST, produced excellent results. This case highlights the possibility of treatment with high-dose fluoroquinolones despite apparent bacterial resistance to these agents. Improved DST methods are necessary for both agents. Development of genotypic approaches may offer a susceptibility profile rapidly, enabling early introduction of individualised treatments.