Nitrofurantoin plasma- and urine exposure in eight healthy beagle dogs following standard nitrofurantoin dosing regimen.

Bacterial cystitis is common in dogs and is usually treated with antibiotics. Nitrofurantoin is used for treatment of bacterial cystitis in humans and might provide a feasible treatment option in dogs. The aim of this study was to investigate the nitrofurantoin plasma concentration-time course and potential adverse effects in dogs. Nitrofurantoin (4.4-5.0 mg/kg) was administered orally to eight healthy beagles every 8 h for five days before repeated plasma and urine samples were collected. An additional four beagles served as untreated controls. The nitrofurantoin plasma and urine concentrations were measured using ultra high precision liquid chromatography coupled to tandem mass-spectrometry and further analysed using a non-compartmental pharmacokinetic model. In plasma, the median Cmax was 2.1 µg/mL, tmax was 2 h, the terminal rate constant was 0.9 per h and the terminal half-life was 0.8 h. In urine, median Cmax was 56 µg/mL, tmax was 1 h and the terminal half-life was 4.3 h. No adverse effects were observed clinically or in haematology or biochemistry. The data presented in this study combined with in vitro sensitivity data from common urine pathogens and the lack of observed adverse effects suggest that nitrofurantoin in a standard dosing regimen could be effective in sporadic bacterial cystitis treatment in dogs. Further clinical studies are highly warranted to verify the effectiveness in clinical cases.

Benzylpenicillin-producing Trichophyton erinacei and methicillin resistant Staphylococcus aureus carrying the mecC gene on European hedgehogs - A pilot-study.

BACKGROUND: A high carriage rate of methicillin-resistant Staphylococcus aureus with the mecC gene (mecC-MRSA) has been described among Wild European hedgehogs (Europeaus erineaus). Due to this frequent occurrence, it has been suggested that hedgehogs could be a natural reservoir for mecC-MRSA. However, the reason why hedgehogs carry mecC-MRSA remains unknown, but it has been hypothesized that mecC-MRSA could have evolved on the skin of hedgehogs due to the co-occurrence with antibiotic producing dermatophytes. The aim of this pilot-study was therefore to investigate if hedgehogs in Sweden carry Trichophyton spp. and to provide evidence that these dermatophytes are able to produce penicillin or similar substances. In addition, the study aimed to identify if dermatophytes co-occurred with mecC-MRSA. METHODS: Samples were collected from hedgehogs (Europeaus erineaus) that were euthanized or died of natural causes. All samples were screened for dermatophytes and mecC-MRSA using selective cultivation methods. Suspected isolates were characterized using PCR-based methods, genome sequencing and bioinformatic analyses. Identification of penicillin was performed by ultra-high-performance liquid chromatography-tandem mass spectrometry. RESULTS: In total 23 hedgehogs were investigated, and it was shown that two carried Trichophyton erinacei producing benzyl-penicillin, and that these hedgehogs also carried mecC-MRSA. The study also showed that 60% of the hedgehogs carried mecC-MRSA. CONCLUSION: The pilot-study demonstrated that Trichophyton erinacei, isolated from Swedish hedgehogs, can produce benzylpenicillin and that these benzylpenicillin-producing T. erinacei co-occurred with mecC-MRSA. The study also reconfirmed the high occurrence of mecC-MRSA among hedgehogs.

Quantification of dimethylsulfoxide (DMSO) in equine plasma and urine using HILIC-MS/MS.

This paper describes quantitative methods for the determination of dimethylsulfoxide (DMSO) in equine plasma and urine based on simple precipitation and dilution followed by hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry (HILIC-MS/MS). DMSO is a polar solvent with analgesic and anti-inflammatory properties. Its pharmacological features make it prohibited in horse racing. However, since DMSO is naturally present in the horses' environment, international threshold values have been implemented for plasma and urine (1 and 15 µg/mL, respectively). Previously presented quantitative methods for the determination of DMSO are based on gas chromatography, thus demanding a tedious extraction step to transfer the analyte from the aqueous bodily fluid to an injectable organic solvent. The column used in the presented method was an Acquity BEH HILIC and the mobile phase was a mixture of ammonium acetate buffer and acetonitrile delivered as a gradient. Hexadeuterated DMSO (2 H6 -DMSO) was used as the internal standard. Validation was performed in the range of the international thresholds concerning selectivity, carry-over, linearity, precision, accuracy, stability and inter-individual matrix variation. The results fulfilled the predefined criteria and the methods were considered fit for purpose. Successful applications on real equine doping control samples were carried out with determined DMSO concentrations exceeding the international thresholds. Copyright © 2016 John Wiley & Sons, Ltd.

A new method for analysis of underivatized free ß-methylamino-alanine: Validation and method comparison.

A new method was developed for analysis of free ß-Methylamino-alanine (BMAA) in biological matrices. The method is based on direct analysis of the underivatized molecule, using an amide column for separation by Hydrophilic Interaction Liquid Chromatography (HILIC) and detection by tandem mass spectrometry (MS/MS) using a deuterium labeled internal standard. The use of Ultra-High Performance Liquid Chromatography (UHPLC) combined with MS/MS detection allowed for high chromatographic resolution and a low limit of detection (0.025 µg/g wet weight (ww) in mussels). The method was validated by analyzing spiked blank mussels from the Baltic Sea (0.15-4.4 µg/g (ww), trueness 99%-105%, RSD 2%-8%). An inter-laboratory comparative analysis of extracts of mussel was performed. The mussels were extracted according to an established protocol for analysis of free BMAA, and the extracts were then analyzed in parallel by the new method and a validated procedure based on detection of BMAA derivatized with dansyl chloride. Both methods detected BMAA in similar concentrations. Thus, derivatization with dansyl chloride did not influence the results compared to direct detection. The new method presents an alternative to the commonly applied derivatization step, and is proved through validation and method comparison to reliably identify and quantify free BMAA at low concentration levels.

Seafood sold in Sweden contains BMAA: A study of free and total concentrations with UHPLC-MS/MS and dansyl chloride derivatization.

ß-N-Methylamino-l-alanine (BMAA) is a potential neurotoxin associated with the aquatic environment. Validated analytical methods for the quantification of both free and total concentrations of BMAA were used in an investigation of seafood purchased from different grocery stores in Uppsala, Sweden. The analysis was performed using ultra high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) and detection of BMAA as a dansyl derivate. The determined concentrations of free BMAA (after a simple trichloroacetic acid extraction) in mussels and scallops were up to 0.46 µg g-1 wet homogenate. The total BMAA (after hydrochloric acid hydrolysis) levels were between 0.29 and 7.08 µg g-1 wet mussel homogenate. The highest concentration of total BMAA was found in imported cooked and canned mussels which contained about ten times the quantity of BMAA measured in domestic cooked and frozen mussels. In this study it was also concluded that BMAA could be detected in seafood origin from four different continents. The risks associated with human exposure to BMAA through food are unknown today. However, the results of this study show that imported seafood in Sweden contain BMAA, indicating that this area needs more investigation, including a risk assessment regarding the consumption of e.g., mussels, scallops and crab.

In vitro formation of phase I and II metabolites of propranolol and determination of their structures using chemical derivatization and liquid chromatography-tandem mass spectrometry.

Derivatization with 1,2-dimethylimidazole-4-sulfonyl chloride (DMISC) has been successfully used as a tool to differentiate between aromatic and aliphatic O-glucuronides of hydroxypropranolol. The analyses were performed with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) with both a triple quadrupole and an ion trap instrument. Hydroxylated forms of propranolol can be glucuronidated in aliphatic as well as aromatic positions. These isoforms are not distinguishable by tandem MS alone, as they both initially lose 176 Da, i.e. monodehydrated glucuronic acid, giving back the aglycone. Two in vitro systems were set up for the production of propranolol metabolites. The obtained isomers of 4'-hydroxypropranolol glucuronide were determined to correspond to one aliphatic and one aromatic form, using chemical derivatization with DMISC and LC-MS(n). DMISC was shown to react with the secondary amine in the case where the naphtol was occupied by the glucuronyl moiety, resulting in a different fragmentation pattern compared with that of the aliphatic glucuronide, where the naphtol group was accessible to derivatization.

Structural evaluation of the glucuronides of morphine and formoterol using chemical derivatization with 1,2-dimethylimidazole-4-sulfonyl chloride and liquid chromatography/ion trap mass spectrometry.

For the first time chemical derivatization of isomeric drug glucuronides with 1,2-dimethylimidazole-4-sulfonyl chloride (DMISC) has been successfully applied as a tool for determining the site of conjugation. This provides a way to differentiate between glucuronide isomers containing aliphatic and phenolic hydroxyl groups. The analyses were performed with liquid chromatography/electrospray ion trap mass spectrometry (LC/ESI-MSn). DMISC has previously been shown to react selectively with phenols in estrogens, thus improving sensitivity in ESI-MS. The model compounds selected for this study were commercially available standards of formoterol, morphine, morphine-3-glucuronide (M3G), and morphine-6-glucuronide (M6G). Formoterol glucuronides were produced with an enzymatic method in house. Both formoterol and morphine possess one phenolic and one aliphatic hydroxyl group where glucuronidation could take place. The product ion mass spectra of the native morphine glucuronides were indistinguishable due to the initial neutral loss of monodehydrated glucuronic acid (176 u). However, a significant difference between the isomers was observed with DMISC derivatization, as only the form with a free phenol, M6G, gave a detectable reaction product. Formoterol formed two detectable glucuronide isomers in the enzymatic reaction. Their respective sites of conjugation could not be directly determined from the product ion spectra. Reaction with DMISC, however, gave a detectable product with only one of the isomers. Based on previous experience of the preferred DMISC reactions with phenols, and interpretation of the fragmentation pattern of the derivative, it was concluded that the reactive isomer had a free phenol, and was thus conjugated on the aliphatic chain.

Differentiation of estriol glucuronide isomers by chemical derivatization and electrospray tandem mass spectrometry.

This paper describes a way of differentiating between the three isomers of estriol glucuronide by the use of chemical derivatization and liquid chromatography/electrospray tandem mass spectrometry (MS/MS). In their native form, these isomers gave rise to almost identical product ion spectra, involving the neutral loss of 176 Da (i.e. monodehydrated glucuronic acid), which made it impossible to determine the position of conjugation by MS/MS alone. In order to change the fragmentation pathways, positive charges were introduced into the analytes by chemical derivatization. The following reagents were tested: 2-chloro-1-methylpyridinium iodide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 2-picolylamine. Interestingly, derivatization using a combination of all three reagents gave a selective fragmentation pattern that could differentiate between the isomers estriol-16-glucuronide and estriol-17-glucuronide. Estriol-3-glucuronide, which lacks a free phenolic group, could be differentiated through a different type of reaction product when exposed to 2-chloro-1-methylpyridinium iodide. Furthermore, in order to assist structural assignment of the fragments, their accurate masses were determined using a hybrid quadrupole time-of-flight mass spectrometer and fragmentation pathways were elucidated by the use of MS3 on an ion trap mass spectrometer.

Detection of altrenogest and its metabolites in post administration horse urine using liquid chromatography tandem mass spectrometry--increased sensitivity by chemical derivatisation of the glucuronic acid conjugate.

Altrenogest (17alpha-allyl-17beta-hydroxyestra-4,9,11-trien-3-one) is a steroid used for the control of estrus in horses. This drug can potentially be abused in racehorses as the occurrence of estrus can alter their performance. This work describes an analytical method based on liquid chromatography-tandem mass spectrometry for the detection of altrenogest in horse urine down to a concentration of 13 pg/mL (0.042 nM). Furthermore, the qualitative aspect of metabolism of altrenogest in the horse has been studied. The main transformations that were found for this species were conjugation with glucuronic acid and sulfate. These phase II metabolites were identified by molecular mass and by comparison of their collision-induced dissociation product ion spectra with that of the synthetic aglycone at positive and negative potential, respectively. No phase I metabolites were discovered. In order to increase the ionisation in positive electrospray, a derivatisation procedure forming a basic oxime was tested. This process significantly increased the detection sensitivity for altrenogest glucuronide in horse urine.