Keith E. Miller

Multi‐class, multi‐residue liquid chromatography/tandem mass spectrometry screening and confirmation methods for drug residues in milk

This paper describes the development and optimization of a multi-residue veterinary drug screening method for whole milk. The drug residues of regulatory interest in milk include beta-lactams, sulfonamides, tetracyclines, fluoroquinolones, and macrolides. Milk samples were extracted with acetonitrile and the samples were then subjected to a clean-up procedure using a bonded solid-phase extraction cartridge and a molecular weight cut-off filter. Liquid chromatography/tandem mass spectrometry (LC/MS/MS) triple quadrupole electrospray methods were developed to monitor for the drugs in milk. Since established tolerance levels are set for most of these drugs in milk, the initial screening procedure was semi-quantitative, where samples were compared to the response of a positive control. The positive control, consisting of an extract from a portion of milk fortified with the drugs at half their allowed levels, was used to set the laboratorys minimum response criteria for unknown samples. Confirmatory analyses, with additional ion transitions for each residue, were performed on the same extracts.

Multiresidue method for the triphenylmethane dyes in fish: Malachite green, crystal (gentian) violet, and brilliant green

Liquid chromatographic methods are presented for the quantitative and confirmatory determination of crystal violet (CV; also known as gentian violet), leucocrystal violet (LCV), brilliant green (BG), and leucobrilliant green (LBG) in catfish. LCV and LBG were oxidized to the chromic CV and BG by reaction with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone, and residues were measured as the combined CV+/-LCV and BG+/-LBG. These methods are extensions of published methods for malachite green (MG) analysis to allow simultaneous determination of MG, CV, and BG. Residues were extracted from muscle with ammonium acetate buffer and acetonitrile, and extracts cleaned up using dichloromethane partitioning and solid-phase extraction. Extracts were analyzed by liquid chromatography with visible detection (LC-VIS). The method was validated for catfish fortified with LCV over the range 0.25-10 ngg(-1) and CV at 2 ngg(-1). Average recoveries were 90.6% (+/-8.1% R.S.D., n=45) for LCV and 84.4% (+/-4.2% R.S.D., n=6) for CV. The average recovery for samples fortified with BG or LBG over the range 0.5-10 ngg(-1) was 67.2% (+/-14.8% R.S.D., n=31). CV and BG were confirmed in fish extracts by ion trap LC-mass spectrometry (LC-MS(n)) with no discharge-atmospheric pressure chemical ionization. Average LC-MS(n) recoveries were 96.5, 96.6, and 70.2% for samples fortified with CV, LCV, and BG or LBG. The limits of detection for CV, BG, and MG were in the range of 0.07-0.24 ngg(-1) (ppb) for the two different instrumental methods. This methodology was applied to the analysis of catfish treated with CV and BG.

Analysis of veterinary drugs and metabolites in milk using quadrupole time-of-flight liquid chromatography-mass spectrometry.

A quadrupole time-of-flight (Q-TOF) liquid chromatography-mass spectrometry (LC-MS) method was developed to analyze veterinary drug residues in milk. Milk samples were extracted with acetonitrile. A molecular weight cutoff filter was the only cleanup step in the procedure. Initially, a set of target compounds (including representative sulfonamides, tetracyclines, β-lactams, and macrolides) was used for validation. Screening of residues was accomplished by collecting TOF (MS(1)) data and comparing the accurate mass and retention times of found compounds to a database containing information for veterinary drugs. The residues included in the study could be detected in samples fortified at the levels of concern with this procedure 97% of the time. Although the method was intended to be qualitative, an evaluation of the MS data indicated a linear response and acceptable recoveries for a majority of target compounds. In addition, MS/MS data were also generated for the [M + H](+) ions. Product ions for each compound were identified, and their mass accuracy was compared to theoretical values. Finally, incurred milk samples from cows dosed with veterinary drugs, including sulfamethazine, flunixin, cephapirin, or enrofloxacin, were analyzed with Q-TOF LC-MS. In addition to monitoring for the parent residues, several metabolites were detected in these samples by TOF. Proposed identification of these residues could be made by evaluating the MS and MS/MS data. For example, several plausible metabolites of enrofloxacin, some not previously observed in milk, are reported in this study.

Analysis of aminoglycoside residues in bovine milk by liquid chromatography electrospray ion trap mass spectrometry after derivatization with phenyl isocyanate

A derivatization procedure using phenyl isocyanate was adapted to liquid chromatography ion trap mass spectrometry (LC-MS(n)) for confirmation and quantification of aminoglycoside residues in milk. Aminoglycoside residues were extracted from milk with acid and isolated from the matrix with a weak cation exchange solid-phase extraction cartridge. After isolating the compounds from the milk, derivatives of gentamicin, neomycin, and tobramycin were formed by reacting the drugs with phenyl isocyanate in the presence of triethylamine. The analytes were separated using a dilute formic acid/acetonitrile gradient on a reversed-phase LC column. The derivatized compounds were analyzed using positive ion electrospray LC-MS(n) with ion trap detection. Product ion spectra were generated from the derivatized protonated molecules. Specific ion transitions were evaluated for quantitative determination and qualitative confirmation of residues in milk. Using this procedure, residues were qualitatively confirmed in milk samples fortified with gentamicin and neomycin at levels ranging from 15 to 300 ng mL(-1). Gentamicin has four major components that were successfully separated and confirmed independently; for quantitative determination the peak areas from the four analogs were summed. Tobramycin was added as an internal standard for quantitation to mitigate the effects of matrix ion suppression and variable recoveries. Overall recoveries for this method ranged from 80% to 120% with relative standard deviations of less than 25%. The method detection limits are 9.8 ng mL(-1) for NEO and 12.8 ng mL(-1) for total GEN residues.

Spark-induced breakdown spectroscopy and multivariate analysis applied to the measurement of total carbon in soil

Identifying and implementing techniques for carbon management has become an important endeavor in the mitigation of global climate change. Two important techniques being pursued are geologic and terrestrial carbon sequestration. With regard to terrestrial sequestration, in order to accurately monitor changes in soil carbon potentially induced by sequestration practices, rapid, cost-effective, and accurate measurements must be developed. Spark-induced breakdown spectroscopy (SIBS) has the potential to be used as a field-deployable method to monitor changes in the concentration of carbon in soil. SIBS spectra in the 248 nm region of eight soils were collected, and the neutral carbon line at 247.85 nm was compared to total carbon concentration determined by standard dry combustion techniques. Additionally, Fe and Si emission lines were evaluated in a multivariate statistical model to evaluate their impacts on the models predictive power for total carbon concentrations. The preliminary results indicate that SIBS is a viable method to quantify total carbon levels in soils, obtaining a correlation of (R(2)=0.972) between measured and predicated carbon in soils. These results show that multivariate analysis can be used to construct a calibration model for SIBS soil spectra.

Determination of oxytocin in a dilute IV solution by LC–MSn

The most common drug prescribed to induce labor in the United States is oxytocin, a peptide hormone composed of nine amino acids. Oxytocin is often reconstituted in intravenous (IV) saline solutions at less than 0.05 units ml(-1) (125 ng ml(-1)) to be delivered at 1-4 drops per minute. Existing LC-UV methods for oxytocin do not have sufficient detection limits to quantitate and/or confirm oxytocin in IV solutions without sample concentration. A determinative and confirmatory method for oxytocin was developed using an LC-MS(n) ion trap instrument with an electrospray ionization (ESI) interface in positive ion mode. Separation was achieved on a C-18 column using an isocratic elution of water with 50% acetonitrile (v/v) and water with 0.05% formic acid (v/v) at a flow rate of 250 microl min(-1). Data was acquired from the selected ion monitoring (SIM) of the precursor ion (m/z 1007.3) and MS(2) scans from the collision induced dissociation of m/z 1007.3 at 30% collision energy. In this method, MS(2) full scans were utilized to obtain three structurally significant ions for the unambiguous identification of oxytocin. Calibration standards, prepared in de-ionized water from 0.006 to 0.046 units ml(-1), were linear with an R(2) value of 0.9983. The methods LOD and LOQ were 0.00084 and 0.0029 units ml(-1) (2 and 7 ng ml(-1)), respectively. This LC-MS(n) method was used to determine the amount of oxytocin in a 0.04 units ml(-1) clinical sample that was prepared in 0.9% sodium chloride IV solution.

Chemical basis for the disparate neuroprotective effects of the anthocyanins, callistephin and kuromanin, against nitrosative stress

ABSTRACT Oxidative and nitrosative stress are major factors in neuronal cell death underlying neurodegenerative disease. Thus, supplementation of antioxidant defenses may be an effective therapeutic strategy for diseases such as amyotrophic lateral sclerosis, Parkinsons disease, and Alzheimers disease. In this regard, treatment with nutraceutical antioxidants has garnered increasing attention; however, the differential neuroprotective effects of structurally similar nutraceuticals, which may affect their suitability as therapeutic agents, has not been directly examined. In this study we compare the ability of two anthocyanins, callistephin (pelargonidin‐3‐O‐glucoside) and kuromanin (cyanidin‐3‐O‐glucoside) to protect cerebellar granule neurons from damage induced by either oxidative or nitrosative stress. These anthocyanins differ by the presence of a single hydroxyl group on the B‐ring of kuromanin, forming a catechol moiety. While both compounds protected neurons from oxidative stress induced by glutamate excitotoxicity, a stark contrast was observed under conditions of nitrosative stress. Only kuromanin displayed the capacity to defend neurons from nitric oxide (NO)‐induced apoptosis. This protective effect was blocked by addition of Cu, Zn‐superoxide dismutase, indicating that the neuroprotective mechanism is superoxide dependent. Based on these observations, we suggest a unique mechanism by which slight structural variances, specifically the absence or presence of a catechol moiety, lend kuromanin the unique ability to generate superoxide, which acts as a scavenger of NO. These findings indicate that kuromanin and compounds that share similar chemical characteristics may be more effective therapeutic agents for treating neurodegenerative diseases than callistephin and related (non‐catechol) compounds. HighlightsAnthocyanins are polyphenolic compounds with neuroprotective properties.Distinct anthocyanins display opposing neuroprotective effects against nitric oxide.Protection from nitric oxide relies on catechol‐dependent superoxide generation.Catecholicanthocyanins may be effective therapeutic agents for neurodegeneration.

Bioaccumulation of cyanuric acid in edible tissues of shrimp following experimental feeding

Due to concerns that cyanuric acid (CYA)-contaminated feed had been used in aquaculture and could enter the human food chain, a method to quantify CYA residues in the edible tissues of fish and shrimp was previously developed and validated. This paper provides further data on the deliberate feeding of CYA to shrimp to determine the extent of residue accumulation in edible tissue. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) was employed for the analysis of CYA in shrimp tissue. Edible tissue of shrimp fed 1666 or 3333 mg kg−1 CYA in their diet (approximately 55 and 124 mg kg−1 body weight) contained 0.767 and 0.406 mg kg−1 CYA, respectively. The residue levels are below the World Health Organization (WHO) tolerable daily intake level for CYA and are generally considered unlikely to pose a human health risk.

An anthocyanin-enriched extract from strawberries delays disease onset and extends survival in the hSOD1G93A mouse model of amyotrophic lateral sclerosis

Objective: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease resulting from the death of motor neurons in the brain, brain stem, and spinal cord. Several processes such as oxidative stress, neuroinflammation, and neuronal apoptosis, contribute to disease progression. Anthocyanins are flavonoid compounds derived from fruits and vegetables that possess antioxidant, anti-inflammatory, and anti-apoptotic abilities. Thus, these unique compounds may provide therapeutic benefit for the treatment of ALS. Methods: We used the G93A mutant human SOD1 (hSOD1G93A) mouse model of ALS to assess the effects of an anthocyanin-enriched extract from strawberries (SAE) on disease onset and progression. Mice were administered SAE orally beginning at 60 days of age until end-stage such that mice received 2 mg/kg/day of the extracts primary anthocyanin constituent. Clinical indices of disease were assessed until mice were sacrificed at end-stage. Histopathological indices of disease progression were also evaluated at 105 days of age. Results: hSOD1G93A mice supplemented with SAE experienced a marked (∼17 day) delay in disease onset and a statistically significant (∼11 day) extension in survival in comparison to their untreated mutant counterparts. Additionally, SAE-treated hSOD1G93A mice displayed significantly preserved grip strength throughout disease progression. Histopathological analysis demonstrated that SAE supplementation significantly reduced astrogliosis in spinal cord, and preserved neuromuscular junctions (NMJs) in gastrocnemius muscle. Discussion: These data are the first to demonstrate that anthocyanins have significant potential as therapeutic agents in a preclinical model of ALS due to their ability to reduce astrogliosis in spinal cord and preserve NMJ integrity and muscle function. Therefore, further study of these compounds is warranted in additional preclinical models of ALS and other neurodegenerative diseases.

NOVEL METHOD FOR TRANSMISSION INFRARED ANALYSIS OF CLAY MINERALS USING SILICON WAFER SUBSTRATES

A novel method for the analysis of clay minerals using Fourier transform infrared spectroscopy is presented. Clay mineral suspensions are dried on a Si wafer substrate for transmission infrared (IR) analysis. Four natural Source Clays from the Source Clays Repository of The Clay Minerals Society, SWy-2, SAz-1, SHCa-1 and KGa-1b, as well as the synthetic hectorite, Laponite RD, were analyzed using the described method with signal to noise (s/n) ratios in excess of 100,000 for the strongly absorbing Si-O stretching frequency. Scanning electron microscopy (SEM) images show that the mineral films possess suitable uniformity and low surface roughness for transmission IR measurements that is confirmed by minimal deviations in the baseline of collected IR spectra. The IR spectra are generated and peak locations are compared to previously reported values, generated from KBr pellet and attenuated total reflectance methods.