Drugs In Animal Tissues Research Articles

Simultaneous determination of fourteen β2-agonist enantiomers in food animal muscles by liquid chromatography coupled with tandem mass spectrometry

Illegal drug residues in animal derived foods are closely related to human's life and health. Studies on illegal drug residues and the metabolism, such as β2-agonists in animals have attracted more and more attention. In most cases, β2-agonists are suppliedand used astheracemate. The metabolic process and distribution of the two enantiomers in animal tissues are different. Therefore, it is very necessary to develop a simple and fast method for chiral resolution of these drugs in animal tissues. In this paper, a reliable resolution and determination method was presented using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for fourteen enantiomers of seven β2-agonist racemates, clenbuterol (CLE), salbutamol (SAL), cimaterol (CIM), terbutaline (TER), clorprenaline (CLO), tulobuterol (TUL), penbuterol (PEN) in pork, beef, and lamb muscle samples. The samples were added the internal standard solution (IS) and extracted in the alkaline medium with acetonitrile. The further sample purification was accomplished through MCX solid phase extraction cartridge. Chromatographic chiral separation was carried out on a VancoShell chiral column (100 mm × 4.6 mm, 2.7 μm) with an isocratic mobile phase consisting of methanol and 10 mmol mL−1 ammonium formate aqueous solution (85:15, v/v). Under the optimized conditions, the resolution (R) of CIM was 2.0, CLE and PEN were 1.5, the others were all greater than 1.0. Enantiomeric determination was performed in the positive electrospray ionization mode using multiple reaction monitoring (MRM). The correlation coefficient (r) in the range of 0.2–25.0 μg L−1 was above 0.993. The average recoveries at the three spiking levels ranged from 95.3% to 117.7% with the relative standard deviation (RSD) lower than 15%. The limit of detection (LOD) and the limit of quantification (LOQ) of β2-agonist enantiomers was 0.2 μg kg−1 and 0.5 μg kg−1 respectively. The method was successfully applied in the analysis and evaluation of β2-agonist enantiomers in positive food animal muscle samples, CLE, SAL, TEB and CIM enantiomers were detected. The concentrations of the corresponding enantiomers were in the range of 1.06–17.3 μg kg−1, the lowest enantiomer fraction (EF) value was 0.42, and the highest value was 0.69. The work is expected to provide a method for chiral separation and enantiomeric determination of the further study of pharmacology, toxicity and residue elimination of β2-agonist enantiomers.

Molecular imaging of small molecule drugs in animal tissues using laser desorption postionization mass spectrometry.

Localization and quantification of the target drug in tissues is a key indicator of efficacy in drug discovery. In contrast to established methods that require matrices and complex sample pretreatment steps, matrix-free and low cost in situ analysis of small molecule drugs by mass spectrometry (MS) remains challenging. Here, we present a novel approach, laser desorption postionization (LDPI), which is coupled to a linear time-of-flight (TOF) MS and used to image the distribution of acriflavine (ACF) directly from a histological section of mouse kidney without any matrix or sample pretreatment. The identification of the mass peaks assigned to ACF was further confirmed by DESI-MS/MS. Moreover, the matrix effect from the tissue section was explored, showing minimal desorption and ionization suppression in the LDPI-MS process. LDPI-MS imaging (LDPI-MSI) was performed on 30 μm kidney sections from mice 15 min postdose that were dosed with 30 mg kg-1 of ACF by monitoring the fragment ion at m/z 209. The LDPI-MS image revealed a global view of the distribution of ACF in the kidney compartments (pelvis, medulla, and cortex). Estimated concentrations of ACF residue in mouse kidney were obtained by LDPI-MSI and LC-MS/MS and a 12.1% difference in measured tissue concentration was found. These results suggest that the use of LDPI-MS in small molecule drug localization and quantification directly from biological tissue at the same time is favorable.

Postmortem Attraction of Sarcosaprophagous Diptera to Tramadol-Treated Rats and Morphometric Aspects of the Developed Larvae.

The presence of some specific drugs in animal tissues may affect the time of minimal postmortem intervals estimated during forensic entomological investigations. To test the effects of a specific drug on decomposition, a field study was conducted at Fayoum University campus, Egypt, from March to May 2013, using tramadol, a synthetic analgesic opioid used to treat moderate to severe pain in humans. Albino rats were used as the animal model during this study. The duration of the fresh stage of tramadol treated rat (Ttr) carcasses was significantly shorter (2.4 ± 0.27days) compared to tramadol free rat (Tfr) carcasses (6.4 ± 0.49days). The dry carcass stage of Ttr lasted longer (10.3 ± 0.99days) as compared to (7.4 ± 0.18days) the Tfr carcass. The decomposition process of the (Ttr) carcass was not significantly faster (24.9 ± 1.58days) as compared to (Tfr) carcasses (29.5 ± 1.69). Lucilia cuprina (Wiedemann), Chrysomya albiceps (Wiedemann), and Musca domestica L. were less attracted to Ttr carcass-baited traps than traps with Tfr carcasses. However, females of Sarcophaga spp. showed a greater attraction to Ttr carcasses. Females of another sarcophagid fly, Wohlfahrtia spp. exhibited similar attraction tendencies to both types of trap baits. Larvae of S. argyrostoma (Robineau-Desvoidy) collected from Ttr carcasses developed to a significantly longer total body length (10.4 ± 0.04mm) as compared to the average length of the larvae collected from Tfr carcasses (8.9 ± 0.34mm). During days 9-13 after rat death, the relative lengths of larvae from Ttr carcasses were not significantly different from Tfr carcasses. Larvae fed on Ttr carcasses pupated 2days later than the control larvae.

Localization and Quantification of Drugs in Animal Tissues by Use of Desorption Electrospray Ionization Mass Spectrometry Imaging

Mass spectrometric imaging (MSI) has emerged as a powerful technique to obtain spatial arrangement of individual molecular ions in animal tissues. Ambient desorption electrospray ionization (DESI) technique is uniquely suited for such imaging experiments, as it can be performed on animal tissues in their native environment without prior treatments. Although MSI has become a rapid growing technique for localization of proteins, lipids, drugs, and endogenous compounds in different tissues, quantification of imaged targets has not been explored extensively. Here we present a novel MSI approach for localization and quantification of drugs in animal thin tissue sections. DESI-MSI using an Orbitrap mass analyzer in full scan mode was performed on 6 μm coronal brain sections from rats that were administered 2.5 mg/kg clozapine. Clozapine was localized and quantified in individual brain sections 45 min postdose. External calibration curves were prepared by micropipetting standards with internal standard (IS) on top of the tissues, and average response factors were calculated for the scans in which both clozapine and IS were detected. All response factors were normalized to area units. Quantifications from DESI-MSI revealed 0.2-1.2 ng of clozapine in individual brain sections, results that were further confirmed by extraction and liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis.

Effect of biological matrix and sample preparation on qPCR quantitation of siRNA drugs in animal tissues

Introduction Quantitative pharmacokinetic measurement of short nucleotide sequences in animal tissues is critical to the successful development of siRNA-based drugs. Stem-loop qRT-PCR is a sensitive and precise methodology, but the effect of biological matrix and purity of the input sample has yet to be investigated. Results The impact of lipid encapsulation, siRNA chemical modification and purity of the biological matrix on the stem-loop qRT-PCR assay was investigated. A comparison of siRNA standard curves in mouse liver homogenates before and after isolation of total RNA uncovered the potential for erroneous measurement due to significant loss of siRNA on purification columns. Recovery of chemically stabilized siRNA was improved by omission of the DNAse I digestion during RNA isolation. The stem-loop qRT-PCR method demonstrated excellent sensitivity and efficiency in mouse liver homogenates, plasma and whole blood. An optimized protocol based on these findings was used to quantitate siRNA in tissues after dosing mice with two different lipid nanoparticle formulations containing siRNA payloads. Conclusions Assay of crude homogenates, whole blood or plasma is more accurate, less resource intensive and more amenable to clinical translation than measurement of column-purified total RNA.

Simultaneous detection of residues of β-adrenergic receptor blockers and sedatives in animal tissues by high-performance liquid chromatography/tandem mass spectrometry

A method using high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC–MS/MS) was developed to screen and confirm residues of multi-class veterinary drugs in animal tissues (porcine kidney, liver, muscle; bovine muscle). Thirty target drugs (19 β-blockers, 11 sedatives) were determined simultaneously in a single run. Homogenized tissue samples were extracted with acetonitrile and purified using a NH 2 solid-phase extraction cartridge. An Acquity UPLC™ BEH C18 column was used to separate the analytes, followed by tandem mass spectrometry using an electrospray ionization source in positive mode. Recovery studies were done at three fortification levels. Overall average recoveries in pig muscle, kidney, and liver fortified at three levels from 76.4% to 118.6% based on matrix-fortified calibration with coefficients of variation from 2.2% to 19.9% ( n = 6). The limit of quantification of these compounds in different matrices was 0.5–2.0 μg/kg. This method was successfully applied in screening and confirming target drugs in >200 samples.

A novel approach for simultaneous determination of 2‐mercaptobenzimidazole and derivatives of 2‐thiouracil in animal tissue by gas chromatography/mass spectrometry

A novel approach for determination of 2-mercaptobenzimidazole (MBI) and other thyreostatic residues in animal tissues by gas chromatography/mass spectrometry (GC/MS) in selected ion monitoring mode was developed. The analytes in animal tissues (including hypothyroid, pork muscle and beef samples) were extracted by acetonitrile, and then purified by a matrix solid-phase dispersion (MSPD) procedure after the extraction residues had been dissolved in water. The thyreostatic residues were derivatized by pentafluorobenzyl bromide (PFBBr) under strong basic conditions and then N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) before GC/MS analysis. Different kinds of solid supports with various polarities for the MSPD procedure were investigated, and it was found that silica gel was suitable for the purpose. The average recoveries of the thyreostatic drugs in animal tissues ranged from 71.5-96.9% with the relative standard deviations below 10%. By using the developed method, the limits of detection were 10 microg/kg for MBI; 5 microg/kg for 6-phenyl-2-thiouracil; and 2 microg/kg for 2-thiouracil, 6-methyl-2-thiouracil and 6-propyl-2-thiouracil. The stability of the thyreostatic drugs in spiked animal tissues was tested, and the results showed that the thyreostatic drugs did not decompose within 3 months if the sample was stored in darkness below -20 degrees C.

Detection, accumulation and distribution of nitrofuran residues in egg yolk, albumen and shell

Nitrofuran antibiotics have been banned for use in food-producing animals in many countries, including the European Union, owing to the threat they pose to human health. Research continues into the accumulation of these drugs in animal tissues and into the appropriate methods for their detection. In this study, an LC–MS/MS method is presented for the detection of the parent compounds, furazolidone, nitrofurantoin, furaltadone and nitrofurazone, in eggs. The parent compounds are first extracted into ethyl acetate, fats are removed by partition between acetonitrile and hexane, and the concentrated sample is analysed by LC–MS/MS. Decision limits (CCα) for the parents were ≤1 µg kg−1 for all four compounds. Within-day and between-day CVs are well within the limits stated in Commission Decision 2002/657/EC. The method provides an alternative to the testing of side-chain metabolites in eggs, which is particularly important in the case of nitrofurazone, where semicarbazide contamination of food has been attributed to sources other than nitrofurazone use. This method was used together with a method for the detection of the side-chain metabolite compounds, 3-amino-2-oxazolidinone (AOZ), 3-amino-5-morpholinomethyl-1,3-oxazolidin-2-one (AMOZ), 1-amino-hydantoin (AHD) and semicarbazide (SEM), to study the accumulation and distribution of nitrofurans in eggs. Eggs were collected from four groups of hens that had been treated with one of the nitrofurans at a feed concentration of 300 mg kg−1 for 1 week. Parent compounds and metabolites were found in the yolk, albumen and shell. Albumen/yolk ratios for the parent compounds were 0.7, 0.82, 0.83 and 0.31 for furazolidone, furaltadone, nitrofurantoin and nitrofurazone, respectively. Ratios for the side-chain metabolites were 1.02, 1.06, 0.83 and 0.55 for AOZ, AMOZ, AHD and SEM, respectively. However, 50% of the total SEM residues were found in eggshell. This may be significant if eggshell products reach the consumer.

Detection of unwanted residues of ivermectin in bovine milk by dissociation-enhanced lanthanide fluoroimmunoassay.

Avermectins are frequently used to control parasitic infestations in many animal species. Previous studies have shown the long-term persistence of unwanted residues of these drugs in animal tissues and fluids. An immunoassay screening test for the detection and quantification of ivermectin residues in bovine milk has been developed. After an extensive extraction procedure, milk samples were applied to a competitive dissociation-enhanced lanthanide fluoroimmunoassay using a monoclonal antibody against an ivermectin-transferrin conjugate. The monoclonal antibody, raised in Balb C mice, showed cross-reactivity with eprinomectin (92%), abamectin (82%) and doramectin (16%). The limit of detection of the assay (mean + 3 SD), calculated from the analysis of 17 known negative samples, was calculated as 4.6 ng/mL. Intra- and inter-assay RSDs were determined as 11.6% and 15.8%, respectively, using a negative bovine milk sample fortified with 25 ng/mL ivermectin. Six Friesian milking cows were treated with ivermectin, three with a pour-on formulation of the drug and three with an injectable solution at the manufacturer's recommended dose rate. An initial mean peak in ivermectin residue concentration was detected at day 4 (mean level = 47.5 ng/mL) and day 5 post-treatment (mean level = 26.4 ng/mL) with the injectable form and pour-on treatment, respectively. A second peak in residue concentration was observed using the DELFIA procedure 28 days post-treatment in both treatment groups (23.1 ng/mL injectable and 51.9 ng/mL pour-on). These second peaks were not confirmed by HPLC and must at this time be considered to be false-positive results. By day 35 after treatment the mean ivermectin residue concentration of both groups fell below the limit of detection of the assay.

Development of multi-residue methodology for the HPLC determination of veterinary drugs in animal tissues.

The application of solid-phase extraction and HPLC with UV-diode array detection to the multi-residue determination of veterinary drugs is described. A two-stage SPE clean-up was employed, using C18 and silica cartridges. HPLC analysis was carried out on a base-deactivated C8 column using gradient elution systems at two pH values. The procedure establishes the basis of a method for routine screening of pig kidney samples for some sulphonamides, benzimidazoles, nitroimidazoles and nitrofurans at concentrations at or below the UK Maximum Residue Limits (MRLs). Limits of detection of 2-18 micrograms/kg could be achieved for these analytes at recoveries of 40-70%. UV spectra measured on-line were used for confirmation of peak identities at these concentrations. The possibility of extension of this procedure to a wider range of analyses is discussed.

Methodology for the Analysis of Benzimidazole Anthelmintics as Drug Residues in Animal Tissues

Methodology for the analysis of 8 benzimidazoles as residues in bovine liver is reported. Spiked tissues were extracted by homogenization in saline and ammonium hydroxide and blending with diatomaceous earth. This matrix was packed into a column, and the benzimidazoles were eluted with ethyl acetate. After the sample was further purified, benzimidazoles were separated and quantitated by liquid chromatography with ultraviolet detection (290 nm). Liver tissue samples obtained from cattle which had undergone a drug depletion study of fenbendazole administered per os were analyzed using these methods. The results of these analyses and the application of this approach to multiresidue analysis of drugs in animal tissues are discussed.

Methods Evaluation in the Chemical Analysis of Drugs in Animal Tissues

Methods Evaluation in the Chemical Analysis of Drugs in Animal Tissues

Acetylation and deacetylation of the p-amino group of sulphonamide drugs in animal tissues

Acetylation and deacetylation of the <i>p</i>-amino group of sulphonamide drugs in animal tissues