Determination of flunixin residues in bovine muscle tissue by liquid chromatography with UV detection.

A new and sensitive liquid chromatography-ultra violet method with a detection limit of 6 ng/g (ppb) and a limit of quantification of 15 ng/g was developed for the determination of flunixin residues in bovine muscle tissue. Flunixin in homogenized animal tissue was extracted with acetonitrile after enzyme digestion. The tissue digest (extract) was then cleaned up on a solid-phase extraction cartridge and eluted with acidified hexane. After the eluate was evaporated to dryness under nitrogen at 55 degrees C, the residue was reconstituted in 1 mL mobile phase solution and analyzed by reversed-phase gradient chromatography with UV detection at 285 nm. The method was then applied in a survey study of slaughter animals to determine whether flunixin is being used in an off-label manner for veal and beef production in Canada.     

The detection of modafinil and its major metabolite in equine urine by liquid chromatography/mass spectrometry

A method has been developed for the detection of modafinil and its major metabolite, modafinil acid, in equine urine by solid-phase extraction and positive ion electrospray ionisation liquid chromatography/mass spectrometry. The method has been applied to the analysis of equine urine samples obtained after the oral administration of modafinil. Modafinil acid was the major component in the urine, and was detected up to 4 days post-administration. Unchanged modafinil was present at substantially lower concentrations, and was detected for only 24 hours.     

Simultaneous Determination of Flunixin,Phenylbutazone, Oxyphenbutazone and γ-Hydroxyphenylbutazone in Equine Plasma by High-Performance Liquid Chromatography: With Application to Pharmacokinetics

Abstract

A high performance liquid chromatographic method was developed for the simultaneous determination of flunixin, phenylbutazone, oxyphenbutazone and γ-hydroxyphenylbutazone in equine plasma. Samples of plasma or sera were deproteinated by addition of acetonitrile containing the internal standard naproxen. The concentration step consisted of taking an aliquot of deproteinated plasma, evaporating under nitrogen to dryness and redissolving in mobile phase. The extracts were chromatographed on a Spherisorb 5 μm ODS column using an isocratic mobile phase of methanol (30% v/v), acetonitrile (20% v/v) and pH 3.0 1% acetate buffer (50% v/v) at a flow rate of 1.2 ml/min using naproxen as the internal standard. The detection limit for flunixin, phenylbutazone, oxyphenbutazone and γ-hydroxyphenylbutazone was 50 ng/ml.

The developed chromatographic method was applied to the determination of equine nonsteroidal anti-inflammatory treatment. Plasma samples from clinically treated horses administered flunixin and phenylbutazone simultaneously are reported. Effect of different anticoagulants used in sampling is reported.     

A broad-spectrum equine urine screening method for free and enzyme-hydrolysed conjugated drugs with ultra performance liquid chromatography/tandem mass spectrometry

The authors' laboratory at one time employed four liquid chromatography/mass spectrometric (LC/MS) methods for the detection of a large variety of drugs in equine urine. Drug classes covered by these methods included anti-diabetics, anti-ulcers, cyclooxygenase-2 (COX-2) inhibitors, sedatives, corticosteroids, anabolic steroids, sulfur diuretics, xanthines, etc. With the objective to reduce labour and instrumental workload, a new ultra performance liquid chromatography/tandem mass spectrometric (UPLC/MS/MS) method has been developed, which encompasses all target analytes detected by the original four LC/MS methods. The new method has better detection limits than the superseded methods. In addition, it covers new target analytes that could not be adequately detected by the four LC/MS methods. The new method involves solid-phase extraction (SPE) of two aliquots of equine urine using two Abs Elut Nexus cartridges. One aliquot of the urine sample is treated with β-glucuronidase before subjecting to SPE. A second aliquot of the same urine sample is processed directly using another SPE cartridge, so that drugs that are prone to decomposition during enzyme hydrolysis can be preserved. The combined eluate is analysed by UPLC/MS/MS using alternating positive and negative electrospray ionisation in the selected-reaction-monitoring mode. Exceptional chromatographic separation is achieved using an UPLC system equipped with a UPLC(?) BEH C18 column (10 cm L×2.1 mm ID with 1.7 μm particles). With this newly developed UPLC/MS/MS method, the simultaneous detection of 140 drugs at ppb to sub-ppb levels in equine urine can be achieved in less than 13 min inclusive of post-run equilibration. Matrix interference for the selected transitions at the expected retention times is minimised by the excellent UPLC chromatographic separation. The method has been validated for recovery and precision, and is being used regularly in the authors' laboratory as an important component of the array of screening methods for doping control analyses of equine urine samples.     

Column-switching techniques for high-performance liquid chromatography of ibuprofen and mefenamic acid in human serum with short-wavelength ultraviolet detection.

Column-switching techniques for high-performance liquid chromatography of two acidic drugs, ibuprofen and mefenamic acid, in human serum with short-wavelength ultraviolet detection are described. The method involved extraction of the analyte from acidified serum followed by the chromatographic analysis using column switching. Three ODS columns were used each with different mobile phase, utilizing the difference of ion-pair formation or of ionization caused by pH change. The method offered high sensitivity and selectivity, with short-wavelength ultraviolet detection at 221 nm for ibuprofen and at 219 nm for mefenamic acid. The detection limits were 0.5 ng/ml (2.4 pmol/ml) for ibuprofen and 0.1 ng/ml (0.4 pmol/ml) for mefenamic acid using 1 ml of serum, both at a signal-to-noise ratio of 3. With some modifications, the principle of the method would be applicable to other acidic compounds in biological fluids.     

Determination of non-steroidal anti-inflammatory drugs and their metabolites in milk by liquid chromatography–tandem mass spectrometry

Non-steroidal anti-inflammatory drugs are widely used for treatment of animals. According to Council Directive 96/23/EC, residues of these drugs must be monitored because of the potential risk they pose to the consumers' health. For this reason an LC-MS-MS method was developed for detection of wide range of NSAIDs, including both "acidic" NSAIDs (carprofen, diclofenac, flunixin, meloxicam, phenylbutazone, oxyphenbutazone, tolfenamic acid, mefenamic acid, naproxen, ketoprofen, ibuprofen, firocoxib, rofecoxib, and celecoxib) and "basic" NSAIDs (four metamizole metabolites). Analytes were extracted from milk samples with acetonitrile in the presence of ammonium acetate. One portion of the extract was directly analyzed for the presence of metamizole metabolites; a second portion was cleaned with an amino cartridge. All NSAIDs were separated on a Phenomenex Luna C8(2) column and analyzed by LC-MS-MS in negative (acidic NSAIDs) and positive (metamizole metabolites) ion modes. The method was validated in accordance with the requirements of Commission Decision 2002/657/EC. Within-laboratory reproducibility was in the range 7-28%, and accuracy was in the range 71-116%. The method enabled detection of all the analytes with the expected sensitivity, below the recommended concentrations. The method fulfills the criteria for confirmatory methods and, because of its efficiency, may also be used for screening purposes. The procedure was also successfully verified in the proficiency test organized by EU-RL in 2010. As far as the authors are aware, this is one of the first methods capable of detecting diclofenac residues below the MRL in milk (0.1 μg kg(-1)). An additional advantage is the possibility of simultaneous determination of "acidic" NSAIDs and metamizole metabolites.     

Doping control analysis of insulin and its analogues in equine urine by liquid chromatography-tandem mass spectrometry

Insulin and its analogues have been banned in both human and equine sports owing to their potential for misuse. Insulin administration can increase muscle glycogen by utilising hyperinsulinaemic clamps prior to sports events or during the recovery phases, and increase muscle size by its chalonic action to inhibit protein breakdown. In order to control insulin abuse in equine sports, a method to effectively detect the use of insulins in horses is required. Besides the readily available human insulin and its synthetic analogues, structurally similar insulins from other species can also be used as doping agents. The author's laboratory has previously reported a method for the detection of bovine, porcine and human insulins, as well as the synthetic analogues Humalog (Lispro) and Novolog (Aspart) in equine plasma. This study describes a complementary method for the simultaneous detection of five exogenous insulins and their possible metabolites in equine urine. Insulins and their possible metabolites were isolated from equine urine by immunoaffinity purification, and analysed by nano liquid chromatography-tandem mass spectrometry (LC/MS/MS). Insulin and its analogues were detected and confirmed by comparing their retention times and major product ions. All five insulins (human insulin, Humalog, Novolog, bovine insulin and porcine insulin), which are exogenous in horse, could be detected and confirmed at 0.05ng/mL. This method was successfully applied to confirm the presence of human insulin in urine collected from horses up to 4h after having been administered a single low dose of recombinant human insulin (Humulin R, Eli Lilly). To our knowledge, this is the first identification of exogenous insulin in post-administration horse urine samples.     

Detection of Quaternary Ammonium Drugs in Equine Urine by Liquid Chromatography-Mass Spectrometry

Quaternary ammonium drugs are anticholinergic agents and some of which have been known to be abused in equine sports. A general screening method for this class of drugs in equine urine by liquid chromatography-mass spectrometry (LC-MS) has not been reported. The paper describes an efficient LC-MS-MS method for the simultaneous detection and confirmation of twenty quaternary ammonium drugs at low ng mL^?1 in equine urine after solid-phase extraction. Quaternary ammonium drugs were extracted from equine urine by solid-phase extraction using ISOLUTE^® CBA SPE Columns and analysed by LC-MS-MS in the positive electrospray ionisation mode. Separation of twenty quaternary ammonium drugs (the quaternary ammonium ions of edrophonium chloride, pyridostigmine bromide, neostigmine bromide, bretylium tosylate, ipratropium bromide, tubocurarine chloride, N-butylscopolammonium bromide, mepenzolate bromide, rocuronium bromide, clidinium bromide, pipenzolate bromide, isopropamide iodide, glycopyrronium bromide, alcuronium chloride, oxyphenonium bromide, propantheline bromide, tridihexethyl chloride, vecuronium bromide, mivacurium chloride and pancuronium bromide) was achieved in a reversed phase column with a mixture of aqueous ammonium formate (pH 3.0, 10 mM) and acetonitrile as the mobile phase. Detection and confirmation of the twenty quaternary ammonium drugs at about 5 ng mL^?1 in equine urine could be achieved within 22 min using product-ion scan MS-MS. The target analytes were detected by examination of extracted-ion chromatograms of their product ions. Drugs spiked in different equine urine (n = 15) were consistently detected. Negative samples (n = 30) of normal post-race equine urine have also been analysed, no matrix interference at the targeted masses and retention times was observed. The method was successfully applied to the analyses of drug-administration samples. Other method validation data including reproducibility and recovery will also be presented. An LC-MS-MS method for the simultaneous detection and confirmation of twenty quaternary ammonium drugs in equine urine was developed. The methodology should be applicable to other biological matrices such as human urine.     

Electromembrane‐surrounded solid‐phase microextraction coupled to ion mobility spectrometry for the determination of nonsteroidal anti‐inflammatory drugs: A rapid screening method in complicated matrices

A new robust method of electromembrane‐surrounded solid‐phase microextraction coupled to ion mobility mass spectrometry was applied for nonsteroidal anti‐inflammatory drugs determination in complex matrices. This is the first time that a graphene/polyaniline composite coating is applied in electromembrane‐surrounded solid‐phase microextraction method. The homemade graphene/polyaniline composite is characterized by a high electrical conductivity and thermal stability. The variables affecting electromembrane‐surrounded solid‐phase microextraction, including extraction time; applied voltage and pH were optimized through chemometric methods, central composite design, and response surface methodology. Under the optimized conditions, limits of detection of 0.04 and 0.05 ng/mL were obtained for mefenamic acid and ibuprofen, respectively. The feasibility of electromembrane‐surrounded solid‐phase microextraction followed by ion mobility mass spectrometry was successfully confirmed by the extraction and determination of low levels of ibuprofen and mefenamic acid in human urine and plasma samples and satisfactory results were obtained.     

Confirmatory identification of sixteen non-steroidal anti-inflammatory drug residues in raw milk by liquid chromatography coupled with ion trap mass spectrometry

The European Council Decision 2002/657/EC established that group B substances detected in foods must be identified and confirmed on the basis of their molecular structure. To this aim, we have developed a panel of methods for unambiguous determination of sixteen non-steroidal anti-inflammatory drugs (NSAIDs) in cattle and buffalo raw milk. A multi-residue reversed-phase high-performance liquid chromatography method with photodiode array detection is described for quantitative screening analysis. For confirmatory purposes, two multi-residue reversed-phase ion trap liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI-MS/MS) methods were developed: the former to identify salicylic acid, naproxen, carprofen, flurbiprofen, ibuprofen, meclofenamic acid, niflumic acid, flunixin and its metabolite 5-hydroxyflunixin in the negative ion mode; the latter to identify ketoprofen, suxibutazone, diclofenac, mefenamic acid, tolfenamic acid, phenylbutazone and its metabolite oxyphenbutazone in the positive ion mode. These drugs are representative of different subclasses of NSAIDs not chemically related. The methods were in-house validated, evaluating specificity and calculating the mean recoveries, repeatability, within-laboratory reproducibility, and limits of quantification. For all the NSAIDs, apart from salicylic acid and 5-hydroxyflunixin, mean recoveries ranging between 69.0% and 96.7% were measured. The qualitative identification of all drugs was attained by their MS/MS spectra in the concentration range studied. Similarly, at 5 microg/kg all NSAIDs, apart from flurbiprofen, were unambiguously confirmed.     

Development and validation of a hydrophilic interaction liquid chromatography with tandem mass spectrometry method for the simultaneous detection and quantification of etilefrine and oxilofrine in equine blood plasma and urine

A sensitive hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry method was developed and validated for the simultaneous detection and quantification of etilefrine and oxilofrine in equine blood plasma and urine. The method is highly sensitive and specific with good precision and accuracy. In plasma the limit of detection and limit of quantification are 0.03 and 0.1 ng/mL, respectively, for both analytes. In urine the limit of detection and limit of quantification are 0.3 and 1 ng/mL, respectively, for both analytes. The suitability of the method for doping control analysis in equine species is demonstrated by analyzing postadministration samples collected after a single intravenous administration of 50 mg etilefrine to a standardbred mare. Etilefrine was detected up to 120 h in urine and up to 48 h in plasma. Etilefrine is highly conjugated in equine urine whereas it exists in the free form in equine plasma. Therefore, enzyme hydrolysis prior to sample preparation is recommended for the detection and quantification of etilefrine and oxilofrine in equine urine.     

A Glassy Carbon Electrode Modified with Multiwalled Carbon Nanotube/Chitosan Composite as a New Sensor for Simultaneous Determination of Acetaminophen and Mefenamic Acid in Pharmaceutical Preparations and Biological Samples

A new chemically modified electrode is constructed based on multiwalled carbon nanotube/chitosan modified glassy carbon electrode (MWCNTs‐CHT/GCE) for simultaneous determination of acetaminophen (ACT) and mefenamic acid (MEF) in aqueous buffered media. The measurements were carried out by application of differential pulse voltammetry (DPV), cyclic voltammetry (CV) and chronoamperometry (CA) methods. Application of DPV method showed that the linear relationship between oxidation peak current and concentration of ACT and MEF were 1 μM to 145 μM, and 4 μM to 200 μM, respectively. The analytical performance of this sensor has been evaluated for detection of ACT and MEF in human serum, human urine and a pharmaceutical preparation with satisfactory results.     

Sensitive liquid chromatographic/tandem mass spectrometric method for the determination of beclomethasone dipropionate and its metabolites in equine plasma and urine

Beclomethasone dipropionate (BDP) is a potent pro-drug to beclomethasone (BOH) and is used in the treatment of chronic and acute respiratory disorders in the horse. The therapeutic dose of BDP (325 microg per horse) by inhalation results in very low plasma and urinary concentrations of BDP and its metabolites that pose a challenge to detection and confirmation by equine forensic laboratories. To solve this problem, a method involving the use of a liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) was developed for the detection, confirmation and quantification of the analytes in equine samples. Ammonium formate or acetate buffer added to LC mobile phase favored the formation of [M + H](+) ions from BDP and its metabolites, whereas formic acid led to the formation of sodium and potassium adduct ions ([M + Na](+), [M + K](+)) together with [M + H](+) ions. Acetonitrile, on the other hand, favored the formation of abundant solvent adduct ions [M + H + CH(3)CN](+) with the analytes under electrospray ionization (ESI) and atmospheric pressure chemical ionization conditions. In contrast, methanol formed much less solvent adduct ions than acetonitrile. The solvent adduct ions were thermally stable and could not be completely desolvated under the experimental conditions, but they were very fragile to collision-induced dissociation (CID). Interestingly, these solvent adduct ions were observed on a triple-quadrupole mass spectrometry but not on an ion trap instrument where helium used as a damping gas in the ion trap might cause the solvent adduct ions desolvated by collision. By CID studies on the [M + H](+) ions of BDP and its metabolites, their fragmentation paths were proposed. In equine plasma at ambient temperature over 2 h, BDP and B21P were hydrolyzed in part to B17P and BOH, respectively, but B17P was not hydrolyzed. Sodium fluoride added to equine plasma inhibited the hydrolysis of BDP and B21P. The matrix effect in ESI was evaluated in equine plasma and urine samples. The method involved the extraction of BDP and its metabolites from equine plasma and urine samples by methyl tert-butyl ether, resolution on a C(8) column with a mobile phase gradient consisting of methanol and ammonium formate (2 mmol l(-1), pH 3.4) and multiple reaction monitoring for the analytes on a triple-quadrupole mass spectrometer. The detection limit was 13 pg ml(-1) for BDP and B17P, 25 pg ml(-1) for BOH and 50 pg ml(-1) for B21P in plasma and 25 pg ml(-1) for BOH in urine. The method was successfully applied to the analysis of equine plasma and urine samples for the analytes following administration of BDP to horses by inhalation. B17P, the major and active metabolite of BDP, was detected and quantified in equine plasma up to 4 h post-administration by inhalation of a very low therapeutic dose (325 microg per horse) of BDP.     

Extraction-spectrophotometric determination of mefenamic acid in pharmaceutical preparations

The optimal conditions of the formation and extraction of mefenamic acid ion associates with the astrafloxin polymethine dye are studied. The extraction of ion associates with isooctane-dichloroethane mixtures attains a maximum at pH 9–11 and dye concentration of (5–7) × 10^?5 M. The Beer law is fulfilled in the range 2.0–21.0 μg/mL; the detection limit for mefenamic acid is 0.72 μg/mL. A method is developed for the extraction-spectrophotometric determination of mefenamic acid in pharmaceutical preparations.     

Spectrophotometric determination of mefenamic acid in pharmaceutical preparations

The present paper describes an effective and low-cost spectrophotometric method for the determination of mefenamic acid in its pure form and pharmaceutical preparations. The method is based on the charge-transfer complexation between mefenamic acid as an n-electron donor and chloranil as a π-acceptor to form a violet chromogen measured at 540 nm. Under the optimum conditions, a linear relationship with a good correlation coefficient (0.9996) was found between the absorbance and concentration of the studied drug in the range of 10–60 μg/mL. The optimal reaction conditions such as reagent concentration, heating time, and stability of the reaction product were determined. The limit of detection (LOD) was 2.16 μg/mL and the limit of quantifycation (LOQ) was 7.15 μg/mL. The method was successfully applied to the determination of mefenamic acid in pharmaceutical preparations without any interference from common excipients. The text was submitted by the author in English.     

Development and validation of a confirmatory method for the determination of 12 non steroidal anti-inflammatory drugs in milk using liquid chromatography-tandem mass spectrometry

A rapid and reliable LC-MS/MS method for the simultaneous confirmation of twelve non steroidal anti-inflammatory drugs (NSAIDs) in bovine milk was developed and fully validated in accordance with the European Commission Decision 2002/657/EC. The validation scheme was built in accordance with the MRLs or target analytical levels (EU-CRL recommended concentrations and detection capabilities) of the analytes, except for diclofenac for which the lower level of validation achieved was 0.5 μg kg(-1) whereas its MRL is 0.1 μg kg(-1). The NSAIDs investigated were as follows: phenylbutazone (PBZ), oxyphenylbutazone (OPB), naproxen (NP), mefenamic acid (MF), vedaprofen (VDP), flunixin (FLU), 5-hydroxyflunixin (FLU-OH), tolfenamic acid (TLF), meloxicam (MLX), diclofenac (DC), carprofen (CPF) and ketoprofen (KTP). Several extraction procedures had been investigated during the development phase. Finally, the best results were obtained with a procedure using only methanol as the extraction solvent, with an evaporation step included and no further purification. Chromatographic separation was achieved on a C18 analytical column and the run was split in 2 segments. Matrix effects were also investigated. Data acquisition implemented for the confirmatory purpose was performed by monitoring 2 MRM transitions per analyte under the negative electrospray mode. Mean relative recoveries ranged from 94.7% to 110.0%, with their coefficients of variation lying between 2.9% and 14.7%. Analytical limits expressed in terms of decision limits (CCα) were evaluated between 0.69 μg kg(-1) (FLU) and 27.54 μg kg(-1) (VDP) for non-MRL compounds, and at 0.10 (DC), 15.37 (MLX), 45.08 (FLU-OH), and 62.96 μg kg(-1) (TLF) for MRL compounds. The validation results proved that the method is suitable for the screening and confirmatory steps as implemented for the French monitoring plan for NSAID residue control in bovine milk.     

Simple,rapid and sensitive method for the determination of mefenamic acid in pharmaceutical preparations

Two simple and fast automated methods for the direct determination of mefenamic acid (MEF) in pharmaceutical samples are described. Continuous flow and stopped-flow systems with spectrophotometric detection of mefenamic acid with sodium nitroprusside and hydroxylamine hydrochloride were developed. Both methods show a good reproducibility (RSD < 1.5 and 1.8%, respectively) and a wide range of linearity (between 10–500 and 3–300 μg/mL). The stopped-flow protocol has a lower detection limit (1.2 μg/mL) with a sensitivity of about two times greater than the continuous flow technique. The proposed procedures are rapid, reliable and can be applied successfully to the estimation of mefenamic acid in different commercial forms.     

Analyses of quaternary ammonium drugs in horse urine by capillary electrophoresis-mass spectrometry

A capillary electrophoresis-mass spectrometry (CE-MS) method for the analysis of quaternary ammonium drugs in equine urine was developed. Quaternary ammonium drugs were first extracted from equine urine by ion-pair extraction and then analysed by CE-MS in the positive electrospray ionization (ESI) mode. Within 12 min, eight quaternary ammonium drugs, each at 1 ng/mL in horse urine, could be detected. The confirmation of these drugs in urine samples was achieved by capillary electrophoresis tandem mass spectrometry (CE-MS/MS). A direct comparison of this method was made with existing liquid chromatography/mass spectrometry (LC-MS) methods in the detection and confirmation of glycopyrrolate and ipratropium bromide in horse urine. While the two drugs could be detected within the same CE-MS run at 1 ng/mL in urine, they could only be detected in separate LC-MS runs at 5 ng/mL in urine. In addition, CE-MS consumed a much smaller volume of extract; the analyte peak widths, in some cases, were much narrower; and as the quaternary ammonium ions were well separated electrophoretically from the mainly neutral urine matrix, a much cleaner background in the CE-MS total ion trace was observed.     

Rapid test by liquid chromatography/tandem mass spectrometry to evaluate equine urine reactivity towards 17beta-OH steroids

Bacteria frequently found in equine urine samples may cause degradation of 17beta-OH steroids. A simple liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed to evaluate the microbiological contamination of equine urine as a marker of poor storage conditions. Norethandrolone was used as the internal standard, and the linearity, sensitivity, precision and accuracy of the method were evaluated. 17beta-OH oxidation was demonstrated for testosterone, nandrolone, trenbolone and boldenone, but did not occur in alpha-epimers such as alpha-boldenone and epitestosterone, demonstrating the stereoselectivity of the reaction. A rapid test was performed by spiking one of the four 17beta-OH steroids in samples of diluted equine urine. The steroids were transformed into their respective ketones in the presence of bacterial activity. The test allows direct injection of diluted samples into the LC/MS system, without the need for prior extraction. Results show that the best method of storage is freezing at -18 degrees C. Urine specimens should be analyzed as soon as possible after thawing. This allows bacterial degradation of equine urine to be arrested temporarily, so that the urine can be used for qualitative or quantitative analysis of 17beta-OH steroids.