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–MSⁿ) 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–MSⁿ 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.     

Screening procedure for the analysis of distigmine bromide in serum by high-performance liquid chromatography-electrospray ionization mass spectrometry

A screening procedure was developed for the identification and quantification of distigmine bromide in serum samples by using liquid chromatography (LC)-electrospray ionization (ESI)-mass spectrometry (MS). In this method, distigmine bromide was analyzed in 0.5 mL serum by using pancuronium bromide as the internal standard, and gradient elution was performed using a reversed-phase column and a mixture of 10 mM-ammonium formate and methanol as the mobile phase. A highly sensitive assay could be performed with simple solid phase extraction using a cation exchange cartridge column by carrying out selected ion monitoring analysis in the positive ion detection mode. The procedure was validated in terms of linearity (0.9973 at 2.5 ng/mL). The inter- and intra-day precisions (coefficient of variation; CV%) were <8.5% and < 9.7%, respectively. The analytes were evaluated for stability and were found to be stable in serum for 1 week at 4 degrees C and 4 weeks at -30 degrees C, and successfully applied to in the analysis of two overdose cases. This method is sensitive and useful for the detection, quantification, and confirmation of distigmine bromide in serum.     

Quantitative analysis of cocaine and its metabolites in whole blood and urine by high-performance liquid chromatography coupled with tandem mass spectrometry

AIM: In forensic toxicology it is important to have specific and sensitive analysis for quantification of illicit drugs in biological matrices. This paper describes a quantitative method for determination of cocaine and its major metabolites (ecgonine methyl ester, benzoylecgonine, norcocaine and ethylene cocaine) in whole blood and urine by liquid chromatography coupled with tandem mass spectrometry LC/MS/MS. METHOD: The sample pre-treatment (0.20 g) consisted of acid precipitation, followed by centrifugation and solid phase extraction of supernatant using mixed mode sorbent columns (SPEC MP1 Ansys Diag. Inc.). Chromatographic separation was performed at 30 degrees C on a reverse phase Zorbax C18 column with a gradient system consisting of formic acid, water and acetonitrile. The analysis was performed by positive electrospray ionisation with a triple quadropole mass spectrometer operating in multiple reaction monitoring (MRM) mode. Two MRM transitions of each analyte were established and identification criteria were set up based on the retention time and the ion ratio. The quantification was performed using deuterated internal analytes of cocaine, benzoylecgonine and ecgonine methyl ester. The calibration curves of extracted standards were linear over a working range of 0.001-2.00 mg/kg whole blood for all analytes. The limit of quantification was 0.008 mg/kg; the interday precision (measured by relative standard deviation-%RSD) was less than 10% and the accuracy (BIAS) less than 12% for all analytes in whole blood. Urine samples were estimated semi-quantitatively at a cut-off level of 0.15 mg/kg with an interday precision of 15%. CONCLUSION: A liquid chromatography mass spectrometric (LC/MS/MS) method has been developed for confirmation and quantification of cocaine and its metabolites (ecgonine methyl ester, benzoylecgonine, norcocaine and ethylene cocaine) in whole blood and semi-quantitative in urine. The method is specific and sensitive and offers thereby an excellent alternative to other methods such as GC-MS that involves derivatisation.     

High-throughput screening of corticosteroids and basic drugs in horse urine by liquid chromatography-tandem mass spectrometry

This paper describes two high-throughput liquid chromatography-tandem mass spectrometry (LC-MS-MS) methods for the screening of two important classes of drugs in equine sports, namely corticosteroids and basic drugs, at low ppb levels in horse urine. The method utilized a high efficiency reversed-phase LC column (3.3 cm L x 2.1 mm i.d. with 3 microm particles) to provide fast turnaround times. The overall turnaround time for the corticosteroid screen was 5 min and that for the basic drug screen was 8 min, inclusive of post-run and equilibration times. Method specificity was assessed by analysing a total of 35 negative post-race horse urine samples. No interference from the matrices at the expected retention times of the targeted masses was observed. Inter-day precision for the screening of 19 corticosteroids and 48 basic drugs were evaluated by replicate analyses (n = 10) of a spiked sample on 4 consecutive days. The results demonstrated that both methods have acceptable precision to be used on a routine basis. The performance of these two methods on real samples was demonstrated by their applications to drug administration and positive post-race urine samples.     

Screening and quantitation of multiclass drugs of abuse and pharmaceuticals in hair by fast liquid chromatography electrospray time-of-flight mass spectrometry

In this work, an automated screening method for the simultaneous identification and quantitation of 30 representative multiclass drugs (including opiates, cocaine and its main metabolite, cannabinoids, amphetamines and other stimulants in hair samples) has been developed using fast liquid-chromatography time-of-flight mass spectrometry (LC-TOFMS). The identification and quantitation of the drugs were carried out by liquid chromatography using a C(18) column (4.6×50 mm) with 1.8 μm particle size. Accurate mass measurements of ions of interest (typically [M+H](+)) by electrospray time-of-flight mass spectrometry in the positive ionization mode were used for unambiguous confirmation of the targeted species. Three sample preparation methodologies were evaluated: (a) direct methanolic extraction by sonication, (b) acidic extraction, and (c) alkaline digestion. Direct methanolic extraction showed better recoveries and cleaner extracts. The limits of detection obtained in hair matrix were as low as 5 pg mg(-1) for cocaine and cannabidiol, ranging from 5 to 75 pg mg(-1) for the studied species while the LOQ ranged from 15 to 250 pg mg(-1). The method has been applied to six hair samples from drug consumer volunteers, where the presence of at least one drug was confirmed by accurate mass measurements within 2 ppm (mass error) in most cases. The present study demonstrates the usefulness of LC-TOFMS for both screening and quantitation purposes in drug testing in hair. In addition, the possibility of non-target or a posteriori data analysis of samples or the extension of the procedure for testing for additional compounds offers interesting features for forensic analysis.     

Confirmation of oxytetracycline, tetracycline and chlortetracycline residues in milk by particle beam liquid chromatography/mass spectrometry.

A method using particle beam liquid chromatography/mass spectrometry was developed for the confirmation of oxytetracycline, tetracycline and chlortetracycline residues in bovine milk. This method is one of the first to apply particle beam technology to the confirmation of animal drug residues in food products for regulatory purposes. The milk is centrifuged, using molecular weight cut-off filters to remove components of 25,000 daltons and above from the milk. The filtrate is passed through a C-18 sample preparation cartridge which retains the tetracyclines. After the columns are washed with water, the tetracyclines are eluted with 0.1 M oxalic acid in methanol and concentrated. The compounds are separated on a Novapak C-18 column with a methanol-oxalic acid-acetonitrile mobile phase. Negative chemical ionization with selective ion monitoring is used to identify the tetracyclines. The procedure was used to confirm the presence of each tetracycline at 100 ng ml-1 in fortified and incurred milk samples.     

Simultaneous high-performance liquid chromatography determination of carbamazepine and five of its metabolites in plasma of epileptic patients

A high-performance liquid chromatographic method with UV detection for the simultaneous analysis of the antiepileptic drug carbamazepine and five of its metabolites in human plasma has been developed. The analysis was carried out on a reversed-phase column (C₈, 150×4.6 mm I.D., 5 μm) using acetonitrile, methanol and a pH 1.9 phosphate buffer as the mobile phase. Under these chromatographic conditions, carbamazepine and its metabolites 10,11-dihydro-10,11-epoxycarbamazepine, 10,11-dihydro-10,11-dihydroxycarbamazepine, 2-hydroxycarbamazepine, 3-hydroxycarbamazepine and 10,11-dihydro-10-hydroxycarbamazepine are baseline separated in less than 18 min. The extraction of the analytes from plasma samples was performed by means of an original solid-phase extraction procedure using Oasis HLB cartridges. The method requires only 250 μl of plasma for one complete analysis. The repeatability (RSD%<2.4), intermediate precision (RSD%<3.5) and extraction yield (84.8–103.0%) were very good for all analytes. The method is suitable for reliable therapeutic drug monitoring of patients undergoing chronic treatment with carbamazepine and for kinetic–metabolic studies of this drug.     

Determination of diclazuril, toltrazuril and its two metabolites in poultry tissues and eggs by gel permeation chromatography-liquid chromatography-tandem mass spectrometry

A new procedure has been described for the extraction of diclazuril (DIZ), toltrazuril (TOZ) and its two main metabolites toltrazuril sulphoxide (TZSO) and toltrazuril sulphone (TZS) from poultry tissues and eggs, using gel permeation chromatography (GPC). The analytes and the deuterated internal standard were extracted from the samples with ethyl acetate. The analytes were measured by LC coupled to an electrospray ionization tandem mass spectrometer operating in the negative ion mode. Excellent linear dynamic range was observed from 1 to 500 μg/L with the correlation coefficients (R(2)) better than 0.99 for all analytes. The method LOQ of the four analytes in real samples was 1.2 μg/kg for DIZ and TOZ, and 1.8 μg/kg for TZSO and TZS. These values are far lower than the maximum residue limits (MRLs) established by several control authorities. The developed method was accurate with overall recoveries in four matrices.     

Determination of methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine and alprazolam in human plasma by liquid chromatography-electrospray ionization mass spectrometry

A fast liquid chromatographic assay with mass spectrometric detection (LC/MS) has been developed and validated for the simultaneous determination of methadone (MT), its primary metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and alprazolam, in human plasma. The extraction procedure was performed with automatic solid phase extraction, and the compounds were separated with a Sunfire column using a gradient mode. Deuterated analogues for all of the analytes of interest were used for quantitation. Limits of detection (LOD) were established between 0.5 and 1 ng/ml. Linearity was obtained over a range of 2-2,000 ng/ml with an average correlation coefficient (R(2)) of >0.99. Intra- and inter-batch coefficients of variation and relative mean errors were less than 10% for all analytes and concentrations. The recoveries were higher than 50.0% in all cases. The method proved to be suitable for evaluation of plasma obtained from patients enrolled in a MT-maintenance program who are frequently treated with alprazolam as a sedative.     

Analysis of eight free progestogens in eggs by matrix solid-phase dispersion extraction and very high pressure liquid chromatography with tandem mass spectrometry

A rapid method to identify and quantify unconjugated progestogens in eggs is presented. Samples were prepared based on matrix solid-phase dispersion (MSPD) using C18 as dispersant, followed by a clean-up step with graphitized carbon black (GCB) solid-phase extraction (SPE) cartridges. The analytes were separated by very high pressure LC on a BEH C18 column for a period of 5 min. Electrospray ionization tandem mass spectrometry (ESI-MS/MS) was operated in the positive time-scheduled multi-reaction monitoring mode. Recovery studies were performed at two fortification levels. Average recoveries of the target compounds varied from 83.8% to 111.2% and relative standard deviations ranged from 10.5% to 23.7%. The limits of detection (LODs) and limits of quantitation (LOQs) were in the range of 0.2-2.0 microg kg(-1) and 0.6-5.0 microg kg(-1), respectively. Investigation of real samples indicated that the range of progesterone in eggs was 9.9-40.0 microg kg(-1).     

Determination of neonicotinoid insecticides residues in bovine tissues by pressurized solvent extraction and liquid chromatography-tandem mass spectrometry

A rapid, sensitive, and environmental-friendly method has been developed for the simultaneous determination of seven neonicotinoid insecticides residues in bovine muscle and liver. The sample preparation procedure was based on a high automated pressurized solvent extraction (PSE) combined with solid-phase extraction (SPE) clean-up. The target compounds were identified and quantitatively determined by liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) operated in multiple reaction monitoring mode. Average recoveries of the seven analytes from fortified samples ranged between 83.2% and 101.9%, with relative standard deviations (RSDs) lower than 10.8%. The limits of detection (LODs) and quantification (LOQs) for neonicotinoids were in the ranges of 0.8-1.5 μgkg?1 and 2.5-5.0 μgkg?1, respectively. This validated method was successively applied to the determination of neonicotinoid insecticides in real samples from markets.     

Accelerated solvent extraction and liquid chromatography–tandem mass spectrometry quantitation of corticosteroid residues in bovine liver

A new method for the rapid extraction and unequivocal confirmation of two highly potent fluorinated synthetic corticosteroids, dexamethasone and its β-epimer betamethasone, in bovine liver was developed. Flumethasone was used as internal standard. An extraction procedure using an accelerated solvent extraction system was employed for the isolation of the analytes in liver samples. The procedure was highly automated, including defatting and extraction steps, sequentially carried out under 1.0·10⁴ kPa in about 35 min. The extracts were then directly analysed by tandem mass spectrometry with on-line liquid chromatography. The analytes were ionised in a heated nebulizer interface operating in the negative ion mode where the molecular related ions [M-H-CH₂O]⁻ were generated for each analyte, at m/z 361 for betamethasone and dexamethasone and at m/z 379 for flumethasone. They served as precursor ions for collision-induced dissociation and three diagnostic product ions for the drugs were identified to carry out analyte confirmation by selected reaction monitoring. Assessment of recovery, specificity and precision for betamethasone, dexamethasone and flumethasone proved the method suitable for confirmatory purposes. The limit of quantification of betamethasone and dexamethasone in liver tissue was 1.0 μg/kg.     

Development and validation of a method for the confirmation of halofuginone in chicken liver and eggs using electrospray tandem mass spectrometry

A method is described for the quantitative confirmation of halofuginone (HFG) residues in chicken liver and eggs. This method is based on LC coupled to positive ion electrospray MS-MS of the tissue extracts, prepared by trypsin digestion of the tissues followed by liquid-liquid extraction and final clean-up using Solid Phase Extraction (SPE). The [M+H](+) ion at m/z 416 is monitored along with four transitions at m/z 398, 138, 120 and 100. The method has been validated according to the draft EU criteria for the analysis of veterinary drug residues at 15, 30 and 45 microg kg(-1) in liver and 5, 15 and 50 microg kg(-1) in eggs. The new analytical limits, CCalpha and CCbeta were calculated for liver and were 35.4 and 43.6 microg kg(-1), respectively.     

Simultaneous determination of five antipsychotic drugs in rat plasma by high performance liquid chromatography with ultraviolet detection

A significant percentage of psychiatric patients who are treated with antipsychotics are treated with more than one antipsychotic drug in the clinic. Thus, it is advantageous to use a rapid and reliable assay that is suitable for determination of multiple antipsychotic drugs in plasma in a single run. A simple and sensitive HPLC-UV method was developed and validated for simultaneous quantification of olanzapine, haloperidol, chlorpromazine, ziprasidone, risperidone and its active metabolite 9-hydroxyrisperidone in rat plasma using imipramine as an internal standard (I.S.). The analytes were extracted from rat plasma using a single step liquid-liquid acid solution back extraction technique with wash procedure, which provided the very clear baseline for blank plasma extraction. The compounds were separated on an Agilent Eclipse XDB C8 (150 mm x 4.6 mm i.d., 5 microm) column using a mobile phase of acetonitrile/30 mM ammonium acetate including 0.05% triethylamine (pH 5.86 adjusted with acetic acid) with gradient elution. All of the analytes were monitored using UV detection. The method was validated and the linearity, lower limit of quantitation (LLOQ), precision, accuracy, recoveries, selectivity and stability were determined. The LLOQ was 2.0 ng/ml and correlation coefficient (R(2)) values for the linear range of 2.0-500.0 ng/ml were 0.998 or greater for all the analytes. The precision and accuracy for intra-day and inter-day were better than 7.44%. The recovery was above 74.8% for all of the analytes. This validated method has been successfully used to quantify the plasma concentration of the analytes for pharmacological and toxicological studies following chronic treatment with antipsychotic drugs in the rat.     

Analysis of veterinary drug residues in shrimp: a multi-class method by liquid chromatography-quadrupole ion trap mass spectrometry

A liquid chromatography-mass spectrometry (LC-MS) method was developed to screen and confirm veterinary drug residues in raw shrimp meat. This method simultaneously monitors 18 drugs of different classes, including oxytetracycline (OTC), sulfonamides, quinolones, cationic dyes, and toltrazuril sulfone (TOLS). The homogenized shrimp meat is extracted with 5% trichloroacetic acid. The extract is further cleaned using polymer-based SPE. A 50 mm phenyl column separates the analytes, prior to analysis with an ion trap mass spectrometer interfaced with an atmospheric pressure chemical ionization source. This method is able to confirm oxytetracycline residues at 200 ng/g, toltrazuril sulfone at 50 ng/g, sulfaquinoxaline at 20 ng/g, and the other 15 drugs at 10 ng/g or lower levels. An estimate of the level of residues can also be made so that only confirmed samples above action levels will be sent for quantitation. The method is validated with both fortified and incurred samples, using multiple shrimp species as well. This multi-class method can provide a means to simultaneously monitor for a wide range of illegal drug residues in shrimp.     

Application of solid-phase microextraction combined with derivatization to the determination of amphetamines by liquid chromatography

This work evaluates the utility of solid-phase microextraction (SPME) in the analysis of amphetamines by liquid chromatography (LC) after chemical derivatization of the analytes. Two approaches have been tested and compared, SPME followed by on-fiber derivatization of the extracted amphetamines, and solution derivatization followed by SPME of the derivatives formed. Both methods have been applied to measure amphetamine (AP), methamphetamine (MA), and 3,4-methylenedioxymethamphetamine (MDMA), using the fluorogenic reagent 9-fluorenylmethyl chloroformate (FMOC) and carbowax-templated resin (CW-TR)-coated fibers. Data on the application of the proposed methods for the analysis of different kind of samples are presented. When analyzing aqueous solutions of the analytes, both approaches gave similar analytical performance, but the sensitivity attainable with the solution derivatization/SPME method was better. The efficiencies observed when processing spiked urine samples by the SPME/on-fiber derivatization approach were very low. This was because the extraction of matrix components into the fiber coating prevented the extraction of the reagent. In contrast, the efficiencies obtained for spiked urine samples by the solution derivatization/SPME approach were similar to those obtained for aqueous samples. Therefore, the later method would be the method of choice for the quantification of amphetamines in urine.     

Multiresidue confirmation of beta-agonists in bovine retina and liver using LC-ES/MS/MS

Misuse of numerous beta-agonist drugs for their growth promoting effects in livestock production requires significant regulatory enforcement activities worldwide. The proof of illegal drug use needed for regulatory action usually requires the high degree of specificity derived from mass spectrometric analysis of suspect tissues and body fluids. In this paper, we describe a multiresidue screening method for confirmation of nine beta-agonist compounds in bovine liver and retina. A wide range of analyte structures was selected in order to demonstrate applicability to other chemically related beta-agonists for which standards are not currently available. The class-specific method, which is based on mixed mode cation exchange/reverse phase solid phase extraction, reverse phase gradient LC separation using a cyanopropyl-silica phase, and tandem mass spectrometry (MS/MS) in the multiple reaction monitoring (MRM) mode, yields high analyte recoveries at the target level of 1 ppb (ng/g). In addition, acquisition of multiple MRM transitions for each analyte permits simultaneous confirmation of beta-agonists at the level of 1 ppb in liver and retina by using intensity ratios between fragment ions and protonated molecules. Estimated values for the limit of quantification (LOQ) for individual beta-agonists were 0.08-0.3 ppb in liver and 0.02-0.5 in retina; the estimated limits of confirmation, using accepted criteria from international regulatory agencies, were 0.25-0.8 ppb in liver and 0.1-1 ppb in retina. This method should be useful in supporting regulatory enforcement programs that monitor beta-agonist misuse.     

Multiresidue determination of sulfonamides in edible catfish, shrimp and salmon tissues by high-performance liquid chromatography with postcolumn derivatization and fluorescence detection

A liquid chromatographic (LC) method for determining 14 sulfonamide (SA) (sulfanilamide, sulfadiazine (SDZ), sulfathiazole, sulfapyridine, sulfamerazine (SMR), sulfamethazine (SMZ), sulfamethizole, sulfamethoxypyridazine, sulfachloropyridazine (SCP), sulfamonomethoxine, sulfadoxine, sulfamethoxazole, sulfadimethoxine (SDM), and sulfaquinoxaline (SQX)) residues in edible catfish, shrimp and salmon tissues was developed and validated at 5, 10 or 20 ng g(-1). The method was then used to determine residues in tissues of catfish, shrimp and salmon dosed with six selected sulfonamides (sulfadiazine, sulfamerazine, sulfamethazine, sulfachloropyridazine, sulfadimethoxine and sulfaquinoxaline). All assays were within U.S. Food and Drug Administration guidelines for recovery and intra-assay variability. The method was developed to determine possible sulfonamide residues in aquacultured catfish, shrimp and salmon produced for food.     

Multi-residue extraction–purification procedure for corticosteroids in biological samples for efficient control of their misuse in livestock production

A fast and efficient multi-residue extraction–purification procedure was developed for 12 corticosteroids in biological matrices (hair, urine and meat), in order to control their illegal use as growth promoters in cattle. Detection and identification of the analytes were achieved using a previously described LC–MS–MS method based on negative electrospray ionisation and a triple quadrupole analyser. The presented procedures included acid (hair) or enzymatic (urine and meat) hydrolysis, C₁₈ reversed-phase SPE, Na₂CO₃ liquid–liquid clean-up and SiOH normal-phase SPE. The detection limits of the developed methods were between 2.9 and 9.3 pg/mg (ppb) for hair samples and in the 40 – 70 pg/g (ppt) range for the urine or meat samples. The acid hydrolysis used for corticosteroid extraction in hair was optimised using an experimental design and response surface methodology. Achieved performances were linked to a physico–chemical approach based on the corticosteroids specific C₁₇ side-chain. This original multi-residue and multi-matrices analytical methodology will be used for further metabolism studies.     

Application of a sensitive liquid chromatographic/tandem mass spectrometric method to a pharmacokinetic study of allylestrenol in healthy Chinese female volunteers

A sensitive, specific and rapid liquid chromatographic/tandem mass spectrometric (LC/MS/MS) assay for the determination of allylestrenol in human plasma was established. Plasma samples were extracted by tert-butyl ether and separated by LC/MS/MS using a Phenomenex Curosil-PFP column (250 mm x 4.6 mm ID, dp 5 microm) with a mobile phase of methanol-water (95:5, v/v). The analytes were monitored with atmospheric pressure chemical ionization (APCI) by selected reaction monitoring (SRM) mode. The linear calibration curves covered a concentration range of 0.04-20.0 ng/mL with lower limit of quantification (LLOQ) at 0.04 ng/mL. The mean extraction recovery of allylestrenol was greater than 81.8%. The intra- and inter-day precisions were less than 1.3% and 3.1% respectively, determined from quality control (QC) samples of three representative concentrations. The method has been successfully applied to determining the plasma concentration of allylestrenol and a clinical pharmacokinetics study in healthy Chinese female volunteers.