Simultaneous determination of alachlor, metolachlor, atrazine, and simazine in water and soil by isotope dilution gas chromatography/mass spectrometry

A multiresidue method was developed for the simultaneous determination of low parts per billion (ppb) concentrations of the herbicides alachlor, metolachlor, atrazine, and simazine in water and soil using isotope dilution gas chromatography/mass spectrometry (GC/MS). Known amounts of 15N,13C-alachlor and 2H5-atrazine were added to each sample as internal standards. The samples were then prepared by a solid phase extraction with no further cleanup. A high resolution GC/low resolution MS system with data acquisition in selected ion monitoring mode was used to quantitate herbicides in the extract. The limit of detection was 0.05 ppb for water and 0.5 ppb for soil. Accuracy greater than 80% and precision better than 4% was demonstrated with spiked samples.     

Method for determination of pentobarbital in dry dog food by gas chromatography/mass spectrometry

A procedure has been developed and validated for measuring the concentration of pentobarbital residues in dry, extruded animal feed in the range of 3-200 ng/g (ppb) with an estimated limit of quantitation of 2 ppb. The method was developed for surveillance purposes: to measure the concentration of euthanizing agent which might be present in feeds incorporating rendered products which themselves might include some fraction of euthanized animals. A previously published qualitative procedure was modified by adding isotopically labelled pentobarbital as an internal standard. Dry feed was ground and extracted with methanol. The extract was loaded on a mixed-mode (C-18, anion exchange) solid-phase extraction cartridge designed for barbiturate residues. Pentobarbital was eluted and derivatized for gas chromatography/mass spectrometry in positive ion chemical ionization mode. Quantitation was based on the ratio of dimethyl-pentobarbital MH+ (m/z 255) vs dimethyl-pentobarbital-d(5) (m/z 260) in standards and extracts. Accuracy ranged from 112% at 3 ppb to 96% at 200 ppb, with relative standard deviations ranging from 4% at 3 ppb to 2% at 200 ppb.     

Development of multiclass methods for drug residues in eggs: hydrophilic solid-phase extraction cleanup and liquid chromatography/tandem mass spectrometry analysis of tetracycline, fluoroquinolone, sulfonamide, and beta-lactam residues

A method was developed for detection of a variety of polar drug residues in eggs via liquid chromatography/tandem mass spectrometry (LC/MS/MS) with electrospray ionization (ESI). A total of twenty-nine target analytes from four drug classes-sulfonamides, tetracyclines, fluoroquinolones, and beta-lactams-were extracted from eggs using a hydrophilic-lipophilic balance polymer solid-phase extraction (SPE) cartridge. The extraction technique was developed for use at a target concentration of 100 ng/mL (ppb), and it was applied to eggs containing incurred residues from dosed laying hens. The ESI source was tuned using a single, generic set of tuning parameters, and analytes were separated with a phenyl-bonded silica cartridge column using an LC gradient. In a related study, residues of beta-lactam drugs were not found by LC/MS/MS in eggs from hens dosed orally with beta-lactam drugs. LC/MS/MS performance was evaluated on two generations of ion trap mass spectrometers, and key operational parameters were identified for each instrument. The ion trap acquisition methods could be set up for screening (a single product ion) or confirmation (multiple product ions). The lower limit of detection for screening purposes was 10-50 ppb (sulfonamides), 10-20 ppb (fluoroquinolones), and 10-50 ppb (tetracyclines), depending on the drug, instrument, and acquisition method. Development of this method demonstrates the feasibility of generic SPE, LC, and MS conditions for multiclass LC/MS residue screening.     

Multiresidue pesticide analysis of agricultural commodities using acetonitrile salt-out extraction, dispersive solid-phase sample clean-up, and high-performance liquid chromatography-tandem mass spectrometry

A multiresidue method analyzing 209 pesticides in 24 agricultural commodities has been developed and validated using the original Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) procedure and high performance liquid chromatography-positive electrospray ionization-tandem mass spectrometry (LC-MS/MS) analysis. Using solvent-only calibration standards (SOCSs) and matrix-matched calibration standards (MMCSs), it was demonstrated that a minimal concentration of 5-10 μg/kg (part per billion, ppb) of analytes in matrix is required for the consistent identification of targeted pesticides with two MRM transitions. Method performance was validated by the precision and accuracy results obtained from fortification studies at 10, 25, 100, and 500 ppb and MMCSs. The method was demonstrated to achieve an average recovery of 100 ± 20% (n = 4) for >75% of evaluated pesticides at the low fortification level (10 ppb) and improved to >84% at the higher fortification concentrations in all 24 matrices. Matrix effects in LC-MS/MS analysis were studied by evaluating the slope ratios of calibration curves (1.0-100 ng/mL) obtained from the SOCSs and MMCSs. Principal component analysis (PCA) of LC-MS/MS and method validation data confirmed that each matrix exerts its specific effect during the sample preparation and LC-MS/MS analysis. The matrix effect is primarily dependent on the matrix type, pesticide type and concentration. Some caution is warranted when using matrix matched calibration curves for the quantitation of pesticides to alleviate concerns on matrix effects. The QuEChERS method with LC-MS/MS was used to identify and quantitate pesticides residues, with concentrations ranging from 2.5 to >1000 ppb in a variety of agricultural samples, demonstrating fitness for screening and surveillance applications.     

Confirmation of phorate, terbufos, and their sulfoxides and sulfones in water by capillary gas chromatography/chemical ionization mass spectrometry

A gas chromatographic/mass spectrometric method capable of confirming phorate, terbufos, their sulfoxides, and sulfones in water is reported. Parents and their metabolites are separated in less than 5 min using a short capillary GC column and high carrier gas linear velocities. Positive ion chemical ionization mass spectrometry generates (M + H) ions indicative of the different molecular weights of the analytes and at least one confirmatory fragment ion for each analyte. Residues have been qualitatively confirmed at the 1 ppb level in fortified water samples from a variety of sources. Apparent residues in control water were less than 0.1 ppb.     

Determination and confirmation of nitrofuran residues in honey using LC-MS/MS

A method was developed for the determination and confirmation of furazolidone, nitrofurazone, furaltadone, and nitrofurantoin as their side-chain residues in honey using liquid chromatography-tandem mass spectrometry (LC-MS/MS). An initial solid-phase extraction cleanup of the honey samples was followed by overnight hydrolysis and derivatization of the nitrofuran side-chain residues with 2-nitrobenzaldehyde. After pH adjustment and liquid-liquid extraction, the extracts were assayed by LC-MS/MS using electrospray ionization in the positive ion mode. The method was validated at concentrations ranging from 0.5 to 2.0 ppb with accuracies of 92-103% and coefficients of variation of < or =10%. The lowest calibration standard used (0.25 ppb) was defined as the limit of quantitation for all four nitrofuran side-chain residues. The extracts and standards were also used for confirmatory purposes. Honey from dosed beehives was assayed to study the stability of the nitrofuran residues and to demonstrate the effectiveness of the method.     

Development of multiclass methods for drug residues in eggs: silica SPE cleanup and LC-MS/MS analysis of ionophore and macrolide residues

A method was developed that is suitable for screening eggs for a variety of nonpolar residues in a single procedure. Residues are extracted by silica solid-phase extraction (SPE). Analysis is conducted via reverse-phase gradient liquid chromatography, electrospray ionization, and tandem ion trap mass spectrometry. For screening purposes (based on a single precursor-product ion transition) the method can detect ionophore (lasalocid, monensin, salinomycin, narasin) and macrolide (erythromycin, tylosin) residues in egg at approximately 1 ng/mL (ppb) and above and novobiocin residues at approximately 3 ppb and above. Conditions are described for confirmatory analysis based on multiple ions in the product ion spectrum. The extraction efficiency for ionophores was estimated at 60-85%, depending on drug. Recovery of macrolides and novobiocin was not as good (estimated at 40-55% after a hexane wash of the final extract was included), but the method consistently screened and confirmed these residues at concentrations below the target of 10 ppb. The method was applied to eggs from hens dosed with each drug individually. Lasalocid was found to have the highest probability of detection in eggs based on its high ionization efficiency and higher rate of deposition relative to the other drugs. The method is part of a larger scheme to provide surveillance methods for a wide variety of drug residues in eggs.     

Quantitative determination and structure elucidation of type A- and B-trichothecenes by HPLC/ion trap multiple mass spectrometry.

A method is presented for the quantification and structure confirmation of trichothecenes in wheat by high-performance liquid chromatography combined with multiple mass spectrometry (MS(n)()). Nine type A- and B-trichothecenes were determined (nivalenol, deoxynivalenol, fusarenon-X, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, neosolaniol, diacetoxyscirpenol, HT-2 toxin, and T-2 toxin). Extraction was carried out with acetonitrile/water. The extract was purified on a MycoSep column. Quantification was based on an internal standard and atmospheric pressure chemical ionization in the positive ion mode. Recoveries from spiked wheat were in the range of 80-106% at levels of 500 ppb. The limits of quantification for the whole method were between 10 and 100 ppb. Ion adduct formation with ammonium and acetate ions and MS(n) experiments provided information about substitution and fragmentation behavior of the mycotoxins. A scheme has been established for the partial structure elucidation of type A- and B-trichothecenes in fungal cultures.     

Use of deuterated internal standards for quantitation of T-2 and HT-2 toxins in human blood by tandem mass spectrometry

Deuterated acetyl derivatives (3-trideutero-acetyl-T-2 and 15-trideutero-HT-2) were prepared for use as internal standards for the quantitation of T-2 and HT-2 in blood by tandem mass spectrometry. The method used was multiple reaction monitoring (MRM), which essentially involves the selection of a parent ion for analysis followed by monitoring of the daughter ions generated by collision activated decomposition. The parent ions chosen for the trifluoroacetate derivative of T-2 and HT-2 were m/z+ 478 and 532, respectively. Both parents yield the same daughter ions, i.e., 180, 138, and 121. HT-2 and T-2 were added to blood extracts in amounts ranging from 1 to 20 ppb. The limit of detection is about 0.5 ppb with an effective detection limit of 1.0 ppb in a range of 1-20 ppb. The recovery is about 90%. This method can be used by veterinarians for purposes of diagnostics. It can be used for urine as well as blood.     

Determination of albendazole and its major metabolites in the muscle tissues of Atlantic salmon,tilapia, and rainbow trout by high performance liquid chromatography with fluorometric detection

A liquid chromatographic procedure for the determination of albendazole ([5-(propylthio)-1H-benzimidazol-2yl]carbamic acid methyl ester) and its major metabolites, albendazole sulfoxide, albendazole sulfone, and albendazole-2- aminosulfone in rainbow trout, tilapia, and salmon muscle with adhering skin tissue is described. The muscle tissue samples are made alkaline with potassium carbonate and extracted with ethyl acetate. The extracts are further subjected to cleanup by utilizing a number of liquid-liquid extraction steps. After solvent evaporation, the residue is reconstituted in mobile phase and chromatographed. The chromatography is carried out on a reversed phase Luna C(18) column, using acetonitrile/methanol/buffer as a mobile phase and a fluorescence detector. The average recoveries from the fortified muscle tissue of the three fish species for albendazole (25-100 ppb), albendazole sulfoxide (15.5-62 ppb), albendazole sulfone (1-10 ppb), and albendazole-2- aminosulfone (10-100 ppb) were 94, 77, 82, and 67%, respectively. The average CV for each compound was < or =10%. The procedure was validated and then applied to the determination of albendazole and its three major metabolites in the muscle tissue of the three fish species obtained after orally dosing with albendazole.     

Determination of ethyl carbamate in distilled alcoholic beverages by gas chromatography with flame ionization or mass spectrometric detection

Quantitative methods are detailed for determination of ethyl carbamate in distilled alcoholic beverages by capillary gas chromatography with flame ionization detection (GC/FID) and by packed-column gas chromatography/mass spectrometry (GC/MS) using selected ion monitoring. Five g samples of distillate of known ethanol concentration are diluted with water to 25% ethanol (v/v), washed with petroleum ether, and extracted with dichloromethane prior to GC/FID or GC/MS analysis. As necessary, sample extracts that exhibit GC/FID interference are passed through alumina for additional cleanup. When internal standards (tert-butyl carbamate and n-butyl carbamate for GC/FID, or ethyl 13C-15N-carbamate for GC/MS) were used for quantitation, the limit of detection for ethyl carbamate was in the range of 5-25 ppb. Coefficients of variation ranged from 3.5 to 6.0% for GC/FID determinations, and from 1.4 to 3.2% for GC/MS. Correlation between methods for 22 random distillate samples ranging in concentration from approximately 40 to 800 ppb gave a correlation coefficient (r) of 0.996.     

Simple and rapid liquid chromatography-tandem mass spectrometry confirmatory assay for determining amoxicillin and ampicillin in bovine tissues and milk

A simple specific and rapid confirmatory method for determining the two amphoteric penicillins, that is, amoxicillin and ampicillin, in bovine muscle, liver, kidney, and milk is presented. This method is based on the matrix solid-phase dispersion technique with hot water as extractant followed by liquid chromatography (LC)-tandem mass spectrometry. With this instrumentation, the selected reaction monitoring acquisition mode with two fragmentation reactions for each analyte was adopted. After acidification and filtration of the aqueous extracts, 25 microL of the tissue final extracts and 50 microL of the milk final extract were injected into the LC apparatus. Absolute recovery of the two analytes in any biological matrix at the 50 ppb level in tissues and the 4 ppb level in milk was 74-95% with relative standard deviations (RSDs) of no larger than 9%. When penicillin V was used as surrogate internal standard, relative recovery of the targeted compounds present in bovine tissues and milk at, respectively, 25 and 2 ppb levels ranged between 100 and 106% with RSDs of no larger than 11%. When fractionation of analytes by using a short chromatographic run was attempted, remarkable signal weakening for the two analytes was experienced. This effect was traced to polar endogenous coextractives eluted in the first part of the chromatographic run that interfered with the gas-phase ion formation of the two penicillins. Slowing the chromatographic run eliminated this unwelcome effect. Limits of quantification of the two analytes in bovine milk were estimated to be <1 ppb, whereas amoxicillin and ampicillin could be quantified in bovine tissues down to 3.1 and 0.8 ppb levels, respectively.     

Preparation of stable isotope-labeled 2-nitrobenzaldehyde derivatives of four metabolites of nitrofuran antibiotics and their comprehensive characterization by UV, MS, and NMR techniques

A convenient method is presented for the preparation of the carbon-13-labeled 2-nitrobenzaldehyde derivatives of the nitrofuran metabolites 3-amino-2-oxazolidinone (AOZ), semicarbazide (SC), 1-aminohydantoin (AH), and 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ), with the purpose of using them as internal standards for the quantification of trace levels of nitrofuran residues by liquid chromatography-tandem mass spectrometry in foods of animal origin. The synthesis encompasses the nitration of [1,2,3,4,5,6-(13)C(6)]toluene prior to chromyl compound-mediated oxidation of the methyl group into the corresponding aldehyde. The four metabolites of nitrofuran antibiotics were derivatized independently with the resulting ring-labeled 2-nitrobenzaldehyde (NBA) to obtain the target compounds. Both the isotopically enriched and native substances were used to perform a comprehensive fragmentation study by electrospray ionization (ESI) collision-induced dissociation (CID) mass spectrometry (MS). Full characterization of the nitrofuran derivatives was accomplished with ultraviolet (UV) and exhaustive nuclear magnetic resonance (NMR) analysis. A major advantage of the described procedure is that it can be extended to the preparation of other carbon-13-labeled derivatives of metabolites of nitrofuran antibiotics.     

Quantitative and confirmatory analyses of malachite green and leucomalachite green residues in fish and shrimp

Liquid chromatographic methods are presented for the quantitative and confirmatory determination of malachite green (MG) and leucomalachite green (LMG) for channel catfish, rainbow trout, tilapia, basa, Atlantic salmon, and tiger shrimp. Residues were extracted from tissues with ammonium acetate buffer and acetonitrile and isolated by partitioning into dichloromethane. LMG was quantitatively oxidized to the chromic MG with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone. Extracts were analyzed for total MG by liquid chromatography with both visible detection (LC-VIS) at 618 nm for routine screening and ion trap mass spectrometry (LC-MSn) with no discharge-atmospheric pressure chemical ionization for residue confirmation. The method was validated in each species fortified with LMG at 1, 2, 4, and 10 ng/g (ppb), and average recoveries ranged from 85.9 to 93.9%. Quantitative data were consistent for the two detection methods, with measured method detection limits of 1.0 ng/g for LC-VIS and 0.25 ng/g for LC-MSn. Incurred tissues from catfish, trout, tilapia, and salmon that had been treated with MG were also extracted and analyzed as part of this study.     

Marker residue determination of tritium-labeled ivermectin in the muscle of aquacultured largemouth bass, hybrid striped bass, and yellow perch following oral treatment

The residue depletion profiles of tritium-labeled ivermectin and its metabolites in the muscle of aquacultured largemouth bass (LMB), hybrid striped bass (HSB), and yellow perch (YP) following oral treatment are reported. Fish were administered 3H-ivermectin at the dose level of 0.1 mg/kg body weight (7-9 μCi) in a gel capsule via stomach tube. At each postdose withdrawal time, six fish of each species were sedated with buffered MS-222 and blood samples taken. Fish were then euthanized, and fillets with adhering skin (scales removed) and bile samples were collected. The muscle fillets were homogenized in dry ice to a fine powder. Aliquots of tissue, plasma, and bile were assayed for total radioactive residue (TRR). The homogenized muscle was extracted in acetonitrile or methanol followed by high-performance liquid chromatographic (HPLC) analysis to determine the presence of parent ivermectin and its potential metabolites. The highest TRR concentrations (ivermectin equivalents) of 53, 45, and 44 ng/g (ppb) were obtained on postdose day 1 for HSB, LMB, and YP, respectively. The TRR depleted most slowly in HSB to 25 ppb at day 91, followed by YP to 19 ppb at day 42 and then by LMB to 22 ppb at day 35. The total residue of ivermectin and its metabolites by HPLC analysis followed the same depletion pattern in the three species. Additionally, the depletion rate of TRR of 3H-ivermectin in the three species followed the pattern bile > plasma > muscle. The results further indicate that one of the polar metabolites of ivermectin could serve as a potential marker residue as an indication of use, rather than the parent ivermectin.     

Quantitative determination of carbaryl in apples, lettuce, and water by densitometry of thin layer chromatograms.

A method for the quantitative determination of carbaryl insecticide by in situ densitometry was developed. After separation on silica gel thin layer plates, carbaryl residues were detected by using p-nitrobenzenediazonium fluoborate reagent and quantitated by scanning the resultant blue spots with a fiber optics densitometer and comparing them with standards. The method was applied to water fortified with carbaryl at 8 ppb and apples and lettuce fortified at 0.10 ppm; all recoveries were greater than 89%. The 2 crop extracts were cleaned up by using the AOAC thin layer chromatographic method for visual estimation of carbaryl. Related carbamate insecticides were detected by using the same reagent, and the potential for quantitation was demonstrated.     

Liquid chromatography-tandem mass spectrometry for the determination of protein-bound residues in shrimp dosed with nitrofurans

An analytical method was developed for the determination of bound residues of the nitrofuran drugs furazolidone, nitrofurazone, furaltadone, and nitrofurantoin with a sensitivity of 1 ppb in shrimp. In this procedure, shrimp tissue is prewashed with solvents followed by overnight acid hydrolysis, during which the side chains of the bound residues are released and simultaneously derivatized with 2-nitrobenzaldehyde. After liquid-liquid extraction cleanup, the derivatives are detected and quantitated using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) with an atmospheric pressure chemical ionization interface. The method was validated using control shrimp fortified with each side-chain analyte at 1, 2, and 4 ppb. Method accuracies were >80% with coefficients of variation of <20% for all four analytes. Tissues from dosed shrimp were assayed to demonstrate the effectiveness of the method for recovering bound residues of nitrofurans. In shrimp dosed with nitrofurans, nitrofurantoin exhibited the lowest level of bound residues.     

Direct aqueous injection LC-ESI/MS/MS analysis of water for 11 chloro- and thiomethyltriazines and metolachlor and its ethanesulfonic and oxanilic acid degradates

A multianalyte method is reported for the determination of atrazine, simazine, propazine, and their respective dealkylated chlorotriazine metabolites; ametryn and prometryn and their respective dealkylated thiomethyltriazine metabolites; and S-metolachlor and its ethanesulfonic and oxanilic acid degradates in deionized, ground, surface, and finished drinking water. Water samples are analyzed using direct aqueous injection (DAI) liquid chromatography-electrospray ionization/mass spectrometry/mass spectrometry (LC-ESI/MS/MS). No preanalysis sample manipulation is required other than transfer of a small portion of sample to an injection vial. The lower limit of the method validation is 0.050 microg/L (ppb) for all analytes except 2,4-diamino-6-chloro- s-triazine (didealkylatrazine, DDA, or G-28273). For this compound the LLMV is 0.50 microg/L (ppb). The overall mean procedural recoveries (and percent relative standard deviations) for all water types for all analytes ranged from 95 to 101% (4.5-11%). The method validation was conducted under U.S. EPA FIFRA Good Laboratory Practice Guidelines 40 CFR 160.     

Gas chromatographic determination of ethylene dibromide in flour products

A method based on gas chromatography with electron capture detection was developed for the determination of ethylene dibromide (EDB) extracted from flour products. The procedure relies on the organic extraction of flour/water mixtures and uses an internal standard, 1-bromo-3-chloropropane. Recoveries of EDB at 10 and 100 ppb were 80.1 +/- 2.8% (SD) and 84.4 +/- 4.3%, respectively; recovery of the internal standard at the working concentration 500 ppb was 98.3 +/- 6.7%. Calibration curves were linear over the range 5-400 ppb, with a mean overall coefficient of variation of less than 5%. The reliability of the procedure was assessed by using gas chromatography combined with mass spectrometry. Results are shown for determination of EDB in locally milled flour products.     

Determination of gentian violet, its demethylated metabolites, and leucogentian violet in chicken tissue by liquid chromatography with electrochemical detection

A method is presented for determination of residues of gentian violet (GV), its demethylated metabolites (pentamethyl and tetramethyl), and leucogentian violet (LGV) in chicken tissue. The analytes are extracted from tissue with acetonitrile/buffer and partitioned into methylene chloride. Polar lipids are removed on an alumina column followed by partitioning into methylene chloride from a citrate buffer. The compounds of interest are isolated on a disposable carboxylic acid cation exchange column and then eluted with 0.02% HCl in methanol. GV, its metabolites, and LGV are determined by liquid chromatography using isocratic elution with a buffered mobile phase from a cyano column and amperometric electrochemical detection at +1.000 V. Average recoveries of GV and LGV from commercially purchased chicken liver fortified with 20 ppb of each compound were 92% [standard deviation (SD) = 7, coefficient of variation (CV) = 7.6%] and 86% (SD = 7, CV = 8.1%), respectively. Average recoveries of GV, LGV, the pentamethyl metabolite, and 1 of the tetramethyl metabolites from control chicken liver (provided by the Center for Veterinary Medicine) fortified with 20 ppb of each compound were 80% (SD = 7, CV = 8.8%), 76% (SD = 3, CV = 3.9%), 83% (SD = 6, CV = 7.2%), and 76% (SD = 8, CV = 10.5%), respectively. Mean results from 10 analyses of residue-incurred chicken liver were 31 ppb GV (SD = 3, CV = 9.7%), 34 ppb pentamethyl metabolite (SD = 3, CV = 8.8%), and 40 ppb tetramethyl metabolite(s) (SD = 2, CV = 5.0%), for an average value of 105 ppb total residues (SD = 6, CV = 5.7%); no LGV was found. Data are also presented to show applicability of the method to muscle tissue.