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.     

Screening and mass spectral confirmation of beta-lactam antibiotic residues in milk using LC-MS/MS.

Milk is typically screened for beta-lactam antibiotics by nonspecific methods. Although these methods are rapid and sensitive, they are not quantitative and can yield false positive findings. A sensitive and specific method for the quantitation and mass spectral confirmation of five beta-lactam and two cephalosporin antibiotics commonly or potentially used in the dairy industry is described using high-performance liquid chromatography with tandem mass spectrometry. The antibiotics studied were ampicillin, amoxicillin, penicillin G, penicillin V, cloxacillin, cephapirin, and ceftiofur. The antibiotics were extracted from milk with acetonitrile, followed by reversed-phase column cleanup. The extract was analyzed by liquid chromatography coupled with a mass spectrometer, using a water/methanol gradient containing 1% acetic acid on a C-18 reversed-phase column. Determination was by positive ion electrospray ionization and ion trap tandem mass spectrometry. Quantitation was based on the most abundant product ions from fragmentation of the protonated ion for amoxicillin, cephapirin, ampicillin, and ceftiofur and on the fragmentation of the sodium adduct for penicillin G, penicillin V, and cloxacillin. The method was validated at the U.S. FDA tolerance or safe level and at 5 or 2.5 ng/mL for these compounds in bovine milk. Theoretical method detection limits in milk based on a 10:1 signal to noise ratio were 0.2 ng/mL (ampicillin), 0.4 ng/mL (ceftiofur), 0.8 ng/mL (cephapirin), 1 ng/mL (amoxicillin and penicillin G), and 2 ng/mL (cloxacillin and penicillin V) using a nominal sample size of 5 mL.     

Determination of five macrolide antibiotic residues in honey by LC-ESI-MS and LC-ESI-MS/MS

Liquid chromatography-electrospray ionization mass spectrometry methods (LC-ESI-MS and LC-ESI-MS/MS) for the determination of five macrolide antibiotics including spiramycin, tilmicosin, oleandomycin, erythromycin, and tylosin in honey are presented. Macrolides were protonated to form singly and/or doubly charged pseudomolecular ions, depending on their chemical structures, in an electrospray positive ionization mode. Data acquisition under MS/MS was achieved by applying multiple reaction monitoring (MRM) of two or three fragment ion transitions to provide a high degree of sensitivity and specificity. The recoveries, that is, determined by LC-ESI-MS/MS, of the five macrolides at fortified levels of 6, 16, 40, and 80 microg/kg ranged from 75.5 to 135.7% in light honey and from 42.1 to 111.0% in dark honey. The ion ratios obtained under MS/MS were key criteria to confirm the identity of macrolides in incurred samples. LC-ESI-MS/MS method detection limits of the five macrolides were <0.1 microg/kg.     

Simultaneous determination of orysastrobin and its isomers in rice using HPLC-UV and LC-MS/MS

Orysastrobin is a new strobilurin-type fungicide to control leaf and panicle blast and sheath blight in rice. An analytical method was developed to determine the residues of orysastrobin and its two isomers, the main metabolite F001 and the major impurity F033, in hulled rice by the use of high-performance liquid chromatography with ultraviolet photometry (HPLC-UV) and liquid chromatography with tandem mass spectrometry (LC-MS/MS). All compounds were extracted with acetone from hulled rice samples. The extract was diluted with saline water, and an extraction step using dichloromethane/n-hexane partition was used to recover analytes from the aqueous phase. An n-hexane/acetonitrile partition and Florisil column chromatography were employed to further remove interfering coextractives prior to instrumental analysis. An octadecylsilyl column was successfully applied to identify orysastrobin and its isomers in sample extracts. Net recovery rates of orysastrobin, F001, and F033 from fortified samples ranged from 80.6 to 114.8% using HPLC-UV and LC-MS/MS. Relative standard deviations for the analytical methods were all <20%, and the quantification limits of the method were in the 0.002-0.02 mg/kg range. The proposed methods were reproducible and sufficiently accurate to evaluate the terminal residue of orysastrobin and its isomers in rice.     

LC-MS/MS analysis of neonicotinoid insecticides in honey: methodology and residue findings in Austrian honeys

An analytical method for the simultaneous determination of residues of eight neonicotinoid insecticides and two metabolites in honey using LC-MS/MS was developed and validated. Two approaches of sample preparation were investigated, with the final method involving acetonitrile extraction and subsequent cleanup by dispersive solid-phase extraction (QuEChERS type). Validation was based on quintuplicate analysis at three fortification levels and showed satisfactory recoveries (60-114%) and high precision (RSDs between 2.7 and 12.8%). Low limits of detection and quantification could be achieved for all analytes ranging from 0.6 to 5 μg/kg and from 2 to 10 μg/kg, respectively. Investigations of Austrian honey samples revealed the presence of acetamiprid, thiacloprid, and thiamethoxam residues in honey; however, no sample exceeded the maximum residue limits. On average, flower honey samples contained neonicotinoid residues in higher quantities compared to forest honey samples.     

Quantitation of total folate in whole blood using LC-MS/MS

An accurate method for measuring whole blood total folate using liquid chromatography with tandem mass spectrometry is described and compared to GC/MS and a chemiluminescence assay. Whole blood from normal adults (n = 15) was fortified with a [(13)C(6)]para-aminobenzoic acid (pABA) internal standard and treated with 12.1 N hydrochloric acid at 110 degrees C for 4 h to hydrolyze all folates to pABA. Contaminants in the hydrolysate were adsorbed onto a C18 SPE cartridge. The eluate containing the folate catabolite pABA was partitioned into ethyl acetate and methylesterified with trimethylsilyldiazomethane. The methyl-pABA derivatives were quantified by positive-ion atmospheric pressure chemical ionization (APCI)LC-MS/MS. An isocratic mobile phase of acetonitrile-water (70:30) (v/v) on a C18 analytical column was used with a postcolumn reagent of 0.025% formic acid. The limit of quantitation for folate was 56.6 nmol/L RBC, and the limit of detection was 22.6 nmol/L RBC. Folate levels as determined by LC-MS/MS correlated well with the chemiluminescence assay and a GC/MS method. This new LC-MS/MS method provides enhanced sample throughput (n = 36 per day) as compared to GC/MS methods. LC-MS/MS will enable accurate measurements of red blood cell (RBC) folate in nutrition surveys and clinical trials.     

Quantitation and confirmation of sulfamethazine residues in swine muscle and liver by LC and GC/MS

The present paper describes a liquid chromatographic (LC) method for purification of crude swine tissue extracts before gas chromatographic/mass spectrometric (GC/MS) quantitation and confirmation of sulfamethazine at low ppb levels. Fractions corresponding to sulfamethazine were collected, evaporated to dryness, N-methylated with diazomethane, concentrated, and analyzed by GC/MS. A mass spectrometer was set to selected ion monitoring (SIM) mode. Ions 233, 227, 228, and 92 m/z were detected. Ratio 227/233 m/z (sulfamethazine/internal standard, [phenyl 13C6] sulfamethazine) was used for quantitation, while ratios 228/227 and 92/227 m/z, respectively, were used for confirmation. Quantitation in spiked blank muscle tissue was tested from 100 to 1 ppb and found acceptable at all concentrations studied; coefficients of variations ranged from 4.9 to 14.4%. Similar results were obtained for liver tissue from 5 to 20 ppb; coefficients of variation ranged from 1.2 to 9.1%.     

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.     

Characterization of triterpene alcohol and sterol ferulates in rice bran using LC-MS/MS

Ferulic acid esters of triterpene alcohols and sterols in rice bran oil have been extensively studied and reported to possess important pharmacological actions. Inconsistent results on the numbers and structures of ferulates have been reported, primarily because of the analytical procedures employed. Conventional methods for analysis of phytosterol content in oil are carried out by characterization of trimethylsilylated derivatives (TMS) using GC-EI-MS after saponification of oils or individual compound isolated from oils. This study developed an LC-MS/MS method for the direct analysis of triterpene alcohol and sterol esters in rice bran oil. In addition to verifying the results of previous research, nine new relatively polar triterpene alcohol and sterol esters were characterized by their retention behaviors in LC and ESI-MS data from both negative- and positive-ion mode. This is the first evidence for the presence of hydroxylated ferulate esters and caffeate esters as part of gamma-oryzanol in rice bran. The method enables rapid and direct on-line characterization of triterpene alcohol and sterol esters in oils. LC-MS/MS equipped with reverse-phase LC and ESI-MS should be well-suited for identification and quantification of the polar metabolites of phytosterols in biological fluids after consumption of rice bran oil or other oils.     

固相萃取-高效液相色谱串联质谱法同时测定有机肥料中13种抗生素残留

建立了同时测定有机肥料中磺胺类、四环素类和喹诺酮类13种抗生素残留的高效液相色谱-串联质谱法,并对样品的前处理方法、仪器分析参数进行了优化。结果表明,13种抗生素在50~1 000 ng/mL间线性关系良好,相关系数均达到0.999以上,检出限在0.02~0.04 mg/kg,样品加标回收率在69%~111%之间,具有良好的重现性。该方法具有灵敏度高、准确度高等优点,可用于有机肥料中13种抗生素残留的同时检测。运用该方法在有机肥料实际检测中发现恩诺沙星、诺氟沙星、土霉素等抗生素残留。     

Methods for determination of ionophore-type antibiotic residues in animal tissues

Methods for the analysis of polyether antibiotics in animal tissues and fluids are described. For monensin and nigericin, only methods based on bioautography are available. For lasalocid, in addition to TLC bioautography for quantitation in chicken skin and fat, LC methods based on fluorescence detection have been developed for quantitation in animal blood, milk, liver, skin, and fat. In addition, a confirmatory method for lasalocid is described; this is based on purification by LC followed by silylation and pyrolysis gas chromatography-positive chemical ionization mass spectrometry.     

Collaborative validation of the QuEChERS procedure for the determination of pesticides in food by LC-MS/MS

Seven FDA pesticide laboratories collaborated to develop and validate an LC-MS/MS method to determine 173 pesticides in <20 min. The average determination coefficient (r2) was >0.99 for all but two compounds tested. The limits of detection were <20 ng/mL for all compounds and <10 ng/mL for 363 of the 368 transitions reported. The method was used to determine pesticides in two AOAC sponsored proficiency samples. The LC-MS/MS determination was used for the analysis of oranges, carrots and spinach using the QuEChERS (Quick, Easy, Cheap, Effective, Rugged, Safe) method. Each matrix was fortified at 20, 100, 400, and 1000 ng/g. No false positive responses were detected in controls of the three matrices. 165 pesticides had recoveries between 70 and 130%, and 161 had minimum detection levels less than 10 ng/g. Recoveries of 169 compounds for the 1000 ng/g spikes were within 50-150%. A matrix effect study indicated all three matrices caused a small net suppressing effect, the most pronounced attributable to the citrus matrix. The procedure proved to be accurate, precise, linear, sensitive and rugged, and adds 100 pesticides to the scope of the FDA pesticide program.     

High-throughput method for the quantitation of total folate in whole blood using LC-MS/MS

A high-throughput liquid chromatography tandem mass spectrometry (HT LC-MS/MS) method for red blood cell (RBC) folate analysis was developed from a previously described manual (M) LC-MS/MS method. The HT LC-MS/MS method used 96-well plates in which RBC folates were hydrolyzed with concentrated HCl in the presence of the [13C6]pABA internal standard (IS). The pH of the hydrolysate was adjusted to 5.0 before cleanup using 96-well plate OASIS HLB SPE cartridges. The analyte and IS were eluted with ethyl acetate/hexane (95:5, v/v) and methylated with methanol and trimethylsilyldiazomethane. The methylated analyte and IS were quantified with LC-MS/MS as previously described. The HT LC-MS/MS method was validated by determining the recovery of six different folate vitamers, which were quantitatively recovered (84-105% with CV<9.0%). RBC folate concentrations in whole blood samples correlated between HT and M LC-MS/MS methods (r=0.922, p<0.0001 for n=43 samples) and between the HT LC-MS/MS method and a chemiluminescence assay (r=0.664, p<0.001 for n=325 samples). Comparison of the results between HT LC-MS/MS and chemiluminescence methods with Bland-Altman difference plots and by ROC curve analysis indicates that the chemiluminescence assay underreports RBC folate concentrations. The HT LC-MS/MS method allows for high-throughput sample preparation for the analysis of RBC folate.     

Screening of foods containing proanthocyanidins and their structural characterization using LC-MS/MS and thiolytic degradation

A normal-phase HPLC-MS/MS method was applied to screen for proanthocyanidins in 88 different kinds of foods. Thirty-nine foods were found to contain proanthocyanidins. These foods include 19 kinds of fruits, eight cereals/beans, seven nuts, two beverages, two spices, and one vegetable. Twenty-five kinds of foods were found to contain both oligomeric (DP 10), and the other 14 foods contained only oligomers. Procyanidins with B-type linkages were detected as the only components in 21 foods and also as principal components in the others. Propelargonidins were identified in pinto bean, raspberry, strawberry, and almond, etc. Plum, avocado, peanut, curry, and cinnamon were identified as potential sources of A-type proanthocyanidins in addition to cranberry. Thiolytic degradation and MS/MS analyses indicated that the A-type linkages are present as a terminal unit in plum or between the extension units in curry, cinnamon, and avocado, whereas A-type linkages exist at both positions in cranberry and peanut.     

Analysis of methoxyfenozide residues in fruits, vegetables, and mint by liquid chromatography-tandem mass spectrometry (LC-MS/MS)

Methoxyfenozide [3-methoxy-2-methylbenzoic acid 2-(3,5-dimethylbenzoyl)-2-(1,1-dimethylethyl) hydrazide; RH-2485], in the formulation of INTREPID, was applied to various crops. Analysis of methoxyfenozide was accomplished by utilizing liquid-liquid extraction and partitioning, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Method validations for fruits, vegetables, and mint are reported. Methoxyfenozide mean recoveries ranged from 72 to 129% over three levels of fortification. The overall average of mean recoveries is 97 +/- 10%. The limit of quantitation for fruits, artichoke, cucumber, squash, and refined sugar was 0.010 ppm, with a detection limit of 0.005 ppm. For all other crops, the limit of quantitation was 0.050 ppm, with a detection limit of 0.025 ppm. No residues were found greater than the limit of quantitation in control samples. Residues above the limit of quantitation were found in all matrices except refined sugar. Foliage (bean, beet, pea, and radish) had greater residue levels of methoxyfenozide residue than their corresponding roots or pods. Other crop matrices contained <1.0 ppm of methoxyfenozide except artichoke, which had a mean of 1.10 ppm.     

Determination of grayanotoxins in biological samples by LC-MS/MS

A rapid LC-MS/MS method was developed for the quantitative determination of grayanotoxins I, II, and III in rumen contents, feces, and urine. The grayanotoxins were extracted from solid samples with methanol. The methanol extract was diluted with water and cleaned up using a reversed phase solid phase extraction column. HPLC separation was performed by reversed phase HPLC using a gradient of water and methanol containing 1% acetic acid. Determination was by positive ion electrospray ionization and ion trap tandem mass spectrometry. Grayanotoxin I quantitation was based on fragmentation of the sodium adduct ion at m/z 435 to a product ion at m/z 375. Grayanotoxins II and III were quantitated on the basis of fragmentation of the ion at m/z 335 to the product ion at m/z 299. The method detection limits were 0.2 microg/g in rumen contents and feces and 0.05 microg/g in urine. Fortifications at the detection limits and 10 times the detection limits of bovine rumen contents, caprine feces, and ovine urine were recovered in the range 80-114%. The diagnostic utility of the method was tested by analyzing samples submitted to the veterinary toxicology laboratory.     

Evaluation of two derivatization reagents for the determination by LC-MS/MS of ammonia in cigarette mainstream smoke

Ammonia in cigarette mainstream smoke was quantified by LC-MS/MS after derivatization. Two different reagents, fluorescamine and dansyl chloride, were investigated, but only the latter gave stable derivatives; therefore, it was considered the most appropriate choice. Smoke samples were collected on a Cambridge filter pad followed by an impinger containing a solution of hydrochloric acid. Ammonia was then derivatized with a 18.5 mM solution of dansyl chloride in acetonitrile at 70 °C for 30 min in a vial with the internal standard, (15)ND(4)Cl. The resulting derivative was analyzed by LC-MS/MS detection with an atmospheric pressure chemical ionization (APCI) interface in the positive ionization mode using multiple-reaction monitoring (MRM). Good linearity was obtained in the concentration range of 0.02-1.65 μg/mL (r(2) ≥ 0.999), and the limit of detection (LOD) was established at 0.006 μg/mL. This method has the advantage of being sensitive, efficient, and reliable and is not hindered by interferences from the sample matrix. It should thus be considered a reference method of choice for the determination of ammonia in smoke.     

Rugged LC-MS/MS survey analysis for acrylamide in foods

The described liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the detection of acrylamide in food entails aqueous room temperature extraction, SPE cleanup, and analysis by LC-MS/MS. The method is applicable to a wide variety of foods. [(13)C(3)]acrylamide is the internal standard. The limit of quantitation is 10 ppb (microg/kg). Data were obtained in duplicate from >450 products representing >35 different food types. The variability in analyte levels in certain food types suggests that it may be possible to reduce acrylamide levels in those foods.     

Determination of strophanthidin in ingesta and plant material by LC-MS/MS

An LC-MS/MS method was developed for the semiquantitative determination of strophanthidin glycosides in ingesta from animals. Strophanthidin glycosides were simultaneously extracted and hydrolyzed to the strophanthidin aglycone using aqueous methanolic hydrochloric acid and the extracts cleaned up using solid-phase extraction. Extracts were analyzed using reverse-phase HPLC coupled with positive ion electrospray mass spectrometry. Characteristic product ion spectra were produced by fragmentation of the [M + H](+) precursor ion for each analyte. Quantitation was performed using the internal standard method with digitoxigenin serving as the internal standard. The method detection limit was calculated to be 0.075 microg/g, and the limit of quantitation was calculated to be 0.24 microg/g for strophanthidin in control rumen samples. This method was used in diagnostic investigations to confirm fatal strophanthidin glycoside poisonings in horses.     

Confirmation of peanut protein using peptide markers in dark chocolate using liquid chromatography-tandem mass spectrometry (LC-MS/MS)

Detection of peptides from the peanut allergen Ara h 1 by liquid chromatography-mass spectrometry (LC-MS) was used to identify and estimate total peanut protein levels in dark chocolate. A comparison of enzymatic digestion subsequent to and following extraction of Ara h 1 from the food matrix revealed better limits of detection (LOD) for the pre-extraction digestion (20 ppm) than for the postextraction digestion (50 ppm). Evaluation of LC-MS instruments and scan modes showed the LOD could be further reduced to 10 ppm via a triple-quadrupole and multiple-reaction monitoring. Improvements in extraction techniques combined with an increase in the amount of chocolate extracted (1 g) improved the LOD to 2 ppm of peanut protein. This method provides an unambiguous means of confirming the presence of the peanut protein in foods using peptide markers from a major allergen, Ara h 1, and can easily be modified to detect other food allergens.