Determination of tetracycline antibiotic residues in edible swine tissues by liquid chromatography with spectrofluorometric detection and confirmation by mass spectrometry

A sensitive and specific method is described for the simultaneous determination of oxytetracycline, tetracycline (TC), and chlortetracycline residues in edible swine tissues, by combining liquid chromatography with spectrofluorometric and mass spectrometry detection. The procedure involved a preliminary extraction with EDTA-McIlvaine buffer acidified at pH 4.0, followed by solid-phase extraction cleanup using a polymeric sorbent. The liquid chromatography analysis was performed with spectrofluorometric detection after postcolumn derivatization with magnesium ions. The limits of quantification were 50 microg/kg for muscle and 100 microg/kg for kidney tissues. The recovery values were greater than 77.8% for muscle and 65.1% for kidney. The method has been successfully used for the quantification of tetracyclines in swine tissues samples. The selective liquid chromatography mass spectrometric analysis for confirmation of oxytetracycline in one positive swine muscle sample was made by atmospheric pressure chemical ionization (APCI). The APCI mass spectra of the TCs gave the protonated molecular ion and two typical fragment ions, required for their confirmation in single ion monitoring scan mode in animal tissues.     

Confirmatory method for sulfamethazine residues in cattle and swine tissues, using gas chromatography--chemical ionization mass spectrometry

A gas chromatographic (GC) method has been reported for the determination of sulfamethazine residues in cattle and swine tissues. The extracts from this procedure were found to be directly amenable to examination by gas chromatography-mass spectrometry (GC-MS), allowing positive confirmation of an apparent residue of sulfamethazine. Chemical ionization mass spectrometry (CIMS) was chosen as the MS technique because it generated an ion indicative of intact sulfamethazine and fragment ions indicative of the amine functionality and sulfanil moiety. Positive ion (PI) chemical ionization mass spectrometry was adequate by itself for a confirmatory technique. Negative ion (NI) chemical ionization mass spectrometry alone could not be used for the confirmatory analysis of sulfamethazine, but it did offer a means to check the quantitative data from the positive ion analyses and provided complementary confirmatory data. Satisfactory recoveries were obtained for sulfamethazine in swine and cattle tissues at the tolerance level of 0.1 ppm. Apparent sulfamethazine residues in control tissues were less than 0.01 ppm.     

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.     

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.     

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.     

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.     

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.     

Determination of desaminosulfamethazine, sulfamethazine, and N4-acetylsulfamethazine by gas chromatography with electron capture detection and confirmation by gas chromatography-chemical ionization mass spectrometry

An electron capture gas chromatographic method for the determination of sulfamethazine was modified to separate and quantitate simultaneously sulfamethazine and 2 of its metabolites, N4-acetylsulfamethazine and desaminosulfamethazine. The modified method was applied to incurred residues in a veal calf depletion study and to incurred residues in swine tissues. With capillary column gas chromatography-positive ion chemical ionization mass spectrometry, confirmation of the identities of incurred desaminosulfamethazine, N4-acetylsulfamethazine, and sulfamethazine in tissues was obtained from a single injection.     

Determination of spinosad and its metabolites in food and environmental matrices. 2. Liquid chomatography-mass spectrometry

A selective and sensitive method utilizing liquid chromatography-mass spectrometry (LC-MS) has been developed for determining residues of the natural insect control agent spinosad in several crop matrices that are difficult to analyze by HPLC with UV detection. The method determines the active ingredients (spinosyns A and D) and three minor metabolites (spinosyns B and K and N-demethylspinosyn D) in alfalfa hay, wheat hay, wheat straw, sorghum fodder, and corn stover. The analytes are extracted from the samples with an acetonitrile/water solution, and the extracts are purified by solid phase extraction with a C(18) disk and a silica cartridge. All five analytes are determined simultaneously in a single injection using positive atmospheric pressure chemical ionization LC-MS with selected ion monitoring. The average recoveries ranged from 69 to 96% with standard deviations ranging from 4 to 15%. The method has a validated limit of quantitation of 0. 01 microgram/g and a limit of detection of 0.003 microgram/g. The LC-MS method can also provide residue confirmation in addition to quantitation.     

Simultaneous identification of soyasaponins and isoflavones and quantification of soyasaponin Bb in soy products,using liquid chromatography/electrospray ionization-mass spectrometry

A method has been developed for simultaneous identification of soyasaponins and soy isoflavones in soy products, based on liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS). Soy-based nutraceutical products were analyzed by LC/ESI-MS with detection of protonated and sodiated molecular ions, as well as characteristic fragment ions for these compounds. Soy isoflavones were characterized by a strong protonated molecular ion in addition to corresponding [aglycone + H](+) ions. Monitoring the soyasaponin-specific protonated aglycone and dehydrated aglycone ions throughout the chromatogram provided a unique fingerprint for soyasaponin content in the samples. This mass spectrometric fingerprint also allowed immediate classification of the soyasaponin analytes as group A or B soyasaponins, based on the unique masses of aglycone ions observed for each class. Quantification of soyasaponin B(b) in soy-derived materials, based on the use of a purified soyasaponin B(b) standard and a glycyrrhizin internal standard, has been accomplished.     

Determination of postharvest fungicides in fruit juices by solid-phase extraction followed by liquid chromatography electrospray time-of-flight mass spectrometry

A multiresidue method using liquid chromatography-time-of-flight mass spectrometry (LC-TOFMS) has been developed for the quantitative analysis of five widely used postharvest fungicides (carbendazim, thiabendazole, imazalil, prochloraz, and iprodione) and two of their transformation products (imazalil and prochloraz metabolites) in fruit juices. LC-TOFMS in positive electrospray ionization mode was used to quantify and confirm trace levels of these fungicides in fruit juices. The proposed method consists of a sample treatment step based on solid-phase extraction using hydrophilic-lipophilic-balanced polymer-based reverse-phase SPE cartridges (Oasis HLB) and methanol as an eluting solvent. Fruit-juice extracts spiked at different fortification levels (10 and 20 microg L(-1)) yielded average recoveries in the range of 71-109% with RSD (%) below 15%. Subsequent identification, confirmation, and quantitation were carried out by LC-TOFMS analysis. The confirmation of the target species was based on accurate mass measurements of protonated molecules ([M+H]+) and fragment ions, obtaining routine accuracy errors lower than 2 ppm in most cases. The obtained limits of detection (LODs) of the proposed method were in the range of 0.08-0.45 microg L(-1). Finally, the proposed method was successfully applied to the analysis of 23 fruit juice samples collected from different European countries and the United States, showing the potential applicability of the method in routine analysis. Over 50% of the samples tested contained pesticide residues, but relatively low concentration levels were found.     

Rapid quantitation and confirmation of aflatoxins in corn and peanut butter, using a disposable silica gel column, thin layer chromatography, and gas chromatography/mass spectrometry

A simple, rapid, and solvent-efficient method for determining aflatoxins in corn and peanut butter is described. Aflatoxins B1, B2, G1, and G2 were extracted from 50 g sample with 200 mL methanol-water (85 + 15). A portion of the extract was diluted with 10% NaCl solution to a final concentration of 50% methanol, and then defatted with hexane. The aflatoxins were partitioned into chloroform. The chloroform solution was evaporated, and the residue was placed on a 0.5 g disposable silica gel column. The column was washed with 3 mL each of hexane, ethyl ether, and methylene chloride. Aflatoxins were eluted with 6 mL chloroform-acetone (9 + 1). The solvent was removed by evaporation on a steam bath, and the aflatoxins were determined using thin layer chromatography (TLC) with silica gel plates and a chloroform-acetone (9 + 1) developing solvent. Overall average recovery of aflatoxin B1 from corn was 82%, and the limit of determination was 2 ng/g. For mass spectrometric (MS) confirmation, aflatoxin B1 in the extract from 3 g sample (20 ng/g) was purified by TLC and applied by direct on-column injection at 40 degrees C into a 6 m fused silica capillary gas chromatographic column. The column was connected directly to the ion source. After injection, the temperature was rapidly raised to 250 degrees C, and the purified extract was analyzed by negative ion chemical ionization MS.     

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.     

GC/MIP/AED method for pesticide residue determination in fruits and vegetables.

This research describes the results of a gas chromatography/microwave induced plasma/atomic emission detection (GC/MIP/AED) method performed on a Hewlett-Packard 5921A system for pesticide residue analysis in fruits and vegetables. A total of 6 experiments were conducted: (1) sensitivity and linearity studies for elements S, P, Cl, and N by analyzing dursban; (2) a study of instrument response to Cl concentration in pesticide molecules; (3) organochlorinated pesticide recoveries; (4) organophosphate pesticide recoveries; (5) carbamate pesticide recoveries; and (6) investigation of metallic pesticides with plictran and vendex as standards. The rank according to sensitivity and linearity was found to be as follows: S-181 greater than P-178 greater than Cl-479 greater than N-174. Instrument response to the concentration of chlorine atoms in the pesticide molecule was linear, with a correlation coefficient of 0.89. Recoveries of organochlorinated pesticides were 91.7-109.3%, with the exception of citrus, whose recovery was affected by coeluting interferences. Organophosphate recoveries were 73.2% or higher, except for the cygon oxygen analog, which degraded in the GC system under all circumstances. Carbamate recoveries were inconsistent quantitatively; however, the information generated from elements N and S were useful for qualitative confirmation of other methods, such as LC postcolumn derivatization analysis. Overall, the GC/MIP/AED method is powerful for qualitative confirmation in pesticide residue analysis. The instrument's capability of acquiring multi-elements (Cl and P) selectively and accurately is an alternative method for organochlorinated and organophosphate pesticide residue analyses. In addition, the GC/MIP/AED system is easy to use, simple to maintain, and its chromatograms can be interpreted by any chromatography analyst without much prior training.     

Evaluation of capillary gas chromatography for pesticide and industrial chemical residue analysis. II. Comparison of quantitative results obtained on capillary and packed columns

Results of pesticide and industrial chemical residue determinations, using both capillary and packed column gas chromatography (GC), in 3 Food and Drug Administration (FDA) laboratories have been compiled and compared. Samples consisted of food products collected for routine residue screening by the respective laboratories. Extracts were prepared by conventional multiresidue methodology. Capillary column systems and operating conditions were selected at the discretion of each laboratory and were therefore variable, although split/splitless injectors in the split mode were used with prescribed precautions in all cases. Packed column systems were operated as specified in the FDA Pesticide Analytical Manual (PAM). Overall correlation between the 2 systems, expressed as the average ratio of packed column result to capillary column result, was 0.99 for 120 determinations in 41 samples. The higher resolving power of the capillary systems allowed quantitation of several residues that were incompletely separated and therefore unquantifiable using the packed columns. Capillary column GC with the split injection technique, used with appropriate precautions, was found to be both reliable and advantageous for regulatory determination of pesticide and industrial chemical residues in foods and feeds.     

Quantitation of ochratoxin A in South African wines

The natural occurrence of the carcinogenic mycotoxin ochratoxin A (OTA) in wines sold in local retail outlets in South Africa and Italy was investigated by HPLC analysis with fluorescence detection following cleanup by immunoaffinity column. All 24 local South African wines tested (15 white and 9 red) were found to contain detectable levels (>0.01 microg/L) of OTA, with a mean of 0.16 microg/L in the white wines and a mean of 0.24 microg/L in the red wines. Results were subsequently confirmed by LC-MS analysis using positive ion electrospray ionization with collision-induced dissociation of the protonated molecular ion [M + H](+) at m/z 404 and selected reaction monitoring of the resultant product ions [M + H - H(2)O - CO](+) at m/z 358 and [M + H - H(2)O](+) at m/z 386. Comparison with the fluorescence method gave a significant correlation (r = 0.87; p < 0.01). Although OTA contamination was present in all of the South African samples analyzed, levels were well below the suggested European Union limit of 0.5 microg/kg. The highest level found in a locally purchased wine was 0.39 microg/L in a blend of local and imported Spanish red wine. Of the eight Italian wines analyzed, only two red wines were contaminated above the suggested maximum level.     

Use of humidified air in optimizing APCI-MS response in breath analysis

An atmospheric-pressure chemical ionization-mass spectrometer (APCI-MS) system was modified to introduce gaseous samples as well as to humidify sheath and auxiliary gases. Solutions of single odorants in pentane were evaporated inside a vessel in a dry environment (relative humidity 0-10%) and in a human-breath-like environment (relative humidity 88-98%) and subsequently analyzed in the positive ion mode. The results indicated that using dry nitrogen as sheath and auxiliary gases led to strong fragmentation of the molecules combined with a low sensitivity of detection. Humidification of both gases not only increased sensitivity but also resulted in the protonated molecular ion as the base peak in 5 of the 6 compounds studied (ethyl butyrate, 3-methyl-3-phenyl glycidic acid ethyl ester, gamma-decalactone, (E)-2-hexenal, and cinnamic aldehyde). Only hexanal, which formed a cluster (m/z 183), did not have the protonated molecular ion as base peak. It was found that the humidity of the sample itself did not have any influence on sensitivity or fragmentation.     

Chloramphenicol extraction from honey, milk, and eggs using polymer monolith microextraction followed by liquid chromatography-mass spectrometry determination

A rapid confirmatory method for monitoring chloramphenicol (CAP) residues in honey, whole milk, and eggs is presented. This method is based on the polymer monolith microextraction (PMME) technique and high-performance liquid chromatography (HPLC)-electrospray ionization mass spectrometry (MS). A poly(methacrylic acid-ethylene glycol dimethacrylate) monolithic capillary column was selected as the extraction medium. To obtain optimum extraction efficiency, several parameters related to PMME were investigated. After dissolution in 20 mM phosphate solution at pH 4.0 and centrifugation, honey, eggs, or milk samples were directly passed through the extraction tube. The LC-MS instrument was equipped with an electrospray ion source and a single quadrupole. The eluates were analyzed by LC-MS in the negative-ion mode and by monitoring a pair of isotopic ions for the target compound. The in-source collision-induced dissociation process produced confirmatory ions. The recoveries of CAP from real samples spiked at 0.1-10 ng/g (honey), 0.2-10 ng/mL (milk), and 0.2-10 ng/g (egg) were in the range of 85-102%, with relative standard deviations ranging between 2.1% and 8.9%. The limits of detection (S/N = 3) were 0.02 ng/g, 0.04 ng/mL, and 0.04 ng/g in honey, milk, and eggs, respectively. The proposed method was proved to be robust in monitoring CAP residue in honey, milk, and eggs.     

Negative ion chemical ionization mass spectrometric method for confirmation of identity of aflatoxin B1: collaborative study

An interlaboratory study of a negative ion chemical ionization mass spectrometric (MS) confirmation procedure for aflatoxin B1 was conducted in laboratories in the United States, England, and West Germany. Twelve partially purified, dry film extracts from naturally and artificially contaminated roasted peanuts, cottonseed, and ginger root containing varying quantities of aflatoxin B1 were distributed to the participating laboratories. The extracts required additional cleanup before MS analysis, using either an acidic alumina column and preparative thin layer chromatography (TLC) or a 2-dimensional TLC procedure. Recovery of purified aflatoxin B1 was influenced by the degree of recovery of sample from acid alumina and/or the TLC plate and incomplete elution of aflatoxin B1 from silica gel. Factors affecting MS confirmation included the purity and recovery of aflatoxin and MS instrument sensitivity. Aflatoxin B1 identity was confirmed in 19.5, 90.9, and 100% of samples containing less than 5, 5-10, and greater than 10 ng aflatoxin B1/g product, respectively, by solid probe introduction using full mass scans. The MS method has been adopted official first action.     

Determination of anabolic steroids in muscle tissue by liquid chromatography-tandem mass spectrometry

A specific and sensitive method based on liquid chromatography-tandem mass spectrometry using atmospheric pressure chemical ionization (LC-APCI-MS/MS) has been developed for the determination of four anabolic steroids [trenbolone, methylboldenone, methyltestosterone, and norethandrolone] in bovine muscle. Methyltestosterone- d 3 was used as internal standard. The procedure involved enzymatic hydrolysis, extraction with tert-butyl methyl ether, defattening, and final cleanup with solid-phase extraction with Oasis HLB cartridges. The analytes were analyzed by reversed-phase LC-MS/MS, acquiring two diagnostic product ions from the chosen precursor [M + H] (+) for the unambiguous confirmation of hormones. The method was validated according to the European Commission Decision 2002/657/EC for the detection and confirmation of residues in products of animal origin. The limits of detection (LOD) and limits of quantitation (LOQ) were found to be 0.3 ng/g and 1.0 ng/g, respectively. The accuracy and precision have been determined, with recoveries ranging from 83% to 104% and the CV factor not exceeding the value of 7%. The decision limits CCalpha were calculated and ranged from 0.05 to 0.15 ng/g while the detection capabilities CCbeta ranged from 0.09 to 0.25 ng/g. The method proved to be sensitive and reliable and thus renders an appropriate means for residue analysis studies.