Determination of N-(carboxymethyl)fumonisin B(1) in corn products by liquid chromatography/electrospray ionization--mass spectrometry

It is well-known that fumonisin B(1) (FB(1)) in corn meal decreases during baking, frying, and cooking, but it is still not exactly clear how heating affects the formation of N-(carboxymethyl)fumonisin B(1) (NCM-FB(1)), the reaction product of FB(1) and reducing sugars. In model experiments corn grits were spiked with FB(1) (2 mg/kg) and D-glucose (50 g/kg) or sucrose (50 g/kg) and manufactured into extrusion products at various temperatures (160--180 degrees C) and moisture levels (16--20%). A liquid chromatography/electrospray ionization-mass spectrometry method using isotopically labeled fumonisin FB(1)-d(6) as an internal standard was developed for the determination of NCM-FB(1). For sample cleanup solid-phase C18 cartridges were used. The detection limit achieved with this method was 10 ng/g (signal-noise ratio = 3:1) using the protonated molecule [M + H](+) signal of NCM-FB(1) (m/z 780) in the selected ion monitoring mode. Low concentrations of NCM-FB(1) (29-97 ng/g) were detected in all samples spiked with D-glucose and FB(1), whereas those spiked with FB(1) and sucrose showed only NCM-FB(1) in samples produced at 180 degrees C (NCM-FB(1) = 27 ng/g). Various corn-containing food samples from the German market were analyzed for the presence of NCM-FB(1), FB(1), and hydrolyzed fumonisin B(1) (HFB(1)). All samples were contaminated with FB(1) (22--194 ng/g) and HFB(1) (5--247 ng/g). Six of nine samples contained NCM-FB(1) in low concentrations ranging from 10 to 76 ng/g. From these data and the low toxicity of NCM-FB(1) it can be concluded that the significance of NCM-FB(1) in food seems to be a minor one.     

Determination of aflatoxin M1 in milk by liquid chromatography with fluorescence detection

A liquid chromatographic (LC) method is proposed for the determination of aflatoxin M1 in milk. The method was successfully applied to both liquid whole and skim milk and also whole and skim milk powder. The samples are initially extracted with acetonitrile-water followed by purification using a silica gel cartridge and a C18 cartridge. Final analysis by LC was achieved using a radial compression module equipped with a 5 micron C18 column and a fluorescence detector. The method was successfully applied to samples at levels of 10 to 0.08 ppb added aflatoxin M1 with recoveries in the range of 70-98%.     

Determination of novobiocin residues in milk, blood, and tissues by liquid chromatography

Residues of novobiocin in milk, blood, and tissues can be detected by microbiological tests but cannot be distinguished from other antibiotics. A simple liquid chromatographic (LC) method was developed for identification of residues. Tissues were blended and milk and blood serum were mixed with 0.2M NH4H2PO4. The mixture was deproteinized by adding aqueous methanol and filtering. The LC apparatus consisted of a variable wavelength detector, set at 340 nm, an automatic loop injector, and a C18 column with guard cartridge. The flow rate was 1 mL/min and the solvent mixture of 0.01M H3PO4-acetonitrile-methanol was programmed from 50 + 0 + 50 (0-1 min) to 20 + 80 + 0 (20 min). Novobiocin was concentrated directly by solid-phase extraction on the analytical column. Five or more 200 microL aliquots of the filtrate in water-methanol (1 + 1) (adjusted if necessary) were injected with the column solvent at 50 + 0 + 50. After the final injection, the program was run to completion. Recoveries were 90-100% with sensitivities of 0.05 ppm or less. The procedure should be adaptable for use with formulations and feeds.     

Determination of fluoroquinolones in milk samples by postcolumn derivatization liquid chromatography with luminescence detection

A liquid chromatography (LC) method with luminescence detection for the determination of eight quinolone antibiotics is reported. The system encompasses three consecutive steps: (a) chromatographic separation using reverse-phase mode (RP-LC), (b) postcolumn derivatization reaction, and (c) luminescence detection by monitoring fluorescence (FL) and time-resolved (TR) signals. The derivatization step is based on the reaction between quinolones and terbium(III) to form luminescent chelates, which were determined at lambda(ex) 340 and lambda(em) 545 nm (FL mode) or at lambda(ex) 281 and lambda(em) 545 nm (TR mode). Dynamic ranges of the calibration graphs, obtained with standard solutions of analytes and FL and TR modes, respectively, were 190-3500 and 316-2000 ng mL-1 for marbofloxacin, 8-3500 and 8.1-1500 ng mL-1 for ciprofloxacin, 6.2-3500 and 13-1500 ng mL-1 for danofloxacin, 7.4-3500 and 8.4-1500 ng mL-1 for enrofloxacin, 14-3500 and 20-2000 ng mL-1 for sarafloxacin, 12.5-3500 and 13.9-1200 ng mL-1 for difloxacin, 7.6-3500 and 13-3000 ng mL-1 for oxolinic acid, and 9-2000 and 130-3000 ng mL-1 for flumequine. Limit of detection values obtained using FL and TR modes, respectively, were 60 and 95 ng mL-1 for marbofloxacin, 2 and 2.4 ng mL-1 for ciprofloxacin, 1.9 and 3.9 ng mL-1 for danofloxacin, 2.2 and 2.5 ng mL-1 for enrofloxacin, 3.8 and 7 ng mL-1 for sarafloxacin, 4 and 4.2 ng mL-1 for difloxacin, 2.3 and 4 ng mL-1 for oxolinic acid, and 2.7 and 40 ng mL-1 for flumequine. The precision was established at two concentration levels of each analyte and expressed as the percentage of relative standard deviation with values ranging between 1.9 and 7.8%. The validation procedure for the analysis of samples was carried out using European Community recommendations, and the decision limit and detection capability were calculated for bovine whole milk. The method was applied to whole, semiskimmed, and skimmed milk samples spiked with the target analytes, and the recoveries ranged between 93.3 and 106.0%.     

Determination of carotenoids in spear shrimp shells (Parapenaeopsis hardwickii) by liquid chromatography

The objectives of this study were to develop a high-performance liquid chromatography method for analysis of carotenoids in spear shrimp shells (Parapenaeopsis hardwickii) and to compare the extraction efficiency of carotenoids by supercritical carbon dioxide (SCD) and solvents. Results showed that the most appropriate HPLC method was accomplished by employing a Cosmosil 5C18-AR-II column and a mobile phase of methanol-dichloromethane-acetonitrile (90:5:5, v/v/v) (A) and water (100%) (B) with the following gradient elution: 92% A and 8% B in the beginning, decreased to 4% B in 9.5 min, 1% B in 26 min, 0% B in 35 min, maintained for 25 min, and returned to 92% A and 8% B in 61 min. All-trans-astaxanthin and its two cis isomers, as well as five astaxanthin monoesters and 11 diesters were resolved within 60 min with a flow rate at 2 mL/min and detection at 480 nm. Astaxanthin diesters were found to contain 12 fatty acids, of which palmitic acid and stearic acid constituted a large portion, whereas astaxanthin monoesters were found to contain 10 fatty acids with arachidonic acid dominating. Solvent extraction could generate a higher content of trans-astaxanthin and astaxanthin esters, while SCD extraction could produce greater levels of 9-cis-astaxanthin and 13-cis-astaxanthin.     

Determination of sulfite in foods by headspace liquid chromatography

Sulfite was determined in a variety of foods by liquid chromatography (LC) after the samples were mixed with a solution containing mannitol, FeSO4, and Na2HPO4, adjusted to pH 11, and left to stand for 15 min at room temperature. An aliquot of the mixture was placed in a headspace vial and mixed with 50% H3PO4. After 15 min, a portion of the headspace was removed with a syringe containing LC mobile phase without acetonitrile. The syringe was shaken and an aliquot of the solution was analyzed on an anion exchange column with a mobile phase of 0.03M methane sulfonate (pH 10.8) containing 5% acetonitrile. Sulfite was detected amperometrically (glassy carbon electrode) at +0.7 V. The method was successfully compared to the FDA-modified Monier-Williams procedure for a variety of foods. Minimum detectable levels were about 1 microgram/g, based on a 15 g sample.     

Determination of thiamphenicol in bovine plasma by liquid chromatography

A liquid chromatographic method is described for the measurement of thiamphenicol in bovine plasma. The plasma (1 mL) is extracted with ethyl acetate. After the solvent is evaporated under a stream of nitrogen, the residue is reconstituted in methanol-water and analyzed by reverse-phase liquid chromatography with UV detection at 224 nm. The intra-day recoveries for bovine plasma spiked with 5 and 50 micrograms/mL of thiamphenicol were 102 and 101%, respectively, with coefficients of variation of 2.40 and 0.28%, respectively. The interday recoveries for the 5 and 50 micrograms/mL samples were 103 and 101%, respectively, with coefficients of variation of 3.40 and 0.94%, respectively. The sensitivity of the method allows quantitation to at least the 100 ng/mL level.     

Determination of N-methylcarbamate pesticides in liver by liquid chromatography

A liquid chromatographic (LC) method using a 2-step purification technique for the simultaneous determination of 10 carbamates in bovine, swine, and duck livers has been developed. Carbamates are extracted from liver samples with methylene chloride. After evaporation, the residues from the extract are dissolved in methylene chloride-cyclohexane (1 + 1) and cleaned up by gel permeation chromatography. The eluate containing carbamate residues is evaporated to dryness, reconstituted in methylene chloride, further purified by passing it through an aminopropyl Bond Elut extraction cartridge, and analyzed by liquid chromatography using post-column derivatization with orthophthalaldehyde and fluorescence detection. Excitation and emission are set at 340 and 418 nm, respectively. Liver samples for beef, pork, and duck were fortified with 5, 10, and 20 ppb of mixed carbamate standards. The average of 10 recoveries of 10 carbamates at all 3 levels of fortification was greater than 80% with coefficients of variation less than 17%.     

Determination of ampicillin residues in fish tissues by liquid chromatography

A liquid chromatographic (LC) method is described for determination of ampicillin residues in fish tissues. The drug is extracted from tissues with methanol, and the extract is evaporated to dryness. This residue is cleaned up by Florisil cartridge chromatography. LC analysis is carried out on a Nucleosil C18 column, and ampicillin is quantitated by ultraviolet detection at 222 nm. Recoveries of ampicillin added to tissues at levels of 0.2 and 0.1 ppm were 73.2 and 61.5%, respectively. The detection limit was 3 ng for ampicillin standard, and 0.03 ppm in tissues.     

Determination of nucleotides in infant formula by ion-exchange liquid chromatography

Nucleotide-supplemented infant formula has been shown to positively modify the composition of intestinal microflora, emulating the attribute of human milk. Quantification of nucleotides in infant formula is of interest because of its applicability in quality and safety assessments. There is no standard method for the analysis of nucleotides in infant formula. In the present study, ion-exchange liquid chromatography (IELC)- and centrifugal ultrafiltration (CUF)-based protocols were developed for routine determination of additive nucleotides in infant formula. Five target nucleotides, guanosine 5'-monophosphate (GMP), inosine 5'-monophosphate (IMP), uridine 5'-monophosphate (UMP), cytidine 5'-monophosphate (CMP), and adenosine 5'-monophosphate (AMP) were measured by IELC with a mobile phase of 50 mM diammonium hydrogen phosphate buffer, pH 4.0, with UV detection at 254 nm. The calibration was linear over the range 0.5-50 microg/mL; R(2) = 0.999. The calculated LOD and LOQ were 0.01-0.05 microg/mL and 0.05-0.5 microg/mL, respectively. Recovery values (spiked concentration levels: 0.5, 5, and 10 microg/mL) ranged from 85.0 +/- 1.4% to 92.3 +/- 2.1% using only CUF preparation. This was applied to measure the concentration of five nucleotides in common infant formulas.     

Determination of olaquindox residues in swine tissues by liquid chromatography

A liquid chromatographic (LC) method is described for determination of olaquindox residues in swine tissues. The drug is extracted from tissues with acetonitrile, and the extract is evaporated to dryness. This residue is cleaned up by alumina column chromatography. LC analysis is carried out on a Nucleosil C18 column, and olaquindox is quantitated by ultraviolet detection at 350 nm. The average recoveries of olaquindox added to tissues at levels of 0.2, 0.1, and 0.05 ppm were 74.0, 68.6, and 66.3%, respectively. The detection limit was 2 ng for olaquindox standard and 0.02 ppm in tissues.     

Determination of sterigmatocystin in barley by acetylation and liquid chromatography

The estimation of sterigmatocystin by fluorescence liquid chromatographic analysis of the acetate derivative has eliminated the background interference normally encountered in analysis of underivatized sterigmatocystin in barley. Barley samples are extracted with acetonitrile-water; the extract is then washed with hexane, transferred to chloroform, and eluted from a silica gel column with cyclohexane-ethyl acetate. The extract is heated with pyridine and acetic anhydride for 3 h to give a stable derivative. Reverse-phase liquid chromatography, using a methanol-water mobile phase gradient and fluorescence detection, is the method of determination. Recoveries from barley samples spiked with 20, 110, 190, and 765 micrograms sterigmatocystin/kg were 31, 69, 75, and 96%, with coefficients of variation between 2.8 and 5.4%. Sterigmatocystin is confirmed by comparing retention times in underivatized extracts of samples and standards, using methanol-water (3 + 2) mobile phase and ultraviolet detection.     

Determination and confirmation of 5-hydroxyflunixin in raw bovine milk using liquid chromatography tandem mass spectrometry

A method was developed and validated to determine 5-hydroxyflunixin in raw bovine milk using liquid chromatography tandem mass spectrometry (LC/MS/MS). The mean recovery and percentage coefficient of variation (%CV) of 35 determinations for 5-hydroxyflunixin was 101% (5% CV). The theoretical limit of detection was 0.2 ppb with a validated lower limit of quantitation of 1 ppb and an upper limit of 150 ppb. Accuracy, precision, linearity, specificity, ruggedness, and storage stability were demonstrated. A LC/MS/MS confirmatory method using the extraction steps of the determinative method was developed and validated for 5-hydroxyflunixin in milk from cattle. Briefly, the determinative and confirmatory methods were based on an initial solvent (acetone/ethyl acetate) precipitation/extraction of acidified whole milk. The solvent precipitation/extraction effectively removed incurred ((14)C) residues from milk samples. The organic extract was then purified by solid phase extraction (SPE) using a strong cation exchange cartridge (sulfonic acid). The final SPE-purified sample was analyzed using LC/MS/MS. The methods are rapid, sensitive, and selective and provide for the determination and confirmation of 5-hydroxyflunixin at the 1 and 2 ppb levels, respectively.     

Determination of nicarbazin in chicken tissues by liquid chromatography and confirmation of identity by thermospray liquid chromatography/mass spectrometry

A liquid chromatographic method is described for the quantitative measurement of nicarbazin in chicken liver, fat, muscle, and skin tissues. The 4,4'-dinitrocarbanilide (DNC) portion of nicarbazin is extracted from tissues with ethyl acetate. After filtration and evaporation, the extract is purified by liquid-liquid partitioning with acetonitrile-hexane and alumina cartridge chromatography. DNC is separated and measured by reverse-phase liquid chromatography (RP-LC) with an octadecylsilyl (ODS) column and a UV detector set at 340 nm. The overall average recovery of DNC added to tissues was 83.4 +/- 3.1%. The lowest level validated in tissues by this procedure was 0.10 ppm. The limit of detection was estimated to be 0.020 ppm. This method provides a sensitive, selective, rapid, and reproducible alternative to existing purification, separation, and detection techniques, such as differential pulse polarography and colorimetry, for determination of nicarbazin in chicken tissues. Identity of DNC is confirmed by subjecting the purified extracts to thermospray-LC/mass spectrometric analysis using negative-ion detection and selected ion monitoring. Three structural-indicating ions at m/z 302, 272, and 164 are monitored in the thermospray-mass spectrum which are characteristic of the DNC molecule.