Gas chromatographic determination of N-nitrosamines, aromatic amines, and melamine in milk and dairy products using an automatic solid-phase extraction system

A reliable analytical method was presented for the simultaneous determination of six N-nitrosamines, nine aromatic amines, and melamine in milk and dairy products using gas chromatography coupled with mass spectrometry. The sample treatment includes the precipitation of proteins with acetonitrile, centrifugation, solvent changeover by evaporation, and continuous solid-phase extraction for cleanup and preconcentration purposes. Samples (5 g) containing 0.15-500 ng of each amine were analyzed, and low detection limits (15-130 ng/kg) were achieved. Recoveries for milk and dairy products samples spiked with 1, 10, and 50 μg/kg ranged from 92% to 101%, with intraday and interday relative standard deviation values below 7.5%. The method was successfully applied to determine amine residues in several milk types (human breast, cow, and goat) and dairy products.     

Naturally occurring estrogens in processed milk and in raw milk (from gestated cows)

The occurrence of the steroid hormones estrone (E1), 17alpha-estradiol (alphaE2), 17beta-estradiol (betaE2), and estriol (E3) in processed bovine milk with different fat contents and in raw milk from (non)gestated cows was investigated. Following liquid extraction, optional enzymatical deconjugation, C18 solid-phase extraction, and derivatization, estrogens were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Free and deconjugated E1 (6.2-1266 ng/L) was the major estrogen followed by alphaE2 (7.2-322 ng/L) and betaE2 (5.6-51 ng/L), whereas E3 was detected regularly at the detection limit of 10 ng/L. The lowest and highest concentrations were determined in raw milk from nonpregnant and from cows in the third trimester of gestation, respectively. The estrogen concentration in processed milk coincides with that of raw milk between first and second trimesters, reflecting the contribution of lactating pregnant cows to the final consumable product. The daily intake of total investigated estrogens through milk is 372 ng, which is dramatically more than currently recognized.     

Determination of dapsone in muscle tissue and milk using high-performance liquid chromatography-tandem mass spectrometry

A precise and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of dapsone in muscle tissue and milk has been developed. The sample preparation was based on extraction with organic solvent and automated solid-phase extraction (SPE) cleanup. At least three product ions were monitored for the analyte. The method was validated according to the European Decision 2002/657/EC. Estimated analytical limits were 0.0018 ng/g for CCα and 0.0031 ng/g for CCβ in meat and milk. An excellent linear concentration range was observed for both matrices with a correlation coefficient better than 0.997. Recoveries were 105-117% in meat and 101-108% in milk, with satisfactory precision and coefficients of variance (CV) less than 8%. Additionally, a simplified quantification approach was successfully evaluated depending only on the response factor (F) without the use of calibration curve. The developed method provides reliable and sensitive identification and quantification of dapsone in meat and milk.     

Rapid determination of estrogens in milk samples based on magnetite nanoparticles/polypyrrole magnetic solid-phase extraction coupled with liquid chromatography-tandem mass spectrometry

In this study, a nanocomposite of polypyrrole-coated magnetite nanoparticles (denoted as MNPs/PPy) was prepared and employed as magnetic solid-phase extraction (MSPE) sorbent for extraction of estrogens from milk samples. Because the polypyrrole coating possessed a highly π-conjugated structure and hydrophobicity, MNPs/PPy showed excellent performance for the estrogen extraction. Estrogens could be captured directly by MNPs/PPy from milk samples without protein precipitation. Moreover, the extraction could be carried out within 3 min. Thus, a rapid, simple, and effective method for the analysis of estrogens in milk samples was established by coupling MNPs/PPy-based MSPE with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The limits of detections for estrogens investigated were in the range of 5.1-66.7 ng/L. The recoveries of estrogens (concentration range of 0.5-20 ng/mL) from milk samples were in the range of 83.4-108.5%, with relative standard deviations ranging between 4.2 and 15.4%.     

Rapid determination of total cholesterol in homogenized milk

A rapid method that is amenable to automation has been developed for the determination of total cholesterol in homogenized milk. The milk sample is saponified in ethanolic KOH in the presence of an internal standard, cholestane. Cholesterol and the internal standard are then isolated by solid-phase extraction on a nonpolar adsorbent and eluted with organic solvent. The evaporated extract is derivatized and analyzed by capillary gas chromatography. Average recovery of cholesterol acetate added to milk prior to saponification was 95%. The average relative standard deviation for repeated analyses was 2%. The limit of detection for this method is 2 mg/100 g. Twenty samples can be analyzed by one analyst in a normal work day if the gas chromatograph is equipped with an autosampler. This method has been compared with a modified AOAC method for the determination of total cholesterol. At a confidence level of 95%, no difference was observed between the 2 methods.     

Determination of five macrolide antibiotic residues in raw milk using liquid chromatography-electrospray ionization tandem mass spectrometry

A confirmatory method using liquid chromatography-electrospray ionization tandem mass spectrometry for determination of five macrolide antibiotics including spiramycin, tilmicosin, oleandomycin, erythromycin, and tylosin in raw milk is presented. Macrolides were extracted from raw milk by acetonitrile, and sample extracts were further cleaned up using solid-phase extraction cartridges. Data acquisition was achieved using multiple reaction monitoring, that is, two transitions, to provide a high degree of sensitivity and specificity. Matrix-matched standard calibration curves with the use of roxithromycin as an internal standard were utilized to achieve the best accuracy of the method. Both a conventional validation procedure and a designed experiment were applied to study the accuracy and precision of the method. The measurement uncertainty arising from accuracy and precision was estimated. The method accuracy, expressed as a percentage of overall recovery, was approximately 100%, and its intermediate precision was <10%. LC-ESI/MS/MS method detection limits (S/N > or = 3:1) of five macrolides were <0.3 microg/kg.     

Gas chromatographic analysis of milk for deoxynivalenol and its metabolite DOM-1

A gas chromatographic method is described for the determination of deoxynivalenol (DON) and its metabolite DOM-1 in milk. Milk samples were extracted with ethyl acetate on a commercially available disposable extraction column, followed by hexane-acetonitrile partitioning. Final purification was accomplished on a reverse phase C-18 cartridge. The trimethylsilyl ether (TMS) derivatives of DON were prepared, chromatographed on an OV-17 column, and quantitated with an electron capture detector. Chromatography of the TMS derivatives of milk extracts was compared to that of the corresponding heptafluorobutyryl derivatives. The limit of detection using TMS derivatives was 1 ng/mL for both toxins with recoveries averaging 82% +/- 9% at 2.5 and 10 ng/mL milk for DON and 85% +/- 6% at 10 ng/mL for DOM-1.     

Determination of nitrofuran residues in milk of dairy cows using liquid chromatography-tandem mass spectrometry

An analytical method has been developed for the determination of total bound and extractable residues of the nitrofuran drugs furazolidone, nitrofurazone, furaltadone, and nitrofurantoin in milk of dairy cows. The method involves overnight acid hydrolysis and simultaneous derivatization of the released side chains with 2-nitrobenzaldehyde. During hydrolysis, the bound metabolites are hydrolyzed to the side chains. After pH adjustment and solid-phase extraction cleanup, the derivatives are detected and quantitated using a liquid chromatography-tandem mass spectrometry system with an atmospheric pressure chemical ionization interface. Validation of the method is accomplished by fortifying control milk with a mixture of side chains at 1, 2, and 4 ng/g. Internal standards are added at the beginning of the procedure to compensate for matrix effects and recovery losses. Method accuracies range from 83 to 104% with coefficients of variation less than 13% for all four analytes. The limits of detection are相似文献   

Determination of perfluorochemicals in cow's milk using liquid chromatography-tandem mass spectrometry

This study describes a new method developed for detection of 10 different perfluorochemicals (PFCs) in cow's milk, seven perfluorinated carboxylates and three perfluorinated sulfonate salts. After attempting multiple methods employing both acidic and basic extractions, a basic extraction using 10 mM sodium hydroxide in methanol digestion along with weak anion-exchange solid-phase extraction was employed. Vortex mixing and varying sonication times were compared as part of sample processing. Results show that sonication during sample processing yield decreased recovery of longer chain perfluorinated carboxylates. The final method developed was used to determine the concentration of PFCs in 12 raw and 49 retail milk samples from across the United States. With the exception of a single raw milk sample obtained from a dairy farm that had applied PFC containing biosolids to its fields, there were no milk samples containing PFCs.     

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.     

Monitoring of five sulfonamide antibacterial residues in milk by in-tube solid-phase microextraction coupled to high-performance liquid chromatography

A simple, rapid, and sensitive method for the quantitative monitoring of five sulfonamide antibacterial residues in milk was developed by coupling in-tube solid-phase microextraction (SPME) to high-performance liquid chromatography with an ultraviolet detector. A poly(methacrylic acid-ethylene glycol dimethacrylate) monolithic capillary column was selected as the extraction medium for this on-line technique. To obtain optimum extraction efficiency, several parameters relating to in-tube SPME were investigated. By simple extraction with ethanol, dilution with phosphate buffer solution, and centrifugation, the sample solution then could be directly injected into the device for extraction. The calculated detection limits for sulfadiazine, sulfamethazine, sulfamethoxazole, sulfamonomethoxine sodium, and sulfacetamide sodium were 2.0, 2.8, 1.7, 2.5, and 22 ng/mL, respectively. The method was linear over the range of 20-5000 ng/mL (100-5000 ng/mL for sulfacetamide sodium) with a correlation coefficient R (2) value >0.9980. Excellent method reproducibility was found by intra- and interbatch precisions, yielding the relative standard deviations of <10.0 and <9.94%, respectively. The proposed method was proved to be robust in monitoring sulfadiazine, sulfamethazine, sulfamethoxazole, sulfamonomethoxine sodium, and sulfacetamide sodium residues in milk.     

Determination of aflatoxin M1 in milk and milk powder using high-flow solid-phase extraction and liquid chromatography-tandem mass spectrometry

Animal feeds occasionally have some degree of contamination by Aspergillus spp. Even pasteurized milk at times contains the toxic liver carcinogen aflatoxin M1 (AFM1). Confirmation of its presence is now done with solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC)-fluorescence, using a small enough sample that SPE time is reasonable. In this study 200 mL of milk was extracted using a C18 disk at a flow rate of approximately 100 mL/min and AFM1 quantified by HPLC-tandem mass spectrometry with negative electrospray ionization. The effectiveness and cleanup efficacy of immunoaffinity columns (IAC) was compared with that of Mycosep multifunctional cleanup columns (MFC). Average recovery and detection limits of whole milk and low-fat milk cleaned up by IAC were significantly superior to those obtained with the MFC (78-87% and 0.59-0.66 ng/L, respectively). The new procedure improves extraction speed, sensitivity, and specificity.     

Rapid assay for analyzing biogenic amines in cheese: matrix solid-phase dispersion followed by liquid chromatography-electrospray-tandem mass spectrometry

A new rapid and sensitive method based on matrix solid-phase dispersion (MSPD) followed by liquid chromatography-electrospray-tandem mass spectrometry was devised for the determination of biogenic amines at trace levels in cheese samples. The method required 0.25 g of sample, CN-bonded silica as a dispersant sorbent, and a formic acid aqueous solution/methanol mixture as an eluting solvent. Extraction recoveries from soft cheese products were calculated in the 98 +/- 4-110 +/- 6% range. A procedure based on solid-phase extraction was also evaluated for the extraction of these compounds in cheese. Chromatographic separation was performed using a C18 column with an aqueous ammonium acetate/methanol mixture as the mobile phase under gradient conditions. The method was validated in terms of detection limits (LOD), quantitation limits (LOQ), linearity, recovery, precision, and trueness. Results in the 0.05-0.25 mg kg(-1) range were obtained for the LOD of histamine, tyramine, and beta-phenylethylamine in soft cheese samples. Linearity was established over 2 orders of magnitude. Excellent precision in terms of intra-day repeatability was calculated (RSD% < 5). The applicability of the method to the determination of biogenic amines in cheese products was demonstrated.     

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.     

Liquid chromatographic determination of zearalenone and alpha- and beta-zearalenols in milk

Previous research has demonstrated transmission of zearalenone and alpha- and beta-zearalenols into the milk of cows and other animals. Since human intake of zearalenone and its metabolites via milk is an unknown factor in risk assessment of zearalenone and because appropriate methodology for their determination in milk is not available, a rapid and sensitive analytical method has been developed. Essentially, the method includes extraction with basic acetonitrile, acidification, partition into methylene chloride on a hydrophilic matrix, cleanup on an aminopropyl solid phase extraction column, and reverse-phase liquid chromatography with fluorescence detection. Recoveries from milk averaged 84% for zearalenone, 93% for alpha-zearalenol, and 90% for beta-zearalenol at spiking levels of 0.5 to 20 ng/mL. As little as 0.2 ng/mL of zearalenone and alpha-zearalenol and 2 ng/mL of beta-zearalenol can be detected in milk. These 3 compounds are stable in refrigerated milk for at least 2 weeks and in milk brought to boiling. Enzymes (beta-glucuronidase and aryl sulfatase) may be added to milk prior to extraction to hydrolyze any conjugates.     

Determination of low levels of perchlorate in lettuce and spinach using ion chromatography-electrospray ionization mass spectrometry (IC-ESI-MS)

A sample preparation method was developed to quantify environmentally relevant (low micrograms per liter) concentrations of perchlorate (ClO4(-)) in leafy vegetables using IC-ESI-MS. Lettuce and spinach were macerated, centrifuged, and filtered, and the aqueous extracts were rendered water-clear using a one-step solid-phase extraction method. Total time for extraction and sample preparation was 6 h. Ion suppression was demonstrated and was likely due to unknown organics still present in the extract solution after cleanup. However, this interference was readily eliminated using a Cl(18)O4(-) internal standard at 1 microg/L in all standards and samples. Hydroponically grown perchlorate-free butterhead lettuce was spiked to either 10.3 or 37.7 microg/kg of fresh weight (FW), and recoveries were between 91 and 98% and between 93 and 101%, respectively. Five types of lettuce and spinach from a local grocery store were then analyzed; they contained from 0.6 to 6.4 microg/kg of FW. Spike recoveries using the store-bought samples ranged from 89 to 100%. The method detection limit for perchlorate in plant extracts is 40 ng/L, and the corresponding minimum reporting limit is 200 ng/L or 0.8 microg/kg of FW.     

Rapid high pressure liquid chromatographic determination of aflatoxin M1 in milk and nonfat dry milk

A modification of the current revised AOAC method, 26.A10-26.A15, is described for the rapid analysis of aflatoxin M1 in milk and nonfat dry milk. The method incorporates chloroform extraction and eliminates the need for column chromatography by using liquid-liquid partition for sample extract cleanup. Quantitation is carried out by using fluorescence detection combined with high pressure liquid chromatography (HPLC) of aflatoxin M1 which has been converted to aflatoxin M2a with trifluoroacetic acid. The method has a detection limit of 0.014 micrograms/L (2 X signal/noise) for whole milk. For 6 samples of naturally contaminated nonfat dry and freeze-dried milk, the modified method gave an average result of 0.698 micrograms/L; the AOAC method gave an average result of 0.386 micrograms/L.     

Simultaneous determination of chloramphenicol and aflatoxin M1 residues in milk by triple quadrupole liquid chromatography-tandem mass spectrometry

A reliable, rapid, and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of chloramphenicol and aflatoxin M(1) in milk has been developed. This method includes simple extraction of sample with acetonitrile, separation on a MGIII-C(18) column using 5 mM ammonium acetate aqueous solution/methanol (60:40, v/v) as mobile phase, and MS/MS detection using multiple reaction monitoring mode. The method was validated according to Commission Decision 2002/657/EC. The limits of detection (LODs) were 0.05 μg/kg for chloramphenicol and 0.005 μg/kg for aflatoxin M(1.) The limits of quantification (LOQs) were 0.2 μg/kg for chloramphenicol and 0.02 μg/kg for aflatoxin M(1). The recovery values ranged from 88.8% to 100.6%, with relative standard deviation lower than 15% in all cases, when samples were fortified at three different concentrations. The decision limits (CCα) and detection capability (CCβ) of the method were also reported. This method has been successfully applied for simultaneous analysis of chloramphenicol and aflatoxin M(1) residues in milk from local supermarkets in China.     

Exposure assessment of prepubertal children to steroid endocrine disruptors. 2. Determination of steroid hormones in milk, egg, and meat samples

In the present study, the occurrence of the main sex steroid hormones in milk, egg, and meat was evaluated on the basis of a highly specific gas chromatography-tandem mass spectrometry measurement method. Globally, the results indicated that targeted estrogens and androgens occurred at similar levels (concentration levels in the 10-100 ng kg (-1) range) in the analyzed muscle and milk samples. The same compounds occurred at about 10-fold higher concentrations (i.e., in the 100-1000 ng kg (-1) range) in eggs and kidney samples. More precisely, egg and milk appeared as a non-negligible sources of estradiol (i.e., 2.2 +/- 0.8 and 3.1 +/- 2.0 ng day (-1), respectively), whereas testosterone exposure is caused by ingestion of meat and/or egg (i.e., 12.2 +/- 48.2 and 5.2 +/- 2.3 ng day (-1), respectively). The provided exposure data will be further exploited in the scope of a risk assessment study regarding endocrine disruption associated with these molecules.     

Direct optical biosensor analysis of folate-binding protein in milk

An automated, rapid, sensitive, and label-free biosensor-based assay for folate-binding protein (FBP) in bovine milk utilizing surface plasmon resonance optical detection is described. The active concentration of FBP is estimated from its specific interaction with a pteroyl-l-glutamic (folic) acid derivative immobilized on the sensor surface in a direct binding assay format. Milk, colostrum, and milk powders are prepared for analysis by dilution into buffer. Analysis conditions, including ligand immobilization, flow rate, contact time, and regeneration, have been defined, and nonspecific binding considerations were evaluated. Performance parameters include a working range for FBP in buffer of 0-200 ng/mL, a method detection limit of 0.13 microg/mL in fluid milk, overall instrument response RSD(R) of 0.64%, a mean interassay RSD(R) of 7.3% for skim milk powder, and surface stability over ca. 200 samples. The technique was applied to the measurement of active FBP content of consumer milks, the heat classification of skim milk powders manufactured under a wide range of thermal processing protocols, and change during early bovine lactation.