Development of an LC-MS/MS method to quantify sex hormones in bovine milk and influence of pregnancy in their levels

Hormones work in harmony in the body, and this status must be maintained to avoid metabolic disequilibrium and the subsequent illness. Besides, it has been reported that exogenous steroids (presence in the environment and food products) influence the development of several important illnesses in humans. Endogenous steroid hormones in food of animal origin are unavoidable as they occur naturally in these products. The presence of hormones in food has been connected with several human health problems. Bovine milk contains considerable quantities of hormones and it is of particular concern. A liquid chromatography–tandem mass spectrometry (LC-MS/MS) method, based on hydroxylamine derivatisation, has been developed and validated for the quantification of six sex hormones in milk [pregnenolone (P₅), progesterone (P₄), estrone (E₁), testosterone (T), androstenedione (A) and dehydroepiandrosterone (DHEA)]. This method has been applied to real raw milk samples and the existence of differences between milk from pregnant and non-pregnant cows has been statistically confirmed. Basing on a revision of existing published data, it could be concluded that maximum daily intakes for hormones are not reached through milk ingestion. Although dairy products are an important source of hormones, other products of animal origin must be considered as well for intake calculations.     

Determination of steroid hormones in bovine milk by LC-MS/MS and their levels in Swiss Holstein cow milk

Synthetic and natural steroid hormones have attracted some attention in recent years as endocrine active substances (EAS) that interact or interfere with the endocrine system. Endogenous hormones occur naturally in food of animal origin, among which bovine milk represents an important source. This study was conducted to determine the occurrence of steroid hormones (oestrogens, androgens, progestogens and glucocorticoids) in cow’s milk samples from three farms in Switzerland. An isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of 12 hormones in milk. Some hormonal levels from individual cows showed large variations. The average levels of the hormones analysed (17α-estradiol = 31 ng kg^–1, 17β-estradiol = 6 ng kg^–1, estrone = 159 ng kg^–1, 4-androstenedione = 684 ng kg^–1, progesterone = 15486 ng kg^–1, 17-hydroxyprogesterone = 214 ng kg^–1, cortisone = 112 ng kg^–1, and cortisol = 235 ng kg^–1) were comparable with literature data. Estriol, testosterone and androstenediols were not detected at their respective limit of quantification. No significant differences of hormonal content among milk from cows at different lactation/calving numbers were evidenced, except for progesterone and 4-androstenedione. Due to confounding parameters linked to the physiological stage of the animal, like pregnancy and gestational stage (pregnancy trimester), the causal correlation between the variation of the levels for these two hormones and the lactation/calving number could not be unambiguously demonstrated.     

Simultaneous determination of chloramphenicol and florfenicol in liquid milk, milk powder and bovine muscle by LC-MS/MS

A validated method based on European and Brazilian legislation is reported. It is applicable to the simultaneous determination of chloramphenicol (CAP) and florfenicol (FF) by LC-MS/MS in liquid milk, milk powder and bovine muscle. The chromatographic analysis is completed in 6 min and the extraction procedure is very simple, involving only one step liquid-extraction with ethyl acetate. Where it proved necessary to include clean-up, an efficient and rapid step using C18-dispersive solid was added. Initially, a complete validation was performed with liquid milk matrix; later the scope was extended to the other matrices through extending the inter-day precision (within laboratory reproducibility) RSD values. An internal standard (d(5)-CAP) was employed for quantitative purposes. The method was shown to have good accuracy and precision for determining CAP residues at the level of 0.3-0.6 g kg(-1) and FF residues at the level of 5-15 μg kg(-1).     

Multiresidue method for pesticides and veterinary drugs in bovine milk using GC/MS and LC/MS/MS

A simple, sensitive and selective method with gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been developed to detect 342 pesticides and veterinary drugs contaminating bovine milk at the maximum residue limits (MRLs) defined in the "positive list system". Sample preparation was performed by extracting the analytes with acetonitrile, followed by salting-out with sodium chloride. For some pesticides, the extract was further cleaned up by n-hexane partitioning and PSA cartridge column chromatography. GC/MS-EI or -NCI was used to determine pesticide residues, while LC/MS/MS-ESI was applicable to the determination of pesticide and veterinary drug residues. The variation of the recoveries of these drugs at MRL was relatively wide; however the relative standard deviations of the recovery of each drug were within 28%, suggesting that the present method is good enough for use as a screening test for contaminants at the MRLs. These results show that this method is useful for multiresidue analysis of numerous pesticides and veterinary drugs in bovine milk.     

Analytical method of cereulide in processed cereal-based foods and milk powder by LC-MS/MS

An LC-MS/MS method for analysis of cereulide, an emetic toxin produced by Bacillus cereus, was developed. Cereulide was extracted from samples, fried rice, pan-fried noodles, red bean paste and baby formula, with methanol and purified using Oasis HLB cartridges. LC separation was performed on a C18 column with a mixture of formic acid solution and methanol containing ammonium formate as a mobile phase, and the mass spectrometer was operated in the positive electrospray ionization mode. Performance evaluation showed that trueness was higher than 70% and repeatability and reproducibility were within 10%. The limits of quantification were lower than 1 μg/kg.     

Quantitative determination of ceftiofur-related residues in bovine raw milk by LC-MS/MS with electrospray ionization

A sensitive and specific liquid chromatography/tandem mass spectrometry (LC-MS/MS) method for the quantitative determination of ceftiofur-related metabolites that retain the ß-lactam structure, expressed as desfuroylceftiofur free acid has been developed and validated for raw bovine milk. The method described is based on the cleavage of the disulfide and/or thioester bonds between the metabolites and their sulfur-containing moiety, using dithioerythritol to yield desfuroylceftiofur and further stabilization to desfuroylceftiofur-acetamide (DCA) using iodacetamide. The final extract containing the DCA derivative was cleaned up by a simple solid-phase extraction step prior to separation on a phenylether reversed phase HPLC-column. Chromatography was performed using a formic acid/methanol gradient. Collision induced dissociation with argon was used for fragmentation of the pseudomolecular ion [M+H]⁺ to achieve the required specificity of the method. The new EU guidelines for identification and quantification were fulfilled.     

Multiresidue automated turbulent flow online LC-MS/MS method for the determination of antibiotics in milk

A fast and reliable multiresidue method is reported for the identification and quantification of 36 different compounds from seven different classes of antibiotics (aminoglycosides, sulfonamides, macrolides, quinolones, tetracyclines, lincosamides and trimethoprim) in milk. Automated online sample cleanup was applied using turbulent flow chromatography (Transcend TLX system), directly coupled to a mass spectrometer (MS/MS) for sensitive and specific detection. The method involved a simple extraction/protein precipitation using acetonitrile, followed by centrifugation and filtration. After this preliminary step, the extract was injected into the TLX-ESI-MS/MS using optimised turbulent flow and liquid chromatography (LC) conditions. Single-laboratory validation of the method was carried out according to the Directive 2002/657/EC, clearly demonstrating the suitability of this method for quantitative determination of this wide range of antibiotics in milk. A small survey, which covered milk samples of different origin and varied fat content, demonstrated the robustness of this method and its suitability for enforcement purposes.     

Determination of androstenone levels in porcine plasma by LC-MS/MS

Androstenone (5α-androst-16-en-3-one) is a major component in boar taint. Here, a liquid chromatography-tandem mass spectrometry method was developed to analyse androstenone in porcine plasma to facilitate studies of its transport from pig testes to adipose tissues. The method used androstanone (5α-andro-stan-3-one) as internal standard, Oasis® MCX solid-phase extraction, C18 reversed-phase column, and API 5000 Triple Quadrupole mass spectrometry with positive electrospray ionisation interface operating in the multiple reaction monitoring mode. Incubation of the plasma samples with β-glucuronidase/arylsulfatase revealed an increase in androstenone yield indicating the presence of a conjugated form of its 3-enol isomer. With this method the limit of detection (LOD) was 1.0 ng and the limit of quantification (LOQ) was 3.3 ng per mL of plasma. In conclusion, the presented method is sensitive and reproducible and thus suitable for accurate quantification of androstenone levels in a small volume of porcine plasma.     

Development and validation of a modified QuEChERS method coupled with LC-MS/MS to determine arbutin in pear peels

A new effective method was developed to determine the concentration of arbutin in pear peels using a modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) method and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The original QuEChERS was modified to enable the extraction of the polar arbutin molecule. Use of an initial 50:50 acetonitrile:water extraction solvent led to the highest extraction efficiency. The arbutin extracted from pear peels was found to be identical to the β-arbutin standard, as confirmed by NMR and LC-MS/MS analyses. For quantitative analysis, the mass spectra of the precursor ion [M+NH₄]⁺ at m/z 290.0 and the product ion of arbutin at m/z 163.0 were used. The limit of detection, limit of quantification, linearity, precision, accuracy, and recovery of the proposed method were evaluated. We successfully applied this method to pear samples and it may be suitable for the quantitative analysis of arbutin in other similar plant materials.     

Analytical method of hydramethylnon in agricultural products by LC-MS/MS

A simple determination method of hydramethylnon in agricultural products by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. The sample was homogenized with phosphoric acid and extracted with acetone. An aliquot of crude extract was re-extracted with hexane and sat. NaCl solution. In the case of tea leaf, solidification processing using ammonium chloride and phosphoric acid was performed before re-extraction with hexane. Clean-up was performed using a silica-gel mini column (500 mg). The LC separation was performed on a C18 column using methanol-water (8 : 2) containing 10 mM ammonium acetate as the mobile phase and MS detection with positive ion electrospray ionization. The calibration curve was linear between 0.002-0.2 μg/mL of hydramethylnon. Recoveries (n=5) of hydromethylnon from 10 kinds of agricultural products fortified at 0.01 μg/g (0.05 μg/g for pineapple) were 82-110%, and their relative standard deviations were 2-12%.     

液相色谱串联质谱法测定食品中红曲色素

采用液相色谱串联质谱法测定了食品中红曲红胺、红曲红素、红曲素、红曲黄素的含量,并选择离子检测进行阳性确证。液体试样用水超声提取,离心定容后,固体和半固体样品采用水溶解定容,提取液再经固相萃取柱净化,样液经洗脱定容后,供液相色谱-质谱/质谱仪测定和确证,外标法定量。该方法的最低检出限、线性范围和方法回收率分别为:1.0 mg/kg、1.0～100.0 mg/kg和89.3%～94.3%。     

液相色谱串联质谱法测定纺织品和皮革中PFOS含量

针对国外对全氟辛烷磺酸(PFOS)的限量要求,建立了纺织品和皮革中PFOS的检测方法.样品使用甲醇快速提取(用ASE300快速溶剂萃取仪提取),提取液经C18固相萃取柱净化,浓缩定容后供液相色谱串联质谱仪(LC-MS/MS)测定,采用多反应监测(MRM)进行确证,外标法定量.本方法的最低检出限是0.10 mg/kg,线性范围为0.1～60.0 ng.方法回收率在80.0%～105.8%.     

Development and validation of an LC-MS/MS method for the simultaneous analysis of 28 specific narcotic adulterants used in dietary supplements

The purpose of this study was to develop a method to analyse the concentration of multiple illegal narcotics present in dietary supplements. To this end, we established and optimised a procedure using LC-MS/MS simultaneously to analyse 28 narcotic compounds in various forms of dietary supplements, including powders, tablets, liquids and capsules. In addition, candy and cookies that have also had detected cases of adulteration were also analysed. The specificity, linearity, accuracy, precision, limit of detection (LOD), limit of quantitation (LOQ), stability and recovery for these methods were validated accordingly. The LOD and LOQ of the LC-MS/MS ranged from 0.01–50.0 to 0.03–100 ng g^?1, respectively. The linearity of these results was good (r² > 0.99), with intra- and inter-day precision values of 0.2–5.2% and 0.2–4.8%, respectively. Further, the intra- and inter-day accuracies of this method were 97.0–103.4% and 94.6–103.1%, respectively. The stability RSD was less than 7.8%. The mean recovery for this LC-MS/MS procedure was 81.1–117.4%, with an RSD less than 9.8%. Following the validation of our method, we analysed 47 commercially available dietary supplements obtained in Korea. Whilst none of these samples had detectable amounts of the 28 specified narcotic adulterants, our novel LC-MS/MS procedure can be utilised comprehensively and continually to monitor illegal drug adulteration in various forms of dietary supplements.     

基于液相色谱-串联质谱法检测多种食品中的9种非食用色素

建立了多种食品(葡萄酒、乳饮料、调制乳、海虾,熟鸡蛋,咸鸭蛋,肉制品,辣椒酱,辣椒粉)中的9种非食用色素酸性红1、酸性橙7、酸性黄36、苏丹红Ⅰ、苏丹红Ⅱ、苏丹红Ⅲ、苏丹红Ⅳ、碱性橙31、罗丹明B的液相色谱-串联质谱定量检测方法。分别对提取方式、不同食品的分类预处理方法和质谱条件进行了优化。并选择离子检测进行阳性验证,外标法定量分析了上述食品样品中的色素含量。该方法对于酸性红的最低检出限、线性范围和方法回收率分别为:0.10 mg/kg、0.10~0.50 mg/L和86.5%~95.3%;对于其他8种非食用色素的最低检出限、线性范围和方法回收率分别为:0.010 mg/kg、0.010~0.10 mg/L和86.5%~95.7%。     

Development and validation of LC-MS/MS method with QuEChERS clean-up for detecting cannabinoids in foods and dietary supplements

ABSTRACT

a rapid and simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the determination of cannabidiol (CBD) and Δ⁹-tetrahydrocannabinol (THC) using a QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) clean-up for a variety of foods and dietary supplements (DS). QuEChERS is widely used in extraction or clean-up procedures to eliminate interference of matrices such as sugars, organic acids, lipids, and fatty acids. The samples were categorised into three types, and various pretreatment methods were compared for each type. In all types, the QuEChERS was superior and selected as the final pretreatment method. The optimised method was validated for specificity, limit of detection (LOD), limit of quantification (LOQ), linearity, recovery, precision and accuracy. All of the validation results met the requirements of the international guidelines for all types of samples. The validated method was applied to 30 commercial food samples, CBD was detected in 17 samples, with 2 of them detected below the LOQ level and the rest detected in a range of 70 μg/kg to 31305 mg/kg (3.1%, w/w). Meanwhile, THC was detected in 14 samples; 2 of them were detected below the LOQ level and the rest detected in a 0.08–98.62 μg/g range. These results indicated that the validated method can be successfully applied for the determination of cannabinoids in a variety of samples. Furthermore, it will be useful for controlling the illegal distribution of cannabinoids.     

Determinative and confirmatory method for residues of tetracyclines in honey by LC-MS/MS

A limited number of substances are authorised for the treatment of bees. Maximum residue limits (MRLs) are set for tetracyclines in several matrices, but not for honey. Nevertheless, tetracycline antibiotics may be used in order to prevent bacterial diseases and the loss of honey bee populations. In this study, a sensitive multi-residue LC-MS/MS method was developed and optimised for the quantitative and qualitative determination of tetracycline residues in honey. Homogenisation of samples under acidic conditions was performed and solid-phase extraction was carried out. The eluate was evaporated under nitrogen and dissolved in an aqueous methanol solution prior to filtration. A mobile phase composed of acetic acid–water and acetic acid–acetonitrile was used. Separation of tetracycline, oxytetracycline, chlortetracycline and doxytetracycline was achieved by using gradient elution on a C18 chromatography column. The analytical method was validated according to Commission Decision 2002/657/EC by the analysis of spiked samples around the recommended concentration of 20 μg kg^?1 by EURL Guidance Paper, December 2007. A matrix effect was observed, so quantification was based on an external matrix calibration curve. Calculated decision limits (CCα) were lower than 10 μg kg^–1 for all tetracyclines. Good linearity, repeatability and within-laboratory reproducibility were achieved.     

Simultaneous determination of four coccidiostats in eggs and broiler meat: validation of an LC-MS/MS method.

A published confirmatory method for the quantitative determination of four ionophoric coccidiostats (lasalocid, monensin, salinomycin and narasin) in eggs and broiler meat has been further developed. It is proposed for replacement of liquid chromatography methods previously used in analysis of ionophoric coccidiostats. The samples were extracted with acetonitrile and purified on a silica solid phase extraction column. Purified samples were analysed by liquid chromatography-mass spectrometry and the method, was validated according to the Commission Decision 2002/657/EC. The validation parameters selectivity, linearity, specificity, precision, recovery, decision limit (CCalpha) and detection capability (CCbeta) were determined. The recoveries of coccidiostats analysed ranged from 64-99% in eggs and 62-100% in broiler meat. CCalpha varied from 0.8-1.4 microg/kg in eggs and from 1.5-2.5 microg/kg in broiler meat. CCbeta varied from 0.9 microg/kg to 2.0 microg/kg in eggs and from 1.7-3.2 microg/kg in broiler meat.     

初乳、常乳及其制品中的雌性激素

简要介绍了人和牛初乳、常乳及其制品中天然雌性激素的种类、结构、含量、存在状态,并讨论了常规乳品加工对不同雌性激素的影响.     

Development and validation of an LC-MS/MS method for the detection of phomopsin A in lupin and lupin-containing retail food samples from the Netherlands

Phomopsins (PHO) are mycotoxins produced by the fungus Diaporthe toxica (also referred to as Phomopsis leptostromiformis). Lupin is the most important host crop for this fungus and PHO are suspected as cause of lupinosis, a deadly liver disease, in sheep. Lupin is currently in use to replace genetically modified soy in many food products available on the European market. However, a validated method for analysis of PHO is not available until now. In this work, a dilute-and-shoot LC-MS/MS-based method was developed for the quantitative determination and identification of phomopsin A (PHO-A) in lupin and lupin-containing food. The method involved extraction by a mixture of acetonitrile/water/acetic acid (80/20/1 v/v), dilution of the sample in water, and direct injection of the crude extract after centrifugation. The method was validated at 5 and 25 µg PHO-A kg^?1 product. The average recovery and RSD obtained were 79% and 9%, respectively. The LOQ (the lowest level for which adequate recovery and RSD were demonstrated) was 5 µg PHO-A kg^?1. Identification of PHO-A was based on retention time and two transitions (789 > 226 and 789 > 323). Using the average of solvent standards from the sequence as a reference, retention times were all within ± 0.03 min and ion ratios were within ± 12%, which is compliant with European Union requirements. The LOD (S/N = 3 for the least sensitive transition) was 1 µg PHO-A kg^?1 product. Forty-two samples of lupin and lupin-containing food products were collected in 2011–2012 from grocery stores and internet shops in the Netherlands and analysed. In none of the samples was PHO-A detected.     

Confirmatory method for the determination of streptomycin and dihydrostreptomycin in honey by LC-MS/MS

A method was specifically developed for the determination and confirmation of streptomycin and dihydrostreptomycin in different types of honey. The method is simple, rapid, sensitive and was validated for streptomycin and dihydrostreptomycin in accordance with Commission Decision 2002/657/EC. After extraction with phosphate buffer and a pH change, clean-up was performed via SPE with polymeric phase. LC-MS/MS analysis was carried out using two different HILIC columns for the separation of the analytes and using a triple quadrupole mass spectrometer in positive ESI mode to measure the transitions of the substances in MRM mode. For the quantification of both substances, matrix calibration curves in a linear range of 5-80 g kg(-1) were used. The validation parameters established for streptomycin and dihydrostreptomycin, CCα (11.8 and 11.5 μg kg(-1), respectively), CCβ (18.9 and 19.9 μg kg(-1), respectively), recovery (97 and 101%, respectively) and the relative within-laboratory reproducibility RSD(wR) (16.4 and 20.8%, respectively) at the recommended concentration of 40 μg kg(-1), fulfil the requirements of Commission Decision 2002/657/EC.