Validation of an ultra high performance liquid chromatography-tandem mass spectrometry method for detection and quantitation of 19 endocrine disruptors in milk

An endocrine disruptor (ED) is an exogenous compound that interferes with the body's endocrine system. Exposure to EDs may result in adverse health effects such as infertility and cancer. EDs are composed of a vast group of chemicals including compounds of natural origin such as phytoestrogens or mycotoxins and a wide range of man-made chemicals such as pesticides. Synthetic compounds may find their way into the food chain where a number of them can biomagnify. Additionally, processing activities and food contact materials may add further to the already existing pool of food contaminants. Thus, our diet is considered to be one of the main exposure routes to EDs. Some precautionary legislation has already been introduced to control production and/or application of some persistent organic pollutants with ED characteristics. However, newly emerging EDs with bioaccumulative properties have recently been reported to appear at lower tiers of the food chain but have not been monitored at the grander scale. Milk and dairy products are a major component of our diet, thus it is important to monitor them for EDs. However, most methods developed to date are devoted to one group of compounds at a time. The UHPLC-MS/MS method described here has been validated according to EC decision 2002/657/EC and allows simultaneous extraction, detection, quantitation and confirmation of 19 EDs in milk. The method calibration range is between 0.50 and 20.0 μg kg with coefficients of determination above 0.99 for all analytes. Precision varied from 4.7% to 23.4% in repeatability and reproducibility studies. Established CCα and CCβ values (0.11-0.67 μg kg) facilitate fast, reliable, quantitative and confirmatory analysis of sub μg kg levels of a range of EDs in milk.

Hydrophilic Interaction Ultra-High Performance Liquid Chromatography Coupled with Triple-Quadrupole Mass Spectrometry for Determination of Nucleosides and Nucleobases in Animal Horns

Traditional Chinese Medicines (TCMs) derived from animal horns are one of the most important types of Chinese medicine. In the present study, a fast and sensitive analytical method was established for qualitative and quantitative determination of 14 nucleosides and nucleobases in animal horns using hydrophilic interaction ultra-high performance liquid chromatography coupled with triple-quadruple tandem mass spectrometry (HILIC-UPLC-QQQ-MS/MS) in selective reaction monitoring (SRM) mode. The method was optimized and validated, and showed good linearity, precision, repeatability, and accuracy. The method was successfully used to determine contents of the 14 nucleosides and nucleobases in 25 animal horn samples. Hierarchical clustering analysis (HCA) and principal component analysis (PCA) were performed and the 25 samples were thereby divided into two groups, which agreed with taxonomy. The method may enable quick and effective search of substitutes for precious horns.

Determination of metformin in plasma using a new ion pair solid phase extraction technique and ion pair liquid chromatography

This article describes the development of the first ion pair solid phase extraction technique (IPSPE), which has been applied to the extraction of metformin from plasma samples. In addition an ion pair chromatographic method was developed for the specific HPLC determination of metformin. Several extraction and HPLC methods have been described previously for metformin, however, most of them did not solve the problems associated with the high polarity of this drug. Drug recovery in the developed method was found to be more than 98%. The limit of detection and limit of quantification was 3 and 5 ng/ml, respectively. The intraday and interday precision (measured by coefficient of variation, CV%) was always less than 9%. The accuracy (measured by relative error, R.E.%) was always less than 6.9%. Stability analysis showed that metformin is stable for at least 3 months when stored at -70degreesC. The method has been applied to 150 patient samples as part of a medication adherence study. (C) 2003 Elsevier B.V. All rights reserved.

Identification of an unknown b-agonist in feed by liquid chromatography/bioassay/quadrupole time-of-flight tandem mass spectrometry with accurate mass measurement.

A new approach to the search for residues of unknown growth promoting agents such as anabolic steroids and -agonists in feed is presented. Following primary extraction and clean-up, samples are separated using gradient liquid chromatography (LC). The effluent is split towards two identical 96-well fraction collectors and an optional electrospray quadrupole time-of-flight mass spectrometry (QTOFMS) system for accurate mass measurement. One 96-well plate is used for a bioassay (enzyme-immuno assay, receptor assay) and will detect the bioactivity and position of the relevant peak in the chromatogram. The positive well in the second 96-well plate is used for identification by LC/QTOFMS/MS. The value of this LC/bioassay/QTOFMS/MS methodology is highlighted by the finding and structure elucidation of a new -agonist in a feed extract.

Dried blood spot liquid chromatography assay for therapeutic drug monitoring of metformin

The use of blood spot collection cards is a simple way to obtain specimens for analysis of drugs for the purpose of therapeutic drug monitoring, assessing adherence to medications and preventing toxicity in routine clinical setting. We describe the development and validation of a microanalytical technique for the determination of metformin from dried blood spots. The method is based on reversed phase high-performance liquid chromatography with ultraviolet detection. Drug recovery in the developed method was found to be more than 84%. The limits of detection and quantification were calculated to be to be 90 and 150 ng/ml, respectively. The intraday and interday precision (measured by CV%) was always less than 9%. The accuracy (measured by relative error, %) was always less than 12%. Stability analysis showed that metformin is stable for at least 2 months when stored at -70 degrees C. The small volume of blood required (10 mu L), combined with the simplicity of the analytical technique makes this a useful procedure for monitoring metformin concentrations in routine clinical settings. The method is currently being applied to the analysis of blood spots taken from diabetic patients to assess adherence to medications and relationship between metformin level and metabolic control of diabetes. (c) 2006 Elsevier B.V. All rights reserved.

The development and validation of liquid chromatography method for the simultaneous determination of metformin and glipizide, gliclazide, glibenclamide or glimperide in plasma

This article describes the development of SPE and HPLC methods for the simultaneous determination of metformin and glipizide, gliclazide, glibenclamide or glimperide in plasma. Several extraction and HPLC methods have been described previously for the determination of each of these analytes in plasma separately. The simultaneous determination of these analytes is important for the routine monitoring of diabetic patients who take combination medications and for studying the pharmacokinetics of the combined dosage forms. In addition this developed method can serve as a standard method for the plasma determination of these analytes therefore saving time, effort and money. The recoveries of the developed methods were found to be between 76.3% and 101.9%. The limits of quantification were between 5 and 22.5 ng/ml. The intraday and interday precision (measured by coefficient of variation, CV%) was always less than 9%. The accuracy (measured by relative error %) was always less than 12%. Stability analysis showed that all analytes are stable for at least 3 months when stored at -70degreesC. (C) 2004 Elsevier B.V. All rights reserved.

Rapid confirmatory method for the determination of sixteen synthetic growth promoters and bisphenol A in bovine milk using dispersive solid-phase extraction and liquid chromatography-tandem mass spectrometry

A rapid liquid chromatographic-tandem mass spectrometric (LC-MS/MS) multi-residue method for the simultaneous quantitation and identification of sixteen synthetic growth promoters and bisphenol A in bovine milk has been developed and validated. Sample preparation was straightforward, efficient and economically advantageous. Milk was extracted with acetonitrile followed by phase separation with NaCl. After centrifugation, the extract was purified by dispersive solid-phase extraction with C18 sorbent material. The compounds were analysed by reversed-phase LC-MS/MS using both positive and negative ionization and operated in multiple reaction monitoring (MRM) mode, acquiring two diagnostic product ions from each of the chosen precursor ions for unambiguous confirmation. Total chromatographic run time was less than 10 min for each sample. The method was validated at a level of 1 mu g L-1. A wide variety of deuterated internal standards were used to improve method performance. The accuracy and precision of the method were satisfactory for all analytes. The confirmative quantitative liquid chromatographic tandem mass spectrometric (LC-MS/MS) method was validated according to Commission Decision 2002/657/EC. The decision limit (CC alpha) and the detection capability (CC beta) were found to be below the chosen validation level of 1 mu g L-1 for all compounds. (C) 2010 Elsevier B.V. All rights reserved.

Towards Surface Plasmon Resonance biosensing combined with bioaffinity-assisted nano HILIC Liquid Chromatography Time-of-flight Mass Spectrometry identification of Paralytic Shellfish Poisons

The potential for coupling technologies to deliver new, improved forms of bioanalysis is still in its infancy. We review a number of examples in which coupling has been successful, with special emphasis on combining surface-plasmon-resonance biosensors with mass spectrometry. We give an overview of current progress towards combining biosensor-based bioanalysis with chemical analysis for confirmation of paralytic shellfish poisons that are marine toxins. This comprehensive approach could be an alternative to the official methods currently used (e.g., animal testing and high-performance liquid chromatography with fluorescence detection) and could serve as a model for many more such applications. (C) 2009 Elsevier Ltd. All rights reserved.

Immunobiosensor detection of domoic acid as a screening test in bivalve molluscs: Comparison with liquid chromatography-based analysis

A rapid and sensitive immuno-based screening method was developed to detect domoic acid (DA) present in extracts of shellfish species using a surface plasmon resonance-based optical biosensor. A rabbit polyclonal antibody raised against DA was mixed with standard or sample extracts and allowed to interact with DA immobilized onto a sensor chip surface. The characterization of the antibody strongly suggested high cross-reactivity with DA and important isomers of the toxin. The binding of this antibody to the sensor chip surface was inhibited in the presence of DA in either standard solutions or sample extracts. The DA chip surface proved to be highly stable, achieving approximately 800 analyses per chip without any loss of surface activity. A single analytical cycle (sample injection, chip regeneration, and system wash) took 10 min to complete. Sample analysis (scallops, mussels, cockles, oysters) was achieved by simple extraction with methanol. These extracts were then filtered and diluted before analysis. Detection limits in the ng/g range were achieved by the assay; however, the assay parameters chosen allowed the test to be performed most accurately at the European Union's official action limit for DA of 20 mu g/g. At this concentration, intra- and interassay variations were measured for a range of shellfish species and ranged from 4.5 to 7.4% and 2.3 to 9.7%, respectively.

Studies on the persistence of estradiol benzoate and nortestosterone decanoate in hair of cattle following treatment with growth promoters, determined by ultra-high-performance liquid chromatography-tandem mass spectrometry

Measurement of steroid esters in bovine hair samples, using sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS), provides a powerful tool for identifying animals treated illicitly with growth promoters. The successful application of such testing requires appropriate sampling of hair from treated animals. This paper describes the results of hair analysis by LC-MS/MS for two animal studies in which animals were treated with estradiol-3-benzoate and nortestosterone decanoate. The results from the first animal study indicate that animals treated with these anabolic steroids may not always be identified from analysis of hair samples; positive test results occur sporadically and only for some of the treated animals. The results from the second animal study identify conditions attaching to positive hair samples, such as, that concentrations of steroid esters in hair are related to distance of sampling from point of injection and to time post-treatment, that concentrations of steroid esters in hair are related to dose given to the animal but that this relationship may vary over time post-treatment, and that steroid esters may be measured in regrowth hair taken some weeks after treatment. Steroid esters are determined along the length of the hair, confirming that accumulation of steroid esters into hair occurs from various sources, including blood, sweat and sebum. The reported research provides some useful insights into the mechanisms governing the persistence of steroid esters in bovine hair following illicit treatment with growth promoters. (C) 2009 Elsevier B.V. All rights reserved.