Microwave-Assisted Extraction of Melamine Residues from Pet Food and Analysis by Ion-Exchange LC–DAD

Melamine is attracting much attention because of its toxicity. In the work discussed in this paper, microwave-assisted extraction in combination with ion-exchange high-performance liquid chromatography with diode-array detection was used for analysis of melamine in pet food. Trichloroacetic acid–N,N-dimethylformamide 10:1 was the best extractant, because of the strong polarity of melamine. Separation was performed on a 250 mm × 4.6 mm i.d., 5-μm particle, cation-exchange column; isocratic elution with a mixture of ammonium formate solution (pH 3.0) and acetonitrile was complete within 10 min. UV absorbance DAD detection was performed at 240 nm. Response was a linear function of melamine concentrations from 0.1 to 50 μg mL^?1, with a detection limit of 1.0 mg kg^?1. Intra-day and inter-day precision, as RSD, was <3% and the recovery of the assay was in the range 95.4–106.8%. In analysis of spiked pet food, the new method yielded satisfactory results. Because of its simplicity and accuracy this straightforward method is particularly suitable for routine melamine analysis.     

Determination of Melamine in Food by SPE and CZE with UV Detection

A novel method for the determination of melamine residue in food was developed using solid-phase extraction and capillary zone electrophoresis with UV detection. Spiked samples were extracted with 1% trichloroacetic acid while 0.03 g sodium deoxycholate was used to precipitate protein in the real samples. After centrifuging and clean-up by solid-phase extraction cartridge, the extract was directly analyzed by CZE–UV. The method was validated and good results were obtained with respect to precision, repeatability and spiked recovery. The limit of detection for melamine varied between 0.25 and 0.5 mg kg^?1. The proposed method was successfully applied for the analysis of melamine in food with total recoveries ranging from 94 to 102% in the spiked range of 0.5–5 mg kg^?1, and the relative standard deviations were between 1.5 and 4.1%.     

Melamine and Cyanuric Acid in Foodstuffs and Pet Food: Method Validation and Sample Screening

Hazardous levels of melamine in food and feed products have been of great concern after the outbreak of contamination reported in Chinese commodities in 2008. Despite the existence of several analytical methods for melamine (MEL) detection in food, few provide a full validation data set, especially when MEL and cyanuric acid (CYA) are analyzed simultaneously. The aim of this study was to validate an analytical methodology for MEL and CYA analysis in foodstuffs by GC-MS after trimethylsilylation with N,O-bis(trimethylsilyl)trifluoroacetamide. Linearity was obtained in the range of 0.4 to 800 mg/kg for both compounds, with limits of detection (LODs) of 0.15 and 0.05 mg/kg for MEL and CYA, respectively. Screening in 20 food products [3 soya milk powder, 1 baby milk powder, 3 soya powder, 13 diversified cookies and biscuits (8 from China and 2 from Portugal), and 3 dog food) revealed MEL incidence in 55% of the cases, with a maximum concentration of 3.4 mg/kg. CYA was not detected in all samples.     

SPE then RP-LC for Simultaneous Analysis of Cyromazine and its Metabolite Melamine in Liquid Milk and Egg

Solid-phase extraction (SPE) and reversed-phase liquid chromatography (RP-LC) have been used for simple, sensitive simultaneous analysis of cyromazine and melamine residues in liquid milk and eggs. The conditions used for SPE and LC were investigated and optimized. A combined cation-exchange–reversed-phase cartridge was used for clean-up, and an ODS (C₁₈) column (150 mm × 4.6 mm i.d., 5-μm particles) with 62:38 (v/v) 5 mm sodium lauryl sulfate (pH 3.4)–acetonitrile as mobile phase was used for RP-LC. Under the optimum conditions the method limit of detection (LOD) for both cyromazine and melamine was 6.2 μg kg^?1 for liquid milk samples, and 11.5 μg kg^?1 for egg samples. Average recovery of cyromazine and melamine from milk samples was 90.3%, RSD 4.6–5.6%, and 99.6%, RSD 3.2–4.7%, respectively. Average recovery of cyromazine and melamine from egg samples was 85.3%, RSD 1.0–4.7%, and 89.6%, RSD 3.1–5.0%, respectively. The method enables detection of melamine and cyromazine at levels as low as 20.7 μg kg^?1 in liquid milk and 38.3 μg kg^?1 in egg.     

SPE then RP-LC for Simultaneous Analysis of Cyromazine and its Metabolite Melamine in Liquid Milk and Egg

Solid-phase extraction (SPE) and reversed-phase liquid chromatography (RP-LC) have been used for simple, sensitive simultaneous analysis of cyromazine and melamine residues in liquid milk and eggs. The conditions used for SPE and LC were investigated and optimized. A combined cation-exchange–reversed-phase cartridge was used for clean-up, and an ODS (C₁₈) column (150 mm × 4.6 mm i.d., 5-μm particles) with 62:38 (v/v) 5 mm sodium lauryl sulfate (pH 3.4)–acetonitrile as mobile phase was used for RP-LC. Under the optimum conditions the method limit of detection (LOD) for both cyromazine and melamine was 6.2 μg kg⁻¹ for liquid milk samples, and 11.5 μg kg⁻¹ for egg samples. Average recovery of cyromazine and melamine from milk samples was 90.3%, RSD 4.6–5.6%, and 99.6%, RSD 3.2–4.7%, respectively. Average recovery of cyromazine and melamine from egg samples was 85.3%, RSD 1.0–4.7%, and 89.6%, RSD 3.1–5.0%, respectively. The method enables detection of melamine and cyromazine at levels as low as 20.7 μg kg⁻¹ in liquid milk and 38.3 μg kg⁻¹ in egg.

     

Development of nanofibrillated cellulose coated with gold nanoparticles for measurement of melamine by SERS

The objective of this study was to develop nanofibrillated cellulose (NFC)-based substrate for rapid detection of melamine in milk by surface-enhanced Raman spectroscopy (SERS). NFC were served as a highly porous platform to load with gold nanoparticles (AuNPs), which can be used as a flexible SERS substrate with nanoscale roughness to generate strong electromagnetic field in SERS measurement. The NFC/AuNP substrate was characterized by UV–Vis spectroscopy and electron microscopy. Milk samples contaminated by different concentrations of melamine were measured by SERS coupled with NFC/AuNP substrate. The spectral data analysis was conducted by multivariate statistical analysis [i.e. partial least squares (PLS)]. Satisfactory PLS result for quantification of melamine in milk was obtained (R = 0.9464). The detection limit for melamine extracted from liquid milk by SERS is 1 ppm, which meets the World Health Organization’s requirement of melamine in liquid milk. These results demonstrate that NFC/AuNP substrate has improved homogeneity and can be used in SERS analysis for food safety applications.     

UPLC–MS–MS Analysis of Quetiapine and Its Two Active Metabolites, 7-Hydroxyquetiapine and 7-Hydroxy-N-dealkylquetiapine, in Rat Plasma and Cerebrospinal Fluid

Quetiapine (QP) is an antipsychotic agent widely used to treat a variety of human psychotic disorders. 7-Hydroxyquetiapine (QPOH) and 7-hydroxy-N-dealkylquetiapine (QPND) are its two active metabolites. A rapid and sensitive ultra-performance liquid chromatographic–tandem mass spectrometric method has been developed for analysis of the three agents in plasma and cerebrospinal fluid (CSF) from rats. The assay was based on liquid–liquid extraction of 100-μL samples. The methods were validated for QP, QPOH, and QPND in both types of sample. Limits of detection (LOD) in plasma were 0.02, 0.01, and 0.02 ng mL^?1 for QP, QPOH, and QPND, respectively; in CSF samples the respective values were 0.02, 0.01, and 0.01 ng mL^?1. The utility of the method was demonstrated by analysis of the pharmacokinetics and CSF distribution properties of QP and its two active metabolites in the plasma and CSF in rats.     

Detection and Confirmation of Ginsenosides in Horse Urine by GC–MS and LC–MS

Ginseng has been used by the Chinese as a traditional herbal medicine for thousands of years. In view of the growing popularity in the use of ginseng preparations as natural remedies and food supplements worldwide, there is an increasing concern for their abuse in both human and animal sports. Ginsenosides are considered the major constituents of ginseng responsible for its pharmacological properties. In this study, a method was developed for the detection and confirmation of a number of ginsenosides in horse urine. The intact ginsenosides were detected and confirmed at 5–100 ng mL^?1 by LC–MS², and two deglycosylation metabolites, namely protopanaxadiol and protopanaxatriol, could both be detected and confirmed at 2 ng mL^?1 by GC–MS² after trimethylsilylation. The above GC–MS and LC–MS methods were then applied to study the in vitro metabolism of ginsenosides Rg1 and Rb1 and the in vivo urinary metabolites after oral administration of Rg1 to horses. Results obtained reveal the very first evidence for the existence of the metabolites, Rg1 and protopanaxatriol, as glucuronides in urine.     

Dabsyl chloride derivatisation of melamine followed by high-performance liquid chromatography determination in water samples

A new and sensitive precolumn derivatisation with dabsyl chloride was developed for the analysis of melamine in water samples by high-performance liquid chromatography (HPLC) with visible detection. Derivatisation with dabsyl chloride leads to improving sensitivity and hydrophobicity of melamine. Under optimum conditions of derivatisation and microextraction, the method yielded a linear calibration curve ranging from 10 to 2000 µg L^?1 with a determination coefficient (R²) of 0.9952. Limit of detection (LOD) and limit of quantification (LOQ) were 2.0 and 6.0 µg L^?1, respectively. The relative standard deviation per cent (RSD%) for intraday and inter-day extraction and determination at 20 and 200 µg L^?1 levels of melamine was less than 8.2% (n = 6). Finally, the proposed method was successfully applied for the determination of melamine in different water samples and satisfactory results were obtained (relative recovery ≥91%).     

Simultaneous determination of cyromazine,melamine and their biodegradation products by ion-pair high-performance liquid chromatography

An ion-pair high-performance liquid chromatography with ultraviolet detection method for the determination of cyromazine, melamine and its biodegradation products (ammeline, ammelide, cyanuric acid and biuret) was developed. C18 column was utilised to separate the six analytes with a mobile phase consisting of perchloric acid-ammonia solution and acetonitrile, under gradient elution and variable flow rate. The detection wavelengths were 205 nm for cyanuric acid and biuret and 222 nm for cyromazine, melamine, ammeline and ammelide. For analysis of sediment samples, the extraction solution containing acetonitrile, ammonia and water (80:10:10 by volume) was used to extract the analytes from sediment matrix. Using the extraction method for the spiked sediment sample, high linearity of matrix-matched standard curve could be obtained for the six analytes. The method detection limit was 0.1 μg g^?1 for melamine and cyromazine, 0.2 μg g^?1 for ammeline and ammelide, 1.2 μg g^?1 for cyanuric acid and 1.0 μg g^?1 for biuret in sediment matrix. The recoveries of these compounds were 70.1–98.3% and the relative standard deviations were 0.5–4.4%. Finally, the proposed method was successfully applied to the analysis of the sediment sample near the wastewater outlet of a melamine-producing factory.     

Rapid determination of melamine in milk and milk powder by surface-enhanced Raman spectroscopy and using cyclodextrin-decorated silver nanoparticles

We have synthesized silver nanoparticles (AgNPs) decorated with α-cyclodextrin (CD) by using the traditional silver mirror reaction in the presence of CD. The CD-AgNPs were used as substrate in surface-enhanced Raman spectroscopy (SERS) for determining melamine. The intensity of the Raman band of melamine at 704 cm^?1 was used to determine melamine in milk and milk powder. The use of CD-AgNPs as the SERS substrate rather than classical silver nanoparticles makes the method more sensitive in giving an enhancement by a factor of up to?~?10⁶ in scattering efficiency. The effects of the volume of solutions (of CD-AgNPs, NaCl, NaOH, melamine) and of mixing time were optimized. The standard addition method was employed for quantitative analysis. The correlation coefficient of the calibration plot is 0.9995, and the limit of detection is 3.0 μg L^?1. The method was successfully applied to the determination of melamine in milk and milk powder, with relative standard deviations of <10 % and recoveries between 89 and 104 %.

Simultaneous LC−UV Analysis of Mirodenafil and Its Two Main Metabolites in Rat Plasma and Urine, and in Tissue Homogenates

A simple, rapid, and reproducible isocratic reversed-phase LC method has been established for simultaneous analysis of mirodenafil and its two main metabolites, SK3541 and SK3544, in rat plasma, urine, and tissue homogenates. Samples were deproteinized with acetonitrile containing sildenafil (internal standard). The compounds were separated on a C₁₈ column with 52:48 (v/v) 0.02 m ammonium acetate buffer (pH 6)—acetonitrile as mobile phase at a flow rate of 1.4 mL min^?1. UV detection was at 254 nm and detection limits of mirodenafil, SK3541, and SK3544 in plasma were 0.03, 0.05, and 0.1 μg mL^?1, respectively. The method is applicable to pharmacokinetic studies of mirodenafil and its metabolites in rats.     

Determination of Melamine Based on the Formation of an Insoluble Copper-Melamine Complex and Flame Atomic Absorption Spectrometry

A copper-melamine complex was optimally synthesized by heating excess copper(II), as 50 m mol L^?1 copper(II) chloride in 50% (v/v) methanol, and melamine at 80°C. The amount of residual copper(II) in solution after removal of the copper-melamine complex was then measured by flame atomic absorption spectrometry. The concentration of depleted copper was proportional to the concentration of melamine, with a linear calibration curve (melamine concentration against copper absorbance) between 0.5 and 2.5 m mol L^?1 (R² = 0.9943) and with a limit of detection of 0.50 m mol L^?1. Although external standard calibration provided poor recoveries in fortified fish flesh (40% to 74% for 5 to 10 mg melamine/g), the method of standard addition provided acceptable values (90% to 93%), with a relative standard deviation of 3% to 10%. The results obtained with the standard addition method were in broad agreement with those obtained by high performance liquid chromatography.     

LC with Fluorimetric Detection for Sensitive Analysis of Reduced Flubendazole in Biological Samples

A validated LC method is proposed for analysis of flubendazole and its metabolites in biological samples of Haemonchus contortus. Two detectors were used—photodiode-array and spectrofluorimetric. The native fluorescence of reduced flubendazole, the key substance investigated during biological experiments, was used for its fluorimetric detection with a very low limit of quantification (0.63 nmol L^?1).     

Microwave-Assisted Solvent Extraction of Melamine from Seafood and Determination by Gas Chromatography–Mass Spectrometry: Optimization by Factorial Design

Melamine attracts considerable attention because of its toxicity. The determination of melamine in seafood was performed by gas-chromatography–mass spectrometry, using an optimized version of a method adopted by the U.S. Food and Drug Administration. The melamine was extracted by closed-vessel microwave-assisted solvent extraction (MAE), as a valid alternative in sample preparation, to reduce analysis time and provide less ambiguous data. The procedure was optimized by means of experimental factorial design considering the three main variables that affect this process: microwave oven power, the maximum temperature inside the extraction tube, and the hold time. The recovery of melamine in spiked samples was used as the elemental response value of the design. Temperature and hold time had a more positive effect on the response than the microwave power. A significant positive interaction was observed between oven power and hold time. A temperature of 70°C and a hold time of 1 min gave a recovery of 92 ± 5% for a microwave power of 600 W. Under these conditions, the total microwave extraction time was approximately 2 minutes, a much shorter time compared to the ultrasonic bath, which required a total time of 40 min. The repeatability of the method was approximately 3%. The limits of quantification were 0.55 mg kg^?1 for MAE and 1.9 mg kg^?1 for the ultrasonic bath; the linearity was confirmed up to 10 mg kg^?1. In conclusion, the MAE procedure was shown to be an excellent alternative to the official method.     

Chromatographic Tandem Mass Spectrometric Detection of Physostigmine and its Major Metabolites in Rat Urine

Abstract

A rapid, sensitive, and specific liquid chromatographic‐electrospray ionization (ESI) tandem ion trap mass spectrometric method has been developed for identification of physostigmine and its metabolites in rat urine. 300 µg kg^–1 of physostigmine were used as a safe oral gavage dose for studies on its metabolites. 0–24 h urine was purified using a C18 solid‐phase extraction cartridge, and then detected by an on‐line MS detector. Identification and structural elucidation of the metabolites were performed by comparing their MSⁿ spectra with physostigmine. Six metabolites and unchanged physostigmine existed in rat urine. All of the metabolites were reported for the first time.     

Simultaneous chromatographic analysis of photoinitiators and amine synergists in food contact materials

Photoinitiators (PIs) are components of UV-cured inks widely used in printing of food packaging. These substances can migrate into food and may be a hazard to human health. High-performance liquid chromatography with diode-array detection (HPLC–DAD) has been used for analysis of PIs and amine synergists in food packaging. Analysis was performed with a Kromasil C18 column (250 mm?×?3.2 mm i.d., 5 μm particle size) with a binary mobile phase gradient prepared from acetonitrile and Milli-Q water. The flow rate was 0.5 mL min^?1. The method enables separation of fourteen PIs and amine synergists in a single run. The method was validated for linearity, repeatability, and limits of detection and quantification. Excellent sensitivity (LODs?≤?1.56 μg dm²) and appropriate repeatability (RSD (n?=?10) <0.9 %) were achieved. Different types of food packaging material including plastic films, cardboard, and cans were analyzed and PIs were detected in 47 % of the samples tested (n?=?17). Positive samples were confirmed by use of LC–MS–MS in positive electrospray ionization (ESI) mode.     

Gold nanoparticles and a glucose oxidase based biosensor: an attempt to follow-up aging by XPS

Amperometric biosensors are widely used for clinical, food industry and environmental control. A universal platform allowing immobilization of different enzymes could provide a fast and easy way to design new sensors, but the main drawback effect with oxidase based biosensors is the production of hydrogen peroxide. The direct electron transfer is a way to limit the H₂O₂ production. A modified electrode described by Zhao et al. (Bioelectrochemistry, 69(2):158, 2006), based on immobilization of glucose oxidase/colloidal gold nanoparticles on a glassy carbon electrode by Nafion film, has been used. Its sensitivity is 0.4 μA mM^?1 cm^?2, reproducibility is 3.0%, detection limit is 0.37 mM, response to glucose is linear up to 20 mM; limit of detection is 0.37 mM and response time is about 1.5 min. This sensor displays a formal redox potential compatible with a direct electron transfer, and has been tested for its response in time and GOx denaturation by X-ray photoelectron spectroscopy. Vanishing of disulphide bonds of GOx has been observed after a period in a saturating solution of glucose but this does not appear determinant in loss of enzyme activity.     

Detection of Melamine in Powdered Milk Using Surface-Enhanced Raman Scattering with No Pretreatment

We detected a trace amount of melamine in powdered milk using surface enhanced Raman scattering (SERS) on gold surfaces at an excitation wavelength of 632.8 nm. A detection limit of ～100 ppm (μg/g) melamine in milk was attained within a few minutes by the gold nanoparticle substrate from chemical reduction; whereas, better sensitivity, as low as ～200 ppb (ng/g), was achieved by the roughened gold substrate. Our method has the advantage of fast and sensitive detection of melamine in powdered milk without pre-treating the samples.     

Simultaneous LC–UV Determination of 5-Methyl-1-(3-fluorophenyl)-2-(1H)- pyridone and Its Two Metabolites in Rat Plasma

A simple, rapid, and reproducible isocratic reverse-phase HPLC method was developed to simultaneously determine AKF-PD and its two oxidized metabolites in rat plasma. 5-Carboxyl-1-phenyl-2-(1H)-pyridone and phenacetin were used as internal standards to ensure the precision and accuracy of the method. The analytes were separated on a C₁₈ reversed-phase column with methanol—phosphate buffer (20 mM, pH 2.5) as mobile phase. The limits of detection for AKF-PD and its two oxidized metabolites was 0.1 μg mL^?1. The method is applicable for the pharmacokinetic studies of AKF-PD and its metabolites in rats.