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.     

Determination of Neomycin in Milk by Reversed Phase Ion-Pairing Liquid Chromatography

Abstract

A fluorometric high performance liquid chromatographic (HPLC) method has been developed for the determination of neomycin in milk. Whole or shelf milk was defatted by initial centrifugation at 4°C. The resulting skim milk was deproteinated with trichloroacetic acid and centrifuged again. The neomycin was determined directly in the supernate by HPLC. The HPLC conditions consisted of an ion-pairing mobile phase, a reversed-phase column, post-column derivatization with o-phthalaldehyde (OPA) reagent and fluorescence detection. The overall recovery of neomycin was 94% (coefficient of variation 6.5%), in whole milk spiked at 0.15–10 ppm and 99% (coefficient of variation 6.4%) in shelf milk spiked at 0.15–5 ppm. The method was used to detect neomycin in milk obtained from cows dosed intramuscularly with neomycin (10 mg/kg). The neomycin concentrations in milk at 8 and 24 h after dosing were 0.3 and 0.2 ug/ml, respectively.     

LC‐MS/MS method for the determination of melamine in rat plasma: Toxicokinetic study in Sprague–Dawley rats

Most recently, melamine has raised international concern for its catastrophic health effects stemming from tainted infant formula. So far there is limited information concerning the pharmacokinetics of melamine in mammals. The present report concerns the development and validation of a sensitive HPLC‐ESI‐MS/MS method for the pharmacokinetic study of melamine in rat. The method employed a simple liquid–liquid extraction process for plasma sample cleanup, and the extraction recoveries of melamine from plasma were consistent at different concentrations. There was a linear relationship between chromatographic area and concentration over the range of 10–5000 ng/mL for melamine in plasma (R = 0.995). In this work, for the first time, melamine was administered intravenously and orally to Sprague–Dawley rats and the pharmacokinetic characteristics of this contaminant were investigated. The mean values of major pharmacokinetic parameters of oral availability, the mean steady‐state distribution volume (V_ss), clearance, and plasma elimination half‐life (T_1/2) of melamine in Sprague–Dawley rats were 72.9 ± 13.2%, 102.5 ± 12.5 mL/kg, 20.1 ± 3.8 mL/h/kg, and 4.9 ± 0.5 h, respectively. The rats pharmacokinetic study results suggested that melamine was predominantly restricted to blood or extracellular fluid and is not extensively distributed to most organ tissues. Meanwhile, melamine should be primarily eliminated by renal filtration for rats and does not undergo significant metabolism. These data should be useful to regulatory for risk assessment.     

Simultaneous derivatization and extraction of iodine from milk samples by hollow fiber liquid-phase microextraction followed by gas chromatography-electron capture detection

A simple, low-cost and sensitive method is demonstrated for derivatization and extraction of iodine from milk samples using hollow fiber liquid-phase microextraction (HF-LPME) and gas chromatography-electron capture detection. Iodide ions are converted to iodine under acidic medium and in the presence of an oxidant. The generated iodine reacted with 3-pentanone in extraction vial to give 2-iodo-3-pentanone and was extracted into 4 μL of 1-octanol located in the lumen of a hollow fiber. Organic solvent was selected using one variable at a time optimization method and the other main factors affecting derivatization and HF-LPME procedures were evaluated using a Taguchi’s L₁₆ (4⁵) orthogonal array. Under optimal conditions, the method showed low limit of detection (0.5 ng mL^?1), wide linear range (1–2,000 ng mL^?1) with good correlation coefficient (0.9997) and acceptable relative standard deviation (4.6 %, n = 5). Finally, the developed method was successfully applied for determination of iodide in real samples including infant milk formulas and cow milk with reasonable relative recoveries (99.8–110.5 %).     

Determination of alkaloids in Corydalis yanhusuo using hollow-fibre liquid phase microextraction and high performance liquid chromatography

Hollow fibre based liquid phase microextraction (HF-LPME) with high performance liquid chromatography (HPLC) was developed for the determination of alkaloids in Corydalis yanhusuo. Three alkaloids (protopine, tetrahydropalmatine, tetrahydroberberine) were extracted from a 10 mL alkaline sample (donor phase) to an organic phase impregnated in the pores of the hollow fibre, and then, they were extracted to an acidic solution (acceptor phase) in the lumen of the fibre. The extract was determined directly by HPLC. Parameters affecting the HF-LPME include the organic solvent, pH of the donor and the acceptor phase, the extraction time and the stirring speed were investigated systematically. To minimize the error of the injection, palmatine was added as an internal standard (I.S.). Under optimal conditions, calibration curves were obtained in the range of 0.1–1.0 mg L⁻¹ with a reasonable linearity (r ² > 0.993) and the limits of detection (LODs) ranged between 10.0 × 10⁻³ mg L⁻¹ and 13.7 × 10⁻³ mg L⁻¹. Additionally, enrichment factors with 100 to 184-fold were obtained. The method was then applied to the crude extract of Corydalis yanhusuo successfully.     

Determination of Trace Bisphenol A in Leachate by Solid Phase Microextraction Coupled with High Performance Liquid Chromatography

Abstract

A rapid determination method for trace bisphenol A in leachate by solid phase microextraction (SPME) coupled with high performance liquid chromatography (HPLC) was developed. The experimental condition of SPME, such as select operation, solid phase microextraction fibers, pH, extraction time, extraction temperature, desorption time, desorption solution, mode, and the analytical conditions of HPLC were optimized. As compared with the graph that was produced by HPLC alone, the graph by only HPLC couldn't analyze bisphenol A and compared to the results of three solid‐phase microextraction fibers. The linear range was between 0.0128 mg/L and 0.192 mg/L in this method, and the correlative coefficient was 0.9975. Limits of detection, repeatability, and reproducibility were also determined. The limit of detection of this method was 3.25 µg/L (3σ, n=11). The relative standard deviation (RSD, n=3) was 4.4%. The method was used for the determination of trace bisphenol A in leachate of Qingshan landfill and leachate of Liufang landfill. The recoveries were between 94.5% and 103.3%. This method is fast, convenient, sensitive, solvent free, and suitable for the determination of trace bisphenol A in leachate.     

高效液相色谱法测定奶粉和液态奶中的三聚氰胺

建立了奶粉和液态奶中三聚氰胺的高效液相色谱（HPLC）检测方法。经阳离子交换固相萃取柱净化后的样品采用HPLC测定。优化的色谱条件：C18柱（4.6 mm ×200 mm,5 μm）,流动相为乙腈-0.01 mol/L庚烷磺酸钠（pH 3.3）（体积比为10∶90）,流速为1.0 mL/min,检测波长为236 nm,柱温为40 ℃,进样量为20 μL。方法的线性范围为1~500 mg/L,检出限为0.2 mg/kg （S/N=3）,定量限为1 mg/kg （S/N=15）,回收率为81.4%~83.7%,相对标准偏差为3.3%~8.5%（n=6）。     

Determination of Melamine Using Magnetic Molecular Imprinted Polymers and High Performance Liquid Chromatography

A novel method using magnetic molecular imprinting polymers (MMIP) specific for recognizing, separating, and extracting melamine in bovine milk was developed with an external additive magnetic shelf in this paper. The MMIP was synthesized with Fe₃O₄ nanoparticles and molecular imprinted polymer synthesis technology. With the help of an elution step using methanol/acetic acid mixture elution, the melamine could be collected and concentrated. By using high performance liquid chromatography combined with this pretreatment technique, the analytes eluted from the magnetic polymers can be determined. This extraction method was validated by analysis of incurred bovine milk matrix. The proposed method simplified the procedures of classical solid-phase extraction method and improved the reliability of method. The obtained recoveries of this method were in the range of 85.04–105.2% in spiked milk samples. The limit of detection (LOD) was below 29.2 ng g^?1.     

Preparation and characterization of molecularly imprinted microspheres for selective extraction of trace melamine from milk samples

We describe molecularly imprinted microspheres (MIMs) for the selective extraction of melamine from milk. The MIMs were made from melamine as the template molecule, methacrylic acid as the functional monomer, and ethylene glycol dimethacrylate as the linking agent. The MIMs were synthesized by suspension polymerization and characterized by rebinding experiments. They displayed high adsorption capacity, fast rebinding kinetics, and highly specific rebinding of melamine. The imprinting factor is 4.1. Scatchard analysis revealed a one-type rebinding behavior, the dissociation constant and maximum rebinding capacity being 37.59 g L^?1 and 30.85 μmol g^?1, respectively. The MIMs exhibited a 25% cross-reactivity towards atrazine, but less than 3.0% towards prometryn, clenbuterol and metronidazole. In addition, a MIM-based solid phase extraction (MISPE) column for melamine was prepared by packing MIMs into a common SPE cartridge. The MISPE extraction gave recoveries of 89.8 to 100.6% of melamine, with relative standard deviations of 5.9 to 7.5%. There was no significant loss of rebinding capacity after more than 60 repeated uses, thus demonstrating the high stability of the MISPE column. The MSPE column also was applied to the extraction of melamine from spiked liquid and powdered milk with satisfying accuracy and precision.

Hydrophilic solid‐phase extraction of melamine with ampholine‐modified hybrid organic–inorganic silica material

In this work, an ampholine‐functionalized hybrid organic–inorganic silica sorbent was successfully used to extract melamine from a milk formula sample by a hydrophilic interaction solid‐phase extraction protocol. Primary factors affecting the extraction efficiency of the material such as extraction solvent, elution solvent, sample loading volume, and elution volume have been thoroughly optimized. Under the optimized hydrophilic solid‐phase extraction conditions, the recoveries of melamine spiked in milk formula samples ranged from 86.2 to 101.8% with relative standard deviations of 4.1–9.4% (n = 3). The limit of detection (S/N = 3) was 0.32 μg/g. The adsorption capacity toward melamine was 30 μg of melamine per grams of sorbent. Due to its simplicity, rapidity and cost effectiveness, the newly developed hydrophilic solid‐phase extraction method should provide a promising tool for daily monitoring of doped melamine in milk formula.     

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%).     

Development of a Reversed-Phase Liquid Chromatographic Assay for the Quantification of Total Persipeptides in Fermentation Broth

The emergence and prevalence of multi-drug-resistant bacterial strains increase the potential for outbreaks of incurable infections. The discovery of novel antibiotics and pharmacological preparations requires the identification of novel bioactive small molecules. A specific, sensitive, and reliable quantification method using high-performance liquid chromatography (HPLC) with UV detection was developed for the determination of total persipeptides (A and B), which are cyclic pentapeptides found in the fermentation broth of Streptomyces zagrosensis UTMC 1154 that exhibit bioactivity against methicillin-resistant Staphylococcus aureus (MRSA). A simple liquid–liquid extraction (LLE) method using butanol was employed to extract persipeptides from the fermentation broth prior to HPLC analysis. The chromatographic separation of persipeptides and the internal standard, virginiamycin, was achieved with a gradient of acetonitrile and water on a C18 reversed-phase analytical column in a 25-min analytical run utilizing a flow rate of 0.8 mL min⁻¹ and detection at 210 nm. The whole assay was validated, and the method presented a linear response range with a regression coefficient of determination R ² of 0.9996 for the quantification of persipeptides in the concentration range of 3.9–250.0 µg mL⁻¹, as well as extraction recoveries ranging from 54.78 ± 9.83 % to 56.45 ± 16.33 %. The bias and the precision of the proposed method were <10 %. The detection and quantification limits for the persipeptides were 27 and 83 µg L⁻¹, respectively.

     

Determination of Imidazole, 4-Methylimidazole,and 2-Methylimidazole in Cigarette Additives by Ultra-High Performance Liquid Chromatography

Ultra-high performance liquid chromatography was employed for the determination of imidazole, 4-methylimidazole, and 2-methylimidazole in cigarette additives. Following solid phase extraction and filtration, the analytes were separated using isocratic elution with 5 mmol/L acetonitrile-ammonium formate (80:20, v/v) at 0.5 mL/min. The quantification of these analytes was achieved with the external standard method on a diode-array detector at 215 nm. Baseline separation was achieved within 1.3 min. The linear dynamic ranges for imidazole, 4-methylimidazole, and 2-methylimidazole were between 0.0375 and 18.0300 mg/kg. The limits of detection for the analytes were 0.0094 mg/kg. The recoveries and the relative standard deviations at fortification levels of 0.1322 mg/kg to 1.6220 mg/kg were 95.20% to 101.93% and 0.55% to 2.54%, respectively. The method offers easy operation, rapid analysis, and accurate results, and is suitable for the determination of imidazole, 4-methylimidazole, and 2-methylimidazole in cigarette additives.     

Hollow Fiber Liquid-Phase Microextraction Combined with Gas Chromatography-Mass Spectrometry for Determination of Root Exudative Potential Allelochemicals in Capsicum Annuum(Chili)

Hollow fiber liquid phase microextraction (HF-LPME) was developed for the determination of potential allelochemicals by gas chromatography-mass spectrometry (GC-MS). Biomass method was applied to study the allelopathic effect. The applicability of the proposed procedure was evaluated through extraction of three potential allelochemicals in recirculating hydroponic culture solution of chili. The parameters affecting extraction. The optimum extraction conditions were obtained with 15 µL toluene as acceptor phase at an extraction time, of 30 min, an extraction temperature of 50°C, a stirring rate of 800 rpm, 9% (w/v) sodium chloride, and pH of 6 in a 10 mL sample. The calibration curves showed a high linearity level in the range of 0.1–-5 µg · mL^?1, relative standard deviations between 5.51% and 8.94%, and enrichment factors that had 6.3–12.5 were obtained for 2,6-DTBP, DPA, and DIBP. Recoveries, ranging from 81.4% to 118.7% and an RSD below 10.3% were obtained when the method was applied to determine three potential allelochemicals in root exudates of real samples of 10 d, 20 d, and 30 d after cultivation.     

Simultaneous Determination of Resibufogenin and Its Major Metabolite 3-epi-Resibufogenin in Rat Plasma by HPLC Coupled with Tandem Mass Spectrometry

A rapid, sensitive and specific method for the simultaneous quantification of resibufogenin (RBG) and 3-epi-resibufogenin (3-ERBG) in rat plasma was developed by using a liquid–liquid extraction procedure and liquid chromatography–electrospray ionization/tandem mass spectrometric (LC–ESI–MS/MS) analysis. The separation was performed by HPLC on a reversed phase C₁₈ HPLC column (150 × 2.1 mm, 3.5 μm) using a mobile phase of acetonitrilel-0.1% formic acid aqueous solution (45:55, v/v). The determination was performed by a triple-quadrupole mass spectrometer in the multiple reaction monitoring using positive mode of electrospray ionization (ESI). The calibration curves were both linear (R > 0.995) over the concentration range of 3.0–5,000 ng mL⁻¹, and the lower limits of quantification were 3.0 ng mL⁻¹ for both RBG and 3-ERBG. The intra-day and inter-day precisions (% RSD) were all less than 15%, and the accuracies (%RE) were within the range of ±15%. The mean recoveries of RBG, 3-ERBG and IS were over 82.7, 84.8 and 90.0% (n = 6), respectively. The method was proved to be rapid, sensitive and specific, and has been successfully applied to determine RBG and its major metabolite 3-ERBG in rat plasma after oral administration of RBG for pharmacokinetic study. Comparison of pharmacokinetic data with anti-tumor activities of RBG and ERBG suggested that 3-ERBG, as a major metabolite of RBG in rats, was perhaps also a bioactive form of RBG in vivo.

     

Determination of Two Pesticides in Soils by Dispersive Liquid–Liquid Microextraction Combined with LC-Fluorescence Detection

In the present work, a simple, rapid and sensitive sample pre-treatment technique, dispersive liquid–liquid microextraction (DLLME) coupled with liquid chromatography-fluorescence detection (LC-FLD), has been developed to determine carbamate (carbaryl) and organophosphorus (triazophos) pesticide residues in soil samples. Methanol was first used as extraction solvent for the extraction of pesticides from the soil samples and then as dispersive solvent in the DLLME procedure. Under the optimum extraction conditions, the linearity was obtained in the concentration range of 0.1–1,000 ng g⁻¹ for carbaryl and 1–5,000 ng g⁻¹ for triazophos, respectively. Correlation coefficients varied from 0.9997 to 0.9999. The limits of detection (LODs), based on signal-to-noise ratio (S/N) of 3, ranged from 14 to 110 pg g⁻¹. The relative standard deviation (RSDs, for 20.0 ng g⁻¹ of each pesticide) varied from 1.96 to 4.24% (n = 6). The relative recoveries of two pesticides from soil A1, A2 and A3 at spiking levels of 10.0, 20.0 and 50.0 ng g⁻¹ were in the range of 88.2–108.8%, 80.8–110.7% and 81.0–111.1%, respectively. The results demonstrated that DLLME was a sensitive and accurate method to determine the target pesticides, at trace levels, in soils.

     

Efficient sample clean‐up and online preconcentration for sensitive determination of melamine in milk samples by capillary electrophoresis with contactless conductivity detection

Based on an efficient sample clean‐up and field‐amplified sample injection online preconcentration technique in capillary electrophoresis with contactless conductivity detection, a new analytical method for the sensitive determination of melamine in milk samples was established. In order to remove the complex matrix interference, which resulted in a serious problem during field‐amplified sample injection, liquid–liquid extraction was utilized. As a result, liquid–liquid extraction provides excellent sample clean‐up efficiency when ethyl acetate was used as organic extraction by adjusting the pH of the sample solution to 9.5. Both inorganic salts and biological macromolecules are effectively removed by liquid–liquid extraction. The sample clean‐up procedure, capillary electrophoresis separation parameters and field‐amplified sample injection conditions are discussed in detail. The capillary electrophoresis separation was achieved within 5 min under the following conditions: an uncoated fused‐silica capillary, 12 mM HAc + 10 mM NaAc (pH = 4.6) as running buffer, separation voltage of +13 kV, electrokinetic injection of +12 kV × 10 s. Preliminary validation of the method performance with spiked melamine provided recoveries >90%, with limits of detection and quantification of 0.015 and 0.050 mg/kg, respectively. The relative standard deviations of intra‐ and inter‐day were below 6%. This newly developed method is sensitive and cost effective, therefore, suitable for screening of melamine contamination in milk products.     

Analysis of melamine in milk powder by using a magnetic molecularly imprinted polymer based on carbon nanotubes with ultra high performance liquid chromatography and tandem mass spectrometry

A new magnetic molecularly imprinted polymer was coupled with ultra high performance liquid chromatography and tandem mass spectrometry for the selective determination of melamine in milk powder. The magnetic molecularly imprinted polymer has been prepared by using carbon nanotubes as the matrix, Fe₃O₄ particles as the magnetic ingredient, melamine as the template molecule, methacrylic acid as the functional monomer, ethylene glycol dimethacrylate as the cross‐linker and polyvinylpyrrolidone as the dispersant. The polymer was characterized with scanning electron microscopy, Fourier transform infrared spectroscopy and a physical property measurement system. The isothermal adsorption, kinetics adsorption, and selectivity were studied to evaluate the rebinding properties of the magnetic molecularly imprinted polymer. Various parameters affecting the extraction efficiency such as the amount of polymer, extraction time, and eluting solution were evaluated. The limit of detection was 0.00075 mg/kg. The relative standard deviations of the intraday and interday precision are 0.4–2.7 and 2.3–5.1%, respectively. The proposed method was successfully applied to determine melamine in different milk powder samples from different provenances, and satisfactory recoveries of 89.0–95.6% were obtained. This method has great significance for quality control and is simple and suitable for the rapid determination of melamine in milk powder.     

Determination of melamine residue in liquid milk by capillary electrophoresis with solid-phase extraction

A novel solid-phase extraction-capillary electrophoresis (SPE-CE) method was developed for the determination of melamine residue in liquid milk. The conditions of SPE and CE were investigated and optimized. A 1% trichloroacetic acid plus 2.2% lead acetate solution were used for the extraction of analyte and the removal of protein. A Cleanert PCX SPE cartridges column was used for clean up. The 50 mM sodium dihydrogenphosphate running buffer (pH adjusted to 3.2 with citric acid) was used as a running buffer. The linearity is satisfactory in the range of 0.8-100 μg/mL with a correlation coefficient of 0.9998. Under the optimal conditions, the method limit of detection (LOD) and method limit of quantification were 0.12 mg/kg and 0.37 mg/kg, respectively. The recovery of melamine from different liquid milk samples was in the range of 89.5-98.5% with a relative standard deviation of 1.8-3.5%. The intra- and inter-day assay precision was 2.8% (n = 6) and 4.1% for five days, respectively. The developed method has been applied successfully for the determination of melamine residue in liquid milk samples. The results obtained by the proposed method agree with those obtained by high-performance liquid chromatographic method. The proposed method enables the quantitative determination of melamine residues at levels as low as 0.37 mg/kg in different liquid milk.