Production of malachite green oxalate and leucomalachite green reference materials certified for purity

Malachite green oxalate (MG oxalate) and leucomalachite green (LMG) have been prepared and certified as pure reference materials. The purities of MG oxalate and LMG were assessed by high-performance liquid chromatography–diode array detection (HPLC–DAD), nuclear magnetic resonance (NMR) spectroscopy, differential scanning calorimetry (DSC), Karl Fischer titration, ashing and thermogravimetric analysis (TGA). MG oxalate was purified by supercritical fluid extraction (SFE). Prior to purification, commercial MG oxalate purity was estimated to be about 90%. The main impurities present in SFE-purified MG oxalate were identified and quantified using HPLC–DAD. The main impurities were found to be monode-MG (monodemethylated MG oxalate synthesis impurity), 4-(dimethylamino)benzophenone (4-DMABP), MG-carbinol and LMG. The homogeneity of both reference materials was also determined. Issues associated with the stability of LMG and MG oxalate in solution forced an extensive study investigating different parameters i.e. solvent, acid, analyte concentration and temperature. MG oxalate (100 μg/mL) was found to be stable in acetonitrile containing 1% v/v glacial acetic acid for at least 155 days and LMG (100 μg/mL) was stable in acetonitrile for at least 133 days. The final purity value for MG oxalate was 94.3 ± 1.4% m/m at the 95% confidence interval (or 67% m/m if MG cation is reported). For LMG, the certified purity was found to be 98.8 ± 0.8% m/m at the 95% confidence interval. Figure Calibration reference materials for malachite green and leucomalachite green, certified for purity, are essential in characterising these key analytes in a fish matrix reference material     

Evaluation of commercial immunoassays for the detection of estrogens in water by comparison with high-performance liquid chromatography tandem mass spectrometry HPLC–MS/MS (QqQ)

In this work four different commercially available enzyme-linked immunosorbent assays (ELISA) (from Japan EnviroChemicals, Ltd., Tokyo, Japan) were evaluated in terms of performance for the rapid screening of estrogens in different water matrices, including natural and spiked samples from urban wastewater, river water and ground water. All four test kits are based on monoclonal antibodies. The compounds detected by these immunoassays are (1) 17-β-estradiol, (2) estrone, (3) 17-α-ethynyl estradiol and (4) estrogens in general, with high recognition properties for 17-β-estradiol, estrone and estriol. Standards were prepared in water containing 10% (v/v) methanol. The IC ₅₀ (corresponding to the 50% of the effective concentration) values, the dynamic ranges, and the limits of detection of the ELISA kits were 0.060–0.304 μg/L, 0.05–5 μg/L and 0.05 μg/L, respectively. All samples were extracted by solid-phase extraction (SPE) beforehand, and the evaluation was carried out by comparing the results obtained by ELISA with those obtained by HPLC–MS/MS using a triple quadrupole (QqQ) instrument. In addition, two different solid-phase extraction procedures were carried out and compared. Except for moderate overestimation in the results observed with the ELISA kits in the analysis of complex wastewater samples, the results obtained using all of the tested techniques were generally in very good agreement.      

Monitoring carbamazepine in surface and wastewaters by an immunoassay based on a monoclonal antibody

The pharmaceutical compound carbamazepine (CBZ) is an emerging pollutant in the aquatic environment and may potentially be used as a wastewater marker. In this work, an enzyme-linked immunosorbent assay (ELISA) for the detection of carbamazepine in surface and sewage waters has been developed. The heterogeneous immunoassay is based on a commercially available monoclonal antibody and a novel enzyme conjugate (tracer) that links the hapten via a hydrophilic peptide (triglycine) spacer to horseradish peroxidase. The assay achieves a limit of detection of 24 ng/L and a quantitation range of 0.05–50 μg/L. The analytical performance and figure of merits were compared to liquid chromatography–tandem mass spectrometry after solid-phase extraction. For nine Berlin surface water samples and one wastewater sample, a close correlation of results was observed. A constant overestimation relative to the CBZ concentration of approximately 30% by ELISA is probably caused by the presence of 10,11-epoxy-CBZ and 2-hydroxy-CBZ in the samples. The ELISA displayed cross-reactivities for these compounds of 83% and 14%, respectively. In a first screening of 27 surface water samples, CBZ was detected in every sample with concentrations between 0.05 and 3.2 μg/L. Since no sample cleanup is required, the assay allowed for the determination of carbamazepine with high sensitivity at low costs and with much higher throughput than with conventional methods.      

Analysis of erythritol in foods by polyclonal antibody-based indirect competitive ELISA

Sugar alcohols are widely used as food additives and drug excipients. Erythritol (INS 968) is an important four-carbon sugar alcohol in the food industry. Erythritol occurs naturally in certain fruits, vegetables, and fermented foods. Currently, HPLC and GC methods are in use for the quantification of erythritol in natural/processed foods. However, an immunoassay for erythritol has not been developed so far. We have utilized affinity-purified erythritol-specific antibodies generated earlier [9] to develop an indirect competitive ELISA. With erythritol–BSA conjugate (54 mol/mol; 100 ng/well) as the coating antigen, a calibration curve was prepared using known amounts of standard meso-erythritol (0.1–100,000 ng) in the immunoassay. Watermelon (Citrullus lanatus) and red wine were selected as the food sources containing meso-erythritol. The amount of meso-erythritol was calculated as 2.36 mg/100 g fresh weight of watermelon and 206.7 mg/L of red wine. The results obtained from the immunoassay are in close agreement with the reported values analyzed by HPLC and GC (22–24 mg/kg in watermelon and 130–300 mg/L in red wine). The recovery analyses showed that added amounts of meso-erythritol were recovered fairly accurately with recoveries of 86–105% (watermelon) and 85–93.3% (red wine). The method described here for erythritol is the first report of an immunoassay for a sugar alcohol. Figure Indirect competitive ELISA for quantitation of erythritol     

Comparison of dispersive liquid–liquid microextraction and hollow fiber liquid–liquid–liquid microextraction for the determination of fentanyl,alfentanil, and sufentanil in water and biological fluids by high-performance liquid chromatography

Dispersive liquid–liquid microextraction (DLLME) and hollow fiber liquid–liquid–liquid microextraction (HF-LLLME) combined with HPLC–DAD have been applied for the determination of three narcotic drugs (alfentanil, fentanyl, and sufentanil) in biological samples (human plasma and urine). Different DLLME parameters influencing the extraction efficiency such as type and volume of the extraction solvent and the disperser solvent, concentration of NaOH, and salt addition were investigated. In the HF-LLLME, the effects of important parameters including organic solvent type, concentration of NaOH as donor solution, concentration of H₂SO₄ as acceptor phase, salt addition, stirring rate, temperature, and extraction time were investigated and optimized. The results showed that both extraction methods exhibited good linearity, precision, enrichment factor, and detection limit. Under optimal condition, the limits of detection ranged from 0.4 to 1.9 μg/L and from 1.1 to 2.3 μg/L for DLLME and HF-LLLME, respectively. For DLLME, the intra- and inter-day precisions were 1.7–6.4% and 14.2–15.9%, respectively; and for HF-LLLME were 0.7–5.2% and 3.3–10.1%, respectively. The enrichment factors were from 275 to 325 and 190 to 237 for DLLME and HF-LLLME, respectively. The applicability of the proposed methods was investigated by analyzing biological samples. For analysis of human plasma and urine samples, HF-LLLME showed higher precision, more effective sample clean-up, higher extraction efficiency, lower organic solvent consumption than DLLME.     

Headspace sorptive extraction using silicone tubes for the determination of chlorobenzenes in water

A new approach for headspace sorptive extraction is presented and demonstrated for the determination of 12 chlorobenzenes in water samples. It consists of a silicone tube (15-mm length) arranged around a stainless steel rod. This device is fixed on a septum cap and exposed to the headspace of 50 mL of a salt-saturated water sample. After extraction (60-min optimized extraction time), thermodesorption is carried out by direct insertion of the silicone tube into the thermodesorption-gas chromatography-mass spectrometry system. Desorption of the analytes is performed at 250 °C for 5 min with a gas flow of 50 mL/min. Repeatability (relative standard deviation 5–10%), extraction yields (9–46%), enrichment factors (129–657), and detection limits (0.002–0.012 μg/L) were determined and four real water samples were analyzed with the headspace tube extraction. The results were verified by standard addition. A comparison of headspace tube extraction with other headspace enrichment techniques underlined the high extraction capacity of the proposed method. A big advantage of tube extraction is the low cost of the silicone material. The tubes can be discarded after single use, avoiding carryover problems and cross-contamination. Figure Scheme of the HS-tube extraction and thermodesorption system     

Quantification of human and veterinary antibiotics in water and sediment using SPE/LC/MS/MS

An analytical method was developed and tested for four different groups of veterinary antibiotics in both river water and sediment matrices. Solid phase extraction (SPE) was used to enrich and to clean up the aqueous sample. Also, Mcllvaine and ammonium hydroxide buffer solutions were used to extract the compounds from the sediment matrix. High performance liquid chromatography (HPLC) equipped with tandem mass spectrometry (MS/MS) was used to separate and quantify the samples. The range of recoveries (in percent) for tetracyclines (TCs), sulfonamides (SAs), macrolides (MLs), and ionophore polyethers (IPs) in the water matrix were 102.2–124.8, 76.6–124.3, 89.5–114.7, 82.7–117.5 with 1–13 (%) of relative standard deviation respectively with three different concentrations. For sediment, the percent recovery ranges were 32.8–114.8, 62.4–108.9, 53.4–128.4 and 51.3–105.4 for TCs, SAs, MLs and IPs, respectively. The relative standard deviation ranged from 16 – 27 (%) over three different concentrations. The limit of quantification (LOQ) was determined by two different methods and calculated to be in the range of 0.01–0.04 μg/l and 0.3–2.5 μg/kg for TCs, SAs, and MLs in water and sediment, respectively. For IPs, the LOQ was 0.001–0.003 μg/l in river water and 0.4–3.6 μg/kg for sediment. The sediment concentration measured in an agriculture-influenced river was much higher than in the overlying water matrix, indicating a high degree of sediment partitioning for these compounds.     

HPLC-ICP-MS and HPLC-ES-MS/MS characterization of synthetic seleno-arsenic compounds

Various toxicological and metabolic interactions have been reported to exist between arsenic and selenium. In the present study, synthetic seleno-arsenic compounds, potentially suitable for probing metabolic interactions between these two elements, were prepared and tentatively characterized by using high-performance liquid chromatography (HPLC)–electrospray tandem mass spectrometry and HPLC–inductively coupled plasma mass spectrometry. In analogy to the recently identified thio-arsenic species, which can be prepared from their corresponding oxo-arsenic species via reaction with H₂S, the seleno-arsenic compounds were also derived from oxo-arsenic compounds via reaction with H₂Se. Figure H₂Se bubbled into solutions containing oxo‐arsenosugars converts them into their seleno‐arsenosugar analogues.     

Multi-component analysis of tetracyclines, sulfonamides and tylosin in swine manure by liquid chromatography–tandem mass spectrometry

A multi-component method focussing on thorough sample preparation has been developed for simultaneous analysis of swine manure for three classes of antibiotic—tetracyclines, sulfonamides, and tylosin. Liquid manure was initially freeze-dried and homogenised by pulverization before extraction by pressurised liquid extraction. The extraction was performed at 75°C and 2,500 psig in three steps using two cycles with 0.2 mol L⁻¹ citric acid buffer (pH 4.7) and one cycle with a mixture of 80% methanol with 0.2 mol L⁻¹ citric acid (pH 3). After liquid–liquid extraction with heptane to remove lipids, the pH of the manure was adjusted to 3 with formic acid and the sample was vacuum-filtered through 0.6 μm glass-fibre filters. Finally the samples were pre-concentrated by tandem SPE (SAX-HLB). Recoveries were determined for manure samples spiked at three concentrations (50–5,000 μg kg⁻¹ dry matter); quantification was achieved by matrix-matched calibration. Recoveries were >70% except for oxytetracycline (42–54%), sulfadiazine (59–73%), and tylosin (9–35%) and did not vary with concentration or from day-to-day. Limits of quantification (LOQ) for all compounds, determined as a signal-to-noise ratio of 10, were in the range 10–100 μg kg⁻¹ dry matter. The suitability of the method was assessed by analysis of swine manure samples from six different pig-production sites, e.g. finishing pigs, sows, or mixed production. Residues of antibiotics were detected in all samples. The largest amounts were found for tetracyclines (up to 30 mg kg⁻¹ dry matter for the sum of CTC and ECTC). Sulfonamides were detected at concentrations up to 2 mg kg⁻¹ dry matter (SDZ); tylosin was not detected in any samples.      

Analysis of captan, folpet, and captafol in apples by dispersive liquid–liquid microextraction combined with gas chromatography

A novel method was developed for the determination of captan, folpet, and captafol in apples by dispersive liquid–liquid microextraction (DLLME) coupled with gas chromatography–electron capture detection (GC–ECD). Some experimental parameters that influence the extraction efficiency, such as the type and volume of the disperser solvents and extraction solvents, extraction time, and addition of salt, were studied and optimized to obtain the best extraction results. Under the optimum conditions, high enrichment factors for the compounds were achieved ranging from 824 to 912. The recoveries of fungicides in apples at spiking levels of 20.0 μg kg⁻¹ and 70.0 μg kg⁻¹ were 93.0–109.5% and 95.4–107.7%, respectively. The relative standard deviations (RSDs) for the apple samples at 30.0 μg kg⁻¹ of each fungicide were in the range from 3.8 to 4.9%. The limits of detection were between 3.0 and 8.0 μg kg⁻¹. The linearity of the method ranged from 10 to 100 μg kg⁻¹ for the three fungicides, with correlation coefficients (r ²) varying from 0.9982 to 0.9997. The obtained results show that the DLLME combined with GC–ECD can satisfy the requirements for the determination of fungicides in apple samples. Figure Dispersive liquid–liquid microextraction (DLLME) coupled with gas chromatography–electron capture detection (GC–ECD) allows satisfactory determination of fungicides in apple samples     

Comparative study of screening methodologies for ochratoxin A detection in winery by-products

The worldwide contamination of winery by-products by mycotoxins may present a serious hazard to human and animal health. Mycotoxins are secondary metabolites of fungi with possible adverse effects on humans, animals, and crops that result in illnesses and economic losses. Mycotoxins are under continuous survey in Europe, but the regulatory aspects still need to be set up for winery by-products, which may be used in animal feed. The aim of this study was to implement a simple but reliable analytical methodology for ochratoxin A (OTA) quantification in grape pomaces in order to perform a survey of samples from the Douro Demarcated Region, Portugal. The method involved a unique preparation step, solvent extraction, followed by high-performance liquid chromatography (HPLC) with fluorescence (FL) detection. A comparative study was performed with two extraction solvents (ethyl acetate and methanol) as well as using extraction on an immunoaffinity column. The linearity range for OTA analysis was 0.05–23.5 μg L⁻¹ with a detection limit of 0.05 μg L⁻¹ and a precision (expressed by the coefficient of variation under repeatability conditions) of 0.4–14.7%. The percentage of recovery was on average 23.5 ± 3.6% (extraction with ethyl acetate) or 70.1 ± 2.5% (extraction with 70% methanol). Accounting for the recovery factor and the chromatographic detection limit, as well as the preconcentration factor, the limit of detection in grape pomaces is 0.04 μg kg⁻¹ (ethyl acetate extraction) and 0.33 μg kg⁻¹ (methanol extraction). Samples from 12 out of 13 sites in the Douro Demarcated Region showed OTA presence with concentrations not exceeding 0.4 μg kg⁻¹. Both developed methods for evaluation of OTA in grape pomace are simple but efficient. Figure Extraction of ochratoxin A (OTA) from grape pomaces allows simple but efficient quantification of OTA in winery by-products by HPLC-FL     

Selective solid-phase extraction of tebuconazole in biological and environmental samples using molecularly imprinted polymers

Molecularly imprinted polymers (MIPs) were prepared by precipitation polymerization using tebuconazole (TBZ) as a template. Frontal chromatography and selectivity experiments were used to determine the binding capabilities and binding specificities of different MIPs. The polymer that had the highest binding selectivity and capability was used as the solid-phase extraction (SPE) sorbent for the direct extraction of TBZ from different biological and environmental samples (cabbage, pannage, shrimp, orange juice and tap water). The extraction protocol was optimized and the optimum conditions were: conditioning with 5 mL methanol:acetic acid (9:1), 5 mL methanol and 5 mL water respectively, loading with 5 mL aqueous samples, washing with 1.2 mL acetonitrile (ACN):phosphate buffer (5:5, pH3), and eluting with 3 mL methanol. The MIPs were able to selectively recognize, effectively trap and preconcentrate TBZ over a concentration range of 0.5–15 μmol/L. The intraday and interday RSDs were less than 9.7% and 8.6%, respectively. The limit of quantification was 0.1 μmol/L. Under optimum conditions, the MISPE recoveries of spiked cabbage, pannage, shrimp, orange juice and tap water were 62.3%, 75.8%, 71.6%, 89% and 93.9%, respectively. MISPE gave better HPLC separation efficiencies and higher recoveries than C18 SPE and strong cation exchange (SCX) SPE. Figure HPLC analysis of spiked pannage after MISPE (A) and after C18 SPE (B). HQ (1), E3 (2), p-NP (3), FTF (4), TBZ (5), PNZ (6), HXZ (7) Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

A new molecularly imprinted polymer for selective extraction of cotinine from urine samples by solid-phase extraction

Cotinine, the main metabolite of nicotine in human body, is widely used as a biomarker for assessment of direct or passive exposure to tobacco smoke. A method for molecularly imprinted solid-phase extraction (MISPE) of cotinine from human urine has been investigated. The molecularly imprinted polymer (MIP) with good selectivity and affinity for cotinine was synthesized using cotinine as the template molecule, methacrylic acid as the functional monomer, and ethylene glycol dimethacrylate as the cross-linker. The imprinted polymer was evaluated for use as a SPE sorbent, in tests with aqueous standards, by comparing recovery data obtained using the imprinted form of the polymer and a non-imprinted form (NIP). Extraction from the aqueous solutions resulted in more than 80% recovery. A range of linearity for cotinine between 0.05 and 5 μg mL⁻¹ was obtained by loading 1 mL blank urine samples spiked with cotinine at different concentrations in acetate buffer of pH 9.0, and by using double basic washing and acidic elution. The intra-day coefficient of variation (CV) was below 7% and inter-day CV was below 10%. This investigation has provided a reliable MISPE–HPLC method for determination of cotinine in human urine from both active smokers and passive smokers. Figure     

Environmental monitoring of phenolic pollutants in water by cloud point extraction prior to micellar electrokinetic chromatography

Many aromatic compounds can be found in the environment as a result of anthropogenic activities and some of them are highly toxic. The need to determine low concentrations of pollutants requires analytical methods with high sensitivity, selectivity, and resolution for application to soil, sediment, water, and other environmental samples. Complex sample preparation involving analyte isolation and enrichment is generally necessary before the final analysis. The present paper outlines a novel, simple, low-cost, and environmentally friendly method for the simultaneous determination of p-nitrophenol (PNP), p-aminophenol (PAP), and hydroquinone (HQ) by micellar electrokinetic capillary chromatography after preconcentration by cloud point extraction. Enrichment factors of 180 to 200 were achieved. The limits of detection of the analytes for the preconcentration of 50-ml sample volume were 0.10 μg L⁻¹ for PNP, 0.20 μg L⁻¹ for PAP, and 0.16 μg L⁻¹ for HQ. The optimized procedure was applied to the determination of phenolic pollutants in natural waters from San Luis, Argentina. Figure Schematic representation of the cloud point extraction process.     

Surface plasmon resonance biosensor based on Hg/Ag–Au film

Mercury or silver was electrodeposited on an Au surface to form an Hg–Au or Ag–Au film. Wavelength-modulation SPR biosensors based on this Hg/Ag–Au film were then used to determine human IgG and rabbit IgG. When direct immunoreactions were performed on the Au sensing surface, the range of concentrations of human IgG and rabbit IgG that could be determined were 2.00–40.00 μg/ml and 2.50–40.00 μg/ml, respectively. When Hg was electrodeposited onto the Au film for 1200 s, the range of concentrations of human IgG and rabbit IgG that could be determined were 0.50–40.00 μg/ml and 0.63–40.00 μg/ml, respectively. When Ag was electrodeposited onto the Au film for 1500 s, the range of concentrations of human IgG and rabbit IgG that could be determined were 0.25–20.00 and 0.42–20.00 μg/ml, respectively. The biosensor based on Ag–Au film was therefore found to be the most sensitive of the three types of biosensor tested, giving limits of determination that were up to eight times lower than those obtained with a biosensor based on Au film alone. Figure The relationship between the concentration of human IgG and the shift in the resonant wavelength Δλ _eq for different Ag electrodeposition times     

Comparison of two solid-phase microextraction methods for the quantitative analysis of VOCs in indoor air

Competitive adsorption on adsorptive solid-phase microextraction (SPME) fibres implies careful determination of operating conditions for reliable quantitative analysis of VOCs in indoor air. With this objective, two analytical approaches, involving non-equilibrium and equilibrium extraction, were compared. The average detection limit obtained for GC-MS analysis of nine VOCs by the equilibrium method is 0.2 μg m⁻³, compared with 1.9 μg m⁻³ with the non-equilibrium method. The effect of the relative humidity of the air on the calibration plots was studied, and shown to affect acetone adsorption only. Hence, the concentrations that can be accurately determined are up to 9 μmol m⁻³. The methods were then applied to indoor air containing different concentrations of VOCs. The non-equilibrium method, involving short extraction time, can be used for detection of pollution peaks whereas equilibrium extraction is preferable for measurement of sub-μg m⁻³ ground concentration levels.      

Study on an on-line molecularly imprinted solid-phase extraction coupled to high-performance liquid chromatography for separation and determination of trace estrone in environment

Estrone is one of the important potential endocrine-disrupting compounds, and the sensitive and reliable analytical methods for the determination of estrone are required for the assurance of human health. In this paper, using estrone as template molecule, 3-aminopropyltriethoxysilane as function monomer, and tetraethoxysilicane as cross-linker, a highly selective molecularly imprinted microsphere was synthesized by surface molecular imprinting technique combined with a sol–gel process. The imprinted material was characterized by the Fourier transform infrared and static adsorption experiments, and the results showed that it exhibited good recognition and selective ability for estrone. A novel method for separation and determination of trace estrone in environmental sample was developed using on-line molecularly imprinted solid-phase extraction coupled to high-performance liquid chromatography. With a sample loading flow rate of 2.6 mL min⁻¹ for a 9.6-min extraction, the enrichment factor obtained by the slopes of the linear portion in comparison with the direct injection of 10 μL standard sample solution was 1,045. The detection limit (S/N = 3) was 5.7 ng L⁻¹, and the relative standard deviations for nine replicate extractions of 5.0 μg L⁻¹ estrone was less than 10.0%. This method was evaluated for quantitative determination of estrone in well and lake water samples spiked at two levels (0.5 and 1.0 μg L⁻¹) with recoveries ranging from 86% to 95%.      

Determination of organochlorine pesticides in propolis by gas chromatography-electron capture detection using double column series solid-phase extraction

A rapid and reliable method was developed and applied for the simultaneous determination of 17 organochlorine pesticides (OCPs) in propolis. After extraction with hexane and acetone (1:1, v/v), four sorbents (florisil, silica, graphitized carbon, and tandem graphitized carbon plus florisil) were assayed for the clean-up step. The elution solvents hexane and ethyl acetate (1:1, v/v), hexane and dichloromethane (3:7, v/v), and ethyl acetate and hexane (2:8, v/v) were studied. The results showed that the combination of the tandem graphitized carbon and florisil cartridge with the elution solvent of 6mL of ethyl acetate and hexane (2:8, v/v), which was capable of eliminating matrix interference and providing colorless eluates, was the most efficient clean-up procedure for propolis extracts when testing for OCPs. The analytical technique employed was gas chromatography with electron capture detection (GC–ECD). The correlation coefficients from linear regression for the analyzed concentrations (5∼100 μg/kg) were >0.9961. The limits of detection (LODs) varied between 0.8 μg/kg for 4,4′-DDE and 11.4 μg/kg for endosulfan II, and the limits of quantitation (LOQs) ranged from 2.6 to 38.1 μg/kg. The average recoveries varied between 62.6 and 109.6%. Relative standard deviations (RSD%) ranged from 0.8 to 9.4%. Sample analysis indicated that 4,4′-DDE was detected more often in propolis than other pesticides, such as β-HCH, δ-HCH and heptachlor. Figure GC-ECD chromatogram of a standard solution with 0.1 mg/L of OCPs     

Determination of trapidil in human serum and urine by derivative UV spectrophotometry after selective solid-phase extraction

A novel analytical technique able to determine the anti-ischemic drug trapidil in human serum and urine is proposed. In order to achieve satisfactory sensitivity and selectivity, an extraction procedure was required to isolate the drug from complex matrixes such as serum and urine. A solid-phase extraction procedure was investigated to both increase the analyte concentration and eliminate the interfering molecules present in large amounts in both matrixes. Optimization of the extraction step was realized by selecting a new polymeric sorbent based on a surface-modified styrene–divinylbenzene polymer which provided fast and efficient drug extraction. Drug quantification was performed by using the third-order derivative spectra of the SPE eluates. Absorbance specific signals at ³D_335,316 and ³D₃₁₆ nm for urine and serum, respectively, were demonstrated to be directly proportional to drug concentration and barely affected by residual matrix interferences. Under the optimized experimental conditions the calibration plots were linear over the concentration range 0.2–50 μg mL⁻¹. The method was validated by analysis of a series of spiked samples. Accuracy (recovery of 95 and 94% for serum and urine, respectively) and precision (RSD below 4%) were good. Figure Assay of Trapidil in biological fluids by SPE and derivative spectrophotometry     

Detection of residual bacitracin A, colistin A, and colistin B in milk and animal tissues by liquid chromatography tandem mass spectrometry

Liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-MS/MS) was applied to the determination of residual bacitracin A, colistin A, and colistin B in milk and animal tissue samples. Prior to instrumental analysis, samples were subjected to acid extraction followed by solid-phase cleanup using Strata-X cartridges. Mass spectral acquisitions were performed under selective multiple reaction monitoring (MRM) mode at m/z 199 and 670 from triply charged precursors of bacitracin A (m/z 475); m/z 385 and 379 from triply charged precursors of colistin A (m/z 391); and m/z 380 and 374 from triply charged precursors of colistin B (m/z 386). Method precision was evaluated from spike recovery of samples fortified at concentrations corresponding to 2/5 of the maximum residue limits (MRLs) for each of the analytes under study. Intra-day and inter-day variations were found to range from 90.9 to 104% with relative standard deviation (RSD) <6.5%, and from 90.1 to 106% with RSD <9.1%, respectively. Limits of quantification (LOQs) were defined as the spiking concentrations at 2/5 MRL, and limits of detection (LODs) were 10–47 μg kg⁻¹ for bacitracin A, 1–16 μg kg⁻¹ for colistin A, and 6–14 μg kg⁻¹ for colistin B in milk and animal tissues. The presented method has good precision and high sensitivity and was applied as a fast screening protocol and a quantitative tool for monitoring of the concerned polypeptides in foods as part of a surveillance program.