Chromogenic platform based on recombinant Drosophila melanogaster acetylcholinesterase for visible unidirectional assay of organophosphate and carbamate insecticide residues

In this study we propose a chromogenic platform for rapid analysis of organophosphate (OP) and carbamate (CM) insecticide residues, based on recombinant Drosophila melanogaster acetylcholinesterase (R-DmAChE) as enzyme and indoxyl acetate as substrate. The visible chromogenic strip had the advantages identical to those of commonly used lateral flow assays (LFAs) with utmost simplicity in sample loading and result observation. After optimization, depending on the color intensity (CI) values, the well-established assay has the capabilities of both qualitative measurement via naked eyes and quantitative analysis by colorimetric reader with the desirable IC₅₀ values against the tested six insecticides (0.06 μg mL⁻¹ of carbofuran, 0.28 μg mL⁻¹ of methomyl, 0.03 μg mL⁻¹ of dichlorvos, 31.6 μg mL⁻¹ of methamidophos, 2.0 μg mL⁻¹ of monocrotophos, 6.3 μg mL⁻¹ of omethoate). Acceptable matrix effects and satisfactory detection performance were confirmed by in-parallel LC–MS/MS analysis in different vegetable varieties at various spiked levels of 10⁻³ to 10¹ μg g⁻¹. Overall, the testified suitability and applicability of this novel platform meet the requirements for practical use in food safety management and environmental monitoring, especially in the developing world.     

Determination of anethole trithione in human plasma using high performance liquid chromatography coupled with tandem mass spectrometric detection

A rapid, sensitive and reliable high performance liquid chromatographic method coupled with tandem mass spectrometry via electrospray ionization (ESI) source (HPLC-MS/MS) has been developed and validated for the determination of anethole trithione (ATT) in human plasma. Diazepam was employed as the internal standard (IS). Sample extracts following liquid-liquid extraction were injected into the HPLC-MS/MS system. The analyte and IS were eluted isocratically on a C₁₈ column, with a mobile phase consisting of methanol and aqueous ammonium acetate solution (5 mM) (80:20, v/v) .The ions were detected by a triple quadrupole mass spectrometric detector in the positive mode. Quantification was performed using selected reaction monitoring (SRM) of the transitions m/z 240.88 → 197.91 and m/z 285.01 → 193.02 for ATT and for the IS, respectively. The analysis time for each run was 5.0 min. The calibration curve fitted well over the concentration range of 0.02-5 ng mL⁻¹, with the regression equation y = 1.1014x + 0.0003631, r = 0.9992. The intra-batch and inter-batch R.S.D.% were less than 15% at all concentration levels within the calibration range. The recoveries were more than 80%. The present method provides a modern, rapid and robust procedure for the pharmacokinetic study of ATT. Some important pharmacokinetic parameters of ATT in healthy Chinese volunteers are also given for the first time.     

Simultaneous determination of salbutamol sulphate, bromhexine hydrochloride and etofylline in pharmaceutical formulations with the use of four rapid derivative spectrophotometric methods

Four simple, rapid, accurate, precise, reliable and economical spectrophotometric methods have been proposed for simultaneous determination of salbutamol sulphate (SS), bromhexine hydrochloride (BH) and etofylline (ET) in pure and commercial formulations without any prior separation or purification. They were first derivative zero crossing spectrophotometry (method 1), simultaneous equation method (method 2), derivative ratio spectra zero crossing method (method 3) and double divisor ratio spectra derivative method (method 4). The ranges for SS, BH and ET were found to be 1-35 μg mL⁻¹, 4-40 μg mL⁻¹ and 5-80 μg mL⁻¹. For methods 1 and 2, the values of limit of detection (LOD) were 0.2314 μg mL⁻¹, 0.4865 μg mL⁻¹ and 0.2766 μg mL⁻¹ and the values of limit of quantitation (LOQ) were 0.7712 μg mL⁻¹, 1.6217 μg mL⁻¹ and 0.9221 μg mL⁻¹ for SS, BH and ET, respectively. For method 3, LOD values were 0.3297 μg mL⁻¹, 0.2784 μg mL⁻¹ and 0.7906 μg mL⁻¹ and LOQ values were 0.9325 μg mL⁻¹, 0.9282 μg mL⁻¹ and 2.6352 μg mL⁻¹ for SS, BH and ET, respectively. For method 4, LOD values were 0.3161 μg mL⁻¹, 0.2495 μg mL⁻¹ and 0.2064 μg mL⁻¹ and LOQ values were 0.9869 μg mL⁻¹, 0.8317 μg mL⁻¹ and 0.6879 μg mL⁻¹ for SS, BH and ET. The precision values were less then 2% R.S.D. for all four methods. The common excipients and additives did not interfere in their determinations. The results obtained by the proposed methods have been statistically compared by means of Student t-test and by the variance ratio F-test.     

Simultaneous screening analysis of multiple beta-blockers in urine by gas chromatography-mass spectrometry in selected ion monitoring mode

A rapid screening procedure is described for the simultaneous determination of 13 β-blockers in urine at the range of 0.010-1.0 μg mL⁻¹. The procedure involves N-ethoxycarbonyl (EOC) derivatization of β-blockers in urine sample, followed by extraction and further conversion to trimethylsilyl (TMS) derivatives for the analysis by gas chromatography-mass spectrometry (GC-MS) in selected ion monitoring (SIM) mode (GC-SIM-MS). The characteristic fragment ions at m/z 260 and m/z 144, and [M − 15]⁺ ions permitted sensitive and selective detection of most of the β-blockers in the presence of co-extracted urinary amino alcohols at much higher levels. The whole procedure of EOC/TMS derivatization with subsequent GC-SIM-MS analysis was linear (r ≥ 0.9988). The LODs were varied from 0.03 to 2.7 ng mL⁻¹. The ranges of precision (%relative standard deviation) and accuracy (%relative error) of the overall procedure at two different added amounts (0.02 and 0.5 μg mL⁻¹) in urine matrix varied from 1.3 to 9.4 and from −9.6 to 9.7, respectively. The recoveries were measured to be ranged from 90.4 to 109.7%.     

Determination of itopride in human plasma by liquid chromatography coupled to tandem mass spectrometric detection: application to a bioequivalence study

A simple method using a one-step liquid-liquid extraction (LLE) with butyl acetate followed by high-performance liquid chromatography (HPLC) with positive ion electrospray ionization tandem mass spectrometric (ESI-MS/MS) detection was developed for the determination of itopride in human plasma, using sulpiride as an internal standard (IS). Acquisition was performed in multiple reaction monitoring (MRM) mode, by monitoring the transitions: m/z 359.5 > 166.1 for itopride and m/z 342.3 > 111.6 for IS, respectively. Analytes were chromatographed on an YMC C₁₈ reverse-phase chromatographic column by isocratic elution with 1 mM ammonium acetate buffer-methanol (20: 80, v/v; pH 4.0 adjusted with acetic acid). Results were linear (r² = 0.9999) over the studied range (0.5-1000 ng mL⁻¹) with a total analysis time per run of 2 min for LC-MS/MS. The developed method was validated and successfully applied to bioequivalence studies of itopride hydrochloride in healthy male volunteers.     

Development of a validated liquid chromatographic method for the quality control of Prunellae Spica: Determination of triterpenic acids

A simple and rapid reversed-phase HPLC-UV method was developed for the determination of triterpenic acids in the crude extract of Prunellae Spica. Five triterpenic acids were extracted and isolated from P. Spica as marker compounds for use in the quality control of herbal medicines. Various solvent extraction techniques were evaluated, and the greatest efficiency was observed with sonication in 100% ethanol. Elemental compositions of the five marker compounds were determined by high-resolution mass spectroscopy. The dynamic range of the HPLC-UV method depended on the specific analyte, and acceptable quantitation was obtained between 10 and 250 μg mL⁻¹ for oleanolic acid, between 10 and 300 μg mL⁻¹ for ursolic acid, between 3 and 75 μg mL⁻¹ for 2α,3α,24-trihydroxyolean-12en-28oic acid, between 5 and 100 μg mL⁻¹ for euscaphic acid, and between 5 and 100 μg mL⁻¹ for 2α,3α-dihydroxyurs-12en-28oic acid. The method was deemed satisfactory by inter- and intra-day validation and exhibited both high accuracy and precision (relative standard deviation <9.4%). Overall limits of quantitation and detection were approximately 0.5-2.5 μg mL⁻¹ at a signal-to-noise ratio (S/N) of 3 and were about 3.0-10.0 μg mL⁻¹ at a S/N of 10. In addition, principal component analysis (PCA) was performed on the analytical data of 15 different P. Spica samples in order to classify samples collected from different regions.     

High extraction efficiency for polar aromatic compounds in natural water samples using multiwalled carbon nanotubes/Nafion solid-phase microextraction coating

A novel solid-phase microextraction (SPME) fiber coated with multiwalled carbon nanotubes (MWCNTs)/Nafion was developed and applied for the extraction of polar aromatic compounds (PACs) in natural water samples. The characteristics and the application of this fiber were investigated. Electron microscope photographs indicated that the MWCNTs/Nafion coating with average thickness of 12.5 μm was homogeneous and porous. The MWCNTs/Nafion coated fiber exhibited higher extraction efficiency towards polar aromatic compounds compared to an 85 μm commercial PA fiber. SPME experimental conditions, such as fiber coating, extraction time, stirring rate, desorption temperature and desorption time, were optimized in order to improve the extraction efficiency. The calibration curves were linear from 0.01 to 10 μg mL⁻¹ for five PACs studied except p-nitroaniline (from 0.005 to 10 μg mL⁻¹) and m-cresol (from 0.001 to 10 μg mL⁻¹), and detection limits were within the range of 0.03–0.57 ng mL⁻¹. Single fiber and fiber-to-fiber reproducibility were less than 7.5 (n = 7) and 10.0% (n = 5), respectively. The recovery of the PACs spiked in natural water samples at 1 μg mL⁻¹ ranged from 83.3 to 106.0%.     

Analysis of microcystins by capillary zone electrophoresis coupling with electrospray ionization mass spectrometry

In this paper, a rapid and effective method based on capillary zone electrophoresis (CZE) coupled with electrospray ionization mass spectrometry (ESI-MS) was established for the trace analysis of microcystin (MC) isomers in crude algae sample. The experimental conditions including the composition, acidity and concentration of buffer, separation voltage, injection time, and MS detection parameters were investigated in detail. A capillary separation system was as follows: a uncoated fused-silica capillary tube (50 μm i.d. × 90 cm), 40 mmol L⁻¹ ammonium acetate solution (pH 9.86) as running buffer, 25 kV as separation voltage, 20 kV × 3 s water first and 20 kV × 20 s for sample injection. Mass analysis was performed in ESI source, with sheath gas temperature 150 °C, sheath gas pressure 10 psi, and sheath gas flow 6 L min⁻¹. And sheath liquid was 7.5 mmol L⁻¹ acetic acid in 50% isopropanol-water (3 μL min⁻¹). Protonation and ammonium adduct molecular ions m/z 506.9 (MC-LR) and 532.0 (MC-YR) were used for the quantification of MCs. Under these conditions, two MCs were baseline separated within 9 min, the calibration curves were obtained in the range of 0.11-10.0 μg mL⁻¹ and 0.16-10.5 μg mL⁻¹ for MC-LR and MC-YR, respectively. Meanwhile, limits of detection were 0.05 and 0.08 μg mL⁻¹ for MC-LR and MC-YR, respectively. The recoveries for the two MCs were in the range of 95.8-108%. The developed approach had been successfully applied to the analysis of MCs in crude algae samples.     

A reversed-phase high performance liquid chromatography coupled with resonance Rayleigh scattering detection for the determination of four tetracycline antibiotics

A new reversed-phase high performance liquid chromatography with resonance Rayleigh scattering detection (HPLC-RRS) was developed for simultaneous separation and determination of four tetracycline antibiotics (TCs). A good chromatographic separation among the compounds was achieved using a Synergi Fusion-RP column (150 mm × 4.6 mm; 4 μm) and a mobile phase consisting of methanol-acetonitrile-oxalic acid (5 mM) at the flow rate of 0.8 mL min⁻¹. Column temperature was 30 °C. The RRS signal was detected at λ_ex = λ_em = 370 nm. The recoveries of sample added standard ranged from 95.3% to 103.5%, and the relative standard deviation was below 2.79%. A detection limit of 2.12-5.12 μg mL⁻¹ was reached and a linear range was found between peak height and concentration in the range of 10.36-518.0 μg mL⁻¹ for oxytetracycline (OTC), 12.11-605.5 μg mL⁻¹ for tetracycline (TC), 11.79-589.5 μg mL⁻¹ for chlortetracycline (CTC) and 10.32-516.0 μg mL⁻¹ for doxycycline (DC). The linear regression coefficients were all above 0.999. The method has been applied successfully to the determination of OTC, TC, CTC, DC in pharmaceutical formulations and in honey. The method was simple, rapid and showed a better linear relation and high repeatability.     

Nanogold-actuated biomimetic peroxidase for sensitized electrochemical immunoassay of carcinoembryonic antigen in human serum

We present a method for the simultaneous determination of guanidinosuccinic acid (GSA) and guanidinoacetic acid (GAA) from urine by protein precipitation and liquid chromatography/tandem mass spectrometry. The chromatographic separation was performed using a cation exchange column with an elution gradient of 0.1 mM and 20 mM ammonium acetate buffers. GSA was detected with the mass spectrometer in negative ion mode monitoring at m/z 174.1, and GAA, creatinine, arginine, and homoarginine were in positive ion mode monitoring at m/z 118.1, 114.1, 175.1, and 189.1, respectively. As an internal standard, l-arginine-¹³C₆ hydrochloride and creatinine-d₃ (methyl-d₃) were used. The calibration ranges were 0.50-25.0 μg mL⁻¹, and good linearities were obtained for all compounds (r > 0.999). The intra- and inter-assay accuracies (expressed as recoveries) and precisions at three concentration levels (1.00, 5.00 and 25.0 μg mL⁻¹) were better than 83.8% and 7.41%, respectively. The analytical performance of the method was evaluated by determination of the compounds in urine from male C57BL/J Iar db/db diabetes mellitus (DM) mice. The values of GSA and GAA corrected by the ratios of the individual compounds to creatinine were significantly increased in DM mice compared with control mice. These results indicated that the newly developed method was useful for determining urinary guanidino compounds and metabolites of arginine.     

Profiling of 19-norsteroid sulfoconjugates in human urine by liquid chromatography mass spectrometry

19-Nortestosterone (nandrolone) major metabolites in human urine are excreted as sulfoconjugated and glucuroconjugated forms. A sensitive and selective liquid chromatography/tandem mass spectrometry (LC/MS/MS) method in negative ESI mode was developed for direct quantification of 19-norandrosterone sulfate (19-NAS) and 19-noretiocholanolone sulfate (19-NES). For both sulfoconjugates, the [M−H]⁻ ion at m/z 355 and the fragment ion at m/z 97 were used as the precursor and product ions, respectively. The purification method involved a complete and rapid separation of sulfates and glucuronides in two extracts after loading the sample on a weak anion exchange solid phase extraction support (SPE Oasis^® WAX). Then, sulfates were separated by LC (Uptisphere^® ODB, 150 mm × 3.0 mm, 5 μm) and analyzed on a linear trap and a triple quadrupole mass spectrometer. The lower limit of detection (LLOD) and lowest limit of quantification (LLOQ) were of 100 pg mL⁻¹ and 1 ng mL⁻¹, respectively. Assay validation demonstrated good performances in terms of trueness (92.0-104.9%), repeatability (0.6-7.2%) and intermediate precision (1.3-10.8%) over the range of 1-2500 ng mL⁻¹. Finally, 19-NAS and 19-NES in urine samples collected after intake of 19-norandrostenedione (nandrolone precursor) were quantified. This assay may be easily implemented to separate glucuronide and sulfate steroids from urine specimens prior to quantification by LC/MS/MS.     

A sensitive sensor for anthraquinone anticancer drugs and hsDNA based on CdTe/CdS quantum dots fluorescence reversible control

Based on CdTe/CdS quantum dots (CdTe/CdS QDs) fluorescence (FL) reversible control, a new and sensitive FL sensor for determination of anthraquinone (AQ) anticancer drugs (adriamycin and daunorubicin) and herring sperm DNA (hsDNA) was developed. Under the experimental conditions, FL of CdTe/CdS QDs can be effectively quenched by AQ anticancer drugs due to the binding of AQ anticancer drugs on the surface of CdTe/CdS QDs and photoinduced electron transfer (PET) process from CdTe/CdS QDs to AQ anticancer drugs. Addition of hsDNA afterwards brought the restoration of CdTe/CdS QDs FL intensity, as AQ anticancer drugs peeled off from the surface of CdTe/CdS QDs and embedded into hsDNA double helix structure. The liner ranges and the detection limits of FL quenching methods for two AQ anticancer drugs were 0.33-9 μg mL⁻¹ and 0.09 μg mL⁻¹ for ADM and 0.15-9 μg mL⁻¹ and 0.04 μg mL⁻¹ for DNR, respectively. The restored FL intensity was proportional to concentration of hsDNA in the range of 1.38-28 μg mL⁻¹and the detection limit for hsDNA was 0.41 μg mL⁻¹. It was applied to the determination of AQ anticancer drugs in human serum and urine samples with satisfactory results. The reaction mechanism of CdTe/CdS QDs FL reversible control was studied.     

Sensitive determination of hydrazine in water by gas chromatography–mass spectrometry after derivatization with ortho-phthalaldehyde

A gas chromatography–mass spectrometric (GC–MS) method has been established for the determination of hydrazine in drinking water and surface water. This method is based on the derivatization of hydrazine with ortho-phthalaldehyde (OPA) in water. The following optimum reaction conditions were established: reagent dosage, 40 mg mL⁻¹ of OPA; pH 2; reaction for 20 min at 70 °C. The organic derivative was extracted with methylene chloride and then measured by GC–MS. Under the established condition, the detection and the quantification limits were 0.002 μg L⁻¹ and 0.007 μg L⁻¹ by using 5.0-mL of surface water or drinking water, respectively. The calibration curve showed good linearity with r² = 0.9991 (for working range of 0.05–100 μg L⁻¹) and the accuracy was in a range of 95–106%, and the precision of the assay was less than 13% in water. Hydrazine was detected in a concentration range of 0.05–0.14 μg L⁻¹ in 2 samples of 10 raw drinking water samples and in a concentration range of 0.09–0.55 μg L⁻¹ in 4 samples of 10 treated drinking water samples.     

A rapid, acetonitrile-free, HPLC method for determination of melamine in infant formula

A simple, precise, accurate and validated, acetonitrile-free, reverse phase high performance liquid chromatography (HPLC) method is developed for the determination of melamine in dry and liquid infant formula. The separation is performed on a Kromasil C18 column (150 mm × 3.2 mm I.D., 5 μm particle size) at room temperature. The mobile phase (0.1% TFA/methanol 90:10) is pumped at a flow rate of 0.3 mL min⁻¹ with detection at 240 nm. Melamine elutes at 3.7 min. A linear response (r > 0.999) is observed for samples ranging from 1.0 to 80 μg mL⁻¹. The method provides recoveries of 97.2-101.2% in the concentration range of 5-40 μg mL⁻¹, intra- and inter-day variation in <1.0% R.S.D. The limit of detection (LOD) and limit of quantification (LOQ) values are 0.1 μg mL⁻¹ and 0.2 μg mL⁻¹, respectively.     

Sensitive quantitative determination of oxymatrine and matrine in rat plasma by capillary electrophoresis with stacking induced by moving reaction boundary

A quantitative method of capillary electrophoresis with sample stacking induced by moving reaction boundary (MRB) was developed for sensitive determination of oxymatrine (OMT) and matrine (MT) in rat plasma. The experimental conditions were optimized firstly. Below are the optimized experimental conditions: 20 mM sodium formate solution (HCOONa, adjusted to pH 10.70 by ammonia) as sample solution, 3 min 14 mbar sample injection, 40 mM formic buffer (HCOOH-HCOONa, pH 2.60) as stacking buffer, 7 min 14 mbar injection of stacking buffer, 100 mM HCOOH-HCOONa (pH 4.80) as separation buffer, 73 cm capillary (effective length 64 cm), 21 kV voltage, 210 nm wavelength. Under the optimized conditions, higher than 60-fold sensitivity improvement of the stacking was simply achieved as compared with capillary zone electrophoresis, and the detectable limits obtained for OMT and MT were 0.26 and 0.19 μg mL⁻¹, respectively. Then, numerous demonstrations were carefully performed for the methodological validations of OMT and MT in rate plasma, including high specificity of method, good linearity (r = 0.9993 for OMT, r = 0.9991 for MT), fair wide linear concentration range (1.30-65.00 μg mL⁻¹ for OMT, 0.84-42.00 μg mL⁻¹ for MT), low limit of detection (1.03 μg mL⁻¹ for OMT, 0.38 μg mL⁻¹ for MT), less than 5% intra- and inter-day variance value, and higher than 96% recovery of OMT and MT in plasma. The developed method could be used for the trace analyses of OMT and MT in plasma and was finally used for the investigation on pharmacokinetic study of OMT in rat plasma.     

Fluorescence enhancement of CdTe/CdS quantum dots by coupling of glyphosate and its application for sensitive detection of copper ion

A novel fluorescent probe for Cu²⁺ determination based on the fluorescence quenching of glyphosate (Glyp)-functionalized quantum dots (QDs) was firstly reported. Glyp had been used to modify the surface of QDs to form Glyp-functionalized QDs following the capping of thioglycolic acid on the core–shell CdTe/CdS QDs. Under the optimal conditions, the response was linearly proportional to the concentration of Cu²⁺ between 2.4 × 10⁻² μg mL⁻¹ and 28 μg mL⁻¹, with a detection limit of 1.3 × 10⁻³ μg mL⁻¹ (3δ). The Glyp-functionalized QDs fluorescent probe offers good sensitivity and selectivity for detecting Cu²⁺. The fluorescent probe was successfully used for the determination of Cu²⁺ in environmental samples. The mechanism of reaction was also discussed.     

A novel application of Onyx™ monolithic column for simultaneous determination of salicylic acid and triamcinolone acetonide by sequential injection chromatography

A novel and fast simultaneous determination of triamcinolone acetonide (TCA) and salicylic acid (SA) in topical pharmaceutical formulations by sequential injection chromatography (SIC) as an alternative to classical high performance liquid chromatography (HPLC) has been developed. A recently introduced Onyx™ monolithic C18 (50 mm × 4.6 mm, Phenomenex^®) with 5 mm monolithic precolumn were used for the first time for creating sequential injection chromatography system based on a FIAlab^® 3000 with a six-port selection valve and 5.0 mL syringe pump in study. The mobile phase used was acetonitrile/water (35:65, v/v), pH 3.3 adjusted with acetic acid at flow rate 0.9 mL min⁻¹. UV detection provided by fibre-optic DAD detector was set up at 240 nm. Propylparaben was chosen as suitable internal standard (IS). There is only simple pre-adjustment of the sample of topical solution (dilution with mobile phase) so the analysis is not uselessly elongated. Parameters of the method showed good linearity in wide range, correlation coefficient >0.999; system precision (relative standard deviation, R.S.D.) in the range 0.45-1.95% at three different concentration levels, detection limits (3σ) 1.00 μg mL⁻¹ (salicylic acid), 0.66 μg mL⁻¹ (triamcinolone acetonide) and 0.33 μg mL⁻¹ (propylparaben) and recovery from the pharmaceutical preparations in the range 97.50-98.94%. The chromatographic resolution between peaks of compounds was more than 4.5 and analysis time was 5.1 min under the optimal conditions. The advantages of sequential injection chromatography against classical HPLC are discussed and showing that SIC can be a method of option in many cases.     

Separation and determination of benzene, toluene, ethylbenzene and o-xylene compounds in water using directly suspended droplet microextraction coupled with gas chromatography-flame ionization detector

The directly suspended droplet microextraction (DSDME) technique coupled with the capillary gas chromatography-flame ionization detector (GC-FID) was used to determine BTEX compounds in aqueous samples. The effective parameters such as organic solvent, extraction time, microdroplet volume, salt effect and stirring speed were optimized. The performance of the proposed technique was evaluated for the determination of BTEX compounds in natural water samples. Under the optimal conditions the enrichment factors ranged from 142.68 to 312.13, linear range; 0.01-20 μg mL⁻¹, limits of detection; 0.8-7 ng mL⁻¹ for most analytes. Relative standard deviations for 0.2 μg mL⁻¹ of BTEX in water were in the range 1.81-2.47% (n = 5). The relative recoveries of BTEX from surface water at spiking level of 0.2 μg mL⁻¹ were in the range of 89.87-98.62%.     

N-alkylpyridinium quaternization combined with liquid chromatography–electrospray ionization-tandem mass spectrometry: A highly sensitive method to quantify fatty alcohols in thyroid tissues

A highly sensitive method was developed for the identification and quantification of fatty alcohols in biological tissues. In the presence of pyridine-d₀ and triflic anhydride (Tf₂O), fatty alcohols were converted into permanently charged N-alkylpyridinium ions. Stable isotope-labeled derivatives were generated by pyridine-d₅ and added as internal standard (IS). The mixture was analyzed by liquid chromatography coupled to positive electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS). This method was optimized and validated in terms of reaction time, derivatization efficiency, stability, desalting, and ion suppression effect. Besides, fatty alcohols exhibited good linear relationship (r² > 0.993) over the concentration range of 10 ng mL⁻¹–1 μg mL⁻¹. The limits of detection (LODs) were lowered from previously reported 0.1 ng mL⁻¹ to 0.25 pg mL⁻¹. Precision (RSD% < 15.6%), accuracy (93.0–107.2%), matrix effect, and recovery (in thyroid tissues) were validated as well. Finally, this method was applied for the analysis of ten even carbon-numbered fatty alcohols (C₈–C₂₄) in human thyroid carcinoma and para-carcinoma tissues, revealing a significant decrease of fatty alcohols (free and esterified) in thyroid carcinoma tissues (p < 0.05).     

Determination of ent-kaurene in subcutaneous fat of Iberian pigs by gas chromatography multi-stage mass spectrometry with the aim to differentiate between intensive and extensive fattening systems

This work presents a gas chromatography multi-stage mass spectrometry (GC-MS³) method for the determination of ent-kaurene in subcutaneous fat of Iberian pig, present in adipose tissue of animals due to pasture ingestion (extensive fattening system). The method comprises a saponification and a liquid-liquid extraction of the unsaponifiable fraction, followed by an isolation of the hydrocarbon fraction by high performance liquid chromatography (HPLC) and analysis by GC-MS³ (ion trap) with electronic ionization. The GC-MS³ analysis allows the isolation and characterization of specific fragments from the original (MS¹) molecular structure, and particularly, those fragments originated from the precursor ion (m/z = 229) characteristic of ent-kaurene. The MS/MS product fragment m/z = 213 is used as a further precursor fragment giving rise to a MS³ spectrum specific for ent-kaurene. The limit of detection of the MS³ technique is lower than 0.2 μg kg⁻¹ and a linear regression has been found between 0.2 and 112 μg kg⁻¹. This method is applicable for the determination of the fattening system of the Iberian pig.