Salting-out Assisted Liquid-liquid Extraction Research Articles

Fast, simple and efficient salting-out assisted liquid-liquid extraction of naringenin from fruit juice samples prior to their enantioselective determination by liquid chromatography

In this study, an easy, simple and efficient method for the determination of naringenin enantiomers in fruit juices after salting-out-assisted liquid-liquid extraction (SALLE) and high-performance liquid chromatography (HPLC) with diode-array detection (DAD) was developed. The sample treatment is based on the use of water-miscible acetonitrile as the extractant and acetonitrile phase separation under high-salt conditions. After extraction, juice samples were incubated with hydrochloric acid in order to achieve hydrolysis of naringin to naringenin. The hydrolysis parameters were optimized by using a half-fraction factorial central composite design (CCD).After sample preparation, chromatographic separation was obtained on a Chiralcel® OJ-RH column using the mobile phase consisting of 10mM aqueous ammonium acetate:methanol:acetonitrile (50:30:20; v/v/v) with detection at 288nm. The average recovery of the analyzed compounds ranged from 85.6 to 97.1%. The proposed method was satisfactorily used for the determination of naringenin enantiomers in various fruit juices samples.

Salting-out assisted extraction method coupled with hydrophilic interaction liquid chromatography for determination of selected β-blockers and their metabolites in human urine.

In this study, a new analytical method was developed and validated for the simultaneous analysis of β-blockers (metoprolol, propranolol, carvedilol) and their metabolites (5'-hydroxycarvedilol, O-desmethylcarvedilol, α-hydroxymetoprolol, O-desmethylmetoprolol, 5-hydroxypropranolol) in human urine. A salting-out assisted liquid-liquid extraction (SALLE) procedure was used for sample preparation. Several parameters affecting the extraction efficiency and method sensitivity including the type and volume of the extraction solvent, the type and quantity of the inorganic salt, extraction time and sample pH were investigated. Hydrophilic interaction liquid chromatography-ultraviolet detection (HILIC-UV) was used for the determination of all analytes. During method development, the effects of mobile phase components (type, pH, concentration of salt, organic modifier type and content, flow rate, column temperature) on the retention and separation of β-blockers and metabolites on the five different HILIC columns were examined. The method was linear for concentrations ranging from 0.1 to 8.0μg/mL, with determination coefficients higher than 0.993 for all analytes. The limits of quantification were in the range from 0.1 to 0.2μg/mL. Intra- and inter-day precision ranged from 0.1 to 8.9%, and accuracy was within±13% interval for all analytes. Under the optimized conditions, extraction efficiency was greater than 83.4% for determined compounds. The validated method was then applied to the measurement of β-blockers and their metabolites in human urine samples.

Liquid chromatography-tandem mass spectrometric assay for the tyrosine kinase inhibitor afatinib in mouse plasma using salting-out liquid-liquid extraction.

A quantitative bioanalytical liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay for afatinib, an irreversible inhibitor of the ErbB (erythroblastic leukemia viral oncogene homolog) tyrosine kinase family, was developed and validated. Plasma samples were pre-treated using salting-out assisted liquid-liquid extraction (SALLE) with acetonitrile, magnesium chloride and a stable isotopically labeled internal standard. After dilution, the extract was directly injected into the reversed-phase liquid chromatographic system. The eluate was transferred into the electrospray interface with positive ionization and compounds were detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer. The assay was completely validated for plasma in a 0.5-500ng/ml calibration range with r(2)=0.995±0.002 (n=6) using linear regression with the inverse square of the concentration as the weighting factor for the calibration. Within-run precisions (n=18) were 2.7-11.7% and between-run (3 runs; n=18) precisions 3.0-14.5%. Accuracies were between 96-109% for the whole calibration range. The drug was sufficiently stable under all relevant analytical conditions. Finally, the assay was successfully applied to determine plasma drug levels and study pharmacokinetics after oral administration of afatinib to female FVB mice.

Emerging flow injection mass spectrometry methods for high-throughput quantitative analysis.

Where does flow injection analysis mass spectrometry (FIA-MS) stand relative to ambient mass spectrometry (MS) and chromatography-MS? Improvements in FIA-MS methods have resulted in fast-expanding uses of this technique. Key advantages of FIA-MS over chromatography-MS are fast analysis (typical run time <60 s) and method simplicity, and FIA-MS offers high-throughput without compromising sensitivity, precision and accuracy as much as ambient MS techniques. Consequently, FIA-MS is increasingly becoming recognized as a suitable technique for applications where quantitative screening of chemicals needs to be performed rapidly and reliably. The FIA-MS methods discussed herein have demonstrated quantitation of diverse analytes, including pharmaceuticals, pesticides, environmental contaminants, and endogenous compounds, at levels ranging from parts-per-billion (ppb) to parts-per-million (ppm) in very complex matrices (such as blood, urine, and a variety of foods of plant and animal origin), allowing successful applications of the technique in clinical diagnostics, metabolomics, environmental sciences, toxicology, and detection of adulterated/counterfeited goods. The recent boom in applications of FIA-MS for high-throughput quantitative analysis has been driven in part by (1) the continuous improvements in sensitivity and selectivity of MS instrumentation, (2) the introduction of novel sample preparation procedures compatible with standalone mass spectrometric analysis such as salting out assisted liquid-liquid extraction (SALLE) with volatile solutes and NH4(+) QuEChERS, and (3) the need to improve efficiency of laboratories to satisfy increasing analytical demand while lowering operational cost. The advantages and drawbacks of quantitative analysis by FIA-MS are discussed in comparison to chromatography-MS and ambient MS (e.g., DESI, LAESI, DART). Generally, FIA-MS sits 'in the middle' between ambient MS and chromatography-MS, offering a balance between analytical capability and sample analysis throughput suitable for broad applications in life sciences, agricultural chemistry, consumer safety, and beyond.

Salting-out-assisted liquid-liquid extraction as a suitable approach for determination of methoxetamine in large sets of tissue samples.

A new designer drug, a dissociative anesthetic, and a putative N-methyl-D-aspartate receptor antagonist, methoxetamine (MXE) noted by the EU Early Warning System has been already identified as a cause of several fatalities worldwide. The primary objective of this work was to develop a suitable sample preparation method allowing for isolation of MXE and its main metabolites in high yields from rat brain, liver, and lungs. For the purpose of the project, MXE and five metabolites were synthesized in-house, specifically O-desmethyl-normethoxetamine, O-desmethylmethoxetamine, dihydro-O-desmethylmethoxetamine, normethoxetamine, and dihydromethoxetamine. A sample preparation procedure consisted in the homogenization of the tissue applying salting-out-assisted liquid-liquid extraction (SALLE). A subsequent liquid chromatography-mass spectrometry (LC-MS) analysis was based on reversed-phased chromatography hyphenated with a triple quad MS system in a positive electrospray mode. Multiple reaction monitoring (MRM) was used for qualification and quantification of the analytes. The quantification was based on the application of an isotopically labeled internal standard, normethoxetamine-d3. The matrix-matched calibrations were prepared for each type of matrix with regression coefficients 0.9943-1.0000. The calibration curves were linear in the concentration range of 2.5-250 ng g(-1). Limits of quantification (LOQs) were estimated as 2.5 and 5 ng g(-1), respectively. Recovery (80-117%) and matrix effect (94-110%) at 100 ng g(-1) and intra- and inter-day accuracy and precision at low (2.5 ng g(-1)), middle (25 ng g(-1)), and upper (250 ng g(-1)) concentration levels for all the analytes in all three types of tissues were also determined. The developed analytical method was applied to a set of real samples gathered in toxicological trials on rats and MXE, and its metabolites were determined successfully.

Ultrasound-assisted chiral derivatization of etodolac with (1R)-(−)-menthyl chloroformate for the determination of etodolac enantiomers

This study presents the first report of an ultrasound-assisted derivatization reaction between a carboxylic acid of etodolac (ETO) and a chiral derivatization reagent, (1R)-(−)-menthyl chloroformate (R-MCF). FiftyμL of deproteinated mouse serum containing ETO enantiomers was derivatized with 125μL of 200mM R-MCF and 17μL of pyridine (a catalyst), with the reaction facilitated by ultrasonic radiation for 13min, which were the optimal conditions as determined by response surface methodology. After quenching the reaction by adding an aqueous L-proline solution, the mixture was subjected to salting-out assisted liquid–liquid extraction (SA-LLE), which provided phase separation for sample concentration as well as cleanup. The ETO diastereomers were separated on a Phenomenex Gemini C18 column (150×4.6mm, 5μm) under a simple gradient elution of a mobile phase containing a mixture of methanol: acetonitrile (10:1, V/V) and 10mM acetic acid at a flow rate of 1.0mLmin−1, followed by fluorescence detection with excitation and fluorescence emission wavelengths of 235nm and 345nm, respectively. The developed method was validated for specificity, sensitivity, linearity, accuracy, precision, stability, and matrix effect. A good linearity in the range of 0.5–50.0μgmL−1 for each ETO enantiomer with r2>0.998 and acceptable values for the intra-day and inter-day accuracy and precision as well as negligible matrix effects supported the suitability and reliability of the method. Finally, this method was used to analyze real samples taken from mice treated with (±)-ETO.

A novel salting-out assisted extraction coupled with HPLC- fluorescence detection for trace determination of vitamin K homologues in human plasma

Recently, new physiological roles of vitamin K homologues have been established in the treatment of rheumatoid arthritis, osteoporosis, hepatocellular carcinoma and leukemia. However, relatively high plasma protein binding, low plasma concentrations and occurrences of interfering lipids make accurate determination of vitamin K homologues a challenging task. Therefore, a sensitive and reliable salting-out assisted liquid/liquid extraction (SALLE) method coupled with HPLC-Fluorescence detection was designed for efficient extraction and quantification of trace levels of vitamin K homologues in human plasma. The investigated vitamin K homologues were phylloquinone (PK, vitamin K1), menaquinone-4 (MK-4) and menaquinone-7 (MK-7). The method employed a new efficient fluorescence derivatization reaction using ethanolic solution of stannous chloride in acidic solution to generate highly fluorescent naphthohydroquinone derivatives. Correlation coefficients were more than 0.998 in the concentration ranges of 0.3–100ngmL−1 with detection limits of 0.1–0.17ngmL−1 in human plasma. The developed HPLC-FL system was successfully applied for sensitive determination of vitamin K homologues in plasma of healthy volunteers. The developed method may provide a valuable tool in the pharmacoinformatic studies concerning the roles of vitamin K homologues.

Salting‐out assisted liquid–liquid extraction coupled to dispersive liquid–liquid microextraction for the determination of chlorophenols in wine by high‐performance liquid chromatography

A novel procedure of sample preparation combined with high-performance liquid chromatography with diode array detection is introduced for the analysis of highly chlorinated phenols (trichlorophenols, tetrachlorophenols, and pentachlorophenol) in wine. The main features of the proposed method are (i) low-toxicity diethyl carbonate as extraction solvent to selectively extract the analytes without matrix effect, (ii) the combination of salting-out assisted liquid-liquid extraction and dispersive liquid-liquid microextraction to achieve an enrichment factor of 334-361, and (iii) the extract is analyzed by high-performance liquid chromatography to avoid derivatization. Under the optimum conditions, correlation coefficients (r) were >0.997 for calibration curves in the range 1-80 ng/mL, detection limits and quantification limits ranged from 0.19 to 0.67 and 0.63 to 2.23 ng/mL, respectively, and relative standard deviation was <8%. The method was applied for the determination of chlorophenols in real wines, with recovery rates in the range 82-104%.

Quantification of nerve agent biomarkers in human serum and urine.

A novel method for rapid and sensitive quantification of the nerve agent metabolites ethyl, isopropyl, isobutyl, cyclohexyl, and pinacolyl methylphosphonic acid has been established by combining salting-out assisted liquid-liquid extraction (SALLE) and online solid phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS). The procedure allows confirmation of nerve agent exposure within 30 min from receiving a sample, with very low detection limits for the biomarkers of 0.04-0.12 ng/mL. Sample preparation by SALLE was performed in less than 10 min, with a common procedure for both serum and urine. Analyte recoveries of 70-100% were obtained using tetrahydrofuran as extraction solvent and Na2SO4 to achieve phase separation. After SALLE, selective analyte retention was obtained on a ZrO2 column by Lewis acid-base and hydrophilic interactions with acetonitrile/1% CH3COOH (82/18) as the loading mobile phase. The phosphonic acids were backflush-desorbed onto a polymeric zwitterionic column at pH 9.8 and separated by hydrophilic interaction liquid chromatography. The method was linear (R(2) ≥ 0.995) from the limits of quantification to 50 ng/mL, and the within- and between-assay repeatability at 20 ng/mL were below 5% and 10% relative standard deviation, respectively.

Development and Validation of LC-MS/MS Assay for the Quantification of Progesterone in Rat Plasma and its Application to Pharmacokinetic Studies.

A sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the determination of progesterone levels in rat plasma. Progesterone-d9 was used as an internal standard (IS). Samples were prepared using salting-out assisted liquid/liquid extraction (SALLE), and the extracts were injected directly onto the LC-MS/MS system. The chromatographic separation was achieved on a CAPCELL PAK C18 MGIII column (100 mm × 2.0 mm, i.d. 5 µm) using methanol and aqueous 0.1% formic acid solution gradient as the mobile phase with a constant flow rate of 0.45 mL/min. Electrospray ionization in the positive-ion mode was employed. Multiple reaction monitoring of the precursor to product ion pairs, from m/z 315.20 to m/z 109.10 for progesterone and from m/z 324.26 to m/z 113.07 for the IS, was used for quantification. Good linearity was observed over the concentration range of 0.05-20.00 ng/mL with a weighted (1/x(2)) linear regression. The intra- and inter-day precision (% relative standard deviation [RSD]) across 3 validation days over the entire concentration range was lower than 6.7%. Accuracy (% nominal) determined at 5 quality control concentrations was between 94.0 and 103.7%. The validation method was applied in a pharmacokinetic study evaluating progesterone levels after intramuscular or vaginal administration to ovariectomized (OVX) rats. The area under the plasma concentration-time curve (AUC) calculated after intramuscular administration was more than 4 times higher than the AUC measured following vaginal administration of a comparable dose.

Liquid chromatography tandem mass spectrometry method for the quantitative analysis of ceritinib in human plasma and its application to pharmacokinetic studies

Ceritinib is a highly selective inhibitor of an important cancer target, anaplastic lymphoma kinase (ALK). Because it is an investigational compound, there is a need to develop a robust and reliable analytical method for its quantitative determination in human plasma. Here, we report the validation of a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the rapid quantification of ceritinib in human plasma. The method consists of protein precipitation with acetonitrile, and salting-out assisted liquid-liquid extraction (SALLE) using a saturated solution of sodium chloride prior to analysis by LC-MS/MS with electrospray ionization (ESI) technique in positive mode. Samples were eluted at 0.800mLmin(-1) on Ascentis Express® C18 column (50mm × 2.1mm, 2.7μm) with a mobile phase made of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The method run time was 3.6min and the low limit of quantification (LLOQ) was estimated at 1.00ngmL(-1) when using 0.100mL of human plasma. The assay was fully validated and the method exhibited sufficient specificity, accuracy, precision, and sensitivity. In addition, recovery data and matrix factor (MF) in normal and in hemolyzed plasmas were assessed, while incurred samples stability (ISS) for ceritinib was demonstrated for at least 21months at a storage temperature of -65°C or below. The method was successfully applied to the measurement of ceritinib in clinical samples and the data obtained on incurred samples reanalysis (ISR) showed that our method was reliable and suitable to support the analysis of samples from the clinical studies.

Determination of ethyl carbamate in spirits using salting-out assisted liquid–liquid extraction and high performance liquid chromatography with fluorimetric detection

In this work, a simple methodology was developed for the determination of ethyl carbamate (EC) in spirits by high performance liquid chromatography with fluorimetric detection. The sample is subjected to salting-out assisted liquid–liquid extraction (SALLE) using a 9-xanthydrol/acetonitrile solution for the simultaneous extraction and derivatisation of EC. Extraction/derivatization occurs under acidic conditions (0.5 mol L−1 HCl) and NaCl was used for phase separation. The methodology showed low limits of detection and quantification (9 μg L−1 and 23 μg L−1, respectively) and good precision (4.4% intraday and 8.7% interday). The determined EC concentrations in the analysed samples varied between 51 and 266 μg L−1. The proposed sample preparation methodology is simple and low cost, it uses limited toxicity solvents and the obtained extract is readily compatible with reversed phase liquid chromatography. Moreover, the stability of the xanthyl-EC product was verified. More importantly it is a more robust alternative to the liquid chromatography based methodologies that do not apply any extraction procedure, which do not consider the problems associated with the long term deterioration of the analytical systems.

Laser diode thermal desorption atmospheric pressure chemical ionization tandem mass spectrometry applied for the ultra-fast quantitative analysis of BKM120 in human plasma.

A sensitive and ultra-fast method utilizing the laser diode thermal desorption ion source using atmospheric pressure chemical ionization coupled to tandem mass spectrometry (LDTD-APCI-MS/MS) was developed for the quantitative analysis of BKM120, an investigational anticancer drug in human plasma. Samples originating from protein precipitation (PP) followed by salting-out assisted liquid-liquid extraction (SALLE) were spotted onto the LazWell™ plate prior to their thermal desorption and detection by tandem mass spectrometry in positive mode. The validated method described in this paper presents a high absolute extraction recovery (>90%) for BKM120 and its internal standard (ISTD) [D8]BKM120, with precision and accuracy meeting the acceptance criteria. Standard curves were linear over the range of 5.00 to 2000ngmL(-1) with a coefficient of determination (R (2)) >0.995. The method specificity was demonstrated in six different batches of human plasma. Intra- and inter-run precision as well as accuracy within ±20% at the lower limit of quantification (LLOQ) and ±15% (other levels) were achieved during a three-run validation for quality control (QC) samples. The post-preparative stability on the LazWell™ plate at room temperature was 72h and a 200-fold dilution of spiked samples was demonstrated. The method was applied successfully to three clinical studies (n = 847) and cross-checked with the validated LC-ESI-MS/MS reference method. The sample analysis run time was 10s as compared to 4.5min for the current validated LC-ESI-MS/MS method. The resultant data were in agreement with the results obtained using the validated reference LC-ESI-MS/MS assay and the same pharmacokinetic (PK) parameters were calculated for both analytical assays. This work demonstrates that LDTD-APCI-MS/MS is a reliable method for the ultra-fast quantitative analysis of BKM120 which can be used to speed-up and support its bioanalysis in the frame of the clinical trials.

Salting-out-assisted liquid-liquid extraction with acetonitrile for the determination of trimetazidine in rat plasma using liquid chromatography-mass spectrometry.

A high-throughout bioanalytical method based on salting-out-assisted liquid/liquid extraction (SALLE) method with acetonitrile and mass spectrometry-compatible salts followed by LC-MS/MS analysis of trimetazidine in rat plasma is presented. It required only 50 μL of plasma and allows the use of minimal volumes of organic solvents. The seamless interface of SALLE and LC-MS eliminated the drying-down step and the extract was diluted and injected into an LC-MS/MS system with a cycle time of 2.5 min/sample. The retention times of trimetazidine and IS were approximately 1.1 and 1.7 min, respectively. Calibration curves were linear over the concentration range of 0.1-100 ng/mL, which can be extended to 500 ng/mL by dilution. The intra- and inter-batch precision, accuracy and the relative standard deviation were all <15%. This method was successfully applied to determine trimetazidine concentrations in rat plasma.

Development and validation of LC-MS/MS method for quantitative determination of (-)-securinine in mouse plasma.

(-)-Securinine (SE) is a major alkaloid found in plant Securinega suffruticosa, which has a wide range of pharmacological activities including anticancer, anti-parasitic and central nervous system stimulating effects, etc. To aid the pharmacological study of SE, we developed an LC-MS/MS method for quantitative determination of SE in mouse plasma. In this method, plasma samples were first prepared with salting-out assisted liquid-liquid extraction using cold acetonitrile (-20°C) and 2.00 M ammonium acetate. Separation of SE and the internal standard (IS) from sample matrix was achieved on a Gemini Nx C18 column using 40% acetonitrile and 60% 10.0mM ammonium acetate at a flow rate of 0.200 mL min(-1). Quantification of SE was accomplished with positive electrospray ionization tandem mass spectrometry using mass transitions m/z 218.1→84.1 for SE, and m/z 204.1→70.2 for the IS. This method has a lower limit of quantitation (LLOQ) of 0.600 ng mL(-1) and a linear calibration range up to 600 ng mL(-1) in mouse plasma. The intra- and inter-run accuracy (%RE) and precision (%CV) were ≤ ± 6% and 6%, respectively. The IS normalized matrix factors from six lots of plasma matrices ranged 0.92-1.07, and the recoveries of plasma SE were 99-109%. The validated method has been applied to the measurement of SE in plasma samples of a mouse study.

Salting-out assisted liquid–liquid extraction combined with capillary HPLC for the determination of sulfonylurea herbicides in environmental water and banana juice samples

A salting-out assisted liquid–liquid extraction (SALLE) combined with capillary high performance liquid chromatography with diode array detector (capillary HPLC-DAD) was proposed for extraction and determination of residues of nine sulfonylurea herbicides (SUHs) in environmental water and banana juice samples. Various parameters affecting the extraction process such as the type and volume of the organic solvent, sample volume, type and amount of salt, pH of the sample and vortex time were optimized. Under optimum conditions, matrix matched calibration curves were established using river water and banana juice samples. Good linear relationships as well as low limits of detection, LODs (0.4–1.3 and 3–13µg/L) and quantification, LOQs (1.3–4.3 and 10–43µg/L) were obtained in water and banana juice samples, respectively. The precision (intra- and inter-day) of the peak areas expressed as relative standard deviations (%, RSD), at two concentration levels were below 10 % in both matrices. Recoveries obtained from spiked environmental waters (river water and groundwater) and banana juice samples, at two concentration levels, ranged from 72 to 115%. The results of the analysis revealed that the proposed SALLE-capillary HPLC method is simple, rapid, cheap and environmentally friendly, being successfully applicable for the determination of SUH residues in waters and banana juices.

Reliable quantification of bisphenol A and its chlorinated derivatives in human urine using UPLC–MS/MS method

Bisphenol A (BPA), a widespread man-made chemical classified as an endocrine disruptor, is increasingly considered as a major cause of concern for human health. Chlorine present in drinking water may react with BPA to form chlorinated derivatives (ClxBPA), which have demonstrated a heightened level of estrogenic activity. If many epidemiological studies report that more than 90% of people have detectable BPA levels in their urine, then no such study has been undertaken regarding ClxBPA. The purpose of this work is to propose a highly sensitive and accurate analytical method adapted to large-scale biomonitoring studies aimed at assessing exposure to BPA and ClxBPA through the use of human urine. To achieve this, we have comprehensively validated a method using salting-out assisted liquid/liquid extraction (SALLE) coupled to UPLC–MS/MS and isotope dilution quantification, to measure unconjugated BPA and ClxBPA in human urine according to the accepted guidelines. Deutered BPA as well as deutered 2,2′-DCBPA was used as internal standards. The matrix calibration curve ranged from 0.05 to 1.60ngmL−1 and from 0.5 to 16.0ngmL−1 for ClxBPA and BPA respectively, and provided good linearity (r²>0.99). This method was precise (the intra- and inter-day coefficients of variation were <20% at three different concentrations: 0.05ngmL−1, 0.2ngmL−1, 0.8ngmL−1 and 0.5ngmL−1, 2ngmL−1, 8ngmL−1 for ClxBPA and BPA, respectively) and accurate (bias ranged from −13% to +12%). The limit of quantification, validated at 0.05ngmL−1 and 0.5ngmL−1 for ClxBPA and BPA respectively when using 300µL of urine, was found to be suitable for the concentration existing in real samples. The matrix effect and the BPA cross-contamination were also investigated in this study. The analytical method developed in this study is in accordance with the requirements applicable to biomonitoring of BPA and ClxBPA in human urine.

Analysis of biogenic amines in wines by salting-out assisted liquid-liquid extraction and high-performance liquid chromatography with fluorimetric detection.

Biogenic amines are nitrogenous organic compounds of low molecular weight that are either formed or metabolized in cells of living organisms and can be found in several food products, being produced mainly by amino acid decarboxylation. When ingested in high concentrations they can induce several health problems in humans. In alcoholic beverages, and especially in wine, they are formed during the vinification process as a result of the action of microorganisms. In this work it is proposed a new methodology for the determination of biogenic amines in wines, which includes a sample preparation approach based on salting-out assisted liquid-liquid extraction, the use of dansyl chloride for the derivatization and chromatographic separation by high-performance liquid chromatography with fluorimetric detection. The salting-out effect is used to promote phase separation between water and a water-miscible organic solvent, while improving the extraction of organic or inorganic species. Several extraction parameters were optimized, such as the dansyl chloride concentration, pH and the effects caused by the order in which the extraction and derivatization were performed. Extraction of amines, and consequent detection, depends on the presence of dansyl chloride in solution prior to extraction. The results showed the possibility to simultaneously perform the extraction and the derivatization, making sample preparation easier and less time-consuming. The methodology was successfully applied to the determination of biogenic amines in five wines (white, red and rosé). This method has the potential to be a good alternative to existing methods since it is cheaper, easier and simplifies the sample preparation step.

Microextraction by packed sorbent and salting-out-assisted liquid–liquid extraction for the determination of aromatic amines formed from azo dyes in textiles

EU legislation prohibits the use of certain azo dyes which, on reduction, form any of 22 aromatic amines listed in Regulation (EC) 1907/2006 at concentrations above the threshold limit of 30 mg Kg(-1). Two different extraction techniques for the determination of aromatic amines formed from azo dyes in textiles in combination with gas chromatography-mass spectrometry (GC-MS) are described. The first one is based on microextraction by packed sorbent (MEPS) and the other approach involves salting-out-assisted liquid-liquid extraction (SALLE). The influence of several parameters on the efficiency of the extraction using MEPS (sorbent material, sample volume, elution solvent, elution volume and washing steps, among others) and SALLE (extraction volume and amount of salt) were investigated. In addition, chromatographic separation was optimized and quadrupole mass spectrometry was evaluated using the synchronous SIM/scan data acquisition mode. The repeatability (n=8, S/N=3) of the methods, calculated as the relative standard deviation (RSD) was below 15 and 11% for all compounds when MEPS and SALLE were used, respectively. Standard additions procedure was used to quantify the aromatic amines in the textil samples. The detection limits in the samples for both methods were lower than the maximum value allowed by legislation. The results obtained in the analysis of textiles revealed the presence of o-anisidine, p-chloroaniline, 4-chloro-o-toluidine, 2-naphthylamine and 3,3'-dimethoxybenzidine in some of them.

Another glimpse over the salting-out assisted liquid–liquid extraction in acetonitrile/water mixtures

The use of the salting-out effect in analytical chemistry is very diverse and can be applied to increase the volatility of the analytes in headspace extractions, to cause the precipitation of proteins in biological samples or to improve the recoveries in liquid–liquid extractions. In the latter, the salting-out process can be used to create a phase separation between water-miscible organic solvents and water. Salting-out assisted liquid–liquid extraction (SALLE) is an advantageous sample preparation technique aiming HPLC-UV analysis when developing analytical methodologies. In fact, some new extraction methodologies like QuEChERS include the SALLE concept. This manuscript discusses another point of view over SALLE with particular emphasis over acetonitrile–water mixtures for HPLC-UV analysis; the influence of the salting-out agents, their concentration and the water–acetonitrile volume ratios were the studied parameters. α-dicarbonyl compounds and beer were used as test analytes and test samples, respectively. The influence of the studied parameters was characterized by the obtained phase separation volume ratio and the fraction of α-dicarbonyls extracted to the acetonitrile phase. Results allowed the distribution of salts within three groups according to the phase separation and their extractability: (1) chlorides and acetates, (2) carbonates and sulfates and (3) magnesium sulfate; of all tested salts, sodium chloride had the highest influence on the α-dicarbonyls fraction extracted.