Salting-out Assisted Liquid-liquid Research Articles

A highly efficient strategy for simultaneous analysis of eleven monomeric hydroxylated-stilbenes in grape wines and juices by a salting-out assisted liquid-liquid (SALLE) extraction combined with a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS).

Wine and grape juice are known to have hydroxylated stilbenes, a group of phytoalexins, health-promoting compounds. The determination of stilbene species in wine and grape juices remains a challenging task. Here, we propose an efficient strategy for the simultaneous determination of eleven hydroxylated stilbenes in grape wines and grape juice by a SALLE in conjunction with an HPLC-ESI-MS/MS. SALLE and HPLC-MS/MS conditions were optimized. The proposed method was validated and found to be a simple, sensitive, and reliable measure for the determination of the stilbene species in wine and grape juice. The SALLE was fast and efficient, taking only 10min. The HPLC-MS/MS was able to quickly separate and quantitate the eleven stilbenes (9min running). This method was successfully applied to determine the contents of stilbene species in commercial wine and grape juice in Korea. This method is advantageous in reduced sample preparation time, low labor, and cost efficiency.

High-throughput salting-out assisted liquid-liquid extraction using a 3D printed device and its application in the quantification of ibrutinib and its metabolite PCI-45227 in human serum

Liquid–liquid extraction methods are widely used for sample treatment in bioanalysis, although their implementation poses a common speed-limiting step in the analytical process when fast separation and detection methods such as UHPLC-MS are used. This study aimed to develop high-throughput salting-out assisted liquid–liquid extraction on a 96-well plate in combination with fast LC-MS analysis of ibrutinib and its active metabolite PCI-45227 (dihydrodiol ibrutinib) in human serum. A specially designed 3D printed extraction device developed in our laboratory allowed for the precise and rapid collection of the organic phase from the 96-well plate using a multichannel pipette, without the risk of aspiration of the bottom aqueous layer. The application of this device significantly accelerated sample preparation and allowed the processing of up to 96 samples in 1 h. The method was successfully validated according to EMA guidelines in the concentration range of 0.1–200 ng/mL for both analytes, providing lower limit of quantification at 100 pg/mL. The intra-day accuracy for IBT was in the range of − 1.67–5.67 %, while the inter-day accuracy was in the range of 0.20–6.90 %. The intra-day precision for IBT was in the range of 3.20–4.37 % and 3.13–4.73 % for the inter-day measurement. PCI-45227 showed intra-day accuracy in the range of − 11.2–3.71 % and the inter-day accuracy in the range of − 5.76–0.92 %. The intra-day precision for PCI was in the range of 3.49–7.64 % and 3.63–8.61 % for the measurement between days.In addition to increasing the speed of sample preparation, this method also offers low consumption of the sample and extraction solvent and can be utilized for other similar small-volume in-well plate extractions where organic solvents of lower density than water are used. The method was successfully applied to the analysis of serum samples (n = 5) of patients with chronic lymphoblastic leukaemia at the trough level (ibrutinib concentration range: 1.63–3.78 ng/mL, PCI-45227 concentration range: 1.84–14.02 ng/mL) and 2 h postdose (ibrutinib concentration range: 7.34–89.0 ng/mL, PCI-45227 concentration range: 5.64–124 ng/mL).

Quantitative extraction of multiclass antibiotic residues in cow milk utilizing salting-out assisted liquid-liquid extraction followed by liquid chromatographic determination

ABSTRACT The salting-out assisted liquid–liquid extraction (SALLE) method with the liquid chromatography ultraviolet-visible detector (LC-UV) has been developed and validated for the simultaneous determination of seven antibiotic compounds of five different therapeutic classes (β-lactams), amoxycillin, ampicillin, penicillin G, (sulphonamides), sulfamethoxazole, (aminoglycosides) gentamicin, (fluoroquinolones) ciprofloxacin and (macrolide) erythromycin, in the cow milk samples. The LC conditions for separation of the selected antibiotic compounds were achieved with the reversed phase isocratic elution. The mobile phase consisted of 0.05 M Na2HPO4, acetonitrile and methanol (70:10:20 v/v/v). The mobile-phase flow rate was 1 mL min−1 with the injection volume of 20 μL and all the seven standard compounds were eluted within 14 min. The developed analytical method has been validated for the linearity, precision, sensitivity, accuracy, specificity, decision limit (CCα), and detection capability (CCβ). Experimental parameters influencing the extraction efficiency were evaluated and optimised. Under the optimum experimental conditions, good linearity over wide concentration ranges was obtained with the coefficient of determination R2 > 0.9989 for all the antibiotics. The limits of detection (LODs) and quantification (LOQs) ranged from 1.28 to 2.46 µg mL−1 and 3.87–7.46 µg mL−1, respectively. The relative standard deviations for the repeated analysis were below 8% indicating acceptable method precision. The accuracy study yielded satisfactory recoveries in the ranges 95.5–103% for the pure standard compounds and 83.8–112% for the spiked matrix-match milk samples. The results of the study revealed that the developed method is simple and fast and the SALLE method is appropriate for the simultaneous determination of targeted five multiclass antibiotic residues in the cow milk samples by LC-UV/Vis with a better efficiency.

A simple and sensitive preconcentration strategy by coupling salting-out assisted liquid-liquid extraction with online three-step stacking for the determination of potent anti-tumour compound vinblastine and its precursor in biological samples by capillary electrophoresis

A sensitive capillary electrophoresis strategy was developed by combining salting-out assisted liquid–liquid extraction and online three-step stacking to detect trace quantities of antitumour indole alkaloids in complex biological samples. The proposed strategy fully exploits these two technologies such that extraction, online stacking and separation are combined in a fast and efficient manner. First, salting-out assisted liquid–liquid extraction was used to extract three indole alkaloids (vinblastine sulfate, catharanthine sulfate and vindoline) from complex biomasses. An appropriate volume of acetonitrile (ACN) was mixed with a faecal aqueous solution to precipitate proteins. The mixed solution was vortexed, followed by the addition of ammonium sulfate ((NH4)2SO4) to induce two-phase separation. Alkaloids were effectively extracted into the organic phase, which was then subjected to capillary electrophoresis (CE) analysis. The sensitivity of capillary electrophoresis was effectively improved by online three-step stacking. Good linearity of the calibration curve for each indole alkaloid was obtained in the concentration range of 0.1–1 μg/mL. Under optimal conditions, the sensitivity of ordinary injection was increased by up to 2366-fold, confirming the applicability of the proposed strategy for the sensitive determination of trace indole alkaloids in complex biological samples.

A simple, rapid and novel method based on salting-out assisted liquid–liquid extraction for ochratoxin A determination in beer samples prior to ultra-high-performance liquid chromatography coupled to tandem mass spectrometry

ABSTRACTA novel, simple, easy and cheap sample treatment strategy based on salting-out assisted liquid-liquid extraction for ochratoxin A (OTA) ultra-trace analysis in beer samples using ultra-high-performance liquid chromatography-tandem mass spectrometry determination was developed. The factors involved in the efficiency of pre-treatment were studied employing factorial design in the screening phase and the optimal conditions of the significant variables on the analytical response were evaluated using a central composite face-centred design. Consequently, the amount of salt ((NH4)2SO4), together with the volumes of sample, hydrophilic (acetone) and nonpolar (toluene) solvents, and times of vortexing and centrifugation were optimised. Under optimised conditions, the limits of detection and quantification were 0.02 µg l−1 and 0.08 µg l−1 respectively. OTA extraction recovery by SALLE was approximately 90% (0.2 µg l−1). Furthermore, the methodology was in agreement with EU Directive requirements and was successfully applied for analysis of beer samples.

Ultra-high performance liquid chromatography with fluorescence detection following salting-out assisted liquid–liquid extraction for the analysis of benzimidazole residues in farm fish samples

Ultra-high performance liquid chromatography (UHPLC) coupled with fluorescence detection (FL) has been proposed for the first time to determine thirteen benzimidazoles (BZs) in farmed fish samples. In order to optimize the chromatographic separation, parameters such as mobile phase composition and flow rate were carefully studied, establishing a gradient mode with a mobile phase consisted of water (solvent A) and acetonitrile (solvent B) at a flow rate of 0.4 mL/min. The separation was performed on a Zorbax Eclipse Plus RRHD C18 column (50 × 2.1 mm, 1.8 μm), involving a total analysis time lower than 12 min. Salting-out assisted liquid-liquid extraction (SALLE) was applied as sample treatment to different types of farmed fish (trout, sea bream and sea bass). To obtain satisfactory extraction efficiencies for the studied analytes, several parameters affecting the SALLE procedure were optimized including the amount of sample, type and volume of the extraction solvent, and the nature and amount of the salt used. Characterization of the method in terms of performance characteristics was carried out, obtaining satisfactory results for the linearity (R2 ≥ 0.997), repeatability (RSD ≤ 6.1%), reproducibility (RSD ≤ 10.8%) and recoveries (R ≥ 79%; RSD ≤ 7.8%). Detection limits between 0.04–29.9 μg kg−1 were obtained, demonstrating the applicability of this fast, simple and environmentally friendly method.

A high-throughput UHPLC method for the analysis of 5-nitroimidazole residues in milk based on salting-out assisted liquid-liquid extraction.

A novel salting-out assisted liquid-liquid extraction (SALLE) method has been developed for the extraction of eight 5-NDZ antibiotics from milk samples prior to their analysis by UHPLC. An exhaustive study of the parameters involved in the SALLE procedure has been carried out, optimizing the extraction solvent volume, the amount of the salt agent, and the centrifugation and vortex timing by an experimental design. After sample treatment, the obtained extract was reconstituted in the mobile phase (6:94 (v/v) acetonitrile/water containing 0.1% (v/v) of formic acid) and analyzed by the proposed UHPLC-UV method. Separation was accomplished in a C18 Zorbax Eclipse Plus (50mm×2.1mm, 1.8μm) column at 45°C within 8min. Gradient mode was considered using a mobile phase consisting of 0.1% (v/v) formic acid aqueous solution and acetonitrile containing 0.1% (v/v) formic acid at a flow rate of 0.45mL/min. Analytical signals were monitored at 320nm. Matrix-matched calibration curves showed satisfactory linearity (R2≥0.996). LODs, ranging from 2 to 4μg/L, were achieved and precision studies showed RSDs lower than 12.8% in terms of peak height. Additionally, recoveries higher than 62.8% were obtained for all studied compounds in milk samples.

Salting-out assisted liquid-liquid extraction coupled to ultra-high performance liquid chromatography-tandem mass spectrometry for the determination of tetracycline residues in infant foods.

The use of salting-out assisted liquid-liquid extraction (SALLE) combined with ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) has been evaluated for the determination of tetracyclines in infant foods based on meat and vegetables or in milk. To obtain satisfactory extraction efficiencies for the studied analytes, several parameters affecting the SALLE procedure were optimized. Analytical performances of the method were satisfactory, obtaining limits of quantification lower than 0.48μgkg-1 in all cases. The precision, expressed as relative standard deviation (%, RSD) was below 11.3%. The extraction efficiency for fortified samples ranged from 89.2 to 96.8%, with RSDs lower than 7.3%. Matrix effect was evaluated for all samples studied, being lower than |21|% in all cases. In relation to the low solvent consumption, the proposed methodology could be considered rapid, cheap and environmentally friendly. Its applicability has been successfully tested in a wide range of infant foods.

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.

Salting-Out Assisted Liquid–Liquid Extraction for Bioanalysis

Salting-out assisted liquid-liquid extraction (SALLE) applies the salting-out effect to separate water-miscible organic solvent such as acetonitrile from plasma or other aqueous biofluids, and can extract a wide range of drug and metabolites, including many hydrophilic compounds. In most cases, the separated organic phase can be directly injected for bioanalysis, or with a simple dilution. SALLE provides similar simplicity to protein precipitation, but cleaner extracts due to a true phase separation. SALLE is also faster, more environmentally friendly and more cost-efficient than conventional liquid-liquid extraction and SPE. Through 96-well automation, SALLE can be easily integrated into the overall high-throughput LC-MS/MS bioanalysis strategy to increase productivity. This article provides a critical overview of the literatures on SALLE and perspectives of the future bioanalytical application of this often overlooked extraction technique. Important parameters impacting SALLE-LC-MS/MS assays are also discussed.