Protocol for preparing cyclic-phospholipid decanoate and glyceryl-didecanoate-phosphate-containing vesicles.

Cyclic-phospholipids-based vesicles can play a role in facilitating the chemical evolution of protocells from the structurally simple to the functionally more complex form. Here, we present a protocol for preparing decanoic acid-derived cyclic phospholipid and glyceryl-diester phosphate-containing vesicles. We describe steps for sample preparation, equilibration, and image acquisition using confocal microscopy. This protocol has the potential for preparing a wide variety of these phospholipid-based artificial cell constructs. For complete details on the use and execution of this protocol, please refer to Pulletikurti et al.1.

Head-to-Head Comparison of Caco-2 Transwell and Gut-on-a-Chip Models for Assessing Oral Peptide Formulations.

Oral delivery of potent peptide drugs provides key formulation challenges in the pharmaceutical industry: stability, solubility, and permeability. Intestinal permeation enhancers (PEs) can overcome the low oral bioavailability by improving the drug permeability. Conventional in vitro and ex vivo models for assessing PEs fail to predict efficacy in vivo. Here, we compared Caco-2 cells cultured in the conventional static Transwell model to a commercially available continuous flow microfluidic Gut-on-a-Chip model. We determined baseline permeability of FITC-Dextan 3 kDa (FD3) in Transwell (5.3 ± 0.8 × 10-8 cm/s) vs Chip (3.2 ± 1.8 × 10-7 cm/s). We screened the concentration impact of two established PEs sodium caprate and sucrose monolaurate and indicated a requirement for higher enhancer concentration in the Chip model to elicit equivalent efficacy e.g., 10 mM sodium caprate in Transwells vs 25 mM in Chips. Fasted and fed state simulated intestinal fluids (FaSSIF/FeSSIF) were introduced into the Chip and increased basal FD3 permeability by 3-fold and 20-fold, respectively, compared to 4-fold and 4000-fold in Transwells. We assessed the utility of this model to peptides (Insulin and Octreotide) with PEs and observed much more modest permeability enhancement in the Chip model in line with observations in ex vivo and in vivo preclinical models. These data indicate that microfluidic Chip models are well suited to bridge the gap between conventional in vitro and in vivo models.

Construction of injectable micron-sized polymorphic vesicles for prolonged local anesthesia with weekly sustained release of ropivacaine.

Currently, to overcome the short half-life of the local anesthetic ropivacaine, drug delivery systems such as nanoparticles and liposomes have been used to prolong the analgesic effect, but they are prone to abrupt release from the site of administration or have poor slow-release effects, which increases the risk of cardiotoxicity. In this study, injectable lipid suspensions based on ropivacaine-docusate sodium hydrophobic ion pairing (HIP) were designed to significantly prolong the duration of analgesia. The resulting ion-paired lipid suspension (HIP/LIPO) had a micrometer scale and a high zeta potential, which facilitates stable in situ retention. The strong interaction between docusate sodium and ropivacaine was verified using thermal and spectroscopic analyses, and the formation of micron-sized polymorphic vesicles was attributed to the mutual stabilizing interactions between ropivacaine-docusate sodium HIP, docusate sodium and lecithin. The HIP/LIPO delivery system could maintain drug release for more than 5 days in vitro and achieve high analgesic efficacy for more than 10 days in vivo, reducing the side effects associated with high drug doses. The stable HIP/LIPO delivery system is a promising strategy that offers a clinically beneficial alternative for postoperative pain management and other diseases.

Enzymatic Synthesis and Characterization of MLM-type Structured Lipid Using Grapeseed Oil and Capric Acid.

The objectives were to optimize the reaction conditions for C10:0 incorporation into grapeseed (GS) oil, characterize the structured lipid (SL) product, and study the changes in antioxidant activity of the SL. Taguchi method was used to optimize C10:0 incorporation by combining parameters in a total of 9 experiments. Lipozyme ® RM IM (Rhizomucor miehei immobilized lipase) and Lipozyme ® 435 (Candida antarctica recombinant immobilized lipase) were used as biocatalysts for the acidolysis reactions. C10:0 incorporation and triacylglycerol (TAG) species of the SL were analyzed to determine optimal conditions and enzyme type that gave higher incorporation. The optimal conditions were the same for both enzymes as follows: substrate molar ratio 1:3 (GS oil: C10:0), enzyme load 5% (w/w) of substrates, temperature 65℃, and time 12 h. HPLC analysis of SL gave MLM-type TAG species of 11.51±0.11 mol% and 12.68±0.34 mol% for Lipozyme ® RM IM and Lipozyme ® 435, respectively. GC analysis indicated that C10:0 incorporated at the sn-1,3 positions of the SL were 46.03±0.55 mol% and 47.28±1.22 mol%, respectively, for Lipozyme ® RM IM and Lipozyme ® 435. However, the total C10:0 incorporated into TAG species with Lipozyme ® RM IM was significantly higher (60.08±0.04 mol%) compared to 50.78±0.44 mol% for Lipozyme ® 435. Scaled-up (300 g) acidolysis reaction and characterization were done on SL synthesized using Lipozyme ® RM IM. SL reaction product was purified using short path distillation and fully characterized in terms of lipid classes, tocopherol, thermal behavior, and oxidative stability. The yield of purified scaled-up SL after short path distillation (SPD) was 72.96 wt%. The antioxidant in SL was reduced after SPD due to loss of tocopherols. This MLM-type-SL synthesized within 12 h using Lipozyme ® RM IM had a high content of C10:0 and may have functional and health benefits.

Preparation and Application of Decanoic Acid/Arginine Hydrogels to a Transdermal Formulation.

We prepared a supramolecular hydrogel composed of decanoic acid and arginine (C10/Arg gel) and evaluated its application to a transdermal formulation. C10/Arg gel adjusted to pH 7 with 1 M NaOH aq or 1 M HCl aq provided a translucent hydrogel with a lamellar liquid crystal structure in the concentration region of decanoic acid ≥12% and arginine ≤9%. Rheological measurements showed that C10/Arg gel is a viscoelastic material with both solid and liquid properties, with elasticity being dominant over viscosity in the low shear stress region. The skin permeability of hydrocortisone (HC) and indomethacin (IM) from C10/Arg gels was investigated in vitro using hairless mouse skin and compared to control formulation drug suspensions (IM or HC) in water. The cumulative permeation amount of HC and IM from the C10/Arg gel at 10 h after application was approximately 16 and 11 times higher than that of the control, respectively. On the other hand, the flux of IM decreased with increasing arginine concentration, likely due to the acid-base interaction between Arg and IM in C10/Arg gel. Adequate drug skin permeation enhancement by C10/Arg gel requires optimizing the gel composition for each specific drug.

Engineering the Spatial Distribution of Amphiphilic Molecule within Complex Coacervate Microdroplet via Modulating Charge Strength of Polyelectrolytes.

The investigation of the interplay between complex coacervate microdroplets and amphiphilic molecules offers valuable insights into the processes of prebiotic compartmentalization on the early Earth and presents a promising avenue for future advancements in biotechnology. Herein, the interaction between complex coacervate microdroplets and amphiphilic molecule (decanoic acid) is systematically investigated by varying charge strengths of negatively charged polyelectrolytes (DNA and PAA) and positively charged polyelectrolytes (PDDA and DEAE-Dextran). It is found that the interaction between amphiphilic molecule and complex coacervate microdroplets depended on the delicate balance between the interaction between decanoic acid and polyelectrolyte and the interaction between two polyelectrolytes. The different spatial distribution of amphiphilic molecule can result in differences in the internal microenvironment, which can further alter the uptake or exclusion of small molecules and biomolecules with different charges and polarities and functional biological process.

Study on multiscale structures and digestibility of cassava starch and medium-chain fatty acids complexes using molecular simulation techniques.

Effect of complexation of three medium-chain fatty acids (octanoic, decylic and lauric acid, OA, DA and LA, respectively) on structural characteristics, physicochemical properties and digestion behaviors of cassava starch (CS) was investigated. Current study indicated that LA was more easily to combine with CS (complex index 88.9%), followed by DA (80.9%), which was also consistent with their corresponding complexed lipids content. Following the investigation of morphology, short-range ordered structure, helical structure, crystalline/amorphous region and fractal dimension of the various complexes, all cassava starch-fatty acids complexes (CS-FAs) were characterized with a flaked morphology rather than a round morphology in native starch (control CS). X-ray diffraction demonstrated that all CS-FAs had a V-type crystalline structure, and nuclear magnetic resonance spectroscopy confirmed that the complexes made from different fatty acids displayed similar V6 or V7 type polymorphs. Interestingly, small-angle X-ray scattering analysis revealed that α value became greater following increased carbon chain length of fatty acids, indicating the formation of a more ordered fractal structure in the aggregates. Changes in rheological parameters G' and G'' indicated that starch complexed with fatty acids was more likely to form a gel network, but difference among three CS-FAs complexes was significant, which might be contributed to their corresponding hydrophobicity and hydrophilicity raised from individual fatty acids. Importantly, digestion indicated that CS-LA complexes had the lowest hydrolysis degree, followed by the greatest RS content, indicating the importance of chain length of fatty acids for manipulating the fine structure and functionality of the complexes.

Combined royal jelly 10-hydroxydecanoic acid and aspirin has a synergistic effect against memory deficit and neuroinflammation.

Alzheimer's disease (AD), the most common form of neurodegenerative dementia among the older population, is associated with acute or chronic inflammation. As a nonsteroidal anti-inflammatory drug, aspirin has recently been widely studied in the prevention and treatment of neurodegenerative diseases. However, there is a controversy about the efficacy as well as the adverse effects of aspirin. 10-Hydroxydecanoic acid (10-HDAA) is a characteristic fatty acid found in the honey bee product royal jelly. In this study, we found that 10-HDAA attenuated the activation of the NF-κB pathway, then targeted Ptgs-1/2, the well-known target of aspirin. Hence, combined therapy of 10-HDAA and aspirin was conducted. In vitro assays suggested that this combinatory group alleviated LPS-induced inflammation in BV-2 cells, as assessed by the downregulation of nitric oxide, COX-2, and IL-6 compared to 10-HDAA or aspirin treatment alone. In vivo assays showed that the combined treatment synergistically inhibited the overactivation of glial cells and decreased the levels of pro-inflammatory mediators. Moreover, 10-HDAA alleviated the adverse effects of aspirin on gastrointestinal injuries and microbiota dysbiosis. The Morris water maze test indicated that neither 10-HDAA nor aspirin effectively improved LPS-induced memory dysfunction, but the combined therapy showed synergistic effects. Altogether, our findings support 10-HDAA and aspirin combinatory therapy as the basis for future therapeutics for AD and other neuroinflammation-related diseases with minimal adverse effects.

Impact of Intestinal Concentration and Colloidal Structure on the Permeation-Enhancing Efficiency of Sodium Caprate in the Rat.

In this work, we set out to better understand how the permeation enhancer sodium caprate (C10) influences the intestinal absorption of macromolecules. FITC-dextran 4000 (FD4) was selected as a model compound and formulated with 50-300 mM C10. Absorption was studied after bolus instillation of liquid formulation to the duodenum of anesthetized rats and intravenously as a reference, whereafter plasma samples were taken and analyzed for FD4 content. It was found that the AUC and Cmax of FD4 increased with increasing C10 concentration. Higher C10 concentrations were associated with an increased and extended absorption but also increased epithelial damage. Depending on the C10 concentration, the intestinal epithelium showed significant recovery already at 60-120 min after administration. At the highest studied C10 concentrations (100 and 300 mM), the absorption of FD4 was not affected by the colloidal structures of C10, with similar absorption obtained when C10 was administered as micelles (pH 8.5) and as vesicles (pH 6.5). In contrast, the FD4 absorption was lower when C10 was administered at 50 mM formulated as micelles as compared to vesicles. Intestinal dilution of C10 and FD4 revealed a trend of decreasing FD4 absorption with increasing intestinal dilution. However, the effect was smaller than that of altering the total administered C10 dose. Absorption was similar when the formulations were prepared in simulated intestinal fluids containing mixed micelles of bile salts and phospholipids and in simple buffer solution. The findings in this study suggest that in order to optimally enhance the absorption of macromolecules, high (≥100 mM) initial intestinal C10 concentrations are likely needed and that both the concentration and total dose of C10 are important parameters.

Prebiotic Membranes and Micelles Do Not Inhibit Peptide Formation During Dehydration.

Cycles of dehydration and rehydration could have enabled formation of peptides and RNA in otherwise unfavorable conditions on the early Earth. Development of the first protocells would have hinged upon colocalization of these biopolymers with fatty acid membranes. Using atomic force microscopy, we find that a prebiotic fatty acid (decanoic acid) forms stacks of membranes after dehydration. Using LC-MS-MS (liquid chromatography-tandem mass spectrometry) with isotope internal standards, we measure the rate of formation of serine dipeptides. We find that dipeptides form during dehydration at moderate temperatures (55 °C) at least as fast in the presence of decanoic acid membranes as in the absence of membranes. Our results are consistent with the hypothesis that protocells could have formed within evaporating environments on the early Earth.

Carboxymethyl chitosan coated medium-chain fatty acid nanoliposomes: structure, composition, stability and in vitro release investigation.

Medium-chain fatty acids (MCFAs) have been proven as an easy energy source and active ingredient to prevent obesity and other metabolic disorders. However, the inherent hydrophobic nature of MCFAs causes poor aqueous solubility and dissolution in the gastrointestinal (GI) tract, thus limiting their applications in aqueous foods. To address these issues, a nutraceutical carrier system was developed by coating nanoliposomes with carboxymethyl chitosan (CMCS) through a series of well-designed processes, including thin-film hydration, dynamic high pressure microfluidization (DHPM) and surface modification. Electron microscopy investigation reveals an obvious morphology evolution from the uncoated nanoliposomes (UC-LPs) to the final CMCS coated nanoliposomes (CMCS-LPs). Together with the FTIR results, it confirms the successful coating of CMCS. More importantly, the resultant CMCS-LPs have a more negatively charged surface with a ζ-potential value of around -18.5 mV, which helps to increase the stability by avoiding severe particle aggregation. Owing to the above benefits, the encapsulated MCFAs can be safely retained in a long storage period of 90 days at 4 °C and the new carrier system also exhibits a more sustained release of MCFAs in the GI fluid.

Ultrasound-assisted dispersive liquid-phase microextraction by solidifying L-menthol-decanoic acid hydrophobic deep eutectic solvents for detection of five fungicides in fruit juices and tea drinks.

An ecofriendly and efficient ultrasound-assisted deep eutectic solvents dispersive liquid-phase microextraction by solidifying the deep eutectic solvents-rich phase was developed to determine azoxystrobin, fludioxonil, epoxiconazole, cyprodinil, and prochloraz in fruit juices and tea drinks by high-performance liquid chromatography. A varieties of environmental hydrophobic deep eutectic solvents serving as extraction agents were prepared using L-menthol and decanoic acid as hydrogen-bond acceptor and hydrogen-bond donor, respectively. The deep eutectic solvents were ultrasonically dispersed in sample solutions, solidified in a freezer and easily harvested. The main variables were optimized by one-factor-at-a-time and response surface test. The new method performs well with relative recovery of 71.75-109.40%, linear range of 2.5-5000 µg/L (r ≥ 0.9968), detection limit of 0.75-8.45 µg/L, quantification limit of 2.5-25 µg/L,, and inter- and intraday relative standard deviations below 13.53 and 14.84%, respectively. As for the extraction mechanism, deep eutectic solvents were disposed into many fine particles in the solution and captured the analytes based on the changes of particle size and quantity in deep eutectic solvents droplets after extraction. The environmental method can successfully detect fungicide residues in real fruit juices and tea drinks.

Formulation of Ocular In Situ Gels with Lithuanian Royal Jelly and Their Biopharmaceutical Evaluation In Vitro.

Royal jelly is a natural substance produced by worker bees that possesses a variety of biological activities, including antioxidant, anti-inflammatory, antibacterial, and protective. Although fresh royal jelly is kept at low temperatures, to increase its stability, it needs to be incorporated into pharmaceutical formulations, such as in situ gels. The aim of this study was to formulate in situ ocular gels containing Lithuanian royal jelly for topical corneal use in order to increase the retention time of the formulation on the ocular surface and bioavailability. Gels were evaluated for physicochemical characteristics (pH, rheological properties, refractive index) and in vitro drug release measuring the amount of 10-hydroxy-2-decenoic acid (10-HDA). An ocular irritation test and cell viability tests were performed using the SIRC (Statens Seruminstitut Rabbit Cornea) cell culture line. Results indicated that all the in situ gels were within an acceptable pH and refractive index range close to corneal properties. Rheology studies have shown that the gelation temperature varies between 25 and 32 °C, depending on the amount of poloxamers. The release studies have shown that the release of 10-HDA from in situ gels is more sustained than royal jelly suspension. All gel formulations were non-irritant according to the short-time exposure test (STE) using the SIRC cell culture line, and long-term cell viability studies indicated that the formulations used in small concentrations did not induce cell death. Prepared in situ gels containing royal jelly have potential for ocular drug delivery, and they may improve the bioavailability, stability of royal jelly, and formation of non-irritant ocular formulations.

Intensification of Enzymatic Synthesis of Decyl Oleate Using Ultrasound in Solvent Free System: Kinetic, Thermodynamic and Physicochemical Study.

The present study evaluates the potential use of ultrasound irradiation to synthesize decyl oleate using Fermase CALBTM10000 under the solvent-free system (SFS). The optimal condition to achieve a maximum yield of 97.14% was found to be 1:2 oleic acid:decanol ratio, 1.8% (w/w) enzyme loading, 45°C temperature, 200 rpm agitation speed, 50 W power input, 50% duty cycle, 22 kHz frequency and reaction time of 25 minutes. The thermodynamic study was done to determine the change in entropy, Gibb's free energy, and change in enthalpy at various temperatures. The experimental results and kinetic study showed that the reaction followed ordered bi-bi model with kinetic parameters as rate of reaction (V max ) = 35.02 M/min/g catalyst, Michaelis constant for acid (K A ) = 34.47 M, Michaelis constant for alcohol (K B ) = 3.31 M, Inhibition constant (Ki) = 4542.4 M and sum of square error (SSE) = 0.000334. The application of ultrasound irradiation combined with biocatalyst and the absence of solvent intensified the process compared to the traditional stirring method using hexane as solvent.

Phase-Transition Thermal Charging of a Channel-Shape Thermal Energy Storage Unit: Taguchi Optimization Approach and Copper Foam Inserts.

Thermal energy storage is a technique that has the potential to contribute to future energy grids to reduce fluctuations in supply from renewable energy sources. The principle of energy storage is to drive an endothermic phase change when excess energy is available and to allow the phase change to reverse and release heat when energy demand exceeds supply. Unwanted charge leakage and low heat transfer rates can limit the effectiveness of the units, but both of these problems can be mitigated by incorporating a metal foam into the design of the storage unit. This study demonstrates the benefits of adding copper foam into a thermal energy storage unit based on capric acid enhanced by copper nanoparticles. The volume fraction of nanoparticles and the location and porosity of the foam were optimized using the Taguchi approach to minimize the charge leakage expected from simulations. Placing the foam layer at the bottom of the unit with the maximum possible height and minimum porosity led to the lowest charge time. The optimum concentration of nanoparticles was found to be 4 vol.%, while the maximu possible concentration was 6 vol.%. The use of an optimized design of the enclosure and the optimum fraction of nanoparticles led to a predicted charging time for the unit that was approximately 58% shorter than that of the worst design. A sensitivity analysis shows that the height of the foam layer and its porosity are the dominant variables, and the location of the porous layer and volume fraction of nanoparticles are of secondary importance. Therefore, a well-designed location and size of a metal foam layer could be used to improve the charging speed of thermal energy storage units significantly. In such designs, the porosity and the placement-location of the foam should be considered more strongly than other factors.

Melting phase change heat transfer in a quasi-petal tube thermal energy storage unit.

In the present study, the thermal energy storage of a hot petal tube inside a shell-tube type Thermal Energy Storage (TES) unit was addressed. The shell is filled with the capric acid Phase Change Material (PCM) and absorbs the heat from a hot U-tube petal. The governing equations for the natural convection flow of molten PCM and phase change heat transfer were introduced by using the enthalpy-porosity approach. An automatic adaptive mesh scheme was used to track the melting interface. The accuracy and convergence of numerical computations were also controlled by a free step Backward Differentiation Formula. The modeling results were compared with previous experimental data. It was found that the present adaptive mesh approach can adequately the melting heat transfer, and an excellent agreement was found with available literature. The effect of geometrical designs of the petal tube was investigated on the melting response of the thermal energy storage unit. The phase change behavior was analyzed by using temperature distribution contours. The results showed that petal tubes could notably increase the melting rate in the TES unit compared to a typical circular tube. Besides, the more the petal numbers, the better the heat transfer. Using a petal tube could increase the charging power by 44% compared to a circular tube. The placement angle of the tubes is another important design factor which should be selected carefully. For instance, vertical placement of tubes could improve the charging power by 300% compared to a case with the tubes' horizontal placement.

Prebiotic Peptide Synthesis and Spontaneous Amyloid Formation Inside a Proto-Cellular Compartment.

Cellular life requires a high degree of molecular complexity and self-organization, some of which must have originated in a prebiotic context. Here, we demonstrate how both of these features can emerge in a plausibly prebiotic system. We found that chemical gradients in simple mixtures of activated amino acids and fatty acids can lead to the formation of amyloid-like peptide fibrils that are localized inside of a proto-cellular compartment. In this process, the fatty acid or lipid vesicles act both as a filter, allowing the selective passage of activated amino acids, and as a barrier, blocking the diffusion of the amyloidogenic peptides that form spontaneously inside the vesicles. This synergy between two distinct building blocks of life induces a significant increase in molecular complexity and spatial order thereby providing a route for the early molecular evolution that could give rise to a living cell.

Chelation Crosslinking of Biodegradable Elastomers.

Widely present in nature and in manufactured goods, elastomers are network polymers typically crosslinked by strong covalent bonds. Elastomers crosslinked by weak bonds usually exhibit more plastic deformation. Here, chelation as a mechanism to produce biodegradable elastomers is reported. Polycondensation of sebacic acid, 1,3-propanediol, and a Schiff-base (2-[[(2-hydroxyphenyl) methylene]amino]-1,3-propanediol) forms a block copolymer that binds several biologically relevant metal ions. Chelation offers a unique advantage unseen in conventional elastomer design because one ligand binds multiple metal ions, yielding bonds of different strengths. Therefore, one polymeric ligand coordinated with different metal ions produces elastomers with vastly different characteristics. Mixing different metal ions in one polymer offers another degree of control on material properties. The density of the ligands in the block copolymer further regulates the mechanical properties. Moreover, a murine model reveals that Fe3+ crosslinked foam displays higher compatibility with subcutaneous tissues than the widely used biomaterial-polycaprolactone. The implantation sites restore to their normal architecture with little fibrosis upon degradation of the implants. The versatility of chelation-based design has already shown promise in hydrogels and highly stretchy nondegradable polymers. The biodegradable elastomers reported here would enable new materials and new possibilities in biomedicine and beyond.

A modified quick, easy, cheap, effective, rugged, and safe method with hydrophobic natural deep eutectic solvent as extractant and analyte protectant for analyzing pyrethroid residues in tomatoes.

In this work, a novel quick, easy, cheap, effective, rugged, and safe technique with hydrophobic natural deep eutectic solvent as both extractant and analyte protectant was developed and combined with gas chromatography-tandem mass spectrometry to analyze pyrethroid residues in tomatoes. Eight hydrophobic natural deep eutectic solvents were first evaluated as analyte protectants and those with decanoic acid or lactic acid as hydrogen bond donor were demonstrated to be effective in compensating for the matrix effects of pyrethroids in the gas chromatography system. Hence, they were added to solvent standards for correcting the quantitation errors instead of matrix-matched calibration standards. Then the abilities of these acid-based deep eutectic solvents to extract pyrethriods from tomatoes were evaluated. Results showed the recoveries of all pyrethroids reached to over 80% with only 5 mL menthol:decanoic acid (1:1) used, and good phase separation was easily achieved without the addition of inorganic salt in the extraction step, indicating hydrophobic natural deep eutectic solvent could be a green substitute for acetonitrile in the quick, easy, cheap, effective, rugged, and safe extraction. Compared with the conventional method, the proposed protocol improved the recoveries, reduced the matrix effects, and simplified the extraction step, demonstrating to be an effective, fast, and green method.

Hydrophobic deep eutectic solvent as a green extractant for high-performance liquid chromatographic determination of tetracyclines in water samples.

A green extractant, hydrophobic deep eutectic solvent was first introduced for extraction of tetracycline, oxytetracycline, and chlortetracycline from environmental water samples prior to high-performance liquid chromatography determination. Deep eutectic solvents consist of methyltrioctylammonium chloride and various medium-chain alcohols/acids, and are easy in preparation, low cost and toxicity, desirably biodegradable, and biocompatible. The overall time required for sample preparation was 6 min and the volume of organic solvent used for extraction was only 400 µL. Under the optimized extraction condition, the present method yielded low limit of detection (0.5-2.0 ng/mL), acceptable precision (relative standard deviations < 9.7%), good linearity from 2.0 to 500 ng/mL (r2  ≥ 0.9991). This optimized procedure was applied for determination of tetracyclines in different water samples with desirable spiked recovery ranged from 77.5 to 87.6%. There is, therefore, a great potential to further expand application of the method for investigation of other ultra-trace analyte(s) in environmental matrixes.