TY - JOUR
AU - Yang,, Yaling
AB - Abstract An environmentally friendly method for the determination of testosterone and methyltestosterone by acid–base-induced deep eutectic solvents liquid–liquid microextraction (DES-ABLLME) combining with high-performance liquid chromatography was established. The deep eutectic solvent (DES) consisting of menthol:lauric acid:decanoic acid (3:1:1) can act as both hydrogen bond donor and hydrogen bond acceptor. In this approach, ammonia solution (NH3•H2O) is used as an emulsifier to react with DESs in the extraction process to generate salt and form milky white solution, achieving high extraction efficiency. Hydrochloric acid was used as a phase separator to change the emulsification state and promote the separation of extraction agent from water phase. A series of parameters were optimized including the volume of DES and the emulsifying agent, glucose concentration as well as hydrochloric acid volume. The method was linear in the range 0.5–100 μg mL−1 with a correlation coefficient (R) of 0.9999, and the limits of detection were 0.067 and 0.2 μg mL−1 for testosterone and methyltestosterone, respectively. This method was applied to analyze testosterone and methyltestosterone in milk samples, and the recoveries were between 89.2 and 108.2%. Introduction Methyltestosterone was first synthesized in the 1950s (1); from then more and more steroid hormones are being synthesized. Steroid hormones can be classified generally in three groups: estrogens, gestagens and androgens by their chemical structure and their pharmacological effects. Steroids have many effects (2), such as anti-inflammatory, antipyretic, immunosuppressive and promoting muscle growth and strength. All androgenic hormones exert both masculinizing and anabolic effects (3). The effects of the hormones ingested in humans by consuming animal products containing steroid hormones are the same as those of endogenous hormones. The sex hormones found in animal products are generally generated spontaneously in their bodies (4). Some manufacturers and farmers abuse drugs in order to increase the weight of animals and milk production for higher commercial profits. This causes abnormally high levels of the sex hormones in the animals’ blood that spread through extracellular pathways into meat and milk (5). It is worth noticing that long-term overuse of steroids, even at low concentrations, can cause many health problems, including early puberty in children, internal and external genital abnormalities, polycystic ovary syndrome and breast cancer and uterine in women, and testicular and prostate cancer in men (6, 7). In order to protect public health, some steroid hormones are prohibited to use in animal husbandry to accelerate animal growth and improve feed conversion efficiency in many countries and regions (8). Many countries and regions and other international organizations have made strict requirements on the residues of hormones in food of animal origin. Milk is an important food in daily life; so the exact amount of steroids in milk has become a focus of attention (4, 6). A highly sensitive detection method is required to detect steroid concentrations. To date, some methods are already been built to detect estrogens in food such as immunological methods, chemiluminescence, high-performance liquid chromatography (HPLC), high-performance liquid chromatography–mass spectrometry (HPLC–MS) and liquid chromatography–tandem mass spectrometry (LC–MS), radioimmunoassay, gas chromatography–mass spectrometer (GC–MS) and gas tandem mass spectrometric methods (9–11). In recent years, some scholars have proposed a liquid–liquid microextraction (LLME) method with the advantages of convenient operation, low solvent consumption and high concentration coefficient (12). The development of an environmentally friendly innoxious solvent is of significance to the LLME technology and is attracting more and more attention (13). Recently, an environment friendly and economic solution, which is called deep eutectic solvent (DES) (13, 14), has been a new generation of ionic liquids. It was obtained by mixing two safe components that are able to form a eutectic mixture with a melting point lower than that of starting components (15). It has been increasingly used by scholars in the extraction of low levels of organic substances and inorganic compounds (16–18). DESs are composed of two or three inexpensive components of hydrogen bond interactions (19). They are the new class of solvents obtained by mixing compounds that are not necessarily salts, where a hydrogen bond connects the donor to the acceptor in accordance with specific molar ratios (20). These components should also be safe, low toxic, renewable, biodegradable and low cost. In the past decades, DESs have been widely used in extraction experiments (21, 22), photocatalysis (23), material synthesis (24, 25) and other fields (26, 27) because of its environmental friendliness and easy access as an alternative to conventional solvents. A group of natural hydrophobic DESs composed of different fatty acids were used in this study for the extraction of the testosterone and methyltestosterone in milk samples that were simply pretreated with acid and centrifugation. The prepared milk does not affect the testosterone and methyltestosterone by acid–base-induced deep eutectic solvents liquid–liquid microextraction (DES-ABLLME). The pH values of the aqueous phase were changed to implement the phase dispersion and separation of the fatty acids eutectics. Experimental section Reagents and solutions Testosterone, methyltestosterone, menthol, lauric acid, octanoic acid (c8), nonanoic acid (c9) and decanoic acid (c10), ammonia solution (NH3•H2O) (25% w/w)，hydrochloric acid (HCl) were obtained from Aladdin Industrial Corporation (Shanghai, China) and Tianjin Chemical Reagent Technology Co., Ltd (Tianjin, China). The other HPLC reagents (acetonitrile) purchased from Merck (Darmstadt, Germany) were of analytical grade and used without further purification. The commercial milk was purchased from a supermarket in Kunming, China. Instruments Vortex mixer (IKA Lab Dancer, China) and digital pH meter (Göttingen, Germany) were used in the experiment. Chromatographic analysis was carried out on an Agilent 1260 series HPLC system equipped with an UV–Vib diode array detector (Agilent Technologies, USA). The chromatographic separation was operated on an Agilent TC-C18 column (150 mm × 4.6 mm, 5 μm) at the column temperature of 35°C. The flow rate was 1.0 mL min−1, and the injection volume was 20 μL. The detection wavelength was set at 250 nm. The mobile phase consisted of 50% acetonitrile and 50% ultrapure water. Preparation process of DES Different kinds of fatty acids can be used as the hydrogen bond donor and the hydrogen bond acceptor in DES (20). A series of DESs were prepared by mixing two or three kinds of fatty acids, including menthol, lauric acid, octanoicacid (c8), nonanoic acid (c9) and decanoic acid (c10) in this work. The fatty acid with different molar ratios were mixed in a sealed glass vial at 60°C until a uniform transparent liquid have been formed. DES-ABLLME procedure In this work， the extraction process is demonstrated in Figure 1A. The first step is 5 mL of deionized water and 1 mL the testosterone (10 μg mL−1) and methyltestosterone (10 μg mL−1) standard solution or 5 mL samples were pipetted into 10 mL centrifuge tubes, and then 600 μL of fatty acids DESs (menthol:lauric acid:decanoic acid = 3:1:1) were injected. The second step is 600 μL of NH3•H2O (1 mol L−1) as an emulsifier agent was injected into the solution and vortex mixed for 60 s to form the uniform emulsion. The third step is 600 μL of HCl (1 mol L−1) was injected into the solution to separate the DES from the solution. After that, the solution was centrifuged at 4000 rpm for 10 min in order to ensure the good distribution of DES droplets in the aqueous phase; the testosterone and methyltestosterone were mostly extracted. The aqueous phase was discarded with a syringe, and finally 0.3 mL of DES was diluted to 0.6 mL with acetonitrile. At last，20 μL of the solution (filtered with 0.45-μm polyether sulfone filters) was analyzed by HPLC. Figure 1 Open in new tabDownload slide (A). Schematic Diagram of the presented DES-ABLLME procedure (B). HPLC chromatograms : (a) milk samples with DES-ABLLME and (b) milk samples spiked with standard testosterone and methyltestosterone with DES-ABLLME. Figure 1 Open in new tabDownload slide (A). Schematic Diagram of the presented DES-ABLLME procedure (B). HPLC chromatograms : (a) milk samples with DES-ABLLME and (b) milk samples spiked with standard testosterone and methyltestosterone with DES-ABLLME. Preparation of the sample solution The commercial milk was bought from a supermarket. Firstly, the milk was heated to 40°C by water bath and the pH of the milk samples was adjusted to ~4.5–5 by adding HCl (1 mol L−1). Secondly, the solution was transferred into polytetrafluoroethylene containers and centrifuged for 10 min at 4000 rpm to form condensed sediments. Thirdly, the resulting supernatant was filtered through a 0.45-μm microporous filter membrane and were subsequently processed by the DES-ABLLME method. Results Analytical performance Under the best experimental conditions, the linearity, limits of detection (LOD), limits of quantification (LOQ) and relative standard deviation (RSD) of this DES-ABLLME technique are evaluated (Table I). The calibration graphs are linear in the concentration and the linear dynamic range (LDR) are from 0.5 to 100 μg mL−1 for testosterone and methyltestosterone; the correlation coefficients are >0.9999 for testosterone and methyltestosterone. The LODs calculated at the signal-to-noise ratio of 3 are 0.067 and 0.2 μg mL−1 for testosterone and methyltestosterone, respectively. Table I Indicators of the Experimental Method Evaluation (n = 5) Sample . Linear regression equation . Correlation coefficient . LDR (μgmL−1) . Recovery (%) . LOD (μg mL−1) . LOQ (μg mL−1) . Testosterone y = 63.049x−30.93 0.9999 0.5–100 97.7–108.2 0.067 0.22 Methyltestosterone y = 62.362x−34.01 0.9999 0.5–100 89.2–99.1 0.2 0.67 Sample . Linear regression equation . Correlation coefficient . LDR (μgmL−1) . Recovery (%) . LOD (μg mL−1) . LOQ (μg mL−1) . Testosterone y = 63.049x−30.93 0.9999 0.5–100 97.7–108.2 0.067 0.22 Methyltestosterone y = 62.362x−34.01 0.9999 0.5–100 89.2–99.1 0.2 0.67 LDR, linear dynamic range. Open in new tab Table I Indicators of the Experimental Method Evaluation (n = 5) Sample . Linear regression equation . Correlation coefficient . LDR (μgmL−1) . Recovery (%) . LOD (μg mL−1) . LOQ (μg mL−1) . Testosterone y = 63.049x−30.93 0.9999 0.5–100 97.7–108.2 0.067 0.22 Methyltestosterone y = 62.362x−34.01 0.9999 0.5–100 89.2–99.1 0.2 0.67 Sample . Linear regression equation . Correlation coefficient . LDR (μgmL−1) . Recovery (%) . LOD (μg mL−1) . LOQ (μg mL−1) . Testosterone y = 63.049x−30.93 0.9999 0.5–100 97.7–108.2 0.067 0.22 Methyltestosterone y = 62.362x−34.01 0.9999 0.5–100 89.2–99.1 0.2 0.67 LDR, linear dynamic range. Open in new tab Table II shows a comparison of the proposed method with other methods in the literature (28–31). As can be seen, this method has high recovery and simple pretreatment. The most noteworthy point is that no toxic solvents were used in DES-ABLLME process. Table II Comparison of the Proposed Method with Reported Methods for the Extraction of Androgen Compounds in Biological Samples Analytical system . Sample preparation . Matrix . Compounds . LOD . Recovery (%) . References . GC–MS SPME MIP Urine, Water ADR, STAN, ADD, MeT, T 0.02–0.1 ng mL−1 80.1–108.4 (28) LC–MS In-tube SPME Urine BOLD, NDL, T, MeT, EADR, ADR, STAN 9–182 pg mL−1 85.7–117.3 (29) LC–MS SBSEM Water NDL, T, DES, MeT, P, T propionate 0.036–0.068 ng mL−1 n.a. (30) LC–DAD SBSEM Wastewater NT, P, DES, T, MeT, NDL phenylpropionate 0.14–0.26 ng−1mL 48.2–110 (31) HPLC–UV DES-ABLLME T, MeT 0.067–0.2 μg mL−1 89.2–108.2 This work Analytical system . Sample preparation . Matrix . Compounds . LOD . Recovery (%) . References . GC–MS SPME MIP Urine, Water ADR, STAN, ADD, MeT, T 0.02–0.1 ng mL−1 80.1–108.4 (28) LC–MS In-tube SPME Urine BOLD, NDL, T, MeT, EADR, ADR, STAN 9–182 pg mL−1 85.7–117.3 (29) LC–MS SBSEM Water NDL, T, DES, MeT, P, T propionate 0.036–0.068 ng mL−1 n.a. (30) LC–DAD SBSEM Wastewater NT, P, DES, T, MeT, NDL phenylpropionate 0.14–0.26 ng−1mL 48.2–110 (31) HPLC–UV DES-ABLLME T, MeT 0.067–0.2 μg mL−1 89.2–108.2 This work ADR, androsterone; STAN, stanolone; ADD, androstenedione; Met, methyltestosterone; T, testosterone; BOLD, boldenone; NDL, nandrolone; EADR, epiandrosterone, P, progesterone, NT, nortestosterone; DES, diethylstilbestrol; HPLC-DAD, high-performance liquid chromatography–diode array detection; n.a., not available; SPME MIP, solid phase microextraction molecular imprinted polymer; in-tube SPME, in-tube solid phase microextraction; SBSE, stir bar sorptive extraction; SBSEM, stir bar sorptive extraction on monolith material. Open in new tab Table II Comparison of the Proposed Method with Reported Methods for the Extraction of Androgen Compounds in Biological Samples Analytical system . Sample preparation . Matrix . Compounds . LOD . Recovery (%) . References . GC–MS SPME MIP Urine, Water ADR, STAN, ADD, MeT, T 0.02–0.1 ng mL−1 80.1–108.4 (28) LC–MS In-tube SPME Urine BOLD, NDL, T, MeT, EADR, ADR, STAN 9–182 pg mL−1 85.7–117.3 (29) LC–MS SBSEM Water NDL, T, DES, MeT, P, T propionate 0.036–0.068 ng mL−1 n.a. (30) LC–DAD SBSEM Wastewater NT, P, DES, T, MeT, NDL phenylpropionate 0.14–0.26 ng−1mL 48.2–110 (31) HPLC–UV DES-ABLLME T, MeT 0.067–0.2 μg mL−1 89.2–108.2 This work Analytical system . Sample preparation . Matrix . Compounds . LOD . Recovery (%) . References . GC–MS SPME MIP Urine, Water ADR, STAN, ADD, MeT, T 0.02–0.1 ng mL−1 80.1–108.4 (28) LC–MS In-tube SPME Urine BOLD, NDL, T, MeT, EADR, ADR, STAN 9–182 pg mL−1 85.7–117.3 (29) LC–MS SBSEM Water NDL, T, DES, MeT, P, T propionate 0.036–0.068 ng mL−1 n.a. (30) LC–DAD SBSEM Wastewater NT, P, DES, T, MeT, NDL phenylpropionate 0.14–0.26 ng−1mL 48.2–110 (31) HPLC–UV DES-ABLLME T, MeT 0.067–0.2 μg mL−1 89.2–108.2 This work ADR, androsterone; STAN, stanolone; ADD, androstenedione; Met, methyltestosterone; T, testosterone; BOLD, boldenone; NDL, nandrolone; EADR, epiandrosterone, P, progesterone, NT, nortestosterone; DES, diethylstilbestrol; HPLC-DAD, high-performance liquid chromatography–diode array detection; n.a., not available; SPME MIP, solid phase microextraction molecular imprinted polymer; in-tube SPME, in-tube solid phase microextraction; SBSE, stir bar sorptive extraction; SBSEM, stir bar sorptive extraction on monolith material. Open in new tab Accuracy of the DES-ABLLME method In order to evaluate the feasibility of the application of DES-ABLLME technology in practical samples, the addition/recovery test of real milk samples was carried out. The pH value of the sample was adjusted between 4.5 and 5, and the sample was filtered with a nylon membrane filter of 0.22 μm. In the subsequent experiments, the samples spiked with different concentration of testosterone and methyltestosterone were assessed by the proposed method. The mean value was calculated after five repeated experiments. The typical HPLC figures are shown in Figure 1B. “a” is the chromatographic figure of the milk sample with DES-ABLLME and “b” is the chromatographic figure of the milk sample spiked with standard testosterone and methyltestosterone with DES-ABLLME. The relevant data results are demonstrated in Table III. The recovery efficiencies of testosterone and methyltestosterone were 89.2–108.2%. The RSD is between 2 and 8%. According to the experimental data, satisfactory results can be obtained by applying this method to the preconcentration and determination of testosterone and methyltestosterone in milk samples. Table III Results of Determination of Testosterone and Methyltestosterone in the Milk Samples (n = 5) Sample . Androgen . Added (μg L−1) . Measured (μg L−1) . RSD (%) . Relative recovery (%) . A Testosterone 0 0 — — 12 1.18 6.3 98.7 24 2.5 4.3 104.1 50 5.11 2.9 102.2 Methyltestosterone 0 1.59 5.4 - 12 2.75 4.9 96.1 24 3.84 3.3 93.4 50 6.17 2 91.4 B Testosterone 0 0 — — 12 1.3 3.1 108.2 24 2.45 5.2 102.1 50 4.89 3.5 97.7 Methyltestosterone 0 1.64 5.4 — 12 2.77 5.3 94.4 24 4.02 4.4 99.1 50 6.49 5.6 97 C Testosterone 0 0 — — 12 1.19 8 99.2 24 2.52 4.6 105.1 50 5.07 4.3 101.5 Methyltestosterone 0 1.58 5.4 — 12 2.71 6.5 92.5 24 3.84 7.6 93.8 50 6.05 4.7 89.2 Sample . Androgen . Added (μg L−1) . Measured (μg L−1) . RSD (%) . Relative recovery (%) . A Testosterone 0 0 — — 12 1.18 6.3 98.7 24 2.5 4.3 104.1 50 5.11 2.9 102.2 Methyltestosterone 0 1.59 5.4 - 12 2.75 4.9 96.1 24 3.84 3.3 93.4 50 6.17 2 91.4 B Testosterone 0 0 — — 12 1.3 3.1 108.2 24 2.45 5.2 102.1 50 4.89 3.5 97.7 Methyltestosterone 0 1.64 5.4 — 12 2.77 5.3 94.4 24 4.02 4.4 99.1 50 6.49 5.6 97 C Testosterone 0 0 — — 12 1.19 8 99.2 24 2.52 4.6 105.1 50 5.07 4.3 101.5 Methyltestosterone 0 1.58 5.4 — 12 2.71 6.5 92.5 24 3.84 7.6 93.8 50 6.05 4.7 89.2 Open in new tab Table III Results of Determination of Testosterone and Methyltestosterone in the Milk Samples (n = 5) Sample . Androgen . Added (μg L−1) . Measured (μg L−1) . RSD (%) . Relative recovery (%) . A Testosterone 0 0 — — 12 1.18 6.3 98.7 24 2.5 4.3 104.1 50 5.11 2.9 102.2 Methyltestosterone 0 1.59 5.4 - 12 2.75 4.9 96.1 24 3.84 3.3 93.4 50 6.17 2 91.4 B Testosterone 0 0 — — 12 1.3 3.1 108.2 24 2.45 5.2 102.1 50 4.89 3.5 97.7 Methyltestosterone 0 1.64 5.4 — 12 2.77 5.3 94.4 24 4.02 4.4 99.1 50 6.49 5.6 97 C Testosterone 0 0 — — 12 1.19 8 99.2 24 2.52 4.6 105.1 50 5.07 4.3 101.5 Methyltestosterone 0 1.58 5.4 — 12 2.71 6.5 92.5 24 3.84 7.6 93.8 50 6.05 4.7 89.2 Sample . Androgen . Added (μg L−1) . Measured (μg L−1) . RSD (%) . Relative recovery (%) . A Testosterone 0 0 — — 12 1.18 6.3 98.7 24 2.5 4.3 104.1 50 5.11 2.9 102.2 Methyltestosterone 0 1.59 5.4 - 12 2.75 4.9 96.1 24 3.84 3.3 93.4 50 6.17 2 91.4 B Testosterone 0 0 — — 12 1.3 3.1 108.2 24 2.45 5.2 102.1 50 4.89 3.5 97.7 Methyltestosterone 0 1.64 5.4 — 12 2.77 5.3 94.4 24 4.02 4.4 99.1 50 6.49 5.6 97 C Testosterone 0 0 — — 12 1.19 8 99.2 24 2.52 4.6 105.1 50 5.07 4.3 101.5 Methyltestosterone 0 1.58 5.4 — 12 2.71 6.5 92.5 24 3.84 7.6 93.8 50 6.05 4.7 89.2 Open in new tab Discussion Hydrophobic DESs selection The solubility and dispersion of DESs are directly related to the extraction efficiency; so it is very important to choose the right extraction agent. Generally, the viscosity, hydrophobicity and melting point of DESs are considered as the main parameters in the extraction process. At first, the viscosity of DESs should be as low as possible. Second, DESs should be as hydrophobic as possible, so that its chemical stability can be guaranteed in contact with the emulsion. As described in the document (13), the longer the fatty acids chains are the higher hydrophobicity of resulting DESs. As a result, different carboxylic acids with ~8–10 carbon atoms in the main chain were selected as the initial materials for the composite of hydrophobic DESs compounds. The experimental results are shown in Figure 2. All the extraction agents have different degrees of extraction effect on target substances. Ternary fatty acid DESs was prepared from menthol, lauric acid and decanoic acid. The optimum molar ratio was determined to be 3:1:1 by the extraction efficiency of testosterone and methyltestosterone. As illustrated in Figure 2, comparing with the single fatty acid and binary fatty acids DESs, it has the maximum extraction efficiency (>95.6%). Figure 2 Open in new tabDownload slide The selection of DESs on recovery. (A: menthol: lauric acid = 1:1; B: menthol:lauric acid = 2:1; C: menthol: lauric acid = 3:1; D: menthol: lauric acid = 4:1; E: menthol: lauric acid: octanoic acid = 3:1:1; F: menthol: lauric acid: pelargonic acid = 3:1:1; G: menthol: lauric acid: decanoic acid. Figure 2 Open in new tabDownload slide The selection of DESs on recovery. (A: menthol: lauric acid = 1:1; B: menthol:lauric acid = 2:1; C: menthol: lauric acid = 3:1; D: menthol: lauric acid = 4:1; E: menthol: lauric acid: octanoic acid = 3:1:1; F: menthol: lauric acid: pelargonic acid = 3:1:1; G: menthol: lauric acid: decanoic acid. Effect of the DESs volume The DESs volume was one of the most important factors, because the volume of DESs has a direct impact on the enrichment factor. In order to optimize the DESs volume, different amounts of DES were used, such as 200, 300, 400, 500, 600, 700 and 800 μL. As can be seen from Figure 3, when the volume of DESs increased from 200 to 600 μL, the analysis signal showed an increasing trend. When the volume of DESs increases >600 μL, the analysis signal showed a stable state and hardly increased. Thus, to obtain the higher analytical signal and better reproducibility, 600 μL was selected as the optimal volume of DESs in this study. Figure 3 Open in new tabDownload slide Effect of the volume of DES on recovery. Figure 3 Open in new tabDownload slide Effect of the volume of DES on recovery. Effect of the NH3•H2O volume When DES is injected into the aqueous phase, a mixed solution is formed. However, it is very difficult to disperse the extractant phase in this mixture, which causes a decrease in the extraction efficiency of the target substance (32). In the present study, NH3•H2O was used as an emulsifier to react with DESs to form a cloudy solution. The absence of NH3•H2O in the solution caused a low recovery of the analyte; the reason of which was that part of the DESs could not be isolated from the sample solution, leading to the reduction of extraction agent. The amount of NH3•H2O volume added ranged from 200- to 800-μL. The effect of NH3•H2O volume on the extraction rates is illustrated in Figure 4. When the NH3•H2O volume exceeds 600 μL, the analysis signal does not increase significantly and remains almost unchanged. Therefore, 600 μL of NH3•H2O was chosen in the subsequent work. Figure 4 Open in new tabDownload slide Effect of NH3·H2O volume on recovery. Figure 4 Open in new tabDownload slide Effect of NH3·H2O volume on recovery. Effect of the glucose concentration Considering that glucose is a common component in milk, a series of experiments was carried out to examine the influence of glucose concentration on DES-ABLLME. Through the experiments, the conclusion that the amount of glucose in the sample has no significant effect on the extraction efficiency of the analyte was drawn. In this DES-ABLLME，a series of glucose amount ranging 5–25 mg mL−1 was checked. The results show (Figure 5) that the extraction efficiency remained almost the same whether the glucose concentration increased or not. The extraction efficiencies of analytes are not influenced by the glucose addition. Figure 5 Open in new tabDownload slide Effect of glucose concentration on recovery. Figure 5 Open in new tabDownload slide Effect of glucose concentration on recovery. Effect of hydrochloric acid volume In extraction studies, the volume of hydrochloric acid in an aqueous phase could be another parameter that can influence the extraction efficiency of an analyte by changing its existing state (13). In this work, the volume of hydrochloric acid was used and volume ranging 200–800 μL was checked. The results show (Figure 6) that the analytical signal is strongest when the volume is 200 μL. The reason for this phenomenon is that some eutectic solvent is not completely separated from the aqueous phase when the content of hydrochloric acid is insufficient. This leads to volume loss of some eutectic solvents, which leads to the extra high extraction rate. In subsequent experiments, 600 μL is selected as the optimal volume of hydrochloric acid. Figure 6 Open in new tabDownload slide Effect of the hydrochloric acid volume on recovery. Figure 6 Open in new tabDownload slide Effect of the hydrochloric acid volume on recovery. Conclusions In this research, a microextraction technique for the acid–base-induced eutectic solvent based on fatty acids was offered and applied to the extraction of testosterone and methyltestosterone from milk samples. 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TI - An Effective Acid–Base-Induced Liquid–Liquid Microextraction Based on Deep Eutectic Solvents for Determination of Testosterone and Methyltestosterone in Milk
JF - Journal of Chromatographic Science
DO - 10.1093/chromsci/bmaa051
DA - 2020-09-29
UR - https://www.deepdyve.com/lp/oxford-university-press/an-effective-acid-base-induced-liquid-liquid-microextraction-based-on-qxhl5RU09v
SP - 880
EP - 886
VL - 58
IS - 9
DP - DeepDyve
ER -