Ultrasound assisted extraction in quantifying lutein from chicken liver using high-performance liquid chromatography

Four sample preparation methods, (1) solvent (SOL), (2) saponification and solvent (SP), (3) ultrasound assisted solvent (UA), and (4) saponification and ultrasound assisted solvent (SP-UA), were used for quantifying lutein in chicken liver samples by HPLC. The lutein concentrations obtained by using SOL, UA, SP, and SP-UA were significantly different with values from 10.4 microg/g (UA) to undetected (SOL). Efficiency of the four different methods for extracting lutein from high to low were the UA, SP, SP-UA, and SOL method. The measured value of lutein in the liver sample using the UA method was approximately two and three times higher than that obtained from the SP and SP-UA method, respectively. The methods with saponification significantly affected the stabilities of lutein in liver samples. The lutein concentration measured with the solvent only method was either much lower than any of the other extraction methods or undetectable. This indicated that little lutein in those samples was in a form that could be extracted directly by solvent. Compared with the saponification method, the ultrasound assisted solvent method could effectively extract lutein from sample matrix and thus avoid chemical degradation reactions, which would be especially important for complex biological tissue such as liver.     

Microwave assisted extraction of ursolic acid and oleanolic acid from Ocimum sanctum

Present study deals with the microwave assisted extraction (MAE) of ursolic acid (UA) and oleanolic acid (OA) from Ocimum sanctum leaves. UA and OA have been reported to possess significant medicinal properties. Various experimental parameters such as selection of solvent, solvent composition, irradiation time, microwave power, solid to solvent ratio, preleaching time and number of cycles were investigated to optimize the extraction process. Under optimum conditions of irradiation time (3 min), microwave power (272 W), solid to solvent ratio (1:30), preleaching time (10 min), maximum UA and OA has been extracted in one extraction cycle with ethanol: water (80:20) as a solvent. Maximum 86.76 and 89.64% of UA and OA was extracted under above mentioned optimized experimental conditions. MAE was also compared with the batch and ultrasound assisted extraction (UAE) method. As compared to batch and UAE, higher extraction yield of these important phytochemicals have been obtained through MAE in only 3 min.     

萱草花瓣中类胡萝卜素分析样品的制备及UPCC-MS检测方法研究

以大苞萱草（Hemerocallis middendorfii Trautv.et Mey.）和‘原谅’萱草（H.‘Pardon Me’）为研究对象,对萱草属植物花瓣中类胡萝卜素的样品制备方法以及UPCC-MS定性和定量检测方法进行了研究。结果表明：（1）萱草花瓣类胡萝卜素样品制备过程中,不同的提取试剂、振荡方法及皂化方法对类胡萝卜素的提取效率均有显著影响,经过对提取结果的方差分析,确定最佳的样品制备方案为：提取试剂B丙酮：正己烷（3：5/V：V）、温控摇床振荡提取,常温皂化16 h;（2） UPCC-MS技术能在10 min内高效分离萱草花瓣中的类胡萝卜素,且使用的有毒化学试剂少,是检测类胡萝卜素的较好选择;（3）大苞萱草和原谅萱草花瓣中共含有20种类胡萝卜素物质,两者颜色不同,类胡萝卜素的组成和含量也存在差异。     

Determination of d-limonene in adipose tissue by gas chromatography-mass spectrometry

We developed a novel method for analyzing d-limonene levels in adipose tissue. Fat samples were subjected to saponification followed by solvent extraction. d-Limonene in the sample extract was analyzed using gas chromatography-mass spectrometry (GC-MS) with selected ion monitoring. Linear calibration curves were established over the mass range of 79.0-2529 ng d-limonene per 0.1g of adipose tissue. Satisfactory within-day precision (R.S.D. 6.7-9.6%) and accuracy (%difference of -2.7 to 3.8%) and between-day precision (R.S.D. 6.0-10.7%) and accuracy (%difference of 1.8-2.6%) were achieved. The assay was successfully applied to human fat biopsy samples from a d-limonene feeding trial.     

Novel HPLC analysis of tocopherols, tocotrienols, and cholesterol in tissue

Tocopherols and tocotrienols are being increasingly recognized to have an important role in the prevention of atherosclerosis. It has been reported that they protect low-density lipoprotein (LDL) and tissues from oxidative stress and that tocotrienols can reduce plasma cholesterol levels. Two isocratic high-performance liquid chromatography (HPLC) methods for simultaneous analysis of tocopherols, tocotrienols, and cholesterol in muscle tissue were developed. Method A involves basic saponification of the sample, but causes losses of the gamma- and delta-homologs of vitamin E. Method B does not involve saponification, thereby protecting the more sensitive homologs. Both permit rapid analysis of multiple samples and neither requires specialized equipment. These methods may provide techniques useful in simultaneous assessment of oxidative stress status (OSS) and cholesterol levels.     

Improved simultaneous determination method of beta-carotene and retinol with saponification in human serum and rat liver

Among the many simultaneous determination methods for carotenoid and retinoid, there are only a few reports including the saponification process. However, the yields of beta-carotene and retinol were higher when using this process. In this study, the analytical conditions, including saponification, were investigated. The extraction solvent was n-hexane and the sample solvent was HPLC mobile phase in the beta-carotene and retinol analysis. BHT as an antioxidant was added at concentrations of 0.125 and 0.025%, respectively, to ethanol and n-hexane phase in the extraction process for serum. The recovery rates were 99.7, 93.7 and 98.3% for beta-carotene, retinol and retinyl palmitate in serum, respectively, and 107.1, 92.8 and 98.8% for beta-carotene, retinol and retinyl palmitate in liver, respectively. The within-day coefficients of variation (C.V.) were 6.0% for serum and 4.7% for liver in the case of beta-carotene, 7.1% for serum, and 5.1% for liver in the case of retinol. The between-day coefficients of variation were 2.7% for serum and 2.7% for liver in the case of beta-carotene, and for retinol, 6.4% for serum and 2.7% for liver.     

A high-throughput method for liquid chromatography–tandem mass spectrometry determination of plasma alkylresorcinols,biomarkers of whole grain wheat and rye intake

Plasma alkylresorcinols are increasingly analyzed in cohort studies to improve estimates of whole grain intake and their relationship with disease incidence. Current methods require large volumes of solvent (>10 ml/sample) and have relatively low daily sample throughput. We tested five different supported extraction methods for extracting alkylresorcinols from plasma and improved a normal-phase liquid chromatography coupled to a tandem mass spectrometer method to reduce sample analysis time. The method was validated and compared with gas chromatography–mass spectrometry analysis. Sample preparation with HybridSPE supported extraction was most effective for alkylresorcinol extraction, with recoveries of 77–82% from 100 μl of plasma. The use of 96-well plates allowed extraction of 160 samples per day. Using a 5-cm NH₂ column and heptane reduced run times to 3 min. The new method had a limit of detection and limit of quantification equivalent to 1.1–1.8 nmol/L and 3.5–6.1 nmol/L plasma, respectively, for the different alkylresorcinol homologues. Accuracy was 93–105%, and intra- and inter-batch precision values were 4–18% across different plasma concentrations. This method makes it possible to quantify plasma alkylresorcinols in 100 μl of plasma at a rate of at least 160 samples per day without the need for large volumes of organic solvents.     

Isolation of chlorophylls and carotenoids from freshwater algae using different extraction methods

Usually marine algae are an excellent source of pigments for different commercial sectors. Freshwater macroalgae can be exploited as a good source of biologically active compounds provided an appropriate extraction method is developed. The efficiency of four methods, like microwave‐assisted (MAE), ultrasound‐assisted extraction (UAE), supercritical fluid extraction (SFE) with ethanol as a co‐solvent, as well as conventional Soxhlet extraction were studied in the same conditions (time, solvent and temperature) for the recovery of chlorophylls and carotenoids from three freshwater green algae species: Cladophora glomerata, Cladophora rivularis and Ulva flexuosa. UV‐Vis spectrophotometry was used to determine chlorophyll a, chlorophyll b and total carotenoid content in obtained extracts. The results of this study showed that the advantages of novel extraction techniques (MAE and UAE) include higher yield and, in consequence, lower costs compared to traditional solvent extraction techniques. These methods were much more efficient in freshwater green algae pigment recovery than the classic Soxhlet extraction as well as SFE.     

Determination of hydroxy metabolites of polychlorinated biphenyls in plasma and tissue by gas chromatography/mass spectrometry

This study examines a novel sample preparation method for the determination of 11 hydroxy metabolites of polychlorinated biphenyls (PCBs) in plasma and organ tissues, followed by gas chromatography with mass spectrometric detection (GC/MS). The clean-up method was optimized to eliminate the interference matter by using a silica column and 10 mL of n-hexane/dichloromethane (4:6, v/v) as an eluent. Solid-phase and solvent extraction procedures were used for the plasma and tissues samples, respectively. Compared to C(18) and C(8) solid-phase, C(2) showed higher extraction efficiency with n-hexane as the eluent for plasma. The hydroxy-PCB extraction recoveries achieved with this combined extraction and clean-up procedure from plasma ranged from 87 to 117%, while those from tissues ranged from 82 to 111%. The linear detector responses for propyl derivatives of hydroxy-PCBs were obtained with the coefficients of determination varying from 0.992 to 0.998 in the concentration range of 0.1-20 ng mL(-1). The method detection limits ranged from 0.1 to 0.5 ng mL(-1) in 1 mL of plasma and from 0.1 to 0.5 ng g(-1) in 1g of tissues. This procedure was successfully applied to the study of 3-OH-2,3',4,4',5-PeCB in rat plasma and liver samples after intraperitoneal injection (20 mg/kg) of 2,3',4,4',5-PeCB.     

A new and simple solid-phase extraction method for LC determination of pyronaridine in human plasma

A new approach using a simple solid-phase extraction technique has been developed for the determination of pyronaridine (PND), an antimalarial drug, in human plasma. After extraction with C18 solid-phase sorbent, PND was analyzed using a reverse phase chromatographic method with fluorescence detection (at lambda(ex)=267 nm and lambda(em)=443 nm). The mean extraction recovery for PND was 95.2%. The coefficient of variation for intra-assay precision, inter-assay precision and accuracy was less than 10%. The quantification limit with fluorescence detection was 0.010 microg/mL plasma. The method described herein has several advantages over other published methods since it is easy to perform and rapid. It also permits reducing both, solvent use and sample preparation time. The method has been used successfully to assay plasma samples from clinical pharmacokinetic studies.     

Algae as production systems of bioactive compounds

Algal extracts are gaining increasing interest due to their unique composition and possibilities of wide industrial applications. Various extraction techniques are used for conversion of algal biomass into extracts. Recently, attention of scientists has been paid to novel methods, such as enzyme‐assisted extraction, microwave‐assisted extraction, pressurized liquid extraction, supercritical fluid extraction, and ultrasound‐assisted extraction, which enable the extraction of biologically active compounds without their degradation. In this review, the properties of biologically active compounds extracted from the biomass of algae reported in the literature are presented in a structured way. Algal extracts contain compounds such as carbohydrates, proteins, minerals, oil, fats, polyunsaturated fatty acids as well as bioactive compounds such as antioxidants (polyphenols, tocopherols [vitamin E], vitamin C, mycosporine‐like amino acids), and pigments, such as carotenoids (carotene xanthophyll), chlorophylls, and phycobilins (phycocyanin, phycoerythrin), which possess antibacterial, antiviral, antifungal, antioxidative, anti‐inflammatory, and antitumor properties. Finally, we assemble a list of applications of algal extracts in different developing branches of agriculture (biostimulants, bioregulators, feed additives) and in pharmaceutical industry.     

Ultrasound-assisted extraction of flaxseed oil using immobilized enzymes

An aqueous enzymatic process assisted by ultrasound extraction (AEP-UE) was applied to the extraction of oil from flaxseed (Linum usitatissimum L.). The highest oil recovery of 68.1% was obtained when ground flaxseed was incubated with 130 U/g of cellulase, pectinase, and hemicellulase for 12 h, at 45 °C and pH 5.0. The IC₅₀ values of oil obtained by AEP-UE and organic solvent extraction (OSE), as measured by DPPH scavenging activity essay, were 2.27 mg/mL and 3.31 mg/mL. The AEP-UE-derived oil had a 1.5% higher content of unsaturated fatty acids than the OSE-derived oil. AEP-UE is therefore a promising environmentally friendly method for large-scale preparation of flaxseed oil.     

Rapid preparation of human urine and plasma samples for analysis of F2-isoprostanes by gas chromatography-mass spectrometry

Reliable MS-based methods have been developed for the measurement of free and esterified F2-isoprostanes. However, prior to sample analysis several steps of purification, including solid-phase extraction followed by TLC or HPLC, are usually required, making it tedious to analyze large sample numbers, e.g., for population studies. We report a quick sample purification method using anion exchange solid phase extraction (SPE), which is highly selective for acidic compounds. Urine and hydrolyzed plasma of healthy individuals were acidified before SPE extraction, washed with 4 different solvent mixtures and finally eluted with ethyl acetate. The eluted samples were first derivatized with pentafluorobenzyl bromide followed by a second derivatization with bis-(trimethylsilyl)trifluoroacetamide. F2-isoprostanes were analyzed by GC-MS-NCI. The method was highly sensitive; the limit of detection at 5:1 signal-to-noise ratio was 0.037 ng/ml and 0.007 ng/mg creatinine for plasma and urine, respectively. Anion exchange SPE extraction for F2-isoprostane showed recovery of 55-65% and high linearity for concentration 0-1.0 ng/ml for urine (CV=4.08%, r2=0.990) and 0-0.5 ng/ml for plasma (CV=4.07%, r2=0.998). Fasting for 6h significantly increased plasma F2-isoprostanes levels, which has implications for the design of intervention studies using this biomarker.     

The simple and sensitive measurement of malondialdehyde in selected specimens of biological origin and some feed by reversed phase high performance liquid chromatography

A method for the determination of malondialdehyde (MDA) concentrations in specimens of animal tissues and feed has been developed using high performance liquid chromatography. The MDA concentration in acidified urine samples was determined after its conversion with 2,4-dinitrophenylhydrazine (DNPH) to a hydrazone (MDA-DNPH). Samples of blood plasma, muscle, liver and feed were prepared by saponification followed by derivatisation with DNPH to MDA-DNPH. The MDA concentration in chicken and hen feed samples was analysed after saponification and derivatisation followed by extractions with hexane. The free MDA in plasma samples was determined after deproteinization followed by derivatisation of MDA with DNPH. The chromatographic separation of MDA-DNPH samples was conducted using Phenomenex C(18)-columns (Synergi 2.5 μm, Hydro-RP, 100 ?, the length of 100mm) with an inner diameter of 2 or 3mm. MDA in processed biological samples was analysed using a linear gradient of acetonitrile in water, and the photodiode detector was set to 307 or 303 nm for detection. The current method that was utilised was based on the high-efficient derivatisation of MDA and was more sensitive compared to previously used methods. The selective and sensitive photodetection of the column effluent was found to be suitable for the routine analysis of MDA in urine, plasma, muscles and liver of animals and some feed samples. Because urine or blood plasma samples can be derivatised in a simple manner, the proposed method can also be suitable for the routine, non-invasive evaluation of oxidative stress in animals and humans.     

Extraction of astaxanthin from Haematococcus pluvialis by supercritical carbon dioxide fluid with ethanol modifier

Conventional solvent extraction methods cannot attain high‐quality antioxidant extracts from microalgae and also require solvent recovery and posttreatment. In this study, we utilized environmental friendly supercritical carbon dioxide fluid extraction (SFE‐CO₂) techniques to obtain pigment (i.e. astaxanthin) from Haematococcus pluvialis. The effects of key operating parameters on the extraction efficiency of astaxanthin were investigated, giving an optimal condition of H. pluvialis weight, 6.5 g; CO₂‐flow rate, 6.0 NL/min; extraction time, 20 min; extraction pressure, 4500 psi; volume of ethanol modifier added, 9.23 mL/g; extraction temperature, 50°C; modifier composition, 99.5%. Under these optimum conditions, the astaxanthin yield was 73.9% (10.92 mg/g dry H. pluvialis powder) after eight cycle of extraction cycles. The saponification index (C_S/C₀, representing the ratio of astaxanthin concentration after and before the saponification procedures) of the extract could be increased from 1 to 12.78 by saponification with 3.5 M NaOH.     

Rapid techniques for the extraction of vitamin E isomers from Amaranthus caudatus seeds: ultrasonic and supercritical fluid extraction

Supercritical fluid extraction (SFE) of seeds of Amaranthus caudatus (Amaranthaceae) and the use of ultrasound as a co-adjuvant in the extraction process were compared with methods traditionally used in the extraction of tocopherols and fatty acids. The use of readily available ultrasound equipment as an adjunct to the classical methods employed for the extraction of tocols provided qualitatively acceptable results more rapidly and more economically. SFE gave quantitatively better yields in shorter times, with solvent-free extracts obtained under conditions that minimised the degradation of thermolabile components. No significant variations were observed in the profile of the fatty acids extracted from amaranth oil by SFE or other methods, thus confirming the qualitative comparability of the faster supercritical extraction with the more time-consuming classical techniques even when processed with the aid of ultrasound.     

Direct liquid chromatography method for retinol, alpha- and gamma-tocopherols in rat plasma

An HPLC method for Vitamins A and E in rat plasma has been developed. The main goals of the method are the small amount of sample, 50 microl, and the direct extraction of analytes in one step with acetone, which is a solvent compatible with the reverse-phase mobile phases. Recoveries, as compared with classical and more tedious methods, were near 100%. The method employs a Supelco Discovery C18 column and methanol/water (95:5, v/v) as mobile phase. After being developed, the method was validated following ICH guidelines, with UV, fluorescence and electrochemical detectors. It proved to be selective, lineal, accurate and precise. This method greatly simplifies sample treatment and that is a critical point when working with a large number of samples.     

Ultrasound-assisted ionic liquid dispersive liquid-liquid microextraction coupled with high performance liquid chromatography for sensitive determination of trace celastrol in urine

Ultrasound-assisted ionic liquid dispersive liquid-liquid microextraction (UA IL-DLLME) coupled with high-performance liquid chromatography (HPLC) has been developed for the determination of celastrol in human urine samples. In the microextraction procedure, ionic liquid (IL) was used as extraction solvent and dispersed into the aqueous sample solution as fine droplets by means of dispersive solvent and ultrasonication which promoted the analyte to migrate into IL phase more easily. Several important parameters affecting the extraction efficiency were studied and optimized, including the type and volume of extraction solvent and dispersive solvent, sample pH, ultrasonication time, cooling time, centrifugation time and salting-out effect. Under the optimized conditions, 110-fold enrichment factor was obtained and the limit of detection (LOD) was 1.6 μg/L at a signal-to-noise ratio of 3. The calibration curve was linear over the range of 10-1000 μg/L for celastrol in human urine sample, with a correlation coefficient of 0.9980. Intra- and inter-assay precision were 0.43% and 2.78%, respectively. The proposed method was successfully applied to the real human urine samples and good spiked recoveries in the range of 93.2-109.3% were obtained.     

Simplified methods for the examination of liver lipids: I determination of vitamin A in liver using a direct solvent extraction technique

A solvent extraction procedure employing acetone and light petroleum, in equal amounts, at room temperature, is described for the determination of vitamin A products in liver. Vitamin A may be determined as a gross value on the solvent residue using the trichloroacetic acid colorimetric method. Elimination of saponification permits the estimation of the ratio of ester to alcohol after their separation by paper chromatography of the solvent residue. Ultraviolet spectrophotometry may be used to check the identity of the eluted bands of vitamin A and other products.     

不同提取溶剂和方法组合从落叶松毛虫蛹中提取的脂肪酸成分和含量的比较

落叶松毛虫Dendrolimus superans (Butler)蛹个体较大, 具有很高利用价值。为明确东北落叶松毛虫蛹中脂肪酸成分, 探讨最佳提取溶剂和提取方法的组合, 分别以正己烷、 石油醚和乙醚为提取溶剂, 结合超声波振荡萃取法、 索氏萃取法及溶剂萃取方法热浸和冷浸4种提取方法提取落叶松毛虫蛹油, 并采用毛细管色谱-质谱法分析提取物的脂肪酸种类和相对含量。结果表明： 正己烷溶剂与4种提取方法的组合中, 溶剂萃取热浸法提取率最高, 为25.60%。索氏萃取及溶剂萃取方法热浸和冷浸均检测到10种脂肪酸, 正己烷-超声波振荡萃取组合检测到9种脂肪酸。石油醚溶剂与4种提取方法的组合中, 索氏萃取提取率最高, 为29.31%, 均检测到10种脂肪酸。乙醚溶剂与4种提取方法的组合中, 溶剂萃取冷浸法提取率最高, 为29.11%, 检测到的脂肪酸种类为溶剂萃取冷浸法（13种）＞索氏萃取法（12种）＞溶剂萃取热浸法（11种）＞超声波振荡萃取法（9种）。在检测到的总脂肪酸中, 63%以上为不饱和脂肪酸, 其含量受提取溶剂和方法的影响不大。因此, 适合东北落叶松毛虫蛹中脂肪酸提取的最佳组合为石油醚溶剂 索氏萃取法。