Rapid determination of natural and synthetic hormones in biosolids and poultry manure by isotope dilution GC–MS/MS

The release of hormones into the environment due to land application of biosolids and manure is a cause of concern for their potential impacts. This paper presents the development of a rapid and sensitive method, based on extraction, for the analysis of 13 hormones in biosolids and poultry manure. A simultaneous derivatization of hydroxyl and ketone groups was carried out for the determination of hormones by GC–MS/MS. The method was validated in three matrices (sewage sludge, manure, and broiler litter). Recoveries from spiked samples at three concentration levels (50, 25, and 10 ng/g) ranged from 76 to 124% with relative SDs ≤ 16%. Method detection limits for the three matrices were in the range of 0.5–3.0 ng/g dry weight. The optimized method was applied to biosolid and poultry manure samples collected in Spain. Only seven of the 13 studied hormones were detected in the different samples. trans‐Androsterone was detected at high levels (up to 3.1 μg/g in biosolid samples). Estrone and estradiol were the two hormones detected at higher levels in layer manure, whereas estrone and 4‐androstene‐3,17‐dione presented the highest levels in broiler litter.     

Analysis of emerging organic contaminants in poultry manure by gas chromatography–tandem mass spectrometry

A multiresidue method was developed for the determination of 19 emerging organic contaminants (pharmaceutical drugs, personal care products, and bisphenol A) in poultry manure. Lyophilized samples of manure were extracted by ultrasound‐assisted matrix solid‐phase dispersion and the extracts were analyzed by gas chromatography with tandem mass spectrometry after derivatization. Analysis of spiked poultry manure samples, at levels ranging from 25 to 150 ng/g, gave satisfactory recovery results for all the compounds, with values from 67 to 106%. The developed procedure provided detection limits that ranged from 0.9 to 2.2 ng/g. Finally, the validated method was applied to poultry manure samples collected from 23 poultry farms in Spain. Salicylic acid was found in most of the samples analyzed at levels up to 2501 ng/g, whereas, methyl paraben, orthophenylphenol, ibuprofen, paracetamol, and carbamazepine were detected at levels up to 250 ng/g. Composting of manure showed an important decrease in the levels of the detected contaminants.     

Ultra‐performance liquid chromatography MS/MS method for the determination of parabens in compost from sewage sludge: Comparison of the efficiency of two extraction techniques

The efficiency of two extraction techniques—ultrasound‐assisted extraction and pressurized liquid extraction—are compared and evaluated in the determination of parabens in compost samples. The extraction parameters for each technique were accurately optimized. The selected compounds were detected and quantified using ultra‐performance LC MS/MS, operating in negative ESI and in SRM mode. The analytes were separated in less than 5 min. Ethylparaben (ring‐¹³C₆ labeled) was used as an internal standard. Two selective, sensitive, and accurate analytical methods were developed and validated. The LODs of the methods ranged from 3 to 7 ng/g and the LOQs from 10 to 23 ng/g, while inter‐ and intraday variability was under 6% in all cases. The methods were validated separately by using matrix‐matched calibration and recovery assays with spiked samples. Recovery rates ranged from 94.0 to 105.0%. Compost samples were taken from different composting plants. Although the statistical comparison demonstrated no statistically significant differences between the two extraction techniques, the method based on pressurized liquid extraction was more sensitive than the ultrasound extraction based method.     

液相萃取-高效液相色谱-串联质谱联用测定污泥中的全氟化合物

全氟化合物是一种新型持久性有机污染物,污水处理厂是其一个主要污染来源。目前还没有建立起一种统一的污泥样品中全氟化合物的分析方法。本文报道了一种基于液相萃取和高效液相色谱-串联质谱联用技术测定污泥中的7种全氟烷基羧酸及其2种不饱和氟调酸前体物、2种全氟烷基磺酸及其5种磺酰胺衍生物前体物的方法。实验对萃取剂(甲醇)的pH值、超声萃取温度与时间、洗脱剂体积进行了优化,确定了中性溶剂、40℃下超声萃取10min,Envicarbon柱净化的前处理方法,并成功地应用于实际污泥样品中全氟化合物的测定。方法的回收率为74%～141%(不饱和氟调酸除外),线性范围为0.1～20μg/L(羧酸系列)及0.25～50μg/L(磺酸系列)内线性关系良好(r20.99),定量限为0.6～30μg/kg(干重)。内标物质的使用可有效消除环境基质引起的仪器离子抑制现象,使定量更加准确。     

Microwave‐assisted extraction combined with on‐line solid phase extraction followed by ultra‐high‐performance liquid chromatography with tandem mass spectrometric determination of benzotriazole UV stabilizers in marine sediments and sewage sludges

Benzotriazole ultra‐violet stabilisers are compounds widely used in personal care products, which can reach the environment after passing through wastewater treatment plants. In this work, we develop a novel method to evaluate the presence of seven compounds in marine sediments and sewage sludges using microwave‐assisted extraction followed by a clean‐up step based in on‐line solid phase extraction coupled to ultra‐high‐performance liquid chromatography with MS/MS detection. This method allows for fast and efficient extraction from the solid matrix, subsequent automatic on‐line purification and preconcentration, and analysis. For the optimised method, LOD were from 53.3 to 146 ng/kg and LOQ were in the range of 176–486 ng/kg. The method was validated for different environmental solid samples with satisfactory recoveries and relative standard deviations, between 46.1 and 83.9 and 7.8 and 15.5% (sludges) and 50.1 and 87.1% and 8.83 and 16.3% (sediments), respectively. Finally, the studied analytes were quantified in concentrations between 0.18 and 24.0 ng/g in real samples of marine sediments and sewage sludges from Gran Canaria Island (Spain).     

Syringe cleanup with UHPLC–MS/MS for nitroimidazoles and steroids detection in manure‐based fertilizers

A syringe‐dispersive solid‐phase extraction method was developed for the determination of seven nitroimidazoles and nine steroids in manure‐based fertilizers by ultra‐high performance liquid chromatography with tandem mass spectrometry. Methanol and acetonitrile were used to extract the sample, and mixed dispersive sorbents dispersed in the syringe were used for purification. The extract was separated with an HSS‐T3 column and detected in positive or negative multiple reaction monitoring mode. Under the optimal conditions, the recoveries of the 16 compounds ranged from 70.3 to 112.3% at the four spiked levels (3, 10, 20, and 50 μg/kg) and the relative standard deviations ranged from 1.0 to 12.4%. The limits of detection and quantification were 0.22–0.86 and 0.73–2.87 μg/kg, respectively. This method is simple, fast, and reliable, and can be used to simultaneously screen and determine nitroimidazoles and steroids in manure‐based fertilizers.     

Novel solvent‐free microwave‐assisted extraction coupled with low‐density solvent‐based in‐tube ultrasound‐assisted emulsification microextraction for the fast analysis of organophosphorus pesticides in soils

A novel and rapid solventless microwave‐assisted extraction coupled with low‐density solvent‐based in‐tube ultrasound‐assisted emulsification microextraction has been developed for the efficient determination of nine organophosphorus pesticides in soils by GC analysis with microelectron capture detection. A specially designed, homemade glass tube inbuilt with a scaled capillary tube was used as an extraction device to collect and measure the separated extractant phase easily. Parameters affecting the efficiencies of the developed method were thoroughly investigated. From experimental results, the following conditions were selected for the extraction of organophosphorus pesticides from 1.0 g of soil sample to 5 mL of aqueous solution under 226 W of microwave irradiation for 2.5 min followed by ultrasound‐assisted emulsification microextraction with 20 μL toluene for 30 s and then centrifugation at 3200 rpm for 3 min. Detections were linear in the range of 0.25–10 ng/g with detection limits between 0.04 and 0.13 ng/g for all target analytes. The applicability of the method to real samples was assessed on agricultural contaminated soils and the recoveries ranged between 91.4 and 101.3%. Compared to other methods, the present method was shown to be highly competitive in terms of sensitivity, cost, eco‐friendly nature, and analysis speed.     

Dispersive Micro Solid‐phase Extraction Coupled with Ultrasound‐assisted Solvent Desorption for Determination of Synthetic Polycyclic and Nitro‐aromatic Musks in Aqueous Samples

An optimized method for the determination of five synthetic polycyclic: celestolide (ADBI), phantolide (AHMI), traseolide (ATII), galaxolide (HHCB), tonalide (AHTN), and two nitro‐aromatic musks: musk xylene (MX) and musk ketone (MK), in water samples is described. The method involves a dispersive micro solid‐phase extraction (D‐μ‐SPE) plus ultrasound‐assisted solvent desorption (UASD) prior to their determination by gas chromatography‐mass spectrometry (GC‐MS) using the selected ion storage (SIS) mode. Factors affecting the extraction efficiency of the target analytes from water samples and ultrasound‐assisted solvent desorption were optimized by a Box‐Behnken design method. The optimal extraction conditions involved immersing 10.1 mg of a typical octadecyl (C18) bonded silica adsorbent (i.e., ENVI‐18) in a 50 mL water sample. After 10.4 min of extraction by vigorously shaking, the adsorbent was collected and dried on a filter, and the target musks were desorbed by ultrasound‐assisted for 38 sec with n‐hexane (200 μL) as the desorption solvent. A 10 μL aliquot was then directly determined by large‐volume injection GC‐MS. The limits of quantitation (LOQs) were 1.2 to 5 ng/L. The precision for these analytes, as indicated by relative standard deviations (RSDs), were less than 11% for both intra‐ and inter‐day analysis. Accuracy, expressed as the mean extraction recovery, was between 74% and 92%. A preliminary analysis of the effluents from municipal wastewater treatment plants (MWTP) and river water samples revealed that HHCB and AHTN were the two most commonly detected synthetic musks; their concentration were determined to range from 88 to 690 ng/L for effluent samples, and 5 to 320 ng/L for river water samples. This is a simple, low cost, effective, and eco‐friendly analytical method.     

Determination of hexabromocyclododecane isomers in sewage sludge by LC‐MS/MS

A method has been developed to determine α, β and γ diastereoisomers of hexabromocyclododecane (HBCD), a brominated flame retardant, in sewage sludge, based on the ultrasonic‐assisted extraction (UAE) of samples with dichloromethane–ACN (1:1) and the subsequent clean‐up of extracts by dispersive solid phase extraction with primary–secondary amine. Levels of HBCD diastereoisomers were determined by LC coupled with ESI MS/MS. Evaluation of the matrix effect showed a high ion suppression for all the diastereoisomers studied, which was counteracted by using a mixture of labelled HBCD diastereoisomers as internal standards. This method yielded recoveries in the range of 79.6–112.5% with SDs equal or lower than 9.1 The limits of detection were 0.3 ng/g for α‐ and β‐HBCD and 0.2 ng/g for γ‐HBCD. The developed method was successfully applied to 19 sludge samples collected from the province of Madrid (Spain). In most of the samples, β‐HBCD was below the method detection limit, whereas α‐ and γ‐HBCD were quantified in all of the sludge samples, and γ‐HBCD was the predominant diastereoisomer in 63% of the analyzed samples and α‐HBCD predominated in the rest.     

Determination of 15 polycyclic aromatic hydrocarbons in aquatic products by solid‐phase extraction and GC–MS

We propose a method for the simultaneous determination of 15 kinds of polycyclic aromatic hydrocarbons in marine samples (muscle) employing gas chromatography with mass spectrometry after saponification with ultrasound‐assisted extraction and solid‐phase extraction. The experimental conditions were optimized by the response surface method. In addition, the effects of different lyes and extractants on polycyclic aromatic hydrocarbons extraction were discussed, and saturated sodium carbonate was first used as the primary saponification reaction and extracted with 10 mL of ethyl acetate and secondly 1 mol/L of sodium hydroxide and 10 mL of n‐hexane were used to achieve better results. The average recovery was 67–112%. Satisfactory data showed that the method has good reproducibility with a relative standard deviation of <13%. The detection limits of polycyclic aromatic hydrocarbons were 0.02–0.13 ng/g. Compared with other methods, this method has the advantages of simple pretreatment, low solvent consumption, maximum polycyclic aromatic hydrocarbons extraction, the fast separation speed, and the high extraction efficiency. It is concluded that this method meets the batch processing requirements of the sample and can also be used to determine polycyclic aromatic hydrocarbons in other high‐fat (fish, shrimp, crab, shellfish) biological samples.     

New analytical method for the determination of musks in personal care products by Quick,Easy, Cheap,Effective, Rugged,and Safe extraction followed by GC–MS

Synthetic musks are organic compounds used as fragrance additives and fixative compounds in a diversity of personal care products. A new method based on quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction followed by GC–MS for the analysis of 12 musks in personal care products was developed and validated. Some experimental parameters, such as total QuEChERS mass, sample mass/solvent volume ratio, type of extraction solvent, as well as salts and sorbents amount were investigated and optimized. The final method involves the musks extraction using acetonitrile, followed by the addition of anhydrous magnesium sulphate and sodium acetate. The clean‐up step was performed using dispersive SPE with primary and secondary amine and octadecyl–silica sorbents. This extraction procedure is fast (about 10 min) when compared to other traditional approaches. The method was robust for the matrices studied and shows a high precision (%RSD < 15%) and accuracy (average recovery of 85%), allowing the detection of musks in minimum concentrations between 0.01 ng/g (galaxolide) and 15.80 ng/g (musk xylene). The developed method was applied to the analysis of 12 samples, which revealed musks concentrations ranging from 2 ng/g (toothpaste) to 882 340 ng/g (perfumed body lotion).     

Determination of main taxoids in Taxus species by microwave‐assisted extraction combined with LC‐MS/MS analysis

A method based on microwave‐assisted extraction (MAE) has been developed for the determination of paclitaxel and five related taxoids, namely 10‐deacetylbaccatin III (10‐DAB III), cephalomannine, 10‐deacetylpaclitaxel (10‐DAT), 7‐xyl‐10‐ deacetylpaclitaxel (7‐xyl‐10‐DAT), and 7‐epi‐10‐deacetylpaclitaxel (7‐epi‐10‐DAT) in Taxus species in this study. The influential parameters of the MAE procedure were optimized, and the optimal conditions were as follows: extraction solvent 80% ethanol solution, solid/liquid ratio 1:10 (g/mL), temperature 50°C, and three extraction cycles, each cycle 10 min. The method validation for LC‐MS/MS analysis was performed. The LOD and LOQ were 3.16–9.20 and 12.20–30.45 ng/mL, respectively. Repeatability and reproducibility for the six taxiods with RSD ranged from 2.78 to 3.85% and from 5.26 to 6.60%. The recoveries of the method for the six taxoids were 92.6–105.6%. The developed MAE‐LC‐MS/MS method was also successfully applied to determine the contents of six taxoids in different Taxus species.     

Determination of fluorotelomer alcohols by liquid chromatography/tandem mass spectrometry in water

Fluorotelomer alcohols (FTOHs) are important polyfluorinated raw materials that belong to the general category of perfluoroalkyl substances (PFAS). PFAS, including perfluoroalkyl carboxylates (PFCAs) and perfluoroalkyl sulfonates, have recently attracted considerable attention because they are persistent and found globally in the environment. FTOHs are precursors that may degrade in the environment to PFCAs. The development of analytical methods for determination FTOHs in environmental samples is necessary to determine the environmental presence of FTOHs. This work presents the development and validation of a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the determination of FTOHs (6-2, 8-2, 10-2) in aqueous samples. Chromatographic conditions were optimized in order to obtain focused FTOH chromatographic peaks. The mobile phase and mass spectrometric conditions were optimized to enable formation of deprotonated FTOH molecules in the negative ion electrospray mode. Two extraction methods were investigated using acetonitrile and methyl tert-butyl ether (MTBE). These methods were validated for a range of environmental water samples fortified with FTOHs at three different levels. Both extraction methods resulted in recoveries from 70 to 120%. Detection limits of FTOHs were estimated to be approximately 0.09 ng/mL for LC/MS/MS detection. An LC/MS method was also developed for FTOH determination with an estimated 1.2 ng/mL limit of detection. Various sample storage scenarios were investigated. It was determined that the aqueous samples of FTOHs are best preserved by storing them frozen in sealed vials with aluminum foil lined septa.     

Determination of three capsaicinoids in Capsicum annuum by pressurized liquid extraction combined with LC‐MS/MS

A new method based on pressurized liquid extraction followed by LC‐MS/MS analysis has been developed for the identification and quantification of three capsaicinoids (capsaicin, dihydrocapsaicin, and nordihydrocapsaicin) in extracts of Capsicum annuum. For the recovery of three capsaicinoids, the efficiency levels of ultrasonic‐assisted extraction, microwave‐assisted extraction, Soxhlet extraction, and pressurized liquid extraction were compared under different conditions. Pressurized liquid extraction resulted in higher yields. Pressurized liquid extractions were performed using methanol; temperature was set at 100°C and pressure at 1500 psi. LC analysis was performed on a Waters XBridge? C₁₈ column (150 × 2.1 mm, id 3.5 μm) eluted by a mobile phase of 0.1% formic acid and ACN. Data acquisition was carried out in multiple reaction monitoring transitions mode, monitoring two‐reaction monitoring transitions to ensure an accurate identification of target compounds in the samples. The proposed method is rapid, simple, and could be utilized for the routine analysis of three capsaicinoids in C. annuum samples.     

Ultrasonic‐assisted derivatization of estrogenic compounds in a cup horn booster and determination by GC‐MS

Cup horn boosters are miniaturized ultrasound baths that maximize efficiency and precision. The optimization of an ultrasonic‐assisted derivatization step by means of a cup horn booster and the determination of estrone, 17β‐estradiol, estriol, 17α‐ethynyl estradiol and mestranol was developed by GC‐MS. Different derivatization reagents and solvents were studied for maximizing the di‐derivatization of 17α‐ethynyl estradiol under ultrasound energy. Only N,O‐bis(trimethylsilyl)trifluoroacetamide with 1% of trimethylchlorosilane in pyridine gave satisfactory results and this mixture was further used in the optimization of the ultrasound assisted derivatization. The experiment designs included sonication time (1–10 min), sonication power (20–80%), sonication cycles (1–9), derivatization reagent volume (25–125 μL) and solvent volume (25–125 μL). Once the optimum conditions were fixed, the effect of organic matter and the frequency of the water bath change were studied. Finally, the validation of the analytical method was carried out using spiked natural and synthetic waters. Recoveries (natural (138–70%) and synthetic (112–89%)), the LODs (0.35–1.66 ng/L), and LOQs (1.16–5.52 ng/L) and the precision (0.2–5.3%) of the method were studied. This is the first work in the literature where a cup horn booster is used with the aim of minimizing derivatization time during the determination of estrogenic compounds.     

Determination of phthalate esters in liquor samples by vortex‐assisted surfactant‐enhanced‐emulsification liquid–liquid microextraction followed by GC–MS

A novel method using vortex‐assisted surfactant‐enhanced‐emulsification liquid–liquid microextraction has been developed for the extraction of phthalate esters (PAEs) in Chinese liquor samples prior to analysis by GC–MS. In the proposed method, a high‐density extraction solvent (carbon tetrachloride) was dispersed into samples with the aid of a surfactant (Triton X‐100) and vortex agitation, resulting in a short extraction equilibrium (30 s). After centrifugation, a single microdrop of solvent was easily collected for GC–MS analysis. Key factors that affected the extraction efficiency were optimized. Under the optimum conditions, linearity was found in the range from 0.05 to 50 μg/L. Coefficients of determination varied from 0.9938 to 0.9971. LODs, based on an S/N of 3, ranged from 4.9 to 13 ng/L. Enrichment factors varied from 140 to 184. Reproducibility and recoveries were assessed by testing a series of three liquor samples that were spiked with different concentration levels. Finally, the proposed method was successfully applied to the determination of PAEs in 16 Chinese liquor samples. In this work, high‐density‐solvent vortex‐assisted surfactant‐enhanced‐emulsification liquid–liquid microextraction was applied for the first time for the extraction of PAEs in Chinese liquor samples and was proved to be simple, rapid, and sensitive.     

Fast quantitative determination of methylphenidate levels in rat plasma and brain ex vivo by MALDI‐MS/MS

This study presents a simple and sensitive high‐throughput matrix‐assisted laser desorption/ionization time‐of‐flight tandem mass spectrometry (MALDI‐MS/MS) method for ex vivo quantification of methylphenidate (MPH) in rat plasma and brain. The common MALDI matrix alpha‐cyano‐4‐hydroxycinnamic acid was used to obtain an optimal dried droplet preparation. For method validation, standards diluted in plasma and brain homogenate prepared from untreated (control) rats were used. MPH was quantified within a concentration range of 0.1–40 ng/ml in plasma and 0.4–40 ng/ml in brain homogenate with an excellent linearity (R² ≥ 0.9997) and good precision. The intra‐day and inter‐day accuracies fulfilled the FDA's ±15/20 critera. The recovery of MPH ranged from 93.8 to 98.5% and 87.2 to 99.8% in plasma and homogenate, respectively. We show that MPH is successfully quantified in plasma and brain homogenate of rats pre‐treated with this drug using the internal standard calibration method. By means of this method, a linear correlation between plasma and brain concentration of MPH in rodents pre‐treated with MPH was detected. The simple sample preparation based on liquid‐liquid extraction and MALDI‐MS/MS measurement requires approximately 10 s per sample, and this significantly reduces analysis time compared with other analytical methods. To the best of our knowledge, this is the first MALDI‐MS/MS method for quantification of MPH in rat plasma and brain. Copyright © 2015 John Wiley & Sons, Ltd.     

Simple method for the determination of personal care product ingredients in lettuce by ultrasound‐assisted extraction combined with solid‐phase microextraction followed by GC–MS

A simple method for the simultaneous determination of personal care product ingredients: galaxolide, tonalide, oxybenzone, 4‐methylbenzyliden camphor, padimate‐o, 2‐ethylhexyl methoxycinnamate, octocrylene, triclosan, and methyl triclosan in lettuce by ultrasound‐assisted extraction combined with solid‐phase microextraction followed by gas chromatography with mass spectrometry was developed. Lettuce was directly extracted by ultrasound‐assisted extraction with methanol, this extract was combined with water, extracted by solid‐phase microextraction in immersion mode, and analyzed by gas chromatography with mass spectrometry. Good linear relationships (25–250 ng/g, R² > 0.9702) and low detection limits (1.0–25 ng/g) were obtained for analytes along with acceptable precision for almost all analytes (RSDs < 20%). The validated method was applied for the determination of personal care product ingredients in commercial lettuce and lettuces grown in soil and irrigated with the analytes, identifying the target analytes in leaves and roots of the latter. This procedure is a miniaturized and environmentally friendly proposal which can be a useful tool for quality analysis in lettuce.     

Trace determination of five triazole fungicide residues in traditional Chinese medicine samples by dispersive solid‐phase extraction combined with ultrasound‐assisted dispersive liquid–liquid microextraction and UHPLC–MS/MS

A novel and reliable method for determination of five triazole fungicide residues (triadimenol, tebuconazole, diniconazole, flutriafol, and hexaconazol) in traditional Chinese medicine samples was developed using dispersive solid‐phase extraction combined with ultrasound‐assisted dispersive liquid–liquid microextraction before ultra‐high performance liquid chromatography with tandem mass spectrometry. The clean up of the extract was conducted using dispersive solid‐phase extraction by directly adding sorbents into the extraction solution, followed by shaking and centrifugation. After that, a mixture of 400 μL trichloromethane (extraction solvent) and 0.5 mL of the above supernatant was injected rapidly into water for the dispersive liquid–liquid microextraction procedure. The factors affecting the extraction efficiency were optimized. Under the optimum conditions, the calibration curves showed good linearity in the range of 2.0–400 (tebuconazole, diniconazole, and hexaconazole) and 4.0–800 ng/g (triadimenol and flutriafol) with the regression coefficients higher than 0.9958. The limit of detection and limit of quantification for the present method were 0.5–1.1 and 1.8–4.0 ng/g, respectively. The recoveries of the target analytes ranged from 80.2 to 103.2%. The proposed method has been successfully applied to the analysis of five triazole fungicides in traditional Chinese medicine samples, and satisfactory results were obtained.     

Application of an efficient strategy based on MAE,HPLC‐DAD‐MS/MS and HSCCC for the rapid extraction,identification, separation and purification of flavonoids from Fructus Aurantii Immaturus

This study presents an efficient strategy based on microwave‐assisted extraction (MAE), HPLC‐DAD‐MS/MS and high‐speed counter‐current chromatography (HSCCC) for the rapid extraction, identification, separation and purification of active components from the traditional Chinese medicine Fructus Aurantii Immaturus. An LC‐DAD‐MS/MS method was applied for the screening and structural identification of main components in crude extract, and five components were preliminarily identified as neoeriocitrin, narirutin, naringin, hesperidin and neohesperidin according to their UV and mass spectra. An efficient MAE method for the extraction of the three most abundant components (narirutin, naringin and neohesperidin) was optimized by the combination of univariate and multivariate approaches. The crude extract was then separated and purified by HSCCC and a total of 61.6 mg of narirutin, 207.3 mg of naringin and 159.5 mg of neohesperidin at high purities of 98.1, 97.2 and 99.5%, respectively, were obtained from 1.42 g of crude extract. The recoveries of these compounds were 86, 93 and 89%, respectively. Copyright © 2009 John Wiley & Sons, Ltd.