Evaluation of green extraction methods on bioactive compounds and antioxidant capacity from Bougainvillea glabra bracts

Bougainvillea glabra is a native plant of South America, and its bracts are still an underexploited source of betalains and phenolic compounds. Microwave (MAE), ultrasound (UAE), conventional aqueous and ethanolic exhaustive extractions were used for the recovery of total phenolic compounds (TPC) and betalains from B. glabra bracts. The highest yields of TPC, betalains, and antioxidant capacity by ABTS were obtained by ethanolic exhaustive extraction followed by MAE at 100 and 600 W. Deoxyribose protection assay measurement by hydroxyl radical scavenging showed high values for extracts obtained by exhaustive extraction and MAE, ranging from 89% to 83%, indicating a high capacity to capture hydroxyl radicals. Agarose gel electrophoresis assay showed that the extracts obtained by UAE, conventional aqueous, and ethanolic extraction were able to protect DNA from free radicals and did not show pro-oxidant effect. Seventeen phenolic compounds and seventeen betalains were identified by HPLC/MS in the extract of B. glabra bracts obtained by exhaustive extraction, composed mainly of kaempferol derivatives (76.5%) and acylated betalains (71.0%), respectively. The results suggest that MAE may be an alternative to recover betalains and phenolic compounds from B. glabra bracts due to the lower extraction times and the absence of organic solvent.     

From agricultural waste to antioxidant-rich extracts: Green techniques in extraction of polyphenols from sugar beet leaves

Different conventional and “green” extraction techniques (solid/liquid extraction, microwave and ultrasound assisted extraction, pressurized liquid extraction and subcritical water extraction) were tested using different operating conditions in the recovery of valuable polyphenol compounds from sugar beet leaves. All tested extraction techniques provided considerable extraction yield in the range from 18.21% to 37.04%. Total phenolic content ranged from 0.4504 up to 1.7171 (g GAE/100 g DW) with the highest values obtained by using microwave and ultrasound assisted extraction. Extraction parameters had highly significant effect on the concentration of bioactive compounds recovered by pressurized liquid extraction and subcritical water extraction. Individual phenolic profile of sugar beet leaves extracts largely depended on the applied extraction technique, however, vitexin was the most abundant phenolic compound present in all extracts regardless of the extraction technique. Investigation of extracts antioxidant activity through DPPH, FRAP and ABTS values indicated the lowest antioxidant activity of extracts obtained by solid/liquid extraction compared to other extraction techniques.     

Chemical characterization of Iraqi propolis samples and assessing their antioxidant potentials

Propolis samples, collected from different geographical locations in Iraq (Baghdad, Dahuk, Mosul and Salah ad-Din), were analyzed and assessed for their anti-oxidant activity. Concentrations of phenolic compounds (flavonoids, phenolic acids and their esters) in propolis were estimated using high performance liquid chromatography coupled to electrospray mass spectrometry. Thirty-eight different compounds were identified and 33 of them were polyphenols. Other compounds were tentatively identified as clerodane diterpenoids, and one was considered unknown. Semi-quantitative measurements showed that phenolic acids and their esters were the predominant constituents in propolis extracts, followed by flavones and flavonols, and then flavanones and dihydroflavonols. Propolis samples were further spectrophotometrically characterized using the Folin-Ciocalteu reagent for the determination of total phenolic compounds. The free radical scavenging activities of propolis samples were also evaluated by using the 2,2-diphenyl-1-picrylhydrazyl assay. The results revealed that propolis extracts exhibited strong free radical scavenging activity.     

Optimization and comparison of three methods for extraction of volatile compounds from Cyperus rotundus evaluated by gas chromatography-mass spectrometry

The essential oil of Cyperus rotundus has multiple pharmacological activities. Therefore, the extraction with high yield and quality is very important for preparation of essential oil of C. rotundus. In this paper, three methods, namely hydrodistillation (HD), pressurized liquid extraction (PLE) and supercritical fluid extraction (SFE), for extraction of volatile compounds from C. rotundus were optimized and compared by gas chromatography-mass spectrometry. Among eight identified compounds in C. rotundus, five components including alpha-copaene, cyperene, beta-selinene, beta-cyperone and alpha-cyperone were quantitatively determined or estimated using alpha-cyperone as standard, which showed that PLE had the highest extraction efficiency, while SFE had the best selectivity for extraction of beta-cyperone and alpha-cyperone. The contents of ingredients from C. rotundus extracted with HD, PLE and SFE are significantly different, which suggest that comparison of chemical components and pharmacological activities of different extracts is helpful to elucidate the active components in C. rotundus and control its quality.     

Metabolic profile of the bioactive compounds of burdock (Arctium lappa) seeds, roots and leaves

In this work the bioactive metabolic profile, the antioxidant activity and total phenolic content of burdock (Arctium lappa) seeds, leaves and roots were obtained. TEAC values and total phenolic content for hydro-alcoholic extracts of burdock ranged from 67.39 to 1.63 μmol Trolox equivalent/100 g dry weight (DW), and from 2.87 to 45 g of gallic acid equivalent/100 g DW, respectively. Phytochemical compounds were analyzed by liquid chromatography coupled to electrospray tandem mass spectrometry (LC/MS/MS) in negative mode. The main compounds of burdock extracts were caffeoylquinic acid derivatives, lignans (mainly arctiin) and various flavonoids.The occurrence of some phenolic acids (caffeic acid, chlorogenic acid and cynarin) in burdock seeds; arctiin, luteolin and quercetin rhamnoside in burdock roots; phenolic acids, quercetin, quercitrin and luteolin in burdock leaves was reported for the first time.     

Characterization and quantification of phenolic compounds of extra-virgin olive oils with anticancer properties by a rapid and resolutive LC-ESI-TOF MS method

The characterization and quantification of extra-virgin olive oil (EVOO) phenolic compounds by a rapid resolution liquid chromatography (RRLC) method coupled to diode-array and time of flight mass spectrometry (TOF) detection systems was developed. The RRLC method transferred from a conventional HPLC one achieved better performance with shorter analysis times. The phenolic compounds were separated with a C18 column (150 mm × 4.6 mm, 1.8 μm) using water with 0.5% acetic acid and acetonitrile as mobile phases. Good peak resolution was obtained and 19 different phenols were identified in less than 20 min providing a new level of information about the samples in shorter time. The applicability of this analytical approach was confirmed by the successful analysis of three different EVOO varieties (Picual, Hojiblanca, and Arbequina) obtained from different trademarks. Besides identification of the most important phenolic compounds and their quantification in three different ways (RRLC-UV, RRLC-MS and a new approach using the total polyphenol content obtained with Folin Ciocalteau, the relative areas and the response factors), we also described the occurrence of correlations between the phenolic composition of EVOO-derived crude phenolic extracts and their anti-proliferative abilities toward human breast cancer-derived cell lines. When compared with lignans-rich EVOO varieties, secoiridoids-rich EVOO had a significantly strong ability to alter cell viability in four different types of human breast carcinoma cells.     

Enhanced antioxidant and antityrosinase activities of longan fruit pericarp by ultra-high-pressure-assisted extraction

The health benefits of fruits acting against chronic diseases are ascribed to their antioxidant activities which are mainly responsible due to the presence of phenolic compounds. The use of ultra-high-pressure-assisted extraction (UHPE) has shown great advantages for the extraction of these phenolic compounds from longan fruit pericarp (LFP). Studies were carried out to investigate the effects of UHPE at pressures of 200, 300, 400 and 500 MPa on total phenolic contents, extraction yield, antioxidant and antityrosinase activities from LFP. The antioxidant activities of these extracts were analyzed, using various antioxidant models like 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, total antioxidant capacity and superoxide anion radical scavenging activity. Extract from ultra-high-pressure-assisted extraction at 500 MPa (UHPE-500) showed the highest antioxidant activities of all the tested models. In addition, it also showed moderate tyrosinase inhibitory activity. Three phenolic acids, namely gallic acid, ellagic acid, and corilagin were identified and quantified by HPLC. Corilagin content was the highest compared to other phenolic acids identified. UHPE-500 obtained the higher phenolic acid contents compared to other high pressure processing and conventional extractions (CE). Compared with CE, UHPE-500 exhibited good extraction effectiveness in terms of higher extraction yields with high phenolic contents and also with higher antioxidant and antityrosinase activities.     

Comparing ultrasonic- and microwave-assisted methods for extraction of phenolic compounds from Kabkab date seed (Phoenix dactylifera L.) and stepwise regression analysis of extracts antioxidant activity

This study aimed to extract bioactive compounds (phenolics and flavonoids and condensed tannins) from roasted date seed (Phoenix dactylifera L. cv Kabkab) using various solvent systems (W: water, AE: aqueous-ethanol, AA: aqueous-acetone) and extraction method (ultrasonic-assisted (UAE), microwave-assisted (MAE) and the combination of these two methods (UMAE) maceration (ME) and Decoction-Infusion (DIE) Extraction). Moreover, the feasibility of antioxidant activity prediction was investigated based on stepwise regression analysis and phytochemical properties. Extraction yield, Total phenolic content (TPC), total flavonoid content (TFD) and antioxidant activity (DPPH*, ABTS*⁺, FRAP and averaged antioxidant activity: AAA) of the extracts were evaluated. The main effect of solvent systems and extraction methods on phytochemical compounds and antioxidant activity were significant (P < 0.01). Water and aqueous-ethanol solvents extracted higher phytochemical compounds than aqueous-acetone (P < 0.05). Although the highest extraction performance was observed for the ME method, the novel methods show an acceptable result in a much shorter time. Among novel methods, the highest and lowest performances were recorded for UAE and MAE. There was no significant difference between novel-green methods (e.g., UAE and UMAE). Although the lowest phytochemical yield was obtained for MAE, this performance was obtained in less than 5 min. The highest and lowest correlation between the phytochemical compositions and antioxidant activity parameters were for DPPH* and AAA, respectively. Stepwise-regression analysis showed the weakest and strongest prediction models for AAA (10-fold S = 0.098 and 10-fold R² = 87.07) and ABTS*⁺ (10-fold S = 0.129 and 10-fold R² = 78.25), respectively.     

Identification of phenolic compounds in rosemary honey using solid-phase extraction by capillary electrophoresis-electrospray ionization-mass spectrometry

Complex extracts of rosemary honey constituents often require very effective separation techniques to allow the identification of different compounds. Capillary electrophoresis (CE) coupled to mass spectrometry (MS) detection can provide structure-selective information about the analytes in such matrices and has turned out to be an attractive alternative to HPLC methods. A simple and cost-effective analytical method involving solid-phase extraction (SPE) and capillary zone electrophoresis coupled to electrospray ionization-ion trap mass spectrometry (CZE-ESI-MS) to identify and characterize phenolic compounds in rosemary honey is described. The SPE, CE and ESI-MS parameters were optimized in order to maximize the number of phenolic compounds detected and the sensitivity of their determination. All CE–ESI-MS experiments were performed with uncoated fused-silica capillaries and an alkaline volatile buffer system consisting of 100 mM NH₄Oac with 10% of 2-propanol at pH 10. Since sheath liquids can made significant effects on the sensitivity in typical CE–ESI-MS application, the effect of type and flow rate of the sheath liquid on the sensitivity of phenolic compounds were investigated. As result, the best sensitivity was obtained with a sheath liquid containing 2-propanol/water 60:40 (v/v) and 0.1% (v/v) of triethylamine at 3 μL/min in the negative ion mode. We describe the first method for the analysis of phenolic compounds in rosemary honey at mg/L levels by using a simple SPE before CE–ESI-MS analysis.     

Subcritical water extraction of nutraceuticals with antioxidant activity from oregano. Chemical and functional characterization

In the present work, oregano leaves (Origanum vulgare L.) are explored as natural source of nutraceuticals with antioxidant activity. To do this, subcritical water extraction (SWE), a new environmentally friendly technique, is employed as extraction procedure and HPLC coupled to DAD is used for the chemical characterization of the extracts. Moreover, the radical scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH) method and the determination of the total phenolic content (measured with the Folin test) are applied to evaluate the antioxidant activity of the extracts. The extraction of antioxidants from oregano leaves by SWE is studied considering different temperatures (25, 50, 100, 150 and 200 °C) to investigate the selectivity of the process. The highest antioxidant activity is observed for the extract obtained at the highest temperature, 200 °C (EC₅₀ equal to 10 μg/ml). Moreover, the extraction yield was also the highest (54% dry weight) at these extraction conditions. The total phenolic content showed no differences among the different extracts, concluding that the amount of phenolic compounds extracted was similar but the type and structure of the phenolics was different, providing in this way different antioxidant activity. Some compounds could be tentatively identified, proposing some probable chemical structures for some of them, such as flavanones, dihydroflavonols, favonols and flavones.     

Comparison of conventional and non conventional methods of extraction of heartwood of Pterocarpus marsupium Roxb

The renewed interest in plant-derived drugs has led to an increased need for efficient extraction methods. The present investigation was an attempt to evaluate and compare the conventional methods of extraction with non conventional methods of extraction, such as ultrasonic-assisted extraction (UAE) and microwave-assisted extraction (MAE) methods. Pterocarpus marsupium Roxb. has been reported to contain bioactive phytochemicals, e.g., pterostilbene (3',5'-dimethoxy-4-stilbenol). The results showed that among the conventional extraction methods, percolation gave the highest yield. The non conventional methods were optimized. The extraction yield was the highest in case of MAE. The phytochemical screening of the extracts indicated similar groups of compounds in all the extracts. The thin layer chromatography showed the presence of pterostilbene in the extracts obtained by using percolation, MAE and UAE. In these extracts the quantification of pterostilbene was conducted by high performance liquid chromatography and the method was validated. The MAE method extracted significantly higher amount of pterostilbene.     

Pressurized liquid extraction of phenolic compounds from Anatolia propolis and their radical scavenging capacities

Propolis samples from important honey producing locations of Anatolia namely; Bingol (BG), Rize (RZ), Tekirdag (TK) and Van (VN), were evaluated for their antiradical capacities, total phenolic contents and individual phenolic compounds which was recovered by means of pressurized liquid extraction (PLE). Several extraction parameters of PLE such as; temperature, pressure, solvent type, extraction time and cell size were investigated for their effects on the extraction performances. The results showed that, 40 °C, 1500 psi, Ethanol:water:HCl; (70:25:5, v/v/v) containing 0.1% tert-butylhydroquinone (tBHQ) as solvent, three extraction cycles within 15 min, and a cell size of 11 mL was the most favorable PLE operating conditions. Results of the tests performed to designate the success of the polyphenol analysis showed that the recovery was in the range of 97.2% and 99.7%.Major phenolic compounds in all samples were found to be gallocatechin (GCT), catechin (CT), epicatechin gallate (ECTG), caffeic acid (CA), chlorogenic acid (ChA), and myricetin (Myr). ChA level of BG propolis was 4.5, 3 and 23 times higher than that of RZ, TK and VN region, respectively. Antiradical tests showed that all propolis samples have superior antiradical capacities up to 500 mg Trolox equivalent activity per gram of extract.     

Antioxidant,anti-microbial and antimutagenicity activities of pistachio (Pistachia vera) green hull extract

Antioxidant, anti-microbial and antimutagenicity activities of pistachio (Ahmadaghaei variety) green hull extracts (crude and purified extracts) were studied. At first, different solvents were compared for determining of the best solvent for extraction of phenolic compounds from pistachio green hull. Water and acetonitrile with 49.32 and 6.22 (mg of gallic acid equivalents/g sample) were the best and the worst solvent in the extraction of phenolic compounds, respectively. The antioxidant capacity of crude and purified extracts were assessed through ABTS assay, DPPH assay and β-carotene bleaching (BCB) method. A concentration-dependent antioxidative capacity was verified in ABTS, DPPH assays and BCB method. The anti-microbial capacity was screened against Gram positive and Gram negative bacteria, and fungi. Aqueous and purified extracts inhibited the growth of Gram positive bacteria; Bacillus cereus was the most susceptible one with MIC of 1 mg/mL and 0.5 mg/mL for the crude and purified extracts, respectively. The results of antimutagenicity test showed that phenolic compounds of pistachio green hull have antimutagenicity activity against direct mutagen of 2-nitrofluorene. The results obtained indicate that pistachio green hull may become important as a cheap and noticeable source of compounds with health protective potential and anti-microbial activity.     

Antioxidant activity and ultra-performance LC-electrospray ionization-quadrupole time-of-flight mass spectrometry for phenolics-based fingerprinting of Rose species: Rosa damascena, Rosa bourboniana and Rosa brunonii

Roses are one of the most important groups of ornamental plants and their fruits and flowers are used in a wide variety of food, nutritional products and different traditional medicines. The antioxidant activity of methanolic extracts from fresh flowers of three rose species (Rosa damascena, Rosa bourboniana and Rosa brunonii) was evaluated by 1,1-diphenyl-2-picryl hydrazyl (DPPH) free-radical method. The ability to scavenge DPPH radical was measured by the discoloration of the solution. The methanolic extract from R. brunonii exhibited maximum free-radical-scavenging activity (64.5 ± 0.38%) followed by R. bourboniana (51.8 ± 0.46%) and R. damascena (43.6 ± 0.25%) at 100 μg/ml. Simultaneously, ultra-performance liquid chromatography coupled with electrospray ionization-quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOF-MS) was used to study phenolic composition in the methanolic extracts from the fresh flowers of rose species. The phenolic constituents were further investigated by direct infusion-ESI-QTOF-MS/MS in negative ion mode. Characteristic Electrospray ionization tandem mass spectrometry (ESI-MS/MS) spectra with other diagnostic fragment ions generated by retro Diels–Alder (RDA) fragmentation pathways were recorded for the flavonoids. Distinct similarities were observed in the relative distribution of polyphenolic compounds among the three species. The dominance of quercetin, kaempferol and their glycosides was observed in all the three species.     

Determination of phenolic compounds in Prunella L. by liquid chromatography-diode array detection

Four species of Prunella L. (Prunella vulgaris L., Prunella laciniata L., Prunella grandiflora L. and Prunella orientalis Bornm.) belong to the family of Lamiaceae and representing popular Western and Chinese herbal medicine were examined for the content of phenolic compounds. Phenolic acids (rosmarinic acid, caffeic acid, ferulic acid, chlorogenic acid, protocatechuic acid), flavonoids (rutin, quercetin) in different quantitative proportions depending on extracts were determined by the rapid, selective and accurate method combining solvent/acid hydrolysis extraction and high performance liquid chromatography-diode array detection (HPLC-DAD). Water, methanol, butanol, acetonitrile, ethyl acetate, hexane and their acidic solutions were used to examine the efficiency of different solvent systems for the extraction of phenolic compounds. Acid hydrolysis extraction was established as the most suitable extraction method for phenolic compounds.     

Are extraction methods in quantitative assays of pharmacopoeia monographs exhaustive? A comparison with pressurized liquid extraction

The extraction methods in selected monographs of the European and the Swiss Pharmacopoeia were compared to pressurized liquid extraction (PLE) with respect to the yield of constituents to be dosed in the quantitative assay for the respective herbal drugs. The study included five drugs, Belladonnae folium, Colae semen, Boldo folium, Tanaceti herba and Agni casti fructus. They were selected to cover different classes of compounds to be analyzed and different extraction methods to be used according to the monographs. Extraction protocols for PLE were optimized by varying the solvents and number of extraction cycles. In PLE, yields > 97 % of extractable analytes were typically achieved with two extraction cycles. For alkaloid-containing drugs, the addition of ammonia prior to extraction significantly increased the yield and reduced the number of extraction cycles required for exhaustive extraction. PLE was in all cases superior to the extraction protocol of the pharmacopoeia monographs (taken as 100 %), with differences ranging from 108 % in case of parthenolide in Tanaceti herba to 343 % in case of alkaloids in Boldo folium.     

Metabolic fingerprinting of different populations of <Emphasis Type="Italic">Phyllanthus niruri</Emphasis> L. from Punjab using electrospray ionization mass spectrometry (ESI–MS)

There is growing interest of healthcare and food industry in ingredients of plant origin as they are potent sources of antioxidant, anti-inflammatory, antimicrobial and anticancer agents. In the present work different extracts of Phyllanthus niruri L. from various regions of Punjab were screened for their phenolic profiles and antioxidant properties. Crude extracts obtained by solid–liquid extraction with different solvents were tested for total anthocyanins, flavonoids, phenolic content, and free radical scavenging activity. Out of all the solvents used, methanol was regarded as best to be used for soxhlet extraction of plant metabolites, as it provided highest phenolic, flavonoid, antioxidant, and anthocyanin contents. Similarly, ESI–MS was employed to obtain mass profiles of phenolic and other metabolites present in various P. niruri populations. Out of 72 compounds detected, 51 are reported for the first time in P. niruri L. Similarly, different populations of P. niruri were discriminated through metabolic fingerprinting using ESI–MS.     

Analysis of bioactive compounds using LC–ESI–MS/MS,cytotoxic, antimicrobial effects,and enzyme activities from Cyclotrichium origanifolium

Cyclotrichium origanifolium is a medicinal plant belonging to the Lamiaceae family. In this study, phenolic content analysis, antimicrobial effects, and cytotoxic effects of extracts of C. origanifolium were investigated. In the extracts, phenolic compound analysis by the liquid chromatography–electrospray ionization–tandem mass spectrometry method, antimicrobial effect by the minimum inhibition concentration method, and cytotoxic effect on human dermal fibroblasts (HDF), glioblastoma cell (U87), ovarian adenocarcinoma cell (Skov-3), and human colorectal adenocarcinoma cell (CaCo-2) cancer cell lines were investigated. Cytotoxicity analyses were performed by the MTT method. In addition, the GST and AChE enzyme activities of the extracts were also measured. Around 18 compounds were detected in both the methanol and ethanol extract. It was found that the best antimicrobial effect on Gram-negative Pseudomonas aeruginosa was on methanol extract, while the ethanol extract was on Candida albicans fungus (respectively, 2.50 mg/ml, 5.0 μg/ml). A 500 μg/ml of methanol extract has been shown to have cytotoxic activity high effect on HDF cells. GST and AChE activity were found to decrease in a concentration-dependent manner.     

Simultaneous analysis of theanine, chlorogenic acid, purine alkaloids and catechins in tea samples with the help of multi-dimension information of on-line high performance liquid chromatography/electrospray-mass spectrometry

A reverse phase high performance liquid chromatography (RP-HPLC) separation coupled with photo diode array detection (DAD) and electrospray ionization mass spectrometry (ESI-MS) detection was established for the analyzing of multiple bioactive compounds in tea and tea extracts. Theanine, chlorogenic acid, purine alkaloids and catechins were identified with authentic standard compounds and with MS-spectra. The content of theanine and catechins was measured by employing DAD and caffeine, chlorogenic acid, theobromine and theopylline by protonated molecular ion on selective ion recording (SIR) mode. The unity of LC/ESI-MS provides more qualitative and quantitative information comparing with general HPLC in the analysis of multi-components in tea, and complex extraction or sample pretreatment is unnecessary. The chromatogram acquired by using this method can be used as a bioactive components fingerprint for the quality control of tea and its extracts. With the help of multi-dimension information of HPLC-DAD-ESIMS, the compounds owning different chemical structure such as amino acid, catechins, etc. in tea and its extracts could be identified and determined in one run successfully.     

Multi‐class analysis of new psychoactive substances and metabolites in hair by pressurized liquid extraction coupled to HPLC‐HRMS

In this paper, an analytical method has been developed and validated for the analysis of new psychoactive substances (NPS) and metabolites in hair samples. The method was based on pressurized liquid extraction (PLE) followed by solid‐phase extraction (SPE) clean‐up and high performance liquid chromatography‐high resolution mass spectrometry (HPLC‐HRMS) analysis. To evaluate extraction efficiency and the applicability of the method, hair samples were fortified by soaking in order to obtain a good surrogate for drug users' hair; the amount of incorporated drugs related to their lipophilicity, similarly to in vivo drug incorporation. To the best of our knowledge, this is the first method that allowed for the analysis of both cathinones (5) and synthetic cannabinoids (7) in hair with a single extraction procedure and chromatographic run. A phenethylamine (2C‐T‐4), 4‐ fluorophenylpiperazine and methoxetamine were also included showing that PLE coupled to SPE clean‐up was suitable for a multi‐class analysis of NPS in hair. In addition, the use of PLE significantly reduced hair analysis time: decontamination, incubation, clean‐up, and liquid chromatography‐mass spectrometry (LC‐MS) analysis were carried out in approximately 45 min. The method was fully validated according to Scientific Working Group for Forensic Toxicology (SWGTOX) and Society of Hair Testing (SoHT) guidelines. Limit of quantification (LOQ) values ranged from 8 to 50 pg mg^‐1 for cathinones, phenetylamines and piperazines, and from 9 to 40 pg mg^‐1 for synthetic cannabinoids (10 pg mg^‐1 for methoxetamine). Matrix effects were below 15% for all the analytes, demonstrating the effectiveness of the clean‐up step. Inaccuracy was lower than 9% in terms of bias. Copyright © 2016 John Wiley & Sons, Ltd.