Application of ultrasound‐assisted liquid–liquid microextraction coupled with gas chromatography and mass spectrometry for the rapid determination of synthetic cannabinoids and metabolites in biological samples

The constant emergence of new psychoactive substances is a challenge to clinical and forensic toxicologists who need to constantly update analytical techniques to detect them. A large portion of these substances are synthetic cannabinoids. The aim of this study was to develop a rapid and simple method for the determination of synthetic cannabinoids and their metabolites in urine and blood using gas chromatography–mass spectrometry. The method involves an ultrasound‐assisted dispersive liquid–liquid microextraction that implies a rapid procedure, giving excellent extraction efficiencies with minimal use of toxic solvents. This is followed by silylation and analysis with gas chromatography–mass spectrometry. The chromatographic method allows for the separation and identification of 29 selected synthetic cannabinoids and some metabolites. The method was validated on urine and blood samples with the ability to detect and quantify all analytes with satisfactory limits of detection (from 1 to 5 ng/mL), limits of quantification (5 ng/mL), and selectivity and linearity (in the range of 5–200 ng/mL). The developed assay is highly applicable to laboratories with limited instrumental availability, due to the use of efficient and low‐cost sample preparation and instrumental equipment. The latter may contribute to enhance the detection of new psychoactive substances in clinical and forensic toxicology laboratories.     

Development and validation of an ultra‐performance convergence chromatography method for the quality control of Angelica gigas Nakai

Ultra‐performance convergence chromatography, which integrates the advantages of supercritical fluid chromatography and ultra high performance liquid chromatography technologies, is an environmentally friendly analytical method that uses dramatically reduced amounts of organic solvents. An ultra‐performance convergence chromatography method was developed and validated for the quantification of decursinol angelate and decursin in Angelica gigas using a CSH Fluoro‐Phenyl column (2.1 mm × 150 mm, 1.7 μm) with a run time of 4 min. The method had an improved resolution and a shorter analysis time in comparison to the conventional high‐performance liquid chromatography method. This method was validated in terms of linearity, precision, and accuracy. The limits of detection were 0.005 and 0.004 μg/mL for decursinol angelate and decursin, respectively, while the limits of quantitation were 0.014 and 0.012 μg/mL, respectively. The two components showed good regression (correlation coefficient (r²) > 0.999), excellent precision (RSD < 2.28%), and acceptable recoveries (99.75–102.62%). The proposed method can be used to efficiently separate, characterize, and quantify decursinol angelate and decursin in Angelica gigas and its related medicinal materials or preparations, with the advantages of a shorter analysis time, greater sensitivity, and better environmental compatibility.     

Fast and sensitive supercritical fluid chromatography – tandem mass spectrometry multi-class screening method for the determination of doping agents in urine

This study shows the possibility offered by modern ultra-high performance supercritical fluid chromatography combined with tandem mass spectrometry in doping control analysis. A high throughput screening method was developed for 100 substances belonging to the challenging classes of anabolic agents, hormones and metabolic modulators, synthetic cannabinoids and glucocorticoids, which should be detected at low concentrations in urine. To selectively extract these doping agents from urine, a supported liquid extraction procedure was implemented in a 48-well plate format. At the tested concentration levels ranging from 0.5 to 5 ng/mL, the recoveries were better than 70% for 48–68% of the compounds and higher than 50% for 83–87% of the tested substances. Due to the numerous interferences related to isomers of steroids and ions produced by the loss of water in the electrospray source, the choice of SFC separation conditions was very challenging. After careful optimization, a Diol stationary phase was employed. The total analysis time for the screening assay was only 8 min, and interferences as well as susceptibility to matrix effect (ME) were minimized. With the developed method, about 70% of the compounds had relative ME within the range ±20%, at a concentration of 1 and 5 ng/mL. Finally, limits of detection achieved with the above-described strategy including 5-fold preconcentration were below 0.1 ng/mL for the majority of the tested compounds. Therefore, LODs were systematically better than the minimum required performance levels established by the World anti-doping agency, except for very few metabolites.     

Simultaneous detection of multiple hydroxylated polychlorinated biphenyls from biological samples using ultra‐high‐performance liquid chromatography with isotope dilution tandem mass spectrometry

We established a method for the separation and detection of nine hydroxylated polychlorinated biphenyls in whole blood and urine samples using ultra high performance liquid chromatography coupled with electrospray negative ionization tandem mass spectrometry. Clean‐up procedures involved a filtration step, and optimization involved a pretreatment step consisting of a simple liquid–liquid extraction using hydrated silica‐gel chromatography (5%). Nine hydroxylated polychlorinated biphenyls were separated on an ultra high performance liquid chromatography HSS T3 column using a gradient elution program of 2 mmol ammonium formate aqueous solution (A) and methanol (B). Recovery ranged from 84.0 to 105.4% for the nine different hydroxylated polychlorinated biphenyls in urine with three spiked levels of 0.1, 1, and 2 ng and from 73.5 to 98.6% for the blood with spiked levels of 0.2, 1, and 2 ng. The relative standard deviations were <8.7% (n = 6), and the limits of detection in urine and whole blood for the nine hydroxylated polychlorinated biphenyls were in the range of 1.5–4 and 20–100 pg/g, respectively. This analytical method may enable the simultaneous detection of various hydroxylated polychlorinated biphenyls from complex tissue matrices.     

Determination of several synthetic cathinones and an amphetamine‐like compound in urine by gas chromatography with mass spectrometry. Method validation and application to real cases

Most routine practices for drugs‐of‐abuse testing do not include screening procedures for new psychoactive substances, despite their increasing diffusion, preventing clear knowledge of the real consumption of these drugs in the populations. To make up for this shortcoming, a gas chromatography with mass spectrometry method was developed for the simultaneous determination of 18 synthetic cathinones and one amphetamine‐like compound in human urine. The sample preparation was based on liquid–liquid extraction under alkaline condition followed by derivatization with trifluoroacetic anhydride. The separation of the 19 analytes was achieved in less than 10 min. The whole methodology was validated according to national and international guidelines. Selectivity, linearity range, limit of detection and limit of quantitation, precision and accuracy were evaluated. For all the analytes, the calibration curve was linear in the 100–1000 ng/mL concentration range. The limits of detection ranged from 10 to 30 ng/mL and limits of quantitation from 30 to 100 ng/mL. Precisions were in the ranges 0.1–10.4%, and 1.0–12.1% for low (100 ng/mL) and high (1000 ng/mL) concentration, respectively. The accuracy, expressed as bias% was within ±20% for all the analytes. The present method was successfully applied to urine samples originating from autopsies, drug abuse/withdrawal controls, clinical investigations, roadside controls, driving re‐licensing, and workplace testing.     

Determination of multi‐class antibiotic residues in compost by microwave‐enhanced accelerated solvent extraction and ultra performance convergence chromatography with tandem mass spectrometry

This work reports the development and application of a multi‐class compound analysis method for the determination of 20 antibiotic residues in compost. Samples were processed by microwave‐enhanced accelerated solvent extraction at 120°C for 7.5 min. Salting‐out homogeneous liquid‐liquid extraction was used to remove water and water‐soluble impurities from the extract before ultra performance convergence chromatography with tandem mass spectrometry analysis. By using the supercritical fluid (carbon dioxide) and organic solvent (methanol) as the mobile phase, the 20 antibiotics and the internal standard were well separated in 8.2 min without obvious matrix effect. Method validation was performed and good trueness (relative error in the range of ±5.0%) and precision (inter‐ and intraday relative standard deviations < 10.8%) were obtained. Method detection and quantitation limits were 0.8–1.9 and 2.7–7.1 ng/g, respectively. Recoveries were assessed at three concentration levels (10, 60, and 400 ng/g) and acceptable mean values (70.4–111.9%) were found. This method has also been used to analyze real samples, and the average concentrations of antibiotics (excepting the concentrations < method quantitation limits) were determined up to 123.6 ng/g. The results showed the method could be helpful for the analysis of multi‐class antibiotics in environmental samples.     

UHPLC coupled with mass spectrometry and chemometric analysis of Kang‐Ai injection based on the chemical characterization,simultaneous quantification,and relative quantification of 47 herbal alkaloids and saponins

Kang‐Ai injection, which is composed of Astragali Radix, Ginseng Radix et Rhizoma, and kushenin, is extensively used in China as an adjuvant therapy for many types of cancer and chronic hepatitis B. In the present study, 47 herbal compounds (11 alkaloids, 8 astragalosides, and 28 ginsenosides), were detected in Kang‐Ai injection by ultra‐high‐performance liquid chromatography coupled to quadrupole time‐of‐flight tandem mass spectrometry, of which 31 were identified using authentic standards. Additionally, a practical ultra‐high‐performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry method was employed for simultaneous quantitative detection (31 available compounds), and relative quantitative detection (16 unavailable compounds) within 10 min. The limit of detection and limit of quantification was 0.11‐2.22 and 0.53‐11.08 ng/mL, respectively. Altogether, content levels of each compound ranged from 0.03 to 9835.57 μg/mL. Furthermore, chemometric analysis indicated oxymatrine, astragaloside IV, ginsenosides Rg1 and Re, and matrine had the greatest effect on concentration fluctuation. Therefore, we suggested these five compounds should be monitored during the manufacturing process. This method can be applied to provide crucial chemical profiles and quality assessments for Kang‐Ai injection, guaranteeing the safety, effectiveness, and controllability of the drug in clinics.     

Towards eco‐friendly secondary plant metabolite quantitation: Ultra high performance supercritical fluid chromatography applied to common vervain (Verbena officinalis L.)

This report presents the first ultra high performance supercritical fluid chromatography diode array detector based assay for simultaneous determination of iridoid glucosides, flavonoid glucuronides, and phenylpropanoid glycosides in Verbena officinalis (Verbenaceae) extracts. Separation of the key metabolites was achieved in less than 7 min on an Acquity UPC² Torus Diol column using a mobile phase gradient comprising subcritical carbon dioxide and methanol with 0.15% phosphoric acid. Method validation for seven selected marker compounds (hastatoside, verbenalin, apigenin‐7‐O‐glucuronide, luteolin‐7‐O‐glucuronide, apigenin‐7‐O‐diglucuronide, verbascoside, and luteolin‐7‐O‐diglucuronide) confirmed the assay to be sensitive, linear, precise, and accurate. Head‐to‐head comparison to an ultra high performance liquid chromatography comparator assay did prove the high orthogonality of the methods. Quantitative result equivalence was evaluated by Passing‐Bablok‐correlation and Bland‐Altman‐plot analysis. This cross‐validation revealed, that one of the investigated marker compound peaks was contaminated in the ultra high performance liquid chromatography assay by a structurally related congener. Taken together, it was proven that the ultra high performance supercritical fluid chromatography instrument setup with its orthogonal selectivity is a true alternative to conventional reversed phase liquid chromatography in quantitative secondary metabolite analysis. For regulatory purposes, assay cross‐validation with highly orthogonal methods seems a viable approach to avoid analyte overestimation due to coeluting, analytically indistinguishable contaminants.     

Ultra‐performance convergence chromatography for the quantitative determination of bioactive compounds in Aralia continentalis Kitagawa as quality control markers

A rapid ultra‐performance convergence chromatography method was developed for the quantitative determination of bioactive compounds in Aralia continentalis as quality control markers. Quantitative analysis indicated the presence of two major bioactive compounds: diterpenoid acids continentalic acid and kaurenoic acid. Using a Torus 1‐aminoanthracene column, continentalic acid and kaurenoic acid were separated in less than 8 min. The method was validated with respect to precision, accuracy, and linearity according to the International Conference on Harmonization guidelines. The optimized method exhibited a good linear correlation (r ² > 0.996), excellent precision (RSD < 1.0%), and acceptable recoveries (99.97–100.26%). Limits of detection for continentalic acid and kaurenoic acid were 0.068 and 0.097 μg/mL, respectively, while their corresponding limits of quantitation were 0.207 and 0.295 μg/mL. The system performance of ultra‐performance convergence chromatography was compared with that of conventional high‐performance liquid chromatography with respect to analysis time and efficiency. The proposed method was found to be reliable and convenient for the quantitative analysis of continentalic acid and kaurenoic acid in A. continentalis from South Korea and A. pubescens from China. This study is expected to serve as a guideline for the quality control of Aralia continentalis .     

Simplifying and expanding the screening for peptides <2 kDa by direct urine injection,liquid chromatography,and ion mobility mass spectrometry

The analysis of low‐molecular‐mass peptides in doping controls has become a mandatory aspect in sports drug testing and, thus, the number of samples that has to be tested for these analytes has been steadily increasing. Several peptides <2 kDa with performance‐enhancing properties are covered by the list of prohibited substances of the World Anti‐Doping Agency including Desmopressin, LH‐RH, Buserelin, Triptorelin, Leuprolide, GHRP‐1, GHRP‐2, GHRP‐3, GHRP‐4, GHRP‐5,GHRP‐6, Alexamorelin, Ipamorelin, Hexarelin, ARA‐290, AOD‐9604, TB‐500 and Anamorelin. With the presented method employing direct urine injection into a liquid chromatograph followed by ion‐mobility time‐of‐flight mass spectrometry, a facile, specific and sensitive assay for the aforementioned peptidic compounds is provided. The accomplished sensitivity allows for limits of detection between 50 and 500 pg/mL and thus covers the minimum required performance level of 2 ng/mL accordingly. The method is precise (imprecision <20%) and linear in the estimated working range between 0 and 10 ng/mL. The stability of the peptides in urine was tested, and –20°C was found to be the appropriate storage temperature for sports drug testing. Finally, proof‐of‐concept was shown by analysing elimination study urine samples collected from individuals having administered GHRP‐6, GHRP‐2, or LHRH.     

Ultra‐high performance supercritical fluid chromatography of lignin‐derived phenols from alkaline cupric oxide oxidation

Traditional chromatographic methods for the analysis of lignin‐derived phenolic compounds in environmental samples are generally time consuming. In this work, an ultra‐high performance supercritical fluid chromatography method with a diode array detector for the analysis of major lignin‐derived phenolic compounds produced by alkaline cupric oxide oxidation was developed. In an analysis of a collection of 11 representative monomeric lignin phenolic compounds, all compounds were clearly separated within 6 min with excellent peak shapes, with a limit of detection of 0.5–2.5 μM, a limit of quantification of 2.5–5.0 μM, and a dynamic range of 5.0–2.0 mM (R² > 0.997). The new ultra‐high performance supercritical fluid chromatography method was also applied for the qualitative and quantitative analysis of lignin‐derived phenolic compounds obtained upon alkaline cupric oxide oxidation of a commercial humic acid. Ten out of the previous eleven model compounds could be quantified in the oxidized humic acid sample. The high separation power and short analysis time obtained demonstrate for the first time that supercritical fluid chromatography is a fast and reliable technique for the analysis of lignin‐derived phenols in complex environmental samples.     

Application of dispersive liquid–liquid microextraction for the preconcentration of eight parabens in real samples and their determination by high‐performance liquid chromatography

A simple and sensitive method for the simultaneous determination of eight parabens in human plasma and urine samples was developed. The samples were preconcentrated using dispersive liquid–liquid microextraction based on the solidification of floating organic drops and determined by high‐performance liquid chromatography with ultraviolet detection. The influence of variables affecting the extraction efficiency was investigated and optimized using Placket–Burman design and Box–Behnken design. The optimized values were: 58 μL of 1‐decanol (as extraction solvent), 0.65 mL methanol (as disperser solvent), 1.5% w/v NaCl in 5.0 mL of sample solution, pH 10.6, and 4.0 min centrifugation at 4000 rpm. The extract was injected into the high‐performance liquid chromatography system for analysis. Under the optimum conditions, the linear ranges for eight parabens in plasma and urine were 1.0–1000 ng/mL, with correlation coefficients above 0.994. The limit of detection was 0.2–0.4 and 0.1–0.4 ng/mL for plasma and urine samples, respectively. Relative recoveries were between 80.3 and 110.7%, while relative standard deviations were less than 5.4%. Finally, the method was applied to analyze the parabens in 98 patients of primary breast cancer. Results showed that parabens existed widely, at least one paraben detected in 96.9% (95/98) of plasma samples and 98.0% (96/98) of urine samples.     

Microextraction by packed sorbent followed by ultra high performance liquid chromatography for the fast extraction and determination of six antidepressants in urine

The growing use of antidepressants in recent years has led to their increasing presence in forensic analyses. In this work, microextraction by packed sorbent followed by ultra‐performance liquid chromatography with photodiode array detection provided a fast method for determining the antidepressants mirtazapine, venlafaxine, escitalopram, fluoxetine, fluvoxamine, and sertraline in human urine. The microextraction conditions (viz., type of sorbent, number of draw–eject extraction cycles or strokes, sample volume and pH, and type and volume of washing solution and eluent) were optimized by using an experimental design. The ensuing analytical method was validated in terms of linearity (25–1000 ng/mL urine), limit of detection (lower than 7.1 ng/mL), limit of quantification (25 ng/mL), precision (4.7–15.1% as relative standard deviation), and accuracy (80.4–126.1% as mean recovery for four replicate determinations). The proposed method allowed the six target antidepressants to be determined at concentrations from therapeutic to toxic levels. The application to small volumes (300 μL) of urine afforded fast extraction of the analytes and provided results on a par with those of existing clinical and forensic alternatives.     

Development of a UHPLC–MS/MS method for the quantitation of bioactive compounds in Phyllanthus species and its herbal formulations

Phyllanthus species are extensively used in traditional medicines for the treatment of hepatic diseases due to their bioactive hypophyllanthin and phyllanthin. This work describes the development and validation of an ultra high performance liquid chromatography with tandem mass spectrometry method in polarity switching multiple reaction monitoring mode for the simultaneous detection and quantitation of 23 compounds using ultra‐performance liquid chromatography coupled with electrospray‐hybrid triple quadrupole‐linear ion trap mass spectrometer. The validated parameters showed good linearity (R ² ≥ 0.996), limit of detection (0.05–1.62 ng/mL), limit of quantitation (0.15–4.95 ng/mL), precisions (intra‐day: RSD ≤ 2.11%), (inter‐day: RSD ≤ 2.91%), stability (RSD ≤ 2.56%) and overall recovery (98.22–104.48%; RSD ≤ 2.93%). The validated method was successfully applied in ethanolic extracts of P. amarus, P. niruri , P. emblica , P. fraternus , fractions of P. amarus and their herbal formulations for quantitation. The maximum content of hypophyllanthin (29.40 mg/g) and phyllanthin (56.60 mg/g) was detected in ethyl acetate fraction of P. amarus . The total content of 23 compounds was abundant in the ethanolic extract of P. emblica fruit. Principal component analysis was used to differentiate the selected Phyllanthus species and their herbal formulations. The results indicated that the present method could be used for quality control of Phyllanthus species and its herbal formulations.     

Comparison of ultra high performance supercritical fluid chromatography,ultra high performance liquid chromatography,and gas chromatography for the separation of synthetic cathinones

A comparison of ultra high performance supercritical fluid chromatography, ultra high performance liquid chromatography, and gas chromatography for the separation of synthetic cathinones has been conducted. Nine different mixtures of bath salts were analyzed in this study. The three different chromatographic techniques were examined using a general set of controlled synthetic cathinones as well as a variety of other synthetic cathinones that exist as positional isomers. Overall 35 different synthetic cathinones were analyzed. A variety of column types and chromatographic modes were examined for developing each separation. For the ultra high performance supercritical fluid chromatography separations, analyses were performed using a series of Torus and Trefoil columns with either ammonium formate or ammonium hydroxide as additives, and methanol, ethanol or isopropanol organic solvents as modifiers. Ultra high performance liquid chromatographic separations were performed in both reversed phase and hydrophilic interaction chromatographic modes using SPP C18 and SPP HILIC columns. Gas chromatography separations were performed using an Elite‐5MS capillary column. The orthogonality of ultra high performance supercritical fluid chromatography, ultra high performance liquid chromatography, and gas chromatography was examined using principal component analysis. For the best overall separation of synthetic cathinones, the use of ultra high performance supercritical fluid chromatography in combination with gas chromatography is recommended.     

Recent advances of online coupling of sample preparation techniques with ultra high performance liquid chromatography and supercritical fluid chromatography

Ultra high performance liquid chromatography and supercritical fluid chromatography techniques are favored because of their high efficiency and fast analysis speed. Although many sample preparation techniques have been coupled with common liquid chromatography online, the online coupling of sample preparation with the two popular chromatography techniques have gained increasing attention owing to the increasing requirements of efficiency and sensitivity. In this review, we have discussed and summarized the recent advances of the online coupling of sample preparation with ultra high performance liquid chromatography and supercritical fluid chromatography techniques. The main sample preparation techniques that have been coupled with ultra high performance liquid chromatography online are solid‐phase extraction and in‐tube solid‐phase microextraction, while solid‐phase extraction and supercritical fluid extraction are the main techniques that have been coupled with supercritical fluid chromatography online. Especially, the strategies for online coupling of sample preparation with chromatography techniques were summarized. Typical applications and growing trends of the online coupling techniques were also discussed in detail. With the increasing demands of improving the efficiency, throughput, and analytical capability toward complex samples of the analysis methods, online coupling of sample preparation with chromatography techniques will acquire further development.     

Simultaneous determination of seven compounds in Chinese patent medicines Chenxiangqu by matrix solid‐phase dispersion coupled with ultra high performance liquid chromatography and quadrupole time‐of‐flight mass spectrometry

A simple, efficient, and sensitive strategy by coupling matrix solid‐phase dispersion with ultra high performance liquid chromatography quadrupole time‐of‐flight mass spectrometry was proposed to extract and determine three types of components (including seven analytes) in Chinese patent medicines Chenxiangqu. The highly ordered mesoporous material Fe‐SBA‐15 synthesized under weakly acidic conditions was selected as a dispersant in matrix solid phase dispersion extraction for the first time. Several parameters including the mass ratio of sample to dispersant, the type of dispersant, the grinding time, and the elution condition were investigated in this work. Under the optimized conditions, 20 compounds were identified by quadrupole time‐of‐flight mass spectrometry and seven analytes were quantified. The results demonstrated that the developed method has good linearity (r > 0.9995), and the limits of detection of the analytes were as low as 0.55 ng/mL. The recoveries of all seven analytes ranged from 97.6 to 104.6% (relative standard deviation < 3.4%). Finally, the improved method was successfully applied to determination of five batches of Chenxiangqu samples, which provided a robust method in quality control of Chinese patent medicines Chenxiangqu. The developed strategy also shows its great potential in analysis of complex matrix samples.     

Biosurfactant trehalose lipid‐enhanced ultrasound‐assisted micellar extraction and determination of the main antioxidant compounds from functional plant tea

Hydrosoluble trehalose lipid (a biosurfactant) was employed for the first time as a green extraction solution to extract the main antioxidant compounds (geniposidic acid, chlorogenic acid, caffeic acid, and rutin) from functional plant tea (Eucommia ulmoides leaves). Single‐factor tests and response surface methodology were employed to optimize the extraction conditions for ultrasound‐assisted micellar extraction combined with ultra‐high‐performance liquid chromatography in succession. A Box‐Behnken design (three‐level, three‐factorial) was used to determine the effects of extraction solvent concentration (1–5 mg/mL), extraction solvent volume (5–15 mL), and extraction time (20–40 min) at a uniform ultrasonic power and temperature. In consequence, the best analyte extraction yields could be attained when the trehalose lipid solution concentration was prepared at 3 mg/mL, the trehalose lipid solution volume was 10 mL and the extraction time was set to 35 min. In addition, the recoveries of the antioxidants from Eucommia ulmoides leaves analyzed by this analytical method ranged from 98.2 to 102%. These results indicated that biosurfactant‐enhanced ultrasound‐assisted micellar extraction coupled with a simple ultra‐high‐performance liquid chromatography method could be effectively applied in the extraction and analysis of antioxidants from Eucommia ulmoides leaf samples.     

A screening method for the simultaneous detection of glucocorticoids,diuretics, stimulants,anti-oestrogens,beta-adrenergic drugs and anabolic steroids in human urine by LC-ESI-MS/MS

This paper presents a general screening method, based on liquid chromatography/mass spectrometry (LC/MS), for the simultaneous detection in human urine of 72 xenobiotics (21 diuretics, 16 synthetic glucocorticoids, 17 beta-adrenergic drugs, 10 stimulants, 5 anti-oestrogens and 3 anabolic steroids), excreted free or as glucuro-conjugates in urine. Although the method has been specifically designed and evaluated in view of its potential application to anti-doping analyses, it can also be effective in other areas of analytical toxicology. Sample preparation was based on two liquid/liquid separation steps (performed at alkaline and at acid pH, respectively) of hydrolyzed human urine, and then an assay by LC/MS-MS in positive and negative ionization mode using an electrospray ionization source (ESI) and multiple reaction monitoring (MRM) as the acquisition mode. The overall time needed for an LC run was less than 15 minutes. All compounds showed good reproducibility in terms of both the retention times (CV%<1) and the relative abundances of the diagnostic transitions (CV%<10). The limits of detection (LOD) were in the range of 1–50 ng/mL for glucocorticoids, anti-oestrogens and steroids, and 50–500 ng/mL for diuretics, beta-adrenergic drugs and stimulants, thus satisfying the minimum required performance limits (MRPL) set by the World Anti-Doping Agency (WADA) for the accredited anti-doping laboratories.     

Supercritical fluid extraction followed by supramolecular solvent microextraction as a fast and efficient preconcentration method for determination of polycyclic aromatic hydrocarbons in apple peels

In this work, reverse micelle‐based supramolecular solvent microextraction method coupled with supercritical fluid extraction and used for determining trace amounts of polycyclic aromatic hydrocarbons in apple peels. The extract was analyzed by high‐performance liquid chromatography equipped with a fluorescence detector. Coupling supramolecular solvent microextraction with supercritical fluid extraction method, resolve low preconcentration factor of supercritical fluid extraction method, improved limit of detection of polycyclic aromatic hydrocarbons and allow the use of supramolecular solvent microextraction in solid matrices. The effective parameters on the supramolecular solvent microextraction and supercritical fluid extraction efficiency were optimized using one variable at a time and face centered design methods, respectively. Under the optimum condition, the limits of detection and limits of quantifications were in the range of 0.34–1.27 and 1.03–3.82 µg/kg, respectively. Analysis of polycyclic aromatic hydrocarbons in apple peels showed that the supercritical fluid extraction/ supramolecular solvent microextraction method provide great potential for trace analysis of polycyclic aromatic hydrocarbons in fruit samples (RSDs < 7.7%).