Determination of cannabinoids in plasma using salting-out-assisted liquid–liquid extraction followed by LC–MS/MS analysis

The detection of the markers of Cannabis consumption in biological specimens is an important task for drug testing laboratories in varous contexts. A simple assay combining salting-out assisted liquid–liquid extraction sample preparation and LC–MS/MS analysis was applied to the measurement of Δ⁹-tetrahydrocannabinol, 11-nor-9-carboxy-Δ⁹-tetrahydrocannabinol (THC-COOH), 11-hydroxy-Δ⁹-tetrahydrocannabinol, cannabinol and cannabidiol concentrations in 100 μl plasma specimens. The assay had linearity of 1–100 ng ml⁻¹ for THC-COOH and 0.5–50 ng ml⁻¹ for the other tested cannabinoids. Assay validation criteria were fulfilled. Extraction yields (88.7–97.3%) and internal-standard correct matrix effects (−9.6 to +5.4%) were acceptable. The assay was applied to 238 clinical specimens from trauma patients, with 19 samples presenting quantifiable concentrations of at least one of the target compounds. The developed assay is a simple and efficient strategy for simultaneous measurement of Δ⁹-tetrahydrocannabinol, THC-COOH, 11-hydroxy-Δ⁹-tetrahydrocannabinol, cannabinol and cannabidiol concentrations in plasma specimens.     

Determination of decabromodiphenyl ether in sediments using selective pressurized liquid extraction followed by GC–NCI-MS

A method based on selective pressurized liquid extraction (SPLE) followed by gas chromatography–negative ion chemical ionization-mass spectrometry (GC–NCI-MS) has been evaluated for analysis of decabromodiphenyl ether (PBDE-209) in sediment samples. Instrumental operating conditions such as source temperature and system pressure were optimized in the NCI-MS system, giving an instrumental detection limit of 2 pg. The limit of determination of the entire SPLE–GC–NCI-MS procedure was around 50 pg g^–1 dry weight (dw), with repeatability of replicates between 4 and 21% relative standard deviation. Application of the method to 13 different river and marine sediment samples collected in Spain revealed that levels of decabromodiphenyl ether ranged between 2 and 132 ng g^–1 dry weight.     

Determination of chlorpyrifos in soils using multi-throughput dynamic microwave-assisted extraction coupled online with salting-out-assisted liquid–liquid extraction followed by LC–MS/MS

A simple and rapid method based on multi-throughput dynamic microwave-assisted extraction coupled online with salting-out-assisted liquid–liquid extraction was developed for the analysis of chlorpyrifos in soil. First, the chlorpyrifos was extracted with acetonitrile aqueous (50%, v/v) under the action of microwave energy. Then the obtained extract was separated clearly and easily into an acetonitrile phase and an aqueous phase with the assistance of ammonium acetate. The acetonitrile phase containing chlorpyrifos was concentrated and determined by liquid chromatography–tandem mass spectrometry. The effects of parameters on extraction efficiency including microwave power, extraction solvent, volumes and flow rate of extraction solvent, sample pH, types and amount of salt were studied and optimised. To eliminate the matrix effect, validation of the method was carried out using the matrix-based calibration curve. The limits of detection and quantification for chlorpyrifos were 0.17 and 0.5 ng g^?1, respectively. The proposed method was applied to analyse chlorpyrifos in five soil samples and verified by the recovery test. The recoveries of chlorpyrifos at three spiked levels (5, 50, 200 ng g^?1) were in the range of 90.0–100.5%, with relative standard deviations varying from 1.3% to 5.7%. Compared with the methods reported previously, the proposed method can simplify the operation procedure and reduce solvent consumption in sample pretreatment.     

Determination of oxygenated and native polycyclic aromatic hydrocarbons in urban dust and diesel particulate matter standard reference materials using pressurized liquid extraction and LC–GC/MS

The objective of this study was to develop a novel analytical chemistry method, comprised of a coupled high-performance liquid chromatography–gas chromatography/mass spectrometry system (LC–GC/MS) with low detection limits and high selectivity, for the identification and determination of oxygenated polycyclic aromatic hydrocarbons (OPAHs) and polycyclic aromatic hydrocarbons (PAHs) in urban air and diesel particulate matter. The linear range of the four OPAHs, which include 9,10-anthraquinone, 4H-cyclopenta[def]phenanthrene-4-one, benzanthrone, and 7,12-benz[a]anthraquinone, was 0.7 pg–43.3 ng with limits of detection (LODs) and limits of quantification (LOQs) on the order of 0.2–0.8 and 0.7–1.3 pg, respectively. The LODs in this study are generally lower than values reported in the literature, which can be explained by using large-volume injection. The recoveries of the OPAHs spiked onto glass fiber filters using two different pressurized liquid extraction (PLE) methods were in the ranges of 84–107 and 67–110 %, respectively. The analytical protocols were validated using the following National Institute of Standards and Technology standard reference materials: SRM 1649a (Urban Dust), SRM 1650b (Diesel Particulate Matter), and SRM 2975 (Diesel Particulate Matter, Industrial Forklift). The measured mass fractions of the OPAHs in the standard reference materials (SRMs) in this present study are higher than the values from the literature, except for benzanthrone in SRM 1649a (Urban Dust). In addition to the OPAHs, 44 PAHs could be detected and quantified from the same particulate extract used in this protocol. Using data from the literature and applying a two-sided t test at the 5 % level using Bonferroni correction, significant differences were found between the tested PLE methods for individual PAHs. However, the measured mass fractions of the PAHs were comparable, similar to, or higher than those previously reported in the literature.     

Determination of basic drugs of abuse in human serum by online extraction and LC–MS/MS

A new quantitation method for the determination of drugs of abuse (opiates, amphetamine and derivatives, cocaine, methadone and metabolites) in serum by using online extraction coupled to liquid chromatography (LC)–mass spectrometry (MS)/MS has been developed. The online extraction is carried out using two extraction columns simultaneously and one analytical column. One extraction column is loaded, while the other one is eluted by a gradient. The elution gradient also separates the analytes in the analytical column. For the sample preparation, serum is spiked with a mixture of deuterated analogues of the drugs. After protein precipitation with methanol/zinc sulphate, centrifugation, evaporation and reconstitution, the sample is injected into the LC system. The quantitation is based on the analysis of two multiple reaction monitoring transitions per drug. The recovery of the protein precipitation step is over 80% for all analytes. Intra- and interday precision, as relative standard deviation, is lower than 6%, and in the case of accuracy, RE is lower than 15%. Only the most polar analytes showed matrix effects. The limits of quantitation for the analysed compounds vary between 0.5 and 2.8 ng/mL. The developed method was used to quantify basic drugs in samples “from driving under the influence of drugs” cases. The results were compared with those obtained by using solid-phase extraction–GC–MS.     

Determination of pesticides in seaweeds by pressurized liquid extraction and programmed temperature vaporization-based large volume injection–gas chromatography–tandem mass spectrometry

A rapid method for the simultaneous identification and quantification of pesticide residues in edible seaweed has been developed. Target analytes were three pyrethroid, a carbamate and two organophosphorus pesticides. The procedure consists of a pressurized liquid extraction (PLE) with integrated clean-up, followed by gas chromatography coupled to tandem mass spectrometry. Five PLE parameters were investigated using a screening design: temperature, static extraction time, number of cycles, percent of flush volume and quantitative composition of the n-hexane/ethyl acetate extraction solvent. The effect of the in-cell clean-up with Florisil^® and graphitized carbon black adsorbents was investigated using a Doehlert response surface design. Large volumes of sample extracts were injected using a programmed-temperature vaporizer (PTV-LVI) to improve both sensitivity and selectivity of measurements. Quantification was carried by the internal standard method with surrogate deuterated standards. The method showed excellent linearity (R² > 0.999) and precision (relative standard deviation, RSD ≤ 8%) for all compounds, with detection limits ranging from 0.3 pg g⁻¹ for chlorpyrifos-ethyl, to 3.0 pg g⁻¹ for carbaryl (23.1 pg g⁻¹ for deltamethrin). Recoveries in real seaweed samples were within the range 82–108%. The method was satisfactory validated for the analysis of wild and cultivated edible seaweeds. The presence of pyrethroid and organophosphorus pesticides in some of the samples was evidenced.     

Determination of Fenofibric Acid in Human Plasma by LC–MS/MS and LC–UV

A sensitive, high-throughput and economic liquid chromatographic method for determination of fenofibric acid in human plasma was developed and validated by ultraviolet detection and tandem mass spectrometry, then applied in pharmacokinetic study to investigate Lipanthyl™ 200 mg MC bioavailability under food and fasting conditions. Fenofibric acid with 2-chloro fenofibric acid-d6 (internal standard) was extracted from 100 µL of human plasma by acetonitrile in a single extraction step. 25 and 2 µL from supernatant were injected onto ACE column, 50 mm, 5 micron with 4.6 mm inner diameter for LC–UV and 2.1 mm for LC–MS/MS, and both systems were eluted isocratically by water:methanol:formic acid (35:65:0.1, v/v/v), with a constant flow rate of 1 mL min⁻¹. The established calibration curve was linear between 0.05–20 µg mL⁻¹, and the within- and between-day precisions were all below 13 % in both LC–MS/MS and LC–UV systems during validation, and accuracies ranged between 91 and 112 %. Twenty-eight healthy adult subjects participated in this clinical study, and the pharmacokinetic parameters including coefficient of variation were calculated and discussed. A dramatic decrease in C _max and AUC0-72 (3.63- and 1.85-fold, respectively) were observed for Lipanthyl™ MC under fasting conditions with more variable inter subject measurements comparing to the fed state.

     

Analysis of pyrethroid pesticides in Chinese vegetables and fruits by GC–MS/MS

A rapid and economical method using modified QuEChERS sample pretreatment coupled with high-sensitivity gas chromatography/triple quadrupole mass spectrometry was established to simultaneously determine ten pyrethroid pesticides in fruits and vegetables. All pesticides were detected within 20 min of one injection. Concurrent backflushing provided column protection, greatly facilitating instrument maintenance. For quantitation, matrix-matched calibration was used to compensate for signal-enhancement effects and to ensure the precision of the method. The limit of detection (LOD) was in the range of 0.3–4.9 μg/kg. The recovery rate was from 78.8 to 118.6%, with relative standard deviation (RSD) below 14.8%. The developed method is suitable for rapid and sensitive multi-residue analysis of pyrethroid pesticides in fruits and vegetables. It is good for users in professional institutions that implement safety controls for testing hundreds of agricultural product samples everyday.     

Determination of Eleutherosides in Dietary Supplements by LC–MS/MS

A simple and specific method for the simultaneous determination of eleutherosides B and E in powdered rhizomes of Eleutherococcus senticosus extract and in solid and liquid dietary supplements was developed and validated. E. senticosus extracts, often mixed with other plants or herbal extracts, are widely used in food supplements because of the tonic and adaptogenic activities referred to the eleutherosides B and E. In this study, samples were analyzed by a liquid chromatography-electrospray tandem mass spectrometry (LC–ESI-MS/MS) method operated in single reaction monitoring (SRM). Validation was carried out in terms of limit of detection (LOD), limit of quantitation (LOQ), linearity, precision and trueness. LOD and LOQ values were fixed at 3 μg L^?1 and 10 μg L^?1, respectively, whereas linearity was established within 10–1,000 μg L^?1 range for both compounds. Good precision was obtained for both eleutherosides in terms of intra-day precision (RSD % lower than 4 %) and inter-day precision (RSD % lower than 6 %). Good percentage recoveries were obtained for both eleutherosides (91.5–103.6 %). Finally, the developed method was successfully applied to analyze a number of solid and liquid commercial dietary supplements containing E. senticosus extracts, also mixed with other herbal extracts.     

Determination of steroid hormones in blood by GC–MS/MS

This paper presents the development, optimization and validation of a methodology to determine nine key steroid hormones (viz. pregnenolone, progesterone, dehydroepiandrosterone, androstenedione, testosterone, dihydrotestosterone, estrone, 17α-estradiol and 17β-estradiol) expressed in the steroidogenesis in biological fluids. The analytical method allows for the determination of steroid hormones in blood plasma and serum down to 0.08–0.16 ng/mL for estrogens, 0.20–0.36 ng/mL for androgens and 0.36–0.43 ng/mL for progestagens. These limits of detection were obtainable using a two-step solid-phase clean-up for fractionation and elimination of interfering lipids (fatty acids, phospholipids, glycerides and sterols) from the steroid hormones. The accuracy of the method was 50–112% in the range 0.10 to 2.00 ng/mL.     

Determination of carbohydrates in tobacco by pressurized liquid extraction combined with a novel ultrasound-assisted dispersive liquid–liquid microextraction method

A novel derivatization-ultrasonic assisted-dispersive liquid–liquid microextraction (UA-DLLME) method for the simultaneous determination of 11 main carbohydrates in tobacco has been developed. The combined method involves pressurized liquid extraction (PLE), derivatization, and UA-DLLME, followed by the analysis of the main carbohydrates with a gas chromatography-flame ionization detector (GC-FID). First, the PLE conditions were optimized using a univariate approach. Then, the derivatization methods were properly compared and optimized. The aldononitrile acetate method combined with the O-methoxyoxime-trimethylsilyl method was used for derivatization. Finally, the critical variables affecting the UA-DLLME extraction efficiency were searched using fractional factorial design (FFD) and further optimized using Doehlert design (DD) of the response surface methodology. The optimum conditions were found to be 44 μL for CHCl₃, 2.3 mL for H₂O, 11% w/v for NaCl, 5 min for the extraction time and 5 min for the centrifugation time. Under the optimized experimental conditions, the detection limit of the method (LODs) and linear correlation coefficient were found to be in the range of 0.06–0.90 μg mL⁻¹ and 0.9987–0.9999. The proposed method was successfully employed to analyze three flue-cured tobacco cultivars, among which the main carbohydrate concentrations were found to be very different.     

Determination of carbamates and organophosphorus pesticides by SDME–GC in natural water

Water contamination due to the wide variety of pesticides used in agriculture practices is a global environmental pollution problem. Analytical methods with low quantification limits are necessary. The application of a new extraction technique, solvent drop microextraction (SDME), followed by gas chromatography with a nitrogen-phosphorus detector, was assessed for determining carbamates and organophosphorus pesticides in natural water. Experimental parameters which control the performance of SDME such as selection of microextraction solvent, optimization of organic drop volume, effects of sample stirring, salt addition, and, finally, sorption time profiles were studied. Once SDME was optimized, analytical parameters such as linearity (r ²>0.99), precision (<13%), and detection limits (0.2 to 5 μg/L), plus matrix effects were evaluated (no matrix effects were found). SDME is a dynamic technique able to extract pesticides from water in 14 min; the use of organic solvents and water samples for SDME is negligible compared to other extraction techniques.     

Determination of chlormequat in pig serum and sow milk by LC–MS/MS

Chlormequat is a plant growth regulator widely used on cereals, and there is general concern that it may impair human fertility. A LC–MS/MS method for the analysis of chlormequat in milk and serum was developed and validated in connection with an investigation on the effect of chlormequat on pig reproduction. Validation of the method was based on recovery tests at three spiking levels, determined as double determinations and repeated at least four times. Samples were extracted with methanol–water–acetic acid, centrifuged, filtrated and determined by LC–MS/MS. The mean recoveries were in the range 80–110%, and the LOD was 0.2 ng/g for serum and 0.3 ng/g for milk. The values for repeatability and reproducibility were within 2/3 of the limits given by the Horwitz equation. Samples of pig serum (59) and sow milk (27) were analyzed using the method. Chlormequat was determined in four milk samples in the range of 0.4 ng/g to 1.2 ng/g and in all serum samples in the range of 0.2 ng/g−4.0 ng/g.     

Determination of Pitavastatin in Human Plasma by LC–MS–MS

A simple and rapid LC–MS–MS assay was developed and validated for the quantitative determination of pitavastatin in human plasma. Sample pretreatment involved simple protein precipitation by addition of acetonitrile. Separation was on an Agilent 1.8 μm Zorbax SB-C18 column (150 mm × 4.6 mm) at 25 °C using isocratic elution with methanol–0.1% formic acid in water (85:15, v/v) at a flow rate of 0.4 mL min^?1. Detection was performed using electrospray ionization in positive ion multiple reaction monitoring mode by monitoring the ion transitions m/z 422.0 → 290.1 for pitavastatin, and m/z 330.1 → 192.1 for paroxetine (IS). LC–MS–MS was found to improve the quantitation of pitavastatin in plasma and was successfully applied in pharmacokinetic studies.     

Determination of antifouling pesticides and their degradation products in marine sediments by means of ultrasonic extraction and HPLC–APCI–MS

A method has been developed for the simultaneous determination of antifouling pesticides and some of their degradation products, e.g. dichlofluanid, diuron, demethyldiuron, 1-(3,4-dichlorophenyl)urea, sea-nine, Irgarol 1051 and one of its metabolites (2-methylthio-4-tert-butylamino-s-triazine) in marine sediments. The determination of these compounds in sediment samples was performed by means of methanolic ultrasonic extraction then clean-up on an Isolute ENV+ solid phase extraction (SPE) cartridge. The resulting extract was then analyzed by reversed-phase high-performance liquid chromatography coupled with atmospheric-pressure chemical-ionization mass spectrometry in negative and positive ion modes (HPLC–APCI–MS). Recovery ranged from 54–109% for the antifouling agents and their degradation products. The determination limits for the different compounds varied between 0.2 and 1.6 μg kg^–1 dry sediment. The analytical procedure was successfully applied to the determination of these pesticides and their degradation products in marine sediment samples from different marinas of the Catalan coast. The compounds detected were: diuron, dichlofluanid, demethyldiuron, sea-nine, and Irgarol 1051. The highest concentrations were those of diuron and Irgarol 1051 – 136 and 88 μg kg^–1, respectively.     

Simultaneous Determination of Tamsulosin and Dutasteride in Human Plasma by LC–MS–MS

A sensitive and selective liquid chromatographic tandem mass spectrometric (LC–MS–MS) method was developed for simultaneous identification and quantification of tamsulosin and dutasteride in human plasma, which was well applied to clinical study. The method was based on liquid–liquid extraction, followed by an LC procedure with a Gemini C-18, 50 mm × 2.0 mm (3 μm) column and using methanol:ammonium formate (97:3, v/v) as the mobile phase. Protonated ions formed by a turbo ionspray in positive mode were used to detect analytes and internal standard. MS–MS detection was by monitoring the fragmentation of 409.1 → 228.1 (m/z) for tamsulosin, 529.3 → 461.3 (m/z) for dutasteride and 373.2 → 305.3 (m/z) for finasteride (IS) on a triple quadrupole mass spectrometer. The lower limit of quantification for both tamsulosin and dutasteride was 1 ng mL^?1. The proposed method enables the unambiguous identification and quantification of tamsulosin and dutasteride for clinical drug monitoring.     

Detection and Confirmation of Ginsenosides in Horse Urine by GC–MS and LC–MS

Ginseng has been used by the Chinese as a traditional herbal medicine for thousands of years. In view of the growing popularity in the use of ginseng preparations as natural remedies and food supplements worldwide, there is an increasing concern for their abuse in both human and animal sports. Ginsenosides are considered the major constituents of ginseng responsible for its pharmacological properties. In this study, a method was developed for the detection and confirmation of a number of ginsenosides in horse urine. The intact ginsenosides were detected and confirmed at 5–100 ng mL^?1 by LC–MS², and two deglycosylation metabolites, namely protopanaxadiol and protopanaxatriol, could both be detected and confirmed at 2 ng mL^?1 by GC–MS² after trimethylsilylation. The above GC–MS and LC–MS methods were then applied to study the in vitro metabolism of ginsenosides Rg1 and Rb1 and the in vivo urinary metabolites after oral administration of Rg1 to horses. Results obtained reveal the very first evidence for the existence of the metabolites, Rg1 and protopanaxatriol, as glucuronides in urine.     

Simultaneous Determination of Atenolol and Chlorthalidone by LC–MS–MS in Human Plasma

A simple, sensitive, selective and rapid liquid chromatography–tandem mass spectrometry method was developed and validated for the simultaneous separation and quantitation of atenolol and chlorthalidone in human plasma using metoprolol and hydrochlorothiazide as internal standard. Following solid phase extraction, the analytes were separated by an isocratic mobile phase on a reversed-phase C₁₈ column and analyzed by MS in the multiple reaction-monitoring mode (atenolol in positive and chlorthalidone in the negative ion mode). The limit of quantitation for this method was 10 and 15 ng mL^?1 and the linear dynamic range was generally 10–2,050 ng mL^?1 and 15–3,035 ng mL^?1 for atenolol and chlorthalidone, respectively.     

Determination of atorvastatin and metabolites in human plasma with solid-phase extraction followed by LC–tandem MS

The aim of the present study was to develop a chromatographic method for the analysis of atorvastatin, o- and p-hydroxyatorvastatin (acid and lactone forms) in human plasma after administration of atorvastatin at the lowest registered dose (10 mg) in clinical studies. Sample preparation was performed by solid-phase extraction and was followed by separation of the analytes on an HPLC system with a linear gradient and a mobile phase consisting of acetonitrile, water and formic acid. Detection was achieved by tandem mass spectrometry operated in the electrospray positive ion mode. Validation of the method for the compounds for which reference compounds were available (acid forms of atorvastatin, o- and p-hydroxyatorvastatin) showed linearity within the concentration range (0.2–30 ng/ml for atorvastatin acid and p-hydroxyatorvastatin acid, and 0.5–30 ng/ml for o-hydroxyatorvastatin acid) (r²0.99, n=5 for all analytes). Accuracy and precision (evaluated at 0.5, 3 and 30 ng/ml for atorvastatin, p-hydroxyatorvastatin and 1, 3 and 30 ng/ml for o-hydroxyatorvastatin) were both satisfactory. The detection limit was 0.06 ng/ml for atorvastatin and p-hydroxyatorvastatin, and 0.15 ng/ml for o-hydroxyatorvastatin. The method has been successfully applied in a clinical study where atorvastatin, o- and p-hydroxyatorvastatin (both acid and lactone forms) could be detected in a 24-h sampling interval after administration of the lowest registered dose of atorvastatin (10 mg) for one week.