Determination of benzimidazole- and bicyclic hydantoin-derived selective androgen receptor antagonists and agonists in human urine using LC–MS/MS

Selective androgen receptor modulators (SARMs) represent a novel class of drugs with tissue-specific agonistic and antagonistic properties, which are prohibited in sports from January 2008 according to the World Anti-Doping Agency. Preventive approaches to restrict the use of SARMs include early implementation of target analytes into doping control screening assays. Five model SARMs were synthesized, four of which are analogs to prostate-specific androgen receptor antagonists with a 5,6-dichloro-benzimidazole nucleus. The fifth SARM is a muscle-tissue specific agonist with a bicyclic hydantoin structure (BMS-564929). Dissociation pathways after negative electrospray ionization were studied using an LTQ-Orbitrap mass analyzer, and diagnostic product ions and common fragmentation patterns were employed to establish a screening procedure that target the intact SARMs as well as putative metabolic products. Sample preparation based on solid-phase extraction and subsequent LC–MS/MS measurement allowed for detection limits of 1–20 ng/mL, intra- and interday precisions of between 2.4 and 13.2% and between 6.5 and 24.2%, respectively. Recoveries varied from 89 to 106%, and tests for ion suppression or enhancement effects were negative for all analytes. Figure Precursor ion scanning as a tool to screen for the common nucleus of benzimidazole-derived SARMs     

Packed in-tube SPME–LC–MS/MS for fast and straightforward analysis of cannabinoids and metabolites in human urine

Cannabinoids are pharmacologically active compounds present in cannabis plants, which have become important research topics in the modern toxicological and medical research fields. Not only is cannabis the most used drug globally, but also cannabinoids have a growing use to treat a series of diseases. Therefore, new, fast, and efficient analytical methods for analyzing these substances in different matrices are demanded. This study developed a new packed-in-tube solid-phase microextraction (IT-SPME) method coupled to liquid chromatography with tandem mass spectrometry (LC–MS/MS), for the automated microextraction of seven cannabinoids from human urine. Packed IT-SPME microcolumns were prepared in (508 µm i.d. × 50 mm) stainless-steel hardware; each one required only 12 mg of sorbent phase. Different sorbents were evaluated; fractional factorial design 2⁴⁻¹ and a central composite design were employed for microextraction optimization. Under optimized conditions, the developed method was a fast and straightforward approach. Only 250 µl of urine sample was needed, and no hydrolysis was required. The sample pretreatment included only dilution and centrifugation steps (8 min), whereas the complete IT-SPME–LC–MS/MS method took another 12 min, with a sample throughput of 3 samples h⁻¹. The developed method presented adequate precision, accuracy and linearity; R² values ranged from 0.990 to 0.997, in the range of 10–1000 ng ml⁻¹. The lower limits of quantification varied from 10 to 25 ng ml⁻¹. Finally, the method was successfully applied to analyze 20 actual urine samples, and the IT-SPME microcolumn was reused over 150 times.     

Determination of vancomycin and meropenem in serum and synovial fluid of patients with prosthetic joint infections using UPLC–MS/MS

Numerous studies have suggested that intra-articular administration of antibiotics following primary revision surgery may be one of the methods for treating prosthetic joint infection (PJI). Vancomycin and meropenem are the two most commonly used antibiotics for local application. Determining the concentrations of vancomycin and meropenem in the serum and synovial fluid of patients with PJI plays a significant role in further optimizing local medication schemes and effectively eradicating biofilm infections. This study aimed to establish a rapid, sensitive, and accurate ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method for determining the concentrations of vancomycin and meropenem in human serum and synovial fluid. Serum samples were processed using acetonitrile precipitation of proteins and dichloromethane extraction, while synovial fluid samples were diluted before analysis. Chromatographic separation was achieved in 6 min on a Waters Acquity UPLC BEH C18 column, with the mobile phase consisting of 0.1% formic acid in water (solvent A) and acetonitrile (solvent B). Quantification was carried out using a Waters XEVO TQD triple quadrupole mass spectrometer with an electrospray ionization (ESI) source in positive ion mode. The multiple reaction monitoring (MRM) mode was employed to detect the following quantifier ion transitions: 717.95–99.97 (norvancomycin), 725.90–100.04 (vancomycin), 384.16–67.99 (meropenem). The method validation conformed to the guidelines of the FDA and the Chinese Pharmacopoeia. The method demonstrated good linearity within the range of 0.5–50 μg/ml for serum and 0.5–100 μg/ml for synovial fluid. Selectivity, intra-day and inter-day precision and accuracy, extraction recovery, matrix effect, and stability validation results all met the required standards. This method has been successfully applied in the pharmacokinetic/pharmacodynamic (PK/PD) studies of patients with PJI.     

Determination of γ-hexachlorocyclohexane and its metabolites in biological samples from rat

We have determined γ-hexachlorocyclohexane (lindane) and its metabolites in urine, serum and feces samples from rats using HPLC-UV-Vis and confirmation of mass with matrix assisted laser desorption/ ionization-time of flight (MALDI-TOF) analysis. Samples were collected from rats treated orally with lindane (17.6 mg/kg; 1/5 of LD₅₀) or vehicle for 2 weeks. Lindane and metabolites were extracted from samples with hexane and analyzed. The HPLC–MALDI-TOF is highly sensitive to the point of detecting very low level (5 ppm) of lindane and metabolites. The HPLC-UV-Vis analysis confirmed the presence of lindane in urine (386–1652 ppm), serum (207–371 ppm) and feces (5–74 ppm). Control samples had no peak corresponding to lindane. MALDI-TOF analysis of urine and serum samples showed a major peak at 293 m/z, whereas feces showed a minor peak at 292–293 m/z, which were consistent with the peak obtained for standard lindane (293 m/z). Our data indicates that HPLC-UV-Vis–MALDI-TOF combo method is sensitive for detecting and quantifying lindane and its metabolites in serum, urine and feces. Our results further showed that minor quantities of lindane and metabolites were excreted through feces confirming that the main pathway for excretion of lindane and metabolites is through urine.     

Simultaneous and sensitive LC–MS/MS determination of tetrahydrocannabinol and metabolites in human plasma

Cannabis is not only a widely used illicit drug but also a substance which can be used in pharmacological therapy because of its analgesic, antiemetic, and antispasmodic properties. A very rapid and sensitive method for determination of ?⁹-tetrahydrocannabinol (THC), the principal active component of cannabis, and two of its phase I metabolites in plasma has been developed and validated. After solid-phase extraction of plasma (0.2 mL), the clean extracts were analyzed by tandem mass spectrometry after a 5-min liquid chromatographic separation. The linear calibration ranges were from 0.05 to 30 ng?mL^?1 for THC and 11-nor-?⁹-carboxy-tetrahydrocannabinol (THC-COOH) and from 0.2 to 30 ng?mL^?1 for ?⁹-(11-OH)-tetrahydrocannabinol (11-OH-THC). Imprecision and inaccuracy were always below 7 and 12 % (expressed as relative standard deviation and relative error), respectively. The method has been successfully applied to determination of the three analytes in plasma obtained from healthy volunteers after oral administration of 20 mg dronabinol.     

Enantioselective CE–MS analysis of ketamine metabolites in urine

The chiral drug ketamine has long-lasting antidepressant effects with a fast onset and is also suitable to treat patients with therapy-resistant depression. The metabolite hydroxynorketamine (HNK) plays an important role in the antidepressant mechanism of action. Hydroxylation at the cyclohexanone ring occurs at positions 4, 5, and 6 and produces a total of 12 stereoisomers. Among those, the four 6HNK stereoisomers have the strongest antidepressant effects. Capillary electrophoresis with highly sulfated γ-cyclodextrin (CD) as a chiral selector in combination with mass spectrometry (MS) was used to develop a method for the enantioselective analysis of HNK stereoisomers with a special focus on the 6HNK stereoisomers. The partial filling approach was applied in order to avoid contamination of the MS with the chiral selector. Concentration of the chiral selector and the length of the separation zone were optimized. With 5% highly sulfated γ-CD in 20 mM ammonium formate with 10% formic acid and a 75% filling the four 6HNK stereoisomers could be separated with a resolution between 0.79 and 3.17. The method was applied to analyze fractionated equine urine collected after a ketamine infusion and to screen the fractions as well as unfractionated urine for the parent drug ketamine and other metabolites, including norketamine and dehydronorketamine.     

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.     

Determination of oleandrin and adynerin in rat plasma by UPLC–MS/MS and their pharmacokinetic study

Oleandrin and adynerin are the main toxic components of oleander, an evergreen shrub or a small tree of the oleander family, which belongs to the class of cardiac glycosides exhibiting delayed action. The pharmacokinetic differences of oleandrin and adynerin in rats were studied by ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) under two different administration modes: oral (5 mg/kg) and sublingual intravenous injection (1 mg/kg). The chromatographic column was UPLC BEH C18 (50 mm × 2.1 mm, 1.7 μm), and the column temperature was set at 40 °C. The mobile phase was acetonitrile–water (containing 0.1 % formic acid), with gradient elution, the flow rate was 0.4 mL/min, and the elution time was 4 min. Electrospray (ESI) positive ion mode detection with multiple reaction monitoring mode (MRM) was used for quantitative analysis: oleandrin m/z 577 → 145, adynerin m/z 534 → 113, and internal standard m/z 237 → 135. The established UPLC–MS/MS method was successfully applied to the pharmacokinetics in rats after administering oleandrin and adynerin. The bioavailability of oleandrin and adynerin was found to be low, 7.0 % and 93.1 %; respectively.     

New UPLC–MS/MS method for simultaneous determination of irbesartan and hydrochlorthiazide in human plasma

Ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) is a preeminent analytical tool for rapid biomedical analysis with the objective of reducing analysis time and maintaining good efficiency. In this study a simple, rapid, sensitive and specific ultra-performance liquid chromatography–tandem mass spectrometry method was developed and validated for quantification of the angiotensin II receptor antagonist, irbesartan and hydrochlorthiazide in human plasma. After a simple protein precipitation using methanol and acetonitrile, irbesartan, hydrochlorthiazide and internal standard (IS) telmisartan were separated on Acquity UPLC BEH? C18 column (50 × 2.1 mm, i.d. 1.7 μm, Waters, USA) using a mobile phase consisting of acetonitrile:10 mM ammonium acetate:formic acid (85:15:0.1 % v/v/v) pumped at a flow rate of 0.3 mL/min and detected by tandem mass spectrometry with negative ion mode. The ion transitions recorded in multiple reaction monitoring mode were m/z 427.2 → 193.08 for irbesartan, m/z 295.93 → 268.90 for hydrochlorthiazide and m/z 513.2 → 287.14 for IS. The assay exhibited a linear dynamic range of 30–500 ng/mL for irbesartan and 1–500 ng/mL in human plasma with good correlation coefficient of (0.996) and (0.997) and with a limit of quantitation of 30  and 1 ng/mL for irbesartan and hydrochlorthiazide, respectively. The intra- and inter-assay precisions were satisfactory; the relative standard deviations did not exceed 10.13 % for irbesartan and 11.14 % for hydrochlorthiazide. The proposed UPLC–MS/MS method is simple, rapid and highly sensitive, and hence it could be reliable for pharmacokinetic and toxicokinetic study in both animals and humans.     

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.     

Identification of in vitro and in vivo metabolites of 12β-hydroxylveratroylzygadenine associated with neurotoxicity by using HPLC–MS/MS

Metabolism study was carried out on 12b-hydroxylveratroylzygadenine(VOG) that is a cevine-type alkaloid existing in Veratrum nigrum L. and a neurotoxic component. In order to better understand the potential mechanism of neurotoxicity of VOG, this study measured VOG-induced DNA damage in the cerebellum and cerebral cortex of mice after 7 days repetitive oral dose by using single-cell gel electrophoresis(Comet assay). High performance liquid chromatography-tandem mass spectrometry(LC–MS/MS) was developed and applied to separate and identify in vitro and in vivo metabolites of VOG for investing the possible relationship of metabolism and neurotoxicity. In vitro experiment was carried out using rat liver microsomes, while the in vivo study was conducted on rats. The obtained results indicated that VOG might cause DNA damage in cerebellum and cerebral cortex of mice in a dosedependent manner. Hydrolysis of ester bond and O-demethylation were proposed to be the main in vivo metabolic pathways of VOG, while the major in vitro metabolic pathways were proposed as methyl oxidation to aldehyde, dehydrogenation, hydrolysis of ester bond, hydrolysis of ester bond together with acetylation, and methoxylation. O-Demethylation reaction was likely to be associated with reactive oxygen species production, leading to the DNA damage.     

Determination of carbofuran, carbaryl and their main metabolites in plasma samples of agricultural populations using gas chromatography–tandem mass spectrometry

A gas chromatography–tandem mass spectrometric (GC-MS/MS) method has been developed for the determination of carbofuran (2,3-dihydro-2,2-dimethylbenzofuran-7-yl methylcarbamate), carbaryl (1-naphthyl-N-methylcarbamate) and their main metabolites in human blood plasma. Optimization of the isolation of the compounds from plasma matrix included the precipitation, denaturation and digestion of plasma proteins. Derivatization was achieved by the use of trifluoroacetic acid anhydride and was optimized for temperature, time and volume of derivatization agent. In the proposed method, a mild precipitation technique was applied using β-mercaptoethanol and ascorbic acid in combination with solid-phase extraction technique using Oasis HLB (Hydrophobic Lipophilic Balance) cartridges for further clean up of samples. Carbamate linkage was not hydrolyzed to its phenol product, but both carbamate phenol and ketones were transformed into trifluoroacetyl derivatives in order to become volatile compounds and were determined using tandem mass spectrometry. The linearity of the method was shown for nine concentrations in the range of 0.50–250 ng mL⁻¹ in fortified plasma aliquots. Limits of detection (LODs) for all compounds ranged from 0.015–0.151 ng mL⁻¹. Inter-day and intra-day assays (RSD) for all compounds, at three concentration levels of 2.5, 25 and 100 ng mL⁻¹ (n=3) in fortified plasma samples were less than 18%. Accuracy (%E _r) was calculated at three concentration levels, 8, 80 and 160 ng mL⁻¹ (n=3), and ranged from −12.0 to 15.0%. Matrix effect was evaluated so mean recoveries were calculated for all compounds and ranged from 81–107%. Specificity for the use of this method to biological monitoring studies was achieved including four main metabolites of CF, 1-naphthol and 2-naphthol from the naphthalene metabolism pathways, and both the parent compound of carbofuran and carbaryl. The proposed method was applied to plasma samples of pesticide users.     

Comparison of LC and UPLC Coupled to MS–MS for the Determination of Sulfonamides in Egg and Honey

Determination of ten sulfonamides (SAs) in egg and honey has been compared using column liquid chromatography (LC) and ultra-performance liquid chromatography (UPLC) coupled to tandem mass spectrometry (MS–MS). A liquid–liquid extraction with acetonitrile followed by solid-phase extraction on a Strata-X cartridge was developed for sample preparation. The analytical performance of both methods was compared applying the alternative matrix-comprehensive in-house validation approach using specially designed software InterVal?. Using UPLC the separation time was shortened about 30% reducing the run time by 8 min and a better resolution was achieved compared to LC. Due to higher peak efficiency achieved with UPLC, the decision limit values obtained by both techniques were almost equal (6.61–9.43 μg kg^?1 and 7.25–11.9 μg kg^?1 for UPLC and LC, respectively), despite the fact that in UPLC twice lower sample volumes were injected. Satisfactory and comparable recoveries (80–110%) were obtained by UPLC and LC for all the SAs, except for sulfacetamide by LC and sulfabenzamide by both methods. For a majority of the spiked compounds, UPLC gave significantly better precision.     

Determination of ecgonine and seven other cocaine metabolites in human urine and whole blood by ultra-high-pressure liquid chromatography–quadrupole time-of-flight mass spectrometry

Ecgonine is suggested to be a promising marker of cocaine (COC) ingestion. A combined mass spectrometry (MS) and tandem MS (MS/MS) method was developed to simultaneously determine ecgonine and seven other metabolites of cocaine in human urine and whole blood with ultra-high-pressure liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. The compounds were extracted from as little as 100 μL of sample by solid-phase extraction with a 96-well μElution solid-phase extraction plate. The protonated molecules or fragment ions at accurate mass acquired in MS mode were used to quantify specific analytes, following by dedicated MS/MS identification. The assay was linear in the range from 5 to 50-100 ng/mL for urine samples, except for ecgonine methyl ester (10-200 ng/mL) and ecgonine (40-400 ng/mL), and was linear from 1-2 to 50 ng/mL for whole blood samples, except for ecgonine methyl ester (20-1,000 ng/mL) and ecgonine (40-2,000 ng/mL). The correlation coefficients were all greater than 0.99. The limits of detection ranged from 0.2 to 16 ng/mL, and the lower limits of quantification ranged from 1 to 40 ng/mL. The repeatability and intermediate precision were 18.1 % or less. The accuracy was in the range from 80.0 to 122.9 %, process efficiencies were in the range from 8.6 to 177.4 %, matrix effects were in the range from 28.7 to 171.0 %, and extraction recoveries were in the range from 41.0 to 114.3 %, except for ecgonine (12.8 % and 9.3 % at low and high concentrations, respectively). This method was highly sensitive in comparison with previously published methods. The validated method was successfully applied to the analysis of real samples derived from forensic cases, and the results verified that, on the basis of data from four positive samples, ecgonine is a promising marker of cocaine ingestion.

Determination of trace elements in human liver biopsy samples by ICP–MS and TXRF: hepatic steatosis and nickel accumulation

Human liver biopsy samples, collected from 52 individuals, were analysed by inductively coupled plasma–mass spectrometry (ICP–MS) and total reflection X-ray fluorescence (TXRF) spectrometry in a retrospective study (i.e. patient selection and liver biopsy were not for the purpose of element analysis). The freeze-dried samples (typically 0.5–2 mg dry weight) were digested in a laboratory microwave digestion system and solutions with a final volume of 1 mL were prepared. The concentrations of Cr, Mn, Fe, Ni, Cu, Zn, Rb, and Pb were determined by use of a Thermo Elemental X7 ICP–MS spectrometer. TXRF measurements were performed with an Atomika Extra IIA spectrometer. Yttrium was employed as an internal standard, prepared by dissolution of 5N-purity yttria (Y₂O₃) in our laboratory. The accuracy was tested by analysis of NIST 1577a Bovine Liver certified reference material. The concentrations of Fe, Cu, Zn, and Rb determined in human liver biopsy samples were in good agreement with data published by other authors. The distribution of nickel in the samples was surprisingly uneven—nickel concentrations ranged from 0.7 to 12 μg g⁻¹ (dry weight) in 38 samples and in several samples were extremely high, 36–693 μg g⁻¹. Analysis of replicate procedural blanks and control measurements were performed to prevent misinterpretation of the data. For patients with steatosis (n=14) Ni concentrations were consistently high except for two who had levels close to those measured for the normal group. As far as we are aware no previous literature data are available on the association of steatosis with high concentration of nickel in human liver biopsies taken from living patients. This paper was presented in part at the 2005 European Winter Conference on Plasma Spectrochemistry Budapest, Hungary.     

SPMMTE and Q-TOF–UPLC–MS for monitoring of atenolol and atorvastatin in human plasma using pentafluoro phenyl column

Iron nanocomposite adsorbent was prepared by green technology with 90% yield. The prepared iron nanocomposite adsorbent was used in solid-phase micromembrane tip extraction (SPMMTE) sample preparation technique. Analysis of atenolol and atorvastatin was performed in human plasma using SPMMTE and Q-TOF–UPLC–MS methods. New generation Acquity UPLC HSS penta fluoro phenyl (2.1?×?75?mm²; 1.8?µm) column was used with acetonitrile–0.1% formic acid in water (50:50 v/v) as mobile phase. The flow rate was 0.2?mL?min^?1 with electrospray mass detection. The limits of detection were 0.2 and 0.4?ng active mass for atenolol and atorvastatin, while the limits of quantification were 1.0 and 2.0?ng active mass, respectively. The values of the retention times were 3.224 and 3.907 for atorvastatin and atenolol. The values of the separation and resolution factors were 1.31 and 1.71, respectively. The peaks were sharp with base lined separation within 4.2?min. The developed SPMMTE and Q-TOF–UPLC–MS methods were reproducible, fast, precise, robust, rugged, and economic for the analyses of atenolol and atorvastatin in human plasma. The reported methods can be applied for monitoring of the reported drugs at trace level.     

UHPLC–MS/MS method for the determination of bisphenol A and its chlorinated derivatives,bisphenol S,parabens, and benzophenones in human urine samples

In the present work, a new method based on a sample treatment by dispersive liquid–liquid microextraction (DLLME) for the extraction of six bisphenols (bisphenol A, bisphenol S, and monochloro-, dichloro-, trichloro-, and tetrachlorobisphenol A), four parabens (methyl-, ethyl-, propyl-, and butylparaben), and six benzophenones (benzophenone-1, benzophenone-2, benzophenone-3, benzophenone-6, benzophenone-8, and 4-hydroxybenzophenone) in human urine samples, followed by ultrahigh-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) analysis, is validated. An enzymatic treatment allows determining the total content of the target EDCs. The extraction parameters were accurately optimized using multivariate optimization strategies. Ethylparaben ring-¹³C₆, benzophenone-d₁₀, and bisphenol A-d₁₆ were used as surrogates. Limits of quantification ranging from 0.1 to 0.6 ng mL^?1 and interday variabilities (evaluated as relative standard deviations) from 2.0 to 13.8 % were obtained. The method was validated using matrix-matched standard calibration followed by a recovery assay with spiked samples. Recovery rates ranged from 94 to 106 %. A good linearity, for concentrations up to 300 ng mL^?1 for parabens and 40 ng mL^?1 for benzophenones and bisphenols, was also obtained. The method was satisfactorily applied for the determination of target compounds in human urine samples from 20 randomly selected individuals.     

Determination of multiclass pesticides in food commodities by pressurized liquid extraction using GC–MS/MS and LC–MS/MS

Pressurized liquid extraction (PLE) was applied to the simultaneous extraction of a wide range of pesticides from food commodities. Extractions were performed by mixing 4 g of sample with 4 g of Hydromatrix and (after optimization) a mixture of ethyl acetate:acetone (3:1, v/v) as extraction solvent, a temperature of 100°C, a pressure of 1000 psi and a static extraction time of 5 min. After extraction, the more polar compounds were analyzed by liquid chromatography (LC), and the apolar and semipolar pesticides by gas chromatography (GC); in both cases LC and GC were coupled with mass spectrometry in tandem (MS/MS) mode. The overall method (including the PLE step) was validated in GC and LC according to the criteria of the SANCO Document of the European Commission. The average extraction recoveries (at two concentration levels) for most of the analytes were in the range 70–80%, with precision values usually lower than 15%. Limits of quantification (LOQ) were low enough to determine the pesticide residues at concentrations below or equal to the maximum residue levels (MRL) specified by legislation. In order to assess its applicability to the analysis of real samples, aliquots of 15 vegetable samples were processed using a conventional extraction method with dichloromethane, and the results obtained were compared with the proposed PLE method; differences lower than 0.01 mg kg⁻¹ were found.     

Simultaneous Determination of Fluoroquinolones, Tetracyclines and Sulfonamides in Chicken Muscle by UPLC–MS–MS

A rapid and sensitive analytical method for the simultaneous determination of four fluoroquinolones, four tetracyclines and six sulfonamides in chicken muscle using ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC–MS–MS) has been developed and validated. Samples were extracted with McIlvaine buffer-acetonitrile, defatted with n-hexane, and analyzed by UPLC–MS–MS. Solvent delay technique was applied in the analysis to remove the non-volatile phosphate and carry out farther on-line SPE clean-up. Satisfactory recoveries (55–110%) of all the veterinary drugs were demonstrated in 1, 10 and 20 μg kg^?1 spiked levels with the overall RSD for intra- and inter-day of 14 analytes less than 18%. The LOD and LOQ were 0.3 and 1.0 μg kg^?1, respectively. Quantitative results of 103 real samples indicated that the present method was suitable for the quantitative analysis of real samples.     

Studies on the metabolism of the α-pyrrolidinophenone designer drug methylenedioxy-pyrovalerone (MDPV) in rat and human urine and human liver microsomes using GC-MS and LC-high-resolution MS and its detectability in urine by GC-MS

Since the late 1990s, many derivatives of the α-pyrrolidinophenone (PPP) drug class appeared on the drugs of abuse market. The latest compound was described in 2009 to be a classic PPP carrying a methylenedioxy moiety remembering the classic entactogens (ecstasy). Besides Germany, 3,4-methylene-dioxypyrovalerone (MDPV) has appeared in many countries in Europe and Asia, indicating its worldwide importance for forensic and clinical toxicology. The aim of the presented work was to identify the phase I and II metabolites of MDPV and the human cytochrome-P450 (CYP) isoenzymes responsible for its main metabolic step(s). Finally, the detectability of MDPV in urine by the authors' systematic toxicological analysis (STA) should be studied. The urine samples were extracted after and without enzymatic cleavage of conjugates. The metabolites were separated and identified after work-up by GC-MS and liquid chromatography (LC)-high-resolution MS (LC-HR-MS). The studies revealed the following phase I main metabolic steps in rat and human: demethylenation followed by methylation, aromatic and side chain hydroxylation and oxidation of the pyrrolidine ring to the corresponding lactam as well as ring opening to the corresponding carboxylic acid. Using LC-HR-MS, most metabolite structures postulated according to GC-MS fragmentation could be confirmed and the phase II metabolites were identified. Finally, the formation of the initial metabolite demethylenyl-MDPV could be confirmed using incubation of human liver microsomes. Using recombinant human CYPs, CYP 2C19, CYP 2D6 and CYP 1A2 were found to catalyze this initial step. Finally, the STA allowed the detection of MDPV metabolites in the human urine samples.