Development of a multianalyte method for the determination of anabolic hormones in bovine urine by isotope-dilution GC–MS/MS

A sensitive, specific and selective multianalyte GC–MS/MS method has been developed for the determination of 11 anabolic hormones in bovine urine. After adjusting the urine pH to 4.8, the samples were spiked with deuterated internal standards and submitted to enzymatic hydrolysis with β-glucuronidase/arylsulfatase. Hormones were eluted with methanol through a C18 solid phase cartridge and submitted to a liquid–liquid extraction. Analytes were derivatized by adding N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane and GC–MS data were obtained in the positive electron impact tandem mass mode. Under these conditions, no matrix effects were observed and limit of detection values were in the range of 0.005 ng/mL (diethylstilbestrol) to 0.38 ng/mL (17α-methyltestosterone and 17α-ethynylestradiol). Recoveries from 81% (α-zeranol) to 149% (17α-methyltestosterone) were found under the selected conditions. These results were better than those found using heptafluorobutyric anhydride (HFBA) as derivative reagent and those measured in full scan and selective ion monitoring modes.     

Mercury speciation analysis in human hair by species-specific isotope-dilution using GC–ICP–MS

We optimized a mercury (Hg) speciation extraction method for human hair in combination with species-specific isotope-dilution analysis by gas chromatography–inductively coupled plasma–mass spectrometry (GC–ICP–MS). The method was validated on human hair reference material RM (IAEA-086), which is recommended for analysis of monomethylmercury (MMHg) and inorganic mercury (IHg). Three reagents, hydrochloric acid (HCl), nitric acid (HNO₃), and tetramethylammonium hydroxide (TMAH), and three extraction procedures, at ambient temperature for 12 h, microwave-assisted at 75 °C for 6 min, and oven heated at 80 °C for 2 h were tested. Extraction efficiency, recovery, and potential species transformations were evaluated for each method. The most efficient procedures, with recovery of ~90 % for each species with limited demethylation (<5 %) and methylation (0 %), were HNO₃ digestion, irrespective of temperature, and microwave-assisted TMAH extraction. Acidic extraction with HCl induces significant demethylation, with production of artifacts. To correct for potential demethylation artifacts we recommend spiking with isotopically enriched standards before the extraction step.     

Comparison of GC–MS and LC–MS methods for the analysis of antioxidant phenolic acids in herbs

Two methods were developed for the quantitative analysis of phenolic acids in herb extracts. The methods were based on liquid chromatography–time-of-flight mass spectrometry (LC–TOFMS) and gas chromatography–mass spectrometry (GC–MS). The methods were compared in terms of their linearity, repeatability, selectivity, sensitivity and the speed of the analysis. The sensitivity was good for both methods, with limits of detection of <80 ng/ml for most of the compounds. The relative standard deviations (RSD) of the peak areas were on average 7.2% for the LC–TOFMS method and 1.4% for the GC–MS method. Both methods were found to be suitable for the determination of the target analytes, although GC–MS was better suited to the quantitative determination of compounds present at low concentrations.     

Analytical development for analysis of pharmaceuticals in water samples by SPE and GC–MS

An analytical procedure involving solid-phase extraction (SPE) and gas chromatography–mass spectrometry (GC–MS) has been developed for determination of pharmaceutical compounds (aspirin, caffeine, carbamazepine, diclofenac, ketoprofen, naproxen, ibuprofen, clofibrate, clofibric acid, and gemfibrozil) in a variety of aqueous samples (wastewater and surface water). After filtration, samples were extracted and concentrated using C₁₈ or HLB cartridges, depending on the type of compound. Sample storage conditions were checked and optimized to ensure preservation of the pharmaceutical substance, taking into consideration environmental sampling conditions. For most of the pharmaceuticals monitored, recovery was in the range 53 to 99% and the variability was below 15% for the complete procedure, with limits of detection ranging from 0.4 to 2.5 ng L⁻¹, depending on the compound. The methods were successfully applied to monitoring of pharmaceutical contamination of the Seine estuary. Concentrations varied from several dozens of nanograms per liter for surface waters to several hundreds of nanograms per liter for wastewaters.     

Determination of seven selected antipsychotic drugs in human plasma using microextraction in packed sorbent and gas chromatography–tandem mass spectrometry

A method using microextraction by packed sorbent (MEPS) and gas chromatography–tandem mass spectrometry (GC-MS/MS) is described for the determination of seven antipsychotic drugs in human plasma. The studied compounds were chlorpromazine (CPZ), haloperidol (HAL), cyamemazine, quetiapine, clozapine, olanzapine (OLZ), and levomepromazine; promazine, protriptyline, and deuterated CPZ were used as internal standards. The validation parameters included selectivity, linearity and limits of detection and quantitation, intra- and interday precision and trueness, recovery, and stability and were studied according to internationally accepted guidelines. The method was found to be linear between the lower limit of quantitation and 1000 ng/mL, except for OLZ and HAL (200 ng/mL), with determination coefficients higher than 0.99 for all analytes, and extraction efficiencies ranged from 62 to 92 %. Intra- and interday precision ranged from 0.24 to 10.67 %, while trueness was within a ±15 % interval from the nominal concentration for all analytes at all studied levels. MEPS has shown to be a rapid procedure for the determination of the selected antipsychotic drugs in human plasma, allowing reducing the handling time and the costs of analysis. Furthermore, GC-MS/MS has demonstrated to be a powerful tool for the simultaneous quantitation of the studied compounds, enabling obtaining adequate selectivity and sensitivity using a sample volume of as low as 0.25 mL.     

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.     

GC–MS method for the determination of paraben preservatives in the human breast cancerous tissue

The general presumption that the preservative laden personal care products may be one of the causative agents for breast cancer, has remained a matter of controversy during this decade. Extensive studies have not been carried out to either prove or disprove the role of preservatives in breast cancer incidences. In this study we have developed a new method for the identification and quantification of the preservatives such as methyl paraben (MeP), ethyl paraben (EtP), propyl paraben (PrP) and butyl paraben (BuP) in breast tissue using Gas Chromatography and Mass Spectrometry (GC–MS). Tissue was extracted by using acetone:n-hexane mixture (1:1 v/v) and derivatized with N-Methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA). The extent of reaction time and the amount of MSTFA to attain greater derivatization were optimized. The developed method yielded good recovery (mean ± SD) of 99.8 ± 5.1, 96 ± 4.4, 107 ± 17 and 113 ± 13% with relative standard deviations (RSDs) of 5.1, 4.6, 15.6 and 13%, and the limits of detection (LOD) of 2.02, 1.05, 1.71 and 3.75 ng g^− 1 for MeP, EtP, PrP and BuP, respectively. The method was successfully validated for the determination of parabens including butyl paraben (log K_ow = 3.57) in cancerous breast tissues; this could be a promising one for screening of breast tissues and also the environment for paraben residues. As far as our knowledge goes this is the first GC–MS method for the determination of parabens in human tissue.     

Application of β-cyclodextrin for the analysis of estrogenic steroids in human urine by high-performance liquid chromatography

Summary A procedure is described for simultaneous determination of estriol, estrone and 17-estradiol in human urine. After acid hydrolysis of the sulphate conjugates, the estrogens were extracted into diethyl ether. The ether extracts were concentrated and applied directly to an HPLC column. Using a 25 cm C-18 column and acetonitrile-water modified by the addition of -cyclodextrin as mobile phase, the separation of estriol, estradiol and estrone was achieved within 20 minutes. The extraction of estrogens from the biological matrix is excellent. Estrogens were detected using a UV-detector (280 nm) or a spectrofluorimetric detector (_exc=280nm, _em=312 nm). The latter detection system has been used for the determination of estrogens in the urine of non-pregnant women. The procedure is simple and can be used in clinical practice.     

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 urinary methylhippuric acids using MIL-53-NH2 (Al) metal–organic framework in microextraction by packed sorbent followed by HPLC–UV analysis

For the analysis of methylhippuric acids (MHAs) in human urine samples, in this study, a new method based on the metal–organic framework (MOF) of MIL-53-NH₂ (Al) in microextraction by packed sorbent (MEPS) was developed. The synthesis of MIL-53-NH₂ (Al) was characterized by Fourier transform infrared spectra, field emission-scanning electron microscopy and X-ray diffraction. Response surface methodology was used to investigate the influences of several parameters including type and volume of elution, type of conditional solvent, sample volume and extraction cycle on MEPS efficiency. The results showed good recoveries (>94%) and excellent extraction efficiencies (>96%) at three different concentrations of 50, 500 and 1500 μg ml⁻¹ (as low, mid and high concentrations, respectively) of MHA isomers. Calibration curves of MHAs were linear over the concentration range of 1–1500 μg ml⁻¹, with high correlation coefficients (r ≥ 0.998). The reproducibility of the proposed MIL-53-MEPS for determination of three isomers of MHA was found to be in the range of 3.5–11.1%. After optimization of the proposed technique, it was used to analyze MHAs in urine samples of workers exposed to xylenes in a petrochemical plant in Asalouyah, Iran. The results indicated that the MOF–MEPS method was selective, sensitive, rapid and efficient for the extraction of urinary MHAs. The technique is also environmentally friendly and inexpensive, and the MOF sorbent is reusable.     

Uncertainty evaluation for the determination of repaglinide in human plasma by LC–MS/MS

Measurement uncertainty although introduced to medical laboratories some years ago, this concept is not familiar to all medical researchers, especially for the measurement of biological samples. Therefore, it is important to highlight the evaluation and expression of measurement uncertainty using a practical example. In accordance with published procedures for evaluating and expressing uncertainty, we analyzed the sources of uncertainty in the determination of repaglinide in human plasma using liquid chromatography–tandem mass spectrometry (LC–MS/MS). We investigated each component of uncertainty and calculated the combined and expanded uncertainties. We evaluated the uncertainty associated with repeatability, weighing, purity, solution and sample preparation, recovery, calibration fitting, and temperature. The expanded uncertainty for low, medium, and high concentrations of repaglinide was 0.090, 0.25, and 3.16 ng/mL, respectively (p = 95 %, k = 2). This example provides an important reference for the evaluation of uncertainty in biological sample determinations using LC–MS/MS and human plasma and will be helpful in explaining the reliability of test results.     

Development of an analytical method for the determination of the residues of four pyrethroids in meat by GC–ECD and confirmation by GC–MS

The development of an analytical method for the determination of four selected pyrethroid insecticides at residue level in beef meat is presented. Acetone and petroleum ether at 40-60 degrees C were chosen as extraction solvents. A two-step clean-up was performed using an Extrelut NT3-C(18) system followed by a Florisil column, with disposable, ready-to-use cartridges. Instrumental analysis was carried out on a gas chromatograph equipped with an electron capture detector (GC-ECD), using matrix-matched and internal standard calibration techniques. Confirmatory analysis by GC-MS was performed. Recoveries at the EU Maximum Residue Limit (MRL), 0.5 x MRL and 1.5 x MRL levels and the repeatabilities were widely satisfactory. The main advantage of the method was the reduction of analysis time as compared with previously published works. The applicability of the method to different matrices and pesticide classes will be investigated.     

GC–MS analysis of organic acids in rat urine: A protocol of direct ultrasound-assisted derivatization

The aim of the present study was to develop a novel ultrasound-assisted derivatization method for analysis of urine that can be used for preliminary screening and monitoring of metabolic disorders. Here we describe an ultrasound-assisted derivatization method followed by GC–MS analysis to quantify 26 organic acids in urine. The optimum levels of the variables affecting the yield of derivatization were investigated, including urease doses, derivatization reagents and derivatization conditions (duration time, reaction temperature and sonic power). The method exhibited the best results with 80 μl urease. The optimal reaction conditions were 100 μl BSTFA, 80% ultrasound power, 70°C and 40 min. This method showed satisfactory linearity, good reproducibility and an acceptable limit of detection and accuracy. Therefore, it could potentially be used to as a standard method to enable comparisons between laboratories. Finally, we applied our method to urine samples from pregnant rats administered 2 or 10 mg/kg folic acid supplementation.     

Review of HPLC–MS methods for the analysis of nicotine and its active metabolite cotinine in various biological matrices

In recent years, tobacco smoking is a risk factor for a series of diseases, including cardiovascular diseases, cerebrovascular diseases, and cancers. Nicotine, the primary component of tobacco smoke, is mainly transformed to its active metabolite cotinine, which is often used as a biomarker for tobacco exposure for its higher blood concentration and longer residence time than nicotine. Various analytical methods have been developed for the determination of nicotine and cotinine in biological matrices. This article reviewed the HPLC–MS based methods for nicotine and/or cotinine analysis in various biological matrices. The sample preparation, mass and chromatographic conditions, and method validation results of these methods have been summarized and analyzed. The sample was mainly pretreated by protein precipitation and/or extraction. Separation was achieved using methanol and/or acetonitrile:water (with or without ammonium acetate) on C18 columns and acetonitrile:water (with formic acid, ammonium acetate/formate) on HILIC columns. Nicotine-d3, nicotine-d4, and cotinine-d3 were commonly used internal standards (ISs). Other non-deuterated ISs such as ritonavir, N-ethylnorcotinine, and milrinone were also used. For both nicotine and cotinine, the calibration range was 0.005–35,000 ng/mL, the matrix effect was 75.96–126.8%, and the recovery was 53–124.5%. The two analytes were stable at room temperature for 1–10 days, at −80°C for up to 6 months, and after three to six freeze–thaw cycles. Comedications did not affect nicotine and cotinine analyses.     

Improved Method for the Determination of Cyanuric Acid in Animal Feed by GC–MS

A specific and sensitive analytical method for the quantitative determination of cyanuric acid in animal feed was developed. Sample preparation involved the diethylamine/acetonitrile/water extraction of feed using sonication and shaking. The extract was subjected to clean-up by dual solid phase extraction using mixed mode anionic and cationic extraction cartridges. After removal of clean-up solvent, cyanuric acid was converted to a tert-butyldimethylsilyl derivative and was determined by gas chromatography–mass spectrometry in the selected ion monitoring mode. ¹³C ₃ ¹⁵ N₃-cyanuric acid was employed as the internal standard. The calibration curve was found to be linear up to 4 mg kg^?1. LOD and LOQ were determined to be 0.06 to 0.4 mg kg^?1 for fish and chicken feed. The mean recovery of cyanuric acid was 96 to 98% with relative pooled standard deviation of 1.8–7.4% in the range of 0.5 to 100 mg kg^?1 for fish and chicken feed. The validated method was applicable for analysing cyanuric acid in animal feed.     

Application of off-line pyrolysis with dynamic solid-phase microextraction to the GC–MS analysis of biomass pyrolysis products

Pyrolysis coupled with dynamic solid-phase micro extraction (Py-SPME) followed by GC–MS analysis was applied to the determination of volatile compounds evolved by a micro-scale off-line pyrolysis apparatus, in order to extend the information affordable with this type of analytical equipment. The Py-SPME method with a carboxen/PDMS fiber working in the retracted mode was tested on four biomass samples (switchgrass, sweet sorghum, corn stalk and poplar) for qualitative analysis of semi-volatile pyrolysis products and quantitative determination of main volatiles (C₁–C₄) pyrolysis products. The developed procedure allowed capturing and analysis of all GC analyzable compounds, without memory effects and with good peak resolution also for early GC-eluting compounds. Twelve main volatile pyrolysis products, including hydroxyacetaldehyde and acetic acid, were successfully quantified; in spite of the intrinsic variability introduced by dynamic SPME sampling, results were relatively accurate and consistent with literature data on bench pyrolysis reactors.     

Extraction and preconcentration of β-blockers in human urine for analysis with high performance liquid chromatography by means of carrier-mediated liquid phase microextraction

A novel method was developed for the analysis of four β-blockers, namely sotalol, carteolol, bisoprolol, and propranolol, in human urine by coupling carrier-mediated liquid phase microextraction (CM-LPME) to high performance liquid chromatography (HPLC). By adding an appropriate carrier in organic phase, simultaneous extraction and enrichment of hydrophilic (sotalol, carteolol, and bisoprolol) and hydrophobic (propranolol) drugs were achieved. High enrichment factors were obtained by optimizing the compositions of the organic phase, the acceptor solution, the donor solution, the stirring rate, and the extraction time. The linear ranges were from 0.05 to 10.0 mg L⁻¹ for sotalol and carteolol, and from 0.05 to 8.0 mg L⁻¹ for bisoprolol and propranolol. The limits of detection (S/N = 3) were 0.01 mg L⁻¹ for sotalol, carteolol, and bisoprolol, and 0.005 mg L⁻¹ for propranolol. The relative standard deviations were lower than 6%. The developed method exhibited high analyte preconcentration and excellent sample clean-up effects with little solvent consumption and was found to be sensitive and suitable for simultaneous determination of the above four drugs spiked in human urine. Furthermore, the successful analysis of propranolol in real urine specimens revealed that the determination of β-blockers in human urine is feasible using the present method.     

Exploratory analysis of human urine by LC–ESI-TOF MS after high intake of olive oil: understanding the metabolism of polyphenols

Olive oil polyphenols have important biological properties which closely depend on their bioavailability; it is, therefore, essential to understand how polyphenols are absorbed, metabolized, and eliminated from the body. An analytical method based on rapid-resolution liquid chromatography (RRLC) coupled with mass spectrometric detection with a time-of-flight analyzer (RRLC–ESI-TOF MS) has been developed for analysis of the main olive oil phenolic compounds and their metabolites in human urine. Urine samples from ten healthy volunteers were collected before and 2, 4, and 6 h after intake of 50 mL extra-virgin olive oil. The proposed method includes liquid–liquid extraction with ethyl acetate, which provides extraction recoveries of the phenolic compounds studied between 35 and 75% from spiked urine samples. Good repeatability was obtained—the relative standard deviations (RSDs) of peak areas in intra-day and inter-day studies were 4.3 and 6.5%, respectively. Statistical studies enabled us to discriminate between urine samples before and after intake, and facilitated the search for m/z values enabling this discrimination. Based on the very accurate mass information and the isotopic pattern provided by the TOF MS analyzer, together with other available information, ten of these biomarkers and more than 50 metabolites, obtained through phase I and phase II biotransformation reactions, were tentatively identified. Additionally, kinetic studies were conducted on the metabolites identified as possible biomarkers; for most of the compounds concentrations were maximum in the first two hours.