In vivo quantification and pharmacokinetic studies of cotinine in mice after smoke exposure by LC–MS/MS

A sensitive analytical method was developed and validated for the quantification of cotinine in mouse plasma after exposure to smoke of 0.5, 1.0, and 1.5 commercially available cigarettes, using liquid chromatography tandem mass spectrometry. The method was validated over a linear concentration range of 0.075–20.0 ng/mL with the R² value being higher than 0.99. Both the precision (coefficient of variation; %) and accuracy (relative error; %) were within acceptable criteria of <15%. The lower limit of quantification (LLOQ) for cotinine was 0.075 ng/mL with sufficient specificity, accuracy, and precision. Following exposure to 0.5, 1.0, and 1.5 cigarette smoke, it was observed that the AUC and the C_max increased linearly as the doses increased. The pharmacokinetics of cotinine was found linear for the range of 0.5–1.5 commercial cigarette smoke. The quantification of the concentration of cotinine in mouse plasma after smoke exposure will facilitate future behavioral and toxicological experiments in animals and may prove useful in predicting cotinine levels in humans during smoking.     

Rapid and simultaneous determination of nonsteroidal anti-inflammatory drugs in human plasma by LC–MS with solid-phase extraction

A rapid, selective and sensitive analytical method for the simultaneous determination of 16 nonsteroidal anti-inflammatory drugs (NSAIDs) in human plasma was carried out using Oasis HLB solid-phase extraction (SPE), followed by reversed-phase high-performance liquid chromatography and quadrupole mass spectrometry with electrospray ionization operated in the negative ion mode. The recoveries of NSAIDs from human plasma by SPE were greater than 76.7%. The use of a short column packed with small particles enabled rapid and simultaneous determination within 7 min. The detection limits for the NSAIDs were 0.01–0.9 μg/ml using the selected ion monitoring mode.     

Molecularly imprinted monolith in-tube solid-phase microextraction coupled with HPLC/UV detection for determination of 8-hydroxy-2′-deoxyguanosine in urine

Urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) has been widely used as a biomarker of oxidative DNA damage. Measurements of 8-OHdG in urinary samples are challenging owing to the low level of 8-OHdG and the complex matrix. In this study, a novel molecularly imprinted polymer (MIP) monolithic column was synthesized with guanosine as a dummy template which was used as the medium for in-tube solid-phase microextraction (SPME). In-tube SPME coupled with HPLC/UV detection for extraction and determination of urinary 8-OHdG was developed. The synthesized MIP monolithic column exhibited high extraction efficiency owing to its greater phase ratio with convective mass transfer and inherent selectivity. The enrichment factor for 8-OHdG was found to be 76 and the limits of detection and quantification of the method for urinary samples were 3.2 nmol/L (signal-to-noise ratio 3) and 11 nmol/L (signal-to-noise ratio 10), respectively. The MIP^’s selectivity also made the sample preparation procedure and chromatographic separation much easier. The linear range of the proposed method was from 0.010 to 5.30 μmol/L (r = 0.9997), with a relative standard deviation of 1.1–6.8%, and the recovery for spiked urine samples was 84 ± 3%. The newly developed method was successfully applied to determine urinary samples of healthy volunteers, coking plant workers, and cancer patients. The 8-OHdG level in cancer patients was significantly higher than that in healthy people.     

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.     

A method for the quantification of biomarkers of exposure to acrylonitrile and 1,3-butadiene in human urine by column-switching liquid chromatography–tandem mass spectrometry

1,3-Butadiene and acrylonitrile are important industrial chemicals that have a high production volume and are ubiquitous environmental pollutants. The urinary mercapturic acids of 1,3-butadiene and acrylonitrile—N-acetyl-S-(3,4-dihydroxybutyl)cysteine (DHBMA) and MHBMA (an isomeric mixture of N-acetyl-S-((1-hydroxymethyl)-2-propenyl)cysteine and N-acetyl-S-((2-hydroxymethyl)-3-propenyl)cysteine) for the former and N-acetyl-S-2-cyanoethylcysteine (CEMA) for the latter—are specific biomarkers for the determination of individual internal exposure to these chemicals. We have developed and validated a fast, specific, and very sensitive method for the simultaneous determination of DHBMA, MHBMA, and CEMA in human urine using an automated multidimensional LC/MS/MS method that requires no additional sample preparation. Analytes are stripped from urinary matrix by online extraction on a restricted access material, transferred to the analytical column, and subsequently determined by tandem mass spectrometry using labeled internal standards. The limits of quantification (LOQs) for DHBMA, MHBMA, and CEMA were 10 μg/L, 2 μg/L, and 1 μg/L urine, respectively, and were sufficient to quantify the background exposure of the general population. Precision within series and between series for all analytes ranged from 5.4 to 9.9%; mean accuracy was between 95 and 115%. We applied the method on spot urine samples from 210 subjects from the general population with no occupational exposure to 1,3-butadiene or acrylonitrile. A background exposure of the general population to acrylonitrile was discovered that is basically influenced by individual exposure to passive smoke as well as active smoking habits. Smokers showed a significantly higher excretion of MHBMA, whereas DHBMA levels did not differ significantly. Owing to its automation, our method is well suited for application in occupational or environmental studies. Figure Boxplots of the results from LC/ESI-MS/MS analysis of urinary excretion of CEMA reveal a strong correlation with nicotine metabolite cotinine, indicating that exposure to passive smoke as well as active smoking is the main source of exposure to acrylonitrile in the general population     

Automated determination of folate catabolites in human biofluids (urine,breast milk and serum) by on-line SPE–HILIC–MS/MS

A rapid, precise and fully-automated method for analysis of folate (vitamin B9) and its catabolites (viz. p-aminobenzoylglutamate and its acetamide derivative) in biofluids is here presented. The method is based on on-line hyphenation of solid-phase extraction (SPE) by a Prospekt 2 system with hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC–MS/MS). The method was analytically characterized by estimation of repeatability (RSD, n = 5, between 0.5 and 4.1%), accuracy (between 96 and 105%), and sensitivity (limits of quantificantion between 0.3 and 8.3 ng/mL (1.1 and 18.8 pmol/mL) or 0.03 and 0.83 ng (0.11 and 1.88 pmol)). The proposed method is suited for routine analysis of folate catabolites as biomarkers to monitor deficiency of vitamin B9.     

Rapid and sensitive HPLC–MS/MS method for the quantification of dopamine,GABA, serotonin,glutamine and glutamate in rat brain regions after exposure to tobacco cigarettes

Tobacco smoking is a preventable main cause of fatal diseases. Accurate measurements of the effects it has on neurotransmitters are essential in developing new strategies for smoking cessation. Moreover, measurements of neurotransmitter levels can aid in developing drugs that counteract the effects of smoking. The aim of this study is to develop and validate a fast, simultaneous and sensitive method for measuring the levels of neurotransmitters in rat brain after the exposure of tobacco cigarettes. The selected neurotransmitters include dopamine, GABA, serotonin, glutamine and glutamate. The method is based on high-performance liquid chromatography–tandem mass spectrometry. Chromatographic separation was achieved within 3 min using a Zorbax SB C₁₈ column (3.0 × 100 mm, 1.8 μm particle size). The mobile phase consisted of HPLC-grade water and acetonitrile each containing 0.3% heptafluorobutyric acid and 0.5% formic acid at gradient conditions. The linear range was 0.015–0.07, 825–7,218, 140–520, 63.42–160.75 and 38.25 × 10³ to 110.35 × 10³ ng/ml for dopamine, GABA, serotonin, glutamine and glutamate, respectively. Inter- and intra-run accuracy were in the range 97.82–103.37% with a precision (CV%) of ≤0.90%. The results revealed that 4 weeks of cigarette exposure significantly increased neurotransmitter levels after exposure to tobacco cigarettes in various brain regions, including the hippocampus and the amygdala. This increase in neurotransmitters levels may in turn activate the nicotine dependence pathway.     

Determination of selected β-receptor antagonists in biological samples by solid-phase extraction with cholesterolic phase and LC/MS

A new method is presented for the determination of five selected β-receptor antagonists by HPLC, which emphasizes sample preparation via retention on a new type of silica gel sorbent used for solid-phase extraction (SPE). Sorbents of this type were obtained by the chemical modification of silica gels of various porosities by cholesterol ligands. The cholesterol-based packing material was investigated by spectroscopic methods and elemental analysis. The recoveries obtained with the extraction procedure were optimum over a relatively broad sample pH range (3.08–7.50). Analytical factors such as the sample loading, the washing step and elution conditions, the concentration of β-receptor antagonists to be extracted, and the type of sorbent were found to play significant roles in the sample preparation procedure and would therefore need to be controlled to achieve optimum recoveries of the analytes. Under optimum conditions, the recoveries of nadolol, acebutolol, esmolol, oxprenolol and propranolol from spiked buffers, blood and urine were reproducible and dependent on the polarity or hydrophilicity of the compounds. The above analytes were determined by reverse-phase high-performance liquid chromatography (HPLC) with UV and ESI-ion trap mass spectrometry (MS) detection. The described method was found to be suitable for the routine measurement of compounds that are both polar and basic, and can be applied for the analysis of biological samples such as urine and blood in clinical, toxicological or forensic laboratories. The recovery measurements were performed on spiked human urine and serum, and on real samples of mouse blood serum.     

Sarcosine as a marker in prostate cancer progression: a rapid and simple method for its quantification in human urine by solid-phase microextraction–gas chromatography–triple quadrupole mass spectrometry

Sarcosine is an amino acid derivative of N-methylglycine and is involved in the amino acid metabolism and methylation processes that are enriched during prostate cancer progression. It could also serve as a new target to be measured during therapeutic interventions and help in the identification of aggressive tumors for radical treatment. In this study, we present a new urine test that can help early diagnosis of prostate cancer. The method for the quantification of sarcosine in urine consists of a solid-phase microextraction (SPME) step followed by gas chromatography–triple quadrupole mass spectrometry analysis. We used a preliminary derivatization step with ethyl chloroformate/ethanol and the corresponding ester was then extracted by SPME in immersion mode. Several fibers were evaluated and the optimization of the parameters affecting the SPME process was carried out using an experimental design. The optimal values were 20 min extraction time, 10% NaCl, and 270°C using a divinylbenzene/Carboxen/polydimethylsiloxane fiber. The triple quadrupole analyzer acquired data in selected reaction monitoring mode, allowing us to obtain reconstructed chromatograms with well-defined chromatographic peaks. The accuracy and precision of this method were evaluated at concentrations of 70, 250, and 800 ng/ml and were found to be acceptable. Very satisfactory values (0.10 and 0.16 ng/ml, respectively) were also achieved for the limit of detection and the limit of quantification. The proposed protocol represents a rapid, simple, selective, and sensitive tool to quantify sarcosine in urine samples for prostate cancer diagnosis and for a screening test.     

Determination of a flame retardant hydrolysis product in human urine by SPE and LC–MS. Comparison of molecularly imprinted solid-phase extraction with a mixed-mode anion exchanger

Diphenyl phosphate is a hydrolysis product and possible metabolite of the flame retardant and plasticiser additive triphenyl phosphate. A molecularly imprinted polymer solid-phase extraction (MISPE) method for extracting diphenyl phosphate from aqueous solutions has been developed and compared with SPE using a commercially available mixed-mode anion exchanger. The imprinted polymer was prepared using 2-vinylpyridine (2-Vpy) as the functional monomer, ethylene glycol dimethacrylate (EGDMA) as the cross-linker, and a structural analogue of the analyte as the template molecule. The imprinted polymer was evaluated for use as a SPE sorbent, in tests with both aqueous standards and spiked urine samples, by comparing recovery and breakthrough data obtained using the imprinted form of the polymer and a non-imprinted form (NIP). Extraction from aqueous solutions resulted in more than 80% recovery. Adsorption by the molecularly imprinted polymer (MIP) was non-selective , but selectivity was achieved by selective desorption in the wash steps. Diphenyl phosphate could also be selectively extracted from urine samples, although the urine matrix reduced the capacity of the MISPE cartridges. Recoveries from urine extraction were higher than 70%. It was important to control pH during sample loading. The MISPE method was found to yield a less complex LC–ESI–MS chromatogram of the urine extracts compared with the mixed-mode anion-exchanger method. An LC–ESI–MS method using a Hypercarb LC column with a graphitised carbon stationary phase was also evaluated for organophosphate diesters. LC–ESI–MS using negative-ion detection in selected ion monitoring (SIM) mode was shown to be linear for diphenyl phosphate in the range 0.08–20 ng L^–1.     

Simultaneous determination of urinary 31 metabolites of VOCs, 8-hydroxy-2′-deoxyguanosine,and trans-3′-hydroxycotinine by UPLC-MS/MS: 13C- and 15N-labeled isotoped internal standards are more effective on reduction of matrix effect

Analytical and Bioanalytical Chemistry - Human beings are inevitably exposed to volatile organic compounds (VOCs) of anthropogenic emissions as they are ubiquitous atmospheric pollutants. Smoking...     

Determination of mitragynine in plasma with solid-phase extraction and rapid HPLC–UV analysis, and its application to a pharmacokinetic study in rat

A new solid phase extraction method for rapid high performance liquid chromatography–UV determination of mitragynine in plasma has been developed. Optimal separation was achieved with an isocratic mobile phase consisting of acetonitrile–ammonium acetate buffer, 50 mM at pH 5.0 (50:50, v/v). The method had limits of detection and quantification of 0.025 and 0.050 μg/mL, respectively. The method was accurate and precise for the quantitative analysis of mitragynine in human and rat plasma with within-day and between-day accuracies between 84.0 and 109.6%, and their precision values were between 1.7 and 16.8%. Additional advantages over known methods are related to the solid phase extraction technique for sample preparation which yields a clean chromatogram, a short total analysis time, requires a smaller amount of plasma samples and has good assay sensitivity for bioanalytical application. The method was successfully applied in pharmacokinetic and stability studies of mitragynine. In the present study, mitragynine was found to be fairly stable during storage and sample preparation. The present study showed for the first time the detailed pharmacokinetic profiles of mitragynine. Following intravenous administration, mitragynine demonstrated a biphasic elimination from plasma. Oral absorption of the drug was slow, prolonged and was incomplete, with a calculated absolute oral bioavailability value of 3.03%. The variations observed in previous pharmacokinetic studies after oral administration of mitragynine could be attributed to its poor bioavailability rather than to the differences in assay method, metabolic saturation or mitragynine dose.     

Simultaneous chiral separation and determination of carvedilol and 5′-hydroxyphenyl carvedilol enantiomers from human urine by high performance liquid chromatography coupled with fluorescent detection

A sensitive and specific high performance liquid chromatography coupled with fluorescent detection (HPLC-FL) and tandem mass spectrometry detection (HPLC-MS/MS) methods for separation and determination of carvedilol (CAR) enantiomers and 5′-hydroxyphenyl carvedilol (5′-HCAR) enantiomers has been developed and validated. The analysed compounds were extracted from human urine by solid phase extraction. Good enantioseparation of the studied enantiomers was achieved on CHIRALCEL® OD-RH column using 0.05% trifluoroacetic acid and 0.05% diethylamine in water and acetonitrile in a gradient elution. The mass spectrometric data were acquired using the multiple reaction monitoring mode by positive electrospray ionisation. The method was validated over the concentration range from 25.0 ng mL^?1 to 200 ng mL^?1 for the analysed compounds. The limit of quantification varied from 14.2 ng mL^?1 to 24.2 ng mL^?1. Both the repeatability and inter-day precisions were below 10.0%, and the accuracy varied from ?13.2% to 3.77%. The extraction recoveries ranged from 79.2% to 108%. The present paper reports the method for the simultaneous determination of CAR enantiomers and their metabolite enantiomers (5′-HCAR) in human urine samples. This newly developed method was successfully used to analyse the aforementioned analytes in human urine samples obtained from patients suffering from cardiovascular disease.      

Simultaneous determination of testosterone, cortisol, and dehydroepiandrosterone in saliva by stable isotope dilution on-line in-tube solid-phase microextraction coupled with liquid chromatography–tandem mass spectrometry

We have developed a simple and sensitive method for the simultaneous determination of testosterone (TES), cortisol (CRT), and dehydroepiandrosterone (DHEA) in saliva by automated online in-tube solid-phase microextraction (SPME) coupled with liquid chromatography–tandem mass spectrometry (LC–MS/MS) using a Discovery HS F5 column. The optimum in-tube SPME conditions were 25 draw/eject cycles of 40 μL of sample at a flow rate of 200 μL/min using a Supel-Q PLOT capillary column as an extraction device. The extracted compounds were easily desorbed from the capillary by passage of the mobile phase, and no carryover was observed. The in-tube SPME LC–MS/MS method showed good linearity with correlation coefficients r?≥?0.9998 for TES, CRT, and DHEA using their respective stable isotope-labeled internal standards. The intra-day and inter-day precisions (relative standard deviations) were below 4.9 and 8.5 % (n?=?5), respectively. This method was successfully utilized to analyze TES, CRT, and DHEA in saliva samples without any other pretreatment or interference peaks, and the quantification limits (S/N?=?10) of TES, CRT and DHEA were about 0.01, 0.03 and 0.29 ng/mL saliva, respectively. The recoveries of these compounds spiked into saliva samples were each above 94 %. This method was applied to analyze changes in salivary TES, CRT, and DHEA levels resulting from stress and fatigue load.     

High-resolution ICP–MS determination of Ti,V, Cr,Co, Ni,and Mo in human blood and urine of patients implanted with a hip or knee prosthesis

The main components (Ti, V, Cr, Co, Ni, and Mo) of metallic alloys currently used in hip and knee articular prostheses have been simultaneously determined in human whole blood and urine of implanted people by a (HR)-ICP–MS method previously developed in our laboratory. The determination of those elements has been carried out in patients with knee and hip prosthesis and in a group of pre-operation patients without any metallic device in their bodies, used as controls, demonstrating the usefulness of this technique to perform multielement analysis at ppt levels in complex matrices. The concentrations of V, Cr, Co, Ni, and Mo in urine and blood of implanted people turned out to be very similar to those obtained in control patients. However, raised Ti levels could be found both in urine and blood of patients with articular prostheses made or coated with a titanium alloy (Ti₆Al₄V).     

On-line SPE LC-MS/MS for the quantification of Δ9-tetrahydrocannabinol (THC) and its two major metabolites in human peripheral blood by liquid chromatography tandem mass spectrometry

A universal and robust analytical method for the determination of Δ9-tetrahydrocannabinol (THC) and two of its metabolites Δ9-(11-OH)-tetrahydrocannabinol (11-OH-THC) and 11-nor-Δ9-carboxy-tetrahydrocannabinol (THC-COOH) in human whole blood was developed and validated for use in forensic toxicology. Protein precipitation, integrated solid phase extraction and on-line enrichment followed by high-performance liquid chromatography separation and detection with a triple quadrupole mass spectrometer were combined. The linear ranges used for the three cannabinoids were from 0.5 to 20 ng/mL for THC and 11-OH-THC and from 2.5 to 100 ng/mL for THC-COOH, therefore covering the requirements for forensic use. Correlation coefficients of 0.9980 or better were achieved for all three analytes. No relevant hydrolysis was observed for THC-COOH glucuronide with this procedure — in contrast to our previous GC-MS procedure, which obviously lead to an artificial increase of the THC-COOH concentration due to the hydrolysis of the glucuronide-conjugate occurring at high pH during the phase-transfer catalyzed methylation step.     

Determination of twelve herbicides in tobacco by a combination of solid–liquid–solid dispersive extraction using multi-walled carbon nanotubes, dispersive liquid-liquid micro-extraction, and detection by GC with triple quadrupole mass spectrometry

We report on a method for the determination of twelve herbicides using solid–liquid–solid dispersive extraction (SLSDE), followed by dispersive liquid-liquid micro-extraction (DLLME) and quantitation by gas chromatography with triple quadrupole mass spectrometric detection. SLSDE was applied to the extraction of herbicides from tobacco samples using multi-walled carbon nanotubes (MWCNTs) as clean-up adsorbents. The effect of the quantity of MWCNTs on SLSDE, and of type and volume of extraction and disperser solvents and of salt effect on DLLME were optimized. Good linearity is obtained in the 5.0 - 500 μg kg^?1 concentration range, with regression coefficients of >0.99. Intra-day and inter-day repeatability, expressed as relative standard deviations, are between 3 and 9 %. The recoveries in case of herbicide-spiked tobacco at concentration levels of 20.0, 50.0 and 100.0 g kg^?1 ranged from 79 to 105 %, and LODs are between 1.5 and 6.1 μg kg^?1. All the tobacco samples were found to contain butralin and pendimethalin at levels ranging from 15.8 to 500.0 μg kg^?1.