Simultaneous quantitative determination of α-ketoglutaric acid and 5-hydroxymethylfurfural in human plasma by gas chromatography–mass spectrometry

α-Ketoglutaric acid (α-KG) and 5-hydroxymethylfurfural (5-HMF) are currently under investigation as promising cancer cell damaging agents. A method for the simultaneous quantitative determination of α-KG and 5-HMF in human plasma was established for screening these compounds in human plasma. Plasma samples were directly treated with O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine hydrochloride to form the corresponding oximes, thus facilitating subsequent liquid–liquid extraction. After formation of the trimethylsilyl ethers, samples were analyzed by gas chromatography with electron ionization mass spectrometry. Stable isotope labeled standards were used, the preparation of ¹³C₆-5-HMF is described. Limits of quantitation were set to 0.938 μg/mL for α-KG and 0.156 μg/mL for 5-HMF. Inter-day accuracy was ≤93.7% (α-KG) and ≤92.8% (5-HMF). Inter-day precision was ≤6.0% (α-KG) and ≤4.6% (5-HMF). The method has been successfully applied to pharmacokinetic profiling of the compounds after intravenous application.     

Optimization of a method for the determination of a mustard gas biomarker in human blood plasma by liquid chromatography–high-resolution mass spectrometry

A method for the determination of a mustard gas biomarker (an S-hydroxyethylthioethyl adduct with albumin) in blood plasma was optimized with the use of HPLC with high-resolution tandem mass-spectrometric detection. This method is based on the hydrolysis of this adduct by the proteinase K enzyme with the formation of the following stable tripeptide with cysteine, proline, and phenylalanine: S-[HETE]-Cys- Pro-Phe. The conditions of the sample preparation of human plasma artificially contaminated by mustard gas (the selection of an aliquot portion volume and an enzyme for the hydrolysis), the mass-spectrometric detection (the selection of optimum pairs of ion reactions and high-resolution detection modes), and the gradient elution program in the HPLC separation of an analyzed mixture were optimized. The detection limit of mustard gas in human blood plasma was 1 ng/mL. The approach proposed was tested in the analysis of human blood plasma samples by the standard addition technique and also within the framework of the first official biomedical test carried out by the Organization for the Prohibition of Chemical Weapons (OPCW) in 2016, and it exhibited a good accuracy, reproducibility, and specificity of determination.     

Sensitive liquid chromatography–tandem mass spectrometry method for the determination of pantoprazole sodium in human urine

A sensitive and selective liquid chromatographic–tandem mass spectrometric (LC–MS–MS) method was developed to determine pantoprazole sodium (PNT) in human urine. After solid-phase extraction with SPE cartridge, the urine sample was analysed on a C₁₈ column (symmetry 3.5 μm; 75 mm × 4.6 mm i.d) interfaced with a triple quadrupole tandem mass spectrometer. Positive electrospray ionization was employed as the ionization source. The mobile phase consisted of acetonitrile–water (90:10, v/v). The method was linear over a concentration range of 1–100 ng mL^?1. The lower limit of quantitation was 1 ng mL^?1. The intra-day and inter-day relative standard deviation across three validation runs over the entire concentration range was <10.5%. The accuracy determined at three concentrations (8.0, 50.0 and 85.0 ng mL^?1 PNT) was within ±1.25% in terms of relative errors.     

Development of a liquid–chromatography tandem mass spectrometry and ultra-high-performance liquid chromatography high-resolution mass spectrometry method for the quantitative determination of zearalenone and its major metabolites in chicken and pig plasma

A sensitive and specific method for the quantitative determination of zearalenone (ZEN) and its major metabolites (α-zearalenol (α-ZEL), β-zearalenol (β-ZEL), α-zearalanol (α-ZAL), β-zearalanol (β-ZAL) and zearalanone (ZAN)) in animal plasma using liquid chromatography combined with heated electrospray ionization (h-ESI) tandem mass spectrometry (LC–MS/MS) and high-resolution Orbitrap^® mass spectrometry ((U)HPLC–HR–MS) is presented. The sample preparation was straightforward, and consisted of a deproteinization step using acetonitrile. Chromatography was performed on a Hypersil Gold column (50 mm × 2.1 mm i.d., dp: 1.9 μm, run-time: 10 min) using 0.01% acetic acid in water (A) and acetonitrile (B) as mobile phases.     

Comparison of liquid chromatography-microchip/mass spectrometry to conventional liquid chromatography–mass spectrometry for the analysis of steroids

The feasibility of a microfluidic-based liquid chromatography-electrospray ionization/mass spectrometric system (HPLC-Chip/ESI/MS) was studied and compared to a conventional narrow-bore liquid chromatography-electrospray ionization/mass spectrometric (LC-ESI/MS) system for the analysis of steroids. The limits of detection (LODs) for oxime derivatized steroids, expressed as concentrations, were slightly higher with the HPLC-Chip/MS system (50–300 pM) using an injection volume of 0.5 μL than with the conventional LC-ESI/MS (10–150 pM) using an injection volume of 40 μL. However, when the LODs are expressed as injected amounts, the sensitivity of the HPLC-Chip/MS system was about 50 times higher than with the conventional LC-ESI/MS system. The results indicate that the use of HPLC-Chip/MS system is clearly advantageous only in the analysis of low-volume samples. Both methods showed good linearity and good quantitative and chromatographic repeatability. In addition to the instrument comparisons with oxime derivatized steroids, the feasibility of the HPLC-Chip/MS system in the analysis of non-derivatized and oxime derivatized steroids was compared. The HPLC-Chip/MS method developed for non-derivatized steroids was also applied to the quantitative analysis of 15 mouse plasma samples.     

Comparison of supercritical fluid chromatography–tandem mass spectrometry and liquid chromatography–tandem mass spectrometry for the stereoselective analysis of chlorfenvinphos and dimethylvinphos in tobacco

This study used reversed-phase liquid chromatography–tandem mass spectrometry and supercritical fluid chromatography–tandem mass spectrometry for determination of the stereoisomers of chlorfenvinphos and dimethylvinphos in tobacco. Tobacco samples were extracted and purified with a modified quick, easy, cheap, effective, rugged, and safe technique using spherical carbon. The performance of both methodologies was comprehensively compared in terms of methods validation parameters (separation efficiency, linearity, selectivity, recovery, repeatability, sensitivity, matrix effect, etc.). Under optimized conditions, the calibration curves of the stereoisomers of chlorfenvinphos and dimethylvinphos in the range of 10–500 ng/mL showed excellent linearity with R² ≥ 0.997 in both methods. The adequate recoveries of analytes from three different spiked tobaccos were obtained using reversed-phase liquid chromatography–tandem mass spectrometry (86.1–95.7%) as well as supercritical fluid chromatography–tandem mass spectrometry (86.5–94.0%). The relative standard deviations for spiked samples were all below 7.0%. Compared with supercritical fluid chromatography–tandem mass spectrometry, lower matrix effects and LODs can be obtained in reversed-phase liquid chromatography–tandem mass spectrometry.     

Trace-level determination of eight cholesterol oxidation products in human plasma by dispersive liquid–liquid microextraction and ultra-performance liquid chromatography–tandem mass spectrometry

7α-Hydroxy cholesterol (7α-OHC), 25-hydroxy cholesterol (25-OHC), 27-hydroxy cholesterol (27-OHC), 4β-hydroxy cholesterol (4β-OHC), 7α-hydroxy-4-cholesten-3-one (7α-C4), 5β-cholestane-3α, 7α, 12α-triol (5β-Triol), cholic acid (CA), and chenodeoxycholic acid (CDCA) are known biomarkers of neurodegenerative diseases. A method for their simultaneous determination in human plasma has been optimized using dispersive liquid–liquid microextraction and ultra-performance liquid chromatography–tandem mass spectrometry. The limits of quantification of the target compounds were in the range of 0.3–3.3?µg/L. The precision achieved by this method was less than 13.4% for intraday and interday analyses. The proposed method was used to analyze eight cholesterol oxidation products in 30 human plasma samples. The analytical results were in a concentration range of 1.6–87.4?µg/L for 7α-OHC, 6.3–58.2?µg/L for 25-OHC, 12.1–98.5?µg/L for 27-OHC, 5.7–64.8?µg/L for 4β-OHC, 1.5–124.1?µg/L for 7α-C4, 0.5–16.5?µg/L for 5β-Triol, 13.1–245?µg/L for CA, and 19.6–487?µg/L for CDCA in the samples. The method may be used for the analysis of biomarkers of neurodegenerative diseases.     

Ultra-high-pressure liquid chromatography–tandem mass spectrometry method for the determination of alkylphenols in soil

A novel method has been developed for the determination of alkylphenols in soil by ultra-high-pressure liquid chromatography employing small particle sizes, combined with tandem mass spectrometry. Soil samples were extracted with pressurized liquid extraction (PLE) and then cleaned with solid-phase extraction (SPE). The extracts were separated on C18 column (1.7 μm, 50 mm × 2.1 mm) with a gradient elution and a mobile phase consisting of water and acetonitrile, and then detected by an electrospray ionization tandem mass spectrometry in negative ion mode with multiple reaction monitoring (MRM). Compared with traditional liquid chromatography, it took ultra-high-pressure liquid chromatography much less time to analyze alkylphenols. Additionally, the ultra-high-pressure liquid chromatography/tandem mass spectrometry method produces satisfactory reliability, sensitivity, and accuracy. The average recoveries of the three target analytes were 74.0–103.4%, with the RSD < 15%. The calibration curves for alkylphenols were linear within the range of 0.01–0.4 μg/ml, with the correlation coefficients greater than 0.99. When 10 g soil sample was used for analysis, the limits of quantification (LOQs) of the three alkylphenols were all 1.0 μg/kg.     

Single-drop microextraction and gas chromatography–mass spectrometry for the determination of volatile aldehydes in fresh cucumbers

Headspace single-drop microextraction (HS-SDME) was used as a rapid and reliable method for the isolation and preconcentration of volatile aldehydes from fresh cucumbers. The utility of this methodology is demonstrated in the determination of (E)-2-nonenal and (E,Z)-2,6-nonadienal. The limit of detection, linearity and repeatability have been determined for 2,6-nonadienal and (E)-2-nonenal. Limits of detection for nonenal and nonadienal were 0.05 and 0.04 mg kg ⁻¹, respectively. The repeatability of extraction was obtained with the RSD values lower than 13%. Concentrations of target aldehydes in fresh cucumbers obtained by means of the HS-SDME method were in the range 9.4–12.5 (nonadienal) and 2.6–3.8 mg kg ⁻¹ (nonenal). The results of the single-drop extraction in combination with gas chromatography show promising potential for the analysis of volatile aldehydes in vegetables. Presented at the 11th International Conference on Chemistry and the Environment, 9-12 September 2007, Torun, Poland.     

Novel derivatisation technique for the determination of chlorophenoxy acid type herbicides by gas chromatography–mass spectrometry

The analytical detection of chlorophenoxycarboxylic-acid-type herbicides (2,4-D, dichloprop, MCPA, etc.) in environmental samples is often a problem in instrumental analysis, as these compounds containing free carboxylic groups require chemical derivatisation prior to gas chromatographic (GC) methods. Nine chlorophenoxy-acid-type herbicide active ingredients have been derivatised successfully with trimethylsilyl N,N-dimethyl carbamate and t-butyldimethylsilyl N,N-dimethyl carbamate by forming their trimethylsilyl (TMS) and t-butyldimethylsilyl (TBDMS) esters, respectively. The detection and determination of the derivatives were performed by capillary gas chromatography–mass spectrometry. The study included determination of retention indices, mass spectral properties and comparison of derivatives produced. The mass spectra of TBDMS derivatives are usually dominated by very characteristic ions [M-57]⁺ resulting from the cleavage of t-butyl moiety during electron impact (EI) ionisation in the mass spectrometer. Limits of detection were 5 to 100 pg applying GC with EI-MS detection in full scan mode. The method, using SPE sample preparation, was applied for the analysis of 115 ground water and surface water samples collected in Békés County, Hungary in 2009.     

Liquid chromatography–mass spectrometric determination of plasmalogens in human plasma

A new liquid chromatography–mass spectrometry (LC/MS) method has been developed for the quantitative analysis of plasmalogens in human plasma using a nonendogenous plasmalogen (1-O-1′-(Z)-tricosenyl-2-oleoyl-rac-glycero-3-phosphocholine, PLS 23:0/18:1) as an internal standard. 1-O-1′-(Z)-Tricosenyl glyceryl ether was prepared by reacting lithioalkoxyallyl with 1-iodoeicosane as the key intermediate in the formation of PLS 23:0/18:1. In LC/MS analyses, PLS 23:0/18:1 generated significant fragment ions in positive and negative modes. In positive ion mode, the [M+H]⁺ of PLS 23:0/18:1 yielded unique fragments with cleavages at the sn-1 and sn-2 positions of the glycerol backbone. In negative ion mode, the [M+CH₃COO]⁻ of PLS 23:0/18:1 resulted in characteristic fragmentation at the sn-2 and sn-3 positions. 1-O-1′-(Z)-Hexadecenyl-2-linoleoyl-rac-glycero-3-phosphocholine (PLS 16:0/18:2) and 2-arachidonoyl-O-1′-(Z)-hexadecenyl-rac-glycero-3-phosphocholine (PLS 16:0/20:4) were chemically synthesized as PLS 23:0/18:1. The calibration curves obtained for PLS 16:0/18:2 and PLS 16:0/20:4 were linear throughout the calibration range (0.04–1.60 pmol). The LOD (S/N = 5:1) was 0.008 pmol and the LOQ (S/N = 6:1) was 0.01 pmol for both PLS 16:0/18:2 and PLS 16:0/20:4. Plasma concentrations of PLS 16:0/18:2 and PLS 16:0/20:4 were 4.0 ± 1.3 μM and 3.5 ± 1.2 μM (mean ± SD), respectively, in five healthy volunteers.     

Dispersive liquid–liquid microextraction followed by gas chromatography–mass spectrometry for the rapid and sensitive determination of UV filters in environmental water samples

The performance of the dispersive liquid–liquid microextraction (DLLME) technique for the determination of eight UV filters and a structurally related personal care species, benzyl salicylate (BzS), in environmental water samples is evaluated. After extraction, analytes were determined by gas chromatography combined with mass spectrometry detection (GC-MS). Parameters potentially affecting the performance of the sample preparation method (sample pH, ionic strength, type and volume of dispersant and extractant solvents) were systematically investigated using both multi- and univariant optimization strategies. Under final working conditions, analytes were extracted from 10 mL water samples by addition of 1 mL of acetone (dispersant) containing 60 μL of chlorobenzene (extractant), without modifying either the pH or the ionic strength of the sample. Limits of quantification (LOQs) between 2 and 14 ng L⁻¹, inter-day variability (evaluated with relative standard deviations, RSDs) from 9% to 14% and good linearity up to concentrations of 10,000 ng L⁻¹ were obtained. Moreover, the efficiency of the extraction was scarcely affected by the type of water sample. With the only exception of 2-ethylhexyl-p-dimethylaminobenzoate (EHPABA), compounds were found in environmental water samples at concentrations between 6 ± 1 ng L⁻¹ and 26 ± 2 ng mL⁻¹.     

Headspace-trap gas chromatography–mass spectrometry for determination of sulphur mustard and related compounds in soil

Methods for trace determination of sulphur mustard (HD) and some related cyclic sulphur compounds in soil samples have been developed using headspace-trap in combination with gas chromatography–mass spectrometry (GC–MS). Two quite different types of soil were employed in the method optimisation (sandy loam and silty clay loam). Prior to analysis, water saturated with sodium chloride was added to the samples, at a water to soil ratio of 1:1. A detection limit of 3 ng/g was achieved for HD, while the cyclic sulphur compounds 1,4-thioxane, 1,3-dithiolane and 1,4-dithiane could be detected at 0.2–0.7 ng/g. The methods were validated in the concentration range from the limit of quantification (LOQ) to hundred times LOQ. The within assay precision at fifty times LOQ was 6.9–7.3% relative standard deviation (RSD) for determination of the cyclic sulphur compounds, and 15% RSD for determination of HD. Recoveries were in the range of 43–60% from the two soil types. As the technique requires very little sample preparation, the total time for sample handling and analysis was less than 1 h. The technique was successfully employed for the determination of cyclic sulphur compounds in a sediment sample from an old dumping site for chemical munitions, known to contain HD degradation products.     

Determination of tandospirone in human plasma by a liquid chromatography–tandem mass spectrometry method

A rapid, sensitive, and selective liquid chromatography–tandem mass spectrometry method for the detection of tandospirone in human plasma is described. It was employed in a pharmacokinetic study. The analyte and internal standard diphenhydramine were extracted from plasma using liquid–liquid extraction, then separated on a Zorbax XDB C₁₈ column using a mobile phase of methanol–water–formic acid (80:20:0.5, v/v/v). The detection was performed with a tandem mass spectrometer equipped with an electrospray ionization source. Linearity was established in the concentration range of 10.0-5,000 pg/ml. The lower limit of quantification was 10.0 pg/ml. The intraday and interday relative standard deviation across three validation runs over the entire concentration range was less than 13%. Accuracy determined at three concentrations (25.0, 200, and 4,000 pg/ml for tandospirone) ranged from 94.4 to 102.1%. Each plasma sample was chromatographed within 3.4 min. The method proved to be highly selective and suitable for bioequivalence evaluation of different formulations containing tandospirone and clinical pharmacokinetic investigation of tandospirone.     

Dried plasma spot–based liquid chromatography–tandem mass spectrometry for the quantification of methotrexate in human plasma and its application in therapeutic drug monitoring

Methotrexate, a folic acid antitumor drug, is widely used to treat childhood acute lymphoblastic leukemia. Therapeutic drug monitoring is crucial for adjusting the dosage of methotrexate according to its plasma concentration and reducing adverse effects. Micro-sampling strategies, like dried plasma spot, is an attractive but underutilized method that has the desired features of easy collection, storage, and transport, and overcomes known hematocrit issues in dried blood spot analysis. This study describes a dried plasma spot–based liquid chromatography–tandem mass spectrometry method for quantification of methotrexate. The assay showed good linearity over 30–2000 ng/mL (R² ≥ 0.995) as well as excellent precision (0.6–9.3%) and accuracy (89.2–108.3%). Methotrexate was extracted from dried plasma spot and wet plasma samples with recoveries greater than 92.1%, and no significant matrix effect was observed. A comparison of dried plasma spot and wet plasma concentrations was assessed in 27 patients treated with methotrexate and Passing–Bablok regression coefficients showed that no significant difference between the two methods. The Bland–Altman plots showed similar agreement between the methods, indicating that the proposed dried plasma spot sampling method is an effective way to monitor the concentration of methotrexate in human plasma.     

Identification and quantification of salinomycin in intoxicated human plasma by liquid chromatography–electrospray tandem mass spectrometry

Salinomycin is a polyether ionophore antibiotic that is widely used in poultry and livestock. Exposure of humans to salinomycin via inhalation or ingestion can cause severe toxicity. The aim of the present work was to develop a simple and sensitive liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the rapid identification and quantification of salinomycin in human plasma. After removing protein using methanol, plasma samples were eluted from a Waters Xterra ^® MS C18 column with an isocratic mobile phase. Detection and quantification of the drug were performed with a triple-quadruple mass spectrometer by monitoring for two specific transitions in the electrospray, positive-ion, multiple-reaction monitoring mode. Assay validation showed good linearity (r ²?=?0.998). The detection and quantification limits of the method were 0.6 and 16 pg/mL, respectively. The inter- and intraday coefficients of variation for the assay were both <15%. Twelve authentic plasma samples from intoxicated patients were analyzed using this method. Salinomycin was detected in six samples, at concentrations of between 0.6 and 46.5 pg/mL. The described assay method allows the sensitive and rapid identification and quantification of salinomycin in human plasma, and thus provides a valuable tool for the specific diagnosis of salinomycin intoxication in clinical and emergency rescue practice.     

Pre-concentration of phenolic compounds in water samples by novel liquid–liquid microextraction and determination by gas chromatography–mass spectrometry

Pre-concentration and determination of 8 phenolic compounds in water samples has been achieved by in situ derivatization and using a new liquid–liquid microextraction coupled GC–MS system. Microextraction efficiency factors have been investigated and optimized: 9 μL 1-undecanol microdrop exposed for 15 min floated on surface of a 10 mL water sample at 55 °C, stirred at 1200 rpm, low pH level and saturated salt conditions. Chromatographic problems associated with free phenols have been overcome by simultaneous in situ derivatization utilizing 40 μL of acetic anhydride and 0.5% (w/v) K₂CO₃. Under the selected conditions, pre-concentration factor of 235–1174, limit of detection of 0.005–0.68 μg/L (S/N = 3) and linearity range of 0.02–300 μg/L have been obtained. A reasonable repeatability (RSD ≤ 10.4%, n = 5) with satisfactory linearity (0.9995 ≥ r² ≥ 0.9975) of results illustrated a good performance of the present method. The relative recovery of different natural water samples was higher than 84%.     

Development of a new ultra-high-performance liquid chromatography–tandem mass spectrometry method for the determination of digoxin and digitoxin in plasma: Comparison with a clinical immunoassay

Cardiac glycosides digoxin and digitoxin are used in therapy for the treatment of congestive heart failure. Moreover, these compounds can be responsible for intoxication cases caused by fortuitous ingestion of leaves of Digitalis. Due to the narrow therapeutic range of these drugs, therapeutic drug monitoring is recommended in the clinical practice. In this context, immunoassays-based methods are generally employed but digoxin- and digitoxin-like compounds can interfere with the analysis. The aim of this study was to develop and validate an original UPLC–MS/MS method for the determination of digoxin and digitoxin in plasma. The method shows adequate sensitivity and selectivity with acceptable matrix effects and very good linearity, accuracy, precision, and recovery. A simple liquid–liquid extraction procedure was used for sample clean-up. The method was applied for the analysis of n = 220 plasma samples collected in two different clinical chemistry laboratories and previously tested by the same immunoassay. The statistical comparison showed a relevant negative bias of the UPLC–MS/MS method versus the immunoassay. These results are consistent with an immunoassay overestimation of digoxin plasmatic levels due to cross-reaction events with endogenous digoxin-like substances.     

Development and validation of a comprehensive two-dimensional gas chromatography–mass spectrometry method for the analysis of phytosterol oxidation products in human plasma

Phytosterol oxidation products (POPs) have been suggested to exert adverse biological effects similar to, although less severe than, their cholesterol counterparts. For that reason, their analysis in human plasma is highly relevant. Comprehensive two-dimensional gas chromatography (GC×GC) coupled with time-of-flight mass spectrometry (TOF-MS) has been proven to be an extremely powerful separation technique for the analysis of very low levels of target compounds in complex mixtures including human plasma. Thus, a GC×GC/TOF-MS method was developed and successfully validated for the simultaneous quantification of ten POPs in human plasma. The calibration curves for each compound showed correlation coefficients (R ²) better than 0.99. The detection limits were below 0.1 ng mL⁻¹. The recovery data were between 71.0% and 98.6% (RSDs <10% for all compounds validated). Good results were obtained for within- and between-day repeatability, with most values being below 10%. In addition, non-targeted sterol metabolites were also identified with the method. The concentrations of POPs found in human plasma in the current study are between 0.3 and 4.5 ng mL⁻¹, i.e., 10–100 times lower than the typical values found for cholesterol oxidation products.