Quantitative determination of taurine and related biomarkers in urine by liquid chromatography–tandem mass spectrometry

Current urinary bladder cancer diagnosis is commonly based on a biopsy obtained during cystoscopy. This invasive method causes discomfort and pain in patients. Recently, taurine and several other compounds such as L-phenylalanine and hippuric acid in urine were found to be indicators of bladder cancer. However, because of a lack of sensitive and accurate analytical techniques, it is impossible to detect these compounds in urine at low levels. In this study, using liquid chromatography–tandem mass spectrometry (LC-MS/MS), a noninvasive method was developed to separate and detect these compounds in urine. ¹⁵N₂-L-glutamine was used as the internal standard, and creatinine acted as an indicator for urine dilution. A phenyl-hexyl column was used for the separation at an isocratic condition of 0.2% formic acid in water and 0.2% formic acid in methanol. Analytes were detected in multiple-reaction monitoring with positive ionization mode. The limit of detection range is 0.18–6 nM and the limit of quantitation ranges from 0.6 to 17.6 nM. The parameters affecting separation and quantification were also investigated and optimized. Proper clinical validation of these biomarkers can be done using this reliable, fast, and simple method. Furthermore, with simple modifications, this method could be applied to other physiological fluids and other types of diseases.     

Metabolomic analysis of amino acids and organic acids in aging mouse eyes using gas chromatography–tandem mass spectrometry

This is a metabolomics study for monitoring altered amino acid (AA) and organic acid (OA) metabolism of in eyes from aging an mouse model at 8 and 18 weeks and 18 months. Simultaneous metabolic profiling analysis of OAs and AAs was performed as ethoxycarbonyl/methoxime/tert-butyldimethylsilyl derivatives by gas chromatography–tandem mass spectrometry. A total of 42 metabolites—24 AAs and 18 OAs—were determined and their composition values were normalized to the corresponding mean values of 8-week-old mice as the control group. Then their normalized values were plotted as star graphs, which were distorted and readily distinguishable for each age-related group. Among the 42 metabolites, 18 AAs and 11 OAs were age dependent and significantly different (p < 0.05). Principal component analysis and partial least squares discriminant analysis showed unclear separation between 8- and 18-week-old mice but clear separation between these and 18-month-old mice. In particular, the variable importance in projection scores of 4-hydroxyproline, cis-aconitic acid, glycine, isocitric acid, leucine, pipecolic acid and lysine from partial least-squares–discriminant analysis were higher than 1.3. A heatmap for the classification and visualization of 42 metabolites showed differences in metabolite changes with aging. Altered AA and OA profiles were monitored, which may explain the metabolic disturbance of AA and OA. These findings are related to mitochondrial dysfunctions related to energy metabolism and the impaired antioxidant system in the aging eye. Therefore, the present metabolomics results of the association between physiological states and altered metabolism of AA and OA will be useful for understanding the aging eye and related diseases.     

Neurosteroid analysis by gas chromatography–atmospheric pressure photoionization–tandem mass spectrometry

A new and simple APPI interface employing commercially available hardware is used to combine GC to MS. The feasibility of the method is demonstrated in the analysis of urine samples for neurosteroids as their trimethylsilyl (TMS) derivatives. The effect of different dopants (chlorobenzene, toluene, anisole) on the ionization of the TMS derivatives was investigated. With chlorobenzene, the TMS derivatives produced intense molecular ions with minimal fragmentation, and chlorobenzene was selected as best dopant. Protonated molecules in addition to intense molecular ions were produced with toluene and anisole. The performance of the method was verified in the analysis of human urine samples. Chromatographic performance was good with peak half-widths of 3.6–4.3 s, linearity (r² > 0.990) was acceptable, limits of detection (LODs) were in the range of 0.01–10 ng mL⁻¹, and repeatability was good with relative standard deviations (rsd%) below 22%. The results show that the method is well suited for the determination of neurosteroids in biological samples.     

Quantitative selenium speciation in human urine by using liquid chromatography–electrospray tandem mass spectrometry

A liquid chromatography–electrospray-tandem mass spectrometry (ES-MS/MS) method was developed for the speciation analysis of four organic selenium species of relevance to human urinary metabolism, namely trimethylselenomium ion (TMSe⁺), selenomethionine (SeMet) and the two selenosugars, methyl 2-acetamido-2-deoxy-1-seleno-β-d-galactos/-glucos-amine (SeGalNAc and SeGluNAc, respectively). Their chromatographic separation was achieved by using a cation exchange pre-column coupled in-series with a reversed-phase high-performance liquid chromatography column, along with an isocratic mobile phase. Online detection was performed using ES-MS/MS in selective reaction monitoring mode. SeGalNAc was detected as the major human urinary metabolite of selenium in the samples analysed, whereas TMSe⁺ was detected in the urine of one volunteer before and after receiving a selenium supplement. SeMet was not detected as a urine excretory metabolite in this study. Spiking experiments performed with the urine samples revealed significant signal suppression caused by coeluting matrix constituents. To overcome such interferences, isotopically labelled ¹³CD₃⁸²SeGalNAc was used as an internal standard, whereas in the absence of an isotopically labelled internal standard for TMSe⁺, the standard addition method was applied. Quality control for the accurate quantitation of TMSe⁺ and SeGalNAc was carried out by analysing spiked human urine samples with appropriate selenium standards over a concentration range of 10–50 μg Se L⁻¹. The method has achieved a limit of detection in the presence of urine matrix comparable to that of HPLC-inductively coupled plasma-mass spectrometry for the four selenium species: 1.0 μg Se L⁻¹ for TMSe⁺, 5.6 μg Se L⁻¹ for SeMet, and 0.1 μg Se L⁻¹ for both SeGalNAc and SeGluNAc.     

Trace analysis of free and combined amino acids in atmospheric aerosols by gas chromatography–mass spectrometry

The analysis of amino acids by gas chromatography mass spectrometry (GC–MS) after their derivatization with N-(t-butyldimethylsilyl)-N-methyltrifluoroacetamide was investigated as an alternative approach for the determination of free (FAA) and combined amino acids (CAA) in aerosols. This technique showed excellent linearity with r² values ranging from 0.9029 to 0.9995 and instrumental limits of detection ranging from 0.3 to 46 pg for the different amino acids. The quality of water used for sample extraction was found to be of utmost importance for achieving low blank levels of FAA and CAA. The addition of isopropanol during the extraction of aerosols was also shown to minimize the coextraction of inorganic salts that interfered with the analysis of FAA, Moreover, the ascorbic acid was found to be the most effective reagent for preventing the oxidative destruction of CAA during the hydrolysis process. By the analysis of spiked aerosol samples, the average recoveries determined for FAA and CAA were higher than 60% and the associated relative standard deviation was lower than 10% for the majority of amino acids. The application of the adopted method in background aerosols of the eastern Mediterranean enabled the unambiguous identification and quantification of 20 amino acids. The total concentration of FAA and CAA in aerosols ranged from 13 to 34 ng m⁻³ and from 29 to 79 ng m⁻³, respectively. The GC–MS based method is proposed to overcome several analytical difficulties usually encountered with the conventional HPLC-fluoresence technique.     

Pharmacokinetics and metabolism of penindolone in rat plasma using liquid chromatography–tandem mass spectrometry

Penindolone (PND) is a novel influenza A virus dual inhibitor that blocks hemagglutinin-mediated adsorption and membrane fusion. A sensitive and specific ultra-performance liquid chromatography–tandem mass spectrometry method was developed and validated to determine PND in rat plasma. Plasma sample preparation was a simple deproteinization with acetonitrile followed by centrifugation. Chromatographic separation was performed on a C₁₈ column with a gradient mobile phase of acetonitrile–water containing 0.1% formic acid. Detection was carried out by electrospray ionization in negative ion multiple reaction monitoring mode. Linear detection responses were obtained for PND ranging from 1 to 1,000 ng/ml. The intra- and inter-day precision (relative standard deviations, RSD) were within 6.5%, and accuracy (relative error, RE) was within ±11.0%. The extraction recovery data for PND and internal standard (IS) were >96.0%. PND was proved to be stable during the sample storage, preparation and analytic procedures. The validated method was successfully applied to pharmacokinetic and bioavailability studies for PND in rats. The results showed the existence of twin peaks, gender difference and nonlinear pharmacokinetics for PND. In addition, two oxidation metabolites and three glucuronidation metabolites of PND were detected by ultra-high-performance liquid chromatography–high resolution mass spectrometry.     

Quantification of atrazine and its metabolites in urine by on-line solid-phase extraction–high-performance liquid chromatography–tandem mass spectrometry

We have developed a method using on-line solid-phase extraction–high-performance liquid chromatography–tandem mass spectrometry (SPE-HPLC-MS/MS) and isotope dilution quantification to measure atrazine and seven atrazine metabolites in urine. The metabolites measured were hydroxyatrazine, diaminochloroatrazine, desisopropylatrazine, desethylatrazine, desethylatrazine mercapturate, atrazine mercaturate and atrazine itself. Our method has good precision (relative standard deviations ranging from 4 to 20% at 5, 10 and 50 ng/mL), extraction efficiencies of 67 to 102% at 5 and 25 ng/mL, relative recoveries of 87 to 112% at 5, 25, 50 and 100 ng/mL limits of detection (LOD) ranging from 0.03 to 2.80 ng/mL. The linear range of our method spans from the analyte LOD to 100 ng/mL (40 ng/mL for atrazine and atrazine mercapturate) with R ² values of greater than 0.999 and errors about the slope of less than 3%. Our method is rapid, cost-effective and suitable for large-scale sample analyses and is easily adaptable to other biological matrices. More importantly, this method will allow us to better assess human exposure to atrazine-related chemicals. Figure A schematic representation showing the elution of the analytes from the solid-phase extraction cartridge onto the analytical column for chromatographic separation prior to MS/MS analysis     

Rapid screening of selective serotonin re-uptake inhibitors in urine samples using solid-phase microextraction gas chromatography–mass spectrometry

In this paper a solid-phase microextraction–gas chromatography–mass spectrometry (SPME–GC–MS) method is proposed for a rapid analysis of some frequently prescribed selective serotonin re-uptake inhibitors (SSRI)—venlafaxine, fluvoxamine, mirtazapine, fluoxetine, citalopram, and sertraline—in urine samples. The SPME-based method enables simultaneous determination of the target SSRI after simple in-situ derivatization of some of the target compounds. Calibration curves in water and in urine were validated and statistically compared. This revealed the absence of matrix effect and, in consequence, the possibility of quantifying SSRI in urine samples by external water calibration. Intra-day and inter-day precision was satisfactory for all the target compounds (relative standard deviation, RSD, <14%) and the detection limits achieved were <0.4 ng mL^–1 urine. The time required for the SPME step and for GC analysis (30 min each) enables high throughput. The method was applied to real urine samples from different patients being treated with some of these pharmaceuticals. Some SSRI metabolites were also detected and tentatively identified.     

Determination of perfluorocarboxylic acids in water by ion-pair dispersive liquid–liquid microextraction and gas chromatography–tandem mass spectrometry with injection port derivatization

A novel technique for derivatization in a gas chromatograph injection port after a one-step extraction of trace perfluorocarboxylic acids (PFCAs) in water with ion pair formation during dispersive liquid–liquid microextraction (DLLME) was investigated. Tetrabutylammonium hydrogen sulfate (TBAHS) was used as the ion pair reagent. PFCA butyl ester derivatives were formed in the GC injection port and then analyzed using gas chromatography coupled to tandem mass spectrometry with negative chemical ionization. According to our analysis, the operative linear range for PFCA detection from 250 pg mL⁻¹ to 2 μg mL⁻¹ with a relative standard derivation (RSD) below 13%. Detection limits were achieved at the level of 37–51 pg mL⁻¹. This method was successfully applied for the analyzing of PFCAs in river water samples from urban and industrial areas without tedious pretreatment. The concentration range over which PFCAs were detected is from 0.6 ng mL⁻¹ to 604.9 ng mL⁻¹.     

Confirmatory method for the determination of nandrolone and trenbolone in urine samples using immunoaffinity cleanup and liquid chromatography–tandem mass spectrometry

A confirmatory method for the simultaneous determination of nandrolone (α and β) and trenbolone (α and β) in urine samples by liquid chromatography electrospray mass spectrometry (LC–MS-MS) was developed. After an enzymatic deconjugation, the urine was subjected to a one-step cleanup on a commercially available immunoaffinity chromatography cartridge. The analytes were detected by liquid chromatography–positive ion electrospray tandem mass spectrometry using deuterium labelled internal standards. The analytical procedure was applicable to bovine and swine urine samples. The procedure was validated as a quantitative confirmatory method according to the Commission Decision 2002/657/EC criteria. The results obtained showed that the method was suitable for statutory residues testing regarding the following performance characteristics: instrumental linearity, specificity, precision (repeatability and intra-laboratory reproducibility), recovery, decision limit (CCα), detection capability (CCβ) and ruggedness. The decision limits (CCα) obtained, were between 0.54 and 0.60 μg L⁻¹; the recovery was above 64% for all the analytes. Repeatability was between 1.6% and 5.7% and within-laboratory reproducibility between 1.6% and 6.0% for all the steroids.     

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%.     

Nonequilibrium hollow-fiber liquid-phase microextraction with in situ derivatization for the measurement of triclosan in aqueous samples by gas chromatography–mass spectrometry

Hollow-fiber liquid-phase microextraction (HF-LPME), a relatively new sample preparation technique, has attracted much interest in the field of environmental analysis. In the current study, a novel method based on hollow-fiber liquid-phase microextraction with in situ derivatization and gas chromatography–mass spectrometry for the measurement of triclosan in aqueous samples is described. Hollow-fiber liquid-phase microextraction conditions such as the type of extraction solvent, the stirring rate, the volume of derivatizing reagent, and the extraction time were investigated. When the conditions had been optimized, the linear range was found to be 0.05–100 μg l⁻¹ for triclosan, and the limit of detection to be 0.02 μg l⁻¹. Tap water and surface water samples collected from our laboratory and Wohushan reservoir, respectively, were successfully analyzed using the proposed method. The recoveries from the spiked water samples were 83.6 and 114.1%, respectively; and the relative standard deviation (RSD) at the 1.0 μg l⁻¹ level was 6.9%.     

Continuous solid-phase extraction and gas chromatography–mass spectrometry determination of pharmaceuticals and hormones in water samples

A semi-automatic flow-based method for the simultaneous determination of 9 pharmaceuticals and 3 hormones in water samples in a single analytical run is proposed. The analytes were retained on a solid-phase extraction sorbent column and 1 μL of the eluate analysed by gas chromatography in combination with electron impact ionization mass spectrometry in the SIM mode. The sorbent used, Oasis-HLB, provided near-quantitative recovery of all analytes. The proposed method was validated with quite good analytical results including low limits of detection (0.01–0.06 ng L⁻¹ for 100 mL of water) and good linearity (r² > 0.993) throughout the studied concentration ranges. The method provided good accuracy (recoveries of 85–103%) and precision (between- and within-day RSD values less than 7%) in the determination of the pharmaceuticals and hormones in tap, river, pond, well, swimming pool and wastewater.     

Trace determination of nine haloacetic acids in drinking water by liquid chromatography–electrospray tandem mass spectrometry

A simple, fast and sensitive liquid chromatography–electrospray tandem mass spectrometry method was established for trace levels of nine haloacetic acids (HAAs) in drinking water. Water samples were removed of residual chlorine by adding l-ascorbic acid, and directly injected after filtered by 0.22 μm membrane. Nine HAAs were separated by liquid chromatography in 7.5 min, and the limits of detection were generally between 0.16 and 0.99 μg/L except for chlorodibromoacetic acid (1.44 μg/L) and tribromoacetic acid (8.87 μg/L). The mean recoveries of nine target compounds in spiked drinking water samples were 80.1–108%, and no apparent signal suppression was observed. Finally, this method was applied to determine HAAs in the tap water samples collected from five waterworks in Shandong, China. Nine HAAs except for monochloroacetic acid, monobromoacetic acid, dibromochloroacetic acid and tribromoacetic acid were detected, and the total concentrations were 7.79–36.5 μg/L. The determination results well met the first stage of the Disinfectants/Disinfection By-Products (D/DBP) Rules established by U.S.EPA and Guidelines for Drinking-water Quality of WHO.