Quantification of plasma homocitrulline using hydrophilic interaction liquid chromatography (HILIC) coupled to tandem mass spectrometry

Homocitrulline (HCit), an amino acid formed by the carbamylation of ε-amino groups of lysine residues, is considered a promising biomarker for monitoring diseases such as chronic renal failure and atherosclerosis. This paper describes a tandem mass spectrometric method for total, protein-bound and free HCit measurement in plasma samples. HCit was separated from other plasma components by hydrophilic interaction liquid chromatography. Detection was achieved by monitoring transitions of 190.1 > 127.1 and 190.1 > 173.1 for HCit, and 183.1 > 120.2 for d₇-citrulline used as internal standard. This method allowed HCit quantification within 5.2 min and was precise (inter-assay CV < 5.85%), accurate (mean recoveries ranging from 97% to 106%), and exhibited a good linearity from 10 nmol/L to 1.6 μmol/L. Plasma samples from control and uremic mice (n = 10) were analyzed. In control mice, mean total plasma HCit concentration was 0.78 ± 0.12 μmol/mol amino acids, whereas it was increased 2.7-fold in uremic mice plasma, reaching 2.10 ± 0.50 μmol/mol amino acids (p < 0.001). In conclusion, this method exhibits good analytical performances and meets the criteria of sensitivity suitable for HCit concentration assessment in plasma samples.     

Automated mass spectrometric analysis of urinary and plasma serotonin

Serotonin emerges as crucial neurotransmitter and hormone in a growing number of different physiologic processes. Besides extensive serotonin production previously noted in patients with metastatic carcinoid tumors, serotonin now is implicated in liver cell regeneration and bone formation. The aim was to develop a rapid, sensitive, and highly selective automated on-line solid-phase extraction method coupled to high-performance liquid chromatography–tandem mass spectrometry (XLC-MS/MS) to quantify low serotonin concentrations in matrices such as platelet-poor plasma and urine. Fifty microliters plasma or 2.5 μL urine equivalent were pre-purified by automated on-line solid-phase extraction, using weak cation exchange. Chromatography of serotonin and its deuterated internal standard was performed with hydrophilic interaction chromatography. Mass spectrometric detection was operated in multiple reaction monitoring mode using a quadrupole tandem mass spectrometer with positive electrospray ionization. Serotonin concentrations were determined in platelet-poor plasma of metastatic carcinoid patients (n = 23) and healthy controls (n = 22). Urinary reference intervals were set by analyzing 24-h urine collections of 120 healthy subjects. Total run-time was 6 min. Intra- and inter-assay analytical variation were <10%. Linearity in the 0–7300 μmol/L calibration range was excellent (R² > 0.99). Quantification limits were 30 and 0.9 nmol/L in urine and plasma, respectively. Platelet-poor serotonin concentrations in metastatic carcinoid patients were significantly higher than in controls. The urinary reference interval was 10–78 μmol/mol creatinine. Serotonin analysis with sensitive and specific XLC-MS/MS overcomes limitations of conventional HPLC. This enables accurate quantification of serotonin for both routine diagnostic procedures and research in serotonin-related disorders.     

Measurement of phospholipids by hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry: the determination of choline containing compounds in foods

A hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC LC-MS/MS) method using multiple scan modes was developed to separate and quantify 11 compounds and lipid classes including acetylcholine (AcCho), betaine (Bet), choline (Cho), glycerophosphocholine (GPC), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphocholine (PCho) and sphingomyelin (SM). This includes all of the major choline-containing compounds found in foods. The method offers advantages over other LC methods since HILIC chromatography is readily compatible with electrospray ionization and results in higher sensitivity and improved peak shapes. The LC-MS/MS method allows quantification of all choline-containing compounds in a single run. Tests of method suitability indicated linear ranges of approximately 0.25-25 μg/ml for PI and PE, 0.5-50 μg/ml for PC, 0.05-5 μg/ml for SM and LPC, 0.5-25 μg/ml for LPE, 0.02-5 μg/ml for Cho, and 0.08-8 μg/ml for Bet, respectively. Accuracies of 83-105% with precisions of 1.6-13.2% RSD were achieved for standards over a wide range of concentrations, demonstrating that this method will be suitable for food analysis. 8 polar lipid classes were found in a lipid extract of egg yolk and different species of the same class were differentiated based on their molecular weights and fragment ion information. PC and PE were found to be the most abundant lipid classes consisting of 71% and 18% of the total phospholipids in egg yolk.     

Evaluation of a novel ELISA for serotonin: urinary serotonin as a potential biomarker for depression

Depression is a common disorder with physical and psychological manifestations often associated with low serotonin. Since noninvasive diagnostic tools for depression are sparse, we evaluated the clinical utility of a novel ELISA for the measurement of serotonin in urine from depressed subjects and from subjects under antidepressant therapy. We developed a competitive ELISA for direct measurement of serotonin in derivatized urine samples. Assay performance was evaluated and applied to clinical samples. The analytical range of the assay was from 6.7 to 425 μg serotonin/g creatinine (Cr). The limit of quantification was 4.7 μg/g Cr. The average recovery for spiked urine samples was 104.4%. Average intra-assay variation was 4.4%, and inter-assay variation was <20%. The serotonin analysis was very specific. No significant interferences were observed for 44 structurally and nonstructurally related urinary substances. Very good correlation was observed between urinary serotonin levels measured by ELISA and liquid chromatography tandem mass spectrometry (LC-MS/MS; ELISA = 1.16 × LC-MS/MS − 53.8; r = 0.965; mean % bias = 11%; n = 18). Serotonin was stable in acidified urine for 30 days at room temperature and at −20 °C. The established reference range for serotonin was 54–366 μg/g Cr (n = 64). Serotonin levels detected in depressed patients (87.53 ± 4.89 μg/g Cr; n = 60) were significantly lower (p < 0.001) than in nondepressed subjects (153.38 ± 7.99 μg/g Cr). Urinary excretion of serotonin in depressed individuals significantly increased after antidepressant treatment by 5-hydroxy-tryptophane and/or selective serotonin re-uptake inhibitor (p < 0.01). The present ELISA provides a convenient and robust method for monitoring urinary serotonin. It is suitable to monitor serotonin imbalances and may be particularly helpful in evaluating antidepressant therapies.     

Semi-automated liquid chromatography-mass spectrometry (LC-MS/MS) method for basic pesticides in wastewater effluents

Effluent from wastewater treatment plants have been identified as an important source of micro-organic contaminants in the environment. An online high-performance liquid chromatography–heated electrospray ionization tandem mass spectrometric method was developed and validated for the determination of basic pesticides in effluent wastewaters. Most available methods for pesticide analysis of wastewater samples are time-consuming, require complex clean-up steps and are difficult to automate. The method developed used a simple solid-phase extraction clean-up for salt and lipid reduction. On-line sample pre-concentration was performed using a reversed phase (C₁₈) column, and analytes were separated by back-flushing onto an analytical column (C₈) with detection using QqQ MS. An option to increase MS resolution was exploited to minimize interference from endogenous compounds in the matrix. A better than unit mass resolution was used (Q1 full width half maximum (FWHM) = 0.2 Da and Q3 FWHM = 0.7 Da), which was as rugged as a unit resolution method, and improved signal/noise and better detection limits were achieved for the targeted basic pesticides. This method was applied to the determination of 11 pesticides, including methoxytriazine, chlorotriazines, chloroacetanilides, phenylurea and carbamate pesticides. The percentage recovery values for these pesticides using the online trapping column were within the range, 73–95%, with relative standard deviation (RSD) values <8.9%. The highest concentrations of these pesticides in wastewater effluents in County Cork, Ireland, were simazine (0.51 μg/L), prometon (0.14 μg/L), diuron (0.21 μg/L) and atrazine (0.19 μg/L).     

Determination of cyanide in blood by electrospray ionization tandem mass spectrometry after direct injection of dicyanogold

An electrospray ionization tandem mass spectrometric (ESI-MS-MS) method has been developed for the determination of cyanide (CN^–) in blood. Five microliters of blood was hemolyzed with 50 μL of water, then 5 μL of 1 M tetramethylammonium hydroxide solution was added to raise the pH of the hemolysate and to liberate CN^– from methemoglobin. CN^– was then reacted with NaAuCl₄ to produce dicyanogold, Au(CN)₂^–, that was extracted with 75 μL of methyl isobutyl ketone. Ten microliters of the extract was injected directly into an ESI-MS-MS instrument and quantification of CN^– was performed by selected reaction monitoring of the product ion CN^– at m/z 26, derived from the precursor ion Au(CN)₂^– at m/z 249. CN^– could be measured in the quantification range of 2.60 to 260 μg/L with the limit of detection at 0.56 μg/L in blood. This method was applied to the analysis of clinical samples and the concentrations of CN^– in the blood were as follows: 7.13 ± 2.41 μg/L for six healthy non-smokers, 3.08 ± 1.12 μg/L for six CO gas victims, 730 ± 867 μg for 21 house fire victims, and 3,030 ± 97 μg/L for a victim who ingested NaCN. The increase of CN^– in the blood of a victim who ingested NaN₃ was confirmed using MS-MS for the first time, and the concentrations of CN^– in the blood, gastric content and urine were 78.5 ± 5.5, 11.8 ± 0.5, and 11.4 ± 0.8 μg/L, respectively.     

Liquid chromatography-tandem mass spectrometry (LC/APCI-MS/MS) methods for the quantification of captan and folpet phthalimide metabolites in human plasma and urine

Captan and folpet are fungicides largely used in agriculture. They have similar chemical structures, except that folpet has an aromatic ring unlike captan. Their half-lives in blood are very short, given that they are readily broken down to tetrahydrophthalimide (THPI) and phthalimide (PI), respectively. Few authors measured these biomarkers in plasma or urine, and analysis was conducted either by gas chromatography coupled to mass spectrometry or liquid chromatography with UV detection. The objective of this study was thus to develop simple, sensitive and specific liquid chromatography–atmospheric pressure chemical ionization-tandem mass spectrometry (LC/APCI-MS/MS) methods to quantify both THPI and PI in human plasma and urine. Briefly, deuterated THPI was added as an internal standard and purification was performed by solid-phase extraction followed by LC/APCI-MS/MS analysis in negative ion mode for both compounds. Validation of the methods was conducted using spiked blank plasma and urine samples at concentrations ranging from 1 to 250 μg/L and 1 to 50 μg/L, respectively, along with samples of volunteers and workers exposed to captan or folpet. The methods showed a good linearity (R ² > 0.99), recovery (on average 90% for THPI and 75% for PI), intra- and inter-day precision (RSD, <15%) and accuracy (<20%), and stability. The limit of detection was 0.58 μg/L in urine and 1.47 μg/L in plasma for THPI and 1.14 and 2.17 μg/L, respectively, for PI. The described methods proved to be accurate and suitable to determine the toxicokinetics of both metabolites in human plasma and urine.     

Determination of dextromethorphan in human plasma using pipette tip solid-phase extraction and gas chromatography-mass spectrometry

Dextromethorphan was extracted from human plasma samples (100 μL) using MonoTip C₁₈ tips, which are packed with C₁₈-bonded monolithic silica gel that is attached to the inside of the tip. The samples, which contained dextromethorphan and trimeprazine as an internal standard (IS), were mixed with 200 μL of distilled water and 50 μL of 1 mol/L glycine–sodium hydroxide buffer (pH 10). The mixture was extracted to the C₁₈ phase of the tip by 20 sequential aspirating/dispensing cycles using a manual micropipettor. The analytes retained on the C₁₈ phase were then eluted with methanol by five sequential aspirating/dispensing cycles. The eluate was injected directly into a gas chromatograph and detected by a mass spectrometer with selected ion monitoring in positive electron ionization mode. An Equity-5 fused silica capillary column (30 m × 0.32 mm i.d., film thickness 0.5 μm) gave adequate separation of the dextromethorphan, IS, and impurities. The recoveries of dextromethorphan and the IS spiked into plasma were >87.4%. The regression equation for dextromethorphan showed excellent linearity from 2.5 to 320 ng/mL of plasma, and the limit of detection was 1.25 ng/mL of plasma. The intraday and interday coefficients of variation were less than 10.5% and 14.7%, respectively. The accuracy ranged from 91.9% to 107%. The validated method was successfully used to quantify the plasma concentration of dextromethorphan in a human subject after oral administration of the drug.     

Solid-phase extraction and GC-MS analysis of potentially genotoxic cleavage products of β-carotene in primary cell cultures

A validated method for the simultaneous determination of prominent volatile cleavage products (CPs) of β-carotene in cell culture media has been developed. Target CPs comprised β-ionone (β-IO), cyclocitral (CC), dihydroactinidiolide (DHA), and 1,1,6-trimethyltetraline (TMT). CPs were extracted by solid-phase extraction applying a phenyl adsorbent, eluted with 10% (v/v) tetrahydrofuran in n-hexane, and identified and quantified by gas chromatography-mass spectrometry with electron impact ionization. Method validation addressed linearity confirmation over two application ranges and homoscedasticity testing. Recoveries from culture media were between 71.7% and 95.7% at 1.0 μg/ml. Precision of recoveries determined in intra-day (N = 5) and inter-day (N = 15) assays were <2.0% and <4.8%, respectively. Limit of detection and limit of quantification of the analysis method were <18.0 and <53.0 ng/ml for β-IO, CC, and TMT, whereas 156 and 474 ng/ml were determined for DHA, respectively. Although extractions of blank matrix proved the absence of interfering peaks, statistical comparison between slopes determined for instrumental and total method linearity revealed significant differences. The method was successfully applied in selecting an appropriate solvent for the fortification of culture media with volatile CPs, including the determination of their availability over the incubation period. For the first time, quantification of volatile CPs in treatment solutions and culture media for primary cells becomes accessible by this validated method.     

Electrochemical immunoassay using quantum dot/antibody probe for identification of cyanobacterial hepatotoxin microcystin-LR

The presence of cyanobacterial hepatotoxins such as microcystin-LR poses health threats to humans due to their potential for causing severe physiological effects when contaminated drinking water is ingested. Here, the electrochemical detection of microcystin-LR is explored using a quantum dot/antibody (QD/Ab) probe for nanoparticle-based amplification and direct electrochemical transduction. The immunological recognition of microcystin-LR using the QD/Ab probe was amplified and converted to an electrochemical signal by measuring the cadmium ions released from QD based on square wave stripping voltammetry under optimized electrochemical factors. Whereas a qualitative analysis for microcystin-LR was achieved using the specific peak potential of the anodic voltammogram at −0.6 ± 0.05 V, concentration of the toxin was quantified based on the charge density of the anodic peak; a dynamic range of 0.227 to 50 μg/L and limit of detection of 0.099 μg/L were obtained with high sensitivity. The extracted microcystin-LR from Microcystis aeruginosa was estimated as 1,944 μg/g of dried weight of the microorganism.     

Visualization of phosphatidylcholine,lysophosphatidylcholine and sphingomyelin in mouse tongue body by matrix-assisted laser desorption/ionization imaging mass spectrometry

The mammalian tongue is one of the most important organs during food uptake because it is helpful for mastication and swallowing. In addition, taste receptors are present on the surface of the tongue. Lipids are the second most abundant biomolecules after water in the tongue. Lipids such as phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and sphingomyelin (SM) are considered to play fundamental roles in the mediation of cell signaling. Imaging mass spectrometry (IMS) is powerful tool for determining and visualizing the distribution of lipids across sections of dissected tissue. In this study, we identified and visualized the PC, LPC, and SM species in a mouse tongue body section with matrix-assisted laser desorption/ionization (MALDI)-IMS. The ion image constructed from the peaks revealed that docosahexaenoic acid (DHA)-containing PC, LPC, linoleic acid-containing PC and SM (d18:1/16:0), and oleic acid-containing PC were mainly distributed in muscle, connective tissue, stratified epithelium, and the peripheral nerve, respectively. Furthermore, the distribution of SM (d18:1/16:0) corresponded to the distribution of nerve tissue relating to taste in the stratified epithelium. This study represents the first visualization of PC, LPC and SM localization in the mouse tongue body.     

Simultaneous determination of catechol and hydroquinone based on poly (diallyldimethylammonium chloride) functionalized graphene-modified glassy carbon electrode

Simultaneous determination of catechol (CC) and hydroquinone (HQ) were investigated by voltammetry based on glassy carbon electrode (GCE) modified by poly (diallyldimethylammonium chloride) (PDDA) functionalized graphene (PDDA-G). The modified electrode showed excellent sensitivity and selectivity properties for the two dihydroxybenzene isomers. In 0.1 mol/L phosphate buffer solution (PBS, pH 7.0), the oxidation peak potential difference between CC and HQ was 108 mV, and the peaks on the PDDA-G/GCE were three times as high as the ones on graphene-modified glass carbon electrode. Under optimized conditions, the PDDA-G/GCE showed wide linear behaviors in the range of 1 × 10⁻⁶−4 × 10⁻⁴ mol/L for CC and 1 × 10⁻⁶−5 × 10⁻⁴ mol/L for HQ, with the detection limits 2.0 × 10⁻⁷ mol/L for CC and 2.5 × 10⁻⁷ mol/L for HQ (S/N = 3) in mixture, respectively. Some kinetic parameters, such as the electron transfer number (n), charge transfer coefficient (α), and the apparent heterogeneous electron transfer rate constant (k _s), were calculated. The proposed method was applied to simultaneous determine CC and HQ in real water samples of Yellow River with satisfactory results.     

Accurate quantification of mercapturic acids of styrene (PHEMAs) in human urine with direct sample injection using automated column-switching high-performance liquid chromatography coupled with tandem mass spectrometry

Styrene is one of the most important industrial chemicals, with an enormously high production volume worldwide. The urinary mercapturic acids of its metabolite styrene-7,8-oxide, namely N-acetyl-S-(2-hydroxy-1-phenylethyl)-l-cysteine (PHEMA 1) and N-acetyl-S-(2-hydroxy-2-phenylethyl)-l-cysteine (PHEMA 2), are specific biomarkers for the determination of individual internal exposure to this highly reactive intermediate of styrene. We have developed and validated a fast, specific and very sensitive method for the accurate determination of the sum of phenylhydroxyethyl mercapturic acids (PHEMAs) in human urine with an automated multidimensional liquid chromatography–tandem mass spectrometry method using ¹³C₆-labelled PHEMAs as internal standards. Analytes were stripped from the urinary matrix by online extraction on a restricted access material, transferred to the analytical column and subsequently determined by tandem mass spectrometry. The limit of quantification (LOQ) for the sum of PHEMAs was 0.3 μg/L urine and allowed us to quantify the background exposure of the (smoking) general population. Precision within series and between series ranged from 1.5 to 6.8% at three concentrations ranging from 3 to 30 μg/L urine; the mean accuracy was between 104 and 110%. We applied the method to spot urine samples from 40 subjects of the general population with no known occupational exposure to styrene. The median levels (range) for the sum of PHEMAs in urine of non-smokers (n = 22) were less than 0.3 μg/L (less than 0.3 to 1.1 μg/L), whereas in urine of smokers (n = 18), the median levels were 0.46 μg/L (less than 0.3 to 2.8 μg/L). Smokers showed a significantly higher excretion of the sum of PHEMAs (p = 0.02). Owing to its automation and high sensitivity, our method is well suited for application in occupational or environmental studies.     

Quantification of femtomolar concentrations of the CYP3A substrate midazolam and its main metabolite 1'-hydroxymidazolam in human plasma using ultra performance liquid chromatography coupled to tandem mass spectrometry

The benzodiazepine midazolam is a probe drug used to phenotype cytochrome P450 3A activity. In this situation, effective sedative concentrations are neither needed nor desired, and in fact the use of very low doses is advantageous. We therefore developed and validated an assay for the femtomolar quantification of midazolam and 1′-hydroxymidazolam in human plasma. Plasma (0.25 mL) and 96-well-based solid-phase extraction were used for sample preparation. Extraction recoveries ranged between 75 and 92% for both analytes. Extracts were chromatographed within 2 min on a Waters BEH C18 1.7 μm UPLC? column with a fast gradient consisting of formic acid, ammonia, and acetonitrile. Midazolam and 1′-hydroxymidazolam were quantified using deuterium- and ¹³C-labeled internal standards and positive electrospray tandem mass spectrometry in the multiple reaction monitoring mode, which yielded lower limits of quantification of 50 fg/mL (154 fmol/L) and 250 fg/mL (733 fmol/L) and a corresponding precision of <20%. The calibrated concentration ranges were linear for midazolam (0.05–250 pg/mL) and 1′-hydroxymidazolam (0.25–125 pg/mL), with correlation coefficients of >0.99. Within-batch and batch-to-batch precision in the calibrated ranges for both analytes were <14% and <12%. No ion suppression was detectable, and plasma matrix effects were minimized to <15% (<25%) for midazolam (1′-hydroxymidazolam). The assay was successfully applied to assess the kinetics of midazolam in two human volunteers after the administration of single oral microgram doses (1–100 μg). This ultrasensitive assay allowed us to quantify the kinetics of midazolam and 1′-hydroxymidazolam for at least 10 h, even after the administration of only 1 μg of midazolam.     

Flow-injection analysis for the determination of total inorganic carbon and total organic carbon in water using the H2O2–luminol–uranine chemiluminescent reaction

In the presence of carbonate and uranine, the chemiluminescent intensity from the reaction of luminol with hydrogen peroxide was dramatically enhanced in a basic medium. Based on this fact and coupled with the technique of flow-injection analysis, a highly sensitive method was developed for the determination of carbonate with a wide linear range. The method provided the determination of carbonate with a wide linear range of 1.0 × 10⁻¹⁰–5.0 × 10⁻⁶ mol L⁻¹ and a low detection limit (S/N = 3) of carbonate of 1.2 × 10⁻¹¹ mol L⁻¹. The average relative standard deviation for 1.0 × 10⁻⁹–9.0 × 10⁻⁷ mol L⁻¹ of carbonate was 3.7% (n = 11). Combined with the wet oxidation of potassium persulfate, the method was applied to the simultaneous determination of total inorganic carbon (TIC) and total organic carbon (TOC) in water. The linear ranges for TIC and TOC were 1.2 × 10⁻⁶–6.0 × 10⁻² mg L⁻¹ and 0.08–30 mg L⁻¹ carbon, respectively. Recoveries of 97.4–106.4% for TIC and 96.0–98.5% for TOC were obtained by adding 5 or 50 mg L⁻¹ of carbon to the water samples. The relative standard deviations (RSDs) were 2.6–4.8% for TIC and 4.6–6.6% for TOC (n = 5). The mechanism of the chemiluminescent reaction was also explored and a reasonable explanation about chemical energy transfer from luminol to uranine was proposed. Figure Chemiluminescence profiles in batch system. 1, Injection of 100 μL of K₂CO₃ into 1.0 mL luminol-1.0 mL H₂O₂ solution; 2-3 and 4-5, Injection in sequence of 100 μL of K₂CO₃ and 100 μL of uranine into 1.0 ml luminol-1.0 mL H₂O₂ solution; C_luminol = 1.0 × 10⁻⁷ mol/L, C_H2O2 = 1.0 × 10⁻⁵ mol/L, C_uranine = 1.0 × 10⁻⁵ mol/L, C_K2CO3 = 1.0 × 10⁻⁷ mol/L except for 4-5 where C_K2CO3 = 1.0 × 10⁻⁴ mol/L     

Preconditioning of Axillary Buds in Thidiazuron-Supplemented Liquid Media Improves In Vitro Shoot Multiplication in <Emphasis Type="Italic">Nyctanthes arbor-tristis</Emphasis> L.

An efficient tissue culture technology has been designed for mass multiplication of Nyctanthes arbor-tristis L. by preculturing nodal explants in thidiazuron (TDZ)-supplemented liquid Murashige and Skoog (MS) media. Direct inoculation of nodal segments on semi-solid MS medium augmented with various concentrations of TDZ (0.1 to 0.9 μM) produced shoots but with low regeneration response and few shoots per explant. Hence, nodal explants were pretreated with greater concentrations of TDZ (5 to 100 μM) in liquid MS media for different durations (4, 8, 12, and 16 days) with the aim of improving shoot regeneration response from cultured explants. After pretreatment, explants were transferred to agar-solidified hormone-free MS medium. Best response in terms of percent regeneration (94%), number of shoots per explant (20.00 ± 1.15), and greatest shoot length (7.23 ± 0.83 cm) were obtained with nodal segments pretreated in75 μM TDZ for 8 days. Similarly, root induction was obtained from pulse-treated microshoots for 24 h with 200 μM indole-3-butyric acid (IBA) followed by their transfer to 1/2 MS medium which produced an average of 5.50 ± 0.92 roots per microshoot. The rooted plantlets were transplanted to soil with 80% success rate.     

Rapid residue analysis of four triazolopyrimidine herbicides in soil, water, and wheat by ultra-performance liquid chromatography coupled to tandem mass spectrometry

A sensitive and effective method for simultaneous determination of triazolopyrimidine sulfonamide herbicide residues in soil, water, and wheat was developed using ultra-performance liquid chromatography coupled with tandem mass spectrometry. The four herbicides (pyroxsulam, flumetsulam, metosulam, and diclosulam) were cleaned up with an off-line C18 SPE cartridge and detected by tandem mass spectrometry using an electrospray ionization source in positive mode (ESI+). The determination of the target compounds was achieved in <2.0 min. The limits of detection were below 1 μg kg⁻¹, while the limits of quantification did not exceed 3 μg kg⁻¹ in different matrices. Quantitation was determined from calibration curves of standards containing 0.05–100 μg L⁻¹ with r ² > 0.997. Recovery studies were conducted at three spiked levels (0.2, 1, and 5 μg kg⁻¹ for water; 5, 10, and 100 μg kg⁻¹ for soil and wheat). The overall average recoveries for this method in water, soil, wheat plants, and seeds at three levels ranged from 75.4% to 106.0%, with relative standard deviations in the range of 2.1–12.5% (n = 5) for all analytes.     

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.     

Development of an LC-MS/MS method for the analysis of serotonin and related compounds in urine and the identification of a potential biomarker for attention deficit hyperactivity/hyperkinetic disorder

Serotonin is a major neurotransmitter and affects various functions both in the brain and in the rest of the body. It has been demonstrated that altered serotinergic function is implicated in various psychiatric disorders including depression and schizophrenia. Serotonin has also been implicated along with dopamine in attention deficit–hyperkinetic disorder (AD-HKD). This study provides a versatile validated method for the analysis of serotonin, hydroxyindole acetic acid and dopamine in urine using LC-MS/MS. This method was then used to quantify these analytes in a test group of 17 children diagnosed with severe AD-HKD. This group was compared to a matched control group to investigate the possibility that one of these compounds may be a potential biomarker for this condition. The developed method provided good linear calibration curves for the multiplex assay of analytes in urine (0.05–3.27 nmol/L; R ² ≥ 0.9977). Acceptable inter-day repeatability was achieved for all analytes with RSD values (n = 9) ranging from 1.1% to 9.3% over a concentration range of 0.11–3.27 μmol/L in urine. Excellent limits of detection (LOD) and limits of quantitation (LOQ) were achieved with LODs of 8.8–18.2 nmol/L and the LOQs of 29.4–55.7 nmol/L for analytes in urine. Recoveries were in the ranges of 98–104%, 100–106% and 91–107% for serotonin, 5-HIAA and dopamine, respectively. An appropriate sample clean-up procedure for urine was developed to ensure efficient recovery and reproducibility on analysis. Evaluation of matrix effects was also carried out and the influence of ion suppression on analytical results reported. Confirmatory analysis was carried out on a linear trap quadrupole-Orbitrap mass spectrometer to obtain high mass accuracy data of the target analytes in the clinical samples.     

Monosegmented flow-analysis of slow enzymatic reactions: Determination of triglycerides in serum

 A monosegmented flow system (MCFA) is proposed to achieve slow enzymatic spectrophotometric determinations, here applied to the determination of triglycerides in blood serum. The sample (4.5 μL), enzymatic reagent (150 μL) and an air plug (100 μL) are simultaneously inserted into a carrier stream buffered to pH 7.9 (Tris ⋅ HCl). In order to avoid the cumbersome step of air removal, a relocating detector was used. This system handles about 60 samples/h, yielding precise results (r.s.d. usually<2.5%). Sensitivity is 56 mAU ⋅ L/mmol up to 6 mmol/L triglycerides. Accuracy was assessed by running 50 samples already analysed by a conventional procedure yielding the equation C_MCFA(mmol/L)=1.00(±0.04) C_Ref(mmol/L)−0.03(±0.08); r=0.990. Received: 22 January 1997/Revised: 12 March 1997/Accepted: 28 March 1997