UHPLC method for the simultaneous determination of β-blockers, isoflavones, and flavonoids in human urine

A simple method using solid-phase extraction (SPE) and ultra high-performance liquid chromatography (UHPLC) for the simultaneous determination of β-blockers, isoflavones, and flavonoids in human urine is developed. A statistical central composite design and response surface analysis is used to optimize the separation of the analytes. These multivariate procedures are efficient in determining the optimal separation condition using resolutions and retention time as responses. A gradient elution using a mobile phase consisting of 0.05% trifluoroacetic acid in water and acetonitrile is applied on a Hypersil GOLD column within a short analysis time of 4.5 min. UV detection was used to monitor the analytes. The suggested method was linear in a concentration range from 0.04-20.00 μg/mL, depending on the compound. The limits of detection ranged from 8.9 to 66.2 ng/mL. The precision was lower than 2.74%, and the accuracy was between 0.01-3.65%. The Oasis HLB column, with the highest recoveries, is selected for the pre-concentration step. This present paper reports, for the first time, a method for the simultaneous determination of β-blockers, isoflavones, and flavonoids in human urine samples. Furthermore, the developed method can also be applied to the routine determination of examined compounds concentrations in human urine.     

Capillary electrophoresis with direct chemiluminescence detection for the analysis of catecholamines in human urine

A rapid and sensitive method for the analysis of three catecholamines by capillary electrophoresis(CE)with directchemiluminescence(CL)detection is described.The detection limits(S/N=3)were 1.3*10-8g/mL for isoprenaline,1.0*10-8g/mL for epinephrine and 2.8*10-8g/mL for dopamine.The proposed method was successfully applied to theanalysis of catecholamines in urine samples of cigarette smokers and nonsmokers.The results showed that there is a close relationbetween the release of dopamine in human body fluids and cigarette smoking/nonsmoking.     

Flow injection chemiluminescence determination of l-cysteine in amino acid mixture and human urine with the BrO3 −–quinine system

In this paper, a novel flow injection chemiluminescence (CL) determination of l-cysteine is proposed. The method is based on the CL reaction of l-cysteine and KBrO₃ in acidic medium. The CL intensity was greatly enhanced in the presence of quinine. The CL intensity was linear with l-cysteine concentration in the range of 0.2–80 g L^–1, and the detection limit was 0.1 g L^–1 (3). A complete analysis, including sampling and injecting, could be performed in 1 min, giving a throughput of about 60 h^–1. The relative standard deviation was 1.6% for 0.8 g L^–1 l-cysteine (n=11). The proposed method was satisfactorily applied to the determination of cysteine in an amino acid mixture and human urine. The mechanism of the CL reaction is also discussed.     

Development of a sensitive enzyme immunoassay for the detection of phenyl-β-d-thioglucuronide in human urine

Immunoassays for the measurement of glucuronides in human urine can be a helpful tool for the assessment of human exposure to toxic chemicals. Therefore an enzyme immunoassay (EIA) for the specific detection of phenyl-β-d-thioglucuronide was developed. The immunoconjugate was formed by coupling p-aminophenyl-β-d-thioglucuronide to the carrier protein thyroglobulin leaving an exposed glucuronic acid. The hapten-protein conjugate was adsorbed to gold colloids in order to enhance the immunogenic effect. Rabbits were injected with the immunogold conjugates to raise polyclonal antibodies. The resulting competitive assay showed an inhibition by phenyl-β-d-thioglucuronide at sample concentrations of 23.0 ± 1.3 ng/mL (50% B/B₀) and a high cross-reactivity to p-aminophenyl-β-D-thioglucuronide (120%). Little cross-reactivities (< 2%) were observed for potential urinary cross reactants. In addition human urine samples were incubated with β-glucuronidase in order to investigate the EIA for specific matrix effects. An integration of high-performance liquid chromatography (HPLC) and EIA was developed in an attempt to decrease the matrix effects and increase the sensitivity of the overall method. The hyphenated technique HPLC-EIA may be used to monitor human exposure to toxic thiophenol which is excreted by mammals as urinary phenyl thioglucuronide.     

A qualitative/quantitative approach for the detection of 37 tryptamine-derived designer drugs, 5 β-carbolines, ibogaine, and yohimbine in human urine and plasma using standard urine screening and multi-analyte approaches

The first synthetic tryptamines have entered the designer drug market in the late 1990s and were distributed as psychedelic recreational drugs. In the meantime, several analogs have been brought onto the market indicating a growing interest in this drug class. So far, only scarce analytical data were available on the detectability of tryptamines in human biosamples. Therefore, the aim of the presented study was the development and full validation of a method for their detection in human urine and plasma and their quantification in human plasma. The liquid chromatography-linear ion trap mass spectrometry method presented covered 37 tryptamines as well as five β-carbolines, ibogaine, and yohimbine. Compounds were analyzed after protein precipitation of urine or fast liquid–liquid extraction of plasma using an LXQ linear ion trap coupled to an Accela ultra ultra high-performance liquid chromatography system. Data mining was performed via information-dependent acquisition or targeted product ion scan mode with positive electrospray ionization. The assay was selective for all tested substances with limits of detection in urine between 10 and 100 ng/mL and in plasma between 1 and 100 ng/mL. A validated quantification in plasma according to international recommendation could be demonstrated for 33 out of 44 analytes.     

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.     

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

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

Development and validation of a liquid chromatography–tandem mass spectrometry assay for the simultaneous quantification of buprenorphine, norbuprenorphine, and metabolites in human urine

A liquid chromatography–tandem mass spectrometry method for the simultaneous quantification of buprenorphine (BUP), norbuprenorphine (NBUP), buprenorphine glucuronide (BUP-Gluc), and norbuprenorphine glucuronide (NBUP-Gluc) in human urine was developed and fully validated. Extensive endogenous and exogenous interferences were evaluated and limits of quantification were identified empirically. Analytical ranges were 5–1,000 ng/mL for BUP and BUP-Gluc and 25–1,000 ng/mL for NBUP and NBUP-Gluc. Intra-assay and interassay imprecision were less than 17% and recovery was 93–116%. Analytes were stable at room temperature, at 4 °C, and for three freeze–thaw cycles. This accurate and precise assay has sufficient sensitivity and specificity for urine analysis of specimens collected from individuals treated with BUP for opioid dependence.     

Identification of the long-acting erythropoiesis-stimulating agent darbepoetin alfa in human urine by liquid chromatography–tandem mass spectrometry

The misuse of recombinant human erythropoietin (rhEPO) increases the proliferation/production of erythrocytes, which enhance oxygen transport capacities, and has grave consequences with respect to human health and fairness in sports. For sports drug testing, the current analytical methods for rhEPOs are mainly gel electrophoretic methods, such as isoelectric focusing–polyacrylamide gel electrophoresis. Mass spectrometry is fundamentally necessary for the reliable identification of rhEPOs in doping control. In this study, a high-sensitivity and high-throughput mass spectrometric qualitative detection method for darbepoetin alfa in human urine was established by a bottom-up approach. The novel method involves the immunopurification of human urine (10 mL), protease digestion with endoproteinase Glu-C (V8-protease) in an ammonium bicarbonate buffer (pH 7.8) and ultra-performance liquid chromatography using a charged surface hybrid C₁₈ column coupled with electrospray-ionisation high-sensitivity tandem mass spectrometry for improved selectivity of the target molecules. The specific fragment digested from darbepoetin alfa was ⁹⁰TLQLHVDKAVSGLRSLTTLLRALGAQKE¹¹⁷ (V₁₁). The lower limit of detection of urinary darbepoetin alfa was 1.2 pg/mL. The limit of detection for the confirmation analysis was estimated to be 5 pg/mL. The developed method allows high-throughput confirmation analysis, namely 6 h for sample preparation and an analytical run time of only 10 min per sample; this high-throughput method dramatically decreases the workload in the laboratory. Darbepoetin alfa could be identified in human urine collected after the intravenous administration of 15 μg darbepoetin alfa (n?=?3). This mass spectrometric method is an innovative and powerful tool for detecting darbepoetin alfa in human urine for doping control testing.

Packed in-tube SPME–LC–MS/MS for fast and straightforward analysis of cannabinoids and metabolites in human urine

Cannabinoids are pharmacologically active compounds present in cannabis plants, which have become important research topics in the modern toxicological and medical research fields. Not only is cannabis the most used drug globally, but also cannabinoids have a growing use to treat a series of diseases. Therefore, new, fast, and efficient analytical methods for analyzing these substances in different matrices are demanded. This study developed a new packed-in-tube solid-phase microextraction (IT-SPME) method coupled to liquid chromatography with tandem mass spectrometry (LC–MS/MS), for the automated microextraction of seven cannabinoids from human urine. Packed IT-SPME microcolumns were prepared in (508 µm i.d. × 50 mm) stainless-steel hardware; each one required only 12 mg of sorbent phase. Different sorbents were evaluated; fractional factorial design 2⁴⁻¹ and a central composite design were employed for microextraction optimization. Under optimized conditions, the developed method was a fast and straightforward approach. Only 250 µl of urine sample was needed, and no hydrolysis was required. The sample pretreatment included only dilution and centrifugation steps (8 min), whereas the complete IT-SPME–LC–MS/MS method took another 12 min, with a sample throughput of 3 samples h⁻¹. The developed method presented adequate precision, accuracy and linearity; R² values ranged from 0.990 to 0.997, in the range of 10–1000 ng ml⁻¹. The lower limits of quantification varied from 10 to 25 ng ml⁻¹. Finally, the method was successfully applied to analyze 20 actual urine samples, and the IT-SPME microcolumn was reused over 150 times.     

Determination of benzimidazole- and bicyclic hydantoin-derived selective androgen receptor antagonists and agonists in human urine using LC–MS/MS

Selective androgen receptor modulators (SARMs) represent a novel class of drugs with tissue-specific agonistic and antagonistic properties, which are prohibited in sports from January 2008 according to the World Anti-Doping Agency. Preventive approaches to restrict the use of SARMs include early implementation of target analytes into doping control screening assays. Five model SARMs were synthesized, four of which are analogs to prostate-specific androgen receptor antagonists with a 5,6-dichloro-benzimidazole nucleus. The fifth SARM is a muscle-tissue specific agonist with a bicyclic hydantoin structure (BMS-564929). Dissociation pathways after negative electrospray ionization were studied using an LTQ-Orbitrap mass analyzer, and diagnostic product ions and common fragmentation patterns were employed to establish a screening procedure that target the intact SARMs as well as putative metabolic products. Sample preparation based on solid-phase extraction and subsequent LC–MS/MS measurement allowed for detection limits of 1–20 ng/mL, intra- and interday precisions of between 2.4 and 13.2% and between 6.5 and 24.2%, respectively. Recoveries varied from 89 to 106%, and tests for ion suppression or enhancement effects were negative for all analytes. Figure Precursor ion scanning as a tool to screen for the common nucleus of benzimidazole-derived SARMs     

Solid-phase extraction liquid chromatography–tandem mass spectrometry analytical method for the determination of 2-hydroxy-4-methoxybenzophenone and its metabolites in both human urine and semen

2-Hydroxy-4-methoxybenzophenone (HMB), which is one of the most commonly used UV filters in sunscreen cosmetics to protect skin from the deleterious effects of the sun, can be percutaneously absorbed, further metabolized, and finally excreted or bioaccumulated. An analytical method for the sensitive determination of HMB and its three metabolites in both human urine and semen is developed. The presented analytical method is based on a solid-phase extraction (SPE) procedure to clean-up and preconcentrate the target analytes from the urine and semen samples followed by liquid chromatography–tandem mass spectrometry (LC-MS/MS) detection. The methodology was fully validated and the standard addition calibration method was used to quantify the target analytes in order to correct the matrix effects observed. Considering this approach, the accuracy of the method was evaluated and the recoveries ranged from 98% to 115% and from 86% to 111% in urine and semen samples, respectively, depending on the analyte. For urine samples, the limits of detection ranged between 0.027 and 0.103 ng mL⁻¹ and the repeatability of the method, expressed as relative standard deviation, was in the range of 7.2–9.2%, depending on the analyte. In the case of semen samples, the limits of detection ranged between 1 and 3 ng mL⁻¹ whereas the repeatability was in the range of 2.2–6.4%, depending on the analyte. The described SPE-LC-MS/MS method was satisfactorily applied to both urine and semen samples from a male volunteer who applied a sunscreen cosmetic product containing HMB. HMB and its metabolites were found and quantified in the low ng mL⁻¹ range in both urine and semen samples, although at a different extent.     

Assay of femtogram level nitrite in human urine using luminol–myoglobin chemiluminescence

In this paper, a sensitive flow injection chemiluminescence system luminol–myoglobin was described for determining femtogram nitrite. Nitrite bound myoglobin producing the ferric heme nitrite complexes, which catalyzed the electron transfer of luminol to myoglobin leading to fast chemiluminescence. The chemiluminescence intensity in the presence of nitrite was remarkably enhanced compared with that in the absence of it. Under the optimum reaction conditions the chemiluminescence increment produced was proportional to the concentration of nitrite in the range of 0.05 pg ml^− 1–1.0 ng ml^− 1 (R² = 0.9991), with a detection limit (3σ) of 20.0 fg ml^− 1. At the flow rate of 2.0 ml min^− 1, the whole process including sampling and washing could be completed in 0.5 min offering the sampling efficiency of 120 h^− 1 accordingly, and the relative standard deviation (RSD) was less than 2.60% (n = 5). It was satisfactory for the application to determine nitrite in human urine samples, and the possible mechanism was proposed.     

A reliable and simple method for the assay of neuroendocrine tumor markers in human urine by solid-phase microextraction–gas chromatography-triple quadrupole mass spectrometry

Homovanillic acid (HVA), vanylmandelic acid (VMA), and 5-hydroxyindoleacetic acid (5-HIAA) are the metabolites of some catecholamines such as epinephrine, nor-epinephrine, dopamine and serotonin and their quantification is used in the diagnosis and management of patients with neurocrine tumors. A novel approach in the assay of these biomarkers in human urine samples by solid phase microextraction (SPME) combined with gas chromatography-triple quadrupole mass spectrometry (GC–QqQ-MS) is presented. A preliminary derivatization with ethyl chloroformate/ethanol was used and the corresponding derivatives were then extracted by SPME in immersion mode. The performance of five SPME fibers and three chloroformates were evaluated in univariate mode and the best results were obtained using the polyacrylate fiber and ethyl chloroformate. The variables affecting the efficiency of SPME analysis were optimized by the multivariate approach of “Experimental design” and, in particular, a central composite design (CCD) was applied. The optimum working conditions in terms of response values were achieved by performing analysis at room temperature with addition of NaCl (9.5%) and with an extraction time of 25.8 min. Identification and quantification of analytes were carried out by using a gas chromatography-triple quadrupole mass spectrometry (GC–QqQ MS) system in multiple reaction monitoring (MRM) acquisition. An evaluation of all analytical parameters shows that the proposed method provides satisfactory results. Very good linearities were, in fact, achieved in the tested calibration ranges with correlation coefficient values >0.99 for all the analytes and accuracies and RSDs calculated for between-run and tested at concentrations of 1, 10, and 80 mg L⁻¹ were ranging from 91.3% to 106.6%, and from 0.5 to 8.9%, respectively. Moreover, the LOD values obtained can be considered very satisfactory (1.3, 0.046 and 24.3 μg L⁻¹ for HVA, VMA and 5-HIAA, respectively). The developed protocol represents, therefore, a simple, rapid and selective tool for assaying these acidic biomarkers in urine samples for neuroendocrine cancer diagnosis.     

Combination of hollow fiber-based liquid-phase microextraction with sweeping techniques in micellar electrokinetic chromatography for the determination of Strychnos alkaloids in human urine

A new method for the enrichment of Strychnos alkaloids in biological samples via liquid-phase microextraction (LPME) based on porous polypropylene hollow fibers in combination with on-line sweeping in micellar electrokinetic chromatography was developed. The calibration curve was linear over the range of 20-200 ng mL-1 for both strychnine and brucine in human urine sample. The detection limits (S/N=3:1) for strychnine and brucine were 1 ng mL-1 and 2 ng mL-1, respectively. The LPME-sweeping method has been successfully applied to the analysis of strychnine and brucine in real urine samples.     

Development of a simple and rapid solid phase microextraction-gas chromatography–triple quadrupole mass spectrometry method for the analysis of dopamine,serotonin and norepinephrine in human urine

The work aims at developing a simple and rapid method for the quantification of dopamine (DA), serotonin (5-HT) and norepinephrine (NE) in human urine. The urinary levels of these biogenic amines can be correlated with several pathological conditions concerning heart disease, stress, neurological disorders and cancerous tumors. The proposed analytical approach is based on the use of solid phase microextraction (SPME) combined with gas chromatography–triple quadrupole mass spectrometry (GC–QqQ-MS) after a fast derivatization of both aliphatic amino and phenolic moieties by propyl chloroformate. The variables influencing the derivatization reaction were reliably optimized by the multivariate approach of “Experimental design”. The optimal conditions were obtained by performing derivatization with 100 μL of propyl chloroformate and 100 μL of pyridine. The extraction ability of five commercially available SPME fibers was evaluated in univariate mode and the best results were obtained using the polyacrylate fiber. The variables affecting the efficiency of SPME analysis were again optimized by the multivariate approach of “Experimental design” and, in particular, a central composite design (CCD) was applied. The optimal values were extraction in 45 min at room temperature, desorption temperature at 300 °C, no addition of NaCl. Assay of derivatized analytes was performed by using a gas chromatography–triple quadrupole mass spectrometry (GC–QqQ-MS) system in selected reaction monitoring (SRM) acquisition. An evaluation of all analytical parameters demonstrates that the developed method provides satisfactory results. Indeed, very good linearities were achieved in the tested calibration range with correlation coefficient values of 0.9995, 0.9999 and 0.9997 for DA, 5-HT and NE, respectively. Accuracies and RSDs calculated for between-run and tested at concentrations of 30, 200, and 800 μg L⁻¹ were in the range from 92.8% to 103.0%, and from 0.67 to 4.5%, respectively. Finally, the LOD values obtained can be considered very good (0.587, 0.381 and 1.23 μg L⁻¹ for DA, 5-HT and NE, respectively).     

Histamine determination in human urine using sub-2 μm C18 column with fluorescence and mass spectrometric detection

A fast, sensitive, and selective method for the determination of histamine in human urine samples by ultrahigh pressure liquid chromatography (LC) with fluorescence and mass spectrometry (MS) detection is investigated. A fluorescent reagent, 4-(1-pyrene) butyric acid N-hydroxysuccinimide ester was conjugated to the primary and secondary amino moieties of histamine. The structure of dipyrene-labeled histamine in human urine was determined by quadrupole time-of-flight MS with electospray ionization interface. The determination of the dipyrene derivative of histamine in urine samples was achieved within 3.9 min on an ultrahigh pressure LC Eclipse Zorbax XDB-C(18) column with 1.8 μm particle diameter. In this work, histamine separation was achieved significantly faster (3.9 min) with improved detection limit (signal-to-noise = 3) of 0.04 nM than 19.5 min with a detection limit of 0.183 nM as reported in a previous method.     

UHPLC–MS/MS method for the determination of bisphenol A and its chlorinated derivatives,bisphenol S,parabens, and benzophenones in human urine samples

In the present work, a new method based on a sample treatment by dispersive liquid–liquid microextraction (DLLME) for the extraction of six bisphenols (bisphenol A, bisphenol S, and monochloro-, dichloro-, trichloro-, and tetrachlorobisphenol A), four parabens (methyl-, ethyl-, propyl-, and butylparaben), and six benzophenones (benzophenone-1, benzophenone-2, benzophenone-3, benzophenone-6, benzophenone-8, and 4-hydroxybenzophenone) in human urine samples, followed by ultrahigh-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) analysis, is validated. An enzymatic treatment allows determining the total content of the target EDCs. The extraction parameters were accurately optimized using multivariate optimization strategies. Ethylparaben ring-¹³C₆, benzophenone-d₁₀, and bisphenol A-d₁₆ were used as surrogates. Limits of quantification ranging from 0.1 to 0.6 ng mL^?1 and interday variabilities (evaluated as relative standard deviations) from 2.0 to 13.8 % were obtained. The method was validated using matrix-matched standard calibration followed by a recovery assay with spiked samples. Recovery rates ranged from 94 to 106 %. A good linearity, for concentrations up to 300 ng mL^?1 for parabens and 40 ng mL^?1 for benzophenones and bisphenols, was also obtained. The method was satisfactorily applied for the determination of target compounds in human urine samples from 20 randomly selected individuals.     

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