Blank Urine Research Articles

Fabric phase sorptive extraction-high performance liquid chromatography-photo diode array detection method for simultaneous monitoring of three inflammatory bowel disease treatment drugs in whole blood, plasma and urine

This paper reports a novel fabric phase sorptive extraction-high performance liquid chromatography-photodiode array detection (FPSE-HPLC-PDA) method for the simultaneous extraction and analysis of three drug residues (ciprofloxacin, sulfasalazine, and cortisone) in human whole blood, plasma, and urine samples, generally administered in human patients to treat inflammatory bowel disease (IBD).The drugs of interest were well resolved using a Luna C18 column (250 mm × 4.6 mm; 5 μm particle size) in gradient elution mode within 20 min. The analytical method was optimized and validated in the range 0.05–10 μg/mL for whole blood, 0.25–10 μg/mL for human plasma, and 0.10–10 μg/mL for human urine. Blank human whole blood, plasma, and urine were used as the sample matrix for the method development and validation; while methyl-p-hydroxybenzoate was used as the internal standard (IS). Weighted-matrix matched standard calibration curves showed a good linearity up to a concentration of 10 μg/mL. The intra- and inter-day accuracy values (precision and trueness) were found in the range from −10.9% to 12.3%, and the performances of the validated FPSE-HPLC-PDA were further tested on real IBD patient samples.This is the first FPSE procedure applied simultaneously to whole blood, plasma, and urine samples for the determination of residual IBD drugs, which possess a wide range of polarity (logP values ranging from 2.30 for Ciprofloxacin, to 1.66 for Cortisone, and 2.92 for Sulfasalazine). The new approach exhibits high potential for immediate adoptation as a rapid, robust and green analytical tool for future clinical and pharmaceutical applications.

Rapid Dynamic Determination of Cetirizine Dihydrochloride in Urine Using Surface Enhanced Raman Scattering with Silver Colloids

ABSTRACTA novel method for the dynamic determination of cetirizine dihydrochloride in urine using surface enhanced Raman scattering (SERS) has been developed. Comparison of SERS activities between gold and silver colloid was made based on the analysis of the transmission electron micrographs and the SERS spectra, revealing that silver colloid is much more efficient on the signal enhancement performance. The primary sites of the adsorption on the nanoparticle surface were represented by the feature peaks of piperazine at ca. 1050 cm−1 and phenyl rings at ca. 1630 cm−1, respectively. The best signal response was extracted at pH 7 at the Cl−:Ag+ molar ratio of 2:1. The matrix effect was eliminated by subtracting the spectral contribution of the blank urine from the sample spectra. Sample preparation procedures were minimized. Quantification could be simply accomplished on the predicted versus actual concentration curve, within the Raman shift range from 500 to 2500 cm−1. Neither modeling nor complicate calculation was required. The method was shown to be specific and applicable for drug urine concentration monitoring in situ.

Magnetic graphene dispersive solid phase extraction-ultra performance liquid chromatography tandem mass spectrometry for determination of β-agonists in urine.

In this study, a magnetic graphene-based dispersive solid phase extraction method was first developed for extraction of β-agonists in urine. During the experiments, the absorbent amount, sample pH, extraction time, elution solution and elution time were optimized respectively. The optimized extraction method was finished within 10min, and showed high enrichment factors for 9 β-agonists (20-26 folds). Furthermore, this absorbent could be reused for at least 60 times. Then this extraction method was combined with ultra performance liquid chromatography triple quadrupole tandem mass spectrometry to determine the 9 drugs in urine. The limits of detection for the 9 drugs were in a range of 0.015-0.023ngmL-1, and the recoveries from the standards fortified blank urine were in a range of 60.2%-109.4%. Therefore, this method could be used as a simple, rapid, sensitive and accurate tool to determine trace level of β-agonists in urine.

Ultra-fast forensic toxicological screening and quantitation under 3 minutes with a QTOF HPLC-MS/MS system

Objectives HPLC-MS/MS technology combines separation (HPLC) and detection (MS, and MS/MS) and is widely used for screening applications. Mostly, the duration of HPLC runtime in these studies vary from 5 to 20 min. For high-throughput laboratories, a fast screening method is desired. There are mainly two approaches to acquire fragmentation information for screening purpose of compounds by HPLC-MS/MS: Information-Dependent-Acquisition (IDA) or Data-Dependent-Acquisition (DDA) and MS/MS All . For IDA-MS/MS, a survey scan is performed to collect the information on the precursor ions, followed by multiple dependent MS/MS scans for a number of most abundant precursor/candidate ions. MS/MS All with SWATH ® acquisition is a novel MS/MS All technique. In every data cycle, the instrument acquires TOF-MS information first, and then it sequentially acquires MSMS information of all precursor ions across a specified mass range in pre-divided mass windows. Both of these two data acquisition approaches are compatible with retrospective data interrogation. In this study, we aimed to develop an ultra-fast screening method in a forensic toxicological setting using the SCIEX X500R QTOF system with SCIEX OS software 1.0. We also aim to compare IDA-MS/MS and MS/MS All with SWATH ® acquisition for this fast screening method. Methods Blank human urine samples were spiked with multiple drugs commonly found in forensics setting at different concentration levels. Typically, samples were diluted 10-fold in 10% methanol and centrifuged. The clear supernatants were transferred to auto-sampler vials and 10 μL samples were injected. HPLC separation was on a 20 × 2 mm cartridge-type column with cartridge holder. Mobile phase A was 10 mm ammonium formatted in water and mobile phase B was 0.1% formic acid in methanol. HPLC runtime was 2.5 min Data was acquired, processed and reported in SCIEX OS software 1.0. Results Good separation for all the analytes tested and good retention for polar compounds. Sample set 1 was blank urine samples spiked with more than 85 compounds at different levels (CO1 means cutoff number for Sample set 1). Despite a very short HPLC runtime (less than 3 min), the method showed excellent sensitivity. The calibration curve for one of the analytes (JWH 210 5-OH metabolite) is showed. Sample set 2 were also urine samples with both calibrators (spiked with a different group of 84 compounds, CO 2 means cutoff number for sample set 2) and unknown samples. It showed true positive rate in the calibrators for set 2. No false positive was identified. It showed the true positive rate with different HPLC methods and MS/MS acquisition methods. Conclusion In this study, we have developed a super-fast screening/quantitation method in forensic setting under 3 minutes using the SCIEX X500R QTOF system. Two non-targeted data acquisition methods: IDA-MS/MS and MS/MS All with SWATH ® acquisition were both tested and compared. Depending on the specific requirement of the screening method, both IDA-MS/MS and SWATH® acquisition have their advantage and disadvantage in certain areas and the user should be flexible to adopt either approach with appropriate method settings. For Research Use Only. Not for use in diagnostic procedures.

A fabric phase sorptive extraction-High performance liquid chromatography-Photo diode array detection method for the determination of twelve azole antimicrobial drug residues in human plasma and urine.

This paper reports a novel fabric phase sorptive extraction-high performance liquid chromatography-photodiode array detection (FPSE-HPLC-PDA) method for the simultaneous extraction and analysis of twelve azole antimicrobial drug residues that include ketoconazole, terconazole, voriconazole, bifonazole, clotrimazole, tioconazole, econazole, butoconazole, miconazole, posaconazole, ravuconazole, and itraconazole in human plasma and urine samples. The selected azole antimicrobial drugs were well resolved by using a Luna C18 column (250mm×4.6mm; 5μm particle size) in gradient elution mode within 36min. The analytical method was calibrated and validated in the range from 0.1 to 8μg/mL for all the drug compounds. Blank human plasma and urine were used as the sample matrix for the analysis; while benzyl-4-hydroxybenzoate was used as the internal standard (IS). The limit of quantification of the FPSE-HPLC-PDA method was found as 0.1μg/mL and the weighted-matrix matched standard calibration curves of the drugs showed a good linearity upto a concentration of 8μg/mL. The parallelism tests were also performed to evaluate whether overrange sample can be analyzed after dilution, without compromising the analytical performances of the validated method. The intra- and inter-day precision (RSD%) values were found ≤13.1% and ≤13.9%, respectively. The intra- and inter-day trueness (bias%) values were found in the range from -12.1% to 10.5%. The performances of the validated FPSE-HPLC-PDA were further tested on real samples collected from healthy volunteers after a single dose administration of itraconazole and miconazole. To the best of our knowledge, this is the first FPSE extraction procedure applied on plasma and urine samples for the simultaneous determination of twelve azole drugs possessing a wide range of logKow values (extending from 0.4 for fluconazole to 6.70 of butoconazole) and could be adopted as a rapid and robust green analytical tool for clinical and pharmaceutical applications.

Detection of the diuretic hydrochlorothiazide in a doping control urine sample as the result of a non-steroidal anti-inflammatory drug (NSAID) tablet contamination

Hydrochlorothiazide (HCTZ, 6-chloro-3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulfonamide-1,1-dioxide) belongs to the class of diuretic agents that represent one of today’s cornerstones of the treatment of hypertensive patients. In addition to its clinical relevance, HCTZ is prohibited in sports according to the regulations of the World Anti-Doping Agency (WADA) at all times and has frequently been detected in sports drug testing urine samples worldwide since its ban was introduced in 1988. Despite these facts, the adverse analytical finding concerning HCTZ in an in-competition routine doping control sample collected in December 2014 was further investigated, particularly motivated by the comparably low urinary concentration of the drug accounting for approximately 5ng/mL.The athlete in question did not declare the use of any nutritional supplement or medication other than the ingestion of a non-steroidal anti-inflammatory drug (NSAID) prior to competition. Hence, the drug (formulated as coated tablet) provided by the athlete as well as the corresponding retention sample of the manufacturer were analyzed. Noteworthy, both samples confirmed the presence of about 2μg of HCTZ per tablet.In order to further probe for the plausibility of the observed urinary HCTZ concentrations with the scenario of drug ingestion and subsequent doping control sample collection, administration studies with produced HCTZ-spiked placebo-tablets (2.5μg of HCTZ/tablet) were conducted. Urine specimens were collected prior to and after ingestion of the drug and subjected to routine doping control analytical procedures employing liquid chromatography/tandem mass spectrometry. While blank urine samples returned negative test results, post-administration specimens were found to contain HCTZ at concentrations of approximately 1–16ng/mL, which supported the athlete’s inadvertent intake of HCTZ via contaminated NSAID tablets.Due to the substantial sensitivity of test methods employed today by doping control laboratories, even drug contaminations ranging within the good manufacturing practice (GMP) limit of 10ppm overall carry-over can evidently lead to adverse analytical findings. This calls into question whether selected (classes of) substances such as diuretics should be reported only when exceeding a defined reporting level and/or whether adverse analytical findings of non-threshold substances should be reported with an estimated semi-quantitative concentration of the identified substance to facilitate the result management by anti-doping organizations.

Long-Term Stability of Volatile Nitrosamines in Human Urine

Volatile nitrosamines (VNAs) are established teratogens and carcinogens in animals and classified as probable (group 2A) and possible (group 2B) carcinogens in humans by the IARC. High levels of VNAs have been detected in tobacco products and in both mainstream and sidestream smoke. VNA exposure may lead to lipid peroxidation and oxidative stress (e.g., inflammation), chronic diseases (e.g., diabetes) and neurodegenerative diseases (e.g., Alzheimer's disease). To conduct epidemiological studies on the effects of VNA exposure, short-term and long-term stabilities of VNAs in the urine matrix are needed. In this report, the stability of six VNAs (N-nitrosodimethylamine, N-nitrosomethylethylamine, N-nitrosodiethylamine, N-nitrosopiperidine, N-nitrosopyrrolidine and N-nitrosomorpholine) in human urine is analyzed for the first time using in vitro blank urine pools fortified with a standard mixture of all six VNAs. Over a 24-day period, analytes were monitored in samples stored at ∼20°C (collection temperature), 4-10°C (transit temperature) and -20 and -70°C (long-term storage temperatures). All six analytes were stable for 24 days at all temperatures (n = 15). The analytes were then analyzed over a longer time period at -70°C; all analytes were stable for up to 1 year (n = 62). A subset of 44 samples was prepared as a single batch and stored at -20°C, the temperature at which prepared samples are stored. These prepared samples were run in duplicate weekly over 10 weeks, and all six analytes were stable over the entire period (n = 22).

Efficient forensic toxicological screening and quantitation workflow using QTOF LC–MS/MS system

Quadrupole time-of-flight (QTOF) mass spectrometry is becoming the desired technology for sensitive and selective screening workflows in a forensic toxicological setting. The technology overcomes many challenges faced when using traditional techniques and more significantly captures all information about the sample in one injection to allow for retrospectively mining the data. Here we describe a new benchtop QTOF system with revolutionary N-optic designed flight path and new, intuitive software for easy adoption of accurate mass technology to forensic testing. Blank human urine was spiked with more than 50 common drugs found in forensics setting at different concentration levels. HPLC separation was performed using the SCIEX ExionLC AC system on a reverse-phase column. Data was collected on a SCIEX X500R QTOF mass spectrometer with SCIEX OS software: TOF – MS survey scan with Information Dependent Acquisition (IDA) – triggering of up to 16 product ion scans; or SWATH® acquisition. For SWATH® acquisition, the precursor isolation window width was varied for each MS/MS experiment. Data processing was also performed in SCIEX OS software with simultaneous identification and quantification being accomplished in single software. Screening: multiple confidence criteria including mass accuracy, retention time, isotope, and most importantly, library matching were used. The availability of MS/MS information for identification was required in this study through library searching the acquired spectra against a SCIEX high resolution forensic library containing MS/MS spectra of 1700 compounds. Excellent true positive rates were accomplished for both acquisition modes (89% with IDA and 98% with SWATH® acquisition). This result is consistent with the expectation that the IDA-MS/MS data acquisition has the risk of occasionally missing acquiring MS/MS. The acquisition of MS/MS data in the screening approach allowed the confident identification of structural isomers that have similar retention times through unique fragment ions and their ratios. Simultaneous Quantification Linearity on the new QTOF system for the majority of compounds tested was four orders linear dynamic range covering 0.1 to 1000 ng/mL. SWATH® acquisition ensures that all data, including MS/MS, is acquired at all times. Due to the fast scanning speed (up to 100 Hz at 35 K resolution) in MS/MS mode, a fast cycletime (0.5 sec) was used for the 6.5-min LC method that allowed for one TOF-MS scan and 16 MS/MS scans that covered 120 to 500 m/z range. A sensitive and selective workflow was developed for toxicological compound screening in a forensic setting using the new X500R QTOF system. The newly designed software that accompanies the new benchtop QTOF instrument allows for simultaneous compound identification and quantification in one step all in one place. SCIEX OS software allows the easy adoption of accurate mass technology for routine forensic workflows.

Analysis of 8-hydroxy-2'-deoxyguanosine in human urine by ultra-high performance liquid chromatography mass spectrometer method

To develop an ultra-high performance liquid chromatography mass spectrometer method for the rapid determination of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in human urines. 8-OHdG standard solution with the concentration from 0.01 to 0.1 μg/ml was formulated. The solution was implanted into ion source with a rate of 7 μl/min, the mass-to-charge ratio of parent ion and product ions, and ion mass to charge ratio was identified. The mass spectrum parameters of each Ion pairs, such as DP, EP and EXP, were gradually optimized. The urine sample with a concentration of 10.0 μg/L was detected, and the pH of the sample was adjusted using 1 mol/L ammonium formate and formic acid solution with a volume ratio of 5∶1, 4∶1, 3∶1, 2∶1, and 1∶1. It was tested using three different polarity of SPE, i.e.: HLB, MCX, and MAX. The elution effect of methanol and water mixture with the proportion of 90∶10, 80∶20, 50: 50, 20: 80, and 10: 90 were tested, and then acetonitrile and water mixture with the proportion of 90∶10, 80∶20, 50∶50, 20∶80, 10∶90 were also tested. The standard curve was constructed using the ratio of a standard series application fluid concentration to corresponding compounds quantitative ion liquid concentration of the peak area. The detection limit was determined as 3 times of the signal to noise ratio corresponding to the concentration of 8-OHdG, and the quantitative lower limit was determined as 10 times of the signal to noise ratio corresponding to the concentration of 8-OHdG. The blank urine spiked recovery method was used to evaluate the precision and recovery rate. The mass to charge ratio of parent ion was 284.1 and the product ions was 168.1, 140.1, 123.0, and 112.0, respectively. The collision voltage of quantitative ion-pair 284.1/168.1 was 18 V, the 284.1/140.1 collision voltage was 42 V, the 284.1/123.0 collision voltage was 48 V, and the 284.1/112.0 collision voltage was 53 V. The recovery rate was the highest (87.9%-104.3%) when the pH of urine was adjusted by a 10 ml 1 mol/L ammonium formate solution, 2 ml of formic acid, 88 ml of water are mixed with the sample solution volume ratio of 1∶5, and then purified with 3 ml of methanol and 3 ml water activated HLB extraction column. Within 1.0-100.0 μg/L concentration range, 8-OHdG standard application solution test results showed a good linear relationship. The regression equation was y= 1.25x+0.74, and the correlation coefficient was r=0.999 5. The detection limit was 0.2 μg/L, and the limit of quantification was 0.7 μg/L. The method of recovery rate was in the range of 87.9% to 104.3%, the precision was in the range from 1.5% to 3.7% and inter-assay precision was in the range from 1.6% to 5.4%. The method developed in this study had high sensitivity, good precision and accuracy, and a wide range of testing concentrations.

Levels of phenolic polycyclic aromatic hydrocarbons in Koreans' urine by LC-MS/MS

Medicinal plants are widely used throughout the world. About 80% of the world's population relies on plant-derived medicines for their primary healthcare [1]. Also some of them are willingly used for culinary purposes or as raw materials for pharmaceutical products, cosmetics and herbal medicinal products. However, the chemistry and efficacy of many of these plants are relatively unknown [2]. Levels of polycyclic aromatic hydrocarbons (PAHs) are generally low in fresh plants, but in grown in close proximity to urban pollution sources, levels of PAHs might be higher [2]. PAHs are products of the incomplete combustion of organic compounds, and man is exposed to PAHs by smoking, diet and environmental pollution. Several PAHs are classified as carcinogens. Because of their global occurrence in food and the environment, they are of toxicological and public concern. Urine samples for this study were collected from 1028 Korean adults. Urine samples were prepared by enzymatic hydrolysis and solid-phase extraction with Sep-pak cartridges. The analysis of PAHs was validated with blank urine by liquid chromatography tandem mass spectrometry; we used blank urine because all the urine samples some metabolites contains of PAHs. The linearity was very satisfying for 17 PAH compounds, with a coefficient of correlation (r2) higher than 0.99. The limit of detections was 0.01 to 0.08 ng/mL, the accuracy was 80% to 120%, and the precision was lower than 20%. Detected PAH metabolites from 1028 adult urine samples included (mean ± SD) 1-naphthol (8.56 ± 20.16 ng/mL), 2-naphthol (8.77 ± 17.46 ng/mL), 2-OH-fluorene (0.81 ± 1.39 ng/mL), 3-OH-fluorene (0.23 ± 0.45 ng/mL) and 1-OH-IP (0.23 ± 0.60 ng/mL), respectively.

High-temperature high-performance liquid chromatography on a porous graphitized carbon column coupled to an Orbitrap mass spectrometer with atmospheric pressure photoionization for screening exogenous anabolic steroids in human urine.

The presence in a urinary matrix of a large number of endogenous steroids and corticosteroids with similar structures can hamper the detection of specific exogenous steroids using liquid chromatography/mass spectrometry (LC/MS) with reversed-phase columns. Therefore, the development of LC/MS methods using alternative columns is of great interest. Porous graphitized carbon is a unique stationary phase for high-performance liquid chromatography (HPLC), with properties differing from traditional silica-based and polymeric stationary phases. The new method involves enzymatic hydrolysis, liquid-liquid extraction, and determination by high-temperature HPLC/Orbitrap mass spectrometry (HTLC/Orbitrap MS) with atmospheric pressure photoionization (APPI). To achieve APPI of doping substances, the mobile phase consisted of 0.1% CF3COOH (A) and a mixture of acetonitrile/2-propanol (25:75 v/v), containing 0.1% CF3COOH (B), which was used as an effective proton source. A screening method for the detection of 57 exogenous steroids has been developed. The method was validated by spiking 10 different blank urine samples at different concentration levels. Validation parameters included limit of detection (LOD), selectivity, ion suppression, extraction recovery, and repeatability. All studied compounds had an LOD lower than the minimum required performance level. Of the 57 steroids studied, 55 showed recovery better than 70%. For all of the analytes, the relative retention times proved to be stable between days, with relative standard deviations (RSDs) smaller than 0.3%. In addition, the interday RSDs of the peak area ratios ranged between 0.7% and 14.5%. The proposed method matches the basic requirements of all methods used to analyze drugs or metabolites in an antidoping laboratory, i.e., sensitivity, selectivity, and specificity. The acquisition of full-scan mass spectra with accurate masses can be a valuable tool in the retrospective evaluation of analyzed samples for anabolic steroids recently added to the prohibited list.

Fenofibric Acid Can Cause False-Positive Urine Methylenedioxymethamphetamine Immunoassay Results.

We present a false-positive result of ecstasy (3,4-methylenedioxy-NN-methylamphetamine) screening due to the therapeutic use of fenofibrate, an antihyperlipidemic drug. Our hypothesis was that the main metabolite of fenofibrate, fenofibric acid, was responsible for this cross-reactivity on a DRI(®) Ecstasy Assay, using a cut-off of 500 ng/mL. We estimated that the addition of 225 µg/mL pure fenofibric acid to blank urine would be sufficient to result in a positive DRI(®) Ecstasy Assay. The results obtained on the urine samples analyses of the patient show that the DRI(®) Ecstasy Assay resulted negative 2 days after discontinuing fenofibrate treatment, when the urine fenofibric acid concentration corrected by creatinine and determinated by gas chromatography-mass spectrometry was 20.3 µg/mg creatinine. The cross-reactivity data for fenofibric acid would seem to indicate that there was insufficient concentration of measured compound to account for the positive immunochemical results for ecstasy. This apparent discrepancy can be explained in several ways, one of them is that the β-glucuronidase-resistent fenofibric acid isomers are responsible. This process could explain the low recovery of free fenofibric acid when we use the developed method for its quantification in urine samples. Positive results on immunoassay screening must be considered presumptive until confirmation with another method based on a different principle, preferably gas chromatography-mass spectrometry or liquid chromatography-mass spectrometry.

Validation of a multi-analyte HPLC-DAD method for determination of uric acid, creatinine, homovanillic acid, niacinamide, hippuric acid, indole-3-acetic acid and 2-methylhippuric acid in human urine

During the last decades exposure sciences and epidemiological studies attracts more attention to unravel the mechanisms for the development of chronic diseases. According to this an existing HPLC-DAD method for determination of creatinine in urine samples was expended for seven analytes and validated. Creatinine, uric acid, homovanillic acid, niacinamide, hippuric acid, indole-3-acetic acid, and 2-methylhippuric acid were separated by gradient elution (formate buffer/methanol) using an Eclipse Plus C18 Rapid Resolution column (4.6mm×100mm). No interfering signals were detected in mobile phase. After injection of blank urine samples signals for the endogenous compounds but no interferences were detected. All analytes were linear in the selected calibration range and a non weighted calibration model was chosen. Bias, intra-day and inter-day precision for all analytes were below 20% for quality control (QC) low and below 10% for QC medium and high. The limits of quantification in mobile phase were in line with reported reference values but had to be adjusted in urine for homovanillic acid (45mg/L), niacinamide 58.5(mg/L), and indole-3-acetic acid (63mg/L). Comparison of creatinine data obtained by the existing method with those of the developed method showing differences from -120mg/L to +110mg/L with a mean of differences of 29.0mg/L for 50 authentic urine samples. Analyzing 50 authentic urine samples, uric acid, creatinine, hippuric acid, and 2-methylhippuric acid were detected in (nearly) all samples. However, homovanillic acid was detected in 40%, niacinamide in 4% and indole-3-acetic acid was never detected within the selected samples.

Development of a SPE-HPLC-MS/MS method for the determination of most prescribed pharmaceuticals and related metabolites in urban sewage samples.

Based on regional prescription data several pharmaceuticals with variable amounts of prescription and corresponding metabolites were selected and analyzed in influent and effluent samples of the sewage treatment plant (STP) in Dresden, Germany. Pharmaceuticals of the following most prescribed therapeutic groups were chosen: antibiotics, antifungals, anticonvulsants, antipsychotics, antidepressants, and cardiovascular active compounds like beta blockers and angiotensin-converting enzyme inhibitors. To analyze the selected compounds, a multi-target method was developed and applied to 24-h composite wastewater samples for three single days in May and June 2014. The method was based on a cleanup of a sample with a volume of 1mL using solid phase extraction followed by a high performance liquid chromatography coupled to a tandem mass spectrometer. Analytes were separated in a 15min chromatographic separation and quantified using 23 Internal Standards and a calibration curve in 40-fold diluted blank urine. The limit of quantification varied between 50 and 200ng/L and for all analytes good accuracy and precision as well as linearity for the calibration curve with the correlation coefficient R(2) higher than 0.99 was reached. A total of 41 and 40 of the selected 55 analytes were detected and quantified in the influent and effluent samples of the studied STP, respectively. Valsartan was the compound with the highest maximum concentration in influent (27.1μg/L) and effluent (15.7μg/L). Furthermore, analytes like bezafibrate, candesartan, carbamazepine, gabapentin, metoprolol, levetiracetam, pregabalin and telmisartan as well as the metabolite O-desmethyl venlafaxine were detectable in influent and effluent samples, respectively, with a concentration higher than 1μg/L.

Effect-based detection of synthetic glucocorticoids in bovine urine

Challenges to testing for the illicit use of anabolic substances in meat-producing animals stem from the production of new synthetic compounds and the administration of low-dose cocktails to circumvent detection by the surveillance schemes of European Union member states. This work evaluated for the first time GR-CALUX, a highly sensitive reporter gene assay, as a screening tool for the detection of synthetic glucocorticoids in bovine urine. In order to verify the effect of natural corticosteroids on the method, the bioassay was tested first using blank urine samples collected at the farm and the slaughterhouse. Next, the dose–response curves were measured for the most commonly used synthetic glucocorticoids. The bioassay’s ability to detect them in spiked and incurred samples of bovine urine was then evaluated. Finally, its performance was compared against a commercially available ELISA kit ordinarily used in screening activities. GR-CALUX performance did not appear to be influenced by physiological levels of endogenous corticosteroids in the farm samples, whereas an increase in these hormones might invalidate the analysis in samples obtained at the slaughterhouse. Using pure compounds, GR-CALUX showed a high sensitivity toward the synthetic glucocorticosteroids tested in order of relative potencies: flumethasone ≫ dexamethasone > betamethasone > methylprednisolone > prednisolone. As expected, the bioassay failed to detect the prohormone prednisone. The results obtained from analysis of the spiked and incurred specimens reproduced those of the blank samples and the pure compounds. GR-CALUX is a promising screening tool for the detection of illicit treatments in meat-producing bovines. Its ability to detect the most commonly used synthetic glucocorticoids was comparable with the ELISA test. Importantly, it appeared to be less susceptible to matrix effects than ELISA.

Urine analysis concerning xenon for doping control purposes.

On September 1(st) 2014, a modified Prohibited List as established by the World Anti-Doping Agency (WADA) became effective featuring xenon as a banned substance categorized as hypoxia-inducible factor (HIF) activator. Consequently, the analysis of xenon from commonly provided doping control specimens such as blood and urine is desirable, and first data on the determination of xenon from urine in the context of human sports drug testing, are presented. In accordance to earlier studies utilizing plasma as doping control matrix, urine was enriched to saturation with xenon, sequentially diluted, and the target analyte was detected as supported by the internal standard d6 -cyclohexanone by means of gas chromatography/triple quadrupole mass spectrometry (GC/MS/MS) using headspace injection. Three major xenon isotopes at m/z 128.9, 130.9 and 131.9 were targeted in (pseudo) selected reaction monitoring mode enabling the unambiguous identification of the prohibited substance. Assay characteristics including limit of detection (LOD), intraday/interday precision, and specificity as well as analyte recovery under different storage conditions were determined. Proof-of-concept data were generated by applying the established method to urine samples collected from five patients before, during and after (up to 48 h) xenon-based general anesthesia. Xenon was traceable in enriched human urine samples down to the detection limit of approximately 0.5 nmol/mL. The intraday and interday imprecision values of the method were found below 25%, and specificity was demonstrated by analyzing 20 different blank urine samples that corroborated the fitness-for-purpose of the analytical approach to unequivocally detect xenon at non-physiological concentrations in human urine. The patients' urine specimens returned 'xenon-positive' test results up to 40 h post-anesthesia, indicating the limits of the expected doping control detection window. Since xenon has been considered a prohibited substance according to WADA regulations in September 2014, its analysis from common specimens of routine sports drug testing is desirable. In previous studies, its traceability in whole blood and plasma was shown, and herein a complementary approach utilizing doping control urine samples for the GC/MS/MS analysis of xenon was reported.

Development and validation of a UPLC-MS/MS method to monitor cephapirin excretion in dairy cows following intramammary infusion.

Cephapirin, a cephalosporin antibiotic, is used by the majority of dairy farms in the US. Fecal and urinary excretion of cephapirin could introduce this compound into the environment when manure is land applied as fertilizer, and may cause development of bacterial resistance to antibiotics critical for human health. The environmental loading of cephapirin by the livestock industry remains un-assessed, largely due to a lack of appropriate analytical methods. Therefore, this study aimed to develop and validate a cephapirin quantification method to capture the temporal pattern of cephapirin excretion in dairy cows following intramammary infusion. The method includes an extraction with phosphate buffer and methanol, solid-phase extraction (SPE) clean-up, and quantification using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The LOQ values of the developed method were 4.02 µg kg−1 and 0.96 µg L−1 for feces and urine, respectively. This robust method recovered >60% and >80% cephapirin from spiked blank fecal and urine samples, respectively, with acceptable intra- and inter-day variation (<10%). Using this method, we detected trace amounts (µg kg−1) of cephapirin in dairy cow feces, and cephapirin in urine was detected at very high concentrations (133 to 480 µg L−1). Cephapirin was primarily excreted via urine and its urinary excretion was influenced by day (P = 0.03). Peak excretion (2.69 mg) was on day 1 following intramammary infusion and decreased sharply thereafter (0.19, 0.19, 0.08, and 0.17 mg on day 2, 3, 4, and 5, respectively) reflecting a quadratic pattern of excretion (Quadratic: P = 0.03). The described method for quantification of cephapirin in bovine feces and urine is sensitive, accurate, and robust and allowed to monitor the pattern of cephapirin excretion in dairy cows. This data will help develop manure segregation and treatment methods to minimize the risk of antibiotic loading to the environment from dairy farms.

Determination of Urinary Excretion of 17 Phenolic Polycyclic Aromatic Hydrocarbons (OH-Pahs) by LC-MS/MS

Background: Polycyclic aromatic hydrocarbons (PAHs) are products of the incomplete combustion of organic compounds, and exposed to human by smoking, diet and environmental pollution. Several PAHs are classified as carcinogens. Because of their global occurrence in different food and environmental media, they are of toxicological and public concern. This study was performed to evaluate analytical method of PAHs in human urine with enzyme hydrolysis and solid-phase extraction coupled with LC-(ESI)-MS/MS technique. Methods: We employed LC-(ESI)-MS/MS(API 4000, AppliedBioscience) techniques with appropriate pretreatment for this biological monitoring and method validation of PAHs analysis in urine samples. Samples were prepared by enzyme hydrolysis (ß-glucuronidase/aryl sulfatase, 37°C, overnight) and solid-phase extraction with Sep-pak cartridge. Analytical method of PAHs were validated with blank urine by liquid chromatography (Shiseido, SI-2) tandem mass spectrometry. Results: The linearity obtained was very satisfying for all 17 targeted PAHs compounds, with a coefficient of determination (R2) higher than 0.99. The limit of detections ranged from 0.01 to 0.08 ng/mL with human urine samples. The accuracies of method ranged between 80% and 120% and precision was lower than 20% RSD for high and low concentrations in PAHs spiked urine samples. Conclusions: Analytical method was validated for low nanogram level of PAHs in human urine samples by HPLC system with triple quadrupole mass spectrometry.

Chitosan/AuNPs Modified Graphene Electrochemical Sensor for Label‐Free Human Chorionic Gonadotropin Detection

AbstractA new immunosensor is presented for human chorionic gonadotropin (hCG), made by electrodepositing chitosan/gold‐nanoparticles over graphene screen‐printed electrode (SPE). The antibody was covalently bound to CS via its Fc‐terminal. The assembly was controlled by electrochemical Impedance Spectroscopy (EIS) and followed by Fourier Transformed Infrared (FTIR). The hCG‐immunosensor displayed linear response against the logarithm‐hCG concentration for 0.1–25 ng/mL with limit of detection of 0.016 ng/mL. High selectivity was observed in blank urine and successful detection of hCG was also achieved in spiked samples of real urine from pregnant woman. The immunosensor showed good detection capability, simplicity of fabrication, low‐cost, high sensitivity and selectivity.

Simultaneous determination of most prescribed antibiotics in multiple urban wastewater by SPE-LC-MS/MS.

A rapid analytical method was developed for the application of a long-term monitoring (>one year) of the most prescribed and often in hospitals used antibiotics in diverse wastewaters of an urban sewage treatment plant (STP). Additionally to the selected multi-class antibiotics amoxicillin, penicillin V and piperacillin (penicillins), cefotaxime and cefuroxime (cephalosporins), azithromycin, clarithromycin and roxithromycin (macrolids), ciprofloxacin and levofloxacin-ofloxacin (fluoroquinolones), clindamycin (lincosamide), doxycycline (tetracycline), sulfamethoxazole (sulfonamide) and trimethoprim (dihydrofolate reductase inhibitor), the bioactive metabolite clindamycin-sulfoxide, the reserve antibiotic vancomycin (glycopeptide) and as tracer of the STP the anticonvulsant carbamazepine and the antifungal fluconazole were involved. The analytical method combines a low-sample-volume solid phase extraction (SPE), followed by a chromatographic separation using a reversed phase (RP) and hydrophilic interaction liquid chromatography (HILIC) technique, respectively, coupled to a triple quadrupole mass spectrometer. Detection was performed with multiple reaction monitoring (MRM) measured with positive electrospray ionization (ESI+). The extraction efficiency of different SPE cartridges and optimized pH-values of the preparation procedure were tested. Finally, the extraction of antibiotics was realized with the Oasis HLB cartridge and a pH adjustment at 3.5. An external calibration curve in diluted blank urine was used for quality control of the sample set of daily composite samples of the STP for the duration of one year monitoring. The squared coefficient of determination (r(2)) in the concentration range (20-20,000ng/L or 100-100,000ng/L) of the calibration curves for the method was higher than 0.99 for all determined substances. The limit of quantification (LoQ) ranged between 0.8ng/L (azithromycin) and 245.1ng/L (vancomycin). Furthermore, a standard addition was used for quantification in wastewater samples. The process efficiencies ranged from 20% (doxycycline) to 134% (cefuroxime) in influent samples and from 31% (doxycycline) to 171% (cefuroxime) in effluent samples of the STP. All selected substances have been found in wastewater samples. Cefuroxime, doxycycline, levofloxacin, piperacillin, sulfamethoxazole and carbamazepine showed highest concentrations up to 6.2μg/L.