Core-shell nanocomposite of flower-like molybdenum disulfide nanospheres and molecularly imprinted polymers for electrochemical detection of anti COVID-19 drug favipiravir in biological samples.

A novel electrochemical sensor is reported for the detection of the antiviral drug favipiravir based on the core-shell nanocomposite of flower-like molybdenum disulfide (MoS2) nanospheres and molecularly imprinted polymers (MIPs). The MoS2@MIP core-shell nanocomposite was prepared via the electrodeposition of a MIP layer on the MoS2 modified electrode, using o-phenylenediamine as the monomer and favipiravir as the template. The selective binding of target favipiravir at the MoS2@MIP core-shell nanocomposite produced a redox signal in a concentration dependent manner, which was used for the quantitative analysis. The preparation process of the MoS2@MIP core-shell nanocomposite was optimized. Under the optimal conditions, the sensor exhibited a wide linear response range of 0.01 ~ 100 nM (1.57*10-6 ~ 1.57*10-2 µg mL-1) and a low detection limit of 0.002 nM (3.14*10-7 µg mL-1). Application of the sensor was demonstrated by detecting favipiravir in a minimum amount of 10 µL biological samples (urine and plasma). Satisfied results in the recovery tests indicated a high potential of favipiravir monitoring in infectious COVID-19 samples.

Structural elucidation of amino amide-type local anesthetic drugs and their main metabolites in urine by LC-MS after derivatization and its application for differentiation between positional isomers of prilocaine.

The demand for clinical toxicology analytical methods for identifying drugs of abuse and medicinal drugs is steadily increasing. Structural elucidation of amino amide-type local anesthetic drugs and their main metabolites by GC-EI-MS and LC-ESI-MS/MS is of great analytical challenge. These compounds exhibit only/mostly fragments/product ions representing the amine-containing residue, while the aromatic amide moiety remains unidentified. This task becomes even more complicated when discrimination between positional isomers of such compounds is required. Here, we report the development of a derivatization procedure for the differentiation and structural elucidation of a mixture of local anesthetic drugs and their metabolites that possess tertiary and secondary amines in water and urine. A method based on two sequential "in-vial" instantaneous derivatization processes at ambient temperature followed by LC-ESI-MS/MS analysis was developed. 2,2,2-Trichloro-1,1-dimethylethyl chloroformate (TCDMECF) was utilized to selectively convert the secondary amines into their carbamate derivatives, followed by hydrogen peroxide addition to produce the corresponding tertiary amine oxides. The resulting derivatives exhibited rich fragmentation patterns, enabling improved structural elucidation of the original compounds. The developed method was successfully applied to the differentiation and structural elucidation of prilocaine and its four positional isomers, which all possess similar GC and LC retention times and four of them exhibit almost identical EI-MS and ESI-MS/MS spectra, enabling their structural elucidation in a single LC-ESI-MS/MS analysis. The developed technique is fast and simple and enables discrimination between isomers based on different diagnostic ions/fragmentation patterns.

Uric Acid Elevation by Favipiravir, an Antiviral Drug.

In light of the recent pandemic, favipiravir (Avigan®), a purine nucleic acid analog and antiviral agent approved for use in influenza in Japan, is being studied for the treatment of coronavirus disease 2019 (COVID-19). Increase in blood uric acid level is a frequent side effect of favipiravir. Here, we discussed the mechanism of blood uric acid elevation during favipiravir treatment. Favipiravir is metabolized to an inactive metabolite M1 by aldehyde oxidase and xanthine oxidase, and excreted into urine. In the kidney, uric acid handling is regulated by the balance of reabsorption and tubular secretion in the proximal tubules. Favipiravir and M1 act as moderate inhibitors of organic anion transporter 1 and 3 (OAT1 and OAT3), which are involved in uric acid excretion in the kidney. In addition, M1 enhances uric acid reuptake via urate transporter 1 (URAT1) in the renal proximal tubules. Thus, favipiravir is thought to decrease uric acid excretion into urine, resulting in elevation of uric acid levels in blood. Elevated uric acid levels were returned to normal after discontinuation of favipiravir, and favipiravir is not used for long periods of time for the treatment of viral infection. Thus, the effect on blood uric acid levels was subclinical in most studies. Nevertheless, the adverse effect of favipiravir might be clinically important in patients with a history of gout, hyperuricemia, kidney function impairment (in which blood concentration of M1 increases), and where there is concomitant use of other drugs affecting blood uric acid elevation.

Determination of primary fatty acid amides in different biological fluids by LC-MS/MS in MRM mode with synthetic deuterated standards: Influence of biofluid matrix on sample preparation.

The recent growing interest in primary fatty acid amides (PFAMs) is due to the broad range of physiological effects they exhibit as bioindicator of pathological states. These bioactive lipids are usually in biological samples at the nanomolar level, making their detection and identification a challenging task. A method for quantitative analysis of seven main PFAMs (lauramide, myristamide, linoleamide, palmitamide, oleamide, stearamide and behenamide) in four human biofluids -namely, urine, plasma, saliva and sweat- is here reported. Two sample preparation procedures were compared to test their efficiency in each biofluid: solid-phase extraction (SPE) and protein precipitation. The latter was the best for plasma and urine, while the analysis of saliva and sweat required an SPE step for subsequent suited determination of PFAMs. Detection of the seven metabolites was performed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring (MRM) mode. Quantitative analysis was supported on the use of stable isotopically labeled internal standards (SIL-ISs) in the calibration method, which required the synthesis of each IS from the precursor deuterated fatty acids. Detection limits for the target analytes were within 0.3-3 ng mL-1. The method was applied to a small cohort of male and female volunteers (n = 6) to estimate the relative concentration profiles in the different biofluids. The analytical features of the method supported its applicability in clinical studies aimed at elucidating the role of PFAMs metabolism.

Identification of Patients with Preeclampsia by Measuring Fluorescence of an Amyloid-Binding Aryl Cyano Amide in Human Urine Samples.

Preeclampsia (PE) is a hypertensive disorder of pregnancy and one of the leading contributors to both maternal and perinatal morbidity and mortality. Reliable diagnostic parameters unique to the disorder that accurately define and diagnose PE are currently unavailable. Recent studies have revealed that PE is accompanied by the accumulation of amyloidogenic deposits in the placenta and the presence of congophilic amyloid-like protein aggregates in the urine. Here, we evaluate the capability of an amyloid-targeting aryl cyano amide (ARCAM-1) fluorophore to identify PE patients from analysis of urine samples. Our results reveal that this probe can distinguish patients with PE from gestationally healthy patients and patients suffering from non-PE hypertension, highlighting the potential for amyloid-targeting fluorophores to help identify PE patients during pregnancy.

Structural elucidation of major selective androgen receptor modulator (SARM) metabolites for doping control.

Selective androgen receptor modulators (SARMs) are a class of androgen receptor drugs, which have a high potential to be performance enhancers in human and animal sports. Arylpropionamides are one of the major SARM classes and get rapidly metabolized significantly complicating simple detection of misconduct in blood or urine sample analysis. Specific drug-derived metabolites are required as references due to a short half-life of the parent compound but are generally lacking. The difficulty in metabolism studies is the determination of the correct regio and stereoselectivity during metabolic conversion processes. In this study, we have elucidated and verified the chemical structure of two major equine arylpropionamide-based SARM metabolites using a combination of chemical synthesis and liquid chromatography-mass spectrometry (LC-MS) analysis. These synthesized SARM-derived metabolites can readily be utilized as reference standards for routine mass spectrometry-based doping control analysis of at least three commonly used performance-enhancing drugs to unambigously identify misconduct.

2-methyl butyramide, a previously identified urine biomarker for Ascaris lumbricoides, is not present in infected Indonesian individuals.

ᅟ: Previous reports suggest that the 2-methyl butyramide and 2-methyl valeramide metabolites of Ascaris lumbricoides in urine of infected individuals could be considered as urinary biomarkers for active infection. We have developed an LC-MS method with a detection limit of 10 ng/mL using synthetic chemicals as reference material. Urine samples (n = 21) of infected individuals were analyzed for the presence of these metabolites, but they were not detected in any of the samples. Furthermore, the recorded 1H-NMR spectrum for reference 2-methyl butyramide did not match with the spectrum that was described for the Ascaris metabolite. Based on these two observations, we concluded that the urinary biomarkers that were detected for A. lumbricoides infection are not 2-methyl butyramide nor 2-methylvaleramide. New discovery efforts will be required to identify the structure of these metabolite biomarkers in urine of infected individuals. TRIAL REGISTRATION: Urine samples used in this study were collected as part of a clinical trial with trial number ISRCTN75636394 (12 November 2013).

Determination of selumetinib, N-desmethyl selumetinib and selumetinib amide in human biological samples by LC-MS/MS.

AIM: Selumetinib is an inhibitor of MEK1/2 in Phase III development that has activity in multiple tumor types. Validated bioanalytical methods were required to quantitate selumetinib and its N-desmethyl and amide metabolites in a variety of human biological matrices. Methodology & results: LC-MS/MS assays were developed and validated that demonstrated acceptable precision, accuracy and selectivity for selumetinib and the two metabolites in human plasma, urine, blood dialysate and plasma ultrafiltrate. Incurred sample re-analysis was acceptable and issues observed in plasma with the amide metabolite, due to potential instability, were addressed. CONCLUSION: Robust and sensitive LC-MS/MS assays for the quantification of selumetinib and two of its metabolites were validated in human biological matrices and are being used to support the clinical development program.

Embutramide, a Component of Tanax(®) (T-61) as a New Drug of Abuse?

Tanax(®) (T-61) is a euthanasia solution commonly used in veterinary medicine in Europe. It consists of three active components: embutramide, mebezonium iodide, and tetracaine hydrochloride. Human consumption of Tanax(®) (T-61) is usually associated with suicide attempts. In our 15-year-long practice, embutramide was detected only three times but within a short period. First, it was found in the urine of a 42-year-old veterinarian, and the other two observations were made in a 16-year-old young man. Urine samples were analyzed using Shimadzu Prominence TOX.I.S.II. HPLC-DAD system with online SPE extraction system. Both of the two patients denied any intention to die. These cases show that this veterinary drug may also be considered as potential drugs of abuse.

Evaluation of a rapid method for the therapeutic drug monitoring of aliskiren, enalapril and its active metabolite in plasma and urine by UHPLC-MS/MS.

Given the increasing popularity of aliskiren, particularly in combination with angiotensin converting enzyme inhibitor (e.g. enalapril), it is important to determine whether its use in combination with these agents is associated with potentially life threatening safety events. Analytical methods for the simultaneous determination of both drugs in plasma and urine utilized in clinical studies on efficacy and safety have not been fully described in the literature. In this work, a new, fast and reliable method using a digitally controlled microextraction by packed sorbent (eVol(®)-MEPS) followed by ultra-high performance liquid chromatography (UHPLC) coupled with tandem mass spectrometry (MS/MS) was developed and validated to quantify an aliskiren, enalapril and its active metabolite in both human plasma and urine. Chromatographic separation was accomplished on a Poroshell 120 EC-C18 column with a gradient elution system consisting of 0.1% formic acid in water and acetonitrile (1.5min of total analysis). Detection was performed by multiple reaction monitoring (MRM) mode using electrospray ionization in the positive ion mode. This assay method has been fully validated in terms of selectivity, linearity, accuracy, precision, stability, recovery and matrix effect. The developed method can be applied to the routine determination of selected compounds in human plasma and urine and can be useful to elucidate the mechanisms of the potential risks triggered by the combination of aliskiren and enalapril as well as its active metabolite enalaprilat.

Urinary bile acids as biomarkers for liver diseases I. Stability of the baseline profile in healthy subjects.

The role of bile acids (BAs) as biomarkers for liver injury has been proposed for decades. However, the large inter- and intra-individual variability of the BA profile has prevented its clinical application. To this end, we investigated the effect of covariates such as food, gender, age, BMI, and moderate alcohol consumption on the BA profile in healthy human subjects. The BA profile was characterized by the calculation of indices that describe the composition, sulfation, and amidation of total and individual BAs. Both inter- and intra-individual variabilities of BA indices were low in serum and even lower in urine compared with those of absolute concentrations of BAs. Serum BA concentrations increased with consumption of food, whereas urinary BA concentrations were mildly affected by food. Gender differences in the urinary and serum BA profile were minimal. The serum and urinary BA profiles were also not affected by age. BMI showed minimal effect on the urine and serum BA profile. Moderate alcohol consumption did not have a significant effect on the BA profile in both urine and serum. When the effect of the type of alcohol was studied, the results indicate that moderate drinking of beer does not affect BA concentrations and has minimal effect on BA indices, whereas moderate wine consumption slightly increases BA concentrations without affecting the BA indices. In summary, urinary BA indices showed lower variability and higher stability than absolute BA concentrations in serum and showed minimal changes to covariate effects suggesting their utility as biomarkers in clinic.

Urinary bile acids as biomarkers for liver diseases II. Signature profiles in patients.

Hepatobiliary diseases result in the accumulation of bile acids (BAs) in the liver, systemic blood, and other tissues leading to an unfavorable prognosis. The BA profile was characterized by the calculation of indices that describe the composition, sulfation, and amidation of total and individual BAs. Comparison of the urinary BA profiles between healthy subjects and patients with hepatobiliary diseases demonstrated significantly higher absolute concentrations of individual and total BAs in patients. The percentage sulfation of some individual BAs were different between the two groups. The percentage amidation of overall and most individual BAs was higher in patients than controls. The percentage of primary BAs (CDCA and CA) was higher in patients, whereas the percentage of secondary BAs (DCA and LCA) was lower in patients. BA indices belonging to percentage amidation and percentage composition were better associated with the severity of the liver disease as determined by the model for end-stage liver disease (MELD) score and disease compensation status compared with the absolute concentrations of individual and total BAs. In addition, BA indices corresponding to percentage amidation and percentage composition of certain BAs demonstrated the highest area under the receiver operating characteristic (ROC) curve suggesting their utility as diagnostic biomarkers in clinic. Furthermore, significant increase in the risk of having liver diseases was associated with changes in BA indices.

Simultaneous quantitative and qualitative analysis of aliskiren, enalapril and its active metabolite enalaprilat in undiluted human urine utilizing LC-ESI-MS/MS.

The benefit-risk ratio of combined blocking by the direct renin inhibitor aliskiren and an angiotensin-converting enzyme inhibitor (e.g. enalapril) on the renin-angiotensin-aldosterone system is discussed. No method was available for simultaneous determination of both drugs in urine. A novel sensitive method for simultaneous quantification in undiluted human urine was developed which enables systematic pharmacokinetic investigations, especially in poorly investigated populations like children. Matrix effects were clearly reduced by applying solid-phase extraction followed by a chromatographic separation on Xselect(TM) C18 CSH columns. Mobile phase consisted of methanol and water, both acidified with formic acid. Under gradient conditions and a flow rate of 0.4 mL/min the column effluent was monitored by tandem mass spectrometry with electrospray ionization. Calibration curves were constructed in the range of 9.4-9600 ng/mL regarding aliskiren, 11.6-12000 ng/mL for enalapril and 8.8-9000 ng/mL for enalaprilat. All curves were analyzed utilizing 1/x(2) -weighted quadratic squared regression. Intra-run and inter-run precision were 3.2-5.8% and 6.1-10.3% for aliskiren, 2.4-6.1% and 3.9-7.9% for enalapril as well as 3.1-9.4% and 4.7-12.7% regarding enalaprilat. Selectivity, accuracy and stability results comply with current international bioanalysis guidelines. The fully validated method was successfully applied to a pharmacokinetic investigation in healthy volunteers.

Effect of para halogen modification of S-3-(phenoxy)-2-hydroxy-2-methyl-N-(4-nitro-3-trifluoromethyl-phenyl)-propionamides on metabolism and clearance.

The purpose of this study was to better understand why para-halogen modifications of S-3-(4-halophenoxy)-2-hydroxy-2-methyl-N-(4-nitro-3-trifluoromethylphenyl) propionamide selective androgen receptor modulators (SARMs) had the opposite of expected effects on total clearance, in which electron-withdrawing groups generally protect benzene ring from hydroxylation. We determined the plasma protein binding of this series of halogen substituted SARMs and characterized the qualitative effects of B-ring halogen substitution on in vivo metabolism. In vivo metabolism of S-9, S-10, and S-11 were determined in rats using LC-MS(n) analysis. Intrinsic clearance was measured by in vitro metabolism using rat liver microsomes. Rat plasma protein binding was measured by equilibrium dialysis and drug concentrations after dialysis were analyzed by LC-MS. The major metabolic pathways of the halogen-substituted SARMs examined were very similar and included three major phase I pathways; (1) hydrolysis of the amide bond, (2) B-ring hydroxylation, and (3) A-ring nitro reduction to an aromatic amine. In plasma protein binding studies, S-1 (F, fu = 0.78 ± 0.17 %) showed the greatest unbound fraction, followed by S-9 (Cl, fu = 0.10 ± 0.04 %), S-10 (Br, fu = 0.03 ± 0.01 %), and S-11 (I, fu = 0.008 ± 0.001 %). The CLint values of S-1, S-9, S-10, and S-11 were 2.4, 2.5, 2.8, and 4.6 µL/min/mg, respectively. These findings suggest that as lipophilicity increased the free fraction was reduced thus compensating for metabolic liability and resulting in the apparent discrepancy between CLint and CL total of halogen-substituted SARMs series.

Investigation of lead concentrations in whole blood, plasma and urine as biomarkers for biological monitoring of lead exposure.

Lead in blood is a major concept in biomonitoring of exposure but investigations of its alternatives are scarce. The aim of the study was to describe different lead biomarkers' variances, day-to-day and between individuals, estimating their fraction of the total variance. Repeated sampling of whole blood, plasma and urine were conducted for 48 lead-exposed men and 20 individuals under normal environmental lead exposure, in total 603 measurements. For lead workers, the fraction of the total variance attributed to differences between individuals was 91% for whole-blood lead (geometric mean 227 µg/l; geometric standard deviation (GSD): 1.55 µg/l); plasma 78% (0.57 µg/l; GSD: 1.84 µg/l); density-adjusted urine 82%; and unadjusted urine 75% (23.7 µg/l; GSD: 2.48 µg/l). For the individuals under normal lead exposure, the corresponding fractions were 95% of the total variance for whole blood (20.7 µg/l; GSD: 8.6 µg/l), 15% for plasma (0.09 µg/l; GSD: 0.04 µg/l), 87% for creatinine-adjusted urine and 34% for unadjusted (10.8 µg/l; GSD: 6.7 µg/l). Lead concentration in whole blood is the biomarker with the best ability to discriminate between individuals with different mean concentration. Urinary and plasma lead also performed acceptably in lead workers, but at low exposures plasma lead was too imprecise. Urinary adjustments appear not to increase the between-individual fraction of the total variance among lead workers but among those with normal lead exposure.

Spectrophotometric determination of 4-acetamidophenyl N'-(sulphanilamide) acetate in biological fluids.

A simple, accurate and low cost spectrophotometric method is proposed for the determination of the synthesized paracetamol derivative; 4-acetamidophenyl N'-(sulphanilamide) acetate (APSA) in biological fluids. The spectrophotometric method is based on a condensation reaction between the alcoholic solution of APSA and acidic solution of p-dimethylaminobenzaldeyde (DPMK) to generate a yellow colored product. The linear range for the determination of APSA was 1-10 µg mL(-1) with molar absorptivity of 3.6877 × 10(4) L mol(-1) cm(-1) and Sandell's sensitivity of 0.001 µg cm-2/0.001 absorbance unit. During the inter-day and intra-day analysis, the relative standard deviation for replicated determination of APSA was found to be less than 2.0% and accuracy was 99.20-101.60% and 99.10-101.30% in blood and urine samples, respectively. There was no interference with commonly used blood and urine sample. The developed spectrophotometric method was successfully applied to assess APSA in biological fluids.

Absorption, distribution, metabolism and excretion of the novel SARM GTx-024 [(S)-N-(4-cyano-3-(trifluoromethyl)phenyl)-3-(4-cyanophenoxy)-2-hydroxy-2-methylpropanamide] in rats.

1. GTx-024, a novel selective androgen receptor modulator, is currently being investigated as an oral treatment for muscle wasting disorders associated with cancer and other chronic conditions. 2. Absorption of GTx-024 was rapid and complete, with high oral bioavailability. A wide tissue distribution of [(14)C]GTx-024 derived radioactivity was observed. [(14)C]GTx-024-derived radioactivity had a moderate plasma clearance (117.7 and 74.5 mL/h/kg) and mean elimination half-life of 0.6 h and 16.4 h in male and female rats, respectively. 3. Fecal excretion was the predominant route of elimination, with ∼70% of total radioactivity recovered in feces and 21-25% in urine within 48 h. Feces of intact rats contained primarily unchanged [(14)C]GTx-024 (49.3-64.6%). Metabolites were identified in urine and feces resulting from oxidation of the cyanophenol ring (M8, 17.6%), hydrolysis and/or further conjugation of the amide moiety (M3, 8-12%) and the cyanophenol ring (M4, 1.3-1.5%), and glucuronidation of [(14)C]GTx-024 at the tertiary alcohol (M6, 3.5-3.7%). There was no quantifiable metabolite in plasma. 4. In summary, in the rat GTx-024 is completely absorbed, widely distributed, biotransformed through several metabolic pathways, and eliminated in feces primarily as an unchanged drug.

Molecularly imprinted polymer in microextraction by packed sorbent for the simultaneous determination of local anesthetics: lidocaine, ropivacaine, mepivacaine and bupivacaine in plasma and urine samples.

This study presents the use of molecularly imprinted polymer (MIP) as packing material for microextraction by packed syringe (MEPS) to achieve higher extraction selectivity. Pentycaine was used as template for MIP. Development and validation of the determination of lidocaine, ropivacaine, mepivacaine and bupivacaine in human plasma and urine samples utilizing MIP-MEPS and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were carried out. The MEPS MIP-cartridge could be used for 100 extractions before it was discarded. The extraction recovery ranged from 60 to 80%. The correlation coefficients values were >0.999 for all assays using lidocaine, ropivacaine, mepivacaine and bupivacaine in the calibration range 5-2000 nmol/L. The accuracy of the studied compounds, given as a percentage variation from the nominal concentration values, ranged from -4.9 to 8.4% using plasma and urine samples. The between-batch precision, given as the relative standard deviation, at three different concentrations (quality control samples) was ranged from -4.7 to 14.0% and from 1.8 to 12.7% in plasma and urine, respectively. The lower limit of quantification and limit of detection of the studied substances were 5.0 and 1.0 nm, respectively.

Selective androgen receptor modulators: in vitro and in vivo metabolism and analysis.

For future targeted screening in National Residue Control Programmes, the metabolism of seven SARMs, from the arylpropionamide and the quinolinone classes, was studied in vitro using S9 bovine liver enzymes. Metabolites were detected and identified with ultra-performance liquid chromatography (UPLC) coupled to time-of-flight mass spectrometry (ToF-MS) and triple quadrupole mass spectrometry (QqQ-MS). Several metabolites were identified and results were compared with literature data on metabolism using a human cell line. Monohydroxylation, nitro-reduction, dephenylation and demethylation were the main S9 in vitro metabolic routes established. Next, an in vivo study was performed by oral administration of the arylpropionamide ostarine to a male calf and urine samples were analysed with UPLC-QToF-MS. Apart from two metabolites resulting from hydroxylation and dephenylation that were also observed in the in vitro study, the bovine in vivo metabolites of ostarine resulted in glucuronidation, sulfation and carboxylation, combined with either a hydroxylation or a dephenylation step. As the intact mother compounds of all SARMs tested are the main compounds present after in vitro incubations, and ostarine is still clearly present in the urine after the in vivo metabolism study in veal calves, the intact mother molecules were selected as the indicator to reveal treatment. The analytical UPLC-QqQ-MS/MS procedure was validated for three commercially available arylpropionamides according to European Union criteria (Commission Decision 2002/657/EC), and resulted in decision limits ranging from 0.025 to 0.05 µg l⁻¹ and a detection capability of 0.025 µg l⁻¹ in all cases. Adequate precision and intra-laboratory reproducibility (relative standard deviation below 20%) were obtained for all SARMs and the linearity was 0.999 for all compounds. This newly developed method is sensitive and robust, and therefore useful for confirmation and quantification of SARMs in bovine urine samples for residue control programmes and research purposes.