Determination of amphetamine enantiomers in urine by conductive vial electromembrane extraction and ultra-high performance supercritical fluid chromatography tandem mass spectrometry

Separation and quantification of amphetamine enantiomers are commonly used to distinguish between consumption of prescription amphetamine (mostly S ‐amphetamine) and illicit forms of the drug (racemate). In this study, electromembrane extraction with prototype conductive vials was combined with ultra‐high performance supercritical fluid chromatography (UHPSFC‐MS/MS) to quantify R ‐ and S ‐amphetamine in urine. Amphetamine was extracted from 100 μL urine, diluted with 25 μL internal standard solution and 175 μL 130 mM formic acid, across a supported liquid membrane (SLM) consisting of 9 μL of a 1:1(w/w) mixture of 2‐nitrophenyloctyl ether (NPOE) and bis(2‐ethylhexyl)phosphite (DEHPi) into an acceptor phase containing 300 μL 130 mM formic acid. The extraction was facilitated by the application of 30 V for 15 min. Enantiomeric separation was achieved using UHPSFC‐MS/MS with a chiral stationary phase. The calibration range was 50–10,000 ng/mL for each enantiomer. The between‐assay CV was ≤5%, within‐assay CV ≤ 1.5%, and bias within ±2%. Recoveries were 83%–90% (CV ≤ 6%), and internal standard corrected matrix effects were 99–105 (CV ≤ 2%). The matrix effects ranged from 96% to 98% (CV ≤ 8%) when not corrected by the internal standard. The EME method was compared with a chiral routine method that employed liquid–liquid extraction (LLE) for sample preparation. Assay results were in agreement with the routine method, and the mean deviation between methods was 3%, ranging from −21% to 31%. Finally, sample preparation greenness was assessed using the AGREEprep tool, which resulted in a greenness score of 0.54 for conductive vial EME, opposed to 0.47 for semi‐automated 96‐well LLE.publishedVersio

Contemporary Medical Management of Primary Hyperparathyroidism:A Systematic Review

IntroductionPrimary hyperparathyroidism is increasingly an asymptomatic disease at diagnosis, but the recognized guidelines for management are based on evidence obtained from studies on patients with symptomatic disease, and surgery is not always indicated. Other patients are unable to undergo surgery, and thus a medical treatment is warranted. This systematic review provides an overview of the existing literature on contemporary pharmaceutical options available for the medical management of primary hyperparathyroidism.MethodsDatabases of medical literature were searched for articles including terms for primary hyperparathyroidism and each of the included drugs. Data on s-calcium, s-parathyroid hormone, bone turnover markers, bone mineral density (BMD) and hard endpoints were extracted and tabulated, and level of evidence was determined. Changes in s-calcium were estimated and a meta-regression analysis was performed.ResultsThe 1,999 articles were screened for eligibility and 54 were included in the review. Weighted mean changes calculated for each drug in s-total calcium (mean change from baseline ± SEM) were pamidronate (0.31 ± 0.034 mmol/l); alendronate (0.07 ± 0.05 mmol/l); clodronate (0.20 ± 0.040 mmol/l); mixed bisphosphonates (0.16 ± 0.049 mmol/l); and cinacalcet (0.37 ± 0.013 mmol/l). The meta-analysis revealed a significant decrease of effect on s-calcium with time for the bisphosphonates (Coef. −0.049 ± 0.023, p = 0.035), while cinacalcet proved to maintain its effect on s-calcium over time. Bisphosphonates improved BMD while cinacalcet had no effect.DiscussionThe included studies demonstrate advantages and drawbacks of the available pharmaceutical options that can prove helpful in the clinical setting. The great variation in how primary hyperparathyroidism is manifested requires that management should rely on an individual evaluation when counseling patients. Combining resorptive agents with calcimimetics could prove rewarding, but more studies are warranted

Overexpression of mmpS5/mmpL5 in Mycobacterium tuberculosis reduces susceptibility to anti-tuberculosis drugs

Thesis (MSc) -- Stellenbosch University, 2022.ENGLISH ABSTRACT: Background: Bedaquiline (BDQ) is the first anti-tuberculosis (anti-TB) drug in recent years with a novel mode of action recommended for the treatment of multi-drug resistant-TB. Rv0678 is the most common genetic determinant of BDQ resistance. It encodes for a regulatory protein (MmpR5) of the MmpS5/MmpL5 efflux system of Mycobacterium tuberculosis. Mutations in Rv0678 lead to the overexpression of the mmpS5/mmpL5 genes and increased efflux of BDQ, resulting in an elevated minimum inhibitory concentration (MIC) or phenotypic resistance. Mutations in Rv0678 are also associated with cross-resistance to clofazimine (CFZ) by the same mechanism of MmpS5/MmpL5 export. As this efflux pump is responsible for hydrophobic siderophore export, we hypothesised that additional drugs are exported, and that drug hydrophobicity may be an important determinant. We hypothesised that a rpoB mutation may exacerbate the effect of a Rv0678 mutation on the expression of mmpS5/mmpL5 as their gene products are both involved in gene transcription. This may be reflected in further alterations in the MICs of drugs exported by the pump. Lastly, due to the overexpression of mmpS5/mmpL5, it was hypothesised that Rv0678 mutations have a fitness cost. Methodology: M. tuberculosis progenitor isolates (a M. tuberculosis H37Rv reference isolate and a Rv0678 mutant harbouring a 193 G deletion) were used to generate rpoB mutants containing a Ser531Leu mutation, using a spontaneous mutagenesis approach. Whole genome sequencing was employed to confirm a clean genetic background of progenitor and rpoB mutant isolates, whereafter MIC assays were performed. Finally, comparative growth curve analysis was conducted between the M. tuberculosis H37Rv and Rv0678 mutant isolates by measuring optical density over time. Results: Spontaneous in vitro rpoB mutants were successfully generated and the mutations of interest were detected with WGS. The Rv0678 mutant showed resistance to BDQ and CFZ, and a slight increase in delamanid MIC within 1-2 doubling dilutions compared to M. tuberculosis H37Rv. The H37Rv rpoB mutant showed resistance to rifampicin (RIF) and rifabutin (RFB). The Rv0678/rpoB mutant showed resistance to BDQ, CFZ, RIF and RFB. This mutant had a higher CFZ MIC within 1- 2 doubling dilutions compared to the Rv0678 mutant. The MICs of other drugs were altered below the epidemiological cut-off (ECOFF) value in the Rv0678 and rpoB mutant isolates and were either raised or decreased, similarly within 1-2 doubling dilutions. The Rv0678 mutant exhibited a similar growth rate to its wildtype M. tuberculosis H37Rv progenitor. Conclusion: There was a clear distinction in resistance levels of BDQ/CFZ, and RIF/RFB in isolates that harboured the Rv0678 and rpoB mutations, respectively. This is supported by literature which shows that a 193 G deletion in Rv0678 is associated with BDQ and CFZ resistance, and that a Ser531Leu rpoB mutation causes high-level RIF and RFB resistance. The remaining drugs showed altered MICs which were below the ECOFF and could not be confirmed as clinically meaningful. The growth curve analysis showed that protein truncating due to the Rv0678 mutation had no significant effect on growth rate and no association with a fitness cost. Further investigation is required to test if a clinical background, different Rv0678 mutations or alternative testing methods would affect the current results.AFRIKAANS OPSOMMING: Agtergrond: Bedaquilien (BDQ) is die eerste tuberkulose middel met ‘n nuwe werkswyse wat in die laaste jare ontwikkel is vir die behandeling van multiweerstandige TB. Die mees algemene genetiese bepaler van BDQ weerstandigheid is Rv0678, wat MmpR5 enkodeer, ‘n regulatoriese proteïen van die MmpS5/MmpL5 uitvloeisisteem van Mycobacterium tuberculosis. Mutasies in Rv0678 veroorsaak verhoogde uitdrukking van die mmpS5/mmpL5 gene, sowel as meer uitvloei van BDQ, wat lei tot verhoogde minimum inhibitoriese konsentrasie (MIK) van BDQ, oftewel fenotipiese weerstandigheid. Mutasies in Rv0678 word ook geassosiëer met weerstandigheid teen clofazimine (CFZ) deur dieselfde meganisme van MmpS5/MmpL5 uitvloei. Ons hipotese is dat addisionele middels uitgevoer word deur hierdie uitvloeipomp, aangesien dit verantwoordelik is vir siderofoor uitvoer, en dat hidrofobisiteit ‘n bepalende faktor is. Verder postuleer ons dat ‘n rpoB mutasie die effek van ‘n Rv0678 mutasie op die uitdrukking van mmpS5/mmpL5 vererger, aangesien beide die geenprodukte betrokke is by transkripsie. Dit mag verder duidelik word uit veranderings in die MIKs van middels wat deur die pomp uitgevoer word. Laastens, postuleer ons dat Rv0678 mutasies ‘n fiksheidskoste dra as gevolg van die oor-uitdrukking van mmpS5/mmpL5. Metodiek: M. tuberculosis voorganger isolate (‘n M. tuberculosis H37Rv indeks stam en ‘n Rv0678 mutant met ‘n 193 G uiwissing) is gebruik om rpoB mutante met ‘n Ser531Leu mutasie te maak deur middel van ‘n spontane mutagenese benadering. Heelgenoom volgordebepaling is gebruik om te bevestig dat beide die voorlooper en die rpoB mutant ‘n overanderde genetiese agtergrond het, waarna MIK bepalings gedoen is. Laastens is vergelykende groeikurwe analise gedoen deur die optiese digtheid van die H37Rv- en Rv0678 mutant-isolate te meet oor tyd. Resultate: Spontane in vitro rpoB mutante is suksesvol gegenereer en die teenwoordigheid van mutasies van belang is met heelgenoom volgordebepaling bevestig. Die Rv0678 mutant het weerstandigheid getoon teen BDQ en CFZ, sowel as ‘n geringe toename in delamanied MIK, binne 1-2 verdubbelde verdunnings, in vergelyking met M. tuberculosis H37Rv. Die H37Rv rpoB mutant het weerstandigheid teen rifampisien (RIF) en rifabutien (RFB) getoon. Die Rv0678/rpoB mutant het weerstandigheid teen BDQ, CFZ, RIF en RFB getoon. Hierdie mutant het ‘n hoër CFZ MIK binne 1- 2 verdubbelde verdunnings, in vergelyking met die Rv0678 mutant. Die MIKs van ander middels het verander in die Rv0678 en rpoB mutante isolate, maar by vlakke onder die epidemiologiese afsnypunt (epidemiological cut-off, ECOFF). Hierdie veranderings sluit verhoging en verlaging van MIK in, maar soos voorheen binne 1-2 verdubbelde verdunnings. Die Rv0678 mutant het ‘n soortgelyke groeitempo getoon as die ongemuteerde M. tuberculosis H37Rv voorloper. Gevolgtrekking: Daar is ‘n duidelike onderskeid tussen BDQ/CFZ en RIF/RFB weerstandigheid in isolate wat onderskeidelik die Rv0678 en rpoB mutasies het. Dit word ondersteun deur literatuur wat toon dat ‘n 193 G uitwissing in Rv0678 geassosiëer is met BDQ en CFZ weerstandigheid en dat ‘n Ser531Leu rpoB mutasie hoëvlak RIF en RFB weerstandigheid veroorsaak. Die res van die middels het veranderde MIKs onder die ECOFF getoon, wat nie as klinies beduidend bevestig kon word nie. Die groeikurwe analise toon dat die verkorting van die proteïen as gevolg van die Rv0678 mutasie geen beduidende effek het op die groeitempo nie, en dat daar geen verband is met fiksheidskoste nie. Verdere ondersoek word benodig om te bepaal of ‘n kliniese agtergrond, verskillende Rv0678 mutasies of alternatiewe toetsmetodes die huidige resultate sou affekteer.Master

Associations between trabecular bone score and biochemistry in surgically vs conservatively treated outpatients with primary hyperparathyroidism:A retrospective cohort study

Purpose: Trabecular Bone Score (TBS) is a software-based method for indirect assessment of trabecular bone structure of the spine, based on analysis of pixels in dual energy x-ray absorptiometry (DXA) images. Few studies describe the use of TBS in patients with primary hyperparathyroidism (PHPT). This study aimed at further describing this relationship, investigating possible correlations between biochemistry, body mass index (BMI), fracture incidence and TBS. Methods: Cross-sectional study of 195 patients with verified PHPT, surgically (27) or conservatively (168) treated at the Department of Endocrinology, Aalborg University Hospital. TBS was acquired by reanalyzing DXA-images of the included subjects from the outpatient clinic. Biochemical variables were obtained from clinical routine blood samples taken in relation to the DXA-scans. History of fractures and medical history was obtained from radiology reports and medical charts. Results: Patients with active PHPT had a TBS-score signifying a partly degraded bone structure, whereas surgically treated patients had a normal bone structure as judged by TBS, though the difference in TBS-score was not statistically significant. Use of antiresorptive treatment was negatively associated with BMD but not TBS. No correlations between the biochemical variables and TBS were found. A negative correlation between TBS and BMI in patients with PHPT was present. Patients experiencing a fragility fracture had a significantly lowered TBS, BMD and T-Score. Conclusion: Biochemistry does not seem to predict bone status in terms of TBS in patients with PHPT. TBS is negatively correlated to BMI, which is also seen in patients not suffering from PHPT. The lack of a predictive value for antiresorptive treatment for TBS may raise concern. TBS appears to have a predictive value when assessing risk of fracture in patients with PHPT. Mini abstract: This cross-sectional study investigates possible correlations between biochemical variables, body mass index (BMI) and trabecular bone score (TBS) in 195 patients with primary hyperparathyroidism. It finds no correlation between biochemical variables and TBS, but finds a negative correlation between TBS and BMI and a clear association between fracture incidence and low TBS-score. Keywords: Primary hyperparathyroidism, Trabecular bone score, Body mass index, Osteoporosis, Osteopenia, Biochemical variable

Conductive vial electromembrane extraction of opioids from oral fluid

The use of oral fluid as sample matrix has gained significance in the analysis of drugs of abuse due to its non-invasive nature. In this study, the 13 opioids morphine, oxycodone, codeine, O-desmethyl tramadol, ethylmorphine, tramadol, pethidine, ketobemidone, buprenorphine, fentanyl, cyclopropylfentanyl, etonitazepyne, and methadone were extracted from oral fluid using electromembrane extraction based on conductive vials prior to analysis with ultra-high performance liquid chromatography–tandem mass spectrometry. Oral fluid was collected using Quantisal collection kits. By applying voltage, target analytes were extracted from oral fluid samples diluted with 0.1% formic acid, across a liquid membrane and into a 300 μL 0.1% (v/v) formic acid solution. The liquid membrane comprised 8 μL membrane solvent immobilized in the pores of a flat porous polypropylene membrane. The membrane solvent was a mixture of 6-methylcoumarin, thymol, and 2-nitrophenyloctyl ether. The composition of the membrane solvent was found to be the most important parameter to achieve simultaneous extraction of all target opioids, which had predicted log P values in the range from 0.7 to 5.0. The method was validated in accordance to the guidelines by the European Medical Agency with satisfactory results. Intra- and inter-day precision and bias were within guideline limits of ± 15% for 12 of 13 compounds. Extraction recoveries ranged from 39 to 104% (CV ≤ 23%). Internal standard normalized matrix effects were in the range from 88 to 103% (CV ≤ 5%). Quantitative results of authentic oral fluid samples were in accordance with a routine screening method, and external quality control samples for both hydrophilic and lipophilic compounds were within acceptable limits.publishedVersio

Prevalence of anticoagulant rodenticides in faeces of wild red foxes (Vulpes vulpes) in Norway

High occurrence of anticoagulant rodenticides (ARs) in wildlife is a rising concern, with numerous reports of secondary exposure through predation. Because of widespread distribution of the red fox (Vulpes vulpes), they may act as sentinels for small mammal-hunting predators in rural, suburban, and urban areas. No AR surveillance in wild mammals with analyses of residues in feces has been conducted throughout a single country. We collected 163 fecal samples from presumed healthy red foxes from 18 out of 19 counties in Norway. The foxes were shot during regular hunting between January and December 2016 and samples collected directly after death. Fecal samples were analyzed for six ARs: brodifacoum, bromadiolone, coumatetralyl, difenacoum, difethialone, and flocoumafen. We detected ARs in 54% (75/139) of the animals. Brodifacoum was most frequently detected (46%; 64/139), followed by coumatetralyl (17%; 23/139), bromadiolone (16%; 22/139), difenacoum (5%; 7/139), difethialone (1%; 2/139), and flocoumafen (1%; 2/139). More than one substance was detected in 40% (30/75) of the positive foxes, and 7% (5/75) of these animals were exposed to four different ARs. There were no statistically significant seasonal, age, or sex differences in foxes after exposure to one AR compound. We found a significant difference in occurrence of brodifacoum and coumatetralyl in foxes from different geographical areas. These findings demonstrate fecal analyses as a valuable method of detecting AR exposure in red foxes. We suggest using direct fecal sampling with analyses as a method to evaluate the occurrence of ARs in live endangered wildlife in connection with radio tagging or collaring operations.acceptedVersio

A monoclonal antibody against 6-acetylmorphine protects female mice offspring from adverse behavioral effects induced by prenatal heroin exposure

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