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Objectives Cystic fibrosis (CF) transmembrane conductance regulator (CFTR) modulators have revolutionized the therapeutic landscape in CF treatment. These vital drugs are extensively metabolized via CYP3A, so caution must be exercised in multimodal CF therapy because of the risk of adverse drug interactions. Our goal was to develop a highly sensitive assay for the purpose of therapeutic drug monitoring (TDM) in diagnostic laboratories. Methods After protein precipitation, the CFTR modulators ivacaftor, lumacaftor, tezacaftor, elexacaftor, and their metabolites ivacaftor-M1, ivacaftor-M6, and tezacaftor-M1 were separated with a two-dimensional chromatography setup within 5 min, and quantified with stable isotope-labeled internal standards. The method was validated according to the European Medicines Agency (EMA) guideline on bioanalytical method validation and applied to CF patient samples. Results Inaccuracy was ≤7.0% and the imprecision coefficient of variation (CV) was ≤8.3% for all quality controls (QCs). The method consistently compensated for matrix effects, recovery, and process efficiency were 105–115 and 96.5–103%, respectively. Analysis of CF serum samples provided concentrations comparable to the pharmacokinetic profile data reported in the EMA assessment report for the triple combination therapy Kaftrio. Conclusions We hereby present a robust and highly selective isotope dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) assay for the simultaneous quantification of the so far approved CFTR modulators and their metabolites in human serum. The assay is suitable for state-of-the-art pharmacovigilance of CFTR modulator therapy in CF patients, in order to maximize safety and efficacy, and also to establish dose-response relationships in clinical trials.

Background Appropriate monitoring of tobacco smoking is extremely important in several areas of medicine, e.g. management of chronic obstructive pulmonary disease (COPD), epidemiological surveys, and allocation of heart or lung transplants. The major metabolite of nicotine is cotinine that is increasingly used as a laboratory parameter for assessing tobacco smoke exposure. Methods Creatinine and cotinine were analyzed simultaneously in urine by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) in one run within 3 min using a biphenyl column. For quantification, the respective stable-isotope-labeled standards were used. Results Detuning and measuring a natural isotope of creatinine as precursor and product ion allowed a simultaneous quantification of creatinine and cotinine. The method revealed robust validation results. For both analytes, inaccuracy and imprecision of the quality control and external quality assessment (EQA) samples were ≤−11.1%. Conclusions One essential novelty of the method presented here is the simultaneous quantification of creatinine and cotinine covered by one analytical method. Despite the very different natural concentrations of creatinine and cotinine, this allows the immediate reporting of the cotinine-to-creatinine ratio without the need for a separate creatinine analysis.

Several studies have addressed the poor stability of meropenem in aqueous solutions, though not considering the main degradation product, the open-ring metabolite (ORM) form. In the present work, we elucidate the metabolic fate of meropenem and ORM from continuous infusion to the human bloodstream. We performed in vitro infusate stability tests at ambient temperature with 2% meropenem reconstituted in 0.9% normal saline, and body temperature warmed buffered human serum with 2, 10, and 50 mg/L meropenem, covering the therapeutic range. We also examined meropenem and ORM levels over several days in six critically ill patients receiving continuous infusions. Meropenem exhibited a constant degradation rate of 0.006/h and 0.025/h in normal saline at 22 °C and serum at 37 °C, respectively. Given that 2% meropenem remains stable for 17.5 h in normal saline (≥90% of the initial concentration), we recommend replacement of the infusate every 12 h. Our patients showed inter-individually highly variable, but intra-individually constant molar ORM/(meropenem + ORM) ratios of 0.21–0.52. Applying a population pharmacokinetic approach using the degradation rate in serum, spontaneous degradation accounted for only 6% of the total clearance.

Severe intravascular hemolysis leads to the simultaneous presence of free heme pigments (oxyhemoglobin, methemoglobin, and methemalbumin) and bilirubin in human plasma. Standard spectrophotometric methods used to assess in vivo hemolysis inadequately address this complex analytical situation. Thus, we propose a novel quantification algorithm to ensure the highest analytical specificity. A corresponding second-derivative fitting algorithm was validated according to the guideline of bioanalytical method validation from the European Medicines Agency using plasma specimens (n = 1759) spiked with different concentrations of oxyhemoglobin and methemoglobin. The results were compared to standard spectrophotometric quantification methods described by Harboe, Noe, and Fairbanks. Based on the second-derivative method, simultaneous quantification of oxyhemoglobin and methemoglobin/methemalbumin in samples with total bilirubin concentrations ≤4.9 mg/dL (83.8 μmol/L) provided robust results (inaccuracy ≤20%, imprecision ≤16%). Analyzing UV/VIS spectra of plasma from patients with confirmed severe intravascular hemolysis evidenced an underestimation of up to 33% for the combined free heme pigment content. The employed second-derivative algorithm allows for automated and highly specific quantification of the free heme pigment content in diluted human plasma, which cannot be realized with standard spectrophotometric evaluation methods. An Excel-based tool readily applicable to clinical datasets accompanies this manuscript.

Background Therapeutic drug monitoring (TDM) of beta-lactam antibiotics and, among them, especially meropenem gains importance in the field of laboratory medicine. Meropenem is known to be unstable, resulting in a degradation product with an open beta-lactam ring. For a more comprehensive TDM of meropenem, the aim was to develop a LC–MS/MS method for the simultaneous quantification of meropenem and its main degradation product, the open-ring metabolite (ORM). Methods The method involves a protein precipitation followed by chromatographic separation using a formic acid-ammonium formate methanol gradient on a pentafluorophenyl column. Multiple reaction monitoring in the positive ion mode and stable isotope labeled internal standards were used for quantification. Validation was performed according to the European Medicines Agency guideline. Results Validation was successful performed within the linear drug concentration range of 1.0–100.0 mg/l for meropenem and 0.62–62.30 mg/l for the ORM. Investigation of selectivity, accuracy and precision showed good results and potential matrix effects were successfully compensated by the internal standards. The suitability of the method was shown by the comparison of 35 anonymized leftover serum samples from intensive care patients with routine analyses. Conclusion For the first time, we herein describe a liquid chromatography tandem mass spectrometry (LC–MS/MS) method for the simultaneous quantification of meropenem and its ORM in human serum. The ratio of active to inactive compound provides valuable pharmaceutical and pharmacokinetic information, which may contribute to therapeutic efficacy.

Highlights • Multi-analyte LC–MS/MS method for the quantification of repurposed COVID-19 drugs in human serum. • Two-dimensional chromatography combined with isotope dilution standardization. • Rapid and robust analysis applicable to therapeutic drug monitoring (TDM)., Background The present COVID-19 pandemic has prompted worldwide repurposing of drugs. The aim of the present work was to develop and validate a two-dimensional isotope-dilution liquid chromatrography tandem mass spectrometry (ID-LC–MS/MS) method for accurate quantification of remdesivir and its active metabolite GS-441524, chloroquine, hydroxychloroquine, lopinavir, ritonavir, favipiravir and azithromycin in serum; drugs that have gained attention for repurposing in the treatment of COVID-19. Methods Following protein precipitation, samples were separated with a two-dimensional ultra-high performance liquid chromatography (2D-UHPLC) setup, consisting of an online solid phase extraction (SPE) coupled to an analytical column. For quantification, stable isotope-labelled analogues were used as internal standards for all analytes. The method was validated on the basis of the European Medicines Agency bioanalytical method validation protocol. Results Detuning of lopinavir and ritonavir allowed simultaneous quantification of all analytes with different concentration ranges and sensitivity with a uniform injection volume of 5 μL. The method provided robust validation results with inaccuracy and imprecision values of ≤ 9.59 % and ≤ 11.1 % for all quality controls. Conclusion The presented method is suitable for accurate and simultaneous quantification of remdesivir, its metabolite GS-441525, chloroquine, hydroxychloroquine, lopinavir, ritonavir, favipiravir and azithromycin in human serum. The quantitative assay may be an efficient tool for the therapeutic drug monitoring of these potential drug candidates in COVID-19 patients in order to increase treatment efficacy and safety.

Background The adrenocorticotropic hormone (ACTH) stimulation test is a widely used diagnostic tool to assess the adrenal gland function. Beyond that the ACTH test can be used in stress research to induce a biochemical stress response under standardized conditions. To study the impact of the stress response on protein metabolism, time-course plasma amino acid profiling in healthy individuals was performed with high performance liquid chromatography tandem-mass spectrometry (HPLC-MS/MS). Methods A set of 39 samples (pre/post 30´ and 60´ IV-ACTH) from 13 healthy individuals (age range 26 - 58, 3 female and 10 male) was investigated. Plasma amino acids were quantified by LC-MS/MS using the AbsoluteIDQ® p180 Kit (Biocrates Life Science, Innsbruck, Austria) including 19 biogenic amino acids, ornithine, and citrulline. Results Statistically significant decreases were observed for 11 proteinogenic amino acids (alanine, asparagine, isoleucine, leucine, tyrosine, phenylalanine, tryptophan, valine, methionine, aspartate, and threonine). The amino acids alanine, asparagine, and isoleucine showed markedly pronounced relative changes with short-term reduction of median inter-individual plasma concentrations of up to 25%. Conclusions Amino acid profiling with LC-MS/MS revealed highly dynamic plasma alterations upon application of exogenous corticotropin as a stress model. Our findings provide novel insights into the biochemical stress response and improve our understanding of short-term metabolic consequences. Further studies should elucidate the impact of corticotropin mediated stress responses on amino acid catabolism.

The synthesis of new analogues of Arcyriaflavin A in which one indole ring is replaced by an aryl or heteroaryl ring is described. These new series of aryl[a]pyrrolo[3,4-c]carbazoles were evaluated as inhibitors of Cyclin D1-CDK4. A potent and selective D1-CDK4 inhibitor, 7a (D1-CDK4 IC(50)=45 nM), has been identified. The potency, selectivity profile against other kinases, and structure-activity relationship (SAR) trends of this class of compounds are discussed.

A simple method for the synthesis of various 3-(4,5-dihydro-1 H -imidazole-2-yl)-1 H -indoles is described. Treatment of different substituted indoles with 1-acetyl-imidazolidin-2-one in the presence of phosphorus oxychloride afforded after hydrolysis in ethanol the corresponding 3-(4,5-dihydro-1 H -imidazole-2-yl)-1 H -indoles in moderate to good yields.

6-[4-Amidinobenzoyl]amino]-tetralone-2-acetic acid is a potent antagonist of GPIIb-IIIa. Substitution in the meta position of the benzamidine, or replacement with a heteroaryl amidine was tolerated in this series. Use of an acyl-linked 4-alkyl piperidine as an arginine isostere also provided active compounds. Compounds from this series provided substantial systemic exposure in the rat following oral administration.

Disubstituted isoquinolones 2 and 3 have affinity for GPIIb-IIIa and represent leads for further structural evaluation. Structure-activity studies centered on the bicyclic beta-turn mimic contained in these molecules indicated that this moiety could accommodate a variety of modifications. Specifically, monocyclic, 6, 5-bicyclic, and 6,7-bicyclic structures provide compounds with affinity for GPIIb-IIIa. Within the 6,6-series, isoquinoline, tetralin, tetralone, and benzopyran nuclei yield potent antagonists that are specific for GPIIb-IIIa. Attachment of the arginine isostere (benzamidine) to the supporting nucleus can be accomplished with an ether or amide linkage, although the latter enhances activity. Several compounds in this series provided measurable blood levels after oral dosing. Conversion of the acid moiety in these molecules to an ester generally provided compounds which gave greater systemic exposure after oral administration. Absolute bioavailabilities in the rat for the ethyl ester prodrug derivatives of the tetralin, tetralone, and benzopyran analogues of 3 were 28%, 23%, and 24%, respectively.

In the presence of trimethylsilyltrifluoromethanesulfonate as Lewis acid catalyst, β-acetates reacted, as glycosyl donors and with neighboring-group participation, with secondary hydroxyl groups of saccharides having low reactivity to give β-glycosidically linked di- and oligo-saccharides in high yields. The protecting groups of both compounds of the reaction had to be stable under acid conditions. Thus, the reaction of 1,2,3,4,6-penta-O-acetyl-β- d -galactopyranose with methyl 2-azido-4,6-di-O-benzoyl-2-deoxy-β- d -galactopyranoside gave, in 83% yield, methyl 2-azido-2,6-di-O-benzoyl-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl-β- d -galactopyranosyl)-β- d -galactopyranoside which is a useful building unit. The glycosidic reactions under Lewis acid catalysis and Koenigs—Knorr conditions are compared. The efficiency of the Lewis acid-catalyzed procedure was demonstrated by the further synthesis of di- and tri-saccharides, such as lactosamine and chitobiose derivatives, whereby hydroxyl groups having low reactivity could be glycosylated in good yields.

The synthesis of the trisaccharides O-beta-D-galactopyranosyl-(1 to 3)-O-(2-acetamido-2-deoxy-alpha-D-galactopyranosyl)-(1 to 6)-D-galactopyranose (15) and O-beta-D-galactopyranosyl-(1 to 3)-O-(2-acetamido-2-deoxy-alpha-D-galactopyranosyl)-(1 to 6)-D-glucopyranose (27) is described and the synthesis of alpha-D-glycosides by reaction of 3,4,6-tri-O-acetyl-2-azido-2-deoxy-beta-D-galactopyranosyl chloride with highly reactive hydroxyl groups is discussed. The trisaccharide 27 was coupled with serum albumin by formation of an imine intermediate and reduced to an amine, to yield a synthetic T-antigen. A similar coupling of 15 was unsuccessful.

It was determined that β-rhodomycin-II (rhodomycin A) (1), β-rhodomycin-I (rhodomycin B, betaclamycin T) (2) and γ-rhodomycin-I (iremycin) (3) may regioselectively be acylated at OH-4′ of the sugar (rhodosamine) moiety(ies), using a two-phase (chloroform/water) solvent system and sodium bicarbonate as a base. This report exemplarily describes the synthesis of the corresponding p-methoxybenzoate esters of 1, 2, and 3, i. e. derivatives 4–7, and highlights the structure elucidation of isomers 5a and 5b.

In the presence of trimethylsilyl triflate, 1,2,3,4,6-penta-O-acetyl-β- d -galactopyranose reacted with benzyl 4-O-acetyl-2,6-di-O-benzyl-β- d -galactopyranoside to give benzyl 2,6-di-O-benzyl-3-O-β- d -galactopyranosyl-β- d -galactopyranoside further converted into the synthetic block 1,2,4,6-tetra-O-acetyl-3-O-(2,3,4,6-tetra-O-acetyl-β- d -galactopyranosyl)-β- d -galactopyranose. This, in the presence of a Lewis acid catalyst and with the corresponding glycosyl acceptors, gave 8-methoxycarbonyloctyl 3-O-β- d -galactopyranosyl-β- d -galactopyranoside and 8-methoxycarbonyloctyl O-β- d -galactopyranosyl-(1→3)-O-β- d -galactopyranosyl-(1→3)-2-acetamido-2-deoxy-α- d -galactopyranoside.

Eine Blocksynthese zu O-Glycopeptiden ist durch Umsetzung des Disaccharidhalogenides 10 mit L-Serin- oder L-Threonin-haltigen Peptid-Derivaten moglich. So ergibt die Umsetzung von 10 mit 8 stereoselektiv das L-Ser-L-Ser-Derivat 15, an das α-glycosidisch zwei Disaccharid-Reste geknupft sind. Diese Methode wird jedoch durch geringe Loslichkeiten von Peptid-Derivaten in Dichlormethan begrenzt. Universeller anwendbar ist eine stufenweise Synthese von O-Glycopeptiden, bei der selektiv entblockierte Derivate von Disaccharid-Aminosauren nach den Methoden der Peptidchemie verknupft werden. So ergibt die Reaktion von 25 mit 29 in Gegenwart von EEDQ das Glycopeptid 30. Als bestes Entacylierungs-Reagenz fur die Deblockierung der Saccharid-Einheiten im letzten Schritt hat sich Kaliumcyanid/Methanol/Ethanol bewahrt. Hiermit erfolgt auch Abspaltung der Benzoylgruppen ohne β-Eliminierung oder Racemisierung des Peptidteiles. Building Units of Oligosaccharides, LXVI. — Synthesis of O-Glycopeptide Blocks of Glycophorine A block synthesis to O-glycopeptides can be realized by reaction of the disaccharide halide 10 with either L-serine- or L-threonine-containing peptide derivatives. Thus, the reaction of 10 with 8 yields stereoselectively the L-Ser-L-Ser derivative 15 in which two disaccharide residues are α-glycosidically linked to the peptide. However, this method is limited by the low solubilities of peptide derivatives in dichloromethane. A more general procedure represents the step-by-step synthesis of O-glycopeptides in which selectively deblocked derivatives of disaccharide amino acids are combined by using the methods of peptide syntheses. An example is the reaction of 25 with 29 in the presence of EEDQ yielding the glycopeptide 30. The reagent potassium cyanide in methanol/ethanol proved to be the most suitable deacylation reagent for the deblocking of the saccharide units in the last step. The benzoyl groups can be removed without β-elimination or racemization of the peptide part.

A regioselective β-glycosidation of lactose derivatives unsubstituted at OH-3′ and -4′ is possible, depending on the catalyst system applied. With silver silicate or silver carbonate as catalyst under heterogeneous conditions, β-glycosidation occurred selectively at the less reactive OH-4′ group of the acceptor, whereas with trimethylsilyl triflate or soluble mercury salt as catalyst under homogeneous conditions, it occurred almost exclusively at the more reactive OH-3′ group. The reactivity of the glycosyl donor, acceptor, and catalyst had to be properly matched for successful regioselective coupling. In this way, the trisaccharide unit of asialo- GM2-ganglioside, O-(2-acetamido-2-deoxy-β- d -galactopyranosyl)-(1→4)-(β- d - galactopyranosyl)-(1→4)- d -glucopyranose, and the tetrasaccharide unit of asialo- GM1-ganglioside, O-(β- d -galactopyranosyl)-(1→3)-(2-acetamido-2-deoxy-β- d -galactopyranosyl)- (1→4)-(β- d -galactopyranosyl)-(1→4)-β- d -glucopyranose were synthesized. This new method constitutes a very useful approach in the field of ganglioside synthesis.

Under the conditions of in situ anomerisation, the 2-azido-4,6-di-O-benzoyl-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl-β- d -galactopyranosyl)-α- d -galactopyranosyl bromide reacted directly and with high selectivity with the reactive hydroxyl groups of l -serine and l -threonine derivatives to form α-glycosidically-linked products. Thus, the glycopeptides containing l -serine and l -threonine are more accessible. The disaccharide block could also be coupled with other reactive hydroxyl compounds to give compounds that contain the T-receptor.

The syntheses of 7-O-α-L-daunosaminyl-∊-isorhodomycinone (6) and 7-O-α-L-rhodosaminyl-∊-isorhodomycinone (7) are described. The glycosyl donors 1,4-di-O-p-nitrobenzoyl-3-N-trifluoroacetyl-α,β-L-daunosamine and 1,4-di-O-acetyl-α,β-L-rhodosamine have proven to be the most suitable for the glycosylation of ∊-isorhodomycinone (∊-iso RMN) (3), using the TMS triflate method. Deblocking (0.5 N NaOH) of the protected glycoside 4 led to the desired 7-O-glycosyl-∊-isorhodomycinones 5 and 6. The daunosaminyl glycoside 6 was methylated (CH2O, NaCNBH3) to provide the rhodosaminyl derivative 7. The photolytic demethylation of this product selectively provided the 7-O-(3′-N-methyl-α-L-daunosaminyl)-∊-isorhodomycinone (8).