Empowering Voices: Inspiring Women in Medicinal Chemistry.

As we celebrate International Women's Day 2024 with the theme "Inspire Inclusion", the women of the ACS Medicinal Chemistry Division (MEDI) want to foster a sense of belonging, relevance, and empowerment by sharing uplifting stories of what inspired them to become medicinal chemists. In this editorial, we are featuring female medicinal chemistry scientists to provide role models, encouragement, and inspiration to others. We asked women medicinal chemists to contribute a brief paragraph about what inspired them to become medicinal chemists or what inspires them today as medicinal chemists. The responses and contributions highlight their passions and motivations, such as their love of the sciences and their drive to improve human health by contributing to basic research and creating lifesaving drugs.

Inhibition of macrophage infectivity potentiator in Burkholderia pseudomallei suppresses pro-inflammatory responses in murine macrophages.

Introduction: Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is a disease endemic in many tropical countries globally. Clinical presentation is highly variable, ranging from asymptomatic to fatal septicemia, and thus the outcome of infection can depend on the host immune responses. The aims of this study were to firstly, characterize the macrophage immune response to B. pseudomallei and secondly, to determine whether the immune response was modified in the presence of novel inhibitors targeting the virulence factor, the macrophage infectivity potentiator (Mip) protein. We hypothesized that inhibition of Mip in B. pseudomallei would disarm the bacteria and result in a host beneficial immune response. Methods: Murine macrophage J774A.1 cells were infected with B. pseudomallei K96243 in the presence of small-molecule inhibitors targeting the Mip protein. RNA-sequencing was performed on infected cells four hours post-infection. Secreted cytokines and lactose dehydrogenase were measured in cell culture supernatants 24 hours post-infection. Viable, intracellular B. pseudomallei in macrophages were also enumerated 24 hours post-infection. Results: Global transcriptional profiling of macrophages infected with B. pseudomallei by RNA-seq demonstrated upregulation of immune-associated genes, in particular a significant enrichment of genes in the TNF signaling pathway. Treatment of B. pseudomallei-infected macrophages with the Mip inhibitor, AN_CH_37 resulted in a 5.3-fold reduction of il1b when compared to cells treated with DMSO, which the inhibitors were solubilized in. A statistically significant reduction in IL-1ß levels in culture supernatants was seen 24 hours post-infection with AN_CH_37, as well as other pro-inflammatory cytokines, namely IL-6 and TNF-α. Treatment with AN_CH_37 also reduced the survival of B. pseudomallei in macrophages after 24 hours which was accompanied by a significant reduction in B. pseudomallei-induced cytotoxicity as determined by lactate dehydrogenase release. Discussion: These data highlight the potential to utilize Mip inhibitors in reducing potentially harmful pro-inflammatory responses resulting from B. pseudomallei infection in macrophages. This could be of significance since overstimulation of pro-inflammatory responses can result in immunopathology, tissue damage and septic shock.

Empowering Voices: Inspiring Women in Medicinal Chemistry.

As we celebrate International Women's Day 2024 with the theme "Inspire Inclusion", the women of the ACS Medicinal Chemistry Division (MEDI) want to foster a sense of belonging, relevance, and empowerment by sharing uplifting stories of what inspired them to become medicinal chemists. In this editorial, we are featuring female medicinal chemistry scientists to provide role models, encouragement, and inspiration to others. We asked women medicinal chemists to contribute a brief paragraph about what inspired them to become medicinal chemists or what inspires them today as medicinal chemists. The responses and contributions highlight their passions and motivations, such as their love of the sciences and their drive to improve human health by contributing to basic research and creating lifesaving drugs.

Combined In-Solution Fragment Screening and Crystallographic Binding-Mode Analysis with a Two-Domain Hsp70 Construct.

Heat shock protein 70 (Hsp70) isoforms are key players in the regulation of protein homeostasis and cell death pathways and are therefore attractive targets in cancer research. Developing nucleotide-competitive inhibitors or allosteric modulators, however, has turned out to be very challenging for this protein family, and no Hsp70-directed therapeutics have so far become available. As the field could profit from alternative starting points for inhibitor development, we present the results of a fragment-based screening approach on a two-domain Hsp70 construct using in-solution NMR methods, together with X-ray-crystallographic investigations and mixed-solvent molecular dynamics simulations. The screening protocol resulted in hits on both domains. In particular, fragment binding in a deeply buried pocket at the substrate-binding domain could be detected. The corresponding site is known to be important for communication between the nucleotide-binding and substrate-binding domains of Hsp70 proteins. The main fragment identified at this position also offers an interesting starting point for the development of a dual Hsp70/Hsp90 inhibitor.

Application of advanced high resolution mass spectrometric techniques for the analysis of losartan potassium regarding known and unknown impurities.

Recalls of medicinal products can cause supply bottlenecks. This is often due to the findings of unexpected impurities that pose a health risk to patients. A recent example is losartan potassium which was contaminated with azido-impurities. The choice of the analytical method determines which substances can be detected and thus controlled. In this study a combination of an untargeted screening approach for impurities and a targeted evaluation of high-resolution mass spectrometry data was applied to search for impurities not described so far, leaving out a precise quantification. Six losartan potassium samples were studied regarding known and unknown impurities and hence highlight the applicability and capability of the approach. For separation a Zorbax RR StableBond C18 column (3.0 ×100 mm, particle size of 3.5 µm, pore size of 80 Å), a gradient elution and an electrospray ionization in positive and negative mode for mass spectrometric detection was used. An information-dependent acquisition method was applied for the measurement of losartan potassium samples. The untargeted data evaluation using general unknown comparative screening revealed the presence of N-methyl-2-pyrrolidone (NMP) and another impurity from synthesis. The identity of NMP was corroborated by a spiking experiment and the amount was estimated by means of standard addition. A targeted data evaluation by generating extracted ion chromatograms resulted in finding of four additional impurities. Combined approaches like this are needed to detect and respond to changes in the quality of drugs precociously.

Do the enantiomers of ketamine bind enantioselectively to human serum albumin?

The binding of drugs to plasma proteins is an important process in the human body and has a significant influence on pharmacokinetic parameter. Human serum albumin (HSA) has the most important function as a transporter protein. The binding of ketamine to HSA has already been described in literature, but only of the racemate. The enantiomerically pure S-ketamine is used as injection solution for induction of anesthesia and has been approved by the Food and Drug Administration for the therapy of severe depression as a nasal spray in 2019. The question arises if there is enantioselective binding to HSA. Hence, the aim of this study was to investigate whether there is enantioselective binding of S-and R-ketamine to HSA or not. Ultrafiltration (UF) followed by chiral capillary electrophoretic analysis was used to determine the extent of protein binding. Bound fraction to HSA was 71.2 % and 64.9 % for enantiomerically pure R- and S-ketamine, respectively, and 66.5 % for the racemate. Detailed binding properties were studied by Saturation Transfer Difference (STD)-, waterLOGSY- and Carr-Purcell-Meiboom-Gill (CPMG)-NMR spectroscopy. With all three methods, the aromatic ring and the N-methyl group could be identified as the structural moieties most strongly involved in binding of ketamine to HSA. pKaff values determined using UF and NMR indicate that ketamine is a weak affinity ligand to HSA and no significant differences in binding behavior were found between the individual enantiomers and the racemate.

The quality of drugs and drug products - Always guaranteed?

To ensure the efficacy, safety, and quality of drugs, several national and international guidelines and regulatory requirements exist. The most important international regulatory framework for quality is the collection of the guidelines ICH Q1-Q14 (International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use), which form the basis for the development and approval of medicinal products. Additionally, international and national pharmacopoeias and national regulatory authorities like Food and Drug Administration (FDA) and European Directory for the Quality of Medicines and HealthCare (EDQM) have to be considered during the lifecycle of a drug. Further, regular updates and optimization of processes and methods together with periodic audits and inspections of the manufacturing plants help to ensure compliance with the complex regulatory requirements for medicinal products. Although the pharmaceutical world seems to be very well regulated and controlled, several drug recalls per year have to be announced and conducted to remove defect products from the market and protect the patient from any potential health risk. This review article provides an overview of the most common reasons for such recalls presenting several historical and current cases with a detailed discussion of root causes. A specific focus lies on quality issues like drug degradation, impurity and nitrosamine contamination, lack of drug stability, occurrence and transformation of polymorphs, contamination with particulates and foreign matters, amongst others. The role of APIs, excipients and packaging will be discussed as well as the analytical challenges to detect, control and mitigate such quality issues. A final chapter will discuss the current situation and an outlook on emerging topics and future challenges for drug quality.

The solvent- and surface-dependent adsorption of the lipopeptide antibiotic daptomycin: The general necessity of adsorption tests.

The impact of poor or non-reproducible analyte recoveries due to non-specific drug adsorption on various analytical assays is often underestimated. Even internationally approved guidelines for pharmaceutical analysis such as the EMA guideline on bioanalytical method validation, the ICH guideline M10 on bioanalytical method validation and study sample analysis or the FDA bioanalytical method validation guidance do not adequately encourage more detailed investigations. Furthermore, other areas of research in which the concentration of active pharmaceutical compounds plays a crucial role, for example screening for minimal inhibitory concentrations of bacterial isolates, are potentially affected as well. The aim of this study was to demonstrate the general necessity of drug adsorption tests, using the lipopeptide antibiotic daptomycin as an example. A wide range of typical materials used in processing samples in pharmaceutical and biological analysis, as well as various solvents and biological matrices were included in the experiments. A fully validated LC-MS/MS method was applied for the determination of daptomycin concentrations, which were subsequently used to calculate the recovery. Recovery results (n = 3) ranged from 0.00% to 102.12% with a maximum relative standard deviation of 12.78%. These findings demonstrate that recovery can vary greatly depending on the solvent and the contact material, indicating the need to be optimized and, if applicable, validated. Hence, high reproducibility can only be achieved if all materials (and their manufacturers) used in a method are specified, not just those used in steps considered critical.

Identification of low-level impurities in drug prototypes of carbocisteine by means of liquid chromatography-high-resolution mass spectrometry and general unknown comparative screening.

High-resolution tandem quadrupole time-of-flight mass analysers enable new automated workflows for untargeted data evaluation of complex samples like drug products. An example of such procedure is the so-called general unknown comparative screening (GUCS), which is used for software-assisted, automated identification of components that are only present in a sample and not in a reference. The GUCS approach has been employed for the first time to detect both degradation products and reaction products in drug products. Two different carbocisteine containing syrup prototypes - one with sucrose and the other with artificial sweeteners - were selected as examples after nine months of storage at 40 °C and 75% relative humidity. The samples were analysed chromatographically using a Coresep SB mixed-mode column and high-resolution MS and MS/MS data were recorded in information dependant acquisition mode on a Sciex X500R quadrupole time-of-flight mass spectrometer. Data analysis was considerably facilitated using the corresponding placebo formulation as reference samples. With the GUCS approach two hitherto unknown degradation products of carbocisteine, i.e. the carbocisteine lactam of the sulfoxides and the disulfide between l-cysteine and thioglycolic acid, were detected at low concentrations in both of the syrup formulations. The presumed structures were confirmed by in silico analysis of the fragment spectra and high-resolution LC-MS experiments with reference substances. Two additional impurities were found in the sucrose-containing sample and identified as the N-glycosides of carbocisteine and its lactam, respectively, using binary mixtures with a 13C-labelled monosaccharide.

Mechanochemical Oxidative Degradation of Thienopyridine Containing Drugs: Toward a Simple Tool for the Prediction of Drug Stability.

The long-term stability of an active-pharmaceutical ingredient and its drug products plays an important role in the licensing process of new pharmaceuticals and for the application of the drug at the patient. It is, however, difficult to predict degradation profiles at early stages of the development of new drugs, making the entire process very time-consuming and costly. Forced mechanochemical degradation under controlled conditions can be used to realistically model long-term degradation processes naturally occurring in drug products, avoiding the use of solvents, thus excluding irrelevant solution-based degradation pathways. We present the forced mechanochemical oxidative degradation of three platelet inhibitor drug products, where the drug products contain thienopyridine. Model studies using clopidogrel hydrogen sulfate (CLP) and its drug formulation Plavix show that the controlled addition of excipients does not affect the nature of the main degradants. Experiments using drug products Ticlopidin-neuraxpharm and Efient show that significant degradation occurs after short reaction times of only 15 min. These results highlight the potential of mechanochemistry for the study of degradation processes of small molecules relevant to the prediction of degradation profiles during the development of new drugs. Furthermore, these data provide exciting insights into the role of mechanochemistry for chemical synthesis in general.

Macrophage infectivity potentiator protein, a peptidyl prolyl cis-trans isomerase, essential for Coxiella burnetii growth and pathogenesis.

Coxiella burnetii is a Gram-negative intracellular pathogen that causes the debilitating disease Q fever, which affects both animals and humans. The only available human vaccine, Q-Vax, is effective but has a high risk of severe adverse reactions, limiting its use as a countermeasure to contain outbreaks. Therefore, it is essential to identify new drug targets to treat this infection. Macrophage infectivity potentiator (Mip) proteins catalyse the folding of proline-containing proteins through their peptidyl prolyl cis-trans isomerase (PPIase) activity and have been shown to play an important role in the virulence of several pathogenic bacteria. To date the role of the Mip protein in C. burnetii pathogenesis has not been investigated. This study demonstrates that CbMip is likely to be an essential protein in C. burnetii. The pipecolic acid derived compounds, SF235 and AN296, which have shown utility in targeting other Mip proteins from pathogenic bacteria, demonstrate inhibitory activities against CbMip. These compounds were found to significantly inhibit intracellular replication of C. burnetii in both HeLa and THP-1 cells. Furthermore, SF235 and AN296 were also found to exhibit antibiotic properties against both the virulent (Phase I) and avirulent (Phase II) forms of C. burnetii Nine Mile Strain in axenic culture. Comparative proteomics, in the presence of AN296, revealed alterations in stress responses with H2O2 sensitivity assays validating that Mip inhibition increases the sensitivity of C. burnetii to oxidative stress. In addition, SF235 and AN296 were effective in vivo and significantly improved the survival of Galleria mellonella infected with C. burnetii. These results suggest that unlike in other bacteria, Mip in C. burnetii is required for replication and that the development of more potent inhibitors against CbMip is warranted and offer potential as novel therapeutics against this pathogen.

Analysis of Structure-Activity Relationships of Novel Inhibitors of the Macrophage Infectivity Potentiator (Mip) Proteins of Neisseria meningitidis, Neisseria gonorrhoeae, and Burkholderia pseudomallei.

The macrophage infectivity potentiator (Mip) protein is a promising target for developing new drugs to combat antimicrobial resistance. New rapamycin-derived Mip inhibitors have been designed that may be able to combine two binding modes to inhibit the Mip protein of Burkholderia pseudomallei (BpMip). These novel compounds are characterized by an additional substituent in the middle chain linking the lateral pyridine to the pipecoline moiety, constituting different stereoisomers. These compounds demonstrated high affinity for the BpMip protein in the nanomolar range and high anti-enzymatic activity and ultimately resulted in significantly reduced cytotoxicity of B. pseudomallei in macrophages. They also displayed strong anti-enzymatic activity against the Mip proteins of Neisseria meningitidis and Neisseria gonorrhoeae and substantially improved the ability of macrophages to kill the bacteria. Hence, the new Mip inhibitors are promising, non-cytotoxic candidates for further testing against a broad spectrum of pathogens and infectious diseases.

An exploratory study on the effect of mechanical stress on particle formation in monoclonal antibody infusions.

Monoclonal antibody infusions (mAb-i) are administered for the treatment of various diseases. They are often transported over long distances from the compounding site to the site of administration. However, transport studies are typically carried out with the original drug product but not with compounded mAb-i. To address this gap, the impact of mechanical stress on the formation of subvisible/nanoparticles in mAb-i was investigated by dynamic light scattering and flow imaging microscopy. Different mAb-i concentrations were subjected to vibrational orbital shaking and stored at 2-8°C up to 35 days. The screening revealed that pembrolizumab and bevacizumab infusions show the highest propensity for particle formation. Especially bevacizumab at low concentrations exhibited an increase in particle formation. Because of the unknown health risks associated with the long-term application of subvisible particles (SVPs)/nanoparticles in infusion bags, stability studies carried out in the frame of licensing application procedures should also focus on SVP formation in mAb-i. In general, pharmacists should minimize the time of storage and mechanical stress during transport, especially in the case of low-concentrated mAb-i. Moreover, if siliconized syringes are used, they should be washed once with saline solution to minimize particle entry.

Hybridization into a Bitopic Ligand Increased Muscarinic Receptor Activation for Isopilocarpine but Not for Pilocarpine Derivatives.

Pilocarpine (1), a secondary metabolite of several Pilocarpus species, is a therapeutically used partial agonist of muscarinic acetylcholine receptors (mAChRs). The available pharmacological data and structure-activity relationships do not provide comparable data for all five receptor subtypes. In this study, pilocarpine (1), its epimer isopilocarpine (2), racemic analogues pilosinine (3) and desmethyl pilosinine (4), and the respective hybrid ligands with a naphmethonium fragment (5-C6 to 8-C6) were synthesized and analyzed in mini-G nano-BRET assays at the five mAChRs. In line with earlier studies, pilocarpine was the most active compound among the orthosteric ligands 1-4. Computational docking of pilocarpine and isopilocarpine to the active M2 receptor suggests that the trans-configuration of isopilocarpine leads to a loss of the hydrogen bond from the lactone carbonyl to N6.52, explaining the lower activity of isopilocarpine. Hybrid formation of pilocarpine (1) and isopilocarpine (2) led to an inverted activity rank, with the trans-configured isopilocarpine hybrid (6-C6) being more active. The hydrogen bond of interest is formed by the isopilocarpine hybrid (6-C6) but not by the pilocarpine hybrid (5-C6). Hybridization thus leads to a modified binding mode of the orthosteric moiety, as the binding mode of the hybrid is dominated by the high-affinity allosteric moiety.

Analytical Quality by Design: Achieving Robustness of an LC-CAD Method for the Analysis of Non-Volatile Fatty Acids.

An alternative to the time-consuming and error-prone pharmacopoeial gas chromatography method for the analysis of fatty acids (FAs) is urgently needed. The objective was therefore to propose a robust liquid chromatography method with charged aerosol detection for the analysis of polysorbate 80 (PS80) and magnesium stearate. FAs with different numbers of carbon atoms in the chain necessitated the use of a gradient method with a Hypersil Gold C18 column and acetonitrile as organic modifier. The risk-based Analytical Quality by Design approach was applied to define the Method Operable Design Region (MODR). Formic acid concentration, initial and final percentages of acetonitrile, gradient elution time, column temperature, and mobile phase flow rate were identified as critical method parameters (CMPs). The initial and final percentages of acetonitrile were fixed while the remaining CMPs were fine-tuned using response surface methodology. Critical method attributes included the baseline separation of adjacent peaks (α-linolenic and myristic acid, and oleic and petroselinic acid) and the retention factor of the last compound eluted, stearic acid. The MODR was calculated by Monte Carlo simulations with a probability equal or greater than 90%. Finally, the column temperature was set at 33 °C, the flow rate was 0.575 mL/min, and acetonitrile linearly increased from 70 to 80% (v/v) within 14.2 min.

Nature-inspired synthesis of antibacterial glucovanillin derivatives.

The ongoing threat of Antimicrobial Resistance (AMR) complicated by the rise of Multidrug-Resistant (MDR) pathogens calls for increased efforts in the search for novel treatment options. While deriving inspiration from antibacterial natural compounds, this study aimed at using synthetic approaches to generate a series of glucovanillin derivatives and explore their antibacterial potentials. Among the synthesized derivatives, optimum antibacterial activities were exhibited by those containing 2,4- and 3,5-dichlorophenylamino group coupled to a glucovanillin moiety (compounds 6h and 8d respectively). In those compounds, the Minimum Inhibitory Concentrations (MIC) of 128-256 µg/mL were observed against reference and MDR strains of Klebsiella pneumoniae, Methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE). Moreover, these findings emphasize the claims from previous reports on the essence of smaller molecular size, the presence of protonatable amino groups and halogens in potential antibacterial agents. The observed moderate and broad-spectrum activities of the stated derivatives point to their suitability as potential leads towards further efforts to improve their antibacterial activities.

Novel Xanomeline-Containing Bitopic Ligands of Muscarinic Acetylcholine Receptors: Design, Synthesis and FRET Investigation.

In the last few years, fluorescence resonance energy transfer (FRET) receptor sensors have contributed to the understanding of GPCR ligand binding and functional activation. FRET sensors based on muscarinic acetylcholine receptors (mAChRs) have been employed to study dual-steric ligands, allowing for the detection of different kinetics and distinguishing between partial, full, and super agonism. Herein, we report the synthesis of the two series of bitopic ligands, 12-Cn and 13-Cn, and their pharmacological investigation at the M1, M2, M4, and M5 FRET-based receptor sensors. The hybrids were prepared by merging the pharmacophoric moieties of the M1/M4-preferring orthosteric agonist Xanomeline 10 and the M1-selective positive allosteric modulator 77-LH-28-1 (1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone) 11. The two pharmacophores were connected through alkylene chains of different lengths (C3, C5, C7, and C9). Analyzing the FRET responses, the tertiary amine compounds 12-C5, 12-C7, and 12-C9 evidenced a selective activation of M1 mAChRs, while the methyl tetrahydropyridinium salts 13-C5, 13-C7, and 13-C9 showed a degree of selectivity for M1 and M4 mAChRs. Moreover, whereas hybrids 12-Cn showed an almost linear response at the M1 subtype, hybrids 13-Cn evidenced a bell-shaped activation response. This different activation pattern suggests that the positive charge anchoring the compound 13-Cn to the orthosteric site ensues a degree of receptor activation depending on the linker length, which induces a graded conformational interference with the binding pocket closure. These bitopic derivatives represent novel pharmacological tools for a better understanding of ligand-receptor interactions at a molecular level.

Fluorescent probe for the identification of potent inhibitors of the macrophage infectivity potentiator (Mip) protein of Burkholderia pseudomallei.

The macrophage infectivity potentiator (Mip) protein belongs to the immunophilin superfamily. This class of enzymes catalyzes the interconversion between the cis and trans configuration of proline-containing peptide bonds. Mip has been shown to be important for the virulence of a wide range of pathogenic microorganisms, including the Gram-negative bacterium Burkholderia pseudomallei. Small molecules derived from the natural product rapamycin, lacking its immunosuppression-inducing moiety, inhibit Mip's peptidyl-prolyl cis-trans isomerase (PPIase) activity and lead to a reduction in pathogen load in vitro. Here, a fluorescence polarization assay (FPA) to enable the screening and effective development of BpMip inhibitors was established. A fluorescent probe was prepared, derived from previous pipecolic scaffold Mip inhibitors labeled with fluorescein. This probe showed moderate affinity for BpMip and enabled a highly robust FPA suitable for screening large compound libraries with medium- to high-throughput (Z factor ∼ 0.89) to identify potent new inhibitors. The FPA results are consistent with data from the protease-coupled PPIase assay. Analysis of the temperature dependence of the probe's binding highlighted that BpMip's ligand binding is driven by enthalpic rather than entropic effects. This has considerable consequences for the use of low-temperature kinetic assays.

Design, synthesis and biological evaluations of quinolone amides against African trypanosomiasis with improved solubility.

The human African trypanosomiasis is a devastating parasitic infection, which is caused by the protozoan Trypanosoma brucei and transmitted by the bite of the tsetse fly. An untreated infection usually results in death and only few drugs with significant drawbacks are currently available for treatment. Previous investigations revealed the quinolone amide MB007 as a lead compound with an excellent selectivity for T. b. brucei. Here, new quinolone amides were synthesized for deeper insights into the structure-activity relationship. Furthermore, the aqueous solubility of the compounds was analyzed, as the poor solubility of previous quinolone amides impeded in vivo studies for target identification. The biological evaluation led to the new lead structure 9f, which exhibits a promising in vitro activity against T. b. brucei (IC50 = 22 nM) and showed no cytotoxicity against macrophages. Moreover, compounds 10b and 10c were discovered, which possessed an improved solubility combined with a decent selectivity.

Development, validation and application of a selective and sensitive LC-MS/MS method for the quantification of daptomycin in a suspension of Mammaliicoccus sciuri in Mueller-Hinton broth.

Reports of therapy failures related to the use of daptomycin (DAP) are steadily increasing. This is mainly due to emerging DAP resistance for which, however, the underlying mechanism is often unknown. To elucidate the mode of action of novel DAP resistance traits it is indispensable to reliably detect and quantify DAP in complex matrices such as bacterial culture media. In this study, we established a selective and sensitive quantification method for DAP upon growth of a DAP resistant Mammaliicoccus sciuri strain in Mueller-Hinton medium. The method combined methanol-induced protein precipitation followed by high performance liquid chromatography with gradient elution coupled to mass spectrometry (LC-MS/MS) using daptomycin-d5 as internal standard. All validation parameters met the acceptance criteria of the European Medicines Agency (EMA) guideline on bioanalytical method validation. We successfully applied this highly selective and sensitive LC-MS/MS method for the quantification of DAP in in vitro studies addressing DAP resistance mechanisms in Gram-positive bacteria.