Quantitative Determination of Water in Organic Liquids by Ambient Mass Spectrometry.

This study uses a rapid tandem mass-spectrometry method to determine water content in complex organic solutions. Emphasis is placed on trace-water analysis by a fast and accurate alternative to the Karl-Fischer method. In this new method, water is captured by a charge-labeled molecular probe. Water binds strongly with high specificity to the strongly electrophilic aldehyde site in a charge-labelled molecule (N-methylpyridinium); competitive binding by other analytes is effectively discriminated against in the mass-measurement step. Quantitative determinations are made over a wide concentration range, 0.001 % (10 ppm) to 99 %, with better than 10 % relative standard deviation, along with short (1 min) analysis times using small sample volumes (several µL). Applications include water measurement in simple organic solvents, for example, deuterated solvents, as well as in complex mixtures, for example, organic reaction mixtures. Additionally, this method allows for water monitoring in levitated droplets. Mechanistic investigations into the impact of water on important chemical processes in organic synthesis and environmental science are reported.

Fragmentation of Polyfunctional Compounds Recorded Using Automated High-Throughput Desorption Electrospray Ionization.

Using desorption electrospray ionization (DESI) as part of an automated high-throughput system, tandem mass spectra of the compounds in a pharmaceutical library were recorded in the positive mode under standardized conditions. Quality control filtering yielded an MS/MS library of 16 662 spectra. Fragmentation of subsets of the compounds in the library chosen to contain a single instance of a particular functional group (amide, piperazine, sulfonamide) was predicted by experts, and the results were compared with the experimental data. Expert performance was good to excellent for all the cases evaluated. Substituents on the functional groups were found to exert important secondary control over the fragmentation, with the main effect observed being product ion stabilization by aromatic substitution, which was consistent across the different groups evaluated. These substituent effects are generally explicable in terms of standard physical organic chemistry considerations of product ion stability as controlling fragmentation. A somewhat unexpected feature was the incidence of homolytic cleavages, driven by the stability of substituted amine radical cations. The findings of this study are intended to lay the groundwork for machine learning approaches to performing MS/MS spectrum → structure and structure → MS/MS spectrum operations on the same experimental data set. The effort involved and the success achieved in computer-aided interpretation, now underway, will be compared with the expert performance as described here.

High-throughput, low-cost reaction screening using a modified 3D printer.

We describe a reaction screening system, based on a 96-well array, and scaled to suit use on the individual scientist's bench. The system was built by modifying a desktop 3D printer and fitting it with a glass syringe and microtiter plate. The effects of experimental variables were characterized, and the performance of the system was optimized. Precise volumes of reaction mixtures (<3% CV) were dispensed into the 96-well array in ca. 40 minutes. The system was used to screen reagents and solvents for the N-alkylation, Katritzky transamination, and Suzuki cross-coupling reactions. Product distributions derived from electrospray mass spectra and represented as heat maps facilitated recognition of optimum conditions. Screening of 96 reaction mixtures was completed in the modest time of approximately 105 minutes (∼65 seconds per reaction mixture). The system is constructed from open-source software and inexpensive 3D printer hardware.

The Degradation Chemistry of Prasugrel Hydrochloride: Part 1-Drug Substance.

Prasugrel hydrochloride is the active ingredient in Effient™, a thienopyridine platelet inhibitor. An extensive study of the degradation chemistry of prasugrel hydrochloride (LY640315 hydrochloride) has been carried out on the drug substance (part I) and on the drug product (part II, future article) using a multidimensional approach including hydrolytic, oxidative, and photolytic stressing, computational chemistry, HPLC analysis, and structure elucidation by various spectroscopic techniques. The major degradation products formed from the drug substance under the various stress conditions have been isolated and structures unambiguously determined, and the pathways leading to these products have been proposed. Fourteen new (not previously disclosed) products were discovered and characterized, in addition to 4 degradation products that had been previously identified in the literature. The pathways indicate that prasugrel is susceptible to hydrolysis, autoxidation (both radical-initiated and single-electron mediated), and peroxide-mediated oxidation; in solution, prasugrel is susceptible to photodegradation.

ReactNMR and ReactIR as reaction monitoring and mechanistic elucidation tools: the NCS mediated cascade reaction of α-thioamides to α-thio-ß-chloroacrylamides.

On-flow ReactIR and (1)H NMR reaction monitoring, coupled with in situ intermediate characterization, was used to aid in the mechanistic elucidation of the N-chlorosuccinimide mediated transformation of an α-thioamide. Multiple intermediates in this reaction cascade are identified and characterized, and in particular, spectroscopic evidence for the intermediacy of the chlorosulfonium ion in the chlorination of α-thioamides is provided. Further to this, solvent effects on the outcome of the transformation are discussed. This work also demonstrates the utility of using a combination of ReactIR and flow NMR reaction monitoring (ReactNMR) for characterizing complex multicomponent reaction mixtures.

Fermentanomics: monitoring mammalian cell cultures with NMR spectroscopy.

As the number of therapeutic proteins produced by mammalian cell cultures in the pharmaceutical industry continues to increase, the need to improve productivity and ensure consistent product quality during process development activities becomes more significant. Rational medium design is known to improve cell culture performance, but an understanding of nutrient consumption and metabolite accumulation within the medium is required. To this end, we have developed a technique for using 1D (1)H NMR to quantitate nonprotein feed components and metabolites in mammalian cell cultures. We refer to the methodology as "Fermentanomics" to differentiate it from standard metabolomics. The method was found to generate spectra with excellent water suppression, signal-to-noise, and resolution. More importantly, nutrient consumption and metabolite accumulation was readily observed. In total, 50 media components have been identified and quantitated. The application of Fermentanomics to the optimization of a proprietary CHO basal medium yielded valuable insight regarding the nutrient levels needed to maintain productivity. While the focus here is on the extracellular milieu of CHO cell cultures, this methodology is generally applicable to quantitating intracellular concentrations and can be extended to other mammalian cell lines, as well as platforms such as yeasts, fungi, and Escherichia coli.

Complete relative stereochemistry of multiple stereocenters using only residual dipolar couplings.

Residual dipolar couplings (RDCs), in combination with molecular order matrix calculations, were used to unambiguously determine the complete relative stereochemistry of an organic compound with five stereocenters. Three simple one-dimensional experiments were utilized for the measurements of (13)C-(1)H, (13)C-(19)F, (19)F-(1)H, and (1)H-(1)H RDCs. The order matrix calculation was performed on each chiral isomer independently. The fits were evaluated by the comparison of the root-mean-square deviation (rmsd) of calculated and measured RDCs. The order tensor simulations based on two different sets of RDC data collected with phage and bicelles are consistent. The resulting stereochemical assignments of the stereocenters obtained from using only RDCs are in perfect agreement with those obtained from the single-crystal X-ray structure. Six RDCs are found to be necessary to run the simulation, and seven are the minimum to get an acceptable result for the investigated compound. It was also shown that (13)C-(1)H and (1)H-(1)H RDCs, which are the easiest to measure, are also the most important and information-rich data for the order matrix calculation. The effect of each RDC on the calculation depends on the location of the corresponding vector in the structure. The direct RDC of a stereocenter is important to the configuration determination, but the configuration of stereocenters devoid of protons can also be obtained from analysis of nearby RDCs.

Characterization by liquid chromatography-nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry of two coupled oxidative-conjugative metabolic pathways for 7-ethoxycoumarin in human liver microsomes treated with alamethicin.

The microsomal metabolism of 7-ethoxycoumarin (7-EC) was investigated using liquid chromatography (LC)-NMR and liquid chromatography-mass spectrometry (LC-MS) to characterize the coupling of oxidative-conjugative metabolism events. Within microsomes, cytochromes P450 (P450s) and UDP-glucuronosyltransferases (UGTs) are spatially disparate, each having surface and luminal localization, respectively. To optimize cofactor and substrate transit to UGT without compromising P450 activity, the pore-forming peptide alamethicin was used for microsomal perforation. Aqueous extracts of microsomal incubations containing NADPH and UDP-glucuronic acid were injected for LC-NMR and LC-MS analysis. The analytical complementarity of LC-NMR and LC-MS permitted the identification of four metabolites (M1 to M4). The metabolites M1 and M2 are novel microsomal metabolites for 7-EC, consistent with 3-hydroxylation and subsequent glucuronidation, respectively. Metabolites M3 and M4 were 7-hydroxycoumarin (7-HC) and 7-HC glucuronide, respectively. Viewed collectively, these results illustrate the utility of alamethicin in the examination of coupled oxidative-conjugative metabolism and the synergy of LC-NMR and LC-MS in metabolite identification.