Unveiling the intricacy of gapmer oligonucleotides through advanced tandem mass spectrometry approaches and scan accumulation for 2DMS.

Antisense oligonucleotides (ASOs) are crucial for biological applications as they bind to complementary RNA sequences, modulating protein expression. ASOs undergo synthetic modifications like phosphorothioate (PS) backbone and locked nucleic acid (LNA) to enhance stability and specificity. Tandem mass spectrometry (MS) techniques were employed to study gapmer ASOs, which feature a DNA chain within RNA segments at both termini, revealing enhanced cleavages with ultraviolet photodissociation (UVPD) and complementary fragment ions from collision-induced dissociation (CID) and electron detachment dissociation (EDD). 2DMS, a data-independent analysis technique, allowed for comprehensive coverage and identification of shared fragments across multiple precursor ions. EDD fragmentation efficiency correlated with precursor ion charge states, with higher charges facilitating dissociation due to intramolecular repulsions. An electron energy of 22.8 eV enabled electron capture and radical-based cleavage. Accumulating multiple scans and generating average spectra improved signal intensity, aided by denoising algorithms. Data analysis utilised a custom Python script capable of handling modifications and generating unique mass lists.

Skeletal muscle index is associated with long term outcomes after lobectomy for non-small cell lung cancer.

BACKGROUND: Skeletal muscle indices have been associated with improved peri-operative outcomes after surgical resection of non-small-cell lung cancer (NSCLC). However, it is unclear if these indices can predict long term cancer specific outcomes. METHODS: NSCLC patients undergoing lobectomy at our institute between 2009-2015 were included in this analysis (N = 492). Preoperative CT scans were used to quantify skeletal muscle index (SMI) at L4 using sliceOmatic software. Cox proportional modelling was performed for overall (OS) and recurrence free survival (RFS). RESULTS: For all patients, median SMI was 45.7 cm2/m2 (IQR, 40-53.8). SMI was negatively associated with age (R = -0.2; p < 0.05) and positively associated with BMI (R = 0.46; P < 0.05). No association with either OS or RFS was seen with univariate cox modelling. However, multivariable modelling for SMI with patient age, gender, race, smoking status, DLCO and FEV1 (% predicted), American Society of Anesthesiology (ASA) score, tumor histology and stage, and postoperative neoadjuvant therapy showed improved OS (HR = 0.97; P = 0.0005) and RFS (HR = 0.97; P = 0.01) with SMI. Using sex specific median SMI as cutoff, a lower SMI was associated with poor OS (HR = 1.65, P = 0.001) and RFS (HR = 1.47, P = 0.03). CONCLUSIONS: SMI is associated with improved outcomes after resection of NSCLC. Further studies are needed to understand the biological basis of this observation. This study provides additional rationale for designing and implementation of rehabilitation trials after surgical resection, to gain durable oncologic benefit.

Association of pre-treatment lymphocyte-monocyte ratio with survival outcome in patients with head and neck cancer treated with chemoradiation.

BACKGROUND: Given the role of systematic inflammation in cancer progression, lymphocyte-monocyte ratio (LMR) from peripheral blood has been suggested as a biomarker to assess the extent of inflammation in several solid malignancies. However, the role of LMR as a prognostic factor in head and neck cancer was unclear in several meta-analyses, and there is a paucity of literature including patients in North America. We performed an observational cohort study to evaluate the association of LMR with survival outcomes in North American patients with head and neck cancer. METHODS: A single-institution, retrospective database was queried for patients with non-metastatic head and neck cancer who underwent definitive chemoradiation from June 2007 to April 2021 at the Roswell Park Comprehensive Cancer Center. Primary endpoints were overall survival (OS) and cancer-specific survival (CSS). The association of LMR with OS and CSS was examined using nonlinear Cox proportional hazard model using restricted cubic splines (RCS). Cox multivariable analysis (MVA) and Kaplan-Meier method were used to analyze OS and CSS. Pre-radiation LMR was then stratified into high and low based on its median value. Propensity scored matching was used to reduce the selection bias. RESULTS: A total of 476 patients met our criteria. Median follow up was 45.3 months (interquartile range 22.8-74.0). The nonlinear Cox regression model showed that low LMR was associated with worse OS and CSS in a continuous fashion without plateau for both OS and CSS. On Cox MVA, higher LMR as a continuous variable was associated with improved OS (adjusted hazard ratio [aHR] 0,90, 95% confidence interval [CI] 0.82-0.99, p = 0.03) and CSS (aHR 0.83, 95% CI 0.72-0.95, p = 0.009). The median value of LMR was 3.8. After propensity score matching, a total of 186 pairs were matched. Lower LMR than 3.8 remained to be associated with worse OS (HR 1.59, 95% CI 1.12-2.26, p = 0.009) and CSS (HR 1.68, 95% CI 1.08-2.63, p = 0.02). CONCLUSION: Low LMR, both as a continuous variable and dichotomized variable, was associated with worse OS and CSS. Further studies would be warranted to evaluate the role of such prognostic marker to tailor interventions.

Theoretical and Experimental Investigation of Autoxidation Propensity of Selected Drugs in Solution State.

The C-H bond dissociation energy (BDE) of drug molecules is often used to estimate their relative propensities to undergo autoxidation. BDE calculations based on electronic structures provide a convenient means to estimate the risk for a given compound to degrade via autoxidation. This study aimed to verify the utility of calculated C-H BDEs of a range of drug molecules in predicting their autoxidation propensities, in the solution state. For the autoxidation study, 2,2'-azobis (2-methylpropionitrile) was employed as the solution state stressor, and the experimental reaction rate constants were determined employing ultraperformance liquid chromatographic (UPLC) methods. Reaction rates in the solution state were compared to the calculated C-H BDE values of the respective compounds. The results indicated a poor correlation for compounds in the solution state, and their relative stabilities could not be explained with C-H BDE. On the other hand, a favorable relationship was observed between the relative extent of ionization and the autoxidation rates of the selected compounds. In the solution state, factors such as the type and extent of drug ionization, degree and type of solvation have been shown to contribute to differences in reactivity. By applying the computational method involving the effect of H-atom abstraction and potential ionization sites in the molecule, the calculated C-H BDE should relate better to the experimental autoxidation rates.

Review of fragmentation of synthetic single-stranded oligonucleotides by tandem mass spectrometry from 2014 to 2022.

The fragmentation of oligonucleotides by mass spectrometry allows for the determination of their sequences. It is necessary to understand how oligonucleotides dissociate in the gas phase, which allows interpretation of data to obtain sequence information. Since 2014, a range of fragmentation mechanisms, including a novel internal rearrangement, have been proposed using different ion dissociation techniques. The recent publications have focused on the fragmentation of modified oligonucleotides such as locked nucleic acids, modified nucleobases (methylated, spacer, nebularine and aminopurine) and modification to the carbon 2'-position on the sugar ring; these modified oligonucleotides are of great interest as therapeutics. Comparisons of different dissociation techniques have been reported, including novel approaches such as plasma electron detachment dissociation and radical transfer dissociation. This review covers the period 2014-2022 and details the new knowledge gained with respect to oligonucleotide dissociation using tandem mass spectrometry (without priori sample digestion) during that time, with a specific focus on synthetic single-stranded oligonucleotides.

Post-Operative Outcomes of Pre-Thoracic Surgery Respiratory Muscle Training vs Aerobic Exercise Training: A Systematic Review and Network Meta-analysis.

OBJECTIVE: To compare the postoperative outcomes of preoperative respiratory muscle training (RMT) with a device to preoperative aerobic exercise training (AET) in patients undergoing thoracic surgeries (cardiac and lung). DATA SOURCES: PubMed, EMBASE, Cochrane, and Web of Science were comprehensively searched upon inception to 9/2020. STUDY SELECTION: All randomized control studies, including preoperative RMT and preoperative AET compared with a non-training control group, were included. DATA EXTRACTION: The meta-analysis was performed for outcomes including postoperative pulmonary complications (PPC), pneumonia, postoperative respiratory failure (PRF), hospital length of stay (HLOS), and mortality. We performed a network meta-analysis based on Bayesian random-effects regression models. DATA SYNTHESIS: A total of 25 studies, 2070 patients were included in this meta-analysis. Pooled data for the patients who performed RMT with a device showed a reduction in PPCs, pneumonia, PRF with odds ratio (OR) of 0.35 (P value .006), 0.38 (P value .002), and 0.22 (P value .008), respectively. Pooled data for the patients who performed AET showed reduction in PPC, pneumonia with a OR of 0.33 (P value <.00001) and OR of 0.54 (P value .01), respectively. HLOS was decreased by 1.69 days (P value <.00001) by performing RMT and 1.79 days (P value .0008) by performing AET compared with the usual group. No significant difference in all-cause mortality compared with usual care in both RMT and AET intervention groups. No significant difference in the incidence of PRF compared with usual group in RMT + AET and AET alone intervention groups (OR 0.32; P=.21; OR 0.94; P=.87). Based on rank probability plots analysis, on network meta-analysis, RMT and AET ranked similarly on the primary outcome of PPC and secondary outcomes of pneumonia, PRF and HLOS. CONCLUSIONS: In thoracic surgeries, preoperative RMT is comparable with preoperative AET to prevent PPC, pneumonia, and PRF and reduce HLOS. It can be considered in patients in resource-limited settings.

Metatarsal fusion resisted bending as jerboas (Dipodidae) transitioned from quadrupedal to bipedal.

Hind limbs undergo dramatic changes in loading conditions during the transition from quadrupedal to bipedal locomotion. For example, the most early diverging bipedal jerboas (Rodentia: Dipodidae) are some of the smallest mammals in the world, with body masses that range between 2-4 g. The larger jerboa species exhibit developmental and evolutionary fusion of the central three metatarsals into a single cannon bone. We hypothesize that small body size and metatarsal fusion are mechanisms to maintain the safety factor of the hind limb bones despite the higher ground reaction forces associated with bipedal locomotion. Using finite-element analysis to model collisions between the substrate and the metatarsals, we found that body size reduction was insufficient to reduce bone stress on unfused metatarsals, based on the scaled dynamics of larger jerboas, and that fused bones developed lower stresses than unfused bones when all metatarsals are scaled to the same size and loading conditions. Based on these results, we conclude that fusion reinforces larger jerboa metatarsals against high ground reaction forces. Because smaller jerboas with unfused metatarsals develop higher peak stresses in response to loading conditions scaled from larger jerboas, we hypothesize that smaller jerboas use alternative dynamics of bipedal locomotion to reduce the impact of collisions between the foot and substrate.

Deletion of arylamine N-acetyltransferase 1 in MDA-MB-231 human breast cancer cells reduces primary and secondary tumor growth in vivo with no significant effects on metastasis.

Arylamine N-acetyltransferase 1 (NAT1) is frequently upregulated in breast cancer. Previous studies showed that inhibition or depletion of NAT1 in breast cancer cells diminishes anchorage-independent growth in culture, suggesting that NAT1 contributes to breast cancer growth and metastasis. To further investigate the contribution of NAT1 to growth and cell invasive/migratory behavior, we subjected parental and NAT1 knockout (KO) breast cancer cell lines (MDA-MB-231, MCF-7, and ZR-75-1) to multiple assays. The rate of cell growth in suspension was not consistently decreased in NAT1 KO cells across the cell lines tested. Similarly, cell migration and invasion assays failed to produce reproducible differences between the parental and NAT1 KO cells. To overcome the limitations of in vitro assays, we tested parental and NAT1 KO cells in vivo in a xenograft model by injecting cells into the flank of immunocompromised mice. NAT1 KO MDA-MB-231 cells produced primary tumors smaller than those formed by parental cells, which was contributed by an increased rate of apoptosis in KO cells. The frequency of lung metastasis, however, was not altered in NAT1 KO cells. When the primary tumors of the parental and NAT1 KO cells were allowed to grow to a pre-determined size or delivered directly via tail vein, the number and size of metastatic foci in the lung did not differ between the parental and NAT1 KO cells. In conclusion, NAT1 contributes to primary and secondary tumor growth in vivo in MDA-MB-231 breast cancer cells but does not appear to affect its metastatic potential.

Effect of musculature on mortality, a retrospective cohort study.

BACKGROUND: While often life-saving, treatment for head and neck cancer (HNC) can be debilitating resulting in unplanned hospitalization. Hospitalizations in cancer patients may disrupt treatment and result in poor outcomes. Pre-treatment muscle quality and quantity ascertained through diagnostic imaging may help identify patients at high risk of poor outcomes early. The primary objective of this study was to determine if pre-treatment musculature was associated with all-cause mortality. METHODS: Patient demographic and clinical characteristics were abstracted from the cancer center electronic database (n = 403). Musculature was ascertained from pre-treatment CT scans. Propensity score matching was utilized to adjust for confounding bias when comparing patients with and without myosteatosis and with and without low muscle mass (LMM). Overall survival (OS) was evaluated using the Kaplan-Meier method and Cox multivariable analysis. RESULTS: A majority of patients were male (81.6%), white (89.6%), with stage IV (41.2%) oropharyngeal cancer (51.1%) treated with definitive radiation and chemotherapy (93.3%). Patients with myosteatosis and those with LMM were more likely to die compared to those with normal musculature (5-yr OS HR 1.55; 95% CI 1.03-2.34; HR 1.58; 95% CI 1.04-2.38). CONCLUSIONS: Musculature at the time of diagnosis was associated with overall mortality. Diagnostic imaging could be utilized to aid in assessing candidates for interventions targeted at maintaining and increasing muscle reserves.

Cross-sectional analysis of myosteatosis and physical function in pretreatment head and neck cancer patients.

BACKGROUND: Head and neck cancer (HNC) and its treatment are associated with muscle weakness and considerable long-term comorbidity. The goal of this study was to determine whether skeletal muscle density (SMD) as quantified from pretreatment computed tomography (CT) scans will correlate with measures of function and strength prior to treatment in physical function in HNC patients. PATIENTS AND METHODS: A cross-sectional analysis was conducted on 90 HNC patients. SMD (myosteatosis vs. normal) was calculated from pretreatment CT scans using SliceOmatic software. Pretreatment physical function was assessed via handgrip strength (HGS), the timed up and go test (TUG), and the short physical performance battery (SPPB). Demographic, cancer, and social characteristics were also collected as confounders. Linear regression models assessed the association between myosteatosis and measures of physical function. RESULTS: The 90 patients were predominately White, male, former smokers with an average BMI of 28.7 ± 5.7 kg/m2. Among men, adjusted models indicate, as compared to those with normal muscle density, the total SPPB score of those with myosteatosis was 1.57 points lower (p = 0.0008), HGS was 0.85 kg lower (p = 0.73), and TUG took 1.34 s longer (p = 0.03). There were no differences in women. CONCLUSION: Myosteatosis is associated with physical function prior to treatment in HNC patients. Larger studies are needed to examine the importance of exercise programs prior to and during treatment to build lean mass and improve long-term prognosis in HNC.

Application of open port sampling interface mass spectrometry (OPSI-MS) to deuterium exchange as an aid for structural elucidation.

RATIONALE: Deuterium exchange has been demonstrated to provide additional information to accurate mass measurement and collision-induced dissociation on unknown chemical structures. An enhanced method for rapid deuterium exchange could make this technique more routine for structural elucidation. Open port sampling interface mass spectrometry (OPSI-MS) with an aprotic solvent offers a rapid method for performing deuterium incorporation. METHODS: Samples of standard drug molecules have been analysed by OPSI-MS directly from solids using a make-up flow of acetonitrile + 0.1% trifluoroacetic acid. The resultant spectra were compared with those obtained by OPSI-MS analysis of the samples dissolved in deuterium oxide (D2 O). Solutions of these molecules in acetonitrile/D2 O were analysed using an Atmospheric Solids Analysis Probe (ASAP) at different temperatures to compare the suitability of this technique. RESULTS: The number of exchangeable hydrogens was obtained through deuterium exchange using the OPSI source, although there was some incomplete exchange or back-exchange observed. Molecules with one to five exchangeable hydrogens were successfully analysed. ASAP analysis produced more complicated spectra with higher levels of incomplete or back-exchanged ions; this was more pronounced at higher temperatures. CONCLUSIONS: The use of OPSI provides a method for the rapid determination of the number of exchangeable hydrogens within a molecule. This yields useful information as an aid to the structural elucidation of unknowns. ASAP produces incomplete exchange and cannot be used for incorporation studies.

Effects of Preoperative Breathing Exercise on Postoperative Outcomes for Patients With Lung Cancer Undergoing Curative Intent Lung Resection: A Meta-analysis.

OBJECTIVE: To determine the benefits of preoperative breathing exercises on hospital length of stay (LOS), pneumonia, postoperative pulmonary complications (PPC), 6-minute walk distance (6MWD), forced expiratory volume in 1 second (FEV1), and health-related quality of life (HRQOL) in patients undergoing surgical lung cancer resection. DATA SOURCES: PubMed, EMBASE, Web of Science Core Collection, and Cochrane Central Register of Controlled Trials were comprehensively searched from inception to March 2021. STUDY SELECTION: Only studies including preoperative inspiratory muscle training (IMT) and/or breathing exercises compared with a nontraining control group were included. The meta-analysis was done using Cochrane software for multiple variables including LOS, pneumonia, PPC, 6MWD, FEV1, mortality, and HRQOL. DATA EXTRACTION: Two authors extracted the data of the selected studies. The primary outcomes were LOS and PPC. DATA SYNTHESIS: A total of 10 studies were included in this meta-analysis, 8 of which had both IMT and aerobic exercise. Pooled data for patients who performed preoperative breathing exercises, compared with controls, demonstrated a decrease in LOS with a pooled mean difference of -3.44 days (95% confidence interval [CI], -4.14 to -2.75; P<.01). Subgroup analysis also demonstrated that LOS was further reduced when breathing exercises were combined with aerobic exercise (χ2, 4.85; P=.03). Preoperative breathing exercises reduce pneumonia and PPCs with an odds ratio of 0.37 (95% CI, 0.18-0.75; P<.01) and 0.37 (95% CI, 0.21-0.65; P<.01), respectively. An increase in 6MWD of 20.2 meters was noted in those performing breathing exercises (95% CI, 9.12-31.21; P<.01). No significant differences were noted in FEV1, mortality, or HRQOL. CONCLUSIONS: Preoperative breathing exercises reduced LOS, PPC, and pneumonia and potentially improved 6MWD in patients undergoing surgical lung cancer resection. Breathing exercises in combination with aerobic exercise yielded greater reductions in LOS. Randomized controlled trials are needed to test the feasibility of introducing a preoperative breathing exercise program in this patient population.

Towards a generic method for ion chromatography/mass spectrometry of low-molecular-weight amines in pharmaceutical drug discovery and development.

RATIONALE: Low-molecular-weight amines are encountered in pharmaceutical analysis, e.g. as reactants in chemical syntheses, but are challenging to analyse using ultrahigh-performance liquid chromatography/mass spectrometry (UHPLC/MS) due to their high polarity causing poor retention. Ion chromatography/mass spectrometry (IC/MS) is an emerging technique for polar molecule analysis that offers better separation. A generic IC/MS method would overcome problems associated with using UHPLC/MS in drug discovery and development environments. METHODS: Amine standards were analysed using IC/MS with gradient elution (variety of column temperatures evaluated). An electrospray ionisation (ESI) quadrupole mass spectrometer was operated in positive ion polarity in scanning mode. The make-up flow composition was evaluated by assessing the performance of a range of organic modifiers (acetonitrile, ethanol, methanol) and additives (acetic acid, formic acid, methanesulfonic acid). The ESI conditions were optimised to minimise adduct formation and promote generation of protonated molecules. RESULTS: The performance attributes were investigated and optimised for low-molecular-weight amine analysis. Organic solvents and acidic additives were evaluated as make-up flow components to promote ESI, with 0.05% acetic acid in ethanol optimal for producing protonated molecules. The hydrogen bonding capability of amines led to abundant protonated molecule-solvent complexes; optimisation of source conditions reduced these, with collision-induced dissociation voltage having a strong effect. The detection limit was ≤1.78 ng for the amines analysed, which is fit-for-purpose for an open-access chemistry environment. CONCLUSIONS: This study demonstrates the value of IC/MS for analysing low-molecular-weight amines. Good chromatographic separation of mixtures was possible without derivatisation. Ionisation efficiency was greatest using a make-up flow of 0.05% acetic acid in ethanol, and optimisation of ESI source conditions promoted protonated molecule generation for easy determination of molecular weight.

On-line reaction monitoring by mass spectrometry, modern approaches for the analysis of chemical reactions.

The application of on-line mass spectrometry for direct analysis of chemical and other types of process continues to grow in importance and impact. The ability of the technique to characterize many aspects of a chemical reaction such as product and impurity formation, along with reactant consumption in a single experiment is key to its adoption and development. Innovations in ionization techniques and mass spectrometry instrumentation are enabling this adoption. An increasing range of ambient ionization techniques make on-line mass spectrometry applicable to a large range of chemistries. The academic development and commercialization of small footprint portable/transportable mass spectrometers is providing technology that can be positioned with any process under investigation. These developments, coupled with research into new ways of sampling representatively from both the condensed and gaseous phases, are positioning mass spectrometry as an essential technology for on-line process optimization, understanding and intelligent control. It is recognized that quantitative capability of mass spectrometry in this application can cause some resistance to its adoption, but research activities to tackle this limitation are on-going.

Multistate occupancy modeling improves understanding of amphibian breeding dynamics in the Greater Yellowstone Area.

Discerning the determinants of species occurrence across landscapes is fundamental to their conservation and management. In spatially and climatologically complex landscapes, explaining the dynamics of occurrence can lead to improved understanding of short- vs. long-term trends and offer novel insight on local vs. regional change. We examined the changes in occupancy for two species of anurans with different life histories over a decade using hundreds of wetland sites in Yellowstone and Grand Teton National Parks. To account for the joint dynamics of wetland drying and amphibian breeding, we adopted a multistate occupancy model as a means to investigate mechanistic relationships of observed occurrence patterns with climatological drivers of wetland hydrologic variability. This approach allowed us to decompose occupancy dynamics into habitat changes caused by wetland drying and amphibian breeding activity, conditional on available water and previous breeding state. Over our 10-yr time series, we observed considerable variability in climate drivers and the proportion of dry wetlands. Boreal chorus frogs (Pseudacris maculata) were more responsive to changes in wetland inundation status than Columbia spotted frogs (Rana luteiventris), as indicated by higher breeding colonization probabilities under favorable (wet) conditions. Both species had high probabilities of breeding persistence in permanently inundated wetlands with prior breeding. Despite the absence of multi-year drought in our time series, mechanistic relationships described here offer insights on how future climate variation may result in reduced and/or shifted occurrence patterns for pond-breeding anurans in the Greater Yellowstone Area. Further, our modeling approach may prove valuable in evaluating determinants of occurrence for other species that are dependent on wetlands or other dynamic habitats.

Regulating RNA Research and CRISPR Gene Drives to Combat Biosecurity Threats.

Recent technological breakthroughs in ribonucleic acid (RNA) research and the creation of synthetic gene drives using CRISPR/Cas9 have increased attention on the ethical and legal regulation of this field. RNA is now perceived as not merely a passive carrier of DNA information but especially through its propensity to mutate as a computation engine of cell biology, developmental biology and evolution. Synthetic Gene drives have been hailed as a potential strategy to reduce climate-change-mediated biosecurity threats such as spreading malaria and have attracted significant investment, with the Gates Foundation pledging US$75 million and the Defense Advanced Research Projects Agency awarding US$65 million. Calls for a global moratorium on RNA-mediated genetic engineering may overstate the potential risks of the developing technology, but form a background to the contest between "process"- and "product" -based approaches to regulation, the former purportedly favoured by the public and regulatory agencies and the latter favoured by the broad scientific community and corporate investors. At stake may be the democratic legitimacy of and equitable access to a technology that could be important to reduce the incidence of biosecurity threats both globally and in Australia.

Coupling and optimisation of online nuclear magnetic resonance spectroscopy and mass spectrometry for process monitoring to cover the broad range of process concentration.

Real time online monitoring of chemical processes can be carried out by a number of analytical techniques, including optical and vibrational spectroscopies, nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). As each technique has unique advantages and challenges, combinations are an attractive option. The combination of a 500-MHz 1 H NMR and a small footprint mass spectrometer to monitor a batch reaction at process concentration was investigated. The mass spectrometer was coupled into the flow path of an online reaction monitoring NMR. Reaction mixture was pumped from a 100-ml vessel to an NMR flow tube before returning to the vessel. Small aliquots were diverted into a sampling make-up flow using an active flow splitter and passed to the mass spectrometer. Advantages of the combination were observed. 1 H NMR was ideal for quantitation of high level components, whereas MS showed a greater capability for detecting those at low level. In preliminary experiments MS produced a limited linear relationship with concentration (0.02% to 2% relative concentration, 0.01 mg/ml-1.25 mg/ml), because of signal saturation at the higher concentrations. NMR was unable to detect components below 0.1% relative to concentration maximum. Optimisation of sample transfer to the MS extended the linearity to 10% relative to the concentration maximum. Therefore, the combination of online NMR and MS allows both qualitative and quantitative analysis of reaction components over the full process range. The application of the combination was demonstrated by monitoring a batch chemical reaction and this is described. Copyright © 2016 John Wiley & Sons, Ltd.

Applicability of gas chromatography/quadrupole-Orbitrap mass spectrometry in support of pharmaceutical research and development.

RATIONALE: Gas chromatography/mass spectrometry (GC/MS) is a fundamental tool used to identify impurities throughout the active pharmaceutical ingredients development process. The coupling of Orbitrap mass spectrometry with GC marks an exciting advance in capability for GC/MS, offering a significant step change in resolving power, mass accuracy, sensitivity and linear range. METHODS: A range of pharmaceutically relevant samples representing typical starting materials has been investigated with particular reference to impurity identification. The mass accuracy in Electron Ionisation (EI) and Chemical Ionisation (CI) was investigated for impurity identification. The linearity and mass accuracy over a wide dynamic range were evaluated. The number of scans obtained across chromatographic peaks was assessed at various resolution settings from 15,000 to 120,000 (full width at half maximum (FWHM) at m/z 200). RESULTS: All the accurate mass measurements for impurities were within <1 ppm of the theoretical m/z value. The scan speed at the highest resolution produced 11 scans across the peak, and the mass accuracy for all scans was consistently <1 ppm - sufficient for impurity investigations and quantitative analysis. Linearity was demonstrated for N,N,N'-trimethylethylenediamine over a concentration range of 0.0001 to 0.1250 µg/mL (w/v) with a correlation coefficient R(2) = 0.9996 and mass accuracy across all concentrations at <1.1 ppm. CONCLUSIONS: GC/Orbitrap MS has been evaluated for both qualitative and quantitative analysis of typical pharmaceutical precursors and impurities. Accurate mass measurement across a wide dynamic range, linearity and the ability to identify impurities in EI and CI illustrate that this instrument is a powerful tool of great benefit to pharmaceutical analysis.