Ion-pair Reversed-phase Liquid Chromatography Research Articles

Vaccine & Immunotherapy Summit October 14-15, 2020 | Brussels, Belgium

Exposure to reactive oxygen species can result in formation of oxidized nucleotides which increase the frequency of mutations, DNA damage, and cell death. MutT Homolog 1 (MTH1) is an enzyme that catalyzes removal of pyrophosphate from the highly mutagenic oxidized nucleoside triphosphate 8-oxo-2'-deoxyguanosine-5'-triphosphate (8-oxo-dGTP). Interest in MTH1 as a potential new target for cancer therapy surged due to reports of MTH1 inhibitors causing DNA damage and inducing cell death in cancer cells. However, questions have been raised about MTH1 target validation. One critical piece of information that is currently lacking is a quantitative understanding of the levels of 8-oxo-dGTP in cancer cells and the effect of MTH1 inhibition on them. In this study, we developed a sensitive and selective method to simultaneously measure the intracellular concentrations of 8-oxo guanosine nucleotides and their unmodified counterparts using liquid chromatography coupled to tandem mass spectrometry (LC-MS/ MS). An ion-pairing reversed phase liquid chromatography method using dimethylhexylamine was employed to quantify the highly polar analytes. The method was validated fit for purpose using a combination of authentic standards and cell samples. The intracellular oxo-nucleotide concentrations in human bone osteosarcoma cell line U2OS were determined using the method. We observed very low levels of 8-oxo nucleotides in untreated and hydrogen peroxide treated cells and MTH1 knockdown in these cells had minimal if any effect on 8-oxo nucleotide levels. This method will allow for a more comprehensive understanding of oxidized nucleotide detoxification pathways and MTH1 as a target for cancer therapy.

Evaluation of hydrophilic interaction liquid chromatography, capillary zone electrophoresis and drift tube ion-mobility quadrupole time of flight mass spectrometry for the characterization of phosphodiester and phosphorothioate oligonucleotides

Oligonucleotide-based medicines that can modulate gene expression have numerous potential applications in targeted therapies. Most of the commercialized therapeutic oligonucleotides are chemically modified to increase their in vivo lifetime. In this work, we studied poly-deoxy(thymidylic) acids (dT) and modified phosphorothioate oligonucleotides (PS). Several analytical techniques, including ion-pair reverse phase liquid chromatography, are described in the literature to assess their quality but most of them present significant drawbacks.In the present study, dT and PS mixtures were analyzed by hydrophilic interaction liquid chromatography (HILIC) and capillary zone electrophoresis (CZE) coupled to ultraviolet detection. In HILIC, the selectivities of three types of stationary phases (dihydroxypropane, phosphorylcholine and amide) were compared. Optimal conditions were determined and consisted of an amide stationary phase with a mobile phase made up of water, acetonitrile and 15 mM ammonium acetate (pH 5.5). In those conditions, high resolving power and good repeatability were achieved. In CZE, the effect of the background electrolyte (BGE), its pH and concentration were evaluated. A BGE made up of 300 mM ammonium acetate adjusted to pH 6.0 was selected. Finally, the two techniques were compared in terms of selectivity, repeatability and peak efficiency.In the second part of the study, HILIC and CZE were both coupled to a drift-tube ion-mobility quadrupole time-of-flight MS detector (DTIMS-QTOF) to assess the added value of this coupling for oligonucleotide characterization. Indeed, by using the measured collision cross section (CCS), the evaluation of the number of nucleotides was performed. Looking across the results, HILIC and CZE coupled to DTIMS-QTOF can be considered as promising tools for the quality control of oligonucleotides.

Development of an IPRP-LC-MS/MS method to determine the fate of intracellular thiamine in cancer cells

Understanding the mechanisms underlying cancer cell survival is critical toward advancing drug discovery efforts in this field. Supplemental vitamins have been proposed to play a role in cancer cell metabolism because the increased supply of nutrients is thought to provide cofactors supporting the higher metabolic rate of cancer cells. Particularly, the role of thiamine (vitamin B1) in many biochemical pathways that supports cancer cell metabolism has been investigated. Consequently, the analysis of thiamine and its derivatives in a manner that reflects its dynamic response to genetic modification and pathophysiological stimuli is essential. In this work, we developed a mass spectrometry based-analytical method to track metabolites derived from stable isotope tracers for a better understanding of the metabolic fate of thiamine in cancer cells. This method used ion-pair reversed phase liquid chromatography to simultaneously quantify underivatized thiamine, thiamine monophosphate (TMP) and thiamine pyrophosphate (TPP) in cells. Hexylamine was used as an ion-pairing agent. The method was successfully validated for accuracy, precision and selectivity in accordance with U.S. FDA guidance. Furthermore, the method was then applied for the determination of thiamine and its derivatives with stable isotope labeling to explore the metabolic fate of intracellular thiamine in cancer cells. The finding shows that thiamine is rapidly converted to TPP however, the TPP does not return to thiamine. It appears that TPP may be utilized for other purposes rather than simply being an enzyme cofactor, suggesting unexplored roles for thiamine in cancer.

Analysis of oligonucleotides by ion-pair reversed-phase liquid chromatography coupled with positive mode electrospray ionization mass spectrometry.

Oligonucleotides are usually analyzed by ion-pair reversed-phase liquid chromatography (IP-RPLC) coupled with negative mode electrospray ionization mass spectrometry (ESI-MS) due to their highly negative charged phosphodiester backbones. Herein, the signal suppression effect of triethylamine (TEA) adducts caused the ion-pair reagent TEA/hexafluoroisopropanol (HFIP) is greatly alleviated after improving the in-source energy in positive mode ESI-MS. This strategy is applied for different RNA sequencing through analyzing their formic acid hydrolysates via IP-RPLC MS. Comparing with negative ion mode, we demonstrate that IP-RPLC MS analysis in positive ion mode is more suitable for RNA sequencing with fewer contaminant interferences. Finally, simultaneous online separation and detection of oligonucleotides and protein digests are achieved in positive ion mode IP-RPLC MS analysis with little interference to each other.

Oligonucleotide analysis by hydrophilic interaction liquid chromatography-mass spectrometry in the absence of ion-pair reagents

Improving our understanding of nucleic acids, both in biological and synthetic applications, remains a bustling area of research for both academic and industrial laboratories. As nucleic acids research evolves, so must the analytical techniques used to characterize nucleic acids. One powerful analytical technique has been coupled liquid chromatography – tandem mass spectrometry (LC–MS/MS). To date, the most successful chromatographic mode has been ion-pairing reversed-phase liquid chromatography. Hydrophilic interaction liquid chromatography (HILIC), in the absence of ion-pair reagents, has been investigated here as an alternative chromatographic approach to the analysis of oligonucleotides. By combining a mobile phase system using commonly employed in liquid chromatography-mass spectrometry (LC–MS) - i.e., water, acetonitrile, and ammonium acetate - and a new, commercially available diol-based HILIC column, high chromatographic and mass spectrometric performance for a wide range of oligonucleotides is demonstrated. Particular applications of HILIC-MS for the analysis of deoxynucleic acid (DNA) oligomers, modified and unmodified oligoribonucleotides, and phosphorothioate DNA oligonucleotides are presented. Based on the LC–MS performance, this HILIC-based approach provides an attractive, sensitive and robust alternative to prior ion-pairing dependent methods with potential utility for both qualitative and quantitative analyses of oligonucleotides without compromising chromatographic or mass spectrometric performance.

Retention of Fluorescent Amino Acid Derivatives in Ion-pairing Reversed-phase Liquid Chromatography.

Recent studies have shown that pillar array columns enable fast and quantitative analysis of amino acids. However, hydrophilic amino acids still cannot be retained on pillar array columns since they have limited retention ability. Ion-pairing liquid chromatography is a promising means of increasing analyte retention. In this study, the effects of ion-pairing reagents on the retention of eight hydrophilic amino acids (histidine, asparagine, glutamine, serine, arginine, aspartic acid, glycine, and glutamic acid) derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) under reversed-phase conditions on a conventional ODS column were studied. Among the ion-pairing reagents investigated, tetraheptylammonium bromide proved to be the most effective for increasing analyte retention. With a mobile phase consisting of 20 mM citrate buffer (pH 6.3)-acetonitrile (100:40, v/v) and 2 mM tetraheptylammonium bromide, the retention times of the eight NBD-amino acids-except NBD-arginine-were longer than 19.4 min, which was the retention time of NBD-valine when eluted without an ion-pairing reagent. As NBD-valine was well retained on pillar array columns, the chromatographic conditions may thus be applied in the analysis of hydrophilic amino acids using pillar array columns.

Monolithic alkylsilane column: A promising separation medium for oligonucleotides by ion-pair reversed-phase liquid chromatography

In this paper, a monolithic octadecylsilane column and particle-packed octadecylsilane columns were used to investigate the retention behaviors of oligonucleotides by ion-pair reversed-phase liquid chromatography (IP-RPLC). Results showed that, with same base composition, hairpin oligonucleotides always had weaker retention than corresponding random coil oligonucleotides on the monolithic column, but not on the particle-packed columns. In addition, the linear correlation between the retention factor k of oligonucleotides and the reciprocal of temperature (1/T), especially for hairpins, was relatively weaker on the particle-packed columns, as compared to the correlation on the monolithic column. The correlation between k and 1/T became weaker with decreasing particle size of the particle-packed columns. Moreover, results revealed that the overall retention order on the particle-packed column with small particles (3 μm) differed greatly from that on the monolithic column. In contrast, the retention order on the 10 μm particle-packed column was very close to that on the monolithic column. From the above, we inferred that oligonucleotides could keep their primary conformations unchanged when passing through the monolithic column, attributed to the special pore structures of the monolith. However, the conformations of oligonucleotides were suppressed or even destroyed when oligonucleotides passed through the particle-packed columns. This because the narrow and tortuous channels created by the stacked stationary phase particles could lead to more complex and unequable retention behaviors. Therefore, the monolithic column exhibited better retention regularity for oligonucleotides of secondary structure especially for hairpins than the particle-packed columns. It is noteworthy that the monolith-based IP-RPLC opens an intriguing prospect in accurately elucidating the retention behaviors of oligonucleotides.

Liquid chromatography–tandem mass spectrometry method for the analysis of N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine, a biocidal disinfectant, in dairy products

A novel and reliable method to quantify residual levels of N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine in dairy products using ion-pairing reversed-phase liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed and fully validated. Sample extraction was done with salting-out technique using acetonitrile and sodium chloride. For LC–MS/MS, the analyte was detected using positive electrospray ionization (ESI+) and two multiple reaction monitoring (MRM) transitions were monitored. The method was validated in the 5–150 µg kg−1 range using total error approach. Thus, performance criteria of the method were evaluated. Relative standard deviations for trueness and precision were lower than 10%; with the exception of hard pressed cheese at 5 µg kg−1 for precision. The limit of quantification (LOQ) was around 5–7 µg kg−1 depending on the matrix of interest. The method was successfully applied to accurately quantify N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine in 146 various dairy products with a maximum contamination level of 225 µg kg−1 in cheese.

Degradation product characterization of therapeutic oligonucleotides using liquid chromatography mass spectrometry.

Synthetic antisense phosphorothioate oligonucleotides (PS) have undergone rapid development as novel therapeutic agents. The increasing significance of this class of drugs requires significant investment in the development of quality control methods. The determination of the many degradation pathways of such complex molecules presents a significant challenge. However, an understanding of the potential impurities that may arise is necessary to continue to advance these powerful new therapeutics. In this study, four different antisense oligonucleotides representing several generations of oligonucleotide therapeutic agents were evaluated under various stress conditions (pH, thermal, and oxidative stress) using ion-pairing reversed-phase liquid chromatography tandem mass spectrometry (IP-RPLC-MS/MS) to provide in-depth characterization and identification of the degradation products. The oligonucleotide samples were stressed under different pH values at 45 and 90°C. The main degradation products were observed to be losses of nucleotide moieties from the 3'- and 5'-terminus, depurination, formation of terminal phosphorothioates, and production of ribose, ribophosphorothioates (Rp), and phosphoribophosphorothioates (pRp). Moreover, the effects of different concentrations of hydrogen peroxide were studied resulting in primarily extensive desulfurization and subsequent oxidation of the phosphorothioate linkage to produce the corresponding phosphodiester. The reaction kinetics for the degradation of the oligonucleotides under the different stress conditions were studied and were found to follow pseudo-first-order kinetics. Differences in rates exist even for oligonucleotides of similar length but consisting of different sequences. Graphical abstract Identification of degradation products across several generations of oligonucleotide therapeutics using LC-MS.

A novel strategy for retention prediction of nucleic acids with their sequence information in ion-pair reversed phase liquid chromatography

In this work, retention behaviors of oligonucleotides and double-stranded deoxyribonucleic acids (dsDNAs) have been investigated in ion-pair reversed-phase liquid chromatography (IP-RPLC). We demonstrated that classic linear solvent strength (LSS) model is applicable for describing isocratic retention of oligonucleotides and dsDNAs, which indicated that nucleic acids share the similar retention mechanism as other common small molecules in IP-RPLC. The separation of nucleic acids in IP-RPLC is driven by both hydrophobic and electrostatic interactions. We defined the parameter lnkw/S obtained from LSS model in IP-RPLC as chromatographic hydrophobic and electrostatic interaction index (CHEI). CHEI of a nucleic acid has been revealed to correlate well with its gradient retention time. Notably, we proposed a strategy for retention prediction based on CHEI and base sequence information of nucleic acids. Corresponding to base locus, each base sequence is converted to a featured locus vector consisting of zeros and ones. CHEI prediction models were established by support vector regression (SVR) algorithm with locus vectors. Predicted CHEI values have been applied to predict retention times under desired gradient elution runs. This protocol is easy to grasp and worth pursuing further development for more precise retention prediction performance of nucleic acids in IP-RPLC.

Liquid Chromatography Separation and Mass Spectrometry Detection of Silver-Lipoate Ag29(LA)12 Nanoclusters: Evidence of Isomerism in the Solution Phase.

Evidence for the existence of condensed-phase isomers of silver-lipoate clusters, Ag29(LA)12, where LA = (R)-α lipoic acid, was obtained by reversed-phase ion-pair liquid chromatography with in-line UV-vis and electrospray ionization (ESI)-MS detection. All components of a raw mixture were separated according to surface chemistry and increasing size via reversed-phase gradient HPLC methods and identified by their corresponding m/z ratio by ESI in the negative ionization mode. Aqueous and methanol mobile-phase mixtures, each containing 400 mM hexafluoroisopropanol (HFIP)-15 mM triethylamine (TEA), were employed to facilitate the interaction between the clusters and stationary phase via formation of ion-pairs. TEA-HFIP (triethylammonium-hexafluoroisopropoxide) had been shown to provide superior chromatographic peak shape and mass spectral signal compared with alternative modifiers such as TEAA (triethylammonium-acetate) for analysis of oligonucleotide samples. Liquid chromatographic separation prior to mass spectrometry detection facilitated sample analysis by production of simplified mass spectra for each eluting cluster species and provided insight into the existence of at least two major solution-phase isomers of Ag29(LA)12. UV-vis detection in-line with ESI analysis provided independent confirmation of the existence of the isomers and their similar electronic structure as judged from their identical optical spectra in the 300-500 nm range. [Diastereomerism provides a possible interpretation for the near-equal abundance of the two forms, based on a structurally defined nonaqueous homologue.].

Development and validation of a bioanalytical method for quantification of LNA-i-miR-221, a 13-mer oligonucleotide, in rat plasma using LC–MS/MS

LNA-i-miR-221, a 13-mer oligonucleotide, is a new miR-221 inhibitor that could be used as a novel drug for multiple myeloma. Herein, an ion-pair reversed phase liquid chromatography–tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the quantification of LNA-i-miR-221 in rat plasma. Plasma samples were prepared with an initial phenol/chloroform/isoamyl alcohol liquid–liquid extraction followed by a solid phase extraction. Chromatographic separation was performed with a gradient system on a HALO C18 column using hexafluoro-2-propanol/triethylamine buffer and methanol as mobile phase at a flow rate of 0.4 mL/min. Under these conditions LNA-i-miR-221 and the analogue internal standard are co-eluted at 1.2 min. The detection was carried out in multiple reaction monitoring (MRM) mode using a negative electrospray ionization (ESI) interface. The assay showed a good linearity within the calibration range 10–10000 ng/mL. The precision, accuracy, and recovery values were found to be <15% (<20% at LLOQ), 100 ± 15%, and 97.6–103.7%, respectively. This method was successfully applied to measure the concentrations of LNA-i-miR-221 in plasma samples following the intravenous administration during a 4-week toxicity study in rats.

Determination of reversed-phase high performance liquid chromatography based octanol-water partition coefficients for neutral and ionizable compounds: Methodology evaluation

Reversed-phase liquid chromatography (RPLC) based octanol-water partition coefficient (logP) or distribution coefficient (logD) determination methods were revisited and assessed comprehensively. Classic isocratic and some gradient RPLC methods were conducted and evaluated for neutral, weak acid and basic compounds. Different lipophilicity indexes in logP or logD determination were discussed in detail, including the retention factor logkw corresponding to neat water as mobile phase extrapolated via linear solvent strength (LSS) model from isocratic runs and calculated with software from gradient runs, the chromatographic hydrophobicity index (CHI), apparent gradient capacity factor (kg’) and gradient retention time (tg). Among the lipophilicity indexes discussed, logkw from whether isocratic or gradient elution methods best correlated with logP or logD. Therefore logkw is recommended as the preferred lipophilicity index for logP or logD determination. logkw easily calculated from methanol gradient runs might be the main candidate to replace logkw calculated from classic isocratic run as the ideal lipophilicity index. These revisited RPLC methods were not applicable for strongly ionized compounds that are hardly ion-suppressed. A previously reported imperfect ion-pair RPLC method was attempted and further explored for studying distribution coefficients (logD) of sulfonic acids that totally ionized in the mobile phase. Notably, experimental logD values of sulfonic acids were given for the first time. The IP-RPLC method provided a distinct way to explore logD values of ionized compounds.

Liquid Chromatographic Analysis of α-Dicarbonyls Using Girard-T Reagent Derivatives.

The measurement of α-dicarbonyls and other degradation products of sugars has become important in view of their toxicity. Although there are several methods used for their analysis, most require long reaction times to form UV absorbing or fluorescent derivatives and the nonpolar nature of commonly used derivatives necessitates relatively high concentrations of organic solvents for elution in reverse phase liquid chromatography. The present method describes the use of Girard-T reagent in a simple, one step derivatization of α-dicarbonyls and conjugated aldehydes and analysis using ion-pair reverse phase liquid chromatography. The limit of detection was in the range of 0.06-0.09 μM (4-12 ng/mL) for glyoxal, methylglyoxal, 3-deoxyglucosone and 5-hydroxymethylfurfural with good linear response and reproducibility using UV detection. The hydrazone derivatives were stable for several days in solution. The method was used to study degradation of several sugars and quantification of the target α-dicarbonyls and 5-hydroxymethylfurfural in several soft drinks.

Novel ribonuclease activity of cusativin from Cucumis sativus for mapping nucleoside modifications in RNA.

A recombinant ribonuclease, cusativin, was characterized for its cytidine-specific cleavage ability of RNA to map chemical modifications. Following purification of native cusativin protein as described before (Rojo et al. Planta 194:328, 17), partial amino acid sequencing was carried out to identify the corresponding protein coding gene in cucumber genome. Cloning and heterologous expression of the identified gene in Escherichia coli resulted in successful production of active protein as a C-terminal His-tag fusion protein. The ribonuclease activity and cleavage specificity of the fusion protein were confirmed with a variety of tRNA isoacceptors and total tRNA. Characterization of cusativin digestion products by ion-pairing reverse-phase liquid chromatography coupled with mass spectrometry (IP-RP-LC-MS) analysis revealed cleavage of CpA, CpG, and CpU phosphodiester bonds at the 3'-terminus of cytidine under optimal digestion conditions. Ribose methylation or acetylation of cytosine inhibited RNA cleavage. The CpC phosphodiester bond was also resistant to cusativin-mediated RNA cleavage; a feature to our knowledge has not been reported for other nucleobase-specific ribonucleases. Here, we demonstrate the analytical utility of such a novel feature for obtaining high-sequence coverage and accurate mapping of modified residues in substrate RNAs. Graphical abstract Cytidine-specific novel ribonuclease activity of cusativin.

Characterization of Apolipoprotein C3 (Apo C3) LNA/DNA Impurities and Degradation Products by LC-MS/MS.

Apolipoprotein C3 (Apo C3) LNA/DNA gapmer was evaluated under various stress and formulation conditions for the purpose of its development as a potential biotherapeutic for low density lipoprotein (LDL) lowering. Using ion-pairing (IP) reversed-phase (RP) liquid chromatography ultra-high resolution (UHR) tandem mass spectrometry (IP-RPLC-MS/MS), a combination of accurate mass measurements and collision-induced dissociation enabled in-depth characterization of Apo C3 LNA/DNA oligonucleotide, in particular the inherent impurities following synthesis and degradation products after exposure to stress conditions. In this study, oligonucleotide samples were stressed under different pH and UV exposure conditions. The primary impurities in Apo C3 LNA/DNA were losses of nucleotide moieties from both the 5'- and 3'-terminus leading to n-1, n-2, etc. species. Desulfurization and depurination were observed in Apo C3 LNA/DNA after a week under UV light stress conditions at low pH. Guanine oxidation and dimerization were the primary degradation products detected under UV light exposure for 1week at high pH. The effect of antioxidants on the levels of these degradation products was evaluated under neutral pH conditions. In the presence of all antioxidants, levels of guanine oxidation and desulfurization under tested conditions were the same as those in the unstressed sample, except for sodium ascorbate. The thorough understanding of the Apo C3 LNA/DNA oligonucleotide structure, its impurities, and degradation products laid the foundation for the successful formulation development of this novel biotherapeutic modality.

Quantitative In Silico Analysis of Retention of Nitrobenzofurazan-Amino Acids in Reversed-Phase Ion-Pair Liquid Chromatography.

The retention mechanisms of nitrobenzofurazan (NBD)-amino acids in reversed-phase ion-pair liquid chromatography were quantitatively analyzed in silico The most contributed interaction for the retention is the Lewis acid-base interaction between an aromatic ring of NBD-amino acids and hydroxyl-group hydrogen of tetra-butyl-ammonium hydroxide coated on the hydrophobic phase. Solvent effects significantly improved the relation between the calculated molecular interaction (MI) energy values using a molecular mechanics program and log k values measured in chromatography. The correlation coefficient between the calculated MI energy values and the log k values was>0.95.

Determination of Trace Amounts of Aluminum in Environmental and Biological Fluids by Reversed-Phase Ion-Pair Liquid Chromatography with Fluorometric Detection Using 5-Sulfoquinoline-8-ol

Determination of Trace Amounts of Aluminum in Environmental and Biological Fluids by Reversed-Phase Ion-Pair Liquid Chromatography with Fluorometric Detection Using 5-Sulfoquinoline-8-ol

Reduced molecular size and altered disaccharide composition of cerebral chondroitin sulfate upon Alzheimer's pathogenesis in mice.

Alzheimer's disease (AD) is a progressive disorder leading to cognitive impairment and neuronal loss. Cerebral extracellular accumulation and deposition of amyloid ß plaques is a pathological hallmark of AD. Chondroitin sulfate (CS) is an extracellular component abundant in the brain. CS is a sulfated glycosaminoglycan covalently attached to a core protein, forming chondroitin sulfate proteoglycan. The structure of CS is heterogeneous with sulfation modification and elongation of the chain. The structural diversity of CS allows it to play various roles in the brain. Increasing evidence has shown that CS promotes aggregation of amyloid ß peptides into higher-order species such as insoluble amyloid ß fibrils. Difficulties in the structural analysis of brain CS, as well as its heterogeneity, limit the study of potential roles of CS in AD pathology. Here we established a microanalysis method with reversed-phase ion-pair high performance liquid chromatography and found that CS in the brains of Tg2576 AD model mice show a lower molecular size and an increased ratio of CS-B motif di-sulfated disaccharide. Our findings provide insight into the structural changes of cerebral CS upon Alzheimer's pathogenesis.

Phenotyping hepatocellular metabolism using uniformly labeled carbon-13 molecular probes and LC-HRMS stable isotope tracing

Metabolite stable isotope tracing is a powerful bioanalytical strategy that has the potential to unravel phenotypic markers of early pharmaceutical efficacy by monitoring enzymatic incorporation of carbon-13 atoms into targeted pathways over time. The practice of probing biological systems with carbon-13 labeled molecules using broad MS-based screens has been utilized for many years in academic laboratories but has had limited application in the pharmaceutical R&D environment. The goal of this work was to establish a LCMS analytical workflow that was capable of monitoring carbon-13 isotope changes in glycolysis, the TCA and urea cycles, and non-essential amino acid metabolism. This work applies a standardized protein precipitation with 80% cold methanol and two distinct reverse-phase ion-pair liquid chromatography methods coupled to either a positive- or negative-ion mode high-resolution accurate mass spectrometry screening method. The data herein combines thousands of single-point peak integrations into a novel metabolite network map as a visualization aid to probe and monitor stable isotope incorporation in murine hepatocytes using uniformly labeled 13C6 glucose, 13C3 lactate, and 13C5 glutamine. This work also demonstrates that nitrogen metabolism may have a large influence on the TCA cycle and gluconeogenic carbon fluxes in hepatocyte cell culture.