Quantitative analysis of intracellular nucleoside triphosphates and other polar metabolites using ion pair reversed-phase liquid chromatography coupled with tandem mass spectrometry

Abstract

Simultaneous, quantitative determination of intracellular nucleoside triphosphates and other polar metabolites using liquid chromatography with electrospray ionization tandem mass spectrometry (LC-MS/MS) represents a bioanalytic challenge because of charged, highly hydrophilic analytes presented at a large concentration range in a complex matrix. In this study, an ion pair LC-MS/MS method using triethylamine (TEA)-hexafluoroisopropanol (HFIP) ion-pair mobile phase was optimized and validated for simultaneous and unambiguous determination of 8 nucleoside triphosphates (including ATP, CTP, GTP, UTP, dATP, dCTP, dGTP, and dTTP) in cellular samples. Compared to the the less volatile ion-pair reagent, triethylammonium acetate (100mM, pH 7.0), the combination of HFIP (100mM) and TEA (8.6mM) increased the MS signal intensity by about 50-fold, while retaining comparable chromatographic resolution. The isotope-labeled internal standard method was used for the quantitation. Lower limits of quantitation were determined at 0.5nM for CTP, UTP, dATP, dCTP, and dTTP, at 1nM for ATP, and at 5nM for GTP and dGTP. The intra- and inter-day precision and accuracy were within the generally accepted criteria for bioanalytical method validation (<15%). While the present method was validated for the quantitation of intracellular nucleoside triphosphates, it had a broad application potential for quantitative profiling of nucleoside mono- and bi-phosphates as well as other polar, ionic metabolic intermediates (including carbohydrate derivatives, carboxylic acid derivatives, co-acyl A derivatives, fatty acyls, and others) in biological samples.

Keywords: Deoxyribonucleoside triphosphate (dNTP); Ion pair chromatography; LC-MS/MS; Metabolomics; Ribonucleoside triphosphate (NTP).

Figure 1

Product ion mass spectra of dATP, dCTP, dGTP, dTTP, ATP, CTP, GTP, and UTP

Figure 2

(A – C) Extracted ion chromatograms of a standard mixture of dATP, dCTP, dGTP, dTTP, ATP, CTP, GTP, and UTP (5 μM in aqueous solution), using the traditional TEAA ion-paring mobile phase (pH 7.0) with different TEAA concentrations: 5 mM (A), 25 mM (B), and 100 mM (C). (D) Extracted ion chromatogram of a standard mixture of dATP, dCTP, dGTP, dTTP, ATP, CTP, GTP, and UTP (1 μM in aqueous solution), using the TEAA ion-paring mobile phase (pH 7.0) with the TEAA concentration at 100 mM. Chromatographic separation was performed on an Atlantis T3 (2.1× 100 mm, 3 μm) column, under a gradient mobile phase consisting of mobile phase A (TEAA, pH 7.0) and mobile phase B (10% acetonitrile in mobile phase A), with gradient B% (min) as 0 (0) → 60 (6.0) → 0 (6.1) → 0 (10) at a flow rate of 0.5 mL/min.

Figure 3

(A – D) Extracted ion chromatograms of a standard mixture of dATP, dCTP, dGTP, dTTP, ATP, CTP, GTP, and UTP (1 μM in aqueous solution), using the TEA-HFIP ion-paring mobile phase with TEA at 8.6 mM and HFIP at different concentrations: 5 mM (A), 20 mM (B), 50 mM (C), and 100 mM (D). (E) Extracted ion chromatograms of dATP, dCTP, dGTP, dTTP, ATP, CTP, GTP, and UTP (at 1 μM in aqueous solution), using a mobile phase containing HFIP only (100 mM, adjusted pH to 8.3 by NH4OH). Chromatographic separation was achieved on an Atlantis T3 (2.1× 100 mm, 3 μm) column, under a gradient mobile phase consisting of mobile phase A (TEA + HFIP or HFIP only) and mobile phase B (10% acetonitrile in mobile phase A), with gradient B% (min) as 0 (0) → 10 (6.0) → 0 (6.1) → 0 (10) at a flow rate of 0.5 mL/min.

Figure 4

Extracted ion chromatograms of a standard mixture of dATP, dCTP, dGTP, dTTP, ATP, CTP, GTP, and UTP (200 nM in aqueous solution), using the TEA-HFIP ion-paring mobile phase with different gradient slope of organic solvent. Chromatographic separation was performed on an Atlantis T3 (2.1× 100 mm, 3 μm) column, under a gradient mobile phase consisting of mobile phase A (8.6 mM TEA + 100 mM HELP) and mobile phase B containing 100% (A), 50% (B), or 10% (C) acetonitrile in mobile phase A. The gradient program was set as B% (min): 0(0) → 10 (6.0) → 0 (6.1) → 0 (10).

Figure 5

Multiple-reaction monitoring (MRM) chromatograms of dATP (490.0 > 392.1), dCTP (466.0 > 367.9), dGTP (506.0 >256.9), dTTP (480.9 > 383.1), ATP (506.0 > 272.9), CTP (482.0 > 384.1), GTP (522.0 > 424.0), and UTP (483.0 > 384.9) for: (A1-A8) a standard mixture (containing 200 nM of ATP, CTP, GTP, UTP, dATP, dCTP, dGTP and dTTP) in aqueous solution; (B1-B8) a cellular extract sample diluted 20-fold with the mobile phase A (8.6 mM TEA + 100 mM HFIP); and (C) the undiluted cellular extract sample (containing ~10 million cells). Chromatographic separation was achieved on an Atlantis T3 (2.1× 100 mm, 3 μm) column, under gradient mobile phase consisting of mobile phase A (8.6 mM TEA + 100 mM HFIP) and mobile phase B (10% acetonitrile in mobile phase A), with gradient B% (min) as 0 (0) → 10 (6.0) → 0 (6.1) → 0 (10) at a flow rate of 0.5 mL/min.

Figure 6

(A and B) Intracellular concentrations of dNTPs and NTPs, and (C) intracellular dNTP/NTP ratios, in H23 non-small cell lung cancer cells with normal and knocked-down expression of RRM1/RRM2.

Figure 7

Partial least square discriminant analysis (PLS-DA) score plots indicating discrimination between the parental A549 cells and A549-Snail cells (with overexpression of Snail). Symbols (▲ or +) represent three biological replicates. Ellipses represent 95% confidence intervals.