Ion-pair HPLC Method Research Articles

Optimization of ion-pairing HPLC method for mutual separation of rare earth elements: unveiling the “diad-effect” appraisal utilizing periodic variations in their properties

A new ion-pairing HPLC method has been developed for the analytical separation of rare earth elements (REEs), utilizing the unique “diad-effect” of the lanthanides. A chemometric approach was used to optimize the separation process, examining the impact of mobile phase pH and ion-pairing reagent concentration on resolution and elution time. A triple gradient elution program was proposed, involving changes in the pH of the mobile phase from 3.5 to 4.5, the ion-pairing reagent concentration from 25 mM to 0.05 M, and the mobile phase complexing additive (hydroxyisobutyric acid) concentration from 0.05 M to 0.5 M. With the proposed method, all REEs, including pairs that are typically difficult to separate (Pr–Nd, Eu–Gd, and Dy–Y), were effectively separated in less than eight minutes. The method validation conducted following ICH guidelines showed accurate results, with an accuracy of 99–108% across a concentration range of 50–150% of the target concentration for REEs. It also exhibited good linearity (0.9998–0.9992) across a concentration range of 5–15 µg/mL for REEs and a detection limit of 0.27–0.98 µg/mL for the various species. The proposed HPLC method proved reliable and successfully separated a commercial sample of yttrium-group oxides.

Development and Validation of an Ion-Pair HPLC-UV Method for the Quantitation of Quinoline and Indoloquinoline Alkaloids in Herbal and Pharmaceutical Antimalarial Formulations

Quinine- and cryptolepine-based antimalarials serve as valuable alternatives to artemisinin-based combination therapies (ACTs) in Ghana. Their use, however, is associated with adulteration and substandard quality challenges. An HPLC method targeting quinoline and indoloquinoline antimalarial alkaloids was developed, validated, and applied to evaluate herbal and pharmaceutical antimalarial formulations (HPAFs) and starting materials (APIs). The separation/quantitation of the alkaloids (including quinine, quinidine, cinchonine, cinchonidine, dihydroquinine, dihydroquinidine, and cryptolepine) was achieved on a Zorbax SB-CN column (250 mm × 4.6 mm, 5 μm), with an isocratic elution system of methanol: trifluoroacetic acid (0.1%, v/v) (15 : 85, v/v) at 1.5 mL/min and 223 nm. Method validation was according to ICH Q2(R1) guidelines. It was then used to assess the quality of APIs (n = 3) and HPAFs (n = 44) including quinine-based pharmaceutical antimalarial formulations (QBPAFs) (n = 23) and herbal antimalarial products (HAMPs). The method was found to be specific, selective, accurate, precise, and robust toward the alkaloids with linearity achieved within specified concentration ranges (r2 > 0.995 for all analytes). Analyte stability ranged between 6 and 12 hours. All the APIs contained quinine <99.0%–101.0%, with dihydroquinine and cinchonidine at levels compliant with the established acceptance criteria. The QBPAFs had quinine content ranging between 50.2% and 151.2%, with 43.5% (n = 10/23) of them complying with the acceptance criteria. The related alkaloids observed in the QBPAFs included quinidine (56.5%, n = 13/23), dihydroquinine (100%, n = 23/23), dihydroquinidine (21.7%, n = 5/23), cinchonine (17.4%, n = 4/23), and cinchonidine (95.7%, n = 22/23). For the HAMPs, 81.0% (n = 17/21) were adulterated with quinine (0.59 ± 0.04 mg/10 mL–86.03 ± 0.02 mg/10 mL). Cryptolepine was identified in 19% (n = 4/21) of the HAMPs with concentration ranging between 43.99 ± 0.43 μg/mL and 747.86 ± 0.34 μg/mL. In conclusion, the application of the ion-pair HPLC method targeting quinoline and indoloquinoline antimalarials has demonstrated the presence of quality and poor-quality HPAFs on the Ghanaian market.

Using a 3-hydroxyflavone derivative as a fluorescent probe for the indirect determination of aminothiols separated by ion-pair HPLC.

Homocysteine, cysteine, cysteinyl-glycine, and glutathione are significant biological aminothiols (ATs) that are marker-molecules in Down syndrome, Alzheimer's disease, or have been implicated as risk factors in atherosclerosis and other vascular diseases, and therefore rapid determination of these molecules is desirable. After reduction of the disulfides, a widely used method utilizes derivatization with ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate (SBD-F) as a fluorogenic probe prior to reversed-phase HPLC separation followed by fluorescence detection. The traditional HPLC determination of ATs is time consuming and economically expensive. We have developed an ion-pair HPLC method coupled with indirect fluorescence detection after post-column reaction with a 2,4-dinitrobenzenesulfonate derivative of a 3-hydroxyflavone. The accuracy, precision, post-column temperature and residence time, and limit-of-detection were evaluated. Sample throughput and reduced sample preparation time of over an hour for the existing methods to less than 20 minutes for the new method is also demonstrated. No statistical differences in HCy, Cys, or Cys-Gly determinations in plasma samples were observed between our method and the traditional HPLC method.

Adenine Nucleotide and Nicotinamide Adenine Dinucleotide Measurements in Plants.

As the principal co-factors of many metabolic pathways, the measurement of both adenine nucleotides and nicotinamide adenine dinucleotide provides important information about cellular energy metabolism. However, given their rapid and reversible conversion as well as their relatively low concentration ranges, it is difficult to measure these compounds. Here, we describe a highly sensitive and selective ion-pairing HPLC method with fluorescence detection to quantify adenine nucleotides in plants. In addition, nicotinamide adenine dinucleotide is a crucially important redox-active substrate for multiple catabolic and anabolic reactions with the ratios of NAD+ /NADH and NADP+ /NADPH being suggested as indicators of the general intracellular redox potential and hence metabolic state. Here, we describe highly sensitive enzyme cycling-based colorimetric assays (with a detection limit in the pmol range) performed subsequent to a simple extraction procedure involving acid or base extraction to allow the measurement of the cellular levels of these metabolites. © 2020 The Authors. Basic Protocol 1: Preparation of plant material for the measurement Basic Protocol 2: Measurement of ATP, ADP, and AMP via HPLC Basic Protocol 3: NAD+ /NADP+ measurements Basic Protocol 4: NADH/NADPH measurements Basic Protocol 5: Data analysis and quality control approaches.

Ionisable substances chromatography: A new approach for the determination of Ketoprofen, Etoricoxib, and Diclofenac sodium in pharmaceuticals using ion – pair HPLC

An ion-pair HPLC method was developed and validated to analyze three of non-steroidal anti-inflammatory drugs (Ketoprofen, Etoricoxib, and Diclofenac sodium) in their pure and pharmaceuticals based on their ionisable characteristics. Cetyltrimethylammonium bromide (Cetrimide) was used as an ion pair reagent since it had not been used before for this purpose. Chromatographic analysis was accomplished using the C18 (250 × 4.6 mm, 5μm) column. Mobile phase consisted of a mixture of 50% Cetrimide10−3 M and 50% acetonitrile to analyze Ketoprofen and Etoricoxib, whereas for Diclofenac sodium, mobile phase was a mixture of 30% Cetrimide10−3 M and 70% acetonitrile. pH value was adjusted if necessary to 10 with ammonium hydroxide. The flow rate was 1mL/min and detection wavelengths were at 254 nm, 234 nm, and 254 nm for Ketoprofen, Etoricoxib, and Diclofenac sodium; respectively under ambient temperature. Retention times (Rt) were 9.41, 7.34, and 6.66 for Ketoprofen, Etoricoxib, and Diclofenac sodium; respectively. The proposed method was evaluated for linearity, accuracy, precision, and specificity according to ICH guidelines. Ketoprofen, Etoricoxib, and Diclofenac sodium were detected in the following linear ranges: (0.031–0.500mg/mL), (0.007–0.110g/mL), and (0.016–0.250mg/mL); respectively with excellent mean recovery values (98.0–102.0%). RSD% was in an acceptable range (less than 2), proving the precision of the developed method. Specificity was proved in the presence of degradation products. Furthermore, a comparison between the results of this study and the reported HPLC methods indicated that this developed method was better in terms of simplicity, analysis time, and no use of buffers in the mobile phase. In conclusion, the developed method can successfully detect Ketoprofen, Etoricoxib, and Diclofenac sodium quantitatively and qualitatively in their dosage forms without any interference with excipients, making this method valuable, reliable, and practical to be applied in quality control laboratories.

Monitoring the Levels of Biogenic Amines in Canned Fish Products Marketed in Ghana

An ion-pair HPLC method with postcolumn o-phthalaldehyde (OPA) derivatization and fluorescence detection was validated for quantitative determination of five biogenic amines (histamine, tyramine, cadaverine, putrescine, and agmatine) in canned fish products (mackerel, sardine, and tuna) marketed in Ghana. The validated method exhibited excellent selectivity and good linearity (R2 > 0.9990) for all the amines. The limits of detection and quantification for studied biogenic amines were in the range of 0.32–0.78 mg·kg−1 and 1.10–2.57 mg·kg−1, respectively. Also, a satisfactory recovery was obtained for all the amines (82.1–101.4%), and the relative standard deviations were lower than 9.3% under repeatability conditions for the studied amines. Subsequently, the method was applied to the analysis of biogenic amines in canned fish products to estimate the safety of Ghanaian consumers. The maximum levels of histamine, tyramine, cadaverine, putrescine, and agmatine detected in the analysed canned fish products were 64.05 mg·kg−1, 27.44 mg·kg−1, 27.23 mg·kg−1, 18.74 mg·kg−1, and 52.72 mg·kg−1, respectively. Thus, the levels of biogenic amines detected in the canned fish products were lower than the acceptable levels and, therefore, can be considered relatively safe for human consumption.

Investigation of biologically active amines in some selected edible mushrooms

Mushrooms are highly valued due to nutritional and functional properties as well as small environmental footprint. However, scarce information is available regarding amines in commercial products. The objective of this study was to investigate the levels of bioactive amines in eight fresh edible commercial mushrooms species. An ion-pair HPLC method with post-column derivatization with o-phthalaldehyde and fluorescence detection was fit for the purpose. Seven out of nine amines were present and levels varied among species. Spermidine was ubiquitous to mushrooms, with highest content in Black Shimeji (12.4 mg/100 g). The levels of spermidine in mushrooms classify them as high polyamines sources, which is valued due to its association with growth, health promotion and antioxidant properties. Agmatine was present in all Pleurotus. Tyramine, tryptamine and phenylethylamine were detected in some species; the levels of cadaverine and putrescine were discrete. A four-principal component model explained 99.4% of the variance and it was able to separate Pleurotus spp. (White shimeji, Hiratake, Black shimeji and Salmon) from Agaricus bisporus (Champignon and Portobello) and Lentinula edodes (Shitake). Hierarchical cluster analysis confirmed the potential of using the occurrence and levels of amines to separate some mushroom species.

An Ultrasound Assessed Extraction Combined with Ion-Pair HPLC Method and Risk Assessment of Nitrite and Nitrate in Cured Meat.

An accurate IPC-UV method was developed and validated for the determination of nitrite (NI) and nitrate (NA) in meat products. The best separation was achieved on a phenyl-hexyl column (150 mm × 4.6 mm, 3 µm) with a mobile phase composed of 25% acetonitrile and 75% buffer (2 mM disodium hydrogen phosphate and 3 mM tetrabutylammonium bromide, pH = 4). Eluents were monitored at 205 nm. Linearity ranges were 1.86 × 10−6–7.5 µg·ml−1 and 0.09–5.0 µg·ml−1 for NI and NA, respectively. The correlation coefficients were greater than 0.999 for NI and NA. This method was applied to a number of processed meat products in Riyadh (n = 155). NI ranged from 1.78 to 129.69 mg·kg−1, and NA ranged from 0.76 to 96.64 mg·kg−1. Results showed extensive use of NI and NA; however, concentrations were within the legal limit of Saudi Arabia except for one sample. Further, the risk assessment and dietary exposure have been estimated for both NI and NA.

Oxygen exchange and energy metabolism in erythrocytes of Rett syndrome and their relationships with respiratory alterations.

Rett syndrome (RTT) is a neurodevelopmental disorder, mainly affecting females, which is associated to a mutation on the methyl-CpG-binding protein 2 gene. In the pathogenesis and progression of classic RTT, red blood cell (RBC) morphology has been shown to be an important biosensor for redox imbalance and chronic hypoxemia. Here we have evaluated the impact of oxidation and redox imbalance on several functional properties of RTT erythrocytes. In particular, we report for the first time a stopped-flow measurement of the kinetics of oxygen release by RBCs and the analysis of the intrinsic affinity of the hemoglobin (Hb). According to our experimental approach, RBCs from RTT patients do not show any intrinsic difference with respect to those from healthy controls neither in Hb's oxygen-binding affinity nor in O2 exchange processes at 37°C. Therefore, these factors do not contribute to the observed alteration of the respiratory function in RTT patients. Moreover, the energy metabolism of RBCs, from both RTT patients and controls, was evaluated by ion-pairing HPLC method and related to the level of malondialdehyde and to the oxidative radical scavenging capacity of red cells. Results have clearly confirmed significant alterations in antioxidant defense capability, adding important informations concerning the high-energy compound levels in RBCs of RTT subjects, underlying possible correlations with inflammatory tissue alterations.

Development and Validation of a Fast Stability-Indicating Ion-Paired Reversed-Phase HPLC Method for the Assay of Thiabendazole and Estimation of Its Related Compounds.

Thiabendazole is a benzimidazole-based anthelmintic active pharmaceutical ingredient for the treatment of intestinal pinworm and Strongyloides infections. Thiabendazole is also used as a fungicide to control fungal diseases in fruits and vegetables. In this paper, we report, for the first time, the development and validation of a novel stability-indicating reversed-phase ion-paired HPLC method for the assay of thiabendazole and estimation of its related compounds. Chromatographic separation was achieved by using an isocratic elution at a flow rate of 1.5 mL/min using an ACE 5 C18 (4.6 × 50 mm, 5 μm particle size) column maintained at 35°C and with UV detection at 300 nm. The mobile phase consisted of 25% acetonitrile and 75% 10 mM 1-octanesulfonic acid sodium salt aqueous solution containing 0.1% methanesulfonic acid. The total run time was only 4 min. The new method was successfully validated according to the International Conference on Harmonization guidelines. The stability-indicating capability of the method was demonstrated through adequate separation of all potential related compounds (from thiabendazole and from each other) present in aged and stressed degraded samples. The ion-paired HPLC method described in this paper is ideal for QC laboratories to conduct routine testing.

A New Validated Stability Indicating Ion-Pair HPLC Method for Evaluation of Impurities of Pramipexole from Low Dose Extended Release Formulation

A New Validated Stability Indicating Ion-Pair HPLC Method for Evaluation of Impurities of Pramipexole from Low Dose Extended Release Formulation

Quality assurance of histamine analysis in fresh and canned fish

Histamine determination is relevant for fish safety, quality and trade. Recently a study by the European Union (EU) compared the Codex and the EU mandated methods for the analysis of histamine and observed that they underestimated and overestimated the results, respectively. To solve this problem, a simple and efficient procedure for the extraction and quantification of histamine by ion-pair HPLC method with post-column derivatization and fluorimetric detection is proposed. It was optimized and validated for the analysis of histamine in fish. The method attended the performance criteria established by Commission Decision 2002/657/CE. The method was also submitted to proficiency testing; uncertainty was calculated; and the stability of solutions and standards was investigated. There was no matrix effect. The LOD, LOQ, CCα and CCβ were fit for the purpose. The method was successfully used in the analyses of freshwater fish and fresh and canned tuna.

Ion-pair HPLC Method for the Quantification of Metformin in Human Plasma and Its Application to a Pharmacokinetic Study

Ion-pair HPLC Method for the Quantification of Metformin in Human Plasma and Its Application to a Pharmacokinetic Study

Purity Determination of Three Curcuminoids Found in Ten Commercially Available Turmeric Dietary Supplements Using a Reverse Phase HPLC Method

The main active component present in turmeric is curcumin and is subsequently responsible for some of its therapeuticeffects. Turmeric, however, contains two other active molecules, namely demethoxycurcumin (DMC) and bisdemethoxycurcumin (BDMC). Collectively, these three molecules are known as curcuminoids. Commercially available dietary supplements vary in these components. In this study the curcuminoids were analyzed using a reverse phase ion-pairing HPLC method. UV detection was used to detect the three curcuminoids at a single wavelength of 430 nm. The validation of the method was carried to ICH guidelines. Good repeatability of the method was achieved at concentrations of curcumin, DMC and BDMC equivalent to 136, 148 and 162 μM, respectively, with R.S.D. values of 2.25, 1.93 and 1.64%, respectively. Similarly, good reproducibility and linearity of the method was obtained for curcumin, DMC and BDMC. On completion of the validation, 10 commercially available turmeric dietary supplements were analyzed for their curcuminoid content. Results showed two of the turmeric dietary supplements to contain near pure curcumin. The other supplements tested contained significant amounts of DMC and BDMC. Similarly, there was a significant increase in curcuminoid content between supplements that contained turmeric extract and those that contained raw turmeric. Interestingly, supplements containing turmeric extract contained a significantly higher proportion of curcumin compared to DMC and BDMC, reflected in the ratio of curcumin: DMC: BDMC.

Novel, Precise, Accurate Ion-Pairing Method to Determine the Related Substances of the Fondaparinux Sodium Drug Substance: Low-Molecular-Weight Heparin.

Fondaparinux sodium is a synthetic low-molecular-weight heparin (LMWH). This medication is an anticoagulant or a blood thinner, prescribed for the treatment of pulmonary embolism and prevention and treatment of deep vein thrombosis. Its determination in the presence of related impurities was studied and validated by a novel ion-pair HPLC method. The separation of the drug and its degradation products was achieved with the polymer-based PLRPs column (250 mm × 4.6 mm; 5 μm) in gradient elution mode. The mixture of 100 mM n-hexylamine and 100 mM acetic acid in water was used as buffer solution. Mobile phase A and mobile phase B were prepared by mixing the buffer and acetonitrile in the ratio of 90:10 (v/v) and 20:80 (v/v), respectively. Mobile phases were delivered in isocratic mode (2% B for 0–5 min) followed by gradient mode (2–85% B in 5–60 min). An Evaporative Light Scattering Detector (ELSD) was connected to the LC system to detect the responses of chromatographic separation. Further, the drug was subjected to stress studies for acidic, basic, oxidative, photolytic, and thermal degradations as per ICH guidelines and the drug was found to be labile in acid, base hydrolysis, and oxidation, while stable in neutral, thermal, and photolytic degradation conditions. The method provided linear responses over the concentration range of the LOQ to 0.30% for each impurity with respect to the analyte concentration of 12.5 mg/mL, and regression analysis showed a correlation coefficient value (r2) of more than 0.99 for all the impurities. The LOD and LOQ were found to be 1.4 µg/mL and 4.1 µg/mL, respectively, for fondaparinux. The developed ion-pair method was validated as per ICH guidelines with respect to accuracy, selectivity, precision, linearity, and robustness.

Development of a Single Ion Pair HPLC Method for Analysis of Terbinafine, Ofloxacin, Ornidazole, Clobetasol, and Two Preservatives in a Cream Formulation: Application to In Vitro Drug Release in Topical Simulated Media-Phosphate Buffer Through Rat Skin.

Present work reports an HPLC method with UV detection for quantification of terbinafine, ofloxacin, ornidazole, and clobetasol in a cream formulation along with two preservatives methyl and propyl paraben. The chromatographic separation and quantification was achieved by an octyl bonded column and a gradient elution program involving an ion-pairing reagent, hexanesulfonic acid (0.2%, pH modified to 2.7 using orthophosphoric acid) and acetonitrile. The method was simple and devoid of buffer salts and therefore advantageous for system and column life. The three step gradient program was initiated with 30% (v/v) acetonitrile for the first 5 min and ramped linearly to 60% in the next 7 min. The mobile phase remained constant for the next 11 min and then concluded at 30% (v/v) of acetonitrile. Flow rate throughout was 0.8 mL/min, and all the signals were monitored at 243 nm. The method was applied for assay of a cream formulation and its in vitro permeation studies to determine the penetration profile of the four drugs and two preservatives. A marketed cream formulation was selected for the permeation study, which was carried out using a diffusion cell consisting of topical simulated media, phosphate buffer (pH=6.8) solution containing 1% sodium lauryl sulfate as a receiver medium.

Development and validation of an ion-pair HPLC method for determination of perchlozone in blood plasma

A procedure involving ion-pair HPLC combined with UV detection was developed for determination of the innovative antituberculosis drug perchlozone in human blood plasma and validated in terms of selectivity, calibration curve, accuracy, precision, sensitivity, and stability. The procedure enables determination of the perchlozone concentration in the blood plasmas of patients taking other antituberculosis medication and can be used for both pharmacokinetic investigations and therapeutic monitoring.

Ion-pair reversed-phase high performance liquid chromatography method for the quantification of isoaspartic acid in a monoclonal antibody

Isomerization of aspartic acid residues is one of the major causes of chemical degradation during the shelf life of biological pharmaceuticals. Monoclonal antibody biopharmaceuticals are typically stored at mildly acidic pH conditions, which can lead to the isomerization reaction. The mechanism of this non-enzymatic chemical reaction has been studied in great detail. However, the identification and quantification of the isomerization sites in a given protein still remains a challenge. We developed an ion-pair reversed-phase HPLC method for the separation of an intact monoclonal antibody variant containing a single isoaspartic acid residue from its native counterpart. We identified and characterized the isomerization site using ion-pair reversed-phase HPLC mass spectrometry methods of the reduced and alkylated antibody and the enzymatically cleaved antibody. Lys-C followed by Asp-N digestion of the antibody was used for the identification of the isomerization site. Electron transfer dissociation (ETD) mass spectrometry was used to confirm the isomerization site at a DY motif at an aspartic acid residue in the CDR-H3 region of the antibody. Tyrosine at the C-terminus of an aspartic acid residue is typically not regarded as a hot spot for isomerization. Our findings suggest that it is not possible to predict isomerization sites in proteins with confidence and all aspartic acid residues located in the CDR regions of antibodies must be considered as potential isomerization site due to the solvent exposure or the flexibility of these regions of the molecule. Additionally, the effect of the pH on the isomerization rate was evaluated using the ion-pair reversed-phase HPLC method, showing that at a lower pH the isomerization rate is faster. Storage at 25°C for 6 months resulted in an increase of the amount of isoaspartic acid to 6.6% at pH 5.4, 6.0% at pH 5.8, and 5.6% at pH 6.2.

Ion-Pair Chromatography for Simultaneous Analysis of Ethionamide and Pyrazinamide from Their Porous Microparticles

Ethionamide (ETA) and pyrazinamide (PZA) are considered the drugs of choice for the treatment of multidrug-resistant tuberculosis. Current methods available in the literature for simultaneous determination of ETA and PZA have low sensitivity or involve column modifications with lipophilic cations. The aim of this study was to develop a simple and validated reversed-phase ion-pair HPLC method for simultaneous determination of ETA and PZA for the characterization of polymeric-based porous inhalable microparticles in in vitro and spiked human serum samples. Chromatographic separation was achieved on a Phenomenex C18 column (250mm × 4.6mm) using a Shimadzu LC 10 series HPLC. The mobile phase consisted of A: 0.01% trifluoroacetic acid in distilled water and B: ACN/MeOH at 1:1 v/v. Gradient elution was run at a flow rate of 1.5mL/min and a fixed UV wavelength of 280nm. The validation characteristics included accuracy, precision, linearity, analytical range, and specificity. Calibration curves at seven levels for ETA and PZA were linear in the analytical range of 0.1-3.0μg/mL with correlation coefficient of r (2) > 0.999. Accuracy for both ETA and PZA ranged from 94 to 106% at all quality control (QC) standards. The method was precise with relative standard deviation less than 2% at all QC levels. Limits of quantitation for ETA and PZA were 50 and 70ng/mL, respectively. There was no interference from either the polymeric matrix ions or the biological matrix in the analysis of ETA and PZA.

Targeted metabolic analysis of nucleotides and identification of biomarkers associated with cancer in cultured cell models

Cancer, like other diseases accompanied by metabolic changes, shows characteristic DNA/RNA modifications and activities of modifying enzymes, resulting in fluctuations in nucleoside levels. In this study, we undertook targeted metabolomic analyses of nucleotides in different cancer cell culture models using a sensitive and reproducible ion-pair HPLC method. The experimental data were analyzed by principal component analysis (PCA) to identify potential biomarkers in cancer cells, and statistical significance was determined by one-way analysis of variance. As a result, a clear differentiation of normal and tumor cells into two clusters was shown, indicating abnormal metabolism of nucleotides in tumor cells. Six variables (AMP, UDP, CTP levels with a significance of P<0.05; ATP, UTP and GMP levels with a significance of P<0.01) were considered as potential biomarkers; the content of AMP, UTP, GMP and ATP was significantly higher in cancer cells. The receiver operating characteristic (ROC) curve analysis allowed us to discriminate normal cells from tumor cells based on area under the curve (AUC). The sequence of their AUC values were: ATP (0.979)>UTP (0.938)>CTP=GMP (0.896)>AMP (0.812)>UDP (0.792), so we conclude that ATP and UTP are the best potential biomarkers in tumor cells. This study may provide a valuable tool for studying minute alterations of intracellular nucleotide pools induced by anticancer/antiviral drugs, diseases or environmental factors.