Ion-pair High-performance Liquid Chromatography Research Articles

A Simple and High Resolution Ion-Pair HPLC Method for Separation and Simultaneous Determination of Nitrate and Thiocyanate in Different Water Samples

An ion-pair reversed-phase high-performance liquid chromatography method with isocratic elution and ultraviolet detection was developed and validated for the separation and simultaneous determination of nitrate and thiocyanate. The separation was performed on a C18 analytical column with mobile phase containing 0.08 mM hexadecyltrimethylammonium bromide as an ion-pair reagent, 40 mM of acetate buffer and 30% methanol at pH 3.2. The detection was monitored at 206 nm. The response was linear from 1 to 10 mM for nitrate, with a detection limit of 0.05 mM, and 1 to 10 mM for thiocyanate, with a detection limit of 0.31 mM. Calibration curves were found to be linear in these concentration ranges with correlation coefficient better than 0.99.

Mitochondrial DNA Variant for Complex I Reveals a Role in Diabetic Cardiac Remodeling

Myocardial remodeling and dysfunction are serious complications of type 2 diabetes mellitus (T2DM). Factors controlling their development are not well established. To specifically address the role of the mitochondrial genome, we developed novel conplastic rat strains, i.e. strains with the same nuclear genome but a different mitochondrial genome. The new animals were named T2DN(mtFHH) and T2DN(mtWistar), where the acronym T2DN denotes their common nuclear genome (type 2 diabetic nephropathy (T2DN) rats) and mtFHH or mtWistar the origin of their mitochondria, Fawn Hooded Hypertensive (FHH) or Wistar rats, respectively. The T2DN(mtFHH) and T2DN(mtWistar) showed a similar progression of diabetes as determined by HbA1c, cholesterol, and triglycerides with normal blood pressure, thus enabling investigation of the specific role of the mitochondrial genome in cardiac function without the confounding effects of obesity or hypertension found in other models of diabetes. Echocardiographic analysis of 12-week-old animals showed no abnormalities, but at 12 months of age the T2DN(mtFHH) showed left ventricular remodeling that was verified by histology. Decreased complex I and complex IV but not complex II activity within the electron transport chain was found only in T2DN(mtFHH), which was not explained by differences in protein content. Decreased cardiac ATP levels in T2DN(mtFHH) were in agreement with a lower ATP synthetic capacity by isolated mitochondria. Together, our data provide experimental evidence that mtDNA sequence variations have an additional role in energetic heart deficiency. The mitochondrial DNA background may explain the increased susceptibility of certain T2DM patients to develop myocardial dysfunction.

DETERMINATION OF FLUOROQUINOLONE RESIDUES IN PENAEUS JAPONICUS BY MICROWAVE-ASSISTED EXTRACTION AND ION-PAIR HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

A multi-residue method developed for the analysis of fluoroquinolone in Penaeus japonicus muscular tissue is presented. The method is based on microwave-assisted extraction (MAE) of Penaeus japonicus muscular tissue using acetonitrile as extractant and analysis of extracts by ion-pair high performance liquid chromatography (HPLC) and fluorescence detection. MAE operational parameters, the solvent type and volume, extraction temperature, and time were optimized in detail with respect to extraction efficiency of the target compounds from Penaeus japonicus muscular tissue. The recoveries of the method at three different spiked levels were assessed by analyzing spiked meat samples and were found to be in the range from 78.40 to 96.62% with good precision (<10%).

Determination of metformin in diabetic rat plasma by an improved ion-pair high-performance liquid chromatography: application to a pharmacokinetic study

Determination of metformin in diabetic rat plasma by an improved ion-pair high-performance liquid chromatography: application to a pharmacokinetic study

Detection of Naphthalene Sulfonates from Highly Saline Brines with High-Performance Liquid Chromatography in Conjunction with Fluorescence Detection and Solid-Phase Extraction

A procedure is developed for the simultaneous determination of 1-naphthalene sulfonate, 2-naphthalene sulfonate, 1,5-naphthalene disulfonate, 1,6-naphthalene disulfonate, 2,6-naphthalene disulfonate and 2,7-naphthalene disulfonate from highly saline geothermal brines using ion-pair high-performance liquid chromatography with fluorescence detection after solid-phase extraction. The substances are baseline separated within 33 min and recoveries in brines with salinities of up to 175g/L NaCl are 100%(±10) by solid-phase extraction. For the overall method, the method quantification limits of the analytes are between 0.05 and 0.4µg/L. The method is also shown to be feasible for matrices encountered in deep geothermal reservoirs.

Determination of Bisphosphonates by Ion-pair HPLC

The optimization of the two bisphosphonates (disodium pamidronate and zoledronic acid) separation by ion-suppression and ion-pair high-performance liquid chromatography is described. The influence of the eluent pH, composition of the mobile phase and the concentration of complexing ion-pairing agents was investigated. This developed method provides high separation and specificity to bisphosphonate analysis. In quantitative analysis in the optimized conditions (10 mM n-amylamine or n-butylamine; 14% acetonitrile in 0.1 M sodium phosphate buffer, pH=7.0), the method showed good linearity (r=0.9999-0.9998) and suffi- cient sensitivity (10 -7 -10 -8 g/ml). It can be easily and conveniently adopted for the routine quality control analysis.

Understanding the Effect of the Counterion on the Reverse-Phase Ion-Pair High-Performance Liquid Chromatography (RPIP-HPLC) Resolution of Heparin-Related Saccharide Anomers

Reverse-phase ion-pair high-performance liquid chromatography (RPIP-HPLC) is an increasingly popular chromatographic technique for the separation of charged compounds, including oligosaccharides derived from the glycosaminoglycans (GAGs) heparin and heparan sulfate (HS). This family of heparin disaccharides has been shown to be useful compounds to probe the details of the RPIP-HPLC separation mechanism, the aspects of which are still being debated. In this manuscript, the effects of ion-pairing reagent (IPR) concentration, counterion, and mobile phase pH on the quality of the RPIP-UPLC separation were examined with particular emphasis on how these factors impact the separation of the disaccharide anomers. These results highlight the role of the IPR counterion and demonstrate that the resolution of the disaccharide anomers can be minimized by conducting the separation at low pH, simplifying chromatographic analysis and improving resolution. The results presented herein can also provide insights into strategies for developing more sensitive and efficient reverse-phase separations for other charged analytes including larger GAG oligosaccharides.

Identification and control of impurities in streptomycin sulfate by high-performance liquid chromatography coupled with mass detection and corona charged-aerosol detection

For the control of impurities in streptomycin sulfate a reversed phase ion-pair high performance liquid chromatography (HPLC) method using charged aerosol detection (CAD) was developed. With this method, 21 impurities could be separated and tentatively identified using a combination of exact mass measurement by TOF-MS and MS/MS experiments with a triple quadrupole MS. For three impurities the suggested structures could be confirmed by in situ formation. The CAD detector response was found to be linear over 2 orders of magnitude allowing a straightforward quantification of all impurities. A limit of quantification of 0.09% for streptomycin sulfate and of 0.008% for streptidine sulfate (referred to the concentration of the 5 mg/ml test solution) could be achieved. The HPLC method was applied to the purity testing of 12 samples of commercially available streptomycin sulfate from different manufacturers. Impurity levels between 4.6% and 16.0% were found. The current European Pharmacopoeia monograph for streptomycin sulfate only limits streptomycin B by a TLC test to 3.0%. Therefore, the results of this study underline the importance of introducing a state-of-the-art test for the control of impurities in the monograph. The new HPLC-CAD method is considered suitable for this purpose.

A novel ion-pairing chromatographic method for the simultaneous determination of both nicarbazin components in feed additives: Chemometric tools for improving the optimization and validation

The development, optimization and validation of an ion-pairing high performance liquid chromatography method for the simultaneous determination of both nicarbazin (NIC) components: 4,4′-dinitrocarbanilide (DNC) and 2-hydroxy-4,6-dimethylpyrimidine (HDP) in bulk materials and feed additives are described. An experimental design was used for the optimization of the chromatographic system. Four variables, including mobile phase composition and oven temperature, were analyzed through a central composite design exploring their contribution to analyte separation. Five responses: peak resolutions, HDP capacity factor, HDP tailing and analysis time, were modelled by using the response surface methodology and were optimized simultaneously by implementing the desirability function. The optimum conditions resulted in a mobile phase consisting of 10.0mmolL−1 of 1-heptanesulfonate, 20.0mmolL−1 of sodium acetate, pH=3.30 buffer and acetonitrile in a gradient system at a flow rate of 1.00mLmin−1. Column was an INERSTIL ODS-3 (4.6mm×150mm, 5μm particle size) at 40.0°C. Detection was performed at 300nm by a diode array detector. The validation results of the method indicated a high selectivity and good precision characteristics, with RSD less than 1.0% for both components, both in intra and inter-assay precision studies. Linearity was proved for a range of 32.0–50.0μgmL−1 of NIC in sample solution. The recovery, studied at three different fortification levels, varied from 98.0 to 101.4 for HDP and from 99.1 to 100.2 for DNC. The applicability of the method was demonstrated by determining DNC and HDP content in raw materials and commercial formulations used for coccidiosis prevention. Assays results on real samples showed that considerable differences in molecular ratio DNC:HDP exist among them.

A New Combination Strategy as Applied in Predicting Chromatographic Retention Times of Oligonucleotides at a Range of Temperatures from 30 °C to 80 °C

AbstractWe develop a new combination strategy to predict retention times of oligonucleotides in ion‐pair reversed‐phase high performance liquid chromatography. The key step of the strategy is to use score of generalized base properties (SGBP) combined with auto cross covariance (ACC) to resolve the feature representation of nucleic acids. This representation characterize physicochemical, quantumchemical, topological, spatial structural features, etc., and their neighboring effect between bases at a certain distance apart in a sequence. The next step is to use the variables selected by genetic algorithm (GA) to construct prediction models of retention times of oligonucleotides based on support vector machine (SVM). Accordingly, GA‐SVM models give different prediction performance using different input descriptors resulting from different step lengths in the ACC transformation, indicating the neighboring effect between bases should not be neglected in the features related to the chromatographic retention of oligonucleotides. As a whole, the GA‐SVM predictors obtained from more than 20 training samples can produce satisfying performance in predicting the chromatographic retention of oligonucleotides at a range of temperatures (30 °C, 40 °C, 50 °C, 60 °C and 80 °C), respectively. The present approach based on the SGBP‐ACC‐GA‐SVM combination shows great application prospect in the field of separation and analysis science, bioinformatics and proteomics.

Abstract P4-02-17: Polymerase Chain Reaction-Liquid Chromatography-Mass Spectrometry — A Convenient and Cost-Effective Strategy for CYP2D6 Genotyping

Abstract Background: Genetic polymorphisms in the drug metabolizing enzyme Cyotchrome P450 2D6 (CYP2D6) gene can significantly influence a person's ability to mediate CYP2D6 dependent metabolism. For example, a notable influence of the CYP2D6 genotype has been described regarding the conversion of tamoxifen to its pharmacologically active metabolites. Thus, the genetic determination of the CYP2D6 metabolizer status has the potential to represent a valuable therapeutic supplement in breast cancer therapy. However, the clinical relevance of the CYP2D6 genotype regarding the treatment of breast cancer with tamoxifen still remains to be elucidated. Due to the high number of relevant polymorphisms to be screened, pharmacogenetic CYP2D6 testing is considered to be time-consuming and laborious and is therefore limited to a few clinical studies only. To enable a more widespread use, we present polymerase chain reaction (PCR) - ion-pair reversed-phase high-performance liquid chromatography — electrospray ionization time of flight mass spectrometry (ICEMS) as fast, convenient and cost-effective strategy for CYP2D6 genotyping. Material and Methods: After DNA extraction from saliva swabs, the CYP2D6 gene was amplified by a long range PCR. Subsequently, polymorphism-specific short amplicons were generated in a multiplexed PCR. The amplicons were directly analysed by ICEMS. The information necessary to determine the allelic state of a PCR-amplified polymorphic locus were obtained from the measured molecular masses. Gene duplications or deletions were analysed by a long range PCR protocol and subsequent agarose gel detection. Results: The developed CYP2D6 genotyping approach enables the reliable determination of the most frequent CYP2D6 alleles in Europe (*1, *2, *3, *4, *6, *7, *8, *9, *10, *11, *12, *17, *41) including whole gene duplications and deletions (*5). The assay involves the following steps: (1) collecting biological material in form of non-invasive saliva swabs; (2) DNA extraction; (3) long range PCR for a specific CYP2D6 amplification; (3) multiplexed PCR for the generation of polymorphism-specific short amplicons; (4) direct analysis of the reaction mixture with ICEMS; (4) determination of the allelic state with the measured molecular masses; (5) analysis of gene duplications or deletions by a long range PCR protocol and subsequent agarose gel detection. Genotyping of a single sample costs about 5-10 USD and can be accomplished within 2-3 days. Parallelization increases the sample throughput and reduces costs. For proof of principle, we have applied the developed method in the context of the PRO-BETh study to determine the CYP2D6 status of 106 breast cancer patients treated with tamoxifen. According to the classification of Gaedik A. et al [1], 19% of the typed breast cancer patients turned out to be “Poor Metabolizers", 51% were “Intermediate Metabolizers", 29% were “Extensive Metabolizers” and 7% turned out to be “Ultrarapid Metabolizers”. Conclusion: PCR-ICEMS represents a cost-effective and convenient tool for pharmacogenetic testing, which enables the determination of the CYP2D6 status using DNA obtained from a non-invasive saliva swab as template. [1] Gaedigk A. et al, J. Clin Pharmacol Ther. 2007 Jun;81(6):817-20. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-02-17.

Heparinase 1 selectivity for the 3,6-di-O-sulfo-2-deoxy-2-sulfamido-α-D-glucopyranose (1,4) 2-O-sulfo-α-L-idopyranosyluronic acid (GlcNS3S6S-IdoA2S) linkages

Porcine intestinal mucosa heparin was partially depolymerized by recombinant heparinase 1 (heparin lyase 1, originating from Flavobacterium heparinum and expressed in Escherichia coli) and then fractionated, leading to the isolation of 22 homogeneous oligosaccharides with sizes ranging from disaccharide to hexadecasaccharide. The purity of these oligosaccharides was determined by gel electrophoresis, strong anion exchange and reversed-phase ion-pairing high-performance liquid chromatography. The molecular mass of oligosaccharides was determined using electrospray ionization-mass spectrometry and their structures were elucidated using one- and two-dimensional nuclear magnetic resonance spectroscopy at 600MHz. Five of the characterized oligosaccharides represent new compounds. The most prominent oligosaccharide comprises the common repeating unit of heparin, ΔUA2S-[-GlcNS6S-IdoA2S-](n)-GlcNS6S, where ΔUA is 4-deoxy-α-l-threo-hex-4-eno-pyranosyluronic acid, GlcN is 2-deoxy-2-amino-d-glucopyranose, IdoA is l-idopyranosyluronic acid, S is sulfate and n=0-7. A second prominent heparin oligosaccharide motif corresponds to ΔUA2S-[GlcNS6S-IdoA2S](n)-GlcNS6S-IdoA-GlcNAc6S-GlcA-GlcNS3S6S (where n=0-5 and GlcA is d-glucopyranosyluronic acid), a fragment of the antithrombin III binding site in heparin. The prominence of this second set of oligosaccharides and the absence of intact antithrombin III binding sites suggest that the -GlcNS3S6S-IdoA2S- linkage is particularly susceptible to heparinase 1.

Ion-pairing and reversed phase liquid chromatography for the determination of three different quinolones: Enrofloxacin, lomefloxacin and ofloxacin

Two simple and sensitive high performance liquid chromatographic (HPLC) methods have been developed for the simultaneous determination of three different quinolones: enrofloxacin, lomefloxacin and ofloxacin in their pure and dosage forms, one with reversed phase HPLC and the other with ion-pair HPLC. In reversed phase HPLC, method (A), the mobile phase consists of 2.18% aqueous solution of KH2PO4 with pH adjusted to 2.4±0.2 with acetonitrile (80:20; v/v), the mobile phase pumped at flow rate of 1.2mlmin−1. A Neucleosil C18 column (10μm, 100Å), 250mm length×4.6mm diameter was utilized as stationary phase. Detection was affected spectrophotometrically at 294nm. While in ion-pair HPLC, method (B), the mobile phase was aqueous solution of 0.65% sodium perchlorate and 0.31% ammonium acetate adjusted to pH 2.2±0.2 with orthophosphoric acid: acetonitrile (81:19; v/v), the mobile phase pumped at flow rate of 1.5mlmin−1. A μ bondapack C18 column (10μm, 100Å), 250mm length×4.6mm diameter was utilized as stationary phase. Detection was affected spectrophotometrically at 294nm. Linearity ranges for enrofloxacin, lomefloxacin and ofloxacin were 4.0–108, 7.0–112 and 8.0–113μgml−1, respectively using method A and 8.0–112, 7.0–112 and 5.0–105μgml−1, respectively applying method B. Minimum detection limits obtained were 0.013, 0.023 and 0.035μgml−1 for enrofloxacin, lomefloxacin and ofloxacin, respectively using method A, and 0.028, 0.023 and 0.011μgml−1 using method B. The proposed methods were further applied to the analysis of enrofloxacin in injection and tablets containing the ofloxacin and lomefloxacin drugs, and the results were satisfied.

Ion-pair high-performance liquid chromatography with diode array detection coupled to dual electrospray atmospheric pressure chemical ionization time-of-flight mass spectrometry for the determination of nucleotides in baby foods

A liquid chromatography with diode array detection coupled to dual electrospray atmospheric pressure chemical ionization time-of-flight mass spectrometry (HPLC/ESI-APCI-TOF-MS) method is described for the rapid determination of five monophosphate nucleotides (cytidine 5′-monophosphate, uridine 5′-monophosphate, adenosine 5′-monophosphate, inosine 5′-monophosphate and guanosine 5′-monophosphate) in baby foods. The method is based on the deproteinisation of foods and direct analysis of nucleotides by ion-pair HPLC using isocratic elution with a mobile phase of 5% (v/v) methanol and 95% (v/v) 0.1 M formate buffer (pH 5.5) containing 0.01 M N,N-dimethylhexylamine (DMHA) at a flow-rate of 0.7 mL min −1. The HPLC was hyphenated with two different detection systems, photodiode-array (DAD) and ESI-APCI-TOF-MS in negative mode. The method was validated for linearity, detection and quantitation limits, selectivity, accuracy and precision. The recoveries obtained for spiked samples were satisfactory for all the analytes. The method was successfully applied to the analysis of nucleotides in different baby and/or functional food samples, as cereals, purees and dairy products. A study was also carried out on the stability of nucleotides in acidified dairy infant food with pasteurized yoghourt and follow-on formulae samples stored at room temperature and at 30 °C.

Determination of 6-Thioguanosine Diphosphate and Triphosphate and Nucleoside Diphosphate Kinase Activity in Erythrocytes: Novel Targets for Thiopurine Therapy?

6-Thioguanine nucleotides are the sum of 6-thioguanosine 5'-monophosphate (TGMP), -diphosphate (TGDP), and -triphosphate (TGTP) representing essential metabolites involved in drug action of thiopurines. Elevated levels of TGDP have been associated with poor response to azathioprine therapy in patients with inflammatory bowel disease. The conversion of TGDP to TGTP is supposed to be catalyzed by nucleoside diphosphate kinase (NDPK). The aim of this work was to investigate simultaneously individual 6-thioguanosine phosphate levels and NDPK activity in red blood cells (RBCs) of patients on azathioprine therapy. Ion-pair high-performance liquid chromatography methods with fluorescence and ultraviolet detection were applied to quantify individual levels of 6-thioguanosine 5'-phosphates and NDPK activity, respectively, in RBCs. Recombinantly expressed NDPK isoforms A and B were unequivocally identified to catalyze the formation of TGTP (30.6 +/- 3.88 nmol x min x mg for NDPK A versus 41.2 +/- 1.05 nmol x min x mg for NDPK B). Comprehensive analyses on the stability of TGMP, TGDP, and TGTP and the reproducibility of NDPK activity in RBCs were performed to provide a reliable sampling protocol for clinical practice. Of note, isolation of RBCs within 6 hours followed by immediate storage at -80 degrees C is crucial for prevention of degradation of 5'-phosphates. In a clinical study of 37 patients on azathioprine, TGTP was the predominant 6-thioguanosine phosphate in RBCs. In contrast, three patients showed TGTP/(TGDP + TGTP) ratios of 57.2%, 64.3%, and 66% corresponding to elevated TGDP levels. NDPK activity ranged from 4.1 to 11.3 nmol x min x mg hemoglobin. No correlation between NDPK activity and the 6-thioguanosine phosphate levels was found. The question whether interindividual variability of NDPK activity may explain differences in 6-thioguanosine 5'-phosphates levels has to be investigated in a prospective large-scale study.

Chemical stability of 4′-azidocytidine and its prodrug balapiravir

Background: R1479, a 4′-azidocytidine nucleoside analog, was developed for the treatment of Hepatitis C virus infection. Balapiravir (R1626) is the tri-isobutyrate ester prodrug of R1479 under clinical development to improve exposure of R1479 upon oral administration. Objective: The chemical stability and the rate of azide release of R1479 and balapiravir were studied. Methods: R1479 and balapiravir solutions were prepared at different pH values and stored at various temperatures. An ion pair high-performance liquid chromatography (HPLC) method with gradient elution was employed to analyze the prodrug, parent, and degradation products. Azide was measured using a reversed phase HPLC method with UV detection after formation of the 3,5-dinitrobenzoyl azide derivative with 3,5-dinitrobenzoyl chloride. The data were analyzed using initial rate and conventional first-order kinetic methods. Results: R1479 degrades to cytosine and azide in aqueous solutions, whereas balapiravir mainly degrades to R1479 and mono- and diesters of R1479. The rates of azide release from R1479 and balapiravir were generally comparable with the corresponding amount formed of cytosine. Conclusion: Azide release is pH dependent and is faster in acidic solutions than in neutral solutions. The amount of azide released is significantly less from balapiravir than that from R1479, suggesting a potential advantage of the prodrug over the parent drug.

Decreased serum essential and aromatic amino acids in patients with chronic pancreatitis

To investigate the influence of chronic pancreatitis (CP) on serum concentrations of amino acids. Thirty-five male patients with alcoholic CP and 21 healthy male subjects were examined. Serum concentrations of amino acids were assayed by ion-pair high-performance liquid chromatography with mass detection. Serum glutamate concentration was increased in CP patients as compared to controls. In contrast, serum concentrations of glutamine, histidine, tyrosine, proline, tryptophan and threonine were significantly decreased in CP patients. A trend towards decreasing concentrations of serum lysine, alanine, methionine and valine as well as for total serum amino acids was observed. The sum of aromatic and the sum of essential amino acid concentrations were significantly lower in CP patients than in controls. CP leads to decreased serum concentrations of several amino acids, such as essential and aromatic serum amino acids, most likely due to decreased exocrine function.

Evaluation of IP-RP-HPLC for Length Determination of the Trinucleotide Repeat Fragments in Huntington's Disease

Expansion of an unstable trinucleotide (CAG)(n) repeat region within exon 1 of the gene IT15 causes autosomal, dominantly inherited Huntington's disease (HD). The number of CAG-repeats varies from 6 to 35 in normal individuals, whereas in affected patients the expanded allele contains 40 or more CAG-repeats. Thus, exact determination of both alleles of the gene (normal and expanded) on the molecular level is of great importance for clinical diagnosis and prognosis regarding the course of the disease. In our study, we optimized and evaluated a highly sensitive, automated, and economical molecular method for length characterization of the trinucleotide fragment expansion such as (CAG)(n) repeat region based on ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC). We found that IP-RP-HPLC can be used for exact fragment length measuring between 60-280 bp as a sensitive and advantageous alternative method to conventional techniques.

Insights into the mechanism of separation of heparin and heparan sulfate disaccharides by reverse-phase ion-pair chromatography

Reverse-phase ion-pair high performance liquid chromatography (RPIP-HPLC) and ultra-performance liquid chromatography (RPIP-UPLC) are increasingly popular chromatographic techniques for the separation of organic compounds. However, the fine details of the RPIP separation mechanism are still being debated. Many factors including type and concentration of the ion-pairing reagent, mobile phase pH, organic modifier, ionic strength, and stationary phase all play a role in the overall efficiency and optimization of ion-pairing separations. This study investigates the role that competition between ion-pairing reagents with different steric bulk and hydrophobicity plays in the separation of structural isomers of heparin and heparan sulfate (HS) disaccharides. In addition to providing insights into the mechanism by which RPIP-HPLC can resolve closely related disaccharides, the use of competition between ion-pairing agents could lead to new methods for the separation of larger heparin and HS oligosaccharides. This approach should also be applicable to the analysis of other compound classes, and could lead to a general approach for the chromatographic resolution of mixtures of charged analytes having similar structures.

Analysis of Fluorescent Dye-labeled Oligonucleotides by Ion-Pair Reversed-Phase High-Performance Liquid Chromatography

Abstract The ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) of fluorescent-labeled oligonucleotides separation was established, and the concentration of triethylamine-acetic acid (TEAA, 0–0.15 M), pH value (4.5–7.0), and gradient strength were optimized. The retention mechanism of fluorescent dye-labeled oligonucleotides was evaluated by comparing the retention of 5, 10, 15-mer unlabeled oligonucleotides with that of 5'end carboxyfluorescein (5'FAM)-labeled oligonucleotides. In addition, TaqMan™ probes as well as other fluorescent dye-labeled oligonucleotides were considered for separation. The results show that the best resolution of different length fluorescent dye-labeled oligonucleotides was observed at a TEAA concentration of 0.01 M and pH 7.0. The retention behavior of fluorescent-labeled oligonucleotides was significantly different from that of the unlabeled isomers; therefore, they can be separated completely. We found that the retention of unlabeled oligonucleotides enhanced with the increase of the length of the molecule. In contrast, the retention of fluorescent-labeled oligonucleotides reduced with the increase of the length of the molecule. Because the hydrophobicity of fluorescent dyes can greatly impact the retention, the oligonucleotides labeled using fluorescent dyes with higher hydrophobicity would have a longer retention time. However, the effect of the hydrophobicity was limited as the length of the oligonucleotide was increased to a certain level.