Ion-pair Reversed-phase HPLC Research Articles

Increased Selectivity, Analytical Precision, and Throughput in Targeted Proteomics

Proteomics is gradually complementing large shotgun qualitative studies with hypothesis-driven quantitative experiments. Targeted analyses performed on triple quadrupole instruments in selected reaction monitoring mode are characterized by a high degree of selectivity and low limit of detection; however, the concurrent analysis of multiple analytes occurs at the expense of sensitivity because of reduced dwell time and/or selectivity due to limitation to a few transitions. A new data acquisition paradigm is presented in which selected reaction monitoring is performed in two ways to simultaneously quantify and confirm the identity of the targeted peptides. A first set of primary transitions is continuously monitored during a predetermined elution time window to precisely quantify each peptide. In addition, a set of six to eight transitions is acquired in a data-dependent event, triggered when all the primary transitions exceed a preset threshold. These additional transitions are used to generate composite tandem mass spectra to formally confirm the identity of the targeted peptides. This technique was applied to analyze the tryptic digest of a yeast lysate to demonstrate the performance of the technique. We showed a limit of detection down to tens of attomoles injected and a throughput exceeding 6000 transitions in one 60-min experiment. The technique was integrated into a linear work flow, including experimental design, data acquisition, and data evaluation, enabling large scale proteomic studies.

Evaluation of cadmium species lability using ion-pair reversed phase HPLC coupled on-line with inductively coupled plasma mass spectrometry

Inductively coupled plasma mass spectrometry (ICP-MS) coupled on-line with an ion-pair reversed phase HPLC (IP-RP HPLC) was developed for determining the lability of Cd species. The IP-RP HPLC–ICP-MS system measures chromatographic behaviors of Cd species in the presence of different model complexing agents (L) with stability constants (logKCdL) from 3.8 to 19.0. Cd species with logKCdL higher than 16, between 8 and 16, and smaller than 8 was then classified into inert, moderately labile, and labile species, respectively. The conditional stability constants and dissociation rate constants were also estimated from their corresponding chromatographic behavior. This method was applied to evaluating the lability-dependent biouptake of different Cd species in Phaeodactylum tricornutum, a typical unicellular marine diatom. IP-RP HPLC–ICP-MS is a useful and promising technique for determining the lability of noncovalent-bonded metal species (such as Cd species) in the environment and for forecasting their corresponding bioavailability especially when their speciation cannot be rigorously controlled and measured.

Reversed-Phase Ion-Pair HPLC and TLC-Densitometric Methods for the Simultaneous Determination of Ciprofloxacin Hydrochloride and Metronidazole in Tablets

Two methods are described for the simultaneous determination of ciprofloxacin HCl (CIP) and metronidazole (MET) in binary mixture. The first method was based on the ion-pair liquid chromatographic separation of the two drugs on reversed-phase, μBondapak C18 column (250 mm × 4.6 mm, 10 μm) Waters. The mobile phase consisted of monobasic potassium phosphate (50 mM, pH 3, adjusted with phosphoric acid) and acetonitrile (65:35, v/v) containing sodium octane sulphonate (50 mM). Flow rate of 1.2 mL min−1 was applied. Quantitation was achieved with UV detection at 280 nm. Linearity, accuracy and precision were found to be acceptable over the concentration range of 3–180 and 15–180 μg mL−1 for CIP and MET, respectively. The second method was based on the TLC separation of the two drugs followed by densitometric measurements of their bands at 280 nm. The separation was carried out on silica gel 60 F254 plates, using acetonitrile, ammonia, methanol, methylene chloride and hexane (1.3:1.1:2.0:3.0:1.0, v/v) as developing system. The linear regression analysis data were used for the regression line in the range of 1.5–10 μg band−1 for CIP and MET. The two proposed methods were successfully applied to the determination of both drugs in laboratory prepared mixtures and in commercial tablets. The optimized methods proved to be specific, robust and accurate for the quality control of the cited drugs in pharmaceutical preparation.

Development of an Ion-Pair Reversed-Phase HPLC Method with Indirect UV Detection for Determination of Phosphates and Phosphites as Impurities in Sodium Risedronate

A method based on RP-HPLC with indirect UV detection was developed for the determination of phosphates and phosphites as impurities in sodium risedronate. RP separation of the phosphates and phosphites was achieved by adding tetrabutylammonium hydroxide as an ion-pairing agent in the mobile phase. Potassium hydrogen phthalate was added to the mobile phase as an ionic chromophore in order to obtain high background absorption of the mobile phase. Separation was performed on a C18 column using a mixture of pH 8.2 buffer (containing 0.5 mM tetrabutylammonium hydroxide and 1 mM phthalate) and acetonitrile (95 + 5, v/v) as the mobile phase, with indirect UV detection at 248 nm. The validation of the method included determination of specificity/selectivity, linearity, LOD, LOQ, accuracy, precision, and robustness. The LOD was 0.86 microg/mL for phosphates and 0.76 microg/mL for phosphites. The LOQ was 2.60 microg/mL for phosphates and 2.29 microg/mL for phosphites. The developed method is suitable for quantitative determination of phosphates and phosphites as impurities in QC of sodium risedronate.

Stereoselective epoxidation of the last double bond of polyunsaturated fatty acids by human cytochromes P450

Cytochromes P450 (CYPs) metabolize polyunsaturated long-chain fatty acids (PUFA-LC) to several classes of oxygenated metabolites. Through use of human recombinant CYPs, we recently showed that CYP1A1, -2C19, -2D6, -2E1, and -3A4 are mainly hydroxylases, whereas CYP1A2, -2C8, -2C9, and -2J2 are mainly epoxygenases of arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), respectively. It is worth noting that the last double bond of these PUFAs, i.e., omega6 in AA or omega3 in EPA and DHA, respectively, was preferentially epoxidized. In this study, we have characterized the stereoselectivity of this epoxidation reaction by comparison with the PUFA-LC epoxide stereoisomers obtained from the enantioselective bacterial CYP102A1 F87V. The stereoselectivity of the epoxidation of the last olefin of AA (omega6), EPA (omega3), or DHA (omega3) differed between the CYP isoforms but was similar for EPA and DHA. These data give additional insight into the PUFA-LC epoxide enantiomers generated by the hepatic CYPs.

Simultaneous determination of nucleotide sugars with ion-pair reversed-phase HPLC

Nucleotide sugars are important in determining cell surface glycoprotein glycosylation, which can modulate cellular properties such as growth and arrest. We have developed a conventional HPLC method for simultaneous determination of nucleotide sugars. A mixture of nucleotide sugars (CMP-NeuAc, UDP-Gal, UDP-Glc, UDP-GalNAc, UDP-GlcNAc, GDP-Man, GDP-Fuc and UDP-GlcUA) and relevant nucleotides were perfectly separated in an optimized ion-pair reversed-phase mode using Inertsil ODS-4 and ODS-3 columns. The newly developed method enabled us to determine the nucleotide sugars in cellular extracts from 1 x 10(6) cells in a single run. We applied this method to characterize nucleotide sugar levels in breast and pancreatic cancer cell lines and revealed that the abundance of UDP-GlcNAc, UDP-GalNAc, UDP-GlcUA and GDP-Fuc were a cell-type-specific feature. To determine the physiological significance of changes in nucleotide sugar levels, we analyzed their changes by glucose deprivation and found that the determination of nucleotide sugar levels provided us with valuable information with respect to studying the overview of cellular glycosylation status.

Using ion-pair reversed-phase HPLC ICP-MS to simultaneously determine Cr(III) and Cr(VI) in urine of chromate workers

Urinary chromium speciation analysis can provide available information of the individual exposure levels of Cr(VI) compounds. An analytical method based on ion-pair reversed-phase HPLC combined with ICP-MS to simultaneously determine Cr(III) and Cr(VI) in human urine has been developed for assessing the occupational exposure to chromate. The separation conditions of the method, including the pH value, the concentrations of ion-pair reagent and methanol in the mobile phase were studied. Specially, a high-speed polyetheretherketone (PEEK) column and a typical sample introduction method were employed to avoid the exogenous chromium contamination during the analysis. The separation of Cr(III) and Cr(VI) could be finished within 4 min with the detection limits as low as 0.03 μg L −1 at 100 μL injections for both of them, providing a convenient method for routine analysis of chromium species. The chromium species in urine of chromate workers were monitored using the developed method. The statistical analysis showed a significant relationship ( n = 32, p < 0.01) between the urinary Cr(VI) and the individual airborne exposure levels, indicating that the urinary Cr(VI) could be used as a convenient and suitable monitor for high level Cr(VI) occupational exposure.

Simultaneous analysis of nucleotide sugars metabolism by ion-pair reversed-phase HPLC and LC-ESI-MS

Simultaneous analysis of nucleotide sugars metabolism by ion-pair reversed-phase HPLC and LC-ESI-MS

Proteome Analysis of Sorangium cellulosum Employing 2D-HPLC-MS/MS and Improved Database Searching Strategies for CID and ETD Fragment Spectra

Shotgun proteome analysis of the myxobacterial model strain for secondary metabolite biosynthesis Sorangium cellulosum was performed employing off-line two-dimensional high-pH reversed-phase HPLC x low-pH ion-pair reversed-phase HPLC and dual tandem mass spectrometry with collision-induced dissociation (CID) and electron transfer dissociation (ETD) as complementary fragmentation techniques. Peptide identification using database searching was optimized for ETD fragment spectra to obtain the maximum number of identifications at equivalent false discovery rates (1.0%) in the evaluation of both fragmentation techniques. In the database search of the CID MS/MS data, the mass tolerance was set to the well-established 0.3 Da window, whereas for ETD data, it was widened to 1.1 Da to account for hydrogen-rearrangement in the radical-intermediate of the peptide precursor ion. To achieve a false discovery rate comparable to the CID results, we increased the significance threshold for peptide identification to 0.001 for the ETD data. The ETD based analysis yielded about 74% of all peptides and about 78% of all proteins compared to the CID-method. In the combined data set, 952 proteins of S. cellulosum were confidently identified by at least two peptides per protein, facilitating the study of the function of regulatory proteins in the social myxobacteria and their role in secondary metabolism.

Improving Peptide Identification in Proteome Analysis by a Two-Dimensional Retention Time Filtering Approach

The combination of a two-dimensional peptide separation scheme based on reversed-phase and ion-pair reversed phase HPLC with a computational method to model and predict retention times in both dimensions is described. The algorithm utilizes statistical learning to establish a retention model from about 200 peptide retention times and their corresponding sequences. The application of retention time prediction to the peptides facilitated an increase in true positive peptide identifications upon lowering mass spectrometric scoring thresholds and concomitantly filtering out false positives on the basis of predicted retention times. An approximately 19% increase in the number of peptide identifications at a q-value of 0.01 was achievable in a whole proteome measurement.

Fluorometric determination of pantothenic acid in human urine by isocratic reversed-phase ion-pair high-performance liquid chromatography with post-column derivatization

We describe here a method for the determination of pantothenic acid, vitamin B5, in human urine by isocratic reversed-phase ion-pair HPLC with post-column derivatization. Pantothenic acid in urine was separated using a Tosoh ODS-80Ts (4.6 i.d.×250mm) column with phosphate–sodium hydroxide buffer (pH 7.0) containing dodecyltrimethylammonium chloride. Following the isolation of pantothenic acid it was decomposed to pantoic acid and β-alanine by alkali treatment. The product β-alanine was post-derivatized to the fluorescent 1-alkylthio-2-alkylisoindole with orthophthaldialdehyde in the presence of 3-mercaptopropionic acid. In the proposed method, a urine sample can be directly injected into a HPLC system without any pre-clean up treatment. The limit of detection was 3pmol (ca. 650pg) per 20μL of urine at a signal-to-noise ratio of 5:1 and the limit of quantification was 5pmol (ca. 1000pg) per 20μL of urine, which was sufficiently sensitive for the determination of pantothenic acid in human urine. The total time required for the analysis was ca. 25min. The proposed method can be used to assess the pantothenic acid content of human urine as an alternative to the standard microbioassay for pantothenic acid.

Solid-Phase Extraction Followed by HPLC for the Determination of Phosphorus(V) and Arsenic(V)

Traces of P(V) and As(V) in sample solutions were converted into heteropoly molybdic acids at pH 1.5 and collected onto polyoxyethylene(20)-4-isononylphenoxy ether-coated Amberlite XAD-4 particles. The desorption was carried out with 0.3 M tetramethylammonium hydroxide solution for the HPLC analysis. Molybdophosphoric and molybdoarsenic acids were separated from excess molybdate by ion-pair reversed phase HPLC, though the peaks of P(V) and As(V) could not be resolved. However, the selective decomposition of molybdoarsenic acid with citric acid allowed the differential determination of P(V) and As(V) at the ng mL(-1) level. The proposed method was applied to the analysis of high-purity iron for P and As.

Ultra performance liquid chromatography analysis of adenine nucleotides and creatine derivatives for kinetic studies

A rapid method for simultaneous quantification of compounds participating in energy metabolism of cardiac muscle cells (creatine (Cr), phosphocreatine (PCr), ADP, and ATP) is described where a conventional ion-pair reversed phase HPLC separation has been improved by introducing the method based on the recently developed ultra performance liquid chromatography (UPLC) technique. In the 0.005–1 mM concentration range, the calibration curves for Cr, ADP, and ATP as pure standard compounds were fitted by the polynomic relationship y = y0 + ax – bx 2 at a high confidence level (R > 0.999 in all cases) and that for PCr by a simple linear relationship with R > 0.998. The method was applied for the study of the kinetics of PCr production by permeabilized cardimyocytes due to the cellular oxydative phosphorylation reactions. The determined steady-state levels of ADP and ATP as well as the rate of PCr production in different conditions can be used for the verification of the results of mathematical modelling of cardiomyocyte functioning.

A sequential extraction procedure for an insight into selenium speciation in garlic

A sequential extraction procedure was developed for the fractionation of different classes of selenium species present in garlic. The consecutive steps included leaching with water, extraction of cell-wall bound species after lysis with a mixture of cellulase, chitinase and β-glucanase completed by a proteolytic attack, extraction with HCl to liberate the residual organic bound species and finally, extractions with sulfite solution and CS 2 to complete the mass balance by the recovery of Se 0 and Se 2−, respectively. Selenium speciation in the aqueous fractions was probed by anion-exchange and ion-pairing reversed-phase HPLC–ICP MS after purification by preparative size-exclusion LC. It was found to be strongly affected by the sample redox conditions. The peak identity was matched with a mixture of 9 compounds expected to be present in allium plants; electrospray QTOF MS turned out to be unsuccessful. Selenite, selenate and selenomethionine were the dominating species present.

Determination of leukocyte DNA 6-thioguanine nucleotide levels by high-performance liquid chromatography with fluorescence detection

A HPLC method for determination of 6-thioguanine nucleotide in DNA was developed. Leukocyte DNA was isolated from peripheral blood, derivatized with chloroacetaldehyde and the formed etheno derivatives N 2,3-etheno 6-thioguanine (ɛ6TG), 1,N 6-etheno adenine (ɛA) and N 2,3-etheno guanine (ɛG) were released from the DNA backbone by hydrolysis at pH 6.0 and 80 °C for 60 min. After extraction of ɛ6TG by immobilized metal ion affinity chromatography (IMAC) the sample was analysed by ion-pair reversed-phase HPLC with fluorescence detection. The limit of quantification was 9.0 nM and the intra- and interday precision ranged from 2.8 to 15.5%. In a small cohort of eight children with acute lymphoblastic leukaemia (ALL), a median of one 6-thioguanine base was found for each 3000 normal bases (range 1:2000–1:11000).

Two-Dimensional Reversed-Phase × Ion-Pair Reversed-Phase HPLC: An Alternative Approach to High-Resolution Peptide Separation for Shotgun Proteome Analysis

A two-dimensional separation scheme for shotgun proteome analysis employing high-pH reversed-phase HPLC in the first and low-pH ion-pair reversed-phase HPLC in the second dimension (RP x IP-RP-HPLC) has been developed and evaluated. Compared to the classical strong cation exchange x ion-pair reversed-phase (SCX x IP-RP-HPLC) approach, the RP x IP-RP-HPLC system was characterized by a lower degree of orthogonality, which was, however, more than counterbalanced by higher separation efficiency, more homogeneous distribution of peptide elution, and easier experimental handling. Peptide fragment fingerprinting by electrospray-ionization tandem mass spectrometry (ESI-MS/MS) was employed for peptide detection and identification. About 13% more peptides and 7% more proteins could be identified with the alternative approach in 30% less analysis time, enabling the analysis of the proteome of Corynebacterium glutamicum with a coverage of 24.9% (745 proteins). Combining the identification results both of the SCX- x IP-RP-HPLC-ESI-MS/MS and RP- x IP-RP-HPLC-ESI-MS/MS methods, a total of 871 proteins were identified in a cytosolic protein preparation, which represented 29.1% of all proteins annotated in the genome of C. glutamicum.

On-Line HPLC/ESI-MS Separation and Characterization of Hyaluronan Oligosaccharides from 2-mers to 40-mers

A new method for the separation and identification of oligosaccharides obtained by enzymatic digestion of hyaluronic acid (HA) with hyaluronidase (EC 3.2.1.35) using on-line high-performance liquid chromatography/electrospray mass spectrometry (HPLC/ESI-MS) is presented. Reversed-phase ion pairing-HPLC, based on tributylamine salts and a volatile mobile phase, provided excellent chromatographic resolution and separation was achieved for HA oligosaccharides containing 2-40 monomers (from 2- to 40-mers). Using the on-line ion trap mass analyzer, complete identification and structural information for each HA oligomer species was obtained. In particular, a series of negatively charged species of different m/z ratios are seen for each oligosaccharide. Smaller HA species, from 2- to 4-mers, exhibit mainly [M-H](-1) anions, whereas the 6-10-mers exist predominantly as the charge state of -2. The HA oligomers from 12- to 18-mers are mainly represented by [M-3H](-3) anions while species from 20- to 28/30-mers are characterized by a charge state of -4. HA oligosaccharides from 32- to 40-mers exist as [M-5H](-5) anions. Furthermore, for smaller HA species, from 4/6- to 18/20-mers, ESI-MS revealed, generally in low relative abundance, anions related to the loss of one/two monosaccharide unit(s) from the oligomers, and no odd-numbered anions were produced for HA species greater than 20-mers.

Comparison between immobilized artificial membrane (IAM) HPLC data and lipophilicity in n-octanol for quinolone antibacterial agents

The membrane phospholipid affinity of ten quinolone antibacterial agents, including both acidic and zwitterionic compounds, was measured by HPLC on two different immobilized artificial membrane (IAM) stationary phases, namely IAM.PC.MG and IAM.PC.DD2; it is expressed as the logarithm of the retention factor measured with (or extrapolated to) 100% aqueous eluent at pH 7.0, log k w IAM . Quinolones are a class of highly potent, orally active, broad-spectrum antibacterial agents. For these compounds, lipophilicity values in n-octanol found in the literature, either calculated or measured, are not consistent with each other and are too low to be compatible with their pharmacokinetic properties. The log k w IAM values obtained in this study showed no relation with any of the lipophilicity values in the literature (clog P(a), clog P(b), MLP, log D 7.4). In contrast, they were collinear with a new lipophilicity scale we had previously obtained by an original ion-pair reversed-phase HPLC method set up to estimate the lipophilicity of the neutral forms, log P N . Moreover, when comparing the retention of quinolones on IAM to the retention of structurally unrelated neutral compounds, we observed that they interact with phospholipids with the same affinity as neutral isolipophilic compounds. The use of an eluent at pH 5.5, instead of pH 7.0, increased the retention on IAM not only for acidic, but also for zwitterionic congeners, indicating that phospholipid affinity is enhanced in the experimental conditions that depress the ionization of the acidic function, even when the ionization of the amino function increases simultaneously. To gain an insight into the mechanism of quinolones/serum-protein interactions, we investigated about possible relationships between quinolones affinity data for serum proteins and IAM data. Quinolone affinity for both HSA and AGP was already demonstrated poorly related to n-octanol lipophilicity values, probably due to the occurrence of electrostatic interactions. Only poor relationships were found between IAM and HSA affinity data, whereas quite good relationships were found with AGP affinity data. However, IAM.PC.DD2 data correlated better than those on IAM.PC.MG with quinolone affinity for both serum-proteins, mainly due to the fact that IAM.PC.MG phase is scarcely discriminative for the compounds with the highest retention values. The results suggest that IAM retention can produce a lipophilicity scale that, unlike solvent/water partition coefficients, is consistent with the pharmacokinetic behaviour of zwitterionic quinolones.

Ion-Pair Reversed-Phase HPLC Method for Determination of Sodium Tanshinone IIA Sulfonate in Biological Samples and Its Pharmacokinetics and Biodistribution in Mice

The ion-pair reversed-phase HPLC method for determination of sodium tanshinone IIA sulfonate (STS) in various biological samples was for the first time developed and validated, and was applied for pharmacokinetics and tissue distribution studies of intravenously administrated STS in mice. A linear relation was found between peak area and STS concentrations within the ranges of 0.1-5 micraog/ml for plasma, 0.1-5 microg/g of tissue for kidney homogenate, 0.1-20 microg/g of tissue for liver homogenate, 0.1-1 microg/g of tissue for heart, spleen and lung homogenates, respectively. In plasma and tissues, the limit of quantification (LOQ) and the limit of detection (LOD) for STS were 100 ng/ml and 20 ng/ml. In all biological specimens, the average inter- and intra-day precision of STS were within 4.9%. The recoveries were more than 92% at all concentration levels in each type of biological specimens. STS plasma concentration-time data were best fitted with a two-compartment model, characterized by an initial rapid phase of drug concentration decrease, and a slower terminal elimination phase. The pharmacokinetics of STS was characterized with a distribution half-life (t(1/2alpha)) of 1.2+/-0.18 min, a terminal half-life (t(1/2beta)) of 21.6+/-2.4 min, a distribution volume (V) of 0.057+/-0.011 l/kg, a plasma clearance (CL) of 0.86+/-0.12 l/h/kg and an AUC(0-infinity) of 58.41+/-6.21 microg x h/ml. STS was widely distributed into most tissues and was obviously accumulated in liver. This results indicated that STS may be promising to treat liver disease.

Enzymatically Active N-Deacetylase/N-Sulfotransferase-2 Is Present in Liver but Does Not Contribute to Heparan Sulfate N-Sulfation

Heparan sulfate (HS) proteoglycans influence embryonic development through interactions with growth factors and morphogens. The interactions depend on HS structure, which is largely determined during biosynthesis by Golgi enzymes. NDST (glucosaminyl N-deacetylase/N-sulfotransferase), responsible for HS N-sulfation, is a key enzyme directing further modifications including O-sulfation. To elucidate the roles of the different NDST isoforms in HS biosynthesis, we took advantage of mice with targeted mutations in NDST1 and NDST2 and used liver as our model organ. Of the four NDST isoforms, only NDST1 and NDST2 transcripts were shown to be expressed in control liver. The absence of NDST1 or NDST2 in the knock-out mice did not affect transcript levels of other NDST isoforms or other HS modification enzymes. Although the sulfation level of HS synthesized in NDST1-/- mice was drastically lowered, liver HS from wild-type mice, from NDST1+/-, NDST2-/-, and NDST1+/- / NDST2-/- mice all had the same structure despite greatly reduced NDST enzyme activity (30% of control levels in NDST1+/- / NDST2-/- embryonic day 18.5 embryos). Enzymatically active NDST2 was shown to be present in similar amounts in wild-type, NDST1-/-, and NDST1+/- embryonic day 18.5 liver. Despite the substantial contribution of NDST2 to total NDST enzyme activity in embryonic day 18.5 liver (approximately 40%), its presence did not appear to affect HS structure as long as NDST1 was also present. In NDST1-/- embryonic day 18.5 liver, in contrast, NDST2 was responsible for N-sulfation of the low sulfated HS. A tentative model to explain these results is presented.