Ion-pair Reversed-phase High-performance Liquid Chromatography Research Articles

分離分析の基礎―ＨＰＬＣを中心に―

High-performance liquid chromatography (HPLC) is one of the most useful methods for both qualitative and quantitative analysis of individual compounds that may be present in a sample. Researchers can select an appropriate method in various combinations of stationary phases with mobile phases depending on an objective compound and other matrix in a sample. This review aims to provide the outline of separation analysis and HPLC, including the history of chromatography, HPLC device configuration, typical separation modes, i.e., reversed-phase, normal-phase, ion-exchanged, ion-pair reverse-phase, and size exclusion HPLC, typical methods for amino acid analysis, key parameters of chromatography performance, typical procedure for optimum separation condition, usefulness of column downsizing, and method robustness, to the beginners of HPLC operations.

Intracellular nucleotide and nucleotide sugar contents of cultured CHO cells determined by a fast, sensitive, and high-resolution ion-pair RP-HPLC

Analysis of intracellular nucleotide and nucleotide sugar contents is essential in studying protein glycosylation of mammalian cells. Nucleotides and nucleotide sugars are the donor substrates of glycosyltransferases, and nucleotides are involved in cellular energy metabolism and its regulation. A sensitive and reproducible ion-pair reverse-phase high-performance liquid chromatography (RP-HPLC) method has been developed, allowing the direct and simultaneous detection and quantification of some essential nucleotides and nucleotide sugars. After a perchloric acid extraction, 13 molecules (8 nucleotides and 5 nucleotide sugars) were separated, including activated sugars such as UDP-glucose, UDP-galactose, GDP-mannose, UDP- N-acetylglucosamine, and UDP- N-acetylgalactosamine. To validate the analytical parameters, the reproducibility, linearity of calibration curves, detection limits, and recovery were evaluated for standard mixtures and cell extracts. The developed method is capable of resolving picomolar quantities of nucleotides and nucleotide sugars in a single chromatographic run. The HPLC method was then applied to quantify intracellular levels of nucleotides and nucleotide sugars of Chinese hamster ovary (CHO) cells cultivated in a bioreactor batch process. Evolutions of the titers of nucleotides and nucleotide sugars during the batch process are discussed.

Selenium speciation in Agaricus bisporus and Lentinula edodes mushroom proteins using multi-dimensional chromatography coupled to inductively coupled plasma mass spectrometry

In this study, selenium species from Se containing proteins in mushrooms ( Agaricus bisporus and Lentinula edodes) were investigated with size-exclusion liquid chromatography coupled to UV and inductively coupled plasma mass spectrometry (ICP-MS). Different protein extraction protocols were investigated. Variability of the fractionation patterns with three extraction media (0.1 M NaOH, 30 mM Tris–HCl, and enzymatic digestions) was evaluated for both mushroom types. A 24 h Tris–HCl extraction followed by acetone addition was found to be optimal for protein precipitation. Presumably protein bound selenoamino acids were released using enzymes (proteinase K, protease XIV and trypsin). The selenium speciation of the proteolytic extract of the water soluble proteins fraction was carried out by using reversed-phase ion-pairing high performance liquid chromatography (RP-HPIPC) coupled on-line to ICP-MS for selenium specific detection. Selenocystine, selenomethionine, methylselenocysteine and inorganic selenium were established in both samples utilizing retention time standards and standard additions to the sample.

Polystyrene/Divinylbenzene Based Monolithic and Encapsulated Capillary Columns for the Analysis of Nucleic Acids by High-Performance Liquid Chromatography-Electrospray Ionisation Mass Spectrometry

Monolithic capillary columns are prepared by copolymerization of styrene and divinylbenzene, encapsulated capillary columns by immobilizing silica particles with different pore sizes inside a 200 μm i.d. fused silica capillary by encapsulation of the derivatized silica sorbent in a poly(styrene/divinylbenzene) (PS/DVB) matrix. Both allow the rapid and highly efficient separation of single- and double-stranded DNA by ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC). The high resolving power of monolithic and encapsulated capillary columns can be utilized for mutation screening in polymerase chain reaction (PCR) amplified polymorphic loci by denaturing HPLC (DHPLC). Recognition of mutations is based on the separation of homo- and heteroduplex species by IP-RP-HPLC under denaturing conditions, resulting in characteristic peak patterns both for homozygous and heterozygous samples. Separations can be readily hyphenated to electrospray ionization-mass spectrometry.

Flow-injection in-line complexation for ion-pair reversed phase high performance liquid chromatography of some metal-4-(2-pyridylazo) resorcinol chelates

Flow injection (FI) was coupled to ion-pair reversed phase high performance liquid chromatography (IP-RPHPLC) for the simultaneous analysis of some metal-4-(2-pyridylazo) resorcinol (PAR) chelates. A simple reverse flow injection (rFI) set-up was used for in-line complexation of metal-PAR chelates prior to their separation by IP-RPHPLC. The rFI conditions were: injection volume of PAR 85 μL, flow rate of metal stream 4.5 mL min −1, concentration of PAR 1.8 × 10 −4 mol L −1 and the mixing coil length of 150 cm. IP-RPHPLC was carried out using a C 18 μBondapak column with the mobile phase containing 37% acetonitrile, 3.0 mmol L −1 acetate buffer pH 6.0 and 6.2 mmol L −1 tetrabutylammonium bromide (TBABr) at a flow rate of 1.0 mL min −1 and visible detection at 530 and 440 nm. The analysis cycle including in-line complexation and separation by IP-RPHPLC was 16 min, which able to separate Cr(VI) and the PAR chelates of Co(II), Ni(II) and Cu(II).

Determination of Ibandronate and its Degradation Products by Ion-Pair RP LC with Evaporative Light-Scattering Detection

A simple method has been developed for analysis of ibandronate and related substances by ion-pair reversed-phase high-performance liquid chromatography (RPIC) with evaporative light-scattering detection (ELSD). After optimization of the chromatographic conditions satisfactory separation of the compounds was achieved on an Intersil C8 column with an isocratic mobile phase—8:4:88 (v/v) acetonitrile–methanol–12 mM ammonium acetate buffer containing 35 mM n-amylamine (pH 7.0). The mobile phase flow rate was 1.0 mL min−1. The calibration plot was linear in the range 352 to 1760 µg mL−1 for ibandronate. The precision and reproducibility were 0.3% and 0.5%, respectively. The average recovery of ibandronate was 100.4% and RSD was 0.6%. The method was validated and shown to be precise, accurate, and specific for assay of ibandronate in bulk material and dosage forms. The proposed liquid chromatographic method can be satisfactorily used for quality control of ibandronate.

Simultaneous determination of adenine and guanine in ruminant bacterial pellets by ion-pair HPLC

An ion-pair reversed-phase high-performance liquid chromatography with gradient elution and UV detection was used to measure adenine (A) and guanine (G) in lyophilized bacterial pellets from ruminants using allopurinol as internal standard. The separation was performed on a Symmetry C18 column and the detection was monitored at 280 nm. Calibration curves were found to be linear in the concentration range from 5 to 50 mg/l with correlation coefficients (r2)>0.999. Mean recoveries of A and G standards added to bacterial samples were 102.2 and 98.2, respectively. The method proposed yielded sharp, well-resolved peaks within 25 min and was successfully applied for the determination of A and G in bacterial pellets.

Detection of DNA Sequence Variations in Homo- and Heterozygous Samples via Molecular Mass Measurements by Electrospray Ionization Time-of-Flight Mass Spectrometry

The potential of ion-pair reversed-phase high-performance liquid chromatography on-line hyphenated to electrospray ionization time-of-flight mass spectrometry for the characterization of polymerase chain reaction (PCR) amplified nucleic acids was evaluated. For that purpose, a "SNP toolbox" was constructed by cloning and PCR-mediated site-directed in vitro mutagenesis at nucleotide position (ntp) 16,519 of a sequence-verified fragment of the human mitochondrial genome (ntps 15,900-599). Confirmatory sequencing demonstrated that within the sequences of the clones one and the same base was mutated to all other bases. Using these clones or equimolar mixtures of these clones as PCR templates, 51-401-bp-long amplicons were generated, which were used to determine the upper size limits of PCR products for the unequivocal detection of sequence variations in homo- and heterozygous samples. Based on the high mass spectrometric performance of the applied time-of-flight mass spectrometer, the unequivocal genotyping of all kinds of single base exchanges in PCR amplicons from heterozygous samples with lengths up to 254 base pairs (bp) was demonstrated. Considering homozygous samples, the successful genotyping of single base substitutions in up to 401-bp-long PCR products was possible. Consequently, the described hyphenated technique represents one of the most powerful mass spectrometric genotyping assays available today.

Identification of Bacteria by Polymerase Chain Reaction Followed by Liquid Chromatography−Mass Spectrometry

Bloodstream infections are an important cause of serious morbidity and mortality. Rapid detection and identification of specific pathogens from blood or other clinical specimens could improve the rational use of antimicrobial therapy in clinical medicine and have a great impact on the outcome of patients with systemic infections. Polymerase chain reaction using generic primers was used to amplify genomic DNA of different bacterial strains. The identification was accomplished by measuring the molecular masses of the PCR products using ion-pair reversed-phase high-performance liquid chromatography hyphenated to electrospray ionization mass spectrometry. DNA from 10 bacterial species was amplified by PCR, and the resulting amplification products were analyzed. In all cases, the measured molecular masses of the PCR products matched the theoretical value for the species-specific DNA sequence. However, three pairs of bacteria could not be distinguished since the theoretical difference in amplicon molecular mass was < 1.0 Da (different sequence, same base composition of amplicon). Determination of intra- and interday mass reproducibility resulted in relative standard deviations of 0.0030 and 0.018%, respectively. The limit of detection of the presented method was shown to be 0.5 genome equivalents/PCR. The specificity of the method in a human background was successfully tested by amplifying and analyzing 1000-10000 genome equivalents of Staphylococcus aureus spiked into human plasma.

Pharmacokinetics of gemcitabine in Chinese patients with non-small-cell lung cancer

To determine the pharmacokinetics of gemcitabine (2',2'-difluorodeoxycytidine) in Chinese non-small-cell lung cancer (NSCLC) patients. Six study subjects were administered gemcitabine at a fixed dose rate of 10 mg/m(2) per min (1200 mg/m(2), two hours infusion), and carboplatin and plasma gemcitabine concentrations were measured by ion-pair reversed-phase high-performance liquid chromatography (HPLC). 3P97 Pharmaceutical Kinetics Software was used for the calculation of pharmacokinetic parameters. The obtained mean parameters, elimination half life (t(1/2)) (10.67+/-3.38 min), area under the curve (AUC) (7.55+/-1.53 (microg x h)/ml), and clearance (CL) (3940.05+/-672.08 ml/min), were consistent with those reported in literature. The hematologic toxicology result showed that the regimen was effective on and tolerated by the patients.

Analysis of Biological and Synthetic Ribonucleic Acids by Liquid Chromatography−Mass Spectrometry Using Monolithic Capillary Columns

Ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) has been evaluated as a method for the fractionation and desalting of ribonucleic acids prior to their characterization by electrospray ionization mass spectrometry. Monolithic, poly(styrene-divinylbenzene)-based capillary columns allowed the rapid and highly efficient fractionation of both synthetic and biological ribonucleic acids. The common problem of gas-phase cation adduction that is particularly prevalent in the mass spectrometric analysis of ribonucleic acids was tackled through a combination of chromatographic purification and the addition of ethylenediaminetetraacetic acid to the sample at a concentration of 25 mmol/L shortly before on-line analysis. For RNA molecules ranging in size from 10 to 120 nucleotides, the mass accuracies were typically better than 0.02%, which allowed the characterization and identification of failure sequences and byproducts with high confidence. Following injection of a 500 nL sample onto a 60 x 0.2 mm column, the limit of detection for a 120-nucleotide ribosomal RNA transcript from Escherichia coli was in the 50-80 fmol range. The method was applied to the analysis of synthetic oligoribonucleotides, transfer RNAs, and ribosomal RNA. Finally, sequence information was derived for low picomole amounts of a 32-mer RNA upon chromatographic purification and tandem mass spectrometric investigation in an ion trap mass spectrometer. Complete series of fragment ions of the c- and y-types could be assigned in the tandem mass spectrum. In conclusion, IP-RP-HPLC using monolithic capillary columns represents a very useful tool for the structural investigation and quantitative determination of RNAs of synthetic and biological origin.

Electrospray ionization mass spectrometric analysis of nucleic acids using high-throughput on-line desalting.

A rapid on-line desalting method utilizing ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) was employed in tandem with negative electrospray ionization mass spectrometry (ESI-MS) for the routine analysis of nucleic acids. Desalting was performed on a short 10 x 2.1 mm guard column packed with 3.5 microm C(18) sorbent. The HPLC system was connected in-line to an orthogonal ESI-TOF mass spectrometer via a six-port, two-position switching valve, allowing desalting followed by mass analysis of nucleic acids. Duty cycle times for the method were as low as 1.5 min per sample. This allowed for the analysis of approximately 950 samples per 24-h time period, which is suitable for medium- to high-throughput applications. Average mass accuracy was determined to be 80 ppm for oligonucleotides up to 110 mer in length with external calibration. The method was utilized for synthetic oligonucleotide quality control and analysis of DNA genotyping fragments.

Stabilization and HPLC Analysis of Betamethasone Sodium Phosphate in Plasma

The analysis of corticosteroid prodrugs in pharmacokinetic (PK) studies poses the risk of overestimation of corticosteroid concentrations due to in vitro hydrolysis of prodrugs after sample collection. This study tests the effectiveness of enzyme inhibitors as stabilizers for betamethasone sodium phosphate (BSP) in pregnant sheep plasma samples collected during PK studies with betamethasone (BET) and provides simultaneous high-performance liquid chromatography analysis of BSP and BET. A rapid, sensitive, and specific ion-paired reversed-phase high-performance liquid chromatography assay for simultaneous measurement of BET and BSP in plasma was developed. This assay was used for analyzing samples from an in vitro prodrug hydrolysis study. Enzyme inhibitors tested were sodium arsenate (Na2HAsO4) and ethylenediaminetetraacetic acid. The BSP was administered intramuscularly to three pregnant sheep to assess in vivo PK. Samples were split with part treated with Na2HAsO4 and part left natural. In vitro hydrolysis of BSP in plasma to BET could be completely inhibited by Na2HAsO4, but not by ethylenediaminetetraacetic acid. The PK study showed lower concentrations of BET in samples with Na2HAsO4 compared with natural samples. This study demonstrates that artifacts in PK profiles of corticosteroids due to in vitro prodrug hydrolysis can be prevented by sample treatment with enzyme inhibitors. © 2004 Wiley-Liss, Inc. and the American Pharmacists Association

Applicability of tandem mass spectrometry to the automated comparative sequencing of long-chain oligonucleotides

An algorithm for the comparative sequencing (COMPAS) of oligonucleotides is shown to be suitable for the sequence verification of nucleic acids ranging in length from a few to 80 nucleotides. The algorithm is based on the matching of a fragment ion spectrum generated by collision-induced dissociation to m/ z values predicted from a known reference sequence employing established fragmentation pathways. Prior to mass spectrometric investigation, the oligonucleotides were on-line purified by ion-pair reversed-phase high-performance liquid chromatography using monolithic separation columns. This study evaluated the potential and the limits of COMPAS regarding the length and the charge state of oligonucleotides, the selected collision energy, and the analyzed amount of sample using a quadrupole ion trap mass spectrometer. The results revealed that oligonucleotides could be very reliably re-sequenced up to 60-mers, although the algorithm was successfully used to even verify sequences up to 80-mers. The relative collision energy was typically in the range between 13 and 20%, which allowed in most cases a verification of the reference sequence in a window of at least three consecutive collision energies. To select a proper charge state for fragmentation, a compromise had to be found between high signal intensity and low charge state. Furthermore, by reducing the eluent flow rate during elution of the oligonucleotide, the sequence of a 50-mer was successfully verified from the analysis of 295 fmol of the raw product. COMPAS was proven to be reproducible and was applied to the genotyping of the polymorphic, Y-chromosomal locus M9 contained in a 62-base pair polymerase chain reaction product.

Systematic study of long-term stability of 3,4-dihydroxyphenylglycol in plasma for subsequent determination with liquid chromatography

The effect of three storage temperature levels (i.e. +4, −20 and −80 °C) and time intervals from sampling (3, 6 and 9 months) on the degradation of 3,4-dihydroxyphenylglycol (DHPG) and norepinephrine (NE) was investigated in a systematic study. Extracted human plasma samples and acidified standard solutions were stored for long periods (up to 9 months) without the addition of any stabilizing agent. DHPG and NE values, determined using a ion-pair reversed-phase high-performance liquid chromatography method with electrochemical detection of coulometric type (IP-RP-HPLC–CD), remained constant over time in those plasma samples and standard solutions that had been stored at the lowest storing temperature (i.e. −80 °C). The expected degradation was observed at higher temperature levels. Plasma and standard DHPG degradation can, therefore, be prevented by storing samples at a lower temperature than previously suggested with no need to add any stabilizing agent.

Identification and characterization of selenium species in enriched green onion (Allium fistulosum) by HPLC-ICP-MS and ESI-ITMS

In this study speciation of selenium in selenium-enriched green onions (Allium fistulosum) was done with reversed-phase ion-pairing high performance liquid chromatography (RP-IP-HPLC) and size-exclusion chromatography (SEC) coupled on-line to ICP-MS for selenium specific detection. The plant extract obtained using sodium hydroxide (0.1 mol l−1) analyzed by SEC (CAPS 10 mmol l−1, pH 10.0) with ICP-MS detection showed the incorporation of selenium in both high (∼12 kDa) and low molecular weight (0.36–2 kDa) fractions. Presumably protein bound selenoamino acids were released using enzymes (Proteinase K and Protease XIV) and selenoamino acids found in cytosol in their free form were extracted using 0.1 M HCl. The extracts were analyzed for speciation studies by RP-IP-HPLC [0.1% (v/v) heptafluorobutyric acid, 5% (v/v) methanol, pH 2.5]. Matching the chromatographic retention times with commercially available standards provided evidence for the presence of Se-cystine, methylselenocysteine, Se-methionine, and inorganic selenium. Electrospray ionization ion trap mass spectrometry (ESI-ITMS) confirmed the presence of γ-glutamyl-Se-methylselenocysteine in green onions as has been reported in other onion types.

SIRPH analysis: SNuPE with IP-RP-HPLC for quantitative measurements of DNA methylation at specific CpG sites.

This chapter describes a detailed protocol using single-nucleotide primer extension (SNuPE) for quantitative analysis of DNA methylation on specific CpG sites. The first step DNA sample to be studied is treated with sodium bisulfite, which converts selectively unmethylated cytosines to uracil, while methylated cytosines remain unconverted. Subsequently, a SNuPE reaction is performed, with an oligo just flanking a CpG site, using a purified polymerase chain reaction product derived from bisulfite-treated DNA as a template. The oligo is extended by either ddCTP or ddTTP depending on whether the site is methylated or unmethylated, respectively. The reaction is quantitative and linear, and two to three sites can be studied simultaneously in a multiple reaction. The SNuPE product, without further purification, is separated by ion-pair reverse-phase (IP RP) high-performance liquid chromatography (using an alkylated nonporous polysterene-divinylbenzene cartridge) that allows an easy, semiautomated method for separation of the extended and unextended products and an accurate quantification of the extended products. The ratio of the ddCTP to the ddTTP gives the fraction of the methylated cytosines at that specific CpG site.

Sulfonylcalix[4]arenetetrasulfonate as pre-column chelating reagent for selective determination of aluminum(III), iron(III), and titanium(IV) by ion-pair reversed-phase high-performance liquid chromatography with spectrophotometric detection

Sulfonylcalix[4]arenetetrasulfonate (SO 2CAS) has been examined as a pre-column chelating reagent for ultratrace determination of metal ions by ion-pair reversed-phase high-performance liquid chromatography with spectrophotometric detection. Metal ions were converted into the SO 2CAS chelates in an acetic buffer solution (pH 4.7). The chelates were injected onto a n-octadecylsilanized silica-type Chromolith™ Performance RP-18e column and were eluted using a methanol (50 wt.%)-water eluent (pH 5.6) containing tetra- n-butylammonium bromide (7.0 mmol kg −1), acetate buffer (5.0 mmol kg −1), and disodium ethylendiamine- N, N, N′, N′-tetraacetate (0.10 mmol kg −1). Under the conditions used, Al(III), Fe(III), and Ti(IV) were selectively detected among 21 kinds of metal ions [Al(III), Ba(II), Be(II), Ca(II), Cd(II), Co(II), Cr(III), Cu(II), Fe(III), Ga(III), Hf(IV), In(III), Mg(II), Mn(II), Mo(VI), Ni(II), Pb(II), Ti(IV), V(V), Zn(II), and Zr(IV)]. The detection limits on a 3σ blank basis were 8.8 nmol dm −3 (0.24 ng cm −3) for Al(III), 7.6 nmol dm −3 (0.42 ng cm −3) for Fe(III), and 17 nmol dm −3 (0.80 ng cm −3) for Ti(IV). The practical applicability of the proposed method was checked using river and tap water samples.

Single nucleotide polymorphisms detected by Temperature-modulated high-performance liquid chromatography

Single nucleotide polymorphisms (SNPs) are new generation markers. An open question in research on SNPs is how to find novel SNPs in a nature population. To this end, we developed an approach of high-throughput genotyping by applying the temperature-modulated heteroduplex analysis (TMHA) based upon ion-pair reversed-phase high-performance liquid chromatography. This approach allowed a very efficient resolution of identically sized PCR products with a single nucleotide change. This approach was also known as denaturing high-performance liquid chromatography (DHPLC). However, the terms of both TMHA and DHPLC should be considered carefully. The term of DHPLC may lead to confusion in annealing and denaturing. Since TMHA can be carried out using gel electrophoresis and HPLC, there is a need to distinguish HPLC from gel electrophoresis. Therefore, we suggest a new term for this technology, which is temperature-modulated high-performance liquid chromatography (TmHPLC). This term not only provides a clear definition for this new method, but also includes both TMHA and HPLC. More importantly, TmHPLC implies that the temperature in HPLC is modulated according to T m of the DNA fragments. Our results show that TmHPLC is a sensitive, accurate and cost effective approach to screening sequence variation in human genome.

Separation of a complex mixture of heparin-derived oligosaccharides using reversed-phase high-performance liquid chromatography

A reversed-phase ion-pairing high-performance liquid chromatography (RP-HPIPC) method for the separation of a complex mixture of heparin-derived oligosacchrides has been developed by a stepwise optimization of the mobile phase, in which the concentration of ion-pairing reagent, mobile phase pH, and acetonitrile concentration were varied. The resolution of more than 30 oligosaccharide components was obtained, under optimized conditions, in an analysis time of less than 30 min. This represents the first RP-HPLC method that can separate a complex mixture of both small and large sulfated oligosaccharides in a single chromatographic step. The heparin-derived oligosaccharides, in this mixture, can also be separated under a second set of RP-HPIPC conditions using a volatile ion-pairing reagent, tributylammonium acetate, to aid in the recovery of individual sulfated oligosaccharides. Moreover, it was possible to replace sodium chloride gradient, required for eluting highly sulfated oligosaccharides, with a fixed, low concentration of a volatile salt, ammonium acetate, by utilizing an acetonitrile gradient. This solvent system might make it possible to directly interface this RP-HPIPC separation with mass spectral analysis.