2D LC Separation and Determination of Bradykinin in Rat Muscle Tissue Dialysate with On-Line SPE-HILIC-SPE-RP-MS

A 2D liquid chromatography (LC) system using hydrophilic interaction chromatography (HILIC) and reversed phase columns has been employed for comprehensive (LC × LC) separation of rat muscle tissue micro-dialysate. Incorporation of an on-line reverse-phase solid phase extraction (SPE) enrichment column in front of the first dimension enabled aqueous samples with high salt concentrations to be injected directly without compromising the chromatographic performance of the HILIC column. Since the SPE enrichment column allowed injection of large sample volumes (e.g. 450 μL), a capillary HILIC column (inner diameter 0.3 mm) could be employed instead of a larger column which is often used in the first dimension to load sufficient amounts of sample. The two chromatographic dimensions were connected using a column selector system with 18, 1.0 mm I.D. C₁₈ “transition” SPE columns. A PLRP C₁₈ column was used in the second dimension. The 2D LC system’s performance was evaluated with a tryptic digest mixture of three model proteins. Good trapping accuracy (HILIC→transition SPE→RP recovery >95%) and repeatability (within-and between day retention time RSDs of first and second dimension chromatography >1%) was achieved. A dialysis sample of rat muscle tissue was separated with the 2D system, revealing complexity and large differences in concentrations of the various compounds present, factors which could potentially interfere with the quantification and monitoring of two target analytes, arg-bradykinin and bradykinin. Subsequently, “Heart-cut” 2D LC-electrospray–mass spectrometry (ESI–MS) with post-column on-line standard injection was employed to monitor arg-bradykinin and bradykinin levels as a function of various muscle conditions. The method’s quantification precision was RSD = 3.4% for bradykinin.     

Comprehensive two-dimensional liquid chromatography

Having nearly exhausted the possibilities for generating peak capacity through improvements in column technology, chromatographers are increasingly looking to alternative ways of maximising chromatographic separation. In recent years there has been increasing activity in the field of comprehensive multidimensional separations to meet analysis demands. Comprehensive two-dimensional liquid chromatography (LC×LC) approaches offer high peak capacity which leads to significantly improved analytical performance over single-column liquid chromatography. There are several closely related avenues available for achieving an LC×LC separation and this review pays special attention to the different valve-based interfaces that have been used to comprehensively couple the first and second dimension columns in LC×LC systems. A brief discussion of column choices for selected applications and the conditions employed is also presented.     

A compact miniaturized continuous flow system for the determination of urea content in milk

A multicommutation-based flow system with photometric detection was developed, employing an analytical microsystem constructed with low temperature co-fired ceramics (LTCC) technology, a solid-phase reactor containing particles of Canavalia ensiformis DC (urease source) immobilized with glutaraldehyde, and a mini-photometer coupled directly to the microsystem which monolithically integrates a continuous flow cell. The determination of urea in milk was based on the hydrolysis of urea in the solid-phase reactor and the ammonium ions produced were monitored using the Berthelot reaction. The analytical curve was linear in the urea concentration range from 1.0 × 10⁻⁴ to 5.0 × 10⁻³ mol L⁻¹ with a limit of detection of 8.0 × 10⁻⁶ mol L⁻¹. The relative standard deviation (RSD) for a 2.0 × 10⁻³ mol L⁻¹ urea solution was lower than 0.4% (n = 10) and the sample throughput was 13 h⁻¹. To check the reproducibility of the flow system, calibration curves were obtained with freshly prepared solutions on different days and the RSD obtained was 4.7% (n = 6). Accuracy was assessed by comparing the results of the proposed method with those from the official procedure and the data are in close agreement, at a 95% confidence level.     

Development of a Validated Stability-Indicating LC Method for Nitazoxanide in Pharmaceutical Formulations

A reversed-phase liquid chromatographic (LC) method was developed for the assay of nitazoxanide (NTZ) in solid dosage formulations. An isocratic LC separation was performed on a Phenomenex Synergi Fusion C₁₈ column (250 mm × 4.6 mm, i.d., 4 μm particle size) using a mobile phase of 0.1% o-phosphoric acid solution, pH 6.0: acetonitrile (45:55, v/v) at a flow rate of 1.0 mL min⁻¹. Detection was achieved with a photodiode array detector at 240 nm. The detector response for NTZ was linear over the concentration range from 2 to 100 μg mL⁻¹ (r = 0.9999). The specificity and stability-indicating capability of the method were proved using stress conditions. The RSD values for intra-day precision were less than 1.0% for tablets and powder for oral suspension. The RSD values for inter-day precision were 0.6 and 0.7% for tablets and powder for oral suspension. The accuracy was 100.4% (RSD = 1.8%) for tablets and 100.9% (RSD = 0.3%) for powder for oral suspension. The limits of quantitation and detection were 0.4 and 0.1 μg mL⁻¹. There was no interference of the excipients on the determination of the active pharmaceutical ingredient. The proposed method was precise, accurate, specific, and sensitive. It can be applied to the quantitative determination of drug in tablets and powder for oral suspension.     

Validated method for the determination of ethylglucuronide and ethylsulfate in human urine

Detection of the alcohol metabolites ethylglucuronide (EtG) and ethylsulfate (EtS) has become routine in many forensic laboratories over the last few years. Most previously published methods using liquid chromatography coupled with electrospray tandem mass spectrometry require a post-chromatographic addition of solvent and/or extensive sample preparation prior to analysis. The aim of the study was to develop a simplified method. To 20 μL urine, internal standard containing EtG-d5 and EtS-d ₅ was added and the mixture was treated with elution buffer internal standard. EtG and EtS were separated using a Shimadzu Prominence high performance liquid chromatography (HPLC) system with a C18 separation column (Restek Ultra Aqueous C18, 4.6 × 150 mm, 5 μm), using isocratic elution with a mobile phase consisting of 10 mM ammonium acetate buffer pH 7 (total run time, 6 min). The compounds were detected using an Applied Biosystems API 5000 liquid chromatography tandem mass spectrometry system (atmospheric pressure chemical ionization, multiple-reaction monitoring mode). The method was fully validated according to international guidelines. The assay was found to be selective for the compounds of interest. It was linear from 0.1 to 10 mg/L for all analytes (R ² > 0.99). Matrix effects studies showed the presence of a slight but consistent ion enhancement (n = 10 different urine samples) at low concentrations and no effects at higher concentrations. Accuracy data were between 0.75% and 8.1% bias for EtG and between −5.0% and −11.3% bias for EtS. Precision data were between 4.3% and 6.9% relative standard deviations (RSD) for EtG and between 6.0% and 7.5% RSD for EtS. No instability was observed after repeated freezing and thawing. This fast, reliable, and accurate method enables the detection and quantification of alcohol metabolites in urine. The method is easier to use and more sensitive than previously published methods.     

Interaction of partially ionized poly(acrylic acid) with a surfactant counterion at constant pH values

Binding of a cationic surfactant ion, dodecylpyridinium ion, to poly(acrylic acids) of low charge densities was examined by potentiometry using surfactant-selective electrodes in the solutions, where the pH was kept constant by employing a pH buffering system. The binding of the surfactant counterions was thus able to be studied at a constant pH during the binding process. The binding took place in two steps, the first cooperative binding step and the second gradual binding step. The critical association concentration decreased as the pH increased, indicating the predominant role of the electric interaction in the binding. The binding isotherms obtained at different but constant pH values were analyzed by the matrix method, taking into account the nearest-neighbor interactions among three different kinds of sites on the polymer: ionized, protonated, and surfactant-bound. The theoretical analysis could describe only the first step but could not explain the second step. A relatively large cooperativity parameter, u, was found for the first step and it can be between 3 × 10³ and 1 × 10⁴. When the ionic strength was decreased tenfold, the cooperativity of the binding decreased (u∼1 × 10³). The binding constants of the isolated site were 5.5–6.0 × 10⁴ kg mol⁻¹ and slightly increased to 6.5 × 10⁴ kg mol⁻¹ as the ionic strength decreased. The deviation of the second step from the theoretical analysis was supposed to arise from a change of proton dissociation constant in the nonpolar space formed by the bound surfactants. Received: 29 November 2000/Accepted: 24 January 2001     

Comprehensive two-dimensional liquid chromatography coupled with mass spectrometry

In this review, instrumental aspects of comprehensive two-dimensional liquid chromatography coupled with mass spectrometry are presented. The milestones of LC×LC are briefly summarized. Instrument configuration, selection of experimental conditions, the different interfaces used in the system and the current applications of LC×LC–MS systems are described.     

Rapid determination of five antibiotic residues in swine wastewater by online solid-phase extraction–high performance liquid chromatography–tandem mass spectrometry

A simple and quick online solid-phase extraction (SPE) coupled to liquid chromatography (LC)/tandem mass spectrometry (MS/MS) for the determination of the five antibiotics (florfenicol, FF; lincomycin, LCM; oxytetracyclin, OTC; tylosin, TS; valnemulin, VLM) in swine wastewater has been developed. After filtration, aliquots (100 μl) of wastewater samples were directly injected to a column-switching LC system. Some matrix interference was removed by washing up SPE column with 0.2% formic acid solution and acetonitrile. Antibiotics eluted from SPE column were separated on analytical column by converting switching valve and were detected by MS/MS. Calibration curves using the method of standard addition had very good correlation coefficients (r > 0.99) in the range of 0.1 to 2 ng/ml. The intra-day precision of the method was less than 12% and the inter-day precision was between 6 to 17%. The detection limits were 0.01–0.1 ng/ml. When this method was applied to wastewater samples in swine facilities, four compounds (LCM, OTC, TS, and VLM) were detected.     

Sensitive determination of nitric oxide in some rat tissues using polymer monolith microextraction coupled to high-performance liquid chromatography with fluorescence detection

A simple, sensitive, selective, and low-cost method is proposed for rapidly determining nitric oxide (NO) in some rat tissues. Polymer monolith microextraction (PMME) using a poly(methacrylic acid–ethylene glycol dimethacrylate) (MAA-EGDMA) monolithic column was combined with derivatization of NO using 1,3,5,7-tetramethyl-8-(3′,4′-diaminophenyl)-difluoroboradiaza-s-indacene (TMDABODIPY), and this was used to analyze the derivatives of NO by high-performance liquid chromatography (HPLC) with fluorescence detection at λ _ex/λ _em = 498/507 nm. The baseline separation of TMDABODIPY and its NO derivative is performed under simple conditions in which a C₁₈ column is used and eluted with 50 mmol L⁻¹ ethanolamine and methanol. The conditions for the extraction of NO derivatives were optimized. The limit of detection of NO was 2 × 10⁻¹² mol L⁻¹ (S/N = 3). The linearity range of the method was 9 × 10⁻¹¹−4.5 × 10⁻⁸ mol L⁻¹. The interday and intraday relative standard deviations were less than 5%. The proposed method was successfully applied to the determination of NO levels in some rat tissue samples including heart, kidney, and liver with recoveries varying from 87.1 to 95.2%.     

Development of a fast and selective method for the sensitive determination of anatoxin-a in lake waters using liquid chromatography–tandem mass spectrometry and phenylalanine-d 5 as internal standard

Anatoxin-a is a potent alkaloid neurotoxin produced by a number of cyanobacterial species and released in freshwaters during cyanobacterial blooms. Its high toxicity is responsible for several incidents of lethal intoxications of birds and mammals around the world; therefore anatoxin-a has to be regarded as a health risk and its concentration in lakes and water reservoirs should be monitored. Phenylalanine is a natural amino acid, also present in freshwaters, isobaric to anatoxin-a, with a very similar fragmentation pattern and LC retention. Since misidentification of phenylalanine as anatoxin-a has been reported in forensic investigations, special care must be taken in order to selectively determine traces of anatoxin-a in the presence of naturally occurring phenylalanine. A fast LC tandem MS method was developed by using a 1.8 μm 50 × 2.1 mm C18 column for the separation of anatoxin-a and phenylalanine, achieving a 3-min analysis time. Isotopically labelled phenylalanine-d ₅ was employed as internal standard to compensate for electrospray ion suppression and sample preconcentration losses. Both compounds were preconcentrated 1,000-fold on a porous graphitic carbon solid-phase extraction (SPE) cartridge after adjustment of sample pH to 10.5. The method was validated by using lake water spiked at four different levels from 0.01 to 1 μg L⁻¹. Anatoxin-a recovery ranged from 73 to 97%, intra-day precision (RSD%) ranged from 4.2 to 5.9, while inter-day precision (RSD%) ranged from 4.2 to 9.1%. Limits of detection and quantification were 0.65 and 1.96 ng L⁻¹ respectively. The method was successfully applied for the detection of anatoxin-a in Greek lakes at concentrations ranging from less than 0.6 to 9.1 ng L⁻¹.     

Adsorptive Voltammetric Studies on the Cerium(III)–Alizarin Complexon Complex at a Carbon Paste Electrode

A novel procedure was developed for the determination of trace cerium on the basis of anodic adsorption voltammetry of the Ce(III)–alizarin complexon (ALC) complex at a carbon paste electrode (CPE). The procedure is convenient to determine cerium individually in the presence of other rare earths because there is a 100 mV difference between the peak potentials of Ce(III)–ALC and other rare earth(III)–ALC complexes in a supporting electrolyte of 0.08 M HAc–NaAc and 0.012 M potassium biphthalate (pH 4.7) when performing linear-scanning from −0.2 to 0.8 V (vs. SCE) at 100 mV/s. The second-order derivative peak currents are directly proportional to the Ce(III) concentration over a range of 6.0 × 10⁻⁹–3.0 × 10⁻⁷ M. The detection limit is as low as 2.0 × 10⁻⁹ M (S/N = 3) for a 120 s preconcentration. An RSD of 3.5% was obtained for 15 determinations of Ce(III) at a concentration of 4.0 × 10⁻⁸ M on the same CPE surface. The method was applied successfully to the determination of cerium in samples of rare earth nodular graphite cast iron.     

A sensitive liquid chromatography–mass spectrometry method for simultaneous determination of alisol A and alisol A 24-acetate from <Emphasis Type="Italic">Alisma orientale</Emphasis> (Sam.) Juz. in rat plasma

A liquid chromatography–mass spectrometry (LC-MS) method was developed and validated for the simultaneous determination of alisol A and alisol A 24-acetate from Alisma orientale (Sam.) Juz. in rat plasma using diazepam as an internal standard. A 200-μl plasma sample was extracted by methyl tert-butyl ether and the separation was performed on Kromasil C₁₈ column (150 × 4.6 mm, 5 μm) with the mobile phase of acetonitrile (containing 0.1% of formic acid)–water (73:27, v/v) at a flow rate of 0.8 ml/min in a run time of 10 min. The two analytes were monitored with positive electrospray ionization by selected ion monitoring mode. The lower limit of quantitation for both alisol A and alisol A 24-acetate were 10 ng/ml. The calibration curves were linear in the measured range 10–1,000 ng/ml for alisol A and 10–500 ng/ml for alisol A 24-acetate. The mean extraction recoveries were above 74.7% for alisol A and above 72.4% for alisol A 24-acetate from biological matrixes. The intra- and inter-day precision for all concentrations of quality controls was lower than 14.1% (RSD %) for each analyte. The accuracy ranged from −12.3% to 9.8% (RE %) for alisol A, and −8.6% to 14.2% (RE %) for alisol A 24-acetate. The method was successfully applied to the study on the pharmacokinetics of alisol A and alisol A 24-acetate in rat plasma.     

HPLC determination of cefotaxime and cephalexine residues in milk and cephalexine in veterinary formulation

An HPLC method was developed and validated for the determination of the cephalosporins cefotaxime and cephalexine in skimmed bovine milk. The analytical column, Kromasil C₁₈ (250 mm × 4.0 mm, 5 μm) was operated at ambient temperature. Mobile phase consisted of CH₃OH-acetate buffer (pH = 4.0) and it was delivered isocratically at a flow rate of 1.0 mL · min⁻¹. Total analysis time was less than 5 min. Caffeine was used as internal standard (5 ng · μL⁻¹). UV detection was performed at 265 nm. Method validation was performed by means of intra-day (n = 5) and inter-day accuracy and precision (n = 8), sensitivity and linearity. Limits of detection (LOD) and limits of quantification (LOQ) were 0.1 and 0.3 ng · μL⁻¹, respectively. The method was applied to the analysis of a veterinary drug (CEPOREX) containing cephalexine. The results were quite accurate with the relative error varying from −8.0 to −3.5%. Solid-phase extraction was applied to remove all matrix interference from milk samples. High extraction recoveries (average 84–121%) were achieved by using Abselut NEXUS cartridges with acetonitrile as eluent and a rinsing step with water and n-butanol. A pre-concentration step was necessary in a 1/10 level to reach the EU MRL concentration level (100 μg · kg⁻¹). RSD values were less than 7% for both cephalosporins. Correspondence: Ioannis N. Papadoyannis, Laboratory of Analytical Chemistry, Department of Chemistry, Aristotle University of Thessaloniki, GR-54124 Thessaloniki, Greece     

Simultaneous Determination of Palladium, Platinum and Rhodium by On-Line Column Enrichment and HPLC with 4-Hydroxy-1-Naphthalthiorhodanine as Pre-Column Derivatization Reagents

In this paper, 4-hydroxy-1-naphthalthiorhodanine (HNTR) was synthesized, and a new method for the simultaneous determination of palladium, platinum and rhodium ions as metal-HNTR chelates was developed using rapid column high-performance liquid chromatography combined with on-line enrichment. The palladium, platinum and rhodium ions were pre-column derivatized with HNTR to form colored chelates. The Pb-HNTR, Pt-HNTR and Rh-HNTR chelates could be absorbed onto the front of the enrichment column when they were injected into the injector and sent to the enrichment column [ZORBAX Stable Bound, 4.6 × 10 mm, 1.8 μm] with a buffer solution of 0.05 mol L⁻¹ sodium acetate-acetic acid (pH 4.0) as mobile phase. After enrichment, and by switching the six-ports switching valve, the retained chelates were back-flushed by mobile phase and traveling towards the analytical column. Separation of these chelates on the analytical column [ZORBAX Stable Bound, 4.6 × 50 mm, 1.8 μm] was satisfactory with 68% acetonitrile (containing 0.05 mol L⁻¹ of pH 4.0 sodium acetate-acetic acid buffer salt and 0.1% of tritonX-100) as mobile phase. Palladium, platinum and rhodium were separated completely within 2 min. The detection limits (S/N = 3) of palladium, platinum and rhodium are 1.2 ng L⁻¹, 1.5 ng L⁻¹ and 1.8 ng L⁻¹, respectively. This method was applied to the determination of palladium, platinum and rhodium in water, urine and soil samples with good results.     

Development and validation of a liquid chromatography–tandem mass spectrometry method for simultaneous quantification of p-aminohippuric acid and inulin in rat plasma for renal function study

The first liquid chromatography–tandem mass spectrometry method was developed and validated for the simultaneous quantification of p-aminohippuric acid and inulin, both typical biomarkers of kidney function. 5-(Hydroxymethyl)furfural, generated from inulin by acid and heat preparation, was used as an inulin substitute for the quantification. Acetaminophen was used as the internal standard. Solid-phase extraction was carried out with 5% methanol as the washing solution to optimize the retention of the analytes and to avoid obstruction of the orifice plate of the mass spectrometer caused by any unreacted inulin residue remaining from the sample preparation process. Chromatography separation was performed on a Symmetry C₁₈ column and a mobile phase composed of 2 mM ammonium formate and 0.1% formic acid in water (solvent A) and 2 mM ammonium formate and 0.1% formic acid in acetonitrile (solvent B) (30:70, v/v). Detection was performed with a triple-quadrupole tandem mass spectrometer using positive ion mode electrospray ionization in the multiple reaction monitoring mode. The selected transitions were m/z 195.2 → 120.2, 127.1 → 109.1, and 152.1 → 110.0 for p-aminohippuric acid, inulin [measured as 5-(hydroxymethyl)furfural], and acetaminophen, respectively. The linearity ranged from 10 to 140 μg/mL and from 100 to 1,400 μg/mL for p-aminohippurric acid and inulin (r > 0.99), respectively. The precisions and accuracies were all within 12 and 11% for the lower limit of quantification and quality control samples, respectively. This application was proven to be reliable and accurate and was successfully applied to a renal function study.     

Epoxycarotenoids esters analysis in intact orange juices using two‐dimensional comprehensive liquid chromatography

In this work, the native carotenoid pattern of different orange juices was studied by LC×LC‐DAD/APCI‐IT‐TOF‐MS for the first time. Special attention was given to the epoxycarotenoids components. It has been already proposed that the relative proportions and composition of these epoxycarotenoids can be used to estimate the age and freshness of an orange juice. Re‐arrangements from 5,6‐ to 5,8‐epoxides can occur with time, partially due to the natural acidity of the juices. Thus, the study of these carotenoids in their intact form, that is, esterified with fatty acids, is of great interest. Besides, other free carotenoid and carotenoids esters were identified in seven different monovarietal orange juices and a commercial orange juice. Moreover, the higher separation power of the present LC×LC approach allowed a clearer identification of the compounds contained in the sample compared to the more commonly used approach which uses C₃₀ stationary phases in conventional LC, thanks to the attainment of clearer MS spectra due to the higher resolution and separation degree obtained in LC×LC. This method could also be used to establish authenticity markers among orange varieties that could be potentially used to prevent or detect adulterations or to establish ripeness indexes.     

Spectrophotometric determination of nitrite in natural waters using diazotization-coupling method with column preconcentration on naphthalene supported with ion-pair of tetradecyldimethylbenzyl-ammonium and iodide

 A column preconcentration method has been established for the spectrophotometric determination of traces of nitrite using diazotization and coupling on an naphthalene-tetradecyldimethylbenzylammonium (TDBA)-iodide (I) adsorbent. Nitrite ion reacts with sulfanilic acid (SA) in the pH range 1.8–3.0 for the SA-1-naphthol system and in the pH range 2.3–3.2 for the SA-1-naphthylamine-4-sulfonate system (SA-NAS system) in hydrochloric acid medium to form water-soluble colourless diazonium cations. These cations were coupled with 1-naphthol in the pH range 1.6–4.6 and with NAS in the pH range 2.6–5.0 to be retained on naphthalene-TDBA-I packed in a column. The solid mass was dissolved from the column with 5 mL of dimethylformamide (DMF) and the absorbance measured at 418 nm for the SA-1-naphthol system and at 485 nm for the SA-NAS system. The calibration curve was linear over the concentration range 0.02–0.87 mg/L for SA-1-naphthol and 0.02–0.80 mg/L in the sample for SA-NAS. The molar absorptivity was calculated to be 1.70×10⁴ L mol^-1 cm^-1 for SA-1-naphthol and 1.66×10⁴ L mol^-1 cm^-1 for SA-NAS. The detection limits were found to be 0.014 and 0.016 mg/L for SA-1-naphthol and SA-NAS, respectively. The preconcentration factors were 8 and 6 for SA-1-naphthol and SA-NAS, respectively. Replicate determinations of seven sample solutions containing 6.6 μg of nitrite for SA-1-naphthol and 5.3 μg of nitrite for SA-NAS gave mean absorbances of 0.486 and 0.382 with relative standard deviations of 0.49 and 0.58%, respectively. Interferences due to various foreign ions have been studied and the method has been applied to the determination of 27–65 μg/L levels of nitrite in natural waters. The recovery and relative standard deviation for water samples were 98–102% and 0.49–0.58% for both systems. Received: 1 December 1995/Revised: 22 April 1996/Accepted: 22 April 1996     

An LC method for monitoring medium-chain fatty acid permeation through CaCo-2 cell monolayers

A simple method was developed for monitoring the permeation of medium-chain fatty acids of C8 (octanoic acid) and C10 (decanoic acid) through CaCo-2 cell monolayers by high-performance liquid chromatography with electrochemical detection (HPLC-ECD). The detection was made based on the electrochemical reduction prepeak of quinone caused by acids, requiring the fabrication of a two-channel HPLC-ECD system. In one channel, acetonitrile–water (7:3, v/v) was used as a mobile phase to separate acids by a C30 column. In the other channel, acetonitrile–water (7:3, v/v) containing 6 mmol/L 3,5-di-t-butyl-1,2-benzoquinone and 20 mmol/L LiClO₄ was used as a quinone solution to detect acids by an electrochemical cell with a glassy carbon working electrode. In this HPLC-ECD system, eluted acids were mixed with the quinone solution in a post column fashion to obtain current signals caused by acids. The peak area was found to be linearly related to the acid amount ranging from 25 to 1,000 pmol (r > 0.992). The detection limits of octanoic acid and decanoic acid were 7.5 and 8.8 pmol, respectively. Octanoic acid and decanoic acid spiked into cell culture media samples were extracted with acetonitrile and their recoveries were more than 89.5% with an RSD of less than 8.2%. This method was applied to the permeation experiment of octanoic acid and decanoic acid with CaCo-2 cell monolayers formed on the Transwell? system. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. This work was partly presented at The Physical Pharma Forum 2008 on March 24, 2008 in Tokyo, Japan.     

A Nano-Chip-LC/MS<Superscript><Emphasis Type="Italic">n</Emphasis></Superscript> Based Strategy for Characterization of Modified Nucleosides Using Reduced Porous Graphitic Carbon as a Stationary Phase

LC/MS analysis of ribonucleosides is traditionally performed by reverse phase chromatography on silica based C18 type stationary phases using MS compatible buffers and methanol or acetonitrile gradients. Due to the hydrophilic and polar nature of nucleosides, down-scaling C18 analytical methods to a two-column nano-flow setup is inherently difficult. We present a nano-chip LC/MS ion-trap strategy for routine characterization of RNA nucleosides in the fmol range. Nucleosides were analyzed in positive ion mode by reverse phase chromatography using a 75 μ × 150 mm, 5 μ particle porous graphitic carbon (PGC) chip with an integrated 9 mm, 160 nL trapping column. Nucleosides were separated using a formic acid/acetonitrile gradient. The method was able to separate isobaric nucleosides as well as nucleosides with isotopic overlap to allow unambiguous MS ⁿ identification on a low resolution ion-trap. Synthesis of 5-hydroxycytidine (oh⁵C) was achieved from 5-hydroxyuracil in a novel three-step enzymatic process. When operated in its native state using formic acid/acetonitrile, PGC oxidized oh⁵C to its corresponding glycols and formic acid conjugates. Reduction of the PGC stationary phase was achieved by flushing the chip with 2.5 mM oxalic acid and adding 1 mM oxalic acid to the online solvents. Analyzed under reduced chromatographic conditions oh⁵C was readily identified by its MH⁺ m/z 260 and MSⁿ fragmentation pattern. This investigation is, to our knowledge, the first instance where oxalic acid has been used as an online reducing agent for LC/MS. The method was subsequently used for complete characterization of nucleosides found in tRNAs using both PGC and C18 chips.     

Ultra-fast two-dimensional microchip electrophoresis using SDS μ-CGE and microemulsion electrokinetic chromatography for protein separations

A poly(methyl methacrylate) microfluidic chip was used to perform a two-dimensional (2-D) separation of a complex protein mixture in short development times. The separation was performed by combining sodium dodecyl sulfate micro-capillary gel electrophoresis (SDS μ-CGE) with microemulsion electrokinetic chromatography (μ-MEEKC), which were used for the first and second dimensions, respectively. Fluorescently labeled Escherichia coli cytosolic proteins were profiled by this 2-D approach with the results compared to a similar 2-D separation using SDS μ-CGE × μ-MEKC (micelle electrokinetic chromatography). The relatively short column lengths (effective length = 10 mm) for both dimensions were used to achieve separations requiring only 220 s of development time. High spot production rates (131 ± 11 spots min⁻¹) and reasonable peak capacities (481 ± 18) were generated despite the fact that short columns were used. In addition, the use of μ-MEEKC in the second dimension was found to produce higher peak capacities compared to μ-MEKC (481 ± 18 for μ-MEEKC and 332 ± 17 for μ-MEKC) due to the higher plate numbers associated with μ-MEEKC.