Anion-exchange High-performance Liquid Chromatography Research Articles

Evaluation of methods for the extraction of nitrite and nitrate in biological fluids employing high-performance anion-exchange liquid chromatography for their determination

Measurements of nitrite (NO 2 −) and nitrate (NO 3 −) in biological fluids are proposed as indices of cellular nitric oxide (NO) production. Determination of NO 2 − and NO 3 − in standard solutions is not difficult, however, determinations which reflect accurately cellular NO synthesis represent a considerable analytical challenge. Problems are often encountered arising from background NO 2 −/NO 3 − contamination in experimental solutions and laboratory hardware, and with methods for sample extraction. We investigated potential procedures for the extraction and determination of NO 2 − and NO 3 − in biological samples. Consequently, a protocol was devised which yielded acceptable results regarding extraction efficiency, assay reproducibility, sample throughput and contaminant minimisation. It entailed rigorous washing of all equipment with water of low NO 2 − and NO 3 − content, sample deproteinisation by centrifugal ultrafiltration through a 3K filter and analysis by high-performance anion-exchange liquid chromatography with UV detection. Retention times for NO 2 − and NO 3 − in standards and plasma were 4.4 and 5.6 min, respectively. Assay linearity for standards ranged between 31 n M and 1 m M. The limit of detection for NO 2 − and NO 3 − in standards was 3 pmol. Recoveries of NO 2 − and NO 3 − from spiked plasma (1–100 μ M KNO 2/KNO 3) and from extracted standards (1–250 μ M) were approximately 100%. Intra-assay and inter-assay RSDs for NO 2 − and NO 3 − in spiked and unspiked plasma were 10.6% or less. Assays on washed platelet supernatants demonstrated collagen-induced platelet generation of NO products and analysis of murine and rat cardiac perfusates was achieved. Our procedure may be suitable for routine determination of NO 2 − and NO 3 − in various biological fluids, e.g., plasma.

EVALUATION OF HPLC SYSTEMS FOR THE SEPARATION AND QUANTIFICATION OF ARSENIC COMPOUNDS FROM APPLE EXTRACTS

Several different high performance liquid chromatography (HPLC) systems were evaluated for the applicability for the determination of trace level arsenic compounds from extracts of freeze-dried apple samples. Various gradients using ion-pair reversed-phase HPLC were explored. Gradient anion-exchange HPLC, as well as a simple isocratic system, were also evaluated in this study. Two extraction procedures were used. Simple sonication by a common laboratory ultrasonic cleaner using a 50/50 (v/v) water/methanol solvent mixture and a two step procedure using initial overnight treatment with α-amylase enzyme followed by sonication with a 60/40 (v/v) acetonitrile/water solvent mixture were evaluated. Inductively Coupled Plasma-Mass Spectrometric (ICP-MS) detection was used to measure the arsenic compounds contained within the apple extracts. The three most abundant arsenic compounds found in the extracts were arsenite (As III), arsenate (As V) and dimethylarsinic acid (DMAA).

Domain Structure of Chondroitin Sulfate E Octasaccharides Binding to Type V Collagen

We demonstrated previously that chondroitin sulfate E (ChS-E) binds to type V collagen (Munakata, H., Takagaki, K., Majima, M., and Endo, M. (1999) Glycobiology 9, 1023--1027). In this study, we investigated the structure and binding of ChS-E oligosaccharides. Eleven oligosaccharides were isolated from ChS-E by gel filtration chromatography and anion-exchange high performance liquid chromatography after hydrolysis with testicular hyaluronidase. Separately, seven oligosaccharides were custom synthesized using the transglycosylation reaction of testicular hyaluronidase. Structural analysis was performed by enzymatic digestions in conjunction with high performance liquid chromatography and mass spectrometry. This library of 18 oligosaccharides was used as a source of model molecules to clarify the structural requirements for binding to type V collagen. Binding was analyzed by a biosensor based on surface plasmon resonance. The results indicated that to bind to type V collagen the oligosaccharides must have the following carbohydrate structures: 1) octasaccharide or larger in size; 2) a continuous sequence of three GlcAbeta1--3GalNAc(4S,6S) units; 3) a GlcAbeta1--3GalNAc(4S,6S) unit, GlcAbeta1--3GalNAc(4S) unit or GlcAbeta1--3GalNAc(6S) unit at the reducing terminal; 4) a GlcAbeta1--3GalNAc(4S,6S) unit at the nonreducing terminal. It is likely that these characteristic oligosaccharide sequences play key roles in cell adhesion and extracellular matrix assembly.

Phosphorylation of beta-D-ribosylbenzimidazoles is not required for activity against human cytomegalovirus.

We have previously reported that 2,5,6-trichloro-1-(beta-D-ribofuranosyl)benzimidazole (TCRB) and its 2-bromo analog (2-bromo-5,6-dichloro-1-(beta-D-ribofuranosy)benzimidazole [BDCRB]) are potent and selective inhibitors of human cytomegalovirus (HCMV) replication that block viral DNA maturation via HCMV gene products UL89 and UL56. To determine if phosphorylation is required for antiviral activity, the in vitro metabolism of BDCRB was examined and the antiviral activities of nonphosphorylatable 5'-deoxy analogs were determined. Reverse-phase high-performance liquid chromatography (HPLC) analysis of extracts from uninfected and HCMV-infected cells incubated with [(3)H]BDCRB revealed two major metabolites. Both were less polar than naturally occurring nucleoside monophosphates, but one peak coeluted with a BDCRB-5'-monophosphate (BDCRB-5'-MP) standard. Further analysis revealed, however, that neither metabolite partitioned with BDCRB-5'-MP on anion-exchange HPLC. Their retention patterns were not affected by incubation with alkaline phosphatase, thereby establishing that the compounds were not nucleoside 5'-monophosphates. Both compounds were detected in uninfected and HCMV-infected cells and in mouse live extracts, but neither has been identified. Like TCRB and BDCRB, the nonphosphorylatable 5'-deoxy analogs were potent and selective inhibitors of HCMV replication. The 5'-deoxy analogs maintained inhibition of HCMV replication upon removal of BDCRB, whereas an inhibitor of DNA synthesis did not. Similar to TCRB, its 5'-deoxy analog (5'-dTCRB) did not affect viral DNA synthesis, but 5'-dTCRB did inhibit viral DNA maturation to genome-length units. Additionally, virus isolates resistant to TCRB were also resistant to 5'-dTCRB and the 5'-deoxy analog of BDCRB. Taken together, these results confirm that TCRB, BDCRB, and their 5'-deoxy analogs have common mechanisms of action and establish that these benzimidazole ribonucleosides, unlike other antiviral nucleosides, do not require phosphorylation at the 5' position for antiviral activity.

Nonradioactive Analysis of Phosphatidylinositides and Other Anionic Phospholipids by Anion-Exchange High-Performance Liquid Chromatography with Suppressed Conductivity Detection

Phosphatidylinositol 4,5-biphosphate (PIP2) modulates the function of numerous ion transporters and channels, as well as cell signaling and cytoskeletal proteins. To study PIP2 levels of cells without radiolabeling, we have developed a new method to quantify anionic phospholipid species. Phospholipids are extracted and deacylated to glycero-head groups, which are then separated by anion-exchange HPLC and detected by suppressed conductivity measurements. The major anionic head groups can be quantified in single runs with practical detection limits of about 100 pmol, and the D3 isoforms of phosphatidylinositol phosphate (PIP) and PIP2 are detected as shoulder peaks. In HeLa, Hek 293 and COS cells, as well as intact heart, PIP2 amounts to 0.5 to 1.5% of total anionic phospholipid (10 to 30 μmol/liter cell water or 0.15 to 0.45 nmol/mg protein). In cell cultures, overexpression of Type I PIP5-kinase specifically increases PIP2, whereas overexpression of Type II PI4-kinase can increase both PIP and PIP2. Phosphatidylinositol 3,4,5-trisphosphate (PIP3) and the D3 isomers of PIP2 are detected after treatment of cells with pervanadate; in yeast, overexpression of a phosphatidylinositol 3-kinase (VPS34) specifically increases phosphatidylinositol 3-phosphate (PI3P). Using isolated cardiac membranes, lipid kinase and lipid phosphatase activities can be monitored with the same methods. Upon addition of ATP, PIP increases while PIP2 remains low; exogenous PIP2 is rapidly degraded to PIP and phosphatidylinositol (PI). In summary, the HPLC methods described here can be used to probe multiple aspects of phosphatidylinositide (Ptide) metabolism without radiolabeling.

Arsenosugar identification in seaweed extracts using high-performance liquid chromatography/electrospray ion trap mass spectrometry.

The development of analytical techniques suitable for providing structural information on a wide range of elemental species is a growing necessity. For arsenic speciation a variety of mass spectrometric techniques, mainly inductively coupled plasma mass spectrometry (ICP-MS) and electrospray tandem mass spectrometry (ES-MS/MS) coupled on-line with high-performance liquid chromatography (HPLC), are in use. In this paper we report the identification of arsenic species present in samples of marine origin (seaweed extracts) using ES ion trap mass spectrometry (IT) multistage mass spectrometry (MS(n)). Both reversed-phase and anion-exchange HPLC have been coupled on-line to ES-ITMS. Product ion scans with multiple stages of tandem MS (MS(n); n=2-4) were used to acquire diagnostic data for each arsenosugar. The spectra contain structurally characteristic fragment ions for each of the arsenosugars examined. In addition it was observed that upon successive stages of collision-induced dissociation (CID) a common product ion (m/z 237) was formed from all four arsenosugars examined. This product ion has the potential to be used as an indicator for the presence of dimethylated arsenosugars (dimethylarsinoylribosides). The HPLC/ES-ITMS(n) method developed allows the sensitive identification of arsenosugars present in crude seaweed extracts without the need for extended sample preparation. In fact, sample preparation requirements are identical to those typically employed for HPLC/ICP-MS analysis. Additionally, the resulting product ions are structurally diagnostic of the arsenosugars examined, and tandem mass spectra are reproducible and correspond well to those obtained using other low-energy CID techniques. As a result, the HPLC/ES-ITMS(n) approach minimises the potential for arsenic species misidentification and has great potential as a means of overcoming the need for characterised standards.

Determination of Inorganic Sb(V) and Methylantimony Species by HPLC with Hydride Generation-Atomic Fluorescence Spectrometric Detection

An on-line method for the separation and analysis of Sb(V) and Me3Sb in the presence of Sb(III) in liquid samples is described. Inorganic and organic antimony species were separated using anion-exchange high-performance liquid chromatography (HPLC) coupled with hydride generation-atomic fluorescence detection (HG-AFS). Optimum conditions for the separation of antimony species by HPLC and the hydride generation conditions for the determination by HG-AFS were established. Matrix interference of the chromatographic determination was studied in relation to MgSO4 and NaCl. The method developed was applied to the separation and determination of antimony species in spiked and natural water samples. The suitability of the method for analysis in microbial growth media and physiological studies involving methylantimony species is discussed.

Reperfusion-induced Ins(1,4,5)P3Generation and Arrhythmogenesis Require Activation of the Na+/Ca2+Exchanger

Reperfusion of globally ischemic rat hearts causes rapid generation of inositol(1,4,5) tris phosphate [Ins(1,4,5)P3] and the development of arrhythmias, following stimulation ofα1 -adrenergic receptors by norepinephrine released from the cardiac sympathetic nerves. The heightened inositol phosphate response in reperfusion depends on the activation of the Na+/H+exchanger, which might reflect a central role for increased Ca2+following reverse mode activation of the Na+/Ca2+exchanger (NCX). Isolated, perfused rat hearts were subjected to 20 min ischemia followed by 2 min reperfusion and the content of Ins(1,4,5)P3measured by mass analysis or by anion-exchange high performance liquid chromatography (HPLC) following [3H]inositol labeling. Reperfusion caused generation of Ins(1,4,5)P3(1266±401 to 3387±256 cpm/g tissue, mean±s.e.m., n=6,P <0.01) and the development of arrhythmias. Inhibition of NCX either by reperfusion at low Ca2+(1133±173 cpm/g tissue, mean±s.e.m., n=6, P<0.01 relative to reperfusion control) or by adding 10 μ m KB-R7943, an inhibitor of reverse mode Na+/Ca2+exchange, prevented the Ins(1,4,5)P3response (1151±243 cpm/g tissue, mean±s.e.m.n=6, P<0.01 relative to reperfusion control) and the development of ventricular fibrillation. Lower concentrations of KB-R7943 were less effective. Reverse mode activation of NCX is therefore required for the enhanced Ins(1,4,5)P3response in early reperfusion, and inhibitors of this transporter may be useful in the prevention of arrhythmias under such conditions.

Inulin determination for food labeling.

Inulin and oligofructose exhibit valuable nutritional and functional attributes, so they are used as supplements as soluble fiber or as macronutrient substitutes. As classic analytical methods for dietary fiber measurement are not effective, several specific methods have been proposed. These methods measure total fructans and are based on one or more enzymatic sample treatments and determination of released sugars. To determine inulin for labeling purposes, we developed an easy and rapid anion-exchange high-performance liquid chromatography (HPLC) method following water extraction of inulin. HPLC conditions included an Aminex HPX- 87C column (Bio-Rad), deionized water at 85 degrees C as the mobile phase and a refractive index detector. The tested foods included tailor-made food products containing known amounts of inulin and commercial products (cookies, milk, ice creams, cheese, and cereal bars). The average recovery was 97%, and the coefficient of variation ranged from 1.1 to 5% in the food matrixes. The obtained results showed that this method provides an easier, faster and cheaper alternative than previous techniques for determining inulin with enough accuracy and precision for routine labeling purposes by direct determination of inulin by HPLC with refractive index detection.

Validation of a high-performance liquid chromatographic assay for the quantification of adenovirus type 5 particles

An anion-exchange–high-performance liquid chromatography (AE–HPLC) method for the quantification of adenovirus type 5 (Ad5) total particles was validated according to performance criteria of precision, specificity, linearity of calibration and range, limit of detection, limit of quantification, accuracy and recovery. The viral particles were detected by absorbance at 260 nm using photodiode array detector (PDA). Cesium chloride (CsCl) purified Ad5 and lysate samples were used for the validation of the method. Relative standard deviations (RSDs) for the inter-day, intra-day precision and reproducibility for both the lysate and the Ad5 standard were less than 10 and 2% for the peak area and retention time, respectively. The method was specific for Ad5 which was eluted at 8.0 min. The presence of DNA does not affect the recovery of Ad5 particles for accurate quantification. Based on the error in prediction to be less than 10%, the working range was established between 2×10 10 and 7×10 11 VP/ml with correlation coefficient of 0.99975, standard deviation of 6.14×10 9 VP/ml and a slope of 3.04×10 5 VP/ml. The recovery of the method varied between 88 and 106% in all of the lysate samples investigated which is statistically similar to 100% recovery at 95% confidence interval.

Cyclic AMP-Dependent Protein Kinase Isoenzymes in Human Myeloid Leukemia (HL60) and Breast Tumor (MCF-7) Cells

Combinations of retinoic acid (RA) and cAMP mediate many biological responses in a large variety of cell types. While the basis for the apparent synergistic effects of RA and cAMP are not clearly defined, it is likely that activation of PKA by cAMP is involved. However, literature reports concerning the identity of PKA isoforms in HL60 and MCF-7 cells are conflicting. The purpose of the present investigation is to identify PKA isoforms in HL60 and MCF-7 cells. Utilization of high-performance anion-exchange liquid chromatography, immunoblotting, and 8-azido-cAMP photoaffinity binding resulted in the finding that HL60 cells contain PKA types Iα and IIα, while MCF-7 cells contain PKA types Iα, IIα, and IIβ. PKA type Iα in both HL60 and MCF-7 cells eluted from columns as two well-separated peaks. One peak eluted at a low salt concentration in agreement with previous reports. The second HL60 PKA type Iα peak eluted at a salt concentration intermediate between that eluting the first peak and that eluting PKA type IIα and contained approximately 62% of the total RIα protein. However, the second MCF-7 PKA type Iα peak contained approximately 66% of the total RIα protein and co-eluted with PKA types IIα and IIβ. This “contamination” of PKA type II fractions with PKA type I has led, in some cases, to interpretations that may need reevaluation.

Analysis of oligonucleotides and unincorporated nucleotides fromin vitro transcription by capillary electrophoresis in Pluronic F127 gels

Small functional RNAs required for structure studies are often prepared by in vitro transcription. Capillary electrophoresis in liquid crystalline gels of Pluronic F127 was used to analyze unfractionated in vitro transcription reactions and anion-exchange high-performance liquid chromatography (HPLC) fractions from transcription reactions. Guanosine monophosphate (GMP), the four nucleoside triphosphates (NTPs), abortive transcripts, and transcripts with lengths near the desired product length were simultaneously resolved and quantified in a single run. Oligonucleotides up to at least 35 nucleotides were resolved to baseline within 10 min using a moderate field (185 V/cm) and short effective capillary length (7.6 cm) for electrophoresis in 20% Pluronic F127 at pH 8.3 in Tris-borate-EDTA (TBE) buffer (30 degrees C). Nucleotide migration times were 4-5 min, in the order UTP+CTP (unresolved) <ATP<GTP<GMP. A single capillary filling was reused for 40-50 runs with little decrease in performance, and for up to 100 runs with performance acceptable for many applications. Higher electric fields appeared to affect the gel structure and necessitated more frequent capillary refilling. Capillary gel electrophoresis (CGE) of HPLC fractions in Pluronic gels facilitates rapid recovery of RNA products and the large remaining amounts of valuable, isotopically labeled NTPs. In addition, comparison of electrophoretic patterns under denaturing and nondenaturing conditions yields insights into potential conformational heterogeneity of the folded nucleic acid states. CGE in Pluronic gels provides a rapid, highly discriminating means for analyzing in vitro transcription reactions.

Analysis of oligonucleotides and unincorporated nucleotides from in vitro transcription by capillary electrophoresis in Pluronic F127 gels.

Small functional RNAs required for structure studies are often prepared by in vitro transcription. Capillary electrophoresis in liquid crystalline gels of Pluronic F127 was used to analyze unfractionated in vitro transcription reactions and anion-exchange high-performance liquid chromatography (HPLC) fractions from transcription reactions. Guanosine monophosphate (GMP), the four nucleoside triphosphates (NTPs), abortive transcripts, and transcripts with lengths near the desired product length were simultaneously resolved and quantified in a single run. Oligonucleotides up to at least 35 nucleotides were resolved to baseline within 10 min using a moderate field (185 V/cm) and short effective capillary length (7.6 cm) for electrophoresis in 20% Pluronic F127 at pH 8.3 in Tris-borate-EDTA (TBE) buffer (30 degrees C). Nucleotide migration times were 4-5 min, in the order UTP+CTP (unresolved) <ATP<GTP<GMP. A single capillary filling was reused for 40-50 runs with little decrease in performance, and for up to 100 runs with performance acceptable for many applications. Higher electric fields appeared to affect the gel structure and necessitated more frequent capillary refilling. Capillary gel electrophoresis (CGE) of HPLC fractions in Pluronic gels facilitates rapid recovery of RNA products and the large remaining amounts of valuable, isotopically labeled NTPs. In addition, comparison of electrophoretic patterns under denaturing and nondenaturing conditions yields insights into potential conformational heterogeneity of the folded nucleic acid states. CGE in Pluronic gels provides a rapid, highly discriminating means for analyzing in vitro transcription reactions.

Anion-exchange high-performance liquid chromatography assays of plasma lipoproteins and modified low-density lipoproteins using a ProtEx-DEAE column

Previously [Anal. Biochem., 232 (1995) 163–171], we reported a high-performance liquid chromatography (HPLC) assay method for human plasma lipoproteins using a diethylaminoethyl (DEAE)-glucomannan column, which is not commercially available. In this study, HPLC assay methods for lipoproteins in plasma samples of human and experimental animals, and modified low-density lipoproteins (LDLs) of rabbits have been developed using a commercially available anion-exchange ProtEx-DEAE column. For the assays of plasma lipoproteins, the method includes complete separation of high-density lipoproteins, LDLs and very low-density lipoproteins within 20 min using stepwise elution, and determination by post-column reaction with an enzymatic cholesterol reagent as the total cholesterol (TC) level. Similarly, mild oxidative and artificially oxidised LDLs were separated into their subfractions using stepwise elution, and determined based on the TC level. The methods using the DEAE-glucomannan and ProtEx-DEAE columns were cross-validated. There was an excellent correlation between the two methods. The obtained results reveal that the anion-exchange HPLC method using the ProtEx-DEAE column could be useful for the assays of plasma lipoproteins and modified LDLs.

Identification of Macrophage Migration Inhibitory Factor as a Potent Endothelial Cell Growth-Promoting Agent Released by Ectopic Human Endometrial Cells1

The growth of endometrial cells in ectopic locations (endometriosis) is dependent on the establishment of an adequate blood supply. Neovascularization (angiogenesis) is therefore a vital step toward the progression of this disease. We first revealed the presence of a potent mitogenic activity for human endothelial cells in the culture medium of immortalized human endometriotic cells. The activity, measured by the level of [(3)H]-thymidine incorporation into the DNA of human coronary artery endothelial cells, was then purified by anion exchange high-performance liquid chromatography. Electrophoretic analysis of one of the bioactive fractions that markedly enhanced endothelial cell proliferation showed three distinct bands with apparent molecular masses of 15.8, 12.6, and 6.5 kDa. N-terminal microsequencing of an internal peptide from the 12. 6-kDa protein showed 100% homology with human macrophage migration inhibitory factor (MIF). The protein was positively identified as MIF by Western blot analysis using a specific anti-MIF antibody. Anti-MIF antibody inhibited the bioactivity found in the evaluated fraction and the conditioned medium of primary endometriotic cell cultures, and commercial recombinant human MIF displayed a high mitogenic activity for endothelial cells. Our findings reveal that MIF is released by endometriotic cells and acts as a potent mitogenic factor for human endothelial cells in vitro. This may have a considerable interest, in view of the crucial role of angiogenesis in ectopic endometrial cell growth and activity and in numerous tissues undergoing dynamic physiological changes, such as human endometrium.

Stability studies of arsenic, selenium, antimony and tellurium species in water, urine, fish and soil extracts using HPLC/ICP-MS.

The stability of arsenic, selenium, antimony and tellurium species in water and urine (NIST SRM 2670n) as well as in extracts of fish and soil certified reference materials (DORM-2 and NIST SRM 2710) has been investigated. Stability studies were carried out with As(III), As(V), arsenobetaine, monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), phenylarsonic acid (PAA), Se(IV), Se(VI), selenomethionine, Sb(III), Sb(V) and Te(VI). Speciation analysis was performed by on-line coupling of anion exchange high-performance liquid chromatography (HPLC) with inductively coupled plasma mass spectrometry (ICP-MS). Best storage of aqueous mixtures of the examined species was achieved at 3 degrees C whereas at -20 degrees C species transformation especially of selenomethionine and Sb(V) took place and a new selenium species appeared within a period of 30 days. Losses and species transformations during extraction processes were investigated. Extraction of the spiked fish material with methanol/water led to partial conversion of Sb(III), Sb(V) and selenomethionine to two new antimony and one new selenium species. The other arsenic, selenium and tellurium species were almost quantitatively extracted. For soil spiked with MMA, PAA, Se(IV) and Sb(III), recoveries after extraction with water and sulfuric acid (0.01 mol/L) were below 20%.

Purification and amino acid sequence of a highly insecticidal toxin from the venom of the Brazilian spider Phoneutria nigriventer which inhibits NMDA-evoked currents in rat hippocampal neurones

A new insecticidal toxin Tx4(5-5) was isolated from the fraction PhTx4 of the venom of the spider Phoneutria nigriventer by reverse phase high performance liquid chromatography (HPLC) and anion exchange HPLC. The complete amino acid sequence determined by automated Edman degradation showed that Tx4(5-5) is a single chain polypeptide composed of 47 amino acid residues, including 10 cysteines, with a calculated molecular mass of 5175 Da. Tx4(5-5) shows 64% of sequence identity with Tx4(6-1), another insecticidal toxin from the same venom. Tx4(5-5) was highly toxic to house fly ( Musca domestica), cockroach ( Periplaneta americana) and cricket ( Acheta domesticus ), producing neurotoxic effects (knock-down, trembling with unco-ordinated movements) at doses as low as 50 ng/g (house fly), 250 ng/g (cockroach) and 150 ng/g (cricket). In contrast, intracerebroventricular injections (30 μg) into mice induced no behavioural effects. Preliminary electrophysiological studies carried out on whole-cell voltage-clamped rat hippocampal neurones indicated that Tx4(5-5) (at 1 μ M) reversibly inhibited the N-methyl- D-aspartate-subtype of ionotropic glutamate receptor, while having little or no effect on kainate-, α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid- or γ-aminobutyric acid-activated currents.

An improved anion-exchange HPLC method for the detection and purification of adenoviral particles

We have developed an anion-exchange high-performance liquid chromatography (HPLC) method using Q Sepharose XL (Amersham Pharmacia Biotech) as adsorbent to analyze samples containing adenovirus. This method has several major advantages over the HPLC method previously described for quantitating particles, namely (1) a >10-fold improvement in the detection limit of adenovirus in crude preparations; (2) absence of interferences originating from nucleic acids and proteins which usually contaminate crude samples; (3) unprecedented sharpness and symmetry of adenovirus peak, rendering the identification of the viral peak unambiguous, even in extremely crude and dilute preparations; and (4) no enzymatic treatment required even for crude samples. This assay was used to quantitate particles in samples taken at the transfection and amplification stages of production of various recombinant adenovirus, and in cultures of wild-type adenovirus of different serotypes. A modification of this analytical method was also developed for the purification of infectious adenovirus particles, including fiber-modified and third-generation recombinant viruses, giving highly purified preparations from low-titer crude lysates with an excellent overall recovery (50-74%).

Anion-exchange high-performance liquid chromatography with conductivity detection for the analysis of phytic acid in food

A sensitive method for the accurate determination of phytic acid in food samples is described. The proposed procedure involves the anion-exchange liquid chromatography with conductivity detection. Initially, two methods of determination of phytic acid were compared: absorptiometry and high-performance ion chromatography (HPIC) with chemically suppressed conductivity detector. Unlike most conventional methods involving precipitation by FeCl 3, the simpler and more reliable HPIC assay avoids the numerous assumptions inherent in the iron precipitation and the accuracy is independent of the phytate content. The protocol was also applied to a survey of phytic acid concentration in some cereal, oil and legume seeds.

Speciation of arsenic in edible algae by bi-dimensional size-exclusion anion exchange HPLC with dual ICP-MS and electrospray MS/MS detection

Anion-exchange (AE) high-performance liquid chromatography (HPLC) with ICP-MS detection was revisited for speciation of arsenic in biomaterials with particular attention given to arsenic-containing ribosides (arsenosugars) in seaweeds. Signal identification by retention time matching and spiking experiments was found to be difficult because of the co-elution of the ribosides with other arsenic species [As(III), monomethylarsonic acid], the medium- and long-term irreproducibility of retention times of arsenic species and an uncontrolled matrix effect on the retention times. Size-exclusion (SE) HPLC was proposed for the fractionation of organoarsenic species and matrix removal prior to detection of arsenosugars by AE-HPLC followed by signal identification by spiking experiments and retention time matching. The results were compared with those obtained by pneumatically-assisted electrospray tandem MS (ESI MS/MS) of the SE-HPLC fractions. A number of 10 commercially available edible algal food samples of different origins available on the French market were investigated. The approaches developed for the identification and determination of arsenosugars (SE-AE-HPLC-ICP-MS and SE-HPLC-ESI MS/MS) were discussed using the example of Hizikia fusiforme.