SAX Column Research Articles

Separation of phenylpropionic acids both by strong anion exchange stationary phase and strong cation exchange stationary phase in supercritical fluid chromatography, using the same additive

This study demonstrates the expanded application of ion-exchange stationary phases (including strong cation exchange, SCX and strong anion exchange, SAX) in supercritical fluid chromatography (SFC), and more importantly, provides a deeper understanding of the retention mechanisms of these two stationary phases when using the same acidic additive. Phenylpropionic acid compounds (belonging to phenolic acids) were selected as probes. On the SCX column, the π-π and polar interactions originating from the bonded benzenesulfonic acid groups were important foundations for prolonging the retention time of solutes, but they were also the main reason for solutes’ tailing profiles. It was found that adding 0.1% phosphoric acid can generate sufficient strength of electrostatic repulsion to obtain satisfactory peak shapes. Here, phosphoric acid can be adsorbed on the surface of the stationary phase to accumulate more negative charges, and at the same time, the phosphate anion and the phenylpropionic acid in mobile phase might combine into the contact ion pair (CIP)- like through the polar interactions to form the apparently negatively charged solute. On the SAX column, phosphate anions generated by ionization of phosphoric acid, were considered as counterions to effectively mask the electrostatic attraction of quaternary ammonium groups, to significantly reduce the retention of phenylpropionic acids, but improve their resolution. Finally, utilizing two developed SFC methods, i.e., SCX with MeOH + 0.1% phosphoric acid in CO2, or SAX with MeOH + 0.3% phosphoric acid in CO2, the baseline separation of the extract of Lonicerae Japonicae Flos was achieved within 6 min and 10 min, respectively.

An off-line three-dimensional liquid chromatography/Q-Orbitrap mass spectrometry approach enabling the discovery of 1561 potentially unknown ginsenosides from the flower buds of Panax ginseng, Panax quinquefolius and Panax notoginseng

To comprehensively elucidate the herbal metabolites is crucial in natural products research to discover new lead compounds. Ginsenosides are an important class of bioactive components from the Panax plants exerting the significant tonifying effects. However, to identify new ginsenosides by the conventional strategies trends to be more and more difficult because of the large spans of acid-base property (the neutral and acidic saponins), molecular mass (400–1400 Da), and rather low content. Herein, an off-line multidimensional chromatography/high-resolution mass spectrometry approach was presented: ion exchange chromatography (IEC) as the first dimension of separation, hydrophilic interaction chromatography (HILIC) in the second dimension, and reversed-phase chromatography (RPC) for the third dimension which was hyphenated to a Q Exactive Q-Orbitrap mass spectrometer. By applying to the flower buds of P. ginseng (PGF), P. quinquefolius (PQF), and P. notoginseng (PNF), IEC using a PhenoSphereTM SAX column could fractionate the total extracts into the neutral (unretained) and acidic (retained) fractions, while HILIC (an XBridge Amide column) and RPC (BEH Shield RP18 column) achieved the hydrophilic interaction and hydrophobic interaction separations, respectively. Q-Orbitrap mass spectrometry offered rich structural information and complementary resolution to the co-eluting components, particular to those minor ones by including precursor ion lists in data-dependent acquisition. We could characterize 803 ginsenosides from PGF, 795 from PQF, and 833 from PNF, and 1561 thereof are potentially unknown. These results can indicate the great potential of this multidimensional approach in the ultra-deep characterization of complex herbal samples supporting the efficient discovery of potentially novel natural compounds.

Studies on analytical methods for determination of potassium bromate in foods

A high performance liquid chromatographic (HPLC) method for the determination of bromate in bread and flour has been developed. Bromate was extracted from samples with water. The extract was cleaned by the use of a LiChrolut SAX cartridge (perchlorate form) with 0.06 M sodium perchlorate solution (pH 3.5) as a fluent. Bromate was separated with Zorbax SAX column by using 0.06 M sodium perchlorate solution (pH 3.5) the mobile phase, and detected at 355 nm after derivatization in the post column 80°C. Recovery studies were performed using bread and flour samples each fortified with bromate at 0.2, 0.5 and 1 μg/ml separately. Recoveries were in the range of 80.0-86.1% with coefficients of variation under 1.9%. The limit of detection was 0.1 ppm. For purposes of comparison, a study on high performance ion chromatography for the determination of bromate in flour was performed. Sample solution was prepared in the same way as in HPLC method, except that the sample was passed through an OnGuard-Ag cartridge instead of a LiChrolut SAX cartridge to remove chloride. Bromate was analyzed with Dionex HPIC AS11 and AG11 column by using 0.25 mM sodium hydroxide solution as an eluent, in combination with a conductivity detector. Recovery studies were performed using flour samples fortified with bromate at 1, 2 and 5 μ g/ml. Recoveries were in the range of 79.7-86.3% with coefficients of variation under 3.9%. The limit of detection was 0.5 ppm. Forty bread and ten flour samples collected from various markets were analyzed. No bromate residue was detected in any samples tested.

High-Yielding Radiosynthesis of [68Ga]Ga-PSMA-11 Using a Low-Cost Microfluidic Device.

Current PET radiotracer production models result in facility and operational costs that scale prohibitively with the number of tracers synthesized, particularly those made as a single dose-on-demand. Short of a paradigm shift in the technology and economics of radiotracer production, the impact of PET on precision medicine will be limited. Inexpensive, microfluidic radiochemistry platforms have the potential to significantly reduce costs associated with dose-on-demand production and expand the breadth of PET tracers accessible for molecular imaging. To produce a miniaturized dose-on-demand device for [68Ga]Ga-PSMA-11 production, a microfluidic chip was assembled in polydimethylsiloxane (PDMS), combining all components of tracer production in an integrated, compact, and easily utilized platform. On-chip radionuclide concentration, as well as radionuclide and precursor starting material mixing and reaction were incorporated. The radionuclide was sourced from a standard, commercially available 68Ge/68Ga generator. Optimal reaction conditions were determined, which included precursor concentration (5μg/mL), temperature (95°C), and reaction time (1min). The total trapping efficiency of combined on-chip SCX and SAX columns was greater than 70% and could be accomplished in ~ 12min. Under optimized conditions, [68Ga]Ga-PSMA-11 could be reliably synthesized starting from a complete generator elution (1100MBq [29.7mCi]) in ~ 12min, with an average radiochemical yield of 70%, radiochemical purity > 99%, and specific activity > 740MBq/μg (20mCi/μg). Quality control testing demonstrated that tracer produced using this platform met or exceeded all typical FDA requirements for human use. A simple, low-cost, dose-on-demand radiosynthesis strategy, such as the chip presented here, represents an opportunity to reduce the financial barriers associated with PET imaging. While this study focused on a device for [68Ga]Ga-PSMA-11, the technology is also applicable to a wide range of other tracers where low-cost, automated, dose-on-demand production is highly desirable.

The use of zirconium stationary phase to determine the four major arsenoriboses concentrations in marine organisms by high-performance liquid chromatography coupled to inductively coupled plasma–mass spectrometry

HPLC-ICP-MS utilising a ZirChrom®-SAX column with an aqueous 10 mM ammonium dihydrogen phosphate mobile phase at pH 7.5 (adjusted with aqueous ammonia) was evaluated for measuring glycerol arsenoriboside (OH-ribose), phosphate arsenoriboside (PO4-ribose), sulfonate arsenoriboside (SO3-ribose) and sulfate arsenoriboside (OSO3-ribose) in marine macroalgae and animals by analysis of seven reference materials. The results obtained were compared to those obtained using PRP-X100 anion-exchange chromatography. Inorganic arsenic species were not eluted from the zirconium column due to the strong interaction with the zirconia stationary phase. Measurements of SO3-ribose and OSO3-ribose concentrations were in close agreement with those obtained using the PRP-X100 column with a 0.005 mg As kg−1 detection limit. Peak shapes of these arsenoriboses were improved allowing lower amounts to be quantified. The quantification of OH-ribose and PO4-ribose, however, was sample dependent and not possible if arsenobetaine (AB) and (DMA) were present at large concentrations in samples because of coelution of these species with OH-ribose and PO4-ribose, respectively. In the absence of OH-ribose, AB can be determined using the zirconium column while AB on the PRP-X100 column coelutes with arsenic cations.

Universal retention standard for peptide separations using various modes of high-performance liquid chromatography

Peptide retention standards are widely used by chromatography specialists. They can be used for quality control of peptide separations (separation efficiency, selectivity, retention values) and for accurate concatenation of retention data from multiple acquisitions in proteomics. So far the repertoire of available retention standards is mostly limited to reversed-phase separations. We introduce a synthetic peptide mixture which can be used in conjunction with the most popular peptide separation techniques: reversed-phase (RPLC), strong-cation exchange (SCX), (strong-anion exchange) SAX and hydrophilic interaction liquid chromatography (HILIC). Target sequences were first designed in-silico using Sequence-Specific Retention Calculator models covering all major peptide separation mechanisms. Peptides were also designed while keeping in mind the simplicity of retention time assignment using MS detection: they all have nearly identical masses and identical intense y3 fragment ions. This contribution demonstrates the application of this mixture for characterization of eight HILIC as well as SAX, SCX and C18 columns.

Applying molecular modelling and experimental studies to develop molecularly imprinted polymer for domoic acid enrichment from both seawater and shellfish

A highly selective sample cleanup method using molecularly imprinted polymers (MIP) was developed for the enrichment of domoic acid (DA, an amnesic shellfish toxin) from both seawater and shellfish samples. Molecular modelling was firstly applied to screening a suitable functional monomer and optimize the polymer preparation. Theoretical results were in a good agreement with those of the experimental studies. MIP was prepared by precipitation polymerization using 1, 3, 5-pentanetricarboxylic acid and 2-(Trifluoromethyl)acrylic acid as the template molecule and functional monomer, respectively. The morphology and molecular structure of MIP were revealed by scanning electron microscope (SEM) and fourier transform infrared spectroscopy (FTIR), respectively. The obtained MIP showed high affinity and selectivity for DA with binding site numbers of 0.875 mg g−1 and an average association constant of 0.219 L mg−1 evaluated by adsorption experiments. The developed molecularly imprinted solid-phase extraction (MISPE) column achieved satisfied adsorption rate (99.2%) and recovery (71.2%) with relative standard deviation (RSD) less than 1.0%, which is more stable and precise than the C18, SAX, and HLB columns. Finally, the determination method for DA in both seawater and shellfish samples was then successfully established and validated using MISPE coupled with high-performance liquid chromatography-ultraviolet detection (HPLC-UV). The method limit of detection was 20 μg L−1 and 50 μg kg−1 for seawater and shellfish, respectively. This study demonstrates that molecular modelling is a useful tool to screening functional monomer and optimize polymer preparation. It provides an innovative polymer for trace DA monitoring in both seawater and shellfish.

Development and validation of a stability indicating method for S-carboxymethyl-l-cysteine and related degradation products in oral syrup formulation

A stability-indicating method for the determination of S-carboxymethyl-l-cysteine and related degradation impurities in Exputex® 250mg/5mL syrup was developed in anion-exchange liquid chromatography mode. A forced degradation study supported the method development to ensure stability indicating conditions. Aqueous solutions of the active pharmaceutical ingredient and syrup samples at different pH-values were stress-tested in different thermal, light exposure and headspace conditions. One degradation product was detected in thermal stress studies at 60°C and 80°C in the pH range 5.0–7.0 and was identified by mass spectrometry as 5-oxo-thiomorpholine-3-carboxylic acid (lactam of carbocysteine). A second degradation product was only generated in moderately strong oxidizing conditions (0.5% H2O2 aqueous solution) and was identified as S-carboxymethyl-l-cysteine-(R/S)-sulphoxide (carbocysteine sulphoxide).The method was developed on a Zorbax SAX column, in isocratic mode. The mobile phase consisted of 200mM phosphate solution at pH 4.0 and acetonitrile (50:50 v/v) and UV detection was performed at a wavelength of 205nm. The method was linear for carbocysteine (R>0.9982) over a concentration range of 2.5–50μg/mL and 0.4–0.6mg/mL. Linearity for the impurities was shown from the LOQ to 50μg/mL. Specificity was verified and accuracy demonstrated for the active ingredient and its degradation products in syrup samples at 3 levels around their respective specification limits. Repeatability, intermediate precision and inter-laboratory reproducibility were assessed on three commercial batches, analyzed in triplicate by two operators at both the transferring and the receiving site and demonstrated a successful method transfer to the manufacturing quality control laboratory.

Simultaneous determination of intracellular UDP-sugars in hyaluronic acid-producing Streptococcus zooepidemicus.

Two chromatographic methods for the quantitative analysis of uridine diphosphate (UDP) sugars involved in hyaluronan pathway of Streptococcus zooepidemicus (SEZ) were developed and compared. The sample preparation protocol using centrifugation and extraction in hot ethanol was employed prior to the analyses. Separation was achieved using an anion exchange Spherisorb SAX column or a Shodex QA-825 column connected with a photodiode array (PDA) detector. To increase the throughput of the chromatography method employing the Spherisorb SAX column, the solid phase extraction (SPE) procedure was introduced. Method validation results displayed that limits of detection (LODs) of UDP-glucose (UDP-Glc), UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-glucuronic acid (UDP-GlcA) calculated according to QC Expert software were in the low micromolar range and the coefficient of correlation (R(2)) was above 0.997. However, the analytical technique using the Spherisorb SAX column resulted in 80-90% recoveries and low LODs (≤6.19μM), the Shodex QA-825 column showed better long-term stability and reproducible chromatographic properties (RSD≤5.60%). The Shodex QA-825 column was successfully used to monitor UDP-sugar levels during the growth rate of SEZ cells.

Online anion exchange column preconcentration and high performance liquid chromatographic separation with inductively coupled plasma mass spectrometry detection for mercury speciation analysis

A hyphenated method for mercury speciation analysis by the coupling of high performance liquid chromatography and inductively coupled plasma mass spectrometry with the online strong anion exchange column (SAX) preconcentration was developed. The Hg analytes (Hg+, MeHg, EtHg and Hg2+) were absorbed on the SAX column preconditioned with sodium 3-mercapto-1-propanesulfonate, and then rapidly eluted (less than 16s) by 5μL 3% (v/v) 2-mercaptoethanol. The enrichment factors of 1025 for Hg+, 1084 for MeHg, 1108 for EtHg and 1046 for Hg2+ were obtained using 6mL sample in a 1.5-min enrichment procedure. Rapid separation of the four mercurial compounds was achieved within 5min on a 50-mm C18 column using 0.5% (v/v) 2-mercaptoethanol as the mobile phase. The detection limits for Hg+, MeHg, EtHg and Hg2+ were 0.015, 0.010, 0.009 and 0.016ngL−1, each, and the relative standard deviations of peak height and peak area (5ngL−1 for each Hg species) were all below 5%. Mercury speciation in three freshwater, two drinking water and two seawater samples were then analyzed by the proposed method. MeHg and Hg2+ concentrations down to 0.14 and 0.56ngL−1 were detected in the drinking waters.

Assessment of dietary intake of fumonisin B1 in São Paulo, Brazil

In this study, fumonisin B1 (FB1) consumption was assessed through determination of FB1 in corn meal, corn flour, corn flakes, polenta, canned corn and popcorn collected from homes of residents of Pirassununga, São Paulo, Brazil, and using a Food Frequency Questionnaire (FFQ) filled out by the residents. One hundred and twenty samples were collected from 39 residents on four separate occasions. FB1 was determined by high performance liquid chromatography using a validated method that uses SAX column clean-up. The highest levels of FB1 were found in corn meal at a mean concentration of 474.6μgkg−1. However, none of the samples tested for FB1 had levels above the tolerance limit established in Brazil. The mean probable daily intake (PDIM) of FB1 was 63.3ngkg−1bodyweightday−1, which is approximately 3% of the provisional maximum tolerable daily intake (PMTDI) recommended for fumonisins.

Strong anion exchange liquid chromatographic separation of protein amino acids for natural 13C‐abundance determination by isotope ratio mass spectrometry

Amino acids are the building blocks of proteins and the analysis of their (13)C abundances is greatly simplified by the use of liquid chromatography (LC) systems coupled with isotope ratio mass spectrometry (IRMS) compared with gas chromatography (GC)-based methods. To date, various cation exchange chromatography columns have been employed for amino acid separation. Here, we report strong anion exchange chromatography (SAX) coupled to IRMS with a Liquiface interface for amino acid δ(13)C determination. Mixtures of underivatised amino acids (0.1-0.5 mM) and hydrolysates of representative proteins (prawns and bovine serum albumin) were resolved by LC/IRMS using a SAX column and inorganic eluents. Background inorganic carbon content was minimised through careful preparation of alkaline reagents and use of a pre-injector on-line carbonate removal device. SAX chromatography completely resolved 11 of the 16 expected protein amino acids following acid hydrolysis in underivatised form. Basic and neutral amino acids were resolved with 35 mM NaOH in isocratic mode. Elution of the aromatic and acidic amino acids required a higher hydroxide concentration (180 mM) and a counterion (NO 3-, 5-25 mM). The total run time was 70 min. The average δ(13)C precision of baseline-resolved peaks was 0.75‰ (range 0.04 to 1.06‰). SAX is a viable alternative to cation chromatography, especially where analysis of basic amino acids is important. The technology shows promise for (13)C amino acid analysis in ecology, archaeology, forensic science, nutrition and protein metabolism.

Explorative Solid-Phase Extraction (E-SPE) for Accelerated Microbial Natural Product Discovery, Dereplication, and Purification

Microbial natural products (NP) cover a high chemical diversity, and in consequence extracts from microorganisms are often complex to analyze and purify. A distribution analysis of calculated pK(a) values from the 34390 records in Antibase2008 revealed that within pH 2-11, 44% of all included compounds had an acidic functionality, 17% a basic functionality, and 9% both. This showed a great potential for using ion-exchange chromatography as an integral part of the separation procedure, orthogonal to the classic reversed-phase strategy. Thus, we investigated the use of an "explorative solid-phase extraction" (E-SPE) protocol using SAX, Oasis MAX, SCX, and LH-20 columns for targeted exploitation of chemical functionalities. E-SPE provides a minimum of fractions (15) for chemical and biological analyses and implicates development into a preparative scale methodology. Overall, this allows fast extract prioritization, easier dereplication, mapping of biological activities, and formulation of a purification strategy.

Molecular Identification of Carnosine Synthase as ATP-grasp Domain-containing Protein 1 (ATPGD1)

Carnosine (beta-alanyl-L-histidine) and homocarnosine (gamma-aminobutyryl-L-histidine) are abundant dipeptides in skeletal muscle and brain of most vertebrates and some invertebrates. The formation of both compounds is catalyzed by carnosine synthase, which is thought to convert ATP to AMP and inorganic pyrophosphate, and whose molecular identity is unknown. In the present work, we have purified carnosine synthase from chicken pectoral muscle about 1500-fold until only two major polypeptides of 100 and 90 kDa were present in the preparation. Mass spectrometry analysis of these polypeptides did not yield any meaningful candidate. Carnosine formation catalyzed by the purified enzyme was accompanied by a stoichiometric formation, not of AMP, but of ADP, suggesting that carnosine synthase belongs to the "ATP-grasp family" of ligases. A data base mining approach identified ATPGD1 as a likely candidate. As this protein was absent from chicken protein data bases, we reconstituted its sequence from a PCR-amplified cDNA and found it to fit with the 100-kDa polypeptide of the chicken carnosine synthase preparation. Mouse and human ATPGD1 were expressed in HEK293T cells, purified to homogeneity, and shown to catalyze the formation of carnosine, as confirmed by mass spectrometry, and of homocarnosine. Specificity studies carried out on all three enzymes were in agreement with published data. In particular, they acted with 15-25-fold higher catalytic efficiencies on beta-alanine than on gamma-aminobutyrate. The identification of the gene encoding carnosine synthase will help for a better understanding of the biological functions of carnosine and related dipeptides, which still remain largely unknown.

Investigation of urinary steroid metabolites in calf urine after oral and intramuscular administration of DHEA

DHEA (3beta-hydroxy-androst-5-en-17-one) is a natural steroid prohormone. Despite a lack of information on the effect, DHEA and other prohormones are frequently used as a food supplement by body-builders. DHEA is suspected for growth promoting abuse in cattle as well. Considering the latter, urine samples from a previous exposure study in which calves were exposed to 1 g DHEA per day for 7 days, were used. The calves were divided in three groups: one orally treated, one intramuscularly injected, and a control group. The effect of this treatment on the urinary profile of several precursors and metabolites of DHEA was investigated. Urine samples were collected several days before and during the 7 days of administration and were submitted to a clean-up procedure consisting of a separation of the different conjugates (free, glucuronidated, and sulfated forms) of each compound on a SAX column (Varian). An LC-MS/MS method was developed for the detection and quantification of several metabolites of the pathway of DHEA including 17alpha- and 17beta-testosterone, 4-androstenedione, 5-androstenediol, pregnenolone, and hydroxypregnenolone. Elevated levels of DHEA, 5-androstenediol, and 17alpha-testosterone were observed in the free and sulfated fraction of the urine of the treated calves, thus indicating that the administered DHEA is metabolized mainly by the Delta(5)-pathway with 5-androstenediol as the intermediate. Sulfoconjugates of DHEA and its metabolites were found to constitute the largest proportion of the urinary metabolites. The free form was also present, but in a lesser extent than the sulfated form, while glucuronides were negligible.

Determination of Ethylglucuronide in Oral Fluid by Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry

An ultra-performance liquid chromatography-tandem mass spectrometry method has been developed and validated for the determination of ethylglucuronide (EtG) in oral fluid. Sample clean-up was achieved by solid-phase extraction with a Hyper-SEP SAX column. Negative ionization was performed in the multiple reaction monitoring mode. Two transitions were monitored for the analyte and one for the internal standard EtG-d(5). The calibration range was 4.4-222 ng/mL. The recovery of the analyte ranged from 86 to 99%, and the between-assay precisions ranged from 5 to 9% RSD. The limit of quantification was found to be 4.4 ng/mL. The concentration of EtG in oral fluid collected 2-14 h after a moderate alcohol intake varied from 13.3 to 57.7 ng/mL.

Studies on Pathway of Producing Terephthalic Acid by Microorganism Coordinated Catalysis

A method was developed for the determination of terephthalic acid, p-tolualdehyde, p-methylbenzyl alcohol, and p-toluic acid by HPLC. Hypersil SAX column was used. The mobile phase was 2.5 M NH 4H 2PO 4 containing 10% acetonitrile, and the pH was 4.32. The flow rate was 0.8 ml min −1, the temperature of column was 30 °C, and the detected wavelength was 254 nm. In this study, each component was completely separated and determined in 7 min. The recovery accorded with the detecting requirement. The method was used to analyze the main metabolites in the fermentation broth at different ferment times, which were produced at the process of biocatalysis producing terephthalic acid by Stenotrophomonas maltophilia and Comamonas testosteroni. The metabolites in cell including organic acids, amino acids, fatty acids, and sugars were detected by GC-MS. According to the analytical results of HPLC and GC-MS, the pathway of producing terephthalic acid by Stenotrophomonas maltophilia and Comamonas testosteroni coordinated catalysis was discussed.

Fractionation of free and conjugated steroids for the detection of boldenone metabolites in calf urine with ultra‐performance liquid chromatography/tandem mass spectrometry

For over a decade there has been an intensive debate on the possible natural origin of boldenone (androst-1,4-diene-17beta-ol-3-one, 17beta-boldenone) in calf urine and several alternative markers to discriminate between endogenously formed boldenone and exogenously administered boldenone have been suggested. The currently approved method for proving illegal administration of beta-boldenone(ester) is the detection of beta-boldenone conjugates. In the presented method the sulphate, glucuronide and free fractions are separated from each other during cleanup on a SAX column to be able to determine the conjugated status of the boldenone metabolites. The sulphate and glucuronide fractions are submitted to hydrolysis and all three fractions are further cleaned up on a combination of C18/NH2 solid-phase extraction (SPE) columns. Chromatographic separation of the boldenone metabolites was achieved with a Waters Acquity UPLC instrument using a Sapphire C18 (1.7 microm; 2x50 mm) column within 5 min. Detection of the analytes was achieved by electrospray ionisation tandem mass spectrometry. The decision limits of this method, validated according to Commission Decision 2002/657/EC, were 0.08 ng mL(-1) for androsta-1,4-diene-3,17-dione, 0.13 ng mL(-1) for androst-4-ene-3,17-dione, 0.11 ng mL(-1) for 17alpha-boldenone, 0.07 ng mL(-1) for 17beta-boldenone, 0.24 ng mL(-1) for 5beta-androst-1-en-17beta-ol-3-one and 0.58 ng mL(-1) for 6beta-hydroxy-17beta-boldenone. Because of the fractionation approach used in this method there is no need for conjugated reference standards which often are not available. The disadvantage of needing three analytical runs to determine the conjugated status of each of the metabolites was overcome by using fast chromatography.

Determination of nitric oxide metabolites, nitrate and nitrite, in Anopheles culicifacies mosquito midgut and haemolymph by anion exchange high-performance liquid chromatography: plausible mechanism of refractoriness

BackgroundThe diverse physiological and pathological role of nitric oxide in innate immune defenses against many intra and extracellular pathogens, have led to the development of various methods for determining nitric oxide (NO) synthesis. NO metabolites, nitrite (NO2-) and nitrate (NO3-) are produced by the action of an inducible Anopheles culicifacies NO synthase (AcNOS) in mosquito mid-guts and may be central to anti-parasitic arsenal of these mosquitoes.MethodWhile exploring a plausible mechanism of refractoriness based on nitric oxide synthase physiology among the sibling species of An. culicifacies, a sensitive, specific and cost effective high performance liquid chromatography (HPLC) method was developed, which is not influenced by the presence of biogenic amines, for the determination of NO2- and NO3- from mosquito mid-guts and haemolymph.ResultsThis method is based on extraction, efficiency, assay reproducibility and contaminant minimization. It entails de-proteinization by centrifugal ultra filtration through ultracel 3 K filter and analysis by high performance anion exchange liquid chromatography (Sphereclone, 5 μ SAX column) with UV detection at 214 nm. The lower detection limit of the assay procedure is 50 pmoles in all midgut and haemolymph samples. Retention times for NO2- and NO3- in standards and in mid-gut samples were 3.42 and 4.53 min. respectively. Assay linearity for standards ranged between 50 nM and 1 mM. Recoveries of NO2- and NO3- from spiked samples (1–100 μM) and from the extracted standards (1–100 μM) were calculated to be 100%. Intra-assay and inter assay variations and relative standard deviations (RSDs) for NO2- and NO3- in spiked and un-spiked midgut samples were 5.7% or less. Increased levels NO2- and NO3- in midguts and haemolymph of An. culicifacies sibling species B in comparison to species A reflect towards a mechanism of refractoriness based on AcNOS physiology.ConclusionHPLC is a sensitive and accurate technique for identification and quantifying pmole levels of NO metabolites in mosquito midguts and haemolymph samples that can be useful for clinical investigations of NO biochemistry, physiology and pharmacology in various biological samples.

Characterization of the Metabolic Activation of Hepatitis C Virus Nucleoside Inhibitor β-d-2′-Deoxy-2′-fluoro-2′-C-methylcytidine (PSI-6130) and Identification of a Novel Active 5′-Triphosphate Species

beta-D-2'-Deoxy-2'-fluoro-2'-C-methylcytidine (PSI-6130) is a potent inhibitor of hepatitis C virus (HCV) replication in the subgenomic HCV replicon system, and its corresponding 5'-triphosphate is a potent inhibitor of the HCV RNA polymerase in vitro. In this study the formation of PSI-6130-triphosphate was characterized in primary human hepatocytes. PSI-6130 and its 5'-phosphorylated derivatives were identified, and the intracellular concentrations were determined. In addition, the deaminated derivative of PSI-6130, beta-d-2'-deoxy-2'-fluoro-2'-C-methyluridine (RO2433, PSI-6026) and its corresponding phosphorylated metabolites were identified in human hepatocytes after incubation with PSI-6130. The formation of the 5'-triphosphate (TP) of PSI-6130 (PSI-6130-TP) and RO2433 (RO2433-TP) increased with time and reached steady state levels at 48 h. The formation of both PSI-6130-TP and RO2433-TP demonstrated a linear relationship with the extracellular concentrations of PSI-6130 up to 100 mum, suggesting a high capacity of human hepatocytes to generate the two triphosphates. The mean half-lives of PSI-6130-TP and RO2433-TP were 4.7 and 38 h, respectively. RO2433-TP also inhibited RNA synthesis by the native HCV replicase isolated from HCV replicon cells and the recombinant HCV polymerase NS5B with potencies comparable with those of PSI-6130-TP. Incorporation of RO2433-5'-monophosphate (MP) into nascent RNA by NS5B led to chain termination similar to that of PSI-6130-MP. These results demonstrate that PSI-6130 is metabolized to two pharmacologically active species in primary human hepatocytes.