Anion-exchange High-performance Liquid Chromatography Research Articles

Abstract 1049: Prognostication of patients with clear cell renal cell carcinomas based on quantification of DNA methylation levels of CpG island methylator phenotype marker genes using anion exchange high performance liquid chromatography

Abstract Background: We have identified CpG island methylator phenotype (CIMP)-positive clear cell renal cell carcinomas (ccRCCs) characterized by accumulation of DNA hypermethylation of CpG islands, tumor aggressiveness and poorer patient outcome. Furthermore, the criteria for prognostication of patients with ccRCCs have been established based on DNA methylation levels of ccRCC-specific CIMP marker genes. Recently, we have developed an anion exchange High Performance Liquid Chromatography (HPLC) column for detection of methylated DNA. To make the prognostication of patients with ccRCCs clinically applicable, the newly developed HPLC column has been applied to DNA methylation analysis of CIMP marker genes. Methods: Genomic DNA extracted from tumorous tissue samples of nephrectomy specimens of 5 patients with CIMP-positive ccRCCs and 5 patients with CIMP-negative ccRCCs was amplified by Polymerase Chain Reaction (PCR) after sodium bisulfite conversion with the EpiTect Bisulfite Kit (QIAGEN). Three-hundred and eighty-four bp-PCR products encompassing the promoter region of the FAM150A gene, a CIMP marker gene, and including 39 CpG sites were subjected to the HPLC column. DNA methylation levels were quantitatively evaluated using 0% and 100% methylated DNA controls (QIAGEN). Results: The analysis time for all samples was within 10 minutes. The retention time difference between 0% and 100% methylated DNA controls was 0.34 min. CIMP-negative ccRCCs were detected as a distinct single peak which had similar retention time with the 0% methylated DNA control, whereas CIMP-positive ccRCCs showed a bimodal peak pattern. Retention time of the later peak of CIMP-positive ccRCCs was similar with the 0% methylated DNA control. On the other hand, an early peak was eluted at various positions depending on its DNA methylation level. Based on the differences of chromatogram patterns, CIMP-positive ccRCCs were completely discriminated from CIMP-negative ccRCCs. DNA methylation levels of the FAM150A gene of each tissue sample calculated from retention time were consistent with those previously evaluated using MassARRAY system (Sequenom). Conclusion: Using newly developed anion exchange HPLC column, tissue specimens of CIMP-positive ccRCCs were readily, quickly and clearly distinguished from those of CIMP-negative ccRCCs, indicating that this HPLC method may be suitable for clinical application of prognostication of patients with ccRCCs based on CIMP diagnosis. Citation Format: Takuya Yotani, Yuriko Yamada, Eri Arai, Ying Tian, Masahiro Gotoh, Motokiyo Komiyama, Hiroyuki Fujimoto, Michiie Sakamoto, Yae Kanai. Prognostication of patients with clear cell renal cell carcinomas based on quantification of DNA methylation levels of CpG island methylator phenotype marker genes using anion exchange high performance liquid chromatography. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1049. doi:10.1158/1538-7445.AM2015-1049

Heparinase Digestion-Based Disaccharide Composition Analysis of Heparin and Heparinoid Drugs

Abstract Heparin and low molecular weight heparin have been used widely in clinical therapy as anticoagulants in cardiovascular disease and in hemodialysis. Crude heparin is usually prepared from porcine intestinal mucosa. Purified heparin is a mixture of polysaccharides consisting mainly of repeating GlcNS(6S)-IdoA2S disaccharides and other disaccharides with different GlcNAc/GlcNS±3S±6S-GlcA/IdoA±2S residues. Heparin injections are prepared by purification and then sterilization of ungraded heparin. Low molecular weight heparins are dominant heparin-based drugs used clinically, which are prepared by degrading heparin into smaller molecules. As a result, low molecular weight heparins share the same major disaccharides with heparin, but may have different reducing and non-reducing ends due to different preparation methods. In the current study, we analyzed the disaccharide composition of clinically used heparin and heparin-based drugs. Heparinase I, II and III were used to degrade all heparin and heparin-based drugs including heparin sodium injection, Enoxaparin sodium injection, Nadroparin calcium injection, Dalteparin sodium injection, and Fondaparinux sodium injection into disaccharides. All the degraded products were analyzed by strong anion exchange-high performance liquid chromatography (SAX-HPLC) equipped with an online UV-detector. Commercially available unsaturated disaccharide standards were then used for structural identification. Furthermore, unusual disaccharides present in Nadroparin, Dalteparin and Fondaparinux were confirmed by reversed-phase ion-pair HPLC coupled with mass spectrometry analysis. The developed method produced detailed structural information, which should be useful for quality control of heparin and heparin-based drugs.

Simultaneous speciation analysis of chromate, molybdate, tungstate and vanadate in welding fume alkaline extracts by HPLC–ICP-MS

A novel analytical procedure was developed for the simultaneous speciation analysis of chromate, molybdate, tungstate and vanadate by anion-exchange high performance liquid chromatography hyphenated to inductively coupled plasma mass spectrometry (HPLC–ICP-MS). Linear gradient elution from 100% water to 100% 0.7M NaCl was applied for chromatographic separation of metal species. In standard aqueous solution at neutral pH molybdate, tungstate and vanadate exist in several aqueous species, while chromate is present as a single CrO42- species. Consequently, only chromate can be separated from this solution in a sharp chromatographic peak. For obtaining sharp chromatographic peaks for molybdate, tungstate and vanadate, the pH of aqueous standard solutions was raised to 12. At highly alkaline conditions single CrO42-, MoO42- and WO42- are present and were eluted in sharp chromatographic peaks, while VO43- species, which predominates at pH 12 was eluted in slightly broaden peak. In a mixture of aqueous standard solutions (pH 12) chromate, molybdate, tungstate and vanadate were eluted at retention times from 380 to 420s, 320 to 370s, 300 to 350s and 240 to 360s, respectively. Eluted species were simultaneously detected on-line by ICP-MS recording m/z 52, 95, 182 and 51. The developed procedure was successfully applied to the analysis of leachable concentrations of chromate, molybdate, tungstate and vanadate in alkaline extracts (2% NaOH+3% Na2CO3) of manual metal arc (MMA) welding fumes loaded on filters. Good repeatability and reproducibility of measurement (RSD±3.0%) for the investigated species were obtained in both aqueous standard solutions (pH 12) and in alkaline extracts of welding fumes. Low limits of detection (LODs) were found for chromate (0.02ngCrmL−1), molybdate (0.1ngMomL−1), tungstate (0.1ngWmL−1) and vanadate (0.2ngVmL−1). The accuracy of analytical procedure for the determination of chromate was checked by analysis of CRM 545, Cr(VI) in welding dust loaded on a filter. Good agreement between determined and reported certified values was obtained. For molybdate, tungstate and vanadate the assessment of accuracy was performed by spiking welding fume filters. Good recoveries for all investigated species (98–101%) confirmed the accuracy of the analytical procedure.

Determination of deoxyribonucleoside triphosphate concentrations in yeast cells by strong anion-exchange high-performance liquid chromatography coupled with ultraviolet detection.

DNA polymerase assays are commonly used for the detection of deoxyribonucleoside triphosphates (dNTPs) in biological samples. For better specificity and accuracy, high-performance liquid chromatography (HPLC) methods have been developed for the analysis of the four dNTPs in complex samples. Here we describe a simple method using isocratic strong anion-exchange (SAX) chromatographic separation coupled with ultraviolet detection (UV) for the analysis of the four dNTPs in budding yeast Saccharomyces cerevisiae. This method can be applied to other species of yeast or bacteria.

Speciation of Selenium in Supplements by High Performance Liquid Chromatography-Inductively Coupled Plasma-Mass Spectrometry

Anion and cation exchange high-performance liquid chromatography (HPLC) combined with inductively coupled plasma-mass spectrometry (ICP-MS) were used for speciation of selenium in supplements. All the parameters in the extraction, separation, and determination procedures were optimized. Recovery studies for the selenium species from the anion and cation exchange columns were performed and there were no analyte losses. Limits of detection for selenium(IV), selenium(VI), Se in selenomethionine, and Se in selenocystine were 0.85, 0.68, 0.84, and 0.99 nanogram per milliliter, respectively. Six brands were analyzed to identify and quantify the selenium species present, and the results found were compared with the values given on the labels. The selenium species matched the labeled species for four brands, whereas two brands were found to contain inorganic Se(VI) in contrast with the labeled claim of selenomethionine.

Liquid chromatography combined with atomic and molecular mass spectrometry for speciation of arsenic in chicken liver

Human exposure to high concentrations of arsenic from water and food is an important health concern. Although 3-nitro-4-hydroxyphenylarsonic acid (Roxarsone) was used for more than 60 years as a feed additive to feed chickens, little is known about the metabolism of this arsenic species in chicken. Determination of potential arsenic metabolites present at trace concentrations is an analytical challenge, requiring efficient separation and sensitive detection. The primary objective of this research is to develop a method that enables the identification and quantification of various arsenic species in chicken liver. This report describes a method of high performance liquid chromatography (HPLC) separation with both inductively coupled plasma mass spectrometry (ICPMS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS) detection. Anion exchange HPLC enabled the separation of Roxarsone and other arsenic species within 12min. Detection with both ICPMS and ESI-MS/MS allowed for identification and quantification of eight arsenic species in chicken livers, including arsenobetaine, inorganic arsenite, dimethylarsinic acid, monomethylarsonic acid, inorganic arsenate, 3-amino-4-hydroxyphenylarsonic acid, N-acetyl-4-hydroxyphenylarsonic acid (N-AHPAA), and Roxarsone. The concentrations of these arsenic species, with the exception of arsenobetaine, are significantly higher in the Roxarsone-fed chickens than in the control chickens. The simultaneous detection by both ICPMS and ESIMS from the same HPLC separation allowed for comparison of peaks in both ICPMS and ESIMS chromatograms. This is advantageous over two separate analyses, particularly when HPLC retention times might fluctuate due to sample matrix effect. HPLC separation with the complementary atomic and molecular mass spectrometry detection prevented misidentification of co-eluting compounds, as demonstrated by the determination of two possible metabolites of Roxarsone, N-AHPAA and 4-amino-phenylarsonic acid (4-APAA). N-AHPAA was confirmed by HPLC separation with simultaneous arsenic-specific detection by ICPMS and multiple reaction monitoring by ESIMS. Although an arsenic-containing compound had identical retention time as 4-APAA in the HPLC–ICPMS chromatogram, it was ruled out as 4-APAA from the simultaneous detection by ESIMS. The identification and quantitation of trace arsenic species present in complex samples demonstrate the potential of HPLC separation with simultaneous ICPMS and ESIMS detection for other speciation applications.

Simple and robust monitoring of ethanol fermentations by capillary electrophoresis.

Free-solution capillary electrophoresis (CE), or capillary zone electrophoresis, with direct UV detection was used for the first time for the determination of mono- and disaccharides, sugar alcohols, and ethanol in fermentation broths. Sample preparation proved to be minimal: no derivatization or specific sample purification was needed. The CE conditions can be adapted to the type of fermentation by simply altering the background electrolyte (BGE). KOH (130 mM) or NaOH (130 mM) as the BGE led to the fastest analysis time when monitoring simple fermentations. A mixture of 65 mM NaOH and 65 mM LiOH led to a 19% improvement in resolution for a complex mixture of carbohydrates. Quantification of a simple carbohydrate fermentation by CE showed values in close agreement with that of high-performance anion exchange chromatography and high-performance liquid chromatography (HPLC) on a cation exchange resin. For complex fermentations, quantification of carbohydrates by HPLC and CE led to similar results, whereas CE requires an injection volume of only 10-20 nL. Analysis of an ethanol fermentation of hydrolyzed plant fiber demonstrated the robustness of the separation and detection of carbohydrates, as well as ethanol. Ethanol determination is achieved by coupling the CE method to pressure mobilization, using the same instrument and the same sample.

Simultaneous LC-MS/MS Analysis of Glyphosate, Glufosinate, and Their Metabolic Products in Beer, Barley Tea, and Their Ingredients

Glyphosate and glufosinate are non-selective herbicides that have been extensively used worldwide. Their ionic and water-soluble characteristics often make it difficult to analyze them, especially in food components. A method was developed in this study for the simultaneous analysis of glyphosate, glufosinate, and three metabolic products in beer, barley tea, and their ingredients (malt and corn). The analytical samples were extracted with H2O, purified with a strong anion-exchange solid-phase extraction (SPE) cartridge, and then analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) with an anion-exchange high-performance liquid chromatography (HPLC) column. This method enabled a rapid and sensitive analysis [limit of quantification (LOQ) = 10 µg/kg] of the herbicides to be achieved.

Borate-aided anion exchange high-performance liquid chromatography of uridine diphosphate-sugars in brain, heart, adipose and liver tissues

In this paper we describe a method optimized for the purification of uridine diphosphate (UDP)-sugars from liver, adipose tissue, brain, and heart, with highly reproducible up to 85% recoveries. Rapid tissue homogenization in cold ethanol, lipid removal by butanol extraction, and purification with a graphitized carbon column resulted in isolation of picomolar quantities of the UDP-sugars from 10 to 30mg of tissue. The UDP-sugars were baseline separated from each other, and from all major nucleotides using a CarboPac PA1 anion exchange column eluted with a gradient of acetate and borate buffers. The extraction and purification protocol produced samples with few unidentified peaks. UDP-N-acetylglucosamine was a dominant UDP-sugar in all the rat tissues studied. However, brain and adipose tissue showed high UDP-glucose levels, equal to that of UDP-N-acetylglucosamine. The UDP-N-acetylglucosamine showed 2.3–2.7 times higher levels than UDP-N-acetylgalactosamine in all tissues, and about the same ratio was found between UDP-glucose and UDP-galactose in adipose tissue and brain (2.6 and 2.8, respectively). Interestingly, the UDP-glucose/UDP-galactose ratio was markedly lower in liver (1.1) and heart (1.7). The UDP-N-acetylglucosamine/UDP-glucuronic acid ratio was also constant, between 9.7 and 7.7, except in liver with the ratio as low as 1.8. The distinct UDP-glucose/galactose ratio, and the abundance of UDP-glucuronic acid may reflect the specific role of liver in glycogen synthesis, and metabolism of hormones and xenobiotics, respectively, using these UDP-sugars as substrates.

Surface chemical composition of chromatographically fractionated graphite nanofiber-derived carbon dots

Carbon dots (C-dots) were synthesized from graphite nanofiber starting material by oxidizing in strong acid (i.e., top-down synthesis). The resulting product was heterogeneous with regard to size and surface functionality. After separating the mixture into fractions by means of anion-exchange (AE) high-performance liquid chromatography (HPLC), fractions with unique photoluminescent properties were selected. We examined the surface of these unique C-dot fractions by means of X-ray photoelectron spectroscopy and infrared spectroscopy and identified the surface functional groups on the C-dot fractions that exhibited higher photoluminescence. Although some surface functionalities were common to all fractions (e.g., carboxylic groups), others were different (e.g., aliphatic CH bonding). Among the luminescent nanoparticles examined, those with the higher luminescence showed less oxygen content. This strongly suggests that in the mixture of the as-synthesized C-dots, all nanoparticles did not have identical surface functionality, and they contributed differently to the observed photoluminescence.

Ultrasound-assisted enzymatic hydrolysis for iodinated amino acid extraction from edible seaweed before reversed-phase high performance liquid chromatography–inductively coupled plasma-mass spectrometry

The combination of reverse phase high performance liquid chromatography (RP-HPLC) with inductively coupled plasma mass spectrometry (ICP-MS) was used for the determination of monoiodotyrosine (MIT) and diiodotyrosine (DIT) in edible seaweed. A sample pre-treatment based on ultrasound assisted enzymatic hydrolysis was optimized for the extraction of these iodinated amino acids. Pancreatin was selected as the most adequate type of enzyme, and parameters affecting the extraction efficiency (pH, temperature, mass of enzyme and extraction time) were evaluated by univariate approaches. In addition, extractable inorganic iodine (iodide) was also quantified by anion exchange high performance liquid chromatography (AE-HPLC) coupled with ICP-MS. The proposed procedure offered limits of detection of 1.1 and 4.3ngg−1 for MIT and DIT, respectively. Total iodine contents in seaweed, as well as total iodine in enzymatic digests were measured by ICP-MS after microwave assisted alkaline digestion with tetramethylamonium hydroxide (TMAH) for total iodine assessment, and also by treating the pancreatin extracts (extractable total iodine assessment). The optimized procedure was successfully applied to five different types of edible seaweed. The highest total iodine content, and also the highest iodide levels, was found in the brown seaweed Kombu (6646±45μgg−1). Regarding iodinated amino acids, Nori (a red seaweed) was by far the one with the highest amount of both species (42±3 and 0.41±0.024μgg−1 for MIT and DIT, respectively). In general, MIT concentrations were much higher than the amounts of DIT, which suggests that iodine from iodinated proteins in seaweed is most likely bound in the form of MIT residues.

Relevance of intermediate-density lipoprotein cholesterol to Framingham risk score of coronary heart disease in middle-aged men with increased non-HDL cholesterol

Cholesterol levels of non-high-density lipoprotein (non-HDL), which contains low-density lipoprotein (LDL), intermediate-density lipoprotein (IDL), very low-density lipoprotein (VLDL) and chylomicron (CM) remnant, have been proven to perform a significant predictor of coronary heart disease (CHD) better than LDL-cholesterol regardless of triglyceride (TG) levels. The present study investigated the relevance of TG-rich lipoproteins (IDL, VLDL, CM) to Framingham risk score (FRS) predictive of 10-year CHD risk. Lipoprotein profiles (cholesterol levels of HDL, LDL, IDL, VLDL, CM) in Japanese men (n = 487) who underwent medical check-up were determined by using our developed anion-exchange high performance liquid chromatography (AEX-HPLC). Total-cholesterol (TC), TG, fasting blood sugar (FBS) and hemoglobin (Hb) A1c were measured by routine methods. The lipoprotein profiles, non-HDL-cholesterol, TC, and TG were examined on these associations with FRS. The lipid levels except for CM-cholesterol were significantly different between two groups (low FRS, < 10%; high FRS, ≥10%) (P < 0.0001), and body mass index (BMI), TC, TG, IDL-, and VLDL-cholesterol were significantly and positively correlated with FRS. Among them, the significant association of non-HDL-cholesterol to FRS was noted (r = 0.411, P < 0.0001). Multiple stepwise regression analysis shows that, in addition to non-HDL-cholesterol, IDL-cholesterol in TG-rich lipoproteins was significantly correlated with FRS in independently of BMI. These correlation results were similarly found even when the part of the study subjects (n = 348) without the drug therapy for hyperlipidemia, diabetes, and hypertension was investigated. These results suggest that IDL-cholesterol may serve as a useful marker for CHD risk in Japanese men with increased non-HDL-cholesterol.

Determination of Inosine Triphosphate Pyrophosphatase Phenotype in Human Red Blood Cells Using HPLC

Thiopurine drugs, widely used in cancer chemotherapy, inflammatory bowel disease, and autoimmune hepatitis, are responsible for common adverse events. Only some of these may be explained by genetic polymorphism of thiopurine S-methyltransferase. Recent articles have reported that inosine triphosphate pyrophosphatase (ITPase) deficiency was associated with adverse drug reactions toward thiopurine drug therapy. Here, we report a weak anion exchange high-performance liquid chromatography method to determine ITPase activity in red blood cells and to investigate the relationship with the occurrence of adverse events during azathioprine therapy. ITPase activity was assessed by the enzymatic conversion of inosine triphosphate (ITP) to inosine monophosphate (IMP). The reaction was stopped by heating for 3 minutes at 120°C. IMP, inosine diphosphate, and ITP were analyzed on a Hypersil APS-2 column, a weak anion exchange phase that exhibits both ionic and hydrophobic properties. The chromatographic method reported allows the analysis of IMP, inosine diphosphate, and ITP in a single run in <12.5 minutes. The method was linear in the range 5-1500 μmole/L of IMP. Intraassay and interassay precisions were <5% for red blood cell lysates supplemented with 50, 500, and 1000 μmole/L IMP. Km and Vmax evaluated by Lineweaver-Burk plot were 677.4 μmole/L and 19.6 μmole·L·min, respectively. The frequency distribution of ITPase from 73 patients was investigated. The method described is useful to determine the ITPase phenotype from patients on thiopurine therapy and to investigate the potential relation between ITPase deficiency and the occurrence of adverse events.

Fractionation of Carbon-Based Nanomaterials by Anion-Exchange HPLC

A sample containing carbon nanoparticles was obtained starting with the soot generated during combustion of inexpensive paraffin oil in a flame. The complexity of the sample, however, required fractionation to isolate its components. Anion-exchange high-performance liquid chromatography (AE-HPLC) was used for the analysis and collection of soot-derived carbon nanoparticles. The fractionated species were monitored by ultraviolet (UV) absorption and laser-induced photoluminescence detection, providing the chromatographic UV absorption and emission profiles of the separated sample. Chromatographic fractionation allowed for bulk measurements of electronic properties for individual fractions and further analysis via transmission electron microscopy (TEM). TEM of fractionated species showed a predominant size of about 4-5 nm diameter particulates. A general trend between photoluminescence and elution time was observed; the later eluting species in the chromatogram exhibited photoluminescence at longer wavelengths than the early eluting species. The AE-HPLC approach can have an immediate impact on the analysis and fractionation of various other nanomaterials, demonstrated here by analyzing samples containing graphitic oxide nanoparticles.

Speciation of trace elements in human serum by micro anion exchange chromatography coupled with inductively coupled plasma mass spectrometry

Speciation analysis of essential trace elements in human serum provides important information on nutritional status and homeostatic mechanisms regulating transport processes, acute phase reactions, and protection against oxidative damage. Anion exchange high-performance liquid chromatography (HPLC) combined with inductively coupled plasma mass spectrometry (ICP–MS) has proved to be a useful tool in speciation. Here we describe a fast method that can be applied to carry out the speciation of Fe, Cu, Zn, and Se in as little as 1 ml of serum. The method employs monolithic anion exchange micro columns installed on a tandem HPLC system coupled on-line with an ICP–MS detector. The chromatographic separation is similar to those reported previously but with considerable gain in terms of time and sample requirement. Reproducibility is acceptable for most species. Using our method, we were able to find species-specific differences between different commercially available trace element reference materials. Because the method chosen to collect blood might interfere with speciation, the proposed methodology was used to compare heparinized plasma, ethylenediaminetetraacetic acid (EDTA) plasma, and serum from adult healthy volunteers. As expected, EDTA strongly affects speciation analysis (especially for Fe and Zn), whereas changes due to the use of lithium–heparin (Li–He) as anticoagulant appear to be minimized.

Anion‐exchange HPLC separation of five major rabbit lipoproteins using a nonporous diethylaminoethyl‐ligated gel with a perchlorate‐containing eluent

Rabbits are often used as experimental animals in studies of atherosclerosis and hyperlipidemia. Although rabbit lipoproteins can be quantitated by sequential ultracentrifugation, a simpler and more reproducible method is desirable for detailed analyses. The current study describes a method to analyze rabbit lipoproteins in plasma by anion-exchange high-performance liquid chromatography using a column filled with nonporous, diethylaminoethyl-ligated polymers. A solution of NaClO₄ was used to adjust the ionic strength of the eluent. The method required only 15 μL of plasma and analysis was completed in 23 min. Five lipoprotein fractions (high-density lipoprotein, low-density lipoprotein, intermediate-density lipoprotein, very-low-density lipoprotein and chylomicrons) were eluted with step-wise increases in a concentration of NaClO₄. The post-column eluate was reacted with an enzymatic reagent to determine total cholesterol, and the lipoprotein-cholesterol fraction was calculated according to relative peak areas in the chromatogram. The within-day and between-day assay coefficients of variation for lipoprotein cholesterol levels ranged between 0.436 and 7.143% and between 2.905 and 10.526%, respectively. Administering a high-fat diet increased lipoprotein-cholesterol concentrations by 6- to 77-fold. The method described here was nevertheless able to quantitate levels of lipoprotein-cholesterol in plasma samples from these rabbits. These results indicate that this method may be applied to lipoprotein studies using hyperlipidemic rabbit models.

Identification of heparin samples that contain impurities or contaminants by chemometric pattern recognition analysis of proton NMR spectral data

Chemometric analysis of a set of one-dimensional (1D) (1)H nuclear magnetic resonance (NMR) spectral data for heparin sodium active pharmaceutical ingredient (API) samples was employed to distinguish USP-grade heparin samples from those containing oversulfated chondroitin sulfate (OSCS) contaminant and/or unacceptable levels of dermatan sulfate (DS) impurity. Three chemometric pattern recognition approaches were implemented: classification and regression tree (CART), artificial neural network (ANN), and support vector machine (SVM). Heparin sodium samples from various manufacturers were analyzed in 2008 and 2009 by 1D (1)H NMR, strong anion-exchange high-performance liquid chromatography, and percent galactosamine in total hexosamine tests. Based on these data, the samples were divided into three groups: Heparin, DS ≤ 1.0% and OSCS = 0%; DS, DS > 1.0% and OSCS = 0%; and OSCS, OSCS > 0% with any content of DS. Three data sets corresponding to different chemical shift regions (1.95-2.20, 3.10-5.70, and 1.95-5.70 ppm) were evaluated. While all three chemometric approaches were able to effectively model the data in the 1.95-2.20 ppm region, SVM was found to substantially outperform CART and ANN for data in the 3.10-5.70 ppm region in terms of classification success rate. A 100% prediction rate was frequently achieved for discrimination between heparin and OSCS samples. The majority of classification errors between heparin and DS involved cases where the DS content was close to the 1.0% DS borderline between the two classes. When these borderline samples were removed, nearly perfect classification results were attained. Satisfactory results were achieved when the resulting models were challenged by test samples containing blends of heparin APIs spiked with non-, partially, or fully oversulfated chondroitin sulfate A, heparan sulfate, or DS at the 1.0%, 5.0%, and 10.0% (w/w) levels. This study demonstrated that the combination of 1D (1)H NMR spectroscopy with multivariate chemometric methods is a nonsubjective, statistics-based approach for heparin quality control and purity assessment that, once standardized, minimizes the need for expert analysts.

Highly Sensitive Potentiometric Strip Test for Detecting High Charge Density Impurities in Heparin

Contamination of heparin with oversulfated chondroitin sulfate (OSCS) became a matter of grave concern in the medical field after many fatal responses to OSCS tainted heparin products occurred during the 2007-2008 period. Even though standard lab-based analytical techniques such as nuclear magnetic resonance (NMR) and strong anion-exchange high performance liquid chromatography (SAX-HPLC) have proven useful for monitoring the OSCS content in heparin products, an easy-to-use, quick, portable, and cost-efficient method is still needed for on-site monitoring during and after the heparin production. In this report, a disposable strip-type electrochemical polyion sensor is described for detection of low levels of OSCS contamination in heparin. A magnetic actuator is incorporated into this simple electrode-based microfluidic device in order to create the mixing effect necessary to achieve equilibrium potential changes of the sensor within a microfluidic channel. The planar membrane electrode detector within the sample channel is prepared with a tridodecylmethylammonium chloride (TDMAC)-doped poly(vinyl chloride) (PVC) membrane essentially equivalent to previously reported polyanion-sensitive electrodes. When the concentration of heparin applied to the single-use strip device is 57 mg/mL (in only 20 μL of sample), the same concentration recommended in the NMR analysis protocol for detecting OSCS in heparin, the detection limit is 0.005 wt % of OSCS, which is ca. 20 times lower than the reported detection limit of the NMR method.

Negatively charged low-density lipoprotein is associated with atherogenic risk in hypertensive patients

Negatively charged low-density lipoprotein (LDL), generated via multiple processes such as oxidation, acetylation, or glycosylation, plays a key role in the initiation and progression of atherosclerosis and related diseases. Anion-exchange high-performance liquid chromatography (AE-HPLC) can subfractionate LDL into LDL-1, LDL-2, and LDL-3 based on LDL particle charge, but the clinical significance of LDL subfractions has not yet been elucidated. The aim of this study was to determine the clinical significance of these fractions with particular regard to atherogenic risk in hypertensive patients. Ninety-eight patients with essential hypertension (age 67.0 ± 10.7 years; 54 males) were enrolled in the present study. The relationships between LDL subfractions and atherogenic risk factors, including lipid profiles, blood pressure and plasma 8-isoprostane as a marker of oxidative stress, were examined. LDL-1 levels were significantly and negatively correlated with body mass index (r = -0.384, p < 0.001), systolic blood pressure (r = -0.457, p < 0.001), non-high-density lipoprotein cholesterol levels (r = -0.457, p < 0.001) and 8-isoprostane levels (r = -0.415, p < 0.001). LDL-3, which is the most negatively charged fraction of total LDL, was significantly and positively correlated with these parameters (r = 0.267, 0.481, 0.357, and 0.337, respectively). LDL-1 levels were significantly lower (p < 0.001), and LDL-2 and LDL-3 levels were significantly higher (each p < 0.001) in patients with poorly controlled hypertension than in patients with well-controlled hypertension. In addition, an increase in the total number of traditional risk factors at time of study participation, but not previous diagnosis, was associated with a decrease in LDL-1 levels and increases in LDL-2 and LDL-3 levels. These data suggest that LDL subfractions are associated with multiple atherogenic risk factors and that treatment to modify these risk factors could result in changes in LDL subfraction levels. In conclusion, LDL subfractions isolated by AE-HPLC may represent a marker of atherogenic risk in patients with hypertension.

Separation and Analysis of Arsenic Species in Dietary Supplements using LC-ICP-MS

Speciation of arsenic is very important as the toxicity and the bioavailability are related to the chemical form. The inorganic species, As III (arsenite) and As V (arsenate), are considered carcinogenic, whereas the toxicity of the organic species is variable. The methylated forms, monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA), are much less toxic. More complex forms as arsenobetaine (AB) and arsenocholine (AC) are considered non-toxic [1]. Arsenic speciation analysis was performed in nine dietary supplements where total arsenic was found to be high. The samples were analyzed using gradient elution anion exchange high-performance liquid chromatography (HPLC) coupled with inductively coupled plasma mass spectrometry (ICP-MS). Solvent extraction with sonication and microwave extraction using various aqueous and aqueous/solvent mixtures was initially evaluated. Six arsenic species, including arsenic acid (As III), arsenous acid (As V), monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), arsenobetaine (AB), and arsenocholine (AC) were determined. The speciation data presented here may be valuable for the quality assurance of analytical method development and surveys of arsenic in dietary supplements. The three most abundant arsenic species found were arsenite, arsenate and dimethylarsinic acid. Total arsenic species concentrations in the commercial products analyzed ranged from 0.1 to 31µg/day. By using HPLC-ICP-MS, the relative amount of inorganic arsenic in the samples ranged from 59 to 89% of the sum of the arsenic species detected in each sample. The maximum tolerable daily intake of inorganic arsenic of 150µg/day for an average body weight of 70kg was established by the Joint FAOWHO Expert Committee on Food Additives (JECFA). All samples analyzed are within limits of JECFA.