High-performance Liquid Chromatography Column Research Articles

Fourth Reported Case of Hemoglobin South Florida: A Rare Hemoglobin Variant That Causes Falsely Elevated HbA1c Values

We report a case of a rare hemoglobin (Hb) variant, Hb South Florida (Hb-SF) with only three cases reported worldwide to our knowledge since initial description in 1985. It causes falsely elevated glycosylated hemoglobin (HbA1c) results during standard laboratory testing in the range of poorly controlled diabetes mellitus. A 42-year-old woman with a history of sclerosing bone dysplasia was noted to have an elevated HbA1c (13.8%) during routine blood work using the ion-exchange high performance liquid chromatography (HPLC) method. The patient did not have a prior diagnosis of diabetes mellitus and denied any symptoms consistent with diabetes. Physical exam was unremarkable. Her blood sugars were normal to minimally elevated. Over several months, she was noted to have persistent elevation in HbA1c in the range of 12-14%. She remained asymptomatic. The patient was referred to endocrinology clinic. Hb electrophoresis was performed due to suspicion for a hemoglobinopathy. The patient was noted to be heterozygous for a rare Hb variant called Hb South Florida that causes falsely elevated HbA1c levels. Her corrected HbA1c, when evaluated by affinity column HPLC, was in normoglycemic range. Hb South Florida was first described in 1985 in Tampa Florida and has since been reported in two other cases worldwide. The Hb is not associated with any clinical disorder reported in literature. There are many commercially available tests for measuring HbA1c levels. Additionally, structural variants in Hb interfere with many of the methods. Ion-exchange HPLC separates Hb based on their charge differences. Hb South Florida variant substitutes a neutral amino acid for a charged one, with valine substituted by methionine. This alteration decreases the transit time in the ion exchange column and causes it to co-elute together with HbA1c, giving a falsely elevated value as they get measured as same species. Affinity chromatography is based on affinity of a species for a particular ligand. It recognizes the structure of the N-terminal glycated amino acids of the beta chain of Hb. In the case of Hb South Florida, it was able to separate the variant Hb from glycated species. This is the fourth case of Hb variant South Florida to our knowledge. It is important for clinicians to keep a wide perspective when making an assessment of diabetes solely based on HbA1c values. Patients who do not present with symptoms consistent with diabetes, and have normal blood glucose should prompt a further workup for abnormal Hb variants. This should be done in order to prevent incorrect diagnosis and treatment. J Med Cases. 2015;6(5):229-231 doi: http://dx.doi.org/10.14740/jmc2134w

Reduced time HPLC analyses for fast quality control of citrus essential oils

Cold-pressed citrus essential oils contain variable amounts of oxygen heterocyclic compounds highly characteristic of each species. Their distribution has been defined by high-performance liquid chromatography (HPLC) analysis with an average time of about 45 minutes. Here are two methods proposed, applying the latest generation HPLC columns based on the fused-core technology. The first method allows the complete resolution of thirty-eight components for a rugged and validated quantitative determination of the oxygen heterocyclic compounds in cold pressed lemon, lime, bergamot grapefruit, sweet orange, bitter orange and mandarin oils, in 10 minutes of total analysis time. The second method is a compromise between chromatographic resolution and analysis time and allows a fingerprinting of each of the abovementioned oils to be obtained, useful for unveiling possible contaminations and frauds in about 3 minutes. This last method can be applied in routine analysis to save time and solvent. The components are sufficiently separated in all the citrus cold pressed oils, permitting detailed profiles of each oil to be acquired and possible contaminations and frauds to be detected.

Development and Validation of a New HPLC Method for In-Vitro Studies of Mebudipine in Pharmaceutical Formulations

A simple and sensitive method has been developed for the analysis of mebudipine in pharmaceutical formulations. According to this, mebudipine is analyzed by reverse phase high performance liquid chromatography (HPLC) using a system equipped with a C18 column and a UV detector operating at a wavelength of 290 nm. The mobile phase is a mixture of methanol, water, and acetonitrile (70 : 25 : 5, v/v/v). Under these conditions, separation of the drug was achieved in less than 20 min. For the assay, one tablet is dissolved in 50 ml methanol and 20 μl of this solution is injected into the HPLC column. The medium for dissolution testing was a mixture of water, 0.1 N HCl, gastric fluid, intestinal fluid, and sodium dodecyl sulfate in various concentrations (1, 2, and 3%). The tests were performed by using 500 ml medium in a paddle apparatus operating at a stirring rate of 75 rpm. Sampling aliquots of 5 ml were withdrawn at 30, 45, 60, 75 and 90 min. The drug release was evaluated by HPLC. It was found that 3% SDS ensured maximum release of mebudipine. The method was linear in the range of 0.5 – 500 μg/mL (R 2 = 0.9992). The limits of detection and quantitation were 0.6 and 0.7 μg/mL for dissolution test and 1.4 and 1.5 μg/mL for assay, respectively.

Phytoecdysones from the Roots of Achyranthes japonica Nakai and their Anti-atopy Activity

The roots of Achyranthes japonica Nakai were extracted with 100% aqueous and concentrated subfraction was separated with ultra-performance liquid chromatography-based activity profiling. Three compounds were isolated from the subfraction 5 through the repeated prep- high performance liquid chromatography column chromatography. According to the results of physico-chemical and spectroscopic data including NMR and MS, the chemical structures of the compounds were determined as ecdysterone (1), 25S-inokosterone (2), and 25R-inokosterone (3). Three phytoecdysones were showed weak inhibitory activity for thymus and activation-regulated chemokine expression levels in tumor necrosis factor (TNF)-α plus IFN-γ induced HaCaT cells, respectively. However, those compounds 1-3 were exhibited the most potent inhibition (80-95% at 200 µg/mL) against TNF-α expression levels in A23187 plus phorbol-myrisrate acetate-induced RBL-2H3 cells. As result, 100% aqueous extract of A. japonica has an excellent anti-atopy activity. It could be used to a large range of functional anti-atopy cosmetics.

Copper sorption efficiency of p-tetranitrocalix[4]arene based high performance liquid chromatographic column

Present study deals with the sorption characteristics of p-tetranitrocalix[4]arene (3) appended silica-based high-performance liquid chromatographic (HPLC) column utilized for preconcentration, determination, and purification of Cu2+ from water. The sorption characteristics of the HPLC column were investigated for Cu2+ in column agreement. The experiments were performed in five steps that were monitored using UV–visible diode-array detector. However, all the HPLC experimental results were reconfirmed by using atomic absorption spectrophotometer. The effect of concentration on the sorption efficiency of the column was evaluated for Cu2+ and data obtained was investigated using Langmuir and Freundlich sorption isotherms. The value of coefficient of determination (R2), i.e. 0.99, suggested that the Freundlich sorption isotherm was found to be the best fit model for Cu2+. The developed method was validated for system efficiency, accuracy, and precision, while the data obtained revealed that the developed column could be applied for the sorption of Cu2+ with adequate efficiency and repeatability.

Surface Characterization of Some Novel Bonded Phase Packing Materials for HPLC Columns Using MAS-NMR Spectroscopy

Information on the surface properties of three novel chemically bonded phase packing materials for High performance liquid chromatography (HPLC) were obtained using spectra obtained by solid state cross-polarization (CP) magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopic experiments for the 29Si, and 13C nuclei. These packing materials were: Cogent bidentate C18 bonded to type-C silica, hybrid packing materials XTerra MS C18, and XBridge Prep. C18. The spectra obtained using cross-polarization magic angle spinning (CP-MAS) on the Cogent bidentate C18 bonded to type-C silica show the surface to be densely populated with hydride groups (Si-H), with a relative surface coverage exceeding 80%. The hybrid packing materials XTerra and XBridge gave spectra that reveal the silicon atoms to be bonded to organic moieties embedded in the molecular structure of these materials with over 90% of the alkyl silicon atoms found within the completely condensed silicon environments. The hydrolytic stability of these materials were investigated in acidic aqueous solutions at pHs of 7.0 and 3.0, and it was found that while the samples of XTerra and XBridge were not affected by hydrolysis at this pH range, the sample of Cogent lost a significant proportion of its Si-H groups after five days of treatment in acidic aqueous solution.

Forward and reverse (retro) iron(III) or gallium(III) desferrioxamine E and ring-expanded analogues prepared using metal-templated synthesis from endo-hydroxamic acid monomers.

A metal-templated synthesis (MTS) approach was used to preorganize the forward endo-hydroxamic acid monomer 4-[(5-aminopentyl)(hydroxy)amino]-4-oxobutanoic acid (for-PBH) about iron(III) in a 1:3 metal/ligand ratio to furnish the iron(III) siderophore for-[Fe(DFOE)] (ferrioxamine E) following peptide coupling. Substitution of for-PBH with the reverse (retro) hydroxamic acid analogue 3-(6-amino-N-hydroxyhexanamido)propanoic acid (ret-PBH) furnished ret-[Fe(DFOE)] (ret-ferrioxamine E). As isomers, for-[Fe(DFOE)] and ret-[Fe(DFOE)] gave identical mass spectrometry signals ([M + H(+)](+), m/zcalc 654.3, m/zobs 654.3), yet for-[Fe(DFOE)] eluted in a more polar window (tR = 23.44 min) than ret-[Fe(DFOE)] (tR = 28.13 min) on a C18 reverse-phase high-performance liquid chromatography (RP-HPLC) column. for-[Ga(DFOE)] (tR = 22.99 min) and ret-[Ga(DFOE)] (tR = 28.11 min) were prepared using gallium(III) as the metal-ion template and showed the same trend for the retention time. Ring-expanded analogues of for-[Fe(DFOE)] and ret-[Fe(DFOE)] were prepared from endo-hydroxamic acid monomers with one additional methylene unit in the amine-containing region, 4-[(6-aminohexyl)(hydroxy)amino]-4-oxobutanoic acid (for-HBH) or 3-(7-amino-N-hydroxyheptanamido)propanoic acid (ret-HBH), to give the corresponding tris(homoferrioxamine E) macrocycles, for-[Fe(HHDFOE)] or ret-[Fe(HHDFOE)] ([M + H(+)](+), m/zcalc 696.3, m/zobs 696.4). The MTS reaction using a constitutional isomer of for-HBH that transposed the methylene unit to the carboxylic acid containing region, 5-[(5-aminopentyl)(hydroxy)amino]-5-oxopentanoic acid (for-PPH), gave the macrocycle for-[Fe(HPDFOE)] in a yield significantly less than that for for-[Fe(HHDFOE)], with the gallium(III) analogue for-[Ga(HPDFOE)] unable to be detected. The work demonstrates the utility and limits of MTS for the assembly of macrocyclic siderophores from endo-hydroxamic acid monomers. Indirect measures (RP-HPLC order of elution, c log P values, molecular mechanics, and density functional theory calculations) of the relative water solubility of the ligands, the iron(III) macrocycles, and the apomacrocycles were consistent in identifying for-DFOE as the most water-soluble macrocycle from for-DFOE, ret-DFOE, for-HHDFOE, ret-HHDFOE, and for-HPDFOE. From this group, only for-DFOE is known in nature, which could suggest that water solubility is an important trait in its natural selection.

Ultra high performance liquid chromatography with tandem mass spectrometry method for the determination of tetrandrine and fangchinoline in rat plasma after oral administration of Fangji Huangqi Tang and Stephania tetrandra S. Moore extracts.

A rapid, sensitive, and reliable analytical method based on ultra high performance liquid chromatography with tandem mass spectrometry has been developed for the simultaneous determination of fangchinoline and tetrandrine in rat plasma. Plasma samples were pretreated by protein precipitation with acetonitrile. The analysis was performed on a C18 reversed-phase ultra high performance liquid chromatography column (2.1 mm × 100 mm, 3.5 μm, Agilent), and the flow rate was set at 0.4 mL/min. Under the experimental conditions, extraction recoveries from plasma were 68.1-72.8% for fangchinoline and 69.2-76.5% for tetrandrine. The plasma concentrations of tetrandrine and fangchinoline in rats at assigned time points were all successfully detected through exactly validated method. Good linearities were obtained in the range of 0.92-184 ng/mL for tetrandrine and in the range of 0.83-166 ng/mL for fangchinoline. The intra- and interday precisions for both analytes were within 11.1% and the accuracies were within the range of -10.7 to 11.3%. The developed method was then successfully applied to investigate the pharmacokinetic properties of these two alkaloids after oral administration of Stephania tetrandra S. Moore extracts and Fangji Huangqi Tang. The comparative results provided a meaningful basis for a better understanding of the practical value of the compatibility theory of traditional Chinese medicine in the clinic.

Stability study of Prulifloxacin and Ulifloxacin in human plasma by HPLC–DAD

A new and specific HPLC–DAD method for the direct determination of Prulifloxacin and its active metabolite, Ulifloxacin, in human plasma has been developed. Plasma samples were analysed after a simple solid phase extraction (SPE) clean-up using a new HILIC stationary phase based high-performance liquid chromatography (HPLC) column and an ammonium acetate buffer (5 mM, pH 5.8)/acetonitrile (both with 1% Et3N, v/v) mobile phase in isocratic elution mode, with Danofloxacin as the internal standard. Detection was performed using DAD from 200 to 500 nm and quantitative analyses were carried out at 278 nm. The LOQ of the method was 1 μg/mL of the cited analytes and the calibration curve showed a good linearity up to 25 μg/mL. For both analytes the precision (RSD%) and the trueness (bias%) of the method fulfil with International Guidelines. The method was applied for stability studies, at three QC concentration levels, in human plasma samples stored at different temperature of + 25, + 4 and −20 °C in order to evaluate plasma stability profiles.

Forced-flow planar chromatography in the rear view mirror.

Mobile phase progress in planar stationary phase can be evoked by either external or internal forces. An internal force is capillarity, while gravity, electric field, a pump and centrifugal forces belong to external forces. Overpressured layer chromatography gives a widely used special chapter of forced-flow planar chromatography, a special bridge between high-performance liquid column chromatography and thin-layer chromatography (TLC). A simple and special rule characterizes the progress of mobile phase. Optimal efficiency is composed by the doubled effect of flow resulting from the pump-forced mobile phase (convex profile of laminar flow) and capillary forces on the dry stationary phase (concave laminar flow). This review describes the most important aspects of forced-flow TLC, including how the set-ups are developed and also the progress of detection methods used.

HPLC Separation of Enantiomers of Some Chiral Carboxylic Acid Derivatives Using Polysaccharide-Based Chiral Columns and Polar Organic Mobile Phases

The separation of enantiomers of 14 chiral carboxylic acid derivatives was studied on six different polysaccharide-based chiral columns in high-performance liquid chromatography with methanol, ethanol and acetonitrile as mobile phases with emphasis on the elution order of enantiomers. Some interesting examples of enantiomer elution order reversal were observed as a function of the nature and composition of chiral selector and mobile phase. For instance, the enantiomer elution order for carprofen, ketorolac, naproxen, proglumide and surprofen reversed with changing the chemical structure of the chiral selector. Also, the enantiomer elution order for carprofen, ketorolac and naproxen changed by varying the composition of the mobile phase. In addition, the interesting effect of column temperature on the retention and separation of some analytes was observed. For instance, the enantiomers of surprofen were only partially resolved at lower temperatures but baseline resolved at higher temperature.

Determining the Isomeric Heterogeneity of Neutral Oligosaccharide-Alditols of Bovine Submaxillary Mucin Using Negative Ion Traveling Wave Ion Mobility Mass Spectrometry

Negative ions produced by electrospray ionization were used to evaluate the isomeric heterogeneity of neutral oligosaccharide-alditols isolated from bovine submaxillary mucin (BSM). The oligosaccharide-alditol mixture was preseparated on an off-line high-performance liquid chromatography (HPLC) column, and the structural homogeneity of individual LC fractions was investigated using a Synapt G2 traveling wave ion mobility spectrometer coupled between quadupole and time-of-flight mass spectrometers. Mixtures of isomers separated by both chromatography and ion mobility spectrometry were studied. Tandem mass spectrometry (MS/MS) of multiple mobility peaks having the same mass-to-charge ratio (m/z) demonstrated the presence of different structural isomers and not differences in ion conformations due to charge site location. Although the oligosaccharide-alditol mixture was originally separated by HPLC, multiple ion mobility peaks due to structural isomers were observed for a number of oligosaccharide-alditols from single LC fractions. The collision-induced dissociation cells located in front of and after the ion mobility separation device enabled oligosaccharide precursor or product ions to be separated by ion mobility and independent fragmentation spectra to be acquired for isomeric carbohydrate precursor or product ions. MS/MS spectra so obtained for independent mobility peaks at a single m/z demonstrated the presence of structural variants or stereochemical isomers having the same molecular formula. This was observed both for oligosaccharide precursor and product ions. In addition, mobilities of both [M - H](-) and [M + Cl](-) ions, formed by adding NH4OH or NH4Cl to the electrospray solvent, were examined and compared for selected oligosaccharide-alditols. Better separation among structural isomers appeared to be achieved for some [M + Cl](-) anions.

Two-dimensional correlation analysis of the reproducibility of high-performance liquid chromatography columns

Two-dimensional (2D) correlation analysis is a well-established tool in spectroscopy. Despite its versatility in various measurement systems, 2D correlation has not yet become popular in separation science. 2D correlation is seldom used in chromatography; only a few a studies can be found on this topic and most of those publications report about gel chromatography. In the present study, 2D correlation analysis is applied to chromatograms. In this study, a simple method is built for studying the similarities and dissimilarities between a number of chromatograms. We present the applicability of the method by two examples, where the repeatability and reproducibility of the analytical and nonlinear measurements in HPLC are evaluated and demonstrated. In order to validate the results of 2D correlation analysis, they are compared to principal component analysis (PCA). We confirm the equivalence in the interpretation of the results obtained with the two methods of calculation. The results confirm that 2D correlation can be a successful chemometric tool in chromatography.

High-performance liquid chromatographic column packings with different particle sizes: chromatographic behavior for the quality analysis of HuanglianShangqing pill.

The chromatographic separation of traditional Chinese medicines is still a highly challenging task in analytical science with respect to its hundreds and thousands of chemical compounds, while increase of separation efficiency can greatly improve the separation power of chromatographic column for traditional Chinese medicine. In this study, 13 bioactive components in HuanglianShangqing pill were selected as an index to optimize the separation conditions and evaluate the system suitability of three commercially available columns packed with 1.8, 3.5, and 5.0 μm particles. The chromatographic separations were obtained by the most appropriate Eclipse Plus C18 column (100 × 2.1 mm, 3.5 μm) within 45 min using gradient elution with aqueous-ammonium acetate (10 mmol/L, pH 5.0) and acetonitrile, at a flow rate of 0.3 mL/min and an operating temperature of 30°C. The quality of HuanglianShangqing pill was assessed through combining simultaneous quantification of 13 compounds with fingerprint analysis. For the qualitative analysis, mass spectrometry was used to confirm the 13 compounds. All the validation data conformed to the acceptable requirements. For the fingerprint analysis, 32 peaks were selected as the common peaks at 254 nm to evaluate the similarities among HuanglianShangqing pills obtained from ten manufacturers.

Synthesis of hierarchically porous polymethylsilsesquioxane monoliths with controlled mesopores for HPLC separation

Sol–gel synthesis of macroporous polymethylsilsesquioxane (PMSQ) monoliths has been successful over the past decade, and applications to separation media have been investigated. However, the control of mesopores to tailor hierarchical porosity, which is promising for improvement of the separation efficiency, remains challenging. In particular, an independent control of meso- and macropores has not been achieved in PMSQ. Herein we present a method to synthesize PMSQ monoliths with well-defined macropores and controlled mesostructure (pore size ranging from 10 to 60 nm, total pore volume from 0.2 to 0.6 cm3 g−1) via sol–gel accompanied by phase separation. Different Pluronic-type nonionic surfactants were used to control phase separation of the hydrophobic PMSQ network in aqueous media. Due to different packing density of the colloidal PMSQ constituents in the continuous skeletons in the micrometer-scale (termed as macropore skeletons) and their rearrangements through the hydrothermal post-treatment under basic conditions, mesopore characteristics have been successfully controlled independently of the preformed macropore structure. Separation columns for high-performance liquid chromatography (HPLC) have been fabricated using the PMSQ monoliths, and acceptable separation performances in both the reversed-phase and normal-phase modes have been demonstrated due to the presence of both hydrophilic silanol groups and hydrophobic methyl groups.

Stability-indicating High-performance Liquid Chromatography Method for Simultaneous Determination of Aminophylline and Chlorpheniramine Maleate in Pharmaceutical Formulations.

The present work deals with the development and validation of method for simultaneous determination of antihistaminic drugs in pharmaceutical formulations. A precise, specific and accurate reverse phase-high-performance liquid chromatography method for the simultaneous measurement of aminophylline and chlorpheniramine maleate was developed. The separation of drugs was achieved on C-18 (5 μm, 250×4.6 mm) high-performance liquid chromatography column. The runtime for analysis was 10 min. Mobile phase is mixture containing dilute H2SO4:methanol (60:40% v/v) with flow rate adjusted at 1.5 ml/min. The detection of components was performed at a wavelength of 264 nm. Retention times of aminophylline and chlorphinramine maleate were found to be 2.00 and 3.25 min, respectively. Linearity was found in the range of 16-24 μg/ml for chlorpheniramine maleate and 102.4-153.6 μg/ml for aminophylline with a correlation coefficient of 0.9998 and 0.9996, respectively. High peak purity index of 99.99% indicated the complete separation of analytes in the presence of degradation products is justification of method stability. Linearity, accuracy, specificity, precision and robustness studies were performed for method validation.

Fourth Reported Case of Hemoglobin South Florida: A Rare Hemoglobin Variant That Causes Falsely Elevated HbA1c Values

We report a case of a rare hemoglobin (Hb) variant, Hb South Florida (Hb-SF) with only three cases reported worldwide to our knowledge since initial description in 1985. It causes falsely elevated glycosylated hemoglobin (HbA1c) results during standard laboratory testing in the range of poorly controlled diabetes mellitus. A 42-year-old woman with a history of sclerosing bone dysplasia was noted to have an elevated HbA1c (13.8%) during routine blood work using the ion-exchange high performance liquid chromatography (HPLC) method. The patient did not have a prior diagnosis of diabetes mellitus and denied any symptoms consistent with diabetes. Physical exam was unremarkable. Her blood sugars were normal to minimally elevated. Over several months, she was noted to have persistent elevation in HbA1c in the range of 12-14%. She remained asymptomatic. The patient was referred to endocrinology clinic. Hb electrophoresis was performed due to suspicion for a hemoglobinopathy. The patient was noted to be heterozygous for a rare Hb variant called Hb South Florida that causes falsely elevated HbA1c levels. Her corrected HbA1c, when evaluated by affinity column HPLC, was in normoglycemic range. Hb South Florida was first described in 1985 in Tampa Florida and has since been reported in two other cases worldwide. The Hb is not associated with any clinical disorder reported in literature. There are many commercially available tests for measuring HbA1c levels. Additionally, structural variants in Hb interfere with many of the methods. Ion-exchange HPLC separates Hb based on their charge differences. Hb South Florida variant substitutes a neutral amino acid for a charged one, with valine substituted by methionine. This alteration decreases the transit time in the ion exchange column and causes it to co-elute together with HbA1c, giving a falsely elevated value as they get measured as same species. Affinity chromatography is based on affinity of a species for a particular ligand. It recognizes the structure of the N-terminal glycated amino acids of the beta chain of Hb. In the case of Hb South Florida, it was able to separate the variant Hb from glycated species. This is the fourth case of Hb variant South Florida to our knowledge. It is important for clinicians to keep a wide perspective when making an assessment of diabetes solely based on HbA1c values. Patients who do not present with symptoms consistent with diabetes, and have normal blood glucose should prompt a further workup for abnormal Hb variants. This should be done in order to prevent incorrect diagnosis and treatment. J Med Cases. 2015;6(5):229-231 doi: http://dx.doi.org/10.14740/jmc2134w

Study on products and reaction paths for synthesis of 3,4-dihydro-2H-3-phenyl-1,3-benzoxazine from phenol, aniline and formaldehyde

To study the synthesis of 3,4-dihydro-2H-3-phenyl-1,3-benzoxazine (benzoxazine), the reaction paths of phenol, aniline and formaldehyde were investigated by analyzing the synthesis crude products. With the aid of high-performance liquid chromatography (HPLC), chromatographic column and preparative HPLC, seven compounds originated from the crude products were obtained and their chemical structures were elucidated. Possible reaction paths are proposed based on these compounds. Results show that N-hydroxymethyl aniline (HMA) derived from the reaction of formaldehyde and aniline is probably the key intermediate during the reaction. HMA can react with itself or other reactants to form other intermediates, such as 1,3,5-triphenyl-1,3,5-triazinane and 2-((phenylamino)methyl)phenol, and further form benzoxazine and byproducts.

Divergence from Hardy Weinberg Equilibrium in Newborn Screening Cohorts for Sickle Hemoglobin

Newborn screening (NBS) for hemoglobinopathies was first introduced in the 1970’s to prevent infectious complications of homozygous sickle cell disease (hemoglobin [Hb] SS). The frequency of HbSS in newborns can be estimated using carrier rates (heterogygotes), assuming Hardy Weinberg Equilibrium (HWE). The current study examined whether NBS cohorts matched expected HWE distribution, and possible reasons for divergence. For the purpose of this study, all variants other than HbS were considered to be HbA. A total of 82 studies (1956 to 2013) were retrieved, of which 7 were excluded (carrier parents selected or screening sickle test used). Distribution of Hb SS, AS and AA was examined in 75 studies, from the following regions: Sub Saharan Africa n=26, Brazil n=12, USA n=12, EU n=10; Middle East/North Africa n=7, Caribbean n=6, India n=1, Canada n=1. The following screening laboratory methods were used: cellulose acetate electrophoresis (n=22), isoelectric focusing (n=21), high performance liquid chromatography (n=16), filter paper or micro column (n=7), other (n=9). A total of16,377,450 newborns or infants were included. In mixed random models, relative risk (RR) of detecting HbSS compared to expected HWE distribution was 2.06 (95% confidence interval [CI] 1.86, 3.66), p<0.001. In cohorts where HbAS carrier rates was >= 15%, RR was 1.44 (95% CI 1.23, 1.70), p=0.005; if carrier rates were between >=5% and <15%, RR was 1.62 (95% CI 1.20, 2.19), p=0.004; if carrier rates were <5%, RR was 5.74 (95% CI 3.4, 9.78), p<0.001. Thirteen studies provided data by subgroup: place of origin, ethnicity or race. Affected individuals were more likely to cluster in studies with lower HbS gene frequency (figure 1). Conversely, HbSS RR observed compared to expected HWE distribution was highest in populations with lowest overall gene frequency (figure 2). Overall RR for detecting HbAS compared to expected was 0.96 (95% CI 95, 97) p<0.001. Controlling for gene frequency, the Caribbean region was least likely to deviate from the expected HWE distribution, compared to other regions (RR 1.05, 95% CI 1.01, 1.05, P=0.007); cellulose acetate electrophoresis based methods were most likely to deviate from expected HWE distribution, compared to other methods (RR 0.97, 95%CI 0.95, 0.100, p=0.038). Deviation from HWE is commonly observed in NBS cohorts. It is an expected finding when smaller populations (e.g.migration) carrying a gene, exist within a larger population less affected with the same gene. However, even when controlling for gene frequency, regional variations were noted. Reasons may include further variations in population homogeneity, non-random mating within smaller groups (consanguinity), variations in hemoglobin variant distribution (alpha, beta thalassemia, fetal hemoglobin expression). Lab methods may also affect agreement with HWE distribution, as correct classification of non-affected individuals may vary between methods (normal homozygotes, variants and carriers). Divergence from HWE, especially when observed HbSS occurrence is less than expected, or when population Hb S gene distribution is known, may serve as a rapid and inexpensive quality control measure. Further examination of possible reasons for HWE deviation and caution when estimating burden of disease using carrier rates derived from NBS cohorts appear warranted. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Protocols for finding the most orthogonal dimensions for two-dimensional high performance liquid chromatography

The selection of two high performance liquid chromatography (HPLC) columns with vastly different retention mechanisms is vital for performing effective two-dimensional (2D-) HPLC. This paper reports on a systematic method to select a pair of HPLC columns that provide the most different separations for a given sample. This was completed with the aid of a HPLC simulator that predicted retention profiles on the basis of real experimental data, which is difficult when the contents of sample matrices are largely-or completely-unknown. Peaks from the same compounds must first be matched between chromatograms to compare the retention profiles and optimised 2D-HPLC column selection. In this work, two methods of matching peaks between chromatograms were explored and an optimal pair of chromatography columns was selected for 2D-HPLC. First, a series of 17 antioxidants were selected as an analogue for a coffee extract. The predicted orthogonality of the standards was 39%, according to the fractional surface coverage ‘bins’ method, which was close to the actual space utilisation of the standard mixture, 44%. Moreover, the orthogonality for the 2D-HPLC of coffee matched the predicted value of 38%. The second method employed a complex sample matrix of urine to optimise the column selections. Seven peaks were confidently matched between chromatograms by comparing relative peak areas of two detection strategies: UV absorbance and potassium permanganate chemiluminescence. It was found that the optimal combinations had an orthogonality of 35% while the actual value was closer to 30%.