Rapid Method Development in Hydrophilic Interaction Liquid Chromatography for Pharmaceutical Analysis Using a Combination of Quantitative Structure-Retention Relationships and Design of Experiments.

A design-of-experiment (DoE) model was developed, able to describe the retention times of a mixture of pharmaceutical compounds in hydrophilic interaction liquid chromatography (HILIC) under all possible combinations of acetonitrile content, salt concentration, and mobile-phase pH with R2 > 0.95. Further, a quantitative structure-retention relationship (QSRR) model was developed to predict retention times for new analytes, based only on their chemical structures, with a root-mean-square error of prediction (RMSEP) as low as 0.81%. A compound classification based on the concept of similarity was applied prior to QSRR modeling. Finally, we utilized a combined QSRR-DoE approach to propose an optimal design space in a quality-by-design (QbD) workflow to facilitate the HILIC method development. The mathematical QSRR-DoE model was shown to be highly predictive when applied to an independent test set of unseen compounds in unseen conditions with a RMSEP value of 5.83%. The QSRR-DoE computed retention time of pharmaceutical test analytes and subsequently calculated separation selectivity was used to optimize the chromatographic conditions for efficient separation of targets. A Monte Carlo simulation was performed to evaluate the risk of uncertainty in the model's prediction, and to define the design space where the desired quality criterion was met. Experimental realization of peak selectivity between targets under the selected optimal working conditions confirmed the theoretical predictions. These results demonstrate how discovery of optimal conditions for the separation of new analytes can be accelerated by the use of appropriate theoretical tools.

Evaluation of desialylation during 2-amino benzamide labeling of asparagine-linked oligosaccharides.

Labeling of released asparagine-linked (N-linked) oligosaccharides from glycoproteins is commonly performed to aid in the separation and detection of the oligosaccharide. Of the many available oligosaccharide labels, 2-amino benzamide (2-AB) is a popular choice for providing a fluorescent product. The derivatization conditions can potentially lead to oligosaccharide desialylation. This work evaluated the extent of sialic acid loss during 2-AB labeling of N-linked oligosaccharides released from bovine fetuin, polyclonal human serum immunoglobulin G (IgG), and human α1-acid glycoprotein (AGP) as well as of sialylated oligosaccharide reference standards and found that for more highly sialylated oligosaccharides the loss is greater than the <2% value commonly cited. Manufacturers of glycoprotein biotherapeutics need to produce products with a consistent state of sialylation and, therefore, require an accurate assessment of glycoprotein sialylation.

Disaccharide analysis of glycosaminoglycans using hydrophilic interaction chromatography and mass spectrometry.

Heparan sulfate (HS) and chondroitin sulfate/dermatan sulfate (CS/DS) glycosaminoglycans (GAGs) participate in many important biological processes. Quantitative disaccharide analysis of HS and CS/DS is essential for the characterization of GAGs and enables modeling of the GAG domain structure. Methods involving enzymatic digestion and chemical depolymerization have been developed to determine the type and location of sulfation/acetylation modifications as well as uronic acid epimerization. Enzymatic digestion generates disaccharides with Δ-4,5-unsaturation at the nonreducing end. Chemical depolymerization with nitrous acid retains the uronic acid epimerization. This work shows the use of hydrophilic interaction liquid chromatography mass spectrometry (HILIC-MS) for quantification of both enzyme-derived and nitrous acid depolymerization products for structural analysis of HS and CS/DS. This method enables biomedical researchers to determine complete disaccharide profiles on GAG samples using a single LC-MS platform.

Reversible interference of Fe³(+) with monoclonal antibody analysis in cation exchange columns.

The effect of Fe³(+) treatment on weak cation exchange column chromatography was demonstrated for monoclonal antibody (MAb) analysis. Fe³(+)-exposed columns showed lowered relative peak areas of the parent MAb peak as well as both acidic and basic variant peaks that could lead to erroneous conclusions. Accurate measurement of relative amounts of variants to the parent MAb is essential for demonstrating the safety and efficacy of therapeutic molecules such as MAbs. Complete reversal of the compromised MAb analysis performance was observed after washing the column with chelating agents, confirming that metal contamination was responsible for the compromised column performance.

Reversed-phase monoliths prepared by UV polymerization of divinylbenzene.

Many different techniques have been developed to prepare monolithic materials specifically for chromatographic techniques. The two most popular polymerization techniques being thermal or via ultra violet (UV) light. Whereas thermal polymerization is easily employed for a whole variety of monomer and porogen systems, UV polymerization has been limited to methacrylate-based systems, and styrenic systems have been avoided due to their strong absorbance at low wavelengths. By careful consideration of wavelength, initiator and other system components, it was proven that reversed-phase columns for the separation of proteins and peptides can be prepared using divinylbenzene through UV initiation of 2-methyl-4'-(methylthio)-2-morpholinopropiophenone at a wavelength of 350 nm.

Towards a more complete glycome: Advances in ion chromatography-mass spectrometry (IC-MS) for improved separation and analysis of carbohydrates.

To date, few tools are available for the analysis of the glycome without derivatization, a process which is known to introduce issues such as differential loss of sialic acid and incomplete labeling. We have previously reported the use of ion chromatography-mass spectrometry (IC-MS) to analyze native sialylated and sulfated glycans. Here, we introduce improvements to IC column technology, enabling the separation of neutral glycans while maintaining charge separation capabilities. When implemented in an IC-MS workflow, this enables the structural characterization of a broad array of chemically distinct glycans. With the newly developed IC column and modified IC-MS instrumentation configuration, we qualitatively investigated O-glycome profiles in bovine fetuin and porcine gastric mucins. The improved chromatographic resolution in combination with high-resolution MS data present a powerful tool for glycan structural identification.

Fast determination of nine haloacetic acids, bromate and dalapon in drinking water samples using ion chromatography-electrospray tandem mass spectrometry.

Ion chromatography-electrospray tandem mass spectrometry (IC-ESI-MS/MS) is used to determine nine haloacetic acids (HAAs), bromate, and dalapon in drinking water samples in U.S. EPA Method 557. In this method, all target analytes are separated and measured with good sensitivity without the need for sample preconcentration or derivatization. However, the separation time is relatively long. In order to reduce the sample analysis time in EPA Method 557, a new anion exchange column has been developed to perform fast separation of the target analytes. Using this new anion exchange column, nine HAAs, bromate, and dalapon can be resolved and separated from interfering matrix ions within 40 minutes, about 33% faster than the analysis time obtained using an earlier anion exchange column reported in EPA Method 557. The new anion exchange column has unique selectivity and high exchange capacity. Method optimization, simplification and improvements in robustness are demonstrated while validating the new column suitability for the determine of HAAs, bromate and dalapon according to EPA Method 557.

Determination of trace concentrations of bromate in municipal and bottled drinking waters using a hydroxide-selective column with ion chromatography.

The International Agency for Research on Cancer determined that bromate is a potential human carcinogen, even at low micro/l levels in drinking water. Bromate is commonly produced from the ozonation of source water containing naturally occurring bromide. Traditionally, trace concentrations of bromate and other oxyhalides in environmental waters have been determined by anion exchange chromatography with an IonPac AS9-HC column using a carbonate eluent and suppressed conductivity detection, as described in EPA Method 300.1 B. However, a hydroxide eluent has lower suppressed background conductivity and lower noise compared to a carbonate eluent and this can reduce the detection limit and practical quantitation limit for bromate. In this paper, we examine the effect of using an electrolytically generated hydroxide eluent combined with a novel hydroxide-selective anion exchange column for the determination of disinfection byproduct anions and bromide in municipal and bottled drinking water samples. EPA Methods 300.1 B and 317.0 were used as test criteria to evaluate the new anion exchange column. The combination of a hydroxide eluent with a high capacity hydroxide-selective column allowed sub-microg/l detection limits for chlorite, bromate, chlorate, and bromide with a practical quantitation limit of 1 microg/l bromate using suppressed conductivity detection and 0.5 microg/l using postcolumn addition of o-dianisidine followed by visible detection. The linearity, method detection limits, robustness, and accuracy of the methods for spiked municipal and bottled water samples will be discussed.

Oligosaccharide analysis by capillary-scale high-pH anion-exchange chromatography with on-line ion-trap mass spectrometry.

A capillary-scale high-pH anion-exchange chromatography (HPAEC) system for the analysis of carbohydrates was developed, in combination with two parallel on-line detection methods of sub-picomolar sensitivity: (1) pulsed amperometric detection (PAD); (2) capillary-scale desalting followed by electrospray ion-trap (IT) mass spectrometry (MS). The capillary chromatographic system combined the superb selectivity of HPAEC that allows routine separation of isomeric oligosaccharides with the information on monosaccharide sequence and linkage positions obtained by MS/MS fragmentation using the IT-MS. The applicability of the system in biomedical research was demonstrated by its use for the analysis of a urine sample of a GM1-gangliosidosis patient. Isomeric glycans in the sample could be resolved by HPAEC and assigned on the basis of the monosaccharide linkage information revealed by on-line IT-MS/MS.

Determination of anions at trace levels in power plant water samples by ion chromatography with electrolytic eluent generation and suppression.

An ion chromatographic method was developed for the determination of nine inorganic and organic acid anions at sub- to low-microg/l levels in power plant water samples. In this method, samples were injected using a large-volume direct injection technique, the analyte anions were separated on a hydroxide-selective anion-exchange column using high-purity hydroxide eluents generated by an on-line electrolytic eluent generator and detected using the suppressed conductivity detection method. The method performance was evaluated by analyzing synthetic water samples containing additives encountered in the power plant water samples and four water samples from a fossil fuel power plant. The relative standard deviations of retention times of analyte ions separated on the hydroxide-selective anion-exchange column were less than 0.4%. The recoveries of analyte ions spiked into the synthetic water samples at concentrations of 0.13-1.0 microg/l were in the range of 70-120%. The method detection limits for analyte ions in deionized water were 0.0099, 0.0056, 0.019, 0.057, 0.0084, 0.023, 0.067, 0.037, and 0.079 microg/l for fluoride, acetate, formate, chloride, nitrite, sulfate, bromide, nitrate, and phosphate, respectively.

Recent developments in electrolytic devices for ion chromatography.

Recent developments in new electrolytic devices that utilize the electrolysis of water and charge-selective electromigration of ions through ion-exchange media have significantly changed the routine operation of ion chromatographic methods. Examples of these new electrolytic devices include on-line eluent generators that produce high-purity electrolyte eluents using deionized water as the carrier stream, continuously regenerated trap columns that remove ionic contaminants in the eluents, and continuously regenerated suppressors that reduce eluent background conductance prior to conductivity detection. The combined use of these electrolytic devices has made it possible to perform various ion chromatographic separations using only deionized water as the mobile phase. This paper reviews the operation principles of these electrolytic devices and their applications in the ion chromatographic determination of anionic and cationic analytes.

Bedside and radiologic procedures in the critically ill obese patient.

Performance of procedures upon the obese critically ill patient in the ICU or in the radiology suite, require certain considerations. Additional staff, equipment and proper ergonomics are often necessary to perform these procedures safely for both patient and staff.

Matrix diversion methods for improved analysis of perchlorate by suppressed ion chromatography and conductivity detection.

Two inline matrix diversion methods were developed for the sensitive analysis of perchlorate in a matrix comprising up to 1000 mg l(-1) of chloride, sulfate and bicarbonate ions using suppressed ion chromatography and conductivity detection. The first method used a cryptand C1 concentrator column, which exhibited a high selectivity for perchlorate ion over the other matrix anions. After retaining the sample anions in a concentrator column derivatized with a crytpand phase, a rinse step was implemented with a weak base to divert the matrix ions to waste while selectively retaining perchlorate in the concentrator column for subsequent analysis. The analysis was done using a 2mm IonPac AS16 or 2 mm IonPac AS20 separator column. The second method was a two-dimensional matrix diversion method with a focus on improving the detection sensitivity. The first dimension was used to achieve some resolution of the matrix ions from perchlorate. The perchlorate ion was then diverted into a concentrator column for subsequent analysis in the second dimension. By pursuing analysis using a 4mm IonPac AS16 or IonPac AS20 column in the first dimension and subsequently pursuing analysis using a 2mm IonPac AS16 or IonPac AS20 column format, excellent sensitivities were achieved when the first and second dimensions were operated at the same linear flow velocity (cm min(-1)). While sensitive detection of perchlorate in the low microg l(-1) regime was achieved by the above methods in the presence of matrix ions, superior recovery for perchlorate was demonstrated under a variety of matrix concentrations by the second method.