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HPLC 2019

48th International Symposium on High Performance Liquid Phase Separations and Related Techniques

Restek at HPLC 2019
Restek at HPLC 2019

ORAL PRESENTATION

Wednesday, June 19, 10:05–10:20 a.m.
A Hybrid HILIC Column for the Separation of Polar Compounds
Ty Kahler, Xiaoning Lu, Vernon Bartlett, Connor Flannery, and Terry Reid
Restek Corporation
For more information, email Ty Kahler.
Read abstract

Hydrophilic interaction liquid chromatography (HILIC) columns have been gaining more and more interest due to their ability to retain and separate polar compounds, such as amino acids and carbohydrates. Additionally, HILIC separations, operated under high organic conditions, offer excellent compatibility with mass spec detection. Various HILIC phase chemistries such as base silica, polyols, amino, zwitterions, and mixed phases, have been reported. Nevertheless, the HILIC retention and separation of highly polar anions, such as glyphosate and its metabolites, remain difficult. Anion-exchange chromatography has been an important means of separating anionic compounds. However, anion-exchange chromatography is not very compatible with MS detection due to the use of non-volatile salts.

This presentation describes the development of a hybrid HILIC and anion-exchange HPLC column. Under MS-compatible conditions, the hybrid column has been demonstrated not only to be useful for the separation of polar anions (including glyphosate and its metabolites, organic acids, and water-soluble vitamins), but also has been shown to be applicable for neutral and positive analytes, such as carbohydrates and peptides. In addition, the column employs superficially porous particles, which provide high speed and efficiency in separations.

RESTEK TECHNICAL POSTERS

Monday, June 17, 11:55 - 14:00
P67: A Novel Solution for Vitamin K1 and K2 Analysis in Human Plasma by LC-MS/MS
Ty Kahler, Shun-Hsin Liang, Ravali Alagandula, Frances Carroll, Paul Connolly
Restek Corporation
For more information, email Ty Kahler.
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Vitamin K is a group of fat-soluble vitamins divided into vitamin K1 (one compound, phylloquinone) and K2 (a group of compounds, menaquinones). Among the K2 family, MK4 and MK7 are the most nutritionally recognized. While K1 plays an important role in controlling blood clotting, studies have shown that MK4 and MK7 have distinct biological function in the regulation of bone metabolism and vascular calcification. As interest for their biological action in extra-hepatic tissues is increasing, an accurate and simple measurement of vitamin K status remains a critical issue for both clinical research and diagnostics. Vitamin K analysis is quite challenging because it is the most lipophilic and least abundant of the fat-soluble vitamins. In this study, a simple and fast plasma sample preparation procedure was developed using phospholipid removal in combination with a chromatographic analysis using a Raptor Biphenyl column. The method provides a novel solution for vitamin K research and high-throughput clinical diagnosis.

P68: Analysis of Phosphatidylethanol (PEth) in Human Whole Blood by LC-MS/MS
Ty Kahler, Shun-Hsin Liang, Ravali Alagandula, Frances Carroll, Paul Connolly
Restek Corporation
For more information, email Ty Kahler.
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Phosphatidylethanol (PEth) is a group of phospholipids formed through an enzymatic reaction between ethanol and phosphatidylcholine on the cell membrane. Among the multiple homologues of PEth, PEth-16:0/18:1 (palmitic acid/oleic acid) is the predominant molecule extracted from human erythrocytes and can be measured in whole blood as a specific biomarker of alcohol consumption with a detection window of up to 3-4 weeks. Previously, PEth was considered a biomarker for high and sustained alcohol consumption, but with the application of highly sensitive LC-MS/MS techniques, it is now possible to use PEth concentration in blood to differentiate chronic drinking from social drinking or as a marker of absolute abstinence. In this study, a fast chromatographic analysis was developed using a Raptor FluoroPhenyl column. Specific and sensitive measurement of PEth-16:0/18:1 in whole blood was achieved using a combination of a simple protein precipitation sample preparation and a fast 3.5-minute LC cycle time.

Monday, June 17, 15:35 - 17:00
P123: Analysis of Bisphenols and Perfluorinated Compounds by LC-MS/MS Using Raptor 1.8 µm Columns
Paul Connolly, Landon A. Wiest, Shun-Hsin Liang, Joe Konschnik, and Ty Kahler
Restek Corporation
For more information, email Paul Connolly.
Read abstract

There are myriad contaminants that have impact on our food and environment. In this poster we present separations performed by the Restek Raptor 1.8 µm column family on two classes of contaminants: bisphenols and perfluorinated alkyl acids (PFAS). Bisphenol A (BPA), widely used in the production of polycarbonate plastic and epoxy resins, is an endocrine disruptor that leads to harmful health effects. As public awareness increases, many products are advertised as “BPA-free,” but still use BPA analogues that have similar harmful properties. An analytical method using a Raptor Biphenyl 1.8 µm column has been developed that resolves BPA and its most prevalent analogues for monitoring human exposure to bisphenols. Similarly, PFAS are widely used across a broad range of products, such as firefighting foams, coating additives, textiles, and cleaning products. PFAS are resistant to degradation and have become persistent, environmental pollutants in soil, air, groundwater, municipal refuse, and landfill leachates. Potential adverse effects on animal and human health has been reported for long-chain PFAS, in particular, perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS). To phase out the application of long-chain PFAS, a wide range of short-chain PFAS are used as replacements, leading to rising levels of these compounds in environmental and human matrices. A Raptor C18 1.8 µm column is well suited to baseline separating many of the most common PFAS residues.

P163: A Perfect Pairing: HILIC and Superficially Porous Particles in Food Analyses
Paul Connolly, Landon A. Wiest, Dan Li, Joe Konschnik, and Ty Kahler
Restek Corporation
For more information, email Paul Connolly.
Read abstract

In residue analyses, separations with good selectivity accompanied by highly sensitive detection are desired. This can be difficult to achieve with polar compounds analyzed by reversed-phase liquid chromatography (RPLC). Hydrophilic interaction liquid chromatography (HILIC) encompasses a wide range of stationary phases that provide suitable retention and selectivity for many classes of polar molecules. When approaching RPLC, there are limited retention mechanisms to consider. Aliphatic hydrocarbon stationary phases (e.g., C18) provide van der Waals interactions between the phase and the analytes, while phenyl-type stationary phases (e.g., Biphenyl) provide primarily π-π interactions. HILIC phases encompass a wider variety of chemical interactions that are used with a high organic (typically buffered) mobile phase consisting primarily of acetonitrile and water. The intermolecular interactions between analytes and stationary phase can include, but are not limited to, hydrogen bonding, strong/weak cation or anion exchange, zwitterionic phases, and many proprietary phases that contain “polar embedded” groups. The objective of this poster is to deconvolute the phase chemistry of several HILIC stationary phases and set some basic ground rules for method development. We will also offer solutions for some common issues that are encountered with the use of HILIC, as well as show some examples of how to improve your HILIC separations.

P164: Optimizing a 190+ Pesticides Multiresidue Screening Workflow for the Preparation and Analysis of Produce by LC-MS/MS.
Paul Connolly, Landon A. Wiest, Dan Li, Alexandria M. Pavkovich, Joe Konschnik and Ty Kahler
Restek Corporation
For more information, email Paul Connolly.
Read abstract

Pesticides are ubiquitously used to help increase crop yields; however, they can pose health risks for the general public and pollinators. Faster multiresidue screening workflows, which combine easier sample preparation techniques that yield higher recoveries with lower instrument detection limits in fruits and vegetables, are often sought. Accomplishing these goals increases sample throughput, and reduces costs for laboratories and their clients. To demonstrate the feasibility of developing improved methods, organic celery and other representative matrices were spiked with pesticides down to 10 ppb. Samples were extracted using QuEChERS salts (AOAC 2007.01 and original unbuffered) and cleaned up with complementary dSPE containing MgSO4 ¬along with appropriate amounts of C18, PSA, and GCB sorbents for each matrix. Each sample was diluted 10x with water prior to analysis. Separations were performed with a sterically protected superficially porous C18 (Raptor ARC-18) column (100 mm x 2.1 mm, 2.7 µm) analyzed by a UHPLC-MS/MS in selected reaction monitoring mode. Optimized LC-MS/MS conditions, pesticide separations, and recovery (accuracy and precision) results from organic celery, spinach, orange, avocado, brown rice flour, and honey will be presented.

P165: Quantitation of Mycotoxins in Four Food Matrices Comparing Stable Isotope Dilution Assay (SIDA) with Matrix-Matched Calibration Methods by LC-MS/MS
Ty Kahler, Dan Li, Justin Steimling, Joseph Konschnik, Paul Connolly
Restek Corporation
For more information, email Ty Kahler.
Read abstract

Mycotoxins are secondary fungal metabolites produced by mold that may be found in food or feed. They can cause severe health problems in humans and animals, and can result in significant economic losses. Among the hundreds of toxic mycotoxins, aflatoxins, fumonisins, deoxynivalenol, ochratoxin A, HT-2 toxin, zearalenone, and T-2 toxin are considered to be a major concern for corn, wheat, peanuts, and other agricultural products. LC-MS has become the standard and is now widely used for routine mycotoxin analysis or identification. One of the challenges faced by LC-MS techniques is the matrix effect caused by the use of electro-spray ionization (ESI). Generally, sample preparation, chromatographic conditions, and calibration techniques are the common strategies for reducing the negative impact of matrix effects. Standard addition, matrix matching, and SIDA are all possible calibration solutions.

In this work, a quick “dilute-filter-shoot” method was used for sample preparation. A 7-minute LC-MS/MS method using a Raptor Biphenyl column was developed and verified for quantifying 12 mycotoxins in four matrices (corn, peanut butter, brown rice, and corn/wheat mix). Both SIDA and matrix-matched calibration methods were applied, compared, and evaluated in terms of recovery, efficiency, advantages, and limitations.

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