Technical Program

TECHNICAL PRESENTATIONS – THURSDAY, MAY 10, 2018

8:40 Introduction / Undergraduate Award / Palmer Award

9:00 Keynote Address: Ravi Ravichandran, PhD

United States Pharmacopeial Convention,

Principal Scientific Liaison

“MODERN SEPARATIONS IN PHARMACEUTICAL QUALITY: A USP PERSPECTIVE”

10:00 Morning Break – EXHIBITS AND POSTERS – CARRIAGE HALL

Time

Captain’s Room

Tack A (lower level)

Tack B (lower level)

Palmer Award Session

Biopharm

Gas Chromatography

10:40 2018 L.S. Palmer Awardee:
Professor Douglas Raynie
South Dakota State Univ.An Evolution of Greener Sample Preparation Technologies 1
11:00
11:20 Extraction of Piperine from Piper Nigrum using Carboxylic Acid-Based Deep Eutectic Solvents (DES)

A. Dwamena 2

Charge Variant Analysis of Protein Biotherapeutics Using CIEF-MS and PK Profiling of Preclinical Catabolites

J. Sausen 4

Wine: When the Vines are Too Cold and Too Warm. Resveratrol Content in Wines from Northern Climate

S. Pandey 6

11:40 Palmer Award Discussion Session:

An Evolution of Greener Sample Preparation Technologies 3

Peptide Purif Utilizing Auto Gradient Optimization & Delay Volume Calibrn for Scale Up from Analyt HPLC to Prep Purif

L. Sandford 5

Turning Up the Heat on Wax GC Columns without Getting Burned

V. Abercrombie 7

12:00 LUNCH (12:00-1:20)

Advanced Stationary Phases

CAPA & Pharma

LC-MS, GC-MS, LC Meth Dvmt

1:20 *** Invited Speaker ***

Advances in Stationary Phases

and Sorbent Materials for Multidimensional Gas Chromatography and Sample Prep

Jared L. Anderson

Iowa State University 8

1:40 Using CAPA to Find, Fix and Monitor Sample Cancellations

K. Rinker 11

Quantitative LC/MS/MS Analysis of THC and Metabolites in Biological Samples

J. Westland 17

2:00 Novel Ligand Functionalized Membranes for Monoclonal Antibody Purification

S. Colak Atan 9

Challenges and Strategies for Leachables and Extractables Identification in Medical Devices

S. Zhang, J. Li 12

Analysis of Phenolic Monomers from the Alkali Lignin using Gas Chromatography Mass-Spectrometry (GC-MS) B. Jadhav 14
2:20 Ionic Liquid-Based Stationary Phases for Gas Chromatographic Separations

He Nan 10

Comparing Silica-Hybrid Based Column to HILIC Column in Separating a Mixture of Analytes

M. Thao, J. Li 13

Dvlpmt of an HPLC Method for the Analysis of a Next-Generation Cyanide Antidote, in Plasma

E. Alzhrani 15

2:45 2017 MCF Undergraduate Research Awardee:

Faith Murphy University of Minnesota – Duluth

Sorption Model: Micropollutant to Polyethylene Terephthalate Film after UV Exposure 16

3:05

AFTERNOON BREAK (3:05)

3:30

Prize Drawing (3:30)

See you at the 2019 MCF Spring Symposium

KEYNOTE PRESENTATION

Abstract

Modern Separations in Pharmaceutical Quality: A USP Perspective

Ravi Ravichandran, Ph.D.

United States Pharmacopeial Convention:
Principal Scientific Liaison

Rockville, Maryland

The US Pharmacopeia is one of the major pharmacopeias, and the oldest in the world. This presentation will focus on the role of USP in pharmcopeial standards and how they are used in the environment regulated by regulatory agencies such as FDA in the US. It will also provide a high-level overview of the standard setting process.

Separations such as HPLC, GC and hyphenated techniques occupy a prominent role in the USP. This talk will focus on the history and role of separation science in pharmacopeial standards, and also address some of the current initiatives underway at USP.

Abstract #1

PRESENTATION BY PALMER AWARDEE

An Evolution of Greener Sample Preparation Technologies

Douglas E. Raynie

Department of Chemistry and Biochemistry,

South Dakota State University

Brookings, South Dakota

Extractions for chromatographic sample preparation represent the largest use of solvent in an analytical procedure. Along with the associated acquisition and disposal costs, most commonly used solvents present concerns associated with health, safety, and energy. In this presentation, an evolution of sample preparation from a green chemistry perspective will be presented. During this talk, the development of a metric to evaluate the green attributes of an analytical method will be presented. Waste generation, safety, toxicity, and reactivity will be considered. Then we will turn to a specific look at extractions used for analytical sample preparation. Newer extraction technologies, starting with solid-phase extraction (SPE) in the 1970s through supercritical fluid extraction (SFE), microwave-assisted extraction (MAE), ultrasound-assisted extraction (UAE), and pressurized solvent extraction (PSE), were developed not necessarily for their green advantages, but for improvements in analytical performance. Each of these techniques has certain advantages in terms of speed, selectivity, or extraction yield, and often possesses an associated cost benefit. However, the reduced solvent use with each of these provides a green advantage. These methods are compared using the hot-ball diffusion model, which shows that, from an efficiency viewpoint, modern extraction methods are equivalent. Finally, we will take a look at solvent alternatives to traditional molecular solvents. While a number of these neoteric solvents exist, we will particularly explore the development of a unique class, deep eutectic solvents, and their properties relevant to developing extraction methods.

Abstract #2

Extraction of Piperine from Piper Nigrum using Carboxylic Acid-Based Deep Eutectic Solvents (DES)

Amos K. Dwamena and DOUGLAS RAYNIE

South Dakota State University

Brookings, South Dakota

Piperine is the major naturally occurring alkaloid in black pepper responsible for pungency and is known for its numerous health and therapeutic effects. The development of greener, cheaper, and efficient solvent system for piperine extraction is necessary for industrial purposes and sustainability. To date, nearly all DES formed for analytical extraction have been hydrophilic. A new carboxylic acid-based DES was prepared using choline chloride with butyric acid ([Ch]Cl:BA), valeric acid ([Ch]Cl:VA), or caprylic acid ([Ch]Cl:CA) as hydrogen bond donors (1:2 molar ratio). The alkyl chain length (C4-C8) of the hydrogen bond donor helped to tailor the polarity of the synthesized DES. From our preliminary data, extracted amounts of piperine with [Ch]Cl:2BA, [Ch]Cl:2VA, and [Ch]Cl:2CA DES yielded 24 mg/g, 22 mg/g, and 14 mg/g, respectively. The mg/g of piperine in peppercorns was found to be higher compared to ground black pepper. DES yielded about 70% of piperine compared to that reported in the literature, calling for further reaction optimization steps. Increasing the degree of hydrophobicity of the DES as HBD chain length reduced the yield of piperine. We are currently comparing the effects of time, temperature, and solvent amount on this extraction.

Abstract #3

PALMER AWARD
DISCUSSION SESSION:

An Evolution of Greener Sample Preparation Technologies

Join the speakers of the Palmer Award Session for an extended discussion on the topic of Greener Sample Preparation Technologies.

Abstract #4

Charge Variant Analysis of Protein Biotherapeutics Using CIEF-MS and PK Profiling of Preclinical Catabolites

John Sausen

Agilent Technologies

Charge variant analysis is a commonly used technique in protein characterization to show the presence of acidic and basic charge variants. This technique historically uses UV detection and lacks specificity in identification. Recently a new online capillary isoelectric focusing-mass spectrometry (cIEF-MS) method was developed providing deconvoluted HRAM for each Charge Variant.1

In Preclinical research, much of the initial PK work done with protein therapeutics utilizes a combination of peptide quantitation with MRM, QQQ and ELISA. A new approach, using a micro-sheath CEMS interface and source design, enables improved selectivity and higher throughput, when profiling both the therapeutic protein, and its associated catabolites (truncations) – measured at either the intact or reduced state over a time course.2

  1. J Dai, J Lamp, Q Xia, Y Zhang – Analytical chemistry, 2017 – ACS Publications. (https://pubs.acs.org/doi/abs/10.1021/acs.analchem.7b04608)
  2. M Han, JT Pearson, Y Wang, D Winters, M Soto, et.al. – Analytical Biochemistry, 2017 – Elsevier (https://www.sciencedirect.com/science/article/pii/S0003269717303901?via%3Dihub)

Abstract #5

Peptide Purification Utilizing Automated Gradient Optimization and Delay Volume Calibration for Scale Up From Analytical HPLC to Preparative Purification with Open Bed Fraction Collection

Lori Sandford

Agilent Technologies, Inc.

Preparative HPLC by reversed phase chromatography is the most widely used method for purification of synthetic and natural peptides. Purification of synthetic biotherapeutic peptides is necessary to remove truncated peptide sequences and other side products resulting from peptide synthesis. Scaling from analytical separations to purification of higher quantities of peptide requires optimization of the mobile phase gradient and compensation for system delay volumes up to the fraction vessel. Automated gradient focusing and delay volume calibration improve throughput of the purification method.

This presentation is a comparison of manual method transfer vs automated analytical to preparative scale-up allowing chromatographers to collect high purity peptides in an open bed, maximum capacity fraction collector. Peptide separations are performed using a robust polymeric reverse phase column chemistry that allows for efficient scale up and compatibility with a broad range of mobile phase conditions. This workflow allows the scientist to easily transfer analytical methods to preparative scale with automated focusing of the solvent gradient to maximize loading using the preparative purification system

Abstract #6

Wine: When the Vines Are Too Cold and Too Warm. Resveratrol Content in Wines from Northern Climate

Sonali R. Pandey and DOUGLAS E. RAYNIE

South Dakota State University

Brookings, South Dakota

Stilbene rich wine is an interesting product due to the added value as a result of the numerous health promoting effects associated with it. Resveratrol is a member of the stilbene family. It is synthesized by plants in response to environmental stress like mechanical injury, bacterial or fungal infections and UV exposure. In the last 2 decades, resveratrol has become an important parameter of wines because of the associated beneficial effects on human health. Evidence from literature demonstrates resveratrol to possess anticancer, cardio-protective, antioxidant and anti-inflammatory activity. Climate conditions affect activities linked to wine production (grape growing, wine making, wine economics, and environmental issues), which eventually alters the resveratrol concentration in red wines. Therefore, its determination and quantification are of high importance. Although several studies have reported resveratrol concentration in wines, none has yet compared the resveratrol level in red wines from grapes grown in different environments. Grapes grown in northern climate like Dakota and Minnesota must contend with extremes of heat in the summer and cold in the winter. The goal of this study is to estimate resveratrol concentration in wines from grapes grown in South Dakota using gas chromatography. Moreover, results from this study will provide knowledge for producing wines that possess functional properties that are gaining increased attention in the eyes of consumers.

Abstract #7

Turning Up the Heat on Wax GC Columns without Getting Burned

Vanessa Abercrombie and DARON DECKER

Agilent Technologies

100 % polyethylene glycol (PEG) columns, also known as WAX columns, are used for a wide variety of applications, such as industrial chemicals, flavors, and fragrances. In comparison to polysiloxane stationary phases, the maximum operating temperature of a WAX GC column is much lower. Traditional WAX columns have a maximum temperature limit of 250 °C isothermal and 260 °C programmed, due to issues with decreased thermal stability, which reduces the potential range of applications. The Agilent J&W DB-HeavyWAX has an extended temperature limit, up to 280 °C isothermal and 290 °C programmed, and increased thermal stability This increases the injection-to-injection retention time reproducibility, as well as column lifetime. The increased upper temperature limit allows for faster analysis while minimizing the possibility of carryover sample to sample.

*** INVITED SPEAKER ***

Abstract #8

Advances in Stationary Phase and Sorbent Materials for Multidimensional Gas Chromatography and Sample Preparation

Jared L. Anderson 1*, KEVIN D. CLARK 1, HE NAN 1 and

MARCELINO VARONA 1

1Department of Chemistry, Iowa State University,

Ames, Iowa 50011

* andersoj@iastate.edu

Ionic liquids (ILs) can be designed to exhibit unique properties for their use in a number of applications in analytical and bioanalytical chemistry. This talk will focus on the design and synthesis of ILs and magnetic ionic liquids (MILs) as well as the use of these materials in a number of applications within multidimensional gas chromatography and sample preparation. A series of monocationic and dicationic ionic liquid (IL)-based stationary phases were evaluated as secondary columns in comprehensive two-dimensional gas chromatography (GC×GC) for the separation of aliphatic hydrocarbons from kerosene. In order to further understand the role that structural features of ILs play on the selectivity of nonpolar analytes, a series of dicationic IL-based stationary phases were evaluated using GC×GC. Finally, nucleic acids are biopolymers that constitute important diagnostic molecules for a broad range of applications from clinical testing to forensic analysis. A major challenge faced by DNA and RNA analysis techniques is the selective extraction of particular nucleic acid sequences using rapid and sensitive methodologies. We will show that ion-tagged oligonucleotides (ITOs) can be used in conjunction with MILs to efficiently capture DNA sequences from complex samples. The ITOs can be created through thio-lene “click” chemistry and the nature of the ion tag can influence the partitioning of the ITO to the hydrophobic MIL. This novel liquid-phase approach towards sequence-selective DNA capture provides superior extraction efficiencies to conventional magnetic bead technology as well as a platform for using external fields to manipulate the liquid droplets.

Abstract #9

Novel Ligand Functionalized Membranes for Monoclonal Antibody Purification

Semra Colak Atan*, JERALD K. RASMUSSEN, ANDREW VAIL,

CATHY A. BOTHOF and GEORGE W. GRIESGRABER

3M Company, Corporate Research Materials Laboratory

3M Center, St.Paul, MN, USA

*scolak@mmm.com

One of the most challenging areas of biopharmaceutical drug production is the filtration/purification process of therapeutic monoclonal antibodies. Removal of contaminants such as aggregates, host cell proteins, nucleic acids and viruses is an essential part of this process. To be able to overcome this filtration problem: fine-tuning of the ligand chemistry on the filtration media is essential. Currently there are no commercial products on the market that specifically target monoclonal antibody aggregate removal. The main objective of this work is to develop a platform of novel mixed-mode functional monomers and investigate their applicability as ligands for functionalized membranes for biopharmaceutical purification. This presentation will summarize our efforts in synthesis of novel multifunctional monomers as ligands, grafting these ligands to various substrates to generate filtration media, and testing their effectiveness in monoclonal antibody purification.

Abstract #10

Ionic Liquid-Based Stationary Phases for Gas Chromatographic Separations

He Nan and JARED L. ANDERSON

Department of Chemistry, Iowa State University,

Ames, Iowa 50011

Ionic liquids (ILs) are a class of molten salts that fulfill many of the requirements of GC stationary phases including high thermal stability, high viscosity, negligible vapor pressure, and tunable selectivity. Compared to polydimethyl(siloxane) and poly(ethyleneglycol) derived GC stationary phases, IL-based columns often provide unique selectivity for the separation of various types of analytes. Lipidic ILs possessing long alkyl chains as well as low melting points have the potential to provide unique selectivity as well as wide operating ranges. A total of eleven lipidic ILs containing various structural features (i.e., double bonds, linear thioether chains, and cyclopropanyl groups) were examined as stationary phases in comprehensive two-dimensional gas chromatography (GC × GC) for the separation of nonpolar analytes in kerosene. Compared to a homologous series of ILs containing saturated side chains, lipidic ILs exhibit improved selectivity toward the aliphatic hydrocarbons in kerosene. The palmitoleyl IL provided the highest selectivity compared to all other lipidic ILs as well as the commercial SUPELCOWAX 10 column. The linoleyl IL containing two double bonds within the alkyl side chain showed the lowest chromatographic selectivity. This study provides the first comprehensive examination into the relation between lipidic IL structure and the resulting solvation characteristics. Silver ion or argentation chromatography utilizes stationary phases containing silver ions for the separation of unsaturated compounds. A mixed-ligand silver-based IL was evaluated for the first time as a GC stationary phase for the separation of light olefin/paraffin mixtures. The selectivity of the stationary phase toward olefins can be tuned by adjusting the ratio of silver ion and the mixed ligands. The maximum allowable operating temperature of these stationary phases was determined to be between 125 ºC and 150 ºC. This study establishes a basis for the use of highly selective silver ILs as GC stationary phases for the separation and identification of various classes of olefins and their isomers.

Abstract #11

Using CAPA to Find, Fix and Monitor Sample Cancellations

Katie Rinker

Minnesota Department of Health

St. Paul, MN

A key component of any robust quality management system is the ability to respond to issues that may arise that compromise meeting data quality objects and to be able to monitor trends to help prevent issues form occurring at a future time. Corrective and Preventive Action (CAPA) is a process used to identify and fix nonconformance that has occurred. The CAPA process should find the root cause of the problem, fix the problem and prevent reoccurrence. The Minnesota Department of Health’s Environmental Laboratory received a client concern that an excessive number samples were being cancelled for a specific analysis. The laboratory used an “Is – Is Not” analysis to find the root cause of increased sample cancellations during 2016. This root cause analysis found several reasons for the sample cancellations. Preventive actions were put in place for the 2017 peak sampling timeframe and the cancelled sample amount went from 11% in 2016 to < 1% in 2017 for the analysis of concern.

Abstract #12

Challenges and Strategies for Leachables and Extractables Identification in Medical Devices

Siyi Zhang and Jianwei Li

Medtronic

Abstract #13

Comparing Silica-Hybrid Based Column to HILIC Column in Separating a Mixture of Analytes

Mai Thao and Jianwei Li

Medtronic

Abstract #14

Analysis of Phenolic Monomers from The Alkali Lignin Using Gas Chromatography Mass-Spectrometry (GC-MS)

Balawanthrao Jadhav, RANEN ROY and DOUGLAS E. RAYNIE

South Dakota State University

Brookings, South Dakota

Lignin is a complex organic biopolymer and the second most abundant biomass type on earth. Lignin is one of the components of lignocellulosic biomass. Lignin comprises 10-20% of lignocellulosic biomass. Lignin is one of the renewable source of producing fuel and aromatic phenolic monomers. It is a copolymer of p-coumaryl (H), coniferyl (G) and sinapyl (S) alcohols. Lignin monomers are used in synthesis of polymers and resins. In this study, we developed a green approach for the depolymerization of alkali lignin in the presence of subcritical water and catalyst. We studied the reactions at 240C for 10 minutes. Reaction mixture was collected in to the glass vial after 10 minutes. Ethyl acetate was used for the extraction of phenolic monomers from the reaction mixture. We separate the organic layer for the analysis of phenolic monomers from the alkali lignin using GC-MS. Identification and quantification of phenolic monomers done using gas chromatography-mass spectrometry (GC-MS). We identified 12 different phenolic monomers and quantified six of these from deoxyhydrogenation of alkali lignin. The main phenolic monomers are guaiacol, vanillin, isoeugenol, acetovanillone, guaiacylacetone, and homovanillic acid were quantified using calibration curves and GC-MS. We found the yield of guiacol is 4.71±0.37 mg/g, vanillin is 20.47±2.3 mg/g, isoeugenol is 4.75±0.08 mg/g, acetovanilone is 3.95±0.16 mg/g, guaiacylacetone is 5.96±0.15 mg/g and homovanillic acid is 23.29±1.6 mg/g from the alkali lignin.

Abstract #15

Development of an HPLC Method for the Analysis of a Next-Generatioin Cyanide Antidote 3-Mercaptopyruvate, in Plasma

Elaf Alzhrani and BRIAN LOGUE

South Dakota State University

Brookings, South Dakota

Cyanide is a highly toxic chemical that causes severe health risks which may eventually result in death. Although current FDA approved cyanide therapies are effective, next generation cyanide antidotes are being investigated to overcome limitations associated with these therapies. 3-Mercaptopyruvate (3-MP) is a next generation sulfur donor therapeutic that has shown promise as cyanide antidote. Analytical methods capable of detecting 3-MP in biological fluids are necessary for the development of 3-MP as a promising antidote. Although 3-MP has been analyzed by LC-MS-MS, this instrument is not widely available. Therefore, a high performance liquid chromatography (HPLC) method with fluorescence detection was developed to analyze 3-MP from swine plasma such that more labs could potentially perform the method. Sample preparation included spiking the plasma with an internal standard (3-mercaptopropionic acid), reaction with monobromobimane to prevent the characteristic dimerization of 3-MP, and mixed mode anion exchange solid phase extraction. The method produced a limit of detection of 0.5 nM and a dynamic range of 0.1-10 µM. The accuracy and precision for the method were good. The validated HPLC-FLD method was capable of detecting 3-MP in swine plasma and can be utilized for further investigations of 3-MP as promising cyanide antidote.

Abstract #16

Sorption of Model: Micropollutant to Polyethylene Terephthalate Film After UV Exposure

Faith Murphy and DR. MELISSA MAURER-JONES

Department of Chemistry and Biochemistry

University of Minnesota-Duluth, Duluth MN 55812

Plastics have proven to be useful in a myriad of applications due to properties like chemical resistance; however, this also makes them a problematic persistent environmental contaminant. In the environment, plastics are known degrade from environmental factors including UV light irradiation. Previous studies have shown that plastics have an ability to sorb organic compounds, yielding a potential for plastics to act as vectors for contaminant transport through the environment to the possibility of bioaccumulation. This work aims to quantify sorption of ibuprofen, a model pollutant, onto photodegraded polymers. The main polymer studied for this work is polyethylene terephthalate, PET, since it is abundantly found in marine environments. Photo-transformations of PET films were characterized by ATR-FTIR after irradiation with 254 nm UV light to understand molecular changes. Ibuprofen sorption to non-photolyzed and photolyzed PE films was evaluated with liquid chromatography (UPLC) and the monitored samples will be fit to a Langmuir isotherm to quantify pollutant partitioning. Through this work, we aim to better the understanding of the interaction between plastic debris and organic pollutants found in water.

Abstract #17

Quantitative LC/MS/MS Analysis of THC and Metabolites in Biological Samples Following Removal of Phospholipids by a Lipid-Specific Sample Preparation Cartridge

Jessica Westland

Agilent Technologies

Phospholipids (PPLs) have been identified as a major cause of ion suppression in LC/MS/MS analysis of whole blood and plasma samples which can result in inaccurate analysis. Efficient extraction, cleanup, and analysis of complex biological samples are extremely beneficial to multiple industries including forensic laboratories.  The forensic analysis for Δ9-THC (THC) and two major metabolites THC-OH and THC-COOH, from plasma or whole blood is a routine assay in which PPLs have been identified as a major cause of matrix effects in their LC-MS/MS analysis.  Prior to LC/MS/MS analysis, an in-well protein precipitation (PPT) followed by PPL removal using Agilent Captiva EMR—Lipid in a pass-through 1 mL cartridge was utilized to extract THC, THC-OH and THC COOH from whole blood and plasma.  The Captiva EMR—Lipid produced clean eluents with removal of over 97 % of the unwanted PPLs from whole blood and plasma, and over 90 % recoveries for target analytes, with RSDs <10 %.  Analysis of THC, THC-OH, and THC-COOH at 1 ng/mL yielded ideal peak shapes with good signal to noise (S/N). Response from 0.5 to 100 ng/mL was linear, with an R2 >0.99. Limits of quantitation of 1.0 ng/g or lower were obtained, with RSD <11.5 %. Results were consistent over three days of experiments.

Poster Abstract # 1

Electrochemical Reduction of Aromatic Nitro Compounds: Strategies for LC-EC Analysis of Sanger Tagged Analytes

Miththira Balasingam, PAULINA GUERRERO, DONG WANG, JOHN BECKER, DR. HAORAN SUN, and DR. MILES KOPPANG

South Dakota State University

Brookings, South Dakota

Analysis of amino acids is crucial to protein structure elucidation. Amino acids are non-volatile and are amenable to separation by liquid chromatography (LC). However, amino acids are not detectable with sensitive detectors typically used in liquid chromatography including absorbance, fluorescence or electrochemical detectors. Derivatization of amino acids using suitable reagents enhances the separation and detection of amino acids using liquid chromatography. Sanger used 2, 4-dinitrofluorobenzene (DNFB) to derivatize amino acids for the structure determination of insulin for which he received the Nobel Prize in 1958. The DNFB makes amino acids suitable for absorbance detection. However, little has been done in terms of liquid chromatography with electrochemical detection (LC-EC) of the DNFB derivatized amino acids. Reduction of aromatic nitro groups has been known for many years but there are disagreements as to the reduction pathway and products. Since aromatic nitro compounds are electrochemically active, electrochemical investigation of nitrobenzene (NB) is essential to determine the reduction pathway. Cyclic voltammetry (CV), rotating disk electrode (RDE) and rotating ring disk electrode (RRDE) experiments have been done on the NB to determine that NB is reduced into phenylhydroxylamine (PHA) by addition of four electrons and four protons and the reduced PHA can be reversibly oxidized into nitrosobenzene removal of two electrons and two protons transfer. Electrochemical detection of the nitro groups will be appropriate for dual electrode detection of DNFB labeled amino acids on which we can observe the reduction on an upstream electrode and corresponding oxidation of the reduction product on the downstream electrode.

Poster Abstract # 2

Analysis of Phenolic Monomers From the Alkali Lignin Using Gas Chromatography Mass-Spectrometry (GC-MS)

Balawanthrao Jadhav, RANEN ROY and DOUGLAS E RAYNIE

South Dakota State University

Brookings, South Dakota

Lignin is a complex organic biopolymer and the second most abundant biomass type on earth. Lignin is one of the components of lignocellulosic biomass. Lignin comprises 10-20% of lignocellulosic biomass. Lignin is one of the renewable source of producing fuel and aromatic phenolic monomers. It is a copolymer of p-coumaryl (H), coniferyl (G) and sinapyl (S) alcohols. Lignin monomers are used in synthesis of polymers and resins. In this study we developed a green approach for the depolymerization of alkali lignin in the presence of subcritical water and catalyst. We studied the reactions at 240C for 10 minutes. Reaction mixture was collected in to the glass vial after 10 minutes. Ethyl acetate was used for the extraction of phenolic monomers from the reaction mixture. We separate the organic layer for the analysis of phenolic monomers from the alkali lignin using GC-MS. Identification and quantification of phenolic monomers done using gas chromatography-mass spectrometry (GC-MS). We identified 12 different phenolic monomers and quantified six of these from deoxyhydrogenation of alkali lignin. The main phenolic monomers are guaiacol, vanillin, isoeugenol, acetovanillone, guaiacylacetone, and homovanillic acid were quantified using calibration curves and GC-MS. We found the yield of guiacol is 4.71±0.37 mg/g, vanillin is 20.47±2.3 mg/g, isoeugenol is 4.75±0.08 mg/g, acetovanilone is 3.95±0.16 mg/g, guaiacylacetone is 5.96±0.15 mg/g and homovanillic acid is 23.29±1.6 mg/g from the alkali lignin.

Poster Abstract # 3

Structural Characterization of Lignin and the Residue by Cuo Oxide Oxidative Degradation

Ranen Roy, JADHAV BALAWANTHRAO, and DOUGLAS E. RAYNIE

South Dakota State University

Brookings, South Dakota

Lignin is a three-dimensional organic biopolymer present in large scale in the secondary cell walls of vascular plants primarily mixed with cellulose and hemicellulose. It is composed of phenylpropanoid units which are linked by ether and carbon-carbon bonds. Its structural determination is very complex because lignin does not have any regular monomeric sequence. Nowadays, lignin is considered as a potential alternative source for different types of value-added chemicals and for renewable energy. Therefore, it is necessary to understand the comprehensive structure and macromolecular features of lignin for its appropriate use, such as catalytic depolymerization to different types of value-added materials. Various chemical degradation methods are used for structural characterization of lignin. Oxidative degradation of lignin produces numerous types of monomeric phenolic compounds. Cupric oxide is a milder oxidant, mainly breaking down the ether linkages among the monomeric units, keeping the aromatic rings intact, which produces different types of phenolic moieties. Hence, the oxidation study explores the lignin structure more understandable. Therefore, in our study, cupric oxide oxidation, followed by GC-MS analysis, was used to determine the structural moieties present in the lignin and the residue from deoxyhydrogenation reaction quantitatively and qualitatively. Our qualitative results showed that lignin contains guaiacol, vanillin, acetovanillone and homovanillic acid moieties and the reaction residue contains vanillin and acetovanillone moieties which all are carbonyl-conjugated guaiacyl (G) moieties. The quantitative result of guaiacol, vanillin, acetovanillone, and homovanillic in lignin are 1.25±0.06, 8.48±0.19, 2.06±0.08, and 4.79±0.13 mg/g respectively. On the other hand, the quantitative result of vanillin and acetovanillone in the residue are 5.22±0.13, and 1.90±0.11 mg/g respectively. Additionally, the results showed residue contains less amount of vanillin and acetovanillone than lignin and no peaks for guaiacol and homovanillic acid. Finally, there is no significant change of acetovanillone moieties in both the samples which represented very small amounts.

Poster Abstract # 4

Extraction of Vanillin from Alkali Lignin by Water-Based Deep Eutectic Solvent (DES)

MD Sajjadur Rahman and DOUGLAS RAYNIE

Department of Chemistry and Biochemistry

South Dakota State University

Vanillin is the major component of vanilla flavoring, a very prominent precursor of many organic syntheses, and a key intermediate for the synthesis of bio-based polymers. It has many applications in various fields like fragrance and flavors, pharmaceuticals, and others. The aim of this study is to develop a simple method to extract vanillin from an available and inexpensive source lignin by dichloromethane (DCM) after exposure to the eco-friendly deep eutectic solvent (DES) (choline chloride: water, 1:4). Sample preparation was carried out by mild heating to less than 80°C of the lignin-DES solution and stirred for a few hours in a covered beaker. After the extraction with DCM, samples were analyzed by GC-MS. This DES system is reported for extraction for the first time. It demonstrates high affinity for vanillin since it has selectively separated vanillin from complex lignin samples. Another advantage is that no further dilution was required for the treated solution since this DES is already less viscous compared to more commonly used DES. No emulsion layer was formed between DES and DCM layers. Both the solvents and source of vanillin are affordable and producing less waste. This extraction method is non-destructive and simple. Although this method still requires optimization, it looks promising.

Poster Abstract # 5

Spectrophotometric Determination of Binding Constants of Bond Elut Emr-Lipid

Shiksha Subedi and DOUGLAS RAYNIE

South Dakota State University

Brookings, South Dakota

Analysis of complex matrices requires extensive sample preparation to extract the analyte of interest at appropriate concentrations. Lipid interferences are a problem for laboratories measuring trace residues in fatty foods. Lipid can build up in the instrument and column, decreasing lifetime and sensitivity. Bond Elut EMR-Lipid is a novel sorbent material that selectively removes the major lipid classes from sample matrices without unwanted analyte loss. Interaction of Bond Elut EMR with lipid forms a stable host-guest complex. However, it does not bind all lipids with equal strength. So, the binding constants of Bond Elut EMR- Lipid with different lipids were determined. Similarly, binding constant of Bond Elut EMR- Lipid with lipid in presence of various percentages of water was also determined. To determine the binding constant, titration with measurement by means of spectrophotometry was carried out.

Poster Abstract #6

Identification of Metabolites From Urine and Plasma by Microflow LC-MS/MS

Khatereh Motamedchaboki and Baljit K. Ubhi

SCIEX

Redwood City, CA

Identification of metabolites from urine and plasma is necessary for validating potential disease biomarkers for research and interrogating the affected metabolic pathways to further understand their biological implications. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis is an essential tool for identification and quantitation of metabolites in complex sample matrices due to its inherent sensitivity gains. Here, we describe a robust and sensitive workflow using a M3 MicroLC coupled to a QTRAP® 6500+ mass spectrometer for qualitative and quantitative analysis of polar metabolites.

Poster Abstract #7

Improving the Analysis of Polar Analytes with a Novel HILIC Phase on Superficially Porous Particle LC Columns

ANNE MACK, WILLIAM LONG, ADAM BIVENS,

Natalie Rasmussen and JASON LINK

Agilent Technologies

The fields of metabolomics, pharmaceuticals, pesticide analysis, and food testing have encountered a major challenge in the separation of the many highly polar analytes of interest in these areas. Adequately retaining and separating small polar molecules with reversed-phase liquid chromatography (RPLC) is a challenging task. Alkyl phase LC columns, like C18, are a common starting point for LC method development, but highly polar analytes are poorly retained on these non-polar stationary phases.

Several techniques have been developed to analyze polar compounds: pH adjustment, ion pairing, normal phase chromatography, derivatization, ion chromatography, and hydrophilic interaction chromatography (HILIC). Of these, HILIC has gained popularity among chromatographers for its ability to retain and separate polar analytes.

The advantage of HILIC mode is that it uses the same solvents and system as RPLC. Unlike ion pairing, HILIC leaves no residue in the system and operates in both positive and negative modes on a mass spectrometer. In HILIC mode, water is the strong eluting solvent, most analytes elute in >50% organic solvent. High levels of organic have advantages over traditional RPLC in both sample preparation and LCMS analytical sensitivity. Samples prepared in organic solvents can be injected directly without drying or solvent transfer, and the higher volatility of organic solvents improves the ionization efficiency and sample recovery on mass spectrometers. Superficially porous particles are known for their ability to generate high efficiency with low back pressure. This high efficiency is critical to resolving closely eluting peaks, while low back pressure allows for flexibility with LC instrumentation. These distinct advantages make superficially porous particles the ideal platform for developing a next generation HILIC chemistry.

This work will demonstrate the direct analysis of highly polar analytes, without requiring the use of ion pairing or derivatization agents, by utilizing a novel HILIC phase on superficially porous particle columns.