Modern Advancements in Elemental Speciation: from Sample Introduction to Chemical Warfare Agent Detection

UNIVERSITY OF CINCINNATI

Date: May 14, 2007

I, Douglas Dennis Richardson II , hereby submit this work as part of the requirements for the degree of: Doctor of Philosophy (Ph.D) in: Chemistry It is entitled: Modern Advancements in Elemental Speciation: From Sample Introduction to Chemical Warfare Agent Detection

This work and its defense approved by:

Chair: Dr. Joseph A. Caruso Dr. William R. Heineman Dr. James Mack

Modern Advancements in Elemental Speciation: From Sample Introduction to Chemical Warfare Agent Detection

A dissertation submitted to the

Division of Research and Advanced Studies of the University of Cincinnati

In partial fulfillment of the requirements for the degree of

Doctor of Philosophy (Ph.D)

In the Department of Chemistry of the McMicken College of Arts and Sciences

2007

Douglas Dennis Richardson II

B.S., Forensic Chemistry, Ohio University, 2003

M.S. Chemistry, University of Cincinnati, 2006

Committee Chair: Dr. Joseph A. Caruso

Abstract of Dissertation

Elemental speciation is the investigation of the chemical form of metal and non- metal containing species in environmental and biological systems for the determination of species specific essentiality and toxicity. Speciation analysis is performed by combining modern separation techniques with state-of-the-art element specific mass spectrometry. Separation techniques used in this work include: capillary electrophoresis

(CE), high performance liquid chromatography (HPLC) and gas chromatography (GC).

Inductively coupled plasma mass spectrometry (ICPMS) is the instrument of choice for ultra-trace elemental speciation analyses due to the excellent sensitivity and selectivity specific to this mass spectrometer.

Modern innovations in analytical instrumentation specific for elemental speciation have provided researchers with resources for the development of new hyphenated techniques and analytical methods. The specific goal of this dissertation is to describe modern advancements specific to elemental speciation.

In the first section a novel interface coupling CE and hydride generation with

ICPMS detection for arsenic speciation is described. The novel concentric tube interface design allowed for the separation, hydride generation, and detection of four arsenic species in less than 10 minutes.

The majority of this dissertation focuses on method development for the analysis of organophosphorus chemical warfare agent (CWA) degradation products. Recent increases in worldwide terrorist activity as well as the threat of chemical weapon attacks have led to the demand for rapid and reliable analytical techniques for CWA analysis.

Methods utilizing both HPLC and GC separation techniques couple with 31P element

specific detection with ICPMS for the analysis of organophosphorus chemical warfare agent degradation products are described. These works are the first to utilize 31P detection with ICPMS for the analysis of chemical warfare agent degradation products.

iii

Acknowledgments

This dissertation is dedicated to my parents Douglas and Rebecca Richardson, without whom none of my educational accomplishments would have been possible. They have always been there to provide spiritual, educational, and monetary support without too many questions. I would also like to acknowledge my siblings Katie, Jon, Chris, and my dog Rocky for their continued support and harassment.

The completion of this degree would not have been possible without the support of my friend and advisor Dr. Joseph Caruso (“Doc”). His willingness to take risks as well as deal with scientific setbacks and breakthroughs in a relaxed manner is a characteristic that I plan to model my career after. Dr. Caruso has provided me with multiple opportunities for both academic and personal growth for which I am forever thankful. In addition Dr. Caruso and his wife Judy have treated me like a son throughout my time in

Cincinnati, and this relationship is more valuable to me than any academic degree.

I would like to thank my committee members Dr. William R. Heineman and Dr.

James Mack for all their scientific challenges and teachings throughout my graduate school experience. Also, thanks to the analytical faculty; Dr. Patrick A. Limbach, Dr.

Tom Ridgway, and Dr. Apryll Stalcup for all their support. None of my research would have been possible without the excellent people behind the scenes Kim Carey, John

Zureick, Betty Ligon, Cassandra McGee, and Jamie Cartwright. A special thanks to Dr.

Sasi Kannamkumarath, Dr. Rodolfo Wuilloud, Dr. Baki Sadi, and Dr. Monika Shah for teaching me everything they (I) know about ICPMS. To the Caruso group members Dr.

Sandra Mounicou, Dr. Juris Meija, Dr. Tyre Grant, Dr. Katie DeNicola, Kevin Kubachka,

Kirk Lokits, Heather Trenary, Scott Afton, Jenny Ellis, Karolin Kroning, and all other

group members past and present, it was a pleasure working with you all. To my fellow graduate students and friends Stuart Willison, Michael Haven, Phil Durham, Kevin

Parker, Dr. Justin Mecomber, Adam Bange, Becky Rolfs, Kady Krivos, Susan Russell, and Colette Castleberry you have made my time in the chemistry department fun and exciting and you all will be missed. To my Australian friends Emeritus Professor David

Knowles and Dr. Anne McLachlan (Macca) it has been my pleasure getting to know you both. You have helped opened my eyes to the entire world and I value our friendship deeply.

A special thanks to Agilent Technologies specifically Toshiaki Matsuda, Steve

Wilbur, Don Potter, Steve Miller, and Ron Sanderson. The University of

Cincinnati/Agilent Metallomics Center of the Americas is alive and well thanks to your support. Other scientist and collaborators with whom I have had the pleasure of interacting include; Dr. Fred Fricke (FDA), Dr. Doug Heitkemper (FDA), Dr. Jack Creed

(EPA), Dr. Brian Gamble (FDA), Dr. Ray Warren (P&G), Dr. Wendy Qin (P&G), Brian

Gray (P&G), and Dr. John Hammons (P&G).

Last but certainly not least I must thank my best friend Daisy-Malloy Hamburg.

You have supported me throughout the emotional rollercoaster ride of graduate school allowing our relationship to blossom along the way. Remember we are like electrons of opposite spin impossible to keep apart and I look forward to many more adventures together. I love you babe!

-DDR

Table of Contents

Abstract of Dissertation ii

Acknowledgments iv

Table of Contents 1

Figures 4

Tables 7

Chapter 1 – Elemental Speciation with Inductively Coupled Plasma Mass Spectrometry

1.1 Overview 1.2 Inductively Coupled Plasma Mass Spectrometry (ICPMS) 1.2.1 Sample Introduction 1.2.1.1 Liquid Sample Introduction 1.2.1.2 Hydride Generation 1.2.2 Plasma Ionization Source 1.2.3 Interference Removal with Collision/Reaction Cell 1.2.3 Mass Spectrometric Detection 1.3 Elemental Speciation Techniques 1.3.1 Capillary Electrophoresis (CE) with ICPMS 1.3.2 High Performance Liquid Chromatography (HPLC) with ICPMS 1.3.2.1 Reversed Phase Chromatography 1.3.2.2 Ion-Pairing Chromatography 1.3.3 Gas Chromatography (GC) with ICPMS 1.3.3.1 Interfacing GC with ICPMS 1.3.3.2 Sample Introduction Techniques for GC with ICPMS 1.4 References

Chapter 2- Hydride Generation Interface for Speciation Analysis Coupling Capillary Electrophoresis to Inductively Coupled Plasma Mass Spectrometry

2.1 Abstract 2.2 Introduction 2.3 Experimental 2.3.1 Reagents 2.3.2 Instrumentation 2.3.3 Interface Design 2.3.4 Coupling Hydride Generation Interface with ICPMS 2.4 Results and Discussion 2.4.1 Optimization of Hydride Generation Conditions 2.4.2 Arsenic Species Separation by Capillary Electrophoresis

2.4.3 Analytical Performance of CE-HG-ICPMS 2.4.4 Arsenic Speciation in Water Samples 2.5 Conclusion 2.6 Acknowledgments 2.7 References

Chapter 3- Reversed phase ion-pairing HPLC-ICPMS for analysis of organophosphorus chemical warfare agent degradation products

3.1 Abstract 3.2 Introduction 3.3 Experimental 3.3.1 Reagents 3.3.2 Instrumentation 3.3.2.1 HPLC 3.3.2.2 ICPMS 3.3.2.3 Electrospray Mass Spectrometry (ESI-MS) 3.4 Results and Discussion 3.4.1 Ion-pairing HPLC 3.4.2 ICPMS Detection 3.4.3 Analytical Figure of Merit 3.4.4 Complex Samples 3.4.5 Characterization Efforts 3.5 Conclusion 3.6 Acknowledgments 3.7 References

Chapter 4- Derivatization of Organophosphorus Nerve Agent Degradation Products for Gas Chromatography with ICPMS and GC-ToF Detection

4.1 Abstract 4.2 Introduction 4.3 Experimental 4.3.1 Reagents 4.3.2 Derivatization 4.3.3 Environmental Samples 4.4 Instrumentation 4.4.1 Gas Chromatography (GC) 4.4.2 ICPMS 4.4.3 Gas Chromatography-Time of Flight Mass Spectrometry (GC-ToF) 4.5 Results and Discussion 4.5.1 GC-ICPMS 4.5.2 GC-ToF 4.5.3 Analytical Figures of Merit 4.5.4 Phosphate and Environmental Samples 4.6 Conclusion

4.7 Acknowledgments 4.8 References

Chapter 5- Screening Organophosphorus Nerve Agent Degradation Products in Pesticide Mixtures by GC-ICPMS

5.1 Abstract 5.2 Introduction 5.3 Experimental 5.3.1 Reagents 5.3.2 Sample Preparation 5.4 Instrumentation 5.4.1 Gas Chromatography (GC) 5.4.2 ICPMS 5.5 Results and Discussion 5.5.1 GC-ICPMS 5.5.2 Pesticide Analysis in Nerve Agent Degradation Product Mixture 5.5.3 Degradation Product Analysis in Pesticide Mixtures 5.6 Conclusion 5.7 Acknowledgments 5.8 References

Chapter 6- Conclusions and Future Directions

Figures

1.1- Experimental considerations for elemental speciation analysis.

1.2- Instrument schematic for an Agilent 7500ce ICPMS.

1.3- Concentric nebulizer design for liquid sample introduction.

1.4- General “ABC” pathway for hydride generation by borohydride reduction.

1.5- Schematic of an inductively coupled plasma torch.

1.6- Plasma ionization pathway.

1.7- Agilent Technologies collision/reaction cell for 7500ce ICPMS.

1.8- Interference removal through collision induced dissociation and KED.

1.9- Electron multiplier (detector) for Agilent 7500ce ICPMS.

1.10- Equations for calculating electrophoretic mobility of analytes in CE.

1.11- Diagram of capillary wall and bulk electrolyte flow (EOF).

1.12- Interface coupling CE with ICPMS.

1.13- Bench top instrumental configuration for LC-ICPMS.

1.14- Ion-pairing chromatography.

1.15- Agilent GC-ICPMS interface.

1.16- SPME sampling for GC-ICPMS.

2.1- Computer aided drawing (CAD) of CE-HG-ICPMS interface.

2.2- CE-HG interface transfer line utilized for removal of hydride condensation and more efficient analyte transport to ICP-MS.

2.3- Optimization of hydrochloric acid (A) and sodium borohydride (B) for standard arsenic mixture based upon peak area during CE separation. Instrumental parameters are presented in Table 2.2.

2.4- Separation of standard arsenic mixture (500 μg L -1) under optimum CE and HG conditions.

2.5- Arsenic mixture (100 μg L -1 ) spiked (A) tap water sample and (B) Miami River water sample.

3.1- Helium collision gas flow rate optimization.

3.2- Separation comparison of three ion-pairing reagents: tetrabutylammonium hydroxide (TBAH), hexadecyltrimethylammonium bromide (HAB), myristyltrimethylammonium bromide (TTAB).

3.3- Separation of 100 ng mL-1 mixture of MPA, EMPA, and IMPA.

3.4- Separation of blank and 100 ng mL-1 spiked Little Miami River (Ohio) water.

3.5- Separation of blank and 100 ng mL-1 spiked laboratory tap water.

3.6- Separation of blank and 100 ng mL-1 spiked top soil.

3.7- Separation of blank and 100 ng mL-1 spiked potting soil.

3.8- A) Time elapsed analysis of EMPA (10 μg mL-1) in DDI water after 2.5 hours. B) 10 μg mL-1 MPA spike.

3.9- Separation of 1.85 μg mL-1 ammonium phosphate, 5 μg mL-1 potassium phosphate, and 5 μg mL-1 sodium phosphate.

4.1 A&B- Hydrolysis pathway of G and V-Type nerve agents.

4.2- TBDMS esterification of alkyl phosphonic acids.

4.3 A&B- Optimization of derivatization time and temperature.

4.4- Separation of 5 ng mixture of seven TBDMS derivatives.

4.5 A- GC-ToF-MS analysis of EMPA-TBDMS.

4.5 B- GC-ToF-MS analysis of IMPA-TBDMS.

4.5 C- GC-ToF-MS analysis of IBMPA-TBDMS.

4.5 D- GC-ToF-MS analysis of PMPA-TBDMS.

4.5 E- GC-ToF-MS analysis of MPA-TBDMS.

4.5 F- GC-ToF-MS analysis of CMPA-TBDMS.

4.5 G- GC-ToF-MS analysis of EDAP-TBDMS.

4.6- GC-ToF-MS identification of trace doubly derivatized phosphate in standard mix (Figure 4.4).

4.7- Standard separation of 30 ng mixture with phosphate-TBDMS

4.8 A&B- Spiked and derivatized river water sample.

4.9 A&B- Spiked and derivatized soil samples.

5.1- Five common organophosphorus pesticides (OPP’s).

5.2- 1 ng degradation product mixture w/ 25 ng degraded dichlorvos.

5.3- 1 ng degradation product mixture w/ 25 ng degraded ethion.

5.4- 1 ng degradation product mixture w/ 25 ng degraded parathion ethyl.

5.5- 5 ng isopropyl methylphosphonic acid-TBDMS in mixture with 50 ng dichlorvos, ethion, and parathion ethyl.

5.6- 5 ng pinacoyl methylphosphonic acid-TBDMS in mixture with 50 ng dichlorvos, chlorpyrifos, and ethion.

5.7- 5 ng cyclohexyl methylphosphonic acid-TBDMS in mixture with 50 ng acephate, parathion ethyl, dichlorvos.

5.8- 5 ng methylphosphonic acid-TBDMS in mixture with 25 ng acephate, ethion, and chloryprifos.

Tables

2.1- Summary of As(III), As(V), MMA and DMA detection limits by various analysis techniques.

2.2- CE-HG-ICPMS instrumental parameters.

3.1- Chemical warfare agents and degradation products.

3.2- HPLC-ICPMS instrumental parameters.

3.3- Chemical warfare degradation product detection limits.

3.4- Analytical figures of merit based on 20 ng mL-1 mixture.

4.1 A - Chemical properties of G-Type nerve agents and degradation products.

4.1 B- Chemical properties of V-Type nerve agents and degradation products.

4.2- Instrumental parameters for GC-ICPMS.

4.3- Figures of merit based upon seven replicate injections of a 5ng mixture.

4.4- Detection limit comparison of the developed GC-ICPMS method with LC-ICPMS.

5.1- ICPMS and GC instrumental parameters.

CHAPTER 1

Elemental Speciation with Inductively Coupled Plasma Mass Spectrometry

1.1 OVERVIEW

Over the past few decades a shift in the research focus for the analysis of metal and non- metal containing samples has resulted in the evolution of ‘elemental speciation’.

Previously, total content of various metals and non-metals was utilized to determine both environmental and health implications from release and exposure. Elemental speciation has led to development of analytical techniques that provide a more detailed look into the chemistry of metal and non-metal containing samples. This species specific chemistry takes into account oxidation state, covalent organic and inorganic ligands, non-covalent complexes, as well as metabolic pathways of specific elements in biological systems. The

International Union for Pure and Applied Chemistry (IUPAC) defines a ‘chemical species’ as a specific form of an element defined as to isotopic composition, electronic or oxidation state, and/or complex or molecular structure1, 2. Therefore, ‘elemental speciation’ is the development of species-specific analytical techniques for the differentiation between the essentiality and toxicity of these elemental species in environmental and biological systems. Typically elemental speciation can be broken down into four main steps: sampling, sample preparation, chromatographic separation, and detection (Figure 1.1). Elemental speciation analyses are performed through coupling analytical separation techniques with element specific detection3-5. These separation techniques include capillary electrophoresis (CE), liquid chromatography

(LC), gas chromatography (GC), and gel electrophoresis. Element specific detection is accomplished utilizing an inductively coupled plasma mass spectrometer (ICPMS).

Element specific mass spectrometry with ICPMS has become the benchmark technique for speciation analysis due to the sensitivity, selectivity, multielement detection

capability, wide dynamic range, and ease of chromatographic hyphenation that accompanies this detection system. Figure 1.2 provides an instrument schematic for a

Agilent 7500ce ICPMS.

Chromatographic Separation Detection

•Capillary Electrophoresis (CE) •Inductively Coupled Plasma Mass Spectrometry (ICP-MS) •Liquid Chromatography (LC) •Electrospray Mass Spectrometry (ESI-MS) •Gas Chromatography (GC) •Matrix Assisted Laser Desorption Ionization •Gel Electrophoresis (GE) Mass Spectrometry (MALDI-MS) •Ultra-violent/Visual Absorbance (UV-Vis)

Elemental Speciation

Sample Preparation Sampling

•Extraction/Digestion •Collection •Derivitization •Storage •Preconcentration

Figure 1.1- Experimental considerations for elemental speciation analysis.

Hyphenated elemental speciation techniques with ICPMS detection provides species identification through retention/migration time matching with commercial or synthetic standards. This harsh ionization process causes a loss of structural information making identification of unknown species impossible. The complementary use of alternative mass spectrometric techniques such as ESI and MALDI provide confirmation and identification for both standards and unknown species. This chapter provides a detailed introduction to ICPMS instrumentation as well as the most commonly used separation

techniques: capillary electrophoresis (CE), liquid chromatography (LC), and gas chromatography (GC).

Agilent 7500ce

Figure 1.2- Instrument schematic for an Agilent 7500ce ICPMS.

1.2 INDUCTIVELY COUPLED PLASMA MASS SPECTROMETRY (ICPMS)

1.2.1 Sample Introduction

Sample introduction with ICPMS varies widely depending upon the physical state of the sample of interest. Techniques for the introduction of liquid, gas, and solid samples have been well studied through the literature6-9. These techniques include concentric and crossflow pneumatic nebulization, ultrasonic nebulization, electrothermal vaporization, hydride generation, direct insertion, and laser ablation10. This section describes the two types of sample introduction used in this dissertation research, liquid sample introduction through concentric nebulization and hydride generation.

1.2.1.1 Liquid Sample Introduction

Liquid sample introduction through aerosol production is the most common technique for elemental speciation. Pneumatic nebulizers are the most commonly used devices for liquid aerosol generation and are typically coupled to a cooled spray chamber to remove large droplets from the aerosol stream.

Figure 1.3- Concentric nebulizer design for liquid sample introduction11.

The most common design for pneumatic nebulizers with ICPMS is the concentric type, consisting of an internal capillary for the sample solution surrounded by a high velocity gas stream (Figure 1.3). Liquid solution is pumped through the internal capillary which terminates at the end of the nebulizer where the solution is exposed to a high velocity gas resulting in solution “breakup” and generation of aerosol droplets10. Efficiency of liquid sample introduction with concentric nebulization ranges from 30-70% depending on solution flow rates. Solution flow rates with concentric nebulizers vary depending upon the design with normal flow from 0.1-2 mL min-1, capillary flow from 10-100 μL min-1, and nanoflow12 of less than 500 nL min-1. Typically when the solution flow is less than

10 μL min-1 the system operates in a total consumption mode due to the efficiency of the aerosol formation.

1.2.1.2 Hydride Generation

Hydride generation (HG), the most popular alternative sample introduction technique, has been utilized for nearly 40 years as a derivatization method for the analysis of trace elements capable of forming hydrides (As, Bi, Ge, Pb, Sb, Se, Sn, Te, In, and Tl).

Generation of volatile metal hydrides can be accomplished in a variety of ways including electrochemical generation, photoinduced generation, thermochemical generation, metal- acid reduction, and sodium borohydride reduction13-15. These techniques allow for improved analyte transport efficiency, separation from matrix interferences, and improved detection limits of by a factor of 10 or more compared to conventional pneumatic nebulization. Aqueous acid reduction through reaction with sodium borohydride provides the simplest route for elemental hydride generation, making this the method of choice for HG-ICPMS experiments. Figure 1.4 provides a general description of the “ABC” (analyte-borane complex) pathway for hydride generation through borohydride reduction16.

MYn + L3BH “ABC” Intermediates b

Yn-1MH b Yn-xMHx b MHn

Figure 1.4- General “ABC” pathway for hydride generation by borohydride reduction16.

1.2.2 Plasma Ionization Source

The inductively coupled plasma (ICP) ionization source is generated through electrical excitation of an argon gas stream within a concentrically designed quartz torch.

Surrounding the end of the torch is a water-cooled copper induction coil (load coil) which is connected to an rf generator typically operated at 27MHz. The rf generator provides

constant operating power ranging from 550-1550 W resulting in a stable plasma while argon gas is present. The resulting annular shaped plasma is due to higher gas flow on the external most section of the concentric torch (Figure 1.5).

Figure 1.5- Schematic of an inductively coupled plasma torch11.

Typical argon gas flow rates for plasma generation are15 L min-1 for the plasma and 1 mL min-1 for auxiliary and sample gas respectively. Sample introduction of a liquid aerosol occurs in the central channel of the torch resulting in desolvation, vaporization, atomization, and ionization (single positive charge) (Figure 1.6). Ionization of gas and solid samples follow the same pathway without the need for the desolvation and vaporization steps.

Figure 1.6- Plasma ionization pathway11.

1.2.3 Interference Removal with Collision/Reaction Cell

Method development for elemental speciation with ICPMS requires the consideration of interferences common with this analytical instrumentation. These interferences include physical, chemical, ionization, ion transmission, and spectral17-19. Modern instrumental advancements have focused upon the removal of spectral interferences which include isobaric overlap from an isotope with the same mass ( i.e. 64Ni+ and 64Zn+) and formation of polyatomic species at the target mass (i.e. 40Ar40Ar+ on 80Se+). Sources of these spectral interferences include the plasma ionization source (argon), sample solution

(oxygen source), and the sample matrix (salt source). Addition of a collision/reaction cell between the ion lens focusing region of the ICPMS and the mass analyzer has provided a tool for spectral interference removal (Figure 1.7). In this research an octopole ion guide, operated in rf only mode, enclosed within a collision/reaction cell (Agilent Technologies) was used for interference removal19.

Gas Inlet

Octopole Ion Energy Barrier Guide

Mass Analyzer

Figure 1.7- Agilent Technologies collision/reaction cell for 7500ce ICPMS11.

This system is then purged with an inert collision/reaction gas typically hydrogen or

helium, and a kinetic energy discrimination (KED) barrier is created between the cell exit

and the mass analyzer. Two mechanisms of interference removal, reaction and collision

induced dissociation, are observed depending upon the gas used19. Hydrogen gas is

typically used for removal of argon based polyatomic species through a reaction pathway

(i.e. 40Ar40Ar+ b 40Ar40ArH+). Helium gas is used for the removal of solution and matrix

interferences through a combination of collision induced dissociation and KED.

Monatomic ions are able to keep enough transitional energy to overcome the KED barrier

at the cell exit and therefore reach the mass analyzer. Figure 1.8 provides a description of

the removal of nitrogen based polyatomic species through collision induced dissociation

and energy discrimination.

Collision (energy discrimination)

+ 3 aSP 31P 12 aSP 15 16 + N O He 1 2 + aSP

+ 31P

15N16O

Ion Energy Reaction cell

Figure 1.8- Interference removal through collision induced dissociation and KED.

1.2.4 Mass Spectrometric Detection

Mass analyzers frequently used with an ICP ionization source are quadrupole mass filter, magnetic sector (sector field), and time of flight (ToF)11, 20. Quadrupole mass filters are the most common (>90%) and least expensive mass analyzers that accompany commercial ICPMS instruments. Consisting of two pairs of parallel hyperbolic rods arranged in a square geometry on axis with the ion beam, ions are separated based upon their mass to charge (m/z) ratios11, 20 . Separation or filtering of the ion beam is accomplished through application of a high frequency AC voltage (out of phase between two rod pairs) and DC voltage (one pair positive the other negative) resulting in a hyperbolic electric field forcing an unstable trajectory and eventual loss of ions outside the specified mass range of the quadrupole11, 20 . Constant variation in the AC and DC fields at a constant ratio creates a narrow bandpass filter allowing selective mass transmission at low resolution (0.5-1 amu) for quadrupole systems11, 20 . Rapid voltage adjustment allows scanning of the entire mass range (2-260) within one-tenth of a second, while providing mass spectrum for all isotopes with that mass range.

Figure 1.9- Electron multiplier (detector) for Agilent 7500ce ICPMS11.

Detection of the filtered ion beam occurs through the use of an electron multipler (EM).

Positively charges ions reach the entrance of the detector where they deflect off the first dynode (high negative voltage) and release secondary electrons from the surface (Figure

1.9)11, 20. The secondary electrons continue the cascade effect by striking the remaining dynodes resulting in an exponential increase in free electrons and a measurable ion pulse at the final dynode. Duel mode detectors (electron multipliers) have provided ICPMS instruments with an extended dynamic range (9 orders of magnitude) through the ability to switch from a pulse counting (digital) mode at lower analyte concentrations to an electron current monitoring (analog) mode at higher concentrations11, 20. The combination of a quadrupole mass filter in conjunction with a duel mode electron multiplier provides the sensitivity and selectivity required for elemental speciation.

1.3 ELEMENTAL SPECIATION TECHNIQUES

1.3.1 Capillary Electrophoresis with ICPMS

Capillary electrophoresis (CE) is a rapid, high resolution separation technique that separates species based on their charge and size. This technique is capable of separating both positively charged, negatively charged, and a single neutral species in a one run.

Separations with CE are performed in a fused silica capillary (length 20-100cm and i.d.

50-75 micrometers) filled with an electrolyte buffer. The negatively charged surface of the capillary is coated with cations from the electrolyte buffer creating a static layer along the capillary surface. Adjacent to the static layer is the mobile layer (Outer Helmholtz layer) comprised of bulk solution with both cations, anions and neutral species. CE injection volumes range from 10-100 nL and can be done using hydrodynamic (pressure), siphoning, or electrokinetic injection. An applied voltage ranging from 5-30 kV provides

an electric field through which the species of interest migrate as a function of the size and charge (Figure 1.10). Migration of the electrolyte (buffer solution) is termed electroosmotic flow (EOF) while analyte migration is referred to as electrophoretic mobility (Figure 1.11). In contrast to hydrodynamic flow commonly observed with pressure driven separation techniques such as liquid chromatography (LC), plug flow of the electrolyte solution minimizes band broadening resulting in a narrower peak shape and detection window with CE. Fast analysis times, small sample volumes, minimal solvent waste, and inexpensive capillaries have made CE a popular separation technique. q M (4 )r 3V 6r 3

w HOHFWURSKRUHWLFPRELOLW\ 0 PDVVRIWKHLRQ T FKDUJHRQWKHSDUWLFOH 9 SDUWLDOVSHFLILFYROXPHRIWKHVROXWH VROXWLRQYLVFRVLW\ U 6WRNHVUDGLXVRIWKHSDUWLFOH Figure 1.10- Equations for calculating electrophoretic mobility of analytes in CE.

Anode Cathode

Figure 1.11- Diagram of capillary wall and bulk electrolyte flow (EOF).

In the past, coupling CE with ICPMS element specific detection was a difficult task due

to the low flow and high electrical potential that accompanies this separation technique.

Olesik et al. were the first to couple CE with ICPMS detection for speciation analyses20.

This work provided an improvement in detection limits of 60 times compared to ICPMS

without speciation (no separation)21. Applications of speciation analyses by CE-ICPMS

have expanded dramatically over the last decade with multiple literature reviews within

the past three years22-25. Advancements in hyphenating CE with ICPMS have also been

accomplished with two commercial interfaces currently available (CETAC CEI-100 and

Burgener Mira Mist CE). Figure 1.12 provides a schematic of an in-house interface

design coupling CE with ICPMS. Chapter 2 provides a detailed description for a novel

interface coupling CE with hydride generation for speciation analysis26.

Microconcentric Nebulizer Make-up Electrolyte Cyclonic Spray Chamber ICP torchtorch Q-pole

CE capillary

Turbo Turbo pump pump

Waste Rotary pump Ground connection Argon gas controller

Figure 1.12- Interface coupling CE with ICPMS.

1.3.2 High Performance Liquid Chromatography with ICPMS

High Performance Liquid Chromatography (HPLC, sometimes referred to as High

Pressure) is the most popular separation technique for elemental speciation with ICPMS.

HPLC is commonly used for separating thermally labile polar, non-polar, hydrophobic, hydrophilic, and charged species which are all non-volatile in nature. Depending upon the analyte of interest, multiple types of HPLC separation techniques are available including normal phase (polar stationary phase with non-polar mobile phase, reversed phase (non- polar stationary phase with polar mobile phase), ion-exchange, ion-pairing, and size exclusion. Coupling HPLC with ICPMS is a simple task consisting of connecting a tube from the chromatographic column exit to the entrance of the ICPMS nebulizer. Figure

1.13 provides a picture of typical bench top HPLC-ICPMS instrumentation.

Figure 1.13- Bench top instrumental configuration for LC-ICPMS11.

Typically, this instrumental configuration allows for simultaneous UV absorbance

(providing the analytes of interest possess a chromophore) data collection and element specific ion traces. Thompson and Houk were the first to couple HPLC with ICPMS for elemental speciation analysis27. Following in the footsteps of this work multiple HPLC separation methods with ICPMS detection have been developed for a variety of biological and environmental applications1, 5, 22, 28-31. This section focuses upon reversed phase and ion-pairing chromatography.

1.3.2.1 Reversed Phase Chromatography

Reversed phase chromatography is the most common type of liquid chromatography utilized for elemental speciation analyses with ICPMS. The separation platform of this technique consists of non-polar stationary phases of varying hydrophobicity (C2-C18) bonded to a solid support (silica). Mobile phase composition consists of an aqueous buffer, typically deionized water, with various types of organic modifiers (methanol, ethanol, acetonitrile) usually less than 10%. Organic modifier concentrations exceed 10% pose problems with ICPMS instrument stability due to the excess carbon present following ionization. Owing to this fact gradient separations are restricted to low flow chromatographic systems in which oxygen is mixed into the argon plasma. Separation occurs based upon partitioning of the analytes in the mobile into the stationary phase through hydrophobic interactions (like-like). Special considerations such as analyte pKa values and buffer pH should be taken when choosing a mobile phase in order to ensure metal analyte stability. Mobile phase flow rates are isocratic in reversed-phase separations with ICPMS typically between 0.5-1 mL min-1 for conventional nebulization.

1.3.2.2 Ion-Pairing Chromatography

An alternative form of reversed phase chromatography, ion-pairing chromatography, is typically used to separate non-volatile charged analytes with varying hydrophobicity.

Mobile phases for ion-pairing chromatography are typically buffered at a pH to create a completely charged analyte of interest. In addition to the buffer pH an ion-pairing agent of opposite charge to the analyte of interest is added to the mobile phase. Common ion- pairing agents include tetralkylammonium salts, triethylalkyl ammonium salts, and alkylsulfonates. Separation occurs through the formation of a neutral ion-pair between the ion-pairing agent and analyte of interest. The resulting neutral ion-pair interacts with the stationary phase through hydrophobic interactions. Figure 1.14 provides a schematic of separation using ion-pairing chromatography. Chapter 3 describes a reversed phase ion- pairing separation for the analysis of organophosphorus chemical warfare agent degradation products with ICPMS32. + IPA A - + A - IPA + + IPA IPA A -

A - + IPA - + - A IPA A Analyte + IPA Ion-Pairing Agent

- + A IPA Si Si Si Si Si Si Si Si Si Si Si Si Si Ion-Pair

Figure 1.14- Ion-pairing chromatography.

1.3.3 Gas Chromatography with ICPMS

Gas chromatography is a vapor (gas) phase separation technique used for the analysis of volatile compounds. GC coupled with ICPMS for elemental speciation has grown in popularity since it inception in 1986. Van Loon et al.33 and Chong et al.34 were the first to couple this gas phase separation technique with element specific detection with ICPMS.

GC separation techniques with ICPMS are typically utilized for analysis of volatile organometallic or heteroatom containing species. Thermally stable analytes can also be studied by GC-ICPMS following a derivatization procedure (silylation, methylation, pentylflorobenzyl, or para-bromophenol) to produce volatile species. The high resolving power (chromatographic resolution) of GC and excellent sensitivity of ICPMS detection has provided a powerful analytical tool for elemental speciation. Coupling GC with

ICPMS detection has evolved along with the applications in industrial, environmental, and biological settings22, 35. Multiple review articles summarizing these applications have emerged within the past five years5, 22, 35, 36. Chapters 4 and 5 describe the use of GC-

ICPMS for the analysis of organophosphorus nerve agent degradation products37.

1.3.3.1 Interfacing GC with ICPMS

Interfacing GC with ICPMS consists of removing the spray chamber and connecting the capillary GC column exit to a heated transfer line which runs down the central channel of the ICP torch (Figure 1.15). The heated transfer line keeps the column eluent in the gas phase, preventing any condensation from forming in the interface region. Owing to the fact that the analytes reach the plasma ionization source in the gas phase, the desolvation and vaporization steps common to liquid sample introduction are eliminated. The resulting gas phase sample introduction (without nebulization) provides nearly 100%

transport efficiency of the sample into the ICPMS while at the same time reducing the background due to a reduction in spectral and matrix interferences. In the past, packed

GC columns were used in the hyphenated technique however a shift toward capillary columns has been observed due to the higher efficiency and better resolution that these columns provide. The ability to optimize ICPMS instrumental parameters in the gas phase has also recently been observed. Mixed GC carrier gases such as hydrogen or helium with a small percentage (<1%) of xenon allows for optimization of ICPMS parameters prior to sample analysis.

Figure 1.15- Agilent GC-ICPMS interface11.

1.3.3.2 Sample Introduction Techniques for GC with ICPMS

In the past GC-ICPMS sample introduction was limited to solution injection, however a the use of a variety of preconcentration techniques has recently emerged. Microwave assisted extraction (MAE), solid-phase microextraction (SPME; headspace or solution), stir bar sorptive extraction (SBSE), and purge and trap sample introduction techniques have been used to achieve increased sensitivity with GC-ICPMS35, 36. SPME, the most popular sample technique, is a simple, inexpensive, and solvent free preconcentration method which allows sorption of analytes onto the surface of a modified fused microfiber from a variety of sample matrices (Figure 1.16).

Solid-Phase Microextraction (SPME)

GC Inlet

Sample Desorption

Headspace Sampling Solution Sampling

Figure 1.16- SPME sampling for GC-ICPMS.

Online thermal desorption of analytes from the microfiber is accomplished in the injection port of the GC through the use of a specially designed microsyringe.

Optimization of the analyte sorption process with SPME is based upon equilibrium between the analyte in the sample (solution or headspace) and on the fiber, requiring the consideration of fiber type, pH, ionic strength, sampling time, agitation, and desorption temperature. These parameters can vary widely depending upon analyte polarity, volatility, and size resulting in a competitive sorption process. Figure 1.16 provides a description of sampling with SPME.

1.4 REFERENCES

(1) Lobinski, R. Handbook of Elemental Speciation II: Species in the Environment, Food, Medicine and Occupational Health by R. Cornelis, J. Caruso, H. Crews, and K. Heumann (Eds.), 2006. (2) Chassaigne, H.; Vacchina, V.; Lobinski, R. TrAC, Trends in Analytical Chemistry 2000, 19, 300-313. (3) Wuilloud, R. G.; Altamirano, J. C. Current Analytical Chemistry 2006, 2, 353- 377. (4) Bettmer, J.; Jakubowski, N.; Prange, A. Analytical and Bioanalytical Chemistry 2006, 386, 7-11. (5) Shah, M.; Caruso, J. A. Journal of Separation Science 2005, 28, 1969-1984. (6) McLeod, C. W., Routh, M.W., Tikkanen, M.W. In Inductively Coupled Plasmas in Analytical Atomic Spectrometry, 2nd ed.; Montaser, A., Golightly, D.W., Ed.; VCH: New York, 1992. (7) Heitkemper, D. T., Wolnik, K.A., Fricke, F.L., and Caruso, J.A. In Inductively Coupled Plasmas in Analytical Atomic Spectrometry, 2nd ed.; Montaser, A., Golightly, D.W., Ed.; VCH: New York, 1992. (8) Gustavsson, A. In Inductively Coupled Plasmas in Analytical Atomic Spectrometry, 2nd ed.; Montaser, A., Golightly, D.W., Ed.; VCH: New York, 1992. (9) Greenfield, S., Montaser, A. In Inductively Coupled Plasmas in Analytical Atomic Spectrometry, 2nd ed.; Montaser, A., Golightly, D.W., Ed.; VCH: New York, 1992. (10) Montaser, A.; Minnich, M. G.; McLean, J. A.; Liu, H.; Caruso, J. A.; McLeod, C. W. Inductively Coupled Plasma Mass Spectrometry 1998, 83-264. (11) Inductively Coupled Plasma Mass Spectrometry: A Primer; Agilent Technologies, 2006. (12) Giusti, P.; Lobinski, R.; Szpunar, J.; Schaumloeffel, D. Analytical Chemistry 2006, 78, 965-971. (13) Sturgeon, R. E.; Mester, Z. Applied Spectroscopy 2002, 56, 202A-213A. (14) Howard, A. G. Journal of Analytical Atomic Spectrometry 1997, 12, 267-272. (15) Olson, L. K.; Vela, N. P.; Caruso, J. A. Spectrochimica Acta, Part B: Atomic Spectroscopy 1995, 50B, 355-368. (16) D'Ulivo, A.; Mester, Z.; Meija, J.; Sturgeon, R. E. Analytical Chemistry 2007, 79, 3008-3015. (17) Gray, A. L. Fresenius' Zeitschrift fuer Analytische Chemie 1986, 324, 561-570. (18) Douglas, D. J.; Tanner, S. D. Inductively Coupled Plasma Mass Spectrometry 1998, 615-679. (19) McCurdy, E.; Woods, G. Journal of Analytical Atomic Spectrometry 2004, 19, 607-615. (20) Turner, P. J.; Mills, D. J.; Schroder, E.; Lapitajs, G.; Jung, G.; Iacone, L. A.; Haydar, D. A.; Montaser, A. Inductively Coupled Plasma Mass Spectrometry 1998, 421-501. (21) Olesik, J. W.; Kinzer, J. A.; Olesik, S. V. Analytical Chemistry 1995, 67, 1-12.

(22) Rao, R. N.; Talluri, M. V. N. K. Journal of Pharmaceutical and Biomedical Analysis 2007, 43, 1-13. (23) Prange, A.; Proefrock, D. Analytical and Bioanalytical Chemistry 2005, 383, 372- 389. (24) Michalke, B. Electrophoresis 2005, 26, 1584-1597. (25) Alvarez-Llamas, G.; Fernandez de laCampa, M. d. R.; Sanz-Medel, A. TrAC, Trends in Analytical Chemistry 2005, 24, 28-36. (26) Richardson, D. D.; Kannamkumarath, S. S.; Wuilloud, R. G.; Caruso, J. A. Analytical Chemistry 2004, 76, 7137-7142. (27) Thompson, J. J.; Houk, R. S. Analytical Chemistry 1986, 58, 2541-2548. (28) Wang, T. Journal of Liquid Chromatography & Related Technologies 2007, 30, 807-831. (29) Butler, O. T.; Cook, J. M.; Harrington, C. F.; Hill, S. J.; Rieuwerts, J.; Miles, D. L. Journal of Analytical Atomic Spectrometry 2007, 22, 187-221. (30) Montes-Bayon, M.; DeNicola, K.; Caruso, J. A. Journal of Chromatography, A 2003, 1000, 457-476. (31) Sutton, K. L.; Caruso, J. A. Journal of Chromatography, A 1999, 856, 243-258. (32) Richardson, D. D.; Sadi, B. B. M.; Caruso, J. A. Journal of Analytical Atomic Spectrometry 2006, 21, 396-403. (33) Van Loon, J. C.; Alcock, L. R.; Pinchin, W. H.; French, J. B. Spectroscopy Letters 1986, 19, 1125-1135. (34) Chong, N. S.; Houk, R. S. Applied Spectroscopy 1987, 41, 66-74. (35) Wuilloud, J. C. A.; Wuilloud, R. G.; Vonderheide, A. P.; Caruso, J. A. Spectrochimica Acta, Part B: Atomic Spectroscopy 2004, 59B, 755-792. (36) Bouyssiere, B.; Szpunar, J.; Lobinski, R. Spectrochimica Acta, Part B: Atomic Spectroscopy 2002, 57B, 805-828. (37) Richardson, D. D., Caruso, J.A. Analytical and Bioanalytical Chemistry 2007, Online First, March 14, 2007.

CHAPTER 2

Hydride Generation Interface for Speciation Analysis Coupling Capillary Electrophoresis to Inductively Coupled Plasma Mass Spectrometry

2.1 ABSTRACT

A novel hydride generation (HG) interface for coupling capillary electrophoresis

(CE) with inductively coupled plasma mass spectrometry (ICPMS) is presented in this work. The CE-HG-ICPMS interface was applied to the separation and quantitation of common arsenic species. Lack of a commercially available HG interface for CE-ICPMS led to a three concentric tube alleviating the back pressure commonly observed in CE-

HG-ICPMS. Due to the high sensitivity and element specific detection of ICPMS, quantitative analysis of As(III), As(V), monomethylarsonic acid, and dimethylarsinic acid was achieved. Optimization of CE separation conditions resulted in the use of 20 mmol

L-1 sodium borate with 2% osmotic flow modifier (pH = 9.0) and -20 kV applied potential for baseline resolution of each arsenic species in less than 8 minutes. Hydride generation conditions were optimized through multiple electrophoretic separation analyses: with 5% HCl and 3% NaBH4 (in 0.2% NaOH) were found to be the optimum conditions. After completion of system optimization, detection limits obtained for the arsenic species were less than 40 ng L-1 with electromigration time precision less than 1% within a total analysis time of 9.0 min. The interface was used for speciation analysis of arsenic in river and tap water samples.

Keywords: Arsenic speciation, Hydride Generation, Interface, Capillary Electrophoresis, ICPMS

2.2 INTRODUCTION

Elemental speciation is defined as the determination of the distribution of a specific element in different chemical species in a sample1-4. Information obtained from speciation analysis of trace elements allows better understanding of the chemical/biochemical processes, environmental availability, and toxicological risks associated with different species1, 5. However, speciation analysis can be a difficult task considering the various steps involved in finding the distribution of metal and non-metal species without altering the original chemical form. Speciation analyses can be affected by complex sample matrices, low natural occurrence, and the usually extensive sample preparation1-3. Sample preparation methods such as solid phase microextraction (SPME)6 and solid phase extraction (SPE)7 have been commonly used to overcome some of these difficulties. To overcome the problems associated with complex sample matrices, improved sample collection, extraction, and purification techniques can be used. Sample introduction systems such as electrothermal vaporization (ETV)8 , laser ablation (LA)9, and hydride generation (HG)10-14 have been used to compensate for the low natural occurrence of species studied. These methods have resulted in improved detection limits due to their higher sample transport efficiency, selective sample preconcentration, and better separation from complex sample matrices.

Hydride generation (HG) has been recognized for more than 35 years as a valuable sample derivatization technique for the analysis of trace elements such as As,

Se, Sn, Sb, Te, Bi, and Ge, whose hydrides are readily volatile. Typical HG techniques include metal-acid reduction, thermochemical generation, electrochemical generation, photo-induced generation, and sodium borohydride reduction 12, 13, 15, 16. Reduction of an

acid with aqueous sodium borohydride is the most commonly used technique due to its ability to reduce multiple hydride forming elements and for the production of volatile analyte products from complex matrices 12, 13. The use of HG as a sample introduction method with inductively coupled plasma mass spectrometric detection (ICPMS) has improved the detection limits by a factor of ten or more. Due to the improved analytical performance that accompanies this technique, it has been successfully applied with separation methods such as high performance liquid chromatography (HPLC)17or capillary electrophoresis (CE)18 coupled to element specific detectors such as atomic fluorescence spectroscopy (AFS)18 and ICPMS19-21 (Table 2.1).

Table 2.1- Summary of As(III), As(V), MMA and DMA detection limits by various analysis techniques.

Detection Limit Analysis Technique Reference (μg L-1 )

HPLC-ICP-MS 1.0 – 3.0 27

HPLC-HG-ICP-MS 0.011 – 0.051 15

CE-ICP-MS 1.0-2.0 26

CE-HG-AFS 9.0 – 18.0 18

CE-HG-ICP-MS 0.03 – 0.04 this study

As a result of its ease of usage, HPLC is reported to be the most popular analytical separation technique used in conjunction with hydride generation for elemental speciation

17, 22-24. Although the HG technique is relatively easy to adapt for HPLC separations, it is also susceptible to various problems associated with the HG interface. Mester et. al. 22 reported an improvement in detection limit of one order of magnitude with an HPLC-

HG-AFS system using an ultrasonic nebulizer (USN). In this work, the use of several

homemade gas liquid separators was also attempted, however higher noise was observed due to pulsation caused by the hydride generation reactions 22. In their review of hydride generation techniques, Story and Caruso reported a decrease in detection limits of one to three orders of magnitude for hydride forming elements compared to conventional nebulization techniques25.

CE is the other separation technique that is effectively coupled to element specific detectors. However, little is available in literature for the coupling of CE to element specific detectors with a hydride generation interface. Recently, Yin et. al. 18 proposed a novel CE-HG interface for arsenic speciation with AFS, which resulted in improved detection limits compared to conventional CE-ICPMS. Magnuson et. al. 19, 20 reported two similar CE-HG-ICPMS interfaces for selenium and arsenic speciation. Both interface designs utilized multiple peristaltic pumps, which isolated the CE from the HG system in order to overcome the back-pressure and capillary suction induced from the excess hydrogen generated from the acid-borohydride reaction 19,20. Despite the improvement in sensitivity that HG provides to element-specific detectors, the lack of a simple HG interface has slowed the application of CE-HG-ICPMS for speciation analyses.

In this work a simple CE-HG interface coupled to ICPMS is proposed and successfully applied for arsenic speciation in tap and river water samples. A detailed description of the HG interface design is given. CE separation and HG conditions are carefully optimized to obtain the best resolution and signal to noise ratio. Analytical figures of merit for each of the four species studied, As(III), arsenate (As(V)), monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA) are presented.

2.3 EXPERIMENTAL

2.3.1 Reagents

-1 • A 20 mmol L sodium tetraborate (Na2B4B O7 10 H2O) solution (Fisher Scientific, Fair

Lawn, NJ, USA) with 2% CIA-Pak OFM Anion Bt (Waters, Milford, MA, USA) was used as the electrolyte buffer. The buffer was prepared fresh from the stock solution before starting the experiment. A 0.1 mol L-1 NaOH (Fisher Scientific, Fair Lawn, NJ,

USA) solution was used to adjust the pH of the electrolyte buffer solution. Analytical reagent grade HCl (12 mol L-1 ) (J.T. Baker, Phillipsburg, NJ, USA) and sodium borohydride (Fluka, Buchs, Switzerland) were used for preparing hydride generation solutions. The optimum hydride generation conditions required 5% HCl and 3% sodium borohydride (0.2 % NaOH); these were prepared by diluting/dissolving calculated quantities of the reagent in doubly deionized (DDI) water (Table 2.2).

The reagents utilized throughout this experiment were of analytical grade and prepared fresh daily through dilution of stock standards with DDI water. All water was prepared by passing through a NanoPure (18 M.cm) treatment system (Barnstead,

Boston, MA, USA).

Individual arsenic stock solutions of 1000 g mL-1 Arsenic were kindly provided by U.S. Food and Drug Administration. Inorganic arsenic standards, As2O3 in 2% (v/v)

HCl and H3AsO441/2 H2O in 2% (v/v) HNO3 were purchased from Spex Industries

(Metuchen, NJ, USA) whereas dimethylarsinic acid (Me2AsO(OH), DMA) and disodium methylarsenate (MeAsO(ONa)2, MMA) were from Chem Service (West Chester, PA,

USA). The stock solutions were stored at 4 oC and further standards of lower concentrations were prepared by serial dilution of the stock solution with DDI water.

Table 2.2- CE-HG-ICPMS instrumental parameters.

ICP-MS parameters

Forward power 1400 W (with shielded torch)

Plasma gas flow rate 15.6 L min-1

Auxiliary gas flow rate 1.0 L min-1

Make up gas flow rate 0.66 L min-1

Carrier gas flow rate 0.1 L min-1

Nebulizer None

Spray chamber Cyclonic ( 0 C)

Sampling Depth 6 mm

Sampling and Skimmer Cones Nickel

Dwell time 0.1 s

Isotopes monitored (m/z) 75 (As+), 77 (ArCl+, Se+)

Octopole Reaction System not used in this study

CE and Hydride Generation operating conditions

CE instrument Waters Quanta 4000 Capillary Ion Analyzer

Power supply -20 kV

Injection 30 s electromigration: -15 kV

Capillary i.d. 75 m; o.d. 365 m; 75 cm long

Temperature 25 °C

Electrolyte solution 20 mmol L-1 Sodium Borate 2 % OFM, pH = 9.0

Hydride Generation Conditions 5% HCl and 3% NaBH4 (in 0.2% NaOH)

2.3.2 Instrumentation

An Agilent 7500c ICPMS (Agilent Technologies, Tokyo, Japan) equipped with shielded torch technology was used for the element specific detection of 75As. Signals at m/z 75 and 77 were both monitored in order to study the chlorine interference on the arsenic signal (40Ar35Cl+ and 40Ar37Cl+). No detectable chlorine interference was observed during the separation. The octopole reaction cell was not used in this study. The instrumental parameters are presented in Table 2.2.

CE separations were performed on a Waters Quanta 4000 capillary ion analysis system (Waters Corporation, Milford, MA, USA). CE operating conditions are presented in Table 2.2. A 75 cm fused silica capillary (75 m i.d., 365 m o.d.) purchased from

Polymicro Technologies (Phoenix, AZ, USA) was used for the separation. The capillary was conditioned through purging with 0.1 mol L-1 NaOH solution for 10 min followed by washing with DDI water for 10 min. Finally the electrolyte buffer solution was passed through the capillary for a minimum of 30 min before starting the electrophoretic separation.

2.3.3 Interface Design

The interface used in this experiment was based upon a three concentric tube design illustrated in Figure 2.1. The interface components were made of PEEK finger tight fittings, three-way tee, and four-way tee (Upchurch Scientific, Oak Harbor, WA, USA).

The design consists of two tees, one four way followed by a three way union. In the four way tee union, a platinum electrode connected at one of the side arms served as a ground connection and its opposite end was connected to a Teflon tubing 30.0 cm in length for

the purpose of introducing hydrochloric acid. The other end of the Teflon tubing was connected to a Tygon tube (3.0 mm o.d., 1.02 mm i.d.) attached to a Gilson Miniplus 3

(Gilson, Villiers Le Bel, France) peristaltic pump. The flow rate through this tube was calibrated at different rpm values of the peristaltic pump. A flow rate of 0.285 mL min-1 for both acid and reducing agent was found to be the optimum for the introduction into the HG system. The remaining two ends of the four way tee were used for the introduction of the capillary into the interface. PEEK tubing (0.50 mm i.d., 1.6 mm o.d.) was used as a sleeve around the CE capillary allowing an air tight seal during its insertion into the four way tee union. The final connection to the four way tee consisted of a 20 cm long Teflon tube (1.6 mm o.d., 0.55 mm i.d.). This Teflon tube served as the innermost tube in the interface design and allowed HCl sheath flow around the CE capillary providing a constant current during the eletrophoretic separation. This Teflon tube was then passed through the three way tee as shown in the Figure 2.1.

Figure 2.1- Computer aided drawing (CAD) of CE-HG-ICPMS interface.

The length of the Teflon tube between the two tee connections was 6.0 cm. The side arm of the three-way tee was connected to a Teflon tubing 30.0 cm in length for the purpose of introducing the NaBH4 flow. The remainder of the connection used was similar to the one for introducing HCl in the four way tee. The flow of NaBH4 through this tubing was found to be lower compared to HCl. This difference is due to slight differences in the tubing internal diameters and the presence of bubbles generated in a fresh solution of

NaBH4. The final connection to the three way tee consisted of a 15.0 cm glass tube (5.0 mm o.d., 3.0 mm i.d.) which concentrically covered the internal Teflon tube allowing sheath flow of the sodium borohydride around the internal Teflon tube carrying HCl. The final stage of the interface had a 9.0 cm glass tube (10.5 mm o.d., 7.5 mm i.d.) placed 6.0 cm from the three way tee and consisted of a side arm (1.0 cm) for the introduction of argon carrier gas spaced 2.0 cm from the beginning of the tube. This tube was attached to the internal glass tube through the use of a rubber fitting and Teflon tape to ensure a proper seal and complete the interface design. Figure 2.1, drawn to scale, shows the complete design of this interface.

2.3.4 Coupling the Hydride Generation Interface with ICPMS

The experimental setup used for coupling the interface to ICPMS is shown in Figure 2.2.

A cyclonic spray chamber was used as a gas-liquid separator for the HG interface. In order to decrease the dead volume in the gas-liquid separator, the spray chamber was filled with glass beads. Two separate flows of argon gas were used to carry the hydrides formed in the interface to the ICPMS instrument. Initial experimental observations of the interface coupled with ICPMS showed high back-pressure due to the hydrogen gas generated during the reaction. In order to overcome the back pressure in the HG interface,

Stainless Steel Transfer Tube Argon Make-Up Gas

Tygon Tubing Torch Tygon Tubing

Cold Finger (0 ºC)

Cyclonic Spray Chamber (0 oC)

Glass Beads Waste Figure 2.2- CE-HG interface transfer line utilized for removal of hydride condensation and more efficient analyte transport to ICP-MS.

the carrier and make-up gas flow rates were first optimized through continuous

introduction of 10 g L-1 As(III) in 5% (v/v) HCl solution followed by optimization of

the interface parameters. Optimum values for the carrier and make-up gases were found

to be 0.10 L min-1 and 0.66 L min-1 respectively (Table 2.2). Thus, a low carrier gas flow

of 0.10 L min-1 was used to carry the hydrides formed during the derivatization reaction.

Higher carrier gas flow decreased the dispersion of the peaks but at the same time

increased the back-pressure in the gas-liquid separator. On the other hand, lower carrier

gas flow rates increased the dispersion of the transient signals and thereby affected peak

separation. A stainless steel transfer tube, which was passed through the central channel

of the torch, along with a glass make-up gas connector surrounding it up to the beginning

of the torch was an integral part in connecting the interface to the ICPMS (Figure 2.2).

The stainless steel transfer tube was grounded securely to avoid rf problems in lighting the plasma, however this design was affected by the condensation of water in the transfer line due to the lack of a proper system to remove the moisture. Efforts to remove condensation led to the use of a cold finger between the gas-liquid separator and the metal transfer tube that was used for the introduction of the hydrides into the plasma.

Thus, the transfer line consisted of two pieces of Tygon tubing, one running from the cyclonic spray chamber to a cold finger and the second running from the cold finger to a stainless steel transfer tube (Figure 2.2). Finally, improved hydride transport resulting from the removal of condensation was achieved through cooling both the cyclonic spray chamber and cold finger in an ice water bath. The use of a small gas-liquid separator with minimum dead volume allowed the use of lower flow for both HCl and NaBH4, which in turn lowered the production of the hydrogen gas. Having lower hydrogen gas generation during the derivatization reaction aided in a more efficient stabilization of the plasma.

Also, the use of lower carrier gas flow not only decreased the back pressure but improved the signal by lowering the dilution factor. Through the reduction in backpressure and removal of condensation, optimum CE-HG interface conditions were achieved and applied for all additional experiments.

2.4 RESULTS AND DISCUSSION

2.4.1 Optimization of Hydride Generation Conditions

The optimization of the HG conditions consisted of determining the optimum HCl and

NaBH4 concentrations for maximum formation of hydride species without affecting the normal instrumental run parameters. The use of high NaBH4 concentrations is typically limited by plasma extinguishing due to the excess formation of hydrogen during the HG

reaction. Due to the difference in the instrumental conditions for continuous sample introduction and transient injection all the optimizations were performed by injecting the arsenic standards in the CE-HG-ICPMS system. The optimization of HCl consisted of multiple CE separations of the standard arsenic mixture with 1%, 2%, 4%, and 5% (v/v)

HCl reacted with 2% NaBH4 (in 0.2% NaOH). Figure 2.3 shows resulting peak areas of each arsenic species plotted versus HCl concentration with 5% HCl established as the optimum concentration. The plot shows a consistent increase in peak area for each arsenic species over concentrations of 1, 2, and 4% HCl (Figure 2.3). Minimal peak area increases were seen for MMA and DMA between 4 and 5% HCl, however significant increases were observed for As(III) and As(V) resulting in 5% HCl as the optimum concentration. Once the optimum concentration of HCl (5%) was obtained, optimization of NaBH4 was performed with multiple CE separations of the standard arsenic mixture over concentrations of 1, 2, and 3% NaBH4 (in 0.2% NaOH). Plots obtained for the resulting peak areas of each arsenic species versus the concentration of NaBH4 showed a consistent increase in peak area for As(III), As(V), and DMA with 3% chosen as the optimum concentration (Figure 2.3). All hydride generation optimization trials utilized a

Gilson Minipuls 3 peristaltic pump with a flow rate of 0.285 mL min-1 each for both HCl

-1 and NaBH4. Flow rate values higher than 0.285 mL min produced a significant backpressure, whereas lower flow rates lead to suction at the interface, which often resulted in loss of current. Therefore, a mobile phase flow rate of 0.285 mL min-1 was used for the remaining experiments. In previous publications involving the use of HG

19,20 interface for CE-ICPMS systems , a higher flow was used for both HCl and NaBH4

250000 As (III) A

200000

DMA

150000

As (V) 100000 Peak Area

50000 MMA

0 1% 2% 3% 4% 5% HCl Concentration

250000 As (III)

200000 B

150000 DMA

As (V) 100000 Peak Area

50000 MMA

0 1% 2% 3% NaBH Concentration 4

Figure 2.3- Optimization of hydrochloric acid (A) and sodium borohydride (B) for standard arsenic mixture based upon peak area during CE separation. Instrumental parameters are presented in Table 2.2.

leading to a major dilution of the transient signals generated in the CE separation and significantly lowering the sensitivity. On the other hand, the use of a lower flow rate for the HG reagents in this work was found to improve the signal to noise ratio. Additionally, the low flow rate of the HG reagents alleviated the backpressure by diminishing the excess H2 gas in the CE-HG-ICPMS experiments performed. The reduction of the H2 gas produced not only reduced the noise, but also increased the signal by lowering the dilution of the hydrides formed.

2.4.2 Arsenic Species Separation by Capillary Electrophoresis

The electrophoretic buffer used in this work has previously been reported for the separation of four anionic arsenic species; As(III), As(V), MMA, and DMA by Van

Holderbeck et. al 26. This work utilized co-electroosmotic flow as the mode of separation through the use of an osmotic flow modifier (OFM) for the analysis of the previously mentioned arsenic species by CE-ICPMS 26. It was also reported that 20 mmol L-1 sodium borate buffer over an optimum buffer pH range of 8-10, with 9.3 the optimum value, allowed complete baseline CE separation of these four anionic arsenic species 26.

The current experiment confirmed the previously reported CE separation conditions after optimization experiments with 20 mmol L-1 sodium borate buffer at pH 9.0 utilized for the CE separation of As(III), As(V), MMA, and DMA (Table 2.2). The charge on these different arsenic species over various buffer pH values were calculated from the

26 previously reported pKa values . At pH 9.0, all the arsenic species are negatively charged and therefore the maximum resolution is obtained. The resolution achieved was sufficient to compensate for the dispersion of the peaks due to the dead volume in the CE-HG

interface and the transfer line from the CE-HG interface to the ICPMS instrument

(Figure 2.2).

75As MMA 1500

Response) (Counts s As (V) As (III) -1) -1

1000 DMA

500 Response (Counts s

0 02 468 10 Time (min)

Figure 2.4- Separation of standard arsenic mixture (500 μg L -1) under optimum CE and HG conditions.

Both hydrodynamic and electrostatic injections of the samples were performed to obtain the maximum sample transfer into the capillary without affecting the resolution of the peaks. Electrostatic injection was found to be better when compared with hydrodynamic injection, which seemed to be affected more by the backpressure resulting in an unreliable sample injection. An injection time of 30 seconds at -20 kV gave the best results for the given capillary dimensions (Table 2.2, Figure 2.4).

2.4.3 Analytical Performance of the CE-HG-ICPMS

The detection limits (3) based on peak area for the analysis of As(III), As(V), MMA, and DMA by CE-HG-ICPMS were found to be 35, 35, 27, and 39 ng L-1 respectively.

The detection limits of the four As species obtained by this novel CE-HG-ICPMS system are comparable to those previously reported for CE-HG-ICPMS20. The complexity of the earlier interfaces was overcome in this work through the application of the concentric tube interface. The use of reduced reagent flow rates allowed lower consumption of chemicals and decreased the generation of hydrogen gas resulting in higher hydride transport efficiency and plasma stability. The precision for repeated injections of a 100

μg L-1 standard mixture were within the range of 0.3-0.9% for migration times and 3-9% for peak areas.

2.4.4 Speciation Analysis in Water Samples

Demonstration of the application of the CE-HG-ICPMS interface was accomplished through the analysis of tap and Little Miami River (Ohio) water samples. Little Miami

River water samples were collected in 1000 mL borosilicate glass bottles. Immediately after sampling the samples were analyzed for arsenic. None of the samples analyzed showed detectable amounts of arsenic. Lack of any detectable arsenic species in the samples led to spiking of both the tap and river water samples with a 100 μg L-1 mixture of As(III), As(V), MMA, and DMA standards (Figure 2.5). When spiked samples were stored at 4 0C, conversion between the various arsenic species over a period of time was observed. In the case of tap water samples there was nearly 80% conversion from As(III) to As(V)

75As

120 As (III) MMA 100 ) -1 DMA

80 As (V)

Response (Counts s (Counts s Response (Counts 40

02468 Time (min) 75As 70 MMA DMA

60 As (V) As (III) ) -1

Response (Counts s (Counts Response 30

02468 Time (min)

Figure 2.5- Arsenic mixture (100 μg L -1 ) spiked (A) tap water sample and (B) Miami River water sample.

whereas in the Little Miami River water samples no conversion was observed over the same period of time. The remaining two arsenic species MMA and DMA were found to be the same in both samples. This conversion can be attributed to matrix differences between the two samples. In the river water samples the matrix stabilized the arsenic species and prevented them from converting. In the case of tap water sample (Figure

2.5), base line resolution was not achieved between As(V) and MMA. Unfortunately, the presence of various matrix components is the most likely cause in the lack of complete baseline separation between these two species. However, this lack of resolution could be effectively solved by changing the electrophoretic separation conditions while the generation of hydrides is assured with the proposed HG system. Species oxidation state changes are most likely due to oxidizing cations. However, this would naturally happen in drinking water as As(III) was introduced.

2.5 CONCLUSION

In this work an HG interface was designed and successfully coupled to CE-

ICPMS for the analysis of As(III), As(V), MMA, and DMA species. Coupling CE with

ICPMS using the HG interface was demonstrated as an efficient separation technique with short analysis time while considerably increasing the efficiency of analyte introduction into the plasma resulting in improved signal to noise ratio. Electrophoretic separation of the four species was achieved in less than 9.0 min with detection limits less than 40 ng L-1 for each species. This novel interface design, when compared with previous HG interfaces, simplified the coupling process, while at the same time problems such as high backpressure associated with the previous interfaces were largely overcome.

Application of the novel interface to the analysis of arsenic species in real sample

matrices demonstrated the proposed HG interface to be a capable technique for the speciation analysis by CE-HG-ICP-MS.

2.6 ACKNOWLEDGMENTS

I would like to give special thanks to Gregory J. De Nicola for providing the CAD drawing of the interface. Also I would like to thank Agilent Technologies for continuing support of my work. We also acknowledge NIEHS grant #ES04908 for partial funding of this research.

2.7 REFERENCES

(1) Caruso, J. A.; Klaue, B.; Michalke, B.; Rocke, D. M. Ecotoxicology and Environmental Safety 2003, 56, 32-44. (2) Caruso, J. A.; Montes-Bayon, M. Ecotoxicology and Environmental Safety 2003, 56, 148-163. (3) Caruso, J. A. Elemental speciation new approaches for trace element analysis: By J.A. Caruso, K.L. Sutton and K.L. Ackley - eds, 2002. (4) Bettmer, J. Analytical and Bioanalytical Chemistry 2002, 372, 33-34. (5) Kannamkumarath, S. S.; Wrobel, K.; Wrobel, K.; B'Hymer, C.; Caruso, J. A. Journal of Chromatography, A 2002, 975, 245-266. (6) Wrobel, K.; Kannamkumarath, S.; Wrobel, K.; Caruso, J. A. Green Chemistry 2003, 5, 250-259. (7) Wrobel, K.; Wrobel, K.; Kannamkumarath, S. S.; Caruso, J. A. Analytical and Bioanalytical Chemistry 2003, 377, 670-674. (8) Bettinelli, M.; Spezia, S.; Terni, C.; Ronchi, A.; Balducci, C.; Minoia, C. Rapid Communications in Mass Spectrometry 2002, 16, 579-584. (9) Heinrich, C. A.; Pettke, T.; Halter, W. E.; Aigner-Torres, M.; Audetat, A.; Guenther, D.; Hattendorf, B.; Bleiner, D.; Guillong, M.; Horn, I. Geochimica et Cosmochimica Acta 2003, 67, 3473-3497. (10) Chen, C.-S.; Jiang, S.-J. Spectrochimica Acta, Part B: Atomic Spectroscopy 1996, 51B, 1813-1821. (11) Mester, Z.; Sturgeon, R. E. Journal of Analytical Atomic Spectrometry 2001, 16, 470-474. (12) Sturgeon, R. E.; Mester, Z. Applied Spectroscopy 2002, 56, 202A-. (13) Howard, A. G. Journal of Analytical Atomic Spectrometry 1997, 12, 267-272. (14) Wolnik, K. A.; Fricke, F. L.; Hahn, M. H.; Caruso, J. A. Analytical Chemistry 1981, 53, 1030-1035. (15) Olson, L. K.; Vela, N. P.; Caruso, J. A. Spectrochimica Acta, Part B: Atomic Spectroscopy 1995, 50B, 355-368. (16) Campbell, A. D. Pure and Applied Chemistry 1992, 64, 227-244. (17) Schmeisser, E.; Goessler, W.; Kienzl, N.; Francesconi, K. A. Analytical Chemistry 2004, 76, 418-423. (18) Yin, X.-B.; Yan, X.-P.; Jiang, Y.; He, X.-W. Analytical Chemistry 2002, 74, 3720-3725. (19) Magnuson, M. L.; Creed, J. T.; Brockhoff, C. A. Analyst (Cambridge, United Kingdom) 1997, 122, 1057-1061. (20) Magnuson, M. L.; Creed, J. T.; Brockhoff, C. A. Journal of Analytical Atomic Spectrometry 1997, 12, 689-695. (21) Magnuson, M. L.; Creed, J. T.; Brockhoff, C. A. Journal of Analytical Atomic Spectrometry 1996, 11, 893-898. (22) Mester, Z.; Woller, A.; Fodor, P. Microchemical Journal 1996, 54, 184-194. (23) Hwang, C.-j.; Jiang, S.-J. Analytica Chimica Acta 1994, 289, 205-213. (24) Yang, H.-J.; Jiang, S.-J. Journal of Analytical Atomic Spectrometry 1995, 10, 963-967.

(25) Story, W. C.; Caruso, J. A. In Preconcentration Techniques for Trace Elements; CRC Press: Boca Raton, FL, 1992. (26) Van Holderbeke, M. Z., Yining; Vanhaecke, Frank; Moens, Luc; Sandra, Pat Journal of Analytical Atomic Spectrometry 1999, 14, 229-234. (27) Thomas, P. S., K Journal of Analytical Atomic Spectrometry 1995, 10, 615-618.

CHAPTER 3

Reversed Phase Ion-Pairing HPLC-ICPMS for Analysis of Organophosphorus Chemical Warfare Agent Degradation Products

Journal of Analytical Atomic Spectrometry, 2006; 21, 396-403 Reproduced by permission of The Royal Society of Chemistry

3.1 ABSTRACT

In this chapter, the separation and analysis of three organophosphorus chemical warfare agent degradation products is described. Ethyl methylphosphonic acid (EMPA, the major hydrolysis product of VX), isopropyl methylphosphonic acid (IMPA, the major hydrolysis product of Sarin (GB)), and methylphosphonic acid (MPA, the final hydrolysis product of both) were the analytes and were separated by reversed phase ion- pairing high performance liquid chromatography (RP-IP-HPLC) with the use of myristyltrimethylammonium bromide as ion-pairing reagent and ammonium acetate/ acetic acid buffer system (pH 4.85). An Agilent 7500ce inductively coupled plasma mass spectrometer (ICPMS) equipped with collision/reaction cell technology was coupled to the chromatographic system for detection of 31P and 47PO+. Historically, ICPMS detection of phosphorous has been limited due to the high first ionization potential (10.5 eV) and presence of severe nitrogen polyatomic interferences (such as 14N16O1H+ and

15N16O+) overlapping its only isotope at m/z = 31. Implementation of an octopole reaction cell with helium as the cell gas allowed for removal of the nitrogen polyatomic interferences and reduction of background signal. Detection limits for EMPA, IMPA, and

MPA were found to be 260, 180, and 140 pg mL-1, respectively, with separation in less than 15 minutes. The developed method was successfully applied to the analysis of spiked environmental water and soil samples.

3.2 INTRODUCTION

Recent increases in terrorist activity and the threat of chemical weapon attacks have lead to the demand of a rapid and reliable method for the analysis of chemical warfare agents (CWA) and their degradation products. As a result of the Chemical

Weapons Conventions (CWC), which banned the production, acquisition, retention, and direct and/or indirect transfer of chemical weapons, destruction of all chemical weapons held in reserve was mandated1, 2. These chemicals, which include nerve and vesicant agents, pose a deadly threat not only to the human population but also to vital aqueous and agricultural resources (Table 3.1)1, 3-6. Based on these facts the development of a sensitive and selective analytical technique for the analysis of CWA and their degradation products is of high importance to ensure homeland security.

Table 3.1- Chemical Warfare Agents and Degradation Products

Chemical Degradation Agent Liquid Chemical Warfare Degradation Product Oral- LD Warfare Agent 50 Degradation Product pKa Human LD (mg kg-1)1 LO Products (mg kg-1)

0.14 2.16

VX EMPA

24 2.24 143-428* Sarin (GB) IMPA

pKa = 2.41 See Above 1 pKa2 = 7.54

MPA VX and Sarin (GB) 1 3 Vapor form LD50 values range from ~0.09-2 mg-min/m (Agent MSDS) *Cerilliant MSDS

Phosphorus containing nerve agents along with their degradation products present difficulties for ultra-trace analysis due to their high polarity, low volatility, and lack of a good chromophore. Direct analysis of CWA degradation products provides an indirect technique for CWA detection. Previous studies have successfully utilized methods such as gas chromatography/mass spectrometry (GC-MS), ion mobility/mass spectrometry

(IMMS), and liquid chromatography/mass spectrometry (LC-MS) for the analysis of organophosphorus containing degradation products with detection limits in the ng mL-1 range4, 5, 7. However, considering the lethal doses as reported in Table 3.1, lower detection limits in the pg mL-1 range are desirable for such nerve agents and their degradation products. To achieve this lower level detection requires a more selective analytical detection technique such as inductively coupled plasma mass spectrometry

(ICPMS).

Elemental speciation analysis by ICPMS allows for high sensitivity, low level detection, and elemental selectivity, making it the ideal for ultra-trace elemental speciation studies8-14. Phosphorous (m/z = 31) analysis by ICPMS until recently was limited due to its high first ionization potential (10.5 eV) and polyatomic interferences, including 14N16O1H+ and 15N16O+ (m/z = 31). Sector-field MS detection with ICP sources do provide a potential resolution enhancement but at the expense of losing part of the beam. For elements with high ionization potentials the throughput is clearly diminished.

Recent developments in collision/reaction cell technology15, 16 have allowed for the analysis of elements prone to isobaric and polyatomic interferences through removal by collisional dissociation (collision energy >> bond energy), chemical reaction, and/or energy discrimination9. Recently Sadi et al.9 applied ICPMS detection to phosphorus

containing herbicides through the use of state of the art collision/reaction cell technology.

Separation of two phosphorous based general purpose herbicides, as well as aminomethylphosphonic acid (AMPA) by reversed phase ion-pairing high performance liquid chromatography (RP-IP-HPLC)17 achieved detection limits of less than 32 pg mL-1

9. In this study reversed phase ion-pairing chromatography was coupled with ICPMS detection for the analysis of three organophosphorus degradation products of Sarin (GB) and VX. A detailed description of the ion-pairing chromatography employed as well as the selection of optimum ion-pairing reagent is provided. Helium collision/reaction cell optimization experiments for the removal of polyatomic interferences through collisional processes and by application of an appropriate energy barrier are also described.

Analytical figures of merit for each species studied, namely, ethyl methylphosphonic acid

(EMPA), isopropyl methylphosphonic acid (IMPA), and methylphosphonic acid (MPA) are presented. Finally, the developed technique was applied to spiked samples of Little

Miami (Ohio) River water, tap water, and soil samples.

3.3 EXPERIMENTAL

3.3.1 Reagents

The three chemical warfare degradation products (ethyl methylphosphonic acid (EMPA), isopropyl methylphosphonic acid (IMPA), and methylphosphonic acid (MPA)) used were obtained from Cerilliant (Austin, TX) as 1 mg mL-1 certified reference materials (CRMs).

CRMs are used as standard analytical solutions for analysis of Schedule 1, 2, or 3 toxic chemicals, their precursors, and/or degradation products as mandated by the CWC for verification1, 5. Stock solutions of 10 mg mL-1 for each degradation product were prepared through dilution in HPLC buffer. Further dilution of these stock solutions in

HPLC buffer as well as preparation of standard mixtures over the range 20-400 ng mL-1 were performed as needed. Instrument tuning was accomplished through the use of a 30 ng mL-1 adenosine 5’-triphosphate (Sigma, St.Louis, MO) corresponding to a phosphorus concentration of 5 ng mL-1 .

The reagents utilized throughout this experiment were of analytical grade prepared fresh daily through dilution of stock standards with DDI water. All water was prepared by passing through a NanoPure (18 M cm ) treatment system (Barnstead,

Boston, MA).

A 50 mmol L-1 ammonium acetate (Fisher Scientific, Fairlawn, NJ) solution with

5 mmol L-1 ion pairing agent and 2% methanol (TEDIA, Fairfield, OH) at pH 4.85 was used as the chromatographic buffer. Ion pairing agents used in these experiments consisted of tetrabutylammonium hydroxide (Aldrich, Milwaukee, WI), myristyltrimethylammonium bromide (Aldrich, Milwaukee, WI), and hexadecyltrimethylammonium bromide (Sigma, St.Louis, MO). The buffer was prepared fresh from stock solution before starting the experiments. Adjustment of the pH was accomplished through addition of glacial acetic acid (Fisher Scientific, Fairlawn, NJ).

Helium gas (Matheson Gas Products, Parisppany, NJ), with a purity of 99.999% was used as the collision/reaction gas throughout all experiments. Gas flow rate was optimized during instrument tuning prior to each experiment and controlled by the mass flow controller provided with the instrument (Figure 3.1)

1E7 5 ng/mL 31P in Buffer

1000000

100000

Blank 10000

CPS (m/z = 31) 1000

100

10 012345 He Flow (mL/min)

100000

PO+

10000 = 47) = m/z

CPS ( Blank

1000

012345 He Flow (mL/min)

Figure 3.1- Helium collision gas flow rate optimization.

Environmental water samples used in this experiment were collected in polypropylene bottles from laboratory tap water and the Little Miami River in Cincinnati,

OH. Both water samples were filtered through 0.20 μm Nalgene nylon/cellulose acetate

(CA) syringe filters (Nalge Nune International Corporation, Rochester, NY) to remove unwanted particulate matter from entering the system. Environmental soil samples were collected from top soil outside of the laboratory and potting soil kindly provided by the greenhouse at the University of Cincinnati. Soil samples were prepared by placing 1.0 g solid material in 5.0 mL DDI water and stirring for 15 minutes. The resulting solution was filtered through 0.20 μm Nalgene nylon/cellulose acetate syringe filters (Nalge Nune

International Corporation, Rochester, NY). All environmental samples were analyzed as blanks and 100 ng mL-1 spiked mixtures (spiked prior to filtration) of ethyl methylphosphonic acid, isopropyl methylphosphonic acid, and methylphosphonic acid.

3.3.2 Instrumentation

3.3.2.1 HPLC Conditions

An Agilent 1100 (Agilent Technologies, Palo Alto, California) high performance liquid chromatograph (HPLC) equipped with a binary pump, autosampler, vacuum degasser, thermostated column compartment, and diode array detector was used for the separation of the three chemical warfare degradation products. A C8 column (Alltima C8,100 Å, 3.2 x 150 mm, 5 μm, Alltech Associates Inc, Deerfield, IL) with a guard column (Alltima C8,

7.5 x 3.0 mm, 5 μm, Alltech Associates Inc, Deerfield, IL) was used for all separation experiments. A detailed description of the HPLC separation conditions is provided in

Table 3.2.

Table 3.2- HPLC-ICPMS Instrumental Parameters.

ICPMS parameters

Forward power 1500 W (with shielded torch)

Plasma gas flow rate 15.6 L min-1

Auxiliary gas flow rate 1.0 L min-1

Carrier gas flow rate 1.20 L min-1

Nebulizer Glass Expansion Microconcentric

Spray chamber 2 C (Scott Double Channel)

Sampling Depth 6 mm

Sampling and Skimmer Cones Nickel

Dwell time 0.1 s

Isotopes monitored (m/z) 31P and 47PO+

Octopole Reaction System He (Flow Optimized Prior to Experiment)

HPLC parameters

Instrument Agilent 1100 HPLC

Flow Rate 0.5 mL min-1

Injection Volume 100 μL

50 mM Ammonium Acetate; 2% Methanol Buffer 5 mM Myristyltrimethylammonium Bromide

pH 4.85

Column Alltima C8 (3.2 x 150 mm) 5 μm

3.3.2.2 Inductively coupled plasma mass spectrometer (ICPMS)

An Agilent 7500ce (Agilent Technologies, Tokyo, Japan) ICPMS equipped with shielded torch and collision/reaction cell technology was used for the element specific detection of

31P and 47 PO+ in this work. The collision/ reaction cell, consisting of an octopole ion guide operated in rf only mode, served for the removal of polyatomic interferences.

Electronic coupling of the ICPMS with the HPLC was accomplished through the use of a remote cable which allowed for simultaneous starting prior to each chromatographic run.

A detailed description of ICPMS operating conditions is provided in Table 3.2.

3.3.2.3 Electrospray mass spectrometry (ESI-MS)

The ESI-MS spectra were acquired in negative ion mode using a Finnigan LTQ (Thermo

Electron Corporation, San Jose, CA) linear ion trap mass spectrometer equipped with an

Ion Max source and Xcalibur (version 1.4 SR1) data software. Parameters were optimized by infusion of an EMPA (10 μg mL-1) standard solution at 3 μL min-1 using a syringe pump. The source parameters were as follows: sheath gas flow of 35 units auxiliary and sweep gas flow of 0 units; spray voltage of 4.00 kV; and capillary temperature of 300 °C. Full scan MS and MS/MS spectra were acquired by infusion of the sample solutions at 3 μL min-1. The collision energy for MS/MS experiments was

41%. Lyophilized peak fractions were dissolved in DDI water (14 mg mL-1) and analyzed following the procedure described above.

3.4 RESULTS AND DISCUSSION

3.4.1 Ion-Pairing HPLC

Due to the charged nature of the compounds of interest, ion-pairing chromatography was investigated as the chromatographic separation technique. The acid dissociation constants for the chemical warfare degradation products are: methylphosphonic acid (MPA) pKa1

2.41, pKa2 7.54; ethyl methylphosphonic acid pKa1 2.16, and isopropyl methylphosphonic acid pKa1 2.24 (Table 1). Based upon the acid dissociation constants a buffer system (acetic acid/ammonium acetate; pKa 4.8) at pH 4.85 was used in the separation experiments. It was hypothesized that the hydrophobicity and difference in effective charges of the different species would allow for separation by the proposed chromatography.

31P

TTAB

TBAH

HAB

0 200 400 600 800 Time (Sec)

Figure 3.2- Separation comparison of three ion-pairing reagents: tetrabutylammonium hydroxide (TBAH), hexadecyltrimethylammonium bromide (HAB), myristyltrimethylammonium bromide (TTAB).

Tetrabutylammonium hydroxide (TBAH) was first investigated as the ion-pairing reagent for the separation of the three species of interest. Baseline resolution was not achieved between the ion-pairs formed with this compound.

Hexadecyltrimethylammonium bromide (HAB) was investigated next as an ion-pairing reagent due to its longer alkyl chain however, baseline resolution was again not achieved

. Myristyltrimethylammonium bromide (TTAB), consisting of an intermediate length alkyl chain, was then investigated as an ion-pairing reagent. A comparison of the separation achieved with each ion-pairing reagent is provided in Figure 3.2.

Myristyltrimethylammonium bromide along with an ammonium acetate/acetic acid buffer

(pH 4.85) and 2% methanol for the mobile phase allowed separation of methylphosphonic acid, ethyl methylphosphonic acid, and isopropyl methylphosphonic acid with the selected column in less than fifteen minutes (Figure 3.3)

600

550

500

450

400

Response (CPS) 350

300

250

200 0 200 400 600 800 Time (sec)

Figure 3.3- Separation of 100 ng mL-1 mixture of MPA, EMPA, and IMPA.

3.4.2 ICPMS Detection

Element specific detection by ICPMS is a popular analytical technique based on the high sensitivity and selectivity offered by this instrument. In this experiment instrument selectivity was vital because of the need for element specific detection of phosphorus

(m/z = 31) and the complex nature of the environmental matrices analyzed. Recently phosphorus analysis by ICPMS has grown in popularity due to the ability to remove nitrogen-based polyatomic interferences to 31P and the ability to ionize phosphorous sufficiently in spite of its high first ionization potential. Other researchers depended upon the formation of PO+ (m/z = 47)18, 19, or the use of high-resolution mass spectrometers to differentiate between the polyatomic interferences and the phosphorus signal at m/z =

3112, 20-22. Monitoring PO+ in these experiments was performed to ensure no loss of 31P signal by oxide formation.

This work consisted of the use of helium collision cell for the removal of

14N16O1H+ and 15N16O+ interferences through a collision/energy discrimination process.

Any fragmentation of the polyatomic interferences would need to overcome the nitrogen- oxygen bond energy by using helium9, 20. After overcoming the polyatomic interferences with collisional dissociation selective ion transmission through adjustment of the pole bias plays a vital role in analyte response. Helium was chosen as the collision gas for all experiments due to its light/non-reactive nature to allow for reduction of the background signal at m/z 31. Optimization of the helium gas flow rate was accomplished through the use of a mass flow control valve and constant introduction of 30 ng mL-1 adenosine 5’- triphosphate (corresponding to 5 ng mL-1 phosphorus) in buffer . Phosphorus response versus helium flow rate was plotted and the flow rate corresponding to optimal signal

with minimal background (buffer signal m/z = 31) was selected (Figure 3.1, Table 3.2).

The gas flow used ranged from 3.5-4.0 mL min-1 helium for all experiments based upon the optimization results.

3.4.3 Analytical Figures of Merit

Calibration curves were prepared through the use of standard mixtures ranging from 20-400 ng mL-1 for the degradation products of interest . All regression coefficients

(r2) values were acceptable with the lowest value being 0.993. Detection limits (3) based on three times the standard deviation of seven replicates of the blank peak areas (IUPAC) for the analysis of MPA, EMPA, and IMPA were found to be 140, 260, and 180 pg mL,-1 respectively. The detection limits for these three species are an improvement of at least one order of magnitude compared with those reported in other analytical techniques

(Table 3.3), although these detection limits were calculated based upon a concentration that would give a signal three times that of the noise.

Table 3.3- Chemical Warfare Degradation Product Detection Limits.

Chemical Warfare Detection Limits Analytical Method Degradation Products ng mL-1 Ion Mobility Mass 560-17005 Spectrometrya

EMPA LC-ESI-TOFb 80-10003

Electrophoresis Microchip IMPA with Contactless 48-8623 Conductivity Detectorc

MPA RP-IP-HPLC-ICPMSd 0.140-0.260* aBased on concentration producing a signal three times that of the noise. b Estimated in SIM mode at concentrations down to 50 ng mL-1 for signal-to-noise ratio of 3:1. c Estimated from signal-to-noise characteristics (S/N = 3) of the response for 150 ng mL-1 mixture. d Based on IUPAC. *This Work

The precision for repeated injections of a 20 ng mL-1 standard mixture was less than 1% for retention times and less than 6% for peak areas. Percent recovery was calculated to evaluate the extraction efficiency for the sample preparation and separation techniques.

These values ranged from 69-86%. The analytical figures of merit are summarized in

Table 3.4.

Table 3.4- Analytical figures of merit based on 20 ng mL-1 mixture.

Chemical Detection RSD (%) Warfare Column RSD (%) Limit Retention Degradation Recovery Peak Area pg mL-1 Time Product MPA 140 86.2 2.75 0.38 EMPA 260 69.2 5.39 0.55 IMPA 180 73.0 5.96 0.65

3.4.4 Complex Samples

To investigate complex sample matrix effects on the method, Little Miami (Ohio)

River water, tap water, top soil, and potting soil samples were analyzed. Samples were treated with the sample preparation procedure described in the Experimental section.

Figures 3.4 and 3.5 show blank and spiked Little Miami River and laboratory tap water samples. An unknown peak around 600 seconds is observed in both samples and overlaps with the EMPA spike resulting in an increased peak area. Sadi et al. reported unknown organophosphorus compounds in raw river water samples, however they were not identified9. Figures 3.6 and 3.7 show both blank and spiked top soil and potting soil samples. The blank chromatograms do not show the presence of any unknown peaks; however, the trend of the spiked top soil sample shows an increase in the EMPA peak compared to the standard sample chromatogram (Figure 3.3).

1600 ?

1400

1200

1000

800 Blank Little Miami River Water

600 Response (CPS)

400

200

0 0 200 400 600 800 1000 Time (sec)

1800

1600

1400 Spiked Little Miami River Water 1200

1000

800

Response (CPS) 600

400

200

0 0 200 400 600 800 1000 Time (sec)

Figure 3.4- Separation of blank and 100 ng mL-1 spiked Little Miami River (Ohio) water.

31P 800 ? Blank Tap Water 700

600

500

400

300 Response (CPS)

200

100

0 0 200 400 600 800 1000 Time (sec)

31P 800 Spiked Tap Water 700

600

500

400

300 Response (CPS)

200

100

0 0 200 400 600 800 1000 Time (sec)

Figure 3.5- Separation of blank and 100 ng mL-1 spiked laboratory tap water.

? 320 300 280 260 240 220 200 180 160 Blank Top Soil 140 120 Response (CPS) Response 100 80 60 40 20 0 0 200 400 600 800 1000 Time (sec)

500

450 Spiked Top Soil 400

350

300

250

200 Response (CPS) 150

100

0 0 200 400 600 800 1000 Time (sec)

Figure 3.6- Separation of blank and 100 ng mL-1 spiked top soil.

550 31P 500

450

400 Blank Potting Soil 350 ?

300

250

200 Response (CPS)

150

100

0 0 200 400 600 800 1000 Time (sec)

550 31 P 500

450 Spiked Potting Soil

400

350

300

250

200 Response (CPS) Response

150

100

0 0 200 400 600 800 1000 Time (sec)

Figure 3.7- Separation of blank and 100 ng mL-1 spiked potting soil.

3.4.5 Characterization Efforts

The unknown peak overlapping EMPA in the standard separation was characterized initially using electrospray ionization mass spectrometry (ESI-MS) and time elapsed hydrolysis experiments. The interfering peak from blank Little Miami River water injections (retention time 600s) was collected multiple times (>15), filtered through a strong cation exchange column to remove the ion pairing agent, and lyophilized. Mass spectrometric analysis of standard EMPA showed a strong molecular ion peak at m/z 123

(data not shown). Analysis of the concentrated river water fractions did not show the presence of EMPA (no m/z 123 peak). Based upon the mass spectrometric data EMPA was determined not to be the unknown species present in water and soil samples.

Time elapsed experiments consisted of spiking DDI water with 10 μg mL-1

EMPA at room temperature in a clear (not UV protected) sample vial resulting in slow hydrolysis to MPA. The sample was then analyzed by the developed method 2.5 hours after spiking (Figure 3.8A). A large peak for EMPA was observed at 660 seconds with a minor peak for MPA seen at 450 seconds. The resulting sample was spiked with 10 μg mL-1 MPA and an increase in peak area was observed at 450 seconds (Figure 3.8B). The shift in retention times for the water spiked samples is attributed to their difference in ionic strength and pH. Data collected from the time elapsed experiments confirms previous predictions that the unknown and interfering species in river water and soil samples was not EMPA due to the lack of hydrolysis to MPA in these complex samples.

31P A) 30000

25000 2000 1800

1600

20000 1400

1200

1000 15000 800 Response (CPS) 600 Response (CPS) 10000 400

200 0 5000 300 330 360 390 420 450 480 Time (sec)

0 0 100 200 300 400 500 600 700 800 900 Time (sec)

B) 31 P 40000

35000

30000

25000

20000

15000 Response (CPS) 10000

5000

0 0 100 200 300 400 500 600 700 800 900 Time (sec)

Figure 3.8- A) Time elapsed analysis of EMPA (10 μg mL-1) in DDI water after 2.5 hours. B) 10 μg mL-1 MPA spike.

7000

31P 6000 O

+ HO P O NH 5000 4 OH 4000

3000

Response (CPS) 2000

1000

0 0 100 200 300 400 500 600 700 800 900 Time (sec) 16000 31P 14000 O

+ 12000 HO P O K

10000 OH

8000

Response (CPS) 6000

4000

2000

0 0 100 200 300 400 500 600 700 800 900 14000 Time (sec) 31P 12000 O

+ 10000 HO P O Na

8000 OH

6000 Response (CPS) Response 4000

2000

0 0 100 200 300 400 500 600 700 800 900 Time (sec)

Figure 3.9- Separation of 1.85 μg mL-1 ammonium phosphate, 5 μg mL-1 potassium phosphate, and 5 μg mL-1 sodium phosphate.

After investigating other phosphorous containing species commonly found in environmental sample matrices, monobasic phosphate (pKa1 2.12, pKa2 7.2, pKa3 12.3) was identified as the interfering species. Ammonium phosphate (SCP Science, Quebec

Canada), potassium phosphate (Fisher Scientific, Fairlawn, NJ), and sodium phosphate

(Fisher Scientific, Fairlawn, NJ) were analyzed by the developed separation method (pH

4.85; Figure 3.9). All three phosphate salts showed retention times of 600 seconds overlapping with the EMPA standard. It is interesting to note that the net charges of

EMPA and monobasic phosphate are nearly identical based upon their pKa1 values of

2.16 and 2.12 respectively. Identical net charges of these species at the buffer pH of 4.85 can account for their overlapping migration times through the ion-pairing separation.

3.5 CONCLUSION

In this work the coupling of ion-pairing reversed phase HPLC with ICPMS equipped with collision/reaction cell allowed for trace analysis of three organophosphorus chemical warfare degradation products: MPA, EMPA, and IMPA.

Ion-pairing chromatography offered the best separation based on interactions of the analyte between the stationary and mobile phases as well as slight charge differences between the species of interest. This method provides a highly sensitive and selective technique with baseline separation of the three species within 15 minutes and detection limits of less than 260 pg mL-1 . Application of the developed method to environmental water and soil samples demonstrated the RP-IP-HPLC-ICPMS technique as high potential for complex sample speciation analysis. Improvements in the chromatographic resolution through investigation of alternative liquid and gas chromatographic separation techniques coupled with atomic mass spectrometric detection are currently underway.

3.6 ACKNOWLEDGMENTS

I would like to thank Bryan M. Gamble of the FDA Forensic Chemistry Center

(Cincinnati, OH) for ESI-MS support. I also acknowledge Agilent Technologies for continued support of our work as well as NIEHS Grant ES04908 (supplement 3P42-

E5004908-15S4) for partial funding of this research.

3.7 REFERENCES

(1) OPCW, Washington D.C., April 29, 1997; United States Bureau of Arms Control and Disarmament Agency (2) Weimaster, J. F.; Beaudry, W. T.; Bossle, P. C.; Ellzy, M. W.; Janes, L. G.; Johnson, D. W.; Lochner, J. M.; Pleva, S. G.; Reeder, J. H.; et al. Journal of Chemical Technology & Biotechnology 1995, 64, 115-128. (3) Liu, Q.; Hu, X.; Xie, J. Analytica Chimica Acta 2004, 512, 93-101. (4) Smith, J. R.; Shih, M. L. Journal of Applied Toxicology 2001, 21, S27-S34. (5) Steiner, W. E.; Clowers, B. H.; Matz, L. M.; Siems, W. F.; Hill, H. H., Jr. Analytical Chemistry 2002, 74, 4343-4352. (6) Wester, R. M.; Tanojo, H.; Maibach, H. I.; Wester, R. C. Toxicology and Applied Pharmacology 2000, 168, 149-152. (7) Black, R. M.; Read, R. W. Journal of Chromatography, A 1998, 794, 233-244. (8) Caruso, J. A.; Klaue, B.; Michalke, B.; Rocke, D. M. Ecotoxicology and Environmental Safety 2003, 56, 32-44. (9) Sadi, B. B. M., Vonderheide, A.P., and Caruso, J.A. Journal of Chromatography, A 2004, In Press. (10) J.A. Caruso, K. L. S. a. K. L. A. Elemental speciation new approaches for trace element analysis: By J.A. Caruso, K.L. Sutton and K.L. Ackley - eds, 2002. (11) Montaser, A. Inductively Coupled Plasma Mass Spectrometry; Wiley-Vch: New York, 1998. (12) Becker, J. S.; Boulyga, S. F.; Pickhardt, C.; Becker, J.; Buddrus, S.; Przybylski, M. Analytical and Bioanalytical Chemistry 2003, 375, 561-566. (13) Wilber, S.; McCurdy, E. Agilent Application Note 2001, 5988-4286EN. (14) Leonhard, P.; Pepelnik, R.; Prange, A.; Yamada, N.; Yamada, T. Journal of Analytical Atomic Spectrometry 2002, 17, 189-196. (15) Tanner, S. D.; Baranov, V. I.; Bandura, D. R. Spectrochimica Acta Part B 2002, 57, 1361-1452. (16) Bandura, D. R.; Baranov, V. I.; Tanner, S. D. Analytical and Bioanalytical Chemistry 2001, 370, 454-470. (17) Bossle, P. C.; Reutter, D. J.; Sarver, E. W. Journal of Chromatography 1987, 407, 399-404. (18) Kudzin, A. H.; Gralak, D. K.; Drabowicz, J.; Luczak, J. Journal of Chromatography A 2002, 947, 129-141. (19) Kudzin, Z. H.; Gralak, D. K.; Andrijewski, G.; Drabowicz, J.; Luczak, J. Journal of Chromatography A 2003, 998, 183-199. (20) Siethoff, C.; Feldmann, I.; Jakubowski, N.; Linscheid, M. Journal of Mass Spectrometry 1999, 34, 421-426. (21) Wind, M.; Feldmann, I.; Jakubowski, N.; Lehmann, W. D. Electrophoresis 2003, 24, 1276-1280. (22) Kozono, S.; Takahashi, S.; Haraguchi, H. Analytical and Bioanalytical Chemistry 2002, 372, 542-548. (23) Wang, J.; Pumera, M.; Collins, G. E.; Mulchandani, A. Analytical Chemistry 2002, 74, 6121-6125.

CHAPTER 4

Derivatization of Organophosphorus Nerve Agent Degradation Products for Gas Chromatography with ICPMS and TOF-MS Detection

Reproduced in part from: Analytical and Bioanalytical Chemistry, March 14th 2007, Online First, Copyright Springer 2007

4.1 ABSTRACT

This chapter describes the separation and detection of seven V (venomous) and G

(German) type organophosphorus nerve agent degradation by gas chromatography with inductively coupled plasma mass spectrometry (GC-ICPMS) is described. The non- volatile alkyl phosphonic acid degradation products of interest included ethyl methylphosphonic acid (EMPA, VX Acid), isopropyl methylphosphonic acid (IMPA, GB

Acid), ethyl hydrogen dimethylamidophosphate sodium salt (EDPA, GA Acid), isobutyl hydrogen methylphosphonate (IBMPA, RVX Acid), and pinacolyl methylphosphonic acid (PMPA), methylphosphonic acid (MPA), cyclohexyl methylphosphonic acid

(CMPA, GF Acid). N-(tert-Butyldimethylsilyl)-N-methyltrifluroacetamide with 1%

TBDMSCl was used to form the volatile TBDMS derivatives of the nerve agent degradation products for separation by GC. Exact mass confirmation for the formation of all seven TBDMS derivatives was obtained by GC- time of flight mass spectrometry

(TOF-MS). The developed method allowed for the separation and detection of all seven

TBDMS derivatives as well as phosphate in less than 10 minutes. Detection limits for the developed method were less than 5 pg with retention time and peak area precision of less than 0.01 and 6%, respectively. The developed method was successfully applied to river water and soil matrices. To date this is the first work describing the analysis of chemical warfare agent (CWA) degradation products by GC-ICPMS.

4.2 INTRODUCTION

The Chemical Weapons Convention (CWC), which began enforcement April 29,

1997, banned the production, acquisition, and direct or indirect transfer of chemical weapons, as well as mandated the destruction of all chemical weapons held in reserve for member states1. The state members of the CWC created the Organization for the

Prohibition of Chemical Weapons (OPCW) to help reach the objectives of the CWC.

Membership as of November 2006 in the OPCW consisted of 180 member states, six signatory states, and nine non-signatory states corresponding to 98% of the global population2. These toxic chemicals, which include schedule 1 blood, choking, blistering, and nerve agents threaten not only the human population but also important food and water resources. According to the OPCW the United States recently met a treaty deadline for the destruction of 50% of their chemical weapons held in reserve.3 Due to the large worldwide reserves as well as the threat of terrorists attacks, development of new analytical techniques for the analysis of chemical warfare agents (CWA) directly or through the trail left by their degradation metabolites is of vital importance to bolster our capabilities to deal with CWA storage/destruction as well as homeland security.

Organophosphorus nerve agent degradation products are nonvolatile, highly polar species with no chromophore for UV detection (Figures 4.1 A&B). Due to the highly specific hydrolysis pathway of the parent CWA, analysis of the less toxic degradation products provides an alternative technique for CWA detection (Tables 4.1 A&B).

Previous studies have successfully utilized analytical methods such as gas chromatography/mass spectrometry (GC-MS)4-10, gas chromatography/ flamephotometric detection (GC-FPD)11, ion mobility/mass spectrometry (IMMS)12, 13, capillary

81 G-Type

O O CH O O 3 CH O P CN H 3 H C P O C CH H C P O H C P O C . 3 3 3 3 N H CH 3 F H3C CH3 F F TAbun (GA) Soman (GD) Cyclosarin (GF) Sarin (GB)

H O H O H O H O 2 Fast 2 Fast 2 Fast 2 Fast

H2O H2O H2O H2O

O CH O O O 3 CH H H 3 + H C P O C CH H C P O C Na O P O CH CH 3 3 H C P O 3 2 3 3 CH OH H C CH OH 3 N 3 3 OH H C CH 3 3 PMPA CMPA (GF Acid) IMPA EDPA (GA Acid)

Slow H O Slow 2 Slow

H2O

H2O H2O H O H2O 2

H2O H2O H O O 2 H C P OH 3 OH MPA

V-Type O

O CH3 H3C P O H C P O CH CH 3 2 3 S CH S 3

N N

VX Russian VX (RVX)

H2O H O H2O Fast H O 2 Fast Fast H O H O 2 H2O 2 H O H O 2 H2O 2

O S O CH H C P O 3 H C P O CH CH 3 H3C P O CH2 CH3 3 2 3 OH OH OH CH 3 EMPTA EMPA (VX Acid) IBMPA (RVX Acid)

Slow Slow

H2O

H O H O 2 2 O H2O H2O

H2O H C P OH 3 OH MPA

Figures 4.1 A&B- Hydrolysis pathway of G and V-Type Nerve Agents.

Table 4.1 A- Chemical properties of G-Type Nerve Agents and Degradation Products

Chemical Agent Liquid Chemical Degradation Degradation

Warfare LD50 Warfare Product pKa Product Oral Agent (mg kg-1)1 Degradation Human LDLO Products (mg kg-1)

O O + Na O P O CH CH O P CN 2 3 57 N pKaacidic=-0.41 N H C CH 3 3 pKaBasic= 7.54 Tabun (GA) EDPA (GA Acid)

O CH O CH3 3 H H H C P O C CH H C P O C CH 3 3 5 3 3 2.17 OH H C CH F H3C CH3 3 3

Soman (GD) PMPA (GD Acid) O O 143-428* H C P O H C P O 3 3 2.41 F 0.35 OH

Cyclosarin (GF) CMPA (GF Acid) O CH O 3 CH H C P O C H 3 3 H C P O C H CH 3 F 3 24 CH 2.24 OH 3 Sarin (GB) IMPA (GB Acid) O H C P OH Common Above 3 pKa = 2.41 OH 1 pKa2= 7.54 MPA electrophoresis/ flame photometric detection (CE-FPD)14, 15, 1D 1H-31P inverse NMR spectroscopy16, 17, and liquid chromatography/mass spectrometry (LC-MS)18 for the analysis of CWA agent degradation products with detection limits in the ng mL-1 range.

Chemical warfare degradation product analysis by inductively coupled plasma mass spectrometry (ICPMS) coupled with ion-pairing reversed phase high performance liquid chromatography (IP-RP-HPLC) has recently been shown as a rapid and reliable speciation technique with detection limits in the pg/mL range19.

A major advantage of element specific detection with ICPMS is the ability to couple this detector with multiple separation techniques including liquid chromatography, capillary electrophoresis, and gas chromatography19-22. Recently, gas chromatography

with ICPMS detection has proven to be a powerful technique for phosphorus speciation studies20-22. Shah et al. recently investigated the use of solid phase microextraction with

GC-ICPMS for the analysis of phosphoric acid triesters in human plasma with detection limits in the ng L-1 range23.

Table 4.1 B- Chemical properties of V-Type Nerve Agents and Degradation Products.

Chemical Warfare Degradation Chemical Agent Degradation Degradation Product Oral Warfare Liquid LD Products Product Human LD 50 LO Agent (mg kg-1)1 pKa (mg kg-1)

O H C P O CH CH 3 2 3 O S H C P O CH CH 0.14 3 2 3 2.16 OH N

EMPA (VX Acid) VX

CH3 O H3C P O CH 143-428* S H C P O 3 CH3 3 0.14 OH 2.25 CH3 N

RVX IBMPA (RVX Acid)

H C P OH pKa = 2.41 Common Above 3 1 OH pKa = 7.54 2 MPA

Gas chromatography for the separation of chemical warfare agent degradation products has been well documented for more than fifteen years.5, 7, 9, 24-30 Due to the non-volatile nature of these moderate to highly polar phosphonic acid degradation products, multiple types of derivatization techniques including pentafluorobenzyl esters, methyl esters, trimethylsilyl, and tert-butyldimethylsilyl esters have been studied for the generation of

more volatile species amenable for GC separation28. Black et al. reviewed derivatization reactions for the analysis of chemical warfare agents and their degradation products28.

Black’s review describes both trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBDMS) derivatives, the most popular derivatives for phosphonic acids28. This continues to state that the TBDMS derivatives are predicted to be the most stable and least sensitive to moisture28. Typically, detection of these CWA degradation product derivatives separated by GC has been accomplished with FPD, NPD, AED, Quad MS, and Ion-Trap-MS.28 To date, the use of GC-ICPMS for the analysis of CWA degradation product derivatives has never been reported in the literature.

In this work the use of GC-ICPMS for the analysis of common degradation products of Soman, Sarin, Tabun, RVX and VX chemical warfare agents is described.

Optimization of temperature and time for the formation TBDMS derivatives of seven organophosphorus nerve agent degradation products is described. Analytical figures of merit for each species studied including ethyl methylphosphonic acid, isopropyl methylphosphonic acid, methylphosphonic acid, cyclohexyl methylphosphonic acid, ethyl hydrogen dimethylamidophosphate sodium salt, isobutyl hydrogen methylphosphonate, and pinacolyl methylphosphonic acid are presented. Finally, the develop method was applied to river water and soil matrices to determine the sensitivity of ICPMS for these organophosphorus nerve agent degradation product derivatives.

4.3 EXPERIMENTAL

4.3.1 Reagents

Seven chemical warfare degradation products were obtained from Cerilliant (Austin, TX) as 1 mg mL-1 certified reference materials (CRMs). CRMs are used as standard analytical

solutions for analysis of Schedule 1, 2, or 3 toxic chemicals, their precursors, and/or degradation products as mandated by the CWC for verification 1. The degradation products utilized for these experiments included ethyl methylphosphonic acid (EMPA,

VX Acid), isopropyl methylphosphonic acid (IMPA, GB Acid), methylphosphonic acid

(MPA), cyclohexyl methylphosphonic acid (CMPA, GF Acid), ethyl hydrogen dimethylamidophosphate sodium salt (EDPA, GA Acid), isobutyl hydrogen methylphosphonate (IBMPA, RVX Acid), and pinacolyl methylphosphonic acid

(PMPA). N-(tert-Butyldimethylsilyl)-N-methyltrifluroacetamide with 1% TBDMSCl

(Sigma-Aldrich, St. Louis, MO) was used as the derivatizing reagent. Derivatization reactions were performed with distilled acetonitrile (Tedia, Cincinnati, OH).

Helium gas (Matheson Gas Products, Parisppany, NJ), with a purity of 99.999% with 1% Xe was used as the GC carrier gas throughout all experiments. Xenon served as the tuning gas for optimization of the GC-ICPMS parameters prior to analysis.

4.3.2 Derivatization

Due to the nonvolatile nature of these CWA degradation products derivatization was required in order to achieve volatile species for separation with gas chromatography.

TBDMS derivatives were formed by creating a 2:1 mixture of acetonitrile with derivatizing agent. Figure 4.2 describes the esterification reaction for the formation of the TBDMS derivatives. Stock solutions of 40 μg mL-1 mixture of each CWA degradation product was created through dilution of the stock degradation products into the acetonitrile/derivatizing agent mixture.

O R1 P O R2 O O 2:1 ACN/TBDMSCl R1 P O R2 H3C Si CH3 80 ºC 45min OH H C CH 3 CH 3 3

H C CH O 3 3 O H C Si O P R1 3 2:1 ACN/TBDMSCl HO P R1 H C CH O 3 3

OH 80 ºC 45min H C Si CH 3 3

H C CH 3 CH 3 3

R1- CH3, ONa* R2- CH2CH3, CH(CH3)2, CH2CH(CH3)2, CH(CH3)C(CH3)3, C6H11 * EDPA replaces –OH with N(CH3)2

Figure 4.2- TBDMS esterification of alkyl phosphonic acids.

Optimization of the derivatization time was based upon peak area of duplicate injections of a 40 μg mL-1 mixture heated at 60 oC for 15, 30, 45, and 60 minutes with 45 minutes chosen as optimum (Figure 4.3A). Temperature optimization was then preformed at room temperature, 40, 60, and 80 ºC for 45 minutes with 80 ºC chosen as the optimum temperature for the derivatization (Figure 4.3B).

A) Derivatization Time

12000000

10000000

EMPA 8000000 IMPA EDPA 6000000 IBMPA PMPA Peak Area 4000000 MPA CMPA

2000000

0 15 30 40 45 60

Time

B) Derivatization Temperature

10000000

9000000

8000000

7000000 EMPA IMPA 6000000 EDPA 5000000 IBMPA PMPA 4000000 Peak Area MPA 3000000 CMPA 2000000

1000000

0 RT 40 60 80

Temperature

Figure 4.3 A&B- Optimization of derivatization time and temperature.

4.3.3 Environmental Samples

To determine the method usefulness in a real world setting investigation into the tolerance of the method for complex environmental sample matrices led to the analysis of river water and soil samples. Typically in a real world setting this method would only be required for monitoring a few of the degradation products for confirmation of proper storage and disposal. Development of a more versatile method capable of separating degradation products from multiple CWA provides future researchers with the flexibility of adjusting the method as desired.

River water samples were collected in polypropylene bottles from the Little

Miami River in Cincinnati Ohio. Initial experiments consisted of drying a 1 mL aliquot of river water under nitrogen. The dried river water was reconstituted in 250 μL of acetonitrile, vortexed, and spiked with a derivative mixture of degradation products to yield a concentration of 20 μg mL-1.The resulting solution was filtered with 0.20 μm nylon-nitrocelluose acetate (CA) filters prior to analysis. Additional experiments consisted of taking 100 μL of river water and spiking with 50 μL aliquots of each 1000

μg mL-1 stock degradation product. The resulting solution was dried and reconstituted with 600 μL acetonitrile and 300 μL derivatizing agent to yield a 55.5 μg mL-1 mixture.

The mixture was heated at 80 ºC for 45 minutes and filtered prior to analysis.

Soil samples were collected from ground outside the laboratory at the University of Cincinnati. Initial experiments consisted of taking 1 g soil and adding 1 mL of distilled deionized water. The resulting slurry was stirred followed by removal of the water extract which was then dried under nitrogen. The dried soil extract was reconstituted in 250 uL of acetonitrile, vortexed and spiked with a derivative mixture of degradation products to

yield a concentration of 20 μg mL-1 . The mixture was then filtered with 0.20 um nylon cellulose syringe filters prior to analysis. Additional experiments for determination of matrix effects on derivatization consisted of taking a 100 μL of soil extract and spiking with 50 μL aliquots of each 1000 μg mL-1 stock degradation product. The spiked sol ution was dried and then reconstituted with 600 μL acetonitrile and 300 μL derivatizing agent to yield a 55.5 μg mL-1 mixture. The mixture was heated at 80 ºC for 45 minutes and filtered prior to analysis.

4.4 INSTRUMENTATION

4.4.1 Gas Chromatography (GC)

GC with ICPMS detection is a highly desirable mode of operation since the gaseous argon plasma detector is optimum for gaseous sample introduction. While liquid sample introduction is the most widely used, the plasma must desolvate the aerosol prior to analyte ionization, requiring time and energy that might have been used to form analyte ions. With gaseous sample introduction 10x lower detection levels are routinely obtained over liquid sample introduction. An Agilent 6890 (Agilent Technologies, Palo Alto,

California) gas chromatograph with helium carrier gas with 1% Xenon was used to separate seven chemical warfare degradation products. Xenon gas allowed for instrument parameter optimization daily prior to analysis. An HP-5 (5% -phenyl-methyl- polylsiloxane) capillary column (30 m, 0.32 mm i.d., 0.25 μm film thickness) was used for all separation experiments. A detailed description of the GC parameters is provided in

Table 4.2.

4.4.2 Inductively Coupled Plasma Mass Spectrometer (ICPMS)

An Agilent 7500ce (Agilent Technologies, Tokyo, Japan) ICPMS equipped with shield torch and collision/reaction cell technology was used for the element specific detection of

31P and 47 PO+ throughout method development. The reason for monitoring 47PO+ is to ensure no loss of 31P signal due to oxide formation, however no significant oxide formation was observed throughout this experiment. Due to the gaseous sample introduction technique that accompanies GC-ICPMS the atmospheric polyatomic interferences (14N16O1H+ and 15N16O+ ) common with LC-ICPMS for 31P are not as abundant, resulting in a lower background signal at m/z = 31. Due to the lower background signal the collision/reaction cell was not needed for reduction of the m/z = 31 background in these experiments. Instrument parameters were optimized daily prior to analysis through optimization of Xe signal at m/z 124. A detailed description of the

ICPMS parameters is provided in Table 4.2.

4.4.3 Gas Chromatography – Time of Flight Mass Spectrometry (GC-TOF-MS)

An Agilent 6890 (Agilent Technologies, Palo Alto, California) gas chromatograph equipped with a Micromass GCT (Waters Corporation, Milford, MA) time of flight mass spectrometer (TOF-MS) was utilized for exact mass confirmation of the TBDMS derivatives. A J&W Scientific (Agilent Technologies, Palo Alto, California) capillary column (D-XLB; 30 m, 0.25 mm i.d., 0.25 μm film thickness) was utilized for all separation experiments. Detector parameters consisted of a source temperature of 180 ºC, electron energy of 70 eV, trap current of 150 μamps, and MCP voltage of 2350. Tuning of the TOF-MS was accomplished through the use of perfluorotributylamine ( FC-43;

Scientific Instrument Services, Ringoes, N.J.).

Table 4.2- Instrumental parameters for GC-ICPMS

ICPMS parameters

Forward power 950 W (with shielded torch)

Plasma gas flow rate 15.6 L min-1

Auxiliary gas flow rate 1.0 L min-1

Carrier gas flow rate 0.90 L min-1

QP Bias -4 eV

Octopole -6 eV

Sampling Depth 6 mm

Sampling and Skimmer Cones Nickel

Dwell time 0.1 s

Isotopes monitored (m/z) 31P and 47PO+

Octopole Reaction System None

GC parameters

Instrument Agilent 6890 GC

Carrier Gas 99.999% He w/ 1% Xe (Constant Pressure 10psi)

Injection Splitless

Purge Time 0.75 min

Injection Volume 1 μL

80 ºC (1 min) 20 ºC/min b 280 ºC Hold (4min) Oven Program

Column HP DB-5 (5% -phenyl-methyl-polylsiloxane)

4.5 RESULTS AND DISCUSSION

4.5.1 GC-ICPMS

Elemental speciation analysis by ICPMS is a powerful analytical technique due to the high sensitivity and selectivity offered by this instrument. Phosphorus analysis by ICPMS provides an ionization source capable of over coming the high first ionization potential

(10.5 eV) of this element as well as state of the art ion optics and octopole interference reduction system.

Previous analysis of organophosphoru s CWA degrad ation products by LC-

ICPMS provided improved sensitivity, howev er lack of sufficient chromatographic resolution resulted in interferences from phosphate in environmental sample matrices19.

Efforts to improve the chromatographic resolu tion have led to th e investigation of gas chromatography followed by 31P element spec ific detection with ICPMS. Due to the nonvolatile nature of the organophosphorus C WA degra dation products formation of the volatile TBDMS ester was required. TBDMS was chose n as the derivatization method of choice due to its stability and tolerance to trac e amo unts of moisture28. Due to the selectivity of ICPMS for 31P, efficiency of derivatization reaction was vital in order to prevent intermediate species formation. Optimum reaction conditions for the formation of the TBDMS derivatives of alkyl phosphonic CWA degradation were determined to be 80

C for 45 minutes (Figure 4.3 A&B). These conditions allowed for the separation of

EMPA (VX Acid), IMPA (GB Acid), EDPA (GA Acid), IBMPA (RVX Acid), PMPA,

MPA, and CMPA (GF Acid) in less than 9 minutes. Figure 4.4 shows the separation and detection of a 5 ng mixture of the seven CWA degradation products. To date this is the

first work utilizing GC-ICPMS for the analysis of TBDMS derivatives of organophosphorus CWA.

5 ng Mixture O H3C P O CH2 CH3 O O CH 3 CH3 31 H C P O Si CH 35000 H C Si CH 3 3 P 3 3 O O CH H CH 3 CH H C P O C 3 3 H C CH3 3 3 CH CH H C Si CH 3 O 3 3 3 H C Si CH 3 3 H C CH 30000 3 CH 3 3 H C CH 3 3 CH3 CH H3C 3 O H C Si O P O CH CH 25000 3 2 3 H C N 3 CH3 H3C CH3 O CH 3 H3C P O O CH 20000 3 H3C Si CH3 O H C CH 3 CH 3 H C P O 3 3 O

15000 CH H C Si CH O 3 3 3 H H C P O C CH 3 3 H C CH 3 CH 3 O H C CH 3

Response (CPS) 3 3 H C Si CH 10000 3 3 CH H3C 3 CH3

5000

0 56789 Time (min)

Figure 4.4- Separation of 5 ng mixture of seven TBDMS derivatives

4.5.2 GC-TOF-MS

Confirmation of the formation of TBDMS derivatives for six of the CWA degradation products of interest was determined by GC-TOF-MS as described in the experimental section. Typically, silyl derivatives (TBDMS and TMS) of alkyl phosphonic acids in positive mode electron impact (EI) ionization results in the formation of a base peak at

+ +28 + m/z 153 corresponding to [M-CnH2n-Me] and [M-CnH2n-Bu] . Loss of [M-CH3] and

+ 28 [M-C4H9] also provides higher mass ions with weak to moderate intensity . Figures 4.5

A-G shows the exact mass confirmation for the formation of TBDMS derivatives of

EMPA (VX Acid), IMPA (GB Acid), IBMPA (RVX Acid), PMPA, MPA, CMPA (GF

Acid), and EDPA (GA Acid) with good correlation to their calculated exact mass. GC-

TOF-MS also allowed identification of doubly derivatized phosphate as the unknown

trace phosphorus peak present at approximately 8 minutes in the standard separation

(Figures 4.4 and 4.6).

TOF MS EI+ 100 153.0129

O H C P OOCHCH CH [M-C H ]+ 3 2 3 4 9 O 181.0459

H C Si CH 3 3

CH H3C 3 CH3 Mass Difference ± 0.0009

% EMPA (VX Acid) Exact Mass – 238.1154

+ [M-CH3 ] – 223.0919 + [M-C4H9] – 181.0450

154.0136

155.0133

120.9858 182.0471 195.0662

136.9950 90.9457 106.9734 156.9933 281.0352 68.0293 53.9960 209.0157 237.1055 257.9796 0 m/z 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200210 220 230 240 250 260 270 2 80 290 300 310

Figure 4.5 A- GC-ToF-MS analysis of EMPA-TBDMS.

TOF MS EI+ 152.9978 100 O H CH3 H3C P O C CH O 3 H C Si CH 3 3 Mass Difference CH + H3C 3 [M-CH3] ± 0.0035 CH3 + [M-C4H9] ± 0.0037

IMPA (GB Acid) % Exact Mass – 252.1311

+ [M-CH3] – 237.1076 153.8540 [M-C H ]+ –195.0606 4 9 + [M-C4H9] + 195.0569 [M-CH3]

237.1041

151.0333

193.0842 154.0146 43.0545 196.0641 238.1138 221.0825 251.1237 79.0568 92.0557 136.0097 179.0339 267.1084 56.0159 97.0073 108.9981 121.0745 165.0583 211.0076 222.0860 0 m/z 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280

Figure 4.5 B- GC-ToF-MS analysis of IMPA-TBDMS.

TOF MS EI+ 152.9977 100 O CH 3 H3C P O O CH 3 H C Si CH 3 3

H C CH 3 3 CH3 Mass Difference ± 0.0061

IBMPA (RVX Acid)

% Exact Mass – 266.1467

+ [M-CH3] – 251.1232 + [M-C4H9] –209.0763

153.8530 120.9825 75.0240 [M-C4H9]+ 195.0567 154.0136 209.0702

155.0140 211.0882 73.0463 151.0339 122.9676 193.0786 77.0101 136.9921 57.0705 78.9930 106.9735 138.9974 168.0381 196.0655 59.0321 90.9406 119.9824 124.9833 99.0459 140.0067 179.0375 223.0920 0 m/z 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220

Figure 4.5 C- GC-ToF-MS analysis of IBMPA-TBDMS.

O CH3 H H C P O C CH 3 3 O H C CH TOF MS EI+ 3 3 153.0039 100 H C Si CH 3 3 Mass Difference H C CH 3 CH 3 3 + [M-CH3] ± 0.0024 + PMPA [M-C4H9] ±0.0002 + Exact Mass – 294.1780 [M-C4H9]

+ 237.1078 [M-CH3] – 279.1545 154.0142 + 120.9850 [M-C4H9] – 237.1076

% 151.0357 75.0286

182.0506

211.0910 73.0494 108.1386 195.0617 155.0166 69.0708

197.0827 122.9720 77.0157 137.0051 136.0118 238.1161 138.0185 197.1273 57.0692 + 106.9785 [M-CH ] 84.0989 179.0324 230.6175 3 55.0557 239.1188 279.1569 90.9420 167.0271 240.1151 267.0959 280.1517 294.1862 0 m/z 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310

Figure 4.5 D- GC-ToF-MS analysis of PMPA-TBDMS.

[M-C H ]+ 4 9 TOF MS EI+ 267.0966 100

O CH CH 3 3 CH H3C P O Si 3

O CH3 CH 3 H C Si CH 3 3 Mass Difference

H C CH + 3 CH 3 [M] ±0.0000 3 + [M-C4H9] ±0.0000 MPA % Exact Mass – 309.1477

+ [M-C4H9] – 267.0966 268.1053

195.0072 269.0993 [M]+ 212.0455 225.0606 309.1477 210.0343 226.0641 134.0679 180.9957 196.0104 213.0384 251.0781 270.1010 310.1440 287.0182 164.9566 177.0070 235.0462 0 m/z 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 Figure 4.5 E- GC-ToF-MS analysis of MPA-TBDMS

97 O

H 3 C P O

O TOF MS EI+ 152.9803 100 H C Si CH 3 3

H C CH 3 CH 3 3

CMPA (GF Acid) 153.8391

Exact Mass – 292.1624 211.0723

+ [M-CH3] – 277.1389 + [M-C H ] – 235.0919 195.0479 4 9 Mass Difference ± 0.0049 75.0187

120.9814 73.0441 + 122.9639 [M-C4H9] 67.0530 155.0078 77.0095 136.9887 235.0870 138.0096 212.0907 82.0793 106.9712 196.0622 124.9822 179.0277 213.0901 236.0954 180.0338 267.0984 177.0513 263.1214 291.1571 309.1533 0 m/z 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310

Figure 4.5.5 F- GC-ToF-MS analysis of CMPA-TBDMS.

TOF MS EI+ 100 68.9955

H C CH 3 3 O H C Si O P O CH CH 3 2 3 H C N 3 CH 3H C CH 3 3

EDAP (GA Acid) 182.0378 Mass Difference ± 0.0002 Exact Mass – 267.1420

+ [M-CH3] – 252.1185 % + [M-C4H9] – 210.0715 + [M-C4H9] 218.9856 210.0713 130.9916

75.0278 138.9984

77.0240 99.9969

118.9933 263.9883 59.0317 184.0402 81.0025 113.9981 207.0514 149.9914 220.0175 168.9873 229.1653 0 m/z 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280

FFigureigure 4.5 G- GC-ToF-MS analysis of EDAP-TBDMS.

TOF MS EI+ 297.0946 100 O CH 3 CH3 HHCHO P O Si CH Phosphate-TBDMS(2x) 3 O CH Exact Mass – 326.1499 3 CH3 H C Si CH + 3 3 [M-CH3] – 311.1265 + CH [M-C H ] – 269.0795 H3C 3 4 9 CH3

+ [M-C4H9]

269.0804

253.0509 % Mass Difference

+ [M-CH3] ± 0.0007 + [M-C4H9] ± 0.0009 298.1074

267.1064 299.1088 254.0531 270.0864 + 263.9819 256.9127 271.0831 [M-CH3] 261.7401 276.0260 295.0955 300.1113 311.1272 251.0752 281.0793 291.3061 309.1479 272.0909 284.9271 293.1659 313.9766 0 m/z 248 250 252 254 256 258 260 262 264 266 268 270 272 274 276 278 280 282 284 286 288 290 292 294 296 298 300 302 304 306 308 310 312 314 316 318

Figure 4.6- GC-ToF-MS identification of trace doubly derivatized phosphate in standard mix (Figure 4.4).

4.5.3 Analytical Figures of Merit

Calibration curves ranging from 0.156-20 ng (nerve agent derivative injected on column)

were prepared through serial dilution of a standard mixtures of EMPA, IMPA, EDPA,

IBMPA, PMPA, MPA, and CMPA TBDMS derivatives. Correlation coefficients (r2)

ranged from 0.998-0.999 and demonstrated excellent linearity for each species analyzed.

Detection limits (3 ) were calculated based upon three times the standard deviation of

seven replicates of blank peak area (IUPAC) ranged from 1.7-5.0 pg for all seven

TBDMS derivatives. The precision based upon seven replicate injections of a 5 ng

mixture of TBDMS derivatives on column was less than 0.01% for retention time and

less than 5.7% for peak area. A summary of the analytical figures of merit is provided in

Table 4.3. Table 4.4 provides a detection limit comparison of LC-ICPMS to the developed GC-ICPMS method19. The improvement in detection limits for the developed

GC-ICPMS are attributed to the lower background associated with this gaseous sample introduction technique compared to conventional liquid introduction (Table 4.4).

Table 4.3- Figures of merit based upon seven replicate injections of a 5ng mixture. Degradation RSD (%) RSD (%) D.L. Derivatives D.L Phosphorus Product r2 Retention Time Peak Area (picograms) (picograms) EMPA 0.999 0.000 2.4 3.4 0.44 IMPA 0.999 0.009 2.8 2.1 0.26 EDPA 0.998 0.008 1.9 1.7 0.20 IBMPA 0.999 0.008 2.3 4.0 0.47 PMPA 0.998 0.007 5.7 4.4 0.42 MPA 0.999 0.007 3.0 5.0 0.52 CMPA 0.998 0.006 5.7 3.8 0.38

Table 4.4- Detection limit comparison of the developed GC-ICPMS method with LC-ICPMS*. Degradation Product Detection Limits Detection Limits HPLC-ICPMS (Picograms)19 GC-ICPMS (Picograms)

EMPA (VX Acid) 26 3.4

IMPA (Sarin) 18 2.1

MPA (Common) 14 5.0

* IUPAC 3

4.5.4 Phosphate and Environmental Samples

Application of the develop method to complex sample matrices consisted of preliminary experiments to determine derivatization, separation, and detection of phosphate-TBDMS in a mixture with the seven degradation products. Figure 4.7 shows the separation of a 30 ng on column mixture of the seven degradation products and phosphate-TBDMS in less

100

than 10 minutes. The ability to derivatize, separate, and detect inorganic phosphate allowed for the application of the developed method to river water and soil matrices.

Investigation of the developed method to river water consisted of the preparation of two separate sample types. First, a standard derivative mixture was spiked into dried river water sample to determine the sensitivity of the developed method to a complex matrix. Figure 4.8A demonstrates the separation and detection of all seven degradation products spiked into the river water matrix. Following the confirmation of minimal matrix effects on derivative response, investigation into derivatizing within the complex

30 ng Mixture w/ Phosphate-TBDMS

31P 1200000 8

1000000 Separation Order 1. EMPA 800000 2. IMPA 3. EDPA 6 4. IBMPA 600000 5. PMPA

onse (CPS) onse 1 6. MPA 2 7. CMPA

Resp 400000 3 4 8. Phosphate

5 200000 7

0 5678910 Time (min)

Figure 4.7- Standard separation of 30 ng mixture with phosphate-TBDMS

matrix was explored. River water extracts were prepared, spiked and derivatized following the procedure described in the experimental section. Figure 4.8B illustrates the

101

separation and detection of the seven degradation products as well as phosphate from the river water in less than 10 minutes.

Application of the developed method to soil samples followed the same procedure as that of the river water samples. Soil extracts were prepared in two separate sample types. First, investigation into the sensitivity of the developed method for the complex soil matrix was accomplished through spiking the soil extract with a standard derivatized mixture as described in the experimental section. Figure 4.9A shows the separation of a

20 ng on column mixture of the seven degradation products. Confirmation of minimal matrix in the spiked sample led to the analysis of matrix effects on the derivatization reaction. Soil extracts were spiked and derivatized following the procedure described in the experimental section. Figure 4.9B show the separation and detection of all seven degradation product and phosphate in less than 10 minutes.

102

20 ng Little Miami River Water Spike

31P 350000 1 300000 2 Separation Order 250000 6 1. EMPA 2. IMPA S 4 3 3. EDPA 200000 4. IBMPA 5. PMPA 150000 6. MPA 7. CMPA Response (CPResponse ) 100000 5 7

50000

0 56789 Time (min)

Derivatized River Water Spike 31 2000000 P 6 8

Separation Order 1500000 1. EMPA 2. IMPA 3. EDPA 4. IBMPA 1000000 1 5. PMPA 4 6. MPA 2 7. CMPA Response (CPS) 8. Phosphate 500000

5 7 3 0 5678910 Time (min)

Figures 4.8 A&B- Spiked and derivatized river water sample.

103

20 ng Soil Extract Spike

31 P 300000 6 1 250000 2 Separation Order 4 1. EMPA 200000 2. IMPA 3 3. EDPA 4. IBMPA 150000 5. PMPA pons P 6. MPA

Res e (C S) 7. CMPA 100000 5 7

50000

0 56789 Time (min)

Derivatized Soil Spike 31P 2000000 6 Separation Order 8 1. EMPA 1500000 2. IMPA 3. EDPA 4. IBMPA 1 5. PMPA 1000000 4 6. MPA 2 7. CMPA 8. Phosphate Response (CPS) Response

500000

5 7 3 0 5678910 Time (min)

Figure 4.9 A&B- Spiked and derivatized soil samples

104

4.6 CONCLUSION

In this work gas chromatography coupled with ICPMS allowed for the separation and detection of seven organophosphorus nerve agent degradation products and phosphate in less than 10 minutes. Through a simple esterification reaction the non-volatile degradation products were converted into their TBDMS derivatives which were ideal for

GC separation. The developed method provides a highly sensitive and selective technique yielding detection limits less than 5 pg with precision of less than 0.01 and 6% for retention time and peak area, respectively. The developed method was successfully applied to complex river water and soil matrices. Future studies currently underway include the investigation of alternative derivatization techniques including TMS, p- bromophenol, and pentafluorbenzyl esters as well as the use of solid phase microextraction (SPME) preconcentration for analysis of these CWA degradation products.

4.7 ACKNOWLEDGMENTS

The authors would like to thank Agilent technologies for their continued support of our research as well as NIEHS Grant ES04908 (supplement 3P42-E5004908-15S4).

105

4.8 REFERENCES

(1) OPCW, Washington D.C., April 29, 1997; United States Bureau of Arms Control and Disarmament Agency (2) OPCW (2006) Weapons. Accessed September 27, 2006, www.opcw.org (3) Ember, L. R. In Chemical and Engineering News; American Chemical Society: Chemical and Engineering News, 2006; Vol. 84, pp 87-89. (4) Tornes, J. A.; Opstad, A. M.; Johnsen, B. A. Science of the Total Environment 2006, 356, 235-246. (5) Barr, J. R.; Driskell, W. J.; Aston, L. S.; Martinez, R. A. Journal of Analytical Toxicology 2004, 28, 372-378. (6) Smith, J. R.; Schlager, J. J. Journal of High Resolution Chromatography 1996, 19, 151-154. (7) Vasilevskii, S. V.; Kireev, A. F.; Rybal'chenko, I. V.; Suvorkin, V. N. Journal of Analytical Chemistry (Translation of Zhurnal Analiticheskoi Khimii) 2002, 57, 491-497. (8) Saradhi, U. V. R. V.; Prabhakar, S.; Reddy, T. J.; Vairamani, M. Journal of Chromatography, A 2006, 1129, 9-13. (9) Gupta, A. K.; Pardasani, D.; Kanaujia, P. K.; Tak, V.; Dubey, D. K. Rapid Communications in Mass Spectrometry 2006, 20, 2115-2119. (10) Lee, H. S. N.; Basheer, C.; Lee, H. K. Journal of Chromatography, A 2006, 1124, 91-96. (11) Logan, T. P.; Allen, E. D.; Way, M. R.; Swift, A. T.; Soni, S.-D.; Koplovitz, I. Toxicology Mechanisms and Methods 2006, 16, 359-363. (12) Steiner, W. E.; Clowers, B. H.; Matz, L. M.; Siems, W. F.; Hill, H. H., Jr. Analytical Chemistry 2002, 74, 4343-4352. (13) Steiner, W. E.; Harden, C. S.; Hong, F.; Klopsch, S. J.; Hill, H. H.; McHugh, V. M. Journal of the American Society for Mass Spectrometry 2006, 17, 241-245. (14) Hooijschuur, E. W. J.; Kientz, C. E.; Brinkman, U. A. T. Journal of Chromatography, A 2001, 928, 187-199. (15) Pumera, M. Journal of Chromatography, A 2006, 1113, 5-13. (16) Meier, U. C. Analytical Chemistry 2004, 76, 392-398. (17) Koskela, H.; Grigoriu, N.; Vanninen, P. Analytical Chemistry 2006, 78, 3715- 3722. (18) Wada, T.; Nagasawa, E.; Hanaoka, S. Applied Organometallic Chemistry 2006, 20, 573-579. (19) Richardson, D. D.; Sadi, B. B. M.; Caruso, J. A. Journal of Analytical Atomic Spectrometry 2006, 21, 396-403. (20) Shah, M.; Meija, J.; Cabovska, B.; Caruso, J. A. Journal of Chromatography, A 2006, 1103, 329-336. (21) Vonderheide, A. P.; Meija, J.; Montes-Bayon, M.; Caruso, J. A. Journal of Analytical Atomic Spectrometry 2003, 18, 1097-1102. (22) Proefrock, D.; Leonhard, P.; Wilbur, S.; Prange, A. Journal of Analytical Atomic Spectrometry 2004, 19, 623-631. (23) Shah, M.; Caruso, J. A. Journal of Separation Science 2005, 28, 1969-1984.

106

(24) Purdon, J. G.; Pagotto, J. G.; Miller, R. K. Journal of Chromatography 1989, 475, 261-272. (25) Black, R. M.; Clarke, R. J.; Read, R. W.; T.J. Reid, M. Journal of Chromatography, A 1994, 662, 301-321. (26) Creasy, W. R.; Rodriguez, A. A.; Stuff, J. R.; Warren, R. W. Journal of Chromatography, A 1995, 709, 333-344. (27) Sng, M. T.; Ng, W. F. Journal of Chromatography, A 1999, 832, 173-182. (28) Black, R. M.; Muir, B. Journal of Chromatography, A 2003, 1000, 253-281. (29) Crenshaw, M. D.; Cummings, D. B. Phosphorus, Sulfur and Silicon and the Related Elements 2004, 179, 1009-1018. (30) Wang, Q.; Xie, J.; Gu, M.; Feng, J.; Ruan, J. Chromatographia 2005, 62, 167- 173.

107

CHAPTER 5

Scre ening Organophosphorus Nerve Agent Degradation Products in Pesticide Mixtures by GC-ICPMS

108

5.1 ABSTRACT

In this chapter, gas chromatography inductively coupled plasma mass spectrometry (GC-ICPMS) was used for the analysis of four organophosphorus nerve agent degradation products in the presence of mixtures of common organophosphorus pesticides. The first degradation products of sarin (isopropyl methylphosphonic acid, GB acid), cyclosarin (cyclohexyl methylphosphonic acid, GF acid), and soman

(pinacolylmethylphosphonic acid) as well as their common final hydrolysis product methyl phosphonic acid were used in these experiments. Due to the non-volatile nature of these alkyl phosphonic acid degradation products, derivatization was performed to generate the volatile tert-butyl dimethylsilyl species. Degraded organophosphorus pesticide standards were obtained for acephate, chlorpyrifos, dichlorvos, ethion, and parathion ethyl. Mixtures consisting of three pesticides in the presence of a single nerve agent degradation product were prepared. GC-ICPMS allowed for the separation and detection of all four degradation products in the presence of pesticide mixtures in slightly longer than 12 minutes. This is the first study analyzing pesticides as interfering species for analysis of nerve agent degradation products by GC-ICPMS.

109

5.2 INTRODUCTION

The Organization for the Prohibition of Chemical weapons (OPCW), which is an international regulatory group that monitors storage, transfer, and disposal of chemical warfare agents (CWAs), recently condemned the use of chemical weapons in Iraq1.

Established as a result of the Chemical weapons convention (CWC), the OPCW’s sole pursuit is worldwide eradication of all chemical weapons1. As of March 2007, the OPCW consisted of 182 member states, six signatory states, and seven non-signatory states1.

CWAs are nerve, vesicant, and choking compounds that p ose risks to both human populations as well as the environment. Increases in the threat of terrorist attacks require the development of state-of-art analytical methods for rapid identification of these toxic species.

Organophosphorus nerve agents are categorized as some of the most toxic species ever created. The compounds hydrolyze in the environment into specific alkyl phosphonic acids. These alkyl phosphonic acid degradation products provide an indirect means for monitoring release of the parent nerve agents. Typically, analytical method development is performed with the less toxic degradation products, which are commercially available as certified reference materials for the analysis of compounds related to the CWC.

Recently, 31P element specific detection with inductively coupled plasma mass spectrometry (ICPMS) has been performed for the analysis of organophosphorus nerve agent degradation products2, 3. These studies used liquid and gas chromatography coupled with ICPMS for the analysis of the degradation products from sarin, cyclosarin, soman, tabun, VX, and RVX. Due to the selectivity of 31P detection with ICPMS, the ability

110

chromatographically resolve interfering species such as phosphate salts and organophosphorus pesticides (OPPs) is a necessity for method development.

Organophosphorus pesticide are the most widely used type of chemical for insecticides, herbicides, and fungicide applications4-8. The popularity of OPPs rose following the ban on organochlorine species in the 1970’s4-8. OPPs are less persistent in the environment, while at the same time possessing a greater acute toxicity towards weeds and insects. Figure 5.1 provides depicts the structures of five common OPPs.

Analysis of OPPs has previously been accomplished through coupling CE5, LC9, and

GC4, 6, 10 with ICPMS element specific detection. Due to the extensive use of these OPPs as agricultural insecticides, their presence has been detected in ground water, drinking water, fruits, and vegtables4, 6, 10. Due to this high environmental presence, OPPs must be considered as a possible interfering species for analysis of organophosphorus nerve agent degradation products with ICPMS.

S O O

CLC C O P O CH OOOP O H C O P O NHAc 2 3 3 H O O S CH 3 O N CH 2 3 Dichlorvos Parathion Acephate ethyl

S S S N O P O Cl O P S C S PO O H O 2 O Cl Cl

Chlorpyrifos Ethion

Figure 5.1- Five common organophosphorus (OPPs).

111

This work describes the application of a previously described GC-ICPMS method for analysis of organophosphorus nerve agent degradation products, but now in the presence of mixtures of OPPs2.

5.3 EXPERIMENTAL

5.3.1 Reagents

Four chemical warfare degradation products were obtained from Cerilliant (Austin, TX) as 1 mg mL-1 certified reference materials (CRMs). The degradation products utilized for these experiments included isopropyl methylphosphonic acid (IMPA, GB Acid), methylphosphonic acid (MPA), cyclohexyl methylphosphonic acid (CMPA, GF Acid) and pinacolyl methylphosphonic acid (PMPA). The five expired (1995) pesticide standards used (1 mg mL-1, Absolute Standards Inc, Hamden, CT) included dichlorvos, chloryprifos, acephate, ethion, and parathion ethyl (Figure 5.1). N-(tert-

Butyldimethylsilyl)-N-methyltrifluroacetamide with 1% TBDMSCl (Sigma-Aldrich, St.

Louis, MO) was used as the derivatizing reagent for the non-volatile alkyl phosphonic acids. Derivatization reactions were performed with distilled acetonitrile (Tedia,

Cincinnati, OH) and have been described elsewhere2.

5.3.2 Sample Preparation

Individual stock standards of the derivatized (TBDMS) chemical warfare degradation products were prepared at a concentration of 100 μg mL-1. Working solutions were prepared through both 20 and 40x dilution of the stock TBDMS derivatives with distilled

112

acetonitrile to yield 5 and 1 μg mL-1 samples respectively. Pesticide working solutions of

25 and 50 μg mL-1 were prepared through dilution of individual stock standards. Each standard was processed individually prior to mixture analysis. Mixtures of three pesticides were added to the working solutions at a 10x higher concentration (50 μg mL-

1) relative to the IMPA, PMPA, and CMPA-TBDMS derivatives. The MPA-

TBDMS/pesticides mixture was prepared with a 5x higher concentration of pesticide relative to the derivative. Because MPA is the final hydrolysis product for the alkyl phosphonic acid degradation products, a smaller concentration difference relative to pesticides species would be expected.

5.4 INSTRUMENTATION

5.4.1 Gas Chromatography (GC)

An Agilent 6890 (Agilent Technologies, Palo Alto, California) gas chromatograph with helium carrier gas with 1% Xenon was used for separation of the seven chemical warfare degradation products. Xenon gas allowed for instrument parameter optimization daily prior to analysis. An HP-5 (5% -phenyl-methyl-polylsiloxane) capillary column (30 m,

0.32 mm i.d., 0.25 μm film thickness) was used for all separation experiments. A detailed description of the GC parameters is provided in Table 5.1.

113

Table 5.1- ICPMS and GC instrumental parameters.

ICPMS parameters

Forward power 950 W (with shielded torch)

Plasma gas flow rate 15.6 L min-1

Auxiliary gas flow rate 1.0 L min-1

Carrier gas flow rate 0.90 L min-1

QP Bias -4 eV

Octopole -6 eV

Sampling Depth 6 mm

Sampling and Skimmer Cones Nickel

Dwell time 0.1 s

Isotopes monitored (m/z) 31P and 47PO+

Octopole Reaction System None

GC parameters

Instrument Agilent 6890 GC

Carrier Gas 99.999% He w/ 1% Xe (Constant Pressure 10psi)

Injection Splitless

Purge Time 0.75 min

Injection Volume 1 μL

80 ºC (1 min) 20 ºC/min b 280 ºC Hold (4min) Oven Program

Column HP DB-5 (5% -phenyl-methyl-polylsiloxane)

114

5.4.2 Inductively Coupled Plasma Mass Spectrometer (ICPMS)

An Agilent 7500ce (Agilent Technologies, Tokyo, Japan) ICPMS equipped with shield torch and collision/reaction cell technology was used for the element specific detection of

ICPMS with GC was accomplished through the use of a remote cable to allow a simultaneous data acquisition start prior to each separation experiment. Instrument parameters were optimized daily prior to analysis through optimization of Xe signal at m/z 124. A detailed description of the ICPMS parameters is provided in Table 5.1.

5.5 RESULTS AND DISCUSSION

5.5.1 GC-ICPMS

Screening organophosphorus nerve agent degradation product by GC-ICPMS is a reliable technique due to the excellent selectivity provided by this element specific detection system2. Separation and detection of seven organophosphorus nerve agent degradation products was previously accomplished in less than 9 minutes2. Elemental speciation with

GC-ICPMS requires high chromatographic resolution in order to ensure no interfering species overlap the degradation product standards, leading to false positives.

Identification of possible interfering species (i.e. volatile and phosphorus containing) led to the analysis of degraded organophosphorus pesticides.

5.5.2 Individual Pesticide Analysis in Nerve Agent Degradation Product Mixture

Initial experiments consisted of the analysis of individual pesticide standards of dichlorvos, ethion, and parathion ethyl in the presence of a mixture of seven

115

organophosphorus nerve agent degradation products (Figures 5.2-5.4). Figure 5.2 shows the separation of a 1ng mixture of seven organophosphorus nerve agent degradation products in the presence of 25 ng of degraded dichlorvos. The degraded dic hlorvos provided three new phosphorus containing pe aks correspon ding to various degradation products. Similar results were observed for both ethion and parathion ethyl in the presence of the organophosphorus nerve agent degradation product mixture (Figures 5.3 and 5.4).

31P

20000 Degraded Dichlorvos 1. EMPA-TBDMS 2. IMPA-TBDMS 3. EDAP-TBDMS 15000 4. IBMPA-TBDMS 5. PMPA-TBDMS 6. MPA-TBDMS 6 7. Phosphate-TBDMS(2x) 10000 4 8. CMPA-TBDMS Response (CPS)

1 3 5000 2

5 8 7 0 4 6810 Time (min)

Figure 5.2- 1 ng degradation product mixture w/ 25 ng degraded dichlorvos.

116

31P 90000

80000 Degraded Ethion 70000 1. EMPA-TBDMS 2. IMPA-TBDMS 20000

S) 3. EDAP-TBDMS 4. IBMPA-TBDMS 5. PMPA-TBDMS 6 6. MPA-TBDMS 7. Phosphate-TBDMS(2x) 10000 8. CMPA-TBDMS Response (CP 4

3 1 2 5 8 7 0

4681012 Time (min) Figure 5.3- 1 ng degradation product mixture w/ 25 ng degraded ethion.

31P 250000

Degraded Parathion Ethyl 1. EMPA-TBDMS 200000 2. IMPA-TBDMS 3. EDAP-TBDMS 4. IBMPA-TBDMS 5. PMPA-TBDMS 6. MPA-TBDMS 7. Phosphate-TBDMS(2x) 20000 8. CMPA-TBDMS Response (CPS) 6

10000 4

1 23 5 8 7 0 4681012 Time (min)

Figure 5.4- 1 ng degradation product mixture w/ 25 ng degraded parathion ethyl.

117

5.5.3 Degradation Product Analysis in Pesticide Mixtures

The likelihood of the simultaneous release of all six parent nerve agents is suggested to be highly unlikely. A more probable scenario would be the release of a well concentrated single nerve agent in an environment with mixtures of organophosphorus pesticides and their hydrolysis products. Figure 5.5 shows the separation of IMPA-TBDMS (Sarin, GB

Acid) in the presence of a 10x higher concentration of dichlorvos, ethion, and parathion ethyl. The multiple phosphorus containing peaks correspond to various hydrolysis products of the three pesticides with the highlighted peak specific to IMPA-TBDMS.

Similar results were obtained for CMPA and PMPA-TBDMS (cyclosarin-GF Acid and soman) derivatives in the presence of pesticide mixtures (Figures 5.6 & 5.7). Parent pesticides peaks are still observed at the longer retention times, however due to the age and storage of the standards degradation would be expected. Figure 5.8 shows the analysis of the final hydrolysis product, MPA-TBDMS, in the presence of a 5x higher concentration of acephate, ethion, and chloryprifos. MPA is typically utilized to verify release of all organophosphorus nerve agents. Due to the highly specific hydrolysis pathway it is also predicted that MPA will be present at higher concentration relative to the other alkyl phosphonic acid degradation products following release into the environment.

118

O H CH 31 H C P O C 3 P 3 CH 200000 O 3 H C Si CH 150000 3 3 IMPA-TBDMS H C CH 100000 3 CH 3 (Sarin) 3 50000

10000

Response (CPS) 5000

2 4 6 8 10 12

Time (min) Figure 5.5- 5 ng isopropyl methylphosphonic acid-TBDMS in mixture with 50 ng dichlorvos, ethion, and parathion ethyl.

31 P 120000 100000 80000 O CH3 60000 H 40000 H C P O C CH 3 3 O H C CH 3 3 H C Si CH 7000 3 3 H C CH 3 CH 3 6000 3 PMPA-TBDMS 5000 (Soman)

4000 Response (CPS) 3000 2000

1000

24681012 Time (min) Figure 5.6- 5 ng pinacoyl methylphosphonic acid-TBDMS in mixture with 50 ng dichlorvos, chlorpyrifos, and ethion.

119

31 P 200000 150000 100000

CMPA-TBDMS 9000 (Cyclosarin) 8000 O H C P O 7000 3 O 6000 H C Si CH 3 3 H C CH 5000 3 CH 3 3 Response (CPS) 4000

3000 2000 1000

24681012 Time (min)

Figure 5.7- 5 ng cyclohexyl methylphosphonic acid-TBDMS in mixture with 50 ng acephate, parathion ethyl, dichlorvos. CH O 3 CH 3 H C P O Si CH 31 3 3 P O CH3 CH 60000 3 3 H3C Si CH3 MPA-TBMDS H C CH 3 CH 3 40000 3

14000

12000

10000

8000 Response (CPS) 6000

4000

2000

24681012 Time (min)

Figure 5.8- 5 ng methylphosphonic acid-TBDMS in mixture with 25 ng acephate, ethion, and chloryprifos.

120

5.6 CONCLUSION

This study describes the analysis of organophosphorus nerve agent degradation products in the presence of pesticides. Both mixtures and individual organophosphorus nerve agent degradation products were analyzed in the presence of pesticides, either individually or in a mixture of three, following a previously described method utilizing gas chromatography with ICPMS detection2. Initial results demonstrated the ability to identify individual degraded pesticides (dichlorvos, ethion, and parathion ethyl) in the presence of seven organophosphorus nerve agent degradation products. Due to the more probable scenario of a single nerve agent being released in the presence of multiple pesticides led to an alternative experimental approach. Individual degradation products of sarin, cyclosarin, and soman as well as their common final hydrolysis product (MPA) were detected in mixtures of three pesticides in approximately 12 minutes. Pesticide mixture concentrations ranging from 5-10x greater than those of the degradation products had no effect on the identification of the nerve agent degradation product of interest. This application demonstrated the chromatographic resolution of the GC separation method was sufficient to resolve possible interfering species from the nerve agent degradation products of interest.

5.7 ACKNOWLEDGMENTS

I would like to thank Dr. Jack Creed of the U.S. Environmental Protection

Agency (EPA) for providing the degraded pesticides. Also I would like to than Dr. Anne

Vonderheide of the EPA for useful discussions on organophosphorus pesticides.

121

5.8 REFERENCES

(1) http://www.opcw.org/index.html, 2007. (2) Richardson, D. D., Caruso, J.A. Analytical and Bioanalytical Chemistry 2007, Online First, March 14, 2007. (3) Richardson, D. D.; Sadi, B. B. M.; Caruso, J. A. Journal of Analytical Atomic Spectrometry 2006, 21, 396-403. (4) Vonderheide, A. P.; Meija, J.; Montes-Bayon, M.; Caruso, J. A. Journal of Analytical Atomic Spectrometry 2003, 18, 1097-1102. (5) Wuilloud, R. G.; Shah, M.; Kannamkumarath, S. S.; Altamirano, J. C. Electrophoresis 2005, 26, 1598-1605. (6) Fidalgo-Used, N.; Montes-Bayon, M.; Blanco-Gonzalez, E.; Sanz-Medel, A. Journal of Analytical Atomic Spectrometry 2005, 20, 876-882. (7) Pehkonen, S. O.; Zhang, Q. Critical Reviews in Environmental Science and Technology 2002, 32, 17-72. (8) Lacorte, S.; Lartiges, S. B.; Garrigues, P.; Barcelo, D. Environmental Science and Technology 1995, 29, 431-438. (9) Sadi, B. B. M.; Vonderheide, A. P.; Caruso, J. A. Journal of Chromatography, A 2004, 1050, 95-101. (10) Proefrock, D.; Leonhard, P.; Wilbur, S.; Prange, A. Journal of Analytical Atomic Spectrometry 2004, 19, 623-631.

122

CHAPTER 6

Conclusions and Future Directions

123

6.1 CONCLUSIONS AND FUTURE DIRECTIONS

This dissertation has described modern advancements for elemental speciation analyses ranging from novel sample in troduction techniques to method development for analysis of chemical warfare agent degrad ation products. These modern advancements have provided a significant scientific contribution to a rapidly g rowing field while at the same time expanding the scope of current appl ications. The ability to couple modern separations techniques such as CE, HPLC and GC with state-of-the-art elemental mass spectrometry (ICPMS) has provided t he technology for theses modern advancements.

Applications described in the previous c hapters have demonstrated that multidisciplinary analytical techniques are required in order for continued advancements in elemental speciation. These multidisciplinary approa ches include the complementary use of alternative mass spectrometric ionization and mass spectrometric detection techniques such as ESI, MALDI, Ion-trap, and TOF, in parallel with the modern separation methods previously described. This multidimensional approach is the key for future scientific advancement in the areas of proteomics, metabolomics, metallomics, homeland security, and elemental speciation.

There are no limitations for the future directions of elemental speciation with hyphenated ICPMS. Advancements in instrumentation and development of capillary and nanoflow sample introduction techniques have paved the way for new speciation analysis in biological and environmental sample matrices. One possible future direction would be the coupling of ion mobility separation (gas phase CE) with ICPMS elements specific detection. Individually these techniques are well established and the combination would be a perfect example of innovation. Another area of future research includes the

124

expanded use of ICPMS for sulfur speciation analyses in chemical and biological research fields. Disulfide bond formation in protein and peptide research poses difficulties for analysis with conventional mass spectrometry. Elemental speciation techniques provide the tools necessary for detection and quantification of sulfur containing biological species. Sulfur containing chemical warfare agents (i.e. sulfur mustards) as well as their degradation products are ideal species for analysis by hyphenated ICPMS. Homeland security related speciation methods will continue to grow, resulting in the wide spread use of element specific detec tion as a preliminary screening tool for harmful chemical agents.

In summary, the work presented here demonstrates that elemental speciation is a powerful analytical tool for analysis of environmental and chemical warfar e related species. Recent advancements have only begun to scratch the surface for the potential applications of these state-of-the-art analytical techniques. Owing to the complexity of current scientific research, modern-day analytical chemists must employ multidisciplinary approaches in their experimental design. Providing this takes place continued modern scientific advancements are inevitable.

125