Detection of Cocaine and Its Interferents by Ion Mobility Spectrometry Coupled with SIMPLSMA and ALS

Home , Benzocaine, Lidocaine

Detection of Cocaine and its Interferents by Ion Mobility Spectrometry coupled with SIMPLSMA and ALS

A thesis presented to the faculty of the College of Arts and Sciences of Ohio University

In partial fulfillment of the requirements for the degree Bachelor of Science with Honors

Detection of Cocaine and its Interferents by Ion Mobility Spectrometry coupled with SIMPLSMA and ALS

by ANNE MARIE ESPOSITO

has been approved for the Department of Chemistry and Biochemistry and the College of Arts and Sciences by

Peter de B. Harrington Director of the Center for Intelligent Chemical Instrumentation

Robert Frank Dean, College of Arts and Sciences 3

Detection of Cocaine and its Interferents by Ion Mobility

Spectrometry coupled with SIMPLSMA and ALS

ESPOSITO, ANNE MARIE, B.S., April 2017, Forensic Chemistry

Director of Thesis: Dr. Peter de B. Harrington

ABSTRACT

Ion mobility spectrometry (IMS) is used by law enforcement for fast, easy- to-use detection of explosives and drugs of abuse. The Barringer IonScan

350® has been designed for use by nonscientists. Interferents can cause false positive or negative errors. The use of reduced mobility can provide more information about the peaks detected by IMS. The use of Simple-to- use interactive self-modeling mixture analysis (SIMPLISMA) can separate the components of a sample to help determine the identity of the interferent and limit the number of false negative errors. For the detection of illicit drugs, it is important to correctly report the likelihood of errors so that appropriate further testing can be performed. In the case of a

Barringer IonScan 400®, a 1% false positive rate is reported by Smiths in their advertising. Based on the drift time and the reduced mobility of benzocaine and lidocaine, they easily could be mistaken for cocaine during field testing.

DEDICATION

To the beloved, Joseph T. Otto

ACKNOWLEDGMENTS

I would like to acknowledge Dr. Peter de B. Harrington and Dr. Jixin Chen for their assistance in writing this work as well as Xinyi Wang and Ahmet

Aloglu for their assistance in developing my research skills. I would like to thank Paul Schmittauer for assistance with instrument operation.

TABLE OF CONTENTS

Page

Abstract...... 3

Dedication...... 4

Acknowledgments...... 5

List of Tables...... 7

List of Figures...... 8

Introduction……………………………………………………………...... 9

a. Importance and Novelty………………………………………………………….9

b. IMS Theory…………………………………………….………………………………10

c. SIMPLISMA Theory………………………………………………………………..13

d. ALS Theory……………………………………………….……………………………17

e. Methods and Materials……………………………….………………………….18

Results and Discussion……………………………...... 22

Conclusion……………………………………………………………………………………………..33

Future Work…………………………………………………………………………………………..34

References...... 35

LIST OF TABLES

Page

Table 1. Operating parameters for the IonScan 350®…………………………21

LIST OF FIGURES

Page

Figure 1. Skin swipe with fiber glass circle.……………………………………………….19

Figure 2. Fiber glass circle loaded into the cartridge. Cartidges are provided with the IonScan® 350…………………………………………………………………20

Figure 3. Cartridge loaded into the catridge holder……………………………..……20

Figure 4. Cartridge after sliding into the thermal desorption unit…………….21

Figure 5. IMS average spectrum of lidocaine………………………………………….…22

Figure 6. IMS average spectrum of benzocaine………………………………………..23

Figure 7. IMS average spectrum of cocaine…………………………..………………….23

Figure 8. SIMPLISMA processed spectrum for lidocaine………………….……….25

Figure 9. SIMPLISMA processed spectrum for benzocaine……………………….26

Figure 10. When using SIMPLISMA input of a third component chooses noise…………………………………………………………………………………………………….……..27

Figure 11. ALS concentration profile of cocaine……………………….………….……28

Figure 12. ALS concentration profile of lidocaine…………………..…………….…..29

Figure 13. ALS concentration profile of benzocaine……………..……………….….29

Figure 14. IMS spectrum of a blank arm swipe after washing………….……..30

Figure 15. IMS spectrum for an arm swipe after application of lidocaine..31

Figure 16. IMS spectrum for an arm swipe after application of lidocaine…31

1. Introduction

a. Importance and Novelty

Different changes to IMS operation have been tested to improve the detection of illicit substances1,2,3. Cocaine has multiple commercially available analogs, including lidocaine and benzocaine. They can both be found in topical pain medications available over the counter. There have been reports of cocaine being cut with both of these substances9. Based on my experiments, benzocaine and lidocaine have reduced mobilities of

1.15 ±.01 cm2 V-1 s-1 as compared to a literature value of 1.16 cm2 V-1 s-1 for cocaine2. It is possible that these substances would be mistaken for cocaine. The variability of reduced mobility has not been reported with use of a commercial instrument4. The IonScan 350® uses drift time windows, peak width, and amplitude to perform its analysis, not reduced mobility. Lidocaine or benzocaine may interfere with the cocaine peak and elicit a false negative or false positive. An additional peak around the same drift time could widen the peak and fall outside of the parameters set by the instrument. Lidocaine or benzocaine alone may also elicit a false positive result. The use of SIMPLISMA would allow for complete separation of these peaks and remove the effect of the interferent.

Swipes of various surfaces, including skin, have been used to detect explosives and illicit drugs. The persistence of these substances on surfaces could help law enforcement determine the time-frame that the 10

drug was present. Because lidocaine is sold as a skin anesthetic, swiping someone’s hands could elicit a positive response for cocaine.

b. IMS Theory

Ion mobility spectrometry5 uses drift time and an internal calibrant to identify ionized species. The IonScan 350® uses a thermal desorption sample inlet. This enables nonvolatile substances to be analyzed by the

IMS. A run begins by sliding the sample tray into the heating unit. The glass fiber sample circles are snapped into a sample holder and the sample holder is placed on the sample tray. The analyte travels from the glass fiber circle into the instrument to be ionized. In the IonScan 350®, ions are formed from a 63Ni radioactive source. Air is ionized first by the following reaction:

(1) N +β →N +e +β

For which is the beta particle emitted from the 63Ni radioactive source,

N2 is the major component of the drift gas and , is the beta particle after some of its energy is lost to ionization of the drift gas5.

(2) N + (HO) → (HO)H +N

(3) (HO)H +CHNO→CHNO + HO

The ionization will transfer to the other present compounds in the drift gas. The reagent ion, which in our case is nicotinamide, will become charged and reach the detector. This reaction is driven by entropy.

Unless the instrument is saturated with the analyte, some reagent ion will always be present in the ion mobility spectrum. When the sample being 11

tested is introduced, the proton will transfer to the sample if it is more basic. By using the weakly basic nicotinamide, non-amine containing compounds that are less basic will not contribute to the signal, while more basic drug compounds will effectively compete for the charge and appear as peaks in the spectrum. Cocaine and its analogs are more basic than nicotinamide; therefore, the proton transfer is energetically favored.

After ionization, the ion gate opens by dropping the potential to ground for a brief period of time and the ions are propelled by the linear voltage drop towards the detector. The mobility of ions K is derived from:

(4) = × × × for which q is the charge on the ion, Ν is the number density of the drift gas, k is Boltzmann's constant, Τ is absolute temperature, µ is represented by:

(5) = /( + ) for which m is the mass of the ion, M is the mass of the drift gas, and Ω is the collisional cross section of the ion in the drift gas. When the instrument holds temperature, pressure, and number density of the drift gas are constant, we can reduce the relationship of ion mobility to:

(6) = × × for which number density is

(7) = 12

for which n is the number of neutral buffer gas molecules in the drift volume V of the instrument.

In the case that the detected compounds are much larger than the drift gas, µ approaches the mass of the drift gas, which is also a constant.

Therefore, the ion will travel at a velocity based on its charge and collisional cross section. The collisional cross section depends on the ion’s size, shape, orientation, and polarizability.

A drift tube under a constant electric field allows the ionized compounds to migrate. Charged rings connected by electrical resistors provide a linear voltage drop so that the ions will travel at a constant velocity. In an

IonScan 350®, air is used as the ambient-pressure drift gas to keep the instrument from accumulating impurities. The air is routed through a tube of Drierite® and activated charcoal to keep the drift gas clean.

In an IonScan 350®, a charged gate is present at the entrance to the drift tube to keep ions out of the drift tube. The voltage on the wires that make up the gate can be represented by:

(8) ±

For which Vr is the voltage on the drift tube rings and Vc is the voltage needed to close the gate. When the gate is closed, all the ions collects on the gate. To release the ions, the gate opens by a voltage pulse to Vr. In a single gate system, the pulse is between 0.2-0.5 ms. 13

In the IonScan 350®, ions terminate at the Faraday plate which is the detector. The resulting ion current detected at the Faraday plate with respect to transit time comprises the ion mobility spectrum.

To account for the difference in operating temperature and pressure of different instruments and at different times of the day, the reduced ion mobility is used.

(9) × = ×

2 -1 -1 For which K0 is the reduced ion mobility in cm V s , and tdrift is the drift time in milliseconds. The K0 of the reagent ion is a known value and the drift times are determined experimentally. This equation can be used here because all ions are travelling the same distance and the product of time and velocity is distance.

Ion mobility spectrometers make measurements in real time which are traditionally averaged over the entire analysis time to yield the spectrum.

However, the increase and decrease of individual peaks over time can provide useful information. If peaks increase and decrease independently with respect to analysis time, then they are unrelated. Multivariate curve resolution can be used to separate the components based on independent changes in the data with respect to acquisition time. Simple-to-use interactive self-modeling mixture analysis (SIMPLISMA), developed by

Windig and Guilment6, is one method to model the temporal response of the signal.

c. SIMPLISMA Theory 14

The IonScan 350® uses the drift time windows to determine the peak identity. The IonScan 400® manual10 uses a window of ±0.050 ms to determine whether the peak is in an acceptable range. However, the

IonScan 350® manual11 allows for the operator to change these ranges.

Too large an acceptable range would increase the number of false positive errors and too small a range would increase the number of false negative errors.

Because the instrument is designed to be user-friendly, there are no scientists required to use this instrument for the data to be presented in court. A sample that was a mixture of cocaine and an interferent may obscure the cocaine peak and lead to a false negative. SIMPLISMA can be used to separate a single peak of a mixture into its pure components, and create a profile of the changing concentration over time. SIMPLISMA can prevent a false negative or false positive error and identify the resolved peak.

SIMPLISMA is a pure variable-based multivariate curve resolution method developed by Windig and Guilment6 and previously applied to IMS data by

Harrington et al.7 Multivariate curve resolution methods are based on a bilinear model such as the one in (10)8.

(10) = +

The data set can be organized into a data matrix D for which each row is a spectral scan at a specific time and each column is a drift time measurement, the elements are the ion current at a specific time or 15

spectral scan and drift time. You can decompose data matrix D into two profiles; the matrix C which contains the resolved concentration profiles and the matrix ST which contains the resolved spectra profiles. E is the matrix of residuals that fall outside the model and are considered experimental error.

Spectra are obtained by regressing the concentration profiles onto the data matrix. SIMPLISMA will normalize each component’s spectra by:

(11) = ∑ ,

(12) = × for which is used for the diagonal elements of the matrix N which then normalizes the spectra P. Normalization is important so that that concentration profiles are quantitative and the spectra are qualitative.

Normalization of the spectra is required to remove ambiguity from the two terms of the product.

SIMPLISMA analyzes every drift time point to determine its purity. The purity, Pi is calculated based on:

(13) = × ̅

The first term measures the relative standard deviation (RSD) because the algorithm seeks drift times for which there is a change in intensity with time. The standard deviation and the mean ̅ are obtained from m time points or number of spectral scans at a specific drift time measurement.

The column of the m×n spectral matrix is a concentration profile at a drift time measurement, and is a damping factor to suppress concentration 16

profiles that have no signal and comprise only noise from yielding high purities P. Note an RSD can be large, when the mean intensity is close to zero. When the mean is large, will have little effect but when the mean is approaching the noise level, it will decrease the purity. The determinant is a measure of the independence between profiles measured at two drift times. The elements of the determinant are correlation coefficients for the concentration profiles. The diagonal elements will be unity because a concentration profile will be perfectly correlated with itself. If the two concentration profiles are perfectly correlated with each other, in that they may be two neighboring points of a peak, then the off-diagonal elements will be unity as well and the determinant will be zero. If the two concentration profiles are independent their cross-correlation rij will be zero and the determinant will be unity. Concentration profiles are selected that maximize the purity by the product of these two terms in (13).

SIMPLISMA requires an input for the number of pure variables. When unsure of the number of components that are present in the mixture, choosing an arbitrary, large, number allows SIMPLISMA to overfit the data.

SIMPLISMA will consider noise and small variation components. By visual examination of the SIMPLISMA spectra and concentration profiles, the number of components that have peaks above the noise level is used.

SIMPLISMA is then rerun with the correct number of components.

When applied to ion mobility spectra, drift time points are analyzed to determine the points with the largest purity. The pure component peaks 17

may overlap so much as to produce a single peak; therefore, the points on either edge of the peak are most likely to contain a pure variable. With

IMS data the spectra are measured with respect to time and the concentration of the mixture components will vary as the sample concentrations change with respect to the thermal desorption. These spectra are typically averaged over a particular range of scan numbers, to give a spectrum that is at the very simplest a mixture of the reagent and analyte ions (figure 5).

d. ALS Theory

Alternating least squares (ALS) data processing takes the information produced by SIMPLISMA analysis and adds the information about the measurement taken over time. SIMPLISMA gives us information about the number of components, and by which peak(s) they are represented.

However, sometimes we have negative peaks in our concentration profiles and spectra. ALS is an iterative refinement method that minimizes any negative features. It uses a special constrained least squares regression that ensures that the projection has no negative values. ALS iteratively solves (10) which calculates matrices C and ST to optimally fit matrix D8.

The initial values for matrices C and ST can be obtained from SIMPLISMA.

In the case that two iterations have a difference in standard deviation of the residuals less than a predetermined percentage, convergence is achieved. This percentage is known as lack of fit calculated by: 18

∑, (14) (%) = 100× ∑,

for which dij uses an element of the data matrix D and eij is the difference between the data matrix element D and ALS reproduction CST.

e. Methods and Materials

For the lidocaine, benzocaine, and cocaine experiments a Barringer

Ionscan® 350® (Smiths Detection, Toronto, Canada) was used. All spectra were acquired in positive, or narcotics, mode; because the target analytes all contained basic amine groups. The acquisition rate was 80 kHz and the number of points per spectrum was 2000. The scan period was 20 ms.

The external data acquisition used a homebuilt LabVIEW 5.1 virtual instrument.

The cocaine hydrochloride standard was obtained from Sigma AldrichTM, lot number: SLBD7073V, lidocaine was purchased as a 4% pain relief cream distributed by CVS Pharmacy, Inc., and benzocaine was purchased as a

20% oral pain reliever solution distributed by CVS Pharmacy, Inc. Glass fiber filter circles were obtained from Fisher ScientificTM, catalogue number: 09-804-42D. These were significantly easier to load into the sample cartridges provided with the instrument.

A 1 mg/mL solution of cocaine hydrochloride standard was prepared in 18

Ω deionized water. A 4x10-4 g/mL lidocaine solution and an 8x10-4 g/mL benzocaine solution were made by diluting with the deionized water. A 19

0.5-mL aliquot of hydrochloric acid was added to the benzocaine and lidocaine solutions to increase their water solubility.

Solutions were aliquoted onto the glass fiber filter circles in amounts between 50.0-10.0 µL and allowed to air dry for 30 min. Skin swipes were performed on washed skin. A 1.000 mL - .125 mL aliquot of lidocaine cream was applied to the skin as instructed. The skin was immediately swiped with a glass fiber filter circle and tested. Images of the skin swipe and sample loading are shown in figures 1, 2, 3, 4.

Figure 1. Skin swipe with fiber glass circle. 20

Figure 2. Fiber glass circle loaded into the cartridge. Cartidges are provided with the IonScan® 350.

Figure 3. Cartridge loaded into the catridge holder. 21

Figure 4. Cartridge after sliding into the thermal desorption unit.

The operating parameters for the IonScan 350® are as follows:

Tube Temperature 234 °C

Inlet Temperature 295 °C

Desorber Temperature 284 °C

Drift Flow 297 cc/min

Exhaust Flow 199 cc/min

Sample Flow 512 cc/min

Table 1. Operating parameters for the IonScan 350®

2. Results and Discussion

The ion mobility spectra for lidocaine, benzocaine, and cocaine are given below (figures 5, 6, 7).

Figure 5. IMS average spectrum of lidocaine. 23

Figure 6. IMS average spectrum of benzocaine.

Figure 7. IMS average spectrum of cocaine. 24

In each spectrum, the left-most peak corresponds to the reagent ion peak

(nicotinamide). The peak at a reduced mobility of 1.15 and 1.14 cm2 V-1 s-

1 is the ion peak for the protonation of lidocaine and benzocaine respectively that have a reduced mobility similar to cocaine. Lidocaine and benzocaine both produce a second ion peak at a reduced mobility of 1.29 and 1.28 cm2 V-1 s-1 respectively. Based on the work by Warnke et. al9, benzocaine can protonate at either the nitrogen or the carbonyl oxygen.

Lidocaine has a second nitrogen and a carbonyl oxygen that could also protonate. Different protonation could explain the second peaks that are part of the lidocaine/benzocaine component. By looking at the SIMPLISMA analysis of the lidocaine and benzocaine spectra over time (figure 8, 9), both of those peaks are the product of one component; therefore, this is not a contaminant peak. The IonScan 350® detects peaks that appear in specific drift time windows to identify ions; therefore, the additional peak would not exclude lidocaine or benzocaine from eliciting a positive response for cocaine, but could yield an additional false positive alarm.

Buxton et. al3 had a similar outcome with multiple peaks for different explosives due to differential protonation and thermal degradation.

According to the IonScan® 400 manual10, a peak that overlaps with a drift time window of ± 0.05 ms is an adequate range to identify cocaine. The drift time for lidocaine and benzocaine fall outside of this range. However, the IonScan 350® manual11 does not provide recommended ranges for drift time. It is up to the operator to decide what those should be. You 25

are permitted to adjust the range in drift time to 0.2 ms, which would include the lidocaine and benzocaine peaks.

Figure 8. SIMPLISMA processed spectrum for lidocaine.

Figure 9. SIMPLISMA processed spectrum for benzocaine.

When you attempt to process the data with three components, noise is chosen as the third component (figure 10). 27

Figure 10. When using SIMPLISMA input of a third component chooses noise.

ALS can be used to view the changing concentrations of the nicotinamide ion and the ion of interest over measurement time. The ALS analysis clearly shows the introduction of each analyte of interest. The increase in the nicotinamide profile around the time of analyte introduction (around 3 seconds in figure 11) is due to the sliding of the sample tray. The vibrations of that movement cause erroneous peaks.

The ALS spectra also give us an idea of the extent of instrument saturation. When the nicotinamide profile reaches zero integrated intensity, the instrument is completely saturated. The nicotinamide ions 28

have transferred all of their charges to the analytes, and any additional analyte will remain unionized and undetected.

Figure 11. ALS concentration profile of cocaine. 29

Figure 12. ALS concentration profile of lidocaine.

Figure 13. ALS concentration profile of benzocaine. 30

IonScan instrumentation is used to swipe objects and people for the presence of narcotics and explosives. The persistence of these substances on various objects is of interest. Because lidocaine is a commonly used as a topical anesthetic, it would commonly be found on the skin. The peak at a reduced mobility of 1.15 cm2 V-1 s-1 for lidocaine has disappeared (figure

15, 16) even at higher concentrations than seen in the solution testing. It is possible that the alternative protonation that leads to the peak at a reduced mobility of 1.15 cm2 V-1 s-1, only occurs in water. Because the peak the interferes with cocaine is no longer present, lidocaine on skin or other surfaces will not be mistaken for cocaine by IMS. Only the reagent ion peak is visible from a skin swipe of washed skin.

Figure 14. IMS spectrum of a blank arm swipe after washing. 31

Figure 15. IMS spectrum for an arm swipe after application of lidocaine.

Figure 16. IMS spectrum for an arm swipe after application of lidocaine.

3. Conclusion

SIMPLISMA can be used to identify components of a mixture when tested by IMS. Only using data produced by the IonScan 350® is not always adequate to identify your sample. Although SIMPLISMA has not been applied to a cocaine/interferent mixture, it has been applied to mixtures of explosives3. ALS concentration profiles are useful for visualizing the rate of proton transfer from the reagent ion to the analyte(s) of interest.

SIMPLISMA and ALS use the measurement time data to exploit changes in concentration that occur with time to resolve overlapping peaks and resolve pure component spectra.

Analysis of skin swipes highlights the influence of surrounding materials on the ions that form. Experiments and discussion on the effects of the environment on drift time and reduced mobility were reported by Forbes et. al7 Because of the lack of peak at the known reduced mobility for cocaine, lidocaine would not be mistaken for cocaine when swiped off the skin.

4. Future work

Testing mixture samples of cocaine with lidocaine and benzocaine would be useful for testing the ability of SIMPLISMA. In addition, testing seized samples that have unknown components would be another good test of this method. Application of this method to other known cutting agents for cocaine and perhaps other drugs and their interferents would help further the knowledge of IMS and its possible issues.

Another application of this work could be in testing the persistence of substances on skin. We have shown that lidocaine can be detected on a glass fiber circle after swiping the skin. The effect of surrounding materials is another area that needs further work. The effects of cocaine on the protonation of lidocaine or benzocaine will be important to their identification.

5. References

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