Determination of the Identity and Purity of Illicit Drugs in an Unknown White Powder

Julia DiOrio, Austin Riggins, and Danya Batallas

University of Virginia: Chem 3721

Abstract

Research by the National Institute on Drug Abuse gives testament to the high prevalence

of illicit drugs of psychostimulants of the amphetamine class and other substances of similar

chemical structure such as powdered cocaine. Forensic labs require a method to determine both

the identity and the purity of illicit drugs for white powders found at crime scenes. This

investigation sought to develop a method using liquid chromatography – mass spectrometry (LC-

MS) using four major illicit drugs: amphetamine, methamphetamine,

methylenedioxymethamphetamine, and cocaine. The overall scheme of the method separated

each component by high performance liquid chromatography (HPLC), determined identity by the

mass to charge ratios observed in the mass spectrum generated using electrospray ionization, and

determined purity as concentration from the peak area under a photodiode array (PDA)

chromatogram using a calibration curve of standard solutions. Challenges arose in the

quantification of cocaine as the standards and unknown samples were unexpectedly cut with

sodium bicarbonate. The success of the developed method was assessed by analysis of three

unknown samples containing any combination of the four illicit drugs. The method successfully

identified and determined the concentration of each drug in all unknowns within a 15% error of

the true value and showed a limit of quantification (LOQ) for each drug ranging from 2.79 to

8.92 mg mL-1, well below the concentrations being investigated.

Introduction

Studies conducted by the National Institute on Drug Abuse show that 23.5 million people

over the age of 12 (9.3% of the general population) required treatment for illicit drug or alcohol

abuse in 2009.1 Of this 23.5 million, 6.5% were treated for the use of psychostimulants of the

amphetamine class and 3.4% were treated for the use of powdered cocaine.1 The prevalence of

these drugs in today’s market presents a particular challenge to police departments as they must

attempt to both identify drugs and determine the purity of a given white powder sample. Liquid

chromatography - mass spectrometry (LC-MS) combines the analytical techniques of separation

by high performance liquid chromatography (HPLC or LC) and mass analysis by mass

spectrometry (MS).2 By using both LC and MS in conjunction, the parts of complex mixtures

may be separated and analyzed independently. Forensic laboratories analyze unknown white

powders potentially containing illicit drugs by LC-MS, allowing for both qualitative and

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quantitative data to be gathered. This study aims to develop a LC-MS technique to identify and

determine purity of three unknown samples containing some combination of amphetamine

(AMP), methamphetamine (MA), methylenedioxymethamphetamine (MDMA), or cocaine

(COC).

In liquid chromatography, separation occurs by distributing analytes between a liquid

mobile phase and a solid stationary phase.2 This research is conducted using reverse phase LC

such that the stationary phase consists of a nonpolar silicate bead as shown in Appendix 1 and

the liquid mobile phase uses a more polar liquid.2 The most nonpolar analytes spend more time

interacting with the nonpolar stationary phase than analytes with a higher polarity; therefore,

reverse phase LC elutes from the most polar to the least polar over time.2 The four compounds in

question for this study are expected to elute in the order of amphetamine, methamphetamine,

methylenedioxymethamphetamine, then cocaine according to the compound structures shown in

Appendix 2.

Liquid chromatography alone simply separates the compounds; therefore, LC must be

performed with a detector. The most popular detectors used with LC include Ultraviolet-Visible

(UV-Vis) spectroscopy and MS. UV-Vis spectroscopy detects wavelength absorbance using a

photodiode array (PDA), and the presence of double bonds in all four drugs allow them to seen

in the 254 nm region. A PDA chromatogram shows a plot of absorbance vs. time that can be

used to calculate concentration from the peak area under the curve. In contrast, MS ionizes

compounds before detecting the mass to charge (m/z) ratios using a quadrupole.2 This protocol

uses electrospray ionization (ESI) to produce both positive and negative ions. The possible m/z

ratios of the analyte are determined by the molecular weight and available protonation sites as

described in Appendix 3.3 ESI applies a high voltage to the liquid analyte to produce large,

charged droplets that then undergo coulombic explosions to produce a fine spray of ionized gas.4

The quadrupole mass analyzer consists of 4 parallel metal rods with each pair of opposite rods

connected electrically to produce a magnetic field that affects the movement of analytes based on

their m/z ratio.4 The resulting mass spectrum shows relative intensity as a function of m/z ratio.

Although both UV-Vis spectroscopy and MS show good linearity in response to

concentration and have similar precision, accuracy, and sensitivity, MS has the advantage of

shorter analytical times and higher selectivity.5 However, the instrument used in this

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investigation utilizes both MS to identify and UV-Vis spectroscopy to determine the purity of the

unknown samples.

Experimental Methods

Chemicals and Materials

Four drugs were selected for analysis: amphetamine, methamphetamine, 3,4-

methylenedioxymethamphetamine, and cocaine. HPLC-grade methanol (MeOH), formic acid

and deionized water were purchased to use for dissolution and the mobile phase. Standard stock

solutions of each compound were prepared by dissolving each sample in MeOH:water (50:50,

v/v). Concentrations were dependent on the amount of available sample, ranging from 359 mg

L-1 to 289 mg L-1. Working solutions for each compound were prepared from stock solutions by

dilution with MeOH:water (50:50, v/v) solution. For the purpose of generating a 6-point

calibration curve, 1:1, 1:10, 2.5:10, 3:10, 1:2, and 7:10 dilutions were made for each standard.

Instrumentation

A Shimadzu Prominence-I LC-2030C was used with a PDA detector at 254 nm and a

LCMS-2020 quadrupole mass spectrometer equipped with an electrospray ionization interface

operated in positive-ion mode. Separation by chromatography was carried out using a C18

column, 1.7 m, 50 mm x 2.1 mm, at a flow rate of 0.25 mL min-1 with the column maintained at

40C. The mobile phase consisted of water with 0.01% formic acid (solvent A) and MeOH with

0.01% formic acid (solvent B). The mobile phase used a gradient changing the percentage of

solvent B as follows: 0 min, 5%; 5 min, 5%; 12 min, 15%; 12.50 min, 15%; equilibration of

column (5%) to 15 min. This program is visually depicted in Appendix 4. Injection volume was

set to 5 L for all samples, and nitrogen was used as the desolvation gas. All data was acquired

and analyzed using LabSolutions software.

Results and Discussion

HPLC and MS optimization

Individual standard solutions were used to optimize HPLC and MS conditions. ESI was

operated in positive ionization mode using the protonated ion [M+H]+ to determine the m/z ratio

of each compound and confirm the order of elution. The expected positive m/z ratios as well as

the actual mass spectrums for all four illicit drugs are depicted in Figure 1. This figure shows that

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AMP, MA, and MDMA mass analysis confirmed the calculated m/z ratios; however, the mass

spectrum of cocaine showed an unexpected variability in m/z ratios between 290 and 304. After

running multiple standards, no consistent ratio between these two major peaks could be

determined.

HPLC program optimization began with analysis of the effect polarity of the mobile

phase be varying the percentage of MeOH in isocratic runs. Figure 2 shows the improved peak

shape as a result of

decreasing MeOH from

35% to 10%. This figure

suggests that the higher

polarity of the mobile

phase in the 35% run did

not allow the molecules

to interact and equilibrate

with the column, causing peak

broadening. Decreasing the percentage of MeOH to 10% allowed molecules to more successfully

interact with the column, resulting in a sharper peak. However, using a flow rate of 0.400 mL

min-1 on a shorter column intended for proteomics did not appropriately resolve amphetamine

and methamphetamine peaks as the smaller molecules of this investigation were easily swept

away from the column. Therefore, the flow rate was significantly reduced to 0.250 mL min-1 to

resolve these peaks. As a result of this change, the peak began to demonstrate significant tailing.

In order to optimize the chromatographic separation and improve peak shape, different

mobile phase compositions were tested. In order to reduce tailing of peaks, 0.01% formic acid

was included in both water and MeOH to favor the formation of the protonated form of each

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drug and to protonate the silica silanols of the column. The protonation of both the molecules

and the column reduced extraneous intramolecular reactions that serve to broaden peaks.6

Additionally, as each drug only contained one protonation site, the formic acid encouraged

formation of the positive ion through electrospray ionization, allowing the MS program to be

developed specifically to detect these ions.

In addition to the inclusion of formic acid, a choice had to be made between the use of

acetonitrile (ACN) or MeOH. Figure 3 details the differences between the two choices of mobile

phase. Using ACN rather than

MeOH on a 10% isocratic run

showed improvement in the

tailing effect of the peak;

however, the slightly more polar

ACN also caused the retention

time to shorten to about 1.25

minutes. As methamphetamine

is the second drug to elute, this

short retention time would not provide the necessary resolution of the first peaks. With the

percentage of ACN decreased to 5%, the peak eluted with a similar retention time to the run of

10% MeOH with the disadvantage of an increased tailing effect. Based on these results, MeOH

with 0.01% formic acid was chosen as the mobile phase.

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This program designed for the

best peak shape was tested on a

solution containing the same

concentration of all four standards to

check for peak separation and

resolution. Ultimately, the percentage

of MeOH with 0.01% formic acid was

decreased to 5% and the injection

volume needed to be decreased from

20 L to 5 L to avoid overloading the column and providing the best resolution between AMP,

MA, and MDMA. With the first three peaks well resolved, the final challenge was to

successfully elute the last peak, cocaine. Figure 4 illustrates the effects of the gradient described

in Appendix 4. In order to preserve the resolution of the first three peaks, the linear gradient was

set to begin at 5 min such that it could decrease the tailing of the MDMA peak and drastically

sharpen the peak of cocaine without altering the retention times of AMP, MA, or MDMA. By

increasing the polarity of the mobile phase (5-15% MeOH with 0.01% formic acid), there was an

increase in the interactions of cocaine with the mobile phase, allowing cocaine to elute faster and

with better peak shape.

Method Validation

Good linearity was shown for the five- or six-point calibration curves in the range or 25

to 300 mg mL-1for all compounds as shown by correlation coefficients greater than 0.99. All four

calibration curves graphing peak area vs. concentration are included in Appendix 5. The limit of

detection (LOD) and limit of quantification (LOQ) were calculated using the standard deviation

of the response (Sy) of the curve and the slope (S) of the calibration curve such that: LOD =

3(Sy/S) and LOQ = 10(Sy/S). The results of linear regression, correlation coefficients, LOD, and

LOQ are shown for each compound in Table 1. Appendix 6 shows a sample calculation for

methamphetamine.

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Application to unknown samples

The developed method was applied to

the three unknown samples dissolved in the

same MeOH:water (50:50, v/v) solution as the

standards. MS specifications were set such that

selected ion monitoring (SIM) mode scanned

for the

specific

m/z

ratios of

each

standard (136, 150, 194, 290, and 304) for the entire run time. Figure 5 shows the raw data from

this program for the first unknown. The SIM TIC (+) shows peaks for m/z ratios of 136 and 150,

indicating the presence of AMP and MA. The corresponding PDA chromatogram validates this

analysis as the two peaks shown agree with the standard solution retention times, and there are

no other peaks indicating the presence of MDMA or COC. The linear equations determined from

the calibration curves were used to calculate the concentration of each drug from the peak area of

the PDA chromatogram.

Table 2 represents the data analysis of all three unknown samples as well as the actual

results and statistical analysis. Appendix 7 shows sample calculations for unknown 1. For

methamphetamine and MDMA, the amount in each unknown was correct within 10% of the true

value. Amphetamine was within 15%, an increase most likely due to the use of a graduated

cylinder to dissolve the original sample rather than a pipette. The results for unknown three

should a particularly unusual error of 372% considering the correlation coefficient of the linear

regression was 0.99992.

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The particular case of cocaine

To account for the extraneous m/z ratio of 290 in the cocaine standard, the original

explanation was the loss of a methyl group on the amine that was then replaced with a hydrogen,

leading to a decrease in molecular weight of 14 g/mol. However, it was assumed that all standard

samples provided were in their pure form; however, this was not the case for the cocaine

standard. The sample used to create standard solutions was cut with sodium bicarbonate,

explaining the presence of a m/z ratio of 290 in addition to the expected m/z ratio of 304

associated with cocaine. In the investigation, the variability of the presence of 290 or 304 was

due to the inhomogeneity of the solution such that more or less sodium bicarbonate could be

present in an injection. In the analysis to determine the concentration of cocaine, the peak area

was representative of sodium bicarbonate rather than cocaine. Therefore, the calculation for the

amount of cocaine in the unknown sample would be the original weight minus the weight of the

sodium bicarbonate, giving a calculated amount of 1.46 mg. This gives a percent error of 0.69%

in comparison to the true amount of 1.45 mg.

Comparison to Literature

In comparison to the main article used to determine a starting point for the parameters,

this investigation produced significantly larger limits of detection and quantification, a difference

of about nine orders of magnitude. This investigation’s limits of quantitation were on the order of

5 mg mL-1; whereas, the article reported limits of detection as low as 5 x 10-9 mg mL-1.6 The

study in question aimed to determine the concentration of illicit drugs in wastewater. To that end,

experimentation used a triple quadrupole tandem MS-MS for analysis and a newly designed

Step-wave ion guide in the LC-MS/MS instrument, which could explain the disparity in

sensitivity.6 However, the overall results of this investigation satisfied the specifications of the

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problem, determining all the correct identities and most of the purities within 15% of the true

values.

Conclusions

This investigation successfully developed a LC-MS method to determine both the

identity and purity of illicit drugs in an unknown sample. Separation was achieved by HPLC

using a linear gradient of methanol with 0.01% formic acid, and compounds were detected using

UV spectroscopy at 254 nm and quadrupole mass spectroscopy using electrospray ionization set

to SIM positive ion mode. Using the SIM TIC (+) to identify the type of drug, a set of calibration

curves for each standard was able to successfully calculate the concentrations of all drugs in each

unknown within 15% of the true value. In future work, accuracy may be increased by using more

accurate volumetric instruments, peak shape could be improved by using a longer column meant

for smaller molecules, and sensitivity could be increased using more advanced instrumentation

such as a tandem MS/MS.

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References1National Institute on Drug Abuse, 2011. https://www.drugabuse.gov/publications/drugfacts

/treatment-statistics (accessed May 1, 2016)2Niessen, W. M. A. Liquid Chromatography – Mass Spectrometry, 2nd ed.; Marcel Dekker, Inc.:

New York, NY, 1999; p 3. 3Arnquist, I. J.; Beussman, D.J. Incorporating Biological Mass Spectrometry into Undergraduate

Teaching Labs, Part 2: Peptide Identification via Molecular Mass Determination. Journal

of Chem. Education, 2009, 86(3), 382-384.4Ho, C. S.; Lam, C. W. K.; Cheung, R. C. K.; Law, L. K.; Ng, K. F.; Suen, M. W, M.; Tai, H.L.

Electrospray Ionisation Mass Spectrometry: Principles and Clinical Applications. The

Clinical Biochemist Reviews, 2003, 24(1), 3–12.5Georgita, C.; Sora, I; Albu, F.; Monciu, C. M. Comparison of a LC/MS Method with a LC/UV

Method for the Determination of Metformin in Plasma Samples. Farmacia. 2010, 58(2),

158-169.6Bijlsma, L.; Betran, E.; Boix, C.; Sanch, J. V.; Hernandez, F. Improvements in analytical

methodology for the determination of frequently consumed illicit drugs in urban

wastewater. Anal. Bioanal. Chemistry, 2014, 406, 4261-4272.

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Appendices

Appendix 1: C-18 column silicate bead

Appendix 2: Illicit Drug Structures and Molecular Weights

Appendix 3: Ion Formation and m/z Ratios

M +nH+¿ yields

→[M+nH ]n+¿¿ ¿

M yields→

[ M−nH ]n−¿+nH+¿¿ ¿

Appendix 4: LC program

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Appendix 5: Calibration Curves of all four standards

***Included error bars of data collected in duplicate are smaller than the area of the dot

displaying the averaged data.

Appendix 6: Determination of LOD and LOQ for methamphetamine

Sy=1135.1

S=594.60

LOD=3(S y

S)=3( 1135.1

594.60 )=5.7272

L OQ=10 ( 1135.1594.60 )=19.091

From Peak Area to Concentration: LOD=5.7272+4313.1594.6

=7.26 mg /mL

Appendix 7: Calculations of for methamphetamine in unknown 1

Peak Area=595.11 (Conc . )−4357.1

45918=595.11 (Conc . )−4357.1

Concentration=84.48 mg /mL

Amount=(84.48mg /mL )/(10 mL)

¿0.85 mg

Percent Error=¿

¿¿

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