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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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