A look at how the separation and identification of pseudoephedrine from illegal drug mixtures can help to identify the sources and the manufacturing pathway of methamphetamine seized in the illicit market.

Methamphetamine (commonly known as "meth") is a powerful and highly addictive stimulant that can irrevocably damage people, families, and communities. A Schedule II controlled substance, it is a white, bitter-tasting powder that can be snorted, injected, smoked, or dissolved in water or alcohol. As with most long-term hard drug use, chronic meth abuse quite literally rots the body and the brain. It causes aggression and violence, psychotic behavior, memory loss, heart damage, severe dental problems, and malnutrition, not to mention the increased transmission of infectious diseases such as HIV and hepatitis among users, and a variety of knock-on social effects including crime and unemployment.

Worryingly, meth production and abuse in the United States is on the rise; what was once a problem limited to Hawaii and western parts of the United States is now spreading eastwards to both rural and urban areas.1 According to the 2005 National Survey on Drug Use and Health, an estimated 10.4 million people aged 12 or older4.3% of the populationhave tried meth at least once. Approximately 1.3 million reported using meth in the past year, and 512,000 reported current (past-month) use. In 1998, meth abuse contributed to nearly 500 deaths. And between 1995 and 2002 there was a 50% increase in the number of emergency department visits linked to meth abuse, with a total of 73,000 meth-related emergency visits (4% of all drug-related visits) in 2004.1

Of the 1,490 clandestine drug synthesis laboratories seized by the Drug Enforcement Administration (DEA) in 2001, 97% of them were being used to make meth.2 While most of the meth in the United States comes from foreign or domestic "superlabs" that are concentrated in southern California and Mexico, there has been a dramatic increase in the number of mom-and-pop outfits run by amateur meth "cooks" out of garages and homes. The DEA reported that in 2001 over 7,700 laboratories had meth-producing capacities of less than ten pounds.3 These home-made laboratories pose dangerous risks not only to those running them, but also to neighbors and the environment. Because the chemicals involved are highly toxic and the gases produced very flammable, the manufacturing processes pose substantial risks of explosions, fires, chemical burns, and toxic fume inhalation. For every pound of meth that is produced, about five to seven pounds of toxic waste are generated, most of which is dumped into rivers, fields, and sewage systems. In California alone, the clean up of more than 2,000 meth laboratories and dumpsites in 2001 cost nearly $5.5 million.4

The Pseudoephedrine Problem
Key to the illegal manufacture of meth are pseudoephedrine and ephedrine, both List I chemicals, which are commonly used as decongestants in over-the-counter cough, cold, and allergy medicines. Pseudoephedrine and ephedrine are similar in structure to amphetamine and methamphetamine and can be reduced to these compounds relatively easily; as such they are highly coveted by drug traffickers. In fact, the diversion of over-the-counter pseudoephedrine-containing products is one of the major contributing factors to the meth situation in the United States.

Pseudoephedrine hydrochloride and sulfate salts are found in cold and allergy medicines as single-ingredient preparations or, more commonly, in combination with antihistamines, naproxen, paracetamol (acetaminophen), and/or ibuprofen. Typically a meth cook extracts pseudoephedrine by placing the over-the-counter product in a solution of water, alcohol, or another solvent for several hours and then converts it to high-quality meth using a variety of common household products and equipment. For drug enforcement agencies to get to the root of this production chain, they need to be able to identify the exact sources and manufacturing pathways of meth seized from clandestine laboratories. Crucial to this effort is the accurate separation and identification of pseudoephedrine from illegal drug mixtures and in particular from the meth group compounds including amphetamine, methamphetamine and 3,4-methylenedioxy-Nmethamphetamine (3,4-MDMA).

Straight to the Source
The analysis of most illicit drug mixtures begins by separating out the individual components using classical techniques such as gas or liquid chromatography. The combination of gas chromatography and mass spectrometry (GC/MS), which is extremely sensitive and provides outstanding identification power, is the typical screening method used for controlled substances. GC/MS relies on the selective adsorption and desorption of volatile components in a stationary phase. Different components are carried through a column by an inert gas to a detector (in this case amass spectrometer), and are identified based on their retention time and mass spectrum.

Such an approach, however, does not provide molecular structural information and cannot be used as a principle means of identification. GC/MS cannot distinguish pseudoephedrine from its sister ephedrine; their GC retention times and mass spectra are essentially the same because the two compounds differ only in their stereochemistry (they possess exactly the same chemical make-up and differ only in the orientation of a side chain on one carbon atom).

In order to achieve structural discrimination, scientists have combined infrared spectroscopy and GC/MS. In this method, the effluent of the GC column, with the separation complete, is directed onto a light pipe through which infrared light is shone. The infrared radiation provides structural information about the intact form of the molecules under study and is able to pick up subtle variations in different structural forms. In this way, pseudoephedrine can be distinguished from ephedrine. While highly selective, this approach is still not as sensitive as GC/MS.

High-performance liquid chromatography (HPLC) has been used to successfully profile pseudoephedrine content in meth.6,7 HPLC uses a liquid mobile phase to transport and separate the various constituents of a mixture. The mixture is injected into a column packed full of a stationary phase under high pressure and is resolved into its various components via absorption and desorption. While useful for separating out compounds that are too non-volatile to be separated by GC, traditional HPLC suffers from a lack of resolving power as compared to conventional GC.

The advent of ultra-high-performance liquid chromatography (UHPLC) heralded in a competitive alternative to traditional GC and a new tool for forensic scientists. This approach employs columns packed with particles that have diameters less than 2μm, and ultra-high-pressure instrumentation. The relatively high mobile phase flow rates associated with UHPLC enable separations to be performed up to 10 times faster and with improved resolution. Combined with mass spectrometry, UHPLC offers significant improvements in sensitivity over conventional HPLC.

The following analysis describes how UHPLC/MS can be used to perform high-throughput separation, identification and quantitation of pseudoephedrine.

chromarograms of drug standards

chromatograms of four pseudoephedrine standard solutions

Analysis of Pseudoephedrine: Experimental Conditions
Standard Preparation
Ephedrine, pseudoephedrine, amphetamine, methamphetamine, and 3,4-MDMA standards (1 mg/mL in methanol) were used. Calibration standards were prepared by mixing all five compounds and then diluting the stock solution with methanol to a series of concentrations before analysis.

Sample Preparation
Three common over-the-counter cold products containing pseudoephedrine as one of the active ingredients (labeled A, B, and C) were analyzed.

Assay solution A: Cold tablet A was disintegrated and then dissolved in 20 mL methanol. The mixture was sonicated for 15 minutes. A portion of the supernatant was centrifuged at 12,000 RPM for 3 minutes and the clear supernatant was diluted to approximately 120 ng/mL with methanol.

Assay solutions B and C: The appropriate cold tablet (B or C) was disintegrated and then dissolved in 20 mL methylene chloride. The mixture was sonicated for 15 minutes. A 1-mL sample of the slurry was diluted to 20 mL with methanol. An aliquot of this solution was centrifuged at 12,000 RPM for three minutes and a portion of the clear supernatant was diluted to approximately 120 ng/mL with methanol.

Chromatographic Conditions
Instrument: Thermo Scientific Accela UHPLC system
Column: Hypersil GOLD™ PFP (perfluorinated phenyl) 1.9 μm,
100×2.1 mm
Flow Rate: 1 mL/min
Mobile phase: A:Water with 0.06% acetic acid
...........................B:Acetonitrile (ACN) with 0.06% acetic acid
Injection volume: 1-μL partial loop injection, 25-μL loop size
Column Temperature: 45°C
Maximum column back pressure observed: 750 bar

Mass Spectrometer Conditions
Instrument: Thermo Scientific MSQ Plus single quadrupole mass spectrometer
Ionization: Electrospray (ESI)
Polarity: Positive
Probe Temperature: 450°C
Cone Voltage: 55.0 V
ScanMode: Full scan with mass range of 100 to 200 m/z or selected
ion monitoring (SIM) at m/z 150.18, 166.18, 177.15, and 194.15.
ESI Voltage: 4.5 kV
Scan Time: 0.2 s

LOQ and LOD of 5 drug compounds

Results and Discussion
Separation, MS Detection, and Pseudoephedrine Confirmation
The separation of the five drug compounds is shown in Figure 1A. Ephedrine elutes first at 2.41 minutes, followed by pseudoephedrine at 2.60 minutes, amphetamine at 2.93 minutes, methamphetamine at 3.38 minutes, and 3,4-MDMA at 3.64 minutes. The analytes are baseline separated with excellent peak efficiency and resolution. The MS spectra of the drug standards show both [M+ACN+H]+ and [M+H]+ion signals. The most abundant ions are the [M+H]+ions at m/z 166.18, 166.18, 150.18, and 194.15 for ephedrine, pseudoephedrine, methamphetamine, and 3.4-MDMA, respectively. The [M+ACN+H]+ion has the most intense signal at m/z 177.15 for amphetamine. Full scans (100 to 200 m/z) were employed for the confirmation of the five compounds and SIM modes were used for sensitivity and quantitation studies.

Pseudoephedrine was identified as the major active ingredient for all three brand-name drugs by the UHPLC/MS method. The chromatograms are illustrated in Figure 1B-D. The retention times of 2.62 minutes for all three samples matched very well with the retention time of the pseudoephedrine standard at 2.60 minutes. The confirmation of pseudoephedrine at 2.6 minutes was further assured by the match of the M'S spectra of the three samples with the pseudoephedrine standard.

calibration curve for pseudoephedrine quantitation

Calibration Curve and Sensitivity
Calibration curves of the five drug standards were constructed over the concentration range of 1.25 to 1667 ng/mL (equivalent to 1.25 to 1667 pg on column) with 10 calibration levels. Each calibration level was injected three times and the mean area responses were plotted against the concentrations. Correlation coefficients with R2 = 0.996 or better were achieved for the five drug standards.

The limit of quantitation (LOQ) and the limit of detection (LOD) of the five drug compounds were determined based on the calibration curve of signal-to-noise ratio versus concentration and the definitions of LOQ and LOD using s/n = 10 and 3. LOQs for all five drugs ranged from 0.96 to 1.7 ng/mL, while LODs ranged from 0.29 to 0.53 ng/mL (Table 1). The outstanding sensitivity of this method was highlighted by the achievement of pg-level quantitation for all five analytes.

Quantitation Using Internal Standard
An internal standard method was used for the quantitative determination of pseudoephedrine in its tablet form. Amphetamine was used as an internal standard (100 ng/mL). It was added to the freshly prepared assay sample solutions and pseudoephedrine standard solutions at 50 ng/mL, 100 ng/mL, 150 ng/mL, and 200 ng/mL. Six repeated injections for each standard and assay solution were conducted. Pseudoephedrine and amphetamine were well separated in four standard solutions at 1.73 minutes and 2.13 minutes (Figure 2A-D). The chromatograms of three assay samples are shown in Figure 2E-G. The concentrations of the assay samples were determined based on the calibration curve of peak area ratio against concentration (Figure 3). Excellent linearity with a correlation coefficient of R2 = 0.997 was obtained. The experimental concentrations of the assay solutions were in good agreement with the reported values (Table 2).

Meth abuse, arguably a scourge on society, is on the increase in the United States. The diversion of over-the-counter pseudoephedrine-derived products into illegal meth production laboratories is an important contributor to the problem and presents drug enforcement agencies with a major challenge. Separating and identifying pseudoephedrine from seized drug samples represents a key step in the fight against meth.

A simple, fast, and reliable method based on UHPLC/MS has been developed to effectively separate and identify pseudoephedrine, ephedrine, amphetamine, methamphetamine and 3,4-MDMA. The ppb-level (ng/mL) sensitivity and accuracy of this method offers an ideal opportunity to identify and quantify pseudoephedrine and/or other components in illicit drug samples. It also offers an efficient tool to determine the source and manufacturing pathway of drugs seized in the illicit market.


  1. National Institute on Drug Abuse, Research Report Series, Methamphetamine Abuse and Addiction.
  2. Drug Data Summary, Office of National Drug Control Policy (2003).
  3. Drug Enforcement Administration, "Drug Trafficking in the United States."
  4. National Drug Threat Assessment, NDIC/USDOJ (2003).
  5. Pseudoephedrine Notice, Office of Division Control, US Department of Justice, Drug Enforcement Administration.
  6. "Impurity profiling of ephedrines in methamphetamine by high-performance liquid chromatography", Yukiko Makino, Yasutera Urano & Tetsuo Nagano, Journal of Chromatography A, 947, 1, 151-154 (2002).
  7. "Separation of enantiomeric ephedrine and pseudoephedrine - high pressure liquid chromatography and capillary electrophoresis," R. M. Iwanicki et al., Journal of Forensic Sciences, 44, 3 (1999).


Guifeng Jiang, Ph.D., earned a B.S. and M.S. in Chemistry from Fudan University, Shanghai, China and her Ph.D. degree in Chemistry from the University of Alberta. She is currently a Strategic Marketing Specialist in the Clinical Research and Toxicology Group in SID Division, Thermo Scientific.

Her research interests have focused on the analytical system developing and prototyping, assay validation and chemical/biochemical analyses utilizing HPLC, mass spectrometry, and capillary electrophoresis.