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 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.
- National Institute on Drug Abuse, Research Report Series, Methamphetamine Abuse and Addiction.
- Drug Data Summary, Office of National Drug Control Policy (2003).
- Drug Enforcement Administration, "Drug Trafficking in the United States."
- National Drug Threat Assessment, NDIC/USDOJ (2003).
- Pseudoephedrine Notice, Office of Division Control, US Department of Justice, Drug Enforcement Administration.
- "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).
- "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.