Application

Note: 419
Quantitative Analysis of Pseudoephedrine
Tablets by UHPLC/MS
Guifeng Jiang, Ray Chen, and Chris Loran; Thermo Fisher Scientific, San Jose, CA, USA

Introduction Experimental Conditions

Key Words Pseudoephedrine is frequently used as a decongestant in Standard Preparation
• Accela™ common cold products including Sudafed®, Advil® Cold, Ephedrine, pseudoephedrine, amphetamine, methampheta-
Drixoral®, Benadryl®, Aleve®, and Claritin®. Pseudoephedrine mine, and 3,4-MDMA standards (1 mg/mL in methanol)
• MSQ Plus™ hydrochloride and sulfate salts are found in these products were purchased from Alltech-Applied Science (State College,
• Illicit Drug either as single-ingredient preparations or, more com- PA, USA). Figure 1 displays the chemical structures of these
monly, in combination with antihistamines, naproxen, five compounds. Calibration standards were prepared by
• MDMA paracetamol (acetaminophen), and/or ibuprofen. mixing all five compounds and then diluting the stock
• Methamphetamine Pseudoephedrine and ephedrine, both List I chemicals, solution with methanol to a series of concentrations
are highly coveted by drug traffickers who use them to before analysis.
manufacture methamphetamine, a Schedule II controlled
substance, for the illicit market. The diversion of over-the- Sample Preparation
counter pseudoephedrine-containing products is one of the Three common over-the-counter cold products (brand
major contributing factors to the methamphetamine situa- names A, B, and C) containing pseudoephedrine as one
tion in the United States.1 The separation and identification of the active ingredients were analyzed.
of pseudoephedrine from illicit drug mixtures, especially Assay solution A: The cold tablet A was disintegrated
the methamphetamine group compounds including amphet- and then dissolved in 20 mL methanol. The mixture was
amine, methamphetamine, and 3,4-methylenedioxy-N- sonicated for 15 minutes. A portion of the supernatant
methamphetamine (3,4-MDMA), will help to identify the was centrifuged at 12,000 RPM for 3 minutes and the
sources and the manufacturing pathway of the methamphet- clear supernatant was diluted to approximately 120
amine seized in the illicit market. ng/mL with methanol.
The Thermo Scientific Accela ultra high performance Assay solutions B and C: The appropriate cold tablet
liquid chromatograph (UHPLC) performs separations 5 (B or C) was disintegrated and then dissolved in 20 mL
to 10 times faster than conventional HPLC by employing methylene chloride. The mixture was sonicated for 15
sub-2 µm diameter stationary phases and ultra high minutes. A 1 mL sample of the slurry was diluted to
pressure instrumentation. The 1 to 2 second peak widths 20 mL with methanol. An aliquot of this solution was
and relatively high mobile phase flow rates that are typical centrifuged at 12,000 RPM for 3 minutes and a portion
of UHPLC methods demand a robust, fast scanning MS of the clear supernatant was diluted to approximately
detector. The fast scanning Thermo Scientific MSQ Plus 120 ng/mL with methanol.
single quadrupole mass spectrometer meets this UHPLC
Chromatographic Conditions
requirement and has been used for similar applications.2
Instrument: Thermo Scientific Accela UHPLC system
This application note describes an effective UHPLC/MS
Column: Hypersil GOLD™ PFP (perfluorinated phenyl)
method for high throughput separation, identification,
1.9 µm, 100 × 2.1 mm (Thermo Fisher Scientific,
and quantitation of pseudoephedrine.
Bellefonte, PA)
Flow Rate: 1 mL/min
Mobile phase: A: Water with 0.06% acetic acid
B: Acetonitrile (ACN) with 0.06% acetic acid

Ephedrine (165.23) Pseudoephedrine (165.23) Amphetamine (135.21) Methamphetamine (149.23) MDMA (193.24)

Figure 1: Chemical structures of the five drug compounds

Gradient: Pseudoephedrine was identified as the major
Gradient I: Separation of five drugs active ingredient for all three brand name drugs by the
t (min) 0 1.5 3.5 3.51 3.99 4.0 5.0 UHPLC/MS method. The chromatograms are illustrated
A (%) 99 99 60 5 5 99 99 in Figure 2B-D. The retention times of 2.62 minutes for
B (%) 1 1 40 95 95 1 1 all three samples matched very well with the retention
time of the pseudoephedrine standard at 2.60 minutes.
Gradient II: Sensitivity evaluation and quantitation
The confirmation of pseudoephedrine at 2.6 minutes was
t (min) 0 3.0 3.1 3.9 4.0 5.0
further assured by the match of the MS spectra of the
A (%) 90 75 5 5 90 90
three samples (Figure 3B-D) with the pseudoephedrine
B (%) 10 25 95 95 10 10
standard (Figure 3A).
Injection volume: 1 µL partial loop injection,
25 µL loop size
Column Temperature: 45°C
Maximum column backpressure observed: 750 bar

Mass Spectrometer Conditions

Instrument: Thermo Scientific MSQ Plus
Ionization: Electrospray (ESI)
Polarity: Positive
Probe Temperature: 450°C
Cone Voltage: 55.0 V
Scan Mode: 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

Results and Discussion

Separation, MS Detection, and Pseudoephedrine
Confirmation
The separation of the five drug compounds is shown in
Figure 2A. Ephedrine elutes first at 2.41 minutes, followed Figure 2: Chromatograms of drug standards (A), with the elution order
of ephedrine, pseudoephedrine, amphetamine, methamphetamine and
by pseudoephedrine at 2.60 minutes, amphetamine at
3,4-MDMA, and assay samples (B-D), extracted from over-the-counter
2.93 minutes, methamphetamine at 3.38 minutes, and cold products with brand names A, B, and C.
3,4-MDMA at 3.64 minutes. The
analytes are baseline separated with
excellent peak efficiency and resolu-
tion. 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, pseudo-
ephedrine, 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.

Figure 3: MS spectra of pseudoephedrine standard (A) and assay samples (B-D) from over-the-counter (OTC)
drugs with brand names A, B, and C. Molecular ion of pseudoephedrine at m/z 166.18 was the major MS
peak. Fragmentation ion of pseudoephedrine at m/z 148.17, created by the in-source CID of the MSQ Plus
with a cone voltage of 55 volts, provided the additional MS confirmation of the assay samples.
Calibration Curve and Sensitivity Quantitation Using Internal Standard
Calibration curves of the five drug standards were An internal standard method was used for the quantitative
constructed over the concentration range of 1.25 to 1667 determination of pseudoephedrine in its tablet form.
ng/mL (equivalent to 1.25 to 1667 pg on column) with Amphetamine was used as an internal standard (100
10 calibration levels (Figure 4). Each calibration level ng/mL). It was added to the freshly prepared assay sample
was injected three times and the mean area responses solutions and pseudoephedrine standard solutions at 50
were plotted against the concentrations. Correlation ng/mL, 100 ng/mL, 150 ng/mL, and 200 ng/mL. Six
coefficients with R2 = 0.996 or better were achieved for repeated injections for each standard and assay solution
the five drug standards. were conducted. Pseudoephedrine and amphetamine were
The limit of quantitation (LOQ) and the limit of well separated in four standard solutions at 1.73 minutes
detection (LOD) of the five drug compounds were deter- and 2.13 minutes (Figure 5A-D). The chromatograms of
mined based on the calibration curve of signal-to-noise three assay samples are shown in Figure 5E-G. The
ratio versus concentration and the definitions of LOQ and concentrations of the assay samples were determined
LOD using s/n = 10 and 3. LOQs for all five drugs ranged based on the calibration curve of peak area ratio against
from 0.96 to 1.7 ng/mL, while LODs ranged from 0.29 to concentration (Figure 6). Excellent linearity with a
0.53 ng/mL (Table 1). The outstanding sensitivity of this correlation coefficient of R2 = 0.997 was obtained. The
method was highlighted by the achievement of picogram experimental concentrations of the assay solutions were
level quantitation for all five analytes. in good agreement with the reported values (Table 2).

Figure 5: Chromatograms of four pseudoephedrine standard bracket solutions

at 50 ng/mL (A), 100 ng/mL (B), 150 ng/mL (C) and 200 ng/mL (D), and three
assay sample solutions from cold products with brand name A (E), brand name
B (F), and brand name C (G). Pseudoephedrine was eluted at 1.73 min. Amphet-
amine (100 ng/mL), the internal standard, was added and eluted at 2.13 min.
Figure 4: Calibration curves constructed for five drug standards at 1.25, 2.5,
5.0, 10, 25, 100, 250, 500, 1000 and 1667 ng/mL (ppb).

LOQ (ng/mL) LOD (ng/mL)

ephedrine 1.21 0.36
pseudoephedrine 1.25 0.38
amphetamine 1.78 0.53
methamphetamine 0.96 0.29
3,4-MDMA 1.09 0.33
Table 1: LOQ and LOD of the five drug compounds with 1 µL sample injection

Figure 6: Calibration curve for pseudoephedrine quantitation using

internal standard method.
 In addition to these
Experimental Reported
value value %RSD offices, Thermo Fisher
(mg/tablet) (mg/tablet) % Recovery (n = 6) Scientific maintains
Brand name A 120.09 120 100.1 1.9 a network of represen-
Brand name B 112.33 120 93.6 5.6 tative organizations
Brand name C 104.49 120 87.1 2.3 throughout the world.

Table 2: Pseudoephedrine quantitation using amphetamine as internal standard

Conclusions
A simple, fast, and reliable separation and identification Africa
method for five drugs (pseudoephedrine, ephedrine, +43 1 333 5034 127
Australia
amphetamine, methamphetamine, and 3,4-MDMA) +61 2 8844 9500
using UHPLC/MS was developed. The ppb (ng/mL) level Austria
sensitivity and accuracy of this method provides great +43 1 333 50340
opportunity to identify and quantify pseudoephedrine Belgium
+32 2 482 30 30
and/or other components in illicit drug samples. It also
Canada
offers an efficient tool to determine the source and manu- +1 800 530 8447
facturing pathway of drugs seized in the illicit market. China
+86 10 8419 3588
Denmark
References +45 70 23 62 60
1
Pseudoephedrine Notice, Office of Division Control, US Department of Europe-Other
Justice, Drug Enforcement Administration. +43 1 333 5034 127
2
G Jiang, R. Chen, and C. Loran, Quantitative Measurement of Simvastatin France
Using High Speed LC/MS, Application Note 402, Thermo Fisher Scientific, +33 1 60 92 48 00
San Jose, CA, USA. Germany
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