J Forensic Sci, July 2016, Vol. 61, No.

4
doi: 10.1111/1556-4029.13095
CASE REPORT Available online at: onlinelibrary.wiley.com

CRIMINALISTICS

Maria Cristina Gaudiano,1 Ph.D.; Anna Borioni,1 Ph.D.; Eleonora Antoniella,1 Ph.D.; and
Luisa Valvo,1 Ph.D.

Counterfeit Adderall Containing Aceclofenac

from Internet Pharmacies*

ABSTRACT: A nontargeted approach based on liquid chromatography equipped with a quadrupole time-of-flight mass detector (LC-MS Q-
TOF) joined to nuclear magnetic resonance (NMR) analysis allowed rapid identification and quantification of the anti-inflammatory drug ace-
clofenac in illegal Adderall tablets. The largest chromatographic peak had m/z = 354.030 and m/z = 376.012 matching, respectively, the ionic
structures (M + H)+ and (M + Na)+ of a molecule M. The accurate mass data generated the molecular formula C16H13Cl2NO4. A screening of
the pharmaceutical active substances having that molecular formula together with the MS/MS fragmentation pattern suggested aceclofenac. Ace-
clofenac structure was unambiguously confirmed by 1H and 13C NMR experiments. The aceclofenac content was 90 mg/tablet (RSD 2%) as
detected by quantitative NMR. Information on the identity and content of illegal drugs is required for legal purposes; it supports in evaluating
the effective impact on users safety, and it is useful for control laboratories using a targeted approach in their analytical activities.

KEYWORDS: forensic science, counterfeit medicine, Adderall, mass spectrometry, nuclear magnetic resonance, aceclofenac

Counterfeit drugs are products deliberately and fraudulently
manufactured to be mistaken for legitimate drugs. Counterfeiting
of medicinal products has rapidly become a global public health
threat owing to a number of factors including the proliferation of
illegal online drug sellers.
Counterfeit drugs may contain only excipients, incorrect ingre-
dients, inappropriate dosages, subpotent or superpotent active
substances, or be contaminated. Consumers face copious risks as
a result of taking counterfeit drugs: even if a counterfeit drug is
not intrinsically dangerous, it may still cause tremendous damage
upon patients in terms of treatment failure (1–9).
Adderallâ is a prescription-only medicine approved for the
treatment of attention-deficit/hyperactivity disorders (ADHD)
and narcolepsy in children and adults; it contains a combination
of nervous system stimulants such as amphetamine and dex-
troamphetamine (10,11).
The illicit demand for amphetamines is mainly concentrated
among younger people who often believe, as revealed by several
blogs, that they increase mental activity and give a general sense
of euphoria while being harmless (12).
In 2011, Adderall was inserted in the USA Food and Drug
Administration (FDA) website shortages list owing to increased
demand and to active ingredients supply issues and, in 2012, the
FDA first warned about a counterfeit version of Teva Adderall
30 mg tablets bought over the Internet (13).
1
Dipartimento del Farmaco, Istituto Superiore di Sanita, Viale Regina
Elena 299, 00161 Rome, Italy.
*Supported by a grant of the Italian Medicines Agency (Convenzione
AIFA-ISS).
Received 23 Dec. 2014; and in revised form 30 Sept. 2015; accepted 24
Oct. 2015.
The investigation conducted on a suspicious parcel containing
a ten tablets blister labeled as Teva Adderall 30 mg is here
reported. The parcel, mailed from an Asian country to a private
citizen, was seized by the Italian customs during the interna-
tional operation PANGEA (1,14). To better identify the risks
from the counterfeit drug, the content of tablets was investigated
by a nontargeted approach based on liquid chromatography
equipped with a quadrupole time-of-flight mass detector (LC-MS
Q-TOF) and nuclear magnetic resonance (NMR). The tablets
resulted to contain a therapeutic quantity of aceclofenac (per
tablet) in place of the declared amphetamines.
Aceclofenac is a nonsteroidal anti-inflammatory drug; it is a
prescription-only medicine indicated for the relief of pain and
inflammation in osteoarthritis, rheumatoid arthritis, and ankylos-
ing spondylitis. Aceclofenac leaflet reports several contraindica-
tions, side effects, and interactions with other medicinal
products. In contrast to Adderall, aceclofenac is not recom-
mended for use in children and adolescents (15).
Materials and Methods
LC-MS Q-TOF analysis was performed using a Fast HPLC
Mod.1290 Infinity equipped with a diode array detector and a
Dual ESI source Q-TOF mass spectrometer detector Mod.
G6520B (Agilent Technologies, Santa Clara, CA). Data were
processed using the MassHunterâ Workstation Software Qualita-
tive Analysis, version B.03.01 (2009). NMR experiments were
recorded on a Bruker Avance instrument (Bruker Italia, Milan,
Italy) operating at 400 MHz equipped with a 5-mm 1H/13C BBI
probe. Data were processed by the Bruker Topspin 1.2 software.
LC-MS grade solvents and aceclofenac reference standard
were purchased from Sigma (Sigma-Aldrich, St. Louis, MO).
Deuterated NMR solvents were purchased from Eurisotop
(Saint-Aubin, France).

1126 © 2016 American Academy of Forensic Sciences

 GAUDIANO ET AL. . NEW COUNTERFEIT ADDERALL FROM ILLEGAL ON-LINE PHARMACIES 1127

Visual Inspection of the Sample completely removed by evaporation in a nitrogen flow. The
whole procedure was repeated on three independent weightings
A careful visual inspection of the labels of the packaging and to obtain three samples for quantitative NMR (qNMR)
of the shape and color of the seized tablets was carried on. analysis.
Adderall is not available on the Italian market; therefore, a com- Deuterated DMSO and an accurately weighted quantity of
parison with samples of the authentic product was not feasible. 3,5-dinitrobenzoic acid (DNB) TraceCERTâ CRM were added
However, images and information on the physical characteristic to each sample. The signal at 6.25 ppm (1H, doublet) was
of the authentic Adderall were obtained from the US FDA warn- integrated for aceclofenac and the signals at about 8.88 ppm
ing report. (3H, doublet and singlet) of DNB were integrated as quantita-
tive reference. 3,5-DNB was selected as reference standard
Sample Preparation and LC-MS Q-TOF Analysis because it exhibits deshielded NMR signals (8.9 ppm) not
interfering with the aceclofenac signals. Aceclofenac signal at
One tablet (about 380 mg) was ground and extracted with 6.2 ppm was the only signal in the spectrum adequately
100 mL methanol using stirring and sonication. After centrifuga- resolved for quantification. Aceclofenac content was calculated
tion, the supernatant was removed, diluted 50 times by addition from spectral data according to the relevant qNMR formula
of a 1:1 v/v acetonitrile/water solution containing 0.1% formic (16).
acid, and used for LC-MS Q-TOF analysis. A nontargeted drug
screening was operated using a quadrupole time-of-flight mass
spectrometer, the mass detector was set in both positive and neg-
ative ions conditions. (a)
The mass instrumental parameters were as follows: nitrogen
temperature 300°C, drying gas 10 L/min, nebulizer 40 psig, and
fragmentor 100 V. The LC analysis was accomplished using a
linear gradient program (starting with 0.1% formic acid in water/
acetonitrile 95:5 v/v to 0.1% formic acid in water/acetonitrile
5:95 v/v in 15 min) at a flow rate of 0.4 mL/min and an injec-
tion volume of 1 lL on a C18 column (Extend, 2.1 9 50 mm,
1.8 micron, Agilent).
The chromatogram was recorded in total ion count and in UV
at 220, 240, 254, 280, and 350 nm wavelengths.
The MS/MS spectrum of the peak was acquired at the colli-
sion offset voltage of 10, 30, and 40 V in positive ions condi-
tions (ESI+) and at the collision offset voltage of 30 V in
negative ions conditions (ESI
). (b)

Sample Preparation and NMR Analysis

The NMR experiments were performed on deuterated metha-
nol extracts of the ground tablets. Structure determination was
based on the results of 1H and 13C monodimensional experi-
ments, 2D 1H–1H magnitude COSY, 2D 1H–1H phase sensitive
NOESY, 2D 1H–13C HSQC with improved sensitivity, and mag-
nitude 2D 1H–13C HMBC experiments. Spectra were recorded
on 4 mg of powder dissolved in 0.7 mL of deuterated methanol
at a temperature of 298 K. Residual methanol signal was par-
tially suppressed by presaturation. The NOESY experiment was
recorded using 600 ms mixing time; a 1H–13C coupling constant
of 145 Hz was applied in HSQC sequence; magnitude HMBC (c)
was recorded in non decoupled mode with long range 1H–13C
coupling constants of 10 Hz.
For quantitative analysis, three tablets were accurately
weighted and ground together to obtain a homogeneous powder.
About 30 mg of the powder was accurately weighted and trans-
ferred to a centrifuge tube. After addition of acetonitrile, the
resulting suspension was sonicated, vortexed and centrifuged,
then the supernatant was quantitatively transferred to a second
test tube. To optimize quantitative recovery of the active ingredi-
ent, the extraction procedure was repeated two more times on
the same portion of the insoluble residue. All the supernatants
were pooled and evaporated to dryness.
To eliminate any acetonitrile interference in the NMR spec-
tra, the solid extract was treated with 1 mL of deuterated ace- FIG. 1––Counterfeit adderall seized by customs at Malpensa Airport in
tonitrile and gently vortexed for 5 min. Finally, acetonitrile was Italy. The low quality of blister printing was a characteristic of the sample.
1128 JOURNAL OF FORENSIC SCIENCES

After the visual inspection, chemical analysis was required to

Results and Discussion
The careful visual inspection of the product packaging, label-
ing, and appearance is the first step in the classification of a
medicinal product as genuine or counterfeit. In the present case,
the visible markers on the drug packaging—hardly readable
chemical composition on the right side of the blister and blurry
illegible writings on the left side—indicated that the medicinal
product was a counterfeit (Fig. 1a, b). Moreover, the required
information on packaging for licensed medicines such as the
product manufacturing country was missing, and no leaflet was
available.
The counterfeit tablets had a white tinge and no debossing
(Fig. 1c), while the authentic Teva Adderall 30-mg tablets are
round, orange/peach in color, and embossed with the letters “dp”
on one side and the number “30” on the other side. The suspi-
cious sample showed different also in comparison with the coun-
terfeit Adderall reported by the US FDA which contained the
anti-inflammatory active substances tramadol and acetamino-
phen. In fact, in the present case, the National Drug Code (NDC
0555-0768-02) and the words “Aspartate” and “Single” were
correctly printed on the blister while the US FDA warning
reported misspelling of the same words as “NDS”, “Aspartrte,”
and “Singel” (13).
determine the chemical composition of the tablets to evaluate the
potential danger for the patients to establish an adequate penalty
for suppliers and also to follow the trend on the illegal market.
Accurate LC-MS Q-TOF and NMR analyses for rapid
unambiguous identification and quantification of the content of
the seized material were carried on.
No amphetamines, as well as no tramadol nor acetaminophen
were detected in the tablets by LC-MS Q-TOF analysis (12).
Instead, the mass spectrum of the largest chromatographic peak
in the total ions count chromatogram displayed m/z = 354.030
and m/z = 376.012 corresponding, respectively, to the ionic
structures (M + H)+ and (M + Na)+ of a molecule (M) matching
the chemical formula C16H13Cl2NO4. A screening of pharmaceu-
tical active substances based on the results of the accurate MS
of the molecular ion suggested the presence of aceclofenac;
interpretation of the MS/MS fragmentation spectrum and the
comparison with literature reports further supported the presence
of aceclofenac in the seized tablets. (17–20). The MS results are
reported in Table 1.
In the first stage of identification, NMR experiments were per-
formed together with the LC-MS Q-TOF analysis to confirm
chemical structure beyond misinterpretations owing to the poten-
tial presence of isomers of aceclofenac. Hydrogen and carbon

TABLE 1––MS and MS-MS data of aceclofenac from the seized sample.

Cl Cl

NH O
NH
Cl O
OH Cl OH
O
O
A
Aceclofenac Diclofenac

MS ionic Molecular Masses Main MS/MS Ionic Fragments

10 V* (ESI+): 30 V* (ESI+):

Positive hydrogenated-ion molecular mass (ESI+): 250.018 (base peak) C13H10Cl2N+‡ 215.049 (base peak) C13H10ClN+
(M + H)+ = 354.030
Cl
+ + .
NH NH

Cl 2
Cl
+
Positive sodium-ion molecular mass (ESI ): Other fragments: Other fragments:
(M + Na)+ = 376.012 278.013 (~80%) C14H10Cl2NO+ (A) 214.041 (~80%)
215.049 (~50%) 186.911 (~10%)
40 V*(ESI+): 30 V*(ESI
):

Negative dehydrogenated-ion molecular mass (ESI
): 214.042 (base peak) C13H9ClN+§ 78.959 (base peak)
(M
H)
= 352.015
Suggested formula Other fragments: Other fragments:
(M) = C16H13Cl2NO4 215.049 (~80%) 242.881 (~99%)
186.911 (~10%) 75.009 (~90%)
96.970 (~22%)
220.899 (~18%)
*Collision offset voltage.
‡
The structures in the table were proposed in reference 18; moreover, other possible structures were reported in the literature.
§
For fragment at m/z = 214, the structure C10H10Cl2N+ proposed in reference 20 appears less probable than that here proposed on the base of the
measured accurate mass.
 GAUDIANO ET AL. . NEW COUNTERFEIT ADDERALL FROM ILLEGAL ON-LINE PHARMACIES 1129

TABLE 2––NMR data of aceclofenac from the seized sample. tablets thus providing valuable information as regards the actual
impact on users’ safety. Such information is also a benefit for
3"
2" Cl those laboratories that use a targeted approach to defeat counter-
4" feiting.
1"
5"
NH O Conclusions
6"
2'
Cl 3 O Counterfeiting is widespread worldwide. Accordingly, there is
3' 4 1 OH
1' 2 an increasing need for a wide-ranging straightforward approach
4' 6' O to identify and quantify substances in counterfeit drugs. Informa-
5' tion on the identity and content of illegal drugs is required to
1
help in determining sentences for offenders and in evaluating the
H NMR Experiments
effective impact on users safety; moreover, information sharing
1D 1H 2D 1H,1H 2D 1H,1H is highly convenient for control laboratories using a targeted
COSY NOESY approach in their analytical activities. In this case, aceclofenac
Position d (ppm)* Integral, Cross-peak Cross-peak was detected in a nearly therapeutic dosage (90 mg/tablet) in
multiplicity
pattern counterfeit Adderall tablets by a general nontargeted analytical
approach.
4-CH2 3.96 2H,s This work showed that, once more, an unexpected active sub-
2-CH2 4.69 2H,s stance was added to a counterfeit medicinal product. The una-
30 -H 6.44 1H,d 40 -H 40 -H
50 -H 6.95 1H,t 60 -CH2 ware intake of aceclofenac according to Adderall indications for
40 -H and 4″-H 7.11–7.08 2H,m 30 -H; use could cause severe side effects in terms of gastrointestinal
3″-H, 5″-H bleeding or perforation. Worse still, something not recommended
60 -H 7.28 1H,t 4-CH2 for use in children and adolescents was illegally introduced in a
3″-H, 5″-H 7.42 2H,d 4″-CH2
drug approved for use in children over 3 years of age. Finally,
13
C NMR Experiments the results of this case report rise some points that physicians
could consider in their activity. Is there a high percentage of
13
1D C 2D 1H,13C 2D 1H,13C patients buying psychotropic medicines without any prescription
HSQC HMBC over the Internet? To what extent is Adderall purchased over the
Position d (ppm)* Cross-peak Cross-peak
4-CH2 37.2 4-CH2 with 60 -H Internet not for ADHD or narcolepsy treatment, but because it
2-CH2 60.9 2-CH2 contains neurologic stimulants as amphetamines? Is there an
C30 117.5 30 -H increasing risk of misuse of nootropics to enhance cognitive per-
C50 121.6 50 -H formances?
C10 124.2 4-CH2
C40 124.5 50 -H and 60 -H
C4″ 127.7 4″-H Acknowledgments
C3″ + C5″ 128.7 3″-H. 5″-H
C6″ + C2″ 129.9 3″-H. 5″-H The Authors thank the Italian Medicines Agency and the
C60 130.8 60 -H 4-CH2 national taskforce IMPACT Italia for their fruitful cooperation.
C1″ 137.9 3″-H. 5″-H
C20 142.9 4-CH2, H-40 ,
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Additional information and reprint requests:
Maria Cristina Gaudiano, Ph.D.
Dipartimento del Farmaco
Istituto Superiore di Sanita
Viale Regina Elena 299
00161 Rome
Italy
E-mail: mariacristina.gaudiano@iss.it
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