Compact mass spectrometer (CMS) analysis of uniformly C-labeled amino acids
to identify authentic therapeutic drugs and protect against counterfeits
13Results:

Overview:

Methods and Materials:

Cost effective, multidimensional GRAS
(generally recognized as safe) counterfeit
protection of drugs based on uniformly 13Clabeled amino acids with compact mass
spectrometry detection.

Uniformly 13C labeled glutamine and cysteine were selected as GRAS compliant amino acids. They were dissolved inIntroduction:

ethanol at one mg/mL and Tic Tacs® (Ferrero Inc.) were placed shortly into the mixture, air dried and stored in an Eppendorf
tube for further processing (Figure 2). Samples were generated by dissolving the Tic Tac® in 0.1 N HCl at 50 mg/mL. 2-200µL sample was injected onto an Intrada Amino Acid 100 mm x 3 mm column (Imtakt USA, OR) with the following LC gradient

Authors: Daniel Eikel(1), Chris Beecher(2) and Simon J Prosser(1)Affiliations: (1) Advion Inc., Ithaca, NY, USA (2) IROA Technologies, Bolton, MA, USA

Conclusions:• Relative abundance ratios are defined by theuniformity of the label and the mixture ratio of the twobatches of e.g. Glutamine and can be used toserialize tablets by various combinations.

• LC/CMS shows the unique isotopic pattern andenriched A+1 signal, suggesting a 5% level
enrichment can reliably be distinguished by IROA
analysis.

• SIM mode shows higher sensitivity of the AA
detection and simpler visualization of the isotopicpattern.

• Example Tic Tac® tablets coated with uniformly 13C

settings: 0-3 min at 5 %B; 3-20 min from 5 to 95 %B; 20-22 min from 95 to 5 %B; 22-25 min at 5 %B, with Solvent Alabeled Glutamine and/or Cysteine can easily be
distinguished from each other by using only two
different levels of enrichment.

(acetonitrile with 0.1 vol% formic acid) and Solvent B (100 mM ammoniumformate) at a total flow rate of 500 µL/min.

• Counterfeit drugs are a significant healthrisk to consumers and an economic
burden to the pharmaceutical industry.

• Isotopic ratio outlier analysis (IROA)
utilizing compact mass spectrometry(CMS) is a simple and cost effective
analysis approach (Figure 1).

acids (95% U-13C Glutamine and 95% U-13C Cysteine).

Literature and Acknowledgements:
Figure 2: Tic Tac® example tablets treated with uniformly 13C labelled
Glutamine and Cysteine.

Figure 4: Isotopic distribution of glutamine with (A) 5 % and (B) 95 % uniform
C label. This specific pattern and relative abundance (IROA) allows forserialization of the drug coating, could be unique to the production batch, andcan not be mimicked by other means.

13

Figure 6:LC/CMS IROA analysis of a Tic Tac® coated with a mixture of 5% U-13C Glutamine and 95% U-13C

Under these conditions, the amino acid Glutamine eluted at Rt

Glutamine in (A) full scan and (B) SIM scan.

oxidation product Cystine at Rt 12.7 min (Figure 3) withcharacteristic 13C enriched isotopic patterns (example forFigure 8: Six Tic Tac® Tablets (T1 to T6) analyzed by LC/CMS for 13C-labeled Glutamine(red, left side) and 13C- labeled Cysteine (blue, right side).

Glutamine shown in Figure 4). The expression CMS wasL

operated in scan mode from m/z 120 to 250 with a scanFigure 3: Example of a 20 AA mixture (500 nmol each on column) separatedusing the expressionL CMS system in SIM detection mode. The discovery ion
chromatogram shows Glutamine and Cysteine (as Cys2 - Cystine) elute at Rt
11.4 and 12.7 min, respectively, (compare [3] for method development and[4] for discovery ion chromatogram (DIC) analysis).
mass spectrometer and gradient pump LC system (AdvionInc.) for IROA amino acid analysis from tablet examples.

time of 300 ms or in SIM mode with m/z 147.1, 148.1,149.1, 150.1, 151.1, 152.1 for Glutamine and 241.1,242.1, 243.1, 244.1, 245.1, 246.1 and 247.1 forCysteine (as oxidized Cystine) at dwell times of 50 ms andspan of m/z 0.3 each.

US Patent 8,969,251 Generation and use of isotopic
patterns in mass spectral phenotypic comparison oforganisms.
[2]
US Patent 8,536,520 Method for generation and use of
isotopic patterns in mass spectral data of simple
organisms.
[3]
Eikel D, Prosser SJ and Henion JD: Determination ofamino acids using a novel liquid chromatography
compact mass spectrometry system (LC/CMS) with
mixed-mode separation media. 63rd Meeting of theAmerican Society for Mass Spectrometry (ASMS) 2015,St. Louis, MO, USA[4]
Klecha L and Eikel D: Discovery Ion Current – A novel
approach to plot total ion current real-time byenhancing signal from newly detected ions. 65th
Meeting of the American Society for Mass Spectrometry(ASMS) 2017, Indianapolis, IN, USA[1]

of 11.4 min and Cysteine eluted and was detected as theFigure 1: LC/CMS set up utilizing an expressionL compact• Tic Tac® example tablets can easily be
distinguished using just two different
enrichment levels of the same AA or
using two different uniformly 13Clabeled AA, resulting in uniqueisotopicpatternsandrelativeabundances.
• Initial studies of different enrichmentlevels suggest that LC/CMS candistinguish 5% different enrichmentlevels which would translate into
millions of serialization steps based on
20 amino acids and 20 enrichmentlevels.

Figure 5: LC/CMS IROA analysis of a Tic Tac coated with a mixture of two different amino• A novel strategy for the uniqueserialization of genuine drugs can beachieved by addition of uniformly 13Clabelled amino acids (AA) with highly
patterned isotopic m/z signals [1,2].

• LC/CMS is a cost-effective IROAanalysis approach for uniformly 13Clabeled amino acids.

Figure 7: Isotopic distribution pattern of Glutamine with (A) natural abundance of 13C and (B)

5% uniform 13C label. This specific pattern and relative abundance (IROA) allows forserialization of the drug coating, could be unique to the production batch, and can not be
mimicked by other means.

We would like to thank Imtakt USA Corp. for thegenerous donation of an Intrada Amino Acid LC column.
This poster was presented at the 65th annual
conference of the American Society of Mass
Spectrometry (ASMS) 2017 in Indianapolis, IN, USA