Analytical Applications - Pharmaceutical Technology

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PharmTech Europe

Analytical Applications
Developing analytical methods and performing related testing is crucial for ensuring the quality of a pharmaceutical product. This analysis is applied to identify and characterize the active ingredient, the finished drug product, and impurities that may be present in the drug substance and finished drug product. In this technical forum, several industry experts offer case studies in pharmaceutical analysis.


Pharmaceutical Technology
pp. s32-s37, s41-43

Sharanya Reddy, PhD, application scientist with PerkinElmer, describes the use of time-of-flight–mass spectrometry for impurity analysis of an over-the-counter drug product. Robert Mattes, application scientist with Foss NIRSystems, and Stephen Hoag, professor, and Ravikanth Kona, PhD candidate, both in the Department of Pharmaceutical Sciences, University of Maryland, explain the use of in-line moisture analysis in a laboratory-scale fluid-bed dryer using diode array near-infrared spectroscopy. Jeffrey P. Kiplinger, president, Paul M. Lefebvre, director of laboratory operations, Michael J. Rego, staff scientist, and John H. Tipping, staff scientist, all with Averica Discovery Services, explain a chiral biotransformation analysis using supercritical fluid chromatography–mass spectrometry.

Impurity analysis with time-of-flight mass spectrometry

Sharanya Reddy, PhD, application scientist, PerkinElmer

Time-of-flight (TOF) mass spectrometry (MS) provides high resolution and exact mass information over a wide mass range to identify unknown compounds. This case study describes a workflow to identify impurities in over-the-counter (OTC) drugs using proprietary technology (TrapPulse, PerkinElmer) of the AxION 2 TOF mass spectrometer (PerkinElmer). In conventional orthogonal TOF instruments, the ions are lost between pulses. In the TrapPulse mode operation of the AxION 2 TOF mass spectrometer, the ions are collected as packets before they are pulsed, which results in signal enhancement. Analysis in the TrapPulse mode prevents potential "wraparound" of higher mass ions into subsequent spectra, thereby eliminating spurious peaks in the spectrum and potentially incorrect mass assignments. Using this technology, the author improved detection limits and identified 10 impurities in OTC melatonin, including previously uncharacterized ones.

The AxION 2 TOF mass spectrometer also provides mass accuracy and isotope ratio-profile accuracy, both of which are required for elemental composition identification of unknowns. The author predicted the elemental composition of several unknown compounds in melatonin using information provided by the TOF in conjunction with elemental composition matching software (AxION EC ID, PerkinElmer). Accurate monoisotopic mass and isotope ratio information is used by the AxION EC ID software to search against databases, such as the PubChem database (National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health) or other databases for potential molecular formula matches. The software provides a ranked summary of the potential matches and suggestions for possible compound structures for a given elemental composition.

Materials and methods. The following materials and conditions were applied for liquid chromatographic (LC) analysis:

  • LC system: Flexar FX-10 high-performance LC
  • LC column: PerkinElmer Brownlee Supra column C18 (1.9 μm, 2.1 50 mm)
  • Column temperature: 25 C
  • Flow rate: 0.4 mL/min
  • Mobile Phase A: water with 0.1% formic acid
  • Mobile Phase B: acetonitrile with 0.1% formic acid
  • Injection volume: 2 μL
  • Gradient: 20% B to 40% B over 5 min, to 70% B over 2 min.

The following were applied for mass spectrometric analysis:

  • Mass spectrometer: PerkinElmer AxION 2 TOF
  • Ionization source: PerkinElmer Ultraspray 2 (Dual electrospray ionization [ESI]source)
  • Ionization mode: ESI positive mode
  • Capillary exit voltage: + 100 V
  • Pulse mode: mass-to-charge ratio (m/z) 100–700
  • TrapPulse mode: m/z 100–700.


Figure 1 (TOF MS): (a) Analysis of melatonin in conventional pulse mode; (b) Analysis of melatonin in TrapPulse (PerkinElmer) mode. (FIGURES 1–3 (TOF–MS) ARE COURTESY OF THE AUTHOR (REDDY))
Sample preparation. An OTC melatonin tablet containing 1 mg of melatonin was crushed by a mortar and pestle and dissolved in 10 mL of water. The mixture was vortexed and centrifuged at 6000 rpm for 10 min. The supernatant was collected and injected on the column.


Figure 2 (TOF MS): (a) Base peak ion chromatogram (BIC) (mass-to-charge ratio (m/z) of 265.132 0.050 Da) shows the elution position of the Impurity Isomers A and D in a melatonin tablet. (b) BIC (m/z of 265.132 0.050 Da) shows the co-elution of N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) with Peak D when spiked into a melatonin tablet extract. (c) Spectrum of Peak A Impurity. The accurate mass of [M + H]+ and the fragments of Peak A are within 2.0 ppm of theoretical value. (d) The mass spectrum of Peak D. The accurate mass of [M + H]+ and the fragments are within 2.5 ppm of theoretical value. (FIGURES 1–3 (TOF–MS) ARE COURTESY OF THE AUTHOR (REDDY))
Results and discussion. Initial analysis in traditional pulse-mode operation showed few impurities in the melatonin tablet present at a low signal-to-noise ratio (S/N) (see Figure 1a [TOF MS]). When the analysis was performed in TrapPulse mode, the increase in S/N was almost fivefold to sevenfold higher, which resulted in the identification of 10 different impurities in melatonin (see Peaks A through J, see Figure 1b [TOF MS]). The comparison highlights the advantage of analyzing the sample in TrapPulse mode versus pulse mode.


Figure 3 (TOF MS): Software (AxION EC ID, PerkinElmer) lists the elemental composition of C13H16N2O4 with the top score for the accurate mass 265.1188 and the observed relative isotope abundances. (FIGURES 1–3 (TOF–MS) ARE COURTESY OF THE AUTHOR (REDDY))
Using accurate mass, isotope-profile information, and EC ID software, the author was able to identify all unknown drug impurities. The workflow used for identification of unknowns with the example of the drug impurities labeled Peak A and D are highlighted (see Figure 2 [TOF MS]). Both Peaks A and D (see Figure 2a [TOF MS]) have identical accurate masses, but have different spectra and elution times, which suggest that they are most likely isomers (see Figures 2c and 2d [TOF MS]). The elemental composition predicted by the EC ID software is C13H16N2O4 (see Figure 3 [TOF MS]). In vivo studies with melatonin have shown a minor metabolite, N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), to be produced, which has an elemental composition and exact mass identical to Peak D. For these reasons, the author suggests that the structure of Peak D to be AFMK. The structure of Peak D was confirmed by analyzing a synthesized standard of AFMK, which matched both the retention time and accurate mass spectrum of Peak D (see Figures 2a, 2b, and 2d[TOF MS]). Using a similar approach, the author identified the remaining impurities Peaks B to J as summarized in Table I (TOF MS).


Table I (TOF MS): Structures of impurities identified by time-of-flight mass spectrometer (AxION, PerkinElmer).
Conclusion . Several impurities in OTC tablets of melatonin that were not visible in the conventional pulse mode were identified using the AxION 2 TOF in TrapPulse mode. High mass accuracy and accurate isotope-profile ratio information provided by the TOF and the elemental composition matching software (AxION EC ID) facilitated the identification of several unknown drug impurities.


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