Report on the Comparison of Arbidol Formulations: “1970 & 2005”
University of Washington Mass Spectrometry Center
This report characterizes and compares two Arbidol monohydrate hydrochloride salt
(ArbidolH2OHCl) formulations of differing manufacture. The dry, amorphous, offwhite samples identical in appearance were submitted in screw capped plastic vials
labeled "1970 original formulation" and "2005 new formulation," August 28, 2006,
by Dr. Gary Rorabaugh, Good Earth Medicine LLC, Bellingham, WA. Both samples
were subjected to nuclear magnet resonance (NMR) and liquid chromatograph/mass
spectrometric (LC/MS) analysis as described below.
NMR Analyses - Approximately one milligram of each sample was dissolved in
deutero-methanol (CD3OD) and analyzed by proton NMR using the Bruker DRX
499 500 MHz NMR located in the University of Washington Department of
Chemistry.
Figures 1a and 2a present the NMR spectra acquired for "Arbidol 1970" and
"Arbidol 2005," respectively, with proposed assignments for observed chemical
shifts. Additional spectra, Figures 1b-e and 2b-f, showing peak fine structure
may be found in the "Arbidol 1970" and "Arbidol 2005" appendices, respectively. As
may be seen the NMR spectra, these samples appear very clean and virtually
identical, not only to each other, but also to that of the reference spectra for
ArbidolH2OHCl in DMSO, Figure 3 (Courtesy Dr. Vasily Kazey, Head of
Bioscreening Department, Chemical Diversity Research Institute, Russia). The
most noticable are differences in the chemical shifts between the sample and
reference spectra. These are attributable to the solvent effects of deutero-methanol
causing an up-field shift in the spectra relative to that of DMSO. Also of note is the
presence of a doublet at 4.90 in the sample spectra due deuterium exchange
between CD3OD solvent and H2O in the samples. The only observed difference
between the spectra for "Arbidol 1970" and "Arbidol 2005" is the small peak in the
latter up-field from the singlet for N(CH3) 3 at 2.994, Fig. 2a & f.
MS Analyses - Electrospray liquid chromatograph/mass spectrometric (ESILC/MS) analysis was performed using a Waters Micromass Quattro II tandem
quadrupole mass spectrometer (LC-MS/MS) directly interfaced to a Shimadzu
solvent delivery system with autoinjector. Fifty microliters of each sample
dissolved methanol (1mg/ml) was applied to a Phenomenex Luna Phenyl-Hexyl
HPLC column (150mm x 2.0mm I.D., 5m) operating at a flow rate of 0.300 l/min.
Upon injection the mobile phase (acetonitrile:water:0.1% formic acid) linearly
programmed from 5% to 95% acetonitrile over a 35 min period after a 2 min hold
under initial conditions. Mass spectrometric data was acquired in the scan mode
over a mass range from 50 to 1000 Da and the processed using Water Micromass
MassLynx 4.0 software.
Figures 4a & b compare the total ion current chromatograms (TICC) obtained for
the “Arbidol 1970” formulation to that of “Arbidol 2005,” respectively. As may been
seen the TICC’s are remarkably similar to each other in the number of components,
their retention times and their relative abundances. While the lesser components
have not been identified, the mass spectra associated with most of them indicate the
presence of bromine. These data imply that both formulations share similar
synthetic and product purification pathways resulting in their having common
contaminates (e.g. synthetic impurities and/or excipients, etc.).
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William N. Howald
Report on the Comparison of Arbidol Formulations: “1970 & 2005”
University of Washington Mass Spectrometry Center
Figures 5a & b compare the spectra obtained by averaging across the major peak
in each chromatogram shown in Fig 4a & b at ~14.9 and ~14.8 min, respectively.
These are identical to each other and to that of reference spectra for Arbidol,
Figure 6. (Courtesy Dr. Vasily Kazey, Head of Bioscreening Department, Chemical
Diversity Research Institute, Russia). As may been seen the TICC’s are remarkably
similar to each other in the number of components, their retention times and their
relative abundances.
Conclusions - The claim for the physical and chemical equivalency of the two
Arbidol formulations, “1970” and “2005,” is strongly supported by their common
nuclear magnetic resonance and mass spectra as presented here. In addition to
sharing these common spectral properties, the formulations also demonstrate
virtually identical liquid chromographic behavior and are indistinguishable in
appearance. Based on the analytical evidence obtained from the two submitted
samples I believe the Arbitol formulations may be considered for all intended
purposes identical to each other.
FIGURES
Figure 1a - Annotated NMR Spectra of Arbidol “1970 Formulation”
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William N. Howald
Report on the Comparison of Arbidol Formulations: “1970 & 2005”
University of Washington Mass Spectrometry Center
Figure 2a - Annotated NMR Spectra of Arbidol “2005 Formulation”
Figure 3 - ArbidolH2OHCl in DMSO Reference Spectra, (Courtesy Dr. Vasily Kazey, Head of Bioscreening
Department, Chemical Diversity Research Institute, Russia)
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William N. Howald
Report on the Comparison of Arbidol Formulations: “1970 & 2005”
University of Washington Mass Spectrometry Center
Figure 4a and b – Comparison of the ESI Total Ion Current Chromatograms (TICC) of Arbidol Formulations:
“1970” (Upper) and “2005” (Lower)
Figures 5a and b.- Flow Injection Analysis Electrospray Spectra of Arbidol Formulations:
“1970” (Upper) and “2005” (Lower)
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William N. Howald
Report on the Comparison of Arbidol Formulations: “1970 & 2005”
University of Washington Mass Spectrometry Center
Figures 6.- Flow Injection Analysis “Turbo Spray” Reference Spectrum for Arbidol (Coutesy Dr. Vasily Kazey,
Head of Bioscreening Department,Chemical Diversity Research Institute, Russia)
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William N. Howald