investigation of ion mobility mass spectrometry … · 2015. 7. 28. · confidence in structural...
TRANSCRIPT
©2015 Waters Corporation
INTRODUCTION
Metabolite ID can be challenging and time-consuming -
particularly when products are only generated at low
concentrations relative to the substrate. We propose that
in silico collisional cross sectional area (CCS) prediction
coupled with ion mobility measurements can help build
confidence in structural assignments, facilitate the
characterization of isobaric products and help confirm
identity during method development. In this study we
have applied LC/IMS/MS to the separation of mixtures of
oxidation products of a model pharmaceutical compound,
naloxone and it’s degradants produced by an
electrochemical flow cell fitted with a glassy carbon
electrode. We compare the accuracy of predicted versus
measured CCS values for mixtures of isobaric oxidation
products of naloxone.
INVESTIGATION OF ION MOBILITY MASS SPECTROMETRY ANALYSIS OF ELECTROCHEMICALLY GENERATED OXIDATION PRODUCTS OF OPIATES AND COMPARISON WITH THEORETICAL CCS VALUES Cris Lapthorn1; Frank Pullen1; Susana da Silva Torres2; Mark R Taylor2; Russell Mortishire-Smith3; Jayne Kirk3; Andrew Baker4
1 University of Greenwich, Greenwich, UK ; 2 Pfizer Sandwich, UK; 3 Waters, Wilmslow, UK; 4 Waters Pleasanton, USA
ELECTROCHEMICAL DEGRADATION
RESULTS
PREDICTION OF DEGRADATION
PRODUCTS
Figure 5. Measured CCS for degradation products with elemental
composition consistent with M+O-H2
Right Panel Component at 1.96 Minutes.
Left Panel Component at 4.26 Minutes.
Row A N2 at 90 ml/min and 1100 m/sec 40 V Wave Height. Row B CO2 at 75 ml/min 950 m/sec 40 V Wave Height.
Row C CO2 at 60 ml/min 950 m/sec 40 V Wave Height.
A CCS Calibration with performed using standard procedures (IntelliStart Routine). Calibrants were PolyAlanine oligomers and
Acetaminophen; CCS values for each gas were obtained using a prototype IMS-MS system fitted with a linear drift tube.
A
B
C
The theoretical CCS (tCCS) for naloxone and naloxone despropyl
appear to show overestimation of tCCSs as both structures largely consist of rigid ring systems, consistent with previous
studies. The quinone and amide naloxone products gave large tCCSs than experimental CCS (eCCS) eliminating them as viable
candidates for the M+O-H2 target.
By comparison with the known elution order for M+O-H2 degrada-tion products of naltrexone it is known that the Hydroxy de-
gradant elutes later than the Keto degradant under similar chro-matographic conditions as shown here. The assignment from ac-
curate mass of the product ions makes a positive assignment non-trivial.
The in silico calculation enables elimination of candidates and cor-
rectly predicts the rank order of the Keto and RSS Hydroxy
Naloxone, consistent with the elution order of corresponding known related degradant products in Naltrexone. Further work is
ongoing to investigate prediction of tCCS in CO2 to compare with experimental data obtained.
There is an improved separation in CO2 which may provide in-
creased separating power for these degradant products. Further work is ongoing to investigate tCCS in CO2 to compare with eCCS
data obtained.
The potential energy surfaces of target ions were initially investi-
gated using Spartan ‘10 using molecular mechanics (MMFF). Likely candidate conformers, typically within 5kcal/mol of the lowest en-
ergy conformer, were selected as the starting geometry for further geometry optimisation and DFT calculations of structures, and asso-
ciated energies were then carried out with the Gaussian 09 program using the hybrid SCF-DFT B3LYP method and 6-31+G(d,p) basis set
and additional keywords pop= (mk,dipole) to generate Merz-Singh-Kollman electrostatic potential partial atomic charges.
Chemcraft software was used to convert the minimised structures
and associated partial atomic charges to MFJ files which were then used as input for a modified version of MOBCAL parameterised for a
nitrogen gas as the buffer gas
Figure 3. UNIFI Report for Degradation Product Naloxone +O-H2
Lower Right Panel: Component chromatogram. Lower Left Panel: Low and High energy MSE Spectra.
Figure 1. LC-IMS Plot for electrochemically synthesized degradation products of naloxone.
Figure 4. Right Panel Arrival Time distributions for Despropenyl
Naloxone +O-H2 and two isobaric Naloxone +O-H2 Products. Left Panel High Energy spectra.
Figure 2. Major degradation products consistent with M+O-H2 predicted by
Lhasa Zeneth software.
CONCLUSIONS
Orthogonal dimension of separation for maximizing peak
capacity
Enhanced confidence in peak purities when developing
separations of complex mixtures such as drug
degradants and metabolites
CCS can be a robust experimental parameter for
identifying and tracking components across different chromatographic systems
Theoretical CCS can be a useful tool to utilise for
identification in addition to, or in the absence of other
identification parameters including retention time and accurate mass assignments.
Column BEH C18 2.1x100 mm 1.7 um dp Mass Spectrometer SYNAPT G2-Si
Mobile Phase A 0.1 % Aqueous NH4OH Ionization Mode ESI +ve
Mobile Phase B Acetonitrile HDMSE Low E 4 V
Flow Rate 400µl/min HDMSE High E Ramp from 30 to 55 V
Gradient Ramp from 5 to 50% B in 7 minutes
Ramp to 100% B in 1.2 minutes
Hold 0.5 minutes
Return to Initial Conditions Mobility Gas N2 at 90 mL/min
Injection Volume 1 µL CO2 at 60 or 75 ml/min
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LC/IMS/MS CONDITIONS