©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