TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS ©2017 Waters Corporation

INTRODUCTION

Delivering a drug via polymeric implant provides

extended and tunable release rates tailored to

therapeutic need and, more importantly, leads to

improved patient compliance.1

Therapy development benefits from understanding the

uniformity of drug distribution in the implant and how it

changes as the implant ages. Mass spectrometry (MS)

detects charged molecules based on their ratio of

molecular mass to charge (m/z). MS used as a

chemical detector can create a spatial map of the

molecular species in a sample via mass spectrometry

imaging (MSI). Thus, MSI of a coated implant provides a

visual map of the chemical distribution across it.

To image the implant, Desorption Electrospray Ionization

(DESI) directly sampled and ionized the surface at

atmospheric pressure for rapid analysis in the MS with

essentially no sample preparation required. The ESI

mechanism works well with Active Pharmaceutical

Ingredients (APIs) and does not require a flat surface,

ideal for cylindrical implants.

In this work, DESI MS Imaging with structure-based pre-

separation (ion mobility) prior to the MS (HDMS Imaging)

detects the differences in drug distribution for control

(untreated) vs. controlled release (treated) drug implants

made from PLA polymer and entecavir API.

THE USE OF DESORPTION ELECTROSPRAY IONIZATION (DESI) MASS SPECTROMETRY IMAGING (MSI) FOR DIRECT DRUG ANALYSIS IN POLYMERIC IMPLANTS Elizabeth E. Pierson,1 William P. Forrest,1 Vivek Shah,1 Roy Helmy,1 Hernando J. Olivos,2 Michael A. Batey, Anthony J. Midey,2 and Bindesh Shrestha2 1Analytical Sciences, Pharmaceutical Sciences and Clinical Supply, Merck Research Laboratories, Rahway NJ. 2Waters Corporation, Beverly, MA 3Waters Corporation, Manchester, UK

DRUG COATED IMPLANTS

Entecavir API standard solutions

Entecavir standard (US Pharmacopeia) used as provided.

Stock solution prepared in methanol to 1 mg/mL; further diluted in methanol to the desired concentrations.

Entecavir coated implant treatment - (Merck)

Continuous Flow-through Cell Method (closed loop configuration)

Flow rate: 16 mL/min

Media: 50:50 MeOH/H2O (v/v) or acid dissociation (PBS, pH 2.5)

Temperature: 37ºC

Implant dimensions: 18.5 mm x 2.2 mm

Drug implants used as received from Merck. References

1. J. Arps, Med. Design Technol., July 2013.

RESULTS

DESI HDMS detection of entecavir drug standard

Figure 6 shows DESI HDMS imaging of 200 ng and 5 ng of entectavir

drug standard spotted on a Prosolia well plate. DESI HDMS detected

the drug to the single ng level with high mass accuracy . Moreover,

DESI and ESI produced the same [M+H]+ and [M+Na]

+ adducts of the

drug (M) with the same drift time, illustrating that the ion mobility

separation does not depend on the ion source.

CONCLUSIONS

DESI HDMS imaging measured the distribution differences of drug API on the exterior and interior surfaces of untreated vs. treated (aged) coated polymeric implants without sample prep.

HDI software designed the imaging experiment and visualized the results, including tools for find spatially co-located ions to a target compound

Ion mobility shape/structure pre-separation prior to MS confirmed the identity of API related peaks and revealed compound classes present.

Entecavir: C12H15N5O3 Average MW: 277.279 Figure 2. Illustration of how DESI works as a charged particle source for

Figure 6. DESI HDMS imaging of dried 200 and 5 ng spots of entecavir

drug standard (M) for the [M+H]+

(left) and [M+Na]+ (right) adducts.

DESI DESI

Desorption Electrospray Ionization (DESI) source for MS

Figure 2 illustrates how Desorption Electrospray Ionization (DESI) works

as a source for introducing ionized (i.e., charged) molecular samples

into the MS. A shower of charged ESI solvent droplets is focused into a

beam that washes a surface to desorb any of the analytes at the surface

of the sample. The desorbed analytes are then ionized and carried into

an inlet capillary that transfers the ions to the MS for analysis. DESI is

minimally destructive and allows multiple imaging analyses of the same

sample. Compounds best detected with ESI such as active drug

ingredients are well suited to DESI sampling.

Figure 5. Illustration of how to do Mass Spectrometry Imaging (MSI).

DESI HDMS Imaging - entecavir distribution (untreated vs. treated)

Figure 4 (see left) shows how the implants were mounted to perform

DESI HDMS Imaging. The implant was attached to a standard glass

slide using adhesive tape (Scotch brand) as shown. For the radial cross

sections of the implants, the sections were simply attached to double-

sided tape on a standard glass slide. The surfaces were then imaged

directly without further treatment and data visualized with High Definition

Imaging (HDI) software ver. 1.4

Figure 7 shows MS images of [M+H]+ and [M+Na]

+ API ion distributions

from HDI for the untreated implant overlaid on the actual implant (left

side). A red-green overlay of the [M+H]+ and red ink standard ions

shows how the distributions aligned physically. A third ion at m/z

299.110 appeared in all implant samples, but not in the standards (right

side). Figure 8 shows MS images of 3 main ions distributed over the

acid dissociated (a) and 50:50 MeOH:H2O (b) treated implants. With the

same intensity scale (Fig. 9), the drug decreased on the surface in both

treated samples, with the greatest decrease using 50:50 treatment.

Figure 7. DESI HDMS Images of untreated implants overlaid on photo of

implant (left); MS images of 3 main ions on same intensity scale (right).

Figure 8. DESI HDMS Images of acid dissociated (a) and 50:50

MeOH:H2O (b) treated implants for the 3 main ions.

Untreated

Figure 9. API [M+H]+ and [M+Na]

+ ion distribution in initial untreated vs.

acid dissociated, MeOH:H2O treated implants (same intensity scale).

Spatial correlation of other ions with drug - untreated implant

HDI imaging software can find the other ions with the same spatial

distribution as a chosen target ion using a built-in Pearson’s moment

calculation of the intensity distribution vs. spatial location to get the R

value. R values closer to 1 are closely spatial correlated to the target.

Figure11. Main ion distributions in initial untreated (a), acid dissociated (b), and

MeOH:H2O (c) treated implant radial sections.

DESI HDMS Imaging - radial implant sections (untreated vs. treated)

MS images of 3 main ions show internal distribution over radial sections

of the initial untreated (a), acid dissociated (b), and 50:50 MeOH:H2O

treated implants. With the same intensity scale (Fig. 12), the drug

concentrates more strongly in the center of the 50:50 treated implant.

Figure 10. Spatial correlation (R) of other ions co-distributed with [M+H]+

API ion in untreated implant calculated with HDI software

Figure 12. Internal distributions in radial sections: initial untreated (top), acid

dissociated (middle), MeOH:H2O (bot.) treated (same int. scale).

HDMS Imaging with ion mobility - identification and confirmation

Different classes of compounds group along trend lines in ion mobility

plots of drift time vs. m/z (IMS; Fig. 14). The MS in Fig. 14 corresponds

to a series of material compounds from the implant having m = 138 Da

with mobility slope highlighted in red. This allows the ions from the

background material to be quickly identified vs. the drug compounds and

related compounds of interest.

Figure 14. IMS plot of drift time vs. m/z (right) indicating compound

(bins)

(bins) (bins)

Mass Spectrometry (MS) - Time-of-Flight (ToF) mass separation

The type of mass spectrometer (MS) used as the detector in the current

experiments determines the ratio of molecular mass to charge (m/z)

based on the time of arrival at a charged particle detector. As illustrated

schematically in Fig.1, all of the ions entering the ToF MS receive the

same push (same energy of motion; kinetic energy). Therefore, the

lightest ion will move fastest, arriving at the detector first. Similarly, the

heaviest ion moves slowest, arriving last.

Figure 1. Principles of Time-of-Flight Mass Spectrometry

Mass Spectrometry Imaging (MSI)

Figure 5 illustrates how MS Imaging was performed. A “grid” of x and y

coordinates was “overlaid” on a sample to image. At each (x,y)

coordinate (i.e, one pixel), a mass spectrum was measured. HDI

software processed the MS data to construct a map of the ion intensity

for a chosen mass-to-charge (m/z) peak across this “grid” mapped to

the sample. The ion distribution was correlated by HDI to other sample

images including digital photos.

Figure 4. Direct DESI HDMS Imaging analysis of a drug coated implant using

SYNAPT G2-Si Ion Mobility Q-ToF MS powered by High Definition Imaging

(HDI) 1.4 software.

DESI

MS

Figure 3. Schematic of DESI SYNAPT G2-Si mass spectrometer with

ion mobility shape/structure separation prior to ToF MS (HDMS).

DESI

Ion mobility

TOF MS

Shape/structure separation with ion mobility before MS

Ion mobility separation is based on ion’s structural size and shape so it is a complementary separation method to MS. Ion mobility spectrometry (IMS) is a gas-phase separation of ions under the influence of a field during collisions with a neutral. As seen in Figure 13, larger, bulkier molecular structures (orange) will not move as easily through the gas flow as smaller, more compact ones (red). Therefore, the smaller structures arrive earlier. Using ion mobility with MS imaging (HDMS Imaging) resolves MS

separation issues such as isomers or isobaric (same m/z) peaks. The

mobility drift time is also an identifying property of a molecule because it

is determined by structure, which is useful in confirming detection of a

targeted compound.

Figure 13. How ion mobility spectrometry separates based on structure

Structure separation

Mass separation

For research use only, not for diagnostic use