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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. Applying mass

spectrometry imaging (MSI) to an implant creates a

spatial map of chemical species in the sample.

Desorption Electrospray Ionization (DESI) directly

samples and ionizes at atmospheric pressure for rapid

analysis with essentially no sample preparation required.

The ESI mechanism works well with Active

Pharmaceutical Ingredients (APIs). DESI MSI of drug

implants gives spatial distributions from non-flat

surfaces quickly with little preparation.

In this work, DESI MS Imaging with ion mobility pre-

separation 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.

DIRECT DRUG ANALYSIS IN POLYMERIC IMPLANTS USING DESI MASS SPECTROMETRY IMAGING (MSI) Elizabeth E. Pierson,1 William P. Forrest,1 Vivek Shah,1 Roy Helmy,1 Anthony J. Midey,2 Hernando J. Olivos,2 and Bindesh Shrestha2 1Analytical Sciences, Pharmaceutical Sciences and Clinical Supply, Merck Research Laboratories, Rahway NJ. 2Waters Corporation, Beverly, MA

Figure 1. Direct DESI HDMS analysis of a drug coated implant using

SYNAPT G2-Si Ion Mobility Q-ToF MS

METHODS

Entecavir 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.

Sample mounting for DESI HDMS Imaging analysis:

Whole implants were mounted to a standard glass slide with adhesive tape (Scotch brand). (See Figure 1).

Radial sections attached to a standard glass slide with double sided tape. References

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

RESULTS

DESI HDMS detection of entecavir drug standard

Figure 3 shows the ESI mass spectrum (left) and ion mobility spectrum

(right) for electrospray ionization (ESI) HDMS of 5 ng/µL entecavir drug

standard detected to high mass accuracy. Similarly, Figure 4 shows

DESI HDMS imaging of 200 ng and 5 ng of entectavir 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 with the same drift time, illustrating

that the ion mobility separation does not depend on the ion source.

CONCLUSION

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.

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

Ion mobility with MS/MS proved that a m/z 299.11 peak only found in the implants was distinct from the API.

Multivariate statistical analyses proved that a 50:50 MeOH/H2O treatment removed the most drug from the implant surface.

Entecavir: C12H15N5O3 Average MW: 277.279

Ion mobility-mass spectrometry (HDMS) MSI

Source: Waters modified 2D DESI stage (Prosolia, US)

Mass Spectrometer: SYNAPT G2-Si ion mobility QToF (Figure 2).

DESI conditions:

95:5 methanol:water with 0.1% formic acid (v) at 5 µL/min

Nebulizing gas pressure of 4.5 bar nitrogen

4.5 kV sprayer voltage

Polarity: Positive

Mass range: 50 -1,200 m/z; 0.5 s per MS scan

MS Imaging Pixel size: 50 µm

Figure 2. Schematic of the DESI SYNAPT G2-Si QToF mass spectrom-

eter with ion mobility shape/structure separation prior to ToF MS

Data management

MSI data were acquired using MassLynx 4.1. Experimental parameters

were defined, raw files processed, and HDMS data visualized using

High Definition Imaging (HDI) 1.4 software for detailed analysis. All ion

images were TIC normalized. Multivariate analysis (MVA) was done

with Progenesis QI 2.3 and EZ-Info 3.0.2.0.

Figure 3. ESI HDMS mass spectrum (left) and ion mobility spectrum

(right) for entecavir drug standard at 5 ng/µL.

Figure 4. DESI HDMS imaging of dried 200 and 5 ng spots of entecavir

drug standard for the [M+H]+

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

ESI

DESI DESI

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

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

Figure 6 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 7 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. 8), the drug decreased on the surface in both

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

Figure 6. DESI HDMS Images of untreated implants overlaid on photo of

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

Figure 7. DESI HDMS Images of acid dissociated (a) and 50:50

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

Untreated

Figure 8. 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 [M+H]+ - untreated implant

Figure 9. 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. 10), the drug

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

Figure 9. Spatial correlation (R) of other ions co-distributed with [M+H]+

API ion in untreated implant calculated with HDI software

Figure 10. 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. 11). The MS in Fig. 11 corresponds

to a series of compounds on the implant having m = 138 Da with

mobility slope highlighted in red. Using HDMS/MS aligned precursor

ions and their fragments by their drift time (Fig. 12). Performing HDMS/

MS on m/z 299.1 resolved and assigned its unique fragments, proving it

was a different species than the m/z 300.1 [M+Na]+ API adduct.

Figure 11. IMS plot of drift time vs. m/z (right) indicating compound class

mobility trends. MS of molecular series with m 138 Da (left).

Multivariate analysis: region of interest (ROI) - untreated vs. treated

The region of interest (ROI) tool created user-defined areas for extraction and exporting of MS image data for multivariate statistical analyses. Principal component analysis (PCA) using the ROI MS data in Fig. 13 did unsupervised calculation of the greatest differences amongst the m/z peaks (PCs) for the initial untreated, acid dissociated, and 50:50 MeOH/H2O treated data. PCA found clear differences between the treated vs. untreated data, and between the two different treatments. Orthogonal Projections to Latent Structures for Discriminant Analysis (OPLS-DA) from the PCA data identified “target” peaks with maximum differences between the two treatments’ ROI MS data. It removed MS variations not related to true group differences to find the most relevant peaks (extremes of the quadrants in an S-plot). As seen in the S-plot in Fig. 13, the biggest differences were in the two drug peaks, confirming the visual observation that the 50:50 treatment removes more drug from the surface than acid dissociation.

Figure 12. HDMS/MS mobility plot for m/z 299.1 selected pre-cursor (drift time

vs. m/z; top). Extracted mobility peak and its MS for m/z 299 (left) and 300

(right) where fragments align by pre-cursor drift time.

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