This presentation provides a summary of recent work by my research group on the use of methylene blue staining as a method for characterizing pharmaceutical glass vials.

1. Quantitative methylene blue staining of
pharmaceutical glass vials
Matthew M. Hall, Associate Professor of Biomaterials & Glass Science
Kazuo Inamori School of Engineering at Alfred University
E-mail: hallmm@alfred.edu

2. Methylene Blue (MB) staining can been used to
qualitatively evaluate surface defects in glass vials
Sulfur-treated Type IA vials autoclaved
and then stained with MB solution
SEM image of corroded surface in heel
region after autoclaving
Images taken from presentation by Daniele Zuccato (Stevanato Group) to PDA Metro Chapter
Available at http://www.pda.org/docs/default-source/website-document-library/chapters/presentations/metro/
glass-delamination-risks-reality-and-regulatories.pdf?sfvrsn=6

3. Methylene Blue (MB) staining can been used to
qualitatively evaluate surface defects in glass vials
• There is no standard procedure for performing MB staining of glass vials
• Typical methods use concentrated MB solution (1% (w/v) is common) with
staining times ranging from 15 minutes to 24 hours
• Can identify regions of selective staining within a single vial (e.g., heel versus
body) and perhaps make observations about relative staining behavior among
different vials
• Qualitative information may be helpful, but does not necessarily produce
data that can be readily documented and acted upon

4. Quantitative results may also be achieved with MB
staining under appropriate conditions
5
4
3
2
1
0
400 450 500 550 600 650 700
Optical Absorption (cm-1)
Wavelength (nm)
1) 1:50,000
2) 1:100,000
3) 1:150,000
4) 1:200,000
5) 1:300,000
6) 1:400,000
1 g MB/100 mL WFI
stock solution
1:400,000 dilution
of stock with WFI
• UV-Vis spectroscopy was used to measure the optical absorption spectra for a series of MB solutions prepared
by serial dilution of a 1% (w/v) stock solution
• 1:200,000 MB solution was found to be suitable for staining; avoids noisy spectrum (see spectrum #1 above)
and provides adequate detection capabilities when used to stain vials in the 8 to 10 mL size range

5. Quantitative results may also be achieved with MB
staining under appropriate conditions
• An experiment was designed to evaluate if MB staining could discriminate
between acceptable and defective glass vials
• Type 1 glass vials provided by supplier on condition of anonymity
• Two lots of vials – one passed a standard hydrolytic resistance test (“Pass”
vials);
the other lot failed the same test (“Fail” vials)
• Chemical stability behavior of same vials previously reported in: Kucko et
al. (2013). Fill volume as an indicator of surface heterogeneity in glass
vials for parenteral packaging. Journal of Pharmaceutical Sciences, 102:
1690-1695.
• Pass and Fail vials were filled to 90% maximum capacity with MB solution
prepared by 1:200,000 volumetric dilution of a 1% (w/v) stock solution
• Vials exposed in triplicate to dilute MB solution for time periods ranging up to
24 hours
• Optical absorption of dilute MB solution measured after each incubation
period

6. Quantitative results may also be achieved with MB
staining under appropriate conditions
1.20
1.15
1.10
1.05
1.00
0.95
0.90
0.85
0.80
0.75
0.70
Decrease in optical absorption
occurs when vial is stained by
MB solution.
Error bars represent 2 standard
deviation of result measured in
triplicate and are smaller than
symbol when not visible.
0 3 6 9 12 15 18 21 24
Optical Absorption of Post-Staining
1:200,000 MB Solution (cm-1)
Time (hr)
Pass
Fail
• Both vial types exhibited detectable
staining by dilute MB solution
• Staining was visible to the unaided eye in
the heel region of the vials
• Staining behavior is time dependent
and unique to vial type
• Staining is similar within 1st hour
• Optical absorption behavior reaches
a local minimum at short times, followed
by an increase and then gradual decline
• Behavior suggests that interior surface of
vial is being altered while exposed to MB
solution, thereby influencing extent of
MB absorption/adsorption
• Fail vials always stained more than Pass
vials after 1 hour of exposure
• Quantitative MB staining can be used as
a simple method for identifying drifts
in manufacturing process

7. Quantitative results may also be achieved with MB
staining under appropriate conditions
Electron microscopy was used to probe origins of different MB staining behavior
in the heel region of Pass and Fail vials
Pass vial, As received
(15,000)
Pass vial, 24 hr stain
(15,000)
Pass vial, 24 hr stain
(~107,000)
• Pass vials show circular pitting in heel region after 24 hr exposure to dilute MB solution
• Circular pitting is characteristic feature of many vials produced from converted tubing

8. Quantitative results may also be achieved with MB
staining under appropriate conditions
Electron microscopy was used to probe origins of different MB staining behavior
in the heel region of Pass and Fail vials
Fail vial, As received
(15,000)
Fail vial, 24 hr stain
(9,900)
Fail vial, 24 hr stain
(30,000)
• Fail vials also showed circular pitting although larger structures were apparent, including semi-circular
cracks

9. Quantitative results may also be achieved with MB
staining under appropriate conditions
Combination of FIB and STEM used to further examine heel region of Fail vials
Platinum layers associated with
sample preparation procedure
Foamed glass shell of blister
Void within blister
Bulk glass below blister
• FIB used to generate cross-section that was subsequently imaged by STEM
• Cross-section shows blister consisting of void surrounded by thin shell of foamed glass, perhaps due to
overheating during conversion process; shell was rich in alkali, which explains poor corrosion resistance
• Outer perimeter of blister faintly visible by FESEM (see middle image of last slide); cracking occurred within shell
• Combination of void and/or high surface blisters presumably contributed to increased MB staining

10. Summary
• Quantitative MB staining may be achieved using appropriately diluted MB
solutions
• In this case, acceptable and defective glass vials were clearly detectable
by differences in quantitative staining behavior
• The degree to which these vials were acceptable or defective is not known.
Further method development is needed to determine if vials that are barely
acceptable or defective by standard measures can be resolved
• If found to be sufficiently sensitive, quantitative MB staining could provide a
simple and inexpensive alternative method for detecting defective vials and
monitoring the vial manufacturing process

11. Acknowledgements
• UV-Vis results and lower magnification SEM results were generated by
undergraduate and graduate students (Tim Keenan, Kelsey Gloss, and
Simon Chon) working in my research group at Alfred University
• High magnification imaging of blister cross-sections were provided in
collaboration with Jeff Shallenberger at Evans Analytical Group
Contact information
• Office phone: 607-871-3143
• Assistant’s phone: 607-871-2486
• Email: hallmm@alfred.edu