Common Extractable and Leachable Project Questions

November 30, 2018

The following are questions that are commonly asked about forced extraction studies. These questions were answered by Pine Lake Laboratories CEO, Dr. Kurt Moyer.

Overview

  1. What sample should I use?
  2. Should the sample be pre-treated?
  3. What size sample will be extracted?
  4. How do you correct for contamination?
  5. What analytical methods do you use?
  6. How do you determine which chromatographic peaks are extractables?
  7. How do you identify extractables?
  8. Can you show me an example study?

Questions and Answers

What sample should I use?

Samples should be identical to the intended marketed products as well as have a unique name and lot number. Each sample should be prepared as it will be in the final product (no more, no less). Moreover, there should be no additional cleaning. Furthermore, samples may be cut as needed to enter extraction apparatus.

What size sample will be extracted?

For exaggerated extractions, I recommend sample size to be selected on targeted limit of detection of extractables in the component.

For simulated use extractions, set sample size based on ratio of surface area/solvent following:

  • ISO 10993-12
  • USP (2mL/cm2)
  • Exact ratio in final product if applicable

How do you correct for laboratory contamination?

Cleanliness and an appropriate blank, the closer the blank matches the sample extract, the better. An example for a reflux extraction is as follows:

  • Fill apparatus with extraction solvent plus an additional 10mL
  • Reflux for 30 minutes
  • Stop reflux and remove 10 mL to be blank
  • Add sample and reflux

During analysis, inject blank immediately before sample extract when possible.

What analytical methods do you use?

  • Volatile organic extractables by headspace GC-MS
  • Semi-volatile organic extractables by direct inject GC-MS
  • Non-volatile organic extractables by LC-UV/MS
  • Inorganic extractables by ICP-MS
  • Additional methods for select compounds like PAHs may be needed

How do you determine which chromatographic peaks are extractables?

Exclude peaks observed in blank, unless sample extract peak is more than 3x larger. Include peaks with a S/N above 10 or above determined LOD of method.

How do you identify extractables?

First Pass Identification

  1. Extract MS
  2. Background corrected and averaged if needed
  3. Compare to library
  4. NIST for GC
  5. Internal library of LC
  6. Interpretation of MS
  7. Check isotopic distribution for CI and Br
  8. Look for signature fragments and patterns (e.g. aromatics)
  9. Attempt identification of extractable
  10. Material information
  11. Comparison between extractions and analyses

Confirmation and Quantitation

  1. Analyze authentic material of compound identified in 1st pass identification under the same chromatographic conditions
  2. If retention time and MS match, identification confirmed
  3. Calculate response factor for authentic material to standards used in analysis of sample extracts
  4. Quantitate extractables using response factor

Executing a Successful Extraction Study and an Example Study:

Example Study Overview

             Sample: 13mm butyl rubber serum stoppers with a laminated coating

             Extraction Solvents: 50 mm sodium phosphate buffer and isopropanol

             Extraction Techniques: Reflux and Headspace

             Analytical Methods: Screening methods for organic leachables

(Headspace- GC/MS, direct injection GC/MS and LC/MS)

Volatile Extractables by Headspace GC-MS

  1. Extraction- neat sample incubated in sealed headspace vial for 30 minutes at 90°C
  2. Inject headspace onto HP-5MS capillary column (30m x 0.25mm, 0.25mm)
  3. Temperature gradient from 40°C to 300°C at 10°C/min
  4. Scan MS from 50-650 amu

Volatile Extractables from Serum Stopper

Identification of Extractables:

Peak 6.40 minutes

Peak 7.78 minutes

Peak 8.73 minutes

Peak 10.55 minutes

Peak 12.94 minutes

Volatile Extractables from Headspace GC-MS

Semi-Volatile Extractables by Direct Injection GC-MS

  1. Aqueous extracts were extracted with equal parts of methylene chloride. Organic extracts were injected neat.
  2. HP-5MS capillary column (30m x 0.25mm, 0.25mm)
  3. Temperature gradient from 40°C to 300°C at 10°C/min
  4. Scan MS from 50-650 amu

Semi Volatile Extractable

Identification of Extractables- peak at 16.48 minutes

Non-Volatile Extractables by LC-MS

  1. Buffer extract injected neat. IPA extract evaporated under nitrogen then reconstituted in ACN:H20
  2. BEH C18 (2.1mm x 50mm, 1.7 mm) column
  3. APCI in positive and negative ionization
  4. Scanning 200-2000 m/z
  5. UV Scan 200-200 nm

Non-Volatile Extractables

Study Conclusion

Results in the Final Report include

  1. Identification of as many extractables as possible
  2. Unknowns described as appropriate to results
  3. Quantitation of all extractables, usually as ug/g of the material

Next Steps for Example Study

  1. Toxicological assessment of extractables
  2. Select target leachables from the extractables
  3. Develop and validate analytical methods for leachables in representative drug product
  4. Leachables study in drug product