Presentation made by Nikki Schopp at Interphex 2014. “USP 233: Sample Preparation" this covers the importance of sample preparation, and looking at each sample on a case by case basis, in applying the US Pharmacopeial Convention’s (USP) chapter on limits <232> and procedures for elemental impurities <233>. Read more http://bit.ly/RCkMpk

1. Sample Preparation
for Elemental
Impurities
Nikki Schopp, 3/19/14
SGS Life Science Services

2. Overview of Methods
• Heavy Metals testing, currently performed per USP <231>
– established over 100 years ago
– undergoing a much needed update
– toxicological data is being used to establish limits
– Both toxic metals and metals used in processing are being
addressed.
• Elemental Impurities
– USP <232>,<233> & <2032>;ICH Q3D (Draft)
– USP, EP and ICH all expressed desire to be harmonized prior to
implementation
– Both metals addressed and limits are concerns.

3. Getting Started
• When analyzing for trace metals using
Inductively Coupled Plasma (ICP), sample
preparation is critical.
– The solubility of the test article is crucial, but not
the primary factor when choosing a sample
preparation method.

4. Sample Preparation Options
Four sample preparation methods are typically
used when performing Elemental Impurities
testing.
• Neat
• In aqueous solutions
• In organic solutions
• Digested in a closed-vessel apparatus

5. Standard Preparation
• The blank and standard solutions should be
prepared in the same matrix as the sample,
– solvent, acid concentration and stabilizers
• Spike recovery studies need to be performed
– Ensures method accuracy, specificity and precision
when used as a quantitative method.

6. • Recommended Stabilizing Acids
– Hydrochloric Acid- Osmium and Ruthenium
– Nitric Acid – Cd, Pb, Hg, As, In, Cr, Ni and Cu
– Nitric Acid/Hydrochloric Mixture- Pd, Pt, Rh, V
– Hydrofluoric – Molybdenum
• Other stabilizers – Gold for Hg
Choosing a Diluent (1)

7. Choosing a Diluent (2)
• Samples analyzed neat – choose a diluent for
standards
• Samples diluted in an acid – correct acid and
concentration needs to be chosen
• Consideration: Just because a test article is
water soluble does not mean that the direct
aqueous solution method is the best choice

8. Choosing a Diluent (3)
• Organic solvents are not the easiest to run on
ICP (spray chamber, nebulizer, instrument
settings)
• MeOH, EtOH, Chloroform
• Concentration of organic solvent

9. Microwave digestion
• Choosing the correct acid and heat profile
• Interferences
• Determing the proper mixture of acids to
obtain stability and recovery (i.e. Os)

10. Validation Requirements
• Accuracy – average % Recoveries must be
– 70-150% at the limit
– half the limit
– 1.5x the limit
• Precision – %RSD of calculated concentration
NMT 20%
• Ruggedness (Intermediate Precision)
– two analyst
– 3 different days
– %RSD of calculated concentration NMT 25%

11. Case Study I – Direct Aqueous
• Injectable Drug Product- 3 different sample
preparations were needed
• Solution 1 – 2% Nitric Acid Diluent for As, Hg,
Ru, Cd, Pb, Cr, Mo, Pd and V
• Solution 2 – 12% Nitric/8% Hydrochloric acid
for Ni, Cu, Ir, Pt, Rh
• Solution 3 – 2% Hydrochloric acid for Osmium

12. Case Study I (2)
• Test Solution 1 weigh and dilute – with all
injectables this is the first attempt during
development. As, Hg, Ru, Cd, Pb, Cr, Mo, Pd
and V all met requirements
• Elements that did not meet requirements
Level Ni Cu Rh Os Ir Pt
50% 63 58 59 62 59 65
100% 66 62 61 66 62 69
150% 66 61 56 62 59 66

13. Case Study I (3)
• Test solution 2 used microwave digestion
Remaining elements passed requirements,
except Os
• Test Solution 3 weigh and dilute, different
acid- since Os is known to behave better with
HCl, this was used as the diluent for Os.

14. Materials used for
Development vs. Validation
• Development and validation should be done on
the final stage of the material of interest
• Changing the process of manufacturing and
purification of the material of interest can change
recoveries, especially if acids and/or bases are
used in the purification process
• Development done on a material, all
requirements are met, then validation begins on
a purified product, some recoveries did not meet

15. Case Study II
• Development done on unpurified material
– sample preparation was weigh and dilute, all criteria
were met
• Validation performed on purified material
• All recoveries were met except for Palladium
-50% the limit average = 65%
-100% the limit average= 67%
-150% the limit= 66%

16. Case Study II (2)
• Purification of the material inhibited the
Palladium from being recovered
• Currently in the process of re-developing a
method

17. Case Study III
• Development sample preparation on unpurified
material
– nitric acid, hydrogen peroxide heat in oven at 105°C
• All recoveries meet criteria during development
• Validation on purified material-all elements meet
criteria except Palladium
50% limit avg recovery= 50.1%
100% limit avg recovery= 82.1%
150% limit avg recovery= 89.5%

18. Case Study III (2)
• Re-developed the method using microwave
digestion
• All Elements meet criteria, even Palladium
Pd 50% limit avg recovery= 101%
Pd 100% limit avg recovery= 103%
Pd 150% limit avg recovery= 99%

19. Summary
• USP will not be giving specific sample
preparations in the monograph
• Sample Preparation for Elemental impurities
will need to be determined on a case by case
basis
• Solubility plays a role however, it is not the
final step in determining a sample preparation

20. Contact Information
• Nikki Schopp, Team Leader Analytical Services,
Life Science Services, SGS
• Nikki.Schopp@SGS.com
• 616 Heathrow Dr. Lincolnshire IL 60069
• (847) 821-8900