Developing Your USP <232> Compliance Project Plan: Analytical Challenges

Samina Hussain Metals Department Manager/Senior Chemist Exova Inc., Health and Sciences, Santa Fe Springs, CA

Finding an appropriate sample preparation technique for

common matrices

Common misconceptions

Practical method development approaches

Analytical Challenges

Neat – Liquids that can be introduced without further dilution Direct aqueous solution – Sample is soluble in aqueous

solvent such as dilute acids and bases Direct organic solution – Sample is soluble in an organic

solvent Indirect solution/Closed vessel digestion – Digestion is

required using concentrated acids

Complete sample dissolution is preferred Leachate extraction methods can be justified with validated metal

disposition studies

Sample Preparation Techniques

Neat Appropriate for samples that are predominately water with

very little dissolved solid matrix Provides the best detection limits since sample does not

need to be diluted May cause enhancement or suppression of internal

standard due to matrix interference Material may not be sampled uniformly (e.g. if matrix is

viscous) May cause heavy deposits on cones, torch, lens

Advantages and Limitations

Direct aqueous solution Sample dissolved in dilute acid or base solution Quick sample preparation: “dilute and shoot” Cleaner sample preparation since digestion vessels, hoods are

not required Lower sample detection limits – concentrated acids do not need

to be diluted down Sample that appears dissolved at the time of preparation may

precipitate out of solution prior to analysis May cause suppression of sample signal and internal standard

response due to undissolved, unseen particles Certain elements prepared in base do not have long term stability

Ex. Ag, Hg, Au, Ir, Os, Pd, Pt, Rh, Ru, Sn Conduct standard stability studies (e.g. 2 weeks expiration)

Advantages and Limitations

Peptides - Soluble in dilute acid solution, however: Sample stability may be limited to a few hours or a few days –

peptide may form a precipitate and fall out of solution Samples will need to be prepared and analyzed within a

predetermined time period Peptides may complex in the instrument – drift in continuing

calibration verification (CCV) of greater then ±20% Higher instrument background for Hg, Au, and Tl after CCV - not

observed with other sample types Elements such as Hg, Au, and Tl may not be stable for a long

duration of time (evident by matrix spike recoveries) Available in limited quantities (only a few milligrams for method

development and method validation)’

Acid digestion may be required in these cases

Examples

Cell Culture Media containing proteins Very common for industry to add acids:

• Acid denatures the protein forming a precipitate • Force this precipitate back into solution by digestion in the

microwave (microwave vessels not always clean) • Using concentrated acids during this process, these acids have

to be diluted down. Increasing the sample dilution factor and thus the sample detection limit

• These detection limits may be too high for the required ppb LODs

Solution: • Prepare the cell culture media in a dilute base solution to

prevent denaturing the protein • 1:10 dilutions work best and provide lower detection limits • Sample stability will may be limited to a few days

Examples

Direct organic solution May be optimal prep. for APis that are soluble in organic solvents Assure organic solvents used are free of elemental impurities Dilutions of 1:100x: LODs low enough for all 24 ICHQ3D elements

(Table A.2.2) Standard may not be stable for more than a few weeks

Conduct standard stability studies (Os is more stable in organic solvents vs acid solutions)

Works very well for Os – sample spike recoveries within 85 -115% Watch final acid concentrations during spike recovery studies for samples

that form a ppt upon addition of acids. Prepare 2- 3 mixed standard cocktails instead of spiking large volumes of acidic standard solutions

Use diluted organic solvent for rinse if sample forms a ppt upon the addition of acids

Requires special instrument configuration: oxygen, Pt cones, silicone tubing, torch (1.5 mm), spray temperature - 2 °C

Advantages and Limitations

Open vessel/Closed vessel digestion Use of high temperature and pressure can completely dissolve the

sample and effectively extract the metals present Handling concentrated acids such as nitric, hydrochloric, sulfuric,

perchloric, hydrofluoric is a safety concern Staff must be trained on HF handling, safety, and first aid.

Sample detection limits may be higher: Larger volumes of acid are needed to cover sample during digestion Diluting down concentrated acids for digestion results in higher detection limits Limited amount of sample can be digested (under 1 g) – higher detection limits

Re-use of digestion vessels may lead to trace contamination Closed vessel digestion - Works well for hard to dissolve matrices such

as tablets containing silicone dioxide or titanium dioxide (Composite 20 tablets and crush to a fine powder – use an aliquot of this composite)

Open vessel digestion - Oligonucleotide – Dissolve in water, add acids may form a cloudy solution, clears up with mild digestion on hot block.

Advantages and Limitations

Do not need to validate for this element if not used in manufacturing process. Avoid concentrated nitric oxide

However, the absence of an oxidizer may inhibit complete sample digestion. Forms volatile and toxic OsO4 – very poisonous and may cause blindness. Enhanced signal due to volatility, high spike recoveries

Do not use a closed vessel microwave If validation is needed, a separate method development/validation will be required:

Prepare samples in hydrochloric acid (5%) Analyze against matrix matched standards Digest standards along with samples needing digestion Working standards are not stable and should be prepared the day of use Allocate a different sample introduction system: torch, spray chamber, nebulizer,

sample and waste tubing dedicated to osmium determination Add a stabilizer during digestion to prevent the Os from volatilizing

Collect and dispose of waste separately

Osmium

Referencing USP <730> and/or USP <233> in place of validation: The older version of USP <730> is a guidance that briefly discusses standards, sample

preparation, ICP-OES, and ICP-MS components ICP-MS and ICP-OES are not compendial methods under USP <233>:

“Before initial use, the analyst should verify that the procedure is appropriate for the instrument and sample used (procedural verification) by meeting the alternative procedure validation requirements below.”

Method is not validated: each material will require its own validation System suitability is not the same as for HPLC methods

System suitability is established with daily tune for ICP-OES, ICP-MS Mass resolution is established with mass tune – recommended on a monthly basis 5 – 6 standard injections with an RSD requirement of NMT 2.0% is not obtainable for

the determination of trace elemental impurities ICP-OES and ICP-MS have a wide dynamic range, single standard analysis at 0.5J

and 2J are sufficient, do not need to analyze 5 standards

Common Misconceptions

Contamination prevention An ISO 14644-1 Class Cleanroom is not necessary Separate areas for sample preparation and instrument analysis Avoid preparing and analyzing samples in areas that use chemicals

containing high levels of metal content such as a general chemistry lab Hoods need to be acid resistant (PVS) with acid resistant ducting (ABS

plastic) Air conditioning should have HEPA filters Metal cabinets can be used if sealed with epoxy paint Use clear, powder-free gloves Use colorless spatulas for sample handling

Common Misconceptions

Contamination prevention Evaluate ceiling tiles Purchase auto-sampler covers Purchase an acid distiller to reduce cost

Ultrex II nitric acid – $300/500 mL verses Reagent grade $150/10,000 mL Pre-screen supplies and reagents before use Dilute sample preparations gravimetrically instead of volumetrically –

higher precision, avoids use of glass Wipe down hoods and work areas daily before commencing work Rinse digestion vessels, bottles, auto-sampler tubes with dilute acid prior

to use

Common Misconceptions

Validating all 15 or 24 elements in elemental impurity chapters: Elements requiring validation must be based on risk assessment Exclude elemental impurities for which there is no potential for

introduction into the final drug product (catalyst metals not used in the synthetic process)

Exclude elemental impurities that could potentially occur but are sufficiently controlled through normal GMP practices (only possible during equipment malfunction or process deviations)

Costly and time consuming Risk one or more element failing validation criteria

Common Misconceptions

Specifications set at the lowest target limits: Set realistic specifications based on dose amount Use equations provided in the chapters Not feasible for:

Samples that are difficult to dissolve Samples that have limited sample availability Samples that require organic solvent preparation Samples that require microwave digestion

Common Misconceptions

ElementCdPbAsHgCoVNiTlAuPdIrOsRhRuSeAgPtLiSbBaMoCuSnCr

*Acetaminophen Max. dose/day is 3000 mg

35

Oral Conc(µg/g)

0.50.51.5

10

10200.81010101010101515

1100

Conc(µg/g)*

1.51.54.59153060

55120140300300600

360

2.4303030303030454530

165

42090090018003300

An optimized method: One sample prep for all the elements of interest One analytical run for all the elements of interest Sample preparation used should yield a clear solution

Method Development

If solubility of sample is known, use this solvent as the diluent as long as it contains low levels of elemental impurities

If solubility of the sample is unknown, proceed with preparation attempts in the following order:

• Dissolve in dilute acid solution (1 – 5% solids) • Dissolve in dilute base solution (<10% solids) • Dissolve in organic solvent • Digest the sample in an open-vessel hot block

Spiking studies monitor loss of volatile elements Recoveries need to be within limits set in USP <233>

• Digest samples in a closed-vessel microwave Keep acid concentration in sample prep <5% for nitric, hydrochloric

and <2% for hydrofluoric

Method Development

Once solubility has been determined

Prepare sample at a dilution that yields a detection limit low enough for a limit test or a quantitative method validation

Example: Specification is 0.5 µg/g for Pb – Limit Test • Instrument detection limit is 0.1 µg/L • Detection limit of 0.25 µg/g is needed • A maximum dilution factor of 2500x will be required • (e.g. 40 mg sample diluted to final mass of 100 g)

Example: Specification is 0.5 µg/g for Pb – Quantitative Method Validation

• Instrument detection limit is 0.1 µg/L • Detection limit of 0.125 µg/g is needed (based on 50% spike at 0.25 µg/g) • A maximum dilution factor of 1250x will be required • (e.g. 80 mg sample diluted to final mass of 100 g)

Method Development

Prepare a sample, duplicate, spike, spike duplicate; use individual sample

portions for each preparation Spike the matrix spike and matrix spike duplicate at the specifications prior to

sample preparation If spiking for a wide range of specifications (ex. 0.5 µg/g – 130 µg/g)

• Prepare sample to obtain detection limits low enough for 0.5 µg/g • Dilute this sample preparation to bring the 130 µg/g spike within the linear

range of the standards Spike recoveries between sample and spike preparations need to be:

• Limit test: 85 – 115% and RSD < 20% • Quantitative test: 70 – 150% and RSD < 20%

Detection limits need to be no more than: • Limit test: 50% of the specification • Quantitative test: 25% of the specification

Internal standard responses should not be suppressed or enhanced • Recoveries of 50 -125% are suggested

Method Development

Specification is “Total Heavy Metals NMT x ppm” Divide the number of elements by the specification. Example: 15 elements require validation with a total specification of 10 ppm

• Spike each element at 0.6 µg/g • A method detection limit of NMT 0.2 µg/g is needed for each

element. • With a 1 ppb instrument detection limit, a dilution factor

should be NMT 1:200 in the sample prep to ensure the sum of the detection limits are < the 10 ppm spec.

Method Development

Parameters not met:

Prepare a more dilute sample Try an alternative preparation technique Analyze sample preparations against matrix matched standards Use a different internal standard Are the specifications set unrealistically low for the matrix?

Method Development

If all parameters have been met:

Scan the un-spiked sample preparation (metals scan of 60+ elements) • Decide if other impurities are present that need to be included in the

validation • Provides information on potential interferences from the sample

matrix • Account for these interferences during sample analysis • Address in specificity section of method validation

Method Development

Choose a limit test or a quantitative method validation (if not specified in monograph) Limit test

• Appropriate when the elements of interest are not detected or trend at levels well below the specification

• Raw materials where catalysts are not used in manufacturing

Quantitative method validation • Appropriate when elements of interest trend above the limit of

quantitation, especially when they are close to the specification • Final drug products

Draft a method validation protocol for review by all parties involved

Method Development

Provide information on sample solubility, if available Provide SDSs Set realistic specifications Conduct a risk assessment: a full metals scan by the contract lab

should be provided at time of method development Communicate which elements require method validation based on

risk assessment Submit samples in plastic containers known to be free of metal

contaminants Provide enough sample for method development and method

feasibility Pre-screen the lot of material prior to commencing method validation

Working with a Contract Lab

Estimated timeline: 1 – 2 weeks for method development 1 – 2 weeks to draft method validation protocol and perform internal

technical and QA review before sending to client for comment and signature

4 – 6 weeks for method validation Lapse of one week for intermediate precision for quantitative method

validations to establish digestion stability (based on 10 day routine TAT) Draft SOP for the validated method, specific for the material

• Prepare QC (Sx, Dup, MS, MSD) every 1 -10 samples • Sample and duplicate or matrix spike and matrix spike duplicate

RPDs must be <20% • Matrix spike recoveries must be within 85-115% (or 70-150%)

Create custom worksheet to prevent deviations during routine sample submission under the validated method.

Working with a Contract Lab

Thank You

Samina Hussain Metals Department Manager/Senior Chemist Exova Inc., Health and Sciences, Santa Fe Springs, CA