Elemental Impurities Testing: ICP-MS and USP 232/233

Elemental impurities testing determines how much of specific inorganic elements, such as lead, arsenic, cadmium, mercury or palladium, is present in a material. For synthetic peptides and other laboratory reagents, these elements can come from catalysts, reagents, water, equipment and packaging. This article explains how the modern compendial framework works, how ICP-MS and ICP-OES compare, and how to read the numbers on a certificate of analysis.

From “heavy metals” to element-specific testing

For decades the standard approach was USP <231> Heavy Metals, a colorimetric test in which metals were precipitated as sulfides and the color of the sample was compared with a lead standard. The result was a single “not more than” value expressed as lead.

The method had well-known weaknesses. It could not tell one element from another, several important elements respond poorly or not at all, and volatile elements such as mercury could be lost during sample ignition. USP omitted <231> and moved to instrumental, element-specific methods described in <232> and <233>, aligned with ICH Q3D.

When you see “heavy metals” on an older document, it usually refers to the sulfide test. A modern report names each element separately.

USP <232>, USP <233> and ICH Q3D: how they fit together

The three documents divide the work cleanly:

• ICH Q3D (current revision R2) is the guideline. It classifies elements by toxicity and likelihood of occurrence and asks manufacturers to perform a risk assessment of where elements could enter a product.
• USP <232> Elemental Impurities—Limits lists the elements and the limits that apply to compendial drug products.
• USP <233> Elemental Impurities—Procedures describes two reference procedures (ICP-OES and ICP-MS) and, importantly, the validation criteria any alternative procedure must meet.

Research reagents are not drug products, so the <232> limits are not a formal requirement for them. Many laboratories still use the Q3D element list and the <233> analytical approach as a sound, widely understood benchmark.

ICH Q3D element classes

Class	Elements	Basis for inclusion
Class 1	As, Cd, Hg, Pb	Significant toxicity; should be evaluated in all risk assessments
Class 2A	Co, Ni, V	Relatively high probability of occurrence
Class 2B	Ag, Au, Ir, Os, Pd, Pt, Rh, Ru, Se, Tl	Lower natural abundance; assessed when intentionally added (for example, catalysts)
Class 3	Ba, Cr, Cu, Li, Mo, Sb, Sn	Relatively low toxicity concern in the guideline’s framework

For peptide synthesis, a practical example is palladium: Pd(0) reagents are sometimes used to remove allyl-based protecting groups during solid-phase synthesis, which makes Pd a reasonable element to include in a risk assessment.

ICP-MS vs ICP-OES for elemental impurities testing

Both techniques use an argon inductively coupled plasma at roughly 6,000–10,000 K to atomize and ionize the sample. They differ in what they detect.

• ICP-OES measures light emitted at element-specific wavelengths.
• ICP-MS extracts ions from the plasma into a mass spectrometer and counts them by mass-to-charge ratio.

Attribute	ICP-OES	ICP-MS
Typical detection limits in solution	Low µg/L (ppb) for many elements	ng/L (ppt) for many elements
Main interferences	Spectral overlap of emission lines, background shifts	Polyatomic ions (e.g., 40Ar35Cl+ on 75As), isobars, doubly charged ions
Interference control	Alternative lines, background correction	Collision/reaction cell (e.g., helium with kinetic energy discrimination), alternative isotopes
Tolerance for dissolved solids	Higher	Lower; samples are usually diluted further
Best fit	Higher limits, larger samples, challenging matrices	Trace levels, small sample amounts, Class 1 elements

Because peptide samples are often available only in milligram quantities, ICP-MS is commonly preferred: its sensitivity compensates for the small mass digested. Internal standards (for example, Sc, Ge, Rh, In or Bi, chosen to bracket the analyte masses) are added to correct for signal drift and matrix suppression.

Sample digestion and contamination control

Peptides are organic matrices, so the sample is normally digested before analysis. Closed-vessel microwave digestion with concentrated nitric acid, sometimes with hydrogen peroxide, is the most common approach. Closed vessels retain volatile elements and reduce contamination from the laboratory air.

Practical points that affect results

• Mercury stability. Hg can adsorb to surfaces or be lost from dilute nitric solutions; adding hydrochloric acid or a gold solution is a common stabilization approach.
• Platinum-group elements. Elements such as Pt and Pd are better kept in solution with some hydrochloric acid present. Osmium can volatilize as OsO₄ under strongly oxidizing conditions.
• Residual carbon. Incomplete digestion leaves carbon that can enhance the ICP-MS signal of As and Se, biasing results high.
• Blanks. Trace-metal-grade acids, PFA or quartz vessels and method blanks carried through the whole procedure are essential when results are close to the quantitation limit.
• Weighing and handling. Stainless steel spatulas can contribute Fe, Cr and Ni; plastic or ceramic tools are often used for trace work.

Hygroscopic lyophilized powders should be weighed quickly and consistently, because absorbed water changes the mass basis of the result. The handling notes in our article on storage and stability of lyophilized peptides apply here too.

How USP <233> validates a procedure

USP <233> frames validation around a target concentration, J: the concentration of an element at its limit, expressed in the final test solution after dilution. For a quantitative procedure, the chapter’s acceptance criteria include:

• Accuracy: spiked samples at 50%–150% of J, with mean results within 70%–150% of the spiked amount.
• Repeatability: six independent samples spiked at J, with a relative standard deviation not more than 20%.
• Intermediate precision (ruggedness): repeated on a different day, instrument or analyst, with a combined RSD not more than 25%.
• Specificity: the procedure must unequivocally assess each target element in the presence of the matrix and other elements.
• Quantitation limit and range appropriate to J.

These wide windows reflect the realities of trace analysis in complex matrices. General validation characteristics are covered in more depth in our sibling article on analytical method validation under ICH Q2(R2).

How results appear on a certificate of analysis

A well-written elemental impurities result lets you see exactly what was measured. Look for:

1. Element list. Each element is named, not grouped as “heavy metals”.
2. Units. Usually µg/g (ppm) of sample; some reports use ng/g (ppb) for very low levels.
3. Censored values. “< 0.05 µg/g” means the element was below the stated quantitation limit, not that it is absent. The LOQ itself tells you how sensitive the test was.
4. Technique and method reference. ICP-MS or ICP-OES, and whether the method follows or was validated against USP <233>.
5. Mass basis. Whether the result refers to the material as received (including water and counter-ions) or is corrected.

When comparing two lots, compare LOQs as well as results. A result of “< 1 µg/g” and one of “< 0.01 µg/g” are both passing values but represent very different levels of sensitivity. Veridryx publishes lot-level COAs in its Testing Library, and our guide on how to read a certificate of analysis walks through the other sections.

Frequently asked questions

Is “heavy metals” the same as elemental impurities?

No. “Heavy metals” usually refers to the retired USP <231> sulfide precipitation test, which gave one combined value. Elemental impurities testing quantifies each element individually by ICP-OES or ICP-MS.

Why is ICP-MS usually chosen for peptides?

Peptide samples are often small, and ICP-MS has detection limits low enough to quantify Class 1 elements from milligram-scale digests. ICP-OES remains a valid choice when limits are higher or more material is available.

Does a “< LOQ” result mean the element is not present?

It means the concentration was below the lowest level the method can quantify with acceptable accuracy and precision. The element could still be present at a lower concentration.

Do USP <232> limits apply to research reagents?

The limits are written for drug products. For research materials they serve as a reference point, and the <233> procedures and validation criteria are a widely used analytical benchmark.

References

1. USP General Chapter <232> Elemental Impurities—Limits.
2. USP General Chapter <233> Elemental Impurities—Procedures.
3. ICH Q3D(R2) Guideline for Elemental Impurities.
4. USP General Chapter <730> Plasma Spectrochemistry.