How Laboratories Prepare Hair Samples for HTMA

Introduction

Hair Tissue Mineral Analysis (HTMA) is not only dependent on analytical instrumentation such as ICP-OES or ICP-MS. The reliability of laboratory results is also strongly influenced by pre-analytical processing — including sample collection, washing, digestion and contamination control.

Different laboratories may use different preparation protocols, which can influence mineral concentrations and reproducibility. Understanding these procedures is important when interpreting HTMA data scientifically.

Why sample preparation matters

Hair is continuously exposed to the external environment. Cosmetic products, shampoos, swimming pools, dust, sweat and airborne particles may all alter surface mineral content.

Before elemental analysis can occur, laboratories attempt to reduce external contamination while preserving endogenous mineral content inside the hair shaft.

The balance between “over-cleaning” and “under-cleaning” remains one of the most debated aspects of HTMA methodology. For more on this topic, see external contamination in HTMA.

Sample collection procedures

Most HTMA laboratories request hair from the occipital region of the scalp because:

• growth tends to be more uniform,
• environmental exposure may be lower,
• sampling reproducibility is improved.

Typical recommendations include:

• untreated or minimally treated hair,
• avoiding hair products immediately before sampling,
• sufficient sample mass (usually 100–150 mg).

The proximal portion of the hair strand is generally analyzed because it reflects more recent mineral deposition.

Washing protocols in HTMA laboratories

Washing protocols are designed to remove:

• sweat residues,
• environmental particles,
• cosmetic residues,
• oils and external metals.

Common laboratory washing agents include:

• nonionic detergents,
• acetone,
• deionized water,
• Triton X solutions.

However, washing itself may influence results. Excessive washing can potentially reduce endogenous minerals such as potassium or sodium. For this reason, laboratories often use standardized multistep washing procedures designed to improve reproducibility.

For related discussion on heavy metal measurement, see heavy metal interpretation in HTMA.

Drying and weighing

After washing:

• samples are dried under controlled conditions,
• hair is weighed precisely,
• laboratories standardize sample mass before digestion.

Analytical precision during weighing is critical because elemental concentrations are later expressed relative to sample weight.

Acid digestion before ICP analysis

Before elemental measurement, solid hair samples must be converted into liquid form.

This is typically achieved through acid digestion using:

• nitric acid,
• hydrogen peroxide,
• microwave digestion systems.

Digestion breaks down keratin structures and releases trace elements into solution.

Incomplete digestion may influence:

• recovery rates,
• detection limits,
• reproducibility.

ICP-OES and ICP-MS measurement

After digestion, laboratories analyze the solution using elemental spectroscopy platforms.

The two most common technologies are:

• ICP-OES (Inductively Coupled Plasma Optical Emission Spectrometry),
• ICP-MS (Inductively Coupled Plasma Mass Spectrometry).

ICP-OES is widely used in commercial HTMA laboratories because:

• it supports multi-element analysis,
• operational costs are lower,
• throughput is high.

ICP-MS offers:

• lower detection limits,
• higher sensitivity,
• improved trace metal detection.

For a detailed comparison, see ICP-OES vs ICP-MS.

Sources of laboratory variability

Differences between laboratories may result from:

• washing protocols,
• digestion chemistry,
• calibration standards,
• instrumentation,
• quality control procedures,
• reference ranges.

This explains why direct comparison between laboratories may sometimes produce inconsistent values.

Scientific interpretation therefore requires:

• methodological transparency,
• reproducibility studies,
• quality assurance systems.

Contamination control and quality assurance

Professional laboratories typically use:

• blanks,
• certified reference materials,
• duplicate measurements,
• calibration verification,
• contamination-controlled environments.

Quality assurance protocols are essential because trace mineral analysis involves extremely low elemental concentrations.

Laboratory accreditation standards may also improve analytical consistency.

Scientific limitations

HTMA preparation protocols remain heterogeneous across laboratories.

Current scientific debates include:

• optimal washing procedures,
• preservation of endogenous minerals,
• standardization between laboratories,
• interpretation of toxic element levels.

HTMA should therefore be understood as a laboratory methodology with both strengths and limitations rather than a universally standardized diagnostic system. For broader context, see what HTMA can and cannot show.

Key takeaways

• HTMA reliability depends heavily on sample preparation quality.
• Washing and digestion protocols influence mineral results.
• ICP-OES and ICP-MS require standardized laboratory procedures.
• Contamination control is critical in trace element analysis.
• Inter-laboratory variability remains an important scientific limitation.

Frequently Asked Questions

References

1. Kempson IM, Lombi E. Hair analysis as a biomonitor for toxicology, disease and health status. Chem Soc Rev. 2011;40(7):3915-3940.
2. Rodushkin I, Axelsson MD. Application of double focusing sector field ICP-MS for multielemental characterization of human hair and nails. Part III. Analyst. 2000;125(2):353-360.
3. Pozebon D, Dressler VL, Curtius AJ. Hair analysis: a review of the bioanalytical methods used for the assessment of trace elements. Anal Bioanal Chem. 1999;364(4):370-374.
4. Eastman RR, Jursa TP, Benedetti C, Lucchini RG, Smith DR. Hair as a biomarker of environmental manganese exposure. Environ Sci Technol. 2013;47(3):1629-1637.
5. Mikulewicz M, Chojnacka K, Gedrange T, Górecki H. Reference values of elements in human hair: a systematic review. Environ Toxicol Pharmacol. 2013;36(3):1077-1086.
6. Morton J, Carnahan J, Affleck S. Manganese in hair: environmental exposure and laboratory considerations. Biomarkers. 2002;7(4):299-310.
7. Chojnacka K, Górecka H, Górecki H. The effect of hair treatments on trace element concentrations in hair. Environ Toxicol Pharmacol. 2006;22(1):32-36.
8. Iyengar GV, Woittiez J. Trace elements in human clinical specimens: evaluation of literature data. Crit Rev Clin Lab Sci. 1988;26(2):135-157.
9. Baranowska I, Baranowski A, Wójcik G, Jarek U. The influence of sample preparation on trace element determination in hair samples. Pol J Environ Stud. 2004;13(4):389-394.
10. International Atomic Energy Agency (IAEA). Elemental analysis of biological materials. Laboratory quality guidance. IAEA-TECDOC-426. 1987.