Speciation Analysis - Striving for Quality

Speciation analysis is aiming to define and quantify the distribution of a target element between the different species in which it occurs. While it is well accepted that information provided by such speciation analysis is mandatory for a complete understanding of processes in biological systems or the environment, the lack of proven quality of such data is seriously limiting its value. 

Unfortunately, concepts for quality control and quality assurance widely implemented in the field of trace element analysis are not often applied to speciation analysis. The higher complexity of speciation analysis in comparison to total element analysis is often used as an excuse for the lacking validation. However, apart from the higher complexicity, speciation analysis shares most problems also known in the field of trace element analysis and therefore can also gain from such quality concepts.

While the final steps of the analytical procedure including the analytical measurement itself receive the greatest attention, actually the first steps of analysis including sampling, sample storage and sample preparation are the most problematic ones. While this is true not only for speciation analysis but for all chemical analysis procedures, speciation analysis has additional problems in these steps.  The major problem of speciation analysis in the course of these steps is to maintain the original distribution of species (speciation) through all the steps until the final step of determination and quantitation.  The ideal solution to this problem would be in situ analysis using sensors. However, very few sensors provide the necessary selectivity and sensitivity required for trace element speciation analysis.

A specific problem of speciation analysis is that original information (molecular integrity) of the species is partially being lost as the sample preparation progresses. Therefore, the best sample preparation for speciation analysis is no preparation at all.

Unfortunately, only few techniques such as EXAFS/XANES provide species data from solid samples. For all other techniques the reduction of the sample preparation to the absolutely necessary number of steps remains the appropriate strategy.

Most often the selectivity and sensitivity required for trace elemental speciation analysis calls for the use of hyphenated techniques combing the separation power of a chromatographic or equivalent separation technique with the detection power of the most sensitive atomic spectroscopic techniques (e.g. ICP-MS, MIP-AES, AFS). These techniques call for liquid samples and therefore sample preparation can seldom be avoided. In these cases obtaining meaningful results requires careful validation of each of the steps of the sample preparation in order to avoid artefacts by species transformation or losses during analysis.

• Sampling: Changes of pH, pE should be avoided; microbial activity should be stopped, stability of species should be verified
• Preservation: Stability of species during sample storage should be verified; absence of analyte losses by sorption to container walls or volatilization should be proven
  
• Extraction: Excessive energy input (e.g. heating etc.) and harsh conditions (oxidants etc.)  must be avoided; stability of species during extraction must be verified; extraction yield should be monitored
• Derivatization: Absence of species transformation during derivatization should be proven; quantitative derivatization yield should be proven
• Separation: absence of species transformation during separation should be proven; column yield should be checked by mass balance with total element concentration; appropriate separation power must be verified;
• Detection/Identification: Unambiguous identification of species (e.g. via molecular mass and retention time) should be verified; "identification" of species in the void volume of chromatograms should be avoided;
  
• Calibration/Quantification: calibration should be done by using reference standards or reference materials; absence of interferences should be proven; factors influencing the result should be assessed systematically; uncertainty of the result should be estimated based on scientific understanding of the theoretical principles of the method and practical experience; results should be compared to those achieved with other methods
  

Meaningful validation for speciation analysis

By carefully looking at the above list of necessary tests it becomes clear that total element analysis should be an integral part of speciation analysis. Without knowing the part of the analyte that was determined, the whole speciation analysis becomes very questionable. A meaningful quality assurance and quality control (QA/QC) frequently includes analysis of a Certified Reference Material (CRM), comparison of results from independent techniques, or analysis of a proficiency test sample. The validation should be as extensive as is necessary to meet the needs of the given application (fit-for-purpose) and the range and accuracy of the values obtainable are all relevant figures of merit (e.g. the uncertainty of the results, detection limit, selectivity of the method, linearity, repeatability and/or reproducibility, robustness against external influences and/or cross-sensitivity against interference
from the matrix of the sample or test object), as assessed for the intended use.

While most peer reviewers expect to see such corroboration of results and many journal editors are at least aware of the importance of doing so for total element analysis, this is not yet the case for speciation analysis. While it is true that the number of CRMs for speciation analysis is rather limited (see our list), many of the above mentioned tests improving the credibility of results can be applied even when no appropriate CRM is available. An alternative way is the use of a primary method, such as species-specific isotope dilution analysis (SIDMS). SIDMS allows corrections for extraction yields, species inter-conversions and quantitation based on an ideal internal standard when suitably
characterised enriched species are available.

Interestingly, journal editors have started to recognize the lack of quality related strategies implemented in submitted papers. As one of the first journals Environmental Chemistry has now published rules for reporting speciation data as a part of their notice to authors.  We encourage all editors but also reviewers to request the performance of such procedures especially when authors suggest new speciation analytical methods.

Publications discussing reliability of speciation analysis (general discussion)

Heidi Goenaga Infante, Quality Control in Speciation Analysis Uasing HPLC with ICP-MS and ESI-MS/MS: Focus on Quantification Strategies Using Isotope Dilution Analysis. In: Bernhard Michalke (ed.), Metallomics: Analytical Techniques and Speciation Methods, Wiley-VCH, Weinheim, 2016, 69-82. DOI: 10.1002/9783527694907.ch3

Claudia Swart, Metrology for metalloproteins - where are we now, where are we heading ?, Anal. bioanal. Chem., 405/17 (2013) 5697-5723. DOI: 10.1007/s00216-013-6933-y

OECD: Series on Principles of Good Laboratory Practice (GLP) and Compliance Monitoring

LGC/VAM: Best Practice Guide for Generating Mass Spectra

LGC/VAM: Best Practice Guide for Meeting the traceability requirements of ISO17025

LGC/VAM: Best Practice Guide for Preparation of Caqlibration Curves

Chapter 15: Liquid-liquid extraction in speciation analysis
Chapter 16: Solid-phase extraction in speciation analysis

Chapter 17: Mercury 'speciation analysis' by using thermal desorption analysis
Chapter 18: Multielement speciation analysis

Chapter 19: Chemical speciation modelling