Understanding the numbers

Issues to consider

  • Sample selection
  • Sampling procedures
  • Nature of the sample

General workflow

  • Extraction (solvent, solid or liquid phase extraction, selectivity, matrix, interfering compounds)
  • Clean-up (selectivity, interference, matrix, solid or liquid phase)
  • Concentration (evaporation or extraction, method of evaporation [e.g. heat or vacuum], extent of concentration, limit of quantification)
  • Analysis (method, instrumentation, interpretation)

Aim: to introduce as much of the analyte (chemical of interest) to the measurement system as possible

Chromatography - resolution in time

  • Sample introduction
  • Carrier gas (helium, nitrogen or hydrogen) or solvents
  • Resolution - separation of molecules (column phase and length, temperature, carrier gas or solvent composition)
  • Detection (broad spectrum, e.g. flame ionization detector [FID], electron capture detector [ECD], nitrogen-phosphorus detector [NPD] or selective detectors, e.g. ultra-voilet [UV] or mass spectroscopy [MS])

Mass spectroscopy - resolution in space

  • Highly selective 
  • Very sensitive
  • Versatile, especially in combination with liquid (LC) or gas chromatography (GC)
  • Different ionisation techniques
  • Different mass filters (e.g. Quadruploe [Q], sector instrument, orbitrap, time-of-flight [TOF])
  • Hyphenation (i.e. MS-MS, Q-TOF)

Sample reduction (mills, fraction collecters, mixers, shakers, tablet press, microwave, high pressure asher, automatic bead machine, bomb combustion)

Analytic challenges

  • Low levels of most allergens
  • Limits of detection (LOD)
  • Limits of quantification (LOQ)
  • Background concentrations
  • Composite samples (dilution)
  • Natural variation
  • Particulates and homogeneity 
  • Comparibility of methods

Quality Assurance

  • ISO 17025
  • Method validation
  • Quality assurance in practice

ISO 17025

  • General requirement for competance testing and calibration
  • ISO9001 management plus technical requirements (i.e. equipment, sampling, technical competancy)

Good laboratory practice

  • Specific to safety (planning, execution, monitoring, data recording, data storage, reporting)

Method validation
Process of providing documented evidence that a method does what it is intended to do

  • Ensure that an analytical method is accurate, reproducible and rugged over the concentration range analysed
  • Validation provides assurance of reliability and comparability

Analytical performance characteristics

  • Precision
  • Accuracy (trueness)
  • Limit of Detection (LOD)
  • Limit of Quantification (LOQ)
  • Specificity / Selectivity
  • Linearity and Range
  • Robustness
  • Method uncertainty

Precision - degree of agreement amongst test results when the method is applied repeatedly to multiple samplings of a homogeneous sample

Expressed as percentage (%) relative standard deviation (RSD) for a statistically significant number of samples

Precision should (ideally) be performed at three levels (increasing complexity)

  • Repeatability
  • Intermediate precision
  • Reproducibility

In practice: 1 sample, 3 replicates a day for 5 days

Accuracy - closeness of test results obtained by the method to the true value

  • Established across the range (ideally)
  • Analysis of (certified) reference material
  • Compare results to a second, well-characterized method
  • Analysis of spiked samples with known quantities of components (recovery)

Selectivity - extent to which a method can determine particular analytes in mixtures or matrices without interferences from other component
IUPAC: “Specificity is the ultimate selectivity” and “Sometimes the term specificity is used. This usage of specificity suggests that no component other than the analyte contributes to the result. Hardly any method is that specific (sic!) and, in general, the term should be avoided

Method validation

  • Detection Limit (LOD) - lowest concentration of analyte in a sample that can be detected (not necessarily quantitated) (limited value in practice)
  • Quantification Limit (LOQ) - lowest concentration of analyte in a sample that can be determined with acceptable precision and accuracy
  • Linearity - ability of the method to elicit test results that are directly proportional to the mass fraction within a given range (variance of the slope of the regression line)
  • Range - interval between upper and lower levels of analyte demonstrated by the method

Robustness - measure of the capacity to remain unaffected by small (deliberate) variations in method parameters

  • Indication of reliability during normal use

If measurements are susceptible to variations in analytical procedures, these conditions should be controlled and a precautionary statement included

Quality assurance in practice - Analytica

  • Selection of reference materials (standards)
    • Internal standard(s)
    • External standard(s)
    • Syringe standard(s)
    • Calibration standards
  • (Certified) reference materials
  • QC-samples
  • QC-charts
  • Proficiency tests
  • Sample preparation
    • Record everything that happens
    • Balance: calibration and handling
    • Samples (randomise)
    • Method blanks (1/5, 1/10, …)
    • Control samples (replicates, (C)RM, …)
  • Instrumental analysis
    • Instrument condition (tune file, peak shape, baseline, …?)
    • Group calibration – group “samples”
    • Randomise within each group
    • Include quality control steps
      • Solvent blanks
      • Replicate standard injections (drift control)

Reference materials - sufficiently homogeneous and stable with respect to one or more specified properties, which has been established to be fit-for-purpose

  • Reference material is a generic term
  • It can only be used for a single purpose in a given measurement
  • Properties can be quantitative or qualitative, e.g. identity of substances or species
  • Uses may include the calibration of a measurement system, assessment of a measurement procedure, assigning values to other materials, and quality control

Certified reference material - reference material characterised by a metrologically valid procedure for one or more specified properties, accompanied by a certificate that provides the value of the specified property, its associated uncertainty, and a statement of metrological traceability

  • Concept of values includes qualitative attributes, such as identity or sequence. Uncertainties for such attributes might be expressed as probabilities.
  • Metrologically valid procedures for the production and certification of reference materials are given in, amongst others, ISO Guides 34 and 35
  • ISO Guide 31 gives guidance on the contents of certificates

Traceability is defined variously as the ability to trace and follow food, feed and ingredients through all stages of production, processing and distribution compared with Metrological traceability, which is the property of a measurement result, whereby the result can be related to a stated metrological reference through a documented unbroken chain of comparisons, all having stated measurement uncertainties.

  • Methods are not traceable
  • Traceability is closely linked to measurement uncertainty
    • Uncertainty plus traceability are the basis of measurement reliability
  • Traceability to a common reference facilitates comparability of results and ensures defined and constant measurement conditions
  • Traceability chain is the sequence of standards and comparisons that relate the result to the stated reference, which should be linked to:
    • Measurement unit
    • Measurement proceedure
    • Reference material

Traceability to a unit

  • Preferably to the SI unit
    e.g. 5 kg means the measurement result of the property “mass” has been obtained by comparing the mass of the item with the mass of the kilogram in Paris (FR)
  • Exists for most physical measurements,  - independent of the method
  • Not (yet) available for many chemical measurements
    • kg ≠ kg depending on the method employed
    • SI-unit is not always relevant for the application (mass vs. catalytic concentration of an enzyme)

With regard to traceability, a certifed reference material has:

  • Certified value with uncertainty
  • Stated traceability (to a final reference)

CRMs ensure traceability of:

  • Measurement conditions, classical calibration
  • Results of final quantification (through calibration)
  • Results of overall method (through validation)
  • Result in daily work

With regard to sample preparation, every sample preparation step breaks the traceability chain (not sure what went in or comes out)

Method assumes correctness, absence of losses, etc. Method validation restores the traceability chain using matrix CRMs.

Measurement uncertainty - degree of doubt about the correctness of a measurement result

Standard uncertainty (“small u”) - format of a standard deviation

Expanded uncertainty (“capital U”) - interval that includes the true value with a high probability

  • If interval incorrectly too small: wrong conclusions
  • If interval incorrectly too large: useless result

CRM are linked to uncertainty through:

  • Validation - proving method trueness - use of CRMs requires uncertainty information
  • Calibration - contributes to uncertainty - CRMs contribute to uncertainty information

Knowledge of the uncertainty of a measurement result is essential for the correct interpretation of results

  • In-depth knowledge of procedure and transparency
  • Demonstration of competence: procedure is under control
  • Identification of (the main) variation sources in a procedure: hints for improvements
  • Confidence: “with x% probability my result is y ± U”
  • Necessary for ISO 17025 accreditation
  • Comparability of data
  • End user has obtained result with adequate confidence interval
  • Audits
  • Required by external contractors

Two uncertainty types

  • Type A : the best estimate of input quantity has been obtained by repeat measurements
  • Type B:  obtained by other means (certificate of analysis, previous measurement data, manufacturer’s specifications, etc.) 

Measurement uncertainty - summary bottom up

  • Modelling of the measurement
  • Identification of all uncertainty sources
  • Evaluation of uncertainties of the input quantities, type A and B
  • Combination of uncertainties

In a measurement, some effects concern:

  • Only the individual measurement - dilutions, pipetting, injections, weighing ...
  • All measurements in a particular series - measurement standard, temperature in the lab, batch of reagents …
  • All measurements with a particular method - deviation from the “true” value

Measurement uncertainty - bottom up

  • Guide for method improvement
  • Evidence that method is fully understood
  • Scientifically elegant
  • Difficult to know all factors
  • Difficult to define equation comprising all factors

Measurement uncertainty - top down

  • Easy to implement
  • Easier to cover all effects
  • No information for method improvement
  • Crude approach

Expanded uncertainty

Combined uncertainty uc : format of a standard deviation 

  • Range ± s covers two-thirds of possible outcomes
  • Need expansion factor to cover “a large fraction of” cases

Expansion factor is called “coverage factor” k, which has the same purpose as t-factor for confidence intervals

Expanded uncertainty U = k x uc

Choose coverage factor k according to level of confidence required.

For normally distributed values and sufficient degrees of freedom: k = 2 (95 % conf.), k = 3 (99 % conf.)

Few values (n < 6) only: recommendation is to set k equal to the two-tailed value of Student’s test for the number of degrees of freedom associated with that contribution, and for the level of confidence required (normally 95 %).

Scruitinising analytical data

Step 1: Determine your need

Step 2: See what you have

Step 3: Use common sense

References

International Vocabulary of Metrology – Vocabulaire International de Métrology (VIM)

ISO guide 17025

ISO guide 5725

Hauck et al. (2008) Making sense of trueness, precision, accuracy and uncertainty. Pharmacopeial Forum 34(3): 838-842

Vessman et al. (2001) Selectivity in analytical chemistry (IUPAC Recommendations 2001) Pure Appl. Chem. 73(8): 1381-1386

GFox (2003) GLP regulations vs. ISO 17025 requirements: How do they differ? Accred. Qual. Assur 8:303 | doi:10.1007/s00769-003-0623-y

ISO/IEC 17025: 2005 General requirements for the competence of testing and calibration laboratories

ISO/IEC Guide 99: 2007 International vocabulary of metrology - basic and general concepts and associated terms

EURACHEM/ CITAC Guide (2003) (eds. Ellison, King, Rösslein, Salit & Williams) Traceability in chemical measurement: A guide to achieving comparable results in chemical measurement

Guide to the expression of uncertainty in measurement, ISO, Geneva, 1995, ISBN 92-67-10188-9

EURACHEM / CITAC Guide CG4 (2012) (3rd Edition): Quantifying uncertainty in analytical measurement

Teemu (2012) Handbook for calculation of measurement uncertainty in environmental laboratories, 2nd Edition, Nordtest, Espoo, FI

ISO 21748:2010 Guidance for the use of repeatability, reproducibility and trueness estimates in measurement uncertainty estimation

Trappmann et al. (2007) Guidance document on measurement uncertainty for GMO testing laboratories, EUR - Scientific and Technical Research Reports ISBN 978-92-79-05566-9

http://www.nordtest.org

http://www.measurementuncertainty.org

Meyer (2007) Measurement uncertainty.  J Chromatogr. A 1158, 15 | DOI:10.1016/j.chroma.2007.02.082 | PMID: 17359984