Top Three Challenges in External Accreditation Audits

Coping with the requirements of customers and external accreditation authorities is the day-to-day business of a lab manager. The three points outlined in this article spell the key to producing reliable results.

By Elisabeth PICHLER, Head of Quality Management, Romer Labs®

External audits for ISO 17025 accreditation can be a true test of expertise. The auditors typically work in the same field and are themselves genuine experts. They assess the technical competence of the laboratory in-depth. For most labs traceability, measurement uncertainty and matrix effects pose the biggest challenges during an external accreditation audit.

Traceability

This is one issue every laboratory struggles with. An accredited laboratory is required to show that a result on one of its test reports can be traced back to international standard (SI) base units. In the case of a result stated in µg/kg (microgram per kilogram) obtained by HPLC (High Performance Liquid Chromatography) or LC-MS (liquid chromatography with mass spectrometry detection) this implies clear documentation. Both use a liquid standard for calibration, and in order to claim full traceability back to the SI base unit kilogram, auditors will want a certification report stating the full procedure of preparation and all measures taken by the supplier. (For more, see the box text on 'Traceability and Certified Reference Materials')

Measurement uncertainty

Under ISO 17025 requirements, the measurement uncertainty (mu) of every accredited method must be calculated and included in the test report. There are many ways to estimate or calculate measurement uncertainty and which way is accepted depends a lot on the preference of the national accreditation body. A simple and practical way for small laboratories to estimate measurement uncertainty is through the use of control charts. It is good laboratory practice to use a matrix-based control sample that ideally is naturally contaminated with or has been spiked with the analyte of interest. This sample is then added to each sequence run.

Results of the control samples are plotted on control charts that are used for long-term assessment and identification of trends for each method. The measurement uncertainty is derived from a two-sigma standard deviation of all results. In Europe, a valid approach is the “fitness for purpose” approach published in Com-

Traceability and Certified Reference Materials

Full is a key factor in audit situations. Certified reference materials, accompanied by full and clear documentation, are one way to accomplish this.

By Lilian KUSTER, Product Manager, Romer Labs®

Many terms describe reference standards in analytical methods, like reference materials, certified reference materials, calibrator, standard, etc.

Certified reference materials are defined as reference materials characterized by metrological traceability, a certified value and an uncertainty budget. It complies with all requirements of ISO 17025, GLP, etc. and comes with complete documentation on:

• purity assessment of the raw material

• traceability back to national standards

• measurement uncertainty and its calculation

• a homogeneity study

• a long- and short-term stability study

• description of intended use

Certified reference materials are intended for the verification of in-house standards such as reference materials routinely used in the lab. They are used for the calibration of equipment especially in ISO 17025 accredited testing laboratories where traceability and high quality of results are of great importance. solutions considered as homogeneous)

Table 1 illustrates the differences between a reference material and a certified reference material for BiopureTM calibrants. A certified reference material has full proof of traceability which is important for audit situations. Furthermore, it has a stated uncertainty and full transparency of the preparation and certification process.

Only an accredited institution which strictly follows ISO Guide 34 is allowed to issue certified reference materials. ISO Guide 34 defines the “General requirements for the competence of reference material producers” and is built upon ISO 17025, including additional technical requirements for certified reference materials.

Full set of homogeneity data according to ISO Guide 35 is available and statistically evaluated

Approved by national accrediting body Processes and calculations

Competence of producer No formal proof of competence

Not 100 % transparent

Equipment Use of calibrated standard equipment

Fully traceable and described in the certification report

Use of equipment with external calibration certificates and lower uncertainty

Packing/Filling Screw vial with high quality brown glass Crimp vial with inert glass of highest quality mission Regulation (EC) No 401/2006 which details the methods of sampling and analysis for the official control of mycotoxin levels in foodstuffs. The following formula presents a way of calculating the maximum standard uncertainty:

1. Describe the measured value in terms of your measurement process (model the measurement)

2. List the input quantities

3. Determine the uncertainty for each input quantity

4. Evaluate any covariances/correlations in input quantities

5. Calculate the measured value to report

6. Correctly combine the uncertainty components

7. Multiply the combined uncertainty by a coverage factor

8. Report the result in the proper format where:

• Uf is the maximum standard uncertainty (μg/kg)

• LOD is the limit of detection of the method (μg/kg)

• α is a constant, numeric factor to be used depending on the value of C.

• C is the concentration of interest (μg/kg).

1. Incomplete definition of the quantity being measured

2. Imperfect realization of the definition of the quantity being measured

3. Non-representative sampling

4. Inadequate knowledge of the effects of environmental conditions on the measurement or imperfect measurement of environmental conditions

5. Personal bias in reading analog instruments, including the effects of parallax

6. Finite resolution or discrimination threshold

7. Inaccurate values of measurement standards and reference materials

8. Inexact values of constants and other parameters obtained from external sources and used in the data-reduction algorithm

Another approach is to follow the steps described in JCGM 100:2008 Guide to the Expression of Uncertainty in Measurement (GUM) shown in Table 2. Accordingly, uncertainty in a measurement is a result of our incomplete knowledge of the value of the measured quantity in combination with the factors influencing it. Table 3 lists many possible sources of uncertainty in measurement.

Matrix effects

Accredited service labs typically face the daily challenge of receiving samples of various matrices. These laboratories validate their methods for the most common matrices. However, even the analysis of an apparently simple matrix like maize is highly influenced

Certification ≠ Accreditation

The terms certification and accreditation are often confused. Whoever wants to certify something, whether it is a management system, a product or a person, needs to be accredited for this task by a national accreditation body. Acquiring ISO 17025 accreditation allows for the issuance of relevant certifications in many jurisdictions.

9. Approximations and assumptions incorporated in the measurement method and procedure

10. Variations in repeated observations of the measure and under apparently identical conditions by different corn varieties. More complex samples like compound feed or highly processed food can alter analysis results tremendously. Using 13C-labeled internal standards for every analyte and a calculation based on an internal calibration are the most accurate, state-of-the-art approaches in use today.

(For more, see the box text on 'Overcoming LC-MS/ MS Matrix Effects for Maximum Reliability', page 7)

Conclusion

Traceability, measurement uncertainty and matrix

effects pose the biggest challenges for most labs during an external accreditation audit. A certification report from an accredited supplier goes a long way to demonstrate traceability. Several methods are available to calculate measurement uncertainty. However, only the 13C-labeled internal standards can fully correct for matrix effects. With these tools in hand, laboratories will be better positioned to successfully navigate the audit process.

Overcoming LC-MS/MS Matrix Effects for Maximum Reliability

As in any analytical technique, liquid chromatography-tandem mass spectrometry (LC-MS/MS) results are influenced by the matrix analyzed. These matrix effects – caused by ion enhancement or suppression – can produce overestimates or underestimates of measured values of the target analyte. Validation of routinely used methods may eliminate the most severe effects in common matrices. However, even in very simple matrices like corn, different varieties might have different influences on ionization, therefore leaving significant room for unreliability.

By Lilian KUSTER, Product Manager, Romer Labs®

Common approaches to counter matrix effects include matrix-matched calibration, standard addition to each sample and the application of internal standards. The first two strategies entail additional work in terms of sample preparation or additional runs, incurring further costs. Importantly, matrix-matched calibration and standard addition cannot fully compensate for matrix effects. The effectiveness of internal standards in compensating for matrix effects depends on the choice of calibrants.

Calibrant choice matters

Internal standards can be chemically-related compounds such as derivatives (e.g. zearalanone for zearalenone) or similar compounds with identical behavior during the ionization process that differ only in terms of the mass of the atoms (stable isotopes). LC-MS/MS analyses rely on stable isotope dilution assays to overcome matrix effects by the addition of known amounts of stable isotope-labeled standards to the analyzed sample. Stable isotope labeling involves the use of non-radioactive isotopes like ²H, 13C or 15N to replace the naturally occurring atom. Using Deuterium (²H) to replace the naturally occurring 1H doubles the mass, which is the reason why deuterated labeled standards might show retention time shifts resulting in less accurate LC-MS/MS results.

Using a nitrogen isotope is impractical because it does not naturally occur in all substances.

Carbon, on the other hand, is naturally present in most compounds and mycotoxins. Replacing naturally-occurring 12C by 13C changes the total mass of the atom only slightly, unlike deuterated labeled standards. Added 13C-labeled standards, e.g. 13C-labeled mycotoxins, retain the same characteristics as their 12C analogues, eluting at the same retention time from the separation column. The difference in mass between 12C and 13C mycotoxins, as shown for deoxynivalenol in Figure 1, allows the separation and identification of both eluted forms when the detection is performed with mass spectrometry.

The 13C peak represents the known quantity of the added standard. This peak can then be used to calculate the unknown amount of the analyte and thereby compensate for different ionization efficiencies. This explains how innovative 13C standards eliminate matrix effects.