Spot On: Challenges in External Accreditation Audits

Issue 1

A magazine of Romer Labs®

3

Creating Liquid Mycotoxin Calibrants: A Behind-the-Scenes Look

Photo: alphaspirit

Top Three Challenges in External Accreditation Audits

Content

4-6

Top Three Challenges in External Accreditation Audits Successfully navigate the biggest challenges for most labs: traceability, measurement uncertainty and matrix effects.

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

Traceability and Certified Reference Materials Full traceability 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, PhD, Product Manager, Romer Labs®

Overcoming LC-MS/MS Matrix Effects for Maximum Reliability Spot On is a quarterly publication of Romer Labs Division Holding GmbH, distributed free-of-charge.

5 7

Validation of routinely used methods may eliminate severe effects in common matrices, but still leave significant room for unreliability.

By Lilian KUSTER, PhD, Product Manager, Romer Labs®

ISSN: 2414-2042

For a digital copy and details, visit: http://magazine.romerlabs.com Editor: Cristian Ilea, Simone Schreiter

Contributors: Elisabeth Pichler, Lilian Kuster, Anna Lilek Graphic: Reinhold Gallbrunner Research: Kurt Brunner

Publisher: Romer Labs Division Holding GmbH Erber Campus 1 3131 Getzersdorf, Austria Tel: +43 2782 803 0 www.romerlabs.com

©Copyright 2015, Romer Labs® All rights reserved. No part of this publication may be reproduced in any material form for commercial purposes without the written permission of the copyright holder except in accordance with the provisions of the Copyright, Designs and Patents Act 1998.

All photos herein are the property of Romer Labs or used with license.

2

Creating Liquid Mycotoxin Calibrants: A Behind-the-Scenes Look

8-11

A step-by-step walk through the production path of a key reference material for the food and feed industries.

By Anna LILEK, Quality Management, Romer Labs®

Spot On Issue 1

Editorial Threading the Needle Analytical service laboratories work under a lot of pressure. Being able to submit results of the highest quality for various matrices is an important pre-requisite for potential customers. However, customers are often not able to evaluate the quality of a result and can only rely on external accreditation according to ISO 17025 to assess the competence of a laboratory. Their most important criteria for choosing a certain lab are usually turnaround time and price. In contrast to these market pressures, the national accreditation body asks for elaborate documentation, validations for all matrices, control samples, full traceability, participation in international ring trials and regular trainings for all lab employees - all factors that add costs and reduce speed in the lab. In this highly competitive environment it is crucial to find ways to fulfill contradicting requirements most efficiently.

In this inaugural issue of Spot On, our magazine for analytical industry professionals, we address the three biggest challenges that labs encounter during the external audit process: traceability, measurement uncertainty and matrix effects. Certified reference materials with a certificate of analysis can go a long way towards fulfilling the first point. Several different methods are available to determine the second.

Regarding the third point, 13C-labeled standards can fully correct matrix effects in LC-MS/MS analysis. Finally, to further explore traceability we walk through the full production process from the initial mold culture to the delivery of a liquid mycotoxin calibrant to clients.

We hope you enjoy this first issue of our quarterly Spot On magazine.

Happy reading!

Elisabeth Pichler Head of Quality Management, Romer Labs®

A magazine of Romer Labs®

3

Photo: alphaspirit

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.

4

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

Spot On Issue 1

E

xternal 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.

➊

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')

➋

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 ComA magazine of Romer Labs®

Traceability and Certified Reference Materials Full traceability 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.

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.

Table 1. Differences between BiopureTM reference and certified reference materials Purity of raw material Documentation

Stability data

Homogeneity data Competence of producer Processes and calculations Equipment Packing/Filling

BiopureTM Reference Material

BiopureTM Certified Reference Material

Assessed by HPLC-UV

Assessed by accredited qNMR method

Certificate of Analysis Available, but no systematic approach

Not available (liquid solutions considered as homogeneous) No formal proof of competence

Not 100 % transparent Use of calibrated standard equipment

Screw vial with high quality brown glass

Certificate of Analysis and a full certification report

Long- and short-term stability data according to ISO Guide 35 are available and statistically evaluated Full set of homogeneity data according to ISO Guide 35 is available and statistically evaluated Approved by national accrediting body

Fully traceable and described in the certification report

Use of equipment with external calibration certificates and lower uncertainty Crimp vial with inert glass of highest quality

5

Table 2. The eight-step GUM method

With these tools in hand, laboratories will be better positioned to successfully navigate the audit process.

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: !" =

(

!"# ! ) + (! ×!)!

2

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).

Table 1. Values to be used for α Concentration (μg/kg)

α

≤ 50

0,2

51-500

0,18

1 001-10 000

0,12

501-1 000

0,15

> 10 000

0,1

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.

➌

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.

6

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 Table 3. Possible sources of uncertainty in measurement

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 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 13 C-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.

Spot On Issue 1

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.

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 13 Natural DON 296 amu → +15 amu → 13C DON C-labeled standards, e.g.

13 C-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.

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.

Figure 1. Full scan MS spectra of (13C15)-Deoxynivalenol (Biopure) x 10 4 2.5

Naturally occurring DON �M-H�- m/z = 295 amu

13

13

295.12

Intensity

2.0

0.5 0.0

13

296.12

295

300

15 amu

309.17 308.17 307.16

305

Red line: Naturally-occurring DON. Blue line: BiopureTM 13C internal standard.

A magazine of Romer Labs®

310.17

C14-DON 15.8%

C13-DON 2.8%

297.12 298.12

290

C15-DON 81.4% 13

1.5 1.0

C-DON isotope labeled deoxynivalenol �M-H�- m/z = 310 amu

310

315

m/z

7

8

Spot On Issue 1

Creating Liquid Mycotoxin Calibrants: A Behind-the-Scenes Look Many accredited labs use reference materials to show traceability and reliability of their analysis results. What’s less well-known is how reference materials are manufactured.

By Anna LILEK, Quality Manager, Romer Labs®

A magazine of Romer Labs®

9

R

eference materials, or calibrants describe substances or objects with one or more defined characteristic property value(s) that are used as a measure or as a benchmark for measurement methods. Given the importance of consumer safety for the food and feed industry, mycotoxin testing involves the use of reference materials in order to obtain accurate and reliable results.

Ever-present danger

Some toxins have favorable molecular

Mycotoxins are naturally-occurring secondary fungal metabolites toxic to animals and humans. Mold fungi grow on the field and during storage. Found in almost all agricultural commodities worldwide, more than 380 mycotoxins have been identified and the toxicity of each substance varies greatly. The predetermined maximum permitted concentrations of various mycotoxins in vegetable raw materials such as grain, wheat, corn – to name just a few – have forced commodity producers to examine their samples carefully in an analytical laboratory to be sure about the quality of their products.

structures, and can be crystallized from a supersaturated solution of polar or apolar organic solvents.

Gravimetric preparation Frequently, a fungus that grows under optimal conditions – sufficiently warm temperatures, high humidity and a suitable substrate – can produce mycotoxins. In the lab, the story of a reference material begins with the attempt to adjust artificial growth conditions in order to obtain optimal mycotoxin yields. This includes the use of a suitable fungal genus – as every fungus produces its own characteristic pattern of metabolites that can

number into the several hundreds. The maintenance of fungal strains for production is crucial. Their vitality and functionality are constantly being monitored, since this forms the basis of all activities for the reference material production processes. Molds are living organisms that might mutate over time, or even degenerate and result in decreasing mycotoxin yields. New metabolites may form after a certain storage period, which can also influence the isolation process immensely. Strains must be renewed regularly in order to counteract mutations, impurities or other undesirable characteristics.

The first step, fermentation, hinges on making the

Table 1. Key challenges in the production of reference materials Challenge

Solution

Mutations of fungal strains (formation of other metabolites, decreases/increases in yields, formation of impurities)

Regular review of the strains.

Check for productivity and vitality.

Renew strains as needed.

Standardization of fermentation and isolation processes

Undetected impurities by superimposing Finding external certified reference materials for process and quality controls

10

Use state-of-the-art techniques, materials and equipment.

Ensure a robust clean up strategy.

Seek other solutions for the identification of the substance to be isolated – e.g. by analyses of accredited service laboratories that can perform an identification and traceable purity determination (quantitative NMR, LC-MS / MS, elemental analysis, etc.)

Spot On Issue 1

Figure 1. Production path of a liquid mycotoxin calibrant

Gravimetric Preparation

• Tempering of solid mycotoxin to room temp.

• Weighing • Dissolving • Homogenizing

Quality Control

• HPLC • UV Photometer • HPLC-MS

Bottling and CoA

• Filling to separate units • Certificate of Analysis (CoA) • Shipping to customer

lab environment literally as tasty as possible for the fungus to promote its growth. The optimal media also vary from strain to strain, so components such as salts and minerals are provided as a source of nutrients. The mold is allowed to grow for a certain time – anywhere from a few days to a few weeks – during which time the fungus metabolizes its medium.

After completion of the fermentation and a careful control of the process the mycotoxin is extracted from the culture material using a suitable organic solvent.

Depending on the molecular structure, these can be polar or non-polar organic solvents. During fermentation, molds mostly produce impurities in addition to the toxin of interest, e.g. other metabolites, colorings, oils, etc.

The resulting crude extract often contains many impurities. During isolation or purification, the mycotoxin is brought step-by-step closer to the target purity of >98% through various chromatographic and preparative applications with different selectivity.

Some toxins have favorable molecular structures, and can be crystallized from a supersaturated solution of polar or apolar organic solvents. This happens, for example, by cooling the solution, by evaporating the crystallization solvent or by mixing several solvents of different polarity. Other toxins may be rendered in crystalline, powdery form by freeze-drying. By crystallization the purity of the toxin is thereby increased again until the target purity is obtained.

A magazine of Romer Labs®

Quality control HPLC, UV Photometer and HPLC-MS are used to determine/confirm the purity of the raw material produced. Depending on the toxin, this can be done for example by High Performance Liquid Chromatography in combination with Diode Array Detection, Fluorescence detection or similar, with UV Photometer (qualitative and quantitative analysis of the compound) or by High Performance liquid Chromatography combined with mass spectrometry detection. MS is particularly required for the above described determination of the isotopic purity (e.g. > 98% 13C atoms) of 13C isotope-labeled mycotoxins. Mycotoxin reference materials have a niche position on the market. Therefore, it is sometimes difficult to find comparative substances that are commercially available and can be used for process control during production and quality controls.

Table 1 illustrates a number of challenges that can occur during the production and QC stages, along with solutions implemented at Romer Labs.

Delivery After passing the final quality control, the solid mycotoxin is liquefied for use as a liquid mycotoxin reference material. The liquid calibrant solution is then bottled and a certificate of analysis is created, stating the property value and its uncertainty, which accompanies every single calibrant.

11

Your copy of Spot On

Innovative Testing Solutions.

Contact us for more information: www.romerlabs.com | solutions@romerlabs.com