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Uncertainty Measurement

Hello, 

I would like to know if the extended measurement uncertainty of 50% according to SANTE/11945/2015 is valid only for multi-residue-method? How do i evaluate findings of residues above the MRL of single-residue-methods like glyphosat or chlorate? Because in this case the extended measurement uncertainty is not valid, isn´t? Example: the MRL for glyphosat in Honey is 0.05 mg/kg. Findings above this MRL of 0.05 mg/kg are not according to EU-Regulation 396/2005. Is this correct?

Thank you for help,

Katharina Schmidt
Intertek Food Services GmbH Bremm
Sri Lanka
 

 

 

Dear Katharina, 

Let me start with your example.

Even if the 50% uncertainty concept was not applying for all or certain SRM compounds or compound/matrix combinations still another appropriate and justifiable uncertainty figure needs to be applied when judging MRL-violations. The 0% uncertainty assumption is judicially not defendable for regulatory laboratories. For private laboratories that test market-products it depends on the risk policy how uncertainty is to be applied. If the analytical value coincides with the MRL-figure as in your example, taking the nominal value for judgment, as you have suggested, would give you a 50% statistical confidence that the MRL is not exceeded. Given the sampling uncertainty this approach would be too risky. If after deducting the uncertainty from the analytical result you end up with a value equal to the MRL you will have a 2.5% statistical certainty whereas adding the uncertainty would give you a 97.5% statistical certainty. In the case of regulatory laboratories the uncertainty is typically deducted to receive a 97.5% certainty that the violation judgment is safe. 

Now let me continue with your question on the applicability or not of the 50% default uncertainty figure in the case of SRM-compounds.

By nature, uncertainty is not a constant figure and difficult to fix with a reasonable number of experiments. A multitude of factors have an influence on it including pesticide, matrix, method, instrument condition and operator. This applies both SRM and MRM pesticides.

For the sake of uniformity and simplicity the 50% default uncertainty concept has been established within the SANCO quality control procedures using data from proficiency tests over many years. This uncertainty concept can be applied to all compound/matrix combinations for which a laboratory has demonstrated that the uncertainty does not exceed 50%. In general, laboratories are on the safe side if the validation figures pass the SANCO criteria (20% RSD and 70-120% recovery) and PT results are acceptable. 

The EURLs are working on an online-tool for the calculation of the within-laboratory uncertainty using both validation and PT data. 

The main problem is the lack of sufficient data, especially PT-data, to support the calculations down to the matrix/compound combination level. To overcome the lack of sufficient data in most cases laboratories calculate a generic uncertainty combining data on many pesticides assuming that all "MRM-pesticides" behave similarly. For practical reasons many labs even mix data from different matrix-groups (such as high water content, high acid content and dry). Needless to say that mixing data leads to overestimated uncertainty for some and underestimated uncertainty of other pesticide/matrix combinations. Even within the group of MRM-pesticides there is compounds that are highly challenging (e.g. captan and folpet). If evaluated separately such pesticides may not pass the 50% uncertainty threshold, whereas other compounds that do not pose analytical problems may show calculated uncertainties of 30%.

For the so-called "SRM-pesticides" a more judicious grouping of compounds is surely needed compared to MRM-compounds. Fortunately, experience from PTs has shown that for most "SRM-pesticides" the variability of PT-results is similar to that of MRM-compounds. Glyphosate and dithiocarbamates, however, tentatively show higher variabilities, but this very much depends on the methods applied by the laboratories. An important factor reducing uncertainty is the use of isotope labelled internal standards. This has been repeatedly shown in PT-data evaluations.

Overall, the 50% default uncertainty concept can also be applied to SRM-compounds provided that the above criteria are met. The SANCO guidelines are in principle open for applying lower or higher uncertainty values if these are supported by data. For SRM compounds it is explicitly stated that: “For results obtained with single-residue methods, particularly if stable isotopically labelled internal standards have been used, lower expanded MU can be justified, especially if supported by correspondingly lower between-laboratory reproducibility RSDR (< 25%).” 

I hope this answers your question. 

Best regards 

Tuija Pihlström
AQC Coordinator

 

 

Validation Requirement

We are now developing pesticide residue analysis using GC-MS/MS, for method validation I am referring to SANTE/12571/2015, there I cannot understand one identification requirement for GC- MS/MS it says "ion ratio within ±30%(relative)" could you please describe that requirement using a example.
Thank you very much in advance.

Thushara Guruge
Government Analyst's Department
Sri Lanka
 

 

Dear Thushara Guruge,
The relative ion ratio between two ion transitions of a compound in a sample compared to the same compound in standard solutions (preferably injected in the same run) is calculated using the following formula: 

Relative ion ratio (%) = 100 x (ion ratio, sample-ion ratio, standard)/ion ratio, standard)

Example, Acrinathrin:
Transition m/z 181>152 (quantitation)
Transition m/z 289>93 (confirmation)
Average ion ratio in standard (response, confirmation/response, quantitation): 0.24 (24 %)
Ion ratio in sample (response, confirmation/response, quantitation): 0.263 (26.3 %)
Relative ion ratio (%) = 100 x (0.263-0.24)/0.24) = 9.58 %
And the relative ion ratio is 30>=9.58<=-30 %, i.e. within the acceptable range.
  

Tuija Pihlström
AQC Coordinator

 

 

MRLS for fruit juices

I woud like to know where I can get the MRLS for fruit juices.

Vijayan Anayath Pisharath
Dubai Central Laboratory
United Arab Emirates

 

Dear Vijayan,

Thank you for question. To answer your question I would like to refer to EU legislation:

According to the Reg. 396/2005:
Article 20
MRLs applicable to processed and/or composite products
1. Where MRLs are not set out in Annexes II or III for processed and/or composite food or feed, the MRLs applicable shall be those provided in Article 18(1) for the relevant product covered by Annex I, taking into account changes in the levels of pesticide residues caused by processing and/or mixing.
2. Specific concentration or dilution factors for certain processing and/or mixing operations or for certain processed and/or composite products may be included in the list in Annex VI in accordance with the procedure referred to in Article 45(2).

This regulation sets no specific MRLs for juices which means that MRL applied for juice is the corresponding MRL for raw agricultural commodity.

Tuija Pihlström
AQC Coordinator

 

How to get the LOQ

I cannot find a detail procedure for how to get the LOQ. For Limit of detection, sometimes abbre. as LOD also, how to avoid confusion?


PAN C
China Agricutual University
CHINA

 

Dear Pan C,

To answer your question you can find the definition of LOQ (Limit of Quantification) also known as limit of determination (LOD) in the Doc SANCO 10684/2009 in Appendix C, Glossary. The latest revised version is available on the website EU RL Pesticides www.crl-pesticides.eu or directly http://www.eurl-pesticides.eu/library/docs/fv/guidefinalversion.pdf or on the http://ec.europa.eu/food/plant/protection/resources/publications_en.htm#residues.

Best regards


Tuija Pihlström
AQC Coordinator

 

 

Method validation for pesticide residues in food stuff

I need some clarification on method validation for pesticide residues in food stuff. I wanted to know if the method validation of these are to be carried out as per 2002-657-EC or there are some other protocols for the same.


Vishal Arora
AES Laboratories (P) Ltd.
INDIA

 

Dear Vishal,

Thank you for your question.

The validation and analysis of pesticide residues in food and feed is described in the SANCO document 10684/2009 "Method validation and quality control procedures for pesticide residue analysis in food an feed" . The latest revised version is available on the website EU RL Pesticides www.crl-pesticides.eu or directly http://www.eurl-pesticides.eu/library/docs/fv/guidefinalversion.pdf or on the http://ec.europa.eu/food/plant/protection/resources/publications_en.htm#residues.

For any information please don´t hesitate to contact us.

Best regards


Tuija Pihlström
AQC Coordinator

 

 

Traceability of the results

I am interested to know how the results are stored at the laboratories. This is interesting not only considering the environment but also considering the huge amount of paper which must be printed out if each result (and chromatogram) has to be stored as paper.

Do you print out all chromatograms (negative and positive results) at your laboratory or to keep an electronic copy is enough?

Tuija Pihlström
National Food Administration
Sweden

I will answer to your question making some reference to the international standard for accreditation (ISO/IEC 17025: 2005). In this standard, it is said that both quality records and technical records (such as chromatograms) of the laboratory may be in any media, such as hard copy or electronic media (see the NOTE at the point 4.13.1.2 of this standard).

In the case that the laboratory has some records in electronic media, it is very important to cumply with the point 4.13.1.4 of the standard, where it is said that the laboratory shall have procedures to protect and back-up records stored electronically and to prevent unauthorized access to or amendmentof these records.

I hope this answer is suitable for you.

Kindest regards,

Antonio Valverde
University of Almería
Spain

 

For Multiresidue Method

For Multiresidue Method (MRM about 300 compound) Quechers: Cheap fast...
Please witch instrument is recommended ? GC-MS or GC-MS/MS (single or triple quad).
Thank you for you collaboration

Ahmed Zouaoui
NOfficial Laboratory For Chemical Analysis and Research LOARC Casablanca
SMorocco

Dear Ahmed,

Triple quad instruments (LC-MS/MS and GC-MS/MS) are recommended to be used for quantification and also for conincident confirmation.
There are more analytes with quantification by using LC-MS/MS than GC-MS/MS in the multimethods. Therefore LC-MS/MS should be the first choice. I am afraid that the singel quad instruments are not sensitive enough to fulfill the requiremnets of 0.01 mg/kg and even lower.

Best regards

Tuija Pihlström
National Food Administration
Sweden
AQC Guidelines Coordinator

 

Point 57 du guide

Monsieur,

Tout d´abord je tiens à vous remercier pour votre aide pour ma demande.

Je souhaite un renseignement sur le point 57 du guide:

57. If the analytical method does not permit determination of recovery (for example, direct analysis of liquid samples, SPME, or headspace analysis), the precision is determined from repeat analyses of calibration standards. The bias is usually assumed to be zero, although this is not necessarily so. In SPME and headspace analysis, the trueness and precision of calibration may depend on the extent to which the analyte has equilibrated, particularly with respect to the sample matrix. If these methods depend upon equilibrium, this must be demonstrated during method development.

Nous sommes en train de metre au point une méthode de dosage des pesticides sur les huiles essentielles. Notre outils de mesure est le CPG-FPD et CPG-XSD, détecteurs spécifiques pour les composes organo-phosphorés et organo-chlorés. II s´avére que pour la détermination du recouvrement, la majorité des matrices ne permettent pas d´obtenir un résultat dans les 70 á 120 % car les composants de la matrice (en moyenne une centaine) interagissent avec le pesticide si ce dernier est ajouté directement dans I´huile essentielle.
D´autre part il existe un très grand nombre d´huiles essentielles et leur composition respective varie selon la provenance.

D´après le point 57 cité plus haut, il est possible de ne pas effectuer le recouvremente classique mais avec des solutions étalons. Dans quels cas est-ce vraiment possible?

Si je vous pose cette question c´est que nous avons eu un avis du COFRAC France qui nous dit que: "ce point 57 n´est recevable en l´état d´autant plus que nous n´avons pas d´autres moyens d´évaluer la justesse de notre méthode."
Comme nous souhaitons à terme que notre méthode soit accréditée, nous souhaitons écarter tout malentendu.

Question1:
Pourriez-vous nous apporter des precisions sur ce point 57 et savoir s´il est légitime ou va être retire?

Question2:
Si applicable, pour quells exemples précis peut-il être utilisé?

Question3:
Si applicable, comment justifier le recours à cet article 57?

Question4:
Nous ne comprenons pas la phrase: If these methods depend upon equilibrium, this must be demostrated Turing method development.

Daniel Dantin / Didier Bozonnet
Pyénéssences Analyses
La Gineste
France

 

Dear Mr Dantin,

1.- Paragraph 57 discusses extraction of volatile organic compounds from liquid samples using SPME or headspace analysis. The technique is based on the equilibrium of the volatile analyte between compounds in gas phase-liquid phase or in liquid phase-fiber. Therefore and equilibrium time is necessary to obtain a good repeatability. Sampling time and temperature are important parameters affecting the analytical response. Therefore, the use of internal standards technique is used to generate response factors and to control the analyte response.

2.- Generally, matrix effects in gas chromatography might become a problem during the transfer of the analyte from injector to the column. Especially, the contribution of essential oils and waxes result in enhanced recoveries.

3.- Since the measured analytical response reflects the contribution of matrix as well, the matrix effect for each compound should be eliminated. Presumably, the matrix protects the pesticide from thermal degradation and prevents analyte adsorption by covering active sites in the gas chromatographic system, resulting in a higher response compared with the same amount in matrix free standard solution. Such effects are often the most important source of error in GC analysis. Since the matrix effect are shown to be dependent on the type of pesticide and/or type of matrix , the accurate quantification of the samples should be done using standards prepared in the corresponding residue free matrix.

Answer to question 1: See paragraph 1.

Answer to question 2:

With SPME:

(i) Determination of pesticide residues in fruit juice.
(ii) Determination of volatile pesticides in waters.

With Headspace: (i) Determination of very volatile pesticides (such as Dichlorvos) compounds in oil.

Answer to question 3: See paragraph 1, 2 and 3

Answer to question 4:. It has been demonstrated that in certain experimental condition the equilibrium is achieved obtaining reproducible results

 

Tuija Pihlström
National Food Administration
Sweden
AQC Guidelines Coordinator

 

Method Validation

We are a private laboratory providing analytical services including pesticide residues to whoever needs them. We would like to base our LC-MS/MS pesticide residues analyses upon the SANCO guidelines. Our question to the QC Panel concerns method validation. Since the SANCO guidelines do not define exactly how to actually prepare the reference standards in matrix and our LC-MS/MS is highly sensitive to matrix effects we have developed a method in which standards are added to a blank organic matrix at the initial step. Following that, the spiked blank (standard) is treated as a routine sample, exactly as outlined in chapter 47 of SANCO/2007/3131. Can we use these standards (different amounts added to a blank in order to get a calibration curve) to calculate the recovery results during method validation?. We would like to indicate that an addition of a known quantity of standard to an aliquot of an organic sample extract (in the last step) prior to the final determination in the LC-MS/MS and calibrating against it failed to give us the required recovery limits defined by SANCO for some of the pesticides concerned.


Uri Golner
Head, Pesticide and Mycotoxin Division
Aminolab
Weizmann Science Park
Ness-Ziona
ISRAEL

 

Dear Uri,

The addition of Analyte Protectants is a concept developed for GC-applications. In my lab we use them in routine and are quite satisfied. Previously we were using the "generic matrix" concept and were also doing quite well. Of course when using a "generic matrix" one should always be aware of incorrect quantifications that will appear when matrix matching is not optimal.

For LC-MS/MS we use standards in neat solvent.
One difference between GC and LC-MS(/MS) as regards matrix effects is that in GC the use of a "generic matrix" will more or less positively influence the quantification of all susceptible compounds. So using a "generic matrix" you are usually better off in GC.
In LC-MS(/MS) however, different matrices may influence different compounds in a different manner as the effects only appear when there is a co-elution of matrix components with pesticides. So, for example banana may contain components that suppress compound A, cucumber compounds B and D and lemon compounds A, B, C, and E.

So, if you want to avoid matrix-matching with the same matrix out of practicability reasons, you have the difficulty of choice. A "mixed matrix" as you suggested might be useful but has to be tested.
I think it is probably better to use pure solvent and at least know what you have. Then you could perform some experiments comparing standards in neat solvent against standards made using blank extracts and develop your own database of matrix-effect observations. This way you surely don't compensate for the effects but at least you know which matrix/pesticide-combinations are unacceptably affected and can react accordingly.
There are of course other solutions for compensating or reducing matrix effects such as extract dilution, standard additions method, isotopically labelled standards, but not always applicable in routine analysis.
I hope this helps.

Best wishes


Dr. Michelangelo Anastassiades
CVUA Stuttgart
EU-CRL for Pesticide Residues using Single Residue Methods Schaflandstrasse 3/2
D-70736 Fellbach

 

Sample Preparation

Dear Sir or Madam
My question is about sample preparation and extration of blood or viscera (stomach content either). Do you use liquid liquid extration, SPE, SPME, or other precedure?
I work at a Forensic Medical Laboratory in BELO HORIZONTE, MG. Thank you in advance.

Marcus Penido
Laboratorio de Toxicologia Inst Medicina Legal Minas Gerais
Brasil

 

Dear Marcus,

Thank you for your question. I regert to inform to you that the AQC panel is primary directed at the discussion/ interpretation of the AQC document: "Method validation and Quality Control Procedurres for Pesticide Residue Analysis in Food and Feed (SANCO/2007/3131)". The quidane is intended for the monitoring of pesticide rersidues in food and feed, therefore the methods used are not applied in blood or viscera. I suggest that you turn your question to the National Laboratories of Forensic Science.

Best wishes

Tuija Pihlström
National Food Administration
Sweden

AQC Guidelines Coordinator

 

Dithiocarbamates

Analytical method for detection of Dithiocarbamates group using GC-MSD (SIM mode) as CS2.
Details, Method Development, Calculations, and related regulations

Moustapha N.M.
Central Lab of Residue Analysis of Pesticides and Heavy Metals in Food
Egypt

 

Dear Mustafa,

I forward your request to the Community Reference Laboratory for Single Residue Methods.
Best regards

Tuija Pihlström
National Food Administration
Sweden

AQC Guidelines Coordinator

 

 

Confirmation of positive results

I would like to know what degree of confirmation is necessary for positive results obtained by selective detectors such as GC-NPD or HPLC-UV etc. In the guidelines the paragraph 69 states: "Confirmation is not mandatory for all positive results, and must be decided by the laboratory on a case-by-case basis". and paragraph 71: "Such limitations in the degree of confirmation should be acknowledged when reporting the results". What factors should be taken into account when deciding on confirmation of positive results (the measured concentration with respect to MRL, expected or frequent residue, etc.)? In what way this should be acknowledge in the report? '

Darinka Stajnbaher
IPH Maribor
Slovenia

 

Dear Darinka,

The acknowledgement in the report is needed since it explains the limited confirmation of the techniqiues used (NPD, ECD, UV etc) and the awareness of it. This is due to the lack of specifity or selectivity towards endogenous peaks etc. As it is stated in para 71, the results have of course a certain confirmation due to the experince and frequency of such findings.

Best regards,

Tuija Pihlström
National Food Administration
Sweden

AQC Guidelines Coordinator

 

Method performance acceptability criteria

G6 states: "Recovery rates outside the range of 70-120% can be accepted if they are consistent (RSD ≤ 20%) and the basis for this is well established (e.g. due to analyte distribution in a partitioning step) ,but the mean recovery should not be lower than 30% or above 140%. However, in these cases a correction for recovery is required or a more accurate method should be used, if practicable." 
What is meant by "in these cases"? 
Is the correction required if the recovery is in the range of 30-70% and 120-140%%? 
Or is it required only if the recovery is below 30% and above 140%?
Thank you very much
 

Dr. Nadja Buchner
Federal Office of Consumer Protection and Food Safety (BVL)

Germany

 

Dear Nadja,

Thank you for your e-mail and thank you also for the information about the AQC Panel.

To answer your question - the correction is needed in the range of 30-70% and 120-140%%. Background to this addition was the discussion about acceptable lowest (and highest) limits for the recoveries. We agreed to accept recoveries at 30% at lowest and 140% at highest. Values below/above are not fit for purpose. So as before we have to correct values up to 70% now we even have defined the lowest acceptable. I hope my answer helps you.

Kind regards

Tuija Pihlström
National Food Administration
Sweden
 
AQC Guidelines Coordinator

 

 

 

Published 19-02-2008, 13:57:38

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