Final Report of APMP.QM-S6 - BIPM - BIPMFinal Report of APMP.QM-S6 APMP Supplementary Comparison...

of 46

Final Report of APMP.QM-S6

APMP Supplementary Comparison

Clenbuterol in Porcine Meat

August 2015

Coordinated by: Della Wai-mei Sin, Clare Ho and Yiu-chung Yip

Government Laboratory, Hong Kong, China (GLHK)

With contributions from

Laly Samuel

Measurement Standards Laboratory of New Zealand (MSL)

Eliane Cristina Pires do Rego

National Institute of Metrology, Quality and Technology, Brazil (INMETRO)

Preeyaporn Pookrod

National Institute of Metrology, Thailand (NIMT)

Xiuqin Li

National Institute of Metrology, P.R. China (NIM)

Pui-kwan Chan

Government Laboratory, Hong Kong (GLHK)

Joachim Polzer and Wolfgang Radeck

German Federal Office of Consumer Protection and Food Safety (BVL)

Elias Kakoulides and Vasiliki Schoina

Chemical Metrology Laboratory, Greece (ΕΧΗΜ/ GCSL-ΕΙΜ)

Claudia Marcela Salazar Arzate

National Metrology Centre of Mexico (CENAM)

APMP.QM-S6: Clenbuterol in Porcine Meat Draft B Report

of 46

Contents

Introduction ................................................................................................................ 3

Measurand .................................................................................................................. 3

Study Material ............................................................................................................ 4

Sample distribution...................................................................................................... 8

Instruction to participants ............................................................................................ 8

Reporting instructions.................................................................................................. 9

Programme Schedule ................................................................................................... 9

Reference materials used by the participating laboratories ........................................... 10

Method applied by the participating laboratories ......................................................... 11

Results reported by the participating laboratories ........................................................ 16

Calculation of the SCRV and associated uncertainty ................................................... 21

Degree of equivalence calculation .............................................................................. 22

Scope of the Supplementary Comparison and “How Far does the Light Shine”? .......... 24

Acknowledgement ..................................................................................................... 26

Annex I: Details of moisture content determination by NIM…………..……………….. 27

Annex II: Measurement uncertainties reported by participants…………………………..29

Annex III: Core competency tables reported by participants……………………………..41


of 46

Introduction

Clenbuterol belongs to the family of β-agonists. It has been medicinally used as a

bronchospasmolytic agent in humans. When the drug is administered to animals at a

higher dosage, it can produce growth-promoting effects and cause a shift from fat to lean,

thus making a higher gain in the meat industry. Excessive administration of clenbuterol

would lead to accumulation of the drug in animal bodies. The presence of clenbuterol in

meat for human consumption poses a health risk for the consumer and is therefore

forbidden in many countries. Systematic monitoring and control of the drug residue are

necessary to safeguard the public health.

At the CCQM Organic Analysis Working Group (OAWG) Meeting held in April 2012

and the APMP TCQM Meeting held in November 2012, an APMP supplementary

comparison (APMP.QM-S6) on the determination of clenbuterol in porcine meat was

supported by the OAWG and APMP TCQM. This study provided the means for

assessing the measurement capabilities for determination of low-polarity measurands in a

procedure that requires extraction, clean-up, analytical separation, and selective detection

in a food matrix. This comparison was organized by the Government Laboratory, Hong

Kong. In order to accommodate a wider participation, a pilot study (APMP.QM-P22)

was run in parallel to APMP.QM-S6.

Measurand

The measurand of this study was the mass fraction of clenbuterol in porcine meat on dry

mass basis. The analyte is clenbuterol (4-amino-alpha-[(tert-butylamino) methyl]- 3,5-

dichlorobenzyl alcohol).

Clenbuterol

Formula C12H18Cl2N2O

CAS Number 37148-27-9

Molar mass g/mole 277.19

-log P (octanol-water) - 2.00


of 46

Structure

Study Material

Preparation

The test material was prepared by the Shanghai Institute for Food and Drug Control

(SIFDC) of P. R. China. The preparation was under the supervision of personnel trained

by GLHK. In brief, the porcine meat containing the incurred analyte of clenbuterol was

freeze-dried, powdered, sieved and homogenized. The level of clenbuterol in the test

material was adjusted by mixing with blank porcine meat. The homogenized powder was

separately dispensed into clean glass bottles with content of about 2 g each. The samples

were then sealed and disinfected by γ-irradiation at a dose of 12 kGy for 18 hours.

Homogeneity Study

Upon receipt of the samples from SIFDC, all samples were kept at the storage condition

of 4 °C by GLHK. For the homogeneity study, 12 bottles of samples were taken

randomly from 500 bottles of sample and analyzed in duplicate at room temperature

(about 20 °C) for determining the sample inhomogeneity. Data were evaluated according

to ISO Guide 35 and the relative standard uncertainty due to between bottle

heterogeneity, ubb, was found to be 1.2% which indicated that the material was

sufficiently homogeneous. The sample size of 0.5 g was used in the homogeneity study.


of 46

Figure 1: Graphical presentation of homogeneity study of clenbuterol in the testing material

Table 1: Summary of ANOVA for homogeneity study of clenbuterol in the testing material

ANOVA

Source of Variance SS DF MS F P-value F Crit

Between Bottles 0.11805 11 0.01073 2.52616 0.06316 2.71733

Within Bottle 0.05098 12 0.00425

Total 0.16902 23

Stability Study

The trend-analysis technique proposed by ISO Guide 35 was applied to assess the

stability of the testing material. Eight bottles of samples were taken randomly from the lot

of 500 bottles and analysed in duplicate at an elevated temperature for monitoring the

sample instability by means of isochronous approach. Samples were conditioned at the

elevated temperature 40 ± 5 °C for 1, 2 and 4 weeks before analysis. Data were evaluated

according to ISO Guide 35. The effect of time on the stability was evaluated using a

linear approximation model by fitting linear regression lines to the data set. The p-value

0.75

0.80

0.85

0.90

0.95

1.00

1.05

263 1593 1309 1591 479 1494 872 1180 1090 2106 389 688

Nor

mal

ized

mas

s fr

acti

on o

f C

len

bute

rol

Bottle No.

Homogeneity test of clenbuterol in porcine meat


of 46

for the significance test of the regression coefficient was found to be 0.3474, which

indicated that the slope was not significantly deviated from zero at 95% confidence level.

In other words, no instability was observed for the testing material at 40 °C during the

testing period and hence the testing material could be dispatched at room temperature

and is fit for the purpose of the comparison in terms of stability.

Figure 2: Graphical presentation of short term stability study of clenbuterol in the testing material

Table 2: Summary of ANOVA test for stability study of clenbuterol in the testing material at 40

°C for a month

SUMMARY OUTPUT

Regression Statistics

Multiple R 0.65263

R Square 0.42593

Adjusted R Square 0.13889

Standard Error 0.04425

Observations 4

ANOVA

df SS MS F Significance F

Regression 1 0.00291 0.00291 1.48387 0.34737

Residual 2 0.00392 0.00196

Total 3 0.00682

0.75

0.80

0.85

0.90

0.95

1.00

1.05

0 1 2 3 4 5

Nor

mal

ized

Mas

s F

ract

ion

of

Cle

nbu

tero

l

No. of weeks

Short term stability study of clenbuterol in porcine meat


of 46

Coefficients Standard Error t Stat P-value Lower 95% Upper 95%

Intercept 5.097 0.034 148.690 0.000 4.949 5.244

X Variable 1 0.018 0.015 1.218 0.347 -0.0461 0.083

The stability of the study material in storage condition was also studied. Six bottles of

sample were randomly selected and analysed in duplicate in classical approach. Samples

were tested (i) after the study material was prepared, (ii) before sample dispatch to

participants in March 2013 and (iii) after the result submission deadline in October 2013.

The result was also evaluated by trend analysis techniques as the short term stability

study. The p-value for the significance test of the regression coefficient was found to be

0.217, which indicated that the slope was not significantly deviated from zero at 95%

confidence level. In other words, no instability was observed for the testing material at 4

°C during the testing period and hence the testing material is fit for the purpose of the

comparison in terms of stability. The result of ANOVA test was summarized in Table 3.

Table 3: Summary of ANOVA test for stability study of clenbuterol in the testing material in

storage condition, at 4 °C, for 18 months.

SUMMARY OUTPUT

Regression Statistics Multiple R : 0.9427 R Square : 0.8887

Adjusted R Square : 0.7773

Standard Error : 0.0318

Observations : 3

ANOVA

df SS MS F Significance F

Regression 1 0.008 0.008 7.982 0.217

Residual 1 0.001 0.001 Total 2 0.009

Coefficients Standard Error t Stat P-value Lower 95% Upper 95%

Intercept 5.187 0.030 171.933 0.004 4.803 5.570

X Variable 1 0.007 0.002 2.825 0.217 -0.024 0.038


of 46

Sample distribution

8 NMIs/DIs have participated in the APMP.QM-S6 programme. Participating

laboratory received a sample pack which contained (i) six bottles of sample, each bottle

containing about 2 g of lyophilized porcine meat; (ii) a sample receipt form; and (iii) a

material safety data sheet that were sent to each of participant via courier in early March

and April 2013. A temperature strip was attached to the sample, which recorded the

highest temperature experienced during the transport. The technical protocol, result

report form and the core competency table were sent to the participants by e-mail. All

samples were well received in April 2013 or before except the sample sent to INMETRO.

The sample were sent thrice, due to the problem of the import license for the sample, and

received by the participant in June 2013.

Instruction to participants

Participants were recommended to use their own reference standards for preparation of

standard solutions for calibration purposes. Participants were requested to determine the

mass fraction (in ng/g) of clenbuterol in the test sample on a dry mass basis using the

analytical method of their choice. The analysis should be conducted with a

recommended sample size of at least 0.5 g. Participants should report the mean value of

replicate measurements of the test material. The analyte and the expected range of mass

fraction are listed as follows:

Measurand Matrix Expected mass fraction, (ng/g)

Clenbuterol Porcine meat 1-50

Participants should carry out the dry mass correction. The determination of dry mass

correction should be conducted with a recommended size of at least 0.5 g. The test

sample portion taken for dry mass correction should be placed over anhydrous calcium

sulphate (DRIERITE®) in a desiccator at room temperature for a minimum of 20 days

until a constant mass is reached. Dry mass correction should be carried out at the same

time as the test sample portion is to be analyzed in the same bottle of sample.

The test sample should be stored at 4 °C or below. The samples should be warmed up to

room temperature (at 20 ± 5 °C and relative humidity <85%) before opening for analysis

and should be shaken thoroughly before use. Participants were reminded that frequent

freeze-thaw cycles of the samples should be avoided, the samples should be processed as


of 46

soon as possible and carefully re-sealed after use and stored in refrigerators at about 4 °C

or below.

Reporting instructions

Participants should report the results by completing the Result Report Form. The

following information should be provided:

(a) Report the mass fraction of clenbuterol in ng/g on dry mass basis;

(b) Report the mean value of all replicate measurements and its associated uncertainty;

and

(c) Description of analytical methods used.

The completed form was requested to be sent to the coordinator (E-mail:

APMP.QM_S6@govtlab. gov.hk) on or before the final deadline for result submission on

30 September 2013. In addition, participants were also requested to analyse their

competency underpinning the measurement and return the completed competency

template to the organizers.

Programme Schedule

The progress for the various phases of the programmes was as follows:

Date Status

January 2013 Call for Participation

28 February 2013 Deadline for registration

March 2013 Distribution of APMP.QM-S6 samples

April 2013 Distribution of APMP.QM-P22 samples

6 September 2013 Deadline for submission of results

30 September 2013 Extended deadline for submission of results

The organizer announced on 5 September 2013 to extend the deadline for submission of

results for APMP.QM-S6 and APMP.QM-P22 from 6 to 30 September 2013 upon the

requests of some participants from the APMP.QM-S6.


of 46

Reference materials used by the participating laboratories

The summary of the reference standards and internal standards used in the programmes

by the participating laboratories was showed in Table 4. All participants used clenbuterol-

d9 as internal standard from different commercial sources. NIM used its own certified

reference material. For GLHK, BVL, EXHM, CENAM and NIMT, purity analysis on

various commercial clenbuterol standards were performed and used as calibrants. MSL

used a commercial clenbuterol standard but the metrological traceability was not

substantiated.

Table 4: Summary of reference standards, and purity assessments and internals standards

materials used by the participating laboratories

NMIs/ DIs Calibration standard Internal standard

Source Purity Purity assessment

Conducted?

Source and purity

MSL Cerilliant Analytical Reference Standards

99.8% No Cerilliant Analytical Reference Standards;

99.7%

INMETRO Result was not submitted.

NIMT Dr. Ehrenstorfer GmbH

99.32 ±0.13% Yes Witega;

>99.7% purity,

>98 atom% D enrichment

NIM NIM 99.3% ±0.5% Yes Witega;

>99.7% purity

GLHK USP Neat Standard (Batch No.: F0I214)

886mg/g ± 6 mg/g as clenbuterol free base

Yes Witega;

>99.7% purity

BVL Sigma-Aldrich GmbH

93.4 % Yes Witega Berlin-Adlershof GmbH;

97.24%

EXHM Sigma-Aldrich 995.6 ± 1.3 mg/g, traceable to SRM (NIST 350b)

Yes Sigma-Aldrich;

99.8%

CENAM Sigma-Aldrich 99.71 g/100 g Yes Sigma-Aldrich;

99.8 g/100 g


of 46

Details of in house purity assessment

Direct estimate of principal

component Estimate of impurities

NMIs/ DIs

Organic Water/ moisture

Residual solvent

Inorganic

MSL Information was not provided

INMETRO Result was not submitted

NIMT

HPLC-UV Karl

Fischer Titration

TGA TGA

NIM

HPLC-UV Karl

Fischer Titration

GC-FID ICP-MS

GLHK

HPLC and LC-

MS/MS

Karl Fischer

coulometry

Headspace GC-MSD

ICP MS and IC

BVL LC-UV/ Vis and

HPLC-QToF

LC-UV/ Vis and HPLC-QToF#

Karl Fischer

Titration

EXHM

qNMR using maleic acid

certified by BAM as the internal

standard

CENAM DSC GC-FID# Karl

Fischer Titration*

HPLC/ conductiv-ity detector and ICP-MS#

* Additional information provided by participants after the issue of result summary report in April 2013.# Further clarified information in OAWG CCQM meeting in April 2014 and subsequent correspondences.

Method applied by the participating laboratories

Participants are encouraged to determine the mass fraction (in ng/g) of clenbuterol in the


of 46

test sample on a dry mass basis using the analytical method of their choice. The details of

method for sample extraction, clean-up, instrumental analysis and quantitation were

summarized in Table 5.

All participating laboratories applied Isotope Dilution Liquid Chromatography-Tandem

Mass Spectrometry (ID-LCMS/MS) technique with clenbuterol-d9 as internal standard

spiked before sample extraction for quantitation in this programme. However, various

extraction techniques and clean-up methods were used. 4 out of 7 submitted results

namely MSL, NIMT, EXHM and BVL used enzymatic digestion method at elevated

temperature with sonication, shaking to extract the analyte from the matrix. EXHM used

acid hydrolysis in addition to enzymatic digestion. Solvent extraction with sonication

and shaking at room temperature was performed by NIM, CENAM and GLHK. All

laboratories used SPE for sample clean-up. Different SPEs such as mixed mode SPE,

strong cation exchange SPE and molecularly imprinted polymer (MIP) SPE were applied.

For measurement, all laboratories, except MSL which did not provide information on the

instrument used, applied triple quadruple mass spectrometry operated under the multiple

reaction monitoring mode (MRM) using the electrospray ionization technique. The

MRM transition 277/203 for clenbuterol and 286/204 or 286/132 for isotopic internal

standard were monitored and used for quantitation. MSL, NIM, GLHK and EXHM

used matrix-matched calibrant and single-point calibration at exact matching and NIMT

applied single point, bracketing calibration as well as matrix-matched calibration blend.

CENAM applied bracketing calibration and also internal calibration for quantitation.

BVL is the only laboratory applied internal calibration with multi-level calibration curve

for quantitation.

All participants applied the suggested protocol for moisture determination and results

were reported in dry mass basis. However, CENAM reported that the weight of the

sample in the 20th day was increased relative to the mass of the first day of moisture

content determination, thus no dry mass correction was made.


of 46

Table 5: Summary of methods applied by the participating laboratories

NMI/ DI Sample

size

Extraction method Extraction condition Clean up method

Analytical instrument

(s) used

Chromatograp-hic column

Type of calibration

Method of quantification

Ions/ MRM transitions

Solvent Technique Temp,

°C Duration,

hours

MSL 1 g

Sodium acetate buffer, pH 5.2

Sonication, shaking, enzyme

digestion and vortex

37°C 28 hrs

SCX solid phase

extraction and sample extracted in isopropanol

-ethyl acetate

(60:40) (v:v)

Agilent 6410 Triple

quad#

XBD-C18 (150 × 4.6 mm,

5 µm) IDMS

Single-point calibration

Clenbuterol 277/132 277/203* 277/ 259

IS 286/132* 286/169

INMETRO Result is not submitted

NIMT 0.5 g Tris

buffer (pH 8)

Enzyme digestion, vortex ,

shaking and centrifuga-

tion

55°C 16 hrs

Liquid-liquid

extraction with ethyl acetate:2-propanol, 99:1 (v:v) , centrifuga-tion and

SPE using VertiPakTM C8/SCX

SPE cartridges

Shimadzu LC system equipped with API 4000 Q TRAP

MS/MS (ESI

Positive mode)

Phenomenex Gemini, C18, (110 Å, 150 ×

4.6 mm, 5 µm) with

Phenomenex C18 Security

Guard column (4.0×2.0 mm)

IDMS

Single-point calibration, bracketing calibration

matrix-matched

calibration blend

Clenbuterol 277.1/203.0* 277.1/168.1

IS 286.2/204.1* 286.2/169.1


of 46

NMI/ DI Sample

size



(s) used


Type of calibration




°C Duration,

hours

NIM 0.5 g

Water : 0.1%

formic acid in acetoni-trile (1:1)

Sonication, shaking and

vortex

Room tempera

-ture

Sonication (1 hr) and shaking (1

hr)

Cleanert PCX SPE column

Shimadzu 30A UFLC system with AB SCIEX Qtrap 5500 LC-MSMS

System (ESI mode)

Acquity CSH C18 (3.0×100 mm, 1.7 µm)

IDMS Single-point calibration

Clenbuterol 277/203* 277/168 277/132

IS 286/204

GLHK About 0.5 g

0.05 M Perchlo-ric acid

Sonication Ambient

temperatu-re

60 mins

Waters Oasis MCX

LP Extraction Cartridges

and Supelco

SupelMIP Clenbuterol

SPE Column

AB Sciex 4000

QTRAP LC-MSMS

System (ESI

Positive mode)

Restek Allure Biphenyl,

(2.1 x 150 mm, 5 µm)


Clenbuterol 277/203* 277/132 277/168 279/205 279/132 279/170

IS 286/204* 286/133 286/169

BVL 0.5 g Tris

buffer Enzyme digestion

55°C Overnight

Clean-Screen Dau Cartridges

(mixed mode

C8/benzene sulfonic

acid)

UPLC Waters Aquity (XEVO

TQ) (ESI mode)

Zorbax Eclipse XDB C18 (4.6 x 100

mm,1.8 µm)

Internal calibra-

tion

Multi-level calibration

curve

Clenbuterol 277.07/203.1 277.07/132.09 277.07/168.05

IS 286.13/204.09


of 46

NMI/ DI Sample

size



(s) used


Type of calibration




°C Duration,

hours

ΕΧΗΜ 0.5 g

Tris buffer

(pH 9.5 in 0.2 M

CaCl2), pH 5.2 acetate buffer

and 0.1 M HClO4

Enzyme digestion,

acid hydrolysis sonication,

shaking, and vortex

55 °C, 37 °C 40 °C

16 hrs, 2 hrs, 0.5 hrs

Defatted with n-

heptane and centrifuged twice prior

to SPE using

molecularly imprinted polymers

Thermo Finnigan,

TSQ Quantum Ultra AM

(ESI Positive mode)

Thermo Hypersil Gold

C18, (2.1 mm x 10

cm, 5 µm)

IDMS

Matrix-matched

calibration and single-point

calibration at exact matching

Clenbuterol 277→203*

277 → 140

IS 286 → 204*

286 → 140

CENAM 0.4 g

0.1 M HCl/ 5% NH4OH in 60:40 ACN/ MeOH

Sonication, shaking and

vortex 20 °C 24 hrs

Centrifuga-tion at

10000rpm, 4 °C,10min

and SPE using

Microplate OASIS MCX

HPLC, Waters, XEVO TQS (ESI

positive mode)

Acquity UPLC HSS T3,

(2.1x100mm, 1.8µm)

Internal calibra-

tion, IDMS

Bracketing calibration

Clenbuterol 277/203* 277/ 259

IS 286/204* 286/268

* MRM transition used for quantitation

# Additional information provided by participants after the issue of result summary


of 46

Results reported by the participating laboratories

7 participating laboratories submitted their results by the extended deadline. The results

were summarized in Table 6 and plotted in Figure 3. INMETRO did not submit their

result due to instrumental problems. BVL reported that there was an error on the unit of

the expanded uncertainty where percentage values was reported instead of the ng/g and a

revised report form was sent to the organizer on 2 October 2013.

Table 6: Summary of results reported by the participating laboratories

NMI/ DI Moisture Content (w/w%)

Mass fraction (ng/g) on dry

mass basis

Combined uncertainty

(ng/g)

Coverage factor (k)

Expanded uncertainty

(ng/g)

MSL 0.635 2.457 0.022 2 0.044


NIMT 1.07 5.0 0.12 2.03 0.25

NIM 9.9 5.18 0.25 2.0 0.50

GLHK 1.6 5.3 0.18 2 0.4

BVL 1.49 5.30 0.34 2 0.68

EXHM 0.80 5.3 0.13 2.31 0.29

CENAM Nil* 9.97 0.45 2 0.89

*CENAM reported that the weight of the sample in the 20th day was increased relative to the mass of the first day of moisture content determination, thus no dry mass correction was made.

The measurement results of NMIs/ DIs with italic fonts were excluded on technical grounds in the SCRV calculation.


of 46

Figure 3 : Summary of the participants’ result and associated reported standard uncertainties

After the distribution of the Initial Summary Report in late October 2013 for discussion

in the OAWG meeting, participants were requested to check their own results and inform

the coordinating laboratory if they identified any measurement problems relating to their

reported results.

MSL informed the coordinator on 30 October 2013 and 19 November 2013 that they

identified a calculation error in their first reported results based on the reasons stated

below:

“After receiving the draft report we have re-evaluated our results and have discovered an error. The

error was made when the technician loaded the sample vials in the wrong order. Although our

initial results were believable we realize this has created a large systematic error which we have now

corrected.”

The mass fraction of clenbuterol on dry mass basis was 4.43 ng/g and expanded

uncertainty was 0.82 ng/g.

CENAM informed the coordinator on 5 November 2013 at the CCQM OAWG meeting

that calculation error was found in the submitted result. CENAM performed a follow up

study and result was submitted on 25 March 2014. The reported moisture content was

1.5775% and the mass fraction of clenbuterol on dry mass basis was 5.42 ng/g and

expanded uncertainty was 0.56 ng/g.

2

4

6

8

10

12M

ass

frac

tion

of c

lenb

uter

ol, n

g/g


of 46

Corrected results submitted by MSL and CENAM would not be included for SCRV

calculation and included in the report for information only.

The issue of high moisture content, 9.9%, reported by NIM was discussed in the APMP

TCQM meeting in October 2013. NIM reported that the recommended drying protocol

was followed; however the weight of sample showed a trend of increasing with the

duration of the sample in desiccator filled with anhydrous calcium sulphate. Therefore,

the moisture content of the sample was measured by the vacuum drying method instead

(reference standard GBW 5009.3-2010) and further verified by the Karl Fischer method.

Further experimental details were provided from NIM as detailed in Annex I.

NIM clarified that as the weights of the sample in all of the four bottles were increased by

about 9% after placed over anhydrous calcium sulphate in desiccator for 22 days,

vacuum drying method was used instead. The pre-treated sample, by placing in a

desiccator for 22 days, was used for subsequent analysis including determination of

clenbuterol and moisture content by vacuum drying method. The reported mass fraction

of clenbuterol in porcine meat on a dry mass basis (5.18 ng/g) was dry mass corrected by

using the reported moisture content of 9.9% measured by vacuum dry method.

In the TCQM meeting in September 2014, the issue was thoroughly discussed and it was

generally agreed that as the method of moisture content determination was different, the

results could not be compared. NIM agreed that their result would not be included in

SCRV calculation.


of 46

The relative standard uncertainties of the results and the major contributions in the

uncertainty budgets were summarized in Annex II and Table 7.

Table 7: Approaches to uncertainty estimation from participants

NMI/ DI Relative standard uncertainty (%)

Contribution to the measurement uncertainty budget

MSL 0.9% Concentration of the clenbuterol standard Mass of isotopically enriched clenbuterol

spike (Balance weighing) Mass of sample (Balance weighing) Mass of clenbuterol standard added to the

calibration blend (Balance weighing) Mass of isotopically enriched clenbuterol

standard (Balance weighing) Ratio of the MS signals for clenbuterol/

clenbuterol-d9 in the sample blend (Instrument response)

Ratio of the MS signals for clenbuterol/ clenbuterol-d9 in the calibration blend (Instrument response)


NIMT 2.5% Mass fraction of analyte in the calibration solution used to prepare the calibration blend. The value was estimated from the purity of clenbuterol standard, masses weighed for preparation of stock solutions and uncertainty using different standards (standard comparison).

Bias and precision effect of the balance. Precision factor. This value was estimated

from standard deviation of the multiple IDMS results.

Interference effect. This value was estimated from potential bias between primary ion pair and secondary ion pair of the MRM program.

Extraction efficiency factor which was estimated from the liquid-liquid extraction and solid-phase – extraction.

Dry mass correction factor which was estimated from the moisture content analysis


of 46



NIM 4.8% Mass fraction of clenbuterol in the sample Matrix effect interference factor Method precision factor Extraction efficiency factor Mass of clenbuterol-d9 added to the sample

blend Mass of clenbuterol added to the calibration

blend Peak area ratio of clenbuterol /isotope in

sample blend Mass of sample Dry mass correction factor obtained from

moisture content analysis Mass of clenbuterol-d9 added to the

calibration blend Peak area ratio of clenbuterol /isotope in

calibration blend

GLHK 3.8% Factor of repeatability Mass fraction of analytes in calibration blend Mass of clenbuterol-d9 added to the sample

blend Mass of sample Mass of clenbuterol standard solution added

to the calibration blend Mass of clenbuterol-d9 added to the

calibration blend Isotope amount ratio in sample blend Isotope amount ratio in calibration blend Dry mass correction factor

BVL 6.4% Within-laboratory reproducibility Calibration solution Sample weight Sample spike Dry mass

ΕΧΗΜ 2.4% Method precision Mass fraction of clenbuterol in calibration

solution Sample moisture content Mass of clenbuterol-d9 solution added to the

sample blend Mass of test material in sample blend


of 46



Mass of clenbuterol-d9 solution added to the calibration blend

Mass of calibration solution added to the calibration blend

Measured peak area ratio of the selected ion in the sample blend

Measured peak area ratio of the selected ion in the calibration blend

CENAM 4.5% Mass fraction of the clenbuterol Mass of internal standard Mass of sample Calibrant purity Repeatability of the sample Reproducibility

Calculation of the SCRV and associated uncertainty

Initial Summary Report and Draft A Report were sent to participants in October 2013

and early April 2014 for the discussion at the OAWG meeting in Pretoria and in Paris.

During the meeting in Pretoria, different approaches of SCRV calculation were discussed.

In view of the errors identified in the results from MSL and CENAM, in addition, the

metrological traceability of the result from MSL was not substantiated; therefore, the

measurement results reported by MSL and CENAM were excluded in the calculation of

SCRV. Due to different drying method was applied, it was agreed in TCQM APMP

meeting hold in September 2014 that the measurement result from NIM was also

excluded from the calculation of SCRV. Finally, only 4 valid results would be used for

SCRV calculation and the provisional SCRVs were summarized in Table 8. Considering

there is no significant difference by using the arithmetic mean and median approach to

calculate the SCRV and the four valid data (n=4) were mutually agreed and with similar

uncertainties, it was generally agree in the OAWG meeting that the simplest arithmetic

mean approach would be preferred.

Table 8: Results of provisional SCRVs and the associated uncertainties calculated by arithmetic

mean and median approaches.

SCRV u(SCRV)* N Standard

Uncertainty

Arithmetic Mean 5.225 0.150 4 0.0750

Median 5.300 0.000 4 0.0000


of 46

*For “arithmetic mean” and “median” approaches, the respective u(SCRV) is based on the

corresponding standard deviation (SD) and MADe.

Figure 4 : SCRV by arithmetic mean approach (green line) and its standard uncertainty (red dotted

line) with participants’ results and the associated reported standard uncertainties.

◆ Data included for SCRV calculation

◆ Data excluded from SCRV calculation

Degree of equivalence calculation

The degrees of equivalence (DoE) of the measurement results submitted by the

participants was established by the Supplementary Comparison Reference Value (SCRV)

as a consensus value of the reported results which were measured by an analytical

method with high metrological order by using primary standard with a metrological

traceable assigned purity and/or by in house purity assessment method.

The DoE (Di, U(Di)) for each participant was calculated according to the following equation:

Di = Xi - Xref

2

4

6

8

10

12

Mas

s fra

ctio

n of

cle

nbut

erol

, ng/

g

APMP.QM-S6: Clenbuterol in Porcine Meat

SCRV by arithmetic mean approach

Standard uncertainty of the SCRV


of 46

where Di is the degree of equivalence of participant i; Xi is the reported result of participant i; and Xref is the proposed SCRV value

The expanded uncertainty of the Di [U(Di)] for each participant was estimated as follows:

U(𝐷𝑖) = �ki2 × u2(Xi) + kref

2 × u2(Xref)

where ki is the coverage factor of participant i; kref is the coverage factor of the proposed SCRV

The kref for the estimation of the expanded uncertainty was chosen as k = 3.18 (t

(0.05;3) ), since there were only 4 valid results.

Table 9: Degrees of equivalence [Di] and their expanded uncertainties at 95% level of confidence

[U (Di)].

NMI/ DI ki Di U(Di) %Di % U(Di) Di/U(Di)

MSL 2 -2.768 0.2427 -52.98 4.64 -11.41

NIMT 2.03 -0.225 0.3410 -4.31 6.53 -0.66

NIM 2.0 -0.045 0.5540 -0.86 10.60 -0.08

GLHK 2 0.075 0.4319 1.44 8.27 0.17

BVL 2 0.075 0.7207 1.44 13.79 0.10

ΕΧΗΜ 2.31 0.075 0.3836 1.44 7.34 0.20

CENAM 2 4.745 0.9311 90.81 17.82 5.10

The measurement results of NMIs/DIs with italic fonts were excluded on technical grounds in the

SCRV calculation.


of 46

Figure 5: APMP.QM-S6: Plot of degrees of equivalence [Di] and their expanded uncertainties

with kref =3.18 at 95% level of confidence [U(Di)].

◆ Data included for SCRV calculation

◆ Data excluded from SCRV calculation

Scope of the Supplementary Comparison and “How Far does the Light Shine”?

The supplementary comparison for this measurement will assist in ensuring the

comparability of reference measurement procedures, as well as to support the CMC

claims of NMIs/DIs in this measurement field.

APMP.QM-S6 provides the means for assessing measurement capabilities for (i) value

assignment of primary references; (ii) extraction of analytes of interest from matrix; (iii)

clean-up and separation of analytes of interest from other undesirable interfering matrix

or extract components; and (iv) analytical separation and specificity.

After thorough discussion in the meeting, it was generally agreed that this study provides

the means for assessing measurement capabilities for determination of low-polarity

measurands in a procedure that requires extraction, clean-up, analytical separation, and

selective detection. Generally, it provides demonstration of a laboratory’s capabilities in

determining the mass fraction in range from 1 to 50 ng/g of stable and non-volatile drug

-60

-20

20

60

100

140D

i (%

)


of 46

residues of low molecular weight analytes (mass range 100-500) and low polarity (pKOW

≤-2) in unprocessed meat.

The successful participation in this supplementary comparison can demonstrate the

ability to apply IDMS in a mass fraction range of 1 to 50 ng/g (applying the respective

analytical technique, i.e. LC/MSMS or GC/MS). An extension to other stable and non-

volatile residues of low molecular weight analytes can be justified, presuming that there is

an isotopically labelled standard available; since the ability to apply effective sample

preparation steps for complex matrices and selective detection techniques has been

demonstrated.

The AOAC food-matrix triangle is shown in Figure 6 for reference. This porcine meat

matrix would be expected to fall into segment “4”.

Figure 6: AOAC Food-matrix Triangle (by courtesy of NIST)


of 46

Acknowledgement

The contribution from the contact persons and also the analysts of the participating

laboratories, as listed below, are highly appreciated.

Laly Samuel Measurement Standards Laboratory of New Zealand (MSL)

Eliane Cristina Pires do Rego National Instuitue of Metrology, Quality and Technology, Brazil (INMETRO)

Preeyaporn Pookrod National Institute of Metrology, Thailand (NIMT)

Xiuqin Li National Institute of Metrology, P.R. China (NIM)

Joachim Polzer and Wolfgang Radeck German Federal Office of Consumer Protection and Food Safety (BVL)

Elias Kakoulides / Vasiliki Schoina Chemical Metrology Laboratory, Greece (ΕΧΗΜ/ GCSL-ΕΙΜ)

Mariana Arce/ Yoshito Mitani National Metrology Centre of Mexico (CENAM)

of 46

Annex I: Details of Moisture Content Determination by NIM

The sample pre-treatment

The test sample portion was placed over anhydrous calcium sulphate (DRIERITE®) in a

desiccator at room temperature for 22 days and the results were listed as follow:

Bottle No. Moisture content increased,

% %RSD

1549 8.901 4.1

991 9.069 3

86 9.098 5.2

435 8.846 2.5

Determining Method: Vacuum drying method

Weighed about 0.5 g sample to weighing bottle which dried to constant weight, put it to

vacuum drying oven that connected to vacuum pump, drawn out the air and heat to 60℃ at

the same time. Turned off the vacuum pump valve, stopped to draw air, and kept vacuum

drying oven at constant temperature and pressure. After 4 hours, turned on the valve and let

the drying air flow into the vacuum drying oven, didn’t open the oven until the pressure

become normal. Put the weighing bottle to the desiccators and weighed the sample after 0.5

h. Repeated the operations until constant weight which meant the difference result between

the first and the second do not exceed 2 mg. The result was shown in the table listed below.

The result of water content determined by vacuum drying method

NO. sample weight/g water content%

1 0.55076 10.1

2 0.57352 10.0

3 0.52444 9.7

9.9% Validation method: Karl Fischer method

Open the moisture meter and wait several minutes for becoming stable. And then, precisely

weighed the sample by the reducing heavy law, added to the moisture meter. The sample

mixing time was set to 150 second. The results calculated by software when the

determination finished. The results shown in the table listed.

of 46

The result of water content determined by Karl Fischer method

NO. sample weight/g water content/%

1 0.02558 10.38

2 0.04595 10.33

3 0.03822 10.47

4 0.02705 10.67

5 0.05089 10.17

6 0.03863 10.57

7 0.05919 10.71

8 0.05023 10.99

9 0.0535 10.73

10 0.03467 10.53

AVE 10.56

of 46

Annex II: Measurement Uncertainties Reported by Participants

1. MSL

The measurement equations used to calculate the mass fraction of the measurand. Please provide details of all

the factors listed in the equations and indicate how these values were determined.

%𝑤𝑤

= 𝐶𝑧 ×𝑚𝑦

𝑚𝑥×𝑚𝑧𝑧

𝑚𝑦𝑧×𝑅𝑏𝑅𝑏𝑧

Cz is the concentration (ng.g-1) of the clenbuterol standard (CoA) My is the mass of isotopically enriched clenbuterol spike (Balance weighing) Mx is the mass of sample (Balance weighing) Mzc is the mass of clenbuterol standard added to the calibration blend (Balance weighing) Myc is the mass of isotopically enriched clenbuterol standard (Balance weighing) Rb is the ratio of the MS signals for clenbuterol/clenbuterol-D9 in the sample blend (Instrument response) Rbc is the ratio of the MS signals for clenbuterol/clenbuterol-D9 in the calibration blend (Instrument response)

Estimation of uncertainties for each factor. Give a complete description of how the estimates were obtained

and combined to calculate the overall uncertainty. Please provide a table detailing the full uncertainty budget.

Parameter (x) u(x) units Sensitivity

Cz 1.06E+00 mg.Kg-1 1.59E-01

My 2.12E-05 Kg 4.76E+03

Mx 2.121E-05 Kg 9.95E-15

Myc 2.121E-05 Kg -4.61E+03

Mzc 2.121E-05 Kg 2.23E-01

Rb 9.38E-02 1.22E-01

Rbc 2.11E-02 1.91E-01

of 46

2. NIMT



Measurement equation: Where; wx = mass fraction of clenbuterol in pork meat wz = mass fraction of analyte in the calibration solution used to prepare the calibration blend my = mass of spike solution added to sample blend myc = mass of spike solution added to calibration blend mzc= mass of standard solution added to calibration blend mx = mass of sample added to sample blend FE = extraction efficiency factor FI = interference effect FP = method precision factor F drymass = drymass correction factor obtained from moisture content analysis R’b and R’bc = observed isotope amount ratios in the sample blend and the calibration blend, respectively

Estimation of uncertainties for each factor. Give a complete description of how the estimates were obtained and combined to calculate the overall uncertainty. Please provide a table detailing the full uncertainty budget.

222222222)()()()()()()()()()(

+

+

+

+

+

+

+

+

=

P

P

E

E

I

I

drymass

drymass

x

x

zc

zc

yc

yc

y

y

zc

zc

FFu

FFu

FFu

FFu

mmu

mmu

mmu

mmu

wwu

xxu

bc

b

ycxdrymass

zcyzIEPx R

RmmF

mmwFFFw

''

.... ⋅

⋅

⋅⋅=

−=

100%1 moistureFdrymass

of 46

Where; u(wz,c) is the standard uncertainty of the mass fraction of analyte in the calibration solution used to prepare the calibration blend. The value was estimated from the purity of clenbuterol standard, masses weighed for preparation of stock solutions and uncertainty using different standards (standard comparison). u(my), u(my,c), u(mx) and u(mz,c) are standard uncertainties of the masses. These values were estimated from the bias and precision effect of the balance. u (FP) is the standard uncertainty of the precision factor. This value was estimated from standard deviation of the multiple IDMS results. u(FI) is the standard uncertainty of the interference effect. This value was estimated from potential bias between primary ion pair and secondary ion pair of the MRM program. u(FE) is the standard uncertainty of the extraction efficiency factor which was estimated from the liquid-liquid extraction and solid-phase –extraction. u(Fdrymass) is the standard uncertainty of the dry mass correction factor which was estimated from the moisture content analysis. Note: For the uncertainty contributing to the R'B and R'B,C ,the precision in measuring the isotope amount ratios of the analyte and the internal standard in the sample and calibration blends was assumed to be incorporated in the overall method precision. The effect of any biases on these ratios was assumed to be negligible as any systematic biases should cancel out since the calibration blends and sample blends were exact-matched for analyte concentration and isotope ratio. Other biases that may arise from interferences, extractions are captured in other factors.

of 46

Measurement Uncertainty budget:

Uncertainty budget of clenbuterol Factor Values Uncertainties

x u(x) u(x)/(x)

Parameter (unit)

Method Precision, FP(1) 1.0000 0.01697 1.697%

mz,c (g) 0.28933 0.000049 0.0171%

my (g) 0.28809 0.000049 0.0172%

my,c (g) 0.29174 0.000049 0.0170%

Fdrymass (1) 0.98928 0.000061 0.0062%

mx (g) 0.51371 0.000049 0.0096%

wz,c (mg/kg) 8.3414 0.076265 0.9143%

Additional Factors

Extraction effects, FE (1) 1.000 0.0100 1.000%

Interference from two different ion pairs, FI (1) 1.000 0.0110 1.097%

Uncertainty Analysis Results wx= 4.99 ng/g

u(x) = 0.12 ng/g

u(x)/x = 2.43%

Veff(total) = 34.863

k= 2.03 (@ 95% level)

U(x) = 0.247

%U(x) = 4.94%

of 46

3. NIM China The measurement equations used to calculate the mass fraction of the measurand. Please provide details of all the factors listed in the equations and indicate how these values were determined.

The expanded measurement equation given was used to calculate the mass fraction of the measurand. The additional factors (F) in the expanded measurement equation represent aspects of the measurement procedure that may influence the measured mass fraction value. They are given a value of 1 but they add an uncertainty component to the uncertainty budget. Expanded measurement equation Cx = FI×FP×FE ×(My×Mzc×Rb)/(Mx×Fdrymass×Myc×Rbc) Where

Cx is the mass fraction of clenbuterol in the sample (ng/g) FI is the matrix effect interference factor FP is the method precision factor FE is the extraction efficiency factor My is mass of clenbuterol-D9 added to the sample blend (g) Mzc is mass of clenbuterol added to the calibration blend (g) Rb is peak area ratio of clenbuterol /isotope in sample blend Mx is mass of sample (g) Fdrymass is the drymass correction factor obtained from moisture content analysis Myc is mass of clenbuterol-D9 added to the calibration blend (g) Rbc is peak area ratio of clenbuterol /isotope in calibration blend

of 46


Uncertainties

Source of uncertainty Parameter x u(x) u(x)/(x)

My (g) 0.1 9.20E-05 0.092%

Mx (g) 0.5 9.20E-05 0.018%

Myc (g) 0.1 9.29E-04 0.929%

Mzc(g) 0.1 9.93E-04 0.993%

Fdrymass (1) 1 1.57E-02 1.570%

Extraction effects, FE (1) 1 2.00E-02 2.000%

Interference from matrix effect , FI (1) 1 1.00E-02 1.000%

Method Precision, FP (1) 1 1.76E-01 3.690%

Relative combined standard uncertainty (uc) 4.8 %

Coverage factor , K 2.00

Relative expanded uncertainty ( Uc) 9.6 %

Uncertainty Analysis Results

Cx= 5.18 µg/kg u(x) = 0.25 µg/kg

k = 2 (@ 95% level)

U(x) = 0.5 µg/kg

of 46

4. GLHK

The measurement equations used to calculate the mass fraction of the measurand. Please provide details of all the factors listed in the equations and indicate how these values were determined.


of 46

5. EXHM



The measurement equation is:

𝑤𝑀,𝑆 = 𝑤𝑀,𝐶

1 −𝐻 ×

𝑚𝑖𝑖,𝑆

𝑚𝑆×𝑚𝑀,𝐶

𝑚𝑖𝑖,𝐶×𝑅𝑆𝑅𝐶

where wM,S = dry mass fraction of measurand in the sample, (ng/g)

wM,C = mass fraction of measurand solution in the calibration blend, (ng/g)

H = sample moisture content (g/g)

mis,S = mass of internal standard solution added to the sample blend, (ng)

mS = mass of test material in the sample blend, (g)

mM,C = mass of measurand solution added to the calibration blend, (g)

mis,C = mass of internal standard solution added to calibration blend, (g)

RS = measured peak area ratio of the selected ions in the sample blend

RC = measured peak area ratio of the selected ions in the calibration blend

Estimation of uncertainties for each factor. Give a complete description of how the estimates were obtained

and combined to calculate the overall uncertainty. Please provide a table detailing the full uncertainty budget.

The equation used to estimate standard uncertainty is :

𝑢(𝑤𝐵𝑆) = ��𝑆𝑆𝑆√𝑛� �

2+ ��𝐶𝑗𝑢(𝑚𝑖)�

2 + ��𝐶𝑗𝑢(𝑅𝑖)�2 + �𝐶𝑗𝑢(𝑤𝑀𝐶)�2 + �𝐶𝑗𝑢(𝐻)�2

where SDr is the standard deviation under reproducibility conditions, n the number of determinations

(8) and Cj the sensitivity coefficients associated with each uncertainty component. The uncertainties

of the peak area ratios were considered to have been included in the estimation of method precision.

Uncertainty estimation was carried out according to JCGM 100: 2008. The standard uncertainties were

combined as the sum of the squares of the product of the sensitivity coefficient (obtained by partial

differentiation of the measurement equation) and standard uncertainty to give the square of the

combined uncertainty. The square root of this value was multiplied by a coverage factor (95%

confidence interval) from the t-distribution at the total effective degrees of freedom obtained from

the Welch-Satterthwaite equation to give the expanded uncertainty.

of 46

6. BVL



( )x y a b= −

( ) ( ){ }b n x y x y n x xi i i i i i= − ∑∑∑ − ∑∑ 2 2

( )a y b x ni i= − ∑∑

x: analyte concentration in the sample (µg/ml) xi: analyte concentration of the i-th standard (µg/ml) a: intercept of the calibration curve b: slope of the calibration curve y: area of the analyte peak of the sample yi: area of the analyte peak of the i-th standard n: number of analyses per concentration range

The measurement equations used to calculate the mass fraction of the measurand. Please provide details of all the factors listed in the equations and indicate how these values were determined.

of 46

7. CENAM



w1 Mass fraction of the solution calibration standard (low level) (µg/kg)

w2 Mass fraction of the solution calibration standard (high level) (µg/kg)

R1 Response relationship of low solution

R2 Response relationship of high solution

mi1

Mass of the isotope solution added o the low level solution calibration standard

(g)

m1

Mass of the mesurand standard solution of low level calibration

standard (g)

mi2

Mass of the isotope solution added o the high level solution calibration standard

(g)

m2

Mass of the mesurand standard solution of high level calibration

standard (g)

mx Mass of sample to be measured (g)

m1x

Mass isotope of the solution added to the sample (g)

Rx

Instrument response relationship between the analyte in the ample and its isotope added

(dimensionless)

x

Ixx

Ix

Ix m

mRR

RRwmmRRw

mm

w ⋅

−

−⋅⋅

−−⋅⋅

=12

211

112

2

2 )()(

of 46

Estimation of uncertainties for each factor. Give a complete description of how the estimates were obtained and

combined to calculate the overall uncertainty. Please provide a table detailing the full uncertainty budget.

Uncertainty source value units information source Original uncertainty units Distribution Relative

uncertainty mass fraction of

CLB 9.97 g experimental 0.44 g 0.045

Mass of IS 0.3 g experimental 6.22495E-05 0.000207498Repeatability 0.00005 g CNM-CC-730-026/2013 2.88675E-05 g B rectangular

Linearity 0.000095 g CNM-CC-730-026/2013 5.48483E-05 g B rectangularResolution 0.00001 g CNM-CC-730-026/2013 5.7735E-06 g B rectangular

Mass of sample 0.4 g experimental 6.22495E-05 0.000155624Repeatability 0.00005 g CNM-CC-730-026/2013 0.000029 g B rectangular

Linearity 0.000095 g CNM-CC-730-026/2013 0.000055 g B rectangularResolution 0.00001 g CNM-CC-730-026/2013 0.000006 g B rectangular

Uncertainty of the curve 10.19 ng/g experimental 0.29 ng/g A normal 0.0287

Calibrant purity 99.71 % experimental 0.05 % A normal 0.0005Repeatability of sample (M-737) 10.63 ng/g experimental 0.36 ng/g A normal 0.034

Reproducibility 9.97 ng/g experimental 0.40 ng/g A normal 0.040

CLB = 9.97 ng/g ± 0.89 ng/g

% Relative uncertainty 8.

clenbuterol

of 46

Annex III: Core competency tables reported by participants

CCQM OAWG: Competency Template for Analyte(s) in Matrix

APMP.QM-S6 MSL Clenbuterol in Porcine Meat Scope of Measurement: This study provides demonstration of a laboratory’s capabilities in determining the mass fraction in range from 1 to 50 ng/g of stable and non-volatile drug residues of low molecular weight analytes (mass range 100-500) and low polarity (pKow<-2) in unprocessed meat.

Competency

Tick, cross, or “N/A” Specific Information as Provided by NMI/DI

Competencies for Value-Assignment of Calibrant Calibrant: Did you use a “highly-pure substance” or calibration solution?

Pure material Cerilliant Analytical Reference Standards

Identity verification of analyte(s) in calibration material.#

N/A

For calibrants which are a highly-pure substance: Value-Assignment / Purity Assessment method(s).#

N/A

For calibrants which are a calibration solution: Value-assignment method(s).#

N/A

Sample Analysis Competencies Identification of analyte(s) in sample Chromatographic retention time and MRM mode with

two ion pairs for identification Extraction of analyte(s) of interest from matrix

Liquid/ liquid extraction, sonication, shaking, enzymatic digestion and vortex

Cleanup - separation of analyte(s) of interest from other interfering matrix components (if used)

Liquid/ Liquid extraction and SPE

Transformation- conversion of analyte(s)of interest to detectable/measurable form (if used)

N/A

Analytical system LC-MS/MS Calibration approach for value-assignment of analyte(s) in matrix


Verification method(s) for value-assignment of analyte(s) in sample (if used)

N/A

Other N/A

of 46

APMP.QM-S6 NIMT Clenbuterol in Porcine Meat Scope of Measurement: This study provides demonstration of a laboratory’s capabilities in determining the mass fraction in range from 1 to 50 ng/g of stable and non-volatile drug residues of low molecular weight analytes (mass range 100-500) and low polarity (pKow<-2) in unprocessed meat.

Competency


Competencies for Value-Assignment of Calibrant

Calibrant: Did you use a “highly-pure substance” or calibration solution?

Pure material Dr. Ehrenstorfer GmbH


LC-UV/Vis and LC-MS/MS


Mass balance approach Organic: HPLC-UV Water/ moisture: Karl-Fischer Titration Residual solvent: TGA Inorganic:TGA


N/A

Sample Analysis Competencies Identification of analyte(s) in sample Chromatographic retention, MRM mode with two ion

pairs for identification Extraction of analyte(s) of interest from matrix

Liquid/ liquid extraction, protein precipitation, defattening, enzymatic digestion, vortex, shaking and centrifugation


SPE and liquid chromatographic separation


Alkaline hydrolysis


IDMS Bracketed exact-matching double IDMS


N/A

Other N/A

of 46

APMP.QM-S6 NIM Clenbuterol in Porcine Meat Scope of Measurement: This study provides demonstration of a laboratory’s capabilities in determining the mass fraction in range from 1 to 50 ng/g of stable and non-volatile drug residues of low molecular weight analytes (mass range 100-500) and low polarity (pKow<-2) in unprocessed meat.

Competency



Highly-pure substance NIM


LC-MS, IR, IC, LC-UV/Vis


Mass balance approach Organic: HPLC-UV Water/ moisture: Karl-Fischer method Residual solvent: GC-FID Inorganic: ICP-MS


N/A

Sample Analysis Competencies Identification of analyte(s) in sample Retention time,Mass spec ion

Extraction of analyte(s) of interest from matrix

Liquid/ liquid extraction, sonication, shaking and vortex


SPE

Transformation - conversion of analyte(s) of interest to detectable/measurable form (if used)

N/A




Other N/A

of 46

APMP.QM-S6 GLHK Clenbuterol in Porcine Meat Scope of Measurement: This study provides demonstration of a laboratory’s capabilities in determining the mass fraction in range from 1 to 50 ng/g of stable and non-volatile drug residues of low molecular weight analytes (mass range 100-500) and low polarity (pKow<-2) in unprocessed meat.

Competency



Pure material USP


LC-MS/MS


Mass balance approach Organic: HPLC and LC-MS/MS Water/ moisture: Karl-Fischer coulometry Residual solvent: Headspace GC-MSD Inorganic:ICP MS and IC


N/A

Sample Analysis Competencies Identification of analyte(s) in sample Retention time and mass spec ion ratios Extraction of analyte(s) of interest from matrix

Sonication


SPE


N/A




N/A

Other N/A

of 46

APMP.QM-S6 BVL Clenbuterol in Porcine Meat Scope of Measurement: This study provides demonstration of a laboratory’s capabilities in determining the mass fraction in range from 1 to 50 ng/g of stable and non-volatile drug residues of low molecular weight analytes (mass range 100-500) and low polarity (pKow<-2) in unprocessed meat.

Competency




Pure material from Sigma-Aldrich GmbH


LC-UV/ Vis and HPLC-QToF


Mass balance approach Organic: LC-UV/ Vis and HPLC-QToF Water/ moisture: Karl-Fischer Titration Residual solvent: Nil Inorganic: Nil


N/A

Sample Analysis Competencies Identification of analyte(s) in sample Identification by retention time, by mass spec ion

ratios (3 ion transitions) and by exact mass (TOF).

Extraction of analyte(s) of interest from matrix

Sonication, shaking, enzymatic digestion and vortex


SPE

Transformation - conversion of analyte(s) of interest to detectable/measurable form (if used)

N/A


Internal standard Multi-level calibration curve


N/A

Other N/A

of 46

APMP.QM-S6 CENAM Clenbuterol in Porcine Meat Scope of Measurement: This study provides demonstration of a laboratory’s capabilities in determining the mass fraction in range from 1 to 50 ng/g of stable and non-volatile drug residues of low molecular weight analytes (mass range 100-500) and low polarity (pKow<-2) in unprocessed meat.

Competency




Pure material Sigma-Aldrich


HPLC-MS-MS


Mass balance approach DSC Organic: GC-FID Water/ moisture: Karl-Fischer Titration Residual solvent: Nil Inorganic: HPLC/ conductivity detector and ICP-MS


N/A

Sample Analysis Competencies Identification of analyte(s) in sample Chromatographic retention time and MRM mode with

two ion pairs for identification Extraction of analyte(s) of interest from matrix

Acid digestion, sonication, shaking, vortex and centrifugation


Microplate elution and centrifugation


N/A


IDMS Bracketing calibration


N/A

Other N/A

Final Report of APMP.QM-S6 - BIPM - BIPMFinal Report of APMP.QM-S6 APMP Supplementary Comparison...

Documents

Transcript of Final Report of APMP.QM-S6 - BIPM - BIPMFinal Report of APMP.QM-S6 APMP Supplementary Comparison...