Final Report CCQM Track C Key Comparison (CCQM-K126) · 2017. 9. 18. · Final Report CCQM Track C...

Final Report

CCQM Track C Key Comparison (CCQM-K126)

Low Polarity Organic in Water: Carbamazepine in Surface Water

March, 2017

Coordinated by: Dr. Della Wai-mei Sin and Dr. Yiu-chung Wong

Government Laboratory, Hong Kong, China (GLHK)

With contribution from

Dr. Andreas Lehmann and Dr. Rudolf J. Schneider

BAM Federal Institute for Materials Research and Testing (BAM), Germany

Dr. Elias Kakoulides

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

Dr. Teo Tang Lin and Dr. Liu Qinde

Health Sciences Authority (HSA), Singapore

Dr. Julie Cabillic and Dr. Sophie Lardy-fontan

Laboratoire National de Metrologie et d'Essais (LNE), France

Dr. Jintana Nammoonnoy

National Institute of Metrology Thailand (NIMT), Thailand

Ms. Désirée Prevoo-Franzsen

National Metrology Institute of South Africa (NMISA), South Africa

Miss Chan Pui Kwan

Government Laboratory, Hong Kong (GLHK), Hong Kong, China

Dr. Eduardo Emilio López and Dr. Cecilia Alberti

Instituto Nacional de Tecnología Industrial (INTI), Argentina

Dr. Fuhai Su

National Institute of Metrology (NIM), People's Republic of China

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CCQM-K126


Content

Abstract ...................................................................................................................... 3

Introduction ................................................................................................................ 4

Samples and Preparation ............................................................................................. 4

(a) Measurand ......................................................................................................... 4

(b) Preparation of Test Material ................................................................................ 5

(c) Homogeneity and Stability Studies ...................................................................... 5

(d) Registration and Sample Distribution .................................................................. 8

(e) Reporting and Submission of Results ................................................................... 9

(f) Programme Schedule ........................................................................................ 10

Result Submitted by Participants ................................................................................ 10

Reference Materials Used by the Participating Laboratories ........................................ 12

Methods Applied by the Participants .......................................................................... 14

Approaches to Uncertainty Estimation ....................................................................... 20

Estimation of Key Comparison Reference Value (KCRV) ........................................... 22

Degree of equivalence (DoE) calculation .................................................................... 25

Core Competent and How Far does the Light Shine? .................................................. 28

Acknowledgement ..................................................................................................... 29

Reference .................................................................................................................. 30

Appendix I ................................................................................................................ 31

Appendix II .............................................................................................................. 47

2

Abstract

This study aimed to assess the measurement capabilities of participating National

Metrology Institutes/ Designated Institutes (NMIs/DIs) and expert laboratories in

determining of low-polarity organics in surface water. This comparison was organized by

Government Laboratory, Hong Kong (GLHK).

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

2012 in Hong Kong, GLHK initially proposed a CCQM key comparison and a parallel

pilot study programme on pharmaceuticals in surface water. Further discussion at the

CCQM Meeting held in April 2014 in Paris, the OAWG approved a CCQM Track C

comparison (CCQM-K126) on low polarity pharmaceuticals in surface water. In the

meeting, the programme was supported by more than six National Metrology Institutes/

Designated Institutes (NMIs/ DIs).

CCQM-K126 officially commenced in July 2014 and had registration from nine

NMIs/DIs. Participants were provided two bottles (40 mL each) of surface water and

were requested to determine the mass fraction of spiked carbamazepinein in surface

water. The coordinator received nine sets of results from eight NMIs/DIs in February

2015. Apart from one using an immunoassay technique, all participants applied isotope

dilution liquid chromatography-tandem mass spectrometry (ID-LCMS/MS) technique as

their determination technique.

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CCQM-K126


Introduction

Chemical pollution with pharmaceutical residues in surface water poses a threat to the

aquatic environment and human health, and this phenomenon has become one of the

major emerging environmental issues in recent decades. In order to protect and restore

the water quality in Europe and ensure its sustainable use, the European Water

Framework Directive (Directive 2000/60/EC of the European Parliament and of the

Council establishing a framework for the Community action in the field of water policy)

commits to achieve good qualitative and quantitative status of all water bodies within

Europe by 2015. The framework has laid down a strategy which involves the

identification of priority substances that pose a significant risk to the aquatic environment

and establishment of environmental quality standard (EQS) for the priority substances at

union level. In addition, the list will be renewed by means of a prioritisation mechanism

to identify new hazardous substances. Therefore accurate assessment methods for

measuring the hazardous substances in surface waters are indispensable tools to

safeguard the environment and the public health.

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

2012 in Hong Kong, GLHK initially proposed a CCQM key comparison and a parallel

pilot study programme on pharmaceuticals in surface water. Further discussion at the

CCQM Meeting held in April 2014 in Paris, the OAWG approved a CCQM Track C

comparison (CCQM-K126) on low polarity pharmaceuticals in surface water. In the

meeting, the programme was supported by several NMIs/DIs.

Samples and Preparation

(a) Measurand

The mass fraction of carbamazepine (CBZ) in surface water was the low polarity

pharmaceutical to be determined. The approximate mass fraction range of CBZ in this

programme study material was approximately 50 to 2000 ng/kg. The general chemical

and physical information of CBZ was as follows:

Formula C15H12N2O

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Molecular weight 236.269

CAS registry number: 298-46-4

Molecular Structure

-log P (octanol-water) -2.45

(b) Preparation of Test Material

Around 15 litres of surface water sample was collected in Hong Kong. The collected

water sample was immediately filtered by fluted filter paper (Whatman No.1, 24 cm) and

followed by membrane filter (Millipore, 0.45µm). The sample was autoclaved at 121 ±

0.5 oC for 15 minutes and stored in a refrigerator at 4 ⁰C overnight.

The refrigerated autoclaved sample was gravimetrically spiked with standard solution of

CBZ (United States Pharmacopeial Convention (USP) standard, 99.9%) at 255 ng/kg of

sample and was then stirred in a chilled water bath for an hour. In order to mimic a

genuine surface water matrix, a known amount of other commonly occurring

pharmaceutical standard solutions were also spiked in to the study material. About 40

mL of the well mixed sample solution were separately dispensed into 50 mL-deactivated

amber glass bottles. A total of 250 bottles were prepared and stored in a refrigerator at 4

⁰C.

(c) Homogeneity and Stability Studies

For the homogeneity study of the test material, twelve bottles were taken randomly from

the bulk and were conditioned to the room temperature (about 20 ⁰C). A sample size of

3.0 grams was used for the determination of mass fraction (ng/kg) of CBZ in each

sample. Analysis was performed in duplicate using LC-MS/MS with an IDMS technique

and all data obtained were statistically evaluated for homogeneity status as stipulated in

ISO Guide 35. The relative standard uncertainty due to between bottle heterogeneity (ubb)

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of CBZ was found to be 0.13% as summarized in Table 1. As Fcalculated is less than Fcritical, it

can be concluded that the material is considered to be adequately homogeneous for use

in the programme.

Table 1: Summary of ANOVA for homogeneity study of carbamazepine in the test material

ANOVA

Source of

Variance SS DF MS Fcalculated p-value F Critical

Between

Bottles

1.65E-05 11 1.50E-06 1.184 0.386 2.717

Within

Bottle

1.52E-05 12 1.26E-06

Total 3.16E-05 23

The short- and long-term stability studies of the test material were analysed at the same

conditions as those described for the homogeneity study and then evaluated by trend-

analysis technique as stipulated in ISO Guide 35. For the short-term stability study, eight

bottles were taken randomly from the bulk and two of the respective bottles were

isochronously conditioned at an elevated temperature of 40 ± 2 ⁰C for 1, 2, 3 and 4

weeks before analysis. The effect of time on analyte stability was assessed by fitting linear

regression lines to the data set. Two additional random bottles at the storage condition

were treated as reference samples. The p-value for the significance test of the regression

coefficient and the relative uncertainty associated with slope of CBZ were found to be

0.24 and 0.05% respectively in short-term stability study. The result is shown in Table 2.

The results indicated that the slope was not significantly deviated from zero at 95%

confidence level and no instability was observed for the test material at 40 ± 2 ⁰C for 1

month duration, the time frame covered the period needed for sample distribution.

Table 2: Summary of ANOVA test for short-term stability study of carbamazepine in the test

material at 40°C for 4 weeks

SUMMARY OUTPUT

Regression Statistics

Multiple R 0.64689

R Square 0.41847

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Adjusted R Square 0.22462

Standard Error 0.00039

Observations 5

ANOVA

df SS MS F Significance F

Regression 1 3.230E-07 3.230E-07 2.16 0.24

Residual 3 4.488E-07 1.496E-07

Total 4 7.718E-07

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

Intercept 0.2532 0.0003 845.0487 0.0000 0.2522 0.2541

X Variable 1 -0.0002 0.0001 -1.4693 0.2381 -0.0006 0.0002

The stability of the study material in storage condition at 4 °C was also studied. Eight

bottles of sample were randomly selected and analysed in duplicate in classical approach.

Samples were tested (i) after the study material was prepared in February 2014, (ii) before

sample dispatch to participants in March and July 2014 and (iii) after the result

submission deadline in March 2015. The p-value for the significance test of the regression

coefficient was found to be 0.94, which indicated that the slope was not significantly

deviated from zero at 95% confidence level which was showed in Table 3. In other

words, no instability was observed for the test material at 4 °C during the test period. It

can be concluded that the test material is considered to be adequately stable for use in the

programme.

Table 3: Summary of ANOVA test for long-term stability study of carbamazepine in the test

material in storage condition, at 4 °C, for 58 weeks

SUMMARY OUTPUT

Regression Statistics

Multiple R : 0.0437

R Square : 0.0019

Adjusted R Square : -0.3308

Standard Error : 0.0026

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Observations 5

ANOVA

df SS MS F Significance F

Regression 1 3.74E-08 3.74E-08 5.74E-03 9.44E-01

Residual 3 1.95E-05 6.51E-06

Total 4 1.96E-05

Coefficients Standard

Error t Stat p-value Lower 95% Upper 95%

Intercept 2.56E-01 1.51E-03 1.69E+02 4.54E-07 2.51E-01 2.61E-01

X Variable 1 -4.00E-06 5.28E-05 -7.58E-02 0.94 -1.72E-04 1.64E-04

(d) Registration and Sample Distribution

A total of ten participants from nine NMIs/DIs participated in the CCQM-K126 programme.

(Note: BAM had registered to submit two sets of results using LC-MS/MS and ELISA

techniques).

Table 4: List of participants

NMIs/ DIs

1 BAM Federal Institute for Materials Research and Testing, Germany*

2 BAM Federal Institute for Materials Research and Testing, Germany^

3 Chemical Metrology Laboratory (EXHM/GCSL-EIM), Greece

4 Health Sciences Authority (HSA), Singapore

5 Laboratoire National de Metrologie et d'Essais (LNE), France

6 National Institute of Metrology Thailand (NIMT), Thailand

7 National Metrology Institute of South Africa (NMISA), South Africa

8 Government Laboratory, Hong Kong (GLHK), Hong Kong, China

9 Instituto Nacional de Tecnología Industrial (INTI), Argentina

10 National Institute of Metrology (NIM), China

Determination using *ELISA and ^ LC-MS/MS techniques respectively.

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Two sample packs were sent to all participants in September and November 2014. An

additional sample pack was sent to all participants after the request for extra samples

from one participant. The sample pack contained (i) two bottles of about 40 mL of test

material and (ii) Material Safety Data Sheet detailing the general precaution and

information of CBZ. To monitor the highest temperature that the test material would be

exposed to during the transportation, temperature recording strips were sent along with

the test material to the participating institutes. Upon receipt of the sample pack,

participants were advised to (i) inspect the physical conditions of the sample bottles; (ii)

check and record the highest temperature being shown in the temperature strip and (iii)

complete and return the Sample Receipt Form to the coordinator as soon as they could.

Sample Receipt Form, Result Report Form and Core Competency Table were sent to

participants by email. All samples were informed to be well received in September and

early December 2014 except the one sent to INTI which was rejected by local customs in

Argentina and returned to GLHK in February 2015. According to the information

provided by the participants in the Sample Receipt Forms, the maximum temperatures

that the test material experienced were all below 29 ºC.

Participants were informed to store the test material at 4 ⁰C when not in use, and to move

to normal room conditions (at 20 ± 5 ⁰C and relative humidity <85%) and vortex

thoroughly prior to analysis. The test materials should be processed as soon as possible

and carefully re-sealed and stored in refrigerators at about 4 ⁰C after use.

Participants were requested to determine the mass fraction (in ng/kg) of CBZ in one of

the sample bottles using the analytical method of their choice and to report in the Result

Report Form. Analysis should be carried out with a recommended sample size of at least

3 g.

Certified reference material of CBZ was not available and participants had to carry out

purity analysis on commercial materials, including CRMs from non-NMI/DIs, and use

as calibrants, in order to establish the metrological traceability.

(e) Reporting and Submission of Results

Participants should report the following technical information in the Result Report Form:

The mass fraction of CBZ in ng/kg;

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The mean value of all replicate measurements and its associated uncertainty;

Description of analytical methods used; and

Description of the calibration standard used.

(f) Programme Schedule

Period Event

Apr. 2014 Presentation on homogeneity and stability results

Jul. 2014 Call for Participation

Aug. 2014 Deadline for registration

Sept. and Nov. 2014 Distribution of samples

Feb. 2015 Deadline for submission of results

Apr. 2015 Presentation of results at the CCQM OAWG Meeting

Result Submitted by Participants

Nine results were submitted from eight NMIs/DIs before the scheduled deadline. The

results reported by participants were summarized in Table 5 and the summary plot was

given in Figure 1. INTI informed the coordinator of their decision to withdraw from the

programme on 9 February 2015 due to the problems in purchasing the internal standard

for analysis and also the second sample pack sent in November 2015 was rejected by

local customs in Argentina.

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Table 5: Results submission by participants

NMIs/ DIs

Mass

fraction No. of replicate

measurements

Combined

Standard

Uncertainty

(ng/kg)

Coverage

Factor k

Expanded

Uncertainty

(ng/kg) (ng/kg)

BAM* 247.93 8 5.71 2 11.41

BAM^ 241.49 6 5.09 2 10.20

ΕΧΗΜ/GCSL-ΕΙΜ 255.4 5 5.1 2.23 11.3

HSA 250.2 12 2.44 2.0 4.9

LNE 246.6 3 2.5 2 5.0

NIMT 215.6 4 3.6 2.03 7.4

NMISA# 262 13 23 2 47

GLHK 256.83 6 4.83 2 9.66

INTI Withdrawn

NIM 244.9 5 3.2 2 6.4

# NMISA notified the coordinators in January 2016 that there was an error in their calculations and that the

Combined Uncertainty and Expanded Uncertainty should be 12 and 25 ng/kg respectively.

Note that after discussions at the OAWG meetings the final result for BAM was determined to be the BAM^

IDMS result and the BAM* ELISA result is provided here purely for information.

Figure 1: Summary of the participants’ result and associated reported standard uncertainties.

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CCQM-K126


Reference Materials Used by the Participating Laboratories

The summary of the reference standards and internal standards used by the participating

laboratories is shown in Table 6. Four participants including GLHK, HSA, NMISA and NIM

used the standard produced by United States Pharmacopeial Convention (USP). BAM and

EXHM/GCSL-EIM used the standard from Aldrich. LNE and NIMT used the standard from

LGC and Dr. Ehrenstorfer GmbH respectively.

As certified reference material of CBZ was not available and participants had to carry out purity

analysis on commercial materials, including CRMs from non-NMI/DIs, and use as calibrants, in

order to establish the metrological traceability. Details of purity assessment conducted by

participating laboratories is shown in Table 7. BAM, EXHM/GCSL-EIM and LNE had applied a

quantitative nuclear magnetic resonance (q-NMR) technique to directly estimate the principal

component. NIM applied q-NMR technique to directly estimate the principal component and also

used a mass balance approach. HSA, NIMT, NMISA, GLHK and NIM applied the mass balance

approach for purity assignment in which four classes of impurities including organic, water/

moisture, residual solvent and inorganic were determined.

Table 6: Reference materials and internal standards used by the participants

NMI/ DI Calibration standard Internal Standard

Source Purity Source

BAM* Aldrich, Lot: SLBH

2762V 99.375 % N/A

BAM^ Aldrich, Lot: SLBH

2762V

99.375 %

Carbamazepine – d2,

Toronto Research, no

native CBZ, c < 1ng/kg

EXHM/GCSL-EIM Sigma Aldrich, Lot:

MKBS4853V# 99.75%

Carbamazepine – d8,

Sigma Aldrich, 99.9%

HSA

United States

Pharmacopeial

Convention (USP)

999.6 ± 3.0 mg/g

Carbamazepine – d2-15N1,

Synfine Research

Limited, 988mg/g

LNE LGC MM0076.00 99.9% Carbamazepine–d8, TRC

Chemicals

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NIMT Dr. Ehrenstorfer GmbH 99.88 ± 0.07%

Carbamazepine–13C6,

Cerillant, Sigma-Aldrich;

99.58%

NMISA USP (Lot K1K264) 99.69%

Carbamazepine–13C6;

Cerilliant, 100.0 ± 0.5

µg/ mL

GLHK USP 99.9%

Carbamazepine –d10,

CIL; 100µg/ml in ACN-

D3

INTI Withdrawn

NIM USP 99.74% Carbamazepine –d8, TRC;

98.5% (d8 % was 50.2% )

# Information provided after distribution of Initial Summary Report.

Table 7: Details of purity assessments

NMI/ DI

Direct estimate

of principal

component

Estimate of impurities

Organic Water/

moisture

Residual

solvent Inorganic

BAM* q-NMR N/A N/A N/A N/A

BAM^ q-NMR N/A N/A N/A N/A

EXHM/GCSL

-EIM q-NMR N/A N/A N/A N/A

HSA

Qualitative 1H

NMR

spectroscopy

and [M+H]+

ion using LC-

MS/MS

LC-DAD Karl-Fischer

coulometry GC-MS

Thermogravim-

etric analysis

(TGA)

LNE q-NMR N/A N/A N/A N/A

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NIMT HPLC-MS/MS HPLC-UV KFT TGA TGA

NMISA

LC-MS and

LC-UV

Coulometric

Karl Fischer

Headspace GC-

TOFMS N/A

GLHK IR, MS and

MSMS HPLC-UV Karl Fischer

Headspace

GCMS

ICP MS and

ion

chromatograph

-y

INTI Withdrawn

NIM LC-UV and q-

NMR HPLC-UV Karl Fischer

Headspace-Gas

chromatograph

-y

ICP-MS

For the participants who applied LC-MS/MS technique, an internal standard was used. Five

different kinds of internal standards including carbamazepine-d2, d8, d2-15N1,

13C6 and d10 were

used and the structures of the internal standards are summarized in Table 8.

Table 8: Structures of internal standard used by the participating laboratories

Carbamazepine –

d2

Carbamazepine –

d8

Carbamazepine –

d2-15N1

Carbamazepine –

13C6

Carbamazepine –

d10

BAM^

EXHM/GCSL-

EIM, LNE and

NIM

HSA NIMT and

NMISA GLHK

Methods Applied by the Participants

Participants were encouraged to determine the mass fraction of carbamazepine in the test

sample using the analytical method of their choice. The details of method for sample

extraction, clean-up, instrumental analysis and quantitation are summarized in Table 9.

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Different kinds of solid phase extraction cartridges were applied by LNE, NIMT,

NMISA and NIM. BAM^, HSA and GLHK did not use any clean-up procedures.

For the instrumental analysis, one participant, BAM*, applied ELISA technique. This

used a multi-level calibration curve with external standard for quantitation. The rest of

the participants employed liquid chromatographic technique for chromatographic

separation, followed by triple quadrupole mass spectrometry operated under the multiple

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

MRM transition 237/194 was the common transition monitored and selected for

quantitation.

All LC-MS/MS analysis applied isotope dilution mass spectrometry (IDMS) with the

corresponding isotopic compounds as internal standard for calibration. BAM^, HSA and

LNE quantified the analyte by using multi-level external standard calibration.

EXHM/GCSL-ΕΙΜ and NIM applied single point calibration while NIMT and NMISA

used both single point and bracketing calibration methods. GLHK used only bracketing

calibration method for quantification. ΕΧΗΜ/GCSL-ΕΙΜ, NIMT and NIM used matrix

matched calibration blends but BAM, HSA, LNE, NMISA and GLHK did not.

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Table 9: Methods applied by the participants

NMI/ DI

Experimental details

Sample

size, g

Extraction

method

Extraction

condition

Clean up

method

Analytical

instrument (s) used

Chromatograp-

hic column

Type of

calibration

Method of

quantification Ions/ MRM transitions

BAM* 40 N/A N/A N/A ELISA, modular

workstation N/A

multi-level

calibration

curve

(sigmoid

calibration

curve)

External

standard N/A

BAM^

20 N/A N/A N/A

Agilent 1260 + AB

SCIEX TSQ 6500

ESI Positive

Kinetex XB-C18

150×3 mm

analytical column

(with pre-column)

with a particle

size of 2.6 µm

(Phenomenex,

Aschaffenburg,

Germany)

Multi-level

calibration

curve

calibration with

internal

standard (CBZ-

d2 )

Carbamazepine:

237 to 194

IS:

239 to 196

16

ΕΧΗΜ/GCSL-

ΕΙΜ 5

Agitated for 1

h in shaker,

vortexed for 1

min

N/A N/A

Thermo TSQ

Quantum Ultra AM

coupled to Thermo

Surveyor MS Pump

Plus and

Autosampler Plus

ESI Positive

Thermo Hypersil

Gold C18 (100 ×

2.1 mm, 3 μm)

Single-point

calibration

Isotope dilution

mass

spectrometry

Carbamazepine:

237 -> 194 (q)

237 -> 192

IS:

245 -> 204 (q)

245 -> 202

HSA 3.0

Vigorous

vortexing for

at least 1 min

before

sampling

N/A Vortexed

thoroughly

AB Sciex Qtrap®

5500 MS/MS

instrument coupled

with

Shimadzu

Prominence UFLC

XR LC system

ESI Positive

Agilent Zorbax

RX-SIL, 2.1×150

mm, 3 μm; and

Agilent Zorbax

SB-Aq, 2.1×100

mm, 3.5 μm

Multi-level

calibration

curve

Isotope dilution

mass

spectrometry

Carbamazepine:

237/194

IS:

240/196

LNE 10

Acidification

pH=3 Formic

acid

N/A Solid Phase

Extraction

HPLC system -TSQ

QUANTUM

Discovery max

(Thermo Fisher

France) TSQ

Quantum Discovery

triple quadripole

mass spectrometer

(Thermo Fisher,

France)

ESI Positive

Symmetry shield

RP18 (5 µm, 3.0 ×

250 mm)

Multi-level

calibration

curve

Isotope dilution

mass

spectrometry

Carbamazepine:

236.9 > 194.2 ;

236.9 > 192.0

IS:

245.1>202.1 ;

245.1>200.1

17

NIMT 5 N/A N/A

VertiPak™

HCP-SC

SPE

Cartridges

Shimadzu LC

System 20A series

coupled with AB

SCIEX API 4000

MS/MS System

ESI Positive

Luna C18, 100Å,

150 × 4.60 mm

dia., 5 µm

Single point

calibration;

Bracketing

calibration

Isotope dilution

mass

spectrometry

Carbamazepine:

Primary ion

pair237.1>194.3; ;

Secondary ion

pair237.1>179.3

IS:

Primary ion

pair243.0>200.3 ;

Secondary ion

pair243.0>185.2

NMISA 3 N/A N/A

Oasis HLB

3cc (60mg)

extraction

cartridges

Waters Acquity

UPLC coupled to a

Waters Micromass

Quattro Premier XE

triple quadrupole MS

ESI Positive

Waters Acquity

UPLC BEH C18

1.7 µm, 2.1 × 100

mm

Single point

calibration;

Bracketing

calibration

Isotope dilution

mass

spectrometry

Carbamazepine:

237.1>179.0;

237.1>192.0;

237.1 > 194.0

quantification ion

IS:

243>184.9 ;243>198.0 ;

243>200.0

quantification ion

GLHK 3

Vortexed

thoroughly

prior to

analysis

Sonication

and vortex;

for 30 min

N/A

Espert ultra LC

coupled with

ABSCIEX Qtrap#

6500

ESI Positive

Phenomenex

Luna 5µm

Phenyl-Hexyl

Bracketing

calibration

Isotope dilution

mass

spectrometry

Carbamazepine:

237/194

IS:

247/204

INTI Withdrawn

18

NIM 4.5

The sample

was adjusted

to pH 7.0 with

10%

ammonium

hydroxide

Vortex at

25℃ for

1hr

3-mL

disposable

Lichrolut EN

(200mg,

Merck,

Darmstadt,

Germany)

Waters XevoTQ-S

ESI Positive

Acquity

UPLC○,R BEH

C18, 1.7 m, 2.1

× 100 mm

Single-point

calibration

Isotope dilution

mass

spectrometry

Carbamazepine:

237.1->194.1

IS:

245.1->200.1

Determination using *ELISA and ^LC-MS/MS techniques respectively.

# Information provided after result submission.

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Approaches to Uncertainty Estimation

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

uncertainty budgets are summarized in Table 10 and details are shown in Appendix I.

There was a discussion on NMISA’s uncertainty for their reported value in the OAWG

meeting in October 2015. It was worthwhile to investigate the reason for the reported

uncertainty. NMISA replied in January 2016 that all the uncertainty contributors were

re-evaluated and this reduced the uncertainty of the peak area ratio of the native/labelled

analytes in the samples (RsB). Using the Grubb’s test one of the ratios was identified as an

outlier. This almost halved the uncertainty for NMISA which still covers all the repeat

measurements performed. The revised result is shown below for reference.

NMI/

DI

Mass

fraction No. of replicate

measurements

Combined

Standard

Uncertainty

(ng/kg)

Coverage

Factor k

Expanded

Uncertainty

(ng/kg)

% Relative

Standard

Uncertainty (ng/kg)

NMISA 262 13 12 2 25 4.6

Table 10: Approaches to uncertainty estimation from participants

NMI/ DI

Relative

Standard

Uncertainty

Contributions to the measurement uncertainty budget

BAM* 2.3%

Mass fraction of native standard by NMR results

Volumetric handling, class of the glassware × sqrt

(number of utilisations)

Run-to-run variability, repeatability by ELISA

Calibration uncertainty

BAM^ 2.1%

Mass fraction of native standard by NMR results

Volumetric handling, class of the glassware × sqrt

(number of utilisations)

Run-to-run variability, repeatability by LCMS

Calibration bias, bias at the content level of the

unknown

Mass of int std in aliquot, combined contribution from

all weighings

Rel calibration uncertainty

Consolidated result with and without ISTD

20

CCQM-K126


EXHM-GCSL-

EIM 2.0%

Method precision

Mass fraction of CBZ in calibration solution

Mass of CBZ-d8 added to sample blend

Mass of test material in sample blend

Mass of CBZ solution added to calibration blend

Mass of CBZ-d8 added to calibration blend

Peak area ratio in sample blend

Peak area ratio in calibration blend

HSA 1.0%

Mass of sample (determined by weighing)

Mass of isotope labeled standard solution (determined

by weighing)

Concentration of carbamazepine in the calibration

standard solution

Isotope mass ratio in sample blend

The standard deviation of the results was used as the

standard uncertainty of method precision

Average of the difference of the results using two

columns divided by 2

LNE 1.0%

Measurement model

Intermediate precision

Preparation of calibration standard

Preparation of sample (weightings)

NIMT 1.7%

Mass fraction of carbamazepine in the calibration

solution used to prepare the calibration blend

Standard uncertainties of the masses

Interference factor

Precision

Sample cleaning up efficiency factor

NMISA# 8.8%

[Native] solution used to prepare calibration blend

Weight native solution added to calibration blend

Weight of isotope solution added to sample

Weight of isotope solution added to calibration blend

Weight of sample analysed

Ratio of peaks areas of native/ labelled in the repeat

samples

Ratio of peaks areas of RM native/ labelled in the

calibration blend -repeat injects

Precision, repeat measurements

GLHK 1.9% Factor of repeatability

21

CCQM-K126


Mass fraction of carbamazepine in calibration blend

Mass of carbamazepine–d10 added to the sample blend

Mass of sample

Mass of carbamazepine standard solution added to the

calibration blend

Mass of carbamazepine–d10 added to the calibration

blend

Isotope amount ratio in sample blend

Isotope amount ratio in calibration blend

INTI Withdrawn

NIM 1.3%

Solution preparation

Weighing of samples

Purity

Method precision (repeatability)

# NMISA replied in January 2016 that the Relative Standard Uncertainty should be 4.6%

Estimation of Key Comparison Reference Value (KCRV)

Results of NMIs/DIs in the key comparison with metrologically traceable calibrants

were considered for inclusion in the calculation of the key comparison reference value

(KCRV). In this programme, BAM had registered as 2 participants in which 2 different

analytical techniques were applied. In result submission, the ELISA result was selected to

be included in the KCRV calculation. The nine submitted results ranged from 240 to 260

ng/kg except the result from NIMT which was 216 ng/kg. An Initial Summary Report

was sent to participants in April for discussion in the OAWG meeting.

As this was the first time to consider incorporating an ELISA result into the KCRV, the

specificity of the method was discussed at OAWG meetings. BAM reported that the

method was validated versus a wide range of other pharmaceuticals and three relevant

literature papers 1-3 were provided. In brief, the ELISA method has been developed and

applied to determine the environmental carbamazepine concentration in different kinds

of water samples. The method has been validated and compared to liquid

chromatography-tandem mass spectrometry after solid phase extraction. It was reported

that cross-reactivities of this ELISA method could be due to (i) tricyclic antidepressant

(such as protriptyline, opipramol, amitriptyline, imipramine and doxepine) (ii)

22

CCQM-K126


carbamazepine metabolites (such as 10,11-dihydro-carbamzxepine and 10,11-

epoxycarbamzxepine (EP-CBZ)) and (iii) structurally not obviously related antihistamine

(such as cetirizine, hydroxyzine and norchlorcyclizine). In general, the cross-reactivities

for tricyclic antidepressants have been determined to be less than 10%. The molar cross-

reactivity of structurally not obviously related antihistamines, such as cetirizine, was

around 22%. The remaining cross-reactivities, originating from EP-CBZ, was found to be

about 6%. BAM confirmed that the influence of these compounds on the assay results

was not expected, because in order to exert a measurable effect they would have to be

present in the sample in amounts equal or higher than CBZ itself which has never been

reported for real samples in the literature. BAM also commented that no “cross-

reactivity” from other compounds in surface water samples would be observed in this

uncontaminated surface water sample spiked solely with carbamazepine.

Validity of ELISA method was thoroughly discussed in the OAWG meeting held in

October 2015, there was consensus that the ELISA methodology did not appear to be a

higher-order technique and that it required a separate assessment of potential

contaminants. In January 2016 BAM agreed to use the IDMS result as their traceable

method to be included in the key comparison and the calculation of the KCRV instead of

the result by ELISA method.

The result from NIMT deviated from most of the participants and they were advised to

review the applied methodology. NIMT confirmed that the method used in the report

was correct and additional samples were sent to NIMT as per their request in order to

investigate the cause of deviation. A second result was submitted in August 2015 as

below.

Table 11: Summary of results submitted by NIMT.

Mass fraction (Mean value of replicate measurements,

ng/kg)

No. of replicate

measurements

Combined standard

Uncertainty

(ng/kg)

Coverage Factor k (95%

level of

confidence)

Expanded Uncertainty

(ng/kg)

235.2 3 5.5 2.03 11.1

Result originally submitted

215.6 4 3.6 2.03 7.4

23

CCQM-K126


As the originally reported result from NIMT was doubtful, NIMT agreed to withdraw

their result and KCRV was estimated based on excluding NIMT.

The OAWG agreed that the median approach was appropriate for estimation of the

KCRV and the associated uncertainty. The summary of the KCRV and standard

uncertainty are listed in Table 12 and the plot is shown in Figure 2.

Table 12: Results of KCRVs and the associated uncertainties calculated by median approaches

based on excluding the result from NIMT and BAM (ELISA method).

Approach Median

KCRV 250.2 ng/kg

u(KCRV)* = MADe

= 7.7 ng/kg

No. of data, N 7

Standard Uncertainty =

N

MADe25.1

= 3.6 ng/kg

24

CCQM-K126


Determination using *ELISA and ^ LC-MS/MS techniques respectively.

Data included for KCRV calculation

◆ Data excluded from KCRV calculation

Figure 2: KCRV by median approach (green line) and its standard uncertainty (red dotted line)

with participants’ results and the associated reported standard uncertainties excluding the result

from NIMT.

Degree of equivalence (DoE) calculation

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

participants was established from the KCRV determined 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. Detailed information is shown in where Di is the

degree of equivalence of participant i;

Xi is the reported result of participant i; and

Xref is the proposed KCRV value

Table 13 and plotted in Figure 3 and Figure 4.

25

CCQM-K126


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

equation:

Di = Xi - Xref

The expanded uncertainty of the Di [U(Di)] (k = 2) for each participant at a 95% level of

confidence was estimated as follows:

)(X2

)(X2

(Di) refiuu2U

where Di is the degree of equivalence of participant i;

Xi is the reported result of participant i; and

Xref is the proposed KCRV value

Table 13: Degrees of equivalence [Di] and their expanded uncertainties at 95% level of confidence

[U (Di)].

NMI/ DI Mass

fraction (ng/kg)

ki Di U(Di) %Di %

U(Di) Di/U(Di)

NIMT 215.6 2.03 -34.600 10.343 -13.83 4.1 -3.35

BAM^ 241.49 2 -8.710 12.600 -3.48 5.0 -0.69

NIM 244.9 2 -5.300 9.803 -2.12 3.9 -0.54

LNE 246.6 2 -3.600 8.951 -1.44 3.6 -0.40

H S A 250.2 2.0 0.000 8.885 0.00 3.6 0.00

EXHM 255.4 2.2 5.200 12.616 2.08 5.0 0.41

GLHK 256.83 2 6.630 12.184 2.65 4.9 0.54

NIMSA 262 2 11.800 46.595 4.72 18.6 0.25

The measurement results of NMIs/DIs with italic fonts were not included in the KCRV estimation.

26

CCQM-K126


◆ Data included for KCRV calculation


Figure 3: CCQM-K126: Plot of degrees of equivalence [Di] and their expanded uncertainties with

kref = 2 at 95% level of confidence [U(Di)] in relative terms.

27

CCQM-K126


◆ Data included for KCRV calculation


Figure 4: CCQM-K126: Plot of percentage of degrees of equivalence [%Di] and their expanded

uncertainties with kref=2 at 95% level of confidence [%U(Di)] in ng/kg.

Core Competent and How Far does the Light Shine?

Filtered surface water sample was tested in this key comparison. This study provided the

means for assessing measurement capabilities for determination of low-polarity analyte in

water. Generally, it provided demonstration of a laboratory’s capabilities in determining

the mass fraction in range from 50 to 2000 ng/kg of drug residues of low molecular

weight analytes (mass range 100-500) and low polarity (pKOW ≤ -2) in aqueous matrix by

LC-MS/MS. The competency of participating laboratories to measure carbamazepine in

surface water has been demonstrated in this key comparison. The successful laboratories

demonstrate the ability to quantitatively determine carbamazepine in surface water by

applying LC-MS/MS technique at a very low level with KCRV of 250.2 ng/kg with an

expanded uncertainty of 3.0%.

28

CCQM-K126


Acknowledgement

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

laboratories, as listed below, were highly appreciated.

With contribution from

Dr. Andreas Lehmann (LC-MS/MS), Dr. Kristin Hoffmann and Dr. Rudolf J. Schneider (ELISA)

BAM Federal Institute for Materials Research and Testing, Germany

Dr. Elias Kakoulides

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

Dr. Teo Tang Lin and Liu Qinde

Health Sciences Authority (HSA), Singapore

Cabillic Julie and Lardy-fontan Sophie

Laboratoire National de Metrologie et d'Essais (LNE), France

Dr. Jintana Nammoonnoy

National Institute of Metrology Thailand (NIMT), Thailand

Ms. Désirée Prevoo-Franzsen, Dr. Laura Quinn and Dr. Maria Fernandes-Whaley

National Metrology Institute of South Africa (NMISA), South Africa

Dr. Lai-ming Ella Wong, Miss Pui-kwan Chan, Miss Wai-yin Fok and Mr. Chi-chung Koo

Government Laboratory, Hong Kong (GLHK), Hong Kong, China

Dr. Eduardo Emilio López and Cecilia Alberti

Instituto Nacional de Tecnología Industrial (INTI), Argentina

Dr. Fuhai Su

National Institute of Metrology (NIM), People's Republic of China

29

CCQM-K126


Reference

1. Bahlmann A, Falkenhagen J, Weller MG, Panne U and Schneider RJ, Analyst, 2011, 136,

1357-1364

2. Bahlmann A, Weller MG, Panne U and Schneider RJ, Anal. Bioanal. Chem., 2009, 395, 1809-

1820

3. Bahlmann A, Carvalho JJ, Weller MG, Panne U, and Schneider RJ, Chemosphere, 2012, 89,

1278-1286

30

CCQM-K126 Appendix I: Measurement Uncertainties Reported by Participants

1. BAM (ELISA)

The measurement equation(s) 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.

C analyte = C ELISA * purity factor NMR

C analyte : mass fraction of the measurand

C ELISA : mass fraction of the measurand, determined by ELISA

purity factor NMR : purity of the calibrant used, determined by NMR

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 budget:

F_purity: mass fraction of native standard by NMR results 0.00040252

F_V: volumetric handling, class of the glassware * sqrt(number of untilisations) 0.014

F_rep: run-to-run variability, repeatability by ELISA 0.010409

F_cal: calibration uncertainty 0.015

total combined standard uncertainty (%) (root of square sums) : 0.023011

expansion factor k = 2

expanded relative uncertainty (%) : 0.046022

31

1. BAM (LC-MS/MS)




C analyte = C LC/MS * purity factor NMR * dilution factor ISTD

C analyte : mass fraction of the measurand

C LC/MS : mass fraction of the measurand, determined by LC – MS/MS

purity factor NMR : purity of the calibrant used, determined by NMR

dilution factor ISTD : dilution factor for the sample in the vial after the internal

standard was added, determined by weighing




uncertainty budget:

F_purity: mass fraction of native standard by NMR results 0.00040252

F_V: volumetric handling, class of the glassware * sqrt(number of untilisations) 0.014

F_rep: run-to-run variability, repeatability by LCMS 0.006359

F_b: calibration bias, bias at the content level of the unknown 0.012978

m_is: mass of intstd in aliquot, combined contribution from all weighings 0.005

ic: rel calibration uncertainty 0.002688

x_sample: consolidated result with and without ISTD 0.0029312

total combined standard uncertainty (%) (root of square sums) : 0.0211

expansion factor k = 2

expanded relative uncertainty (%) : 0.0422

32

2. EXHM

The measurement equation(s) 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 measurement equation is:

𝑤𝐶𝑀𝑍,𝑆 = 𝑤𝑀,𝐶 ×𝑚𝑖𝑠,𝑆

𝑚𝐶𝑀𝑍,𝑆

×𝑚𝐶𝑀𝑍,𝐶

𝑚𝑖𝑠,𝐶

×𝑅𝑆

𝑅𝐵

where wCMZ,S = dry mass fraction of CMZ in the sample, (mg/kg)

wCMZ,C = mass fraction of CMZ in the calibration solution, (mg/kg)

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

mM,S = mass of test material in sample blend, (g)

mCMZ,C = mass of CMZ solution added to 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) =√

(𝑆𝐷𝑟

√𝑛⁄ )2 + ∑(𝐶𝑗 𝑢(𝑚𝑖))2 + ∑(𝐶𝑗 𝑢(𝑅𝑖))2 + (𝐶𝑗𝑢(𝑤𝑀𝐶))2

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

determinations and Cj the sensitivity coefficients associated with each uncertainty

component. The uncertainty of the peak area ratios was considered to have been

33

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.

34

3. HSA




The mass fraction of carbamazepine in the sample was calculated based

on the IDMS calibration curve as follows:


were obtained and combined to calculate the overall uncertainty. Please provide a table

detailing the full uncertainty budget.

35

Carbamazepine reference standard from Sigma-Aldrich was purity assessed by quantitative 1H

NMR, and was used to spike into filtered surface water collected in Singapore. The spiked

material was used as quality control sample. The quality control sample was measured together

with the comparison sample. The results obtained from the quality control sample deviated

37

from the targeted value by -0.60% to 1.15%, with an average deviation of 0.24%,

which was well within the range of the relative measurement uncertainty of 1.95% of the

reporting result.

38

4. LNE




Cwater Clab mlab

(a Rwater b) f f m

s tan dard F water

Cwater: mass fraction of CBZ in water in µg/kg

Clab: mass fraction of labeled CBZ solution in µg/kg

mlab: mass of labeled solution added in the water sample in kg

mwater: mass of water sample in kg

Rwater: unlabeled/labeled ion peak area ratio in the water sample

a: slope of the linear regression plot

b: y- intercept of the linear regression plot

f standard: correction factor due to standards solutions uncertainty

f F: correction factor due to measurement intermediate precision




Measurement model : 78.5%

Intermediate Precision: 12.12%

Preparation of calibration standard: 7.89%

Preparation of sample (weightings): 1.49%

39

5. NIMT


Please provide details of all the factors listed in the equations and indicate how

these values were determined.

w F

.F .F .w

m .m .

y z,c .

R'B

x P C I z,c mx .m

y ,c

R'B,C

where;

wX = mass fraction of carbamazepine in the sample (ng/kg)

wZ ,C =

mass fraction of carbamazepine in the calibration solution used to prepare the calibration blend

(ng/kg)

mY = mass of 13C6-carbamazepine solution added to the sample blend (g)

mY ,C = mass of 13C6-carbamazepine solution added to the calibration blend (g)

mX = mass of water sample used (g)

mZ ,C = mass of carbamazepine solution added to the calibration blend

R'B

R'B,C =

, observed isotope amount ratios in the sample blend and the calibration blend, respectively

FI = interference effect given a value of 1

FP = method precision factor given a value of 1

FC = Sample clean up effect given a value of 1




𝑢 (𝑤𝑥)

𝑤𝑥

= √(𝑢(𝑤𝑍,𝐶)

𝑤𝑍,𝐶

)2 + (𝑢(𝑚𝑌)

𝑚𝑌

)2 + (𝑢(𝑚𝑌,𝐶)

𝑚𝑌,𝐶

)2 + (𝑢(𝑚𝑋)

𝑚𝑋

)2+(𝑢(𝑚𝑍,𝐶)

𝑚𝑍,𝐶

)2 + (𝑢(𝐹𝑃)

𝐹𝑃

)2 + (𝑢(𝐹𝐼)

𝐹𝐼

)2 + (𝑢(𝐹𝐼)

𝐹𝐼

)2

where;

𝑢𝑢(𝑤𝑤𝑧𝑧,𝑐𝑐) is the standard uncertainty of the mass fraction of carbamazepine in the

calibration solution used to prepare the calibration blend. The value was estimated

from the purity of carbamazepine standard and dilutions processes.

𝑢(𝑚𝑌), 𝑢(𝑚𝑌,𝑐), 𝑢(𝑚𝑥) and 𝑢(𝑚𝑧,𝑐) are standard uncertainties of the masses.

40

These values were estimated from the bias and precision effects of the balance. 𝑢(𝐹𝐼) is

the standard uncertainty of interference factor. It was estimated from potential bias

between primary ion pair and secondary ion pair.

𝑢(𝐹𝑃) is the standard uncertainty of the precision factor which is estimated from

standard deviation of the mean of the multiple IDMS results.

𝑢(𝐹𝐶) is the standard uncertainty of the sample cleaning up efficiency factor which is

estimated from SPE clean-up.

Note: The precision in measuring the isotope amount ratios of carbamazepine and

13C6-carbamazepine standard in the sample and calibration blends was assumed to be

incorporated in the overall method precision. Any systematic biases on these ratios

were assumed to be negligible since the calibration blends and sample blends were

exactly-matched for carbamazepine concentration and its isotope ratio. Other biases

that may arise from interferences and sample clean-up are captured in other factors.

41

Uncertainty budget for carbamazepine

Factor Values Uncertainties

Measurement equation factors x u(x) u(x)/(x)

mzc (g) 0.51937 0.000055 0.0106%

my (g) 0.51291 0.000055 0.0108%

myc (g) 0.51456 0.000055 0.0107%

mx

(g)

4.97439

0.000055

0.0011%

wz (ng/kg) 2040.151

9

12.548679 0.6151%

Additional Factors

Method Precision 1.0000 0.01145 1.145%

Sample clean up effect 1.000 0.0100 1.000%

Interference from two different ion

pairs

1.000

0.0029

0.286%

2. Uncertainty Analysis Results

wx = 215.6 ng/kg

u(x) = 3.6 ng/kg

u(x)/x = 1.66%

Veff(total) = 33.567

k = 2.03 (at the 95% CI)

U(x) = 7.4

%U(x) = 3.4%

42

6. NIMSA

43

CRM purity

NMISA (residual solvents, water content,

related impurities)

uncertainty of the 6 decimal place balance

uncertainty of the mass balance

0.9969 0.01150 0.011535761 0.000133074

g 0.019938 0.000007 0.000351088 1.23263E-07

g 100 0.0001 0.000001 1E-12

NMISA

Revised: Measurement equation used to calculate the mass fraction of carbamazepine in the

sample replied in January 2016

𝑤𝑥 = 𝑤𝑧 ×𝑚𝑛𝑎𝑡𝑖𝑣𝑒 𝐶𝐵

𝑚𝑖𝑠𝑜𝑡𝑜𝑝𝑒 𝐶𝐵×

𝑚𝑖𝑠𝑜𝑡𝑜𝑝𝑒 𝑆𝐵

𝑚𝑡𝑒𝑠𝑡×

𝑅𝑆𝐵

𝑅𝐵𝐶

Where:

Wx = Mass fraction of carbamazepine in test portion (ng/kg)

WZ = Mass fraction of carbamazepine solution prepared (ng/kg)

mnative CB = Mass of native carbamazepine solution added to calibration blend (g)

misotope CB = Mass of isotopically labelled carbamazepine added to calibration blend (g)

misotope SB = Mass of isotopically labelled carbamazepine added to sample blend (g)

𝑚test = m test = Mass of test portion (g)

𝑅SB = Peak area ratios, native/labelled, in the sample blend

𝑅BC = Peak area ratios, native/labelled, in calibration blend

Estimation of uncertainties for each factor

x u u/x u/x2

ng/kg

g

g

g

g

0.00219684

12 u

25 U (k=2)

WHERE 9 Rel U

Wz x u u/x u/x2

g

g

90.259 ng/kg

Wz

[native] solution used to

prepare calibration blend (ng/ kg)

7821 90.258822

(calculated below) 0.011541102 0.000133197

mnativeCB

weight native solution added to calibration blend (g)

0.108 0.000100

(u of mass balance) 0.000927816 8.60842E-07

misotopeSB

weight of Isotope solution added to sample

(g) 0.095

0.000100 (u of mass balance)

0.001053408 1.10967E-06

misotopeCB

weight of Isotope solution added to

calibration blend (g) 0.095

0.000100 (u of mass balance)

0.001056859 1.11695E-06

mtest weight of sample analysed

3.022 0.000100

(u of mass balance) 3.30897E-05 1.09493E-09

RsB

ratio of peaks areas of

native/ labelled in the repeat samples

0.743 0.0059

(ESDM across ratio of 4 repeat measurements) 0.007948421 6.31774E-05

RBC

ratio of peaks areas of

RM native/ labelled in the calibration blend - repeat injects

0.794 0.0339

(ESDM across ratio of 4 injections of Cal blend)

0.042717415

0.001824778

Precision Repeat measurements (13)

262 3.4405 0.013137786 0.000172601

44

7. GLHK




𝐶𝑥 = 𝐷 × 𝐶𝑧 × 𝑀𝑧𝑐 ×𝑚𝑦

𝑚𝑦𝑐×

𝑅𝐵

𝑅𝐵𝐶/𝑀𝑥

D = Factor of repeatability (Assume =1)

Cz = Mass fraction of carbamazepine in calibration blend (ng/kg)

My = Mass of carbamazepine –d10 added to the sample blend (ng)

Mx = Mass of sample (g)

Mzc = Mass of carbamazepine standard solution added to the calibration blend (g)

Myc = Mass of carbamazepine –d10 added to the calibration blend (g)

RB = Isotope amount ratio in sample blend

RBC = Isotope amount ratio in calibration blend




Value x

Standard uncertainty

u(xi)

Relative standard

uncertainty u(xi)/xi

Contribution to

total uc (%)

cz (ng/g) 3646.27 20.1003 0.0055 8.40

my (g) 0.20203 0.0001 0.0005 0.07

mx (g) 2.99982 0.0001 0.0000 0.00

myc (g) 0.94213 0.0001 0.0001 0.00

mz (g) 0.98298 0.0001 0.0001 0.00

Rb 0.999 0.0073 0.0073 14.61

Rbc 1.008 0.0093 0.0092 23.13

D 1.00 0.0140 0.0140 53.79

cx,i 256.83 ng/g

uc(cx,i) 4.83 ng/g

U (k=2) 9.66 ng/g

45

8. NIM

The measurement equation(s) 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.

𝐶𝑥 =𝑚2

𝑚′2×

𝑚′1𝑀

×𝑅1

𝑅2

where Cx is the mass fraction of the carbamazepine in the water, m2 is the mass of

the labeled carbamazepine added to the sample, m2' is the mass of the labeled

carbamazepine added to the calibration solution, m1' is the mass of the carbamazepine

added to the calibration solution, M is the mass of the water sample, R1 is the measured

ratio (peak area of carbamazepine/peak area of labeled carbamazepine) of the spiked sample,

and R2 is the average measured ratio (peak area of carbamazepine standard/peak area of

labeled carbamazepine) of the calibration solutions.


were obtained and combined to calculate the overall uncertainty. Please provide a table detailing

the full uncertainty budget.

Factors Uncertainty

Solution Preparation 0.90%

Weighing of Samples 0.06%

Purity 0.35%

Method Precision (repeatability) 0.85%

combined uncertainty 1.3%

Expanded combined uncertainty 2.6%

Expanded Uncertainty (ng/kg) 6.4

46

CCQM-K126 Appendix II: CCQM OAWG: Core Competency Table for Analyte(s) in Matrix

CCQM-K126 BAM

(KCRV)

Low Polarity Organic in Water:

Carbamazepine in Surface Water Scope of Measurement: This study provides the means for assessing measurement capabilities for

determination of low polarity measurand in a procedure that may require extraction, clean-up, analytical separation, and selective detection in water. Generally, it provides demonstration of a laboratory’s capabilities

in determining the mass fraction in range from 50 to 2000 ng/kg of pharmaceutical residue of low molecular

weight analytes (mass range 100-500 a.m.u.) and having low polarity (pKOW <-2) in aqueous matrix.

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?

Carbamazepine, Aldrich, Lot: SLBH 2762V

Purity: 99.375 % Identity verification of analyte(s) in

calibration material.#

estimated by 1H-qNMR

For calibrants which are a highly-pure substance: Value-Assignment / Purity

Assessment method(s).#

estimated by 1H-qNMR, reference: Benzoic acid

NIST SRM 350b

For calibrants which are a calibration

solution: Value-assignment method(s).#

N/A

Sample Analysis Competencies Identification of analyte(s) in sample The determination was performed by an ELISA

(heterogeneous, competitive enzyme

immunoassay) performed in microtiter plates

using commercially available monoclonal anti-

carbamazepine antibodies and an enzyme tracer

prepared from horseradish peroxidase and a

carbamazepine derivate carrying a triglycine

spacer.

Extraction of analyte(s) of interest from matrix

N/A

Cleanup - separation of analyte(s) of

interest from other interfering matrix components (if used)

N/A

Transformation - conversion of analyte(s)

of interest to detectable/measurable form (if used)

N/A

Analytical system ELISA, modular workstation

Calibration approach for value-assignment of analyte(s) in matrix

a) calibration with external standard b) multi-level calibration curve (sigmoid calibration

curve)

Verification method(s) for value-

assignment of analyte(s) in sample (if

used)

N/A

Other N/A

47

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

CCQM-K126 BAM Low Polarity Organic in Water:





Competency

Tick,

cross,

or

“N/A”




Carbamazepine, Aldrich, Lot: SLBH 2762V

Purity: 99.375 % Identity verification of analyte(s) in


estimated by 1H-qNMR



estimated by 1H-qNMR, reference: Benzoic acid

NIST SRM 350b



N/A

Sample Analysis Competencies Identification of analyte(s) in sample Retention time (LC) and multiple reaction monitoring

(MS/MS)


N/A


interest from other interfering matrix

components (if used)

N/A


of interest to detectable/measurable form

(if used)

N/A

Analytical system LC – MS/MS


a) calibration with internal standard ( CBZ-d2 ) b) multi-level calibration curve


assignment of analyte(s) in sample (if used)

N/A

Other N/A

48


CCQM-K126 EXH M Low Polarity Organic in Water:


determination of low polarity measurand in a procedure that may require extraction, clean-up, analytical

separation, and selective detection in water. Generally, it provides demonstration of a laboratory’s capabilities in determining the mass fraction in range from 50 to 2000 ng/kg of pharmaceutical residue of low molecular


Competency

Tick,

cross,

or

“N/A”




carbamazepine

Identity verification of analyte(s) in


qNMR



qNMR



gravimetric

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


N/A

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


N/A

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

(if used)

N/A

Analytical system LC-MS/MS


IDMS, single-point calibration, matrix matched


assignment of analyte(s) in sample (if used)

standard additions

Other N/A

49


CCQM-K126 HSA Low Polarity Organic in Water:





Competency

Tick,

cross,

or

“N/A”




High purity reference standard of carbamzepine from

the United States Phamacopeial Convention (USP)

was used as the calibrant. The purity of the calibrant

was determined from in-house purity assessment

using mass balance approach



Qualitative 1H NMR was performed to verify the

identity of the calibration material and [M+H]+ ion

using LC-MS/MS.



The purity of the carbamazepine reference standard

from USP was assessed using mass balance

approach. The four classes of the impurities namely,

the structurally related organic impurities, water,

residual solvent and inorganic impurities were

determined by LC-DAD, Karl-Fisher coulometry,

GC-MS and thermogravimetric analysis,

respectively.



N/A

Sample Analysis Competencies Identification of analyte(s) in sample The analyte in the sample was identified against two

sources of carbamazepine reference standards (from

USP and Sigma Aldrich) by comparing their MRM

transition and retention time on the LC-MS/MS.

Their retention time was the same even when two

columns with different properties (RX-SIL normal

phase and SB-Aq reverse phase) were used. The

identity of the carbamazepine reference standards

from USP and Sigma-Aldrich were in turn confirmed

by 1H NMR spectroscopy.


N/A

50



The bottle was warmed up to ambient room

temperature (18 – 25 oC), thoroughly vortexed,

sampled, then spiked with appropriate amount of

isotope labelled internal standard. The resulting

sample blends were vortexed and allowed to stand at

4 oC for at least 2 hours for equilibration. The sample

blends were vortexed thoroughly again before LCMS/

MS measurement.



N/A

Analytical system AB Sciex Qtrap® 5500 MS/MS instrument coupled

with Shimadzu Prominence UFLC XR LC system.


Four-point calibration curve IDMS method was used

for the quantification of carbamazepine in surface

water. Four calibration blends with isotope ratios

being close to 0.8, 0.9, 1.1, and 1.2 were prepared,

and the isotope ratio in the sample blends were

controlled to be close to 1.0.



used)

Carbamazepine reference standard from Sigma

Aldrich was purity assessed by quantitative 1H NMR,

and was used to spike into filtered surface water

collected in Singapore. The spiked material was used

as quality control sample. The quality control sample

was measured together with the comparison sample.

Other N/A

51


CCQM-K126 LNE Low Polarity Organic in Water:


determination of low polarity measurand in a procedure that may require extraction, clean-up,

analytical separation, and selective detection in water. Generally, it provides demonstration of a

laboratory’s capabilities in determining the mass fraction in range from 50 to 2000 ng/kg of

pharmaceutical residue of low molecular weight analytes (mass range 100-500 a.m.u.) and having

low polarity (pKOW <-2) in aqueous matrix.

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? Carbamazepine LGC MM0076.00



Mass spectrum, abundance of characteristics ions

(transitions), comparison with the bibliography



q NMR



N/A

Sample Analysis Competencies Identification of analyte(s) in sample Retention time, specific transitions and ratio of specific

transitions

Extraction of analyte(s) of interest from

matrix Solid Phase Extraction


interest from other interfering matrix


N/A


of interest to detectable/measurable form

(if used)

N/A

Analytical system LC/ESI/MSMS

Calibration approach for value-assignment

of analyte(s) in matrix a) quantification mode used : IDMS

b) calibration mode used: 7 points calibration curve



used)

N/A

Other N/A

52


CCQM-K126 NIMT Low Polarity Organic in Water:





Competency

Tick,

cross,

or

“N/A”




Pure carabamazepine powder was obtained from Dr.

Ehrenstorfer GmbH Identity verification of analyte(s) in


LC-MS/MS



Mass balance( HPLC-PDA, TGA, KFT)



N/A

Sample Analysis Competencies Identification of analyte(s) in sample LC-MS/MS (Chromatographic retention, MRM mode

with two ion pairs for identification)


N/A



VertiPak™ HCP-SC SPE Cartridges

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

(if used)

N/A



a) bracketed exact- matching IDMS, b) Single-point calibration

c) Matrix-matched calibration blend



used)

N/A

Other N/A

Remarks:

The DoE result for NIMT for carbamazepine does not cross zero. The reported value was not consistent

with the KCRV. No specific competency in the Table above was identified as the reason.

53


CCQM-K126 NMISA Low Polarity Organic in Water:





Competency

Tick,

cross,

or

“N/A”




USP Reference standard Lot K1K246 highly

pure substance. In-house purity determination



LC-UV, LC-MS and LC-MS/MS



LC-UV quantification of structurally related impurities.

Coulometric Karl Fischer (direct insertion) for water content

determination. Headspace GCTOFMS for residual solvent

determination.



N/A

Sample Analysis Competencies Identification of analyte(s) in sample Mass transitions and ratios and retention time


N/A


interest from other interfering matrix components (if used)

Solid phase extraction



N/A



Single point and bracketing IDMS

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

used)

N/A

Other N/A

54


CCQM-K126 GLHK

Low Polarity Organic in Water:





Competency

Tick,

cross,

or

“N/A”


Competencies for Value-AssignmentofCalibrant


“Pure material” Produced by the U.S. Pharmacopeial Convention

Identity verification of analyte(s)in


IR, MS and tandem MS.

For calibrants which area highly-pure substance: Value-Assignment/Purity


Mass balance approach. - Organic impurity: HPLC-UV - Water/ moisture: Karl Fischer - Residual solvent: Headspace GCMS - Inorganic impurities: ICP MS and ion

chromatography

For calibrants which area calibration


SampleAnalysis Competencies Identification of analyte(s) in sample Retention time and the MRM transition ratio


Sonication and vortex

Cleanup- separation of analyte(s)of

Interest from other interfering matrix components(if used)

Liquid chromatographic separation

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

(if used)

NA


Calibration approach for value-assignment of analyte(s)in matrix

a) IDMS b) Bracketing

Verification method(s) for value- Assignment of analyte(s) in sample

(if used)

The samples were tested on 2 columns namely a Phenomenex Luna Phenyl-Hexyl and Waters Atlantis T3 columns.

Besides, the calibration approach was verified by using 5-point calibration curve as well as gravimetric standard addition method.

Other

55


CCQM-K126 NIM Low Polarity Organic in Water:





Competency

Tick,

cross,

or

“N/A”


Competencies forValue-Assignment of Calibrant


We used a “pure material”, purchased from USP.

Identity verification of analyte(s)in


√ We used LC-MS and NMR to identify analyte(s)

For calibrants which area highly-pure substance: Value-Assignment/Purity


√ Mass balance(HPLC-UV, Karl Fischer, ICP-

MS and HS-GC), and q-NMR,

For calibrants which area calibration


N/A

SampleAnalysis Competencies Identification of analyte(s) in sample

√

Retention time, mass spec ion ratios


N/A

Cleanup- separation of analyte(s)of

Interest from other interfering matrix

components(if used)

√

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

√

a) quantification mode is IDMS with internal standard b) calibration mode is single-point calibration


Assignment of analyte(s) in sample (if used)

N/A

Other N/A

56

Final Report CCQM Track C Key Comparison (CCQM-K126) · 2017. 9. 18. · Final Report CCQM Track C...

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