Choice of anticoagulant and Comparison of counter ionsPart II. Counter ion comparison Study design...

Choice of anticoagulant

and

Comparison of counter ions

- Results from two EBF surveys

Presenter: Carl Sennbro on behalf of EBF

Presented at: EBF open meeting , 02 December 2010, Barcelona

http://www.europeanbioanalysisforum.eu 2 02-Dec-2010

Aim

To present results from two EBF surveys

Part I: Anticoagulant mini-survey

– Overview of the policy on and choice of anticoagulant within EBF companies

Part II: Counter ion data comparison

– Collection of data in order to compare LC-MS assay performance using different counter ions


Mini-survey design Part A: General questions on policy for usage of anticoagulants and

counter ions within various bioanalytical issues

Part B. Choice of anticoagulant in various applications

Part I. Anticoagulant mini-survey

Outcome

Survey distributed in September – 2009

Responder rate 25/27 companies

Results presented and discussed at EBF closed meetings 2010

Aim To map the company policy on usage of anticoagulants and counter ions

To map the company choice of anticoagulant


Conclusions I – anticoagulant policy

Calibrator/QC samples in Method validation versus Method application

– Most companies (21/25) do not allow different anticoagulants

– Most companies (18/25) do allow different counter ions (same

anticoagulant)

Study samples versus Calibrator/QC samples

– Most companies (21/25) do not allow different anticoagulants

– Most companies (16/25) do allow different counter ions (same

anticoagulant)

10 of 25 companies cover these issues in an SOP.



Conclusions II – anticoagulant choice

EDTA is the most commonly used anticoagulant


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6

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12

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20

EDTA Heparin

Clinical studies

16

9

0

2

4

6

8

10

12

14

16

EDTA Heparin

Preclinicalstudies

In clinical studies:

For one company – different preferences in Europe vs US

For two companies – the choice is project dependent

For all companies:

Same choice in non-regulated studies


Conclusions III – anticoagulant choice

The rationale for the choice of anticoagulant


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20

Practical Scientific Econonic Other

Rationale

Main specified rationale:

Less problems with clotting using EDTA

Sadagopan et al., Rapid Comm Mass Spectrom

2003;17;1065


EDTA Heparin

Ethylenediaminetetraacetic acid

Commercially synthesized

Chelating agent

Mw 292 g/mol

Various salts available

Variably-sulfated glycosaminoglycan

Extracted from mammals (porcine)

Medical agent

Mw 12-15 kDa (average)

Various salts available

n


Part II. Counter ion comparison

Study design To analyze one batch of spiked Calibrator/QC plasma samples including

– Calibration curve in plasma with validated anticoagulant and counter ion

– One set of QCs in plasma with validated anticoagulant and counter ion

– One set of QCs in plasma with validated anticoagulant but different counter ion

– 2-3 levels of QCs, 3-6 replicates of each level

– Results delivered as calculated Precision and Accuracy

Each company to provide experimental data for at least one compound

– Also, description of the assay and analyte(-s) delivered

Pair-wise comparison of calculated precision and accuracy

Aim To compare experimental data on counter ion impact in LC-MS assays


Conclusions

Results within acceptance criteria regardless of counter ion and anticoagulant

No significant differences (neither statistical nor practical) for Precision nor Accuracy

No specific parameter (e.g. sample prep, internal standard, etc) affected the results




Summary of assay and analyte data Exclusively LC-MS/MS assays (n=42) for 34 compounds

Stable isotope labelled IS for 32 of 42 assays

Species: Human (n=32), rat (n=5), dog (n=4), primate (n=1)

Sample preparation techniques

– PP, SPE, LLE, DBS, Derivatization, Hydrolyzation

Mw = 206 – 1069 Da (average 461 Da)

Analyte type: acids (n=7), neutrals (n=6), bases (n=12), unspecified (n=9)

Log P 1.1 – 7.1 (average 3.9)

Survey outcome

Survey distributed in June – 2009

Responder rate 15/27 companies

Results presented and discussed at EBF closed meetings 2010


Precision & Accuracy data for comparison

Data for EDTA counter ion comparison; 43 pairs of measurements for 15 compounds

For Heparin counter ion comparison; 51 pairs of measurements for 15 compounds

-----------------------------------------------------------------------

Data also included comparison of different anticoagulants: 21 pairs of measurements for 9 compounds – data not shown in this presentation



Results, Precision EDTA

Pair-wise comparison (n=43)

All measurements within acceptance criteria of 15%

0.0

5.0

10.0

15.0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43

Measurement No

CV

(%

)

CV (%) validated ion

CV (%) other ion

EDTA


-15.0

-10.0

-5.0

0.0

5.0

10.0

15.0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43

Measurement No

CV

(%

) v

ali

da

ted

io

n -

CV

(%

) o

the

r io

n CV (%) validated ion - CV (%) other ion

EDTA

Results, Precision difference EDTA

EDTA. Paired t-test: p = 0.060

Average diff: - 0.7 %

Values close to 0 (zero) indicate no difference between counter ions

Negative values indicate worse precision for non-validated ion


Results, Accuracy bias EDTA

Pairwise comparison (n=43)

All measurements within acceptance criteria +/- 15%

-15.0

-10.0

-5.0

0.0

5.0

10.0

15.0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43

Measurement no

Bia

s (

%)

Bias (%) validated ion

Bias (%) other ion

EDTA


Results, Accuracy bias difference EDTA

EDTA. Paired t-test: p = 0.31

Average diff: + 0.6 %

-15.0

-10.0

-5.0

0.0

5.0

10.0

15.0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43

Measurement No

Bia

s (

%)

vali

date

d io

n -

Bia

s (

%)

oth

er

ion Bias (%) validated ion - Bias (%) other ion

EDTA




Results, Precision Heparin


All measurements within acceptance criteria of 15%

0.0

5.0

10.0

15.0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51

Measurement No

CV

(%

)

CV (%) validated ion

CV (%) other ion

Heparin


Results, Precision difference Heparin

Heparin. Paired t-test: p = 0.26

Average diff: + 0.5 %

-15.0

-10.0

-5.0

0.0

5.0

10.0

15.0

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51

Measurement No

CV

(%

) v

ali

da

ted

io

n -

CV

(%

) o

the

r io

n

CV (%) validated ion - CV (%) other ion

Heparin




Results, Accuracy bias Heparin


96 % of measurements within acceptance criteria +/- 15%

-15.0

-10.0

-5.0

0.0

5.0

10.0

15.0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51

Measurement no

Ac

cu

racy

bia

s (

%)

Bias (%) validated ion

Bias (%) other ion

Heparin19.7 20.3 16.5 15.8


Results, Accuracy bias difference Heparin

Heparin. Paired t-test: p = 0.15

Average diff: - 0.9 %

-15.0

-10.0

-5.0

0.0

5.0

10.0

15.0

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51

Measurement No

Accu

racy 1

- A

ccu

racy 2

Bias (%) validated ion - Bias (%) other ion

Heparin




Conclusions, counter ion comparisons

Results within acceptance criteria regardless of counter ion and anticoagulant

No significant differences (neither statistical nor practical) for Precision nor Accuracy

No specific parameter (e.g. sample prep, internal standard, etc) affected the results

Results in compliance with previous findings by Bergeron et al (2009); Bioanalysis 1(3); 537-48.

These observations justify an EBF suggestion/recommendation


Main conclusion, counter ion comparisons

EBF suggestion/recommendation

In regulated bioanalysis, collected plasma samples containing different counter ions but same anticoagulant may be regarded as equal matrices


Acknowledgement

Magnus Knutsson, Ferring A/S

Peter van Amsterdam, Abbott

All contributing EBF companies


Back-up slide 1a: Different sample preps

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-6.00

-4.00

-2.00

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10.00

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Measurement No

% P

recis

ion

1 -

Pre

cis

ion

2

Protein Prec SPE LLE DBS

Average CV Difference

PP: -0.6 % SPE: +0.1 % LLE: -0.0 % DBS: +0.8 %


Back-up slide 1b: Different sample preps

-15.00

-10.00

-5.00

0.00

5.00

10.00

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65 67 69 71 73 75 77 79 81 83 85 87 89 91 93

Measurement No

% A

cc

ura

cy

bia

s 1

- A

ccu

racy

bia

s 2

_

Protein Prec SPE LLE DBS

Average Bias Difference

PP: -0.9 % SPE: +1.1 % LLE: -1.3 % DBS: -0.6 %


Back-up slide 2: Internal standard

Compared assays with or without SIL IS.

CV difference

With SIL IS +0.7 %

Without SIL IS -0.3 %

Bias difference

With SIL IS -0.4 %

Without SIL IS -0.1 %


Back-up slide 3a: PLS model,

-2.5

-2.0

-1.5

-1.0

-0.5

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0.5

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-4.0 -3.5 -3.0 -2.5 -2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

t[2]

t[1]

Evaluation_Heparin_data_survey_20101119.M5 (PLS), PLS prec diff

t[Comp. 1]/t[Comp. 2]

Colored according to Obs ID (Sample prep)

R2X[1] = 0.234553 R2X[2] = 0.103526

Ellipse: Hotelling T2 (0.95)

DBS

LLE

PP

PP + SPE

SPE

SIMCA-P+ 12.0.1 - 2010-11-19 10:35:21 (UTC+1)


Back-up slide 3b: PLS model

Choice of anticoagulant and Comparison of counter ionsPart II. Counter ion comparison Study design...

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