European interlaboratory comparison of breath ¹³CO₂ analysis

Abstract

The BIOMED I programme Stable Isotopes in Gastroenterology and Nutrition (SIGN) has focused upon evaluation and standardisation of stable isotope breath tests using ¹³C labelled substrates. The programme dealt with comparison of ¹³C substrates, test meals, test conditions, analysis techniques, and calculation procedures. Analytical techniques applied for ¹³CO₂ analysis were evaluated by taking an inventory of instrumentation, calibration protocols, and analysis procedures. Two ring tests were initiated measuring ¹³C abundances of carbonate materials.
Evaluating the data it was found that seven different models of isotope ratio mass spectrometers (IRMS) were used by the participants applying both the dual inlet system and the continuous flow configuration. Eight different brands of certified ¹³C reference materials were used with a ¹³C abundance varying from δ¹³C_PDB−37.2 to +2.0 ‰. CO₂ was liberated from certified material by three techniques and different working standards were used varying from −47.4 to +0.4 ‰ in their δ¹³C_PDB value.
The standard deviations (SDs) found for all measurements by all participants were 0.25 ‰ and 0.50 ‰ for two carbonates used in the ring tests. The individual variation for the single participants varied from 0.02 ‰ (dual inlet system) to 0.14 ‰ (continuous flow system). The measurement of the difference between two carbonates showed a SD of 0.33 ‰ calculated for all participants. Internal precision of IRMS as indicated by the specifications of the different instrument suppliers is <0.05 ‰ for dual inlet systems and <0.3‰ for continuous flow systems. In this respect it can be concluded that all participants are working well within the instrument specifications even including sample preparation. Increased overall interlaboratory variation is therefore likely to be due to non-instrumental conditions. It is possible that consistent differences in sample handling leading to isotope fractionation are the causes for interlaboratory variation. Breath analysis does not require sample preparation. As such, interlaboratory variation will be less than observed for the carbonate samples and within the range indicated as internal precision for continuous flow instruments. From this it is concluded that pure analytical interlaboratory variation is acceptable despite the many differences in instrumentation and analytical protocols. Coordinated metabolic studies appear possible, in which different European laboratories perform ¹³CO₂ analysis. Evaluation of compatibility of the analytical systems remains advisable, however.



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Figure 1 .

Overall test results of ¹³C abundance measurements in CO₂ liberated from two carbonate sources (BG03 N and BG02 K) (n=9). 


Figure 2 .

Interlaboratory comparison of ¹³C abundance measurements of CO₂ liberated from two carbonate sources (BG03 N and BG02 K): (A) individual δ¹³C_PDB values and (B) difference between the values for carbonates 1 and 2.