Analysis of Caffeine by Immunoassay - Caffeine: Chemistry, Analysis, Function and Effects - page 222

Table 12.2 Key facts for the division ''Immunoanalytics''.

N Division ''Immunoanalytics'' is a division within the Department of Analytical
Chemistry; Reference Materials of BAM Federal Institute for Materials Research
and Testing; Head of Division: Dr. Rudolf J. Schneider.
N The group is dedicated to the development of immunoanalytical methods, especially
immunoassays.
N The group develops applications for enzyme-linked immunosorbent assay (ELISA)
in the fields of food analysis, environmental monitoring and in vitro diagnostics
with a focus on quality assurance and uncertainty assessment for these methods.
N We developed an ELISA for caffeine based on a commercial monoclonal antibody.
Enzyme conjugates will be available in the BAM webshop http://
www.webshop.bam.de/.

d n 0 t 2 n g | 3

Table 12.3 Cross-reactivity of the immunoassay with caffeine related

compounds.

Substance CAS number Cross-reactivity

Caffeine (1,3,7-trimethylxanthine) 58-08-02 100%

Caffeine-trimethyl- 13 C 78072-66-9 93%

Theophylline (1,3-dimethylxanthine) 58-55-9 12%

Theobromine (3,7-dimethylxanthine) 83-67-0 0.13%

Paraxanthine (1,7-dimethylxanthine) 611-59-6 0.08%

Xanthine 69-89-6 , 0.002%

CR describes the cross-reactivity in percent; C caffeine is a parameter of the
4PL giving the caffeine concentration at the inflection point and C Test refers to
the concentration of the cross-reacting compound at its inflection point. The
results are summarized in Table 12.3.
The assay shows very low cross-reactivity overall, theophylline being the
only cross-reactant worth mentioning. Samples containing theophylline in
equal or higher concentration than caffeine are an obvious limitation of the
assay. For concentrations of theophylline 10-fold below caffeine, for example,
an overestimation of only 1.2% of the final concentration is expected and when
ratios of caffeine : theophylline are higher than 10, the assay can be used
without major concern.

12.2.5 Comparing the Results with an LC-MS/MS Method
The samples were analyzed on the same day by immunoassay and by LC-MS/
MS and the results obtained by both methods compared as presented in
Figure 12.4. Moreover, plotting caffeine concentration by immunoassay vs.
caffeine concentration by LC-MS/MS produces a regression line with a slope
of 1.004 and a correlation coefficient of 0.9946.