Analysis of Caffeine by Immunoassay - Caffeine: Chemistry, Analysis, Function and Effects - page 219

previously diluted 1 : 50 000 in assay buffer. The plate is incubated for 30
min under the same conditions as mentioned for the antibody;

N

Plate washing (3x) with washing buffer;

N

Substrate solution is added into the plate (200 mL per well) and incubated
for 30 min. A blue color gradation should be observed within the plate: dark
blue

d n 0 t 2 n g | 3

for the blanks and fading according to caffeine concentration

increments;

N

Sulfuric acid 1 M solution added (100 mL per well) producing a yellow-
colored solution in the wells;

N

Spectrophotometric read-out at two fixed wavelengths: 450 nm and 620 nm;

N

Plotting caffeine concentration versus OD signal difference 450 nm-620 nm
and fitting the sigmoidal curve to the calibrators using a 4-parameter
logistic model (Rodbard 1974).

12.2.3 Quantifying Caffeine in Beverages, Tablets and Shampoo

12.2.3.1 Sample Pre-treatment
Samples containing CO 2 must be degasified before analysis. Briefly, 5 mL of
the sample can be transferred into a 10 mL glass tube and degassed by
immersing a metal tube dispensing argon into the sample for some minutes.
Coffee and tea can be used without any pre-treatment but they must be diluted
in ultrapure water so their caffeine concentration will fall within the
immunoassay calibration range. Caffeine in caffeine-containing tablets has
to be brought into solution prior to analysis. A typical protocol encompasses a
3 hour dissolution of the tablets in 1 liter of acidified (pHy3) ultrapure water,
under stirring (Carvalho et al 2010).
The shampoo samples should be dissolved in ultrapure water prior to
analysis. For shampoos containing about 5 g L 21 caffeine, a dilution of 0.5 g
shampoo in a liter of ultrapure water is suggested.
Because the assay is measuring caffeine concentration in the mgL 21 range,
samples containing caffeine in the mg L 21 or even g L 21 range have to be
diluted. Caffeine-free beverages like Coca-Cola 1 caffeine-free and some
intensely colored fruit infusions, e.g. raspberry tea, can also be measured after
a 10-fold dilution. Thus, a caffeine concentration under 0.010 mg L 21

(10 x

LOQ) can be attributed to such samples (Carvalho et al 2010).

12.2.3.2 Determining Caffeine Concentration
Depending on the accuracy and the precision desired, or legally required, the
number of standards to be used, as well as the number of replicates should be
established by the analytical laboratory or industrial plant. For the results
discussed within this chapter, standards ranging from 1 to 100 mgL 21 caffeine
in ultrapure water were used (1, 1.5, 2.5, 5, 10, 15, 25, 50, 75 and 100 mgL 21 ),
assayed in triplicate, the same as for the samples. A typical calibration curve is