1. The major outcome variable of our study was the
amount of native bilirubin left over after exposure to light. We
consciously avoided using the amount of isomers formed as the primary
outcome variable as we did not characterize them. We therefore used the
term photoconversion rather than photodegradation or photoisomerization.
On the other hand, we would also like to point out that the technique
used by us (LC-MS/MS in a highly efficient Multiple Reaction Monitoring
mode [MRM] along with hydrophilic interaction chromatography) separates
bilirubin from its isomers having similar molecular weights. So, we do
not agree with the reader’s comment that we ‘focused largely on
photodegradation and not photoisomerization’.
2. We agree with the reader that the photochemistry
of bilirubin in organic solvents could be different from serum/aqueous
albumin solutions. Still for the comparative evaluation of different
light sources under controlled experimental conditions, we opted for the
methanolic solution of bilirubin at the concentration of 1µg/ml because
of the following factors: (a) lack of aqueous solubility of
bilirubin (b) concerns over availability of unbound fraction of
bilirubin from plasma for photoreactions and (c) the risk of
interferences in estimation by the biomatrix. Usage of organic solvents
for water insoluble drugs for photodegradation analysis is not uncommon.
For the preparation of stock concentration of bilirubin, dilute ammonia
solution of methanol was used and it was serially diluted to reach the
concentration of 1µg/mL with methanol.
3. It is true that over-irradiated samples are
capable of producing more and more photoconversion products. However,
the method adopted by us for determination of bilirubin concentration
(LC-MS/MS) is the gold standard for measuring compounds with higher
precision. As it is quantifying the compounds based on their molecular
weight, color of the compound is immaterial. The standard methanolic
bilirubin appearing at 1.23 min and the formation of a photoisomer
product at 1.9 min can very well be seen in the accompanying web
figure. Moreover, we have used more time points for quantification.
4. We have shown the separation of peaks within the
period of 3 min in LC-MS/MS using the method reported in the manuscript
(Figure available on request). For in vivo quantification process
(ongoing study), the method was optimized to include the extraction
solvent with an internal standard in the composition of 70% acetonitrile
containing 0.1% formic acid. Therefore, the same method was adopted for
this in vitro study. From the observed data using the method, it is
convincing that the photoisomers formed and survived the experimental
conditions. However, we did not isolate any photoisomer for further
characterization. Further studies are in progress to isolate and
characterize the photoisomers for their quantification in vivo
conditions.