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Indian Pediatr 2011;48: 656-657


T Velpandian and AK Deorari

Email: ashokdeorari_56@hotmail.com

We thank the reader for the interest shown in our article published recently in the Journal (Epub). The questions raised by the reader are indeed valid. However, they would probably not have arisen if all our original figures and the text were retained in the final published article (the figures have to be removed because of space constraints). Nevertheless, we have tried to address the concerns here:

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.


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