April 2014
Volume 55, Issue 13
ARVO Annual Meeting Abstract  |   April 2014
Validation of a method for determination of cyclosporine in rabbit tissues in a pharmacokinetic study
Author Affiliations & Notes
  • Hai Tang
    shenyang sinqi, Shenyang, China
  • Footnotes
    Commercial Relationships Hai Tang, None
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Investigative Ophthalmology & Visual Science April 2014, Vol.55, 3691. doi:
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      Hai Tang; Validation of a method for determination of cyclosporine in rabbit tissues in a pharmacokinetic study. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3691.

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      © ARVO (1962-2015); The Authors (2016-present)

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To validate a simplified liquid chromatograph coupled with tandem mass spectrometry (LC-MS-MS) method with sensitivity and specificity for determination of Cyclosporine A (CSA) in rabbit tissues and application to the pharmacokinetic studies.


A 50μl of 0.05% CSA was instilled into the left eyes of 20 rabbits and the animals divided evenly into four groups. From each group, tear specimens were collected at 0.5, 2, 12, 24 hours and the animals sacrificed immediately to collect aqueous humor from the anterior chamber, and subsequently the cornea and conjunctival tissues. CSA and internal standard (IS, Etofesalamide) were extracted from specimens by protein precipitation with methonal. Chromatographic separation was carried out on a Diamonsil C8 column with a mobile phase of methanol-water (including 25mM ammonium acetate) using gradient elution. Mass spectrometric detection was achieved by a triple-quadrupole mass spectrometer equipped with an ESI interface operating in positive ionization mode. Quantitation was performed using multiple reaction monitoring (MRM) of precursor-product ion transition at m/z 1219.8→m/z 1202.8 for CSA.


The LC-MS-MS method allowed CSA quantification as low as 1.0 ng/ml (R>0.99) with a sample run time of 10 minutes, for up to 24 hours after a single administration of 0.05% CSA to the eye. The highest concentration of CSA found was at 0.5 hour in tear (8988.8 ng/ml), and at 2 hours in cornea and conjunctiva (513.6 ng/ml and 451.2 ng/ml, respectively). The CSA levels reduced to 449 ng/ml, 420.6 ng/ml and 391.4 ng/ml at 24 hour, respectively. The elimination rates of the CSA in cornea and conjunctiva were much lower than that in the tear. CSA levels were not detectable in aqueous humor (< 1 ng/ml) at all time points.


Our simplified method achieved the required sensitivity and specificity for a pharmacokinetic study in rabbit specimens after a topical administration of CSA. Lack of detectable level of CSA in the aqueous humor most likely is due to the highly lipophilic nature of CSA. Corneal stroma, a highly hydrophilic layer, is an impermeable barrier to the absorption of CSA into the aqueous humor. The presence of high level CSA in tear, conjunctiva and cornea 24 hours after topical administration suggests that a once a day dosing should be adequate for the treatment of ocular surface inflammation seen in keratoconjunctivitis sicca.

Keywords: 496 detection  

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