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Quantitative LC Analysis of Cyclosporine A in Ocular Samples

Article  in  Chromatographia · September 2011

DOI: 10.1007/s10337-011-1963-0

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Chromatographia (2011) 73:817–821
DOI 10.1007/s10337-011-1963-0

LIMITED SHORT COMMUNICATION

Quantitative LC Analysis of Cyclosporine A in Ocular Samples

Bizhan Malaekeh-Nikouei • Toka Banaee •
Javad Aghamohammadian • Navid Mosallaei •

Seyed Ahmad Mohajeri

Received: 14 November 2010 / Revised: 17 January 2011 / Accepted: 4 February 2011 / Published online: 25 February 2011
Ó Springer-Verlag 2011

Abstract An isocratic reversed-phase HPLC method The method is therefore suitable for analysis of CyA in
with ultraviolet detection at 205 nm has been developed for ocular samples.
analysis of cyclosporine A (CyA) in rabbit ocular samples.
Neither internal standard nor extraction was needed for Keywords Column liquid chromatography  Ocular
sample preparation. Acetonitrile (ACN; 1 mL) was added samples  Cyclosporine A
to 250 lL aqueous and vitreous samples to precipitate
proteins. The supernatant was dried and the residue was
reconstituted in mobile phase and injected for HPLC Introduction
analysis. Chromatography was performed on an octadecyl
silane-A (ODSA) C18 (4.6 9 250 mm, 5 lm) column. The Cyclosporine A (CyA) is a vital immunosuppressive drug
column temperature was fixed at 70 °C and the mobile widely used to prevent rejection of transplanted organs and
phase was ACN 65%, methanol 20% and water 15% at a for the treatment of autoimmune diseases [1–3]. It has also
flow rate of 1.5 mL min-1. The calibration curve for CyA been used for new applications, for example reversal of
in rabbit ocular samples was linear over the concentration multi drug resistance, herpes virus infection, rheumatoid
range 0.2 and 10 lg mL-1 with a correlation coefficient of arthritis, type I diabetes, and as a potent anti-human
0.9992. Intra-day and inter-day precision were 4.61–7.83% immunodeficiency virus 1 (HIV-1) agent [4–6]. The drug
and 5.27–10.70%, respectively. Intra-day and inter-day has some serious side effects, including chronic nephro-
accuracy were 89.2–108% and 83.4–111%, respectively. toxicity, increased blood pressure, dyslipidemia, and renal
The limits of detection (LOD) and quantification (LOQ) hypoperfusion [7, 8]. The therapeutic window of CyA is
were 5.7 and 38 ng mL-1, respectively. The method was narrow and the consequences of therapeutic and toxic
successfully used for analysis of CyA in real aqueous and levels are graft rejection on the one hand and nephrotoxi-
vitreous humor samples from New Zealand albino rabbits. city on the other [9, 10]. Because of poor correlation
between dose administered and clinical response and large
inter-subject variability in the bioavailability and metabo-
lism of this drug, therapeutic monitoring of CyA is of
B. Malaekeh-Nikouei  N. Mosallaei critical importance [9, 11]. Systemically administered CyA
Nanotechnology Research Center, School of Pharmacy, penetrates the eye only if an inflammation such as severe
Mashhad University of Medical Sciences, Mashhad, Iran uveitis occurs [12]. Because of the adverse effects and low
ocular delivery of the drug after systemic administration,
T. Banaee
Khatam-al-Anbia Eye Hospital and Eye Research Center, many efforts have been made to prepare an ophthalmic
Mashhad University of Medical Sciences, Mashhad, Iran CyA delivery system which improves the efficacy of drug
in different ocular diseases [13–15]. These studies include
J. Aghamohammadian  S. A. Mohajeri (&)
the use of drug-delivery systems such as nanoparticles [13]
Pharmaceutical Research Center, School of Pharmacy,
Mashhad University of Medical Sciences, Mashhad, Iran and nanocapsules [16]. Therefore, analysis and quantifi-
e-mail: mohajeria@mums.ac.ir cation of CyA in ophthalmic fluids is very important for

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818 B. Malaekeh-Nikouei et al.

evaluation of ocular bioavailability from drug-delivery Precision and Accuracy

systems. Many papers have been published on the analysis
of CyA in biological fluids [17–20]. Analytical methods Assay precision was determined by analysis of ocular
such as HPLC [17, 21, 22], liquid chromatography–mass samples spiked with known concentrations of CyA. Drug
spectrometry [8], capillary electrophoresis [18], and concentrations were determined in batches of four on the
immunoassay [19, 23] have been reported for this purpose. same day and on different days. Intra and inter-day assay
Sample preparation and extraction is usually needed in precision was determined by calculating relative standard
analytical methods [17, 21]. deviation (RSD, %) of the results obtained.
The objective of this study was to develop a simple and Relative accuracy was determined by use of the
reliable liquid chromatographic method for quantitative equation:
analysis of CyA in aqueous and vitreous humor samples Observedconcentration
with high linearity. The method was validated by deter- Accuracy ð%Þ ¼  100
Nominal concentration
mination of the concentration of the drug in rabbit ocular
samples. Chromatographic Conditions

High-performance liquid chromatography of CyA was

Experimental performed with a Young Lin (Anyang, South Korea) Acme
9000 system, consisting of an SP930D solvent-delivery
Materials module, an SDV50A solvent-mixing vacuum degasser, a
CTS30 column oven, a UV730 dual-wavelength UV–visi-
CyA was purchased from LC Laboratories (Woburn, MA, ble detector, and an ODSA C18 (4.6 9 250 mm, 5 lm)
USA). Acetonitrile (ACN) and methanol were of HPLC- column. Data were analyzed by use of Autochro-3000
grade and obtained from Duksan (Ansan, South Korea). software supplied by Young Lin. The injection volume was
20 lL, the flow rate was 1.5 mL min-1, and the column
Preparation of Stock and Standard Solutions temperature was fixed at 70 °C. The UV detector was set to
205 nm. An isocratic method was used for chromato-
Stock solution (1 mg mL-1) of CyA was prepared by graphic analysis of CyA. The composition of the mobile
dissolving 10 mg CyA in 10 mL methanol. Standard phase was: ACN 65%, methanol 20%, and water 15%.
solutions (0.2, 0.5, 1, 2.5, 5 and 10 lg mL-1) were pre-
pared from the stock solution by dilution with mobile phase Application of Analytical Method for Ocular Samples
and a calibration curve was plotted over this concentration
range. To test the applicability of the assay method for analysis of
CyA in ocular samples, New Zealand albino rabbits were
Calibration chosen. Nanoliposomes containing CyA were prepared
according to our previous study [24]. The nanoliposomal
Standard solutions (2–100 lg mL-1) in water, used for formulation applied topically to the rabbit ocular surface
spiking calibration samples, were also prepared from stock every day. After 1 month, rabbits were killed by intrave-
solution. For calibration, 25 lL standard solutions were nous pentobarbital overdose.
added to 225 lL vitreous humor to obtain 0.2–10 lg mL-1 Both eyes were immediately enucleated, the corneas
calibration standards. The ocular samples were frozen and were cut off, the lenses were removed, the posterior cap-
stored at -20 °C. Quantification of the CyA was per- sule of the lens and anterior hyaloid face was severed, and
formed by plotting nominal CyA concentrations against the the whole of the vitreous was aspirated. The vitreous
corresponding area under UV absorption peak for spiked samples were kept at -80 °C for quantitative analysis.
samples. Before analysis, the eye samples were defrosted overnight
at 4 °C. The animal protocol was approved by the local
Sample Preparation ethics committee.

ACN (1 mL ) was added to 250 lL ocular sample in a

1.5 mL microtube to precipitate proteins. After centrifu- Results and Discussion
gation at 11,337g, 0.5 mL supernatant was dried under a
stream of nitrogen and reconstituted in 100 lL mobile Several reports have been published on HPLC analysis of
phase, and 20 lL of this solution was injected for HPLC CyA in blood or other biological fluids [8, 9, 12, 14, 21].
analysis. Most of these have focused on HPLC analysis of CyA in

123
Analysis of Cyclosporine in Ocular Samples 819

blood samples rather than ocular fluids. In previous works,

extraction was usually performed and an internal standard
was necessary to increase the precision and accuracy of the
method [9, 25]. Many of the methods used in these studies
were expensive and complicated. Sometimes the lack of
availability of an internal standard was a problem.
In this study we developed an HPLC method for anal-
ysis of CyA in ocular samples. We found that HPLC
analysis could be performed with good accuracy and pre-
cision without adding an internal standard or use of an
extraction procedure. Aqueous and vitreous humor are not
as complicated as blood. For example, the protein content
of aqueous humor is both quantitatively and qualitatively
different from that of plasma. The protein content of
aqueous humor and vitreous humor is a factor of 200 and
10, respectively, less than that of plasma [26, 27]. This
means that ocular humors are dilute aqueous solutions,
which could be a reason for not adding an internal standard
or using an extraction procedure. Thus, simple protein
precipitation was sufficient before HPLC. Sometimes low
sample volume is a problem. Most reported methods for
analysis of CyA required 1–2 mL sample [21]. In this study
the volume of some ocular samples was no more than
250 lL. Thus, we calibrated the method for a sample
volume as low as 250 lL. ACN (1 mL) was used for Fig. 1 Chromatograms obtained from a blank aqueous humor sample
protein precipitation. After centrifugation, 500 lL super- (a) and from an aqueous humor sample spiked with a standard
natant was dried and re-dissolved in 100 lL mobile phase solution of CyA (b)
before injection for HPLC analysis. This concentrating step
was performed to improve the quantification and detection
limits of the method. As described in other studies, rela- concentration that produces a signal-to-noise ratio of 3,
tively high temperature (70 °C) affected the results [9]. was 5.7 ng mL-1 and the limit of quantification (LOQ),
When the temperature is reduced the CyA peaks become defined as the concentration that produces a signal-to
wider and the resolution of the peaks decreases. Thus, we noise ratio of 20, was 38 ng mL-1. In a study by
fixed the column temperature at 70 °C. Under these con- Khoschsorur et al. [21], the LOD and LOQ for analysis of
ditions no interfering peaks were observed in the chro- CyA in whole blood were 15 and 31 ng mL-1, respec-
matogram (Fig. 1). tively. In another study, the LOQ obtained for determi-
nation of CyA in rat plasma was 50 ng mL-1 [8].
Method Validation Recovery of CyA in this calibrated analytical method was
between 77 and 101%.
Linearity
Precision and Accuracy
A calibration curve for standard solutions (y =
40.705x - 0.5018, R2 = 0.999) was plotted over the Table 1 shows the precision and accuracy of the assay for
range 0.2–10 lg mL-1. A calibration curve for assay of ocular samples. Intra-day and inter-day precision were
CyA in rabbit ocular samples (y = 35.243x ? 2.6793, determined as the relative standard deviation (RSD%) of
R2 = 0.9992) was plotted over the same range. This range the results obtained. Intra-day precision for CyA concen-
of concentrations was within the acceptable limit for trations of 0.2, 0.5, and 1 lg mL-1 ranged from 4.61 to
analysis of real ocular samples. Linearity was tested using 7.83%, and inter-day precision for these concentrations was
aqueous and vitreous humor spiked with standard solu- between 5.27 and 10.70%. In other studies, intra-day and
tions containing known concentrations of the drug. The inter-day precision ranged between 0.75 and 21.87% [9, 17,
correlation coefficient obtained for the calibration curve 21]. Accuracy, calculated to reflect the difference between
demonstrated the linearity of the results from in this nominal and observed concentrations, was in the range
analysis. The limit of detection (LOD), defined as the 83.4–111%, which is acceptable.

123
820 B. Malaekeh-Nikouei et al.

Table 1 Intra-day and inter-day precision and accuracy of determination of CyA in rabbit ocular samples
Nominal concentration Intra-day Inter-day
(lg mL-1)
Observed mean SD RSD (%) Accuracy (%) Observed mean SD RSD Accuracy
concentration (lg mL-1) concentration (lg mL-1) (%) (%)

0.2 0.194 0.01 5.47 97.19 0.222 0.02 9.03 111.09

0.5 0.54 0.024 4.61 108 0.517 0.027 5.27 103.53
1 0.892 0.069 7.83 89.25 0.834 0.089 10.7 83.4

Stability of the Analyte Application of the Analytical Method to Ocular

Samples
CyA was stable in solutions at room temperature for at
least 12 h; it also remained intact at -20 °C for up to The optimized and calibrated method was used for analysis
4 weeks. With regard to the run-time stability of processed of CyA in aqueous and vitreous humor samples from New
samples, no significant loss of CyA was observed at room Zealand albino rabbits. After topical administration of a
temperature. Thus, no degradation was observed during the nanoliposomal formulation of CyA to the rabbit ocular
day of an experiment. This information suggests repeated surface for 1 month the concentrations of the drug in ocular
analysis of samples within 12 h can be performed with samples were determined by HPLC. Fourteen aqueous and
confidence. 14 vitreous samples were tested in this study. The con-
centration of CyA in aqueous humor was significantly
Ruggedness higher than that in vitreous humor (P = 0.01 \ 0.05). The
average CyA concentration in aqueous humor was
The ruggedness of the method was evaluated as the 0.412 ± 0.157 lg mL-1 whereas that in vitreous humor
repeatability of results obtained by analysis of the same was 0.291 ± 0.093 lg mL-1.
sample under a variety conditions, for example different
experimenters or instruments. No significant difference
was observed between results, thus proving the method was Conclusion
rugged.
In this study a simple, fast, and reliable HPLC method
Robustness of the Method was developed for analysis CyA in ocular samples. CyA
was not extracted from ocular samples and no internal
To study the robustness of the method, deliberate slight standard was needed. Because of the low volume of some
variations were made in method conditions, for example ocular samples, the assay was calibrated for a volume as
changes of flow rate, ACN content of the mobile phase, and low as 250 lL. The intra-day and inter-day precision and
column temperature. These slight variations had no sig- accuracy results were within acceptable limits. Concen-
nificant effect on the area and shape of the peak. The trations of CyA as low as 0.2 lg mL-1 in ocular samples
results indicated that the resolution of the peak is more were measured. The calibrated HPLC method was used
sensitive to changes in column temperature than to changes for analysis of drug concentration in real rabbit ocular
in the other conditions. samples.

Specificity
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Analysis of Cyclosporine in Ocular Samples 821

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