ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Oct. 2010, p. 4467–4470 0066-4804/10/$12.00 doi:10.1128/AAC.00509-10 Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 54, No. 10
Rapid and Sensitive Liquid Chromatography/Mass Spectrometry Assay for Caspofungin in Human Aqueous Humor䌤 Chin Fen Neoh,1,2 Hui He,2§ Jian Li,1,2 Robert O. Fullinfaw,3 Lok Leung,4 Anant Misra,5¶ Rasik B. Vajpayee,5,6 Geoffrey E. Davies,7 Kay Stewart,1 and David C. M. Kong1,2* Department of Pharmacy Practice, Centre for Medicine Use and Safety, Faculty of Pharmacy and Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australia1; Facility for Anti-infective Drug Development and Innovation (FADDI), Monash Institute of Pharmaceutical Sciences, Faculty of Pharmacy and Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australia2; Special Chemistry, Melbourne Health Pathology, the Royal Melbourne Hospital, Grattan Street, Parkville, Victoria 3050, Australia3; Department of Pharmacy, Royal Victorian Eye and Ear Hospital (RVEEH), 32 Gisborne Street, East Melbourne, Victoria 3002, Australia4; Corneal and Cataract Surgery Unit, Royal Victorian Eye and Ear Hospital (RVEEH), 32 Gisborne Street, East Melbourne, Victoria 3002, Australia5; Centre for Eye Research Australia, University of Melbourne, Royal Victorian Eye and Ear Hospital (RVEEH), Locked Bag 8, East Melbourne, Victoria 3002, Australia6; and Health Systems Program Office, Land Systems Division, Defence Materiel Organisation, Victoria Barracks Melbourne HW1, 256-310 St. Kilda Road, Southbank, Victoria 3006, Australia7 Received 15 April 2010/Returned for modification 25 May 2010/Accepted 20 July 2010
A rapid, precise, and sensitive liquid chromatography/mass spectrometry (LC/MS) method to quantify the caspofungin concentration in human aqueous humor was developed and validated. Sample preparation involved simple dilution of aqueous humor samples with acetonitrile. Azithromycin was the internal standard. Good linearity over 10 to 5,000 ng/ml was observed. The lower limit of quantification was 10 ng/ml. The intra- and interday accuracies (percent bias) were within 11%, while the intra- and interday precisions were within 6%. caspofungin that involves simple preparation and low sample volumes. Caspofungin diacetate was provided by Merck Sharp & Dohme. Azithromycin dihydrate (internal standard [IS]) was purchased from Kopran Ltd. (Maharashtra, India). Calibration standard and quality control (QC) stock solutions of caspofungin (1 mg/ml) were prepared separately in water and stored at ⫺80°C. IS stock solution (1 mg/ml) was freshly prepared in acetonitrile for each analysis. Working solutions of caspofungin and IS were freshly prepared by diluting the stock solutions with water and acetonitrile, respectively. The calibration curve was constructed using water due to the limited availability of blank aqueous humor (6, 10). Calibration standards were prepared by adding 30 l IS working solution to 30 l caspofungin working solutions. The final concentrations of calibration standards were 10, 50, 100, 500, 1,000, 2,000, and 5,000 ng/ml with the IS (500 ng/ml). All QC samples (30, 300, and 4,000 ng/ml) were freshly prepared. The blank aqueous humors were pooled to prepare aqueous humorbased low (30 ng/ml; n ⫽ 3) and high (4,000 ng/ml; n ⫽ 3) QC samples (30 l each) to validate the water-based calibration curve. The Shimadzu LC system (Shimadzu, Japan) comprised a high-pressure gradient unit (LC-20AD pump and LC-20ADsp pump), DGU-20A3 degasser, and CTO-10A column oven with a FCV-12AH switching valve. A Synergi Hydro-RP C18 column (80 Å, 50 by 2 mm, 4 m; Phenomenex) and guard column (4 by 2 mm, 4 m; Phenomenex) were used. The column temperature was 30°C, and the flow rate was 0.5 ml/
Current topical treatment for fungal keratitis is inadequate (13) given the lack of favorable outcomes with existing antifungal eye drops (i.e., fluconazole, amphotericin B, and natamycin) (7, 14). Accordingly, it is important to investigate the topical application of newer antifungal agents that are available only as injections. In rabbit models (5, 8), caspofungin eye drops were effective in inhibiting the progression of fungal keratitis. It remains unknown if caspofungin eye drops are able to penetrate the human eye. Studies in this area are impeded by the absence of a simple and sensitive analytical assay to quantify caspofungin in human aqueous humor. Currently, high-performance liquid chromatography (HPLC) with fluorescence or amperometric detection (4, 11, 12, 15) and liquid chromatography/tandem mass spectrometry (LC/ MS/MS) (1–3, 9, 16) are used to quantify caspofungin in biological samples. The majority of these methods, however, involve liquid-liquid extraction and require large sample volumes. Because aqueous humor samples usually have small volumes (⬍150 l), these methodologies are not suitable. This study aimed to develop a rapid and sensitive LC/MS assay for * Corresponding author. Mailing address: Department of Pharmacy Practice, Centre for Medicine Use and Safety, Faculty of Pharmacy and Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australia. Phone: 61-3-9903-9035. Fax: 61-39903-9629. E-mail:
[email protected]. § Present address: Department of Physiology, University of Tennessee Health Science Center, 894 Union Avenue, Memphis, TN 38163. ¶ Present address: Department of Ophthalmology, Southern Health, 246 Clayton Road, Clayton, Victoria 3168, Australia. 䌤 Published ahead of print on 26 July 2010. 4467
4468
NEOH ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 1. Representative chromatogram of blank aqueous humor sample (A) or two aqueous humor samples containing a caspofungin concentration of 95.1 ng/ml or 63.8 ng/ml (with IS at 500 ng/ml) (B1 and B2, respectively) from two consenting patients who were administered one drop of 0.5% caspofungin eye drop hourly for 4 h prior to the surgery. The aqueous humor samples were removed from the participants 1.67 h and 1.17 h after the last caspofungin eye drop was administered for B1 and B2, respectively.
min. The mobile phase was Milli-Q water–formic acid (A; 100:0.1 [vol/vol]) and methanol-formic acid (B; 100:0.1 [vol/ vol]). The gradient elution program was as follows: 0.0 to 2.8 min from 20% B to 30% B, 2.8 to 3.0 min from 30% B to 95% B, 3.0 to 5.0 min at 95% B, 5.0 to 5.5 min from 95% B to 20% B, and 5.5 to 8.0 min at 20% B. The mobile phase was directed to an MS system between 3.0 and 6.0 min. The run time was 8.0 min. The injection volume was 10 l. An MS-2010EV single-quadrupole mass analyzer coupled with
an electrospray ionization interface (Shimadzu, Japan) was used. The tuning voltages were fixed for the interface, the curved desolvation line (CDL), and Q-array. The detector voltage was 1.5 kV. Flow rates were 1.5 liters/min for nitrogen gas and 10 liters/min for both the nebulizer and the drying gas. The temperatures for CDL and heat block were 200°C. Data were acquired and processed using the LCMSsolutions software program (version 3.4). The protonated ions of caspofungin (m/z ⫽ 547.5 [M⫹2H]2⫹) and IS (m/z ⫽ 749.4 [M⫹H]⫹) were monitored.
VOL. 54, 2010
LC/MS ASSAY FOR CASPOFUNGIN IN HUMAN AQUEOUS HUMOR
4469
TABLE 1. Accuracy and precision data for assay of caspofungin QC concn Intraday (n ⫽ 6)
Parameter
Mean concn (ng/ml) CV (%)
Interday (n ⫽ 5)
30 ng/ml
300 ng/ml
4,000 ng/ml
30 ng/ml
300 ng/ml
4,000 ng/ml
26.9 2.52
280 3.88
3,986 5.43
31.4 3.71
282 2.71
3,965 2.39
Caspofungin eluted at 4.68 ⫾ 0.01 min, and the IS eluted at 4.48 ⫾ 0.01 min (Fig. 1). Blank aqueous humor samples (n ⫽ 6) showed no interfering peaks with endogenous products or coadministered drugs used in eye surgery (phenylephrine, cyclopentolate, tropicamide, and oxybuprocaine). Calibration curves plotted the peak area ratio of caspofungin to IS (y) against the caspofungin concentration (x), with y ⫽ 0.005x ⫺ 0.074 (n ⫽ 5; r2 ⫽ 0.999 ⫾ 0.003). Good linearity over 10 to 5,000 ng/ml was observed, with 1/x weighting employed. The limit of detection (LOD) calculated using the equation LOD ⫽ 3.3/S was 2.11 ng/ml ( is the standard deviation of the calibration curve, and S is the slope of the calibration curve). The lower limit of quantification (LLOQ), defined as the reproducible peak of the lowest concentration in the calibration curve, was 10 ng/ml (n ⫽ 6), with an accuracy of 105% and a coefficient of variation (CV) of 5.44%. For intraday analysis, six determinations of each QC concentration were assayed. For interday assessment, three replicates at each QC concentration were run on five separate days. The intra- and interday accuracies (percent bias) were within 11.0%. The intra- and interday precisions (CV) were within 6% (Table 1). The aqueous humor-based low (mean ⫽ 28.8 ng/ml; CV ⫽ 4.21%) and high (mean ⫽ 4,111 ng/ml; CV ⫽ 3.03%) QC samples gave results similar to those of the corresponding water-based QC samples. The retention times for caspofungin in aqueous humor- or water-based samples were identical. Caspofungin was stable in water (i.e., it remained at ⫾15% of the initial concentration) under the following conditions: (i) after two freeze-thaw cycles, (ii) at ⫺80°C for 60 days (Fig. 2), and (iii) at 2 to 8°C for 3 days. Caspofungin and the IS in a 1:1 mixture of acetonitrile-water were stable for 24 h when left in
FIG. 2. Stability of caspofungin in samples and stock solution when stored at ⫺80°C.
the LC/MS autosampler at 4°C. The caspofungin stock solution was stable for 2 months when stored at ⫺80°C (Fig. 2). Although our LLOQ was not as low as that reported by Rochat et al. (9), it was impossible to adapt their LC/MS/MS method, which involved liquid-liquid extraction, given the limited volume of aqueous humor. The sensitivity of the current assay could be further improved with tandem MS. Caspofungin could nonspecifically adsorb to plastic and glass surfaces, thus reducing assay recovery (9). A simple sample pretreatment with an equal volume of acetonitrile, as per the current assay, prevents the nonspecific adsorption. This is in contrast to published methods which required additional preanalysis processing (2, 9, 11, 15, 16). This study has established a simple and rapid LC/MS assay which is sensitive, accurate, and reproducible for quantifying caspofungin in a limited volume of biological samples. The support from study participants and the nursing staff at the RVEEH is gratefully acknowledged. We thank Vishal Jhanji for his contribution at the early stages of this project in facilitating access to some blank aqueous humor required for the development of this assay. We thank Merck Sharp & Dohme (Australia) Pty. Limited for the caspofungin pure substance used in the analytical assay. This study was supported by a grant from the Contributing to Australian Scholarship and Science (CASS) Foundation. This study was partly funded by a University of Technology MARA (UiTM) scholarship to C.F.N. J.L. is an Australian National Health and Medical Research Council R. Douglas Wright Research Fellow. REFERENCES 1. Chavez-Eng, C. M., M. S. Schwartz, M. L. Constanzer, and B. K. Matuszewski. 1999. Determination of a cyclic hexapeptide, a novel antifungal agent, in human plasma by high-performance liquid chromatography with ion spray and turbo ion spray tandem mass spectrometric detection. J. Chromatogr. B Biomed. Sci. Appl. 721:229–238. 2. Egle, H., R. Trittler, and K. Kummerer. 2004. An advanced double columnswitching technique (LC-LC) for liquid chromatography/electrospray ionisation tandem mass spectrometry for fully automated analysis of caspofungin. Rapid Commun. Mass Spectrom. 18:2871–2877. 3. Farowski, F., O. A. Cornely, J. J. Vehreschild, P. Hartmann, T. Bauer, A. Steinbach, M. J. G. T. Ruping, and C. Muller. 2010. Quantitation of azoles and echinocandins in the compartments of peripheral blood by liquid chromatography tandem mass spectrometry. Antimicrob. Agents Chemother. 54:1815–1819. 4. Goldblum, D., K. Fausch, B. E. Frueh, R. Theurillat, W. Thormann, and S. Zimmerli. 2007. Ocular penetration of caspofungin in a rabbit uveitis model. Graefes Arch. Clin. Exp. Ophthalmol. 245:825–833. 5. Goldblum, D., B. E. Frueh, G. M. Sarra, K. Katsoulis, and S. Zimmerli. 2005. Topical caspofungin for treatment of keratitis caused by Candida albicans in a rabbit model. Antimicrob. Agents Chemother. 49:1359–1363. 6. Keski-Rahkonen, P., O. Parssinen, E. Leppanen, T. Mauriala, M. Lehtonen, and S. Auriola. 2007. Determination of tamsulosin in human aqueous humor and serum by liquid chromatography-electrospray ionization tandem mass spectrometry. J. Pharm. Biomed. Anal. 43:606–612. 7. O’Day, D. M., G. Foulds, T. E. Williams, R. D. Robinson, R. H. Allen, and W. S. Head. 1990. Ocular uptake of fluconazole following oral administration. Arch. Ophthalmol. 108:1006–1008. 8. Ozturk, F., G. F. Yavas, T. Kusbeci, Z. Cetinkaya, U. U. Inan, S. S. Ermis, and N. Kiraz. 2007. Efficacy of topical caspofungin in experimental Fusarium keratitis. Cornea 26:726–728. 9. Rochat, B., S. Bolay, A. Pascual, T. Calandra, and O. Marchetti. 2007.
4470
NEOH ET AL.
Liquid chromatography-mass spectrometry method for quantification of caspofungin in clinical plasma samples. J. Mass Spectrom. 42:440–449. 10. Schenkel, F., S. Rudaz, Y. Daali, M. K. Oestreicher, J. L. Veuthey, and P. Dayer. 2005. Development and validation of a new reversed-phase ion pairing liquid chromatographic method with fluorescence detection for penciclovir analysis in plasma and aqueous humour. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 826:1–7. 11. Schwartz, M. S., W. Kline, and B. K. Matuszewski. 1997. Determination of a cyclic hexapeptide (L-743 872), a novel pneumocandin antifungal agent in human plasma and urine by high-performance liquid chromatography with fluorescence detection. Anal. Chim. Acta 352:299–307. 12. Sheng, B., M. S. Schwartz, R. B. Desai, A. R. Miller, and B. K. Matuszewski. 2005. A semi-automated procedure for the determination of caspofungin in human plasma using solid-phase extraction and HPLC with fluorescence
ANTIMICROB. AGENTS CHEMOTHER.
13. 14. 15.
16.
detection using secondary ionic interactions to obtain a highly purified extract. J. Liq. Chromatogr. Relat. Technol. 28:2895–2908. Srinivasan, M. 2004. Fungal keratitis. Curr. Opin. Ophthalmol. 15:321–327. Thomas, P. A. 2003. Current perspectives on ophthalmic mycoses. Clin. Microbiol. Rev. 16:730–797. Traunmu ¨ller, F., I. Steiner, M. Zeitlinger, and C. Joukhadar. 2006. Development of a high-performance liquid chromatography method for the determination of caspofungin with amperometric detection and its application to in vitro microdialysis experiments. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 843:142–146. Vorwerk, C. K., S. Tuchen, F. Streit, L. Binder, W. Hofmu ¨ller, and W. Behrens-Baumann. 2009. Aqueous humor concentrations of topically administered caspofungin in rabbits. Ophthalmic Res. 41:102–105.
