Rapid and Sensitive Liquid Chromatography/Mass Spectrometry Assay for Caspofungin in Human Aqueous Humor

Abstract

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%.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 (Ͻ150 l), these methodologies are not suitable. This study aimed to develop a rapid and sensitive LC/MS assay for 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 Ϫ80°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 ϭ 3) and high (4,000 ng/ml; n ϭ 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-20A 3 degasser, and CTO-10A column ...

Penetration of Topically Administered 0.5-Percent Caspofungin Eye Drops into Human Aqueous Humor

“…The difference could be due to the use of a more sensitive analytical assay (limit of quantification [LOQ], 10 ng/ml) (17) than that used in the earlier study (LOQ, 50 ng/ml) (10).…”

“…The samples were then divided into 30-l aliquots, stored at Ϫ80°C, and analyzed within 1 week using a validated liquid chromatography/mass spectrometry (LC/MS) assay (17).…”

An alternative capillary electrophoresis method for the quantification of caspofungin in lyophilisate powder and its measurement uncertainty

“…Despite the importance of echinocandins, there are a few number of methods described in literature and none in most important official compendia and pharmacopeias. Caspofungin in biological samples and in pharmaceutical products is often analyzed by high performance liquid chromatography with mass spectrometry (Egleet al, 2004;Rochatet al, 2007;Neoh et al, 2010), electrochemical (Traunmuller et al, 2006) and UV detector (Ghisleni et al, 2014b) or by microbiological assay (Ghisleni et al, 2014a). However, a capillary electrophoresis method of analysis of echinocandins, including caspofungin, was not found in literature.…”