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Background Luliconazole is a novel antifungal agent, in the class of imidazoles, being developed for the treatment of onychomycosis. Currently approved therapies are either ineffective or are complicated by the potential of liver toxicity requiring hepatic monitoring. Topical application of luliconazole has demonstrated rapid penetration and therapeutically effective levels in the nail bed. (Jones T and Tavakkol A, AAC 2013 Jun; 57(6):2684-9) A sensitive and selective LC-MS/MS method was developed to determine the concentrations of luliconazole in human toenails to support clinical trials. Challenges encountered during method development included separation of isomeric forms of the drug, and the homogenization and processing of a solid matrix (i.e. toenails). Sample Preparation Published methods for toenail sample preparation typically include acid or alkaline digestion. These methods are not practical for luliconazole due to compound degradation, therefore mechanical homogenization was evaluated. Different homogenizing mills (Figure 1) and sample vial/crusher systems (Figure 2) were tested. The optimized sample processing procedure is listed below. 1. Transfer the entire toenail sample into a reinforced polypropylene vial and add a 7-mm stainless steel bead (Figure 2D). Cap the vials and place into the tube block of a Model TK-AM5-B Auto-Mill, Tokken Inc. (Figure 1B). 2. Dip the tube block into liquid nitrogen for 1 minute. 3. Place the super-cooled tube block onto the Auto-Mill and run the homogenization program (1200 r/min for 180 seconds). 4. Repeat steps 2–3 to ensure complete homogenization. T2309. Development of an LC-MS/MS Method for the Determination of the Antifungal Luliconazole in Human Toenails M. K. Leahy 1 , L. Geisler 1 , J. Wang 1 , K. Morton 2 , N. Nakamura 2 and M. Wolter 2 1 Covance Laboratories Inc., Madison, WI; 2 Topica Pharmaceuticals, Inc., Los Altos, CA Diseased toenails are often non-uniform in shape and consistency. To avoid possible bias from subdividing the sample, the entire nail clipping sample provided is processed. Calibrators are prepared based on a theoretical sample size of 5.00 mg. Unknown samples are weighed and then the entire sample is assayed. A post-analysis correction factor (CF) is applied to convert the measured concentrations into μg/g (CF = 5 ÷ sample weight, CF * calculated value = sample concentration in μg/g). Extraction 1. Remove the samples from the Auto-Mill and add 1.00 mL of Methanol:DMSO (1:1, v:v) to each vial. Leave the stainless steel beads in the vials. 2. Vortex-mix for 10 minutes, then sonicate the samples for 30 minutes. 3. Centrifuge the samples at a minimum of 8320 x g for 5 minutes. 4. Transfer 50.0 μL aliquots of the supernatant to polypropylene tubes. Note: After sample aliquots are taken, the remaining homogenized nail sample may be stored frozen for possible reassay. 5. Add 50.0 μL of Working Internal Standard Solution to the samples. Vortex-mix for 1 minute. 6. Dilute the samples with Methanol:Water (1:1, v:v) and transfer into a 96-well collection plate. 7. Store at refrigerated conditions if needed prior to injection. Chromatography It is known that luliconazole can convert to a (Z)-luliconazole metabolite, both of which share the same precursor and product ions or MRM transitions. Baseline chromatographic separation was therefore required to resolve luliconazole from (Z)-luliconazole. The best resolution was obtained using a Kinetex 2.6μm PFP column (Phenomenex). HPLC Parameters HPLC System: Shimadzu Prominence Column: Phenomenex, Kinetex PFP, 50 x 2.1 mm, 2.6 μm particle size Pre-filter: Phenomenex, KrudKatcher 0.5 μm Column Temp.: 40°C Mobile Phase: A: 50 mM Ammonium Formate in Water B: 50 mM Ammonium Formate in Methanol:Water (90:10) Gradient Program: Time (minute) Action Value 0.01 %B 53 0.50 %B 53 1.00 %B 69 3.20 %B 69 3.21 %B 95 3.80 %B 95 3.81 %B 53 4.50 Stop MS Parameters Mass Spectrometer: Sciex API 4000 Ionization: Positive Ion Electrospray (ESI+) Results The calibration curves were linear in the range of 100 to 10000 μg/g based on a 5 mg toenail sample size for calibration standards. The overall precision and accuracy values, based upon relative standard deviation (RSD) and relative error (RE) of quality control (QC) samples, were ≤5.1% and +6.2%, respectively. Luliconazole was stable in homogenized nail clippings subjected to 6 freeze/thaw cycles, at least 24 hour storage at room temperature, and at least 29 days stored at -20°C. Luliconazole was stable in fortified nail clippings (prior to homogenization) for at least 489 days stored at -20˚C. Processed samples are stable in injection solvent for up to 4 days. Toenail clippings from 6 individual healthy donors not currently undergoing an antifungal regimen were obtained from Bioreclamation for selectivity testing. No significant interference was found in the MRM chromatograms of the six lots of nail clippings tested. Additionally, no matrix effect was detected in these six lots of nail clippings. An estimated recovery is performed since it is not known whether the blank matrix is penetrated by the analyte after spiking. Recoveries were determined by comparing the mean peak area of processed samples with the mean peak area of recovery samples prepared by adding luliconazole and lanoconazole (internal standard) to blank human toenail extracts. The overall recoveries for luliconazole and lanoconazole were 91.6 and 98.7%, respectively. Conclusion Method development for the quantitation of luliconazole in human toenails using LC-MS/MS presented unique challenges in sample preparation due to an atypical matrix and in chromatography due to the requirement for baseline separation of luliconazole and (Z)-luliconazole. The method has been successfully validated and used in the analysis of unknown samples. Figure 1. Tissue homogenizers. Figure 2. Vial/crusher systems. Figure 3. (Z)-Luliconazole/luliconazole resolution. Table 1. Mass Spectrometer Parameters Compound Name MRM Transition Monitored Approximate RT (min) Luliconazole 354.1 → 150.3 2.7 (Z)-Luliconazole 354.1 → 150.3 2.4 Lanoconazole (Internal Standard) 320.1 → 150.3 2.1 Figure 4. Chromatograms of extracted toenails. Table 2. Quality Control Data for Luliconazole in Human Toenails LLOQ QC 100 µg/g LQC 300 µg/g MQC 1500 µg/g HQC 7500 µg/g DQC 50000 µg/g (10X Dilution) Mean Measured Concentration (μg/g) 97.6 288 1430 7070 46900 Inter-run SD 4.96 10.6 47.8 303 568 Inter-run RSD (%) 5.1 3.7 3.3 4.3 1.2 Inter-run RE (%) -2.4 -4.0 -4.7 -5.7 -6.2 n 18 18 18 18 6 Flow Rate: 0.5 mL/min Back Pressure: 300 bar (Typical) Injection Volume: 5 μL Cycle Time: Approximately 5 min Figure 5. Calibration curve. A: FastPrep-24 Homogenizer, MP Biomedicals (Reference: www.ads-img.co.jp/product/english/pdf/tk_am5.pdf). B: Auto-Mill, Model TK-AM5-B, Tokken Inc. (Reference: www.mpbio.com/product.php?pid=116004500&country=223). Mode: MRM IonSpray Voltage: 2000 V TurboIon Spray Temp: 450°C
