The protocol was approved by the institutional animal care and use committee. The Animal Facility of the Institute for Neurodegenerative Diseases provided animal samples from 2 different strains of mice, FVB and CD1. Twenty four animals of each strain (24 FVB and 24 CD1 strain animals) were fed 37.5 mg/kg/D and 24 animals of each strain were fed 75 mg/kg/D of oral quinacrine over a 4-week period (weeks 1–4), given ad lib as a chocolate flavored liquid diet. From weeks 5–8 animals were given regular feed without quinacrine. At weekly intervals, 3 animals of each strain were euthanized and tissues were collected for analysis.
Quinacrine level analysis
Preparation of standard solutions
Quinacrine dihydrochloride (purity: 98.6%) was purchased from Fluka, while sulfadimethoxine sodium salt (purity: 98%) which served as the internal standard (IS) was obtained from Sigma. Quinacrine stock solution was prepared as 1 mg/ml in methanol. Sulfadimethoxine stock solution was prepared as 1 mg/ml in 50% methanol. The working solutions were prepared by diluting the respective standard and control stock solutions with 50% methanol and 0.1% formic acid to 2 μg/ml and 100 ng/ml, respectively. All solutions were stored in a 4°C refrigerator in silinized brown glass containers.
LC/MS/MS system and conditions
The HPLC system employed a Shimadzu LC-10 AD pump and a Waters intelligent Sample Processor 717 Plus autosampler/injector. A BDS Hypersil C18 column, 50 × 4.60 mm, 5 μm particle size was directly coupled to a Micromass Quattro LC Ultima triple quadrupole tandem mass spectrometer using electrospray ionization in positive ion mode. The sample cone voltage and collision energy were 25 V and 20 eV respectively for both quinacrine and the internal standard and the source block and desolvation temperature were 100°C and 400°C, respectively. The mass scanning mode employed multiple reaction monitoring (MRM) with the singly charged quinacrine ion selected at m/z 400.5 giving a fragment ion at m/z/142.0, and the internal standard at m/z 311.0→156.0. The mobile phase consisted of CH3OH/H2O/TFA (45:55:0.05) with 1 mM ammonium formate. The flow rate was 0.8 ml/min with 1/4 split into the mass spectrometer. The injection volume was 5–10 μl with a run time of 3.5 min.
All samples were stored at -70°C until analyzed. Each tissue sample was subjected to a specific method, as described below, for drug extraction and for the determination of concentration. All samples were analyzed by LC/MS/MS.
Accuracy and precision
Accuracy and precision was demonstrated throughout the working range with interday and intraday coefficient of variation and relative error <10%.
Each plasma sample was thawed at room temperature for 10–15 min; then 20 μl of plasma was aliquotted to a new test tube. To each tube 200 μl of 70% acetonitrile solution, containing 0.1% formic acid and 50 ng/mL of internal standard, was added. The test tubes were vortexed at high speed for 1 min and centrifuged at 10,000 rpm for 10 min. The supernatant was transferred into the autosampler for LC/MS/MS analysis. A set of standard curves with a duplicate set of quality control (QC) samples was generated for sample analysis.
Prior to the in vivo study, the stability of quinacrine in mouse brain tissue was determined. Using 3 different doses of quinacrine (4 mg/kg/d, 80 mg/kg/d, 160 mg/kg/d), mouse brain tissue was soaked in 100% methanol for 7 days. Samples were taken on days 0,1,2,3, and 7 to analyze quinacrine concentrations using LC/MS/MS. Complete equilibration was observed by Day 1 and no degradation in quinacrine was noted through day 7.
Brain samples were thawed (in plastic tubes) at room temperature for 15–20 min and weighed. To each sample 0.5 ml of 0.9% NaCl was added followed by incubation at room temperature for 1 h. For the standard curve and quality control samples, a brain tissue sample from an untreated mouse was spiked with different amounts of quinacrine and incubated at room temperature for 1 h. After the addition of 100 μl of 1 μg/ml internal standard solution in 50% methanol and 0.1% formic acid, 5 ml of 100% methanol was added into each sample and soaked at 4°C for two weeks. On the last day, 200 μl was aliquotted from each brain sample and placed into the autosampler for analysis via LC/MS/MS.
Liver samples were thawed at room temperature for 15–30 min and weighed. Considering the increased size of the liver samples and that the increased connective tissue in liver samples could potentially prevent the complete distribution of quinacrine into methanol, homogenization was used for these samples. To each sample 100% methanol was added (10 ml/g of tissue) and the tissue was homogenized in ice water for 1 min at speed 3 (Tissue Tearor, model 985-370, Biospec Products, Inc). The internal standard (100 μl of 10 μg/ml in 50% methanol, 0.1% formic acid) was added to 0.2 ml of each homogenized liver sample in a glass test tube. Samples were vortexed for 1 min, centrifuged at 3000 rpm for 10 min and 20 μl of each supernatant was transferred into a new test tube. Each sample was further diluted with 4 ml of 50% methanol, vortexed, and 200 μl was transferred to the autosampler for LC/MS/MS analysis. A set of standard curve with a duplicate set of QC samples was generated for sample analysis.
Spleen sample preparation was similar to the preparation of brain samples using a methanol soak. Prior to the in vivo study, the stability of quinacrine in mouse spleen tissue was determined. Using 3 different doses of quinacrine (4 mg/kg/d, 80 mg/kg/d, 160 mg/kg/d), mouse spleen tissue was soaked in 100% methanol for 7 days. Samples were taken on days 0, 1, 2, 3, and 7 to analyze quinacrine concentrations using LC/MS/MS. Complete equilibration took place by Day 1 and no degradation in quinacrine was observed through day 7.
Samples from the in vivo analysis were soaked for 14 days at 4°C. On day 14, 50 μl was aliquotted from each spleen sample and diluted with 1 ml of 50% methanol. Each sample was vortexed for 1 min and 200 μl was transferred to the autosampler for LC/MS/MS analysis. A set of standard curves with a duplicate set of QC samples was generated for sample analysis.