2.1. Chemicals and reagents
Glucoraphanin sodium salt (purity 99.1%) was obtained from the Chemopreventive Agent repository maintained by the Chemopreventive Agent Development Research Group, Division of Cancer Prevention, National Cancer Institute (Bethesda, MD). Acetonitrile, ammonium acetate and formic acid (all HPLC grade) were purchased from Fisher Scientific (Fair Lawn, NJ). HPLC grade water was generated using a PURELAB Ultra system from ELGA (Lowell, MA) followed by filtration with a Millipore (Billerica, MA) system using a 0.25 µm filter. Blank plasma was obtained from otherwise untreated animals and frozen at −20 °C until analysis.
Three (3) male and 3 female non-naïve beagle dogs (approximately two and a half years of age; Covance Inc., Cumberland, VA) and twenty-five (25) male and 25 female CD rats [Crl:CD(SD) IGS BR] (received at approximately six weeks of age; Charles River Laboratories, Portage, MI) were used in this study. Prior to experimental initiation the attending veterinarian certified that the animals were healthy and free from disease and parasites.
Dogs were housed individually in pens and rats were housed individually in stainless steel cages. The animals were housed in accordance with standards set forth in the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996) and by the U.S. Department of Agriculture though the Animal Welfare Act ( 7 USC 2131, 1985) and Animal Welfare Standards incorporated in Title 9, Part 3 of the Code of Federal Regulations, 1991.
Animal rooms were held within a temperature range of approximately 18–29°C and a humidity range of approximately 30–70%. Fluorescent lighting in the animal rooms was provided for 12 hours followed by 12 hours of darkness.
Certified commercial dog or rodent diet was provided once daily (dogs) or ad libitum (rats). City of Chicago municipal water was available ad libitum by automatic watering systems in all pens and cages.
2.3. Preparation of stock solutions and standards
Stock solution of glucoraphanin (1 mg/mL) was prepared by dissolving glucoraphanin in acetonitrile/water (50:50, v/v). Stock solution was stored at −20 °C when not in use. Working standards were prepared by further diluting the stock solution in acetonitrile/water (50:50, v/v) to prepare concentrations ranging from 100 to 20000 ng/mL. Calibrators, ranging from 10 to 2000 ng/mL, were prepared by adding 10 µl of the working standards to 100 µl of plasma on each day of analysis.
2.4. Sample preparation
Plasma samples were thawed at room temperature before processing. In a microcentrifuge tube, 1 mL of acetonitrile was added to 100 µl of plasma, to which 10 µl of acetonitrile/water, [50:50, v/v; or a standard or quality control (QC) solution] was also added. The samples were vortex mixed for 1 min. and centrifuged in a Sorvall RC 5C Super Speed centrifuge (Thermo Fisher Scientific, Waltham, MA) at the highest setting for 5 min. The supernatant layer was transferred to a vial for injection into the LC-MS/MS.
Samples were analyzed on an API 3000 LC-MS/MS system (Applied Biosystems/MDS Sciex, Foster City, CA) equipped with an Agilent 1100 HPLC (Agilent Technologies, Wilmington, DE). Analyst™ 1.3.2 was used to control the HPLC and mass spectrometer and to capture the mass spectrometer data, perform linear regression analysis and calculate sample concentrations. Separation of glucoraphanin from plasma components was achieved using HILIC with a Luna 5 µm Silica (2) 100 Å column 50 × 2.0 mm (Phenomenex, Torrance, CA). The column temperature was maintained at 25 °C, and a flow rate of 0.3 ml/min. was used. The mobile phase consisted of Solvent A: 200 mM ammonium acetate and formic acid (99:1, v/v) and Solvent B : acetonitrile. The mobile phase gradient was as follows: after injection, initial conditions with Solvent B at 90% were held for 0.1 min., decreased to 40% in 0.5 min. and held constant for 2.5 min., returning to initial conditions for another 3 min. of reequilibration time. Retention time of glucoraphanin was approximately 2.9 min. Total run time was 6 min. A turbo ion spray interface was used as the ion source operating in negative ion mode. Acquisition was performed in multiple reaction monitoring mode using m/z 435.80 (deprotonated molecule, [M-H]−) → 96.70 ([SO3H]−) at low resolution. Ion spray voltage was −4200 V, ion spray temperature was 500 °C, and dwell time was 300 ms. The collision gas was nitrogen and the collision energy was set at −40 V.
2.6. Method validation
Method validation in dog plasma was performed following the FDA’s Guidance for Industry: Bioanalytical Method Validation
] after a full validation in rat plasma. The following factors were used to assess assay performance: selectivity, linearity, precision, accuracy, recovery and stability. The selectivity of the method was assessed by analyzing extract from six individual animals for the presence of analytical interferences and comparing the results to those obtained from spiking the blank plasma sources with glucoraphanin at the lower limit of quantitation (LLOQ; 10 ng/mL). Linearity was assessed using the external standard method and up to eight calibrators with analyte concentrations in the 10 to 2000 ng/mL range. The curves were built from peak areas using least-squares linear regression with (1/x2
) weighting factor. The weighting factor was chosen based on goodness-of-fit criteria including coefficient of determination (r2
), the back-calculated concentration of individual calibrators, and minimizing intercept value. Precision and accuracy of the method were determined from three validation runs with QC samples (n
= 6, 3, 3 for runs 1, 2 and 3, respectively) prepared at the LLOQ (10 ng/mL), low (25.0 ng/mL), mid (800 ng/mL), and high (1600 ng/mL) concentrations. Within-run precision and accuracy was assessed from the results from a single day, while between-run precision and accuracy were determined from the results from the three validation runs. Extraction recovery of glucoraphanin was determined by comparison of peak area results of the QC samples to peak area results of extracted blank plasma spiked post extraction with glucoraphanin at the same levels. Bench-top stability was determined by analyzing the low and high level QC samples for glucoraphanin concentration after 24 hours of storage at ambient temperature and comparing the results to those obtained from freshly thawed samples. Freeze-thaw stability of the low and high level QC samples was determined over three freeze-thaw cycles. Stability was also determined for low and high level QC samples that were extracted and stored in the autosampler; samples were analyzed and the concentrations determined after approximately one day of storage. To evaluate the impact of dilution on the analyses, six samples were prepared at 8000 ng/mL and diluted 5-fold with blank plasma prior to analysis.
2.7. Application to pre-clinical toxicological study
Glucoraphanin was administered orally by capsule to three male and three female non-naïve beagle dogs once daily for three consecutive days at a dose level of 200 mg/kg/day of body weight. Plasma samples were obtained from each dog pre-dose and approximately 60 minutes following dosing on the last day (Day 3) of the three-day dosing period.
To evaluate the toxicity of glucoraphanin following oral administration (gavage) to rats for fourteen days, five groups of rats (five males and 5 females per group) were dosed at 0 (control), 10, 50, 100, or 500 mg/kg of body weight daily. Dose formulations were prepared using ASTM Type 1 water as the vehicle. Glucoraphanin was measured in plasma samples collected approximately 60 min. after dosing on Day 13 of the study.
The dog and rat plasma samples were analyzed for levels of glucoraphanin using the analytical method presented here.