Dexamethasone is a corticosteroid commonly used in anti-cancer therapy. For patients who have brain tumors, dexamethasone is used to reduce inflammation caused by the tumor
Dexamethasone is a corticosteroid commonly used in anti-cancer therapy. For patients who have brain tumors, dexamethasone is used to reduce inflammation caused by the tumor
Dexamethasone and phenytoin were purchased from Sigma Aldrich Inc (St. Louis, MO, U.S.A.). A Shimadzu HPLC system is used (Kyoto, Japan). It consists of a photodiode array (Kyoto, Japan, SPD-M 10A VP), two pumps (LC-10AT), and a controller (SCL-10A VP). It is coupled to a C18 column (Phenomenex, 250 × 4.6 mm, 5-micron particles) through a high-pressure injection valve from Rheodyne Inc. (Cotati, CA, USA, model RH-7725i) with a 10 µL sample loop. The detection wavelength for phenytoin and dexamethasone is 219 nm and 241 nm respectively.
The mobile phase consists of a mixture of 0.001 M KH2PO4, acetonitrile, and methanol adjusted to pH 5.6 (48:32:20) and is pumped at a flow rate of 1.0 mL/min. Retention time for dexamethasone and phenytoin was determined by injecting each compound individually to the HPLC system. The identification of dexamethasone and phenytoin from the extracted samples was determined by the corresponding retention time.
Initially, the detection wavelength for both dexamethasone and phenytoin was set at 230 nm. After further study, it was determined that the optimum detection wavelength for phenytoin and dexamethasone is 219 nm and 241 nm respectively.
A phenytoin stock solution (concentration = 504 µg/mL) and a dexamethasone stock solution (concentration = 598 µg/mL) were prepared by accurately weighing out the corresponding substance and using methanol as the solvent. Five standard solutions were prepared from these two stock solutions with a concentration range from 25.2 - 1.57 µg/mL for phenytoin and 29.9 - 1.87 µg/mL for dexamethasone. Each standard solution was injected three times.
20 mL of urine sample was spiked with 1 mL of the phenytoin stock solution (concentration = 563 µg/mL) and 1 mL of the dexamethasone stock solution (concentration = 578 µg/mL). The resulting phenytoin and dexamethasone concentrations in the spiked sample are 28.15 µg/mL and 28.90 µg/mL respectively. The spiked sample was vortexed for 7 min. A blank was prepared similarly with 20 mL of distilled water.
Extractions were performed with the 3 mL Strata C-18-E cartridges (Phenomenex) and the 24-port, vacuum manifold (Phenomenex).
The cartridge is conditioned with: 1) 3.0 mL of methanol (MeOH), 2) 2.0 mL of DI water, and 3) 2.0 mL of 0.1 M phosphate buffer at pH 6. The extraction procedure is as follows:
1) 3 mL of the spiked urine sample is loaded onto the cartridge, 2) washed with 3.0 mL of distilled water, 3) washed with 2.0 mL of 20% MeOH (v/v), and 4) finally eluted with 3.0 mL of MeOH.
Eluted samples were dried under a steady stream of nitrogen gas. Each dried sample was re-dissolved in 1.0 mL of methanol. The dissolved sample was then filtered through a 0.45 µm syringe filter prior to injection into the HPLC system. Each dissolved sample was injected 3 times.
20 mL of the urine sample was spiked with 1 mL of the phenytoin stock solution (concentration = 112.6 µg/mL) and 1 mL of the dexamethasone stock solution (concentration = 115.6 µg/mL). The resulting phenytoin and dexamethasone concentrations in the spiked sample were 5.630 µg/mL and 5.780 µg/mL respectively.
Percent recovery studies for the simultaneous extraction of dexamethasone and phenytoin were performed by spiking urine samples and blanks at two concentration levels as described above. Precision was evaluated by inter-day (3 days and intra-day (3 samples) studies.
Calibration curves for phenytoin and dexamethasone are shown in
Chromatograms of the extracted urine sample are shown in
Percent recovery was calculated as the ratio between the amount extracted from the spiked urine samples and the amount extracted from the blank. Results of the inter-day and intra-day studies at the two spiking levels are shown in
Percent Recovery |
|||||
SpikedConcentration |
Day 1 Intra-day average (n = 3) and RelativeStandard Deviation(RSD) |
Day 2 Intra-day average (n = 3) and RelativeStandard Deviation(RSD) |
Day 3 Intra-day average (n = 3) and RelativeStandard Deviation(RSD) |
Inter-DayAverage |
Inter-day RelativeStandardDeviation(RSD) |
Phenytoin 28.15 µg/mL |
93.4% (RSD = 1.42%) |
94.5% (RSD = 3.63%) |
92.1% (RSD = 2.99%) |
93.3% |
2.68% |
Dexamethasone 28.90 µg/mL |
78.3% (RSD = 2.63%) |
77.8% (RSD = 3.36%) |
78.3% (RSD = 2.48%) |
78.1% |
2.82% |
Percent Recovery |
|||||
SpikedConcentration |
Day 1 Intra-day average(n = 3) and Relative Standard Deviation (RSD) |
Day 2 Intra-day average(n = 3) and RelativeStandard Deviation(RSD) |
Day 3 Intra-day average(n = 3) and Relative Standard Deviation (RSD) |
Inter-Day Average |
RelativeStandardDeviation(RSD) |
Phenytoin 5.630 µg/mL |
96.7% (RSD = 1.59%) |
95.5% (RSD = 1.27%) |
94.0% (RSD = 0.583%) |
95.4% |
1.15% |
Dexamethasone 5.780 µg/mL |
82.8% (RSD = 1.96%) |
78.3% (RSD =7.95%) |
82.2% (RSD =0.769%) |
81.1% |
3.56% |
An efficient and simple solid-phase extraction method was developed for simultaneous extraction of phenytoin and dexamethasone from urine example was developed. Percent recovery for phenytoin and dexamethasone was 95.4% and 81.1%. Similar percent recovery for both drugs was observed at a different spiking level. Results from the inter-day and intra-day studies show that the extraction method has good precision (RSD < 4%).
The HPLC conditions used for this study effectively separate phenytoin and dexamethasone. Calibration curves for phenytoin and dexamethasone show excellent linearity (r2 > 0.99). Analysis was performed in less than 11 minutes in an isocratic mode on a reversed-phase C18 column using a mobile phase that is composed of 0.001 M KH2PO4, acetonitrile, and methanol adjusted to pH 5.6 (48:32:20).
*Corresponding author.