Abstract

RAPID DETERMINATION OF METHOTREXATEMETHOTREXATE METHOTREXATE METHOTREXATEMETHOTREXATE LEVEL IN HUMAN PLASMA BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Ahmed Yusuf, Syed N. Alvi, and Muhammad M. Hammami* 

ABSTRACT

A simple, precise, and rapid reversedA simple, precise, and rapid reversedA simple, precise, and rapid reversed A simple, precise, and rapid reversed A simple, precise, and rapid reversed A simple, precise, and rapid reversedA simple, precise, and rapid reversedA simple, precise, and rapid reversed A simple, precise, and rapid reversedA simple, precise, and rapid reversed A simple, precise, and rapid reversedA simple, precise, and rapid reversedA simple, precise, and rapid reversed A simple, precise, and rapid reversed A simple, precise, and rapid reversed A simple, precise, and rapid reversed A simple, precise, and rapid reversed-phase high performance liquid phase high performance liquid phase high performance liquid phase high performance liquid phase high performance liquid phase high performance liquid phase high performance liquid phase high performance liquid phase high performance liquid phase high performance liquid phase high performance liquid phase high performance liquid phase high performance liquid phase high performance liquid phase high performance liquid chromatography (HPLC) method for the determination of methotrexatechromatography (HPLC) method for the determination of methotrexate chromatography (HPLC) method for the determination of methotrexate chromatography (HPLC) method for the determination of methotrexatechromatography (HPLC) method for the determination of methotrexate chromatography (HPLC) method for the determination of methotrexate chromatography (HPLC) method for the determination of methotrexatechromatography (HPLC) method for the determination of methotrexatechromatography (HPLC) method for the determination of methotrexate chromatography (HPLC) method for the determination of methotrexatechromatography (HPLC) method for the determination of methotrexatechromatography (HPLC) method for the determination of methotrexatechromatography (HPLC) method for the determination of methotrexatechromatography (HPLC) method for the determination of methotrexatechromatography (HPLC) method for the determination of methotrexate chromatography (HPLC) method for the determination of methotrexate chromatography (HPLC) method for the determination of methotrexatechromatography (HPLC) method for the determination of methotrexate chromatography (HPLC) method for the determination of methotrexatechromatography (HPLC) method for the determination of methotrexate chromatography (HPLC) method for the determination of methotrexate chromatography (HPLC) method for the determination of methotrexate chromatography (HPLC) method for the determination of methotrexatechromatography (HPLC) method for the determination of methotrexatechromatography (HPLC) method for the determination of methotrexate chromatography (HPLC) method for the determination of methotrexate chromatography (HPLC) method for the determination of methotrexate chromatography (HPLC) method for the determination of methotrexatechromatography (HPLC) method for the determination of methotrexate chromatography (HPLC) method for the determination of methotrexatechromatography (HPLC) method for the determination of methotrexatechromatography (HPLC) method for the determination of methotrexatechromatography (HPLC) method for the determination of methotrexate (MTX) (MTX) level in human plasma level in human plasma level in human plasma level in human plasma level in human plasma level in human plasma using using sulfamethoxazole as an internal standard (IS) was developed and validated. Plasma samples containing was developed and validated. Plasma samples containing was developed and validated. Plasma samples containing was developed and validated. Plasma samples containing was developed and validated. Plasma samples containing was developed and validated. Plasma samples containing was developed and validated. Plasma samples containing was developed and validated. Plasma samples containing was developed and validated. Plasma samples containing was developed and validated. Plasma samples containing was developed and validated. Plasma samples containing was developed and validated. Plasma samples containing was developed and validated. Plasma samples containing was developed and validated. Plasma samples containing was developed and validated. Plasma samples containing was developed and validated. Plasma samples containing was developed and validated. Plasma samples containing was developed and validated. Plasma samples containing was developed and validated. Plasma samples containing was developed and validated. Plasma samples containing was developed and validated. Plasma samples containing was developed and validated. Plasma samples containing was developed and validated. Plasma samples containing was developed and validated. Plasma samples containing was developed and validated. Plasma samples containing was developed and validated. Plasma samples containing MTX were spiked with the IS, vortex MTX were spiked with the IS, vortex MTX were spiked with the IS, vortex MTX were spiked with the IS, vortexMTX were spiked with the IS, vortex MTX were spiked with the IS, vortexMTX were spiked with the IS, vortex MTX were spiked with the IS, vortex MTX were spiked with the IS, vortex MTX were spiked with the IS, vortexMTX were spiked with the IS, vortexMTX were spiked with the IS, vortex MTX were spiked with the IS, vortex MTX were spiked with the IS, vortex-mixed with methanol mixed with methanolmixed with methanolmixed with methanol mixed with methanol mixed with methanol mixed with methanol , and and centrifuged. centrifuged. centrifuged. centrifuged. centrifuged. centrifuged. After evaporation After evaporation After evaporationAfter evaporationAfter evaporationAfter evaporationAfter evaporation After evaporation After evaporation After evaporation , supernatant supernatantsupernatantsupernatant residue was dissolved residue was dissolved residue was dissolved residue was dissolved residue was dissolved residue was dissolved residue was dissolved residue was dissolved residue was dissolved in 0.01 M HCl 0.01 M HCl 0.01 M HCl 0.01 M HCl and and injected into the HPLC system. The The compounds of of interest were efficiently separated on Symmetry C interest were efficiently separated on Symmetry Cinterest were efficiently separated on Symmetry C interest were efficiently separated on Symmetry C interest were efficiently separated on Symmetry C interest were efficiently separated on Symmetry C interest were efficiently separated on Symmetry Cinterest were efficiently separated on Symmetry Cinterest were efficiently separated on Symmetry Cinterest were efficiently separated on Symmetry C interest were efficiently separated on Symmetry C interest were efficiently separated on Symmetry Cinterest were efficiently separated on Symmetry Cinterest were efficiently separated on Symmetry C interest were efficiently separated on Symmetry C interest were efficiently separated on Symmetry C interest were efficiently separated on Symmetry Cinterest were efficiently separated on Symmetry Cinterest were efficiently separated on Symmetry C interest were efficiently separated on Symmetry C interest were efficiently separated on Symmetry Cinterest were efficiently separated on Symmetry Cinterest were efficiently separated on Symmetry C interest were efficiently separated on Symmetry Cinterest were efficiently separated on Symmetry C interest were efficiently separated on Symmetry Cinterest were efficiently separated on Symmetry Cinterest were efficiently separated on Symmetry C-8 column at roomcolumn at room column at room column at roomcolumn at room column at room temperature, and detected with a photodiode array detector wavelength temperature, and detected with a photodiode array detector wavelength temperature, and detected with a photodiode array detector wavelength temperature, and detected with a photodiode array detector wavelengthtemperature, and detected with a photodiode array detector wavelength temperature, and detected with a photodiode array detector wavelengthtemperature, and detected with a photodiode array detector wavelength temperature, and detected with a photodiode array detector wavelength temperature, and detected with a photodiode array detector wavelength temperature, and detected with a photodiode array detector wavelength temperature, and detected with a photodiode array detector wavelength temperature, and detected with a photodiode array detector wavelength temperature, and detected with a photodiode array detector wavelengthtemperature, and detected with a photodiode array detector wavelengthtemperature, and detected with a photodiode array detector wavelength temperature, and detected with a photodiode array detector wavelength temperature, and detected with a photodiode array detector wavelengthtemperature, and detected with a photodiode array detector wavelengthtemperature, and detected with a photodiode array detector wavelength temperature, and detected with a photodiode array detector wavelengthtemperature, and detected with a photodiode array detector wavelengthtemperature, and detected with a photodiode array detector wavelengthtemperature, and detected with a photodiode array detector wavelength temperature, and detected with a photodiode array detector wavelength temperature, and detected with a photodiode array detector wavelengthtemperature, and detected with a photodiode array detector wavelength temperature, and detected with a photodiode array detector wavelength temperature, and detected with a photodiode array detector wavelength temperature, and detected with a photodiode array detector wavelengthtemperature, and detected with a photodiode array detector wavelengthtemperature, and detected with a photodiode array detector wavelengthtemperature, and detected with a photodiode array detector wavelengthtemperature, and detected with a photodiode array detector wavelength at 303 nm. The mobile phase consisted of 0.03 M dibasic potassium . The mobile phase consisted of 0.03 M dibasic potassium . The mobile phase consisted of 0.03 M dibasic potassium . The mobile phase consisted of 0.03 M dibasic potassium . The mobile phase consisted of 0.03 M dibasic potassium . The mobile phase consisted of 0.03 M dibasic potassium . The mobile phase consisted of 0.03 M dibasic potassium . The mobile phase consisted of 0.03 M dibasic potassium . The mobile phase consisted of 0.03 M dibasic potassium . The mobile phase consisted of 0.03 M dibasic potassium . The mobile phase consisted of 0.03 M dibasic potassium . The mobile phase consisted of 0.03 M dibasic potassium . The mobile phase consisted of 0.03 M dibasic potassium . The mobile phase consisted of 0.03 M dibasic potassium . The mobile phase consisted of 0.03 M dibasic potassium . The mobile phase consisted of 0.03 M dibasic potassium . The mobile phase consisted of 0.03 M dibasic potassium . The mobile phase consisted of 0.03 M dibasic potassium phosphate (pH adjusted t phosphate (pH adjusted t phosphate (pH adjusted t phosphate (pH adjusted tphosphate (pH adjusted tphosphate (pH adjusted t phosphate (pH adjusted tphosphate (pH adjusted t phosphate (pH adjusted to 6.5 with phosphoric acid), methanol, and o 6.5 with phosphoric acid), methanol, and o 6.5 with phosphoric acid), methanol, and o 6.5 with phosphoric acid), methanol, and o 6.5 with phosphoric acid), methanol, and o 6.5 with phosphoric acid), methanol, and o 6.5 with phosphoric acid), methanol, and o 6.5 with phosphoric acid), methanol, and o 6.5 with phosphoric acid), methanol, and o 6.5 with phosphoric acid), methanol, and o 6.5 with phosphoric acid), methanol, and o 6.5 with phosphoric acid), methanol, and o 6.5 with phosphoric acid), methanol, and acetonitrile (82:14:4, vacetonitrile (82:14:4, vacetonitrile (82:14:4, vacetonitrile (82:14:4, v acetonitrile (82:14:4, v acetonitrile (82:14:4, v acetonitrile (82:14:4, v:v :v ), and was delivered at a flow rate of 1 ), and was delivered at a flow rate of 1), and was delivered at a flow rate of 1 ), and was delivered at a flow rate of 1), and was delivered at a flow rate of 1), and was delivered at a flow rate of 1 ), and was delivered at a flow rate of 1 ), and was delivered at a flow rate of 1 ), and was delivered at a flow rate of 1 ), and was delivered at a flow rate of 1 ), and was delivered at a flow rate of 1 ), and was delivered at a flow rate of 1), and was delivered at a flow rate of 1 ), and was delivered at a flow rate of 1), and was delivered at a flow rate of 1), and was delivered at a flow rate of 1 ), and was delivered at a flow rate of 1), and was delivered at a flow rate of 1), and was delivered at a flow rate of 1), and was delivered at a flow rate of 1 ), and was delivered at a flow rate of 1 ), and was delivered at a flow rate of 1), and was delivered at a flow rate of 1.0 ml/min. No interference in blank plasma or of commonly used drugs ml/min. No interference in blank plasma or of commonly used drugs ml/min. No interference in blank plasma or of commonly used drugs ml/min. No interference in blank plasma or of commonly used drugs ml/min. No interference in blank plasma or of commonly used drugs ml/min. No interference in blank plasma or of commonly used drugs ml/min. No interference in blank plasma or of commonly used drugs ml/min. No interference in blank plasma or of commonly used drugs ml/min. No interference in blank plasma or of commonly used drugs ml/min. No interference in blank plasma or of commonly used drugs ml/min. No interference in blank plasma or of commonly used drugs ml/min. No interference in blank plasma or of commonly used drugs ml/min. No interference in blank plasma or of commonly used drugs ml/min. No interference in blank plasma or of commonly used drugs ml/min. No interference in blank plasma or of commonly used drugs ml/min. No interference in blank plasma or of commonly used drugs ml/min. No interference in blank plasma or of commonly used drugs ml/min. No interference in blank plasma or of commonly used drugs ml/min. No interference in blank plasma or of commonly used drugs ml/min. No interference in blank plasma or of commonly used drugs ml/min. No interference in blank plasma or of commonly used drugs ml/min. No interference in blank plasma or of commonly used drugs ml/min. No interference in blank plasma or of commonly used drugs ml/min. No interference in blank plasma or of commonly used drugs ml/min. No interference in blank plasma or of commonly used drugs ml/min. No interference in blank plasma or of commonly used drugs ml/min. No interference in blank plasma or of commonly used drugs ml/min. No interference in blank plasma or of commonly used drugs ml/min. No interference in blank plasma or of commonly used drugs ml/min. No interference in blank plasma or of commonly used drugs ml/min. No interference in blank plasma or of commonly used drugs ml/min. No interference in blank plasma or of commonly used drugs was observed was observed was observed was observedwas observedwas observed, and the detection limit of MTX was 0.02 , and the detection limit of MTX was 0.02, and the detection limit of MTX was 0.02 , and the detection limit of MTX was 0.02, and the detection limit of MTX was 0.02 , and the detection limit of MTX was 0.02, and the detection limit of MTX was 0.02, and the detection limit of MTX was 0.02 , and the detection limit of MTX was 0.02 , and the detection limit of MTX was 0.02 , and the detection limit of MTX was 0.02 , and the detection limit of MTX was 0.02 , and the detection limit of MTX was 0.02 , and the detection limit of MTX was 0.02 , and the detection limit of MTX was 0.02, and the detection limit of MTX was 0.02 , and the detection limit of MTX was 0.02 , and the detection limit of MTX was 0.02 , and the detection limit of MTX was 0.02 µg/ml. Theµg/ml. Theµg/ml. The µg/ml. The µg/ml. The relationshi relationshi relationshi p between p between p between p between MTX MTX concentration in plasma and peak area ratioconcentration in plasma and peak area ratio concentration in plasma and peak area ratioconcentration in plasma and peak area ratio concentration in plasma and peak area ratioconcentration in plasma and peak area ratio concentration in plasma and peak area ratio concentration in plasma and peak area ratioconcentration in plasma and peak area ratio concentration in plasma and peak area ratio concentration in plasma and peak area ratioconcentration in plasma and peak area ratio concentration in plasma and peak area ratioconcentration in plasma and peak area ratioconcentration in plasma and peak area ratioconcentration in plasma and peak area ratio concentration in plasma and peak area ratioconcentration in plasma and peak area ratio concentration in plasma and peak area ratio of of MTX /IS was linear (R/IS was linear (R/IS was linear (R/IS was linear (R/IS was linear (R /IS was linear (R/IS was linear (R/IS was linear (R /IS was linear (R /IS was linear (R/IS was linear (R/IS was linear (R/IS was linear (R/IS was linear (R2 ≥ 0.9997) in the range of 0.05 ≥ 0.9997) in the range of 0.05 ≥ 0.9997) in the range of 0.05 ≥ 0.9997) in the range of 0.05 ≥ 0.9997) in the range of 0.05 ≥ 0.9997) in the range of 0.05 ≥ 0.9997) in the range of 0.05 ≥ 0.9997) in the range of 0.05 ≥ 0.9997) in the range of 0.05 ≥ 0.9997) in the range of 0.05 ≥ 0.9997) in the range of 0.05 ≥ 0.9997) in the range of 0.05 ≥ 0.9997) in the range of 0.05 – 20 µg/ml 20 µg/ml20 µg/ml20 µg/ml 20 µg/ml. I . Intrantra - and interand inter and inter and interand inter-day day coefficient of variations (CV) were ≤ coefficient of variations (CV) were ≤ coefficient of variations (CV) were ≤ coefficient of variations (CV) were ≤ coefficient of variations (CV) were ≤ coefficient of variations (CV) were ≤ coefficient of variations (CV) were ≤ coefficient of variations (CV) were ≤ coefficient of variations (CV) were ≤ coefficient of variations (CV) were ≤ coefficient of variations (CV) were ≤ coefficient of variations (CV) were ≤ coefficient of variations (CV) were ≤ coefficient of variations (CV) were ≤ coefficient of variations (CV) were ≤ coefficient of variations (CV) were ≤ coefficient of variations (CV) were ≤ coefficient of variations (CV) were ≤ 8.2 %. Extraction recoveries xtraction recoveries xtraction recoveries xtraction recoveries xtraction recoveries xtraction recoveries xtraction recoveries xtraction recoveries xtraction recoveries xtraction recoveries of of MTX and the IS and the IS and the IS and the IS and the IS and the IS were were were were ≥ 93%, whereas IntraIntra -daydayday and interand inter and inter and interand inter-day day day bias (relative recovery) of MTX measurement was -8.0% to 0.8% using quality control samples and -4.0% to 1.2% as determined by back calculation from peak area ratios of the calibration curves. The method was used to assess the stability of MTX in human plasma under conditions generally encountered in clinical laboratory. Further, it was successfully applied to measure MTX level in samples obtained from a patient on MTX therapy.

Keywords: Methotrexate (MTX) Methotrexate (MTX)Methotrexate (MTX)Methotrexate (MTX)Methotrexate (MTX) Methotrexate (MTX) , Sulfamethoxazole, Human plasma, HPLC.