Comparison Of Conventional Packed Bed, Extraction Disk, And 96-well Solid Phase Extraction Formats For The Determination Of Methotrexate In Plasma
E. C. Hernandez, C. E. Serpe and A. Witkowski*
BASi Analytics, 2701 Kent Avenue, West Lafayette, IN, 47906, USA
Phone: 765-463-4527 ext. 271
Fax: 765-463-1309
OBJECTIVE
To compare the relative merits of conventional packed bed, extraction disk, and 96-well solid phase extraction (SPE) formats for the determination of methotrexate in plasma.ABSTRACT
Methotrexate (MTX) is a competitive dihydrofolate reductase inhibitor that is used to treat a variety of human cancers. Recently, a study was undertaken to compare three solid phase extraction (SPE) formats for use in the LC-UV analysis of MTX in plasma. For each assay, the sample was extracted using a strong cation exchange (SCX) sorbent, and the SPE eluate was injected directly into the LC system. Linear calibration curves were obtained for all extraction formats in the range of 1000 to 10.0 ng/mL, and in all cases the inter-day accuracy and precision were within (15%/15%/20% at the high, middle and low concentrations, respectively.EXPERIMENTAL
Conventional Packed Bed SPE (Manual)
Sample: 1.0 mL plasma and 50µL ISTD
Sorbent: SCX (1 mL, 100 mg, Varian)
Procedure: acidify sample with 50µL 5 N HCl, precondition SPE tube with 2 x 1 mL methanol and 2 x 1 mL deionized water, apply sample, rinse with 2 x 1 mL 0.01 M HCl, add 200µL 0.05 M sodium citrate pre-elution solution, elute with 750µL 3:97 ACN:0.1 M citric acid (pH 6.5)
SPEC Disk SPE (Automated)
Sample: 250µL sample and 50µL ISTD
Sorbent: SCX (3 mL, SPEC·PLUS, 15 mg, Ansys)
Automation Equipment: RapidTraceTM
SPE Workstation
Procedure: dilute sample with 0.2 mL deionized water, precondition SPE tube with 0.5 mL ACN and 0.5 mL 0.01 M HCl, apply sample, rinse with 2 mL 0.01 M HCl, elute with 250µL 10:90 ACN:0.1 M citric acid (pH 6.5)
96-Well SPE (Manual)
Sample: 250µL sample and 50µL ISTD
Sorbent: SCX (MicroluteTM, 25 mg, Varian)
Procedure: dilute sample with 200µL deionized water, precondition SPE tube with >= 250µL ACN and >= 250µL 0.5 M HCl, apply sample, elute with 250µL 10:90 ACN:0.1 M citric acid (pH 6.5)
1000 ng/mL Methotrexate Extract
10.0 ng/mL Methotrexate Extract
Blank Plasma Extract
Conventional Packed Bed
| . | nominal MTX concentration (ng/mL) | ||
|---|---|---|---|
| . | 550 | 90.0 | 15.0 |
| mean | 583 | 92.3 | 15.2 |
| std dev | 27.4 | 4.29 | 0.579 |
| rsd | 4.7% | 4.6% | 3.8% |
| accuracy | 106.0% | 102.5% | 101.2% |
Extraction Disk
| . | nominal MTX concentration (ng/mL) | ||
|---|---|---|---|
| . | 800 | 200 | 30.0 |
| mean | 815 | 211 | 30.8 |
| std dev | 65.7 | 22.0 | 2.43 |
| rsd | 8.1% | 10.4% | 7.9% |
| accuracy | 101.8% | 105.4% | 102.6% |
96 - Well Format
| . | nominal MTX concentration (ng/mL) | ||
|---|---|---|---|
| . | 800 | 200 | 30.0 |
| mean | 863 | 208 | 29.5 |
| std dev | 66.9 | 27.4 | 3.60 |
| rsd | 7.7% | 13.2% | 12.2% |
| accuracy | 107.8% | 103.8% | 98.3% |
CONCLUSIONS
* All 3 Formats Can Be Used For The Analysis Of MTX In Plasma.* The Conventional Packed Bed Column Performs Well But Can Be More Time Consuming.
* The Automated Rapidtrace Extraction Takes About As Long As Conventional Packed Bed Sample Preparation, But It Frees Personnel For Other Tasks .
* The 96 Well Plate Approach Allows For Faster Sample Preparation, But When Done Manually It Is More Susceptible To Technique Variability (Ex. Recovery Problems, Etc). This Limitation Would Be Reduced By Automating The Reagent Delivery And By Better Control Of The Flow Rates.
* Reduced Elution Volume Afforded By SPEC Discs And Microlute Plates Gives A Concentration Enhancement For The "Elute And Shoot" Method, Improving The Signal To Noise Ratio Near The LLOQ And Allowing For A Reduced Sample Volume To Be Used.
