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*
BAS 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 50 µL ISTD
Sorbent: SCX (1 mL, 100 mg, Varian)

Procedure: acidify sample with 50 µL 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 200 µL 0.05 M sodium citrate pre-elution solution, elute with 750 µL 3:97 ACN:0.1 M citric acid (pH 6.5)


SPEC Disk SPE (Automated)

Sample: 250 µL sample and 50 µL 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 250 µL 10:90 ACN:0.1 M citric acid (pH 6.5)


96-Well SPE (Manual)

Sample: 250 µL sample and 50 µL ISTD
Sorbent: SCX (MicroluteTM, 25 mg, Varian)

Procedure: dilute sample with 200 µL deionized water, precondition SPE tube with >= 250 µL ACN and >= 250 µL 0.5 M HCl, apply sample, elute with 250 µL 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.


ACKNOWLEDGEMENT

The authors wish to acknowledge Angela Johnson and Abe Brubaker for their contributions to this work.