Cynthia Coulter, Catherine Castro
Immunalysis Corporation, Pomona, CA, U.S.A.
Objective
Tramadol, a CNS depressant and analgesic is used in the treatment of moderate to severe pain. The objective was to determine whether semi-quantitative data collected with HEIA has a strong correlation with routine immunoassay, ELISA.
Relevance
Since the main issues in clinical practice are the time and expense of sample processing, the development of a rapid semi-quantitative method for the screening of pain management drugs is necessary.
Methodology
® Oral fluid samples collected in the Immunalysis Quantisal® device were screened at a cut-off concentration of 50µg/L using ELISA and HEIA.
® Controls for ELISA and HEIA were diluted 1+3 with Quantisal® buffer to achieve neat oral fluid concentration.
® Positive samples were extracted using a previously published and validated solid phase extraction and analyzed using GC-MS. 1
Validation
Both assays: Calibration standards: 25, 50, 100, 200, 500µg/L
HEIA: Instrument: Olympus AU400e; Sample volume: 25µL; R1 and R2 volumes: 100µL Primary wavelength: 340nm; secondary wavelength: 410nm Measuring points 15 th -19 th rotation.
Inter-day precision was performed over 20 days with 2 replicates per day (N=40)
Coefficient of variation (CV): 150µg/L and 250µg/L - 0.65% and 0.64% respectively.
ELISA: Sample volume: 10µL. Intra-assay precision CV% for 150µg/L and 250µg/L - 5.35% and 4.05%, respectively Linearity r 2=0.9794 for ELISA semi-quantitative curve.
Results
® Thirty-three (33) oral fluid specimens previously found to be positive for tramadol using GC-MS were analyzed using HEIA and ELISA in the semiquantitative mode.
® Comparison of the data with the quantitative GC-MS data showed samples in the linear range of the assay to be within +/-20% of the confirmed concentration. Samples outside the upper limits of the curve and causing maximum absorbance readings were later diluted 1:10 or 1:100 and re-plated to fit within the standard curve range.
® Samples with screen values above the upper limits of the curve could be further diluted prior to extraction to eliminate the need for re-analysis, ultimately decreasing extraction costs and increasing throughput.
Conclusion
® Tramadol can be determined in oral fluid specimens using semi-quantitative screening modes on routine chemistry analysers or ELISA platforms.
® Both assays were rapid and simple to operate, showing a high degree of quantitative correlation with GC-MS.
Reference
Moore C, Rana S, Coulter C. Determination of meperidine, tramadol and oxycodone in human oral fluid using solid phase extraction and gas chromatography-mass spectrometry. J Chromatogr B. Biomed Applns 2007; 850: 370 - 375
SOFT, Oklahoma City, 2009
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