Determination of tramadol and O-desmethyltramadol in human plasma by high-performance liquid chromatography with mass spectrometry detection

• Published in: Talanta (Oxford) Vol. 75; no. 4; pp. 1104 - 1109
• Main Authors: Vlase, Laurian, Leucuta, Sorin E., Imre, Silvia
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 30.05.2008; Oxford Elsevier
• Subjects: Administration, Oral; Analytical chemistry; Chemistry; Chromatographic methods and physical methods associated with chromatography; Chromatography, High Pressure Liquid; Exact sciences and technology; Humans; LC/MS; O-Desmethyltramadol; Other chromatographic methods; Pharmacokinetics; Reproducibility of Results; Spectrometric and optical methods; Spectrometry, Mass, Electrospray Ionization; Time Factors; Tramadol; Tramadol - analogs & derivatives; Tramadol - blood; Tramadol - metabolism; LC/MS; O-Desmethyltramadol; Pharmacokinetics; Tramadol; Water; Metabolite; Chromatographic retention; HPLC chromatography; Mobile phase; Retention time; Electrospray; Ion trap; Precipitation; Accuracy; Sample preparation; Detection limit; Standard curve; Tablet; Quantitative analysis; Correlation coefficient; Human; Stability; Coupled method; Secondary ion mass spectrometry; Liquid chromatography; Isocratic condition; Chemical ionization; Protein; Reversed phase chromatography; Perchloric acid; Acetonitrile; Mass spectrometry
• ISSN: 0039-9140; 1873-3573
• DOI: 10.1016/j.talanta.2008.01.006

A new simple, sensitive and selective liquid chromatography coupled with mass spectrometry (LC/MS) method for quantification of tramadol and its active metabolite O-desmethyltramadol in human plasma was validated. The tramadol and its metabolite were separated on a reversed phase column (Zorbax SB-C18, 100 mm × 3.0 mm I.D., 3.5 μm) under isocratic conditions using a mobile phase of a 10:90 (v/v) mixture of acetonitrile and 0.2% (v/v) trifluoroacetic acid in water. The flow rate was 1 ml/min at the column temperature 45 °C. In these chromatographic conditions, the retention times were 2.3 min for O-desmethyltramadol and 3.5 min for tramadol, respectively. The detection of both analytes was in SIM mode using an ion trap mass spectrometer with electrospray positive ionisation. The monitored ions were m/ z 264 for tramadol and m/ z 250 for its metabolite. The sample preparation was very simple and rapid and consisted in plasma protein precipitation from 0.2 ml plasma using 0.2 ml solution of perchloric acid 7%. Calibration curves were generated over the range of 2–300 ng/ml for both analytes with values for coefficient of correlation greater than 0.998 and by using a weighted (1/ y) quadratic regression. The values of precision and accuracy for tramadol at quantification limit were less than 10.9% and 5.1, respectively, both for within- and between-run. For O-desmethyltramadol, precision and accuracy at quantification limit were 10.1% and −9.9% for within-run determinations and 6.7% and 10.4% for between-run determinations, respectively. The mean recovery for both analytes was 96%. Both tramadol and its metabolite demonstrated good short-term, long-term, post-preparative and freeze–thaw stability. This is the first reported method for analysis of tramadol and O-desmethyltramadol in human plasma that uses protein precipitation as sample processing procedure. The method is very simple and allows obtaining a very good recovery of both analytes. The validated LC/MS method has been applied to a pharmacokinetic study of 50 mg tramadol tablets on healthy volunteers.