Quantitative Determination of 6ß-Naltrexol in the Femtogram/mL Range from EDTA Human Plasma by LC-MS-MS
Authors
Klaus-Peter Adam, Michael P. Sullivan, Christopher J.L. Buggé
Introduction
6ß-Naltrexol is the main phase I metabolite of the opioid antagonist naltrexone. After oral administration of naltrexone, plasma concentrations of 6ß-naltrexol appear to be approximately 10 fold higher than its parent drug naltrexone with a similar blood level profile. Therefore, monitoring 6ß-naltrexol plasma levels allows monitoring of naltrexone levels that are about one order of magnitude lower. For this purpose we have developed a very sensitive (0.250 pg/mL) and rapid LC-MS-MS assay to measure 6ß-naltrexol in human plasma. The method presented here describes a sensitive, accurate, and rugged procedure for measuring 6ß-naltrexol in plasma from dosed human subjects.
Experimental
Chemicals
6ß-Naltrexol and the internal standard 6ß-naltrexol-D 3 were obtained commercially with a reported chemical purity of greater than 99%. All other chemicals were AR grade and all solvents used were HPLC grade.
Standards/Solutions
Stock solutions of 6ß-naltrexol and its stable labelled internal standard, were prepared in acetonitrile/water (1:1). Combined Intermediate solutions were prepared from these stock solutions and were corrected for their salt content to provide known free base concentrations. A series of spiking solutions was made to cover the required quantitation range.
Quality control (QC) solutions were prepared in K 3 EDTA human plasma at three different concentrations (0.750, 4.00 and 8.00 pg/mL 6ß-naltrexol). These QC Low, QC Medium and QC High samples were stored in 1.00 mL aliquots at –20 °C. QC samples were prepared from different 6ß-naltrexol stock solutions than the standard spiking solutions.
LC-MS-MS
The LC-MS-MS system consisted of a Shimadzu LC pump, Perkin-Elmer autosampler and SCIEX API 4000 mass spectrometer with an TurboIonSpray interface. The mass spectrometer was optimized for the intensity of the 6ß-naltrexol signal in positive ion mode. The ion transitions monitored were as follows:
| 6ß-Naltrexol | m/z 344 --> 326 |
| 6ß-Naltrexol - D3 (IS) | m/z 347 --> 329 |
Extracts were injected on the HPLC system flowing at a rate of 1.00 mL/min. The extract was separated on a 2.0 x 50 mm HPLC column with a mobile phase consisting of acetonitrile/water/acetic acid/ammonium formate and analyzed by LC-MS-MS.
Results
The linear quantitative range was established from 0.250 to 10 pg/mL using freshly fortified calibration samples. Precision and accuracy of the method was determined to be acceptable after evaluating LLOQ samples and QC samples over four days of validation. Inter–day precision (%CV) at the LLOQ (0.250 pg/mL) was 6.8%. Accuracy (% of theoretical concentration) for the LLOQ was 102%. Precision of QC samples prepared at 0.750, 4.00 and 8.00 pg/ml ranged from 1.9% to 5.3%, with accuracy ranging from 94.1% to 96.5%. Stability of QC samples in plasma during normal handling was determined to be acceptable after stressing with multiple freeze-thaw cycles, room temperature exposure, and an extended term in –20 °C frozen storage. Extraction recovery was acceptable. Furthermore, no significant chromatographic interferences of 18 common OTC drugs were detected at the retention times of 6ß-natlrexol or Internal Standard. There were no matrix effects or chromatographic interferences observed within varying lots of plasma.
Conclusions
This method demonstrates good ruggedness over the stated quantitation range coupled with excellent sensitivity. Being a simple liquid-liquid extraction, we have automated the extraction process using a robotics system (Packard MultiPROBE II). Using this method, one analyst can extract and analyze more than 400 samples per LC-MS-MS instrument per day. Furthermore, no significant chromatographic interferences of 18 common OTC drugs were detected at the retention times of 6ß-naltrexol or Internal Standard.