This application note presents the procedure for the analysis of morphine and its metabolites in blood plasma using electrospray LC/MS, a method which does not require derivatization.
This document details an analytical method for the determination of opiates and their metabolites in blood samples using electrospray LC/MS. The method utilizes the Agilent 1100 Series LC/MS system, which includes a binary pump, vacuum degasser, autosampler, thermostatted column compartment, diode-array detector, and the LC/MSD VL quadrupole mass spectrometer. The analysis is performed using electrospray ionization (ESI) in positive mode without the need for derivatization.
The procedure involves solid-phase extraction (SPE) for sample preparation, followed by chromatographic separation using a Supelco Discovery HSC18 column and a gradient mobile phase of water with 0.1% formic acid and methanol. The method achieves excellent recovery rates and accuracy, with coefficients of variation typically 5% or less. It allows for the identification and quantitation of various opiates, including morphine, hydromorphone, codeine, hydrocodone, 6-acetylmorphine, and oxycodone, as well as the internal standard nalorphine.
| Mobile phase A | 0.1% formic acid in water |
|---|---|
| Mobile phase B | methanol |
| Flow rate | 0.5 ml/min |
| Column temp | 50°C |
| Veap | 3000 V |
| Nebulizer | 40 psig |
| Drying gas flow | 13 l/min |
| Drying gas temp | 350°C |
| Mass range | m/z 100-650 |
| Fragmentor | 120 V |
The Agilent 1100 Series LC/MS system, including a binary pump, vacuum degasser, autosampler, thermostatted column compartment, diode-array detector, and the LC/MSD VL quadrupole mass spectrometer.
The flow rate for the analysis is set to 0.5 ml/min.
The eluate is evaporated to dryness with a stream of nitrogen at 40°C.