Opioids assayed by GC after extraction by SPE

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  • Published: Oct 1, 2017
  • Author: Ryan De Vooght-Johnson
  • Channels: Gas Chromatography
thumbnail image: Opioids assayed by GC after extraction by SPE

Accurate measurement of opioid levels in blood required

Synthetic opioid compounds are widely used for pain relief. However, some can give rise to dangerous irregularities in heart rhythms, along with other serious side effects. One opioid, propoxyphene, has now been phased out in the UK, although it is still used in some other countries. It is important that opioid concentrations in blood can be accurately and conveniently measured so that adequate pain relief can be given while minimising dangerous side effects.

Analysis of opioids in plasma can be carried out by LC-MS, but GC-MS has the advantages of fewer interfering peaks, cheaper instrumentation and minimal solvent use. The Buenos Aires researchers developed a method using SPE of plasma samples, followed by GC-MS. The mass spectrometry used electron ionisation (EI) in SIM mode, in which only data from particular ions of interest are collected.

Five species were examined: propoxyphene, norpropoxyyphene amide, tramadol, meperidine (also known as pethidine) and normeperidine (also known as norpethidine). Normeperidine is a metabolite of meperidine (loss of a methyl group), while norpropoxyphene amide is formed by the rearrangement of norpropoyxphene under basic conditions, the main metabolite of propoxyphene (loss of a methyl group). Norpropoxyphene was rearranged to its amide because the former compound tends to decompose during GC.

SPE and GC-MS used to measure opioids in plasma

Plasma samples were taken and spiked with known amounts of the species of interest, with norpropoxyphene being added rather than its amide. Deuterated internal standards, dissolved in methanol or acetonitrile depending on the compound, were added (the tramadol internal standard contained 13C in addition to deuterium). The samples were treated with aqueous sodium hydroxide to rearrange the norpropoxyphene to norpropoxyphene amide. Sodium dihydrogen phosphate and phosphate buffer were then added to give a pH of 6, and solids were removed by centrifugation. The compounds of interest were adsorbed onto an SPE cartridge (UCT Clean Screen® CSDAU303). The cartridge was washed with water, aqueous acetic acid and n-hexane to remove impurities. The compounds were extracted from the cartridge using dichloromethane:isopropanol (4:1 v/v containing 2% NH4OH). The solvent was evaporated and the residue taken up into ethyl acetate.

GC was carried out with an HP 6890N instrument fitted with a J&W HP-5MS column. The temperature was taken from 130 to 280 °C in a series of ramps, with a flow rate of 1.3 mL/min. The chromatogram gave clear separation of the five compounds, the final one appearing after 15 minutes. Mass spectrometry employed a HP 5973 quadrupole instrument in SIM mode. Appropriate quantification and qualifying ions were selected for each species, with accurate quantification being achieved by comparison with the deuterated internal standards.

The method gave good accuracy and precision, as well as adequate recovery from the spiked solutions. The limit of quantification (LOQ) for each compound was only 25 ng/ml. The method was validated and then successfully applied to plasma samples from patients being treated with the opioids concerned.

New GC method can be used for three different opioids

Although a refinement of a previous protocol for propoxyphene determination in urine, the new method has the advantage of being able to deal with three different opioids by GC-MS relatively quickly. The use of a base-catalysed rearrangement neatly solves the problem of norpropoxyphene instability on the GC. It is likely that other drugs could also be detected by this method or a modified version of it.

Related Links

Rapid Communications in Mass Spectrometry, 2017, 31, 1519-1533. Fernández et al. Simultaneous quantitation of meperidine, normeperidine, tramadol, propoxyphene and norpropoxyphene in human plasma using solid-phase extraction and gas chromatography/mass spectrometry: Method validation and application to cardiovascular safety of therapeutic doses.

Journal of Analytical Toxicology, 1996, 20, 547-554. Amalfitano et al. Gas chromatographic quantitation of dextropropoxyphene and norpropoxyphene in urine after solid-phase extraction.

Wikipedia, Mass Chromatogram

Article by Ryan De Vooght-Johnson

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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