(W0930-06-32) A Sensitive Method for the Detection and Quantitation of Nalmefene in Human Plasma Samples for Pharmacokinetic Analysis

Purpose: Nalmefene is an opioid receptor antagonist that was originally approved by the FDA in 1995 for the complete or partial reversal of opioid overdose effects. However, in 2008 the US marketing authorization holder discontinued the distribution of nalmefene for reasons not related to safety or effectiveness of the drug. In February 2022, the FDA approved a generic nalmefene hydrochloride injection vial (2 mg in 1 mg/mL solution) for the complete or partial reversal of opioid drug effects, including respiratory depression, induced by either natural or synthetic opioids and for the management of known or suspected opioid overdose. Nalmefene is known to have high affinity for mu-opioid receptors, a long elimination half-life compared to naloxone and may provide advantages as a potential reversal agent for opioid overdose effects in the United States. A sensitive method for the detection and quantitation of nalmefene was developed and validated in human plasma samples at Syneos Health Clinical to support clinical pharmacokinetic studies. The analyte was extracted from plasma by a liquid-liquid extraction (LLE) procedure and separated by reversed phase HPLC followed by MS/MS in positive mode ESI detection. The method proved to be rapid, robust, sensitive, and met FDA validation criteria.

Methods: Nalmefene and an analog internal standard (IS), d₃-nalbuphine, were extracted from human plasma by liquid-liquid extraction method in a 96-well plate. Internal standard working solution was added to all wells except the matrix blank. A volume of 25.0 µL of methanol/water (25/75; v/v) was added to all matrix blank samples. The plate was covered and vortexed followed by the addition of 200 µL of 0.2M ammonium hydroxide to each well and vortexed. One milliliter of MtBE was added to the extracts and mixed followed by a 5-minute pause for the organic layer and aqueous layer to separate. An aliquot (900 µL) was transferred to 96-well polypropylene plate and the samples were evaporated to dryness under nitrogen at 40°C. The dried samples were reconstituted in 100 µL of methanol/water (25/75; v/v) and analyzed using reversed phase HPLC conditions on a Waters X-Bridge C18 5 µm, 50.0 x 2.1 mm column, 0.1% Formic Acid in Water as MPA and Methanol as MPB. Nalmefene and IS were detected and quantified by tandem mass spectrometry in positive ion mode on an MDS Sciex API 4000 equipped with a Turbo Ionspray^® probe. Calibration curves were generated by performing a linear regression (weighted 1/x²) on the calibration standards.

Results: Under the chromatographic conditions used, nalmefene and IS were well separated with an approximate retention time of 1.60 min and 0.98 min, respectively. Nalmefene was quantified by comparing the peak area of m/z 340.2 → 322.2 to that of IS m/z 361.4 → 343.5. The method was validated over a range of 0.10–100 ng/mL as the lower and upper limits of quantitation for nalmefene. At all QC levels (low (0.30 ng/mL), mid (5.00 ng/mL) and high (75.0 ng/mL), within-run precision and within-run accuracy values were ≤ 3.55% and -10.3 to 5.80%, respectively. At all QC levels, between-run precision and between-run accuracy values were ≤ 5.17% and -5.20 to 1.40%, respectively. All experiments met FDA validation criteria. Short-term stability was established for 28.5 hours at ambient temperature, freeze/thaw for 8 cycles and long-term stability for 519 days at -20ºC and -80ºC. Selectivity was performed in six normal individual lots of plasma plus one lot of 2% hemolyzed plasma and one lot of lipemic plasma. No matrix effects were observed.

Conclusion: A sensitive method for the quantitation of nalmefene concentrations in human plasma samples was developed and proven to be precise, accurate, and selective over a wide range. The method is reliable and robust, and the analyte and IS are stable under all conditions studied. This method allows to detect and quantitate low levels of nalmefene in human plasma. It is being used for the quantitation of nalmefene concentrations in human plasma samples from pharmacokinetic studies.


Precision and Accuracy Table


Precision and Accuracy Table