Determination of phentermine, N-hydroxyphentermine and mephentermine in urine using dilute and shoot liquid chromatography–tandem mass spectrometry

•We developed a reliable method for determination of phentermine, N-hydroxyphentermine and mephentermine in urine using LC–MS/MS.•The method was validated and its applicability was confirmed by analysis of real urine samples.•The method is considered to be effective for differentiating whether PT or MPT is ingested.

Nonmedical use of prescription stimulants such as phentermine (PT) has been regulated by law enforcement authorities due to its euphorigenic and relaxing effects. Due to high potential for its abuse, reliable analytical methods were required to detect and identify PT and its metabolite in biological samples. Thus a dilute and shoot liquid chromatography–tandem mass spectrometric (LC–MS/MS) method was developed and validated for simultaneous determination of PT, N-hydroxyphentermine (NHOPT) and mephentermine (MPT) in urine. A 5 μL aliquot of diluted urine was injected into the LC–MS/MS system. Chromatographic separation was performed by reversed-phase C18 column with gradient elution for all analytes within 5 min. Identification and quantification were based on multiple reaction monitoring (MRM) detection. Linear least-squares regression with a 1/x2 weighting factor was used to generate a calibration curve and the assay was linear from 50 to 15000 ng/mL (PT and MPT) and 5 to 750 ng/mL (NHOPT). The intra- and inter-day precisions were within 8.9% while the intra- and inter-day accuracies ranged from −6.2% to 11.2%. The limits of quantification were 3.5 ng/mL (PT), 1.5 ng/mL (NHOPT) and 1.0 ng/mL (MPT). Method validation requirements for selectivity, dilution integrity, matrix effect and stability were satisfied. The applicability of the developed method was examined by analyzing urine samples from drug abusers.