Revealing Pharmacokinetic Interactions of Tramadol, Tapentadol, and Venlafaxine: A Cutting-Edge Liquid Chromatography–Tandem Mass Spectrometry Analytical Approach in Rat Plasma

The detection and quantification of opioid analgesics adulterated with serotonin reuptake inhibitors are critical in forensic toxicology due to their significant clinical and legal implications. This study focuses on developing and validating a highly sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the simultaneous quantification of tramadol (TMD), tapentadol (TAP), and venlafaxine (VEN) in small volumes of rat plasma. The method employs a straightforward single-step protein precipitation technique for sample pretreatment. Utilizing a Shim-pack Velox SP-C18 column (2.7 μm, 2.1 × 150 mm, Shimadzu, Japan), a 12 min gradient elution was performed with a mobile phase of 0.1% formic acid in water (solvent A) and acetonitrile (solvent B). Detection and quantification were achieved in multiple-reaction-monitoring mode, with ion transitions of m/z 264.3 → 58.1 for TMD, m/z 222.3 → 107.1 for TAP, m/z 278.2 → 58.2 for VEN, and m/z 195.5 → 138.1 for caffeine, which served as the internal standard (IS). The method exhibited excellent linearity, with concentration ranges of 1–500 ng/mL for TMD (r² = 0.9981), 1–1000 ng/mL for TAP (r² = 0.9972), and 1–900 ng/mL for VEN (r² = 0.9987) in rat plasma using only 50 μL of sample. This validated method was applied to a pharmacokinetic interaction study, revealing significant drug interactions: the maximum observed plasma concentration (C_max) and area under the curve (AUC) for both TMD and VEN decreased. For TMD, C_max decreased by 2.58-fold from 276.25 to 106.99, and AUC_0–t decreased by 1.4-fold from 3005.8 to 2159.3. For VEN, C_max decreased by 2.51-fold from 494 to 191, and AUC_0–t decreased by 2.3-fold from 4988 to 2114. However, for TAP, C_max increased by 3.4-fold from 138.54 to 471.85, and AUC_0–t increased by 2.66-fold from 1060.1 to 2826.8. The concurrent administration likely creates metabolic competition at CYP2D6 and UDP glycosyltransferase enzyme sites, affecting the pharmacokinetic parameters. These findings underscore the importance of further studies to monitor the simultaneous presence of these drugs and their metabolites in plasma, especially when coadministration occurs unintentionally.