Development, Validation, and Application of a Novel Multi-Analyte High-Resolution Gas Chromatography-Mass Spectrometry (HRGC-MS) Method for the Quantification of Opioid Derivatives, Including Heroin, Morphine, and Fentanyl, in Biological Matrices with Enh

Abstract

critical demand for analytical methods capable of detecting opioids with exceptional sensitivity and specificity in complex biological matrices. Traditional gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) methods often encounter challenges such as matrix interference, low volatility of analytes, and limited multi-analyte capability.

Objectives : This study aimed to develop, validate, and apply a novel multi-analyte high-resolution gas chromatography–mass spectrometry (HRGC–MS) method for the simultaneous quantification of heroin, morphine, and fentanyl in biological matrices (plasma, urine, and tissue). The method was designed to provide enhanced sensitivity, reproducibility, and selectivity, following international guidelines (FDA, 2018; ICH, 2022).

Methods : Sample preparation employed solid-phase extraction (SPE) for efficient analyte recovery, followed by derivatization using BSTFA + 1% TMCS to improve volatility and stability. Chromatographic separation was achieved using a DB-5 ms Ultra Inert column (30 m × 0.25 mm, 0.25 µm) with helium as the carrier gas (1.0 mL min⁻¹). The HRGC–MS operated under electron ionization (70 eV) and high-resolution mode (≥ 60,000 FWHM) to ensure precise mass accuracy (< 2 ppm). Validation parameters—linearity, accuracy, precision, recovery, matrix effects, stability, and sensitivity—were assessed according to FDA (2018) and ICH Q2(R2) criteria.

Results : The method exhibited excellent linearity (R² ≥ 0.998) across wide calibration ranges: heroin (0.5–500 ng mL⁻¹), morphine (1–1000 ng mL⁻¹), and fentanyl (0.1–100 ng mL⁻¹).

Accuracy and precision were within ±5% and <6% RSD, respectively.

Limits of detection (LODs) reached 0.15 ng mL⁻¹ for heroin, 0.30 ng mL⁻¹ for morphine, and 0.03 ng mL⁻¹ for fentanyl, representing a 3–6-fold improvement over conventional GC–MS (Valdez et al., 2022).

Mean recovery ranged from 89% to 96%, with matrix effects below ±10%. Application to authentic forensic and clinical samples confirmed reliable detection of opioids and metabolites (6-acetylmorphine, morphine) with minimal interference.

Conclusions: The validated HRGC–MS method provides superior sensitivity, specificity, and reproducibility for simultaneous opioid analysis. Its multi-analyte capability, short run time (13.8 min), and robust matrix tolerance make it a powerful tool for forensic toxicology and clinical drug monitoring. This approach establishes a scalable framework for detecting emerging synthetic opioids and can be expanded through automation or hybrid HRGC–MS/MS coupling to further enhance analytical throughput and scope.