Analysis of Major Psychoactive Compounds in Nutmeg Using GC-MS/MS

Importance of the Topic

Nutmeg, a common culinary spice derived from Myristica fragrans seeds, contains psychoactive constituents safrole, myristicin, and elemicin. Excessive consumption for recreational purposes can lead to hallucinations and a range of toxic effects, including nausea, tachycardia, and neurological symptoms. Reliable quantification of these compounds in biological fluids is essential for forensic investigations, clinical management of poisoning cases, and advancing our understanding of nutmeg’s toxicokinetics.

Objectives and Study Overview

This study aimed to develop and validate a sensitive and selective gas chromatography-tandem mass spectrometry (GC-MS/MS) method for simultaneous quantification of safrole, myristicin, and elemicin in human serum. The method was then applied to a documented case of nutmeg overdose to determine serum concentrations over time and calculate elimination parameters.

Methodology and Instrumentation

Sample Preparation and Reagents:

• Human serum spiked with known concentrations of safrole, myristicin, elemicin (0.5–300 ng/mL) and an internal standard (meconin-d3).
• Extraction using MonoSpin® C18 cartridge: sequential conditioning with ethyl acetate, methanol, and water; sample loading; washing; elution with ethyl acetate.

Instrumentation and Analytical Conditions:

• GC–MS/MS system: Shimadzu GCMS-TQ™ 8040 NX with SH-I 5Sil MS capillary column (30 m × 0.25 mm, 0.25 µm).
• Oven program: 60 °C (1 min) to 320 °C at 10 °C/min, hold 10 min.
• Injector and transfer line temperatures: 280 °C; splitless injection; helium carrier gas at 1.8 mL/min.
• Ion source: EI at 70 eV, 200 °C; MRM transitions: safrole m/z 162 > 104/131, myristicin m/z 192 > 161/91, elemicin m/z 208 > 193/177, meconin-d3 m/z 162 > 104.

Validation Procedures:

• Selectivity assessed in pooled human sera.
• Linearity checked over 0.5–300 ng/mL with weighted (1/x²) regression.
• Limits of detection (LOD) and quantification (LOQ) determined by signal-to-noise and calibration slope (LOD 0.14–0.16 ng/mL; LOQ 0.5 ng/mL).
• Intra- and inter-day precision and accuracy evaluated at LOQ, low (1 ng/mL), medium (120 ng/mL), and high (240 ng/mL) concentration levels.
• Matrix effects, recovery, process efficiency, and stability (freeze/thaw, long-term, processed samples) were assessed.

Main Results and Discussion

Validation Outcomes:

• Excellent linearity (R² 0.996–0.997) across calibration range.
• Precision within 2.4–11 % RSD and accuracy bias between –2.6 and 2.1 %.
• Matrix effects corrected by internal standard, recoveries 79–95 %.
• Analytes stable under all tested conditions.

Application to Nutmeg Poisoning Case:

• Serum collected at admission (approx. 8 h post-ingestion) and at 8.7, 21.2, 45.7, and 93.7 h.
• Initial concentrations: safrole 16.7 ng/mL, myristicin 388 ng/mL, elemicin 844 ng/mL.
• By 45.7 h: 2.7, 65.2, and 30.6 ng/mL respectively; by 93.7 h: 0.5, 7.1, and < LOQ.
• Elimination half-lives calculated as 19.2 h (safrole), 16.9 h (myristicin), and 8.5 h (elemicin), correlating with symptom resolution.

Benefits and Practical Applications

This straightforward GC-MS/MS protocol enables high sensitivity and specificity for key nutmeg toxins in serum. It can be adopted by forensic and clinical laboratories for rapid diagnosis of nutmeg intoxications, therapeutic monitoring, and supporting legal casework. The low LOQ allows detection of trace exposure levels, while reliable pharmacokinetic data inform treatment strategies.

Future Trends and Potential Applications

Potential extensions include adaptation to other biological matrices (urine, hair), integration with high-resolution mass spectrometry for broader screening of natural toxins, automation of sample preparation, and development of rapid point-of-care assays. Expanded toxicokinetic studies in larger cohorts could refine risk assessments and regulatory guidelines for nutmeg-containing products.

Conclusion

A validated GC-MS/MS method using MonoSpin® C18 extraction demonstrates robust quantification of safrole, myristicin, and elemicin in human serum with high sensitivity and accuracy. Application to a real poisoning case provided the first human toxicokinetic profiles for these compounds, underscoring the method’s value in forensic toxicology and clinical toxicology.

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