Evaluation of the programmed temperature vaporiser for large-volume injection of biological samples in gas chromatography

Importance of the Topic

The analysis of trace-level drugs in biological fluids demands highly sensitive and selective techniques. Programmed temperature vaporiser (PTV) large-volume injection in gas chromatography (GC) offers a route to enhance detection limits by introducing tens to hundreds of microlitres of sample into the GC system. This approach is particularly important for therapeutic drug monitoring, forensic toxicology and pharmacokinetic studies, where analyte concentrations in plasma or other matrices often fall below conventional detection thresholds.

Objectives and Study Overview

This application note evaluates the use of a PTV fitted with a packed liner for the large-volume injection of plasma extracts into GC. The study aims to:

• Assess solvent and packing purity requirements for volumes up to 100 µl;
• Investigate liquid capacity and inertness of several liner packings;
• Compare flame ionisation detection (FID) and mass-selective detection (MSD) in total ion current (TIC) and selected ion monitoring (SIM) modes;
• Determine achievable detection limits for secobarbital, lidocaine, phenobarbital and diazepam in plasma.

Methodology and Instrumentation

• Sample preparation by solid-phase extraction (SPE) on C18 cartridges; plasma (1 ml) loaded in pH 6 phosphate buffer; acidic and alkaline drug fractions eluted, evaporated, reconstituted in ethyl acetate.
• GC system: Hewlett-Packard 5890 II for FID and HP 5971 MSD for mass spectrometry.
• Columns: HP-5 (30 m × 0.32 mm, 0.25 µm) for FID; HP-5 MS (30 m × 0.25 mm, 0.25 µm) for MSD.
• PTV injector: OPTIC 2 with 80 mm × 3.4 mm liners packed with either ATAS “A” treated material, silanised glass wool or capillary fragments.
• Temperature program: injector 40 °C (solvent vent) then ramp to 290 °C; column from 40 °C up to 290 °C in three stages; helium carrier gas flow 1.35 ml/min (FID) or 0.48 ml/min (MSD).

Main Results and Discussion

• Solvent purity: high-grade ethyl acetate (residue-analysis grade) required to avoid background peaks with 100 µl injections.
• Liner capacity: ATAS “A” liners tolerated up to ~115 µl, glass wool ~65 µl.
• Packing inertness: fresh ATAS “A” liners showed minimal adsorption for all test drugs; glass wool retained basic drugs but adsorbed acidic barbiturates.
• Liner ageing: repeated injections of plasma extracts (>15 injections) gradually reduced inertness of ATAS “A” packing, indicated by brown coloration and reduced recoveries, especially for phenobarbital.
• Linearity: excellent correlation (R²> 0.998) over 20–100 µl injection volumes and 5–2000 ng/ml concentration range without internal standard correction.
• Detection limits: GC-FID detected 5–10 ng per 50 µl extract (5–10 ng/ml plasma); GC-MSD TIC improved to ~4 ng; MSD-SIM achieved 0.25–0.5 ng (250 pg/ml plasma) in alkaline fraction.
• Matrix effects: non-selective FID exhibited higher background from co-injected matrix; MSD-SIM suppressed background interference effectively.

Benefits and Practical Applications

The PTV-GC approach with large-volume injection:

• Extends sensitivity of routine GC for bioanalysis without complex coupling to LC.
• Enables detection of drugs at sub-ng/ml levels in plasma using common SPE and GC-MS equipment.
• Reduces sample preparation while maintaining quantitative performance.

Future Trends and Potential Uses

Advances may include:

• Automated on-line SPE–PTV-GC interfaces to minimize manual handling and increase throughput.
• Exploration of novel inert packing materials or deactivation protocols to extend liner lifetime under biological matrices.
• Application to a broader range of compounds (e.g., steroids, illicit substances) and alternative matrices (urine, tissue homogenates).

Conclusion

The study demonstrates that a PTV with ATAS “A” packing allows reliable large-volume injections of plasma extracts, yielding detection limits up to 50-fold lower than standard injections. Mass spectrometric detection in SIM mode further enhances sensitivity and selectivity for trace drug analysis. Regular monitoring of liner inertness is essential to maintain performance over multiple injections.

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