Systematic SPE Method Development

Significance of the Topic

Accurate and efficient extraction of diazepam and its metabolites from biological matrices is crucial for therapeutic drug monitoring, pharmacokinetic studies and quality control in pharmaceutical and clinical laboratories. Solid phase extraction (SPE) streamlines sample preparation, removes endogenous interferences and enhances method sensitivity and reproducibility.

Objectives and Study Overview

This study aimed to develop a streamlined SPE-HPLC-UV protocol for simultaneous extraction of diazepam and its three major metabolites (temazepam, oxazepam and desmethyl diazepam) from goat serum. Key objectives included achieving ≥90% recovery, relative standard deviations below 3.5% at a 0.5 µg/mL spike level, detection limits ≤0.5 µg/mL, an analytical run time under 8 minutes and elimination of eluate evaporation and reconstitution steps.

Methodology and Instrumentation

A systematic SPE development workflow was applied: defining sample prep objectives, selecting retention mechanism and sorbent chemistry, optimizing load pH, wash and elution profiles, and validating under real-matrix conditions. Discovery DSC-8 monomeric C8 SPE 96-well plates (100 mg/well) were compared to conventional C18 plates. Goat serum samples (0.5 mL) spiked with analytes were diluted 1:1 in 25 mM ammonium formate pH 7.1 before loading. Wash and elution solvents varied in methanol content and pH to map analyte retention and release. HPLC-UV analysis employed a Discovery C18 column (5 cm × 4.6 mm, 5 µm) with mobile phase 45:55 methanol/10 mM ammonium acetate pH 4.5, flow rate 1.5 mL/min, column temperature 35 °C and detection at 240 nm.

Main Results and Discussion

Load optimization at neutral and high pH showed complete retention of all analytes. Wash/elution profiling revealed that at low pH (2.75) and 20% methanol non-target interferences were effectively removed, while full analyte recovery was achieved at 60% methanol. The systematically developed DSC-8 method delivered absolute recoveries between 90.0% and 99.9% with RSDs below 3.5%. In contrast, the generic C18 method yielded lower recoveries (68.5%–89.1%) and higher variability (RSDs up to 9.1%). Chromatograms from the DSC-8 extracts exhibited minimal background and well-resolved peaks within a 6 minute run time.

Practical Benefits and Applications

• Simplified workflow without evaporation and reconstitution steps
• Cleaner extracts with reduced matrix effects
• Shortened analytical runtime (6 min)
• High and reproducible recoveries (>90%)
• Compatibility with direct injection for high-throughput analysis

Future Trends and Potential Applications

The systematic SPE approach can be extended to other drug classes and biological matrices. Integration with LC-MS/MS platforms and automation in 96-well formats will further enhance throughput and sensitivity. Emerging sorbent chemistries and mixed-mode phases may offer tailored selectivity for complex analyte panels.

Conclusion

A structured SPE method development strategy using monomeric C8 Discovery DSC-8 plates has been demonstrated for efficient extraction of diazepam and its metabolites from goat serum. The optimized protocol delivers robust recoveries, simplified sample preparation and rapid HPLC-UV analysis, making it well suited for bioanalytical and QA/QC laboratories.