HPLC-UV Method for the Fractionation of Basic and Neutral Compounds Extracted from Human Plasma and Urine using SOLA CX

Significance of the Topic

Solid phase extraction and HPLC-UV analysis of basic and neutral compounds in plasma and urine is critical for bioanalytical, clinical and QA/QC laboratories where high throughput, reproducibility and low solvent consumption are required.

Aims and Study Overview

The study aims to demonstrate the use of Thermo Scientific SOLA CX SPE cartridges to fractionate neutral and basic analytes from human plasma and urine, followed by separation on an Accucore RP-MS column with UV detection. Six model compounds (hydrocortisone, corticosterone, progesterone, procainamide, propranolol, amitriptyline) are evaluated.

Methodology and Instrumentation

Solid Phase Extraction:

• Cartridge: SOLA CX 10 mg/1 mL
• Conditioning: 1 mL methanol, 1 mL water
• Loading: 350 µL spiked plasma or urine
• Washing: 350 µL water with 2% formic acid
• Elution: two steps—350 µL methanol; 350 µL methanol with 5% ammonia
• Post-processing: add internal standard and dilute with water

HPLC-UV Analysis:

• Instrument: Thermo Scientific HPLC system, UV detection at 254 nm
• Column: Accucore RP-MS 2.6 µm, 50 × 3 mm
• Mobile phases: 20 mM ammonium acetate (A) and acetonitrile (B)
• Gradient: 5% B to 95% B over 5 min, hold, then return to 5%
• Flow rate: 0.8 mL/min; Temperature: 25 °C; Injection: 10 µL

Main Results and Discussion

Initial co-elution of hydrocortisone and propranolol when analyzing all analytes together was resolved via SPE fractionation. Neutral steroids and basic drugs were collected separately, yielding clear chromatograms. Recoveries ranged from 87% to 102% with RSDs below 12% for all compounds in both matrices, demonstrating high accuracy and precision.

Benefits and Practical Applications

• High recoveries and reproducibility for neutral and basic analytes
• Reduced solvent usage and simplified workflow
• Cleaner extracts yielding improved sensitivity
• Adaptable to high-throughput bioanalytical and clinical assays

Future Trends and Potential Applications

Future developments may include coupling SPE fractionation with mass spectrometry for enhanced selectivity, miniaturized automated SPE for microsampling, expansion to diverse analytes and matrices, and integration with UHPLC or online SPE for further throughput gains.

Conclusion

The combination of SOLA CX SPE cartridges and Accucore RP-MS HPLC columns offers a robust, efficient method for fractionating and quantifying basic and neutral compounds in human plasma and urine, delivering excellent recoveries, precision and clean chromatograms suitable for routine bioanalysis.

References

• Thermo Fisher Scientific. ANCCSBAS&NEUT: HPLC-UV Method for the Fractionation of Basic and Neutral Compounds Extracted from Human Plasma and Urine using SOLA CX. 2011.