LC-MS/MS method for the determination of raloxifene and its glucuronide metabolites from human plasma using SPE micro elution for rapid, high-throughput sample processing

Significance of the Topic

Raloxifene is a widely used selective estrogen receptor modulator for the prevention and treatment of postmenopausal osteoporosis. Its extensive first-pass metabolism yields two major glucuronide conjugates (raloxifene-4-glucuronide and raloxifene-6-glucuronide) whose quantification in human plasma is crucial for pharmacokinetic studies, therapeutic monitoring, and assessment of inter-patient variability. A rapid, sensitive, and high-throughput analytical approach is therefore essential to support clinical research and drug development.

Goals and Overview

The study aimed to develop and validate a streamlined LC-MS/MS workflow for simultaneous determination of raloxifene and its two glucuronide metabolites in human plasma. Key objectives included minimizing sample volume, enhancing throughput via micro-elution solid-phase extraction (SPE), achieving baseline separation of analytes, and demonstrating robust linearity, accuracy, and precision over relevant concentration ranges.

Methodology and Used Instrumentation

A 96-well micro-elution SPE plate (SOLAµ SCX) was employed for plasma cleanup and concentration, followed by reversed-phase gradient separation on a Hypersil GOLD PFP column (100 × 3 mm, 3 µm). Tandem mass spectrometric detection was performed on a Thermo Scientific TSQ Vantage with heated electrospray ionization operated in positive SRM mode.

• Sample preparation: 295 µL plasma acidified with formic acid, spiked with internal standard (d4-raloxifene) and analyte standards, loaded onto SPE, washed, eluted in low volumes of methanol/ammonia, then diluted.
• Chromatography: Mobile phase A = water + 0.1% formic acid; B = acetonitrile + 0.1% formic acid; gradient from 20% to 80% B over 6 min, flow 0.5 mL/min, 30 °C, 10 µL injection.
• MS settings: HESI-2 source, spray voltage 4,000 V, vaporizer 400 °C, collision pressure 1.5 mTorr; monitored transitions m/z 474.2→112.1 (RAL), 650.2→112.0 (R4G/R6G), 478.2→116.1 (IS).

Used Instrumentation:

• Thermo Scientific SOLAµ SCX 96-well micro-elution SPE plate
• Thermo Scientific Hypersil GOLD PFP HPLC column
• Thermo Scientific Dionex UltiMate 3000 RS UHPLC system
• Thermo Scientific TSQ Vantage triple quadrupole mass spectrometer

Main Results and Discussion

Calibration curves were linear with R2 ≥ 0.995 across 0.02–2 ng/mL (raloxifene), 3–300 ng/mL (R4G), and 0.6–60 ng/mL (R6G). Limits of quantitation provided clear, well-resolved SRM peaks at the lowest concentrations. Accuracy deviations were within ±12% for all analytes over the dynamic ranges. Intra- and inter-day precision (RSD) at low, medium, and high QC levels remained below 12% for peak area ratios. Recoveries averaged 112% for raloxifene, and 56–60% for the glucuronides. Matrix effects were minimal for raloxifene (±5%), with moderate suppression/enhancement observed for conjugates (±15%). Baseline separation of all three analytes and internal standard was achieved within a 7.5-min run time.

Benefits and Practical Applications

• High sensitivity and low limits of quantitation suitable for pharmacokinetic profiling.
• Reduced solvent consumption and sample volume via micro-elution SPE.
• Improved throughput for large clinical studies with 96-well plate format.
• Reproducible extractions and robust retention of analytes on a PFP stationary phase for challenging polar metabolites.

Future Trends and Potential Applications

Advances may include further miniaturization of SPE workflows, integration with automated liquid handlers, and extension to additional conjugated metabolites or structurally related compounds. Emerging high-resolution mass spectrometers and multiplexed separation platforms could enhance specificity and throughput. Real-time therapeutic drug monitoring and application in special populations (e.g., pediatric, hepatic impairment) represent promising areas for method adaptation.

Conclusion

The described LC-MS/MS method combining SOLAµ micro-elution SPE, a PFP HPLC column, and TSQ Vantage detection provides a rapid, sensitive, and reproducible workflow for quantifying raloxifene and its glucuronide metabolites in human plasma. It meets stringent criteria for linearity, accuracy, precision, and throughput, making it suitable for clinical pharmacokinetic and bioavailability studies.

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