Quantification of EPA 1694 Pharmaceuticals and Personal Care Products in Water at the ng/L Level Utilizing Online Sample Preparation with LC-MS/MS

Significance of the Topic

Trace levels of pharmaceuticals and personal care products (PPCPs) in water pose emerging risks to ecosystems and human health. Regulatory methods such as EPA 1694 require labor-intensive offline solid-phase extraction (SPE) of large sample volumes. A streamlined, sensitive approach enables routine monitoring at ng/L concentrations while reducing time, labor, and cost.

Objectives and Overview

This study demonstrates an online sample preparation workflow using the Thermo Scientific EQuan system coupled to LC-MS/MS for the quantification of 67 PPCPs, including antibiotics, beta-blockers, antifungals, and benzodiazepines. The aim was to replace manual SPE of 1 L water with direct injection of 0.5 mL–20 mL samples, achieve ng/L limits of quantitation, and shorten analysis to a single 10-minute run.

Methodology and Instrumentation

An online two-column strategy was implemented: a Hypersil GOLD aQ trapping column (2.1 × 20 mm, 12 µm) for pre-concentration and a Betasil C18 analytical column (2.1 × 100 mm, 3 µm) for separation. The system layout combined an Accela 600 pump, Accela 1250 pump, and six-port valves. Detection was performed on a TSQ Vantage triple Stage quadrupole mass spectrometer operating in positive electrospray SRM mode with time-scheduled windows. Sample pH (2.9, 6.6, 11.3) and acetonitrile content (5–20%) were optimized for retention and sensitivity. Metformin, a highly polar analyte, was separated by HILIC as per EPA 1694 guidelines.

Main Results and Discussion

• Single 10-min LC-MS/MS method quantified 67 PPCPs at or below 10 ng/L (0.5 mL injection).
• Sample pH critically influenced analyte stability and solubility: chlorotetracycline converted to 4-epi form at pH 11.3; penicillin derivatives showed pH-dependent recoveries.
• Increasing acetonitrile improved signal for lipophilic macrolides (e.g., tylosin, roxithromycin) but reduced trapping of certain fluoroquinolones and sulfonamides when > 10% ACN.
• Optimal compromise: acidified (pH 2.9) and basified (pH 11.3) water with 10% ACN delivered broad retention and recoveries.
• Time-scheduled SRM windows maximized dwell time, yielding low- or sub-ng/L limits of quantitation on the TSQ Vantage.

Benefits and Practical Applications

• Eliminates laborious offline SPE of large volumes, reducing cold-storage requirements.
• Shortens total analysis time from hours to minutes per sample.
• Uses small sample volumes (0.5–20 mL), lowering waste and solvent consumption.
• Maintains ng/L sensitivity and selectivity across diverse chemical classes.

Future Trends and Opportunities

Continued method expansion may include additional PPCP classes, integration of high-resolution mass spectrometry for non-target screening, fully automated robotic sampling workflows, and real-time field-deployable devices. Advances in HILIC and mixed-mode chromatography could further improve coverage of ultra-polar analytes. Data-driven environmental monitoring networks will benefit from rapid, high-throughput online sample preparation platforms.

Conclusion

An online EQuan LC-MS/MS approach offers a rapid, sensitive, and high-throughput alternative to traditional SPE for quantifying PPCPs at ng/L levels in water. By optimizing pH and organic content, a single 10-minute method covers 67 compounds with minimal sample handling, demonstrating a practical workflow for environmental laboratories and regulatory monitoring.

References

1. EPA Method 1694: Pharmaceuticals and personal care products in water, soil, sediment, and biosolids by HPLC/MS/MS, December 2007, EPA-821-R-08-002.
2. Loftin KA, Adams CD, Meyer MT, Surampalli R. Effects of Ionic Strength, Temperature, and pH on Degradation of Selected Antibiotic. J Environ Qual. 2008;37:378–386.