Using ¹³C-labeled Standards and ID-HRMS for Low-Level PAH Determination in Water and Tissue Samples

David Hope | Pacific Rim Laboratories 

Introduction

Polycyclic aromatic hydrocarbons (PAHs) are multi-ringed aromatics with two to six fused benzene rings. Traditional methods of analysis (EPA 8270, 625) involve full-scan GC-MS, however, for more sensitivity (10-100 ng/L or 1-10 ng/g) one can either use selected ion monitoring (SIM) or HPLC with diode array and fluorescence detectors. In each case, surrogates (deuterated PAHs) are added to monitor extraction efficiency, and final quantitation is done by internal standard methods. 

In order to achieve the very lowest detection limits, we utilize isotope dilution high resolution mass spectrometry (ID-HRMS) with ¹³C-labeled internal standards. This technique allows for detection limits reaching parts-per-trillion (ppt) levels in tissue, and parts-per-quadrillion (ppq) levels in water. 

Methodology

Some deuterated PAH standards can undergo deuterium exchange, which may compromise the method’s ability to achieve extremely low limits of detection. Therefore, we turned to ¹³C isotopically labeled standards from Cambridge Isotope Laboratories, Inc. (CIL) as these compounds: 1) do not undergo exchange; and 2) typically have undetectable or negligible amounts of native content. The method uses ¹³C-labeled analogs of the analytes of interest, added to the sample before extraction and then used as internal standards for quantitation. The ¹³C standards can also be used as surrogates and their recoveries calculated. 

In order to reduce PAH detection limits, we set up an analytical method using ¹³C-labeled PAH standards. 50 ng each of 16 ¹³C-PAHs (CIL catalog no. ES-4087) was added to 12 real-world river water samples that had been adjusted to pH 10. The samples were extracted with dichloromethane and the extract concentrated to a final volume of 1 mL prior to GC-HRMS analysis (Thermo DFS). Isotope dilution calculations were used whereby the native PAH was calculated relative to its ¹³C-analog (e.g., ¹³C₆-naphthalene was used to quantify naphthalene). Concentrations for the 16 native PAHs ranged from 0.14-2.28 ng/L. The data was used to calculate method detection limits (as defined in 40 CFR 136 Appendix B) ranging from 0.08-0.85 ng/L. Instrument detection limits, based on a peak area with signal/noise ratio of 4, were 0.023-0.26 ng/mL. 

Tissue samples can have significant lipid concentrations that must be removed prior to any GC analysis. We use base saponification for this purpose. Eight 10 g sole filets were fortified with 5 ng PAH and 50 ng each of 16 ¹³C PAHs. The tissue was placed in a boiling flask with 100 mL of methanol and 10 g of NaOH and refluxed for 1 hour. Water was added and the solution again refluxed for 1 hour. The solution was extracted with hexane and washed with water prior to cleanup on silica gel. Recovery of the PAH ranged from 86-121%, with MDLs calculated at 0.07-0.23 ng/g. 

Conclusion

Low-concentration PAH analysis for environmental and food samples can be achieved using ¹³C-labeled standards coupled with GC-HRMS. Detection limits are <1 ng/L for water and <0.3 ng/g for tissue. Recovery standards can be added prior to HRMS analysis in order to quantify the recovery of the ¹³C-PAH internal standards, thus giving the analyst additional information on the quality of the data.