WCPS: Application of Compound Independent Calibration (CIC) Software for the Quantitation of As-species in Undiluted Urine by LC-ICP-MS

Significance of Topic

Effective quantification of arsenic species in biological fluids is vital for clinical diagnostics, toxicological investigations and environmental exposure assessments. Liquid chromatography coupled with inductively coupled plasma mass spectrometry (LC-ICP-MS) has become a gold standard for arsenic speciation due to its sensitivity and selectivity. Yet the reliance on individual compound calibration standards can be limiting when pure species are unavailable or costly. Compound independent calibration (CIC) offers a streamlined alternative by leveraging the element-specific response of ICP-MS to arsenic, reducing dependence on expensive and scarce standards.

Objectives and Overview of Study

The primary goal of the presented work is to compare the performance of compound independent calibration (CIC) against traditional compound specific calibration (CSC) for the quantitation of five arsenic species—arsenobetaine (AB), monomethylarsonic acid (MMA), arsenite (As(III)), dimethylarsinic acid (DMA) and arsenate (As(V))—in undiluted urine samples. The authors aim to demonstrate CIC’s feasibility, assess its robustness over extended runs and illustrate potential for simplifying routine speciation workflows.

Methodology and Instrumentation

Samples comprised twelve patient urine specimens, each injected directly without dilution. Calibration standards were prepared for CSC and CIC using only As(V) for compound independent calibration. The Agilent 1260 HPLC system equipped with an arsenic speciation column and guard column was interfaced to an Agilent 7700x ICP-MS. Key operating conditions included:

• Mobile phase: 2.0 mM phosphate buffer/0.2 mM EDTA/10 mM acetate/3.0 mM nitrate/1% ethanol at pH 11.00
• Flow rate: 1.0 mL/min, injection volume: 5 μL
• ICP-MS: RF power 1550 W, sample depth 9.0 mm, spray chamber at 2 °C, carrier gas 1.04 L/min, makeup gas 0.3 L/min

Main Results and Discussion

Over a continuous 13-hour analytical run with 36 repeated urine injections and 12 standard injections, CIC and CSC delivered closely matching results. The ratio CIC/CSC for each species ranged from 0.89 to 1.16, indicating agreement within 16%. As(V) quantitation remained consistent throughout the run, with no observable drift in retention time or signal intensity. Chromatographic overlays of a 50 μg/L mixed standard over the entire sequence confirmed stable separation and reproducible peak integration windows.

The direct comparison highlights that CIC can substitute for CSC without significant loss of accuracy. The slightly higher variance observed for MMA and As(III) suggests that combining multiple species for calibration or optimizing As(V) standard concentration could further refine the approach.

Benefits and Practical Application

Compound independent calibration offers several advantages:

• Elimination of expensive or hard-to-obtain pure standards for each species
• Simplified calibration workflow using a single, readily available arsenic standard
• Capability to quantify unexpected or unknown arsenic compounds by relying on elemental response
• Robustness over extended analytical sequences, reducing downtime for recalibration

These benefits facilitate routine monitoring of arsenic exposure in clinical laboratories and support high-throughput environmental screening.

Future Trends and Opportunities

Advancements may include development of multi-element CIC strategies to cover broader speciation targets simultaneously. Integration of automated data processing and machine-learning algorithms could enhance peak recognition of unknown arsenic species. Further research into CIC calibration across different matrices (water, soil extracts, food) will extend its applicability. Collaboration between instrument vendors and regulatory bodies could establish CIC protocols as standardized methods for trace element speciation.

Conclusion

This study demonstrates that compound independent calibration using As(V) as a universal calibrant yields quantitation of urinary arsenic species comparable to compound specific calibration. The approach streamlines calibration procedures, reduces reliance on multiple standards and maintains analytical robustness. CIC represents a practical and cost-effective solution for routine arsenic speciation in clinical and environmental laboratories.

Reference

Routine Analysis of Toxic Arsenic Species in Urine Using HPLC with ICP-MS; T. Sakai, S. Wilbur. Agilent Technologies Application Note 5989-5505EN, January 2011.