Using Heteroatoms as “Natural Labels” in the Quantitative Analysis of Active Pharmaceutical Ingredients by HPLC-ICP-MS

Significance of the Topic

Quantitative analysis of active pharmaceutical ingredients (APIs) is critical throughout drug development and quality control. Conventional LC-MS/MS methods offer excellent molecular selectivity but often require compound-specific calibration and are subject to matrix effects. Inductively Coupled Plasma Mass Spectrometry (ICP-MS), particularly in triple quadrupole (ICP-QQQ) MS/MS mode, provides structure-independent elemental quantification at trace levels. By exploiting heteroatoms such as sulfur, phosphorus or chlorine naturally present in APIs, HPLC-ICP-QQQ enables accurate, sensitive, and interference-free quantification without derivatization or isotopic labels.

Objectives and Study Overview

This work demonstrates the application of HPLC-ICP-QQQ in MS/MS mass-shift mode to quantify small-molecule APIs (sulfonamides, zoledronic acid, clonidine hydrochloride) and a monoclonal antibody (mAb) using their intrinsic heteroatoms (S, P, Cl) as natural labels. Key aims were to:

• Establish mass-shift methods for S, P and Cl using O₂ or H₂ cell gases.
• Determine method detection limits (MDLs) and recoveries for each API.
• Assess applicability to both small molecules and large biomolecules.

Methodology

Samples of three sulfonamide APIs were dissolved, filtered, and injected in an isocratic mobile phase (13% acetonitrile/0.1% formic acid). Zometa® zoledronic acid hydrate was diluted 2000-fold in a buffered organic mobile phase. Catapres® clonidine hydrochloride tablets were sonicated, filtered and analyzed in an aqueous/acetonitrile isocratic system. A monoclonal antibody (IgG2a) was diluted in ultrapure water and separated by size exclusion chromatography.

Instrumentation

An Agilent 1260 Infinity Bio-inert HPLC system with quaternary pump and autosampler was interfaced to an Agilent 8800 Triple Quadrupole ICP-MS operated in MS/MS mass-shift mode. Small molecules were separated on a ZORBAX Plus C18 column; the mAb was analyzed on a Bio SEC-3 column. Cell gases were O₂ for oxidation of S and P precursors and H₂ for conversion of Cl to ClH₂⁺. Internal standards (Co, Y) were infused online for tuning and stability monitoring, with no drift correction required.

Key Results and Discussion

• Sulfonamide APIs yielded MDLs around 23 nM (1.5 ppb S) with clear, interference-free chromatograms at 100 ppb S load.
• The mAb was quantified via its ~1% sulfur content, achieving an MDL of 14 nM (40 ng) with baseline separation.
• Zoledronic acid hydrate in Zometa® recovered 102% at 426 µg/L API with an MDL of 25 nM (1.5 ppb P).
• Clonidine hydrochloride from Catapres® was quantified with 96% recovery at 75 µg API per tablet and an MDL of 146 nM (15 ppb Cl).

The MS/MS mass-shift capability effectively eliminated polyatomic interferences by filtering the precursor ion in Q1 and detecting only the mass-shifted product in Q2.

Practical Implications and Benefits

HPLC-ICP-QQQ offers:

• Structure-independent quantification eliminating the need for compound-specific molecular standards.
• High selectivity through element-specific detection and immuno- or chemical tagging options.
• Robust performance in complex matrices due to the high-temperature plasma and MS/MS filtering.
• Applicability to both small APIs and large biotherapeutics.

Future Trends and Opportunities

Ongoing developments aim to:

• Lower detection limits further by optimizing reaction chemistries and instrument sensitivity.
• Expand the range of detectable heteroatoms and explore novel cell gases.
• Integrate rare earth element tagging for non-naturally heteroatom-containing compounds.
• Apply HPLC-ICP-QQQ in bioanalysis, immunoassays, and advanced QA/QC workflows in pharmaceutical manufacturing.

Conclusion

HPLC-ICP-QQQ in MS/MS mass-shift mode enables accurate, sensitive quantification of heteroatom-containing pharmaceuticals without derivatization. The method complements molecular MS techniques by providing elemental specificity, minimal matrix effects, and reliable performance across diverse compound classes, making it a valuable tool for drug development, biotherapeutics analysis, and quality control.

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