Quantitation Of Pamoic Acid In Human, Rat, And Canine Plasma #2496

N. Simmons
Bioanalytical Systems, Inc., 2701 Kent Avenue, West Lafayette, IN, 47906



To develop a method for the quantitation of pamoic acid in human, rat and canine plasma.


Pamoic acid is an aromatic dicarboxylic acid used as an adjuvant to prolong therapeutic action. Pamoic acid and internal standard are extracted from plasma by protein precipitation. The supernatant is evaporated to dryness, reconstituted in a buffer, and then loaded onto SPE cartridges. Extracts are injected onto an HPLC system utilizing a YMC Basic column with a buffer/ion pair reagent/acetonitrile mobile phase.


The three different matrices yielded successfully validated methods. The resulting assays were sensitive, selective, accurate and precise. Inter-assay precision and accuracy for pamoic acid ranged from: 5.3% to 8.5% and -9.6% to -6.2%, respectively (rat plasma) and 7.8% to 11.4% and -1.4% to 0.3%, respectively (human plasma). The method was subsequently cross-validated into canine plasma.


This assay serves as a specific and sensitive analytical method to aid in pharmacokinetic measurements of pamoic acid in three different matrices.


  • To develop a method for the quantitation of pamoic acid in human, rat, and
    canine plasma
  • Extraction and chromatographic conditions will be based on previous method for pamoic acid in dog and rat serum*
  • Achieve a lower limit of quantitation of 2 ng/mL for human and canine plasma and 10 ng/mL for rat plasma
  • Maximize the extraction efficiency to hit the required lower limits

METHOD - Validation Scheme

  • Fresh Calibration Line
  • QCs at the Limits of Quantitation (n = 6)
  • 3-Day Inter-Assay Precision and Accuracy (Human and Rat Plasma)
  • 1-Day Cross-validation (Canine Plasma)

*Jorgensen, Martin. “Quantitative determination of pamoic acid in dog and rat serum by automated ion-pair solid-phase extraction and reversed-phase high-performance liquid chromatography”; J. Chrom. B, 716 (1998), 315-323.

Chemical Structures

General Assay Procedure

  • Precipitate proteins with acetonitrile
  • Transfer supernatant, evaporate acetonitrile, and reconstitute residue in pH 6 phosphate buffer
  • Extract samples using C18 SPE cartridges
  • Evaporate solvent and reconstitute before injecting on HPLC system equipped with fluorescence detector

Assay Specifics

Sample volume: 0.5 mL
Column: YMC Basic 150 x 4.6 mm, 5 µm (and guard)
Mobile phase: 52% acetonitrile / 48% phosphate buffer (10 mM),
cetyltrimethylammonium hydrogensulfate (5 mM)
Flow rate: 0.8 mL/min
Fluorescence detection: λex = 240 nm, λem = 523 nm
Injection volume: 35 µL

Calibration Standard Statistics

Inter-Assay Quality Control Sample Statistics

Representative Human Plasma Chromatograms

Representative Dog Plasma Chromatograms

Representative Rat Plasma Chromatograms

Typical Calibration Line


  • All three matrices validated with extraction efficiencies >95%
  • Selectivity/specificity: One method for human, canine, and rat plasma
  • Ruggedness: >5 months of preclinical sample analysis