Method Development And Validation Of An Hplc Assay For Choline In Carbachol Formulations Using A Post Column Reactor And A Chemically Modified Glassy Carbon Electrode

R.L. Steffen1 , L. Liu1 , G. Chong1 , R. Frank1 , T. Gibson2

1Bioanalytical System Inc, West Lafayette, IN USA
2Alcon Laboratories, Inc, Ft.Worth, TX, USA


Carbachol (carbamylcholine chloride) is the active ingredient in some ophthalmic formulations used to induce miosis (pupil contraction) during eye surgeries. The miotic reduces the intraocular pressure in the eye by increasing the amount of fluid the eye drains. A diminished concentration of carbachol in the formulation may prevent the effective reduction of intraocular pressure, which may have several deleterious effects such as iris prolapse. To ascertain whether the concentration of carbachol remains at a therapeutically active level under different storage conditions in a stability program, a method to selectively detect and quantitate choline, a hydrolytic degradation product of carbachol, was developed and validated at low levels of degradant (<1 µg/mL). Choline is neither UV-absorbing nor electroactive under reasonable conditions. However, via enzyme catalyzed reaction, it is converted to hydrogen peroxide, which can be readily oxidized or reduced.


Choline is separated from other components in the matrix by ion exchange using a BASi Acetylcholine/Choline analytical column (#MF-6150). The isocratic separation is accomplished by using:

  • 50 mM sodium phosphate (pH 8.0) mobile phase
  • column/cell temperature: 30 °C
  • flow rate: 1 mL/min
  • injection volume: 20 µL

A BASi immobilized enzyme reactor (IMER, #MF-6149) containing choline oxidase covalently attached to a polymeric substrate is placed after the analytical column and is used to convert choline to hydrogen peroxide by the following enzyme catalyzed reaction:

The resultant H O is reduced at the surface of a glassy carbon electrode, modified with a film of osmium poly(vinylpyridine) redox polymer sandwiched between horseradish peroxidase and the glassy carbon surface. The redox polymer effectively "wires" the horseradish peroxidase to the glassy carbon surface. The electrode is operated at only +100 mV (vrs Ag/AgCl) with a gain of 500 nA full scale.

The combination of an enzyme catalzyed reaction and a "wired" electrode operated at low potential creates a highly sensitive and selective detector.

Validation Results


System Suitability


Response is linear from LOQ to 1% of nominal analytical concentration of carbachol.


Method yields acceptable results for recovery and accuracy for sample preparations at 80, 100, and 120 percent of nominal levels of choline degradant.

Solution Stability

With the addition of preservative (1% ProClin®), standard solutions are stable for 7 days at room temperature or under refrigeration.


For reproducible peak area and retention time, flow rate and buffer concentration are critical parameters.

Ruggedness (Intermediate Precision)

Analytical method yields comparable results when different analysts run the same samples on different instruments with different solution preparations.

Multiple Injections

Multiple injections do not significantly deteriorate the performance of the column/post column reactor/modified glassy carbon electrode combination.


The results indicate that a rugged, selective, and sensitive method has been developed and validated for determining the concentration of choline at low-levels (ppb) in an ophthalmic formulation containing carbachol in a stability program.