Page 104 - Application Handbook - Liquid Chromatography

LAAN-A-LC-E234

Application High Performance Liquid Chromatography

News Ion Analysis in Drugs (Part 4)

Determination of Counterions (Anions) by Ion
No.L457 Chromatography

In Application News No.L387, we introduced examples Table 2 Analytical Conditions
of impurity ion analysis and counterion analysis in Column : Shim-pack IC-A3 (150 mm L.×4.6 mm I.D.)
pharmaceuticals using ion chromatography. Typically, a Mobile Phase : A : 8.0 mmol/L p-Hydroxybenzoic acid
variety of counterions are used to selecting the 3.2 mmol/L Bis-Tris
optimum salts in the development stage of a 50 mmol/L Boric acid
B : Acetonitrile
pharmaceutical product because the physicochemical A : B = 95:5 (v/v)
and pharmacokinetic properties associated with the Flowrate : 1.2 mL/min
active pharmaceutical ingredients (API) will vary Column Temp. : 40 ˚C
depending on differences in counterions. Furthermore, Injection Volume : 50 μL
: Conductivity (Non-suppressor mode)
Detection
as inorganic substances such as catalysts and ions used
during synthetic process may affect such properties as
solubility and stability, it is very important to conduct
analysis of ionic contaminants. In order to eliminate the uV
impurities from the synthesized API, HPLC is commonly
used for fractionation and preparative purification. For 1000 ˙Peaks
the preparation of mobile phase, acetic acid, formic 1. Acetate
acid, trifluoroacetic acid, and their salts are used as 2. Formate
additives for easier post-processing. Additionally, in the 3. Chloride
solid phase synthesis of peptides, trifluoroacetic acid or 800 4. TFA
its salt is used for the isolation of the synthesized
peptide from the solid phase, so trifluoroacetate
becomes the counterion for the peptide that is the
principle component. In many cases, after that, salt 600
replacement by means of acetate or chloride ions takes
place before use. Consequently, such ions can become
counterions or impurity ions in the API. 2
Here, we introduce some examples of the analysis of 400
acetate, formate, chloride, and trifluoroacetate ions in a
pharmaceutical product. 3

200
n Analysis of Trace Amounts of Anions 1 4
A low-concentration analysis of acetate, formate,
chloride, and trifluoroacetate (TFA) ions was carried out.
Fig. 1 shows the chromatogram that was obtained from 0
the standard solution. The retention time and the area
repeatability (n = 6) are shown in Table 1, and the 0.0 2.5 5.0 7.5 10.0 min
analytical conditions used are shown in Table 2.
Fig. 1 Chromatogram of a Four-Anion Standard Mixture

Table 1 Repeatability
Conc (mg/L) R.T. %RSD Area %RSD
Acetate 2.5 0.05 0.54
Formate 2.5 0.04 0.68
Chloride 0.5 0.03 0.78
TFA 10 0.02 0.85