Clavulanic acid is commonly used in combination with penicillin and cephalosporin antibiotics to overcome bacterial resistance.
Clavulanic acid is produced and isolated from Streptomyces clavuligeru with ethyl acetate and then converted to a stable form. One secondary purification approach uses nonaqueous precipitation
of potassium clavulanate by adding the potassium salt of 2-ethylhexanoic acid in isopropanol to the primary isolation extract.
While 2-ethylhexanoic acid is soluble in the organic solvent, a small fraction may co-precipitate with the potassium clavulanate.
The United States Pharmacopeia (USP) monograph for potassium clavulanate describes a GC–FID method to determine 2-ethylhexanoic
acid in the active pharmaceutical ingredient (API). This contribution describes a simpler approach for determining 2-ethylhexanoic
acid in clavulanate with a Reagent-Free Ion Chromatography (RFIC) system. USP clavulanate reference standards were prepared
at a 150-fold lower concentration in deionized water than the current USP method, spiked with 2-ethylhexanoic acid, and injected
directly without additional sample pretreatment. The target analyte was separated from clavulanate-related peaks using electrolytically
generated potassium hydroxide eluent and measured by suppressed conductivity detection.
Analyses are performed using a Dionex ICS-2100, AS Autosampler and Chromeleon Chromatography Workstation. Separations use
an IonPac AG11/AS11 column set (USP L61) with electrolytically generated KOH eluent.1
Results and Discussion
Figure 1 compares 500 µg/mL clavulanate with and without 2-ethylhexanoic acid spiked at the 0.8% acceptance criterion cutoff
value specified in the USP monograph. As shown, 2-ethylhexanoic acid is separated in less than 10 min without any interference
from minor components in the sample matrix. The method demonstrated good sensitivity with a low limit of detection (LOD) for
2-ethylhexanoic acid of 0.036 µg/mL, which corresponds to a concentration of 0.008% in the API. This represents a 100-fold
lower concentration than the 0.8% acceptance criterion cutoff value in clavulanate. Results for linearity, spike recovery,
retention time precision and peak area precision determinations show that IC is an accurate and reproducible technique to
determine 2-ethylhexanoic acid in clavulanate below the 0.8% acceptance criterion. The lower LOD achieved by this method allows
a 150-fold lower API concentration, thereby reducing cost. In addition, this proposed method eliminates the need for two solvent
extractions required in the GC–FID method in the current USP monograph, thus significantly simplifying sample preparation,
reducing waste, and further improving cost efficiency.
Figure 1: Chromatographic comparison of clavulanate without [trace (a)] and with [trace (b)] 4 µg/mL (0.8%) 2-ethylhexanoic
acid. Peak 1: 2-ethylhexanoic acid (4 µg/mL – 0.8%). Eluent: 3 mM KOH from 0–10 min; 3–60 mM KOH from 10–10.1 min; 60 mM KOH
from 10.1–20.1 min. Flow-rate: 0.25 mL/min. Temp.: 30 °C. Inj. Vol.: 5 µL. Detection: suppressed conductivity, ASRS 300 2
mm, recycle mode,2 mA suppressor current during 3 mM KOH, switch to 38 mA at 10.1 min.
1. Dionex Corporation. Determination of 2-Ethylhexanoic Acid Impurity in Clavulanate Using a Reagent-Free Ion Chromatography
System. Application Note 262 (LPN 2608, Oct., 2010); Sunnyvale, California, USA
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