Separation of Macrocyclic Lactones

Avermectins are a series of 16-membered macrocyclic lactone derivatives that are used extensively in animal and crop protection. They occur in nature and can be produced as fermentation by-products by the micro-organism, streptomyces avermitilis. Examples of well-known avermectins and derived products include ivermectin, eprinomectin, selamectin, doramectin, and abamectin. All the avermectins have high anthelmintic and insecticidal properties even at low dose levels and residues of these veterinary drug components reach the environment through manufacturing and animal waste and may potentially affect terrestrial and aquatic life forms.

Figure 1: Structures of selected macrocyclic lactones.

Several crop protection companies have strong interest in the synthesis, production, and analysis of these compounds. Therefore, there exists a need to develop a fast and efficient analytical method capable of determining avermectin residues on animals, in food, and in the environment. The HPLC method presented here is fast and efficient and can be used for residual analysis as well as for quality control of avermectins in drug formulations. The peaks produced are sharp resulting in high sensitivity.

Figure 2: Separation of macrocyclic lactones using FLARE C18 MM column.

The mixture can be baseline separated using gradient elution with an MS-compatible mobile phase containing acetonitrile, methanol, and water. What is even more interesting is that the four avermectin products considered and up to 20 separate degradation products could be identified.

Figure 3: Exploded view showing peaks between 8 and 12 min.

HPLC Conditions

Column name: FLARE C18 MM
Column dimensions: 4.6 × 150 mm (15698-14-2, 3.6 µm, 120 Å)
HPLC system: Agilent 1200
Inj. vol. and conc.: 5.0 µL, ~0.2 mg/mL of each major analyte in MeOH
Detection: UV at 244 nm
Flow rate: 1.0 mL/min
Solvents: A: 50 mL ACN, 100 mL MeOH, 850 mL H₂O; B: 600 mL ACN, 200 mL MeOH, 200 mL H₂O
Gradient: 0.00 min, 10% B; 10.00 min, 90% B; 10.01 min, 10% B; 18.00 min, 10% B
Temperature: 35 °C

The FLARE C18 MM column is manufactured with 3.6 µm diamond core-shell particles that have 120 Å pores. The particle geometry and tight particle size distribution contribute to high packing density and column efficiency with reduced plate height, h, of ~2. The columns come in various lengths and diameters and are compatible with 100% aqueous to 100% organic solvents. They are pH and temperature stable and available to ship worldwide.

Diamond Analytics
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Website: www.diamond-analytics.com