Development and Validation of a Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) Method for Identification, Assay and Estimation of Related Substances of Ivermectin in Bulk Drug Batches of Ivermectin Drug Substance.

A reversed-phase high-performance liquid chromatography (RP-HPLC) method has been developed and validated for the identification and assay of Ivermectin, including the identification and estimation of its process-related impurities and degradation products in bulk drug substance of Ivermectin. Analytes were separated on a HALO C18 column (100 mm × 4.6 mm I.D., 2.7 µm particle size) maintained at 40 °C (column temperature) with gradient elution. All analytes of interests were adequately separated within 25 min. All degradation products, process-related impurities and assay were monitored by ultraviolet detection at 254 nm. The new HPLC method described here successfully separated an isomer peak of the active pharmaceutical ingredient (API) from the major API peak. This newly separated isomer peak is around 1.2 to 1.5% (peak area) in typical API samples, and coelutes with the major API peak by all current HPLC methods. Quantitation limit of the HPLC method is 0.1% of target analytical concentration (~1.0 µg/mL). This method has been demonstrated to be accurate, robust, significantly higher degree of selectivity compared to the HPLC methods of Ivermectin drug substance reported in the literature and in the compendial HPLC methods prescribed in the current USA and European Pharmacopeia.

Development and Validation of a Stability-Indicating Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) Method for Identification, Assay, and Estimation of Related Substances of Ivermectin Drug Substance.

BACKGROUND: Ivermectin is a potent semi-synthetic antiparasitic drug used in veterinary medicine. It is widely used for the treatment of parasites. OBJECTIVE: This study aimed to develop a stability-indicating reversed-phase HPLC (RP-HPLC) method for assay and identification of ivermectin including identification and estimation of its related substances in bulk drug substance batches of ivermectin. METHOD: Ivermectin and its related substances were separated on an Ascentis Express C18 column (100 mm × 4.6 mm id, 2.7 µm particle size) maintained at 45°C (column temperature) on an HPLC system with gradient elution. The mobile phase was composed of water - acetonitrile (ACN; 50 + 50, v/v) as mobile phase A, and isopropanol - ACN (15 + 85, v/v) as mobile phase B. Analytes were detected with a detection wavelength of 252 nm and quantitated against an external reference standard of ivermectin with a quantitation limit of 0.1% of the target (analytical) concentration. RESULTS: The HPLC method was able to separate all analytes of interest by gradient elution within 25 min. The method was validated according to the guidelines described in the International Conference on Harmonization guideline Q2(R1). CONCLUSIONS: The HPLC method for assay of ivermectin and estimation of its related substances was successfully developed, validated, and demonstrated to be accurate, robust, specific, and stability indicating. HIGHLIGHTS: The performance of the HPLC method is significantly faster and possesses a higher degree of selectivity. Implementation of this method for routine analysis in QC laboratories would save significant time, resources and solvents.

Development and Validation of a Stability-Indicating Reversed-Phase High-Performance Liquid Chromatography Method for Assay of Doramectin and Estimation of its Related Substances in Commercial Batches of Doramectin Drug Substance.

BACKGROUND: Doramectin is a broad-spectrum antiparasitic drug used in veterinary medicine. It belongs to the family of avermectins, which possess a macrocyclic lactone structure, and is widely used for the treatment of parasites. OBJECTIVE: This study aimed to develop a stability-indicating reversed-phase (RP) HPLC method for the assay and identification of doramectin including identification and estimation of its related substances in commercial batches of doramectin drug substance. METHOD: Methanol was used to dissolve and prepare doramectin samples. Doramectin and its related substances were separated on a HALO C8 (100 mm × 4.6 mm i.d., 2.7 µm particle size) column maintained at 40 °C using an isocratic HPLC method with a mobile phase composed of acetonitrile-water (70 + 30, v/v). Analytes were detected with UV detection at 245 nm and quantitated against a single point external reference standard of doramectin. The LOQ of the method is 0.1% of the target concentration as described in the method. RESULTS: The HPLC method can separate all analytes of interest by an isocratic elution within 10 min. The method was validated according to the guidelines described in the International Conference on Harmonization Q2(R1). CONCLUSIONS: The HPLC method for assay of doramectin and estimation of its related substances was successfully developed, validated, and demonstrated to be accurate, robust, specific, and stability-indicating. HIGHLIGHTS: This is the first known paper to report an HPLC method for assay of doramectin and estimation of its related substances in commercial batches of doramectin drug substance.

Geopolymers as a New Class of High pH Stable Supports with Different Chromatographic Selectivity.

Geopolymers belong to an interesting class of X-ray amorphous polycondensed aluminosilicate ceramic solids. The high mechanical strength, chemical stability in basic conditions, and water insolubility make geopolymers a unique solid support in separation science. This work describes a new straightforward synthetic procedure for making spherical porous geopolymer particles with high surface area which are amenable for chromatographic purposes. In-depth physicochemical evaluation of geopolymers is conducted via particle size distribution, porosity measurements, X-ray diffraction, pH titration, and energy-dispersive spectroscopy and compared with silica, titania, and zirconia. Chromatographic selectivity shows that the surface chemistry of geopolymers has strong hydrophilic and electrostatic character, which makes it different from 36 chromatographic columns. Hydrophilic interaction liquid chromatography in columns packed with geopolymer particles shows different selectivity than that in silica columns, with excellent peak shapes. Phosphate or fluoride additives are not required as they are for zirconia or titania phase.

Fundamental and Practical Insights on the Packing of Modern High-Efficiency Analytical and Capillary Columns.

New stationary phases are continuously developed for achieving higher efficiencies and unique selectivities. The performance of any new phase can only be assessed when the columns are effectively packed under high pressure to achieve a stable bed. The science of packing columns with stationary phases is one of the most crucial steps to achieve consistent and reproducible high-resolution separations. A poorly packed column can produce non-Gaussian peak shapes and lower detection sensitivities. Given the ever larger number of stationary phases, it is impossible to arrive at a single successful approach. The column packing process can be treated as science whose unified principles remain true regardless of the stationary phase chemistry. Phenomenologically, the column packing process can be considered as a constant pressure or constant flow high-pressure filtration of a suspension inside a column with a frit at the end. This process is dependent on the non-Newtonian suspension rheology of the slurry in which the particles are dispersed. This perspective lays out the basic principles and presents examples for researchers engaged in stationary phase development. This perspective provides an extensive set of slurry solvents, hardware designs, and a flowchart, a logical approach to optimal column packing, thus eliminating the trial and error approach commonly practiced today. In general, nonaggregating but high slurry concentrations of stationary phases tend to produce well packed analytical columns with small particles. Conversely, C18 packed capillary columns are best packed using agglomerating solvents.

Separation of peptides on superficially porous particle based macrocyclic glycopeptide liquid chromatography stationary phases: consideration of fast separations.

Macrocyclic glycopeptide based liquid chromatography stationary phases are known for their highly selective peptide separations. Fast and ultrafast (t R < 1 min) high-efficiency separations were achieved with superficially porous particle (SPP)-based stationary phases. Separations of pharmaceutically important classes of peptides such as enkephalins and bradykinins have been achieved in less than 5 min in isocratic elution modes. Selectivity for peptides structurally similar to one another was increased with use of teicoplanin-based stationary phases compared with commercial C18 stationary phases. Ultrafast isocratic separations of structurally related peptides were achieved with teicoplanin- and vancomycin-based short SPP columns. Acidic mobile phases produced better separations. Ammonium formate was the optimal mobile phase buffer additive. Use of an appropriate combination of a macrocyclic glycopeptide stationary phase and a mobile phase permits faster and more electrospray ionization mass spectrometry compatible isocratic separations than previous gradient approaches. The tryptic peptide separation characteristics of the teicoplanin stationary phase are demonstrated. Additionally, compared with commercial C18 stationary phases, teicoplanin showed tryptic peptide separations with different selectivities. Graphical Abstract Ultrafast separation of enkephalin peptide epimers.

Salient Sub-Second Separations.

Sub-second liquid chromatography in very short packed beds is demonstrated as a broad proof of concept for chiral, achiral, and HILIC separations of biologically important molecules. Superficially porous particles (SPP, 2.7 µm) of different surface chemistries, namely, teicoplanin, cyclofructan, silica, and quinine, were packed in 0.5-cm-long columns for separating different classes of compounds. Several issues must be addressed to obtain the maximum performance of 0.5 cm columns with reduced plate heights of 2.6 to 3.0. Modified UHPLC hardware can be used to obtain sub-second separations provided extra-column dispersion is minimized and sufficient data acquisition rates are used. Further, hardware improvements will be needed to take full advantage of faster separations. The utility of power transform, which is already employed in certain chromatography detectors, is shown to be advantageous for sub-second chromatography. This approach could prove to be beneficial in fast screening and two-dimensional liquid chromatography.