Development and Validation of a Stability-Indicating Reversed-Phase HPLC Method for Assay and Estimation of Related Substances of Ivermectin in an Oral Paste.

Ivermectin is a potent semi-synthetic antiparasitic drug used in veterinary medicine. A reversed-phase high performance liquid chromatography (HPLC) method has been developed and validated for the identification and assay of Ivermectin, including the identification and estimation of its related impurities in an oral paste. Analytes were separated using a gradient elution at a flow rate of 1.5 mL/min on a Zorbax Extend-C18 column (150 mm × 4.6 mm i.d., 3.5-µm particle size) maintained at 30 °C. The mobile phase was composed of water as mobile-phase A and acetonitrile/methanol (85/15, v/v) as mobile-phase B. Ultraviolet detection at 245 nm was employed to monitor the analytes. Limit of quantitation (LOQ) and limit of detection (LOD) of the method are 0.6 and 0.2 µg/mL, respectively. The validation results demonstrated excellent linearity of the method in the range of 0.1-150% of the analytical concentration (0.6 mg/mL) of the method. The stability-indicating capability of the method has been demonstrated by adequately separating the degradation products from the stress degraded samples of the oral paste as per method validation requirements prescribed in the current International Council for Harmonisation guidelines.

Simultaneous Determination of Esafoxolaner, Eprinomectin, Praziquantel, BHT and Their Related Substances in a Topical Finished Product by a Single Reversed-Phase High-Performance Liquid Chromatography Method.

The topical product with three active pharmaceutical ingredients (APIs), namely, esafoxolaner, eprinomectin and praziquantel has demonstrated its efficacy in the treatment of cats with mixed infections with ectoparasites and nematodes and cestodes. A reversed-phase high-performance liquid chromatography (RP-HPLC) method has been developed and validated for assay and determination of related substances peaks of three APIs including the assay of antioxidant butylated hydroxytoluene (BHT) in the finished product. Analytes were separated on a short Zorbax SB-C18 column (50 × 4.6 mm I.D., 5 µm particle size, pore size: 80 Å) with gradient elution at 40 °C column temperature. Analytes were detected at 245 nm for praziquantel, esafoxolaner, eprinomectin and their degradation products. BHT and eprinomectin degradation product 8a-oxo-B1a were detected at 280 nm. All analytes of interest were adequately separated within 40 min. The assay for praziquantel, esafoxolaner, eprinomectin and BHT was conducted against their corresponding external reference standards. The related substances peaks of each API were determined by peak area and relative response factor against total peak area of their corresponding API peak in sample solution. This method has been demonstrated to be accurate, robust, specific and stability indicating.

Simultaneous determination of ivermectin, clorsulon and their related substances in an injectable finished product by a stability-indicating RP-HPLC method.

A reversed-phase high performance liquid chromatography (RP-HPLC) method has been developed for the determination of ivermectin and clorsulon, including the identification and estimation of their related impurities in an ivermectin and clorsulon injectable finished product. Chromatographic separation was achieved using a gradient elution on a Supelco Ascentis® Express C18 column (150 mm × 4.6 mm i.d., 5 µm particle size) maintained at 55 °C. Mobile phase-A is composed of water and mobile phase-B is composed of acetonitrile/methanol (65/35, v/v). Analytes were monitored by UV detection at 245 nm. The stability-indicating capability of this method has been demonstrated by the adequate separation of all the process related impurities and degradation products of ivermectin and clorsulon from the stress degraded samples of the injectable product. This method was also successfully validated as per the current ICH guidelines with respect to specificity, linearity (R2> 0.999), limit of detection (0.3 µg/mL), limit of quantitation (1.0 µg/mL), accuracy, precision, and robustness for both APIs. This proposed method can significantly benefit the end-users in QC laboratories with laboratory efficiency and throughput during routine analysis and stability monitoring of the injectable product.

Assay of afoxolaner and determination of its related substances in commercial bulk batches of afoxolaner by reversed-phase HPLC method based on a short octadecyl column.

Afoxolaner is a new insecticidal and acaricidal active pharmaceutical ingredient (API) belonging to the isoxazoline family, widely prescribed for the control of fleas and ticks in dogs. A stability-indicating reversed-phase high performance liquid chromatography (RP-HPLC) method has been developed for the assay of afoxolaner and determination of its related compounds in bulk API lots of afoxolaner. The chromatographic separation of afoxolaner and its related compounds was achieved by gradient elution on a Zorbax-SB C18 column (50 mm × 4.6 mm i.d., 5 µm particle size) maintained at 40 °C. Mobile phase-A is composed of water and mobile phase-B is composed of acetonitrile/methanol (50/50, v/v). Analytes were monitored by UV detection at 225 nm with a flow rate of 2.0 mL/min. The stability-indicating capability of the method has been demonstrated by adequate separation of all the process related impurities and degradation products of afoxolaner generated by stress degradation of afoxolaner bulk drug substance under various stress conditions. This method was also successfully validated as per the current ICH guidelines for afoxolaner and Q6S07 (afoxolaner related substance) with respect to specificity, linearity (R2 > 0.999), detection limit (∼0.21 µg/mL), quantitation limit (∼0.70 µg/mL), accuracy, precision, and robustness. Due to its speed, high degree of selectivity, and accuracy, the proposed method is suitable and highly desirable in quality control laboratories for routine analysis of afoxolaner.

Alternative and Improved Stability-Indicating HPLC Method for the Assay of Eprinomectin and Determination of Its Related Compounds in Bulk Batches of Eprinomectin Drug Substance.

BACKGROUND: Eprinomectin is used as an active pharmaceutical ingredient (API) in various drug products by the animal health industry. Several major related impurities of eprinomectin API are not separated and coelute by the current United States Pharmacopeia (USP) method for eprinomectin. OBJECTIVE: The aim was to develop and validate a true and reliable stability-indicating reversed-phase (RP) HPLC method for assay and determination of related substances of eprinomectin in bulk batches of eprinomectin API. METHOD: HPLC analysis is carried out using a Kinetex C8 column (100 mm × 4.6 mm i.d. , 2.6 µm particle size) maintained at 30°C with water-acetonitrile-isopropanol (48 + 42 + 10, v/v/v) as mobile phase A and 100% acetonitrile as mobile phase B. Analytes are separated by gradient elution at a flow rate of 0.7 mL/min and detected by UV at 252 nm. The total run time of the method is 30 min. Eprinomectin assay and estimation of all eprinomectin-related substances are obtained in a single HPLC run. RESULTS: The HPLC method was able to separate all analytes of interest by gradient elution. The new method was successfully validated according to current The Internal Council for Hamonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) and International Cooperation on Hamonisation of Technical Requirements for Registration of Veterinary Medicinal Products (VICH) guidelines and was found to be specific, linear, accurate, precise, robust, and sensitive. CONCLUSIONS: An HPLC method for assay of eprinomectin and estimation of its related substances was successfully developed, validated, and demonstrated to be accurate, robust, specific, and stability-indicating. HIGHLIGHTS: The HPLC method presented in this paper is more desirable as compared to USP and suitable for routine analysis of bulk batches of eprinomectin API and stability samples in QC laboratories.

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.

Development and Validation of a Reversed-Phase Chiral HPLC Method to Determine the Chiral Purity of Bulk Batches of (S)-Enantiomer in Afoxolaner.

Afoxolaner is a new antiparasitic molecule from the isoxazoline family that acts on insect acarine g-aminobutyric acid and glutamate receptors. Afoxolaner is a racemic mixture, which has a chiral center at the isoxazoline ring. A reversed-phase chiral HPLC method has been developed to determine the chiral purity of bulk batches of (S)-enantiomer in afoxolaner for the first time. This method can also be used to verify that afoxolaner is a racemic mixture, which was demonstrated by specific rotation. ChromSword, an artificial intelligence method development tool, was used for initial method development. The column selected for the final method was CHIRALPAK AD-RH (150 × 4.6 mm, 5 µm particle size), maintained at 45°C, and isocratic elution using water-isopropanol-acetonitrile (40 + 50 + 10, v/v/v) as the mobile phase with a detection wavelength of 312 nm. The run time for the method was 11 min. The resolution and selectivity factors of the two enantiomers were 2.3 and 1.24, respectively. LOQ and LOD of the method were 1.6 and 0.8 µg/mL, respectively. This method was appropriately validated according to International Conference on Harmonization guidelines for its intended use.

PREPARATION AND EVALUATION OF HPLC CHIRAL STATIONARY PHASES BASED ON CATIONIC/BASIC DERIVATIVES OF CYCLOFRUCTAN 6.

The cyclofructan 6 (CF6) macrocyclic-oligosaccharide was derivatized with five different substituents able to bear positive charges: propyl imidazole (IM) methyl benzimidazole (BIM), dimethyl aminopropyl (AP), pyridine (PY) and dimethyl aminophenyl (DMAP). The derivatized cyclofructans were reacted with triethoxysilyl-propylisocyanate as a linker to bond them to 5 µm spherical silica gel particles and then used to prepare HPLC columns. The bonded silica particles were analyzed to establish the bonding densities. A set of 34 chiral compounds including acids, neutral compounds and bases was tested with nine different mobile phase compositions including two reverse phase (RP) acetonitrile/pH 4 buffer, three polar organic (PO) acetonitrile/methanol and four normal phase (NP) heptane/ethanol mobile phases. No compounds could be separated in the RP mode. Eight compounds only could be enantioseparated in the PO mode and 21 compounds in the NP mode. The most effective chiral stationary phase was the propyl imidazole derivatized CF6 phase, provided that no more than six imidazole substituents and two linkers are attached per CF6 unit.

Enantiomeric separations of illicit drugs and controlled substances using cyclofructan-based (LARIHC) and cyclobond I 2000 RSP HPLC chiral stationary phases.

Recently a novel class of chiral stationary phases (CSPs) based on cyclofructan (CF) has been developed. Cyclofructans are cyclic oligosaccharides that possess a crown ether core and pendent fructofuranose moieties. Herein, we evaluate the applicability of these novel CSPs for the enantiomeric separation of chiral illicit drugs and controlled substances directly without any derivatization. A set of 20 racemic compounds were used to evaluate these columns including 8 primary amines, 5 secondary amines, and 7 tertiary amines. Of the new cyclofructan-based LARIHC columns, 14 enantiomeric separations were obtained including 7 baseline and 7 partial separations. The LARIHC CF6-P column proved to be the most useful in separating illicit drugs and controlled substances accounting for 11 of the 14 optimized separations. The polar organic mode containing small amounts of methanol in acetonitrile was the most useful solvent system for the LARIHC CF6-P CSP. Furthermore, the LARIHC CF7-DMP CSP proved to be valuable for the separation of the tested chiral drugs resulting in four of the optimized enantiomeric separations, whereas the CF6-RN did not yield any optimum separations. The broad selectivity of the LARIHC CF7-DMP CSP is evident as it separated primary, secondary and tertiary amine containing chiral drugs. The compounds that were partially or un-separated using the cyclofructan based columns were screened with a Cyclobond I 2000 RSP column. This CSP provided three baseline and six partial separations.

Separation of nucleotides by hydrophilic interaction chromatography using the FRULIC-N column.

A stationary phase composed of silica-bonded cyclofructan 6 (FRULIC-N) was evaluated for the separation of four cyclic nucleotides, six nucleoside monophosphates, four nucleoside diphosphates, and five nucleoside triphosphates via hydrophilic interaction chromatography (HILIC) in both isocratic and gradient conditions. The gradient conditions gave significantly better separations by narrowing peak widths. Sixteen out of nineteen nucleotides were baseline separated on the FRULIC-N column in one run. Unlike other known HILIC stationary phases, there can be dual-retention mechanisms unique to this media. Traditional hydrogen bonding/dipolar interactions can be supplemented by dynamic ion interaction effects for anionic analytes. This occurs because the FRULIC-N stationary phase is able to bind certain buffer cations. The extent of the ion interaction is tunable, in comparison to stationary phases with embedded charged groups, where the inherent ionic properties are fixed. The best mobile phase conditions were determined by varying the organic modifier (acetonitrile) content, as well as salt type/concentration and electrolyte pH. The thermodynamic characteristic of the FRULIC-N column was investigated by evaluating the column temperature effect on retention and utilizing van't Hoff plots. This study shows that there is a greater entropic contribution for the retention of nucleotide di and triphosphates, whereas there is a greater enthalphic contribution for the cyclic nucleotides with the FRULIC-N column.

Ammonium chloride salting out extraction/cleanup for trace-level quantitative analysis in food and biological matrices by flow injection tandem mass spectrometry.

A sample extraction and purification procedure that uses ammonium-salt-induced acetonitrile/water phase separation was developed and demonstrated to be compatible with the recently reported method for pesticide residue analysis based on fast extraction and dilution flow injection mass spectrometry (FED-FI-MS). The ammonium salts evaluated were chloride, acetate, formate, carbonate, and sulfate. A mixture of NaCl and MgSO4, salts used in the well-known QuEChERS method, was also tested for comparison. With thermal decomposition/evaporation temperature of <350°C, ammonium salts resulted in negligible ion source residual under typical electrospray conditions, leading to consistent method performance and less instrument cleaning. Although all ammonium salts tested induced acetonitrile/water phase separation, NH4Cl yielded the best performance, thus it was the preferred salting out agent. The NH4Cl salting out method was successfully coupled with FI/MS/MS and tested for fourteen pesticide active ingredients: chlorantraniliprole, cyantraniliprole, chlorimuron ethyl, oxamyl, methomyl, sulfometuron methyl, chlorsulfuron, triflusulfuron methyl, azimsulfuron, flupyrsulfuron methyl, aminocyclopyrachlor, aminocyclopyrachlor methyl, diuron and hexazinone. A validation study was conducted with nine complex matrices: sorghum, rice, grapefruit, canola, milk, eggs, beef, urine and blood plasma. The method is applicable to all analytes, except aminocyclopyrachlor. The method was deemed appropriate for quantitative analysis in 114 out of 126 analyte/matrix cases tested (applicability rate=0.90). The NH4Cl salting out extraction/cleanup allowed expansion of FI/MS/MS for analysis in food of plant and animal origin, and body fluids with increased ruggedness and sensitivity, while maintaining high-throughput (run time=30s/sample). Limits of quantitation (LOQs) of 0.01mgkg(-1) (ppm), the 'well-accepted standard' in pesticide residue analysis, were achieved in >80% of cases tested; while limits of detection (LODs) were typically in the range of 0.001-0.01mgkg(-1) (ppm). A comparison to a well-established HPLC/MS/MS method was also conducted, yielding comparable results, thus confirming the suitability of NH4Cl salting out FI/MS/MS for pesticide residue analysis.

Metal cation detection in positive ion mode electrospray ionization mass spectrometry using a tetracationic salt as a gas-phase ion-pairing agent: evaluation of the effect of chelating agents on detection sensitivity.

RATIONALE: The detection of metal cations continues to be essential in many scientific and industrial areas of interest. The most common electrospray ionization mass spectrometry (ESI-MS) approach involves chelating the metal ions and detecting the organometallic complex in the negative ion mode. However, it is well known that negative ion mode ESI-MS is generally less sensitive than the positive ion mode. To achieve greater sensitivity, it is necessary to examine the feasibility of detecting the chelated metal cations in positive ion mode ESI-MS. METHODS: Since highly solvated native metal cations have relatively low ionization efficiency in ESI-MS, and can be difficult to detect in the positive ion mode, a tetracationic ion-pairing agent was added to form a complex with the negatively charged metal chelate. The use of the ion-pairing agent leads to the generation of an overall positively charged complex, which can be detected at higher m/z values in the positive ion mode by electrospray ionization linear quadrupole ion trap mass spectrometry. RESULTS: Thirteen chelating agents with diverse structures were evaluated in this study. The nature of the chelating agent played as important a role as was previously determined for cationic pairing agents. The detection limits of six metal cations reached sub-picogram levels and significant improvements were observed when compared to negative ion mode detection where the metal-chelates were monitored without adding the ion-pairing reagent (IPR). Also, selective reaction monitoring (SRM) analyses were performed on the ternary complexes, which improved detection limits by one to three orders of magnitude. CONCLUSIONS: With this method it was possible to analyze the metal cations in the positive ion mode ESI-MS with the advantage of speed, sensitivity and selectivity. The optimum solution pH for this type of analysis is 5-7. Tandem mass spectrometry (MS/MS) further increases the sensitivity. Speciation is straightforward making this a broadly useful approach for the analysis of metal ions.

Rapid determination of sample purity and composition by nanopore stochastic sensing.

A rapid highly sensitive method to determine sample composition is reported. By monitoring the interaction between the sample of interest and a properly engineered nanopore, information regarding the identities and concentrations of the sample components could be revealed via their characteristic signatures, e.g., blockage amplitudes and event residence times. The sample composition method should be applicable in various research areas, including analytical chemistry, organic synthesis, pharmaceutical industry, etc.

Sulfonated cyclofructan 6 based stationary phase for hydrophilic interaction chromatography.

A stationary phase composed of silica-bonded sulfonated cyclofructan 6 (SCF6) was synthesized and evaluated for hydrophilic interaction chromatography (HILIC). The separation of a large variety of polar compounds was evaluated on different versions of the stationary phase and compared with the same separations obtained with commercially available HILIC columns. The new columns successfully separate polar and hydrophilic compounds including ß blockers, xanthines, salicylic acid related compounds, nucleic acid bases, nucleosides, maltooligosaccharides, water soluble vitamins and amino acids. The separation conditions were optimized by changing the composition and the pH of the mobile phase. The dependence of analyte retention on temperature was studied using van't Hoff plots. The newly synthesized stationary phase showed broad applicability for HILIC mode separations.

Evaluation of aromatic-derivatized cyclofructans 6 and 7 as HPLC chiral selectors.

The two best aromatic-functionalized cyclofructan chiral stationary phases, R-naphthylethyl-carbamate cyclofructan 6 (RN-CF6) and dimethylphenyl-carbamate cyclofructan 7 (DMP-CF7), were synthesized and evaluated by injecting various classes of chiral analytes. They provided enantioselectivity toward a broad range of compounds, including chiral acids, amines, metal complexes, and neutral compounds. It is interesting that they exhibited complementary selectivities and the combination of two columns provided enantiomeric separations for 43% of the test analytes. These extensive chromatographic results provided useful information about method development of specific analytes, and also gave some insight as to the enantioseparation mechanism.