Development and Validation of a Chiral Liquid Chromatographic Assay for Enantiomeric Separation and Quantification of Verapamil in Rat Plasma: Stereoselective Pharmacokinetic Application

Abstract

A novel, fast and sensitive enantioselective HPLC assay with a new core–shell isopropyl carbamate cyclofructan 6 (superficially porous particle, SPP) chiral column (LarihcShell-P, LSP) was developed and validated for the enantiomeric separation and quantification of verapamil (VER) in rat plasma. The polar organic mobile phase composed of acetonitrile/methanol/trifluoroacetic acid/triethylamine (98:2:0.05: 0.025, v/v/v/v) and a flow rate of 0.5 mL/min was applied. Fluorescence detection set at excitation/emission wavelengths 280/313 nm was used and the whole analysis process was within 3.5 min, which is 10-fold lower than the previous reported HPLC methods in the literature. Propranolol was selected as the internal standard. The S-(−)- and R-(+)-VER enantiomers with the IS were extracted from rat plasma by utilizing Waters Oasis HLB C18 solid phase extraction cartridges without interference from endogenous compounds. The developed assay was validated following the US-FDA guidelines over the concentration range of 1–450 ng/mL (r2 ≥ 0.997) for each enantiomer (plasma) and the lower limit of quantification was 1 ng/mL for both isomers. The intra- and inter-day precisions were not more than 11.6% and the recoveries of S-(−)- and R-(+)-VER at all quality control levels ranged from 92.3% to 98.2%. The developed approach was successfully applied to the stereoselective pharmacokinetic study of VER enantiomers after oral administration of 10 mg/kg racemic VER to Wistar rats. It was found that S-(−)-VER established higher Cmax and area under the concentration-time curve (AUC) values than the R-(+)-enantiomer. The newly developed approach is the first chiral HPLC for the enantiomeric separation and quantification of verapamil utilizing a core–shell isopropyl carbamate cyclofructan 6 chiral column in rat plasma within 3.5 min after solid phase extraction (SPE).

Separation and quantitation of valacyclovir enantiomers using stability‐indicating chiral liquid chromatography method with a chiral stationary phase of amylose tris‐(3,5‐dimethylphenylcarbamate)

“…High-performance liquid chromatography (HPLC) is one of the most widely used key separation techniques in liquid chromatography and is currently operated in two different modes: normal phase HPLC with chiral columns and reverse phase HPLC with achiral columns. Pharmaceutical firms are widely using it today and getting great benefits from it for qualitative and quantitative analysis, isolation, and purification of organic compounds [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18]. The chiral columns used in normal-phase HPLC are made of silica gel and polar polymers (such as polysaccharides like cellulose or its derivatives, cyclodextrin and its derivatives, proteins, helical polymers, and ligand exchange polymers).…”

“…The chiral columns used in normal-phase HPLC are made of silica gel and polar polymers (such as polysaccharides like cellulose or its derivatives, cyclodextrin and its derivatives, proteins, helical polymers, and ligand exchange polymers). Nowadays, different types of chiral columns are commercially available to withstand both polar and non-polar organic solvents [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18].…”

“…In this study, the stereo-selective isomers of VAL were well separated, and their amounts were measured using a newly developed chiral HPLC method that showed stability-indicating properties. The chiral isomers must be continuously monitored and controlled well within the permitted range using chromatography methods, since the biological effects of chiral isomers (such as stereo-selective isomers and optical isomers like R-and S-enantiomers), achiral isomers (diastereomers like cis-and trans-isomers), and racemates are very different, and it is mandatory to estimate the amount of each isomer in a drug substance or drug product [9][10][11][12][13][14][15][16][17][18].…”

A Liquid Chromatography Tandem Mass Spectrometry Method for the Simultaneous Estimation of the Dopamine Receptor Antagonist LE300 and Its N-methyl Metabolite in Plasma: Application to a Pharmacokinetic Study

“…Analytical method validation is necessary to demonstrate the suitability for any desired pharmacokinetic application. The method has to produce precise, reproducible, and accurate results; this is necessary for bioavailability, pharmacodynamics, pharmacokinetics, bioequivalence, or toxicological studies, since such methods are used in analyte quantification of various biological matrices, such as plasma or urine [ 10 , 11 , 12 , 13 ].…”

Separation and simultaneous estimation of enantiomers and Diastereomers of muscarinic receptor antagonist Solifenacin using stability‐indicating Normal‐phase HPLC technique with chiral stationary phase amylose tris‐(3,5‐dimethylphenylcarbamate)

“…[5][6][7][8][9][10][11][12][13][14][15][16] LC techniques are very frequently used to separate target analytes from a complex sample mixture without any interference. [17][18][19][20][21][22][23][24][25][26][27] In this study, the solifenacin (S, R-enantiomer) and its chiral impurities or stereoisomer (R,S-isomer, an enantiomer of solifenacin) and diastereomers (S,S-isomer and R,R-isomer) were separated well from each other and measured using normal-phase highperformance liquid chromatography (NP-HPLC) with the chiral stationary phase made of amylose tris (3,5-dimethylphenylcarbamate) coated on silica gel (Chiralpak, AD-H). The chiral isomers (stereoisomers) and other organic impurities have different biological effects, and they must be measured in medicines, [17][18][19][20][21][22][23] therefore, chromatography methods have been used to quantify impurities and keep them well within permitted limits.…”