Maternal Ezetimibe Concentrations Measured in Breast Milk and Its Use in Breastfeeding Infant Exposure Predictions.

BACKGROUND: Lactating mothers taking ezetimibe, an antihyperlipidemic agent, may be hesitant to breastfeed despite the known benefit of breastfeeding to both mother and infant. Currently, no data exist on the presence or concentration of ezetimibe and its main active metabolite, ezetimibe-glucuronide (EZE-glucuronide), in human breast milk. METHODS: Voluntary breast milk samples containing ezetimibe and EZE-glucuronide were attained from lactating mothers taking ezetimibe as part of their treatment. An assay was developed and validated to measure ezetimibe and EZE-glucuronide concentrations in breast milk. A workflow that utilized a developed and evaluated pediatric physiologically based pharmacokinetic (PBPK) model, the measured concentrations in milk, and weight-normalized breast milk intake volumes was applied to predict infant exposures and determine the upper area under the curve ratio (UAR). RESULTS: Fifteen breast milk samples from two maternal-infant pairs were collected. The developed liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay showed an analytical range of 0.039-5.0 ng/mL and 0.39-50.0 ng/mL for ezetimibe and EZE-glucuronide, respectively. The measured concentrations in the breast milk samples were 0.17-1.02 ng/mL and 0.42-2.65 ng/mL of ezetimibe and EZE-glucuronide, respectively. The evaluated pediatric PBPK model demonstrated minimal exposure overlap in adult therapeutic dose and breastfed infant simulated area under the concentration-time curve from time zero to 24 h (AUC24). Calculated UAR across infant age groups ranged from 0.0015 to 0.0026. CONCLUSIONS: PBPK model-predicted ezetimibe and EZE-glucuronide exposures and UAR suggest that breastfeeding infants would receive non-therapeutic exposures. Future work should involve a 'mother-infant pair study' to ascertain breastfed infant plasma ezetimibe and EZE-glucuronide concentrations to confirm the findings of this work.

Ezetimibe: A Review of Analytical Methods for the Drug Substance, Pharmaceutical Formulations and Biological Matrices.

Ezetimibe (EZM) is a selective inhibitor of the sterol transporter Niemann-Pick C1-Like 1 in the small intestine used as an adjunctive therapy to lower cholesterol levels in cases of hyperlipidemia. The goal of this work was to summarize the main physical-chemical, pharmacological and pharmacokinetic characteristics of EZM, as well as to describe the main analytical methodologies for the quantification of the drug. Methods described in the United States Pharmacopeia for EZM raw material and tablets were also presented. The drug has a large number of process-related impurities and degradation products and needs strict quality control of its impurities. Specific chiral methods for the evaluation of its chiral impurities are also a need for EZM. The main advantages and disadvantages of the compiled analytical methods were presented, as well as the limits of detection and quantitation. The fastest and most efficient methods were highlighted. Most methods for analyzing EZM used C8 or C18 stationary phases in gradient mode with binary mobile phases containing acetonitrile and an acidic buffer solution with ultraviolet detection. For analysis of EZM in biological matrices, liquid chromatography-tandem mass spectrometry is generally employed using electron spray ionization in negative ionization mode using multiple reaction monitoring. Different methods in the literature evaluate a large number of impurities for EZM, however new stability-indicating high-performance liquid chromatography methods for the drug are still needed.

Sustainable environment-friendly quantitative determination of three anti-hyperlipidemic statin drugs and ezetimibe in binary mixtures by first derivative Fourier transform infrared (FTIR) spectroscopy.

FTIR spectrometry is considered a sustainable green analytical chemistry procedure. Its use in quantitative analysis of pharmaceutical compounds in their raw resources and in their dosage forms is growing currently. The current research offers an environment-friendly, speedy, cost-effective, reliable and easy method for the simultaneous estimation of anti-hyperlipidemic drugs. No sample preparation was required except for grinding and mixing with KBr for making pellets used for acquisition of the FT-IR spectra. First-derivative FTIR spectroscopy is used to assess quantitatively atorvastatin (ATR), rosuvastatin (RSV) and simvastatin (SMV) in their binary mixtures with ezetimibe (EZT). For the first mixture, EZT and ATR were determined at 1733.18 cm-1 and 1647.74 cm-1, respectively. In the second mixture, the zero-crossing wave numbers selected for the determination of EZT and RSV were 1733.18 cm-1 and 955.69 cm-1, correspondingly. Whereas, the third mixture was quantified at the wavenumbers of 1520.93 and 3569.68 cm-1 for EZT and SMV, respectively. Validation of the procedure has been performed complying with recommendations of the International Conference of Harmonization (ICH) presenting linearity, accuracy, precision, robustness and selectivity. The linear range for all drugs was 2-30 mg/g. It was found that the LOD was 0.607, 0.311, 0.491 and 0.395 mg/g and the LOQ was found to be 1.839, 0.942, 1.490 and 1.190 mg/g for EZT, ATR, RSV, and SMV, correspondingly. The proposed technique was found to be accurate and precise in terms of percentage error and percentage relative standard deviation among intraday and interday measurements. It was also found selective through comparison of the results of standard drugs with results of binary mixtures and of pharmaceutical tablets. It was found robust through making slight variations in the working conditions and the results obtained remained statistically equivalent. The technique was applied effectively for the estimation of the binary mixtures under study in their tablets. Comparing the found outcomes to those of reference derivative UV spectrophotometric methods gave no significant difference between them. Analytical eco-scale and the scale of Green Analytical Procedure Index (GAPI) are the two scales utilized for evaluation of the greenness of the technique and it was found to be excellent green.

Synthesis of magnetic nanoparticle-based molecularly imprinted polymer as a selective sorbent for efficient extraction of ezetimibe from biological samples.

In the present study, a novel and efficient adsorbent constructed of molecularly imprinted polymer on the surface of modified magnetic nanoparticles with oleic acid (MNPs) was applied for the selective extraction of ezetimibe. The magnetic molecularly imprinted polymer (MMIP) was polymerized at the surface of modified MNPs using methacrylic acid as functional monomer, ezetimibe as template and ethylene glycol dimethacrylate as cross-linker. The resulting MMIP showed high adsorption capacity, good selectivity and fast kinetic binding for the template molecule. It was characterized by Fourier transform infrared analysis, scanning electron microscopy and transmission electron microscopy methods. The maximum adsorption capacity of MMIP was obtained as 137.1 mg g-1 and it took about 20 min to achieve the equilibrium state. The adsorption model of the adsorbent was fitted with the Freundlich and Langmuir isotherm equations. The assay exhibited a linear range of 0.003-20.000 mg L-1 for ezetimibe with a correlation coefficient of 0.995. The relative standard deviations for the recoveries were <5.2. The method was also examined for the analysis of ezetimibe in the biological samples.

Development of a Simple, Highly Selective RP-LC Method for the Quantification of Diastereomers and Other Related Substances of Ezetimibe Using Multivariate Analysis.

A simple reverse phase method for the selective quantification of ezetimibe (EZM), its diastereomers and other related substances was developed. The method demonstrated an excellent separation between each of the 14 impurities (including diastereomers, specified impurities and degradation products) and EZM within a runtime of 45 min. The developed method was evaluated against the reported USP method, other literature methods found that none of them was able to separate/show the absence of all the diastereomers and degradation products. The critical method parameters were optimized using central composite design. Forced degradation studies proved the method to be highly specific and the structure of all the major degradation products were confirmed by LC-MS study. The results of validation proved the method to be precise (% RSD < 4), accurate (recoveries in range of 100 ± 6%), linear (R2 > 0.999) and sensitive (LOQ ≤ 0.04% and LOD ≤ 0.01%) for all the impurities and drug. The method is suitable for both drug substance and oral solid dosage form.

Chemometric-Assisted Spectrophotometric Method for the Simultaneous Quantitative Determination of Ezetimibe and Simvastatin in Their Combined Dosage Forms.

The multivariate method, partial least-squares (PLS), was used as a calibration procedure for the simultaneous UV spectrophotometric determination of ezetimibe and simvastatin in their pharmaceutical forms. The method was developed and satisfactorily validated according to International Conference on Harmonization guidelines with respect to specificity, linearity, precision, accuracy, and robustness. In this study, the PLS algorithms are based on the absorption spectra of 25 different mixtures of drugs obtained by a multilevel factorial design. The method was linear in the concentration range of 2-8 µg/mL for ezetimibe and 4-16 µg/mL for simvastatin (r2 > 0.99; n = 7) at wavelengths of 238 and 247 nm, respectively. The LOD and LOQ were 0.28 and 0.93 µg/mL for ezetimibe and 0.16 and 0.53 µg/mL for simvastatin, respectively. Precision and accuracy data, evaluated by RSD, were lower than 2%. The method, which proved to be robust, was performed with a 2n full-factorial design. The validated method is simple and low cost, has a low use of polluting reagents, and is environmental friendly. Therefore, the proposed method was successfully applied for the simultaneous quantitative analysis of ezetimibe and simvastatin in commercial formulations.

A Versatile Liquid Chromatographic Method for the Simultaneous Determination of Metformin, Sitagliptin, Simvastatin, and Ezetimibe in Different Dosage Forms.

A new LC method is introduced with the concept of its versatile application to widely used drugs from different pharmacological classes. Metformin hydrochloride (MTF), sitagliptin phosphate (SIT), simvastatin (SIM) and ezetimibe (EZB) were simultaneously determined with a simple reversed-phase LC method in which a SIT-SIM binary mixture, present in a dosage form brand, was considered central for its development. Chromatographic separation was achieved with a mobile phase of acetonitrile and 0.02 M potassium dihydrogen phosphate (pH 5.2) (77 + 23, v/v) flowing through a C18 column (BDS Hypersil, 250 × 4.6 mm, 5 µm) at 1.2 mL/min at ambient temperature. UV detection was programmed to be carried out at 210 nm for EZB, SIT, and MTF, whereas SIM was detected at 240 nm. The method was validated according to International Conference on Harmonization guidelines. Linearity, accuracy, and precision were satisfactory over concentration ranges 4-40 µg/mL for EZB and SIM, 0.5-50 µg/mL for SIT, and 5-500 µg/mL for MTF. Coefficients of determination were >0.99 for the four drugs. LOQs found were 0.01 µg/mL for EZB, 0.02 µg/mL for SIT, 0.2 µg/mL for MTF, and 0.02 µg/mL for SIM. The developed method is simple, rapid, accurate, precise, and suitable for the routine QC analysis of the cited drugs in pharmaceutical products by conventional HPLC systems.

Development and Validation of an LC-MS-MS Method for the Simultaneous Determination of Simvastatin, Simvastatin Acid and Ezetimibe in Human Plasma and Its Application to Pharmacokinetic Study in the Indian Population.

A simple, selective, sensitive and high-throughput liquid chromatography-tandem mass spectrometry (LC-MS-MS) method has been developed and validated for the simultaneous quantification of simvastatin (SS), simvastatin acid (SSA, active metabolite of SS) and ezetimibe (EZM) in K2 EDTA containing human plasma, using simvastatin D6, simvastatin acid D3 and ezetimibe D4 as internal standards (ISTDs), respectively. A volume of plasma sample of only 400 µL was processed by the solid phase extraction technique; then 20 µL of processed sample was run on a Phenomenex, Kinetix XB C18, 150 × 4.6 mm, 5 µm column using an isocratic mobile phase consisting of 10 mM ammonium formate buffer (pH 4.0 ± 0.3): acetonitrile (27 : 73, v/v) with a run time of 6.3 min. The precursor and product ions of SSA, EZM and their ISTDs were monitored on a triple quadrupole instrument operated in the negative ionization mode, and SS was monitored in the positive mode. The method was validated over a concentration range of 0.2-80 ng/mL for SS, 0.1-60 ng/mL for SSA and 0.05-15 ng/mL for EZM. The method has been successfully applied in clinical pharmacokinetic study in the Indian population. The Cmax, AUC0-inf and Tmax values obtained in our study were 10.61 ± 5.287, 77.58 ± 29.367 and 1.62 ± 0.436 for EZM; 69.74 ± 45.274, 190.71 ± 107.271 and 1.74 ± 0.480 for SS; and 25.36 ± 23.576, 139.24 ± 131.653 and 3.95 ± 0.671 for SSA, respectively.

DEVELOPMENT OF CHROMATOGRAPHIC METHOD FOR DETERMINATION OF DRUGS REDUCING CHOLESTEROL LEVEL--STATINS AND EZETIMIBE.

The presented developed HPLC method and GC method may be used to separate and determine all analyzed 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) and ezetimibe using a single columns and a uniform methodology. In order to perform qualitative and quantitative tests of statins and ezetimibe the Symmetry C18 column 250 mm x 4.6 mm, 5 µm, the mobile phase: acetonitrile:water (70:30, v/v), adjusted to pH = 2.5 and a spectrophotometric detector for the HPLC method were used. For GC method column HP-1; 30 m x 0.25 mm x 0.25 µm and FID detector were selected. All results and statistical data obtained indicate good method sensitivity and precision. The RSD values are appropriate for both newly developed methods.

Application of new spectrofluorometric techniques for determination of atorvastatin and ezetimibe in combined tablet dosage form.

Two accurate, reliable, and highly sensitive spectrofluorometric methods were developed for simultaneous determination of the binary mixture of Atorvastatin and Ezetimibe without prior separation steps. The first method is based on double scan synchronous fluorescence spectrometry. Each of Atorvastatin and Ezetimibe can be determined independent of the other when scanned at Δλ=100 nm and 40 nm, respectively. The relative fluorescence intensity-concentration plots at two wavelengths, 272 (Δλ=100 nm) and 266 nm (Δλ=40 nm) were rectilinear over the range of 0.4-8 µg/mL (for Atorvastatin) and 0.6-8 µg/mL (for Ezetimibe), respectively. The second method is based on the technique of simultaneous equations (Vierodt's method), in which two equations are solved simultaneously after using a single excitation wavelength of 273 nm and λEm1=380 nm of Atorvastatin and λEm2=301 nm of Ezetimibe. Under the optimum conditions, linear relationships were found between the relative fluorescence intensity and the concentrations of the investigated drugs in the range of 0.4-8 µg/mL (for Atorvastatin) 0.6-8 µg/mL (for Ezetimibe). The different experimental parameters affecting the fluorescence intensities of the two drugs were carefully studied and optimized. The proposed methods were successfully applied for the determination of the investigated drugs in pure form, dosage form and in synthetic mixtures with good recovery and the results obtained were favorably compared to those obtained with a reference method.

Development and validation of a novel stability-indicating HPLC method for the quantitative determination of eleven related substances in ezetimibe drug substance and drug product.

Ezetimibe is a novel lipid-lowering agent that inhibits intestinal absorption of dietary and biliary cholesterol. In the present work, a simple, sensitive and reproducible gradient reverse phase high performance liquid chromatographic (RP-HPLC) method for separation and determination of the related substances of ezetimibe was developed and validated. Eleven potential process-related impurities (starting materials, (3S,4S,3'S)-isomer, degradants and byproducts) were identified in the crude samples. Tentative structures for all the impurities were assigned primarily based on comparison of their retention time and mass spectrometric data with that of available standards and references. This method can be applied to routine analysis in quality control of both bulk drugs and commercial tablets. Separation of all these compounds was performed on a Phenomenex Luna Phenyl-Hexyl (100mm×4.6mm, 5µm) analytical column. The mobile phase-A consists of acetonitrile-water (pH adjusted to 4.0 with phosphoric acid)-methanol at 15:75:10 (v/v/v), and mobile phase-B contains acetonitrile. The eluted compounds were monitored at 210nm. Ezetimibe was subjected to hydrolytic, acid, base, oxidative, photolytic and thermal stress conditions as per ICH serves to generate degradation products that can be used as a worst case to assess the analytical method performance. The drug showed extensive degradation in thermal, acid, oxidative, base and hydrolytic stress conditions, while it was stable to photolytic degradation conditions. The main degradation product formed under thermal, acid, oxidative, base and hydrolytic stress conditions corresponding to (2R,3R,6S)-N, 6-bis(4-fluorophenyl)-2-(4-hydroxyphenyl)-oxane-3-carboxamide (Ezetimibe tetrahydropyran impurity) was characterized by LC-MS/MS analysis. The degradation products were well resolved from the main peak and its impurities, thus proved the stability-indicating power of the method. The developed method was validated as per international conference on harmonization (ICH) guidelines with respect to specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, precision and robustness.

New Validated RP-HPLC Analytical Method for Simultaneous Estimation of Atorvastatin and Ezetimibe in Bulk Samples as Well in Tablet Dosage Forms by Using PDA Detector.

This paper describes a new RP-HPLC method for simultaneous quantification of these compounds in the bulk sample drug as well as in tablet dosage forms. The chromatographic separation was performed on an XTerra C8 (4.6 x 250 mm; 5 µm), with phosphate buffer [pH 3.5] and acetonitrile in the ratio of 40:60 (v/v) as mobile phase. The detection was carried out at 240 nm. The accuracy was found to be 99.59% and 98.98% for atorvastatin and ezetimibe respectively. The linearity was 5-25 µg/ml for both the drugs. The intra-day RSD was 0.57% and inter-day RSD was 0.13% for atorvastatin calcium and intra-day RSD was 0.56% and inter-day RSD was 0.09% for ezetimibe. The validation of method was carried out utilizing ICH-guidelines.