An Isocratic HPLC Method for the Simultaneous Determination of Cholesterol, Cardiolipin, and DOPC in Lyophilized Lipids and Liposomal Formulations

Abstract

Phospholipids, such as 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,1',2,2'-tetramyristoyl cardiolipin, along with cholesterol, form liposomes in aqueous media and have been investigated at NeoPharm (Lake Bluff, IL) as drug-delivery systems. To accurately assess the effectiveness of various formulations involving the use of aforementioned phospholipids and cholesterol, their quantitative determination is essential. An isocratic high-performance liquid chromatographic method for the simultaneous determination of cholesterol, cardiolipin, and DOPC in various pharmaceutical formulations containing the active drug substance has consequently been developed and is presented here. The current method utilizes an ASTEC-diol analytical column and is shown to be stability-indicating and free from interference from any of the formulation excipients, such as sucrose, sodium chloride, and sodium lactate. The analytes are detected using an evaporative light scattering detector (Alltech or Polymer Laboratories). The quantitation of each lipid component is performed using non-linear regression analysis. The retention characteristics of the analytes are examined as a function of eluent composition (e.g., pH, salt content, organic to aqueous phase ratio) and column temperature. The method was validated and was found to be sensitive, specific, rugged, and cost-effective. The current method provides enhanced chromatographic separation for lipid components as well as degradation products as compared to similar methods reported in the literature. It is also inherently simpler than other similar methods reported in the literature that typically use complex gradient elution.

Analysis of cationic liposomes by reversed-phase HPLC with evaporative light-scattering detection

“…An evaporative lightscattering detector (ELSD) was used to monitor separation and sensitively detect the lipids as well as their degradation products. The ELSD is a quasi-universal mass detector that can detect any analyte less volatile than the mobile phase and has been proven to be effective in detecting poor UV-absorbers, such as most lipids [22,23,[27][28][29][30]. Unlike refractive index detector (RID), another commonly used mass-detection method, ELSD is compatible with gradient elution and insensitive to ambient temperature variation, making it apparently the detection method of choice for lipid analysis.…”

A Reliable Quantification of Cholesterol and 25-Hydroxycholesterol in Liposomal Adjuvant Formulation by Liquid Chromatography High-Resolution Tandem Mass Spectrometry

“…Multiple analytical techniques to determine cholesterol in foods and pharmaceutical samples, including the classical chemical method, gas chromatography, spectrophotometry, capillary electrophoresis, liquid chromatography, and direct mass spectrometry (MS), have been reported. − Due to the complexity of the liposomal matrix, a more accurate and reliable quantitative analysis of cholesterol and its degradation product requires a separation step combined with an appropriate detection system. To date, cholesterol in liposome formulations and the related lipid nanoparticles were detected and quantified using LC coupled with UV–vis, , charged aerosol detection (CAD), evaporative light scattering detection (ELSD), , and MS with different ionization strategies. ,, The LC-MS/MS-based method has been shown to be reliable for detecting cholesterol and its degradation products in complex matrices. ESI is one of the widely employed MS ionization techniques for quantitative analyses; however, it is not effective in ionizing cholesterol due to its poor proton affinity and low acidity.…”

“… 30 − 34 Due to the complexity of the liposomal matrix, a more accurate and reliable quantitative analysis of cholesterol and its degradation product requires a separation step combined with an appropriate detection system. To date, cholesterol in liposome formulations and the related lipid nanoparticles were detected and quantified using LC coupled with UV–vis, 35 , 36 charged aerosol detection (CAD), 37 evaporative light scattering detection (ELSD), 38 , 39 and MS with different ionization strategies. 31 , 40 , 41 The LC-MS/MS-based method has been shown to be reliable for detecting cholesterol and its degradation products in complex matrices.…”

Lipid loss and compositional change during preparation of liposomes by common biophysical methods

“…Inspired by methods to study liposome-based nutritional supplements and drug formulations (27)(28)(29)(30)(31), we have developed a method to quantify the lipid composition of liposomes using high-performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD). Peak identification in our HPLC-ELSD approach is relatively straightforward since the lipid components are known a priori, and the retention time of each peak can be compared with a standard.…”