Distearoyl phosphatidylglycerol and dioleoyl phosphatidylglycerol increase the retention and reduce the toxicity of amphotericin B-loaded in nanoemulsions.

Aim: To develop nanoemulsions (NEs) loading amphotericin B (AmB) and to evaluate the influence of different excipients on the stability and the supramolecular organization, retention and toxicity of AmB. Materials & methods: The NEs were developed from different oils, surfactants, external media and anionic lipids (disteaoryl phosphatidylglycerol [DSPG] and dioleoyl phosphatidylglycerol [DOPG]). Their impact on the size, pH, zeta potential, AmB encapsulation efficiency, AmB retention and hemolytic potential of the NEs was evaluated. Results & conclusion: The use of soybean oil (lipid matrix), Span 80 (surfactant), phosphate buffer (external phase) and DSPG or DOPG (hydrophobic ion pair) provided better NE stability, higher AmB retention within the NEs and a safer formulation profile in hemolysis tests.

The Adaptation of the QV600 LLI Milli-Fluidics System to House Ex Vivo Gastrointestinal Tissue Suitable for Drug Absorption and Permeation Studies, Utilizing MALDI MSI and LC-MS/MS.

Careful formulation of pharmaceuticals for oral delivery is essential to ensure that the optimal amount of the active ingredient reaches its intended site of action. This chapter demonstrates how mass spectrometry can be used in conjunction with ex vivo tissue and an adapted milli-fluidics system to carry out a drug absorption study. MALDI MSI is used to visualize the drug within the small intestine tissue from the absorption experimentation. LC-MS/MS is used to complete a mass balance of the experiment and quantify the amount of drug that has permeated through the tissue.

Adaptation of the Kirkstall QV600 LLI Microfluidics System for the Study of Gastrointestinal Absorption by Mass Spectrometry Imaging and LC-MS/MS.

Absorption studies on oral drugs can be difficult due to the challenge of replicating the complex structure and environment of the GI tract. Drug absorption studies can be conducted using in vivo and ex vivo animal tissue or animal-free techniques. These studies typically involve the use of Caco-2 cells. However, Caco-2 cells do not incorporate all the cell types found in intestinal tissue and lack P450 metabolizing enzymes. The QV600 LLI system is a microfluidics system designed for use with cell culture. Here, it has been adapted to house appropriate sections of ex vivo porcine tissue to act as a system that models the duodenum section of the small intestine. A pH regulated solution of Atorvastatin was flowed over the apical layer of the GI tissue and a nutrient solution flowed over the basal layer of the tissue to maintain tissue viability. The tissue samples were snap-frozen, cryosectioned, and imaged using MALDI Mass Spectrometry Imaging (MSI). A proof-of-concept study on the effect of excipients on absorption was conducted. Different concentrations of the solubilizing agent were added to the donor circuit of the QV600 LLI. The amount of Atorvastatin in the acceptor circuit was determined to study the effect of the excipient on the amount of drug that had permeated through the tissue. Using these data, Papp, pig values were calculated and compared with the literature.

Evaluating a Modified High Purity Polysorbate 20 Designed to Reduce the Risk of Free Fatty Acid Particle Formation.

PURPOSE: To evaluate a modified high purity polysorbate 20 (RO HP PS20)-with lower levels of stearate, palmitate and myristate esters than the non-modified HP PS20-as a surfactant in biopharmaceutical drug products (DP). RO HP PS20 was designed to provide functional equivalence as a surfactant while delaying the onset of free fatty acid (FFA) particle formation upon hydrolytic degradation relative to HP PS20. METHODS: Analytical characterization of RO HP PS20 raw material included fatty acid ester (FAE) distribution, higher order ester (HOE) fraction, FFA levels and trace metals. Functional assessments included 1) vial and intravenous bag agitation; 2) oxidation via a placebo and methionine surrogate study; and 3) hydrolytic PS20 degradation studies to evaluate FFA particle formation with and without metal nucleation. RESULTS: Interfacial protection and oxidation propensity were comparable between the two polysorbates. Upon hydrolytic degradation, FFA particle onset was delayed in RO HP PS20. The delay was more pronounced when HOEs of PS20 were preferentially degraded. Furthermore, the hydrolytic degradants of RO HP PS20 formed fewer particles in the presence of spiked aluminum. CONCLUSION: This work highlights the criticality of having tighter control on long chain FAE levels of PS20 to reduce the occurrence of FFA particle formation upon hydrolytic degradation and lower the variability in its onset. By simultaneously meeting compendial PS20 specifications while narrowing the allowable range for each FAE and shifting its composition towards the shorter carbon chain species, RO HP PS20 provides a promising alternative to HP PS20 for biopharmaceutical DPs.

Challenges in polysorbate characterization by mass spectrometry.

Polysorbates are used in a variety of applications over a wide range of markets. Simple in concept, these products are complex in actual composition. Mass spectrometry and related techniques have been effectively used to characterize these products, from the major components to the minor residual production byproducts and degradation species. In this paper we review the use of MALDI-MS, LC/MS, GC/MS, and SFC/MS in the analysis of these materials. The wealth of information provided by MALDI is presented, using Polysorbate 60 as an example. Limitations are described, with the impact of matrix selection and cationization agent demonstrated. Furthermore, unique challenges of MALDI analysis of Polysorbate 80 are shown. Polysorbates have been extensively analyzed, especially by the biopharmaceutical industry, to better understand the impact of various grades of purity and manufacture on the stability of formulations. Using Polysorbate 80 as an example, we illustrate some of the more advanced techniques used to more fully characterize these complex molecules using high-resolution LC/MS and LC/MS/MS. Finally, the use of other techniques (such as GC/MS and SFC/MS) is briefly reviewed.

Quantitative Investigation of Terbinafine Hydrochloride Absorption into a Living Skin Equivalent Model by MALDI-MSI.

The combination of microspotting of analytical and internal standards, matrix sublimation, and recently developed software for quantitative mass spectrometry imaging has been used to develop a high-resolution method for the determination of terbinafine hydrochloride in the epidermal region of a full thickness living skin equivalent model. A quantitative assessment of the effect of the addition of the penetration enhancer (dimethyl isosorbide (DMI)) to the delivery vehicle has also been performed, and data have been compared to those obtained from LC-MS/MS measurements of homogenates of isolated epidermal tissue. At 10% DMI, the levels of signal detected for the drug in the epidermis were 0.20 ± 0.072 mg/g tissue for QMSI and 0.28 ± 0.040 mg/g tissue for LC-MS/MS at 50% DMI 0.69 ± 0.23 mg/g tissue for QMSI and 0.66 ± 0.057 mg/g tissue for LC-MS/MS. Comparison of means and standard deviations indicates no significant difference between the values obtained by the two methods.

Increasing UV detection sensitivity in the supercritical fluid chromatographic analysis of alcohol polyethers.

Alcohol ethoxylates (AEOs) that contain a wide distribution of oligomers pose a challenge for ultraviolet (UV) absorbance detection due to the fact that the AEOs absorb strongly only in the range of commercial UV detectors between 190 and 200 nm. Most mobile phase components, with the exceptions of water and carbon dioxide, also absorb in this region. Ethoxylated hexadecanol and octadecanol were derivatized with disilazane-chlorosilane mixtures for the formation of phenyl containing silylethers. Derivatized samples were analyzed by supercritical fluid chromatography (SFC) coupled with both electrospray ionization mass spectrometry and UV absorbance detection. An increase in the number of phenyl groups incorporated into the derivatives increased the number of oligomers observed by UV detection. An increase in the number of oligomers detected increased the calculated average molar ethoxylate values. The average molar oligomer values calculated by SFC-UV for these alcohols were consistent with the nominal reported values.

Determination of alcohol polyether average molar oligomer value/distribution via supercritical fluid chromatography coupled with UV and MS detection.

Supercritical fluid chromatography (SFC) was used for the analysis of ethoxylated and propoxylated surfactants. Samples were derivatized to phenylated silyl ethers with a disilazane-chlorosilane mixture. Addition of a phenyl group to the surfactant allowed UV-absorbance detection of each oligomer. Acetonitrile and methanol were evaluated as mobile phase modifiers. Better peak shape was realized with methanol-modified CO2 on an octadecyl silica bonded phase than with acetonitrile-modified CO2. Peak assignments were made via SFC coupled with electrospray ionization-mass spectrometry (ESI-MS) in the positive ion mode. A sulfonamide-embedded alkyl stationary phase was also evaluated for separation of the derivatized samples. SFC-UV and SFC-ESI-MS data were jointly used for calculation of average molar oligomer values which were then compared to values calculated from 1H NMR data of non-derivatized samples. The derivatization or separation method using the sulfonamide embedded phase required no preliminary cleanup and yielded reproducible oligomer values that were consistent with those of the manufacturer's nominal values.

Separation of derivatized alcohol ethoxylates and propoxylates by low temperature packed column supercritical fluid chromatography using ultraviolet absorbance detection.

Supercritical fluid chromatography (SFC) is capable of separating oligomers of alcohol ethoxylates (AEOs) and propoxylates (APOs) samples with pure carbon dioxide. The instrumental conditions, however, needed for separation necessitate both high temperature and high pressure. Derivatization of alcohol polyether samples with an UV absorbing agent has been achieved with a phenylated disilazane in hopes of employing a solvent-modified CO2 mobile phase in conjunction with both lower CO2 pressure and lower temperature for oligomer separation. A silylether containing a single phenyl group was formed via the derivatization of the hydroxyl termini of AEO and APO samples. The derivatized polyethers were detected at 215 nm with little or no interference from the mobile phase. Octadecylsilica (ODS) and a polar embedded alkyl bonded silica stationary phase were studied with the organic solvent-modified CO2 mobile phase. The combination of an ODS phase and the polar embedded phase, tandemly stacked, produced the best chromatographic separation of oligomeric species. Data from SFC-UV separations combined with peak assignments from SFC with electrospray ionization-mass spectrometric (ESI-MS) detection produced average molar oligomer values for each surfactant sample.