Liposomal Phytosterols as LDL-Cholesterol-Lowering Agents in Diet-Induced Hyperlipidemia.

The high blood level of low-density lipoprotein cholesterol (LDL-C) is a primary risk factor for cardiovascular disease. Plant sterols, known as phytosterols (PSs), can reduce LDL-C in a range of 8-14%. The extent of LDL-C reduction depends on its formulation. Encapsulation into liposomes is one formulation strategy to enhance the efficiency of PSs. PSs (campesterol, stigmasterol, and ß-sitosterol) have frequently been assessed alone or in combination for their LDL-C-lowering ability. However, one naturally abundant PS, brassicasterol, has not yet been tested for its efficacy. We have previously developed a novel liposomal formulation containing the PS mixture present naturally in canola that is composed of brassicasterol, campesterol, and ß-sitosterol. In this work, the efficacy of our novel liposomal PS formulation that includes brassicasterol was assessed in a hamster model. Animals were divided into five groups: (i) liposomal PS in orange juice, (ii) liposomal PS in water, (iii) marketed PS in orange juice, (iv) control orange juice, and (v) control water. The animals were fed a high-fat, cholesterol-supplemented (0.5%) diet to induce hypercholesterolemia. The treatment was administered orally once daily for 4 weeks. Fasting blood samples were collected at baseline, week 2, and week 4. The extent of the reduction of total cholesterol, LDL-C, high-density lipoprotein cholesterol (HDL-C), and triglycerides was compared among the groups. Liposomal PSs in both orange juice and water significantly reduced LDL-C compared to their controls. Furthermore, the liposomal PS was as effective as a marketed PS-containing product in reducing LDL-C. Liposomal PSs in both orange juice and water showed similar efficacy in LDL-C reduction, highlighting that these vehicles/food matrices do not affect the efficacy of PSs. The liposomal formulation of a natural PS mixture extracted from canola oil, with brassicasterol as a major component, exhibited a significant LDL-C reduction in a hamster model.

A comparative metabolomics investigation of flavonoid variation in faba bean flowers.

INTRODUCTION: Faba bean (Vicia faba L.) flowers are edible and used as garnishes because of their aroma, sweet flavor and attractive colors. Anthocyanins are the common plant pigments that give flowers their vivid colors, whereas non-anthocyanin flavonoids can serve as co-pigments that can modify the color intensity of flowers. OBJECTIVES: To explore the polyphenol diversity and differences in standard and wing petals of faba bean flowers; and identify glycosylated flavonoids that contribute to flower color. METHODS: Flower standard and wing petals from 30 faba bean genotypes (eight color groups with a total of 60 samples) were used for polyphenol extraction. Samples were analyzed using a targeted method and a semi-untargeted analysis using liquid chromatography-high resolution mass spectrometry (LC-HRMS) combined with photodiode array (PDA) detection. Compound Discoverer software was used for polyphenol identification and multivariate analysis. RESULTS: The semi-untargeted analysis guided by the PDA detected 90 flavonoid metabolites present in faba bean flower petals. Ten anthocyanins largely influenced the flower colors, but other flavonoids (63 flavonols and 12 flavones) found with variable levels in different flower color groups appeared to also influence color, especially in mixed colors. CONCLUSION: Analysis of the different colored faba bean flowers confirmed that the color variation between the flowers was mainly controlled by anthocyanins in brown, red and purple-red flowers. Of the other flavonoids, multiglycosylated kaempferols were abundant in white and brown flowers, monoglycosylated kaempferols were common in red and purple-red flowers, and quercetin and apigenin glycosides were abundant co-pigments in purple-red flowers.

Mass Spectrometry-Based Untargeted Metabolomics Reveals the Importance of Glycosylated Flavones in Patterned Lentil Seed Coats.

Lentil seed coats are rich in antioxidant polyphenols that are important for plant defense and have potential as valorized byproducts. Although biochemical differences among lentil seed coat colors have been previously studied, differences among seed coat patterns remain largely unexplored. This study used mass spectrometry-based untargeted metabolomics to investigate polyphenol differences among lentil seed coat patterns to search for biochemical pathways potentially responsible for seed coat pattern differences. Comparing patterned with non-patterned green lentil seed coats, 28 significantly upregulated metabolites were found in patterned seed coats; 19 of them were identified as flavones. Flavones were virtually absent in non-patterned seed coats, thereby strongly suggesting a blockage in their flavone biosynthetic pathway. Although the black pattern is not readily discernible on black seed coats, many of the same flavones found in green marbled seed coats were also found in black seed coats, indicating that black seed coats likely have a marbled pattern.

Development of a liquid chromatography-tandem mass spectrometry method for the analysis of docetaxel-loaded Poly(lactic-co-glycolic acid) nanoparticles.

Docetaxel is among the most effective chemotherapeutic agents used for the treatment of solid tumors, such as breast cancer. Targeting docetaxel to the tumor site would increase the safety and efficacy of the treatment. The focus of this work was to develop an efficient liquid chromatography tandem mass spectrometry (LC-MS/MS) method to quantify docetaxel entrapped in optimized poly lactic-co-glycolic acid (PLGA) nanoparticles. Several nanoparticle formulations were prepared to optimize the nanoparticles based on their size and yield percentage using a modified solvent evaporation technique. The MS/MS fingerprints of docetaxel and paclitaxel (as internal standard) were used to identify diagnostic product ion for developing a multiple reaction monitoring (MRM) LC-MS/MS method for the quantification of docetaxel in the PLGA nanoparticles. A triple quadrupole linear ion trap instrument (AB Sciex 4000 QTRAP) equipped with electrospray ionization was used. The optimized nanoparticles had a zeta potential of -23.2 ± 1.4 mV and mean particle sizes of 202.2 ± 4.7 nm and 251.7 ± 8.2 nm before and after freeze-drying, respectively. Polydispersity index values of the nanoparticles confirmed their uniform size distribution. The developed LC-MS/MS method could quantify docetaxel in the PLGA matrix with accuracy and precision covering a broad linear range of 15.6-4000 ng/mL. Method validation was conducted using the regulatory guidelines of the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) and showed acceptable values for all the tested criteria. The developed LC-MS/MS method with the novelty of using a phenyl column will be beneficial for future analysis of docetaxel loaded polymeric nano-delivery systems.

Determination of phytosterol oxidation products in pharmaceutical liposomal formulations and plant vegetable oil extracts using novel fast liquid chromatography - Tandem mass spectrometric methods.

Phytosterol oxidation products (POPs) formed by the auto-oxidation of phytosterols can lead to negative health consequences. New liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantitative and qualitative approaches were developed. For quantification, sixteen phytosterol oxidation products (POPs) in liposomal formulations; namely 7-keto, 7-hydroxy, 5,6-epoxy, and 5,6-dihydroxy derivatives of brassicasterol, campesterol, stigmasterol, and ß-sitosterol were quantified. The method has a short run time of 5 min, achieved on a poroshell C18 column, using isocratic elution. To the best of our knowledge, this is the shortest run time among reported methods for the quantitative analysis of POPs. Atmospheric pressure chemical ionization (APCI) was used, and the mobile phase was composed of acetonitrile/methanol (99:1 v/v). The quantitative method was validated as per the FDA guidelines for linearity, accuracy, precision, selectivity, sensitivity, matrix effect, dilution integrity, and stability. The method was applied for the quantification of POPs in liposomal phytosterol formulations prepared with and without tocopherols, as antioxidants. The formulation process had little impact on the formation of POPs as only 7-ketobrassicasterol was quantified in tested samples. The quantified value of POPs in liposomal samples was insignificant to impart any toxicological effects. Other degradation products such as 7-hydroxy, 5,6-epoxy and 5,6-dihydroxy derivatives of brassicasterol, campesterol and ß-sitosterol were below the lower limit of quantification. Phytosterol-containing formulations were then assessed for their oxidative stability after microwave exposure for 5 min. The incorporation of tocopherols significantly increased the stability of phytosterols in the liposomal formulations. Finally, LC-MS/MS qualitative identification of phytosterols obtained from extra virgin olive oil was performed. New POPs, namely 7-ketoavenasterol, and 7-ketomethylenecycloartenol were putatively identified, illustrating the applicability of the method to identify POPs with varying structures present in various phytosterol sources. In fact, it is the first time that 7-ketomethylenecycloartenol is reported as a POP.

Development and validation of patient-community pharmacist encounter toolkit regarding substance misuse: Delphi procedure.

BACKGROUND: Pharmacists' roles and services for patients with substance use are not well defined and inconsistent from site to site. Several barriers have been identified that hinder pharmacists' care for people who use substances, such as a lack of training and resources. Clinical practice tools can aid in transferring evidence-based approaches to the practice sphere. OBJECTIVES: The aim of the study was to develop a substance misuse management toolkit for community pharmacists to help them manage their encounters with people who use substances. METHODS: A focused literature review was conducted and 2 needs assessment studies, one for community pharmacists and one for patients informed the development of the toolkit. The toolkit is an adaption of the screening, brief intervention, and referral to treatment (SBIRT) approach, which is one of the most well-defined and effective strategies for substance use management. However, SBIRT is a novel care model in community pharmacy settings. Therefore, a substance misuse management toolkit with 20 items was created for community pharmacists incorporating evidence-based strategies and clinical algorithms. Delphi procedure was used to validate the toolkit. RESULTS: Two rounds of questions were sent to experts in the field of substance misuse, some of whom were pharmacists. In both rounds, these experts were asked to rate the appropriateness and clarity of items in the toolkit and provide comments and suggestions. Items with a median rating of 7 or more out of 10 were included in the toolkit. In the second round, the experts were asked to rerate the revised version and provide additional feedback. After the second round, agreement was reached for almost all items of the toolkit. CONCLUSION: A Delphi procedure was successfully used to provide evidence of the validity of the new guiding toolkit for community pharmacists. The toolkit will be implemented and evaluated to provide additional evidence of validity in practice.

An Untargeted Metabolomics Approach for Correlating Pulse Crop Seed Coat Polyphenol Profiles with Antioxidant Capacity and Iron Chelation Ability.

Pulse crop seed coats are a sustainable source of antioxidant polyphenols, but are typically treated as low-value products, partly because some polyphenols reduce iron bioavailability in humans. This study correlates antioxidant/iron chelation capabilities of diverse seed coat types from five major pulse crops (common bean, lentil, pea, chickpea and faba bean) with polyphenol composition using mass spectrometry. Untargeted metabolomics was used to identify key differences and a hierarchical analysis revealed that common beans had the most diverse polyphenol profiles among these pulse crops. The highest antioxidant capacities were found in seed coats of black bean and all tannin lentils, followed by maple pea, however, tannin lentils showed much lower iron chelation among these seed coats. Thus, tannin lentils are more desirable sources as natural antioxidants in food applications, whereas black bean and maple pea are more suitable sources for industrial applications. Regardless of pulse crop, proanthocyanidins were primary contributors to antioxidant capacity, and to a lesser extent, anthocyanins and flavan-3-ols, whereas glycosylated flavonols contributed minimally. Higher iron chelation was primarily attributed to proanthocyanidin composition, and also myricetin 3-O-glucoside in black bean. Seed coats having proanthocyanidins that are primarily prodelphinidins show higher iron chelation compared with those containing procyanidins and/or propelargonidins.

Qualitative assessment of patients' perspectives and needs from community pharmacists in substance use disorder management.

BACKGROUND: Non-medical use of psychoactive substances is a common harmful behavior that leads to the development of Substance Use Disorders (SUDs). SUD is a significant health concern that causes adverse health consequences and elevates the economic burden on the health care system. SUD treatment plans that utilize a patient-centered approach have demonstrated improved treatment outcomes. It is essential for health care providers, including community pharmacists, to understand patients' needs and prioritize them. Therefore, this study was conducted to explore the perspective of patients living with SUDs or who used substances non-medically regarding community pharmacist services and the delivery of services in a community pharmacy setting. The study took place in Saskatoon, a small urban center of Saskatchewan, Canada. METHODS: Qualitative methodology was used for this research inquiry. Four focus groups were conducted, with a total of 20 individuals who had experienced substance use and accessed community pharmacy services. The discussion of the four focus groups was transcribed verbatim and analyzed independently by two researchers. Agreement on the emergent themes was reached through discussion between the two researchers. RESULTS: Data analysis resulted in four themes that described participants' perspectives about community pharmacists. The four emergent themes are: 1) conflicted experiences with community pharmacists, 2) lack of knowledge concerning community pharmacists' extended services, 3) negative experiences in Opioid Agonist Therapy (OAT) program, and 4) needs from community pharmacists. CONCLUSION: There is significant potential for the patient-pharmacist relationship to address the varying needs of patients who use substances and improve their overall health care experience. Patients who use substances are receptive to pharmacists' services beyond dispensary; however, respectful communication, provision of drug-related information, and counseling are among the primary demands. Future research should focus on studying the impact of meeting the needs of patients on their treatment outcomes.

Novel Fast Chromatography-Tandem Mass Spectrometric Quantitative Approach for the Determination of Plant-Extracted Phytosterols and Tocopherols.

Phytosterols and tocopherols are commonly used in food and pharmaceutical industries for their health benefits. Current analysis methods rely on conventional liquid chromatography, using an analytical column, which can be tedious and time consuming. However, simple, and fast analytical methods can facilitate their qualitative and quantitative analysis. In this study, a fast chromatography-tandem mass spectrometric (FC-MS/MS) method was developed and validated for the quantitative analysis of phytosterols and tocopherols. Omitting chromatography by employing flow injection analysis-mass spectrometry (FIA-MS) failed in the quantification of target analytes due to analyte-to-analyte interferences from phytosterols. These interferences arise from their ambiguous MS fingerprints that would lead to false identification and inaccurate quantification. Therefore, a C18 guard column with a 1.9 µm particle size was employed for FC-MS/MS under isocratic elution using acetonitrile/methanol (99:1 v/v) at a flow rate of 600 µL/min. Analyte-to-analyte interferences were identified and eliminated. The false peaks could then be easily identified due to chromatographic separation. In addition, two internal standards were evaluated, namely cholestanol and deuterated cholesterol. Both internal standards contributed to the observed analyte-to-analyte interferences; however, adequate shift in the retention time for deuterated cholesterol eliminated its interferences and allowed for an accurate quantification. The method is fast (1.3 min) compared to published methods and can distinguish false peaks observed in FIA-MS. Seven analytes were quantified simultaneously, namely brassicasterol, campesterol, stigmasterol, ß-sitosterol, α-tocopherol, δ-tocopherol, and γ-tocopherol. The method was successfully applied in the quantitative analysis of phytosterols and tocopherols present in the unsaponifiable matter of canola oil deodorizer distillate (CODD). ß-sitosterol and γ-tocopherol were the most abundant phytosterols and tocopherols, respectively.

Establishment of the tandem mass spectrometric fingerprints of taxane-based anticancer compounds.

RATIONALE: Compounds in the taxane drug family are among the most successful and effective chemotherapeutic agents used in the treatment of solid tumors, such as breast, ovarian, and prostate cancers. The tandem mass spectrometric (MS/MS) fragmentation behavior of these compounds is described in detail, and a generalized MS/MS fingerprint is established for the first time. METHODS: Five compounds, namely paclitaxel, docetaxel, cabazitaxel, cephalomannine, and baccatin III, were evaluated. A hybrid quadrupole orthogonal time-of-flight (Q-TOF) mass spectrometer was used to obtain accurate mass measurements, whereas MS/MS and second-generation MS/MS (MS3 ) analyses were performed using a triple quadrupole-linear ion trap mass spectrometer. Both instruments were equipped with an electrospray ionization source operated in the positive ion mode. RESULTS: All taxanes showed an abundant singly charged [M + H]+ species in the single-stage analysis with mass accuracies less than 3 ppm. The evaluated compounds exhibited common fragmentation behavior in their MS/MS analysis, which allowed for the production of a universal fragmentation pattern. MS3 experiments confirmed the genesis of the various product ions proposed in the fragmentation pathway. In addition, diagnostic product ions were originated from a cleavage in the ester bond between the core diterpene ring structure and the side chain. CONCLUSIONS: Varying functional groups present in these compounds resulted in unique product ions that are specific to each structure. The established MS/MS fingerprints will be used in the near future for identification and for the development of multiple reaction monitoring liquid chromatography-MS/MS quantification methods.

Analytical Strategies to Analyze the Oxidation Products of Phytosterols, and Formulation-Based Approaches to Reduce Their Generation.

Phytosterols are a class of lipid molecules present in plants that are structurally similar to cholesterol and have been widely utilized as cholesterol-lowering agents. However, the susceptibility of phytosterols to oxidation has led to concerns regarding their safety and tolerability. Phytosterol oxidation products (POPs) present in a variety of enriched and non-enriched foods can show pro-atherogenic and pro-inflammatory properties. Therefore, it is crucial to screen and analyze various phytosterol-containing products for the presence of POPs and ultimately design or modify phytosterols in such a way that prevents the generation of POPs and yet maintains their pharmacological activity. The main approaches for the analysis of POPs include the use of mass spectrometry (MS) linked to a suitable separation technique, notably gas chromatography (GC). However, liquid chromatography (LC)-MS has the potential to simplify the analysis due to the elimination of any derivatization step, usually required for GC-MS. To reduce the transformation of phytosterols to their oxidized counterparts, formulation strategies can theoretically be adopted, including the use of microemulsions, microcapsules, micelles, nanoparticles, and liposomes. In addition, co-formulation with antioxidants, such as tocopherols, may prove useful in substantially preventing POP generation. The main objectives of this review article are to evaluate the various analytical strategies that have been adopted for analyzing them. In addition, formulation approaches that can prevent the generation of these oxidation products are proposed.

STRATEGIES AND CHALLENGES IN METHOD DEVELOPMENT AND VALIDATION FOR THE ABSOLUTE QUANTIFICATION OF ENDOGENOUS BIOMARKER METABOLITES USING LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY.

Metabolomics is a dynamically evolving field, with a major application in identifying biomarkers for drug development and personalized medicine. Numerous metabolomic studies have identified endogenous metabolites that, in principle, are eligible for translation to clinical practice. However, few metabolomic-derived biomarker candidates have been qualified by regulatory bodies for clinical applications. Such interruption in the biomarker qualification process can be largely attributed to various reasons including inappropriate study design and inadequate data to support the clinical utility of the biomarkers. In addition, the lack of robust assays for the routine quantification of candidate biomarkers has been suggested as a potential bottleneck in the biomarker qualification process. In fact, the nature of the endogenous metabolites precludes the application of the current validation guidelines for bioanalytical methods. As a result, there have been individual efforts in modifying existing guidelines and/or developing alternative approaches to facilitate method validation. In this review, three main challenges for method development and validation for endogenous metabolites are discussed, namely matrix effects evaluation, alternative analyte-free matrices, and the choice of internal standards (ISs). Some studies have modified the equations described by the European Medicines Agency for the evaluation of matrix effects. However, alternative strategies were also described; for instance, calibration curves can be generated in solvents and in biological samples and the slopes can be compared through ratios, relative standard deviation, or a modified Stufour suggested approaches while quantifying mainly endogenous metabolitesdent t-test. ISs, on the contrary, are diverse; in which seven different possible types, used in metabolomics-based studies, were identified in the literature. Each type has its advantages and limitations; however, isotope-labeled ISs and ISs created through isotope derivatization show superior performance. Finally, alternative matrices have been described and tested during method development and validation for the quantification of endogenous entities. These alternatives are discussed in detail, highlighting their advantages and shortcomings. The goal of this review is to compare, apprise, and debate current knowledge and practices in order to aid researchers and clinical scientists in developing robust assays needed during the qualification process of candidate metabolite biomarkers. © 2019 John Wiley & Sons Ltd. Mass Spec Rev.

Fast Quantification Without Conventional Chromatography, The Growing Power of Mass Spectrometry.

Mass spectrometry (MS) in hyphenated techniques is widely accepted as the gold standard quantitative tool in life sciences. However, MS possesses intrinsic analytical capabilities that allow it to be a stand-alone quantitative technique, particularly with current technological advancements. MS has a great potential for simplifying quantitative analysis without the need for tedious chromatographic separation. Its selectivity relies on multistage MS analysis (MSn), including tandem mass spectrometry (MS/MS), as well as the ever-growing advancements of high-resolution MS instruments. This perspective describes various analytical platforms that utilize MS as a stand-alone quantitative technique, namely, flow injection analysis (FIA), matrix assisted laser desorption ionization (MALDI), including MALDI-MS imaging and ion mobility, particularly high-field asymmetric waveform ion mobility spectrometry (FAIMS). When MS alone is not capable of providing reliable quantitative data, instead of conventional liquid chromatography (LC)-MS, the use of a guard column (i.e., fast chromatography) may be sufficient for quantification. Although the omission of chromatographic separation simplifies the analytical process, extra procedures may be needed during sample preparation and clean-up to address the issue of matrix effects. The discussion of this manuscript focuses on key parameters underlying the uniqueness of each technique for its application in quantitative analysis without the need for a chromatographic separation. In addition, the potential for each analytical strategy and its challenges are discussed as well as improvements needed to render them as mainstream quantitative analytical tools. Overcoming the hurdles for fully validating a quantitative method will allow MS alone to eventually become an indispensable quantitative tool for clinical and toxicological studies.

The simultaneous quantification of phytosterols and tocopherols in liposomal formulations using validated atmospheric pressure chemical ionization- liquid chromatography -tandem mass spectrometry.

A novel liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and validated to simultaneously quantify phytosterols (brassicasterol, campesterol, stigmasterol and ß-sitosterol) and tocopherols (alpha, beta, gamma and delta) entrapped in the lipid bilayer of a liposomal formulation. Apart from liposomes (a pharmaceutical product), the developed method was able to quantify target analytes in agricultural products, thus showing wide applications. Atmospheric pressure chemical ionization (APCI) was employed due to the enhanced ionization of phytosterols and tocopherols in comparison to electrospray ionization. Unlike published work, the chromatographic conditions were modified to simplify the analytical approach. For the first time, a simple isocratic elution (acetonitrile:methanol 99:1 v/v) was utilized for the separation of four phytosterols and four tocopherols in a single run. A substantially better baseline separation of phytosterols were obtained in comparison to reported methods by using poroshell C18 column. The method has a total run time of 7 min, which is the shortest run time among all reported quantitative methods for the simultaneous determination of four phytosterols and four tocopherols. Calibration curves for all phytosterols were linear in the range of 0.05-10 µg/mL. In the case of tocopherols, alpha tocopherol showed linear response in the range of 0.25-10 µg/mL. However, gamma and delta tocopherols exhibited quadratic relationship in the same concentration range (0.25-10 µg/mL). Validation parameters met the International Conference on Harmonization (ICH) guidelines in terms of selectivity, accuracy, precision, repeatability, sensitivity, matrix effects, dilution integrity and stability. The method was, for the first time, successfully applied for the quantifying phytosterols and tocopherols entrapped inside liposomes. An interesting chromatographic phenomenon was observed during sample analysis. Alpha tocopherol (entrapped in the liposomal lipid bilayer) was found to elute at two retention times, 2.53 min and 3.60 min. Such dual separation was not observed in calibration standards and quality controls. It was concluded that the chiral recognition ability of liposomes made up of phosphatidylcholine separated the enantiomers of alpha tocopherol, giving rise to two peaks at two different retention time. To sum, the reported novel LC-MS/MS method addresses three major analytical shortcomings, namely i)longer run time, ii)complex gradient elution and iii)poor baseline separation of phytosterols and tocopherols.

Mass Spectrometric Detection and Characterization of Metabolites of Gemini Surfactants Used as Gene Delivery Vectors.

Gemini surfactants are a class of lipid molecules that have been successfully used in vitro and in vivo as nonviral gene delivery vectors. However, the biological fate of gemini surfactants has not been well investigated. In particular, the metabolism of gemini surfactants after they enter cells as gene delivery vehicles is unknown. In this work, we used a high-resolution quadrupole-Orbitrap mass spectrometry (Q-Exactive) instrument to detect the metabolites of three model gemini surfactants, namely, (a) unsubstituted (16-3-16), (b) with pyridinium head groups (16(Py)-S-2-S-16(Py)), and (c) substituted with a glycyl-lysine di-peptide (16-7N(GK)-16). The metabolites were characterized, and structures were proposed, based on accurate masses and characteristic product ions. The metabolism of the three gemini surfactants was very different as 16-3-16 was not metabolized in PAM 212 cells, whereas 16(Py)-S-2-S-16(Py) was metabolized primarily via phase I reactions, including oxidation and dealkylation, producing metabolites that could be linked to its observed high toxicity. The third gemini surfactant 16-7N(GK)-16 was metabolized mainly via phase II reactions, including methylation, acetylation, glucose conjugation, palmityl conjugation, and stearyl conjugation. The metabolism of gemini surfactants provides insight for future directions in the design and development of more effective gemini surfactants with lower toxicity. The reported approach can also be applied to study the metabolism of other structurally related gemini surfactants.

Liquid chromatography-tandem mass spectrometry bioanalytical method for the determination of kavain in mice plasma: Application to a pharmacokinetic study.

A simple and fast bioanalytical method for the quantification of kavain in mice plasma was developed using liquid chromatography (LC)-tandem mass spectrometry (MS/MS). A full method validation was performed, according to regulatory guidelines, employing isotopically labeled kavain as the internal standard (racemic-kavain-d3). For the quantification, [M+H]+ was formed using an electrospray ionization (ESI) source in the positive ion mode and multiple reaction monitoring (MRM) was employed using a quadrupole-linear ion trap (4000 QTRAP®) instrument. The monitored MRM transitions were 231.0 â†’ 115.1 and 231.0 â†’ 152.8 for kavain; and 234.2 â†’ 199.2 for the internal standard. A linear response was obtained at the concentration range of 10 to 200 ng/mL with intra- and inter-day variations within the acceptable criteria for all quality control samples. After validation, the method was successfully applied for the quantification of kavain in mice plasma after oral administration of the kavain standard and Kava-kava extract. The plasma concentration over time results were applied for a pharmacokinetics study. The obtained pharmacokinetic parameters indicated a considerably higher bioavailability for kavain when Kava-kava extract was administered due to a pharmacokinetic synergism between the analyte and the other compounds present in the extract.

Cellular Uptake and Distribution of Gemini Surfactant Nanoparticles Used as Gene Delivery Agents.

Gemini surfactants are promising molecules utilized as non-viral gene delivery vectors. However, little is known about their cellular uptake and distribution after they release their therapeutic cargo. Therefore, we quantitatively evaluated the cellular uptake and distribution of three gemini surfactants: unsubstituted (16-3-16), with pyridinium head groups (16(Py)-S-2-S-16(Py)) and substituted with a glycyl-lysine di-peptide (16-7N(GK)-16). We also assessed the relationship between cellular uptake and distribution of each gemini surfactant and its overall efficiency and toxicity. Epidermal keratinocytes PAM 212 were treated with gemini surfactant nanoparticles formulated with plasmid DNA and harvested at various time points to collect the enriched nuclear, mitochondrial, plasma membrane, and cytosolic fractions. Gemini surfactants were then extracted from each subcellular fraction and quantified using a validated flow injection analysis-tandem mass spectrometry (FIA-MS/MS) method. Mass spectrometry is superior to the use of fluorescent tags that alter the physicochemical properties and pharmacokinetics of the nanoparticles and can be cleaved from the gemini surfactant molecules within biological systems. Overall, a significantly higher cellular uptake was observed for 16-7N(GK)-16 (17.0%) compared with 16-3-6 (3.6%) and 16(Py)-S-2-S-16(Py) (1.4%), which explained the relatively higher transfection efficiency of 16-7N(GK)-16. Gemini surfactants 16-3-16 and 16(Py)-S-2-S-16(Py) displayed similar subcellular distribution patterns, with major accumulation in the nucleus, followed by the mitochondrion, cytosol, and plasma membrane. In contrast, 16-7N(GK)-16 was relatively evenly distributed across all four subcellular fractions. However, accumulation within the nucleus after 5 h of treatment was the highest for 16(Py)-S-2-S-16(Py) (50.3%), followed by 16-3-16 (41.8%) and then 16-7N(GK)-16 (33.4%), possibly leading to its relatively higher toxicity. Graphical Abstract.

Tandem mass spectrometric analysis of novel caffeine scaffold-based bifunctional compounds for Parkinson's disease.

RATIONALE: Novel bifunctional compounds composed of a caffeine scaffold attached to nicotine (C8 -6-N), 1-aminoindan (C8 -6-I), or caffeine (C8 -6-C8 ) were designed as therapeutics or diagnostics for Parkinson's disease (PD). In order to probe their pharmacological and toxicological profile, an appropriate analytical method is required. The goal of this study is to establish a tandem mass spectrometric fingerprint for the development of quantitative and qualitative methods that will aid future assessment of the in vitro and in vivo absorption, distribution, metabolism, excretion (ADME) and pharmacokinetic properties of these lead bifunctional compounds for PD. METHODS: Accurate mass measurement was performed using a hybrid quadrupole orthogonal time-of-flight mass spectrometer while multistage MS/MS and MS3 analyses were conducted using a triple quadrupole linear ion trap mass spectrometer. Both instruments are equipped with an electrospray ionization (ESI) source and were operated in the positive ion mode. The source and compound parameters were optimized for all three tested bifunctional compounds. RESULTS: The MS/MS analysis indicates that the fragmentation of C8 -6-N and C8 -6-I is driven by the dissociation of the nicotine and 1-aminoindan moieties, respectively, but not caffeine. A significant observation in the MS/MS fragmentation of C8 -6-C8 suggests that a previously reported loss of acetaldehyde during caffeine dissociation is instead a loss of CO2 . CONCLUSIONS: The collision-induced tandem mass spectrometry (CID-MS/MS) analysis of these novel bifunctional compounds revealed compound-specific diagnostic product ions and neutral losses for all three tested bifunctional compounds. The established MS/MS fingerprint will be applied to the future development of qualitative and quantitative methods.

Peptide-Modified Gemini Surfactants: Preparation and Characterization for Gene Delivery.

Diquaternary ammonium-based gemini surfactants have been investigated widely as nonviral gene delivery systems. These unique cationic lipids have versatility in their chemical structure, show relatively low toxicity, are able to compact genetic material (pDNA, RNA) into nano-sized lipoplexes, and can be easily produced. In addition, the gemini surfactants show significant improvement in the transfection activity and biocompatibility compared to other cationic lipids used as nonviral gene delivery agents. The successful applications of gemini surfactant-based lipoplexes as topical gene delivery systems in animal models indicate their potential as noninvasive carriers for genetic immunization, theranostic agents, and in other gene therapy treatments. Detailed physicochemical characterization of gemini surfactant lipoplexes is a key factor in terms of formulation optimization and elucidation of the cellular uptake and stability of the lipoplexes system. In this chapter, we describe in detail different formulation methods to prepare gemini surfactant lipoplexes and comprehensive physicochemical characterization. In addition, we illustrate general protocols for in vitro evaluations.