Pharmacy

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Integration of medicinal chemistry in therapeutic decision-making: A way forward?
(Currents in Pharmacy Teaching and Learning, 2024) Krol, Ed; Albon, Simon P
Many attempts have been made to integrate medicinal chemistry knowledge into therapeutic decision-making in pharmacy programs across North America. Examples include the use of Structure-Based Therapeutic Evaluations, alignment of medicinal chemistry content with courses in pharmacology, pharmaceutics and pharmacotherapeutics, and team-based or problem-based learning methods. The majority of these approaches indicate that students have greater confidence or comfort with medicinal chemistry, but there remain few cases where an improvement in performance has been measured. This is especially challenging for assessing a student’s ability to implement medicinal chemistry learning in pharmacotherapeutic decision-making. Building upon our national special interest group’s recent environmental scan of medicinal chemistry instruction in Canadian Pharmacy programs, we are investigating strategies for integration of medicinal chemistry learning in therapeutic decision-making. This commentary will discuss methods and evidence to support medicinal chemistry integration, relevant assessment strategies, and potential paths forward.
Medical cannabis in schools: The experiences of caregivers
(Oxford University Press, 2023-05) Mansell, Holly; Zaslawski, Zina; Mbabaali, Sophia; King, Patricia M.; Kelly, Lauren E.; Lougheed, Taylor; Anderson, Jennifer; Huntsman, Richard J.; Alcorn, Jane
Abstract Objectives Implementing medical cannabis (MC) into a child’s daily routine can be challenging and there is a lack of guidance for its therapeutic use in schools in Canada. Our objective was to learn about the experiences of caregivers of school-aged children who require MC. Methods Qualitative description was used and caregivers were interviewed about MC in schools and in general. The transcripts were entered into Dedoose software for qualitative analysis and content analysis was performed. Sentences and statements were ascribed line by line into meaning units and labelled with codes, and organized according to categories and subcategories. Results Twelve caregivers of school-aged children who take MC participated. The most common reasons for treatment were drug-resistant epilepsy (DRE), autism, or other developmental disorders. Approximately half of the participants’ children (n = 6) took MC during the school day and most (5/6) perceived their experiences to be positive or neutral but reported a lack of knowledge about MC. While data saturation was not reached regarding MC in schools, rich dialogues were garnered about MC in general and three categories were identified: challenges (subcategories stigma, finding an authorizer, cost, dosing, and supply); parents as advocates (subcategories required knowledge, attitudes, skills, and sources of information); and caregiver relief for positive outcomes. Conclusions Caregivers demonstrate remarkable tenacity despite the many challenges associated with MC use. Education and practice change are needed to ensure that children using MC can benefit from or continue to experience its positive outcomes within the school environment and beyond.
Nutritional Status of Preschool Children (aged 24 TO 59 months) and Chickpea Production and Consumption in Ethiopia
(African Journal of Food, Agriculture, Nutrition and Development, 2024-10) Asrat, R. T; Girma, M; Tafesse, E; Whiting, S. J; Henry, Carol
Child under-nutrition is a major public health concern in developing countries including Ethiopia. Poor quality diet is the major determinant factor contributing to child undernutrition. Pulse crop production and consumption can improve children’s nutrient quality and dietary diversity. This study aimed to compare the nutritional status and child health, dietary intake, and wealth index of preschool children among pulse crop producer and non-producer families in Ethiopia. The study hypothesized that children living in families who grew pulses and consumed pulses (rather than selling their crop) would have better nutritional status due to availability of this protein-rich food source. A cross-sectional survey was conducted to determine demographic background, child feeding and caring practice and varieties and amount of pulse crop produced at the household level. A simple random sampling technique was used to select a sample of 432 children aged 24-59 months from central Ethiopia the Data was entered to SPSS v 20, WHO Anthro-plus software used to calculate the anthropometry indices, child dietary intake, level of chickpea production and consumption and wealth index, were computed using logistic regression. Chickpea (cicer arietinum L) was produced by 55.3% of the participants. Most (60.7%) of the children from chickpea producer families were high pulse consumers whereas only 15.5% of children from non-producers were high chickpea consumers (the difference in proportion is significant at p-value<0.001). Close to 60% of the children of the non-producer families had a low dietary diversity score (that is <3) compared with 18.8 % from the producer families (p-value <0.001). The study revealed that nutritional status of children in both producer and non-producer families were 41.0 % stunted, 16.1 % underweight and 6.1 % wasted. Stunting and underweight were significantly inversely associated with chickpea production at pvalue <0.001whereas for chickpea consumption, both stunting and underweight was significant at P < 0.05. However, wasting was not significantly associated with either production or consumption of chickpeas. In conclusion, chickpea production and consumption by families had a positive relationship with nutritional status of children. The authors recommend that engaging smallholder farmers in both chickpea production and consumption has potential in improving the dietary diversity and in turn benefit nutritional status of preschool children at the household level and therefore supporting smallholder farmer in chickpea production and consumption will help to contribute to decreasing the prevalence of under nutrition in the country.
Two-Spirit Peoples’ experiences accessing and receiving care from community pharmacies
(SAGE Publications, 2024) Marissa Pirlot; Swidrovich, Jaris
Background: Two-Spirit Peoples face unique challenges in accessing and receiving health care in Canada due to health services, including community pharmacy services, being built on hetero- and cis-normative models that impede appropriate care for this group. Currently, there is limited published information on Two-Spirit Peoples’ experiences accessing and receiving care in community pharmacy settings. Methods: To address the lack of published information, 21 Two-Spirit individuals shared their experiences in a focus group setting. Four different focus groups were held across Canada, including 1 in Saskatoon, Vancouver, Edmonton, and Toronto. Informed by Indigenous methodologies, data were recorded via audio recording and notetaking, and the audio was transcribed and then analyzed for themes using the Voice-Centred Relational Method. Results: Three major structural systems that affect the experiences of Two-Spirit Peoples in community pharmacies were identified: 1) white supremacy, 2) capitalism, and 3) heteronormativity. These 3 systemic issues presented themselves via racism, homophobia, transphobia, pharmacists’ lack of knowledge about Two-Spirit individuals and their health and lack of time spent educating or building relationships with Two-Spirit Peoples. Participants provided suggestions for how community pharmacists can better serve the Two-Spirit community, such as using inclusive language, adding pronouns and preferred names to patient files, increasing knowledge about Two-Spirit health and advocating for Two-Spirit Peoples. Discussion: The results suggest that dismantling current structures and ideologies in community pharmacy and society are required to overcome the identified issues. Conclusion: Two-Spirit Peoples face barriers when it comes to accessing and receiving care in community pharmacies, resulting in many Two-Spirit individuals avoiding health care to save themselves from unsafe and uncomfortable interactions. Pre- and postlicensure pharmacy education about Two-Spirit Peoples is required to improve Two-Spirit Peoples’ experiences accessing and receiving care in community pharmacies.
Cannabinoid Therapy in Athletics: A Review of Current Cannabis Research to Evaluate Potential Real-World Cannabinoid Applications in Sport
(Springer, 2024-08-21) Thompson, Elizabeth S.; Alcorn, Jane; Neary, J. Patrick
The increasing legalization of Cannabis sativa plant products has sparked growing interest in their therapeutic applications. Prohibition laws established in 1937 hindered formal research on cannabis, a plant with cultural and medicinal roots dating back to 2700 BC in Chinese history. Despite regulatory hurdles, published research on cannabis has emerged; yet elite athletes remain an underrepresented population in these studies. Athletes, known for exploring diverse substances to optimize performance, are drawn to the potential benefits of cannabinoid therapy, with anecdotal reports suggesting positive effects on issues ranging from anxiety to brain injuries. This review aims to evaluate empirical published cannabis research with a specific focus on its potential applications in athletics. The changing legal landscape, especially the removal of cannabis from drug testing programs in leagues such as the National Basketball Association (NBA), and endorsements by Major League Baseball (MLB) for cannabinoid products and the National Football League (NFL) for cannabis research, reflects a shift in the acceptability of such substances in sports. However, stigma, confusion, and a lack of education persist, hindering a cohesive understanding among sports organizations, including business professionals, policymakers, coaches, and medical/training staff, in addition to athletes themselves. Adding to the confusion is the lack of consistency with cannabinoid regulations from sport to sport, within or out of competition, and with cannabis bioactive compounds. The need for this review is underscored by the evolving attitudes toward cannabinoids in professional sports and the potential therapeutic benefits or harms they may offer. By synthesizing current cannabis research, this review aims to provide a comprehensive understanding of the applications and implications of cannabinoid use in the realm of athletics.
Development of a liquid chromatography-tandem mass spectrometry method for the analysis of docetaxel-loaded Poly(lactic-co-glycolic acid) nanoparticles
(2023) Khajavinia, Amir; El-Aneed, Anas
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 to 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.
Carbon-based nanoparticles and their surface-modified counterparts as MALDI matrices
(2023) Khajavinia, Amir; El-Aneed, Anas
The Effect of Diphenylethane Side-chain Substituents on Dibenzocyclohexadiene Formation and their Inhibition of α-Synuclein Aggregation in vitro
(2023-01-15) Dalio Bernardes da Silva, Gabriel; Munir, Omer; Krol, Ed
The naturally-occurring di-catechol lignan nordihydroguaiaretic acid (NDGA) and an analog without methyl groups on the butyl linker both undergo intramolecular cyclization at pH 7.4 to form dibenzocyclooctadienes. Both NDGA and these dibenzocyclooctadienes have been shown to prevent in vitro aggregation of α-synuclein, an intrinsically disordered protein associated with Parkinson's disease. NDGA possesses two vicinal methyl groups on the butyl linker and the presence of these methyl groups attenuates the rate of intramolecular cyclization versus the unsubstituted analog, in opposition to the anticipated Thorpe-Ingold effect, likely due to steric repulsions during cyclization. Numerous 1,2-bis-ethane di-catechols are known to inhibit α-synuclein aggregation in vitro and we hypothesize that these compounds undergo a similar intramolecular cyclization and the cyclized products may be responsible for the activity. To test this hypothesis we prepared a series of 1,2-bis-ethane di-catechols with 0, 2 and 4 methyl substituents on the linker. We have confirmed that these compounds undergo intramolecular cyclization to form dibenzocyclohexadienes and that steric interactions between the methyl substituents leads to an increase in the rate of intramolecular cyclization, which is in contrast to what was observed for lignan di-catechols. The rate of cyclization to form six-membered rings is 10-30 times more rapid than formation of eight membered rings and the dibenzocyclohexadienes also prevent in vitro aggregation of α-synuclein.
Tandem mass spectrometric analysis of novel caffeine scaffold-based bifunctional compounds for Parkinson's disease
(2019) Nwabufo, Chukwunonso; El-Aneed, Anas; Krol, Ed
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.
Mass Spectrometric Detection and Characterization of Metabolites of Gemini Surfactants Used as Gene Delivery Vectors
(2020) Jin, Wei; Purves, Randy; Krol, Ed; Badea, ildiko; El-Aneed, Anas
Gemini surfactants are a class of lipid molecules that have been successfully used in vitro and in vivo as non-viral 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 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 PAM212 cells, whereas 16(Py)-S-2-S-16(Py) was metabolized primarily via phase I reactions, including oxidation and de-alkylation, 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.
Cellular Uptake and Distribution of Gemini Surfactant Nanoparticles Used as Gene Delivery Agents
(SpringerLink, 2019-08-06) Jin, Wei; Al-Dulaymi, Mays; Badea, Ildiko; Leary, Scot; Rehman, Jeveria; El-Aneed, Anas
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.
Fast Quantification Without Conventional Chromatography, The Growing Power of Mass Spectrometry
(ACS Publications, 2020-06-08) gachumi, george; Purves, Randy; Hopf, Carston; El-Aneed, Anas
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.
Employing in vitro metabolism to guide design of F-labelled PET probes of novel alpha-synuclein binding bifunctional compounds
(Taylor & Francis, 2021-06-30) Nwabufo, Chukwunonso; Aigbogun, Omozojie; Allen, Kevin; Madeline N. Owens; Jeremy S. Lee; Christopher P. Phenix; Krol, Ed
1. A challenge in the development of novel 18F-labelled positron emission tomography (PET) imaging probes is identification of metabolically stable sites to incorporate the 18F radioisotope. Metabolic loss of 18F from PET probes in vivo can lead to misleading biodistribution data as displaced 18F can accumulate in various tissues. 2. In this study we report on in vitro hepatic microsomal metabolism of novel caffeine containing bifunctional compounds (C8-6-I, C8-6-N, C8-6-C8) that can prevent in vitro aggregation of -synuclein, which is associated with the pathophysiology of Parkinson’s disease. The metabolic profile obtained guided us to synthesize stable isotope 19F-labelled analogues in which the fluorine was introduced at the metabolically stable N7 of the caffeine moiety. 3. An in vitro hepatic microsomal metabolism study of the 19F-labelled analogues resulted in similar metabolites to the unlabelled compounds and demonstrated that the fluorine was metabolically stable, suggesting that these analogues are appropriate PET imaging probes. This straightforward in vitro strategy is valuable for avoiding costly stability failures when designing radiolabelled compounds for PET imaging.
The Health-Care Provider’s Perspective of Education Before Kidney Transplantation
(Sage, 2016-08-22) Trivedi, Paraag; Rosaasen, Nicola; Mansell, Holly
CONTEXT: Adequate patient education is essential for preparing potential recipients for kidney transplantation. Health-care providers play a vital role in education and can identify gaps in patient understanding. OBJECTIVE: To identify deficits in patient knowledge from the perspective of a transplant multidisciplinary care team and determine whether their perceptions align with patients who have previously undergone a transplant. DESIGN: An open call was advertised for health-care providers to attend a focus group discussion regarding the educational needs of pretransplant patients in 1 Canadian center. A predetermined, semistructured set of questions was used to collect the views of transplant caregivers. A moderator, assistant moderator, and research assistant facilitated the discussion, which was transcribed verbatim. Paper surveys were distributed to collect opinions of those unable to attend or uncomfortable to voice their opinion in an open forum. Qualitative analysis software was used to identify any emergent themes. Results were compared to a previous study undertaken in transplant recipients. RESULTS: Despite pre- and posttransplant education, specific themes emerged including misconceptions about the assessment process and time on the wait list and the surgery, incongruency between patient expectations and outcome, and confusion regarding medications. Health-care provider perceptions were remarkably consistent with transplant recipients. CONCLUSION: Health-care providers identified gaps in patient understanding indicating that transplant candidates may not be internalizing what is taught. Innovative educational approaches may be needed to provide more successful patient education. Similarities between health-care provider and patient perceptions suggest that care providers are a valuable source of information.
Education Before Kidney Transplantation: What Do Patients Need to Know?
(Sage, 2017-01-11) Rosaasen, Nicola; Mainra, Rahul; Shoker, Ahmed; Wilson, Jay; Blackburn, David; Mansell, Holly
CONTEXT: Poor knowledge about immunosuppressive (IS) medications remains a major problem for patients in the posttransplant setting. Therefore, more effective educational strategies in the pretransplant setting are being considered as a possible method to improve knowledge and readiness for the challenges of posttransplant care. However, the most effective/relevant content of a pretransplant educational program is yet to be determined. OBJECTIVE: To identify pretransplant education topics from the posttransplant patient perspective. DESIGN: A focus group meeting was conducted among 7 high-functioning, stable adult kidney transplant recipients recruited from the Saskatchewan Transplant Program. Demographic information including age, gender, occupation, background/ethnicity, and time since transplant were recorded. A moderator, assistant moderator, and research assistant facilitated the 90-minute focus group meeting using a predetermined semistructured interview guide. The session was audio recorded and transcribed verbatim. Nvivo software was used to code the data and identify emerging themes exploring views of participants relating to the educational information required for pretransplant patients. RESULTS: Patients were satisfied with the education they had received. Ideas were classified into the following major themes-patient satisfaction, transplant waitlist, surgery, medications, posttransplant complications, lifestyle and monitoring, knowledge acquisition, illusion of control, and life changes posttransplant. Knowledge gaps were identified in all areas of the transplantation process and were not exclusive to IS medications. CONCLUSION: Misconceptions regarding transplantation were identified by a group of high-functioning, stable adult recipients who were satisfied with their clinical care. Future educational strategies should aim to address the entire transplantation process and not be limited to medications.
Comparative analysis of creatinine and osmolality as urine normalization strategies in targeted metabolomics for the differential diagnosis of asthma and COPD
(Springer Link, 2018-09-01) Khamis, Mona M.; Holt, Teagan; Awad, Hanan; El-Aneed, Anas; Adamko, Darryl
Introduction Urine is an ideal matrix for metabolomics investigation due to its non-invasive nature of collection and its rich metabolite content. Despite the advancements in mass spectrometry and 1H-NMR platforms in urine metabolomics, the statistical analysis of the generated data is challenged with the need to adjust for the hydration status of the person. Normalization to creatinine or osmolality values are the most adopted strategies, however, each technique has its challenges that can hinder its wide application. Objective Assessment of whether the statistical model established using a targeted urine metabolomics dataset for the differential diagnosis of asthma and chronic obstructive pulmonary disease (COPD) would be improved by normalization to osmolality instead of creatinine. Methods A metabolomics dataset from 51 patient urine samples previously analyzed using two liquid chromatography-tandem mass spectrometry methods was used. The data was normalized to creatinine and osmolality. The statistical analysis was achieved using partial least square discriminant analysis and models of separation were generated and compared. Results Creatinine and osmolality values in asthma and COPD patients were strongly correlated. Using the same metabolites, we found that normalization to osmolality did not significantly change the results. The metabolites of importance in separation remained the same for both methods. The statistical strength of the creatinine model was somewhat better than the osmolality normalized model (R2Q2=0.919, 0.705 vs R2Q2 =0.929, 0.671). Conclusion Our findings suggest that targeted urine metabolomics data can be normalized to creatinine or osmolality with no significant impact on the diagnostic accuracy of the model.
The development of simple flow injection analysis tandem mass spectrometric methods for the cutaneous determination of peptide-modified cationic gemini surfactants used as gene delivery vectors.
(Elsevier, 2018-09-10) Al-Dulaymi, Mays; Michel, Deborah; Chitanda, Jackson M; Badea, ildiko; El-Aneed, Anas
Diquaternary ammonium gemini surfactants are a class of non-viral gene delivery vectors, primarily studied for their dermal applications. However, their biological fate has rarely been investigated. In this work, we developed simple flow injection analysis tandem mass spectrometric methods, (FIA)-MS/MS, to understand the fate and biodistribution of topically applied gemini surfactant-based therapeutics in an ex-vivo skin model. Three peptide-modified gemini surfactants with varied structures and transfection efficiencies were evaluated. For each compound, two methods were developed to quantify their presence in skin tissue and in phosphate buffered saline (PBS). The methods were developed using single-point calibration mode. Skin penetration was assessed on CD1 mice dorsal skin tissue mounted in a Franz diffusion cell after extraction. Amongst the five evaluated liquid-liquid extraction protocols, the Folch method provides the highest extraction efficiency for all compounds. Weak cationic exchange solid phase extraction was also used to further isolate gemini surfactants from endogenous skin lipids. FIA–MS/MS analysis of the skin revealed that all compounds were detected in the skin with minimal partition into the PBS compartment, which represents circulation. Interestingly, the detected amounts of gemini lipids in the skin were correlated with their transfection efficiencies.
Quantitative determination of potential urine biomarkers of respiratory illnesses using new targeted metabolomic approach
(Elsevier, 2019-01-24) Khamis, Mona M.
The diagnosis of asthma and chronic obstructive pulmonary disease (COPD) can be challenging due to the overlap in their clinical presentations in some patients. There is a need for a more objective clinical test that can be routinely used in primary care settings. Through an untargeted 1H NMR urine metabolomic approach, we identified a set of endogenous metabolites as potential biomarkers for the differentiation of asthma and COPD. A subset of these potential biomarkers contains 7 highly polar metabolites of diverse physicochemical properties. To the best of our knowledge, there is no liquid chromatography-tandem mass spectrometry (LC-MS/MS) method that evaluated more than two of the target metabolites in a single analytical run. The target metabolites belong to the families of monosaccharides, organic acids, amino acids, quaternary ammonium compounds and nucleic acids, rendering hydrophilic interaction liquid chromatography (HILIC) an ideal technology for their quantification. Since a clinical decision is to be made from patients data, a fully validated analytical method is required for biomarker validation. Method validation for endogenous metabolites is a daunting task since current guidelines were designed for exogenous compounds. As such, innovative approaches were adopted to meet the validation requirements. Herein, we describe a sensitive HILIC-MS/MS method for the quantification of the 7 endogenous urinary metabolites. Detection was achieved in the multiple reaction monitoring (MRM) mode with polarity switching, using quadrupole-linear ion trap instrument (QTRAP 6500) as well as single ion monitoring in the negative-ion mode. The method was fully validated according to the regulatory guidelines. Linearity was established between 6 and 21000 ng/mL and quality control samples demonstrated acceptable intra- and inter-day accuracy (85.7%-112%), intra- and inter-day precision (CV% <11.5%) as well as stability under various storage and sample processing conditions. To illustrate the method's applicability, the validated method was applied to the analysis of a small set of urine samples collected from asthma and COPD patients. Preliminary modelling of separation was generated using partial least square discriminant analysis (R2 0.752 and Q2 0.57). The adequate separation between patient samples confirms the diagnostic potential of these target metabolites as a proof-of-concept for the differentiation between asthma and COPD. However, more patient urine samples are needed in order to increase the statistical power of the analytical model.
The determination of gemini surfactants used as gene delivery agents in cellular matrix using validated tandem mass spectrometric method
(Elsevier, 2018-10-03) El-Aneed, Anas
A simple, reliable flow injection analysis (FIA)-tandem mass spectrometric (MS/MS) method was developed for the determination of gemini surfactants, designated as 16-3-16, 16(Py)-S-2-S-(Py)16 and 16-7N(GK)-16, as gene delivery agents in cellular matrix. 16-3-16 is a conventional gemini surfactant bearing two quaternary amines, linked by a 3-carbon spacer region, 16(Py)-S-2-S-(Py)16 contains two pyridinium head groups, while 16-7N(GK)-16 bears a glycine-lysine di-peptide in the space region. The method was fully validated according to USFDA guidelines. It is the first time that FIA-MS/MS method was developed for the quantification of gemini surfactants, belonging to different structural families. The method was superior to existing liquid chromatographic (LC)-MS/MS methods in terms of sensitivity and time of analysis. Positive electrospray ionization (ESI) in the multiple reaction monitoring (MRM) mode were used on a triple quadrupole-linear ion trap (4000 QTRAP®) instrument. Deuterated internal standards were used to correct for matrix effects and variations in ionization within the ESI source. Isotope dilution standard curves were established in cellular matrix, with a linear range of 10nM-1000nM for 16-3-16 and 16(Py)-S-2-S-(Py)16, and 20nM-2000nM for 16-7N(GK)-16. The precision, accuracy, recovery and stability were all within the acceptable ranges as per the USFDA guidelines. The method was successfully applied for the quantification of target gemini surfactants in the nuclear fraction of PAM 212 keratinocyte cells treated with nanoparticles, which varied significantly and may explain differences in the observed efficiency and/or toxicity of these gemini surfactants in gene delivery.
Comparison of accuracy and precision between multipoint calibration, single point calibration and relative quantification for targeted metabolomic analysis
(SpringerLink, 2018-09) Khamis, Mona M.; Klemm, Nancy; Adamko, Darryl; El-Aneed, Anas
Targeted metabolomics requires accurate and precise quantification of candidate biomarkers, often through tandem mass spectrometric (MS/MS) analysis. Differential isotope labeling (DIL) improves mass spectrometric (MS) analysis in metabolomics by derivatizing metabolites with two isotopic forms of the same reagent. Despite its advantages, DIL-liquid chromatographic (LC)-MS/MS can result in substantial increase in workload when fully validated quantitative methods are required. To decrease the workload, we hypothesized that single point calibration or relative quantification could be used as alternative methods. Either approach will result in significant saving in resources and time. To test our hypothesis, six urinary metabolites were selected as model compounds. Urine samples were analyzed using a fully-validated multipoint dansyl chloride-DIL-LC-MS/MS method. Samples were reprocessed using single point calibration and relative quantification modes. Our results demonstrated that the performance of single point calibration or relative quantification was inferior, for some metabolites, to multipoint calibration. The lower limit of quantification failed in the quantification of ethanolamine in most of participant samples using single point calibration. In addition, its precision was not acceptable in one participant during serine and ethanolamine quantification. On the other hand, relative quantification resulted in the least accurate data. In fact, none of the data generated from relative quantification for serine was comparable to that obtained from multipoint calibration. Finally, while single point calibration showed an overall acceptable performance for the majority of the model compounds, we cannot extrapolate the findings to other metabolites within the same analytical run. Analysts are advised to assess accuracy and precision for each metabolite in which single point calibration is the intended quantification mean.

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