College of Pharmacy and Nutrition

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Development and validation of fast and simple flow injection analysis-tandem mass spectrometry (FIA-MS/MS) for the determination of metformin in dog serum
(Elsevier, 2015-03-25) El-Aneed, Anas; Arnason, Terra; Michel, Deborah; Gaunt, Matthew
A simple, fast and sensitive quantification method for the drug metformin in dog serum was developed using flow injection analysis (FIA)- tandem mass spectrometry (MS/MS). The method was fully validated according to industry standards. It is the first time that FIA-MS/MS for metformin was developed surpassing all existing methods in terms of time of analysis. The quantification method was dependent on the formation of [M+H]+ using electrospray ionization (ESI) and employing multiple reaction monitoring (MRM) using quadrupole- linear ion trap (4000 QTRAP®) instrument. A deuterated internal standard (IS) of metformin bearing six deuterium atoms was used to compensate for matrix effects and for variation in ion current within the ESI source. The ion transitions that were monitored were m/z 130.1 -> m/z 71.0 and m/z 130.1-> m/z 60.1 for metformin and m/z 136.0 -> m/z 77.0 for the internal standard. A linear response (r = 0.9966) was established for a range of concentrations of 5- 2340 ng/ml. The inter- and intra-day variations were within the acceptable criteria for all quality control samples. The method was successfully applied for measurement of serum metformin concentration in dogs after intravenous injection.
Mass Spectrometric Based Approaches in Urine Metabolomics and Biomarker Discovery
(Wiley, 2015-04-15) Khamis, Mona; Adamko, Darryl; El-Aneed, Anas
Urine metabolomics has recently emerged as a prominent field for the discovery of non-invasive biomarkers that can detect subtle metabolic discrepancies in response to a specific disease or therapeutic intervention. Urine, compared to other biofluids, is characterized by its ease of collection, richness in metabolites and its ability to reflect imbalances of all biochemical pathways within the body. Following urine collection for metabolomic analysis, samples must be immediately frozen to quench any biogenic and/or non-biogenic chemical reactions. According to the aim of the experiment; sample preparation can vary from simple procedures such as filtration to more specific extraction protocols such as liquid-liquid extraction. Due to the lack of comprehensive studies on urine metabolome stability, higher storage temperatures (i.e. 4 °C) and repetitive freeze-thaw cycles should be avoided. To date, among all analytical techniques, mass spectrometry (MS) provides the best sensitivity, selectivity and identification capabilities to analyze the majority of the metabolite composition in the urine. Combined with the qualitative and quantitative capabilities of MS, and due to the continuous improvements in its related technologies (i.e. ultra high-performance liquid chromatography [UPLC] and hydrophilic interaction liquid chromatography [HILIC]), liquid chromatography (LC)-MS is unequivocally the most utilized and the most informative analytical tool employed in urine metabolomics. Furthermore, differential isotope tagging techniques has provided a solution to ion suppression from urine matrix thus allowing for quantitative analysis. In addition to LC-MS, other MS-based technologies have been utilized in urine metabolomics. These include direct injection (infusion)-MS, capillary electrophoresis-MS and gas chromatography-MS. In this article, the current progresses of different MS-based techniques in exploring the urine metabolome as well as the recent findings in providing potentially diagnostic urinary biomarkers are discussed.
Establishment of Tandem Mass Spectrometric Fingerprint of Novel Antineoplastic Curcumin Analogues using Electrospray Ionization
(Wiley, 2015-07-30) Awad, Hanan; Das, Umashankar; Dimmock, Jonathan; El-Aneed, Anas
RATIONALE: Curcumin analogues are antineoplastic agents, designed based on the structure of the spice turmeric with structural modifications aiming at enhancing potency. The goal is to identify the common tandem mass spectrometric (MS/MS) behavior of 13 novel curcumin analogues. Such knowledge is critical for their biological assessment, including metabolite identification and pharmacokinetic evaluation. METHODS: Both detection of the protonated molecules [M + H](+) of the synthesized compounds and determination of their exact molecular masses were achieved with hybrid quadrupole orthogonal time-of-flight mass spectrometry (QqTOF-MS). Low-energy collision-induced dissociation (CID)-MS/MS analysis was performed using triple quadrupole linear ion trap mass spectrometry (QqLIT-MS). Both instruments were equipped with an electrospray ionization (ESI) source. MS(3) and neutral loss experiments were performed using QqLIT-MS to confirm the genesis of the observed product ions. RESULTS: Abundant [M + H](+) molecules were formed using the QqTOF-MS hybrid instrument with mass accuracies below 6 ppm. CID-MS/MS dissociation studies were centered on the piperidone ring of curcumin analogues; twelve common product ions have been identified from the fission of the various bonds within the piperidone moiety. There was a tendency for the formation of highly conjugated product ions, stabilized via resonance. The variety of the side-chain substituents at the nitrogen atom resulted in side-chain-specific product ions. CONCLUSIONS: The ESI-CID-MS/MS analysis of curcumin analogues revealed a common fragmentation behavior of all tested compounds, which gave diagnostic product ions identified for each molecule. The established MS/MS behavior will be applied to determine metabolic by-products of curcumin analogues as well as to develop targeted identification/quantification methods within biological extracts.
Hydrophilic interaction liquid chromatography–tandem mass spectrometry quantitative method for the cellular analysis of varying structures of gemini surfactants designed as nanomaterial drug carriers
(Elsevier, 2016-05-13) Donkuru, McDonald; Michel, Deborah; Awad, Hanan; Katselis, George; El-Aneed, Anas
Diquaternary gemini surfactants have successfully been used to form lipid-based nanoparticles that are able to compact, protect, and deliver genetic materials into cells. However, what happens to the gemini surfactants after they have released their therapeutic cargo is unknown. Such knowledge is critical to assess the quality, safety, and efficacy of gemini surfactant nanoparticles. We have developed a simple and rapid liquid chromatography electrospray ionization-tandem mass spectrometry (LC-ESI–MS/MS) method for the quantitative determination of various structures of gemini surfactants in cells. Hydrophilic interaction liquid chromatography (HILIC) was employed allowing for a short simple isocratic run of only 4 minutes. The lower limit of detection (LLOD) was 3 ng/mL. The method was valid to 18 structures of gemini surfactants belonging to two different structural families. A full method validation was performed for two lead compounds according to USFDA guidelines. The HILIC-MS/MS was compatible with the physicochemical properties of gemini surfactants that bear a permanent positive charge with both hydrophilic and hydrophobic elements within their molecular structure. In addition, an effective liquid-liquid extraction method (98% recovery) was employed surpassing previously used extraction methods. The analysis of nanoparticle-treated cells showed an initial rise in the analyte intracellular concentration followed by a maximum and a somewhat more gradual decrease of the intracellular concentration. The observed intracellular depletion of the gemini surfactants may be attributable to the biotransformation into metabolites and exocytosis from the host cells. Obtained cellular data showed a pattern that grants additional investigations , evaluating metabolite formation and assessing the subcellular distribution of tested compounds.
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.
Analysis of a Series of Chlorogenic Acid Isomers using Differential Ion Mobility and Tandem Mass Spectrometry
(Elsevier, 2016-08-24) Willems, Jamie; Khamis, Mona, M.; Saeid, Waleed Mohammed; Purves, Randy, W.; Katselis, George; Low, Nicholas; El-Aneed, Anas
Chlorogenic acids are among the most abundant phenolics found in the human diet. Of these, the mono-caffeoylquinic acids are the predominant phenolics found in fruits, such as apples and pears, and products derived from them. In this research, a comprehensive study of the electrospray ionization (ESI) tandem mass spectrometric (MS/MS) dissociation behavior of the three most common mono-caffeoylquinic acids, namely 5-O-caffeoylquinic acid (5-CQA), 3-O-caffeoylquinic acid (3-CQA) and 4-O-caffeoylquinic acid (4-CQA), were determined using both positive and negative ionization. All proposed structures of the observed product ions were confirmed with second-generation MS3 experiments. Similarities and differences between the dissociation pathways in the positive and negative ion modes are discussed, confirming the proposed structures and the established MS/MS fingerprints. MS/MS dissociation was primarily driven via the cleavage of the ester bond linking the quinic acid moiety to the caffeic acid moiety within tested molecules. Despite being structural isomers with the same m/z values and dissociation behaviors, the MS/MS data in the negative ion mode was able to differentiate the three isomers based on ion intensity for the major product ions, observed at m/z 191, 178 and 171. This differentiation was consistent among various MS instruments. In addition, ESI coupled with high-field asymmetric waveform ion mobility spectrometry-mass spectrometry (ESI-FAIMS-MS) was employed for the separation of these compounds for the first time. By combining MS/MS data and differential ion mobility, a method for the separation and identification of mono-caffeoylquinic in apple/pear juice samples was developed with a run time of less than one minute. It is envisaged that this methodology could be used to identify pure juices based on their chlorogenic acid profile (i.e., metabolomics), and could also be used to detect juice-to-juice adulteration (e.g., apple juice addition to pear juice).
The Development of Novel Nanodiamond Based MALDI Matrices for the Analysis of Small Organic Pharmaceuticals
(Springer, 2016-10-27) Chitanda, Jackson M; Zhang, Haixia; Pahl, Erica; Purves, Randy; El-Aneed, Anas
The utility of novel functionalized nanodiamonds (NDs) as matrices for matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) is described herein. MALDI-MS analysis of small organic compounds (<1000 Da) is typically complex due to interferences from numerous cluster ions formed when using conventional matrices. To expand the use of MALDI for the analysis of small molecules, , novel matrices were designed by covalently linking conventional matrices (or a lysine moiety) to detonated NDs. Four new functionalized NDs were evaluated for their ionization capabilities using five pharmaceuticals with varying molecular structures. Two ND matrices were able to ionize all tested pharmaceuticals in the negative ion mode, producing the deprotonated ions [M-H]-. Ion intensity for target analytes was generally strong with enhanced signal-to-noise ratios compared with conventional matrices. The negative ion mode is of great importance for biological samples as interference from endogenous compounds is inherently minimized in the negative ion mode. Since the molecular structures of the tested pharmaceuticals did not suggest that negative ion mode would be preferable, this result magnifies the importance of these findings. On the other hand, conventional matrices primarily facilitated the ionization as expected in the positive ion mode, producing either the protonated molecules [M+H]+ or cationic adducts (typically producing complex spectra with numerous adduct peaks). The data presented in this study suggests that these matrices may offer advantages for the analysis of low molecular weight pharmaceuticals/metabolites.
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.
Tandem Mass Spectrometric Analysis of Novel Peptide-Modified Gemini Surfactants Used as Gene Delivery Vectors.
(Wiley, 2017-05-31) El-Aneed, Anas; Al-Dulaymi, Mays
Diquaternary ammonium gemini surfactants have emerged as effective gene delivery vectors. A novel series of 11 peptide-modified compounds was synthesized, showing promising results in delivering genetic materials. The purpose of this work is to elucidate the tandem mass spectrometric (MS/MS) dissociation behavior of these novel molecules establishing a generalized MS/MS fingerprint. Exact mass measurements were achieved using a hybrid quadrupole orthogonal time-of-flight mass spectrometer, and a multi-stage MS/MS analysis was conducted using a triple quadrupole-linear ion trap mass spectrometer. Both instruments were operated in the positive ionization mode and are equipped with electrospray ionization. Abundant triply charged [M+H]3+ species were observed in the single-stage analysis of all the evaluated compounds with mass accuracies of less than 8 ppm in mass error. MS/MS analysis showed that the evaluated gemini surfactants exhibited peptide-related dissociation characteristics because of the presence of amino acids within the compounds' spacer region. In particular, diagnostic product ions were originated from the neutral loss of ammonia from the amino acids' side chain resulting in the formation of pipecolic acid at the N-terminus part of the gemini surfactants. In addition, a charge-directed amide bond cleavage was initiated by the amino acids' side chain producing a protonated α-amino-ε-caprolactam ion and its complimentary C-terminus ion that contains quaternary amines. MS/MS and MS3 analysis revealed common fragmentation behavior among all tested compounds, resulting in the production of a universal MS/MS fragmentation pathway.
Rapid and Simple Flow Injection Analysis-Tandem Mass Spectrometric (FIA-MS/MS) Method for the Quantification of Melphalan in Lipid-Based Drug Delivery System
(Wiley, 2017-07-01) Mohammed-Saeid, Waleed; Michel, Deborah; El-Aneed, Anas
The use of the anticancer drug melphalan is limited due to its poor water solubility. To address such limitation, it is incorporated within a novel delivery system using β-cyclodextrin-gemini surfactants (18:1βCDg). Herein, two fast and simple FIA-MS/MS methods are developed for the quantification of melphalan (Mel) within the drug delivery system so that the solubilization efficiency of the system can be assessed. FIA-MS/MS methods are developed using a triple quadrupole-linear ion trap mass spectrometer, equipped with electrospray ionization (ESI) in the positive ion mode. A deuterated form of melphalan (melphalan-d8) was used as an internal standard (IS). The methods were validated according to the FDA guidance. A linearity in the range of 2–100 ng/mL and accuracy and precision below 15% were observed for all standard points and quality control samples. The intra- and inter-day variations, freeze-thaw stability were within the acceptable range according to the criteria set by regulatory guidelines. On the other hand, other stability measures, such as room temperature stability and long term stability did not meet the required guidelines in some cases, indicating the need for quick sample analysis upon preparation. Such a fact could have been overlooked if full method validation was not performed. The developed methods were applied to determine the encapsulation/solubilisation of [18:1βCDg\Mel] delivery system. 18:1βCDg enhances the aqueous solubility of melphalan without the need for co-solvent. The highest melphalan solubility was observed at the 18:1βCDg\Mel complex molar ratio of 2:1. This study demonstrated that a fast analysis for the purpose of quantifying a chemically unstable drug, such as melphalan is feasible and important for the development of commercial dosage forms.
Development of a validated LC- MS/MS method for the quantification of 19 endogenous asthma/COPD potential urinary biomarkers
(Elsevier, 2017-10-09) Khamis, Mona; Adamko, Darryl; El-Aneed, Anas
Obstructive airways inflammatory diseases sometimes show overlapping symptoms that hinder their early and correct diagnosis. Current clinical tests are tedious and are of inadequate specificity in special population such as the elderly and children. Therefore, we are developing tandem mass spectrometric (MS/MS) methods for targeted analysis of urine biomarkers. Recently, proton-nuclear magnetic resonance (1H-NMR) analysis proposed 50 urinary metabolites as potential diagnostic biomarkers among asthma and chronic obstructive pulmonary disease (COPD) patients. Metabolites are divided into 3 groups based on chemical nature. For group 1 (amines and phenols, 19 urinary metabolites), we developed and validated a high performance liquid chromatographic (HPLC)-MS/MS method using differential isotope labeling with dansyl chloride. Method development included the optimization of the derivatization reaction, the MS/MS conditions, and the chromatographic separation. Linearity varied from 2 to 4800 ng/mL and the use of 13C2-labeled derivatives allowed for the correction of matrix effects as well as the unambiguous confirmation of the identity of each metabolite in the presence of interfering isomers in urine. Despite the challenges associated with method validation, the method was fully validated as per the food and drug administration (FDA) and the European medicines agency (EMA) recommendations. Validation criteria included linearity, precision, accuracy, dilution integrity, selectivity, carryover, and stability. Challenges in selectivity experiments included the isotopic contributions of the analyte towards its internal standard (IS), that was addressed via the optimization of the IS concentration. In addition, incurred sample analysis was performed to ensure that results from patient samples are accurate and reliable. The method was robust and reproducible and is currently being applied in a cohort of asthma and COPD patient urine samples for biomarker discovery purposes.
Mass spectrometric approaches for the analysis of phytosterols in biological samples.
(ACS Publications, 2017-10-23) Gachumi, George N.; El-Aneed, Anas
Plant sterols (phytosterols) are important structural components of plant cellular membranes and they play a major role during development and metabolism. They have health-associated benefits, especially in lowering blood cholesterol levels. Due to their many health claims, there is a growing interest in their analysis. Although various analytical strategies have been employed in analyzing phytosterols, chromatography linked to mass spectrometry (MS) is superior due to its sensitivity. Furthermore, specificity and selectivity are enhanced by utilizing tandem mass spectrometry (MS/MS). This article reviews the various mass spectrometric strategies used for the analysis of phytosterols. It highlights the applications and limitations associated with each MS strategy in various sample matrices such as plant, human, animal, food, and dietary supplements. GC-MS was historically the method of choice for analysis; however, the derivatization step rendered it tedious and time-consuming. On the other hand, liquid chromatography coupled to MS (LC-MS) simplifies the analysis. Many ionization techniques have been used namely electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI). APCI showed superiority in terms of ion intensity and consistency in ion formation, primarily forming [M+H-H2O]+ ions rather than [M+H]+. In addition, matrix assisted laser desorption ionization (MALDI) as well as ambient mass spectrometry such as direct analysis in real time (DART) have also been evaluated.
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.
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.
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.
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.
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.
Development and Characterization of Liposomal Formulations Containing Phytosterols Extracted from Canola Oil Deodorizer Distillate along with Tocopherols as Food Additives
(MDPI, 2019-04-16) Poudel, Asmita; Gachumi, George; Wasan, Kishor M.; Bashi, Zafer Dallal; El-Aneed, Anas
Phytosterols are plant sterols recommended as adjuvant therapy for hypercholesterolemia and tocopherols are well-established anti-oxidants. However, thermo-sensitivity, lipophilicity and formulation-dependent efficacy bring challenges in the development of functional foods, enriched with phytosterols and tocopherols. To address this, we developed liposomes containing brassicasterol, campesterol and β-sitosterol obtained from canola oil deodorizer distillate, along with alpha, gamma and delta tocopherol. Three approaches; thin film hydration-homogenization, thin film hydration-ultrasonication and Mozafari method were used for formulation. Validated liquid chromatographic tandem mass spectrometry (LC-MS/MS) was utilized to determine the entrapment efficiency of bioactives. Stability studies of liposomal formulations were conducted before and after pasteurization using high temperature short time (HTST) technique for a month. Vesicle size after homogenization and ultrasonication (<200 nm) was significantly lower than by Mozafari method (>200 nm). However, zeta potential (-9 to -14 mV) was comparable which was adequate for colloidal stability. Entrapment efficiencies were greater than 89% for all the phytosterols and tocopherols formulated by all three methods. Liposomes with optimum particle size and zeta potential were incorporated in model orange juice, showing adequate stability after pasteurization (72 °C for 15 s) for a month. Liposomes containing phytosterols obtained from canola waste along with tocopherols were developed and successfully applied as a food additive using model orange juice.
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.

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