Publications by authors named "Anas El-Aneed"

52 Publications

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

Anal Chim Acta 2022 Feb 29;1194:339404. Epub 2021 Dec 29.

Drug Discovery and Development Research Group, College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, SK, Canada. Electronic address:

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 β-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 β-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.
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http://dx.doi.org/10.1016/j.aca.2021.339404DOI Listing
February 2022

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

J Am Pharm Assoc (2003) 2022 Jan-Feb;62(1):176-186. Epub 2021 Aug 21.

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.
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http://dx.doi.org/10.1016/j.japh.2021.08.018DOI Listing
January 2022

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

Molecules 2021 Jun 23;26(13). Epub 2021 Jun 23.

College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada.

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--glucoside in black bean. Seed coats having proanthocyanidins that are primarily prodelphinidins show higher iron chelation compared with those containing procyanidins and/or propelargonidins.
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http://dx.doi.org/10.3390/molecules26133833DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8270320PMC
June 2021

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

Subst Abuse Treat Prev Policy 2021 05 1;16(1):38. Epub 2021 May 1.

College of Pharmacy and Nutrition, University of Saskatchewan, 107 Wiggins Road, Room 3D01.3, Saskatoon, Saskatchewan, S7N 5E5, Canada.

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.
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http://dx.doi.org/10.1186/s13011-021-00374-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8088612PMC
May 2021

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

Rapid Commun Mass Spectrom 2021 Jun;35(13):e9107

College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.

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 (MS ) 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. MS 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.
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http://dx.doi.org/10.1002/rcm.9107DOI Listing
June 2021

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

Molecules 2021 Mar 5;26(5). Epub 2021 Mar 5.

Drug Design and Discovery Group, College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada.

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 /) 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, β-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). β-sitosterol and γ-tocopherol were the most abundant phytosterols and tocopherols, respectively.
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http://dx.doi.org/10.3390/molecules26051402DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7961602PMC
March 2021

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

Pharmaceutics 2021 Feb 16;13(2). Epub 2021 Feb 16.

Drug Discovery and Development Research Group, College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada.

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.
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http://dx.doi.org/10.3390/pharmaceutics13020268DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7920278PMC
February 2021

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

Anal Chem 2020 07 24;92(13):8628-8637. Epub 2020 Jun 24.

College of Pharmacy and Nutrition, University of Saskatchewan, 107 Wiggins Road, Saskatoon, Saskatchewan Canada, S7N 5E5.

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 (MS), 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.
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http://dx.doi.org/10.1021/acs.analchem.0c00877DOI Listing
July 2020

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

J Pharm Biomed Anal 2020 May 9;183:113104. Epub 2020 Jan 9.

Drug Design and Discovery Group, College of Pharmacy and Nutrition, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK, S7N 5E5, Canada. Electronic address:

A novel liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and validated to simultaneously quantify phytosterols (brassicasterol, campesterol, stigmasterol and β-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.
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http://dx.doi.org/10.1016/j.jpba.2020.113104DOI Listing
May 2020

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

J Am Soc Mass Spectrom 2020 Feb 10;31(2):366-378. Epub 2020 Jan 10.

Drug Design & Discovery Group, College of Pharmacy and Nutrition , University of Saskatchewan , 107 Wiggins Road , Saskatoon , Saskatchewan Canada , S7N 5E5.

Gemini surfactants are a class of lipid molecules that have been successfully used and 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.
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http://dx.doi.org/10.1021/jasms.9b00004DOI Listing
February 2020

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

J Chromatogr B Analyt Technol Biomed Life Sci 2020 Jan 13;1137:121939. Epub 2019 Dec 13.

Departamento de Produtos Farmacêuticos, Faculdade de Farmácia, Universidade Federal de Minas Gerais, Belo Horizonte, MG 31270-901, Brazil. Electronic address:

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 → 115.1 and 231.0 → 152.8 for kavain; and 234.2 → 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.
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http://dx.doi.org/10.1016/j.jchromb.2019.121939DOI Listing
January 2020

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

Mass Spectrom Rev 2021 01 15;40(1):31-52. Epub 2019 Oct 15.

College of Pharmacy and Nutrition, University of Saskatchewan, 107 Wiggins Rd, Saskatoon, Saskatchewan, S7N 5E5, Canada.

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.
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http://dx.doi.org/10.1002/mas.21607DOI Listing
January 2021

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

AAPS J 2019 08 6;21(5):98. Epub 2019 Aug 6.

Drug Design & Discovery Group, College of Pharmacy and Nutrition, University of Saskatchewan, 107 Wiggins Road, Saskatoon, Saskatchewan, S7N 5E5, Canada.

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.
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http://dx.doi.org/10.1208/s12248-019-0367-1DOI Listing
August 2019

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

Rapid Commun Mass Spectrom 2019 Dec;33(23):1792-1803

Drug Discovery and Development Research Group, College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, SK, Canada.

Rationale: Novel bifunctional compounds composed of a caffeine scaffold attached to nicotine (C -6-N), 1-aminoindan (C -6-I), or caffeine (C -6-C ) 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 MS 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 C -6-N and C -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 C -6-C suggests that a previously reported loss of acetaldehyde during caffeine dissociation is instead a loss of CO .

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.
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http://dx.doi.org/10.1002/rcm.8540DOI Listing
December 2019

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

Methods Mol Biol 2019 ;2000:203-225

College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, SK, Canada.

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.
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http://dx.doi.org/10.1007/978-1-4939-9516-5_14DOI Listing
March 2020

The Establishment of Tandem Mass Spectrometric Fingerprints of Phytosterols and Tocopherols and the Development of Targeted Profiling Strategies in Vegetable Oils.

J Am Soc Mass Spectrom 2019 Sep 20;30(9):1700-1712. Epub 2019 May 20.

College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, SK, Canada.

Phytosterols and tocopherols are essential for plant biochemistry, and they possess beneficial health effects for humans. Evaluating the tandem mass spectrometric (MS/MS) behavior of phytosterols and tocopherols is needed for the development of a qualitative and quantitative method for these biologically active plant metabolites. Herein, the MS/MS dissociation behavior of phytosterols and tocopherols is elucidated to establish generalized MS/MS fingerprints. MS/MS and multistage (MS) analysis revealed common fragmentation behavior among the four tested phytosterols, namely β-sitosterol, stigmasterol, campesterol, and brassicasterol. Similar analysis was conducted for the tocopherols (i.e., alpha (α), beta (β), gamma (γ), and delta (δ)). As such, a universal MS/MS fragmentation pathway for each group was successfully established for the first time. Based on the generalized MS/MS fragmentation behavior of phytosterols, diagnostic product ions were chosen for the development of profiling methods for over 20 naturally occurring phytosterols. A precursor ion scan-triggered-enhanced product ion scan (PIS-EPI) method was established. Due to enhanced chromatographic peaks, multiple ion monitoring-triggered-enhanced product ion scan (MIM-EPI) was employed for confirmation. The screening approach was applied successfully to identify blinded samples obtained from standard mixtures as well as sesame and olive oils. The oil samples contain other phytosterols, and their successful identification indicates that, the generalized MS/MS fragmentation behavior is applicable to various structures of phytosterols. A similar approach was attempted for tocopherols and was only hindered by the low concentration of these bioactive metabolites present in the oil samples.
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http://dx.doi.org/10.1007/s13361-019-02242-2DOI Listing
September 2019

Development and Characterization of Liposomal Formulations Containing Phytosterols Extracted from Canola Oil Deodorizer Distillate along with Tocopherols as Food Additives.

Pharmaceutics 2019 Apr 16;11(4). Epub 2019 Apr 16.

Drug Design and Discovery Group, College of Pharmacy and Nutrition, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK S7N 5E5, Canada.

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.
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http://dx.doi.org/10.3390/pharmaceutics11040185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6523737PMC
April 2019

Qualitative exploration of the education and skill needs of community pharmacists in Saskatoon concerning substance use disorder.

Can Pharm J (Ott) 2019 Mar-Apr;152(2):117-129. Epub 2019 Feb 7.

College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, Saskatchewan.

Background: Identifying the skills and educational needs of community pharmacists concerning addiction is critical to improving the services provided for people who suffer from addiction disease (PWSAD).

Methods: Eleven one-to-one semi-structured interviews were conducted with community pharmacists practising in the Saskatoon Health Region, Canada. The interviews were recorded and transcribed verbatim and verified with the participants. Thematic analysis was employed to analyze the transcripts.

Results: Four major themes were identified: 1) effect of the work setting on pharmacists' encounters with PWSAD, 2) pharmacists' knowledge of key aspects of addiction, 3) level of support within the health care system, and 4) educational and training needs.

Conclusion: Participants indicated that a lack of knowledge and training were major hindrances to improving the quality of the services provided to people who suffer from addiction disease. Additional practicum experience at the undergraduate level and interprofessional interactive educational sessions at the continuing educational level were key recommendations emerging from the study.
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http://dx.doi.org/10.1177/1715163518816726DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6410432PMC
February 2019

Investigation into spinal anesthetic failure with hyperbaric bupivacaine: the role of cold exposure on bupivacaine degradation.

Can J Anaesth 2019 07 15;66(7):803-812. Epub 2019 Mar 15.

Department of Anesthesiology, College of Medicine, University of Saskatchewan and Royal University Hospital, Saskatoon, SK, Canada.

Purpose: Hyperbaric bupivacaine (0.75% in dextrose) is used for spinal obstetric anesthesia. Occasional clusters of anesthetic failures occur in this setting, not readily attributable to clinical factors. We hypothesized that cold temperature exposure is related to bupivacaine instability.

Methods: An electronic survey was distributed to Canadian anesthesiologists to determine consistencies in spinal anesthesia practice, and to invite submission of failed bupivacaine samples for analysis. Another survey for hospital pharmacists focused on bupivacaine logistics. Ultraviolet (UV) spectrometry, differential scanning calorimetry, and high performance liquid chromatography were used to evaluate the effect of temperature on bupivacaine chemical stability. Mass spectrometry (MS) was used to observe bupivacaine and dextrose degradation in laboratory samples of hyperbaric 0.75% bupivacaine in dextrose. Hyperbaric bupivacaine that failed to produce adequate anesthesia in labour and delivery patients was subject to tandem MS/MS analysis on commonly observed ions to look for ion patterns consistent with bupivacaine degradation products and to compare with laboratory samples subjected to cold temperatures.

Results: Canadian obstetric anesthesiologists report similar practices and use hyperbaric bupivacaine for spinal anesthesia. Pharmacists surveyed indicated facility storage at room temperature but variable temperatures during shipping. No standard procedure for failure reporting was identified. Analysis of bupivacaine showed a slight decrease in bupivacaine concentration or UV spectral changes after incubation at temperatures ≤ 4°C. Mass spectrometric analysis of hyperbaric bupivacaine from failed spinal anesthesia cases showed complex and inconsistent patterns of ion formation, and different from the ion patterns observed for cooled vs uncooled bupivacaine solutions. Temperature-related changes were noted for dextrose in cooled samples in which dextrose-related ions were formed.

Conclusions: Canadian clinical practice and handling of hyperbaric bupivacaine is consistent. Most respondents indicated an interest in a formal reporting and collection process. Cold exposure did not degrade bupivacaine. A complex and possibly inconsistent reaction involving dextrose was identified that requires further analysis of a larger sample size to elucidate the mechanisms.
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http://dx.doi.org/10.1007/s12630-019-01343-6DOI Listing
July 2019

Comparative analysis of creatinine and osmolality as urine normalization strategies in targeted metabolomics for the differential diagnosis of asthma and COPD.

Metabolomics 2018 08 29;14(9):115. Epub 2018 Aug 29.

Department of Pediatrics, College of Medicine, University of Saskatchewan, Saskatoon, SK, Canada.

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 H-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 wider application. We have been developing targeted urine metabolomic methods to differentiate two important respiratory diseases, namely asthma and chronic obstructive pulmonary disease (COPD).

Objective: To assess whether the statistical model of separation of diseases using targeted metabolomic data would be improved by normalization to osmolality instead of creatinine.

Methods: The concentration of 32 metabolites was previously measured by two liquid chromatography-tandem mass spectrometry methods in 51 human urine samples with either asthma (n = 25) or COPD (n = 26). The data was normalized to creatinine or osmolality. Statistical analysis of the normalized values in each disease was performed using partial least square discriminant analysis (PLS-DA). Models of separation of diseases were compared.

Results: We found that normalization to creatinine or osmolality did not significantly change the PLS-DA models of separation (RQ = 0.919, 0.705 vs RQ = 0.929, 0.671, respectively). The metabolites of importance in the models remained similar for both normalization methods.

Conclusion: Our findings suggest that targeted urine metabolomic data can be normalized for hydration using creatinine or osmolality with no significant impact on the diagnostic accuracy of the model.
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http://dx.doi.org/10.1007/s11306-018-1418-9DOI Listing
August 2018

Quantitative determination of potential urine biomarkers of respiratory illnesses using new targeted metabolomic approach.

Anal Chim Acta 2019 Jan 25;1047:81-92. Epub 2018 Sep 25.

College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, SK, Canada. Electronic address:

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 H 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 (R 0.752 and Q 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.
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http://dx.doi.org/10.1016/j.aca.2018.09.035DOI Listing
January 2019

The determination of gemini surfactants used as gene delivery agents in cellular matrix using validated tandem mass spectrometric method.

J Pharm Biomed Anal 2019 Feb 3;164:164-172. Epub 2018 Oct 3.

Drug Design & Discovery Group, College of Pharmacy and Nutrition, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK, S7N 5E5, Canada. Electronic address:

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 10 nM-1000 nM for 16-3-16 and 16(Py)-S-2-S-(Py)16, and 20 nM-2000 nM 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.
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http://dx.doi.org/10.1016/j.jpba.2018.10.003DOI Listing
February 2019

Molecular Engineering as an Approach To Modulate Gene Delivery Efficiency of Peptide-Modified Gemini Surfactants.

Bioconjug Chem 2018 10 13;29(10):3293-3308. Epub 2018 Sep 13.

Canadian Light Source , Saskatoon , Saskatchewan S7N 4L5 , Canada.

The unique molecular structure confers the diquaternary ammonium gemini surfactants with enhanced nucleic acid complexation ability, bottom-up design flexibility, and relatively low cytotoxicity. To capitalize on their potential as gene delivery vectors, novel structural modifications should be explored. In this work, 22 novel peptide-modified gemini surfactants with various alkyl tails and peptide spacer modifications were evaluated. This work represents the first report of dendrimer-like gemini surfactants and first evaluation of the impact of incorporating a hydrocarbon linker into the peptide chain. Our aim was to establish a structure activity relationship of the peptide-modified gemini surfactants and to identify the fundamental architectural requirements needed for the ultimate gene delivery systems. In vitro assessment revealed that the highest transfection efficiency and lowest cytotoxicity were associated with the glycyl-lysine modified gemini surfactants having the hexadecyl tail, 16-7N(G-K)-16. In fact, it showed an 8-fold increase in secreted protein with 20% increase in cell viability relative to the first-generation unsubstituted gemini surfactants. Further increase in the size of the attached peptides resulted in a decrease in the transfection efficiency and cell viability. Whereas the incorporation of a hydrocarbon linker into the peptide chain decreased the transfection efficiency of compounds with dipeptides, it increased the transfection efficiency of compounds with larger peptide chains. Such an increase was more prominent with the incorporation of a longer hydrocarbon linker. We conclude that a balance between the hydrophilic and hydrophobic characteristics of the compound is necessary since it results in physicochemical parameters conducive to the gene delivery process.
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http://dx.doi.org/10.1021/acs.bioconjchem.8b00480DOI Listing
October 2018

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.

J Pharm Biomed Anal 2018 Sep 2;159:536-547. Epub 2018 Jul 2.

College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, SK, Canada. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jpba.2018.06.063DOI Listing
September 2018

Comparison of accuracy and precision between multipoint calibration, single point calibration, and relative quantification for targeted metabolomic analysis.

Anal Bioanal Chem 2018 Sep 13;410(23):5899-5913. Epub 2018 Jul 13.

College of Pharmacy and Nutrition, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK, S7N 5E5, Canada.

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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http://dx.doi.org/10.1007/s00216-018-1205-5DOI Listing
September 2018

Mass Spectrometric Approaches for the Analysis of Phytosterols in Biological Samples.

J Agric Food Chem 2017 Nov 8;65(47):10141-10156. Epub 2017 Nov 8.

College of Pharmacy and Nutrition, University of Saskatchewan , Saskatoon, Saskatchewan, Canada , S7N 5E5.

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. Because of 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 matrixes 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 - HO] 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.
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http://dx.doi.org/10.1021/acs.jafc.7b03785DOI Listing
November 2017

Development of a validated LC- MS/MS method for the quantification of 19 endogenous asthma/COPD potential urinary biomarkers.

Anal Chim Acta 2017 Oct 16;989:45-58. Epub 2017 Aug 16.

College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, SK, Canada. Electronic address:

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 (H-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 (DIL) 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 C-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.
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http://dx.doi.org/10.1016/j.aca.2017.08.007DOI Listing
October 2017

Rapid and simple flow injection analysis tandem mass spectrometric method for the quantification of melphalan in a lipid-based drug delivery system.

Rapid Commun Mass Spectrom 2017 Sep;31(18):1481-1490

College of Pharmacy & Nutrition, University of Saskatchewan, Saskatoon, SK, Canada, S7N 5E5.

Rationale: The use of the anticancer drug melphalan is limited due to its poor water solubility. To address this limitation, it is incorporated within a novel delivery system using β-cyclodextrin-gemini surfactants (18:1βCDg).

Methods: Herein, two fast and simple flow injection analysis/tandem mass spectrometric (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.

Results: 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 and 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 had not been performed.

Conclusions: The developed methods were applied to determine the encapsulation/solubilization of the [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 a melphalan18:1βCDg/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.
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http://dx.doi.org/10.1002/rcm.7926DOI Listing
September 2017

Analysis of a series of chlorogenic acid isomers using differential ion mobility and tandem mass spectrometry.

Anal Chim Acta 2016 Aug 1;933:164-74. Epub 2016 Jun 1.

Drug Design & Discovery Group, College of Pharmacy and Nutrition, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK, S7N 5E5, Canada. Electronic address:

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 MS(3) 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, 179 and 173. 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 1 min. 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).
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http://dx.doi.org/10.1016/j.aca.2016.05.041DOI Listing
August 2016

The Development of Novel Nanodiamond Based MALDI Matrices for the Analysis of Small Organic Pharmaceuticals.

J Am Soc Mass Spectrom 2016 10 3;27(10):1686-93. Epub 2016 Aug 3.

University of Saskatchewan, Saskatoon, Canada.

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 because of 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. Graphical Abstract ᅟ.
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http://dx.doi.org/10.1007/s13361-016-1454-5DOI Listing
October 2016
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