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Anabolic Androgenic Steroids (AAS) are a group of compounds with high potential for abuse in sports including horse racing. Within the last three decades, both gas chromatography –mass spectrometry (GC-MS) and liquid chromatography – mass spectrometry (LC-MS) methods have been developed for routine qualitative and quantitative detection of endogenous or exogenous AAS in urine and serum to address their misuse in sports. However, most current screening methods for the detection of AAS require using enzymatic or chemical hydrolysis to detect free steroids rather than their phase II conjugates which constitute the majority of excreted drug for most anabolic agents. In recent decades, new long-term phase II metabolites of AAS that were previously not detected by GC-MS have been identified by LC-MS methods, including glucurono-conjugates and sulfo-conjugates that are difficult to cleave using enzymatic hydrolysis. This research project focuses on the development and validation of a method capable of the simultaneous detection of 32 steroid phase II metabolites using LC-MS/MS, with prior clean-up steps including protein precipitation and Solid Phase Extraction (SPE) through weak anion exchange cartridges. All compounds, including isomers and epimers, were chromatographically separated over a 20-minute run using a reversed phase C18 column using methanol and water as the organic and aqueous mobile phases. The mass spectrometer data acquisition was set to use selected reaction monitoring scans following introduction via electrospray ionization in both positive and negative modes. The method was validated, and the following parameters were determined: linearity, accuracy, precision, limit of detection (LOD), limit of quantitation (LOQ), recovery, matrix effect, and stability. Finally, a population study using gelding, mare, and stallion urine samples from horses actively competing was conducted to evaluate the endogenous production of the targeted compounds.

Atmospheric aerosols are generated by the millions globally from the bursting of ocean bubbles, the action of wind on dust, burning processes, and many other sources. Aerosol particles have high surface area to volume ratios and typically contain chemicals in significantly greater concentrations compared to the bulk source. From serving as the seeds for clouds to promoting multiphase chemistry and interacting with cells, aerosols have profound impacts on the climate and human health. Among the many properties of aerosols, one of the most important is pH, a measure of the particle’s acidity. Low pH aerosols have detrimental effects on the lungs, and many atmospheric transformations exhibit pH dependence. Although aerosol pH is critically important, it is challenging to measure due to the very small volumes individual aerosols occupy. As a result, prior to the work presented here, the pH of nascent sea spray aerosol (the largest source of aqueous particles globally) was unknown. Here, a novel method for the determination of aerosol pH and its application to the measurement of sea spray aerosol acidity is presented. It is found that, in just two minutes, these aerosols are acidified to pH levels ranging from 2 to 4 depending on particle size. Following this result, several key causes and effects of aerosol pH are discussed. Acidity is shown to impact the extent of acceleration of S(IV) oxidation in aerosols, an important chemical reaction for the fate of sulfur dioxide emissions and air quality. A unique type of buffering to aerosols is presented next, where the titration of the particle results in a decrease in the organic content of the aerosol, instead of a drop in pH, due to partitioning of acids. Following this, the kinetics of nitrate and chloride depletion for single aerosols is shown for the first time. These processes both modulate aerosol pH and change particle hygroscopicity, which is a central parameter for atmospheric cloud formation. Finally, the link between aerosol acidity and surface chemistry is shown by an exploration of the pH-dependent surface propensity of amino acids in aqueous environments. Together, these studies shed light on the drivers of aerosol pH and the kinetics of critical aerosol-phase reactions. Ultimately, this research improves understanding of the complex ocean-climate system and how invisible particles present health challenges for populations globally.

Understanding biomolecular interactions and cellular activities based on these interactions are important research topics in bioanalytical chemistry that can have a large impact on key fields such as drug discovery, biomedical sciences, and environmental monitoring. High performing biosensors are an essential part for this task. Effective sensing platforms with improved analytical performance and reduced cost are actively sought after and remain a major area of intense research endeavors. The goal of this thesis is to develop novel biosensing systems enabled by 3D printing technology and nanoscience for probing biochemical interactions and achieving cell lipidomic analysis.Surface plasmon resonance (SPR) biosensors have been wildly applied for biomolecular assays in a label-free fashion. The sensitivity of SPR sensors, however, still needs signal enhancement when dealing with trace amounts of analytes. Chapter 2 describes the fabrication and application of gold nanoparticles-coupled POPC liposomes nanoclusters for signal amplification for SPR sensors and the study of protein-membrane recognition on a biomimetic interface. By combining the large mass of liposomes and plasmonic coupling of the noble metal nanoparticles, the highly stable POPC-gold nanoparticles showed a large amplification effect and a 0.1 ng/mL LOD was achieved for cholera toxin detection.3D printing technology has greatly impacted the bioanalytical fields by providing the fabrication capability with almost no geometric restriction, rapid prototyping, and low cost. Chapter 3 describes the design and fabrication of a Dove prism by 3D printing with transverse micropatterns for multiplexed sensing with SPR imaging. The sensitivity of the system was evaluated by bulk refractive index change and protein binding assays; both showed similar performance as compared to the SPR configuration using high end glass optics.3D printing technology has been further applied to fabricating microfluidic chips for cell analysis. Chapter 4 demonstrates the design and fabrication of a 3D printed microfluidic device for on-chip cell lysis, lipid extraction and phase separation for lipidomic study of algae C. reinhardtii cells by the microchip enhanced MALDI-MS. Compared with conventional bulk methods, extraction by the microfluidic device showed higher efficiency and cell lysis capability.

Herein, we reported an HPLC method for the simultaneous determination of tibezonium iodide (TBN) and lignocaine hydrochloride (LGN). The method was developed according to the International Conference for Harmonization guidelines (ICH) Q2R1 using Agilent® 1260 with a mobile phase consisting of acetonitrile and phosphate buffer (pH 4.5) in a volumetric ratio of 70:30 and flowing through a C8 Agilent® column at 1 mL/min. The results revealed that TBN and LGN peaks were isolated at 4.20 and 2.33 min, respectively, with a resolution of 2.59. The accuracy of TBN and LGN was calculated to be 100.01 ± 1.72% and 99.05 ± 0.65% at 100% concentration, respectively. Similarly, the respective precision was 100.03 ± 1.61% and 99.05 ± 0.48%. The repeatability for TBN and LGN was found to be 99.05 ± 0.48% and 99.19 ± 1.72%, respectively, indicating that the method was precise. The respective regression co-efficient (r2) for TBN and LGN was found to be 0.9995 and 0.9992. Moreover, the LOD and LOQ values for TBN were 0.012 and 0.037 µg/mL, respectively, while for LGN, they were 0.115 and 0.384 µg/mL, respectively. The calculated greenness of the method for ecological safety was found to be 0.83, depicting a green contour on the AGREE scale. No interfering peaks were found when the analyte was estimated in dosage form and in volunteers’ saliva, depicting the specificity of the method. Conclusively, a robust, fast, accurate, precise and specific method was successfully validated to estimate TBN and LGN.

Tributylamine (TBuA) and triethylamine (TEtA) are the most commonly used ion pair reagents in ion pair chromatography especially for the analysis of oligonucleotides. In order to improve the understanding of the retention and separation mechanism of oligonucleotides in ion pair chromatography, it is important to understand the retention mechanism and the nature of interaction of these ion pair reagents with the stationary phase in the chromatographic column. Adsorption isotherm is helpful in evaluating such interactions, and subsequently predicting the retention mechanism. Alkylamines are very polar molecules which lack suitable chromophore and are commonly present in charged forms. Therefore, their determination and the subsequent acquisition of their adsorption isotherms using traditional liquid chromatography is very difficult. In this study, we first developed an analytical method for the determination of TBuA and TEtA in a typical chromatographic mobile phase (acetonitrile-water) and then used the same method to acquire the adsorption isotherms for tributylammonium acetate (TBuAA) and triethylammonium acetate (TEtAA). This method started with the conversion of the alkylammonium ions to free neutral forms by treating the sample with a strong base, followed by pentane-mediated extraction and finally the analysis of the extracts using gas chromatography-flame ionization detector (GC-FID). This three-step method was validated for parameters like range, linearity, intra-day and inter-day precision and accuracy, limit of detection and limit of quantitation. For the adsorption isotherms, the C18 column was first equilibrated with the solutions having different concentrations of alkylammonium ions and then stripped with eluent devoid of alkylammonium ions. Several stripping eluents were investigated and it was discovered that the eluent requirement could be decreased by the addition of sodium chloride. The effluents from the stripping phase were collected and analyzed using the developed analytical method to acquire the adsorption data. Under the investigated conditions, adsorption of TBuAA and TEtAA showed type III and type I isotherm behavior respectively.

Multiparametric flow cytometry is a powerful cellular analysis tool used in various stages of drug development. In adoptive cell therapies, the flow cytometry methods are used for the evaluation of advanced cellular products during manufacturing and to monitor cellular kinetics after infusion. In this report, we discussed the bioanalytical method development challenges to monitor cellular kinetics in CAR-T cell therapies. These method development challenges include procuring positive control samples for the development of the method, flow cytometry panel design, LLOQ, prestain sample stability, staining reagents and data analysis.

Objective: This work aimed to validate a simple UV-Visible spectrophotometric method for estimating Cefixime trihydrate in bulk and to produce an accurate, precise, repeatable, and cost-effective method. Material and Methods: The pH 7.4 Phosphate buffer was utilized as the solvent throughout the experiment. The drug’s absorption maxima (max) were discovered to be at 288 nm. Beer’s law was found to be obeyed in the range of 10-45 μg/mL during the quantitative analysis of the substance at 288 nm. In the doses tested, the approach was found to be linear, with the line equation y = 0.035x – 0.002 and a correlation coefficient of 0.999. Results and Discussion: Cefixime Trihydrate recovery values varied from 99.656 percent to 101.825 percent. Six duplicates of the experiment had a relative standard deviation of less than 2%. The interday precision range was 0.52-1.02%, and the intraday precision range was 0.57-0.995 percent relative standard deviation (RSD percent). The detection and quantification limits were 0.914 and 3.142 μg/mL, respectively. The method’s robustness and ruggedness had a percent relative standard deviation of 0.532-0.827 percent. Conclusion: As a result, the proposed procedure was precise, accurate, and economical. This method could be used to determine the quantity of medicine in bulk.

In this research work, an isocratic, reversed-phase high-performance liquid chromatography-ultraviolet/visible detector method was developed for analysis of voriconazole standard (stock-solution) and in plasma samples. Optimization and validation of the method was carried out as per international guidelines. The method offered a simple liquid–liquid extraction technique, which exhibited best recovery of voriconazole along with fluconazole, i.e., internal standard. Different experimental conditions were tried and ultimately, the best outcomes were accomplished utilizing C-18 Perkin-Elmer® column with particulars of 150 mm length, 4.6 mm inner diameter and 5 μm particle size, protected by an RP-18 Perkin-Elmer® Pre-column guard cartridge with specifications of 10 μm particle size, 30 mm length and 4.6 mm inner diameter, utilizing mobile-phase of acetonitrile-water (ACN: H2O) in proportion of 60: 40 v/v, having a flow rate of 1.5 mL/min, and wavelength of 254 nm. All the analytes were observed to be separated in ≤7 min. A linear calibration curve was obtained at concentration range of 01–10 μg/mL of voriconazole. The correlation coefficient of voriconazole was observed to be 0.999, and average recovery (in percent) was 97.4%, whereas the relative standard deviation value was ≤2%. The lower limit of detection was 0.01 μg/mL, whereas, lower limit of quantification was 0.03 μg/mL, respectively. This developed method provided outstanding results of all validation parameters, i.e., recovery, accuracy, selectivity, precision and reproducibility. The method proposed for voriconazole analysis was applied effectively for further research investigation of voriconazole in human-plasma samples (to assess the pharmacokinetic parameters), pharmaceutical formulations and pharmacokinetic drug–drug interaction’s.

Capmatinib, a recently approved tyrosine kinase inhibitor, is used for the treatment of non-small cell lung cancer. We describe two new HPLC methods for capmatinib quantification in vivo and in vitro. HPLC with a fluorescence detection method was used to quantify capmatinib in plasma for the first time. The method was successfully applied in a pharmacokinetic study following a 10 mg/kg oral dose of capmatinib given to rats. The chromatographic separation was performed using a Eurospher II 100-3 C18H (50 × 4 mm, 3 µm) column and a mobile phase containing 10 mM of ammonium acetate buffer (pH 5.5): acetonitrile (70:30, v/v), at a flow rate of 2.0 mL min−1. The study also describes the use of HPLC-PDA for the first time for the determination of capmatinib in human liver microsomes and describes its application to study its metabolic stability in vitro. Our results were in agreement with those reported using LC-MS/MS, demonstrating the reliability of the method. The study utilized a Gemini-NX C18 column and a mobile phase containing methanol: 20 mM ammonium formate buffer pH 3.5 (53:47, v/v), delivered at a flow rate of 1.1 mL min−1. These methods are suitable for supporting pharmacokinetic studies, particularly in bioanalytical labs lacking LC-MS/MS capabilities.

In the pharmaceutical field, and more precisely in quality control laboratories, robust liquid chromatographic methods are needed to separate and analyze mixtures of compounds. The development of such chromatographic methods for new mixtures can result in a long and tedious process even while using the design of experiments methodology. However, developments could be accelerated with the help of in silico screening. In this work, the usefulness of a strategy combining response surface methodology (RSM) followed by multicriteria decision analysis (MCDA) applied to predictions from a quantitative structure–retention relationship (QSRR) model is demonstrated. The developed strategy shows that selecting equations for the retention time prediction models based on the pKa of the compound allows flexibility in the models. The MCDA developed is shown to help to make decisions on different criteria while being robust to the user’s decision on the weights for each criterion. This strategy is proposed for the screening phase of the method lifecycle. The strategy offers the possibility to the user to select chromatographic conditions based on multiple criteria without being too sensitive to the importance given to them. The conditions with the highest desirability are defined as the starting point for further optimization steps.

Continued adoption of two-dimensional liquid chromatography (2D-LC) in industrial laboratories will depend on the development of approaches to make method development for 2D-LC more systematic, less tedious, and less reliant on user expertise. In this paper, we build on previous efforts in these directions by describing the use of multifactorial modeling software that can help streamline and simplify the method development process for 2D-LC. Specifically, we have focused on building retention models for second dimension (2D) separations involving variables including gradient time, temperature, organic modifier blending, and buffer concentration using LC simulator (ACD/Labs) software. Multifactorial retention modeling outcomes are illustrated as resolution map planes or cubes that enable straightforward location of 2D conditions that maximize resolution while minimizing analysis time. We also illustrate the practicality of this approach by identifying conditions that yield baseline separation of all compounds co-eluting from a first dimension (1D) separation using a single combination of 2D stationary phase and elution conditions. The multifactorial retention models were found to be very accurate for both the 1D and 2D separations, with differences between experimental and simulated retention times of less than 0.5%. Pharmaceutical applications of this approach for multiple heartcutting 2D-LC were demonstrated using IEC-IEC or achiral RPLC-chiral RPLC for 2D separations of multicomponent mixtures. The framework outlined here should help make 2D-LC method development more systematic and streamline development and optimization for a variety of 2D-LC applications in both industry and academia.

Methods for the detection and quantification of food allergens in complex matrices are necessary to ensure compliance with labeling regulations and assess the effectiveness of food allergen preventive controls. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) has emerged as an orthogonal technique in complement to immunochemical-based assays. However, the absence of established guidelines for MS-based quantification of allergens in food has limited harmonization among the method development community. In this study, different quantification strategies were evaluated using a previously developed multiplexed LC-MS/MS method for the detection of egg, milk, and peanut. Peptide performance criteria (retention time, signal-to-noise ratio, and ion ratio tolerance) were established and quantification approaches using varying calibrants, internal standards, background matrices, and calibration curve preparation schemes were systematically evaluated to refine the previous method for routine laboratory use. A matrix-matched calibration curve using allergen ingredients as calibrants and stable isotope–labeled peptides as internal standards provided the most accurate quantitative results. The strategy was further verified with commercially available reference materials and allowed for the confident detection and quantification of food allergens. This work highlights the need for transparency in calibration strategy and peptide performance requirements for effective evaluation of mass spectrometric methods for the quantification of food allergens.

Quantitative proteomics/metabolomics investigation of laser-capture-microdissection (LCM) cell populations from clinical cohorts affords precise insights into disease/therapeutic mechanisms, nonetheless high-quality quantification remains a prominent challenge. Here, we devised an LC/MS-based approach allowing parallel, robust global-proteomics and targeted-metabolomics quantification from the same LCM samples, using biopsies from prostate cancer (PCa) patients as the model system. The strategy features: (i) an optimized molecular weight cutoff (MWCO) filter-based separation of proteins and small-molecule fractions with high and consistent recoveries; (ii) microscale derivatization and charge-based enrichment for ultrasensitive quantification of key androgens (LOQ = 5 fg/1k cells) with excellent accuracy/precision; (iii) reproducible/precise proteomics quantification with low-missing-data using a detergent-cocktail-based sample preparation and an IonStar pipeline for reproducible and precise protein quantification with excellent data quality. Key parameters enabling robust/reproducible quantification have been meticulously evaluated and optimized, and the results underscored the importance of surveying quantitative performances against key parameters to facilitate fit-for-purpose method development. As a proof-of-concept, high-quality quantification of the proteome and androgens in LCM samples of PCa patient-matched cancerous and benign epithelial/stromal cells was achieved (N = 16), which suggested distinct androgen distribution patterns across cell types and regions, as well as the dysregulated pathways involved in tumor-stroma crosstalk in PCa pathology. This strategy markedly leverages the scope of quantitative-omics investigations using LCM samples, and combining with IonStar, can be readily adapted to larger-cohort clinical analysis. Moreover, the capacity of parallel proteomics/metabolomics quantification permits precise corroboration of regulatory processes on both protein and small-molecule levels, with decreased batch effect and enhanced utilization of samples.

Aim: For decades, the traditional approach for ligand-binding assays has been to generate two measurements from adjacent wells on the plate. In recent years, scientists have investigated the true benefit of this ‘duplicate analysis’ by looking back at previously generated bioanalytical data with the conclusion that the benefits are negligible. Materials & methods: We demonstrated a method development approach to determine the best number of replicate measurements of an immunogenicity assay. We used an anti-pembrolizumab immunogenicity assay on Gyrolab® to challenge the traditional use of duplicate measurements as we compare it to singlet measurement and show a balanced design for assessing the cut-point in singlet. Results & conclusion: We introduced the concept of calculating the maximum drug tolerance during method development. In this method, we found no practical benefit for duplicate analysis and go further in recommending that singlet analysis should be considered the default for all ligand-binding assays.

Although commercially available software provides options for automatic peak detection, visual inspection and manual corrections are often needed. Peak detection algorithms commonly employed require carefully written rules and thresholds to increase true positive rates and decrease false positive rates. In this study, a deep learning model, specifically, a convolutional neural network (CNN), was implemented to perform automatic peak detection in reversed-phase liquid chromatography (RPLC). The model inputs a whole chromatogram and outputs predicted locations, probabilities, and areas of the peaks. The obtained results on a simulated validation set demonstrated that the model performed well (ROC-AUC of 0.996), and comparably or better than a derivative-based approach using the Savitzky-Golay algorithm for detecting peaks on experimental chromatograms (8.6% increase in true positives). In addition, predicted peak probabilities (typically between 0.5 and 1.0 for true positives) gave an indication of how confident the CNN model was in the peaks detected. The CNN model was trained entirely on simulated chromatograms (a training set of 1,000,000 chromatograms), and thus no effort had to be put into collecting and labeling chromatograms. A potential major drawback of this approach, namely training a CNN model on simulated chromatograms, is the risk of not capturing the actual "chromatogram space" well enough that is needed to perform accurate peak detection in real chromatograms.

A method using ion chromatography coupled to high-resolution Orbitrap mass spectrometry was developed to quantify highly-polar organic compounds in aqueous filter extracts of atmospheric particles. In total, 43 compounds, including short-chain carboxylic acids, terpene-derived acids, organosulfates, and inorganic anions were separated within 33 min by a KOH gradient. Ionization by electrospray was maximized by adding 100 µL min-1 isopropanol as post-column solvent and optimizing the ion source settings. Detection limits (S/N ≥ 3) were in the range of 0.075-25 μg L-1 and better than previously reported for 22 compounds. Recoveries of extraction typically range from 85 to 117%. The developed method was applied to three ambient samples, including two arctic flight samples, and one sample from Melpitz, a continental backround research site. A total of 32 different compounds were identified for all samples. From the arctic flight samples, organic tracers could be quantified for the first time with concentrations ranging from 0.1 to 17.8 ng m-3 . Due to the minimal sample preparation, the beneficial figures of merit, and the broad range of accessible compounds, including very polar ones, the new method offers advantages over existing ones and enables a detailed analysis of organic marker compounds in atmospheric aerosol particles.

Biomarker assay method development is a multistep rigorous process and calibrant material selection is integral to ensuring the quality of such assays. However, the impact of selection of calibrator material may often get overlooked. In this article, we highlight three case studies where biomarker calibrant material selection was deemed an essential criterion for consideration. Through these case studies we highlight challenges faced, steps taken and discuss the impact on assay-related decision-making. We also provide additional perspectives for selection and characterization of calibrant proteins in the setting of an evolving biomarker context of use.

Analysis of acyl coenzyme A thioesters (acyl-CoAs) is crucial in the investigation of a wide range of biochemical reactions and paves the way to fully understand the concerned metabolic pathways and their superimposed networks. We developed two methods for suspect screening of acyl-CoAs in bacterial cultures using a high-resolution Orbitrap Fusion tribrid mass spectrometer. The methods rely on specific fragmentation patterns of the target compounds, which originate from the coenzyme A moiety. They make use of the formation of the adenosine 3′,5′-diphosphate key fragment (m/z 428.0365) and the neutral loss of the adenosine 3′-phosphate-5′-diphosphate moiety (506.9952) as preselection criteria for the detection of acyl-CoAs. These characteristic ions are generated either by an optimised in-source fragmentation in a full scan Orbitrap measurement or by optimised HCD fragmentation. Additionally, five different filters are included in the design of method. Finally, data-dependent MS/MS experiments on specifically preselected precursor ions are performed. The utility of the methods is demonstrated by analysing cultures of the denitrifying betaproteobacterium “Aromatoleum” sp. strain HxN1 anaerobically grown with hexanoate. We detected 35 acyl-CoAs in total and identified 24 of them by comparison with reference standards, including all 9 acyl-CoA intermediates expected to occur in the degradation pathway of hexanoate. The identification of additional acyl-CoAs provides insight into further metabolic processes occurring in this bacterium. The sensitivity of the method described allows detecting acyl-CoAs present in biological samples in highly variable abundances. Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03318-3.

Aromas can give smell and/or flavor to a variety of products in the cosmetic and food industry. They are also used as e-cigarette additives. Some of those substances are recognized as fragrance allergens and can cause allergic reactions so there is a need to control their use. Gas chromatography is the method of choice for analyzing fragrance allergens because of their low boiling points. This study aimed to develop and validate a robust and simple method for the analysis of fragrance allergens in aromas for e-cigarettes. A method using gas chromatography coupled with a flame ionization detector was developed for 25 fragrance allergens. Optimized parameters were sample diluent, internal standard, and carrier gas. The output method was the one previously developed and optimized. The linearity of the method was >0.994 over the range of 0.5-40 μg/mL. Accuracy and precision were within the acceptance range. Limits of detection and quantification were determined, and calibration curves were plotted. The method was applied to three real samples of aromas. Thirteen fragrance allergens were detected. Concentrations varied in the range of dozens to thousands of μg/mL showed that concentrations of fragrances in aromas for e-cigarettes can be high and varies among products.

Economical and fast analytical methods are required to determine the content of herbs and herbal medicine. With the increasing interest in rose tea and products prepared from rose flower extracts, it has emerged that products prepared from dried rose petals and rose flower extracts should have certain characteristics. In the European Pharmacopeia, there is no specific method to meet the specific needs of the industry. Therefore, in this study, a simple, cheap, fast and reliable RP-HPLC-DAD method was developed and validated for the identification and quantification of flavonoids in the methanol extract of Rosa damascena Mill. (RD) petals. 10 different RD petal samples obtained from Turkey (Isparta), Pakistan and different parts of Iran were studied and compared with fingerprinting. Several trials were conducted to identify the most robust and consistent HPLC method. Separation of flavonoids was achieved on a reversed-phase C-18 column kept at 25 degrees C by gradient elution with a flow rate of 1.2 mL/min, injection volume of 10 mu L and detection at 330 nm. Six different flavonol glycosides were identified which are kaempferol 3-O-glucoside (K3GLU), kaempferol 3-O-galactoside (K3GAL), kaempferol 3-O-rhamnoside (K3RHA), quercetin 3-O-glucoside (Q3GLU), quercetin 3-O-galactoside (Q3GAL), quercetin 3-O-rhamnoside (Q3RHA). K3GLU was determined to be the most suitable compound as a marker and was used for quantification and validation studies. Standard curve of this compound was plotted to calculate quantification. K3GLU amount was found the highest in Isparta rose with 0.7047%. Various validation parameters were done, and the method was found to be specific, stable, linear, sensitive, accurate, precise and robust to determine the chemical specification of RD petals.

A rapid GC-FID method was developed to simultaneously determine residual levels of triethylamine (TEA), 1,1,3,3-tetramethylguanidine (TMG), and diisopropylamine (DIPA) in the synthetic route of an active pharmaceutical ingredient (API). Due to the severe absorption of amines on GC stationary phases, GC columns with various stationary phases were evaluated for optimal peak shape and reproducibility. The final conditions used the Agilent CP-Volamine column to resolve the three amines in 12 min. Various inlet liners were also screened to further improve the sensitivity of the analysis. The Restek Siltek® liner was selected to achieve the desired detectability for the method. The quantitation limits were 4, 3, and 4 μg/mL for TEA, DIPA, and TMG in the presence of API, respectively. All three amines showed good linearity (r > 0.999) and recoveries (> 90%) over the concentration range of 3 to 16 μg/mL. The testing of residual amines was initially performed at the penultimate stage of the synthesis. However, this work demonstrates that TMG can act as a proton sponge to react with salicylic acid, the counter ion of the penultimate, to form a volatile component that elutes at a different retention time. Consequently, in the final method, these three amines were monitored in the final API to circumvent the matrix interference. Key parameters of the method were qualified per method validation requirements in ICH guidelines. The method was successfully applied for batch testing during development and implemented as an in-process control procedure at manufacturing sites., Graphical abstract Image 1, Highlights • A fast and sensitive GC-FID method was developed to monitor the residue of three volatile amines in an API. • The detection of 1,1,3,3-Tetramethylguanidine (TMG) at trace level is reported the first time. • The method circumvented the interference of sample matrix since TMG can react with carboxylic acid in penultimate. • The method was qualified per ICH guideline and implemented as an in-process-control procedure.

Present study reports design of experiment (DoE) based development and validation of a simple, rapid and sensitive reversed-phase high-performance liquid chromatography (RP-HPLC) method for estimation of efavirenz (EFZ), a non-nucleotide reverse transcriptase inhibitor (NNRTs), used in the treatment of acquired immunodeficiency syndrome (AIDS). Plackett–Burman design was explored to screen the critical method variables (CMVs) for the RP-HPLC method. A response surface Box–Behnken design was employed to optimize the screened CMVs which affect the analytical responses (ARs) of RP-HPLC method. Using the optimized CMVs the HPLC method was developed and validated according to International Conference on Harmonization (ICH) guidelines. EFZ in marketed formulation was estimated using the validated method. Acetonitrile proportion, pH of the phosphate buffer and mobile phase flow rate were the CMVs and retention time and number of theoretical plates were the ARs for the study. The optimized chromatographic parameters were acetonitrile proportion in mobile phase: 51.17%v/v, pH of phosphate buffer: 4.04 and flow rate: 1.25 mL/min. Use of these optimized parameters resulted in retention time of 11.031 min and 9,498.787 number of theoretical plates as ARs of the HPLC method. The method was further validated in harmony with current ICH guidelines Q2 (R1). The method was capable of the successful estimation of EFZ in marketed formulation. The study depicts successful development and validation of a simple RP-HPLC method of EFZ using DoE approach.

Background: Over the last few decades, there has been a growing interest in Two- Dimensional Liquid Chromatography (2D-LC). This advanced technology has become a relevant solution for solving all the analytical challenges encountered in the pharmaceutical industry. The primary purpose of this review is to give a brief account of the developing techniques notable in 2D-LC. Methods: Review of recent literature on advanced separation techniques for complex mixtures revealed that 2D-LC is the most widely used technique in the pharmaceutical laboratory for the separation of complex samples. Among the various techniques used in 2D-LC, comprehensive 2D-LC and heart-cutting 2D-LC are highly used techniques. To enhance the resolution, further development has been made to heart-cutting 2D-LC called multiple heart-cutting 2D-LC, which has recently become a very popular technique in the pharmaceutical industry. Therefore research articles mentioning the use of multiple heart-cutting 2D-LC for separation of complex mixtures were reviewed. Results: The most crucial benefit of multiple heart-cutting 2D-LC is its reduced effort in method development and also the storage of multiple sample sections for further analysis. The multiple heartcutting 2D-LC is suited, preferably for method development and impurity analysis of pharmaceutical substances, fine chemicals and offers better peak separation, which is otherwise not possible by comprehensive 2D-LC. Conclusion: The advantage of two-dimensional liquid chromatography over one-dimensional liquid chromatography is its high resolving power. Further improvement in resolution was achieved by using the advanced multiple heart-cutting 2D-LC which appears to have an imperative role in pharmaceutical and food analysis.

Background Dronedarone HCl (DRN) is an anti-arrhythmic drug indicated for atrial fibrillation. DRN has a low solubility of 2 µg/mL and 4% bioavailability, thus it is formulated as a co-amorphous system to enhance its solubility by using quercetin (QCT) as a co-former. A sensitive, accurate, and economic method for the simultaneous quantification of DRN and QCT in formulation is not found in the literature. Objective To develop a Reverse Phase -HPLC method for the simultaneous estimation of DRN and QCT in a DRN–QCT co-amorphous system. Method The co-amorphous system was prepared using a solvent evaporation technique with DRN and QCT in a 1:1 molar ratio. The separation was achieved on a Purospher® STAR C18 (250 mm × 4.6 mm × 5 μm id (internal diameter)) column with the mobile phase comprising of acetonitrile and a 25 mM phosphate buffer pH 3.6 (60:40%, v/v). Results DRN and QCT were retained on the column for 6.7 and 3.5 min, respectively. For both molecules, the method was developed with a wide linearity range of 0.2–500 µg/mL. The LOD for DRN was found to be 0.0013 µg/mL and for QCT it was found to be 0.0026 µg/mL. The LOQ for DRN was found to be 0.0041 µg/mL, and for QCT it was 0.0078 µg/mL. Conclusions The method was validated as per International Conference on Harmonization (ICH) guidelines for linearity, precision, accuracy, and robustness. The method was used in simultaneous quantification of DRN and QCT in co-amorphous samples. Highlights The method developed was used for the analysis of content uniformity and solubility samples of co-amorphous system, where the method was able to successfully quantify DRN and QCT. Low detection and quantification limits contribute to the sensitivity of the method and wide linearity range assures the robust and precise quantification of molecules.

We have revamped a classic analytical chemistry laboratory experiment, “Determination of an Unknown Acid”, to provide students an opportunity to learn about new advances in the material sciences and their applications in analytical chemistry in an effort to reinforce the key concepts of chemical analysis. Specifically, students were given the opportunity to develop their own low-cost, low-maintenance, ease-of-use, multidimensional pH sensor with intrinsically conducting polyaniline material. The students tested their polyaniline pH sensor against the traditional glass pH electrode to determine an unknown acid concentration and performed statistical tests to validate the analytical capabilities of their electrochemical polyaniline pH sensor. Thus, the revamped laboratory experiment allows for the reinforcement of several fundamental analytical chemistry lecture concepts, including chemical analysis, method development, validation, and statistical practice utilizing a more cohesive hands-on laboratory learning approach. Furthermore, this revamped lab gives students the opportunity to evaluate and compare the analytical performance of the polyaniline pH sensor, such as its sensor lifetime against the glass pH electrode, and learn polyaniline’s electrochromic properties. This laboratory experiment can be easily disseminated to advance the objective of analytical curriculum, provide students an opportunity for multidimensional learning and well-connected critical thinking in analytical chemistry, and stimulate students’ creativity in developing next generation analytical sensor technology.

Capillary electrophoresis is a powerful technique for the analysis of polar chiral compounds and has been widely accepted for analytical enantioseparations of drug compounds in pharmaceuticals and biological media. In addition, many mechanistic studies have been conducted in an attempt to rationalize enantioseparations in combination with spectroscopic and computational techniques. The present review will focus on recent examples of mechanistic aspects and summarize recent applications of stereoselective pharmaceutical and biomedical analysis published between January 2017 and November 2020. Various separation modes including electrokinetic chromatography in combination with several detection modes including laser-induced fluorescence, mass spectrometry and contactless conductivity detection will be discussed. A general trend also observed in other analytical techniques is the application of quality by design principles in method development and optimization.

The aim of this study was to develop an analytical method, which separates selected chlorinated polycyclic aromatic hydrocarbons (Cl-PAHs) from fat, and fat-free or vegetable matrices. The method contains extraction-, cleanup-, and quantification steps. Integration of automated analysis actions, as in extraction and cleanup, should enhance the reproducibility, precision, and efficiency of the method. This was confirmed by validation of the overall analytical process. In the end, as a performance check, the developed method was applied on different matrices, e.g., tea, rice, grilled pork, and eel and predator eggs, as a non-food example. An inter-laboratory check was initiated as replacement for the lack of proficiency tests. Due to the high level of automation, both personnel and time effort are very low. In addition, the method is very robust with regard to the variability of the solvent selection and the loss of analytes by evaporation to dryness. It could be demonstrated that the developed method is applicable to different matrices with reproducible and precise results. This applies also to low-fat food and feed.

The human body comprises rich populations of cells, which are arranged into tissues and organs with diverse functionalities. These cells exhibit a broad spectrum of phenotypes and are often organized as a heterogeneous but sophisticatedly regulated ecosystem - tissue microenvironment, inside which every cell interacts with and is reciprocally influenced by its surroundings through its life span. Therefore, it is critical to comprehensively explore the cellular machinery and biological processes in the tissue microenvironment, which is best exemplified by the tumor microenvironment (TME). The past decade has seen increasing advances in the field of spatial proteomics, the main purpose of which is to characterize the abundance and spatial distribution of proteins and their post-translational modifications in the microenvironment of diseased tissues. Herein, we outline the achievements and remaining challenges of mass spectrometry-based tissue spatial proteomics. Exciting technology developments along with important biomedical applications of spatial proteomics are highlighted. In detail, we focus on high-quality resources built by scalpel macrodissection-based region-resolved proteomics, method development of sensitive sample preparation for laser microdissection-based spatial proteomics, and antibody recognition-based multiplexed tissue imaging. In the end, critical issues and potential future directions for spatial proteomics are also discussed.

In this study, a new size exclusion chromatographic method has been developed and validated for the analysis of mucopolysaccharide polysulfate used as an anti-inflammatory and antithrombotic agent in topical formulations. Mucopolysaccharide polysulfate was analyzed in Repromer OH-4000 (10 μm, 8.0 × 300 mm) and Repromer OH-5000 (10 μm, 8.0 × 300 mm) columns using a 0.05 M sodium sulfate isocratic elution mobile phase system at 40 °C with a flow rate of 1 mL min−1 and detected by using refractive index detection. The method was validated by means of the limit of quantification, limit of detection, linearity, robustness, recovery, precision and accuracy using the Bioanalytical Method Validation Guidance. The calibration curve showed linearity in the 0.090–1.575 mg mL−1 range. The limits of detection and quantification were found to be 45.000 and 90.000 μg mL−1, respectively. Assay recovery and precision of mucopolysaccharide polysulfate from topical formulations at 0.450, 0.900 and 1.350 mg mL−1 concentrations were evaluated. Intra-day and inter-day relative standard deviation values were calculated to be less than 2.46%. The mean recovery was calculated as 96.64%. The validated method was successfully applied to the determination of mucopolysaccharide polysulfate in cream and gel formulations.

Stable isotopic analysis is an important branch of analytical chemistry. Accurate determination of the stable isotopic compositions of substances is critical for tracing their sources and investigating their transformation processes. The new generation of mass spectrometry technology has greatly facilitated the development of high-precision stable isotopic analysis methods. In particular, multi-collector inductively coupled plasma mass spectrometry (MC-ICP-MS) has emerged as a powerful tool for isotopic composition determination. However, isotopic analysis by MC-ICP-MS is highly sensitive to the sample matrix, which may compromise the precision and accuracy of the analysis. Therefore, it is particularly important to reduce the sample matrix effect using efficient sample purification techniques. This article summarizes the recent progress in sample purification and instrument hyphenation methods for MC-ICP-MS, and provides perspectives on the future application of MC-ICP-MS in different fields.

The present work aims at developing a QbD assisted RP-HPLC method for the estimation of individual curcuminoids in Curcuma longa rhizomes along with its quality assessment. The quality of crude C. ...

This study presents the determination of toxic and nutrient elements in vinegar samples using inductively coupled plasma – atomic emission spectrometry (ICP-AES). The determined elements were Pb, N...

An important aspect to understand about an experimental molecule in drug discovery is its stability in solution. A compound that degrades might be eliciting its apparent effect via a degradation product, so it is important to understand the solution stability profile of a compound early on in the drug discovery process. Improvements and application of a streamlined, higher-throughput method for testing solution stability to support drug discovery are described. Mass spectrometry detection has been incorporated into the screen to allow for the identification of degradation products. The amount of compound needed for the assay has been significantly reduced using 10 mM DMSO solutions instead of solid material. The buffers used in the screen provide the stability-pH profile of compounds with additional variations to assess liabilities under oxidizing and reducing conditions. In this article, we discuss the method development, screen validation, guidelines for result interpretation, and results for a set of marketed drugs to illustrate the application of the screen.

Curcuma longa (turmeric) has traditionally been used in Ayurvedic, Unani and herbal drugs to cure numerous ailments. Due to the high demand, the quantitative standardization of herbal products is challenging to maintain their quality. We aim to develop a rapid, sensitive and validated high-performance thin-layer chromatography (HPTLC) method for the simultaneous determination and quantification of curcumin I, curcumin II and curcumin III in C. longa and herbal formulation. The three standards were separated using centrifugal preparative thin-layer chromatography (CPTLC) silica gel and identified by different spectroscopic methods. The developed HPTLC method was validated by following ICH guidelines (linearity; limit of detection, LOD; limit of quantitation; accuracy; precision; and robustness). The calibration curves of both the compounds were linear (50–500 ng/spot), with a correlation coefficient (r2) of >999. The developed HPTLC method was effectively applied to the concurrent detection and quantification of curcumins I–III in fresh, dry rhizomes and the herbal formulation of C. longa extracts was obtained by hot and cold extraction methods.