NMR Longitudinal Rotating Frame Relaxation Time (T1ρ) with a Weak Spin Locking Field as an Approach to Characterize Solid-State Active Pharmaceutical Ingredients: Proof of Concept.

Nuclear magnetic resonance (NMR) longitudinal rotating frame relaxation time (T1ρ), rarely used in low-field NMR, can be more effective than conventional T1 and T2 relaxation times to differentiate polymorphic forms of solid pharmaceuticals. This could be attributed to T1ρ sensibility to structural and molecular dynamics that can be enhanced by changing the strength of the oscillating magnetic field (B1) of spinlock pulses. Here, we compared the capacity of T1, T2, and T1ρ to differentiate inactive (A) and active (C) crystalline forms of the World Health Organization essential drug Mebendazole. The results showed that T1 and T2 values of both forms were statistically identical at 0.47 T. Conversely, T1ρ of both forms measured with weak spinlock B1 fields, ranging from 0.08 to 0.80 mT were statistically different in the same spectrometer. The T1ρ also has the limit of detection to detect the presence of at least 10% of inactive A form in the active C form. Therefore, T1ρ, measured with weak spinlock B1 fields can be an effective, streamlined, and complementary approach for characterizing not only solid active pharmaceutical ingredients but other solid-state materials as well.

Analytical Methodologies to Detect N-Nitrosamine Impurities in Active Pharmaceutical Ingredients, Drug Products and Other Matrices.

Since 2018, N-nitrosamine impurities have become a widespread concern in the global regulatory landscape of pharmaceutical products. This concern arises due to their potential for contamination, toxicity, carcinogenicity, and mutagenicity and their presence in many active pharmaceutical ingredients, drug products, and other matrices. N-Nitrosamine impurities in humans can lead to severe chemical toxicity effects. These include carcinogenic effects, metabolic disruptions, reproductive harm, liver diseases, obesity, DNA damage, cell death, chromosomal alterations, birth defects, and pregnancy loss. They are particularly known to cause cancer (tumors) in various organs and tissues such as the liver, lungs, nasal cavity, esophagus, pancreas, stomach, urinary bladder, colon, kidneys, and central nervous system. Additionally, N-nitrosamine impurities may contribute to the development of Alzheimer's and Parkinson's diseases and type-2 diabetes. Therefore, it is very important to control or avoid them by enhancing effective analytical methodologies using cutting-edge analytical techniques such as LC-MS, GC-MS, CE-MS, SFC, etc. Moreover, these analytical methods need to be sensitive and selective with suitable precision and accuracy, so that the actual amounts of N-nitrosamine impurities can be detected and quantified appropriately in drugs. Regulatory agencies such as the US FDA, EMA, ICH, WHO, etc. need to focus more on the hazards of N-nitrosamine impurities by providing guidance and regular updates to drug manufacturers and applicants. Similarly, drug manufacturers should be more vigilant to avoid nitrosating agents and secondary amines during the manufacturing processes. Numerous review articles have been published recently by various researchers, focusing on N-nitrosamine impurities found in previously notified products, including sartans, metformin, and ranitidine. These impurities have also been detected in a wide range of other products. Consequently, this review aims to concentrate on products recently reported to contain N-nitrosamine impurities. These products include rifampicin, champix, famotidine, nizatidine, atorvastatin, bumetanide, itraconazole, diovan, enalapril, propranolol, lisinopril, duloxetine, rivaroxaban, pioglitazones, glifizones, cilostazol, and sunitinib.

Advances in Nanoplatforms for Immunotherapy Applications Targeting the Tumor Microenvironment.

Cancer immunotherapy is a treatment method that activates or enhances the autoimmune response of the body to fight tumor growth and metastasis, has fewer toxic side effects and a longer-lasting efficacy than radiotherapy and chemotherapy, and has become an important means for the clinical treatment of cancer. However, clinical results from immunotherapy have shown that most patients lack responsiveness to immunotherapy and cannot benefit from this treatment strategy. The tumor microenvironment (TME) plays a critical role in the response to immunotherapy. The TME typically prevents effective lymphocyte activation, reducing their infiltration, and inhibiting the infiltration of effector T cells. According to the characteristic differences between the TME and normal tissues, various nanoplatforms with TME targeting and regulation properties have been developed for more precise regulation of the TME and have the ability to codeliver a variety of active pharmaceutical ingredients, thereby reducing systemic toxicity and improving the therapeutic effect of antitumor. In addition, the precise structural design of the nanoplatform can integrate specific functional motifs, such as surface-targeted ligands, degradable backbones, and TME stimulus-responsive components, into nanomedicines, thereby reshaping the tumor microenvironment, improving the body's immunosuppressive state, and enhancing the permeability of drugs in tumor tissues, in order to achieve controlled and stimulus-triggered release of load cargo. In this review, the physiological characteristics of the TME and the latest research regarding the application of TME-regulated nanoplatforms in improving antitumor immunotherapy will be described. Furthermore, the existing problems and further applications perspectives of TME-regulated platforms for cancer immunotherapy will also be discussed.

The Use of Systemically Absorbed Drugs to Explore An In Vitro Bioequivalence Approach For Comparing Non-Systemically Absorbed Active Pharmaceutical Ingredients in Drug Products For Use in Dogs.

PURPOSE: Currently, for veterinary oral formulations containing one or more active pharmaceutical ingredient (API) that are not systemically absorbed and act locally within the gastrointestinal (GI) tract, the use of terminal clinical endpoint bioequivalence (BE) studies is the only option for evaluating product BE. This investigation explored the use of a totality of evidence approach as an alternative to these terminal studies. METHODS: Three formulations of tablets containing ivermectin plus praziquantel were manufactured to exhibit distinctly different in vitro release characteristics. Because these APIs are highly permeable, plasma drug concentrations served as a biomarker of in vivo dissolution. Tablets were administered to 27 healthy Beagle dogs (3-way crossover) and the rate and extent of exposure of each API for each formulation was compared in a pairwise manner. These results were compared to product relative in vitro dissolution profiles in 3 media. In vivo and in vitro BE predictions were compared. RESULTS: In vivo/in vitro inconsistencies in product relative performance were observed with both compounds when considering product performance across the 3 dissolution media. Formulation comparisons flagged major differences that could explain this outcome. CONCLUSIONS: The finding of an inconsistent in vivo/in vitro relationship confirmed that in vitro dissolution alone cannot assure product BE for veterinary locally acting GI products. However, when combined with a comparison of product composition and manufacturing method, this totality of evidence approach can successfully alert scientists to potential therapeutic inequivalence, thereby supporting FDA's efforts to Replace, Reduce, and/or Refine terminal animal studies.

Potential and performance of anisotropic 19F NMR for the enantiomeric analysis of fluorinated chiral active pharmaceutical ingredients.

Controlling the enantiomeric purity of chiral drugs is of paramount importance in pharmaceutical chemistry. Isotropic 1H NMR spectroscopy involving chiral agents is a widely used method for discriminating enantiomers and quantifying their relative proportions. However, the relatively weak spectral separation of enantiomers (1H Δδiso(R, S)) in frequency units at low and moderate magnetic fields, as well as the lack of versatility of a majority of those agents with respect to different chemical functions, may limit the general use of this approach. In this article, we investigate the analytical potential of 19F NMR in anisotropic chiral media for the enantiomeric analysis of fluorinated active pharmaceutical ingredients (API) via two residual anisotropic NMR interactions: the chemical shift anisotropy (19F-RCSA) and dipolar coupling ((19F-19F)-RDC). Lyotropic chiral liquid crystals (CLC) based on poly-γ-benzyl-L-glutamate (PBLG) show an interesting versatility and adaptability to enantiodiscrimination as illustrated for two chiral drugs, Flurbiprofen® (FLU) and Efavirenz® (EFA), which have very different chemical functions. The approach has been tested on a routine 300 MHz NMR spectrometer equipped with a standard probe (5 mm BBFO probe) in a high-throughput context (i.e., ≈10 s of NMR experiments) while the performance for enantiomeric excess (ee) measurement is evaluated in terms of trueness and precision. The limits of detection (LOD) determined were 0.17 and 0.16 µmol ml-1 for FLU and EFA, respectively, allow working in dilute conditions even with such a short experimental duration. The enantiodiscrimination capabilities are also discussed with respect to experimental features such as CLC composition and temperature.

Cyclodextrin-based nanosponges as promising carriers for active pharmaceutical ingredient.

Effective drug distribution at the intended or particular location is a critical issue that researchers are now dealing. Nanosponges have significantly increased in importance in medication delivery using nanotechnology in recent years. An important step toward solving these problems has been the development of nanosponges. Recently created and proposed for use in drug delivery, nanosponge is a unique type of hyper-crosslinked polymer-based colloidal structures made up of solid nanoparticles with colloidal carriers. Nanosponges are solid porous particles that may hold pharmaceuticals and other actives in their nanocavities. They can be made into dosage forms for oral, parenteral, topical, or inhalation use. The targeted distribution of drugs in a regulated manner is greatly aided by nanosponge. The utilization of nanosponges, their benefits, their production processes, the polymers they are made of, and their characterization have all been covered in this review article.

Knowledge-Based Design of Multifunctional Polymeric Nanoparticles.

Conventional drug delivery systems (DDS) today still face several drawbacks and obstacles. High total doses of active pharmaceutical ingredients (API) are often difficult or impossible to deliver due to poor solubility of the API or undesired clearance from the body caused by strong interactions with plasma proteins. In addition, high doses lead to a high overall body burden, in particular if they cannot be delivered specifically to the target site. Therefore, modern DDS must not only be able to deliver a dose into the body, but should also overcome the hurdles mentioned above as examples. One of these promising devices are polymeric nanoparticles, which can encapsulate a wide range of APIs despite having different physicochemical properties. Most importantly, polymeric nanoparticles are tunable to obtain tailored systems for each application. This can already be achieved via the starting material, the polymer, by incorporating, e.g., functional groups. This enables the particle properties to be influenced not only specifically in terms of their interactions with APIs, but also in terms of their general properties such as size, degradability, and surface properties. In particular, the combination of size, shape, and surface modification allows polymeric nanoparticles to be used not only as a simple drug delivery device, but also to achieve targeting. This chapter discusses to what extent polymers can be designed to form defined nanoparticles and how their properties affect their performance.

Comparison of permitted daily exposure (PDE) values for active pharmaceutical ingredients (APIs) - Evidence of a robust approach.

Permitted Daily Exposure Limits (PDEs) are set for Active Pharmaceutical Ingredients (APIs) to control cross-contamination when manufacturing medicinal products in shared facilities. With the lack of official PDE lists for pharmaceuticals, PDEs have to be set by each company separately. Although general rules and guidelines for the setting of PDEs exist, inter-company variations in the setting of PDEs occur and are considered acceptable within a certain range. To evaluate the robustness of the PDE approach between different pharmaceutical companies, data on PDE setting of five marketed APIs (amlodipine, hydrochlorothiazide, metformin, morphine, and omeprazole) were collected and compared. Findings show that the variability between PDE values is within acceptable ranges (below 10-fold) for all compounds, with the highest difference for morphine due to different Point of Departures (PODs) and Adjustment Factors (AFs). Factors of PDE variability identified and further discussed are: (1) availability of data, (2) selection of POD, (3) assignment of AFs, (4) route-to-route extrapolation, and (5) expert judgement and differences in company policies. We conclude that the investigated PDE methods and calculations are robust and scientifically defensible. Additionally, we provide further recommendations to harmonize PDE calculation approaches across the pharmaceutical industry.

Simultaneous and trace-level quantification of four benzene sulfonate potential genotoxic impurities in doxofylline active pharmaceutical ingredients and tablets using high-performance liquid chromatography with ultraviolet detection.

In the production of doxofylline, the common occurrence of toxic p-toluene sulfonate generation prompted the development and validation of a method using HPLC with ultraviolet detection (HPLC-UV). This method is designed for detecting four potential genotoxic impurities (PGIs) present in both doxofylline drug substance and tablets, with a focus on the UV-absorbing group p-toluene sulfonate. The four impurities were methyl 4-methylbenzenesulfonate (PGI-1), ethyl 4-methylbenzenesulfonate (PGI-2), 2-hydroxyethyl 4-methylbenzenesulfonate (PGI-3), and 2-(4-methylphenyl)sulfonyloxyethyl 4-methylbenzenesulfonate (PGI-4). In this method, chromatographic separation was achieved using a Waters Symmetry C18 column (250 mm × 4.6 mm, 5 µm). The mobile phases consisted of 20% acetonitrile as mobile phase A and pure acetonitrile as mobile phase B, operating in gradient elution mode at a flow rate of 1.0 mL/min. According to the guidelines of the International Conference on Harmonization, it was determined that this method could quantify four PGIs at 0.0225 µg/mL in samples containing 60 mg/mL. The validated approach demonstrated excellent linearity (R2 > 0.999) across the concentration range of 30%-200% (relative to 0.075 µg/mL doxofylline) for the four PGIs. The accuracy of this method for the four PGIs ranged from 94.8% to 100.4%. The reverse-phase-HPLC-UV analytical method developed in this study is characterized by its speed and precision, making it suitable for the sensitive analysis of benzene sulfonate PGIs in doxofylline drug substances and tablets.

Triboelectrification of Active Pharmaceutical Ingredients: Amines and Their Hydrochloride Salts.

The triboelectric properties of active pharmaceutical ingredients (APIs) contribute to problems during the manufacturing of pharmaceuticals. However, the triboelectric properties of APIs have not been comprehensively characterized. In this study, the effect of salt formulation on the triboelectric properties of APIs was investigated. The triboelectric properties of three groups of amines, namely tertiary amines, purine bases, and amino acids, and their hydrochlorides were evaluated using a suction-type Faraday cage meter. Most of the hydrochloride salts exhibited more negative charges than the corresponding free bases, and the degree by which the triboelectric property changed upon hydrochlorination depended on the structural groups of the compounds. In the case of tertiary amines, the change in the zero-charge margin upon hydrochlorination was negatively correlated with the zero-charge margin of the free base. In contrast, hydrochlorination of the amino acids led to a significant change in the zero-charge margin. In most cases, salt formation also affected the triboelectric properties of API powders. Controlling the triboelectric properties of APIs solves various problems caused by the electrification of raw material powders and granules during the production of pharmaceuticals, thereby increasing the quality of produced pharmaceuticals.

Non-destructive Raman Method Development for Quantifying Active Pharmaceutical Ingredient in an Oral Suspension Through Plastic Dosing Syringes.

For liquid drug products, e.g., solutions or suspensions for oral or parenteral dosing, stability needs to be demonstrated in primary packaging during storage and in dosing devices during in-use periods per quality guidelines from the International Conference on Harmonisation (ICH) and the European Agency for the Evaluation of Medicinal Products (EMEA). One aspect of stability testing for liquid drug products is in-use stability, which typically includes transferring the liquid samples into another container for further sample preparation with extraction diluent and necessary agitation. Samples are then analyzed with traditional chromatography methods, which are laborious, prone to human errors, and time-consuming, especially when this process needs to be repeated multiple times during storage and in-use periods. Being able to analyze the liquid samples non-destructively would significantly improve testing efficiency. We investigated different Raman techniques, including transmission Raman (TRS) and back scatter Raman with a non-contact optic (NCO) probe, as alternative non-destructive tools to the UHPLC method for API quantitation in in-use liquid samples pulled into plastic dosing syringes. The linearity of the chemometric methods for these two techniques was demonstrated by cross-validation sample sets at three levels over an API concentration range of 60 to 80 mg/mL. The accuracy of the chemometric models was demonstrated by the accurate prediction of the API concentrations in independent samples from four different pilot plant batches manufactured at different sites. Both techniques were successful in measuring a signal through a plastic oral dosing syringe, and predicting the suspension API concentration to within 4% of the UHPLC-measured value. For future work, there are opportunities to improve the methodology by exploring additional probes or to expand the range of applications by using different sample presentations (such as prefilled syringes) or formulation matrices for solutions and suspensions.

Potential of Powder Rheology for Detecting Unforeseen Cross-Contamination of Foreign Active Pharmaceutical Ingredients.

Unexpected cross-contamination by foreign components during the manufacturing and quality control of pharmaceutical products poses a serious threat to the stable supply of drugs and the safety of customers. In Japan, in 2020, a mix-up containing a sleeping drug went undetected by liquid chromatography during the final quality test because the test focused only on the main active pharmaceutical ingredient (API) and known impurities. In this study, we assessed the ability of a powder rheometer to analyze powder characteristics in detail to determine whether it can detect the influence of foreign APIs on powder flow. Aspirin, which was used as the host API, was combined with the guest APIs (acetaminophen from two manufacturers and albumin tannate) and subsequently subjected to shear and stability tests. The influence of known lubricants (magnesium stearate and leucine) on powder flow was also evaluated for standardized comparison. Using microscopic morphological analysis, the surface of the powder was observed to confirm physical interactions between the host and guest APIs. In most cases, the guest APIs were statistically detected due to characteristics such as their powder diameter, pre-milling, and cohesion properties. Furthermore, we evaluated the flowability of a formulation incorporating guest APIs for direct compression method along with additives such as microcrystalline cellulose, potato starch, and lactose. Even in the presence of several additives, the influence of the added guest APIs was successfully detected. In conclusion, powder rheometry is a promising method for ensuring stable product quality and reducing the risk of unforeseen cross-contamination by foreign APIs.

Brazilian national production of active pharmaceutical ingredients: regulatory and strategic framework.

Active Pharmaceutical Ingredient (API) is any component of the final pharmaceutical product that serves as the active ingredient. The goal of the API Manufacturing is to produce APIs that are competitively priced and meet the quality standards with the least possible impact on the environment. The global API market is expected to experience massive growth in the coming years reaching the size of USD 355.94 billion. The global Pharmaceutical Industry is facing a new scenario in 2023 after responding to the COVID-19 pandemic. In this new panorama, rethinking the pharmaceutical production and market is necessary. Despite Brazil's prominence in terms of worldwide pharmaceutical spending, only 5% of the APIs required by local pharmaceutical companies are produced domestically. Therefore, Brazil is an untapped field for APIs' manufacturing and faces a scenario of health vulnerability associated with the reliance on foreign API imports to ensure the viability of national Pharmaceutical Production and Services. Huge investments are required to boost the growth of the API Manufacturing sector. Herein is presented a critical analysis of the current regulatory and strategic status of Brazilian national production and/or acquisition of APIs, which represent the key starting materials for the Pharmaceutical Industry.

Removal of two antidepressant active pharmaceutical ingredients from hospital wastewater by polystyrene-coated magnetite nanoparticles-assisted batch adsorption process.

This study employed simple polystyrene-coated magnetite nanoparticles (PS@MNPs)-assisted batch adsorption process for the removal of two antidepressant active ingredients (amitriptyline HCl and sertraline HCl) from hospital wastewater. Dominant parameters of the adsorption process including pH, adsorbent amount, and contact period were optimized through the univariate approach to enhance the adsorption efficiency. Upon reaching optimum adsorption conditions, equilibrium experiments were performed by spiking the adsorbates in hospital wastewater in the concentration range of 100-2000 µg/L. The concentrations of the adsorbates in the effluent were calculated using the matrix-matching calibration strategy to enhance the accuracy of quantification. A validated switchable solvent-based liquid phase microextraction (SS-LPME) method was employed to enrich the two active pharmaceutical ingredients (APIs) prior to sensitive determination with GC-MS (gas chromatography-mass spectrometry). The equilibrium data were mathematically modeled employing the Langmuir and Freundlich adsorption isotherm models. The isotherm constants were calculated, and the results showed that both the isotherm models fitted well with the experimental data. The efficient and simple batch adsorption strategy reported in this study was successfully employed to remove amitriptyline HCl and sertraline HCl from hospital wastewater at low concentrations.

Global Production of Active Pharmaceutical Ingredients for US Generic Drugs Experiencing Shortages.

This study evaluates the characteristics of generic active pharmaceutical ingredients (APIs) used to manufacture drugs with shortages in the US and facilities producing APIs worldwide.

Microencapsulation for Pharmaceutical Applications: A Review.

In order to improve bioavailability, stability, control release, and target delivery of active pharmaceutical ingredients (APIs), as well as to mask their bitter taste, to increase their efficacy, and to minimize their side effects, a variety of microencapsulation (including nanoencapsulation, particle size <100 nm) technologies have been widely used in the pharmaceutical industry. Commonly used microencapsulation technologies are emulsion, coacervation, extrusion, spray drying, freeze-drying, molecular inclusion, microbubbles and microsponge, fluidized bed coating, supercritical fluid encapsulation, electro spinning/spray, and polymerization. In this review, APIs are categorized by their molecular complexity: small APIs (compounds with low molecular weight, like Aspirin, Ibuprofen, and Cannabidiol), medium APIs (compounds with medium molecular weight like insulin, peptides, and nucleic acids), and living microorganisms (such as probiotics, bacteria, and bacteriophages). This article provides an overview of these microencapsulation technologies including their processes, matrix, and their recent applications in microencapsulation of APIs. Furthermore, the advantages and disadvantages of these common microencapsulation technologies in terms of improving the efficacy of APIs for pharmaceutical treatments are comprehensively analyzed. The objective is to summarize the most recent progresses on microencapsulation of APIs for enhancing their bioavailability, control release, target delivery, masking their bitter taste and stability, and thus increasing their efficacy and minimizing their side effects. At the end, future perspectives on microencapsulation for pharmaceutical applications are highlighted.

Differentiation of solvatomorphs of active pharmaceutical ingredients (API) by solid-state vibrational circular dichroism (VCD).

Here, we present the new application of solid-state Vibrational Circular Dichroism (VCD) spectroscopy to differentiate several dutasteride (DS) solvatomorphs - the model active pharmaceutical ingredient (API). Several crystalline DS hydrochloride hydrates solvated with methanol, ethanol, acetonitrile, acetone, and acetic acid were prepared. In contrast to almost identical IR spectra, the VCD ones were very sensitive to changes in the sample composition. We marked significant differences in the shape of VCD spectra of studied DS solvatomorphs, DS hydrates, and DS polymorphic forms. Our findings, supported by DFT calculations, show that VCD spectroscopy has the pronounced ability to distinguish their crystal arrangements. We believe that this contribution will extend the use of VCD in the pharmaceutical industry for developing and designing new chiral drug products for the identification, description, and in-depth probing of several pharmaceutical solvatomorphs in the future.

Continuous flow reactions in the preparation of active pharmaceutical ingredients and fine chemicals.

Herein, we present an overview of continuous flow chemistry, including photoflow and electroflow technologies in the preparation of active pharmaceutical ingredients (APIs) and fine chemical intermediates. Examples highlighting the benefits and challenges associated with continuous flow processes, mainly involving continuous thermal, photo- and electrochemical transformations, are drawn from the relevant literature, especially our experience and collaborations in this area, with emphasis on the synthesis and prospective scale-up.

Nanoneuroscience: Cutting-edge Approach for Disease Management.

Neurological disorders (ND) have affected a major part of our society and have been a challenge for medical and biosciences for decades. However, many of these disorders haven't responded well to currently established treatment approaches. The fact that many active pharmaceutical ingredients can't get to their specified action site inside the body is one of the main reasons for this failure. Extracellular and intracellular central nervous system (CNS) barriers prevent the transfer of drugs from the blood circulation to the intended location of the action. Utilizing nanosized drug delivery technologies is one possible way to overcome these obstacles. These nano-drug carriers outperform conventional dosage forms in many areas, including good drug encapsulation capacity, targeted drug delivery, less toxicity, and enhanced therapeutic impact. As a result, nano-neuroscience is growing to be an intriguing area of research and a bright alternative approach for delivering medicines to their intended action site for treating different neurological and psychiatric problems. In this review, we have included a short overview of the pathophysiology of neurological diseases, a detailed discussion about the significance of nanocarriers in NDs, and a focus on its recent advances. Finally, we highlighted the patented technologies and market trends, including the predictive analysis for the years 2021-2028.

Quantification of residual organic bases in an active pharmaceutical ingredient using mixed-mode chromatography and UV detection.

The use of organic bases is ubiquitous in chemical synthesis, yet quantifying these compounds with traditional HPLC methodologies is often hampered by poor peak shape, low retention, and limited UV absorption. When employed in the manufacture of an active pharmaceutical ingredient (API), these compounds must be controlled to levels that are safe for human consumption, requiring robust analytical methods with sufficiently low quantification limits. This work describes the development of an HPLC method for the quantification of imidazole and 1,8-Diazabicyclo[5.4.0]undec­7-ene (DBU) in an API using mixed-mode chromatography. Through control of the pH and organic modifier gradients, the retention of the basic analytes and API can be tuned independently to achieve desirable retention and sensitivity for each compound. The resulting HPLC method exhibits good performance in linearity, accuracy, sensitivity, specificity, and solution stability. Notably, these conditions avoid more complex detection modalities, such as mass spectrometry, while maintaining a system pressure below 400 bar, making the method compatible with a broad range of instruments. This approach to mixed-mode chromatography method development could be extended to different organic bases in the presence of complex molecules to fit the needs of projects in an academic or industrial environment.