Universal ion chromatography method for anions in active pharmaceutical ingredients enabled by computer-assisted separation modeling.

Ion Chromatography (IC) is one of the most widely used methods for analyzing ionic species in pharmaceutical samples. A universal IC method that can separate a wide range of different analytes is highly desired as it can save a lot of time for method development and validation processes. Herein we report the development of a universal method for anions in active pharmaceutical ingredients (APIs) using computer-assisted chromatography modeling tools. We have screened three different IC columns (Dionex IonPac AS28-Fast 4 µm, AS19 4 µm and AS11-HC 4 µm) to determine the best suitable column for universal IC method development. A universal IC method was then developed using an AS11-HC 4 µm column to separate 31 most common anionic substances in 36 mins. This method was optimized using LC Simulator and a model which precisely predicts the retention behavior of 31 anions was established. This model demonstrated an excellent match between predicted and experimental analyte retention time (R2 =0.999). To validate this universal IC method, we have studied the stability of sulfite and sulfide analytes in ambient conditions. The method was then validated for a subset of 29 anions using water and organic solvent/water binary solvents as diluents for commercial APIs. This universal IC method provides an efficient and simple way to separate and analyze common anions in APIs. In addition, the method development process combined with LC simulator modeling can be effectively used as a starting point during method development for other ions beyond those investigated in this study.

Biocatalytic cascade process of islatravir: Analytical and regulatory control strategy of minor enantiomer.

Commercial process of islatravir (MK-8591, EFdA) utilizes biocatalytic cascade reactions to construct the ribose moiety of the molecule which bears three chiral centers. However, this biocatalytic process also brought analytical challenges where all stereoisomers and process related compounds are controlled in one isolated intermediate, the final drug substance. A chiral LC method was developed to resolve all those compounds from islatravir and its minor enantiomer by thorough column screening and careful optimization. Detail of designing key method validation components such as method linearity, precision and robustness is discussed, and their results were presented. The method was successfully validated to fulfill various expectation from each individual health authority including FDA, EMA, PMDA, and ANVISA.

Development and validation of ion-pairing HPLC-CAD chromatography for measurement of Islatravir's phosphorylated intermediates.

Biocatalytic processes have become more prevalent in the pharmaceutical industry, leading to analytical challenges not faced when characterizing more traditional synthetic routes. A novel one-pot biocatalytic process has been established for Islatravir, an HIV reverse transcriptase translocation inhibitor for the treatment and prevention of HIV-1. As a one-pot reaction, the Islatravir chemistry contains multiple intermediates that are not isolated. Additionally, these unisolated intermediates have no chromophores, making traditional LC-UV techniques ineffective for characterization. A hydrophilic interaction chromatography (HILIC) method with a charged aerosol detector (CAD) was initially developed, however numerous inorganic species present in the one-pot reaction were retained; this led to co-elution of compounds and poor peak shapes. An innovative ion-pairing LC method was developed in order to resolve inorganic species, intermediates, and the API, for use during in-process control of the Islatravir biocatalytic reaction. Aided by a volatile ion-pairing reagent compatible with the CAD, this method successfully retains and resolves the highly polar intermediates of interest and Islatravir API. This novel method was successfully validated and has allowed the Islatravir biocatalytic process to be fully characterized from the early intermediates through the final API within the one-pot reaction without the need for isolations. This novel ion-pairing HPLC-CAD technique lays the groundwork for method development on current and future biocatalytic-produced drug substances.

Development of a Robust Manufacturing Route for Molnupiravir, an Antiviral for the Treatment of COVID-19.

Herein is described the development of a large-scale manufacturing process for molnupiravir, an orally dosed antiviral that was recently demonstrated to be efficacious for the treatment of patients with COVID-19. The yield, robustness, and efficiency of each of the five steps were improved, ultimately culminating in a 1.6-fold improvement in overall yield and a dramatic increase in the overall throughput compared to the baseline process.

Generic gas chromatography flame ionization detection method using hydrogen as the carrier gas for the analysis of solvents in pharmaceuticals.

Within the pharmaceutical industry, the determination of residual solvents by Gas Chromatography Flame Ionization Detection (GC-FID) is a highly utilized analytical test that often employs helium (He) as the carrier gas. However, many do not realize that helium is a non-renewable resource that will eventually become progressively more difficult to source. In recent years, analytical chemists are increasingly adopting hydrogen (H2) in place of helium for routine GC analysis. In this study, a simple and efficient generic/universal GC-FID method using H2 as the carrier gas has been developed with the capability of baseline resolution of over 30 of the most commonly used solvents in development and manufacturing with a method run time of less than eight minutes. The use of this method for the separation and analysis of solvents within a pharmaceutical manufacturing process is demonstrated with additional method validation data presented using five different diluents as a means to increase flexibility for the chromatographer. Furthermore, it is the recommendation of the authors that the current compendia for residual solvent analysis be updated to allow for hydrogen as a carrier gas. The similarity between He and H2 observed within this study supports the use of hydrogen as a suitable replacement for helium, and an update of the EU and USP compendia for residual solvent analysis should be made to reflect this.

Supercritical fluid chromatography-photodiode array detection-electrospray ionization mass spectrometry as a framework for impurity fate mapping in the development and manufacture of drug substances.

Impurity fate and purge studies are critical in order to establish an effective impurity control strategy for approval of the commercial filing application of new medicines. Reversed phase liquid chromatography-diode array-mass spectrometry (RPLC-DAD-MS) has traditionally been the preferred tool for impurity fate mapping. However, separation of some reaction mixtures by LC can be very problematic requiring combination LC-UV for area % analysis and a different LC-MS method for peak identification. In addition, some synthetic intermediates might be chemically susceptible to the aqueous conditions used in RPLC separations. In this study, the use of supercritical fluid chromatography-photodiode array-electrospray ionization mass spectrometry (SFC-PDA-ESIMS) for fate and purge of two specified impurities in the 1-uridine starting material from the synthesis of a bis-piv 2'keto-uridine, an intermediate in the synthesis of uprifosbuvir, a treatment under investigation for chronic hepatitis C infection. Readily available SFC instrumentation with a Chiralpak IC column (4.6 × 150 mm, 3 µm) and ethanol: carbon dioxide based mobile phase eluent enabled the separation of closely related components from complex reaction mixtures where RLPC failed to deliver optimal chromatographic performance. These results illustrate how SFC combined with PDA and ESI-MS detection can become a powerful tool for direct impurity fate mapping across multiple reaction steps.

Generic gas chromatography-flame ionization detection method for quantitation of volatile amines in pharmaceutical drugs and synthetic intermediates.

Volatile amines are among the most frequently used chemicals in organic and pharmaceutical chemistry. Synthetic route optimization often involves the evaluation of several different amines requiring the development and validation of analytical methods for quantitation of residual amine levels. Herein, a simple and fast generic GC-FID method on an Agilent J&W CP-Volamine capillary column (using either He or H2 as the carrier gas) capable of separating over 25 volatile amines and other basic polar species commonly used in pharmaceutical chemistry workflows is described. This 16min method is successfully applied to the analysis and quantitation of volatile amines in a variety of pharmaceutically-related drugs and synthetic intermediates. Method validation experiments showed excellent analytical performance in linearity, recovery, repeatability, and limit of quantitation and detection. In addition, diverse examples for the application of this method to the simultaneous determination of other amine-related chemicals in reaction mixtures are illustrated, thereby indicating that these GC-FID method conditions can be effectively used as starting point during method development for the analysis of other basic polar species beyond the validated list of amines described in this study.

Ultrafast chiral separations for high throughput enantiopurity analysis.

Recent developments in fast chromatographic enantioseparations now make high throughput analysis of enantiopurity on the order of a few seconds achievable. Nevertheless, routine chromatographic determinations of enantiopurity to support stereochemical investigations in pharmaceutical research and development, synthetic chemistry and bioanalysis are still typically performed on the 5-20 min timescale, with many practitioners believing that sub-minute enantioseparations are not representative of the molecules encountered in day to day research. In this study we develop ultrafast chromatographic enantioseparations for a variety of pharmaceutically-related drugs and intermediates, showing that sub-minute resolutions are now possible in the vast majority of cases by both supercritical fluid chromatography (SFC) and reversed phase liquid chromatography (RP-LC). Examples are provided illustrating how such methods can be routinely developed and used for ultrafast high throughput analysis to support enantioselective synthesis investigations.

Covalent functionalization of silica surface using "inert" poly(dimethylsiloxanes).

Methyl-terminated poly(dimethylsiloxanes) (PDMSs) are typically considered to be inert and not suitable for surface functionalization reactions because of the absence of readily hydrolyzable groups. Nevertheless, these siloxanes do react with silica and other oxides, producing chemically grafted organic surfaces. Known since the 1970s and then forgotten and recently rediscovered, this reaction provides a versatile yet simple method for the covalent functionalization of inorganic surfaces. In this work, we have explored the reactions of linear methyl-terminated and cyclic PDMS and bis-fluoroalkyl disiloxanes for the surface functionalization of mesoporous silica (Dpore ≈ 30-35 nm). The optimal reaction conditions included 24 h of contact of neat siloxane liquids and silica at 120-250 °C (depending on the siloxane). A study of the reactions of silicas with different extents of hydration demonstrated the critical role of water in facilitating the grafting of the siloxanes. The proposed reaction mechanism involved the hydrolysis of the adsorbed siloxanes by the Lewis acidic centers (presumably formed by water adsorbed onto surface defects) followed by the coupling of silanols to the surface to produce grafted siloxanes. For rigorously dehydrated silicas (calcination ∼1000 °C), an alternative pathway that did not require water and involved the reaction of the siloxanes with the strained siloxane rings was also plausible. According to FTIR and chemical analysis, the reactions of bis-fluoroalkyl disiloxanes and cyclic PDMS (D3-D5) produced covalently-attached monolayer surfaces, and the reactions of high-MM methyl-terminated PDMS produced polymeric grafted silicas with a PDMS mass content of up to 50%. As evidenced by the high contact angles of ∼130°/100° (adv/rec) and the negligible amount of water adsorption over the entire range of relative pressures, including saturation (p/p0 → 1), the siloxane-grafted porous silicas show uniform, high-quality hydrophobic surfaces. An overall comparison of siloxanes with classical silane coupling agents (i.e., silanes with readily hydrolyzable functionalities such as chloro, amino, etc.) demonstrated that the reactions of siloxanes produced surfaces of similar quality and, although requiring higher temperatures, used noncorrosive, less hazardous reagents, thereby providing an environmentally benign alternative to the chemical functionalization of metal oxide surfaces.

Development of an automated system for preparation of liquid scintillation counting samples for radiolabeled pharmaceuticals.

Radiolabeled compounds are essential tools in drug development used to obtain critical metabolism and safety information. To support the synthesis and ensure quality of radiolabeled compounds for all programs, bench automation has been implemented in our laboratories. The concept of a platform technology for bench-top automation is not new. A considerable investment in the automation of various critical analytical laboratory workflows to both harmonize the efforts of a large and diverse global organization and minimize capital footprint has been made on our part. Various custom automation techniques and applications have been developed to increase capabilities and productivity of radiochemical analyses at Merck. In this paper, we will present a novel system that is capable of automating the liquid scintillation counting procedure. The system has handled multiple radiolabeled ((3)H, (14)C, and (35)S) pharmaceutical compounds with an accuracy of 5% with a standard deviation of 2% and a cycle time of ~10 min per analysis.

Adsorption and wetting characterization of hydrophobic SBA-15 silicas.

This work describes adsorption and wetting characterization of hydrophobic ordered mesoporous silicas (OMSs) with the SBA-15 motif. Three synthetic approaches to prepare hydrophobic SBA-15 silicas were explored: grafting with (1) covalently-attached monolayers (CAMs) of C(n)H(2)(n+1)Si(CH(3))(2)N(CH(3))(2), (2) self-assembled monolayers (SAMs) of C(n)H(2)(n+1)Si(OEt)(3), and (3) direct ("one-pot") co-condensation of TEOS with C(n)H(2)(n+1)Si(OEt)(3) in presence of P123 (n=1-18). The materials prepared were characterized by nitrogen adsorption, TEM, and chemical analysis. The surface properties of the materials were assessed by water contact angles (CAs) and by BET C constants. The results showed that, while loadings of the alkyl groups (%C) were comparable, the surface properties and pore ordering of the materials prepared through different methods were quite different. The best quality hydrophobic surfaces were prepared for SBA-15 grafted with CAMs of alkylsilanes. For these materials, the water CAs were above ∼120°/100° (adv/rec) and BET C constants were in the range of ∼15-25, indicating uniform low-energy surfaces of closely packed alkyl groups on external and internal surfaces of the pores respectively. Moreover, surfaces grafted with the long-chained (C(12)-C(18)) silanes showed super-hydrophobic behavior (CAs∼150-180°) and extremely low adhesion for water. The pore uniformity of parental SBA-15 was largely preserved and the pore volume and pore diameter were consistent with the formation of a single layer of alkylsilyl groups inside the pores. Post-synthesis grafting of SBA-15 with SAMs worked not as well as CAMs: the surfaces prepared demonstrated lower water CAs and higher BET C constants, thereby indicating a small amount of accessible polar groups (Si-OH) related to packing constrains for SAMs supported on highly curved surfaces of mesopores. The co-condensation method produced substantially more disordered materials and less hydrophobic surfaces than any of the grafting methods. The surfaces of these materials showed low water CAs and high BET C constants (∼100-200) thereby demonstrating a non-uniform surface coverage and presence of unmodified silica. It is concluded that CAMs chemistry is the most efficient approach in preparation of the functionalized OMS materials with uniform surfaces and pores.

Microscale HPLC enables a new paradigm for commercialization of complex chiral stationary phases.

The small column size (0.3 mm i.d. x 15 cm) used in microscale HPLC contains only a small fraction (<1%) of the chromatographic packing material of a typical analytical HPLC column. Consequently, chromatographic stationary phases that are prohibitively expensive in conventional HPLC, owing either to synthetic complexity or costly starting materials, may become commercially viable in the microscale format. To illustrate this point, a previously described, synthetically complex, crown ether chiral stationary phase was prepared and evaluated in the microscale format, showing excellent separation of the enantiomers of underivatized amine analytes.