Synthesis Of Active Pharmaceutical Ingredients Research Articles

Modification of B-Nor Steroids Mediated by Filamentous Fungus Fusarium culmorum: Focus on 15α-Hydroxylase Activity

The metabolic activities of microorganisms to modify the chemical structures of organic compounds are an effective tool for the production of high-value steroidal drugs or active pharmaceutical ingredients (APIs). The integration of biotransformation into the synthesis of APIs can greatly reduce the number of reaction steps and achieve higher process efficiency, thus enabling their greener production. The current research efforts are focused on either the optimization of existing processes or identification of new potentially useful bioconversions. This study aimed to assess the catalytic abilities of the filamentous fungus Fusarium culmorum AM 282 to transform B-nor analogues (5(6→7)abeo compounds) of steroid hormones: androstenedione (AD), dehydroepiandrosterone (DHEA) and its acetate. Our previous studies have demonstrated that this strain is an active hydroxylating catalyst for many steroidal compounds with diverse structures. The results presented in this work showed that the hydroxylation of B-nor steroids occurred with the regio- and stereoselectivity typical of this strain in relation to the corresponding natural hormones of the standard 6:6 A/B series. After the transformations of B-nor-DHEA and its acetate, 15α-hydroxy-B-nor-DHEA was obtained as the sole product of the reaction, while the transformation of the AD analogue resulted in a mixture of its 15α- and 6α-hydroxy derivatives. A detailed analysis of the transformation course indicated that all the obtained hydroxy derivatives could be the result of the activity of the same enzyme. The presented results may provide a basis for research aimed at understanding the molecular nature of cytochrome P-450 monooxygenase from F. culmorum AM 282 with its ability for 15α-hydroxylation of steroidal compounds. An analysis of the pharmacokinetic and pharmacodynamic properties of the obtained metabolites with cheminformatics tools suggests their potential biological activity.

Pd-Catalyzed Borylation in Water and Its Application to the Synthesis of Active Pharmaceutical Ingredient (API) Intermediates.

An efficient catalytic borylation reaction of aryl bromides in water based on Pd catalysis under micellar conditions is presented. The peculiar combination of the proper Pd precursor with a Sphos ligand and a hindered lipophilic base ensures good yields in the synthesis of a wide range of boronic esters, even for heteroaryl derivatives with a good purity profile. The method is specifically developed for the in situ preparation of boronic esters that are directly converted into examples of relevant active pharmaceutical ingredient intermediates through cross-coupling reactions or via oxidation to phenols.

Candidaantarctica Lipase B mediated kinetic resolution: A sustainable method for chiral synthesis of antiproliferative β-lactams

Biocatalysis is a valuable industrial approach in active pharmaceutical ingredient (API) manufacturing for asymmetric induction and synthesis of chiral APIs. Herein, we investigated synthesis of a panel of microtubule-destabilising antiproliferative β-lactam enantiomers employing a commercially available immobilised Candida antarctica lipase B enzyme together with methanol and MTBE. The β-lactam ring remained intact during chiral kinetic resolution reactions, plausibly due to a bulky N-1 phenyl substituent on the β-lactam ring substrate. The predominant reaction mediated by CAL-B was methanol catalysed conversion of the β-lactam 3-acetoxy substituent to a 3-hydroxyl group, with preferential methanolysis of the 3S, 4S enantiomer. The unreacted substrate underwent progressive enantioenrichment to the 3R, 4R enantiomer. Substitution patterns on the B ring C3 meta position of the β-lactam scaffold greatly affected the rate of reaction. Halo substituents (fluoro-, chloro- and bromo-) reduced the rate of conversion compared to unsubstituted analogues, which in turn increased enantiomeric excess (ee). Ee values up to 86 % for the 3S, 4S 3-hydroxyl enantiomer were achieved. A double resolution approach for unreacted substrate yielded high ee values (>99 %) for the 3R, 4R 3-acetoxy enantiomer. CAL-B mediated methanolysis is a more sustainable method for resolution of racemic antiproliferative β-lactams compared to a previous technique of chiral diastereomeric resolution. Yields of β-lactams obtained using CAL-B are far superior than previously described, which will facilitate progression toward pre-clinical and clinical development. Biocatalysis is a useful tool in the toolbox of the medicinal chemist.

Advantages and drawbacks of life cycle assessment application to the pharmaceuticals: a short critical literature review.

Pharmaceuticals are among the most challenging products to assess by life cycle assessment (LCA). The main drawback highlighted by LCA practitioners is the lack of inventory data, both regarding the synthesis of active pharmaceutical ingredient (API) precursors (upstream) and the details concerning the downstream phases (use and end of life). A short critical review of pharma-LCAs found in the literature is here proposed, with discussion of several tools and models used to predict the environmental impacts derived from the life cycle of pharmaceuticals, emphasizing current strengths and weaknesses, and exploring the possibilities for improvements. The case of antibiotics is selected as a representative class of pharmaceuticals, due to their massive use worldwide and the growing related issue of antimicrobial resistance enrichment, which is generally not included in most of LCAs. Also, we comment on drafting product category rules (PCRs) in the relevant field to develop standard methodologies and enhance the comparability of the studies, ultimately advocating collaboration with companies and improving inventory data quality and availability for the whole value chain of products.

Copper-Catalyzed Chemoselective Nitro Reduction

AbstractThe reduction of nitro compounds into the highly valuable anilines is reported using a Cu catalyst and B2Pin2. The reactions proceed under very mild conditions and showcase excellent functional group tolerance. This method is applied to a large panel of nitro derivatives, including biorelevant molecules and important synthetic intermediates, toward the synthesis of active pharmaceutical ingredients (APIs). This novel reaction manifold intends to provide a complementary approach to the existing portfolio of nitro-reduction methods.

Diaminated Cellulose Beads as a Sustainable Support for Industrially Relevant Lipases.

Environmentally persistent polystyrene or polyacrylic beads are used as supports in enzyme large-scale bioprocesses, including conversion glucose isomerization for high-fructose corn syrup production, hydrolysis of lactose, and synthesis of active pharmaceutical ingredients. In this paper, we report the development of a novel sustainable and scalable method to produce diaminated cellulose beads (DAB) as highly efficient alternative supports for industrially relevant lipases. Regenerated cellulose beads were grafted with diaminated aliphatic hydrocarbons via periodate oxidation and reductive amination. The oxidation step indicated that aldehyde content can be easily tuned through the reaction time and concentration of reactants. Reductive amination of dialdehyde cellulose was more efficient as the length of the diaminated hydrocarbon compound increased. Morphological analysis of DAB showed that cellulose chemical grafting enabled the preservation of the bead shape and internal structure upon freeze-drying. Enzymatic degradability studies demonstrated that chemical functionalization did not undermine enzyme cellulose hydrolysis. The addition of aminated moieties on cellulose dramatically increased absorption efficiency for all industrially relevant lipases used, reaching 100% for Thermomyces lanuginosus lipase (TLL). Storage and recyclability experiments demonstrated that enzymes were retained and recyclable for at least nine cycles, although the activity gradually declined after each cycle. Medium chain triacylglycerol hydrolysis in a SpinChem reactor using TLL immobilized on 1,6 DAB exhibited higher activity compared to acrylic beads (588 vs 459 U/g) suggesting that biodegradable cellulose-based materials could be a valid and attractive alternative to plastics carriers.

Process intensification in flow chemistry using micro and millidevices: Numerical simulations of the syntheses of three different intermediate API compounds for diabetes mellitus drug production

Microreactor Technology (MRT) appears as an interesting alternative for process intensification, including the development of continuous synthesis of fine chemicals and Active Pharmaceutical Ingredients (API). The inherent proposal of MRT is to reduce the scale-up time by simply increasing the number of microreactors in parallel, the numbering-up concept using optimal designed micro or millidevices. Among the API, rosiglitazone and lobeglitazone are derivatives of thiazolidine-2,4-dione (TZD), which is used as a building block, for molecules with biological activity, sensitive to insulin and, thus, with its administration the body makes better use of the insulin it produces, being an important structural element in medicinal chemistry, especially for type 2 diabetes mellitus, one of the biggest concerns in the health area worldwide, affecting nowadays more than 180 million people. The numerical assessment of MRT for various API synthesis related to type 2 diabetes mellitus drugs indicates its comparable or superior performance compared to traditional batch process. Specifically, proposed micromixer designs facilitate successful scale-up to milliscale while maintaining microscale performance, with significantly lower pressure drops than commercial Asia microreactors. The proposed flow distributors offer good performance in ensuring uniform distribution across outlets due to their conical shape design and laminar flow regime, with low pressure drops. The flow distributor with eights outlet is preferred for higher flow rates due to its superior performance, minimizing the pressure drops. Efficient flow rate and molar concentration control at each reactor unit can be ensured by using one distributor for each reactant's feed stream. This approach guarantees accurate concentration, flow rate control and ideal residence time, ultimately facilitating higher total throughput for scale-up. The potential use of micromixer actuators lies in its ability to maintain or enhance performance while transitioning from micro to milli-scale, offering promising alternatives for process intensification and scale-up in pharmaceutical synthesis, particularly for medications targeting type 2 diabetes mellitus.

Chemo-Enzymatic Strategy for the Efficient Synthesis of Steroidal Drugs with 10α-Methyl Group and a Side Chain at C17-Position from Biorenewable Phytosterols.

Steroidal pharmaceuticals with a 10α-methyl group or without the methyl group at C10-position are important medicines, but their synthesis is quite challenging, due to that the natural steroidal starting materials usually have a 10β-methyl group which is difficult to be inverted to 10α-methyl group. In this study, 3-((1R,3aS,4S,7aR)-1-((S)-1-hydroxypropan-2-yl)-7a-methyl-5-oxooctahydro-1H-inden-4-yl) propanoic acid (HIP-IPA, 2e) was demonstrated as a valuable intermediate for the synthesis of this kind of active pharmaceutical ingredients (APIs) with a side chain at C17-position. Knockout of a β-hydroxyacyl-CoA dehydrogenase gene and introduction of a sterol aldolase gene into the genetically modified strains of Mycobacterium fortuitum (ATCC 6841) resulted in strains N13Δhsd4AΩthl and N33Δhsd4AΩthl, respectively. Both strains transformed phytosterols into 2e. Compound 2e was produced in 62% isolated yield (25 g) using strain N13Δhsd4AΩthl, and further converted to (3S,3aS,9aS,9bS)-3-acetyl-3a,6-dimethyl-1,2,3,3a,4,5,8,9,9a,9b-decahydro-7H-cyclopenta[a]naphthalen-7-one, which is the key intermediate for the synthesis of dydrogesterone. This study not only overcomes a challenging synthetic problem by enabling an efficient synthesis of dydrogesterone-like steroidal APIs from phytosterols, the well-recognized cheap and readily available biobased raw materials, but also provides insights for redesigning the metabolic pathway of phytosterols to produce other new compounds of relevance to the steroidal pharmaceutical industry.

Electric-field-assisted in situ separation of mandelic acid in an enzymatic milli-reactor improves the control of reaction conversion and enhances the enantiomeric ratio

Enantiopure or enantiomerically enriched chemicals are often used as active pharmaceutical ingredients (APIs). However, API synthesis and purification on a preparative scale is usually costly, time-consuming, and difficult to scale up in conventional batch reactors. To develop an alternative solution, we applied kinetic enantioselective synthesis with simultaneous product separation. More specifically, we constructed a continuously operated packed-bed milli-reactor (PBR) with an orthogonally applied electric field for in situ separation. We tested our concept in the enzymatic production of mandelic acid (MA) enantiomers from racemic methyl mandelate (rMM) by immobilised Aspergillus niger lipase. The electric field selectively extracted the product (mandelate anions) whilst leaving almost 100 % of the electroneutral substrate in the reaction mixture. Such selective product separation significantly enhanced MA formation by shifting the reaction toward product synthesis. The applied electric current efficiently controlled the overall rMM conversion, reaching more than 90 % compared to 30% without current. We identified the electric current providing the highest (R)-(-)-MA to (S)-(+)-MA enantiomeric ratio equals to 5.20. This ratio was above 3.86 achieved in an ordinary PBR, albeit subject to further optimization. Our PBR with in situ product separation provided superior process control and significantly enhanced enantiomerically enriched chemical synthesis.

Deep Eutectic Solvents as Catalysts in the Synthesis of Active Pharmaceutical Ingredients and Precursors

Deep Eutectic Solvents (DESs) have appeared in recent years as an appealing alternative to classical organic solvents, due to their valuable environmental properties. In addition, these compounds, formed by the combination of one hydrogen bond donor with a hydrogen bond acceptor at a defined stoichiometric ratio, present other valuable activities not only as a reaction medium. DESs can also be employed as catalysts through hydrogen-bond interactions in different chemical transformations, thus substituting hazardous reagents and solvents. The search for novel and more environmentally friendly catalysts is an area of interest of pharmaceutical chemists, and therefore, the efforts made in the application of DESs as catalysts in the synthesis of APIs or its precursors are described, focusing mainly on condensations, nucleophilic additions to carbonyl moieties, and multicomponent reactions.

Biocatalysis for the Synthesis of Active Pharmaceutical Ingredients in Deep Eutectic Solvents: State-of-the-Art and Prospects

Biocatalysis holds immense potential for pharmaceutical development as it enables synthetic routes to various chiral building blocks with unparalleled selectivity. Therein, solvent and water use account for a large contribution to the environmental impact of the reactions. In the spirit of Green Chemistry, a transition from traditional highly diluted aqueous systems to intensified non-aqueous media to overcome limitations (e.g., water shortages, recalcitrant wastewater treatments, and low substrate loadings) has been observed. Benefiting from the spectacular advances in various enzyme stabilization techniques, a plethora of biotransformations in non-conventional media have been established. Deep eutectic solvents (DESs) emerge as a sort of (potentially) greener non-aqueous medium with increasing use in biocatalysis. This review discusses the state-of-the-art of biotransformations in DESs with a focus on biocatalytic pathways for the synthesis of active pharmaceutical ingredients (APIs). Representative examples of different enzyme classes are discussed, together with a critical vision of the limitations and discussing prospects of using DESs for biocatalysis.

Continuous Manufacturing of Active Pharmaceutical Ingredients: Current Trends and Perspectives

AbstractThe pharmaceutical industry has traditionally employed batch processing as a means of synthesizing active pharmaceutical ingredients (APIs) on a commercial scale. Batch manufacturing enables API synthesis in large quantities in order to meet regulations. Yet, this strategy remains cumbersome, is labor‐intensive, and creates complex logistical networks. In recent decades, batch API processing has gradually eroded American economic competitiveness and has led to the offshoring of API manufacturing from the United States. Today it is estimated that +80% of APIs are manufactured overseas.[1] Meanwhile, disruptions from the Coronavirus Disease (SARS‐CoV‐2) have exacerbated weaknesses in the global supply chain, culminating with widespread shortages of critical life‐saving drugs.[2] These shortages have emphasized the importance of reshoring drug manufacturing to the U.S. and fostered a regulatory landscape that seeks advanced methods of API manufacturing in order to bolster America's supply chain resiliency.[3] Recently, increased attention has been directed towards continuous drug manufacturing to enable nonstop API production within modular stand‐alone flow systems. Continuous manufacturing (CM) facilitates modular automation, offers new avenues towards process intensification, and even enables in‐line analytical monitoring from raw materials to packaged products. This strategy offers better conversion, increased safety, reduced waste, and overall cost savings versus traditional batch‐style processing. The rise of advanced API manufacturing, coupled with the current regulatory landscape, offers a rare window of opportunity to convert traditional batch pharmaceutical processes into continuous, streamlined production systems to on‐shore API manufacturing and eliminate drug shortages. This review will outline the current state‐of‐the‐art in the CM of APIs and will highlight the translation of chemistry and unit operations from batch processes to modular CM systems.

β-(2R,3S,5R)-2-(hydroxymethyl)-6-(5-phenyl-<i>2Н</i>-tetrazole-2-yl) tetrahydro-<i>2Н</i>-piran-3,4,5-triol. Synthesis and computer prediction of biological activity

Background. An urgent problem of creating a domestic production of modern medicines under the conditions of sanctions pressure is the molecular design and development of rational methods for the synthesis of active pharmaceutical ingredients of original drugs and young generics.Objective. In this work, a computer prediction of the biological activity of β-(2R,3S,5R)-2-(hydroxymethyl)-6-(5-phenyl-2H-tetrazole-2-yl) tetrahydro-2H- piran-3,4,5-triol 1 — compounds, in the molecule of which there are two promising pharmacophore fragments, was performed: tetrazolyl and galactopyranosyl. Both fragments of the studied molecule are actively used by leading scientific centers for the molecular design and synthesis of promising active pharmaceutical ingredients (API) of modern medicines.Design and methods. For this purpose, the latest versions of the PASS computer complex were used in combination with molecular docking and scoring in the AutoDoc Vina program.Results. Based on computer prediction data, it is shown that this compound may have multi-target biological activity.Conclusion. A rational, scalable method for the synthesis of compound 1, which is recommended for testing in vitro, in vivo activity, is proposed.

Preparation of Intermediates for Organic Synthsis

Several synthetic methods are described for the preparation of compounds such as derivatives of natural amino acids, unnatural amino acids, amino esters, oxime esters and amino amides that may serve as intermediates in the synthesis of active pharmaceutical ingredients (APIs) or drugs. The compounds described herein are produced in high yield and purity and the synthetic procedures are detailed along with the NMR spectra of the compounds and useful chemical data relating to these compounds.

A Commentary on Co-Processed API as a Promising Approach to Improve Sustainability for the Pharmaceutical Industry

Pharmaceutical products represent a meaningful target for sustainability improvement and emissions reduction. It is proposed here that rethinking the standard, and often linear, approach to the synthesis of Active Pharmaceutical Ingredients (API) and subsequent formulation and drug product processing will deliver transformational sustainability opportunities. The greatest potential arguably involves API that have challenging physico-chemical properties. These can require the addition of excipients that can significantly exceed the weight of the API in the final dosage unit, require multiple manufacturing steps to achieve materials amenable to delivering final dosage units, and need highly protective packaging for final product stability.Co-processed API are defined as materials generated via addition of non-covalently bonded, non-active components during drug substance manufacturing steps, differing from salts, solvates and co-crystals. They are an impactful example of provocative re-thinking of historical regulatory and quality precedents, blurring drug substance and drug product operations, with sustainability opportunities. Successful examples utilizing co-processed API can modify properties with use of less excipient, while simultaneously reducing processing requirements by delivering material amenable to continuous manufacturing. There are also opportunities for co-processed API to reduce the need for highly protective packaging. This commentary will detail the array of sustainability impacts that can be delivered, inclusive of business, regulatory, and quality considerations, with discussion on potential routes to more comprehensively commercialize co-processed API technologies.

Absolute configuration assignment of highly fluorinated carboxylic acids via VCD and MRR spectroscopy

Chiral analysis has become a crucial step in studying the stereospecific synthesis of Active Pharmaceutical Ingredients (APIs). Both Vibrational Circular Dichroism (VCD) and Molecular Rotational Resonance (MRR) spectroscopy are capable of determining absolute configurations (ACs) via comparison of experimental and calculated data. In this regard, each technique has its own caveats. In VCD analysis, accurate prediction of the normal modes as well as rigorous conformational searches of both the analyte and potential (self-)aggregation products are required to optimally match experimental spectra. In MRR analysis, chiral species are resolved through complexation with a chiral tag to prepare spectrally distinct diastereomeric complexes. Although individual complex isomers can be distinguished, spectral assignments need to be matched to unique isomer geometries for unambiguous AC assignment. In this work, the ACs of two highly fluorinated carboxylic acids were successfully assigned using VCD and MRR spectroscopy. In the VCD analysis, the M06-2X functional was demonstrated to be superior to B3LYP and B3LYP-GD3 in accurately predicting the C–F normal modes and both monomeric and dimeric spectral contributions were observed. In a similar analysis with broadband MRR, most experimentally identified geometries had more than one possible computational match. Nevertheless, careful consideration of the chiral tag, as well as additional isomer assignments, resulted in successful assignment of the AC. This comparative study demonstrates the power of contemporary VCD analysis and the unique contributions of MRR to the analytical toolbox.

Continuous Synthesis of Cinnarizine Salt with Malic Acid by Applying Green Chemistry Using Water-Assisted Twin Screw Extrusion.

Organic solvent-free process or green chemistry is needed for manufacturing pharmaceutical salts to avoid various environmental, safety, and manufacturing cost issues involved. In this study, a cinnarizine (CNZ) salt with malic acid at a 1:1 molar ratio was successfully prepared by twin screw extrusion (TSE) with water assistance. The feasibility of salt formation was first evaluated by screening several carboxylic acids by neat grinding (NG) and liquid-assisted grinding (LAG) using a mortar and pestle, which indicated that malic acid and succinic acid could form salts with CNZ. Further studies on salt formation were conducted using malic acid. The examination by hot-stage microscopy revealed that the addition of water could facilitate the formation and crystallization of CNZ-malic acid salt even though CNZ is poorly water-soluble. The feasibility of salt formation was confirmed by determining the pH-solubility relationship between CNZ and malic acid, where a pHmax of 2.7 and a salt solubility of 2.47 mg/mL were observed. Authentic salt crystals were prepared by solution crystallization from organic solvents for examining crystal properties and structure by differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD), Fourier transform infrared (FTIR) spectroscopy, solid-state 13C and 15N nuclear magnetic resonance (NMR), and single-crystal X-ray diffraction (SXD). These techniques also established that a salt, and not a cocrystal, was indeed formed. The CNZ salt crystals were then prepared by TSE of a 1:1 CNZ-malic acid mixture, where the addition of small amounts of water resulted in a complete conversion of the mixture into the salt form. The salts prepared by solvent crystallization and water-assisted TSE had identical properties, and their moisture sorption profiles were also similar, indicating that TSE is a viable method for salt preparation by green chemistry. Since TSE can be conducted in a continuous manner, the results of the present investigation, if combined with other continuous processes, suggest the possibility of continuous manufacturing of drug products from the synthesis of active pharmaceutical ingredients (APIs) to the production of final dosage forms.

Green Oxidative Catalytic Processes for the Preparation of APIs and Precursors

Asymmetric oxidation processes have constituted a valuable tool for the synthesis of active pharmaceutical ingredients (APIs), especially for the preparation of optically active sulfoxides, compounds with interesting biological properties. Classical approaches for these oxidative procedures usually require the application of non-sustainable conditions that employ hazardous reagents and solvents. In the last decades, chemists have tried to combine the preparation of valuable compounds of high yields and selectivities with the development of more sustainable protocols. To achieve this objective, greener solvents, reagents, and catalysts are employed, together with the use of novel chemical techniques such as flow catalysis or photocatalysis. The last efforts in the development of greener approaches for the preparation of APIs and their intermediates using oxidative procedure will be reviewed herein. Most of these approaches refer to biocatalytic methods, in which mild reaction conditions and reagents are employed, but other novel techniques such as photocatalysis will be described.

Compact capillary high performance liquid chromatography system for pharmaceutical on-line reaction monitoring.

Due to their size, conventional high performance liquid chromatographs (HPLCs) are difficult to place close to a reaction vessel within a pharmaceutical manufacturing or development site. Typically, long transfer lines are required to move sample from the reactor to the HPLC for analysis and high solvent usage is required. However, herein a compact and modular separation system has been developed to enable co-location of an HPLC with a small-scale reactor for reaction monitoring in the synthesis of active pharmaceutical ingredients. Using a framework based on capillary HPLC, a compact gradient separation system with a fully modular architecture is described. A custom miniature diode-array detector with a linear dynamic range (up to 1500 mAU at 210nm) was integrated and evaluated for on-line reaction monitoring. In evaluating system suitability, average peak area %RSD of <3%, and an average retention time %RSD of <0.7%, were achieved. To demonstrate practical utility, the compact system was coupled directly to an on-line lab-scale flow through reactor for continuous reaction monitoring in the laboratory fume hood, where a study of the 3rd Bourne reaction was used to compare the performance of the compact system with a commercially available process HPLC instrument (Waters PATROL UPLC). Further, 33 off-line samples from a continuous crystallization reactor were analysed and it was found that the developed compact HPLC system showed equivalent quantitative performance to an Agilent 1290 Infinity II HPLC system.

HPLC-UV Method Development and Validation to Monitor Difluprednate Synthesis.

During the synthesis of active pharmaceutical ingredients (APIs) there is a need for the development and validation of a simple and rapid high performance liquid chromatography (HPLC) method for the determination and quantification of the synthesized product and related by-products. An HPLC method gives a better understanding of how a synthesis is proceeding. A rapid and easy to use HPLC-UV (ultraviolet) method for the determination of difluprednate and monitoring of impurities generated during synthesis was developed and validated. A Shimadzu VP Series HPLC equipped with a LabSolutions software and UV detector set at 240nm was used for analysis. The mobile phase consisted of phosphate buffer (pH6) and acetonitrile 50:50 (v/v) and was eluted at a flow rate of 1.2mL/min. Separation took place on a reversed-phase Kinetex C18 column (150×4.60mm; 5μm i.d.). Column temperature was set at 40°C. The developed method was found to have good linearity and acceptable accuracy and precision. The developed method may be effectively applied to determine products and by-products formed during synthetic reactions of steroids and to calculate the yield of the products obtained during each step of the synthesis.