Halo C18 Research Articles

Diatom biosilica for liquid chromatography.

This work presents, for the first time, the preparation method and subsequent use of biosilica in column liquid chromatography in reverse-phase mode. Diatom biosilica consists of the siliceous exoskeletons (frustules) of unicellular algae. Controlled cultivation of Pseudostaurosira trainorii diatoms resulted in frustules with an average diameter of approximately 4 µm, sidewall thickness of 1 µm, and a bottom thickness of 110-150 nm. These frustules contained pores (holes) with diameters ranging from 150 to 300 nm. XRD measurements revealed an opal A silica structure, with some lamellar features, and the material was characterized by a surface area of 21.1 m²/g. The raw material required careful preparation to remove residual organics by heating it. Following this, the surface was modified with octadecyldimethylchlorosilane to create a reverse-phase chromatographic adsorbent. The resulting columns demonstrated good chromatographic performance, with a theoretical plate number (N) of 22,000 plates for alkylbenzenes on a 160 mm long column, and permeability (KF) of 5.33 × 10⁻¹⁵ m². The prepared material exhibited lower hydrophobicity compared to the commercially available HALO C18 stationary phase, which can be attributed to its lower surface area and high number of silanol groups. As a result, only partial separation of six polyaromatic hydrocarbons was achieved due to excessive tailing. However, five anti-inflammatory drugs and two veterinary antibiotics were successfully separated.

Development of a Reversed-Phase UPLC Method for Assay of Fipronil Including Determination of Its Related Substances in Bulk Batches of Fipronil Drug Substance.

Fipronil is a commonly used pesticide in the agricultural and animal health industries for the protection of crops and control of fleas, ticks, and chewing lice. It is difficult to obtain reproducible retention time and relative retention time (RRT) for a common hydrolytic degradation product of fipronil with the current European Pharmacopeia (EP) monograph for assay and estimation of related substances of fipronil. This situation causes misidentification, mislabeling, and/or false out-of-specification results for this hydrolytic degradation product of fipronil in bulk commercial batches during batch release and/or in the stability samples during the shelf life of a released batch. This study aimed to develop a reversed-phase ultra performance liquid chromatography (UPLC) method for assay and identification of fipronil including identification and estimation of its related substances in bulk drug substance batches of fipronil and provide consistent retention time of the hydrolytic degradation product. Fipronil and its related substances were separated by gradient elution on a Halo C18 column (50 mm × 2.1 mm id, 2.0 µm particle size) maintained at 40°C with 0.1% H3PO4 in H2O as mobile phase-A and acetonitrile-methanol (50 + 50, v/v) as mobile phase-B. Fipronil and its related substances were detected and quantified at 280 nm with a quantitation limit of 0.05% of the target (analytical) concentration. The UPLC method was able to separate all analytes of interest by gradient elution with a total run time of 7 min (approximately 40% faster than EP). In this paper, we report the development and validation of a fast, stability-indicating reversed-phase UPLC method for assay and estimation of related substances of fipronil in stability samples and bulk batches of fipronil. The new UPLC method is approximately 40% faster than the current Ph. Eur. monograph for fipronil assay and the new method provides reproducible retention of a common hydrolytic degradation product of fipronil.

Simultaneous determination of praziquantel, fipronil, eprinomectin, (S)-methoprene, their key related substances and butylated hydroxytoluene (BHT) in a topical veterinary drug product by a single stability indicating high performance liquid chromatography method

A simple, robust and QC (quality control) friendly high performance liquid chromatography (HPLC) method was developed and validated for simultaneous determination of four active pharmaceutical ingredient [namely fipronil, (S)-methoprene, eprinomectin, and praziquantel] and their key degradation products in a broad-spectrum topical finished product. Typical sample of the finished product contains a total of 30-plus peaks of interest. Analytes were separated on a HALO C18 column (150 mm × 4.6 mm i.d., 2.7 µm particle size) with a gradient elution at 50 °C column temperature and 0.6 mL/min flow rate. Detection wavelength of 245 nm is used for praziquantel, eprinomectin and their degradation products, 265 nm for (S)-methoprene and its degradation products and 280 nm for fipronil and its degradation products and for the antioxidant, BHT. Mobile phase of the method is composed of 10 mM potassium phosphate buffer and 1,4- Dioxane (98/2, v/v, pH 5.0) as mobile phase-A, and EtOH/MeOH/MeCN/IPA (86/4/6/4, v/v/v/v) as mobile phase-B. All analytes of interest were adequately separated by this single HPLC method. The stability-indicating capability of the method has been demonstrated by successfully separating the degradation products in the stressed degraded samples of the finished product. Limit of quantitation (LOQ) and limit of detection (LOD) of the method is 0.3% and 0.1% of target analytical concentration for each individual API in the finished product. This method has been demonstrated to be sensitive, robust, specific, accurate and stability-indicating for analysis of the topical drug product containing praziquantel, fipronil, eprinomectin and (S)-methoprene.

Unique Quality by Design Approach for Developing HPLC and LC-MS Method for Estimation of Process and Degradation Impurities in Pibrentasvir, Antiviral Agent for Hepatitis C.

Pibrentasvir (PIB) was approved for treating hepatitis C patients. A specific, accurate, linear, robust, and stability-indicating method was developed and validated for determining degradation impurities present in the PIB drug substance by studying the quality by design (QbD) principles. All identified degradation impurities were separated with the stationary phase HALO C18, 150 mm × 4.6 mm, 2.7 μm. Mobile phase A contains pH 2.5 phosphate buffer and acetonitrile in the ratio of (70:30, v/v), and mobile phase B contains water and acetonitrile in the ratio of (30:70, v/v), respectively. The chromatographic conditions were optimized, such as flow rate of 0.8 mL/min, UV detection at 252 nm, injection volume of 20 μL, and column temperature of 40 °C. The proposed method was validated per the current ICH Q2 (R1) guidelines. The recovery study and linearity ranges were established from limit of quantification (LOQ) to 300% optimal concentrations. The method validation results were between 98.6% and 106.2% for recovery, and linearity r 2 was more than 0.999 for all identified impurities. The method precision results achieved below 5% relative standard deviation (RSD). The forced degradation results demonstrated that the drug was sensitive to chemical stress conditions. During the stress study, degrading impurities were identified by the LC-MS technique and the mechanism pathway. A QbD-based experimental design (DoE) approach was used to establish the robustness of the method.

Comprehensive study on degradation profile of firocoxib and structural elucidation of its key degradation products

Firocoxib is widely used in veterinary medicine as a non-steroidal analgesic and anti-inflammatory drug substance. Herein, a comprehensive study on the degradation profile of firocoxib was performed through force degradation studies to understand its degradation profile and characterize its major degradation products (DPs). Firocoxib drug substance was subjected to acidic, alkaline, oxidation (H2O2, KMnO4, and K2Cr2O7), thermal (solid and solution state), and photolytic (solid and solution state) stress degradation, as recommended in the ICH guidelines. Firocoxib and its major DPs were adequately separated by investigational HPLC method, which utilized a HALO C18 (100 × 2.1 mm, 2.0 µm) column. Mobile phase-A for the HPLC method is composed of 0.1% formic acid in water and mobile phase-B acetonitrile. A total of six major DPs were observed for firocoxib drug substance under these stress degradation conditions. Structural elucidation of the DPs performed using liquid chromatography-high resolution mass spectrometry and comparison of their fragmentation profile with that of the parent compound. For further structural confirmation of two major DPs, DP-2 and DP-6 were isolated and purified from the stressed samples using a preparative HPLC and analyzed by comprehensive nuclear magnetic resonance (NMR) spectroscopy studies. Most probable mechanistic pathways for the formation of DPs of firocoxib under various stress degradation conditions were postulated to understand its degradation profile. Based on the results from forced degradation, firocoxib was found to be quite stable under basic and thermal conditions, and somewhat unstable under acidic, oxidative, and photolytic conditions. The results of this study should facilitate quality monitoring and establish a stability profile of firocoxib drug substance and drug products. These results may also assist in the design and development of new formulations made with firocoxib drug substance with desired shelf life.

Structural elucidation of major degradation products of milbemycin oxime drug substance using LC-MS and NMR

Milbemycin oxime (MO) drug substance is a 16-membered macrocyclic lactone that exhibits a broad spectrum of biological activity and high potency towards parasites. In this study, a comprehensive forced degradation study was carried out on MO drug substance to identify and characterize its major degradation products (DPs). MO drug substance was subjected to acid, base, oxidation (H2O2), heat (solid and solution state), and photolytic (solid and solution state) stress degradation as per the ICH guidelines. Chromatographic separation of the drug substance (MO A3 and MO A4) and its DPs was achieved using a gradient elution on a HALO C18 column (100 × 4.6 mm, 2.7 µm). Mobile phase A consisted of water/acetonitrile (60/40, v/v) and mobile phase B consisted of ethanol/isopropanol (50/50, v/v). A total of twelve major DPs were observed for MO drug substance under various stress conditions. These DPs were further identified and characterized using liquid chromatography-high resolution mass spectrometry and comparison of their fragmentation profile with MO A4 and MO A3 using tandem mass spectrometry. Of these, H2O2 induced oxidative degradation product (3,4-dihydroperoxide MO A4) was isolated using semi-preparative HPLC and characterized by comparison of its nuclear magnetic resonance spectroscopy data with MO A4. The proposed structures of the DPs have been rationalized by appropriate degradation pathways for MO A4 and MO A3.

Identification and characterization of major degradation products of Eprinomectin drug substance including degradation pathways using LC-HRMS and NMR

Eprinomectin (EPM) is a semi-synthetic potent antiparasitic drug widely used in veterinary medicine. In this study, a comprehensive forced degradation study was carried out on EPM drug substance as per ICH guidelines. Generation of adequate quantities of major degradation products of EPM via forced degradation studies was necessary for identification, structure elucidation, and understanding its degradation mechanism and degradation pathways. EPM drug substance was subjected to acid, base, oxidation (H2O2 and K2Cr2O7), thermal (solid and solution state), and photolytic (solid and solution state) stress degradation. The degradation products (DPs) formed in the stressed degraded samples were successfully separated using a gradient elution on a HALO C18 column (100 × 4.6 mm, 2.7 µm). Mobile phase A consisted of water and mobile phase B consisted of ethanol/isopropanol (98/2, v/v). A total of six major DPs of EPM drug substance formed under various stress conditions. The chemical structures of DPs were determined using liquid chromatography-high resolution mass spectrometry (LC-HRMS) and characterized through comparison of their fragmentation profile with EPM B1a using tandem mass spectrometry (MS/MS). Additionally, two solvates (methanol adduct B1a #1 and methanol adduct B1a #2) were observed during the acid-stressed degradation study of EPM in presence of methanol. To confirm the chemical structure, these products were isolated with semi-preparative HPLC and characterized by using a combination of LC-MS/MS and nuclear magnetic resonance spectroscopy. The elucidated chemical structure of the degradation products of EPM was also justified through mechanistic explanations. Identification and characterization of the DPs including degradation mechanism(s) of EPM should facilitate the understanding of the stability behavior of EPM drug substances as well as aid in the design of new formulations made with EPM.

A comprehensive study to identify and characterize major degradation products of Ivermectin drug substance including its degradation pathways using LC-HRMS and NMR

Ivermectin (IVM) drug substance is a semi-synthetic macrocyclic lactone that exhibits a broad spectrum of activity and high potency towards endo- and ectoparasites. In this study, a comprehensive forced degradation study was carried out on IVM drug substance (under the conditions recommended in the ICH guidelines) to identify and characterize its major degradation products (DPs). IVM drug substance was subjected to acidic, alkaline, oxidation (H2O2 and K2Cr2O7), thermal (solid and solution state), and photolytic (solid and solution state) stress degradations. Chromatographic separation of the drug substance and its DPs was achieved using a gradient elution on a HALO C18 column (150 × 4.6 mm, 2.7 µm). A total of five major DPs were observed for IVM drug substance under various stressed conditions. Additionally, ivermectin API lots exhibited instability when stored under room temperature and at 45% relative humidity for two years. These DPs were identified and characterized using liquid chromatography-high resolution mass spectrometry (LC-HRMS) and a comparison of their fragmentation profile with IVM H2B1a using tandem mass spectrometry. Of these, H2O2 induced oxidative degradation product (3,4-epoxide H2B1a) was isolated using semi-preparative HPLC and its structure was elucidated comprehensively using LC-HRMS and nuclear magnetic resonance spectroscopy. The proposed structures of the DPs have been rationalized by appropriate degradation pathways of IVM H2B1a. Comprehensive degradation profile of IVM drug substance should facilitate the understanding of the stability profile of IVM drug substance, setting the specification of DPs in finished products as well as aid in the design of generic formulation made with IVM.

A Novel, Simple Rapid Reverse-Phase HPLC-DAD Analysis, for the Simultaneous Determination of Phenolic Compounds and Abscisic Acid Commonly Found in Foodstuff and Beverages.

A novel, simple, rapid, 7-minute HPLC-DAD method for the determination of 10 phenolic compounds and abscisic acid commonly found in teas, wines, fruit and honey was successfully developed and validated according to the International Council of Harmonization (ICH) guidelines. This reverse-phase (RP) HPLC-DAD method boasts rapid separation and excellent resolution while introducing green chemistry techniques. The Agilent 1200 series diode array detector SL coupled with a reverse-phase Advanced Materials Technology Halo C18 column (100 × 3.0mm i.d., 2.7μm) contributed to the rapid analyses. This, together with a 0.1% formic acid in water (v/v) and methanol mobile phase, a flow rate of 0.8mL/min and the utilization of a meticulous gradient elution resulted in a validated method for the determination of 10 phenolic compounds and abscisic acid commonly found in various foodstuffs. The resulting method proved to be rapid, accurate, precise and linear with sensitive detection limits from 0.025μg/mL to 0.500μg/mL and percentage recoveries of 98.07%-101.94%. Phenolic compounds have been acknowledged throughout literature for their therapeutic properties, interalia, antioxidant, anti-inflammatory and antiaging due to free radical scavenging potentials. However, resulting analysis, can be frequently complicated and long and very often discounts green chemistry techniques. The developed and validated method successfully and rapidly analyses, gallic acid, caffeic acid, trans-p-coumaric acid, rutin, myricetin, abscisic acid, trans-cinnamic acid, quercetin, luteolin, kaempferol and chrysin with excellent resolution and precision.

Development and Validation of a Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) Method for Identification, Assay and Estimation of Related Substances of Ivermectin in Bulk Drug Batches of Ivermectin Drug Substance.

A reversed-phase high-performance liquid chromatography (RP-HPLC) method has been developed and validated for the identification and assay of Ivermectin, including the identification and estimation of its process-related impurities and degradation products in bulk drug substance of Ivermectin. Analytes were separated on a HALO C18 column (100mm × 4.6mm I.D., 2.7μm particle size) maintained at 40 °C (column temperature) with gradient elution. All analytes of interests were adequately separated within 25min. All degradation products, process-related impurities and assay were monitored by ultraviolet detection at 254nm. The new HPLC method described here successfully separated an isomer peak of the active pharmaceutical ingredient (API) from the major API peak. This newly separated isomer peak is around 1.2 to 1.5% (peak area) in typical API samples, and coelutes with the major API peak by all current HPLC methods. Quantitation limit of the HPLC method is 0.1% of target analytical concentration (~1.0μg/mL). This method has been demonstrated to be accurate, robust, significantly higher degree of selectivity compared to the HPLC methods of Ivermectin drug substance reported in the literature and in the compendial HPLC methods prescribed in the current USA and European Pharmacopeia.

Rapid Nanomolar Detection of Guaiacol from its Precursors Using a Core‐shell Reversed‐phase Column Coupled with a Boron‐doped Diamond Electrode

AbstractThe electrooxidation mechanisms of guaiacol (2‐methoxyphenol), a food and beverage spoilage metabolite, and its precursors; vanillic acid (VA), vanillin, and ferulic acid (FA) were investigated by cyclic voltammetry (CV) with a boron‐doped diamond (BDD) electrode. Reversed‐phase liquid chromatography (RPLC) together with a BDD electrode poised at +1.6 V vs. Pd/H2, was optimized for their sensitive detection. The separation was achieved in 60 s with a core‐shell column (HALO C18). The detection limits of these analytes ranged from 10–30 nM. The method was applicable for the analysis of guaiacol and its precursors from a popular commercial drink.

Development and validation of bioanalytical methods for LNA-i-miR-221 quantification in human plasma and urine by LC-MS/MS

LNA-i-miR-221, a 13-mer oligonucleotide, has proved favorable efficacy and safety profiles in the preclinical studies, leading to being approved for use in clinical trials by regulatory authorities. The objective of this study was to develop and validate LC–MS/MS methods to quantify LNA-i-miR-221 in human plasma and urine. Chromatographic separation was performed with a gradient system on HALO C18 column using hexafluoro-2-propanol/triethylamine buffer and methanol as mobile phase. LNA-i-miR-221 was detected on tandem mass spectrometer with electrospray ionization source in negative ion mode. The methods showed good linearity within the calibration range of 50−25000 ng/mL and 50−50000 ng/mL for human plasma and urine, respectively. The methods proved to be accurate, precise and selective in both human matrices. These validated methods are reliable and are currently in use to support a first-in-human clinical trial of LNA-i-miR-221 in patients affected by refractory multiple myeloma and advanced solid tumors.

Profiling of phenolic flavorings using core-shell reversed-phase liquid chromatography with electrochemical detection at a boron-doped diamond electrode

A high-performance liquid chromatography (HPLC) method equipped with a boron-doped diamond (BDD) electrode was established for the simultaneous determination of phenol, 4-ethylphenol (4-EP), guaiacol, 4-ethylguaiacol (4-EG), 4-vinylguaiacol (4-VG), eugenol, and o-, m- and p-cresol. The separation was performed on a reversed-phase HALO C18 core-shell column (3.0 × 50 mm, 2.7 µm) with a mobile phase comprising 10 mM formate, pH 3, and 15% acetonitrile (ACN) (v/v), a flow rate of 1.5 mL/min, corresponding to a total run time of 9 min. The electrochemical detection (ECD) was set at +1.5 V vs. Pd/H2 in oxidative mode. Under optimized operating conditions, good linearity was obtained for the nine phenolics with corresponding coefficients of determination (R2) above 0.998. The limits of detection (LODs, S/N = 3) were 10 nM–1 µM, with an 80-fold increase in sensitivity for guaiacol achieved with ECD over ultraviolet (UV) detection. The sensitive and selective HPLC-ECD method was successfully applied for the identification and quantification of the nine phenolics in Islay, Irish, Scotch, and Highland whiskey samples, with significantly higher concentrations of the flavorings determined in Islay whiskey.

국산 아카시아꿀 내 Abscisic acid의 UPLC 정량분석법 개발

Acacia honey, which was collected from nectar of Robinia pseudoacacia by honeybee, is produced more than any other honey in Korea. It is known to have antibacterial activity against Helicobacter pylori that induced gastric disease. In present study, quantitative analysis for abscisic acid which was reported as anti-H. pylori compound in acacia honey was developed and validated by UPLCDAD. The UPLC method was established using Halo C18 column (2.1×100mm, 2μm) that eluted gradient system by acetonitrile and 0.1% phosphoric acid at 263nm of detection wavelength. The method was completely validated by specificity, linearity, limit of detection, limit of quantification, precision and accuracy. Also, the established UPLC method was possible to quantitative analysis for abscisic acid in Korean acacia honeys collected from 8 different areas. These results suggest that the developed UPLC method could be sufficiently applied for quality evaluation of Korean acacia honeys.

Simultaneous Determination of Chlorogenic Acid Isomers and Metabolites in Rat Plasma Using LC-MS/MS and Its Application to A Pharmacokinetic Study Following Oral Administration of Stauntonia Hexaphylla Leaf Extract (YRA-1909) to Rats.

Stauntonia hexaphylla leaf extract (YRA-1909), which is widely used for the antirheumatic properties, has been under phase 2 clinical trials in patients with rheumatoid arthritis since April 2017. Liquid chromatography-tandem mass spectrometric method while using liquid–liquid extraction with ethyl acetate was validated for the simultaneous determination of the major active components of YRA-1909, including chlorogenic acid (CGA), neochlorogenic acid (NCGA), cryptochlorogenic acid (CCGA), and their metabolites (i.e., caffeic acid (CA), caffeic acid 3-O-glucuronide (CA-3-G), caffeic acid 4-O-glucuronide (CA-4-G), and ferulic acid (FA)) in rat plasma and applied to a pharmacokinetic study of YRA-1909 in rats. Seven analytes were separated on Halo C18 while using gradient elution of formic acid and methanol, and then quantified in selected reaction monitoring mode whle using negative electrospray ionization. Following oral administration of YRA-1909 at doses of 25, 50, and 100 mg/kg to male Sprague-Dawley rats, CGA, NCGA, and CCGA were rapidly absorbed and metabolized to CA, CA-3-G, and CA-4-G. The area under the plasma concentration-time curve (AUClast) of CGA, NCGA, CCGA, and three metabolites linearly increased as the YRA-1909 dose increased. Other pharmacokinetic parameters were comparable among three doses studied. AUClast values for CA, CA-3-G, and CA-4-G exceeded those for CGA, NCGA, and CCGA.

Development, validation and application of a novel HPLC-MS/MS method for the quantification of atorvastatin, bisoprolol and clopidogrel in a large cardiovascular patient cohort

Cardiovascular disease is a leading cause of morbidity, mortality, and healthcare expenditure worldwide. Importantly, there is interindividual variation in response to cardiovascular medications, leading to variable efficacy and adverse events. Therefore a rapid, selective, sensitive and reproducible multi-analyte HPLC-MS/MS assay for the quantification in human plasma of atorvastatin, its major metabolites 2-hydroxyatorvastatin, atorvastatin lactone and 2-hydroxyatorvastatin lactone, plus bisoprolol and clopidogrel-carboxylic acid has been developed, fully validated, and applied to a large patient study. Fifty microliter plasma samples were extracted with a simple protein precipitation procedure involving acetonitrile with acetic acid (0.1%, v/v). Chromatographic separation was via a 2.7 μm Halo C18 (50 × 2.1 mm ID, 90 Å) column and gradient elution at a flow rate of 500 μL/min consisting of a mobile phase of water (A) and acetonitrile (B), each containing 0.1% formic acid (v/v), over a 6.0 min run time. The six analytes and their corresponding six deuterated internal standards underwent positive ion electrospray ionisation and were detected with multiple reaction monitoring. The developed method was fully validated with acceptable selectivity, carryover, dilution integrity, and within-run and between-run accuracy and precision. Mean extraction recovery for the analytes was 92.7–108.5%, and internal standard-normalised matrix effects had acceptable precision (coefficients of variation 2.2–12.3%). Moreover, all analytes were stable under the tested conditions. Atorvastatin lactone to acid interconversion was assessed and recommendations for its minimisation are made. The validated assay was successfully applied to analyse 1279 samples from 1024 patients recruited to a cardiovascular secondary prevention prospective study.

Quantitative Method Development and Validation of Naloxone and 6α‐naloxol from Male African Green Monkey Plasma.

Theopioid epidemic is a rapidly growing crisis around the world. Current models forpharmacokinetic assessment of countermeasures use African green monkeys (AGM). Arapid, sensitive and selective method was developed and validated usingLC‐MS/MS for the determination of naloxone and 6α‐naloxol in AGM plasma. Samplepreparation involved a simple solid phase extraction (SPE) using ahydrophilic‐lipophilic‐balanced reversed‐phase cartridge. Separation of naloxoneand 6α‐naloxol was achieved on a Halo C18, 2.1 × 50 mm, 2.7μ analytical columnusing an Agilent 1290 Infinity HPLC. A solvent system of methanol‐water‐formicacid was used for the elution of analytes. The analytes were detected and monitoredby a SCIEX QTRAP 6500+ triple quadrupole tandem mass spectrometer operatedin electrospray ionization (ESI) positive mode and multiple reaction monitoring(MRM). Molecular ion transitions for naloxone, 6α‐naloxol and naloxone‐D5 were 328.0/212.0,330.0/185.0, and 333.1/212.0 m/z, respectively. The method was validated usingcalibration and recovery assays with spiked samples. Linear calibration curveswere generated over the range of 1.5–100 ng/ml with values for the coefficientof determination (R2) of >0.9950. The values for both inter‐dayand intra‐day precision and accuracy were well within the generally acceptedcriteria for analytical methods (≤15%). This method was subsequentlyused to measure plasma concentrations of naloxone and 6α‐naloxol in male AGMs administerednaloxone subcutaneously and intravenously. The developed method demonstratedexcellent recovery, repeatability, accuracy and sensitivity.Support or Funding InformationDISCLAIMERS. The views expressed in this abstractare those of the authors (s) and do not reflect the official policy of theDepartment of Army, Department of Defense, or U. S. Government. This researchwas supported by the Defense Threat Reduction Agency – Joint Science andTechnology office, Medical S&T Division.Thisproject was supported in part by an appointment to the Research ParticipationProgram for the U.S. Army Medical Research and Materiel Command administratedby the Oak Ridge Institute for Science and Education through an agreementbetween the U.S. Army Medical Research and Materiel Command.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Development and validation of a bioanalytical method for quantification of LNA-i-miR-221, a 13-mer oligonucleotide, in rat plasma using LC–MS/MS

LNA-i-miR-221, a 13-mer oligonucleotide, is a new miR-221 inhibitor that could be used as a novel drug for multiple myeloma. Herein, an ion-pair reversed phase liquid chromatography–tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the quantification of LNA-i-miR-221 in rat plasma. Plasma samples were prepared with an initial phenol/chloroform/isoamyl alcohol liquid–liquid extraction followed by a solid phase extraction. Chromatographic separation was performed with a gradient system on a HALO C18 column using hexafluoro-2-propanol/triethylamine buffer and methanol as mobile phase at a flow rate of 0.4 mL/min. Under these conditions LNA-i-miR-221 and the analogue internal standard are co-eluted at 1.2 min. The detection was carried out in multiple reaction monitoring (MRM) mode using a negative electrospray ionization (ESI) interface. The assay showed a good linearity within the calibration range 10–10000 ng/mL. The precision, accuracy, and recovery values were found to be <15% (<20% at LLOQ), 100 ± 15%, and 97.6–103.7%, respectively. This method was successfully applied to measure the concentrations of LNA-i-miR-221 in plasma samples following the intravenous administration during a 4-week toxicity study in rats.

Development of UPLC Fingerprint with Multi-Component Quantitative Analysis for Quality Consistency Evaluation of Herbal Medicine "Hyangsapyeongwisan".

Hyangsapyeongwisan (HSPWS), known as traditional herbal medicine, has been used in the treatment of gastric disease. Standardization of HSPWS is a necessary step for the establishment of a consistent biological activity for the production and manufacturing of HSPWS herbal preparations. A simple, sensitive and accurate method using ultra-performance liquid chromatography (UPLC) fingerprinting with a diode array detector (DAD) was developed and validated for systematic quality evaluation of HSPWS. Separation conditions were optimized using a Halo C18 2.7 µm, 4.6 × 100 mm column with a mobile phase of 0.1% phosphoric acid and acetonitrile, and detection wavelengths of 215, 250 and 350 nm. Validation of the analytical method was evaluated by tests of linearity, precision, accuracy and robustness. All calibration curves of components showed good linearity (R(2) > 0.9996). The limit of detection (LOD) and limit of quantification (LOQ) were within the ranges of 0.004-0.134 and 0.012-0.406 µg/mL, respectively. The relative standard deviation (RSD) values of intra- and inter-day testing were within the range of 0.01-3.84%. The result of the recovery test was 96.82-104.62% with an RSD value of 0.14-3.84%. Robustness values of all parameters as well as the stability test of analytical solutions were within the standard limit. It showed that the developed method was simple, specific, sensitive, accurate, precise, reproducible and robust for the quantification of active components of HSPWS. Chromatographic fingerprinting with quantitative analysis of marker compounds in HSPWS prepared by different methods and commercial formulation was also evaluated successfully.

Stability Indicating Reverse Phase Chromatographic Method for Estimation of Related Substances in Voriconazole Drug Substance by Ultra Performance Liquid Chromatography

A simple, sensitive and accurate analytical method for related substances of Voriconazole has been developed and validated by using RP-UPLC technique. The developed analytical procedure was validated as per ICH recommendations. Analysis was performed on a HALO C18 (100 × 2.1 2.7 μ) column. 20 mM ammonium formate adjusted the pH to 4.5 with formic acid was selected as buffer. Acetonitrile was used as organic modifier in the mobile phase composition. A simple gradient was applied in the method. Flow rate of mobile phase was kept at 0.4 ml per min. Column compartment temperature was maintained at 45°C. Injection volume was set at 1 μL with an auto sampler maintained the temperature at 10°C. Detection of all the components was monitored at 254 nm by photodiode array detector. Developed method satisfies the system suitability criteria, peak integrity, and resolution for the parent drug and its related substances. The proposed method was validated for Specificity, precision, accuracy, linearity, limit of detection and quantification. Forced degradation studies were conducted to assess stability indicating nature of the method under acidic, basic, oxidative and photolytic conditions. Run time less than 7.0 minutes indicating that the method is cost effective and productive; it can be successfully applied for testing of related substances in drug substance and assay of drug substance in routine quality control analysis and stability testing.