The Development and Full Validation of a Novel Liquid Chromatography Electrochemical Detection Method for Simultaneous Determination of Nine Catecholamines in Rat Brain.

Objectives: Chemical neurotransmission, managed by neurotransmitters, has a crucial role in brain processes such as fear, memory, learning, and pain, or neuropathology such as schizophrenia, epilepsy, anxiety/depression, and Parkinson's disease. The measurement of these compounds is used to elucidate the disease mechanisms and evaluate the outcomes of therapeutic interventions. However, this can be quite difficult because of various matrix effects and the problems of chromatographic separation of analysts. In the current study; for the first time, an optimized and fully validated high-performance liquid chromatography-electrochemical detection (HPLC-EC) method according to Food and Drug Administration and European Medicines Agency Bioanalytical Validation Guidance was developed for the simultaneous analysis of nine neurotransmitter compounds, namely dopamine, homovanilic acid, vanilmandelic acid, serotonin (SER), 5-hydroxyindole-3-acetic acid, 4-hydroxy-3-methoxyphenylglycol, norepinephrine, 3,4 dihydroxyphenylacetic acid, and 3-methoxytyramine and simultaneously determined in rat brain samples. Materials and Methods: Separation was achieved with 150 mm x 4.6 mm, 2.6 µm Kinetex F5 (Phenomenex, USA) column isocratically, and analysis was carried out by HPLC equipped with a DECADE II EC detector. Results: The method exhibited good selectivity, and the correlation coefficient values for each analyte's calibration curves were > 0.99. The detection and quantification limits ranged from 0.01 to 0.03 ng/mL and 3.04 to 9.13 ng/mL, respectively. The stability of the analyses and method robustness were also examined in detail in the study, and the obtained results are presented statistically. Conclusion: The developed and fully validated method has been successfully applied to actual rat brain samples, and important results have been obtained. In the rat brain sample analysis, the lowest number of SER and the highest amount of noradrenaline were found.

Liquid chromatographic determination of lumacaftor in the presence of ivacaftor and identification of five novel degradation products using high-performance liquid chromatography ion trap time-of-flight mass spectrometry.

Lumacaftor is a transmembrane conductance regulator potentiator drug, prescribed for the treatment of cystic fibrosis in patients who are homozygous for the F508del mutation. Quantitation of lumacaftor besides its degradation products and ivacaftor was achieved on a fused-core silica particle column packed with pentafluorophenylpropyl stationary phase (Ascentis Express F5, 2.7 µm particle size 100 mm × 4.6 mm; Supelco) using gradient elution (A: 0.1% [v/v] formic acid in water, B: 0.1% [v/v] formic acid in acetonitrile [the mobile phase pH 2.5]). A constant flow rate at 1 mL/min was applied, and the detection was realized using a photodiode array detector set at 216 nm. The pseudo tablet formulation of the lumacaftor/ivacaftor fixed-dose combination preparation, namely, Orkambi®, was prepared in vitro and used for the analytical performance validation and method application studies. In addition, five novel degradation products, four of which even have no Chemical Abstracts Services registry number, were identified using high-resolution mass spectrometry instrument, and their possible mechanisms of formation were proposed. According to current literature, this paper can be regarded as the most comprehensive liquid chromatographic study on lumacaftor determination, among its counterparts.

A Different Perspective on the Characterization of a New Degradation Product of Flibanserin With HPLC-DAD-ESI-IT-TOF-MSn and Its Pharmaceutical Formulation Analysis With Inter-Laboratory Comparison.

BACKGROUND: Flibanserin (FLB) was first synthesized as an antidepressant drug; however, due to its enhancing effects on sexual activity, it was approved for treatment of hypoactive sexual desire disorder in women in 2015. OBJECTIVE: The aim of this study was to develop a new and fully validated HPLC method for analysis of FLB in pharmaceutical formulations besides its degradation products, and identification of possible formation mechanisms by using HPLC-DAD-ESI-IT-TOF-MSn. METHOD: The HPLC separation was achieved in a Supelco Ascentis® Express series phenyl hexyl column (100 × 4.6 mm, ID 2.7 µm). The mobile phase was acetonitrile-ammonium acetate solution (50:50, v/v, 10 mM, pH 5.4) mixture, which was pumped at the rate of 0.5 mL/min. Chromatography, detection, and structural identification was performed by using a LCMS-IT-TOF instrument (Shimadzu, Japan). RESULTS: 1-(2-(4-(3-hydroxy-5-(trifluoromethyl)phenyl)piperazine-1-yl)ethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one is proposed as a novel degradation product, with a mass of 407.1695 and a formula of C20H21F3N4O2 with a margin of error about 0.001 ppm. The developed method is applicable with 98% accuracy within the 2.5-50.0 µg/mL range. The LOD and LOQ were about 500 ng/mL and 1.50 µg/mL, respectively. The transferability and variation between laboratories were tested by inter-laboratory comparison and evaluated with one-way analysis of variance. CONCLUSIONS: A novel FLB degradation product, which was produced under oxidative forced degradation conditions was observed and identified for the first time; in addition, the formation kinetics of the degradation product besides decomposition of FLB was studied. Furthermore, an inter-laboratory comparison was carried out, and application of the proposed method on a pseudo Addyi® (Sprout Pharmaceuticals, Inc.) sample was tested using both instrument configurations. HIGHLIGHTS: A novel stability-indicating assay method was developed and fully validated according to the International Council on Harmonization (Q2) R1 for the analysis of FLB in the pharmaceutical preparations. A new degradation product was identified in the oxidative forced degradation condition and characterized using HPLC-DAD-ESI-IT-TOF-MS3. Moreover, the possible mechanism and the formation kinetic of the degradation product were revealed. In addition, the developed method was transferred to another LC-PDA instrument for inter-laboratory comparison. Finally, the current method was applied to a pseudo formulation of Addy in both instruments, and ANOVA was applied for evaluation.

Determination of ivacaftor by liquid chromatography techniques in pharmaceutical formulation with interlaboratory comparison and characterization of five novel degradation products by high-performance liquid chromatography ion trap time-of-flight mass spectrometry.

Cystic fibrosis is a life-threatening genetic disease that causes damage to the lungs. Ivacaftor, the first drug to target the underlying defect of the disease caused by specific mutations, improves outcomes and reduces hospitalizations. In this study, quantitative determination of ivacaftor was performed by liquid chromatography, while high-resolution mass spectrometric analyses were performed for qualitative determination. The validation studies of the developed methods were performed according to International Conference on Harmonisation Q2(R1) guideline. Ivacaftor was separated from its degradation product by using Phenomenex Kinetex C18 (150 × 3 mm, 2.6 µm) column. The isocratic mobile phase for binary pump configuration was 0.1% (v/v) formic acid in water and 0.1% (v/v) formic acid in acetonitrile (27:63) (v/v), pH = 2.5; the flow rate of 0.25 mL/min was used in all methods. In the degradation studies, five degradation products were identified using high-performance liquid chromatography ion trap time-of-flight mass spectrometric analyses: three of them have never been reported up to date; whereas the other two were existing in the literature and they were having Chemical Abstracts Services registry numbers since they were synthesized before for various other purposes. Also, analysis of an in-lab prepared chemical equivalent of Kalydeco® and interlaboratory comparison were performed.

A Novel HPLC Method for Simultaneous Determination of Methyl, Ethyl, n-propyl, Isopropyl, n-butyl, Isobutyl and Benzyl Paraben in Pharmaceuticals and Cosmetics.

INTRODUCTION: The alkyl esters of p-hydroxybenzoic acid at the C-4 position, "the parabens," including methyl, ethyl, propyl, and butyl, are widely used as antimicrobial preservatives in foods, cosmetics, and pharmaceuticals. Official regulations on the use of these compounds make their analysis essential for the estimation of their exposure. METHODS: On this basis, the presented study was realized to develop a simple, selective and cheap high-performance liquid chromatographic method for the quantitative determination of methylparaben, ethylparaben (EP), n-propyl paraben (NPP), isopropyl paraben (IPP), n-butyl paraben (NBP), isobutyl paraben (IBP) and benzyl paraben (BP) in pharmaceuticals and cosmetic products. RESULTS: The chromatographic separation of the analytes was achieved under flow rate gradient elution conditions using a C18-bonded core-shell silica particle column (2.6 µm particle size, 150 × 3.0 mm from Phenomenex Co.). The samples were injected into the system as aliquots of 1.0 µL, and the compounds were detected by using a photodiode array detector set at 254 nm wavelength. With this technique, seven paraben derivatives can be determined in the concentration range of 250-2000 ng/mL. The recovery of the method is in the range of 99.95-13.84%, and the RSD is at a maximum value of 3.95%. CONCLUSION: The proposed method was fully validated and successfully applied to different pharmaceutical and cosmetic samples (n=16), including syrups, suspensions, oral sprays, gels, etc. At least one paraben derivative was detected in six samples and was determined quantitatively. The maximum amount of a paraben derivative found in the analyzed samples was 321.7 ng/mL, which was MP. To the best of our knowledge, this is the first LC method, which is applicable both on pharmaceutical and cosmetic samples.

A Novel and Sensitive LC-MS/MS Method for the Quantitation of Ceftiofur in Pharmaceutical Preparations and Milk Samples.

INTRODUCTION: In the present study, a sensitive and selective liquid chromatographytandem mass spectrometry (LC-MS/MS) method was described for the determination of ceftiofur (CEF) in cow milk and pharmaceutical preparations. CEF is an antibiotic compound, which is commonly used in the treatment of animal diseases such as respiratory system, soft tissue, and foot infections, as well as postpartum acute puerperal metritis. One of the critical features of CEF is its prescription while breastfeeding cows; in accordance, its quantitative estimation is essential to assess its residual amounts. METHODS: In the method reported herein, after simple protein precipitation using acetonitrile, the pre-treated samples were introduced into an LC-MS/MS instrument equipped with a Chromolith® High-Resolution RP-18 series HPLC column (100 mm × 4.6 mm from Merck KGaA, Germany). Electrospray ionization was employed as the ionization source in the triple-quadrupole tandem mass spectrometer. RESULTS: For the calibration method using solvent-based standards, LOQ was 3.038 ng/mL, 12.15 ng/mL, and LOD was 1.215 ng/mL and 6.076 ng/mL for ESI+ and ESI- modes, respectively. On the other hand, for the method of matrix-matched standards, LOQ was 1.701 ng/mL, 10.13 ng/mL, and LOD was 0.486 ng/mL and 5.929 ng/mL for ESI+ and ESI- modes, respectively as obtained from signal to noise ratio. CONCLUSION: Applicability of both positive and negative ion modes was tested, and the analyte was detected via multiple reaction monitoring. The distorting effects of the milk matrix on the MS ionization and quantitation of CEF were overcome by using matrix-matched calibration for the first time.

Stability-indicating LC-MS/MS and LC-DAD methods for robust determination of tasimelteon and high resolution mass spectrometric identification of a novel degradation product.

Novel stability-indicating HPLC methods have been presented for the analysis of Tasimelteon (TSM) besides its main degradation products in bulk and pseudo tablet formulations in this study. For the LC-DAD method; quantitation of TSM and its separation with other degradation products were achieved on an Ascentis ExpressTM pentafluorophenylpropyl (F5)-bonded fused-core silica particle column (2.7 µm particle size 100 × 4.6 mm, Supelco) using the mobile phase consisted of acetonitrile: acetate buffer (0.025 M, pH 4.5): water (40:10:50, v/v/v); the elution was performed at 0.8 mL min-1 flow rate, detecting the compounds at 281 nm. In addition to regular in- house validation studies, the developed method was tested on another UPLC instrument to assess its ruggedness for possible method transfer demands. For the LC-MS/MS method, analyte quantitation was achieved on a second-generation monolithic silica column (Chromolith™ High-Resolution RP-18e, 100 × 4.6 mm from Merck KGaA, Germany); the mobile phase was a mixture with 0.1 % (v/v) formic acid in water and 0.1 % (v/v) formic acid in acetonitrile (60: 40 (v/v), pH = 2.5). The instrumental and analytical performances of all three instrumental systems were compared with each other in terms of the working range, LOD, and LOQ. In addition to the above, a new degradation product was identified using LCMS-IT-TOF system. It can be concluded that among the similar ones in the literature, this study is the most comprehensive method optimization and validation study to date about TSM, in which a novel degradation product was also reported.

Development of Validated and Stability-Indicating LC-DAD and LC-MS/MS Methods for Determination of Avanafil in Pharmaceutical Preparations and Identification of a Novel Degradation Product by LCMS-IT-TOF.

Avanafil (AVA), one of the most effective drugs prescribed for erectile dysfunction, is a pyrimidine-derivative PDE5 inhibitor. In the current work, new LC methods were developed and validated for quantitative determination of avanafil and qualitative determination of its degradation products. The quantitative determination of avanafil was carried out using liquid chromatography with photodiode array detection (LC-DAD) and liquid chromatography-tandem mass spectrometry LC-MS/MS methods, and fully validated according to the ICH Q2 (R1) guideline, while qualitative determination was performed using a liquid chromatography mass spectrometry-ion trap-time of flight (LCMS-IT-TOF) instrument. The separation of avanafil and its degradation products was carried out using the same reversed-phase chromatographic conditions, in which a second-generation C18-bonded monolithic silica column (Chromolith® High Resolution RP-18e, 100 × 4.6 mm, Merck KGaA) was used as stationary phase. Briefly, the methods enable quantitation of avanafil with high accuracy (recovery > 95%) and precision (RSD% < 2.0), within the ranges of 0.5⁻20 µg/mL for LC-DAD and 150⁻6000 ng/mL for LC-MS/MS. In the forced degradation studies, over and above currently existing data, a new oxidation-based degradation product, whose predicted m/z is 367.1168, was identified and its structure was confirmed by high-resolution mass spectrometric analysis. As the main advantage, either an LC-DAD or LC-MS/MS instrument can be chosen for interference-free quantitation of AVA, according to the facilities in quality-control laboratories.

Synthesis of Some Novel Thiadiazole Derivative Compounds and Screening Their Antidepressant-Like Activities.

Novel thiadiazole derivatives were synthesized through the reaction of acetylated 2-aminothiadiazole and piperazine derivatives. The chemical structures of the compounds were clarified by Infrared Spectroscopy (IR), ¹H Nuclear Magnetic Resonance Spectroscopy (¹H-NMR), 13C Nuclear Magnetic Resonance Spectroscopy (13C-NMR) and Electronspray Ionisation Mass Spectroscopy (ESI-MS) spectroscopic methods. Antidepressant-like activities were evaluated by the tail-suspension (TST) and modified forced swimming (MFST) methods. Besides, possible influence of the test compounds on motor activities of the animals were examined by activity cage tests. In the TST, administration of the compounds 2c, 2d, 2e, 2f, 2g and 2h significantly decreased the immobility time of mice regarding the control values. Further, in the MFST, the same compounds reduced the total number of immobility behaviors while increasing swimming performance. However, no change was observed in the total number of climbing behaviors. These data suggested that compounds 2c, 2d, 2e, 2f, 2g and 2h possess notable antidepressant-like activities. Reference drug fluoxetine (10 mg/kg) was also exhibited its antidepressant activity, as expected. No significant difference was seen between the locomotor activity values of the test groups signifying that observed antidepressant-like activities are specific. Theoretical calculation of absorption, distribution, metabolism, excretion (ADME) properties for the obtained compounds were performed and obtained data supported the antidepressant-like potential of these novel thiadiazole derivatives.

Design, synthesis and biological assessment of new thiazolylhydrazine derivatives as selective and reversible hMAO-A inhibitors.

In the recent works, it was shown that numerous thiazolylhydrazine derivatives display hMAO inhibitory activity in the range of micromolar concentration. Hence, in the present study a new series of new thiazole-hydrazines (3a-3n) were designed, synthesized, characterized and screened for their hMAO-A and hMAO-B inhibitory activity by an in vitro flurometric method. The enzyme inhibition assay revealed that most of the synthesized compounds have selective inhibition potency against hMAO-A. The compounds 3f and 3h showed promising hMAO-A inhibition with an IC50 values of 0.012 µM and 0.011 µM and significant selectivity indexes of 1214 and 1601 towards hMAO-A, respectively. The mechanism of hMAO-A inhibition of compounds 3f and 3h was investigated by Lineweaver-Burk graphics and reversible-competitive inhibition of hMAO-A was determined. Cytotoxicity and genotoxicity studies were carried out and the compound 3h was found as non-cytotoxic and non-genotoxic. Theoretical calculation of ADME properties suggested that synthesized compounds may have a good pharmacokinetic profile. The docking study of compound 3f and 3h revealed that there is a strong interaction between the active sites of hMAO-A and analyzed compound.

Pharmacological and Toxicological Screening of Novel Benzimidazole-Morpholine Derivatives as Dual-Acting Inhibitors.

The aim of this study was to investigate acetylcholinesterase (AChE), monoamine oxidase A (MAO-A), monoamine oxidase B (MAO-B), cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) enzyme inhibitory, and antimicrobial activities of a new series of 2-(4-substituted phenyl)-1-[2-(morpholin-4-yl)ethyl]-1H-benzimidazole derivatives, for their possible use as multi-action therapeutic agents. Target compounds (n = 15) were synthesized under microwave irradiation conditions in two steps, and their structures were elucidated by FT-IR, ¹H-NMR, 13C-NMR and high resolution mass spectroscopic analyses. Pharmacological screening studies revealed that two of the compounds (2b and 2j) have inhibitory potential on both COX-1 and COX-2 enzymes. In addition, cytotoxic and genotoxic properties of the compounds 2b, 2j and 2m were investigated via the well-known MTT and Ames tests, which revealed that the mentioned compounds are non-cytotoxic and non-genotoxic. As a concise conclusion, two novel compounds were characterized as potential candidates for treatment of frequently encountered inflammatory diseases.

Synthesis of New Hydrazone Derivatives for MAO Enzymes Inhibitory Activity.

In the present work, 14 new 1-substituted-2-phenylhydrazone derivatives were synthesized to evaluate their inhibitory activity against hMAO enzymes. The structures of the newly synthesized hydrazones 2a-2n were characterized by IR, 1H-NMR, 13C-NMR, HR-MS spectroscopic methods. The inhibitory activity of compounds 2a-2n against hMAO-A and hMAO-B enzymes was elucidated by using an in-vitro Amplex Red® reagent assay based on fluorometric methods. According to the activity studies, 2a and 2b were found to be the most active compounds against hMAO-A enzyme, with IC50 values of 0.342 µM and 0.028 µM, respectively. The most active compounds 2a-2b were evaluated by means of enzyme kinetics and docking studies. Moreover, these compounds were subjected to cytotoxicity and genotoxicity tests to establish their preliminary toxicological profiles and were found to be non-cytotoxic and non-genotoxic. Consequently, the findings of this study display the biological importance of compounds 2a, 2b as selective, irreversible and competitive inhibitors of hMAO-A. Docking studies revealed that there is a strong interaction between hMAO-A and the most active compound 2b.

A novel dilute and shoot HPLC assay method for quantification of irbesartan and hydrochlorothiazide in combination tablets and urine using second generation C18-bonded monolithic silica column with double gradient elution.

Irbesartan (IRB) and hydrochlorothiazide (HCT) are angiotensin-II receptor antagonist and thiazide-class diuretic compounds, respectively, which are in use in the treatment of hypertension. A novel dilute-and-shoot HPLC assay method for simultaneous quantification of IRB and HCT in fixed-dose combination tablets and urine samples was described. The separation of IRB, HCT and agomelatine (internal standard) was carried out using a second generation C18-bonded monolithic silica column (Chromolith(®) High Resolution RP-18e, 100×4.6mm, Merck KGaA), utilizing both mobile phase and flow rate gradient elution programs. The analytes were detected at 230 nm wavelength using photodiode array detector within 24 minutes with high resolution, observing about 50 percent more peak capacity when using second generation C18-bonded monolithic silica column. Urine samples were introduced into the system effortlessly, with only filtration and subsequent dilution. Validation studies were performed according to the official recommendations of USP and ICH, and the developed method was successfully applied to pharmaceutical tablets and urine samples.