Method development for quantification of quizartinib in rat plasma by liquid chromatography/tandem mass spectrometry for pharmacokinetic application.

Quizartinib is a highly potent inhibitor of the fms-like tyrosine kinase receptor, which is one of the most commonly mutated genes in acute myeloid leukemia. Quizartinib has shown a significant antileukemic clinical influence among relapsed/refractory acute myeloid leukemia patients. This study aimed at developing and validating an analytical method for the measurement of quizartinib in rat plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method was validated according to US Food and Drug Administration guidelines, and the results obtained in this work met the set criteria. Liquid-liquid extraction was used and chromatographic separation was achieved on a BEHTM C18 column. Detection of quizartinib was achieved in multiple reaction monitoring mode using positive-ion mode electrospray ionization. The MS/MS ion transitions at mass-to-charge ratios (m/z) of 561.129/114.09 and 441.16/84.03 were monitored for quizartinib and ibrutinib, respectively. The linear detection range was 2-1000 ng/mL (r > 0.998), with intra- and inter-day assay precisions ≤13.07 and 13.17%, respectively. This rapid, simple and sensitive method was validated and successfully applied to the pharmacokinetic study of quizartinib in rat samples.

Cancer stem cells CD133 inhibition and cytotoxicity of certain 3-phenylthiazolo[3,2-a]benzimidazoles: design, direct synthesis, crystal study and in vitro biological evaluation.

Cancer stem cells (CSCs) have been objects of intensive study since their identification in 1994. Adopting a structural rigidification approach, a novel series of 3-phenylthiazolo[3,2-a]benzimidazoles 4a-d was designed and synthesised, in an attempt to develop potent anticancer agent that can target the bulk of tumour cells and CSCs. The anti-proliferative activity of the synthesised compounds was evaluated against two cell lines, namely; colon cancer HT-29 and triple negative breast cancer MDA-MB-468 cell lines. Also, their inhibitory activity against the cell surface expression of CD133 was examined. In particular, compound 4b emerged as a promising hit molecule as it manifested good antineoplastic potency against both tested cell lines (IC50 = 9 and 12 µM, respectively), beside its ability to inhibit the cell surface expression of CD133 by 50% suggesting a promising potential of effectively controlling the tumour by eradicating the tumour bulk and inhibiting the proliferation of the CSCs. Moreover, compounds 4a and 4c showed moderate activity against HT-29 (IC50 = 21 and 29 µM, respectively) and MDA-MB-468 (IC50 = 23 and 24 µM, respectively) cell lines, while they inhibited the CD133 expression by 14% and 48%, respectively. Finally, a single crystal X-ray diffraction was recorded for compound 4d.

Synthesis, biological evaluation and 2D-QSAR study of halophenyl bis-hydrazones as antimicrobial and antitubercular agents.

In continuation of our endeavor towards the development of potent and effective antimicrobial agents, three series of halophenyl bis-hydrazones (14a-n, 16a-d, 17a and 17b) were synthesized and evaluated for their potential antibacterial, antifungal and antimycobacterial activities. These efforts led to the identification of five molecules 14c, 14g, 16b, 17a and 17b (MIC range from 0.12 to 7.81 µg/mL) with broad antimicrobial activity against Mycobacterium tuberculosis; Aspergillus fumigates; Gram positive bacteria, Staphylococcus aureus, Streptococcus pneumonia, and Bacillis subtilis; and Gram negative bacteria, Salmonella typhimurium, Klebsiella pneumonia, and Escherichia coli. Three of the most active compounds, 16b, 17a and 17b, were also devoid of apparent cytotoxicity to lung cancer cell line A549. Amphotericin B and ciprofloxacin were used as references for antifungal and antibacterial screening, while isoniazid and pyrazinamide were used as references for antimycobacterial activity. Furthermore, three Quantitative Structure Activity Relationship (QSAR) models were built to explore the structural requirements controlling the different activities of the prepared bis-hydrazones.

Multistage fragmentation of ion trap mass spectrometry system and pseudo-MS3 of triple quadrupole mass spectrometry characterize certain (E)-3-(dimethylamino)-1-arylprop-2-en-1-ones: a comparative study.

A new approach was recently introduced to improve the structure elucidation power of tandem mass spectrometry simulating the MS(3) of ion trap mass spectrometry system overcoming the different drawbacks of the latter. The fact that collision induced dissociation in the triple quadrupole mass spectrometer system provides richer fragment ions compared to those achieved in the ion trap mass spectrometer system utilizing resonance excitation. Moreover, extracting comprehensive spectra in the ion trap needs multistage fragmentation, whereas similar fragment ions may be acquired from one stage product ion scan using the triple quadrupole mass spectrometer. The new strategy was proven to enhance the qualitative performance of tandem mass spectrometry for structural elucidation of different chemical entities. In the current study we are endeavoring to prove our hypothesis of the efficiency of the new pseudo-MS(3) technique via its comparison with the MS(3) mode of ion trap mass spectrometry system. Ten pharmacologically and synthetically important (E)-3-(dimethylamino)-1-arylprop-2-en-1-ones (enaminones 4a-j) were chosen as model compounds for this study. This strategy permitted rigorous identification of all fragment ions using triple quadrupole mass spectrometer with sufficient specificity. It can be used to elucidate structures of different unknown components. The data presented in this paper provide clear evidence that our new pseudo-MS(3) may simulate the MS(3) of ion trap spectrometry system.

5,6-Dimethyl-4-(thio-phen-2-yl)-1H-pyrazolo-[3,4-b]pyridin-3-amine.

In the title mol-ecule, C(12)H(12)N(4)S, the thio-phene ring is disordered over two orientations with a refined site-occupancy ratio of 0.777 (4):0.223 (4). The pyrazolo-pyridine ring system is essentially planar with an r.m.s. deviation of 0.0069 (3) Šand makes dihedral angles of 82.8 (2) and 72.6 (5)°, respectively, with the major and minor components of the thio-phene ring. In the crystal, mol-ecules are linked into a chain along the a axis by a pair of N-H⋯N(pyrazole) hydrogen bonds and a pair of N-H⋯N(pyridine) hydrogen bonds, both having a centrosymmetric R(2) (2)(8) graph-set motif. A C-H⋯π inter-action is also present.

(Z)-1-(4-Methyl-phen-yl)-2-(phenyl-sulfon-yl)ethanone oxime.

The mol-ecule of the title compound, C(15)H(15)NO(3)S, has a twisted U-shaped conformation: the twist occurs at the central C-S(=O)(2)-C-C-C unit and the benzene ring makes a dihedral angle of 28.74 (7)° with the phenyl ring. The S-C-C=N torsion angle is -88.95 (13)°. In the crystal, inversion dimers linked by pairs of O-H⋯N hydrogen bonds generate R(2) (2)(6) loops, and C-H⋯O hydrogen bonds connect the dimers into a three-dimensional network.

3-(1H-Imidazol-1-yl)-1-phenyl-propan-1-ol.

In the title compound, C(12)H(14)N(2)O, the imidazole ring forms a dihedral angle of 66.73 (5)° with the phenyl ring. In the crystal, mol-ecules are linked via O-H⋯N and C-H⋯O hydrogen bonds into sheets lying parallel to (100). The crystal structure is further consolidated by C-H⋯π inter-actions.

1-(4-Methyl-phen-yl)-2-(phenyl-sulfon-yl)ethanone.

In the title compound, C(15)H(14)O(3)S, the benzene and phenyl rings make a dihedral angle of 33.56 (16)°. In the crystal, mol-ecules are linked by C-H⋯O hydrogen bonds into a layer parallel to the ab plane.

Ethyl 1-(4-methyl-phen-yl)-5-phenyl-4-phenyl-sulfon-yl-1H-pyrazole-3-carboxyl-ate.

The title compound, C(25)H(22)N(2)O(4)S, features a tetra-substituted pyrazole ring. The dihedral angles formed between the five-membered ring (r.m.s. deviation = 0.007 Å) and the N- and C-bound phenyl rings are 48.10 (7) and 72.01 (7) °, respectively, indicating that the planes through the residues are significantly twisted from the plane through the heterocycle. The ester-CO(2) group is also twisted out of this plane, with an O-C-C-N torsion angle of -29.04 (11)°. The sulfonyl-O atoms lie to one side of the pyrazole plane and the sulfonyl-phenyl ring to the other. The dihedral angle between the two ring planes is 70.63 (7) °. Supra-molecular arrays are formed in the crystal structure sustained by C-H⋯O and C-H⋯π(pyrazole) inter-actions and methyl-C-H⋯π(N-bound benzene) contacts.

Thiazolo[3,2-a]benzimidazoles: synthetic strategies, chemical transformations and biological activities.

The present review covers the recent synthetic strategies and chemical transformations of thiazolo[3,2-a]benzimidazoles and it also presents the highlights of the biological activities of these compounds.

Simultaneous Detection and Quantification of Three Novel Prescription Drugs of Abuse (Suvorexant, Lorcaserin and Brivaracetam) in Human Plasma by UPLC-MS-MS.

Suvorexant (SVR), lorcaserin (LCR) and brivaracetam (BVR) have been recently approved for the treatment of insomnia, obesity and epilepsy, respectively. Despite their clinical uses, these drugs have some abuse potential and have been enlisted under the schedule IV (SVR, LVR) and schedule V (BVR) categories of the Controlled Substances Act. A sensitive UPLC-MS-MS assay was developed for simultaneously determining SVR, LCR and BVR in human plasma. The liquid-liquid extraction method, using tert-butyl methyl ether as an extracting solvent, was used for sample preparation. Chromatographic separation was performed by using the Acquity BEH C18 column, using 10 mM ammonium acetate/acetonitrile/formic acid (15/85/0.1%; v/v/v) as the mobile phase. For sample ionization, electrospray ionization was used in the positive-ion mode. The multiple-reaction monitoring mode was used for detecting and quantifying analytes by using separate precursor-to-product ion transitions. The assay was validated following the SWGTOX guidelines, and all validation results were within the acceptable limits. The calibration curves of the analytes in the plasma were found to be linear, and the coefficient of determination (R2) was ≥ 0.992 for all the three analytes. The limit of detection values for SVR, LCR and BVR were 0.08, 0.11 and 0.26 ng/mL, respectively, whereas the limit of quantification values were 0.16, 0.27 and 0.65 ng/mL, respectively. The assay developed in this study is suitable for the identification and quantification of SVR, LCR and BVR in the forensic laboratory.

UPLC-MS/MS determination of suvorexant in urine by a simplified dispersive liquid-liquid micro-extraction followed by ultrasound assisted back extraction from solidified floating organic droplets.

Suvorexant is a novel sedative/hypnotic drug approved for treatment of insomnia. It has significant forensic importance due to its hypnotic and depressant effects on central nervous system. In this study, a highly sensitive UPLC-MS/MS assay was developed and validated for the determination of suvorexant in urine sample. A simplified dispersive liquid-liquid microextraction followed by ultrasound assisted back extraction from solidified floating organic droplets was employed for sample preparation. The 20 µL of 1-undecanol and 200 µL of acetonitrile were used as extraction solvent and dispersive solvent, respectively. An ultrasound assisted back extraction step was employed to enable the cleanup procedure compatible with mass spectrometric detection. Acquity CSH™C18 column with mobile phase composition of 15 mM ammonium acetate: acetonitrile: formic acid (15:85:0.1%; v/v/v) were used for chromatographic separation. The multiple reaction monitoring transition of 451.12 →104.01 and 451.12→186.04 were used for identification and quantification of suvorexant, respectively, whereas 237.06→194.1 was used for IS in positive mode. The assay demonstrated good linearity in the range of 0.27-1000 ng mL-1 with limit of detection (LOD) and quantification (LOQ) of 0.10 and 0.27 ng mL-1, respectively. Assay validation was performed by following SWGTOX guidelines and all validation results were found to be within acceptable limits. This is the first report of dispersive liquid-liquid microextraction based on solidification of floating organic droplets employed to UPLC-MS/MS for application in biological fluids.

High throughput µ-SPE based elution coupled with UPLC-MS/MS for determination of eluxadoline in plasma sample: Application in pharmacokinetic characterization of PLGA nanoparticle formulations in rats.

Eluxadoline is a novel µ- and κ-opioid receptor (OR) agonist and δ-OR antagonist, recently approved as a first line therapy for the treatment of irritable bowel syndrome. Due to abuse potential, poor bioavailability and high intersubject variability, a sensitive and reliable assay is prerequisite for its determination in biological samples. This work first time report the development and validation of UPLC-MS/MS assay for determination of eluxadoline in rat plasma sample using risperidone as an internal standard (IS). A high-throughput 96-well plate format µ-SPE technique was used for plasma sample extraction. The extracted samples were separated on Acquity BEH™ C18 column (100×2.1mm, 1.7µm) using mobile phase elution of acetonitrile: 20mM ammonium acetate (80:20, v/v) at a flow rate of 0.3mLmin-1. The precursor to product ion transition of m/z 570.16→118.12 (qualifier), 570.16→171.08 (quantifier) for eluxadoline, and m/z 411.18→191.07 for IS were used for MRM monitoring. The calibration curves were linear in concentration range of 0.15-50ngmL-1 with LOD and LOQ of 0.07 and 0.15ngmL-1, respectively. The validation results satisfied the criteria of USFDA and SWGTOX guidelines and were within the acceptable limit. Finally, the method was successfully applied in bioavailability enhancement study of the newly developed PLGA nanoparticles and Eudragit coated PLGA nanoparticles of eluxadoline in rats.

A validated UPLC-MS/MS method for flibanserin in plasma and its pharmacokinetic interaction with bosentan in rats.

AIM: The purpose of this study was development, validation and application of ultra-performance liquid chromatography (UPLC)-ESI-MS/MS method for quantitation of flibanserin in plasma samples. METHOD & RESULTS: After extraction of analyte from plasma by diethyl ether, separation was performed on UPLC C18 column using mobile phase composition of 10 mM ammonium formate-acetonitrile (30:70, v/v) by isocratic elution of 0.3 ml/min. The multiple reaction monitoring transitions of m/z 391.13 → 161.04 and 384.20 → 253.06 were used for detection of analyte and internal standard (quetiapine), respectively. The calibration curves were linear (r ≥ 0.995) between 0.22 and 555 ng/ml concentration and all validation results were within the acceptable range as per US FDA guidelines. CONCLUSION: The assay procedure was fully validated and successfully applied in pharmacokinetic interaction study of flibanserin with bosentan in rats.

UPLC/MS-MS assay development for estimation of mozavaptan in plasma and its pharmacokinetic study in rats.

AIM: Mozavaptan is a nonpeptide vasopressin receptor antagonist approved for the treatment of ectopic antidiuretic hormone secretion syndrome. METHODS & RESULTS: A simple, rapid and fully validated UPLC/MS-MS method was developed for the quantitation of mozavaptan in rat plasma. The chromatographic separation was conducted on an Acquity UPLC BEH™ C18 column with an optimum mobile phase of 10 mM ammonium acetate buffer and 0.1% formic acid in acetonitrile (30:70 v/v) at a flow rate of 0.3 ml/min. The multiple reaction monitoring transitions were performed at m/z 428.16→119.03 for mozavaptan and m/z 237.06→179.10 for carbamazepine (internal standard). CONCLUSION: The method was effectively applied for the determination of mozavaptan pharmacokinetic parameters after the oral administration of 3 mg/kg mozavaptan in rats.

UPLC-MS/MS assay for identification and quantification of brivaracetam in plasma sample: Application to pharmacokinetic study in rats.

Brivaracetam (BVR) is a novel antiepileptic drug (AED), approved clinically for the treatment of partial onset seizures in adults and adolescents. It has some abuse potential and assigned to Schedule V category under the Controlled Substance Act by the Drug Enforcement Administration. Being an AED and drug of abuse, a sensitive and robust assay is necessary for determination of BVR in biological fluids. Herein, we report a sensitive and validated UPLC-MS/MS assay for identification and quantification of BVR in plasma samples. The samples were prepared by one step liquid liquid extraction method using tert-Butyl methyl ether as extracting solvent. BVR and internal standard (carbamazepine) were separated on Aquity BEH™ C-18 column and eluted by using gradient mobile phase combination of acetonitrile and 0.1% formic acid in water. The precursor to product ion transition of m/z 213.12→54.95 (qualifier), 213.12→168.10 (quantifier) for BVR, and m/z 237.06→193.25 for IS were used for MRM detection and quantification. The assay was validated according to USFDA and SWGTOX guidelines for method validation and all parameter results were within the acceptable limits. The calibration curves were found to be linear in concentration range of 1.98-2000ng/mL (r2≥0.995) having LOD and LLOQ of 0.80 and 1.98ng/mL, respectively. The assay was successfully employed in BVR pharmacokinetic study in rats and can be suitable for therapeutic drug monitoring, pharmacokinetic study and forensic analysis.

Simple and Highly Sensitive UPLC-ESI-MS/MS Assay for Rapid Determination of Suvorexant in Plasma.

Suvorexant is a dual orexin receptor antagonist, recently approved by USFDA for the treatment of insomnia. It is drug-of-abuse and listed in Schedule IV drug of the Controlled Substances Act. In this study, a simple and highly sensitive UPLC-MS/MS assay was developed and fully validated for the determination of suvorexant in rat plasma. Both suvorexant and internal standard (rivaroxaban; IS) were separated on Aquity BEHTM C18 column after the extraction form plasma using diethyl ether as extracting agent. An isocratic mobile phase, consisting of acetonitrile and 10 mM ammonium acetate in composition ratio of 85:15 was eluted at flow rate of 0.3 mL/min. Both suvorexant and IS were eluted within one min with total run time of 1.5 min only. The ionization was performed on electrospray ionization interface in positive mode by multiple reaction monitoring. Precursor to product ion transition of 451.12 > 104.01 for qualifier and 451.12 > 186.04 for quantifier were used for suvorexant whereas 436.10 > 144.93 for IS, respectively. The calibration curves in plasma were linear in the concentration range of 0.33-200 ng/mL (r2 ≥ 0.995) having limit of detection and limit of quantification of 0.10 and 0.33 ng/mL, respectively. All the validation parameters results were found to be within the acceptable limits according to the "Scientific Working Group for Forensic Toxicology (SWGTOX) guidelines" and can be implemented for forensic analysis.

A Validated UPLC-MS-MS Assay for the Rapid Determination of Lorcaserin in Plasma and Brain Tissue Samples.

Lorcaserin is a novel, potent and highly efficacious 5-HT2C receptor agonist, recently approved by US Food and Drug Administration for the treatment of obesity. It has some abuse potential also and is listed as a Schedule IV drug in the Controlled Substances Act. Herein, a sensitive, selective and reliable UPLC-MS-MS assay was developed and validated for the quantitative analysis of lorcaserin in rat plasma and brain tissue using carbamazepine as an internal standard (IS). After the extraction of samples by protein precipitation, both lorcaserin and IS were separated on an Acquity BEH™ C18 (50 × 2.1 mm, 1.7 µm) column using a mobile phase consisting of acetonitrile-10 mM ammonium acetate-formic acid (85:15:0.1, v/v/v) at a flow rate of 0.25 mL/min. Detection and quantification were performed on a positive electrospray ionization interface in the multiple-reaction monitoring (MRM) mode. The MS-MS ion transitions were monitored at m/z 195.99 > 143.91 for lorcaserin and m/z 237.00 > 178.97 for IS, respectively. The calibration curves were linear over a concentration range of 1.08-500 ng/mL in plasma and 3.07-500 ng/mL in brain tissue homogenates, respectively. All the validation parameters results were within the acceptable range described in guidelines for bioanalytical method validation. The assay was successfully applied in a pharmacokinetic study of lorcaserin after oral administration in rats.

Preclinical pharmacokinetics, tissue distribution and excretion studies of a novel anti-candidal agent-thiosemicarbazide derivative of isoniazid (TSC-INH) by validated UPLC-MS/MS assay.

A simple and sensitive UPLC-MS/MS assay was developed and validated for rapid determination of thiosemicarbazide derivative of isoniazid (TSC-INH), a potent anti-candidal agent in rat plasma, tissues, urine and feces. All biological samples were prepared by protein precipitation method using celecoxib as an internal standard (IS). Chromatographic separation was achieved on Acquity BEH™ C18 (50×2.1 mm, 1.7 µm) column using gradient mobile phase of acetonitrile and water (containing 0.1% formic acid) at flow rate of 0.3 mL/min. The MRM transitions were monitored at m/z 305.00→135.89 for TSC-INH and m/z 380.08→316.03 for IS in ESI negative mode. All validation parameter results were within the acceptable range described in guideline for bioanalytical method validation. The pharmacokinetic study showed that the compound TSC-INH was orally active with 66% absolute bioavailability in rats. It was rapidly absorbed with peak plasma concentration of 1985.92 ng/mL achieved within 1 h after single oral dose (10 mg/kg) administration. TSC-INH exhibited rapid distribution across the body with highest levels in liver and lungs. Penetration in brain tissues suggests that TSC-INH crossed the blood brain barrier. Only 5.23% of the orally administered drug was excreted as unconverted form in urine and feces implying that TSC-INH was metabolized extensively before excretion. With the preliminary knowledge of in vivo pharmacokinetics and disposition properties, this study will be beneficial for further development of compound TSC-INH in future studies.

Determination of apremilast in rat plasma by UPLC-MS/MS in ESI-negative mode to avoid adduct ions formation.

BACKGROUND: Quantification of target analyte by LC-MS/MS is sometimes hampering due to competitive adduct ions formation (sodium and/or ammonium) in positive ionization mode. A UPLC-MS/MS assay was developed for the determination of apremilast in rat plasma using ESI-negative mode to avoid adduct ions formation. METHOD & RESULTS: After extraction from plasma by ethyl acetate, analyte and IS were separated on Aquity BEH C18 column using acetonitrile-10 mM ammonium acetate (85:15) as mobile phase. The calibration curve was linear between 3.04 and 1000 ng/ml with correlation coefficients (r2) of ≥0.995 and lower limit of quantification of 3.04 ng/ml. All validation parameter results were within the acceptable range. The assay was successfully employed in oral PK study with Cmax of 584.29 ng/ml and AUC0-20 of 6530 ng.h/ml after apremilast (2 mg/kg) administration. CONCLUSION: This result suggests that ESI in negative mode would be an alternative approach for LC-MS/MS quantification of analytes, which produce competitive adducts in positive mode.