Accumulation of oxysterols in the erythrocytes of COVID-19 patients as a biomarker for case severity.

BACKGROUND: Due to the high risk of COVID-19 patients developing thrombosis in the circulating blood, atherosclerosis, and myocardial infarction, it is necessary to study the lipidome of erythrocytes. Specifically, we examined the pathogenic oxysterols and acylcarnitines in the erythrocyte homogenate of COVID-19 patients. These molecules can damage cells and contribute to the development of these diseases. METHODS: This study included 30 patients and 30 healthy volunteers. The erythrocyte homogenate extract was analyzed using linear ion trap mass spectrometry combined with high-performance liquid chromatography. The concentrations of oxysterols and acylcarnitines in erythrocyte homogenates of healthy individuals and COVID-19 patients were measured. Elevated levels of toxic biomarkers in red blood cells could initiate oxidative stress, leading to a process known as Eryptosis. RESULTS: In COVID-19 patients, the levels of five oxysterols and six acylcarnitines in erythrocyte homogenates were significantly higher than those in healthy individuals, with a p-value of less than 0.05. The mean total concentration of oxysterols in the red blood cells of COVID-19 patients was 23.36 ± 13.47 µg/mL, while in healthy volunteers, the mean total concentration was 4.92 ± 1.61 µg/mL. The 7-ketocholesterol and 4-cholestenone levels were five and ten times higher, respectively, in COVID-19 patients than in healthy individuals. The concentration of acylcarnitines in the red blood cell homogenate of COVID-19 patients was 2 to 4 times higher than that of healthy volunteers on average. This finding suggests that these toxic biomarkers may cause the red blood cell death seen in COVID-19 patients. CONCLUSIONS: The abnormally high levels of oxysterols and acylcarnitines found in the erythrocytes of COVID-19 patients were associated with the severity of the cases, complications, and the substantial risk of thrombosis. The concentration of oxysterols in the erythrocyte homogenate could serve as a diagnostic biomarker for COVID-19 case severity.

Intra-tumoral drug concentration mapping within solid tumor micro-milieu using in-vitro model and doxorubicin as a model drug.

In contrast to plasma pharmacokinetics, intratumoral pharmacokinetics of doxorubicin (DOX) determines its spatial anti-tumoral activity. Three-dimensional multicellular layers (MCL) model for solid tumors present optimum experimental platform for studying the intratumoral pharmacokinetics of DOX. This might imply new insights for understanding intratumoral pharmacokinetic parameters with realistic clinical implications. Herein, we are presenting simplified method for the spatial in-situ concentration assessment of DOX within the avascular simulating MCL solid tumor model of DLD-1 and HT-29 cell lines. DLD-1 and HT-29 formed viable well-structured MCL model abundant in extracellular matrix component (fibronectin). DOX (100 µM) showed stronger anti-proliferative effect against MCL of DLD-1 compared to HT-29 MCL (38.8% and 27.9%, respectively). The differential potencies of DOX closely correlate to the intratumoral pharmacokinetics within MCL's of both cell lines. DOX penetrated faster and washed out slower through the MCL of DLD-1 compared to HT-29 MCL. Distribution of DOX within MCL of DLD-1 was more homogenous compared to HT-29 MCL. Tissue concentration of DOX within MCL of DLD-1 was significantly higher than HT-29 MCL's after 96 h exposure (0.7 and 0.4 µmole/gm tissue, respectively). Concentration of DOX within MCL of both cell lines exceeded the IC50 under monolayer conditions (2.3 ± 0.6 µM and 0.6 ± 0.1 µM, respectively). In addition, DOX was extensively metabolized to less active metabolites (doxorubicinol and doxorubicinone) through the thickness of both MCL's. In conclusion, Intratumoral pharmacokinetic barriers to DOX might be key determinant in drug resistance on the tissue level, despite cellular and molecular events.

Multi-Steps Fragmentation-Ion Trap Mass Spectrometry Coupled to Liquid Chromatography Diode Array System for Investigation of Olaparib Related Substances.

A high-performance liquid chromatography-diode array-mass spectrometric (LC-DAD-MS) method was developed and validated to investigate the related substances of olaparib (OLA) in bulk form. OLA was exposed to acid⁻base hydrolysis, boiling, oxidation with hydrogen peroxide, and UV light followed by LC-DAD-MS analysis. OLA and OLA-related substances were simultaneously and quantitatively monitored by DAD at 278 nm and triple quadrupole mass spectrometry (QQQ-MS). The investigated compounds were auto-scanned by an ion trap MS which applied positive and negative modes separately. The fragmentation pathway was confirmed by applying multi-steps fragmentation to identify the resulted cleaved ions and their parent ion. OLA was found to be sensitive to the alkaline hydrolysis and less sensitive to UV light. Two major hydrolytic degradation products, including the protonated molar ions m/z 299 and m/z 367, were identified. Three potential impurities were also characterized. The LC-MS limit of detection (LOD) and limit of quantification (LOQ) were 0.01 and 0.05 ng/µL, respectively. The quantitative results obtained by LC-DAD was comparable with that of LC-QQQ-MS. The proposed method shows good intra-day and inter-day precision with relative standard deviation (RSD) <2%.

Profiling of esterified fatty acids as biomarkers in the blood of dengue fever patients using a microliter-scale extraction followed by gas chromatography and mass spectrometry.

An improved gas chromatography with mass spectrometry procedure was developed to highlight the esterified fatty acids in 100 µL blood of dengue fever patients in the early febrile phase versus healthy volunteers. 24 adult patients and 24 healthy volunteers were included in this study. The recoveries of targeted esterified fatty acids content were in the range of 92.10-101.00% using methanol/dichloromethane (2:1, v/v) as the extraction solvent. An efficient chromatographic separation of targeted 17 esterified fatty acid methyl esters was obtained. The limits of detection and quantification were within the range of 16-131 and 53-430 ng/mL, respectively. The relative standard deviation of intraday and interday precision values ranged from 0.4 to 5.0%. The statistical data treatment showed a significant decrease of the content of four saturated fatty acids, C14:0, C15:0, C16:0, and C18:0 (P value < 0.05), and also showed a decrease of the content of eight unsaturated fatty acids, C16:1, C18:3n6, C18:2n6, C18:1n9, C20:3n3, C20:4n6, C20:2, and C22:6n3 (P value < 0.05) in dengue fever patients. Moreover, the amount of three omega-6 fatty acids including C18:3n6, C18:2n6, and C20:4n6 was dramatically decreased in the blood of dengue fever patients to a limit of 50 ± 10%.

Bioactive secondary metabolites from the Red Sea marine Verongid sponge Suberea species.

In a continuation of our efforts to identify bioactive compounds from Red Sea Verongid sponges, the organic extract of the sponge Suberea species afforded seven compounds including two new dibrominated alkaloids, subereamollines C and D (1 and 2), together with the known compounds aerothionin (3), homoaerothionin (4), aeroplysinin-1 (5), aeroplysinin-2 (6) and a revised subereaphenol C (7) as ethyl 2-(2,4-dibromo-3,6-dihydroxyphenyl)acetate. The structures of the isolated compounds were assigned by different spectral data including optical rotations, 1D (1H and 13C) and 2D (COSY, multiplicity-edited HSQC, and HMBC) NMR and high-resolution mass spectroscopy. Aerothionin (3) and subereaphenol C (7) displayed potent cytotoxic activity against HeLa cell line with IC50 values of 29 and 13.3 µM, respectively. In addition, aeroplysinin-2 (6) showed potent antimigratory activity against the human breast cancer cell line MDA-MB-231 with IC50 of 18 µM. Subereamollines C and D are new congeners of the previously reported compounds subereamollines A and B with methyl ester functionalities on the side chain. These findings provide further insight into the biosynthetic capabilities of members of the genus Suberea and the chemical diversity as well as the biological activity of these compounds.

The Efficacy of Muscle Energy and Mulligan Mobilization Techniques for the Upper Extremities and Posture after Breast Cancer Surgery with Axillary Dissection: A Randomized Controlled Trial.

Background: Breast cancer surgeries affect the upper extremities and posture. This study aimed to examine the efficacy of muscle energy and Mulligan mobilization techniques on the upper extremities and posture after breast cancer surgery with axillary dissection. Methods: A total of 90 female participants who had undergone breast cancer surgery with axillary dissection were recruited and randomly assigned to three groups. Group A received a combination of the Mulligan and muscle energy techniques, while Groups B and C received either the Mulligan or muscle energy techniques for six weeks, respectively. The study measured the shoulders' range of motion, posture, and upper-extremity disabilities. Outcome measurements were taken at three different time points: baseline, post-intervention, and at eight-week follow-up. Results: All the interventions significantly improved the study outcomes. The combination of the Mulligan and muscle energy techniques was significantly better than a single intervention. Mulligan mobilization was superior to the muscle energy techniques in terms of improving the shoulders' range of motion and disability. The interventions showed a significant effect pre-post-treatment and pre-follow-up but not post-follow-up. Conclusions: The Mulligan mobilization and muscle energy techniques have been found beneficial in improving the postural changes and shoulder outcomes after breast cancer surgery with axillary dissection. The superior effectiveness of the combined interventions points out the importance of integrating multiple therapeutic approaches for optimal outcomes. Regular examination and long-term follow-up assessment are important for studying the effect of rehabilitation interventions in people after the late stages of breast surgery.

A Sensitive Liquid Chromatography-Mass Spectrometric Method for Determination of Bisoprolol in Rat Serum after Pre-Column Derivatization.

Two derivatization regents were reacted with bisoprolol (BIS), followed by liquid-chromatography-mass spectrometric analysis. 3-Bromomethyl-propyphenazone (BMP) and dansyl-chloride (Dns-Cl) were reacted via the secondary amino group using a catalyst to accelerate the reaction progress and completeness with minimal reaction byproducts. The sensitivity and the ionization efficiency of both BIS-methyl-propyphenazone (BIS-MP) and BIS-Dns via electrospray ionization were studied. The sensitivity of BIS-MP was superior to BIS-Dns. The derivatization procedure, extraction procedure, and LC-MS method were optimized and validated to achieve the monitoring of BIS in rat serum at a picogram scale. The calibration curve showed a regression coefficient value of 0.999 within a concentration spanning a range of 10-500 pg/mL. The detection limit and quantitation were 4 and 10 pg/mL, respectively. The intraday and inter-day precision values (% relative standard deviation) ranged from 0.53 to 6.91%, whereas the accuracy values (expressed as % error) ranged from -4.20 to -0.77%. The pharmacokinetic parameters were: 15,280 pg/mL for a maximum concentration of BIS (Cmax) at the maximum time (Tmax) of 1 h. BIS's elimination half-life (t1/2) was determined to be 3 h. The value of the area under the concentration-time curve (AUC0 - t) was 34,370 pg/mL h.

Local inflammation influences oestrogen metabolism in prostatic tissue.

UNLABELLED: What's known on the subject? and What does the study add? The role of oestrogen in prostatic inflammation has been extensively shown. The catechol oestrogens are known to be more potent oestrogenic moieties that not only aggravate the inflammatory response in situ, but are also believed to have oxidative stress and genotoxic effects. The present study highlights a significant role of inflammation in oestrogen metabolism and, particularly, in generating 'bad' oestrogen metabolites. This finding may pave the way for new therapeutic methods for the treatment and/or prevention of prostate diseases. OBJECTIVE: • To investigate the impact of experimentally induced inflammation on oestrogen metabolism in rat prostate. MATERIALS AND METHODS: • Prostatitis was induced in normal and oestrogen-treated male rats by injecting 2% carrageenan solution into the ventral prostate. After 48 h, the rats were killed and the ventral prostate was collected. • Prostatic inflammation and proliferation were confirmed by gross visual evidence, histology and elevated tumour necrosis factor-α, prostaglandin E(2) and cyclin-D(1) . • Expression of oestrogen-metabolizing enzymes was assessed using capillary electrophoresis, and oestrogen metabolites within prostate tissue were assayed using liquid chromatography mass spectrometry. RESULTS: • Animals exposed to carrageenan insult combined with oestrogen treatment showed the most prominent inflammatory and proliferative response. • Treatment of animals with oestrogen alone induced moderate inflammation and proliferation. • Oestrogen-metabolizing enzymes were overexpressed in animals with experimental prostatitis with sequential accumulation of catechol oestrogens within prostatic tissues. • Oestrogen receptor-α was underexpressed in the prostatitis with oestrogen group, while oestrogen receptor-ß was overexpressed. CONCLUSIONS: • The present work provides experimental evidence that local inflammation enhances oestrogen synthesis and directs oestrogen metabolism to generate catechol oestrogens within prostatic tissues. • This may contribute, at least partly, to enhanced prostatic cell proliferation.

Utility of Kolliphor RH 40 in micellar sensitized fluorescence of the novel tyrosine kinase inhibitor "Erdafitinib": Application to human plasma.

Erdafitinib is the first treatment targeting susceptible fibroblast growth factor receptor (FGFR) genetic alterations in patients with locally advanced or metastatic urothelial carcinoma. A simple and precise spectrofluorimetric method was developed for its determination depending on fluorescence enhancement using Kolliphor RH 40 micellar medium at pH 10. The fluorescence intensity was measured at 495 nm after excitation at 410 nm. Different experimental parameters affecting the fluorescence intensity, including type of organized medium, diluting solvent, buffer type and pH were studied during optimization phase. Validation according to ICH Q2(R1) guidance was fully fulfilled. The method was linear over the range of 50 - 800 ng/mL. The lower limit of detection (LOD) and lower limit of quantitation (LOQ) were 14.36 and 43.50 ng/mL, respectively. The relative standard deviation values of intraday and interday precisions were less than 1.93 %. The proposed method was successfully applied to laboratory-prepared tablets. Furthermore, the high sensitivity of the method allowed its application on spiked human plasma samples with a high percent of recovery. The greenness of the method was investigated as an Eco-friendly alternative for erdafitinib determination with minimal organic solvent consumption.

LC-MS/MS monitoring of the colorectal carcinoma cellular uptake and entrapment of sorafenib and its N-oxide active metabolite.

Sorafenib (SOR) is a multikinase inhibitor with a mild activity against colorectal cancer cells due to multi-drug resistance mechanisms. Potentiated SOR activity was expected upon combination with some ginger derived compounds due to their interference with intracellular drug metabolism. Studying such combination necessitates the development of a sensitive validated LC-MS/MS method for the determination of intra and extracellular concentration of SOR and its N-oxide metabolite (SNX) in colorectal cancer cells. SOR, SNX and the internal standard (diclofenac sodium) were efficiently separated on Eclipse plus C18 column (3.0 ×150 mm, 5 µm) using isocratic elution with acetonitrile and 0.01 M ammonium formate aqueous solution containing 0.1% formic acid (69:31, v/v). Sample pretreatment using solid phase extraction was optimized and the mean percent recoveries were more than 97.01% for both analytes. Detection was conducted at positive ion multiple reaction monitoring (MRM) mode and the monitored mass transitions were 465.2 → 252.2 for SOR and 481.1 → 286.0 for SNX. The method was linear over the range 0.25 - 200.00 ng/mL (r2 ≥ 0.9992) for SOR and 0.10 - 125.00 ng/mL (r2 ≥ 0.9990) for SNX in both intra and extracellular matrices. The lower limits of quantification (LLOQ) were 0.25 and 0.10 ng/mL for SOR and SNX, respectively. Accuracies were within 94.25 - 109.45% and precision CV values did not exceed 7.63%. The method was able to monitor the cellular uptake and entrapment of both analytes and to prove the positive effect of the ginger derived compounds on SOR activity.

Association of cytochromes P450 3A4*22 and 3A5*3 genotypes and polymorphism with response to simvastatin in hypercholesterolemia patients.

BACKGROUNDS: Inter-individual variability in response to statin was mainly due to genetic differences. This study aimed to investigate the association of CYP3A4*22 (rs35599367), CYP3A5*3 (rs776746) single nucleotide polymorphism (SNP) with response to simvastatin in hypercholesterolemia patients conducted at King Abdulaziz University hospital (KAUH) in Jeddah, Saudi Arabia. PATIENTS AND METHODS: A total of 274 participants were registered in the current study. Hypercholesterolemic patients taking simvastatin 20 mg (n = 148) and control subjects (n = 126) were tested for rs35599367 and rs776746 genotypes using Custom Taqman ® Assay Probes. Response to simvastatin in these patients was assessed by determination of low density lipoprotein (LDL-C), total cholesterol (TC) and by measuring statin plasma levels using Liquid Chromatography-Mass Spectrometry (LC-MS). RESULTS: None of the participants carried a homozygous CYP3A4*22 mutant genotype, while 12 (4.4%) individuals had a heterozygous genotype and 262 (95.6%) had a wild homozygous genotype. The CYP3A5*3 allele was detected in the homozygous mutant form in 16 (5.8%) individuals, while 74 (27.0%) individuals carried the heterozygous genotype and 184 (67.2%) carried the wildtype homozygous genotype. Of the patient group, 15 (11%) were classified as intermediate metabolizers (IMs) and 133 (89%) as extensive metabolizers (EMs). Plasma simvastatin concentrations for the combined CYP3A4/5 genotypes were significantly (P<0.05) higher in the IMs group than in the EMs group. TC and plasma LDL-C levels were also significantly (P<0.05) higher in IMs than in EMs. CONCLUSION: The present study showed associations between CYP3A4*22 (rs35599367) and CYP3A5*3 (rs776746) SNP combination genotypes with response to statins in hypercholesterolemia. Patients who had either a mutant homozygous allele for CYP3A5*3 or mutant homozygous and heterozygous alleles for CYP3A4*22 showed increased response to lower TC and LDL-C levels.

Etodolac Fortified Sodium Deoxycholate Stabilized Zein Nanoplatforms for Augmented Repositioning Profile in Human Hepatocellular Carcinoma: Assessment of Bioaccessibility, Anti-Proliferation, Pro-Apoptosis and Oxidant Potentials in HepG2 Cells.

This work aimed to enhance the purposing profile of Etodolac (ETD) in Human Hepatocellular Carcinoma (HCC) HepG2 cells using sodium deoxycholate stabilized zein nanospheres (ETD-SDZN NSs). ETD-SDZN NSs were formulated using the nan-precipitation method and were characterized, in particular, in terms of mean particle size, zeta potential, encapsulation efficiency, colloidal stability and bioaccessibility. Estimations of cytotoxicity, cellular uptake, cell cycle progression, Annexin-V staining, mRNA expression of apoptotic genes and oxidative stress evaluations were conducted. The ETD-SDZN NSs selected formula obtained an average particle size of 113.6 ± 7.4 nm, a zeta potential value of 32.7 ± 2.3 mV, an encapsulation efficiency of 93.3 ± 5.2%, enhanced bioaccessibility and significantly reduced IC50 against HepG2 cells, by approximately 13 times. There was also enhanced cellular uptake, accumulation in G2-M phase and elevated percentage cells in pre-G1 phase, significant elevated mRNA expression of P53, significant reduced expression of Cyclin-dependent kinase 1 (CDK1) and Cyclooxygenase-2 (COX-2) with enhanced oxidative stress by reducing glutathione reductase (GR) level, ameliorated reactive oxygen species (ROS) generation and lipid peroxidation outputs. ETD-SDZN NSs obtained a supreme cell death-inducing profile toward HepG2 cells compared to free ETD. The method of formulation was successful in acquiring the promising profile of ETD in HCC as a therapeutic molecule due to ameliorated cellular uptake, proapoptotic and oxidant potentials.

HPLC-UV determination of erdafitinib in mouse plasma and its application to pharmacokinetic studies.

Erdafitinib is a recently approved fibroblast growth factor receptor (FGFR) inhibitor. It is the first treatment targeting susceptible FGFR genetic alterations for patients with metastatic bladder cancer. A simple validated HPLC-UV method was developed for the determination of erdafitinib in mouse plasma. Erdafitinib and internal standard (rivaroxaban) were efficiently separated on Eclipse plus C18 column (4.6 × 100 mm, 3.5 µm). The mobile phase consisted of acetonitrile and 0.01 M ammonium acetate aqueous solution, adjusted to pH 4.4 with acetic acid (26:74, v/v) and it was eluted isocratically at a flow rate of 1.2 mL/min. The UV detection was at 292 nm and the total run time for each sample was 11 min. The method linearity was validated over the range of 0.05-2.00 µg/mL (r2 ≥ 0.9992) and the lower limit of quantification (LLOQ) was 0.05 µg/mL. The within-run and between-run accuracies were 98.56 and 99.24%, respectively while the CV of the method precision did not exceed 6.52%. Plasma samples were extracted using a solid phase extraction procedure and the extraction recoveries were 97.90 ± 4.58%. The method was optimized for the sensitive determination of the studied drug in mouse plasma and was successfully applied to its pharmacokinetic studies.

Formulation, optimization, and nephrotoxicity evaluation of an antifungal in situ nasal gel loaded with voriconazole‒clove oil transferosomal nanoparticles.

Fungal infections of the paranasal cavity are among the most widely spread illnesses nowadays. The aim of the current study was to estimate the effectiveness of an in situ gel loaded with voriconazole‒clove oil nano-transferosomes (VRC-CO-NT) in enhancing the activity of voriconazole against Aspergillus flavus, which causes rhinosinusitis. The nephrotoxic side effects of voriconazole may be reduced through the incorporation of the clove oil, which has antioxidant activity that protects tissue. The Box‒Behnken design was applied to formulate the VRC-CO-NT. The particle size, entrapment efficiency, antifungal inhibition zone, and serum creatinine concentration were considered dependent variables, and the soybean lecithin, VRC, and CO concentrations were considered independent ones. The final optimized formulation was loaded into a deacetylated gellan gum base and evaluated for its gelation, rheological properties, drug release profile, permeation capabilities, and in vivo nephrotoxicity. The optimum formulation was determined to be composed of 50 mg/mL lecithin, 18 mg/mL VRC, and 75 mg/mL CO, with a minimum particle size of 102.96 nm, an entrapment efficiency of 71.70%, an inhibition zone of 21.76 mm, and a serum creatinine level of 0.119 mmol/L. The optimized loaded in situ gel released 82.5% VRC after 12 hours and resulted in a 5.4-fold increase in drug permeation. The in vivo results obtained using rabbits resulted in a nonsignificant differentiation among the renal function parameters compared with the negative control group. In conclusion, nasal in situ gel loaded with VRC-CO-NT is considered an efficient novel carrier with enhanced antifungal properties with no signs of nephrotoxicity.

A new approach for characterization of phosphatidylcholines and lysophosphatidylcholine in human plasma.

Aim: Characterization of phosphatidylcholines (PCs) and lysophosphatidylcholine in human plasma using LC-IT-MSn. The characterization approach was based on trapping the eluted positive ions and applying low voltage for fragmentation to MS2 and further fragmentation of the most abundant two peaks to obtain MS3. This approach allowed linking the MS3 data to MS2 and precursor ion. Methodology: The fatty acid part, at sn-1 and sn-2 of the glycerol backbone, could be identified based on the favored cleavage pathway. Conclusion: The dysregulated PCs and lysophosphatidylcholines in human plasma obtained from acute coronary syndrome cases, and Type 2 diabetes patients suffering no coronary syndromes were estimated and matched versus healthy volunteers. An epoxide form of 16:0-18:2 PC was confirmed, m/z 774.6.

LC-MS/MS determination of avanafil and its metabolites in rat plasma and brain: pharmacokinetic study after oral administration and transdermal film application.

Avanafil (AVA) has been FDA approved in 2012 as a phosphodiesterase-type five inhibitor drug (PDE-5), for the treatment of erectile dysfunction (ED). It was necessary to study the pharmacokinetics and bioavailability parameters of AVA since it exhibits side effects, a long time from drug administration. As a result of this, we described a sensitive high-performance-liquid chromatography-triple quad-mass spectrometric method (LC-QqQ-MS) for the analysis of AVA in rat plasma and brain. Furthermore, the concentrations of AVA and its primary metabolites were determined in rat brain since it is known that PDE-5 inhibitor drugs are capable of crossing the blood-brain barrier (BBB). The liquid-liquid extraction method was developed, optimized, and applied for maximum recovery of AVA from plasma and brain homogenates. The percentage of recovery was 96.60 ± 2.44% and 94.50 ± 1.86%, in rat plasma and brain homogenate, respectively. The separation was performed on a Nucleodur C18 column, with mobile phase composed of 0.1% formic acid and acetonitrile (29 : 71, v/v), at flow rate 0.5 mL min-1, and monitored with QqQ-MS applying positive multiple reaction monitoring (MRM) mode. The calculated pharmacokinetic parameters, noncompartmental model, were: C max 1503.82 ± 354.11 ng mL-1 with a t 1/2 value of 4.87 ± 0.42 h and C max 141.94 ± 22.57 ng mL-1 with a t 1/2 value of 7.05 ± 1.59 h, for oral AVA suspension and transdermal film, respectively. The average percentage of total metabolites in plasma and brain was 27.1 ± 2.2% and 7.0 ± 1.0%, respectively.

A sensitive liquid chromatography-tandem mass spectrometric method for determination of five ß-blockers after labeling with either hydrazonoyl chloride or dansyl chloride reagent.

A sensitive liquid chromatography-tandem mass spectrometry (LC-MS) method was developed for the screening of five ß-blockers (BBs), including atenolol, metoprolol, bisoprolol, propranolol, and betaxolol, in rabbit plasma. An inhouse prepared hydrazonoyl chloride compound (UOSA54) and dansyl chloride (DNS) were successfully coupled with BBs via the amino functional group and analyzed by LC-MS/MS. The excess hydrazonoyl chloride was characterized by a negligible ionization suppression at the electrospray-ionization (ESI) source, which enables the analysis of BBs at a low concentration level. The relative ESI-MS response of derivatized to underivatized BBs was enhanced 2.3 to 3.7-fold. The developed method could be applied for screening of BBs in samples by searching the most abundant MS product ions, including m/z 169 and 211, in addition to the precursor ion and the cleavage of ether moiety. The method could be applied for trace analysis and screening of BBs abuse. The use of UOSA54 was adventitious over dansylated derivatives because of minimal reaction by-products and the negligible ionization suppression effect. The extraction efficiency of BBs from rabbit plasma was improved to reach 77.5-93.9% using tert-butylamine and Chromabond® C18ec-100 mg column. The optimal reaction conditions were optimized and validated. The linear range of analyzed BBs in rabbit plasma was within the range of 0.1 to 25.0 ng/mL, while the limit of quantification (LO Q) was ranged from 0.10 to 0.25 ng/mL.

Ultra-performance liquid chromatography-tandem mass spectrometric method for quantitation of the recently Food and Drug Administration approved combination of vaborbactam and meropenem in human plasma.

A parenteral medical combination containing vaborbactam and meropenem is used mainly to treat complicated urinary tract infections. A novel ultra-performance liquid chromatography tandem mass spectrometric method was developed for the sensitive determination of both compounds in human plasma. Sample preparation was performed by precipitation technique. The chromatographic separation was accomplished using the Acquity C18-BEH column, 0.01 M ammonium formate: acetonitrile (47 : 53, v/v) as a mobile phase with a flow rate of 0.2 ml min-1. Analytes were monitored by applying multiple reaction monitoring. The bioanalytical validation criteria were conducted following the Food and Drug Administration recommendations. The method was linear within range 0.5 to 50 µg ml-1, for both drugs. The intra-day and inter-day precision, as coefficient variation (% CV) and the accuracy, as % bias did not exceed 15% for both drugs. The percentage recovery of targeted analytes was not less than 77%, calculated at three quality control levels. The proposed method showed a suitable lower level of quantification value of 0.50 µg ml-1 for both analytes, which is far lower than the expected C max, which permits the use of this method for pharmacokinetic studies. The proposed method proved to be useful for the evaluation of this combination in both human plasma and pharmaceutical formulation.

Sensitive determination of ranitidine in rabbit plasma by HPLC with fluorescence detection.

A sensitive high-performance liquid chromatographic method for determination of ranitidine (RAN) in rabbit plasma is described. The method is based on liquid-liquid extraction, labeling with dansyl chloride and monitoring with fluorescence detector at 338nm (ex)/523nm (em). Plasma samples were extracted with diethyl ether alkalinized with 1M sodium hydroxide. Ephedrine HCl (EPH-HCl) was used as internal standard. Both, RAN and EPH were completely derivatized after heating at 60 degrees C for 10min in sodium bicarbonate solution (pH 9.5). The derivatized samples were analyzed by HPLC using Agilent Zorbax Extended C18 column (150mmx4.6mm i.d.) and mobile phase consists of 48% acetonitrile and 52% sodium acetate solution (0.02M, pH 4.6). The linearity of the method was in the range of 0.025-10microg/ml. The limits of detection (LOD) and quantification (LOQ) were 7.5+/-0.18 and 22.5+/-0.12ng/ml, respectively. Ranitidine recovery was 97.5+/-1.1% (n=6; R.S.D.=1.8%). The method was applied on plasma collected from rabbits at different time intervals after oral administration of 5mg/kg ranitidine HCl.

Stress degradation studies on betahistine and development of a validated stability-indicating assay method.

The purpose of this work was to study the stability of betahistine (BET) at different stress conditions and to develop a sensitive stability-indicating high-performance liquid chromatographic (HPLC) assay method. The stress conditions applied were including the effect of heat, moisture, acid-base, and ultra-violet (UV) light. Betahistine and its decomposition products were derivatized by reaction with dansyl chloride (Dan-Cl) and analyzed by HPLC equipped with fluorescence detector (FL) set at 336 and 531 nm as excitation and emission wavelengths, respectively. The drug was particularly labile at UV light and oxygen rich media. Two potential degradation products could be separated and identified by spectral methods. The chromatographic method involved Zorbax Eclipse XDB-C(18) column kept at 30+/-2 degrees C and a gradient elution with mobile phase composed of acetonitrile and 0.02 mol L(-1) sodium acetate. The response factor of dansylated BET monitored by fluorescence detection was 32 times more than its UV response. The calibration curve of BET in bulk form was linear from 0.005 to 4.2 ng microL(-1). Intraday and interday precision were less than 0.04% (CV), and accuracy was between 99.2% and 100.9% over 2.0 ng microL(-1). The limit of detection was 0.002 ng microL(-1). The method was also validated for sample stability during reaction, robustness and selectivity. The method was applied for purity testing of betahistine in tablet form.