Development and validation of an ultra-performance liquid chromatography-tandem mass spectrometry method for the quantification of the antimalarial drug pyronaridine in human whole blood.

Malaria remains a major health concern, aggravated by emerging resistance of the parasite to existing treatments. The World Health Organization recently endorsed the use of artesunate-pyronaridine to treat uncomplicated malaria. However, there is a lack of clinical pharmacokinetic (PK) data of pyronaridine, particularly in special populations such as children and pregnant women. Existing methods for the quantification of pyronaridine in biological matrices to support PK studies exhibit several drawbacks. These include limited sensitivity, a large sample volume required, and extensive analysis time. To overcome these limitations, an ultra-performance reversed-phase liquid chromatography tandem-mass spectrometry method to determine pyronaridine was developed and validated according to international guidelines. The method enabled fast and accurate quantification of pyronaridine in whole blood across a clinically relevant concentration range of 0.500-500 ng/mL (r2 ≥ 0.9963), with a required sample volume of 50 µL. Pyronaridine was extracted from whole blood using liquid-liquid extraction, effectively eliminating the matrix effect and preventing ion enhancement or suppression. The method achieved a satisfactory reproducible sample preparation recovery of 77%, accuracy (as bias) and precision were within ±8.2% and ≤5.3%, respectively. Stability experiments demonstrated that pyronaridine was stable for up to 315 days when stored at -70°C. Adjustments to the chromatographic system substantially reduced carry-over and improved sensitivity compared to prior methods. The method was successfully applied to quantify pyronaridine in whole blood samples from a selection of pregnant malaria patients participating in the PYRAPREG clinical trial (PACTR202011812241529) in the Democratic Republic of the Congo, demonstrating its suitability to support future PK studies. Furthermore, the enhanced sensitivity allows for the determination of pyronaridine up to 42 days post-treatment initiation, enabling assessment of the terminal elimination half-life.

Penetration, distribution, and elimination of remofuscin/soraprazan in Stargardt mouse eyes following a single intravitreal injection using pharmacokinetics and transmission electron microscopic autoradiography: Implication for the local treatment of Stargardt's disease and dry age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of blindness in older people in the developed world while Stargardt's disease (SD) is a juvenile macular degeneration and an orphan disease. Both diseases are untreatable and are marked by accumulation of lipofuscin advancing to progressive deterioration of the retinal pigment epithelium (RPE) and retina and subsequent vision loss till blindness. We discovered that a small molecule belonging to the tetrahydropyridoether class of compounds, soraprazan renamed remofuscin, is able to remove existing lipofuscin from the RPE. This study investigated the drug penetration, distribution, and elimination into the eyes of a mouse model for increased lipofuscinogenesis, following a single intravitreal injection. We measured the time course of concentrations of remofuscin in different eye tissues using high-performance liquid chromatography combined with mass spectroscopy (HPLC-MS). We also visualized the penetration and distribution of 3 H-remofuscin in eye sections up to 20 weeks post-injection using transmission electron microscopic (TEM) autoradiography. The distribution of silver grains revealed that remofuscin accumulated specifically in the RPE by binding to the RPE pigments (melanin, lipofuscin and melanolipofuscin) and that it was still detected after 20 weeks. Importantly, the melanosomes in choroidal melanocytes only rarely bind remofuscin emphasizing its potential to serve as an active ingredient in the RPE for the treatment of SD and dry AMD. In addition, our study highlights the importance of electron microscopic autoradiography as it is the only method able to show drug binding with a high intracellular resolution.

First-in-Human Evaluation of the Safety, Tolerability, and Pharmacokinetics of a Neuroprotective Poly (ADP-ribose) Polymerase-1 Inhibitor, JPI-289, in Healthy Volunteers.

BACKGROUND: Poly (ADP-ribose) polymerase-1 (PARP-1) inhibitor has therapeutic potential for acute ischemic stroke by suppressing microglial activation and facilitating neuroprotection. In this first-in-human study, we investigate the safety, tolerability and pharmacokinetics (PK) of JPI-289 in healthy male volunteers. SUBJECTS AND METHODS: In single ascending dose (SAD) study, 35, 75, 150, 300, 600 mg JPI-289 or placebo was infused intravenously over 30 minutes to 40 subjects. In multiple ascending dose (MAD) study, 150, 300, 450 mg JPI-289 or placebo was infused over 1 hour every 12 hours to each of 24 subjects for 3.5 days (7 times). The plasma and urine concentrations of JPI-289 and its metabolites were determined. RESULTS: In the SAD study, AUClast and Cmax tended to increase supra-proportionally especially at higher doses in SAD study. However, Cmax showed dose-proportionality in the range of 75-600mg. JPI-289 reached a mean Tmax within 0.50 hour after dosing and a mean elimination half-life (t1/2) was 2.18 to 3.21 hours. In the MAD study, observed accumulation index ranged from 1.52 to 1.76. The effective half-life of JPI-289 was 1.88 to 3.05 hours, indicating that the plasma JPI-289 concentration rapidly reaches steady state. % recovered of JPI-289 measured in urine was 1.59-9.05%. In both studies, concentration of metabolites was less than 10% of JPI-289. Adverse events reported in the study were all mild in intensity and resolved without any sequelae. CONCLUSION: The tolerable dose ranges and pharmacokinetic characteristics of JPI-289 evaluated in these studies will be useful in further clinical development of JPI-289.

LC-MS/MS method for quantitation of the CK2 inhibitor silmitasertib (CX-4945) in human plasma, CSF, and brain tissue, and application to a clinical pharmacokinetic study in children with brain tumors.

Silmitasertib (CX-4945) as a potent and selective inhibitor of CK2 exhibited promising in vitro and in vivo anti-cancer activity. An assay employing cation-exchange solid phase extraction (SPE) followed by LC-MS/MS analysis was successfully developed and validated for the quantitation of silmitasertib in human plasma, brain tissue, and human cerebrospinal fluid (CSF). Reverse phase chromatographic separation was achieved using Synergi™ hydro-RP column (4 µm, 75 × 2.0 mm) and gradient elution with 5 mM ammonium formate aqueous solution (pH 6.5) as mobile phase A and 0.1% formic acid in acetonitrile as mobile phase B. Multiple reaction monitoring (MRM) transition of m/z 350.2 â†’ 223.2 and m/z 316.2 â†’ 223.2 were chosen for detection of silmitasertib and internal standard (CX-4786) respectively. Since silmitasertib concentration in patient plasma is expected to be in a wide range due to the study design, two calibration curves with range 0.2-125 ng/ml and 32-20,000 ng/ml were established. A different curve ranging from 2 to 40 ng/g was used for measurement of silmitasertib in brain tissue, while another calibration curve ranging from 0.2 to 20 ng/ml was established for CSF. All these calibration curves corresponding to different matrices showed good linearity (R2 > 0.99) over the concentration range. This assay demonstrated excellent precision below 15% and accuracies between 85% and 115% within-day and between-day for all the concentration levels in each matrix. This assay was also validated for each matrix for selectivity, sensitivity, matrix effects, recovery, and stability. We applied the validated method to the analysis of plasma silmitasertib for a clinical study.

A high-performance liquid chromatography-tandem mass spectrometry method for the determination of lifrafenib, a novel RAF kinase and EGFR inhibitor, in human plasma and urine and its application in clinical pharmacokinetic study.

Lifirafenib (BGB-283), a dual inhibitor trageting BRAF kinase and EGFR, showed favorable efficacy and safety in treating patients with different cancer types harboring mutations in BRAF, KRAS and NRAS. In order to support the clinical pharmacokinetic study, a sensitive high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed and validated to quantify lifirafenib concentration in human plasma and urine. Plasma samples were purified using protein precipitation. Urine samples were pre-treated by adding tween 80 with the purpose of preventing non-specific adsorption, then extracted by centrifugation. Chromatographic separation was achieved on Phenomenex Luna C18 column with a gradient elution. The mass detection was performed using electrospray ionization (ESI) source under multiple reaction monitoring (MRM) in positive ionization mode. The method was fully validated, and the result of inter-assay and intra-assay precisions were less than 15% and the accuracy within the scope of ±15%. The linear range for plasma and urine covered from 10 to 10,000 ng/mL and 1 to 200 ng/mL, respectively, with correlation coefficients of 0.99. The validation for matrix effect, recovery, stability and carryover were met the acceptance criteria. The method showed robust and sensitive, it successfully fulfilled the requirement of clinical pharmacokinetic study of lifirafenib in Chinese patients with locally advanced or metastatic solid tumors.

New spectrophotometric/chemometric assisted methods for the simultaneous determination of imatinib, gemifloxacin, nalbuphine and naproxen in pharmaceutical formulations and human urine.

In this paper, novel univariate and multivariate regression methods along with model-updating technique were developed and validated for the simultaneous determination of quaternary mixture of imatinib (IMB), gemifloxacin (GMI), nalbuphine (NLP) and naproxen (NAP). The univariate method is extended derivative ratio (EDR) which depends on measuring every drug in the quaternary mixture by using a ternary mixture of the other three drugs as divisor. Peak amplitudes were measured at 294nm, 250nm, 283nm and 239nm within linear concentration ranges of 4.0-17.0, 3.0-15.0, 4.0-80.0 and 1.0-6.0µgmL-1 for IMB, GMI, NLP and NAB, respectively. Multivariate methods adopted are partial least squares (PLS) in original and derivative mode. These models were constructed for simultaneous determination of the studied drugs in the ranges of 4.0-8.0, 3.0-11.0, 10.0-18.0 and 1.0-3.0µgmL-1 for IMB, GMI, NLP and NAB, respectively, by using eighteen mixtures as a calibration set and seven mixtures as a validation set. The root mean square error of predication (RMSEP) were 0.09 and 0.06 for IMB, 0.14 and 0.13 for GMI, 0.07 and 0.02 for NLP and 0.64 and 0.27 for NAP by PLS in original and derivative mode, respectively. Both models were successfully applied for analysis of IMB, GMI, NLP and NAP in their dosage forms. Updated PLS in derivative mode and EDR were applied for determination of the studied drugs in spiked human urine. The obtained results were statistically compared with those obtained by the reported methods giving a conclusion that there is no significant difference regarding accuracy and precision.

Identification and validation of novel prognostic markers in Renal Cell Carcinoma.

Kidney cancer (Renal Cell Carcinoma (RCC)) is one of the most deadly malignancies due to frequent late diagnosis and poor treatment options. Histologically, RCC embraces a wide variety of different subtypes with the clear cell variant (ccRCC) being the most common, accounting for 75-90% of all RCCs. At present, the surveillance protocols for follow-up of RCC patients after radical nephrectomy are based on the American Joint Committee on Cancers (AJCC) pathological tumor-node-metastasis (TNM) classification system. Other comprehensive staging modalities have emerged and have been implemented in an attempt to improve prognostication by combining other pathological and clinical variables, including Fuhrman nuclear grade and Leibovich score. However, even early stage tumors remain at risk of metastatic progression after surgical resection and 20-40% of patients undergoing nephrectomy for clinically localized RCC will develop a recurrence. Identifying this high-risk group of RCC patients remains a challenge. Hence, novel molecular prognostic biomarkers are needed to better predict clinical outcomes. An intensive search within this field has been ongoing in the past few years, and the three main predictive and prognostic markers validated in RCC are Von Hippel Lindau (VHL), vascular endothelial growth factor (VEGF) and carbonic anhydrase IX (CAIX). Nonetheless, the use of these is still debated and none of them have yet been implemented in clinical routine. RCC is resistant to conventional oncological therapies, such as chemotherapy and radiation. The availability of novel targeted therapies directed against tumorigenic and angiogenic pathways have increased over the last years, and the outcome of patients with advanced RCC has significantly improved as a consequence. Unfortunately, all patients eventually become resistant. Thus, the development of novel targeted therapies is of great importance. The aim of this thesis was therefore to contribute in the search for novel prognostic molecular markers in RCC and to identify novel targeted therapies by in-vitro studies. This was specifically conducted by investigating; 1) The impact of symptom presentation of RCC on prognosis, 2) The expression of Calcium-activated potassium channels in RCC, the correlation of KCa3.1 to prognosis in ccRCC and the ability of TRAM-34, RA-2 and Paxilline to inhibit the proliferation of ccRCC cell lines in-vitro, 3) The gene expression and prognostic value of 19 selected genes in ccRCC and 4) The expression of the protein kinase CK subunits in subtypes of RCC, the prognostic impact of high protein expression of the CK2α subunit in ccRCC and the ability of CX-4945 and E9 to inhibit ccRCC growth in-vitro. Our molecular study cohort consisted of 155 patients with different subtypes of RCC and the benign renal neoplasm, oncocytoma. They were diagnosed in Region of Southern Denmark in 2001-2013. Frozen tissue from tumor and normal renal cortex parenchyma, together with paraffin-embedded tissue was available for every patient. We performed gene expression analysis by qRT-PCR, immunohistochemical staining of Tissue Micro Arrays, protein kinase activity analysis and functional studies. Study I was performed as a descriptive observational study focusing on the prognostic impact of symptom presentation in RCC. We included 204 patients with renal neoplasms diagnosed in 2011-2012. Incidentally discovered RCC without symptomatic presentation had overall a better prognosis, and presented with smaller tumors, a lower T-stage, lower Fuhrman grade and lower Leibovich score. In addition, the non-symptomatic patient group experienced metastatic disease less frequently. In study II we focused on the expression of two calcium-activated potassium channels in ccRCC and oncocytoma. Both KCa3.1 and KCa1.1 were higher expressed in ccRCC compared to oncocytoma. High expression of KCa3.1 was moreover correlated with poor progression free survival of ccRCC. Functional studies provided new insights since we could detect currents compatible with KCa3.1 and KCa1.1 in the cell membrane of primary and commercial ccRCC cell lines. Nonetheless, we were not able to show any significant inhibition of cell growth by the selective inhibitors of KCa3.1 and KCa1.1, TRAM-34, RA-2 and Paxilline. In study III our aim was to investigate the prognostic role of 19 genes selected on the basis of an earlier study done by the group. We used Taqman® Low Density Array to perform a quantitative real-time PCR analysis. By selecting an optimal cut-point and correct for overestimation of the p-value, we could identify three genes with impact on prognosis of ccRCC in both univariate and multivariate analysis. High expression of the genes SPP1 and CSNK2A1 (encoding Osteopontin and CK2α respectively) correlated with poor prognosis while high expression of DEFB1 (encoding ß-Defensin) correlated with better prognosis. Study IV focused on validating the results obtained in Paper III by investigating the protein expression of CK2α (Protein kinase 2, alpha subunit) in the different subtypes of RCC and oncocytoma. Furthermore, we investigated whether protein expression of CK2α in ccRCC correlated with prognosis. Here we could show, that a positive nuclear staining was a marker of poor prognosis in high-stage ccRCC. Moreover, enzyme activity analysis revealed a higher activity of the protein kinase in tumor tissue of ccRCC than in normal renal cortex. Novel insights were provided in a proliferation study where we investigated the selective inhibitors of CK2α, CX-4945 and E9. CX-4945 was able to inhibit ccRCC cell growth by nearly 50%. All together the studies presented in this thesis add additional information to the ongoing research within identification of novel prognostic markers in ccRCC. We have discovered four new molecular markers, which reliably can predict prognosis at the time of diagnosis. Additionally, we identified CK2α as a novel therapeutic target of ccRCC. The studies suggest further research to validate the findings on larger cohorts and thereby obtain more insight into the involved pathways. Future research initiatives based on the results presented in this thesis could clarify the potential role of CX-4945 as a novel targeted treatment of ccRCC patients.

Ecological risk assessment of pharmaceuticals in the receiving environment of pharmaceutical wastewater in Pakistan.

The pharmaceutical industry of Pakistan is growing with an annual growth rate of 10%. Besides this growth, this industry is not complying with environmental standards, and discharging its effluent into domestic wastewater network. Only limited information is available about the occurrence of pharmaceutical compounds (PCs) in the environmental matrices of Pakistan that has motivated us to aim at the occurrence and ecological risk assessment of 11 PCs of different therapeutic classes in the wastewater of pharmaceutical industry and in its receiving environmental matrices such as sludge, solid waste and soil samples near the pharmaceutical formulation units along Shiekhupura road, Lahore, Pakistan. Target PCs (paracetamol, naproxen, diclofenac, ibuprofen, amlodipine, rosuvastatin, ofloxacin, ciprofloxacin, moxifloxacin, sparfloxacin and gemifloxacin) were quantified using in-house developed HPLC-UV. Ibuprofen (1673µg/L, 6046µg/kg, 1229µg/kg and 610µg/kg), diclofenac (836µg/L, 4968µg/kg, 6632µg/kg and 257µg/kg) and naproxen (464µg/L, 7273µg/kg, 4819µg/kg and 199µg/kg) showed the highest concentrations among 11 target PCs in wastewater, sludge, solid waste and soil samples, respectively. Ecological risk assessment, in terms of risk quotient (RQ), was also carried out based on the maximum measured concentration of PCs in wastewater. The maximum RQ values obtained were with paracetamol (64 against daphnia), naproxen (177 against fish), diclofenac (12,600 against Oncorhynchus mykiss), ibuprofen (167,300 against Oryzias latipes), ofloxacin (81,000 against Pseudomonas putida) and ciprofloxacin (440 against Microcystis aeruginosa). These results show a high level of ecological risk due to the discharge of untreated wastewater from pharmaceutical units. This risk may further lead to food web contamination and drug resistance in pathogens. Thus, further studies are needed to detect the PCs in crops as well as the government should strictly enforce environmental legislation on these pharmaceutical units.

Structural elucidation of gemifloxacin mesylate degradation product.

Gemifloxacin mesylate (GFM), chemically (R,S)-7-[(4Z)-3-(aminomethyl)-4-(methoxyimino)-1-pyrrolidinyl]-1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboxylic acid methanesulfonate, is a synthetic broad-spectrum antibacterial agent. Although many papers have been published in the literature describing the stability of fluorquinolones, little is known about the degradation products of GFM. Forced degradation studies of GFM were performed using radiation (UV-A), acid (1 mol L(-1) HCl) and alkaline conditions (0.2 mol L(-1) NaOH). The main degradation product, formed under alkaline conditions, was isolated using semi-preparative LC and structurally elucidated by nuclear magnetic resonance (proton - (1) H; carbon - (13) C; correlate spectroscopy - COSY; heteronuclear single quantum coherence - HSQC; heteronuclear multiple-bond correlation - HMBC; spectroscopy - infrared, atomic emission and mass spectrometry techniques). The degradation product isolated was characterized as sodium 7-amino-1-pyrrolidinyl-1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboxylate, which was formed by loss of the 3-(aminomethyl)-4-(methoxyimino)-1-pyrrolidinyl ring and formation of the sodium carboxylate. The structural characterization of the degradation product was very important to understand the degradation mechanism of the GFM under alkaline conditions. In addition, the results highlight the importance of appropriate protection against hydrolysis and UV radiation during the drug-development process, storage, handling and quality control.

Simultaneous determination of fluoroquinolones in environmental water by liquid chromatography-tandem mass spectrometry with direct injection: A green approach.

This work describes an on-line multi-residue method for simultaneous quantification of ciprofloxacin, enrofloxacin, gemifloxacin, moxifloxacin, norfloxacin and ofloxacin in superficial and wastewater samples. For that, an octyl restricted-access media bovine serum albumin column (RAM-BSA C8) was used for sample clean-up, enrichment and analysis with quantitation carried out by tandem mass spectrometry. For water samples volumes of only 500µL the method provided good selectivity, extraction efficiency, accuracy, and precision with quantification limits in the order of 20-150ngL(-1). Out of the six fluoroquinolones only ciprofloxacin (195ngL(-1)) and norfloxacin (270ngL(-1)) were quantified in an influent sample of the wastewater treatment plant (WWTP) of São Carlos (SP, Brazil). None were found in the superficial water samples analyzed. The capability of injecting native sample in an automated mode provides high productivity and represents a greener approach in environmental sample analysis.

Supramolecular interaction of gemifloxacin and hydroxyl propyl ß-cyclodextrin spectroscopic characterization, molecular modeling and analytical application.

The solid inclusion complex of gemifloxacin (GFX) and hydroxyl propyl ß-cyclodextrin (HPß-CD) was prepared and examined by UV-visible, FTIR, NMR, electrospray ionization mass spectrometry (ESI-MS) and fluorescence spectroscopy. The formation of inclusion complex has been confirmed on the basis of changes of spectroscopic properties. Further the interaction between GFX and HPß-CD was studied using molecular modeling approaches. The results showed that HPßCD reacted with GFX to form a 1:1 host-guest inclusion complex. Based on the enhancement of the fluorescence intensity of GFX produced through complex formation, a simple, accurate, rapid and highly sensitive spectrofluorometric method for the determination of GFX in pharmaceutical formulation was developed. The linear relationships between the intensity and GFX concentration was obtained in the concentration range of 20-140 ng/mL with good correlation coefficients (0.9997). The limit of detection (LOD) was found to be 4 ng/mL. The proposed method was successfully applied to the analysis of GFX in pharmaceutical preparation.

Utility of gold nanoparticles in luminescence determination of trovafloxacin: comparison of chemiluminescence and fluorescence detection.

Two novel sensitive sequential injection chemiluminescence analysis and fluorescence methods for trovafloxacin mesylate detection have been developed. The methods were based on the enhancement effect of gold nanoparticles on luminol-ferricyanide-trovafloxacin and europium(III)-trovafloxacin complex systems. The optimum conditions for both detection methods were investigated. The chemiluminescence signal was emitted due to the enhanced effect of gold nanoparticles on the reaction of luminol-ferricyanide-trovafloxacin in an alkaline medium. The response was linear over a concentration range of 1.0 × 10(-9) to 1.0 × 10(-2) mol/L (%RSD = 1.3), (n = 9, r = 0.9991) with a detection limit of 1.7 × 10(-10) mol/L (S/N = 3). The weak fluorescence intensity signal of the oxidation complex of europium(III)-trovafloxacin was strongly enhanced by gold nanoparticles and detected at λex = 330 and λem = 540 nm. Fluorescence detection enabled the determination of trovafloxacin mesylate over a linear range of 1.0 × 10(-8) to 1.0 × 10(-3) mol/L (%RSD = 1.2), (n = 6, r = 0.9993) with a detection limit of 3.3 × 10(-9) mol/L. The proposed methods were successfully applied to the determination of the studied drug in its bulk form and in pharmaceutical preparations. The results were treated statistically and compared with those obtained from other reported methods.

A very simple high-performance liquid chromatographic method with fluorescence detection for the determination of gemifloxacin in human breast milk.

A high-performance liquid chromatographic method with fluorescence detection was developed and validated for the determination of gemifloxacin in human breast milk. The proposed method allows the determination of gemifloxacin in breast milk samples without complex sample preparation. The samples were mixed with a mobile phase and filtered with a 0.45 µm polytetrafluoroethylene filter before analysis. Chromatographic separation was carried out on a C18 column (150 × 4.6 mm, 5 µm I.D.) using methanol:50 mM ortho-phosphoric acid solution (40:60) as the mobile phase with a 1.0 mL/min flow rate. Quantitation was performed using fluorescence detection with an excitation wavelength at 272 nm and an emission wavelength at 395 nm. The linear range was found to be 0.1-2.5 µg/mL. The method was applied successfully for the determination of gemifloxacin in breast milk obtained from a breastfeeding mother after oral administration of a single tablet that included 320 mg gemifloxacin per gemifloxacin tablet.

Gemifloxacin mesylate (GFM): dissolution test based on in vivo data.

Gemifloxacin mesylate (GFM) is a synthetic, broad-spectrum, fluoroquinolone antibacterial agent. It is different from other class members because it achieves adequate plasma concentrations to inhibit both topoisomerase IV and gyrase. The aim of this study was to develop and validate a dissolution test for GFM in coated tablets, using a simulated absorption profile based on in vivo data obtained from the literature. The fraction and percentage of the dose absorbed were calculated using model-dependent Loo-Riegelman approach for two compartments. The best in vitro dissolution profile was obtained using 900 mL of pH 6.0 phosphate buffer as a dissolution medium at 37 °C ± 0.5 °C and paddles at 50 rpm. The in vitro dissolution samples were analyzed using a liquid chromatography method, and the validation was performed according to USP 34 (2011). The method showed specificity, precision, accuracy, robustness and linearity. Under these conditions, a level-A in vitro-in vivo correlation was suggested (r = 0.9926). The prediction errors were calculated to determine the validity and accuracy of the suggested correlation. The dissolution test can be used to evaluate the dissolution profile of GFM-coated tablets and minimize the number of bioavailability studies as part of new formulation development.

Spectrophotometric method for the determination of Gemifloxacin mesylate in pure and tablet dosage form.

A spectrophotometric method for the determination of Gemifloxacin mesylate (GFX) is developed and validated according to ICH guidelines. GFX is a fluoroquinolone that is used in the treatment of pneumonia. The analysis of the pure drug was carried out at its λmax 270 nm. The method was linear from 0.5-5µg/mL, r(2) 0.999 and equation is 0.102-0.000. The % RSD for inter-day (0.969%) and intra-day (0.714%) assuring a good precision and accuracy was close to 100%. Limit of detection and Limit of quantification were 0.197 and 0.599µg/mL, respectively. The validation results and statistical data demonstrate that the method is accurate, sensitive, cost effective and reproducible and has an importance in quality assurance of GFX analysis. The developed method was proved suitable for analysis of GFX in the pure and tablet dosage forms without interference of excepients.

[Mn2+-cTMAB-sensitized fluorescent microscopic determination of tosufloxacin tosylate based on a self-ordered ring].

With the development of antimicrobial drugs, drug residue in animal products has a serious potential hazards for the environment and public health, it is urgent to set up drug residue detection method with low detection limit and good selectivity. In our paper, with poly (vinyl alcohol)-124 (PVA-124) and NH3-NH4Cl (pH 10.50) as the medium, Mn2+ and cetyltrimethyl ammonium bromide (CTMAB) as sensitizer, tosufloxacin tosylate (TSFX) formed a self-ordered ring (SOR) on a hydrophobic glass slide support. When the droplet volume is 0.20 microL, the TSFX can be detected in the range of 4.05 x 10(-14) approximately 4.28 x 10(-13) mol x ring(-1) (2.02 x 10(-7) approximately 2.14 x 10(-6) mol x L(-1)), and the limit of detection (LOD) can reach 4.1 x 10(-15) mol x ring(-1) (2.0 x 10(-8) mol x L(-1)). The established method had been applied satisfactorily to determine the content of TSFX in tablet, which close to the marked value of 0.15 g x piece(-1)) found value: 0.144 g x piece(-1)) and the results of TSFX concentrations in rabbit serum at different time after dosing with the recoveries of 90.0% - 105.0% and RSDs 1.9% - 3.3% were satisfactory. Xilinguole of Inner Mongolia is the national important livestock products ground, whose lamb is the main suppling source for Beijing-Tianjin-Tangshan region. It is very urgent to detect residue of antibacterial drug in its sheep tissue samples (meat, liver, kidney). In this paper, when acetonitrile was used as extraction agent in pretreatment of sheep tissue samples in six rangelands including Sonid Right Banner, Xiwuzhumuqin Banner, Xilinhot City, Duolun County, The white Flag Town and The Blue Flag Town, the extract can be directly determined with SOR technology without filtering process, the sample standard addition recovery of sheep tissues were 92.0%-101.0% and RSDs were less than 2.7%. The results indicated that the SOR technique can be successfully applied to pharmaceuticals and biological samples, which broaden the applied range of SOR technique. The assay would provide reliable experimental data and theoretical basis for the relevant departments.

Spectrofluorimetric determination of gemifloxacin mesylate and linezolid in pharmaceutical formulations: Application of quinone-based fluorophores and enhanced native fluorescence.

Quinone-based fluorophores and enhanced native fluorescence techniques were applied for a fast quantitative analysis of gemifloxacin mesylate (GEM) and linezolid (LIN) in pharmaceutical formulations. For this purpose, three sensitive, accurate and precise spectrofluorimetric methods were developed. GEM, as an n-electron donor, reacts with 7,7,8,8-tetracyanoquinodimethane (method A) and 2,5-dichloro-3,6-dihydroxy-p-benzoquinone (method B) as п-electron acceptors, forming charge transfer complexes that exhibit high fluorescence intensity at 441 and 390 nm upon excitation at 260 and 339 nm, respectively. Method C depends on measurement of enhanced native fluorescence of LIN in phosphate buffer (pH 5) at 380 nm upon excitation at 260 nm. Experimental factors affecting fluorescence intensity were optimized. Linearity was obtained over concentration ranges 50-500, 10-60 and 20-400 ng mL-1 for methods A, B and C, respectively. The developed methods were validated and successfully applied for determination of the cited drugs in tablets.

Spectrofluorimetric analysis of gemifloxacin mesylate in pharmaceutical formulations.

A simple, rapid and highly sensitive spectrofluorimetric method was developed for determination of gemifloxacin mesylate (GFX) in tablets. The method is based on measuring the native fluorescence of GFX in isopropanol at 400 nm after excitation at 272 nm. The fluorescence-concentration plot was rectilinear over the range of 0.01-0.50 µg/mL with a lower detection limit of 1.19 ng/mL and quantification limit of 3.6 ng/mL. The method was fully validated and successfully applied to the determination of GFX tablets with an average percentage recovery of 99.65 ± 0.532. The method was extended to the stability study of GFX. The drug was exposed to acidic, alkaline, oxidative and photolytic degradation according to International Conference on Harmonization guidelines. The rate of GFX degradation was found at its highest in acidic conditions, and in its lowest in the neutral one. However, it was stable under dry heat and photolytic degradation conditions.

Feasibility of multiphoton microscopy-based quantification of antibiotic uptake into neutrophil granulocytes.

Antibiotic levels in livestock are usually evaluated through destructive analysis. Taking advantage of the fluorescent properties of marbofloxacin (MBX) and trovafloxacin (TVX), multiphoton microscopy (MPM) was evaluated as a minimally invasive and nondestructive method to determine the penetration of TVX and MBX into sheep neutrophils. Standard curves were measured with drug-only solutions and suggested that MBX was more suited for this type of analysis. The intracellular concentration of both TVX and MBX was higher than the extracellular concentration after incubating neutrophils for 30 min at concentrations ranging from 0.1 to 100 µg/ml for both the drugs. The intracellular concentration of TVX increased with the extracellular concentration but was always greater than the extracellular concentration, suggesting active internalization. On the other hand, intracellular/extracellular ratio (I/E) peaked at 1.6-fold I/E for 1 µg/ml and then gradually decreased with increased concentration to 1.2-fold I/E at 100 µg/ml. For the first time, this study showed the use of MPM to quantify antibiotic uptake by sheep neutrophils and observed that both antibiotics were taken up by sheep neutrophils beyond extracellular levels.

Validation of an efficient LC-microdialysis method for gemifloxacin quantitation in lung, kidney and liver of rats.

A liquid chromatography method has been established for the reliable determination of unbound gemifloxacin concentrations in kidney, lung and liver microdialysates of rats. Microdialysis probes were inserted into tissues of rats, and then dialysates were collected at regular time intervals after intravenous administration of gemifloxacin (40 mg kg(-1)). A pilot study was performed to assess gemifloxacin penetration in lung, kidney and liver of rats. Gemifloxacin was separated on a C(18) column eluted using triethylamine solution (0.5%, v/v), adjusted to pH 3.0±0.1 with 85% phosphoric acid, methanol and acetonitrile (71:15:14, v/v/v) as mobile phase at a flow rate of 1.1 mL min(-1). The fluorescence detector was set at excitation and emission wavelengths of 344 nm and 399 nm, respectively. The limit of quantitation was found to be 50 ng mL(-1). Linearity was found to be over a concentration range of 50-2000 ng mL(-1). The intra-assay and inter-assay precision and accuracy values were determined from the analysis of six quality control samples. The results obtained at three concentration levels showed R.S.D. values lower than 6.06% and 4.10% for repeatability and intermediate precision, respectively. The accuracy (R.E.%) ranged from 90.0 to 106.5%. The chromatographic run time of each sample was performed in 9 min. Drug stability in microdialysates was shown at room temperature for 8h, after three freeze-thaw cycles, in freezer at -80 °C for 14 days, and in the autosampler after processing for 8h. The relative recoveries determined by extraction efficiency (EE) and retrodialysis (RD) in vitro employing a flow rate of 1.5 µL min(-1) were 29.24±3.67% and 23.67±3.31%, respectively. In vivo recoveries determined by RD in Wistar rats' kidney, lung and liver were 27.69±2.09%, 23.12±3.79% and 17.38±0.68%, respectively. The method was successfully applied to investigate tissue penetration of unbound gemifloxacin into the kidney, lung and liver of rats.