Stability-indicating assay method for determination of actarit,its process related impurities and degradation products:Insight into stability profile and degradation pathways

The stability of the drug actarit was studied under different stress conditions like hydrolysis（acid,alkaline and neutral）,oxidation,photolysis and thermal degradation as recommended hy International Conference on Harmonization（ICH） guidelines.Drug was found to be unstable in acidic,basic and photolytic conditions and produced a common degradation product while oxidative stress condition produced three additional degradation products.Drug was impassive to neutral hydrolysis,dry thermal and accelerated stability conditions.Degradation products were identified,isolated and characterized by different spectroscopic analyses.Drug and the degradation products were synthesized by a new route using green chemistry.The chromatographic separation of the drug and its impurities was achieved in a phenomenex luna C18 column employing a step gradient elution by high performance liquid chromatography coupled to photodiode array and mass spectrometry detectors（HPLC-PDAMS）.A specilic and sensitive stability-indicating assay method for the simultaneous determination of the drug actarit.its process related impurities and degradation products was developed and validated.

A Validated Stability-Indicating UHPLC Method for Determination of Naproxen and Its Related Compounds in Bulk Drug Samples

A simple, rapid, precise, accurate, rugged and robust stability-indicating ultra-fast high performance liquid chromatographic (UHPLC) method has been developed for the estimation of related compounds (imp-A, imp-B, imp-C, imp-D and imp-E) in Naproxen and also the assay of Naproxen from bulk drug samples. The stability indicating capability of the method was proven by subjecting the samples to stress conditions such as acid, base, oxidation, photolysis and thermal degradation. The efficient chromatographic separation was achieved using mobile phase solution A prepared as buffer solution 10 mM monobasic potassium phosphate pH 4.0 ± 0.05 adjusted with diluted ortho phosphoric acid solution and solution B acetonitrile with linear gradient elution on poroshell 120 EC-C18 shot column (50 mm × 4.6 mm, 2.7 μm) and UV detection at 235 nm at a flow rate 1.0 mL/min, column oven temperature was set to 25?C. The above are all known impurities and degradation impurities are well resolved with Naproxen peak and these are eluted within a 10 min runtime of HPLC. The photo diode array detector was used for peak homogeneity testing during stress study experiments and the overall mass balance was found to be 99.2% to 100.2% in all stress conditions. The linear calibration range was found to be 0.05 μg/mL to 0.75 μg/mL for related compounds and 50 μg/mL to 150 μg/mL for Naproxen and the accuracy of the method was found to be 91.5% to 98.5% recovery for the related substance method and 95.4% to 97.4% recovery for the assay method. The Naproxen and related compounds were found to be stable up to 48 hours and the method validation data show excellent results for precision, linearity, specificity, limit of detection, limit of quantitation and robustness. The present method can be successfully used for routine QC and stability studies and it will help to reduce the analysis cost, time and effluent load compared to conventional HPLC methods.

A SENSITIVE PROTEIN ASSAY BY Co(II)-BIURET METHOD

A new and senseitive Co (II)-biuret method for determination of protein has for the first time been established. In visible wavelength region, the Co (II) -protein complexes had anabsorption peak at 360 nm. -Under the present conditions. the assay had a linear range of 0-120 μg/ml of protein with a detection limit of about 2 μg/ml, and similar calibration curves were obtained for BSA. and BγG. Compared with Cu (II) -biuret method, the present Co (II) -biuret method was more simple, sensitive, and tolerant of interferences from non-protein molecules.

New Colorimetric Method for Lipases Activity Assay in Microbial Media

A simple, rapid and precise method has been developed for determination of lipase activity in microbial media. The method is based on using phenyl acetate as substrate for lipase and determination of liberated phenol by Folin Ciocalteu reagent. Reaction mixture containing substrate 2.4 ml of phenyl acetate 165 μM in Tris HCl buffer, 0.1 M and pH 7, with 1% (v/v) Triton X-100) and 0.1 ml lipase is incubated at 40?C during 10 minutes and the absorbance was measured at 750 nm. Linearity was observed in the concentration range 0-0.8 g/L lipase.

Validated stability indicating methods for determination of nitazoxanide in presence of its degradation products

Three sensitive,selective and reproducible stability-indicating methods are presented for determination of nitazoxanide (NTZ),a new anti-protozoal drug,in presence of its degradation products.Method A utilizes the first derivative of ratio spectra spectrophotometry by measurement of the amplitude at 364.4 nm using one of the degradation products as a divisor.Method B is a chemometric-assisted spectrophotometry,where principal component regression (PCR) and partial least squares (PLS) were applied.These two approaches were successfully applied to quantify NTZ in presence of degradation products using the information included in the absorption spectra in the range 260-360 nm.Method C is based on the separation of NTZ from its degradation products followed by densitometric measurement of the bands at 254 nm.The separation was carried out on silica gel 60F 254,using chloroform-methanol-ammonia solution-glacial acetic acid (95:5:1:1 by volume,pH=5.80) as a developing system.These methods are suitable as stability-indicating methods for the determination of NTZ in presence of its degradation products either in bulk powder or in pharmaceutical formulations.Statistical analysis of the results has been carried out revealing high accuracy and good precision.

A novel and rapid microbiological assay for ciprofoxacin hydrochloride

The present work reports a simple, fast and sensitive microbiological assay applying the turbidimetric method for the determination of ciprofloxacin hydrochloride （CIPRO HC1） in ophthalmic solutions. The validation method yielded good results and included excellent linearity, precision, accuracy and specificity. The bioassay is based on the inhibitory effect of CIPRO HC1 upon the strain of Staphylococcus epMermidis ATCC 12228 used as the test microorganism. The results were ar, ated statistically by analysis of variance （ANOVA） and were found to be linear （r=0.9994, in the range of 14.0-56.0 lag/mL）, precise （intraday RSD %=2.06; interday RSD%=2.30） and accurate （recovery = 99.7%）. The turbidimeaic assay was compared to the UV spectrophotometric and I-IPIX2 methods for the same drug. The tuIbidimetric bioassay described on this paper for determination of ciprofioxacin hydrochloride in ophthalmic solution is an alternative to the physicochemical methods disclosed in the literature and can be used in quality control routine.

Long-term stability of gentamicin sulfate-ethylenediaminetetraacetic acid disodium salt (EDTA-Na_2) solution for catheter locks

A lock solution composed of gentamicin sulfate(5 mg/mL) and ethylenediaminetetraacetic acid disodium salt(EDTA-Na2, 30 mg/mL) could fully eradicate in vivo bacterial biofilms in totally implantable venous access ports(TIVAP). In this study, fabrication, conditioning and sterilization processes of antimicrobial lock solution(ALS) were detailed and completed by a stability study. Stability of ALS was conducted for12 months in vial(25 °C 7 2 °C, 60% 7 5% relative humidity(RH), and at 40 °C 7 2 °C, RH 75% 7 5%)and for 24 h and 72 h in TIVAP(40 °C 7 2 °C, RH 75% 7 5%). A stability indicating HPLC assay with UV detection for simultaneous quantification of gentamicin sulfate and EDTA-Na2 was developed. ALS was assayed by ion-pairing high performance liquid chromatography(HPLC) needing gentamicin derivatization, EDTA-Na2 metallocomplexation of samples and gradient mobile phase. HPLC methods to separate four gentamicin components and EDTA-Na2 were validated. Efficiency of sterility procedure and conditioning of ALS was confirmed by bacterial endotoxins and sterility tests. Physicochemical stability of ALS was determined by visual inspection, osmolality, pH, and sub-visible particle counting. Results confirmed that the stability of ALS in vials was maintained for 12 months and 24 h and 72 h in TIVAP.

Formulation, stability testing, and analytical characterization of melatonin-based preparation for clinical trial

A new institutional clinical trial assessed the improvement of sleep disorders in 40 children with autism treated by immediate-release melatonin formulation in different regimens(0.5 mg, 2 mg, and 6 mg daily) for one month. The objectives of present study were to(i) prepare low-dose melatonin hard capsules for pediatric use controlled by two complementary methods and(ii) carry out a stability study in order to determine a use-bydate. Validation of preparation process was claimed as ascertained by mass uniformity of hard capsules.Multicomponent analysis by attenuated total reflectance Fourier transformed infrared(ATR-FTIR) of melatonin/microcrystalline cellulose mixture allowed to identify and quantify relative content of active pharmaceutical ingredients and excipients. Absolute melatonin content analysis by high performance liquid chromatography in 0.5 mg and 6 mg melatonin capsules was 93.6% ± 4.1% and 98.7% ± 6.9% of theoretical value, respectively. Forced degradation study showed a good separation of melatonin and its degradation products. The capability of the method was 15, confirming a risk of false negative < 0.01%. Stability test and dissolution test were compliant over 18 months of storage with European Pharmacopoeia. Preparation of melatonin hard capsules was completed manually and melatonin in hard capsules was stable for 18 months, in spite of low doses of active ingredient. ATR-FTIR offers a real alternative to HPLC for quality control of highdose melatonin hard capsules before the release of clinical batches.

Production and stability study of a hospital parenteral nutrition solution for neonates

Standard parenteral nutrition solutions are mixtures comprising interacting components that may degrade themselves over time. The objective of this study was to investigate the physicochemical and microbiological stability of a hospital preparation for parenteral nutrition in neonatology. The analyses were performed throughout the storage of the preparations at 2–8 °C(up to 4 months). The extent of stability was based on the determination of amino acids dosage, visual and physicochemical properties(glucose and electrolytes concentrations, pH and osmolality measurements, particle counting) and microbiological analysis(sterility test). A thermal degradation of ascorbic acid was conducted to evaluate the antioxidant properties of the parenteral mixture. Physicochemical and microbiological controls were found to comply with the specifications. Amino acids showed a good stability throughout the 4 months storage except for cysteine, which was progressively degraded to cystine, conferring a yellow coloration to parenteral solutions. Parenteral nutrition standards solutions remain stable for 4 months at 2–8 °C,ensuring safe administration in preterm infants.

Selective separation, detection of zotepine and mass spectral characterization of degradants by LC–MS/MS/QTOF

A simple, precise, accurate stability-indicating gradient reversed-phase high-performance liquid chromatographic （RP-HPLC） method was developed for the quantitative determination of zotepine （ZTP） in bulk and pharmaceutical dosage forms in the presence of its degradation products （DPs）. The method was developed using Phenomenex C18 column （250 mm ~ 4.6 mm i.d., 5 mm） with a mobile phase containing a gradient mixture of solvents, A （0.05%trifluoroacetic acid （TFA）, pH ？ 3.0） and B （acetonitrile）. The eluted compounds were monitored at 254 nm;the run time was within 20.0 min, in which ZTP and its DPs were well separated, with a resolution of 41.5. The stress testing of ZTP was carried out under acidic, alkaline, neutral hydrolysis, oxidative, photolytic and thermal stress conditions. ZTP was found to degrade significantly in acidic, photolytic, thermal and oxidative stress conditions and remain stable in basic and neutral conditions. The developed method was validated with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision and robustness as per ICH guidelines. This method was also suitable for the assay determination of ZTP in pharmaceutical dosage forms. The DPs were characterized by LC-MS/MS and their fragmentation pathways were proposed.

Anti-proliferative and anti-angiogenic activities of ion-channel modulators:In-ovo,in-vitro and in-vivo study

Objective:Angiogenesis is the development of new blood vessels.The ion channels on endothelium play a vital action in cell proliferation and so in the related angiogenesis.We aimed to investigate the anti-angiogenic effects of Mefloquine(Cl-channel blocker) and4-Aminopyridine(K+ channel blocker).Methods:The anti-angiogenic activities of Mefloquine and 4-Aminopyridine(4-AP)were investigated by in-vivo(sponge implantation method),in-vitro(aortic ring assay)and in-ovo(CAM,Chick Chorioallantoic membrane) methods.The standard antiangiogenic drug used was Bevacizumab.Results:In the CAM assay,both the ion channel blockers exhibited noticeable antiangiogenic activity at the concentrations of 10-5M and 10-4M where they significantly exhibited ant proliferative activity by inhibiting the new blood vessel formation.For the further confirmation anti-angiogenic activity was evaluated in vitro and in vivo.In Rat aortic ring assay reduction in the area of sprouts were observed with 40 m M of 4-AP and7 m M of Mefloquine.A significant reduction in weight of sponges,number of blood vessels formed and hemoglobin content were observed at 4.2 mg/kg of 4-AP and 20 mg/kg and 30 mg/kg of Mefloquine.Conclusions:These scientific findings indicate the use of Mefloquine and 4-Aminopyridine in pathological situations involving excessive angiogenesis.Negative regulation of cell volume,cell migration and proliferation of blood vessels may be the underlying molecular mechanisms.

Optima of Trypsin-Catalyzed Hydrolysis and Its Inhibition Determined by SDS-PAGE

SDS-PAGE was applied to determine trypsin activity and inhibition. After the hydrolysis by trypsin to substrate bovine serum albulnin (BSA) under different temperatures and pH, the hydrolysis degree of BSA was conducted using SDS-PAGE. From the quantitative analysis to the electrophoresis bands of BSA and its hydrolysis products in SDS-PAGE pattern, the change of trypsin activity was determined, and then the optimum temperature at 40°C and the optimum pH at pH 8.5 - 8.7 for trypsin activity were obtained. All the target bonds in BSA molecule could be hydrolyzed at the same time by trypsin. The inhibition was due to the binding of inhibitor to trypsin, which made it impossible for trypsin to touch the substrate protein. SDS-PAGE was demonstrated to be also an effect method for assaying the characteristics of trypsin activity and its inhibition.

指示稳定性的高效液相色谱方法和强制降解研究片剂型马西替坦中的杂质（英文）

Macitentan (MAC) is a pulmonary arterial hypertension (PAH) drug marketed as a tablet and often has stability issues in the final dosage form. Quantitative determination of MAC and its associated impurities in tablet dosage form has not been previously reported. This study quantified impurities present in Macitentan tablets using a binary solvent-based gradient elution method using reversed phase-high performance liquid chromatography.The developed method w as validated per International Conference on Harmonization (ICH) guidelines and the drug product w as subjected to forced degradation studies to evaluate stability. The developed method efficiently separated the drug and impurities (48 min) w ithout interference from solvents,excipients,or other impurities. The developed method met all guidelines in all characteristics w ith recoveries ranging from 85%-115%,linearity w ith r 2≥0. 996 6,and substantial robustness. The stability-indicating nature of the method w as evaluated using stressed conditions (hydrolysis:1 N HCl at 80℃/15 min; 1 N NaOH at 25℃/45 min; humidity stress (90%relative humidity) at 25℃for 24 h,oxidation:at 6%(v/v) H2O2,80℃/15 min,thermolysis:at105℃/16 h and photolysis:UV light at 200 Wh/m2; Fluorescent light at 1. 2 million luxh). Forced degradation experiments show ed that the developed method w as effective for impurity profiling. All stressed samples w ere assayed and mass balance w as> 96%. Forced degradation results indicated that MAC tablets w ere sensitive to hydrolysis (acid and alkali) and thermal conditions. The developed method is suitable for both assay and impurity determination,w hich is applicable to the pharmaceutical industry.

Antibiotic Effect on Planktonic and Biofilm-Producing Staphylococci

The pathogenic effect of Staphylococci is due to extra-cellular factors and properties such as adherence and biofilm production. The nature of the biofilm and the physiological properties of biofilm-producing bacteria result in an inherent antibiotic resistance and require further investigation. Two hundred and sixty Staphylococcal strains were cultured from 600 clinical specimens obtained from hospitalized patients. Among these, 155 were identified as coagulase-positive (CPS) and 105 as coagulase-negative (CNS) staphylococci. Staphylococcal strains were tested for biofilm production using the tissue culture plate (TCP) method. TCP detection showed that of the 155 CPS, 124 (80%) were biofilm producers, while 63 (60%) of the 105 CNS were biofilm producers. Biofilm-producing strains were scanned by scanning electron microscope (SEM) to confirm biofilm formation, study biofilm production, and examine antibiotic effects on biofilm formation. Disc diffusion method was used to study resistance of planktonic and biofilm-forming cells to antibiotics. Planktonic cells were less resistant to antibiotics than biofilm-forming cells. Microbroth dilution method and a new BioTimer assay were used to determine antibiotic MICs affecting planktonic and biofilm cells. Both methods showed that the MICs for planktonic cells were less than that for biofilm cells. The BioTimer assay was therefore found to be sensitive, accurate, and reliable, with results in agreement with those from the broth dilution method and SEM.

Cytotoxic, Antimicrobial and Antioxidant Properties of <i>Commelina diffusa</i>Burm. F.

Commelina diffusa Burm. F. is a herbaceous tropical plant with different traditional medicinal uses. Present study is aimed to isolate the bioactive compounds from DCM-Methanol extract of the powdered whole plant of Commelina diffusa and to investigate the cytotoxic, antimicrobial and antioxidant activities of the crude extract and its twelve vacuum liquid chromatographic fraction (CD1-12). Vacuum Liquid Chromatography (VLC) was employed to isolate bioactive metabolites. The physicochemical properties of the isolated compound were examined and the molecular structure was elucidated by NMR spectroscopy.?Cytotoxic, antibacterial, antioxidant activities were evaluated following brine shrimp lethality bioassay, disc diffusion method and DPPH free radical scavenging assay respectively. The isolated compound was identified as steroid (stigmasterol) which had significant cytotoxic effect on vero cell line. The crude extract and its fractions (CD8-CD12) exhibited strong cytotoxicity in brine shrimp lethality bioassay having LC50 values 3.79, 9.19, 29.49, 16.60, 19.36, 44.58 μg/mL respectively. The crude extract showed mild to strong antibacterial activity. Fractions (CD5-CD12) showed mild to strong antibacterial activity against Escherichia coli in comparison with Kanamycin (standard). Strong antioxidant activities were found in crude extract (IC50 value 30.52 μg/ml), CD11 (IC50 value 39.27 μg/ml) and CD12 (IC50 value 19.50 μg/ml). The study suggests Commelina diffusa plant extract to have strong antioxidant and cytotoxic activity which is indicative of presence of compounds with broad spectrum of curative applications. One compound namely stigmasterol has been isolated from the plant.

The activity of <i>Rhaphidophora pinnta</i>Lf. Schott leaf on MCF-7 cell line

Ekor naga’s leaf (Rhaphidophora pinnata (Lf) Schott) is a type of vines and climbing plant. The leaves are elongated round and hollowed inside. This plant had been using for the treatment of breast cancer. Extraction with percolation method has been done in ekor naga’s leaves with ethanol, and fractionated by nhexane, chloroform and ethyl acetate using liquid-liquid extraction (LLE). Cytotoxicity assay of ethanol extract, n-hexane fraction, chloroform fraction, ethyl acetate fraction and water fraction against MCF-7 cells were done using MTT method (3-(4,5-dimetiltiazol-2-il)-2,5-diphenyl tetrazolium bromide). Phytochemical screening results showed the presence of the compounds such as triterpenoida/steroid, alkaloid, flavonoid, tannin, and saponin. n-hexane fraction was positive for the presence of triterpenoida/steroid, chloroform fraction containing alkaloids, saponin and triterpenoid;ethyl acetate fraction contained, flavonoid, tannin, and the fraction of water indicated the presence of tannin and saponin. Secondary metabolite compounds in ethanol extract, chloroform fraction and ethyl acetate fraction gave positive results against MCF-7 cells. Cytotoxicity assay of MCF-7 cell line showed that crude ethanol extracts had 112.240 mcg/ml IC50 chloroform fraction IC50 was 59.082 mcg/ml, and ethyl acetate fraction IC50 was 812.663 mcg/ml.

An analytical method for quantitative estimation of the cytotoxicity of dental alloys using MTT assay,ICP analysis and effective cytotoxicity calculation

The two most important criteria for dental materials are their biofunctional and biocompatible endurance within the anticipated life-span of the dental restoration in the mouth. Biocompatibility relates mainly to the allergenicity and the toxicity of the material. To test the non-specific toxicity of dental materials, in vitro cell culture assays have been developed. For in vitro screening, such tests are recommended to check the cytotoxicity of dental materials (ISO 10993 5). Various studies have already been performed to quantitatively determine the cytotoxicity level of dental alloys. However, as long as only dental alloys and the cell culture technique are applied, it is not possible to determine which of the alloying elements cause the cytotoxicity. Therefore, an analytical method is needed. Wataha et al determined in 1991 the TC50 values of 9 metal cations of various dental casting alloys, using cell culture methods. Kapert et al reported in 1994 a complex in vitro test concept, where the ICP analysis (inductively coupled plasma emission spectroscopy) was introduced to measure the trace elements extracted from various alloys. Experimentelle Zahnheilkunde, Universitts ZMK Klinik Freiburg, Germany (Lü XY and Kappert HF) The aim of the present study was to find a relation between the ICP results, the TC50 value of metal cations, and the cytotoxicity of dental alloys. The cytotoxicity levels of various dental alloys and the TC50 values of 10 metal cations were established using the MTT assay, an effective cell culture of method. Then, the concentrations of the corrosively soluted metal cations in the extracts of the alloys were measured using the ICP method. From all these experimental results it was found that the relation between the effective cytotoxicity Z eff of an alloy, the concentrations C i of i th trace element and the TC50 values T Ci of the i th metal cation can approximately be expressed by Z eff =∑iC i2·T Ci . Two significant applications of this expression are a) The cytotoxicity of an alloy can be estimated by ICP analysis of the extract if the TC50 values of the trace elements are know. b) The cytotoxicity of a new-developed-alloy can be estimated in advance, according to the alloying components.

A sensitive and convenient enzyme-linked immunosorbent assay method in serum MG7 antigen detection in gastric cancer

Objective To explore a highly sensitive and highly specific method to detect the serum MG7 antigen(Ag)level for early gastric cancer diagnosis.Methods The serum MG7-Ag level was detected by enzyme-linked immunosorbent assay(ELISA)method in 116 preoperative gastric cancer patients,63 postoperative gastric cancer patients,41 patients with precancerous lesion,37 pa-

A reporter gene assay for determining the biological activity of therapeutic antibodies targeting TIGIT

T cell immunoglobulin and ITIM domain(TIGIT)is a novel immune checkpoint that has been considered as a target in cancer immunotherapy.Current available bioassays for measuring the biological activity of therapeutic antibodies targeting TIGIT are restricted to mechanistic investigations because donor primary T cells are highly variable.Here,we designed a reporter gene assay comprising two cell lines,namely,CHO-CD112-CD3 scFv,which stably expresses CD 112(PVRL2,nectin-2)and a membranebound anti-CD3 single-chain fragment variable(scFv)as the target cell,and Jurkat-NFAT-TIGIT,which stably expresses TIGIT as well as the nuclear factor of activated T-cells(NFAT)response element-controlled luciferase gene,as the effector cell.The anti-CD3 scFv situated on the target cells activates Jurkat-NFATTIGIT cells through binding and crosslinking CD3 molecules of the effector cell,whereas interactions between CD 112 and TIGIT prevent activation.The presence of anti-TIGIT mAbs disrupts their interaction,which in turn reverses the inactivation and luciferase expression.Optimization and validation studies have demonstrated that this assay is superior in terms of specificity,accuracy,linearity,and precision.In summary,this reliable and effective reporter gene assay may potentially be utilized in lot release control,stability assays,screening,and development of novel TIGIT-targeted therapeutic antibodies.