10-μl Volume Research Articles

Research on detection methods of related substances and degradation products of the antitumor drug selpercatinib.

Selpercatinib, a selective RET kinase inhibitor, is approved for treating various cancers with RET gene mutations such as RET-rearranged thyroid cancer and non-small cell lung cancer. The presence of process-related and degradation impurities in its active pharmaceutical ingredient (API) can significantly affect its safety and effectiveness. However, research on detecting these impurities is limited. This study developed and systematically validated a High-Performance Liquid Chromatography (HPLC) method for identifying selpercatinib and its related impurities. The method utilized a 4.6mm × 250mm chromatographic column with 5μm particles, employing a flow rate of 1.0mL/min, a detection wavelength of 235nm, an injection volume of 10μL, and a column temperature of 35°C. Mobile phase A was composed of a 9:1 ratio of water to acetonitrile, with the aqueous component adjusted to pH 2.5 and containing 2mM potassium dihydrogen phosphate (KH2PO4) and 0.4% triethylamine. Mobile phase B was pure acetonitrile. The gradient elution program was as follows: 0-2min, 5%B; 2-15min, 5% to 15%B; 15-30min, 15% to 35%B; 30-35min, 35% to 45%B; 35-36min, 45% to 5%B; 36-45min, 5%B. The chromatographic method established in this study was validated according to the ICH Q2 (R1) guidelines. The developed HPLC method demonstrated excellent specificity, sensitivity, stability, linearity, precision, accuracy, and robustness. It efficiently separated the impurities present in selpercatinib, thereby confirming the method's efficacy in ensuring the purity and quality of the drug. The chromatographic method established in this study can be used for the detection of selpercatinib and its impurities, providing significant reference value for the quality research of selpercatinib bulk drug and its preparations, and ensuring the safety of medication for patients.

Development and Validation of Fast and Sensitive RP-HPLC Stability-Indicating Method for Quantification of Piroxicam in Bulk Drug.

A simple, rapid, sensitive, and cost-effective green solvent-assisted reverse-phase high-performance liquid chromatographic technique, coupled with a photodiode array detector, was developed and validated for the estimation of piroxicam (PRXM). The chromatographic separation was achieved by using a C-18 (250 × 4.6) mm, 5-μm stationary phase and a mobile phase consisting of methanol and 0.1% ortho-phosphoric acid in water in a ratio of (80:20) v/v at a flow rate of 1ml/min. The detection was carried out at a wavelength of 254nm with a constant injection volume of 10μL throughout the analysis. The calibration curve was observed to be linear over the optimum concentration range of 50-300μgmL-1, with an R2 value of 0.9995. The developed method was validated as per the International Council for Harmonisation (ICH) Q2 (R1) guideline. Various parameters like selectivity/specificity, accuracy/recovery, linearity, precision, detection limit, quantitation limit, robustness and stability of analyte in solution were performed for the method validation. The PRXM was evaluated under stressed conditions, including acidic, basic, oxidative, thermal and photolytic, as per ICH Q1 (R2) guidelines. Significant degradation was observed in acidic and basic degradation conditions. Conversely, the drug substance showed stability when exposed to oxidative, photolytic and thermal degradation conditions.

Evaluation of a new portable analyzer, HemoCue® Hb801, for hemoglobin point-of-care testing

The use of portable hemoglobin measuring devices is widespread. In this context, the company HemoCue® has put on the market a new device, the Hb801. It uses a whole blood absorbance measurement method and not the azidmethemoglobin measurement method used by HemoCue's older devices. We evaluated this new equipment on EDTA venous blood. Hb801 is lightweight, compact, requires a volume of 10μL of blood and renders its result in less than a second. The repeatability and intermediate precision are close to the values expected according to Ricos, with coefficients of variation respectively for a low level of hemoglobin: 2.1% and 1.9%, for an average level: 0.8% and 1.5% and for a high level: 1.5% and 1.3%. Comparison to our laboratory reference method (XN-10 Sysmex®) and HemoCue® Hb201+ was performed on 96 samples. Bias (SD) found were: XN-10: +0.42g/dL (0.17), HemoCue® Hb201+: +0.17g/dL (0.41). Clinically acceptable performance (within ± 1g/dL of reference hemoglobin) was high: 93.8%. In the end, this device seems to us to be suitable for hemoglobin point-of-care testing.

Using a developed co-culture device to evaluate the proliferation of bone marrow stem cells by stimulation with platelet-rich plasma and electromagnetic field

BackgroundsBone marrow stem cell can differentiate to osteoblast by growth factors, pulsed low-intensity ultrasound and electric magnetic field. In the research, bone marrow stem cells were cultured; bone marrow stem cells in culture can be stimulated by platelet-rich plasma and electric field.MethodsThe culture well of the co-cultivation device has a radius of 7.5 mm and a depth of 7 mm. It is divided into two sub-chambers separated by a 3 mm high and 1 mm wide barrier. The bone marrow stem cells were seeded at a density of 2 × 104 cells and the medium volume was 120μl. Platelet-rich plasma (PRP) or platelet-poor plasma (PPP) was added to the other sub-chamber at a volume of 10μl. The bone marrow stem cells were subjected to different electric fields (0 ~ 1 V/cm) at a frequency of 70 kHz for 60 min.ResultsThe highest osteogenic capacity of bone marrow stem cells was achieved by addition of PRP to electric field stimulation (0.25 V/cm) resulted in a proliferation rate of 599.78%. In electric field stimulation (0.75 V/cm) with PPP, the proliferation rate was only 10.46%.ConclusionsBone marrow stem cell with PRP in the co-culture device combined with electric field at 0.25 V/cm strength significantly promoted the growth of bone marrow stem cells.

Identification and Structural Characterization of Forced Degradation Impurities of Sacubitril by HRMS – Development and Validation of a Stability indicating RP-HPLC method

The current work identifies and characterises the stress degradation products of the anti-hypertensive drug sacubitril. The stability profile of sacubitril was evaluated under several stress settings including hydrolytic, oxidative, thermal and photolytic conditions in accordance with ICH recommendations. The degradation products of sacubitril were separated using reverse phase liquid chromatography and structural characterization was carried out using HRMS. The chromatographic separation was carried out by Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) using a Fortis C18 column (150 mm X 4.6 mm; 5μm particle size) with mobile phase A (consisting of 0.1% TFA in Milli-Q water) and mobile phase B (100% Acetonitrile) in gradient mode. With a sample injection volume of 10μL, the mobile phase flow rate was 0.7 mL/min. A quadrupole time-of-flight (Q-TOF) mass spectrometer was used for the LC-MS analysis. Sacubitril is relatively stable in oxidative, thermal and photolytic conditions; however, considerable degradation was observed in acid, base and neutral hydrolysis. Three degradation products were identified and the structural characterization of these impurities was performed using the HRMS data and mass fragmentation. The proposed RP-HPLC method was robust, precise and specific in determining the impurity profile of sacubitril under different stress conditions.

New validated Ultra High Performance Liquid Chromatographic method for Estimation of Quercetin

Background: A new Ultra performance liquid chromatographic analytical method was developed and validated for estimation of Quercetin in bulk powder. The reverse phase chromatographic elution using UPLC was carried out in gradient mode on C-18 column (Phenomenex Luna 5 µm, 150 mm X 4.6 mm) as stationary phase utilizing a mobile phase composed of Acetonitrile: 0.1 % formic acid buffer (50:50 v/v) with a flow rate of 1 ml/min and injection volume of 10µl. The analysis was performed at temperature of 400C and detection of eluent was carried out using photo diode array at 371 nm. The newly developed Ultra performance liquid chromatography method was validated in terms of linearity and range, system suitability, specificity, precision, sensitivity, robustness, ruggedness and accuracy as per ICH guidelines. Results: The retention time of quercetin was found to be at 3.9 min with total run time of analysis is 7 minutes. The linearity was observed between the concentration ranges from 0.5 to 16µg/ml with correlation coefficient 0.999. The precision assays values were found to be less than 2% for both the drugs. The Limit of Detection and Limit of Quantification were 0.47µg/ml and 1.44µg/ml for Quercetin. The mean percentage recovery values were found to be within the range of 90-110 %. Conclusion: The proposed method was found to be simple, specific, precise, sensitive, robust, rugged and accurate and can be used for routine quality control analysis of Quercetin.

Bioanalytical RP-HPLC Method for Simultaneous Quantification of Rivaroxaban and Ticagrelor in Spiked Human Plasma: Validation and Stability

For the effective assessment of preclinical, biopharmaceutical and clinical pharmacological investigations, selective and sensitive analytical methods for the quantitative evaluation of drugs and their metabolites (analytes) are essential. Using Rilpivirine as an internal standard (IS), a simple, fast, sensitive, precise and accurate high performance liquid chromatographic method for simultaneous determination of Rivaroxaban and Ticagrelor in human plasma was developed. The analytes were extracted using a direct protein precipitation method with acetonitrile from 500μL aliquots of human plasma. The content of the drugs was assessed using a mobile phase consisting of acetonitrile and 0.1% orthophosphoric acid buffer in a 40:60 v/v ratio and with a flow rate of 1.0ml/min, and an injection volume of 10μL. A Kromasil C18 (250×4.6mm; 5µm) analytical column was used for chromatographic separation and the effluents were monitored at 265nm using a photo diode array (PDA) detector.Rivaroxaban, Ticagrelor and Rilpivirine had retention times of 4.716min, 6.396min and 5.405 min, respectively. Rivaroxaban was shown to be linear at concentrations ranging from 0.018µg/mL - 1.80µg/mL, whereas Ticagrelor was found to be linear between 0.020µg/Ml - 2.00µg/mL. The method was validated according to US-FDA guidelines, and the findings satisfied the acceptance criteria. It was successfully used to determine Rivaroxaban and Ticagrelor in human plasma at the same time and this method is applicable for the pre-clinical and clinical estimation of drugs in biological fluids.

Stability-Indicating Method Development and Validation for Quantitative Estimation of Assay and Organic Impurities of anti-viral drug Baloxavir-Marboxil in Drug Substance and Pharmaceutical Dosage Form using HPLC and LC-MS Methods.

Baloxavir-Marboxil (BXM) is a polymerase-acidic-endonuclease inhibitor used as an anti-viral drug. A simple, reliable, and robust liquid chromatographic method was developed and validated per ICH Q2(R1) for estimating the assay and impurities of BXM in drug substance and pharmaceutical formulations. The chromatographic separation was carried-out on C18 (100 x 4.6 mm, 5-μm) with binary solvent delivery system (A:0.1% trifluoroacetic-acid in water; B:0.1% trifluoroacetic-acid in acetonitrile) along with detection wavelength of 260 nm, column temperature of 57°C, flow of 1.2-mL/min and injection volume of 10-μL. All five known impurities and unknown impurities were separated well with resolution >1.7, and were estimated accurately without any interference. Recovered values and regression value were 99.5-101.2% and R2 >0.999, respectively. The recovery and linearity studies covered from 50-150% for assay, and quantitation limit (QL) - 120% for five BXM impurities. Stability-indicating property of the HPLC developed method was assessed from the forced degradation studies. The mass spectral data of unknown impurity formed under oxidation stress condition was discussed. The developed method was also successfully utilized for stability samples analysis of drug substance and tablets dosage form.

Analysis of Fixed-Dose Combination of Three Antihypertensive Drugs by a Green and Quality by Design Approach.

This paper presents the result of a combined employment of Analytical Quality-by-Design and Green Analytical Chemistry principles for the development of a robust high-performance liquid chromatography method for simultaneous determination of fixed-dose combination of three drugs, perindopril tert-butylamine, amlodipine besylate and indapamide. Optimum conditions were achieved on ZORBAX Eclipse XDB-C18 column (150mm × 4.6mm, 5μm particle size), the mobile phase comprising acetonitrile and phosphate buffer (30mM, pH2.7) in the ratio 34:66 (v/v), the flow rate of 1mLmin-1, injection volume of 10μL and UV detection at 210nm. By assigning the design space from the overlay plot, the regions within which the robustness of the method is achieved were defined and confirmed by Dong's algorithm calculations. The proposed method was validated and shown to be applicable for the determination of the three drugs in commercially available tablets. In addition, the impact of the method on the environment was assessed through four different analytical tools: National Environmental Methods Index, Analytical Eco-Scale, Green Analytical Procedure Index and Assessment of Green Profile. The proposed method was determined to be greener, with minimal impact on the environment with regard to waste production, energy consumption and use of hazardous chemicals.

A Distance-Based Microfluidic Paper-Based Biosensor for Glucose Measurements in Tear Range.

The prevalence of diabetes has increased over the past years. Therefore, developing minimally invasive, user-friendly, and cost-effective glucose biosensors is necessary especially in low-income and developing countries. Cellulose paper-based analytical devices have attracted the attention of many researchers due to affordability, not requiring trained personnel, and complex equipment. This paper describes a microfluidic paper-based analytical device (μPAD) for detecting glucose concentration in tear range with the naked eye. The paper-based biosensor fabricated by laser CO2; and glucose oxidase/horseradish peroxidase (GOx/HRP) enzyme solution coupled with tetramethylbenzidine (TMB) were utilized as reagents. A sample volume of 10μl was needed for the biosensor operation and the results were observable within 5min. The color intensity-based and distance-based results were analyzed by ImageJ and Tracker to evaluate the device performance. Distance-based results showed a linear behavior in 0.1-1.2mM with an R2 = 0.9962 and limit of detection (LOD) of 0.1mM. The results could be perceived by the naked eye without needing additional equipment or trained personnel in a relatively short time (3-5min).

Development and Validation of new RP-HPLC Method for simultaneous Estimation of Methylcobalamin, Epalrestat and Pregabalin in bulk and Pharmaceutical dosage form

A new rapid, accurate, precise and economical reverse phase high performance liquid chromatographic method has been developed and validated for simultaneous estimation of Methylcobalamin, Epalrestat and Pregabalin in bulk and pharmaceutical dosage form. The separation was accomplished utilizing Agilent C18, 150 x 4.6mm, 5 column at a detection wavelength of 210nm utilizing the mobile phase water and acetonitrile 60: 40 v/v at the flow rate of 0.8ml/min and injection volume of 10µl. The total run time was 6.0 min. Validation discovered the method was specific, rapid, accurate, precise, reliable and reproducible. The calibration curve was linear over the concentration range of 0.37 – 2.25μg/ml of Methylcobalamin, 37.5-225μg/ml of Epalrestat and 37.5-225μg/ml of Pregabalin respectively with correlation coefficient of 0.999. The accuracy was determined by recovery studies and was found to be 99.5-100%. The precision of the results stated that the %RSD was <2%. The limits of detection for Methylcobalamin, Epalrestat and Pregabalin were 0.2, 0.9 and 1.2μg/ml, while the limits of quantification were 0.5, 1.5 and 0.9μg/ml respectively. Forced degradation study was carried out under acidic, alkaline, oxidative, photolytic and thermal conditions to prove the stability-indicating ability of the developed HPLC method. The high recovery confirms the suitability of developed method and can be further used in routine analysis.

Simultaneous Determination of Abamectin and Closantel in Veterinary Formulation by Validated HPLC Method.

A rapid and accurate high-performance liquid chromatographic method was developed for the determination of both abamectin and closantel in the veterinary formulation. The chromatographic separation was conducted on an Agilent 1200 with a UV detector using Waters C18 (4.6mm×50mm; 2.7μm). The mobile phase consisted of acetonitrile:water (80:20v/v) adjusts pH3.0 using diluted phosphoric acid. The flow rate of 1.5mLmin-1 was used. An injection volume of 10μL was used The calibration curve of abamectin B1b was linear with a correlation coefficient (r2)=0.9996; over a concentration range of 2.0-8.0μg/mL, abamectin B1a was linear with a correlation coefficient (r2)=0.9997; over a concentration range of 8.0-32.0μg/mL; with a retention time of 2.18 and 3.72minutes for avermectin B1b and avermectin B1a, respectively. While the calibration curve of closantel was linear with a correlation coefficient (r2)=0.99929; over a concentration range of 250.0-1,000.0μg/mL for; with a retention time of 5.84minutes. Correlation coefficient was r2≥0.999. The relative standard deviation was found to be ≤ 2. The proposed method was validated and successfully applied for the simultaneous determination of abamectin and closantel in the veterinary formulation.

Validated HPLC method for paclitaxel determination in PLGA submicron particles conjugated with α-fetoprotein third domain: Sample preparation case study

The goal of this study was to develop sample preparation method and validate the HPLC method for precise determination of paclitaxel (Ptx) in PLGA submicron particles conjugated with protein vector molecule. Ptx loaded PLGA submicron particles were formulated by a single emulsification method. PLGA submicron particles were conjugated with alpha fetoprotein third domain (rAFP3d) via standard carbodiimide technique. The obtained conjugate was analyzed using 1525 binary pump and 2487 UV-VIS detector system (Waters, USA) and Reprosil ODS C-18 analytical column with the dimensions of 150mm×4.6mm ID×5μm (Dr. Maisch GmbH, Germany). Sample preparation method was developed utilizing guard cartridge with С18 stationary phase (Phenomenex, USA). HPLC method was validated according to the international conference on harmonization guidelines. Efficient sample preparation was achieved using 4% of DMSO pre-dissolution, following by 10min of centrifugation at 4500g. Ptx determination was performed using acetonitrile/0.1% phosphoric acid (50:50 v/v) mobile phase at a flow rate of 1.0mL/min, injection volume of 10μL, and at 227nm. The developed method showed linearity, accuracy and precision in the range from 0.03 to 360μg/mL, with LOD and LOQ values of 0.005 and 0.03μg/mL, respectively. The intra- and inter-day precisions presented RSD values of lower than 2%. The validated method was successfully applied to calculate Ptx encapsulation efficacy and drug loading in the developed formulation.

28 Evaluation invitro of two protocols of vitrification from alpaca (Vicugna pacos) embryos

The reproductive efficiency of South American camelids as the alpaca is low, with a few number of animals having a good genetic characteristic. The transfer of cryopreserved embryos has great potential to disseminate valuable genetic, but the suitable protocol for such cryopreservation still needs to be developed. In this study, two protocols of vitrification of alpaca embryos were tested. Day 6.5 post-mating, embryos (n=66) were recovered from 14 female alpacas through a non-surgical technique and classified according to the characteristics of old world camelids reported by Skidmore et al. 2004 (Reprod. Fertil. Dev. 16, 605–609). Only quality 1 and 2 embryos were used for the study. They were placed together in 50-µL drops of holding medium for 30min and transferred to a 100-µL drop of equilibration solution 1, consisting of 7.5% (v/v) ethylene glycol (EG) + 0.25M sucrose. After 1min, embryos were transferred to equilibration solution 2, consisting of 15% (v/v) EG + 0.5M sucrose. After 2min, embryos were transferred into 2 consecutive drops of vitrification solutions A [SA: 30% (v/v) EG + 1M sucrose] for 20s each, then in 2 other drops of vitrification solution B [SB: 30% (v/v) EG + 3% glycerol + 1M sucrose] for 20s each. Thereafter, embryos were quickly loaded into open pulled straws (OPS) in a volume of 10µL and then plunged into liquid nitrogen. For warming, the OPS were held in air for 5s and subsequently thawed at 37°C for 50s. Straws were emptied into 1mL of prewarmed holding medium solution (HMS1) containing 1M sucrose for wash and the thawed blastocysts were transferred into a second 1mL of prewarmed HMS1. After 5min incubation at 37°C, the blastocysts were transferred into 1mL of warmed Holding medium solution 2 (HMS2) containing 0.5M sucrose maintained at room temperature (∼24°C) for evaluation. Data were analysed by the Chi-squared test. Post-thaw embryo expansion results were 81.3% and 58.8% for SA and SB (P<0.05), respectively. Post-thaw embryo quality (1 and 2) were found at 62.5% and 29.1% with SA and SB, respectively (P<0.05). In conclusion, the vitrification of alpaca embryos with the ethylene glycol:sucrose solution results in better post-thaw outcomes than the ethylene glycol:sucrose:glycerol. Further experiments with embryo transfer are needed. This research was funded by FONDECYT project no. 149-2017.

A novel RP- HPLC method development and forced degradation studies for semaglutide in active pharmaceutical ingredients and pharmaceutical dosage form

This research objective is for the development of a specific and simple method to trace Semaglutide presence in active pharmaceutical ingredient and pharmaceutical dosages. As part of a study on Semaglutide drug, solvents of HPLC grade waters HPLC instrument (Empower software) with PDA detector, ultrasonicator (Make: Labman) and pH meter (Make: Adwa) are used. The Method was optimized with mobile phase with a composition of buffer and solvent were of 60:40%v/v, flow maintained was 1.0ml/min, the injection volume of 10µl, run time was 5min. All separations were performed with PDA detector and column used was Discovery C18 150 x 4.6mm, 5m. Results for the developed method are accurate and specific. The detection wavelength was 292 nm, the retention time for Semaglutide was 2.689min, linearity resulted with r2= 0.9998, % RSD for precision was 1.0; %mean recovery for accuracy was in the range of 99.73 to 100.29. This study report is for industrial application for determining Semaglutide presence in pharmaceutical ingredient and dosages.

The effect of oral spray mist containing pine needle extract in reducing halitosis and compounds inducing halitosis

This study aimed to assess the effect of oral spray containing pine needle extract in suppressing halitosis, by analyzing the concentration of volatile sulfur compounds(VSCs) inducing halitosis and organic acids within saliva. The concentration of VSCs inside the oral cavity after the spray usage was measured using Sensor Gas Chromatograph ODSA-P2, and the analysis of organic acids within saliva was performed using HPLC Dionex ion chromatography system (GP50 gradient pump, ED 50 conductivity detector) with mobile phase under following conditions: NaOH 100mM with flow rate of 1.0mL/min, injection volume of 10㎕, and temperature of 20℃. In the experimental group, the levels of H2S and CH3SH were remarkably reduced by 80.87% and 89.42% respectively. In addition, the analysis of organic acids within saliva showed suppressive effect by 63.46–97.05% after 4 weeks. Therefore, it seems that using oral spray mist containing pine needle extract can partially suppress VSCs and organic acids within saliva that are associated with halitosis.

PDTM-17. IDENTIFICATION OF HUMAN PEDIATRIC BRAIN TUMORS CELLS IN PATIENT DERIVED ORTHOTOPIC XENOGRAFT MOUSE MODELS BY USING SPECIES-SPECIFIC PTGER2 GENE qPCR

AbstractBACKGROUNDPediatric brain tumors continue to be among the most difficult to cure and are the leading cause of cancer-related deaths in children. Research investigating pediatric brain cancer is still impaired by the lack of cancer models. We established a large panel of PDOX models as well as several paired permanent cell lines using and culture techniques. However, an inherent risk of xenograft models is the contamination of isolates of human tumors with host cells. Therefor strategies to identify the human/mouse ratio are essential. In this study we investigated Prostaglandin Receptor EP2 (PTGER2) as a useful target to identifying mouse cell contamination in PDOX models.METHODSpecies specific primers based on Alcoster et al., 2011 were used to detect PTGER2 in xenograft and cultured cells. RT-qPCR was carried out using a StepOnePlus™ RT-PCR System. Reaction mix contained SYBR Green master mix, respective primers and 5ng of total gDNA in a total reaction volume of 10μL. The qPCR conditions were as follows: 50°C-2min, 95°C-10min, 40 cycles of (95°C-15sec, 61°C-1min). Primers were additionally controlled by analysis of amplified products on a 1.2% agarose gel. FACS was used to isolate human cells according to anti-Human HLA-ABC Ab and mouse-specific Ab cocktail surface staining and subjected to qPCR.RESULTSVarious models of pediatric brain cancer established in our group were investigated for mouse cell contamination. Xenograft as well as cultured cells were routinely analyzed. Our study confirms PTGER2 as a useful target for accurately estimate mouse cell in PDOX models. This enabled us to improve detection and characterization of HLA-ABC/mouse-cocktail double negative population as seen in FACS.CONCLUSIONIn conclusion we have successfully implemented an reliable qRT-PCR approach to determine the human/mouse cell ration in any mixed PDOX cell population.

Universal efavirenz determination in transport study, rat placenta perfusion and placenta lysate by HPLC-UV

Efavirenz is an antiretroviral drug used in the treatment of HIV-positive patients. A simple, fast and sensitive high-performance liquid chromatography (HPLC) method was developed in order to determine efavirenz in three types of samples provided from pharmacokinetic studies. The analysis took 5min and was performed using a C18 analytical column (Discovery HS C18, 150×4.6mm, particle size of 5μm) in isocratic mode with a mobile phase containing acetonitrile and water (65:35, v/v), a flow rate of 1.6mLmin−1, a sample volume of 10μL and UV detection at 245nm. Three different sample matrices (Opti-MEM medium, Krebs perfusion liquid and tissue lysate) and their treatment (dilution, SPE) were considered. The validated method was applied for the analysis of 805 real samples arising from in vitro transcellular transport assays and in vivo organ perfusion experiments in order to evaluate the interaction of efavirenz with ATP-dependent drug efflux transporters. The lack of interaction of efavirenz with ABCB1, ABCG2 and ABCC2 transporters as well as technical aspects of this analysis, including the adhesion of efavirenz to the plastic materials and the stability of the drug during different tissue lysis approaches are discussed.

Method Development and Validation of Fingolimod in Bulk and Tablet Dosage Form by RP-HPLC

A facile and rapid isocratic reverse phase high performance liquid chromatography assay method has been developed and validated for the determination of fingolimod in bulk and tablet dosage form. The column was equilibrated for at least 30 min and separation was achieved by using kromofil-ODS C18 (150 x 4.6 mm, particle size 5 μm). The column was maintained at ambient temperature (25°C). The mobile phase employed was methanol: water (60: 40 v/v). The eluent was monitored using UV detector at 220 nm. A volume of 10μL of standard and sample solutions was injected in to the HPLC. The flow rate was 1.0 mL/min. The retention times were 4.525 min for fingolimod. The method was found to be linear in the range of 25–150 μg/mL. The developed method was validated as per ICH guidelines. The developed method was found to be simple, precise, accurate, selective and sensitive.

LC-ESI-MS/MS estimation of loratadine-loaded self-nanoemulsifying drug delivery systems in rat plasma: Pharmacokinetic evaluation and computer simulations by GastroPlus™

A rapid, sensitive, and accurate bioanalytical method was established for the quantitation and pharmacokinetic investigation of loratadine (LTD) in rat plasma by liquid chromatography–electrospray ionization mass spectrometry (LC-ESI-MS/MS) using loratadine-d5 as internal standard (ISTD). The analyte and ISTD were extracted by solid-phase extraction and chromatographic separation was achieved on Gemini NX- Reverse Phase C18 (50×4.6mm;5μ) using mobile phase mixture of 5mM ammonium formate buffer in water (pH 3.5±0.1 with formic acid), and acetonitrile (20:80v/v), at a flow rate of 0.400mL/min with injection volume of 10μL. LTD and ISTD were detected at m/z 383.3→337.4 and 388.4→337.3 with retention time of 2.62 and 2.59min, respectively. High sensitivity (1.0ng/mL) was achieved using small volume of rat plasma (20μL) and the method was validated over a linearity range of 1.05–405.41ng/mL with high correlation coefficient (r=0.9998). The extraction method displayed a mean process efficiency of 63.25 and 65.47% for LTD and ISTD, respectively. The validated method when successfully applied for quantification of LTD in rat plasma revealed enhanced bioavailability of orally administered LTD-loaded self-nanoemulsifying drug delivery system (SNEDDS) (Cmax, 466.65±18.94ng/mL and AUC0-t 633.00±12.44 ng-h/mL) over LTD-suspension (Cmax, 104.75±2.87ng/mL and AUC0-t 287.00±9.11 ng-h/mL). The in vivo-in silico prediction by the GastroPlus™ software showed good prediction accuracy for LTD-SNEDDS (fold error<2). The Loo-Reigelman method (2-compartment) presented best model-fitting indicating adequate in vitro–in vivo correlations. Conclusively, the developed sensitive analytical method displayed enhanced systemic availability of LTD-SNEDDS, and the in vivo in silico approach revealed sufficiently good GI simulation.