Spiked Plasma Samples Research Articles

Reproducible Quantification of Unbound Fractions of Four Beta-Lactam Antibiotics: Ultrafiltration Versus Microdialysis of Spiked Healthy Donor Plasma.

Ultrafiltration (UF) is a conventional method for isolating the protein-unbound plasma fractions of therapeutic drugs. However, the ideal UF conditions for specific compounds remain largely unexplored. By comparing UF-derived unbound concentrations with the corresponding results obtained using a reference method, the authors sought to identify appropriate UF conditions for cefotaxime, cloxacillin, flucloxacillin, and piperacillin. In vitro microdialysis (MD) with a no-net-flux approach was used as a reference method for plasma protein separation, for which UF performance was assessed. Four levels of relative centrifugal force (2500-11,290 g ) and 2 levels of temperature (37 vs. 22°C) during 10 minutes of UF centrifugation were evaluated. Ultrafiltrates and reference microdialysates were analyzed using liquid chromatography-tandem mass spectrometry to obtain unbound concentrations. After identifying the appropriate UF conditions in the spiked plasma samples, exploratory analyses of clinical samples (n = 10 per analyte) were performed. Of the evaluated UF alternatives, the best overall agreement with the MD-derived reference concentrations was obtained with 11,290 g UF performed at 22°C. For cloxacillin specifically, 37°C UF yielded better agreement than 22°C UF at 11,290 g. Clinical sample analyses indicated minimal differences between 22°C and 37°C at 11,290 g UF for cefotaxime and piperacillin. However, consistently lower levels of unbound cloxacillin (median: -23%, IQR: -19% to -24%) and flucloxacillin (median: -27%, IQR: -21 to -34%) were observed after UF at 22°C versus 37°C. For the evaluated UF device, 10 minutes of 11,290 g UF at 22°C is appropriate for flucloxacillin, cefotaxime, and piperacillin, and can arguably be justified for cloxacillin as well for laboratory practice purposes. Maintenance of 37°C during high-centrifugal UF may lead to overestimation, particularly for unbound flucloxacillin.

Ultrafast detection of folic acid in nanomolar levels and cancer cell imaging using hydrothermally synthesized carbon dots

The development of an ultrafast folic acid (FA) sensor (with no incubation time), having a detection limit and detection range in the nanomolar levels is of high importance in the field of biomedical FA sensing. For that, highly luminescent carbon dots (CDs) are synthesized by hydrothermal method with a lower growth duration of ‘1 h’ using the precursors, anhydrous citric acid as the carbon source and ethylenediamine as the nitrogen dopant material. TEM images confirmed a spherical morphology for the prepared CDs with an average size of nearly 2.8 nm. A photoluminescence quenching-based technique is used for FA detection using the prepared CDs. The detection limit is 6 nM, the lowest reported for CDs made from these precursors and the detection range is 0–50 nM. The mode of quenching is found to be static, based on the lifetime measurements and UV–vis spectroscopy. The mechanism of static quenching due to the formation of a ground state complex between the synthesized CDs and FA is confirmed by Raman spectroscopy and dynamic light scattering (DLS) study, and the binding mechanism of CDs and FA is explained in detail using FTIR and Raman spectroscopy for the first time. The fabricated CDs are used as biocompatible probes to image the N2a cells. A quantitative reduction in fluorescence intensity was observed for CD + FA mixtures internalized into the cells with incremental addition of FA. Furthermore, the practicality of the CD probe as an FA sensor is evaluated by testing the amount of FA in spiked plasma samples from albino rats, and the results are validated using a clinical method.

Magnetofluid-integrated biosensors based on DNase-dead Cas12a for visual point-of-care testing of HIV-1 by an up and down chip.

The CRISPR Cas system, as a novel nucleic acid detection tool, is often hindered by cumbersome experimental procedures, complicated reagent transfer processes, and associated aerosol pollution risks. In this study, an integrated nucleic acid detection platform named "up and down chip" was developed, which combined RT-RAA technology for nucleic acid amplification, DNase-dead Cas12a-modified magnetic beads for specific recognition of target nucleic acid, and HRP-TMB chromogenic reaction for signal output in different chambers of a single microfluidic chip. The magnetic beads were migrated in an up-and-down manner between different chambers through magnetic driving, achieving a "sample-in, result-out" detection mode. By introducing a homemade heating box for temperature control during the reaction and using the naked eye or a smartphone APP for color-based signal reading, no professional or precise instruments were required in this platform. Using this platform, highly sensitive detection of the HIV-1 genome as low as 250 copies (CPs) per mL was achieved within 100 min while maintaining good detection performance against common variants as well as excellent specificity and anti-interference ability. In addition, compared with qRT-PCR, it also exhibited good accuracy for 56 spiked plasma samples, indicating its promising potential for clinical application.

Simultaneous spectrofluorimetic determination of remdesivir and simeprevir in human plasma

As new infectious mutations of SARS-CoV-2 emerged throughout the world, innovative therapies to counter the virus-altered drug sensitivities were urgently needed. Several antiviral options have been in clinical trials or in compassionate use for the treatment of SARS-CoV-2 infections in an attempt to minimize both clinical severity and viral shedding. Recent research indicated that simeprevir acts synergistically with remdesivir, allowing for a multiple-fold decrease in its effective dose when used at physiologically acceptable concentrations. The goal of this work is to develop a sensitive synchronous spectrofluorimetric approach to simultaneously quantify the two drugs in biological fluids. Using this method, remdesivir and simeprevir could be measured spectrofluorimetrically at 283 and 341 nm, respectively, without interference from each other using Δλ of 90 nm. The effect of various experimental parameters on the fluorescence intensity of the two drugs was extensively explored and optimized. For each of remdesivir and simeprevir, the method exhibited a linearity range of 0.10–1.10 μg/mL, with lower detection limits of 0.01 and 0.02 μg/mL and quantification limits of 0.03 and 0.05 μg/mL, respectively. The high sensitivity of the developed method permitted the simultaneous determination of both drugs in spiked plasma samples with % recoveries ranging from 95.0 to 103.25 with acceptable standard deviation values of 1.92 and 3.04 for remdesivir and simeprevir, respectively. The validation of the approach was approved by the International Council of Harmonization (ICH) guidelines.

Computational and experimental studies of magnetic molecularly imprinted sorbent with high specificity towards aceclofenac

In this paper, the comprehensive studies were carried out to fabricate specific magnetic molecularly imprinted polymer (MMIP) towards the analysis of aceclofenac from spiked plasma samples. For that purpose, the optimization step, consisting of the analysis of binding capacities of bulk polymers synthesized from ethylene glycol dimethacrylate (cross-linker) and independently used five various functional monomers, in the presence of three different templates was performed. The results revealed that the polymers build-up from allylamine or 4-vinylpyridine molecularly imprinted by aceclofenac were characterized by the highest binding capacities towards target analyte (844 ± 101 and 673 ± 56 ng g−1, respectively) but with the highest specificity for the latter one (IF = 4.42 and 8.63, respectively) when compare to non-imprinted counterparts. The theoretical analysis revealed that hydrophobic interactions between the analyte and the polymer matrix were responsible for the highest binding capacity. Then, the MMIP, containing optimized molecularly imprinted external layer, was fabricated to facilitate the process of magnetic solid phase extraction (m-SPE). The physicochemical characterization confirmed the structure of MMIP and the morphology analysis showed the specific surface area equal to 16.42 m2 g-1. The effect of various loading solutions for the total recovery of aceclofenac was studied in the m-SPE. The analysis revealed that the total recoveries of aceclofenac from standard solutions were higher than from spiked human plasma. However, in the latter one, the total recoveries were significantly improved when the plasma was diluted by water. The analysis of total recoveries verified the capability of new MMIP to serve as the selective clean-up sorbent for the determination of aceclofenac from complex samples such as human plasma.

Determination of rivaroxaban by utilizing its quenching effect on acetoxymercuric fluorescein reagent in pharmaceutical preparations and in spiked biological matrices

A simple, precise and inexpensive spectrofluorimetric method has been developed for assay of rivaroxaban raw material and its tablets. The method depends on the quenching effect of rivaroxaban on the fluorescence intensity of acetoxymercuric fluorescein (AMF). Parameters that may affect the reaction such as pH, AMF solution concentration, reaction time and diluting solvents were studied and optimized. The proposed method was applied for determination of rivaroxaban in tablets with percentage recovery of 100.4 ± 0.28, and in organic extract of spiked plasma samples with percentage recovery of 98.40 ± 1.08. The developed method was validated according to ICH guidelines in terms of accuracy, precision, linearity, range, limit of detection (LOD) and limit of quantification (LOQ).

An electrochemical genosensing platform for the relative quantification of the circulating long noncoding RNA CCAT1 to aid in the diagnosis of colorectal cancer

Long noncoding RNAs (lncRNAs) are promising biomarkers for the noninvasive diagnosis of colorectal cancer, but detection of cancer-associated changes in their expression levels is now restricted to the use of RT-qPCR. To extend the clinical applicability of these biomarkers, we developed an electrochemical platform, integrated by two different sandwich genosensors, for the relative quantification of a lncRNA called colon cancer associated transcript-1 (CCAT1) with respect to the mRNA GAPDH as an endogenous control. To achieve highly sensitive detection, and in contrast to previous sensors of this class, we use multiple fluorescein-tagged hybridization assistant probes allowing the incorporation of multiple redox enzymes per target molecule, in turn favoring the selective capture of these highly-structured targets onto the sensing platform. The resultant biosensors show high sensitivity (9.7 ± 0.3 µA nM−1 for CCAT1 and 7.3 ± 0.1 µA nM−1 for GAPDH) and low limits of detection (990 fM and 1830 fM for CCAT1 and GAPDH, respectively). Coupled to a PCR assay, we detect the lncRNA at clinically relevant concentrations. Preliminary results using total RNA extracted from the colorectal cancer cell line HT29 and spiked plasma samples from healthy individuals suggest that this new sensing architecture is sensitive and selective enough to obtain the expression level of CCAT1 simply from the ratio of outputs from the two platform sensors. We envision that the same design could be applied to detect other lncRNAs associated to various malignancies, facilitating early cancer diagnosis.

Voltammetric Determination of Oxybutynin Hydrochloride Utilizing Pencil Graphite Electrode Decorated with Gold Nanoparticles

AbstractA novel voltammetric method was successfully applied for the determination of an anticholinergic drug, oxybutynin hydrochloride (OXB). The method is concerned with electrooxidation of the drug on the surface of pencil graphite electrode (PGE). In order to enhance the electrode sensitivity and peak current, the electrode was coated with gold nanoparticles (Au‐NPs) via electrochemical deposition using cyclic voltammetry from gold salt solution. The surface of Au‐NPs modified PGE has been characterized using scanning electron microscopy and X‐ray photoelectron spectroscopy. Various experimental variables were studied and optimized to enhance the sensor's response towards OXB. Quantitative determination of the drug was achieved in phosphate buffer pH 7.5 using differential pulse voltammetry by scanning the potential over range of 0.00 to 2.20 V with scan rate of 40 mV s−1. Validation of the method was achieved according to ICH guidelines. The method was found to be linear over concentration range (2.0×10−7–1.0×10−6 M). The suggested sensor was efficiently developed for the quantitative determination of OXB in pure form, pharmaceutical dosage form and spiked plasma samples.

Identifying the minimum concentrations of cell-free fetal DNA in maternal blood required for bovine fetal sexing using PCR

We evaluated the feasibility of cffDNA extraction from the maternal blood samples regarding the threshold concentrations required for fetal sexing in pregnant cattle by PCR. In four trials, we 1) compared the extraction efficiency of seven methods using freshly harvested plasma/blood of cows carrying male fetii (150–240 d gestation) bovine amelogenin (bAML) and Y-specific gene sequences, 2) identified the minimum amounts of spiked cffDNA needed for a PCR for fetal sexing, 3) determined the most optimal protocol among three commercial kits for cffDNA extraction from neat and spiked plasma samples (181–240 d gestation) for PCR detection of Y-specific sequence and 4) tested Y-specific sequence PCR on pregnant cows at different stages of gestation (60–150 versus 151–240 d pregnant). In these experiments, blood samples from unbred dairy heifers (Canadian Holstein, n = 10), pregnant dairy cows (Canadian Holstein, 60–240 d gestation, n = 25 with male fetii), and aborting beef cows (Angus cross, n = 5, 100–150 d pregnant) were used for DNA extraction, spiking, and PCR. Extracted DNA from the blood samples of unbred heifers (n = 5) and bull calves (n = 5) served as controls in all trials. In the first trial, DNeasy Blood and Tissue, Qiagen DSP Virus, and NucleoMag cfDNA isolation kits were relatively successful among seven methods to isolate cffDNA from freshly harvested maternal plasma/blood of pregnant cows. In trial 2, using serial dilutions of cffDNA from male fetii spiked in cow plasma samples, a positive and unambiguous detection by PCR targeting Y-specific sequence and bAML gene was observed when spiked cffDNA concentration in plasma was >31.3 pg/ml and >2 ng/ml, respectively. In the third trial, the DNeasy Blood and Tissue kit was most successful in extracting cffDNA spiked at the minimum concentrations in maternal plasma and subsequent PCR for Y-specific sequence. In our fourth trial, more cows in the second half (9/10) of gestation showed a positive Y-specific PCR outcome than those in the first half (3/9, Fischer's exact test; P < 0.05, 90%; CI: 55.5–99.75 vs 33%; CI: 7.5–70.1). In conclusion, we observed variability between different DNA extraction methodologies and stages of gestation results in the PCR for prenatal sexing. Thus, the current PCR methodologies are unreliable for detecting cffDNA in pregnant cows. Additionally, ≥10 (≥31.3 pg/ml of cffDNA) and ≥648 (≥2 ng/ml of cffDNA) copies of the whole fetal genome in bovine maternal plasma are needed for Y-specific PCR and bAML PCR, respectively.

Point-of-care diagnostics for therapeutic monitoring of levofloxacin in human plasma utilizing electrochemical sensor mussel-inspired molecularly imprinted copolymer

• Fabrication of MIP electrochemical sensor to personalize the Levofloxacin dose. • Bio-inspired molecularly imprinted copolymer was synthesized utilizing Levodopa as a monomer. • Molecularly imprinted copolymer was electropolymerized on Au-NPs decorated pencil graphite electrode as a substrate. • The sensor was employed successfully for levofloxacin detection in spiked human plasma. Therapeutic drug monitoring is mandatory for drugs with narrow therapeutic index and is an integral part of the patient care standards, where drug concentration is measured in biological fluids to “ personalize ” patient’s dosage. In this work a disposable electrochemical sensor has been developed for Levofloxacin (LEV) determination in human plasma. The selected substrate was pencil graphite electrode (PGE). To enhance the sensor performance, the PGE has been modified with Au-NPs via electrochemical deposition. To achieve high selectivity towards levofloxacin, the PGE/Au-NPs electrode has been further modified with molecularly imprinted copolymer; poly( l -dopa)/poly( o -phenylenediamine), by anodic electropolymerization of the corresponding monomers. The Levodopa not only acts as a functional monomer but moreover, a substrate for mussel-inspired polymer. The sensor surface has been characterized using both scanning electron microscopy (SEM) imaging and X-ray photoelectron spectroscopy (XPS) to investigate the morphology and elemental composition of the modified PGE, respectively. To optimize the sensor performance, experimental variables were studied and tuned to improve the outcomes. The sensor has a linear response in two concentration windows (1.0 × 10 −6 –1.0 × 10 −4 mol/L) and (1.0 × 10 −4 –1.0 × 10 −2 mol/L) Levofloxacin, with a limit of detection of 4.62 × 10 −7 mol/L. Selectivity, repeatability, and low manufacturing cost are some benefits of the proposed sensor. The sensor was successfully used to determine Levofloxacin in Tavanic® tablets and spiked plasma samples.

Unique Solid Dispersions by Microwave Fusion Technique with Etoricoxib and Thiocolchicoside: In vivo Evaluation and Comparison with Marketed Formulation

Aim: The primary objective of the study is to evaluate solid dispersion (SDs) made into tablets containing Etoricoxib (ECB) and Thiocolchicoside (TCS) for different in vivo parameters and in vitro drug release as an extension of the work described by Annepogu et al., in 2018. Methods: The in vitro study was constructed on various evaluation parameters of SDs of ECB and TCS. From earlier study results, we have selected formula-5 SDs of ECB and TCS for in-vivo study and comparison with marketed ones. Results: Plasma drug availability was performed on 6 healthy rabbits of both sexes (weighing 2.0-2.5 kg). They were aimlessly alienated into 2 groups of alike size by parallel design. Later, the study was HPLC analysis of the drug in the plasma, which involved preparation of the spiked plasma sample, and an analytical technique was established (simple and cost-effective method for ECB and TCS simultaneous estimation). The plasma samples were assessed for pharmacokinetic parameters. Finally, the F-5 formulation was assessed for in vitro drug release by comparison with the marketed preparation (Recox tablets-Hochest Biotech India). Conclusion: The study summarizes that TCS and ECB reached systemic circulation better than the pure drugs, and the kinetic values were appreciable. The drug discharge from the SDs was identical to the marketed one.

Synthesis, characterization, and the investigation of the applicability of citric acid functionalized Fe2O3 nanoparticles for the extraction of carvedilol from human plasma using DFT calculations and clinical samples analysis

A novel citric acid-functionalized Fe2O3 magnetic nanoparticle was used to extract carvedilol from human plasma. The developed method was used to determine carvedilol concentrations in hospitalized cardiovascular patients. The surface of the magnetic nanoparticles produced was modified with (3-amino propyl) tri-ethoxy-silane and citric acid. We utilized combined multi-spectroscopic, microscopic, and molecular modeling approaches to evaluate the nanoparticles' physicochemical characteristics. Using density functional theory (DFT) simulations, the probable mechanism of carvedilol interaction with the generated nanoparticle was investigated. Characterization data confirmed the production of magnetic nanoparticles and the surface modification by citric acid.The amount of CA-FSLN.NPs, pH, and composition of the extraction and elution solvents were optimized using spiked plasma samples. The FDA guideline for bioanalytical method validation was used to validate the applicability of the developed SPE-HPLC-UV method for carvedilol analysis. Based on the data, the calibration curvewas linear in the range of 0.02–0.8 µg.mL−1 (R2 = 0.99 andLLOQ = 0.02 µg.mL−1). The outcomes showed that the described method could be utilized foraccurate carvedilol analysesin clinical plasma samples. Carvedilol concentrations ranged from 0.06 to 0.26 µg.mL−1 in studied plasmasamples from seven patients. According to DFT calculations, the adsorption energy of carvedilol on CA-FSLN.NPs was 298.13 KJmol−1, with hydrogen bonding and hydrophobic interactions playing a significant role in the interaction of nonionized carvedilol with CA-FSLN.NPs.

The use of laser-based diagnostics for the rapid identification of blood borne viruses in human plasma samples

To demonstrate the use of a laser-based method of detection as a potential diagnostic test for the rapid identification of blood borne viruses in human plasma. In this study, using light emissions from laser sparks on plasma samples, the successful differentiation of both human immunodeficiency virus (HIV) and hepatitis C virus (HCV) in both residual de-identified plasma samples and plasma samples spiked to clinically relevant levels with each virus were demonstrated using plasma from more than 20 individuals spanning six different blood types (O+, O-, A+, A-, B+, B-). These experiments demonstrate that mathematical analysis of spectral data from laser sparks can provide accurate results within minutes. This capability was demonstrated using both spiked laboratory plasma samples and clinical plasma samples collected from infected and uninfected individuals. There is an ongoing need to rapidly detect viral infections and to screen for multiple viral infections. A laser-based approach can achieve sensitive, multiplex detection with minimal sample preparation and provide results within minutes. These properties along with the flexibility to add new agent detection by adjusting the detection programming make it a promising tool for clinical diagnosis. The potential for a laser-based approach has been previously demonstrated using pathogens spiked into human blood to clinically relevant levels. This study demonstrates this same ability to detect infections in clinical and laboratory spiked plasma samples. The ability to differentiate between plasma samples from infected and uninfected donors and determine the virus type using a laser-based diagnostic has not been previously demonstrated. Furthermore, this study is the first demonstration of the capability to differentiate viral infections in clinical plasma samples whereas previously published work used laboratory samples spiked with a virus or dealt with the detection of cancer in clinical plasma samples.

Simultaneous Determination of Pentoxifylline and Melatonin in Spiked Plasma Samples by Reverse Phase High Performance Liquid Chromatography Method

Due to their anti-inflammatory and antioxidant properties, pentoxifylline and melatonin have been considered by many authors as potential adjuvants in coronavirus disease infection. The present study describes the development of a high performance liquid chromatography method with ultraviolet detection for the simultaneous determination of pentoxifylline and melatonin in spiked plasma samples. The chromatographic separation was performed isocratically at a temperature of 25°, using a LiChrosorb® reverse phase-18 (125×4.0 mm, 5 μm) as stationary phase. The mobile phase consists of water and acetonitrile in a ratio of 80:20 v/v. Under optimal chromatographic conditions, for both pentoxifylline and melatonin, the number of theoretical plates was >2000, peak asymmetry was <1.3 and resolution factors were >2. The relative standard deviations for pentoxifylline and melatonin for both intra-day and interday not exceeded 4 % and recoveries were in the ranges 99.46 %-102.51 % for pentoxifylline and 96.02 %-102.85 % for melatonin.

SERS based Y-shaped aptasensor for early diagnosis of acute kidney injury.

Considering the pivotal role of biomarkers in plasma, the development of biomarker specific sensing platforms is of great significance to achieve accurate diagnosis and monitor the occurrence and progress in acute kidney injury (AKI). In this paper, we develop a promising surface-enhanced Raman scattering-based aptasensor for duplex detection of two protein biomarkers in AKI. Exploiting the base-pairing specificity of nucleic acids to form a Y-shaped self-assembled aptasensor, the MGITC labelled gold nanoparticles will be attached to the surface of magnetic beads. In the presence of specific AKI-related biomarkers, the gold nanoparticles will detach from magnetic beads into the supernatant, thus leading to a SERS signal increase, which can be used for the highly sensitive analysis of target biomarkers. In addition, the limit of detection calculated for each biomarker indicates that the SERS-based aptasensor can well meet the detection requirements in clinical applications. Finally, the generality of this sensor in the early diagnosis of AKI is confirmed by using a rat model and spiked plasma samples. This sensing platform provides a facile and general route for sensitive SERS detection of AKI-related biomarkers, which offers great promising utility for in vitro and accurate practical bioassay in AKI early diagnosis.

Development of a fast, online three-phase electroextraction hyphenated to fast liquid chromatography–mass spectrometry for analysis of trace-level acid pharmaceuticals in plasma

Sample preparation is a challenge for high-throughput analysis, especially for volume-limited samples with low-abundant analytes. Ideally, sample preparation enriches the analytes of interest while removing the interferents to reduce the matrix effect and improve both sensitivity and quantification. In this study, a three-phase electroextraction (EE) method hyphenated to fast online liquid chromatography–mass spectrometry (LC-MS) was developed. Four model acidic drugs of relevance for drug monitoring in plasma, i.e. naproxen, fenoprofen, flurbiprofen, and ibuprofen, were utilized for the optimization and evaluation of the method. A Design of Experiment approach (Box-Behnken design) was used to optimize the critical parameters of the method, i.e., the type of organic solvent, pH of the sample and acceptor phase, and the extraction voltage and time. Good fitness (P < 0.02, R2 > 0.95) was observed for the developed quadratic model. Extraction could be achieved in less than 2 min (115 s) with enrichment factors (EF) up to 190 and extraction recoveries (ER) up to 38% for academic samples. Additionally, the optimized three-phase EE method was successfully applied to spiked plasma samples with low-abundant (50 ng mL−1) analytes and a low sample volume of 15 μL plasma in 10-fold diluted samples. Finally, two crucial contributors to the matrix effect of three-phase EE application on plasma samples were determined. Specifically, the ion-suppression effect in the MS source was reduced by the fast LC separation, and the matrix effect during extraction was negligible for the diluted protein-precipitated plasma samples. The developed three-phase EE method is easy to operate and provides fast and online extraction of trace-level acidic analytes from volume-limited biological samples. Therefore, this method can provide a potential solution for sample-preparation bottlenecks in high-throughput bioanalysis workflows.

The influence of storage time on ponazuril concentrations in feline plasma.

BackgroundThe pharmacokinetics of ponazuril have been determined in several species; however, there is very little information on the stability of the drug after storage for long periods of time. This study was undertaken to determine the stability of ponazuril in plasma samples stored at −80 °C, which is the temperature most commonly used in the author’s laboratory.MethodSpiked plasma samples (0.3, 7.5, and 15 µg/mL) were stored at −80 °C for three months. Analysis occurred on the first day and then once a week for the following twelve weeks. The drug was extracted using a chloroform extraction and separated by high performance liquid chromatography using ultraviolet detection.ResultsThere was no loss of drug for any concentration for the first four weeks of storage. There was an average loss of less than 5% from day 35 through day 70 and an average loss of 6% on day 77 and 84. The data suggest that ponazuril is stable for 4 weeks when stored at −80 °C and undergoes minimal loss in the remaining 8 weeks.

Impedimetric Sensors for Cyclocreatine Phosphate Determination in Plasma Based on Electropolymerized Poly(o-phenylenediamine) Molecularly Imprinted Polymers.

Cyclocreatine and its water-soluble derivative, cyclocreatine phosphate (CCrP), are potent cardioprotective drugs. Based on recent animal studies, CCrP, FDA-awarded Orphan Drug Designation, has a promising role in increasing the success rate of patients undergoing heart transplantation surgery by preserving donor hearts during transportation and improving the recovery of transplanted hearts in recipient patients. In addition, CCrP is under investigation as a promising treatment for creatine transporter deficiency, an X-linked inborn error resulting in a poor quality of life for both the patients and the caregiver. A newly designed molecularly imprinted polymer (MIP) material was fabricated by the anodic electropolymerization of o-phenylenediamine on screen-printed carbon electrodes and was successfully applied as an impedimetric sensor for CCrP determination to dramatically reduce the analysis time during both the clinical trial phases and drug development process. To enhance the overall performance of the proposed sensor, studies were performed to optimize the electropolymerization conditions, incubation time, and pH of the background electrolyte. Scanning electron microscopy, electrochemical impedance spectroscopy, and cyclic voltammetry were used to characterize the behavior of the developed ultrathin MIP membrane. The CCrP-imprinted polymer has a high recognition affinity for the template molecule because of the formation of 3D complementary cavities within the polymer. The developed MIP impedimetric sensor had good linearity, repeatability, reproducibility, and stability within the linear concentration range of 1 × 10–9 to 1 × 10–7 mol/L, with a low limit of detection down to 2.47 × 10–10 mol/L. To verify the applicability of the proposed sensor, it was used to quantify CCrP in spiked plasma samples.

Bio-Analytical Method Development for Estimation of Levofloxacin: Application in Estimation of Drug in Nano-formulations and Pharmacokinetic Studies

Abstract: Aim/Background: Levofloxacin is a safe antibiotic for various inflammatory disorders and a rapid analytical method for various samples reduces the estimation time as well as injects efficiency in the system. The developed method uses minimal solvents with least preparation time also helps in reducing the errors generated by the analyst during processing. Materials and methods: RP-HPLC method was developed for estimation of levofloxacin using Box Behnken Design. The PDA detector was set at 295nm with a run time of 6 mins. The samples were run at binary gradient mode with 35% phase B. The mobile phase consisted of acetate buffer, acetonitrile and methanol. Linearity was established for pure drug as well as spiked plasma samples. Drug extraction from plasma samples was done by liquid-liquid extraction using acetonitrile and methanol. Results: The developed method has a retention time of 3.1± 0.41 mins and is validated as per ICHQ2R1 guidelines. The standard curve indicates high correlation with a R2 value of 0.9999. The robustness (RSD<2) and system suitability studies indicate that it is a stable method with high specificity as determined with different samples. The LOD and LOQ were found to be 0.08ppm and 0.25ppm respectively indicating estimations of samples with a low drug content. Conclusion: A rapid, robust, selective, simple and sensitive method was developed and validated for quick estimation of levofloxacin in different sample. The significant reduction in retention time obtained by the developed method proves its efficiency in tapping the mobile phase costs as well as expediting the analytical process. The DOE optimised method is suitable for rapid estimation of levofloxacin in plasma as well as nanoformulations. Key words: Bioanalytical method, Robustness, Specificity, Stability, ICHQ2R1.

A Portable Digital Loop-Mediated Isothermal Amplification Platform Based on Microgel Array and Hand-Held Reader.

Digital polymerase chain reaction (dPCR) has found widespread applications in molecular diagnosis of various diseases owing to its sensitive single-molecule detection capability. However, the existing dPCR platforms rely on the auxiliary procedure to disperse DNA samples, which needs complicated operation, expensive apparatus, and consumables. Besides, the complex and costly dPCR readers also impede the applications of dPCR for point-of-care testing (POCT). Herein, we developed a portable digital loop-mediated isothermal amplification (dLAMP) platform, integrating a microscale hydrogel (microgel) array chip for sample partition, a miniaturized heater for DNA amplification, and a hand-held reader for digital readout. In the platform, the chip with thousands of isolated microgels holds the capability of self-absorption and partition of DNA samples, thus avoiding auxiliary equipment and professional personnel operations. Using the integrated dLAMP platform, λDNA templates have been quantified with a good linear detection range of 2-1000 copies/μL and a detection limit of 1 copy/μL. As a demonstration, the epidermal growth factor receptor L858R gene mutation, a crucial factor for the susceptibility of the tyrosine kinase inhibitor in non-small-cell lung cancer treatment, has been accurately identified by the dLAMP platform with a spiked plasma sample. This work shows that the developed dLAMP platform provides a low-cost, facile, and user-friendly solution for the absolute quantification of DNA, showing great potential for the POCT of nucleic acids.