Porphyrin-based hypercrosslinked polymers as sorbents for efficient extraction of nitroimidazoles from water, honey and chicken breast.

In this work, a series of 5,10,15,20-tetraphenylporphyrin (TPP)-based hyper crosslinked polymers were prepared by Friedel-Crafts reaction. Among them, the HCP-TPP-BCMBP, which was prepared by using TPP as the monomer and with 4,4'-Bis(chloromethyl)-1,1'-biphenyl (BCMBP) as the cross-linking agent, had the best adsorption capability for the enrichment of the nitroimidazoles of dimetridazole, ronidazole, secnidazole, metronidazole, and ornidazole. Then, a solid-phase extraction (SPE) method with the HCP-TPP-BCMBP as adsorbent coupled with HPLC-UV detection for the determination of nitroimidazole residues in honey, environmental water, and chicken breast samples was established. The influence of the main factors that affect the SPE, i.e., sample solution volume, sample loading rate, sample pH, and eluent and its volume, were studied. Under the optimal conditions, the limits of detection (S/N = 3) for the nitroimidazoles were measured to be in the range of 0.02-0.04 ng mL-1, 0.4-1.0 ng g-1 and 0.5-0.7 ng g-1 for environmental water, honey, and chicken breast samples, with the determination coefficients being in the range of 0.9933-0.9998. The analytes recoveries by the method in fortified samples fell in the range from 91.1% to 102.7% for environmental water, from 83.2% to 105.0% for honey, and from 85.9% to 103.0% for chicken breast samples, and the relative standard deviations for the determination were less than 10%. It shows that the HCP-TPP-BCMBP has a strong adsorption capability for some polar compounds.

[Analysis of parabens in environmental water samples by covalent organic framework-based magnetic solid-phase extraction-high performance liquid chromatography].

Parabens are a class of antimicrobial preservatives that are widely used in cosmetics, pharmaceuticals, and food products because of their ease of production, antimicrobial effect, and low price. The widespread use of these parabens, poses potential risks to human health. Therefore, it is necessary to establish a simple and rapid method for the detection of parabens. The large number of substrate interferences in complex samples is an important factor affecting the sensitivity of analytical methods. Magnetic solid-phase extraction (MSPE) has received much attention because of its advantages of easy operation, short extraction time, small sample amount, low cost, and environmental friendliness. Covalent organic frameworks (COFs) with high crystallinity, high specific surface area, adjustable pore size, regular porosity, as well as high chemical and thermal stability are now widely used in separation and analysis. Therefore, a sample pretreatment method combining MSPE and COF for the analysis of parabens in complex matrices is very promising. A magnetic covalent organic framework, Fe3O4@TbBd, was successfully synthesized by the Schiff base reaction of 1,3,5-triformylbenzene (Tb) and benzidine (Bd) at room temperature using Fe3O4 nanoparticles as magnetic cores. Characterization by scanning electron microscopy (SEM), thermogravimetric analysis (TGA), X-ray diffraction (XRD), vibrating sample magnetometer (VSM) measurements, etc. revealed that the magnetic COF has high magnetic responsiveness, as well as good thermal and chemical stability, which make it an ideal adsorbent for the MSPE of parabens. Some factors related to the extraction efficiency, including the amount of adsorbent, extraction time, pH, desorption solvent, desorption time, and number of desorption were systematically investigated. A method involving MSPE and high performance liquid chromatography-ultraviolet detection (HPLC-UV) based on the Fe3O4@TbBd was developed for the determination of four parabens (ethylparaben, propylparaben, butylparaben, and benzylparaben) in environmental water samples. Under the optimal extraction conditions, the method showed good linearities. The limits of detection and limits of quantification were 0.2-0.4 µg/L and 0.7-1.4 µg/L for the four analytes, respectively. The recoveries at three spiked levels were in the range of 86.1%-110.8% with intra-day and inter-day RSDs of less than 5.5% and 4.9%, respectively. The method was successfully applied to the determination of parabens in East Lake water, Yangtze water, and domestic wastewater. Ethyl paraben and propyl paraben were detected in domestic wastewater at the levels of 1.8 µg/L and 0.4 µg/L, respectively. The recoveries of the parabens at different spiked levels ranged from 80.7% to 117.5%, with RSDs of 0.2%-8.8%. The method has good potential for the determination of parabens in environmental water samples because of its operational simplicity, short extraction time, high sensitivity, and environmental friendliness.

Determination of gibberellic acid and abscisic acid in (Zea mays L.) (ICA-V305) seeds germinated using dynamic sonication assisted solvent extraction and maceration.

Phytohormones are molecules responsible for growth, development, and metabolism regulation in plants. Gibberellic acid (GA3) and abscisic acid (ABA) are the main phytohormones involved in seed germination. Notably, it should be a highlight that GA3 induces germination, whereas ABA inhibits it. For this reason, it is important to calculate the concentration of these two phytohormones during seeds germination. Firstly, the maize seeds (MS) were germinated and samples of these were taken at different imbibition times, after that, methanol extracts were obtained using two methods of dynamic solvent extraction assisted by sonication (DSASE) and a traditional extraction method (maceration); finally, to estimate the concentration of GA3 and ABA a high performance liquid chromatographic method was used. The results of this study showed that the three extraction methods used, allowed quantifying GA3 and ABA during the maize germination time studied. However, of the two extraction methods employed, DSASE was the best technique because higher concentrations of GA3 and ABA were found. Therefore, it is important to continue using this green chemistry methodology for these and other analyses.•The extraction protocol developed was based on dynamic sonication-assisted solvent extraction.•The chromatographic method used allowed the simultaneous determination of two phytohormones with different physicochemical properties in maize seeds.•This methodology offers good sensitivity, linearity, precision, reproducibility and suitable detection and quantification limits.

Development of an UHPLC-diode arrays detector (DAD) method for the analysis of polydatin in human plasma.

A new chromatographic method by Ultra High Performance Liquid Chromatographic (UHPLC) technology, has been developed and validated for the determination of polydatin and resveratrol, as potential metabolite, in human plasma. After the optimization of the chromatographic conditions, the method has been validated on spiked human plasma samples. The optimized extraction allowed to obtain analytes recovery up to 98.48 ± 4.03 %. Then, the isocratic elution in reversed phase mode, provides the separation of polydatin and resveratrol in less than 10.0 min. Chromatographic analysis was performed on a C18, 10 cm x 3.0 mm, 2.7 µm stationary phase, by using triethanolamine phosphate solution (0.1 M, pH = 3.7) and ACN 85:15 (v/v) as mobile phase at a flow rate of 0.5 mL/min. The UV detector was set at 306 nm for the analysis of both polydatin and resveratrol. The limit of detection (LoD) and the limit of quantification (LoQ) for polydatin in plasma samples were found to be 7.82 ± 0.38 nM and 26.06 ± 1.28 nM respectively. The method was found to be accurate and precise with a coefficient for intra- and inter-day variation below 5 %. All the reported data demonstrate how the developed method is rapid and sensitive. Moreover, results of the analysis of plasma samples, obtained from orally treated volunteers with nutritional supplements containing polydatin, have shown the method to be suitable for the pharmacokinetic characterization of polydatin and resveratrol, as metabolite, in humans.

High performance liquid chromatographic assay of amlodipine, valsartan and hydrochlorothiazide simultaneously and its application to pharmaceuticals, urine and plasma analysis.

A Simple, Specific, Precise, Accurate, Linear, Rugged, Robust High Performance Liquid Chromatographic method of analysis for simultaneous determination of assay of Amlodipine, Valsartan and Hydrochlorothiazide drugs in the pharmaceuticals tablet formulations using Pioglitazone as a common internal standard was developed and validated. The assay was accomplished using a mixture of acetonitrile & methanol in the volume ratio of 20:80 v/v (mobile phase B) and Ammonium acetate buffer (Mobile phase A) in gradient flow as mobile phase on an Hibar RP-18e, 250 × 4.6 mm, 5µ as chromatographic column at a flow rate of 1.300 mLmin-1, injection volume 10 µL and at a wavelength 235 nm with UV detector. Linearity of the analytical method was evaluated at a concentration range of 2.5-45.3 µg/ml for Amlodipine, 32.0-720.1 µg/ml for valsartan and 5.0-112.6 µg/ml for Hydrochlorothiazide respectively with Correlation coefficient (r) value more than 0.9997. The limit of detection (LOD) for Amlodipine, Valsartan and Hydrochlorothiazide was found to be 1.1 µg/ml, 8.0 µg/ml & 1.0 µg/ml respectively. Specificity, Method Precision, System Precision, Ruggedness, Robustness, Recovery, Stability of analytical solution, Filter paper selection study, Stress testing (Force Degradation) at various conditions were performed as per the ICH (Q2) recommendations. The chromatographic method may also be applied for simultaneous estimation of analytes in plasma and urine.

Investigation of the Presence of Sildenafil in Herbal Dietary Supplements by Validated HPLC Method.

OBJECTIVES: As the first FDA-approved phosphodiesterase type 5 inhibitor, sildenafil (SDF) is widely used in the treatment of erectile dysfunction due to its strong pharmacodynamic activity. Since many food supplements are now involved in illegal adulteration, the presence of SDF in food supplements is very important because of their toxicological risks. In this study a simple fast, reliable high-performance liquid chromatography method with ultraviolet (UV) detector has been developed and validated for SDF analysis in herbal dietary supplements (HDSs). MATERIALS AND METHODS: 10 mM phosphate buffer containing 0.1% triethylamine (pH 3.5) and acetonitrile (65:35, v/v), as mobile phase was applied isocratically to a reverse phase C18 analytical (4.6×250 mm, 5 µm) column. Chromatographic separation was achieved by a C18 reverse-phase analytical column 4.6×250 mm, 5 µm particle size, using acetonitrile, with 10 mM phosphate buffer containing 0.1% triethylamine (65:35, v/v, pH 3.5) as a mobile phase. The mobile phase flow rate was 1 mL min-1 and the column temperature was 35°C. The UV detector was set at 293 nm. The liquid-liquid extraction method used in the study provided a simple and practical method for the recovery of SDF in HDSs and their obtained values ranged from 87.6 to 111.7%. RESULTS: The method showed linearity with an excellent correlation coefficient (r2>0.999). Moreover, it was specific and sensitive with the limit of quantification, 6.5 ng mL-1. Intraday and interday method precision was ≤8.2 (relative standard deviation %). Intraday and interday method accuracy was between -4.0 and 7.1 (RE%). The method was strong according to the robustness test results obtained from UV detection, mobile phase buffer pH, column temperature, and flow rate changes. The described procedure was simple, fast, precise, and feasible for routine adulteration analysis of SDF, especially in food control or toxicology laboratories. This method was successfully applied to 50 individual solid and liquid form HDSs. CONCLUSION: The results showed that 37 out of 50 samples of HDSs (represented 74.0%) examined contained SDF between 0.01 and 465.47 mg/g, 150.87±127.48 (mean ± standard deviation), which could lead to serious health problems and might even be fatal for consumers. The described procedure was found to be simple, rapid, precise and feasible for routine adulteration analysis of SDF, especially in food control or toxicology laboratories.

Reversed-phase high-performance liquid chromatographic method development and validation for allyl isothiocyanate estimation in phytosomes of Brassica nigra extract.

A simple, specific, accurate, precise, and robust reverse-phase high-performance liquid chromatographic method has been developed and validated for the estimation of allyl isothiocyanate (AITC) in phytosomes of black mustard extract (Brassica nigra). The method was validated with respect to specificity, linearity, accuracy, precision, and robustness. The linearity was achieved over a range of 10-18 µg/mL and regression coefficient was obtained 0.9961. Accuracy of chromatographic method was evaluated by standard addition method; percentage recovery attained was 97.07 ± 0.008-103.66 ± 0.013. Relative standard deviation for intraday and interday precision was 0.02% and 0.22%, respectively. The limit of detection and limit of quantification of the AITC were found to be 0.0043 µg/mL and 0.01324 µg/mL, respectively. This result shows that the method was well validated. In the present study, the AITC content was found 0.0009% ± 0.014% in black mustard. This study reveals that the proposed high-performance thin-layer chromatography method is accurate, fast, and cost-effective for the routine estimation of AITC in the phytosome formulation.

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 4months 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.

Production and stability study of a hospital parenteral nutrition solution for neonates / 药物分析学报

Standard parenteral nutrition solutions are mixtures comprising interacting components that may de-grade 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 mi-crobiological 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 4months 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.

Modified Nucleosides - Molecular Markers Suitable for Small-volume Cancer?

BACKGROUND: Modified nucleosides (mNS) in urine are shown to be encouraging markers in cancer, mostly in patients presenting with high tumor mass such is breast and lung cancer. To our knowledge, mNS have not been investigated in head and neck squamous cell carcinoma (HNSCC). HNSCC is characterized by early metastasis into locoregional lymph nodes and slow infiltrating growth, but even in the advanced stage exhibits only a relatively low cancer volume. Therefore, reliable distinction between HNSCC and healthy controls by urinary mNS might pose substantial analytical problems and even more as patients with HNSCC mostly have an increased exposure to tobacco smoke and excessive alcohol consumption which affect the renal mNS pattern. MATERIALS AND METHODS: Urinary mNS in samples of 93 therapy-naive patients with HNSCC and 242 healthy controls were quantified by reversed-phase high-performance liquid chromatography. Considering that the circadian rhythm causes diuresis-induced variations in concentration, the mNS-to-creatinine ratio was chosen to compare patients and controls. For sensitivity and specificity in discriminating between patients and controls, the corresponding curve was plotted. Additionally, logistic regression was carried out and a multilayer perceptron neuronal network (NN) was created. RESULTS: Fifteen mNS were detectable in cases and controls; concentrations of 11 were found to be significantly different. The sensitivity and specificity depend on the total volume of the lesion; HNSCC with volume <20 ml was reliably detected, but those with a volume of 20 ml or greater produced amounts of mNS which led to the most accurate detection of HNSCC based on HNSCC-specific mNS patterns. CONCLUSION: Analysis of urinary mNS allows for detection of small-volume HNSCC, with acceptable specificity and sensitivity if the tumor volume exceeds 20 ml.

Medical Emergencies Related to Ethanol and Illicit Drugs at an Annual, Nocturnal, Indoor, Electronic Dance Music Event.

Introduction Medical problems are frequently encountered during electronic dance music (EDM) events. Problem There are uncertainties about the frequencies and severity of intoxications with different types of recreational drugs: ethanol, "classical" illicit party drugs, and new psychoactive substances (NPS). METHODS: Statistical data on the medical problems encountered during two editions of an indoor electronic dance event with around 30,000 attendants were retrieved from the Belgian Red Cross (Mechelen, Belgium) database. Data on drug use were prospectively collected from the patient (or a bystander), the clinical presentation, and/or toxicological screening. RESULTS: In the on-site medical station, 487 patients were treated (265 in 2013 and 222 in 2014). The most frequent reasons were trauma (n=171), headache (n=36), gastro-intestinal problems (n=44), and intoxication (n=160). Sixty-nine patients were transferred to a hospital, including 53 with severe drug-related symptoms. Analysis of blood samples from 106 intoxicated patients detected ethanol in 91.5%, 3,4-methylenedioxymethamphetamine (MDMA) in 34.0%, cannabis in 30.2%, cocaine in 7.5%, amphetamine in 2.8%, and gamma-hydroxybutyric acid (GHB) in 0.9% of patients (alone or in combination). In only six of the MDMA-positive cases, MDMA was the sole substance found. In 2014, the neuroleptic drug clozapine was found in three cases and ketamine in one. Additional analyses for NPS were performed in 20 cases. Only in one agitated patient, the psychedelic phenethylamines 25B-NBOMe and 25C-NBOMe were found. CONCLUSIONS: At this particular event, recreational drug abuse necessitated on-site medical treatment in one out of 350 attendants and a hospital transfer in one out of 1,000. Ethanol remains the most frequently abused (legal) drug, yet classical illicit recreational drugs are also frequently (co-) ingested. The most worrying observation was high-risk poly-drug use, especially among MDMA users. Regarding NPS, the number of cases was low and the clinical presentations were rather mild. It should be stressed that these observations only apply to this particular event and cannot be generalized to other EDM events. Calle P , Sundahl N , Maudens K , Wille SMR , Van Sassenbroeck D , De Graeve K , Gogaert S , De Paepe P , Devriese D , Arno G , Blanckaert P . Medical emergencies related to ethanol and illicit drugs at an annual, nocturnal, indoor, electronic dance music event. Prehosp Disaster Med. 2018;33(1):71-76.

[A novel pattern for predicting the quality of Chinese herba preparation intelligently by high performance liquid chromatographic formula fingerprints].

In order to build the fusion models, the high performance liquid chromatographic (HPLC) fingerprints of scutellariae radix (SR), rhei radix et rhizoma (RRR), coptidis rhizoma (CR) and their synthesizing fingerprints were developed in this study. After exploring the consistency between the fingerprints of compound synthesizing fingerprints (CSF) and the sample, the quality of traditional Chinese medicine preparation was predicted intelligently using CSF. HPLC coupled with diode array detector was used to obtain chromatograms of SR, RRR, CR and Yi Qing Tablet (YQT) samples at 268 nm. Meanwhile, the quality of CSF and the 15 batches of YQT samples was evaluated by systematically quantified fingerprint method (SQFM) qualitatively and quantitatively. The chromatograms showed that CSF covered the main fingerprints' information of each herb and the 55 common peaks of CSF covered the main information of the 50 common peaks in YQT sample. The evaluation results showed that among the 15 batches of YQT samples, only YQT-S01 was grade 5 and the others were all above grade 3. Most of the CSFs were grade 2 or grade 1 except CSF-2 which was grade 6. The fingerprints of Chinese herba preparation could be replaced by CSF to achieve a novel pattern for predicting the quality of TCM preparation intelligently by studying the relationship between the standard fingerprints and the CSF, and simultaneously developing first-class evaluation software.

High-performance liquid chromatographic cartridge with gradient elution capability coupled with UV absorbance detector and mass spectrometer for peptide and protein analysis.

We discuss the construction and performance of a high-performance liquid chromatography cartridge that we developed that resulted from a culmination of previous research. We have recently developed an innovative approach to creating gradient elutions using dual electroosmotic pumps and a series of three valves. This method has been proved to be the most reproducible and robust in producing gradients compared to our previously tested methods. Using this approach, we have assembled a high-performance liquid chromatography cartridge powered and controlled via a computer. We have successfully coupled the cartridge with an ultraviolet absorbance detector and a mass spectrometer for separating complex protein/peptide samples. The cartridge is readily coupled with other detectors such as electrochemical detector and laser-induced fluorescence detector.

Determination of glycerol in propofol medium and long chain fat emulsion injection by HPLC / 中国生化药物杂志

Objective A high performance liquid chromatographic (HPLC) method was established for the determination of glycerol in propofol medium and long chain fat emulsion injection.MethodsThe chromatographic conditions were as follows: Kromasil 100-5-NH2 column(4.6×250mm,5μm) with the column temperature was 40℃,acetonitrile-water(8515)as mobile phase with flow rate of 1.0mL/min.Glycerol was detected by refractive index (RI) detector at 40℃.ResultsThe linear range of glycerol was 455.3916-2276.9580μg/mL(r=0.9999,n=7),the average recovery rate was 99.5%,RSD was 0.6%(n=9),the limit of detection(LOD) was 121ng and the limit of quantification(LOQ)was 364ng.ConclusionThe method was simple, rapid, strong specifity and accurate with good reproducibility, which is suitable for the content determination of glycerol in propofol medium and long chain fat emulsion injection.

Quantification of DOX bioavailability in biological samples of mice by sensitive and precise HPLC assay.

CONTEXT: Doxorubicin (DOX)-loaded folate-targeted poly(3-hydroxybutyrate-co-3-hydroxyoctanoate) [P(HB-HO)] nanoparticles [DOX/FA-PEG-P(HB-HO) NPs] were prepared by the W1/O/W2 solvent extraction/evaporation method for applications in cancer treatment. However, the biodistribution, pharmacokinetics, and targeting of the nanoparticles (NPs) have not yet been studied. OBJECTIVE: The biodistribution, pharmacokinetics, and targeting of DOX/FA-PEG-P(HB-HO) NPs were evaluated in female BALB/c nude mice bearing HeLa tumors. MATERIALS AND METHODS: Three DOX formulations were injected into the tail vein of the mice at a dosage of 5 mg/kg. At each time point, blood and various tissues were collected. All samples were then processed and analyzed by a validated high performance liquid chromatographic (HPLC) method. RESULTS: The t1/2 values of DOX/P(HB-HO) NPs and DOX/FA-PEG-P(HB-HO) NPs were 2.7- and 3.5-times higher than that of free DOX. No significant difference (p > 0.05) was found in Cmax between the NPs and free DOX. The Tmax values of the two NPs were prolonged from 0.25 to 1 h. The AUC0-t values were 1.55- and 3.05-folds higher than that of free DOX, and MRT increased to 15.99 h for DOX/P(HB-HO) NPs and 25.14 h for DOX/FA-PEG-P(HB-HO) NPs. For DOX/FA-PEG-P(HB-HO) NPs, the DOX content in the tumors were 10.81- and 3.33-times higher than those for free DOX and DOX/P(HB-HO) NPs at 48 h, respectively. DISCUSSION AND CONCLUSIONS: DOX/FA-PEG-P(HB-HO) NPs displayed reduced cardiac toxicity and improved bioavailability. Moreover, the NPs exhibited a significant extent of DOX accumulation in the tumors, thus suggesting that folate-targeted NPs could effectively transport into HeLa tumors with satisfying targeting.

Computerized optimization of high-performance liquid chromatographic method by using DryLab to test and isolate six cytochrome P450 probe substrates / 国际中医中药杂志

Objective DryLab software was used to assist high performance liquid chromatography (HPLC) method to test and isolate six Cytochrome P450 (CYP450) probe substrates.Methods Six CYP450 probe substrates were selected and the right HPLC method was developed and validated with the assistance of DryLab software.Results The new HPLC method with the assistance of DryLab software could test and isolate six probe substrates with degrees of isolation more than 2.00. The correlation coefficients (R> 0.999 8) indicated high linear correlation between the concentrations and the peak areas among six probe substrates. Recovery studies showed good results for all the probe substrat from 86.38% to 110.29%. And therelative standard deviation (RSD) ranged from 1.69% to 3.80% with its intra-day and inter-day precision ranging from 0.42% to 2.01%, and 1.36% to 2.29%, respectively.Conclusions The developed HPLC method with the assistance of DryLab could test and isolate six probe substrates with shortertime than the HPLC method alone.

Simultaneous determination of xylitol and L-xylulose in fermentation broth with high performance liquid chromatography / 中国生化药物杂志

Objective A high performance liquid chromatographic (HPLC) method was established for the simultaneous determination of xylitol and L-xylulose in fermentation broth.Methods The chromatographic conditions were as follows:C18 column (250 mm ×4.6 mm) with the temperature 35℃, acetonitrile-water (85∶15,v/v)as mobile phase with the flow rate of 0.8 mL/min.Xylitol was detected by refractive index (RI) detector at 33℃and L-xylulose was determined by ultraviolet ( UV) detector at 210 nm at room temperature.Results This method showed good linearity over the range from 0.50~30.00 g/L with a correlation coefficient of 0.9995 for xylitol and 0.30~30.00 g/L with a correlation coefficient of 0.9986 for L-xylulose. Moreover, the limit of quantification (LOQ) for xylitol and L-xylulose were 0.58 and 0.40,respectively.The limit of determination (LOD) for xylitol and L-xylulose were 0.18 and 0.15,respectively.The relative standard deviations (RSDs) of intraday and interday for xylitol were less than 0.64%and 0.80%,respectively.The intraday and interday RSDs for L-xylulose were less than 0.31%and 0.59%.The recoveries of xylitol and L-xylulose in fermentation broth were between 99.00%-101.00%.Conclusion There was no interference from other constitutes in the fermentation broth by this method.The methods were suitable for the simultaneous determination of the substrate xylitol and the product L-xylulose in fermentation process.

Quantification of alpha-amanitin in biological samples by HPLC using simultaneous UV- diode array and electrochemical detection.

α-Amanitin is a natural bicyclic octapeptide, from the family of amatoxins, present in the deadly mushroom species Amanita phalloides. The toxicological and clinical interests raised by this toxin, require highly sensitive, accurate and reproducible quantification methods for pharmacokinetic studies. In the present work, a high-performance liquid chromatographic (HPLC) method with in-line connected diode-array (DAD) and electrochemical (EC) detection was developed and validated to quantify α-amanitin in biological samples (namely liver and kidney). Sample pre-treatment consisted of a simple and unique deproteinization step with 5% perchloric acid followed by centrifugation at 16,000×g, 4°C, for 20min. The high recovery found for α-amanitin (≥96.8%) makes this procedure suitable for extracting α-amanitin from liver and kidney homogenates. The resulting supernatant was collected and injected into the HPLC. Mobile phase was composed by 20% methanol in 50mM citric acid, and 0.46mM octanessulfonic acid, adjusted to pH 5.5. The chromatographic runs took less than 22min and no significant endogenous interferences were observed at the α-amanitin retention time. Calibration curves were linear with regression coefficients higher than 0.994. The overall inter- and intra-assay precision did not exceed 15.3%. The present method has low interferences with simple and fast processing steps, being a suitable procedure to support in vivo toxicokinetic studies involving α-amanitin. In fact, the validated method was successfully applied to quantify α-amanitin in biological samples following intraperitoneal α-amanitin administration to rats. Moreover, human plasma was also used as matrix and the purposed method was adequate for detection of α-amanitin in that matrix. The results clearly indicate that the proposed method is suitable to investigate the pharmacokinetic and tissue distribution of α-amanitin. Additionally, the method will be very useful in the development of novel and potent antidotes against amatoxins poisoning and to improve the knowledge of α-amanitin toxicity.

A novel, simple and inexpensive procedure for the simultaneous determination of iopamidol and p-aminohippuric acid for renal function assessment from plasma samples in awake rats.

The purpose of the current study was to design, validate and implement a novel analytical method for the simultaneous plasma measurement of iopamidol and p-aminohippuric acid (PAH) to estimate renal function in awake rats. A reverse-phase high performance liquid chromatographic (RP-HPLC) method for the simultaneous measurement of iopamidol (for glomerular filtration rate estimation, GFR) and PAH (for tubular secretion determination, TS) was designed and validated using a C-18 column, 0.1M acetic acid-10% acetonitrile (90:10, v/v) as mobile phase, at a flow rate of 0.3 ml/min, and UV detection at 270 nm. Iopamidol (244.8 mg/kg) was administered intravenously followed immediately by sodium PAH (100 mg/kg) to healthy female Sprague-Dawley rats. Plasma samples obtained at 2.5, 5, 10, 15, 20, 30, 45, 60, 90, and 120 min after drug administration were deproteinized with 2.5% trichloroacetic acid containing p-aminobenzoic acid as internal standard, and separated by the validated RP-HPLC method described above. The iopamidol and PAH chromatographic data were analyzed using a non-compartmental model. The results demonstrated that the RP-HPLC method was linear in ranges between 15-120 µg/ml and 2.5-120 µg/ml for iopamidol and PAH, respectively. Precision and accuracy were within 15% for both drugs. Recovery of iopamidol and PAH was 92% and 100%, respectively. Plasma iopamidol and PAH clearances in awake rats, estimates for GFR and TS, respectively, were 1.49±0.20 ml/min and 3.73±0.38 ml/min. In conclusion, the method here described is a simple and reliable procedure, for the simultaneous and time-saving determination of GFR and TS from plasma samples in the conscious rat.

Development and Validation of Stability-indicating High Performance Liquid Chromatographic Method for the Estimation of Everolimus in Tablets.

The present study depicts the development of a validated reversed-phase high performance liquid chromatographic method for the determination of the everolimus in presence of degradation products or pharmaceutical excipients. Stress study was performed on everolimus and it was found that it degrade sufficiently in oxidizing and acidic conditions but less degradation was found in alkaline, neutral, thermal and photolytic conditions. The separation was carried out on Hypersil BDS C18 column (100×4.6 mm, 5 µ) column having particle size 5 µ using acetate buffer:acetonitrile (50:50 v/v) with pH 6.5 adjusted with orthophosphoric acid as mobile phase at flow rate of 1 ml/min. The wavelength of the detection was 280 nm. A retention time (Rt) nearly 3.110 min was observed. The calibration curve for everolimus was linear (r(2)=0.999) from range of 25-150 µg/ml with limit of detection and limit of quantification of 0.036 µg/ml and 0.109 µg/ml, respectively. Analytical validation parameters such as selectivity, specificity, linearity, accuracy and precision were evaluated and relative standard deviation value for all the key parameters were less than 2.0%. The recovery of the drug after standard addition was found to be 100.55%. Thus, the developed RP-HPLC method was found to be suitable for the determination of everolimus in tablets containing various excipients.