Herbal medicine has been proven to be an effective therapy offering a variety of benefits, such as moderate reduction in hypoglycemia, in the treatment and prevention of obesity and diabetes. Phyllanthus niruri has been used as a treatment for diabetes mellitus. Herein, the induction of type 2 diabetes in Sprague-Dawley rats was achieved by a low dose of streptozotocin (STZ) (25mg/kgbw). Here, we evaluated the in vivo antidiabetic properties of two concentrations (250 and 500mg/kg bw) of P. niruri via metabolomics approach. The administration of 500mg/kgbw of P. niruri extract caused the metabolic disorders of obese diabetic rats to be improved towards the normal state. The extract also clearly decreased the serum glucose level and improved the lipid profile in obese diabetic rats. The results of this study may contribute towards better understanding the molecular mechanism of this medicinal plant in managing diabetes mellitus.
In this study a sensitive and selective gradient reverse phase UPLC-MS/MS method was developed for the simultaneous determination of six process related impurities viz., Imp-I, Imp-II, Imp-III, Imp-IV, Imp-V and Imp-VI in darunavir. The chromatographic separation was performed on Acquity UPLC BEH C18 (50 mm×2.1mm, 1.7μm) column using gradient elution of acetonitrile-methanol (80:20, v/v) and 5.0mM ammonium acetate containing 0.01% formic acid at a flow rate of 0.4mL/min. Both negative and positive electrospray ionization (ESI) modes were operated simultaneously using multiple reaction monitoring (MRM) for the quantification of all six impurities in darunavir. The developed method was fully validated following ICH guidelines with respect to specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, precision, robustness and sample solution stability. The method was able to quantitate Imp-I, Imp-IV, Imp-V at 0.3ppm and Imp-II, Imp-III, and Imp-VI at 0.2ppm with respect to 5.0mg/mL of darunavir. The calibration curves showed good linearity over the concentration range of LOQ to 250% for all six impurities. The correlation coefficient obtained was >0.9989 in all the cases. The accuracy of the method lies between 89.90% and 104.60% for all six impurities. Finally, the method has been successfully applied for three formulation batches of darunavir to determine the above mentioned impurities, however no impurity was found beyond the LOQ. This method is a good quality control tool for the trace level quantification of six process related impurities in darunavir during its synthesis.
Baeckea frutescens or locally known as Cucur atap is used as antibacterial, antidysentery, antipyretic and diuretic agent. In Malaysia and Indonesia, they are used as an ingredient of the traditional medicine given to mothers during confinement. A three-steps infra-red (IR) macro-fingerprinting method combining conventional IR spectra, and the secondary derivative spectra with two dimensional infrared correlation spectroscopy (2D-IR) have been proved to be effective methods to examine a complicated mixture such as herbal medicines. This study investigated the feasibility of employing multi-steps IR spectroscopy in order to study the main constituents of B. frutescens and its different extracts (extracted by chloroform, ethyl acetate, methanol and aqueous in turn). The findings indicated that FT-IR and 2D-IR can provide many holistic variation rules of chemical constituents. The structural information of the samples indicated that B. frutescens and its extracts contain a large amount of flavonoids, since some characteristic absorption peaks of flavonoids, such as ∼1600cm(-1), ∼1500cm(-1), ∼1450cm(-1), and ∼1270cm(-1) can be observed. The macroscopical fingerprint characters of FT-IR and 2D-IR spectra can not only provide the information of main chemical constituents in medicinal materials and their different extracts, but also compare the components differences among the similar samples. In conclusion, the multi-steps IR macro-fingerprint method is rapid, effective, visual and accurate for pharmaceutical research.
A micellar electrokinetic chromatography method for the determination of sumatriptan succinate in pharmaceutical formulations was developed. The effects of several factors such as pH, surfactant and buffer concentration, applied voltage, capillary temperature, and injection time were investigated. Separation took about 5 min using phenobarbital as internal standard. The separation was carried out in reversed polarity mode at 20 °C, 26 kV and using hydrodynamic injection for 10s. Separation was achieved using a bare fused-silica capillary 50 μm×40 cm and background electrolyte of 25 mM sodium dihydrogen phosphate-adjusted with concentrated phosphoric acid to pH 2.2, containing 125 mM sodium dodecyl sulfate and detection was at 226 nm. The method was validated with respect to linearity, limits of detection and quantification, accuracy, precision and selectivity. The calibration curve was linear over the range of 100-2000 μg mL(-1). The relative standard deviations of intra-day and inter-day precision for migration time, peak area, corrected peak area, ratio of corrected peak area and ratio of peak area were less than 0.68, 3.48, 3.28, 2.97 and 2.83% and 2.01, 5.50, 4.46, 4.92 and 4.07%, respectively. The proposed method was successfully applied to the determinations of the analyte in tablet. Forced degradation studies were conducted by introducing a sample of sumatriptan succinate standard solution to different forced degradation conditions using neutral (water), basic (0.1 M NaOH), acidic (0.1 M HCl), oxidative (10% H(2)O(2)) and photolytic (exposure to UV light at 254 nm for 2 h). It is concluded that the stability-indicating method for sumatriptan succinate can be used for the analysis of the drug in various samples.
A cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC) method with hydroxypropyl-gamma-cyclodextrin (HP-gamma-CD) as chiral selector for the enantiomeric separation of econazole is reported. Enantioseparation of econazole was successfully achieved by the optimized CD-MEKC system containing 40mM HP-gamma-CD, 50mM SDS and 20mM phosphate buffer (pH 8) solution with an analysis time of less than 9min. Calibration curves were linear for the two stereoisomers of econazole (r(2)>0.998). Good repeatabilities in the migration time, peak area and peak height were obtained in terms of RSD% ranging from 0.30 to 7.67%. Combination of solid-phase extraction (SPE) procedure using diol column and the CD-MEKC method was successfully applied to the determination of econazole in a formulated cream sample.
In this study, the development and validation of a high-performance liquid chromatography (HPLC) assay for determination of repaglinide concentration in human plasma for pharmacokinetic studies is described. Plasma samples containing repaglinide and an internal standard, indomethacin were extracted with ethylacetate at pH 7.4. The recovery of repaglinide was 92%+/-55.31. Chromatographic separations were performed on Purospher STAR C-18 analytical column (4.8 mm x 150 mm; 5 microm particle size). The mobile phase composed of acetonitrile-ammonium formate (pH 2.7; 0.01 M) (60:40, v/v). The flow rate was 1 ml/min. The retention time for repaglinide and indomethacin were approximately 6.2 and 5.3 min, respectively. Calibration curves of repaglinide were linear in the concentration range of 20-200 ng/ml in plasma. The limits of detection and quantification were 10 ng/ml and 20 ng/ml, respectively. The inter-day precision was from 5.21 to 11.84% and the intra-day precision ranged from 3.90 to 6.67%. The inter-day accuracy ranged 89.95 to 105.75% and intra-day accuracy ranged from 92.37 to 104.66%. This method was applied to determine repaglinide concentration in human plasma samples for a pharmacokinetic study.
A simple, sensitive and specific reversed phase high performance liquid chromatographic (RP-HPLC) method with UV detection at 251 nm was developed for simultaneous quantitation of buparvaquone (BPQ), atenolol, propranolol, quinidine and verapamil. The method was applicable in rat in situ intestinal permeability study to assess intestinal permeability of BPQ, a promising lead compound for Leishmania donovani infections. The method was validated on a C-4 column with mobile phase comprising ammonium acetate buffer (0.02 M, pH 3.5) and acetonitrile in the ratio of 30:70 (v/v) at a flow rate of 1.0 ml/min. The retention times for atenolol, quinidine, propranolol, verapamil and BPQ were 4.30, 5.96, 6.55, 7.98 and 8.54 min, respectively. The calibration curves were linear (correlation coefficient > or =0.996) in the selected range of each analyte. The method is specific and sensitive with limit of quantitation of 15 microg/ml for atenolol, 0.8 microg/ml for quinidine, 5 microg/ml for propranolol, 10 microg/ml for verapamil and 200 ng/ml for BPQ. The validated method was found to be accurate and precise in the working calibration range. Stability studies were carried out at different storage conditions and all the analytes were found to be stable. This method is simple, reliable and can be routinely used for accurate permeability characterization.
The applicability of microwave non-destructive testing (NDT) technique in characterization of matrix property of pharmaceutical films was investigated. Hydroxypropylmethylcellulose and loratadine were selected as model matrix polymer and drug, respectively. Both blank and drug loaded hydroxypropylmethylcellulose films were prepared using the solvent-evaporation method and were conditioned at the relative humidity of 25, 50 and 75% prior to physicochemical characterization using microwave NDT technique as well as ultraviolet spectrophotometry, differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FT-IR) techniques. The results indicated that blank hydroxypropylmethylcellulose film exhibited a greater propensity of polymer-polymer interaction at the O-H and C-H domains of the polymer chains upon conditioned at a lower level of relative humidity. In the case of loratadine loaded films, a greater propensity of polymer-polymer and/or drug-polymer interaction via the O-H moiety was mediated in samples conditioned at the lower level of relative humidity, and via the C-H moiety when 50% relative humidity was selected as the condition for sample storage. Apparently, the absorption and transmission characteristics of both blank and drug loaded films for microwave varied with the state of polymer-polymer and/or drug-polymer interaction involving the O-H and C-H moieties. The measurement of microwave NDT test at 8GHz was sensitive to the chemical environment involving O-H moiety while it was greatly governed by the C-H moiety in test conducted at a higher frequency band of microwave. Similar observation was obtained with respect to the profiles of microwave NDT measurements against the state of polymer-polymer and/or drug-polymer interaction of hydroxypropylmethylcellulose films containing chlorpheniramine maleate. The microwave NDT measurement is potentially suitable for use as an apparent indicator of the state of polymer-polymer and drug-polymer interaction of the matrix.
Pogostemon cablin, originating in Malaysia and India, is cultivated in southern China including Guangdong and Hainan Province, which was called GuangHuoXiang to differentiate it from the HuoXiang of the north, the species Agastache rugosa, that it resembles. Essential oil of P. cablin mainly contributes to the pharmacological activities and the therapeutic properties of the essential oils are directly correlated with their qualitative and quantitative composition. For controlling the quality, standard fingerprint of P. cablin collected from different regions was developed by using GC-MS. Nine compounds including beta-patchoulene, caryophyllene, alpha-guaiene, seychellene, beta-guaiene, delta-guaiene, spathulenol, patchouli alcohol and pogostone were identified among 10 main peaks in P. cablin. Hierarchical clustering analysis based on characteristics of 10 investigated peaks in GC profiles showed that 18 samples were divided into three main clusters, patchouliol-type, pogostone-type and an interim-type, which was the one between the two chemotypes. The simulative mean chromatogram for the three types P. cablin was generated using the Computer Aided Similarity Evaluation System. The fingerprint can help to distinguish the substitute or adulterant, and further assess the differences of P. cablin grown in various areas of China.
Application of gas chromatography-triple quadrupole mass spectrometry for identification, confirmation and quantification of 6 phosphodiesterase-5 (PDE-5) inhibitors (sildenafil, dimethylsildenafil, homosildenafil, thiosildenafil, thiodimethylsildenafil and thiohomosildenafil) in dietary supplements was investigated. The MS was operated in multiple reaction monitoring mode, for better sensitivity and selectivity. In this manner, the method is adequate to reduce background noise with less interference from co-eluting compounds in the samples. Two different ionisation techniques, electron ionisation (EI) and chemical ionisation (CI), were studied and compared. The chromatographic separation was performed on a short 10 m non-polar capillary column without any derivatisation step. This permitted fast analysis for all analogues with retention time less than 11 min, for both techniques. Use of backflushing can aid method retention time reduction and improves column maintenance. Evaluation of method validation included limit of detection (LOD), lower limit of quantitation (LLOQ), linearity, precision and recovery were performed for both EI and CI techniques. The LOD obtained varied from 0.03 to 1.50 μg/g and the LLOQ ranged from 0.10 to 5.00 μg/g. Good calibration linearity was obtained for all analogues for both techniques, with correlation coefficients (r(2)) higher than 0.99. Mean recoveries of all analogues using CI show higher values (83.4-108.8%) than that of EI (61.9-91.1%). The intra- and inter-assay precisions were evaluated for all analogues at spiked concentration of 10 μg/g and the relative standard deviation was less than 15% for both methods. These methods were then successfully applied to dietary supplement samples without prior derivatisation, confirming that the samples were adulterated with sildenafil and/or its analogues.
A quantitative structure-permeability relationship was developed using Artificial Neural Network (ANN) modeling to study penetration across a polydimethylsiloxane membrane. A set of 254 compounds and their experimentally derived maximum steady state flux values used in this study was gathered from the literature. A total of 42 molecular descriptors were calculated for each compound. A genetic algorithm was used to select important molecular descriptors and supervised ANN was used to correlate selected descriptors with the experimentally derived maximum steady-state flux through the polydimethylsiloxane membrane (log J). Calculated molecular descriptors were used as the ANN's inputs and log J as the output. Developed model indicates that molecular shape and size, inter-molecular interactions, hydrogen-bonding capacity of drugs, and conformational stability could be used to predict drug absorption through skin. A 12-descriptor nonlinear computational neural network model has been developed for the estimation of log J values for a data set of 254 drugs. Described model does not require experimental parameters and could potentially provide useful prediction of membrane penetration of new drugs and reduce the need for actual compound synthesis and flux measurements.
A simple high-performance liquid chromatographic method was developed for the determination of omeprazole in human plasma. Omeprazole and the internal standard, chloramphenicol, were extracted from alkalinized plasma samples using dichloromethane. The mobile phase was 0.05 M Na2HPO4-ACN (65:35, v/v) adjusted to pH 6.5. Analysis was run at a flow rate of 1.0 ml/min at a detection wavelength of 302 nm. The method was specific and sensitive with a detection limit of 2.5 ng/ml at a signal-to-noise ratio of 4:1. The limit of quantification was set at 5 ng/ml. The calibration curve was linear over a concentration range of 5-1280 ng/ml. Mean recovery value of the extraction procedure was about 96%, while the within and between day coefficient of variation and percent error values of the assay method were all less than 14%.
A quantitative structure-human intestinal absorption relationship was developed using artificial neural network (ANN) modeling. A set of 86 drug compounds and their experimentally-derived intestinal absorption values used in this study was gathered from the literature and a total of 57 global molecular descriptors, including constitutional, topological, chemical, geometrical and quantum chemical descriptors, calculated for each compound. A supervised network with radial basis transfer function was used to correlate calculated molecular descriptors with experimentally-derived measures of human intestinal absorption. A genetic algorithm was then used to select important molecular descriptors. Intestinal absorption values (IA%) were used as the ANN's output and calculated molecular descriptors as the inputs. The best genetic neural network (GNN) model with 15 input descriptors was chosen, and the significance of the selected descriptors for intestinal absorption examined. Results obtained with the model that was developed indicate that lipophilicity, conformational stability and inter-molecular interactions (polarity, and hydrogen bonding) have the largest impact on intestinal absorption.
This paper investigates the use of a neural-network-based intelligent learning system for the prediction of drug release profiles. An experimental study in transdermal iontophoresis (TI) is employed to evaluate the applicability of a particular neural network (NN) model, i.e. the Gaussian mixture model (GMM), in modeling and predicting drug release profiles. A number of tests are systematically designed using the face-centered central composite design (CCD) approach to examine the effects of various process variables simultaneously during the iontophoresis process. The GMM is then applied to model and predict the drug release profiles based on the data samples collected from the experiments. The GMM results are compared with those from multiple regression models. In addition, the bootstrap method is used to assess the reliability of the network predictions by estimating confidence intervals associated with the results. The results demonstrate that the combination of the face-centered CCD and GMM can be employed as a useful intelligent tool for the prediction of time-series profiles in pharmaceutical and biomedical experiments.
The identification, characterization and quantification of crystal forms are becoming increasingly important within the pharmaceutical industry. A combination of different physical analytical techniques is usually necessary for this task. In this work solid-state techniques, diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) and X-ray powder diffractometry (XRPD) were combined to analyze polymorphic purity of crystalline ranitidine-HCl, an antiulcer drug, H2 receptor antagonists. A series of 12 different mixtures of Form 1 and 2 was prepared by geometric mixing and their DRIFT spectra and XRD powder patterns were obtained and analyzed, either alone or combined together, using Artificial Neural Networks (ANNs). A standard feed-forward network, with back-propagation rule and with multi layer perceptron architecture (MPL) was chosen. A working range of 1.0-100% (w/w) of crystal Form 2 in Form 1 was established with a minimum quantifiable level (MQL) of 5.2% and limit of detection of 1.5% (w/w). The results demonstrate that DRIFTS combined with XRPD may be successfully used to distinguish between the ranitidine-HCl polymorphs and to quantify the composition of binary mixtures of the two.
The aim of the present study is to investigate the influence of the CYP2D6*10 allele on the disposition of tramadol hydrochloride in Malaysian subjects. A single dose of 100 mg tramadol was given intravenously to 30 healthy orthopaedic patients undergoing various elective surgeries. After having obtained written informed consents, patients were genotyped for CYP2D6*10: the most common CYP2D6 allele among Asians by means of allele-specific polymerase chain reaction. The presence of other mutations (CYP2D6*1, *3, *4, *5, *9 and *17) was also investigated. Tramadol was extracted from 1 ml serum with an n-hexane: ethylacetate combination (4:1) after alkalinisation with ammonia (pH 10.6). Serum concentrations were measured by means of high-performance liquid chromatography. The pharmacokinetics of tramadol was studied during the 24 h after the dose. As among other Asians, the allele frequency for CYP2D6*10 among Malaysians was high (0.43). Subjects who were homozygous for CYP2D6*10 had significantly (P=0.046) longer mean serum half-life of tramadol than subjects of the normal or the heterozygous group (Kruskal-Wallis test). When patients were screened for the presence of other alleles, the pharmacokinetic parameter values were better explained. CYP2D6 activity may play a main role in determining tramadol pharmacokinetics. The CYP2D6*10 allele particularly was associated with higher serum levels of tramadol compared with the CYP2D6*1 allele. However, genotyping for CYP2D6*10 alone is not sufficient to explain tramadol disposition.
Quantitative structure-retention relationship(QSRR) method was used to model reversed-phase high-performance liquid chromatography (RP-HPLC) separation of 18 selected amino acids. Retention data for phenylthiocarbamyl (PTC) amino acids derivatives were obtained using gradient elution on ODS column with mobile phase of varying acetonitrile, acetate buffer and containing 0.5 ml/l of triethylamine (TEA). Molecular structure of each amino acid was encoded with 36 calculated molecular descriptors. The correlation between the molecular descriptors and the retention time of the compounds in the calibration set was established using the genetic neural network method. A genetic algorithm (GA) was used to select important molecular descriptors and supervised artificial neural network (ANN) was used to correlate mobile phase composition and selected descriptors with the experimentally derived retention times. Retention time values were used as the network's output and calculated molecular descriptors and mobile phase composition as the inputs. The best model with five input descriptors was chosen, and the significance of the selected descriptors for amino acid separation was examined. Results confirmed the dominant role of the organic modifier in such chromatographic systems in addition to lipophilicity (log P) and molecular size and shape (topological indices) of investigated solutes.
A simple, rapid, sensitive, and reproducible LC-MS/MS method was developed for simultaneous quantification of flavoxate and 3-methyl-flavone-8-carboxylic (MFCA) in human plasma, using diphenhydramine HCl as internal standard (IS). The chromatographic separation was achieved using Agilent Poroshell 120 EC-C18 - Fast LC column (100 × 2.1mmID, 2.7 μm) fitted with UHPLC Guard Poroshell 120 EC-C18 (5 × 2.1 mmID, 2.7 μm). The mobile phase consisted of 0.1 % v/v formic acid and acetonitrile (30:70, v/v) run at a flow rate of 0.40 mL/min. The standard calibration curve was linear over the concentration range of 2.00 - 2,000.31 ng/mL and 240.00 - 24,000.04 ng/mL for flavoxate and MFCA. For flavoxate and MFCA, the within-run precision was 0.81-6.67 % and 1.68-4.37 %, while accuracy was 100.21-108.25 % and 103.99-110.28 %. The between-run precision was 2.01-9.14 % and 2.31-11.11 %, and accuracy was 96.09-103.33 % and 102.37-109.52 %. The extended run precision was 7.78-11.04 % and 2.22-3.33 %, while accuracy was 100.72-101.88 % and 102.34-105.60 %. Flavoxate and MFCA in plasma were stable 4 h at bench top (short term), 24 h in autosampler and instrumentation room (post-preparative), after 7 freeze-thaw cycles, and 89 days in the freezer. Both analytes and IS stock solutions were stable for 31 days when kept at room temperature (25 ± 4 °C) and refrigerated (2-8 °C). The validated method was successfully applied to a bioequivalence study of two flavoxate formulations involving 24 healthy volunteers.
Atovaquone (ATQ) is a poorly soluble drug. Therefore, formulating ATQ into its supersaturated state through solid dispersion for bioavailability enhancement can be of great value. However, due to fast crystallising properties of ATQ, the quantification of ATQ in a supersaturated solid dispersion system can be complicated. Therefore, in pursuit of accurate quantification of such sample, a simple HPLC analytical method utilising a C18 column (250 × 4.6 mm ID, 5 μm) for the quantitation of ATQ has been developed and validated. Atovaquone elution using the proposed method demonstrated a retention time around 7.6 min with good linearity (R2 > 0.999). The system suitability is also detailed with the tailing factor at 1.365 ± 0.002. The addition of solubilising agent as sample treatment step aided in ensuring the accurate quantitation of the fast crystallising ATQ. The developed HPLC quantitation method has been successfully employed in the analysis of ATQ from solid dispersion samples in in vitro dissolution as well as ex vivo permeation studies for formulation development.
A facile electrochemical sandwich immunosensor for the detection of a breast cancer biomarker, the human epidermal growth factor receptor 2 (HER2), was designed, using lead sulfide quantum dots-conjugated secondary HER2 antibody (Ab2-PbS QDs) as a label. Using Ab2-PbS QDs in the development of electrochemical immunoassays leads to many advantages such as straightforward synthesis and well-defined stripping signal of Pb(II) through acid dissolution, which in turn yields better sensing performance for the sandwiched immunosensor. In the bioconjugation of PbS QDs, the available amine and hydroxyl groups from secondary anti-HER2 and capped PbS QDs were bound covalently together via carbonyldiimidazole (CDI) acting as a linker. In order to quantify the biomarker, SWV signal was obtained, where the Pb2+ ions after acid dissolution in HCl was detected. The plated mercury film SPCE was also detected in situ. Under optimal conditions, HER2 was detected in a linear range from 1-100 ng/mL with a limit of detection of 0.28 ng/mL. The measures of satisfactory recoveries were 91.3% to 104.3% for the spiked samples, displaying high selectivity. Therefore, this method can be applied to determine HER2 in human serum.