Crystalline constituents of euphorbiaceae—XII : Isolation and structural elucidation of three new lignans from the leaves of Phyllanthus niruri Linn.

Three more new lignans from the hexane extract of Phyllanthus niruri Linn. are now reported besides phyllanthin and hypophyllanthin. The structure of niranthin is now established as 3,3′,4′,9,9′-pentamethoxy-4,5-methylenedioxy-8,8′-butyrolignan (2). Nirtetralin and phyltetralin are shown to be 1-phenyl tetralins. On the basis of NMR and mass spectra, nirtetralin is assigned the structure (7) and phyltetralin (11).     

Separation and quantification of lignans in Phyllanthus species by a simple chiral densitometric method

A sensitive, selective, and robust high-performance TLC (HPTLC) method using chiral TLC plates for qualitative and quantitative analysis of phyllanthin (A), hypophyllanthin (B), niranthin (C), and nirtetralin (D), the active lignans of Phyllanthus species, was developed and validated. The effectiveness and role of various stationary phases viz TLC silica gel 60F(254), HPTLC silica gel 60F(254), and chiral TLC plates in the quantitation were evaluated. A precoated chiral TLC plate was found suitable for the simultaneous analysis of four pharmacologically active lignans. For achieving good separation, the optimized mobile phase of n-hexane/acetone/1,4-dioxane (9:1:0.5 by volume) was used (R(f) = 0.30, 0.36, 0.41, and 0.48 for compounds A, B, C, and D, respectively). A densitometric determination of the above compounds was carried out in reflection/absorption mode at 620 nm. Optimized chromatographic conditions provide well-separated compact bands for the tested lignans. The calibration curves were found linear in the concentration range of 100-500 ng/band. Recoveries of A-D were 99.98, 100.51, 99.22, and 98.74%, respectively. The method was validated according to ICH guidelines. The method reported here is reproducible and applied for the quantitative analysis of the above lignans in the leaves of four Phyllanthus species, i. e., P. amarus, P. maderaspatensis, P. urinaria, and P. virgatus.     

Development of a UHPLC–MS/MS method for the quantitation of bioactive compounds in Phyllanthus species and its herbal formulations

Phyllanthus species are extensively used in traditional medicines for the treatment of hepatic diseases due to their bioactive hypophyllanthin and phyllanthin. This work describes the development and validation of an ultra high performance liquid chromatography with tandem mass spectrometry method in polarity switching multiple reaction monitoring mode for the simultaneous detection and quantitation of 23 compounds using ultra‐performance liquid chromatography coupled with electrospray‐hybrid triple quadrupole‐linear ion trap mass spectrometer. The validated parameters showed good linearity (R ² ≥ 0.996), limit of detection (0.05–1.62 ng/mL), limit of quantitation (0.15–4.95 ng/mL), precisions (intra‐day: RSD ≤ 2.11%), (inter‐day: RSD ≤ 2.91%), stability (RSD ≤ 2.56%) and overall recovery (98.22–104.48%; RSD ≤ 2.93%). The validated method was successfully applied in ethanolic extracts of P. amarus, P. niruri , P. emblica , P. fraternus , fractions of P. amarus and their herbal formulations for quantitation. The maximum content of hypophyllanthin (29.40 mg/g) and phyllanthin (56.60 mg/g) was detected in ethyl acetate fraction of P. amarus . The total content of 23 compounds was abundant in the ethanolic extract of P. emblica fruit. Principal component analysis was used to differentiate the selected Phyllanthus species and their herbal formulations. The results indicated that the present method could be used for quality control of Phyllanthus species and its herbal formulations.     

High-performance thin-layer chromatography densitometric method for simultaneous quantitation of phyllanthin, hypophyllanthin, gallic acid, and ellagic acid in Phyllanthus amarus

Whole plant of Phyllanthus amarus Linn. is a reputed drug of the Indian systems of medicine that is used as hepatoprotective agent. A simple high-performance thin-layer chromatography (HPTLC) densitometric method has been developed for the simultaneous quantitation of phyllanthin, hypophyllanthin, gallic acid, and ellagic acid in the whole plant of P. amarus. They were found at levels of 0.37, 1.16, 0.36, and 0.17% (w/w), respectively. The method was validated for precision, repeatability, and accuracy. Instrumental precision was found to be 0.54, 0.93, 0.08, and 0.78% (coefficient of variation, CV); repeatability of the method was 1.01, 0.79, 0.98, and 1.06% (CV) for phyllanthin, hypophyllanthin, gallic acid, and ellagic acid, respectively. Accuracy of the method was determined by a recovery study conducted at 3 different levels, and the average recovery was found to be 99.09% for phyllanthin, 99.27% for hypophyllanthin, 98.69% for gallic acid, and 100.49% for ellagic acid. The proposed HPTLC method was found to be simple, precise, specific, sensitive, and accurate and can be used for routine quality control of raw material of P. amarus and formulations containing P. amarus. It also has the applicability in quantitating any of these marker compounds in other drugs.     

Determination of bupropion using liquid chromatography with fluorescence detection in pharmaceutical preparations, human plasma and human urine

A novel pre-column derivatization reversed-phase high-performance liquid chromatography with fluorescence detection is described for the determination of bupropion in pharmaceutical preparation, human plasma and human urine using mexiletine as internal standard. The proposed method is based on the reaction of 4-chloro-7-nitrobenzofurazan (NBD-Cl) with bupropion to produce a fluorescent derivative. The derivative formed is monitored on a C18 (150 mm × 4.6 mm i.d., 5 μm) column using a mobile phase consisting of methanol-water 75:25 (v/v), at a flow-rate of 1.2 mL/min and detected fluorimetrically at λ(ex) = 458 and λ(em) = 533 nm. The assay was linear over the concentration ranges of 5-500 and 10-500 ng/mL for plasma and urine, respectively. The limits of detection and quantification were calculated to be 0.24 and 0.72 ng/mL for plasma and urine, respectively (inter-day results). The recoveries obtained for plasma and urine were 97.12% ± 0.45 and 96.00% ± 0.45, respectively. The method presents good performance in terms of precision, accuracy, specificity, linearity, detection and quantification limits and robustness. The proposed method is applied to determine bupropion in commercially available tablets. The results were compared with an ultraviolet spectrophotometry method using t- and F-tests.     

Simultaneous determination of tandospirone and its active metabolite, 1‐[2‐pyrimidyl]‐piperazine in rat plasma by LC–MS/MS and its application to a pharmacokinetic study

A rapid, sensitive and selective liquid chromatography–tandem mass spectrometry method for the detection of tandospirone (TDS) and its active metabolite 1‐[2‐pyrimidyl]‐piperazine (1‐PP) in Sprague–Dawley rat plasma is described. It was employed in a pharmacokinetic study. These analytes and the internal standards were extracted from plasma using protein precipitation with acetonitrile, then separated on a CAPCELL PAK ADME C₁₈ column using a mobile phase of acetonitrile and 5 mm ammonium formate acidified with formic acid (0.1%, v/v) at a total flow rate of 0.4 mL/min. The detection was performed with a tandem mass spectrometer equipped with an electrospray ionization source. The method was validated to quantify the concentration ranges of 1.000–500.0 ng/mL for TDS and 10.00–500.0 ng/mL for 1‐PP. Total time for each chromatograph was 3.0 min. The intra‐day precision was between 1.42 and 6.69% and the accuracy ranged from 95.74 to 110.18% for all analytes. Inter‐day precision and accuracy ranged from 2.47 to 6.02% and from 98.37 to 105.62%, respectively. The lower limits of quantification were 1.000 ng/mL for TDS and 10.00 ng/mL for 1‐PP. This method provided a fast, sensitive and selective analytical tool for quantification of tandospirone and its metabolite 1‐PP in plasma necessary for the pharmacokinetic investigation.     

Method for simultaneous analysis of eight vitamin E isomers in various foods by high performance liquid chromatography and fluorescence detection

The objective of this study was to optimize a method to investigate the occurrence and to quantify the full isomeric composition of vitamin E (α-, β-, γ- and δ-tocopherols and tocotrienols) in 6 vegetables (raw and cooked), 3 herbs/spices, raw and cooked eggs, vegetable oils (canola, olive and soybean), flaxseed and sorghum (flour and seeds) and soy (flour) by HPLC with fluorescence detection. Different conditions of extraction and analysis were tested. The optimized method consisted of direct extraction with solvent (hexane:ethyl acetate, 85:15, v/v). For analysis normal phase column was used with mobile phase consisting of hexane:isopropanol:acetic acid (98.9:0.6:0.5) with isocratic elution and fluorescence detection. Excellent separation of all isomers was obtained along with adequate quantification in the foods analyzed. Recovery rates of standards ranged from 91.3 to 99.4%. The linearity range for each isomer varied from 2.5 to 137.5 ng/mL (R2 greater than 0.995 in all cases). Detection limits ranged from 21.0 to 48.0 ng/mL for tocopherols and from 56.0 to 67.0 ng/mL for tocotrienols, while quantification limits ranged from 105.0 to 240.0 ng/mL for tocopherols and from 280.0 to 335.0 ng/mL for tocotrienols. The optimized method was considered simple, fast and reliable, and also preserved vitamin E isomers when compared to validated methods involving saponification.     

HPLC method for determination of diclofenac in human plasma and its application to a pharmacokinetic study in Turkey

A simple high-performance liquid chromatography (HPLC) method has been developed for determination of diclofenac in human plasma. The method was validated on Ace C(18) column using UV detection. The mobile phase consisted of 20 mM phosphate buffer (pH 7) containing 0.1% trifluoroacetic acid-acetonitrile (65:35, v/v). Calibration curve was linear between the concentration range of 75-4000 ng/mL. Intra- and inter-day precision values for diclofenac in plasma were less than 3.6, and accuracy (relative error) was better than 5.3%. The limits of detection and quantification of diclofenac were 25 and 75 ng/mL, respectively. Also, this assay was applied to determine the pharmacokinetic parameters of diclofenac in healthy Turkish volunteers who had been given 50 mg diclofenac.     

Qualification and quantification of seventeen natural steroids in plasma by GC-Q-MS and GC-IT-MS/MS

Studying the plasma steroid profile offers information about the possible existence of endocrinological alterations. This study describes the development and validation of gas chromatographic-mass spectrometric and gas tandem mass spectrometric methods for the simultaneous identification of 17 steroid hormones in human plasma using five different solvents. The n-hexane/ethyl acetate solvent mixture, in a proportion of 70/30 (v/v) provided the best results. The extracts were derivatized with N-methyl-N-trimethylsilyl-trifluoroacetamide. The obtained limits of detection were below 1 ng/mL in the majority of the studied steroids and the limits of quantification were below 5 ng/mL; the method obtained good linearity, reproducibility, repeatability, accuracy and recoveries above 95% in most cases.     

Simultaneous Determination of Hydrochlorothiazide and Reserpine in Human Urine by LC with a Simple Pre-Treatment

A simple, selective and sensitive reversed-phase high performance liquid chromatography method for simultaneous analysis of hydrochlorothiazide and reserpine in human urine was developed and subjected to primary pharmacokinetic study. After a simple protein precipitation using methanol and extraction with ethyl acetate, the analytes were separated on an Elite C(18) column at a flow rate of 0.8 mL min(-1). The mobile phase was composed of acetonitrile (A) and 0.2% ammonium chloride solution (B) for a gradient elution starting at A:B at 30:70, v/v for 0~6 min, linearly raising the percent of A from 30% to 50% (6~9 min) and ending at 50:50, v/v (9~25 min). The standard curves were linear over the range of 0.05-20 μg mL(-1) for hydrochlorothiazide and 0.02-5.0 μg mL(-1) for reserpine, respectively (r > 0.999). The limit of detection (LOD) and the limit of quantification (LOQ) were 5.5 ng mL(-1) and 18.2 ng mL(-1) for hydrochlorothiazide, and 7.1 ng mL(-1) and 23.6 ng mL(-1) for reserpine, respectively. The recoveries for both analytes were above 89.0±1.35%. The intra-day and inter-day precision for hydrochlorothiazide were less than 1.91% and 1.38%, and those for reserpine were below 1.61% and 2.64%, respectively. The method indicated good performance in terms of specificity, linearity, detection and quantification limits, precision and accuracy, and it was employed successfully for the simultaneous determination of hydrochlorothiazide and reserpine in human urine samples.     

Simultaneous determination of olmesartan medoxomil and irbesartan and hydrochlorothiazide in pharmaceutical formulations and human serum using high performance liquid chromatography

 A simple, selective, sensitive, precise, simultaneous high performance liquid chromatographic analysis of serum samples and commercial tablet formulation containing hydrochlorothiazide, olmesartan medoxomil and irbesartan are reported. Good chromatographic separation was achieved using a μ-Bondapak, C18 column (15 cm×4.6 mm, 5 μm), and a mobile phase consisting of acetonitrile-0.2% acetic acid aqueous solution (50∶50, v/v) at a flow rate of 1.0 mL/min. The ultraviolet detector was set at a wavelength of 260 nm. Hydrochlorothiazide, olmesartan medoxomil, and irbesartan were eluted at 1.2, 3.8, and 4.4 min, respectively. No extraneous materials were found to interfere. The method uses protein precipitation with acetonitrile for the preparation of serum sample. The linear ranges for hydrochlorothiazide, olmesartan medoxomil, and irbesartan were 6.25-18.75, 20-60, and 75-225 ng/mL, respectively. The recoveries of hydrochlorothiazide, olmesartan medoxomil, and irbesartan in spiked samples were all greater than 98%, and their relative standard deviations were less than 2.0%. The limits of detection were 1, 2, and 2 ng/mL for hydrochlorothiazide, olmesartan medoxomil, and irbesartan, respectively, and the limits of quantification were 3 ng/mL, which allow their determination at the expected serum concentration levels.     

Stereoselective liquid chromatographic determination of 1'-oxobufuralol and 1'-hydroxybufuralol in rat liver microsomal fraction using hollow-fiber liquid-phase microextraction for sample preparation

A three-phase hollow-fiber liquid-phase microextraction (HF-LPME) method for the stereoselective determination of bufuralol metabolites 1'-oxobufuralol (1'-Oxo-BF) and 1'-hydroxybufuralol (1'-OH-BF) in microsomal preparations is described for the first time. The HPLC analysis was carried out using a Chiralcel OD-H column with hexane/2-propanol/methanol (97.5:2.0:0.5, v/v/v) plus 0.5% diethylamine as the mobile phase, and UV detection at 248 and 273 nm. The HF-LPME optimized conditions involved: n-octanol as the organic solvent, 0.2 mol/L acetic acid as the acceptor phase, donor phase pH adjusted to 13, sample agitation at 1500 rpm and extraction for 30 min. By using this extraction procedure, the recovery rates were in the range of 63-69%. The method was linear over the concentration range of 100-5000 ng/mL for each enantiomer of 1'-Oxo-BF (r>0.9978) and of 100-2500 ng/mL for each stereoisomer of 1'-OH-BF (r>0.9957). The quantification limits were 100 ng/mL for all analytes. The validated method was used to assess the in vitro biotransformation of bufuralol using rat liver microsomal fraction that demonstrated predominant formation of (S)-1'-Oxo-BF and (R,R)-1'-OH-BF.     

Analysis of metformin dosage formulations by capillary electrophoresis at nano scale detection

An inexpensive, rapid and reproducible capillary electrophoretic method has been developed and validated for the determination of metformin in pharmaceutical preparations. The method was developed utilizing a fused silica capillary (60 cm x 50 microm I.D.), phosphate buffer (50 mM, 3.0 pH)-acetonitrile (95:5, v/v) as background electrolyte (BGE), 20 kV applied voltage with UV detection at 254 nm and at a working temperature of 23 +/- 1 degrees C. Linearity was observed in the concentration range from 100 ng/L to 5 microg/L, with a correlation coefficient (R2) of 0.9998. The limits of detection and quantification achieved were 60 and 100 ng/mL, respectively. The recovery of metformin from pharmaceutical preparations was 99.1%. These validation parameters demonstrate the precision of the method and its suitability for the determination of metformin in pharmaceutical tablet formulations.     

Quantification of cephalexin as residue levels in bovine milk by high-performance liquid chromatography with on-line sample cleanup

A multidimensional, selective and precise high performance liquid chromatography (HPLC) method based on direct injection of biological samples has been developed for the determination of cephalexin in skimmed and whole bovine milk. The cephalosporin antibiotic was extracted from bovine milk using an octyl restricted access medium bovine serum albumin column (C₈-RAM-BSA) and analyzed on an octadecyl column using phosphate buffer (pH 7.5, 0.01 M): ACN (92:8, v/v) and ultraviolet detection at 260 nm. The calibration graph was linear in the concentration range of 25-1600 ng/mL and this is in accordance with the tolerance level of 100 ng/mL set by the FDA (Food and Drug Administration) and EU (European Union) for cephalexin as residue in bovine milk. The intra- and inter-day coefficients of variation for the replicate analysis at the quality control levels were less than 15% while the transfer efficiency was in the range of 90.2-92.3%. The limits of detection and quantification were 10 and 20 ng/mL, respectively.     

LC-ESI-MS/MS determination of paclitaxel on dried blood spots

A simple and rapid high‐performance liquid chromatography–tandem mass spectrometric assay for determination of paclitaxel on rat dried blood spots was developed and validated. The extracted sample was chromatographed without further treatment using a reverse‐phase Oyster ODS3, 4.6 × 50 mm, 3 µm column with mass spectrometry detection. The mobile phase comprised of acetonitrile–water, 60:40 v/v, with a flow rate of 0.4 mL/min was used. The calibration was linear over the range 0.2–20 ng/mL. The limits of detection and quantification were 0.08 and 0.2 ng/mL, respectively. The intra‐ and inter‐day precision (CV%) and accuracy (relative error %) were less than 10 and 12%, respectively. Copyright © 2011 John Wiley & Sons, Ltd.     

Simultaneous quantification of oxysophocarpine and its active metabolite sophocarpine in rat plasma by liquid chromatography/mass spectrometry for a pharmacokinetic study

A sensitive, rapid and specific method for the simultaneous quantification of oxysophocarpine (OSC) and its active metabolite sophocarpine (SC) in rat plasma was developed and validated, using a liquid-liquid extraction procedure followed by liquid chromatography/electrospray ionization mass spectrometric (LC/ESI-MS) analysis. The separation was performed on a Zorbax Extend-C(18) column (2.1 mm i.d. x 50 mm, 5 microm) with a C(18) guard column using methanol-water containing 5 mm ammonium acetate (15:85, v/v) as mobile phase. Analysis was performed in selected ion monitoring (SIM) mode with an electrospray ionization (ESI) interface. [M + H](+) at m/z 263 for OSC, [M + H](+) at m/z 247 for SC and [M + H](+) at m/z 249 for matrine (internal standard) were selected as detecting ions, respectively. The method was linear in the concentration ranges 10-1000 ng/mL for OSC and 5-500 ng/mL for SC. The intra- and inter-day precisions (coefficient of variation) were within 7% for both analytes. Their accuracy (relative error) ranged from -6.4 to 1.5%. The limits of detection for OSC and SC were 3 and 1.5 ng/mL, respectively. The limits of quantitation for OSC and SC were 10 and 5 ng/mL, respectively. Recoveries of both analytes were greater than 85% at the low, medium and high concentrations. Both analytes were stable during all sample storage, preparation and analytic procedures. The method was successfully applied to a pharmacokinetic study after an oral administration of OSC to rats with a dose of 15 mg/kg.     

Resolution, quantification and confirmation of betamethasone and dexamethasone in equine plasma by liquid chromatography/tandem mass spectrometry

This method describes the simultaneous separation, identification, quantification and confirmation of betamethasone (BTM) and dexamethasone (DXM) in equine plasma by liquid chromatography (LC) integrated with multidimensional tandem mass spectrometry. Analytes were directly extracted from equine plasma by methyl tert-butyl ether (MTBE). The residues were reconstituted with sample solvent. LC separation of the analytes was performed on a Hypercarb column using acetonitrile/water/formic acid (95:5:0.5, v/v/v) as the mobile phase. Sample screening, quantification and confirmation were performed in multiple reaction monitoring (MRM) mode. The method was linear over the concentration range of 0.1-75 ng/mL for both analytes. Limit of detection (LOD) was 50 pg/mL and that of quantification (LOQ) was 100 pg/mL for both analytes. The limit of confirmation (LOC) for the presence of BTM or DXM by MRM was 0.5 ng/mL. The intra-and inter-day precisions expressed as coefficient of variation (CV) for quantification of DXM and BTM from 0.1 to 50 ng/mL were less than 7% and the accuracy was in the range of 97-105%. This method is capable of distinguishing BTM from DXM when both analytes are simultaneously present in equine plasma. Measurement uncertainty for both analytes was estimated at less than 16%. The method is rapid, specific, selective, sensitive, simple and reliable. The importance of this method is its usefulness in directly identifying and differentiating BTM from DXM without derivatization.     

GC-FID/MS method for the impurity profiling of synthetic d-allethrin

A GC/FID/MS method was developed for the identification and quantification of d-allethrin (DA) and its major impurities in commercial samples. Optimisation of the experimental conditions was carried out considering such important requirements as resolution, reproducibility, detection limits of 0.1% (m/m) for the impurities, and short analysis time. Under the optimised final conditions the method was validated for specificity, precision (CV% = 0.133 at 2.10 mg/mL and CV% = 0.035 at 3.00 mg/mL), linearity (0-3.00 microg injected), limits of detection (0.09 ng injected) and quantitation (0.28 ng injected), and robustness. The DA related impurities were identified by using a GC/MS method with ion trap mass detection and also by comparison with synthesised standards. The most abundant impurities were crysolactone, allethrolone, chrysanthemic acid, and chloro-derivatives of DA.     

Simple-QuEChERS Nano结合GC-MS/MS同时检测全血中的97种农药

建立了Simple-QuEChERS Nano结合气相色谱-串联质谱(GC-MS/MS)同时检测血液中97种农药的方法,并对基质条件、提取溶剂以及净化材料进行了优化。0.5 mL血液样品经3倍水稀释混匀,使用2.0 mL乙酸乙酯提取后振荡、离心,过Simple-QuEChERS Nano净化柱及0.22μm有机微孔滤膜后,采用多反应监测模式(MRM)进行分析。结果显示,97种农药在一定质量浓度范围内线性关系良好(r2≥0.9873),除丙烯菊酯(检出限和定量下限分别为11.03、36.76 ng/mL)外,其余农药的检出限为0.06~4.27 ng/mL,定量下限为0.18~14.24 ng/mL。采用空白全血进行加标回收实验,97种农药在100、200、400 ng/mL 3个加标水平下的回收率为32.2%~120%,日内精密度为1.9%~11%,日间精密度为3.6%~13%。该方法由传统QuEChERS方法改进,绿色环保、操作简单、快速高效,可用于血液中多种农药的同时检测,用于实际案件血液样品中农药的筛查与定性定量检验,获得了良好的结果。     

Development and full validation of an HPLC methodology to quantify atorvastatin and curcumin after their intranasal co‐delivery to mice

There is increasing interest in atorvastatin and curcumin owing to their potential anticancer activity. A new, accurate and sensitive HPLC method was developed, for the first time, to simultaneously quantify atorvastatin and curcumin in mouse plasma and brain, liver, lung and spleen tissues following protein precipitation sample preparation. The chromatographic separation was achieved in 13 min on a C₁₈ column, at 35°C, using a mobile phase composed of acetonitrile–methanol–2% (v/v) acetic acid (37.5:2.5:60, v/v/v) at a flow rate of 1.0 mL/min. The detection of analytes and internal standard was carried out at 247, 425 and 250 nm, respectively. According to international guidelines, the method was shown to be selective, with lower limits of quantification ranging from 10 to 500 ng/mL for curcumin, and from 100 to 600 ng/mL for atorvastatin, linear over a wide concentration range (r² ≥ 0.9971) and with acceptable accuracy (bias ± 12.29%) and precision (coefficient of variation ≤13.15%). The analytes were reproducibly recovered at a percentage >81.10% and demonstrated to be stable under various experimental conditions in all biological matrices. This method can be easily applied to in vivo biodistribution studies related to the intranasal administration of atorvastatin and curcumin, separately or simultaneously.