Development of gas chromatography/mass spectrometry following headspace solid-phase microextraction for fast determination of asarones in plasma

Asarones (alpha-asarone and beta-asarone) are the active components in the traditional Chinese medicine (TCM) of Acorus tatarinowii Schott, which has been used to treat epilepsy for several thousand years. To perform the pharmacokinetics (PK) study of alpha- and beta-asarone from the TCM essential oil, a simple, rapid and sensitive method was developed for the determination of asarones from the TCM in rabbit plasma, based on headspace solid-phase microextraction (HS-SPME) followed by gas chromatography/mass spectrometry (GC/MS) with electron ionization (EI). The extraction parameters of headspace volume, fiber coating, sample temperature, extraction time, stirring rate and ion strength were systemically optimized. Furthermore, the method linearity, detection limit and precision were also investigated. It was shown that the proposed method provided a good linearity (0.02-20 microg/mL, R(2) > 0.99), low detection limit (<2.0 ng/mL) and good precision (RSD < 7.0%). Finally, HS-SPME followed by GC/MS was applied to fast determination of alpha- and beta-asarone in rabbit plasma at different time points after oral adminstration of the essential oil from A. tatarinowii. The experimental results suggest that the proposed method provides an alternative approach to the PK studies of volatile compounds in TCMs.     

Determination of the stabilizer sucrose in a plasma-derived antithrombin process solution by hydrophilic interaction chromatography with evaporative light-scattering detection

Hydrophilic interaction chromatography (HILIC) is used for the quantitation of sucrose in the range of 10-100 micro g/mL. A poly-2-hydroxyethylaspartamide column is eluted with 25% water-75% acetonitrile, and evaporative light scattering is utilized for detection. A process sample of antithrombin (Atenativ) from Octapharma AB (Stockholm, Sweden) containing 20% sucrose is analyzed. The precision for this high-performance liquid chromatographic method is a percent relative standard deviation (%RSD) of 4, limit of detection (s/n=3) of 1 microg/mL, and mean recovery of spiked samples of 101% (RSD% of 3, n=6). Analysis time is 10 min/sample. Glucose, fructose, sodium citrate, sodium phosphate, Triton X-100, and tri-n-butyl phosphate do not interfere with the method.     

Chemical composition and antimicrobial activity of the volatile oil from Fusarium tricinctum, the endophytic fungus in Paris polyphylla var. yunnanensis

The volatile oil, obtained by hydro-distillation from Fusarium tricinctum, the endophytic fungus isolated from Paris polyphylla var. yunnanensis, was analyzed by gas chromatography-mass spectrometry (GC-MS). trans-1,2,3,3a,4,7a-Hexahydro-7a-methyl-5H-inden-5-one (73.1%), 2-methylene-4,8,8-trimethyl-4-vinyl bicyclo [5.2.0] nonane (12.0%), and 2,6-dimethyl-6-(4-methyl-3-pentenyl) bicyclo [3.1.1] hept-2-ene (4.5%) were the major compounds of the 15 identified components accounting for 95.4% of the volatile oil. The antimicrobial activity of the volatile oil was assayed against eight bacteria and two fungi. The minimum inhibitory concentration (MIC) values of the volatile oil against the test bacteria ranged from 25 to 45 microg/mL. The MIC values against the fungi Candida albicans and Magnaporthe oryzae were 100 and 225 microg/mL, respectively. The mean inhibitory concentration (IC50) values of the volatile oil against the test bacteria ranged from 17.8 to 31.6 microg/mL, and those of the volatile oil against C. albicans and M. oryzae were 84.3 and 204.3 microg/mL, respectively.     

Essential oil from the leaves of Annona vepretorum: chemical composition and bioactivity

The essential oil from the leaves of Annona vepretorun was obtained by hydrodistillation using a Clevenger-type apparatus and analyzed by GC-MS and GC-FID. Eighteen compounds representing 98.1% of the crude essential oil were identified. The major compounds identified were bicyclogermacrene (43.7%), spathulenol (11.4%), alpha-felandrene (10.0%), alpha-pinene (7.1%), (E)-beta-ocimene (6.8%), germacrene D (5.8%), and p-cymene (4.2%). The trypanocidal activity against Trypanosoma cruzi epimastigote forms, as well as, the antimicrobial and antioxidant proprieties was investigated. The essential oil showed a potent trypanocidal activity with IC50 value of 31.9 +/-1.3 microg x mL(-1). For antimicrobial activity, the best result was observed against Candida tropicalis with a MIC value of 100 microg x mL(-1). For antioxidant capacity the essential oil showed weak activity.     

Quantitative analysis of aglycone quercetin in mulberry leaves (Morus alba L.) by capillary zone electrophoresis.

A capillary zone electrophoresis method was established for analysis of aglycone quercetin in mulberry leaves (Morus alba L.). The influence of, e.g., background electrolyte concentrations and pH, surfactant concentrations, organic solvents, temperature, and voltage on the separation of aglycone quercetin, rutin, quercitrin, kaempferol, catechin, and gallic acid was systematically investigated. The optimum condition providing baseline separation of all compounds within 16.5 min was obtained in 150 mM boric acid (pH 10.0) using a fused-silica capillary with an effective length of 42.5 cm (50 microm inner diameter), temperature of 32 degrees C, and voltage of 15 kV. Method assessment was performed by standard addition method using rutin as an internal standard. Linearity of the method was excellent (r(2) > 0.999) over the concentration tested (40-160 microg/mL). The relative standard deviations (%RSDs) from injection, intraday, and interday precision were less than 2.5%. Recoveries were good (asymptotically equal to 100.0%,%RSD = 0.8%) with a limit of detection (LOD) and limit of quantitation (LOQ) of 0.86 and 3.16 microg/mL (%RSD = 1.8%), respectively. The aglycone quercetin found in the mulberry leaves was 0.452 g/100 g (%RSD = 0.6%) on dry weight.     

A high-performance liquid chromatographic method for saikosaponin a quantification in rat plasma

A high-performance liquid chromatographic method with UV detection has been developed for the determination of saikosaponin a in rat plasma. Saikosaponin a and internal standard jujuboside A were isolated from plasma samples by solid-phase extraction. The chromatographic separation was achieved on a reversed-phase C(18) column with the mobile phase of acetonitrile-water (35:65, v/v) at a flow rate of 1 mL/min and UV detection was set at 205 nm. The standard curve for saikosaponin a was linear over the concentration range 0.25-10 microg/mL and the limit of detection was 0.05 microg/mL. The absolute recovery was greater than 82%. The precision and accuracy ranged from 3.05 to 9.59% and 95.61 to 110.00%, respectively. The validated method was used to determine saikosaponin a in plasma samples in a pharmacokinetic study of saikosaponin a administered to Sprague-Dawley rats.     

Determination of didanosine in maternal plasma, amniotic fluid, fetal and placental tissues by high-performance liquid chromatography-tandem mass spectrometry

A rapid and efficient high-performance liquid chromatography (HPLC)-tandem mass spectrometry method for the determination of didanosine concentrations in maternal rat plasma, amniotic fluid, placental and fetal tissue samples has been developed and validated. Tissue samples were homogenized in optima water and centrifuged. The supernatant was subjected to solid-phase extraction (SPE) prior to analysis. Plasma and amniotic fluid samples were extracted without pretreatment. An Agilent 1100 Series HPLC coupled with a Micromass Quattro II triple quadrupole mass spectrometer was used for all analyses. Chromatographic resolution was achieved on a Nova-Pak phenyl analytical column (2.0 x 150 mm, 4 microm particle size) equipped with a Phenomenex Security-guard phenyl guard cartridge (2.0 x 4.0 mm) using 60% methanol in 10 mm ammonium acetate buffer mobile phase for all matrices at a flow rate of 0.15 mL/min. The method yields retention times of 2.9 min for didanosine and 3.0 min for the internal standard, stavudine. Limits of detection were 1 ng/mL for all matrices. Recoveries were 70% or greater for both compounds in the different matrices. Within- and between-run precision (%RSD) and accuracy (%error) was less than 15% for all matrices.     

Development and validation of a HPLC method for 4,7-phenanthroline-5,6-dione I and identification of its major impurity by HPLC-MS-APCI

In this study, the development and validation of an analytical method for the assay of 4,7-phenanthroline-5,6-dione I (dione I) using high-performance liquid chromatography (HPLC) and the determination of its synthetic impurities by employing the method in HPLC-mass spectrometry with atmospheric-pressure chemical ionization and photodiode-array UV detection is reported. The results show that dione I is eluted as a spectrally pure peak resolved from its impurities. 5-Bromo,4-7-phenanthroline is identified as the main impurity. This is supported by elemental analysis of the dione I, which demonstrated the presence of bromine. Validation parameters such as specificity and selectivity, linearity, accuracy, precision, limit of detection (LOD), limit of quantitation (LOQ), ruggedness, stability, and system suitability, which are evaluated for this method. The LOD and LOQ are 2.0 microg/mL and 50 microg/mL with a 0.50% relative standard deviation (%RSD), respectively. The calibration curves showed good linearity over the concentration range of 0.05-1.50 mg/mL. The correlation coefficient is > 0.9991 in each case. The %RSD values for intra- and interday precision studies are < 0.40%.     

NH2-SEP-PAK cartridges for enrichment of Aromatic Sulfur Compounds from sea water: determination by GC-FID and GC-MS

The Aromatic Sulfur Compounds (ASCs) are considered as "markers" of the crude oil age. In this frame, we have considered ASCs as "markers" of remote crude oil sea water pollution for their chemical characteristics, good solubility in water and resistance to the biodegradation. Gas chromatography of ASCs in distilled water and sea water samples has been performed after adsorption from a 80 mL water sample on to a cartridge containing 100 mg NH2-bonded porous silica. The ASCs are desorbed with 2 mL acetone-trichloromethane (1:1), which is concentrated and analysed by GC-FID or GC-MS in SIM mode. The average recovery of 0.1 microg mL(-1) of each ASC from distilled water and 0.04-0.2 microg mL(-1) from sea water samples is > or = 96.3% with a standard deviation < or = 2.2. The limits of detection are 0.06-0.05 ng mL(-1) for thiophen-2-carboxaldehyde and benzothiophene, and 0.006-0.004 ng mL(-1) for dibenzothiophene and thiantrene in GC-FID whereas they are 0.02 ng mL(-1) for thiophen-2-carboxaldehyde and benzothiophene, and 0.003 ng mL(-1) for dibenzothiophene and thiantrene in GC-MS (SIM) with a relative standard deviation < or =7.     

Determination of L-carnitine in food supplement formulations using ion-pair chromatography with indirect conductimetric detection

A novel method for the determination of L-carnitine in food supplement formulations was developed and validated, using ion-pair chromatography with indirect conductimetric detection. The chromatographic method was based on a non-polar (C18) column and an aqueous octanesulfonate (0.64 mM) eluent, acidified with trifluoroacetic acid (5.2 mM). The retention time was 5.4 min and the asymmetry factor 0.65. A linear calibration curve from 10 to 1000 microg/ml (r= 0.99998), with a detection limit of 2.7 microg/ml (25 microl injection volume), a repeatability %RSD of 0.8 (40 microg/ml, n = 5) and reproducibility %RSD of 2.6 were achieved. The proposed method was applied for the determination of carnitine in oral solutions and capsules. No interference from excipients was found and the only pretreatment step required was the appropriate dilution with the mobile phase. Recovery from spiked samples was ranged from 97.7 to 99.7% with a precision (%RSD, n = 3) of 0.01-2.1%.     

Precision improvement for the analysis of flavonoids in selected Thai plants by capillary zone electrophoresis

A capillary zone electrophoresis (CZE) method for the analyses of kaempferol in Centella asiatica and Rosa hybrids and rutin in Chromolaena odorata was developed. The optimization was performed on analyses of flavonoids (e.g., rutin, kaempferol, quercetin, myricetin, and apigenin) and organic carboxylic acids (e.g., ethacrynic acid and xanthene-9-carboxylic acid) by investigation of the effects of types and amounts of organic modifiers, background electrolyte concentrations, temperature, and voltage. Baseline separation (R(s) = 2.83) of the compounds was achieved within 10 min in 20 mM NaH2PO4 - Na2HPO4 (pH 8.0) containing 10% v/v ACN and 6% v/v MeOH using a voltage of 25 kV, a temperature of 30 degrees C, and a detection wavelength set at 220 nm. The application of the corrected migration time (t(c)), using ethacrynic acid as the single marker, was efficient to improve the precision of flavonoid identification (% relative standard deviation (RSD) = 0.65%). The method linearity was excellent (r2 > 0.999) over 50-150 microg/mL. Precision (%RSD < 1.66%) and recoveries were good (> 96% and %RSDs < 1.70%) with detection and quantitation limits of 2.23 and 7.14 microg/mL, respectively. Kaempferol in C. asiatica and R. hybrids was 0.014 g/100 g (%RSD = 0.59%) and 0.044 g/100 g (%RSD = 1.04%), respectively, and rutin in C. odorata was 0.088 g/100 g (%RSD = 0.06%).     

Development of a novel method based on liquid chromatography-evaporative light scattering detection for the direct determination of streptomycin and dihydrostreptomycin in raw materials, pharmaceutical formulations, culture media and plasma

A novel method for the non-derivatization liquid chromatographic determination of streptomycin (STR) and dihydrostreptomycin (DHSTR) was developed and validated based on evaporative light scattering detection (ELSD). Utilizing a ThermoHypersil BetaBasic C18 analytical column, evaporation temperature of 50 degrees C and pressure of nebulizing gas (nitrogen) of 3.5 bar, the optimized mobile phase was 1.25 mL L(-1) TFA aqueous solution, in an isocratic mode at a rate of 1.0 mL min(-1). STR was eluted at 5.6 min and DHSTR at 7.8 min with a resolution of 4.4. Linear calibration curves were obtained from 2 to 120 microg mL(-1) (r > 0.9990) for STR and 2-75 microg mL(-1) (r > 0.9994) for DHSTR, with a LOD equal to 0.7 and 0.5 microg mL(-1), respectively. The developed method was applied for the assay of STR and DHSTR (sulfate) in pharmaceutical raw materials and formulations, while the simultaneous direct determination of sulfate was feasible (tR = 2.5 min, LOD = 1.4 microg mL(-1), double logarithmic calibration curve in the range of 4-50 microg mL(-1), r > 0.9998). Modified isocratic mobile phase (H2O-ACN, 90:10, v/v, containing 1.25 mL L(-1) TFA), was used for the determination of streptomycin B impurity in STR sulfate raw material and a gradient mobile phase (H2O-ACN containing TFA) was used for the determination of DHSTR in the presence of penicillinG procaine. The developed method was also applied for the assay of commercial formulations (STR powder and DHSTR injection solution and suspension) (%recovery 98-102, %RSD < 1.3, n = 3 x 3), for the determination of STR in bacteria culture medium (%recovery 99.6, %RSD = 0.8, n = 3 x 3), and for the determination of DHSTR in human plasma (2.0-23.0 microg mL(-1)) after solid phase extraction using carboxylate cartridges (%recovery 98.4-101.8, %RSD = 3.2, n = 3 x 3).     

Simultaneous quantitation of enalapril and enalaprilat in human plasma by 96-well solid-phase extraction and liquid chromatography/tandem mass spectrometry

A sensitive and rapid method based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) combined with rapid solid-phase extraction (SPE) has been developed and validated for the quantitative determination of enalapril and its active metabolite enalaprilat in human plasma. After addition of internal standard to human plasma, samples were extracted by 96-well SPE cartridge. The extracts were analyzed by HPLC with the detection of the analyte in the multiple reaction monitoring (MRM) mode. This method for the simultaneous determination of enalapril and enalaprilat was accurate and reproducible, with respective limits of quantitation of 0.2 and 1.0 ng/mL in plasma. The standard calibration curves for both enalapril and enalaprilat were linear (r(2) = 0.9978 and 0.9998) over the concentration ranges 0.2-200 and 1.0-100 ng/mL in human plasma, respectively. The intra- and inter-day precision over the concentration range for enalapril and enalaprilat were lower than 13.3 and 15.4% (relative standard deviation, %RSD), and accuracy was between 89.2-105.0 and 91.9-104.7%, respectively.     

Improved gas chromatography methods for micro-volume analysis of haloacetic acids in water and biological matrices

A fast headspace solid-phase microextraction gas chromatography method for micro-volume (0.1 mL) samples was optimized for the analysis of haloacetic acids (HAAs) in aqueous and biological samples. It includes liquid-liquid microextraction (LLME), derivatization of the acids to their methyl esters using sulfuric acid and methanol after evaporation, followed by headspace solid-phase microextraction with gas chromatography and electron capture detection (SPME-GC-ECD). The derivatization procedure was optimized to achieve maximum sensitivity using the following conditions: esterification for 20 min at 80 degrees C in 10 microL methanol, 10 microL sulfuric acid and 0.1 g anhydrous sodium sulfate. Multi-point standard addition method was used to determine the effect of the sample matrix by comparing with internal standard method. It was shown that the effect of the matrix for urine and blood samples in this method is insignificant. The method detection limits are in the range of 1 microg L(-1) for most of the HAAs, except for monobromoacetic acid (MBAA) (3 microg L(-1)) and for monochloroacetic acid (MCAA) (16 microg L(-1)). The optimized procedure was applied to the analysis of HAAs in water, urine and blood samples. All nine HAAs can be separated in < 13 min for biological samples and < 7 min for drinking water samples, with total sample preparation and analysis time < 50 min. Analytical uncertainty can increase dramatically as the sample volume decreases; however, similar precision was observed with our method using 0.1 mL samples as with a standard method using 40 mL samples.     

Rapid determination of volatile constituents in safflower by microwave distillation and simultaneous solid-phase microextraction coupled with gas chromatography-mass spectrometry

In this paper, microwave distillation and solid-phase microextraction coupled with gas chromatography-mass spectrometry (MD-SPME/GC-MS) was developed for the analysis of essential components in safflower. Using the MD-SPME technique, the isolation, extraction and concentration of volatile compounds in safflower were carried out in only one step. Some parameters affecting the extraction efficiency such as SPME fiber coating, microwave power, irradiation time and the volume of water added were optimized. The optimal experiment parameters obtained were: 65 microm CW/DVB SPME fiber, a microwave power of 400 W, an irradiation time of 3 min and water volume of 1 mL. The proposed method has been compared with conventional steam distillation (SD) for extraction of essential oil compounds in safflower. Using MD-SPME followed by GC-MS, 32 compounds in safflower were separated and identified, which mainly included paeonol, alpha-asarone, beta-asarone, 1-methyl-4-(2-propenyl)-benzene and diethenyl-benzene, whereas only 18 compounds were separated and identified by conventional SD followed by GC-MS. The relative standard deviation (R.S.D.) values of less than 10% show that the proposed method has good reproducibility. The results show that MD-SPME/GC-MS is a simple, rapid, effective method for the analysis of volatile oil components in safflower.     

Determination of calycosin-7-O-beta-D-glucopyranoside in rat plasma and urine by HPLC

A reversed-phase high-performance liquid chromatographic (HPLC) assay for calycosin-7-O-beta-D-glucopyranoside in rat plasma and urine with solid-phase extraction (SPE) was developed. Rutin was employed as an internal standard. The mobile phase consisted of acetonitrile-water (16:84, v/v) at a flow rate of 1.0 mL/min. Detection was set at 280 nm. The limit of quantitation of calycosin-7-O-beta-D-glucopyranoside was 0.2 microg/mL in both plasma and urine. The standard curve was linear from 0.2 to 10.0 microg/mL in plasma, and 0.2 to 5.0 microg/mL in urine. Both intra- and inter-day precision of the calycosin-7-O-beta-d-glucopyranoside were determined and their RSD did not exceed 10%. The method was successfully applied to the analysis of samples obtained from a basic pharmacokinetic study, in which calycosin-7-O-beta-d-glucopyranoside was administered orally to rats.     

Convergence chromatographic determination of camphor in the essential oil of Tanacetum parthenium L.

Feverfew (Tanacetum parthenium L., Asteraceae) is a perennial medicinal plant which has been used to alleviate the symptoms of migraine, headache and rheumatoid arthritis and possesses numerous pharmacological activities. An ultra‐high‐performance supercritical fluid chromatographic method (UHPSFC) was developed and validated in accordance with the International Conference on Harmonization guidelines in order to determine the camphor content of the volatile oil, which was accurate, precise, robust and selective. The method was validated for specificity, accuracy (100.2%), repeatability and intermediate precision, linearity (r² > 0.999), limit of detection (2.055 μg/mL), limit of quantification (6.228 μg/mL) and robustness. The common range of accuracy and linearity was between 0.125 and 1.000 mg/mL. Steam distillation was carried out in order to study the essential oil yield of three different T. parthenium L. samples originating from Hungarian medicinal herb collections. The camphor content of the essential oils from the aerial parts of feverfew samples from different origin was compared. Although the composition of the essential oil is well reported, a validated quantitative UHPSFC method for the determination of the constituents is presented herein for the first time.     

The use of graphene as an adsorbent for the extraction of permethrin from urine

A nonmagnetically modified graphene was employed as the adsorbent in the dispersive solid-phase extraction (d-SPE) for the isolation of permethrin from urine samples that were further analyzed by the HPLC/UV method. The following four key factors influencing permethrin extraction efficacy were studied: 1. graphene suspension volume, 2. adsorption time, 3. desorbent (acetonitrile) volume, and 4. desorption time. Under the optimized conditions of the d-SPE (graphene) procedure the main validation parameters of the analytical method were determined as follows: extraction recovery (%) 71.2, 63.5, and 95.4; intra-day repeatability (%RSD) 14.7, 11.5, and 15.2; inter-day repeatability (%RSD) 16.2, 11.9, and 19.1 for 0.125, 0.250, and 1.0 µg/mL permethrin concentration levels, respectively. Limits of detection (LOD) and quantification (LOQ) were 0.045 and 0.125 µg mL^?1, respectively. The proposed method may be useful for the monitoring of permethrin in human urine taken from people dressed in uniforms disinfected with this pesticide.     

Development and validation of a high performance liquid chromatography-diode array detection method for the determination of aging markers in tequila

A solid-phase extraction procedure followed by analysis by high performance liquid chromatography (HPLC) with UV-vis photodiode array detection (DAD) is proposed to simultaneously determine 11 aging markers in tequila. The method showed good intraday (n=5) and interday (n=3) precision, RSD<1.6% in both cases, for each of the identified compounds. The calibration curves were linear at the tested ranges (R(2)>0.999). Good recoveries (84.2-108.5%) were obtained for 10 of the 11 compounds studied; and the LOD and LOQ ranged from 0.62 to 4.09 microg/mL and 1.9-12.4 microg/mL, respectively. The proposed methodology was applied to a set of 15 authentic tequila samples grouped by aging state (blanco, reposado and a?ejo). An ANOVA analysis combined with discriminant analysis with stepwise backward variable selection was used to differentiate between the various aging groups based on their oak related compounds content.     

Evaluation of the mixed aggregate method as an alternative to the bismuth active substances and cobalt thiocyanate active substances procedures for the determination of nonionic surfactants in raw and treated sewage

The mixed aggregate (MA) method was evaluated for the routine determination of total ethoxylated nonionic surfactants in municipal raw and treated sewage. Nonionics in wastewater samples were enriched with a C8 solid-phase extraction cartridge, eluted with ethyl acetate-methanol, and separated from interferences with stacked strong anionic (quaternary ammonium form) and cationic (benzenesulfonic acid form) exchange columns. Sample extracts were evaporated to dryness and redissolved in distilled water. The total amount of ethoxylates, on a weight basis, was determined by measuring the critical micelle concentration of a mixed micelle made up of ethoxylates and the anionic surfactant sodium dodecyl sulfate, used as the reagent. The dye Coomassie Brilliant Blue G was used as a photometric probe. The minimum amount of ethoxylate required for a single determination was 1 microg. For raw sewage samples, mean recoveries and standard deviations were 98 +/- 6 and 98 +/- 4% at fortification levels of 0.3 and 0.6 microg/mL, respectively. For treated sewage samples, mean recoveries and standard deviations were 95 +/- 2 and 100 +/- 2% at fortification levels of 0.03 and 0.06 microg/mL, respectively. The MA method and the classical bismuth active substances and cobalt thiocyanate active substances procedures were compared. The former offers some significant advantages over the classical procedures in terms of sensitivity, selectivity, precision, simplicity, and rapidity; the type of response is independent of both the molecular weight and the chain length of the surfactant.