The use of high-flow high performance liquid chromatography coupled with positive and negative ion electrospray tandem mass spectrometry for quantitative bioanalysis via direct injection of the plasma/serum samples

Two bioanalytical methods have been developed and validated utilizing high flow high performance liquid chromatography (HPLC) for on-line purification of plasma and serum samples and electrospray tandem mass spectrometry for detection and quantitation. Each plasma or serum sample, after mixing with an aqueous solution of the internal standard, was injected into a small diameter (1 x 50 mm) column packed with large particles of OASIS (30 microns), with a 100% aqueous mobile phase at a high flow rate (3-4 mL/min). The combination of the high linear speed (6-8 cm/s) of the aqueous mobile phase and the large particle size resulted in the rapid passage of the proteins and other large biomolecules through the column while the small-molecule analytes were retained on the column. During this purification period, the HPLC effluent was directed to waste. After the purification step, the HPLC mobile phase was rapidly changed from 100% aqueous to < or = 100% organic, the flow was reduced to 0.5-0.8 mL/min, and the column effluent was directed towards the mass spectrometer. The small molecule analytes were eluted during this period. In the method developed and validated for the quantitative determination of compound I in rat plasma (method A), the same OASIS column (1 x 50 mm, 30 microns) served as the purification and analytical (elution) column. In the method developed for the simultaneous determination of pravastatin and its positional isomer biotransformation product (SQ-31906) in human serum (method B), the purification column was connected to a conventional C18 analytical column (3.9 x 50 mm, 5 microns) to achieve the required chromatographic separation between the two isomers. For method A, where 50 microL of rat plasma mixed 1:1 with water containing the internal standard was injected, the standard curve range was 1 to 1,000 ng/mL. For method B, where 200 microL of a human serum sample mixed 4:1 with water containing the internal standard was injected, the standard curve range was 0.5 to 100 ng/mL. The total analysis time for each method was < or = 5 min per sample. The accuracy, inter-day precision and intra-day precision were within 10% for both methods.     

Ischemic preconditioning: a long term survival study using behavioural and histological endpoints

Simple procedures for the extraction and chromatographic determination of benomyl and carbendazim in honey, bees wax, larvae, bees and pollen are proposed. The fungicides were extracted from honey, larvae and bees using ethyl acetate, while methanol was more suitable for wax and pollen samples. Pollen extracts need a further clean-up step with n-hexane. The determination is carried out by reversed-phase high-performance liquid chromatography (HPLC) with fluorescence detection. The procedures have been applied to the analysis of benomyl on honey and larvae samples from hives whose bees were nourished with artificial food mixed with benomyl.     

Ascorbic acid is a stimulatory cofactor for mitochondrial glycerol-3-phosphate dehydrogenase

We describe in this report a sensitive and direct method for the analysis of tamoxifen (TAM) in microsamples of plasma. The drug and internal standard (quinine bisulfate, I.S.) were separated on a 10-microm particle, 10 cm X 8 mm CN cartridge in conjunction with a radial compression system. The mobile phase was a mixture of 0.1 M sodium acetate in 0.001 M tetrabutylammonium phosphate solution (pH 6) and methanol (30:70, v/v) at a flow-rate of 4 ml/min. After addition of I.S. and o-phosphoric acid in acetonitrile (0.6 M) to the plasma (30 microl), the mixture was placed in an ultraviolet shortwave transluminator for 2 min prior to injection into the chromatograph. The compounds were detected in the effluent fluorometrically at excitation and emission wavelengths of 258 and 378 nm, respectively. Under these conditions, no interference in the assay from any endogenous substance or other concurrently used drugs was observed and the retention times of I.S. and TAM were 4.4 and 10.15 min, respectively. The concentration of TAM in plasma was linearly (r>0.9983) related to the peak height ratio (TAM/I.S.) in the range 0.01-2.0 microg ml(-1) and C.V. at 0.075, 0.4 and 1.2 microg ml(-1) was < or = 4.96%. We are currently using this assay for monitoring TAM in plasma and investigating its pharmacokinetics in cancer patients receiving cytotoxic drugs in addition to TAM as a multi-drug resistance modifier.     

Effects of organic acid and monensin treatment on in vitro mixed ruminal microorganism fermentation of cracked corn

The objective of this study was to determine the effects of organic acids and monensin on the in vitro fermentation of cracked corn by mixed ruminal microorganisms. Ruminal fluid was collected from a steer fed 36.3 kg of wheat silage and 4.5 kg of concentrate supplement once daily. Mixed ruminal microorganisms were incubated in anaerobic media that contained 20% (vol/vol) ruminal fluid and .4 g of cracked corn. Incubations were carried out in batch culture for 24 h at 39 degrees C. Organic acids (L-aspartate, fumarate, and DL-malate) were added to serum bottles (n = 4) to achieve final concentrations of 0, 4, 8, or 12 mM. Monensin, dissolved in ethanol, was included in serum bottles at a final concentration of 0 or 5 ppm of culture fluid. The addition of 8 and 12 mM organic acids to cracked corn fermentations increased final pH (P < .05), tended to increase total gas production and CO2 concentration, and decreased the acetate:propionate ratio (P < .05). Organic acids tended to decrease methane concentrations and hydrogen concentration was not altered. DL-Malate addition at all levels reduced (P < .05) lactate accumulation. Additive effects of monensin and organic acids were observed in some fermentations. In conclusion, organic acid addition to in vitro mixed ruminal microorganism fermentations yielded beneficial results independent of monensin treatment by decreasing the acetate: propionate ratio and increasing final pH.     

Determination of 13-cis-3-hydroxyretinoic acid, all-trans-3-hydroxyretinoic acid and their 4-oxo metabolites in human and animal plasma by high-performance liquid chromatography with automated column switching and UV detection

A high-performance liquid chromatographic method with automated column switching was developed for the simultaneous determination of 13-cis-3-hydroxyretinoic acid, all-trans-3-hydroxyretinoic acid and their metabolites 13-cis-3-hydroxy-4-oxo-retinoic acid and all-trans-3-hydroxy-4-oxo-retinoic acid in plasma samples from man, rat, dog, rabbit and mouse. The method consists of deproteination of plasma (0.4 ml) with ethanol (1.5 ml), containing the internal standard Ro 12-7310. After centrifugation, 1.4 ml of the supernatant was directly injected onto the precolumn (PC) (4 x 4 mm) packed with LiChrospher 100 RP-18 (5 microm). Ammonium acetate (0.02%)-acetic acid-ethanol (100:3:4, v/v/v) was used as mobile phase M1A during injection, as well as to decrease the elution strength of the injection solution by on-line addition using a T-piece (M1B). After valve switching, the retained components were transferred to the analytical column (AC), separated by gradient elution and detected at 360 nm. Two coupled Purospher 100 RP-18 endcapped columns (both 250 x 4 mm) were used for the separation, together with a mobile phase consisting of acetonitrile-water-10% ammonium acetate-acetic acid, (A), 540:450:2:30 (v/v/v/v), (B), 600:350:2:30 (v/v/v/v), and (C), 950:40:2:30 (v/v/v/v). The method was linear in the range 1-500 ng ml(-1), at least, with a quantification limit of 1 ng ml(-1). The mean recoveries from human plasma were 100-107% and the mean inter-assay precision was 2.0-4.7% (range 1-500 ng ml(-1)). Similar results were obtained for animal plasma. The analytes were stable in the plasma of all investigated species stored at -20 degrees C for 3 months, at least. The method was successfully applied to clinical and toxicokinetic studies.     

Bile acid analysis in biological fluids: a novel approach

In contrast to current methods of bile acid analysis that require the separation of bile acids into different groups prior to their analysis, the HPLC method using a reverse phase column and gradient elution that we developed permits the separation and detection of nonconjugated, glycine-conjugated, and esterified bile acids as their fluorescent dimethoxycoumarin esters. The mild conditions for ester formation make possible the identification of allylic bile acids characteristic of metabolic errors in bile acid synthesis. Quantification is obtained using 7 alpha,12 alpha-dihydroxy-5 beta-cholanoic acid as an internal standard. In addition to identification based on retention time, peak-shift strategy is used by treatment of aliquots with cholyglycine hydrolase and/or solvolysis. Loss of the parent peak and appearance of the derivative provide further assurance of the identity of each bile acid in biologic fluids that contain other organic acids.     

Extraction of gold(III) in hydrochloric acid solution using monoamide compounds

The extraction and separation properties of Au(III) using two monoamide compounds, N,N-di-n-octylacetamide (DOAA) and N,N-di-n-octyllauramide (DOLA), which have different side chain lengths attached to the carbonyl carbon (CH₃ for DOAA and n-C₁₁H₂₃ for DOLA), were investigated. The solvent extraction of some precious and base metals (Au(III), Pd(II), Pt(IV), Rh(III), Fe(III), Cu(II), Ni(II) and Zn(II)) in HCl solutions was carried out using DOAA and DOLA diluted with n-dodecane and 2-ethylhexanol. A good selectivity for Au(III) extraction with 0.5 M extractant is obtained at lower HCl concentrations (< 3.0 M) in both systems. The extractability of Au(III) with DOAA is greater than that with DOLA. In the 0.5 M DOAA–3.0 M HCl system, a third phase is formed when the Au(III) concentration in the initial aqueous phase is over 39 g/L. In contrast, third phase formation is not found in the 0.5 M DOLA–3.0 M HCl system, and its loading capacity of Au(III) is about 79 g/L. The Au(III) extracted in the organic phase is effectively back-extracted by 1.0 M thiourea in 1.0 M HCl solution in both systems, while some thiourea is precipitated using the organic phase containing 20 g/L of Au(III). The back extraction of Au(III) using water is poor in the DOAA system, but possible in the DOLA system.     

Urban malaria and its vectors Anopheles stephensi and Anopheles culicifacies (Diptera : Culicidae) in Gurgaon, India

A simultaneous assay for moricizine, its two sulphoxidation metabolites, moricizine sulphoxide and moricizine sulphone, using high-performance liquid chromatography (HPLC) is described. The drug and metabolites and clozapine (internal standard) in biological fluids were extracted using pentanesulphonic acid into diethyl ether. The ethereal extract was evaporated to dryness and the residue was redissolved in the mobile phase (methanol-water-triethylamine, 65:35:0.5, v/v). The analyses were performed on a microBondapak reversed-phase C18 column housed in a Waters Z-module, linked to a C18 pre-column, with a run-time of 12 min. The retention times were 2.7, 3.5, 6.2 and 9.7 min for moricizine sulphone, moricizine sulphoxide, moricizine and clozapine, respectively. The recovery of the compounds from plasma ranged from 89.9% for the sulphoxide to 98.1% for clozapine. The limits of detection of the assay for moricizine, moricizine sulphoxide and moricizine sulphone were 20, 10 and 5 ng/ml, respectively.     

Determination of nalidixic acid and its two major metabolites in human plasma and urine by reversed-phase high-performance liquid chromatography

This paper describes a precise and sensitive method for analysis of nalidixic acid and its two major metabolites in plasma and urine following the oral administration of a therapeutic dose in humans. After addition of an internal standard (oxolinic acid), 1-ml samples of plasma or urine are extracted at acidic pH with chloroform. The extracts are purified by re-extraction with sodium hydroxide solution and then chloroform. The final extracts are evaporated to dryness, reconstituted in mobile phase and injected into a high-performance liquid chromatograph equipped with RP-8 column and UV detector operating at 254 nm. The limit of sensitivity of the method is lower than 0.5 micrograms/ml of plasma or urine for each compound. The applicability of the method to pharmacokinetic studies of nalidixic acid in humans is demonstrated.     

Big and little forms of osteoclast activating factor

We have further characterized osteoclast activating factor (OAF) using a bioassay for bone resorption which utilizes the release of previously incorporated (45)Ca from fetal rat long bones in organ culture. When supernatant media from activated leukocyte cultures were concentrated on Amicon PM10 membranes (assigned molecular weight cutoff 10,000 daltons) and chromatographed on Sephadex G-50 columns, the bone-resorbing activity eluted between the molecular weight markers chymotrypsinogen (25,000 daltons) and cytochrome c (12,500 daltons). This peak of biological activity has been called big OAF. When filtrates from the PM10 membranes were concentrated on Amicon UM2 membranes (assigned molecular weight cutoff 1,000 daltons) and chromatographed on Sephadex G-50 columns, some of the biological activity eluted between the molecular weight markers chymotrypsinogen and cytochrome c (big OAF), but there was a separate peak of biological activity which eluted with [(3)H]proline (140 daltons). This second peak has been called little OAF. Little OAF was eluted from Bio-Gel P6 columns between the molecular weight markers calcitonin (approximately 3,500 daltons) and vitamin B(12) (1,330 daltons), but was retained by Spectrapor dialysis tubing (nominal molecular weight cutoff 3,500 daltons). Big OAF was converted to little OAF by equilibration in 1 M NaCl or 2 M urea. Little OAF was self-associated back to big OAF by equilibration in buffers of low ionic strength (Tris-HCl 10-50 mM). Little OAF was extracted into the organic phase in ethyl acetate after acidification of the sample to pH 3.5. The biological activity remained in the aqueous phase after ethyl acetate extraction at pH 7.5-8.4. Little OAF has been purified more than 6,000-fold compared with the original material so that bone-resorbing activity is maximal in a sample with a protein concentration of 80 ng/ml.     

P204从高铝粉煤灰硫酸浸液中萃取除铁

主要针对高铝粉煤灰酸浸液中铁铝萃取分离性能进行研究,通过模拟高铝粉煤灰酸浸液(C(Al3+)=37.1 g/L,C(Fe3+)=5.1 g/L),采用P204+260#溶剂油萃取体系,对组成液中铁铝进行萃取分离,系统研究了P204体积分数、温度、反应时间、溶液初始pH值、相比V(O):V(A)等因素对萃取和反萃的影响,...     

Development and validation of a chiral high-performance liquid chromatography assay for rogletimide and rogletimide-N-oxide isomers in plasma

The purpose of the present study was to develop and validate a stereo-specific high-performance liquid chromatography (HPLC) assay for rogletimide (Rog) and rogletimide-N-oxide (Nox) isomers in plasma. The assay was performed with a chiral cellulose-[4-methylbenzoate]ester column (Chiracel OJ). Optimal separation was achieved isocratically with a mobile phase consisting of n-hexane/anhydrous ethanol (65/35, v/v) at a flow rate of 0.9 ml/min, with the column being thermostated at +35 degrees C (UV detection at 257 nm). Under these conditions, retention times were approximately 17, 28, 31 and 76 min for R-Rog, S-Rog, R-Nox and S-Nox, respectively. S-aminoglutethimide (S-Ag) served as the internal standard (retention time 70 min). An extraction procedure from plasma samples was developed on Bond Elut RP8 500-mg cartridges; conditioning was performed with 5 ml methanol and 5 ml water, after which 1 ml plasma that had previously been spiked with 5 microM S-Ag was applied. Washing was done with 6 ml water and elution, with 4 ml methanol. After evaporation to dryness, residues were dissolved in 400 microliters anhydrous ethanol and 12-48 microliters was injected onto the HPLC system. Blank plasma from healthy donors showed the random presence of a small interference eluting at the retention time of R-Rog, precluding the accurate quantification of R-Rog concentrations below 2.5 microM. Reproducibility assays demonstrated the need to use an internal standard. Taking into account the internal standard, at 2.5 microM the intra- and inter-assay coefficients of variation were 10.5% and 21.0% for R-Rog 5.5% and 8.7% for S-Rog, 7.6% and 20.8% for R-Nox and 11.7% and 6.4% for S-Nox, respectively. The detection limit was 2.5 microM for R-Rog, 0.5 microM for S-Rog, 0.25 microM for R-Nox and 0.5 microM for S-Nox. Linearity was satisfactory at concentrations ranging from 2.5 to 10 microM for R-Rog, from 0.5 to 10 microM for S-Rog, from 0.25 to 2.5 microM for R-Nox and from 0.50 to 2.5 microM for S-Nox. This assay was applied to plasma obtained from rog-letimide-treated breast cancer patients receiving conventional oral doses and demonstrated its feasibility with regard to sensitivity. The preliminary pharmacokinetic results reported herein suggest for the first time that both the R-Rog and S-Rog isomers are metabolized into rogletimide-N-oxide.     

Administrative issues in health team development: report of a workshop

Conventional microbiological assay procedures for cephalosporins in serum do not allow the determination of serum concentrations if more than one cephalosporin is present in a single sample. An HPLC procedure has been developed which permits the simultaneous quantitative determination of cefazolin sodium and cephalothin sodium in serum. Reverse phase chromatography using methanol in 0.2 M ammonium acetate as the mobile phase was employed to separate and quantitate the two cephalosporins in a trichloroacetic acid supernatant solution prepared from serum.     

电感耦合等离子体原子发射光谱法测定钛白废酸萃取相中6种微量元素

建立了一种用电感耦合等离子体原子发射光谱法(ICP-AES)同时测定钛白废酸萃取相中主要微量元素(钛、铝、钪、铁、钙和镁)方法。试样中大部分溶剂煤油经低温加热已挥发,剩余有机相用硝酸-硫酸混合酸在低温下消解,分解完全后冒三氧化硫白烟赶尽硝酸,冷却后,在选定的仪器工作条件下,以铟作为内标元素,对试液中钛、铝、钪、铁、钙和镁进行ICP-AES测定,共存元素之间基本没有干扰。方法的的检出限(μg/mL)分别为0.001(钛)、 0.016(铝)、0.004(铁)、0.015(钙)、0.002(钪)和0.005(镁)。方法用于钛白废酸萃取相样品中上述6种元素测定,相对标准偏差在0.35％～0.76％之间,加标回收率为97％～102％。     

Stereoselective determination of unchanged and glucuroconjugated eliprodil, a new anti-ischaemic drug, in human plasma and urine by precolumn derivatization and column-switching high-performance liquid chromatography with fluorescence detection

An HPLC method was developed and validated for the determination in human plasma and urine of the enantiomers of eliprodil, (+/-)-alpha-(4-chlorophenyl)-4[(4-fluorophenyl) methyl]piperidine-1-ethanol hydrochloride, a new anti-ischaemic agent administered as a racemate. Both enantiomers are present in human plasma in unchanged and glucuroconjugated form, whereas only the glucuroconjugated form is excreted into urine; as a consequence, such metabolites in human plasma and urine should be submitted to enzymatic deconjugation with beta-glucuronidase (Escherichia coli) before being extracted. The general method involves a liquid-liquid extraction of eliprodil and internal standard from alkalinized plasma or urine with n-hexane, evaporation of the organic phase and derivatization with (S)-(+)-naphthylethyl isocyanate to give carbamate diastereoisomeric derivatives of (S)-(+)- and (R)-(-)-eliprodil and internal standard; after evaporation of the derivatizing mixture and dissolution of the residue in a small volume of phosphate buffer-acetonitrile (60:40, v/v), an aliquot is injected into a column-switching HPLC system. The derivatized sample extract is purified on a precolumn filled with C8-bonded silica material, which is flushed with acetonitrile-water, then diastereoisomers of eliprodil and the internal standard are automatically transferred by the mobile phase to the analytical column. The analytical column is a C8 type, specially deactivated for basic compounds, the mobile phase is 0.025 M phosphate buffer (pH 2.6)-methanol-acetonitrile (42:2:56) at a flow-rate of 1.2 ml min-1 and fluorimetric detector operating at lambda ex = 275 nm and lambda em = 336 nm is used. The retention times, under these conditions, are about 16 and 17 min for (S)-(+)- and (R)-(-)-eliprodil diastereoisomers, respectively, and about 19 min for the first-eluted diastereoisomer of the internal standard. During the analysis time, the precolumn, reset in a different path from that of the analytical column, is back-flushed with different solvents, then re-equilibrated with acetonitrile-water before the next injection. Linearity in plasma, for unchanged eliprodil enantiomers, was assessed in the range 0.15-10 ng ml-1 and for total eliprodil enantiomers (unchanged + conjugated) in the range 0.75-500 ng ml-1; the limit of quantitation (LOQ) is 0.15 ng ml-1 for each unchanged enantiomer and 0.75 ng ml-1 for each total enantiomer. Linearity was also assessed in urine for total (conjugated) eliprodil enantiomers in the range 50-25 000 ng ml-1; the LOQ is 50 ng ml-1 for each enantiomer. The intra- and inter-day precision and accuracy of the method were investigated in plasma and urine and found to be satisfactory for pharmacokinetic studies. The method has been extensively used in pharamcokinetic studies in man treated with a 20-mg dose of eliprodil racemate and some results of this application are reported.     

Determination of 3,4-dihydroxyphenylacetic acid and 5-hydroxyindoleacetic acid in human plasma by a simple and rapid high-performance liquid chromatography assay

In order to study the pharmacodynamic effects of drugs on dopamine and serotonin metabolism, a reversed-phase HPLC assay coupled with electrochemical detection (ECD) for measuring plasma concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindoleacetic acid (5-HIAA) was developed. The system was operated isocratically using a mobile phase of aqueous 0.03 M KH2PO4 buffer containing 0.15 mM EDTA in methanol (8.75:1.25), with a final pH of 4.0. The flow rate was set at 1.5 mL/min, and potentials at +450 mV. Using a signal to noise ratio of > 3, the minimum detection limit assessed by direct on-column injection of a standard solution for DOPAC and 5-HIAA was < 1 pg. The assays were linear from basal concentrations (1-10 ng/mL) to 100 ng/mL. The intra- and interassay variations were < 10% and < 20%, respectively.     

Liquid chromatographic determination of nalidixic acid in pharmaceutical preparations

A simple, rapid, and environmentally safe liquid chromatographic (LC) method was developed for the qualitative and quantitative determination of pharmaceutical preparations of nalidixic acid. The new method was applied to commercial preparations of tablets and a suspension of nalidixic acid and found to be satisfactory for both quantitative and qualitative determinations. Previous LC methods either used chloroform to extract, which we were trying to eliminate, or used a mobile phase of about pH 2.5, which will destroy the column coating. The LC system for the new method uses sulfanilic acid as internal standard, a mu-Bondapak C18 column, and a mobile phase of methanol, 0.0045M dibasic potassium phosphate, and 0.0072M hexadecyltrimethylammonium bromide. The detection wavelength is 254 nm. The sample is dissolved in methanol, and an aliquot is injected through a 20 microL injection loop. Average recoveries ranged from 99.4 to 101.3%.     

Rapid and Sensitive Liquid Chromatography-Tandem Mass Spectrometry for Quantification of Glycyrrhetic Acid in Human Plasma

A simple, rapid and sensitive liquid chromatography-tandem mass spectrometry(LC-MS/MS) for the determination of glycyrrhetic acid in human plasma with ginsenoside Rh2 as internal standard was developed and validated. The plasma samples were prepared via liquid-liquid extraction with ethyl acetate. Chromatographic separation was accomplished on a Venusil MP-C18(50 mm×2.1 mm, 5μm i.d.) column at 25℃. The mobile phase consisted of acetonitrile/5 mmol·L-1 ammonium acetate(10:90, volume ratio) at a flow rate of 0.4 mL/min. Negative electrospray ionization was utilized as the ionization source. Glycyrrhetic acid and internal standard were determined via the mutiple reaction monitoring of precursor→production ion transitions at m/z 469→425, 409 and m/z 621→161,respectively. Each sample was chromatographed within 2.5 min. The lower limit of uantification was 0.50 ng/mL for 200 μL of plasma sample and the linear range was from 0.50 ng/mL to 800 ng/mL. The intra-and inter-day precisions were less than 8.76% in terms of relative standard deviation(RSD), and the accuracy was within a range of -3.25%-1.32% in terms of relative error(RE). The method was successfully applied to the pharmacokinetic studies of glycyrrhetic acid in healthy male Chinese volunteers after a single oral administration of 75 mg of glycyrrhizin.     

The solvent extraction separation of bismuth and molybdenum from a low grade bismuth glance flotation concentrate

The main purpose of this study was to characterize and to extract bismuth and molybdenum from a low grade bismuth glance concentrate. Selective leaching of bismuth could be obtained at a temperature range 60 to 85 °C for a leaching duration of 2 h with hydrochloric acid concentration of 150 gpl, lignin calcium concentration of 0.02 gpl and using a solid–liquid ratio 1/4 g/cc. Treatment of leach liquor for the solvent extraction of bismuth with N235 showed that 8.0 × 10^− 2 M N235 in kerosene, a 3 min period of equilibration and a pH 0.2 were sufficient for the extraction of Bi(III). This bismuth-loaded organic phase was almost completely stripped using 0.5 M EDTA solution. Treatment of leached residue was dealt with by roasting in the presence of slaked lime, and followed by hydrometallurgical treatment of the roasted products. In the lime roasting process, molybdenum recoveries of around 99% were achieved when an excess of 50% lime over stoichiometric requirement was roasted at 700 °C for 2 h and the calcine was leached with 4 M HCl, at 70–80 °C for 2 h. Molybdenum then was effectively extracted from the leached residual solution with N235. An optimum pH of 0.5 was determined for molybdenum extraction. From loaded solvent, this metal was easily stripped with ammonia solutions to give a pregnant solution suitable for final recovery of metal by salt precipitation. Under the optimized conditions, the ultimate recovery rate of bismuth and molybdenum was more than 99% and 98% respectively.     

Microvascular transfer of the combined gastrocnemius-soleus muscle flap in the rat

A convenient high-performance liquid chromatography (HPLC) was developed for the rapid assay of granisetron (GRN) in biological fluids, such as serum, urine, and pleural effusion, from cancer patients. Extrelut-1 was used for the solid-phase extraction. HPLC was carried out using a LiChroCART cartridge column packed with Lichrospher 100 CN and a mobile phase consisting of 0.1 M acetate buffer (pH 3.5) and acetonitrile (7:3). A fluorescence detector of 290 nm for excitation and 365 nm for emission was used. The standard curve was linear over the range of 2 to 100 ng/ml of GRN. Assay precision, expressed as a coefficient of variation (C.V.), was in the range of 0.9-5.4% in the within-day assay and 2.5-6.9% in the between-day assay, respectively. GRN was well separated on the HPLC chromatogram from drugs such as etoposide, metclopramide, ondansetron, and domperidone which are often used together with GRN. It was suggested that the present method is useful for the rapid monitoring of GRN in the serum, urine, and pleural effusion of patients undergoing cancer chemotherapy.