Determination of ascorbic acid in soft drinks, preserved fruit juices and pharmaceuticals by flow injection spectrophotometry: Matrix absorbance correction by treatment with sodium hydroxide

Two flow injection systems for the spectrophotometric determination of ascorbic acid at 245 nm have been described. On treatment with sodium hydroxide a fraction of the ascorbic acid was decomposed into substances, which do not absorb in UV region, and the decrease in signal measured. This was directly related to the amount of ascorbic acid present. The calibration graph was linear over the range 1-25 and 1-50 microg/ml in the two methods with a correlation coefficient of 0.9981 and 0.9994, respectively. The detection limit (2sigma) was 0.5 and 0.2 microg/ml, respectively. The RSD for 1 microg/ml standard was 2.5 and 1.8% (n = 6) in the two methods, and the sampling throughput 30/hr. The methods permitted the use of 6 microg/ml of 2-mercaptoethanol as an anti-oxidant and stabilizer for ascorbic acid, which is difficult to handle at its microg/ml level. Upon matrix absorbance correction, spiked samples that are known to contain UV-absorbing substances produced an average recovery of 101% with a RSD of 1.2%. The methods were used for the rapid and simple determination of ascorbic acid in soft drinks, preserved fruit juices and pharmaceuticals and the results thus produced compared with those obtained by previously checked methods involving titration with iodine, chloranil 2,6-dichlorophenolindophenol, and HPLC. When there was a disagreement between the results, this was traced to the presence of substances which are known to interfere in comparison methods.     

Determination of molybdenum in ores, iron and steel by atomic-absorption spectrophotometry after separation by alpha-benzoinoxime extraction or further xanthate extraction

A simple and moderately rapid method for determining 0.001% or more of molybdenum in ores, iron and steel is described. After sample decomposition, molybdenum is separated from the matrix elements, except tungsten, by chloroform extraction of its alpha-benzoinoxime complex from a 1.75 M hydrochloric-0.13 M tartaric acid medium. Depending on the amount of tungsten present, molybdenum, if necessary, is back-extracted into concentrated ammonia solution and subsequently separated from coextracted tungsten by chloroform extraction of its xanthate complex from a 1.5M hydrochloric-0.13M tartaric acid medium. It is ultimately determined by atomic-absorption spectrophotometry, at 313.3 nm, in a 15% v/v hydrochloric acid medium containing 1,000 microg/ml of aluminium as the chloride, after evaporation of either extract to dryness with nitric, perchloric and sulphuric acids and dissolution of the salts in dilute ammonia solution.     

Kinetic method for determination of ascorbic acid on flow injection system by using its catalytic effect on the complexation reaction of an ultra sensitive colorimetric reagent of porphyrin with Cu(II)

A kinetic method performed on a flow injection system is described for the determination of ascorbic acid by using its catalytic effect on the complexation reaction of Cu(II) with 5,10,15,20-tetrakis(4-N-trimethyl-aminophenyl)porphyrin. The characteristic spectrum of porphyrin (Soret band), which shows intense absorption around 400 nm (epsilon>2.0 x 10(5) cm(-1)M(-1)), was used first time for determining ascorbic acid. By incorporating the complexation reaction into a flow injection system, ascorbic acid could be determined either over a broad dynamic range of 0.1-1000 microg/ml or at a trace level below 5 ng/ml. Good repeatability was also achieved by testing a working standard of 0.1 microg/ml with 10 injections at a throughput of 35 h(-1), obtaining a relative standard deviation of 0.11%. Substances like amino acids, vitamins, sugars, organic acids and metal ions, showed no or little interference even present at high concentrations. The method was validated in the determination of ascorbic acid contents of some commercially available soft drinks by comparison with the official 2,6-dichloroindophenol method with reasonable agreement.     

Spectrophotometric determination of rifampicin through chelate formation and charge transfer complexation in pharmaceutical preparation and biological fluids

Two simple and accurate spectrophotometric methods for determination of Rifampicin (RIF) are described. The first method is based on charge transfer (CT) complex formation of the drug with three pi-electron acceptors either 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), 7,7,7,8-Tetracyanoquinodimethane (TCNQ) or 2,3,5,6-Tetrachloro-1,4-benzoquinone (p-chloranil) in acetonitrile. The method is followed spectrophotometrically by measuring the maximum absorbance at 584 nm, 761 nm (680 nm) or 560 nm for DDQ, TCNQ and p-chloranil, respectively. Under the optimized experimental conditions, the calibration curves showed a linear relationship over the concentration ranges of 5-140 microg/ml, 2-45 microg/ml (5-120 microg/ml) and 15-200 microg/ml, respectively. The second method is based on the reaction of RIF with iron(III) forming a water insoluble violet complex which is extracted into chloroform. The method determines RIF in concentration range of 10-240 microg/ml at 540 nm. The proposed methods applied to determination of RIF in capsule, human serum and urine samples with good accuracy and precision. The results were compared statistically with the official method and showed no significant different between the methods compared in terms of accuracy and precision.     

Spectrophotometric determination of platinum in glass after extraction with polyurethane foam

A spectrophotometric method has been developed for determination of trace amounts of platinum in glass. The method is based on the extraction of platinum(II) from 1M hydrochloric acid containing 0.2M stannous chloride and 4 x 10(-4)M dithizone onto polyurethane foam, elution with acetone (containing 3% v/v concentrated hydrochloric acid) and measurement of the absorbance of the eluate at 530 nm. Beer's law is obeyed up to 10.0 microg/ml Pt. The minimum platinum level in the eluate that can be determined by this method is 0.1 microg/ml.     

Kinetic spectrophotometric method for the determination of oxalic acid by its catalytic effect on the oxidation of safranine by dichromate

A new catalytic kinetic spectrophotometric method for the determination of oxalic acid has been described based on its catalytic effect on the redox reaction between safranine and dichromate in dilute sulfuric acid media. The reaction is monitored photometrically by measuring the decrease in absorbance of safranine at the maximum wavelength of 530 nm. Under the optimum conditions, a calibration graph from 0.10 to 10.00 microg ml(-1) of oxalic acid with a detection limit of 0.08 microg ml(-1) was obtained. The relative standard deviation (R.S.D.) for ten replicate measurements of 1.0 and 5.0 microg ml(-1) oxalic acid was 2.7 and 2.5%, respectively. The purposed method is simple, sensitive, selective and inexpensive. The applicability of the proposed method was determined by the determination of oxalic acid in spinach and wastewater samples with satisfactory results.     

Flow injection chemiluminescence analysis of some penicillins by their sensitizing effect on the potassium permanganate-glyoxal reaction.

A new chemiluminescence method using flow injection is described for the determination of four penicillins, namely: phenoxymethylpenicillin potassium, amoxicillin, ampicillin, and ampicillin sodium. The method is based on sensitizing effect of these drugs on the chemiluminescence reaction of potassium permanganate in sulfuric acid with glyoxal. The different experimental parameters affecting the chemiluminescence intensity were carefully studied and incorporated into the procedure. The method allows the determination of 0.1-1.0 microg/ml phenoxymethylpenicillin potassium, 0.1-1.0 microg/ml amoxicillin, 0.1-1.0 microg/ml ampicillin, and 0.1-1.0 microg/ml ampicillin sodium. The method was successfully applied to the determination of four penicillin antibiotics in pharmaceutical preparations.     

Solid-liquid extraction and preconcentration of trace nickel using 2-nitroso-1-naphthol-4-sulfonic acid (Nitroso-S) and TDBA onto benzophenone and determination by atomic absorption spectrometry.

Nickel was quantitatively retained by 2-nitroso-1-naphthol-4-sulfonic acid (Nitroso-S) and tetradecyldimethylbenzylammonium chloride (TDBA Cl) onto benzophenone in the pH range 5.0-6.0 from large volumes of aqueous solutions of various samples. After filtration, each solid mass consisting of a nickel complex and benzophenone was dissolved with 5 ml of dimethylformamide (DMF) and the metal was determined by atomic absorption spectrometry (AAS). About 0.6 microg of nickel could be concentrated from 200 ml of an aqueous sample, where its concentration was as low as 3.0 ng/ml. Eight replicate determinations of 2.5 microg/ml of nickel in the final DMF solution gave a mean absorbance of 0.112 with a relative standard deviation of 1.9%. The sensitivity for 1% absorption was 98 ng/ml. The interference of a number of anions and cations was studied and the developed optimized conditions were utilized for the trace determination of nickel in various alloys and biological samples.     

Determination of sulfonated azo dyes in river water samples by capillary zone electrophoresis

A method based on capillary zone electrophoresis coupled with photodiode-array detection has been developed to determine several sulfonated dyes, including a sulfonated dye (acid yellow 1), and the sulfonated azo dyes acid orange 7, acid orange 12, acid orange 52, acid red 26, acid red 27 and acid red 88. A CElect-FS75 CE column is used. The electrophoresis buffer contains a 1:5 dilution of 10 mM phosphoric acid and tetrabutylammonium hydroxide buffer (pH 11.5), and 25 mM of triethylamine, the final pH being 11.55. The detection limits for the seven dyes ranged from 0.1 to 4.53 microg/ml. Spiked river water samples (100 ml), containing different concentration levels (0.025-0.150 microg/ml) of the dyes were analyzed after acidification (pH 3) and pre-concentration in disposable SPE Oasis HLB, 1 ml cartridges.     

Determination and pharmacokinetic analysis of salvianolic acid B in rat blood and bile by microdialysis and liquid chromatography

Salvianolic acid B is an herbal ingredient isolated from Salvia miltiorrhiza. An in vivo microdialysis sampling method coupled to high-performance liquid chromatography has been developed for continuous monitoring of protein-unbound salvianolic acid B in rat blood and bile. Microdialysis probes were inserted into the jugular vein/right atrium and bile duct of Sprague-Dawley rats, and a dose of 100 mg/kg salvianolic acid B was then administered via the femoral vein. Dialysates were collected and directly injected into a liquid chromatographic system. Salvianolic acid B was eluted using a microbore reversed-phase ODS 5 microm (150 mm x 1 mm I.D.) column. Isocratic elution of salvianolic acid B was achieved within 10 min using the liquid chromatographic system. The chromatographic mobile phase consisted of acetonitrile-methanol-20 mM monosodium phosphoric acid (pH 3.5) (10:30:60, v/v/v) containing 0.1 mM 1-octanesulfonic acid with 0.05 ml/min. The wavelength of the UV detector was set at 290 nm. Salvianolic acid B in both blood and bile dialysates was adequately determined using the liquid chromatographic conditions described, although the blank bile pattern was more complex. The retention times of salvianolic acid B in rat blood and bile dialysates were found to be 7.2 min. Peak-areas of salvianolic acid B were linear (r2 > 0.995) over a concentration range of 0.1-50 microg/ml. In vivo recoveries of microdialysis probes of salvianolic acid B in rat blood and bile averaged 22 +/- 2% and 41 +/- 1%, respectively. This study indicates that salvianolic acid B undergoes hepatobiliary excretion.     

Simultaneous determination of sildenafil, vardenafil and tadalafil as forbidden components in natural dietary supplements for male sexual potency by high-performance liquid chromatography-electrospray ionization mass spectrometry

A high-performance liquid chromatographic method coupled with ultraviolet detection and electrospray ionization mass spectrometry (HPLC-UV-ESI-MS) was developed for simultaneous determination of banned additives-sildenafil, vardenafil and tadalafil in dietary supplements for male sexual potency. The separation was achieved on a C18 column with acetonitrile and aqueous solution (20 mmol ammonium acetate, 0.2% formic acid) as mobile phase at a flow rate of I ml/min with a linear gradient program. UV detection was at 292 nm. Identification of drugs was accomplished using ESI-MS. Good linearity between response (peak area) and concentration was found over a concentration range of 0.8-80 microg/ml for sildenafil; 2.25-225 microg/ml for vardenafil; and 1.1-110 microg/ml for tadalafil, with regression coefficient is better than 0.999. The recovery of the method ranged from 93.3 to 106.1%, and the relative standard deviation varied from 2.0 to 5.6% (n = 6). The method has been successfully applied to the analysis of practical samples of natural dietary supplements.     

Determination of tin by resin-suspension electrothermal atomic absorption spectrometry after enrichment as the complex with ammonium pyrrolidinedithiocarbamate.

A resin-phase extraction method has been optimized for the trace determination of tin(II) by ETAAS. Tin(II) was extracted on a finely divided anion exchange resin as the complex with ammonium pyrrolidinedithiocarbamate (APDC). The resin was collected on a membrane filter and then dispersed in 1.00 ml of 1 mol l(-1) nitric acid containing 100 microg of Pd(II) and 60 microg of Ni(II). The resulting resin suspension was subjected to GFAAS. The proposed method was applied to the determination of tin(II) in hydrochloric acid.     

Extractive photometric determination of gold(III) with 1-(2',4',6'-trichlorophenyl)-4,4,6-trimethyl-(1H,4H)-2-pyrimidinethiol in presence of tri-iso-octylamine

Tervalent gold was determined spectrophotometrically as its anionic 1:4 gold-thiol complex extracted into chloroform from aqueous acidic medium (1.5M sulphuric acid) in the presence of tri-iso-octylamine. The complex exhibits maximum absorption at 480 nm (molar absorptivity 4.60 x 10(3) l.mole(-1).cm(-1)) and Beer's law is obeyed in the concentration range 5-50 microg of gold(III) per ml. The relative standard deviation and relative error, calculated from ten determinations of solutions containing 15 microg of gold(III) per ml were 1.0% and 0.8%. The method is simple, selective and reproducible. It permits separation of gold(III) from associated elements and its determination in synthetic mixtures.     

Characterization of the interactions in polymer-filler systems by inverse gas chromatography

Chromium is a primary drinking water contaminant in the USA with hexavalent chromium, Cr(VI), being the most toxic form of the metal. As a required step in developing a revised state drinking water standard for chromium, the California Department of Health Services recently issued a new Public Health Goal (PHG) of 2.5 microg/l for total chromium and 0.2 microg/l for Cr(VI). Hexavalent chromium can be determined (as chromate) by ion chromatography, as described in US Evironmental Protection Agency Method 218.6; however, the method as originally published does not allow sufficient sensitivity for analysis at the California PHG level of 0.2 microg/l. Modification of the conditions described in Method 218.6, including the use of a lower eluent flow-rate, larger reaction coil, and larger injection volume, significantly increases the method sensitivity. The modified method, which uses IonPac NG1 and AS7 guard and analytical columns, an eluent of 250 mM ammonium sulfate-100 mM ammonium hydroxide operated at 1.0 ml/min, a 1000 microl injection volume, and postcolumn reaction with 2 mM diphenylcarbazide-10% methanol-0.5 M sulfuric acid (using a 750 microl reaction coil) followed by UV-Vis detection at 530 nm, permits a method detection limit for chromate of 0.02 microg/l. This results in a quantitation limit of 0.06 microg/l, which is more than sufficient for analysis at the California PHG level. Calibration is linear over the range of 0.1-10 microg/l and quantitative recoveries (>80%) are obtained for chromate spiked at 0.2 microg/l in drinking water. The modified method provides acceptable performance, in terms of chromate peak shape and recovery, in the presence of up to 1000 mg/l chloride or 2000 mg/l sulfate.     

Kinetic spectrofluorimetric determination of trace ascorbic acid based on its inhibition on the oxidation of pyronine Y by nitrite

A highly sensitive spectrofluorimetric method is proposed for the determination of trace amount of ascorbic acid using a new indication. The method is based on the inhibition of ascorbic acid on the oxidation of pyronine Y (PRY) by nitrite. The detection limit for ascorbic acid is 0.012 microg ml(-1), the linear range of the determination is 0.02-0.36 microg ml(-1). Analytical parameters, such as reagent concentration, pH, reaction temperature and time, were optimized. The relative standard deviations of eleven replication determinations of 0.12 and 0.24 microg ml(-1) ascorbic acid were 1.4 and 0.72%, respectively. This method has been used to determine ascorbic acid in pharmaceuticals, vegetables, fruits and soft drink with satisfactory results.     

Kinetic spectrophotometric determination of ascorbic acid by reduction of toluidine blue

A simple kinetic method is described for the determination of ascorbic acid. The procedure is based on the reduction of toluidine blue with ascorbic acid. The rate of reaction is followed by measuring the decrease in absorbance of toluidine blue (lambda(max) = 600 nm) as a result of its decolorization upon reduction by ascorbic acid. Ascorbic acid in the range of 3-35 microg/ml was determined using slope and fixed time methods of analysis, while the variable time method allowed the determination of 5-50 microg/ml of ascorbic acid. The percent relative standard deviation of the method varied from 0.78 to 1.32% depending on the kinetic method used. The high sensitivity of the method also allows determination of low levels of ascorbic acid in some fruits and vegetables such as dew melon, water melon, parsley and coriander.     

Determination of aristolochic acid in botanicals and dietary supplements by liquid chromatography with ultraviolet detection and by liquid chromatography/mass spectrometry: single laboratory validation confirmation

The presence of aristolochic acid in some dietary supplements is a concern to regulators and consumers. A method has been developed, by initially using a reference method as a guide, during single laboratory validation (SLV) for the determination of aristolochic acid I, also known as aristolochic acid A, in botanical species and dietary supplements at concentrations of approximately 2 to 32 microg/g. Higher levels were determined by dilution to fit the standard curve. Through the SLV, the method was optimized for quantification by liquid chromatography with ultraviolet detection (LC-UV) and LC/mass spectrometry (MS) confirmation. The test samples were extracted with organic solvent and water, then injected on a reverse phase LC column. Quantification was achieved with linear regression using a laboratory automation system. The SLV study included systematically optimizing the LC-UV method with regard to test sample size, fine grinding of solids, and solvent extraction efficiency. These parameters were varied in increments (and in separate optimization studies), in order to ensure that each parameter was individually studied; the test results include corresponding tables of parameter variations. In addition, the chromatographic conditions were optimized with respect to injection volume and detection wavelength. Precision studies produced overall relative standard deviation values from 2.44 up to 8.26% for aristolochic acid I. Mean recoveries were between 100 and 103% at the 2 microg/g level, between 102 and 103% at the 10 microg/g level, and 104% at the 30 microg/g level.     

Separation and determination of abused drugs clenbuterol and salbutamol from complex extractants in swine feed by capillary zone electrophoresis with simple pretreatment

A simple but efficient capillary zone electrophoresis (CZE) method was developed for the fast separation and determination of two misused beta2-agonists clenbuterol (CLB) and salbutamol (SAL) from complex background extractants existing in swine feed samples. The proper experimental conditions were achieved as 20.0 mmol/l pH 10.5 Na(2)HPO(4)-NaOH buffer, 20 kV applied voltage, fused-silica capillary of 60.5 cm x 75 microm i.d. (50 cm to detector). Under the proper conditions, the two abused drugs can be online isolated from the complex extractants and the separation between CLB and SAL is good, all of the target analytes can be detected within 4.5 min. The linear response of CLB and SAL concentration ranges from 2.0 to 100.0 microg/ml with high correlation coefficient (R(2)=0.9990) and (R(2)=0.9986), respectively. Limit of detection (LOD) and limit of quantification (LOQ) was calculated to be 0.95 and 3.17 microg/ml for CLB, 1.07 and 3.57 microg/ml for SAL. The precision values (expressed as R.S.D.) of intra- and inter-day were 1.24-2.36% and 0.90-3.85% for CLB, 0.47-1.64% and 0.91-3.46% for SAL. Recoveries spiked at levels 5.0, 25.0, 80.0 microg/ml ranged between 93.30% and 104.33% with R.S.D. less than 5%. Finally, the developed method has been applied to the analysis of real swine feed samples and has achieved satisfactory results.     

Rapid determination of underivatized pyroglutamic acid, glutamic acid, glutamine and other relevant amino acids in fermentation media by LC-MS-MS

Determination of amino acids in a complex matrix without derivatization is advantageous, however, difficulties are found in both the detection and the separation of those compounds. In this study, a rapid and reliable LC-MS-MS method for the quantitation of underivatized amino acids in exocellular media was established. Injections were made directly after centrifugation of the samples, without further preparation. The separation of seven underivatized amino acids was achieved on a reversed-phase C18 column with pentadecafluorooctanoic acid as a volatile ion-pair reagent, and the specific detection of most amino acids was achieved by MS-MS of the specific transitions [M + H]+-->[M + H - 46]+. The calibration curves of all analytes were linear over the range of 1.0-1000 microg ml(-1) and the detection limits ranged from 0.1 to 5 ng ml(-1), with an injection volume of 20 microl. The inter-day and intra-day precisions ranged from 2.6 to 5.7% and 4.8 to 8.2%, respectively; the mean recoveries of the seven analytes were 81-104%, 91-107% and 93-101% respectively at the spiked level of 10, 40 and 200 microg ml(-1). A large number of fermentation samples were analysed using this method. The technique is simple, rapid, selective and sensitive, and shows potential for the high-throughput quantitation of amino acids from other biological matrices.     

Determination of quinolizidine alkaloids in traditional Chinese herbal drugs by nonaqueous capillary electrophoresis.

A rapid method for the determination of quinolizidine alkaloids by nonaqueous capillary electrophoresis was developed. A total of 10 alkaloids (matrine, sophocarpine, oxymatrine, oxysophocarpine, sophoridine, cytisine, sophoramine, aloperine, lehmannine and dauricine) could be easily separated within 18 min. A running buffer composed of 50 mM ammonium acetate, 10% tetrahydrofuran and 0.5% acetic acid in methanol was found to be the most suitable for this separation. Five of these alkaloids were selected for further studies. The linear calibration ranges were 2.51-50.1 microg/ml for sophoridine and sophocarpine, 2.71-54.2 microg/ml for matrine, 3.30-65.9 microg/ml for oxymatrine, and 3.10-62.0 microg/ml for oxysophocarpine. The recovery of the five alkaloids was 98.0-101.3% with relative standard deviations from 1.03 to 2.68% (n=5). The limits of detection for all 10 alkaloids were over the range 0.93-2.31 microg/ml. The method was successfully applied to the phytochemical analysis of alkaloid extracts from three commonly used traditional Chinese herbal drugs: Sophora flavescens Ait. (Kushen), S. alopecuroides L. (Kudouzi or Kugancao) and S. tonkinensis Gapnep (Shandougen).