Flow injection spectrophotometric determination of lactic acid in skimmed milk based on a photochemical reaction

A spectrophotometric method for the determination of lactic acid in milk samples based on the use of a photochemical reaction carried out in a Flow Injection System is proposed. Determination is based on the reaction between lactic acid and Fe(III), which is reduced to Fe(II) in the presence of UV light, being the latter made to react with o-phenanthroline. The complex formed between Fe(II) and o-phenanthroline, Fe(o-phen)(3)(2+) (ferroin) is a coloured compound and it can be spectrophotometrically monitored at 512 nm. The method shows a linear range between 0.5 and 50 mug ml(-1) with a limit of detection of 0.16 mug ml(-1). The precision was +/-2.15 expressed as relative standard deviation (n=11) and the sample throughput of 30 samples h(-1). Also non-linear adjustments have been made and validated by ANOVA. The proposed method has been applied to the determination of lactic acid in both synthetic and milk samples.     

Determination of tazobactam and piperacillin in human plasma, serum, bile and urine by gradient elution reversed-phase high-performance liquid chromatography

A gradient elution high-performance liquid chromatographic method is described for the analysis of the beta-lactamase inhibitor tazobactam (YTR-830H) and a semi-synthetic parenteral penicillin, piperacillin, in human plasma, serum, bile and urine. The assay for plasma, serum and bile involves deproteinization with acetonitrile and the removal of lipids with dichloromethane; urine is diluted with buffer. Separation and quantitation are achieved using a mobile phase based on ion-suppression chromatography on a C18 reversed-phase column with ultraviolet detection at 220 nm. The limit of quantitation for both compounds is 1.0 microgram/ml in plasma, serum and bile using a 0.2-ml sample and 50.0 micrograms/ml in urine using a 0.1-ml sample. The method has been validated by preparing and analyzing a series of fortified samples (range 1.0-200 micrograms/ml for each compound in plasma, serum and bile and 50.0-10,000 micrograms/ml for each compound in urine). Excellent linearity, accuracy, precision and recovery were obtained. The method was not interfered with by other endogenous components, nor by other commonly administered antibiotics such as amoxicillin, mezlocillin, cefometazole and cefotaxime. The assay has been successfully applied to the analysis of samples from pharmacokinetic studies in man and animals.     

Long-path photometry in clinical analysis : I. The determination of chromium using diphenylcarbazide

The diphenylcarbazide method for determining chromium in biological material was reinvestigated and scaled down to require only 0.2 ml (or 0.2 g) of sample. The samples are digested at 300 °C within 1 hr by a mixture of concentrated sulfuric, nitric, and perchloric acids in a volume ratio 75:15:10. Chromium is oxidized to dichromate with permanganate and the excess of the latter is destroyed by azide. The color reagent is added and the absorbance is measured in a 20-cm long-path microcell requiring less than 0.3 ml of solution. Results on serum pools shown a standard deviation of about ±1.1 ng of Cr/ml of serum for contents in the range of 25 ng/ml. The results compare very favorable with those obtained by flameless atomic absorption. The determinations can be made by using a simple, dedicated photometer consisting of the long-path cell with a green LED attached to one window and a phototransistor to the other and needing only very simple circuits and low battery power.     

酶荧光毛细管分析法测定微量血清中乳酸脱氢酶活性

基于丙酮酸/还原型辅酶I/乳酸脱氢酶(LDH)/乳酸/氧化型辅酶I荧光猝灭体系和荧光毛细管分析技术,建立了可用于微量样品中LDH酶活性测定的方法。优化的测定条件为:激发及发射波长分别为350和460nm;测定温度为25℃;酸度为pH 6.5;NADH浓度为300μmol/L;丙酮酸浓度为1.2mmol/L。本方法的测定范围为50～1500IU/L,检出限为30IU/L,相对标准偏差2.1%～2.2%(n=10),回收率在96.4%～105%范围内。本方法操作简单,每次测定仅需样品2.0μL、试剂18.0μL,分析速度约为30样/h,利用本方法测定了微量血清中LDH的活性。     

A simple and fast liquid–liquid extraction method for the determination of 2,2′,4,4′,5,5′-hexachlorobiphenyl (CB-153) and 1,1-dichloro-2,2-bis(p-chlorophenyl)-ethylene (p,p′-DDE) from human serum for epidemiological studies on type 2 diabetes

A simple and fast method is presented to be used for example in studies on the relationship between serum levels of persistent organic pollutants and type 2 diabetes mellitus. Method is based on liquid–liquid extraction and gas chromatography coupled with high-resolution mass spectrometry. In the sample pre-treatment special attention was paid to minimize the number of sample manipulation steps and the amounts of organic solvents needed. Compounds analyzed were 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB-153) and 1,1-dichloro-2,2-bis (p-chlorophenyl)-ethylene (p,p′-DDE), the major metabolite of DDT. The method included extraction and cleanup of 0.2 ml of serum in a single test tube and subsequent analysis of the extract from 0.2 ml final volume. Validation was conducted to explore the performance of the method. The limits of detection for p,p′-DDE and PCB-153 based on the standard deviation of the blank samples were 4.3 and 3.1 pg/ml, respectively. Repeatability was less than 2.5% at three concentration levels tested and recovery from Certified Reference Material SRM 1589a was 84% for p,p′-DDE and 87% for PCB-153 of the certified values, respectively. Serum samples from the AMAP intercalibration round 2008-2 were also analyzed, and results were 101–116% of the assigned values. The presented method was used for an epidemiological study with more than 700 serum samples from a type 2 diabetes cohort from Sweden.     

Amperometric determination of lactate dehydrogenase based on a carbon fiber microcylinder electrode modified covalently with Toluidine Blue O by acylation

A method has been developed for the modification of a carbon fiber microcylinder electrode with acylation. The stability and surface coverage of the Toluidine Blue O-modified microelectrode were studied by cyclic voltammetry. The modified electrode showed significant activity for the electrocatalytic oxidation of NADH in pH 6.8-7.8 solution. The catalytic current increased linearly with increasing concentration of NADH from 4.0 x 10(-5) to 1.5 x 10(-3) M. A simple amperometric determination based on electrochemical detection of NADH produced from the enzymatic reaction of lactate with NAD(+) under the catalysic effect of lactate dehydrogenase (LDH) is reported. The experimental factors which had primary influence on the analytical performance were studied. The sensor had a linear response over a range of LDH concentrations from 5.0 U l(-1) to 200 U l(-1) at -0.2 V vs. SCE under optimum conditions. A satisfactory result was obtained for the determination of LDH in clinical blood samples.     

Dispersive solid phase micro-extraction of dopamine from human serum using a nano-structured Ni-Al layered double hydroxide, and its direct determination by spectrofluorometry

We report on a simple method for dispersive solid-phase micro-extraction of dopamine (DA) from human serum. It is based on a layered double hydroxide (LDH) of nickel-aluminum, which acts as the extractant. DA is extracted at pH 8 using the LDH sol solution as a dispersed solid-phase extractor. The extracted DA is quantified by spectrofluorometry at 285?nm excitation and 315?nm emission wavelengths. A comparison of the fluorescence of DA in bulk solution and that of DA intercalated in the LDH revealed a nearly 5-fold improvement in intensity. Factors such as pH, concentration of LDH in the sol solution, and temperature were optimized. Under these conditions, the limit of detection is 0.015?μg?L^?1. The inter- and intra-day relative standard deviations for six replicate determinations of 1?μg?L^?1 DA were 1.7 and 1.1?%, respectively. The method was successfully applied to the determination of DA in human serum samples.

Determination of ivermectin B(1a) in animal plasma by liquid chromatography combined with electrospray ionization mass spectrometry

A novel, sensitive and specific method for the quantitative determination of ivermectin B(1a) in animal plasma using liquid chromatography combined with positive electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) is presented. Abamectin was used as the internal standard. Extraction of the samples was performed with a deproteinization step using acetonitrile. Chromatographic separation was achieved on a Nucleosil ODS 5 microm column, using gradient elution with 0.2% (v/v) acetic acid in water and 0.2% (v/v) acetic acid in acetonitrile. The method was validated according to the requirements defined by the European Community. Calibration curves using plasma fortified between 1 and 100 ng ml(-1) showed a good linear correlation (r > or = 0.9989, goodness-of-fit coefficient < or =8.1%). The trueness at 2 and 25 ng ml(-1) (n = 6) was +4.2 and -17.1%, respectively. The trueness and between-run precision for the analysis of quality control samples at 25 ng ml(-1) was -4.0 and 11.0%, respectively (n = 16). The limit of quantification of the method was 1.0 ng ml(-1), for which the trueness and precision also fell within acceptable limits. Using a signal-to-noise ratio of 3 : 1, the limit of detection was calculated to be 0.2 ng ml(-1). The specificity was demonstrated with respect to ivermectin B(1b).The method was successfully used for the quantitative determination of ivermectin B(1a) in plasma samples from treated bovines, demonstrating the usefulness of the developed method for application in the field of pharmacokinetics.     

Determination of oxytetracycline in blood serum by high-performance liquid chromatography with direct injection.

A direct injection analysis by high-performance liquid chromatography has been developed for oxytetracycline in serum of animals and fish. A Hisep shielded hydrophobic phase column (15 cm x 4.6 mm I.D.) and a mobile phase of methanol-0.2 M oxalic acid (10:90, v/v, pH 7.0) with ultraviolet detection at 360 nm were used. The standard calibration curves in serum of chicken, hog, cattle and rainbow trout were linear over the range 0.1-20 micrograms/ml. The recoveries of oxytetracycline from all serum samples determined at two different concentrations (0.5 and 2.0 micrograms/ml) were 88-103%. The detection limit was 0.05 micrograms/ml for every serum sample.     

The promotion effect of titania nanoparticles on the direct electrochemistry of lactate dehydrogenase sol-gel modified gold electrode

The promotion effect of titania nanoparticles (nano-TiO(2)) on the direct electron transfer between lactate dehydrogenase (LDH) and the silica sol-gel modified gold electrode was investigated by adding nano-TiO(2) (50 nm) in the modification process. This nano-TiO(2)-LDH electrode showed a pair of quasi-reversible cyclic voltammetry peaks with the formal potential of 70 mV (vs. SCE). Compared to the previous result of LDH modified electrode with only an irreversible cathodic peak, an anodic peak appeared and the cathodic peak potential shifted to the positive direction on this nano-TiO(2)-LDH electrode, which demonstrated that the direct electrochemistry of LDH was enhanced by nano-TiO(2). We supposed that the direct electrochemistry of LDH may be due to the redox reaction of some electroactive amino acids in the LDH molecule. The surface morphologies of electrodes characterized by SEM indicated that LDH was successfully immobilized on the sol-gel matrix and also had some interactions with nano-TiO(2). This electrode can be used as a biosensor for the determination of lactic acid. The calibration range of lactic acid was from 1.0 to 20 micromolL(-1) and the detection limit was 0.4 micromolL(-1). Meanwhile, the small K(m)(app) value (2.2 micromolL(-1)) suggested that LDH possessed high enzymatic activity and good affinity to lactic acid owing to the promotion effect of nano-TiO(2).     

微量乳酸脱氢酶毛细管电泳在线反应电化学检测方法的研究

以乳酸脱氢酶催化乳酸与NAD⁺反应生成丙酮酸与NADH和安培法检测NADH为基础,利用电泳中介微分析(EMMA)技术,研究了超微量乳酸脱氢酶的毛细管电泳在线反应的电化学检测方法,并从理论上对EMMA电泳图中的平台宽度和高度作了初步探讨.结果表明,在EMMA的恒高压和零高压两种模式下,使用直径为150μm和束状碳纤维电极,在+0.8V检测电位下,对LDH活性检测灵敏度分别为1.1nU和0.6nU;所导出的平台高度、宽度与实验条件的关系式对提高毛细管电泳分离效率和检测灵敏度有一定指导意义.     

Immobilization enzyme fluorescence capillary analysis for determination of lactic acid

A new method for the determination of lactic acid based on the immobilization enzyme fluorescence capillary analysis (IE-FCA) was proposed. Lactic dehydrogenase (LDH) was immobilized on inner surface of a capillary with glutaraldehyde, and an immobilized enzyme lactate capillary bioreactor (IE-LCBR) was formed for the determination of lactic acid. After nicotinamide adenine dinucleotide (NAD⁺) is mixed with lactic acid solution, it was sucked into the IE-LCBR and was detected at λ_ex 353 nm/λ_em 466 nm. Optimized conditions are as follows: the temperature is 38 °C; the reaction time is 15 min; the concentrations of Tris buffer (pH 8.8) and NAD⁺ are 0.1 mol L⁻¹ and 4 mmol L⁻¹, respectively; the concentration of LDH used for immobilization is 15 kU L⁻¹. The concentration of lactic acid is directly proportional to the fluorescence intensity measured from 0.50 to 2.0 mmol L⁻¹; and the analytical recovery of added lactic acid was 99–105%. The minimum detection limit of the method is 0.40 mmol L⁻¹ and sensitivity of the IE-CBR is 4.6 F mmol⁻¹ L⁻¹ lactate. Its relative standard deviation (R.S.D.) is ≤2.0%. This IE-FCA method was employed for determination of lactate in milk drink.     

Determination of anethole in serum samples by headspace solid-phase microextraction-gas chromatography-mass spectrometry for congener analysis

A rapid headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) method has been developed for the determination of anethole in serum samples. Anethole is a characteristic marker for the consumption of aniseed spirits. This method enabled the detection of anethole with a limit of detection (LoD) of 3.6 ng/ml and a limit of quantification (LoQ) of 5.3 ng/ml in serum samples with a good degree of precision intraday (2.8%) and interday (4.5%). Experiments were conducted with one volunteer, in which the subject consumed the alcoholic drink ouzo on 3 different days under controlled conditions. At defined intervals, blood samples were taken from the subject. Using these blood samples, the concentration-time profiles for anethole were determined. In blood samples taken from 50 drivers who claimed to have consumed drinks containing anethole (ouzo, raki and the German aniseed liqueur "Küstennebel") before the taking of the blood sample, anethole was detected in the serum in concentrations of between 5.4 and 17.6 ng/ml in 10 cases. This is the first report describing the qualitative and quantitative determination of a beverage-characteristic aroma compound - in this case anethole - in serum samples after consumption of alcoholic beverages.     

Determination of cetirizine in serum using reversed-phase high-performance liquid chromatography with ultraviolet spectrophotometric detection.

A method using reversed-phase high-performance liquid chromatography with ultraviolet detection for the determination of ceterizine in serum is described. The method is sensitive down to 50 ng/ml (250-microliter loop). Sample preparation involves only serum deproteination with perchloric acid and injection of the centrifuged supernatant. Elution is at pH 2.5 with acetonitrile-methanol-0.05 M phosphate buffer (33:9:58, v/v) on a 25 cm x 4.6 mm I.D. Spherisorb S5 ODS2 column. Detection is at 211 nm, its lambda max. For levels above 300 ng/ml the serum sample size is 100 microliter and a 200-microliter sample is necessary for concentrations less than 300 ng/ml. At the 2 micrograms/ml concentration the intra-assay relative standard deviation is better than 2.2%, whilst the inter-assay deviation is 2.6% over eight samples. At 200 ng/ml the intra-assay relative standard deviation is 6% over seven samples. Detector response is linear from 100 ng/ml to 10 micrograms/ml (100-microliter loop).     

Simultaneous determination of nefiracetam and its metabolites by high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatographic assay was developed to simultaneously quantitate nefiracetam (NEF), a novel nootropic agent, and its three known oxidized metabolites (N-[(2,6-dimethylphenylcarbamoyl)methyl]succinamic acid (5-COOH-NEF), 4-hydroxy-NEF and 5-hydroxy-NEF) in human serum and urine. The quantitative procedure was based on solid-phase extraction with Sep-Pak C18 and ultraviolet detection at 210 nm. The calibration curves of NEF and the metabolites were linear over a wide range of concentrations (0.5-21.5 nmol/ml for NEF and 0.4-9.5 nmol/ml for metabolites in serum and 4-86 nmol/ml for NEF and 8-190 nmol/ml for metabolites in urine). Intra- and inter-day assay coefficients of variation for the compounds were less than 10%. The limit of detection was 0.1 nmol/ml for NEF, 5-COOH-NEF and 4-hydroxy-NEF, and 0.2 nmol/ml for 5-hydroxy-NEF in both serum and urine. This method is applicable for the determination of NEF and its metabolites in human serum and urine with satisfactory accuracy and precision.     

Sensitive method for the determination of the antiarrhythmic drug detajmium in serum by solid-phase extraction and high-performance liquid chromatography with fluorimetric detection.

The objective of this study was to develop a very sensitive and selective method for the determination of detajmium (4-3-diethylamino-2-hydroxypropyl-ajmaline), a sodium-channel-blocking drug with antiarrhythmic properties, in serum. A high-performance liquid chromatography (HPLC) method with solid-phase extraction and fluorimetric detection has been applied. Serum samples were diluted with phosphate buffer (pH 3.5) and the extraction of detajmium and ajmaline, which was used as an internal standard, was carried out with Oasis cartridges (Waters). The chromatographic separation was performed on a RP18 column. The limit of quantification for serum samples of detajmium was 1 ng/ml with good reproducibility (R.S.D. < 15%) and a linear response from 1 to 200 ng/ml. The described method is highly sensitive and specific for the determination of detajmium in serum of patients and volunteers.     

High-performance liquid chromatographic method for the determination of a potential memory-enhancing compound (CL 275,838) and its desbenzyl metabolite in rat plasma or serum.

A rapid, selective, precise reversed-phase liquid chromatographic method has been developed for the determination of a potential memory-enhancing agent (CL 275,838) and its main metabolite (CL 286,527) in plasma and serum. The procedure includes isolation of compounds from proteins precipitated with acetonitrile, subsequent resolution by reversed-phase (Whatman Partisphere C8) high-performance liquid chromatography and ultraviolet detection. The assay was linear over the range 0.12-1.25 micrograms/ml of plasma or serum. The detection limit was 0.12 micrograms/ml, using 0.2 ml of plasma or serum. Intra- and inter-day validation studies indicated an acceptable precision and reproducibility of the method within the concentration range investigated, the overall coefficient of variation being less than 10%. The method is currently applied in support of pharmacological and toxicity studies of the compound in rodents.     

High-performance liquid chromatographic methods for the determination of the penems SCH 29482 and FCE 22101 in human serum and urine.

High-performance liquid chromatographic methods have been developed for the determination of two 6-(1-hydroxyethyl)penems, SCH 29482 (I) and FCE 22101 (II), in serum and urine. Serum samples were combined with an equal volume of methanol to remove proteins and, after centrifugation, an aliquot of the supernatant was analysed by ion-pair chromatography on a reversed-phase C18 column with hexadecyltrimethylammonium bromide as the ion-pairing agent. The compounds were detected by their ultraviolet absorbance at 305 nm for II and 322 nm for I. Urine samples were diluted, filtered and analysed by the same chromatographic procedure. At concentrations of 1-500 micrograms/ml of each compound, the within- and between-day precisions were 1.8-3.6 and 2.6-5.1%, respectively. The detection limit was 0.2 micrograms/ml for I and 0.3 micrograms/ml for II.     

Spectrofluorimetric determination of drugs containing active methylene group using N1-methyl nicotinamide chloride as a fluorigenic agent

A spectrofluorimetric method was described for the determination of drugs containing active methylene groups adjacent to carbonyl groups. The method was applied successfully to the determination of three life saving cardiovascular drugs, with narrow therapeutic indices: pentoxifylline (I), propafenone hydrochloride (II) and acebutolol hydrochloride (III), in laboratory-prepared mixtures, in commercial tablets and in plasma samples. The method involved the reaction of each of the tested drugs with N1-methyl nicotinamide chloride (NMNCl) in the presence of alkali, followed by addition of formic acid, where highly fluorescent reaction products were produced. The produced fluorescence were measured quantitatively at 472 nm (lambdaex 352 nm), 409 nm (lambdaex 310 nm) and 451 nm (lambdaex 266 nm) for (I), (II), and (III) respectively. The method was linear over concentration ranges of 10-1000 microg/ml , 0.2-12 microg/ml and 0.08-10 microg/ml in standard solutions for (I), (II), and (III) respectively. In spiked human plasma samples, calibration graphs were linear over concentration ranges of 20-1000 microg/ml, 0.2-15 microg/ml and 0.08-10 microg/ml for (I), (II), and (III) respectively. The method showed good accuracy, specificity and precision in both laboratory-prepared mixtures and spiked human plasma samples. The proposed method is simple, with low instrumentation requirements, suitable for quality control application, bioavailability and bioequivalency studies.     

Headspace solid-phase microextraction-gas chromatography-mass spectrometry for the quantitative determination of the characteristic flavouring agent eugenol in serum samples after enzymatic cleavage to validate post-offence alcohol drinking claims

A rapid headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) method has been developed for the determination of eugenol in serum samples after enzymatic cleavage. Eugenol is a characteristic marker for the consumption of certain alcoholic beverages including some digestif bitters and herbal liqueurs as well as wood-cask-aged spirits. This method enables the detection of eugenol with a limit of detection (LOD) of 3.2 ng/ml and a limit of quantification (LOQ) of 4.8 ng/ml in serum samples with excellent precision (5.3% intraday, 6.9% interday) and linearity (correlation coefficient R2=0.992). Our findings confirm that eugenol undergoes a rapid phase II metabolism as it occurs completely conjugated as eugenol glucuronide in serum. Free eugenol was not detectable in any of our samples, which necessitated enzymatic cleavage with beta-glucuronidase prior to HS-SPME sampling. In vivo experiments were conducted with a volunteer, who consumed a digestif bitter beverage on three different days under controlled conditions. At defined intervals, blood samples were taken from the subject. Using these blood samples, concentration/time profiles for serum eugenol glucuronide were determined. A rapid resorption leads to a peak eugenol glucuronide concentration directly after drinking (up to 1742 ng/ml if 78 mg of eugenol are ingested) followed by a decrease during the next 3h. Blood samples were also taken from 20 drivers claiming to have consumed drinks containing eugenol. In five of the samples, eugenol glucuronide was detected at serum concentrations ranging from 12.1 to 172.3 ng/ml. These test results, in particular, confirm that the analysis of volatile compounds can be useful in forensic toxicology for the verification of post-offence alcohol consumption claims.