Comparison of Reagents for Lipid and Phospholipid Detection and Densitometric Quantitation on Silica Gel and C18 Reversed Phase Thin Layers

Abstract

A variety of chromogenic and fluorogenic detection reagents were evaluated for detection and densitometric quantification of lipids and phospholipids on silica gel and chemically bonded octadecylsilane reversed phase thin layer plates. Phosphosphomolybdic acid (PMA) was found to be the most generally favorable reagent, followed by cupric acetate and ethanolic sulfuric acid. The quantification of lipids in blood serum by chemically bonded RP-TLC was demonstrated using PMA with direct sample application to preadsorbent plates.     

Isclation of Lecithin and Sphingomyelin from Lipid Mixtures for Quantitation by Infrared Spectroscopy

Abstract

A new procedure for the determination of lecithin and sphingomyelin in amniotic fluid is described. rapid column chromatography in the sample preparation steps with computer-assisted infrared spectroscopy in the final determination. Lipids are extracted from amniotic fluid with a chloroform/methanol solvent. The organic layer is separated and evaporated to dryness. The residue is The new method couples the use of simple, redissolved i n chloroforn and Fassed through a hydroxyapatite column to remove interfering constituents. The complete isolation cflecithin and sphingomyelin fror other li Fidsis confimec by both infrared spectroscopy and thin layer chronatcgrayhy.     

An Extraction Method to Quantitate Serotonin in Human Serum,Amniotic Fluid and Urine Samples by HPLC Using 6-Hydroxy Tryptamine as Internal Standard

Abstract

A method for extracting serotonin (5-HT) from human serum, amniotic fluid and urine samples is described. A mixture of sample, 6-hydroxy tryptamine (6-HT) as internal standard and sodium carbonate is extracted with ethyl acetate. The ethyl acetate layer is mixed with 200 ul of triethylamine phosphate buffer (pH 3.0) used for mobile phase and centrifuged. An aliquot of the aqueous layer was used for HPLC quantitation with amperometric detection. With this method 30 samples can be assayed in 6 hours.     

Derivative Spectrophotometry: Determination of Bilirubin in Amniotic Fluid

Abstract

Spectrophotometric analysis presents very often the disadvantage of interferences by substances which can absorb near the substance of interest. The use of derivative spectra allows to overcome this difficulty. In this paper we describe a method for determination of bilirubin in amniotic fluid based on use of derivative spectrophotometry. This method presents some advantages of accuracy and quickness in comparison with the ones normally used.     

Analysis of Lipid Classes by HPLC with the Evaporative Light Scattering Detector

Abstract

The evaporative light scattering detector enables the detection and quantitation of all relatively non-volatile lipids. The mixtures of polar and non-polar lipids were separated in one run, in 20 to 25 minutes on Silica Si-100 columns, using consecutive gradients of pentane to diethylether, to chloroform, to methanol containing a large concentration of ammonia.

The flexibility of the method is illustrated by the change in elution patterns following the treatment of the packing material by ammonia. For example, the elution order of phosphatidyl inositol and phosphatidyl choline is reversed and the separation of the former compound from phosphatidyl serine, which is generally difficult, is now accomplished readily.

The weak dependence of the detector sensitivity on the nature of the analytes permits an easy quantitation, as illustrated by the results of the analyses of lipid classes in blood serum, amniotic fluid, beef brain and other natural samples.

The method is particularly useful for the analysis of lecithin and sphingomyelin in the amniotic fluid. The ratio of the concentration of these two compounds is an indicator of lung maturity and could permit an early diagnosis of the respiratory stress syndrome of neonates.     

Direct and Rapid Electrochemical Determination of Total Surfactants in Amniotic Fluid for Assessment of Foetal Lung Maturity

Abstract

The characteristic response in the polarographic current-time curves, observed by the inhibition of the oxygen reduction process by adsorption of phospholipids at the mercury-solution interface, is used for the direct and rapid analysis of surfactants content in amniotic fluid samples. Good correlations were obtained between the described method and two well established routine methods used for estimation of foetal lung maturity in prenatal diagnosis of the respiratory distress syndrome.     

Prenatal diagnosis of methylmalonic aciduria by measuring methylmalonic acid in dried amniotic fluid on filter paper using gas chromatography-mass spectrometry

Methylmalonic aciduria is a common inherited metabolic disorder. Methylmalonic acid (MMA), a key indicator of methylmalonic aciduria, increases in the amniotic fluid of affected fetuses. For prenatal diagnosis, the MMA in amniotic fluid can be measured by stable-isotope dilution gas chromatography-mass spectrometry. Here, we quantified the MMA in cell-free amniotic fluid samples that had been dried on filter paper and transported at ambient temperatures, and compared the results with data obtained from the original amniotic fluid. Our results indicated that the filter paper method was reproducible and accurate enough to be applied to clinical analysis. We also used the filter paper method to screen at-risk fetuses and obtained a clear diagnosis in each case. We conclude that our method enables the prenatal diagnosis of methylmalonic aciduria using practical procedures and a simplified method for transporting the samples.     

Tryptophan Determination in Tissue Homogenates and Biological Fluids Using HPLC-EC

Abstract

A method for quantitating tryptophan in tissue homogenates and biological fluids using 6-hydroxy-tryptamine as internal standard is described. Tryptophan in CSF and free tryptophan in serum or plasma can be quantitated by directly injecting CSF or ultrafiltrate into the HPLC system with the internal standard. Serum, plasma, amniotic fluid, and saliva are deproteinized by addition of equal volume of acetonitrile. Urine samples are processed through Biorex-70 columns. Cross validation was done by comparing the values of column procedures with direct injection method.     

Amniotic fluid uric acid levels determined by reversed-phase liquid chromatography with spectrophotometric and electrochemical detection

A rapid and precise, reversed-phase high-performance liquid chromatography method for amniotic fluid uric acid is described. Detection of uric acid and other naturally occurring constitutents is based on UV absorption at a wavelength of 280 nm and direct electrochemical oxidation at a potential of +0.800 V. The total analysis time is short (20 min) and the assay requires only filtration of the samples. Uric acid levels were determined in 14 samples of amniotic fluid obtained during the 15th to 24th week of gestation. Results ranged from 0.897 to 4.39 mg per 100 ml of amniotic fluid.     

Effect of Poly(methacrylic acid) on Photoinduced Processes with Cyanine Dyes in Aqueous Solutions

The cyanine dyes thiamonomethinecyanine (I), thiacarbocyanine (II), and thiadicarbocyanine (III) in an aqueous poly(methacrylic acid) (PMA) solution form different molecular species: monomers anchored to PMA, dimers, and H aggregates, in proportions determined by the [dye]/[PMA] concentration ratio. Each molecular species is characterized by its own probabilities of radiative and nonradiative transitions: the absence of photoisomerization for I–IIIdimers and H aggregates, the absence of fluorescence for H aggregates of IIand III, the capability of transition to a triplet state for PMA-anchored monomers I–III, and considerable enhancement of intersystem crossing to the triplet state for the dimers of II.     

Simultaneous Determination of Fourteen Catecholamines and Their Metabolites by High Performance Liquid Chromatography with Electrochemical Detection

Abstract

A simple method has been developed for simultaneous determination of 14 catecholamines and their metabolites in cerebrospinal fluid and brain tissue by reversed-phase, ion-pair high-performance liquid chromatography with electrochemical detection. The time required for complete separation and analysis of all compounds was less than 35 min. Quantitation was based on the use of an internal standard isoproterenol. The mobile phase consisted of a 91:9 (v/v) mixture of 0.1 M formic acid and acetonitrile containing sodium-1-octane sulfonic acid. Using this method, analysis of neurotransmitters in brain tissue can be accomplished without a clean-up procedure.     

Rapid Determination of Methotrexate and 7-Hydroxymethotrexate in Serum and Cerebrospinal Fluid by Radial Compression Liquid Chromatography

Abstract

A rapid, specific and sensitive radial compression reverse phase liquid chromatographic method for the analysis of methotrexate and 7-hydroxymethotrexate in serum and cerebrospinal fluid is reported. A mobile phase consisting of acetonitrile-methanol-pH 3 phosphate (8:15:77) at 6 ml/min flow rate was employed. The U.V. detector was set at 317 nm, and folic acid was used as an internal standard. A rapid extraction of methotrexate and 7-hydroxymethotrexate was performed using Sep-Pak cartridges with high extraction efficiency for both compounds. Patients serum and cerebrospinal fluid samples were analyzed by the described method and the concentrations of methotrexate were compared to those obtained by an enzyme immunoassay. No interference from other metabolites or anticancer drugs in the described assay was observed.     

12-钼磷酸的BrÖnsted酸位和Lewis酸位

研究了加热、抽空等预处理对固态12-钼磷酸(PMA)酸位的影响。发现了PMA上除存在B-酸位外,还可生成L-酸位。研究了PMA上L-酸位的成因,水在两类酸位形成过程中的作用,L-酸位与B-酸位的相对强度,以及PMA上酸强度的分布。据此,对Miscno等认为PMA中只存在质子酸和酸强度分布均匀的观点提出了不同的看法。     

Capillary electrophoretic determination of oxalate in amniotic fluid

A capillary electrophoretic (CE) assay for oxalate has been applied to the quantitative determination of free oxalate in amniotic fluid. Indirect absorbance detection of oxalate is accomplished with a chromate-based background electrolyte modified with ethylenediaminetetraacetic acid (EDTA). Detection interference due to the presence of high levels (≈4 mg/ml) of inorganic chloride is eliminated through a direct sample clean-up procedure based on cation (Ag⁺-form) resins. Separation interference from amniotic fluid proteins is prevented through the use of a simple aqueous-based dilution procedure. This method for the determination of oxalate in amniotic fluid provides precision of ≈5% relative standard deviation (RSD). Within-day precisions for the oxalate response and migration time are better than 3% RSD and 1% RSD, respectively. Between-day precisions for the oxalate response and migration time are better than 6% RSD and 3% RSD, respectively. The analytical recovery of oxalate (1000 ng/ml) spiked into amniotic fluid was better than 96%. The limit of detection (LOD) for the method is ≈100 ng/ml oxalate. This method also shows promising results for the determination of oxalate in human blood plasma samples.     

Analysis of perchlorate, thiocyanate, nitrate and iodide in human amniotic fluid using ion chromatography and electrospray tandem mass spectrometry

Because of health concerns surrounding in utero exposure to perchlorate, we developed a sensitive and selective method for quantifying iodide, as well as perchlorate and other sodium-iodide symporter (NIS) inhibitors in human amniotic fluid using ion chromatography coupled with electrospray ionization tandem mass spectrometry. Iodide and NIS inhibitors were quantified using a stable isotope-labeled internal standards (Cl¹⁸O₄⁻, S¹³CN⁻ and ¹⁵NO₃⁻ with excellent assay accuracy of 100%, 98%, 99%, 95% for perchlorate, thiocyanate, nitrate and iodide, respectively, in triplicate analysis of spiked amniotic fluid sample). Excellent analytical precision (<5.2% RSD for all analytes) was found when amniotic fluid quality control pools were repetitively analyzed for iodide and NIS-inhibitors. Selective chromatography and tandem mass spectrometry reduced the need for sample cleanup, resulting in a rugged and rapid method capable of routinely analyzing 75 samples/day. Analytical response was linear across the physiologically relevant concentration range for the analytes. Analysis of a set of 48 amniotic fluid samples identified the range and median levels for perchlorate (0.057-0.71, 0.18 μg/L), thiocyanate (<10-5860, 89 μg/L), nitrate (650-8900, 1620 μg/L) and iodide (1.7-170, 8.1 μg/L). This selective, sensitive, and rapid method will help assess exposure of the developing fetus to low levels of NIS-inhibitors and their potential to inhibit thyroid function.     

Simultaneous multiresponse optimization of an HPLC method to separate seven cephalosporins in plasma and amniotic fluid: Application to validation and quantification of cefepime, cefixime and cefoperazone

An HPLC method for the separation of seven cephalosporins [Cefepime (CEP), ceftazidime (CTA), ceftizaxime (CTI), ceftriaxone (CTR), cefotaxime (COT), cefixime (CIX) and cefoperazone (COP)] in human plasma and amniotic fluid has been developed. Optimization of the chromatographic method was performed in three steps: a series of initial experiments followed by two sets of experiments based on different experimental designs. The initial experiments were performed to decide the basic analytical requirements of the method. Then screening experiment fractional factorial design was used in order to decrease the number of parameters by eliminating parameters which having insignificant effect on responses. The parameters having significant effect were further optimized through a full factorial design. Having studied two responses (retention times and resolutions), a desirability function that assess the responses together, was used to find experimental conditions where the system generated desirable results. The desirable results were obtained with XTerra C18 (250 mm × 4.6 mm, 5 μm i.d.) column, 40 mM phosphate buffer, pH 3.2, 18% MeOH, 0.85 mL min⁻¹ flow rate and 32 °C column temperature. Gradient elution with MeOH was applied. A simple and efficient solid-phase extraction was applied for the preparation of plasma and amniotic fluid samples. The validation parameters of the method were evaluated in accordance with ICH guideline. The method validated was applied to the analysis of CEP and COP in maternal venous, fetal venous and fetal arterial plasma, and to the analysis of CIX in maternal venous plasma and amniotic fluid.     

Spectrophotometric determination of microquantities of frusemide using iso- and heteropolyanions of molybdenum(VI) as oxidizing agents

Summary A spectrophotometric method for the determination of microquantities of frusemide (FRS) is described. It is based on the oxidation of the latter by the paramolybdate anion (PMA) or by the molybdatophosphoric anion (MPA). The oxidation is carried out in acid medium at 98° C ±0.5° C for a fixed time of 25 min. The blue coloured reaction product can be measured spectrophotometrically at 690 nm and 700 nm, respectively. Beer's law is obeyed over the range of 5–200 ppm of FRS. The apparent molar absorptivities and Sandell's sensitivities (in l mol^–1 cm^–1 and ng cm^–2 per 10^–3 absorbance unit, respectively) are 2.8×10³ and 118.2 using PMA as oxidizing agent and 2.16×10³ and 153 using MPA for the same purpose. Six replicate analyses of solutions containing seven different concentrations of FRS were realized and gave a mean correlation coefficient of 0.9999 using the first oxidant (PMA) and 0.9998 using the second one (MPA), while the slope and intercept of the regression line equation were calculated. FRS was successfully determined both in the pure form and in pharmaceutical preparations. The results demonstrated that the proposed procedure is at least as accurate, precise and sensitive as the official method, while a statistical analysis indicated that there was no significant difference between the results obtained by the recommended procedure and those of the official method.

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Spektralphotometrische Bestimmung von Mikromengen Frusemid unter Verwendung von Iso- und Heteropolyanionen des Mo(VI) als Oxidationsmittel

     

High pressure liquid chromatography of standard and amniotic fluid phospholipids

A method for the HPLC separation of phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylcholine (PC), and sphingomyelin (SPH) was achieved using five in-series columns packed with LiChrosorb, Partisil, and μ-Porasil adsorbents, a solvent mixture of chloroform/methanol/ammonium hydroxide (50 : 36 : 6.7, by volume), and a Pye LCM2 Moving Wire (FID) detector. The same phospholipid mixture was also separated using four μ-Porasil columns with the same eluent and detector. The latter conditions were found to be suitable for the analysis of phospholipids obtained after centrifuging, extraction, and precipitation of surface-active lipid components of patient amniotic fluid collected at amniocentesis section. The lecithin/sphingomyelin (L/S) ratios, determined by the HPLC method, correlated well with those determined by the TLC technique in four normal pregnancies, whereas results of shake tests did not correlate too well with L/S ratios determined by the above two chromatographic methods. Besides the lecithin/sphingomyelin ratio, the present method was able to supply additional information: the concentrations of phosphatidylglycerol and phosphatidylinositol, for prediction of fetal lung maturity, and the palmitic acid content of amniotic fluid phosphatidylcholine.     

An evaluation of acid digestion methods for amniotic fluid phospholipids

Five methods of acid digestion of amniotic fluid phospholipids for inorganic phosphate release were evaluated at a temperature of 225 °C and a heating time of 30 min. The methods used the following digestants: (A (perchloric acid), B (perchloric acid containing molybdate), C (sulfuric acid, 30% hydrogen peroxide, 5% urea), D (sulfuric-perchloric acids), and E (sulfuric-perchloric acids with vanadium pentoxide). After digestion and dilution with water, a hydrogen ion concentration of about 2.5 mol/ liter was obtained that permitted the use of stannous chloride-hydrazine sulfate as the reducing agent for phosphate color development. Recoveries of the different amniotic fluid phospholipids and aqueous phosphate standards were quantitative for all methods. A comparative study with amniotic fluids showed similar results for all methods. Since all methods were found to be equivalent under the specified conditions described, the choice was dependent upon the user's preference.     

A semi-automated and manual method for serum phospholipids

Five methods of acid digestion of amniotic fluid phospholipids for inorganic phosphate release were evaluated at a temperature of 225 °C and a heating time of 30 min. The methods used the following digestants: (A (perchloric acid), B (perchloric acid containing molybdate), C (sulfuric acid, 30% hydrogen peroxide, 5% urea), D (sulfuric-perchloric acids), and E (sulfuric-perchloric acids with vanadium pentoxide). After digestion and dilution with water, a hydrogen ion concentration of about 2.5 mol/ liter was obtained that permitted the use of stannous chloride-hydrazine sulfate as the reducing agent for phosphate color development. Recoveries of the different amniotic fluid phospholipids and aqueous phosphate standards were quantitative for all methods. A comparative study with amniotic fluids showed similar results for all methods. Since all methods were found to be equivalent under the specified conditions described, the choice was dependent upon the user's preference.