Disopyramide analysis using REMEDi: comparison with EMIT and conventional high performance liquid chromatographic methods.

REMEDi (Rapid EMErgency Drug identification; Bio-Rad) is an automated high performance liquid chromatographic (HPLC) system designed to detect, identify and measure a range of basic and neutral drugs in 0.5-1.0 mL of urine or plasma/serum. We have evaluated REMEDi in the analysis of the antiarrhythmic drug disopyramide in patient samples. The specimens were also analysed by a conventional HPLC method, based on solvent extraction and UV detection (254 nm), and by EMIT. There were good correlations between the results obtained with each method (r = 0.91 or greater). REMEDi gave a lower mean result than EMIT [means +/- SD (mg/L): REMEDi 2.64 +/- 1.10, EMIT 3.14 +/- 1.51; t = 4.0, p less than 0.01; n = 25], but there were no other significant differences in mean results. The principal disopyramide metabolite, mono-N-desalkyldisopyramide, did not interfere in any method. Clearly REMEDi can be used for therapeutic drug monitoring of disopyramide provided enough sample is available.     

Automated direct high-performance liquid chromatographic assay for estetrol, estriol, cortisone and cortisol in serum and amniotic fluid.

An automated direct assay for the simultaneous determination of unconjugated estetrol, estriol, cortisone and cortisol in serum and amniotic fluid, using high-performance liquid chromatography with electrochemical detection and ultraviolet detection, has been developed. The analysis time is ca. 1 h. This system offers good reproducibility with low coefficients of variation (estetrol, 2.3%; estriol, 2.3%; cortisone, 2.6%; cortisol, 1.9%). Detection limits are low enough for routine determinations (estetrol and estriol, 150 pg; cortisone and cortisol, 5 ng). Comparison of the values measured by the present method and by radioimmunoassay revealed significant correlations for estetrol (r = 0.787, p less than 0.01), estriol (r = 0.957, p less than 0.01), cortisone (r = 0.956, p less than 0.01) and cortisol (r = 0.865, p less than 0.01). This system proved to be valuable in monitoring feto-placental function.     

High-performance liquid chromatographic method for the simultaneous determination of monoethylglycinexylidide and lignocaine.

An accurate and sensitive high-performance liquid chromatographic method with UV detection was developed for the simultaneous measurement of monoethylglycinexylidide (MEGX) and lignocaine in human plasma and serum, using organic solvent extraction and trimethoprim (TMP) as an internal standard. The mean recoveries for MEGX, TMP and lignocaine were 86.1 +/- 3.7, 98.3 +/- 1.8 and 77.0 +/- 4.7%, respectively (n = 6). The relative standard deviations for MEGX concentrations of 10 and 200 ng/ml were less than 4% and for lignocaine concentrations of 200 and 1200 ng/ml they were less than 8%.     

Determination of megestrol acetate and cyproterone acetate in serum of patients with advanced breast cancer by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method with ultraviolet detection of megestrol acetate and cyproterone acetate in human sera is described. The proposed assay is linear up to 1400 ng/ml (r = 0.999) and has a detection limit of 5 ng/ml. Recoveries of both compounds in spiked sera were ca. 95%; inter-assay coefficients of variation were 4.0 and 3.1% and intra-assay values were 1.3 and 1.4%, respectively. For validation of the method we also developed a gas chromatographic-mass spectrometric method for both steroids. The results obtained by the two methods showed good correlation: for megestrol acetate r = 0.98, n = 31, p less than 0.0001, and for cyproterone acetate r = 0.94, n = 0, p less than 0.0001. Large inter-individual differences in the serum concentrations of both substances were found in groups of patients with metastatic breast cancer receiving the same oral load of either steroid.     

Determination of pyridoxal 5'-phosphate in human serum by reversed phase high performance liquid chromatography combined with spectrofluorimetric detection of 4-pyridoxic acid 5'-phosphate as a derivative

A very sensitive spectrofluorimetric method for the determination of pyridoxal 5'-phosphate (PLP) in human serum is described. The specificity is based on the selective oxidation of PLP to 4-pyridoxic acid 5'-phosphate with potassium cyanide. Separation of the highly fluorescent 4-pyridoxic acid 5'-phosphate is achieved by reversed-phase high-performance liquid chromatography. Specificity is improved by a careful choice of fluorescence filters, maximised at the excitation (325 nm) and emission (418 nm) wavelengths of 4-pyridoxic acid 5'-phosphate. The detection limit for the reaction is 0.22 ng ml(-1). For quantification, the serum is spiked with PLP before protein precipitation with 3.3% m/V trichloroacetic acid. The method can be used for the determination of PLP in serum, even in vitamin B6 deficient patients. The mean value for human serum PLP from 30 healthy adults was found to be 14.6 +/- 4.8 ng ml(-1) (mean +/- standard deviation).     

Determination of Enzymatic Activities Based on an Optical Flow-Through p-Nitrophenol Sensor

Abstract

An optical flow-through biosensor based on the transient retention of p-nitrophenol on an ion-exchange support packed in the flow cell is presented. The approach was applied to the determination of the activity of some enzymes which catalyze the conversion of p-nitrophenyl-derivatives into p-nitrophenol. The method was first developed for determination of p-nitrophenol yielding a linear range between 0.1–5 μg/mL (r² = 0.9922, r.s.d.% less than 2.5), then, it was also applied to the determination of β-D-glucuronidase activity in serum, with a linear range between 0.1–20 U/L (r² = 0.9976, r.s.d.% less than 3.0), and a sampling frequency of 20 h^?1. The application of the method to the determination of the enzyme activity in serum samples provided results consistent with those obtained by a conventional method and recoveries within 95–104%.     

毛细管电泳接触反应法测定血清中铁传递蛋白的浓度

建立了一种测定血清中铁传递蛋白的毛细管电泳接触反应方法，利用血清中铁传递蛋白计算化学结合铁的性质，用标准铁饮和铁传递蛋通过对铁准确定量，测定血清铁伟递蛋白的有浓度。     

A rapid radioimmunoassay procedure for thyroxin

A sensitive thyroxin /T₄/ radioimmunoassay procedure with a short incubation time /10 min/ similar to a conventional stat test in clinical chemistry is described. The assay parameters such as concentration of antisera and¹²⁵I-T₄, and incubation temperature that affect the kinetics of antigen-antibody reaction have been studied. Within- and between-assay variations were less than 7% CV. Analysis of 45 serum samples by the proposed method and by a conventional assay gave similar results /Y=1.05X–0.187; r=0.990/.     

Correlation between glycated lipoproteins and fructosamine level in serum.

The correlation between the level of fructosamine and glycated proteins, including glycated lipoproteins, in serum from diabetic and nondiabetic subjects was studied. Assay of glycated proteins in serum was performed using an agarose gel film electrophoresis with nitroblue tetrazolium coloration. Glycated albumin correlated well with the fructosamine level in the diabetics (r = 0.83-0.92, p less than 0.01) but showed no correlation with the nondiabetics (r = 0.25-0.26). Also, a high correlation between the glycated beta-lipoprotein and fructosamine levels was observed in diabetic patients with hyperglycemia and in nondiabetic subjects with a high risk of atherogenesis (atherogenic index, low-density lipoprotein-cholesterol/high-density lipoprotein-cholesterol greater than 2.8) (r = 0.51-0.66, p less than 0.01). Nondiabetics with a high level of beta-lipoprotein, which is well known to cause high atherogenesity, showed a high level of glycated beta-lipoprotein similar to that in the diabetic groups with hyperglycemia; therefore, the high level of glycated beta-lipoprotein seems to be attributable not only to the hyperglycemia-accelerated glycation of beta-lipoprotein but also to an increase in the level of beta-lipoprotein in serum. Consequently, the present results show that the fructosamine level in serum reflects not only the glycation of albumin but also that of lipoproteins which are known to increase in diabetes mellitus.     

Stereospecific high-performance liquid chromatographic assay of acebutolol in human plasma and urine

A sensitive high-performance liquid chromatographic technique is described for the separation of R- and S-acebutolol in human plasma and urine. The procedure involves derivatization with the chiral reagent S-(+)-1-(1-naphthyl)ethyl isocyanate. The resulting diastereoisomers are quantified using normal-phase high-performance liquid chromatography with fluorescence detection (220/389 nm). Virtual baseline separation, free from interference, with achieved (resolution factor = 1.45). Excellent linearity (r greater than 0.998) was observed throughout the range 10-500 ng/l and 2-100 mg/l in plasma and urine, respectively. Inter-assay variability was less than 5% for each enantiomer at concentrations of 10 ng/ml. This method is applicable for the determination of the pharmacokinetics, in man, of acebutolol enantiomers in plasma and urine.     

A Sensitive Colorimetric Reagent for the Determination of Cyanide and Hydrogen Cyanide in Various Environmental Samples

A sensitive reagent system is proposed for the determination of cyanide and hydrogen cyanide in various environmental samples. The method is based on the conversion of cyanide into cyanogen bromide followed by its reaction with pyridine to form glutaconic aldehyde. The glutaconic aldehyde so formed is coupled with p‐aminoacetophenone forming yellow‐orange polymethine dye measured at 445 nm. The colour system obeys Beer's law in the range of 0.01–0.16 ppm of cyanide inaqeous phase and 0.002–0.03 ppm in extracting system. The molar absorptivity and Sandell's sensitivity were found to be 6.51 × 10⁵ l mol^?1 cm^?1 and 0.0001 μg cm^?2, respectively. The method has been successfully applied for the determination of cyanide in air, industrial effluent, biological samples, and in the pesticide acrylonitrile.     

气相色谱/质谱法测定大鼠脑中5-羟色胺的含量

采用气相色谱／质谱法（ＧＣ／ＭＳ）测定了正常大鼠和服药大鼠脑组织中５－羟色胺（５－ＨＴ）的浓度。大鼠脑组织制成匀浆后,先将５－ＨＴ酰化,酰化物经乙酸乙酯提取后,再经七氟丁酸酐衍生化,用ＧＣ／ＭＳ测定。ＭＳ采用电子捕获负离子化学电离（ＥＣＮＩＣＩ）模式。方法的线性范围为０．５０～５０．０μｇ／Ｌ,回归方程为Ｙ＝０．１３４８Ｘ－０．０７９９５（ｒ＝０．９９９６）;平均回收率为９８．２％±３．８％（ｎ＝１０）;检测限为０．５μｇ／Ｌ;相对标准偏差小于１０％。     

High performance liquid chromatographic determination of methylxanthines in canine serum, gastric and pancreatic juices

A convenient high performance liquid chromatographic method for the determination of methylxanthines in biological samples is described. Separation was achieved by reversed phase chromatography using a mobile phase consisting of tetrahydrofuran + methanol + 0.01M potassium dihydrogen phosphate, pH 3.5 (1:20:79, v/v/v), on a 7 microns C18 column and a C18 Lichrosorb precolumn at a flow rate of 0.8 mL/min. Levels varying from 0.25-16 mg/L could be detected by UV at 280 nm. In this range, standard curves were established for 4 methylxanthines: theobromine, paraxanthine, theophylline and caffeine in 4 media: mobile phase, serum, gastric and pancreatic juices, and were found to be linear (r greater than or equal to 0.9975). Overall characteristics of the method were determined as: percent recovery (89.54%), accuracy (greater than or equal to 99.4%) and reproducibility (greater than or equal to 95%). Retention times ranged from 4.21 +/- 0.01 (1-methyluric acid) to 10.8 +/- 0.03 min (caffeine). Animal experiments (5 and 10 mg/kg boluses) were used to determine caffeine half life in dog's blood (310 +/- 46 and 453 +/- 59 min, respectively) and its secretion into pentagastrin stimulated gastric juice (mean concentrations 2.51 and 6.04 mg/L; mean outputs 351 and 1206 micrograms/2.25 h; both statistically different at p less than 0.001 level).     

Kinetic and thermodynamic studies on ligand substitution reactions and base-on/base-off equilibria of cyanoimidazolylcobamide, a vitamin B12 analog with an imidazole axial nucleoside

Ligand substitution reactions of the vitamin B12 analog cyanoimidazolylcobamide, CN(Im)Cbl, with cyanide were studied. Cyanide substitutes imidazole (Im) in the alpha-position more slowly than it substitutes dimethylbenzimidazole in cyanocobalamin (vitamin B12). The kinetics of the displacement of Im by CN- showed saturation behaviour at high cyanide concentration; the limiting rate constant was found to be 0.0264 s(-1) at 25 degrees C and is characterized by the activation parameters: DeltaH(not =) = 111 +/- 2 kJ mol(-1), DeltaS(not =) = +97 +/- 6 J K(-1) mol(-1), and DeltaV(not =) = +9.3 +/- 0.3 cm3 mol(-1). These parameters are interpreted in terms of an I(d) mechanism. The equilibrium constant for the reaction of CN(Im)Cbl with CN- was found to be 861 +/- 75 M(-1), which is significantly less than that obtained for the reaction of cyanocobalamin with CN- (viz. 10(4) M(-1)). pKbase-off for the base-on/base-off equilibrium was determined spectrophotometrically and found to be 0.99 +/- 0.05, which is about 0.9 pH units higher than that obtained previously in the case of cyanocobalamin. In addition, the kinetics of the base-on/base-off reaction was studied using a pH-jump technique and the data obtained revealed evidence for an acid catalyzed reaction path. The results obtained in this study are discussed in reference to those reported previously for cyanocobalamin.     

Fluorimetric assay for argininosuccinate lyase in human plasma and erythrocytes with a benzoin reagent

A fluorimetric method for the assay of argininosuccinate lyase in human plasma (15–150 units) and erythrocytes (40–1200 units) is established. The arginine formed enzymatically is quantified by means of its fluorescent reaction with benzoin. The method is rapid, simple and sensitive enough to allow the enzyme activity to be determined in as little as 100 μl of plasma or 12.5 μl of packed erythrocytes.     

Investigation of multiaxis molecular motion by off-magic angle spinning deuteron NMR

The relatively new deuteron NMR method of off-axis-magic angle spinning (OMAS) has been extended and used to investigate multiaxis rotational jump motion. Floquet theory is developed for simulating deuteron OMAS spectra with multisite jumps at different rates about noncoincident axes, and efficient procedures are presented for computing the sideband line shapes. It is demonstrated experimentally that reproducible adjustment of the angle between the rotor axis and the static magnetic field is feasible with precision approaching +/- 0.01 degrees. This leads to the reintroduction of a scaled, first-order quadrupole coupling that defines a new kinetic window and makes deuteron OMAS much more sensitive than ordinary magic angle spinning to motion on the kilohertz time scale. Temperature-dependent deuteron OMAS line shapes of octanoic acid/urea-d4 inclusion compound have been recorded and fitted, using least-squares procedures, to provide rates of rotation about both CN and CO bonds. The Arrhenius activation parameters for rotation about CN bonds, Ea = 60.4+/-2.4 kJ/mol and ln(A) = 24.9+/-0.3, agree well with previous values determined by selective inversion experiments. However, OMAS yields Ea = 26.3+/-0.4 kJ/mole and ln(A) = 24.9+/-0.3 for whole-body rotation about the CO bond axis in contrast to previous analysis of static quadrupole echo (QE) line shapes which gave Ea = 22.3+/-0.3 kJ/mole and ln(A) = 24.8+/-0.6 for the same sample. The underlying homogeneous linewidths of OMAS spectra are much smaller than those of QE spectra, and this provides higher precision and less systematic error in the determination of rates.     

Determination of pioglitazone in dog serum using solid-phase extraction and high-performance liquid chromatography with ultraviolet (229 nm) detection

An analytical method is described for the determination of the free base of pioglitazone hydrochloride (U72, 107A, AD-4833) in dog serum. The method used solid-phase extraction of pioglitazone from serum followed by high-performance liquid chromatographic analysis on an octadecylsilane column with an eluent of acetonitrile-water (41:59, v/v) containing 1.2 ml/l acetic acid (pH 6.0 +/- 0.05). The column effluent was monitored at 229 nm. The analytical procedure has a linear range of 25 ng/ml to 20 micrograms/ml, a minimum quantifiable level of 25 ng/ml, absolute recovery of greater than 90% (n = 15), and precision of less than or equal to 8.8% (n = 45). The method was used in a preliminary dose proportionality study in the dog.     

Sensitive assay for triazolam in plasma following low oral doses.

At low doses of triazolam currently recommended increased assay sensitivity is required for measurement of low plasma concentrations. A highly sensitive capillary gas chromatographic analytical method with a limit of detection of 0.02 ng/ml was developed and used to describe the pharmacokinetics of triazolam following the oral intake of 0.125, 0.250 and 0.375 mg. Six male subjects were studied with blood sampling at the following times: 0, 15, 30 and 45 min and 1, 1.5, 2.0, 2.5, 3, 4, 5, 6 and 8 h. The mean pharmacokinetic parameters for the three doses, respectively, were as follows: half-life, 2.7 +/- 0.4, 3.2 +/- 0.5 and 3.2 +/- 0.6 h; apparent oral clearance, 302.3 +/- 59.0, 260.2 +/- 67.9 and 328.6 +/- 77.8 ml/min; apparent volume of distribution, 64.3 +/- 9.6, 62.0 +/- 12.6 and 73.3 +/- 7.7 l; time to maximum concentration, 0.7 +/- 0.2, 0.6 +/- 0.1 and 0.8 +/- 0.3 h; maximum concentration, 2.2 +/- 0.3, 4.3 +/- 0.6 and 5.0 +/- 0.5 ng/ml; and the area under the concentration-time curve (AUC) up to 8 h, 6.8 +/- 1.2, 16.8 +/- 2.9 and 19.6 +/- 3.5 ng/ml h; and AUC extrapolated to infinity, 8.5 +/- 1.7, 21.4 +/- 4.4 and 26.3 +/- 7.2 ng/ml h. There were no significant differences in the half-life, clearance, volume of distribution and time to maximum concentration among the three doses. The AUC was significantly different on the three occasions and was linearly correlated with dose: r = 0.64 (p less than 0.005).     

Determination of natural levels of lithium and strontium in human blood serum by discrete injection and atomic emission spectrometry with a nitrous oxide—acetylene flame

A sensitive technique is described for the rapid, direct determination of normal levels of lithium and strontium in 100-μl samples of human blood serum without separation or preconcentration. Nitrous oxide—acetylene flame emission spectrometry, using conventional atomic absorption apparatus is used, with discrete sample injection. Lithium and strontium standards were prepared in 21% (v/v) glycerol which approximates the viscosity of serum. It is recommended that serum samples be analyzed by either calibration with artificial serum and glycerol standards or by immediate standard microaddition procedures. Results for pooled human serum indicate accuracy and precision of better than 6% at the 10 ng ml^-1 level. The method is free from nebulizer clogging and matrix interferences and should be useful as a routine clinical laboratory procedure.     

A Rapid and Simple High Pressure Liquid Chromatographic Method for Pharmacokinetic Study of Ciprofloxacin in Human Serum

Abstract

A rapid, accurate and sensitive method has been developed for the quantitative determination of ciprofloxacin, a new second-generation quinolone carboxylic acid antimicrobial agent, with high in vitro activity against a wide range of Gram- negative pathogens and Gram-positive cocci.

A Lichrosorb RP-18 250 x 4.0 mm, 5 μm analytical column was used with an eluting system consisting of a mixture of CH₃CN-CH₃OH-Citric acid 0.4 M (1:3:6 v/v). Detection was performed with a variable wavelength UV-visible detector at 275 nm resulting in a limit of detection of 0.2 ng per 20 μl injection. For the quantitative determination theophylline was used as chromatographic internal standard at a concentration of 1.56 ng/μl. A rectilinear relationship was observed up to 20 ng/μl. Analysis time was less than 6 min. The statistical evaluation of the method was examined performing intra-day (n=8) and inter-day calibration (n=8) and was found to be satisfactory with highly accurate and precise results. The method was applied to the direct determination of ciprofloxacin in human blood serum. Sample pretreatment involved only protein precipitation with acetonitrile. Recovery of ciprofloxacin in spiked samples was 98 ± 4% over the range of 0.5–5 mg/μl.