Small Plasma Samples Research Articles

Development and validation of an automated, enzyme-mediated colorimetric assay of salicylate in serum

This salicylate-specific assay can be adapted for use with most discrete analyzers, for rapid emergency or routine testing with small serum or plasma sample volumes and a single calibration. The basis of this method is as follows: salicylate monooxygenase (EC 1.14.13.1) converts salicylate to catechol in the presence of NADH; the catechol then reacts with 4-aminophenazone under alkaline conditions, catalyzed by manganese ions, to produce a red dye. Incorporation of an NADH-regenerating system, involving glucose and glucose dehydrogenase, into the enzyme reagent ensures that the working reagent is stable for more than two weeks. The standard curve is linear over the drug concentration range 0 to 5 mmol/L. The CV was less than 4% over 20 days. Results correlated well with those by the Trinder colorimetric method and an HPLC method. We saw no interference by any of 80 drugs we tested at therapeutic concentrations or by endogenous compounds in serum.

Determination of zonisamide (3-sulphamoylmethyl-1,2-benzisoxazole) in plasma at therapeutic concentrations by high-performance liquid chromatography

A selective and sensitive liquid chromatographic method for the determination of zonisamide in small (0.1 ml) plasma samples is described. After adding internal standard, a direct solvent extract of the sample is examined by reversed-phase liquid chromatography with ultra-violet spectrophotometric detection. The method is rapid, simple and capable of determining plasma levels after therapeutic ingestion of zonisamide. Some results from a dose-ranging clinical trial are presented.

Isolation of Human Fibrinogen and its Derivatives by Affinity Chromatography on Gly-Pro-Arg-Pro-Lys-Fractogel

With an immobilized synthetic pentapeptide GlyProArgProLys comprising the N-terminal sequence GlyProArg of the alpha-chain of fibrin, a new affinity method for the quantitative isolation of fibrinogen out of anticoagulated plasma was developed. The method proved to be superior to all known isolation methods in respect to ease of use and yield, since fibrinogen could be isolated in one step out of plasma with a recovery of more than 95% when compared to the immunologically measurable amounts of fibrinogen. Moreover the amounts of contaminating proteins such as fibronectin, factor XIII or plasminogen were negligible and the purity of the isolated fibrinogen was higher than 95% as measured by polyacrylamide gel electrophoresis. The clottability was 90% and more. Another advantage of this affinity purification method is the possibility to isolate fibrinogen quantitatively out of small plasma samples (less than 5 ml). Further, abnormal fibrinogen molecules, provided their complementary binding site for GlyProArg is preserved, may also be quantitatively isolated independent of any solubility differences as compared to normal fibrinogen. In addition fibrin(ogen) fragments originating from plasmic digestion can be separated on the basis of their affinity to GlyProArg. The described affinity gel can be used more than 50 times without any loss of capacity.

Further analysis of the disturbed adrenocortical function in the cerebro‐hepato‐renal syndrome of zellweger

Plasma aldosterone, corticosterone, 11-deoxycorticosterone, progesterone, 17-hydroxyprogesterone, 11-desoxycortisol, cortisol and cortisone were determined simultaneously in small plasma samples obtained during an i.v. ACTH (Synacthen) stimulation test in five patients (age 4-7 years) with Zellweger syndrome. The response of all ACTH-dependent steroids to Synacthen was severely impaired in all patients, despite normal basal levels. It can be concluded that the biochemical defect in adrenal steroidogenesis causing the inadequate response to ACTH injection is located proximal to progesterone. We propose that the lack of responsiveness to ACTH is secondary to an abnormality of ACTH receptors on the adrenocortical cell. The extremely low levels of non-specific lipid transfer protein may be a contributing factor.

High-performance liquid chromatographic analysis of desferrioxamine : Pharmacokinetic and metabolic studies

A high-performance liquid chromatographic method has been developed that permits determination and quantitation of desferrioxamine and metabolites as their iron (III) complexes in small samples of mammalian plasma at levels encountered with ion-specific chelation treatments. The technique permits measurement of desferrioxamine and metabolite concentrations which can be used in pharmacokinetic studies. A human study is presented as an example.

A Micromethod for Determination of Plasma Pyridoxal Phosphate and Its Use in Assessment of Storage Stability of the Vitamer

SummaryA widely used macromethod employing tyrosine apodecarboxylase for measurement of pyridoxal phosphate (PLP) concentration in 0.5–1.0‐ml plasma was modified to a microscale utilizing 0.1‐ml plasma. Mean PLP levels in 12 plasma samples were 160.6 ± 32.8 pmol/ml (mean ± SD) when analyzed by the macro‐method, and were not significantly different compared to those obtained by the micromethod (158.4 ± 28.2 pmol/ ml). Results of the two methods were significantly correlated (r = +0.97, p < 0.001). Plasma PLP concentrations of 11 samples determined by the micromethod ( = 151.8 ± 30.0 pmol/ml) were similar and significantly correlated (r = +0.95, p < 0.001) to levels measured in the same samples 1–2 years earlier ( = 145.1 ± 26.2 pmol/ml). This suggests that plasma PLP content of the samples was stable for up to 2 years of storage when the micro‐method was utilized for analysis. The strong significant correlation between macro‐ and micromethods attests that the micromethod is a reliable alternative to the macromethod. The micromethod is useful in instances where only small samples of plasma are available for measurement of PLP.

Hepatic blood flow determination: A comparison of 99mTc-diethyl-IDA and indocyanine green as hepatic blood flow indicators in man

99mTc-diethyl-acetanilide-iminodiacetic acid (IDA) was compared with indocyanine green (ICG) as an indicator of hepatic blood flow (HBF). Twelve subjects (8 with cirrhosis, 2 with fatty liver, one with pancreatitis, and one with intestinal angina) were studied during hepatic vein catheterization. In 9 subjects the HBF measurements (indirect Fick-principle) were within 0.8-1.9 l/min, and no significant difference was observed between the values obtained by ICG and 99mTc-diethyl-IDA (mean 1.24 vs 1.26 l/min, P greater than 0.4). In 2 subjects with cirrhosis very high but almost identical values were found with the two indicators. In one subject ICG could not be measured in plasma because of hyperlipidaemia, but HBF was easily determined by 99mTc-diethyl-IDA. The results indicate that 99mTc-diethyl-IDA can be used as an indicator of HBF. This indicator is not superior to ICG in patients with decreased liver function, but offers advantages in that it can be used with small plasma samples and permits the determination of HBF in the presence of hyperlipidaemia.

Analysis of submicromolar concentrations of adenosine in plasma using reversed phase high-performance liquid chromatography

A method is described for the determination of adenosine in small samples of plasma (< 1 ml) using reversed-phase high-performance liquid chromatography (HPLC) in either a simple isocratic or a gradient elution system which gives a clear separation of adenosine from other plasma constituents. Acetone is used to deproteinize plasma and chloroform to remove unwanted lipid soluble material prior to HPLC. 6-Methyladenosine is used as an internal standard for making corrections for changes in concentration during sample processing. Adenosine in plasma could be reliably detected at concentrations lower than its minimum effector concentration as a vasodilator (4 × 10 −8 Mol l −1 using the isocratic system and 1.9 × 10 −8 Mol l −1 with gradient elution). The recoveries of adenosine added to blood at concentrations ranging from 2 × 10 −8 Mol l −1 to 1.4 × 10 −6 Mol l −1 were from 101.4 ± 16.9% ( n = 4) to 100.0 ± 3.6% ( n = 5). The present method provides a simple, sensitive and selective assay for submicromolar concentrations of adenosine in plasma with good recovery.

High-performance liquid chromatographic assay for morphine with electrochemical detection using an unmodified silica column with a non-aqueous ionic eluent

A quick, sensitive and accurate high-performance liquid chromatographic method for morphine measurement in plasma has been developed using an unmodified silica column with a non-aqueous ionic eluent. Small plasma samples (0.4 ml) were extracted with a liquid—liquid extraction column and dihydromorphine was used as internal standard. The limit of quantitation was 2 ng/ml. The electrochemical detector was capable of detecting 150 pg of morphine. A calibration line with a correlation of 0.9994 ± 0.00026 was produced over the range 2–80 ng/ml.

High-speed bubble-segmented blood sampler for the rat.

An arterial blood sampler for use in the conscious rat is described. With this apparatus it is possible to obtain small (10 microliter) whole-blood or plasma samples as frequently as 1/s and to derive accurate arterial time-concentration curves in the first 60-120 s after compounds are injected for regional blood flow or pharmacokinetic measurements. The blood is withdrawn from an implanted arterial catheter into polyethylene tubing at a constant rate while bubbles are introduced at regular intervals via a side channel into the column of blood. Although some dispersion of blood samples occurs as the tubing is traversed, this can be mathematically corrected. However, correction is unnecessary if the information of interest is the area under the time-concentration curve.

Sensitive mass fragmentographic determination of acidic catecholamine metabolites in human body fluids

A sensitive and simple method for the simultaneous quantitation of homovanillic acid and vanilmandelic acid in small samples (0.1–1.0 ml) of urine, serum, plasma and cerebrospinal fluid is described. The stable dimethylthiophosphinyl methyl ester derivatives are detected specifically by mass fragmentography using the respective deuterated compounds as internal standards. Gas chromatographic separation is performed on a fused-silica DB-1 capillary column combined with a cold injection system for large sample volumes. Linear response curves and a detection limit of 1 ng/ml are obtained. The method has been applied to the localization of pheochromocytoma by selective determination of venous vanilmandelic acid.

Direct measurement of apoprotein C-III specific activity in 125I-labeled very low density lipoproteins using immunoaffinity chromatography.

We have developed a technique for isolating apoprotein C-III by immunoaffinity chromatography, allowing the measurement of its specific radioactivity in lipoprotein fractions from small plasma samples. IgG specific for apoC-III was purified from goat antisera and bound to Sepharose. One ml of this gel (5 mg of IgG) bound 80-90 micrograms of apoC-III. The specific activity of apoC-III was determined by application of delipidated very low density lipoproteins to 1-ml columns and analysis of the protein eluted at pH 2.5 for mass and radio-activity. The coefficient fo variation for apoC-III specific activity determination from 125I-labeled VLDL was 4.3%. Minimal contamination of the eluates by apoproteins B, E, and C-II was confirmed by radioimmunoassay (0.3-1.2%). Following the injection of autologous 125I-labeled VLDL, specific activity decay curves for VLDL apoC-III were biexponential, with the clearance of apoC-III being slower in hypertriglyceridemic subjects. These affinity columns can be used repeatedly and yield reproducible results. This technique should be useful for simultaneous studies of the turnover of several apoproteins in the same individual following a single injection of labeled autologous lipoprotein.

Rapid gas chromatographic measurement of diazepam and its metabolites desmethyldiazepam, oxazepam, and 3-hydroxydiazepam (temazepam) in small samples of plasma.

A rapid method was developed for the determination of diazepam, desmethyldiazepam, oxazepam, and 3-hydroxydiazepam (temazepam) in human plasma using electron capture gas chromatography. Following a single extraction with benzene from 0.2 ml plasma, diazepam and its metabolites were quantitated on a SP-2250 column with medazepam as internal standard. The method has a sensitivity limit of 4 ng diazepam, oxazepam, and desmethyl-diazepam, and 15 ng 3-hydroxydiazepam per ml of plasma.

Microprocedure to determine the polymorphic forms of acid α 1-glycoprotein in plasma : Application to depressive patients treated with amitriptyline

The polymorphic forms of α 1-acid glycoprotein have been determined by isoelectric focusing of small samples of whole plasma, without prior isolation of the protein. The results obtained by this technique confirm the microheterogeneity of this glycoprotein, which is not due to artefacts. Densitometric measurements of the polymorphic forms of this protein, which binds antidepressive drugs, have been performed in twelve depressive patients.

Celite multi-column chromatography for the simultaneous separation of progesterone, deoxycorticosterone and 17α-hydroxyprogesterone from small plasma or tissue samples

A rapid, inexpensive and reliable chromatographic method has been developed for the simultaneous separation of progesterone, deoxycorticosterone and 17α-hydroxyprogesterone on 15-cm Celite columns using n-heptane—benzene—methanol—water (100:35:80:20) as mobile phase. Six columns can be run in parallel within an hour with the assistance of nitrogen pressure. There is negligible column-to-column or batch-to-batch variation with consistent high recoveries. Columns are easily and quickly prepared and being inexpensive are disposable which is important when working with radioactivity. Using this method three important corticosteroids can be rapidly and reliably isolated from 20–30 small plasma or tissue samples per day.

Simultaneous determination of blood concentrations of methohexital and its hydroxy metabolite by gas chromatography and identification of 4′-hydroxymethohexital by combined gas—liquid chromatography—mass spectrometry

A simple, sensitive and selective method is described for the simultaneous determination of low concentrations (less than 50 ng/ml) of underivatized methohexital and its hydroxy metabolite in small (0.1 ml) samples of human and rat plasma or whole blood by gas chromatography with nitrogen-selective detection. Moreover, the main metabolite in rat and man was identified as 4′-hydroxymethohexital by comparison of chromatograms from gas—liquid chromatography (GLC) with data obtained from GLC—mass spectrometry and 1H-nuclear magnetic resonance spectrometry of this metabolite, produced both by incubating methohexital with isolated rat liver microsomes and by isolating this metabolite from rat urine.

A rapid, simple, and sensitive procedure for the determination of free fatty acids in plasma using glass capillary column gas-liquid chromatography.

A rapid and sensitive method for the analysis of plasma free fatty acids with glass capillary column gas-liquid chromatography and flame ionization detection is described. The plasma sample, together with n-pentadecanoic acid as an internal standard, was treated with 2,2-dimethoxypropane and hydrochloric acid. 2,2-Dimethoxypropane serves as water scavenger, deproteinizing agent, and as a methylating agent. Under the assay conditions, only free fatty acids were converted to their methyl esters; esterified fatty acids, such as those in triglycerides and phospholipids, were not significantly transmethylated. This advantage eliminated the need for thin-layer chromatography for the separation of free and esterified fatty acids. The methyl esters of fatty acids were then extracted into isooctane and analyzed with a 10-meter glass capillary column coated with SP-2100. Splitless mode of injection was used to increase the sensitivity. Only 20 microliter or less of plasma was required for analysis. The coefficient of variation was 4.6%, which was better than the conventional gas-liquid chromatographic methods. These latter methods require 20 to 50 times larger samples, as compared with the present assay. This method is suitable for the measurement of both total free fatty acids and individual fatty acid patterns in small plasma samples.

Measurements of the degradation products of radioiodinated proteins

This paper presents evidence that the fraction of radioactive iodide ( ∗I −) present in a sample of radioiodinated protein is grossly underestimated if determined by trichloroacetic acid precipitation in the absence of excess unlabeled iodide ion. In addition, the widely used separation of ∗I − from radioactive iodine-labeled iodotyrosine in supernatants of trichloroacetic acid precipitates, which proceeds by oxidation of ∗I − and subsequent extraction into chloroform (24), is dependent on the concentration of trichloroacetic acid used. Reproducibility and accuracy appear to be obtainable at concentrations of 10% (w/v) or higher. The modification of Goldberg's thin-layer chromatographic method (25) presented as an alternative method in the current paper offers the simplicity of separating ∗I −, ∗I-Tyr, and radioiodinated protein in a single step. The method can be used for small samples of urine, plasma, or tissue homogenates in which quantification of the labeled by-products from radioiodinated proteins is sought.

Changing patterns of steroid production in the fetus and placenta and their effects on development.

A variety of radioimmunoassays specifically characterized for use with fetal and maternal ovine plasma have been used to measure steroid hormone concentrations in small plasma samples drawn simultaneously from mother and fetus. By repeated sampling from the same animal preparation the changes over time of plasma concentrations of cortisol, pregnenolone and its sulphate, and oestrone and oestrone sulphate have been systematically studied. In animals delivering spontaneously at term the fetal plasma cortisol concentration rises between 120 and 130 days gestation and plays an important part in the maturation of several vital physiological systems, including the fetal thyroid axis. In normal term deliveries and premature deliveries induced by synthetic adrenocorticotropin (ACTH(1-24), 1 microgram h-1), the concentration of oestrone sulphate in maternal plasma increases before the fetal plasma concentration. Fetal and maternal oestrogens are probably important in the control of the low-grade tonic myometrial activity that occurs throughout gestation, in the initiation of labour and in the control of uterine blood flow. Low-grade tonic myometrial activity affects fetal oxygenation, fetal breathing and the fetal sleep state and may constitute a pathway through which the mother influences fetal development, with important physiological and possibly pathological consequences.

Quantitative high-performance liquid chromatography determination of ICI 125,211 in plasma.

A rapid, specific and quantitative high-performance liquid chromatography assay procedure is described for the determination of ICI 125, 211, (I), a guanidino-thiazole H2-receptor antagonist, in small samples of animal plasma. A 0.2 mL aliquot of plasma is extracted at pH 9 with ethyl acetate. After reconstitution in mobile phase, the sample is run on a silica column using ultraviolet detection at 280 nm. The procedure utilizes ICI 125, 253, (II), an analogous compound as the internal standard. The assay is linear (r2 = 0.99) and the recovery is 97.6 +/- 6.38% over a concentration range of 0.050-5.00 microgram/mL. Detection of levels as low as 0.025 microgram/mL has been documented in some animal study samples. Typical day-to-day precision is 3-9%, relative, for triplicate determinations of four concentrations in the 0.050-0.500 microgram/mL concentration range. Compound (I) is stable in whole blood for at least two hours at room temperature. Plasma concentrations remain constant for at least 48 hours at room temperature and six months at -5 degrees C. The whole blood/plasma ratio averages 0.97 +/- .073 for dog blood (N = 5). Pharmacokinetic data gathered for two dogs dosed orally with an HPMC-TWEEN 80 suspension of (I) at 40 mg/kg gave elimination half-life values of 4.3 and 7.4 hours. The specificity of the HPLC procedure has been confirmed by mass spectrometry.