Small Volumes Of Biological Fluids Research Articles

ANALYsis oF RYRRoLIDiNopHENoNE DERivATivEs iN BioLoGiOAL FLuiDs

Pyrrolidinophenone derivatives are the group of narcotic drugs controlled in the Russian Federation. The review presents the trends of biotransformation of а-pyrrolidinovalerophenone and 3,4-methylenedioxypyrovalerone, the data about their primary metabolites is provided. Various techniques of the sample preparation of biological fluids for analytical toxicology studies for substances of the pyrrolidinophenone derivative group are discussed. The use of enzymatic hydrolysis followed by solid-phase extraction (sorption) provides low detection limits for native sub- PHARMACY ФАРМАЦИЯ stances of this group and primary metabolites using small volumes of biological fluids (0.5 and 1.0 ng/ml blood for a-pyrrolidinovalerophenone and 3,4-methylenedioxypirovalonone respectively). The main characteristics of pyrro-lidinophenone derivatives (Kovac’s retention indices in nonpolar stationary liquid phases and the main characteristic ions in the mass spectra of electron impact) are presented. They allow to identify pyrrolidinophenone derivatives and their primary metabolites in biological fluids during chromatographic-mass spectrometric screening. Analytical possibilities of an alternative variant of screening for biological fluids i.e. analysis by using current immunochemical test systems, including “biochips” are discussed. The main methods of reliable identification and quantitative determination of pyrrolidinophenone derivatives are chromatography-mass spectrometry and high-performance liquid chromatography-tandem mass spectrometry. The detection limit of 3,4-methylenedioxypyralovalone in blood by gas chromatography-mass spectrometry is 1.0 ng/ml. The ranges of the determined concentrations of the method of quantitation by gas chromatography-mass spectrometry are 2.0-2000.0 ng/ml for blood and 0.05-50.0 ng/10 mm for hair. The high-performance chromatography-tandem mass spectrometry method with a triple quadrupole in the monitoring mode of multiple molecular reactions makes it possible to achieve a nearly complete suppression of analytical background “noise” for a sample, and to obtain detection and quantification limits for 3,4-methylenedioxypy-raloperone in cadaveric blood at a level of 10.0-100.0 pg/ml and 1.0-10.0 ng/ml, respectively. One of the advantages of the high-performance chromatography-tandem mass spectrometry system is the screening possibility.

Portable capillary electrophoresis instrument with contactless conductivity detection for on-site analysis of small volumes of biological fluids

A novel, easy to use and portable capillary electrophoretic instrument for injection of small volumes of biological fluids equipped with contactless conductivity detection was constructed. The instrument is lightweight (<5 kg), all necessary parts including a tablet computer are accommodated in a plastic briefcase with dimensions 20cm×33cm×17cm (w×l×h), allows hydrodynamic injection of small sample volumes and can continuously operate for at least 10hours. The semi-automated hydrodynamic sample injection is accomplished via a specially designed PMMA interface that is able to repeatedly inject sample aliquots from a sample volume as low as 10μL, with repeatability of peak areas below 5%. The developed interface and the instrument were optimized for the injection of biological fluids. Practical utility was demonstrated on the determination of formate in blood serum samples from acute methanol intoxication patients and on the analysis of ionic profile (nitrosative stress markers, including nitrite and nitrate) in the exhaled breath condensate from one single exhalation.

Improved high sensitivity analysis of polyphenols and their metabolites by nano-liquid chromatography–mass spectrometry

This study was conducted to assess the value of a high resolution, high mass accuracy time-of-flight analyzer in combination with nanoliquid chromatography for the analysis of polyphenols and their metabolites. The goal was to create a method that utilizes small volumes of biological fluids and provides a significant improvement in sensitivity compared with existing methods. Accordingly, nanoLC–MS and nanoLC-pseudo-multiple reaction monitoring (MRM) methods were developed that had a lower limit of quantification of 0.5nM for several polyphenols and were linear over 2–3 orders of magnitude (R2>0.999). Using urine samples, the ability to observe and quantify polyphenols in such a complex biological fluid depended on much narrower mass windows (0.050amu or less) on a TOF analyzer than those used on a quadrupole analyzer (0.7amu). Although a greater selectivity was possible with the low mass resolution of a triple quadrupole instrument using the MRM approach, for the daidzein metabolite O-DMA, a chromatographically resolvable second peak could only be substantially reduced by using a 0.01amu mass window. The advantage of a TOF analyzer for product ion data is that the whole MSMS spectrum is collected at high mass accuracy and MRM experiments are conducted in silico after the analysis.

Age-Dependent Changes in the Cerebrospinal Fluid Proteome by Slow Off-Rate Modified Aptamer Array

An important precondition for the successful development of diagnostic assays of cerebrospinal fluid (CSF) biomarkers of age-related neurodegenerative diseases is an understanding of the dynamic nature of the CSF proteome during the normal aging process. In this study, a novel proteomic technology was used to quantify hundreds of proteins simultaneously in the CSF from 90 cognitively normal adults 21 to 85 years of age. SomaLogic's highly multiplexed proteomic platform can measure more than 800 proteins simultaneously from small volumes of biological fluids using novel slow off-rate modified aptamer (SOMAmer) protein affinity reagents with sensitivity, specificity, and dynamic ranges that meet or exceed those of enzyme-linked immunosorbent assays. In the first application of this technology to CSF, we detected 248 proteins that possessed signals greater than twofold over background. Several novel correlations between detected protein concentrations and age were discovered that indicate that both inflammation and response to injury in the central nervous system may increase with age. Applying this powerful proteomic approach to CSF provides potential new insight into the aging of the human central nervous system that may have utility in discovering new disease-related changes in the CSF proteome.

Breakup of a liquid bridge as a method of rheological testing of biological fluids

A method for the on-line testing of small volumes of biological fluids is presented. The method is based on the ability of some liquids to form, when stretched, long thinning filaments, liquid bridges. Measuring the filament thinning rate makes it possible to determine the rheological parameters of the fluid. The method was tested on synthetic polymer solutions and biological fluids, such as saliva, bronchial sputum, follicular fluid, and chicken egg albumen and yolk. The results evidence that this technique can be used in various biomechanical applications, in particular, for disease diagnostics and treatment monitoring.

Miniaturization of a chloride ion assay for use in a microtiter format

We have miniaturized a common assay for the spectrophotometric determination of chloride in a variety of sample substrates, including biological fluids. The modified assay requires only 2 μL of serum, making it ideal for use with small animal systems for which only small volumes of biological fluids can be obtained. The assay utilized small amounts of inexpensive chemicals, and was shown to provide linear results for chloride up to 150 ppm, and the sensitivity was measured as low as 1 ppm. The assay was employed to measure chloride ion in the serum of alligators, thus allowing for the determination of dehydration status in animals that were exposed to saltwater for prolonged periods of time.

Determination of Volatile Organic Compounds in Biological Samples Using Headspace Solid-Phase Microextraction and Gas Chromatography: Toluene and Styrene

Epidemiological and laboratory investigations have shown that toluene and styrene are toxic compounds that lead to impairment of the nervous system. To quantitate toluene and styrene in biological samples, liquid-liquid phase, headspace (HS), and solid-phase microextraction (SPME) methods are generally used. Most of these methods are not sensitive enough for applications involving small sample volumes. Here, we present a method for quantitative analysis of low concentrations of styrene and toluene in very small volumes of biological samples using HS-SPME and gas chromatography (GC) equipped with a flame-ionization detector. The method was developed by optimizing operating parameters that affect the HS-SPME-GC process [i.e., desorption time (30 s), depth of the fiber in the GC injection port (3.7 cm), adsorption time (4 min), and adsorption temperature (room temperature)]. It has a wide range of linearity (0.5-500 ng/10 microL), high precision (coefficient of variation < 5%), good accuracy (deviation < 11%), and low detection limits of 0.13 and 0.08 ng/10 microL for styrene and toluene in serum, respectively. This analytical technique can be applied to the estimation of styrene and toluene in small volumes of biological fluids (blood, serum, and perilymph) and tissues of low lipid content (cochlea).

Measurement of cytokines in sweat patches and plasma in healthy women: Validation in a controlled study

Cytokines have been detected by ELISA in a variety of body fluids. Recycling immunoaffinity chromatography (RIC) coupled with laser-induced fluorescence detection is a highly sensitive and specific method, which allows simultaneous measurements of many analytes in small volumes of biological fluids. This method has been applied to plasma, cervical secretions and other body fluids, but has not previously been applied to sweat. The aim of this study was to validate the RIC methodology in sweat for measurements of IL-1α, IL-1β, IL-6, TNF-α, IL-8 and TGF-β. Two sweat patches were applied for 24 h on the torso, and blood was collected at one time point during this period in nine healthy women. Cytokines were measured in paired samples of plasma and sweat. Cytokines were detected in sweat in similar concentrations to plasma. Linear regression analysis confirmed that sweat levels of these cytokines accounted for a large percentages of variance in plasma levels: IL-1α ( R 2 = 0.70, p = 0.005), IL-1β ( R 2 = 0.79, p = 0.003), IL-6 ( R 2 = 0.52, p = 0.03), TNF-α ( R 2 = 0.95, p < 0.0001), IL-8 ( R 2 = 0.81, p = 0.001) and TGF-β ( R 2 = 0.94, p = 0.0003). These findings indicate that cytokine levels measured in sweat are informative of circulating levels and that sweat patches combined with RIC represents a viable non-invasive method to measure cytokines in ambulatory settings over time. This method is unobtrusive and requires minimal active compliance on the part of the subjects being studied, without pain or stress. This approach can open a new generation of studies to address the effects of environmental factors on immune responses in a wide range of different settings.

Donald Dexter Van Slyke (1883–1971): An Oral Biography

At the start of the 20th century, clinical chemistry emerged into its own space on the mosaic of medical practice. The pattern of its future growth and development took shape during the first two decades of the new century. Before that time, the United States had played no role in the growth or development of clinical chemistry, but at the turn of the century important work began in the US. One of the names that dominated this period was that of Donald Dexter Van Slyke. His systematic explorations on blood and urine led to practical and clinically applicable methods of analysis based on a new approach to methodology: analysis of small volumes of biological fluids. Using colorimetric and gasometric techniques, his work influenced the practice of medicine and played a major role in guiding the direction of clinical chemistry for the remainder of the century. I met Donald Van Slyke in August 1960 at the Fourth International Congress on Clinical Chemistry in Edinburgh, Scotland. I had presented a paper at an afternoon session after an earlier paper by Dr. Van Slyke. During a break for afternoon tea, a friend and I spotted Dr. Van Slyke sitting alone at a table and asked if we could join him. He welcomed us and started to tell us about his visits to various laboratories on the Continent before the Congress, especially about his reception at a laboratory in Germany. When introduced to the supervisor, he was greeted by shock and surprise: “Ach, du lebst noch!” (What! You’re still alive?), she exclaimed. Van Slyke could barely suppress his laughter. He thought this was a very funny story! This history, dealing mainly with Donald Van Slyke’s career at the Rockefeller Institute for Medical Research in New York, is derived from the transcript of a 5-h …

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of proteins in human cerebrospinal fluid.

Matrix-assisted laser desorption/ionization time-of-flight mass spectra of proteins in cerebrospinal fluid analyzed without prior purification are presented. Less than 100 fmol amounts of proteins in the 10,000 to 20,000 u mass range and linked to human disease (multiple sclerosis, Alzheimer's disease, and stroke) were detected in a complex mixture of proteins and peptides, in the presence of high concentrations of salts, lipids and free amino acids. The mass resolution was sufficient to distinguish between the non-hydroxylated and hydroxylated forms of a 13,400 u protein. Simple fractionation of the cerebrospinal fluid using microbore-reversed phase high performance liquid chromatography improved signal-to-noise ratios in the mass spectra. High-accuracy peptide mass mapping and database searching were utilized to confirm the identity of several proteins. The presented results show that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry could be used as a tool to perform rapid screening of chemically altered proteins in small volumes of biological fluids.

Enhanced chemiluminescent assay for measuring the total antioxidant capacity of serum, saliva and crevicular fluid.

This paper reports the development of an enhanced chemiluminescent (ECL) assay for measuring the total antioxidant (AO) capacity of serum, saliva and a fluid collectable from the gum margin called gingival crevicular fluid (GCF). The theory behind the assay is explained, and the optimum conditions for the assay, and for storage of reagents and clinical samples is described. Calibration lines were linear (R > or = 0.99; P < 0.0001) and the within batch coefficient of variations for a water soluble vitamin E analogue (Trolox), serum and saliva samples were < 5%. In saliva and GCF, a characteristic AO response not seen in serum of the same patients, was identified. Total peripheral (serum) and local (saliva) AO capacities (mumol/L Trolox) were investigated in patients with (n = 18) and without (n = 16) adult periodontitis. Serum AO status did not differ between groups. Salivary total AO concentrations were lower in the peridontitis (P) group [175 (53) mumol/L] than in the non-periodontitis (NP) group [254 (110) mumol/L1: P < 0.01], as were saliva:serum AO ratio's [0.37 (0.11) versus 0.5 (0.18): P < 0.01]. Periodontitis patients may have a reduced salivary AO concentration, which could result from, or predispose to, the damaging effects of reactive oxygen species (ROS). The potential for ROS production in the oral and periodontal environment may explain the presence of a specific antioxidant in oral fluids that is not detectable in serum. The ECL assay described provides a rapid, simple and reproducible method of measuring total antioxidant defence in small volumes of biological fluids.

The Use of Bamifylline as Internal Standard in the Reversed Phase HPLC Analysis of Mefenamic Acid in Pharmaceuticals and Small Volumes of Biological Fluids

Abstract The reversed-phase liquid chromatographic determination of mefenamic acid in pharmaceutical preparations and small volumes of biological fluids, blood serum (40 μl) and urine (100 μl) is developed. The biological fluids containing internal standard passed through C18 cartridges, Bond Elut, for sample clean-up and subsequent separation of bamifylline and mefenamic acid from endogenous interfering compounds. HPLC analyses are performed with a Lichrosorb-RP 18, 10 μm, 250x4 mm I.D. column, employing gradient elution for the development of the chromatogram. The gradient system is formed with the following solutions: A = 0.05 M ammonium acetate and B = methanol. The chromatogram is started with solvent A containing 78% solvent B, followed by a sharp decrease from 78 to 72% in 5 min. The flow rate is 0.81 ml/min and the detection is achieved at 285 nm. The retention time is 3.90 min for mefenamic acid and 4.93 min for the internal standard, bamifylline. Thus the problems existing with caffeine in blood...

Quantitative Trace Element Analysis of Micro-Samples by SIMS

A number of researchers have applied secondary ion mass spectrometry (SIMS) in various areas of medical research. The potential of SIMS as an analytical tool in elemental microanalysis of biological specimens lies in its sub-part per million (ppm) elemental detection sensitivities and its ability to generate two and three dimensional distributions of the intensities of mass selected secondary ions. Established techniques such as inductively coupled plasma (ICP) and electron probe microanalysis (EPMA) are also capable of sub-ppm elemental detection limits. ICP detection limits are adversely affected as the sample volume decreases below 1 milliliter. The EPMA technique can achieve ppm detection limits with volumes in the 100 picoliter range but its detection limits do not decrease as the sample volume increases. SIMS combines excellent detection sensitivities with small sample masses (picogram) which makes it a very useful complementary technique to ICP and EPMA especially for minor and trace element level analyses in small volumes of biological fluids.

Blood steroid levels in the goldfish: Measurement of six ovarian steroids in small volumes of serum by reverse-phase high-performance liquid chromatography and radioimmunoassay

A reliable and rapid technique for the measurement of estradiol-17β, testosterone, progesterone, 17α-hydroxyprogesterone, 17α,20β-dihydroxy-4-pregnen-3-one, and cortisol by reverse-phase high-performance liquid chromatography and radioimmunoassay in single female goldfish serum samples of 50 μl was developed. The steroids were extracted with Sep-Pak C18 cartridges after heat treatment. While estradiol-17β was assayed directly after extraction, the other steroids were separated on a μBondapak C18 stainless-steel column with acetonitrile:water (53:47, v/v) in 17 min under isocratic conditions, and then quantitated by specific radioimmunoassays. The technique was validated and was shown to be highly accurate, precise, sensitive, and specific for measuring those particular ovarian steroids in goldfish serum. The steroid levels measured in this way were not significantly different from those measured after separation on a Nova-Pak C18 column with methanol:water (56:44, v/v), where all the individual steroids could be completely resolved during a 40-min run. With this technique, the steroid levels in the serum of the goldfish during the secondary yolk stage, the tertiary yolk stage, and at 0 hr after ovulation were determined. While estradiol-17β was found to be significantly higher in the tertiary yolk stage, testosterone was significantly higher in the secondary yolk stage than in the other two stages. There was no significant difference in the levels of cortisol, 17α-hydroxyprogesterone, 17α,20β-dihydroxy-4-pregnen-3-one, and progesterone among the three stages. We conclude that the technique is particularly useful for assaying multiple steroids in very small volumes of biological fluids.

Volume and density changes of biological fluids with temperature.

High-precision (10(-5) g/ml) mass density measurements on human blood, plasma, plasma ultrafiltrate (using PM-10 membranes), and erythrocyte concentrate samples were performed with the mechanical oscillator technique. Measurement temperatures varied between 4 and 48 degrees C and were accurate to +/- 1 X 10(-2) K. The coefficient of thermal expansion (beta), defined as relative volume change with temperature, was calculated. It was shown that beta increases with temperature in these fluid samples over the entire temperature range investigated; the magnitude of this increase declines with increasing temperature; beta increases with density at temperatures below 40 degrees C but is independent of density above 40 degrees C; and the beta of the intracellular fluid has about twice the value of the beta for extracellular fluid at low (4-10 degrees C) temperatures but is equal for both fluids at greater than or equal to 40 degrees C. The mechanical oscillator technique provides data with an accuracy sufficient to perform precise (10(-5) K) calculations of beta of small volumes of biological fluids.

X-ray microanalysis of picoliter microdroplets: Improvement of the method for quantitative X-ray microanalysis of samples of biological fluids

Abstract A new practical approach to quantitative X-ray microanalysis of very small volumes of biological fluids, combining wavelength dispersive analysis and the use of thin films as supports is presented. The results from this technique are compared with those obtained using earlier methods and were found as predicted from theoretical considerations to be very accurate. Because of the favourable ratios between the detected characteristic radiation and the background radiation coupled with the resultant shorter periods of analysis, the use of this new technique of X-ray microanalysis for liquid samples not only saves time, but also smaller elemental concentrations can be detected. Suitable procedures to subtract background radiation are presented and discussed.

Column elution and concentration of volatile compounds in biological fluids

A new solvent elution method is described for the isolation and concentration of volatile metabolites in biological fluids. The procedure involves elution of the sample with a solvent through a micro-column of an adsorbent into glass-wool and evaporation of the excess solvent in a stream of helium at room temperature. The volatiles are recovered by subsequent heat desorption into a chromatographic system. Both elution and evaporation are made in a single step using a novel sampling device. Since the technique is based on liquid-solid adsorption chromatography, the adsorbent-solvent system which gives the maximum yield of volatile constituents was studied. The method is simple, highly efficient and reproducible, requiring only small volumes of biological fluids (less than 100 μl).

Laboratory Manual of Pediatric Microbiochemical Techniques, ed. 4, by Donough O'Brian, M.D., F.R.C.P.E., Frank A. Ibbott, Ph.D., F.I.M.L.T., and Denis O. Rogerson, M.S., F.I.M.L.T. New York/Evanston/London: Hoeber Medical Division, Harper and Row, 1968, 367 pp., $11.90

This manual of micro-techniques is one of the finest compendiums of clinical laboratory methods which is currently available in the English language. The analytical procedures have been selected with consistent care to ensure their reliability as well as their applicability to the relatively small volumes of biological fluids which can be obtained from infants and young children. The procedural instructions are written clearly and succinctly, so that they can readily be followed by experienced laboratory workers.