Maciej Bogusz

The determination of drugs of abuse in whole blood by means of FPIA and EMIT-dau immunoassays — A comparative study

Six groups of common drugs of abuse (cannabinoids, benzoylecgonine, opiates, barbiturates, benzodiazepines and amphetamines) were determined in whole blood after acetone precipitation, using enzyme multiplied immunoassay (EMIT dau) and fluorescence polarisation immunoassay (FPIA--Abbott TDx and ADx) methods. Both methods, designed primarily for urine, allowed the determination of all above mentioned class of drugs but amphetamine. Only 1 ml of a pre- or postmortem blood sample was needed. The sensitivity of cannabinoids determination was higher by FPIA. The FPIA method gave more precise results, particularly in the case of autopsy blood. The method was applied for drug screening in autopsy and police blood samples. The results (both positive and negative) were in agreement with those obtained with chromatographic methods.

Improved standardization in reversed-phase high-performance liquid chromatography using 1-nitroalkanes as a retention index scale.

A retention index scale based on 1-nitroalkanes was introduced for reversed-phase high-performance liquid chromatography. The retention behaviour of nitroalkanes was examined using two different ODS-silica columns. The nitroalkanes are stable compounds having high UV absorbances at 200-220 nm. The correlation between the logarithm of the capacity factor and the carbon number was linear from the second homologue. The compounds up to 1-nitrohexane are easily available commercially, and they cover the retention range for the majority of toxicologically relevant substances. The retention indices of several test compounds showed that the scale based on 1-nitroalkanes is comparable to that based on alkyl aryl ketones, and additionally may cover the early eluted compounds, for which reference compounds on the alkyl aryl ketone scale are not available.

Determination of serum vitamins 25-OH-D2 and 25-OH-D3 with liquid chromatography-tandem mass spectrometry using atmospheric pressure chemical ionization or electrospray source and core-shell or sub-2 μm particle columns: a comparative study.

OBJECTIVE Develop a simple LC-MS-MS method for determination of 25-hydroxymetabolites of vitamin D2 and D3 in serum. DESIGN AND METHODS 0.1 mL serum was mixed with 0.3 mL deproteinizing mixture containing two deuterated internal standards. Two LC-MS-MS systems were used: Waters Premier with an ESI source and a Hypersil Gold RP 18 50 × 2.1 mm column, and a Thermo Quantum, with an APCI source and a Kinetex RP 18 core-shell 50 × 2.1 mm column. Certified reference material 972 from NIST was used for method calibration. The samples were additionally analyzed with commercial HPLC procedure. RESULTS Baseline separation of compounds was achieved in 6 min run time. Both systems have limits of detection of 0.5 nmol/L and recovery ranging from 86% to 112%. The measurement of 25-OH-D3 gave comparable results for all procedures; the results of 25-OH-D2 measurement obtained with the HPLC often differed from those obtained with LC-MS-MS methods. CONCLUSIONS Established procedure performed well for ESI and APCI sources and totally porous and core-shell LC columns. The use of two internal standards enhanced the accuracy of the measurements.

Use of Corrected Retention Indices Based on 1-Nitroalkane and Alkyl Arylketone Scales for HPLC Identification of Basic Drugs

Sixteen basic drugs were examined by HPLC (gradient elution in acetonitrile/phosphate buffer, pH 3.2, containing 0.05% nonylamine) with six different ODS-silica columns. The retention indices (RI) were calculated with alkyl arylketone and 1-nitroalkane scales and were subjected to correction, which enabled comparison of results from commercially different column packing materials. The correction procedure was successful for the 1-nitroalkane scale. The scale based on alkyl arylketones was of less use for basic drugs, because some of them eluted earlier than the first reference homologue. This made impossible the proper calculation and correction of RI values for drugs such as cocaine, diphenhydramine, doxepin, and promethazine. The correction procedure of RI values calculated against the series of 1-nitroalkanes is recommended as a method of standardization of HPLC data.

Studies on the formation of ethanol and of pyruvate as its precursor from some di- and tricarbonic compounds in putrefying blood in vitro

Abstract The study carried out on putrefying post-mortem blood under anaerobic conditions at room temperature showed that dicarbonic compounds (glycine, ethanolamine, glycollate and glyoxalate) added in vitro to blood samples cannot be converted to ethanol during putrefaction. Tricarbonic, glucogenic amino acids (alanine, serine and cysteine) also had no influence on pyruvate or ethanol formation. Deamination and transamination of these amino acids is limited by anaerobic conditions. Alanine could be converted to pyruvate in the course of transamination only after addition of exogenous α-oxoglutarate. The experiments reported demonstrate that, despite high transaminase activity in putrefying blood, the conversion of glucogenic amino acids into pyruvate and ethanol under anaerobic conditions is practically impossible, owing to the lack of α-oxoglutarate as acceptor of amino groups.

ISOLATION OF DRUGS FROM BLOOD AND TISSUES WITH XAD-2 BAGS

Nylon bags containing 2-g portions of Amberlite XAD-2 resin were used for systematic analysis of drugs in biosamples. The procedure requires 10 or less grams of material, two XAD-2 bags, and enables rapid and economical isolation of most common drugs. The method was demonstrated on autopsy blood spiked withe 19 of the most common drugs, and was routinely used in cases of fatal and non-fatal poisoning. The eluates were clean and suitable for direct gas chromatographic and ultraviolet spectrophotometric analysis. The procedure used appeared more convenient than XAD-2 column extraction procedures. Classic solvent extraction methods were usually less efficient.

The usefulness of the enzymatic tests in acute poisonings

In the group of 107 patients poisoned by carbon monoxide (18 patients), ethanol (10), barbiturates (18), glutethimide (10), tranquilizers (19), organic solvents (10), salicylates (3), organochlorines (8), and sulfonamides (5) — the activities of 8 serum enzymes were determined for 6 consecutive days of treatment, the enzymes being as follows: aminotransferases, cholinesterase, alkaline phosphatase, lactate, α-hydroxybutyrate, glutamate, and sorbitol dehydrogenase. The antipyrine half-life was also assayed.It has been shown that the poisonings by particular groups of poisons do not bring about characteristic changes in the activity of enzymes that might be of any diagnostic value. The intensity of changes was connected with the depth and duration of toxic coma. Most frequently an increase ensued in the activity of AspAt and AlAt in the third 24-hrs period, and an increase in the activity of SDH in the first 24-hrs period.In the group under examination there were 26 drug abusers in whom a shortening of the antipyrine half-life was discovered. They were less responsive to toxic doses of drugs, and the enzymatic changes in them were less distinct. No changes in the activity of tested enzymes, which are characteristic of toxicomania, were found.ZusammenfassungIn einer Gruppe von 107 Kranken, mit akuter Kohlenmonoxydvergiftung (N=18), Vergiftung durch Äthanol (N=10), Barbiturate (N=18), Gluthetimid (N=10), Tranquilizer (N=19), organischen Lösungsmitteln (N=10), Salicylaten (N=9), chlorierten Insecticiden (N=8) und Sulfonamiden (N=5), wurden die Aktivitäten von 8 Serumenzymen an 6 aufeinanderfolgenden Tagen nach Vergiftungsbeginn bestimmt. Es wurden folgende Enzyme bestimmt: Aminotransferasen, Cholinesterase, alkalische Phosphatase, Lactat-, Hydroxybutyrat-, Glutamat- und Sorbitol-Dehydrogenase. Außerdem wurde die Antipyrin-Halbwertzeit bestimmt.Vergiftungen mit einer bestimmten Gruppe von Giften rufen keine charakteristischen Veränderungen der Enzymaktivitäten hervor. Die Intensität der Veränderungen war mit der Tiefe und Zeitdauer des toxischen Coma korreliert. Am häufigsten wurde die Aktivitätserhöhung von AspAt und AlAt während des dritten Tages und der SDH während des ersten Tages der Behandlung beobachtet.In der beobachteten Krankengruppe gab es 26 Drogensüchtige, bei denen eine Verkürzung der Antipyrinhalbwertszeit nachgewiesen wurde. Diese Kranken waren weniger empfindlich gegenüber toxischen Dosen, die Enzymanstiege waren bei ihnen weniger prägnant. Es wurden keine für Toxikomanie charakteristischen Veränderungen der Aktivitäten der untersuchten Enzyme gefunden.

Rapid determination of ethylene glycol in biological material

SummaryA rapid and simple modification of a gas chromatographic (AFID) method of ethylene glycol determination, based on derivation with n-butylboronic acid, was developed.ZusammenfassungEs wird eine Modifikation zur schnellen Bestimmung von Glykol mit der Methode der Gaschromatographie mit Hilfe von Veresterung der n-Butylboronsäure dargestellt.

Enzymic hydrolysis of tissues before XAD-2 extraction in poisoning cases

Samples of blood, liver and brain taken from 13 cases of fatal poisoning with barbiturates, antidepressants, phenothiazines and anticonvulsants were digested with trypsin and beta-glucuronidase. The concentrations of drugs were higher in hydrolyzed livers in barbiturate and phenothiazine poisonings. In the case of diazepam, phenytoin, carbamazepine, amitriptyline, imipramine and chloroprothixene poisonings the results obtained after enzymic hydrolysis were the same or worse than in controls. The blood levels of drugs in non-fatal poisonings with diazepam, doxepin and chlorpromazine were similar in samples digested with trypsin and in controls. All samples were extracted with Amberlite XAD-2 resin by XAD-2 Bag technique.

Isolation of drugs from autopsy material by XAD-2 adsorption-elution technique. A routine procedure.

The method of adsorption of drugs on Amberlite XAD-2 resin, followed by differential elution was under study during 1 1/2 year of routine use. The method allows a separation of acidic and basic drugs and — due to acid hydrolysis step before adsorption — assures better recovery of conjugated and proteinbound drugs. The amounts of various drugs, found in autopsy cases by the XAD-2 method were usually higher than those found by solvent extraction. The method applied requires 20 g of sample (biofluid or tissue) for general toxicological analysis.ZusammenfassungDie Methode der Arzneiadsorption mittels des XAD-2-Harzes mit der nachfolgenden differenzierten Elution wurde in 1 1/2 Jahre dauerndem Gebrauch untersucht. Diese Methode ermöglicht es sauere und basische Arzneien abzufremmen und ergibt eine bessere Ausbeute für die gebundene Drogen, weil der sauren Hydrolyse die Adsorption vorangeht. Die Ausbeute der bei letalen Vergiftungen anhand der XAD-2-Methode ausgemittelten Arzneisubstanzen war in der Regel höher als bei der Extraktion mit Lösungsmitteln. Die hier verwendete Methode erfordert 20 g des biologischen Materials für eine allgemeine toxikologische Analyse.