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Quaternary nitrogen muscle relaxants pancuronium, rocuronium, vecuronium, gallamine, suxamethonium, mivacurium, and atracurium and its metabolites were extracted from whole blood and other biological fluids and tissues by using a solid-phase extraction procedure. The extracts were examined by using high-performance liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS). The drugs were separated on a ODS column in a gradient of ammonium acetate buffer (pH 5.0) and acetonitrile. Full-scan mass spectra of the compounds showed molecular ions, and MS-MS spectra showed fragments typical of the particular compounds. LC-ESI-MS allowed an unequivocal differentiation of all muscle relaxants involved. The method was applied in a case of rocuronium and suxamethonium administration in a Caesarian section and in a case of intoxication by pancuronium injection. In both cases, the administered drugs could be detected and identified in the supplied samples.

Broad-spectrum drug screening requires that all relevant substances be isolated, detected, and identified, regardless of their structure and/or polarity. To this end, systematic solid-phase extraction (SPE) approaches for drug isolation from biological fluids are required. Because speed and cost effectiveness are key issues in analytical toxicology, we have evaluated a disc-format extraction device for this purpose and compared the latter with an existing packed-bed column-format method. The discs were SPEC.PLUS.C18AR/MP3 cartridges with 10-mL solvent reservoirs, providing hydrophobic and cation exchange interactions. Blank human urine was spiked at 2 microg/mL with a selection of acidic, neutral, and basic drugs representing a variety of relevant drug classes. Urine specimens (2 mL) were diluted with 2 mL 0.1 M phosphate buffer (pH 5.0) and then applied to the preconditioned disc. Washing was done with 1 mL water. Acidic and neutral drugs were eluted with 1 mL ethyl acetate/acetone (1:1), and basic drugs were eluted with 1 mL ammoniated ethyl acetate. The eluates were collected separately, evaporated down to about 0.1 mL, and analyzed by gas chromatography-flame-ionization detection to check cleanliness, recoveries, and reproducibilities. The discs showed good extraction properties for all drugs and were easy to handle. Recoveries were 75-100% with coefficients of variation of around 5%. The resulting eluates showed only a few matrix interferences. As compared to our standard SPE method with packed-bed columns, the disc procedure allowed reductions in elution volumes and total processing time of approximately 60-65%.

In the present paper, disk extraction was evaluated for the rapid isolation of clenbuterol from human and calf urine, followed by high-performance liquid chromatography analysis with UV detection. A method was developed for the extraction with standard density C8 disks. The disks could be washed with 25% methanol in 0.01M sodium hydroxide without significant losses of clenbuterol. The recovery of denbuterol was about 85%, and the extracts were clean. The detection limit was about 10 ng/mL. The main advantages of these disks were the saving of time and the reduced amounts of organic solvents needed.

During our evaluation of ion-mobility spectrometry (IMS) screening for the abuse of the beta-agonist clenbuterol during fattening in cattle, we found that clenbuterol could not be detected with the instrument in extracts obtained by solid-phase extraction or ion-pair extraction, although the recovery for these procedures was at least 85%. Several experiments showed that the signal loss occurred mainly during the evaporation of the organic solvent. Again, it could be shown that no clenbuterol was lost during this step. On further investigation, we found that so-called solvent residues, which are left after the evaporation of the organic solvent, caused a significant reduction of the clenbuterol signal. Other reagents used in our sample pretreatment procedures were also found to reduce the signal. For example, the signal reductions caused by the different reagents used for the ion-pair extraction of clenbuterol from human urine were determined. We think that IMS is only useful for our purpose if a chromatographic preseparation is performed.

Analysis of liver specimens is an important issue in forensic toxicology, but suitable workup and extraction methods for general screening purposes have been lacking until now. A workup and extraction scheme based on a recently developed procedure for the screening of biological fluids was developed that can be used for the screening of acidic, neutral, and basic drugs in liver. This method uses a single solid-phase extraction (SPE) column and gas chromatography-flame ionization detection (GC-FID) for the final analysis. First, the homogenized liver sample is sonicated and centrifuged; the resulting supernatant is applied to the SPE column. Elution of acidic, neutral, and some weakly basic drugs is then performed with acetone-chloroform and analyzed by GC-FID. Next, the pellet of tissue material obtained from the centrifugation is enzymically digested by subtilisin Carlsberg. This frees the drugs bound to the liver tissue. The resulting clear liquid is brought to the reconditioned SPE column. A wash step is introduced to remove acidic and neutral interferences and the basic drugs can then be eluted with ammoniated ethyl acetate. Using 100-mg wet liver samples spiked with 2 micrograms of amounts of various drugs, recoveries were 70-102% with relative standard deviations less than 9%. The resulting GC-FID chromatograms were virtually free of endogenous interferences. GC-nitrogen-phosphorous detection detected smaller amounts of nitrogen-containing drugs, again without endogenous interferences. With the SPE columns currently used, which contain a bed mass of 130 mg, the liver samples should be smaller than 200 mg because the endogenous compounds obtained after the digestion of the tissue will overload the column, which results in a lower recovery of the drugs of interest. Drugs that decompose under the digestion conditions (pH 10.5 at 60 degrees C for 1 h) may be lost in the present procedure. This phenomenon is being investigated further.

Because thin-layer chromatography can be affected by climatic conditions, we investigated the impact of high temperatures (33-38 degrees C) and high relative humidities (80-100%) on the applicability of Toxi-Lab thin-layer chromatographic systems for the screening of basic and neutral drugs during a 6-month climatological cycle in Jakarta, Indonesia that can be characterized as hot and humid. In general, the Rf values as observed on the plate showed substantial deviations from those in the literature obtained in moderate climates. Most substances gave higher Rf values, and the deviations (sometimes amounting to 20 Rf units) increased with increasing humidities. However, when the observed Rf values were corrected by means of a reference mixture of standard drugs that was run on each plate, much better accuracy and reproducibility were obtained. Thus, these corrected Rcf values were compatible with the existing databases developed in temperate climates. These results are in line with earlier studies carried out under hot and dry conditions. In the earlier studies, the observed Rf values tended to be lower than those present in other databases, but here too, the Rf correction procedure corrected this phenomenon.

Determination of basic drugs from biological matrices at concentration levels of 100-200 ng/mL was accomplished by using mixed-mode Bond Elut Certify columns and a gas chromatograph equipped with a nitrogen-phosphorus detector. The extraction procedure developed for general drug screening on a GC-FID system was found suitable for the extraction of basic drugs from plasma and whole blood for GC-NPD analysis. For urine samples, an extra wash step with 20% acetonitrile in water was required to obtain clean extracts. The recoveries of 18 basic drugs ranged from 77.7 to 104.5%, with relative standard deviations less than 7.0%.

A fully automated solid-phase extraction (SPE) method for drug screening is described. The extraction of 19 toxicologically relevant drugs from pretreated plasma and pretreated whole blood was accomplished automatically by a Gilson ASPEC system equipped with disposable 2.8-mL Bond Elut Certify columns. The automated extraction procedure includes 11 fundamental steps: column preconditioning; sample application; column washing; pH adjustment; elution of drugs by two eluents, which were collected into two separated tubes; addition of the chromatographic standard solution; and several SPE column rack movement steps. After evaporation, the drugs were quantitated by gas chromatography. Water was chosen as the transfer liquid in the ASPEC system because it was cheap and, more importantly, because it caused no protein precipitation problem. The effects of the sample and eluent flow rates were investigated, and it was found that low flow rates were necessary to recover the drugs maximally. In the study, the optimal flow rates of sample application, acetone-chloroform elution, and ammoniated ethyl acetate elution were 1.5, 0.72, and 0.33 mL/min, respectively. The absolute recoveries of 19 drugs from whole blood exceeded 82%, with relative standard deviations less than 5% at 2 micrograms/mL.

A solid-phase extraction procedure was developed for the isolation of acidic, neutral, and basic drugs from whole blood. A blood sample was sonicated, diluted with phosphate buffer, and the drugs were extracted on a mixed-mode bonded-phase silica column at pH 6.0. The extraction system was adjusted to pH 3.3 with acetic acid. After column drying, the drugs were selectively eluted from the column by two different eluates, which were collected separately. Acidic, neutral, and weakly basic drugs with lower pKa values (e.g., benzodiazepines) were present in the first acetone-chloroform (1:1) fraction. The other basic drugs were present in the second fraction (basic ethyl acetate). The drugs with pKa's close to the pH of the extraction system (pH 3.3) appeared in both fractions. The two fractions were evaporated until approximately 100 microL of solvent remained in the tube and were then analyzed on a gas chromatograph equipped with a wide-bore capillary column and flame ionization detector. The absolute recoveries of all tested drugs exceeded 81% at a concentration of 2 micrograms/mL.

Over the years, numerous TLC systems have been developed, each of them having their special advantages. Yet little or no attention has been paid to the time-factor needed to obtain sufficiently reliable Rf-values, combined with satisfactory resolution. As speed remains an important factor in toxicology, the applicability of shorter development distances have been investigated, resulting in a considerable gain in time as the development times increase exponentially with developing distance. In this paper, the effects of shorter development distances on resolution, spot size, reproducibility of Rf-values, and sensitivity of detection are described.

Four column packings for screening volatiles in biological material by gas chromatography are evaluated. Retention data are standardized by the calculation of retention indices, and packing materials are compared by discriminating power and identification power. A combination of 5% Carbowax 20M on Carbopack B and 0.3% Carbowax 20M on Carbopack C appears to be best suited for screening. Hydroxy-n-alkanes are used as reference substances for the calculation of retention indices.

Samples of autopsy blood and liver were spiked individually with aminophenazone, p-aminosalicylic acid, clordiazepoxide, clonazepam, cyclobarbital, furosemide, medazepam, phenacetin, phenazone, and phenobarbital and extracted with diethyl ether at pH 5. The extracts, as well as solutions of pure drugs, were developed in three thin layer chromatographic systems: chloroform:acetone (80:20), ethyl acetate:methanol:ammonia (85:10:5) and chloroform:methanol (90:10). The investigations performed in parallel in two laboratories showed that the intra- and inter-laboratory variability of RF values is larger for drugs extracted from liver. The biological matrix affected both precision and accuracy of results. The number of analysts involved in TLC procedures also affected the intra-laboratory precision.

The retention indices of eight basic and neutral drugs extracted from plasma, blood, or liver homogenate by six different methods were calculated using temperature-programmed gas chromatography on OV-1 and SE-30 columns. Two concentrations were applied: 1 and 50 mg/L, respectively. The type of extraction had great influence on recovery and analytical background. The retention indices tended to be slightly concentration-dependent in that they were somewhat lower at higher drug concentration. Neither the biological sample nor the type of extraction exerted significant influence on scatter and mean value of the retention indices.

The retention behavior for a selection of acidic, neutral, and basic drugs was examined on four fused silica capillary columns in a splitless injection mode. Mixtures of n-alkanes and diisopropylamino-alkanes were also analyzed. The concentrations used ranged from 0.25-1000 ng/microL and injection volumes were either 2 or 4 microL. This corresponded to quantities of 1-4000 ng injected on the column. All substances showed concentration-dependent behavior: after being nearly constant or showing a decrease at low concentrations, retention times markedly increased when concentrations exceeded about 100 ng/microL (400 ng injected). At very high concentrations, peak splitting occurred. In the more pronounced cases, the differences caused by this concentration effect could reach values of over 100 retention-index units.

The retention behavior of eight basic and neutral drugs, extracted from plasma, blood, and liver by five different methods (XAD-2, Extrelut, Elut-X, Elut-C18, chloroform) and developed in three chromatographic systems [MeOH, Me OH:BuOH:NaBr, CHCl3:MeOH (KOH)] was observed in parallel in two laboratories. The corrected Rf-values were compared with reference data from a data base with data for pure drugs. The biological matrix and/or the extraction severely lowered the precision and, to a lesser extent, the accuracy of the Rf-values as compared to pure drug data and to reference Rf-values. The intra- and interlaboratory variation was smallest in the MeOH system and largest in the CHCl3:MeOH (KOH) system. The observed irreproducibility is caused by the biological matrix (extraction has a large negative impact on the potentials of TLC in identification procedures). Precision and accuracy of extracted drugs were independent of the biological matrix and on the extraction method used.

A wide-bore capillary column with a methylsilicone stationary phase has been evaluated with regard to its ability to identify substances by means of their retention indices. Column efficiency was compared with a normal packed column and a medium-bore capillary column, and the impact of carrier gas flow and load capacity were investigated. It was shown that under defined conditions retention indices on the wide-bore capillary column were comparable to those available in a data bank obtained on packed columns.

Correction of crude thin layer chromatographic data usually enables a reliable comparison with reference data bases. However, when the correction standards are not spotted in the right positions, the calculation may produce false results if anomalous migration occurs. This was observed in the case of acidic and neutral drugs developed in the chloroform-methanol (9:1) system.

In systematic toxicological analyses (STA), analytical methods are needed with a high Identification Power. The mean list length (MLL) approach, which is an improved and extended version of the Identification Power concept, provides an objective tool for the evaluation and optimum choice of analytical methods for STA. The MLL approach is elucidated and applied to the screening for basic drugs by thin layer chromatography (TLC) and gas-liquid chromatography (GLC). Its usefulness for TLC and GLC systems for the identification of these basic drugs has been evaluated for both single systems and combinations of systems. The MLL approach is also substance directed and can be applied to computerized data searches and substance identification. The method is applicable for single analytical systems and combinations of systems. Mixtures of substances can also be handled.