SPEC disc solid-phase extraction for rapid broad-spectrum drug screening in urine.

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%.

An enzymic digestion and solid-phase extraction procedure for the screening for acidic, neutral, and basic drugs in liver using gas chromatography for analysis.

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.

Determination of basic drugs extracted from biological matrices by means of solid-phase extraction and wide-bore capillary gas chromatography with nitrogen-phosphorus detection.

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%.

Pitfalls and solutions in the development of a fully automated solid-phase extraction method for drug screening purposes in plasma and whole blood.

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.

Reusability of Bond Elut Certify columns for the extraction of drugs from plasma.

The reusability of Bond Elut Certify columns for the extraction of toxicologically relevant drugs from plasma has been evaluated. Pentobarbital, hexobarbital, mepivacaine, trimipramine and clonazepam were selected as test drugs to represent various classes of drugs. The columns were regenerated immediately after an extraction by washing with methanol, hydrochloric acid (1%), water and methanol, sequentially. These regenerated columns were found to be reusable for plasma. More than 85% of the test drugs were recovered when the columns were used three times. However, the extraction power of the regenerated columns decreased slightly with the number of reuses, so that a column should not be used more than two or three times.

Study of lot-to-lot reproducibilities of Bond Elut Certify and Clean Screen DAU mixed-mode solid-phase extraction columns in the extraction of drugs from whole blood.

The lot-to-lot reproducibilities of Bond Elut Certify and Clean Screen DAU columns are described. The recoveries of five test drugs obtained from twelve lots of Bond Elut Certify columns ranged from 84 to 104% with standard deviations of less than 9%. The recoveries of five test drugs obtained from six lots of Clean Screen DAU columns ranged from 81 to 103% with standard deviations of less than 7%. The 95% confidence intervals of the means as obtained by ANOVA demonstrate that there are no significant differences between the tested lots of Bond Elut Certify and Clean Screen DAU columns. Comparison of the two brands shows that both Bond Elut Certify and Clean Screen DAU columns are well acceptable for routine drug screening in systematic toxicological analysis, with a slightly higher overall recovery for the former.

Semi-automated solid-phase extraction procedure for drug screening in biological fluids using the ASPEC system in combination with Clean Screen DAU columns.

The use of a semi-automated solid-phase extraction system (ASPEC) for the screening of drugs in plasma and urine on a single mixed-mode column (Clean Screen DAU) is described. The processes of column preconditioning, sample application, column wash, pH adjustment and elution of the drugs were accomplished by the ASPEC. After off-line evaporation, the residues were injected into a wide-bore capillary gas chromatograph. The recoveries of the tested drugs were in the range of 73-96%, with relative standard deviations less than 5% at a concentration level of 2 micrograms/ml.

Isolation of acidic, neutral, and basic drugs from whole blood using a single mixed-mode solid-phase extraction column.

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.

A single-column procedure on Bond Elut Certify for systematic toxicological analysis of drugs in plasma and urine.

A single-column solid-phase extraction procedure was developed for the screening of acidic, neutral, and basic drugs from plasma. The recoveries of all 25 tested drugs exceeded 82%. After the plasma had been diluted with phosphate buffer (pH 6.0), the drugs were extracted using a single Bond Elut Certify column. The acidic and most of the neutral drugs were eluted by acetone/chloroform (1:1) and the basic drugs were eluted by 2% ammoniated ethyl acetate. Some neutral drugs appeared in both fractions. The two fractions were collected separately and evaporated until approximately 100 microL of solvent remained in the tube. Both fractions were analyzed separately on a gas chromatograph equipped with a wide-bore capillary column and a flame ionization detector. The procedure could also be used for urine samples.

[Analysis of residues of organochlorine compounds in plant drugs. 3. Identification of residues of polychlorobiphenyl compounds by comparison of gas chromatography on packed and capillary columns and GCMS coupling]. / Untersuchung pflanzlicher Drogen auf Rückstände von Organochlorverbindungen. 3. Mitteilung: Identifizierung von Rückständen polychlorierter Biphenyle durch Vergleich von Gaschromatographie an gepackter und Kapillarsäule sowie GC/MS-Kopplung.

The identification of residues of polychlorinated biphenyls in a test sample of Flores Chamomillae could be achieved by the retention behavior at gas chromatographic analyses on packed and capillary columns compared with reference standard Clophen A 60, respectively as well as well by capillary GC/MS using single ion monitoring of substance-characteristic ion mass.

Systematic analysis of solvents and other volatile substances by gas chromatography.

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.

Impact of urine matrix and isolation procedure on retention behaviour of basic drugs in thin-layer chromatography.

The influence of three different isolation procedures, namely liquid-liquid extraction, Extrelut column extraction and XAD-2 column extraction, and of the urine matrix on the standardised Rf values and variability of Rf values of some selected basic drugs was investigated. It appears that the liquid-liquid extraction may give a significant deviation of standardised Rf values in respect of pure drugs, which is dependent on the TLC system. For all three isolation procedures, the search window for substance identification by means of data collection based on standardised Rf values of pure drugs should be slightly wider after extraction than when using pure drugs. The TLC system cyclohexane-toluene-diethylamine (75:15:10, v/v/v) showed the best accuracy and precision of Rf values.

Influence of biological matrix on retention behaviour and identification possibilities of selected neutral and acidic drugs in thin layer chromatography. An interlaboratory investigation.

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.

Potentials of wide-bore fused silica capillary columns for substance identification by means of retention indices.

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.

[Plant drugs with residues of organochlorine compounds. 2. Identification of residues of DDT and its analogs by comparison of gas chromatography on packed and capillary columns and GC/MS coupling]. / Untersuchung pflanzlicher Drogen auf Rückstände von Organochlorverbindungen. 2. Mitteilung: Identifizierung der Rückstände von DDT und seinen Analogen durch Vergleich von Gaschromatografie an gepackter und Kapillarsäule sowie GC/MS-Kopplung.

For the GC analysis of DDT isomers and metabolites in extracts of Flores Chamomillae end Radix Valerianae the separation on a packed QF-1/OV-17 column was compared with various capillary columns of the CP-Sil type. Identification of the individual compounds could be achieved by comparing the retention behavior, chemical transformation of DDT and DDE, as well as by capillary GC-MS using single ion monitoring of substance-characteristic ion mass. In this way, residues of p,p'-DDT, o,p'-DDE and p,p'-TDE could be identified.

Multicenter evaluation of ultrafiltration, dialysis, and thermal coagulation as sample pretreatment methods for the colorimetric determination of paraquat in blood and tissues.

Three methods of sample pretreatment for the rapid colorimetric determination of paraquat were compared: ultrafiltration, dialysis, and thermal coagulation. Spiked autopsy blood and tissue samples were examined in parallel in Groningen and Krakow and some samples were interchanged. All three methods gave recoveries between 87 to 102%; accuracy at the 20-mg/L level was within 10% of the target value and coefficients of variation in the 10 to 60-mg/L range were between 3 to 15%. Determinations in blood and liver in a fatal case of Gramoxone poisoning showed excellent agreement. Because of its reliability, speed, and simplicity, ultrafiltration is the method of choice.

Determination of carbisocaine, heptacaine and pentacaine in plasma by capillary gas chromatography with nitrogen-selective detection.

A gas-liquid chromatographic method is described for the determination of the local anaesthetics carbisocaine, heptacaine and pentacaine in plasma. A C(18) solid-phase extraction was used in a modification to increase selectivity. Following on-column derivatization with trimethylanilinium hydroxide, the analytes were determined by means of capillary gas chromatography and nitrogen-phosphorus selective detection. In comparison with flame ionization detection, the sensitivity of NPD was 20 times higher with a limit of determination in plasma of 10 ng ml(-1).

Impact of biological matrix, drug concentration, and method of isolation on detectability and variability of retention index values in gas chromatography.

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.

Impact of biological matrix and isolation methods on detectability and interlaboratory variations of TLC Rf-values in systematic toxicological analysis.

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.