Evaluation of six methods for extraction and purification of viral DNA from urine and serum samples

The sensitivity and reliability of PCR for diagnostic and research purposes require efficient unbiased procedures of extraction and purification of nucleic acids. One of the major limitations of PCR-based tests is the inhibition of the amplification process by substances present in clinical samples. This study used specimens spiked with a known amount of plasmid pBKV (ATCC 33-1) to compare six methods for extraction and purification of viral DNA from urine and serum samples based on recovery efficiency in terms of yield of DNA and percentage of plasmid pBKV recovered, purity of extracted DNA, and percentage of inhibition. The most effective extraction methods were the phenol/chloroform technique and the silica gel extraction procedure for urine and serum samples, respectively. Considering DNA purity, the silica gel extraction procedure and the phenol/chloroform method produced the most satisfactory results in urine and serum samples, respectively. The presence of inhibitors was overcome by all DNA extraction techniques in urine samples, as evidenced by semiquantitative PCR amplification. In serum samples, the lysis method and the proteinase K procedure did not completely overcome the presence of inhibitors.     

Gas chromatographic determination of guanadrel in plasma and urine

To evaluate the pharmacokinetics and drug availability from various dosage formulations, a method for the determination of guanadrel, (1,4-dioxaspiro[4,5]dec-2-ylmethyl)guanidine, in plasma and urine was required. a gas chromatographic procedure, based on formation of a hexafluoroacetylacetone derivative in a two-phase system of water and toluene, was developed. The limit of determination of the method is 5 ng/ml guanadrel in plasma and 15 ng/ml guandrel in urine. Statistical analyses indicate average recoveries of 98.1 ± 18.0 and 104.4 ± 15.6% from plasma and urine, respectively. Mass spectrometric analyses, in conjunction with gas chromatography, confirmed the specificity of the method for intact drug. The procedure was applied successfully to drug absorption studies in humans.     

Determination of monoacetyldiamines and polyamines in urine by high-performance liquid chromatography

A procedure is described for the determination of monoacetylputrescine, N¹-acetylspermidine and N⁸-acetylspermidine in human urine. The procedure is based on the high-performance liquid chromatographic separation of the 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) derivatives of these amines using two different chromatographic modes. Monoacetyl-1,6-diaminohexane was used as an internal standard. The amines were extracted from urine using a silica gel cartridge. The dansyl monoacetylpolyamines were separated from the mixture of dansyl derivatives of urinary amines on a bonded-phase CN column using a programmed solvent gradient elution. The dansyl acetylpolyamines were rechromatographed on a silica gel column.This chromatographic procedure was used for the determination of the concentration of N¹-acetylspermidine, N⁸-acetylspermidine and monoacetylputrescine in the urine of healthy volunteers and cancer patients.     

Toxicological detection of the new designer drug 1-(4-methoxyphenyl)piperazine and its metabolites in urine and differentiation from an intake of structurally related medicaments using gas chromatography-mass spectrometry

Studies are described on the toxicological analysis of the piperazine-derived designer drug 1-(4-methoxyphenyl)piperazine (MeOPP) in rat urine using gas chromatography-mass spectrometry (GC-MS). The authors' systematic toxicological analysis (STA) procedure using full-scan GC-MS after acid hydrolysis, liquid-liquid extraction and microwave-assisted acetylation allowed the detection of MeOPP and its metabolites 1-(4-hydroxy phenyl)piperazine and 4-hydroxyaniline in rat urine after administration of a single dose corresponding to doses commonly taken by drug users. Therefore, this procedure should also be suitable for detection of a MeOPP intake in human urine. However, the metabolites of MeOPP are not unique and can be produced from other drugs. Therefore, differentiation of use of this designer drug from use of the medicaments dropropizine, oxypertine or others, which are metabolized to the MeOPP isomer 1-(2-methoxyphenyl)piperazine, is discussed.     

Determination of trenbolone and its metabolite in bovine fluids by liquid chromatography-tandem mass spectrometry

A liquid chromatographic-tandem mass spectrometric (LC-MS-MS) method has been developed for the determination of trenbolone in bovine urine and serum. The aim was a control of the misuse of trenbolone in food-producing animals. The procedure involved, in both cases, a preliminary solid-phase clean-up followed by a liquid-liquid extraction for urine samples after a preliminary enzymatic hydrolysis. The extracts have been directly analysed by reversed-phase LC-MS-MS in selected reaction monitoring (SRM), acquiring two diagnostic product ions from the chosen precursor [M+H](+). The procedures were validated across the concentration range of 1-1500 ng/ml. The linearity, the inter- and intra-day accuracy and precision have been determined. The procedure was specific and the accuracy values were better than 20% at the limit of quantitation of spiked samples. The limit of quantification (LOQ) and the limit of detection (LOD) were, respectively, 1 ng/ml and 350 pg/ml for urine and serum. According to the draft, SANCO/1805/2000, we determined the decision limit CCalpha and the detection capability CCbeta. The recovery values for urine ranged from 87 to 128%, and for plasma the recovery was 70+/-4%. The procedure proved to be simple and suitable for routine and confirmatory purposes such as those developed for residue studies.     

A simple modification of the Salmonella liquid-incubation assay. Increased sensitivity for detecting mutagens in human urine

A simple modification of the Salmonella/microsome liquid-incubation procedure improves the sensitivity of the assay for detecting mutagens in human urine. Extracts from cigarette smokers' urine were used as a model complex mutagenic mixture for validation of the assay. The modification consists of adding increased numbers of bacterial cells (approximately 10(9] in a concentrated suspension to liver homogenate mix and urine extract, all in 0.2-ml volume. After 90 min incubation at 37 degrees C, the mixture is processed according to the standard Ames test protocol. This procedure is 20 times more sensitive than the standard plate-incorporation test and 13 times more sensitive than a previously reported liquid-incubation protocol. The number of spontaneous revertants did not increase under these conditions and, compared to the plate-incorporation test, 10-fold less liver homogenate and 5-fold less enzymatic cofactors were needed per plate. The procedure was approximately 14 times more sensitive in detecting the mutagenic activity of benzo[ a ]pyrene. We also used the modification to determine mutagenic activity in urine from a group of nonsmokers. The method may be generally useful for investigations of mutagenic activity in human urine samples.     

Determination of the anti-platelet-activating factor BN-50727 and metabolites in human urine by high-performance liquid chromatography using solid-phase extraction

A sensitive and selective HPLC solid-phase extraction procedure was developed for the determination of platelet-activating factor antagonist BN-50727 and its metabolites in human urine. The procedure consisted in a double solid-phase extraction of the urine samples on cyanopropyl and silica cartridges, followed by an automated solid-phase extraction of the drug and metabolites on CBA cartridges and posterior elution on-line to the chromatographic system for its separation. The method allowed quantitation in the concentration range 10–2400 ng/ml urine for both BN-50727 and the main metabolite, the O-demethylated BN-50727 product. The limit of quantitation for both compounds was 10 ng/ml. The inter-assay precision of the method, expressed as relative standard deviation, ranged from 1.9 to 4.5% for BN-50727 and from 2.5 to 9.0% for the metabolite. The accuracy, expressed as relative error, ranged from −2.4 to 4.2% and from 0.2 to 6.2%, respectively. This paper describes the validation of the analytical methodology for the determination of BN-50727 in human urine and also for its metabolites. The method has been used to follow the time course of BN-50727 and its metabolites in human urine after single-dose administration.     

Isolation and identification of S-adenosylmethionine from human urine

Procedures are described for the isolation and identification of adenosylmethionine from human urine. Previously described preliminary separative procedures using anion and cation exchange columns and an XAD-4 resin column have been extended to permit the separation of adenosylmethionine. The adenosylmethionine has been identified by conversion to methylthioadenosine followed by rechromatography of the latter compound with three different types of columns and elution systems. Mean adenosylmethionine values for urine were as follows: adults, 0.26; children, 0.36 nmole/mumole creatinine. Recovery of adenosylmethionine added to urine and determined by this separative procedure was 52%.     

Development of a headspace gas chromatographic test for the quantification of 1- and 2-bromopropane in human urine

A test procedure was developed for the detection and quantification of 1- and 2-bromopropane in human urine. 1-Bromopropane (1-BP) is a commonly used industrial solvent, and 2-bromopropane (2-BP) is often found as an impurity component in industrial grade 1-BP. Both compounds are a health concern for exposed workers due to their chronic toxicity. Bromopropanes have been associated with neurological disorders in both animals and humans. Sample preparation consisted of diluting urine with water and fortification with 1-bromobutane (1-BB), which was used as an internal standard; then each sample was sealed in a headspace vial. A static-headspace sampler (Teledyne-Tekmar Model 7000) was used to heat each sample at 75 degrees C for a 35-min equilibrium time. Quantification was by means of a gas chromatograph (GC) equipped with an electron capture detector (ECD) and a dimethylpolysiloxane (DB-1) capillary column. A recovery study using fortified urine samples at multiple concentrations (0.5-8 microg/ml) demonstrated full recovery; 104-121% recovery was obtained. Precision ranged from 5 to 17% for the 15-20 spiked samples used at each concentration, which were analyzed over multiple experimental trial days. The limit of detection (LOD) for this test procedure was approximately 2 ng/ml 1-BP and 7 ng/ml 2-BP in urine. A recovery study of 1- and 2-BP from fortified urine stored in vials appropriate for field collection was also completed. These results and other factors of the development and validation of this test procedure will be discussed.     

Stereoselective and simultaneous measurement of cis- and trans-isomers of doxepin and N-desmethyldoxepin in plasma or urine by high-performance liquid chromatography

Doxepin is a tricyclic antidepressant marketed as an irrational mixture of cis- and trans-geometric isomers in the ratio of 15:85. A convenient high-performance liquid chromatographic (HPLC) procedure for simultaneous quantitation of geometric isomers of doxepin and N-desmethyldoxepin in plasma and urine is described. The HPLC procedure employed a normal phase system with a silica column and a mobile phase consisting of hexane-methanol-nonylamine (95:5:0.3, v/v/v), a UV detector and nortriptyline as the internal standard. The liquid-liquid extraction solvent was a mixture of n-pentane-isopropanol (95:5, v/v). The limit of quantitation was 1 ng/ml for each isomer. The calibration curves were linear over the ranges 1–200 ng/ml (plasma) and 1–400 ng/ml (urine). In plasma, the accuracy (mean±S.D.) (97.53±1.67%) and precision (3.89±1.65%) data for trans-doxepin were similar to corresponding values for urine, i.e., 97.10±2.40 and 3.82±1.14%. Accuracy and precision data for trans-N-desmethyldoxepin in plasma were 97.57±2.06 and 4.38±3.24%, and in urine were 97.64±3.32 and 5.26±1.83%, respectively. Stability tests under three different conditions of storage indicated no evidence of degradation. The recovery of doxepin was 61–64% from plasma and 63–68% from urine. The method has been applied to analyses of plasma and urine samples from human volunteers and animals dosed with doxepin.     

Determination of diphenylpyraline in plasma and urine by high-performance liquid chromatography

A rapid, reliable and specific reversed-phase high-performance liquid chromatographic procedure is described for the determination of diphenylpyraline hydrochloride at nanogram concentrations in plasma and urine. After extraction of the drug with n-pentane-2-propanol (50:1) from alkalinized samples, the organic extract was evaporated to dryness, reconstituted with methanol and chromatographed using a 5-μm Asahipak ODP-50 C₁₈ column with UV detection at 254 nm. The elution time for diphenylpyraline was 7.9 min. The overall recovery of diphenylpyraline from spiked plasma and urine samples at concentrations ranging from 53 to 740 ng/ml were 94.65% and 92.29%, respectively. Linearity and precision data for plasma and urine standards after extraction were acceptable. The limit of detection was 15 ng/ml for both plasma and urine samples at 0.002 AUFS.     

Simultaneous detection of methylphenidate and its main metabolite, ritalinic acid, in doping control

Two analytical methods for the simultaneous detection in urine of methylphenidate and its main metabolite, ritalinic acid, are described. Both procedures are based on solid-phase extraction of urine samples on Bond Elut Certify columns, and capillary gas chromatographic—mass spectrometric detection of O-trimethylsilyl, N-trifluoroacetyl derivatives. The former method is used as a general screening procedure for the detection of basic polar nitrogen-containing compounds in urine such as stimulants, narcotic and adrenergic drugs. The latter procedure is proposed as a specific method to confirm methylphenidate ingestion. The two methods are sensitive enough to detect methylphenidate and ritalinic acid in urine at least for 24 h after administration of a therapeutic dose (20 mg oral dose) of methylphenidate.     

Metabolism and toxicological detection of the designer drug 4-iodo-2,5-dimethoxy-amphetamine (DOI) in rat urine using gas chromatography-mass spectrometry

The amphetamine-derived designer drug 4-iodo-2,5-dimethoxy-amphetamine (DOI) is an upcoming substance on the illicit drug market. In the current study, the identification of its metabolites in rat urine and their toxicological detection in the authors' systematic toxicological analysis (STA) procedure were examined. DOI is extensively metabolized by O-demethylation and beside small amounts of parent compound it was found to be excreted mainly in form of metabolites. The STA procedure using full-scan GC-MS allowed proving an intake of a common drug users' dose of DOI by detection of the two O-demethyl metabolite isomers in rat urine. Assuming similar metabolism, the described STA procedure should be suitable for proof of an intake of DOI in human urine.     

Molecular forms of beta-hexosaminidase and cathepsin D in serum and urine of healthy subjects and patients with elevated activity of lysosomal enzymes.

A procedure is described that allows the characterization of the molecular forms of beta-hexosaminidase and cathepsin D in controls and pathological specimens of human serum and human urine. The following observations were made. (1) In human serum, beta-hexosaminidase (alpha- and beta-chain) and cathepsin D are present predominantly in their high-molecular-weight precursor forms. In human urine, these enzymes exist as both precursor and mature forms. (2) Cathepsin D precursor from serum and urine differs in the number of oligosaccharides that are sensitive to endo-beta-N-acetylglucosaminidase H. Therefore the urine enzyme is not likely to originate from the serum. (3) The presence exclusively of precursors of beta-hexosaminidase and of cathepsin D in the sera of patients with hepatitis suggests that in hepatitis secretion of lysosomal enzymes is elevated, rather than the enzymes leaking from damaged cells. (4) In the urine of patients with nephrotic syndrome, beta-hexosaminidase and cathepsin D are present in grossly elevated amounts, but do not differ in the polypeptide patterns from controls. (5) In urine from a patient with mucolipidosis II, the elevated activity of beta-hexosaminidase is accounted for mainly by the precursor forms. Mature beta-chain of beta-hexosaminidase is lacking, and incompletely processed beta-hexosaminidase polypeptides are present. Both the precursor and the mature forms of cathepsin D are increased. They contain only complex oligosaccharides.     

Diagnostic cystocentesis: technique and considerations

Cystocentesis, the aspiration of urine from the urinary bladder, has both diagnostic and therapeutic uses. This month, we discuss the diagnostic reasons for using this technique and summarize the steps necessary to safely perform the procedure in laboratory animals.     

New extraction procedure and high-performance liquid chromatographic method for analyzing polyethylene glycol-400 in urine

We describe a new, highly efficient method for extracting polyethylene glycol-400 from urine and for its analysis by isocratic reverse-phase high-performance liquid chromatography. This method is an improvement over previously published methods in that it does not require the use of ion-exchange resins and lyophilization prior to extraction, nor does it require the separation and analysis of the individual polymers of polyethylene glycol. The procedure described in this report entails extraction with a salt—solvent combination of ammonium sulfate and dichloromethane and analysis by reversed-phase high-performance liquid chromatography. The lower limit of detection was approximately 0.25 g/l with a 2-ml urine sample. Analytical recoveries of polyethylene glycol-400 added to urine at 2.5 and 5.0 g/l averaged 97 and 96%, respectively (n = 10). Within- and between-day coefficients of variation were less than 5% at 2.5 and 5.0 g/l. Studies of various urine samples from patients receiving polyethylene glycol-400 revealed no interferences from other urine substances.     

Changes in urinary level and configuration ratio of D-lactic acid in patients with short bowel syndrome

The present study showed that the D-lactic acid configuration ratio in the urine rose earlier than that in blood or the urinary or blood D-lactic acid levels upon disease onset, and that the D-lactic acid measurement in urine is more sensitive and useful than that in blood. As this result, a prediction of a D-lactic acidosis may be possible. To simplify the procedure for detecting D-lactic acid, we first showed a correlation between the D-lactic acid configuration ratio in urine and blood, indicating urine could be used. To separate the optical isomers of lactic acid, we simplified our previous procedure. For chiral recognition, we chose O-acetyl-(-)-menthylation and analyzed the samples under GC/MS by capillary gas chromatography on a DB-5 MS column. This procedure is less sensitive than the former method, but it is faster and simpler, requiring only one derivatization step. This method may be useful for predicting D-lactic acidosis in patients with short bowel syndrome.     

Development of a metabolomic approach based on urine samples and direct infusion mass spectrometry

The analysis of urine by direct infusion mass spectrometry suffers from ion suppression due to its high salt content and inter-sample variability caused by the differences in urine volume between persons. Thus, urine metabolomics requires a careful selection of the sample preparation procedure and a normalization strategy to deal with these problems. Several approaches were tested for metabolomic analysis of urine samples by direct infusion electrospray mass spectrometry (DI–ESI–MS), including solid phase extraction, liquid–liquid extraction, and sample dilution. In addition, normalization of results based on conductivity values and statistical treatment was performed to minimize sample variability. Both urine dilution and solid phase extraction with mixed mode sorbent considerably reduced the salt content in urine, providing comprehensive metabolomic fingerprints. Moreover, statistical data normalization enabled the correction of inter-sample physiological variability, improving the quality of results obtained. Therefore, high-throughput DI–ESI–MS fingerprinting of urine samples can be achieved with simple pretreatment procedures allowing the use of this noninvasive sampling in metabolomics. Finally, the optimized approach was tested in a pilot metabolomic investigation of urine samples from transgenic mice models of Alzheimer’s disease (APP/PS1) in order to illustrate the potential of the methodology.     

Extraction and separation of urinary catecholamines as their diphenyl boronate complexes using C18 solid-phase extraction sorbent and high-performance liquid chromatography

The clinical utility of a one-step extraction procedure based on the retention of a diphenyl boronate-catecholamine complex on a C18 solid-phase extraction sorbent was investigated for the measurement of urinary catecholamines. Although recoveries with the extraction procedure were optimal over a relatively broad pH range (7.5-9.5), analytical factors such as sample loading and elution flow-rates, wash step and elution conditions, the concentration of catecholamines in urine to be extracted and the type of C18 sorbent used for extraction were found to influence the efficiency of this procedure and would therefore need to be controlled for optimal recoveries. Under optimal conditions the recovery of noradrenaline, adrenaline and dopamine from spiked urine was high and reproducible (mean recoveries were >85% for all catecholamines). The effectiveness of sample clean-up step was demonstrated by reverse phase, ion pair high-performance liquid chromatography with electrochemical detection. The method described was found to be suitable for the routine measurement of catecholamines in urine in clinical biochemistry laboratories. It has a high sample extraction throughput (40/h) and has adequate precision (between batch CV<8%) and sensitivity (LOD<30 nmol/l; LOQ<65 nmol/l) for all the catecholamines measured. The method has acceptable accuracy, showing a mean bias of 6.6% for noradrenaline, 7.3% for adrenaline and 6.8% for dopamine from the mean value of laboratories (N=69) participating in an External Quality Assurance scheme for greater than 12 months.     

Single-step procedure for gas chromatography-mass spectrometry screening and quantitative determination of amphetamine-type stimulants and related drugs in blood, serum, oral fluid and urine samples

We describe a rapid GC/MS assay for amphetamine-type stimulant drugs (ATSs) and structurally related common medicaments in blood, serum, oral fluid and urine samples. The drugs were extracted from their matrices and derivatized with heptafluorobutyric anhydride (HFBA) in a single step, using the following procedure: 100 microl (oral fluid) or 200 microl (blood, serum, urine) of the sample were mixed with 50 microl of alkaline buffer and 500 microl of extraction-derivatization reagent (toluene + HFBA + internal standard), centrifuged, and injected into a GC/MS apparatus. As revealed by the validation data this procedure, with its limit of quantitation being set at 20 ng/ml for oral fluid, 25 ng/ml for blood or 200 ng/ml for urine, is suitable for screening, identification and quantitative determination of the ATSs and related drugs in all the matrices examined. Thus, time-consuming and expensive multiple analyses are not needed, unless specifically required.