Edelfosine protects precultured heart fragments against the invasion of malignant cells through altered sialylation.

1-O-octadecyl-2-O-methylglycero-3-phosphocholine (ET-18-OMe)-treated precultured heart fragments (PHF) are resistant to the invasion of malignant cells. Previous studies have demonstrated that this effect is due to the alterations of the N-linked glycoproteins in PHF after 48-h ET-18-OMe treatment. Moreover, the observed effect was still present seven days after ET-18-OMe was omitted. The present study reveals that approximately 13.4% of the administered ET-18-OMe was taken up by PHF and about 75% of the initial uptake was still present after ET-18-OMe was removed. In addition, we found significant changes in the sialic acid content and sialyltransferase activities in both conditions. Overall, these results clearly demonstrate that the uptake and retention of ET-18-OMe are responsible for the resistance to the invasion of malignant cells due to the altered sialylation of the cell surface glycoproteins in PHF.

Evaluation of automated single mass spectrometry to tandem mass spectrometry function switching for comprehensive drug profiling analysis using a quadrupole time-of-flight mass spectrometer.

A liquid chromatographic mass spectrometric strategy for systematic toxicological analysis (STA) is presented using the automatic 'on-the-fly' single mass spectrometry mode to tandem mass spectrometry mode (MS to MS/MS) switching abilities of a quadrupole time-of-flight (Q-TOF) instrument. During the chromatographic run, the quadrupole is initially set to transmit all masses until (an) ion(s) reaches a certain set threshold. Thereupon, the quadrupole automatically switches to the MS/MS mode, selecting the ion(s), which are subsequently fragmented in the high-efficiency hexapole collision cell, thus generating product ions that are further mass analyzed by the TOF. By limiting the TOF spectral accumulation time in the MS/MS mode to a statistically acceptable minimum, the quadrupole almost instantly switches back to the MS mode. Qualitative information, comprising the complementary MS ([M + H](+) ion mass) and MS/MS (informative product ion profile) data, as well as quantitative information obtained by integration of the MS extracted ion chromatogram(s), can be obtained in one single acquisition. Optimization of the automatic switching parameters, such as threshold, TOF spectral accumulation time, detection window and collision energy, was carried out by injection of a mix of 17 common drugs which were not necessarily baseline separated in the chromatographic system used. Indeed, the complete separation of the drugs is not deemed necessary since up to 8 different ions can 'simultaneously' be selected for MS/MS if they reach the preset criteria. In addition, the quantitative performance of the method was defined. In a second phase, the developed method was field-tested. To that end, the resulting data from extracts of urine samples were compared with and found to be in close concordance with those obtained by a standard toxicological analysis. This innovative approach clearly holds the potential for a substantial advance in the introduction of LC/MS in STA.

Liquid chromatography-mass spectrometry in forensic toxicology.

Liquid chromatography-mass spectrometry has evolved from a topic of mainly research interest into a routinely usable tool in various application fields. With the advent of new ionization approaches, especially atmospheric pressure, the technique has established itself firmly in many areas of research. Although many applications prove that LC-MS is a valuable complementary analytical tool to GC-MS and has the potential to largely extend the application field of mass spectrometry to hitherto "MS-phobic" molecules, we must recognize that the use of LC-MS in forensic toxicology remains relatively rare. This rarity is all the more surprising because forensic toxicologists find themselves often confronted with the daunting task of actually searching for evidence materials on a scientific basis without any indication of the direction in which to search. Through the years, mass spectrometry, mainly in the GC-MS form, has gained a leading role in the way such quandaries are tackled. The advent of robust, bioanalytically compatible combinations of liquid chromatographic separation with mass spectrometric detection really opens new perspectives in terms of mass spectrometric identification of difficult molecules (e.g., polar metabolites) or biopolymers with toxicological relevance, high throughput, and versatility. Of course, analytical toxicologists are generally mass spectrometry users rather than mass spectrometrists, and this difference certainly explains the slow start of LC-MS in this field. Nevertheless, some valuable applications have been published, and it seems that the introduction of the more universal atmospheric pressure ionization interfaces really has boosted interests. This review presents an overview of what has been realized in forensic toxicological LC-MS. After a short introduction into LC-MS interfacing operational characteristics (or limitations), it covers applications that range from illicit drugs to often abused prescription medicines and some natural poisons. As such, we hope it can act as an appetizer to those involved in forensic toxicology but still hesitating to invest in LC-MS.

DNA typing of fingerprints using capillary electrophoresis: effect of dactyloscopic powders.

DNA typing is a useful tool in crime solving, not only for blood samples, sperm, or saliva but also for traces of DNA left on tools or pieces of clothing used in burglaries or thefts. On these kinds of samples, the sources of DNA are extremely small amounts of skin debris left after gripping tools. When a sensitive technique such as polymerase chain reaction (PCR) coupled with capillary electrophoresis is used, it is possible to get a profile from these low amounts of DNA. The classic technique in such cases, used in forensic sciences, is to reveal fingerprints by different dactyloscopic powders. Therefore, DNA profiling was performed on physical fingerprints left on glass and wooden plates, in order to establish eventual problems or interferences involved by using both techniques simultaneously. Eleven dactyloscopic powders were investigated on their influence on DNA typing. The results show that some can be used together with DNA profiling but that serious precautions have to be taken to avoid contamination.

Cellular uptake and retention measurements of alkylphosphocholines in the SK-BR-3 breast cancer and Molt-4 leukemia cell line using capillary gas chromatography.

The determination of cellular content of octadecylphosphocholine (D-19391) and hexadecylphosphocholine (HePC, D-18506), two anticancer agents of the alkylphosphocholine group, using capillary gas chromatography is described. The compounds' cytotoxicity was first determined by the MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium] assay, being indicative for the concentration used in the uptake and retention measurements. D-19391 was added to the SK-BR-3 breast cancer cell line and HePC to the Molt-4 leukemia cell line in concentrations of, respectively, 18.6 and 15.0 microM, during a 36-h incubation period at 37 degrees C, 5% CO2. HePC uptake in the leukemia cells was followed by a 24-h reversibility test in drug-free medium. Subsequently, sample clean-up was performed on a weak cation-exchange column. For the quantitative analysis, HePC was used as internal standard for the D-19391 measurements and vice versa. Derivatization of the samples with trimethylsilylbromide was followed by capillary gas chromatographic analysis. From these data we conclude that our uptake results are quite similar with those of a previous study of HePC cellular uptake in the more resistant Caco-2T colon cancer cell line. Without having investigated the mechanism that underlies the cellular uptake results obtained, our study points to no direct correlation between the compounds' cellular uptake and their cytotoxic effects.

Analysis of melphalan adducts of 2'-deoxynucleotides in calf thymus DNA hydrolysates by capillary high-performance liquid chromatography-electrospray tandem mass spectrometry.

Melphalan is a bifunctional alkylating agent that covalently binds with intracellular nucleophilic sites. A methodology using electrospray mass spectrometry was developed to detect and identify DNA adducts. Alkylation sites within a particular nucleotide were examined using electrospray tandem mass spectrometry hyphenated to capillary liquid chromatography in combination with a column switching system. In the reaction mixtures resulting from the interaction of 2'-deoxynucleotides and melphalan several base-aklylated adducts were found. In the case of 2'-deoxyadenosine monophosphate, thymidine monophosphate and 2'-deoxyguanosine phosphate alkylation was observed in the mononucleotide reaction mixtures but not in the DNA-hydrolysates. Calf thymus DNA was reacted in vitro with melphalan. The DNA pellet was isolated and enzymatically hydrolyzed with the aid of Nuclease P1. In this hydrolysate both mono-alkylated 2'-deoxynucleotides and dinucleotides were found. The most important adduct found was identified as the N-7 alklylated dGMP adduct. The alkylated dinucleotides were identified as a pdApdT/melphalan and pdGpdC/melphalan the latter being the most important.

Preparative capillary zone electrophoresis in combination with off-line graphite furnace atomic absorption for the analysis of DNA complexes formed by a new aminocoumarine platinum (II) compound.

Calf thymus DNA was incubated in vitro with a new aminocoumarin platinum (II) complex in order to study its interaction with DNA. The platinated DNA was hydrolyzed enzymatically to the 5'-mononucleotide level using DNAase I and nuclease P1. Analysis of the DNA hydrolysate with capillary zone electrophoresis (CZE), using sample stacking, revealed the presence of unhydrolyzed oligonucleotides in the platinated DNA. A homemade system, using only some plastic pipet tips, was constructed to collect the oligonucleotide fraction during CZE analysis. The platinum content of this fraction was determined using graphite furnace atomic absorption with Zeeman background correction. This system proved to be a useful tool to detect platinated DNA species (with a quantifiable detection limit for the detection of platinum of 0.78 ng). Subsequent gel filtration experiments confirmed the presence of high molecular weight oligonucleotides that were platinated. This was proven by reversal of the platination using thiourea and subsequent enzymatic hydrolysis to 5'-mononucleotides.

Analysis of the DNA adducts of phenyl glycidyl ether in a calf thymus DNA hydrolysate by capillary zone electrophoresis-electrospray mass spectrometry: evidence for phosphate alkylation.

Calf thymus DNA was reacted in vitro with phenyl glycidyl ether (PGE) and was hydrolysed enzymatically, to the 5'-monophosphate nucleotides using deoxyribonuclease I (DNA-ase I) and nuclease P1. The adducts were concentrated using solid phase extraction (SPE), on a polystyrene divinylbenzene copolymer in order to remove the unmodified nucleotides. The adducts could be identified using capillary zone electrophoresis-electrospray tandem mass spectrometry (CZE ES-MS/MS), using sample stacking. In addition to the base alkylated 2'-deoxynucleotides present in the DNA-hydrolysate, also phosphate alkylated 2'-deoxynucleotide adducts were identified for TMP and dAMP. An additional adduct, dUMP alkylated on the uridine moiety was found originating from the hydrolytic deamination of dCMP alkylated on N3 of the cytosine moiety. Enzymatic hydrolysis using nuclease P1 was incomplete as shown by the presence of dinucleotides alkylated on the base moiety. They were successfully hydrolysed to the corresponding 2'-deoxynucleotides by snake venom phosphodiesterase (SVP). Data are shown indicating that alkylations on the pyrimidine bases were more resistant to enzymatic hydrolysis with nuclease P1 than the purine alkylated products.

Population genetic study of four short tandem repeat loci in the Belgian population, using capillary electrophoresis.

Allele frequencies of four short tandem repeat loci (HumCD4, HumTH01, HumD21S11 and HumSE33) were investigated in a sample of 395 unrelated Belgian individuals using multiplex polymerase chain reaction and capillary electrophoresis. Automated laser fluorescence was used to detect four fluorescent dyes, enabling the use of an internal standard within each lane. With this method rapid typing with high resolution was obtained and the different alleles were grouped on a statistical base. All loci meet Hardy-Weinberg expectations. The allelic frequency data, together with the constructed allelic ladder, can be used in paternity testing and personal identification in the medical and forensic sciences.

Comparison of slab gel electrophoresis and capillary electrophoresis for the detection of the fluorescently labeled polymerase chain reaction products of short tandem repeat fragments.

The sizing capability of slab gel electrophoresis for short tandem repeat (STR) fragments was compared to the sizing capability of capillary electrophoresis (CE). Both systems used automated laser fluorescence detection to detect four fluorescent dyes, enabling the use of an internal lane standard within each sample. The STR fragments were amplified using a multiplex polymerase chain reaction (PCR) in which the STR fragments Hum CD-4, Hum TH01, Hum D21S11 and Hum SE33 were amplified simultaneously. The reproducibility of the size calling was determined for both systems. The average standard deviation obtained for the slab gel system was 0.2, which was comparable to the standard deviation of 0.12 obtained for the CE system. The CE system produced results comparable to those obtained on the slab gel system, with a level of precision of +/- 1.0 bp (between instruments).

Narrow-bore HPLC in combination with fluorescence and electrospray mass spectrometric detection for the analysis of cocaine and metabolites in human hair.

A simple, but sensitive and specific, high-performance liquid chromatographic assay for cocaine, cocaethylene, and benzoylecgonine is described. Using direct fluorometric detection, the procedure is particularly interesting for the routine analysis of human hair samples. In the sample preparation part, the hair samples are cut and washed and two internal standards with close structural resemblance to benzoylecgonine and cocaine as well as to cocaethylene are added. Subsequently, the hair samples are homogenized, hydrolyzed overnight in a 0.1 M HCl solution at 56 degrees C, and extracted on IST Confirm HCX solid-phase extraction columns. Chromatographic separation is achieved on a narrow-bore Hypersil BDS C18 column (125 x 2.1 mm, 3 microns) by gradient elution with an ammonium acetate buffer-methanol/acetonitrile mixture. For the fluorometric detection, excitation and emission wavelengths of 242 and 315 nm, respectively, are used. This analysis protocol affords a method of high sensitivity and specificity which has been fully evaluated and validated. The data presented show good accuracy and linearity with excellent reproducibility and recovery. Because unequivocal identity confirmation is mandatory in forensic applications, an extension of the analysis protocol was accomplished toward mass spectrometric detection. We succeeded in a simple methodological transfer from LC/FL to LC/ESI-MS/MS, thus providing two complementary approaches after a single, common sample-processing step. Hair samples from 29 fatalities, all known drug users and suspected victims from a drug overdose, were analyzed in this way. Of the investigated samples, 12 were positive and the concentrations found range from 0.98 to 938 ng/mg of hair for cocaine and from 1.45 to 388 ng/mg of hair for benzoylecgonine. Traces of cocaethylene were also found in two of the hair samples. The results obtained with LC/ESI-MS/MS were in close agreement with those obtained with LC/FL, positively confirming the isolates' identity and structure by means of the resulting MS/MS spectra.

Analysis of the DNA damage induced by phenyl glycidyl ether using capillary zone electrophoresis-electrospray mass spectrometry.

The in vitro adduct formation between phenyl glycidyl ether (PGE) and calf thymus DNA was investigated. Agarose slab gel electrophoresis of DNA incubated with PGE revealed that nearly all high-molecular-weight species were degraded after 10 h of incubation. After DNA precipitation the reaction products present in the supernatant were subjected to a solid-phase extraction on a polystyrene divinylbenzene copolymer, enabling analysis on capillary zone electrophoresis (CZE), using sample stacking. These reaction products were mainly produced during the first 10 h of incubation, indicating that these products result from the DNA degradation. On the other hand, analysis of the adducts present in the enzymatic digest of the DNA pellet revealed that these adducts were formed only after 10 h of incubation. The reaction products present in the DNA supernatant were identified by on-line coupling of CZE to electrospray tandem mass spectrometry. Three major reaction products resulted from phosphate alkylation, as proven by the analysis of the corresponding low-energy CAD product ion mass spectra. This phosphate alkylation results in phosphotriesters which readily hydrolyze, resulting in DNA strand breaks.

Analysis of DNA adducts in DNA hydrolysates by capillary zone electrophoresis and capillary zone electrophoresis-electrospray mass spectrometry.

The in vitro adduct formation with phenyl glycidyl ethers (PGEs) was studied on 2'-deoxynucleotides and DNA. The modified DNA was hydrolyzed enzymatically, and the mixtures consisting of unmodified 2'-deoxynucleotide adducts were analyzed by capillary zone electrophoresis (CZE), CZE-electrospray mass spectrometry (CZE/ES-MS) and CZE-electrospray tandem mass spectrometry (CZE/ES-MS/MS) using sample stacking. For the CZE analyses, a homemade system was developed in order to enhance the reproducibility of the retention times. This modification enabled the total comparison of the electropherograms obtained for the analysis of 2'deoxynucleotides mixtures with the electropherograms obtained for the DNA hydrolysates both treated with PGEs. The assignment of adducted and nonadducted 2'-deoxynucleotide peaks was unambiguous. Analysis of the CZE/ES-MS data gave the necessary structural information and revealed the presence of mono- and dialkylated 2'-deoxynucleotides. Interpretation of the CZE/ES-MS/MS data of the monoalkylated products allowed differentiation between purine or pyrimidine alkylation and alkylation of the 5-phosphate moiety. Recording of full-scan mass spectra during CZE/ES-MS/MS analysis of 2'-deoxynucleotide reaction mixtures and DNA hydrolysates was possible, using the described CZE sample stacking technique.

Capillary gas chromatography of hexadecylphosphocholine in Caco-2T cells and cell culture media.

The gas-chromatographic determination of hexadecylphosphocholine (HePC), an experimental antitumor agent of the alkyllysophospholipid group, in Caco-2T cell culture and cell culture media is described. The Caco-2T cells were treated with HePC at a concentration of 40 micrograms/ml (9.8 microM) and the uptake of the drug into the cells (calculated per milligram protein) was measured after 48 h culture (37 degrees C, 10% CO2). Also, a reversibility test for another 48 h was carried out in which the retention of the drug was measured. The toxicity of HePC on Caco-2T cells in viability assays was determined. Before the capillary gas-chromatographic determination, sample cleanup was performed by solid-phase extraction (SPE) on a weak cation-exchange column of the CBA (carboxylic acid) type. For quantitation, racemic 1-O-octadecyl-2-O-methylglycero-3-phosphorylcholine (ET-18-OMe) was added as internal standard, followed by derivatization with trimethylsilylbromide. The results showed that HePC taken up by the cells during 48 h of treatment was still detectable 48 h after removal of the drug from the medium.