Determination of antidepressants in human postmortem blood, brain tissue, and hair using gas chromatography-mass spectrometry.

A gas chromatographic-mass spectrometric (GC-MS) method in positive ion chemical ionization mode in combination with a solid phase extraction was optimized for new-generation antidepressants and their metabolites in postmortem blood, brain tissue, and hair. Twelve antidepressants and their active metabolites (i.e., mirtazapine, viloxazine, venlafaxine, citalopram, mianserin, reboxetine, fluoxetine, fluvoxamine, sertraline, maprotiline, melitracen, paroxetine, desmethylfluoxetine, desmethylmianserin, desmethylmirtazapine, desmethylsertraline, desmethylmaprotiline, desmethylcitalopram, and didesmethylcitalopram) could be quantified. In this article, in addition to the validation of the GC-MS method, four postmortem cases are discussed to demonstrate the usefulness of the described method in forensic toxicology. In these cases, sertraline, fluoxetine, citalopram, and trazodone in combination with their active metabolites were quantified. Blood concentrations ranged from subtherapeutic to toxic concentrations, while brain to plasma ratios ranged from 0.8 to 17. Hair concentrations ranged from 0.4 to 2.5 ng/mg depending on the compound and hair segment.

Comparison of electron and chemical ionization modes by validation of a quantitative gas chromatographic-mass spectrometric assay of new generation antidepressants and their active metabolites in plasma.

A gas chromatographic-mass spectrometric method (GC-MS) for the simultaneous determination of the 'new' antidepressants (mirtazapine, viloxazine, venlafaxine, trazodone, citalopram, mianserin, reboxetine, fluoxetine, fluvoxamine, sertraline, maprotiline, melitracen, paroxetine) and their active metabolites (desmethylmirtazapine, O-desmethylvenlafaxine, m-chlorophenylpiperazine, desmethylcitalopram, didesmethylcitalopram, desmethylmianserin, desmethylfluoxetine, desmethylsertraline, desmethylmaprotiline) in plasma using different ionization modes was developed and validated. Sample preparation consisted of a strong cation exchange mechanism and derivatisation with heptafluorobutyrylimidazole. The GC separation was performed in 24.8 min. Identification and quantification were based on selected ion monitoring in electron (EI) and chemical ionization (CI) modes. Calibration by linear and quadratic regression for electron and chemical ionization, respectively, utilized deuterated internal standards and a weighing factor 1/x(2). Limits of quantitation were established between 5 and 12.5 ng/ml in EI and positive ionization CI (PICI), and 1 and 6.25 ng/ml in negative ionization CI (NICI). During validation stability, sensitivity, precision, accuracy, recovery, and selectivity were evaluated for each ionization mode and were demonstrated to be acceptable for most compounds. While it is clear that not all compounds can be quantitated either due to chromatographic (trazodone) or derivatisation problems (O-desmethylvenlafaxine), this method can quantitate most new antidepressants (ADs) in the therapeutic range using EI. PICI and NICI lead to higher selectivity. Moreover, NICI is of interest for small sample volumes and high sensitivity requirements. This paper draws the attention to the pros and cons of the different ionization modes in the GC-MS analysis of these antidepressants in plasma.

Development of a solid phase extraction for 13 'new' generation antidepressants and their active metabolites for gas chromatographic-mass spectrometric analysis.

A solid phase extraction procedure (SPE) for 13 'new' antidepressants (venlafaxine, fluoxetine, viloxazine, fluvoxamine, mianserin, mirtazapine, melitracen, reboxetine, citalopram, maprotiline, sertraline, paroxetine and trazodone) together with eight of their metabolites (O-desmethylvenlafaxine, norfluoxetine, desmethylmianserine, desmethylmirtazapine, desmethylcitalopram, didesmethylcitalopram, desmethylsertraline and m-chlorophenylpiperazine) from plasma is optimized using HPLC-DAD as monitoring system. Special attention has been paid to the choice of washing and eluting solvent, resulting in a highly concentrated, clean and moisture free extract, also suitable for GC-MS. A total number of 10 sorbents (apolar, polymeric, ion-exchange and mixed mode) was evaluated. Based on recovery, reproducibility and absence of interfering substances the strong cation exchanger gave the best results. Recoveries were determined at low and high therapeutic and toxic levels and ranged between 70 and 109% for all compounds, except for trazodone (39%).

Development of an immunoassay-based lateral flow dipstick for the rapid detection of aflatoxin B1 in pig feed.

The aim of this work was to develop an immunoassay-based lateral flow dipstick for the rapid detection of aflatoxin B(1) in pig feed. The test consisted of three main components: conjugate pad, membrane, and absorbent pad. The membrane was coated with two capture reagents, that is, aflatoxin B(1)-bovine serum albumin conjugate and rabbit anti-mouse antibodies. The detector reagent consisted of colloidal gold particles coated with affinity-purified monoclonal anti-aflatoxin B(1) antibodies, which saturated the conjugate pad. A comparison of several extraction methods for the pig feed matrix is presented. A mixture of methanol/water (80:20, v/v) gave the best recoveries. After sample extraction and dilution, the dipstick was put in the sample solution at the conjugate pad side and developed for 10 min. Analyte present in the sample competed with the aflatoxin B(1) immobilized on the membrane for binding to the limited amount of antibodies in the detector reagent. Thus, the line color intensity of an aflatoxin B(1)-positive dipstick is visually distinguishable from that of an aflatoxin B(1)-negative sample. The visual detection limit for aflatoxin B(1) is 5 microg/kg. The major advantages of this one-step striptest are that results can be obtained within 10 min and that all reagents are immobilized on the lateral flow dipstick.

Confirmation of synthetic glucocorticoids with liquid chromatography/mass spectrometry: organization and results of an international interlaboratory comparison test.

Within the framework of a European Union (EU) research project entitled "Food Safety Screening: Synthetic Glucocorticoids (QLK1-1999-00122)," an international interlaboratory ring test was organized to compare and evaluate different liquid chromatography/mass spectrometry (LC/MS) confirmatory methods that are applied in European monitoring programs for detecting the use of synthetic glucocorticoids. Liver and urine samples of bovines treated with synthetic glucocorticoids were collected and sent to the participants of the study for analysis. Participants received 3 liver and 3 urine samples and were free to use either their own LC/MS method or an LC/MS-based method developed during the EU research project. The residue concentrations in the samples were calculated as the mean of the concentrations reported by each laboratory. The mean dexamethasone concentration of liver sample L1 was calculated as 2.27 microg/kg [relative standard deviation (RSD) 43%, n = 9], which exceeds the maximum residue level (MRL) of 2 microg/kg. Three of the 9 laboratories (33%) reported concentration levels less than 2 microg/kg, resulting in obviously false compliant results. The overall mean concentration of flumethasone in liver sample L2 was calculated as 3.27 microg/kg (RSD 33%, n = 8). Applying a comparable limit for flumethasone of 2 microg/kg, 8 of the 9 laboratories would have obtained a correct noncompliant result. As for the blank liver sample, 1 participant found a false noncompliant result. The urine sample U1 contained prednisolone residues at a mean concentration of 1.58 microg/kg (RSD 43%, n = 9). Four out of 9 results were less than a theoretical minimum required performance level (MRPL) of 2 microg/kg. The calculated concentration of dexamethasone in urine sample U3 was 5.21 microg/kg (RSD 62%, n = 9). One of the 9 results was lower than 2 microg/kg. Urine sample U2 was correctly reported as blank by all participants.

Adduct formation in quantitative bioanalysis: effect of ionization conditions on paclitaxel.

Quantitative analysis of target compounds with liquid chromatography atmospheric pressure ionization mass spectrometry is sometimes hampered by adduct formation. In these situations, cationization with alkali metal ions instead of proton addition is often observed in the positive ion mode. This work studies the process of adduct formation and investigates potential strategies to control this phenomenon. Paclitaxel, a pharmaceutical chemotherapeutic agent, was used as a model compound. Electrospray (ESI), atmospheric pressure chemical ionization (APCI) and sonic spray ionization (SSI) are evaluated and compared. The work was performed on two different instruments, allowing the evaluation of different ionization behavior for different source design for electrospray, if any. Different mobile phase additives were compared, including acetic acid, formic acid, ammonium formate, and a range of primary amines. Continuous infusion was used for a fast screening, to detect optimal conditions. These were then further investigated in detail by LC-MS. The results indicate that electrospray is the more sensitive interface for this compound on the investigated apparatus. Unacceptable quantitative data were acquired without additives in the mobile phase. Generally, additives increased the reproducibility significantly. A response of mainly one ion was achieved with dodecylamine/acetic acid and acetic acid/sodium acetate. The data also point out the importance of evaluating adduct formation for compounds prone to this phenomenon during method development, especially in view of accurate quantitation.

Optimization of solid-phase extraction for a liquid chromatographic-tandem mass spectrometric general unknown screening procedure by means of computational techniques.

A solid-phase extraction (SPE) method was optimized to suit the particular demands of an information-dependent acquisition LC-MS-MS procedure for general unknown screening in a forensic toxicology setting. In a first phase, a Plackett-Burman screening design with fold-over was carried out to distinguish the significant factors affecting the extraction procedure. This part eventuated in the determination of only three statistically relevant parameters, requiring consecutive optimization. To that end, in phase II of this study, a rotatable central composite design was applied to define the response surface as a function of the significant parameters and to choose the optimal conditions for the SPE.

Rapid characterization of oligonucleotides by capillary liquid chromatography-nano electrospray quadrupole time-of-flight mass spectrometry.

A fast quality control method is developed allowing the desalting and characterization of oligonucleotides by capillary liquid chromatography and on-line nano-electrospray ionization quadrupole time-of-flight mass spectrometry using column switching. The influence of addition of ammonium acetate, trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid, formic acid or acetic acid to the sample, addition of ammonium acetate to the trapping solvent and variation of the trapping time on the further reduction of cation adduction was studied. Final conditions were the addition of 0.1 M ammonium acetate to the sample, the use of a trapping solvent consisting of 0.4 M aqueous 1,1,1,3,3,3-hexafluoro-2-propanol (HFLP) adjusted to pH 7.0 with triethylamine plus 10 mM ammonium acetate during 8 min and the elution of the oligonucleotides with 0.4 M HFIP in 50% methanol. The potential of the optimized procedure is demonstrated for different synthetic oligonucleotides.

Suitability testing of commercial solid-phase extraction sorbents for sample clean-up in systematic toxicological analysis using liquid chromatography-(tandem) mass spectrometry.

An entire series of SPE sorbents, classified into three different categories (apolar, mixed-mode and polymeric) was evaluated for sample preparation of a data-dependent LC-MS-MS "general unknown" screening procedure. An extraction procedure was formulated for each individual column, in agreement with the enclosed instructions, according to the characteristics of each packing. For conciseness, only neutral and basic compounds were chosen for this sorbent suitability test. Thus, the goal of our research was to select the best sorbent with regard to extraction yield and cleanliness of the extracts, all with respect to data-dependent acquisition (DDA) mediated LC-MS-MS general unknown screening. We conclude that for that purpose an Isolute C(8) sorbent performs best in terms of extraction yield and clean-up potential.

Simultaneous, quantitative determination of opiates, amphetamines, cocaine and benzoylecgonine in oral fluid by liquid chromatography quadrupole-time-of-flight mass spectrometry.

A method using liquid chromatography coupled to tandem mass spectrometry is described for the determination of drugs of abuse in oral fluid. The method is able to simultaneously quantify amphetamines (amphetamine, methamphetamine, MDA, MDMA and MDEA), opiates (morphine and codeine), cocaine and benzoylecgonine. Only 200 micro of oral fluid is spent for analysis. The sample preparation is easy and consists of mixed mode phase solid-phase extraction. Reversed-phase chromatography is carried out on a narrow bore phenyl type column at a flow-rate of 0.2 ml/min. A gradient is applied ranging from 6 to 67.6% methanol with ammonium formate (10 mM, pH 5.0) added to the mobile phase. The column effluent was directed into a quadrupole-time-of-flight instrument by electrospray ionization, without the use of a splitter. A validation study was carried out. Recovery ranged from 52.3 to 98.8%, within-day and between-day precision expressed by relative standard deviation were less than 11.9 and 16.8%, respectively, and inaccuracy did not exceed 11.6%. The limit of quantification was 2 ng/ml (0.66 x 10(-5)-1.48 x 10(-5) M) for all compounds. Internal standards were used to generate quadratic calibration curves (r(2)>0.999). The method was applied to real samples obtained from suspected drug users. An interference was observed from the device used to sample the oral fluid, consequently this was excluded from the method which was validated on oral fluid obtained by spitting in a test-tube.

Immunochemical screening and liquid chromatographic-tandem mass spectrometric confirmation of drug residues in edible tissues of calves injected with a therapeutic dose of the synthetic glucocorticoids dexamethasone and flumethasone.

A field study was performed to assess the drug residue level in edible tissues after a therapeutic application of the synthetic glucocorticoids dexamethasone and flumethasone. Three diseased calves were injected intramuscularly with a commercial batch of dexamethasone esters and slaughtered 72 h after treatment. Another three calves were injected intramuscularly with an aqueous flumethasone preparation and slaughtered 24 h later. Residues of synthetic glucocorticoids in liver, muscle, kidney, and urine were assessed by competitive enzyme immunoassay. All dexamethasone concentrations exceeded the maximal residue level of 0.75 microg/kg in muscle and kidney and 2 microg/kg in the liver. The presence of both dexamethasone and flumethasone in the liver was confirmed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). These results indicate that liver tissue provides a suitable matrix to monitor the presence of illegal residues of synthetic glucocorticoids in slaughtered animals.

Aconitine involvement in an unusual homicide case.

We describe a homicide complicated by an aconitine poisoning, which was initially thought to be a strangulation case. Routine toxicological analyses demonstrated only a small amount of alcohol in the blood and the urine. The case could not be clarified until 5 years after the event. A new element in the investigation made the wife the prime suspect, and finally, after thorough interrogation, she confessed her crime. She had mixed a decoction of three plants of Aconitum with red wine. Additional toxicological analyses, using the liquid chromatography-tandem mass spectrometry (LC-MS-MS) technique demonstrated 810 ng/ml of aconitine in urine, 6.5 ng/g in liver and 1.3 ng/g in the kidneys. Even though aconitine poisoning is still rare in Europe, it should be taken into account in suicides and homicides, particularly in unclarified cases.

Analysis of nucleic acid constituents by on-line capillary electrophoresis-mass spectrometry.

This review is focused on the capillary electrophoresis-mass spectrometric (CE-MS) analysis of nucleic acid constituents in the broadest sense, going from nucleotides and adducted nucleotides over nucleoside analogues to oligonucleotides. These nucleic acid constituents play an important role in a variety of biochemical processes. Hence, their isolation, identification, and quantification will undoubtedly help reveal the process of life and disease mechanisms, such as carcinogenesis, and can also be useful for antitumor and antiviral drug research to provide valuable information about mechanism of action, pharmacokinetics, pharmacodynamics, toxicity, therapeutic drug level monitoring, and quality control related to this substance class. Fundamental investigations into their structure, the search for modifications, the occurrence and biochemical impact of structural variation amongst others, are therefore of great value. In view of the related bioanalytical procedures, the coupling of CE to MS has emerged as a powerful tool for the analysis of the complex mixtures of nucleic acid constituents: CE confers rapid analysis and efficient resolution, while MS provides high selectivity and sensitivity with structural characterization of minute amounts of compound. After an introduction about the biochemical and analytical perspectives on the nucleic acid constituents, the different modes of CE used in this field of research as well as the relevant CE-MS interfaces and the difficulties associated with quantitative CE-MS are briefly discussed. A large section is finally devoted to field-oriented applications.

Development of a quality control method for the characterization of oligonucleotides by capillary zone electrophoresis-electrospray ionization-quadrupole time of flight-mass spectrometry.

A capillary zone electrophoresis-negative electrospray ionization-quadrupole time of flight-mass spectrometric method was developed for the characterization of oligonucleotides after synthesis, using model compounds. The major difficulty is the adduction of metal cations to the polyanionic backbone of the oligonucleotide sample, resulting in complex spectra and decreased sensitivity. Several approaches were investigated to circumvent this problem. Separation was performed in an ammonium carbonate buffer. During separation, the interfering metal ions were exchanged for ammonium ions, which are less tightly bound to the oligonucleotide when ionized. The influence of the addition of piperidine and imidazole or trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) to the running buffer for further reduction of cation adduction was investigated. Addition of CDTA to the buffer system resulted in a deconvoluted spectrum with very little adducts. On-line sample stacking proved vital to preconcentrate the samples. The pH and the concentration of the ammonium carbonate buffer as well as the electrophoresis voltage were optimized to achieve the best signal response for the oligonucleotides and a maximum reduction of the cation adducts as well as a short analysis time. Finally, the sheath liquid composition was examined for further improvement of the signal. The developed method was used to analyze different oligonucleotides (5000-9200 Da) in light of its use as a final quality control method for oligonucleotides in terms of purity and sequence homogeneity of the synthesized products. In all cases, very little adducts were observed in the deconvoluted spectra, and the relative errors of the measured molecular masses ranged from 3 to 35 ppm.

Pharmacokinetics and Tissue Residues of Sulfathiazole and Sulfamethazine in Pigs.

The plasma pharmacokinetics and tissue penetration of sulfathiazole (ST) and sulfamethazine (SM) after intravenous and intramuscular injection in pigs were studied. Following a single intravenous dose of 40 mg ST/kg of bodyweight or 80 mg SM/kg of bodyweight, the plasma ST and SM concentrations were best fitted to a two-compartment model. The areas under the curve were 447 ± 39 and 1485 ± 41 mg/h/L, clearances were 0.090 ± 0.007 and 0.054 ± 0.001 L/kg/h, volumes of distribution were 1.16 ± 0.16 and 0.77 ± 0.06 L/kg, half-lifes in distribution phase were l.18 ± 0.57 and 0.23 ± 0.16 h and half-lifes in eliminations phase were 9.0 ± l.6 and 9.8 ± 0.6 h. When the two compounds were administered simultaneously as a single intravenous injection, the pharmacokinetic parameters for ST were not significantly different. The values for SM show statistical differences for some important parameters: α, ß and the AUC0->∞ were significantly decreased and t1/2α, Vd and CIB were significantly increased. It can be concluded that after a single intravenous injection of 40 mg/kg, sulfathiazole has a high tl/2ß resulting in higher tissue concentrations. This half-life, which is higher than what is reported in the literature, is not influenced by the simultaneous presence of sulfamethazine. The tl/2ß for sulfamethazine after a single intravenous injection of 80 mg/kg is comparable to the data from the literature and is not influenced by the presence of sulfathiazole. Sulfathiazole and SM were also administered simultaneously as an intramuscular injection to healthy pigs at a dosage of 40 and 80 mg/kg bodyweight. Pharmacokinetic experiments were conducted on three pigs. From this pharmacokinetic study it can be concluded that upon a single intramuscular administration of 40 mg/kg of ST and 80 mg/kg of SM the absolute bioavailability in pigs is 0.92 ± 0.04 for ST and l.01 ± 0.07 for SM. Six pigs received five intramuscular im) injections as a single dose of ST and SM every 24 h for five consecutive days for the residue study. The pigs were slaughtered at different times after the last dose was given and samples were taken from various tissues and organs. Concentrations were determined by a microbiological method and a HPTLC method. No edible tissue contained more than 100 µg/kg of the individual sulfonamides after 10 days of withdrawal. It means that adult animals which have a shorter half-life and thus lower tissue concentrations will certainly meet the economic community EC) maximum residue limits after a 10 days withdrawal period.

Development of a dual luciferase reporter screening assay for the detection of synthetic glucocorticoids in animal tissues.

Synthetic glucocorticoids belong to the most frequently administered drugs in livestock production. These synthetic hormones are employed for therapeutic purposes against inflammatory reactions, disorders of the musculoskeletal system, bovine ketosis and many other diseases of farm animals. A widespread illegal use of synthetic glucocorticoids to improve feed intake and weight gain has also been observed. To enforce the residue limits imposed on glucocorticoid drugs and preclude their illicit administration as growth promoters, it is necessary to establish high throughput analytical methods that can be applied to the screening of animal tissues. Here, we developed a dual luciferase reporter assay that detects residues or contaminants with glucocorticoid activity. This screening assay is performed by transfection of human cell lines with two reporter constructs followed by the measurement of two distinct luminescence signals, one of which serves as internal control to correct for assay variabilities and unspecific matrix effects. The limit of detection (1.25 microg for dexamethasone in liver) depends on the biological potency of each synthetic glucocorticoid but, with all drugs tested, the maximal response reaches a 20 to 30 fold induction of luciferase activity. In combination with an appropriate sample clean-up method (recovery of 82%), this luciferase assay has been applied to the analysis of liver samples from calves treated with a single therapeutic injection of either dexamethasone or flumethasone. Thus, the dual luciferase reporter assay provides a new screening tool to detect unwanted glucocorticoid activities in animal tissues or other crude biological samples without knowledge of the precise chemical entity of the parent compounds or their metabolites.

Information-dependent acquisition-mediated LC-MS/MS screening procedure with semiquantitative potential.

The development of a LC-MS/MS general unknown screening procedure for toxicologically relevant substances in blood samples by means of information-dependent acquisition on a Q-TOF is reported. IDA is an artificial intelligence-based product ion scan mode providing automatic "on-the-fly" MS to MS/MS switching. By performing information-dependent scanning at two different fragmentation energies, two collision-induced dissociation product ion spectra for each of the detected compounds are generated. As such, information-rich MS/MS spectra are obtained from precursor ions not known beforehand. In addition, limitation of the MS/MS acquisition time to an acceptable minimum resulted in an almost instantaneous switch back to the MS mode. As such, this approach provided MS chromatograms that still could be of use for semiquantitative purposes. Since the switching intensity threshold, unequivocally related to the background noise, proved a critical parameter, the solid-phase extraction procedure, the liquid chromatographic conditions, and the mass spectrometric parameters all were optimized to the advantage of information-dependent acquisition. Finally, the screening procedure we developed was benchmarked, on one hand, qualitatively against the results obtained from traditional GUS approaches in a number of routine toxicological laboratories (20 samples) and, on the other hand, quantitatively with respect to its potential against established LC-MS/MS methods (7 samples). The procedure performed very well from a qualitative point of view; almost all of the drugs detected by the conventional techniques were identified, as well as additional drugs that were not previously reported. The procedure proved well-suited for an initial semiquantitative assessment, as is customary in, for example, forensic toxicology before accurate intoxication levels are determined using targeted analytical analyses.

Analysis of benzo[a]pyrene diol epoxide-DNA adducts by capillary zone electrophoresis- electrospray ionization-mass spectrometry in conjunction with sample stacking.

Benzo[a]pyrene diol epoxide (BPDE) was reacted in vitro with (2'-deoxy)nucleotides and with calf thymus DNA. The modified DNA was enzymatically hydrolyzed to the 5'-monophosphate nucleotides using deoxyribonuclease I (DNA-ase I), nuclease P1 and snake venom phosphodiesterase (SVP). Most of the unmodified nucleotides were removed using solid phase extraction (SPE) in a polystyrene divinylbenzene copolymer. Three adducts could be detected and identified using capillary zone electrophoresis(negative)-ion electrospray ionization-mass spectrometry (CZE-(-)-ESI-MS) in conjunction with sample stacking. This way, not only a BPDE-dGMP adduct [(M-H)(-) at m/z 648], a well-known nucleotide adduct, could be retrieved, but also a BPDE-dAMP [(M-H)(-) at m/z 632] and a BPDE-dCMP adduct [(M-H)(-) at m/z 608] could be detected for the first time. The presence of the prominent ion at m/z 195 (the deoxyribose-phosphate ion) in the three low-energy collision-activated decomposition (CAD) spectra indicated that the adducts were formed through base-alkylation. CZE-positive ion ESI-MS/MS experiments were performed to obtain further information on base-alkylation. The absence of the loss of NH(3) from the nucleobase in each CAD spectrum points to an alkylated exocyclic NH(2) position of the nucleobase. So, the three adducts could be identified as BPDE-N(2)-dGMP, BPDE-N(6)-dAMP and BPDE-N(4)-dCMP using CZE-ESI-MS and CZE-ESI-MS/MS.