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When developing an LC-MS/MS-method matrix effects are a major issue. The effect of co-eluting compounds arising from the matrix can result in signal enhancement or suppression. During method development much attention should be paid to diminish matrix effects as much as possible. The present work evaluates matrix effects from aqueous environmental samples in the simultaneous analysis of a group of nine specific pharmaceuticals with LC-ESI/MS/MS: flubendazole, propiconazole, pipamperone, cinnarizine, ketoconazole, miconazole, rabeprazole, itraconazole and domperidone. Solutions to diminish signal suppression were examined: optimisation of the sample preparation, decrease of the flow rate, and the use of appropriate internal standards. Several SPE-stationary phases were tested in view of retention of the analytes: Oasis HLB, C8, Phenyl, Strata X-polymer RP sorbent and Strata X-polymeric SCX/RP sorbent. Oasis HLB showed the best retention for all analytes. The Oasis HLB SPE-method was optimised, but analyses showed high matrix suppression. Therefore, a second SPE-method, on a phenyl stationary phase (the second best option), was also optimised. A comparison of the matrix effect was made between the two procedures: the phenyl-method was less subject to matrix effects, however, the average matrix effect (ME%) of 46% indicated that matrix effects where still present. Several optimisation options for the phenyl-method were evaluated: addition of a ferric nitrate solution before extraction, application of an alkaline wash step, and use of a second SPE-cartridge, either a NH2-column or a florisil column. A more efficient sample clean-up was achieved by applying the extract after extraction on the phenyl column and after dilution with chloroform, onto a NH2-column (average ME%: 53%). In addition, applying a post-column split (1:5), further reduced matrix effects (average ME%: 65%). Labelled internal standards are the best way to tackle matrix effects, but no such internal standards were commercially available for the analytes of interest. The thorough search and application of four internal standards (structural analogues) was beneficial and compensates the matrix effect partially (average ME%: 83%). In an attempt to reduce the analysis time Speedisk phenyl columns were applied. Under these conditions matrix effects decreased even more while recoveries were between 91 and 109%. Different kinds of surface water samples were analyzed, and different matrix effects were observed. For this reason, standard addition will be used to perform quantitative analysis.

This work describes the development of a straightforward method for the determination of free docetaxel and paclitaxel in plasma. The separation of bound and unbound drug was performed with ultrafiltration. Different ultrafiltration devices were evaluated, especially regarding non-specific binding to the device. The most appropriate device for this application was selected and a procedure to counteract non-specific binding to the ultrafiltrate collection cup was developed. This consisted of a wash procedure with methyl t-butyl ether. A liquid/liquid extraction with methyl t-butyl ether was performed and samples were analysed with a previously developed liquid chromatography-tandem mass spectrometry procedure. The method used a Merck Purospher Star RP-18 column (55 mm x 2.0 mm, 3-microm particle size) and electrospray in the positive mode. A triple quadrupole instrument was used to monitor MRM transitions. Small modifications to this procedure were made to ensure adequate sensitivity. Within- and between-day reproducibility did not exceed 15% and accuracy ranged between 94.4 and 102.5%. The calibration range of the method was from 0.4 to 100 ng/ml both for paclitaxel and docetaxel. Finally, a fast and relatively simple method could be developed.

A quantitative method for the simultaneous determination of docetaxel (Taxotere), paclitaxel (Taxol), 6alpha-hydroxypaclitaxel, and p-3'-hydroxypaclitaxel in human plasma and oral fluid is developed and validated. Oral fluid (this term is now preferred to saliva) was sampled with a Salivette collection device. The procedure used a simple liquid/liquid extraction with methyl tert-butyl ether followed by LC-ESI-MS/MS. Gradient elution was applied and provided increased robustness to ion suppression by the drug formulation vehicle (polysorbate 80 and Cremophor EL). Adduct ion formation with sodium and potassium was noticed and controlled by mobile-phase optimization. The protonated analytes generated in the positive ion mode were monitored through multiple reaction monitoring. Calibration was performed by internal standardization with cephalomannine, and regression curves were constructed ranging between 2 and 1000 ng/mL in plasma and 0.125 and 62.5 ng/mL in oral fluid, using a weighing factor of 1/x2. The regression curves were quadratic for paclitaxel and docetaxel and linear for the paclitaxel metabolites. Accuracy varied from 91.3 to 103.6%, and imprecision did not exceed 12.7% for all analytes in plasma and oral fluid. In conclusion, a sensitive and robust method was obtained, which fulfilled all validation criteria.

para-Aminobenzoic acid (PABA), a precursor in the synthesis of folates in plants, is determined by liquid chromatography/tandem mass spectrometry (LC/MS/MS). In plants PABA can be converted into its beta-D-glucopyranosyl ester (PABA-Glc) and can also exist in its free form. In this work, we developed and validated a quantitative method to study free and total PABA in plants. The total PABA (free PABA plus PABA-Glc) can be evaluated after acid hydrolysis at 80 degrees C for 2 hours. The plant material is homogenized and the PABA content is quantified using the standard addition procedure. The validated method is selective, sensitive, simple, accurate, has a recovery between 99.6 to 102.5%, is reproducible (RSD between 1.4 and 4.4%), and is linear between 2.5 and 1538 ng/mL. Free and total PABA determinations in five vegetables showed that different plant species had different amounts of free and total PABA, and that the ratios of total versus free PABA were also variable. This new method could be valuable for studies of folate synthesis in plants., (Copyright 2005 John Wiley & Sons, Ltd.)

Liquid chromatography-tandem mass spectrometry (LC-MS-MS) is often performed in a high-throughput environment. Unfortunately, with atmospheric pressure ionization (API) techniques, shorter run-times or reduced sample clean-up often result in ion or matrix suppression, which can lead to erroneous results. The present work on the analysis of paclitaxel compares ion suppression, sensitivity and linearity of a high-throughput LC-MS-MS method (0.8 min run-time, method B) to a method with increased separation (2.0 min run-time, method A). An atmospheric pressure chemical ionization (APCI) interface was used for both methods. The high-sample-throughput method uses an increased amount of organic solvent in the mobile phase (isocratic, 85% versus 70% of methanol) and a higher flow-rate (600 microl/min versus 400 micro/min). As a result, internal standard (docetaxel) and target analyte (paclitaxel) co-elute, close to, although separated from the solvent front. Ion suppression of both methods was evaluated by comparing pure standard to plasma and plasma containing a vehicle. Sensitivity and linearity were compared by injecting matrix matched calibration samples with both methods. Ion suppression by the vehicle Cremophor EL led to poor data quality for the standard method (A), while for the short method (B), ion suppression was compensated for by the co-eluting internal standard. The short method showed similar linearity but increased sensitivity by at least a factor five. This work provides a strategy to compensate for ion suppression without the use of labeled internal standards. In addition, a better sensitivity and a shorter run-time are noted.

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.

A procedure involving chemical conversion of all forms of folate present in plant material into para-aminobenzoic acid (PABA) and a liquid chromatographic-fluorimetric determination with on-line postcolumn derivatization is reported. All folates are cleaved with liberation of PABA by hydrogen peroxide followed by acid hydrolysis using concentrated hydrochloric acid (37%) at 110 degrees C for 6 h. The reaction yield for individual folates conversion to PABA ranged from 44.4 to 97.3%. PABA could be determined sensitively by on-line postcolumn derivatization with fluorescamine, the detection limit for PABA being 3.02 nM. On the basis of this principle, a method for the determination of total folate in plant material, including a purification step on an affinity column, is presented, which offers a sufficient sensitivity and selectivity for routine analysis of total folate in natural samples. The total folate contents of tomatoes, carrots, white cabbage, and spinach were determined, and the results were quite comparable to the data reported. The recovery of PABA and the comparison of total folate analysis in spinach on different occasions (over 6 months) are also reported. The method is reliable, universal for all folates, including polyglutamate and monoglutamate forms, and eliminates the need for a deconjugation step and multiple conversion reactions.

We present a fatal case involving the combined ingestion of amphetamine, 3,4-methylenedioxymethylamphetamine, 3,4-methylenedioxyamphetamine, and paramethoxyamphetamine. Various postmortem specimens (e.g., several blood samples, urine, and tissue samples) were analyzed to study the distribution of the compounds and their metabolites in the human body. Quantitation took place using liquid chromatography-sonic spray ionization-mass spectrometry after pretreatment with a liquid-liquid extraction. The medico-legal findings were compatible with a disseminated intravascular coagulation induced by hyperthermia caused by the simultaneous intake of the amphetamine analogues.

Abuse of amphetamine derivatives such as 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyamphetamine (MDA) is an important issue in current forensic practice and fatalities are not infrequent. Therefore, we investigated an immunohistochemical method to detect the amphetamine analogues MDMA and MDA in human tissues. For the staining procedure, the Catalysed Signal Amplification (CSA) method using peroxidase (HRP) provided by Dako and specific monoclonal antibodies were used. Appropriate controls for validation of the technique were included. The distribution of these designer drugs was studied in various brain regions including the four lobes, the basal ganglia, hypothalamus, hippocampus, corpus callosum, medulla oblongata, pons, cerebellar vermis and, additionally, in the pituitary gland. A distinct positive reaction was observed in all cortical brain regions and the neurons of the basal ganglia, the hypothalamus, the hippocampus and the cerebellar vermis but in the brainstem, relatively weak staining of neurons was seen. The reaction presented as a mainly diffuse cytoplasmic staining of the perikaryon of the neurons, and often axons and dendrites were also visualised. In addition, the immunoreactivity was present in the white matter. In the pituitary gland, however, distinct immunopositive cells were observed, with a prominent heterogeneity. The immunohistochemical findings were supported by the toxicological data. This immunostaining technique can be used as evidence of intake or even poisoning with MDMA and/or MDA and can be an interesting tool in forensic practice when the usual samples for toxicological analysis are not available. Furthermore, this method can be used to investigate the distribution of these substances in the human body.

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.

Paramethoxyamphetamine (PMA) is an amphetamine-like designer drug that has emerged recently on the European illicit drug market. This drug has a wicked reputation, as a number of lethal intoxications have occurred. A method using high-performance liquid chromatography coupled to ion trap based mass spectrometry (LC/MS) is described for the determination of this compound together with 3,4-methylenedioxymethamphetamine (XTC or MDMA), amphetamine and 3,4-methylenedioxyamphetamine (MDA) in human matrices. A liquid/liquid extraction (LLE) was applied to whole blood, urine and postmortem tissues. Reversed-phase liquid chromatography was performed on a narrow-bore phenyl-type column at a flow rate of 0.3 mL/min. A switch box allowed disposal of early-eluting irrelevant material to waste, protecting the mass spectrometer from contamination. The column effluent was directed into an ion trap mass spectrometer by a sonic spray ionization (SSI) interface. The method was validated for all three matrices, proving the applicability of SSI even when dealing with complex biological matrices. The within-and between-day precisions were less than 17.5% and accuracy was below 16.2%. Weighted (1/x) quadratic calibration curves were generated ranging from 10 to 1000 ng/mL (blood and urine) or 20 to 2000 ng/g (tissue) and correlation coefficients (r(2)) always exceeded 0.995. In addition, the mass spectrum of PMA is given together with a proposed fragmentation pattern for the obtained LC/MS spectrum. This information can be useful for future identification of PMA with LC/MS in biological matrices as well as in confiscated powders or tablets., (Copyright 2002 John Wiley & Sons, Ltd.)