Journal of Chromatography B (v.773, #1)

Determination of clenbuterol in human urine by GC–MS–MS–MS: confirmation analysis in antidoping control by Luca Amendola; Cristiana Colamonici; Francesca Rossi; Francesco Botrè (7-16).
This work presents a GC–MS–MS–MS method for the direct determination of clenbuterol in human urine. The method comprises a pretreatment procedure and the instrumental analysis of the derivatives performed by GC–MS3 (ion trap) with electron impact ionization. The GC–MS3 analysis allows isolation and characterization of specific fragments from the original (MS1) molecular structure, and in particular, those fragments originating from the precursor ion cluster (m/z=335–337) characteristic of clenbuterol. The MS2 product fragment m/z=300 is in turn used as a further precursor fragment giving rise to a MS3 spectrum specific for clenbuterol. MS4 fragmentation spectra were also investigated. However, further fragmentation of MS3 product ions does not lead to functional MS4 spectra nor to any significant increase in the signal-to-noise ratio. The sensitivity limit of the MS3 technique is lower than 0.2 μg/l, with a linear range between 0.5 and 5 μg/l, thus matching the basic requirements for antidoping analysis according to the guidelines of the International Olympic Committee. Due to its overall analytical performance, the method is presently being evaluated as a confirmation protocol to be followed to detect illicit clenbuterol administration to the athletes, and compared with reference GC–MS and GC–MS–MS techniques.
Keywords: Clenbuterol;

Selective serotonin reuptake inhibitors (SSRIs), serotonin noradrenergic reuptake inhibitors (SNaRIs) and noradrenergic and specific serotoninergic antidepressant (NaSSA) are widely used in the treatment of depression. An increase in antidepressant intoxications led to the development of reliable analytical methods for their analysis. A new determination procedure for these compounds (milnacipran, venlafaxine, desmethylvenlafaxine, mirtazapine, desmethylmirtazapine, citalopram, desmethylcitalopram, fluvoxamine, paroxetine, sertraline and fluoxetine) was developed by micellar electrokinetic capillary chromatography (MEKC) with diode array detection (DAD). Separation and determination were optimised on an uncoated fused-silica capillary (600 mm, 75 μm I.D.). The migration buffer consisted of 20 mM sodium borate, pH 8.55, with 20 mM SDS and 15% isopropanol, at an operating voltage of 25 kV. The column temperature was maintained at 40 °C. Injection in the capillary was performed in the hydrodynamic mode (0.5 p.s.i., 15 s). In these conditions, the migration time of the antidepressants was less than 11 min. In most cases, calibration curves were established for 30–2000 ng/ml (r>0.995). The limit of detection and the limit of quantification were ranged between 10 and 20 and between 20 and 30 ng/ml, respectively, for all the molecules. This method allowed the determination of some of these compounds in biological fluids (blood, urine) in post-mortem cases. Samples (1 ml) were extracted with diethyl ether (5 ml) at pH 9.6 and reconstituted in diluted migration buffer. Similar results were obtained by a HPLC–DAD determination, performed as a reference method. These results suggest that this MEKC method can be useful for the determination of new antidepressants in post-mortem cases.
Keywords: Antidepressants;

4′-Methyl-α-pyrrolidinopropiophenone (MPPP) is a new designer drug which has appeared on the illicit drug market. The aim of our study was to identify the MPPP metabolites and to develop a toxicological detection procedure in urine using solid-phase extraction, ethylation and GC–MS. In urine samples of rats treated with MPPP, MPPP was found to be completely metabolized by oxidative desamination, hydroxylation of the 4′-methyl group followed by oxidation finally to the corresponding carboxy compound and/or by hydroxylation of the pyrrolidine ring followed by dehydrogenation to the corresponding lactam. The carboxy groups were found to be partly conjugated. Based on these data, MPPP could be detected in urine via its metabolites by GC–MS using mass chromatography for screening and library search for identification.
Keywords: 4′-Methyl-α-pyrrolidinopropiophenone; Designer drug; Metabolism;

Studies are described on the metabolism and on the toxicological analysis of the piperazine-like designer drug N-benzylpiperazine (BZP, scene name “A2”) in rat and human urine using gas chromatography–mass spectrometry (GC–MS). The identified metabolites indicated that BZP was hydroxylated at the aromatic ring and that the piperazine moiety is metabolically degraded. Our systematic toxicological analysis (STA) procedure using full-scan GC–MS after acid hydrolysis, liquid–liquid extraction and microwave-assisted acetylation allowed the detection of the parent compound as well as of the above mentioned metabolites in rat urine after single administration of a dose calculated from the doses commonly taken by drug users. It has also proved to be applicable in authentic clinical or forensic cases. However, it should be considered that BZP is also a metabolite of the medicament piberaline.
Keywords: N-Benzylpiperazine (BZP); A2;

Ion suppression effects during electrospray-ionsation mass spectrometry (ESI-MS) caused by different sample preparation procedures for serum were investigated. This topic is of importance for systematic toxicological analysis for which LC–ESI-MS has been developed with transport-region collision-induced dissociation (ECI-CID) and mass spectra library searching. With continuous postcolumn infusion of two test compounds—codeine and glafenine—the ion suppression effects of extracted biological matrix obtained after a standard liquid–liquid extraction, a mixed-mode solid-phase extraction (SPE) method, a protein precipitation method and a combination of precipitation with polymer-based mixed-mode SPE have been investigated. Extracted ion chromatograms of codeine ([M+H]+, m/z 300) and glafenine ([M−H], m/z 371) were used for monitoring ion suppression. Severe ion suppression effects for codeine and glafenine were detected in positive and in negative ionisation modes, respectively, in the LC-front peak after serum clean-up with SPE (acid/neutral fraction) and protein precipitation as well as with protein precipitation combined with SPE. Less ion suppression of codeine in positive mode was found with liquid–liquid extraction of serum samples. No ion suppression was detected with the second fraction of the mixed-mode SPE (using RP-C8 and cation-exchange phase) in both ionisation modes. All suppression effects were caused by polar and unretained matrix components, which were present after extraction and/or protein precipitation. However, no specific ion suppression was seen after elution of the polar LC-front throughout the whole gradient. It could be demonstrated, that ion suppression is not generally present at any retention time when using reversed-phase HPLC with rather long gradient programs, but may play an important role in case of high-throughput LC–MS analysis, when the analyte is not separated from the LC-front, or in flow injection analysis without chromatographic separation.

A fast liquid chromatographic method with tandem diode array–fluorescence detection for the simultaneous determination of in total 17 opium alkaloids and opioids is presented. Blank blood and urine samples (1 ml) were spiked with different concentrations of a standard mixture, as well as with the internal standard, butorphanol (2000 ng/ml). After solid-phase extraction, based on weak cation exchange (Bond Elut® CBA SPE columns), the extracts were examined by HPLC–DAD–FL. By using a “high-speed” phenyl column (53×7.0 mm I.D., particle size 3 μm) eluted with a gradient system (A: water–methanol (90:10, v/v), B: methanol, both containing 25 mM triethylammoniumformate (pHA=4.5)) all compounds could be baseline separated within 12 min. The method was validated and its applicability was demonstrated by the analysis of real-time forensic cases.
Keywords: Opium alkaloids; Opioids;

An atmospheric pressure chemical ionization liquid chromatographic–mass spectrometric (APCI–LC–MS) LC–MS assay is presented for fast and reliable screening and identification as well as precise and sensitive quantification of oral antidiabetics of the sulfonylurea-type (OADs) in plasma. It allowed the specific diagnosis of an overdose situation or a Munchausen syndrome caused by ingestion of OADs. After liquid–liquid extraction, the OADs glibenclamide, glibornuride, gliclazide, glimepiride, glipizide, gliquidone, glisoxepide, tolazamide and tolbutamide were separated using fast gradient elution. After screening and identification in the scan mode using our new LC–MS library, the OADs were quantified in the selected-ion mode. The quantification assay was validated according to the criteria established by the Journal of Chromatography B. All validation data were inside the required limits. The assay is part of a general LC–MS procedure for fast screening, identification and quantification of different toxicologically relevant compounds in plasma and has proven to be appropriate for OADs.
Keywords: Antidiabetics; Sulfonylurea-type;

A procedure for the determination of acetaldehyde, acetone, methanol, ethanol, 1-propanol and 2-propanol in blood was developed. Separation of analytes was carried out on DB-wax capillary column (l=30 m, I.D.=0.32 mm, dF=0.5 μm) at 40 °C, hydrogen was used as a carrier gas (at 30 kPa) and FID as a detector. Quantification was performed with the use of 2-butanol as an internal standard. Headspace solid-phase microextraction was applied as the sample preparation technique. The usefulness of most commercially available fiber coatings was checked and 65 μm Carbowax/DVB proved most effective. Microextraction was carried out from the headspace at 60 °C for 10 min. The sample was stirred at 750 rpm. In order to improve the extraction efficiency of analytes, salting-out agents were also applied. Potassium carbonate turned out to be the most efficient. A 1.0-g amount of this salt and 0.1 ml of I.S. were added to 0.5 ml of sample. Validation of the worked-out method was performed. For each analyte, the limits of detection and quantification, linearity, working range, accuracy and precision were determined or tested.
Keywords: Ethanol; Volatiles;