Journal of Chromatography B (v.767, #2)

News Section (N1-N3).

An assay was developed to determine concentrations of modafinil (dl-2-[(diphenylmethyl)sulfinyl]acetamide; Provigil®) and its two major circulating metabolites, modafinil acid and modafinil sulfone, in human plasma. The assay utilized liquid–liquid extraction of the analytes and an internal standard, (phenylthio)acetic acid, from plasma into a mixture of hexane–dichloromethane–glacial acetic acid (55:45:2, v/v). The analytes were resolved isocratically on a narrow-bore phenyl column at a mobile phase flow-rate of 0.3 ml/min and were monitored by UV detection at 235 nm. The method reported herein reduces the required sample volume of previously reported methods from 1.00 to 0.200 ml of plasma while lowering the limit of quantification (LOQ). The linear range of the assay was from 0.100 to 20.0 μg/ml for each of the three compounds.
Keywords: Modafinil; Modafinil acid; Modafinil sulfone;

Control of propranolol intake by direct chromatographic detection of α-naphthoxylactic acid in urine by M.J. Ruiz-Ángel; P. Fernández-López; J.A. Murillo-Pulgarı́n; M.C. Garcı́a-Alvarez-Coque (277-283).
A rapid chromatographic procedure with a C18 column, a mobile phase of 0.15 M sodium dodecyl sulfate (SDS)–10% (v/v) 1-propanol at pH 3 (0.01 M phosphate buffer), and fluorimetric detection, is reported for the control of propranolol (PPL) intake in urine samples, which are injected directly without any other treatment than filtration. The peak of PPL was only observed in samples taken a few hours after ingestion of the drug due to its extensive conjugation and metabolisation. The detection of several unconjugated PPL metabolites was therefore considered: desisopropylpropranolol (DIP), propranolol glycol (PPG), α-naphthoxylactic acid (NLT) and α-naphthoxyacetic acid (NAC). NLT showed the best characteristics; it eluted at a much shorter retention time than PPL, its concentration in urine samples was greater and it did not present any interference from endogeneous compounds in urine, common drugs or drugs administered in combination with PPL. The limit of quantification, measured as the concentration of analyte providing a relative standard deviation of 20%, was 24 ng/ml, and the day-to-day imprecision was below 4% for concentrations above 200 ng/ml. The procedure allows the routine control of PPL at therapeutic urine levels. Urinary excretion studies showed that the detection of NLT is possible at least up to 20–30 h after oral administration.
Keywords: Propranolol; α-Naphthoxylactic acid;

Ergosteroids by Ashok Marwah; Padma Marwah; Henry Lardy (285-299).
Because relatively large amounts of dehydroepiandrosterone (DHEA) are required to demonstrate its diverse metabolic effects, it is postulated that this steroid may be converted to more active molecules. To search for the possible receptor-recognized hormones, DHEA was incubated with whole rat liver homogenate and metabolite appearances were studied by LC–MS as a function of time to predict the sequence of their formation. An array of metabolites has been resolved, identified and characterized by highly specific and accurate technique of LC–MS, and several of these steroids were analyzed quantitatively. Their identities were established by comparison with pure chemically synthesized compounds and by chemical degradation of isolated fractions. In the present study, we have reasonably established that DHEA was converted to 7α-OH-DHEA, 7-oxo-DHEA, and 7β-OH-DHEA in sequence. These metabolites were further reduced at position 7 and/or 17 to form their respective diols and triols, which were also sulfated at 3β-position. DHEA and its 7-oxygenated derivatives were also converted to their respective 3β-sulfate esters. Several of these steroids are being reported for the first time. 16α-Hydroxy-DHEA, androst-5-ene-3β,16α,17β-triol, androst-4-ene-3,17-dione, 11-hydroxyandrost-4-ene-3,17-dione, androst-5-ene-3,17-diol and testosterone were also identified and characterized. In all, 19 metabolites of DHEA are being reported in this extensive study. We have also detected the formation of 12 additional metabolites including several conjugates, which are the subject of current investigation.
Keywords: Ergosteroids; Dehydroepiandrosterone;

The major urinary metabolite of selenium (Se) in rats was identified by HPLC–inductively coupled argon plasma mass spectrometry (ICP–MS) and –electrospray tandem mass spectrometry (ESI–MS/MS). As the urine sample was rich in matrices such as sodium chloride and urea, it was partially purified to meet the requirements for ESI–MS. The group of signals corresponding to the Se isotope ratio was detected in both the positive and negative ion modes at m/z 300 ([M+H]+) and 358 ([M+CH3COO]) for 80Se, respectively. These results suggested that the molecular mass of the Se metabolite was 299 Da for 80Se. The Se metabolite was deduced to contain one methylselenyl group, one acetyl group and at least two hydroxyl groups from the mass spectra of the fragment ions. The spectrum of the Se metabolite was completely identical to that of the synthetic selenosugar, 2-acetamide-1,2-dideoxy-β-d-glucopyranosyl methylselenide. However, the chromatographic behavior of the Se metabolite was slightly different from that of the synthetic selenosugar. Thus, the major urinary Se metabolite was assigned as a diastereomer of a selenosugar, Se-methyl-N-acetyl-selenohexosamine.
Keywords: Selenium; 2-Acetamide-1,2-dideoxy-β-d-glucopyranosyl methylselenide;

This paper describes a method for residue analysis of difloxacin and sarafloxacin in chicken muscle. Clean-up and preconcentration of the samples are effected by solid-phase extraction (C18) and the determination is carried out by capillary electrophoresis using a photodiode array detection system. The method was validated with satisfying results. The calibration graphs are linear for difloxacin and sarafloxacin from 50 to 300 μg/kg. The limit of detection obtained for difloxacin and sarafloxacin are 10 and 25 μg/kg, respectively, which allows the detection of positive muscle samples at the required maximum residue limits of European Union.
Keywords: Difloxacin; Sarafloxacin; Quinolones;

Endogenous alkaloids in man by Gerhard Bringmann; Miriam Münchbach; Doris Feineis; Kim Messer; Stefanie Diem; Markus Herderich; Hans-Willi Clement; Christine Stichel-Gunkel; Wilfried Kuhn (321-332).
An improved sensitive assay for the determination of the dopaminergic and serotonergic neurotoxin 1-trichloromethyl-1,2,3,4-tetrahydro-β-carboline (TaClo) is presented, based upon on-line coupling of high-performance liquid chromatography with electrospray ionization tandem mass spectrometry (HPLC–ESI-MS–MS). Applying synthetic [D4]TaClo as a fourfold deuterated internal standard, TaClo was detected and reliably quantified as a trace constituent of blood samples (0.5 up to 70 ng g−1 of clot) obtained from six patients orally treated with the hypnotic chloral hydrate. Unambiguous identification of this tricyclic “endogenous alkaloid” was achieved by selected reaction monitoring (SRM) experiments. The molecular ion peaks of TaClo, m/z 289 (for [35Cl3]TaClo) and m/z 291 (for its [37Cl35Cl2]isotopomer), were both monitored to undergo a retro-Diels–Alder fragmentation by loss of a CH2NH portion (−29 u) as typical of a tetrahydropyrido ring system of tetrahydro-β-carbolines. Detection of the resulting fragments, m/z 260 and m/z 262, with the expected statistical chlorine isotopic intensities of 100:96 confirmed the identity of the TaClo molecule. In addition, an enantiomer-specific device was developed for TaClo, by employing a chiral reversed-phase HPLC column in combination with circular dichroism (CD) spectroscopy and MS–MS analysis (LC–CD and LC–MS–MS coupling). In a human clot sample, both TaClo enantiomers were found in equimolar concentration (i.e., as a racemate) corroborating a spontaneous, non-enzymatic formation of TaClo from biogenic tryptamine and therapeutically administered chloral. In urine samples of TaClo-treated rats, by contrast, the (S)-antipode was found to predominate, hinting at an enantiomer-differentiating metabolism of the compound.
Keywords: Alkaloids; 1-Trichloromethyl-1,2,3,4-tetrahydro-β-carboline;

Capillary electrophoresis with electroosmotic flow reversed by cationic surfactant for diagnosis of purine and pyrimidine inherited enzyme deficiencies is reported. Final separation conditions consist of 45 mM borate, 55 mM N-tris[hydroxymethyl]methylglycine, 10 mM tartrate, 1 mM cetyltrimethylammonium bromide and 0.44% tetrabutylammonium hydroxide-2-amino-2-methyl-1,3-propanediol (pH 8.6). Average sensitivity (2.51 μM), reproducibility of migration times (run-to-run C.V.≤0.6%, day-to-day C.V.≤2.5%), linearity (R 2>0.994) and imprecision (mean intra-assay RSD 4.7% and inter-assay RSD 6.6%) of the method are acceptable for diagnostic purposes. Applicability of the method is demonstrated on urine samples from patients with enzymatically proven enzyme deficiences.
Keywords: Purine; Pyrimidine;

An experimental approach for the validation of chromatographic qualitative methods and its application in an antidoping control laboratory is described. The proposed strategy for validation of qualitative methods consists of the verification of selectivity/specificity, limit of detection (LOD), extraction recovery and repeatability (intra-assay precision). A one-day assay protocol, based on the analysis of five blank samples obtained from different sources and four replicates of control samples at two different concentrations of the analytes, has been defined to evaluate the validation parameters. The following evaluation criteria have been applied: absence of interfering substances at the retention time of the analytes in the blank samples to check the selectivity/specificity of the method, the LOD recommended by international sports authorities has to be attained, and for repeatability, the relative standard deviation should be <25% for the low concentration control sample and <15% for the high concentration control sample. Qualitative screening procedures are able to detect a great number of analytes so that extraction and analysis conditions are always a compromise for the different analytes. For this reason, no minimum acceptance criteria have been defined for data of extraction recoveries. The proposed protocol has been used for the validation of the screening and confirmation qualitative methods included in the scope of the accreditation of an antidoping control laboratory according to ISO quality standards.

Direct cocktail analysis of drug discovery compounds in pooled plasma samples using liquid chromatography–tandem mass spectrometry by Yunsheng Hsieh; Matthew S Bryant; Jean-Marc Brisson; Kwokei Ng; Walter A Korfmacher (353-362).
Direct plasma injection technology coupled with a LC–MS/MS assay provides fast and straightforward method development and greatly reduces the time for the tedious sample preparation procedures. In this work, a simple and sensitive bioanalytical method based on direct plasma injection using a single column high-performance liquid chromatography (HPLC) and tandem mass spectrometry (MS/MS) was developed for direct cocktail analysis of double-pooled mouse plasma samples for the quantitative determination of small molecules. The overall goal was to improve the throughput of the rapid pharmacokinetic (PK) screening process for early drug discovery candidates. Each pooled plasma sample was diluted with working solution containing internal standard and then directly injected into a polymer-coated mixed-function column for sample clean-up, enrichment and chromatographic separation. The apparent on-column recovery of six drug candidates in mouse plasma samples was greater than 90%. The single HPLC column was linked to either an atmospheric pressure chemical ionization (APCI) or electrospray ionization (ESI) source as a part of MS/MS system. The total run cycle time using single column direct injection methods can be achieved within 4 min per sample. The analytical results obtained by the described direct injection methods were comparable with those obtained by semi-automated protein precipitation methods within ±15%. The advantages and challenges of using direct single column LC–MS/MS methods with two ionization sources in combination of sample pooling technique are discussed.
Keywords: Drug discovery compounds;

A selective and simple HPLC procedure has been developed to determine histidine (His) and histidinol (HDL) in liver supernate. The separation was performed on a column, Mightysil RP-18 GP. The eluted analytes were measured with UV detection without derivatization which provided detection limits of 1.1 and 2.0 μM for His and HDL (S/N ratio, 3:1), respectively. Recovery of the analytes added to liver sample was 98.3–101.6% within a 1-day study and 95.7–98.6% on different day (6 days) studies. The apparent histidinol dehydrogenase activities (nmol/g wet tissue) at pH 8, 9, 10, 11, and 12 were 38.6, 50.4, 160.3, 274.3, and 185.6 for liver; 90.6, 132.2, 30.7, 22.1, and 6.76 for kidney; 0.0, 0.0, 38.2, 20.1, and 12.9 for pancreas; 0.0, 0.0, 0.0, 14.7, and 6.8 for spleen; 0.0, 0.0, 4.2, 6.8, and 0.0 for muscle; and 0.0, 0.0, 4.9, 1.8, and 0.0 for small intestine, respectively. On the basis of optimum pH values, histidinol dehydrogenase activity in the organs was in the following order: liver>kidney>pancreas>spleen>muscle>small intestine.
Keywords: Histidine; Histidinol dehydrogenase;