Journal of Chromatography B (v.775, #1)

News Section (N1-N2).

Validation of the determination of oxymetholone in human plasma analysis using gas chromatography–mass spectrometry by Carla Rodrigues Cardoso; Marlice A Sı́poli Marques; Ricardo Carvalho Caminha; Maria Christina Maioli; Francisco R Aquino Neto (1-8).
A simple and rapid procedure for extraction of oxymetholone in human plasma using gas chromatography coupled with quadrupole mass spectrometric was evaluated. The method involves analyte extraction with tert.-butylmethylether after alkalinization of the plasma and derivatization with MSTFA–NH4I–2-mercaptoethanol before the high resolution gas chromatographic–mass spectrometry separation. Methyltestosterone was used as internal standard. The calibration curves were linear, with typical r 2 values >0.995 and F table>F calculated (α=0.05). Recovery from plasma proved to be more than 70%. The method was accurate and reproducible, and was successfully applied to determine the pharmacokinetic parameters of oxymetholone for healthy volunteers after oral administration of 50 mg of the compound. The (C max) and (T max) were 18.8±0.4 ng/ml and 210±42.4 min, respectively.
Keywords: Oxymetholone;

Isolation of melatonin by immunoaffinity chromatography by Jakub Rolc̆ı́k; René Lenobel; Vĕra Siglerová; Miroslav Strnad (9-15).
A single-step, highly specific and easy-to-use method was developed for isolation and purification of melatonin from complex biological matrices. Polyclonal antibodies highly specific against melatonin (with cross-reactivities with related compounds below 0.02%, except for 6-hydroxymelatonin) were raised, characterised by enzyme-linked immunosorbent assay (ELISA) and used for preparation of immunoaffinity gel. Melatonin recovery by the immunoaffinity method was ∼95%, allowing single-step processing of samples prior to electrospray HPLC–MS analysis (with detection limit 10 fmol). The method was successfully used for determining melatonin in human serum and turned out to be better than the non-specific solid-phase extraction published earlier.
Keywords: Melatonin;

Capillary electrophoresis with laser-induced fluorescence detection for fast and reliable apolipoprotein E genotyping by G.W Somsen; H.T.M.E Welten; F.Ph Mulder; C.W Swart; I.P Kema; G.J de Jong (17-26).
The use of capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection for the rapid determination of apolipoprotein E (apoE) genotypes was studied. High resolution and sensitive detection of the concerned DNA restriction fragments was achieved using CE buffers with hydroxypropylmethylcellulose (HPMC) as sieving polymer and ethidium bromide (EB) as fluorescent intercalating agent. In order to achieve adequate resolutions in short analysis times, parameters such as concentration of HPMC and EB, separation voltage, and length and coating of the capillary were evaluated. Using a separation buffer with 0.8% (w/w) HPMC and 7 μM EB, characteristic DNA-fragment profiles could be obtained for all common apoE genotypes at an overall rate of ten samples per hour. The method allows direct injection of untreated PCR samples and the use of standard fused-silica capillaries which are effectively coated following a short, one-step rinse procedure. With a simple computerized algorithm based on migration-time ratios for pattern assignment, highly reliable apoE genotyping was achieved. Overall, in terms of speed, ease of use and objectivity the presented method provides a significant improvement over previously reported CE-based procedures for apoE genotyping.
Keywords: Apolipoprotein E;

A robust tool is proposed for the rapid at-line verification of the identity and integrity of (recombinant) proteins, namely the hyphenation of multidimensional chromatography and mass spectrometry (MS). A recombinant human antibody produced in Chinese hamster ovary cells is taken as pertinent example. The recombinant human antibody is first captured from the production environment by affinity chromatography (rProtein A, isolation/concentration of the target molecule) and automatically transferred to an enzyme reactor (immobilized trypsin column) for digestion, thereby yielding different peptides corresponding to the protein sequence. The peptides are then separated on a reversed-phase column before being analyzed and identified by MS. This step does not require a fine resolution since the mass spectrometer can identify a variety of substances at the same time. The results are then analyzed in silico with suitable bio-informatic tools. When the gene sequence of the protein product is known, proteolytic cleavages can be predicted and the exact mass and hence the amino acid sequence of each peptide can thereby be deduced. Fitting experimental data and reference peptide sequences then provides important information about the integrity of the protein and more particularly about its sequence. In our case, the integrity of 45% of the light and 75% of the heavy chain sequences of the antibody could be verified within minutes.
Keywords: Recombinant protein drugs;

The separation and on-line concentrations of lysergic acid diethylamide (LSD), iso-lysergic acid diethylamide (iso-LSD) and lysergic acid N,N-methylpropylamide (LAMPA) in human urine were investigated by capillary electrophoresis–fluorescence spectroscopy using sodium dodecyl sulfate (SDS) as an anionic surfactant. A number of parameters such as buffer pH, SDS concentration, Brij-30 concentration and the content of organic solvent used in separation, were optimized. The techniques of sweeping-micellar electrokinetic chromatography (sweeping-MEKC) and cation-selective exhaustive injection-sweep-micellar electrokinetic chromatography (CSEI-sweep-MEKC) were used for determining on-line concentrations. The advantages and disadvantages of this procedure with respect to sensitivity, precision and simplicity are discussed and compared.
Keywords: Lysergic acid diethylamide;

Internal standard method for the measurement of choline and acetylcholine by capillary electrophoresis with electrochemical detection by Dana D Wise; Tatyana V Barkhimer; Pierre-Alexandre Brault; Jon R Kirchhoff; William S Messer; Richard A Hudson (49-56).
An internal standard method has been developed for the determination of the neurotransmitter acetylcholine and/or its metabolic precursor choline. This approach couples the high separation efficiency of capillary electrophoresis with the sensitivity and selectivity of electrochemical detection at an enzyme-modified electrode. Indirect electrochemical detection is accomplished at a 25 μm platinum electrode modified by cross-linking the enzymes choline oxidase and acetylcholinesterase with glutaraldehyde. Although in this simple form of electrode fabrication there is a gradual loss of response from the electrochemical detector with time, accurate quantitation is achieved by the addition of butyrylcholine, which is also a substrate for acetylcholinesterase, as an internal standard. A linear response is achieved between 0 and 125 μM with a limit of detection of 2 μM (25 fmol). The utility of this method was demonstrated by monitoring the kinetics of choline uptake in synaptosomal preparations.
Keywords: Choline; Acetylcholine;

A sensitive and convenient high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) assay for the opioid receptor agonist–antagonist butorphanol in human plasma is described. BC-2605, a cyclopropyl analogue of butorphanol, was employed as an internal standard. Butorphanol was recovered from plasma (84.4±10.9%) by liquid–liquid extraction. The mobile phase flow-rate was 0.3 ml/min and consisted of methanol–water–formic acid (90:10:0.1, v/v/v). The analytical column (4.6×100 mm) was packed with Partisil C8 (5 μm). The standard curve was linear from 13.7 to 1374 pg/ml (r 2>0.99). The lower limit of quantitation was 13.7 pg/ml. The assay was specific, accurate (% deviation from nominal concentrations were <15%), precise and reproducible (within- and between-day coefficients of variation <7%). Butorphanol in plasma was stable over 3 freeze/thaw cycles and at room temperature for 1 day. The utility of the assay was demonstrated by following butorphanol plasma concentrations in two healthy subjects for 24 h following a 1 mg intranasal dose.
Keywords: Butorphanol;

Determination of poorly separated monoclonal serum proteins by capillary zone electrophoresis by Thierry Le Bricon; Erika Launay; Pascal Houze; Djaouida Bengoufa; Bernard Bousquet; Bernard Gourmel (63-70).
A capillary zone electrophoresis (CZE) technique was developed for the determination of poorly separated monoclonal serum proteins by agarose gel electrophoresis (AGE). A P/ACE 5500 capillary instrument (Beckman) was used under the following conditions: 57 cm×50 μm I.D. fused-silica capillary, pH 9.6 borate buffer, and 214 nm on-line detection. Sixty patients (61±13 years) with a well isolated (n=24, group A) or poorly separated monoclonal band(s) by AGE (n=36, group B) were included in this study. Within- and between-run precision for CZE was below 4% for albumin and 7% for γ-globulin. A 100% (group A) or 61% agreement (group B, more bands detected by CZE in 10 cases) was obtained between CZE and AGE for the number of monoclonal bands. In group B, quantification was possible in 92% of samples by CZE vs. 64% by AGE (P<0.05, chi-square). The proposed CZE method appears as an additional helpful technique for the determination of poorly separated monoclonal serum proteins by AGE.
Keywords: Monoclonal proteins;

The present investigation was designed to develop an assay suitable for pharmacokinetic studies of new compounds, i.e. the novel 7,8-methylenedioxy-4H-2,3-benzodiazepin-4-one derivatives (2a and 2b), acting as non-competitive AMPA-receptor antagonists. A reversed-phase high-performance liquid chromatographic method has been developed to determine the time-course of plasma concentrations of derivatives 2a and 2b administered intraperitoneally to Sprague–Dawley rats. The separation of compounds studied and a N-methyl-2,3-benzodiazepin-4-one derivative as internal standard (I.S.) from plasma, were carried out by liquid–liquid extraction using diethyl ether. The samples were injected onto the analytical column (Partisil 10 ODS) eluted with acetonitrile/0.01 M acetate buffer (pH 5.3) at a flow-rate of 2 ml/min and detected at 240 nm. Compounds 2a, 2b and I.S. gave retention times of 8.5, 5.25 and 11.1 min, respectively. The selectivity of the method was satisfactory. The mean recovery from spiked rat plasma ranged from 86.7 to 91.6% for 2a, and from 85.1 to 87.0% for 2b. The procedures were validated with a good reproducibility and linear response from 0.0625 to 2 μg/ml, with a regression coefficient of 0.9932 for 2a and 0.9854 for 2b. The lower limit of quantification (LOQ) was taken as 15 ng/ml for the two compounds. 2a and 2b showed no signs of significant degradation in rat plasma during storage at −20 °C and following freeze/thaw cycles. Moreover, plasma levels of the tested compounds have been correlated with their anticonvulsant activity, determined in vivo in genetically epilepsy-prone rats. Due to its sensitivity, the method was suitable for application to pharmacokinetic study.
Keywords: 7,8-Methylenedioxy-4H-2,3-benzodiazepin-4-ones; Anticonvulsant; Pharmacokinetic;

An on-line coupling of microdialysis to a packed capillary column switching liquid chromatographic system (0.2 mm I.D.) and mass spectrometric detection was developed. The microdialysates were collected in the loop of the first of three valves, coupled in direct series. A deuterated internal standard was added on-line by the middle valve and the third valve operated both a pre-column, for desalting of the physiological buffer used in the sampling procedure, and a separation column. The on-line system was used to study free concentrations of ropivacaine and its metabolite (PPX) in human plasma samples. The analytes were detected by electrospray ionization in a tandem mass spectrometer operating in multiple reaction monitoring mode. The free fractions of ropivacaine (200 nM total concentration) and PPX (20 nM total concentration) in spiked plasma samples were 12±3 and 47±5% (±standard deviation for day-to-day variations, n=3), respectively.
Keywords: Ropivacaine;

A large amount of oxolinic acid administered in freshwater fish farms reaches the environment. In order to allow environmental monitoring, an HPLC method to determine oxolinic acid in the bryophyte Fontinalis antipyretica was developed. Nalidixic acid was used as an internal standard. Oxolinic and nalidixic acids were extracted by cracking the bryophytes in liquid nitrogen with 0.1 M acid oxalic solution in ethyl acetate and a liquid–liquid clean up procedure was then performed. Mobile phase was a 0.02 M orthophosphoric acid aqueous solution–acetonitrile mixture (70:30, v/v). The stationary phase was 5 μm PuroSpher RP18e and quinolones were detected by fluorescence. The linearity, accuracy and precision of this method were demonstrated by a validation assay. The limits of detection and quantitation were 1 and 10 ng/g respectively. The linearity range was 10 to 500 ng/g. This method was applied to a 25 days experimental study performed with the bryophyte Fontinalis antipyretica.
Keywords: Quinolones; Bryophytes; Fontinalis antipyretica;

A validated method for the determination of remifentanil in human blood, applicable to all therapeutic concentrations, using capillary GC with nitrogen-specific detection and fentanyl as the internal standard has been developed. Citrated whole blood samples were extracted into 1-chlorobutane following precipitation of proteins with methanol. The drugs were back extracted into 10 mM HCl and re-extracted into methanol–1-chlorobutane. The extracts were reconstituted in methanol and injected onto a 25-m BPX-5 column. The lower limit of quantitation was 0.2 ng/ml with within- and between-day coefficients of variation of less than 15%.
Keywords: Remifentanil;

A method for the high-performance liquid chromatography (HPLC) determination of nicarbazin uptake and excretion in ducks is presented. The method uses few clean-up steps and provides a rapid assessment of nicarbazin excretion by measuring the analyte 4,4′-dinitrocarbanalide (DNC). During method development the effect of extraction volume, number of extractions, mobile phase composition, column temperature, and injection volume were varied to optimize sensitivity and achieve as short a run time as possible. For our purposes, a 2×5.0 ml 1:1 dimethyl formamide (DMF):acetonitrile (ACN) extraction injected (40 ml) into an HPLC system equipped with a Keystone octadecylsilyl (ODS) C18 column and a UV variable wavelength detector (λ=347 nm) with a mobile phase of 60:40 (v/v) ACN–H2O, at a flow-rate of 1.0 ml/min at a column temperature of 35 °C provided adequate resolution and an acceptable total run time. Studies conducted during method development for inter-day recovery efficiencies for 0.46, 1.8 and 88.5 μg/g fortified samples (n=3) had mean recoveries of 91, 94 and 97% and intra-day (n=3) recoveries at the same fortification levels of 103, 94, and 92%. The method has been used successfully in excretion studies of nicarbazin in ducks.
Keywords: Nicarbazin;

A simple and fast HPLC system is presented for quantifying paraquat in human plasma and serum using 1,1′-diethyl-4,4′-bipyridyldiylium (diethyl paraquat) as an internal standard. An octadecyl–silica column is used with an eluent of 10% acetonitrile (v/v) containing sodium 1-octanesulphonic acid (3.0 mM) and a diethylamine–orthophosphoric acid buffer (pH 3). Unlike with other techniques, sample treatment requires only the precipitation of protein contents by 6% perchloric acid (v/v) in methanol. The method has a limit of detection of 0.1 μg/ml and is linear up to 10 μg/ml. The serum of four patients and the plasma of one patient with paraquat intoxication’s were analysed and positive identification and quantification was readily achieved. One of those patients survived, partially given the rapid disclosure of his levels of paraquat. Therefore, this method is suitable for quantification of paraquat in toxicological samples. It may be used as a prognostic tool in critical case detoxification and to quickly identify potentially salvageable patients for enrolment in new hemofiltration studies.
Keywords: Paraquat;

A new method for quantifying specific amino acids in small volumes of plasma and whole blood has been developed. Volatile derivatives of amino acids are analyzed by gas chromatography–mass spectrometry. The method only takes a few minutes to perform and requires minimal sample preparation. The accurate assay of phenylalanine, tyrosine and other amino acids in dried blood spots could be used for neonatal screening for phenylketonuria and other aminoacidemias. Because of the low cost, this neonatal screening method is suited to application in developing countries such as China.
Keywords: Amino acids;

Current chromatographic methods of estimation of malondialdehyde, a marker of oxidative lipid injury, often require extensive extraction procedures, column cleaning or specialized equipment. A rapid and sensitive HPLC method is described for the determination of MDA in plasma and urine. The mobile phase consisted of 40:60 ratio (v/v) of methanol to 50 mM potassium monobasic phosphate at pH 6.8, pumped at a rate of 1.0 ml/min on a Hewlett-Packard Hypersil 5 μ ODS 100×4.6 mm placed in a column warmer set to 37 °C. Samples of plasma and urine were treated with the antioxidant, butylated hydroxytoluene and heat derivatized at 100 °C for 1 h with thiobarbituric acid at an acid pH. Samples were extracted with n-butanol and 10 μl of the extract was injected at 1 min intervals using an autosampler. The Hewlett-Packard model 1046A programmable fluorescence detector was set at excitation of 515 nm and emission of 553 nm. Retention time was 1.87 min, however absence of interfering peaks, allowed analysis to be carried out in increments of 1 min per sample. Within day variability in estimation was between 8.6% and 10.3%. Between days variability was 3.6–7.9%. Recovery was between 88 and 101%. Samples of urine and plasma from ten normotensive volunteers were 1.94±0.79 μmol/g creatinine and 0.69±0.13 μmol/l respectively and were similar to those reported in the literature. MDA degrades at room temperature at a rate of 10% per hour. It is therefore, suggested that the total assay time be limited to 1 h beginning with sample preparation.
Keywords: Malondialdehyde;