Journal of Chromatography B (v.780, #2)

News Section (N1-N2).

In vitro cytochrome P450 assays are used in metabolism studies in support of early phases of drug discovery to investigate, e.g., metabolic stability, enzyme inhibition and induction by new chemical entities. LC–UV and LC–fluorescence are traditional analytical tools in support of such studies. However, these tools typically comprise different methods of relatively low throughput for the various metabolites of probe reactions. In recent years, LC–MS methods have been developed to increase throughput. Increased throughput can also be achieved by means of modern chromatographic tools in combination with UV and fluorescence detection. This approach is especially suitable when cytochrome P450 isoforms are investigated by means of single probe incubations. Here, an LC–UV/fluorescence system based on a monolithic porous silica column is described for the analysis of metabolites of nine cytochrome P450 marker reactions [phenacetin to paracetamol (CYP1A2), coumarin to 7-hydroxycoumarin (CYP2A6), paclitaxel to 6α-hydroxypaclitaxel (CYP2C8), diclofenac to 4-hydroxydiclofenac (CYP2C9), mephenytoin to 4-hydroxymephenytoin (CYP2C19), bufuralol to 1-hydroxybufuralol (CYP2D6), chlorzoxazone to 6-hydroxychlorzoxazone (CYP2E1), midazolam to 1-hydroxymidazolam (CYP3A4), and testosteron to 6β-hydroxytestosteron (CYP3A4)]. While offering sensitivities and linear ranges comparable to previously reported methods, the set-up described here provides ease of use and increased throughput with maximum cycle times of 4.5 min.
Keywords: Cytochrome P450;

A sensitive, accurate, and reliable method is described for the quantitative determination of chloral hydrate (CH) and its metabolites in blood plasma of mice and rats. Metabolites of CH include trichloroacetic acid (TCA), trichloroethanol (TCE), and trichloroethanol glucuronide (TCE-Glu). This new method uses capillary gas chromatography with electron-capture detection (GC/ECD). Procedures for improving sample stability and quality assurance are also described that were not mentioned in previous literature. Rat or mouse plasma (50 μl) is acidified (or treated enzymatically for TCE-Glu determination) and extracted with peroxide free methyl t-butyl ether. Distilled diazomethane (CH2N2) is added to derivatize TCA to its methyl ester. Detection limits were estimated at 0.2 μg/ml for CH and TCE, and 0.1 μg/ml for TCA. Detector response to TCA and TCE were shown to be linear in the range of 3.125–200 μg/ml (r≥0.9996). For CH, the response fits a second-order equation in this same range (r=0.99994)
Keywords: Chloral hydrate;

A gas chromatographic method with mass selective detection was developed which enables the simultaneous determination of the urinary hydroxy terpenes cis-verbenol, α-terpineol, myrtenol, carveol, perillyl alcohol and trans-sobrerol. The sample preparation consisted of enzymatic hydrolysis, solid phase extraction (SPE) with RP-C18 SPE material and clean up with silica gel cartridges. Large volume injection was used and the mass selective detection was done in the single ion modus. Low detection limits in the range of 1.0–4.5 μg/l for the terpene metabolites in urine and mean recoveries of 100% were achieved.
Keywords: Monoterpenes; Hydroxyterpenes;

Affinity chromatography of bull seminal proteins on mannan–Sepharose by J Liberda; H Ryšlavá; P Jelı́nková; V Jonáková; M Tichá (231-239).
The interaction of bull seminal plasma proteins and sperm with mannan was investigated using an enzyme-linked binding assay (ELBA). A high mannan-binding activity was found in the protein fraction interacting with heparin. Mannan binding to seminal plasma proteins was inhibited by d-mannose and d-fructose, but not by d-mannose-6-phosphate, d-glucose-6-phosphate, ovalbumin and ovomucoid. Mannan inhibited the binding of bovine zona pellucida glycoproteins both to bull sperm and seminal plasma proteins. Yeast mannan immobilized to divinyl sulfone-activated Sepharose was used for the isolation of mannan-binding proteins. The protein components of this fraction were identified on the basis of relative molecular mass determination and N-terminal amino acid sequencing: RNAase dimer, PDC-109 and a protein homologous to BSP-30K (relative molecular mass 14 500). The isolated proteins were characterized by a high zona pellucida binding activity.
Keywords: Proteins; Mannan;

Direct injection micellar liquid chromatographic determination of benzodiazepines in serum by Maria-Elisa Capella-Peiró; Devasish Bose; Adrià Martinavarro-Domı́nguez; Mayte Gil-Agustı́; Josep Esteve-Romero (241-249).
A simple micellar liquid chromatographic (MLC) procedure is reported for the determination of several benzodiazepines in serum: bromazepam, diazepam, flunitrazepam, halazepam, medazepam, nitrazepam, oxazepam and tetrazepam. The optimization studies have been made in C18 and C8 columns, using solutions containing sodium dodecyl sulphate (SDS) modified with butanol or pentanol as mobile phases. The method proposed for the determination of the benzodiazepines uses a hybrid micellar mobile phase of 0.06 M SDS–5% butanol–0.01 M phosphate buffer (pH 7) at 25 °C, and UV detection (230 nm) in a C18 column. The serum samples were injected directly, without any pretreatment, and eluted in less than 22 min, in accordance with their relative polarities, as indicated by their octanol–water partition coefficients. The limits of detection (ng ml−1) were within the ranges of 2–6 and 4–18 for aqueous and serum samples, respectively. Repeatability and intermediate precision were tested for three different concentrations of the drugs, and RSD (%) was below 10 for most of the assays. The MLC results were compared with those obtained from a conventional HPLC method using methanol–water 5:5 (v/v) which requires a previous extraction procedure.
Keywords: Benzodiazepines;

A high-performance liquid chromatographic (HPLC) method with electrochemical detection and solid-phase extraction (SPE) using cartridges of weak cation-exchange capacity as the primary retention mechanism is described for the separation and determination of methylnaltrexone (MNTX) in small clinical samples of plasma or urine. The procedure was performed using a Phenomenex Prodigy ODS-2, 5 μm, 150×3.2 mm analytical column and 50 mM potassium acetate buffer, with 11% methanol as organic modifier at pH* 4.5 at a flow-rate of 0.5 ml/min. The detection potential was 700 mV. The six-point standard calibration curves were linear over three consecutive days in the range from 2 to 100 ng/ml. The average goodness of fit (r) was 0.9993. The lower limit of detection (LOD) and limit of quantification (LOQ) were found to be 2.0 and 5.0 ng/ml, respectively. At the LOQ, the coefficient of variation for the entire method was 8.0% and the accuracy was 10.0% (n=10). Recovery of the drug from plasma was in the region of 94%. The method was applied to a pharmacokinetics study of methylnaltrexone after subcutaneous administration and in numerous assays of analytes in blood plasma and urine. The pharmacokinetics parameters for a single dose of 0.1 or 0.3 mg/kg in plasma were C max=110 (±55) and 287 (±101) ng/ml and t max=16.7 (±10.8) and 20.0 (±9.5) min, respectively. The method is simple, yet sensitive for the detection and determination of methylnaltrexone in biological samples at the level of the physiological response.
Keywords: Methylnaltrexone; Naltrexone;

A sensitive HPLC assay for all-trans-retinol, α-tocopherol, and γ-tocopherols in human serum and plasma is reported. Sample preparation is performed in one step and involves precipitation of proteins and extraction of lipids with two volumes of an ethanol–chloroform mixture (3:1, v/v) without I.S. addition. After removal of the precipitated protein, 20 μl aliquots of the supernatant (equivalent to 6.7 μl of serum or plasma) were injected into the HPLC system and analyzed using fluorometric detection. RP-HPLC was performed using a C18 S3 ODS2 column with a methanol–water step gradient (97:3 to 100) at 1.0 ml/min. The quantification limit expressed as nanograms of analyte per milliliter of serum or plasma was approximately 30 ng for all-trans-retinol, 300 ng for α-tocopherol and 250 ng for γ- and δ-tocopherol. The method was validated and applied to human serum and plasma from a total of 120 subjects. This procedure requires a small volume of serum or plasma and can therefore be a valuable tool for measuring low concentrations of these vitamins in preterm infants with sensitivity, precision and accuracy.
Keywords: Retinol; Tocopherols;

Two analytical methods were developed for quantitative determination of DADLE (H2N–Tyr–d-Ala–Gly–Phe–d-Leu–COOH) and its two cyclic prodrugs in rat plasma. For high-performance liquid chromatography with fluorescence detection (LC–FLU), precolumn derivatization of DADLE was accomplished by labeling the N-terminal amino group with the reagent naphthalene-2,3-dicarboxaldehyde in the presence of cyanide (NDA/CN) to form a highly fluorescent 1-cyanobenz[f]isoindole (CBI) derivative. A multi-dimensional LC system was employed to improve selectivity, and solid-phase extraction (SPE) was used for plasma sample preparation. The cyclic prodrugs were converted to DADLE prior to their derivatization. With fluorescence detection after derivatization, the limit of quantitation (LOQ) was 6 ng ml−1 for the analysis of DADLE, and good linearity was observed up to 6000 ng ml−1 in rat plasma. Quantitative analysis of DADLE and its cyclic prodrugs was also performed using liquid chromatography interfaced to electrospray ionization tandem mass spectrometry (LC–ESI-MS–MS). Chromatographic separation was achieved on a C18 column using gradient elution in a water–acetonitrile system containing 0.1% (v/v) formic acid. The tandem mass spectrometric analysis was performed in the multiple reaction monitoring mode using internal standardization to improve assay precision and accuracy. For plasma sample pretreatment, acetonitrile was added first to precipitate proteins and SPE was used to minimize matrix effects. Using LC–ESI-MS–MS, the LOQ was 0.5 ng ml−1 for DADLE and 2 to 5 ng ml−1 for its prodrugs. Good linearity was observed from the LOQ up to 1000 ng ml−1 for all compounds. For the analysis of DADLE, both analytical methods showed good precision, accuracy and stability. However, for prodrug analysis, LC–FLU showed some sensitivity and accuracy problems, while the LC–ESI-MS–MS method provided consistent and satisfactory results. In conclusion, LC–ESI-MS–MS is the method of choice for the analysis of DADLE and its cyclic prodrugs in rat plasma samples due to its good selectivity, high sensitivity, and fast analysis. Its application was demonstrated through biodisposition and bioconversion studies of the coumarinic acid-based prodrug after intravenous administration in rats.
Keywords: Opioid peptides; DADLE; Cyclic prodrugs;

Heterogeneity of protein labeling with a fluorogenic reagent, 3-(2-furoyl)quinoline-2-carboxaldehyde by Alexander V Stoyanov; Hossein Ahmadzadeh; Sergey N Krylov (283-287).
Fluorogenic reagents are used for protein labeling when high-sensitivity fluorescence detection is required. Similar to traditional labeling with activated fluorescent dyes, such as fluorescein isothiocyanate, a fluorogenic reaction is expected to change the physical–chemical properties of proteins. Knowledge of these changes may be essential for efficient separation and identification of labeled proteins. Here we studied the effect of labeling of myoglobin with a fluorogenic reagent on the acid–base properties of the protein. The fluorogenic reagent used was 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ). In slab-gel isoelectric focusing, we found that the labeling reaction generated at least six species with pI values lower than that of non-labeled myoglobin. These species can be identified as products of progressive labeling of myoglobin with one to six FQ molecules. The same series of FQ-labeled species were observed when the reaction products were analyzed by capillary zone electrophoresis. The comparison of experimental and theoretical pI values allowed us to elucidate the labeling pattern—the number of FQ molecules corresponding to each labeled product detected by isoelectric focusing.
Keywords: Proteins; 3-(2-furoyl)quinoline-2-carboxaldehyde;

Liquid chromatographic method for the determination of ganciclovir and/or acyclovir in human plasma using pulsed amperometric detection by Satoshi Kishino; Yoh Takekuma; Mitsuru Sugawara; Tsuyoshi Shimamura; Hiroyuki Furukawa; Satoru Todo; Katsumi Miyazaki (289-294).
We have developed a simple, rapid and highly sensitive method for determining plasma concentrations of ganciclovir and/or acyclovir by using reversed-phase chromatography followed by pulsed amperometric detection. A linear relationship between the amount of ganciclovir (0.05–10 μg/ml plasma) or acyclovir (0.1–20 μg/ml plasma) and peak height ratio was obtained. The relative standard deviations of all standard curves were greater than or equal to 0.999. The limits of detection for ganciclovir and acyclovir quantitation were 10 ng/ml and 50 ng/ml (signal/noise >3), respectively. Daily fluctuations of plasma standard curves (n=5) for the ganciclovir and acyclovir samples were small, with relative standard deviations (RSD) of 3.3 and 4.5% (n=5), respectively. The intra-assay precision for the ganciclovir and acyclovir samples were 6.9 (n=5) and 5.5% (n=5), respectively. Inter-assay precision of ganciclovir (n=3) and acyclovir (n=3) ranged from 2.6 to 6.8% and 3.5 to 5.0%, respectively. Using this method, the pharmacokinetics and removal of ganciclovir during continuous hemodiafiltration (CHDF) in a liver transplant recipient being treated for severe cytomegalovirus infection was investigated. The mean (±SD) ratio of ganciclovir concentrations at the inlet and outlet of the dialyzer (C outlet/C inlet) was 0.56±0.09. The areas under the curves of ganciclovir up to 12 h postdosing (AUC0→12) at the inlet and outlet of the dialyzer were 12.54 μg h/ml and 7.16 μg h/ml, respectively. The ultrafiltrate of ganciclovir was 16.6 mg. The terminal elimination half-life (T 1/2) of ganciclovir during CHDF was 3.6 h. These results demonstrate that CHDF effectively removes ganciclovir. Until formal guidelines have been established, ganciclovir or acyclovir dosage should be adjusted according to the results of monitoring of plasma drug concentration. The method described here is suitable for clinical monitoring of plasma ganciclovir or acyclovir levels in solid organ transplant recipients and for use in studies involving pharmacokinetics.
Keywords: Ganciclovin; Acyclovir;

A rapid and specific liquid chromatographic mass spectrometric (LC–MS–MS) method has been developed for the determination of paroxetine in human plasma. The procedure involves a liquid–liquid extraction of paroxetine and fluoxetine (internal standard) with cyclohexane–ethyl acetate. The standard curve was linear over a working range of 0.2–50 ng/ml. The lower limit of quantitation was 0.2 ng/ml. No endogenous compounds were found to interfere with the analysis. The absolute recovery was 70.8% for paroxetine and 84.1% for the internal standard. The accuracy of inter-assay and intra-assay accuracy was in the ranges −4.8 to −0.5% and −3.4 to 4.8%, respectively. This method proved to be suitable for bioequivalence studies by being simple, selective and reproducible.
Keywords: Paroxetine;

High-performance liquid chromatographic separation of renin–angiotensin system peptides and most of their metabolic fragments by Adriana Pelegrini-da-Silva; Wiliam A Prado; Maria A Juliano; Sherwin Wilk; Antonio R Martins (301-307).
We describe here a gradient HPLC procedure for the separation, and quantification by UV absorption of renin tri- and tetradecapeptide substrates, angiotensins I, II, III, IV and V, angiotensin-derived peptides, and peptidase inhibitors including amastatin, bestatin, pepstatin, lisinopril, a renin peptide inhibitor, Z-Pro-prolinal, N-[1-(R,S)-carboxy-2-phenylethyl]-l-Ala-l-Ala-l-Phe-p-aminobenzoate, and phosphoramidon. Most peptides and peptidase inhibitors were baseline-resolved within 32 min. The overall intra- and inter-assay precisions ranged from 0.8 to 5.9 (n=6) and 2 to 13% (n=6), respectively. There was a linear relationship (correlation coefficients≥0.9660) between peak height and peptide amount injected. In conclusion, the present method when combined with a peptidase-inhibitor paradigm can lead to the identification of renin–angiotensin system metabolizing enzymes, and when combined with radioimmunoassay can enhance the specificity of angiotensin measurement.
Keywords: Peptides; Renin–angiotensin;

A simple, low-cost, sensitive and selective LC method was developed for the determination of enrofloxacin and ciprofloxacin in chicken blood. The method was applied to whole blood from a chicken using dried blood spots on filter paper disks. The detection limits of enrofloxacin and ciprofloxacin (100 μl of whole blood on a disk) were 0.005 and 0.01 μg/ml, respectively. The whole procedure was verified in intra-laboratory studies (recoveries of both compounds were above 90%), and its applicability was tested with blood from the chicken receiving enrofloxacin in a single oral dose at a level of 10 mg/kg body mass. The method permits the use of a small volume of blood from a chicken and should be useful for pharmacokinetic studies.
Keywords: Enrofloxacin; Ciprofloxacin;

A sensitive, precise and accurate stable isotope dilution high-performance liquid chromatography–electrospray ionization mass spectrometry method has been developed for measuring endogenous 2- and 4-hydroxyestrones, the main catechol estrogens in human urine. Compared to the published methods using gas chromatography–mass spectrometry, this approach simplifies sample preparation and increases the throughput of analysis. The unique part of our method is the use of a simple and rapid derivatization step that forms a hydrazone at the C-17 carbonyl group of catechol estrogens. This derivatization step has greatly enhanced method sensitivity as well as HPLC separability of 2- and 4-hydroxyestrones. Standard curves were linear over a 100-fold calibration range with correlation coefficients for the linear regression curves typically greater than 0.996. The lower limit of quantitation for each catechol estrogen is 1 ng per 10-ml urine sample, with an accuracy of 97–99% and overall precision, including the hydrolysis, extraction and derivatization steps, of 1–3% for samples prepared concurrently and 2–11% for samples prepared in several batches. This method is adequate for measuring the low endogenous levels of catechol estrogens in urine from postmenopausal women.
Keywords: Hydroxyestrones; p-Toluenesulfonhydrazide;

Determination of histamine in the whole blood of colon cancer patients by Maurizio Previati; Andrea Raspadori; Lucia Bertolaso; Alina Parmeggiani; Debora Bindini; Cristina Vitali; Irene Lanzoni; Elisa Corbacella; Massimo Saviano; Francesco Fagioli; Gabriella Blo; Silvano Capitani (331-339).
The aim of the present work is to investigate whether histamine assay could be useful in detecting the presence of primary cancer. The high-performance liquid chromatographic (HPLC)-based o-phthalaldialdehyde (OPA) histamine derivatization assay was investigated with respect to several variables, dramatization reagent concentration, organic solvent requirement, derivatization time and counter-ion effect on chromatographic separation.The OPA histamine assay, in the absence of added –SH groups, was found to detect histamine in whole blood samples with relative standard deviations <14% and recoveries not less than 90%. The assay showed high selectivity towards other aminic-containing compounds and a detection limit of 18 nM of histamine was evaluated. Calibration curves in the range 50–500 nM were obtained by using histamine standards in 0.1 M HCl with a regression coefficient value (r 2) of 0.9969. In order to assess the usefulness of this assay in primary tumor monitoring, two groups of individuals, 29 controls and 29 colon cancer patients were selected, and serum levels of histamine, carcinogen embrionary antigen (CEA), carcinogen antigen 19.9 (CA19.9), and tumor staging, were determined. A significant histamine reduction (P=0.028) between controls (180.12±70.4 nM) and patients (134.5±90.3 nM) was found, and a cut-off value of 157.5 nM was extrapolated as intercept point of sensitivity and specificity curves. Fifty percent of patients showed a histamine value below the cut-off, while 45.8 and 8.3% of patients were positive for CEA and CA19.9, respectively. No correlation was found between Tumor Node Metastasis staging and histamine amount, indicating that this marker is not related to the tumor mass. Our data suggest that histamine level, together with other classical tumor markers, could be a potentially interesting tumor marker in colon cancer monitoring.
Keywords: Histamine; OPA;

A simple, rapid method of determining the ibuprofen concentration in small volumes of human plasma (50 μl) by HPLC was developed. The sample was prepared for injection using a solid-phase extraction method, with naproxen as the internal standard. A 96-well extraction plate was used, easing sample preparation and allowing the simultaneous extraction of multiple plasma samples directly into the HPLC injection vials. Samples were stable at room temperature for at least 48 h prior to injection. The HPLC method used an ultraviolet detector with a 5-min run time and measured concentrations across the range typically seen with the clinical use of this drug. The calibration curve was linear across the concentration range of 0.78–100 μg/ml with a limit of quantitation (LOQ) of 1.56 μg/ml. The coefficient of variation for intra-day and inter-day precision was 6% or less with accuracies within 2% of the nominal values for low (4.5 μg/ml), medium (40 μg/ml) and high (85 μg/ml) ibuprofen concentrations. For ibuprofen concentrations at the LOQ, the intra-day and inter-day precision and accuracy were within 10 and 15%, respectively. Recovery was 87% or greater for ibuprofen. This method was used to analyze plasma samples for unknown ibuprofen concentrations in bioequivalence and limited food effect studies of different formulations of ibuprofen. Thus, this method has been fully validated and used in the analysis of unknown plasma samples for ibuprofen.
Keywords: Ibuprofen;

A simple and sensitive high-performance liquid chromatographic (HPLC) method is established for the trace determination of tobramycin in human plasma by derivatization. The method is based on the chemical derivatization of aminoglycoside antibiotic, tobramycin in human plasma, with 1-naphthyl isothiocyanate (NITC) in pyridine at 70 °C. After derivatization reaction, a methylamine/acetonitrile solution was added to the reaction mixture to eliminate the excess derivatizing agent and shorten the analysis time. The resulting derivative was separated using a Purospher® STAR RP-18e column and a water–acetonitrile (50:50, v/v) mobile phase (detection at 230 nm). Optimization conditions for the derivatization of tobramycin were investigated by HPLC. The linear range for the quantitation of tobramycin in spiked plasma was over 0.93–9.34 mg/l; the detection limit (signal-to-noise ratio=3; injection volume, 10 μl) was about 0.23 mg/l. The relative standard deviation was less than 2.1% for intra-day assay (n=6) and 5.2% for inter-day assay (n=6) and relative recoveries were found greater than 99%.
Keywords: Tobramycin;

Evaluation of lipophilicity and antitumour activity of parallel carboxamide libraries by Ferenc Hollósy; János Seprödi; László Örfi; Dániel Erös; György Kéri; Miklós Idei (355-363).
Searching for molecules possessing antitumour activity, a parallel molecule library of aromatic carboxamides has been designed and synthesised. This work resulted in a “thiophene” sub-library containing a thiophene core and of a “furoyl” sub-library with a furoyl core, respectively. In both sub-libraries substitutions were carried out with six different groups resulting in six pairs of compounds differing in only the heteroatom of aromatic ring of the cores. To study the importance of the type of cores and the specific substitutions in relation to their lipophilicity and antitumour activity, lipophilicity of carboxamides was determined by chromatographical data (log k′) and by software calculated parameters (CLOGP). Pairs of compounds were tested for their ability to inhibit the proliferation of the A431 cells by MTT assay. The isosteric molecule pairs were successfully separated. Our results showed that the experimentally determined (log k′) and the calculated (CLOGP) lipophilicity parameters correlated well with each other. Furthermore, lipophilicity values of the thiophene sub-library were always higher than those in the furoyl sub-library. Moreover, compounds of the thiophene sub-library were more active than their respective furoyl pairs in our MTT antiproliferative assay. From these observations we can conclude that the higher the lipophilicity values the higher the antitumour activity of the carboxamides synthesised. Therefore, determination of lipophilicity by measuring the log k′ or by calculating the CLOGP values of the carboxamide sub-libraries may help to predict their biological activities.
Keywords: Carboxamide parallel library;

Environmental exposure levels of bisphenol A (BPA) in human were investigated by measuring BPA glucuronide (BPA-G) in urine. After enzymatic hydrolysis of glucuronide substances in urine, BPA was extracted with diethyl ether. The extract was analyzed using a column-switching HPLC system employing a C8 and a C18 column with multi-channel coulometric electrochemical detection (ECD). The sensitivity and selectivity provided with redox mode of ECD allowed measurement of low level BPA in hydrolyzed urine. The quantification limit of BPA-G in urine was 0.2 ng/ml. RSDs of the intraassay precision were less than 3% and recoveries of the method were over 96% when analyzing BPA spiked urine samples (1.0 and 10 ng/ml). In a group of 48 women students, the level of BPA-G in urine ranged from 0.2 to 19.1 ng/ml with a median concentration of 1.2 ng/ml. Normalized against urinary creatinine, BPA-G ranged from 0.1 to 11.9 ng/mg creatinine with a median of 0.77 ng/mg creatinine.
Keywords: Bisphenol A;

A rapid quantitative assay method, developed by combining fast gradient liquid chromatography and electrospray ionization-ion trap mass spectrometry, is described for the simultaneous determination of CYP450 probe substrate metabolites (4-aminophenol for CYP2E1, acetaminophen for CYP1A2, dextrorphan for CYP2D6, 4′-hydroxymephenytoin for CYP2C19, 4-hydroxytolbutamide for CYP2C9 and 6β-hydroxytestosterone for CYP3A4) in microsomal incubations. Using this method Michaelis–Menten kinetic parameters K m and V max for the probe substrates in human liver microsomes were obtained. This LC–MS–MS method, developed with the use of LC–ESI-ion trap MS instrumentation, can efficiently be used to improve the throughput and cost-effectiveness in the preclinical drug metabolism studies.
Keywords: 4-Aminophenol; Acetaminophen; Dextrorphan; 4′-Hydroxymephenytoin; 4-Hydroxytolbutamide; 6β-Hydroxytestosterone;

Concentrations of tryptophan and its metabolites in plasma are of great interest in determining proper diagnosis and medication of several neurological diseases like, for example, Alzheimer’s disease. A method of standard addition was developed to determine total level of tryptophan and two of its metabolites, kynurenine and kynurenic acid, in human plasma by capillary liquid chromatography–electrospray ionization tandem mass spectrometry. Plasma samples were simply deproteinized by addition of diluted perchloric acid. Samples were then mixed with trichloroacetic acid and injected onto a capillary column. Analytes were separated by a fast gradient elution of the injected samples. Detection was performed by sheathless electrospray tandem mass spectrometry in the multiple reaction monitoring mode. Linear calibration curves were obtained for spiked plasma sample with up to 100% of the expected analytes concentrations. The determined concentrations were well within ranges previously reported (i.e., 6 nM–95 μM) and limit of detections were around 3 nM for each analyte.
Keywords: Tryptophan; Kynurenine; Kynurenic acid;

The partitioning of alpha-1-antitrypsin was assayed in biphasic aqueous systems containing potassium phosphate and two polyethyleneglycols of molecular mass 600 and 1000, respectively. In order to isolate the alpha-1-antitrypsin from serum plasma, the partitioning behaviour of human serum albumin, its principal contaminant, was also studied. Several aqueous two-phase systems with different partitioning properties were obtained by varying the PEG1000/PEG600 mass proportion. In systems with PEG1000/PEG600 mass ratio of 8, the optimal difference between the partition coefficients of both proteins was found. Under such conditions, a satisfactory purification was carried out by a three-step extraction procedure. By applying this method the alpha-1-antitrypsin specific activity increased severalfold (nearly 10 times) with a yield of 43%.
Keywords: α-1-antitrypsin;

We have developed two solid-phase microextraction (SPME) methods, coupled with gas chromatography, for quantitatively analysing the major Eucalyptus leaf terpene, 1,8-cineole, in both expired air and blood from the common brushtail possum (Trichosurus vulpecula). In-line SPME sampling (5 min at 20 °C room temperature) of excurrent air from an expiratory chamber containing a possum dosed orally with 1,8-cineole (50 mg/kg) allowed real-time semi-quantitative measurements reflecting 1,8-cineole blood concentrations. Headspace SPME using 50 μl whole blood collected from possums dosed orally with 1,8-cineole (30 mg/kg) resulted in excellent sensitivity (quantitation limit 1 ng/ml) and reproducibility. Blood concentrations ranged between 1 and 1380 ng/ml. Calibration curves were prepared for two concentration ranges (0.05–10 and 10–400 ng/50 μl) for the analysis of blood concentrations. Both calibration curves were linear (r 2=0.999 and 0.994, respectively) and the equations for the two concentration ranges were consistent.
Keywords: 1,8-Cineole;

In this paper we developed a simple, rapid and sensitive method for the quantitative analysis of phenylalanine (Phe) and tyrosine (Tyr) in dried blood spots of newborns by gas chromatography–mass spectrometry (GC–MS). Phe and Tyr in blood samples were reacted with N-methyl-N-(tert.-butyldimethylsilyl)trifluoroacetamide at 120 °C for 30 min and their corresponding single derivatives were obtained. Phe and Tyr were determined by measurement of their derivatives by GC–MS in the selected ion monitoring mode. Contents of Phe and Tyr in blood spots were calculated by external standard method. The ratio of Phe to Tyr was used for neonatal screening for phenylketonuria. The present method only took a few minutes to perform and required minimal sample preparation. In addition it provided low detection limits of 1.2 μmol l−1 for Phe and 1.6 μmol l−1for Tyr.
Keywords: Phenylalanine; Tyrosine;

An analytical method to improve the characterization of lipoprotein fractions is presented. Human plasma samples were treated with Sudan Black B to stain the lipid component in lipoproteins, then the stained lipoproteins were separated by frit inlet asymmetrical flow field-flow fractionation (FI-AFlFFF), according to the lipoprotein particle sizes, with the selective detection of eluting lipoprotein fractions, high-density lipoproteins (HDL), low-density lipoproteins (LDL) and very-low-density lipoproteins (VLDL), at 610 nm. The capability of this technique has been evaluated with plasma samples obtained from patients with coronary artery disease (CAD), and it showed that the retention profile of patients’ lipoprotein samples was clearly distinct from those of healthy persons. The potential of this technique comes with the direct injection of a stained lipoprotein sample without a prior procedure such as ultracentrifugation for sample preparation, and the size calculation of lipoprotein particles from the experimental retention time by theory. Since sample relaxation was achieved hydrodynamically in an FI-AFlFFF channel, sample injection and separation processes were continuously made without stopping the separation flow. This study demonstrated the potential of the FI-AFlFFF technique to be utilized as a powerful tool for the determination of the LDL profiles of patients with CAD.
Keywords: Lipoproteins; Low-density lipoproteins;

Isocratic ion-exchange chromatographic assay for the nucleotide gemcitabine triphosphate in human white blood cells by Rolf W Sparidans; Mirjam Crul; Jan H.M Schellens; Jos H Beijnen (423-430).
An isocratic bio-analytical assay for the nucleotide gemcitabine triphosphate (2′,2′-difluorodeoxycytidine 5′-triphosphate, dFdCTP) in human white blood cells (leukocytes) has been developed and validated. The method is based on ion-exchange liquid chromatography and ultraviolet detection (275 nm). dFdCTP is isolated from the matrix by extraction with perchloric acid while the sample is chilled on ice. After neutralization with potassium hydroxide and removal of the potassium perchlorate precipitate, with the sample still chilled on ice, the mixture is injected into the chromatograph. The method has been validated in the range 0.4–20 μM, 0.4 μM (∼20 pmol/106 cells) being the lower limit of quantification, using erythrocytes as a substitute for leukocytes. Precisions and accuracies both meet the current requirements for a bioanalytical assay. The stability of dFdCTP in intact mononuclear blood cells on ice is strongly limited (half-life ∼100 min) and after freezing the half-life of the analyte in the cellular lysate is ∼30 min. On the other hand, no degradation was observed for dFdCTP for at least ∼24 h in perchloric acid extracts on ice or in neutralized extracts at ambient temperature. The applicability of the assay was demonstrated in white blood cells of a patient with advanced non-small cell lung cancer receiving i.v. gemcitabine.
Keywords: Nucleotide; Gemcitabine triphosphate;

We describe an analytical method that allows the determination of organophosphorus pesticides (OPs) in different human tissues. It involves an extraction procedure with ethanol–ethyl acetate, followed by gel permeation chromatography clean-up step and analysis by capillary gas chromatography–negative chemical ionization mass spectrometry in the selected ion monitoring mode. The method was tested for 37 OPs and the recoveries obtained vary between 60 and 106% with standard deviations ranging between ±2 and ±10. These values are independent of the analyzed tissue. Peak area repeatability as RSD for some OPs was ≤4.8% while a good linear relationship in the range 1.0–500 pg μl−1 with r 2≥0.9878 was obtained. The limit of detection for the 37 OPs falls between 0.01 and 0.09 ng g−1 with an RSD≤9.5%. The analytical set up in this paper has been used to analyze different samples of human tissues (liver, healthy kidney, cancer kidney and adipose tissue) of 24 patients. The number of the identified OPs in the tissue samples is different (max. 20) according to the sample while their concentration ranges between the limit of detection and 28.0 ng g−1. The highest concentrations have been determined in liver samples without any pathology (0.4–28.0 ng g−1) while the lowest concentrations have been determined in healthy kidney samples (0.01–1.50 ng g−1). In the cancer kidney samples OP concentrations vary between 0.03 and 4.6 ng g−1: these concentrations are more elevated than those determined in healthy kidney samples. The comparison between the concentration of OPs determined in the healthy part, when possible, and those determined in the cancer part of the same kidney sample are very interesting: in fact, in the latter the OP concentration is generally 1–2-times higher than that in the former, an index of lower enzymatic activity in the cancer tissue.
Keywords: Organophosphorus pesticide residues;

The separation of the major stratum corneum lipids, i.e., ceramides, fatty acids, cholesterol and its esters by means of high-performance thin-layer chromatography is hereby presented. The used automated multiple development technique allows the reproducible development of a 17-step solvent gradient also capable of separating seven ceramide classes in the same run. Reliable quantification has been performed after visualisation and densitometric scanning. The present approach is less time and solvent-consuming than previously described procedures. The application to samples obtained by in vivo skin surface extraction with hexane–ethanol (2:1) demonstrates that the method can be routinely used for diagnostic purposes.
Keywords: Lipids; Ceramides; Fatty acids; Cholesterol;

Semi-automated determination of plasma stability of drug discovery compounds using liquid chromatography–tandem mass spectrometry by Gangfeng Wang; Yunsheng Hsieh; YauYi Lau; K.-C Cheng; Kwokei Ng; Walter A Korfmacher; Ronald E White (451-457).
A simple procedure for the measurement of stability of drug candidates in plasma was developed to eliminate the traditional labor-intensive and time-consuming sample preparation procedures that are typically used for these studies. The procedure makes use of a thermostatic autosampler as an incubator combined with the direct plasma injection method based on high-performance liquid chromatography (HPLC) coupled to tandem mass spectrometry (MS–MS). Untreated human, monkey, mouse and rat plasma containing the test compound was directly injected into a mixed-function column for on-line protein removal and chromatography. The test compound and its biotransformation product were separated via HPLC and monitored using the tandem mass spectrometer. The need for adequate chromatographic separation of the test compound (M) from its carboxylic acid metabolite (M+1) is demonstrated. Plasma samples from four different species at specified incubation temperatures were sequentially assayed in one analytical procedure. The injection-to-injection time was about 6 min. The peak responses of the test compound in individual plasma samples were repeatedly determined every 24 min. The retention times and peak shape of all analytes were found to be consistent throughout the experiments. The stability of the test compound in plasma was found to be a function of animal species, incubation time and temperature. The test compound was rapidly degraded in rat plasma at 37 °C, but it could be stabilized by adding sodium thiosulfate.

Phenotype of CYP2C19 and CYP3A4 by determination of omeprazole and its two main metabolites in plasma using liquid chromatography with liquid–liquid extraction by Héctor M González; Elba M Romero; Teresa de J Chavez; A.Aaron Peregrina; Vı́ctor Quezada; Carlos Hoyo-Vadillo (459-465).
We present a new simple and reliable HPLC method for measuring omeprazole and its two main metabolites in plasma. This can be used for studying CYP2C19 and CYP3A4 genetic polymorphisms using omeprazole as the probe drug. Omeprazole, hydroxyomeprazole and omeprazole sulfone were extracted from plasma samples with phosphate buffer and dichloromethane–ether (95:5). HPLC separation was achieved using an Ultrasphere ODS C18 (Beckman) column. The mobile phase was acetonitrile–phosphate buffer (24:76, pH 8), containing nonylamine at 0.015%. Retention times were 9.5 min for omeprazole, 3.25 min for hydroxyomeprazole, 7.4 min for omeprazole sulfone and 6.27 min for internal standard (phenacetine). Detection (UV at 302 nm) of analytes was linear in the range from 96 to 864 ng/ml. This is useful for calculating metabolic index for CYP2C19 and CYP3A4 in adults and children. This method is stable, reproducible, improves resolution and has practical advantages such as low cost.
Keywords: Omeprazole; Hydroxyomeprazole; Omeprazole sulfone;

Triazine–human serum albumin association: thermodynamic approach and sodium effect by Lhassane Ismaili; Claire André; Laurence Nicod; Tong Thanh Truong; Joëlle Millet; Mireille Thomassin; Eric Cavalli; Jean Pierre Chaumont; Alain Xicluna; Yves Claude Guillaume (467-474).
Human serum albumin (HSA) serves as a carrier protein to transport triazine herbicides to molecular targets. In this paper, a theoretical treatment was developed to describe the HSA–triazine herbicides association. A determination of the association constant, K, as well as the degree of complexation n c (the percent of complex guest) was carried out. Enthalpy–entropy compensation was also analyzed in relation to this mathematical model to confirm the herbicide complexation behavior with HSA. The role of the sodium cation (Na+) on this association was investigated. It was expected that the sodium ion would act on the herbicide–HSA association process by modifying the surface tension of the bulk solvent and increase the K and n c values. The results showed that for patients who suffer from Na+ desequilibrium, the triazine–HSA binding would change and as well the toxicological effect of these herbicides.
Keywords: Triazine; Human serum albumin;

Measurement of resiniferatoxin in cerebrospinal fluid by high-performance liquid chromatography by Andrew J Mannes; Dorothy Cimino Brown; Sandra Z Perkowski; Jason Keller; Robert M Caudle; Michael J Iadarola; Qing C Meng (475-479).
A sensitive and simple high-performance liquid chromatographic (HPLC) assay was developed for the quantification of resiniferatoxin (RTX) in canine cerebrospinal fluid (CSF). A reversed-phase C18 column and acetonitrile in 0.02 M NaH2PO4 as mobile phase provided satisfactory resolution for RTX analysis. Direct HPLC analysis of the CSF samples without sample extraction or preparation improves the accuracy and detection limits of this assay. This assay was applied to measure CSF RTX content to test this method for research and clinical applications related to studies examining its analgesia effects.
Keywords: Resiniferatoxin;

A previously published liquid chromatographic method for determining residues of nine quinolones in chicken, porcine, bovine and ovine muscle was adapted and applied to fish tissue for simultaneous determination of three quinolones (flumequine, oxolinic acid and sarafloxacin). The analytes were extracted from homogenised muscle using an acetonitrile basic solution. After centrifugation, partial evaporation and cleaning with hexane, direct injection was possible. Separation was achieved on PLRP-S column and detection was performed with a programmable fluorescence detector. Chromatographic conditions were optimised to be compatible with the determination of the three quinolones in a single run. The linearity, recovery, accuracy and precision of the method were evaluated from fortified tissue samples at concentration levels ranging from 15 to 120 μg kg−1 for sarafloxacin and 75 to 600 μg kg−1 for oxolinic acid and flumequine according to the EU maximum residue limit of each quinolone. The limits of detection were estimated to be 2, 5 and 7 μg kg−1, respectively, for sarafloxacin, oxolinic acid and flumequine. The limits of quantification were validated at 15 μg kg−1 for sarafloxacin and 75 μg kg−1 for oxolinic acid and flumequine. Mean extraction recoveries of quinolones in fish ranged from 56.9 to 71.0%. This simple and rapid method is suitable for residue control.
Keywords: Oxolinic acid; Flumequine; Sarafloxacin;