Journal of Chromatography B (v.817, #2)

News Section (N1-N2).

The accuracy of two clinical assays, the enzyme-multiplied immunoassay (EMIT) and fluorescence polarization immunoassay (FPIA2), universally employed for measurement of plasma levels of methotrexate (MTX) in children administered a high dose of this drug for treatment of acute lymphoblastic leukemia was evaluated here. Because of its superior specificity, sensitivity, and precision, high performance liquid chromatography (HPLC) was selected as the reference method with which the other two procedures were compared using approximately 420 different plasma samples for method comparison. 7-Hydroxymethotrexate (7-OHMTX), the major plasma metabolite of MTX, that can be detected in plasma at relatively high concentrations for long periods following infusion of a high dose of MTX, was also quantitated by HPLC. Forty-two and 66 h after infusion, the plasma level of MTX was overestimated in 2% and 3% of the samples by the FPIA2 procedure in 5% and 31% by the EMIT assay. The overall correlation coefficients (r 2) for the values obtained by FPIA2 or EMIT versus those based on HPLC were 0.989 and 0.663, respectively. The presence of 7-OHMTX exerted a highly significant influence (p  = 0.0007 as determined by the unpaired t-test) on MTX measurement by the EMIT assay. We conclude that the rapid automated procedures routinely used at present and in particular EMIT, suffer from cross-reactivity with metabolites of MTX. Thus, the relatively high percentage of samples in which the level of MTX is overestimated at check-points by EMIT may result in longer periods of hospitalization, higher costs and prolonged administration of elevated doses of “rescue” leucovorin with an increased risk for relapse.
Keywords: Methotrexate; 7-Hydroxymethotrexate; Enzyme-multiplied immunoassay; Fluorescence polarization immunoassay; High performance liquid chromatography; Leukemia;

Isolation of plasma albumin by ethanol extraction is inappropriate for isotope ratio measurements during the acute phase response by Rita Jacobs; Hans Demmelmair; Peter Rittler; Josef Kellermann; Berthold Koletzko; Maximiliane Krick; Karl-Walter Jauch; Wolfgang H. Hartl (145-151).
Isolation of high-purity albumin from plasma is essential to study albumin kinetics in vivo with tracer techniques. Because of its simplicity ethanol extraction has been repeatedly used for albumin purification. However, it cannot be excluded that this single-step procedure completely prohibits contamination by other proteins, especially those known to be produced at an accelerated rate during the acute phase response. In the present study, we wanted to examine the reliability of ethanol extraction in different clinical conditions and to study the effects of potential impurities on albumin enrichment during stable isotope tracer studies. SDS-PAGE revealed a contaminating protein band at about 25,000 Da in healthy subjects and postoperative patients during the acute phase response, but not in critically ill patients. According to densitometry about 8% of proteins after ethanol extraction were contaminants. To examine potential contaminant effects on tracer enrichment 1-[13C]-leucine was given to healthy subjects and postoperative patients. Blood samples were taken after various amounts of time, and albumin enrichments (tracer/tracee ratios) were determined from isotope ratios obtained by mass spectrometry. Irrespective of the magnitude of tracer enrichment, postoperative tracer/tracee ratios were significantly higher (on average +10%) in samples exclusively analysed by ethanol extraction than in samples which had undergone additional electrophoretic purification. No significant effect of the contaminant was seen in healthy subjects. N-terminal protein sequencing revealed contaminants to mainly consist of apolipoprotein A-1. Its physiology and pathophysiology may sufficiently explain its variable effects of albumin enrichment. Our findings suggest that exclusive ethanol extraction is inappropriate for albumin isolation in tracer studies performed during the acute phase response. Ethanol extraction may also not be advisable in all other situations known to be associated with a rise in apolipoprotein A-1 turnover.
Keywords: Albumin; Ethanol extraction; Mass spectrometry; Acute phase response;

An analytical method for simultaneous determination of erythromycin propionate and its active metabolite, erythromycin base, in human plasma by high-performance liquid chromatography–electrospray mass spectrometry (HPLC–ESI-MS) was developed and validated. Roxithromycin was selected as the internal standard. The samples were directly injected after simple deproteinized procedure only. The separation was achieved on a Johnson Spherigel analytical column packed with 5 μm C18 silica, employing acetonitrile −0.1% formic acid aqueous solution (50:50) as mobile phase. The quantification of target compounds was obtained by using a selected ion monitoring (SIM) at m/z 790.7 for erythromycin propionate, m/z 734.7 for erythromycin base and m/z 837.8 for roxithromycin. The correlation coefficients of the calibration curves were better than 0.997 (n  = 6), in the ranges from 2 ng/ml to 1 μg/ml, and from 1 to 10 μg/ml for erythromycin propionate and base. The method can provide the necessary sensitivity, precision and accuracy to allow the simultaneous determination of both compounds in a patient's plasma following a single administration of erythromycin stinoprate capsule (500 mg erythromycin base equivalent).
Keywords: Erythromycin base; Erythromycin propionate; Roxithromycin; HPLC–ESI-MS;

The development of new pharmaceutical forms with classical active compounds generates new analytical problems. That is the case of sugar-free sachets of cough-cold products containing acetaminophen, phenylephrine hydrochloride and chlorpheniramine maleate. Two cyanopropyl stationary phases have been employed to tackle the problem. The Discovery cyanopropyl (SUPELCO) column permitted the separation of the three actives, maleate and excipients (mainly saccharine and orange flavour) with a constant proportion of aqueous/ organic solvent (95:5, v/v) and a pH gradient from 7.5 to 2. The run lasted 14 min. This technique avoids many problems related to baseline shifts with classical organic solvent gradients and opens great possibilities to modify selectivity not generally used in reversed phase HPLC. On the other hand, the Agilent Zorbax SB-CN column with a different retention profile permitted us to separate not only the three actives and the excipients but also the three known related compounds: 4-aminophenol, 4-chloracetanilide and 4-nitrophenol in an isocratic method with a run time under 30 min. This method was validated following ICH guidelines and validation parameters showed that it could be employed as stability-indicating method for this pharmaceutical form.
Keywords: Common cold drugs; Impurities; pH gradient; HPLC;

In this study, a solid-phase microextraction (SPME) method based on poly(acrylate)-coated fibres has been developed for detection and quantification of chlorinated bisphenol A in human plasma due to the need for an assessment of human exposure to them. After desorption of the analytes for 7 min at 300 °C, they were directly derivatized in the GC injector port by injection of 2 μL of diluted bis(trimethylsilyl)trifluoroacetamide (BSTFA). The formation of trimethylsilylate derivatives improves the selectivity, sensitivity and performance of the chromatographic properties obtained when the analytes are directly separated. Quantification was carried out using single-ion monitoring (SIM). The respective chloroderivative molecular ions appear at 406, 440, 474 and 508 m/z; whereas the base peaks corresponding to a loss of a methyl group in all cases appear at 391, 425, 459 and 493 m/z for mono-, di-, tri- and tetrabisphenol A, respectively. Deuterated bisphenol A (BPA-d16) was used as an internal standard. The method was applied to the determination of Cl-BPA, Cl2-BPA, Cl3-BPA and Cl4-BPA at very low concentration levels in plasma. Recovery efficiencies were close to 100% in all cases.
Keywords: Chlorinated bisphenol A; Solid-phase microextraction; Column silylation; GC–MS; Human plasma;

DNA is a universal analyte found in almost every organism. It is the code that dictates our genetic make-up and it provides a vast library of information. DNA sequences can indicate genetic modification of foodstuffs, how we may metabolise pharmaceuticals and the likelihood of suffering particular diseases. The basis for many of these genetic tests would benefit greatly from procedures that can accurately quantitate DNA in an absolute manner. This would then provide a sound and universally consistent foundation for regulatory and diagnostic decision making. This work compares two different enzymatic digestion systems as precursor steps to high accuracy isotope dilution mass spectrometry (IDMS) quantitation of a 20mer oligonucleotide. In the first approach, snake venom phosphodiesterase (SVP) digests the oligonucleotide to its constituent deoxynucleotides (dNMPs), followed by liquid chromatography–IDMS (LC–IDMS) quantitation. The second enzyme digestion approach used a combination of snake venom phosphodiesterase and shrimp alkaline phosphatase (SAP) which reduces the oligonucleotide to its constituent deoxynucleosides (dNs). This was then followed by an alternative LC separation and equivalent IDMS measurements. Total phosphorous content of the 20mer oligonucleotide was measured by inductively coupled plasma optical emission spectroscopy (ICP-OES). This provided independent data for comparison with the two enzyme digestion–IDMS based procedures. The most appropriate method of quantitation was found to be the combined SVP and SAP digestion. This approach negates the need to consider and/or account for the lack of a 5′ terminal phosphate residue. It also enables the use of positive ion mass spectrometry which simplifies the chromatographic requirements. Based on the exact matched IDMS of the adenine deoxynucleoside, the concentration of the original 20mer oligonucleotide was found to be 110 ± 9 μg g−1. This showed good agreement with the ICP-OES data based on the measurement of phosphorus which gave an equivalent value for the original 20mer oligonucleotide of 108 ± 5 μg g−1 (uncertainties at the 95% confidence interval). It is intended that this high accuracy methodology should be used to produce high calibre reference standards. These, in turn, could then be used to underpin the quality and consistency of routine measurements involving a variety of more commonly encountered methodologies. It should be noted that the IDMS procedures are equally applicable to both sequenced and non-sequenced oligonucleotide materials.
Keywords: Oligonucleotide; Quantitation; Liquid chromatography–isotope dilution mass spectrometry; Digestion; Enzyme;

Liquid chromatographic assay of abouthiouzine in plasma and its application to pharmacokinetic studies by Khalid M. Alkharfy; Rao Muzaffar A. Khan; Badraddin M. Al-Hadiya; Hisham S. Abou-Auda; Rafiq R. Abou-Shaaban (183-186).
Abouthiouzine is a newly synthesized antithyroid agent with a proposed less adverse effects profile than other currently used drugs. A simple and rapid reversed phase high performance liquid chromatography assay was developed to determine the concentration of abouthiouzine in human plasma. The procedure involved extraction of the drug and propranolol (internal standard) from the plasma using ethylacetate. The extract was evaporated under nitrogen and the residue was constituted with the mobile phase and injected onto μ-Bondapack phenyl column (10 μm, 3.9 mm × 150 mm). The mobile phase consisted of 10 mM potassium dihydrogen phosphate buffer, acetonitrile, and methanol in the ratio of 60:25:15 (v/v/v, pH = 3.0), which was delivered at a rate of 1.5 ml/min. Abouthiouzine and the internal standard were monitored using UV detection at 240 nm; the run time was less than 5 min. The detection limit of abouthiouzine is 0.5 μg/ml. The within- and between-day coefficients of variation were less than 7%. Our method has been successfully used to measure abouthiouzine plasma concentrations in a rabbit model following an intravenous administration of the drug.
Keywords: Abouthiouzine; HPLC; Assay;

Huperzine-A (Hup-A), a biologically potent, reversible acetylcholinesterase inhibitor for the treatment of Alzheimer disease (AD) in China, has very low blood concentration. In order to study the pharmacokinetics of newly developed Hup-A transdermal patches in animal, a rapid and sensitive ion-pair reverse-phase high performance liquid chromatography (RP-HPLC) method for the determination of Hup-A in beagle dog serum using mebendazole as internal standard has been developed and validated. The analyte and internal standard were extracted from serum using chloroform–isopropanol (95:5, v/v), analyzed on a C (18) column (5 μm, 150 mm × 4.6 mm i.d.) with a mobile phase consisting of methanol–water–glacial acetic acid (50:48.5:1.5, v/v/v), using sodium dodecylsulfonate as an ion-pair reagent, and detected with UV detector at 313 nm. The chromatographic run time was within 15 min. The assay was linear over the concentration range of 1–12 ng/ml and intra- and inter-day precision over this range was not more than 12.8%. The limit of quantification in serum was 1 ng/ml. The method was successfully applied to characterize the Hup-A concentration–time profiles and study the single and multiple doses phamacokinetics of Hup-A transdermal patches in beagle dogs. The pharmacokinetic study results showed that Hup-A patches has the characteristic of sustained or controlled drug release in vivo.
Keywords: Ion-pair RP-HPLC; Huperzine-A; Dog serum;

A rapid, selective and sensitive high-performance liquid chromatographic method with spectrophotometric detection was developed for the determination of clarithromycin in human plasma. Liquid–liquid extraction of clarithromycin and norverapamil (as internal standard) from plasma samples was performed with n-hexane/1-butanol (98:2, v/v) in alkaline condition followed by back-extraction into diluted acetic acid. Chromatography was carried out using a CN column (250 mm × 4.6 mm, 5 μm) under isocratic elution with acetonitrile–50 mM aqueous sodium dihydrogen phosphate (32:68, v/v), pH 4.5. Detection was made at 205 nm and analyses were run at a flow-rate of 1.0 ml/min at 40 °C. The analysis time was less than 11 min. The method was specific and sensitive with a quantification limit of 31.25 ng/ml and a detection limit of 10 ng/ml in plasma. The mean absolute recovery of clarithromycin from plasma was 95.9%, while the intra- and inter-day coefficient of variation and percent error values of the assay method were all less than 9.5%. Linearity was assessed in the range of 31.25–2000 ng/ml in plasma with a correlation coefficient of greater than 0.999. The method was used to analyze several hundred human plasma samples for bioavailability studies.
Keywords: Clarithromycin; Ultraviolet detection;

Determination of lamotrigine in small volumes of plasma by high-performance liquid chromatography by Ching-Ling Cheng; Chen-Hsi Chou; Oliver Yoa-Pu Hu (199-206).
Lamotrigine is a broad-spectrum antiepileptic agent. This study describes a simple and sensitive high-performance liquid chromatographic method for the determination of lamotrigine in 50 μl of plasma. Lamotrigine and the internal standard guanabenz were extracted with 1.2 ml of diethyl ether, after the samples alkalinized with 10 μl of sodium hydroxide solution (1N). Chromatographic separation was achieved on a silica column with the mobile phase of acetonitrile–water containing 0.2% phosphoric acid and 0.3% triethylamine (pH 2.7) (84:16, v/v), at a flow-rate of 1 ml/min. The eluant was detected at 225 nm. The retention time was about 6 min for lamotrigine and 7 min for guanabenz. No endogenous substances and concomitant anticonvulsants were found to interfere. Calibration curves were linear from 0.1 to 5   μg/ml. The relative recovery of lamotrigine averaged about 80%. The limit of quantitation was 0.1   μg/ml. The intra- and inter-day precision (expressed as coefficient of variation, CV) was 8.1%, or less, and the accuracy was within 11.5% deviation of the nominal concentration. The method is suitable in pharmacokinetic investigation and monitoring lamotrigine concentration.
Keywords: Lamotrigine; Antiepileptive; HPLC; Pharmacokinetics;

A simple high-performance liquid chromatographic (HPLC) method was developed for the assay of total and free mycophenolic acid (MPA) in human plasma. Prior to analysis, total mycophenolic acid was extracted by protein precipitation and free drug was isolated from plasma samples using ultrafiltration. The extracts were injected onto a Kromasil C8 column at 30 °C with excitation and emission wavelengths set at 342 and 425 nm, respectively. The mobile phase was consisted of acetonitrile-32 mM glycine buffer, pH 9.2 (20:80, v/v), at a flow rate of 1.0 ml/min. The method was found to be linear over the concentration range investigated, 0.05–40 mg/l for total mycophenolic acid (r  > 0.999) and 5–1000 μg/l (r  > 0.99) for free drug. The percentage error of the analytical method was below 10.9%. The intra- and inter-day reproducibility was adequate with the coefficients of variation of 8.28% or below. The run time were 4 and 6 min for free and total MPA, respectively. The method thus can be effectively applied to measure mycophenolic acid concentrations in clinical samples.
Keywords: Mycophenolic acid;

Detection of synthetic glucocorticoid residues in cattle tissue and hair samples after a single dose administration using LC–MS/MS by Olivia Van den hauwe; Frederic Dumoulin; Chris Elliott; Carlos Van Peteghem (215-223).
A sensitive and specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay for the detection of several synthetic glucocorticoids in kidney, muscle and hair samples of cattle after a single intramuscular injection is described. After a dichloromethane wash of the hair samples, analytes were released from the hair matrix by enzymatic digestion. Muscle samples were also digested enzymatically using proteinase, while kidney samples were deconjugated by Helix pomatia juice. These preliminary steps were followed by a methanol extraction and a solid phase extraction (SPE) clean up step for all matrices. Chromatographic separation was achieved on a Hypersil Hypercarb column and MS/MS data were obtained in the multiple reaction monitoring mode using negative electrospray ionization. The developed protocols were evaluated by assessing residue concentrations in muscle, kidney and hair samples of thirteen calves, treated with a particular intramuscular injection of glucocorticoid. The lowest residue levels were found in muscle samples (approximately 5% of the residue levels in kidney), while high residue levels were obtained in hair samples. Hair is an interesting matrix since the sampling is non-invasive and the drugs may stay incorporated for a longer period of time.
Keywords: Glucocorticoids; LC–MS/MS; Food safety; Growth-promoters;

Database independent detection of isotopically labeled MS/MS spectrum peptide pairs by Frank Potthast; Jiri Ocenasek; Dorothea Rutishauser; Martin Pelikan; Ralph Schlapbach (225-230).
Mass spectrometry data generated in differential profiling of complex protein samples are classically exploited using database searches. In addition, quantitative profiling is performed by various methods, one of them using isotopically coded affinity tags, where one typically uses a light and a heavy tag. Here, we present a new algorithm, ICATcher, which detects pairs of light/heavy peptide MS/MS spectra independent of sequence databases. The method can be used for de novo sequencing and detection of posttranslational modifications. ICATcher is distributed as open source software.
Keywords: Proteomics; ICATcher; ICAT; Peptide sequencing; PTM detection; Differential analysis; Chromatography;

Polystyrene-type resin used for peptide synthesis: application for anion-exchange and affinity chromatography by Regina S.H. Carvalho; Danielle A. Ianzer; Luciana Malavolta; Mauricio M. Rodrigues; Eduardo M. Cilli; Clovis R. Nakaie (231-238).
This paper deals with an unusual application for a copolymer of styrene–1% divinylbenzene bearing high amount of aminomethyl groups for anion-exchange and affinity chromatography. The so-called aminomethyl resin (AMR), to date only employed for peptide synthesis, swelled appreciably in water and was used successfully to purify negatively charged peptides. By correlating swelling degree of beads with pH of the media, it was possible to estimate that the AMR amino group pK a is approximately 5.5. In addition, the synthesized acetyl–(NANP)3–AMR succeeded in the affinity interaction with large antibody molecules related to malaria transmission and raised previously against this dodecapeptide sequence.
Keywords: Resin; Peptide; Polymer; Ion-exchange chromatography; Affinity chromatography;

In this paper, multi-wall carbon nanotubes (MWNTs)/Pt microparticles nanocomposite was prepared by electrodepositing Pt microparticles onto the MWNTs matrix. The surface of glassy carbon electrode was modified with this kind of nanocomposite for measurement of thiols, such as l-cysteine (l-Cys) and glutathione (GSH). Compared with the MWNTs or Pt microparticles modified electrode, the nanocomposite modified electrode exhibited high sensitivity and good stability for detection of thiols. According to the results of experiments, the peak currents of l-Cys and GSH are linear with their concentrations and the detection limits (S/N = 3) are 2.9 × 10−8  mol/L and 4.5 × 10−8  mol/L, respectively. Coupled with microdialysis, the method has been successfully applied to the determination of these two thiols in rat striatal microdialysates.
Keywords: Multi-wall carbon nanotubes; Pt microparticles; l-Cysteine; Glutathione;

A simple ion-pairing reverse-phase HPLC method, with UV diode array detection, was developed and validated for quantitation of the urinary niacin metabolites 1-methylnicotinamide and l-methyl-2-pyridone-5-carboxamide in a single run. Urine samples were purified using a polymer-based mixed mode anion exchange reverse-phase cartridge. Analysis was performed on a reverse-phase C18 column, using a methanol gradient elution system, containing phosphate buffer pH 7.0, 1-heptanesulphonic acid as the ion-pairing agent and trimethylamine as a modifier. The assay was applied to the measurement of the niacin status of two subjects using spot urine samples. The samples were collected over 4 consecutive days and at four time points during 1 day. Status, expressed as the concentration ratios (2-PYR or 1-MN)/creatinine and 2-PYR/l-MN, varied within and between days and was least for fasting samples. This work illustrates the potential of spot urine sampling for niacin status assessment, but highlights the need for further validation prior to its use in field nutritional surveys.
Keywords: Niacin; Metabolism; Human; Urine; Spot sampling; 1-Methylnicotinamide; l-Methyl-2-pyridone-5-carboxamide; HPLC;

A sensitive and reproducible method is described for the analysis of trichloroacetic acid in urine and 1,1,1-trichloroethane in blood using dynamic headspace GC/MS. Samples were analyzed using the soil module of a modified purge and trap autosampler to facilitate the use of disposable purging vessels. Coefficients of variation were below 3.5% for both analytes, and response was linear in the range of 0.01–7.0 μg/ml for trichloroacetic acid and 0.9 ng/ml–2.2 μg/ml for 1,1,1-trichloroethane. Attempts at using dynamic headspace for the analysis of trichloroethanol in urine were unsuccessful.
Keywords: Methyl chloroform; Trichloroacetic acid; Trichloroethanol; Dynamic headspace; Purge and trap;

A rapid and sensitive method for the simultaneous determination of paracetamol and guaifenesin in human plasma was developed and validated, using high-performance liquid chromatographic separation with tandem mass spectrometric detection. After extracted from plasma samples by diethyl ether–dichloromethane (3:2, v/v), the analytes and internal standard osalmide were chromatographed on a C18 column. Detection was performed on a triple quadrupole tandem mass spectrometer by selected reaction monitoring (SRM) mode via atmospheric pressure chemical ionization (APCI). The method was linear in the concentration range of 0.05–20.0 μg/ml for paracetamol and 5.0–2000.0 ng/ml for guaifenesin. The intra- and inter-day precision was within 14% for both paracetamol and guaifenesin. The assay accuracy was within ±2.4% for the analytes. This is the first assay method described for the simultaneous determination of paracetamol and guaifenesin in plasma using one chromatographic run. The method was successfully employed in a pharmacokinetic study after an oral administration of a multicomponent formulation, containing 650 mg paracetamol, 200 mg guaifenesin, 60 mg pseudoephedrine and 20 mg dextrorphan.
Keywords: Paracetamol; Guaifenesin;

Determination of dioscin in rat plasma by liquid chromatography–tandem mass spectrometry by Ke Li; Yingwu Wang; Jingkai Gu; Xiaoyan Chen; Dafang Zhong (271-275).
A sensitive and specific high-performance liquid chromatography–tandem mass spectrometric (LC–MS/MS) assay for dioscin in rat plasma was developed. Ginsenoside Rh2 was employed as an internal standard. Dioscin is a naturally occurring saponin present in many traditional Chinese medicinal plants. Dioscin was determined after the acetonitrile-mediated plasma protein precipitation. The mobile phase consisted of acetonitrile:10 mmol/l aqueous ammonium acetate (95:5, v:v), which was pumped at 0.8 ml/min. The analytical column (100 mm × 4.6 mm i.d.) was packed with Hypersil ODS material (5 μm). The standard curve was linear from 1 to 100 ng/ml. The assay was specific, accurate (percentage deviations from nominal concentrations were <10%), precise and reproducible (within- and between-day coefficients of variation <10%). Dioscin in rat plasma was stable over three freeze–thaw cycles and at ambient temperatures for 24 h. The utility of the assay was demonstrated by determining dioscin plasma concentrations in five rats for 120 h following a single oral gavage dose of 90 mg/kg.
Keywords: Dioscin;

This article describes the development of SPE and HPLC methods for the simultaneous determination of metformin and glipizide, gliclazide, glibenclamide or glimperide in plasma. Several extraction and HPLC methods have been described previously for the determination of each of these analytes in plasma separately. The simultaneous determination of these analytes is important for the routine monitoring of diabetic patients who take combination medications and for studying the pharmacokinetics of the combined dosage forms. In addition this developed method can serve as a standard method for the plasma determination of these analytes therefore saving time, effort and money. The recoveries of the developed methods were found to be between 76.3% and 101.9%. The limits of quantification were between 5 and 22.5 ng/ml. The intraday and interday precision (measured by coefficient of variation, CV%) was always less than 9%. The accuracy (measured by relative error %) was always less than 12%. Stability analysis showed that all analytes are stable for at least 3 months when stored at −70 °C.
Keywords: HPLC; Metformin; Glipizide; Gliclazide; Glimperide; Glibenclamide;

To support pharmacokinetic studies, a selective and sensitive liquid chromatography/tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the simultaneous determination of a novel KDR kinase inhibitor (1) and its active metabolite (2) in human plasma. The method is fully automated using a Packard MultiPROBE II system and a TomTec Quadra 96 liquid handling workstation to perform sample preparation and solid-phase extraction (SPE). Following the extraction on a mixed-mode SPE using Oasis MCX 96-well plate, the analytes were separated on a Aquasil C18 column (50 mm × 2.1 mm, i.d., 3 μm) with a mobile phase consisting of acetonitrile/ammonium acetate buffer (5 mM, pH 5.0) (60/40, v/v). The run time for each injection was 4.5 min with the retention times of approximately 2.0 and 2.7 min for 1 and 2 respectively, at a flow rate of 0.25 mL/min. A tandem mass spectrometric detection was conducted using multiple reaction monitoring (MRM) under the positive ion mode with a turbo ion-spray interface. The linear ranges of the calibration curves were 0.05–400 ng/mL for 1 and 0.1–400 ng/mL for 2 on a PE Sciex API 4000 LC–MS/MS system. The lower limits of quantitation (LLOQ) of the assay were 0.05 and 0.1 ng/mL for 1 and 2 respectively, when 0.4 mL of plasma was processed. Intra-day assay precision (using five standard curves prepared by spiking compounds to five lots of plasma) was less than 4.9% for 1 and less than 9.6% for 2 on each concentration. Assay accuracy was found to be 95.1–104.6% of nominal for 1 standards and 93.5–105.6% for 2 standards. QC samples were stable when kept at room temperature for 4 h, at −70 °C for 10 days, and after three freeze–thaw cycles. The extraction recoveries were 80%, 83% and 84% for 1 and 2 and I.S. respectively, and no significant matrix effects were observed. The method was successfully applied to plasma samples from clinical studies after oral administration of compound 1.
Keywords: KDR kinase inhibitor; N-Oxide metabolite; LC–MS/MS; Oasis MCX solid-phase 96-well extraction plate;

High performance liquid chromatography analysis of a 4-anilinoquinazoline derivative (PD153035), a specific inhibitor of the epidermal growth factor receptor tyrosine kinase, in rat plasma by Silvana Aparecida Rocco; Jesus Antonio Velho; Rodrigo Miguel Marin; Luís de Arruda Rolim Filho; Anibal Eugênio Vercesi; Roberto Rittner; Kleber Gomes Franchini (297-302).
The quinazoline derivative, 4-N-(3′-bromo-phenyl)amino-6,7-dimethoxyquinazoline (PD153035), has recently been identified as a potential drug for the treatment of proliferative disease. Here, we report a sensitive high performance liquid chromatography (HPLC)-based quantitative detection method for measurement of PD153035 levels in rat plasma. Sample pretreatment involved a two-step extraction with chloroform. The analytes were separated on a column packed with OmniSpher C18 material and eluted with acetonitrile–0.1 M ammonium acetate, pH 7.2 (70:30, v/v). The column effluent was monitored by UV detection at 330 nm. A linear response was achieved over the concentration range 0.50–100.00 μM using multilevel calibration with an internal standard. The analytical method inter- and intra-run accuracy and precision were better than ±15%. The lower limit of quantification was 0.50 μM. The method has been applied to study the preclinical pharmacokinetics of this compound in rats.
Keywords: 4-Anilinoquinazoline derivatives; Liquid–liquid extraction; High performance liquid chromatography;

Roscovitine, a purine analogue that selectively inhibits cyclin-dependent kinases, has been considered as a potential anti-tumor drug. The determination of roscovitine in plasma and urine was performed using microextraction in packed syringe as on-line sample preparation method with liquid chromatography and tandem mass spectrometry. The sampling sorbent utilized was polystyrene polymer. 2H3-lidocaine was used as internal standard. The limit of detection for roscovitine was as low as 0.5 ng/mL and the lower limit of quantification was 1.0 ng/mL. The accuracy and precision values of quality control samples were between ±15% and ≤11%, respectively. The calibration curve was obtained within the concentration range 0.5–2000 ng/mL in both plasma and urine. The regression correlation coefficients for plasma and urine samples were ≥0.999 for all runs. The present method is miniaturized and fully automated and can be used for pharmacokinetic and pharmacodynamic studies.
Keywords: Roscovitine; Purine analogues; MEPS; 2H3-lidocaine; LC–MS/MS;

Determination of urinary ortho- and meta-cresol in humans by headspace SPME gas chromatography/mass spectrometry by Silvia Fustinoni; Rosa Mercadante; Laura Campo; Licia Scibetta; Carla Valla; Vito Foà (309-317).
ortho-Cresol (o-C) and meta-cresol (m-C) are minor urinary metabolites of toluene, a widely used chemical with neurotoxicological properties. A new assay for their determination in human urine is here proposed. Urinary cresol sulphates and glucuronates are submitted to acid hydrolysis, urine is neutralized, added with o-cresols-d8, and analytes are sampled in the headspace of urine by SPME using a polydimethylsiloxane fiber. Analysis is performed by GC/MS using, for separation, either a SupelcoWax10 (for o-C) or a chiral CP Cresol (for o-C and m-C) column. The method is very specific, with a range of linearity 0–5.0 mg/l, within- and between-run precision, as coefficient of variation, <15% and <19%, limit of detection of 0.006 mg/l for o-C and 0.007 mg/l for m-C. The procedure is applied to the quantification of cresols in urine from workers exposed to toluene and from subjects belonging to the general population.
Keywords: Urinary cresols; Biological monitoring; Occupational exposure to toluene;

A simple, rapid and reliable method was developed for the quantification of oxymatrine (OMT) and its metabolite matrine (MT) in beagle dog plasma using a liquid–liquid extraction procedure followed by liquid chromatography–electrospray ionization mass spectrometric (LC–ESI-MS) analysis. Extend-C18 column (2.1 mm i.d. × 50 mm, 5 μm) with a C18 guard column (2.1 mm i.d. × 12.5 mm) was used as the analytical column. Linear detection responses were obtained for OMT concentration ranging from 5 to 4000 ng/ml and for MT concentration ranging from 5 to 2000 ng/ml. The precision and accuracy data, based on intra- and inter-day variations over 5 days, were lower than 5%. The limit of quantitation for OMT and MT were 2 and 1 ng/ml, respectively, and their recoveries were greater than 90%. Pharmacokinetic data of OMT and its active metabolite MT obtained with this method following a single oral dose of 300 mg OMT capsules to six beagle dogs was also reported for the first time.
Keywords: Oxymatrine; Matrine; Liquid chromatography–mass spectrometry; Dog plasma; Metabolite; Pharmacokinetics;

Simultaneous determination of mycophenolic acid (MPA) and mycophenolic acid glucuronide (MPAG) in plasma was accomplished by isocratic HPLC with UV detection. After protein precipitation and phase separation with saturated sodium dihydrogenphosphate, chromatographic separation was achieved on a monolithic column “Chromolith Performance RP-18e”, with acetonitrile/0.01 M phosphate buffer, pH 3, (25:75, v/v), as the mobile phase; flow rate 3.3 ml/min and measurement at 214 nm. Linearity was verified up to 40 mg/l for MPA and up to 400 mg/l for MPAG. Detection limits based on the analysis of 50 μl plasma were 0.05 and 0.5 mg/l for MPA and MPAG, respectively. Accuracy was 99.6–104% for MPA and 95.6–105% for MPAG and total imprecision (CV) was <7% for both compounds. Analytical recovery was >95% for MPA and MPAG. The method is simple, rapid, accurate and suitable for routine determination of MPA and MPAG in plasma.
Keywords: Mycophenolic acid; Mycophenolic acid glucuronide;

Simple determination of riluzole in rat brain by high-performance liquid chromatography and spectrophotometric detection by Adriana Maltese; Francesco Maugeri; Filippo Drago; Claudio Bucolo (331-334).
A simple method was developed for separation and quantification of riluzole in rat brain. The analyses were performed by high-performance liquid chromatography using a C18 reversed-phase column (Hypersil ODS) with UV detection at 264 nm. The mobile phase consisted of methanol–water containing 1% triethylamine adjusted with orthophosphoric acid to pH 3.2. The retention time was 8.6 min. A simple liquid–liquid extraction with ethyl acetate was used to obtain riluzole from brain samples. The limit of quantification was 10 ng/g. The recovery was about 80%. The relationship between peak areas and concentrations was linear over the range between 0.01 and 0.8 μg/g, with r 2 value over 0.99. The assay provided good reproducibility and accuracy and proved to be suitable for pharmacokinetic studies of riluzole.
Keywords: Riluzole; Spectrophotometric detection; Brain; Rat;

Determination of bromide in whole blood and urine from humans using gas chromatography–mass spectrometry by Shigetoshi Kage; Keiko Kudo; Hideaki Ikeda; Akira Tsujita; Noriaki Ikeda (335-339).
We devised a sensitive and simple method for determination of bromide in whole blood and urine from humans using gas chromatography–mass spectrometry. Bromide was alkylated with pentafluorobenzyl p-toluenesulphonate in the mixture of acetone and phosphate buffer (pH 6.8). The derivative obtained was analyzed using gas chromatography–mass spectrometry with the positive-ion EI mode. The lower limit of detection for the compound was 1 mg/l. The calibration curve for bromide was linear over the concentration range from 2 to 100 mg/l. With use of this method, levels of bromide in whole blood and urine were determined in cases of poisoning by inhaled brominated hydrocarbons.
Keywords: Bromide; Methyl bromide; Toxicology; Gas poisoning; Metabolite; Gas chromatography–mass spectrometry;

Author Index (341-343).

Keyword Index (345-351).