Journal of Chromatography B (v.814, #1)

News Section (N1-N4).

l-Tyrosine and l-tyrosine residues in proteins are attacked by various reactive-nitrogen species (RNS) including peroxynitrite to form 3-nitrotyrosine (NO2Tyr) and protein-associated 3-nitrotyrosine (NO2TyrProt). Circulating NO2Tyr and NO2TyrProt have been suggested and are widely used as biomarkers of oxidative stress in humans. In this article the mass spectrometry (MS)-based analytical methods recently reported for the quantification of circulating levels of NO2Tyr and NO2TyrProt are discussed. These methodologies differ in sensitivity, selectivity, specificity and accessibility to interferences with the latter mainly arising from artifactual formation of NO2Tyr and NO2TyrProt during sample treatment such as acidification and chemical derivatization. Application of these methodologies to healthy normal humans revealed basal circulating levels for NO2Tyr which range between 0.7 and 64 nM, i.e. by two orders of magnitude. Application of gas chromatography–tandem mass spectrometry (GC–tandem MS) methods by two independent research groups by using two different protocols to avoid artifactual nitration of l-tyrosine revealed almost identical mean plasma levels of the order of 1.0 nM in healthy humans. The lower limits of quantitation (LOQ) of these methods were 0.125 and 0.3 nM, respectively. This order of magnitude for basal NO2Tyr is supported by two liquid chromatography–tandem mass spectrometry (LC–tandem MS) methods with LOQ values of 4.4 and 1.4 nM. On the basis of the data provided by GC–tandem MS and LC–tandem MS the use of a range of 0.5–3 nM for NO2Tyr and of 0.6 pmol/mg plasma protein or a molar ratio of 3-nitrotyrosine to tyrosine in plasma proteins of the order of 1:106 for NO2TyrProt in plasma of healthy humans as reference values appear reasonably justified. Recently reported clinical studies involving 3-nitrotyrosine as a biomarker of oxidative stress are discussed in particular from the analytical point of view.
Keywords: Reviews; Biomarker; Oxidative stress;

Proteomic approaches in the search for disease biomarkers by A. Vlahou; M. Fountoulakis (11-19).
Significant technological advances in protein chemistry, physics and computer sciences in the last two decades have greatly improved protein separation methodologies, such as electrophoresis and chromatography, and have established mass spectrometry (MS) as an indispensable tool for protein study. The goal of this review is to provide a brief overview of the recent improvements in these methodologies and present examples from their application in proteome analysis and search for disease biomarkers.
Keywords: Mass spectrometry; Proteomics; Electrophoresis; Liquid chromatography; Biomarkers;

A reversed phase HPLC method was developed for the simultaneous analysis of different phospholipids and lysophospholipids in human bronchoalveolar lavage fluid (BALF). Separation was achieved using a pellicular C8 column at elevated temperatures with an increasing gradient of acetonitrile containing 0.1% formic acid. Detection was carried out by electrospray ionization ion-trap mass spectrometry. Calibration graphs for selected phospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and lysophosphatidylcholine) showed linearity up to 50 ng allowing quantitative determinations. Identification of the individual species within each class was possible with tandem mass spectrometry. Analysis of BALF phospholipids was performed after liquid/liquid extraction with a mixture of chloroform/methanol/acetic acid. Recoveries ranged from 69 to 97% with standard deviations of less than 6%. The limit of detection varied slightly between different classes but was in the range 0.05–0.25 ng total injected amount.
Keywords: Phospholipids; Lysophospholipids; BALF; HPLC/MS;

Turbulent flow chromatography (TFC) is a technique for the direct and efficient analysis of drugs and metabolites in biological matrices. We report here TFC on-line with an HPLC–MS/MS assay for the determination of 5-[2,4-dioxothiazolidin-5-yl)methyl]-2-methoxy-N-[[(4-trifluoromethyl)phenyl]methyl]benzamide (I, MK-0767, KRP297, Fig. 1) in plasma. Samples were transferred using an automated system followed by the addition of internal standard (II), prepared in 0.1 M ammonium acetate (pH 4.0). The plasma samples were directly injected onto a C18 turbulent flow column on-line with an HPLC–MS/MS system, and the analytical column used was a ThermoHypersil Keystone C18. Detection was achieved by MS/MS, using positive ionization on a TurboIonSpray® probe, operated in multiple reaction monitoring (MRM) mode. The linear range was 4–2000 ng/mL for I when using 50 μL of plasma. The method exhibited good linearity and reproducibility. The method also showed good selectivity and ruggedness when applied to clinical samples, and was successfully cross-validated with a conventional off-line SPE, LC–MS/MS method.
Keywords: Turbulent flow chromatography; Sample preparation; Validation; MK-0767; KRP 297;

In this study, a high-performance liquid chromatographic method with pre-column derivatization and fluorescence detection was optimised and validated for the quantification of azithromycin (AZM) in plasma. Clarithromycin (CLM) was used as an internal standard. Pre-column derivatization was done with 9-fluorenylmethyloxycarbonyl-chloride. Recovery from blood and polymorphonuclear neutrophils (PMNNs) isolated by a gravity separation procedure was also assessed. Analytical separation was carried out using a C18 column as stationary phase and acetonitril–phosphatebuffer as mobile phase. Peak quantification was carried out by excitation at 267 nm and detection at 317 nm. A lower limit of quantitation of 0.042 ± 0.017 mg/l in plasma, 0.119 ± 0.065 mg/l in blood and 0.072 ± 0.036 in water was achieved. Linearity was assessed from 0 to 1.5 mg/l in plasma and blood and from 0–9 mg/l in water. The analytical method proved to be applicable in a pharmacokinetic study of AZM in a Cystic Fibrosis patient.
Keywords: Azithromycin; Derivatization; LC;

Urinary electrophoretic profiles from chronic fatigue syndrome and chronic fatigue syndrome/fibromyalgia patients: a pilot study for achieving their normalization by Begoña Casado; Chiara Zanone; Laura Annovazzi; Paolo Iadarola; Gail Whalen; James N. Baraniuk (43-51).
Aim of our study was to determine if there were distinct, disease-related patterns of urinary analytes in chronic fatigue syndrome (CFS) and chronic fatigue syndrome/fibromyalgia (CFS/FM) compared to normal controls (NC). Urine was collected from these subjects for two consecutive 24 h periods and aliquots were submitted to micellar electrokinetic chromatography (MEKC). To compensate for the differences in peak migration times, these were normalized from the 35 min duration of run to a 100-point scale, and each peak was assigned its normalized time measure. Peak heights were also normalized by dividing the mAU by that of the internal standard (creatinine) and multiplying by 100. MEKC with normalization for peak height and migration time generated comparable results within each of the patient groups. CFS/FM and CFS had significant differences in peaks compared to NC that may be of significance as biomarkers of illnesses.
Keywords: Chronic fatigue syndrome; Fibromyalgia;

We found a new variant of human growth hormone (hGH) from the recombinant hGH expression process in Saccharomyces cerevisiae. The variant was identified as N α-acetyl methionyl hGH which may be formed by N α-acetylation of met-hGH during the intracellular expression of hGH in S. cerevisiae. The variant was isolated from manufacturing process of LG Life Sciences’ hGH product. The variant was subjected to trypsin digestion and RP-HPLC analysis, resulting in a delayed retention time and an increased mass (173 Da) of T1 tryptic peptide. The amino acid composition and amino acid sequence of the peptide showed the same result with T1 peptide of met-hGH except the N-terminal modification on methionine in the variant peptide. With collision induced dissociation (CID) experiments of the variant T1 tryptic peptide, we found the sequence and the a1 fragment of N-terminal residue matched with those of acetyl-methionyl hGH. Within our production process, we produce the methionyl hGH first and then use the aminopeptidase to cut the N-terminal methionine. So the acetylation may inhibit the aminopeptidase to remove methionine and produces N α-acetyl methionyl hGH. And the biological activity of the variant was comparable to one of the unmodified hGH when tested by rat weight gain bioassay.
Keywords: Human growth hormone; Modification; Variant; Mass spectrometry; Aminopeptidase;

Simplified ultraviolet liquid chromatographic method for determination of sertindole, dehydrosertindole and norsertindole, in human plasma by Mireille Canal-Raffin; Evelyne Déridet; Karine Titier; Eric Frakra; Mathieu Molimard; Nicholas Moore (61-67).
An ultra-violet high-performance liquid chromatographic method was developed for the determination of sertindole, an atypical antipsychotic drug and its main metabolites dehydrosertindole and norsertindole, in human plasma. With a small sample volume, after a single-step liquid–liquid extraction, the compounds were separated on a reversed-phase XTerra® RP18 column, eluted with 45% of acetonitrile and 55% of ammonium acetate buffer (0.05 M, adjusted pH 8) and detected at 256 nm within 11 min. This method shows a good linearity for plasma concentration between 5–100 ng/ml and 100–1000 ng/ml, a good precision (inter and intra day CV < 11%) and a good inter-assay accuracy (bias < 11%). The limit of quantification concentration was 5 ng/ml. The absolute recovery of sertindole was higher than 99%. This rapid and sensitive method could be used for therapeutic drug monitoring as well as for overdose management.
Keywords: Sertindole; Human plasma; Liquid–liquid extraction; Drug monitoring; Overdose;

Quantitative determination of amygdalin epimers from armeniacae semen by liquid chromatography by Ja-Yong Koo; Eun-Young Hwang; Sonhae Cho; Je-Hyun Lee; Yong-Moon Lee; Seon-Pyo Hong (69-73).
d-Amygdalin and its conversion product, neoamygdalin, were quantitatively analyzed on reverse-phase, high-performance liquid chromatography with an optimized eluent of 10 mM sodium phosphate buffer (pH 3.1) containing 8.5% acetonitrile. Linearity between concentrations and detector responses was obtained in the range from 0.05 to 0.5 mM. The detection limits for d-amygdalin and neoamygdalin were approximately 5 μM per injected amount. Armeniacae semen contains not only amygdalin but also emlusin, which is an enzyme that hydrolyzes amygdalin. When extracting amygdalin from a whole piece of armeniacae semen in boiling water, there was almost no influence of emulsin; which increased the extraction efficiency. However, conversion of d-amygdalin into neoamygdalin at high temperature was found. In this report, we solved this problem by using 4% citric acid as an extractant. This solution also prevented the extraction process from being affected by emulsin. In addition, the extraction efficiency remained the same as that when methanol was used as an extractant, regardless of the cutting size.
Keywords: d-Amygdalin; Neoamygdalin; Epimer separation; Armeniacae semen; Citric acid; Epimerization inhibition;

Analysis of benidipine enantiomers in human plasma by liquid chromatography–mass spectrometry using a macrocyclic antibiotic (Vancomycin) chiral stationary phase column by Wonku Kang; Dong-Jun Lee; Kwang-Hyeon Liu; Yu Eun Sunwoo; Kwang-il Kwon; In-June Cha; Jae-Gook Shin (75-81).
We used a novel chromatographic method to rapidly and simply characterize the pharmacokinetics of benidipine enantiomers in human plasma. The stereoisomers of benidipine were extracted from plasma using diethylether under alkaline conditions. After evaporating the organic layer, the residue was reconstituted in the mobile phase (methanol:acetic acid:triethylamine, 100:0.01:0.0001, v/v/v). The enantiomers in the extract were separated on a macrocyclic antibiotic (Vancomycin) chiral stationary phase column. The mobile phase was eluted at 1 ml/min and was split by an interface. One-fifth of the eluent was used to quantify both isomers in a tandem mass spectrometer in multiple reaction-monitoring mode. The coefficient of variation of the precision of the assay was less than 8%, the assay accuracy was between 93.4 and 113.3%, and the limit of detection was 0.05 ng/ml for 1 ml of plasma. The method described above was used to measure the concentration of both benidipine enantiomers in plasma from healthy subjects who received a single oral dose of a racemate of 8 mg benidipine. The C max and AUCinf values of (+)-alpha benidipine were higher than those of (−)-alpha benidipine by 1.96- and 1.85-fold, respectively (p  < 0.001), whereas, the T max and t 1/2 for each of the benidipine stereoisomers were not significantly different.
Keywords: Benidipine; Enantiomers; Chiral stationary phase; Tandem mass spectrometry;

The distribution of silver, arsenic, cadmium, cobalt, chromium, copper, iron, manganese, nickel, lead, selenium and zinc binding to species with different molecular weight in aqueous extract of krill was studied by on-line size-exclusion chromatography (SEC)/inductively coupled plasma mass spectrometry (ICP-MS). The extract was fractionated in three fractions with different molecular weight (MW) ranges (>20,000 relative molecular mass (rel. mol. mass), 2000–20,000 rel. mol. mass and <2000 rel. mol. mass), which were further analyzed by SEC with columns having different optimum fractionation ranges in order to obtain more detailed information about the MW distribution of the elements. Various distribution profiles for the target elements among different MW ranges were observed. The results obtained indicated that manganese, zinc, silver, cadmium and lead species were mostly distributed in the higher MW range (>20,000 rel. mol. mass). In the case of chromium, iron, cobalt, arsenic and selenium, most of them bind to species with lower MW (<2000 rel. mol. mass). Only copper and nickel species was predominantly present in middle MW range (2000–20,000 rel. mol. mass). Further speciation of arsenic compounds in the small MW fraction was carried out with anion exchange chromatography (AEC) coupled with ICP-MS. The results showed that the dominant arsenic species in this fraction is As(III) (63% of extractable arsenic), while As(V) (13%) and two unknown arsenic species (19% and 5%, respectively) are present in lower amounts.
Keywords: Fractionation; Elements; Antarctic krill; Size-exclusion chromatography; Inductively coupled plasma-mass spectrometry;

Determination of amphetamine-derived designer drugs in human urine by SPE extraction and capillary electrophoresis with mass spectrometry detection by Gianpiero Boatto; Maria Nieddu; Antonio Carta; Amedeo Pau; Michele Palomba; Battistina Asproni; Riccardo Cerri (93-98).
In recent years, a number of newer designer drugs have entered the illicit drug market. The methylenedioxy-derivates of amphetamine represent the largest group of designer drugs. This paper describes a method for screening for and quantification of ten 2,5-methylenedioxy-derivates of amphetamine and phenylethylamine in human urine, using capillary electrophoresis coupled to electrospray ionisation–mass spectrometry (CE–ESI–MS). Prior to CE–MS analysis, a simple solid-phase extraction (SPE) was used for sample cleanup. The method was validates according to international guidelines.
Keywords: Methylenedioxy-derivates; Amphetamine; Phenylethylamine; Designer drug; Urine; Capillary electrophoresis; Mass spectrometry; Solid-phase extraction (SPE);

The study on the interactions between two anti-human immunodeficiency virus type 1 (anti-HIV-1) active compounds with trans-activation response (TAR) RNA by affinity capillary electrophoresis (ACE) with UV absorbance detection is presented. The results showed that the novel active molecules could interact with TAR RNA and inhibit the reproduce process of HIV-1. The binding constants were estimated by the change of migration time of the analytes through the change of concentrations of TAR RNA in the buffer solution. The yielded binding constants of 8.87 × 103  M−1 for active compound C3 and 8.42 × 103  M−1 for MC3 at 20.0 °C, 0.626 × 103  M−1 and 0.644 × 103  M−1 at 37.0 °C, respectively. The thermodynamic parameters ΔH and ΔS were obtained and shown that both hydrophobic and electrostatic interaction played roles in the binding processes. The results showed that the presented method was an easy and simple method to evaluate the interaction of small molecules with some bioactive materials.
Keywords: Binding constants; Capillary electrophoresis; Thermodynamic parameters; TAR RNA;

A high-throughput bioanalytical method based on automated sample transfer, automated solid phase extraction, and hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC–MS/MS) analysis, has been developed for the determination of cetirizine, a selective H1-receptor antagonist. Deuterated cetirizine (cetirizine-d8) was synthesized as described and was used as the internal standard. Samples were transferred into 96-well plates using an automated sample handling system. Automated solid phase extraction was carried out using a 96-channel programmable liquid-handling workstation. Solid phase extraction 96-well plate on polymer sorbent (Strata X) was used to extract the analyte. The extracted samples were injected onto a Betasil silica column (50 × 3, 5 μm) using a mobile phase of acetonitrile–water–acetic acid–trifluroacetic acid (93:7:1:0.025, v/v/v/v) at a flow rate of 0.5 ml/min. The chromatographic run time is 2.0 min per injection, with retention time of cetirizine and cetirizine-d8 both at 1.1 min. The system consisted of a Shimadzu HPLC system and a PE Sciex API 3000 or API 4000 tandem mass spectrometer with (+) ESI. The method has been validated over the concentration range of 1.00–1000 ng/ml cetirizine in human plasma, based on a 0.10-ml sample size. The inter-day precision and accuracy of the quality control (QC) samples demonstrated <3.0% relative standard deviation (R.S.D.) and <6.0% relative error (RE). Stability of cetirizine in stock solution, in plasma, and in reconstitution solution was established. The absolute extraction recovery was 85.8%, 84.5%, and 88.0% at 3, 40, and 800 ng/ml, respectively. The recovery for the internal standard was 84.1%. No adverse matrix effects were noticed for this assay. The automation of the sample preparation steps not only increased the analysis throughput, but also increased method ruggedness. The use of a stable isotope-labeled internal standard further improved the method ruggedness. Practical issues of analyzing incurred samples were discussed. This HILIC–MS/MS method for analysis of citirizine in human plasma was successfully used to support clinical studies.
Keywords: Cetirizine; Synthesis of cetirizine-d8; HILIC–MS/MS; Method validation;

Chromatographic evaluation of the toxicity in fish of pesticides by José María Bermúdez-Saldaña; Laura Escuder-Gilabert; María José Medina-Hernández; Rosa María Villanueva-Camañas; Salvador Sagrado (115-125).
Ecotoxicity assessment is essential before placing new chemical substances on the market. An investigation of the use of the chromatographic retention (log  k) in biopartitioning micellar chromatography (BMC) as an in vitro approach to evaluate the toxicity in fish of pesticides (acute toxicity levels as pLC50) is proposed. A heterogeneous data set of 85 pesticides from six chemical families with available experimental fish toxicity data (ECOTOX database from U.S. Environmental Protection Agency (EPA)) was used. For pesticides exhibiting non-polar narcosis mechanism in fish (non-specific toxicity), more reliable models and precise pLC50 estimations are obtained from log  k (quantitative retention–activity relationships, QRAR) than from log  P (quantitative structure-activity relationships, QSAR) or ECOSAR (ECOSAR program from U.S. EPA).
Keywords: Fish toxicity; Non-polar narcosis mechanism of action; Pesticides; Biopartitioning micellar chromatography;

A sensitive and specific method was developed for quantification of alprazolam and its two metabolites 4-hydroxyalprazolam and α-hydroxyalprazolam in plasma. The work up procedure was solid phase extraction. Liquid chromatography–mass spectrometry (LC–MS) was used for separation, detection and quantification of the analytes. The limit of quantitation (LOQ) was 0.05 ng/mL for alprazolam and the two metabolites. The extraction recovery was more than 82% for alprazolam and its metabolites. The within- and between-assay coefficients of variation were in the range of 1.9–17.9%. The method was used for determination of the pharmacokinetics parameters of alprazolam and its two metabolites in healthy Caucasian subjects who ingested 1 mg of alprazolam.
Keywords: Liquid chromatography–mass spectrometry; Alprazolam; 4-Hydroxyalprazolam; α-Hydroxyalprazolam; Pharmacokinetics;

The clinical development of a sensitizer for photodynamic therapy (PDT) requires the structural identification of the photoproducts and their quantification in biological fluids and tissues. We describe the LC–MS identification of the most important photoproducts of a cationic phthalocyanine sensitizer (RLP068/Cl) and a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the determination of the main photoproduct (the cationic phthalimide derivative 3-[(1,3-dioxo-2,3-dihydro-1H-isoindol-4-yl)oxy]-N,N,N-trimethylbenzenaminium chloride) in rabbit plasma. The tri-deuterated product was used as co-eluting internal standard. The cationic photoproduct was isolated from plasma samples by protein precipitation with perchloric acid in methanol (7%, v/v). HPLC step was performed on a Phenomenex Synergi Hydro-RP column (20 mm × 2.0 mm, 2 μm particles) with a mobile phase of 0.5% (v/v) aqueous TFA/methanol (85:15, v/v). Flow rate was 0.2 mL/min and 40 μL injection were performed. Run time was 10 min. Detection was achieved by means of a Bruker Esquire 3000+ ion trap mass spectrometer equipped with an ESI source working in positive mode. A multiple reaction monitoring method following the transitions 297.1 → 282.1 for the analyte and 300.1 → 282.1 + 285.1 for the internal standard was used. The analytical method was validated over the concentration range 0.46–91.2 ng/mL and lower limits of detection (LLOD) and quantification (LLOQ) respectively of 0.2 and 0.5 ng/mL were found.
Keywords: Phthalocyanine; Photodynamic therapy (PDT); Photoproduct; Ion trap mass spectrometer;

Simultaneous determination of OSI-774 and its major metabolite OSI-420 in human plasma by using HPLC with UV detection by Wenjiang Zhang; Lillian L. Siu; Malcolm J. Moore; Eric X. Chen (143-147).
A new method was developed and validated for quantitating OSI-774 and its metabolite OSI-420 in human plasma. Sample preparation involved initial extraction with methyl t-butyl ether followed by back extraction with HCl and re-extraction with methyl t-butyl ether. This extraction process resulted in significant improvement in the specificity, reproducibility and sensitivity. The analytes were separated on a Water Symmetry C18 analytical column and the mobile phase consisted of acetonitrile–0.05 M potassium phosphate buffer (42:58, v/v) (pH 4.8), and monitored at a wavelength 345 nm. Values of between- and within-day precision and accuracy for both OSI-774 and OSI-420 were <20%. This method was successfully applied to study steady-state pharmacokinetics of OSI-774 and OSI-420 in a phase II clinical trial.
Keywords: OSI-774; OSI-420; HPLC;

Measurement of serum pralidoxime methylsulfate (Contrathion®) by high-performance liquid chromatography with electrochemical detection by Pascal Houzé; Stephen W. Borron; François Scherninski; Bernard Bousquet; Bernard Gourmel; Frédéric Baud (149-154).
Pralidoxime methylsulfate (Contrathion®) is widely used to treat organophosphate poisoning. Despite animal and human studies, the usefulness of Contrathion® therapy remains a matter of debate. Therapeutic dosage regimens need to be clarified and availability of a reliable method for plasma pralidoxime quantification would be helpful in this process. We here describe a high-performance liquid chromatography technique with electrochemical detection to measure pralidoxime concentrations in human serum using guanosine as an internal standard. The assay was linear between 0.25 and 50 μg mL−1 with a quantification limit of 0.2 μg mL−1. The analytical precision was satisfactory, with variation coefficients lower 10%. This assay was applied to the analysis of a serum from an organophosphorate poisoned patient and treated by Contrathion® infusions (100 and 200 mg h−1) after a loading dose (400 mg).
Keywords: Pralidoxime methylsulfate; Electrochemical detection; Human serum;

Reactive α,β,γ,δ-unsaturated aldehydes and oxo-acids produced by marine diatoms upon cell damage interfere negatively with the reproduction success of their grazers. A simple, sensitive and specific method based on gas-chromatography coupled to mass spectrometry (EI or CI/EC) was developed for the quantification of these deleterious substances in laboratory diatom cultures and in natural phytoplankton populations. For aldehyde quantification, diatom containing samples are damaged in the presence of O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine hydrochloride (PFBHA·HCl) which leads to an in situ derivatisation without inhibition of the biosynthesis of the aldehydes. The oxime derivates of oxo-acids were in addition reacted with N-tert-butyldimethylsilyl-N-methyl-trifluoracetamide (MTBSTFA).
Keywords: Decadienal; Oxo-acids; Marine plankton; Chemical defence; GC/MS;

Identification, separation and quantitation of iodoaminoacids, is essential for the biological research and the clinical diagnosis of thyroid gland disease. Under this aspect a reversed-phase high-performance liquid chromatographic method was developed for the determination of thyroid gland hormones and some of their primary metabolites, 3,3′,5,5′-tetra-iodo-l-thyronine (l-thyroxine), 3,3′,5-tri-iodo-l-thyronine, 3,5-di-iodo-l-thyronine, l-thyronine, 3,5-di-iodo-l-tyrosine, 3-iodo-l-tyrosine and l-tyrosine. Analysis was performed on an Inertsil C18 column with photodiode-array detection, using a 25 min gradient scale program of a binary mobile phase consisted of 0.1% aqueous solution of trifluoroacetic acid at pH 3 as solvent A and acetonitrile as solvent B, at a flow rate of 1 mL/min. Quantitation was performed using were obtained using theophylline as internal standard. The method was applied to commercial pharmaceuticals and biological samples (serum, urine and tissue). Drug-free urine and serum samples were spiked with known concentrations of the analytes standards and pretreated by solid phase extraction to remove matrix interferences. C18 cartridges were used, yielding recoveries ranging from 87.1% to 107.6% for serum samples and from 92.1% to 98.7% for urine samples. With regard to total-T4 concentrations in serum samples, results are cross-validated with RIA and found to agree well.
Keywords: Thyroid gland hormones; Iodotyrosines; Iodothyronines; Pharmaceuticals; HPLC; SPE biological samples; Serum; Urine; Tissue;

Optimization and validation of an ion chromatographic method for the simultaneous determination of sodium, ammonium and potassium in exhaled breath condensate by Sophie Svensson; Ann-Charlotte Isacsson; Göran Ljungkvist; Kjell Torén; Anna-Carin Olin (173-177).
An ion chromatographic method with conductivity detection for the simultaneous quantification of sodium, ammonium and potassium in exhaled breath condensate (EBC) was developed and validated. A factorial design was used to optimize the chromatographic conditions, which resulted in baseline separations of the cations within 6 min. The method requires no pre-treatment of EBC samples. The optimized method was used for the intra-individual screening of cations in EBC of 10 healthy volunteers. The LOQs were low (0.3, 0.1 and 0.2 μM for sodium, ammonium and potassium, respectively), compared with levels detected in healthy volunteers. The responses were linear with good precision, and samples could be stored for at least 10 weeks at refrigerating conditions.
Keywords: Ion chromatography; Exhaled breath condensate; Sodium; Ammonium; Potassium; Chemometrics;

A cost-effective HPLC method for determination of pyrimethamine (PYR) in human whole blood samples dried on filter paper (Whatman®) is reported. Trimethoprim (TMP) was used as an internal standard. Whole blood spiked with PYR was transferred (100 μl) onto filter paper and dried at room temperature. Capillary blood samples (100 μl) after ingestion of three tablets of sulfadoxine-pyrimethamine (SP) by one subject were also tested. PYR and an internal standard (IS) TMP were extracted into di-isopropyl ether as bases and then re-extracted with 150 μl mobile phase. A C-18 column was used and the mobile phase consisted of phosphate buffer (0.05 M, pH 5):acetonitrile:concentrated perchloric acid (750:300:2.5, v/v/v).The absorbances of PYR and IS were monitored at 270 nm. The limit of quantification was 40 ng/ml. The within- and between-assay coefficient of variations were <10% at the limit of quantification.
Keywords: Pyrimethamine; Sulfadoxine; Trimethoprim; Internal standard; Filter paper method;

A test procedure was developed for the detection and quantification of 1- and 2-bromopropane in human urine. 1-Bromopropane (1-BP) is a commonly used industrial solvent, and 2-bromopropane (2-BP) is often found as an impurity component in industrial grade 1-BP. Both compounds are a health concern for exposed workers due to their chronic toxicity. Bromopropanes have been associated with neurological disorders in both animals and humans. Sample preparation consisted of diluting urine with water and fortification with 1-bromobutane (1-BB), which was used as an internal standard; then each sample was sealed in a headspace vial. A static-headspace sampler (Teledyne-Tekmar Model 7000) was used to heat each sample at 75 °C for a 35-min equilibrium time. Quantification was by means of a gas chromatograph (GC) equipped with an electron capture detector (ECD) and a dimethylpolysiloxane (DB-1) capillary column. A recovery study using fortified urine samples at multiple concentrations (0.5–8 μg/ml) demonstrated full recovery; 104–121% recovery was obtained. Precision ranged from 5 to 17% for the 15–20 spiked samples used at each concentration, which were analyzed over multiple experimental trial days. The limit of detection (LOD) for this test procedure was approximately 2 ng/ml 1-BP and 7 ng/ml 2-BP in urine. A recovery study of 1- and 2-BP from fortified urine stored in vials appropriate for field collection was also completed. These results and other factors of the development and validation of this test procedure will be discussed.
Keywords: 1-Bromopropane; 2-Bromopropane;