Journal of Chromatography B (v.839, #1-2)

Foreword by G.J. de Jong (1).

Direct analysis of bromide in human serum by capillary electrophoresis by Jennifer P. Pascali; Maristella Trettene; Federica Bortolotti; Giorgia de Paoli; Rossella Gottardo; Franco Tagliaro (2-5).
The purpose of the present work was the development and validation of a simple, rapid and reliable method for direct bromide quantification in serum based on capillary electrophoresis (CE). The analysis was carried out with an automated capillary electropherograph. Analytical conditions were as follows. Capillary: uncoated fused silica, effective length 50 cm, internal diameter 50 μm; voltage: 20 kV in reverse polarity mode; temperature: 25 °C; running buffer: 90 mmol/L sodium tetraborate decahydrate and 10 mmol/L NaCl, pH 9.24; detection: direct UV absorption at 200 nm; sample treatment: dilution of serum 1:10 with the internal standard solution (2 mmol/L thiocyanate). Under the described conditions, bromide ions and internal standard were baseline separated in 7 min. No interferences from other serum components were observed. The analytical sensitivity was characterized by a LOD: 0.05 mmol/L and a LOQ of 0.1 mmol/L. Excellent linearity was verified in the range from 2.5 to 60 mmol/L [y  = 0.0746x  − 0.0372; R 2  = 0.9995 (x  = bromide concentration; y  = bromide peak area/internal standard (I.S.) peak area)]. Quantitative imprecision in intra-day (n  = 7) and day-to-day (n  = 7) experiments was always within R.S.D. values <2%. Recovery was quantitative throughout the range of linearity of the method. Clinical cases of infants undergoing potassium bromide therapy for refractory epilepsy were analyzed with results in agreement with literature data. On the basis of these considerations, capillary electrophoresis can be proposed as the method of choice for bromide analysis in serum samples, especially for therapeutic drug monitoring purposes.
Keywords: Serum bromide analysis; Capillary electrophoresis; Potassium bromide therapy; Epilepsy;

High sensitivity analysis of oxprenolol in spiked human urine has been performed by capillary zone electrophoresis (CZE) in ammonium formate buffer pH 2.5 using an uncoated capillary with 1 cm length C18 on-capillary preconcentrator at the inlet side. The preconcentrator was fabricated in laboratory using the packing method and not encapped C18 5 μm particles as stationary phase material. The packed path was retained into the capillary by sintered stationary phase frits. Before running the CZE analysis, the oxprenolol was eluted from the preconcentrator by injecting a short plug of acetonitrile/water mixtures. With respect to classical CZE, the use of on-line preconcentrator widely increased the method sensitivity allowing the detection of the drug at 0.5 ng/mL (injected concentration). The method showed a linear response in the range of 1–150 ng/mL oxprenolol standard compound. The intra-day repeatability (n  = 11) R.S.D. values for migration time, peak area and normalized peak area were 0.72%, 3.96% and 3.66%, respectively, while inter-day repeatability (n  = 5 days) R.S.D. values were 2.74%, 9.41% and 9.83%, respectively. The method was successfully applied to the analysis of oxprenolol in extracted urine spiked at 250 pg/mL (oxprenolol LOQ concentration in urine).
Keywords: Capillary zone electrophoresis; On-line preconcentrator; Oxprenolol; Urine; High sensitivity;

A capillary electrophoretic (CE) method with contactless conductivity detection (CCD) has been developed for the determination of free amino acids (AAs) in the amniotic fluid. Apart from 20 proteinogenic AAs, 12 other biogenic compounds have been identified including ethanolamine, choline, β-alanine, 2-aminobutyric acid, 4-aminobutyric acid, creatinine, ornithine, carnitine, citrulline, 4-hydroxyproline, 1-methylhistidine and 3-methylhistidine. The running electrolyte consisted of 1.7 M acetic acid and 0.1% hydroxyethyl-cellulose (pH 2.15). An addition of acetonitrile to the sample improved the separation of AAs significantly and permitted an increase in the amount of the sample injected. As a result, the sensitivity of the determination increased and the limit of detection (LOD) decreased by a factor of ca. 4, as compared with our previous study. The LOD values were between 1.5 μM (arginine) and 6.7 μM (aspartic acid). The CE/CCD method has then been applied to clinical analyses of the amniotic fluid collected from 20 pregnant women aged over 35 years and 24 pregnant women with whom abnormal foetus development was suspected. The latter group of women was found to exhibit systematically enhanced amniotic levels of most of the AAs studied.
Keywords: Amino acid; Contactless conductivity detection; Acetonitrile; Amniotic fluid;

Three preparation batches of the recombinant birch pollen allergen Bet v 1a have been analyzed by capillary zone electrophoresis (CZE) using a separation electrolyte consisting of 100 mmol L−1 phosphate at pH 6.50 with 2.0 mmol L−1 tetraethylenepentamine (TEPA) added. TEPA improved the resolution by wall shielding and selective attachment to allergens, but reduced migration repeatability at concentrations >2.0 mmol L−1. Heterogeneity of preparations determined by CZE and electrospray ionization-quadrupole-time-of flight-MS were in accordance and revealed chemically modified (carbamylated) allergens in one of the preparations. The method was validated according to the ICH-guidelines. Repeatability of effective electrophoretic mobility (μ eff) was <0.55% R.S.D. (n  = 5). Migration time corrected peak areas were used for quantification. Limit of quantification (LOQ) was 25 μg mL−1 for the major isoform Bet v 1a, based on a signal-to-noise ratio of 10, and detector response was linear between LOQ and 0.90 mg mL−1. Purity of the different rBet v 1a preparations was determined to be between 40 and 92% depending on the manufacturing protocol.
Keywords: Recombinant allergens; Capillary zone electrophoresis; Dynamic coating with tetraethylenepentamine; ESI-Q-TOF-MS; Validation;

On-line coupling of size exclusion chromatography and capillary electrophoresis via solid-phase extraction and a Tee-split interface by F.W. Alexander Tempels; Gerard Wiese; Willy J.M. Underberg; Govert W. Somsen; Gerhardus J. de Jong (30-35).
An on-line size exclusion chromatography (SEC)–solid-phase extraction (SPE)–capillary electrophoresis (CE) system using a Tee-split interface has been developed for the analysis of peptides in biological fluids. The SEC column fractionates the sample by molecular size and the low-molecular-weight fraction, which contains the peptides, is directed to a C18 SPE microcolumn, where the peptides are trapped and concentrated. The SPE column is desorbed with 425 nL acetonitrile and the effluent is sent to the Tee-split interface, which hydrodynamically splits (1:40) the flow and, thus, allows appropriate injection of analytes into the CE system. The performance of the system is investigated by the analysis of enkephalins in cerebrospinal fluid (CSF). It is demonstrated that the SEC step efficiently removes potentially interfering proteins, permitting reproducible SPE and CE. The total system provides efficient separations of the enkephalins with plate numbers up to 100,000. Concentration limits of detection (S/N = 3) for the peptides are about 100 ng/mL for injection of 20 μL spiked CSF samples. Plots of enkephalin peak areas versus concentration showed good linearity over the 0.25–10 μg/mL range (R 2  ≥ 0.985). Repeatability of migration time and peak area was within 2% and 10% R.S.D., respectively.
Keywords: Size exclusion chromatography; Solid-phase extraction; Capillary electrophoresis; Tee-split interface; Enkephalin; Cerebrospinal fluid;

An HPLC method has been developed and validated for the determination of spironolactone, 7α-thiomethylspirolactone and canrenone in paediatric plasma samples. The method utilises 200 μl of plasma and sample preparation involves protein precipitation followed by Solid Phase Extraction (SPE). Determination of standard curves of peak height ratio (PHR) against concentration was performed by weighted least squares linear regression using a weighting factor of 1/concentration2. The developed method was found to be linear over concentration ranges of 30–1000 ng/ml for spironolactone and 25–1000 ng/ml for 7α-thiomethylspirolactone and canrenone. The lower limit of quantification for spironolactone, 7α-thiomethylspirolactone and canrenone were calculated as 28, 20 and 25 ng/ml, respectively. The method was shown to be applicable to the determination of spironolactone, 7α-thiomethylspirolactone and canrenone in paediatric plasma samples and also plasma from healthy human volunteers.
Keywords: Spironolactone; 7α-Thiomethylspirolactone; Canrenone; HPLC; Pharmacokinetics; Children;

A liquid chromatography–tandem mass spectrometric assay for the determination of uracil, 5,6-dihydrouracil and β-ureidopropionic acid in urine was developed to measure the activities of enzymes involved in pyrimidine breakdown. The assay was required to investigate the relation between the uracil–dihydrouracil ratio and toxicities observed after treatment with fluoropyrimidines drugs. After addition of stable isotopically labelled internal standards, the analytes were isolated from a 100-μl urine sample using liquid–liquid extraction with ethyl acetate–2-propanol. Compounds were separated on an Atlantis dC18 column, using ammonium acetate–formic acid in water as the eluent. The eluate was totally led into an electrospray interface with positive ionisation and the analytes were quantified using triple quadrupole mass spectrometry. The assay was validated in the range 1.6–1600 μM, using both, artificial urine and pooled urine as matrices. Intra-day precisions were ≤8% and inter-day precisions were ≤10%. Accuracies between 91 and 108% were found. The analytes were chemically stable under all relevant conditions and the assay was successfully applied in two clinical studies of cancer patients treated with 5-fluorouracil or capecitabine.
Keywords: Uracil; 5,6-Dihydrouracil; β-Ureidopropionic acid; Pyrimidines; Liquid–liquid extraction; LC–MSMS;

A new indirect RP-HPLC method was developed for determination of small, ng/ml, concentrations of triamcinolone (TMC) in human plasma, in presence of endogenous corticosteroids: cortisol (hydrocortisone, F), cortisone (E) and their metabolites, after administration of TMC in a free alcohol form. After solid phase extraction (SPE) in cartridges with octadecyl phase, TMC and prednisolone (I.S.) were derivatized by treatment with 9-anthroyl nitrile (9-AN) in a basic mixture, consisting of triethanolamine and quinuclidine, to receive fluorescent esters at 21-hydroxyl group of the steroid chain. Optimal conditions were also established to purify fluorescent TMC and I.S. derivatives before injection into HPLC column. The fluorescent esters were determined using an isocratic RP-HPLC technique in a C18 column. The mobile phase consisted of acetonitrile and 0.3 mM ortho-phoshoric acid. The method was validated before using to pharmacokinetic studies. Calibration curve of TMC was linear in the range of 2.5–100.0 ng/ml. Intra- and inter-day measurement precision and accuracy were equal to or lower than 15%. Percent recovery, and limits of detection (LOD) and quantification (LOQ) of TMC were also determined. The method was applied for in vivo conditions after administration of tablets containing TMC to healthy volunteers. Moreover, the method provided potential to determine TMC and, simultaneously, other glucocorticoids: E, F and their metabolites in one analytical run. Column interactions were observed between endogenous metabolites of E. Usefulness of the elaborated method was confirmed in pharmacokinetic studies following administration of a small (4 mg) dose of TMC to human volunteers. The method can provide an alternative to HPLC coupled with RIA in determination of small quantities of TMC.
Keywords: Triamcinolone free alcohol form; Solid phase extraction; Derivatization; Endogenous steroid metabolites; Interaction; Validation; Tablets;

An isocratic high-performance liquid chromatographic method with detection at 240 nm was developed, optimized and validated for the determination of ketoconazole in canine plasma. 9-Acetylanthracene was used as internal standard. A Hypersil BDS RP-C18 column (250 mm × 4.6 mm, 5 μm particle size), was equilibrated with a mobile phase composed of methanol, water and diethylamine 74:26:0.1 (v/v/v). Its flow rate was 1 ml/min. The elution time for ketoconazole and 9-acetylanthracene was approximately 9 and 8 min, respectively. Calibration curves of ketoconazole in plasma were linear in the concentration range of 0.015–10 μg/ml. Limits of detection and quantification in plasma were 5 and 15 ng/ml, respectively. Recovery was greater than 95%. Intra- and inter-day relative standard deviation for ketoconazole in plasma was less than 3.1 and 4.7%, respectively. This method was applied to the determination of ketoconazole plasma levels after administration of a commercially available tablet to dogs.
Keywords: Ketoconazole; RP-HPLC with UV detection; Canine plasma; Pharmacokinetic profile;

Selective protein removal and desalting using microchip CE by L.H.H. Silvertand; E. Machtejevas; R. Hendriks; K.K. Unger; W.P. van Bennekom; G.J. de Jong (68-73).
This paper describes the on-line sample pretreatment and analysis of proteins and peptides with a poly(methylmethacrylate) (PMMA) microfluidic device (IonChip™). This chip consists of two hyphenated electrophoresis channels with integrated conductivity detectors. The first channel can be used for sample preconcentration and sample clean-up, while in the second channel the selected compounds are separated. Isotachophoresis (ITP) combined with zone electrophoresis (CZE) was used to preconcentrate a myoglobin sample by a factor of about 65 before injection into the second dimension and to desalt a mixture of six proteins with 100 mM NaCl. However, ITP–CZE could not be used for the removal of two proteins from a protein/peptide sample since the protein zone in the ITP step was too small to remove certain compounds. Therefore, we used CZE–CZE for the removal of proteins from a protein/peptide mixture, thereby injecting only the peptides into the second CZE separation channel.
Keywords: 2D microfluidic device; Isotachophoresis-zone electrophoresis; Sample depletion; On chip sample pretreatment; Proteins and peptides;

A sensitive method for the determination of glutathione (GSH) and glutathione disulfide (GSSG) in human saliva was developed and validated. GSH was captured and stabilized by the addition of N-ethylmaleimide (NEM). Solid-phase extraction (SPE) using an Oasis MAX® extraction cartridge was employed for sample preparation and analysis was performed on a Shimadzu LCMS-2010 A that was operated in the single ion monitoring mode using positive ion electrospray ionization (ESI) as the interface. The monitored ion for GSH-NEM was m/z 433 and that for GSSG was m/z 613. Chromatography was carried out on an Atlantis HILIC silica column (150 mm × 2.1 mm, 5 μm) with acetonitrile and formate buffer as the mobile phase at the flow rate of 0.2 ml/min. The calibration curve was linear over the range of 0.1–100 μM for GSH-NEM. The extraction recoveries of GSH-NEM spiked at concentrations of 25 and 50 μM were 97.1 and 104.4%, respectively. Similar results were obtained for GSSG. The newly developed hydrophilic interaction chromatography with mass spectrometry (HILIC/MS) method showed superior sensitivity for the determination of GSH and GSSG in human saliva samples.
Keywords: Hydrophilic interaction chromatography; Saliva; Mass spectrometry; Glutathione; Glutathione disulfide;

Blackcurrant (Ribes nigrum) seed oil is rich in α- and γ-linolenic acids, the latter in particular being of potential use in medicine. The enzymatic hydrolysis of the oil was carried out in supercritical carbon dioxide using lipase Lipozyme as catalyst and changes in the composition of acylglycerols were recorded. Mono-, di-, and triacylglycerols and free fatty acids were separated by non-aqueous high-performance liquid chromatography in reversed phase mode and detected by UV diode array and 1H NMR detectors. Lipozyme was found to exert low specificity to individual fatty acids in the hydrolysed oil.
Keywords: RP-HPLC; NMR; Oil hydrolysis; Supercritical carbon dioxide; Enzyme;

Development of an HPLC method for the determination of nifedipine in human plasma by solid-phase extraction by Dragica Zendelovska; Suzana Simeska; Olgica Sibinovska; Elena Kostova; Kalina Miloševska; Krume Jakovski; Emilija Jovanovska; Igor Kikerkov; Jasmina Trojačanec; Dimče Zafirov (85-88).
Nifedipine, a dihydropyridine calcium channel antagonist, is widely used in the treatment of hypertension and other cardiovascular disorders. A selective, sensitive and accurate high-performance liquid chromatographic method has been developed, validated and applied for determination of nifedipine in human plasma samples. A series of studies were conducted in order to investigate the effects of mobile phase composition, buffer concentration, mobile phase pH and concentration of organic modifiers, and to develop a convenient and easy-to-use method for quantitative analysis of nifedipine. The method involves solid-phase extraction on C18 cartridges. The chromatographic separation was accomplished on a Lichrocart Lichrospher 60 RP selectB column with a mobile phase composed of 0.020 mol/L KH2PO4 (pH 4.8) and acetonitrile (42:58, v/v). UV detection was set at 240 nm. The calibration curve was linear in the concentration range of 5.0–200.0 ng/mL for nifedipine in plasma and the limit of quantification was 5.0 ng/mL.
Keywords: Nifedipine; Solid-phase extraction; HPLC;

Direct determination of verapamil in urine and serum samples by micellar liquid chromatography and fluorescence detection by M. Rambla-Alegre; M.T. Gil-Agustí; M.E. Capella-Peiró; S. Carda-Broch; J.S. Esteve-Romero (89-94).
Verapamil, a calcium channel antagonist, is one of the most commonly prescribed drugs in the treatment of hypertension. In this work, it was determined in serum and urine samples by a sensitive and precise chromatographic procedure without any pre-treatment step in a C18 column using a micellar mobile phase of 0.15 M sodium dodecyl sulfate and 5% pentanol at pH 7. Fluorescence detection set at 230 nm (excitation) and 312 nm (emission) was used. Verapamil is eluted at 12.5 min with no interference by the protein band or endogenous compounds. Linearities (r  > 0.998), as well as intra- and inter-day precision, were studied in the validation of the method. LODs were also calculated to be 11.0, 18.5 and 20.2 ng/mL in micellar solution, serum and urine, respectively. Recoveries in the biological matrices were in the 97–99% range. Drug excretion in urine was studied in a volunteer receiving treatment for hypertension, and verapamil, as an unchanged drug, was separated from other metabolites. The procedure developed can be useful in the field of toxicology and clinical analysis.
Keywords: Verapamil; Micellar mobile phase; Direct injection; Serum; Urine;

Capillary zone electrophoresis was optimized to quantitatively determine codeine and paracetamol via central composite factorial design. Critical parameters (concentration, buffer, pH, voltage) assessed effects on resolution, analysis time and efficiencies. Optimum separation conditions were achieved using phosphate buffer 20 mM (pH 6.8) and voltage (15 kV). The optimized procedure easily determined codeine and paracetamol with separation in less than 3 min. Calibration curves (R  > 0.999) were prepared, with LODs of 13.5 and 340 ng mL−1 for codeine and paracetamol, respectively, and a good R.S.D.% (<3%). This method was applied to determine codeine and paracetamol in pharmaceutical formulations; recoveries coincided with stated contents.
Keywords: Central composite design; Capillary zone electrophoresis; Codeine; Paracetamol; Pharmaceuticals;

Sensitive quantification of chosen drugs by reversed-phase chromatography with electrochemical detection at a glassy carbon electrode by Aleksandra Chmielewska; Lucyna Konieczna; Alina Plenis; Henryk Lamparczyk (102-111).
Reversed-phase-high-performance liquid chromatographic method with electrochemical detection has proven to be a highly sensitive and selective method for determination of trace components in complex biological samples, and the electrochemical detector becomes an important alternative tool to ultraviolet and fluorescence detectors. A rapid and sensitive method for the accurate determination of metoclopramide, hydrochlorothiazide, imipramine and diclofenac in serum or plasma samples is described. The method is based on liquid–liquid extraction. The compounds were separated on C-18 column as stationary phase with a different binary mixture as mobile phase. Proposed method was validated with respect to specificity, linearity range, limit of detection and quantitation, precision, accuracy and successfully applied in a pharmacokinetic studies.
Keywords: RP-HPLC/ECD; Metoclopramide; Hydrochlorothiazide; Imipramine; Diclofenac; Pharmacokinetics;

Capillary electrophoresis of peptides and proteins with plug of Pluronic gel by P. Sedlakova; J. Svobodova; I. Miksik (112-117).
Electromigration capillary methods are promising techniques in proteomics and they are still under research. We used a partial filling approach, i.e. a combination of gel and non-gel separation mechanisms in a single dimension. We tried using an interesting gel, Pluronic F 127, which can be considered as a surfactant capable of self-association both with isotropic and anisotropic gels. The Pluronic was inserted inside the capillary as a plug at the start of the capillary, and it provided separation at the first time. Separation by this gel was achieved according to molecular weight and/or hydrophobicity. The applicability of this method was demonstrated in the separation of real samples—peptides arising from collagen after CNBr or collagenase cleavage and albumin after trypsin cleavage (peptide mapping). Some peptides and proteins were selectively retained by the Pluronic gel. These interactions with the gel did not depended on their molecular weight alone, but they probably depend on a combination of both principles. It was confirmed that capillary electrophoresis with Pluronic plug can give us another new separation option, complementary to free solution capillary electrophoresis. The CE method presented here, consisting of a partial filling approach with combine gel and non-gel separation mechanisms seemed to be a promising method for the separation of complex mixtures of peptides.
Keywords: Pluronic; Partial filling; Proteins; Peptide;

Pharmacokinetic study of orphenadrine using high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) by Soo-Youn Lee; Hyeon Ju Oh; Jong Won Kim; Yoon Gyoon Kim; Chul Jin Moon; Eun Hee Lee (118-123).
We developed and validated a simple, rapid, and accurate HPLC–MS/MS method with simple protein precipitation for the determination of orphenadrine. Injection-to-injection running time was 3 min with a retention time of orphenadrine of 1.1 min. The linear assay range was 1–200 ng/mL (r 2  > 0.99). The intra- and inter-assay imprecisions were CV 0.6–4.2% and CV 1.6–6.1%, respectively. The accuracy, extraction recovery, specificity and stability were satisfactory. Using the measured plasma concentrations of orphenadrine in 24 healthy subjects, pharmacokinetic profiles of orphenadrine were evaluated (AUC0–72, 1565 ± 731 ng h/mL, C max 82.8 ± 26.2 ng/mL, T max 3.0 ± 0.9 h, elimination half-life 25.8 ± 10.3 h).
Keywords: Orphenadrine; HPLC–MS/MS; Pharmacokinetic;

We have developed a simple, rapid, and accurate HPLC-MS/MS method for the determination of iohexol in serum. The column used was a Zorbax Eclipse XDB-C8 (100 mm × 2.1 mm i.d., 3.5 μm). Mobile phases consisted of water containing 2 mM ammonium acetate and 0.1% formic acid (A) and methanol containing 2 mM ammonium acetate and 0.1% formic acid (B). After simple protein precipitation with ZnSO4, serum samples were mixed with I.S. (bromperidol) and centrifuged for 3 min. The obtained extraction recovery at three levels was 94.6–107.4%. Quantitative analysis was performed in the multiple reaction-monitoring mode (m/z 822.0 → 804.0 for iohexol, 420.1 → 122.7 for I.S.) with the total running time of 3 min for each sample. The assay was linear between 0.5 and 1500 μg/mL (r 2  > 0.997). The intra- and inter-assay coefficient of variations were 2.4 – 6.2% and 5.5 – 6.5%, respectively. Our method provided sufficient analytical range and specificity for the 210 clinical samples analyzed.
Keywords: Iohexol clearance; HPLC-MS/MS;