Journal of Chromatography B (v.809, #1)

News Section (N1).

A new on-line, rapid and sensitive column-switch LC/MS/MS method to measure nelfinavir (NFV), an HIV-1 protease inhibitor, and its major metabolite (M1) in rat plasma was developed. Rat plasma containing the analytes and the internal standard was treated with acetonitrile and the supernatant was processed through an on-line extraction and an analytical columns, with a column-switch device. ESI-LC/MS with multiple reaction monitors for appropriate analytes was performed. This assay gave a limit of quantitation (LOQ) of <1 ng/mL for the analytes with 5 min run time. The within-run and between-run precisions were <12 and <10%, respectively. This analytical method was successfully applied to a study to correlate changes in maternal and placental NFV plasma concentrations in rats following NFV exposure in utero.
Keywords: Nelfinavir;

Determination of metoprolol, and its four metabolites in dog plasma by Jim Fang; Hugh A Semple; Jiuxue Song (9-14).
Metoprolol and its metabolites α-hydroxymetoprolol, O-desmethylmetoprolol, metoprolol acid and deaminated metoprolol were quantified in dog plasma using an isocratic high-performance liquid chromatographic method (with a BetaBasic Cyano column) with fluorescence detection. A solid phase extraction technique (Oasis HLB, Waters) was used to extract metoprolol and its four metabolites from dog plasma with high recovery rates. The method was shown to be sensitive and reproducible and was used to determine the pharmacokinetic profile of metoprolol in dog. To the best of our knowledge, this is the only method that can extract and analyze metoprolol and its four metabolites in a single assay.
Keywords: Metoprolol;

Use of reversed phase HP liquid chromatography to assay conversion of N-acylglycines to primary fatty acid amides by peptidylglycine-α-amidating monooxygenase by Tara Carpenter; Derek D Poore; Andrew J Gee; Pallavi Deshpande; David J Merkler; Mitchell E Johnson (15-21).
Primary fatty acid amides (R-CO-NH2) and N-acylglycines (R-CO-NH-CH2-COOH) are classes of compounds that have only recently been isolated and characterized from biological sources. Key questions remain regarding how these lipid amides are produced and degraded in biological systems. Relative to the fatty acids, little has been done to develop methods to separate and quantify the fatty acid amides and N-acylglycines. We describe reversed phase HPLC methods for the separation of C2–C12 primary fatty acid amides and N-acylglycines and also C12–C22 fatty acid amides. Separation within each class occurs primarily on the basis of simple interactions between the acyl chain and the chromatographic stationary phase, but the polar headgroups on these and related fatty acids and N-acylethanolamides modulate the absolute retention in reversed phase mode. We use these methods to measure the enzyme-mediated, two-step conversion of N-octanoylglycine to octanoamide.
Keywords: N-Acylglycines; Fatty acid amides; Peptidylglycine-α-amidating monooxygenase;

A selective, accurate and reproducible high-performance liquid chromatographic (HPLC) method for the separation of individual enantiomers of DRF 2725 [R(+)-DRF 2725 and S(−)-DRF 2725 or ragaglitazar] was obtained on a chiral HPLC column (Chiralpak®). During method optimization, the separation of enantiomers of DRF 2725 was investigated to determine whether mobile phase composition, flow-rate and column temperature could be varied to yield the base line separation of the enantiomers. Following liquid–liquid extraction, separation of enantiomers of DRF 2725 and internal standard (I.S., desmethyl diazepam) was achieved using an amylose based chiral column (Chiralpak® AD™) with the mobile phase, n-hexane-propanol-ethanol-trifluoro acetic acid (TFA) in the ratio of 89.5:4:6:0.5 (v/v). Baseline separation of DRF 2725 enantiomers and I.S., free from endogenous interferences, was achieved in less than 25 min. The eluate was monitored using an UV detector set at 240 nm. Ratio of peak area of each enantiomer to I.S. was used for quantification of plasma samples. Nominal retention times of R(+)-DRF 2725, S(−)-DRF 2725 and I.S. were 15.8, 17.7 and 22.4 min, respectively. The standard curves for DRF 2725 enantiomers were linear (R 2 > 0.999) in the concentration range 0.3–50 μg/ml for each enantiomer. Absolute recovery, when compared to neat standards, was 70–85% for DRF 2725 enantiomers and 96% for I.S. from rat plasma. The lower limit of quantification (LLOQ) for each enantiomers of DRF 2725 was 0.3 μg/ml. The inter-day precisions were in the range of 1.71–4.60% and 3.77–5.91% for R(+)-DRF 2725, S(−)-DRF 2725, respectively. The intra-day precisions were in the range of 1.06–11.5% and 0.58–12.7% for R(+)-DRF 2725, S(−)-DRF 2725, respectively. Accuracy in the measurement of quality control (QC) samples was in the range 83.4–113% and 83.3–113% for R(+)-DRF 2725, S(−)-DRF 2725, respectively. Both enantiomers and I.S. were stable in the battery of stability studies viz., bench-top (up to 6 h), auto-sampler (up to 12 h) and freeze/thaw cycles (n = 3). Stability of DRF 2725 enantiomers was established for 15 days at −20 °C. The application of the assay to a pharmacokinetic study of ragaglitazar [S(−)-DRF 2725] in rats is described. It was unequivocally demonstrated that ragaglitazar does not undergo chiral inversion to its antipode in vivo in rat plasma.
Keywords: Enantiomer separation; Ragaglitazar;

MDL 28170, Cbz-(l)–Val-(d,l)–Phe-H, which exists as a mixture of l,l- and l,d-diastereoisomers, is a calpain inhibitor currently investigated as a novel therapeutic agent for the treatment of ischemic stroke and head and spinal trauma. This report describes a capillary electrophoresis (CE) method that uses sodium dodecyl sulfate (SDS) micellar electrokinetic conditions for the separation of the l,l- and l,d-diastereoisomers of MDL 28170. The report also describes the applications of this CE method to the study of epimerization of the l,l- and l,d-diastereoisomers in pH 7.4 phosphate buffered saline solution (PBS), rat and human plasma at 37 °C. The relative percent-time courses obtained showed interconversion of the diastereoisomers in all three matrices studied. However, the epimerization process in rat and human plasma was found to be at least 50 times faster than that in PBS. The epimerization half-life of the l,l-diastereoisomer in rat plasma was approximately 30 min, which is about three-fold faster than the observed elimination half-life of the l,l-diastereoisomer reported in a pharmacokinetic study following intravenous bolus dosing.
Keywords: Epimerization; MDL 28170; Calpain inhibitor;

Colloidal gold-based immunochromatographic assay for detection of botulinum neurotoxin type B by Der-Jiang Chiao; Rong-Hwa Shyu; Chieh-Shen Hu; Hsien-Yuan Chiang; Shiao-Shek Tang (37-41).
A rapid immunochromatographic assay was developed to detect botulinum neurotoxin type B (BoNT/B). The assay was based on the sandwich format using polyclonal antibody (Pab). The thiophilic gel purified anti-BoNT/B Pab was immobilized to a defined detection zone on a porous nitrocellulose membrane and conjugated to colloidal gold particles that served as a detection reagent. The BoNT/B-containing sample was added to the membrane and allowed to react with Pab-coated particles. The mixture was then passed along the porous membrane by capillary action past the Pab in the detection zone, which will bind the particles that had BoNT/B bound to their surface, giving a red colour within this detection zone with an intensity proportional to BoNT/B concentration. In the absence of BoNT/B, no immunogold was bound to the solid-phase antibody. With this method, 50 ng/ml of BoNT/B was detected in less than 10 min. The assay sensitivity can be increased by silver enhancement to 50 pg/ml. The developed BoNT/B assay also showed no cross reaction to type A neurotoxin (BoNT/A) and type E neurotoxin (BoNT/E).
Keywords: Immunochromatography; Botulinum neurotoxin type B; Colloidal gold;

A bioanalytical method for the determination of piperaquine in 100 μL blood applied onto sampling paper, by solid-phase extraction and liquid chromatography, has been developed and validated. Blood spots were cut into small pieces prior to addition of 0.3 M perchloric acid, acetonitrile and phosphate buffer containing an internal standard. The liquid phase was loaded onto a mixed phase cation-exchange (MPC) solid-phase extraction column. Piperaquine and the internal standard were analysed by liquid chromatography and separated on a Chromolith Performance (100 mm × 4.6 mm) column with acetonitrile:phosphate buffer pH 2.5, I = 0.1 (8:92, v/v) at the flow of 3.5 mL/min. The UV detection was performed at 345 nm. The intra-assay precision was 12.0% at 0.150 μM, 7.3% at 1.25 μM and 7.3% at 2.25 μM. The inter-assay precision was 1.8% at 0.150 μM, 5.2% at 1.25 μM and 2.8% at 2.25 μM. The lower limit of quantification (LLOQ) was determined to 0.050 μM where the precision was 14.7%.
Keywords: Piperaquine;

Capecitabine (N 4-pentoxycarbonyl-5′-deoxy-5-fluorocytidine, Xeloda®), a prodrug of 5-fluorouracil (5-FU), is an oral tumor-selective fluoropyrimidine carbamate approved in the treatment of colorectal and breast cancer. It has a preferential activation to 5-FU by thymidine phosphorilase (TP) in target tumor tissues through a series of three metabolic steps minimizing the exposure of normal tissues to 5-FU. It offers the potential of less gastrointestinal toxicity and advantages in terms of convenience and quality of life for the patient, in addition to cost-effectiveness as compared with intravenous 5-FU chemotherapy. We developed a high performance liquid chromatography assay for the determination of plasma capecitabine and its nucleoside metabolite concentrations and 5-FU catabolite dihydro-5-fluorouracil in a single step extraction and a single HPLC injection. The retention times of dihydro-5-fluorouracil, 5-FU, 5′-deoxy-5-fluorouridine (5′-DFUR) and capecitabine were 3.6, 4.4, 11.4 and 20.4 min, respectively and the internal standard retention times were 8.7 and 12.2 min for 5-bromouracil (5-BU) and tegafur, respectively. The limit of detection was 0.01 μg/ml for capecitabine and its nucleoside metabolites and the limit of quantification was 0.025 μg/ml. Extraction efficiency was >80% with a single solvent mixture extraction step for all analytes of interest. The assay had good precision, the within-day and between-day standard deviation of the mean (R.S.D.) being <10% in the linear range 0.025–10 μg/ml. The authors conclude that the method described here is ideally suited for the therapeutic monitoring of capecitabine and its metabolites.
Keywords: Capecitabine; 5-Fluorouracil; Dihydro-5-fluorouracil;

Determination of N G,N G-dimethyl-l-arginine, an endogenous NO synthase inhibitor, by gas chromatography–mass spectrometry by Jennifer Albsmeier; Edzard Schwedhelm; Friedrich Schulze; Mariola Kastner; Rainer H Böger (59-65).
A fully validated gas chromatographic–mass spectrometric (GC–MS) method for the accurate and precise quantification of N G,N G-dimethyl-l-arginine (asymmetric dimethylarginine, ADMA), an endogenous inhibitor of the NO synthase, in cell culture supernatants and in small volumes of plasma is described. ADMA was concentrated by solid phase extraction and converted to its methyl ester pentafluoropropionic amide derivative. The derivatives were analyzed without any further purification. Using gas chromatography–chemical ionization mass spectrometry, fragment ions at m/z 634 and m/z 640 were obtained for ADMA and for N G,N G-[2H6]-dimethyl-l-arginine ([2H6]-ADMA) as internal standard, respectively. [2H6]-ADMA was synthesized by reaction of l-ornithine fastened at bromcyan-agarose with dimethylamine. The limit of detection of the method was 2 fmol, while the limit of quantitation for cell culture supernatants was 0.05 μM. The method was validated in a concentration range of 0–1.2 μM in cell culture medium and 0–2 μM in 50 μl aliquots of human plasma. The precision was ≥97% and the accuracy was determined to be ≥94%. This method is fast, rugged and an alternative to high performance liquid chromatography (HPLC) analysis of ADMA in cell culture supernatants and small volumes of human plasma.
Keywords: N G,N G-Dimethyl-l-arginine;

An HPLC/MS based method was used for fast and convenient determination of drug plasma–protein interactions in early drug discovery screening by employing a human serum albumin affinity column. Results from this methodology were compared with data from ultrafiltration or dialysis methods, and good agreement was observed. A compound not suitable for ultrafiltration due to the very high non-specific binding to artificial membrane of ultrafiltration device was also successfully analyzed by this method, and the protein binding determined by this chromatography method was very similar to data obtained by dialysis technique employing biological membranes. The immobilized HSA column LC/MS method also proved to be more reproducible and precise compared to ultrafiltration method in drug protein binding measurements.
Keywords: Drug–protein binding; Human serum albumin;

SCH 201781 is a direct thrombin inhibitor recently under study in clinical trials to determine its safety and efficacy for the treatment of venous and arterial thrombosis. In aqueous solution, SCH 201781 exists as three forms, a ring-opened hydrated form and two ring-closed diastereomers. An automated solid-phase extraction LC-MS/MS method that chromatographically separates and measures each form was developed and validated from 1 to 1000 ng/mL in human plasma. For calibration curve standards, within- and between-run precision (%CV) ranged from 0.6 to 13.7%, while accuracy (%bias) ranged from −4.8 to 13.1%. For quality control samples, within- and between-run %CV ranged from 1.5 to 9.9% while %bias ranged from −9.1 to 4.9%. The method requires a sample volume of 0.8 mL and utilizes 2H6-labeled SCH 201781 as the internal standard. For sample processing, an Isolute C-8 96-well solid phase extraction plate and a Tomtec Quadra 96 sample processor is employed. Separation of the three forms of SCH 201781 is achieved using a 5 μm, 2 mm × 100 mm Asahipak C8 HPLC column and gradient elution. A Sciex API 365 equipped with a turbo ionspray source is used in the selected reaction monitoring mode for detection. The validated method was used to support clinical studies.
Keywords: SCH 201781;

Determination of the cyclic depsipeptide FK228, a histone deacetylase inhibitor, by liquid chromatography–mass spectrometry by Kyunghwa Hwang; Richard L Piekarz; Susan E Bates; William D Figg; Alex Sparreboom (81-86).
An analytical method was developed for the quantitative determination of the novel histone deacetylase inhibitor, depsipeptide FK228 (formerly FR901228; NSC 630176), in human plasma. Calibration curves were constructed in the range of 0.5–100 ng/ml, and were analyzed using a weight factor proportional to the nominal concentration. Sample pretreatment involved a liquid–liquid extraction with ethyl acetate using 500 μl aliquots of plasma. The analyte was separated on a column (50 mm × 4.6 mm i.d.) packed with 3.5 μm C8 material, and eluted with methanol—10 mM ammonium formate (55:45; v/v; pH 8). The column effluent was monitored by mass spectrometry with electrospray ionization. The values for precision and accuracy were always ≤7.88% and <3.33% relative error, respectively. The method was successfully applied to examine the pharmacokinetics of FK228 in a cancer patient.
Keywords: Cyclic depsipeptide; FK228;

5,6-Dimethylxanthenone-4-acetic acid (DMXAA) is a potent cytokine inducer, with a bioavailability of >70% in the mouse. The aim of this study was to develop and validate HPLC methods for the determination of DMXAA and DMXAA acyl glucuronide (DMXAA-G) in the human intestinal cell line Caco-2 monolayers. The developed HPLC methods were sensitive and reliable, with acceptable accuracy (85−115% of true values) and precision (intra- and inter-assay CV < 15%). The total running time was within 6.8 min, with acceptable separation of the compounds of interest. The limit of quantitation (LOQ) values for DMXAA and DMXAA-G were 14.2 and 24 ng/ml, respectively. The validated HPLC methods were applied to examine the epithelial transport of DMXAA and DMXAA-G by Caco-2 monolayers. The permeability coefficient (P app) values (overall mean ± S.D., n = 3–9) of DMXAA over 10–500 μM were independent of concentration for both apical (AP) to basolateral (BL) (4.0 ± 0.4 × 10−5  cm/s) and BL-AP (4.3 ± 0.5 × 10−5  cm/s) transport, and of similar magnitude in either direction, with net efflux ratio (R net) values of 1–1.3. However, the P app values for the BL to AP transport of DMXAA-G were significantly greater than those for the AP to BL transport, with R net values of 17.6, 6.7 and 4.5 at 50, 100 and 200 μM, respectively. Further studies showed that the transport of DMXAA-G was Na+- and energy-dependent, and inhibited by MK-571 [a multidrug resistance associated protein (MRP) 1/2 inhibitor], but not by verapamil and probenecid. These data indicate that the HPLC methods for the determination of DMXAA and DMXAA-G in the transport buffer were simple and reliable, and the methods have been applied to the transport study of both compounds by Caco-2 monolayers. DMXAA across Caco-2 monolayers was through a passive transcellular process, whereas the transport of DMXAA-G was mediated by MRP1/2.
Keywords: 5,6-Dimethylxanthenone-4-acetic acid; Caco-2 cells;

BAY 43-9006 is a selective Raf-1 kinase inhibitor with antitumor activity against a variety of human cancers. A highly sensitive HPLC method for determination of BAY 43-9006 in small volumes of serum (30 μl) was developed. Sample preparation involved a liquid–liquid extraction procedure with tolnaftate as internal standard followed by linear gradient elution at a reversed phase C18 column and UV detection. The method was selective and the calibration curves were linear over the concentration range of 80–2000 ng/ml. The intra-day accuracy ranged from 99.9 to 107.6% and the inter-day accuracy from 94.6 to 115%. The lower limit of quantitation (LOQ) was 80 ng/ml with an accuracy of 105.8%. Thus, this method has been validated and can be applied for the drug monitoring or pharmacokinetic studies of BAY 43-9006 in small volumes of serum samples.
Keywords: BAY 43-9006; Raf-1 kinase;

Liquid chromatography determination of the anti-androgen vinclozolin and its metabolites in rat serum by Adolfo Sierra-Santoyo; Hugh A Barton; Michael F Hughes (105-110).
The objective of this study was to develop a chromatographic method for the analysis of the anti-androgen vinclozolin (V) and its metabolites 2-[[(3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3-butenoic acid (M1), 3′,5′-dichloro-2-hydroxy-2-methylbut-3-enanilide (M2) and 3,5-dichloroaniline (M3) in rat serum. V, M1–M3 were resolved using an HPLC gradient program with a mobile phase consisting of 60–75% methanol:acetonitrile (70:30) and 0.05 M monobasic sodium phosphate buffer pH 3.3 at 1 ml/min, a C18 column, and monitored at 212 nm. Incubates of 0.01 M monobasic potassium phosphate buffer (PB) pH 7.4 and rat serum were spiked with V and its metabolites and processed by diluting samples (1:4) with 0.1 M PB pH 3.3, to limit methodological hydrolysis of analytes, followed by addition of acetonitrile. Recoveries of V, M1 and M2 ranged from 85 to 105%, whereas recovery of M3 was <25%. V was hydrolyzed to M1 and M2 after incubation in PB pH 7.4 and rat serum, with M1 the predominant metabolite. This method was successfully applied in the analysis of V and its metabolites in the serum of a male rat after oral administration of V (100 mg/kg).
Keywords: Vinclozolin;

A method of field-amplified sample stacking in capillary electrophoresis is described for the simultaneous determination of clozapine (CZP) and its metabolites, clozapine N-oxide (CNO), and desmethylclozapine (DMC), in human plasma. Plasma (0.2 mL) was extracted with organic solvents (ethyl acetate/n-hexane/isopropyl alcohol, 8/1/1 by volume) and centrifuged. An aliquot of supernatant was evaporated and suitably reconstituted with water for CE analysis. An untreated fused-silica capillary was used (31.2 cm; effective length, 20 cm; 50 μm i.d.) for the analysis. The background buffer was phosphate buffer (400 mM, pH 3.0) containing 50% ethylene glycol. The separation voltage was 25 kV with a detection wavelength of 214 nm. In the method validation, the calibration curves were linear (r ≧ 0.98) over a range of 50–800 ng/mL for CZP, 30–180 ng/mL for CNO, and 25–600 ng/mL for DMC. The relative standard deviation (R.S.D.) and relative error (R.E.) were all less than 11% for the intra- and inter-day assays. The limits of detection (S/N = 3, electric-driven injection, 99.9 s) of CZP, DMC, and CNO were 5, 5, and 10 ng/mL, respectively. After continuing treatment with the CZP tablets, a blood sample from one male schizophrenic patient (41-year-old, 62 kg) who had been receiving ongoing treatment with the CZP tablets was prepared and analyzed. The levels of CZP, DMC, and CNO were determined and the feasibility of the method’s application in clinical treatment was proven.
Keywords: Field-amplified sample stacking; Clozapine; Clozapine N-oxide; Desmethylclozapine;

Selective and rapid liquid chromatography–tandem mass spectrometry assay of dutasteride in human plasma by N.V.S Ramakrishna; K.N Vishwottam; S Puran; M Koteshwara; S Manoj; M Santosh (117-124).
A simple, rapid, sensitive and specific liquid chromatography–tandem mass spectrometry method was developed and validated for quantification of dutasteride (I), a potent and the first specific dual inhibitor of 5α-reductase, in human plasma. The analyte and internal standard (finasteride (II)) were extracted by liquid–liquid extraction with diethyl ether/dichloromethane (70/30, v/v) using a Glas-Col Multi-Pulse Vortexer. The chromatographic separation was performed on a reverse phase Xterra MS C18 column with a mobile phase of 10 mM ammonium formate/acetonitrile (15/85, v/v, pH adjusted to 3.0 with formic acid). The protonated analyte was quantitated in positive ionization by multiple reaction monitoring with a mass spectrometer. The mass transitions m/z 529.5 → 461.5 and m/z 373.3 → 317.4 were used to measure I and II, respectively. The assay exhibited a linear dynamic range of 0.1–25.0 ng/mL for dutasteride in human plasma. The lower limit of quantitation was 100 pg/mL with a relative standard deviation of less than 15%. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. A run time of 1.2 min for each sample made it possible to analyze a throughput of more than 400 human plasma samples/day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.
Keywords: Dutasteride;

Alkylresorcinols, phenolic lipids present in high amounts in wholegrain wheat and rye, are of interest as potential biomarkers of the intake of these cereals. Alkylresorcinols are known to be absorbed by humans and animals, but little is known about their metabolism or resulting metabolites. A preliminary human study was carried out to identify alkylresorcinol metabolites in human urine. Urine samples, collected before and after a wheat-bran based meal, were deconjugated with β-glucuronidase/sulphatase and then extracted with ethyl acetate. Extracts were separated by thin-layer chromatography, and fractions containing alkylresorcinols and possible metabolites were identified by retention on the plate compared to standard compounds, and staining with fast blue B. These fractions were further analysed by gas chromatography-mass spectrometry. Deconjugated human urine after the wheat-bran based meal contained two alkylresorcinol metabolites, 3,5-dihydroxybenzoic acid and 3-(3,5-dihydroxyphenyl)-1-propanoic acid, as well as smaller amounts of unchanged alkylresorcinols, confirming the hypothesis that alkylresorcinols are metabolised in humans via β-oxidation of their alkyl chain.
Keywords: Biomarkers; Alkylresorcinol;

Determination of tetrabromobisphenol A in human serum by liquid chromatography–electrospray ionization tandem mass spectrometry by Tadashi Hayama; Hideyuki Yoshida; Suzuko Onimaru; Sayuri Yonekura; Hiroaki Kuroki; Kenichiro Todoroki; Hitoshi Nohta; Masatoshi Yamaguchi (131-136).
A method for the determination of tetrabromobisphenol A (TBBPA) in human serum utilizing solid-phase extractions (SPEs) and liquid chromatography (LC) with electrospray ionization tandem MS (MS/MS) has been developed. After purification and concentration of TBBPA using consecutive SPEs on reversed-phase and normal-phase cartridges, the serum sample was subjected to LC. TBBPA was separated on a C18 reversed-phase column by gradient elution with a mixture of water, methanol, and acetonitrile as the mobile phase, and then detected with electrospray ionization MS/MS in negative ion mode. 13 C 12 -TBBPA was suitable as an internal standard for the reproducible determination of TBBPA in human serum samples (5 g). The method has been validated in TBBPA concentration range of 5–100 pg per g serum, and the recoveries in the concentration range were higher than 83.3%. The repeatabilities of the proposed method of non-spiked control serum (6.3 pg per g serum) and spiked serum (added 5–100 pg per g serum) were within 10.0% as relative standard deviations. The limit of quantification (LOQ) for TBBPA was 4.1 pg per g serum, which was corresponded to 0.63 fmol on column.
Keywords: Tetrabisphenol A;

High-performance affinity chromatography was used to study the binding of phenytoin to an immobilized human serum albumin (HSA) column. This was accomplished through frontal analysis and competitive binding zonal elution experiments, the latter of which used four probe compounds for the major and minor binding sites of HSA injected into the presence of mobile phases containing known concentrations of phenytoin. It was found that phenytoin can interact with HSA at the warfarin-azapropazone, indole-benzodiazepine, tamoxifen, and digitoxin sites of this protein. The association constants for phenytoin at the indole-benzodiazepine and digitoxin sites were determined to be 1.04 (±0.05) × 104  M−1 and 6.5 (±0.6) × 103  M−1, respectively, at pH 7.4 and 37 °C. Both allosteric interactions and direct binding for phenytoin appear to take place at the warfarin-azapropazone and tamoxifen sites. This rather complex binding system indicates the importance of identifying the binding regions on HSA for specific drugs as a means for understanding the transport of such substances in blood and in characterizing their potential for drug–drug interactions.
Keywords: Phenytoin; Human serum albumin;

Large-volume sample stacking using the electroosmotic flow (EOF) pump technique has been investigated for the quantification of 3-nitrotyrosine in urine of diabetic rats. The best separation conditions for these highly complex samples were obtained using capillary electrophoresis (CE) in the reversed polarity mode (i.e., injecting at the cathode and detecting at the anode) using cetyltrimethylammonium bromide (CTAB) in the running buffer. The optimum CE separation conditions were achieved using a phosphate buffer prepared with 0.15 M phosphoric acid and 0.5 mM CTAB adjusted to pH 6.4 with sodium hydroxide. In such CE conditions, the limit of detection (LOD) was 1.77 μM for 3-nitrotyrosine with normal injection mode, meanwhile with the large-volume sample stacking technique a more than 20-fold improvement was observed (i.e., LOD = 0.08 μM was obtained) without noticeable loss of resolution. This value allowed the detection of 3-nitrotyrosine in urine from diabetic rats. To our knowledge, this work is one of the few applications showing the great possibilities of these stacking procedures to analyse biological samples by CE.
Keywords: 3-Nitrotyrosine;

Discovery stage pharmacokinetics using dried blood spots by Patrick Beaudette; Kevin P Bateman (153-158).
Early in the discovery stage, the measurement of drug candidates in biological fluids as a function time provides important information used in decision making for lead optimization. The detection methodology primarily used is liquid chromatography coupled to triple quadrupole mass spectrometry (LC–MS). Sample preparation is an important aspect of these experiments and robotic-based automation is commonly used. The often overlooked aspect of these experiments is the sample collection itself. Typically, several hundred microliters of whole blood is collected and the plasma fraction separated for each time-point. The plasma is then transferred to an appropriate vessel for subsequent aliquoting and processing. We describe a method for performing discovery stage pharmacokinetic analysis using whole blood dried onto filter paper. The use of dried blood spots is a well established technique for neo-natal screening, and its application to early screening of drug candidates proves to be robust, reliable and reproducible.
Keywords: Discovery stage pharmacokinetic analysis; Dried blood spots;

A selective, sensitive and precise HPLC method with fluorimetric detection has been developed for the assay of lisinopril in human plasma and urine. The clean up of the sample was carried out by solid-phase extraction, firstly with C18-cartridge and secondly with a silica-cartridge. After a pre-column derivatization with fluorescamine, the reaction mixture was chromatographed on C18-column with gradient elution, using methanol and 0.02 M phosphate buffer (pH = 3.2). The fluorescamine–lisinopril derivative was detected fluorimetrically by monitoring the emission at 477 nm, with excitation at 383 nm. Linear quantitative response curve was generated over a concentration range of 5–200 ng/ml and 25–1000 ng/ml for plasma and urine samples, respectively. The mean recovery of lisinopril from plasma and urine was 63.41 and 74.08%, respectively. Intra-day and inter-day R.S.D. and R.M.E. values at three different concentrations were assessed. The method was applied for pharmacokinetic study in a healthy volunteer after a single oral dose of 20 mg of the drug.
Keywords: Lisinopril; Fluorescamine;

Determination of tetracationic zinc(II) phthalocyanine derivative RLP068 in rabbit serum by liquid chromatography–tandem mass spectrometry by Giacomo Chiti; Moira Municchi; Valentina Paschetta; Daniele Nistri; Gabrio Roncucci (167-174).
The development and validation of a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the determination of the tetracationic zinc(II) phthalocyanine derivative RLP068 in rabbit serum is described. The dodecadeuterated product (RLP068-D12) was used as co-eluting internal standard. RLP068 was isolated from serum samples by solid-phase extraction using weak cationic exchange cartridges (WCX). An oxidative derivatisation was used in order to simplify the peculiar HPLC and MS behaviour of the analyte and thus increasing sensitivity. Liquid Chromatography was carried out on a Polaris C18 Ether column (50 mm × 2.0 mm) with an isocratic run of 0.5% aqueous TFA/methanol. 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 2–65 ng/mL. lower limits of detection (LLOD) and quantification (LLOQ) were respectively 1 and 2 ng/mL. The method is innovative and applicable to pharmacokinetic studies.
Keywords: Zinc(II) phthalocyanine; RLP068;

The use of a monolithic column (Chromolith®, SpeedROD RP-18e, by Merck) was studied on the determination of cephalosporin antibiotics. Results were compared with those from a previously developed analytical method using conventional silica-based analytical column. A rapid, accurate and sensitive method has been developed and validated for the quantitative simultaneous determination of four cephalosporins: Cephalexine and Cephadroxil (first generation), Cefaclor (second generation) and Cefotaxim (third generation) in pharmaceuticals as well as in human blood serum and urine. Hydroflumethiazide (HFM) (3,4-dihydro-6(trifluoromethyl)-2H-1,2,4-benzothiadiazine-7-sulfonamide-1,1-dioxide) was used as an internal standard at a concentration of 1.5 ng/μL. A rectilinear relationship was observed up to 5 ng/μL for the four compounds. Analysis time was less than 4 min. The statistical evaluation of the method was examined by means of within-day repeatability (n = 8) and day-to-day precision (n = 8) and was found to be satisfactory with high accuracy and precision results. The method was applied to the determination of the cephalosporins in commercial pharmaceuticals and in biological fluids: human blood serum after solid phase extraction and urine simply after filtration and dilution. Recovery of analytes in spiked serum samples was in the range from 88.7 to 107.8%, while for urine samples recovery was from 98.0 to 105.6%. By comparing the figures of merit for the monolithic column and the silica-based one, regarding the determination of the four cephalosporins investigated in the present study, the outstanding efficiency of the monolithic column can be noticed.
Keywords: Monolithic columns; Cephalosporins; Cephadroxil; Cefaclor; Cefotaxime; Cephalexine;

Frequency of intentional exposure to organic solvents has been increasing among children and adolescents in Brazil. Analysis of benzene, toluene and xylenes (BTX) in human blood is necessary to diagnose the intentional and accidental exposure to these solvents. A method for BTX determination in blood samples by gas chromatography preceded by solid phase microextration (SPME) from headspace (HS) has been described. SPME has several advantages when compared to other extraction techniques such as simplicity, low cost and solvent-free extraction. The method presents good repeatability (precision was of 2.2–8.0%), accuracy from −4.7 to −9.4%, limit of detection <1.0 ug/mL, linearity from 1.0 to 100 ug/mL for toluene and from 5.0 to 100 ug/mL for the other solvents (R 2 > 0.99), which shows to be efficient and adequate for the detection of exposure to BTX in blood samples.
Keywords: Benzene; Toluene; Xylene;