Journal of Chromatography B (v.832, #2)

High-performance liquid chromatography spectrometric analysis of trans-resveratrol in rat plasma by Hui He; Xijing Chen; Guangji Wang; Jiping Wang; Andrew Keith Davey (177-180).
A HPLC method for determination of trans-resveratrol concentrations in rat plasma was developed. Plasma samples were treated with acetonitrile to deposit proteins. The analysis used a Hypersil ODS2 C18 column (5 μm, 4.6 mm × 250 mm) and methanol/distilled water as the mobile phase (flow-rate = 1 mL/min). The UV detection wavelength was 303 nm, and chlorzoxazone was used as the internal standard. The calibration curve was linear over the range of 0.02–40 μg/mL with a correlation coefficient of 0.9997. This concentration range corresponds well with the plasma concentrations of resveratrol in pharmacokinetic studies. There was 98.7%, 91.3% and 84.4% recovery from 0.02, 0.4 and 40 μg/mL plasma samples respectively. The R.S.D. of intra- and inter-day assay variations were all less than 12%. This HPLC assay is a quick, precise and reliable method for the analysis of resveratrol in pharmacokinetic studies.
Keywords: Resveratrol; HPLC; Rat; Plasma concentration;

An HPLC method for the determination of ng mifepristone in human plasma by Zhiyong Guo; Chengding Chu; Gengxin Yin; Mingli He; Keqin Fu; Jianli Wu (181-184).
An HPLC method was developed and validated for the determination of mifepristone in human plasma. C18 solid-phase extraction cartridges were used to extract plasma samples. Separation was by C18 column; mobile phase, methanol–acetonitrile–water (50:25:25, v/v/v); flow rate, 0.8 ml/min; UV detection at 302 nm. The calibration curve was linear in the concentration range of 10 ng/ml to 20 μg/ml (r  = 0.9991). Within- and between-day variability were acceptable. The limit of detection for the assay was 6 ng/ml. Plasma samples were stable for at least 7 days in the state of plasma or residue treated at −20 °C. The method was simple, sensitive and accurate, and allowed to determine ng mifepristone in human plasma. It could be applied to assess the plasma level of mifepristone in women receiving low oral doses of mifepristone.
Keywords: Mifepristone; High performance liquid chromatography;

A simple, rapid and reliable method was developed for the identification and quantification of helicidum and its metabolites in beagle dog plasma by liquid chromatography/ultra-violet/electrospray ionization-ion trap mass spectrometry (LC/UV/ESI-ITMS). Two metabolites were identified by MS: formylphenyl-O-β-d-pyranosyl alloside (I) and hydroxylmethylphenyl-O-β-d-pyranosyl alloside (II). UV was used for concentration determination with the wavelength of 270 nm. Liquid–liquid extraction was used and the extraction recovery exceeded 90%. Kromacil C18 column (5 μm, 4.6 mm i.d. × 250 mm) was used as the analytical column. Linear detection responses were obtained for helicidum concentration ranging from 1.76 × 10−4 to 70.4 × 10−4  μmol/mL (0.050–2.00 μg/mL). The precision and accuracy data, based on intra- and inter-day variations over 3 days, were less than 5%. The limit of determination and quantitation (LOD, LOQ) for helicidum was 0.010 and 0.030 μg/mL, respectively. Pharmacokinetic data of helicidum and the two metabolites were obtained with this method after administration of intravenous injection and a single oral dose of tablets to six beagle dogs, respectively.
Keywords: Helicidum; Metabolite; HPLC/UV/ESI-ITMS; Dog plasma; Pharmacokinetics;

Application of microbore HPLC in combination with tandem MS for the quantification of rosuvastatin in human plasma by Kathalijne A. Oudhoff; Timothy Sangster; Elizabeth Thomas; Ian D. Wilson (191-196).
The potential of microbore high-performance liquid chromatography (HPLC) in combination with tandem mass spectrometry (MS/MS) for the sensitive detection of rosuvastatin (Crestor™) in human plasma was investigated. Three microbore HPLC columns with internal diameters (i.d.) of 0.5, 1.0 and 2.0 mm were evaluated for column efficiency and mass sensitivity, and compared to a conventional 4.6 mm i.d. column. The 2.0 and 1.0 mm i.d. columns performed very well while the 0.5 mm i.d. column was slightly less efficient, this is probably due to a lower packing density. Good results with respect to gains in mass sensitivity compared to the conventional analytical column were achieved with the 2.0 and 1.0 mm columns. Thus, the 2.0 mm i.d. column had an improved signal-to-noise (S/N) ratio of 16 whilst the 1.0 mm i.d. column had an improved S/N ratio of greater than 70. Experiments with the 1.0 mm i.d. HPLC column were performed to determine the robustness of the microbore method for human plasma extracts after sample preparation using solid-phase extraction (SPE). A number of problems were encountered with extracts including high backgrounds, the blocking of the column and a rapid deterioration in column performance. The blocking of the column by particulates was solved by off-line filtration of the sample extracts. Peak tailing of the analytes and high background, both of which were due to endogenous interferences in the extracts, were eliminated using gradient elution. Using these approaches over 500 injections of plasma extracts were achieved without significant deterioration in assay performance. Quantities of rosuvastatin of 0.3 pg on-column could be detected and cross-validation experiments demonstrated that the conventional and the microbore HPLC-MS/MS methods provided similar information on the concentration of rosuvastatin but with greatly reduced sample consumption using the microbore method.
Keywords: Microbore HPLC; Rosuvastatin; Human plasma; Bioanalysis;

A simple and ultra rapid high performance liquid chromatographic (HPLC) method coupled with alumina extraction and fluorescence detection was described for determination of methyldopa in human serum. The drug and an internal standard were adsorbed onto alumina and eluted using acidic methanol. The eluate was directly injected onto ODS reverse phase column using a mixture of phosphate buffer (0.05 M) containing triethylamine (100 μl/l, v/v; pH 2.3) and methanol (92:8, v/v) at a flow rate of 2.1 ml/min as the mobile phase. The fluorescence detector excitation and emission wavelengths were set at 270 and 320 nm, respectively. No interference in the assay from any endogenous substances or other concurrently used drugs was observed and the retention times of I.S. and the drug were 1.7 and 2.4 min, respectively with total run time (injection to injection) of less than 3.5 min. The limit of quantification was evaluated to be 20 ng/ml. Validity of the method was studied and the method was precise and accurate with a linearity range from 20 ng/ml to 5000 ng/ml. This method has been used in a randomized crossover bioequivalence study of two different methyldopa preparations in 24 healthy volunteers.
Keywords: Reverse phase chromatography; HPLC; Methyldopa; Bioequivalence study;

The use of blood spot collection cards is a simple way to obtain specimens for analysis of drugs for the purpose of therapeutic drug monitoring, assessing adherence to medications and preventing toxicity in routine clinical setting. We describe the development and validation of a microanalytical technique for the determination of metformin from dried blood spots. The method is based on reversed phase high-performance liquid chromatography with ultraviolet detection. Drug recovery in the developed method was found to be more than 84%. The limits of detection and quantification were calculated to be to be 90 and 150 ng/ml, respectively. The intraday and interday precision (measured by CV%) was always less than 9%. The accuracy (measured by relative error, %) was always less than 12%. Stability analysis showed that metformin is stable for at least 2 months when stored at −70 °C. The small volume of blood required (10 μL), combined with the simplicity of the analytical technique makes this a useful procedure for monitoring metformin concentrations in routine clinical settings. The method is currently being applied to the analysis of blood spots taken from diabetic patients to assess adherence to medications and relationship between metformin level and metabolic control of diabetes.
Keywords: HPLC; Metformin; Blood spot; Therapeutic drug monitoring;

A practical approach to optimization and validation of a HPLC assay for analysis of polyribosyl-ribitol phosphate in complex combination vaccines by Mary Belfast; Rong Lu; Robert Capen; Jinglin Zhong; Mai-Anh Nguyen; Juan Gimenez; Robert Sitrin; Ralph Mancinelli (208-215).
The use of multi-factor statistical experimental design methodology minimized the vaccine material and laboratory resources required for optimization and validation of an HPLC assay for quantitation of deploymerized and total PRP. Components of the assay selected for optimization were adjuvant dissolution, ultracentrifuge conditions including ultracentrifuge model, sample diluent, mobile phase and column oven temperature. Previous experience has shown these components of the assay to be most troublesome and therefore required optimization prior to validation. Specificity, linearity, precision, accuracy and ruggedness were confirmed through a validation of the optimized assay. The validation also established the assay to be stability indicating, by showing that changes to the integrity of the PRP-OMPC conjugate could be detected.
Keywords: PRP; Assay validation; Assay optimization; Combination vaccine; HPLC; Design of experiment;

Efficient removal of albumin from human serum by monosize dye-affinity beads by Evrim Banu Altıntaş; Adil Denizli (216-223).
Cibacron Blue F3GA was covalently attached onto monosize poly(glycidyl methacrylate) [poly(GMA)] beads for removal of human serum albumin (HSA) from human serum. Monosize poly(GMA) beads, 1.6 μm in diameter, were produced by dispersion polymerization. Cibacron Blue F3GA loading was 1.73 mol/g. HSA adsorption experiments were performed by stirred-batch adsorption. The non-specific adsorption of HSA was low (0.8 mg/g polymer). Dye attachment onto the monosize beads significantly increased the HSA adsorption (189.8 mg/g). The maximum HSA adsorption was observed at pH 5.0. With an increase of the aqueous phase concentration of sodium chloride, the adsorption capacity decreased drastically. The equilibrium adsorption of HSA significantly decreased with increasing temperature. The elution studies were performed by adding 0.1 M Tris/HCl buffer containing 0.5 M NaSCN to the HSA solutions in which adsorption equilibria had been reached. The elution results demonstrated that the adsorption of HSA to the adsorbent was reversible. The depletion efficiencies for HSA were above 87% for all studied concentrations. To test the efficiency of HSA removal from human serum, proteins in the serum and eluted portion were analyzed by two-dimensional gel electrophoresis. Eluted proteins include mainly albumin, and a small number of nonalbumin proteins such as apo-lipoprotein A1, sero-transferrin, haptoglobulin and α1-antitrypsin were bound by the dye-affinity beads. IgA was not identified in eluted fraction.
Keywords: Cibacron Blue F3GA; Monosize beads; Poly(GMA); Albumin removal; Dye-ligand chromatography; Affinity depletion;

Stability of glufosfamide in phosphate buffers and in biological samples by Yuming Sun; Xiaoyan Chen; Haiyan Xu; Zhongmin Guan; Dafang Zhong (224-230).
Glufosfamide is a new, potential chemotherapeutic agent currently under investigation. Stability of glufosfamide was investigated in sodium phosphate buffers with different pH and temperature and in biological samples. Glufosfamide and isophosphamide mustard were quantified simultaneously using a liquid chromatography–ion trap mass spectrometric method; precision and accuracy were within 15% for each analyte. Glufosfamide was stable in neutral buffers, but decomposed to form isophosphoramide mustard under acidic and basic conditions, which was pH- and temperature-dependent. The stability of glufosfamide varied in different biological samples. Results indicated that glufosfamide was unstable in some biological samples, such as the small intestine, smooth muscles, pancreas and urine, especially in the small intestine homogenate, with a half-life of 1.1 h. But the pH (<8) and β-glucosidase of the tissue homogenate was found to have negligible contribution to the degradation of glufosfamide. The enzymatic inhibition experiment with the specific inhibitor, saccharo-1,4-lactone, demonstrated that it was glucuronidase that resulted in the degradation of glufosfamide in small intestine homogenate. Methanol was recommended to be used to homogenize the tissue in an ice water bath, and the container for urine collection should also be maintained in an ice water bath, and all the biological samples collected should be preserved in frozen condition until analysis.
Keywords: Stability; Glufosfamide; β-Glucosidase; β-Glucuronidase;

A new simple HPLC assay for the quantification of ertapenem in human plasma, lung tissue, and broncho-alveolar lavage fluid by Ralf G. Mundkowski; Jolanta Majcher-Peszynska; Olaf Burkhardt; Tobias Welte; Bernd Drewelow (231-235).
Ertapenem is an important newer broad-spectrum carbapenem antibiotic covering various infections caused by common gram-positive and -negative aerobes and anaerobes. Due to its physicochemical peculiarities, pharmacokinetic data of other carbapenems are of limited value in predicting ertapenem distribution into particular compartments of the body. This raises demand for detailed pharmacokinetic studies and, as a consequence, rapid and specific ways of analysis. The HPLC assays for the quantification of ertapenem in biological matrices reported so far are based on columns of 4.6 mm I.D. and involve pre-concentration by use of column-switching. However, automated column-switching technique is not standard equipment with all analytical laboratories. Furthermore, signal-to-noise ratios are likely not to be sufficient for quantification of specimens of low concentration. Therefore, a new HPLC/UV method based on narrow-bore column design using sample pre-cleaning by liquid–liquid extraction has been developed. The assay is rapid for specimen concentrations ≥1 mg/l and is easily tuned to achieve low quantification limits at high chromatographic resolution for lower concentrated samples. The method has been successfully applied to plasma, serum, lung tissue or cell homogenates, and broncho-alveolar lavage fluid with lower limits of quantification of 40 and 20 μg/l, respectively. It was also used for the pharmacokinetic monitoring of ertapenem in humans.
Keywords: Cell; Ertapenem; HPLC; Lavage fluid; LLE; Plasma; Serum; Tissue; UV;

Determination of ranitidine in urine by capillary electrophoresis-electrochemiluminescent detection by Ying Gao; Yiling Tian; Xiuhua Sun; Xue-Bo Yin; Qian Xiang; Ge Ma; Erkang Wang (236-240).
The fast analysis of ranitidine is of clinical importance in understanding its efficiency and a patient's treatment history. In this paper, a novel determination method for ranitidine based on capillary electrophoresis-electrochemiluminescence detection is described. The conditions affecting separation and detection were investigated in detail. End-column detection of ranitidine in 5 mM Ru(bpy)3 2+ solution at applied voltage of 1.20 V was performed. Favorable ECL intensity with higher column efficiency was achieved by electrokinetic injection for 10 s at 10 kV. The R.S.D. values of ECL intensity and migration time were 6.38 and 1.84% for 10−4  M and 6.01 and 0.60% for 10−5  M, respectively. A detection limit of 7 × 10−8  M (S/N = 3) was achieved. The proposed method was applied satisfactorily to the determination of ranitidine in urine in 6 min.
Keywords: Capillary electrophoresis; Electrochemiluminescence; Ranitidine;

A simple and sensitive column-switching high-performance liquid chromatographic method was developed for the simultaneous determination of omeprazole and its two main metabolites, 5-hydroxyomeprazole and omeprazole sulfone, in human plasma. Omeprazole, its two metabolites and lansoprazol as an internal standard were extracted from 1 ml of alkalinized plasma sample using diethyl ether–dichloromethane (45:55, v/v). The extract was injected into a column I (TSK-PW precolumn, 10 μm, 35 mm × 4.6 mm i.d.) for clean-up and column II (Inertsil ODS-80A column, 5 μm, 150 mm × 4.6 mm i.d.) for separation. The mobile phase consisted of phosphate buffer–acetonitrile (92:8 v/v, pH 7.0) for clean-up and phosphate buffer–acetonitrile–methanol (65:30:5 v/v/v, pH 6.5) for separation, respectively. The peak was detected with an ultraviolet detector set at a wavelength of 302 nm, and total time for chromatographic separation was ∼25 min. The validated concentration ranges of this method were 3–2000 ng/ml for omeprazole, 3–500 ng/ml for 5-hydroxyomeprazole and 3–1000 ng/ml for omeprazole sulfone. Mean recoveries were 84.3% for omeprazole, 64.3% for 5-hydroxyomeprazole and 86.1% for omeprazole sulfone. Intra- and inter-day coefficient variations were less than 5.1 and 6.6% for omeprazole, 4.6 and 5.0% for 5-hydroxyomeprazole and 4.6 and 4.9% for omeprazole sulfone at the different concentrations. The limits of quantification were 3 ng/ml for omeprazole and its metabolites. This method was suitable for use in pharmacokinetic studies in human volunteers, and provides a useful tool for measuring CYP2C19 activity.
Keywords: Omeprazole; 5-Hydroxyomeprazole; Omeprazole sulfone; CYP2C19; HPLC;

A simple and fast procedure was developed for the simultaneous determination of eight benzodiazepines (BZDs) in whole blood using liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS). Sample pretreatment was carried out using a simple liquid–liquid extraction (LLE) with n-butylchloride, and chromatographic separation was performed using a monolithic silica column to speed up the analytical process. APCI and electrospray ionization (ESI) were compared. Whereas both ionization techniques appeared suitable for BZDs, APCI was found to be slightly more sensitive, especially for the determination of frequently low-dosed compounds. The method was validated according to the guidelines of the “Société Française des Sciences et Techniques Pharmaceutiques” (SFSTP) in the concentration range of 2.5–500 μg/L. The limit of quantification (LOQ) was 2.5 μg/L for all the compounds. Validation data including linearity, precision, and trueness were obtained, allowing subtherapeutic quantification of frequently low-dosed BZDs. The high selectivity of the mass spectrometer, along with the properties of the monolithic support, allowed unequivocal analysis of the eight compounds in less than 5 min. To demonstrate the potential of the method, it was used for the analysis of benzodiazepines in postmortem blood samples.
Keywords: APCI; Benzodiazepines; LC–MS; Monolithic column; Validation;

Chiral evaluation of fluvastatin in human plasma by high-performance liquid chromatography electrospray mass spectrometry by Giuliano Di Pietro; Eduardo Barbosa Coelho; Tufik Magalhães Geleilete; Maria Paula Marques; Vera Lucia Lanchote (256-261).
The report describes for the first time the enantioselective analysis of fluvastatin in plasma using LC–MS–MS. The enantiomers of fluvastatin (FV) were extracted from plasma with diisopropyl ether at pH 5.0. The enantiomers were separated on a ChiralCel® OD-R column with a mobile phase consisting of a mixture of acetonitrile, methanol and water (24:36:40) containing 0.1% formic acid. The protonated ions and their respective product ions were monitored in two functions, 410.6 > 348.2 for FV enantiomers and 307.1 > 161.6 for the internal standard (warfarin). Recoveries were higher than 90% and the quantitation limit was 1.5 ng mL−1 plasma for both enantiomers. The coefficients of variation and the relative errors obtained for the validation of the intra- and interassay precision and accuracy were less than 10%. The method was applied to the investigation of the enantioselective pharmacokinetics of FV administered in a single dose of 40 mg (Lescol®, Novartis, São Paulo, SP, Brazil) to a patient with primary hypertension and hypercholesterolemia and genotyped as CYP2C9*1/*1. The data showed higher plasma concentrations of the (−)-3S,5R-fluvastatin enantiomer, with an AUC (−)/(+) of 1.84. Oral clearance values (CL/F) were 29.27 and 49.58 L/h, respectively, for the (−)-3S,5R- and (+)-3R,5S-fluvastatin enantiomers.
Keywords: Fluvastatin; Pharmacokinetics; Enantiomers; LC–MS–MS;

Purification of capsular polysaccharide from Neisseria meningitidis serogroup C by liquid chromatography by Tânia Pinheiro Pato; Antonio de Pádua R. Barbosa; José Godinho da Silva Junior (262-267).
Neisseria meningitidis serogroup C capsular polysaccharide (MenCPS) is an important antigen against meningococcal infection. This paper describes a new purification methodology employing liquid chromatography that resulted in a polysaccharide showing the characteristics recommended by the World Health Organization for vaccine purposes. In this method, steps of the traditional procedure that yield low recovery and use toxic materials were modified. The present process consists in the following steps: (1) continuous flow centrifugation of the culture for removal of the cells; (2) supernatant concentration by tangential filtration (100 kDa cutoff); (3) addition of 0.5% DOC, heating to 55 °C during 30 min and tangential filtration (100 kDa cutoff); (4) anion exchange chromatography (Source 15Q) and (5) size exclusion chromatography (Sepharose CL-4B). The polysaccharide C fraction obtained in that way was dialyzed and freeze-dried. The structural identity of the polysaccharide was demonstrated by 1H-NMR spectrometry.
Keywords: Neisseria meningitidis; Capsular polysaccharide; Purification by liquid chromatography;

Quantitative analysis of Variolin analog (PM01218) in mouse and rat plasma by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry by Jianming Yin; Pablo Aviles; Carl Ly; William Lee; Maria Jose Guillen; Simon Munt; Carmen Cuevas; Glynn Faircloth (268-273).
PM01218 is a novel marine-derived alkaloid and has shown potent growth inhibitory activity against several human cancer cell lines. A rapid and sensitive high performance liquid chromatography/tandem mass spectrometry (HPLC–MS/MS) method was developed and validated to quantify PM01218 in mouse and rat plasma. The lower limit of quantitation (LLOQ) was 0.05 ng/mL. The calibration curve was linear from 0.05 to 100 ng/mL (R 2  > 0.999). The assay was specifically based on the multiple reaction monitoring (MRM) transitions at m/z 278.4 → 184.2, no endogenous material interfaced with the analysis of PM01218 and its internal standard from blank mouse and rat plasma. The mean intra- and inter-day assay accuracy remained below 15 and 8%, respectively, for all calibration standards and QC samples. The intra- and inter-day assay precision was less than 12.8 and 8.5% for all QC levels, respectively. The utility of the assay was demonstrated by pharmacokinetics studies of i.v. (bolus) PM01218 on SD rats.
Keywords: PM01218; HPLC–MS/MS; Pharmacokinetics;

Analysis of expired air of fasting male monks at Mount Athos by M. Statheropoulos; A. Agapiou; A. Georgiadou (274-279).
Expired air chemical analysis is investigated as a search and locate method for the early detection of entrapped people under the ruins of collapsed buildings after an earthquake. Fasting individuals were examined as a group that simulates the medical status of some of such victims. Exhaled air from seven fasting male monks (after 63 h) was analysed using thermal desorption–gas chromatography–mass spectrometry (TD–GC–MS) analysis. Over 150 volatile organic compounds (VOCs) were identified and the 43 most frequent are presented. Acetone showed by far the highest “positive alveolar gradient”. Other compounds included phenol, di-limonene, 2-pentanone, isoprene and acetaldehyde. Quantitative results showed a 30-fold increase of acetone concentration (5.8 ppmv) compared to control measurements of a volunteer. Breath acetone was also identified through a portable gas chromatography-ion mobility spectrometer showing possible, under certain conditions, effectiveness of the method in the field.
Keywords: Expired air; VOCs; Fasting; Breath; Earthquake; TD–GC–MS; Ion mobility spectrometry;

A liquid chromatographic–tandem mass spectrometric method (LC–MS/MS) for the determination of ulifloxacin, the active metabolite of prulifloxacin, in human plasma is described. After sample preparation by protein precipitation with methanol, ulifloxacin and ofloxacin (internal standard) were chromatographically separated on a C18 column using a mobile phase consisting of methanol, water and formic acid (70:30:0.2, v/v/v) at a flow rate of 0.5 ml/min and then were detected using MS/MS by monitoring their precursor-to-product ion transitions, m/z 350 →  m/z 248 for ulifloxacin and m/z 362 →  m/z 261 for ofloxacin, in selected reaction monitoring (SRM) mode. Positive electrospray ionization was used for the ionization process. The linear range was 0.025–5.0 μg/ml for ulifloxacin with a lower limit of quantitation of 0.025 μg/ml. Within- and between-run precision was less than 6.6 and 7.8%, respectively, and accuracy was within 2.0%. The recovery ranged from 92.1 to 98.2% at the concentrations of 0.025, 0.50 and 5.0 μg/ml. Compared with the reported LC method, the present LC–MS/MS method can directly determine the ulifloxacin in human plasma without any need of derivatization. The present method has been successfully used for the pharmacokinetic studies of a prulifloxacin formulation product after oral administration to healthy volunteers.
Keywords: Prulifloxacin; Ulifloxacin; Liquid chromatography–tandem mass spectrometry; Human plasma;

Determination of platinum derived from cisplatin in human tissues using electrospray ionization mass spectrometry by Kayoko Minakata; Hideki Nozawa; Naoko Okamoto; Osamu Suzuki (286-291).
Determination of platinum (Pt) derived from cisplatin in tissues was performed by electrospray ionization mass spectrometry (ESI-MS) using silver (Ag) as the internal standard. Pt and Ag reacted with diethyldithiocarbamate (DDC), and were extracted using isoamylalcohol and acidified with oxalic acid. The compounds were termed Pt(DDC)3 + and Ag(DDC)2 +, based on their m/z values exhibiting the highest peaks at m/z 639 and m/z 405, respectively. The limit of detection was 30 pg and the quantitation range was from 100 to 10,000 pg using 5 mg tissue. The present method allowed the determination of Pt in wet-ashed tissue in 10 min.
Keywords: Platinum; Electrospray ionization; Mass spectrometry Diethyldithiocarbamate; Silver;

Quantification of 31 volatile organic compounds in whole blood using solid-phase microextraction and gas chromatography–mass spectrometry by Benjamin C. Blount; Robert J. Kobelski; David O. McElprang; David L. Ashley; John C. Morrow; David M. Chambers; Frederick L. Cardinali (292-301).
The prevalence of exposure to volatile organic compounds (VOCs) has raised concern about possible health effects resulting from chronic human exposure. To support studies exploring the relation between VOC exposure and health effects, we developed an automated analytical method using solid-phase microextraction (SPME), capillary gas chromatography (GC), and quadrupole mass spectrometry (MS). This method quantifies trace levels (low parts per trillion) of 14 halogenated alkanes, 5 halogenated alkenes, 10 aromatic compounds, and 2 other VOCs in human blood. Detection limits for the SPME–GC–MS method range from 0.005 to 0.12 μg/L, with linear calibration curves spanning three orders of magnitude. The improved throughput of this method will enable us to expand biomonitoring efforts to assess nonoccupational VOC exposure in large epidemiological studies.
Keywords: Volatile organic compound; VOC; Solid-phase microextraction; Mass spectrometry; Human; Whole blood;

Quantification of isosorbide 5-mononitrate in human plasma by liquid chromatography–tandem mass spectrometry using atmospheric pressure photoionization by Lara C. Silva; Lina S.O.B. Oliveira; Gustavo D. Mendes; Gabriel Garcia; Alberto dos Santos Pereira; Gilberto De Nucci (302-306).
Isosorbide 5-mononitrate (5-ISMN) is an organic nitrate widely used for its vasodilating properties in the treatment of angina pectoris. In the present study, an efficient, sensitive, robust method was developed for the determination and quantification of isosorbide 5-mononitrate, in human plasma, by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS), using photospray ionization. Isosorbide 5-mononitrate was extracted from 0.5 mL human plasma by liquid–liquid extraction (LLE). The method had a chromatographic run of 2.0 min using a C8 analytical column (100 mm × 2.1 mm i.d.) and the linear calibration curve over the range was linear from 20 to 2000 ng mL−1 (r 2  > 0.995). The between-run precision, based on the relative standard deviation replicate quality controls, was 7.9% (60 ng mL−1), 5.2% (300 ng mL−1) and 7.0% (1800 ng mL−1). The between-run accuracy was 94.9%, 94.1% and 88.8% for the above-mentioned concentrations, respectively. The method herein described was employed in a bioequivalence study of two tablet formulations of isosorbide 5-mononitrate 40 mg.
Keywords: Isosorbide 5-mononitrate; Photospray; Human plasma;

A liquid chromatographic method is described for the simultaneous determination of cefalexin and trimethoprim in dog plasma. A simple protein precipitation procedure was adopted for the sample preparation with satisfactory extraction recoveries for both analytes. Chromatographic separation of the analytes was achieved on a C18 column using a mixture of 2 mol/l formate buffer (pH 3.5), methanol and acetonitrile (22:7:7, v/v/v) containing a 0.002 mol/l sodium dodecyl sulfate as mobile phase and detection was performed at 240 nm. The linearity was obtained over the concentration ranges of 1.0–100.0 μg/ml for cefalexin and 0.5–50.0 μg/ml for trimethoprim. For each level of QC samples including the lower limit of quantification, both inter- and intra-day precisions (R.S.D.) were ≤14.0% for cefalexin and ≤11.4% for trimethoprim, and accuracy (RE) was −1.4% for cefalexin and −3.0% for trimethoprim. The present LC method was successfully applied to the pharmacokinetic studies of coformulated cefalexin dispersible tablets after oral administration to beagle dogs.
Keywords: Cefalexin; Trimethoprim; Liquid chromatography; Dog plasma;

In the present study, we developed a fast and reliable HPLC assay for the determination of the new triazole antifungal agent voriconazole in plasma, using a Chromolith® RP 18e (100 mm × 4.6 mm) monolithic silica rod HPLC column. After liquid–liquid extraction, plasma samples were separated with a mobile phase consisting of ammoniumdihydrogencarbonate buffer (pH 5.8)–acetonitrile–tetrahydrofuran (72:25:3) at a flow-rate of 3.5 mL/min and UV detection at 255 nm. The retention times for voriconazole and internal standard (UK-115794) were 2.3 and 2.7 min, respectively, and total run time was 4 min. The calibration curves were linear between 0.05 and 10 μg/mL, and within-assay and between-assay coefficients of variation were <4%. The proposed assay for voriconazole in plasma is fast, sensitive and reliable, and, thus, well suited for routine therapeutic monitoring of patients and for pharmacokinetic studies. It can be predicted that the use of monolithic silica rod chromatography will substantially shorten the turn-around time in the therapeutic drug monitoring laboratory.
Keywords: Voriconazole; Monolithic columns; HPLC; Therapeutic drug monitoring;

A simple HPLC assay to determine plasma concentration of tipranavir is presented. A liquid/liquid extraction of the drugs in ethyl acetate/hexane from 250 μL of plasma is followed by a reversed phase isocratic HPLC assay with UV detection at 205 nm. The imprecision and inaccuracy are lower than 10%, the low limit of quantitation is 0.4 mg/L. Thus, this method can be used for therapeutic drug monitoring of tipranavir in HIV infected patients
Keywords: Tipranavir;

Author Index (321-324).

Keyword Index (325-332).