Journal of Chromatography B (v.806, #2)

News Section (N1-N2).

The aim of this paper was to develop LC/MS/MS methodology for the determination of methamphetamine (METH) and amphetamine (AMP) using low microliter volumes (20–150 μl) of rat serum and demonstrate the use of this method for the study of serum pharmacokinetics in the rat. The analytes were extracted from rat serum using solid-phase extraction followed by an isocratic separation on a narrow-bore Hypersil C18 column. Lower limits of quantitation for METH and AMP were 0.3 ng/ml using positive ion electrospray tandem mass spectrometry. The accuracy of the method was within 20% of the actual values over a wide range of serum concentrations. The within-day and between-day precision was better than 20% (R.S.D.). Ion-suppression matrix effects on electrospray ionization were evaluated for extracted rat serum. The LC/MS/MS method was further validated by comparing serum concentrations of METH and AMP to serum concentrations previously determined using an LC/[ 3 H ]-METH assay with radiochemical detection. Finally, the LC/MS/MS method was used to study the pharmacokinetics of METH and AMP after a 1 mg/kg intravenous bolus dose of METH to female Sprague–Dawley rats.
Keywords: Methamphetamine; Amphetamine;

A solid phase microextraction (SPME)–HPLC–UV method for the determination of the immunosuppressant mycophenolic acid (MPA) in human serum samples was developed for the first time. The procedure, that employed a carbowax/templated resin (Carbowax/TPR-100) as fiber coating, required a very simple sample pretreatment, an isocratic elution, and provides an highly selective extraction. The linear range was 0.2–100 μg ml−1. Recovery was practically unchanged (63±4%) passing from 0.2 to 100 μg ml−1 level. Within-day and between-days coefficient of variation ranged from 5.9 to 6.5% and from 8.8 to 9.2%, respectively. A detection limit of 0.05 μg ml−1 was estimated in spiked serum. The method was successfully applied to the determination of MPA in serum of a patient under mycophenolate mophetil ester (MMF) therapy, as demonstrated by the relevant concentration-time profiles.
Keywords: Mycophenolic acid;

Heterogeneity of mitochondrial creatine kinase by Fusae Kanemitsu; Takeshi Kageoka; Shohei Kira (95-100).
The heterogeneity of cardiac sarcomeric mitochondrial creatine kinase (creatine N-phosphotransferase, EC 2.7.3.2, sMi-CK), namely, brain ubiquitous Mi-CK (uMi-CK) and an atypical Mi-CK detected in the serum of a patient with ovarian cancer, was studied by isoelectric focusing. These Mi-CKs were found to be slightly different from each other with respect to their pIs under the examined conditions. The atypical Mi-CK was found to be an atypically oxidized form of uMi-CK. Results suggest that these heterogeneities of Mi-CK are caused by the genotypes, structures, biological functions and metabolism/dissimilation of Mi-CKs in the mitochondria and intravascular circulation.
Keywords: Creatine kinase;

Determination of isoflavones in soybean food and human urine using liquid chromatography with electrochemical detection by Bořivoj Klejdus; Jan Vacek; Vojtěch Adam; Josef Zehnálek; René Kizek; Libuše Trnková; Vlastimil Kubáň (101-111).
A highly sensitive high-performance liquid chromatographic method with electrochemical detection (HPLC-ED) was developed for the determination of isoflavones. Electrochemical behaviour of daidzein and genistein was studied on carbon paste electrode (CPE) by adsorptive transfer stripping square wave voltammetry. The obtained electrochemical results were used for the development of HPLC-ED method. Furthermore, isoflavones were separated on an Atlantis dC18 column using a mobile phase consisting of acetonitrile (solvent A) and 0.15 M acetate buffer of pH 5.5 (solvent B) at a flow rate 0.4 mL/min. A linear gradient profile (solvent B) was at 0–2 min 87%; 22 min 60%; 27 min 50%; 31 min 45%; 47 min 87%. Full scan of multi-channel coulometric detection was tested and optimal potential at 450 mV was chosen for our purposes. Calibration curves were linear (daidzein R 2=0.9993 and genistein R 2=0.9987). The detection limit for daidzein/genistein was 480/394 pg/mL (1.8/1.5 nM) and per column 2.4/1.9 pg. Isoflavones extracted from soybean products (farina, meat, milk) by the accelerated solvent extraction (ASE) procedure and isoflavones present in human urine were determined by the HPLC-ED method.
Keywords: Isoflavones;

A sensitive, rapid and reproducible assay for the determination of glutaminyl cyclase activity is reported. This method is based on the monitoring of the absorption of l-pyroglutamic acid β-naphthylamide at 235 nm, enzymatically formed from the substrate l-glutaminyl-β-naphthylamide, after separation by high-performance liquid chromatography using a C-18 reversed-phase column by isocratic elution. The detection limit of this method is at a level as low as 0.08 nmol/ml and, the time consumed for analysis is <6.5 min per sample for separation and quantification. The optimum pH for glutaminyl cyclase activity was 8.0–8.5. The K m and V max values were 100.2±2.9 μM and 332±21.7 pmol/(h μg protein), respectively, with the use of enzyme extract obtained from bovine pituitary. Glutaminyl cyclase activity was strongly inhibited by zinc(II) ion and 1,10-phenanthroline. By using this assay, the stimulatory effect of bacterial lipopolysaccharide on this enzyme activity was observed in macrophage cell line RAW 264.7. Our newly developed assay would be useful for clarification of the physiological role of this enzyme.
Keywords: Glutaminyl cyclase; Enzymes;

A method is described for on-line high-speed extraction of microsomal samples and analysis by capillary liquid chromatography–mass spectrometry (LC–MS) for the determination of metabolic stability in connection with the development of positron emission tomography (PET) tracers. The method allowed direct injections of large sample volumes at a fast extraction rate, providing a gain in both sensitivity and sample preparation time. The calibration curve of the test compound flumazenil (Ro 15–1788) was linear in the concentration range of 1–150 nM, with a correlation coefficient exceeding 0.999. The accuracy of the method ranged from 98 to 101%. A high precision was obtained, with mean intra-assay and inter-assay relative standard deviations of at most 1.4 and 1.5%, respectively, for quality control (QC) samples. The extraction efficiency was determined to be 99.4%, the total recovery 96% and the carryover to ≤0.23%. Extractions were performed in a concentration interval of 30–3000 nM without any sign of column overload. The method was successfully used for determining the microsomal metabolic stability of flumazenil. As a result, the described analysis system is currently used for metabolic screening of PET tracer candidates in our laboratory.
Keywords: High-flow-rate extraction; Metabolic stability; Positron emission tomography;

A sensitive and rapid high-performance liquid chromatography (HPLC) method with solid-phase extraction (SPE) to simultaneously determine albiflorin and paeoniflorin in rat serum was described. Serum samples were pretreated with solid-phase extraction using Extract-Clean™ cartridges, and the extracts were analyzed by HPLC on a reversed-phase C18 column and a mobile phase of acetonitrile-0.03% formic acid (17:83 (v/v)) with ultraviolet detection at 230 nm. Pentoxifylline was used as the internal standard (IS). The linear ranges of the calibration curves were 29–1450 ng/ml for albiflorin and 10–2000 ng/ml for paeoniflorin. The intra- and inter-day precisions (R.S.D.) were ≤10.49% for albiflorin and ≤11.29% for paeoniflorin, respectively. Mean recovery was determined to be 89.75% for albiflorin and 85.82% for paeoniflorin. The limit of quantification was 29 ng/ml for albiflorin and 10 ng/ml for paeoniflorin, respectively. The validated method was applicable to pharmacokinetic studies of albiflorin and paeoniflorin from rat serum after oral administration of Si–Wu decoction. The pharmacokinetic study indicated that albiflorin and paeoniflorin had poor absorption and rapid elimination. This assay result was necessary for the pharmacokinetic evaluation of Si–Wu decoction.
Keywords: Albiflorin; Paeoniflorin;

Liquid chromatographic–fluorimetric method for the estimation of nitric oxide biosynthesis in the central nervous system by Iván Pérez-Neri; Sergio Montes; Marie-Catherine Boll; Jesús Ramı́rez-Bermúdez; Camilo Rı́os (133-139).
Some amino acids are involved in the biosynthesis of nitric oxide (NO), which has a physiological and pathophysiological role. To study NO biosynthesis, we compared arginine and citrulline levels in the cerebrospinal fluid (CSF) from patients with infectious and/or inflammatory processes within the central nervous system (CNS), with those from patients without those disorders. Arginine concentration was not significantly different between the groups (P=0.115), whereas citrulline was significantly elevated in the first group (P=0.020). We propose a simple chromatographic method to estimate NO biosynthesis ex vivo within the CNS, that may be applicable for the study of neurodegenerative and psychiatric diseases such as Parkinson’s disease and schizophrenia.
Keywords: Nitric oxide; Arginine; Citrulline;

Demonstrating the presence or absence of cocaine (COC) and COC-related molecules in postmortem fluids and/or tissues can have serious legal consequences and may help determine the cause of impairment and/or death. We have developed a simple method for the simultaneous determination of COC and the COC metabolites benzoylecgonine (BE), norbenzoylecgonine (NBE), ecgonine methyl ester (EME), ecgonine (E), and norcocaine (NCOC), as well as anhydroecgonine methyl ester (AEME) (a unique byproduct of COC smoking), cocaethylene (a molecule formed by the concurrent use of COC and ethanol) and their related metabolites, anhydroecgonine (AE), norcocaethylene (NCE), and ecgonine ethyl ester (EEE). This method incorporates a Zymark® RapidTrace™ automated solid-phase extraction (SPE) system, gas chromatography/mass spectrometry (GC/MS) and 2,2,3,3,3-pentafluoro-1-propanol (PFP)/pentafluoropropionic anhydride (PFPA) derivatives. The lower limits of detection ranged from 0.78 to 12.5 ng/mL and the linear dynamic range for most analytes was 0.78–3200 ng/mL. The extraction efficiencies were from 26 to 84% with the exception of anhydroecgonine and ecgonine, which were from 1 to 4%. We applied this method to five aviation fatalities. This method has proven to be simple, robust and accurate for the simultaneous determination of COC and 11 COC metabolites in postmortem fluids and tissues.
Keywords: Cocaine; Cocaine, pyrolysis products; Cocaine-ethanol adducts;

The presence of electrical charges on the surface of an organelle is the source of the organelle’s electrophoretic mobility. Recently, we reported that capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) can be used to determine the electrophoretic mobility of individual mitochondria. Here, we describe the use of CE-LIF to monitor changes in the electrophoretic mobility distributions of: (i) mitochondria isolated from cultured NS-1 mouse hybridoma cells disrupted by nitrogen cavitation or mechanical homogenization; (ii) mitochondria isolated from rat liver and purified by gradient centrifugation before and after being frozen in liquid nitrogen; and (iii) mitochondria chemically transformed into mitoplasts. These results indicate that the organelle electrophoretic mobility observed by researchers is affected by preparation procedures and that CE-LIF is a complementary technique for monitoring the quality of mitochondrial preparations.
Keywords: Electrophoretic mobility; Mitochondrial preparations;

A method coupled with microdialysis technique and liquid chromatography was applied in the continuous and concurrent in vivo monitoring of extracellular hesperidin in the blood and bile of anaesthetized rats. Hesperidin was intravenously administered via the femoral vein. Sampling was achieved using two microdialysis probes, which were implanted into the jugular vein and into the bile duct. Dialysates of blood and bile were both directly injected onto the liquid chromatographic system, so no further clean-up procedures were required. Separation was performed using a reversed phase ODS-2 microbore column 150  mm×1  mm i.d., particle size 5 μm with mobile phase of acetonitrile–0.1 M ammonium acetate (30:70, v/v) at flow-rate of 0.05 ml/min. The UV detection for hesperidin was set at a wavelength of 283 nm. This method was used to determine the pharmacokinetics of hesperidin and its interaction in the presence of cyclosporin A, which is a P-glycoprotein modulator. The results indicate that the curve of area under the concentration versus time (AUC) for hesperidin in bile was significantly greater than that for hesperidin in blood at the dose of 30 mg/kg. The blood-to-bile distribution ratio (k=AUCbile/AUCblood) was 8.9±2.5 for hesperidin at 30 mg/kg. Following cyclosporin A treatment, the distribution ratio was reduced to 3.2±0.6. In conclusion, hesperidin goes through hepatobiliary elimination against the concentration gradient from blood to bile, and this hepatobiliary excretion of hesperidin may be regulated by the P-glycoprotein.
Keywords: Hesperidin;

High throughput LC–MS/MS assays to quantitate a new ανβ3 bone integrin antagonist (I) in human plasma and urine have been developed using instruments programmed to automate sample preparation procedures. Packard liquid handling system-MultiPROBE® II EX was programmed for preparing calibration standards in control plasma and urine, acidifying all standards, quality control (QC), and clinical samples with necessary dilutions, and adding the internal standard to the acidified samples. TOMTEC Quadra 96™ was programmed to perform the solid phase extraction (SPE) process on a 3 M 96-well mixed phase cation standard density (MPC-SD) plate to isolate the analytes from the sample matrix. The extract collected from both types of matrices was directly injected into reversed-phase LC–MS/MS system with a Turbo Ion Spray (TIS) interface in the positive ionization mode. The plasma and urine assays have the calibration range of 0.5–1500 and 2–6000 ng/mL, respectively. Validation of the automated and the manual plasma assays showed that application of MultiPROBE II to sample preparation gave comparable accuracy and precision. Overall, the automated approaches with minimum manual intervention enhanced the throughput of sample preparation.
Keywords: Integrin;

A novel method was developed and assessed to extend the lifetime of extraction columns of high-throughput liquid chromatography (HTLC) for bioanalysis of human plasma samples. In this method, a 15% acetic acid solution and 90% THF were respectively used as mobile phases to clean up the proteins in human plasma samples and residual lipids from the extraction and analytical columns. The 15% acetic acid solution weakens the interactions between proteins and the stationary phase of the extraction column and increases the protein solubility in the mobile phase. The 90% THF mobile phase prevents the accumulation of lipids and thus reduces the potential damage on the columns. Using this novel method, the extraction column lifetime has been extended to about 2000 direct plasma injections, and this is the first time that high concentration acetic acid and THF are used in HTLC for on-line cleanup and extraction column lifetime extension.
Keywords: Drug analysis; Column lifetime;

Purification of recombinant bovine normal prion protein PrP(104–242) by HPHIC by Chaozhan Wang; Xindu Geng; Dawei Wang; Bo Tian (185-190).
Purification of the prion protein (PrP) is a major concern for biological or biophysical analysis as are the structural specificities of this protein in relation to infectivity. A simple and efficient method for purification of recombinant bovine normal prion protein containing residues 104–242, PrP(104–242) expressed in Escherichia coli by high performance hydrophobic interaction chromatography (HPHIC) was presented in this work. The solution containing denatured and reduced protein in 8.0 mol/L urea extracted from the inclusion body was directly injected into the HPHIC column, aggregates were prevented by the interaction between the denatured PrP(104–242) molecules and the stationary phase during the chromatographic process, the soluble form of PrP(104–242) in aqueous solution was obtained after desorbed from the column. Several factors, including pH value, types of stationary phase and salt, and gradient mode, influencing the purification results were investigated. Optimal conditions were obtained for the purification of PrP(104–242) by HPHIC. This procedure yield PrP(104–242) of a purity of 96% with a recovery of 87%, respectively, for a single step purification of 40 min.
Keywords: Protein refolding; Bovine normal prion protein;

A high-throughput method for quantitative determination of methadone enantiomers in human plasma was developed and validated by liquid chromatography/tandem mass spectrometry. The effects of pH and of types and concentrations of mobile-phase modifiers on the enantioselectivity of (R)- and (S)-methadone were investigated on a Chiral-AGP column. A baseline separation of the enantiomers was achieved with a retention time of less than 5 min. Ionization suppression and other matrix effects were evaluated. Morphine, cocaine, 6-monoacetylmorphine, benzoylecgonine and ecgonine methyl ester did not interfere with the performance of the assay. The specificity, linearity, intra- and inter-assay precision and accuracy, and extraction recovery were fully evaluated. The method showed excellent reproducibility (overall coefficient of variance < 8%) and accuracy (overall bias < 2.7%) with a broad linear range. The enantiomers were stable in human plasma after five freeze-thaw cycles, under bench-top storage at room temperature (RT) for 6 h, in the extract reconstitution solution at RT for 17 h, and in processed-extracts stored at RT for 142 h. This validated LC/MS/MS assay offers high-throughput and improved specificity, sensitivity, linear range and ruggedness over previously published methods and has been successfully applied to the analysis of clinical samples.
Keywords: Enantiomer separation; Methadone;

A sensitive and selective method for the determination of hydrochlorothiazide (HCTZ) concentrations in rat plasma was developed using high performance liquid chromatography–electrospray ionization tandem mass spectrometry (LC–MS/MS). An aliquot of plasma (50 μl) was mixed with the solution of internal standard, hydrofluorothiazide (HFTZ), and extracted with tert-butyl methyl ether. The reconstituted extract was applied to the LC–MS/MS system with a reversed phase C8 column and eluted with distilled water/acetonitrile (85/15, v/v). To enhance negative ionization of HCTZ and HFTZ in the multiple reaction monitor (MRM), the solution consisting of acetonitlile/1% (v/v) ammonia solution (95/5, v/v) was delivered after column separation. This additional technique, so-called the post-column addition, increased sensitivity of HCTZ and HFTZ about 500- and 200-fold, respectively. The calibration curve showed good linearity (r=0.999) over the range of 4–1000 ng/ml. Acceptable accuracy (100.8–113.1%) and precision (0.28–16.4%) were confirmed in the intra- and the inter-day analyses. It is indicated that this LC–MS/MS method is useful for pharmacokinetic studies of HCTZ in small animals, because it enabled the serial determination of plasma level of HCTZ in rats.
Keywords: Hydrochlorothiazide;

A comprehensively validated procedure is presented for simultaneous semiquantitative/quantitative screening of 51 drugs of abuse or drugs potentially hazardous for traffic safety in serum, plasma or whole blood. Benzodiazepines (12), cannabinoids (3), opioids (8), cocaine, antidepressants (13), antipsychotics (5) and antiepileptics (2) as well as zolpidem, zaleplon, zopiclone, meprobamate, carisoprodol, tizanidine and orphenadrine and internal standard flurazepam, were isolated by high-yield liquid-liquid extraction (LLE). The dried extracts were derivatized by two-step silylation and analyzed by the combination of two different gas chromatographic (GC) separations with both electron capture detection (ECD) and mass spectrometry (MS) operating in a selected ion-monitoring (SIM) mode. Quantitative or semiquantitative results were obtained for each substance based on four-point calibration. In the validation tests, accuracy, reproducibility, linearity, limit of detection (LOD) and limit of quantitation (LOQ), selectivity, as well as extraction efficiency and stability of standard stock solutions were tested, and derivatization was optimized in detail. Intra- and inter-day precisions were within 2.5–21.8 and 6.0–22.5%, and square of correlation coefficients of linearity ranged from 0.9896 to 0.9999. The limit of quantitation (LOQ) varied from 2 to 2000 ng/ml due to a variety of the relevant concentrations of the analyzed substances in blood. The method is feasible for highly sensitive, reliable and possibly routinely performed clinical and forensic toxicological analyses.
Keywords: Screening; Derivatization, GC; Drugs of abuse;

Milnacipran is an antidepressant drug belonging to the class of serotonin and noradrenaline reuptake inhibitors. A sensitive high performance liquid chromatographic during the development method coupled with a fluorimetric detection was set up, validated and then used routinely of the drug. After liquid–liquid extraction, milnacipran and its internal standard were analyzed by reversed-phase liquid chromatography (LC). The drug was derivatized with fluorescamine for fluorescence detection. The identity of the liquid chromatography peaks was controlled using mass spectrometry. The assay linearity was validated up to 1000 ng/ml. The limit of quantification was set at 5 ng/ml. Precision values (relative standard deviations) were lower than 5.4%, whereas the mean accuracy was higher than 95%. The extraction recoveries were higher than 70% for both milnacipran and the internal standard. In clinics, the LC-fluorescence method was routinely used to investigate the pharmacokinetics of milnacipran in patients and proved to be robust and capable of quantifying milnacipran in plasma for at least 36 h (four- to five-fold the elimination half-life).
Keywords: Milnacipran;

Interval immobilization technique for recognition toward a highly hydrophilic cyanobacterium toxin by Takuya Kubo; Ken Hosoya; Yoshiyuki Watabe; Nobuo Tanaka; Hiroo Takagi; Tomoharu Sano; Kunimitsu Kaya (229-235).
A novel adsorption medium containing selective molecular recognition site for one of the powerful cyanobacterium toxins, Cylindrospermopsin (CYN) was developed using a special technique, namely interval immobilization technique. The adsorption medium was prepared using molecular assembly derived from an alternative-template molecule coupled with functional monomers for fixing the interval between the ionic functional groups in CYN. As results of liquid chromatographic evaluations, selective molecular recognition ability for CYN was observed as expected. Further studies proved that the association constant for CYN on this medium was slightly higher than that on blank polymer.
Keywords: Interval immobilization technique; Cylindrospermopsin;

Capillary electrophoresis with laser-induced fluorescence detection (CE-LIFD) coupled to in vivo microdialysis sampling was used in order to monitor simultaneously a drug and several neurotransmitters in the brain extracellular fluid. Determination of the antiepileptic drug vigabatrin and the amino acid neurotransmitters glutamate (Glu), l-aspartate (l-Asp) and γ-aminobutyric acid (GABA) was performed on low-concentration samples which were derivatized with naphthalene-2,3-dicarboxaldehyde (NDA) and separated using a pH 9.2 75 mM sodium borate running buffer containing 60 mM sodium dodecyl sulfate (SDS) and 5 mM hydroxypropyl-β-cyclodextrin (HP-β-CD). Glu, l-Asp and vigabatrin derivatized at a concentration of 1.0×10−9  M, and GABA derivatized at a concentration of 5.0×10−9  M, produced peaks with signal-to-noise ratios of 8:1, 8:1, 4:1 and 5:1, respectively. The nature of the neurotransmitter peaks found in rat brain microdialysates was confirmed by both electrophoretic and pharmacological validations. This method was used for monitoring vigabatrin and amino acid neurotransmitters in microdialysates from the rat striatum during intracerebral infusion of the drug and revealed rapid vigabatrin-induced changes in GABA and Glu levels. This original application of CE-LIFD coupled to microdialysis represents a powerful tool for pharmacokinetic/pharmacodynamic investigations.
Keywords: Vigabatrin; Amino acid neurotransmitters;

Described is a liquid chromatography–mass spectrometry (LC–MS) procedure for the determination of hydroxylated biotransformation products of polycyclic aromatic hydrocarbons (PAH) in the human gastrointestinal tract. The formation of hydroxylated PAHs was monitored upon incubation of PAHs with colon microbiota from the Simulator of the Human Intestinal Microbial Ecosystem (SHIME). The analytical method consisted of a biomass removal step followed by a solid phase extraction (SPE) step using C18 packed columns to remove non-digested food compounds and microbial metabolites that interfere with the detection of the target compounds. For quantification, 9-hydroxyphenanthrene 13 C 6 was used as the internal standard. The detection limits of the hydroxylated PAHs were generally in the range 0.36–14.09 μg l−1, based on a signal/noise ratio of 3:1. The recovery of hydroxylated PAHs in intestinal suspension was variable ranging from 45 to 107%, with relative standard deviation (R.S.D.) between 5 and 17%. The analytical procedure was used to show the microbial production of 1-hydroxypyrene and 7-hydroxybenzo(a)pyrene, metabolites that may give colon incubated PAHs bioactive properties.
Keywords: Polynuclear aromatic hydrocarbons;

Determination of fosmidomycin in human serum and urine by capillary electrophoresis by Stéphane Bronner; Corinne Renault; Martin Hintz; Jochen Wiesner; Hassan Jomaa; Henri Monteil; François Jehl (255-261).
A capillary electrophoresis method with direct UV detection was developed for the determination of fosmidomycin, a promising new anti-malarial drug, in human serum and urine. Optimization of the separation parameters resulted in a buffer system adjusted to pH 10.8 containing a cationic reagent and an organic modifier. Under these conditions, the migration time of fosmidomycin was 5.2 min with serum and 7.4 min with urine samples. Validation of the method revealed good recoveries, precision and accuracy. The limit of quantification was 0.5 μg/ml in serum and 10 μg/ml in urine. The determination of fosmidomycin in serum was linear over a range of 0.1–150 μg/ml. Short and long-term stability tests resulted in no significant loss of fosmidomycin. The described technique will provide a fast and accurate analytical method for future pharmacokinetic studies.
Keywords: Fosmidomycin;

HPLC methodology was investigated for the simultaneous determination of cisapride and ranitidine in small volume paediatric plasma samples. Such a simultaneous determination proved difficult due to the small sample volumes, the low concentrations of the drugs and the different log  P values of the two compounds. The two drugs and their respective internal standards were separated “on-cartridge” using HLB Solid Phase Extraction cartridges and the samples quantified by individual HPLC methodologies. The technique has been applied successfully to 60 paediatric plasma samples containing both cisapride and ranitidine.
Keywords: Ranitidine; Cisapride;

A simple, rapid and sensitive method for determination of rivastigmine in plasma samples was developed using headspace solid-phase microextraction (HS-SPME) and gas chromatography with mass spectrometry (GC–MS). The optimum conditions for the SPME procedure were: headspace extraction on a 65-μm polydimethylsiloxane/divinylbenzene (PDMS/DVB) fiber; 0.5 ml of plasma modified with 1.0 ml of sodium hydroxide-sodium carbonate solution (0.7 M:0.5 M); extraction temperature of 100 °C, with stirring at 2000 rpm for 30 min. The calibration curve showed linearity in the range from 0.2 to 80 ng/ml with regression coefficient corresponding to 0.9965 and coefficient of the variation of the points of the calibration curve lower than 10%. The quantification limit for rivastigmine in plasma was 0.2 ng/ml. The method was applied to determination of rivastigmine in canine plasma samples from animals after a single oral administration.
Keywords: Rivastigmine;

A simple, rapid, sensitive and specific reversed-phase high performance liquid chromatographic (RP-HPLC) method involving ultraviolet detection (λ=210 nm) was developed for analysis of indinavir along with propranolol in samples obtained from ex vivo intestinal permeability studies. Chromatography was carried out on C-18 column with mobile phase comprising of phosphate buffer-acetonitrile (68:32, v/v) pumped at flow rate of 1 ml/min. The proposed method has a short run time of 12 min and involves a simple sample preparation for the purpose of reducing permeability model artifacts and to concentrate the samples. Fluorescein was used as internal standard. The proposed method has been validated with regard to specificity, detection limit, recovery, accuracy and precision. For both the drugs, method was found to be selective, linear (R 2≈0.999), accurate (recovery = 100–105%) and precise (<3% R.S.D.) in the range of 2–20 μg/ml. The limit-of-detection and limit-of-quantification of the method were 40 ng/ml and 100 ng/ml for indinavir, and 30 and 80 ng/ml for propranolol, respectively. Indinavir, a widely prescribed HIV protease inhibitor, suffer from bioavailability problems where involvement of P-glycoprotein mediated drug efflux may play a significant role. The proposed method was successfully applied for intestinal permeability of indinavir to estimate the contribution of P-glycoprotein in limiting its oral bioavailability. The advantage of the developed method lies in the simultaneous determination of propranolol, a passive integrity marker, routinely employed in permeability studies and its selectivity in presence of various P-gp modulators and permeability markers.
Keywords: Indinavir; Propranolol;

Solid phase extraction procedure for urinary organic acid analysis by gas chromatography mass spectrometry by Aiping Liu; Mark M. Kushnir; William L. Roberts; Marzia Pasquali (283-287).
We have developed a solid phase extraction procedure for the detection of organic acids by GC–MS using a strong anion exchange column (Sep-Pak Vac RC, Accell Plus QMA cartridge). Extraction efficiencies of 25 organic acids were established by analyzing standards in water based solutions. High extraction efficiencies (90 to 100%) were found for many of the compounds studied. We estimated the limit of detection for 48 organic acids and glycine conjugates. They were below 5 nmole with the exception of malonic and oxalic acids and mevalonic acid lactone. This method provides the advantage of higher recoveries for a wide range of compounds of interest and therefore can be a potential alternative to liquid–liquid extraction for organic acid screening. It is especially sensitive for the detection of some polar compounds, such as 3-OH-glutaric and N-acetylaspartic acids.
Keywords: Organic acids;

Determination of the antiangiogenesis agent 2-methoxyestradiol in human plasma by liquid chromatography with ultraviolet detection by Nehal J Lakhani; Alex Sparreboom; William L Dahut; Jürgen Venitz; William D Figg (289-293).
A high-performance liquid chromatography (HPLC) assay was developed for the quantitative determination of 2-methoxyestradiol (2ME2) in human plasma. Sample pretreatment involved a solid-phase extraction of 1 ml aliquots of plasma with C18 micro-columns. Separation was achieved on a Novapak C18 column (300  mm×3.9  mm i.d.; 4 μm PS) at room temperature at an isocratic flow rate of 1 ml/min with 50% acetonitrile in water. Detection was performed at a UV wavelength of 205 nm. Calibration curves were linear in the concentration range of 1–50 ng/ml. The accuracy and precision values obtained from three different sets of quality controls analyzed in replicates of four on four separate occasions ranged from 90.7 to 105.2 and 3.17 to 8.27%, respectively.
Keywords: 2-Methoxyestradiol;

A previously described method of measurement of malondialdehyde (MDA) in human urine after derivatisation with 2,4-dinitrophenylhydrazine (DNPH) was tested for a possibility of using methyl malondialdehyde (MeMDA) as an internal standard. Despite structural similarity, those compounds were found to produce different yields of derivatisation under the same conditions depending on urine matrix. We conclude, that MeMDA is not suitable as an internal standard for the measurement of MDA in urine under previously reported conditions when DNPH is used as a deriviatising agent.
Keywords: Derivatisation, LC; Methyl malondialdehyde; 2,4-Dinitrophenylhydrazine;

A rapid and sensitive high performance liquid chromatography (HPLC) method with fluorescence detection has been developed for the determination of sumatriptan in human plasma. The procedure involved a liquid–liquid extraction of sumatriptan and terazosin (internal standard) from human plasma with ethyl acetate. Chromatography was performed by isocratic reverse phase separation on a C18 column. Fluorescence detection was achieved with an excitation wavelength of 225 nm and an emission wavelength of 350 nm. The standard curve was linear over a working range of 1–100 ng/ml and gave an average correlation coefficient of 0.9997 during validation. The limit of quantitation (LOQ) of this method was 1 ng/ml. The absolute recovery was 92.6% for sumatriptan and 95.6% for the internal standard. The inter-day and intra-day precision and accuracy were between 0.8–3.3 and 1.1–6.3%, respectively. This method is simple, sensitive and suitable for pharmacokinetics or bioequivalence studies.
Keywords: Sumatriptan;

Determination of midazolam in human plasma by liquid chromatography with mass-spectrometric detection by Erin R. Lepper; J.Kevin Hicks; Jaap Verweij; Suoping Zhai; William D. Figg; Alex Sparreboom (305-310).
A liquid chromatographic assay with mass-spectrometric detection was developed for the quantitative determination of the cytochrome P450 3A phenotyping probe midazolam in human plasma. Sample pretreatment involved a one-step extraction of 600 μl aliquots with ethyl acetate. Midazolam and the internal standard, lorazepam, were separated on a column (150  mm×4.6  mm , i.d.) packed with 5 μm Zorbax Eclipse XDB-C8 material, using a mobile phase composed of methanol and 10 mM aqueous ammonium acetate (60:40, v/v). Column effluents were analyzed using mass-spectrometry with an atmospheric pressure chemical ionization source. Calibration curves were linear in the concentration range of 1.00–200 ng/ml. The accuracy and precision ranged from 92.8 to 112% and 0.056 to 13.4%, respectively, for four different concentrations of quality control samples analyzed in triplicate on eight separate occasions. The developed method was subsequently applied to study the pharmacokinetics of midazolam in a group of 35 human subjects at a single dose of 25 μg/kg.
Keywords: Midazolam;

Fumonisin-ortho-phthalaldehyde derivative is stabilized at low temperature by Lonnie D Williams; Filmore I Meredith; Ronald T Riley (311-314).
Fumonisins are water soluble mycotoxins produced by the fungus Fusarium verticillioides (formerly F. moniliforme). Fumonisin B1 (FB1) is a diester of propane-1,2,3-tricarboxylic acid and 2-amino-12, 16-dimethyl-3,5,10,14,15-pentahydroxyeicosane, and is the most abundant of the naturally occurring fumonisins. Upon removal of the two tricarballylic acid side chains, the structure is referred to as hydrolyzed FB1 (HFB1). FB1 and HFB1 are structurally similar to sphinganine, a sphingoid base. The fumonisins do not absorb UV light or fluoresce; therefore, derivatizing reagents are used for detection when separation is by high performance liquid chromatography (HPLC). The standard derivatizing reagent used for HPLC is ortho-phthalaldehyde (OPA) plus 2-mercaptoethanol (ME) reaction partner, however, the OPA-FB1 derivative is not stable at room temperature. The objectives of this study were to: (1) determine the effect of temperature on the stability of the OPA-FB1 derivative and (2) determine which structural characteristics of FB1 contribute to the instability of the OPA-FB1 derivative. The results indicate that OPA-FB1, OPA-FB3 and OPA-HFB1 derivatives are unstable at 24 °C but that their stability improves significantly at 4 °C. The OPA-sphinganine derivative is stable for at least 24 h at 24 °C. Thus, the instability of the OPA-FB1 derivative may be attributed to its lack of a hydroxyl group at the carbon 1 position.
Keywords: Derivatization, LC; Fumonisin;

Author Index (315-317).

Compound Index (319-320).