Journal of Chromatography B (v.854, #1-2)

A rapid, sensitive and specific liquid chromatographic-electrospray ionization (ESI) tandem ion trap mass spectrometric method has been developed for identification of dauricine and its metabolites in rat urine. Six healthy rats were administrated a single dose (100 mg/kg) of dauricine by oral gavage. The urine were sampled from 0 to 24 h and purified by using a C18 solid-phase extraction (SPE) cartridge, then the purified urine samples were separated on a reversed-phase C18 column using methanol/2 mmol/L ammonium acetate (70:30, v/v, adjusted to pH 3.5 with formic acid) as mobile phase and detected by an on-line MS detector. Identification and structural elucidation of the metabolites were performed by comparing their changes in molecular mass (Δm) and full scan MS n spectra with those of the parent drug. At least eight metabolites (such as N-demethyl, dehydrogenate, demethoxyl, hydroxyl, glucuronide conjugated and sulfate conjugated metabolites) and the parent drug were found in rat urine.
Keywords: Dauricine; HPLC–MS n ; Metabolite;

A novel mixed-mode high performance liquid chromatographic system (HPLC) interfaced with an atmospheric pressure chemical ionization (APCI) source and a tandem mass spectrometer (MS/MS) was developed for the determination of cytarabine (ara-C) in mouse plasma to support pharmacodynamic studies. The mixed-mode reversed-phase ion-exchange chromatography column was adapted for sufficient retention and separation of a small and polar analyte. The impact of the mobile phase composition on both chromatographic separation and the ionization efficiency of the test compound in the positive mode was investigated. The potential of ionization suppression from endogenous biological matrices on the mixed-mode LC–APCI/MS/MS method was evaluated using the post-column infusion technique. Furthermore, the feasibility of using the mixed-mode HPLC–MS/MS method for the determination of the plasma concentrations of cytarabine in mice was demonstrated by comparing those obtained by the ion-pairing HPLC–MS/MS method.
Keywords: Mixed-mode liquid chromatography; Tandem mass spectrometry; Cytarabine; Matrix ionization suppression;

Biotechnology industry has recently been demanding nanoparticulate products (20–200 nm) such as viruses, plasmids, virus-like particles and drug delivery assemblies. These products are mainly used as gene delivery systems in gene therapy protocols. During the process development for the manufacture of these products, it is crucial to optimize the recovery and purification steps. Unfortunately, the high value of some bio-nanoparticles complicates the optimization studies. The solvent extraction method with aqueous two-phase systems (ATPS) has been used to successfully recover bioproducts on a large scale. In this study, the potential miniaturization of ATPS is presented. The partition behavior of pure bovine serum albumin (BSA) in PEG-800-phosphate and bacteriophage T4 in PEG 8000-phosphate and PEG 600-sulphate systems were studied at three different scales (10 g, 2 g and 300 μl). The results obtained showed that the volume ratio (V R) for BSA (V R  = 1.0) was comparable to the blank systems at the scales studied. Additionally, the partition coefficient (K) was also similar (K  = 0.05) with more than 82% of BSA concentrated in the bottom phase. Same system was challenged with bacteriophage T4 showing a V R  = 1.0 and K greater than 5 with the infective particles concentrated in the top phase. The bacteriophage T4 was concentrated in opposite phase in the PEG-600-sulfate system with a consistent V R  = 0.8 and K  < 0.2 for the scales analyzed. The partition behavior the bacteriophage T4 was comparable to that reported previously for adenoviral vectors in same system at 15 ml scale. The results obtained demonstrated that the miniaturization of ATPS is feasible and reproducible for the two models selected. This provides significant information about the miniaturization process of such ATPS for their potential generic applications in the recovery of different bio-nanoparticle products.
Keywords: Nanoparticulate; Aqueous two-phase system; Recovery; Bacteriophage T4;

GC/MS analysis of the rat urine for metabonomic research by Qi Zhang; Guangji Wang; Yu Du; Lingling Zhu; Jiye A (20-25).
In this paper, an optimized protocol was established and validated for the metabonomic profiling in rat urine using GC/MS. The urine samples were extracted by methanol after treatment with urease to remove excessive urea, then the resulted supernatant was dried, methoximated, trimethylsilylated, and analyzed by GC/MS. Forty-nine endogenous metabolites were separated and identified in GC/MS chromatogram, of which 26 identified compounds were selected for quantitative analysis to evaluate the linearity, precision, and sensitivity of the method. It showed good linearity between mass spectrometry responses and relative concentrations of the 26 endogenous compounds over the range from 0.063 to 1.000 (v/v, urine/urine + water) and satisfactory reproducibility with intra-day and inter-days precision values all below 15%. The metabonomic profiling method based on GC/MS was successfully applied to urine samples from hyperlipidemia model rats. Obviously, separated clustering of model rats and the control rats were shown by principal components analysis (PCA); time-dependent metabonomic modification was detected as well. It was suggested that metabonomic profiling based on GC/MS be a robust method for urine samples.
Keywords: Metabonomics; Urine; GC/MS;

Ultra-high pressure liquid chromatography (UHPLC) is a relatively new technology which utilizes chromatographic media with a 1.7 μm particle size. This technology has the potential to offer significant advantages in resolution, speed, and sensitivity for analytical determinations, particularly when coupled with mass spectrometric detection. Drug Candidate A, under development at Merck Research Laboratories, contains two chiral centers which have the absolute configuration R, S. Under in vivo and ex vivo conditions, one of the chiral centers readily epimerizes to produce the R, R diastereomer. Initially, a traditional high performance liquid chromatography/tandem mass spectrometry (HPLC–MS/MS) method was developed to separate and quantify these two diastereomers in rat plasma. The lower limit of quantification (LOQ) of the two analytes was 2 ng/mL, and a chromatographic run time of approximately 11 min was needed to separate R, S-(A) and R, R-(A). In this study, we explored a simple and robust UHPLC–MS/MS method in order to increase sample throughput and productivity. We were able to achieve a two-fold reduction in the lower limit of quantification and a three-fold reduction in retention time utilizing the UHPLC method, while keeping the same sample extraction procedure and similar MS/MS methodology. The new method exhibited good intra- and inter-day accuracy and precision, and was linear over a dynamic range of 1–500 ng/mL for each diastereomer. The method was successfully applied for the determination of R, S-(A) and R, R-(A) concentrations for in vitro and in vivo studies of epimerization of A in Sprague-Dawley rats.
Keywords: Diastereomers; UHPLC–MS/MS; Quantitation;

An efficient and reliable micellar electrokinetic capillary chromatography (MEKC) method has been developed for the simultaneous determination of isoniazid (ISO) and pyridoxine hydrochloride (PYR) in pharmaceutical formulations. A chemometric two level full factorial design approach was used to search for the optimum conditions of separation. Three parameters were selected for this study: the buffer pH, the buffer concentration and sodium dodecyl sulphate (SDS) concentrations. Resolution, peak symmetry and analysis time were established as response. The two analytes were separated within 6 min with the optimized conditions: 50 mM borate buffer, 25 mM SDS pH 7.8, 35 °C, at 50 mbar 4 s injection and 30 kV by using a fused silica capillary (72 cm effective length, 50 μm i.d.). The detection wavelength was set to 205 nm. Meloxicam was used as internal standard. The method was validated with respect to stability, linearity range, limit of quantitation and detection, precision, accuracy, specificity and robustness. The detection limits of the method were 1.0 μg mL−1 for ISO and 0.40 μg mL−1 for PYR and the method was linear at least in the range of 3.0–100 μg mL−1 for ISO and 1.0–100 μg mL−1 for PYR with excellent correlation coefficients (0.9995 for ISO and 0.9998 for PYR). Relative standard deviations (R.S.D.s) of the described method ranged between 0.54 and 2.27% for intra-day precision and between 0.65 and 2.69% for inter-day precision. The developed method was applied to the tablet form of ISO and PYR-containing the pharmaceutical preparations and the data were compared with obtained from the standard addition method. No statistically significant difference was found.
Keywords: Experimental design; Validation; Pharmaceutical; Isoniazid; Pyridoxine hydrochloride; Micellar electrokinetic capillary chromatography (MEKC);

A rapid, sensitive and accurate high-performance liquid chromatographic method with UV detection was developed and validated for the quantification of gabapentin in human plasma. Gabapentin was quantified using pre-column derivatization with 1-fluoro-2,4-dinitrobenzene following protein precipitation of plasma with acetonitrile. Amlodipine was used as internal standard. The chromatographic separation was carried out on a Nova-Pak C18 column using a mixture of 50 mM NaH2PO4 (pH = 2.5)–acetonitrile (30:70, v/v) as mobile phase with UV detection at 360 nm. The flow rate was set at 1.5 ml/min. The method was linear over the range of 0.05–5 μg/ml of gabapentin in plasma (r 2  > 0.999). The within-day and between-day precision values were in the range of 2–5%. The limit of quantification of the method was 0.05 μg/ml. The method was successfully used to study the pharmacokinetics of gabapentin in healthy volunteers.
Keywords: Gabapentin; Epilepsy; 1-Fluoro-2,4-dinitrobenzene; HPLC; UV-detection;

A new drug, quick-acting anti-motion capsule (QAAMC) composed of d-amphetamine sulfate, dimenhydrinate and ginger extraction has been studied for anti-motion-sickness use. We have developed a sensitive, specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) for the quantitative determination of d-amphetamine and diphenhydramine, the main effective components of the QAAMC, using pseudoephedrine as the internal standard. The analytes and internal standard were isolated from 200 μL plasma samples by a simple liquid–liquid extraction (LLE). Reverse-phase HPLC separation was accomplished on a Zorbax SB-C18 column (100 mm × 3.0 mm, 3.5 μm) with a mobile phase composed of methanol–water–formic acid (65:35:0.5, v/v/v) at a flow rate of 0.2 mL/min. The method had a chromatographic total run time of 5 min. A Varian 1200 L electrospray tandem mass spectrometer equipped with an electrospray ionization source was operated in selected reaction monitoring (SRM) mode with the precursor-to-product ion transitions m/z 136.0 → 91.0 (d-amphetamine), 256.0 → 167.0 (diphenhydramine) and 166.1 → 148.0 (IS) used for quantitation. The method was sensitive with a lower limit of quantitation (LLOQ) of 0.5 ng/mL for d-amphetamine and 1 ng/mL for diphenhydramine, with good linearity in the range 0.5–200 ng/mL for d-amphetamine and 1–500 ng/mL for diphenhydramine (r 2  ≥ 0.9990). All the validation data, such as accuracy, precision, and inter-day repeatability, were within the required limits. The method was successfully applied to pharmacokinetic study of the QAAMC in beagle dogs.
Keywords: Motion sickness prevention; d-Amphetamine; Diphenhydramine; LC–MS/MS; Pharmacokinetics;

Application of liquid chromatography–mass spectrometry to measure the concentrations and study the synthesis of short chain fatty acids following stable isotope infusions by R.J.W. Meesters; H.M.H. van Eijk; G.A.M. ten Have; A.A. de Graaf; K. Venema; B.E.J. van Rossum; N.E.P. Deutz (57-62).
A new method involving zinc sulphate deproteinization was developed to study short chain fatty acids (SCFA) production in the colon and subsequent occurrence of SCFA in blood. SCFA were baseline separated in a 30 min cycle using ion-exclusion chromatography and detected by mass spectrometry. Concentrations could be measured down to 10 μM and isotopomeric distributions could be assessed, enabling the conduction of tracer studies to study changes in SCFA synthesis. The applicability of the method was tested in an extensively characterized pig model yielding portal SCFA concentrations ranging from 70 μM (butyric acid) to 150 μM (propionic acid) to 440 μM (acetic acid) prior to butyrate tracer infusion, reaching butyric acid isotopic steady state within 2 h.
Keywords: Short chain fatty acids; LC–MS; SCFA; Stable isotope;

Stereoselective determination of vigabatrin enantiomers in human plasma by high performance liquid chromatography using UV detection by Valentina Franco; Iolanda Mazzucchelli; Cinzia Fattore; Roberto Marchiselli; Giuliana Gatti; Emilio Perucca (63-67).
A rapid and simple high-performance liquid chromatographic method for the determination of the R-(−)- and S-(+)-enantiomers of the antiepileptic drug vigabatrin in human plasma is described. After adding the internal standard (1-aminomethyl-cycloheptyl-acetic acid), plasma samples (200 μL) are deproteinized with acetonitrile and the supernatant is derivatized with 2,4,6 trinitrobenzene sulfonic acid (TNBSA). Separation is achieved on a reversed-phase cellulose-based chiral column (Chiralcel-ODR, 250 mm × 4.6 mm i.d.) using 0.05 M potassium hexafluorophosphate (pH 4.5)/acetonitrile/ethanol (50:40:10 vol/vol/vol) as mobile phase at a flow-rate of 0.9 mL/min. Chromatographic selectivity is improved by concentrating the derivatives on High Performance Extraction Disk Cartridges prior to injection. Detection is at 340 nm. Calibration curves are linear (r 2  ≥ 0.999) over the range of 0.5–40 μg/mL for each enantiomer, with a limit of quantification of 0.5 μg/mL for both analytes. The assay is suitable for therapeutic drug monitoring and for single-dose pharmacokinetic studies in man.
Keywords: Vigabatrin; Enantiomers; Enantioselective assay; HPLC; UV detection;

A simple, sensitive and robust liquid chromatography/electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS) method was developed and validated for quantification of chlorpromazine in rat plasma and brain tissue. Chlorpromazine was extracted from rat plasma and brain homogenate using liquid–liquid extraction. The compounds were separated on a Waters Atlantis™ dC-18 (30 mm × 2.1 mm i.d., 3 μm) column using a mobile phase of acetonitrile/20 mM ammonium formate (pH 4.25 adjusted with formic acid) with gradient elution. Chlorpromazine was detected in positive ion mode using multiple reaction monitoring (MRM). The method was validated and the specificity, linearity, lower limit of quantitation (LLOQ), precision, accuracy, recoveries and stability were determined. The LLOQ was 0.2 ng/ml for plasma and 0.833 ng/g for brain tissue. The method was linear over the concentration range from 0.2 to 200.0 ng/ml for plasma and from 0.833 to 833.3 ng/g for brain tissue. The correlation coefficient (R 2) values were more than 0.998 for both plasma and brain homogenate. The precision and accuracy for intra-day and inter-day were better than 7.54%. The relative and absolute recovery was above 84.9% and matrix effects were lower than 5.6%. This validated method has been successfully used to quantify the rat plasma and brain tissue concentration of chlorpromazine after chronic treatment.
Keywords: Chlorpromazine; Plasma; Brain tissue; LC–MS/MS;

In vivo pharmacokinetic and metabolism studies of ginsenoside Rd by Liu Yang; Yuanhui Deng; Shunjun Xu; Xing Zeng (77-84).
A high-performance liquid chromatography–electrospray ionisation–tandem mass spectrometry (LC–ESI–MSn) method has been developed to determine ginsenoside Rd in human plasma and to identify its metabolites in rat urine. The plasma and urine samples were pretreated by solid phase extraction (SPE) prior to analyses. In this work, gentiopicroside was used as the internal standard. The lower limit of quantification (LLOQ) for Rd in human plasma was 3 ng/ml. The average half-life time in plasma was detected as 19.29 h, when 10 mg of ginsenoside Rd was administrated intravenously to the volunteers. Seven metabolites including three oxygenated, two combined and two hydrolyzed components were identified in rat urine samples by using LC–MS and MS–MS, when ginsenoside Rd administered either orally or intravenously.
Keywords: In vivo; Pharmacokinetics; Metabolism; Ginsenoside Rd; Liquid chromatography–mass spectrometry;

A weak ion exchange monolithic column prepared by modifying the GMA–MAA–EDMA (glycidyl methacrylate–methacrylic acid–ethylene glycol dimethacrylate) monoliths with ethylenediamine was applied to remove matrix compounds in biological fluid. Using this monolithic column, on-line clean-up and screening of oxacillin and cloxacillin in human urine and plasma samples had been investigated. Chromatography was performed by reversed-phase HPLC on a C18 column with ultraviolet detection at 225 nm. Results showed that the ion exchange monolithic column could be used for deproteinization and retaining oxacillin and cloxacillin in human urine and plasma, which provided a simple and fast method for assaying drugs in human urine and plasma.
Keywords: Oxacillin; Cloxacillin; On-line clean-up; Weak ion exchange monolithic column;

A selective and sensitive high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (ESI-MS/MS) method for simultaneous determination of metformin and rosiglitazone in human plasma using phenformin as internal standard (IS) has been first developed and validated. Plasma samples were precipitated by acetonitrile and the analytes were separated on a prepacked Phenomenex Luna 5u CN 100A (150 mm × 2.0 mm I.D.) column using a mobile phase comprised of methanol:30 mM ammonium acetate pH 5.0 (80:20, v/v) delivered at 0.2 ml/min. Detection was performed on a Finnigan TSQ triple-quadrupole tandem mass spectrometer in positive ion selected reaction monitoring (SRM) mode using electrospray ionization. The ion transitions monitored were m/z 130.27 → 71.11 for metformin, m/z 358.14 → 135.07 for rosiglitazone and m/z 206.20 → 105.19 for the IS. The standard curves were linear (r 2  > 0.99) over the concentration range of 5–3000 ng/ml for metformin and 1.5–500 ng/ml for rosiglitazone with acceptable accuracy and precision, respectively. The within- and between-batch precisions were less than 15% of the relative standard deviation. The limit of detection (LOD) of both metformin and rosiglitazone was 1 ng/ml. The method described is precise and sensitive and has been successfully applied to the study of pharmacokinetics of compound metformin and rosiglitazone capsules in 12 healthy Chinese volunteers.
Keywords: Metformin; Rosiglitazone; Liquid chromatography/tandem mass spectrometry; Pharmacokinetics; Quantitation;

A sensitive and reproducible high performance liquid chromatography (HPLC)-UV method for determination of nicousamide, an inhibitor of rennin and transforming growth factor-beta1 (TGF-β1) type II receptors, has been developed and validated. Following acetonitrile deproteiniation, samples were separated by isocratic reversed-phase HPLC on an Aichrom Bond-AQ C18 column and quantified using UV detection at 320 nm. The mobile phase was acetonitrile/water (ratio 62:38 containing 0.1% H3PO4), with a flow-rate of 1.0 ml/min. A linear curve over the concentration range 5–200 ng/ml (r 2  = 0.9978) was obtained. The coefficients of the variation for the intra- and inter-day precisions ranged from 1.4–10.7% and 1.8–7.1%, respectively. The percentage of relative recovery was 91.56–105.45%. The method was used to determine the plasma concentration–time profiles for nicousamide after oral doses of 30, 100 and 300 mg/kg in dogs. A nonlinear pharmacokinetics was found in dogs at doses from 30 to 300 mg/kg. Following 30 mg/kg oral dose, the C max and AUC in females were lower than that in male. There is a potential for accumulation in dogs following multiple doses.
Keywords: Nicousamide; HPLC; Pharmacokinetic; Dog; Diabetic nephropathy;

Determination of linezolid in growth media by high-performance liquid chromatography with on-line extraction by Boubakar B. Ba; Branly Bikie Bi Nso; Claudine Quentin; Marie-Claude Saux (104-108).
An isocratic high-performance liquid chromatography (HPLC) method with on-line extraction has been developed to determine linezolid in Mueller-Hinton broth. The loading mobile phase consisting of water–acetonitrile 99:1 (v/v) allowed retention of the analyte on a LiChrocart 4-4 pre-column filled with a LiChrospher 100 RP-8, 5 μm. The transfer of the analyte by a backflush mode to a 150 mm × 4.6 mm I.D. Kromasil C8 5 μm column was performed using a mobile phase of water–acetonitrile 80:20 (v/v). UV detection at 254 nm allowed a quantification limit of 0.39 μg/mL with a 50-μL sample size. The method was successfully applied to in vitro pharmacokinetic–pharmacodynamic studies.
Keywords: Linezolid; Growth media; HPLC; On-line extraction;

Simultaneous determination of α-lipoic acid and its reduced form by high-performance liquid chromatography with fluorescence detection by Soichiro Satoh; Toshimasa Toyo’oka; Takeshi Fukushima; Shinsuke Inagaki (109-115).
The simultaneous determination of α-lipoic acid (LA) and DHLA (reduced form of LA) was carried out by HPLC with fluorescence detection. DHLA in the sample was first labeled with ABD-F at room temperature for 10 min and then the LA was labeled with SBD-F at 50 °C for 1 h after conversion to DHLA using the reducing agent, TCEP. The resulting fluorophores, ABD-DHLA and SBD-DHLA, were separated by reversed-phase chromatography and detected at 510 nm (excitation at 380 nm). Both fluorophors were completely separated without any interference of endogenous thiols and disulfides in the sample and sensitively detected by fluorimetry. The proposed method was applied to the assay of the LA supplement and the determination in human plasma after the oral administration of LA tablets. The concentration (%) of LA in the tablet was reasonable to the stated amount. Furthermore, the result of a time course study in the plasma after the administration of LA did not differ from a previous report. Thus, the present method seems to be applicable to the simultaneous determination of LA and DHLA in various biological specimens.
Keywords: α-Lipoic acid (LA); Dihydrolipoic acid (DHLA); Thiols; Disulfides; 4-(Aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F); Ammonium 4-fluoro-2,1,3-benzoxadiazole-7-sulfate (SBD-F); Fluorescence; HPLC;

Simple and simultaneous determination for 12 phenothiazines in human serum by reversed-phase high-performance liquid chromatography by Einosuke Tanaka; Takako Nakamura; Masaru Terada; Tatsuo Shinozuka; Chikako Hashimoto; Katsuyoshi Kurihara; Katsuya Honda (116-120).
A high-performance liquid chromatographic method has been developed for the simultaneous analysis of the 12 phenothiazines (chlorpromazine, fluphenazine, levomepromazine, perazine, perphenazine, prochlorperazine, profenamine, promethazine, propericiazine, thioproperazine, thioridazine and trifluoperazine) in human serum using HPLC/UV. The separation was achieved using a C18 reversed-phase column (250 mm × 4.6 mm I.D., particle size 5 μm, Inersil ODS-SP). The mobile phase, consisting of acetonitrile–methanol–30 mM NaH2PO4 (pH 5.6) (300:200:500, v/v/v), was delivered at a flow rate of 0.9 mL/min and UV detection was carried out at 250 nm. The recoveries of the 12 phenothiazines spiked into serum samples were 87.6–99.8%. Regression equations for the 12 phenothiazines showed excellent linearity, with detection limits of 3.2–5.5 ng/mL for serum. The inter-day and intra-day coefficients of variation for serum samples were commonly below 8.8%. The selectivity, accuracy and precision of this method are satisfactory for clinical and forensic purposes. This sensitive and selective method offers the opportunity for simultaneous screening and quantification of almost all phenothiazines available in Japan for the purposes of clinical and forensic applications.
Keywords: Phenothiazine; HPLC; Serum; Human;

Separation of topological forms of plasmid DNA by anion-exchange HPLC: Shifts in elution order of linear DNA by Clara R. Smith; Randolph B. DePrince; Jennifer Dackor; Debra Weigl; Jack Griffith; Magnus Persmark (121-127).
We sought to establish a single anion-exchange HPLC method for the separation of linear, open circular and supercoiled plasmid topoisomers using purified topoisomeric forms of three plasmids (3.0, 5.5 and 7.6 kb). However, finding one condition proved elusive as the topoisomer elution order was determined to depend on salt gradient slope. The observed change in selectivity increased with plasmid size and was most pronounced for the linear form. Indeed, the elution order of the linear 7.6 kb plasmid was reversed relative to the supercoiled form. This observation may have implications for methods used in quality control of plasmid DNA.
Keywords: Plasmid; Topoisomer separation; Anion-exchange HPLC; Slalom chromatography;

A HPLC method with UV detection (210 nm) was developed and validated for the quantification of atomoxetine, a new medication for the treatment of attention deficit/hyperactivity disorder, in human plasma. Following a two-step liquid–liquid extraction with diethyl ether, the analyte and internal standard (maprotiline) were separated using an isocratic mobile phase of acetonitrile/phosphate buffer (39/61, v/v, pH 6.6) on a reverse phase Inertsil C18 column. Linearity was verified over the range of 3.12–200 ng/mL atomoxetine in plasma. The lowest limit of detection is 2.5 ng/mL (S/N = 10). This HPLC method was validated with within- and between-batch precisions of 4.9–14.4% and 4.7–13.1%, respectively. The within- and between-batch biases were −1.9 to 1.4% and 0.1–13.8%, respectively. Commonly used psychotropic drugs and frequently coadministered drugs did not interfere with the drug and internal standard. This method is simple, economical and specific, and has been used successfully in a pharmacokinetic study of atomoxetine.
Keywords: Atomoxetine; Liquid–liquid extraction; HPLC; Pharmacokinetic;

Nitric oxide (NO) is a bioactive molecule that has recently emerged as a cellular messenger in numerous physiological processes in plants. A novel high-performance liquid chromatography (HPLC) method combined with poly(methacrylic acid-ethylene glycol dimethacrylate) (MAA-EGDMA) monolith microextraction (PMME) is developed for sensitive determination of NO in hydrophytes. NO is derivatized using a fluorescent probe, 1,3,5,7-tetramethyl-8-(3′,4′-diaminophenyl)-difluoroboradiaza-s-indacene (DAMBO), and then the derivatives are extracted with PMME and analyzed by high-performance liquid chromatography (HPLC) with fluorescence detection. The conditions for the derivatization and the subsequent extraction of NO derivatives are optimized in detail. The detection limit (S/N = 3) of NO is determined to be 2 × 10−12  mol L−1. Close correlation coefficient and excellent method reproducibility are obtained for the analyte over a linear range of 9 × 10−11–4.5 × 10−8  mol L−1. The inter- and intraday relative standard deviations (R.S.D.s) are less than 5%. The proposed method is successfully applied to the determination of NO levels in hydrophytes samples.
Keywords: High-performance liquid chromatography; Nitric oxide; Poly(methacrylic acid-ethylene glycol dimethacrylate) monolith microextraction; 1,3,5,7-Tetramethyl-8-(3′,4′-diaminophenyl)-difluoroboradiaza-s-indacene;

A highly sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for simultaneous determination of the prodrug fosinopril and its major active metabolite fosinoprilat for pharmacokinetic studies in healthy subjects. In order to get the lower limit of quantification (LLOQ), especially for analysis of fosinopril, key points of the method have been investigated including chromatographic conditions and selection of LC–MS/MS conditions. The analytes were extracted from plasma samples by liquid–liquid extraction, separated on a reversed-phase C8 column using gradient procedure, and detected by tandem mass spectrometry with a triple quadrupole ionization interface. The analytes and internal standard zaleplon were detected using positive electrospray ionization (ESI) in the SRM mode. The LLOQ of the method down to 0.1 ng mL−1 for fosinopril and 1.0 ng mL−1 for fosinoprilat were identifiable and reproducible. The standard calibration curves for both fosinopril and fosinoprilat were linear over the ranges of 0.1–15.0 and 1.0–700 ng mL−1 in human plasma, respectively. The within- and between-batch precisions (relative standard deviation (RSD)%) and the accuracy were acceptable. The validated method was successfully applied to reveal the pharmacokinetic properties of fosinopril and fosinoprilat after oral administration.
Keywords: Fosinopril; Fosinoprilat; Liquid chromatography–tandem mass spectrometry; Human plasma; Pharmacokinetics;

Development and validation of a stability-indicating high performance liquid chromatographic (HPLC) assay for biperiden in bulk form and pharmaceutical dosage forms by A. Mohammadi; A. Mehramizi; F. Aghaee Moghaddam; L. Erfani jabarian; M. pourfarzib; H.N. Kashani (152-157).
Current compendial (USP) methods of assay for the analysis of biperiden in bulk form and pharmaceutical dosage forms involve the use of titrimetric and spectrophotometric procedures, respectively. These are non-selective and non-stability-indicating techniques. In this work, a stability-indicating high performance liquid chromatographic assay procedure has been developed and validated for biperiden. The liquid chromatographic separation was achieved isocratically on a symmetry C8 column (150 mm × 3.9 mm i.d., 5 μm particle size) using a mobile phase containing methanol-buffer (50:50, v/v, pH 2.50) at a flow rate of 1 ml/min and UV detection at 205 nm. The buffer was composed of sodium dihydrogen phosphate (50 mM) and 1-heptanesulfonic acid sodium salt (5 mM). The method was linear over the concentration range of 0.5–25 μg/ml (r  = 0.9998) with a limit of detection and quantitation 0.03 and 0.1 μg/ml, respectively. The method has the requisite accuracy, selectivity, sensitivity and precision to assay biperiden in bulk form and pharmaceutical dosage forms. Degradation products resulting from the stress studies did not interfere with the detection of biperiden and the assay is thus stability-indicating.
Keywords: Biperiden; Stability-indicating; HPLC-UV;

An HPLC method for determination of inosine and hypoxanthine in human plasma from healthy volunteers and patients presenting with potential acute cardiac ischemia by Don Farthing; Domenic Sica; Todd Gehr; Bill Wilson; Itaf Fakhry; Terri Larus; Christine Farthing; H. Thomas Karnes (158-164).
A simple and sensitive high-performance liquid chromatography (HPLC) method utilizing ultraviolet (UV) detection was developed for the determination of inosine and hypoxanthine in human plasma. For component separation, a monolithic C18 column at a flow rate of 1.0 mL/min with an aqueous mobile phase of trifluoroacetic acid (0.1% TFA in deionized water pH 2.2, v/v) and methanol gradient was used. The method employed a one-step sample preparation utilizing centrifugal filtration with high component recoveries (∼98%) from plasma, which eliminated the need of an internal standard. The method demonstrated excellent linearity (0.25–5 μg/mL, R  > 0.9990) for both inosine and hypoxanthine with detection limits of 100 ng/mL. This simple and cost effective method was utilized to evaluate potential endogenous plasma biomarker(s), which may aid hospital emergency personnel in the early detection of acute cardiac ischemia in patients presenting with non-traumatic chest pain.
Keywords: Inosine; Hypoxanthine; Biomarker; HPLC; Acute cardiac ischemia; Monolithic column;

A high-performance liquid chromatographic method coupled with tandem mass spectrometry detection has been developed for the determination of propofol and its main glucuroconjugate metabolites (propofol–glucuronide (PG), 1-quinol-glucuronide (1-QG) and 4-quinol-glucuronide (4-QG) in human plasma. All compounds were extracted with a single solid phase extraction procedure using Max Oasis© cartridges. Propofol and thymol (internal standard) were analyzed using a C8 reversed-phase column with a mobile phase consisting of methanol–water (75:25, v/v) containing 0.025% NH4OH. Chromatography of glucuroconjugate metabolites and phenyl-β-d-glucuronide (internal standard) was performed using a hydrophilic interaction liquid chromatography (HILIC) and a mixture of acetonitrile/water/ammonium acetate buffer (100 mM, pH 5, 87/1/12, v/v/v). Both chromatographic separations were achieved in isocratic mode allowing a rapid analysis without re-equilibration of the phase. The method is specific and sensitive with a range of 10–1500 ng mL−1 for propofol and 1-QG, 20–3000 ng mL−1 for PG and 25–3750 ng mL−1 for 4-QG. The regression curves were linear for all compounds. The method is accurate and precise with intra-assay and inter-assay precision <8% and bias ≤6% for all compounds. This assay has allowed the successful measurement of propofol and its main glucuroconjugate metabolites in human plasma from 24 patients undergoing anaesthesia for elective partial hepatectomy surgery.
Keywords: Propofol; Metabolites; Tandem mass spectrometry; Blood;

The Mass Distance Fingerprint: A statistical framework for de novo detection of predominant modifications using high-accuracy mass spectrometry by Frank Potthast; Bertran Gerrits; Jari Häkkinen; Dorothea Rutishauser; Christian H. Ahrens; Bernd Roschitzki; Katja Baerenfaller; Richard P. Munton; Pascal Walther; Peter Gehrig; Philipp Seif; Peter H. Seeberger; Ralph Schlapbach (173-182).
We describe a statistical measure, Mass Distance Fingerprint, for automatic de novo detection of predominant peptide mass distances, i.e., putative protein modifications. The method’s focus is to globally detect mass differences, not to assign peptide sequences or modifications to individual spectra. The Mass Distance Fingerprint is calculated from high accuracy measured peptide masses. For the data sets used in this study, known mass differences are detected at electron mass accuracy or better. The proposed method is novel because it works independently of protein sequence databases and without any prior knowledge about modifications. Both modified and unmodified peptides have to be present in the sample to be detected. The method can be used for automated detection of chemical/post-translational modifications, quality control of experiments and labeling approaches, and to control the modification settings of protein identification tools. The algorithm is implemented as a web application and is distributed as open source software.
Keywords: Mass Distance Fingerprint; Mass Distance Histogram; Post-translational modification; Protein identification;

Partial within-animal calibration: A new calibration approach in lead optimisation high-throughput bioanalysis by Walthère Dewé; Michaël De Smet; Nathalie Evrard; Benoit Culot; Marc Lastelle; Gaëlle Saucez; Dominique Ingels; Xavier Taillieu (183-191).
The lead optimization phase of drug discovery requires high-throughput analyses for quantification in biological matrices and in plasma in particular. Over the last decade, some technical innovations allowed the pharmaceutical industry to improve the quality of the results. However, there is room for improvement. In this context, a new calibration strategy is proposed in this paper. Experiments were performed on dog plasma samples and it was showed that a within-animal calibration strategy can reduce the bias up to 20% and improve the precision up to 20%. However, a partial within-animal calibration is preferred to the full approach in order to avoid to many sample preparations.
Keywords: Bioanalysis; High-throughput; Calibration; Validation; Accuracy; Bias; Precision;

A sensitive high-performance liquid chromatography method coupled to UV and single mass spectrometry (MS) detection was developed for the determination of tenofovir in human plasma. A solid phase extraction procedure (Bond-Elut® C18 Varian cartridges) provided high extraction efficiency (91% for tenofovir and 68.8% for the internal standard, 3-methylcytidine). An atlantis®-dC-18 analytical column is used with an isocratic mode elution of a mixture (pH 2.5) of ammonium acetate/methanol (98.5:1.5, v/v). Detection was performed at 260 nm and by using the ion at m/z 288. The signals from both detectors were validated over the range of 10–1000 ng mL−1 and were found to be linear, accurate and precise. At the lowest limit of quantification, 10 ng mL−1 for UV and 5 ng mL−1 for MS, the average coefficient of variation was 6.9 and 3.9%, respectively. To investigate the potential of the validated method for clinical studies, more than 170 samples from HIV-infected adult patients were then analyzed with this assay. A good correlation was observed between the results obtained with both detectors. However, in several cases discordant results were observed between UV and MS detections. Therefore, tenofovir can sometimes suffer from interferences using either UV or single MS detection. We concluded that the double detection allows to obtain a more specific quantification of tenofovir. The present assay is sound and can be used for therapeutic drug monitoring allowing a higher reliability of the results which are transmitted to the medical team.
Keywords: Tenofovir (TNF); Mass spectrometry; UV; HPLC;

An alkaline protease from a salt-tolerant alkaliphilic Streptomyces clavuligerus was purified to homogeneity by 141-fold with a yield of 12% using two-step method of salt precipitation and ion exchange chromatography on DEAE cellulose. The apparent molecular mass was 49 ± 2 kDa and the enzyme appeared as monomer based on SDS and Native-PAGE. The temperature optimum was 70 °C with significant stability at 60–80 °C for more than 60 min. The enzyme was active over the pH range of 8.5–11, with an optimum at 10–11. The serine nature of the protease was confirmed by PMSF inhibition. The enzyme was highly resistant against chemical denaturation and displayed varied effects towards metal ions. The results are significant as extremozymes are difficult to purify and therefore, a two-step purification of alkaline protease from relatively less explored group of actinomycetes is quite appealing.
Keywords: Two-step purification of alkaline protease; Salt-tolerant alkaliphilic actinomycete; Streptomyces clavuligerus; Thermostable alkaline protease;

SPE–GC/FTD determination of N-methyl-2-pyrrolidone and its metabolites in urine by Ryuichi Kubota; Yoko Endo; Akito Takeuchi; Yoshinori Inoue; Hiroko Ogata; Masanori Ogawa; Tomoo Nakagawa; Nobuhiko Onda; Ginji Endo (204-210).
An analytical method using a combination of solid-phase extraction (SPE) and gas chromatography with a flame thermionic detector (GC/FTD) was developed for determination of N-methyl-2-pyrrolidone (NMP), N-methylsuccinimide (MSI), and 2-hydroxy-N-methylsuccinimide (2-HMSI) in human urine. The SPE cartridge of poly(divinylbenzene/hydroxymethacrylate) used was directly loaded with urine sample, followed by elution with methyl isobutyl ketone (MIBK) and subsequent centrifugation, and the supernatant was injected into the capillary GC using a DB1701. This method allowed efficient separation of NMP, MSI, and 2-HMSI, which were nearly free of interference by other GC peaks arising from urine. Recoveries of NMP, MSI, and 2-HMSI from the SPE cartridge were about 98, 101, and 67%, respectively, with limits of detection of 0.04, 0.02, and 0.06 mg/L, respectively, which met the regulatory requirements. The present method was used for assay in biological monitoring of workers exposed to NMP in their occupational environment.
Keywords: NMP; N-Methyl-2-pyrrolidone; N-Methylsuccinimide; 2-Hydroxy-N-methylsuccinimide; Solid-phase extraction; Flame thermionic detector; Gas chromatography;

Separation of metformin and glibenclamide was achieved within a single chromatographic run on a Zorbax CN column, under isocratic conditions, using acetonitrile and aqueous component (0.01 moles/L ammonium acetate adjusted at pH 3.5 with acetic acid) in volumetric ratio 1/1. Plasma sample preparation is based on protein precipitation by means of organic solvent addition. 1,3,5-Triazine-2,4,6-triamine (IS1) was used as internal standard for metformin, while gliquidone (IS2) played the same role for glibenclamide. Detection was performed with an ion trap mass analyzer, using atmospheric pressure chemical ionization (APCI). A single MS stage was used for detection of metformin and IS1, by extracting ion chromatograms corresponding to molecular ions. MS/MS detection in the SRM mode was used for glibenclamide (m/z transition from 494 to 369 Da) and IS2 (m/z transition from 528 to 403 Da). The method produces linear responses up to 2000 ng/mL for metformin and 400 ng/mL for glibenclamide, respectively. Low limits of quantification were found in the 40 ng/mL range for metformin and at the 4 ng/mL level for glibenclamide. Precision was characterized by relative standard deviations (RSD%) below 9%. The analytical method was successfully applied to a single dose, open-label, randomized, two-period, two-sequence, crossover bioequivalence study of two commercially available anti-diabetic combinations containing 400 mg metformin and 2.5 mg of glibenclamide per coated tablet.
Keywords: Metformin; Glibenclamide; Anti-diabetic combination; Simultaneous assay; Human plasma; Extraction-less sample preparation; APCI; MS detection; Method validation; Bioequivalence; Pharmacokinetic parameters;

A sensitive and selective high-performance liquid chromatographic method was developed for simultaneous determination of trans-resveratrol-3-O-glucoside (TRG) and its metabolites, trans-resveratrol-3-O-glucuronide (TRN) and trans-resveratrol (TR) in rat plasma. The plasma proteins were precipitated with acetonitrile and supernatant was evaporated to dryness. The analytes and internal standard baicalin were chromatographed on a C18 column. The mobile phase consisted of 25% acetonitrile and 75% H2O adjusted with formic acid to pH 3.5. The flow-rate was 1.0 ml/min and ultraviolet detection was set at 320 nm. Standard curves were linear over the concentration range of 0.04–40 μg/ml for TRG and TRN, and 0.04–10 μg/ml for TR, respectively. The precision, expressed as the intra-day R.S.D. and inter-day R.S.D., was below 9.3% for TRG, TRN and TR. The accuracy, expressed as the relative error (RE) was within ±7.4% for all analytes. The mean recoveries of TRG, TRN, TR and I.S. were 93.6%, 93.1%, 91.0% and 87.9%, respectively. This method was successfully applied to a pharmacokinetic study of TRG after an oral dose of 150 mg/kg to Wistar rats.
Keywords: Trans-resveratrol-3-O-glucoside; Trans-resveratrol-3-O-glucuronide; Trans-resveratrol;

This paper proposed a method with capillary zone electrophoresis (CZE) for analysis of eight heterocyclic amines in a commercial meat matrix. The influence of composition, pH and concentration of buffer, and applied voltage were investigated. A 5 mmol/L formic acid–ammonium formate solution at pH 2.20 was chosen as the running electrolyte. Also several solid-phase extraction actions were performed for the clean-up of the samples. With 3 s hydrodynamic injection, detection limits ranging from 0.554 to 1.783 μg/g was obtained. To improve sensitivity, field-amplified sample injection (FASI) was used with the conditions of 3 s hydrodynamic injection of a water plug and 25 s electrokinetic injection of the sample. And methanol–water (1:1 in volume) was applied as the sample solvent. Under above conditions, detection limits ranged from 1.329 to 19.39 ng/g, which were at least 50 times lower than those with hydrodynamic injection.
Keywords: Capillary electrophoresis; Heterocyclic amines; On-line concentration; UV-DAD detection;

Development of a SPE/HPLC/DAD method for the determination of antileishmanial 2-substituted quinolines and metabolites in rat plasma by Julie Desrivot; Fathi Moussa; Pierre Champy; Alain Fournet; Bruno Figadère; Christine Herrenknecht (230-238).
A SPE/HPLC/DAD method was developed for the in vivo monitoring of three new antileishmanial 2-substituted quinolines under study in our laboratory for the development of an oral treatment. Three phase I metabolites were included in this work for the optimization of the method. Trifunctional tC18 cartridges (resulting from the reaction of trifunctional silanes with silica surface) were selected among four sorbents tested. Two linear gradients were developed to ensure resolution of metabolites. Recovery of quinolines from rat plasma was comprised between 80.6 and 88.2%. In a drug development perspective, apparent pK a, lipophilicity and solubility were determined, as well as the extent of plasma protein or albumin binding.
Keywords: Leishmaniasis; Solid phase extraction; 2-Substituted quinoline; Protein binding; Lipophilicity; Solubility;

Based on the sensitizing effect of formaldehyde on the chemiluminescence (CL) reaction of propylthiouracil (PTU) and methylthiouracil (MTU) with acidic potassium permanganate and the combination technique of high-performance liquid chromatography (HPLC), a sensitive, selective and simple post-column CL detection method for determining PTU and MTU is described. The optimal conditions for the CL detection and HPLC separation were carried out. The linear ranges were 0.1–20 μg mL−1 for MTU and 0.1–10 μg mL−1 for PTU, the detection limits were 0.03 μg mL−1 for PTU, 0.03 μg mL−1 for MTU and the quantification limits were 0.1 μg mL−1 for PTU, 0.1 μg mL−1 for MTU. The method has been satisfactorily applied for the determination of MTU and PTU in human serum samples.
Keywords: HPLC; Chemiluminescence; Propylthiouracil; Methylthiouracil;

The use of enzymatic digests of the peptide HIV-1 fusion inhibitor enfuvirtide as a tool for the absolute quantification of this polypeptide (MW 4492 Da) in human plasma by LC–MS/MS has been evaluated. Two different methods applying digestion of enfuvirtide with chymotrypsin after solid phase extraction (SPE) of the plasma samples have therefore been developed and validated. One method used a stable isotopically labeled analog of the complete peptide (d60-enfuvirtide) as internal standard (IS) and could use as much as four different chymotryptic fragments for the quantification of enfuvirtide in a range of 100–10,000 ng/ml. Intra- and inter-assay precisions and deviations from the nominal concentrations varied for the different fragments, but were below 9% when the four results were averaged. The other method used a stable isotopically labeled chymotryptic fragment of the peptide (d10-ASLW) as IS. Although this IS does not correct for variations in digestion recovery, it allows the selective quantification of enfuvirtide (100–10,000 ng/ml), besides the quantification of the sum of enfuvirtide and its de-amidated metabolite M-20 (120–12,000 ng/ml). Both methods were suitable for the absolute quantification of enfuvirtide and M-20 in plasma, but proper selection of the fragment(s) used for the quantification appeared crucial when the deuterated fragment was used as IS.
Keywords: Peptides; Enzymatic digestion; Quantification; LC–MS/MS;

Caco-2 cells, the human colon carcinoma cells, are typically used for screening compounds for their permeability characteristics and P-glycoprotein (P-gp) interaction potential during discovery and development. The P-gp inhibition of test compounds is assessed by performing bi-directional permeability studies with digoxin, a well established P-gp substrate probe. Studies performed with digoxin alone as well as digoxin in presence of test compounds as putative inhibitors constitute the P-gp inhibition assay used to assess the potential liability of discovery compounds. Radiolabeled 3H-digoxin is commonly used in such studies followed by liquid scintillation counting. This manuscript describes the development of a sensitive, accurate, and reproducible LC–MS/MS method for analysis of digoxin and its internal standard digitoxin using an on-line extraction turbulent flow chromatography coupled to tandem mass spectrometric detection that is amendable to high throughput with use of 96-well plates. The standard curve for digoxin was linear between 10 nM and 5000 nM with regression coefficient (R 2) of 0.99. The applicability and reliability of the analysis method was evaluated by successful demonstration of efflux ratio (permeability B to A over permeability A to B) greater than 10 for digoxin in Caco-2 cells. Additional evaluations were performed on 13 marketed compounds by conducting inhibition studies in Caco-2 cells using classical P-gp inhibitors (ketoconazole, cyclosporin, verapamil, quinidine, saquinavir etc.) and comparing the results to historical data with 3H-digoxin studies. Similarly, P-gp inhibition studies with LC–MS/MS analytical method for digoxin were also performed for 21 additional test compounds classified as negative, moderate, and potent P-gp inhibitors spanning multiple chemo types and results compared with the historical P-gp inhibition data from the 3H-digoxin studies. A very good correlation coefficient (R 2) of 0.89 between the results from the two analytical methods affords an attractive LC–MS/MS analytical option for labs that need to conduct the P-gp inhibition assay without using radiolabeled compounds.
Keywords: Caco-2; LC–MS/MS; Turbulent flow chromatography; P-glycoprotein inhibition; Digoxin; Permeability;

Lymphoma is the most common hematopoietic tumor in dogs and manifests as a proliferation of malignant lymphoid cells primarily affecting the lymph nodes or solid visceral organs. We describe the use of capillary gel electrophoresis (CGE) with a laser-induced fluorescence (LIF) detector based on polymerase chain reaction (PCR) to rapidly detect a disorder of the canine T-cell receptor γ (TCRγ) gene. After the PCR amplification of the specific TCR( gene in dogs, the 90-bp DNA fragment amplified was separated in a fused-silica capillary by CGE–LIF. Under an electric field of 375 V/cm and with a sieving matrix of 1.5% poly (ethyleneoxide) (M r 600,000), the amplified PCR products were analyzed within 4 min by CGE separation. When the CGE–LIF method was applied to real clinical samples of the specific DNA fragment of the TCR( gene, the migration time and the corrected peak area showed relative standard deviations (n  = 5) of 0.29% and 0.58%, respectively. Both methods of CGE–LIF and slab gel electrophoresis showed same results for nine clinical samples. This PCR/CGE–LIF technique may prove to be a new fast and simple tool for the rapid diagnosis of the PCR-amplified DNA of canine T-cell lymphoma.
Keywords: Canine T-cell lymphoma; Fast detection; Capillary gel electrophoresis; Polymerase chain reaction;

This study presents the partitioning and purification of recombinant Bacillus badius phenylalanine dehydrogenase (PheDH) in aqueous two-phase systems (ATPS) composed of polyethylene glycol 6000 (PEG-6000) and ammonium sulfate. A single-step operation of ATPS was developed for extraction and purification of recombinant PheDH from E. coli BL21 (DE3). The influence of system parameters including; PEG molecular weight and concentration, pH, (NH4)2SO4 concentration and NaCl salt addition on enzyme partitioning were investigated. The best optimal system for the partitioning and purification of PheDH was 8.5% (w/w) PEG-6000, 17.5% (w/w) (NH4)2SO4 and 13% (w/w) NaCl at pH 8.0. The partition coefficient, recovery, yield, purification factor and specific activity values were of 92.57, 141%, 95.85%, 474.3 and 10424.97 U/mg, respectively. Also the K m values for l-phenylalanine and NAD+ in oxidative deamination were 0.020 and 0.13 mM, respectively. Our data suggested that this ATPS could be an economical and attractive technology for large-scale purification of recombinant PheDH.
Keywords: Aqueous two-phase systems (ATPS); Ammonium sulfate; Phenylalanine dehydrogenase (PheDH); Partition; Purification; PEG-6000;

A sensitive and specific high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS–MS) method has been developed and validated for the determination of palmatine in canine plasma. Palmatine and jatrorrhizine (internal standard, I.S.) were extracted from plasma samples by solid-phase extraction (SPE) using Oasis HLB cartridges. The chromatographic separation was performed on a Waters XTerra MS C18 reversed-phase column at 30 °C. The gradient mobile phase, delivered at 0.25 mL/min, was composed of a mixture of acetonitrile −0.1% (v/v) acetic acid aqueous solution adjusted to pH 2.8 with triethylamine. Positive electrospray ionization was utilized as the ionization source. Palmatine and the internal standard (I.S.) were determined using multiple reaction monitoring (MRM) of precursor → product ion transitions at m/z 352 → 336 and m/z 338 → 322, respectively. The lower limit of quantification (LLOQ) was 0.1 ng/mL using 100 μL plasma samples and the linear calibration range was from 0.1 to 500 ng/mL. The inter-day and intra-day RSDs were lower than 9.9% and the recoveries of palmatine ranged from 87.3 to 100.9%. The mean extraction recoveries of palmatine and the I.S. were 99.2 and 96.8%, respectively. The method has been successfully applied to the pharmacokinetic studies of palmatine in beagle dogs after oral administration and intramuscular injection of palmatine.
Keywords: Palmatine; LC–MS–MS; Pharmacokinetics;

An HPLC protocol for sugar microanalysis based on the formation of ultraviolet-absorbing benzoyl chloride derivatives was improved. Here, samples were prepared with a C-8 cartridge and analyzed with a high efficiency ODS column, in which porous spherical silica particles 3 μm in diameter were packed. These devices allowed us to simultaneously quantify multiple sugars and sugar alcohols up to 10 ng/ml and to provide satisfactory separations of some sugars, such as fructose and myo-inositol and sorbitol and mannitol. This protocol, which does not require special apparatuses, should become a powerful tool in sugar research.
Keywords: Sugar microanalysis; Benzoylation; Solid phase extraction;

Glucosamine is an amino monosaccharide reagent. It is difficult to assay using typical reversed-phase column due to the early elution, by optimizing the chromatographic conditions, especially the analytical column and the mobile phase composition, an improved analytical method was developed and validated, which offers rapid, sensitive and specific determination of glucosamine in human plasma. Following protein precipitation, the analyte and internal standard (valibose) were separated using an isocratic mobile phase on an Inertsil CN-3 column and detected by mass spectrometry in the multiple reaction monitoring mode using the respective precursor to product ion combinations of m/z 180/72 for glucosamine and m/z 252/198 for valibose. The chromatographic time was just 4.2 min for each sample, which made it possible to analyze more than 120 human plasma samples per day. The method exhibited a linear dynamic range of 4.00–4000 ng/mL for glucosamine in human plasma. The lower limit of quantification (LLOQ) was 4.00 ng/mL with a relative standard deviation of less than 10.9%. Acceptable precision and accuracy were obtained for the plasma concentrations over the standard curve range. By monitoring the two different MRM transitions, it was proved that no endogenous glucosamine was found in human plasma. The validated method has been successfully used to analyze human plasma samples for application in a bioequivalence study.
Keywords: Glucosamine; Chromatography optimization; LC/MS/MS; Plasma concentration;

The report presents a segmental hair analysis for the retrospective multi-parameter evaluation of drugs of abuse including opioids, cocainics and amphetamines. The analysis was carried out with the use of liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry (LC–APCI-MS-MS). The authors have evaluated the differences in the contents of particular opiates in the hair as related to the origin of a sample taken from Polish drug users taking “Polish heroin”, and also from heroin abusers from Western European countries taking “Western heroin”. The results indicate distinct differences in the 6-MAM concentration values in the Polish and foreigners, suggesting that the foreigners take products containing high concentrations of heroin and the Polish take the poppy product “compote” characterized by its variable and low heroin content. An additional argument for a different abuse profile in the Polish and Western drug users is found in the presence of cocaine detected in hair samples originating from the latter, while cocaine is much less frequently detected in Polish drug users.
Keywords: Opiates; Hair analysis; LC–APCI-MS-MS;

HPLC quantification of perphenazine in sheep plasma: Application to a pharmacokinetic study by Cecilia Pedernera; José Luis Ruiz; Glòria Castells; Xavier Manteca; Carles Cristòfol (308-312).
A high performance liquid chromatographic (HPLC) method for the determination of perphenazine (PPZ) in sheep plasma was developed and validated. The separation was achieved using a 5 μm C18 column (125 mm × 4 mm) with a mobile phase composed of acetonitrile and an aqueous solution of H3PO4 and TBA (inverse gradient). The flow rate was 1.5 mL/min and the UV detection was performed at 258 nm. The method was validated with respect to linearity, intra and inter-day precision and accuracy, limit of quantification, limit of detection and storage stability. This method was used to perform a pilot pharmacokinetic study of PPZ after subcutaneous administration to one ewe.
Keywords: Perphenazine; HPLC; UV; Pharmacokinetic; Sheep;

Quantitative HPLC-UV method for the determination of firocoxib from horse and dog plasma by Valerie Kvaternick; Thomas Malinski; Jill Wortmann; James Fischer (313-319).
A sensitive reversed-phase HPLC-UV method was developed for the determination of firocoxib, a novel and highly selective COX-2 inhibitor, in plasma. A 1.0 mL dog or horse plasma sample is mixed with water and passed through a hydrophobic–lipophilic copolymer solid-phase extraction column to isolate firocoxib. Quantitation is based on an external standard curve. The method has a validated limit of quantitation of 25 ng/mL and a limit of detection of 10 ng/mL. The validated upper limit of quantitation was 2500 ng/mL for horses and 10,000 ng/mL for dogs. The average recoveries ranged from 88–93% for horse plasma and 96–103% for dog plasma. The coefficient of variation in all cases was less than 10%. This method is suitable for the analysis of clinical samples from pharmacokinetic and bioequivalence studies and drug monitoring.
Keywords: Firocoxib; HPLC-UV; Plasma; Method validation; Coxib; NSAID; Horse; Dog;

4-Anilinoquinazolines (e.g. Iressa and Glivec) are a class of epidermal growth factor receptor tyrosine kinase (EGFR-TK) inhibitors widely used to treat non-small cell lung cancer and other tumors. However, low clinical response rate, resistance, and host toxicity of currently available EGFR-TK inhibitors prompt the development of second generation of TK inhibitors with improved efficacy, selectivity, and less resistance. CH330331 is a recently synthesized novel 4-anilinoquinazoline analog with confirmed anticancer activity in vitro and in vivo. To predict its oral pharmacokinetic behavior and transport nature in the intestine before entering clinical trials, we have developed and validated a high performance liquid chromatographic (HPLC) method for the determination of CH330331 in Caco-2 (a human colon cancer cell line) monolayers. The developed HPLC method was sensitive and reliable, with acceptable accuracy (90–110% of nominal values) and precision (intra- and inter-assay R.S.D.  <  10%). The total running time was within 10 min, with acceptable separation of the target analytes. The lower limit of quantitation (LLOQ) value for CH330331 was 200 ng/ml when an aliquot of 100 μl sample was injected onto the HPLC. The validated HPLC method was applied to characterize the epithelial transport of CH330331 in Caco-2 monolayers. The transport of CH330331 across the Caco-2 monolayers from the apical to basolateral side was 8- to 10-fold higher than that from the basolateral to apical side. Co-incubation of sodium azide or MK-571, but not verapamil, significantly inhibited the apical to basolateral transport of CH330331. These findings provide initial evidence that the intestinal absorption of CH330331 is mediated by an active mechanism. Further studies are required to explore the interaction of CH330331 with ATP-binding cassette transporters and the possible influence on its pharmacokinetics and pharmacodynamics.
Keywords: HPLC; Validation; CH330331; Caco-2 cells;

In this paper, a rapid method based on high-performance liquid chromatography/electrospray–mass spectrometry (HPLC/ESI-MS) method for the quantitative determination of andrographolide (AND) in human plasma has been developed and validated. A liquid–liquid extraction (LLE) procedure was selected to isolate AND from biological matrixes. Isosorbide-5-mononitrate (IS-5-MN) was selected as the internal standard (IS). The correlation coefficient of the calibration curve was 0.998, in the range of 9.9–320.0 ng/mL. The validated method may be used to assess the bioavailability and pharmacokinetics of the drug.
Keywords: Andrographolide; HPLC/ESI-MS; Human plasma;

High performance liquid chromatography with ultraviolet detection for the determination of SYUIQ-5, a novel telomerase inhibitor for cancer therapy: Application to an enzyme kinetic study in rat liver microsomes by Qi-Biao Su; Fan He; Su Guan; Yu-Jing Lu; Lian-Quan Gu; Zhi-Shu Huang; Xiao Chen; Min Huang; Chun-Guang Li; Balram Chowbay; Shu-Feng Zhou (332-337).
A sensitive assay for the determination of SYUIQ-5, a novel telomerase inhibitor and anti-tumor drug, in rat liver microsomes was developed by using high-performance liquid chromatography with ultraviolet detection. SYUIQ-5 was incubated in vitro with liver microsomes from rats pre-treated with control vehicle, β-naphthofIavone, phenobarbital, 20% ethanol or dexamethasone. The analytes were extracted with diethyl ether and separated a C18 5-μm analytical column. Elution was conducted with 30 mM dipotassium hydrogen phosphate (pH 8.0)–methanol–triethylamine (30:70:0.05, v/v/v) at a flow-rate of 1.0 ml/min and the detection of UV absorbance was conducted at 278 nm. Intra-day and inter-day precision and accuracy of the method were within 10%. The mean analytical recoveries of SYUIQ-5 ranged from 78.8 to 95.3%. The linearity of the calibration curve was in the range of 1.0–80.0 μM. The lower limit of quantification (LOQ) was 1.0 μM. Kinetic analysis showed that β-naphthofIavone and dexamethasone significantly induced SYUIQ-5 metabolism, suggesting that cytochrome P450 1A and 3A are the major contributor to SYUIQ-5 metabolism in rat liver microsomes.
Keywords: SYUIQ-5; HPLC; Rat liver microsomes; Metabolism;

Using isocratic column-switching high-performance liquid chromatography (HPLC) we established a group method for automated quantitative analysis of the antiepileptic drugs lamotrigine, oxcarbazepine and its metabolite 10-monohydroxycarbazepine (MHD) that are also used in psychiatry as mood stabilizers. Samples were cleaned from interfering proteins and lipids by transfer onto a pre-column, using a PerfectBond® C-8 material, with 8% acetonitrile in water as a pre-column eluent. Separation was performed by elution onto the analytical column (Betasil® C6 5 μm, 250 mm × 4.6 mm) at a flow rate of 1.0 ml/min with potassium dihydrogenphosphate buffer (20 mmol/l, pH3.0)/acetonitrile (70/30; v/v) as analytical eluent. UV-spectrophotometric detection was set to 215 nm for all three compounds. The analytical run was finished within 18 min. Detection limit was 30 ng/ml for lamotrigine, 35 ng/ml for oxcarbazepine and 25 ng/ml for 10-monohydroxycarbazepine. The method was found to be suitable for automated analysis of serum samples of patients treated with lamotrigine and oxcarbazepine.
Keywords: Column-switching; Lamotrigine; Oxcarbazepine; 10-monohydroxycarbazepine; Therapeutic drug monitoring;

Cyclophosphamide is a cytotoxic prodrug with a very narrow therapeutic index. To study the clinical pharmacology of cyclophosphamide in a large cohort of patients a previously published method for the simultaneous quantitative determination of cyclophosphamide and 4-hydroxycyclophosphamide in human plasma using liquid chromatography tandem mass spectrometry (LC–MS/MS) was optimized. Addition of an isotopically labelled internal standard and adaptation of the gradient resulted in a fast, robust and sensitive assay. Because 4-hydroxycyclophosphamide is not stable in plasma, the compound is derivatized with semicarbazide immediately after sample collection. Sample preparation was carried out by protein precipitation with methanol–acetonitrile (1:1, v/v), containing isotopically labelled cyclophosphamide and hexamethylphosphoramide as internal standards. The LC separation was performed on a Zorbax Extend C18 column (150 mm × 2.1 mm ID, particle size 5 μm) with 1 mM ammonium hydroxide in water–acetonitrile (90:10, v/v) as the starting gradient, at a flow-rate of 0.40 mL/min with a total run time of 6 min. The lower limit of quantification (LLQ, using a 100 μL sample volume) was 200 ng/mL and the linear dynamic range extended to 40,000 ng/mL for cyclophosphamide and 50–5000 ng/mL for 4-hydroxycyclophosphamide. Accuracies as well as precisions were lower than 20% at the LLQ concentration and lower than 15% for all other concentrations. This method has been successfully applied in our institute to support ongoing studies into the pharmacokinetics and pharmacogenetics of cyclophosphamide.
Keywords: LC–MS/MS; Cyclophosphamide; 4-Hydroxycyclophosphamide;

Determination of oxalyl-coenzyme A decarboxylase activity in Oxalobacter formigenes and Lactobacillus acidophilus by capillary electrophoresis by Claudia Bendazzoli; Silvia Turroni; Roberto Gotti; Stefano Olmo; Patrizia Brigidi; Vanni Cavrini (350-356).
Oxalyl-coenzyme A decarboxylase (OXC) is a key enzyme in the catabolism of the highly toxic oxalate, catalysing the decarboxylation of oxalyl-coenzyme A (Ox-CoA) to formyl-coenzyme A (For-CoA). In the present study, a capillary electrophoretic (CE) method was proposed for the assessment of the activity of recombinant OXC from two bacteria, namely Oxalobacter formigenes DSM 4420 and Lactobacillus acidophilus LA 14. In particular, the degradation of the substrate Ox-CoA occurring in the enzymatic reaction could be monitored by the off-line CE method. A capillary permanently coated with polyethylenimine (PEI) was used and in the presence of a neutral background electrolyte (50 mM phosphate buffer at pH 7.0), a reversal of the electroosmotic flow was obtained. Under these conditions, the anodic migration of Ox-CoA (substrate) and For-CoA (reaction product) occurred and their separation was accomplished in less than 12 min. The CE method was validated for selectivity, linearity (range of Ox-CoA within 0.005–0.650 mM), sensitivity (LOD of 1.5 μM at the detection wavelength of 254 nm), precision and accuracy. Steady state kinetic constants (V max, K m or k′) of OXC were finally estimated for both the bacteria showing that although L. acidophilus LA 14 provided a lower oxalate breakdown than O. formigenes DSM 4420, it could be a potentially useful probiotic in the prevention of diseases related to oxalate.
Keywords: Oxalyl-coenzyme A decarboxylase; Oxalobacter formigenes; Lactobacillus acidophilus; Capillary electrophoresis; Polyethylenimine; Kinetic parameters;