Journal of Chromatography B (v.858, #1-2)

A simple and rapid analytical method for the simultaneous quantification of zidovudine (AZT) and its monophosphate (AZTMP) in cell extracts has been developed using high-performance liquid chromatography (HPLC) with on-line solid-phase extraction and 2-aminoethyl-3′-azido-2′,3′-dideoxythymidin-5′-yl phosphodiester sodium salt as internal standard (IS). The cell extract samples were directly injected on a short reversed-phase precolumn using an aqueous buffer containing an ion-pairing reagent as a mobile phase. Under these conditions, the analytes were retained on the precolumn whereas the proteins were discarded. The analytes were then transferred onto the analytical column by increasing the strength of the eluent. The calibration curve was linear over a concentration range of 0.5–100 μg/ml. Inter- and intra-day accuracy and precision results satisfied the accepted criteria for bioanalytical validation. This method was used to study the decomposition pathway of a model pronucleotide in an in vitro approach.
Keywords: Zidovudine; Zidovudine monophosphate; On-line solid-phase extraction; Ion pair; Cell extracts;

Alkaline saponification of entire sample matrixes for quantification of α-, γ-, δ-tocopherols (α-T, γ-T, δ-T) and α-tocopherol acetate (α-TAc) was examined. High-performance liquid chromatography was used to measure α-T, γ-T, δ-T and α-TAc in tocopherol standard solutions, milk and ovine blood plasma. Saponification in the presence of vitamin C decreases the concentration of tocopherols, especially α-T and γ-T. The poor recovery of tocopherols is due to the decomposition of tocopherols in saponified standard solutions, milk or plasma. Saponification of samples in the presence of 2,[6]-ditertbutyl-p-cresol or flushed only with a stream of Ar resulted in a major decrease in the concentrations of α-T, γ-T, δ-T and α-TAc in comparison with saponification in the presence of vitamin C.
Keywords: Tocopherols; Alkaline saponification; HPLC;

Liquid chromatography tandem mass spectrometry method for simultaneous determination of metoprolol tartrate and ramipril in human plasma by K. Veeran Gowda; Uttam Mandal; P. Senthamil Selvan; W.D. Sam Solomon; Animesh Ghosh; Amlan Kanti Sarkar; Sangita Agarwal; T. Nageswar Rao; Tapan Kumar Pal (13-21).
A simple, rapid, sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for quantification of metoprolol tartrate (MT) and ramipril, in human plasma. Both the drugs were extracted by liquid–liquid extraction with diethyl ether–dichloromethane (70:30, v/v). The chromatographic separation was performed on a reversed-phase C8 column with a mobile phase of 10 mM ammonium formate–methanol (3:97, v/v). The protonated analyte was quantitated in positive ionization by multiple reaction monitoring with a mass spectrometer. The method was validated over the concentration range of 5–500 ng/ml for metoprolol and ramipril in human plasma. The precursor to product ion transitions of m/z 268.0–103.10 and m/z 417.20–117.20 were used to measure metoprolol and ramipril, respectively.
Keywords: Metoprolol tartrate; Ramipril; LC–MS/MS; Validation;

Scutellarin, a flavone glycoside, popularly used in the treatment of heart disease, has been efficiently separated using macroporous resins from crude extracts of Chinese medicinal plant Erigeron breviscapus (vant.) Hand. Mazz. HPD-800 resin offered the best adsorption and desorption capacity for scutellarin among the eight macroporous resins tested, and its adsorption data at 25 °C fit best to the Langmuir isotherm. The dynamic adsorption and desorption experiments have been carried out on a HPD-800 resin packed column to optimize the separation process of scutellarin from the crude extracts of E. breviscapus. After one run treatment with HPD-800 resin, the scutellarin content in the product was increased 15.69-fold from 2.61% to 40.96% with a recovery yield of 95.01%. The preparative separation process via adsorption–desorption method developed in this study provides a new approach for scale-up separation and purification of scutellarin for its wide pharmaceutical use.
Keywords: Adsorption; Desorption; Erigeron breviscapus (vant.) Hand. Mazz.; Macroporous resins; Scutellarin;

A simplified method for the measurement of urinary free cortisol using LC–MS/MS by Joanne E. Wear; Laura J. Owen; Katherine Duxbury; Brian G. Keevil (27-31).
The measurement of 24 h urinary free cortisol is used in the investigation of patients with symptoms of hypercortisolism. Many different methods have been published for the measurement of cortisol, but most of these methods involve cumbersome pre-extraction of the cortisol prior to analysis. We have developed a method using in-well protein precipitation which serves to clean up the sample without requiring lengthy sample preparation. A Shimadzu SIL-HT autosampler was used to inject 50 μL of extract onto a Phenomemex® Gemini C18 guard column attached to a Waters™ Xbridge C18 column. The eluant was introduced directly into a Waters™ Quattro Micro tandem mass spectrometer. The method was found to be linear up to 3448 nmol/L with a lower limit of detection of 5.3 nmol/L. Precision and accuracy were acceptable, and no interference was noted from compounds such as prednisolone or fenofibrate. This assay was compared to a previously published method, which uses solid phase extraction prior to LC–MS/MS analysis. We have developed a simplified, robust assay for the quantitation of urinary free cortisol that will increase the throughput of the assay and avoid the use of neurotoxic solvents such as dichloromethane.
Keywords: Urinary free cortisol; Liquid chromatography tandem mass spectrometry;

Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthesis, is hydrolyzed to dimethylamine (DMA) and l-citrulline by the enzyme dimethylarginine dimethylaminohydrolase (DDAH). In the present article we report on a GC–MS assay for DDAH activity in rat liver homogenate in phosphate buffered saline. The method is based on the quantitative determination of ADMA-derived DMA by GC–MS as the pentafluorobenzamide derivative. Quantification was performed by selected-ion monitoring of the protonated molecules at m/z 240 for DMA and m/z 246 for the internal standard (CD3)2NH in the positive-ion chemical ionization mode. The assay was applied to determine the enzyme kinetics in rat liver, the hepatic DDAH activity in streptozotocin-induced (50 mg/kg) diabetes in rats, and to evaluate the importance of S-nitrosothiols as DDAH inhibitors. The K M and V max values were determined to be 60 μM ADMA and 12.5 pmol DMA/min mg liver corresponding to 166 pmol DMA/min mg protein. Typical DDAH activity values measured in rat liver homogenate were 8.7 pmol DMA/min mg liver at added ADMA concentration of 100 μM. DDAH activity was found to be 1.7-fold elevated in diabetic as compared to non-diabetic rats (P  = 0.01). The SH-specific agents HgCl2, S-nitrosocysteine ethyl ester (SNACET), a synthetic lipophilic S-nitrosothiol, S-nitrosoglutathione (GSNO), S-nitrosocysteine (CysNO) and S-nitrosohomocysteine (HcysNO) were found to inhibit DDAH activity in rat liver homogenate. The IC50 values for HcysNO, SNACET, CysNO and GSNO were estimated to be 300, 500, 700 and 1000 μM, respectively. Oral administration of 15N-labelled SNACET to two healthy volunteers (1 μmol/kg) resulted in elevated urinary excretion of 15N-labelled nitrite and nitrate, but did not reduce creatinine-corrected excretion of DMA in the urine. Our results suggest that inhibition of DDAH activity on the basis of reversible nitros(yl)ation or irreversible N-thiosulfoximidation of the sulfhydryl group of the cysteine moiety involved in the catalytic process is most likely not a rationale design of DDAH inhibitors. A major advantage of the present GC–MS assay over other assays is that DDAH activity is assessed by measuring the formation of the specific enzymatic product DMA but not the formation of unlabelled or (radio)labelled l-citrulline or the decay of the substrate ADMA. The GC–MS assay reported here should be suitable to probe for DDAH activity in various disease models.
Keywords: Diabetes; Enzyme inhibition; Michaelis–Menten; Nitric oxide; S-Nitrosothiols; SNACET;

A new method was developed to analyze three cardiovascular drugs in rat plasma, Mexiletine hydrochloride (MXL), Methoxamine hydrochloride (MTX), and Metaraminol bitartrate (MTR), by high-performance liquid chromatography (HPLC) using 9,10-anthraquinone-2-sulfonyl chloride (ASC) as the derivatization reagent. The derivatization modes and conditions for this method were optimized. The quantitative analysis was achieved using a C18 column at room temperature (25 °C), with various volume ratios of methanol–water as the mobile phase and a detection wavelength at 256 nm. Analytical linearity was obtained for the method over the concentration range of 0.04–8.0 μg mL−1 for all the three drugs. The lower limit of quantification (LLOQ) was 0.04 μg mL−1. This method was successfully applied to the analysis of the three drugs in rat plasma and their pharmacokinetic studies. The t 1/2 values of the three drugs in rats were found to be 5.38 ± 0.61, 4.49 ± 0.53, and 3.70 ± 0.19 h for MXL, MTX, and MTR, respectively.
Keywords: High-performance liquid chromatography; 9,10-Anthraquinone-2-sulfonyl chloride; Derivatization; Mexiletine hydrochloride; Methoxamine hydrochloride; Metaraminol bitartrate; Rat pharmacokinetics;

Cytochrome P450 bio-affinity detection coupled to gradient HPLC: On-line screening of affinities to cytochrome P4501A2 and 2D6 by Jeroen Kool; Sebastiaan M. van Liempd; Stefan Harmsen; Joran Beckman; Danny van Elswijk; Jan. N.M. Commandeur; Hubertus Irth; Nico P.E. Vermeulen (49-58).
Here we describe novel on-line human CYP1A2 and CYP2D6 Enzyme Affinity Detection (EAD) systems coupled to gradient HPLC. The use of the systems lies in the detection of individual inhibitory ligands in mixtures (e.g. metabolic mixtures or herbal extracts) towards two relevant drug metabolizing human CYPs. The systems can rapidly detect individual compounds in mixtures with affinities to CYP1A2 or 2D6. The HPLC–EAD systems were first evaluated and validated in flow injection analysis mode. IC50 values of known ligands for both CYPs, tested both in flow injection and in HPLC mode, were well comparable with those measured in microplate reader formats. Both EAD systems were also connected to gradient HPLC and used to screen known compound mixtures for the presence of CYP1A2 and 2D6 inhibitors. Finally, the on-line CYP2D6 EAD system was used to screen for the inhibitory activities of stereoisomers of a mixture of five methylenedioxy-alkylamphetamines (XTC analogs) on a chiral analytical column.
Keywords: Bio-affinity detection; On-line; HPLC; Cytochrome P450;

Confirmation of cannabinoids in meconium using two-dimensional gas chromatography with mass spectrometry detection by Stephanie J. Marin; Rebecka Coles; Francis M. Urry; Gwendolyn A. McMillin (59-64).
Meconium has become the specimen of choice for determining fetal exposure to drugs of abuse, but its physical complexity can cause interferences from matrix effects. A new method to determine 9-carboxy-11-nor-Δ9-THC (9-THCA) and 11-hydroxy-Δ9-THC (11-OH-THC) using two-dimensional (2D) GC–MS was developed to reduce interferences and carryover. The method was validated using 70 spiked samples prepared in drug-free meconium and 46 residual patient specimens that were confirmed to contain cannabinoids. Ten patient specimens that failed to confirm due to interferences using the previous GC–MS method were analyzed using the new 2D method and 9-THCA was quantitated in all ten samples. The 2D GC–MS method improved chromatography which significantly reduced interferences and carryover when compared to the previous GC–MS method.
Keywords: Cannabinoids; THC; Meconium; Two-dimensional GC; Mass spectrometry; Deans switch;

A sensitive and specific liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the investigation of the pharmacokinetics of 20(R)-dammarane-3β,12β,20,25-tetrol (25-OH-PPD) in rat. Ginsenoside Rh2 was employed as an internal standard. The plasma samples were pretreated by liquid–liquid extraction and analyzed using LC/MS/MS with an electrospray ionization interface. The mobile phase consisted of methanol–acetonitrile–10 mmol/l aqueous ammonium acetate (42.5:42.5:15, v:v:v), which was pumped at 0.4 ml/min. The analytical column (50 mm × 2.1 mm i.d.) was packed with Venusil XBP C8 material (3.5 μm). The standard curve was linear from 10 to 3000 ng/ml. The assay was specific, accurate (accuracy between −1.19 and 2.57% for all quality control samples), precise and reproducible (within- and between-day precisions measured as relative standard deviation were <5% and <7%, respectively). 25-OH-PPD in rat plasma was stable over three freeze–thaw cycles and at ambient temperatures for 6 h. The method had a lower limit of quantitation of 10 ng/ml, which offered a satisfactory sensitivity for the determination of (25-OH-PPD) in plasma. This quantitation method was successfully applied to pharmacokinetic studies of 25-OH-PPD after both an oral and an intravenous administration to rats and the absolute bioavailability is 64.8 ± 14.3%.
Keywords: 25-OH-PPD; LC/MS/MS; Pharmacokinetics;

Simultaneous determination of gemcitabine and gemcitabine-squalene by liquid chromatography–tandem mass spectrometry in human plasma by Hania Khoury; Alain Deroussent; L. Harivardhan Reddy; Patrick Couvreur; Gilles Vassal; Angelo Paci (71-78).
Gemcitabine-squalene is a new prodrug that self-organizes in water forming nanoassemblies. It exhibits better anti-cancer properties in vitro and in vivo than gemcitabine. A liquid chromatography/tandem mass spectrometry assay of gemcitabine-squalene and gemcitabine was developed in human plasma in order to quantitate gemcitabine and its squalene conjugate. After protein precipitation with acetonitrile/methanol (90/10, v/v), the compounds were analyzed by reversed-phase high performance liquid chromatography and detected by tandem mass spectrometry using multiple reaction monitoring. The method was linear over the concentration range of 10–10,000 ng/ml of human plasma for both compounds with an accuracy lower than 10.4% and a precision below 14.8%. The method showed a lower limit of quantitation of 10 ng/ml of human plasma for dFdC and dFdC-SQ. A preliminary in vivo study in mice was shown as application of the method as no significant difference between human and mice plasma for the analysis of dFdC and dFdC-SQ was demonstrated.
Keywords: Gemcitabine; Gemcitabine-squalene; Mass spectrometry; Cancer; Nucleosides analogues;

There is a confusion in the application of circular dichroism (CD) spectroscopy in analyzing collagen's structure for the overlapping of the spectral shapes and positions of the collagen triple helix and poly(proline-II)-like structure. The unique repetitive sequence of the collagen triple helix is susceptible to misalignment during the spontaneous assembly. Such misaligned structures are usually difficult to be characterized by CD or NMR spectroscopy. Here, RP-HPLC was developed as a conformational characterization technique for synthetic collagen-like peptides based on the different hydrophobicities exhibited by the triple-helical and unassembled peptides. RP-HPLC was also used to study thermal transitions and to measure melting point temperatures (T m) of the collagen-like peptides.
Keywords: Collagen; Triple helix; RP-HPLC; Melting analysis;

A novel HPLC fluorescence method for the quantification of methylphenidate in human plasma by Hao-Jie Zhu; Jun-Sheng Wang; Kennerly S. Patrick; Jennifer L. Donovan; C. Lindsay DeVane; John S. Markowitz (91-95).
A number of analytical methods have been established to quantify methylphenidate (MPH). However, to date no HPLC methods are applicable to human pharmacokinetic studies without the use of mass spectrometry (MS) detection. We developed a sensitive and reliable HPLC-fluorescence method for the determination of MPH in human plasma using 4-(4,5-diphenyl-1H-imidazol-2-yl) benzoyl chloride (DIB-Cl) as the derivatizing agent. An established GC-MS method was adopted in this study as a comparator assay. MPH was derivatized using DIB-Cl, and separated isocratically on a C18 column using a HPLC system with fluorescence detection (λ ex  = 330 nm, λ em  = 460 nm). The lower limit of quantification was found to be 1 ng/mL. A linear calibration curve was obtained over the concentrations ranging from 1 ng/mL to 80 ng/mL (r  = 0.998). The relative standard deviations of intra-day and inter-day variations were ≤9.10% and ≤7.58%, respectively. The accuracy ranged between 92.59% and 103.06%. The method was successfully applied to the pharmacokinetic study of a subject who received a single oral dose (0.3 mg/kg) of immediate-release MPH and yielded consistent results with that of the GC-MS method. This method is the first HPLC assay with non-MS detection providing sufficient reliability and sensitivity for both pre-clinical and clinical studies of MPH.
Keywords: Methylphenidate; Pharmacokinetics; HPLC; Fluorescence detection; GC-MS;

Liquid chromatography-electrospray ionization-tandem mass spectrometry for simultaneous analysis of chlorogenic acids and their metabolites in human plasma by Yuji Matsui; Shun Nakamura; Naoki Kondou; Yoshio Takasu; Ryuji Ochiai; Yoshinori Masukawa (96-105).
A method using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) was developed for the simultaneous analysis of nine chlorogenic acids (CGAs), three isomers each of caffeoylquinic acids (CQAs), feruloylquinic acids (FQAs) and dicaffeoylquinic acids (dCQAs), and their two metabolites, caffeic acid (CA) and ferulic acid (FA), in human plasma. In simultaneous multiple reaction monitoring (MRM) measurements using ESI-MS/MS with a negative ion mode, a deprotonated molecular ion derived from each of the 11 molecules was used as a precursor ion while three diagnostic product ions characteristic for each were selected for the qualitative analysis. To obtain maximal intensities for all diagnostic product ions, the collision energy was optimized for each one. LC separation was achieved under conditions of a reversed-phase Inertsil ODS-2 column combined with a gradient elution system using 50 mM acetic acid with 3% acetonitrile aqueous solution and 50 mM acetic acid with 100% acetonitrile. In the quantitative analysis, one of the three diagnostic product ions for each of the 11 molecules was selected. Application of simultaneous LC-ESI-MS/MS MRM measurements to analyze the 11 standards spiked into blank human plasma indicated that all diagnostic product ions were detected without any interference, and that the sensitivity, linearity and recovery of this method were acceptable. When using this method to analyze those 11 molecules in the plasma after oral ingestion of 250 ml of a drink containing a green coffee bean extract (300 mg CGAs), all 11 molecules were identified and CQAs, FQAs and FA were quantified. CQAs, FQAs and dCQAs in human plasma were detected for the first time. This method should be useful to understand the biological and pharmacological effects of CGAs, such as improvement of human hypertension.
Keywords: Chlorogenic acids; Human plasma; LC-ESI-MS/MS; Metabolite; Simultaneous analysis;

Normal phase and reverse phase HPLC-UV-MS analysis of process impurities for rapamycin analog ABT-578: Application to active pharmaceutical ingredient process development by Yong Chen; Gregory M. Brill; Nancy J. Benz; M. Robert Leanna; Madhup K. Dhaon; Michael Rasmussen; Casey Chun Zhou; James A. Bruzek; John R. Bellettini (106-117).
ABT-578, an active pharmaceutical ingredient (API), is a semi-synthetic tetrazole derivative of the fermented polyene macrolide rapamycin. Reverse phase (RP)-HPLC-UV-MS and normal phase (NP)-HPLC-UV-MS methods employing an LC/MSD trap with electrospray ionization (ESI) have been developed to track and map all significant impurities from the synthetic process. Trace-level tracking of key impurities occurring at various process points was achieved using complimentary methodologies, including a stability indicating reverse phase HPLC method capable of separating at least 25 starting materials and process-related impurities from the API (YMC-Pack Phenyl column, UV-MS, 210 nm) and a targeted reverse phase HPLC method capable of separating very polar compounds from crude reaction mixtures (Phenomenex Synergi Polar RP column, UV, 265 nm). In addition, a normal phase HPLC method condition with post-column modifier infusion is described for the separation of epimeric impurities, and analysis of aqueous-sensitive reactive species (YMC-Pack SIL column, UV-MS, 278 nm). Process control strategies were established with these combinations of analytical technologies for impurities analyses to enable a rich understanding of the ABT-578 process.
Keywords: Drug-eluting stent; Zotarolimus; ABT-578; Rapamycin; Sirolimus; RP-HPLC-UV-MS; NP-HPLC-UV-MS; Process impurities; API process analytical development; Impurities tracking and mapping; HPLC impurity profile;

Microdose clinical trial: Quantitative determination of fexofenadine in human plasma using liquid chromatography/electrospray ionization tandem mass spectrometry by Naoe Yamane; Zenzaburou Tozuka; Yuichi Sugiyama; Toshiko Tanimoto; Akira Yamazaki; Yuji Kumagai (118-128).
A sample treatment procedure and high-sensitive liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method for quantitative determination of fexofenadine in human plasma was developed for a microdose clinical trial with a cold drug, i.e., a non-radioisotope-labeled drug. Fexofenadine and terfenadine, as internal standard, were extracted from plasma samples using a 96-well solid-phase extraction plate (Oasis HLB). Quantitation was performed on an ACQUITY UPLC system and an API 5000 mass spectrometer by multiple reaction monitoring. Chromatographic separation was achieved on an XBridge C18 column (100 mm × 2.1 mm i.d., particle size 3.5 μm) using acetonitrile/2 mM ammonium acetate (91:9, v/v) as the mobile phase at a flow rate of 0.6 ml/min. The analytical method was validated in accordance with the FDA guideline for validation of bioanalytical methods. The calibration curve was linear in the range of 10–1000 pg/ml using 200 μl of plasma. Analytical method validation for the clinical dose, for which the calibration curve was linear in the range of 1–500 ng/ml using 20 μl of plasma, was also conducted. Each method was successfully applied for making determinations in plasma using LC/ESI-MS/MS after administration of a microdose (100 μg solution) and a clinical dose (60 mg dose) in eight healthy volunteers.
Keywords: Microdosing; Fexofenadine; Electrospray ionization; Tandem mass spectrometry; Validation;

A rapid, sensitive and selective LC–MS/MS method was developed and validated for the quantification of aniracetam in human plasma using estazolam as internal standard (IS). Following liquid–liquid extraction, the analytes were separated using a mobile phase of methanol–water (60:40, v/v) on a reverse phase C18 column and analyzed by a triple-quadrupole mass spectrometer in the selected reaction monitoring (SRM) mode using the respective [M + H]+ ions, m/z 220 → 135 for aniracetam and m/z 295 → 205 for the IS. The assay exhibited a linear dynamic range of 0.2–100 ng/mL for aniracetam in human plasma. The lower limit of quantification (LLOQ) was 0.2 ng/mL with a relative standard deviation of less than 15%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The validated LC–MS/MS method has been successfully applied to study the pharmacokinetics of aniracetam in healthy male Chinese volunteers.
Keywords: Aniracetam; Liquid chromatography–tandem mass spectrometry; Human plasma; Pharmacokinetic;

Liquid chromatography–tandem mass spectrometric method for determination of mosapride citrate in equine tissues by Yoichi Aoki; Hideki Hakamata; Yu Igarashi; Kazunari Uchida; Hisato Kobayashi; Norio Hirayama; Akira Kotani; Fumiyo Kusu (135-142).
A simple method for determination of mosapride citrate and its metabolite, des-p-fluorobenzyl mosapride (M-1), in equine muscle, liver, kidney, adipose tissue and intestine by liquid chromatography–tandem mass spectrometry has been developed. (±)-4-Amino-5-chloro-2-ethoxy-N-[[4-(2-chlorobenzyl)morpholinyl]methyl]benzamide was used as an internal standard. The analytes and internal standard were spiked and extracted from tissues by acetonitrile. The chromatographic separation was performed on a reversed-phase TSK-GEL SUPER ODS column with a mobile phase of acetonitrile–0.05% (v/v) formic acid containing 5 mmol/L nonafluoropentanoic acid (2:3, v/v). The method exhibited a large linear range from 0.0005 to 0.2 μg/mL for both mosapride citrate and M-1 (r  > 0.9976). In the intra-day assay (n  = 5), the relative standard deviations (RSDs) ranged from 1.1 to 7.8% for mosapride citrate and 1.6 to 7.2% for M-1. In the inter-day assay (n  = 3), the RSDs ranged from 1.0 to 13% for mosapride citrate and 0.8 to 11% for M-1. The extraction recovery at 1.28 μg/g of mosapride citrate from equine tissues ranged from 97 to 107%. The lower limit of quantification for mosapride citrate was found to be 0.004 μg/g. Stability studies were carried out at different storage conditions. The method reported is reliable, precise, and accurate and it has the capacity to be used for determination of mosapride citrate and its metabolite in tissue samples.
Keywords: Mosapride; Equine tissues; LC/MS/MS;

A rapid, sensitive and accurate liquid chromatographic-tandem mass spectrometry method is described for the simultaneous determination of nebivolol and valsartan in human plasma. Nebivolol and valsartan were extracted from plasma using acetonitrile and separated on a C18 column. The mobile phase consisting of a mixture of acetonitrile and 0.05 mM formic acid (50:50 v/v, pH 3.5) was delivered at a flow rate of 0.25 ml/min. Atmospheric pressure ionization (API) source was operated in both positive and negative ion mode for nebivolol and valsartan, respectively. Selected reaction monitoring mode (SRM) using the transitions of m/z 406.1 →  m/z 150.9; m/z 434.2 →  m/z 179.0 and m/z 409.4 →  m/z 228.1 were used to quantify nebivolol, valsartan and internal standard (IS), respectively. The linearity was obtained over the concentration range of 0.01–50.0 ng/ml and 1.0–2000.0 ng/ml and the lower limits of quantitation were 0.01 ng/ml and 1.0 ng/ml for nebivolol and valsartan, respectively. This method was successfully applied to the pharmacokinetic study of fixed dose combination (FDC) of nebivolol and valsartan formulation product after an oral administration to healthy human subjects.
Keywords: Nebivolol; Valsartan; LC–API-MS–MS;

A fast and efficient purification method for recombinant glucose oxidase (rGOx) for flask fermentation scale (up to 2 L) was designed for the purposes of characterization of rGOx mutants during directed protein evolution. The Aspergillus niger GOx was cloned into a pYES2-αMF-GOx construct and expressed extracellularly in yeast Saccharomyces cerevisiae. Hydrophobic interaction (HIC)/size exclusion (SEC)-tandem chromatographic system was designed for direct purification of rGOx from a conditioned complex expression medium with minimum preceding sample preparation (only adjustments to conductivity, pH and coarse filtering). HIC on Butyl 650s (50 mM ammonium acetate pH 5.5 and 1.5 M ammonium sulphate) absorbs GOx from the medium and later it is eluted by 100% stepwise gradient with salt free buffer directly into SEC column (Sephadex 200) for desalting and final polishing separation. The electrophoretic and UV–vis spectrophotometric analyses have proven enzyme purity after purification.
Keywords: Recombinant glucose oxidase; Purification; Hydrophobic interaction chromatography; Size exclusion chromatography;

Sensitive high-performance liquid chromatography–tandem mass spectrometry method for quantitative analysis of mycophenolic acid and its glucuronide metabolites in human plasma and urine by Marie-Odile Benoit-Biancamano; Patrick Caron; Éric Lévesque; Robert Delage; Félix Couture; Chantal Guillemette (159-167).
A method to determine total and free mycophenolic acid (MPA) and its metabolites, the phenolic (MPAG) and acyl (AcMPAG) glucuronides, using HPLC and mass spectrometry was developed. Mean recoveries in plasma and urine samples were >85%, and the lower limits of quantification for MPA, MPAG and AcMPAG were 0.05, 0.05 and 0.01 mg/L, respectively. For plasma, the assay was linear over 0.05–50 mg/L for MPA and MPAG, and from 0.01 to 10 mg/L for AcMPAG. A validation study demonstrated good inter- and intra-day precision (CV ≤ 11%) and accuracy (bias ≤ 16%) and satisfactory specificity and stability. Pharmacokinetic parameters were assessed in plasma and urine from healthy volunteers after an oral dose of mycophenolate mofetil.
Keywords: HPLC; Mass spectrometry; Mycophenolic acid; MPAG; AcMPAG; Glucuronide;

A selective and sensitive hydrophilic interaction liquid chromatography tandem mass spectrometric bioanalytical method for the quantitative determination of gaboxadol in human heparinized plasma was developed and validated. Gaboxadol and the stable isotope labeled internal standard were extracted from plasma by mixed mode solid phase extraction and analyzed on an Asahipak NH2P HPLC column with a mobile phase composed of 70% acetonitrile and 30% ammonium acetate (20 mM, pH 4). The analytes were detected by a SCIEX API 4000 triple quadropole instrument using turbo electrospray ionization and multiple reaction monitoring negative mode. The method was validated over the concentration range of 0.5–100 ng/mL. The intra-day precision of the assay, as measured by the coefficient of variation (CV%), was within 4%. The intra-day assay accuracy was found to be within 2.2% of the nominal concentration for all the standards. The average recovery of gaboxadol was about 87% and the ion suppression was approximately 8%. To eliminate late eluters including the glucuronides, a “front cut” column switching procedure was added to the chromatographic system. The effectiveness of the column switching in eliminating the absolute matrix effect caused by late eluters was demonstrated by the low variation (CV < 3.5%) in the peak areas of the internal standard during the assessment of the inter-day precision and accuracy and no significant relative matrix effect was observed as illustrated by the excellent intra-day precision (CV < 1.5%) from the assessment of standard samples prepared in five different lots of control plasma. The described bioanalytical method has been successfully utilized for the analysis of gaboxadol in post-dose samples (>8000) from various clinical studies. Inter-day precision and accuracy were assessed from the daily mean (n  = 2) of QC values from 52 runs, i.e. more than 3000 samples. The inter-day precision of the assay, based on the coefficient of variation of QC, ranged from 2.1 to 5.1%. The inter-day assay accuracy was found to be within 4% of the nominal concentration for all QC samples.
Keywords: Gaboxadol; HILIC; LC–MS/MS; SPE; Matrix effect; Validation;

SPE/SPME–GC/MS approach for measuring musk compounds in serum and breast milk by Zsuzsanna Kuklenyik; Xavier A. Bryant; Larry L. Needham; Antonia M. Calafat (177-183).
Musks can be used to provide distinctive odor or scent in many personal care products. Musk compounds have received growing attention in recent years by environmental scientists and regulatory agencies because of their increasing production volume and widespread environmental presence. A combined separation approach using solid phase extraction (SPE) and solid phase micro extraction (SPME) coupled to detection by gas chromatography mass spectrometry was developed for measuring four polycyclic musk compounds (Galactoside®, Tonalide®, Muskene®, Celestolide®) in serum and milk. The SPE and SPME separation steps were fully automated and required minimal sample handling. The method, which requires only 1 mL serum or breast milk to achieve limits of detection of 0.03–0.3 ng/mL, is applicable in biomonitoring studies for human internal dose measurement of polycyclic musk compounds.
Keywords: Musks; SPE; SPME; Gas chromatography; Mass spectrometry;

Structural characterization of metabolites of salvianolic acid B from Salvia miltiorrhiza in normal and antibiotic-treated rats by liquid chromatography–mass spectrometry by Man Xu; Hui Guo; Jian Han; Shi-feng Sun; Ai-hua Liu; Bao-rong Wang; Xiao-chi Ma; Peng Liu; Xue Qiao; Zi-chuan Zhang; De-an Guo (184-198).
This study was conducted to compare the in vivo metabolites of salvianolic acid B (Sal B) between normal rats and antibiotic-treated rats and to clarify the role of intestinal bacteria on the absorption, metabolism and excretion of Sal B. A valid method using LC–MS n analysis was established for identification of rat biliary and fecal metabolites. And isolation of normal rat urinary metabolites by repeated column chromatography was applied in this study. Four biliary metabolites and five fecal metabolites in normal rats were identified on the basis of their MS n fragmentation patterns. Meanwhile, two normal rat urinary metabolites were firstly identified on the basis of their NMR and MS data. In contrast, no metabolites were detected in antibiotic-treated rat urine and bile, while the prototype of Sal B was found in antibiotic-treated rat feces. The differences of in vivo metabolites between normal rats and antibiotic-treated rats were proposed for the first time. Furthermore, it was indicated that the intestinal bacteria showed an important role on the absorption, metabolism and excretion of Sal B. This investigation provided scientific evidence to infer the active principles responsible for the pharmacological effects of Sal B.
Keywords: Salvianolic acid B; In vivo metabolites; LC–MS n ; Intestinal bacteria; Antibiotic-treated rats;

Determination of aesculin in rat plasma by high performance liquid chromatography method and its application to pharmacokinetics studies by QiuHong Chen; ShiXiang Hou; Jia Zheng; YueQi Bi; YuanBo Li; XiaoJiao Yang; Zheng Cai; XiangRong Song (199-204).
A sensitive and reproducible high performance liquid chromatography method with UV detection was described for the determination of aesculin in rat plasma. After deproteinization by methanol using metronidazole as internal standard (I.S.), solutes were evaporated to dryness at 40 °C under a gentle stream of nitrogen. The residue was reconstituted in 100 μl of mobile phase and a volume of 20 μl was injected into the HPLC for analysis. Solutes were separated on a Diamonsil C18 column (250 mm × 4.6 mm i.d., 5 μm particle size, Dikma) protected by a ODS guard column (10 mm × 4.0 mm i.d., 5 μm particle size), using acetonitrile–0.1% triethylamine solution (adjusted to pH 3.0 using phosphoric acid) (10:90, v/v) as mobile phase (flow-rate 1.0 ml/min), and wavelength of the UV detector was set at 338 nm. No interference from any endogenous substances was observed during the elution of aesculin and internal standard (I.S., metronidazole). The retention times for I.S and aesculin were 10.4 and 12.4 min, respectively. The limit of quantification was evaluated to be 57.4 ng/ml and the limit of detection was 24.0 ng/ml. The method was used in the study of pharmacokinetics of aesculin after intraperitoneal injection (i.p.) administration in rats.
Keywords: Aesulin; Reversed-phase HPLC; Pharmacokinetics; Metronidazole; Plasma;

Partitioning of caseinomacropeptide in aqueous two-phase systems by César A. Sodré da Silva; Jane S.R. Coimbra; Edwin E. Garcia Rojas; Luis A. Minim; Luis Henrique Mendes da Silva (205-210).
This study evaluates the influence of type of salt and temperature on the partition coefficient of caseinomacropetide (CMP) to determine the best conditions for the recovery of CMP in aqueous two-phase systems (ATPS) composed by poly(ethylene glycol) (PEG) 1500 and an inorganic salt (potassium phosphate, sodium citrate, lithium sulfate or sodium sulfate). In all systems, CMP presented affinity for the PEG-rich phase. The PEG1500 + lithium sulfate showed the highest values of partitioning coefficient. In addition, thermodynamic parameters (ΔH°, ΔS°, ΔG°) as a function of temperature, were calculated for the system PEG1500-sodium citrate at different PEG concentrations and the results imply thermodynamic differences between partitioning of CMP in this system.
Keywords: Aqueous two-phase systems; Caseinomacropetide; Hydrophobicity; Salt; Temperature; Thermodynamic parameters;

Extracts from Swertia chirata (family Gentianaceae) have antidiabetics and antioxidant activity, largely attributed to the flavonoids and secoiridoids, which are a major class of functional components in methanolic extracts from aerial part of plants. In order to facilitate analysis of systemic exposure to S. chirata derived products in animals, we developed a liquid chromatography tandem mass spectrometry (LC–MS/MS) based method that is capable of routinely monitoring plasma levels of flavonoids and secoiridoids. An LC–MS/MS-based method has been developed for the simultaneous estimation of two bioactive markers, mangiferin and amarogentin along with three other components, amaroswerin, sweroside and swertiamarin in rat plasma. All the analytes including the internal standard (kutkoside) were chromatographed on RP-18 column (250 mm × 4 mm i.d., 5 μm.) coupled with guard column using acetonitrile: 0.5 mM ammonium acetate buffer, pH ∼3.0 as mobile phase at a flow rate of 1 ml/min in gradient mode. The final flow to source was splitted in 1:1 ratio. The detection of the analytes was performed on API 4000 LC–MS/MS system in the multiple reaction-monitoring (MRM) mode. The quantitation for analytes other than the pure markers was based on relative concentration. The method was validated in terms of establishing linearity, specificity, sensitivity, recovery, accuracy and precision (Intra- and Inter-day), freeze-thaw stability, peltier stability, dry residue stability and long-term stability. The recoveries from spiked control samples were >90% for all analytes and internal standard except mangiferin where recovery was >60%. Intra- and inter-day accuracy and precision of the validated method were within the acceptable limits of <15% at low and <10% at other concentrations. The quantitation method was successfully applied to generate pharmacokinetic (PK) profile of markers as well as to detect other components in plasma after intravenous dose administration of herbal preparation in male Sprague-Dawley (SD) rats.
Keywords: LC–MS/MS; Bioanalysis; Swertia chirata; Mangiferin and secoiridoid glycosides;

Direct process integration of extraction and expanded bed adsorption in the recovery of crocetin derivatives from Fructus Gardenia by Min Zhang; Ping Hu; Qionglin Liang; Huihua Yang; Qingfei Liu; Yiming Wang; Guoan Luo (220-226).
A process that integrated an extraction tank (EXT) and an expanded bed adsorption (EBA) into a new system EXT-EBA for direct purifying crocetin derivatives from Fructus Gardenia was described. Conditions were set to allow the extraction and purification in a single step. A comparison between the integrated process and the conventional process to purify crocetin derivatives was presented. The integrated process resulted in 52.79% recovery of crocin compared to 24.12% in the conventional process. The process time and solvent used were decreased in the integrated process. The result suggests that the EXT-EBA integrates extraction, clarification, and purification in a single step, greatly simplifying the process flow and reducing the cost and time of extraction and purification of crocetin derivatives from Fructus Gardenia.
Keywords: Crocetin derivatives; Fructus Gardenia; Process integration; Expanded bed adsorption;

Continuous extraction of α- and β-amylases from Zea mays malt in a PEG4000/CaCl2 ATPS by J.P.M. Biazus; J.C.C. Santana; R.R. Souza; E. Jordão; E.B. Tambourgi (227-233).
In the present work, α- and β-amylase enzymes from Zea mays malt were recovered by continuous extraction in a PEG/CaCl2 aqueous two-phase system (ATPS). The influences of the flux rate (R Q), free area of vane (A free) and vane rotation (R V) on enzyme recovery were studied by optimization using response surface methodology (RSM). The protein content and enzyme activity were measured from time to time in the extract and refined fluxes. RSM curves showed a squared dependence of recovery index with the R Q, A free and R V. The best system for recovering the maize malt enzymes was with low vane rotation and flux rate and high free area of vane. α- and β-amylases were purified 130-fold in the salt-rich phase.
Keywords: Continuous extraction; α- and β-amylases; PEG/CaCl2 ATPS; Zea mays malt; Maize seeds;

Amino acids partitioning in aqueous two-phase system of polypropylene glycol and magnesium sulfate by Alireza Salabat; Mohammad H. Abnosi; Azadeh R. Bahar (234-238).
The counter-current chromatography method using aqueous two-phase systems, which is a form of liquid–liquid partition chromatography, could be applied for separation of the amino acids. This method needs some information about the partition coefficient of the amino acids in such systems. In this work, partitioning of amino acids d-alanine, l-valine and l-leucine was investigated in aqueous two-phase system of polypropylene glycol (PPG425) + MgSO4  + H2O at 298.15 K. The results showed that increasing the amino acid hydrophobicity lead to a corresponding increase in the partition coefficients and increasing tie line length lead to decreasing partition coefficients. The effect of the pH on amino acids partitioning was also determined. The experimental data are correlated using a modified virial-type model. The comparisons between the correlation and the experimental data reveal a good agreement.
Keywords: Aqueous two-phase system; Polypropylene glycol; Amino acids; Partition coefficient; Magnesium sulfate;

Jones oxidation and high performance liquid chromatographic analysis of cholesterol in biological samples by Jun Dong; Wenxiang Chen; Shu Wang; Jiangtao Zhang; Hongxia Li; Hanbang Guo; Yong Man; Baosheng Chen (239-246).
A simple pre-column derivatization procedure for HPLC analysis of cholesterol in biological samples was developed. Cholesterol was treated with chromic acid and sulfuric acid in acetone (the Jones oxidation) and cholest-4-en-3,6-dione was formed. The reaction was finished in 5 min at room temperature and the product showed a strong UV absorbance at 250 nm that enabled an HPLC detection limit of 0.2 pmol. With stigmasterol as an internal standard, the reaction was applied to the analysis of total and free cholesterol in serum and high-density lipoproteins and the analysis showed a within-run and total coefficient of variation of about 0.2% and 0.5%, respectively.
Keywords: Cholesterol; Cholest-4-en-3,6-dione; Stigmasterol; Chromic acid; HPLC;

Partitioning of six typical globular proteins with molecular weights ranging from 12.6 to 250 kDa was investigated using an aqueous two-phase system formed by heating a solution containing the individual proteins and n-dodecyldimethylphosphine oxide (APO12) above the cloud point of the nonionic surfactant (approximately 40 °C). The partition coefficient, K p, was much greater at 55 than 45 °C and depended on both APO12 and protein concentrations. The value of K p for bovine β-lactoglobulin (β-L) varied from 2 to 60, and was larger for 1.0 mg/mL solutions than for ovalbumin (2× greater), bovine serum albumin (3× greater) and lysozyme (12× greater). Catalase and cytochrome c were apparently denatured in the presence of 20 mg/mL of APO12 and were not investigated. Large values of K p for β-L resulted when the pH of APO12 mixtures containing phospholipids and either a cationic or anionic surfactant in molar ratios of 10:0.5:1.0 was partitioned above or below the isoelectric point of the protein, respectively. The affinity of the proteins for the APO12 micelle was responsible for partitioning of the proteins into the upper phase. Finally, DSC studies with β-L showed that the denaturing action of n-decyldimethylphosphine oxide (APO10) below 61 °C and APO12 at 22 °C was reversed by dilution or dialysis, respectively.
Keywords: Two-phase partitioning; Micelles; Nonionic surfactant; Proteins; ATPS;

Structural effect of a recombinant monoclonal antibody on hinge region peptide bond hydrolysis by Tao Xiang; Edwin Lundell; Zuping Sun; Hongcheng Liu (254-262).
IgG hinge region peptide bonds are susceptible to degradation by hydrolysis. To study the effect of Fab and Fc on hinge region peptide bond hydrolysis, a recombinant humanized monoclonal IgG1 antibody, its F(ab′)2 fragment, and a model peptide with amino acid sequence corresponding to the hinge region were incubated at 40 °C in formulation buffer including complete protease inhibitor and EDTA for 0, 2, 4, 6 and 8 weeks. Two major cleavage sites were identified in the hinge region of the intact recombinant humanized monoclonal antibody and its F(ab′)2 fragment, but only one major cleavage site of the model peptide was identified. Hinge region peptide bond hydrolysis of the intact antibody and its F(ab′)2 fragment degraded at comparable rates, while the model peptide degraded much faster. It was concluded that Fab region of the IgG, but not Fc portion had significant effect on preventing peptide bond cleavage by direct hydrolysis. Hydrolysis of hinge region peptide bonds was accelerated under both acidic and basic conditions.
Keywords: Recombinant monoclonal antibody; Hydrolysis; Hinge region; Mass spectrometry;

A sensitive and reliable method was developed to quantitate phenylephrine in human plasma using liquid chromatography–electrospray tandem mass spectrometry. The assay was based on solid-phase extraction with C18 cartridges and hydrophilic interaction chromatography performed on a pentafluorophenylpropylsilica column (50 mm × 4 mm, 3 μm particles), the mobile phase consisted of methanol–10 mM ammonium acetate (90:10, v/v). Quantification was through positive-ion mode and selected reaction monitoring at m/z 168.1 → 135.0 for phenylephrine and m/z 182.1 → 135.0 for internal standard etilefrin, respectively. The lower limit of quantitation was 51 pg/ml using 0.25 ml of plasma and linearity was observed from 51 to 5500 pg/ml. Within-day and between-day precision expressed by relative standard deviation was less than 12% and inaccuracy did not exceed 8% at all levels. The assay was applied to the analysis of samples from a pharmacokinetic study.
Keywords: Phenylephrine; Etilefrin; Mass spectrometry; HILIC; Liquid chromatography;

A rapid, sensitive and accurate liquid chromatographic–tandem mass spectrometry (LC–MS–MS) method is described for the determination of duloxetine in human plasma. Duloxetine was extracted from plasma using methanol and separated on a C18 column. The mobile phase consisting of a mixture of acetonitrile and 5 mM ammonium acetate (45:55, v/v, pH 3.5) was delivered at a flow rate of 0.3 ml/min. Atmospheric pressure ionization (API) source was operated in positive ion mode. Multiple reaction monitoring (MRM) mode using the transitions of m/z 298.1 →  m/z 44.0 and m/z 376.2 →  m/z 123.2 were used to quantify duloxetine and internal standard (I.S.), respectively. The linearity was obtained over the concentration range of 0.1–50.0 ng/ml and the lower limit of quantitation (LLOQ) was 0.1 ng/ml. This method was successfully applied to pharmacokinetic study of a duloxetine formulation product after oral administration to healthy human subjects.
Keywords: Duloxetine; LC-API–MS–MS;

One prerequisite for therapeutic effects of psychiatric drugs is the ability to pass the blood brain barrier. Hence, it is important to know the concentration of antipsychotic drugs in brain tissue. In general, determinations of lipophilic compounds from lipophilic matricies such as the brain are a challenge. Here we have adapted a plasma assay for antipsychotics for the target organ the brain. Using modified sample preparation and chromatographic strategies, the analytes were extracted from rat brain homogenate and analyzed by LC-MS/MS. The method used a Waters Atlantis™ dC-18 (30 mm × 2.1 mm i.d., 3 μm) column with a mobile phase of acetonitrile/5 mM ammonium formate (pH 6.1 adjusted with formic acid) and gradient elution. All analytes were detected in positive ion mode using multiple-reaction monitoring. The method was validated and the linearity, lower limit of quantitation, precision, accuracy, recoveries, specificity and stability were determined. This method was then successfully used to quantify the rat brain tissue concentration of the analytes after chronic treatment with these antipsychotic drugs.
Keywords: Brain; Risperidone; 9-Hydroxyrisperidone; Olanzapine; Clozapine; Ziprasidone; Haloperidol;

A rapid LC–MS/MS method was developed and partially validated for the quantitation of montelukast in spiked sheep plasma. A total run time of 1.5 min was achieved using a short monolithic column and employing a rapid gradient. Sample preparation involved protein precipitation with twofold acetonitrile by volume during which a deuterated internal standard (montelukast D-6) was incorporated. The MRM transitions for montelukast and the deuterated internal standard were 586/422 and 592/427, respectively. A linear dynamic range of 0.25–500 ng/mL with a correlation coefficient of 0.9999 was achieved. Precision was below 5% at all levels except at the LOQ (0.36 ng/mL) which demonstrated an overall of R.S.D. of 8%. Post-column infusion experiments were performed with precipitated plasma matrix and showed minimal interference with the peaks of interest.
Keywords: Montelukast; Sheep; Plasma; Mass spectrometry;

Determination of serum lysophosphatidic acid as a potential biomarker for ovarian cancer by Marija Meleh; Barbara Požlep; Anita Mlakar; Helena Meden-Vrtovec; Lucija Zupančič-Kralj (287-291).
A fast and selective analytical method, used to determine the different lysophosphatidic acid (LPA) species in serum, has been developed and validated. LPA species were quantitatively extracted from serum using methanol–chloroform (2:1, v/v). The proteins were precipitated by this solvent mixture and separated by centrifugation in one step. LPA levels were determined in clear extracts using the HPLC-MS/MS method. The linearity of this method was established in the concentration range between 0.1 and 16 μM for all LPA species with a correlation coefficient greater than 0.99. Recovery of all LPA species determined by the serum, fortified at approximately 1 μM and 2–3 μM, was between 93% and 111% with an average R.S.D. of less than 8%. This method was used to determine LPA in numerous sera of healthy controls, patients with benign ovarian tumours and ovarian cancer at different stages. Significantly higher total LPA levels were determined in the sera of patients with different types of tumours (benign and malignant).
Keywords: Lysophosphatidic acid; Ovarian cancer; Biomarker; Serum; High performance liquid chromatography; Tandem mass spectrometry;

Determination of derivatized amino acids in human embryo culture media by gas chromatography by Mitja Križman; Irma Virant-Klun; Mirko Prošek (292-295).
An adequate analytical method for determination of amino acids can provide a better insight in the metabolism of in vitro human embryo cultures, increasing the success rate of embryo implantation. Since individual amino acid amounts per embryo occur in the nanogram range, GC was the technique of choice, due to its inherent sensitivity and high sample throughput. Amino acids were analyzed as alkyl formate derivatives. The limits of detection (LOD) of all amino acids involved were in the sub-nmol range. The high risk of sample contamination proved to be the major analytical issue, but it could be overcome. For an extended method sensitivity, a simple preconcentration step could also be used.
Keywords: Amino acids; Embryo; Derivatization; Gas chromatography;

A two-dimensional column-switching system without sample loop trapping, where two columns were switched directly via a six-port two-position switching valve, was successfully applied for the first time to the isolation and purification of six iridoid glycosides including geniposide, gardenoside, shanzhiside, scandoside methyl ester, deacetyl-asperulosidic acid methyl ester and genipin-1-β-d-gentiobioside from Gardenia jasminoides Ellis, a plant used in the traditional Chinese medicine. The introduction of the six-port switching valve instead of sample loop assured 100% recovery from the first dimension to the second, and the injection volumes of the second dimension could reach 20 ml. In this mode of operation, the sample size of the two-dimensional approach was more than 1.3 times that of conventional gradient methods with even less solvent consumption. And the simultaneous operations of the two dimensions allowed the cycle time to be less than 19 min, compared with that (90 min) in the gradient elution single-dimension mode of operation. All of the six isolated iridoid glycosides were isolated at high purities of over 99% with approximately 96% recoveries.
Keywords: Preparative chromatography; Column switch; Gardenia jasminoides Ellis; Iridoid glycoside; Two-dimensional chromatography;

Determination of 17-dimethylaminoethylamino-17-demethoxygeldanamycin in human plasma by liquid chromatography with mass-spectrometric detection by Xiaohong Chen; Erin R. Gardner; Martin Gutierrez; Shivaani Kummar; William D. Figg (302-306).
An analytical method was developed and validated for the quantitative determination of 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG; NSC707545), a novel heat shock 90 inhibitor, in human plasma. Calibration curves were linear in the concentration range of 1–500 ng/mL. Sample pretreatment involved a liquid–liquid extraction of 0.2 mL aliquots of plasma with ethyl acetate. 17-DMAG and the internal standard, beclomethasone, were separated on a Zorbax SB C18 column (75 mm × 2.1 mm, 3.5 μm), using a mobile phase composed of methanol and 0.2% formic acid (55:45, v/v). The column effluent was monitored by mass spectrometry with electrospray ionization. For the quality control samples at four different concentrations that were analyzed in quintuplicate, on four separate occasions, the accuracy and precision ranged from 93.8% to 99.5% and 1.4% to 3.3%, respectively. The assay modifications significantly improve upon our original, validated method. The developed method was subsequently applied to study the pharmacokinetics of 17-DMAG in a group of 23 patients.
Keywords: 17-DMAG; LC/MS;

Liquid chromatography method for detecting native fluorescent bioamines in urine using post-column derivatization and intramolecular FRET detection by Makoto Yoshitake; Hitoshi Nohta; Susumu Ogata; Kenichiro Todoroki; Hideyuki Yoshida; Takashi Yoshitake; Masatoshi Yamaguchi (307-312).
Liquid chromatography (LC) with fluorescence detection is described for simultaneous determination of native fluorescent bioamines (indoleamines and catecholamines). This is based on intramolecular fluorescence resonance energy transfer (FRET) in an LC system following post-column derivatization of native fluorescent bioamines’ amino groups with o-phthalaldehyde (OPA). OPA fluorescence was achieved through an intramolecular FRET process when the molecules were excited at maximum excitation wavelength of the native fluorescent bioamines. Bioamines separated by reversed-phase LC on ODS column were derivatized with OPA and 2-mercaptoethanol. This method provides sufficient selectivity and sensitivity for the determination of normetanephrine, dopamine, tyrosine, 5-hydroxytryptamine, tryptamine, and tryptophan in healthy human urine without prior sample purification.
Keywords: FRET; Liquid chromatography; Post-column derivatization; Indoleamines; Catecholamines; o-Phthalaldehyde;