Journal of Chromatography B (v.842, #1)

Reason: This article has been published without consent of all authors. P.N Khalil and M.N. Khalil have submitted the paper without knowledge or consent of the other co-authors, N. Erb, G. Escherich and G.E. Janka-Schaub.

A simple, rapid, selective and specific high-performance liquid chromatography (HPLC) method with fluorescence detection was developed for determination of glucosamine sulfate in human plasma and application to a bioequivalence in healthy volunteers. Precipitation of plasma was accomplished with acetonitrile to separate interfering endogenous products from the compound of interest. After vortex mixing and centrifugation, the supernatant was transferred and derivatized with 9-fluorenylmethoxycarbonyl chloride–acetonitrile solution in borate buffer (pH = 8.0) at 30 °C for 30 min. The chromatographic separation was performed on a Diamonsil™ C18 column (150.0 mm × 4.6 mm, 5 μm) with a mobile phase gradient consisting of water and acetonitrile at a flow rate of 1 mL/min. The method was linear in the range of 0.1–10.0 μg/mL with a correlation coefficient (r) of 0.9996. The limit of detection was 15 ng/mL. Inter- and intra-day precisions were ≤6.28 and 7.41%, respectively, and the accuracy ranged from 95.20 to 104.92%. Extraction recoveries of glucosamine sulfate from plasma were more than 90.4%. Plasma samples containing glucosamine sulfate were stable for 40 days at −20 °C and for 24 h after derivatization at 4 °C. The method was successfully applied to the bioequivalence study of glucosamine sulfate in healthy volunteers.
Keywords: Glucosamine sulfate; High performance liquid chromatography; Derivatization; Fluorescence; Bioequivalence;

A multi-bed sorption trap designed to quantitatively collect volatile organic compounds from large-volume vapor samples and inject them into a gas chromatograph is combined with a comprehensive two-dimensional gas chromatograph (GC × GC) for the analysis of organic compounds in human breath samples. The first-column effluent of the GC × GC is modulated by a single-stage, resistively-heated and air-cooled segment of 0.18-mm i.d. stainless steel column using the same stationary phase as the first column. Cooling gas is provided by a two-stage conventional refrigeration system, and thus no consumables other than carrier gas and electric power are required. The sorption trap uses four discreet beds, three containing different grades of graphitized carbon and one containing a carbon molecular sieve. The ordering of the beds in the trap tube is from the weakest to strongest adsorbent during sample collection. Breath samples are collected in gas sampling bags, and samples are passed through the trap at a flow rate of about 50 cm3/min. After sample collection, hydrogen carrier gas flow is initiated in the direction opposite to the sample collection flow, and the metal trap tube is resistively heated to inject a sample plug into the GC × GC. Performance data for the combined GC × GC/sorption-trap instrument is described, and human breath-sample chromatograms are presented.
Keywords: GC × GC; Human breath analysis; Instrument design; FID;

A simple, rapid, and specific analytical method for simultaneous determination of geniposide, baicalin, cholic acid and hyodeoxycholic acid in 50 μL samples of rat serum was developed by high performance liquid chromatography–tandem mass spectrometry. The quantification of the target compounds was determined by multiple reaction monitoring (MRM) mode using electrospray ionization (ESI). The correlation coefficients of the calibration curves were better than 0.997. The intra- and inter-day accuracy, precision, and linear range had been investigated in detail. This method was subsequently applied to pharmacokinetic studies of geniposide, baicalin, cholic acid and hyodeoxycholic acid in rats successfully.
Keywords: HPLC–MS–MS; Geniposide; Baicalin; Cholic acid; Hyodeoxycholic acid; Pharmacokinetics;

The interest of many investigators in naphthoquinones is due to their broad-range of biological actions from phytotoxic to fungicidal. The main aim of this work was to investigate the influence of different pH values of cultivation medium on naphthoquinone content in Dionaea muscipula. For this purpose, we optimized the simultaneous analysis of the most commonly occurring naphthoquinones (1,4-naphthoquinone, lawsone, juglone and plumbagin) by high performance liquid chromatography coupled with diode array detector (HPLC-DAD). The most suitable chromatographic conditions were as follows: mobile phase: 0.1 mol l−1 acetic acid:methanol in ratio of 33:67 (%, v/v), flow rate: 0.75 ml min−1 and temperature: 42 °C. Moreover, we looked for the most suitable technique for preparation of plant samples (D. muscipula, Juglans regia, Paulownia tomentosa, Impatience glandulifera, Impatience parviflora, Drosera rotundifolia, Drosera spathulata and Drosera capensis) due to their consequent analysis by HPLC-DAD. It clearly follows from the results obtained that sonication were the most suitable technique for preparation of J. regia plants. We also checked the recoveries of the determined naphthoquinones, which were from 96 to 104%. Finally, we investigated the changes in content of plumbagin in D. muscipula plants according to different pH of cultivation medium. The content increased with increasing pH up to 5 and, then, changed gradually. The lower content of plumbagin at lower pH values was of interest to us. Therefore, we determined the content of this naphthoquinone in the cultivation medium, what has not been studied before. We discovered that the lower tissue content of plumbagin was due to secretion of this naphthoquinone into the cultivation medium.
Keywords: Naphthoquinones; High performance liquid chromatography; Diode array detector; Plumbagin; 1,4-Naphthoquinone; Lawsone; Juglone; Plants;

A rapid, sensitive, selective and specific HPLC/ESI-MS/MS assay method was developed and validated for the simultaneous quantitation of α-/β-diastereomers of arteether (AE), sulphadoxine (SDX) and pyrimethamine (PYR) in rat blood plasma using propyl ether analogue of β-arteether as internal standard. The method involved a single-step, liquid–liquid extraction with ethyl acetate and the analytes were chromatographed on a C18 chromatographic column by isocratic elution with methanol:ammonium acetate buffer (10 mM, pH 4) (90:10%, v/v) and analyzed by tandem mass spectrometry. The run time was 4.5 min and the weighted (1/x 2) calibration curves were linear over a range of 0.78–400 ng ml−1. The method was validated fully and the lower limit of quantification (LLOQ) in plasma was 0.78 ng ml−1 for all the analytes. The intra- and inter-day precision and accuracy were found to be well within the acceptable limits (<15%) and the analytes were stable after three freeze–thaw (f–t) cycles. The absolute recoveries were consistent and reproducible. The assay method was applied to pre-clinical pharmacokinetic interaction studies of α-/β-AE, SDX and PYR in rats.
Keywords: Tandem mass spectrometry; Blood plasma; Sulphadoxine; Pyrimethamine; α-/β-Arteether;

Simultaneous determination of plasmatic phytosterols and cholesterol precursors using gas chromatography–mass spectrometry (GC–MS) with selective ion monitoring (SIM) by H.S. Mohamed Ahmida; Pierfrancesco Bertucci; Letizia Franzò; Renato Massoud; Claudio Cortese; Alberto Lala; Giorgio Federici (43-47).
Phytosterols (β-sitosterol, cholestanol and campesterol) and cholesterol precursors (desmosterol and lathosterol), have been suggested as important biochemical markers of intestinal cholesterol absorption and liver biosynthesis, respectively, and as useful clinical parameters in the study of hypercholesterolemia, β-sitosterolemia, atherosclerosis and cardiovascular disease, including pharmacological response to hypolipidemic agents. We developed an optimised analytical method for the simultaneous analysis of cholestanol, desmosterol, lathosterol, campesterol and β-sitosterol in plasma using capillary gas chromatography coupled to mass spectrometry (GC–MS) with multiple selected ion monitoring (SIM). This method is based on the alkaline hydrolysis of sterol esters, extraction of free sterols and derivatization. The recovery of all sterols was in the range 76–101%. Within-day relative standard deviations (R.S.Ds.) and the between-day R.S.Ds. of cholestanol, desmosterol, lathosterol, campesterol and β-sitosterol were less than 8%, and their plasma levels in 161 normal subjects were (mean ± S.D.) 4.73 ± 2.57, 2.37 ± 1.04, 6.23 ± 3.14, 3.67 ± 1.95 and 5.92 ± 3.62 μmol/l, respectively.
Keywords: Phytosterols; Cholesterol precursors; Gas chromatography–mass spectrometry;

Rotavirus-like particles primary recovery from insect cells in aqueous two-phase systems by Jorge Benavides; Jimmy A. Mena; Mayra Cisneros-Ruiz; Octavio T. Ramírez; Laura A. Palomares; Marco Rito-Palomares (48-57).
Virus-like particles have a wide range of applications, including vaccination, gene therapy, and even as nanomaterials. Their successful utilization depends on the availability of selective and scalable methods of product recovery and purification that integrate effectively with upstream operations. In this work, a strategy based on aqueous two phase system (ATPS) was developed for the recovery of double-layered rotavirus-like particles (dlRLP) produced by the insect cell-baculovirus expression system. Polyethylene glycol (PEG) molecular mass, PEG and salt concentrations, and volume ratio (Vr, volume of top phase/volume of bottom phase) were evaluated in order to determine the conditions where dlRLP and contaminants concentrated to opposite phases. Two-stage ATPS consisting of PEG 400-phosphate with a Vr of 13.0 and a tie-line length (TLL) of 35% (w/w) at pH 7.0 provided the best conditions for processing highly concentrated crude extract from disrupted cells (dlRLP concentration of 5 μg/mL). In such conditions intracellular dlRLP accumulated in the top phase (recovery of 90%), whereas cell debris remained in the interface. Furthermore, dlRLP from culture supernatants accumulated preferentially in the interface (recovery of 82%) using ATPS with a Vr of 1.0, pH of 7.0, PEG 3350 (10.1%, w/w) and phosphate (10.9%, w/w). The purity of dlRLP from culture supernatant increased up to 55 times after ATPS. The use of ATPS resulted in a recovery process that produced dlRLP with a purity between 6 and 11% and an overall product yield of 85% (w/w), considering purification from intracellular and extracellular dlRLP. Overall, the strategy proposed in this study is simpler than traditional methods for recovering dlRLP, and represents a scalable and economically viable alternative for production processes of vaccines against rotavirus infection with significant scope for generic commercial application.
Keywords: Aqueous two-phase systems; Rotavirus like-particles; Insect cells;

Simple, sensitive and rapid LC–MS method for the quantitation of indapamide in human plasma—application to pharmacokinetic studies by Wei-Dong Chen; Yan Liang; Hong Zhang; Hao Li; Ye Xiong; Guang-Ji Wang; Lin Xie (58-63).
A sensitive and specific method using liquid chromatography with electrospray ionization mass spectrometry (LC–ESI–MS) has been developed and validated for the identification and quantification of indapamide in human plasma. A simple liquid–liquid extraction procedure was followed by injection of the extracts on to a C18 column with gradient elution and detection using a single quadrupole mass spectrometer in selected ion monitoring (SIM) mode. The method was tested using six different plasma batches. Linearity was established for the concentration range 0.5–100.0 ng/ml, with a coefficient of determination (r) of 0.9998 and good back-calculated accuracy and precision. The intra- and inter-day precision (RSD%) was lower than 10%, and accuracy ranged from 85 to 115%. The lower limit of quantification was reproducible at 0.2 ng/ml with 0.2 ml plasma. The proposed method enables the unambiguous identification and quantification of indapamide for pre-clinical and clinical studies.
Keywords: Indapamide; HPLC–ESI–MS; Human plasma; Pharmacokinetics;

A new method for determination of plasma homocystine by isotope dilution and electrospray tandem mass spectrometry by Michela Tomaiuolo; Gennaro Vecchione; Elvira Grandone; Nicola Cocomazzi; Bruno Casetta; Giovanni Di Minno; Maurizio Margaglione (64-69).
A new analytical determination method of homocystine in human plasma has been developed. The method utilises liquid chromatography coupled to ionspray tandem mass spectrometry. Quantitative analysis was achieved using as an internal standard homocystine-d8. Mass spectrometer operated in the multiple reaction mode: homocystine and homocystine-d8 were detected through the transition from the precursor to the product ion (from m/z 269.3 to 90.0, and m/z 277.3 to 94.0, respectively). The method is extremely sensitive, with limit of detection in the range of 6 fmol/L. The interassay and intraassay coefficients of variation for homocystine were 6.22% and 3.4%, respectively. The accuracy for the added homocystine ranged from 85% to 110%. High specificity of tandem mass spectrometry coupled with a fast chromatographic process is suitable for a rapid and reliable assay of homocystine.
Keywords: Homocystine; Electrospray tandem mass spectrometry;

On-line biological sample cleanup for electrospray mass spectrometry using sol–gel columns by Nina Johannesson; Evan Pearce; Maria Dulay; Richard N. Zare; Jonas Bergquist; Karin E. Markides (70-74).
Using a slight overpressure, a urine sample is loaded onto a monolithic photopolymerized sol–gel column that has been derivatized with hydrophobic carbon chains and then the complex urine matrix is washed with aqueous solution. A buffer containing organic solvent is used to elute the adsorbed peptides by an applied voltage and the sample is then introduced into a mass spectrometer by sheath flow electrospray. The importance of desalting this type of sample is demonstrated by an experiment that shows that the signal intensity of a test solution with neurotensin, sprayed directly into the mass spectrometer, decreased from 4.5 × 104  cps to no detectible signal when just 10% urine is added to the sample solution. We suggest that this procedure may find general application for desalting biological samples prior to mass spectrometric analysis.
Keywords: Desalting; On-line; Preconcentration; Biological sample; Mass spectrometry; Electrophoresis; Octadecyltrimethoxysilane; Sol–gel;