Journal of Chromatography B (v.809, #2)
News Section (N1-N2).
OFC: Update (OFC).
Investigation of the use of immobilised metal affinity chromatography for the on-line sample clean up and pre-concentration of nucleotides prior to their determination by ion pair liquid chromatography-electrospray mass spectrometry: a pilot study by Robin Tuytten; Filip Lemière; Walter Van Dongen; Herman Slegers; Russell P Newton; Eddy L Esmans (189-198).
This study explored an alternative way to enrich and pre-purify biological samples containing nucleoside mono-, di- and triphosphates. These compounds were trapped by immobilised metal affinity chromatography (IMAC) on a Poros® 20 MC IMAC-column, which was conditioned with Fe3+. The IMAC-column was implemented in a column switching set-up separating nucleoside mono-, di- and triphosphates on a Hypersil ODS 35 mm × 0.3 mm capillary column hyphenated to electrospray mass spectrometry resulting in the first miniaturised column switching liquid chromatography–mass spectrometry (LC–MS) system for nucleotides.
Keywords: Immobilised metal affinity chromatography; Nucleotides;
Determination of hydrogen peroxide in exhaled breath condensate by flow injection analysis with fluorescence detection by Sophie Svensson; Anna-Carin Olin; Mona Lärstad; Göran Ljungkvist; Kjell Torén (199-203).
A method for the determination of hydrogen peroxide in exhaled breath condensate (EBC) by automated flow injection analysis (FIA) with fluorescence detection was developed and validated. In the enzymatic assay a fluorescent dimer of para-hydroxyphenyl acetic acid (HPAA) was formed by the redox coupling reaction between hydrogen peroxide and horseradish peroxidase (HRP). The calibration curve of hydrogen peroxide was linear over a range of 40–5000 nM. The coefficient of variation (CV) for within-day precision was 1–3%; for between-day precision, it was 2–5% over the validated range. The assay requires a small sample aliquot (150 μl) and no incubation time, and has an analytical runtime of <2 min. It is therefore suitable for larger studies. The method was used to detect hydrogen peroxide in EBC of asthmatic patients and healthy volunteers. A statistically significant difference was found between patients with asthma (n = 19) and control subjects without asthma (n = 19), 780 nM versus 480 nM (P = 0.03).
Keywords: Hydrogen peroxide; Exhaled breath condensate;
Determination of SCH 211803 by nanoelectrospray infusion mass spectrometry: evaluation of matrix effect and comparison with liquid chromatography–tandem mass spectrometry by Jiwen Chen; Liyu Yang; James T Kapron; Li Ma; Ellan Pace; Colleen K Van Pelt; Patrick J Rudewicz (205-210).
A high throughput assay for SCH 211803, an M2 muscarinic receptor antagonist in human plasma using nanoelectrospray infusion tandem mass spectrometry is described. Sample processing consisted of protein precipitation followed by solid phase extraction using octadecasilyl resin-filled pipette tips on a liquid handling robotic system. The sample extracts were infused directly to the mass spectrometer using a nanoelectrospray interface in a silicon chip format. SCH 211803 was quantified in plasma over the concentration range of 1–1000 ng/mL. In comparison with a liquid chromatography–tandem mass spectrometry assay, the nanoelectrospray method has comparable accuracy, precision and limit of quantitation, with a nine-fold improvement in sample throughput. Using the nanoelectrospray assay, ion suppression was evaluated and found to be 15%. This represented a four-fold reduction in matrix suppression when compared to a conventional electrospray source operating in the flow injection analysis mode at a flow rate common for LC–MS/MS analysis.
Keywords: Matrix effects; Nanoelectrospray infusion; SCH 211803;
Lisinopril quantification in human plasma by liquid chromatography–electrospray tandem mass spectrometry by Ana A.F Padua; Rafael E Barrientos-Astigarraga; Vinicius M Rezende; Gustavo D Mendes; Gilberto De Nucci (211-216).
An analytical method based on liquid chromatography with positive ion electrospray ionization (ESI) coupled to tandem mass spectrometry detection was developed for the determination of Lisinopril in human plasma using Enalaprilat as internal standard. The analyte and internal standard were extracted from the plasma samples by solid-phase extraction using Waters HLB Oasis® SPE cartridges and chromatographed on a C8 analytical column. The mobile phase consisted of acetonitrile/water (60:40, v/v) + 20 mM acetic acid + 4.3 mM of triethylamine. The method had a chromatographic total run-time of 6.5 min and was linear within the range 2.00–200 ng/ml. Detection was carried out on a Micromass triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM). The precision (CV%) and accuracy, calculated from limit of quantification (LOQ) samples (n=8), were 8.9 and 98.9%, respectively. The method herein described was employed in a bioequivalence study of two tablet formulations of Lisinopril 20 mg.
Determination of phenprocoumon, warfarin and their monohydroxylated metabolites in human plasma and urine by liquid chromatography–mass spectrometry after solid-phase extraction by Mike Ufer; Bernd Kammerer; Julia Kirchheiner; Anders Rane; Jan-Olof Svensson (217-226).
A high-performance liquid chromatography–mass spectrometry (HPLC–MS) method for the quantification of phenprocoumon, warfarin, and their known monohydroxylated metabolites in human plasma and urine was developed using a simple, selective solid-phase extraction scheme. Chromatographic separation was achieved on a reversed-phase Luna C18 column and step gradient elution resulted in a total run time of about 13 min. Limits of quantification (LOQ) were ≤40 nM for the parent compounds and ≤25 nM for the metabolites and the limit of detection (LOD) was ≤2.5 nM for all analytes. Average recovery was 84% (±3.7) and 74% (±13.2) in plasma and urine, respectively. Intra- and inter-day coefficients of variation were ≤8.6 and ≤10.6% in plasma and urine, respectively. The method was successfully applied to the analysis of phenprocoumon samples from four healthy volunteers and should prove useful for future comparative studies of warfarin and phenprocoumon pharmacokinetics.
Keywords: Phenprocoumon; Warfarin;
Rapid determination of loratadine in small volume plasma samples by high-performance liquid chromatography with fluorescence detection by Hossein Amini; Abolhassan Ahmadiani (227-230).
A simple and rapid high-performance liquid chromatographic method with fluorescence detection was developed for the determination of loratadine in small volume plasma samples. Liquid–liquid extraction of loratadine and diazepam (as internal standard) from plasma samples was performed with n-butyl alcohol/n-hexane (2:98, v/v) in alkaline condition followed by back-extraction into diluted perchloric acid. Chromatography was carried out using a C8 column (250 × 4.6 mm, 5 μm) under isocratic elution with acetonitrile-20 mM sodium dihydrogen phosphate-triethylamine (43:57:0.02, v/v), pH 2.4. Analyses were run at a flow-rate of 1.0 ml/min at room temperature. The method was specific and sensitive with a quantitation limit of 0.62 ng/ml and a detection limit of 0.2 ng/ml at a signal-to-noise ratio of 3:1. The mean absolute recovery of loratadine from plasma was 84%, while the intra-and inter-day coefficient of variation and percent error values of the assay method were all less than 9.7%. Linearity was assessed in the range of 0.62–20 ng/ml in plasma with a correlation coefficient of greater than 0.999. The method has been used to analyze several hundred human plasma samples for bioavailability studies.
Rapid, automated screening method for enzymatic transformations using a robotic system and supercritical fluid chromatography by Li Di; Oliver J. McConnell; Edward H. Kerns; Alan G. Sutherland (231-235).
An automated screening method was developed for enzymatic transformations using a robotic system and rapid chiral supercritical fluid chromatography (SFC) analysis with a run time of 1.5 min. The method accelerates the enzyme selection process for screening biocatalysts, where a large number of enzymes are evaluated for activity and enantioselectivity. Kinetic resolution of secondary alcohols by enzymatic transesterification was used as a prototype for method development. The rapid automated method can be used effectively for screening enzymes and optimizing reaction conditions in biocatalysis.
Keywords: Enzyme screening; Biocatalysis; Hydrolases;
Study of the determination and pharmacokinetics of Compound Danshen Dripping Pills in human serum by column switching liquid chromatography electrospray ion trap mass spectrometry by Wei-Jing Pei; Xin-Feng Zhao; Zhong-Min Zhu; Chang-Zheng Lin; Wen-Ming Zhao; Xiao-Hui Zheng (237-242).
Compound Danshen Dripping Pill (CDDP) is an important drug widely used for the treatment of cardiovascular diseases. An active component Danshensu (DS) of CDDP was separated by reversed-phase high performance liquid chromatography using column-switching system and analyzed by electrospray mass spectrometry. With this validated assay the pharmacokinetics of CDDP was studied in 10 healthy volunteers after a single oral administration of 250 mg. After trichloroacetic acid precipitation of serum proteins, the analytes were preconcentrated and black-flushed on a reversed-phase column for separation using a switching valve. The analytes were ionized using negative electrospray ionization (ESI) mode. The precursor ion of m/z 196.6 was used to quantify DS in serum. The linear calibration curve ranged from 1.25 to 175 μg/mL. The limit of quantification (LOQ) for DS was 0.15 μg/mL. The intra-day and inter-day precision (R.S.D.) was less than 7.4 and 7.9%, respectively.
Keywords: Compound Danshen Dripping Pill;
Quantitation of tadalafil in human plasma by liquid chromatography–tandem mass spectrometry with electrospray ionization by N.V.S. Ramakrishna; K.N. Vishwottam; S. Puran; M. Koteshwara; S. Manoj; M. Santosh; J. Chidambara; S. Wishu; B. Sumatha (243-249).
A simple, rapid, sensitive and specific liquid chromatography–tandem mass spectrometry method was developed and validated for quantitation of tadalafil (I) in human plasma, a new selective, reversible phosphodiesterase 5 inhibitor. The analyte and internal standard (sildenafil, II) were extracted by liquid–liquid extraction with diethyl ether/dichloromethane (70/30, v/v) using a Glas-Col Multi-Pulse Vortexer. The chromatographic separation was performed on reverse phase Xterra MS C18 column with a mobile phase of 10 mM ammonium formate/acetonitrile (10/90, v/v, pH adjusted to 3.0 with formic acid). The protonate of analyte was quantitated in positive ionization by multiple reaction monitoring with a mass spectrometer. The mass transitions m/z 390.4 → 268.0 and m/z 475.5 → 58.3 were used to measure I and II, respectively. The assay exhibited a linear dynamic range of 10–1000 ng/mL for tadalafil in human plasma. The lower limit of quantitation was 10 ng/mL with a relative standard deviation of less than 15%. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. Run time of 1.2 min for each sample made it possible to analyze a throughput of more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.
Assay of collagenase activity for native triple-helical collagen using capillary gel electrophoresis with laser-induced fluorescence detection by Masatake Sano; Ikuko Nishino; Kyoji Ueno; Hiroshi Kamimori (251-256).
Three collagenase assays for two native triple-helical collagens have been developed using capillary gel electrophoresis (CGE) with laser-induced fluorescence (LIF) detection in order to discover the matrix metalloproteinases (MMPs) inhibitors. These collagenase assays include measurement of the activities of interstitial collagenase (MMP-1) and neutrophil collagenase (MMP-8) against type I collagen, and collagenase-3 (MMP-13) against type II collagen, and the enzyme activities could be readily measured by determining the 3/4 fragments produced from the cleavage of the native collagens. The highly desired sensitivity of the assays could be achieved, employing a dynamic fluorescence labeling technique with the running buffer containing 0.05% sodium dodecylsulfate and non-covalent fluorescent dye for protein, NanoOrange. The collagen, its 1/4 and 3/4 fragments of type I or II collagen could be separated and detected within the run time of 20 min by CGE mode using the gel buffer (pH 8.8) containing 4% polyacrylamide. Good linearity of the peak area of the 3/4 fragment was obtained over each assay range of collagenase (15–150 ng/tube for MMP-1, 3–30 ng/tube for MMP-8, and 1.5–30 ng/tube for MMP-13, respectively). The relative standard deviation of the peak areas of the 3/4 fragment produced from type II collagen by MMP-13 cleavage was calculated to be less than 13.4%, indicating that the assay was reproducible. Also, IC50 values of three MMPs inhibitors, which were calculated for estimation by the variation of the peak areas of the 3/4 fragments using 90 ng/tube for MMP-1, 30 ng/tube for MMP-8 or 15 ng/tube for MMP-13, were almost consistent with data from other assays. The CGE-LIF method is expected to be very useful for proteinase assay and its application to the estimation of inhibitors because this method enables an assay of collagenase activity using native substrate to be conducted without experimentally troublesome procedure such as preparation of antibody or fluorescence-labeled substrate.
Keywords: Collagenase; Collagen;
Simultaneous determination of gentisic, salicyluric and salicylic acid in human plasma using solid-phase extraction, liquid chromatography and electrospray ionization mass spectrometry by R. Pirker; C.W. Huck; M. Popp; G.K. Bonn (257-264).
A method is developed for the simultaneous extraction of gentisic (GA), salicyluric (SUA) and salicylic acid (SA) in human plasma from Willow Bark extract, by solid phase extraction (SPE) using Waters Oasis HLB (divinylbenzene–n-vinylpyrrolidone copolymer) cartridges. Also, a method is optimized comprising of reversed-phase (RP) high-performance liquid chromatography (HPLC) in connection with electrospray ionization mass spectrometry (ESI-MS), fluorescence detection (FLD) and photo diode array detection (DAD) to identify and quantify GA, SUA and SA in the SPE effluents. An improved sensitivity regarding the lower detection limit (LOD) of <7 ng/ml, the limit of quantitation (LOQ) of 20 ng/ml and short analysis times of <15 min is required. The validated SPE method shows linearity in the range of 9.0–58.2 ng/ml for GA, 9.4–191.5 ng/ml for SUA and 12.8–1101.6 ng/ml for SA. The correlation coefficient values are >0.9994 and 0.99 for fluorescence detection (FLD) and electrospray ionization mass spectrometry (ESI-MS), respectively. The recoveries are from 91.3–102.1% for gentisic acid (GA), 86.8–100.5% for salicyluric acid (SUA) and 75.8–81.4% for salicylic acid (SA) depending on the starting concentrations. RP-LC–ESI-MS/MS studies using collision induced dissociation (CID) confirm that the investigated analytes are not artifacts and facilitate further specific identification in addition to the determination of the parent ion mass even in the presence of co-eluting peaks. The established method is also used to analyze gentisic (GA), salicyluric (SUA) and salicylic acid (SA), not only after intake of Willow Bark capsules (Assalix®, BNO 1455) but also as naturally occurring constituents in human plasma after the intake of salicylic acid containing foods.
Keywords: Gentisic acid; Salicyluric acid; Salicylic acid;
Study of stereoselective pharmacokinetics of anisodamine enantiomers in rabbits by capillary electrophoresis by G.R. Fan; Z.Y. Hong; M. Lin; X.P. Yin; Y.T. Wu (265-271).
The purpose of this study was to determine the pharmacokinetics of anisodamine enantiomers in plasma after oral and intravenous administration of racemic anisodamine in rabbits. A capillary electrophoresis method for the simultaneous separation of two pairs of enantiomers in plasma has been firstly developed and validated. Using a 75 mM phosphate buffer containing 25 mM carboxymethylated-γ-cyclodextrin at pH 2.5, good resolution was achieved on a 45-cm uncoated fused-silica capillary at the voltage of 20 kV and 25 °C. The pharmacokinetics of individual anisodamine enantiomers were characterized using the CE assay, the sole method of enantiomeric separation for anisodamine. Pharmacokinetic analysis of results indicated that anisodamine enantiomers showed non-stereoselective disposition or stereoselective disposition in different rabbits. For the rabbits with non-stereoselective disposition, similar pharmacokinetic characteristics were observed between (6S, 2′S)- and (6R, 2′R)-, or (6S, 2′R)- and (6R, 2′S)-anisodamine. For the rabbits with stereoselective disposition, (6S, 2′S)- and (6R, 2′S)-anisodamine were below the established LOD, while the two remaining enantiomers also had similar pharmacokinetic profiles. Further investigations remain necessary to find out the underlying mechanism about the stereoselective disposition of (6S, 2′S)- and (6R, 2′S)-anisodamine.
Keywords: Enantiomer separation; Anisodamine;
Determination of vincristine in mouse plasma and brain tissues by liquid chromatography–electrospray mass spectrometry by Ping Guo; Xiaomin Wang; Feng Zhou; James M. Gallo (273-278).
Vincristine is an anticancer agent that continues to be examined in preclinical models even though it is used in a variety of human neoplastic disorders. We developed a sensitive liquid chromatography–mass spectrometry (LC–MS) method for the determination of vincristine in plasma and in brain tissues that would support investigations on drug distribution into tissues in animal models. The procedure required only a small sample volume (10 μl) of plasma, which circumvented a limitation of most other assays that were developed for human samples. A solid-phase extraction procedure was employed that enabled the eluent to be directly injected onto a reversed-phase chromatographic HPLC system using positive electrospray ionization followed by mass spectrometric detection. The extraction recoveries of vincristine were 57 and 60% from plasma and brain tissues, respectively. The mobile phase consisted of methanol and 15 mM ammonium acetate in 0.02% formic acid (70:30) that was pumped at 0.2 ml/min to yield retention times of 1.6 and 1.8 min for vincristine and vinblastine, the internal standard, respectively. The method was validated at vincristine plasma concentrations from 0.01 to 2 μg/ml, and from 0.01 to 1 μg/g in brain tissue. The advantage of the method enabled the quantitation of vincristine in multiple plasma samples obtained from a single mouse, which permitted the accurate estimation of its pharmacokinetic properties.
Application of inductively coupled plasma mass spectrometry and high-performance liquid chromatography—with parallel electrospray mass spectrometry to the investigation of the disposition and metabolic fate of 2-, 3- and 4-iodobenzoic acids in the rat by Berit Packert Jensen; Christopher J. Smith; Christopher Bailey; Colin Rodgers; Ian D. Wilson; Jeremy K. Nicholson (279-285).
ICP-MS, HPLC-ICP-MS and HPLC-ICP-MS/ESI-MS have been applied to determine the disposition and metabolic fate of 2-, 3- and 4-iodobenzoic acids following intraperitoneal administration at 50 mg kg−1 to male bile duct cannulated rats. Quantitative excretion balance studies based on the determination of the total iodine content of urine and bile showed that all three iodobenzoic acids were rapidly excreted. Recoveries ranging from 95 to 105% of the administered doses were achieved within 24 h of administration. Metabolite profiles for urine and bile showed extensive metabolism with unchanged iodobenzoic acids forming a minor part of the total. A combination of alkaline hydrolysis and MS enabled the identification of the major metabolites of all three iodobenzoic acids as glycine and ester glucuronide conjugates with very little if any of the parent compounds excreted unchanged.
Keywords: Iodobenzoic acids;
Evaluation of a cyano stationary phase for the determination of tacrolimus, sirolimus and cyclosporin A in whole blood by high-performance liquid chromatography–tandem mass spectrometry by Panos Hatsis; Dietrich A. Volmer (287-294).
The potential of a cyano HPLC column for the analysis of three immunosuppressants is investigated. Tacrolimus, sirolimus and cyclosporin A, were used to probe differences in the retention and efficiency of a cyano column compared to the more widely used C18 column. The cyano column showed comparable retention for all three compounds, whereas the C18 column showed stronger retention, especially for cyclosporin A. Furthermore, the efficiencies at 50 °C were up to 12 times higher on the cyano column. As a result, a baseline separation was achieved in less than three minutes with the cyano column, using an isocratic mobile phase of 52/48 (v/v) acetonitrile/water at 0.45 mL/min. The analysis of immunosuppressant drugs in human whole blood was performed with the cyano column using a selected reaction monitoring (SRM) method for each analyte with negative ion mode electrospray ionization on a triple quadrupole mass spectrometer. Detection limits were 0.05 ng/mL for sirolimus, 0.1 ng/mL for cyclosporin A and 0.2 ng/mL for tacrolimus. Calibration curves were linear over three orders of magnitude. Good agreement was obtained with the actual levels of immunosuppressant drugs in patient samples with an average error of less than 10%.
Keywords: Immunosuppressants; Cyano column; Negative ions;
Determination of thiocyl in biological samples by liquid chromatography with ThioGlo™3 derivatization by Wei Wu; Nuran Ercal (295-299).
Thiocyl (sodium thiosalicylate) belongs to a salicylate group of drugs, thus it has analgesic, antipyretic and anti-inflammatory effects. It possesses metal chelating function because it also belongs to a thiol-containing group of compounds which are well-known chelators. The studies of our research group showed that thiocyl is a promising chelator of lead poisoning due to its antioxidant and metal-chelating abilities. To the best of our knowledge, no methods were currently available for measuring thiocyl in biological samples. Therefore, we developed a reversed-phase HPLC method using fluorescence detection (λ ex = 365 nm, λ em = 445 nm) with a one-step derivatizing reaction between thiocyl and a derivatizing agent-ThioGlo™3 (9-acetoxy-2-(4-(2, 5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)pyenyl)-3-oxo-3H-naphtho[2,1-b]pyran). Most biological thiols (such as N-acetylcysteine (NAC), cysteine (CYS), glutathione (GSH) and homocysteine (HCYS)) do not interfere with the detection of thiocyl by using this technique. The linear range of its calibration curve was determined to be 25–2500 nM, and the detection limit of thiocyl was found to be 3 nM with 20 μL injection volume. The coefficients of variation (CV) for within-run precision and between-run precision ranged from 0.93 to 7.21%. This assay proved to be a rapid, sensitive and simple method for determining thiocyl in biological samples.
Influence of high concentration monovalent cations on the protein partitioning in polyethyleneglycol 1500-phosphate aqueous two-phase systems by Beatriz Farruggia; Rubén Rigatuso; Lorena Capezio; Veronica Diez; Guillermo Picó (301-306).
The influence of chloride salts of Na+, Rb+ and Cs+ at concentrations from 0.15 to 1.2 M was studied with bovine albumin, trypsin, ovoalbumin and lysozyme partitioning in an aqueous two-phase system formed by polyethyleneglycol 1500 and potassium phosphate at pH 7.4. Monovalent cations favoured the protein transfer to the polyethyleneglycol rich phase in the following order: Rb+ > Na+ > Cs+. Structure making cations as Na+ induced a poor loss of structured water, producing little diminution of the molar partial specific volume of polyethyleneglycol, while Rb+ and Cs+, structure breaking cations, induced a significant decrease in the specific volume of the polyethylene glycol. The increase of available solution free volume in the top phase favours the protein transfer to the polyethyleneglycol rich phase. Na+ and Rb+ induced a slight decrease in the alpha helix content of the proteins, while Cs+ increased the secondary structure for all the proteins. All the cations induced a decrease in the hydrophobic surface of the proteins, this effect was more significant in the presence of Cs+.
Keywords: Partitioning; Aqueous two-phase systems; Cations;
Rapid determination of tamsulosin in human plasma by high-performance liquid chromatography using extraction with butyl acetate by J. Macek; J. Klíma; P. Ptáček (307-311).
A high-performance liquid chromatographic method with fluorescence detection for the determination of tamsulosin in human plasma is reported. The sample preparation involved liquid–liquid extraction of tamsulosin from alkalised plasma with butyl acetate and back-extraction of the drug to the phosphate buffer (pH 2). Butyl acetate is preferable to more commonly used ethyl acetate because of its much lower solubility in water. Liquid chromatography was performed on an octadecylsilica column (55 mm × 4 mm, 3 μm particles), the mobile phase consisted of acetonitrile–30 mM dihydrogenpotassium phosphate (25:75 v/v). The run time was 3.5 min. The fluorimetric detector was operated at 228/326 nm (excitation/emission wavelength). An analogue of tamsulosin, (R)-5-[2-[(3-(2-ethoxyphenoxy)propyl)amino]-2-methylethyl]-2-methoxybenzensulfonamide was used as the internal standard. The limit of quantitation was 0.4 ng/ml using 1 ml of plasma. Within-day and between-day precision expressed by relative standard deviation was less than 10% and inaccuracy did not exceed 5%. The assay was applied to the analysis of samples from several pharmacokinetic studies.
Keywords: Tamsulosin; Butyl acetate;
Stereoselective determination of methadone and the primary metabolite EDDP in human plasma by automated on-line extraction and liquid chromatography mass spectrometry by Dale Whittington; Pam Sheffels; Evan D. Kharasch (313-321).
A sensitive stereoselective bioanalytical liquid chromatographic assay with mass spectrometric detection (LC–MS) was developed and validated for the on-line extraction and quantification of R- and S-methadone and the primary metabolite R- and S-2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) from human plasma. Deproteinized plasma was injected directly onto a small C8 column, washed and then back-flushed using a column switching valve and a second pump onto an α1-acid glycoprotein analytical column, and enantioselective separation achieved using a mobile phase gradient of methanol and ammonium formate. Analytes were validated over a range of 0.1–25 ng/ml R- and S-EDDP and 0.1–100 ng/ml R- and S-methadone, respectively. Unweighted standard curves were linear over this concentration range (regression coefficients >0.999). Quality control samples were evaluated at 1, 5, 12.5 ng/ml R- and S-EDDP and 1, 10, 50 ng/ml R- and S-methadone. Intra- and inter-day accuracy was >95%, and intra- and inter-day coefficients of variation were less than 10% for all analytes and concentrations. This assay represents the only method currently available which combines on-line extraction and achieves chiral separation of both methadone and EDDP from plasma, and offers improvements in sensitivity over existing methods.
Keywords: Methadone; EDDP; On-line extraction;
Lectin affinity capillary electrophoresis in glycoform analysis applying the partial filling technique by Maria Bergström; Mikael Nilsson; Roland Isaksson; Ingvar Rydén; Peter Påhlsson; Sten Ohlson (323-329).
The study of protein glycosylation and its significance in biological interactions is a field of growing interest. This work demonstrates a lectin-based separation of protein glycoforms of α1-acid glycoprotein (AGP or orosomucoid) with capillary electrophoresis. Glycoform analysis was performed with a “partial filling technique” with the lectin Concanavalin A (Con A) as affinity ligand. Con A separated human AGP into two peaks; the first peak included AGP glycoforms without biantennary glycans, and the second peak represented the fraction that had one or more biantennary glycans. The applicability of the method was demonstrated with the analysis of AGP from clinical samples and AGP treated with N-glycosidase F. The AGP separation was also used as a reporter system to estimate the dissociation constant (K D) between Con A and a competing sugar.
Keywords: Partial filling technique; α1-Acid glycoprotein; Orosomucoid;
Direct liquid chromatography–mass spectrometry method for the detection of glutathione S-transferase isozymes and investigation of their expression in response to dietary flavone by Stephanie A. Burns; Yun-Jeong Hong; Alyson E. Mitchell (331-337).
The cytosolic GSTs were measured directly in tissue homogenates using HPLC interfaced via ESI to a mass spectrometer (LC/MS). Total ion chromatograms were generated and filtered for ion currents corresponding to m/z ratio characteristic of individual GST isozymes. Direct LC/MS has a high degree of precision (8%) and low instrument detection limits (50–100 ng) and offers the advantage of monitoring GST expression at the protein level. In this study we describe the sub-chronic effect of feeding flavone (2500 mg/kg diet) on the expression of mGSTA3, mGSTP1, mGSTM1, and mGSTM2 in male and female mice. Additionally, we tentatively identify mGSTO and its up-regulation by flavone; a result that will facilitate the study of this novel enzyme. Flavone induced mGSTM1 and mGSTP1 in a gender and isozyme specific manner yet had no appreciable effect on the expression of mGSTA3. Male animals (day 5) displayed a 8-fold increase in mGSTM1 and a 2.6-fold increase in mGSTP1 whereas female animals displayed a 5- and 3-fold increase in mGSTM1 and mGSTP1, respectively. The mGSTM2 was detected only in flavone-fed animals, indicating an up-regulation of this isozyme by flavone. Results obtained using direct LC/MS compare favorably to the specific activity of individual isozymes (P = 0.19), and are comparable to GST levels determined using affinity chromatography followed by LC/MS (P = 0.79).
Keywords: Glutathione S-transferase isozymes;
Comparison of conventional and fast gas chromatography in human plasma fatty acid determination by Isabel Bondia-Pons; Ana I. Castellote; M. Carmen López-Sabater (339-344).
A fast gas chromatographic technique for the determination of fatty acids in human plasma was developed. Its validation and comparison to a conventional method are here reported. The fast method significantly reduced the time required for analysis by a factor of 5 (total time of 3.2 min) while maintaining a similar resolution. Reproducibility of qualitative and quantitative data was measured in both applications. The results demonstrated that the applied fast gas chromatography (GC) conditions do not affect the analytical quality of the assays, allowing a short analysis time.
Keywords: Fatty acids;
Hydrophilic interaction liquid chromatography–tandem mass spectrometry for the determination of levosulpiride in human plasma by In Bok Paek; Ya Moon; Hye Young Ji; Hui-Hyun Kim; Hye Won Lee; Yong-Bok Lee; Hye Suk Lee (345-350).
A rapid, sensitive and selective hydrophilic interaction liquid chromatography–tandem mass spectrometric (HILIC–MS/MS) method for the determination of levosulpiride in human plasma was developed. Levosulpiride and internal standard, tiapride were extracted from human plasma with ethyl acetate at pH 11 and analyzed on an Atlantis HILIC silica column with the mobile phase of acetonitrile–ammonium formate (190 mM, pH 3.0) (94:6, v/v). The analytes were detected using an electrospray ionization tandem mass spectrometry in the multiple-reaction-monitoring mode. The standard curve was linear (r = 0.999) over the concentration range of 1.00–200 ng/ml. The lower limit of quantification for levosulpiride was 1.00 ng/ml using 100 μl plasma sample. The coefficient of variation and relative error for intra- and inter-assay at three quality control (QC) levels were 3.8–9.1 and −2.9 to −0.1%, respectively. The recoveries of levosulpiride ranged from 80.5 to 87.4%, with that of tiapride (internal standard) being 84.6%. This method was successfully applied to the pharmacokinetic study of levosulpiride in humans.
New method for the chiral evaluation of mirtazapine in human plasma by liquid chromatography by Fernando José Malagueño de Santana; Evandro José Cesarino; Pierina Sueli Bonato (351-356).
A simple, rapid and sensitive high-performance liquid chromatography (HPLC) method was developed for the enantioselective analysis of the new antidepressant drug mirtazapine in human plasma. The procedure involved liquid–liquid extraction using toluene, followed by liquid chromatography coupled to UV detection at 292 nm. The chromatographic separation of the (+)-(S)- and (−)-(R)-enantiomers of mirtazapine was achieved on a Chiralpak AD column (250 mm × 4.6 mm, 10 μm particle size) protected with a CN guard column, using hexane–ethanol (98:2, v/v) plus 0.1% diethylamine as the isocratic mobile phase, at a flow rate of 1.2 ml/min. The total analysis time was less than 12 min per sample. The recoveries of (+)-(S)- and (−)-(R)-mirtazapine were in the 88–111% range with a linear response over the 6.25–625 ng/ml concentration range for both enantiomers. The quantification limit (LOQ) was 5 ng/ml. Within-day and between-day assay precision and accuracy were studied at three concentration levels (10, 50 and 250 ng/ml). For both mirtazapine enantiomers, the coefficients of variation (CV) and deviation from the theoretical value were lower than 15% at all concentration levels. The method proved to be suitable for pharmacokinetic studies.
Keywords: Enantiomer separation; Mirtazapine; Antidepressant; Chiral stationary phase;
Author Index to Vol. 809 (357-360).
Keyword Index to Vol. 809 (361-364).