Journal of Chromatography B (v.776, #2)

News section (N1-N2).

High-performance affinity chromatography for characterization of human immunoglobulin G digestion with papain by Quanzhou Luo; Xiqin Mao; Liang Kong; Xiaodong Huang; Hanfa Zou (139-147).
Reactive continuous rods of macroporous poly(glycidyl methacrylate-co-ethylene dimethacrylate) were prepared within the confines of a stainless steel column. Then papain was immobilized on these monoliths either directly or linked by a spacer arm. In a further step, a protein A affinity column was used for the characterization of the digestion products of human immunoglobulin G (IgG) by papain. The results showed that papain immobilized on the monolithic rod through a spacer arm exhibits higher activity for the digestion of human IgG than that without a spacer arm. The apparent Michaelis–Menten kinetic constants of free and immobilized papain, K m and V max, were determined. The digestion conditions of human IgG with free and immobilized papain were optimized. Comparison of the thermal stability of free and immobilized papain showed that the immobilized papain exhibited higher thermal stability than the free enzyme. The half-time of immobilized papain reaches about a week under optimum pH and temperature conditions.
Keywords: Immunoglobulin G; Papain;

Application of shielding boronate affinity chromatography in the study of the glycation pattern of haemoglobin by YuCai Li; Jan-Olof Jeppsson; Magnus Jörntén-Karlsson; Eva Linné Larsson; Hans Jungvid; Igor Yu Galaev; Bo Mattiasson (149-160).
Human haemoglobin (Hb) may appear in a number of glycated species. The glycation pattern of Hb using shielding boronate affinity chromatography (SBAC) has been studied in the present work. SBAC is a novel separation technique, which eliminates nonspecific boronate–protein interactions by introducing a so-called shielding reagent . Two samples from Bio-Rad (Lyphochek®)—one from normal persons’ blood with relatively low HbA1c level (HbL) and the other from diabetic patients’ blood with an elevated HbA1c level (HbH)—were used for the investigation. Glycated Hb (GHb) was separated from nonglycated Hb species using Tris as the shielding reagent. Two eluted peaks, eluted peak 1 (E1) and eluted peak 2 (E2), were obtained using a linear gradient elution with Tris. Several bands were observed on isoelectric focusing gel, which showed the same migration positions as Hb adducts, such as HbA0, which is major Hb component containing two α chains and two β chains; HbA1c, which is post-translational glycation on the N-terminus of the β chains of HbA0; Foetal Hb (HbF), consisting of two α chains and two γ chains; and glutathione Hb (also called HbSSG), which is the result from thiol-disulphide interchain exchange during oxidation of the thiol groups of Hb. In both HbL and HbH samples, E2 exhibited slightly higher amounts of HbF than E1. Electrospray-ionisation mass spectrometry showed that: (1) HbL-E1 was glycated with single glucose on both α and β chains while no observable glycated chains were present in HbL-E2; (2) both HbH-E1 and HbH-E2 were glycated with single glucoses on both α and β chains, however, compared with HbH-E1, HbH-E2 showed a higher relative intensity of the glycated β chain and lower relative intensity of the glycated α chain; and (3) the degree of glycation increased with increasing glycation level of the sample. The amount of HbA1c presented in the eluted peaks was further determined using enzymatic digestion of glycated Hb by endoproteinase Glu-C and the subsequent separation and analysis of the digested peptides by reversed-phase high-performance liquid chromatography and capillary electrophoresis. The values of HbA1c/HbA0 of the eluted peaks, i.e. HbL-E1, HbL-E2, HbH-E1 and HbH-E2, were 0.27, 0.19, 0.50 and 0.43, respectively. In both HbL and HbH samples, E1 contained higher amounts of HbA1c than E2. This study demonstrates the structural heterogeneity of GHb as well as the possibility of using SBAC to detect glycated species of Hb.
Keywords: Haemoglobin;

A method is described for the determination of betamethasone in rat plasma by liquid chromatography–tandem mass spectrometry (LC–MS–MS). The analyte was recovered from plasma by solid-phase extraction and subsequently analyzed by LC–MS–MS. A Packard Multiprobe II, an automated liquid handling system, was employed for the preparation and extraction of a 96-well plate containing unknown plasma samples, standards and quality control samples in an automated fashion. Prednisolone, a structurally related steroid, was used as an internal standard. Using the described approach, a limit of quantitation of 2 ng/ml was achieved with a 50 μl aliquot of rat plasma. The described level of sensitivity allowed the determination of betamethasone concentrations and subsequent measurement of kinetic parameters of betamethasone in rat. Combination of automated plasma extraction and the sensitivity and selectivity of LC–MS–MS offers a valuable alternative to the methodologies currently used for the quantitation of steroids in biological fluids.
Keywords: Betamethasone;

A convenient liquid chromatographic–single quadrupole mass spectrometric (LC–MS) method was developed and validated for the determination of chlorpheniramine maleate (INN name: chlorphenamine) in human plasma. The method had advantages of a single liquid–liquid extraction with diethylether and high sensitivity. The linearity was also excellent over the concentration range of 0.52–20.8 ng/ml of chlorpheniramine maleate. The intra- and inter-day precision and accuracy ranged between 0.0 and 13.9%, showing a good reproducibility. This developed method was successfully applied to analysis of chlorpheniramine maleate in clinical studies.
Keywords: Chlorpheniramine maleate;

High-performance liquid chromatographic assay validation of Manumycin A in mouse plasma by Joanne Gonzales; Sai-Ching Jim Yeung; Judith A Smith (177-182).
Manumycin A is a natural antibiotic produced by Streptomyces parvulus that has antineoplastic activity against a variety of human cancers in nude mouse models. We have developed a highly sensitive reverse phase high-performance liquid chromatography (HPLC) method based on ultraviolet (UV) detection for the determination of manumycin A in mouse plasma. Manumycin A was isolated from mouse plasma by solid-phase extraction. A gradient elution of methanol and 0.05 M H3PO4 with 0.2% triethylamine mobile phase was employed and separation was achieved with a C18 analytical column. Manumycin A was detected by UV absorption at 345 nm. Retention time for manumycin A was 8.9±0.2 min. The manumycin A peak was baseline resolved, with the nearest peak at 1.5 min distance and no interfering peaks detected. Inter- and intra-day coefficients of variance were less than 6.1 and 5.1%, respectively. Based on an extracted manumycin A standard plasma sample of 0.25 μg/ml, the assay precision was 99.8% with a mean accuracy of 95.1%. At plasma concentrations of 0.5 and 5 μg/ml, the mean recovery rates of manumycin A were 59.64 and 60.28%, respectively. The lower limit of detection (LLD) for manumycin A was 0.1 μg/ml in mouse plasma. The lower limit of quantification (LLQ) for manumycin A was 0.125 μg/ml. Results of the stability study indicated that when frozen at −80 °C, manumycin A was stable in mouse plasma for up to 2 weeks. This method is useful in quantification of manumycin A in mouse plasma for clinical pharmacology studies in mice.
Keywords: Manumycin A;

pH-peak focusing counter-current chromatography (CCC) was applied to the purification of lactic acid dehydrogenase (LDH) from a crude bovine heart extract using a cross-axis coil planet centrifuge (CPC). The experiment was performed with two sets of polymer phase systems composed of 16% (w/w) polyethylene glycol (PEG) 1000–12.5% (w/w) potassium phosphate buffer and 15% (w/w) PEG 1540–15% (w/w) ammonium sulfate each at various pH values. The best result was achieved from the PEG 1540–ammonium sulfate polymer phase system by adding a retainer (10 mM acetic acid) to the upper stationary phase and an eluter (100 mM sodium hydroxide) to the lower mobile phase. At a flow-rate of 0.5 ml/min, LDH was eluted as a sharp peak which was well resolved from other proteins. Collected fractions were analyzed by the LDH enzymatic activity and by sodium dodecyl sulfate–polyacrylamide slab gel electrophoresis to detect contaminated proteins. LDH was purified directly from crude bovine heart extract in a concentrated state.
Keywords: Lactic acid dehydrogenase;

We have developed a new analytical method to quantify the dl-homoalanine-4-yl(methyl)phosphinate (dl-GLUF) enantiomers in biological specimens using a reversed-phase high-performance liquid chromatography system with a fluorescence detection system. The derivatization of dl-GLUF enantiomers with (+)-1-(9-fluorenyl)ethyl chloroformate was carried out under mild conditions (40 °C for 30 min) without inducing racemization. The lower limit of quantitation was 0.01 μg/ml for both d-GLUF and l-GLUF, and the detection limit was 5 ng/ml. When dl-GLUF enantiomers were added to serum to produce concentrations between 0.1 and 100 μg/ml, the mean recovery rate was at least 93.8%. The recovery rate from urine was also satisfactory.
Keywords: Glufosinate;

A fast automated extraction and clean-up procedure for low-level analysis of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and coplanar polychlorinated biphenyls (cPCBs) in biological fluids is presented. Online extraction of prepared fluids is carried out using disposable octadecyl bonded (C18) solid-phase extraction columns. Extracts are then cleaned up through disposable multi-layer silica (acidic, basic and neutral) and dispersed PX-21 carbon columns. This new methodology is compared with classical Soxhlet extraction and manual solid-phase extraction in terms of repeatability, reproducibility, accuracy and recovery rates for reference and certified materials. Robustness is evaluated on different matrices, such as cow’s milk, breast milk and human serum. As a consequence of the reduced number of reusable glassware used, as well as lowering of solvent consumption, recorded blank levels are decreased in favor of limits of detection (LODs). Total analysis time and cost are further reduced using simultaneous sample preparation units and the sample throughput is increased compared to classical methods. As a result, this new approach appears to be suitable for the fast sample preparation often required for such fluids in case of emergency foodstuffs analysis or during large epidemiological studies.
Keywords: Polychlorinated dibenzo-p-dioxins; Polychlorinated dibenzofurans; Polychlorinated biphenyls;

Determination of voriconazole in aqueous humor by liquid chromatography–electrospray ionization-mass spectrometry by Lei Zhou; Randolph D Glickman; Nancy Chen; William E Sponsel; John R Graybill; Kwok-Wai Lam (213-220).
A novel method based on liquid chromatography–mass spectrometry with electrospray ionization (LC–MS) has been developed for analysis of voriconazole in aqueous humor. The separation was achieved on a reversed-phase C18 column eluted by 70% acetonitrile–30% water–0.01% TFA. The correlation between the concentration of voriconazole to peak area was linear (r 2=0.9990) between 0.04 and 60 ng, with a coefficient of variance of less than 3%. Limit of quantitation (LOQ) was estimated to be 5 ng/ml voriconazole with an injection volume of 2 μl of aqueous humor. Both intra-day and inter-day imprecision were less than 3% over the whole analytical range. Parallel analyses of voriconazole samples by LC–MS and by high-performance liquid chromatography (HPLC)–UV showed that the two methods were highly correlated (r 2=0.9985). LC–MS was used to the determine voriconazole levels achieved in the aqueous humor of the rabbit eye, following topical application of 5 or 10 μg voriconazole in the form of eyedrops for 11 days b.i.d. The lower dosage produced an aqueous humor concentration of 7.29±5.84 μg/ml, while the higher dosage produced a concentration of 14.56±12.90 μg/ml.
Keywords: Voriconazole; Antifungal; Aqueous humor;

Determination of catecholamines and metanephrines in urine by capillary electrophoresis–electrospray ionization–time-of-flight mass spectrometry by Zlatuse D Peterson; David C Collins; Christopher R Bowerbank; Milton L Lee; Steven W Graves (221-229).
A method successfully coupling capillary electrophoretic separation to time-of-flight mass spectrometric (TOFMS) detection for the simultaneous analysis of catecholamines (dopamine, norepinephrine, and epinephrine) and their O-methoxylated metabolites (3-methoxytyramine, normetanephrine, and metanephrine) is described. The inner capillary wall was coated with polyvinyl alcohol in order to obtain baseline resolution of catecholamines and metanephrines and to ensure reproducibility without extensive restorative washing of the capillary. Using electrokinetic injection, detection limits of 0.3 μM for dopamine and norepinephrine, 0.2 μM for 3-methoxytyramine and normetanephrine, and 0.1 μM for epinephrine and metanephrine were achieved with standard solutions. The usefulness of this approach was demonstrated by applying the developed method to the analysis of a spot collection of human urine from a healthy volunteer. The catecholamines and metanephrines were removed from the urine samples and preconcentrated by simultaneous SPE on cation-exchange sorbents. The recoveries of all analytes, with the exception of epinephrine (75%), were over 80%. Catecholamines and metanephrines in the urine samples were quantitated using 3,4-dihydroxybenzylamine as an internal standard. Submicromolar concentrations, consistent with the catecholamine and metanephrine levels reported for normal human urine, were detected.
Keywords: Catecholamines; Metanephrines;

The experimental anti-cancer drug 5,6-dimethylxanthenone-4-acetic acid (DMXAA) is mainly metabolised by acyl glucuronidation and to a lesser degree by 6-methyl hydroxylation. Strain differences in the maximum tolerated dose (MTD) of DMXAA in mice have been observed. The aim of this study was to compare the kinetics of DMXAA acyl glucuronidation and 6-methylhydroxylation in five various mouse strains, and correlate the in vitro metabolism data with MTD observed. In all mouse strains studied, DMXAA acyl glucuronidation and 6-methylhydroxylation in the liver microsomes followed Michaelis–Menten kinetics. Significant strain variations in the kinetic parameters (K m, V max and K m/V max, i.e., CLint) for DMXAA acyl glucuronidation and 6-methylhydroxylation in mouse liver microsomes were observed. A 2–6-fold variation was spanned across strains for K m, V max and CLint, respectively, for DMXAA glucuronidation and 6-methylhydroxylation. The rank order for total CLint by glucuronidation and 6-methylhydroxylation was BDF1 (1.70 ml/min per g)>wild type of mice lacking IFN-γ receptor (0.80 ml/min per g)>nude mice (0.70 ml/min per g)>Swiss CD mice (0.56 ml/min per g)>C57Bl/6 mice (0.46 ml/min per g), with a 4-fold variation between the mouse strain of the highest and lowest CLint. There was no significant correlation between total CLint and MTD (r 2=0.88, P>0.05), but the rank order for CLint was consistent with that for MTD. These results suggested that there were significant strain differences in DMXAA metabolism in mouse liver microsomes and the strain-related differences in the metabolism of DMXAA did not provide an explanation for the strain differences in the MTD.
Keywords: 5,6-Dimethylxanthenone-4-acetic acid;

A reversed-phase HPLC method with fluorescence detection for the quantification of hexafluoroisopropanol (HFIP) in urine is presented. HFIP, a metabolite of the inhalation anesthetic sevoflurane, is excreted mainly in urine as glucuronic acid conjugate. After enzymatic hydrolysis of the glucuronate, primary amino groups of interferent urinary compounds are blocked by reaction with o-phthalic dicarboxaldehyde and 3-mercaptopropionic acid, followed by labeling of HFIP with 9-fluorenylmethyl chloroformate. The derivatization reaction proceeds in a water–acetonitrile (1:1) solution at room temperature with a borate buffer of pH 12.5 as a catalyst. A stable fluorescent derivative of HFIP is formed within 5 min. The HFIP–FMOC derivative is separated by reversed-phase chromatography with isocratic elution on an octadecyl silyl column (33×4.6 mm, 3 μm) and guard column (20×4.0 mm, 40 μm), at 35 °C, and detected by fluorescence detection at an excitation wavelength of 265 nm and an emission wavelength of 311 nm. The method detection limit is 40 pg, per 10-μl injection volume, corresponding to 16 μg/l of HFIP in urine. The among-series relative standard deviation is <6% at 200 μg/l (n=6). As a preliminary application, the method was used to detect HFIP concentration in the urine of two volunteers exposed for 3 h to an airborne concentration of sevoflurane in the order of 2 ppm.
Keywords: Hexafluoroisopropanol; Sevoflurane;

Piperine, a major alkaloid of Piper longum and Piper nigrum has been reported to have several pharmacological/toxicological effects. Though a number of methods for analysis of this omnipresent food component in pepper fruits are available, its analysis in body fluids has been largely neglected. A high-performance liquid chromatography method for the analysis of piperine in rat plasma is presented in this communication. Analysis was performed using a Symmetry® C18 column (250×4.6 mm) by isocratic elution with 25 mM KH2PO4 (pH 4.5)–acetonitrile (35:65) and UV detection at 340 nm. The calibration plot was linear over the range studied (2–2000 ng) with correlation coefficient of 0.9984. Limit of detection and limit of quantitation were 1 ng/ml and 3 ng/ml, respectively. Good overall recovery (85.5±6%) was obtained with 4 ml ethyl acetate and extraction time of 3 min. Intra- and inter-assay coefficient of variation was found to be less than 7.5%. Plasma concentration–time profile of piperine in a conscious rat implanted with jugular vein cannula was obtained using this method. The method is simple, sensitive and reproducible.
Keywords: Piperine;

Rapid determination of mycophenolic acid in plasma by reversed-phase high-performance liquid chromatography by Anna Pastore; Anna Lo Russo; Fiorella Piemonte; Liliana Mannucci; Giorgio Federici (251-254).
A simple, accurate and sensitive high-performance liquid chromatographic method with UV detection was carried out to measure plasma concentrations of mycophenolic acid. Following a simplified acid hydrolysis of the sample, the separation was carried out in 4 min using a Zorbax® Eclipse® C8 reversed-phase column with a flow-rate of 1.5 ml/min, and monitoring the absorbance at 250 nm. Throughput was up to 100 samples in 24 h. Within the investigated concentration ranges of mycophenolic acid (0–100 mg/l), good linearity (r>0.99) was obtained. The method is sensitive (the limit of detection was about 20 μg/l) and precise (for 0.49 mg/l added to plasma, within-run C.V. was 2% and between-run was 4.2%; for 2.88 mg/l, within-run C.V. was 0.35% and between-run C.V. was 0.69%; for 24.38 mg/l, within-run C.V. was 0.77% and between-run C.V. was 3.1%). Analytical recoveries were 96% for 0.5 mg/l mycophenolic acid added to plasma, 100% for 12 mg/l and 102.5% for 24 mg/l.
Keywords: Mycophenolic acid;