Journal of Chromatography B (v.834, #1-2)

Complex polysaccharides, glycosaminoglycans (GAGs), are a class of ubiquitous macromolecules exhibiting a wide range of biological functions. They are widely distributed as sidechains of proteoglycans (PGs) in the extracellular matrix and at cellular level. The recent emergence of enhanced analytical tools for their study has triggered a virtual explosion in the field of glycomics. Analytical electrophoretic separation techniques, including agarose-gel, capillary electrophoresis (HPCE) and fluorophore-assisted carbohydrate electrophoresis (FACE), of GAGs and GAG-derived oligosaccharides have been employed for the structural analysis and quantification of hyaluronic acid (HA), chondroitin sulfate (CS), dermatan sulfate (DS), keratan sulfate (KS), heparan sulfate (HS), heparin (Hep) and acidic bacterial polysaccharides. Furthermore, recent developments in the electrophoretic separation and detection of unsaturated disaccharides and oligosaccharides derived from GAGs by enzymatic or chemical degradation have made it possible to examine alterations of GAGs with respect to their amounts and fine structural features in various pathological conditions, thus becoming applicable for diagnosis. In this paper, the electromigration procedures developed to analyze and characterize complex polysaccharides are reviewed. Moreover, a critical evaluation of the biological relevance of the results obtained by these electrophoresis approaches is presented.
Keywords: Glycosaminoglycans; Heparin; Dermatan sulfate; Chondroitin sulfate; K4 polysaccharide; K5 polysaccharide; Lipopolysaccharide; Electrophoresis;

Hair analysis for veterinary drug monitoring in livestock production by M. Gratacós-Cubarsí; M. Castellari; A. Valero; J.A. García-Regueiro (14-25).
This review summarizes the basic information and applications concerning the use of hair analysis for the detection of misuse of therapeutic and anabolic agents in livestock animals. Hair biology, hair-shaft structure and the mechanisms of drug incorporation are described, considering the different factors which can affect the deposition. Sampling and extraction methods are reviewed with special attention to the particularities of this matrix, while the use of different analytical techniques is discussed, taking into account the concentration and the sensitivity required for drug detection. Advantages, drawbacks, promising prospects and possible applications of this technique in the future are also discussed.
Keywords: Hair analysis; Veterinary drugs; Drug residues; Livestock;

Determination of green tea catechins in human plasma using liquid chromatography–electrospray ionization mass spectrometry by Yoshinori Masukawa; Yuji Matsui; Namii Shimizu; Naoki Kondou; Hidenori Endou; Michiya Kuzukawa; Tadashi Hase (26-34).
A method for the sensitive and specific determination of eight green tea catechins, consisting of catechin (C), epicatechin (EC), gallocatechin (GC), epigallocatechin (EGC), catechin-3-gallate (CG), epicatechin-3-gallate (ECG), gallocatechin-3-gallate (GCG) and epigallocatechin-3-gallate (EGCG), in human plasma was established. For optimization of conditions for LC–ESIMS, the separation of the eight catechins was achieved chromatographically using Inertsil ODS-2 column combined with a gradient elution system of 0.1 M aqueous acetic acid and 0.1 M acetic acid in acetonitrile. Detection using a mass spectrometer was performed with selected ion monitoring at m/z  = 289 for E and EC, 305 for GC and EGC, 441 for CG and ECG, and 457 for GCG and EGCG under negative ESI. A preparative procedure, consisting of the addition of perchloric acid and acetonitrile to the plasma for deproteinizing and the subsequent addition of potassium carbonate solution to remove excess acid, was developed. In six different plasma with the eight catechins spiked at two different concentrations, the average recoveries were in the range between 72.7 and 84.1%, which resulted from the matrix effect and preparative loss, with coefficients of variance being 8.2–19.8% among individuals. The levels of the catechins in prepared plasma solutions that were kept at 5 °C within 24 h were stable, which allows us to simply analyze many prepared plasma solutions using an autosampler overnight. When using this method to analyze the eight catechins in human plasma after oral ingestion of a commercial green tea beverage, we detected all the catechins absorbed into human blood for the first time. This also suggested that extremely small amounts of the eight catechins orally ingested may be absorbed based on each absorptive property for the catechins. The method should enable pharmacokinetic studies of green tea catechins in humans.
Keywords: LC–ESIMS; Green tea; Catechins; Deproteinizing; Human plasma;

A negligible depletion-solid phase microextraction (nd-SPME) method is presented to measure free concentrations of octylphenol in biological samples. Potential confounding factors, such as matrix effects, are studied as well. Fouling of the fibre appears to occur, but it does not seem to reduce or enhance the measured uptake of octylphenol. In the setup applied here, without any agitation, it has also been found that there is a large effect of protein presence on the kinetics of octylphenol uptake. In addition, an apparent affinity constant of octylphenol for bovine serum albumin was determined.
Keywords: nd-SPME; Octylphenol; Matrix effects; Albumin; Fouling; Kinetics; GC–MS; Affinity;

The mechanism of action of α-amylase from Lactobacillus fermentum on maltooligosaccharides by Pascale Talamond; Michel Noirot; Alexandre de Kochko (42-47).
The action pattern of Lactobacillus fermentum α-amylase (FERMENTA) was examined using a series of maltooligosaccharides (G2–G7) as substrates. Structurally, this enzyme has a molecular mass (106 kDa) almost twofold higher than α-amylases from mammalians and cereals. The product pattern was investigated through an analysis of products and substrates using HPAEC with pulsed amperometric detection. FERMENTA was consistent with an endo-type of amylase. The bond cleavage frequencies were studied using maltooligosaccharides of various chain lengths as substrate, i.e. maltose up to maltoheptaose and DP 4900-amylose catalyzed by FERMENTA. The catalytic efficiency (k cat/K m) increased with chain length from maltose (8.7 × 104  M−1  s−1) up to amylose (1 × 109  M−1  s−1). These action pattern results revealed that FERMENTA can readily cleave the third linkage from the reducing end of the maltooligosaccharides (G5–G7).
Keywords: Alpha-amylase; Maltooligosaccharide hydrolysis kinetics; Lactobacillus fermentum; Maltooligosaccharides; Catalytic efficiency;

A sensitive and rapid method for determination of angiotensin converting enzyme (ACE) inhibitory activity was developed based on a combination of enzymatic reaction followed by high performance liquid chromatography/electrospray-mass spectrometry (HPLC-ESI-MS) determination of its product. The most commonly used substrate hippuryl-histidyl-leucine (HHL) or hippuryl-glycyl-glycine (HGG) hydrolysis catalyzed by purified rabbit lung ACE or human plasma ACE was investigated in the presence of benazeprilat. The incubation time was 8 min for purified lung ACE, and 16 min for human plasma ACE. The produced hippuric acid (HA) was separated from substrate HHL or HGG by HPLC on a C18 column with isocratic elution within 6.5 min, and quantified by electrospray ionization mass spectrometry (ESI-MS) with p-phthalic acid as an internal standard (IS). The limit of detection of HA was 6.0 ng/ml. HHL or HGG hydrolysis catalyzed by purified lung ACE displayed excellent accuracy and reproducibility. The small total reaction volume, the low concentration of substrate, and the simple treating procedures present the advantages of the new method. Furthermore, the total time of the whole procedure for one sample with the novel method is less than 1/2 of that of the conventional HPLC or spectrophotometry method, while the accuracy and the precision of the new method are almost the same as the conventional HPLC method with UV detection.
Keywords: ACE; Hippuryl-glycyl-glycine; Hippuryl-histidyl-leucine; Hippuric acid; HPLC-ESI-MS;

Analysis of agaritine in mushrooms and in agaritine-administered mice using liquid chromatography–tandem mass spectrometry by Kazunari Kondo; Asako Watanabe; Yuko Iwanaga; Ikuro Abe; Hideya Tanaka; Megumi Hamano Nagaoka; Hiroshi Akiyama; Tamio Maitani (55-61).
A sensitive and specific method for quantifying a genotoxic hydrazine, agaritine, has been developed using liquid chromatography–electrospray ionization tandem mass spectrometry (MS). Synthetic agaritine was structurally assigned by 1H, 13C and two-dimensional nuclear magnetic resonance (NMR) analysis (heteronuclear multiple-bond correlation [HMBC] and heteronuclear multiple-quantum coherence [HMQC]), high-resolution fast-atom-bombardment (HR-FAB) MS. Agaritine was separated on an ODS column using 0.01% AcOH–MeOH (99:1) as an eluent with a simple solid-phase-extraction cleanup for mushroom samples and with acetonitrile and methanol deprotenization for plasma samples. There were no interference peaks in any of the mushrooms or mouse plasma samples. The recoveries of agaritine from the spiked mushroom samples and spiked mouse plasma were 60.3–114 and 74.4%, respectively. The intra-day precision values for the spiked mushrooms were 5.5 and 4.2%, and the inter-day precision values were 15.0 and 23.0%, respectively. The limit of quantification was 0.01 μg/g (in mushrooms) and 0.01 μg/ml (in plasma). A precursor ion scan confirmed that agaritine derivatives, which can exert a similar toxicity, were absent. These results indicate that this analytical method for quantifying agaritine could help to evaluate the risk of mushroom hydrazines to humans.
Keywords: Phenylhydrazine; Agaritine; Mushroom; Genotoxic; LC–MS–MS;

A new, selective and sensitive high-performance liquid chromatography (HPLC) method with fluorimetric detection was developed for the determination of tianeptine (TIA) in human plasma using solid phase extraction (SPE) procedures. The method is based on the derivatization of TIA with 4-chloro-7-nitrobenzofurazan (NBD-Cl) in borate buffer of pH 8.5 to yield a yellow, fluorescent product. The HPLC separation was achieved on a Phenomenex C18 column (250 mm × 4.6 mm) using a mobile phase of acetonitrile–10 mM orthophosphoric acid (pH 2.5) (77:23, v/v) solvent system at 1 mL/min flow rate. Gabapentin (GA) was used as the internal standard. The fluorometric detector was operated at 458 nm (excitation) and 520 nm (emission). The assay was linear over the concentration range of 5–300 ng/mL. The detection limit (LOD) was found to be 2 ng/mL. The mean recovery was determined to be 88.6%. The proposed method was applied for pharmacokinetic study of 12.5 mg TIA in a healthy volunteer.
Keywords: Tianeptine; 4-Chloro-7-nitrobenzofurazan; High-performance liquid chromatography (HPLC); Pharmacokinetic study;

Evaluation of IDA-PEVA hollow fiber membrane metal ion affinity chromatography for purification of a histidine-tagged human proinsulin by Luciana Cristina Lins de Aquino; Heloisa Ribeiro Tunes de Sousa; Everson Alves Miranda; Luciano Vilela; Sônia Maria Alves Bueno (68-76).
Inabilities to process particulate material and to allow the use of high flow rates are limitations of conventional chromatography. Membranes have been suggested as matrix for affinity separation due to advantages such as allowing high flow rates and low-pressure drops. This work evaluated the feasibility of using an iminodiacetic acid linked poly(ethylenevinyl alcohol) membrane in the immobilized metal ion affinity chromatography (IMAC) purification of a human proinsulin(His)6 of an industrial insulin production process. The screening of metal ions showed Ni2+ as metal with higher selectivity and capacity among the Cu2+, Ni2+, Zn2+ and Co2+. The membrane showed to be equivalent to conventional chelating beads in terms of selectivity and had a lower capacity (3.68 mg/g versus 12.26 mg/g). The dynamic adsorption capacity for human proinsulin(His)6 was unaffected by the mode of operation (dead-end and cross-flow filtration).
Keywords: Proinsulin-(His)6; Purification; Affinity membrane; IMAC;

Thiopurine S-methyltransferase (TPMT; EC is the key enzyme in the metabolism of thiopurine drugs. Determination of TPMT activity has been used for the individualization of thiopurine dose. We developed HPLC-UV assay for the determination of TPMT activity in human erythrocytes using 6-mercaptopurine as a substrate. Various extraction and chromatographic conditions were compared. In-house developed extraction with acetonitrile provided the lowest limit of quantification. TPMT activity was determined in 99 previously genotyped healthy Estonians. TPMT activity was expressed as the formation of 6-methylmercaptopurine ng/ml/h and normalized either to haemoglobin, haematocrit, erythrocyte count or protein content. The receiver-operating characteristic curve analysis revealed similar accuracy values for TPMT activity in predicting heterozygous and wild type individuals for each method of calculation. In healthy Estonians, TPMT activity varied from 21.5 to 129.6 ng/ml/h. For heterozygous individuals (n  = 18), TPMT activity was 48.1 ± 11.7 ng/ml/h. Wild type individuals (n  = 81) revealed significantly higher TPMT activity 79.3 ± 20.7 ng/ml/h (P  < 0.001). This sensitive HPLC assay for quantitative determination of TPMT activity could easily be used in clinical settings. Under constant experimental conditions for haemolysate preparation no normalization is required.
Keywords: Thiopurine S-methyltransferase; 6-Mercaptopurine; Normalization;

A rapid, simple, accurate, and precise reverse-phase high-performance liquid chromatography (HPLC) method for measuring sparfloxacin in human serum was improved, validated, and applied to determine the influence of polymorphisms in MDR1 (exons 12, 21, and 26) gene on sparfloxacin pharmacokinetics. Sparfloxacin and an internal standard, ciprofloxacin, were extracted from human serum by protein precipitation with dilution and analyzed on a Luna C18 5-μm column in a mobile phase of acetonitrile–0.035 M perchloric acid (28:72, v/v, adjusted to pH 2.0 with 0.015 M triethylamine) and UV detection at 300 nm. This analysis was performed at three different laboratories using the same quality control (QC) samples. The chromatograms showed good resolution, sensitivity, and no interference by human serum. The method showed linear responses over a concentration range of 0.05–2 μg/ml, with correlation coefficients of greater than 0.999 at the three laboratories. Intra- and inter-day assay precision and accuracy fulfilled international requirements. The mean absolute recovery for human serum was 98.8 ± 5.7%. Sparfloxacin in human serum was stable during storage and the assay procedure. The lower limit of quantification using 0.2 ml of serum was 0.05 μg/ml, which was sensitive enough for pharmacokinetic studies. This method was used to study the pharmacokinetics of sparfloxacin in human volunteers, following a single oral administration of sparfloxacin (100 mg) two tablets at three different laboratories. MDR1 polymorphisms did not significantly (P  < 0.01) affect the pharmacokinetic parameters (AUC and C max) of sparfloxacin.
Keywords: Sparfloxacin; MDR1; Pharmacokinetics;

During the analytical method development for BAY 11-7082 ((E)-3-[4-methylphenylsulfonyl]-2-propenenitrile), using HPLC-MS–MS and HPLC-UV, we observed that the protein removal process (both ultrafiltration and precipitation method using organic solvents) prior to HPLC brought about a significant reduction in the concentration of this compound. The use of a structurally similar internal standard, BAY 11-7085 ((E)-3-[4-t-butylphenylsulfonyl]-2-propenenitrile), was not effective in compensating for the loss of analyte as the extent of reduction was different to that of the analyte. We present here a systematic investigation of this problem and a new validated method for the determination of BAY 11-7082.
Keywords: BAY 11-7082; BAY 11-7085; HPLC-MS–MS; Protein precipitation; Ultrafiltration;

Keywords: High throughput; Therapeutic drug monitoring; Solid phase extraction; Liquid chromatography–mass spectrometry; Clozapine; Clozapine-N-oxide; Desmethylclozapine; Serum;

An HPLC method employing CHIRAL-I (150 mm × 3 mm), 5 μm column from Chrom. Tech., immobilized with human serum albumin (HSA), was used to determine in vitro protein binding of several compounds. Experimentally obtained plasma protein data exhibited good correlation with the reported values. The method was compared with the conventional ultra filtration technique and both yielded similar results. Proprietary compounds that could not be analyzed by ultra filtration due to high non-specific binding to filter membrane were successfully analyzed by HSA-HPLC method. On the other hand, two proprietary compounds did not elute from HSA column due to strong binding, but were successfully analyzed by ultra filtration. This proves that both the techniques have their own merits and demerits and should be exploited judiciously as per the requirement. The plasma protein binding studies conducted on four gyrase inhibitors in rat and human plasma exhibited no interspecies difference via ultra filtration method. Further, it was also observed that the protein binding obtained for the four gyrase inhibitors by HSA-HPLC method was not only similar to that obtained by ultra filtration in human plasma but was also in accordance with ex vivo and in vitro protein binding obtained for rat plasma after ultra filtration because these compounds predominantly bind to HSA The binding of several compounds to α1-acid glycoprotein (AGP), another important plasma protein, was also examined using AGP immobilized column. However, the data could not be relied upon since some anti-bacterials and non-steroidal anti-inflammatory drugs (NSAIDS), which are known to predominantly bind to HSA, were also found to bind to AGP.
Keywords: Plasma protein binding; Immobilized human serum albumin-HPLC; Ultra filtration;

Distribution of the UV filter 3-benzylidene camphor in rat following topical application by Tue Søeborg; Niels-Christian Ganderup; Jakob Højer Kristensen; Poul Bjerregaard; Knud Ladegaard Pedersen; Peter Bollen; Steen Honoré Hansen; Bent Halling-Sørensen (117-121).
A straightforward analytical method for determination of 3-benzylidene camphor (3-BC) in rat adipose tissue, brain, liver, muscle, plasma and testis following topical application was developed and validated. Three exposure levels (60, 180 and 540 mg kg−1  day−1) were tested for 65 days in male Sprague–Dawley rats (24 days postnatal). Sample preparation involving homogenization and n-heptane or methanol extraction of the tissue was applied before injection into the LC–ESI–MS–MS system. The response was linear from 2 to 100 μg l−1 for the qualifier and the quantifier MRM transitions (R 2 (quantifier) > 0.994). Detection limit of the method corresponded to 0.005 μg g−1 tissue and 12.5 μg l−1 plasma, respectively. Recovery was determined for all tissues (adipose tissue: 40%; all other tissues: 80–100%) at three individual levels. 3-(4-Methyl benzylidene camphor) (4-MBC) was used throughout the study as internal standard. 3-Benzylidene camphor was detected in all tissues at all exposure levels at concentrations between 0.05 μg g−1 (liver) and 36 μg g−1 (adipose tissue) and in plasma at 16–89 μg l−1. The method allowed for the quantification of 3-benzylidene camphor in all tested tissues following topical application. Furthermore, it was shown that 3-benzylidene camphor can be found in various tissues in the rat following topical application. These findings may suggest that following use of 3-benzylidene camphor containing sunscreen, similar disposition and distribution may occur in humans.
Keywords: Sunscreen; Estrogenic effect; In vivo; Muscle; Plasma; Testis; Adipose tissue; Brain; Liver; Mass spectrometry;

Microsatellite instability (MSI) and loss of heterozygosity (LOH), the alteration in length and strength of short tandem repeat sequences are an important molecular characteristic of many human tumors. MSI and LOH analysis has become an attractive method for diagnostic and tumor research purposes. A method for the simultaneous analysis of MSI and LOH at the five microsatellite loci (BAT-26, D17S261, D3S1283, D2S123 and D3S1611) was developed employing a cheap homemade kit to replace the expensive commercial kit on ABI 310 capillary genetic analyzer. After studying the effect of temperature and urea denaturant on microsatellite analysis, 8 mol/L urea and 60 °C were selected for assessing accurately fragment size of microsatellite alleles. Based on this method, 52 sporadic gastric cancers were screened, and MSI and LOH, at least one locus was observed in 15 of 52 (28.8%) patients. Moreover, it is found that a statistically significant association exists between MSI and LOH and tumor-differentiated level.
Keywords: Microsatellite instability; Loss of heterozygosity; Capillary electrophoresis;

Quantification of clotiazepam in human plasma by gas chromatography–mass spectrometry by Sung-Hoon Ahn; Han-Joo Maeng; Tae-Sung Koo; Dae-Duk Kim; Chang-Koo Shim; Suk-Jae Chung (128-133).
An analytical procedure was developed and validated for the quantification of clotiazepam in human plasma. After subjecting plasma samples to solid-phase extraction, the extract was evaporated and the residue re-constituted. An aliquot of the mixture was injected onto a gas chromatography–mass spectrometry system. The detector response was linear for clotiazepam concentrations in the range of 5–200 ng/ml. Intra- and inter-day precision for the assay over the concentration range was below 13.1 and 13.5%, and the accuracy ranged between 99.0–107.9% and 92.4–101.3%, respectively. The drug was found to be stable under various processing conditions used. The method is applicable to human pharmacokinetic studies of clotiazepam.
Keywords: Clotiazepam; Gas chromatography–mass spectrometry; Pharmacokinetics;

A simple and fast method for the measurement of 4-hydroxynonenal (4HNE), a highly toxic end-product of lipid peroxidation, in urine samples is described. The method combines stir bar sorptive extraction (SBSE) with two derivatization steps, followed by thermal desorption and GC/MS. 4HNE is derivatized in situ with O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine and the oxime is extracted from the aqueous phase with SBSE. The 4HNE-oxime is further acylated by headspace derivatization prior to thermal desorption. Derivatization reactions and extraction were optimized in terms of reagent quantities, temperature and time. The method is linear over a concentration range of 0.5–5 ng mL−1 with a correlation coefficient of 0.997. The limit of detection and limit of quantitation are 22 and 75 pg mL−1 urine, respectively. The high sensitivity of the method allows the measurement of physiological concentrations of 4HNE in urine samples.
Keywords: Stir bar sorptive extraction; Gas chromatography–mass spectrometry; 4-Hydroxynonenal; Oxidative stress; Urine samples;

Rapid determination of the synthetic pyrethroid insecticide, deltamethrin, in rat plasma and tissues by HPLC by Kyu Bong Kim; Michael G. Bartlett; Sathanandam S. Anand; James V. Bruckner; Hyo Jung Kim (141-148).
Deltamethrin (DLM), [(S)-α-cyano-d-phenoxybenzyl-(1R,3R)-e-(2,2 dibromovinyl)-2,2-dimethylcyclo-propane-1-carboxylate], is a pyrethroid insecticide widely used in agriculture and households. There are several methods for analysis of DLM in biological fluids and tissues, but these methods are time consuming. They generally involve the extraction of DLM with lipid-soluble solvents such as n-pentane, n-hexane, diethylether or acetone, and subsequent evaporation of the solvent. A more rapid and sensitive high-performance liquid chromatography (HPLC) method to analyze DLM in plasma and tissues (liver, kidney, and brain) was developed and validated according to U.S. Food and Drug Administration (U.S. FDA) and International Conference on Harmonization (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use guidelines. The limit of detection (S/N of 3/1) for DLM was 0.01 μg/ml for plasma, liver, kidney and brain. The method performances were shown to be selective for DLM and linear over the concentration range 0.01–20.0 μg/ml. For five replications of samples at 0.05, 0.1, 0.2, 1.5 and 4.0 μg/ml, intraday precision and accuracy values were in the range of 0.7–13.1% relative standard deviation (%R.S.D.) and 1.8–14.1%Error, respectively. Interday (n  = 15) precision and accuracy values at 0.05, 0.1, 0.2, 1.5, and 4.0 μg/ml were in the range of 3.2–15.2% (%R.S.D.) and 3.7–14.8%Error, respectively. The absolute recoveries of DLM ranged from 93 to 103% for plasma, 95 to 114% for liver, 97 to 108% for kidney, and 95 to 108% for brain. This method can be quite useful for DLM pharmacokinetic and tissue distribution studies, for which multiple plasma and tissue samples have to be analyzed quickly with high reproducibility.
Keywords: Deltamethrin; HPLC; Pyrethroid insecticide; Plasma; Tissues;

Liquid chromatography–tandem mass spectrometry validated method for the estimation of indapamide in human whole blood by Deepak S. Jain; Gunta Subbaiah; Mallika Sanyal; U.C. Pande; Pranav Shrivastav (149-154).
A highly precise and sensitive method for the estimation of indapamide in human whole blood using high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) is described. The method developed is validated in human whole-blood matrix, with a sensitivity of 0.5   ng/ml as lower limit of quantification. The procedure for the extraction of indapamide and glimepiride as internal standard (IS) involves haemolysis and deprotienation of whole blood using ZnSO4 followed by liquid–liquid extraction using ethyl acetate. The sample extracts after drying were reconstituted and analysed by LC–MS/MS, equipped with turbo ion spray (TIS) source, operating in the positive ion and selective reaction monitoring (SRM) acquisition mode to quantify indapamide in human whole blood. The mean recovery for indapamide was 82.40 and 93.23% for IS. The total run time was 2.5 min to monitor both indapamide and the IS. The response of the LC–MS/MS method for indapamide was linear over the range of 0.5–80.0 ng/ml with correlation coefficient, r  ≥ 0.9991. The coefficient of variance (% CV) at 0.5 ng/ml was 4.02% and the accuracy was well within the accepted limit of ±20% at 0.5 ng/ml and ±15% at all other concentrations in the linear range. This method is fully validated for the accuracy, precision and stability studies and also applied to subject-sample analysis of bioequivalence study for 1.5 mg sustained-release (SR) formulations.
Keywords: LC–MS/MS; SRM; Indapamide; Human whole blood;

A fast and selective HPLC-MS–MS method was established to determine l-threonate in human plasma and urine. Plasma and urine samples were extracted by protein precipitation and diluted with water, then chromatographed on an YMC J'Sphere C18 column with methanol–acetonitrile–10 mM ammonium acetate (20:5:75, v/v) as mobile phase, and at a flow rate of 0.2 ml/min. Detection was performed on a triple–quadrupole tandem mass spectrometer using negative electrospray ionization (ESI). Multiple reactions monitoring (MRM) was used and l-threonate was quantified by monitoring the ion transition of m/z 134.5 → 74.7. The linear calibration curves of l -threonate in plasma and urine were obtained over the concentration range of 0.25–50 μg/ml and 2.5–500 μg/ml, respectively. Lower limit of quantitation was 0.25 and 2.5 μg/ml, respectively. Accuracy was within 85–115%, and intra- and inter-batch precision (R.S.D.%) were within ±15%. The method proved to be accurate and specific, and was applied to the pharmacokinetic study of l-threonate in Chinese healthy subjects.
Keywords: l-Threonate; HPLC-MS–MS; MRM;

Selective method for the determination of cefdinir in human plasma using liquid chromatography electrospray ionization tandam mass spectrometry by Zhang-jing Chen; Jing Zhang; Ji-cheng Yu; Guo-ying Cao; Xiao-jie Wu; Yao-guo Shi (163-169).
A sensitive and selective liquid chromatographic–tandem mass spectrometric (LC–MS/MS) method was developed for the determination of cefdinir in human plasma. After a simple protein precipitation using trichloracetic acid, the post-treatment samples were applied to a prepacked RP18 Waters SymmetryShield column interfaced with a triple quadrupole tandem mass spectrometer. Positive electrospray ionization was employed as the ionization source. The mobile phase consisted of methanol–water–formic acid (25:75:0.075, v/v/v). The analyte and I.S. cefaclor were both detected by the use of selected reaction monitoring mode. The method was linear in the concentration range of 5–2000 ng/ml. The lower limit of quantification was 5 ng/ml. The intra- and inter-day relative standard deviation across three validation runs over the entire concentration range was less than 4.3%. The accuracy determined at three concentrations (36, 360 and 1800 ng/ml for cefdinir) ranged from 99.6 to 106.7% in terms of recovery. The chromatographic run time for each plasma sample was less than 3 min. The method herein described was successfully applied for the evaluation of pharmacokinetic profiles of cefdinir capsule in 12 healthy volunteers.
Keywords: Cefdinir; LC; MS/MS; Human plasma; Method validation;

A rapid, robust and sensitive HPLC method for analysis of uracil (U) and dihydrouracil (UH2) in plasma was developed using solid phase extraction and ultraviolet detection. Separation was achieved with a SymmetryShield RP18 column and an Atlantis dC18 column using a 10 mM potassium phosphate buffer as mobile phase. Compounds were eluted within 15 min without interference. Recovery was 80.4 and 80.6% for U and UH2. Calibration curves were linear from 2.5 to 80 ng/mL for U and 6.75 to 200 ng/mL for UH2. The LLQ was, respectively, 2.5 ng/mL for U, and 6.75 ng/mL for UH2. Within-run and between-run precision were less than 5.94% and inaccuracy did not exceed 7.80%. The overall procedure has been applied to correlate UH2/U ratio with dihydropyrimidine dehydrogenase activity in 165 cancer patients.
Keywords: DPD activity; Uracil; Dihydrouracil; HPLC;

A new and simple high-performance liquid chromatography–evaporative light scattering detection (HPLC–ELSD) method for the determination of spectinomycin hydrochloride and its related substances was developed. The column was Agilent SB-C18 (250 mm × 4.6 mm, 5 μm).The mobile phase was 25 mM trifluoroacetic acid. The drift tube temperature was 40 °C. The pressure of nebulizing gas was 3.5 bar. Good separation of spectinomycin from main related substances could be achieved. The standard curve was rectilinear in the range of 0.07–3.8 mg/ml (r  = 0.9997). Precision was 1.0% (R.S.D.). The limit of detection was 6 μg/ml. The method is simple and rapid, and the results are accurate and reproducible. The HPLC–MS n method was used to characterize the structures of impurities contained in the spectinomycin. In positive mode, impurities were elucidated by use of electrospray ion trap mass spectrometry in the multi-stage MS full scan mode. The possible structures of impurities C and D in spectinomycin were deduced based on the HPLC–MS n data.
Keywords: Spectinomycin hydrochloride; Impurities; HPLC–ELSD; HPLC–MS n ;

Lactulose and mannitol intestinal permeability detected by capillary electrophoresis by Rita Paroni; Isabella Fermo; Laura Molteni; Laura Folini; Matteo Rocco Pastore; Andrea Mosca; Emanuele Bosi (183-187).
Aim of this study was to set up a method by capillary electrophoresis to detect lactulose and mannitol in urine after an oral load, and to estimate the intestinal permeability in controls and in type I diabetes patients. The underivatized carbohydrates were monitored by indirect UV detection using sorbate, cetyltrimethylammonium bromide and LiOH as background electrolyte. Urines were purified by solid phase extraction, shaken with cation exchange resin, filtered and analysed. Carbohydrates migrated in <10 min in relation to their pK a and M r. Controls (n  = 33) and patients (n  = 23) had an excretion ratio lactulose/mannitol 0.025 (0.018–0.051) and 0.067 (0.050–0.127), respectively (p  < 0.01, median, interquartile range).
Keywords: Mannitol; Lactulose; Intestinal permeability; Urine; Capillary electrophoresis;

A fast liquid chromatographic assay with mass spectrometric detection (LC/MS) has been developed and validated for the simultaneous determination of methadone (MT), its primary metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and alprazolam, in human plasma. The extraction procedure was performed with automatic solid phase extraction, and the compounds were separated with a Sunfire® column using a gradient mode. Deuterated analogues for all of the analytes of interest were used for quantitation. Limits of detection (LOD) were established between 0.5 and 1 ng/ml. Linearity was obtained over a range of 2–2000 ng/ml with an average correlation coefficient (R 2) of >0.99. Intra- and inter-batch coefficients of variation and relative mean errors were less than 10% for all analytes and concentrations. The recoveries were higher than 50.0% in all cases. The method proved to be suitable for evaluation of plasma obtained from patients enrolled in a MT-maintenance program who are frequently treated with alprazolam as a sedative.
Keywords: Methadone; Metabolite; Alprazolam; LC–MS plasma;

A capillary zone electrophoresis (CZE) method based on systematic one-variable-at-a time approach was developed for the analysis of four important bioactive components (geniposidic acid, ursolic acid, quercetin and p-coumaric acid) in the extract of Hedyotis diffusa (HD). Separations were carried out in a fused-silica capillary tube with peak detection at 214 nm. Good separation was achieved using a 20 mM borate buffer containing 5% acetonitrile as organic modifier and pH adjusted to 10.0. Operating voltage was 15 kV and temperature was maintained at 25 °C while hydrodynamic injection was 5 s. A good linearity, with correlation coefficients in the ranges of 0.997–0.999 was obtained in the calibration curves of each standard. Relative standard deviation (R.S.D.) of migration time was between 0.32 and 0.70% and deviation of corrected peak area was between 8.84 and 11.99%. These results indicate that this method could be used for rapid and simultaneous analysis of the bioactive components in HD and other herbal products.
Keywords: Hedyotis diffusa; Capillary zone electrophoresis; Organic modifier; Geniposidic acid; Ursolic acid; Quercetin; p-Coumaric acid;

We report a HPLC-UV method for determination of p-nitrophenol (PNP) hydroxylation to 4-nitrocatechol (4NC) as a marker for CYP2E1 activity in rat hepatic microsomes. Proteins were precipitated by addition of 50 μL phosphoric acid (50%, v/v in water) to 500 μL microsomal suspensions. Following vortex mixing and centrifugation the supernatant (20 μL) was injected onto a Supelcosil® C18 column (150 mm × 4.6 mm, 5 μm), and mobile phase (22% acetonitrile, 0.1% trifluoroacetic acetic acid, 0.5% triethylamine) delivered at 1.0 mL/min produced resolved peaks for internal standard, 4NC, and PNP in <11 min. Calibration curves were linear (r 2  = 0.999) from 0.1 to 40 μM with intra- and inter-day precision <12% and accuracy >90%. The method's improved sensitivity (LOQ = 0.1 μM) and minimal sample processing allowed rapid monitoring of PNP hydroxylase activity in fetal, neonatal, juvenile, and adult rat livers.
Keywords: p-Nitrophenol; CYP2E1; Microsomes; Metabolism; HPLC; Validation;

A selective and sensitive high-performance liquid chromatography method has been developed and validated for determination of mitiglinide (MGN) in rat plasma using 2-(4-biphenylyl) propionic acid (BPA) as internal standard. Liquid–liquid extraction was used for sample preparation. Chromatographic separation was achieved on a C18 column using acetonitrile and 0.02 mol/l KH2PO4 buffer (pH 4.0) (45:55, v/v) as mobile phase delivered at 1.0 ml/min. The UV detector was set at 210 nm. The assay was linear over the range 0.1–20 μg/ml for MGN. The average extraction recoveries of MGN and BPA from rat plasma were 98.6 and 97.4%, respectively. The developed method has been applied to the pharmacokinetic study of MGN in rats.
Keywords: Mitiglinide; Pharmacokinetic;

Purification of synthetic all-E lycophyll (ψ,ψ-carotene-16,16′-diol) by Cristi L. Braun; Henry L. Jackson; Samuel F. Lockwood; Geoff Nadolski (208-212).
An efficient purification of synthetic all-trans (all-E) lycophyll is described. The synthetic preparation of the rare xanthophyll lycophyll produces a mixture of geometric isomers. Purification by HPLC using reverse-phase C30 silica affords milligram quantities of the desired all-trans isomer in >95% purity, as confirmed by 1H NMR and LC/MS. Most recently, a facile work-up of the geometric mixture formed during total synthesis was found to provide multigrams of the targeted all-E geometric isomer of lycophyll. The acquisition of modest quantities of this specific lycopene analog allows its therapeutic potential to be explored.
Keywords: All-trans lycophyll; Carotenoids; HPLC analysis; Lycophyll; Lycophyll isomers; Semi-preparative chromatography;

A high-performance liquid chromatographic (HPLC) method has been developed for the simultaneous analysis of the local anesthetic amide drugs, bupivacaine, mepivacaine and ropivacaine, belonging to the pipecoloxylidide group using a C18 reversed-phase column (150 × 4.6 mm I.D.) filled with 5-μm particles and attached to a UV detector. The mobile phase was composed of acetonitrile–methanol–30 mM NaH2PO4 (pH 5.6) (100:100:300, v/v/v) and the flow rate was 1 ml/min. The absorbance of the eluate was monitored at 210 nm. The retention times of the three compounds were: 4.6 min (mepivacaine), 9.7 min (ropivacaine) and 16.4 min (bupivacaine). With this sample preparation method, good and consistent recoveries of the three compounds were obtained: 88–91% for mepivacaine, 87–89% for ropivacaine and 88–91% for bupivacaine. The limit of quantification for three compounds in human serum was 2 ng/ml for mepivacaine, 5 ng/ml for bupivacaine and ropivacaine. This method may be useful in clinical and forensic applications for the determination or identification of the local anesthetic drugs: bupivacaine, mepivacaine or ropivacaine.
Keywords: Local anaesthetic drug; Bupivacaine; Mepivacaine; Ropivacaine;

The objective of this study was to establish the possibility of using liquid chromatography coupled to mass spectrometry for the detection of hydroxyethylstarch (a corn starch derived product) in urine as an alternative to the current time consuming GC–MS methods. Analyses were performed using an ion trap instrument after acidic hydrolysis. Ionization was carried out using atmospheric pressure chemical ionisation (APCI) operated in negative ionization mode and detection was performed using MS2. The results indicate that the developed method can successfully be applied as a fast and reliable method for the detection and identification of hydroxyethylstarch.
Keywords: Doping; Liquid chromatography; Mass spectrometry; HES; Urine; Glucose;

Author Index (221-222).

Keyword Index (223-228).