Journal of Chromatography B (v.829, #1-2)

Receptor–ligand binding assays: Technologies and Applications by Lutea A.A. de Jong; Donald R.A. Uges; Jan Piet Franke; Rainer Bischoff (1-25).
Receptor–ligand interactions play a crucial role in biological systems and their measurement forms an important part of modern pharmaceutical development. Numerous assay formats are available that can be used to screen and quantify receptor ligands. In this review, we give an overview over both radioactive and non-radioactive assay technologies with emphasis on the latter. While radioreceptor assays are fast, easy to use and reproducible, their major disadvantage is that they are hazardous to human health, produce radioactive waste, require special laboratory conditions and are thus rather expensive on a large scale. This has led to the development of non-radioactive assays based on optical methods like fluorescence polarization, fluorescence resonance energy transfer or surface plasmon resonance. In light of their application in high-throughput screening environments, there has been an emphasis on so called “mix-and-measure” assays that do not require separation of bound from free ligand. The advent of recombinant production of receptors has contributed to the increased availability of specific assays and some aspects of the expression of recombinant receptors will be reviewed. Applications of receptor–ligand binding assays described in this review will relate to screening and the quantification of pharmaceuticals in biological matrices.
Keywords: Receptor–ligand binding; Assay technologies; High-throughput screening; Quantitative receptor assay; Recombinant receptor expression;

Kinetin (free base and riboside), which was assumed by many scientists to be a synthetic cytokinin plant growth hormone, has been detected for the first time in the endosperm liquid of fresh young coconut fruits (“coconut water”). To facilitate the study, we developed a sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the identification and quantification of kinetin and kinetin riboside in purified coconut water extract sample. Following a solid-phase extraction of cytokinins in coconut water using C18 columns, the samples were further purified by Oasis MCX columns and analyzed by LC–MS/MS for kinetin and kinetin riboside. Detection by mass spectrometry was carried out using selected reaction monitoring (SRM) mode, by identifying the putative kinetin and kinetin riboside based on their characteristic fragments. Based on a signal-to-noise ratio of 3, the limits of detection in SRM mode were 0.02 μM and 0.005 μM for kinetin and kinetin riboside, respectively. Furthermore, optimal conditions for a baseline chromatographic separation of 18 cytokinin standards by high performance liquid chromatography (HPLC) were developed. The HPLC method had been employed for the confirmation and further fractionation of kinetin in coconut water extracts. The confirmation and fractionation of kinetin riboside was carried out using a further modified HPLC program due to the presence of other interfering material(s) in the sample matrix. Finally, fractions of putative kinetin and kinetin riboside collected from HPLC eluate of coconut water sample were further authenticated by independent capillary zone electrophoresis (CZE) experiment.
Keywords: Kinetin; Kinetin riboside; Liquid chromatography; Tandem mass spectrometry; Selected reaction monitoring; Capillary electrophoresis; Coconut water;

The major flavonoids in rat serum after oral administration of Dalbergia odorifera extract were analyzed qualitatively and quantitatively by high performance liquid chromatography (HPLC) and its coupling to mass spectrometry (HPLC–MS). Utilizing HPLC–MS technique, 18 flavonoids including five isoflavones, four isoflavanones, four neoflavones, two flavanones, two chalcones, one isoflavanonol were identified in free form in serum sample based on comparison with the authentic standards. Furthermore, the amounts of the four prominent flavonoids, (3R)-4′-methoxy-2′,3,7-trihydroxyisoflavanone, vestitone, formononetin and sativanone were determined in serum by HPLC–UV with internal standard method. The method was validated and utilized in pharmacokinetic studies of these four analytes. This is the first report on identification and determination of the major flavonoids in rat serum after oral administration of D. odorifera extract and the results provided a firm basis for clarifying the pharmacological effect of D. odorifera and evaluating the clinical applications of this medicinal herb.
Keywords: Dalbergia odorifera; Flavonoids; (3R)-4′-methoxy-2′,3,7-trihydroxy-isoflavanone; Vestitone; Formononetin; Sativanone;

GTI-2040 is a 20-mer phosphorothioate oligonucleotide complementary to the mRNA of the R2 subunit of ribonucleotide reductase (RNR). It is under clinical development as an anti-cancer agent. A reverse phase high-performance liquid chromatograph (HPLC) method was established for the quantitative analysis of GTI-2040 in human plasma. Plasma samples were prepared with an initial solid-phase extraction (SPE) followed by a liquid–liquid extraction step. HPLC analysis was performed with a gradient system on a Waters XTerra®MS C18 column. The mobile phase consisted of acetonitrile–tetrabutyl ammonium hydrogen sulfate (TBAS) buffer (pH 9.0, 20 mM) at a flow rate of 1.0 ml/min, and the detector was set at a wavelength of 260 nm. A cationic pairing reagent, tetrabutyl ammonium hydrogen sulfate was added during plasma sample clean-up with solid-phase extraction, resulting in significant improvement in extraction recovery. In addition, TBAS addition to the mobile phase improved the peak symmetry of GTI-2040. This method was successfully used in the analysis of GTI-2040 in clinical plasma samples.
Keywords: Antisense oligonucleotide; GTI-2040; Solid-phase extraction;

We have developed a rapid, sensitive and selective LC–MS method for the simultaneous assay of bupropion and its metabolite hydroxybupropion during its intestinal absorption, studied with the rat everted gut sac model. The method was validated in the concentration range of 1–15 μM (0.024–3.58 μg/mL) for bupropion and 0.005–1 μM (0.00127–0.25 μg/mL) for hydroxybupropion with 10 μL injected. Bupropion is used as a probe for the activity of the CYP2B6 isoenzyme of the P450 family of enzymes in man. Its major metabolite hydroxybupropion was found in the serosal media of the gut sac showing that the isoenzyme of the 2B group was active in the intestinal mucosa and metabolized bupropion during its passage across the mucosa. The metabolite was also quantified in the mucosal media indicating its ability to cross the apical membrane of the epithelial cells.
Keywords: Bupropion; Hydroxybupropion; LC–MS; Gut sac; Metabolism; Cytochrome P450;

Detection, quantification and confirmation of anabolic steroids in equine plasma by liquid chromatography and tandem mass spectrometry by Fuyu Guan; Cornelius E. Uboh; Lawrence R. Soma; Yi Luo; Jeffery Rudy; Thomas Tobin (56-68).
Anabolic androgenic steroids are related to the male sex hormones and are abused in equine sports. In an effort to deter the abuse of anabolic steroids, a sensitive LC–MS/MS method was developed for detection, quantification and confirmation of eight major anabolic steroids (testosterone, normethandrolone, nandrolone, boldenone, methandrostenolone, tetrahydrogestrinone (THG), trenbolone, and stanozolol) in equine plasma. Formation of solvent adduct ions of the analytes was observed under electrospray ionization (ESI) conditions, and desolvation of the solvent adduct ions by source collision-induced decomposition (CID) increased the abundance of the [M + H]+ ions as well as the multiple-reaction monitoring (MRM) signals. ESI (+) and APCI (+) were compared with respect to sensitivity for the analytes and the former provided better sensitivity. The matrix effect on ion suppression or enhancement was evaluated, and was negligible. Confirmation of the analytes was performed using criteria of three ion transitions and LC retention time of each analyte. The limit of detection (LOD) and quantification (LOQ) was 25 pg/mL. The limit of confirmation (LOC) was 25 pg/mL for boldenone; 50 pg/mL for normethandrolone, nandrolone, and methandrostenolone; and 100 pg/mL for testosterone, THG, trenbolone, and stanozolol. The analytes were evaluated for stability and found to be stable in plasma for 24 h at room temperature, 13 days at 4 °C, and 34 days at −20 and −70 °C. The method was successfully applied to analyses of equine plasma samples for pharmacokinetics study. This method is sensitive and useful for detection, quantification and confirmation of these anabolic steroids in equine plasma.
Keywords: Anabolic steroid; LC–MS; Plasma; Horse; Doping control; THG; Testosterone; Boldenone;

A method for the determination of sertraline, a new antidepressant drug and a selective serotonin reuptake inhibitor (SSRI), in human plasma is described. Therapeutic drug monitoring (TDM) necessitates efficient, fast and reliable analytical methods validated by external quality control. We therefore devised a simple, rapid and sensitive isocratic reversed-phase liquid chromatographic-tandem mass spectrometric method equipped with Turbo Ion spray (TIS) source, operating in the positive ion and selective reaction monitoring (SRM) acquisition mode to quantify sertraline in human plasma. A new and superior procedure of solid-phase extraction (SPE) (compared to liquid–liquid extraction) was followed to extract sertraline and imipramine as internal standard (IS) from the human plasma. Sample preparation was performed using waters hydrophilic–lipophilic balance (HLB) cartridge and this method yielded extremely clean extracts with very good recovery, 81.47 and 85.79% for sertraline and IS, respectively. Both were analyzed by combined reverse phase liquid chromatography and tandem mass spectrometry (LC–MS/MS) with positive ion TIS ionization using SRM acquisition mode. The response of the LC–MS/MS method for sertraline was linear over the dynamic range of 0.5–60.0 ng/ml with correlation coefficient r  ≥ 0.9996. The coefficient of variance (%CV) was 8.53% at 0.5 ng/ml and the accuracy was well within the accepted limit of ±20% at lower limit of quantification (LLOQ) and ±15% at all the other concentrations in the linear range. This method was fully validated for the accuracy, precision and stability studies. The above findings indicate that the method is very much accurate and precise and can be successfully applied for bioequivalence studies in human subjects.
Keywords: Human plasma; Sertraline; Selective reaction monitoring (SRM); Solid-phase extraction; LC–MS/MS;

A rapid and sensitive liquid chromatography–tandem mass spectrometry (LC–MS–MS) method has been developed and validated for simultaneous quantification of venlafaxine (VEN) and O-desmethyl venlafaxine (ODV) in human plasma. The analytes were extracted from human plasma by using solid-phase extraction (SPE) technique. Escitalopram (ESC) was used as the internal standard. A Betasil C18 column provided chromatographic separation of analytes followed by detection with mass spectrometry. The mass transition ion-pair has been followed as m/z 278.27 → 121.11 for VEN, m/z 264.28 → 107.10 for ODV and m/z 325.00 → 262.00 for ESC. The method involves a solid phase extraction from plasma, simple isocratic chromatography conditions and mass spectrometric detection that enables detection at nanogram levels. The proposed method has been validated with linear range of 3–300 ng/ml for VEN and 6–600 ng/ml for ODV. The intrarun and interrun precision and accuracy values are within 10%. The overall recoveries for VEN and ODV were 95.9 and 81.7%, respectively. Total elution time as low as 3 min only.
Keywords: Venlafaxine; O-Desmethyl venlafaxine; LC–MS–MS; Human plasma;

We report a precise and accurate method for simultaneous quantification of protease inhibitors (PIs) amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir in plasma. An internal standard was added to samples prior to protein precipitation with acetonitrile followed by addition of ammonium formate buffer. Analysis was by HPLC-MS/MS. Calibration curves were validated over concentration ranges encompassing both subtherapeutic and potentially ‘toxic’ drug concentrations. Inter- and intra-assay variation were below 11% and PI recovery was above 87%. The bioanalytical method described is successfully applied to measure PI concentrations obtained from clinical pharmacokinetic studies and routine therapeutic drug monitoring (TDM).
Keywords: Protease inhibitors; HPLC-MS/MS; Therapeutic drug monitoring;

NC100668 is being developed as a new tracer for radiopharmaceutical imaging of venous thromboembolism. NC100668 consists of a targeting peptide of 13 amino acids with a 99mTc-binding chelator linked to the C-terminal amino acid. The present report describes a method for quantification of NC100668 in human citrated plasma. The method is based on solid-phase extraction followed by reversed-phase liquid chromatography using a gradient of water and acetonitrile with 0.1% formic acid. The chromatographic system was coupled on-line with an electrospray mass spectrometer. The analyses were performed by selective ion monitoring of the [M + 2H]2+ and the [M + 3H]3+ ions of NC100668 and an internal standard which was identical to NC100668 except for not being iodinated in the tyrosine residue. The limit of quantification of the method was 2 ng NC100668/ml plasma. The calibration curve ranged from 2 to 250 ng NC100668/ml plasma and was fitted to a linear equation with a weighing factor of 1/y 2 and found to be highly reproducible. The total precision of the method, expressed as the relative standard error of the mean, was 23.2, 8.8 and 14.7% for the low, medium and high control samples, respectively. The accuracy of the method was 108.5, 100.0 and 105.0% for the low, medium and high control samples, respectively. NC100668 was stable in human plasma during at least three freeze/thaw cycles, during 30 h on dry ice and up to 3 months when stored in a −20 °C freezer.
Keywords: NC100668; Human plasma; LC–MS; Ion-trap; Imaging; Venous thromboembolism;

Identification of the major metabolites of resveratrol in rat urine by HPLC-MS/MS by Donggeng Wang; Taijun Hang; Chunyong Wu; Wenying Liu (97-106).
To identify the major metabolites of resveratrol in rat, rat urine samples were pretreated by using solid-phase extraction technique (SPE) with polyamide cartridges. And a LC–MS/MS method with electrospray ionisation (ESI), negative ion mode and collision induced dissociation (CID), was used to elucidate the structures of the major metabolites of resveratrol. According to the results of our experiment, we found that the main metabolites of resveratrol were resveratrol monoglucuronide (M1), dihydroresveratrol monosulfate (M2), resveratrol monosulfate (M3) and dihydroresveratrol (M4).
Keywords: Trans-resveratrol; Metabolism; LC–MS/MS; Polyamide SPE;

In the field of proteomics, reproducible liquid chromatographic description of analytes is often a key element for the differentiation or identification of proteins or peptides for clinical or biological research projects. However, analyte identification by retention time can be problematic in proteomics where lack of standardization can result in significantly different chromatography for the same analytes analyzed on different machines. Here we present a novel method of monitoring the mobile phase gradient of LC–MS/MS analyses by monitoring the ion current signal intensities of tracer molecules dissolved in the mobile phase solvents. The tracers’ ion current signal intensities chronicled gradient fluctuations, did not adversely affect the number or quality of CID-based sequence identifications, and had lower run-to-run variance when compared to retention time.
Keywords: Liquid chromatography; Electrospray mass spectrometry; Proteomics; Peptide; Tracer molecules;

Doxil® is a pegylated liposome formulation of the anthracycline doxorubicin. To better explain observed differences in the toxicity of Doxil® and free doxorubicin in solution, the intracellular metabolism of the formulations after treatment in CCRF-CEM and CEM/C2 human leukemia cell lines was investigated. Using micellar electrokinetic capillary chromatography with laser-induced fluorescence detection, with a 63 zepto (10−21) mole doxorubicin limit of detection, five common metabolites and doxorubicin were detected upon treatment with both of these drug delivery systems. Two unique metabolites appeared with the Doxil® and two unique metabolites appeared with the free doxorubicin delivery systems. For common metabolites, the relative amount of metabolite generated from Doxil® was approximately 10 times higher than for free doxorubicin.
Keywords: Doxorubicin; Doxil; Liposome; Micellar electrokinetic capillary chromatography; Capillary; Electrophoresis; Laser-induced fluorescence; Metabolism;

Simultaneous determination of myristyl nicotinate, nicotinic acid, and nicotinamide in rabbit plasma by liquid chromatography–tandem mass spectrometry using methyl ethyl ketone as a deproteinization solvent by Paul Catz; Walter Shinn; Izet M. Kapetanovic; Hyuntae Kim; Moonsun Kim; Elaine L. Jacobson; Myron K. Jacobson; Carol E. Green (123-135).
Myristyl nicotinate (Nia-114) is an ester prodrug being developed for delivery of nicotinic acid (NIC) into the skin for prevention of actinic keratosis and its progression to skin cancer. To facilitate dermal studies of Nia-114, a novel liquid chromatography–tandem mass spectrometry (LC–MS/MS) method using methyl ethyl ketone (MEK) as a deproteinization solvent was developed and validated for the simultaneous determination of Nia-114, NIC, and nicotinamide (NAM) in rabbit plasma. NAM is the principal metabolite of NIC, which is also expected to have chemopreventive properties. The analytes were chromatographically separated using a Spherisorb Cyano column under isocratic conditions, and detected by multiple reaction monitoring (MRM) in positive-ion electrospray ionization mode with a run time of 9 min. The method utilized a plasma sample volume of 0.2 ml and isotope-labeled D4 forms of each analyte as internal standards. The method was linear over the concentration range of 2–1000, 8–1000, and 75–1000 ng/ml, for Nia-114, NIC, and NAM, respectively. The intra- and inter-day assay accuracy and precision were within ±15% for all analytes at low, medium, and high quality control standard levels. The relatively high value for the lower limit of quantitation (LLOQ) of NAM was demonstrated to be due to the high level of endogenous NAM in the rabbit plasma (about 350 ng/ml). Endogenous levels of NIC and NAM in human, dog, rat, and mouse plasma were also determined, and mean values ranged from <2 ng/ml NIC and 38.3 ng/ml NAM in human, to 233 ng/ml NIC and 622 ng/ml NAM in mouse. Nia-114 was generally unstable in rabbit plasma, as evidenced by loss of 44–50% at room temperature by 2 h, and loss of 64–70% upon storage at −20 °C for 1 week, whereas it was stable (<7% loss) upon storage at −80 °C for 1 month.
Keywords: Myristyl nicotinate; Nicotinamide; Nicotinic acid; Cancer chemoprevention; LC–MS/MS; Plasma;

Oligosaccharide analysis by capillary-scale high-pH anion-exchange chromatography with on-line ion-trap mass spectrometry by Cees Bruggink; Manfred Wuhrer; Carolien A.M. Koeleman; Victor Barreto; Yan Liu; Chris Pohl; Arnd Ingendoh; Cornelis H. Hokke; André M. Deelder (136-143).
A capillary-scale high-pH anion-exchange chromatography (HPAEC) system for the analysis of carbohydrates was developed, in combination with two parallel on-line detection methods of sub-picomolar sensitivity: (1) pulsed amperometric detection (PAD); (2) capillary-scale desalting followed by electrospray ion-trap (IT) mass spectrometry (MS). The capillary chromatographic system combined the superb selectivity of HPAEC that allows routine separation of isomeric oligosaccharides with the information on monosaccharide sequence and linkage positions obtained by MS/MS fragmentation using the IT-MS. The applicability of the system in biomedical research was demonstrated by its use for the analysis of a urine sample of a GM1-gangliosidosis patient. Isomeric glycans in the sample could be resolved by HPAEC and assigned on the basis of the monosaccharide linkage information revealed by on-line IT-MS/MS.
Keywords: HPAEC-PAD; Inulin; GM1-gangliosidosis; On-line desalter; Clinical glycomics;

A rapid, sensitive and selective HPLC separation with photodiode array detection was developed for the analysis of the novel pentacyclic triterpenoid acetyl-11-keto-α-boswellic acid. Complete baseline separation of acetyl-11-keto-α-boswellic acid from the corresponding isomer acetyl-11-keto-β-boswellic acid was achieved on a fluorinated stationary phase. The standard curve was linear from 0.98 nmol/l to 196 nmol/l acetyl-11-keto-α-boswellic acid. The compound was isolated from chick embryonic plasma using extraction on diatomaceous earth with an overall average extraction yield of 82%. This method was applied in a kinetic study on the chick chorioallantoic membrane model (CAM) and showed unequivocal separation between acetyl-11-keto-α-boswellic acid and acetyl-11-keto-β-boswellic acid unachievable so far.
Keywords: Pentacyclic triterpenoids; Acetyl-11-keto-α-boswellic acid; Chick chorioallantoic membrane; Pharmacokinetics; Pentafluorophenylpropyl-silica;

We have developed a method that uses on-line microdialysis sampling coupled with high-performance liquid chromatography (HPLC) to determine arbutin in whitening cosmetics. The optimum analytical conditions for microdialysis sampling were a probe length of 10 mm and a dialysis flow-rate of 5 μl min−1. The accuracy (% bias) for intra-day (n  = 6) and inter-day (n  = 30, five consecutive days) analyses ranged from −8.9 to 11.5%, with a precision below 7.64% R.S.D. The calibration curve was linear within the range from 0.1 to 20 mM (R 2  = 0.9989). The detection limit was 15 μM. By comparing the arbutin levels determined this way in the whitening cosmetics with the results obtained from the no-net-flux method, we conclude that our proposed on-line microdialysis–HPLC system displays good accuracy. We evaluated the robustness of our optimum conditions by means of a Plackett–Burman design. Apart from the effect of a low flow-rate of perfusate – an increase of 12.52 ± 2.31% – we observed no significant changes in the analyses upon changing the levels of any other parameter. Because this on-line method offers the advantages of simplicity, reliability, the lack of any tedious sample pretreatment process, and a reduced use of organic solvents, we believe that it is suitable for the routine analysis of commercial cosmetics.
Keywords: Microdialysis sampling; High-performance liquid chromatography; Arbutin; Whitening cosmetics; Validation;

Erratum to “Urinary high performance reverse phase chromatography cortisol and cortisone analyses before and at the end of a race in elite cyclists” by Rosalba Gatti; Enrico Cappellin; Barbara Zecchin; Giorgia Antonelli; Paolo Spinella; Franco Mantero; Elio Franco De Palo (154-159).
A functional and basic method for the quantitative analysis of urine cortisol (F) and cortisone (E) using a solid-phase extraction (SPE) column and HPLC with ultraviolet detection is here described and validated to analyse urine samples. Urine specimens were analysed to study F and E relation and ratio in athletes and healthy sedentary subjects. The F and E concentrations in random urine specimens were significantly higher in the post-exercise versus pre exercise condition in cyclists (F: 136 ± 93 nmol/l versus 67 ± 50 nmol/l (p  < 0.001); E: 797 ± 400 nmol/l versus 408 ± 252 nmol/l (p  < 0.001)). The F/E ratio was 0.18 ± 0.11 versus 0.16 ± 0.07, respectively, and a significant difference was only demonstrated comparing sedentary (0.11 ± 0.07) and cyclist individuals at rest (p  < 0.05).
Keywords: Solid-phase extraction; Exercise; 11β-HSD;

Author Index (160-161).

Keyword Index (162-165).