Journal of Chromatography B (v.860, #2)

Comparison of ESI-MS interfaces for the analysis of UV-crosslinked peptide–nucleic acid complexes by Philip R. Gafken; Catalin E. Doneanu; Samuel E. Bennett; Douglas F. Barofsky (145-152).
In this report, the effectiveness of high performance liquid chromatography (HPLC) in conjunction with electrospray ionization mass spectrometry (ESI-MS) is examined as a tool for identifying the sites of crosslinking in a protein that has been photoreacted with a non-photolabeled oligonucleotide. ESI-MS and MALDI-MS analyses preceded by off-line microflow and nanoflow HPLC, on-line microflow HPLC/ESI, and on-line nanoflow HPLC/ESI interfaces were performed in order to determine their relative effectiveness in separating mixtures of nucleopeptides and identifying sites of crosslinking on the individual components. The characteristics of these four techniques as well as possibilities for improving the analysis of nucleopeptides by ESI-MS are compared and discussed.
Keywords: Nanoscale liquid chromatography; Electrospray ionization mass spectrometry; UV-crosslinking; Peptide–nucleic acid complexes;

A fast, sensitive and specific LC/MS/MS method for the simultaneous analysis of oxcarbazepine (OXC), 10-hydroxycarbazepine (MHD) and trans-diol-carbazepine (DHD), in human serum, has been developed and validated. Serum drugs were extracted by C8 solid-phase cartridges (SPE) and separated in less than 3 min on a C18 reverse-phase column using an isocratic elution. A tandem mass spectrometer, as detector, was used for quantitative analysis in positive mode by a multiple reaction monitoring. Calibration curves, obtained on two ranges of concentration (0.78–50 mg/L for MHD and 0.078–5.0 mg/L for OXC and DHD), showed correlation coefficients (r) better than 0.997. Within day and between days quality controls imprecision, as CV%, ranged from 0.3 to 4.6% and from 1.9 to 5.8%, respectively. Cyheptamide (CYE) was used as internal standard. No detectable carry-over and no relevant cross-talk and matrix effect occurred. Samples from 24 treated patients were analysed and drug serum concentrations obtained by this method are in agreement with those of other methods and also are well correlated (r  = 0.88) in comparison to our routine HPLC-UV method. Based on the analytical results and short run time, the method is suitable to support routine analysis of therapeutic drugs monitoring from human serum of treated patients or for pharmacokinetic studies.
Keywords: Oxcarbazepine; 10-Hydroxycarbazepine; Therapeutic drug monitoring; LC/MS/MS;

A reversed-phase high-performance liquid chromatographic method using a mobile phase of acetonitrile–methanol–trifluoroacetic acid–water (16.1:7.2:0.1:76.6, v/v/v/v) at a flow rate of 1.0 ml min−1 on a LiChrospher™ RP-18 column with UV (254 nm) detection has been developed for the separation of sulfadoxine and its metabolite N-acetyl sulfadoxine in plasma. No interferences due to endogenous compounds or common antimalarial drugs were noticed. The limit of detection for sulfadoxine and N-acetyl sulfadoxine was 0.01 μg ml−1 with a signal-to-noise ratio of 5:1 while the limit of quantification was 2.5 μg ml−1. Intra-day mean relative standard deviations (RSD's) for sulfadoxine and N-acetyl sulfadoxine were 2.6 and 2.8%, respectively, while mean inter-day RSD's for sulfadoxine and N-acetyl sulfadoxine were 2.4 and 2.8%, respectively. Extraction recoveries averaged 90.6% for sulfadoxine and 86.9% for N-acetyl sulfadoxine. The method was applied for the assay of sulfadoxine and its metabolite N-acetyl sulfadoxine in plasma from Plasmodium falciparum malaria patients. Mean plasma sulfadoxine concentrations on day 2 (51 h) from samples collected from sensitive and resistant P. falciparum patients treated with three tablets of Fansidar™ were 62.8 and 60.5 μg ml−1, respectively. Mean ratio of N-acetyl sulfadoxine to sulfadoxine was 9.1% for responders and 13.9% for non-responders which revealed that higher amounts of the metabolite N-acetyl sulfadoxine were present in non-responders. The method described should find an application in the therapeutic monitoring of malaria patients.
Keywords: HPLC; Sulfadoxine; N-acetyl sulfadoxine; Malaria cases;

Epimedium herbs are a type of complex traditional Chinese medicine (TCM) with high estrogenic bioactivity. The Epimedium herbal decoction mixture contains many compounds including icariin that can exert potent effects on numerous physiological processes related to human health. An ultrasensitive liquid chromatography tandem mass spectrometric (LC–MS/MS) method has been developed to determine trace levels of icariin in human serum with dansyl chloride derivatization after oral administration of the Epimedium herbal decoctions. The dansyl-icariin showed an intense protonated molecular ion at m/z 910. The collision-induced dissociation of this ion formed a distinctive product at m/z 764, corresponding to a characteristic removal of a rhamnose sugar moiety of icariin. The selected reaction monitoring, based on the m/z 910 → 764 transition, was highly specific and ultrasenstive for icariin in human serum samples. The lower limit of quantitation was 10 pg/mL icariin spiked into blank serum. The ranges of coefficients of variation for interday assays and intraday assays were 0–15.0% and 1.1–17.5%, respectively, for a wide linear range from 10 pg/mL to 4 ng/mL. This method was successfully applied to measure trace levels of icariin in a human serum after oral administration of Epimedium decoction within 48 h for the first time.
Keywords: Trace analysis; Icariin; Liquid chromatography; Tandem mass spectrometry; Epimedium decoction; Human clinical trial;

Virus analysis by electrophoresis on a microfluidic chip by Victor U. Weiss; Viliam Kolivoška; Leopold Kremser; Bohuslav Gaš; Dieter Blaas; Ernst Kenndler (173-179).
Exploiting the advantages of miniaturization of analytical devices we worked out conditions for the analysis of viruses, subviral particles, and virus–receptor complexes on microfluidic chips. To allow for detection via laser-induced fluorescence, the viral capsids were labelled with the fluorescent dye Cy5. We analyzed human rhinovirus serotype 2 and subviral particles, followed the complexation of the virus with a synthetic fragment of the VLDL-receptor, and tracked the heat-induced conversion of intact virions into empty capsids. In contrast to fused silica capillaries, the glass micro-channels allowed for electrophoresis of the analytes without detergent, and analyses were accomplished within few tens of seconds. This opens the avenue towards the analytics of membrane-enveloped viruses and other biological assemblies that are not stable in the presence of detergent. The chip format has the additional advantage of containment and easy disposal, making it particularly attractive for the analysis of infectious material.
Keywords: Chip electrophoresis; Fluorescence; Virus; Cy5; HRV2; VLDL-receptor; Concatemer; Lab-on-a-chip;

The reaction from retinal to retinoic acid catalyzed by retinal dehydrogenase on a polyvinylidene difluoride (PVDF) membrane was examined using laser desorption ionization time of flight mass spectrometry (LDI-TOF MS) when the enzyme was separated by non-denaturing two-dimensional electrophoresis (2-DE), transferred onto the membrane, and stained without impairing the enzyme activity. Furthermore, the enzyme was analyzed by de novo sequencing using electrospray ionization tandem mass spectrometry (ESI-MS/MS) after proteins from mouse liver were separated by non-denaturing 2-DE, blotted onto the membrane, and stained. The results indicated that the reported methods could be applied for the direct examination of changes in retinoid catalyzed by enzymes on such membranes.
Keywords: Retinal dehydrogenase; Retinal; Retinoic acid; Identification; De novo sequencing;

Biological fingerprinting analysis of the traditional Chinese prescription Longdan Xiegan Decoction by on/off-line comprehensive two-dimensional biochromatography by Yun Wang; Liang Kong; Lianghai Hu; Xiaoyuan Lei; Li Yang; Guixin Chou; Hanfa Zou; Changhong Wang; S.W. Annie Bligh; Zhengtao Wang (185-194).
A comprehensive two-dimensional biochromatography method using a silica-bonded human serum albumin (HSA) column and a RP-HPLC column was developed for the biological fingerprinting analysis of bioactive components in a traditional Chinese medicine (TCM) prescription, Longdan Xiegan Decoction (LXD). The biochromatography with HSA-immobilized stationary phase was applied to study the interaction of multiple components in LXD with HSA in the first dimension, and fractions of HSA column were further separated by a silica monolithic ODS column (on-line)/an ODS column (off-line) coupled with a diode array detector and an atmospheric pressure chemical ionization mass spectrometer (APCI-MS). More than 100 compounds in LXD that interacted with the immobilized HSA were separated and analyzed. Among them 19 compounds were identified based on their retention values, UV spectra, molecular weights and mass spectra. The results show that the developed comprehensive two-dimensional biochromatography system reported here is capable of being used for biological fingerprinting analysis of natural products in complex matrices such as extracts of TCMs and their prescriptions.
Keywords: Comprehensive two-dimensional biochromatography; Longdan Xiegan Decoction; HSA column; Biological fingerprinting analysis; Traditional Chinese medicine;

The butenolide ring is the main common characteristic of all cardenolides. Its formation is supposed to be initiated by the transfer of a malonyl moiety from malonyl-coenzyme A to an appropriate 21-hydroxypregnane. A new, reliable, fast and sensitive method to determine malonyl-coenzyme A: 21-hydroxypregnane 21-O-malonyltransferase activity had to be developed since previous attempts employing HPLC, TLC or GC did not prove successful. A surrogate substrate was synthesized containing a side chain resembling the sugar side chain attached to C-3 of putative cardenolide precursors and containing a chromophor allowing UV detection. 3β-benzoyloxy-5β-pregnane-14β,21-dihydroxy-20-one and its 21-O-malonylated derivative were synthesized, the latter being the expected product of the enzyme reaction. The new substrate was well accepted by the enzyme. An HPLC method has been established to detect and quantify 3β-benzoyloxy-5β-pregnane-14β,21-dihydroxy-20-one and its 21-O-malonylated derivative, 3β-benzoyloxy-5β-pregnane-14β-hydroxy-20-one 21-O-malonylhemiester. The method was validated.
Keywords: HPLC method; Method validation; Benzoylation; Malonyl-coenzyme A: 21-hydroxypregnane 21-O-malonyltransferase; Cardenolides; Cardiac glycosides;

Development and validation of high-throughput liquid chromatography–tandem mass spectrometric method for simultaneous quantification of loratadine and desloratadine in human plasma by G. Srinubabu; Rajaram S. Patel; Vinay P. Shedbalkar; Allam Appa Rao; M. Narasimha Rao; Veera Venkata Ratnam Bandaru (202-208).
As a continuation of effort to improve our high flow on-line bioanalytical approach for high-throughput quantification of drugs and metabolites in plasma by high-throughput liquid chromatography tandem mass spectrometry (HTLC–MS/MS), we have developed a simple, sensitive and reliable method for simultaneous quantification of loratadine and desloratadine in human plasma. We have performed on-line coupling of extraction with Cyclone P 50 mm × 0.5 mm 50 μm HTLC column and chromatographic separation is performed with Zorbax XDB C18 50 mm × 2.1 mm 5 μm, followed by quantification with mass detector. The method is validated and showed good performances in terms of linearity, sensitivity, precision, accuracy and stability. A marked improvement in sample throughput efficiency is realized with this method and the proposed method will be useful for pharmacokinetic and/or bioequivalence studies.
Keywords: Loratadine; Desloratadine; High-throughput liquid chromatography; Mass spectrometric detection; Human plasma; Validation;

Evaluation of a novel agarose-based synthetic ligand adsorbent for the recovery of antibodies from ovine serum by Sunil Chhatre; Richard Francis; Nigel J. Titchener-Hooker; Anthony R. Newcombe; Eli Keshavarz-Moore (209-217).
This paper evaluates a prototype agarose-based affinity adsorbent utilizing a bound synthetic ligand designed to replace Protein A as an IgG-affinity capture resin and compares its purification characteristics with four commercially available matrices for the recovery of polyclonal antibodies from crude hyperimmune ovine serum. The novel adsorbent was found to show the highest dynamic capacity (29.2 mg/mL) of all matrices under evaluation—30% higher than the other commercial adsorbents evaluated. When using a post-load caprylic acid wash, IgG yields of over 85% and purities of over 90% were achieved consistently over multiple loading cycles. To evaluate bead diffusion, inverted confocal microscopy was used to visualise fluorescent antibody binding on to individual adsorbent beads in real time. The results indicate that the binding characteristics of the prototype adsorbent are similar to those obtained with Protein G Sepharose. This study indicates that the high-capacity prototype matrix is a feasible and potentially cost-effective alternative for the direct capture of antibodies from crude ovine serum and may therefore also be applicable to the purification of other complex industrial feedstocks such as transgenic milk or monoclonal antibodies expressed using recombinant technologies.
Keywords: Affinity chromatography; Antibody; Ovine; Polyclonal; Serum; Synthetic;

Gambogic acid (GBA), the main component of Gamboge, possesses significant anti-tumour activity. Due to its structural complexity, little is known about GBA metabolism. Here, we investigate the metabolism of GBA in vivo in rat bile. Identification of the metabolites formed was elucidated using high-performance liquid chromatography (HPLC) with UV–vis detection, HPLC/ion trap electrospray ionization-mass spectrometry, as well as HPLC/nuclear magnetic resonance. Four main metabolites were determined. Two phase I metabolites, 10-hydroxygambogic acid and 9,10-epoxygambogic acid, were oxides on the 9,10-olefinic bond of GBA. The others phase II metabolites, were 9,10-epoxygambogic acid-30-O-glucuronide and 10-hydroxylgambogic acid-30-O-glucuronide.
Keywords: Gambogic acid; Metabolites; Structural determination; 10-Hydroxygambogic acid; 9,10-Epoxygambogic acid;

Chiral bioanalysis of torcetrapib enantiomers in hamster plasma by normal-phase liquid chromatography and detection by atmospheric pressure chemical ionization tandem mass spectrometry by Ravi Kumar Trivedi; Buddhadev Layek; T. Santosh Kumar; Shivva Vittal; Ramesh Ganneboina; P.K. Dubey; Ramesh Mullangi; Nuggehally R. Srinivas (227-234).
A highly sensitive and enantioselective assay has been developed and validated for the estimation of torcetrapib (TTB) enantiomers [(+)-TTB and (−)-TTB] in hamster plasma with chiral liquid chromatography coupled to tandem mass spectrometry with an atmospheric pressure chemical ionization interface in the negative-ion mode. The assay procedure involves liquid–liquid extraction of TTB enantiomers and IS (DRL-16126) from 100 μL hamster plasma with acetonitrile. TTB enantiomers were separated using n-hexane:propanol (80:20, v/v) at a flow rate of 0.7 mL/min on a Chiralpak AD column. The MS/MS ion transitions monitored were 599.2 → 340.2 for TTB and 623.2 → 298.1 for IS. Absolute recovery was found to be between 64 and 68% for TTB enantiomers and >100% for IS. The standard curves for TTB enantiomers were linear (r 2  > 0.995) in the concentration range 5–2500 ng/mL for each enantiomer with an LLOQ of 5 ng/mL for each enantiomer. The inter- and intra-day precisions were in the range of 10.5–12.4 and 9.15–11.5% and 3.75–12.9 and 5.16–12.5% for (+)-TTB and (−)-TTB, respectively. Accuracy in the measurement of quality control (QC) samples was in the range 91.3–105 and 88.6–111% for (+)-TTB and (−)-TTB, respectively. This novel method has been applied to the study of stereoselective oral pharmacokinetics of (−)-TTB.
Keywords: Torcetrapib; Enantioselective; Plasma; Validation; Chiralpak AD; LC–MS/MS; APCI; Pharmacokinetics;

A validated, simple and universal HPLC-UV method for the determination of cimetidine, famotidine, nizatidine and ranitidine in human urine is presented. This is the first single HPLC method reported for the analysis of all four H2 antagonists in human biological samples. This method was also utilized for the analysis of ranitidine and its metabolites in human urine. All calibration curves showed good linear regression (r 2  > 0.9960) within test ranges. The method showed good precision and accuracy with overall intra- and inter-day variations of 0.2–13.6% and 0.2–12.1%, respectively. Separation of ranitidine and its metabolites using this assay provided significantly improved resolution, precision and accuracy compared to previously reported methods. The assay was successfully applied to a human volunteer study using ranitidine as the model compound.
Keywords: H2 antagonists; High performance liquid chromatography; Bioavailability; Metabolism; Ranitidine; Famotidine; Cimetidine; Nizatidine;

Simultaneous determination of cefepime, vancomycin and imipenem in human plasma of burn patients by high-performance liquid chromatography by K.J. Vera López; D. Faria Bertoluci; K.M. Vicente; A.M. Dell’Aquilla; S.R.C. Jorge Santos (241-245).
A liquid chromatographic method with UV detection for simultaneous determination of cefepime, vancomycin and imipenem has been developed. Cefuroxime was used as internal standard. After the clean up of samples by plasma protein precipitation, 5 μl of the extract were injected into the chromatograph and peaks were eluted from the Sulpelcosil™ LC-18 column using a mobile phase consisting of 0.075 M acetate buffer:acetonitrile (92:8, v/v), pH 5.0 at low rate (0.8 ml/min). The detection wavelength was 230 nm. The limit of detection was 0.4 μg/ml for cefepime and 0.2 μg/ml for vancomycin and imipenem. The method was applied to plasma samples of burn patients, and only small volumes of plasma were required for the simultaneous determination of those antimicrobial agents.
Keywords: Cefepime; Vancomycin; Imipenem; Liquid–liquid chromatography;