Journal of Chromatography B (v.878, #5-6)

A new acid mix enhances phosphopeptide enrichment on titanium- and zirconium dioxide for mapping of phosphorylation sites on protein complexes by Michael Mazanek; Elisabeth Roitinger; Otto Hudecz; James R.A. Hutchins; Björn Hegemann; Goran Mitulović; Thomas Taus; Christoph Stingl; Jan-Michael Peters; Karl Mechtler (515-524).
The selective enrichment of phosphorylated peptides prior to reversed-phase separation and mass spectrometric detection significantly improves the analytical results in terms of higher number of detected phosphorylation sites and spectra of higher quality. Metal oxide chromatography (MOC) has been recently described for selective phosphopeptide enrichment (Pinkse et al., 2004 ; Larsen et al., 2005 ; Kweon and Hakansson, 2006 ; Cantin et al., 2007 ; Collins et al., 2007 ). In the present work we have tested the effect of a modified loading solvent containing a novel acid mix and optimized wash conditions on the efficiency of TiO2-based phosphopeptide enrichment in order to improve our previously published method (Mazanek et al., 2007 ). Applied to a test mixture of synthetic and BSA-derived peptides, the new method showed improved selectivity for phosphopeptides, whilst retaining a high recovery rate. Application of the new enrichment method to digested purified protein complexes resulted in the identification of a significantly higher number of phosphopeptides as compared to the previous method. Additionally, we have compared the performance of TiO2 and ZrO2 columns for the isolation and identification of phosphopeptides from purified protein complexes and found that for our test set, both media performed comparably well. In summary, our improved method is highly effective for the enrichment of phosphopeptides from purified protein complexes prior to mass spectrometry, and is suitable for large-scale phosphoproteomic projects that aim to elucidate phosphorylation-dependent cellular processes.
Keywords: Phosphopeptide enrichment; Titanium dioxide; Zirconium dioxide; Spectrum database; APC/C; Condensin-I;

A sensitive method was developed and validated for the measurement of ixabepilone (BMS-247550, Ixempra™) using a demethylated analogue of ixabepilone (BMS-212188) as an internal standard. A 0.050 mL portion of each plasma sample was extracted with 0.450 mL of acetonitrile containing the internal standard via protein precipitation. The supernatant was analyzed on a LC–MS/MS system. Chromatography was carried out on a 2.0 mm × 100 mm YMC ODS-AQ 3 μm column using an isocractic mobile phase consisting of acetonitrile:10 mM ammonium acetate, pH 5.0 (70:30, v/v) at a flow rate of 0.30 mL/min. The mass spectrometer was fitted with a TurboIonSpray® source and operated in negative ionization mode. Detection of ixabepilone and BMS-212188 were accomplished using multiple reaction monitoring (MRM) of precursor > product ion pairs of m/z 505.2 > 405.2, and 492.1 > 392.1, respectively. The assay range was 2.00–500 ng/mL and was fitted to a 1/x 2 weighted quadratic regression model. Replicate sample analysis indicated that intra- and inter-day accuracy and precision are within ±15.0%. The recovery of ixabepilone from 0.050 mL of plasma containing 5.00 and 400 ng/mL was greater than 94%. The method was demonstrated to be sensitive, selective and robust, and was successfully used to support clinical studies. This paper also discussed approaches used for resolving a curve splitting issue observed during quantitative analysis of ixabepilone in biological matrices. Finally, to adapt the methodology to pharmacokinetics of ixabepilone after oral administration, the potential interference of chemical degradants on the determination of ixabepilone was evaluated.
Keywords: Ixabepilone; BMS-247550; Ixempra™; LC–MS/MS; Protein precipitation; Calibration issues; Adsorption; Degradants;

The natural pigment composition of purple bracts of Bougainvillea glabra (Nyctaginaceae) consists of a highly complex mixture of betacyanins solely differing by the substitution with a variety of acyl-oligoglycoside units. This study was focused on a two-dimensional chromatography approach, a combination of preparative high-speed countercurrent chromatography (HSCCC) and analytical C18-HPLC with ESI–DAD–MS/MS detection which finally enabled a more detailed view into the pigment profile and elucidated the existence of an overwhelming amount of varying betacyanin structures occurring in Bougainvillea bracts. The detected molecular weights of the pigments reached so far unknown high values and ranged up to maximum values of 1653 and 1683 Da for the largest molecules due to oligosaccharide linkage and multiple acyl substitutions. The preparative IP–HSCCC separation yielded 15 complex fractions containing betacyanins of enhanced polarity as well as structures with highly increased lipophilicity. Betacyanin structures extended by large oligosaccharide chains with bigger number of glycoside units and also carrying a reduced number of hydroxycinnamic acid substitutions were characteristic for polar pigments occurring mainly in the early eluting CCC fractions. IP–HSCCC was proven to be extremely effective for fractionating this complex crude betalain pigment extract into more defined ‘polarity-windows’. Structural analysis by analytical LC–ESI–MS/MS in the positive ionization mode detected a total sum of 146 different betacyanin pigments in the CCC fractions of reduced complexity.
Keywords: Betanin; Betalains; Acyl-oligosaccharide linked betacyanins; Preparative ion-pair countercurrent chromatography; IP–HSCCC; HPLC–DAD–ESI–MS/MS; Trifluoroacetic acid; Perfluorinated carboxylic acids; Bougainvillea glabra; Nyctaginaceae;

Determination of mildronate by LC–MS/MS and its application to a pharmacokinetic study in healthy Chinese volunteers by Ye Peng; Jing Yang; ZhiRui Wang; Jingwen Wang; Yu Liu; Zhuojing Luo; AiDong Wen (551-556).
A simple, rapid and accurate liquid chromatography–tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the determination of mildronate in human plasma. Following a simple protein precipitation with methanol, the analyte was separated on a C18 column by isocratic elution with methanol and 10 mM ammonium acetate (55:45; v/v), and then analyzed by mass spectrometry in the positive ion MRM mode. Good linearity was achieved over a wide range of 0.01–20 μg/mL. The intra- and inter-batch precisions (as RSD, %) were less than 7.1%. The average extraction recovery was 87.5%. The method described above has been used, for the first time, to reveal the pharmacokinetics of mildronate injection in healthy subjects. After single intravenously administration of 250, 500 and 1000 mg mildronate, the elimination half-life (t 1/2) were (5.56 ± 1.55), (6.46 ± 1.07) and (6.55 ± 1.17) h, respectively. The Student–Newman–Keuls test results showed that peak plasma concentration (C max) and the area under the plasma concentration versus time curve from time 0 to 24 h (AUC0–24) were both linearly related to dose. The pharmacokinetics of mildronate fitted the linear dynamic feature over the dose range studied. The essential pharmacokinetic parameters of multidoses administration intravenously (500 mg, b.i.d) were as follows: t 1/2 was (15.34 ± 3.14) h; C max was (25.50 ± 3.63) μg/mL; AUC0–24 was (58.56 ± 5.57) mg h/L. The t 1/2 and AUC of multidoses administration intravenously were different from those of single-dose administration significantly. These findings suggested that accumulation of mildronate in plasma occurred.
Keywords: Mildronate; Pharmacokinetics; LC–MS/MS;

Purification of human IgG by negative chromatography on ω-aminohexyl-agarose by Maria Cristiane Martins de Souza; Igor Tadeu Lazzarotto Bresolin; Sonia Maria Alves Bueno (557-566).
The ω-aminohexyl diamine immobilized as ligand on CNBr- and bisoxirane-activated agarose gel was evaluated for the purification of human immunoglobulin G (IgG) from serum and plasma by negative affinity chromatography. The effects of matrix activation, buffer system, and feedstream on recovery and purity of IgG were studied. A one-step purification process using Hepes buffer at pH 6.8 allowed a similar recovery (69–76%) of the loaded IgG in the nonretained fractions for both matrices, but the purity was higher for epoxy-activated gel (electrophoretically homogeneous protein with a 6.5-fold purification). The IgG and human serum albumin (HSA) adsorption equilibrium studies showed that the adsorption isotherms of IgG and HSA obeyed the Langmuir–Freundlich and Langmuir models, respectively. The binding capacity of HSA was high (210.4 mg mL−1 of gel) and a positive cooperativity was observed for IgG binding. These results indicate that immobilizing ω-aminohexyl using bisoxirane as coupling agent is a useful strategy for rapid purification of IgG from human serum and plasma.
Keywords: Human IgG; Purification; Aminohexyl; Human serum; Human plasma; Negative chromatography;

Monolithic bioreactors: Effect of chymotrypsin immobilization on its biocatalytic properties by E.A. Ponomareva; V.E. Kartuzova; E.G. Vlakh; T.B. Tennikova (567-574).
The effect of different modes of α-chymotrypsin attachment to the surface of methacrylate-based ultrashort monolithic minicolumns on enzyme activity has been studied. The immobilization of protease was carried out via direct covalent binding of chymotrypsin, as well as via its attachment through small and polymer spacers. It was established that the lowest enzyme activity against N-benzoyl-l-tyrosine ethyl ester was found for bioreactor obtained via direct attachment of chymotrypsin to the surface of GMA–EDMA minidisks, whereas the highest parameter close to that determined for dissolved enzyme was found in the case of bioreactor prepared by the introduction of copolymer of 2-deoxy-N-methacryloylamido-d-glucose with N-vinylpyrrolidone and acrolein as a long and flexible polymer spacer. Additionally, the effect of flow rate of substrate recirculation on bioconversion efficiency was examined. Independently on immobilization method, the increase of flow rate led to the raise of biocatalytic efficiency.
Keywords: Polymer monolithic sorbents; Enzyme immobilization; Polymer spacer;

Simultaneous and sensitive determination of xanthotoxin, psoralen, isoimpinellin and bergapten in rat plasma by liquid chromatography–electrospray ionization mass spectrometry by Wei Yang; Chao Feng; Dezhi Kong; Xiaowei Shi; Yang Cui; Man Liu; Qiao Wang; Yongli Wang; Lantong Zhang (575-582).
A sensitive, specific and rapid liquid chromatography–mass spectrometry (LC–MS) method has been developed and validated for the simultaneous determination of xanthotoxin (8-methoxypsoralen), psoralen, isoimpinellin (5,8-dimethoxypsoralen) and bergapten (5-methoxypsoralen) in rat plasma using pimpinellin as an internal standard (IS). The plasma samples were pretreated by protein precipitation with methanol and chromatographic separation was performed on a C18 column with a mobile phase composed of 1 mmol ammonium acetate and methanol (30:70, v/v). The detection was accomplished by multiple-reaction monitoring (MRM) scanning via electrospray ionization (ESI) source operating in the positive ionization mode. The optimized mass transition ion-pairs (m/z) for quantitation were 217.1/202.1 for xanthotoxin, 187.1/131.1 for psoralen, 247.1/217.0 for isoimpinellin, 217.1/202.1 for bergapten, and 247.1/231.1 for IS. The total run time was 6 min between injections. The calibration curves were linear over the investigated concentration range with all correlation coefficients higher than 0.998. The lower limits of quantitation (LLOQ) of these analytes were less than 1.21 ng/ml. The intra- and inter-day RSD were no more than 9.7% and the relative errors were within the range of −8.1% to 4.5%. The average extraction recoveries for all compounds were between 90.7% and 106.2%. The proposed method was further applied to the determination of actual plasma samples from rats after oral administration of Radix Glehniae extract.
Keywords: HPLC–MS; 8-Methoxypsoralen; Psoralen; 5,8-Dimethoxypsoralen; 5-Methoxypsoralen; Radix Glehniae extract;

Analyte loss due to non-specific binding, especially container surface adsorption, is not uncommon in the quantitative analysis of urine samples. In developing a sensitive LC–MS/MS method for the determination of a drug candidate, BAF312, in human urine, a simple procedure was outlined for identification, confirmation and prevention of analyte non-specific binding to a container surface and to recover the ‘non-specific loss’ of an analyte, if no transfer has occurred to the original urine samples. Non-specific binding or container surface adsorption can be quickly identified by using freshly spiked urine calibration standards and pre-pooled QC samples during a LC–MS/MS feasibility run. The resulting low recovery of an analyte in urine samples can be prevented through the use of additives, such as the non-ionic surfactant Tween-80, CHAPS and others, to the container prior to urine sample collection. If the urine samples have not been transferred from the bulk container, the ‘non-specific binding’ of an analyte to the container surface can be reversed by the addition of a specified amount of CHAPS, Tween-80 or bovine serum albumin, followed by appropriate mixing. Among the above agents, Tween-80 is the most cost-effective. β-cyclodextrin may be suitable in stabilizing the analyte of interest in urine via pre-treating the matrix with the agent. However, post-addition of β-cyclodextrin to untreated urine samples does not recover the ‘lost’ analyte due to non-specific binding or container surface adsorption. In the case of BAF312, a dynamic range of 0.0200–20.0 ng/ml in human urine was validated with an overall accuracy and precision for QC sample results ranging from −3.2 to 5.1% (bias) and 3.9 to 10.2% (CV), respectively. Pre- and post-addition of 0.5% (v/v) Tween-80 to the container provided excellent overall analyte recovery and minimal MS signal suppression when a liquid–liquid extraction in combination with an isocratic LC separation was employed. The compound was stable in 0.5% Tween-80 treated human urine QC samples for at least 24 h at room temperature, after three freeze/thaw cycles with storage at ≤−60 °C and for at least 3 months when stored at ≤−60 °C. The current work could serve as a simple example in trouble shooting non-specific binding or container surface adsorption in quantitative analysis of urine samples.
Keywords: BAF312; Urine assay; Tween-80; CHAPS; Bovine Serum Albumin (BSA); β-cyclodextrin; LC–MS/MS; Container Surface Absorption;

An assay to quantify several possible breast cancer peptide biomarkers in human serum has been developed and validated, using liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). The peptides include bradykinin, Hyp3-bradykinin, des-Arg9-bradykinin and fragments of fibrinogen α-chain (Fib-α[605–629]), inter-α-trypsin inhibitor heavy chain 4 (ITIH4[666–687]) and complement component 4a (C4a[1337–1350]). Ile13-ITIH4[666–687], d20-C4a[1337–1350] and Sar-D-Phe8-des-Arg9-bradykinin were used as internal standards. Bovine plasma, with 2 mM captopril and 2 mM d-l-mercaptoethanol-3-guanidino-ethylthiopropanoic acid (MEGETPA) to prevent rapid degradation of the bradykinins, was used as analyte-free matrix. Recoveries for solid-phase extraction (SPE) on mixed-mode weak cation exchange sorbents were between 62 and 90%. Multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer equipped with a heated electrospray source (H-ESI), operating in the positive ion-mode, was used for detection. The assay was fully validated and stabilities of the peptides were extensively explored. Bradykinin (10–500 ng/ml), Hyp3-bradykinin (4–200 ng/ml), des-Arg9-bradykinin (2–100 ng/ml), Fib-α[605–629] (120–3000 ng/ml), ITIH4[666–687] (0.4–10 ng/ml) and C4a[1337–1350] (1–25 ng/ml) were simultaneously quantified with deviations from the nominal concentrations below 22% and intra- and inter-assay precisions below 15 and 20%, respectively, for all peptides at all concentrations. The method has been successfully applied to several serum samples from breast cancer patients and matched controls.
Keywords: Bradykinins; ITIH4[666–687]; C4a[1337–1350]; Fibrinogen-α[605–629]; quantification; LC–MS/MS; Biomarkers; Breast cancer;

In the present work we have developed a standard-addition HPLC method using a mobile phase containing low concentration of ZnAc2 to determine physiological level of kynurenine (KYN), kynurenic acid (KYNA) and tryptophan (TRP) in human plasma simultaneously. The method greatly improved the sensitivity of KYNA, the resolution of KYNA and TRP, and avoided clotting risk caused by high concentration of ZnAc2 in mobile phase. Samples were deproteinized by addition of equal volume of 0.6 mol/L HClO4. Analytes in supernatants were separated by an Agilent HC-C18 (2) analytical column; an aqueous mobile phase containing 20 mmol/L NaAc, 3 mmol/L ZnAc2 and 7% acetonitrile at flow rate of 1.0 mL/min. Detections were performed by a variable wavelength detector at wavelength 365 nm for KYN and a fluorescence detector at wavelengths excitation 344 nm and emission 398 nm for KYNA and TRP. Good linear responses were found with r 2  > 0.999 for all analytes within the concentration range of physiological levels. The limit of detection of the developed method was 0.03 μmol/L, 0.9 nmol/L and 0.4 μmol/L for KYN, KYNA and TRP respectively. Recoveries from spiked human plasma were 95.4–99.7% for KYN, 98.9–104% for KYNA and 96.5–100.2% for TRP. All CVs for the repeatability and intermediate precision were less than 5%. We conclude that the developed method is helpful for the research investigations in KYN pathway of TRP metabolism.
Keywords: Kynurenine; Kynurenic acid; Tryptophan; Human plasma; High performance liquid chromatography;

The hyphenation of high performance liquid chromatography with inductively coupled plasma mass spectrometry (HPLC–ICP-MS) is proposed in this work as a novel approach for the evaluation of DNA methylation, defined as the ratio between methylated cytosine and total cytosine bases in DNA. In the first part, reversed phase separation of 5-methyl-2′-deoxycytidine monophosphate (5mdCMP) and four deoxynucleotides with specific ICP-MS detection on 31P had been explored. In further development, selective labeling of 5-methylcytosine in ssDNA was carried out using potassium osmate (K2OsO4) in the presence of strong oxidant (K3Fe(CN)6) and N,N,N′,N′-tetramethylethylenediamine (TEMED). The sample was then cleaned up and introduced to size exclusion chromatography–ICP-MS for specific detection at 31P and 189Os and for evaluation of the molar ratio between Os and P eluted in DNA molecular mass fraction. The quantification of the two elemental tags was achieved by external calibration with phosphoric acid and Os(VI)-TEMED, respectively. The amount of Os in DNA fraction corresponded to methylated cytosines, while P signal was directly proportional to the total amount of DNA and could be recalculated to the amount of cytosine bases. The two procedures were tested by analyzing salmon testes DNA and a commercial oligonucleotide of known composition. For comparative purposes, these same samples were digested to deoxynucleosides and analyzed by reversed phase HPLC with spectrophotometric detection (DAD) at 280 nm. The results obtained using two procedures based on ICP-MS detection were in good agreement and also in agreement with the results obtained by HPLC–DAD procedure. In conclusion, ICP-MS specific detection at internal or external element tags seems to be an interesting alternative for the evaluation of global DNA in epigenetic studies.
Keywords: DNA methylation; Phosphorus; Osmium; Elemental tag; HPLC–ICP-MS;

Simultaneous determination of citalopram and its metabolite in human plasma by LC–MS/MS applied to pharmacokinetic study by Tao Jiang; Zhengxing Rong; Liang Peng; Bing Chen; Yifan Xie; Congying Chen; Jing Sun; Yiping Xu; Yang Lu; Hongzhuan Chen (615-619).
A simple sensitive and robust method for simultaneous determination of citalopram and desmethylcitalopram was developed using liquid chromatography tandem mass spectrometry (LC–MS/MS). A 200 μL aliquot of plasma sample was employed and deproteinized with methanol and desipramine was used as the internal standard. After vortex mixing and centrifugation, the supernatant was diluted with water (1:1, v/v) and then directly injected to analysis. Analytes were separated by a Zorbax XDB C18 column with the mobile phase composed of acetonitrile and water (30:70, v/v) with 0.25% formic acid and monitored in MRM mode using a positive electrospray source with tandem mass spectrometry detection. The total run time was 3.5 min. The dynamic range was 0.2–100 ng/mL for citalopram and 0.25–50 ng/mL for desmethylcitalopram, respectively. Compared to the best existing literatures for plasma samples, the same LOQ for CIT (0.5 ng/mL) and lower LOQ for DCIT (0.25 vs 5 ng/mL) were reached, and less sample preparation steps and runtime (3.5 vs 10 min) were taken for our method. Accuracy and precision was lower than 8% and lower than 11.5% for either target. Validation results and its application to the analysis of plasma samples after oral administration of citalopram in healthy Chinese volunteers demonstrated the method was applicable to pharmacokinetic studies.
Keywords: Citalopram; Desmethylcitalopram; LC–MS/MS; Pharmacokinetics;