Phosphoproteome analysis of human liver tissue by long‐gradient nanoflow LC coupled with multiple stage MS analysis

Reversible protein phosphorylation plays a critical role in liver development and function. Comprehensively cataloging the phosphoproteins and their phosphorylation sites in human liver tissue will facilitate the understanding of physiological and pathological mechanisms of liver. Owing to lacking of efficient approach to fractionate phosphopeptides, nanoflow‐RPLC with long‐gradient elution was applied to reduce the complexity of the phosphopeptides in this study. Two approaches were performed to further improve the coverage of phosphoproteome analysis of human liver tissue. In one approach, ten‐replicated long‐gradient LC‐MS/MS runs were performed to analyze the enriched phosphopeptides, which resulted in the localization of 1080 phosphorylation sites from 495 proteins. In another approach, proteins from liver tissue were first fractionated by SDS‐PAGE and then long‐gradient LC‐MS/MS analysis was performed to analyze the phosphopeptides derived from each fraction, which resulted in the localization of 1786 phosphorylation sites from 911 proteins. The two approaches showed the complementation in phosphoproteome analysis of human liver tissue. Combining the results of the two approaches, identification of 2225 nonredundant phosphorylation sites from 1023 proteins was obtained. The confidence of phosphopeptide identifications was strictly controlled with false discovery rate (FDR)≤1% by a MS²/MS³ target‐decoy database search approach. Among the localized 2225 phosphorylated sites, as many as 70.07% (1559 phosphorylated sites) were also reported by others, which confirmed the high confidence of the sites determined in this study. Considering the data acquired from low accuracy mass spectrometer and processed by a conservative MS²/MS³ target‐decoy approach, the number of localized phosphorylation sites obtained for human liver tissue in this study is quite impressive.     

Immuno‐capture as ultimate sample cleanup in LC‐MS/MS determination of the early stage biomarker ProGRP

This paper presents a selective and efficient sample preparation procedure for NLLGLIEAK, signature peptide for the small cell lung cancer (SCLC) biomarker ProGRP, in human serum. The procedure is based on immuno‐capture of ProGRP in 96‐wells microtiter plates coated with the mAb E146. After immuno‐capture and thorough rinse, trypsin was added for in‐well digestion. Subsequently the signature peptide was enriched by SPE and determined by LC‐MS/MS. Various steps in the procedure were optimized to achieve a low LOD such as dilution of sample, tryptic digestion, and SPE cleanup and peptide enrichment conditions. A single quadropole MS was used during optimization of the method. A triple quadropole MS was used in the method evaluation in order to improve sensitivity. The evaluation showed good repeatability (RSD, 11.9–17.5%), accuracy (3.0–6.6%), and linearity (r² = 0.995) in the tested range (0.5–50 ng/mL). LOD and LOQ were in the pg/mL area (0.20 and 0.33 ng/mL, respectively), enabling the determination of clinically relevant concentrations. The method was applied to two patient samples and showed good agreement with an established immunological reference method. The final method was compared to a previous published LC‐MS method for the determination of ProGRP in serum based on protein precipitation and online sample cleanup. Both showed acceptable method performance, however, the immuno‐capture LC‐MS method was superior with respect to sensitivity. This illustrates the large potential of immuno‐capture sample preparation prior to LC‐MS in protein biomarker quantification.     

A single liquid chromatography–quadrupole time‐of‐flight mass spectrometric method for the quantification of total antibody,antibody‐conjugated drug and free payload of antibody–drug conjugates

A single hybrid affinity‐captured‐LC‐TOF‐MS/MS method was developed and applied for the quantification of total antibody, antibody conjugated drug and free payload of antibody drug conjugate (ADC). Adcetris®, a valine–citrulline monomethyl auristatin E conjugated ADC, was used as a model ADC compound. A quadratic regression (weighted 1/concentration) was used to fit calibration curves over the concentration range 30.65–613.00 ng/mL with an equation y = ax² + bx + c for the antibody‐conjugated drug of Adcetris®. The qualification run met the acceptance criteria of ±25% accuracy and precision values for quality control samples. For the analysis of total antibody, a signature peptide (TTPPVLDSDGSFFLYSK, molecular weight 1874) was used after affinity capture using magnetic beads and on‐bead trypsin digestion. A quadratic regression (weighted 1/concentration) was used to fit calibration curves over the concentration range 5.00–100.00 μg/mL with an equation y = ax² + bx + c for total antibody. For free payload analysis of monomethyl auristatin E, a protein precipitation method followed by LC‐TOF‐MS/MS analysis was used. A quadratic regression (weighted 1/concentration) was used to fit calibration curves over the concentration range 1.01–2200 ng/mL with an equation y = ax² + bx + c for free payload. Pharmacokinetic study samples and in vitro stability samples in rat were successfully analyzed by this a hybrid affinity‐captured‐LC‐TOF‐MS/MS method. This single platform method is a useful complementary method for the pharmacokinetics study of ADC with valine–citrulline linker at the early drug discovery stage.     

Two‐step Immobilized Metal Affinity Chromatography (IMAC) for Phosphoproteomics Using Mass Spectrometry

In this study, a new strategy named two‐step IMAC is demonstrated as a novel prelude to MS analysis of phosphoproteome by increasing the enrichment factor of phosphoproteins/phosphopeptides from a protein mixture. In this method, the first IMAC was performed at the protein level to extract the minute amount of phosphoproteins present in the sample. During this step, nonphosphoproteins and other undesired chemicals or inhibitors were excluded. After tryptic digestion, the second IMAC was performed at the peptide level to enrich phosphopeptides present in the tryptic digest, and the eluent from the second IMAC was analyzed by MALDI‐MS. It is particularly noticeable that the eluent from the first IMAC can be directly digested by trypsin without buffer exchange. Our results revealed that β‐casein that was spiked in a protein mixture can be successfully extracted by the first IMAC at a concentration of less than 1–3%, and the two phosphopeptides of β‐casein with single and four phosphorylation sites, respectively, can be captured by the second IMAC. It was found that the two‐step IMAC method could significantly reduce non‐specific bindings from unwanted proteins and greatly enhance the MALDI‐MS signal of phosphopeptide ions compared to the typical one‐step IMAC, by which only IMAC at the peptide level was performed. Two‐step IMAC was also found to tolerate a greater amount and a greater concentration range of proteins than one‐step IMAC, which is especially important when analyzing complicated unknown samples. Furthermore, the MS signal of phosphopeptide ions did not appear to be degraded by the presence of biological matrixes, such as the cell lysate in which the β‐casein was spiked in.     

Ion‐pairing high‐performance liquid chromatography‐mass spectrometry of impurities and reduction products of sulphonated azodyes

An HPLC separation method with triethylammonium acetate mobile phase additive developed for the analysis of impurities in polysulphonated azo dyes provides good separation selectivity and compatibility with electrospray ionisation (ESI) mass spectrometry. The negative‐ion ESI mass spectra containing only peaks of deprotonated molecules [M–H]^– for monosulphonic acids, [M–xH]^x–, and sodiated adducts [M–(x + y)H + yNa]^x– for polysulphonic acids allow easy molecular mass determination of unknown impurities. Based on the knowledge of the molecular masses and of the fragment ions in the MS/MS spectra, probable structures of trace impurities in commercial dye samples are proposed. To assist in the interpretation of the mass spectra of complex polysulphonated azodyes, additional information can be obtained after chemical reduction of azodyes to aromatic amines. The structures of the non‐sulphonated reduction products can be determined by reversed‐phase HPLC/MS with positive‐ion atmospheric pressure chemical ionisation and of the sulphonated products by ion‐pairing HPLC/MS with negative‐ion ESI.     

On‐line 2D‐LC‐ESI/MS/MS determination of rifaximin in rat serum

A highly sensitive and selective on‐line two‐dimensional reversed‐phase liquid chromatography/electrospray ionization–tandem mass spectrometry (2D‐LC‐ESI/MS/MS) method was developed and validated to determine rifaximin in rat serum by direct injection. The 2D‐LC‐ESI/MS/MS system consisted of a restricted access media column for trapping proteins as the first dimension and a Waters C₁₈ column as second dimension using 0.1% aqueous acetic acid:acetonitrile as mobile phase in a gradient elution mode. Rifampacin was used as an internal standard. The linear dynamic range was 0.5–10 ng/mL (r² > 0.998). Acceptable precision and accuracy were obtained over the calibration range. The assay was successfully used in analysis of rat serum to support pharmacokinetic studies. Copyright © 2009 John Wiley & Sons, Ltd.     

Phosphoryl group differentiating α‐amino acids from β‐ and γ‐amino acids in prebiotic peptide formation

In recent years β‐amino acids have increased their importance enormously in defining secondary structures of β‐peptides. Interest in β‐amino acids raises the question: Why and how did nature choose α‐amino acids for the central role in life? In this article we present experimental results of MS and ³¹P NMR methods on the chemical behavior of N‐phosphorylated α‐alanine, β‐alanine, and γ‐amino butyric acid in different solvents. N‐Phosphoryl α‐alanine can self‐assemble to N‐phosphopeptides either in water or in organic solvents, while no assembly was observed for β‐ or γ‐amino acids. An intramolecular carboxylic–phosphoric mixed anhydride (IMCPA) is the key structure responsible for their chemical behaviors. Relative energies and solvent effects of three isomers of IMCPA derived from α‐alanine (2a–c), with five‐membered ring, and five isomers of IMCPA derived from β‐alanine (4a–e), with six‐membered ring, were calculated with density functional theory at the B3LYP/6‐31G** level. The lower relative energy (3.2 kcal/mol in water) of 2b and lower energy barrier for its formation (16.7 kcal/mol in water) are responsible for the peptide formation from N‐phosphoryl α‐alanine. Both experimental and theoretical studies indicate that the structural difference among α‐, β‐, and γ‐amino acids can be recognized by formation of IMCPA after N‐phosphorylation. © 2003 Wiley Periodicals, Inc. Int J Quantum Chem 94: 232–241, 2003     

Identification of phosphoproteins and determination of phosphorylation sites by zirconium dioxide enrichment and SELDI-MS/MS

Reversible protein phosphorylation mediated by protein kinases and phosphatases is the most studied post-translational modification. Efficient characterization of phosphoproteomes is hampered by (1) low stoechiometry, (2) the dynamic nature of the phosphorylation process and (3) the difficulties of mass spectrometry to identify phosphoproteins from complex mixtures and to determine their sites of phosphorylation. Combination of the phosphopeptide enrichment method with MALDI-TOFMS, or alternatively, with HPLC-ESI-MS/MS and MS(3) analysis was shown to be a step forward for the successful application of MS in the study of protein phosphorylation. In our study we used phosphopeptide enrichment performed in a simple single-tube experiment using zirconium dioxide (ZrO(2)). A simple protein mixture containing precipitated bovine milk caseins was enzymatically digested and the mixture of tryptic fragments was analysed before and after enrichment using nanoflow HPLC-ESI-MS/MS and surface-enhanced laser desorption/ionization (SELDI)-MS/MS on QqTOF instruments to compare the efficiency of the two methods in the determination of phosphorylation sites. Both approaches confirm the high selectivity obtained by the use of batch-wise, ZrO(2)-based protocol using di-ammonium phosphate as the eluting buffer. More phosphorylation sites (five for beta-casein and three for alpha(S1)-casein) were characterized by SELDI-MS/MS than by nanoflow HPLC-ESI-MS/MS. Therefore, ZrO(2)-based phosphopeptide enrichment combined with SELDI-MS/MS is an attractive alternative to previously reported approaches for the study of protein phosphorylation in mixtures of low complexity with the advance of fast in situ peptide purification. The method was limited to successful analysis of high-abundance proteins. Only one phosphorylation site was determined for the minor casein component alpha(S2)-casein by ESI-MS/MS and none for kappa-casein. Therefore an improvement in enrichment efficiency, especially for successful phosphoproteomic applications, is needed.     

Comparative pharmacokinetic study of four major bioactive components after oral administration of Zhi‐Zi‐Hou‐Po decoction in normal and corticosterone‐induced depressive rats

A highly selective and efficient LC–MS/MS method was developed to determine the plasma concentration of magnolol, hesperidin, neohesperidin and geniposide following oral administration of Zhi‐Zi‐Hou‐Po decoction in normal and depressed rats. Plasma samples were pretreated by protein precipitation with methanol. Chromatographic separation was performed on an XTerra^® MS C₁₈ column using a gradient elution with a mobile phase composed of acetonitrile–0.1% aqueous formic acid. The proposed method was validated to be specific, accurate and precise for the analytes determination in plasma samples. The calibration curves displayed good linearity over definite concentration ranges for the analytes. The intra‐ and inter‐day precision of the proposed method at three different levels were all within <11.13% and the relative errors ranged from ?8.46 to 8.93%. The recovery of the four compounds ranged from 82.72 to 89.08% and no apparent matrix effect was observed during sample analysis. After full validation, the established method was successfully applied for comparing the pharmacokinetics of four components between normal and depressed rats. The results showed that the AUC and C_max of four analytes in depressed rats were significantly different from those in normal rats and might provide helpful information to guide the clinical use of Zhi‐Zi‐Hou‐Po to treat depression.     

Comparative analysis of salt‐responsive phosphoproteins in maize leaves using Ti4+‐IMAC enrichment and ESI‐Q‐TOF MS

Salinity is one of the most common abiotic stresses encountered by plants. Reversible protein phosphorylation is involved in plant defense processes against salinity stress. Here, we performed global phosphopeptide mapping through enrichment by our synthesized PVA‐phosphate‐Ti⁴⁺ IMAC coupled with subsequent identification by ESI‐Q‐TOF MS. A total of 104 peptide sequences containing 139 phosphorylation sites were determined from 70 phosphoproteins of the control leaves. In contrast, 124 phosphopeptides containing 143 phosphorylated sites from 92 phosphoproteins were identified in salt‐stressed maize leaves. Compared with the control, 47 proteins were phosphorylated, 25 were dephosphorylated, and 45 overlapped. Among the 72 differential phosphoproteins, 35 were known salt stress response proteins and the rest had not been reported in the literature. To dissect the differential phosphorylation, gene ontology annotations were retrieved for the differential phosphoproteins. The results revealed that cell signaling pathway members such as calmodulin and 14‐3‐3 proteins were regulated in response to 24‐h salt stress. Multiple putative salt‐responsive phosphoproteins seem to be involved in the regulation of photosynthesis‐related processes. These results may help to understand the salt‐inducible phosphorylation processes of maize leaves.     

Global quantification of phosphoproteins combining metabolic labeling and gel‐based proteomics in B. pumilus

Differential proteomics targeting the protein abundance is commonly used to follow changes in biological systems. Differences in localization and degree of post‐translational modifications of proteins including phosphorylations are of tremendous interest due to the anticipated role in molecular regulatory processes. Because of their particular low abundance in prokaryotes, identification and quantification of protein phosphorylation is traditionally performed by either comparison of spot intensities on two‐dimensional gels after differential phosphoprotein staining or gel‐free by stable isotope labeling, sequential phosphopeptide enrichment and following LC‐MS analysis. In the current work, we combined in a proof‐of‐principle experiment these techniques using ¹⁴N/¹⁵N metabolic labeling with succeeding protein separation on 2D gels. The visualization of phosphorylations on protein level by differential staining was followed by protein identification and determination of phosphorylation sites and quantification by LC‐MS/MS. This approach should avoid disadvantages of traditional workflows, in particular the limited capability of peptide‐based gel‐free methods to quantify isoforms of proteins. Comparing control and stress conditions allowed for relative quantification in protein phosphorylation in Bacillus pumilus exposed to hydrogen peroxide. Altogether, we quantified with this method 19 putatively phosphorylated proteins.     

Development of a mass spectrometric hydroxyl‐position determination method for the hydroxyindole metabolites of JWH‐018 by GC‐MS/MS

One of the many issues of designer drugs of abuse like synthetic cannabinoids (SCs) such as JWH‐018 is that details on their metabolism has yet to be fully elucidated and that multiple metabolites exist. The presence of isomeric compounds poses further challenges in their identification. Our group has previously shown the effectiveness of gas chromatography‐electron ionization‐tandem mass spectrometry (GC‐EI‐MS/MS) in the mass spectrometric differentiation of the positional isomers of the naphthoylindole‐type SC JWH‐081, and speculated that the same approach could be used for the metabolite isomers. Using JWH‐018 as a model SC, the aim of this study was to differentiate the positional isomers of its hydroxyindole metabolites by GC‐MS/MS. Standard compounds of JWH‐018 and its hydroxyindole metabolite positional isomers were first analyzed by GC‐EI‐MS in full scan mode, which was only able to differentiate the 4‐hydroxyindole isomer. Further GC‐MS/MS analysis was performed by selecting m/z 302 as the precursor ion. All four isomers produced characteristic product ions that enabled the differentiation between them. Using these ions, MRM analysis was performed on the urine of JWH‐018 administered mice and determined the hydroxyl positions to be at the 6‐position on the indole ring. GC‐EI‐MS/MS allowed for the regioisomeric differentiation of the hydroxyindole metabolite isomers of JWH‐018. Furthermore, analysis of the fragmentation patterns suggests that the present method has high potential to be extended to hydroxyindole metabolites of other naphthoylindole type SCs in identifying the position of the hydroxyl group on the indole ring. Copyright © 2016 John Wiley & Sons, Ltd.     

Identification of phosphorylation sites of equine β‐casein isoforms

Equine β‐casein is phosphorylated at variable degrees and isoforms carrying 3 to 7 phosphate groups (3P–7P) have been found in milk, but the phosphorylated amino acid residues of each isoform are not yet identified. In the present work, the different phosphorylation variants were first isolated by ion‐exchange chromatography and then hydrolysed by trypsin to generate caseinophosphopeptides (CPPs), each containing all the potential phosphorylation sites. The equine CPPs were prepared by metal oxide affinity chromatography, a method based on the affinity of phosphate groups towards titanium dioxide immobilized onto a micro‐column. This method turned out to be an efficient tool to separate the CPPs Arg¹–Lys³⁴ and Glu⁴–Lys³⁴ from non‐phosphorylated peptides. Purification was achieved by reversed‐phase high‐performance liquid chromatography (RP‐HPLC) and each CPP was hydrolyzed by endoproteinase Glu‐C. Finally, the digests were analyzed by RP‐HPLC/electrospray ionization mass spectrometry (RP‐HPLC/ESI‐MS) and identified by nano‐electrospray ionization tandem mass spectrometry (nESI‐MS/MS) to locate the phosphorylated sites of the β‐casein isoforms 4P–7P with accuracy. Thus, the isoform 4P was found to be phosphorylated on residues Ser⁹, Ser²³, Ser²⁴, and Ser²⁵. Addition of phosphate groups on Ser¹⁸, Thr¹², and Ser¹⁰ led to the formation of the isoforms 5P–7P, respectively. The results indicated that the in vivo phosphorylation of the equine β‐casein follows a sequential way and is not randomly performed. Copyright © 2010 John Wiley & Sons, Ltd.     

Determination of the chemical constituents of the different processed products of Anemarrhena asphodeloides Rhizomes by high‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry

In this work, high‐performance liquid chromatography (HPLC) coupled with a hybrid quadrupole time of‐flight mass spectrometry (Q‐TOF‐MS/MS) was used to study chemical compositions of different processed products of Rhizoma Anemarrhenae (RA). A Grace Alltima^TM C₁₈ column (250 × 4.6 mm, 5 µm) was used for separation. Mobile phase consisted of 0.1% formic acid and acetonitrile, using gradient elution. ESI‐MS data was acquired in both positive and negative mode. The experiment was established on the basis of a series of reference substances (two xanthone and seven saponins) to qualitatively identify the chemical compounds of different processed products of RA by MS analysis. There was no difference in the type of chemical constituents between different processed products of RA. A total of 25 compounds were identified, including four xanthones, 21 steroidal saponins and eight pairs of isomers. Copyright © 2015 John Wiley & Sons, Ltd.     

Development and validation of a RP‐HPLC method for stability‐indicating assay of gemifloxacin mesylate including identification of related substances by LC‐ESI‐MS/MS, 1H and 13C NMR spectroscopy

A validated stability indicating RP‐HPLC assay of gemifloxacin mesylate was developed by separating its related substances on an Inertsil‐ODS3V‐C₁₈ (4.6 × 250 mm; 5 μm) column using 0.1% trifluoroaceticacid (pH 2.5) and methanol as a mobile phase in a gradient elution mode at a flow rate of 1.0 mL/min at 27°C. The column effluents were monitored by a photodiode array detector set at 287 nm. The method was validated in terms of accuracy, precision and linearity as per ICH guidelines. Forced degradation of gemifloxacin (GFX) was carried out under acidic, basic, thermal, photolysis and peroxide conditions and the degradation products were separated and characterized by ESI‐MS/MS, ¹H and ¹³C NMR spectroscopy. The method was successfully applied to the analysis of bulk drugs and the recoveries of gemifloxacin and impurities were in the range of 97.60–102.90 and 96.99–102.10%, respectively. No previous reports were found in the literature on identification of degradation products of gemifloxacin. Copyright © 2011 John Wiley & Sons, Ltd.     

Validation of a liquid chromatography‐triple quadrupole mass spectrometric method for the determination of 5‐nitro‐5′‐hydroxy‐indirubin‐3′‐oxime (AGM‐130) in human plasma and its application to microdose clinical trial

A liquid chromatography–triple quadrupole mass spectrometric (LC‐MS/MS) method was developed and validated for the determination of 5‐nitro‐5′‐hydroxy‐indirubin‐3′‐oxime (AGM‐130) in human plasma to support a microdose clinical trial. The method consisted of a liquid–liquid extraction for sample preparation and LC‐MS/MS analysis in the positive ion mode using TurboIonSpray^TM for analysis. d₃‐AGM‐130 was used as the internal standard. A linear regression (weighted 1/concentration) was used to fit calibration curves over the concentration range of 10–2000 pg/mL for AGM‐130. There were no endogenous interference components in the blank human plasma tested. The accuracy at the lower limit of quantitation was 96.6% with a precision (coefficient of variation, CV) of 4.4%. For quality control samples at 30, 160 and 1600 pg/mL, the between run CV was ≤5.0 %. Between‐run accuracy ranged from 98.1 to 101.0%. AGM‐130 was stable in 50% acetonitrile for 168 h at 4°C and 6 h at room temperature. AGM‐130 was also stable in human plasma at room temperature for 6 h and through three freeze–thaw cycles. The variability of selected samples for the incurred sample reanalysis was ≤12.7% when compared with the original sample concentrations. This validated LC‐MS/MS method for determination of AGM‐130 was used to support a phase 0 microdose clinical trial. Copyright © 2015 John Wiley & Sons, Ltd.     

A UPLC–MS/MS approach for simultaneous determination of eight flavonoids in rat plasma,and its application to pharmacokinetic studies of Fu‐Zhu‐Jiang‐Tang tablet in rats

The purpose of this study is to establish and validate a UPLC–MS/MS approach to determine eight flavonoids in biological samples and apply the method to pharmacokinetic study of Fu‐Zhu‐Jiang‐Tang tablet. A Waters BEH C₁₈ UPLC column was employed with methanol/0.1% formic acid–water as mobile phases. The mass analysis was carried out in a triple quadrupole mass spectrometer using multiple reaction monitoring with negative scan mode. A one‐step protein precipitation by methanol was used to extract the analytes from blood. Eight major flavonoids were selected as markers. Our results showed that calibration curves for 3′‐hydroxypuerarin, mirificin, puerarin, 3′‐methoxypuerarin, daidzin, rutin, astragalin and daidzein displayed good linear regression (r ² > 0.9986). The intra‐day and inter‐day precisions (RSD) of the eight flavonoids at high, medium and low levels were <8.03% and the bias of the accuracies ranged from −5.20 to 6.75%.The extraction recoveries of the eight flavonoids were from 91.4 to 100.5% and the matrix effects ranged from 89.8 to 103.8%. The validated approach was successfully applied to a pharmacokinetic study in Sprague–Dawley rats after oral administration of FZJT tablet. Double peaks were emerged in curves of mean plasma concentration for 3′‐methoxypuerarin, which was reported for the first time.     

UPLC‐MS/MS based diagnostics for epithelial ovarian cancer using fully sialylated C4‐binding protein

Serum levels of fully sialylated C4‐binding protein (FS‐C4BP) are remarkably elevated in patients with epithelial ovarian cancer (EOC) and can be used as a marker to distinguish ovarian clear cell carcinoma from endometrioma. This study aimed to develop a stable, robust and reliable liquid chromatography–hybrid mass spectrometry (UPLC‐MS/MS) based diagnostic method that would generalize FS‐C4BP as a clinical EOC biomarker. Glycopeptides derived from 20 μL of trypsin‐digested serum glycoprotein were analyzed via UPLC equipped with an electrospray ionization time‐of‐flight mass spectrometer. This UPLC‐MS/MS‐based diagnostic method was optimized for FS‐C4BP and validated using sera from 119 patients with EOC and 127 women without cancer. A1958 (C4BP peptide with two fully sialylated biantennary glycans) was selected as a marker of FS‐C4BP because its level in serum was highest among FS‐C4BP family members. Preparation and UPLC‐MS/MS were optimized for A1958, and performance and robustness were significantly improved relative to our previous method. An area under the curve analysis of the FS‐C4BP index receiver operating characteristic curve revealed that the ratio between A1958 and A1813 (C4BP peptide with two partially sialylated biantennary glycans) reached 85%. A combination of the FS‐C4BP index and carbohydrate antigen‐125 levels further enhanced the sensitivity and specificity.     

A rapid and sensitive LC‐MS/MS method for evaluation of the absolute oral bioavailability of a novel c‐Met tyrosine kinase inhibitor QBH‐196 in rats

A sensitive, selective and high‐throughput UPLC‐MS/MS method was developed and validated for the determination of a novel c‐Met tyrosine kinase inhibitor, QBH‐196, in rat plasma. QBH‐196 and its analog BH357 (IS) were extracted from rat plasma using a mixture of dichloromethane and N‐hexane (2:3, v/v). The chromatographic separation was carried out on Phenomenex C₁₈ column (50 × 2.1 mm, 2.6 µm particle size) with a gradient mobile phase of methanol (A) and water containing 0.05% formic acid (B) at a flow rate of 0.2 mL/min. The assay was performed by positive electrospray ionization in multiple reaction monitoring mode using transitions of m/z 622.68 → 140.41 for QBH‐196 and m/z 591.19 →126.21 for the IS, respectively. Good linearity was obtained over the concentration range of 8.0–4000 ng/mL (r² > 0.99) for QBH‐196 and the lower limit of quantification was 8.0 ng/mL in rat plasma. Validations of the method, including its sensitivity, extraction recovery, matrix effect, intra‐ and inter‐day precision, accuracy and stability, were all within acceptable limits. The established method was successfully applied to determine absolute oral bioavailability of QBH‐196 in rats for the first time. The mean oral absolute bioavailability of QBH‐196 was found to be about 40.8% and the elimination half‐life was 40.0 ± 13.1 h. This result suggested that QBH‐196 exhibits good oral absorption in vivo, which is very important for the further development of QBH‐196 as a new oral anticancer drug. Copyright © 2015 John Wiley & Sons, Ltd.