Journal of Chromatography B (v.981-982, #C)

An accelerated solvent extraction (ASE)-solid-phase extraction (SPE)-liquid chromatography with electrospray ionization-tandem mass spectrometry (ASE-SPE-LC–ESI-MS/MS) methodology was developed for the extraction, cleanup and quantification of ultra-trace organic acids in Masson pine (Pinus massoniana L.) tissues. The separation was carried out on a Bio-Rad Aminex HPX-87H sulfonic column with an eluent containing 5 mmol L−1 H2SO4 at a flow rate of 0.5 mL min−1. A linear ion trap mass spectrometer equipped with electrospray ionization (ESI) source was operated in negative ion mode, and the six organic acids were eluted within 20 min. ASE extraction, SPE cleanup and LC–ESI-MS/MS analysis conditions were optimized to obtain reliable information about plant organic acid composition. Selective reaction monitoring (SRM) was employed for quantitative measurement. Intra-day precisions averaged 6.7%, and inter-day precisions were 2.1–10.7% for organic acid measurements in the pine samples. External standard calibration curves were linear over the range of 16.5–5000 ng L−1, and detection limits based on a signal-to-noise ratio of three were at 0.5–5.0 ng L−1. The results obtained showed the sensibility of the method was better than that of previously described HPLC methodology, and had no significant matrix effect. The proposed ASE-SPE-LC–ESI-MS/MS method is sensitive and reliable for the determination of ultra-trace organic acids in plant samples, despite the presence of the particularly complex matrix.
Keywords: Organic acids; Accelerated solvent extraction; Liquid chromatography–tandem mass spectrometry; Masson pine (Pinus massoniana L.);

Characterization of antibody drug conjugate positional isomers at cysteine residues by peptide mapping LC–MS analysis by Marie-Claire Janin-Bussat; Marina Dillenbourg; Nathalie Corvaia; Alain Beck; Christine Klinguer-Hamour (9-13).
Antibody-drug conjugates (ADCs) are becoming a major class of oncology therapeutics. Because ADCs combine the monoclonal antibody specificity with the high toxicity of a drug, they can selectively kill tumor cells while minimizing toxicity to normal cells. Most of the current ADCs in clinical trials are controlled, but heterogeneous mixtures of isomers and isoforms. Very few protocols on ADC characterization at the peptide level have been published to date. Here, we report on the improvement of an ADC peptide mapping protocol to characterize the drug-loaded peptides by LC–MS analysis. These methods were developed on brentuximab vedotin (Adcetris®), a commercial ADC with an average of four drugs linked to interchain cysteine residues of its antibody component. Because of the drug hydrophobicity, all the steps of this protocol including enzymatic digestion were improved to maintain the hydrophobic drug-loaded peptides in solution, allowing their unambiguous identification by LC–MS. For the first time, the payloads positional isomers observed by RP-HPLC after IdeS-digestion and reduction of the ADC were also characterized.
Keywords: Antibody drug conjugates; Brentuximab vedotin; Drug-loaded peptide mapping; IdeS; Liquid chromatography mass spectrometry; Payload positional isomers;

Determination and pharmacokinetic study of pirfenidone in rat plasma by UPLC–MS/MS by Wei Sun; Zhe-li Jiang; Lei Zhou; Rui-min Chen; Zhe Wang; Wan-shu Li; Shuo-min Jiang; Guo-xin Hu; Rui-jie Chen (14-18).
A rapid, sensitive and selective ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) was developed and validated for the determination and pharmacokinetic investigation of pirfenidone in rat plasma. Sample preparation was accomplished through a simple one-step deproteinization procedure with 0.2 mL of acetonitrile to a 0.1 mL plasma sample. Plasma samples were separated by UPLC on an Acquity UPLC BEH C18 column using a mobile phase consisting of acetonitrile-0.1% formic acid in water with gradient elution. The total run time was 3.0 min and the elution of pirfenidone was at 1.39 min. The detection was performed on a triple quadrupole tandem mass spectrometer in the multiple reaction-monitoring (MRM) mode using the respective transitions m/z 186.2 → 92.1 for pirfenidone and m/z 237.1 → 194.2 for carbamazepine (IS), respectively. The calibration curve was linear over the range of 5–2000 ng/mL with a lower limit of quantitation (LLOQ) of 5 ng/mL. Mean recovery of pirfenidone in plasma was in the range of 80.4–84.3%. Intra-day and inter-day precision were both <12.1%. This method was successfully applied in pharmacokinetic study after oral administration of 10.0 mg/kg pirfenidone in rats.
Keywords: Pirfenidone; UPLC–MS/MS; Rat plasma; Pharmacokinetics;

Pharmacokinetics of Rac inhibitor EHop-016 in mice by ultra-performance liquid chromatography tandem mass spectrometry by Tessa Humphries-Bickley; Linette Castillo-Pichardo; Francheska Corujo-Carro; Jorge Duconge; Eliud Hernandez-O’Farrill; Cornelis Vlaar; Jose F. Rodriguez-Orengo; Luis Cubano; Suranganie Dharmawardhane (19-26).
The Rho GTPase Rac is an important regulator of cancer cell migration and invasion; processes required for metastatic progression. We previously characterized the small molecule EHop-016 as a novel Rac inhibitor in metastatic breast cancer cells and recently found that EHop-016 was effective at reducing tumor growth in nude mice at 25 mg/kg bodyweight (BW). The purpose of this study was to compare the pharmacokinetics and bioavailability of EHop-016 at different dosages in a single dose input scheme (10, 20 and 40 mg/kg BW) following intraperitoneal (IP) and oral gavage (PO) administration to nude mice. We developed and validated a rapid and sensitive method for the quantitation of EHop-016 in mouse plasma by ultra high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (UPLC/MS/MS). Separation was carried out on an Agilent Poroshell 120 EC-C18 column (3.0 mm × 50 mm) using organic and aqueous mobile phases. EHop-016 was identified from its accurate mass and retention time from the acquired full-scan chromatogram and quantified by its peak area. The validated method was linear (R 2  > 0.995) over the range of 5–1000 ng/mL (1/x 2 weighting). Pharmacokinetic parameters were obtained by non-compartmental analysis using WinNonlin®. The area under the curve (AUC0– ) ranged from 328 to 1869 ng h/mL and 133–487 ng h/mL for IP and PO dosing, respectively. The elimination half-life (t 1/2) ranged from 3.8–5.7 h to 3.4–26.8 h for IP and PO dosing, respectively. For both IP and PO administration, the AUC0– values were proportional to the tested doses demonstrating linear PK profiles. The relative bioavailability of EHop-016 after oral gavage administration ranged from 26% to 40%. These results support further preclinical evaluation of EHop-016 as a new anti-cancer therapy.
Keywords: EHop-016; Pharmacokinetics; UPLC/MS/MS; Rac; Cancer;

An efficient method was established by HSCCC combined with prep-HPLC for separating isochlorogenic acid isomers from flower buds of Lonicera japonica. The partially purified sample from the methanol extract of flower buds of L. japonica by silica gel column was separated by HSCCC to result in a fraction containing two isochlorogenic acid isomers. The fraction was further isolated by prep-HPLC to yield isochlorogenic acid A and isochlorogenic acid C with purities of 98% and 96%, and the total recoveries at 80.1% and 79.8%, respectively. The chemical structures of isochlorogenic acid isomers were confirmed by electrospray ionization mass spectrometry (ESI-MS) and 1H nuclear magnetic resonance (NMR).
Keywords: Isochlorogenic acid A; Isochlorogenic acid C; Flower buds of Lonicera japonica; High-speed counter-current chromatography; Preparative high performance liquid chromatography;

Proteomic analysis of individual fruit fly hemolymph by Qi Zeng; David J. Smith; Scott A. Shippy (33-39).
Analysis of blood proteins holds critical promise for in depth understanding of physiological states. Protein content of hemolymph from Drosophila melanogaster is of particular analytical interest because the insect open circulatory system involves chemical signaling through the hemolymph. The challenge of working with this sample, however, is the nanoliter volumes of solution available for analysis. In this study, we developed a novel hyphenated Agilent nano-HPLC chip column-MS method to obtain proteomic information from individual fruit fly hemolymph, using a low-volume sample collection technique established previously. The total amount of individual Drosophila hemolymph protein is determined around 0.798 ± 0.251 μg/100 nL based upon a Bradford assay with BSA. Hemolymph samples around 50 nL were collected from single flies and digested using a customized micro-scale digestion protocol. Mass spectral analysis shows a total of 19 proteins were identified from the hemolymph of individual flies. Of these findings, 6 novel proteins have been identified for the first time with evidence at the translation level. Detection of 13 proteins well-known in the literature speaks to the method's validity and demonstrates the ability to reproducibly analyze volume-limited samples from individual fruit flies for protein content. This nano-scale analysis method will facilitate future study of Drosophila and lead to a more complete understanding of the physiology of the fly model system.
Keywords: Drosophila melanogaster; Hemolymph; Nanoliter samples; Mass spectrometry; On-chip liquid chromatography;

Determination of tapentadol and tapentadol-O-glucuronide in human serum samples by UPLC–MS/MS by Vera Hillewaert; Klaus Pusecker; Luc Sips; Tom Verhaeghe; Ronald de Vries; Manfred Langhans; Rolf Terlinden; Philip Timmerman (40-47).
Tapentadol is a novel, centrally acting analgesic with 2 mechanisms of action, MOR agonism and noradrenaline (NA) reuptake inhibition in a single molecule. It is the first member of a new therapeutic class, MOR-NRI. A high throughput liquid chromatography-tandem mass spectrometric (LC–MS/MS) assay was developed and validated for the quantitative analysis of tapentadol and its O-glucuronide metabolite in human serum. Simultaneous quantification was deemed to be challenging because of the large difference in concentrations between tapentadol and its O-glucuronide metabolite in clinical samples. Therefore, a method was established using a common processed sample, but with different injection volumes and chromatographic conditions for each analyte. Tapentadol and tapentadol-O-glucuronide were determined by protein precipitation of 0.100 ml of the samples with acetonitrile. The internal standards used are D6-tapentadol and D6-tapentadol-O-glucuronide. The validated concentration range was 0.200–200 ng/ml (tapentadol) and 10.0–10,000 ng/ml (tapentadol-O-glucuronide). Chromatographic separation was achieved by gradient elution on a Waters Acquity UPLC BEH C18 (1.7 μm, 2.1 × 50 mm) column, with mobile phase consisting of 0.01 M ammonium formate (adjusted to pH 4 using formic acid) (A) and methanol (B). A separate injection was done for measurement of each analyte, with a different gradient and run time. The analytes were detected by using an electrospray ion source on a triple quadrupole mass spectrometer operating in positive ionization mode. The run time was 1.6 min for tapentadol and 1.5 min for tapentadol-O-glucuronide. The high sensitivity and acceptable performance of the assay allowed its application to the analysis of serum samples in clinical trials. The validated method was used for analysis of tapentadol in over 17,000 samples.
Keywords: Tapentadol; Serum; Mass spectrometry; LC–MS/MS; UPLC; Assay validation;

Establishment of a three-step purification scheme for a recombinant protein rG17PE38 and its characteristics identification by Xiao-Li Feng; Zeng-Shan Liu; Xi-Lin Liu; Shi-Ying Lu; Yan-Song Li; Pan Hu; Dong-Ming Yan; Wei-Hua Tong; Quan Wang; Yu Zhou; Wen Jin; Yan-Xia Ding; Dong-Xue Gai; Hong-Lin Ren (48-56).
A protein with high purity has become an essential pre-requisite for investigating its bioactivity, molecular structure and characteristics. Therefore, the development of technologies for efficient purification of protein is urgently necessary. The objective of this study was to establish a purification protocol for a recombinant protein rG17PE38. Different forms of chromatography such as hydrophobic interaction and ion exchange chromatography were chosen as the core purification steps. The performance of each technique was optimized to meet the requirements and the purification steps were arranged in a logical way of facilitating to operate in next step. In addition, some characteristics of the protein such as stability, bioactivity and cellular location were determined. Finally, whether the protein could induce cell apoptosis was also explored. The results showed the protein purified via the suggested three-step purification scheme could obtain a purity of 95%, and its bioactivity in the form of IC50 was 17.6 ng/mL, furthermore it could keep stable at 4 °C for at least 10 days. The protein could bind on its target cell membrane specifically, and inducing cell apoptosis was demonstrated to be one of the cytotoxicity mechanisms of the protein. Results obtained in our study may provide useful information on strategies of protein purification and lay a substantial foundation for the followed animal or clinical experiments on rG17PE38.
Keywords: Recombinant protein; Purification; Chromatography; Apoptosis;

Simultaneous determination of albumin and low-molecular-mass thiols in plasma by HPLC with UV detection by Kamila Borowczyk; Monika Wyszczelska-Rokiel; Paweł Kubalczyk; Rafał Głowacki (57-64).
In this paper, we describe a simple and robust HPLC based method for determination of total low- and high-molecular-mass thiols, protein S-linked thiols and reduced albumin in plasma. The method is based on derivatization of analytes with 2-chloro-1-methylquinolinium tetrafluoroborate, separation and quantification by reversed-phase liquid chromatography followed by UV detection. Disulfides were converted to their thiol counterparts by reductive cleavage with tris(2-carboxyethyl)phosphine. Linearity in detector response for total thiols was observed over the range of 1–40 μmol L−1 for Hcy and glutathione (GSH), 5–100 μmol L−1 for Cys–Gly, 20–300 μmol L−1 for Cys and 3.1–37.5 μmol L−1 (0.2–2.4 g L−1) for human serum albumin (HSA). For the protein S-bound forms these values were as follows: 0.5–30 μmol L−1 for Hcy and GSH, 2.5–60 μmol L−1 for Cys–Gly and 5–200 μmol L−1 for Cys. The LOQs for total HSA, Cys, Hcy, Cys–Gly and GSH were 0.5, 0.2, 0.4, 0.3 and 0.4 μmol L−1, respectively. The estimated validation parameters for all analytes are more than sufficient to allow the analytical method to be used for monitoring of the total and protein bound thiols as well as redox status of HSA in plasma.
Keywords: Albumin; Endogenic plasma thiols; High performance liquid chromatography; Derivatization; Ultraviolet detection;