Journal of Chromatography B (v.1008, #C)

Determination of mesalazine, a low bioavailability olsalazine metabolite in human plasma by UHPLC–MS/MS: Application to a pharmacokinetic study by Jagadeesh Banda; Ramalingam Lakshmanan; Ramesh Babu Katepalli; Uday Kumar Reddy Venati; Ramesh Koppula; V.V.S. Shiva Prasad (1-10).
Olsalazine sodium, salicylate derivative (prodrug) is effectively bioconverted to mesalazine (5-aminosalicylic acid; 5-ASA), which has an anti-inflammatory activity in ulcerative colitis. In this article, a novel highly sensitive and selective method was developed and validated to determine mesalazine in human plasma using a derivatization technique to enhance the signal intensity by using ultra- high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC–MS/MS) with an electrospray ionization interface. The sample preparation consisted of a derivatization with propionyl anhydride followed by liquid liquid extraction (LLE) to remove the interference and minimize the matrix effect of human plasma. The multiple reaction monitoring (MRM) mode of the negative ion was performed and the transitions of m/z 208.1 → 107.0 and m/z 211.1 → 110.1 were used to measure the derivative of mesalazine and mesalazine-d3 . The chromatographic separation was achieved using kinetex XB-C18 (100 × 4.6 mm 2.6 μ) analytical column with 0.1% formic acid in water and acetonitrile as mobile phase with a gradient elution. Nominal retention times of mesalazine and IS were 3.08 and 3.07 min, respectively. Absolute recovery was found to be between 82–95% for analyte and about 78% for IS. The standard curves was linear (r 2  > 0.995) in the concentration range 0.10 to 12.0 ng/mL with lower limit of quantification (LLOQ) in human plasma was 0.10 ng/mL. The average intra-day/inter-day precision values (%CV) were in the range from 0.6–2.9 % and 1.3–3.8 %, respectively, while the average accuracy value was 103.8–107.2%. This method has been successfully applied to the human pharmacokinetics of olsalazine sodium 250 mg capsules following single oral administration.
Keywords: Mesalazine; Derivatization; Human plasma; Method validation; LC–MS/MS; Pharmacokinetics;

Purification of glycocalicin from human plasma by Basma HadjKacem; Héla Mkaouar; Ikram Ben Amor; Jalel Gargouri; Ali Gargouri (11-14).
Glycocalicin (GC) is a large extracellular proteolytic fragment of glycoprotein Ib, a membrane platelet component playing an essential role in the physiological processes of platelet adhesion and aggregation. GC contains the binding sites for thrombin and von Willebrand factor. GC circulates normally in vivo in significant concentrations and the plasma level of this protein reflects a complex function of factors including platelet count or platelet turnover. It can therefore serve as a good indicator for many diseases like hypoplastic thrombocytopenia and idiopathic thrombocytopenic purpura. For this reason, several purification assays have been previously described. In this work, we describe a novel analytical method for GC purification from human platelets based on preparative HPLC gel filtration followed by immuno-affinity chromatography on NHS activated column conjugated with specific antibody. Pure GC was obtained from tiny amount of starting material. Our protocol of GC purification is simple, fast and provides a pure end product.
Keywords: Glycocalicin purification; HPLC gel filtration; Affinity chromatography; NHS column; Blood purification;

Protocol for quality control in metabolic profiling of biological fluids by U(H)PLC-MS by Helen G. Gika; Chrysostomi Zisi; Georgios Theodoridis; Ian D. Wilson (15-25).
The process of untargeted metabolic profiling/phenotyping of complex biological matrices, i.e., biological fluids such as blood plasma/serum, saliva, bile, and tissue extracts, provides the analyst with a wide range of challenges. Not the least of these challenges is demonstrating that the acquired data are of “good” quality and provide the basis for more detailed multivariate, and other, statistical analysis necessary to detect, and identify, potential biomarkers that might provide insight into the process under study. Here straightforward and pragmatic “quality control (QC)” procedures are described that allow investigators to monitor the analytical processes employed for global, untargeted, metabolic profiling. The use of this methodology is illustrated with an example from the analysis of human urine where an excel spreadsheet of the preprocessed LC–MS output is provided with embedded macros, calculations and visualization plots that can be used to explore the data. Whilst the use of these procedures is exemplified on human urine samples, this protocol is generally applicable to metabonomic/metabolomic profiling of biofluids, tissue and cell extracts from many sources.
Keywords: Quality control; Metabolomics; Metabonomics; Metabolic profiling; Biological samples; Protocols;

A simple and sensitive method for simultaneous derivatization and hollow fiber liquid phase microextraction (HF-LPME) followed by high performance liquid chromatography–fluorescence detection (HPLC–FL) to determine memantine hydrochloride (MT) in human plasma was developed. The derivatization and microextraction was combined to a single step to ensure the precision. What is more, the derivatization reaction accelerated the mass transfer during the process of microextraction. The hollow fiber was filled with cyclohexane and dansyl chloride (derivatization agent) as acceptor phase and submersed in the alkalinized plasma sample. The system was submitted to stirring at 800 rpm for 50 min at 40 °C. Different experimental parameters were systematically evaluated by response surface methodology. Under the optimized conditions, the calibration curve was linear in the range of 1–100 ng/mL (r  = 0.9991) with a limit of detection of 0.1 ng/mL (S/N  = 3). The precision estimated as the relative standard deviation (RSD) was less than 4.5% and the accuracy was 94.3–100.7%. The present method was successfully applied to determine MT in human plasma samples.
Keywords: Hollow fiber liquid phase microextraction; Simultaneous derivatization; Memantine hydrochloride;

We have developed a sensitive and specific LC–MS/MS method for the simultaneous determination of alisol A (A), alisol A 23-acetate (A23) and alisol A 24-acetate (A24), the major active components in Rhizoma Alismatis extract (RAE), in rat plasma. In brief, plasma samples were extracted by methyl tert-butyl ether and chromatographically separated by using a C18 column. A tandem mass spectrometric detection with an electrospray ionization (ESI) interface was conducted via multiple reaction monitoring (MRM) under positive ionization mode. This method was validated for specificity, linearity, accuracy (within ±15.4%), intra- and inter-day precision (CV < 11.4%) over the concentration range of 25–5000 ng/mL for A, and 5–1000 ng/mL for both A23 and A24. The significantly lower detection limit was determined as 25 ng/mL for A, 5 ng/mL for A23 and A24. This validated method of ours was then used to study the pharmacokinetics of RAE in rat. The elimination half-lives (t 1/2) of A, A23 and A24 was determined as 0.75, 0.83 and 0.82 h respectively after intravenous injection, and the oral absolute bioavailability of A, A23 and A24 was 43.1 ± 18.1%, 6.3 ± 1.5% and 7.9 ± 1.2%. This new determination method of us for alisols is proven to very useful to study the pharmacological activities of RAE in future.
Keywords: Alisol A; Alisol A 23-acetate; Alisol A 24-acetate; LC–MS/MS; Rhizoma Alismatis extract; Pharmacokinetics;

Purification and characterization of oligonucleotide binding (OB)-fold protein from medicinal plant Tinospora cordifolia by Mohd Amir; Md. Anzarul Haque; Wahiduzzaman; Mohammad Aasif Dar; Asimul Islam; Faizan Ahmad; Md. Imtaiyaz Hassan (38-44).
The oligonucleotide binding fold (OB-fold) is a small structural motif present in many proteins. It is originally named for its oligonucleotide or oligosaccharide binding properties. These proteins have been identified as essential for replication, recombination and repair of DNA. We have successfully purified a protein contains OB-fold from the stem of Tinospora cordifolia, a medicinal plants of north India. Stems were crushed and centrifuged, and fraction obtained at 60% ammonium sulphate was extensively dialyzed and applied to the weak anion exchange chromatography on Hi-Trap DEAE-FF in 50 mM Tris–HCl buffer at pH 8.0. Eluted fractions were concentrated and applied to gel filtration column to get pure protein. We observed a single band of 20-kDa on SDS-PAGE. Finally, the protein was identified as OB-fold by MALDI-TOF. The purified OB-fold protein was characterized for its secondary structural elements using circular dichroism (CD) in the far-UV region. Generally the OB-fold has a characteristic feature as five-stranded beta-sheet coiled to form a closed beta- barrel. To estimate its chemical stability, guanidinium chloride-induced denaturation curve was followed by observing changes in the far-UV CD as a function of the denaturant concentration. Analysis of this denaturation curve gave values of 8.90 ± 0.25 kcal mol−1 and 3.78 ± 0.18 M for ΔG D° (Gibbs free energy change at 25 °C) and C m (midpoint of denaturation), respectively. To determine heat stability parameters of OB-fold protein, differential scanning calorimetry was performed. Calorimetric values of ΔG D°, T m (midpoint of denaturation), ΔH m (enthalpy change at T m), and ΔC p (constant-pressure heat capacity change) are 9.05 ± 0.27 kcal mol−1, 85.2 ± 0,3 °C, 105 ± 4 kcal mol−1 and 1.6 ± 0.08 kcal mol−1  K−1. This is the first report on the isolation, purification and characterization of OB-fold protein from a medicinal plant T. cordifolia.
Keywords: T. cordifolia; Ion-exchange chromatography; Oligonucleotide binding fold; Circular dichroism; Chemical denaturation; Differential scanning calorimetry;

The ultrasonic assisted temperature-switch ionic liquid microextraction (UATS-ILME) has been successfully applied in extracting of seven lignans from Schisandra. 1-Butyl-3-methylimidazolium tetrafluoroborate ([C4MIM][BF4]) aqueous solution was selected for extracting the target analytes in raw material at 80 °C. The lignans were deposited into a single drop by in situ forming 1-butyl-3-methylimidazolium hexafluorophosphate ([C4MIM][PF6]) by cooling down to 0 °C and centrifuging for 10 min. The extracts were analyzed by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) in a robust multiple-reaction monitoring (MRM) mode in five minutes. Meanwhile, the proposed method was validated and successfully applied to the determination of seven lignans in twelve Schisandra species. The results indicated that UATS-ILME combined with UPLC-MS/MS is a powerful and practical tool, which has great potential for comprehensive quality control of herbal medicines.
Keywords: Ionic liquid microextractoin; Ultrasonic assisted extraction; Liquid chromatography tandem mass spectrometry; Schisandra;

Myrtus communis L. is an aromatic evergreen shrub and its essential oil possesses known powerful antimicrobial activity. However, the contribution of each component of the plant essential oil in observed antimicrobial ability is unclear. In this study, chemical components of the essential oil samples of the plant were identified qualitatively and quantitatively using GC/FID/Mass spectrometry system, antimicrobial activity of these samples against three microbial strains were evaluated and, these two set of data were correlated using chemometrics methods. Three chemometric methods including principal component regression (PCR), partial least squares (PLS) and orthogonal projections to latent structures (OPLS) were applied for the study. These methods showed similar results, but, OPLS was selected as preferred method due to its predictive and interpretational ability, facility, repeatability and low time-consuming. The results showed that α-pinene, 1,8 cineole, β-pinene and limonene are the highest contributors in antimicrobial properties of M. communis essential oil. Other researches have reported high antimicrobial activities for the plant essential oils rich in these compounds confirming our findings.
Keywords: Myrtus communis L.; Antimicrobial activity; Essential oil; GC/FID technique; GC/Mass spectrometry; Orthogonal projections to latent structures (OPLS);

Enrichment and separation of chlorogenic acid from the extract of Eupatorium adenophorum Spreng by macroporous resin by Boyan Liu; Beitao Dong; Xiaofan Yuan; Qirong Kuang; Qingsheng Zhao; Mei Yang; Jie Liu; Bing Zhao (58-64).
A simple and efficient chromatographic method for separation of chlorogenic acid from Eupatorium adenophorum Spreng extract was developed. The adsorption properties of nine macroporous resins were evaluated. NKA-II resin showed much better adsorption/desorption properties. The adsorption of chlorogenic acid on NKA-II resin at 25 °C was well fitted to Langmuir isotherm model and pseudo-second-order kinetic model. The dynamic adsorption and desorption experiments were carried out on columns packed with NKA-II resin to optimize the separation process. The content of chlorogenic acid in the product increased to 22.17%, with a recovery yield of 82.41%.
Keywords: NKA-II macroporous resin; Chlorogenic acid; Eupatorium adenophorum Spreng; Separation;

A rapid, specific and sensitive liquid chromatography–tandem mass spectrometric (LC–MS/MS) method was developed and validated for the simultaneous determination of strychnine, brucine, strychnine N-oxide and brucine N-oxide in rat plasma. Plasma samples were pretreated via simple protein precipitation with methanol and ephedrine hydrochloride was used as internal standard. Chromatographic separation was carried out on an ZORBAX Eclipse XDB-C18 column (2.1 × 150 mm, 3.5 μm) by gradient elution with methanol and 10 mM ammonium acetate (adjusted to pH 4.0 with formic acid). The quantification of the analytes was performed by mass spectrometry with TurboIonSpray ionization (ESI) inlet in the positive ion multiple reaction monitoring (MRM) mode. The results showed that the calibration curve was linear in the concentration range of 0.510∼306.3 ng mL−1 for strychnine, brucine and 0.102∼306.0 ng mL−1 for strychnine N-oxide and brucine N-oxide, respectively. The intra- and inter-day precisions were less than 14.9%, and the accuracy ranged from 89.4 to 113% at three QC levels for the 4 analytes. The validated method was successfully applied to the pharmacokinetic study of strychnine, brucine, strychnine N-oxide and brucine N-oxide in rat plasma after oral administration of each monomer and the total alkaloids from Semen Strychni. After single oral administration of the total alkaloids from Semen Strychni at 4 dose levels, C max, AUC0−t of strychnine and brucine increased and were proportional to the oral doses. In comparative pharmacokinetics studies, no significant difference was found between each monomer and the total strychnos alkaloids on the pharmacokinetic parameters such as C max and AUC. Mean C max and AUC of strychnine and brucine were slight increased in the monomer groups in comparison to the total strychnos alkaloids groups, which suggested that some other alkaloids in the Semen Strychni may decrease the absorption of strychnine and brucine in body.
Keywords: Strychnine; Brucine; Strychnine N-oxide; Brucine N-oxide; LC–MS/MS; Pharmacokinetics; The total strychnos alkaloids; Monomer; Rat plasma;

Simultaneous detection and comparative pharmacokinetics of amoxicillin, clavulanic acid and prednisolone in cows’ milk by UPLC–MS/MS by Yuan Liu; Kui Zhu; Jianfen Wang; Xiaoyong Huang; Guanlin Wang; Congying Li; Jie Cao; Shuangyang Ding (74-80).
Amoxicillin (AMOX), clavulanic acid (CLAV) and prednisolone (PSL) are widely used in combination for the treatment of mastitis in lactating dairy cows. However, no method has been reported to detect these three chemicals in milk in a single assay. In the present work, a reliable and sensitive UPLC–MS/MS method was developed and validated for simultaneous determination of AMOX, CLAV and PSL in cow’s milk. The analytes were determined by a positive and negative ionization electrospray mass spectrometer via multiple reaction monitoring. The linear ranges of AMOX, CLAV and PSL were from 2 to 1000 ng/mL, 20–1000 ng/mL and 1–1000 ng/mL, respectively, with the correlation coefficients greater than 0.999. The limits of quantification (LOQs) were 2 ng/mL (AMOX), 20 ng/mL (CLAV) and 1 ng/mL (PSL). Recoveries of the analytes of interest in milk samples were in the ranges of 84.2–101.4%. The intra-day and inter-day precisions ranged from 1.8% to 11.9%. This method was successfully applied to investigate the pharmacokinetics of AMOX, CLAV and PSL in milk from healthy and mastitic cows. The elimination times of AMOX and PSL in mastitic cows were longer than that in healthy cows, but the elimination times of CLAV did not show significant difference.
Keywords: Amoxicillin; Clavulanic acid; Prednisolone; UPLC–MS/MS; Comparative pharmacokinetics;

A rapid extraction method was developed and validated for levo-tetrahydropalmatine (l-THP) determination in rabbit plasma by liquid chromatography tandem–mass spectrometry (LC–MS/MS). The sample preparation included a single-step acetonitrile extraction and salting out liquid–liquid partitioning from the water in plasma with MgSO4. Berberine was used as internal standard. The mass spectrometry source was negative electrospray ionization. The method showed good performance in the concentration range from 5 to 200 ng mL−1. The limit of quantification (LOQ) was 1 ng mL−1. The method was successfully applied to a pharmacokinetic study in rabbit comparing the two drug formulation of l-THP including the raw material and the self-microemulsifying drug delivery system pellet.
Keywords: Levo-tetrahydropalmatine; l-THP; Liquid chromatography; –tandem mass spectrometry; LC–MS/MS; Self-microemulsifying drug delivery system; Pharmacokinetic study;

The DNA sequence specificity of the cancer chemotherapeutic agent, bleomycin, was investigated in two human mitochondrial DNA sequences. Bleomycin was found to cleave preferentially at 5′-TGT*A-3′ DNA sequences (where * is the cleavage site). The bleomycin analysis using capillary electrophoresis with laser-induced fluorescence was determined on both DNA strands and each strand was independently fluorescently labelled at the 3′- and 5′-ends. There was a high level of correlation between the intensity of bleomycin cleavage sites analysed by 3′- and 5′-end labelling. This is the first occasion that a comprehensive comparison has been made between these two end-labelling procedures to quantify cleavage by a DNA damaging agent and to investigate end-label bias. A comparison was also made between the bleomycin DNA sequence specificity obtained from genome-wide next-generation sequencing with that obtained from purified plasmid DNA sequences. This was accomplished by cloning sections of human mitochondrial DNA and comparing these identical mitochondrial DNA in the human mitochondrial genome. At individual sites, there was a very low level of correlation between bleomycin cleavage in plasmid sequencing and genome-wide sequencing. However, the overall bleomycin DNA sequence specificity was very similar in the two environments, namely 5′-TGT*A-3′.
Keywords: Anti-tumour drug; DNA sequence specificity; End-labelling at 5′- and 3′-ends; Fluorescent labelling; Genome-wide DNA sequencing; Illumina DNA sequencing; Human mitochondrial DNA;

Label-free assay based on immobilized capillary enzyme reactor of Leishmania infantum nucleoside triphosphate diphosphohydrolase (LicNTPDase-2-ICER-LC/UV) by Luana Magalhães; Arthur Henrique Cavalcante de Oliveira; Raphael de Souza Vasconcellos; Christiane Mariotini-Moura; Rafaela de Cássia Firmino; Juliana Lopes Rangel Fietto; Carmen Lúcia Cardoso (98-107).
Nucleoside triphosphate diphosphohydrolase (NTPDase) is an enzyme belonging to the apyrase family that participates in the hydrolysis of the nucleosides di- and triphosphate to the corresponding nucleoside monophosphate. This enzyme underlies the virulence of parasites such as Leishmania. Recently, an NTPDase from Leishmania infantum (LicNTPDase-2) was cloned and expressed and has been considered as a new drug target for the treatment of leishmaniasis. With the intent of developing label-free online screening methodologies, LicNTPDase-2 was covalently immobilized onto a fused silica capillary tube in the present study to create an immobilized capillary enzyme reactor (ICER) based on LicNTPDase-2 (LicNTPDase-2-ICER). To perform the activity assays, a multidimensional chromatographic method was developed employing the LicNTPDase-2-ICER in the first dimension, and an analytical Ascentis C8 column was used in the second dimension to provide analytical separation of the substrates and products. The validated LicNTPDase-2-ICER method provided the following kinetic parameters of the immobilized enzyme: K M of 2.2 and 1.8 mmol L−1 for the ADP and ATP substrates, respectively. Suramin (1 mmol L−1) was also shown to inhibit 32.9% of the enzymatic activity. The developed method is applicable to kinetic studies and enables the recognition of the ligands. Furthermore, a comparison of the values of LicNTPDase-2-ICER with those obtained with an LC method using free enzyme in solution showed that LicNTPDase-2-ICER-LC/UV was an accurate and reproducible method that enabled automated measurements for the rapid screening of ligands.
Keywords: Nucleoside triphosphate diphosphohydrolase; NTPDase-2; Leishmania infantum; Enzyme Immobilization; Multidimensional enzymatic assay;

A sensitive and accurate liquid chromatography–tandem mass spectrometry (LC–MS/MS) method has been developed and validated for determination of tulobuterol in rat plasma for the first time. Plasma samples were extracted by liquid–liquid extraction method with methyl tert-butyl ether and the analyte and clenbuterol (IS) were separated on a Venusil MP C18 column (100 mm × 2.1 mm, 3 μm) using 0.1% formic acid–water–methanol as mobile phase, with a runtime of 5 min. The analyte was detected in multiple reaction monitoring (MRM) mode with positive electrospray ionization. Transitions of m/z 228.2 → 154.0 for tulobuterol and m/z 277.1 → 203.0 for the clenbuterol were monitored. The linear range was 0.5–100 ng/ml (r  = 0.9967) for tulobuterol with the lower limit of quantitation of 0.5 ng/ml. The intra-day and inter-day precisions were less than 10.3% for the analyte and the accuracy was less than −8.6%. The RSD of matrix effect and recovery yield were within ±15% of nominal concentrations and tulobuterol was stable during stability studies. The validated method has been successfully applied to a pharmacokinetic study of three doses of tulobuterol patch in rats for the first time.
Keywords: LC–MS/MS; MR; IS; HPLC; ESI; LLE; LLOQ; QC; RE; RSD; MF; Tulobuterol patch; LC–MS/MS; Transdermal administration; Linear pharmacokinetic study;

The present work proposes the application of bar adsorptive microextraction coated with mixed sorbent phases (n-vinylpyrrolidone and divinylbenzene polymers with strong and weak anion exchangers), combined with liquid desorption followed by capillary electrophoresis with diode array detection (BAμE(PMIX)-LD/CE–DAD) for the determination of trace levels of non-steroidal anti-inflammatory drugs (NSAIDs: salicylic acid, mefenamic acid, diclofenac and naproxen as model compounds) in urine and water matrices. Assays performed on 25 mL of water samples spiked at the 80.0 μg/L level, yielded average recoveries between 86.6 and 104.% for all the NSAIDs under study using optimized experimental conditions. The proposed analytical methodology demonstrated suitable detection limits (0.3 μg/L) and good linear dynamic ranges (2.5–320.0 μg/L) with determination coefficients higher than 0.9981. By using the standard addition methodology, the present analytical approach was applied on urine and water samples, where good selectivity and sensitivity were achieved. The proposed method, which operated under the floating sampling technology, proved to be a suitable sorption-based static microextraction alternative for monitoring trace levels of NSAIDs in urine and water samples. The methodology showed to be easy to implement, demonstrating good reproducibility and robustness, allowing the possibility to choose the most selective sorbent, or mixed sorbent phases, according to the compounds of interest.
Keywords: Bar adsorptive microextraction (BAμE); Capillary electrophoresis; Environmental and biological samples; Floating sampling technology; Mixed sorbent phases; Non-steroidal anti-inflammatory drugs;

Direct injection LC–MS/MS method for the determination of teicoplanin in human plasma by Kwang-Youl Kim; Sang-Heon Cho; Yong-Hyun Song; Moon-Suk Nam; Cheol-Woo Kim (125-131).
A direct injection-based, simple, accurate, and robust LC–MS/MS method was developed and validated for the determination of teicoplanin in human plasma. Patient plasma samples were diluted in an aqueous buffer prior to injection into the LC–MS/MS system. Chromatographic separation was achieved using a Cadenza HS-C18 column and a gradient mixture of acetonitrile–water (both containing 0.1% formic acid) as the mobile phase at a flow rate of 0.5 mL/min. The analytes were detected in multiple reaction monitoring mode with positive ion electrospray ionization. The concentration of teicoplanin was determined as the sum of six components (A3-1, A2-1, A2-2, A2-3, A2-4, and A2-5). The calibration curve was linear over a concentration range of 1–50 mg/L, which covered the clinically accepted trough and therapeutic plasma levels. The intra- and inter-day precision and accuracy values were both less than 15%. This validated method was successfully applied to therapeutic drug monitoring of teicoplanin in routine clinical practice. Thus, we expect it to be useful for the determination of teicoplanin concentration in human plasma.
Keywords: Teicoplanin; Multiple reaction monitoring; Direct injection; Therapeutic drug monitoring;

Direct site-specific immobilization of protein A via aldehyde-hydrazide conjugation by Berlin Zang; Jun Ren; Li Xu; Lingyun Jia (132-138).
Immobilization of affinity ligands on supporting matrices is a key step for the preparation of affinity chromatography resins, and an efficient coupling strategy can significantly improve the validity and cost of the affinity system, especially for systems that employ expensive recombinant proteins or antibodies as affinity ligands. This study described a simple method for obtaining site-specific immobilization of protein A (the ligand) via aldehyde-hydrazide conjugation and its application in antibody purification via protein A chromatography. An aldehyde group was generated at the N-terminus of protein A in vivo by co-expressing a formylglycine-generating enzyme (FGE) and recombinant protein A containing a FGE recognizing sequence (aldehyde tag) in Escherichia coli. The resulting aldehyde allowed direct immobilization of protein A onto the hydrazide-modified agarose matrices under mild condition. We found that 100 mM aniline was most effective for catalyzing the coupling reaction, and the recombinant protein A could be coupled with high selectivity, directly from a crude cell extract. The site-specific immobilized protein A showed good capacity for antibody purification. The specificity of the aldehyde-hydrazide reaction not only allowed site-specific immobilization of affinity ligands, but also improved the cost of the process by employing unpurified ligands, a method that might be of great use to industrial applications.
Keywords: Site-specific immobilization; Aldehyde-tag; Formylglycine-generating enzyme; Protein A chromatography; Ligand immobilization;

In this study, an ultra performance liquid chromatography tandem mass spectrometry method (UPLC–MS/MS) was developed for simultaneous determination of umbelliferone, apigenin, daphnoretin and genkwanin in total (free and conjugated) forms in rat plasma using psoralen as internal standard. Plasma samples were protein precipitated with acetonitrile followed by liquid–liquid extracted with ethyl acetate. Four ingredients were separated on an Acquity UPLC® BEH C18 column using gradient elution with the mobile phase consisting of 0.1% formic acid aqueous solution and acetonitrile, and detected by positive ion electrospray ionization (ESI) in multiple reaction monitoring mode (MRM). The method was linear for all analytes over investigated ranges with all correlation coefficients greater than 0.99. The validated lower limit of quantification was 3 ng/mL for umbelliferone, 3 ng/mL for apigenin, 12 ng/mL for daphnoretin and 2 ng/mL for genkwanin, respectively. Intra- and inter-day precisions (RSD%) were less than 15% and accuracy (RE%) ranged from −1.1% to 15%. The mean absolute extraction recoveries of analytes and IS from rat plasma were all more than 75%. The validated method was firstly and successfully applied to investigate the pharmacokinetics of four chemical ingredients after oral administration of Wikstroemia indica extract (WIE) to rats.
Keywords: UPLC–MS/MS; Wikstroemia indica (L.) C. A. Mey; Coumarin; Flavonoid; Pharmacokinetics;

Benzodiazepines (BDs) are used widely in clinical practice, due to their multiple pharmacological functions. In this study a dispersive nanomaterial-ultrasound assisted- microextraction (DNUM) method followed by high performance liquid chromatography (HPLC) was used for the preconcentration and determination of chlordiazepoxide and diazepam drugs from urine and plasma samples. Various parameters such as amount of adsorbent (mg: ZnS-AC), pH and ionic strength of sample solution, vortex and ultrasonic time (min), and desorption volume (mL) were investigated by fractional factorial design (FFD) and central composite design (CCD). Regression models and desirability functions (DF) were applied to find the best experimental conditions for providing the maximum extraction recovery (ER). Under the optimal conditions a linear calibration curve were obtained in the range of 0.005–10 μg mL−1 and 0.006–10 μg mL−1 for chlordiazepoxide and diazepam, respectively. To demonstrate the analytical performance, figures of merits of the proposed method in urine and plasma spiked with chlordiazepoxide and diazepam were investigated. The limits of detection of chlordiazepoxide and diazepam in urine and plasma were ranged from 0.0012 to 0.0015 μg mL−1, respectively.
Keywords: Benzodiazepines; Dispersive nanomaterial-ultrasound assisted microextraction; Fractional factorial design; Central composite design; Urine; Plasma;

Adenosine triphosphate and its metabolites are involved in the cellular metabolism process in Saccharomyces cerevisiae. It is very important to simultaneously determine the relative contents of ATP and its metabolites in yeast. In this study, an effective capillary zone electrophoresis method with high selectivity was established. The calibration curves were linear in the concentration range from 1 to 20 mg/L (ATP and cAMP) and 2 to 40 mg/L (ADP and AMP) with excellent correlation coefficients (r 2) > 0.999. The recovery of ATP, ADP, AMP, and cAMP were 99.4%, 94.7%, 100.3% and 99.6%, respectively. Simple sample preparation and easy detection of ATP and its metabolites make this method suitable for the study of changes in the four adenine nucleotides levels caused by caloric restriction in yeast. It is expected that the current method may contribute to further energy metabolism and related investigations of yeast.
Keywords: Saccharomyces cerevisiae; Capillary zone electrophoresis; Adenosine triphosphate; Adenosine diphosphate; Adenosine monophosphate; Cyclic adenosine monophosphate;

Comparison of enzyme kinetics of warfarin analyzed by LC–MS/MS QTrap and differential mobility spectrometry by Abdul Naveed Shaik; Richard Grater; Mukesh Lulla; David A. Williams; Lawrence L. Gan; Tonika Bohnert; Barbara W. LeDuc (164-173).
Warfarin is an anticoagulant used in the treatment of thrombosis and thromboembolism. It is given as a racemic mixture of R and S enantiomers. These two enantiomers show differences in metabolism by CYPs: S-warfarin undergoes 7 hydroxylation by CYP2C9 and R-warfarin by CYP3A4 to form 10 hydroxy warfarin. In addition, warfarin is acted upon by different CYPs to form the minor metabolites 3′-hydroxy, 4′-hydroxy, 6-hydroxy, and 8-hydroxy warfarin. For analysis, separation of these metabolites is necessary since all have the same m/z ratio and similar fragmentation pattern. Enzyme kinetics for the formation of all of the six hydroxylated metabolites of warfarin from human liver microsomes were determined using an LC–MS/MS QTrap and LC–MS/MS with a differential mobility spectrometry (DMS) (SelexION™) interface to compare the kinetic parameters. These two methods were chosen to compare their selectivity and sensitivity. Substrate curves for 3′-OH, 4′-OH, 6-OH, 7-OH, 8-OH and 10-OH warfarin formation were generated to determine the kinetic parameters (K m and V max) in human liver microsomal preparations. The limit of quantitation (LOQ) for all the six hydroxylated metabolites of warfarin were in the range of 1–3 nM using an LC–MS/MS QTrap method which had a run time of 22 min. In contrast, the LOQ for all the six hydroxylated metabolites using DMS interface technology was 100 nM with a run time of 2.8 min. We compare these two MS methods and discuss the kinetics of metabolite formation for the metabolites generated from racemic warfarin. In addition, we show inhibition of major metabolic pathways of warfarin by sulfaphenazole and ketoconazole which are known specific inhibitors of CYP2C9 and CYP3A4 respectively.
Keywords: Warfarin; Hydroxywarfarin; Human liver microsomes; Sulfaphenazole; Ketoconazole; Method validation; LC–MS/MS; QTrap; Differential mobility spectroscopy (DMS); Substrate kinetics;

Oxycodone is an opioid agonist largely prescribed for the treatment of moderate to severe pain. Variability in analgesic efficacy could be explained by inter-subject variations in plasma levels of parent drug and its active metabolite, oxymorphone. For this purpose it is necessary to develop and validate a sensitive and selective analytical method for the quantification of oxycodone and its major metabolites, noroxycodone and oxymorphone, in human plasma. The analytical method consisted of a liquid–liquid extraction procedure followed by a high performance liquid chromatography with heated assisted electrospray ionization mass spectrometry (HPLC–HESI–MS/MS). The chromatographic separation was achieved using gradient elution with a mobile phase consisting of ethanol and 10 mM ammonium acetate on a Synergi MAX-RP analytical column (150 × 2 mm, 4 μm) protected by a security guard cartridge (C12 4 × 2 mm) at a flow rate of 300 μL/min.The calibration functions are linear in the range of 300–50,000 pg/mL for oxycodone and noroxycodone and 50 to 10 000 pg/mL for oxymorphone. Intra- and inter-day relative standard deviations are less than 5.5% and 6.4%, respectively for all analytes. The limit of detection was 30 pg/mL for all analytes. We introduce a new HPLC–HESI–MS/MS sensitive and specific analytical method capable to simultaneously quantify oxycodone, noroxycodone and oxymorphone, in human plasma, and suitable for the conduct of pharmacokinetic studies after a single dose administration of the parent compound.
Keywords: Oxycodone; Noroxycodone; Oxymorphone; Mass spectrometry; Liquid chromatography; Plasma; Human;

Simultaneous determination of 3-mercaptopyruvate and cobinamide in plasma by liquid chromatography–tandem mass spectrometry by Michael W. Stutelberg; Joseph K. Dzisam; Alexandre R. Monteil; Ilona Petrikovics; Gerry R. Boss; Steven E. Patterson; Gary A. Rockwood; Brian A. Logue (181-188).
The current suite of Food and Drug Administration (FDA) approved antidotes (i.e., sodium nitrite, sodium thiosulfate, and hydroxocobalamin) are effective for treating cyanide poisoning, but individually, each antidote has major limitations (e.g., large effective dosage or delayed onset of action). To mitigate these limitations, next-generation cyanide antidotes are being investigated, including 3-mercaptopyruvate (3-MP) and cobinamide (Cbi). Analytical methods capable of detecting these therapeutics individually and simultaneously (for combination therapy) are essential for the development of 3-MP and Cbi as potential cyanide antidotes. Therefore, a liquid chromatography–tandem mass-spectrometry method for the simultaneous analysis of 3-MP and Cbi was developed. Sample preparation of 3-MP consisted of spiking plasma with an internal standard (13C3-3-MP), precipitation of plasma proteins, and derivatizing 3-MP with monobromobimane to produce 3-mercaptopyruvate-bimane. Preparation of Cbi involved denaturing plasma proteins with simultaneous addition of excess cyanide to convert each Cbi species to dicyanocobinamide (Cbi(CN)2). The limits of detection for 3-MP and Cbi were 0.5 μM and 0.2 μM, respectively. The linear ranges were 2–500 μM for 3-MP and 0.5–50 μM for Cbi. The accuracy and precision for 3-MP were 100 ± 9% and <8.3% relative standard deviation (RSD), respectively. For Cbi(CN)2, the accuracy was 100 ± 13% and the precision was <9.5% RSD. The method presented here was used to determine 3-MP and Cbi from treated animals and may ultimately facilitate FDA approval of these antidotes for treatment of cyanide poisoning.
Keywords: Cyanide antidote; Sulfanegen; Mass spectrometry;

Xanthine oxidoreductase activity assay in tissues using stable isotope-labeled substrate and liquid chromatography high-resolution mass spectrometry by Takayo Murase; Mai Nampei; Mitsuru Oka; Naoki Ashizawa; Koji Matsumoto; Atsushi Miyachi; Takashi Nakamura (189-197).
Studies of pathological mechanisms and XOR inhibitor characterization, such as allopurinol, febuxostat, and topiroxostat, require accurate and sensitive measurements of XOR activity. However, the established assays have some disadvantages such as susceptibility to endogenous substances such as uric acid (UA), xanthine, or hypoxanthine. Here, we aimed to develop a novel XOR activity assay utilizing a combination of high-performance liquid chromatography (LC) and high-resolution mass spectrometry (HRMS) for tissues such as the liver, kidney, and plasma. Stable isotope-labeled [15N2]-xanthine was utilized as substrate and the production of [15N2]-uric acid was determined. [15N2]-UA production by XOR was dependent on the amounts of [15N2]-xanthine and enzyme and the time of reaction. Because high concentrations of endogenous xanthine and hypoxanthine affect XOR activities, we employed a multi-component analysis using LC/HRMS to improve the accuracy of XOR activity assay. Quantification of [15N2]-UA was validated and showed good linearity, accuracy, and precision. We measured the XOR activities of retired ICR mice using [15N2]-xanthine and LC/MS. The XOR activities in plasma, kidney, and liver samples were 38.1 ± 0.7, 158 ± 5, 928 ± 25 pmol/min/mg of protein, respectively (mean ± SD, n  = 5). Furthermore, we measured the XOR activities in the same samples using the LC/ultraviolet and LC/fluorescence (FL) methods. The level of [15N2]-xanthine oxidation by XOR was equal to that of xanthine oxidation and approximately 7.9–8.9 times higher than that of pterin oxidation. We found a good correlation between XOR activities examined using LC/MS assay with [15N2]-xanthine and those examined using LC/FL assay with pterin. This result suggested that although both the LC/MS assay with [15N2]-xanthine and the LC/FL assay with pterin were useful, the former provided information regarding XOR activities that more directly reflected the physiological condition than the latter.
Keywords: High-resolution mass spectrometry; Xanthine oxidoreductase activity; Stable isotope-labeled substrate; Xanthine oxidoreductase inhibitor;

High-performance thin-layer chromatography as a fast screening tool for phosphorylated peptides by Lena Morschheuser; Sandra Mükusch; Maria Riedner; Harald Seitz; Sascha Rohn (198-205).
This study aimed at developing a rapid chromatographic assay to monitor phosphorylation sites in peptides. For the analysis of nociceptive signal transduction pathways, the detection of phosphorylated proteins/peptides plays a fundamental role. To get further insights in the phosphorylation mechanism of protein kinase C-ε (PKC-ε) and protein kinase A (PKA), potential targets were divided into subsections resulting in peptides that contain only one possible phospho-binding site. The use of high-performance thin-layer chromatography (HPTLC) offers the possibility of a high throughput of samples and the advantage of a quick sample clean-up. A combined strategy of an effect-directed overlay procedure on the TLC plate using specific antibodies (immunostaining, HPTLC-IS) as well as a parallel, direct mass spectrometric methodology by HPTLC-MALDI-TOF-MS was developed. With regard to HPTLC-IS, validation of the data exhibited a lower limit of detection than the traditionally used protein derivatization reagent fluorescamine. Besides the identification of the phosphorylated peptides, a semi-quantitative estimation can be performed with HPTLC-IS.
Keywords: High-performance thin-layer chromatography; Phosphopeptide detection; Nociceptive pathway; HPTLC-MALDI-TOF-MS; HPTLC-immunostaining;

The determination of circulating trimethylamine-N-oxide (TMAO), choline, betaine, l-carnitine and O-acetyl-l-carnitine concentration in different human matrices is of great clinical interest. Recent results highlighted the prognostic value of TMAO and quaternary ammonium containing metabolites in the field of cardiovascular and kidney diseases. Herein, we report a method for the rapid and simultaneous measurement of closely related phosphatidylcholine-derived metabolites in three different biological matrices by stable isotope dilution assay. Plasma, serum and urine samples were simply deproteinized and separated by HILIC-chromatography. Detection and quantification were performed using LC–MS/MS with electrospray ionization in positive mode. For accuracy and precision, full calibration was performed covering more than the full reference range. Assay performance metrics include intra- and interday imprecision were below 10% for all analytes. To exclude matrix effects standard addition methods were applied for all matrices. It was shown that calibration standards and quality control prepared in water can be used instead of matrix-matched calibration and controls. The LC/MS/MS-based assay described in this article may improve future clinical studies evaluating TMAO and related substances as prognostic markers for cardiovascular risk and all-cause mortality in different patient populations.
Keywords: Isotope dilution assay; HILIC-chromatography; LC–MS/MS analysis; TMAO;

Highly sensitive and selective detection of human carboxylesterase 1 activity by liquid chromatography with fluorescence detection by Dan-Dan Wang; Qiang Jin; Jie Hou; Lei Feng; Na Li; Shi-Yang Li; Qi Zhou; Li-Wei Zou; Guang-Bo Ge; Jin-Guang Wang; Ling Yang (212-218).
Human carboxylesterases 1 (hCE1), one of the most important human drug metabolizing enzymes, catalyzes the hydrolysis of a large number of structurally diverse of endogenous and exogenous substrates. However, a practical, reliable and sensitive method for the precise measurement of hCE1 activities in complex biological samples has been rarely reported. In this study, a liquid chromatography-fluorescence detection (LC-FD) based method was developed for highly selective and sensitive measurement of hCE1 activities in human tissue and cell preparations. This method was based on the fluorimetric detection of HMBT, the hydrolyzed product of BMBT which was a newly developed specific probe substrate for hCE1. The developed LC-FD method was fully validated in terms of specificity, sensitivity, linearity, precision, recovery and stability. With the help of LC separation, most polar endogenous compounds in biological samples could be eluted in the column dead time, which is very beneficial for accurate determination of hCE1 activities in complex biological samples. The lower limit of quantification for HMBT (product of hCE1) of this LC-FD based method was as low as 20 nM, which was quite lower than other reported methods. The method also exhibited good precision, both intra- and inter- assay variances were both lower than 2.5%. Furthermore, the newly developed method was successfully applied to measure hCE1 activity in human liver preparations from individual donors (n  = 12), as well as in homogenates from eleven different human cell lines. All these findings combined with this practical method are very helpful for the deep understanding of the expression and function of hCE1 in human biological samples.
Keywords: Liquid chromatography-fluorescence detection (LC-FD); Human carboxylesterase 1 (hCE1); BMBT hydrolysis; Biological samples;

Etanercept is the first tumor necrosis factor inhibitor to be approved for rheumatic disease treatment. Its in vivo concentration is usually detected with commercial enzyme-linked immunosorbent assay (ELISA) kits; specifically, previous researchers have mostly used double-antibody sandwich ELISA technology. Double-antibody sandwich ELISA is employed to detect the total etanercept rather than biologically active etanercept, which is more relevant in terms of therapeutic drug monitoring. In this work, a sensitive ELISA that employed its antigen TNF-α to capture biologically active etanercept for concentration detection was established and validated for etanercept pharmacokinetic (PK) study in patients with ankylosing spondylitis (AS). The proposed assay was demonstrated to be precise and accurate over the linear range of 12.5–400 pg/mL. The intra- and inter-assay relative standard deviation ranged from 3.9 to 12.2% and 6.2 to 11.1%, respectively, and recovery varied between 90.1 and 99.7%, confirming the assay’s reliability. The effectiveness and accuracy of the assay was also validated according to quality samples containing etanercept with different TNF-α concentrations, and with plasma samples from patients with AS. To complete the study, both the proposed assay and double-antibody sandwich ELISA were applied to the PK study of etanercept in patients and compared. The multiple-dose results of both analytical methods were consistent, while the drug exposure of the first dose as-detected by the proposed assay was lower than that detected by double-antibody sandwich ELISA. In conclusion, the proposed ELISA was shown to provide more accurate concentration data for therapeutic drug monitoring in comparison to commercial ELISA kits.
Keywords: Ankylosing spondylitis; Biologically active; Enzyme-linked immunosorbent assay; Etanercept; Pharmacokinetics;

Specific molecularly imprinted polymers for dencichine were developed for the first time in this study by the bulk polymerization using phenylpyruvic acid and dl-tyrosine as multi-templates. The photographs confirmed that molecularly imprinted polymers prepared using N,N′-methylene diacrylamide as cross-linker and glycol dimethyl ether as porogen displayed excellent hydrophilicity. Selectivity, adsorption isotherm and adsorption kinetics were investigated. The sample loading-washing-eluting solvent was optimized to evaluate the property of molecularly imprinted solid phase extract. Compared with LC/WCX-SPE, water-compatible molecularly imprinted solid phase extraction displayed more excellent specific adsorption performance. The extracted dencichine from Panax notoginseng with the purity of 98.5% and the average recovery of 85.6% (n  = 3) was obtained.
Keywords: Molecularly imprinted polymers; HPLC; Natural products; Dencichine; Panax notoginseng;

Highly specific phosphopeptide enrichment by titanium(IV) cross-linked chitosan composite by Ting Wu; Jiani Shi; Chuanjing Zhang; Lingfan Zhang; Yiping Du (234-239).
Natural chitosan was applied as supporting material for Ti(IV) based immobilized metal ion affinity chromatographic (IMAC) material (Ti-CTS). Compared with other polymer based IMAC, Ti-CTS can save the cockamamie synthesis procedures and be easy to obtain. The morphology, surface area, pore volume and elemental composition of Ti-CTS were revealed by scanning electron microscopy (SEM), Brunauer–Emmett–Teller (BET) method and X-ray photoelectron spectroscopy (XPS). Tryptic digest products from several standard proteins and two real samples (non-fat milk and serum) were enriched using Ti-CTS to demonstrate the efficiency of this method. The results showed that this composite enables high sensitive and selective phosphopeptide enrichment from casein variants, non-fat milk and human serum. Furthermore, multi-phosphorylated peptides with three serine phospholated sites (S*S*S*) demonstrated high affinity to Ti-CTS. Hence, this method had great potential for future studies of complex phosphoproteomes and especially multi-phosphorylated peptides.
Keywords: Phosphopeptide enrichment; IMAC; Chitosan; MALDI TOF MS;

Keywords: γ-Hydroxybutyrate; Adduct formation; Buffer concentration; LC–MS/MS; Electrospray ionisation;

A novel liquid chromatographic method with tandem mass spectrometric detection (LC–MS/MS) for the determination of Pasireotide (SOM230) was developed and validated with a dynamic range of 0.5–250 ng/ml using 50 μl of monkey plasma. SOM230 and the internal standard, [M + 6]SOM230, were extracted from monkey plasma via μElution SPE. The acidified sample matrix was loaded onto the preconditioned Waters SPE plate for further processing. The analyte was eluted from the SPE plate using freshly prepared elution solvent, which was followed by dilution and LC–MS/MS analysis. By eliminating a step of evaporation of the SPE eluent, instead, injecting the eluent after a simple dilution, compound loss due to non-specific binding to the 96-well materials was prevented. Furthermore, freshly prepared elution solution was found a key to optimal extraction recovery of the analyte from monkey plasma. The optimal chromatographic separation was achieved on an Atlantis dC18 (50 × 2.1 mm, 5 μm particle size) column using gradient elution with a total analysis cycle time approximately 4 min per injection. The mobile phases were water containing 0.5% acetic acid and 0.05% trifluoroacetic acid (TFA) (mobile phase A) and acetonitrile containing 0.5% acetic acid and 0.05% TFA (mobile phase B). The incorporation of TFA (0.05%, v/v) and acetic acid (0.5%, v/v) into the mobile phases was accompanied by the improved chromatography and minimized carryover due to the HPLC column. The current method was validated for specificity, sensitivity, matrix effect, recovery, linearity, accuracy and precision, dilution integrity, batch size and stability. The accuracy and precision for the LLOQs (0.5 ng/ml) were within ±5.6% bias and ≤7.8% CV, respectively. From the intra-day and inter-day evaluations, the precision of the other QC samples (1.5, 7.5, 75 and 190 ng/ml) ranged from 2.7 to 4.9% CV and the accuracy (% bias) from −1.3 to 7.3%, respectively. Additional assessment of incurred sample reanalysis (ISR) was conducted to demonstrate the ruggedness and robustness of the assay method. The validated method was successfully implemented to support a toxicity study in monkeys administered with 5 and 30 mg of SOM230 in a single intramuscular injection of a long acting release (LAR) formulation.
Keywords: Pasireotide; SOM230; LC–MS/MS; Quantification; Monkey plasma; SPE; μElution;

Study on pharmacokinetics of 3,4-divanillyltetrahydrofuran in rats by ultra-fast liquid chromatography/tandem mass spectrometry by Chen-xiao Shan; Xiao-bing Cui; Sheng Yu; Chuan Chai; Hong-mei Wen; Xin-zhi Wang; Xue Sun (250-254).
3,4-Divanillyltetrahydrofuran is the main active ingredient of nettle root which can increase steroid hormones in the bloodstream for many of bodybuilders. To better understand its pharmacological activities, we need to determine its pharmacokinetic profiles. In this study, a rapid and sensitive ultra-fast liquid chromatography-tandem mass spectrometry (UFLC–MS/MS) method has been developed for the determination of 3,4-divanillyltetrahydrofuran in the plasma of rats. Chromatographic separation was performed on a C18 column at 40 °C, with a gradient elution consisting of methanol and water containing 0.3% (v/v) formic acid at a flow rate of 0.8 mL/min. The detection was performed using an electrospray triple-quadrupole MS/MS via positive ion multiple reaction monitoring mode. The lower limits-of-quantification determined were 0.5 ng/mL. The intra- and inter-day precision (RSD%) was found to be within 15% and the accuracy (RE%) ranged from −4.0% to 7.0%. This simple yet sensitive method was fully validated and could be successfully applied to the study on pharmacokinetics of 3, 4-divanillyltetrahydrofuran.
Keywords: 3,4-Divanillyltetrahydrofuran; Rat plasma; Pharmacokinetics; UFLC–MS/MS;

Simultaneous determination of ledipasvir, sofosbuvir and its metabolite in rat plasma by UPLC–MS/MS and its application to a pharmacokinetic study by Chenwei Pan; Yongping Chen; Weilai Chen; Guangyao Zhou; Lingxiang Jin; Yi Zheng; Wei Lin; Zhenzhen Pan (255-259).
In this work, a rapid and sensitive ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method for the determination of ledipasvir, sofosbuvir and its metabolite GS-331007 in rat plasma was developed. The analytes and the internal standard (diazepam) were separated on an Acquity UPLC BEH C18 chromatography column (2.1 mm × 50 mm, 1.7 μm) using gradient elution with a mobile phase of acetonitrile and 0.1% formic acid in water at a flow rate of 0.4 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode to monitor the precursor-to-product ion transitions of m/z 889.8 → 130.1 for ledipasvir, m/z 530.3 → 243.1 for sofosbuvir, m/z 261.5 → 113.1 for GS-331007 and m/z 285.2 → 193.1 for diazepam (IS) using a positive electrospray ionization interface. The method was validated over a concentration range of 2–500 ng/mL for ledipasvir, 10-2000 ng/mL for sofosbuvir and 10-2000 ng/mL for GS-331007. Total time for each chromatography was 3.0 min. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels exhibited relative standard deviations (RSD) < 10.2% and the accuracy values ranged from −9.8% to 11.2%. The method was successfully applied to a pharmacokinetic study of ledipasvir, sofosbuvir and GS-331007 in rats.
Keywords: Ledipasvir; Sofosbuvir; GS-331007; UPLC–MS/MS; Plasma; Pharmacokinetics;

Purification, identification and structural modelling of DPP-IV inhibiting peptides from barbel protein hydrolysate by Assaad Sila; Oscar Martinez Alvarez; Anissa Haddar; Fakher Frikha; Pascal Dhulster; Naima Nedjar-Arroume; Ali Bougatef (260-269).
Inhibition of DPP-IV may improve glycemic control in diabetics by preventing the rapid breakdown and there by prolonging the physiological action of incretin hormones. Barbel muscle protein hydrolysate (BMPH) was noted to exhibit DPP-IV inhibitory activity, with an IC50 value of 1.94 mg/mL. It was fractionated into five major fractions (F IF V) by size exclusion chromatography using a Superdex peptide. The FIII fraction was noted to display the highest inhibitory activity, with an IC50 value of 1.23 mg/mL, and was, therefore, further fractionated by RP-HPLC. Four major peptide sub-fractions were selected. The results revealed that the SF4 sub-fraction showed the highest DPP-IV inhibitory activity, with an IC50 value of 0.21 mg/mL. This sub-fraction was submitted to RP-HPLC, ESI–MS, and ESI–MS/MS analyses. The findings indicated that SF4 consisted of two peptides (IC50  = 96 μg/mL), namely PP1 and PP2, whose structures were identified as Trp-Ser-Gly (330 Da) and Phe-Ser-Asp (349 Da), respectively. This is the first report of these sequences from barbel proteins.The structural modelling through docking simulations results with DPP-IV showed that the Trp-Ser-Gly peptide bound to DPP-IV with high affinity. Overall, the results suggested that BMPH can be considered as a promising natural source of DPP-IV inhibitory peptides.
Keywords: Purification; HPLC; FPLC; LC/MS/MS; Anti-DPP-IV peptides; Structural modelling;