Journal of Chromatography B (v.971, #C)

A liquid chromatography–tandem mass spectrometry assay for quantification of rilpivirine and dolutegravir in human plasma by M. Grégoire; G. Deslandes; C. Renaud; R. Bouquié; C. Allavena; F. Raffi; P. Jolliet; E. Dailly (1-9).
A liquid chromatography–tandem mass spectrometry assay requiring a 100 μL aliquot of human plasma for simultaneous determination of rilpivirine, a second generation non-nucleoside reverse transcriptase inhibitors of HIV and dolutegravir, a novel integrase stand transfer inhibitors of HIV concentrations has been developed. Sample pre-treatment is limited to protein precipitation with a mixture of methanol and zinc sulfate. After centrifugation the supernatant is injected in the chromatographic system, which consists of on-line solid phase extraction followed by separation on a phenyl-hexyl column. This 2.5 min method, with its simple sample preparation provides sensitive (the limit of quantitation is 25 ng/mL for each compound), accurate and precise (the intra-day and inter-day imprecision and inaccuracy are lower than 15%) quantification of the plasma concentration of these drugs and can be used for therapeutic drug monitoring in patients infected with HIV.
Keywords: Mass spectrometry; Dolutegravir; Rilpivirine;

Antibody purification using affinity chromatography: A case study with a monoclonal antibody to ractopamine by Zhanhui Wang; Qi Liang; Kai Wen; Suxia Zhang; Jianzhong Shen (10-13).
The application of antibodies to small molecules in the field of bioanalytics requires antibodies with stable biological activity and high purity; thus, there is a growing interest in developing rapid, inexpensive and effective procedures to obtain such antibodies. In this work, a ractopamine (RAC) derivative, N-4-aminobutyl ractopamine (ABR), was synthesized for preparing new specific affinity chromatography to purify a murine monoclonal antibody (mAb) against RAC from ascites. The performance of the new specific chromatography was compared with four other purification methods in terms of recovery, purity and biological activity of mAb. These four purification methods were prepared by using specific ligands (RAC and RAC-ovalbumin) and commercial ligands (protein G and protein A), respectively. The results showed that the highest recovery (88.1%) was achieved using the new chromatography; in comparison, the recoveries from the other methods were all below 70%. The purity of the mAbs from the new chromatography was 88.3%, while, the highest purity of 97.6% was from protein G chromatography and the lowest purity of 84.7% was from protein A chromatography. The biological activity of the purified mAb from all of the chromatography methods was comparable in enzyme-linked immunosorbent immunoassay (ELISA).
Keywords: Purification; Ligand; Chromatography; Antibody; Ractopamine; Small molecule;

Analysis of cyclophosphamide and carboxyethylphosphoramide mustard enantiomers in human plasma and application to clinical pharmacokinetics by Francine Attié de Castro; Gabriel dos Santos Scatena; Quézia Bezerra Cass; Belinda Pinto Simões; Vera Lucia Lanchote (14-19).
This study describes for the first time a method for the sequential analysis of the enantiomers of cyclophosphamide (CY) and its metabolite carboxyethylphosphoramide mustard (CEPM) in human plasma. The CY and CEPM enantiomers were extracted from plasma using only ethyl acetate and separated on a Chiralpak® AD-RH column using a mixture of water:acetonitrile:ethanol (45:30:25, v/v/v) plus 0.1% trifluoroacetic acid as the mobile phase at a flow rate of 0.5 mL/min. No matrix effect was observed in the analysis of the enantiomers of both analytes and the analytical method was linear in the range of 0.05–25.0 μg and 250–1000 ng of each enantiomer/mL plasma. The coefficients of variation and relative errors obtained for the assessment of intra- and interassay precision and accuracy were less than 15%. CY and CEPM were found to be stable in human plasma after three successive freeze/thaw cycles, during storage for 4 h at room temperature, and after 24 h inside the autosampler at 4 °C, with deviations less than 15%. The method was applied to the study of the pharmacokinetics of CY and its metabolite CEPM in patients with multiple sclerosis (n  = 10) who received a CY pretransplant conditioning regimen for hematopoietic stem cell transplantation. The pharmacokinetic parameters showed plasma accumulation of the (S)-(−)-CY enantiomer (S/R ratio = 1.3) and lack of enantioselective exposure to the CEPM metabolite (S/R ratio = 1.0).
Keywords: Cyclophosphamide; Carboxyethylphosphoramide mustard; Enantiomers; LC-MS/MS; Plasma; Pharmacokinetics;

A new, sensitive and fast high-performance liquid chromatography–diode-array detection assay based on microextraction by packed sorbent (MEPS/HPLC-DAD) is herein reported, for the first time, to simultaneously quantify carbamazepine (CBZ), lamotrigine (LTG), oxcarbazepine (OXC), phenobarbital (PB), phenytoin (PHT), and the active metabolites carbamazepine-10,11-epoxide (CBZ-E) and licarbazepine (LIC) in human plasma. Chromatographic separation of analytes and ketoprofen, used as internal standard (IS), was achieved in less than 15 min on a C18-column, at 35 °C, using acetonitrile (6%) and a mixture (94%) of water–methanol–triethylamine (73.2:26.5:0.3, v/v/v; pH 6.5) pumped at 1 mL/min. The analytes and IS were detected at 215, 237 or 280 nm. The method showed to be selective, accurate [bias ±14.8% (or ±17.8% in the lower limit of quantification)], precise [coefficient variation ≤9.7% (or ≤17.7% in the lower limit of quantification)] and linear (r 2  ≥ 0.9946) over the concentration ranges of 0.1–15 μg/mL for CBZ; 0.1-20 μg/mL for LTG; 0.1–5 μg/mL for OXC and CBZ-E; 0.2–40 μg/mL for PB; 0.3–30 μg/mL for PHT; and 0.4–40 μg/mL for LIC. The absolute extraction recovery of the analytes ranged from 57.8 to 98.1% and their stability was demonstrated in the studied conditions. This MEPS/HPLC-DAD assay was successfully applied to real plasma samples from patients, revealing to be a cost-effective tool for routine therapeutic drug monitoring of CBZ, LTG, OXC, PB and/or PHT.
Keywords: Antiepileptic drugs; High-performance liquid chromatography; Microextraction by packed sorbent; Human plasma; Bioanalytical method validation; Therapeutic drug monitoring;

A liquid chromatography/tandem mass spectrometry method for determination of obatoclax in human plasma by Ganesh S. Moorthy; Robin E. Norris; Peter C. Adamson; Elizabeth Fox (30-34).
We describe a selective and highly sensitive high-performance liquid chromatography-electrospray ionization-collision induced dissociation-tandem mass spectrometry (HPLC-ESI-CID-MS/MS) assay for the pan-antiapoptotic Bcl-2 family inhibitor, obatoclax, in human plasma. Fifty μL plasma specimens were prepared by addition of extraction solution consisting of Mobile Phase A (10 mM ammonium formate (aq) titrated to pH of 3.0 with formic acid): Mobile Phase B (100% methanol) (20:80, v,v) and internal standard followed by centrifugation. HPLC separations were performed on a Waters, YMC-Pack™, ODS-AQ™ S-3 analytical column with LC mobile phase A and B. Linearity and sensitivity was assessed over a linear range of 0.04–25 ng/ml at eleven concentrations. The lower limit of quantification for obatoclax was 0.04 ng/mL. The intra-day precision based on the standard deviation of replicates of quality control (QC) samples ranged from 0.9 to 5.1% and the accuracy ranged from 98.9 to 106.8%. Stability studies performed replicate sets of QC samples (0.1 ng/mL, 2.5 ng/mL, and 15 ng/mL) showed that obatoclax in human plasma was stable at room temperature for 24 h as well as at −80 °C for 1 m and 2y. Stability was also demonstrated after 3 freeze/thaw cycles (RT to −80 °C). The analytical method showed excellent sensitivity, precision, and accuracy. This method is robust and has been successfully employed in a Children's Oncology Group Phase 1 Consortium study of obatoclax in children with cancer.
Keywords: Obatoclax; LC-MS/MS; Plasma; Pediatric; Cancer;

Development of a UHPLC–MS/MS method for the determination of plasma histamine in various mammalian species by Jia Liu; Lei Wang; Wenjuan Hu; Xiaoyan Chen; Dafang Zhong (35-42).
Histamine is an important mediator of anaphylactic reactions. Although several methods have been developed to measure histamine levels, each has its limitations. In this study, we developed and validated a convenient bioanalytical method for the qualitative and quantitative determination of histamine in plasma samples from humans, beagle dogs, Sprague–Dawley rats, and imprinting control region mice. A simple plasma protein precipitation method using acetonitrile was selected, and hydrophilic interaction liquid chromatography coupled with mass spectrometry was used for sample separation and detection. Histamine was subjected to gradient elution with acetonitrile, ammonium acetate buffer, and formic acid. A mass spectrometer equipped with an electrospray ionization source was operated in the positive-ion multiple reaction monitoring mode for the detection of histamine and the internal standard. The [M + H]+ transitions were m/z 112 → 95 for histamine and m/z 116 → 99 for d4-histamine, which was used as the internal standard. The lower limit of quantification was 0.2 μg/L and the calibration range was 0.2–500 μg/L. The overall recovery ranged from 93.6% to 102.8%. The intra- and inter-run precision and accuracy were <15% for plasma samples from all four species. The method was validated by measuring the plasma histamine concentrations in five healthy human volunteers. In conclusion, we have developed and validated a novel bioanalytical method for the quantification of histamine levels in plasma samples from various mammalian species.
Keywords: Histamine; UPLC HILIC column; LC–MS/MS; Plasma concentration;

Development and validation of an LC–MS/MS method for the toxicokinetic study of deoxynivalenol and its acetylated derivatives in chicken and pig plasma by N. Broekaert; M. Devreese; T. De Mil; S. Fraeyman; S. De Baere; S. De Saeger; P. De Backer; S. Croubels (43-51).
This study aims to develop an LC–MS/MS method allowing the determination of 3-acetyl-deoxynivalenol, 15-acetyl-deoxynivalenol, deoxynivalenol and its main in vivo metabolite, deepoxy-deoxynivalenol, in broiler chickens and pigs. These species have a high exposure to these toxins, given their mainly cereal based diet. Several sample cleanup strategies were tested and further optimized by means of fractional factorial designs. A simple and straightforward sample preparation method was developed consisting out of a deproteinisation step with acetonitrile, followed by evaporation of the supernatant and reconstitution in water. The method was single laboratory validated according to European guidelines and found to be applicable for the intended purpose, with a linear response up to 200 ng ml−1 and limits of quantification of 0.1–2 ng ml−1. As a proof of concept, biological samples from a broiler chicken that received either deoxynivalenol, 3- or 15-acetyl-deoxynivalenol were analyzed. Preliminary results indicate nearly complete hydrolysis of 3-acetyl-deoxynivalenol to deoxynivalenol; and to a lesser extent of 15-acetyl-deoxynivalenol to deoxynivalenol. No deepoxy-deoxynivalenol was detected in any of the plasma samples. The method will be applied to study full toxicokinetic properties of deoxynivalenol, 3-acetyl-deoxynivalenol and 15-acetyl-deoxynivalenol in broiler chickens and pigs.
Keywords: LC–MS/MS; Deoxynivalenol (DON); 3-Acetyl-deoxynivalenol (3ADON); 15-Acetyl-deoxynivalenol (15ADON); Poultry; Pig;

Clevidipine, a vascular selective calcium channel antagonist of the dihydropyridine class, is rapidly metabolized by ester hydrolysis because of incorporation of an ester linkage into the drug molecule. To characterize its pharmacokinetic profiles in dogs, a simple, rapid and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for quantitation of clevidipine in dog blood. After one-step protein precipitation with methanol, the chromatographic separation was carried out on an Ecosil C18 column (150 mm × 4.6 mm, 5 μm) with a gradient mobile phase consisting of methanol and 5 mM ammonium formate at a flow rate of 0.5 mL/min. The quantitation analysis was performed using multiple reaction monitoring (MRM) at the specific ion transitions of m/z 454.1 [M−H]m/z 234.1 for clevidipine and m/z 256.1 [M−H]  →  m/z 227.1 for elofesalamide (internal standard) in the negative ion mode with electrospray ionization (ESI) source. This validated LC–MS/MS method showed good linearity over the range 0.5–100 ng/mL with the lower limit of quantitation (LLOQ) of 0.5 ng/mL together with the satisfied intra- and inter-day precision, accuracy, extraction recovery and matrix effect. Stability testing indicated that clevidipine in dog blood with the addition of denaturant methanol was stable on workbench for 1 h, at −80 °C for up to 30 days, and after three freeze–thaw cycles. Extracted samples were also observed to be stable over 24 h in an auto-sampler at 4 °C. The validated method has been successfully applied to a pharmacokinetic study of clevidipine injection to 8 healthy Beagle dogs following intravenous infusion at a flow rate of 5 mg/h for 0.5 h.
Keywords: Clevidipine; LC–MS/MS; Pharmacokinetics; Dog blood;

Simultaneous determination of flurbiprofen and its hydroxy metabolite in human plasma by liquid chromatography-tandem mass spectrometry for clinical application by Hye-In Lee; Chang-Ik Choi; Ji-Yeong Byeon; Jung-Eun Lee; So-Young Park; Young-Hoon Kim; Se-Hyung Kim; Yun-Jeong Lee; Choon-Gon Jang; Seok-Yong Lee (58-63).
Flurbiprofen (FLB) is one of the phenylalkanoic acid derivatives of non-steroidal anti-inflammatory drugs used for the management of pain and inflammation in patients with arthritis. We developed and validated a rapid and sensitive high-performance liquid chromatography analytical method utilizing tandem mass spectrometry (HPLC–MS/MS) for the simultaneous determination of FLB and its major metabolite, 4′-hydroxyflurbiprofen (4′-OH-FLB), in human plasma. Probenecid was used as an internal standard (IS). After liquid–liquid extraction with methyl t-butyl ether, chromatographic separation of the two analytes was achieved using a reversed-phase Luna C18 column (2.0 mm × 50 mm, 5 μm particles) with a mobile phase of 10 mM ammonium formate buffer (pH 3.5)–methanol (15:85, v/v) and quantified by MS/MS detection in ESI negative ion mode. The flow rate of the mobile phase was 250 μl/min and the retention times of FLB, 4′-OH-FLB, and IS were 1.1, 0.8, and 0.9 min, respectively. The calibration curves were linear over a range of 0.01–10 μg/ml for FLB and 0.01–1 μg/ml for 4′-OH-FLB. The lower limit of quantifications using 100 μl of human plasma was 0.01 μg/ml for both analytes. The mean accuracy and precision for intra- and inter-run validation of FLB and 4′-OH-FLB were all within acceptable limits. The present HPLC–MS/MS method showed improved sensitivity for quantification of the FLB and its major metabolite in human plasma compared with previously described analytical methods. The validated method was successfully applied to a pharmacokinetic study in humans.
Keywords: Flurbiprofen; 4′-Hydroxyflurbiprofen; HPLC–MS/MS; Human plasma; Pharmacokinetics;

Simultaneous determination of puerarin and its active metabolite in human plasma by UPLC-MS/MS: Application to a pharmacokinetic study by Hyo-Rin Jung; Sook-Jin Kim; Seong-Ho Ham; Jung-Hee Cho; Yong-Bok Lee; Hea-Young Cho (64-71).
A rapid, selective and sensitive ultra-performance liquid chromatography (UPLC)-tandem mass spectrometry method about the simultaneous determination of puerarin and its major active metabolite, daidzein, in human plasma was developed and validated in order to investigate the pharmacokinetics (PKs) of Gegen after the usual oral dose administration to human. Chromatography was carried out on a Kinetex C18 column (2.1 mm × 50 mm, 1.7 μm) using 0.05% acetic acid in water and 0.05% acetic acid in methanol as mobile phase with a gradient elution. Liquid–liquid extraction with ethyl acetate in acidic condition could remove the interference and minimize the matrix effect of human plasma. The lower limit of quantification in human plasma was 0.2 ng/mL for both of compounds, puerarin and daidzein. The calibration curves for puerarin and daidzein in human plasma were linear over all the concentration range of 0.2–100 ng/mL with correlation coefficients greater than 0.998. This assay procedure was successfully applied to the PKs of puerarin and daidzein, after the usual oral dose of Gegen extract powder (2.56 g, containing 9.984 mg puerarin) in human subjects.
Keywords: Puerarin; Daidzein; Human; UPLC-MS/MS; Pharmacokinetics;

An ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for the determination of zopiclone and zolpidem in whole blood, for use in cases with suspected driving under influence of drugs (DUID) and autopsy cases. Sample preparation was performed with liquid–liquid extraction (LLE) using ethyl acetate/n-heptane (80:20, v/v) and 0.1 mL whole blood. Deuterated analogues were used as internal standards (IS) for both compounds. The compounds were separated using a reversed phase C18-column (2.1 mm × 100 mm, 1.7 μm), with a flow rate of 0.5 mL/min, 1 μL injected and gradient elution with 5 mM ammonium formate pH 10.2 and acetonitrile. Quantification was done by MS/MS using multiple reaction monitoring (MRM) in positive mode. The run time of the method was 4.5 min including equilibration time. The calibration curves of extracted whole blood standards were fitted by linear-order calibration curves weighted 1/x, with R 2 values above 0.999 for both compounds. Intermediate precision and accuracies (bias) were 2.4–12.9% RSD and from −5.9 to 6.8%, respectively. Recoveries of the compounds were ≥70%. The lower limit of quantification (LLOQ) for zopiclone was 0.50 nmol/L (0.19 ng/mL) or 0.05 pg injected on column, and 3.5 nmol/mL (1.10 ng/mL) for zolpidem, or 0.27 pg injected on column. The limit of detection (LOD) was 0.2 nmol/L (0.08 ng/mL) for zopiclone and 0.3 nmol/L (0.09 ng/mL) for zolpidem. Matrix effects (ME) were between 108 and 115% when calculated against IS. A comparison with former confirmation LC-MS method at the Norwegian Institute of Public Health, Division of Forensic Medicine (NIPH) was performed during method validation. Good correlation was seen for both compounds. The method has been running on a routine basis for two years, and has proven to be very robust and reliable with satisfactory long term precision and bias and with results for external quality samples corresponding well to consensus mean or median. Zopiclone and zolpidem concentrations in post mortem and ante mortem cases were reported. The method also meets the requirements of the legislative limits for driving under the influence of non-alcohol drugs introduced in the Norwegian Road Traffic Act Law from 2012.
Keywords: Zopiclone; Zolpidem; z-hypnotica; Whole blood; Liquid–liquid extraction; Ultra high performance liquid chromatography-tandem mass spectrometry;

Simultaneous quantification of Akebia saponin D and its five metabolites in human intestinal bacteria using ultra-performance liquid chromatography triple quadrupole mass spectrometry by Liang Yan; Xiaolin Yang; Zhaoqing Meng; Yongliang Yuan; Wei Xiao; Zhenzhong Wang; Wenze Huang; Zhonglin Yang; Chunfeng Zhang (81-88).
A rapid and sensitive ultra-performance liquid chromatography triple quadrupole mass spectrometry (UPLC-TQ/MS) method was developed for simultaneous quantification of Akebia saponin D (ASD) and its five metabolites in intestinal mixtures of bacteria from human feces. After protein precipitation, the analytes and internal standard (IS), glycyrrhetinic acid, were determined in selected ion recording (SIR) mode with negative ion ESI source. Chromatographic separation was carried out on an ACQUITY UPLC™ BEH C18 column (100 mm × 2.1 mm, 1.7 μm) using gradient elution. The mobile phase consisted of solvents A (acetonitrile) and B (0.1% aqueous formic acid) at the flow rate of 0.4 mL/min. Each sample was chromatographed within 10.5 min including equilibration time. The linearity ranged from 0.1 to 100 μg/mL for ASD, and 2–1000 ng/mL for five metabolites, Dipsacus saponin A (M1), HN-saponin F (M2), hederagenin-28-O-β-d-glucopyranoside (M3), Akebia saponin PA (M4), hederagenin (M5). The limits of detection (LOD) were 0.41, 0.59, 0.61, 0.55, 0.52 and 0.31 ng/mL for ASD, M1, M2, M3, M4 and M5, respectively. The intra- and inter-day precision was all within 11.1% and accuracy ranged from −8.33% to 12.47%. The conversion rate of five metabolites was 41.21% in 24 h. The method was validated and successfully applied to quantification of ASD and its five metabolites in human intestinal bacteria.
Keywords: Akebia saponin D; Metabolites; Human intestinal bacteria; UPLC-TQ/MS;

The performance of five different C18 chromatography analytical columns with different lengths, particle sizes and porosities were compared for analysis of ochratoxin A (OTA) in fungal cultures and raisin samples. Chromatographic parameters including retention time, limit of detection, limit of quantification, number of theoretical plates and reduced plate height were obtained and compared. This showed that, compared with traditional columns, shorter ones (100 and 75 mm × 4.6 mm) with 2.7 μm solid core particles are suitable for analysis of OTA in different matrices and allows a reduction of the total analysis time by approximately 50% without any detrimental effect on performance. This leads to significant reduction in analysis costs by savings in use of organic solvents and increasing the total number of analyses per day. The capability of these columns for analyzing samples, from different matrices, was assessed by analyzing OTA-contaminated samples from cultures of Aspergillus westerdijkiae and Aspergillus niger grown on a defined nutritional media (yeast extract sucrose agar) and from natural and OTA spiked raisins.
Keywords: Ochratoxin A; Particle size; Column length; Poroshell; Solid core particles; Raisins;

Detection of Aβ-interacting proteins via a novel Aβ-adsorbents that use immobilized regular comb polymer by Li Xu; Conggang Wang; Linli Chen; Jun Ren; Jian Xie; Lingyun Jia (94-98).
A detailed study of individual Aβ-interacting proteins has always been a difficult task because Aβ has a wide range of molecular weights and can easily form aggregates. In this study, we established a novel method for isolating Aβ-interacting proteins by utilizing regular comb polymer immobilized on Sepharose CL-4B. To achieve site-directed ligation of Aβ, a cysteine residue was added at the N-terminus of Aβ. Asp and Asp12, which have 2 and 13 carboxyl groups, respectively, were selected as the carriers for the regular comb polymer. Firstly, the N-termini of Asp and Asp12 were immobilized on Sepharose CL-4B. Next, modified Aβ molecules were coupled to the carboxyl groups of Asp and Asp12 using bromoethylamine as a spacer. To obtain homogeneous comb polymer, the efficiency of the reaction was controlled during the synthesis process. Thioflavin T staining indicated that homogeneous Aβ was achieved. The prepared Aβ-adsorbents were used to isolate Aβ-interacting proteins from mice brain extracts. The results showed that the adsorption capacity of the Aβ-adsorbents for proteins in mice brain extracts increased with the ages of the animals. SDS-PAGE analysis of the Aβ-interacting proteins showed that many kinds of brain proteins were selectively adsorbed by the Aβ adsorbents, and the levels of some of these proteins varied with the ages of the animals. The results indicated that Aβ-interacting proteins could be successfully obtained through the use of immobilized comb polymer. Similar method could also be used to isolate other amyloid-interacting proteins.
Keywords: Aβ; Oligomer; Comb polymer; Interaction protein; Alzheimer's disease;

Protein solubilization: A novel approach by David H. Johnson; W. William Wilson; Lawrence J. DeLucas (99-106).
Formulation development presents significant challenges with respect to protein therapeutics. One component of these challenges is to attain high protein solubility (>50 mg/ml for immunoglobulins) with minimal aggregation. Protein–protein interactions contribute to aggregation and the integral sum of these interactions can be quantified by a thermodynamic parameter known as the osmotic second virial coefficient (B-value). The method presented here utilizes high-throughput measurement of B-values to identify the influence of additives on protein–protein interactions. The experiment design uses three tiers of screens to arrive at final solution conditions that improve protein solubility. The first screen identifies individual additives that reduce protein interactions. A second set of B-values are then measured for different combinations of these additives via an incomplete factorial screen. Results from the incomplete factorial screen are used to train an artificial neural network (ANN). The “trained” ANN enables predictions of B-values for more than 4000 formulations that include additive combinations not previously experimentally measured. Validation steps are incorporated throughout the screening process to ensure that (1) the protein's thermal and aggregation stability characteristics are not reduced and (2) the artificial neural network predictive model is accurate. The ability of this approach to reduce aggregation and increase solubility is demonstrated using an IgG protein supplied by Minerva Biotechnologies, Inc.
Keywords: Self-interaction chromatography; Protein solubility; Physical stability; High-throughput screening; Neural network; Design of experiments;

The characteristic of Listeria monocytogenes’ pyrolysis product was found by fingerprint analysis of high resolution pyrolysis gas chromatography and mass spectrometry (HRPGC/MS), which hold a great potential to rapidly detect L. monocytogenes with the application of selected ion monitoring (SIM). Food products (beef and milk) contaminated by L. monocytogenes and uncontaminated were evaluated. The retention time of the characteristic peak of pyrolysis product was 19.056 min, the ion of m/z were 54, 98. The results showed that the peak at retention time 19.056 min was detected in agricultural products that contaminated by L. monocytogenes, while the result of the uncontaminated food, there is no peak at the retention time 19.056 min. Qualified by the retention time of chromatographic and mass spectrometry, it can eliminate the interference induced by different types of agricultural products. The results prove united technologies of HRPGC/MS and SIM is not only reliable, reproducible, but also a new method for rapid detecting L. monocytogenes in food products.
Keywords: HRPGC/MS; SIM; L. monocytogenes; Characteristic pyrolysis product peak; Rapid detection;

Methyl-tert-butyl ether (MTBE) is widely used as an antiknock additive for increasing octane number of gasoline. Recently, the in vivo studies demonstrated that MTBE has genotoxic potential and able to form adducts with DNA. In the work, the interactions of MTBE with calf thymus DNA (ct-DNA) and the Na+ form of G-quadruplex DNA (wtTel22) were studied by using of head space-solid phase microextraction technique coupled to gas chromatography. The binding equilibrium constants were measured through the equilibriums of a four phase system. In addition, the MTBE Henry's law constants for two different buffers in the temperature range of 283–303 K were measured. Thermodynamic studies revealed that the complexation of MTBE to both DNAs is enthalpy favored and entropy disfavored. The thermodynamic results revealed that MTBE may have interaction with ct-DNA via the minor groove of DNA. Also, MTBE may be complexed into the basket of G-quadruplex structure. In addition, the low difference in the binding constants of MTBE for both different DNA targets may confirm that MTBE is poorly selective for different conformations of DNA.
Keywords: Methyl-tert-butyl ether; Calf thymus DNA; Human telomeric G-quadruplex; Thermodynamic parameters; Solid phase microextraction; Gas chromatography;

Reaction profiling by ultra high-pressure liquid chromatography/time-of-flight mass spectrometry in support of the synthesis of DNA-encoded libraries by Leonard O. Hargiss; G. Greg Zipp; Theodore C. Jessop; Xuejun Sun; Philip Keyes; David B. Rawlins; Zhi Liang; Kum Joo Park; Huizhong Gu (120-125).
An ultra high-pressure liquid chromatography/mass spectrometry (UHPLC/MS) separation and analysis method has been devised for open access analysis of synthetic reactions used in the production of DNA-encoded chemical libraries. The aqueous mobile phase is 100 mM hexafluoroisopropanol and 8.6 mM triethylamine; the organic mobile phase is methanol. The UHPLC separation uses a C18 OST column (50 mm × 2.1 mm × 1.7 μm) at 60 °C, with a flow rate of 0.6 mL/min. Gradient concentration is from 10 to 40% B in 1.0 min, increasing to 95% B at 1.2 min. Cycle time was about 5 min. This method provides a detection limit of a 20-mer oligonucleotide by mass spectrometry of better than 1 pmol on-column. Linear UV response for 20-mer extends from 2 to 200 pmol/μL in concentration, same-day relative average deviations are less than 5% and bias (observed minus expected) is less than 10%. Deconvoluted mass spectra are generated for components in the predicted mass range using a maximum entropy algorithm. Mass accuracy of deconvoluted spectra is typically 20 ppm or better for isotopomers of oligonucleotides up to 7000 Da.
Keywords: UHPLC; Oligonucleotides; Mass spectrometry; Electrospray ionization; Maximum entropy; DNA-encoded libraries;

Deng-yan granule, consisting of Herba Erigerontis Breviscapi, Rhizoma Corydalis Yanhusuo and Radix Astragali Mongolici, is a widely used Traditional Chinese Medicine preparation for treatment of coronary heart disease. Scutellarin and tetrahydropalmatine are main active constituents in Herba Erigerontis Breviscapi and Rhizoma Corydalis Yanhusuo, and have been used as marker components for quality control of Deng-yan preparations. In order to make good and rational use of Deng-yan granule in the future, a rapid, sensitive and high throughput ultra-fast liquid chromatography with tandem mass spectrometry (UFLC-MS/MS) method was developed for the simultaneous determination of scutellarin and tetrahydropalmatine in rat plasma using rutin as internal standard (IS). The plasma samples were extracted by liquid–liquid extraction with ethyl acetate after acidification and separated on a Shim-pack XR-ODS C18 column (75 mm × 3.0 mm, 2.2 μm) with a mobile phase consisting of methanol–0.1% formic acid water (50:50, v/v) at a flow rate of 0.4 mL/min. Mass spectrometric detection was conducted on an API 3200 QTRAP mass spectrometry equipped with electrospray ionization source in positive ionization mode. Quantification was performed using multiple reaction monitoring (MRM) by monitoring the fragmentation of m/z 463.2 → 287.1 for scutellarin, m/z 356.1 → 192.1 for tetrahydropalmatine and m/z 611.2 → 303.2 for IS, respectively. The linear range was 10–5000 ng/mL for both scutellarin and tetrahydropalmatine with lower limit of quantitation (LLOQ) of 10 ng/mL. The intra- and inter-day precisions were below 12.2% for scutellarin and below 9.7% for tetrahydropalmatine in terms of relative standard deviation (RSD), and the accuracy was within ±9.1% for scutellarin and within ±11.2% for tetrahydropalmatine in terms of relative error (RE). Extraction recovery, matrix effect and stability were satisfactory in rat plasma. The validated method was successfully applied to a pharmacokinetic study of scutellarin and tetrahydropalmatine after oral administration of Deng-yan granule to rats.
Keywords: Deng-yan granule; Scutellarin; Tetrahydropalmatine; UFLC-MS/MS; Pharmacokinetics;

Dexpramipexole (DEX) was being investigated in clinical studies for the treatment of amyotrophic lateral sclerosis (ALS). To monitor the potential chiral interconversion of dexpramipexole to pramipexole (PPX) in vivo, a highly sensitive and selective chiral LC–MS/MS assay was developed and qualified for the detection of pramipexole in the presence of dexpramipexole in human plasma. In this assay, plasma samples were extracted by protein precipitation coupled with solid phase extraction (SPE). The analyte PPX was separated from its enantiomer DEX using a chiral HPLC method. The assay was qualified with a dynamic range of 0.150–1.00 ng/mL. The lower limit of quantitation (LLOQ) for PPX was 0.150 ng/mL in the presence of up to 1000 ng/mL of DEX. The qualified method was used to analyze plasma samples from a DEX clinical study. No PPX was detected in humans at pharmacologically significant levels after administration of dexpramipexole at single doses up to 600 mg per day.
Keywords: Chiral HPLC; LC–MS/MS; Bioanalysis; Chiral interconversion; Dexpramipexole;

Rebaudioside A (RA) and stevioside (SS) are the primary effective glycoside components in Stevia Rebaudiana. The RA glycoside is sweeter, and it tastes similarly to sucrose. Because extracts with a high RA content can be used as natural sweeteners for food additives approved by the FAO and FDA, RA should generate high market demand. In this study, an efficient method for separating RA was established based on the synergistic multi-hydrogen bonding interaction between a polymeric adsorbent and the RA glycoside. To overcome the destruction of the hydrophobic affinity required for the selective adsorption of RA, an innovative non-aqueous environment was established for adsorption and separation. To this end, an initial polymeric adsorbent composed of a glycidyl methacrylate and trimethylolpropane trimethacrylate (GMA-co-TMPTMA) copolymer matrix was synthesized, and polyethylene polyamine was employed as a functional reagent designed to react with the epoxy group on GME-co-TMPTMA to form a highly selective macroporous adsorbent. The effects of the different functional reagents and the solvent polarity on the adsorption selectivity for RA and SS, respectively, were investigated. Matching the structure of the polyethylene polyamine and sugar ligand on the glycoside molecule was essential in ensuring that the maximum synergistic interaction between adsorbent and adsorbate would be achieved. Moreover, the hydrogen-bonding force was observed to increase when the polarity of the adsorption solvent decreased. Therefore, among the synthesized macroporous polymeric adsorbents, the GTN4 adsorbent-bonding tetraethylenepentamine functional group provided the best separation in an n-butyl alcohol solution. Under the optimized gradient elution conditions, RA and SS can be effectively separated, and the contents of RA and SS increased from 33.5% and 51.5% in the initial crude extract to 95.4% and 78.2% after separation, respectively. Compared to conventional methods, the adsorption–desorption process is more advanced due to its procedural simplicity, low cost and adaptability for industrial production.
Keywords: Polymeric adsorbent; Multi-hydrogen bond; Synergistic effect; Rebaudiana A; Steviol glycosides; Non-aqueous system;