Journal of Chromatography B (v.1015-1016, #C)

The present study is focused on the development of an analytical method for the simultaneous analysis of seventy-four pesticides belonging to different chemical classes (organochlorines, organophosphates, pyrethroids, dinitroanilines, dicarboximides, triazoles, etc.) in Chinese material medica. The samples were extracted according to the acetate QuEChERS protocol. To reduce the amount of co-extracted compounds, n-hexane instead of acetonitrile was employed as the extraction solvent. To improve the overall recoveries of problematic basic and base-sensitive compounds, sodium acetate was used to adjust the pH to a neutral condition, and florisil combined with octadecyl-modified silica (C18) were utilized in the cleanup step. The samples were analysed by GC–MS/MS, and quantified by matrix-matched calibration. The validation study was carried out on two representative herbs, Chuanxiong Rhizoma and Angelica Sinensis Radix. In two matrices, the linearity of the calibration was good between 5 and 250 ng/mL concentration ranges, and the limits of quantification (LOQs) less than 0.01 mg/kg for most pesticides. At the LOQs and ten times the LOQs, the mean recoveries of almost all pesticides were within 70–120%, with relative standard deviations (RSDs) lower than 10%. The method was applied on twenty real samples. Seven batches of Chuanxiong and five batches of Danggui were found to contain the residues. The combination of modified QuEChERS and GC-MS/MS offers low cost of analysis as well as excellent accuracy and sensitivity. This method could be especially useful for trace analysis of pesticide residues in complex matrices.
Keywords: Chuanxiong rhizoma; Angelica sinensis radix; Pesticide residues; QuEChERS; GC–MS/MS;

Supercritical fluid chromatography/tandem mass spectrometry (SFC-MS/MS) is an effective tool in separation science which uses the nontoxic CO2 fluid for better control of analyte retention. Also the technology of a postcolumn additive to complement MS/MS ensure the high-selectivity determination. In this paper, a green and sensitive analytical method was developed for the enantioselective separation and determination of isofenphos-methyl enantiomers in foodstuff and soil by SFC-MS/MS. The enantioseparation was performed within 3.50 min using Chiralpak IA-3 column with CO2/isopropanol (90:10, v/v) as the mobile phase at 2.2 mL/min flow rate. The postcolumn compensation technology provided with 0.1% formic acid/methanol greatly improved the ionization efficiency of mass spectrometry. Column temperature, auto back pressure regulator pressure, and flow rate of compensation solvent were optimized to 30 °C, 2200 psi, and 0.1 mL/min, respectively. The QuEChERs method was adopted in this study, which mean recoveries of isofenphos-methyl enantiomers ranged from 75.7% to 111.4%, with relative standard deviations less than 11.3% at three concentration levels in all matrices. The limits of detection for both enantiomers varied from 0.02 μg/kg to 0.15 μg/kg, while the limit of quantification did not exceed 0.50 μg/kg. The proposed method was then successfully applied to analyze authentic samples, confirming that it was a versatile strategy for the analysis of isofenphos-methyl enantiomers in food and environmental matrices.
Keywords: Isofenphos-methyl; Enantioseparation; QuEChERs; SFC-MS/MS;

Currently, the interest in microalgae as a source of biologically active components exploitable as supplementary ingredients to food/feed or in cosmetics continues to increase. Existing research mainly aims to focus on revealing and recovering the rare, cost competitive components of the algae metabolom. Because these components could be of very different physicochemical character, a universal approach for their isolation and characterization should be developed. This study demonstrates the systematic development of the extraction strategy that represents one of the key challenges in effective algae bioprospecting, which predefines their further industrial application. By using of Trachydiscus minutus as a model microalgae biomass, following procedures were tested and critically evaluated in order to develop the generic procedure for microalgae bioprospecting: (i) various ways of mechanical disintegration of algae cells enabling maximum extraction efficiency, (ii) the use of a wide range of extraction solvents/solvent mixtures suitable for optimal extraction yields of polar, medium-polar, and non-polar compounds, (iii) the use of consecutive extractions as a fractionation approach. Within the study, targeted screening of selected compounds representing broad range of polarities was realized by ultra-high performance liquid chromatography coupled with high resolution tandem mass spectrometric detection (UHPLC–HRMS/MS), to assess the effectiveness of undertaken isolation steps. As a result, simple and high-throughput extraction-fractionation strategy based on consecutive extraction with water–aqueous methanol–hexane/isopropanol was developed. Moreover, to demonstrate the potential of the UHPLC–HRMS/MS for the retrospective non-target screening and compounds identification, the collected mass spectra have been evaluated to characterize the pattern of extracted metabolites. Attention was focused on medium-/non-polar extracts and characterization of lipid species present in the T. minutus algae. Such detailed information on the composition of native (non-hydrolyzed) lipids of this microalga has not been published yet.
Keywords: Microalgae; Extraction; Fractionation; Bioprospecting; Lipids; Ultra-high performance liquid chromatography–; high resolution mass spectrometry;

The objective of this research is to develop and validate a sensitive and reproducible UPLC–MS/MS method to quantify irinotecan, its active metabolite SN-38 and SN-38 glucuronide (phase II metabolite of SN-38) simultaneously in different bio-matrices (plasma, urine, feces), tissues (liver and kidney) and to use the method to investigate its pharmacokinetic behavior in rats. Irinotecan, SN-38 and SN-38 glucuronide has been resolved and separated by C18 column using acetonitrile and 0.1% formic acid in water used as the mobile phases. Triple quadruple mass spectrometer using multiple reaction monitoring (MRM) with positive scan mode were employed to perform mass analysis. The results showed that the linear response range of irinotecan and SN-38 in plasma, feces, liver and kidney is 4.88–10000 nM, 39–5000 nM, 48.8–6250 nM and 48.8–6250 nM, respectively (R2  > 0.99). In case of SN-38 glucuronide, the standard curves were linear in the concentration range of 6.25–2000 nM, 4.88–1250 nM, 9.8–1250 nM and 9.8–1250 nM in plasma, feces, liver and kidney homogenates, respectively. The lower limit of detection (LLOD) of irinotecan, SN-38 and SN-38 glucuronide was determined to be less than 25 nM in all bio-matrices as well as tissue homogenates. Recoveries of irinotecan, SN-38 and SN-38 glucuronide at three different concentrations (low, medium and high) were not less than 85% at three different concentrations in plasma and feces. The percentage matrix factors in different bio-matrices and tissues were within 20%. The UPLC–MS/MS method was validated with intra-day and inter-day precision of less than 15% in plasma, feces, liver and kidney. Owing to the high sensitivity of this method, only 20 μl of plasma, urine and homogenates of liver, kidney and feces is needed. The validated method has been successfully employed for pharmacokinetic evaluation of irinotecan in male wistar rats to quantify irinotecan, SN-38 and SN-38 glucuronide in plasma, feces, and urine samples.
Keywords: Irinotecan; SN-38; Pharmacokinetics; UPLC–MS/MS; SN-38 glucuronide;

Vonoprazan fumarate (TAK-438) is a potassium-competitive acid blocker which was approved in Japan for a treatment of acid-related diseases.In this study a simple and validated bioanalytical method, which can simultaneously determine vonoprazan (TAK-438F) and its four metabolites (M-I, M-II, M-III and M-IV-Sul) in human plasma, was developed. The method is based on protein precipitation and subsequent ultra-high performance liquid chromatography separation followed by tandem mass spectrometry detection. The mass spectrometric parameters for detection of TAK-438F, M-I, M-III and M-IV-Sul were modified from their optimum values in order to achieve a simultaneous quantification while retaining enough sensitivity and wide dynamic ranges for all the target analytes.The validity and robustness of the method was verified through a validation study as per the regulatory guidance on bioanalytical method validation. The calibration ranges are 0.1–100 ng/mL for TAK-438F and M-III, and 1–1000 ng/mL for M-I, M-II and M-IV-Sul using the 100 μL of human plasma. The total run time per sample is 5 min. The working solution for M-III was recommended to be prepared separately, especially for the long-term use, in order to avoid the instability of M-III in the mixed working solutions, which could cause the high consumption of reference standards.The established method was applied to clinical pharmacokinetic studies and concentrations of all the analytes in human plasma were successfully determined with high reproducibility ensured by incurred sample reanalysis, indicating the suitableness of the established method.
Keywords: Vonoprazan (TAK-438F); Bioanalytical method validation (BMV); Simultaneous quantification; LC/MS/MS; Regulated bioanalysis; Human plasma;

Metabolomics approach to explore the effects of Kai-Xin-San on Alzheimer’s disease using UPLC/ESI-Q-TOF mass spectrometry by Hang Chu; Aihua Zhang; Ying Han; Shengwen Lu; Ling Kong; Jinwei Han; Zhidong Liu; Hui Sun; Xijun Wang (50-61).
Alzheimer’s disease (AD) is a multifactorial neurodegenerative disease that influences elderly populations, with no effective method for its treatment so far. To improve its diagnosis and treatment, changes of small molecule metabolite during AD should be elucidated. Kai-Xin-San (KXS) is an herbal formulae that has been widely used to treat mental disorders, especially amnesia and depression in China. Experimental AD was induced in rats by an intraperitoneal injection of d-galactose (d-gal) and administered intragastrically with aluminum chloride (AlCl3) simultaneously for 105 days. Morris water maze task as a behavior test was used for testing the effects of KXS on AD model and pathological changes to the brain were assessed by hematoxylin-eosin staining and immunohistochemistry. The levels of Bcl-2 and ChAT in hippocampus were evaluated by western-blot. Furthermore, metabolite profiling of AD was performed through ultra-performance liquid chromatography/electrospray ionization quadruple time-of- flight-high-definition mass spectrometry (UPLC/ESI-Q-TOF/HDMS) combined with pattern recognition approaches and pathway analysis. d-gal and AlCl3-treated caused a decline in spatial learning and memory, hippocampal histopathological abnormalities and increased Aβ1-40 levels in the brain cortex and hippocampus along with decreased Bcl-2 and ChAT expression in the hippocampus. KXS significantly improved the cognitive impairment induced by d-gal and AlCl3, attenuated hippocampal histopathological abnormalities, reduced Aβ1-40 levels and increased Bcl-2 and ChAT expression in the hippocampus. A total of 48 metabolites were considered as potential biomarkers of AD, and 36 metabolites may correlate with the regulation of KXS treatment on AD. Changes in AD metabolic profiling were close to normal states through regulating multiple perturbed pathways after KXS treatment. This study has revealed the potential biomarkers and metabolic networks of AD, illuminated the biochemistry mechanism of AD and the metabolic pathways influenced by KXS.
Keywords: Alzheimer’s disease; Metabolomics; Kai-Xin-San; SYNAPT G2-Si HDMS; Traditional chinese medicine;

Determination of American ginseng saponins and their metabolites in human plasma, urine and feces samples by liquid chromatography coupled with quadrupole time-of-flight mass spectrometry by Jin-Yi Wan; Chong-Zhi Wang; Zhi Liu; Qi-Hui Zhang; Mark W. Musch; Marc Bissonnette; Eugene B. Chang; Ping Li; Lian-Wen Qi; Chun-Su Yuan (62-73).
American ginseng is a commonly consumed herbal medicine in the United States and other countries. Ginseng saponins are considered to be its active constituents. We have previously demonstrated in an in vitro experiment that human enteric microbiota metabolize ginseng parent compounds into their metabolites. In this study, we analyzed American ginseng saponins and their metabolites in human plasma, urine and feces samples by liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS). Six healthy male volunteers ingested 1 g of American ginseng twice a day for 7 days. On day 7, biological samples were obtained and pretreated with solid phase extraction. The ginseng constituents and their metabolites were characterized, including 5 ginseng metabolites in plasma, 10 in urine, and 26 in feces. For the plasma, urine and feces samples, the levels of ginsenoside Rb1 (a major parent compound) were 8.6, 56.8 and 57.7 ng/mL, respectively, and the levels of compound K (a major metabolite) were 58.4 ng/mL, 109.8 ng/mL and 10.06 μg/mL, respectively. It suggested that compound K had a remarkably high level in all three samples. Moreover, in human feces, ginsenoside Rk1 and Rg5, Rk3 and Rh4, Rg6 and F4 were detected as the products of dehydration. Further studies are needed to evaluate the pharmacological activities of the identified ginseng metabolites.
Keywords: American ginseng; Ginsenosides; Enteric; Microbiota; Ginsenoside Rb1; Compound K; LC-Q-TOF-MS;

The analysis of blood provides in depth chemical information of physiological states of organisms. Hemolymph (blood) is the fluid in the open circulatory system of Drosophila melanogaster that is the medium for molecules regulating a wide variety of physiological activities and signaling between tissues. Adult Drosophila is typically less than 3 mm in length and, as a consequence, the available volume of hemolymph is usually less than 50 nL from individual flies. Proteomic analysis of volume-limited hemolymph is a great challenge for both sample handling and subsequent mass spectrometry characterization of this chemically diverse biological fluid with a wide dynamic range of proteins in concentrations. Less abundant proteins, in particular, could be easily lost during sample preparation or missed by current mass spectrometry methods. This article describes simple and customized RPLC column and IEX columns to prefractionate volume-limited hemolymph without excessive dilution. Step-gradient elution methods were developed and optimized to enhance the identification of novel proteins from an individual fruit fly hemolymph sample. Fractions from each step gradient was analyzed by an Agilent nano-RPLC chip column and then characterized by high mass resolution and high mass accuracy orbitrap mass spectrometry. As a result, both RPLC (11 proteins) and IEX fractionation approaches (9 proteins) identified more proteins than an unfractionated control approach with higher protein scores, emPAI values and coverage. Furthermore, a significant number of novel proteins were revealed by both RPLC and IEX fractionation methods, which were missed by unfractionated controls. The demonstration of this method establishes a means to deepen proteomic analysis to this commonly used, important biological model system.
Keywords: Drosophila melanogaster; Reverse phase liquid chromatography; Ion exchange chromatography; Nanoliter samples; Shotgun proteomics;

Traditional Chinese medicines (TCMs) bring a great challenge in quality control and evaluating the efficacy because of their complexity of chemical composition. Chemometric techniques provide a good opportunity for mining more useful chemical information from TCMs. Then, the application of chemometrics in the field of TCMs is spontaneous and necessary. This review focuses on the recent various important chemometrics tools for chromatographic fingerprinting, including peak alignment information features, baseline correction and applications of chemometrics in metabolomics and modernization of TCMs, including authentication and evaluation of the quality of TCMs, evaluating the efficacy of TCMs and essence of TCM syndrome. In the conclusions, the general trends and some recommendations for improving chromatographic metabolomics data analysis are provided.
Keywords: Chemometrics; Chromatographic fingerprinting; Metabolomics; Traditional Chinese medicine;

The loading characteristics of recombinant Staphyloccocus aureus protein A (rSPA) on polypropylene (PP) capillary-channeled polymer (C-CP) fibers were investigated through breakthrough curves and frontal analysis. The dynamic adsorption data was fit to various isotherm models to assess the possible mode of rSPA-PP fiber adsorption. Among them, the Langmuir-linear model fit the experimental data best, suggesting a two-stage mechanism of adsorption. The first stage involves the formation of a monolayer coverage, which follows the Langmuir isotherm. When the adsorbate concentration increases, solute starts to adsorb onto the already adsorbed layer, following a linear adsorption response. The relationship between the rSPA loading and flow rate and column length was also investigated. These two parameters are related through the residence time of rSPA in the column. It was determined that loading at the flow rate of 0.5 mL min−1 (∼28 mm s−1) with a 1 × 10−5  M (0.5 mg mL−1) rSPA feed concentration on a 30-cm (0.762 mm i.d.) column could conveniently produce a reasonable binding capacity of rSPA on PP surface within only 6 min. Under those conditions, the rSPA binding at 50% breakthrough was found to be ∼2.1 mg g−1 fiber. Operation of the rSPA-modified columns across ten complete processing cycles using clean-in-place conditions (including urea, guanidine HCl, and NaOH) commonly used in the bioprocessing industry allows assessment of the robustness of the rSPA capture layers. In all cases, the robustness was quite good, with the relative responses providing insights to the rSPA/PP surface structure.
Keywords: Protein A; IgG; Capillary-channeled polymer; Fibers; Downstream processing;

Accurate and reproducible measurement of blood-brain barrier (BBB) integrity is critical in the assessment of the pathophysiology of the central nervous system disorders and in monitoring therapeutic effects. The widely-used low molecular weight marker [14C]sucrose is non-specific in the absence of chromatographic separation. The purpose of this study was to develop and validate a sensitive and reproducible LC–MS/MS method for the analysis of stable isotope-modified [13C12]sucrose in brain, plasma, and blood to determine BBB permeability to sucrose. After addition of internal standard (IS, [13C6]sucrose), the marker and IS were recovered from diluted rat blood, plasma, and brain homogenate by protein precipitation using acetonitrile. The recovery of the marker and IS was almost quantitative (90–106%) for all three matrices. The recovered samples were directly injected into an isocratic UPLC system with a run time of 6 min. Mass spectrometry was conducted using multiple reaction monitoring in negative mode. The method was linear (r2  ≥ 0.99) in the concentration ranges tested for the diluted blood and plasma (10–1000 ng/mL) and brain homogenate (1–200 ng/mL). The lower limit of quantitation of the assay was 0.5 pg injected on column. The assay was validated (n  = 5) based on acceptable intra- and inter-run accuracy and precision values. The method was successfully used for the measurement of serial blood and plasma and terminal brain concentrations of [13C12]sucrose after a single intravenous dose (10 mg/kg) of the marker to rats. As expected, the apparent brain uptake clearance values of [13C12]sucrose were low in healthy rats. The method may be useful for determination of the BBB integrity in animal models.
Keywords: Sucrose; Blood-brain barrier permeability; UPLC–MS/MS; Blood; Plasma; Brain;

From top-down to bottom-up: Time-dependent monitoring of proteolytic protein degradation by LC-MS by Joanna Tucher; Tomas Koudelka; Jana Schlenk; Andreas Tholey (111-120).
The understanding of proteolytic processes includes manifold aspects, ranging from the characterization of proteases and their corresponding substrates to the localization of cleavage sites. The analysis of protease-catalyzed reactions at a single time-point in many cases excludes the identification of intermediate cleavage products of potential biological function. To overcome this problem, proteolysis has to be monitored over time.For that purpose, we established a straight-forward two-step approach. First, Tricine-SDS-PAGE separation of the proteolytic products is applied to survey the proteolytic reaction. In the second step, the reaction mixture is analyzed by an LC-MS set-up. An optimized chromatographic separation coupled to electrospray Orbitrap mass spectrometry allowed the simultaneous monitoring of intact substrates, intermediates and cleavage products of lower molecular weight. The applicability of the strategy was shown on the example of the gastric protease pepsin and its physiologically relevant substrate hen egg white lysozyme, one of the major egg allergens. While lysozyme-derived cysteine-free peptides were cleaved comparatively fast, disulfide bonds protected connected peptides from rapid peptic proteolysis. Two previously identified potential IgE-binding motifs were observed as disulfide-linked cleavage products.In summary, the presented approach is not only ideally suited for the simulation of gastro-intestinal digestion, which is of high interest in food research, but can be transferred to any protease-substrate pair of interest. Furthermore the strategy can be exploited to deduce the effect of post-translational modifications on proteolysis.
Keywords: Proteolysis; Cleavage product; Lysozyme; LC-MS; Disulfide bond; Intermediates;

Development and characteristics of polymer monoliths for advanced LC bioscreening applications: A review by Caleb Acquah; Charles K.S. Moy; Michael K. Danquah; Clarence M. Ongkudon (121-134).
Biomedical research advances over the past two decades in bioseparation science and engineering have led to the development of new adsorbent systems called monoliths, mostly as stationary supports for liquid chromatography (LC) applications. They are acknowledged to offer better mass transfer hydrodynamics than their particulate counterparts. Also, their architectural and morphological traits can be tailored in situ to meet the hydrodynamic size of molecules which include proteins, pDNA, cells and viral targets. This has enabled their development for a plethora of enhanced bioscreening applications including biosensing, biomolecular purification, concentration and separation, achieved through the introduction of specific functional moieties or ligands (such as triethylamine, N,N-dimethyl-N-dodecylamine, antibodies, enzymes and aptamers) into the molecular architecture of monoliths. Notwithstanding, the application of monoliths presents major material and bioprocess challenges. The relationship between in-process polymerisation characteristics and the physicochemical properties of monolith is critical to optimise chromatographic performance. There is also a need to develop theoretical models for non-invasive analyses and predictions. This review article therefore discusses in-process analytical conditions, functionalisation chemistries and ligands relevant to establish the characteristics of monoliths in order to facilitate a wide range of enhanced bioscreening applications. It gives emphasis to the development of functional polymethacrylate monoliths for microfluidic and preparative scale bio-applications.
Keywords: Monoliths; Polymethacrylate; Bio-screening; Chromatography; Polymerisation; Biomolecules;

Adsorption characteristics and preparative separation of chaetominine from Aspergillus fumigatus mycelia by macroporous resin by Changqing Liu; Ruihua Jiao; Lingyun Yao; Yupeng Zhang; Yanhua Lu; Renxiang Tan (135-141).
Chaetominine (CHA) is a quinazolinone alkaloid with strong anti-cancer activity produced by Aspergillus fumigatus CY018. For recovering CHA from A. fumigates efficiently, adsorption and desorption capacities of eight macroporous resins were tested in this work. Based on batch experiments, XAD-16 resin was revealed the best adsorption and desorption performance among all the tested resins. Then, adsorption kinetics and adsorption isotherms were constructed on XAD-16 resin, and the experimental data were fitted well to the pseudo first-order kinetics and Freundlick isotherm model. In the dynamic adsorption and desorption, the purity of CHA increased from 0.0314% (w/w) in the crude extract to 57.86% in the final product with recovery yield of 70.56% by a one-step treatment. Moreover, the experiments were also performed in a lab scale-up scale, in which the purity and recovery of CHA were 56.12% (w/w) and 68.02%, respectively. In addition, XAD-16 resin could be recycled 3 times for CHA separation after regeneration without adverse effects on adsorption/desorption performance. These results suggested that XAD-16 resin adsorption could act as a useful and economic method for recovering CHA from A. fumigatus.
Keywords: Adsorption kinetics and isotherms; Aspergillus fumigates; Chaetominine; Macroporous adsorption resin; Separation;

Ambient ionization based on mesoporous graphene coated paper for therapeutic drug monitoring by Ji Ji; Lei Nie; Lei Liao; Ruijun Du; Baohong Liu; Pengyuan Yang (142-149).
Paper spray (PS), as a new ambient ionization method, has been applied for direct qualitative and quantitative analysis. The high sensitivity and minimum internal energy (low spray voltage) with optimized paper spray conditions is a significant request for real application in POCT. In this study, a simple and efficient ambient ionization method is developed by spraying from a mesoporous graphene foams (MGFs)-modified paper surface. The good electrical conductivity of MGFs results in obvious spray voltage decrease. Meanwhile, the MGFs-paper substrate has a well improvement in separation and elution efficiency ascribing to ultrahigh specific surface area and π–π electrostatic stacking property of graphene. In combination a commercial triple quadrupole mass spectrometer, the paper spray is successfully used for analysis of amphetamine in saliva. The linear dynamic ranges expand 10 fold in comparison with unmodified chromatography papers and the low limit of quantitation (LOQ) is as low as 1 pg/mL. A small sample volume (0.5 μL) could be analyzed immediately after spotting, without any pretreatment. The performance of this method was demonstrated for application in fast point-of-care mass spectrometry.
Keywords: Paper spray; ESI mass spectrometry; Graphene; Amphetamine; Drug monitoring;

Validated UPLC/MS/MS assay for quantitative bioanalysis of elbasvir in rat plasma and application to pharmacokinetic study by Haiyan Liu; Hongjiang Xu; Wei Song; Yinsheng Zhang; Sen Yu; Xin Huang (150-156).
Rapid, sensitive, selective and accurate ultra performance liquid chromatography with tandem mass spectrometry (UPLC–MS/MS) method was developed and validated for the quantification of elbasvir (ELB) in rat plasma with deuterated elbasvir (ELB-D6) as internal standard (IS).Sample preparation was done by protein precipitation using acetonitrile containing 50 ng/mL IS. Chromatographic separation was achieved by an UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm) column with a gradient mobile phase consisting of acetonitrile–water (containing 5.0 mM ammonium acetate with 0.01% acetic acid, pH 4.5) as mobile phase at a flow rate of 0.3 mL/min for 3 min. ELB was monitored using positive electrospray triple quadrupole mass spectrometer (Waters Xevo TQ-S) via multiple reaction monitoring (MRM) mode. The monitored transitions were set at m/z 882.51 → 656.42 and m/z 888.49 → 662.43 for ELB and ELB-D6, respectively. The achieved lower limit of quantification was 1.0 ng/mL. The validated method had an excellent linearity in the range of 1.0–2000 ng/mL (r2  > 0.996). Recovery efficiency at three levels QC concentrations of 2.0 (low), 160 (medium) and 1600 (high) ng/mLwas in the range of 98.29–106.40% for ELB. Matrix effect was found to be minimal. The intra- and inter-day precisions were less than 7.01%. The intra- and inter-day accuracies were determined to be within ±6.23% for all accuracy measurements. The validated simple and rapid UPLC–MS/MS method was successfully used to the pharmacokinetics study of ELB in rats, providing its applicability in relevant preclinical studies.
Keywords: Elbasvir; Deuterated elbasvir; UPLC–MS/MS; Plasma; Pharmacokinetics; Rat;

A lateral flow immunosensor for direct, sensitive, and highly selective detection of hemoglobin A1c in whole blood by Shu Hwang Ang; T. Malathi Thevarajah; Pei Meng Woi; Yatimah binti Alias; Sook Mei Khor (157-165).
An immunosensor that operates based on the principles of lateral flow was developed for direct detection of hemoglobin A1c (HbA1c) in whole blood. We utilized colloidal gold-functionalized antibodies to transduce the specific signal generated when sandwich immuno-complexes were formed on the strip in the presence of HbA1c. The number and intensity of the test lines on the strips indicate normal, under control, and elevated levels of HbA1c. In addition, a linear relationship between HbA1c levels and immunosensor signal intensity was confirmed, with a dynamic range of 4–14% (20–130 mmol mol−1) HbA1c. Using this linear relationship, we determined the HbA1c levels in blood as a function of the signal intensity on the strips. Measurements were validated using the Bio-Rad Variant II HPLC and DCA Vantage tests. Moreover, the immunosensor was verified to be highly selective for detection of HbA1c against HbA0, glycated species of HbA0, and HbA2. The limit of detection was found to be 42.5 μg mL−1 (1.35 mmol mol−1) HbA1c, which is reasonably sensitive compared to the values reported for microarray immunoassays. The shelf life of the immunosensor was estimated to be 1.4 months when stored at ambient temperature, indicating that the immunoassay is stable. Thus, the lateral flow immunosensor developed here was shown to be capable of performing selective, accurate, rapid, and stable detection of HbA1c in human blood samples.
Keywords: Glycated hemoglobin; Diabetes-mellitus; HbA1c; Biosensor; Lateral flow; Whole blood;

Determination of macrocyclic lactones in bovine liver using QuEChERS and HPLC with fluorescence detection by David Pimentel-Trapero; Alicia Sonseca-Yepes; Sonia Moreira-Romero; Miguel Hernández-Carrasquilla (166-172).
A method for the determination of the macrocyclic lactones Abamectin (ABA), Ivermectin (IVER), Doramectin (DORA) and Moxidectin (MOXI) in liver using HPLC with fluorescence detection is presented. The analytes were extracted and purified using a QuEChERS method. Prior to injection into an HPLC system the sample extracts were reacted with a mixture of 1-methylimidazole, triethylamine, and trifluoroacetic anhydride to obtain the fluorescent derivatives of the analytes. The method has been validated according to EU Decision 2002/657. Recovery for the analytes ranged from 85 to 90%. Repeatibility and Trueness varied between 8–18% and −6 to 6%, respectively and were compliant with the requirements stated in EU Decision 2002/657. Limit of Decision (CCα) were: 22.8 μg kg−1 (ABA), 126.3 μg kg−1 (DORA), 118.3 μg kg−1 (MOXI) and 126.9 μg kg−1 (IVER).
Keywords: Macrocyclic lactones; Liver; HPLC-fluorescence; QuEChERS;

Carvedilol is an antihypertensive drug, which is available in clinical practice as a racemic mixture. (S)-(−)-carvedilol is a β- and α1-adrenergic antagonist, while (R)-(+)-carvedilol only acts as an α1-adrenergic antagonist. Carvedilol is metabolized mainly by glucuronidation and, to a lesser extent, by CYP2D6 to hydroxyphenyl carvedilol (OHC) and by CYP2C9 to O-desmethyl carvedilol (DMC). This study describes the development and validation of a method for the sequential analysis of the enantiomers of carvedilol, OHC and DMC in plasma using a Chirobiotic® V chiral-phase column coupled to an LC–MS/MS system. The method was linear in the range of 0.05–100, 0.05–10 and 0.02–10 ng/mL for the carvedilol, OHC and DMC enantiomers, respectively. Application of the method to the investigation of a patient with type 2 diabetes mellitus treated with a single oral dose of 25 mg racemic carvedilol showed plasma accumulation of the (R)-(+)-carvedilol, (R)-(+)-DMC and (R)-(+)-OHC enantiomers. These results suggest that plasma accumulation of (R)-(+)-carvedilol cannot be explained by its oxidative metabolism.
Keywords: Carvedilol; Enantiomers; Pharmacokinetics; Metabolism; Plasma; Diabetes;

Recovery and purification of a Kluyvermyces lactis β-galactosidase by Mixed Mode Chromatography by Micael de Andrade Lima; Maria de Fátima Matos de Freitas; Luciana Rocha Barros Gonçalves; Ivanildo José da Silva Junior (181-191).
Mixed Mode Chromatography (MMC) is a potential separation technique that allows simultaneous ionic and hydrophobic interactions between the adsorbent and the adsorbate. The aim of this work was to assess the recovery and purification of a Kluyveromyces lactis β-galactosidase employing MMC. Protein precipitation and dialysis were performed in order to concentrate the enzyme of interest and eliminate cell debris and other interferences inherent in the fermentation medium. The best conditions for both adsorption and desorption were attained by a non-factorial Central Composite Experimental Design and employed in the chromatographic runs with resin CAPTO MMC. Fermentation yielded mean values of total enzyme concentration of 0.44 mg/mL, enzymatic activity (employing lactose as a substrate) of 74 U/mL and specific activity of 168 U/mg. The Purification Factor (PF) obtained was of 1.17. After precipitation and dialysis, the subsequent chromatographic run resulted in recovery values ​​of 41.0 and 48.2% of total protein concentration and enzymatic activity, respectively. SDS-PAGE electrophoresis confirmed the purification evolution throughout the unit operations employed, attesting the feasibility of the technique to obtain enzymes with not only considerable degree of purity but also possessing high-added value.
Keywords: Mixed Mode Chromatography; Biomolecule recovery and purification; Adsorption;

Development of a readily applied method to quantify ractopamine residue in meat and bone meal by QuEChERS-LC–MS/MS by Vanessa Gressler; Angélica R.L. Franzen; Gustavo J.M.M. de Lima; Fernando C. Tavernari; Osmar A. Dalla Costa; Vivian Feddern (192-200).
A QuEChERS method of ractopamine (RCT) residue detection in swine meat and bone meal (MBM) samples was demonstrated. Samples were hydrolyzed with protease and β-glucuronidase prior to QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) extraction and clean-up. Samples were analyzed in a Liquid Chromatography (equipped with ACE 5 C18 column under gradient elution) coupled with a triple quadrupole mass spectrometer operating in positive electrospray ionization mode (using multiple reaction monitoring, MRM). The method was validated for its specificity, decision limit (CCα), detection capability (CCβ), recovery, repeatability, reproducibility, linearity, limits of detection (LODs), quantification (LOQs), and stability according to international guidelines (European Commission Decision 2002/657/EC). Recoveries ranged from 96.3 to 107.0%. Repeatability and reproducibility showed both RSD < 5.7% and 3.1%, respectively. LODs and LOQs were 1.91 and 6.36 ppb, respectively. CCα and CCβ values were 1.91 and 2.37 ppb, respectively. RCT showed good stability for spiked samples and real samples when the concentration was higher, otherwise at lower concentration stability was lower. The proposed method can be successfully applied on a regular basis for the determination of RCT in MBM, demonstrating the usefulness of the method as a tool for compliance monitoring in regulatory laboratories.
Keywords: Swine; Feed ingredient; β-agonist; Method development; Mass spectrometer; Dispersive solid phase extraction;

Bupropion metabolites formed via oxidation and reduction exhibit pharmacological activity, but little is known regarding their stereoselective disposition. A novel stereoselective liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed to separate and quantify enantiomers of bupropion, 4-hydroxybupropion, and erythro- and threo-dihydrobupropion. Liquid-liquid extraction was implemented to extract all analytes from 50 μL human plasma. Acetaminophen (APAP) was used as an internal standard. The analytes were separated on a Lux 3 μ Cellulose-3 250 × 4.6 mm column by methanol: acetonitrile: ammonium bicarbonate: ammonium hydroxide gradient elution and monitored using an ABSciex 5500 QTRAP triple-quadrupole mass spectrometer equipped with electrospray ionization probe in positive mode. Extraction efficiency for all analytes was ≥70%. The stability at a single non-extracted concentration for over 48 h at ambient temperature resulted in less than 9.8% variability for all analytes. The limit of quantification (LOQ) for enantiomers of bupropion and 4-hydroxybupropion was 0.3 ng/mL, while the LOQ for enantiomers of erythro- and threo-hydrobupropion was 0.15 ng/mL. The intra-day precision and accuracy estimates for enantiomers of bupropion and its metabolites ranged from 3.4% to 15.4% and from 80.6% to 97.8%, respectively, while the inter-day precision and accuracy ranged from 6.1% to 19.9% and from 88.5% to 99.9%, respectively. The current method was successfully implemented to determine the stereoselective pharmacokinetics of bupropion and its metabolites in 3 healthy volunteers administered a single 100 mg oral dose of racemic bupropion. This novel, accurate, and precise HPLC–MS/MS method should enhance further research into bupropion stereoselective metabolism and drug interactions.
Keywords: Bupropion; 4-Hydroxybupropion; threo-Dihydrobupropion; erythro-Dihydrobupropion; Enantiomers; Stereoselective; HPLC–MS/MS;

The main objective of this study was to develop and validate a simpler and less time consuming analytical method for determination of propofol glucuronide from hair sample, by using mixed mode anion exchange cartridge and liquid chromatography-tandem mass spectrometry (LC–MS/MS). The study uses propofol glucuronide, a major metabolite of propofol, as a marker for propofol abuse. The hair sample was digested in sodium hydroxide solution and loaded in mixed-mode anion cartridge for solid phase extraction. Water and ethyl acetate were used as washing solvents to remove interfering substances from the hair sample. Consequently, 2% formic acid in ethyl acetate was employed to elute propofol glucuronide from the sorbent of mixed-mode anion cartridge, and analyzed by LC–MS/MS. The method validation parameters such as selectivity, specificity, LOD, LLOQ, accuracy, precision, recovery, and matrix effect were also tested. The linearity of calibration curves showed good correlation, with correlation coefficient 0.998. The LOD and LLOQ of the propofol glucuronide were 0.2 pg/mg and 0.5 pg/mg, respectively. The intra and inter-day precision and accuracy were acceptable within 15%. The mean values of recovery and matrix effect were in the range of 91.7–98.7% and 87.5–90.3%, respectively, signifying that the sample preparation, washing and extraction procedure were efficient, and there was low significant hair matrix effect for the extraction of propofol glucuronide from hair sample on the mixed mode anion cartridge. To evaluate the suitability of method, the hair of propofol administered rat was successfully analyzed with this method.
Keywords: Propofol glucuronide; Hair analysis; Solid phase extraction; LC–MS/MS;