Journal of Chromatography B (v.942-943, #C)

Dictamnine is an herbal ingredient isolated from the root bark of Dictamnus dasycarpus Turcz. (Rutaceae). The present study was aimed at the development of an ultra-high performance liquid chromatography–tandem mass spectrometry method to quantify the concentration of dictamnine in rat plasma and tissues for the in vivo pharmacokinetics, tissue distribution and excretion study. Biological samples were processed with protein precipitation. Skimmianine was chosen as internal standard. The chromatographic separation was carried out on a Thermo Syncronis C18 column (2.1 mm × 50 mm, 1.7 μm) with an isocratic mobile phase consisting of methanol and 0.1% formic acid water (75:25, v/v). The detection was accomplished by using positive ion electrospray ionization in multiple reaction monitoring (MRM) mode. The MS/MS ion transitions were monitored at m/z 200.0 → 129.0 for dictamnine and 260.3 → 227.1 for IS, respectively. An excellent linearity was observed over the concentration range from 0.5 to 250 ng/mL. The lower limit of quantification (LLOQ) was 0.5 ng/mL for dictamnine. The developed method was rapid, accurate, and highly sensitive and selective. It was successfully applied to the in vivo pharmacokinetics, tissue distribution and excretion study of dictamnine in rats after oral or intravenous administration of dictamnine.
Keywords: Dictamnine; Pharmacokinetics; Bioavailability; Tissue distribution; Excretion; Dictamnus dasycarpus;

Immobilization of trypsin onto the superparamagnetic carboxymethyl chitosan (Fe3O4 (PEG + CM-CTS)) nanoparticles was studied. FTIR and fluorescence spectroscopy data demonstrated that the Fe3O4 (PEG + CM-CTS) nanoparticles were capable of preventing the trypsin unfolding. Due to the large specific surface area and excellent dispersibility, the adsorption equilibrium of trypsin onto the nanoparticles was achieved quickly within 30 min. The results of kinetic parameters (Michaelis constant, Km) with regards to the free trypsin (FT) and immobilized trypsin (IT) were 23.1 and 24.1 mg/mL separately, implying that IT has less affinity to the BAEE used as the substrate. However, the MALDI-TOF MS analysis indicated that, IT could be used for fast and efficient Bovine Serum Albumin (BSA) digestion under very facile processes, thanks to the easy manipulation of the magnetic nanoparticles (MNs), as well as the greatly reduced digestion time (from 12 h to 15 min). IT exhibited a sound stability after re-uses for six times, with 76.3% of the initial activity thereof still retained, thus making it more attractive in the application fields. These results are expected to open up a great potential use of such Fe3O4 (PEG + CM-CTS) nanoparticles as a superior nanosupport for trypsin immobilization.
Keywords: Superparamagnetic nanoparticles; Carboxymethyl c hitosan; Trypsin immobilization; Proteolysis;

Simple segmental hair analysis for α-pyrrolidinophenone-type designer drugs by MonoSpin extraction for evaluation of abuse history by Akira Namera; Kyohei Konuma; Takeshi Saito; Shigenori Ota; Hiroshi Oikawa; Shota Miyazaki; Shumari Urabe; Hiroaki Shiraishi; Masataka Nagao (15-20).
For detection of a history of drug abused, we developed a simple method for extracting pyrrolidinophenone-type designer drugs in human hair by using a MonoSpin® C18 column. Target drugs were extracted from a single alkaline-digested hair segment (length, 10 mm; weight, ca 0.1 mg). The analytes extracted were then analyzed by high-performance liquid chromatography–mass spectrometry without evaporation of the eluent after MonoSpin extraction. Linearity from 0.5 to 500 ng/mg was observed for all the tested drugs using an internal standard method (correlation coefficients >0.998) and the limit of detection was 0.2 ng/mg. The recoveries were between 0.7 and 11.1%. The coefficients for intraday and interday variations at 4, 40, 200, and 400 ng/mg in hair were between 0.7 and 11.1%. This method was successfully applied to the identification of these designer drugs in segmented human hair from drug abusers and indicated their history of drug abuse. The results were consistent with the patients’ statements, indicating that this rapid method can be used to detect a history of drug abuse.
Keywords: MonoSpin; Pyrrolidinophenone; Designer drug; Hair analysis;

This work describes the development of a fast and reliable method based on capillary zone electrophoresis coupled with electrospray ionization–mass spectrometry (CZE–ESI–MS) for the determination of modified nucleosides in untreated human urine. The target compounds were guanine, 1-methyl-guanine, 7-methyl-guanine, 9-methyl-guanine, adenosine, 1-methyl-adenosine, cytidine, guanosine, 7-methyl-guanosine. As internal standards, ribose-2-13C-adenosine and 8-13C-guanine were used. The CZE separation was carried out in acidic medium (pH 2.5). MS detection with a single quadrupole, with ESI operating in positive-ion mode, was optimized. For the analysis of urine samples, owing to the endogenous character of these analytes different quantification strategies were explored. The standard additions method, matrix-matched calibration in synthetic urine and calibration in pure aqueous medium were compared in order to evaluate the endogenous levels of these compounds in human urine. The results obtained showed that calibration in synthetic urine as a surrogate matrix was an appropriate alternative to the method of standard additions for the accurate quantitation of compounds such as guanine, 1-methyl-guanine, 7-methyl-guanine, adenosine, 1-methyl-adenosine and cytidine by CE–ESI–MS directly in the urine matrix; values in the range 0.1 μg/mL for cytidine and 6.4 μg/mL for 7mGua, as the lowest and the highest level, were found in untreated urine from healthy volunteers. These results were confirmed by LC–MS/MS detection. It can be concluded that the electrophoretic CZE–ESI–MS methodology offers a valid and reliable alternative for the determination of urinary nucleosides at naturally occurring levels in healthy individuals.
Keywords: Nucleosides; Direct analysis; Synthetic urine; Surrogate matrix; Capillary electrophoresis; Mass spectrometry;

Cytokinins (CKs), a vital family of phytohormones, play important roles in the regulation of shoot and root development. However, the quantification of CKs in plant samples is frequently affected by the complex plant matrix. In the current study, we developed a simple, rapid and efficient hydrophilic interaction chromatography-solid phase extraction (HILIC–SPE) method for CKs purification. CKs were extracted by acetonitrile (ACN) followed by HILIC–SPE (silica as sorbents) purification. The extraction solution of plant samples could be directly applied to HILIC–SPE without solvent evaporation step, which simplified the analysis process. Moreover, with HILIC chromatographic retention mechanism, the hydrophobic co-extracted impurities were efficiently removed. Subsequently, CKs were separated by RPLC, orthogonal to the HILIC pretreatment process, and detected by tandem mass spectrometry. The method exhibits high specificity and recovery yield (>77.0%). Good linearities were obtained for all eight CKs ranging from 0.002 to 100 ng mL−1 with correlation coefficients (r) higher than 0.9927. The limits of detection (LODs, signal/noise = 5) for the CKs were between 1.0 and 12.4 pg mL−1. Reproducibility of the method was evaluated by intra-day and inter-day measurements and the results showed that relative standard deviations (RSDs) were less than 10.5%. Employing this method, we successfully quantified six CKs in 20 mg Oryza sativa leaves and the method was also successfully applied to Brassica napus (flower and leaves).
Keywords: Cytokinins; Sample preparation; Hydrophilic interaction; Mass spectrometry;

Activity-guided identification of acetogenins as novel lipophilic antioxidants present in avocado pulp (Persea americana) by Dariana Rodríguez-Sánchez; Christian Silva-Platas; Rocío P. Rojo; Noemí García; Luis Cisneros-Zevallos; Gerardo García-Rivas; Carmen Hernández-Brenes (37-45).
Avocado fruit is a rich source of health-related lipophilic phytochemicals such as monounsaturated fatty acids, tocopherols, carotenes, acetogenins and sterols. However, limited information is available on the contribution of specific phytochemicals to the overall antioxidant capacity (AOC) of the fruit. Centrifugal partition chromatography was used as fractionation tool, guided by an in vitro chemical assay of oxygen radical absorbance capacity (ORAC). Subsequent experiments focused on isolation and characterization of the chemical nature of the main contributors to lipophilic AOC of avocado pulp. ORAC values obtained for acetogenins were contrasted with results from an isolated kidney mitochondria membrane lipid peroxidation bioassay. The present study established that lipophilic AOC of the pulp was significantly higher than its hydrophilic AOC. Our results confirmed the presence of acetogenins in the fractions with highest lipophilic AOC, and for the first time linked them as contributors to lipophilic-ORAC values. Further HPLC-PDA/MS-TOF analysis led to structural elucidation of two novel acetogenins, not previously reported as present in avocado pulp, along with five already known related-compounds. Antioxidant properties observed for avocado pulp acetogenins by the ORAC assay suggested that, in the presence of an emulsifying agent, acetogenins could serve as novel lipophilic antioxidants in a food matrix. Results from isolated mitochondria lipid peroxidation bioassay, indicated that L-ORAC values which may have relevance for food matrix applications, should not be interpreted to have a direct relevance in health-related claims, compounds need to be evaluated considering the complexity of biological systems.
Keywords: Acetogenins; Avocado; Antioxidant capacity (AOC); Centrifugal partition chromatography; Activity-guided isolation;

Simultaneous determination of eight illegal dyes in chili products by liquid chromatography–tandem mass spectrometry by Juan Li; Xiao-Ming Ding; Dan-Dan Liu; Fei Guo; Yu Chen; Yan-Bing Zhang; Hong-Min Liu (46-52).
A sensitive and accurate method based on the use of liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed for the simultaneous determination of eight illegal synthetic dyes (Sudan (I–IV), Para Red, Rhodamine B, Chrysoidin and Auramine O) in chili products. A simple sample treatment procedure entailing the use of an extraction step with acetonitrile/H2O (9/1) without further cleanup was developed. HPLC was performed on a C18 column using a multistep gradient elution with 5 mM ammonium acetate (pH 3.0 with formic acid) and methanol as the mobile phase. Mass spectral acquisition was done in multiple reaction monitoring (MRM) mode using positive electrospray ionization (ESI). Linear calibrations were obtained with correlation coefficients R 2  > 0.99. Limit of detection (LOD) and limit of quantification (LOQ) for the studied dyes were in the ranges of 0.05–0.6 μg kg−1 and 0.3–3.0 μg kg−1 depending on matrices, respectively. The recoveries of the eight synthetic dyes in five matrices ranged from 70.5% to 119.2%. The intra- and inter-day precisions (RSDs) were between 2.3–15.8% and 5.7–15.6%, respectively. The applicability of the method to the determination of eight banned dyes in chili products was demonstrated.
Keywords: Food analysis; Adulteration; Illegal dyes; LC–MS/MS; Chili products;

The profile of bile acids and their sulfate metabolites in human urine and serum by Sai Praneeth R. Bathena; Sandeep Mukherjee; Marco Olivera; Yazen Alnouti (53-62).
The role of sulfation in ameliorating the hepatotoxicity of bile acids (BAs) in humans remains unknown due to the lack of proper analytical methods to quantify individual BAs and their sulfate metabolites in biological tissues and fluids. To this end, a simple and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated to characterize the detailed BA profile in human urine and serum. The limit of quantification was 1 ng/mL and baseline separation of all analytes was achieved within in a run time of 32 min. The method was validated over the dynamic range of 1–1000 ng/mL. The LC–MS/MS method was more accurate, precise, and selective than the commercially available kits for the quantification of sulfated and unsulfated BAs, and the indirect quantification of individual sulfated BAs after solvolysis. The LC–MS/MS method was applied to characterize the BA profile in urine and serum of healthy subjects. Thirty three percent of serum BAs were sulfated, whereas 89% of urinary BAs existed in the sulfate form, indicating the role of sulfation in enhancing the urinary excretion of BAs. The percentage of sulfation of individual BAs increased with the decrease in the number of hydroxyl groups indicating the role of sulfation in the detoxification of the more hydrophobic and toxic BA species. Eighty percent of urinary BAs and 55% of serum BAs were present in the glycine-amidated form, whereas 8% of urinary BAs and 13% of serum BAs existed in the taurine-amidated form.
Keywords: LC–MS/MS; Bile acids; Sulfation; Human; Amidation;

A liquid chromatography–tandem mass spectrometry method for simultaneous detection of 22 toxic plant alkaloids, including aconitum alkaloids and their hydrolyzed products (aconitine, hypaconitine, mesaconitine, yunaconitine, crassicauline A, benzoylaconine, benzoylmesaconine, benzoylhypaconine, deacetylyunaconitine, deacetylcrassicauline A), solanaceous tropane alkaloids (atropine, anisodamine, scopolamine, anisodine), sophora alkaloids (matrine, sophoridine, oxymatrine, cytisine, N-methylcytisine), strychnos alkaloids (brucine, strychnine) and colchicine, in herbal and urine samples was developed and validated. Following sample preparation by liquid–liquid extraction, chromatographic separation was achieved on Eclipse XDB C8 column. Identification was based on two multiple reaction monitoring transitions and the relative ion intensity. Method selectivity was demonstrated. The limits of detection were 5 ng/mL for all analytes, except 50 ng/mL for cytisine. The herbal matrix effects ranged from 89% to 118%, whereas the urine matrix effects were between 91% and 109% for all analytes except cytisine (57%) and N-methylcytisine (67%). The urine extraction recovery ranged from 74% to 110% for all analytes, except cytisine (15%) and oxymatrine (30%). With the good extraction efficiency of the other major sophora alkaloids, the relatively low extraction recovery of the minor sophora alkaloids cytisine and oxymatrine did not affect identification of sophora alkaloids as a group. Carry-over was minimal at less than 0.1%. The method was successfully applied in analysis of 170 cases of suspected herbal poisoning, with aconitum alkaloids, sophora alkaloids, solanaceous tropane alkaloids, and strychnos alkaloids being detected in 53, 42, 18, and 6 cases, respectively.
Keywords: Alkaloids; Liquid chromatography; Tandem mass spectrometry; Urine; Herb;

A rapid resolution ultra performance liquid chromatography (UPLC) coupled with electrospray ionization (ESI) mass spectrometry method was developed and validated for the quantitative analysis of corydaline in rats’ plasma and various tissues for pharmacokinetic, tissue distribution and excretion studies of corydaline. The analytes were separated on an Acquity UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm) and detected with a triple quadrupole mass spectrometer using positive ion ESI in the multiple reaction monitoring (MRM) mode. The MS/MS ion transitions monitored were m/z 370.0 → 192.0 for corydaline and 354.1 → 188.0 for IS, respectively. Calibration curves (1/x 2 weighted) offered satisfactory linearity (r 2  > 0.9984) within 1–1000 ng/mL. The accuracy and precision ranged from −7.4% to 8.5% and 3.4% to 12.8%, respectively. The absolute matrix effect (94.2–119.2%), relative matrix effect (1.7–9.6%) and recoveries (81.4–93.7%) were satisfactory in all the biological matrices examined. The assay was successfully applied to the plasma pharmacokinetics, tissue distribution and excretion studies of corydaline in rats. The pharmacokinetic parameters such as half-life (t 1/2), mean residence time (MRT) and maximum concentration (C max) were determined. These preclinical data of corydaline would be useful for the clinical reference.
Keywords: Corydaline; Pharmacokinetic; Tissue distribution; Excretion; Analysis; Determination;

Keywords: Fibrinolytic compound; Pharmacokinetic characters; Tissue distribution; HPLC;

Determination of naloxone-3-glucuronide in human plasma and urine by HILIC–MS/MS by Ji Dong; Shuaibing Liu; Hua Zhang; Qianli Hua; Xuegang Zhao; Liyan Miao (83-87).
A hydrophilic interaction chromatography–tandem mass spectrometric (HILIC–MS/MS) method was developed for the direct determination of naloxone-3-glucuronide (N3G) in human plasma and urine. After a straightforward sample preparation by protein precipitation, N3G was analyzed directly without the need for hydrolysis. Chromatographic separation was performed on a HILIC column. The mobile phase was composed of acetonitrile–10 mmol/L ammonium formate (86:14, v/v), with 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 via positive electrospray ionisation (ESI+) source. The linear calibration range was 0.5 to 200 ng/mL in plasma and 10 to 5000 ng/mL in urine (r 2  > 0.99). The intra- and inter-day precision (relative standard deviation, RSD) values were below 15% and the accuracies (relative error, RE) were −7.1% to 2.8% in plasma and −1.3% to 10.3% in urine at three quality control levels. In human subjects receiving 100 mg tilidine and 8 mg naloxone, mean AUC0–24 of N3G was 160.93 ± 52.77 ng/mL h and mean C max was 75.33 ± 25.27 ng/mL. In 24-h urine samples, 8.0% of the dose was excreted in the form of N3G in urine. These results demonstrated a new method suitable for in vivo pharmacokinetic studies of N3G.
Keywords: Naloxone-3-glucuronide; HILIC–MS/MS; Pharmacokinetics;

A stable and sensitive method has been developed for use in food and livestock product safety for the detection of mycotoxins. This newly developed method allows for the determination of T-2 toxin, HT-2 toxin and diacetoxyscirpenol (DAS) in heart, liver, spleen, lung, kidney, Glandular stomach, muscular stomach, small intestine, muscle, bone and brain samples from broilers using liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). The samples were initially extracted with ethyl acetate before being filtered through a 0.22 μm nylon syringe filter and subjected to chromatographic separation on a reversed-phase C18 (50 × 2.1 mm, 3 μm) column. A mobile phase composed of 0.1% acetic acid and 10 mM ammonium acetate in methanol and water was used in an assay of the levels of T-2 toxin, HT-2 toxin and DAS. For the analysis of the target compounds, the mass spectrometer was operated under positive electrospray ionization conditions in the selected reaction monitoring mode. The limit of detection was in the range of 0.02–0.05 ng/g, whereas the limit of quantification was in the range of 0.08–0.15 ng/g. The extraction recoveries of spiked samples from the high, intermediate and low levels ranged from 58.5% to 110.5%, and the relative standard deviation (RSD (%)) values were less than 17.0%. The results of inter- and intra-day precision (RSD (%)) were within 14.7%. The results revealed that the present method could be successfully applied to the analysis of T-2 toxin, HT-2 toxin and DAS in the real samples
Keywords: T-2 toxin; HT-2 toxin; Diacetoxyscirpenol; LC–MS/MS; Broiler biological matrice;

Analysis of neutral lipids from microalgae by HPLC-ELSD and APCI-MS/MS by F. Donot; G. Cazals; Z. Gunata; D. Egron; J. Malinge; C. Strub; A. Fontana; S. Schorr-Galindo (98-106).
A method was developed to analyze neutral lipids through the use of three triglycerides, four free fatty acids, six di- and four mono-glycerides standards by high performance liquid chromatography (HPLC) normal phase coupled with either with evaporative light scattering detector (ELSD) or with mass spectrometry (MS) operating in atmospheric pressure chemical ionization (APCI) mode. The method was applied to the determination of the neutral lipid fraction from a Botryococcus braunii race A (B. braunii) culture. This method led us to identify neutral lipids synthesized by B. braunii in a single analysis within 45 min through HPLC–APCI-MS/MS technique.
Keywords: Neutral lipids; Microalgae; HPLC; ELSD; APCI-MS/MS; SPE;

A novel uHPLC–MS/MS method for the quantitation of AZD7451 (AZ12607092) in human plasma by Cody J. Peer; Jeffrey L. Brown; Timothy J. Martin; Jeffrey Roth; Shawn D. Spencer; Patrick Brassil; Katharine A. McNeill; Teri N. Kreisl; Howard A. Fine; William D. Figg (107-112).
Tropomyosin-related kinases (Trk) are tyrosine kinase receptors implicated in tumor proliferation, invasion, and survival signaling across a number of tumors, making them potentially attractive targets for the treatment of cancer. AZD7451 is a potent and selective inhibitor of Trk kinases currently undergoing a Phase I dose escalation in glioblastoma multiforme at the National Cancer Institute. A key part of early clinical testing for AZD7451 involves demonstrating that pharmacokinetic half-life and clinical exposures of AZD7451 are sufficient to inhibit Trk receptors in preclinical models. To address this need, an ultra sensitive analytical method was developed to measure the AZD7451 profile in human plasma. A liquid–liquid extraction recovered >80% of AZD7451 before quantitative analysis by ultra HPLC–MS/MS. A Varian Polaris® C18-A column and a mass transition of m/z 383.5 → 340.5 (m/z 389.6 → 342.0 for the internal standard [2H6]-AZD7451) was used, and a dynamic calibration range of 0.5–1000 ng/mL was established, which provided a sensitive (<8.5% deviation), and precise (<6%) quantitative assay for AZD7451. AZD7451 demonstrated stability in human plasma at room temperature for 24 h (<7% change) and after extraction at 4 °C for 24 h (<8% change), and was stable through 4 freeze/thaw cycles (<8% change). This method was used to measure AZD7451 plasma levels in clinical samples to confirm the sensitivity at several time points following AZD7451 treatment in subjects with glioblastoma.
Keywords: Tropomyosin-related kinase; Ultra-high performance liquid chromatography; Tandem mass spectrometry;

A high-throughput method for the simultaneous determination of multiple mycotoxins in human and laboratory animal biological fluids and tissues by PLE and HPLC–MS/MS by Xiaoqin Cao; Shuangchan Wu; Yuan Yue; Shi Wang; Yuting Wang; Li Tao; Hui Tian; Jianmei Xie; Hong Ding (113-125).
A high-throughput method for the determination of 28 mycotoxins involving pressurised liquid extraction (PLE) coupled with liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) has been optimised and validated for determination in various biological fluids and tissues of human and laboratory animals. High-throughput analysis was achieved using PLE pre-treatment and without the need for any cleanup. The extraction solvent was acetonitrile/water/acetic acid (80/19/1, v/v/v). The static extraction time was 5 min. The extraction pressure and temperature were 1500 psi and 140 °C, respectively. The flush volume was 60%. The limits of detection, which were defined as CCα, varied from 0.01 μg/kg (μg/L) to 0.69 μg/kg (μg/L). The recoveries of spiked samples from 0.20 μg/kg (μg/L) to 2 μg/kg (μg/L) ranged from 71% to 100.5% with relative standard deviations of less than 17.5%, except FB1 and FB2 recoveries, which were lower than 60%. The method was successfully applied in real samples, and the data indicate that this technique is a useful analytical method for the determination of mycotoxins from humans and animals. To the best of our knowledge, this method is the first for the large-scale testing of multi-class mycotoxins in all types of biological fluids and tissues that uses PLE and HPLC–MS/MS.
Keywords: Multiple mycotoxins; Humans; Laboratory animals; Biological fluids and tissues; PLE; LC–MS/MS;

We compared classical and multimodal cation exchange resins for the capture of recombinant antibodies from Chinese hamster ovary (CHO) cell culture supernatant. Both Capto S and Capto MMC resins present anionic groups while the multimodal Capto MMC also features a hydrophobic moiety. First we screened optimal binding and elution conditions in microplates with a pure antibody. We validated the results on the lab-scale with columns with a pure antibody and a CHO cell culture supernatant. Both resins achieved good yield and purity for the capture step of an antibody. However, the multimodal resin appeared more efficient and selective. Then we identified proteins in the antibody fraction by mass spectrometry in order to highlight the behavior of host cell proteins (HCPs).
Keywords: Mixed-mode chromatography; Cation exchange chromatography; Antibody; Mass spectrometry; Host cell proteins;

Hollow-fiber liquid-phase microextraction combined with capillary electrophoresis for trace analysis of sulfonamide compounds by Fanghong Tong; Yang Zhang; Fang Chen; Ying Li; Guanhua Ma; Yanping Chen; Kun Liu; Jiaming Dong; Jiannong Ye; Qingcui Chu (134-140).
A hollow-fiber liquid-phase microextraction (HF-LPME) method has been developed for the preconcentration of trace sulfonamides in water samples. Six commonly used sulfonamides including sulfamethazine (SMZ), sulfamerazine (SMR), sulfadiazine (SDZ), sulfadimethoxine (SDM), sulfamethoxazole (SMX), and sulfathiazole (STZ) were determined by CE with electrochemical detection (CE-ED) after microextraction. Several factors that affect extraction efficiency, separation, and detection were investigated. Under the optimum conditions, above sulfonamide compounds could achieve baseline separation within 35 min, exhibiting a linear calibration over three orders of magnitude (r 2  ≥ 0.998); the obtained enrichment factors were between 121 (for SDZ) and 996 (for SDM), and the LODs were in the range of 0.033–0.44 ng/mL. The proposed HF-LPME/CE-ED method has been applied for the sensitive analyses of the real-world water samples with recoveries in the range of 75.1–109%.
Keywords: Hollow-fiber liquid-phase microextraction; Sulfonamides; Capillary electrophoresis; Electrochemical detection; Water analysis;

A simple, RRLC–ESI-MS/MS method was developed for the simultaneous determination of five oleanane pulchinenosides (B3, BD, B7, B10, and B11), in rat plasma following solid-phase extraction (SPE). Detection and quantitation were performed by MS/MS using electrospray ionization (ESI) and multiple reaction monitoring (MRM) mode. The MS/MS transitions of the triterpenoidal saponins: m/z 911.4 → 603.2, 749.4 → 471.3, 895.6 → 733.2, 733.5 → 455.3, and 579.3 → 371.1 were monitored for B3, BD, B7 and B10, B11 and internal standard (Forsythin), respectively. The method was validated in plasma samples, showed good linearity over a wide concentration range (r 2  > 0.99), and with lower limits of quantification of 1.11 (B3), 0.751 (BD), 0.996 (B7), 0.415 (B10), and 0.332 (B11) ng/mL. The intra- and inter-day assay variability was less than 15% for all analytes. The mean extraction recoveries of analytes and IS from rats plasma were all more than 70.0%. The validation results demonstrate that this method is robust and specific. The validated method was successfully applied for the pharmacokinetic and bioavailability studies of the five pulchinenosides which are potentially active saponins present in P. chinensis saponins (PRS) extracts.
Keywords: RRLC–ESI-MS/MS; Oleanane pulchinenosides; Pulsatilla chinensis; Pharmacokinetics; Bioavailability;

This paper described the preparation and application of a new dimethylethanolamine aminated polychloromethyl styrene nano-latex (DMEAPL) coated capillary column (ccc-DMEAPL) in the determination of four tetracycline antibiotics (TCA) including tetracycline (TC), oxytetracycline (OTC), doxycycline (DC) and chlorotetracycline (CTC) in pig plasma. The ccc-DMEAPL column was characterized with steady EOF values of ca. 1.5–5.2 × 10−5  cm2/V s at pH 1.8–6.3. The optimized conditions for field-amplified sample stacking open-tubular capillary electrochromatography (FASS-OT-CEC) were as following: background electrolyte, 10 mmol/L Na2HPO4  + 15 mmol/L citric acid (pH 3.2); ccc-DMEAPL, 50 μm i.d. × 50 cm (effective length 41.5 cm), separation voltage, 18 kV; column temperature, 25 °C; UV detection wavelength, 270 nm; water-plug injection: 30 mbar × 10 s; sample electrokinetic injection, 10 kV × 20 s. The four TCA were extracted with the solution of 10 mmol/L Na2HPO4  + 15 mmol/L citric acid + 4 g/L EDTA-2Na (pH 3.2). The FASS-OT-CEC method was validated in terms of linearity, sensitivity, selectivity, precision and accuracy. The LODs ranged from 3 to 7 ng/mL, the recoveries for the four TCA were all more than 80%. The developed method was successfully applied for the determination of TCs in the actual pig plasma samples.
Keywords: Tetracycline antibiotics; Plasma; Open-tubular capillary electrochromatography; Field-amplified sample stacking; Dimethylethanolamine aminated polychloromethyl styrene; Nano-latex;

A hydrophilic interaction liquid chromatography/positive ion electrospray-mass spectrometry (HILIC-ESI/MS) has been developed and fully validated for the quantification of alprazolam and its main metabolite, α-hydroxy-alprazolam, in human plasma. The assay is based on 50 μL plasma samples, following liquid-liquid extraction. All analytes and the internal standard (tiamulin) were separated by hydrophilic interaction liquid chromatography using an X-Bridge-HILIC analytical column (150.0 mm × 2.1 mm i.d., particle size 3.5 μm) under isoscratic elution. The mobile phase was composed of a 7% 10 mM ammonium formate water solution in acetonitrile and pumped at a flow rate of 0.20 mL min−1. Running in positive electrospray ionization and selected ion monitoring (SIM) the mass spectrometer was set to analyze the protonated molecules [M + H]+ at m/z 309, 325 and 494 for alprazolam, α-hydroxy-alprazolam and tiamulin (ISTD) respectively. The assay was linear over the concentration range of 2.5–250 ng mL−1 for alprazolam and 2.5–50 ng mL−1 for α-hydroxy alprazolam. Intermediate precision was less than 4.1% over the tested concentration ranges. The method is the first reported application of HILIC in the analysis benzodiazepines in human plasma. With a small sample size (50 μL human plasma) and a run time less than 10.0 min for each sample the method can be used to support a wide range of clinical studies concerning alprazolam quantification.
Keywords: Hydrophilic interaction liquid chromatography (HILIC); Liquid chromatography/mass spectrometry; Alprazolam; α-Hydroxy-alprazolam; Tiamulin;