Journal of Chromatography B (v.974, #C)

Molecularly imprinted nanoparticles prepared by miniemulsion polymerization as a sorbent for selective extraction and purification of efavirenz from human serum and urine by Mojgan Pourfarzib; Maryam Shekarchi; Hossein Rastegar; Behrouz Akbari-Adergani; Ali Mehramizi; Rassoul Dinarvand (1-8).
A molecularly imprinted polymer (MIP) has been synthesized in order to specifically extract efavirenz from serum and urine by dispersive solid-phase extraction following by HPLC–UV analysis. The imprinted nanoparticles were prepared by miniemulsion polymerization method using efavirenz as template molecule and methacrylic acid as functional monomer. Molecular recognition properties, binding capacity and selectivity of the MIPs were evaluated and the results revealed that the obtained MIPs had high specific retention for efavirenz in aqueous medium. The MIP was used as a molecular sorbent for the separation of efavirenz from human serum and urine. The extraction of efavirenz by MIP coupled with HPLC analysis showed a linear calibration curve in the range of 50–300 μg/L with exellent precisions (3.66% and 4.6% for 100 and 300 μg/L respectively). The limit of detection (LOD) and limit of quantification (LOQ) were determind in serum (17.3 and 57.5 μg/L) and urine (10.6 and 36.2 μg/L). The maximum recoveries for serum and urine samples were found to be 95.2% and 92.7% respectively. Due to the high precision and accuracy, this method may be the UV–HPLC choice with MIP extraction for bioequivalence analysis of efavirenz in serum and urine.
Keywords: Molecularly imprinted polymer; Nanoparticles; Efavirenz; Human serum; Urine;

Natural products (NPs) are important sources of lead compounds in modern drug discovery. To facilitate the screening of volatile active compounds in NPs, we have developed a new biochromatography method that uses rat vascular smooth muscle cells (VSMC), which are rich in L-type calcium channels (LCC), to prepare the stationary phase. This integrated method, which couples cell membrane chromatography (CMC) with gas chromatography–mass spectrometry (GC–MS) via microextraction by packed sorbent (MEPS) technology, has been termed VSMC/CMC-MEPS-GC–MS. Methodological validation confirmed its specificity, reliability and convenience. Screening results for Radix Angelicae Dahuricae and Fructus Cnidii obtained using VSMC/CMC-MEPS-GC–MS were consistent with those obtained using VSMC/CMC-offline-GC–MS. MEPS connection plays as simplified solid-phase extraction and replaces the uncontrollable evaporation operation in reported offline connections, so our new method is supposed to be more efficient and reliable than the offline ones, especially for compounds that are volatile, thermally unstable or difficult to purify. In application, senkyunolide A and ligustilide were preliminary identified as the volatile active components in Rhizoma Chuanxiong. We have thus confirmed the suitability of VSMC/CMC-MEPS-GC–MS for volatile active compounds screening in NP.
Keywords: Cell membrane chromatography; GC–MS; Microextraction by packed sorbent; Natural product; Screening.;

Pharmacokinetics and metabolism study of firocoxib in camels after intravenous administration by using high-resolution bench-top orbitrap mass spectrometry by Ibrahim A. Wasfi; Hanan M. Saeed; B.A. Agha; Asmaa M. Kamel; Nasreen A. Al Biriki; Khaled M. Al Neaimi; Waleed A. Al Ali; Saeed Mahmoud Sultan (17-23).
In this study, we developed a high-resolution liquid chromatography mass spectrometry method for the pharmacokinetic study of firocoxib followed by full method validation. Following a solid-phase extraction, the firocoxib and internal standard (celecoxib) were separated on an Agilent Zorbax ZDB C18 column (50 mm × 2.1 mm i.d., 3.5 μm) with a gradient elution using methanol and 0.1% aqueous formic acid. Data acquisition was performed at 25,000 resolution with the automatic gain set to 1,000,000 and the maximum injection time of 100 ms. Data were acquired in full-scan mode over a mass range of 100–550 Da in positive electrospray mode. Linear calibration curves were obtained over the concentration ranges of 0.5–200 ng/mL and no interfering peaks were detected at the retention time of firocoxib and internal standard in blank camel plasma samples. The mean extraction recoveries of firocoxib at three concentrations of 5, 25 and 75 ng/mL ranged from 92 to 104%. Coefficient of variation of intra-day and inter-day precision were both <10%. The accuracy of the method ranged from 95 to 107%. The validated method was then successfully applied in evaluating the pharmacokinetics and metabolism of firocoxib in camels (Camelus dromedarus) (n  = 5) following intravenous (i.v.) administration of a dose of 0.1 mg kg/body weight. The results obtained (mean ± SD) were as follows: the terminal elimination half-life (t 1/2β) was 5.75 ± 2.26 h, and total body clearance (ClT) was 354.1 ± 82.6 mL/kg/h. The volume of distribution at steady state (V SS) was 2344.4 ± 238.7 mL/kg. One metabolite of firocoxib was tentatively identified as desalkyl firocoxib (m/z 283). Firocoxib could be detected in plasma 3–5 days following i.v. administration in camels using a sensitive liquid chromatography high-resolution orbitrap mass spectrometry method.
Keywords: Firocoxib; Camels; Clearance; Pharmacokinetics; High resolution; Orbitrap.;

Blood-based diagnosis of Alzheimer's disease using fingerprinting metabolomics based on hydrophilic interaction liquid chromatography with mass spectrometry and multivariate statistical analysis by Koichi Inoue; Hirofumi Tsuchiya; Takahiro Takayama; Hiroyasu Akatsu; Yoshio Hashizume; Takayuki Yamamoto; Noriyuki Matsukawa; Toshimasa Toyo’oka (24-34).
Early and definitive diagnosis of Alzheimer's disease (AD) can lead to a better and more-targeted treatment and/or prevention for patients. In the diagnostic biomarkers of AD, the blood sample represents a more non-invasive, inexpensive and acceptable sources for repeated measurements than the cerebrospinal fluid. In this study, the fingerprinting metabolomics was proposed for the challenge of the blood-based diagnosis of defined AD by hydrophilic interaction liquid chromatography mass spectrometry (HILIC/MS). These plasma samples were selected from postmortem specimens based on these pathological examinations. Firstly, we compared these HILIC columns for the non-targeted metabolic assay using pooled plasma. The principal component analysis plot of these seven columns was performed using the repeatability of these chromatograms, and can be used to visualize trends in data sets by three-dimensional dispersion, contributory standard deviation and the number of detections. Based on these results, TSK-Amide 80 and TSKgel-NH2 columns are used as a reliable HILIC/MS assay of blood-based AD metabolomics that showed metabolic profiling of the AD pathology in MS chromatograms that ranged from 1182 to 2284 compounds. A total of 54 peaks were evaluated in order to identify useful ion signal candidates using an orthogonal partial least-squares-discriminant analysis. These peaks were then specifically analyzed using the HILIC–tandem MS assay by a receiver operating characteristic curve and linear discriminant analysis for the diagnosis of the defined AD. The fingerprinting metabolomics can overcome the limitations of previous challenging blood-based diagnosis of AD, and directly evaluates the specific comparative statistical values from the raw data.
Keywords: HILIC/MS; Alzheimer's disease; Blood; Metabolomics;

Determination of parabens in urine samples by microextraction using packed sorbent and ultra-performance liquid chromatography coupled to tandem mass spectrometry by Valeria Cristina Jardim; Lidervan de Paula Melo; Diego Soares Domingues; Maria Eugênia Costa Queiroz (35-41).
A simple, sensitive, and selective method using ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC–MS/MS) was developed and validated for simultaneous determination of parabens [methyl paraben (MeP), ethyl paraben (EtP), propyl paraben (PrP), butyl paraben (BuP), and benzyl paraben (BzP)] in human urine samples. After microextraction by packed sorbent (MEPS) using a C18 phase, the parabens were separated on a Kinetex C18 column (100 mm × 2.1 mm × 1.7 μm) within 4.6 min using isocratic elution. These compounds were detected on a triple quadrupole tandem mass spectrometer using the multiple reactions monitoring (MRM) mode via an electrospray ionization source operating in the negative ionization mode. Important factors that influence MEPS performance were evaluated, such as the sample pH, draw–eject sample volume, clean-up step, and desorption conditions. The proposed MEPS/UPLC–MS/MS method presented a linear range from 0.5 ng mL−1 (limit of quantification – LOQ) to 50 ng mL−1, and interassay precision with coefficients of variation lower than 15%, and relative standard error values of the accuracy ranged from −8.8% to 15%. The MEPS/UPLC–MS/MS method was applied successfully to determine parabens in urine samples from 30 postpartum volunteers, enabling assessment of human exposure to these compounds.
Keywords: Microextraction by packed sorbent; Parabens; Urine sample; Ultra-performance liquid chromatography coupled to tandem mass spectrometry;

20(S) protopanaxatriol is the main metabolite of notoginsenoside R1, ginsenoside Rg1, ginsenoside Re in Panax notoginseng and has significant activities. A ultra high performance liquid mass spectrometry method has been developed and validated for the simultaneous determination of notoginsenoside R1 (R1), ginsenoside Rg1 (Rg1), ginsenoside Re (Re) and 20(S) protopanaxatriol (PPT) in beagle dog plasma after oral administration of a Panax notoginseng saponin preparation. After the addition of the internal standard (digoxin), plasma samples were subjected to liquid–liquid extraction with acetone and methanol and separated on a 100 × 2.1 mm ACQUITY 1.7 μm C18 column (Waters, USA), with acetonitrile and water as the mobile phase, within a runtime of 7.0 min. The analytes were detected without interference in Selected Reaction Monitoring mode with a change in the electrospray ionization from positive to negative. The detection limits were 0.01 to 0.04 mg/L and the calibration curves of the peak areas for the four ingredients were linear over four orders of magnitude with a correlation coefficient greater than 0.9957. The intra-day and inter-day precision values (relative standard deviation, RSD, %) were within 10.25% and 13.51%, respectively, and the accuracy (relative error, RE, %) was less than 7.81%. The validated method was successfully applied to a comparative pharmacokinetic study of four saponins in beagle dogs after oral administration of a Panax Notoginseng Saponins preparation. The pharmacokinetic parameters were calculated with DAS 3.20. The T max and C max values indicate a dose–dose relationship between the saponins (R1, Rg1, and Re) and their sapogenin (PPT).
Keywords: Ultra high performance liquid mass spectrometry; Notoginsenoside R 1; Ginsenoside Rg1; Ginsenoside Re; 20(S) protopanaxatriol (PPT); Pharmacokinetics;

A generic approach for “shotgun” analysis of the soluble proteome of plant cell suspension cultures by Thomas De Vijlder; Dirk Valkenborg; Debbie Dewaele; Noor Remmerie; Kris Laukens; Erwin Witters (48-56).
Cell suspension cultures from different plant species act as important model systems for studying cellular processes in plant biology and are often used as “green factories” for the production of valuable secondary metabolites and recombinant proteins. While mass spectrometry based proteome analysis techniques are ideally suited to study plant cell metabolism and other fundamental cellular processes from a birds eye perspective, they remain underused in plant studies. We describe a comprehensive sample preparation and multidimensional ‘shotgun’ proteomics strategy that can be generically applied to plant cell suspension cultures. This strategy was optimized and tested on an Arabidopsis thaliana ecotype Landsberg erecta culture. Furthermore, the implementation of strong cation exchange chromatography as a peptide fractionation step is elaborately tested. Its utility in mass spectrometry based proteome analysis is discussed. Using the presented analytical platform, over 13,000 unique peptides and 2640 proteins could be identified from a single plant cell suspension sample. Finally, the experimental setup is validated using Nicotiana tabacum cv. “Bright Yellow-2” (BY-2) plant cell suspension cultures, thereby demonstrating that the presented analytical platform can also be valuable tool in proteome analysis of non-genomic model systems.
Keywords: Arabidopsis thaliana; Nicotiona tabacum cv “Bright Yellow-2”; Proteome analysis; Plant cell suspension cultures; Mass spectrometry;

(2S, 3R)-methyl-2-benzamidomethyl-3-hydroxybutyrate (MBHB) is a key intermediate in the synthesis of 4-aceoxyazetidinone, a building block for the production of penems and carbapenems. More attentions have been paid to screen biocatalysts achieving asymmetric preparation of (2S, 3R)-MBHB. In this study, an improved chiral high-performance liquid chromatographic (HPLC) method was developed for the stereoselective determination of 2-benzamidomethyl-3-oxobutanoate (BMOB) and MBHB, and further employed into the biotransformation of BMOB. Chiral separation was achieved within 12 min on Chiralpak AY-H column, which was faster and more suitable for screening biocatalysts exhibited reduction activity and (2S, 3R)-stereospecificity toward BMOB than on other columns. Ultimately, a new strain, Burkholderia gladioli ZJB-12126 capable of reducing BMOB to (2S, 3R)-MBHB was successfully isolated based on this newly constructed HPLC method. Samples were prepared by liquid–liquid extraction system using ethyl acetate as the extractor solvent. The extraction recoveries of BMOB and MBHB isomers ranged from 91.6 to 94.1% with relative standard deviation (RSD) below 10%. Linear calibration curves were obtained in the concentration range of 50–5000 μg/mL for both BMOB and MBHB isomers, respectively. Intra-day and inter-day precisions and accuracy were below 15% for all isomers evaluated by RSDs and relative errors (REs), respectively. This novel method was demonstrated to be suitable for assessing the biotransformation process of BMOB.
Keywords: Stereoselective determination; Chiralpak AY-H; Biotransformation; 2-Benzamidomethyl-3-oxobutanoate; Methyl-2-benzoylamide-3-hydroxybutanoate.;

In this work, 33 organochlorine pesticides (OCPs) and 9 pyrethroid pesticides (PYPs) in Morinda officinalis were effectively and selectively extracted and cleaned up by accelerated solvent extraction assisted matrix solid phase dispersion (ASE/MSPD) method, followed by gas chromatography-electron capture detection (GC–ECD). Carbophenothion was selected as the internal standard and added into the final extracts to improve the precision and accuracy of the method. Parameters for ASE/MSPD procedure including ratio of acetone to n-hexane, temperature and amount of Florisil were optimized to improve the performance of the method through orthogonal experimental design. Under the optimized conditions, the average recoveries (six replicates) for all pesticides (spiked at 0.05, 0.5 and 1.0 mg kg−1) ranged from 69.3% to 112% with RSD less than 14.14%. A wide linear range of 10–1000 ng mL−1 was observed with r values of 0.9963–0.9999. Meanwhile, the method gave high selectivity and sensitivity (LODs < 3 μg kg−1 and LOQs < 8.0 μg kg −1), good repeatability (RSD of 9.64%, on average) and precision (RSD of 5.48%, averagely) and excellent stability (RSD <9.47%). The feasibility of the proposed method was demonstrated by applying it for preconcentration and determination of OCPs and PYPs in 40 batches of real samples. Four kinds of pesticides (beta-endosulfan, tecnazene, hexachlorobenzene and alpha-BHC) were detected in three batches of samples, which were successfully confirmed by GC–MS. The results indicated that ASE/MSPD is a reliable and half-automated extraction and purification technique, with many advantages over traditional techniques. The combination of ASE/MSPD and GC–ECD could be especially useful for trace analysis of pesticide residues in complex matrices.
Keywords: Accelerated solvent extraction; Matrix solid phase dispersion; Morinda officinalis; GC–ECD; Orthogonal experimental design;

Steroid saponins are a class of naturally existing substances widely distributed in the plants of Smilacaceae. Their biological activities have been attracting the interest of scientists in the chemical field for the past few years. To our best knowledge, there has been no study on structural characterization of steroidal saponins from Smilax trinervula (S. trinervula) using LTQ-Orbitrap mass spectrometry, which could provide an excellent approach for rapid screening of steroidal saponins in other plants of Smilacaceae. An ultra high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC/HRMS) method was therefore developed to characterize steroidal saponins in S. trinervula. This method was operated in both negative and positive ion modes with HRMS, as a result, a total of twenty-two steroidal saponins with three aglycone skeletons were elucidated in the crude extract from the root of S. trinervula. The characteristic-fragment ions could well identify and differentiate the three types of aglycone skeletons (diosgenyl saponins, furostanol saponins, C27-hydroxy diosgenyl saponins).
Keywords: Steroidal saponins; Smilax trinervula; UHPLC/HRMS; LTQ-Orbitrap mass;

Bilirubin oxidation end products (BOXes) appear upon endogenous heme degradation and can be found in the cerebrospinal fluid after hemorrhagic stroke. BOXes are assumed to contribute to delayed cerebral vasospasm and secondary loss of brain tissue. Here, we present a validated LC–ESI-MS/MS method for the sensitive determination of the regio-isomers Z-BOX A and Z-BOX B in human serum. We found that Z-BOX A and Z-BOX B appear in serum of healthy volunteers. The sample preparation includes the addition of 5-bromonicotinamide as internal standard and protein precipitation with acetonitrile. Baseline-separation was achieved on a C-18 column with a binary solvent gradient of formic acid in water/acetonitrile at 1 mL/min within a total analysis time of 17 min. Using single reaction monitoring in the positive ion mode, the linear working ranges were 2.74–163 pg/μL (Z-BOX A) and 2.12–162.4 pg/μL (Z-BOX B) with R 2  > 0.995. Intra- and inter-day precisions were <10%. The inherent analyte concentrations of Z-BOX A (14.4  ±   5.1 nM) and Z-BOX B (10.9 ±   3.1 nM) in pooled human serum were determined by standard addition. The photolability of both analytes was demonstrated. This method enables to monitor Z-BOX A and Z-BOX B as a prerequisite to systematically study the biological significance of higher order metabolites of heme degradation.
Keywords: Quantification; Heme degradation; Bilirubin oxidation end products; Reactive oxygen species; LC–MS/MS;

Liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS) and first-dimensional fractionation is widely used for reducing sample complexity in large-scale proteomic profiling experiments. However, the limited number of proteins identified and the relatively long running time are a barrier to the successful application of this approach. In this study, off-line high pH reversed-phase fractionation (RPF) was combined with nano-LC–MS/MS in order to develop an improved method for global proteomic profiling of different cell lines. In the first dimensional reverse phase HPLC separation, 300 μg of digested cell protein was separated into 78 fractions under high pH conditions and condensed into 26 fractions for the second nano-LC–MS/MS analysis at low pH. The chromatographic conditions for the first and second steps were optimized, and the accuracy and reproducibility of protein quantification were investigated with an average Pearson correlation coefficient of 0.94. The method was then applied in the identification of proteins in six common cell lines (DMS, MFM, HepG2, U2OS, 293T and yeast), which resulted in identification of 7300–8500 and 8956 proteins in heavy/light labeled and label-free cell samples, respectively, in 1.5 days. The performance of the developed method was compared with isoelectric focusing (IEF)-nano-LC–MS/MS and the previously reported method; and off-line high pH RPF-nano-LC–MS/MS proved advantageous in terms of the number of proteins identified and the analytical time needed to achieve a successful global proteomic profiling outcome. The RPF-nano-LC–MS/MS method identified more proteins from low abundance (150 μg) samples with an average sequence coverage for each cell line of 23.4–35.1%. RPF-nano-LC–MS/MS may therefore be an efficient alternative tool for achieving improved proteomic coverage of multiple cell lines.
Keywords: Reversed-phase fractionation; Nano-LC–MS/MS; Off-line; Global proteomics; Cell lines;

A simple, rapid and sensitive high-performance liquid chromatography–tandem mass spectrometric (HPLC–MS/MS) method was developed and validated for determining lycorine in rat plasma. Plasma samples were prepared by a simple protein precipitation with methanol containing dextrorphan as internal standard. The chromatographic separation was performed on a Kromasil 60-5CN column (3 μm, 2.1 mm × 150 mm) with the mobile phase of methanol/water (containing 0.1% formic acid) (40:60, v/v) at a flow rate of 0.2 mL/min, the total analytical runtime was 5 min. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with Electronic Spray Ion by selected reaction monitoring (SRM) of the transitions at m/z 288.1 → 147.1 for lycorine and m/z 258.1 → 157.2 for dextrorphan, respectively. The calibration curve was linear over the range of 1–1000 ng/mL with the lower limit of quantification of 1 ng/mL for lycorine. The intra- and inter-day precision (R.S.D.%) were less than 8.5% and accuracy (R.E.%) was within ±7.0%. Lycorine was sufficiently stable under all relevant analytical conditions. This method was successfully applied to the pharmacokinetic study of lycorine in rats after intraperitoneal administration with different doses of 5, 10 and 20 mg/kg.
Keywords: Lycorine; Plasma concentration; HPLC–MS/MS; Pharmacokinetics;

Determination of fluxes by 13C tracer experiments depends on monitoring the 13C labeling pattern of metabolites during isotope experiments. In metabolome-based 13C metabolic flux analysis, liquid chromatography combined with mass spectrometry or tandem mass spectrometry (LC/MS or LC/MS/MS, respectively) has been mainly used as an analytical platform for isotope pattern studies of central carbon metabolites. However, gas chromatography with mass spectrometry (GC/MS) has several advantages over LC/MS, such as high sensitivity, low cost, ease of operation, and availability of mass spectra databases for comparison. In this study, analysis of isotope pattern for central carbon metabolites using GC/MS was demonstrated. First, a proper set of mass ions for central carbon metabolites was selected based on carbon backbone information and structural isomers of mass fragment ions. A total of 34 mass fragment ions was selected and used for the quantification of 25 central carbon metabolites. Then, to quantify isotope fractions, a natural mass isotopomer library for selected mass fragment ions was constructed and subtracted from isotopomer mass spectra data. The results revealed a surprisingly high abundance of partially labeled 13C intermediates, such as 56.4% of fructose 6-phosphate and 47.6% of dihydroxyacetone phosphate at isotopic steady state, which were generated in the pentose phosphate pathway. Finally, dynamic changes of isotope fragments of central metabolites were monitored with a U-13C glucose stimulus response experiment in Kluyveromyces marxianus. With a comprehensive study of isotope patterns of central carbon metabolites using GC/MS, 25 central carbon metabolites and their isotopic fractions were successfully quantified. Dynamic and precise acquisition of isotope pattern can then be used in combination with proper kinetic models to calculate metabolic fluxes.
Keywords: Metabolome-based mass isotopomer study; Carbon central metabolites; Stimulus response experiment; GC/MS;

Deamidation in ricin studied by capillary zone electrophoresis- and liquid chromatography–mass spectrometry by Tomas Bergström; Sten-Åke Fredriksson; Calle Nilsson; Crister Åstot (109-117).
Deamidation in ricin, a toxin present in castor beans from the plant Ricinus communis, was investigated using capillary zone electrophoresis (CZE) and liquid chromatography coupled to high resolution mass spectrometry. Potential sites for deamidation, converting asparagine (Asn) into aspartic or isoaspartic acid (Asp or isoAsp), were identified in silico based on the protein sequence motifs and tertiary structure. In parallel, CZE- and LC–MS-based screening were performed on the digested toxin to detect deamidated peptides. The use of CZE–MS was critical for the separation of small native/deamidated peptide pairs. Selected peptides were subjected to a detailed analysis by tandem mass spectrometry to verify the presence of deamidation and determine its exact position. In the ricin preparation studied, deamidation was confirmed and located to three asparagine residues: Asn54 in the A-chain, and Asn42 and Asn60 in the B-chain. Possible in vitro deamidation occurring during sample preparation was monitored using a synthetic peptide with a known and rapid rate of deamidation. Finally, we showed that the isoelectric diversity previously reported in ricin is related to the level of deamidation.
Keywords: Ricin; Deamidation; Capillary zone electrophoresis; Liquid chromatography; Mass spectrometry; Isoelectric diversity;

In this study, a modified quick, easy, cheap, efficient, rugged and safe (QuEChERS) method coupled with gas chromatography with flame photometric detection (GC–FPD) was developed for the determination of 44 organophosphorous pesticide (OPP) residues in 44 batches of Pogostemon cablin and its related products for the first time. The QuEChERS extraction conditions were optimized, and the matrix effect that may influence recoveries was evaluated and minimized by matrix-matched calibration curves. Under the optimized conditions, the calibration curves for all OPPs showed good linearities in the concentration range of 0.04–1.5 μg mL−1 with correlation coefficients better than 0.9909. The limits of detection were in the range of 0.004–0.02 μg mL−1 and quantification were 0.01–0.04 μg mL−1, below the regulatory maximum residue limits suggested. Mean recoveries ranged between 76.62 and 113.7% (99.34% on average), and relative standard deviation was 3.71% on average. The validated method was applied on 44 real samples including P. cablin, and P. cablin oil and powder. Two (4.5%) samples were found to be contaminated by chlorpyrifos with levels below the legal limits, which were successfully confirmed by gas chromatography–mass spectrometry (GC–MS). Based on the results, the developed method was proved to be simple, fast, accurate, low cost and environmentally friendly and can be successfully applied in the determination of targeted OPP residues in P. cablin and its related products. Moreover, it also attaches great importance to pesticide monitoring programs in food, soil and air in the future.
Keywords: Pogostemon cablin; OPPs residues; QuEChERS; GC–FPD; Matrix effect.;

A rapid, sensitive and selective high-performance liquid chromatography–tandem mass spectrometric method (HPLC–MS) has been developed and validated for the simultaneous determination of 14-thienyl methylene matrine (TMM) and matrine (MT) in rat plasma in the present study. The analytes were separated on a C18 column (1.9 μm, 2.1 mm × 100 mm) with a security guard C18 column (5 μm, 2.1 mm × 10 mm) and a triple-quadrupole mass spectrometry equipped with an electrospray ionization (ESI) source was applied for detection. With pseudoephedrine hydrochloride as internal standard, sample pretreatment involved in a one-step protein precipitation with isopropanol:ethyl acetate (v/v, 20:80). The method was linear over the concentration ranges of 5–1000 ng/ml for TMM and 10–2000 ng/ml for MT. The intra-day and inter-day relative standard deviations (RSD) were less than 15% and the relative errors (RE) were all within 15%. The proposed method enables unambiguous identification and quantification of TMM and MT in vivo. This was the first report on determination of the TMM and MT in rat plasma after oral administration of TMM. The results provided a meaningful basis for evaluating the clinical applications of the medicine.
Keywords: LC/MS/MS; 14-Thienyl methylene matrine; Matrine; Pharmacokinetics;

Measurement of hydroxysafflor yellow A in human urine by liquid chromatography–tandem mass spectrometry by Chang-Yin Li; Ji-Hong Chu; Jun Zhang; Bing-Ting Sun; Guo-Liang Dai; Shi-Jia Liu; Wen-Zheng Ju (131-137).
A rapid and specific high performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed for the quantification of hydroxysafflor yellow A (HSYA) in human urine with isorhamnetin-3-O-neohespeidoside as internal standard (IS). HSYA and IS were extracted from urine samples by simple solid-phase extraction and separated on an Agilent Zorbax SB C18 column (4.6 mm × 150 mm, 5 μm) with the mobile phase of 0.2 mM ammonium acetate: methanol (30/70, v/v) at a flow rate of 0.4 mL/min. Polar endogenous interferences eluted in 0.1–2.5 min were switched into waste channel by the Valve Valco, to reduce the possible matrix effect for MS detection in each run. The MS detection of analytes was performed on a tandem mass spectrometer equipped with an electrospray ionization source in negative mode using multiple-reaction monitoring. The MS/MS ion transitions monitored were m/z 611.3 → 491.2 for HSYA and m/z 623.2 → 299.2 for IS. The method was fully validated for selectivity, sensitivity, linearity, precision, accuracy, recovery, matrix effect and stability, and then was applied to the urinary excretion study of injectable powder of pure HSYA in healthy Chinese volunteers for the first time. The results suggested that urine was the main excretion way of HSYA in healthy volunteers, further demonstrating the feasibility and necessity of our current method.
Keywords: Hydroxysafflor yellow A; SPE; LC–MS/MS; Human urine; Excretion; Column switching;

A rapid-resolution ultra high-performance liquid chromatography–tandem mass spectrometry separation method (UPLC–MS/MS) was developed for the simultaneous determination of carvedilol, and its three metabolites: 4′-hydroxyphenyl-carvedilol, 5′-hydroxyphenyl-carvedilol, o-desmethyl-carvedilol. The effective UPLC–MS/MS separation of the examined compounds was applied on an Acquity BEH C18 column (2.1 mm × 50 mm, 1.7 μm particle size) column with a gradient mobile phase system. The analysis was performed in less than 6 min with a flow rate of 0.4 mL/min. The assay was validated over concentration ranges of 0.500–100 ng/mL for carvedilol and 0.0500–10.0 ng/mL for its three metabolites. Intra- and inter-assay precision values for replicate quality control samples were within 11.4% for all analytes during the assay validation. Mean quality control accuracy values were within ±11.5% of nominal values for all analytes. Assay recoveries were high (>91%) and internal standard normalized matrix effects were minimal. The four analytes were stable in rat plasma for at least 24 h at room temperature, 89 days at −20 °C and −80 °C, and following at least five freeze–thaw cycles. The validated assay was successfully applied to the quantification of carvedilol and its pharmacologically active metabolites in rat pharmacokinetic study. The accurate and simple method we developed could be applied to human pharmacokinetic study in the near future.
Keywords: Carvedilol; Metabolite; UPLC–MS/MS; Assay;

Ultrasensitive and quantitative gold nanoparticle-based immunochromatographic assay for detection of ochratoxin A in agro-products by Marjan Majdinasab; Mahmoud Sheikh-Zeinoddin; Sabihe Soleimanian-Zad; Peiwu Li; Qi Zhang; Xin Li; Xiaoqian Tang (147-154).
In most cases of mycotoxin detection, quantitation is critical while immunochromatographic strip tests are qualitative in nature. Moreover, the sensitivity of this technique is questioned. In order to overcome these limitations, an ultrasensitive and quantitative immunochromatographic assay (ICA) for rapid and sensitive quantitation of ochratoxin A (OTA) was developed. The assay was based on a competitive format and its sensitivity was improved by using a sensitive and selective OTA monoclonal antibody (OTA-mAb). The visible ICA results were obtained within 15 min, and in addition to visual examination, they were read by the rapid color intensity portable strip reader. The visual and computational detection limits (vLOD and cLOD, respectively) for ochratoxin A were 0.2 and 0.25 ng mL−1, respectively. These values were lower than those reported by previous studies in a range 5–2500 folds. For validation, contaminated samples including wheat, maize, rice and soybean were assayed by ICA and a standard high performance liquid chromatography (HPLC). The results were in good agreement for both ICA and HPLC methods. The average recoveries of the HPLC were in the range 72–120% while the ICA values were from 76 to 104%, confirming the accuracy and sensitivity of this method.
Keywords: Immunochromatographic assay (ICA); Ochratoxin A; Agro-products; Gold nanoparticles;