Journal of Chromatography B (v.967, #C)

Simultaneous determination of 50 residual pesticides in Flos Chrysanthemi using accelerated solvent extraction and gas chromatography by Xiaohui Huang; Xinghui Zhao; Xiaotong Lu; Huaiping Tian; Ajing Xu; Yan Liu; Zhang Jian (1-7).
A gas chromatographic method for simultaneous determination of 50 organochlorine (OCP) and pyrethroid (PP) pesticides in Flos Chrysanthemi was established. Accelerated solvent extraction (ASE) technique was used to extract the target compounds, cleaned with alumina neutral–florisil column, and eluted by mixed solvents of ethyl acetate and hexane (15:85, v/v). Selected pesticides were identified using HP-5 and DB1701 capillary dual column and detected by electron-capture detector. Quantitative analysis was performed using an external standard by HP-5 capillary column. Results showed that recoveries were 73.4–120.1%, and the relative standard deviations (RSDs) were 1.6–12.4%. The limits of detection of the method were 0.0021–0.0069 mg/kg, and the limits of quantity were 0.0064–0.0210 mg/kg.
Keywords: Flos Chrysanthemi; Gas chromatography; Organochlorine pesticides (OCP); Pyrethroid pesticides (PP); Multi-residues;

11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) and formoterol are newly revised prohibited threshold substances (150 ng/mL for THC-COOH and 40 ng/mL for formoterol) by the World Anti-Doping Agency (WADA). In continuation of our direct quantitation work of the prohibited threshold substances, direct LC–MS/MS methods combined with a simple sample preparation procedure have been developed and validated for the measurement of these two threshold substances in urine samples. After the enzymatic hydrolysis of urine samples, the resulting samples were diluted with acetonitrile and centrifuged. The supernatant was directly analyzed by LC–MS/MS using the selected reaction monitoring mode. The calibration curve range of the assay was ranged over 50–200% of the threshold value according to WADA guidelines. The limit of detection and limit of quantification were 6.1 and 18.4 ng/mL for THC-COOH and 2.0 and 6.2 ng/mL for formoterol, respectively. Intra- and inter-day precisions were between 2.08% and 7.28% and the accuracies ranged from 95.16% to 104.49%. The present methods were successfully applied to the analysis of the proficiency test samples.
Keywords: THC-COOH; Formoterol; Quantitative analysis; Liquid chromatography; Tandem mass spectrometry;

Novel regenerative large-volume immobilized enzyme reactor: Preparation, characterization and application by Guihua Ruan; Meiping Wei; Zhengyi Chen; Rihui Su; Fuyou Du; Yanjie Zheng (13-20).
A novel large-volume immobilized enzyme reactor (IMER) on small column was prepared with organic–inorganic hybrid silica particles and applied for fast (10 min) and oriented digestion of protein. At first, a thin enzyme support layer was formed in the bottom of the small column by polymerization with α-methacrylic acid and dimethacrylate. After that, amino SiO2 particles was prepared by the sol–gel method with tetraethoxysilane and 3-aminopropyltriethoxysilane. Subsequently, the amino SiO2 particles were activated by glutaraldehyde for covalent immobilization of trypsin. Digestive capability of large-volume IMER for proteins was investigated by using bovine serum albumin (BSA), cytochrome c (Cyt-c) as model proteins. Results showed that although the sequence coverage of the BSA (20%) and Cyt-c (19%) was low, the large-volume IMER could produce peptides with stable specific sequence at 101–105, 156–160, 205–209, 212–218, 229–232, 257–263 and 473–451 of the amino sequence of BSA when digesting 1 mg/mL BSA. Eight of common peptides were observed during each of the ten runs of large-volume IMER. Besides, the IMER could be easily regenerated by reactivating with GA and cross-linking with trypsin after breaking the –C=N– bond by 0.01 M HCl. The sequence coverage of BSA from regenerated IMER increased to 25% comparing the non-regenerated IMER (17%). 14 common peptides. accounting for 87.5% of first use of IMER, were produced both with IMER and regenerated IMER. When the IMER was applied for ginkgo albumin digestion, the sequence coverage of two main proteins of ginkgo, ginnacin and legumin, was 56% and 55%, respectively. (Reviewer 2) Above all, the fast and selective digestion property of the large-volume IMER indicated that the regenerative IMER could be tentatively used for the production of potential bioactive peptides and the study of oriented protein digestion.
Keywords: Enzyme immobilization; Organic–inorganic hybrid silica particles; Regenerative enzyme reactor; On-column digestion;

Mass spectrometric behavior of phenolic acids standards and their analysis in the plant samples with LC/ESI/MS system by Wojciech Ostrowski; Anna Wojakowska; Magdalena Grajzer; Maciej Stobiecki (21-27).
Liquid chromatography coupled to mass spectrometry (MS) with electrospray ionization (ESI) is one of analytical techniques to obtain accurate results of low molecular weight aromatic compounds in biological samples of different origin. The interpretations of mass spectra of these aromatic compounds in the negative spectra registered in the full scan MS mode may be uneasy due to presence of deprotonated molecules [M−H] from different co-eluting entities, fragment ions created after the break-up of precursor ions and also ions representing modified molecules clusters. Thus, the first aim of this study was to evaluate general parameters during analysis performed in the full scan MS or MS/MS mode. Secondly, to set general fragmentation rules for aromatic compounds and entities in a complex biological matrix. We established that different groups of low molecular weight phenolic acids form unique adduct ions and additionally registration LC/MS/MS spectra with two different collision energies may allow for differentiating isomeric or isobaric molecules. These findings together with some general fragmentation rules can facilitate identifications of aromatic acids as we outlined in the sample of cold-pressed rose-hip oil and lupine leaves extract.
Keywords: Aromatic acids; Cluster ions; Fragmentation pathways; Liquid chromatography/mass spectrometry; Substitution pattern;

Development and validation of an ultrafiltration-UPLC-MS/MS method for rapid quantification of unbound docetaxel in human plasma by Ping Du; Xiaohong Han; Ning Li; Hongyu Wang; Sheng Yang; Yuanyuan Song; Yuankai Shi (28-35).
Docetaxel lipid microsphere (DT-LM) is a novel formulation of docetaxel without Tween-80. A sensitive, robust and reproducible ultrafiltration (UF) followed by UPLC-MS/MS method was developed and validated for the quantification of unbound docetaxel in human plasma using paclitaxel as IS. Ultrafiltrate samples were chromatographed on Acquity UPLC BEH C18 column (50 mm × 2.1 mm, 1.7 μm). The mobile phase was a mixture of 10 mM ammonium formate in water containing 0.2% formic acid (A) and acetonitrile containing 0.2% formic acid (B). The volume of plasma utilized was only 450 μL. The calibration curve was linear over the range of 0.2–200 ng/mL, with LLOQ of 0.2 ng/mL. The method was shown to be reliable and reproducible with intra- and inter-day precision and accuracy <±15%, and extraction recovery of 98.1–104.8%. Docetaxel was stable during stability studies, e.g., short term, post-preparation and freeze–thaw cycles. The validated method was utilized to support the pharmacological study of DT-LM in patients with advanced cancer.
Keywords: Docetaxel lipid microsphere; Unbound analyte; Ultrafiltration; UPLC-MS/MS; Human plasma;

Enzymatic protein digestion in polyacrylamide gel has been used for sample pretreatment in mass spectrometry-based proteomics due to its effectiveness in removing contaminants that interfere with sample ionization. However, the difficulty of recovering the digested peptides from the solid gel matrix has been a drawback of this method. Here we have developed a novel in-gel digestion method to enhance peptide recovery using a dissolvable, bis-acrylylcystamine (BAC)-crosslinked polyacrylamide gel. After enzymatic protein digestion in BAC gel, we completely dissolved the gel by reductive treatment with tris-(2-carboxyethyl) phosphine to release the digested peptides from the gel. Our analysis revealed that the reductive dissolution of the BAC gel enhances the peptide recovery, which has a significantly higher protein identification capability than the conventional method, using an insoluble polyacrylamide gel. In addition, protein samples trapped in dehydrated BAC gel were stable at room temperature and reproducible sample recovery was obtained after storage for one week. These results indicate that the proposed method could be an effective tool for conducting sample pretreatment for mass spectrometry-based protein analysis.
Keywords: Bis-acrylylcystamine; In-gel protein digestion; Protein quantitation; Shotgun proteomics;

A new HILIC-MS/MS method for the simultaneous analysis of carbidopa, levodopa, and its metabolites in human plasma by Raquel de Oliveira Vilhena; Flávia Lada Degaut Pontes; Breno Maurício Marson; Rômulo Pereira Ribeiro; Katherine Athayde Teixeira de Carvalho; Marco André Cardoso; Roberto Pontarolo (41-49).
Monitoring of the plasmatic levels of levodopa (LEV) and carbidopa (CAR) is necessary to adjust the dose of these drugs according to the individual needs of Parkinson's disease patients. To support drug therapeutic monitoring, a method using HILIC mode and LC–MS/MS was developed for the simultaneous determination of carbidopa, levodopa, and its metabolites (3-o-methyldopa (3-OMD) and dopamine (DOPA)) in human plasma. A triple quadrupole mass spectrometry was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. After straightforward sample preparation via protein precipitation, an Atlantis HILIC (150 × 2.1 mm, 3 μm, Waters, USA) column were used for separation under the isocratic condition of acetonitrile/water (79:21, v/v) containing 0.05% formic acid and 3 mmol/L ammonium formate and the total run time was 7 min. Deuterated LEV was used as internal standard for quantification. The developed method was validated in human plasma with a lower limit of quantitation of 75 ng/mL for LEV, 65 ng/mL for CAR and 3-OMD, and 20 ng/mL for DOPA. The calibration curve was linear within the concentration range of 75–800 ng/mL for LEV, 65–800 ng/mL for CAR and 3-OMD, and 20–400 ng/mL for DOPA (r  > 0.99). The assay was accurate and precise, with inter-assay and intra-assay accuracies within ±13.44% of nominal and inter-assay and intra-assay precision ≤ 13.99%. All results were within the acceptance criteria of the US FDA and ANVISA guidelines for method validation. LEV, CAR, 3-OMD and DOPA were stable in the battery of stability studies, long-term, bench-top, autosampler, and freeze/thaw cycles. Samples from patients undergoing treatment were analyzed, and the results indicated that this new method is suitable for therapeutic drug monitoring in Parkinson's disease patients.
Keywords: HILIC-MS/MS; Levodopa; Carbidopa; Parkinson's disease; Drug monitoring;

HS061, a new structure analogue of human insulin, was investigated for the treatment of diabetes. In this study, we developed a simple and accurate UPLC–MS/MS method for the pharmacokinetic studies of HS061 in non-diabetic rats followed by a full method validation. Following a simple protein precipitation with acetonitrile, the analyte and internal standard (Levemir, IS) were separated on a Waters XBridge™ BEH300 C4 column (100 mm × 4.6 mm i.d., 3.5 μm) with a gradient elution using acetonitrile and 0.2% aqueous formic acid. The method was operated under pseudo-multiple reaction monitoring (pseudo-MRM) in the positive electrospray ionization mode. The monitored transitions were set at m/z 1563.4 → 1563.4 for HS061 by pseudo-MRM and m/z 1184.7 → 454.5 for IS by MRM. Linear calibration curves were obtained over the concentration ranges of 10–1000 ng/mL and no interfering peaks were detected at the retention time of HS061 and IS in blank rat plasma. The mean extraction recoveries of HS061 at three concentrations of 20, 100, 800 ng/mL were greater than 95.17%. Stability was assessed under different conditions and no significant degradations were found. The validated method was then successfully applied in measuring HS061 following subcutaneous (0.5, 1.0, 3.0 U/kg) and intravenous (1.0 U/kg) injection in rat plasma to support the pre-clinical pharmacokinetic study. Maximum plasma concentration (C max) and area under the curve (AUC) for the subcutaneous doses of HS061 was approximately dose proportional while other pharmacokinetic parameters showed no significant differences among the three doses (p  > 0.05). The absolute bioavailability of HS061 after subcutaneous administration at 1.0 U/kg was estimated to be 70.40%.
Keywords: Human insulin; Pharmacokinetics; UPLC–MS/MS; Rat plasma;

Chrysotoxine (CTX), a naturally occurring bibenzyl compound isolated from Dendrobium species, has been reported to have neuroprotective effects. To evaluate its pharmacokinetics in rats, a rapid, sensitive and specific high performance liquid chromatography–tandem mass spectrometric (HPLC–MS/MS) method has been developed and validated for the quantification of CTX in rat plasma. Samples were pretreated using a simple liquid–liquid extraction with ethyl acetate and the chromatographic separation was performed on a C18 column with acetonitrile–water (90:10, v/v) as the mobile phase. CTX and the internal standard (wogonin) were detected using a tandem mass spectrometer in positive multiple reaction monitoring mode. Method validation revealed excellent linearity over the range 0.5–1000 ng/mL together with satisfactory intra- and inter-day precision, accuracy and recovery. Stability testing showed that CTX spiked into rat plasma was stable for 8 h at room temperature, for up to two weeks at −20 °C, and during three freeze–thaw cycles. Extracted samples were also observed to be stable over 24 h in an auto-sampler. The method was successfully used to investigate the pharmacokinetic profile of CTX after oral (100 mg/kg) and intravenous (25 mg/kg) administration in rats. CTX showed rapid excretion and low bioavailability in rats.
Keywords: Chrysotoxine; HPLC–MS/MS; Pharmacokinetics; Method validation; Bioavailability;

Metabolite identification of liguzinediol in dogs by ultra-flow liquid chromatography/tandem mass spectrometry by Chen-xiao Shan; Wei Li; Hong-mei Wen; Xin-zhi Wang; Xiao-wen Zhu; Xiao-bin Cui (63-68).
Ultra-flow liquid chromatography/quadrupole-time-of-flight mass spectrometry (UFLC/Q-TOF MS) method combined with metabolitepilotMT software was used for analysis of the metabolites of liguzinediol in dogs. Urine, bile, feces and plasma samples were collected after intravenous administration of 8 mg/kg liguzinediol to healthy dogs. Besides liguzinediol, seven metabolites were detected and identified by UFLC/Q-TOF MS method. The results showed that liguzinediol had some main metabolic pathways in dogs including oxidation, sulfation, cysteine conjugation, N-acetylcysteine conjugation and glucuronidation.
Keywords: Liguzinediol; UFLC/Q-TOF MS; Metabolites; Beagle dog;

Phenylalanine is an essential amino acid and its metabolites relate to various physiological and immune functions of living organisms. To monitor the alteration of concentration of primary and secondary phenethylamines including N-methyltyramine, octopamine, tyramine, tyrosine and phenylalanine in the metabolic pathway of phenylalanine, a sensitive and selective reversed-phase high-performance liquid chromatographic method has been developed in this study. The identification and quantification of phenethylamines were performed by fluorescent detection after pre-column derivatization with 1,3,5,7-tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)difluoroboradiaza-s-indacene, an excellent fluorescent probe which could react with both primary and secondary amino groups simultaneously. The derivatization was carried out at 25 °C for 25 min, and the separation was performed on a C18 column within 20 min. The linear ranges were from 2.0 to 100 nM for phenylalanine and tyramine to 5.0 to 250 for tyrosine and octopamine, with the detection limits of 0.1 nM for octopamine, tyramine, tyrosine and phenylalanine and 0.2 nM for N-methyltyramine (signal-to-noise ratio = 3), which allowed for the sure determination of phenethylamines at trace levels in the real samples without complex pretreatment or enrichment during multitudinous samples analysis. The proposed method has been validated by the analysis of the five target compounds in biological samples with spiked recoveries of 96.4–104.4% and the relative standard deviation of 1.0 and 4.4%.
Keywords: Fluorescence detection; High-performance liquid chromatography; Phenethylamines; 1,3,5,7-Tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)-difluoroboradiaza-s-indacene;

A sensitive, specific and rapid liquid chromatography–mass spectrometry (LC–MS/MS) method has been developed and validated for the simultaneous determination of swertiamarin, gentiopicroside, sweroside, mangiferin, isoorientin and isovitexin in rat plasma, bile, urine and feces after oral administration of Swertia pseudochinensis extract using sulfamethalazole (SMZ) as an internal standard (IS). The samples were pretreated and extracted by liquid-liquid extraction as the sample clean-up procedure. The chromatographic separation was performed on a C18 column with a linear gradient elution using a mobile phase consisted of 0.1% formic acid and methanol at a flow rate of 0.8 mL/min. The total run time was 10 min. Determination and quantification of the analytes were achieved by use of a hybrid quadrupole linear ion-trap mass spectrometer. A multiple-reaction monitoring scanning (MRM) method with electrospray ionization (ESI) source was employed with simultaneously monitoring the positive and negative electrospray ion source polarity in a single run. A full validation of the method was performed. The linearity of the analytical response was good, with correlation coefficients greater than 0.9953. The intra-day and inter-day precisions (RSD %) exhibited within 10.4%, and the accuracy (RE %) ranged from −8.7% to 9.5%. A non-compartmental model was employed to calculate the parameters. The values of elimination rate constants (K e) ranged from 0.0026 to 0.0118 and the elimination half-life (T 1/2) ranged from 58.4 to 263.0 min. This is the first report on pharmacokinetic and excretion study of Swertia pseudochinensis extract after oral administration. The results provided a meaningful basis for the clinical application of this herb.
Keywords: Pharmacokinetics; Excretion study; LC–MS/MS; Secoiridoid glycoside; Flavonoid glycoside; Swertia pseudochinensis extract;

Determination of opiates in human fingernail—Comparison to hair by Min Shen; Hang Chen; Ping Xiang (84-89).
6-Monoacetylmorphine in keratinized matrices can be used to discriminate between heroin users and individuals exposed to other sources of morphine alkaloids. Frozen pulverization is effective in preventing 6-monoacetylmorphine hydrolysis. The main aim of this study was to develop an LC-MS/MS method for the determination of five opiates in human fingernails using a frozen pulverization preparation method and to investigate the correlation between the concentration of opiates in nail and hair samples from subjects whose urine specimens were positive for morphine. Borate buffer (500 μL; pH 9.2) was added to 20 mg of pulverized fingernail, followed by ultrasonication and liquid–liquid extraction. Analytes were analyzed on an Allure PFP propyl column by gradient elution. The mass spectrometer was operated in the positive electrospray ionization mode and multiple reactions monitoring mode. A total of 12 of 18 fingernail samples contained detectable 6-monoacetylmorphine (mean = 0.43 ng/mg, range = 0.10–1.37 ng/mg), morphine (mean = 1.74 ng/mg, range = 0.58–3.16 ng/mg) and codeine (range from <limit of quantification to 0.27 ng/mg). Similarly, 12 of 18 hair samples obtained from the same subjects were positive at the revised Society of Hair Testing cutoff level of 0.2 ng/mg. The concentrations of 6-monoacetylmorphine, acetylcodeine and codeine in hair were significantly higher than those in nails. However, the concentration of MOR in nails was significantly higher than that in hair, except for one sample. All of the ratios of 6-MAM/MOR were below 0.57. It is proposed that nails may be an alternative to hair for documenting heroin abuse.
Keywords: Fingernail; Opiates; Frozen pulverization; LC-MS/MS; Hair.;

A novel micellar per aqueous liquid chromatographic method was investigated to simultaneously determine diltiazem hydrochloride, metoprolol tartrate and isosorbide mononitrate in human serum. Separation and determination of the analytes were performed on a Pinnacle II Cyano column as the stationary phase using the mobile phase consisted of aqueous solution (4.15 × 10−2  mol/L sodium dodecyl sulfate and 0.02 mol/L sodium dihydrogen phosphate) with 10% (v/v) of 1-propanol at pH 7.0. This method was validated by linearity, lower limit of quantification, extraction recovery, stability, precision, and accuracy. The main analytical parameters were linearity (r  > 0.9950), intra- and inter-day precisions (intra-day RSD 2.2–3.5%, and inter-day RSD 3.7–9.5%), lower limit of quantification (20 ng mL−1 for isosorbide mononitrate, metoprolol tartrate and diltiazem hydrochloride). The extraction recovery was 63.3% (0.1 μg/mL), 65.6% (1.0 μg/mL), and 69.5% (25 μg/mL) for isosorbide mononitrate; 65.1% (0.1 μg/mL), 69.5% (1.0  μgmL) and 73.5% (2.5 μg/mL) for metoprolol tartrate; 67 .1% (0.1 μg/mL), 68.8% (1.0 μg/mL) and 73.8 % (2.5 μg/mL) for diltiazem hydrochloride. The relative error of stability was <6.4% at the room temperature for 24 h, <3.8% at 4 °C for 1 week, <4.6% at −20 °C for 1 month, and <6.7% for freeze/thaw cycles (n  = 3). The results indicated that the proposed method was rapid, sensitive, and accurate for determination of the three antianginal drugs in human serum. The possible separation mechanism of the method was also discussed, and a model of separation mechanism for the analytes was established.
Keywords: Micellar per aqueous liquid chromatography; Diltiazem hydrochloride; Metoprolol tartrate; Isosorbide mononitrate; Human serum; Cyano column;

HPTLC method for direct determination of gemifloxacin mesylate in human plasma by W.M. El-Koussi; N.N. Atia; A.M. Mahmoud; S.R. El-Shabouri (98-101).
Novel, simple and sensitive high performance thin-layer chromatography (HPTLC) with fluorescence detection has been successfully developed and validated for determination of gemifloxacin mesylate (GFX) in plasma samples without prior pretreatment. Montelukast (MK) was used as internal standard. GFX and MK in plasma samples were separated using a mobile phase consisting of a mixture of ethyl acetate:methanol:25% ammonia, (8:4.5:3, v/v/v). The emission intensity was measured using optical filter K400 after excitation at 342 nm. The R f values for GFX and MK were 0.45 ± 0.03 and 0.79 ± 0.02, respectively. Under the optimum conditions, a linear relationship with good correlation coefficient (r  = 0.9965, n  = 6) was obtained in concentration range of 3–180 ng/band. The LOD and LOQ of the proposed method were 0.45 and 1.5 ng/band, respectively. The accuracy of the method was proved as the recovery % of GFX from spiked human plasma was 94.21–101.85%. The efficiency of the proposed method was confirmed by in-vivo application on human plasma in real patient samples. Moreover, the stability of GFX in plasma was carefully tested at different conditions and compared to others in aqueous solution.
Keywords: HPTLC; Gemifloxacin mesylate; Fluorescence detection; Human plasma;

Acrylonitrile, acrolein, 1,3-butadiene, benzene, and crotonaldehyde are hazard volatile organic compounds in tobacco smoke, which can be metabolized to mercapturic acids (MAs) excreted in urine. MAs are can be regarded as important and specific biomarkers to evaluate exposure to those carcinogenic volatile organic compounds. A simultaneous determination of N-acetyl-S-2-cyanoethyl-cysteine (CEMA), 3-hydroxypropyl)-l-cysteine (3-HPMA), N-acetyl-S-(3,4-dihydroxybutyl)-l-cysteine (DHBMA), N-acetyl-S-(phenyl)-l-cysteine (SPMA) and 3-hydroxy-1-methylpropylmercapturic acid (HMPMA) derived from five volatile organic compounds by column-switching LC–MS/MS has been described. MAs were concentrated and cleaned up by an online reusable pre-column packed with restricted access material. The intra- and inter-day precisions of the method ranged from 0.7% to 15.2%. The LODs was 0.013–0.053 ng/mL. The recovery of the whole analytical procedure ranged from 79.3% to 116%. After validation, this method was successfully applied to urine samples from smokers (n  = 246) and nonsmokers (n  = 58). The results showed MAs in urine from smokers were significantly higher than that in nonsmoker except for SPMA. Urinary CEMA significantly correlated with 3-HPMA (r  = 0.763, P  < 0.0001) and HMPMA (r  = 0.910, P  < 0.0001). CEMA, 3-HPMA and HMPMA are potential biomarkers to distinguish the differences between smokers and nonsmokers.
Keywords: Column-switching; LC–MS–MS; Urine; Mercapturic acids;

We developed a high-performance liquid chromatography–tandem mass spectrometric method for phospholipid biomarker discovery and applied it to a cell-based assay system for the screening of phospholipidosis-inducing drugs. We studied the compositions of phospholipid molecules exceeding 100 species in cultured cells and found a characteristic alteration in the composition by treatment with cationic amphiphilic drugs possessing phospholipidosis-inducing potency. The compositions of phosphatidylinositol in RAW264 cells were significantly affected by the drug treatment. Similar alterations were also found in THP-1 cells. These phenomena were not observed when cells were treated with warfarin, which does not have phospholipidosis-inducing potency. Structural analysis of the altered phosphatidylinositols by a product ion scan revealed the presence of certain fatty acyl chains. Based on our findings, we proposed a prediction parameter (PP) for phospholipid accumulation calculated from the relative compositions of phosphatidylinositol species. As the dosage of imipramine (a cationic amphiphilic drug) increased, both the PP and cellular phospholipid content increased. Our results suggest that PP has potency as a biomarker for phospholipid accumulation in cells treated with drugs.
Keywords: Phospholipidosis, HPLC-MS/MS; Phosphatidylinositol; Arachidonic acid; Eicosatrienoic acid; RAW264; THP-1; U-937.;

Separation of porcine parvovirus from bovine serum albumin using PEG–salt aqueous two-phase system by K. Saagar Vijayaragavan; Amna Zahid; Jonathan W. Young; Caryn L. Heldt (118-126).
Vaccine production faces a challenge in adopting conventional downstream processing steps that can efficiently purify large viral particles. Some major issues that plague vaccine purification are purity, potency, and quality. The industry currently considers 30% as an acceptable virus recovery for a vaccine purification process, including all downstream processes, whereas antibody recovery from CHO cell culture is generally around 80–85%. A platform technology with an improved virus recovery would revolutionize vaccine production. In a quest to fulfill this goal, we have been exploring aqueous two-phase systems (ATPSs) as an optional mechanism to purify virus. ATPS has been unable to gain wide implementation mainly due to loss of virus infectivity, co-purification of proteins, and difficulty of polymer recycling. Non-enveloped viruses are chemically resistant enough to withstand the high polymer and salt concentrations that are required for effective ATPS separations. We used infectious porcine parvovirus (PPV), a non-enveloped, DNA virus as a model virus to test and develop an ATPS separation method. We successfully tackled two of the three main disadvantages of ATPS previously stated; we achieved a high infectious yield of 64% in a PEG–citrate ATPS process while separating out the main contaminate protein, bovine serum albumin (BSA). The most dominant forces in the separation were biomolecule charge, virus surface hydrophobicity, and the ATPS surface tension. Highly hydrophobic viruses are likely to benefit from the discovered ATPS for high-purity vaccine production and ease of implementation.
Keywords: PPV; ATPS; Non-enveloped virus; Vaccine purification; Hydrophobicity; Hofmeister series;

Targeted metabolite profiling has aided in the understanding of a variety of biological processes in the malaria parasite as well as in drug discovery. A fast and sensitive analytical method, based on ion-pairing reversed phase ultra-high performance liquid chromatography tandem mass spectrometry (IP-RP–UPLC–MS/MS), was optimized for the simultaneous analysis of intracellular levels of 35 purine and pyrimidine nucleobases, nucleosides, and nucleotides. This analytical method allows for chromatographic separation of highly polar metabolites using reverse phase chemistry within 15 min. The analytical performance of the method was evaluated and successfully applied to the quantification of purines and pyrimidines in Plasmodium falciparum and its host cell, the human erythrocyte. In addition, this method can be customized to include other related metabolites such as NADPH and NADP, among others.
Keywords: UPLC–MS; Purines and pyrimidines; Malaria; Plasmodium falciparum; Human erythrocytes;

Direct silylation of Trypanosoma brucei metabolites in aqueous samples and their GC–MS/MS analysis by Peter Podolec; Alexandra Hengerics Szabó; Jaroslav Blaško; Róbert Kubinec; Renáta Górová; Jozef Višňovský; Anna Gnipová; Anton Horváth; Václav Bierhanzl; Tomáš Hložek; Radomír Čabala (134-138).
A simple two-step method for the derivatization of polar compounds (lactate, alanine, glycerol, succinate and glucose) using hexamethyldisilazane (HMDS) and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) was developed. This method allows direct derivatization of aqueous samples wihout sample pretreatment. The method was used for the analysis of the metabolites of the unicellular organism Trypanosoma brucei. The limits of detection by GC–MS/MS analysis were in the range of 0.02 mgL−1 for glucose to 0.85 mgL−1 for lactate.
Keywords: Aqueous samples; GC–MS/MS analysis; Metabolites; Protozoa; Trimethylsilylation;

Two-dimensional paper chromatography-based fluorescent immunosensor for detecting acute myocardial infarction markers by Jung-Hwan Cho; Min-Ha Kim; Rak-Sun Mok; Jin-Woo Jeon; Guei-Sam Lim; Chan-Young Chai; Se-Hwan Paek (139-146).
A novel washing scheme following antigen–antibody reactions with analyte was used during construction of a fluorescent immunosensor to resolve the background problem in the lateral flow assay with human serum. An immuno-membrane strip was devised to simultaneously measure cardiac troponin I (cTnI), creatinine kinase-MB isoform (CK-MB), and myoglobin to diagnose acute myocardial infarction. This strip was then installed within a cartridge containing a built-in washing solution tank, which was used to supply the solution across the signal generation pad of the strip after the immune reactions. Such cross-flow washing was initiated by onset-signaling from the internal control and began to run automatically upon sample addition. Under optimal conditions, the immunosensor displayed a stably suppressed background baseline, enabling us to attain a low detection limit for cTnI (0.05 ng/mL) as well as favorable reproducibility for repetitive measurements (relative standard deviation <10%). No interference was observed among the different complex formations at the respective analyte sites, and no artifacts were caused by sample matrices. We tested the performance relationship with the Pathfast reference system for positive serum samples (36 for cTnI, 58 for CK-MB, and 17 for myoglobin), and the correlation coefficients were >0.98. This result suggests that the new immunosensor system based on two-dimensional chromatography can be used for clinical testing
Keywords: Cross-flow washing; Lateral flow assay; Signal-to-noise ratio; Acute myocardial infarction triple biomarkers; Real human serum samples;

For liquid chromatographic analysis of amino acids involving derivatization and mass-spectrometric detection, it becomes more important to evaluate the presence of matrix effects in complex samples. This is somewhat complicated for amino acid analysis where analyte free sample matrix is often unavailable. In this work, matrix effects were investigated using post-column infusion method for 9-fluorenylmethyl chloroformate (FMOC-Cl) derivatives of β-Ala, Gly and Phe and diethyl ethoxymethylenemalonate (DEEMM) derivative of β-Ala. While for DEEMM derivatives, the main signal suppression was due to the borate buffer, in case of FMOC-Cl, other FMOC-derivatives caused signal suppression.Analysis of amino acids in tea and honey with DEEMM, FMOC-Cl, p-N,N,N-trimethylammonioanilyl N′-hydroxysuccinimidyl carbamate iodide (TAHS) and dansyl chloride (DNS) showed that amino acid concentrations found with different reagents do not agree well. Sample dilution experiments indicated that the sample matrix affected the analysis results obtained with DEEMM the least, but with FMOC-Cl, TAHS and DNS, sample dilution had an influence on the results. When sample dilution and extrapolative dilution approach were applied on the latter results, an agreement of amino acid concentrations measured with different reagents was achieved within relative standard deviation (RSD) of 22% for most cases.
Keywords: Amino acid analysis; Derivatization; Liquid chromatography; Electrospray mass spectrometry; Matrix effects;

Nigella glandulifera Freyn et Sit is a folk medicinal plant, whose seeds show significant anticancer activities attributed to triterpene saponins and volatile oil. In this study, an in vitro cytotoxicity assay demonstrated that Nigella A, the major component of triterpene saponins extracted from N. glandulifera, exhibited growth inhibition in the human lung carcinoma A-549 cell line. Due to this potential activity, a reliable liquid chromatography–tandem mass spectrometry (LC–MS/MS) method to quantify Nigella A in rat plasma for a pharmacokinetic study was developed. Nigella A and pravastatin, as internal standard (IS), were extracted from rat plasma using acetonitrile to precipitate protein. Separation was performed on an Agilent Zorbax SB-Aq (3.0 × 150 mm, 3.5 μm) column using a gradient elution method with acetonitrile–0.1% formic acid in water at a flow rate of 0.35 mL/min. Detection was performed using an electrospray ionization in a negative ion multiple reaction monitoring mode. The deprotonated precursor to product ion transitions monitored for Nigella A and IS was at m/z 1352.7→882.6 and m/z 423.1→321.0, respectively. The linear range was 0.240–120 μg/mL with a square regression coefficient (r  = 0.9996). The intra-day and inter-day precision was less than 6.93%. The simple extraction procedure provided recovery ranged from 92.32 to 95.44% for both analyte and IS. The method was proved to be reliable, precise, and accurate, and was successfully applied to a pharmacokinetic study of Nigella A in rats after i.v. administration via the tail vein at doses of 10, 20, and 30 mg/kg.
Keywords: Nigella A; LC–MS/MS; Pharmacokinetics; Rat plasma;

(−)-Epiafzelechin is a flavan-3-ol commonly found in plant source. Biological studies suggested that (−)-epiafzelechin may have anti-inflammatory, anti-oxidant and bone-protective effect. However, it's in vivo efficacy remains to be demonstrated. A specific detection method for (−)-epiafzelechin was successfully developed by using UPLC–MS/MS to quantify the amount of (−)-epiafzelechin present in mice plasma after a liquid–liquid extraction by ethyl acetate. The separation was achieved by using a reversed-phase C18 column with a 16 min gradient elution protocol consisting of water (0.1%, v/v, formic acid) and 0–70% ACN (0.1%, v/v, formic acid). The lower limit of quantitation for (−)-epiafzelechin was found to be 12.5 ng/mL. This method exhibited a good linearity (r 2  = 0.992). The intra-day and inter-day precision were within 12%, while the accuracy was between 97.6 and 113. 4%. A quantity of 10 mg/kg synthetic (−)-epiafzelechin was administered to C57BL/6J mice by intravenous (i.v.) and intraperitoneal (i.p.) injections and the blood was collected at different time points. The plasma was then analyzed by the UPLC–MS/MS method, and the plasma drug concentration–time curves for i.v. and i.p. (−)-epiafzelechin injection were constructed. The maximum concentrations (C max) of (−)-epiafzelechin in blood by i.v. and i.p. injection were found to be 10.6 and 6.0 μg/mL, respectively, while the time for reaching C max in i.p. injection was found to be 15 min. The distribution half-lives of (−)-epiafzelechin after i.v. and i.p. injection were found to be 7.0 and 12.6 min, respectively. Some of the PK parameters were found to be similar in both i.v. and i.p. injections of (−)-epiafzelechin owing to its high solubility in water.
Keywords: (−)-Epiafzelechin; Intravenous; Intraperitoneal; Pharmacokinetics; UPLC–MS/MS;

In situ monolithic molecularly imprinted polymer sol–gel packed tips (MMSTs) were prepared and evaluated for the extraction of lung cancer biomarker l-tyrosine (Tyr) from human plasma and urine samples. Several extraction parameters such as the conditioning, washing and elution solutions, pH and time were investigated. The enrichment factor (EF) and extraction recovery (ER) were studied. MMST showed good selectivity and a high extraction recovery, and MMST as a sorbent showed good stability and repeatability. The method validation showed good regression correlation coefficients for plasma and urine samples (R 2  ≥ 0.996) within the concentration range of 5–1000 and 1–1000 nmol L−1 in plasma and urine samples, respectively. The lower limits of quantification (LLOQ) in the plasma and urine samples were 5 and 1 nmol L−1, respectively. The between-batch precision for Tyr in plasma ranged from 1.0 to 6.0%, and in urine it was from 1.0 to 7.0%. The results show that the developed method has more facility, stability, durability and repeatability compared with previous similar methods. To the best of our knowledge, this is the first study aimed at the selective separation of Tyr as a lung cancer biomarker by MMSTs from biological matrixes and detection by LC/MS/MS.
Keywords: Monolithic molecularly imprinted polymer sol–gel in-tip; Tyrosine; Plasma; Urine; Liquid chromatography–tandem mass spectrometry;

This paper presents an application of ultrahigh-performance liquid chromatography and quadrupole Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HR MS) for the screening, confirmation and quantification of 11 antidiabetics in herbal medicines and dietary supplements. The mass spectrometer was operated in Full MS/dd-MS2 (data-dependent MS2) mode. The full MS scan acquired data for identification and quantification, and dd-MS2 scan obtained product ion spectra for confirmation. UHPLC-Q-Orbitrap MS quantification was achieved using matrix-matched standard calibration curves with phenacetin as internal standard. The method validation that included selectivity, sensitivity, calibration curve, accuracy and precision, recovery, matrix effect and stability was evaluated. The response showed good linear relationship with the concentrations of analytes over wide ranges (e.g., 0.0004-1 μg/g for metformin) with all the coefficients of correlation (r 2) >0.9991. The detection limits (LODs) were in the range of 0.05-0.5 ng/g for different analytes. The recoveries yielded results higher than 74.3% for all compounds. The accuracy was in the range of -6.75 to 3.85%, while the intra- and inter-day precision ranged from 0.048 to 11.5%. Among 63 batches of herbal medicines and 34 batches of dietary supplements samples, 7 batches of dietary supplements were positive, while all the herbal medicines were negative. Overall, the novel UHPLC-Q-Orbitrap has demonstrated great performance for identification, confirmation and quantification of antidiabetics in herbal medicines and dietary supplements, ensuring food safety and public health
Keywords: Orbitrap; Illegal adulteration; Antidiabetics; Identification; Quantitation.;

This paper describes the development of a novel high-throughput hollow fiber membrane solvent microextraction technique for the simultaneous measurement of the octanol/water distribution coefficient (log  D) for organic compounds such as drugs. The method is based on a designed system, which consists of a 96-well plate modified with 96 hollow fiber membrane tubes and a matching lid with 96 center holes and 96 side holes distributing in 96 grids. Each center hole was glued with a sealed on one end hollow fiber membrane tube, which is used to separate the aqueous phase from the octanol phase. A needle, such as microsyringe or automatic sampler, can be directly inserted into the membrane tube to deposit octanol as the accepted phase or take out the mixture of the octanol and the drug. Each side hole is filled with aqueous phase and could freely take in/out solvent as the donor phase from the outside of the hollow fiber membranes. The log  D can be calculated by measuring the drug concentration in each phase after extraction equilibrium. After a comprehensive comparison, the polytetrafluoroethylene hollow fiber with the thickness of 210 μm, an extraction time of 300 min, a temperature of 25 °C and atmospheric pressure without stirring are selected for the high throughput measurement. The correlation coefficient of the linear fit of the log  D values of five drugs determined by our system to reference values is 0.9954, showed a nice accurate. The −8.9% intra-day and −4.4% inter-day precision of log  D for metronidazole indicates a good precision. In addition, the log  D values of eight drugs were simultaneously and successfully measured, which indicated that the 96 throughput measure method of log  D value was accurate, precise, reliable and useful for high throughput screening.
Keywords: Hollow fiber membrane; Solvent microextraction; Octanol/water distribution coefficient; High-throughput; Drug screening;

A capillary electrophoresis (CE) with chemiluminescence (CL) detection method was developed for the simultaneous quantification of 5-hydroxyindoleacetic acid (5-HIAA) and 5-hydroxytryptamine (5-HT). In this method, CdTe quantum dot (QD) and horseradish peroxidase (HRP) were used as enhancing reagents to co-catalyze the post-column CL reaction between luminol and hydrogen peroxide, achieving highly efficient CL emission. 5-HIAA and 5-HT inhibit the CL emission resulting to the formation of negative peaks in electropherogram. The degree of CL suppression is proportional to the concentration of 5-HT and 5-HIAA. The linear ranges for the determination of 5-HIAA and 5-HT were 2.5 × 10−8–2.5 × 10−6  M and 2.5 × 10−8–5.0 × 10−6  M with detection limits (signal/noise = 3) of 7.0 × 10−9  M and 6.0 × 10−9  M, respectively. Intraday precision do not exceed 5.0%. The accuracy was confirmed by the recoveries ranged from 98% to 104%. The present method was successfully applied for the quantification of 5-HIAA and 5-HT in human urine. The concentrations of 5-HT and 5-HIAA in human urine were found to be in the range of 0.78–1.2 μM and 3.2–5.1 μM, respectively.
Keywords: Capillary electrophoresis; Chemiluminescence; Quantum dot; 5-Hydroxyindoleacetic acid; 5-Hydroxytryptamine; Human urine;

An LC–MS/MS method was developed for simultaneous quantification of 25-hydroxyvitamin D3 (25(OH)D3), 3-epi-25(OH)D3, and 25(OH)D2 in human serum. Methods: Sample preparation consisted of protein precipitation followed by off-line SPE. Calibration curves for each vitamin D metabolite were constructed in phosphate-buffered saline with 60 g/L albumin including its corresponding stable isotope labelled (SIL) internal standard. A pentafluorophenyl (PFP) analytical column was used to resolve 25(OH)D3 from 25(OH)D2 and 3-epi-25(OH)D3, followed by SRM registration using positive ESI-MS/MS. Accuracy was assessed from measurement of samples with NIST reference method procedure (RMP) assigned values. The PFP LC–MS/MS method was compared to an in-house C18 column LC–MS/MS method, not resolving 25(OH)D3 from 3-epi-25(OH)D3, using adult and newborn samples. Results: Intra-assay and inter-assay coefficients of variation were less than 4% and 7.5%, respectively for all three vitamin D metabolites; lower limits of quantification were 1, 1 and 2 nmol/L and linearity of methods were 1–500, 1–200 and 2–500 nmol/L for 25(OH)D3, 3-epi-25(OH)D3 and 25(OH)D2, respectively. The PFP LC–MS/MS method showed minimal bias to the NIST RMP. Method comparison revealed that in the C18 LC–MS/MS method, the 3-epi-25(OH)D3 concentration is overestimated inadvertently not only from co-elution of both analytes, but also by an additional 30–40% higher ionisation efficiency of 3-epi-25(OH)D3 when compared to 25(OH)D3. Conclusion: This accurate LC–MS/MS method allows the simultaneous measurement of 25(OH)D3, 3-epi-25(OH)D3, and 25(OH)D2 in human serum. Due to increased ionisation efficiency, the contribution of the 3-epi-25(OH)D3 metabolite to the total 25(OH)D3 concentration is significantly overestimated in MS methods that do not resolve 3-epi-25(OH)D3 from 25(OH)D3 and may compromise its use in infant samples known to have significant amounts of 3-epi-25(OH)D3.
Keywords: C3-epimer; 3-epi-25-hydroxyvitamin D3; 25-hydroxyvitamin D; LC–MS/MS; MS response factor; Ionisation behaviour;

This paper describes an extension and performance characterization of a quantitative confirmatory multi-residue liquid chromatography–tandem mass spectrometric method for residues of macrolide and lincosamide antibiotics, originally validated for application to bovine kidney tissues, to tissues of salmon, shrimp and tilapia. The 12 analytes include clindamycin, erythromycin A, gamithromycin, josamycin, lincomycin, neospiramycin 1, oleandomycin, pirlimycin, spiramycin 1, tildipirosin, tilmicosin and tylosin A. The limit of detection was 0.5 μg/kg. Within-laboratory precision evaluated over the analytical range of 5.0–50.0 μg/kg ranged from 4 to 17%. The accuracy of the method ranged from 80 to 112%. Recoveries ranged from 47 to 99% with all but one recovery above 60%. This is the first report of a quantitative confirmatory method for gamithromycin, pirlimycin and tildipirosin in fish and shrimp.
Keywords: Fish; Shrimp; Antibiotics; Macrolides; Lincosamides; LC–MS/MS.;

Development of an LC–MS/MS method for high throughput quantification of metformin uptake in transporter inhibition assays by Marianne Vath; Lizbeth Gallagher; Wilson Shou; Harold Weller; Lisa Elkin; Jun Zhang (211-218).
A high throughput LC–MS/MS method for quantification of metformin substrate uptake enables conversion of radiometric transporter inhibition assays for multidrug and toxin extrusion transporters (MATE 1 and 2) and organic cation transporter 2 (OCT2) to a nonradioactive format. Such conversion greatly simplifies assay complexity and reduces assay costs. The development of a quantitative LC–MS/MS method for metformin in support of the high throughput transporter inhibition assays faced specific challenges of achieving both adequate chromatographic retention and rapid analytical turnaround. Here we report a method that circumvents both challenges. The utilization of a porous graphitic carbon column (Hypercarb) ensured adequate retention of highly polar metformin in biological samples. The combined employment of a ballistic gradient on a 3 mm × 30 mm, 5 μm Hypercarb column, and dual staggered chromatography coupled with multiple injection chromatography acquisition, yielded a fast injection-to-injection cycle time of 30 s. The method demonstrated good accuracy, precision and excellent robustness for high throughput applications, and has been successfully implemented in the development and validation of the nonradioactive transporter inhibition assays for MATEs and OCT2.
Keywords: Metformin; LC–MS/MS; Transporter inhibition; Porous graphite carbon; Silica hydride;

A simple LC–MS/MS method for determination of sitafloxacin in human urine by Yuanyuan Wang; Yang Liu; Hongwen Zhang; Yongqing Wang; Yun Liu; Libin Wang; Ning Ou (219-224).
Sitafloxacin is a new fluoroquinolone antimicrobial agent with high activity. In this article, we reported a simple, rapid and specific LC–MS/MS method for accurate determination of sitafloxacin concentrations in human urine from healthy volunteers in detail. A two-step dilution method for the analysis of sitafloxacin in human urine using LC coupled to positive MS/MS has been developed and validated according to US FDA guidelines and Chinese State Food and Drug Administration (CFDA) guidelines for the validation of bioanalytical methods. The method uses 50 μL of urine and covers a working range from 0.025 to 20 μg/mL with a LLOQ of 0.025 μg/mL. This new LC–MS/MS assay is sensitive and specific.
Keywords: Sitafloxacin; LC–MS/MS; Urine; Urine recovery study;

Simultaneous determination of bambuterol and its two major metabolites in human plasma by hydrophilic interaction ultra-performance liquid chromatography–tandem mass spectrometry by Ting Zhou; Qing Cheng; Chengjuan Zou; Ting Zhao; Shan Liu; Marco Pistolozzi; Evina Tan; Ling Xu; Wen Tan (225-234).
In this study, a rapid and sensitive hydrophilic interaction ultra-performance liquid chromatography–tandem mass spectrometry (HILIC–UPLC–MS/MS) method was developed for simultaneous determination of bambuterol and its two major metabolites monocarbamate bambuterol and terbutaline in human plasma. All samples were simply precipitated using acetonitrile and separated on a UPLC-HILIC column under gradient elution with a mobile phase consisting of acetonitrile and water with the addition of 10 mm ammonium acetate and 0.1% formic acid at 0.4 mL/min. The analytes were detected by a Xevo TQ-S tandem mass spectrometer with positive electrospray ionization in multiple reaction monitoring mode. The established method was highly sensitive with the lower limit of quantification (LLOQ) of 10.00 pg/mL for each analyte, and the intra- and inter-day precisions were <12.8%. The analytical runtime within 4.0 min per sample made this method suitable for high throughput determination. The validated method was successfully applied to a clinical pharmacokinetic study of bambuterol in eight healthy volunteers. Furthermore, the effects of the chromatographic conditions on the retention of the analytes on HILIC were investigated, and the benefits of HILIC were evaluated by comparing with a C18 column. The results indicated that liquid–liquid partition and the electrostatic interactions played an important role in the retention of the analytes on HILIC in this study. And HILIC offered particular advantages over RPLC approach in the aspects of the peak symmetry, the column efficiency, and the column pressure.
Keywords: HILIC-UPLC-MS/MS; Bambuterol; Major metabolites; Plasma; Pharmacokinetics.;

UPLC–MS/MS determination of thiamphenicol in human plasma and its application to a pharmacokinetic study by Zhe Wang; Hui Yang; Wei Sun; Cheng-ke Huang; Xiao Cui; Xiang-jun Qiu; Qing-quan Lian; Zeng-shou Wang (235-239).
A sensitive and rapid ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method was developed to determine thiamphenicol (TAP) in human plasma using chlorzoxazone as the internal standard (IS). Sample preparation was accomplished through a liquid–liquid extraction procedure with ethyl acetate to precipitation of plasma protein, and to a 0.1 mL plasma sample. The analyte and IS were separated on an Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm) with the mobile phase of acetonitrile and 1% formic acid in water with gradient elution at a flow rate of 0.40 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with electrospray ionization (ESI) by multiple reactions monitoring (MRM) of the transitions at m/z 354.3 → 185.1 for TAP and m/z 168.1 → 132.1 for IS. The linearity of this method was found to be within the concentration range of 10–8000 ng/mL with a lower limit of quantification of 10 ng/mL. Only 1.5 min was needed for an analytical run. The method herein described was superior to previous methods and was successfully applied to the pharmacokinetic study of TAP in healthy Chinese volunteers after oral administration.
Keywords: Thiamphenicol; UPLC–MS/MS; Human plasma; Pharmacokinetics;

Organic cation transporters are responsible for the disposition of various endogenous and therapeutic agents in humans; thus, there is a great need for the development of a simple assay for simultaneous assessment of the activities of multiple transporters. Using liquid chromatography–mass spectrometry (LC/MS), we developed an assay that allows for simultaneous quantitation of plasma and urinary levels of N 1-methylnicotinamide (a substrate of hOCT2/hMATEs), l-carnitine (a substrate of hOCTN2), and creatinine (an indicator of glomerular filtration). Samples were diluted with ultrapure water, deproteinized with trichloroacetic acid, filtered, and then injected on a cation exchange column. The analytes were separated with a gradient LC technique and detected by MS. The total assay time was less than 8 min. The lower detection limits for N 1-methylnicotinamide, l-carnitine, and creatinine were 2, 10, and 24 ng/mL, respectively. Recovery of the analytes was almost complete. A preliminary clinical study conducted in 25 healthy subjects revealed that the mean ± SD for the renal clearance (CLR) of N 1-methylnicotinamide (272.7 ± 81.0 mL/min) far exceeded the glomerular filtration rate (116.3 ± 19.6 mL/min), indicating the involvement of active tubular secretion, while the mean CLR of clearance of l-carnitine was close to nil (1.5 ± 1.4 mL/min), indicating almost complete tubular reabsorption. The present method is potentially useful for clinical studies on the genetic control of cationic transporter activities and the transporter-mediated drug interactions.
Keywords: N 1-Methylnicotinamide; l-carnitine; Creatinine; LC/MS; Humans; Plasma; Urine;

Ying-zhi-huang injection (YZH-I) is an injectable multi-herbal prescription derived from the ancient Chinese remedy “Yin-chen-hao-tang”, which is widely used in the clinic for the treatment of jaundice and chronic liver diseases. To date, little information is available on the pharmacokinetic properties of this poly-herbal formulation. Herein, we reported a simple, rapid and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for quantitative multiple reaction monitoring (MRM) of eight major ingredients of YZH-I (including baicalin, baicalein, wogonoside, geniposide, geniposidic acid, chlorogenic acid, neochlorogenic acid, and caffeic acid) in rat plasma. A fast single-tube multi-impurity precipitation extraction (“SMIPE”) procedure was introduced for straightforward plasma preparation, based on one-pot deproteinization precipitation with acidified methanol extraction and in-situ multifunction impurity removal by a solid sorbent mixture (anh. magnesium sulfate plus octadecylsilane). Particularly, the addition of ascorbic acid in methanol (10 mg/mL) was found to exhibit a pronounced protective effect and significantly increase extraction effectiveness of the herbal phenolic components. Some pretreatment variables (protein precipitating solvent, acidifying agent and sorbent) were optimized with acceptable matrix effect (−18 to 7.7%), extraction recovery (65–88%) and process efficiency (62–91%) for the SMIPE-based LC-MRM multi-analyte quantitation using matrix-matched calibration (5–1000 ng/mL) without using internal standard. Mean accuracies were obtained in the range of 83–114% at three different fortification levels, with intra- and inter-day variations within 13%. This validated method was successfully applied to the simultaneous measurement and pharmacokinetic investigation of the chemical constituents in rats following an intravenous administration of YZH-I.
Keywords: Yin-zhi-huang formula; Multi-component quantitation; Multiple reaction monitoring; Protein precipitation; Single-tube extraction; Pharmacokinetics;

A liquid chromatography–electrospray ionization–tandem mass spectrometry method was described for the simultaneous determination of resibufogenin, bufalin, gamabufotalin, telibufagin, arenobufagin, cinobufagin and bufotalin in rat plasma. Plasma samples were pretreated by liquid–liquid extraction with ethyl acetate. Chromatographic separation was carried out on an ACQUITY HSS T3 column with gradient elution using mobile phase consisting of acetonitrile–0.1% formic acid in water at a flow rate of 0.3 mL/min. All analytes showed good linearity over a wide concentration range (r  > 0.99). The lower limit of quantification was in the range of 0.5–10 ng/mL for seven bufadienolides. The mean recovery of the analytes ranged from 94.36 to 104.18%. The intra- and inter-day precisions were in the range of 1.74–13.78% and the accuracies were between 89.37 and 101.38%. The validated method was successfully applied to a pharmacokinetic (PK) study of the seven bufadienolides in rat plasma after oral administration of Shexiang Baoxin Pill (SBP). The selected PK marker compounds with typical efficacy/toxicity may provide a practical solution for marker compound selection and dosage design for the therapeutic drug monitoring and PK study of SBP in its clinical applications.
Keywords: Bufadienolides; Shexiang Baoxin Pill; LC–ESI–MS/MS; Pharmacokinetic study;

Chromatographic fingerprint analysis of metabolites in natural and artificial agarwood using gas chromatography–mass spectrometry combined with chemometric methods by Xiaoxia Gao; Mingrong Xie; Shaofeng Liu; Xiaoling Guo; Xiaoying Chen; Zhaojian Zhong; Lei Wang; Weimin Zhang (264-273).
Agarwood is a resinous material formed in wounded Aquilaria sinensis in China, which is widely used as an effective traditional Chinese medicine (TCM). This study is aimed to use gas chromatography–mass spectrometry combined with chemometric methods to create reliable criteria for accurate identification of natural agarwood and artificial agarwood, as well as for quality evaluation of artificial agarwood. Natural agarwood and artificial agarwood (stimulated by formic acid or formic acid plus fungal inoculation) were used as standards and controls for the gas chromatography–mass spectrometry (GC–MS) and multivariate analysis. The identification criteria developed were applied to commercial agarwood. A reliable criteria including correlation coefficient of GC–MS fingerprint of natural agarwood and 22 markers of metabolism in natural and artificial agarwood was constructed. Compared with chemically stimulated agarwood (formic acid) and in terms of the 22 markers, artificial agarwood obtained by formic acid stimulation and fungal inoculation were much closer to natural agarwood. The study demonstrates that the chemical components of artificial agarwood obtained by comprehensive stimulated method (formic acid plus fungal inoculation) are much closer to the natural agarwood than those obtained by chemically stimulated method (formic acid), as times goes by. A reliable criteria containing correlation coefficient of GC–MS fingerprint of natural agarwood and 22 metabolism markers can be used to evaluate the quality of the agarwood. As an application case, three samples were identified as natural agarwood from the 25 commercial agarwood by using the evaluation method.
Keywords: Agarwood; Aquilaria sinensis (Lour.) Gilg; Multivariate analysis; Chromatographic fingerprinting;