Journal of Chromatography B (v.965, #C)

Molecularly imprinted polymers (MIPs) with high selectivity and affinity to glabridin were designed based on the screening results of the library of non-imprinted polymers (NIPs). The NIP library contained 48 polymers that were polymerized with the combinations of different functional monomers, cross-linkers, and porogenic solvents. The distribution coefficient (k) values were used to estimate the affinity of NIPs to glabridin. The corresponding MIPs of the best three NIPs were prepared. After evaluating the imprinting effects and selectivity of the three MIPs, the performance of the best MIP as solid phase extraction sorbent was investigated. Glabridin with percent recovery of 83 was obtained from the extract of Glycyrrhiza glabra L. (G. glabra L.) residues by molecularly imprinted solid phase extraction (MISPE). Thus, this material can be successfully used for the extraction and purification of glabridin from G. glabra L. residues.
Keywords: Imprinting; Glabridin; Polymer; Adsorption; Polymer screening;

Forensic testing for drugs of abuse in hair has become a useful diagnostic tool in determining chronic drug use as well as examining long-term drug history thorough segmental analysis. However, sensitive and specific analytical methods are needed.A simple, rapid and highly sensitive and specific method for the extraction and quantification of 33 opioids, opiates, cocaine, and amphetamines is presented. The method was fully validated according to international guidelines. Twenty milligrams of hair sample was pulverized and then incubated in the same disposable tube with methanol (under sonication at 45 °C) during 4 h. After centrifugation the supernatant was evaporated up to about 100 μL and a solid phase extraction (SPE) followed by separation and quantification using ultra performance liquid chromatography–tandem mass spectrometry (UHLC–MS/MS) were carried out. Chromatographic separation was achieved using a BEH phenyl column eluted with 0.1% formic acid: methanol (0.1% formic acid). Selectivity of the method was achieved by a combination of retention time, and two precursor–product ion transitions. Good intra-assay and inter-assay precision (relative standard deviations (RSDs) were observed (<15%) for most of the compounds. The lower limit of quantification was fixed at the lowest calibrator in the linearity experiments and it ranged from 0.006 to 0.063 ng/mg. No instability was observed in processed samples. Extraction efficiency varied from 37 to 107% (except for EDDP with a recovery of 5%) and matrix effects ranged from 52 to 160%, and for most of the compounds it was compensated by the internal standard (IS).The method was subsequently applied to authentic hair samples obtained from forensic and toxicology cases and to proficiency test (obtaining z-scores lower than 1 for most of the compounds). The validation and actual sample analysis results show that this method is rugged, precise, accurate, and well-suited for routine hair analysis.
Keywords: UPLC–MS/MS; Hair; Opioids; Cocaine; Amphetamines; Proficiency test;

Pharmacokinetic study of multiple active constituents from Kushen–Gancao Decoction after oral administration in rat by HPLC–MS/MS by Qinhui Wang; Lei Shi; Xiuling Tang; Qingwei Wang; Xueliang Dang; Yan Zhang (19-26).
Kushen–Gancao Decoction (KGD) is a classic traditional Chinese herb combination in treating viral hepatitis and chronic liver diseases. This study aims to investigate the pharmacokinetic (PK) study of matrine (MT), oxymatrine (OMT), glycyrrhizic acid (GL) and glycyrrhetinic acid (GA) following oral administration of KGD in rats. A rapid, sensitive and reliable HPLC–MS/MS method was successfully developed for the simultaneous determination of MT, OMT, GL and GA in rat plasma. A Inertsil C18 analytical column was used with a gradient mobile phase system of methanol-ammonium acetate (5 mM) with a flow rate of 0.5 mL/min. The analysis was performed on a positive and negative ionization electrospray mass spectrometer via multi reaction monitoring (MRM). Linear calibration curves were obtained for the following concentration range: 10–5000 ng/mL for MT, OMT and GL, 50–15,000 ng/mL for GA in rat plasma (R 2  > 0.99). The lower limit of quantification (LLOQ) was 5 ng/mL (MT, OMT and GL) and 20 ng/mL (GA). The intra- and inter-day accuracies ranged from −7.91 to 9.10% and precisions (RSD) were within 15%. The analytes were found to be stable under short-term temperature conditions, post-preparative temperature conditions, and after three freeze-thaw cycles conditions. The validated method was successfully applied to a pharmacokinetic study in rats after oral administration of KGD.
Keywords: Matrine; Oxymatrine; Glycyrrhizic acid; Glycyrrhetinic acid; HPLC–MS/MS; Pharmacokinetic;

Scutellaria-coptis herb couple is widely used traditional Chinese medicine (TCM) in treating type 2 diabetes; however, the in vivo integrated metabolism of its main bioactive components in type 2 diabetic rats remains unknown. In this paper, ultra-performance liquid chromatography (UPLC) coupled to quadrupole time-of-flight (Q-TOF) and the MetaboLynx™ software combined with mass defect filtering (MDF) together provided unique high throughput capabilities for drug metabolism study with excellent MS mass accuracy and enhanced MSE data acquisition. This rapid automated analysis method was successfully applied for screening and identification of the absorbed and metabolized constituents after oral administration of scutellaria-coptis extract to rats. The results showed that a total of 14 metabolites of two parent compounds were detected and tentatively identified in vivo based on the characteristics of their protonated ions. Main parent components of scutellaria-coptis extract such as baicalin and berberine were absorbed into the blood circulation of the rats. Differences of metabolite classes were not observed between normal and type 2 diabetic rat plasma and urine samples. However, the concentrations of baicalin and methylated berberine in type 2 diabetic rat plasma were much higher than those in normal sample. While, the concentrations of these two compounds in type 2 diabetic rat urine were remarkably lower than those in normal sample. This helped maintain a high blood drug concentration which might be beneficial for the treatment of type 2 diabetes. Additionally, the developed method was simple and reliable, revealing that it could be used to rapid screen and propose the structures of active components responsible for pharmacological effects of scutellaria-coptis and to better clarify its action mechanism. This work suggests that the integrative metabolism approach makes a useful template for drug metabolism research of TCMs.
Keywords: Scutellaria-coptis herb couple; Baicalin; Berberine; Metabolism; UPLC-ESI-Q-TOF/MS;

Due to its cytotoxicity, genotoxicity, and adipogenicity observed in in vitro studies, bisphenol A diglycidyl ether (BADGE) may pose a health risk to humans. Quantifying BADGE exposure is an essential step to assess potential health risks associated with this ubiquitous compound widely used in certain plastic products. Due to the lack of endogenous sources for BADGE, bio-monitoring of BADGE and/or its hydrolytic metabolites (BADGE·H2O and BADGE·2H2O) can be a useful means to measure exposure. In this study, we developed a highly specific and sensitive method to measure BADGE, BADGE·H2O and BADGE·2H2O in plasma and urine, using a fast liquid–liquid extraction technique followed by a high-performance liquid chromatography and positive electrospray tandem mass spectrometry (LC–ESI-MS/MS) method. The method can quantify BADGE, BADGE·H2O and BADGE·2H2O with lower limits of quantification (LLOQ) of 0.05, 0.05 and 0.2 ng/ml, respectively. The percentage deviation of mean calculated concentrations from target concentrations was within 20%, variations across repeated analyses were within 15%, and mean extraction recovery was higher than 51.4% for all the three analytes in both plasma and urine matrices. The method has been applied to venous blood samples, cord blood samples, and urine samples collected from 9 to 14 adult volunteers. Results showed that concentrations of BADGE were lower than LLOQ in all of these samples except one urine sample. Low levels of BADGE·H2O from 0.108 to 0.222 ng/ml were observed in four venous blood samples and one urine sample (0.187 ng/ml). In contrast, concentrations of BADGE·2H2O were higher than LLOQ, varying from 0.660 to 303.593 ng/ml, in all the 10 venous blood samples and 1 cord blood sample (0.592 ng/ml) and two urine samples (0.200 and 0.306 ng/ml). The results suggest that bio-monitoring of blood and urine for BADGE exposure should focus on the hydrolysis derivatives of BADGE, mainly in the form of BADGE·2H2O.
Keywords: BADGE; Metabolite; Plasma; Urine; LC–MS/MS;

Simultaneous determination of l-tetrahydropalmatine and cocaine in human plasma by simple UPLC–FLD method: Application in clinical studies by Mingming Yu; Hazem E. Hassan; Ahmed Ibrahim; Kenneth S. Bauer; Deanna L. Kelly; Jia Bei Wang (39-44).
Currently, there are no FDA approved medications for treatment of cocaine addiction underscoring the dire need to develop such a product. There is an accumulating body of evidence that l-tetrahydropalmatine (l-THP), a non-selective dopamine antagonist, can be used for the treatment of cocaine addiction. Indeed, the FDA recently approved its usage in a Phase I study in cocaine abusers and it was indispensable to develop a simple and sensitive method for the simultaneous determination of l-THP and cocaine in human plasma. We developed a UPLC–FLD method for quantitation of these molecules using an ACQUITY BEH C18 column (2.1 mm × 50 mm, 1.7 μm) and a mobile phase that consisted of 10 mM ammonium phosphate (pH = 4.75), methanol, and acetonitrile (v:v:v, 78:16:6). Venlafaxine was used as the internal standard while hexane was used for the liquid–liquid extraction. The flow rate was 0.4 mL/min with fluorescence detection using an excitation wavelength of 230 nm and emission detection wavelength of 315 nm. This method was selective, linear and sensitive with a lower limit of quantification of 2.5 ng/mL for both cocaine and l-THP. The intra-day precision of cocaine and l-THP was <9.50% while the accuracy was <4.29%. The inter-day precision of cocaine and l-THP was <9.14%, and the accuracy was <12.49%. The recovery for cocaine and l-THP ranged from 43.95 to 50.02% and 54.65 to 58.31%, respectively. In comparison to forty reported cocaine quantitation methods this method is simple, sensitive and cost-effective and can be used for simultaneous quantitation of l-THP and cocaine. This method meets the FDA guidelines and can be used in current and future clinical studies.
Keywords: Cocaine; l-THP; UPLC; Analysis; Human plasma; Fluorescence;

A high-performance liquid chromatography–tandem mass spectrometry method for the determination of artemether and dihydroartemisinin in human plasma by M.J. Hilhorst; G. Hendriks; R. de Vries; V. Hillewaert; T. Verhaege; N.C. van de Merbel (45-53).
A liquid chromatography–tandem mass spectrometry method is described for the quantitative determination of artemether (ART) and its metabolite dihydroartimisinin (DHA) in human plasma samples. Quantitation of ART and DHA in plasma is challenging due to the presence of malaria related hemolytic products in patient plasma causing degradation of the compounds when organic solvents are used during sample processing. Furthermore, both compounds consist of two epimeric forms that can interconvert both in solution and during chromatographic separation, an effect that is dependent on temperature and solvent properties and needs to be taken into account. This method utilizes micro-elution solid-phase extraction as sample preparation technique to minimize the need for organic solvents. Reversed-phase HPLC using a C18 50 × 2.1 mm column with 3.5 μm particles and a mobile phase of acetonitrile:water (30:70, v/v), followed by a step gradient at 90% acetonitrile, is applied to separate ART from DHA and matrix interferences within a run time of 4 min. Chromatographic conditions were optimized to allow analyte quantitation independent of the (unknown) ratio of the epimers in the injected sample. A triple quadruple mass spectrometer equipped with an atmospheric pressure chemical ionization interface in positive mode was used for detection in order to detect all epimeric forms. The method proved to be linear over a concentration range of 1.00–1000 ng/mL using 50 μL of plasma. Accuracy and precision were within 15% for bias and CV (20% at the lower limit of quantification). ART and DHA were stable (bias <15%) in plasma for 211 days after storage at −20 °C and −70 °C, 17 h on melting ice and 2 h at room temperature. Furthermore, both compounds were stable in whole blood after storage for 2 h on melting ice and at room temperature and after five freeze/thaw cycles. The method was successfully used for the analysis of pharmacokinetic samples originating from a drug–drug interaction study in which the antimalarial drugs artemether/lumefantrine were coadministrated etravirine or darunavir/ritonavir in healthy human immunodeficiency virus (HIV)-negative subjects.
Keywords: Artemether; Dihydryartemisinin; Epimerization; Degradation by malaria related hemolytic products; LC–MS/MS; Validation;

We developed and validated an analytical method for the simultaneous determination of methyl mercapturic acid (MeMA), ethyl mercapturic acid (EtMA), n-propyl mercapturic acid (PrMA) and iso-propyl mercapturic acid (iPrMA) in human urine. These alkyl mercapturic acids are known or presumed biomarkers of exposure to several alkylating agents including methyl bromide, dimethyl sulfate, ethyl bromide, 1-bromopropane and 2-bromopropane. The method involves a column switching arrangement for online solid phase extraction of the analytes with subsequent analytical separation and detection using liquid chromatography and tandem mass spectrometry. Within day and day-to-day imprecision was determined to range from 4.5 to 12.2%. The analytical method is distinguished by its wide linear working range of up to 2500 μg/L with detection limits ranging from 2.0 μg/L (for PrMA) to 5.1 μg/L (for MeMA) that render possible the application in various biomonitoring studies regarding exposure to alkylating agents. The results of a pilot study on urine samples of 30 individuals occupationally non-exposed to alkylating agents using the new procedure confirmed the background excretion of MeMA (<5.1–35.6 μg/L) and PrMA (<2.0–95.7 μg/L).
Keywords: Biomonitoring; Mercapturic acid; Alkylating agents; Carcinogens; LC–MS/MS;

Extraction method for total microcystins in cyanobacteria-laden sludge by Carlos J. Pestana; Petra J. Reeve; Gayle Newcombe (61-64).
Cyanobacteria in water treatment sludge pose a health risk as they continue to be viable, multiply, and produce potentially harmful secondary metabolites. To date, little research has focused on accurately determining cell bound microcystin (MC) concentrations of cyanobacterial cells in water treatment sludge. Three extraction methods (freeze–thaw, lyophilisation, direct methanolic extraction) with three different pre-treatments (homogenisation, (ultra)sonication, combination of both, and controls) were investigated for their MC extraction recovery. It was found that lyophilisation with prior sonication achieved the highest toxin recovery across the two MC analogues (MC-LR, MC-LA) tested. The method was able to extract 69 and 56% of MC-LR and MC-LA, respectively with good reproducibility. Comparable results were also obtained with direct methanolic extraction, with poor reproducibility. The least efficient method was freeze-thawing which achieved poor recoveries and was less reproducible. This study highlights a rapid, efficient, low-cost extraction method for determining total microcystins in cyanobacterial-laden sludge.
Keywords: Microcystin; Sludge; Freeze–thaw; Lyophilisation; Direct methanolic extraction;

Characterization of asparagine 330 deamidation in an Fc-fragment of IgG1 using cation exchange chromatography and peptide mapping by Yonghua Taylor Zhang; Jennifer Hu; Amanda L. Pace; Rita Wong; Y. John Wang; Yung-Hsiang Kao (65-71).
Deamidation is one of the most common degradation pathways for proteins and frequently occurs at “hot spots” with Asn-Gly, Asn-Ser or Asn-Thr sequences. Occasionally, deamidation may occur at other motifs if the local protein structure can participate or assist in the formation of the succinimide intermediate. Here we report the use of a chymotryptic peptide mapping method to identify and characterize a deamidated form of an IgG1 which was observed as an acidic peak in the cation exchange chromatography (CEX). The antibody was formulated in sodium acetate buffer, pH 5.3 and this deamidated form was observed mainly under thermal stress conditions. It was found that the IgG1 molecule with deamidation in the Fc region at asparagine residue 330 (in a Val-Ser-Asn-Lys motif) is the predominant form in this CEX peak, and was missed by tryptic mapping because the peptides are hydrophilic and elute near the void volume. In addition, a domain-based CEX method using papain digestion was developed to monitor the Asn 330 deamidation. These methods revealed that the Fc deamidation occurs mainly at Asn 330 in the VSNK motif at pH 5.3, whereas at pH 7.5, deamidation occurs predominantly at Asn 389 and Asn 394 in the NGQPENNYK motif.
Keywords: Deamidation; Cation exchange chromatography; Peptide mapping; IgG; Digestion strategies;

Protein A affinity precipitation of human immunoglobulin G by Lars Janoschek; Matthias Freiherr von Roman; Sonja Berensmeier (72-78).
The potential of protein A affinity precipitation as an alternative method for traditional antibody purification techniques was investigated. Recombinant produced protein A from Staphylococcus aureus (SpA) was covalently linked to the pH-responsive copolymer Eudragit® S-100 and used for purification of human immunoglobulin G (hIgG). The Eudragit-SpA conjugate had a static binding capacity of 93.9 ± 2.8 mg hIgG per g conjugate and a dissociation constant of 787 ± 67 nM at 7 ± 1 °C. The antibody was adsorbed rapidly onto Eudragit-SpA and reached equilibrium within 5 min. An excess of hIgG binding sites, provided by the conjugate, as well as adjusted elution conditions resulted in an appropriate hIgG purification performance. In summary, Eudragit-SpA was successfully applied to capture hIgG from a protein mixture with 65% antibody yield in the elution step. Nearly 96% purity and a purification factor of 12.4 were achieved. The Eudragit-SpA conjugate showed a stable ligand density over several cycles, which enabled reusability for repeated precipitation of hIgG. According to this, pH induced affinity precipitation can be seen as a potential alternative for protein A chromatography in antibody purification processes.
Keywords: Antibody purification; Eudragit; Affinity precipitation; protein A; Immunoglobulin G;

A selective and sensitive LC–MS/MS method was developed and validated for simultaneous determination of three glucuronide conjugates of scutellarein in rat plasma. Plasma samples were pretreated by protein precipitation with acetonitrile. The analytes (scutellarin, scutellarein-6,7-di-O-β- d -glucuronide and scutellarein-6-O-β- d -glucuronide), together with internal standard (IS, baicalin) were separated on a Diamonsil C18 column (150 mm × 4.6 mm, 5 μm) with an isocratic mobile phase consisting of methanol–water–formic acid (55:45:0.2, v/v/v). Mass spectrometric detection was performed by selected reaction monitoring (SRM) mode via electrospray ionization source operating in negative ionization mode. The method was linear for all the analytes over the investigated concentration ranges with correlation coefficients greater than 0.9954. The intra- and inter-day precisions were less than 9.1% and the relative error was between −1.7% and 4.2%. The extraction recoveries of the analytes and IS from rat plasma were over 63%. The validated method has been successfully applied to a pharmacokinetic study of breviscapine in rats after intragastric administration at a dose of 20 mg/kg. The pharmacokinetic results would be helpful to better understand the pharmacological actions of breviscapine.
Keywords: LC–MS/MS; Scutellarin; Breviscapine; Rat; Metabolite; Pharmacokinetics;

In this study, magnetic single-walled carbon nanotubes (MSWCNTs) were prepared by impregnating magnetic Fe3O4 nanoparticles onto the surfaces of carboxylic single-walled carbon nanotubes based on electrostatic interactions. The prepared MSWCNTs were used as the adsorbent for the dispersive solid-phase extraction (DSPE) of paraquat from human urine. After adsorption, the paraquat was quantitatively desorbed with 5%TFA in acetonitrile and determined by HPLC-MS. Extraction parameters such as the type of CNT adsorbent, extraction time, sample volume, wash solvent, and the type and volume of desorption solvent were optimized to obtain high DSPE recoveries and extraction efficiencies. Under the optimized conditions, the calibration curve was linear in the range 3.75–375.0 μg/L with a correlation coefficient of 0.999 45. The LOD (S/N = 3) and LOQ (S/N = 10) were 0.94 and 2.82 μg/L, respectively. The recoveries ranged from 92.89 to 108.9% for spiked real urine samples with RSDs below 3.21%. Finally, the new method was successfully used to determine paraquat in urine samples of suspected paraquat poisoning patients. The MSWCNTs exhibited suitable properties and a high adsorption capacity for the extraction of paraquat.
Keywords: Magnetic single-walled carbon nanotubes; Dispersive solid-phase extraction; HP LC-MS; Determination; Paraquat;

Identification and elucidation of the structure of in vivo metabolites of diaveridine in chicken by Hui Wang; Bo Yuan; Zhenling Zeng; Limin He; Huanzhong Ding; Chunna Guo; Xiangkai Kong; Wei Wang; Xianhui Huang (91-99).
Diaveridine (DVD) is a popular antibacterial synergist that is widely used in combination with sulfonamide. It has been reported to be genotoxic to mammalian cells, but more studies are required to clarify this. Moreover, there is very little information on its pharmacokinetics, metabolic elimination and mechanism of toxicity. Therefore, in order to gain a better understanding of the metabolism of DVD, we performed high-performance liquid chromatography linear ion trapped orbitrap mass spectrometer (LC-LTQ-Orbitrap). With this approach, we identified 15 metabolites of DVD in chicken after a single oral administration of DVD; 10 of these metabolites have been identified in vivo for the first time. Nine phase I and five phase II metabolites were detected in the plasma, and eight phase I and six phase II metabolites were found in feces. The major phase I metabolites were formed via the O-demethylation and N-oxidation pathways, and the major phase II metabolites were glucuronide conjugates. These results are essential for understanding this compound more clearly and lay the basis for further studies about the metabolism of DVD. Therefore, using this approach, we were able to identify and characterize metabolites of DVD with high sensitivity and resolution. We were able to detect a broad range of metabolites, even some trace ones and some so far unknown metabolites.
Keywords: Diaveridine; Metabolites; LC-LTQ-Orbitrap; Chicken;

Liquid chromatography coupled with tandem mass spectrometry (HPLC–MS/MS) has become the method of choice for analysis in biological matrices, because of its high specificity and sensitivity. However, it should be taken into account that the presence of matrix components coeluting with analytes might interfere with the ionization process and affect the accuracy and precision of the assay. For this reason, the presence of a “matrix effect” should always be evaluated during method development, above all in complex matrix such as urine. In the present work, a HPLC–MS/MS method was developed for the quantification of urinary iPF-III and iPF-VI. A careful assessment of matrix effect and an accurate validation were carried out, in order to verify the reliability of quantitative data obtained. Ion suppression, due to the matrix components, was reduced through optimization of both chromatographic method and sample extraction procedure. Urine samples were purified by solid phase extraction (SPE) and the extracts injected into the HPLC–MS/MS system, equipped with a TurboIonSpray ionization source operated in negative ion mode (ESI). Stable isotope-labeled analogues (iPF-III-d4 and iPF-VI-d4) were used as internal standards, and quantification was performed in multiple reaction monitoring (MRM) mode by monitoring the following mass transitions: m/z 353.4 → 193.2 for iPF-III, m/z 357.2 → 197.0 for iPF-III-d4, m/z 353.4 → 115.1 for iPF-VI, and m/z 357.4 → 115.1 for iPF-VI-d4. The validated assay, applied to the analysis of urinary samples coming from healthy and overweight subjects, resulted suitable for an accurate quantification of iPF-III and iPF-VI in human urine.
Keywords: Liquid chromatography; Mass spectrometry; F2-isoprostanes; Urine; Matrix effect;

UPLC–MS/MS method for determination of avicularin in rat plasma and its application to a pharmacokinetic study by Wei-min Zhang; Rui-fang Li; Ming Sun; Da-ming Hu; Jian-fei Qiu; Yun-hao Yan (107-111).
A rapid, sensitive and selective ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) was developed and validated for the determination and pharmacokinetic investigation of avicularin in rat plasma. Sample preparation was accomplished through a simple one-step deproteinization procedure with 0.2 mL of acetonitrile–methanol (9:1, v/v) to a 0.1 mL plasma sample. Plasma samples were separated by UPLC on an Acquity UPLC BEH C18 column using a mobile phase consisting of acetonitrile–0.1% formic acid in water with gradient elution. The total run time was 1.60 min and the elution of avicularin was at 1.20 min. The detection was performed on a triple quadrupole tandem mass spectrometer in the multiple reaction-monitoring (MRM) mode using the respective transitions m/z 434.1 → 301.3 for avicularin and m/z 237.2 → 194.3 for carbamazepine (IS), respectively. The calibration curve was linear over the range of 10–3000 ng/mL with a lower limit of quantitation (LLOQ) of 10 ng/mL. Mean recovery of avicularin in plasma was in the range of 84.2–89.5%. Intra-day and inter-day precision were both <12%. This method was successfully applied in pharmacokinetic study after intravenous administration of 5.0 mg/kg avicularin in rats.
Keywords: Avicularin; UPLC–MS/MS; Rat plasma; Pharmacokinetics;

Tolvaptan is a competitive vasopressin V2-receptor antagonist that inhibits water reabsorption in the renal collecting ducts. A selective high-performance liquid chromatography method for determining tolvaptan enantiomers in rat and dog sera was developed and validated. Benzyl salicylate was used as an internal standard. Sample preparation involved liquid–liquid extraction with an n-hexane:diethyl ether (1:1, v/v) solution, followed by solid-phase extraction using a silica-gel cartridge. Chromatographic separation was performed on a cellulose-based chiral stationary phase in the reversed phase mode. The analytes were monitored with UV detection. The calibration curve showed linearity over the concentration range from 0.025 to 2.5 μg/mL for each analyte. Precision as the percentage coefficient of variation did not exceed 14.8%, and accuracy as relative error was within ±14.6% for the analytes. The validated method was successfully applied to evaluate the pharmacokinetics of oral tolvaptan enantiomers in rats and dogs, indicating gender and species differences in the systemic exposure to tolvaptan enantiomers.
Keywords: Tolvaptan; OPC-41061; Samska™;

A rapid, specific and sensitive liquid chromatography–tandem mass spectrometric (LC–MS/MS) method was developed and validated for the simultaneous determination of timosaponin B-II (TB-II) and A-III (TA-III) in rat plasma. Plasma samples were pretreated via simple protein precipitation with acetonitrile and ginsenoside Rg2 was used as internal standard. Chromatographic separation was carried out on an Agilent XDB-C8 (150 mm × 2.1 mm i.d., 5 μm) column by isocratic elution with acetonitrile–2 mmol/L ammonium acetate (55:45, v/v). The detection was performed on a Sciex API 4000+ triple-quadrupole tandem mass spectrometer with TurboIonSpray ionization (ESI) inlet via the negative ion multiple reaction monitoring (MRM) mode. The results showed that the calibration curve was linear in the concentration range of 3–3000 ng/mL for TB-II and 0.3–3000 ng/mL for TA-III, respectively. The intra- and inter-day precisions were less than 13.25%, and the accuracy ranged from 100.88% to 104.07% at three QC levels for both. The pharmacokinetic profiles of TB-II and TA-III in timosaponins (total timosaponin) at three dose levels (TB-II 150, 300, 600 mg/kg and TA-III 0.59, 1.17, 2.34 mg/kg, respectively) and in timosaponins–Huangbai alkaloids mixtures (1:1, 1:3, w/w, TB-II 300 mg/kg and TA-III 1.17 mg/kg) were studied for the first time in rats by this LC–MS/MS method. After single oral administration of timosaponins, mean C max and AUC0−t of TB-II and TA-III increased but non-proportional to the oral doses. When timosaponins–Huangbai alkaloids (1:1, 1:3, w/w) mixtures were administered, C max and AUC0−t of TB-II in the mixtures were obviously higher than the corresponding values in timosaponins at the same dose level.
Keywords: Timosaponin B-II; Timosaponin A-III; LC–MS/MS; Pharmacokinetics; Rat plasma;

Quantitative determination of microbicidal spermicide ‘nonoxynol-9’ in rabbit plasma and vaginal fluid using LC–ESI–MS/MS: Application to pharmacokinetic study by Yashpal S. Chhonker; Hardik Chandasana; Veenu Bala; Lokesh Kumar; Vishnu Lal Sharma; Gopal Gupta; Rabi S. Bhatta (127-132).
Nonoxynol-9 (N-9), a microbicidal spermicide, has been in use as an over-the-counter contraceptive since the 1960s. A detailed account of its pharmacokinetic profile using highly sensitive detection method has not been reported yet. We developed and validated a rapid, selective and sensitive high-performance liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) method for N-9 detection in plasma and simulated vaginal fluid. The analytes were quantified using reverse phase Thermo Accucore C18 (150 mm × 4.6 mm, 5 μm) column with isocratic elution using acetonitrile: 0.1% formic acid in triple distilled water (90:10, v/v) as mobile phase. The ionization was optimized using ESI (+) and selectivity was achieved by tandem mass spectrometric analysis using MRM transition, m/z 617.4 → 133.2 for N-9 and m/z 180.1 → 138.1 for phenacetin. The method was linear over the range 0.195–100 ng/mL. The method was accurate and precise with intra-batch and inter-batch accuracy (% bias) of less than ±15% and precision (% CV) of <15% for N-9. The mean peak plasma concentration (C max) 4.87 ± 0.37 ng/mL was achieved 1.0 h after vaginal application with terminal half-life 1.45 ± 0.07 h in rabbits. The validated method was successfully applied for pharmacokinetic study of N-9 in rabbits after vaginal administration.
Keywords: Nonoxynol-9; Spermicide; Microbicide; Pharmacokinetics; LC–MS/MS;

Simultaneous determination of metformin and vildagliptin in human plasma by a HILIC–MS/MS method by Roberto Pontarolo; Ana Carolina Gimenez; Thais Martins Guimarães de Francisco; Rômulo Pereira Ribeiro; Flávia Lada Degaut Pontes; João Cleverson Gasparetto (133-141).
The objective of this work was to develop and validate a HILIC–MS/MS method for the simultaneous determination of metformin and vildagliptin in human plasma. Chromatographic separation was achieved using an Atlantis HILIC Silica 150-mm × 2.1-mm, 3-μm particle size column maintained at 40 °C. The isocratic mobile phase consisted of 20% water and 80% acetonitrile/water solution 95:5 (v/v), containing both 0.1% formic acid and 3 mM ammonium formate. The flow rate was maintained at 400 μL min−1. Data from validation studies demonstrated that the new method is highly selective, sensitive (limits of detection <1.5 ng mL−1) and free of matrix and residual effects. The new method was also precise (RSD < 9.0%), accurate (RE < 11.2%) and linear (r  ≥ 0.99) over the ranges of 5–500 ng mL−1 for each compound. The developed method was successfully applied to determine metformin and vildagliptin in plasma volunteers who orally received a single dose of metformin (850 mg), vildagliptin (50 mg) or drug association (metformin 850 mg + vildagliptin 50 mg). The new method can thus also be used as a tool for the clinical monitoring of metformin and vildagliptin.
Keywords: Metformin; Vildagliptin; LC–MS/MS; Human plasma; HILIC;

Determination of selective serotonin reuptake inhibitors in plasma and urine by micellar liquid chromatography coupled to fluorescence detection by Nitasha Agrawal; Josep Esteve-Romero; Devasish Bose; Neeti Prakesh Dubey; Juan Peris-Vicente; Samuel Carda-Broch (142-149).
Citalopram, paroxetine and fluoxetine are selective serotonin reuptake inhibitor (SSRIs) currently used in the treatment of psychiatric disorders. We present an analytical method using micellar liquid chromatography to quantify these three drugs in pharmaceutical formulations, plasma and urine. The resolution was performed using a mobile phase of 0.075 M SDS – 6% (v/v) butanol buffered at pH 7 running through a C18 column under isocratic mode at 1 mL/min at 25 °C. The analytes were eluted in less than 20 min. The fluorescence detection was programmed at the maximum excitation (236, 295 and 230 nm) and emission (310, 350 and 305 nm) wavelengths for citalopram, paroxetine and fluoxetine, respectively. The experimental procedure was expedited to 1/5 dilution of the sample in the micellar mobile phase and filtration, thus avoiding clean-up and extraction steps. An aliquot of 20 μL was injected after 80 min of preparation, to obtain maximum sensitivity. The method was validated according to the guidelines of the Food and Drug Administration (FDA) in terms of calibration range (20–500 ng/mL; r 2  > 0.999), sensitivity, accuracy (91.3–103.2%), precision (<9.3%), and robustness (<6.1%). The suitability of the method was successfully evaluated by analyzing plasma and urine samples from patients treated with SSRIs and checking the content of the active principle in tablets. Thus, the method can be applied to pharmacokinetics studies and in forensic cases, as well as in quality control of commercial pharmaceutical formulations.
Keywords: Antidepressant; Micellar liquid chromatography; Fluorescence; Plasma; Urine; Validation;

In this study, a simple and efficient preparative procedure was developed for preparation of seven flavonoids from the peel of Trichosanthes kirilowii Maxim. using polyamide resin followed by semi-preparative high performance liquid chromatography (SPHPLC). First, the ethyl acetate fraction from the peel of T. kirilowii Maxim. obtained “prefractionation” using polyamide resin, which yielded two subfractions. And then the two subfractions were isolated by SPHPLC with an isocratic elution of methanol–water. Finally, seven known flavonoids were purified from 35 g of ethyl acetate extract including quercetin-3-O-[α-l-rhamnose (1→2)-β-d-glucopyranosyl]-5-O-β-d-glucopyranoside (19 mg), quercetin-3-O-rutinoside (24 mg), apigenin-7-O-β-d-glucopyranoside (10 mg), diosmetin-7-O-β-d-glucopyranoside (45 mg), luteolin (21 mg), apigenin (15 mg), and diosmetin (56 mg). The purities of the compounds were determined by HPLC and the chemical structures were confirmed by UV and NMR analysis. In the present study, a simple, effective, and rapid procedure was established for preparative separation of multiple components from the peel of T. kirilowii Maxim. Furthermore, it was scalable and economical, so it was a promising basis for large-scale preparation of flavonoids from other plant extracts.
Keywords: T. kirilowii Maxim.; Flavonoids; Polyamide resin; Semi-preparative high performance liquid chromatography;

Quantification of five plasticizers used in PVC tubing through high performance liquid chromatographic-UV detection by Tsanta Radaniel; Stéphanie Genay; Nicolas Simon; Frédéric Feutry; Francesca Quagliozzi; Christine Barthélémy; Marie Lecoeur; Valérie Sautou; Bertrand Décaudin; Pascal Odou (158-163).
Searching for alternatives to di-(2-ethylhexyl)-phthalate, a plasticizer that has been widely used in the manufacturing of PVC medical devices, has become a major challenge since a European regulation underlined some clinical risks. The aim of this study is to develop an HPLC–UV method to quantify the currently used alternative plasticizers to DEHP. Five plasticizers, acetyl tributyl citrate, di-(2-ethylhexyl)-phthalate, di-(ethylhexyl)-terephthalate, di-isononyl-1,2-cyclohexane-dicarboxylate, and trioctyl trimellitate, were separated on a C8 stationary phase (2.6 μm, 100 mm × 4.6 mm) under gradient elution in 13 min. They were detected at 221 nm leading to a quantification threshold from 0.3 to 750 μg/mL as a function of the plasticizer. Within-day and between-day precisions were inferior to 0.9% and 18%, respectively. The assays were validated according to the accuracy profile method. Plasticizers were extracted from PVC-tubing by dissolving PVC in THF then precipitating it in methanol with a yield of over 90% for each plasticizer. This assay could feasibly be used to quantify plasticizers in PVC medical devices.
Keywords: DEHP; HPLC; Plasticizers; Medical devices; PVC (polyvinylchloride);

This study aimed to develop a sensitive and reliable multi-residue method for the determination of trace amounts of endocrine disrupting chemicals including five phthalate esters (PAEs), five monoalky phthalate esters (MPEs), four alkylphenols (APs) and bisphenol A (BPA) in seafood. Ultrasonic liquid extraction was selected for extraction based on acetonitrile, instead of frequently-used n-hexane, due to its lower background of PAEs. Application of solid phase extraction (SPE) with primary secondary amine (PSA, 1 g/6 mL) cartridge achieved the relatively low matrix effects for MPEs and BPA in seafood. To our knowledge, it is the first study reporting about simultaneous extraction and purification of PAEs, MPEs, APs and BPA in biota samples. To obtain the maximum sensitivity, both liquid chromatography tandem mass spectrometry (LC–MS/MS) and gas chromatography tandem mass spectrometry (GC–MS/MS) were applied for analysis. This method was validated and tested on fish, mollusk and prawn. Sufficient linearity was verified by Mandel's fitting test for the matrix-matched calibrations used in this study for MPEs, APs and BPA, between 0.5 ng/g and 200 ng/g or 400 ng/g. And correlation coefficients of all calibrations suppressed 0.99 for all analytes. Good recoveries were obtained, ranging from 60% to 127% for most compounds. The sensitivity was good with method detection limits (MDLs) of 0.015–2.2 ng/g wet weight (ww) for all compounds. Most MDLs are much lower than those in previous reports. The sensitive method was then applied on real fish, mollusk and prawn samples from the Yangtze River Delta sea area (China), and all the target compounds were detected with the maximum concentrations of PAEs, MPEs, APs and BPA up to 219.3 ng/g ww, 51.4 ng/g ww, 62.0 ng/g ww and 8.6 ng/g ww, respectively.
Keywords: Simultaneous pretreatment; Phthalate esters; Monoalky phthalate esters; Alkylphenols; Bisphenol A; Seafood;

In healthy human subjects, less than 0.2% of l-arginine is converted to l-citrulline and nitric oxide (NO) by NO synthases (NOS), a metabolic pathway present in all cell types. Assessment of NOS activity in vitro and in vivo by measuring l-citrulline or NO is difficult. l-citrulline is formed from l-arginine to a much higher extent by other pathways including the urea cycle. Furthermore, NO is a very short-lived gaseous molecule and is oxidized to nitrite and nitrate which are ubiquitous. In fact, nitrite and nitrate are also derived from food and air and are major laboratory contaminants. Further, NOS (in the uncoupled state) are also able to produce superoxide in addition and/or instead of l-citrulline and NO. The difficulties of NOS assays based on l-citrulline and NO measurement can only in part be overcome by sophisticated techniques including use of radio-labeled (3H or 14C) and stable-isotope labeled (15N2 at the guanidine group) l-arginine analogs as substrates for NOS and measurement of radio-labeled l-citrulline and 15N-labeled nitrite and nitrate, respectively. In the present work, we report on the development, validation and application of an UPLC–MS/MS method for the assessment of the activity of recombinant NOS enzymes by using [guanidino-15N2]-l-arginine (20 μM for recombinant NOS, 5 mM in cell systems) as the substrate and by measuring [ureido-15N]-l-citrulline as the reaction product (usually formed at concentrations below 1 μM) using 2H7-l-citrulline as the internal standard. The lower limit of detection of the method is about 80 fmol 2H7-l-citrulline. In cell systems, exceeding [guanidino-15N2]-l-arginine is removed by strong cation exchanger solid-phase extraction. The method was cross-validated by a GC–MS assay that measures simultaneously 15N-nitrite and 15N-nitrate as pentafluorobenzyl derivatives, with unlabeled nitrite and nitrate serving as the internal standards. By means of this UPLC–MS/MS 15N-citrulline assay, N G-nitro-arginine (100 μM) was found to inhibit recombinant inducible NOS (iNOS) activity (by 38%), whereas nitrite and GSSG (each at 500 μM) did not affect iNOS activity at all. Nitrite and GSSG at pathophysiological concentrations are unlikely to uncouple NOS. NOS activity was not detectable in platelets of healthy humans by the UPLC–MS/MS and GC–MS assays.
Keywords: l-Citrulline; Nitric oxide (NO); NO synthase; Quantification; Tandem mass spectrometry; Validation;

An LC–MS/MS method for the simultaneous determination of goserelin and testosterone in rat plasma for pharmacokinetic and pharmacodynamic studies by Shu Zhang; Jiangbin Han; Guangyi Leng; Xin Di; Chunjie Sha; Xuemei Zhang; Youxin Li; Wanhui Liu (183-189).
A liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS) method was developed, using testosterone-d3 as a surrogate analyte, for the simultaneous quantification of goserelin and testosterone in rat plasma. According to this method, the pharmacokinetic and pharmacodynamic data were obtained from a single plasma sample aliquot. The method involved the addition of alarelin as an internal standard (IS) for goserelin and testosterone-13C3 for testosterone or testosterone-d3. The conditions for the separation of these two compounds were achieved on a ZORBAX Eclipse Plus C18 column (Agilent, 2.1 × 50 mm, 1.8 μm, Stockport, UK) in a single chromatographic run at a flow rate of 400 μL/min. In order to minimize interferences of complex matrix, the extraction of plasma consisted of a protein precipitation step using methanol, followed by purification using an Oasis® HLB solid-phase extraction column. The method was validated in the concentration range of 0.01–30.0 ng/mL for goserelin and 0.05–30.0 ng/mL for testosterone-d3, respectively. The within- and between-run precisions were 1.7–9.2% and 2.1–6.9%, respectively. The within- and between-run accuracies were −1.8 to 5.3% and −4.9 to 4.0%, respectively. This accurate and highly specific assay provides a useful method to evaluate the pharmacokinetics and pharmacodynamics of goserelin in rats.
Keywords: Goserelin; Testosterone; LC–MS/MS; Surrogate analyte; Pharmacokinetic; Pharmacodynamic;

Novel magnetic molecularly imprinted polymers based on multiwalled carbon nanotubes (MWNTs@MMIPs) with specific selectivity toward bisphenol A were synthesized using bisphenol A as the template molecule, methacrylic acid, and β-cyclodextrin as binary functional monomers and ethylene glycol dimethacrylate as the cross-linker. The MWNTs@MMIPs were characterized by Fourier transform infrared, vibrating sample magnetometer, and transmission electron microscopy. Batch mode adsorption experiment was carried out to investigate the specific adsorption equilibrium and kinetics of the MWNTs@MMIPs. The MWNTs@MMIPs exhibited good affinity with a maximum adsorption capacity of 49.26 μmol g−1 and excellent selectivity toward bisphenol A. Combined with high-performance liquid chromatography analysis, the MWNTs@MMIPs were employed to extract bisphenol A in tap water, rain water, and lake water successfully with the recoveries of 89.8–95.4, 89.9–93.4, and 87.3–94.1%, respectively.
Keywords: Molecularly imprinted polymers; Bisphenol A; Multiwalled carbon nanotubes; Magnetic; Solid-phase extraction;

A modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) method using multi-walled carbon nanotubes (MWCNTs) as a reversed-dispersive solid phase extraction (r-dSPE) material combined with ultra-high liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) was developed for the simultaneous determination of amantadine, rimantadine and memantine in chicken muscle. The satisfactory separation of isomers (rimantadine and memantine) was obtained on an Acquity BEH C18 column (2.1 mm × 100 mm, 1.7 μm) after optimization of mobile phase composition, column temperature and flow rate. The method involved an acetonitrile-based sample preparation and a dSPE clean-up procedure with MWCNTs material. Variations in the type and amount of MWCNTs, the pH value of the extract, the extraction time for MWCNTs, and the type of eluent were used to determine the optimal parameters for increasing the sample throughput and the sensitivity. The samples were quantified using amantadine-D15, rimantadine-D4 and memantine-D6 as the internal standards. Under the optimized conditions, recoveries of 96.8–104.6% and the values of coefficient of variation (CV) of 3.8–6.4% were obtained for the three drugs in chicken muscle at three spiked levels (0.5, 1.0 and 1.5 μg/kg), and the decision limits (CCα) and detection capabilities (CCβ) were 0.15–0.20 μg/kg and 0.20–0.25 μg/kg, respectively. Positive results were obtained from local supermarket using this method, and the concentrations obtained from the newly developed method compared well to the previously reported method.
Keywords: Amantadine; Rimantadine; Memantine; Chicken muscle; LC–MS/MS; MWCNTs;

Zhi-zi-chi Decoction has been clinically utilized for the treatment of depression for more than thousand years. In order to investigate the possible bioactive components that could pass through the blood brain barrier (BBB) and the mechanism of antidepressant, a sensitive LC–MS method was developed to detect the ingredients (geniposide, scandoside methyl ester, gardenoside, deacetyl asperulosidic acid methyl ester and genipin-1-β-gentiobioside) in rat brain microdialysates and tissue homogenates samples (hippocampus, hypothalamus, premotor cortex, striatum, oblongata and cerebellum). Method development and validation are described in terms of calibration curves, extraction yield, lower limit of quantification (LLOQ), precision, accuracy, intra- and inter-day variability, which are in accordance with the requirements. Microdialysis in hippocampus demonstrated that the five iridoids possessed complete pharmacokinetic process while brain tissue homogenate method testified the distribution regularity in brain. The work clarified that the five iridoids, as antidepressant ingredients, could pass through the BBB, distribute targeted and possess complete pharmacokinetics in brain. These observations, along with the large database of rat brain microdialysates and tissue homogenates data, could enable future efforts aimed to improve our understanding of the relationship between bioactive ingredients and clinical therapy of depression.
Keywords: LC–MS; Zhi-zi-chi Decoction; Iridoids; Brain microdialysate; Brain tissue homogenates;

Determination of total and unbound concentrations of lopinavir in plasma using liquid chromatography–tandem mass spectrometry and ultrafiltration methods by S.M. Illamola; L. Labat; S. Benaboud; R. Tubiana; J. Warszawski; J.M. Tréluyer; D. Hirt (216-223).
Lopinavir is an HIV protease inhibitor with high protein binding (98–99%) in human plasma. This study was designed to develop an ultrafiltration method to measure the unbound concentrations of lopinavir overcoming the non-specific binding issue. A liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the determination of total concentrations of lopinavir in plasma was developed and validated, and an adaptation was also optimized and validated for the determination of unbound concentrations. The chromatographic separation was performed with a C18 column (100 mm × 2.1 mm i.d., 5 μm particle size) using a mobile phase containing deionized water with formic acid, and acetonitrile, with gradient elution at a flow-rate of 350 μL min−1. Identification of the compounds was performed by multiple reaction monitoring, using electrospray ionization in positive ion mode. The method was validated over a clinical range of 0.01–1 μg/mL for human plasma ultrafiltrate and 0.1–15 μg/mL in human plasma. The inter and intra-assay accuracies and precisions were between 0.23% and 11.37% for total lopinavir concentrations, and between 3.50% and 13.30% for plasma ultrafiltrate (unbound concentration). The ultrafiltration method described allows an accurate separation of the unbound fraction of lopinavir, circumscribing the loss of drug by nonspecific binding (NSB), and the validated LC–MS/MS methodology proposed is suitable for the determination of total and unbound concentrations of lopinavir in clinical practice.
Keywords: Lopinavir; Unbound fraction; Ultrafiltration; Nonspecific binding; UPLC–MS/MS;

Enantioselective HPLC determination and pharmacokinetic study of secnidazole enantiomers in rats by Jiangbo Du; Yifan Zhang; Yao Chen; Dongqin Liu; Xiaoyan Chen; Dafang Zhong (224-230).
Secnidazole is a long-lasting nitroimidazole antimicrobial agent that is used as racemic mixture in clinical settings. We developed and validated an enantioselective high-performance liquid chromatography method to determine secnidazole enantiomers in rat plasma. Secnidazole enantiomers and S-(−)-ornidazole (internal standard) were extracted from 50 μL of rat plasma using diethyl ether–dichloromethane (3:2, v/v). Baseline resolution (R s  = 2.45) was achieved within 7.0 min on a Chiral-AGP column (150 mm × 4.0 mm, 5 μm) at 20 °C. The mobile phase consisted of 10 mM ammonium acetate–methanol (96:4, v/v) and was delivered at a flow rate of 0.5 mL/min with ultraviolet detection at 318 nm. The method was linear over the concentration range 0.500–100 μg/mL for both enantiomers. The lower limit of quantification was 0.500 μg/mL for both enantiomers. The relative standard deviation values for intra- and inter-day precision were 0.8–8.6 and 1.8–8.2% for S-(+)-secnidazole and R-(−)-secnidazole, respectively. The relative error values of accuracy ranged from −7.8 to 1.1% for S-(+)-secnidazole and from −7.3 to −0.1% for R-(−)-secnidazole. The method was successfully used to determine the pharmacokinetic properties of secnidazole enantiomers in rats after administration of the racemate and individual enantiomers. The pharmacokinetic results indicate that the disposition of secnidazole enantiomers is not stereoselective and chiral inversion and enantiomer–enantiomer interaction do not occur in rats.
Keywords: Secnidazole; Enantiomers; Enantioselective high-performance liquid chromatography; Pharmacokinetics;

Simulated moving bed purification of flaxseed oil orbitides: Unprecedented separation of cyclolinopeptides C and E by Denis P. Okinyo-Owiti; Peta-Gaye G. Burnett; Martin J.T. Reaney (231-237).
The purification and enrichment of most natural products with potential pharmaceutical applications has been performed mainly employing conventional batch-mode chromatographic processes. There is a growing interest in use of simulated moving bed (SMB) chromatography for natural product enrichment as this method enables conservation of mobile phase, while increasing productivity of chromatography medium. SMB increases yield while decreasing cost. Cyclolinopeptides C ([1-9-NaC],[1-MetO]-CLB, 3) and E ([1-8-NaC],[1-MetO]-CLE, 8) were extracted as a mixture from flaxseed oil and then enriched using a three-zone simulated moving bed. The current research extends the SMB technology to enrichment of cyclolinopeptides (CLs), a group of biologically active hydrophobic cyclic peptides that occur in flaxseed oil. Of interest are [1-9-NaC],[1-MetO]-CLB (3) and [1-8-NaC],[1-MetO]-CLE (8) that provide synthetic scaffolds for modified CLs. The influence of flow rate (feed, desorbent, and extract) on the separation of [1-9-NaC],[1-MetO]-CLB (3) and [1-8-NaC],[1-MetO]-CLE (8) was investigated.
Keywords: Simulated moving bed chromatography; Cyclolinopeptide; Orbitide; Separation; Flow rate;

Determination of LBPT in human plasma by high performance liquid chromatography–tandem mass spectrometry by Ming Liu; Hongyun Wang; Hongzhong Liu; Ao Peng; Fen Yang; Wenjie Wang; Liya Zhu; Haihong Huang; Ji Jiang; Pei Hu (238-243).
A rapid and selective HPLC–MS/MS method was developed for the determination of LBPT in human plasma. The analyte was extracted from plasma samples by solid-phase extraction and then chromatographed on a C18 analytical column. The mobile phase consisted of acetonitrile-10 mM ammonium formate in 0.1% formic acid (30:70, v/v) and the flow rate was 0.2 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer in multiple reactions monitoring (MRM) mode using positive electrospray ionization (ESI). The method was validated over the concentration range of 0.2–100 ng/mL. Inter- and intra-day precision (RSD %) were less than 9.2% and the accuracy (RE %) ranged from 0 to 11.0%. The lower limit of quantitation (LLOQ) was 0.2 ng/mL. The extraction recovery was on average 75% and the detection was not affected by the matrix. The method was successfully applied to the pharmacokinetic study of LBPT in healthy Chinese subjects.
Keywords: LBPT; HPLC–MS/MS; Chinese; Pharmacokinetics;

An assay was developed and validated for the quantification of eight protease inhibitors (indinavir (IDV), ritonavir (RTV), lopinavir (LPV), saquinavir (SQV), amprenavir (APV), nelfinavir (NFV), atazanavir (AZV) and darunavir (DRV)) in dried plasma spots using parallel ultra-high performance liquid chromatography and mass spectrometry detection in the multiple reaction monitoring mode. For each analyte an isotopically labeled internal standard was used and the assay based on liquid-solid extraction the area response ratio (analyte/IS) was found to be linear; from 0.025 μg/ml to 20 μg/ml for IDV, SQV, DRV, AZV, LPV, from 0.025 μg/ml to 10 μg/ml for NFV, APV and from 0.025 μg/ml to 5 μg/ml for RTV using 15 μl of plasma spotted on filter paper placed in a sample tube. The total analysis time was of 4 min and inter-assay accuracies and precisions were in the range of 87.7–109% and 2.5–11.8%, respectively. On dried plasma spots all analytes were found to be stable for at least 7 days. Practicability of the assay to blood was also demonstrated. The sample drying process could be reduced to 5 min using a commercial microwave system without any analyte degradation. Together with quantification, confirmatory analysis was performed on representative clinical samples.
Keywords: Quantification; Plasma; Blood; DBS; LC–MS; Protease inhibitors;

Chiral separation and determination of amino acids in real samples by LE-MEKC using Cu(II)-l-proline as chiral selector by Yujiao Wu; Yuyao Zhai; Yu Zhang; Hongfen Zhang; Huanwang Jing; Anjia Chen (254-259).
This work reports that Cu(II) complexes with l-proline were used as chiral additives for the enantioseparations and determination of three underivatized amino acids by ligand-exchange micellar electrokinetic chromatography (LE-MEKC). Sodium dodecylsulfate (SDS) was shown to be necessary for simultaneous separation of the enantiomeric amino acids. Separation parameters such as SDS concentrations, the Cu(II)-l-proline ratio, the concentration of the copper(II) complex at a specific Cu(II)-l-proline ratio, pH and separation voltage were investigated for the enantioseparation in order to achieve the maximum possible resolution. A good separation was achieved in the BGE composing of 10 mM ammonium acetate, 10 mM Cu(II) and 20 mM l-proline and 30 mM SDS at pH 5.0, and an applied voltage of 15 kV performed. Under above-mentioned optimum conditions, linearity was achieved within concentration ranges of up to two orders of magnitudes for the investigated amino acids with the correlation coefficients ranging from 0.9917 to 0.9984. The proposed method has been successfully applied to the determination of amino acid enantiomers in human urine, compound amino acids injection, and amino acid oral liquid.
Keywords: Chiral separation; LE-MEKC; Amino acids; Real samples;