Journal of Chromatography B (v.879, #24)

Ciprofibrate quantification in human plasma by high-performance liquid chromatography coupled with electrospray tandem mass spectrometry for pharmacokinetic studies by Fabiana D. Mendes; Lu Shi Chen; André Borges; Tainah Babadópulos; Jaime O. Ilha; Khalid M. Alkharfy; Gustavo D. Mendes; Gilberto De Nucci (2361-2368).
A rapid, sensitive and specific method for quantifying ciprofibrate in human plasma using bezafibrate as the internal standard (IS) is described. The sample was acidified prior extraction with formic acid (88%). The analyte and the IS were extracted from plasma by liquid–liquid extraction using an organic solvent (diethyl ether/dichloromethane 70/30 (v/v)). The extracts were analyzed by high performance liquid chromatography coupled with electrospray tandem mass spectrometry (HPLC–MS/MS). Chromatography was performed using Genesis C18 4 μm analytical column (4.6 × 150 mm i.d.) and a mobile phase consisting of acetonitrile/water (70/30, v/v) and 1 mM acetic acid. The method had a chromatographic run time of 3.4 min and a linear calibration curve over the range 0.1–60 μg/mL (r  > 0.99). The limit of quantification was 0.1 μg/mL. The intra- and interday accuracy and precision values of the assay were less than 13.5%. The stability tests indicated no significant degradation. The recovery of ciprofibrate was 81.2%, 73.3% and 76.2% for the 0.3, 5.0 and 48.0 ng/mL standard concentrations, respectively. For ciprofibrate, the optimized parameters of the declustering potential, collision energy and collision exit potential were −51 V, −16 eV and −5 V, respectively. The method was also validated without the use of the internal standard. This HPLC–MS/MS procedure was used to assess the bioequivalence of two ciprofibrate 100 mg tablet formulations in healthy volunteers of both sexes. The following pharmacokinetic parameters were obtained from the ciprofibrate plasma concentration vs. time curves: AUClast, AUC0–168 h, C max and T max. The geometric mean with corresponding 90% confidence interval (CI) for test/reference percent ratios were 93.80% (90% CI = 88.16–99.79%) for C max, 98.31% (90% CI = 94.91–101.83%) for AUClast and 97.67% (90% CI = 94.45–101.01%) for AUC0–168 h. Since the 90% CI for AUClast, AUC0–168 h and C max ratios were within the 80–125% interval proposed by the US FDA, it was concluded that ciprofibrate (Lipless® 100 mg tablet) formulation manufactured by Biolab Sanus Farmacêutica Ltda. is bioequivalent to the Oroxadin® (100 mg tablet) formulation for both the rate and the extent of absorption.
Keywords: Ciprofibrate; Healthy volunteer; Plasma; Pharmacokinetic; HPLC–MS/MS;

Metabolomics study of alcohol-induced liver injury and hepatocellular carcinoma xenografts in mice by Shangfu Li; Hongxia Liu; Yibao Jin; Shuhai Lin; Zongwei Cai; Yuyang Jiang (2369-2375).
Alcohol abuse is one of the major causes of liver injury and a promoter for hepatocellular carcinoma (HCC). To understand the disease-associated metabolic changes, we investigated and compared the profiles of metabolites in nude mice with alcohol-induced liver injury or bearing a HCC xenograft (HCCX). Alcohol-induced liver injury was achieved by daily administration of grain liquor, and HCC xenografts were generated by subcutaneous inoculation of HepG2 cells in nude mice. Metabolites in serum samples were profiled by ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS). The acquired data was analyzed by principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) to identify potential disease-specific biomarkers. Results showed that the phosphatidylcholine (PC) levels were significantly higher in both liver injury and HCCX mice compared with the control. Interestingly, lysophosphatidylcholines (LPCs) that contain saturated or monounsaturated fatty acids were reduced in both liver injury and HCCX mice, but polyunsaturated fatty acids LPCs were elevated in liver injury mice only. These data delineated the disease-related metabolic alterations of LPCs in liver injury and HCC, suggesting that the LPC profile in serum may be biomarkers for these two common liver diseases.
Keywords: Metabolomics; Alcohol liver injury; Hepatocellular carcinoma xenograft; Phospholipid; Liquid chromatography–mass spectrometry;

A high-performance liquid chromatography–tandem mass spectrometric (LC–MS/MS) method has been developed and validated for the quantitative analysis of NIM811, a cyclophilin inhibitor, in human dried blood spot (DBS) samples, which were produced by spotting 20 μl whole blood onto FTA cards. A 3 mm disc was cut from the DBS samples and extracted using methanol, followed by liquid–liquid extraction with MTBE. The reconstituted extracts were chromatographed using a Halo C18 column and gradient elution for MS/MS detection. The possible impact of hematocrit, blood sample volume and punching location on DBS sampling was investigated. The results showed that blood sample volume or punching location has no impact on assay performance, but the presence of a high hematocrit resulted in significantly increased analyte concentrations measured from the high QC samples. The current method was fully validated over the range of 10.0–5000 ng/ml with correlation coefficients (r 2) for three validation batches equal to or better than 0.991. The accuracy and precision (CV) at the LLOQ were −0.7 to 6.0% bias of the nominal value (10.0 ng/ml) and 10.2–2.3%, respectively. For the balance of QC samples (20.0, 50.0, 750, 1500 and 3750 ng/ml), the precision (CV) ranged from 3.2 to 11.7% and from 5.6 to 10.2%, respectively, for the intra-day and inter-day evaluations. The accuracy ranged from −6.8 to 8.5% and −0.2% to 2.7% bias, respectively, for the intra-day and inter-day batches. NIM811 is stable in the DBS samples for at least 24 h at room temperature and 4 h at 60 °C. Interestingly, the long term stability (LTS) assessment showed that the stability of the analyte is better when the DBS samples were stored at a lower storage temperature (e.g. ≤−60 °C) compared to storage at room temperature. This is probably due to the interaction of the additives and/or other materials (e.g. cellulose, etc) on the DBS card with NIM811, a cyclic peptide. The current methodology has been applied to determine the NIM811 levels in DBS samples prepared from a clinical study.
Keywords: Dried blood spot (DBS); NIM811; Punching location; Blood volume; Hematocrit effect; Stability; LC–MS/MS;

Nitrogen mustards (NMs) are known to have DNA alkylation and strong vesicant properties. Their availability to terrorist organizations makes them a potential choice for chemical attacks on civilian populations. After an exposure, it is difficult to measure NMs directly because of their rapid metabolism in the human body. Therefore to determine an individual's level of exposure to NMs, it is necessary to analyze for NM metabolites being excreted by the body. The metabolites of NMs are generated by a hydrolysis reaction, and are easily detectable by liquid chromatography tandem mass spectrometry (LC–MS/MS). This work is focused on the development of a high-throughput assay for the quantitation of N-ethyldiethanolamine (EDEA) and N-methyldiethanolamine (MDEA) metabolites of bis (2-chloroethyl) ethylethanamine (HN1) and bis (2-chloroethyl) methylethanamine (HN2), respectively. The method uses automated 96-well plate sample preparation of human urine samples and a 2-position 10-port switching valve to allow for simultaneous regeneration of the liquid chromatography (LC) columns. Using this method, over 18 h was saved through the reduction of sample preparation and analysis time when compared to a conventional method for 96 samples. The validated method provided excellent accuracy for both EDEA (100.9%) and MDEA (100.6%) with precision better than 5.27% for each analyte.
Keywords: HTP-LC–MS/MS; LC-column switching; High-throughput extraction; N-ethyldiethanolamine; N-methyldiethanolamine;

A LC–MS/MS method for the specific, sensitive, and simultaneous quantification of 5-aminolevulinic acid and porphobilinogen by Jinglan Zhang; Makiko Yasuda; Robert J. Desnick; Manisha Balwani; David Bishop; Chunli Yu (2389-2396).
Accurate determinations of 5-aminolevulinic acid (ALA) and porphobilinogen (PBG) in physiologic fluids are required for the diagnosis and therapeutic monitoring of acute porphyrias. Current colorimetric methods are insensitive and over-estimate ALA and PBG due to poor specificity, while LC–MS/MS methods increase sensitivity, but have limited matrices. An LC–MS/MS method was developed to simultaneously determine ALA and PBG concentrations in fluids or tissues which were solid phase extracted, butanol derivatized, and quantitated by selective reaction monitoring using 13C5, 15N-ALA and 2,4-13C2-PBG internal standards. ALA was separated from interfering compounds on a reverse phase C8-column. For ALA and PBG, the matrix effects (87.3–105%) and process efficiencies (77.6–97.8% and 37.2–41.6%, respectively) were acceptable in plasma and urine matrices. The assay was highly sensitive for ALA and PBG (LLOQ = 0.05 μM with 25 μL urine or 100 μL plasma), and required ∼4 h from extraction to results. ALA and PBG accuracy ranged from 88.2 to 110% (n  = 10); intra- and inter-assay coefficients of variations were <10% for urine and plasma. In clinical applications, patients with mutation-confirmed acute porphyrias had normal to slightly increased urinary ALA and PBG levels when asymptomatic, and high levels during acute attacks, which decreased with hemin therapy. In AIP mice, baseline ALA and PBG levels in urine, plasma, and liver were increased after phenobarbital induction 28-/63-, 42-/266-, and 13-/316-fold, respectively. This LC–MS/MS method is rapid, specific, highly sensitive, accurate, and simultaneously measures ALA and PBG in urine, plasma, and tissues permitting porphyria clinical diagnoses, therapeutic monitoring, and research.
Keywords: 5-Aminolevulinic acid; Porphobilinogen; Acute intermittent porphyria; Tandem mass spectrometry;

An on-line method based upon dynamic microwave-assisted extraction (DMAE) coupled with high-speed counter-current chromatography (HSCCC) was developed for continuous isolation of nevadensin from Lyeicnotus pauciflorus Maxim. The DMAE parameters were optimized by means of the Box–Behnken design. The maximum extraction yield was achieved using 30:1 ml/g of liquid–solid ratio, 10 ml/min of solvent flow rate and 200 W of microwave power. The crude extracts were then separated by HSCCC with a two-phase solvent system composed of n-hexane–ethyl acetate–methanol–water (7:3:5:5, v/v/v/v). 13.0 mg of nevadensin was isolated from 15.0 g original sample by HSCCC with five times sample injection in 12 h, and the isolation yield of nevadensin was 0.87 mg/g. The average purity of nevadensin was higher than 98.0%. The chemical structure of collected fraction was identified by HPLC, ESI-MS and 1H NMR. The results indicated that this on-line method was effective and fast for high-throughput isolation of nevadensin from L. pauciflorus Maxim.
Keywords: Dynamic microwave-assisted extraction; High-speed counter-current chromatography; On-line; Nevadensin; Lyeicnotus pauciflorus Maxim.; Isolation;

Quantitative determination of T-2 toxin, HT-2 toxin, deoxynivalenol and deepoxy-deoxynivalenol in animal body fluids using LC–MS/MS detection by S. De Baere; J. Goossens; A. Osselaere; M. Devreese; V. Vandenbroucke; P. De Backer; S. Croubels (2403-2415).
A sensitive and specific method for the quantitative determination of deoxynivalenol (DON), deepoxy-deoxynivalenol (DOM-1), T-2 toxin (T-2) and HT-2 toxin (HT-2) in animal body fluids (plasma and bile) using liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS) is presented. The extraction of plasma consisted of a deproteinization step using methanol, followed by a clean-up using an Oasis® HLB solid-phase extraction column. For bile analysis, an extraction using a methanol/water mixture (70/30, v/v), followed by a liquid–liquid extraction using ethyl acetate, was performed. Chromatographic separation was achieved on a reversed-phase Nucleosil (100-5 C18 G100 × 3.0 mm) column. For the analysis of DON and DOM-1, a mixture of 0.1% acetic acid in water and methanol was used as the mobile phase. T-2 and its metabolite HT-2 were separated using 5 mM ammonium acetate in a mixture of water/methanol/acetic acid. The mass spectrometer was operated in the negative or positive ESI selected reaction monitoring mode for DON and T-2 analysis, respectively. Calibration graphs (1–250 ng mL−1) were prepared for all matrices and correlation and goodness-of-fit coefficients were between 0.9978–1.000 and 2.96–11.77%, respectively. Limits of quantification were between 1 and 2.5 ng mL−1 for all compounds. Limits of detection ranged from 0.01 to 0.63 ng mL−1. The results for the within-day precision and accuracy fell within the ranges specified. The method has been successfully used for the quantitative determination of DON, DOM-1, T-2 and HT-2 in plasma and the semi-quantitative determination of the same compounds in bile from broiler chickens and pigs, respectively.
Keywords: Mycotoxins; Trichothecenes; Deoxynivalenol; T-2 toxin; LC–MS/MS; Plasma; Bile;

With the increasing presence of illegal dyes, such as sudan reds and malachite green, in animal feeds and food products during the last few years, there is an urgent need of accurate quantitative determination methods for these illicit compounds. Here we established an accurate method for the simultaneous determination of 15 illegal dyes in animal feeds, meat, eggs and other food products using ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS). The samples were extracted with a simple procedure using acetonitrile and solid phase extraction cleaning up. The application of C18 rapid column can achieve satisfactory separation of the 15 dyes within 16 min; and multiple reaction monitoring of positive ions ensure confirmative detection of these illegal dyes. With the developed method, a sample can be analyzed in less than 2 h. Dyes spiked in feeds, poultry meat and eggs in the range of 0.1–5.0 mg kg−1 were tested in terms of linearity, sensitivity, repeatability and recovery. Recoveries for the compounds ranged from 60 to 140%. Intra- and inter-day precisions (RSDs) were less than 15%. Limit of quantification ranged from 0.01 to 5.61 μg kg−1 for different dyes. The developed UHPLC–MS/MS method could be used as a qualitative and quantitative technique for the simultaneous determination of illegal dyes in animal feeds and poultry products.
Keywords: Dyes; Animal Feed; Poultry products; UHPLC–MS/MS;

A novel isocratic reversed-phase high performance liquid-chromatography/ultraviolet detection method for simultaneous determination of cefdinir and cefixime in human plasma was developed and validated after optimization of various chromatographic conditions and other experimental parameters. Sample preparation based on a simple extraction procedure consisting of deproteination and extraction with 3 parts of 6% trichloroacetic acid aqueous solution followed by volume make up with the aqueous component of the mobile phase obtained best recoveries of the two analytes. Samples were separated on a Supelco Discovery HS C18 (150 mm × 4.6 mm, 5 μm) analytical column protected by a Perkin Elmer C18 (30 mm × 4.6 mm, 10 μm) guard cartridge. The mobile phase, methanol/acetonitrile (50/50, v/v):0.05% trifluoroacetic acid (19:81, v/v), operated at 50 °C column oven temperature was pumped at a flow rate of 2.0 mL min−1 and the column eluents were monitored at a wavelength of 285 nm. When Sample was injected into the Perkin Elmer high performance liquid-chromatography system through Rheodyne manual (or auto-sampler) injector equipped with 20 μL loop, separation was achieved within 4 min. The present method demonstrated acceptable values for selectivity, linearity within the expected concentration range (0.004–5.0 μg mL−1; r 2  > 0.999 for both analytes), recovery (>95% for cefdinir and >96% for cefixime), precision (%RSD < 2.0 for cefdinir and <2.2 for cefixime), sensitivity (limit of detection: 1 ng mL−1 and lower limit of quantification: 4 ng mL−1 for both analytes), stability of solutions, and robustness. The method was efficiently applied to a pharmacokinetic study in healthy volunteers.
Keywords: Cefixime; Cefdinir; Reversed-phase high performance liquid-chromatography; Optimization; Validation; Pharmacokinetic study;

Zaltoprofen, available commercially as a racemic mixture, is a propionic acid derivative of non-steroidal anti-inflammatory drugs (NSAIDs). Firstly, (+)- and (−)-zaltoprofen glucuronide was biosynthesized and purified. Then a simple and rapid RP-HPLC analysis method for direct determination of (+)- and (−)-zaltoprofen glucuronide in rat hepatic microsomes was developed and validated. The calibration curves of (+)- and (−)-zaltoprofen glucuronide both showed good linearity in the concentration range from 0.15 to 31.13 μM. The lower limit of quantification was 0.15 μM. Finally, this method was used to investigate the enantioselectivity of zaltoprofen glucuronidation in rat hepatic microsomes. The kinetics of zaltoprofen glucuronidation in rat hepatic microsomes for 40 min incubation fit the Michaelis–Menten model. Kinetic analysis indicated that (−)-zaltoprofen had a higher glucuronidation rate in rat liver microsome than that of (+)-zaltoprofen. The catalyzing efficiency (V max/K m ) ratio of (+)-zaltoprofen to (−)-enantiomer is 0.8 times in rat liver microsomes.
Keywords: Zaltoprofen; Glucuronide; RP-HPLC; Kinetic analysis; Stereoselectivity;

Efficient and inexpensive method for purification of heparin binding proteins by Sumit Batra; Nilesh Sahi; Kristen Mikulcik; Heather Shockley; Camille Turner; Zachary Laux; Vivek D. Badwaik; Eric Conte; Dakshinamurthy Rajalingam (2437-2442).
Heparin binding (HB) proteins mediate a wide range of important cellular processes, which makes this class of proteins biopharmaceutically important. Engineering HB proteins may bring many advantages, but it necessitates cost effective and efficient purification methodologies compared to currently available methods. One of the most important classes of HB proteins are fibroblast growth factors (FGFs) and their receptors (FGFRs). In this study, we report an efficient off-column purification of FGF-1 from soluble fractions and purification of the D2 domain of FGFR from insoluble inclusion bodies, using a weak Amberlite cation (IRC) exchanger. FGF-1 and the D2 domain have been expressed in Escherichia coli and purified to homogeneity using IRC resin. This approach is an alternative to conventional affinity column chromatography, which exhibits several disadvantages, including time-consuming experimental procedures for purification and regeneration and results in the expensive production of recombinant proteins. Results of the heparin binding chromatography and steady state fluorescence experiments show that the FGF-1 and the D2 are in a native conformation. The findings of this study will not only aid an in-depth investigation of this class of proteins but will also provide avenues for inexpensive and efficient purification of other important biological macromolecules.
Keywords: Heparin binding proteins; Fibroblast growth factor; Biopharmaceuticals; Protein purification; Inclusion bodies; Affinity chromatography; Thermal denaturation;

A simple, precise and rapid ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method has been developed and validated for the quantification of darunavir, a protease inhibitor, using darunavir-d9 as internal standard (IS). The method involved liquid–liquid extraction of darunavir and IS in methyl-tert-butyl ether from 50 μL human plasma. The chromatographic separation was achieved on an Acquity UPLC BEH C18 (50 mm × 2.1 mm, 1.7 μm particle size) analytical column under gradient conditions, in a run time of 1.6 min. The precursor → product ion transitions for darunavir (m/z 548.1 → 392.0) and IS (m/z 557.1 → 401.0) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) and positive ion mode. The method was extensively validated for its selectivity, sensitivity, carryover check, linearity, precision and accuracy, reinjection reproducibility, recovery, matrix effect, ion suppression/enhancement, stability and dilution integrity. The linearity of the method was established in the concentration range of 1.0–5000 ng/mL. The mean relative recovery for darunavir (100.8%) and IS (89.8%) from spiked plasma samples was consistent and reproducible. The application of this method for routine measurement of plasma darunavir concentration was demonstrated by a bioequivalence study conducted in 40 healthy Indian subjects for a 600 mg tablet formulation along with 100 mg ritonavir as booster under fast and fed conditions. To demonstrate the reproducibility in the measurement of study data, an incurred sample reanalysis was done with 400 subject samples and the % change in concentration was within ±12%.
Keywords: Darunavir; UPLC–MS/MS; Human plasma; Bioequivalence study; Incurred sample reanalysis;

A sensitive and automated method is described for determination of rifampicin in plasma samples for therapeutic drug monitoring by in-tube solid-phase microextraction coupled with liquid chromatography (in-tube SPME/LC). Important factors in the optimization of in-tube SPME are discussed, such as coating type, sample pH, sample draw/eject volume, number of draw/eject cycles, and draw/eject flow rate. Analyte pre-concentrated in the polyethylene glycol phase was directly transferred to the liquid chromatographic column by percolation of the mobile phase, without carryover. The method was linear over the 0.1–100 μg/mL range, with a linear coefficient value (r 2) of 0.998. The inter-assay precision presented coefficient of variation ≤1.7%. The effectiveness and practicability of the proposed method are proven by analysis of plasma samples from ageing patients undergoing therapy with rifampicin.
Keywords: Rifampicin; Liquid chromatography; Plasma sample; In-tube solid-phase microextraction;

Rapid UHPLC determination of polyphenols in aqueous infusions of Salvia officinalis L. (sage tea) by Benno F. Zimmermann; Stephan G. Walch; Laura Ngaba Tinzoh; Wolf Stühlinger; Dirk W. Lachenmeier (2459-2464).
Sage tea, the aqueous infusion of dried sage leaves (Salvia officinalis L.), is used as a form of food as well as a form of traditional herbal medicine. Several in vivo and in vitro studies point to sage polyphenols as active principles that may inhibit lipid peroxidation and improve antioxidant defences. This study describes an UHPLC methodology with MS/MS and UV detection, which allows the separation, identification and quantification of the major phenolic constituents in sage tea within 34 min, and was used to characterize 16 commercial brands of sage tea. The quantitatively dominating compounds were either rosmarinic acid (12.2–296 mg/L) or luteolin-7-O-glucoside (37.9–166 mg/L). In general, considerable differences in polyphenolic composition between the brands were detected, leading to the demand for quality standardization and control, especially if these sage teas are to be used for therapeutic purposes.
Keywords: Sage; Salvia officinalis L.; Tea infusion; Liquid chromatography–mass spectrometry (LC–MS); UHPLC; Polyphenols;

Chiral assay of omeprazole and metabolites and its application to a pharmacokinetics related to CYP2C19 genotypes by Hideo Shiohira; Norio Yasui-Furukori; Tomonori Tateishi; Tsukasa Uno (2465-2470).
Studies investigating the relationship between CYP2C19 genotype and the stereoselective metabolism of omeprazole have not been reported. In the present study, we developed a simple and sensitive analytical method based on column switching reversed phase high-performance liquid chromatography (HPLC) with UV detection to determine the concentrations of (R)- and (S)-omeprazole and of its principal metabolites, (R)- and (S)-5-hydroxyomeprazole, and the non-chiral, omeprazole sulfone, in human plasma. Sample preparation involved liquid–liquid extraction with diethyl ether:dichloromethane (60:40, v/v) followed by clean-up on a TSK BSA-ODS/S column (5 μm, 10 mm × 4.6 mm i.d.) using phosphate buffer:acetonitrile (97:3, v/v, pH 6.4). After column switching, separation was performed on a Shiseido CD-ph chiral column (5 μm, 150 mm × 4.6 mm i.d.) using phosphate buffer:methanol (45:55, v/v, pH 5.0) as mobile phase. The limit of quantitation (LOQ) was 5 ng/mL for all analytes with intra- and inter-day precisions (as coefficient of variation) of <9.5% and <9.6%, respectively for all analytes. The present method was successfully applied to a chiral pharmacokinetic study of omeprazole in human volunteers with different CYP2C19 genotypes. The results show that the formation of (R)-5-hydroxyomeprazole gives the best correlation with CYP2C19 genotype.
Keywords: (R)-Omeprazole; (S)-Omeprazole; (R)-5-Hydroxyomeprazole; (S)-5-Hydroxyomeprazole; Omeprazole sulfone; CYP2C19;