Journal of Chromatography B (v.878, #28)

Echinacoside is an important bioactive compound extracted from Cistanche tubulosa which was endangered by overexploitation. It is imperative to find an alternative source. Echinacoside was isolated from Penstemon barbatus (Can.) Roth for the first time. The peak contents of echinacoside are 9.09 ± 0.32 mg/g and 7.25 ± 0.36 mg/g respectively in the leaves and roots annually. The methanolic extracts from 20 g of dried powder of the roots of P. barbatus were pre-purified by AB-8 resin and the fraction containing echinacoside was further purified by conventional high-speed counter-current chromatography (HSCCC) and recycling HSCCC with the solvent system n-butanol–water (1:1, v/v). Totally 42.0 mg echinacoside with a purity of 96.3% was recovered. The recovery rate of echinacoside by recycling HSCCC reached 91.0%. The structure of our echinacoside confirmed by IR, 1H NMR and 13C NMR is identical to the standard sample. This indicates that P. barbatus might be ideal source for preparation of large scale of echinacoside.
Keywords: Penstemon barbatus (Can.) Roth; Echinacoside; HSCCC; HPLC; Characterization;

Hydroxyapatite as a concentrating probe for phosphoproteomic analyses by Gabriella Pinto; Simonetta Caira; Marina Cuollo; Sergio Lilla; Olga Fierro; Francesco Addeo (2669-2678).
A novel method for the selective enrichment of casein phosphoproteins/phosphopeptides (CPP) from complex mixtures is reported herein. This method employs ceramic hydroxyapatite (HA) as a solid-phase adsorbent to efficiently capture phosphoproteins and CPP from complex media. Casein was chosen as the model phosphoprotein to test the protocol. CPP immobilized on HA microgranules formed a complex that was included in the matrix-assisted laser desorption/ionization mass spectrometry (MALDI) matrix before desorbing directly from the well plate. Casein fractions with different levels of phosphorylation were desorbed based upon the specific concentration of trifluoroacetic acid (TFA) included in the MALDI matrix. The HA-bound casein enzymolysis was performed in situ with trypsin to remove non-phosphorylated peptides and isolate the immobilized CPP. The latter were recovered by centrifugation, dried, and co-crystallized with a 1% phosphoric acid (PA) solution in the matrix that was appropriate for detecting CPP in MALDI-MS spectra. This approach for the selection of casein/CPP resulted in the identification of 32 CPP by MALDI-time of flight (TOF). The analytical process involved two steps requiring ∼2 h, excluding the time required for the enzymatic reaction. The alkaline phosphatase (AP)-assisted de-phosphorylation of tryptic CPP allowed the phosphorylation level of peptides to be calculated concurrently with MALDI-TOF MS and liquid chromatography-electrospray ionization–mass spectrometry (LC–ESI–MS/MS). The effectiveness of the extraction procedure assayed on eggshell phosphoproteins resulted in the identification of 5 phosphoproteins and 14 derived phosphopeptides with a phosphoprotein global recovery of ∼70% at least.
Keywords: Direct MALDI detection; Hydroxyapatite enrichment; Phosphopeptides (CPP); Phosphoproteins; Proteomics;

The alpha (α) and beta (β) subunits of buffalo pituitary luteinizing hormone (LH) were chromatographed on Cibacron Blue 3GA agarose and their immunoreactivity was quantitated using anti-α and anti-β anti sera. Subsequent analyses showed α subunits were relatively more hydrophilic than β subunits. Further, the naturally occurring free α and β subunits were more hydrophobic than their native counterparts which were dissociated and isolated from heterodimeric LH. The lesser sugar content in freely occurring α and beta subunits may be attributed for increased hydrophobicity and consequent upon the existence of their uncombined free forms. In order to ascertain putative sugar–dye interaction, crude LH carrying free subunits, pure LH, and non-glycosylated recombinant β subunit of LH were loaded separately on Cibacron Blue. Methyl mannoside was able to elute 33% of the bound protein in case of crude and pure LH, whereas there was little (3%) elution in case of recombinant LH β subunit. This study suggests a compositional heterogeneity in free and native subunits of LH from the buffalo pituitary. In addition, our findings reveal the pseudolectin-like behavior of Cibacron Blue.
Keywords: Buffalo; Cibacron Blue; Free subunits; Glycoprotein hormones; Pseudolectin;

LC–MS/MS method for the determination of several drugs used in combined cardiovascular therapy in human plasma by Oskar Gonzalez; Gorka Iriarte; Estitxu Rico; Nerea Ferreirós; Miren Itxaso Maguregui; Rosa Maria Alonso; Rosa Maria Jiménez (2685-2692).
A simple, fast and validated method is reported for the simultaneous analysis, in human plasma, of several drugs usually combined in cardiovascular therapy (atenolol, bisoprolol, hydrochlorothiazide, chlorthalidone, salicylic acid, enalapril and its active metabolite enalaprilat, valsartan and fluvastatin) using high performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) with electrospray ionization (ESI), working in multiple reaction monitoring mode (MRM). Separation of analytes and internal standard (pravastatin) was performed on a Luna C18(2) (150 mm × 4.6 mm, 3 μm) column using a gradient elution mode with a run time of 15 min. The mobile phase consisted of a mixture of acetonitrile and water containing 0.01% formic acid and 10 mM ammonium formate at pH 4.1. Sample treatment consisted of a simple protein precipitation with acetonitrile, enabling a fast analysis. The method showed good linearity, precision (RSD% values between 0.7% and 12.7%) and accuracy (relative error values between 0.9% and 14.0%). Recoveries were within 68–106% range and the ion-suppression was not higher than 22% for any analyte. The method was successfully applied to plasma samples obtained from patients under combined cardiovascular treatment.
Keywords: Metabolic syndrome; Bioanalysis; LC–MS/MS; Validation; Cardiovascular therapy;

Isolation of lignans from Schisandra chinensis with anti-proliferative activity in human colorectal carcinoma: Structure–activity relationships by John Gnabre; Irem Unlu; Tso-cheng Chang; Paul Lisseck; Bryan Bourne; Ryan Scolnik; Neil E. Jacobsen; Robert Bates; Ru Chih Huang (2693-2700).
Separate benzocyclooctadiene lignans were isolated from the berries of Schisandra chinensis in milligram quantities on analytical reverse phase (RP) HPLC by an automated repeat-injection method and shown to have anti-proliferative activity against human colorectal cancer cells. Structures of the compounds were determined by a combination of NMR and mass spectrometry. Stereospecific NMR assignments for gomisin-N and deoxyschisandrin, gave more complete and accurate data than previously reported, based on 600 MHz 2D HSQC, DQF-COSY and HMBC data. Comparison of coupling constants and HMBC crosspeak intensities with calculated and X-ray crystal structures confirmed their stereochemistry and conformation. Analysis of structure–activity relationships revealed the importance of key structural determinants. The S-biphenyl configuration of gomisin N, the most active lignan, correlated with increased anti-proliferative activity, while the presence of a hydroxyl group at the C7 position reduced or abolished this activity. Increased activity was also observed when a methylenedioxy group was present between C12 and C13. The percent yield of the most active compounds relative to the starting plant materials was 0.0156% for deoxyschisandrin and 0.0173% for gomisin N. The results of these studies indicate that automated repeat-injection method of analytical HPLC may provide a superior alternative to the standard semi-preparative HPLC techniques for separation of complex mixtures.
Keywords: Schisandra chinensis; Lignans; Anti-proliferation; Automated repeat-injection; Conformation;

A highly sensitive HPLC–ESI-MS method has been developed and validated for the quantification of ginkgolic acid (15:1) in a small quantity of rat plasma (50 μL) using its homologous compound ginkgolic acid (17:1) as an internal standard. GA (15:1) and GA (17:1) were extracted from biological matrix by direct protein precipitation with 5-fold volume of methanol and separated on an Elite hypersil BDS C18 column (2.1 × 100 mm, 3 μm), eluted with acetonitrile:water (92:8, v/v, containing 0.3% glacial acetic acid). Linear range was 8–1000 ng/mL with the square regression coefficient (r 2) of 0.996. The lowest concentration (8 ng/mL) in the calibration curve was estimated as LLOQ with both deviation of accuracy and RSD of precision <20% (n  = 6). The intra- and inter-day precision ranged from 3.6% to 9.9%, and the intra- and inter-day accuracy was between 89.9% and 101.3%. This method was successfully applied to study pharmacokinetics of GA (15:1) in rats after oral administration at a dose of 10 mg/kg. GA (15:1) pharmacokinetic parameters C max, T max, t 1/2, AUC0–12h are 1552.9 ± 241.0 ng/mL, 0.9 ± 0.7 h, 5.5 ± 2.6 h, 3356.0 ± 795.3 ng h/mL, respectively.
Keywords: Ginkgolic acid (15:1); Ginkgolic acid (17:1); Rat; Validation; HPLC–MS; Pharmacokinetics;

Determination of the major constituents in fruit of Arctium lappa L. by matrix solid-phase dispersion extraction coupled with HPLC separation and fluorescence detection by He Liu; Yupu Zhang; Yantao Sun; Xue Wang; Yujuan Zhai; Ye Sun; Shuo Sun; Aimin Yu; Hanqi Zhang; Yinghua Wang (2707-2711).
The arctiin and arctigenin in the fruit of Arctium lappa L. were extracted by matrix solid-phase dispersion (MSPD) and determined by high-performance liquid chromatography (HPLC) with fluorescence detection. The experimental conditions for the MSPD were optimized. Silica gel was selected as dispersion adsorbent and methanol as elution solvent. The calibration curve showed good relationship (r  > 0.9998) in the concentration range of 0.010–5.0 μg mL−1 for arctiin and 0.025–7.5 μg mL−1 for arctigenin. The recoveries were between 74.4% and 100%. The proposed method consumed less sample, time and solvent compared with conventional methods, including ultrasonic and Soxhlet extraction.
Keywords: Arctiin; Arctigenin; Matrix solid-phase dispersion extraction (MSPD); High-performance liquid chromatography (HPLC); Fluorescence detection;

We have developed an online analytical method that combines A431 cell membrane chromatography (A431/CMC) with high performance liquid chromatography and mass spectrometry (LC/MS) for identifying active components from Radix Caulophylli acting on human EGFR. Retention fractions on A431/CMC model were captured onto an enrichment column and the components were directly analyzed by combining a 10-port column switcher with an LC/MS system for separation and preliminary identification. Using Sorafenib tosylate as a positive control, taspine and caulophine from Radix Caulophylli were identified as the active molecules which could act on the EGFR. This A431/CMC-online-LC/MS method can be applied for screening active components acting on EGFR from traditional Chinese medicines exemplified by Radix Caulophylli and will be of great utility in drug discovery using natural medicinal herbs as a source of novel compounds.
Keywords: Cell membrane chromatography (CMC); EGFR; High performance liquid chromatography/mass spectrometry (LC/MS); Radix Caulophylli;

Anion exchange monolithic chromatography is increasingly becoming a prominent tool for plasmid DNA purification but no generic protocol is available to purify all types of plasmid DNA. In this work, we established a simple framework and used it to specifically purify a plasmid DNA model from a clarified alkaline-lysed plasmid-containing cell lysate. The framework involved optimising ligand functionalisation temperature (30–80 °C), mobile phase flow rate (0.1–1.8 mL/min), monolith pore size (done by changing the porogen content in the polymerisation reaction by 50–80%), buffer pH (6–10), ionic strength of binding buffer (0.3–0.7 M) and buffer gradient elution slope (1–10% buffer B/min). We concluded that preferential pcDNA3F adsorption and optimum resolution could be achieved within the tested conditions by loading the clarified cell lysate into 400 nm pore size of monolith in 0.7 M NaCl (pH 6) of binding buffer followed by increasing the NaCl concentration to 1.0 M at 3%B/min.
Keywords: Process optimisation; Anion exchange; Monolithic chromatography; Plasmid DNA; pcDNA3F;

An automated screening method for drugs and toxic compounds in human serum and urine using liquid chromatography–tandem mass spectrometry by Stefan Sturm; Felix Hammann; Juergen Drewe; Hans H. Maurer; André Scholer (2726-2732).
A fully automated screening using liquid chromatography–mass spectrometric method applying data-dependent acquisition was developed to identify toxicologically relevant substances in serum and urine. A library including more than 405 spectra of about 365 compounds (main drugs and important metabolites) was established. An easy to use program was created to automate and accelerate library search. Drugs were identified based on their relative retention times, molecular ions and fragment ions. Limits of detection were tested with 100 of the 365 compounds the majority of these were lower than 100 μg/l (67%). The developed LC–MS–MS system seems to be a valuable alternative to other general unknown screening methods allowing fast and specific identification of drugs in serum and urine samples.
Keywords: General unknown screening; Data-dependent acquisition; Toxic compounds;

Previously, multivariate calibration techniques have been successfully applied to model and predict the antioxidant activity of green tea from its chromatographic fingerprint. Since the selectivity differences between dissimilar chromatographic systems have already been valuably used in several applications, in this paper it is studied whether combining the complementary information contained in two dissimilar fingerprints can improve the predictive capacity of the multivariate calibration model. The simplest way of combining the data is concatenating both fingerprints for each sample. The resulting matrix can then be subjected to Orthogonal Projections to Latent Structures (O-PLS). Unfortunately, this approach resulted in a more complex model with a prediction error of about the average of the errors obtained with the individual fingerprints. Secondly, only the peaks with high loading and low orthogonal loading from both chromatograms were included in the O-PLS model. This resulted in a reduced complexity, but not in better predictions, probably due to a lack of complementarity of the information concerning the antioxidant capacity. Finally, the concatenated fingerprints were subjected to stepwise multiple linear regression (MLR) in order to build a model based on the variables most correlated with the antioxidant capacity. The obtained prediction error was lower than those of both previous approaches, but still higher than the error of the model based on a single analysis. This is probably again caused by a lack of complementarity in the variables. Nevertheless, it was advantageous to develop fingerprints on dissimilar system, because it enables to choose the most suited chromatographic profile to build a multivariate calibration model for the considered purpose. In contrast to what was expected, the study showed that the most simple (so the worst separated) fingerprints resulted in the best predictions. On the other hand, a more complex fingerprint in which more compounds are separated is still important to improve the interpretability of the model.
Keywords: Green tea; Fingerprints; Multivariate calibration; Dissimilar chromatographic systems; Orthogonal chromatographic systems; Orthogonal Projections to Latent Structures;

Glechoma longituba is a widely used traditional Chinese medicine (TCM) in treating various diseases; however, the in vivo integrated metabolism of its multiple bioactive components remains unknown. In this paper, ultra-performance liquid chromatography (UPLC) coupled to quadrupole time-of-flight (QTOF) and the MetaboLynx™ software combined with mass defect filtering (MDF) together provide 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 constituents absorbed and metabolized studies of G. longituba extract after oral administration to rats. The results showed that 21 parent components of G. longituba extract were absorbed into the blood circulation of the rats and a total of 80 metabolites of 9 parent compounds were tentatively detected in vivo by their MS spectra obtained at low or high collision energy scan with the comparison of the authentic standards and literature data. The developed method was simple and reliable, revealing that it could be used to rapid screen and identify the structures of active components responsible for pharmacological effects of G. longituba and to better clarify its action mechanism. This work suggests that the integrative metabolism approach makes a useful template for drug metabolism research of TCM.
Keywords: Glechoma longituba; Metabolite; UPLC–QTOF/MS; MDF; MSE;

Liquid chromatography-tandem mass spectrometric assay for pravastatin and two isomeric metabolites in mouse plasma and tissue homogenates by Rolf W. Sparidans; Dilek Iusuf; Alfred H. Schinkel; Jan H.M. Schellens; Jos H. Beijnen (2751-2759).
A bioanalytical assay for pravastatin and two isomeric metabolites, 3′α-isopravastatin and 6′-epipravastatin, was developed and validated. Mouse plasma and tissue homogenates from liver, kidney, brain and heart were pre-treated using protein precipitation with acetonitrile containing deuterated internal standards of the analytes. The extract was diluted with water and injected into the chromatographic system. This system consisted of a polar embedded octadecyl silica column using isocratic elution with formic acid in a water–acetonitrile mixture. The eluate was transferred to an electrospray interface using negative ionization and the analytes were detected and quantified with the selected reaction monitoring mode of a triple quadrupole mass spectrometer. The assay was successfully validated in a 3.4–7100 ng/ml concentration range for pravastatin, 1.3–2200 ng/ml for 3′α-isopravastatin and 0.5–215 ng/ml for 6′-epipravastatin using only plasma for calibration. For plasma samples, subjected to full validation, within and between day precisions were 1–7% (9–18% at the LLQ level) and accuracies were between 91% and 103%. For tissue homogenates, subjected to partial validation, within and between day precisions were 2–12% (6–19% at the LLQ level) and accuracies were between 87% and 113% (81 and 113% at the LLQ level). Drug and metabolites were shown to be chemically stable under most relevant analytical conditions. Finally, the assay was successfully applied for a pilot study in mice. After intravenous administration of the drug, all isomeric compounds were found in plasma; however, in liver and kidney homogenate only the parent drug showed levels exceeding the LLQ.
Keywords: Pravastatin; 3′α-Isopravastatin; 6′-Epipravastatin; LC/MS/MS; Plasma; Mouse tissue homogenate;

High-yield expression and purification of the Hsp90-associated p23, FKBP52, HOP and SGTα proteins by Zacariah L. Hildenbrand; Sudheer K. Molugu; Atanu Paul; Gustavo A. Avila; Nadia Herrera; Chuan Xiao; Marc B. Cox; Ricardo A. Bernal (2760-2764).
Hsp90 is a ubiquitous molecular chaperone that plays a key role in the malignant development of hormone-dependent pathologies such as cancer. An important role for Hsp90 is to facilitate the stable binding of steroid hormones to their respective receptors enabling the ligand-based signal to be carried to the nucleus and ultimately resulting in the up-regulation of gene expression. Along with Hsp90, this dynamic and transient process also involves the recruitment of additional proteins and co-chaperones that add further stability to the mature receptor–chaperone complex. In the work presented here, we describe four new protocols for the bacterial over-expression and column chromatographic purification of the human p23, FKBP52, HOP and SGTα proteins. Each of these proteins plays a distinct role in the steroid hormone receptor regulatory cycle. Affinity, ion-exchange and size-exclusion techniques were used to produce target yields greater than 50 mg/L of cultured media, with each purified sample reaching near absolute sample homogeneity. These results reveal a reliable system for the production of p23, FKBP52, HOP and SGTα substrate proteins for use in the investigation of the Hsp90-associated protein interactions of the steroid hormone receptor cycle.
Keywords: Affinity; Ion-exchange; Size-exclusion chromatography;

8-Hydroxy-2′-deoxyguanosine (8-OHdG) and 5-methyl-2′-deoxycytidine (5-mdC) are utilized as useful biomarkers not only for early diagnosis but also for the detection and assessment of high-risk individuals. In the present study, a sensitive and specific method was developed for simultaneous determination of 8-OHdG and 5-mdC in DNA by high performance liquid chromatography/positive electrospray ionization tandem mass spectrometry. The limits of quantification for 8-OHdG and 5-mdC were 80 and 40 pg/ml, respectively. The calibration curves of 8-OHdG and 5-mdC were linear over the concentration range of 0.02–100 ng/ml and the correlation coefficients were higher than 0.9990. The intra-day and inter-day relative standard derivative values were in the range of 0.70–7.47% for 8-OHdG and 1.07–7.06% for 5-mdC, respectively. The recoveries were 93.4–108.5% for 8-OHdG and 87.4–104.9% for 5-mdC, respectively. This method was validated by determination of the background levels of 8-OHdG and 5-mdC in calf thymus DNA, and satisfactory results were obtained.
Keywords: 8-Hydroxy-2′-deoxyguanosine (8-OHdG); 5-Methyl-2′-deoxycytidine (5-mdC); Electrospray tandem mass spectrometry; Oxidative DNA damage; DNA methylation;

Fascioliasis is a zoonotic disease of considerable public health and great veterinary significance and new drugs are needed. OZ78 is a promising fasciocidal drug candidate. In order to support the development of OZ78, including pharmacokinetic (PK) studies an accurate, precise, and selective liquid chromatography/mass spectrometry (LC/MS) method for OZ78 was developed for sheep plasma and validated in accordance with the US Food and Drug Administration Guidance on Bioanalytical Method Validation. Protein precipitation was used for sample clean up. Separation was performed through a Phenomenex C8(2) analytical column (50.0 mm × 2.0 mm, 5 μm) with a mobile phase of acetonitrile (buffer B) and 5 mM ammonium formate (buffer A) at a flow-rate of 0.3 mL/min and a gradient from 20% to 95% acetonitrile. The mass spectrometer was operated under selected ion monitoring, and orifice voltage set to −4.1 kV and ion spray temperature to 400 °C. Nitrogen was used as a nebulizer, curtain, and collision gas. OZ78 was monitored at 321.4  m/z (deprotonated parent compound, M-). The validated linear dynamic range was between 156.25 ng/mL and 5 μg/mL and the achieved correlation coefficient (r 2) was greater than 0.99. The validation results demonstrated that the developed LC/MS method is precise, accurate, and selective for the determination of OZ78 in sheep plasma. The method was successfully applied to the evaluation of the PK profile of OZ78 in sheep.
Keywords: OZ78; Fascioliasis; Secondary ozonides; Trioxolanes; Liquid chromatography; Mass spectrometry;

Chromatographic analysis of acetohexamide binding to glycated human serum albumin by K.S. Joseph; Jeanethe Anguizola; Abby J. Jackson; David S. Hage (2775-2781).
Acetohexamide is a drug used to treat type II diabetes and is tightly bound to the protein human serum albumin (HSA) in the circulation. It has been proposed that the binding of some drugs with HSA can be affected by the non-enzymatic glycation of this protein. This study used high-performance affinity chromatography to examine the changes in acetohexamide–HSA binding that take place as the glycation of HSA is increased. It was found in frontal analysis experiments that the binding of acetohexamide to glycated HSA could be described by a two-site model involving both strong and weak affinity interactions. The average association equilibrium constant (K a ) for the high affinity interactions was in the range of 1.2–2.0 × 105  M−1 and increased in moving from normal HSA to HSA with glycation levels that might be found in advanced diabetes. It was found through competition studies that acetohexamide was binding at both Sudlow sites I and II on the glycated HSA. The K a for acetohexamide at Sudlow site I increased by 40% in going from normal HSA to minimally glycated HSA but then decreased back to near-normal values in going to more highly glycated HSA. At Sudlow site II, the K a for acetohexamide first decreased by about 40% and then increased in going from normal HSA to minimally glycated HSA and more highly glycated HSA. This information demonstrates the importance of conducting both frontal analysis and site-specific binding studies in examining the effects of glycation on the interactions of a drug with HSA.
Keywords: Acetohexamide; Human serum albumin; Glycation; Drug–protein binding; High-performance affinity chromatography;

A rapid, inexpensive, sensitive and specific HPLC-ECD method for the determination of lipoic acid in human plasma was developed and validated over the linearity range of 0.001–10 μg/ml using naproxen sodium as an internal standard (IS). Extraction of lipoic acid and IS from plasma (250 μl) was carried out with a simple one step liquid–liquid extraction using dichloromethane. Similarly solid-phase extraction was carried out using dichloromethane as extraction solvent. The separated organic layer was dried under the stream of nitrogen at 40 °C and the residue was reconstituted with the mobile phase. Complete separation of both lipoic acid and IS at 30 °C on Discovery HS C18 RP column (250 mm × 4.6 mm, 5 μm) was achieved in 6 min using 0.05 M phosphate buffer (pH 2.5 adjusted with phosphoric acid):acetonitrile (50:50, v/v) as a mobile phase pumped at the rate of 1.5 ml/min using electrochemical detector in DC mode at the detector potential of 1.0 V. The limit of detection and limit of quantification of lipoic acid were 200 pg/ml and 1 ng/ml, respectively. While on column limit of detection and limit of quantification of lipoic acid were 10 and 50 pg/ml, respectively. The absolute recoveries of lipoic acid with liquid–liquid and solid-phase extraction were 98.43, 95.65, 101.45, and 97.36, 102.73, 100.17% at 0.5, 1 and 5 μg/ml levels, respectively. Coefficient of variations for both intra-day and inter-day were between 0.28 and 4.97%. The method is validated and will be quite suitable for the analysis of lipoic acid in the plasma of human volunteers as well as patients with diabetes and cardiovascular diseases.
Keywords: Lipoic acid; Liquid–liquid extraction; Separation; Solid-phase extraction; Plasma;

A new one-step liquid chromatography–electrospray tandem MS/MS method is described to quantify ezetimibe (EZM) a novel lipid lowering drug in human serum. Also using collision-induced dissociation (CID) of the analyte, identification and chromatographic separation of its major metabolite, ezetimibe glucuronide (EZM-G) is achieved in this study. A thawed serum aliquot of 100 μL was deproteinated by addition of 500 μL methanol containing omeprazole as internal standard (I.S.). Separation of the drug, its metabolite and the I.S. were achieved using acetonitrile–water (70:30, v/v) as mobile phase at flow rate of 0.5 mL/min on a MZ PerfectSil target C18 column. Multiple reaction monitoring (MRM) mode of precursor–product ion transition (408.7 → 272.0 for EZM and 345 → 194.5 for the I.S.) was applied for detection and quantification of the drug while, EZM-G was chromatographically separated and identified using CID. The analytical method was linear over the concentration range of 1–32 ng/mL of EZM in human serum with a limit of quantification of 1 ng/mL. The coefficient variation values of both inter- and intra-day analysis were less than 8% whereas the percentage error was less than 3.7. The validated method was applied in a randomized cross-over bioequivalence study of two different EZM preparations in 24 healthy volunteers.
Keywords: HPLC; Ezetimibe; Ezetimibe glucuronide; MRM; LC–MS/MS bioequivalence study;

Curcumin and its derivatives generally display favorable cytotoxic activities against a number of cancer cell types. We focus our rational antineoplastic drug design program on curcumin analogues containing the 1,5-diaryl-3-oxo-1,4-pentadienyl pharmacophore. Favorable outcomes from pharmacological screens of this series demanded further pharmacokinetic evaluations to determine their suitability as effective compounds in vivo. To allow such evaluations and to provide a general, sensitive, rapid and simple method for the analysis of compounds containing the 1,5-diaryl-3-oxo-pentadienyl scaffold, we developed an HPLC method with ultraviolet detection for their detection in various biological matrices of a relevant preclinical species, i.e. the rat. Our HPLC method is specific for the analysis of many members in this series in rat blood, plasma, serum and hepatic microsomes following liquid–liquid extraction with TBME (1:30, v/v). The assay procedure involves chromatographic separation on a Zorbax-Eclipse C-18 column under isocratic conditions with the mobile phase consisting of acetonitrile and ammonium acetate buffer (pH 5.0, 10 mM) in different ratios depending upon the compound. The method was validated for NC 2083 in rat serum and rat liver microsomes, a potential lead compound, to demonstrate its applicability. The standard curve was linear (r 2  ≥ 0.997) from 50 to 5000 ng/mL. Intra- and inter-day precision and accuracy of the method were within USFDA specified limits. The stability of NC 2083 was established in an auto-injector, on bench-top, during freeze–thaw cycles and long-term stability at −80 °C for 40 days. The method is suitable for a number of compounds containing the 1,5-diaryl-3-oxo-pentadienyl scaffold with divergent log  P values with only minor adjustments in the buffer to acetonitrile ratio of the mobile phase.
Keywords: Curcumin analogues; HPLC; Microsomal stability; Piperidones; Rat biomatrices; Validation;

A microfluidic structure is presented where selective capture of proteins in complex samples, followed by clean-up, enzymatic processing, and MALDI-MS sample preparation of peptides generated, can be performed. The structure uses an affinity column to capture the protein while all other components in the sample are disposed of. The protein of interest is then eluted from the affinity column and captured on a second column on which the enzymatic processing is performed. Salts and hydrophilic contaminants are then removed before the products from the enzymatic reaction are eluted together with a suitable MALDI matrix and the solvent evaporated in a designated MALDI target structure. All steps can be performed automatically in 54 parallel microstructures on a microfluidic compact disc. The process is demonstrated by the selective capture and tryptic digest of recombinant IgG molecules from samples containing other proteins: an excess of bovine serum albumin or spent cell culture media.
Keywords: Microfluidics; Microfluidic CD; MALDI-MS; Recombinant therapeutic antibodies; Tryptic digestion; Peptide mapping;

Hollow fiber liquid-phase microextraction (HF-LPME) coupled with high-performance liquid chromatography was used to simultaneously determine three Aconitum alkaloids, including aconitine (AC), hypaconitine (HA) and mesaconitine (MA) in human urine sample. Analytes were extracted from 5 mL urine sample containing 1.0 mmol/L NaOH into 1-octanol membrane phase impregnated in the pores of hollow fiber wall, and then back extracted into acidified aqueous solution in the lumen of the hollow fiber. After extraction, 10 μL of the acceptor phase was analyzed directly by HPLC. In this method, some important extraction parameters, such as organic solvent, extraction time, stirring rate, pH of donor phase and acceptor phase, temperature, and the volume of acceptor phase were optimized. This method provided 98- to 288-fold enrichment factors within 60 min of extraction and good repeatability with RSDs of 0.99–7.22%. The calibration curves were linear over the ranges of 16.0–128.0 μg/L for AC, 11.0–88.0 μg/L for HA and 8.1–64.8 μg/L for MA in human urine sample, with correlation coefficients of 0.9949, 0.9969 and 0.9904, respectively. Limits of detection were from 0.7 to 1.5 μg/L, and recoveries from spiked urine sample varied from 84.4% to 106.2% for AC, 77.3% to 85.6% for HA and 90.1% to 100.8% for MA.
Keywords: Hollow fiber liquid-phase microextraction; High-performance liquid chromatography; Aconitine; Hypaconitine; Mesaconitine; Urine sample;

Isolated post-challenge diabetes (IPD, 2h-PG ≥11.1 mmol/L and FPG <7.0 mmol/L) is often ignored in screening for diabetes by fasting plasma glucose (FPG) levels. The aim of this study was to investigate the metabolic profiles of serum free fatty acids (FFAs) and to identify biomarkers that can be used to distinguish patients with IPD from those with type 2 diabetes mellitus (T2DM) or healthy control individuals. FFA profiles of the subjects were investigated using gas chromatography–mass spectrometry (GC–MS). Principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) were used for classification and prediction among the three groups. The predictive correct rates were 92.86% for IPD and healthy control individuals and 90.70% for T2DM and healthy control individuals, indicating that PLS-DA could satisfactorily distinguish IPD individuals from healthy controls and those with T2DM. Finally, palmitic acid, stearic acid, oleic acid, linoleic acid and α-linolenic acid were identified as potential biomarkers for distinguishing IPD from healthy control and T2DM individuals. These potential biomarkers might be helpful for diagnosis and characterization of diabetes.
Keywords: Free fatty acid; Biomarker; Isolated post-challenge diabetes; Type 2 diabetes mellitus; Gas chromatography–mass spectrometry;

Experimental studies of OH° radical/pressure dependence of arginase activity using a molecular chromatography approach by Claire André; Firas Ibrahim; Tijani Gharbi; Guillaume Herlem; Yves Claude Guillaume (2826-2830).
Arginase is an enzyme which plays a role in pathophysiology such as hypertension. Here we demonstrated for the first time the direct implication of pressure and OH° radical formation on the arginase activity via a novel analytical procedure. Pressure increased arginase activity in the range 12–52 bars. Activation by OH° radical showed a hyperbolic response. The OH° radicals produced were significantly inhibited by sulfasalazine (SAZ) and the inhibition of OH° radicals parallels the inhibition of arginase activity.
Keywords: Arginase; IMER; OH° radical; Pressure;

LC–MS/MS method development and validation for the determination of polymyxins and vancomycin in rat plasma by Chang Cheng; Shaorong Liu; Deqing Xiao; John Hollembaek; Lili Yao; Jing Lin; Steven Hansel (2831-2838).
Simple, sensitive and robust liquid chromatography–tandem mass spectrometer (LC–MS/MS) methods were developed and validated for the determination of lipopeptide polymyxins and glycopeptide vancomycin in rat plasma. The effect of trichloroacetic acid (TCA) concentration on sample recoveries (peak area of sample recovered from plasma/peak area of sample from neat solvent solutions) was studied and an optimized concentration of 30% TCA were determined that gives the best sample recovery for the peptides from rat plasma. The effect of the TCA concentration on the chromatographic behavior of peptides was studied on a Phenomenex Jupiter C18 5 μ 300 Å 50 mm × 2 mm column using a mobile phase with a pH of 2.8. Other than protein precipitation, TCA also acted as ion pairing reagent and was only present in the samples but not in the mobile phases. The data demonstrated that by increasing the TCA concentration, the analyte retention and sensitivity were improved. The absence of TCA in mobile phase helped to reduce the ion source contamination and to achieve good reproducibility. The plasma method was linearly calibrated from 5 to 5000 ng/mL for polymyxins with precisions to be of 2.3–10.8%, and accuracies to be 91.7–107.4% for polymyxin B1, B2, E1, E2, respectively. For vancomycin the calibration is from 1 to 5000 ng/mL with precisions to be of 7.8–10.3 and accuracies to be 96.2–102.0%. The LLOQs corresponding with a coefficient of variation less than 20% were 7.5, 18.1, 7.3, 5.0 and 1.0 ng/mL for polymyxin B1, B2, E1, E2 and vancomycin, respectively.
Keywords: LC–MS/MS; Trichloroacetic acid; Polymyxin; Colistin; Vancomycin; Sample recovery; Antibacterial; Peptide drugs;

Utilizing a solid phase extraction column (MCT) containing mixed hydrophilic functional gel and cation exchange sorbent, a sensitive and rapid HPLC–MS/MS method for simultaneously determining the residues of melamine (MEL) and cyanuric acid (CYA) in human foodstuffs was developed. MEL and CYA in egg, pork, liver, kidney and pork, shrimp, sausage casing, honey, soybean milk, soybean powder and dairy product were extracted using acetonitrile/water, defatted with hexane and isolated using MCT solid phase extraction column. The residues were separated upon a hydrophilic interaction liquid chromatography (HILIC) column and analyzed by electrospray ionization under negative–positive switched mode on a triplequadrupole mass spectrometry. The selected reaction monitoring was performed on [M+H]+ of m/z 127.9 to provide the transition of 127 > 85 and 127 > 68 (MEL) while the [M−H] of m/z 127.1 was selected as the precursor ion for CYA resulting in product ions m/z 85 and 42. Isotope labeled internal standard (15N3-MEL and 13C3-CYA) and matrix-matched calibration were both used to observe the recovery to be 70.0–129.6% and 70.0–128.9% with RSD of 1.4–23.3% and 1.5–21.7% for MEL and CYA, respectively (n  = 6). All the LODs and LOQs of MEL and CYA were less than 39.4 and 99.1 μg kg−1, respectively, in 18 matrices, which were sensitive enough for quantitative analysis. This method has been proven effective in simultaneous determination of melamine and cyanuric acid when inspecting unknown and positive samples.
Keywords: Melamine; Cyanuric acid; Mixed-mode SPE; HILIC–MS/MS; Simultaneous determination;

The dependence of the incorporation of methamphetamine into rat hair on dose, frequency of administration and hair pigmentation by Eunyoung Han; Yonghoon Park; Eunmi Kim; Sooyeun Lee; Hwakyung Choi; Heesun Chung; Joon Myong Song (2845-2851).
In this paper, the incorporation of methamphetamine (MA) into rat hair was studied. The main purpose of this study was to investigate whether MA can be detected or positive hair results can be obtained in hair of rats administered a single dose of MA. The relationship between dose and frequency of administration and the concentrations of MA and its metabolite, amphetamine (AP), in rat hair were evaluated and the MA and AP concentrations in white and pigmented hair were compared. MA was administered to rats as follows: low dose (0.5 mg/kg/day), medium dose (2 mg/kg/day) and high dose (10 mg/kg/day). The frequency of administration was one time per day for 1, 2, 3, 4, 5, 15 and 30 days. Hair and urine samples were collected from rats and analyzed by gas chromatography/mass spectrometry (GC/MS). MA could be identified in pigmented rat hair when MA was administered for 4 or more days at low daily dose and on day 1 following administration of medium and high daily doses. Positive results for MA were obtained from pigmented rat hair when MA was administered for 30 days at low daily dose, for 4 or more days at medium daily dose, or for 2 or more days at high daily dose. The concentrations of MA and AP found in rat hair were proportional to the dose and frequency of administration. The concentrations of MA and AP in pigmented rat hair were 2–10 times higher than those in white rat hair. The results of this study on the incorporation of MA into rat hair can serve as a model to better understand the incorporation of MA into human hair even though there are differences between animal models and human hair.
Keywords: Hair analysis; Methamphetamine; Incorporation; GC/MS;

Simultaneous production of immunoaffinity membranes by Youji Shimazaki; Masayuki Miyamoto (2852-2856).
We simultaneously separated antibodies for transferrin, the third component of complement (C3), haptoglobin and transthyretin by multi-sample non-denaturing two-dimensional electrophoresis (2-DE), transferred them to a polyvinylidene difluoride (PVDF) membrane and then stained them using direct blue 71 to obtain membrane-immobilized antibodies. The antigens, transferrin, C3, haptoglobin and transthyretin were specifically bound to the membrane-immobilized antibodies and were eluted only after rinsing the membrane with acid solution. The antigens specifically bound to the membrane-immobilized antibodies were separated by SDS-PAGE and identified by peptide mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Furthermore, transferrin and transthyretin were trapped and eluted by each membrane-immobilized antibody and detected by MALDI-TOF MS directly without separations. Using membrane-immobilized anti-transferrin antibody, transferrin in flowing blood was directly trapped and analyzed. The results indicated that membrane-immobilized antibodies are simultaneously produced, and that the immunoaffinity membranes can capture specific substances in flowing fluids.
Keywords: Non-denaturing 2-DE; Transferrin; C3; Haptoglobin; Transthyretin; Immobilization;

A simple, sensitive, and inexpensive singe-drop microextraction (SDME) followed by gas chromatography and flame-ionization detection (GC-FID) was developed for determination of nicotine, anabasine, and cotinine in human urine and saliva samples. The target compounds were extracted from alkaline aqueous sample solution into an organic acceptor drop suspended on the tip of a 25-μL GC microsyringe in the aqueous sample solution. This microsyringe was also used for direct injection after extraction. Under optimized experimental conditions, calibration plots were found to be linear in the range of 0.5–25.0, 0.5–65.0, and 0.5–45.0 mg L−1 for nicotine, anabasines and cotinine, respectively. The method detection limit values were in the range of 0.33–0.45 mg L−1. Intra-day and inter-day precisions for peak area ratios were in the range of 1.3–9.2% and 2.0–7.0%, respectively. The proposed procedure was successfully applied to the determination of analytes in spiked urine and saliva samples with satisfactory results. The mean relative recoveries of spiked water samples ranged over 71.2–111.0%, with relative standard deviations varying from 2.3% to 10.0%.
Keywords: Nicotine; Anabasine; Cotinine; Urine; Saliva; Single-drop microextraction;

We describe an ultra high performance liquid chromatography–tandem mass spectrometry (UHPLC MS/MS) method suitable for a routine laboratory to determine endogenous and exogenous glucocorticoids in plasma, plasma ultrafiltrate, urine and saliva in a single analytical run. After addition of a multi-analyte internal standard, a standardised sample preparation procedure with solid phase extraction followed, before injecting into a tandem mass spectrometer with positive mode electron spray ionisation and multiple reactant monitoring acquisition. The chromatography time was 3 min. The limit of quantitation for cortisol and cortisone in plasma was 3.75 nmol/L and linearity extended to 2000 nmol/L. The limit of quantitation for cortisol in plasma ultrafiltrate and saliva was 0.6 nmol/L. The limit of quantitation for 11-deoxycortisol and prednisolone was 5 nmol/L and for dexamethasone 1 nmol/L. The intra-assay CV was <5% and the inter-assay CV <10% for all analytes in all matrices. Comparison with an immuno-assay (IA) plasma cortisol method resulted in a regression equation of UHPLC = 0.79 × IA + 31.12 with R 2  = 0.960 (p  < 0.0001). Comparison with a high performance liquid chromatography (HPLC) cortisol method yielded a regression equation of UHPLC = 1.06 × HPLC + 9.82, R 2  = 0.992 (p  < 0.0001). The simultaneous measurement of endogenous and exogenous glucocorticoids contributed to patient care in cases with dexamethasone and metyrapone dynamic tests and unsuspected therapeutic glucocorticoid use.
Keywords: Tandem mass spectrometry; Ultra high performance liquid chromatography; Cortisol; Cortisone; Dexamethasone; Free hormones; 11β-Hydroxysteroid dehydrogenase;

Bile acids are increasingly gaining attention since they were discovered to be activators of the transcription factor farnesoid X receptor (FXR) in addition to their well-established role in dietary lipid emulsification. Moreover, the differential activation potency of bile acids on FXR, which is due to structural variation of the ligands, generates the need for new analytical tools that are sensitive and specific enough to quantify the individual species of this complex class of compounds. Because bile acids undergo enterohepatic circulation, the additional assessment of a bile acid precursor as a marker for bile acid biosynthesis is used to differentiate between newly synthesised bile acids and bile acids reabsorbed from the intestine. This paper describes two new methods using liquid chromatography–tandem mass spectrometry (LC–MS/MS) for the quantification of the major unconjugated bile acids in human serum (cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid and ursodeoxycholic acid) with their glycine- and taurine-conjugates as well as their precursor 7α-hydroxy-4-cholesten-3-one (C4). Intra- and inter-day variation was less than 12% and accuracy was between 84% and 102% for all analytes. Extraction recovery was between 78% and 100% for the bile acids whereas it was 62% for C4 and limit of quantification values ranged from 2 nmol/l to 50 nmol/l for all compounds. These two methods have the practical advantage of requiring low sample volume (100 μl serum for each method) and identical eluents, stationary phase as well as ionisation technique, so that they can be used in a combined way. Moreover, they provide information on the composition of the bile acid pool on one hand and on the relative amount of newly synthesised bile acids on the other, which taken together, gives new insights in the investigation of bile acid metabolism.
Keywords: 7α-hydroxy-4-cholesten-3-one; Bile acids; Electrospray ionisation; Liquid chromatography–mass spectrometry; Method validation; Quantification;

Monacolin K from red yeast rice was extracted by accelerated solvent extraction (ASE). The effects of various extraction parameters including extraction temperature, static extraction time and cycle index on yield were investigated using a DIONEX ASE 300 system to select the optimal conditions by an orthogonal test design L9 (3)3. The optimum extraction conditions were determined as follows: extraction temperature 120 °C, static extraction time 7 min, and cycle index 3. Under the optimal conditions, the yield of ASE extract and monacolin K was 5.35% and 9.26 mg/g of dry red yeast rice, respectively. A separation and purification method of monacolin K was then established using high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane–ethyl acetate–methanol–water (8:2:5:5, v/v/v/v). From 300 mg of crude extract, 51.2 mg of monacolin K was obtained with the purity of 98.7%. The chemical structure of isolated compound was identified by UV, ESI-MS and 1H NMR.
Keywords: Accelerated solvent extraction (ASE); High-speed counter-current chromatography (HSCCC); Red yeast rice; Monacolin K;

Determination of salivary efavirenz by liquid chromatography coupled with tandem mass spectrometry by Anri Theron; Duncan Cromarty; Malie Rheeders; Michelle Viljoen (2886-2890).
A novel and robust screening method for the determination of the non-nucleoside reverse transcriptase inhibitor, efavirenz (EFV), in human saliva has been developed and validated based on high performance liquid chromatography tandem mass spectrometry (LC–MS/MS). Sample preparation of the saliva involved solid-phase extraction (SPE) on C18 cartridges. The analytes were separated by high performance liquid chromatography (Phenomenex Kinetex C18, 150 mm × 3 mm internal diameter, 2.6 μm particle size) and detected with tandem mass spectrometry in electrospray positive ionization mode with multiple reaction monitoring. Gradient elution with increasing methanol (MeOH) concentration was used to elute the analytes, at a flow-rate of 0.4 mL/min. The total run time was 8.4 min and the retention times for the internal standard (reserpine) was 5.4 min and for EFV was 6.5 min. The calibration curves showed linearity (r 2, 0.989–0.992) over the concentration range of 3.125–100 μg/L. Mean intra- and inter-assay relative standard deviation, accuracy, mean extraction recovery, limit of detection (LOD) and limit of quantification (LOQ) were 0.46–9.43%, 80–120%, 60% (±7.95), 1.84 and 6.11 μg/L respectively. The working range was defined as 6.25–100 μg/L. This novel LC–MS/MS assay is suitable for reliable detection of low EFV concentrations in saliva and can be used as a screening tool for monitoring EFV compliance.
Keywords: Efavirenz; Saliva; LC–MS/MS;

A simple and sensitive HPLC fluorescence method for determination of tadalafil in mouse plasma by Christine A. Farthing; Don E. Farthing; Saisudha Koka; Terri Larus; Itaf Fakhry; Lei Xi; Rakesh C. Kukreja; Domenic Sica; Todd W.B. Gehr (2891-2895).
A simple and sensitive high-performance liquid chromatographic (HPLC) method utilizing fluorescence detection was developed for the determination of the phosphodiesterase type 5 inhibitor tadalafil in mouse plasma. This method utilizes a simple sample preparation (protein precipitation) with high recovery of tadalafil (∼98%), which eliminates the need for an internal standard. For constituent separation, the method utilized a monolithic C18 column and a flow rate of 1.0 mL/min with a mobile phase gradient consisting of aqueous trifluoroacetic acid (0.1% TFA in deionized water pH 2.2, v/v) and acetonitrile. The method calibration was linear for tadalafil in mouse plasma from 100 to 2000 ng/mL (r  > 0.999) with a detection limit of approximately 40 ng/mL. Component fluorescence detection was achieved using an excitation wavelength of 275 nm with monitoring of the emission wavelength at 335 nm. The intra-day and inter-day precision (relative standard deviation, RSD) values for tadalafil in mouse plasma were less than 14%, and the accuracy (percent error) was within −14% of the nominal concentration. The method was utilized on mouse plasma samples from research evaluating the potential cardioprotective effects of tadalafil on mouse heart tissue exposed to doxorubicin, a chemotherapeutic drug with reported cardiotoxic effects.
Keywords: Tadalafil; Doxorubicin; HPLC; Fluorescence; Monolithic column;

Determination of the unstable drug otilonium bromide in human plasma by LC–ESI-MS and its application to a pharmacokinetic study by Yan-rong Zhao; Li Ding; Hong-wei Fan; Yong Yu; Xie-min Qi; Ye Leng; Ya-kun Rao (2896-2900).
Otilonium bromide (OB) degrades rapidly in plasma and readily undergoes hydrolysis by the plasma esterase. In this paper, an LC–ESI-MS method has been developed for the determination of OB in human plasma. The rapid degradation of OB in plasma was well prevented by immediate addition of potassium fluoride (KF, an inhibitor of plasma esterase) to the freshly collected plasma before prompt treatment with acetonitrile. The method was validated over the concentration range of 0.1–20 ng/ml. The data of intra-run and inter-run precision and accuracy were within ±15%. The mean extraction recoveries for OB and the internal standard were higher than 93.0% and the matrix effects were negligible. The method has been successfully used in a pharmacokinetic study.
Keywords: Otilonium bromide; LC–ESI-MS; Sample preparation; Stability; Pharmacokinetics;

A novel capillary electrophoresis (CE) with chemiluminescence (CL) detection method for the determination of mitoxantrone (MTX) has been developed, which based on the CL reaction of potassium ferricyanide with luminol in sodium hydroxide medium sensitized by MTX. Under optimum analytical conditions, MTX is determined over the range of 7.0 × 10−8–1.0 × 10−6  M with a detection limit of 1.0 × 10−8  M. The relative standard deviation (RSD) was 3.7%, 2.6% and 3.0% for 7.0 × 10−8, 5.0 × 10−7 and 1.0 × 10−6  M MTX (n  = 11), respectively. In laboratory-built CE–CL apparatus, the proposed method has been applied to determination of MTX in commercial drug and spiked in human urine and plasma with satisfactory results.
Keywords: Mitoxantrone; Capillary electrophoresis; Chemiluminescence; Human urine; Human plasma;

Determination of 3,4-methylenedioxymethamphetamine and its five main metabolites in rat urine by solid-phase extraction and high performance liquid chromatography with on line mass spectrometry by Marie-Claude Menet; Julien Fonsart; Françoise Hervé; Dominique Fompeydie; Martine Galliot-Guilley; Florence Noble; Jean-Michel Scherrmann (2905-2910).
The consumption of psychostimulant amphetamine-like drugs has increased significantly in recent years. Some MDMA metabolites are probably involved in the neurotoxicity and neurodegeneration caused by prolonged use rather than MDMA itself. We recently developed a method to analyze MDMA and its five main metabolites in rat plasma . We have now fully validated this method to the quantification of these drugs in rat urine. We extracted MDMA and its metabolites with Oasis WCX cartridges, separated them on a Nucleodur C18 analytical column and quantified them by ion-trap mass spectrometry. Linearity was excellent: 12.5–1250 ng/mL urine for HMA, HMMA, MDA and MDMA, 25–2500 ng/mL for HHMA, and 150–7500 ng/mL for HHA (r 2  > 0.993 for all analytes). The lower limits of quantification were 12.5 ng/mL urine for MDMA, MDA, HMA and HMMA, 25 ng/mL for HHMA and 150 ng/mL for HHA. Reproducibility was good (intra-assay precision = 1.7–6.1%; inter-assay precision = 0.6–5.7%), as was accuracy (intra-assay deviation = 0.1–4.8%; inter-assay deviation = 0.7–7.9%). Average recoveries were around 85.0%, except for HHMA (66.2%) and HHA (53.0%) (CV < 8.3%). We also checked the stability of stock solutions and the internal standards after freeze-thawing and in the autosampler. Lastly, we measured the MDMA, MDA, HHMA, HHA, HMMA and HMA in urine samples taken over 24 h from rats given subcutaneous MDMA.
Keywords: 3,4-Methylenedioxymethamphetamine; Metabolites; Rat urine; Solid-phase extraction; Liquid chromatography/electrospray ionization/mass spectrometry; Validation;

A rapid, sensitive and simple high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method was developed for determination of cefazedone in human plasma using metronidazole as internal standard (IS). The chromatographic separation was achieved on an Ultimate XB-CN column (2.1 mm × 150 mm, 5 μm) with an isocratic mobile phase of acetonitrile and 20 mM ammonium acetate in 0.1% formic acid in water (15:85, v/v). Detection was performed using electrospray ionization in positive ion multiple reaction-monitoring mode (SRM), monitoring the transitions m/z 548.2 → 344.1 for cefazedone and m/z 172.2 → 128.1 for IS. Calibration curves were linear over a wide range of 0.20–401.12 μg/mL for cefazedone in plasma. The lower limit of quantification (LLOQ) was 0.20 μg/mL. The intra- and inter-day precisions were less than 7.2%. The average recovery of cefazedone was 90.8–91.0%. The validated method was successfully applied to the pharmacokinetic study of cefazedone in Chinese healthy volunteers following intravenous (IV) administration of 500, 1000 and 2000 mg cefazedone injection.
Keywords: Cefazedone; LC–MS/MS; Pharmacokinetics;

Quantification of cyanuric acid residue in human urine using high performance liquid chromatography–tandem mass spectrometry by Parinya Panuwet; Erin L. Wade; Johnny V. Nguyen; M. Angela Montesano; Larry L. Needham; Dana Boyd Barr (2916-2922).
Concern has increased about the resulting health effects of exposure to melamine and its metabolic contaminant, cyanuric acid, after infants in China were fed baby formula milk products contaminated with these compounds. We have developed a selective and sensitive analytical method to quantify the amount of cyanuric acid in human urine. The sample preparation involved extracting free-form cyanuric acid in human urine using anion exchange solid phase extraction. Cyanuric acid was separated from its urinary matrix components on the polymeric strong anion exchange analytical column; the analysis was performed by high performance liquid chromatography–tandem mass spectrometry using negative mode electrospray ionization interface. Quantification was performed using isotope dilution calibration covering the concentration range of 1.00–200 ng/mL. The limit of detection was 0.60 ng/mL and the relative standard deviations were 2.8–10.5% across the calibration range. The relative recovery of cyanuric acid was 100–104%. Our method is suitable to detect urinary concentrations of cyanuric acid caused by either environmental exposures or emerging poisoning events.
Keywords: Cyanuric acid; Human urine; Biomonitoring; Isotope dilution technique; Tandem mass spectrometry; High performance liquid chromatography;

Simple, sensitive and rapid HPLC–MS/MS method for the determination of cepharanthine in human plasma by Guangtao Hao; Haixia Liang; Yuanyuan Li; Haiyan Li; Hongzhi Gao; Guang Liu; Zeyuan Liu (2923-2927).
A rapid, sensitive and specific method for the determination of cepharanthine in human plasma using high performance liquid chromatography coupled with tandem mass spectrometry (HPLC–MS/MS) was described. Cepharanthine and the internal standard (I.S.), telmisartan, were extracted from human plasma by methanol to precipitate the protein. A centrifuged upper layer was then evaporated and reconstituted with 100 μL methanol. Chromatographic separation was performed on an AGILENT XDB-C8 column (150 mm × 2.1 mm, 5.0 μm, Agilent, USA) using a gradient mobile phase with 1 mmol/L ammonium acetate in water with 0.05% formic acid and methanol. Detection and quantitation was performed by MS/MS using electrospray ionization (ESI) and multiple reaction monitoring (MRM) in the positive ion mode. The most intense [M+H]+ MRM transition of cepharanthine at m/z 607.3 → 365.3 was used for quantitation and the transition at m/z 515.5 → 276.4 was used to monitor telmisartan. The calibration curve was linear within the concentration range of 0.5–200.0 ng/mL (r  = 0.9994). The limit of quantification (LOQ) was 0.5 ng/mL. The extraction recovery was above 81.1%. The accuracy was higher than 92.3%. The intra- and inter-day precisions were less than 9.66%. The method was accurate, sensitive and simple and was successfully applied to a pharmacokinetic study after single intravenous administration of 50 mg cepharanthine in 12 healthy Chinese volunteers.
Keywords: Cepharanthine; HPLC–MS/MS;

Development of a high-performance liquid chromatography method to monitor the residues of benzimidazoles in bovine milk by Dongmei Chen; Yanfei Tao; Zhaoying Liu; Zhenli Liu; Lingli Huang; Yulian Wang; Yuanhu Pan; Dapeng Peng; Menghong Dai; Zonghui Yuan (2928-2932).
A reversed-phase high-performance liquid chromatography with ultraviolet (UV) detection was developed that can determine 11 benzimidazole (BZDs) and 10 metabolites of albendazole, fenbendazole and mebendazole in bovine milk. Samples were extracted with acetonitrile and purified by mixed-mode cation exchange (MCX) solid phase extraction cartridges. LC separations were performed on a C18 column with gradient elution using acetonitrile and ammonium acetate solution. The UV-detection was set at 292 nm. The method is very sensitive to each analyte with limits of quantification (LOQs) of lower than 10 μg kg−1. The recoveries of the BZDs and their metabolites spiked in milk were more than 78% with between-day relative standard deviation values less than 16% at the concentration of 10, 50 and 100 μg kg−1. The method developed has been successfully applied to monitoring real samples containing BZDs, which demonstrated that it is a simple, fast and robust method, and could be used as a regulatory toll to determine the residues of BZDs in milk.
Keywords: Benzimidazoles; High-performance liquid chromatography; Residues; Milk;

A novel noncovalently bilayer-coated capillary using cationic polymer polybrene (PB) and anionic polymer (sodium 4-styrenesulfonate) (PSS) as coatings was prepared. This PB–PSS coating showed good migration-time reproducibility for proteins and high stability in the range of pH 2–10 and in the presence of 1 M NaOH, acetonitrile and methanol. Capillary electrophoresis with PB–PSS coated capillaries was successfully applied to quantitatively investigate the stability of bovine serum albumin, ovomucoid, β-lactoglobulin and lysozyme in simulated gastrointestinal fluids. β-lactoglobulin A and β-lactoglobulin B were both stable in simulated gastric fluid with degradation percentages of 34.3% and 17.2% after 60 min of incubation, respectively. Bovine serum albumin, ovomucoid and lysozyme were stable in simulated intestinal fluid with degradation percentages of 17.7%, 23.4% and 22.8% after 60 min of incubation, respectively. The superiority of the proposed method over sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and capillary electrophoresis with untreated fused silica capillaries was demonstrated and emphasized.
Keywords: Capillary electrophoresis; Capillary coating; Allergenic proteins; Simulated gastrointestinal fluids;

Enantiomers of lomefloxacin hydrochloride were separated by high-speed counter-current chromatography (HSCCC) using sulfated-β-cyclodextrin as a chiral selector (CS). The separation was performed with a two-phase solvent system composed of ethyl acetate–methanol–water (10:1:10, v/v) containing CS at 0–60 mmol/l in a head-to-tail elution mode, while obtained fractions were identified by polarimeter and spectropolarimeter. The results show that the concentration of the CS in the system strongly affects the peak resolution (Rs). As the concentration of CS increases, the Rs first increases reaching the maximum at 50 mmol/l and then decreases. When the CS concentration is kept constant in the solvent systems, the Rs decreases as the concentration of the lomefloxacin hydrochloride increases. The overall results of our studies indicated that sulfated-β-cyclodextrin is very useful for the chiral separation by HSCCC.
Keywords: High-speed counter-current chromatography; Enantioseparation; Lomefloxacin hydrochloride; Sulfated-β-cyclodextrin;