Journal of Chromatography B (v.873, #2)

Pepsin extraction from bovine stomach using aqueous two-phase systems: Molecular mechanism and influence of homogenate mass and phase volume ratio by Natalia Imelio; Analía Marini; Darío Spelzini; Guillermo Picó; Beatriz Farruggia (133-138).
Pepsin partitioning, a gastric acid protease, in aqueous two-phase systems of polyethyleneglycol/potassium phosphate, sodium citrate and ammonium sulphate was assayed using polyethylenglycol of different molecular mass. Pepsin was found to be partitioned towards the polymer-rich phase in all the systems, which suggests an important protein–polymer interaction due to the highly hydrophobic character of the protein surface exposed to the solvent. The pepsin partitioning behavior was explained according to Timasheff's preferential interaction theory. The process was driven entropically with participation of structured water around the polyethyleneglycol ethylenic chains. The best pepsin recovery was observed in the systems polyethyleneglycol molecular mass 600. These systems were chosen in order to assay the bovine stomach homogenate partition and to compare different working conditions such as the top-bottom phase volume ratio and homogenate proportions in the total system. The best purification factors were obtained with PEG600/potassium phosphate with low top-bottom volume ratio using 15% of bovine stomach homogenate in the system total mass.
Keywords: Pepsin; Aqueous two-phase system; Partition; Bovine stomach;

Complex formation between protein and poly vinyl sulfonate as a strategy of proteins isolation by Mauricio Braia; María Cecilia Porfiri; Beatriz Farruggia; Guillermo Picó; Diana Romanini (139-143).
The complex formation between the basic protein trypsin and the strong anionic polyelectrolyte poly vinyl sulfonic acid was studied by using turbidimetric and isothermal calorimetric titrations. The trypsin–polymer complex was insoluble at pH lower than 5, with a stoichiometric ratio polymer mol per protein mol of 1:136. NaCl, 0.5 M inhibited the complex precipitation in agreement with the proposed coulombic mechanism of complex formation. The protein structure and its thermodynamic stability were not significantly affected by the presence of the polyelectrolyte. The enzymatic activity of trypsin increases throughout time, even in the presence of the polymer.
Keywords: Trypsin; Poly vinyl sulfonate; Polyelectrolyte;

A novel method for the determination of 1,5-anhydroglucitol, a glycemic marker, in human urine utilizing hydrophilic interaction liquid chromatography/MS3 by Joelle M. Onorato; Robert A. Langish; Petia A. Shipkova; Mark Sanders; Jennifer Wang; Jae Kwagh; Sanjoy Dutta (144-150).
Plasma levels of 1,5-anhydroglucitol (1-deoxyglucose), a short-term marker of glycemic control, have been measured and used clinically in Japan since the early 1990s. Plasma levels of 1,5-anhydroglucitol are typically measured using either a commercially available enzymatic kit or GC/MS. A more sensitive method is needed for the analysis of 1,5-anhydroglucitol in urine, where levels are significantly lower than in plasma. We have developed a sensitive and selective LC/MS3 assay utilizing hydrophilic interaction liquid chromatography and ion trap mass spectrometry for the quantitative determination of 1,5-anhydroglucitol in human urine. Diluted human urine samples were analyzed by LC/MS3 using an APCI source operated in the negative ionization mode. Use of an ion trap allowed monitoring of MS3 transitions for both 1,5-anhydroglucitol and the internal standard which provided sufficient selectivity and sensitivity for analysis from 50 μL of human urine. Quantitation of 1,5-anhydroglucitol levels in urine was accomplished using a calibration curve generated in water (calibration range 50 ng/mL to 10 μg/mL). Method ruggedness and reproducibility were evaluated by determining the intra- and inter-day accuracies and precision of the assay, as well as the bench–top and freeze–thaw stability. For both inter- and intra-assay evaluations, the accuracy of the assay was found to be acceptable, with the concentrations of all QCs tested not deviating more than 8% from theoretical. Four-hour bench–top and freeze–thaw stabilities were also evaluated; 1,5-anhydroglucitol was found to be stable at room temperature (<18% deviation from theoretical) and during 3 freeze–thaw cycles (<1% deviation from theoretical, except at the lowest QC level). The LC/MS3 assay was then used to successfully determine the concentration of 1,5-AG in more than 200 urine samples from diabetic patients enrolled in a clinical study.
Keywords: 1,5-Anhydroglucitol; HILIC; LC/MS3; Ion trap mass spectrometry; Human urine; Diabetes; Glycemia;

Xindi soft capsule is a traditional Chinese medicine preparation which consists of sea buckthorn flavonoids and sea buckthorn berry oil. In this study, a urinary metabonomics method based on the ultra-performance liquid chromatography combined with quadrupole time-of-flight tandem mass spectrometry (UPLC Q-TOF MS) was used to evaluate the efficacy and study the mechanism of traditional Chinese medicine preparation to blood stasis. With pattern recognition analysis (principal component analysis and partial least squares-discriminate analysis) of urinary metabolites, a clear separation of acute blood stasis model group and healthy control group was achieved, the dose groups were located between acute blood stasis model group and healthy control group showing a tendency of recovering to healthy control group, high dose and middle dose were more effective than low dose. Some significantly changed metabolites like cholic acid, phenylalanine and kynurenic acid have been found and identified and used to explain the mechanism. The work shows that the metabonomics method is a valuable tool in the research mechanism of traditional Chinese medicine.
Keywords: Sea buckthorn; Metabonomics; Urine; UPLC Q-TOF MS; Biomarker; Acute blood stasis;

This study presents a high-performance liquid chromatography–positive/negative electrospray ionization tandem mass spectrometric (LC–ESI(+/−)–MS–MS) method for the determination of betamethasone (BOH) and betamethasone 17-monopropionate (B17P) in human plasma using beclomethasone dipropionate as the internal standard (I.S.). Both compounds were extracted from human plasma with ether–cyclohexane (4:1, v/v) and were separated by HPLC on a Hanbon Lichrospher C18 column with a mobile phase of methanol–water (85:15, v/v) at a flow rate of 0.7 ml/min. Calibration curves were linear over the range of 0.10–50 ng/ml for BOH and 0.050–50 ng/ml for B17P. The inter-run relative standard deviations were less than 14.4% for BOH and 12.3% for B17P. The intra-run relative standard deviations were less than 9.3% for BOH and 7.9% for B17P. The mean plasma extraction recovery for BOH and B17P were in the ranges of 82.7–85.9% and 83.6–85.3%, respectively. The method was successfully applied to study the pharmacokinetics of a new formulation of betamethasone phosphate/betamethasone dipropionate injection in healthy Chinese volunteers.
Keywords: Electrospray ionization (+/−) tandem mass spectrometry; Betamethasone; Betamethasone 17-monopropionate; Pharmacokinetics;

Liquid chromatography–tandem mass spectrometry characterization of ergocristam degradation products by Jana Olšovská; Miroslav Šulc; Petr Novák; Sylvie Pažoutová; Miroslav Flieger (165-172).
The UPLC method with diode array UV detection was developed for qualitative determination of ergocristine and ergocristam including degradation products. The mechanism of the ergocristam disruptive reaction was described based on MS/MS characterization of ammonolytic product, N-(d-lysergyl)-l-valinamide (A1) and two methanolytic products, methyl ester of N-(d-lysergyl)-l-valine (M2), and N-[N-(d-lysergyl)-l-valyl]-l-phenylalanyl-d-prolyl methyl ester (M1). The influence of extraction conditions on epimerization and degradation of ergocristine and ergocristam was tested and conditions for reproducible decomposition of ergocristam were found. The presented method could potentially be applied for ergot alkaloids determination in sclerotia, fermentation broth, mycelium, and possibly contaminated food products, i.e. corn, flour, bread, etc., and feeding stuffs containing ungrounded cereals.
Keywords: Ergopeptams; UPLC; MS/MS;

A rapid, specific and sensitive liquid chromatography–tandem mass spectrometry (LC/MS/MS) method was developed for simultaneous quantitation of six Aconitum alkaloids, i.e. aconitine (AC), mesaconitine (MA), hypaconitine (HA), benzoylaconine (BAC), benzoylmesaconine (BMA) and benzoylhypaconine (BHA) in human plasma collected from 18 healthy volunteers after intravenous drop infusion of “SHEN-FU” injectable powder in three different dosages. Lappaconitine was selected as the internal standard (IS). LC/MS/MS system coupled with an electrospray ionization (ESI) source was performed in multiple-reaction monitoring (MRM) mode. The transitions of the Aconitum alkaloids executed as following: m/z 646.3 → 586.0 for AC; m/z 632.4 → 573.1 for MA; m/z 616.2 → 556.1 for HA; m/z 604.2 → 104.8 for BAC; m/z 590.1 → 104.8 for BMA; m/z 574.1 → 104.8 for BHA; m/z 585.2 → 161.8 for IS. Sample preparation was performed with solid-phase extraction (SPE) on a 1 mL HLB cartridge prior to analysis. The separation was applied on a Waters C18 column (1.7 μm, 2.1 mm × 100 mm) and a gradient elution of methanol and 0.1% formic acid–water was used as mobile phase. The retention time was less than 4.5 min. The concentrations ranged from 0.1 to 1000 ng/mL for all six Aconitum alkaloids and showed a good linearity with the correlation coefficient (r 2) >0.995. The validated method was employed to simultaneous quantitation and successfully used for the first time for the pharmacokinetic evaluation of the six Aconitum alkaloids after intravenous drop administration of “SHEN-FU” injectable powder in phase I clinical trial.
Keywords: LC/MS/MS; “SHEN-FU” injectable powder; Aconitum alkaloids; Pharmacokinetics;

Cross validation and ruggedness testing of analytical methods used for the quantification of urinary phthalate metabolites by Manori J. Silva; James L. Preau; Larry L. Needham; Antonia M. Calafat (180-186).
Since the publication of our first analytical method in 2000 to detect and quantify phthalate metabolites in human urine, we have modified the method several times to improve performance, reduce the volume of matrix and solvents used, and to increase the number of analytes in one analytical run. We performed cross method validation and ruggedness testing after each modification to ensure that the analytical method adopted is robust and produces accurate and reproducible data when compared to the previously used method. Here, we present the results from the evaluation of the ruggedness of our analytical approach under variable experimental conditions, using the current analytical method. Minor deviations of the standard experimental conditions, i.e., pH, incubation time, amount of deconjugation enzyme, and incubation temperature, had no effect on final analyte concentrations. Furthermore, we validated the method to ensure accuracy at concentrations beyond the highest calibration standard. The concentrations obtained by using a lower volume of urine agreed well with original levels, suggesting broad linear calibration range as well as complete hydrolysis of the glucuronide conjugates with the standard amount of β-glucuronidase used for deglucuronidation; also, the time of incubation (90 min) was adequate regardless of the amount of glucuronide present. We also summarize the precision of concentration data acquired by the five different analytical approaches we have used since 2000. The correlation plots of concentration data for each analyte obtained from split sample analysis, using three of these approaches, produced linear curves (R 2  > 0.98) with slopes and intercepts that were not statistically different (p  > 0.05) from 1 and 0, respectively. These results suggest that the data are reproducible and accurate, regardless of the analytical method used. Furthermore, analysis of quality control urine samples made over the years confirmed the stability of the phthalate metabolites in urine at −70 °C for several years and the consistency of the analytical measurements obtained by using various methodological approaches over time.
Keywords: Analytical method validation; Ruggedness resting; Phthalate metabolites; Cross method validation; HPLC–MS/MS;

Simultaneous determination of 11 designated hallucinogenic phenethylamines by ultra-fast liquid chromatography with fluorescence detection by Jun Zhe Min; Yoshiha Shimizu; Toshimasa Toyo’oka; Shinsuke Inagaki; Ruri Kikura-Hanajiri; Yukihiro Goda (187-194).
To avoid the spreading of illegal drugs, a designated drug regulation system was introduced along with revision of the Pharmaceutical Affairs Law in Japan in 2006, and 32 substances including phenethylamine-type drugs were listed in April 2007. In this study, a new simultaneous determination method, based on ultra-fast liquid chromatography coupled with fluorescence detection (UFLC–FL), was developed for the 11 designated phenethylamine drugs. The phenethylamines were labeled with 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) at 60 °C for 2 h in 0.1 M borax (pH 9.3). The resulting 11 fluorophores were completely separated by reversed-phase chromatography using an ACQUITY UPLC BEH C18 column (2.1 mm × 100 mm 1.7 μm) and fluorometrically detected at 550 nm (excitation at 450 nm). The calibration curves obtained from the peak areas versus the injection amounts of the phenethylamines showed a good linearity. The limits of detection (signal-to-noise ratio of 3: S/N = 3) on the chromatogram were in the range from 10 fmol (PMMA) to 2.5 pmol (MMDA-2). Good accuracy (%) and precision (CV) by intra-day assay and inter-day assay were also obtained using the present procedure. The method was applied to the qualitative and quantitative analyses of phenethylamine in real products obtained from the Japanese market. As the results, BDB (0.24 mg/mg), MMDA-2 (0.98 mg/mL) and 2C-I (0.016 mg/mg) were identified from the different products (powder, liquid and mushroom like). Because the procedure is simple, selective and sensitive, the present method seems to be useful for the qualitative and quantitative analyses of the designated phenethylamines in various samples including biological specimens.
Keywords: Designated hallucinogenic phenethylamines; Fluorescence labeling; 4-(N,N-Dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole; Ultra-fast liquid chromatography;

MK-0974 (1a), N-[(3R,6S)-6-(2,3-difluorophenyl)-2-oxo-1-(2,2,2-trifuoroethyl)azepan-3-yl]-4-(2-oxo-2,3-dihydro-1H-imidazo-[4,5-B] pyridine-1-yl)piperidine-1-carboxamide, is a novel calcitonin gene-related peptide (CGRP) receptor antagonist with two chiral centers. Direct separation of its four stereoisomers (1ad) was achieved using a cellulose chiral stationary phase, a Chiralcel OJ-RH column (150 mm × 4.6 mm), under reversed-phase condition, following the extraction of 0.2 mL plasma on Oasis μElution HLB 96-well solid-phase-extraction (SPE) plate. The tandem mass spectrometric detection was conducted in the positive-ion mode with a turbo-ion-spray (TIS) interface using multiple-reaction-monitoring on a Sciex API3000. Addition of ammonium trifluoroacetate to low-organic mobile phase improved detection sensitivity by more than 30-fold. The simultaneous quantification of the four stereoisomers in human plasma was validated over the ranges of 0.5–5000 nM for 1a and 0.5–500 nM for its three isomers (1bd). Intraday validation, conducted with five lots of human control plasma, resulted in <12.4% (% coefficient of variation, CV) precision and 96.3–105.4% accuracy for all four stereoisomers. Further evaluation indicated that the assay was specific, the samples were stable after three freeze/thaw cycles, the recovery was reasonable (above 65%) and no matrix effect was observed for all four isomers. Investigation on the chiral integrity of 1a indicated that the diastereomer 1c, inversion at azepinone-3 carbon, was the only isomer observed in the post-dose clinical samples and accounted for 2.4–5.2% of MK-0974 exposure in the circulatory system. The possibility of inversion during blood collection, plasma storage and sample preparation was ruled out, while inversion was observed in the clinical formulation accounting for ∼0.12% of 1a in a 100-mg capsule.
Keywords: Reversed-phase chiral assay; CGRP receptor antagonist; MK-0974 (1a); Diastereomer; Enantiomer; Solid-phase extraction (SPE); LC–MS/MS;

A highly sensitive liquid chromatography-tandem mass spectrometric (LC–MS/MS) method has been developed to determine rasagiline in human plasma. The analytical method utilized liquid–liquid extraction of plasma with n-hexane–dichloromethane–isopropanol (20:10:1, v/v/v). Separation of analyte and the internal standard (IS) pseudoephedrine was performed on a Zorbax Extend C18 column (150 mm × 4.6 mm, 5 μm) with a mobile phase consisting of acetonitrile–5 mM ammonium acetate–acetic acid (40:60:0.05, v/v/v) at a flow rate of 0.5 mL/min. The API 4000 triple quadrupole mass spectrometer was operated in multiple reaction monitoring mode via positive electrospray ionization interface using the transitions m/z 172.1 →  m/z (117.1 + 115.1) for rasagiline, and m/z 166.0 →  m/z 148.1 for the internal standard. The method was linear over the concentration range of 0.020–50.0 ng/mL. The intra- and inter-day precisions were less than 11.2% in terms of relative standard deviation (R.S.D.), and the accuracy was within ±6.4% in terms of relative error (RE). The lower limit of quantification (LLOQ) was identifiable and reproducible at 0.020 ng/mL with acceptable precision and accuracy. The mean extraction-efficiency at three concentrations was 95.6 ± 7.0%, 97.9 ± 3.0% and 95.3 ± 8.3%. The validated method offered increased sensitivity (10 times higher than those reported) and wide linear concentration range. This method was successfully applied for the evaluation of pharmacokinetics of rasagiline after single oral doses of 1, 2 and 5 mg rasagiline to 12 Chinese healthy volunteers.
Keywords: Liquid chromatography–tandem mass spectrometry; Rasagiline; Pharmacokinetics;

The differences among individual bile acids (BAs) in eliciting different physiological and pathological responses are largely unknown because of the lack of valid and simple analytical methods for the quantification of individual BAs and their taurine and glycine conjugates. Therefore, a simple and sensitive LC–MS/MS method for the simultaneous quantification of 6 major BAs, their glycine, and taurine conjugates in mouse liver, bile, plasma, and urine was developed and validated. One-step sample preparation using solid-phase extraction (for bile and urine) or protein precipitation (for plasma and liver) was used to extract BAs. This method is valid and sensitive with a limit of quantification ranging from 10 to 40 ng/ml for the various analytes, has a large dynamic range (2500), and a short run time (20 min). Detailed BA profiles were obtained from mouse liver, plasma, bile, and urine using this method. Muricholic acid (MCA) and cholic acid (CA) taurine conjugates constituted more than 90% of BAs in liver and bile. BA concentrations in liver were about 300-fold higher than in plasma, and about 180-fold higher in bile than in liver. In summary, a reliable and simple LC–MS/MS method to quantify major BAs and their metabolites was developed and applied to quantify BAs in mouse tissues and fluids.
Keywords: HPLC; Mass spectrometry; Bile acids; Mouse; bile; Quantification;