Journal of Chromatography B (v.878, #29)

Qualitative and quantitative determination of major saponins in Paris and Trillium by HPLC-ELSD and HPLC–MS/MS by Shuli Man; Wenyuan Gao; Yanjun Zhang; Jieyin Wang; Wanshun Zhao; Luqi Huang; Changxiao Liu (2943-2948).
High-performance liquid chromatographic (HPLC) with evaporative light scattering detection (ELSD) and HPLC with electrospray ionization multistage tandem mass spectrometry (HPLC–ESI-MS n ) were used to identify and quantify steroid saponins in Paris and Trillium plants. The content of the known saponins such as Paris I, II, III, V, VI, VII, H, gracillin and protodioscin in Paris and Trillium plants was determined simultaneously using the developed HPLC-ELSD method. Furthermore, other 12 steroid saponins were identified by HPLC–ESI(+/−)-MS n detection. In the end, a developed analytical procedure was proved to be a reliable and rapid method for the quality control of Paris and Trillium plants. In addition, the alternative resources for Paris yunnanensis used as a traditional Chinese medicine were discovered according to the hierarchical clustering analysis of the saponin fraction of these plants.
Keywords: HPLC; ELSD; ESI-MS/MS; Steroid saponins; Paris;

Folate deficiency is a prevalent phenomenon worldwide especially in underprivileged countries. Polyglutamyl 5-methyltetrahydrofolate (5MTHF) species are the naturally occurring principle folate in store-bought vegetables. Here we report a simple and complete extraction method for the determination of native polyglutamyl 5-methyltetrahydrofolate in vegetables using high performance liquid chromatography with tandem mass spectrometric detection (HPLC–MS/MS). Coarsely chopped samples (18 different vegetables) were steamed to inactivate glutamylase enzymes and liberate folate from binding proteins and extracted in a reducing buffer with 13C5 5MTHF stable isotope added as internal standard. The polyglutamyl 5-methyltetrahydrofolate species were separated in 9 min on a C18 column using a reversed phase system. HPLC eluate was interfaced with a triple quadrupole mass spectrometer operated in electrospray positive mode. The respective pseudomolecular cation of each polyglutamyl 5-methyltetrahydrofolate species was selected for fragmentation to a common daughter ion for detection. We quantitated polyglutamyl 5-methyltetrahydrofolate in store-bought vegetables from families Brassicaceae, Asteraceae and Amaranthaceae (including mustard greens, romaine lettuce and Swiss chard) of which most have not been quantitated previously. Most vegetables from Asteraceae and those from Amaranthaceae contained similar amounts of monoglutamyl 5MTHF and polyglutamyl 5MTHF while Brassicaceae were dominated by polyglutamyls and endive species (Asteraceae) contained mainly monoglutamyl 5MTHF. The precision of the method for the various polyglutamyl 5-methyltetrahydrofolate forms was 1–9% RSD, recovery 84–91%, limit of detection 64–658 fmol and limit of quantitation 193–1994 fmol. Herein we describe a rapid, sensitive and selective HPLC–MS/MS technique to quantitate polyglutamyl 5-methyltetrahydrofolate species. This method may be suitable for analyzing the polyglutamyl 5-methyltetrahydrofolate profile of inherent folates in a wide range of leafy green vegetables.
Keywords: Polyglutamyl 5-methyltetrahydrofolate; Vegetable analysis; HPLC–MS/MS; γ-Glutamyl hydrolase inactivation;

A hydrophilic interaction liquid chromatography–time-of-flight mass spectrometry (HILIC–TOFMS) method for the quantification and confirmation of morphine (M), codeine (C), morphine-3-glucuronide (M3G), morphine-6-glucuronide (M6G) and codeine-6-glucuronide (C6G) is presented. The method was validated in terms of specificity, selectivity, extraction recovery, accuracy, repeatability, linearity and matrix effect. After a straightforward sample preparation by solid phase extraction (SPE) the compounds were analyzed directly without the need for hydrolysis, solvent transfer, evaporation or reconstitution. The HILIC technique provided good chromatographic separation which was critical for isomers M3G and M6G. The analytes were detected after electrospray ionization (ESI) in positive mode with mass accuracies below 2 mDa using a 5-mDa window. A measurement range of 50–5000 ng/ml was applied for calibration using deuterated analogs as internal standards. The precision of the method was 5.7% and 10.2% (RSD) within and between days, respectively. The applicability of the method was demonstrated with authentic urine samples known to contain codeine and/or morphine and their intact glucuronide conjugates. Identification of the analytes was based on in-source collision induced dissociation (ISCID), applying three diagnostic ions with accurate mass.
Keywords: Glucuronide conjugate; HILIC–TOFMS; Quantification; Confirmation; Morphine; Codeine;

A specific and reliable HPLC-PDA method for the quantitative determination of triamcinolone acetonide, budesonide and fluticasone propionate (as internal standards) in small volumes of microdialysate and rat plasma was developed. An efficient solid-phase extraction (SPE) procedure for plasma samples yielded extremely clean extracts with overall recovery of 104.3% and 95.7% for triamcinolone acetonide (TA) and fluticasone propionate, respectively. Plasma extracts obtained after SPE and microdialysis samples were directly injected on a C18 columm to separation. The method has been validated with good linearity, sensitivity, specificity and high accuracy (RE −5.28% to 9.14%) and precision (CV 0.50% to 6.62%) on both matrices. In stability studies, TA and budesonide were stable during storage and assay procedures. The method was applied to a pharmacokinetic study in rodents using microdialysis to determine protein unbound TA concentrations in blood and muscle.
Keywords: Triamcinolone acetonide; Budesonide; HPLC; Microdialysis; Solid-phase extraction;

A sensitive, selective and quantitative method for the simultaneous determination of casopitant, a potent and selective antagonist of the human Neurokinin 1 (NK-1) receptor, and its three major metabolites M12, M13 and M31 was developed and validated in dog and rat plasma. Acetonitrile containing stable labeled internal standards for the four analytes was used to precipitate proteins in plasma. Chromatographic separation was obtained using a reversed phase column with multiple reaction monitoring turboionspray positive ion detection. The lower and upper limits of quantification for casopitant and its metabolites were 15 and 15,000 ng/mL, using a 50 μL of dog or rat plasma aliquot, respectively. The inter-day precision (relative standard deviation) and accuracy (relative error) in dog plasma, derived from the analysis of validation samples at 5 concentrations, ranged from 4.1% to 10.0% and −10.8% to 8.7%, respectively, for casopitant and its 3 major metabolites. The intra-day precision (relative standard deviation) and accuracy (relative error) in rat plasma, derived from the analysis of validation samples at 5 concentrations, ranged from 3.9% to 6.6% and −9.6% to 8.3%, respectively, for casopitant and its three metabolites. All analytes were found to be stable in analytical solutions for at least 43 days at 4 °C, in dog and rat plasma at room temperature for at least 24 h, at the storage temperature of −20 °C for at least 6 months, and following the action of three freeze–thaw cycles from −20 °C to room temperature. All analytes were also found to be stable in processed extracts at 4 °C for at least 72 h. This assay proved to be accurate, precise, fast and was used to support long-term toxicology studies in dog and rat.
Keywords: Casopitant; LC–MS/MS; Protein precipitation; Validation;

Gas chromatography coupled with time-of-flight mass spectrometry and principal component analysis were used to obtain the metabolite profiles of guava (Psidium guajava) fruits. Results with two types of data-processing software, ChromaTOF and AMDIS, were compared to explain the differences between the samples. There were some differences in score and loading plot patterns of PCA as well as in the composition of the metabolites. However, little difference was observed in the type of metabolites detected and identified using either type of software. Both the flesh and peel of premature and mature white guava fruits were compared for the analysis of the metabolite profiles. Malic acid, aspartic acid, and glucose were the major metabolites distinguishing the different parts of guava fruits in the PCA loading plot. In addition, the metabolic profiles of the fruits revealed significant changes in some metabolites during ripening. The major components contributing to the separation were serine, citric acid, fructose, sucrose, and some unknowns. In particular, sucrose, fructose, serine and citric acid were related to the ripening of guava fruits. Fructose and sucrose were increased whereas citric acid was decreased during guava fruit ripening.
Keywords: Guava fruit; Ripening; Flesh/peel; Gas chromatography time-of-flight mass spectrometry (GC-TOF/MS); Principal component analysis (PCA); Data processing;

Simultaneous determination of m-nisoldipine and its three metabolites in rat plasma by liquid chromatography–mass spectrometry by Dezhi Kong; Sanni Li; Xiaowei Zhang; Jianmin Gu; Man Liu; Yan Meng; Yan Fu; Xiaojin La; Gangqiang Xue; Lantong Zhang; Qiao Wang (2989-2996).
A simple, sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the simultaneous determination of m-nisoldipine and its three metabolites in rat plasma has been developed using nitrendipine as an internal standard (IS). Following liquid–liquid extraction, the analytes were separated using an isocratic mobile phase on a reverse phase C18 column and analyzed by MS in the multiple reaction monitoring (MRM) mode. To avoid contamination by residual sample in the injection syringe, a special injection protocol was developed. We found that m-nisoldipine, metabolite M1 and IS could be ionized under positive or negative electrospray ionization conditions, whereas metabolite M and M2 could only be ionized in the positive mode. The mass spectrometry fragmentation pathways for these analytes are analyzed and discussed herein. The total analysis time required less than 5 min per sample. We employed this method successfully to study the metabolism of m-nisoldipine when it was orally administered to rats at a dose of 9 mg/kg. Three metabolites of m-nisoldipine and an unknown compound of molecular weight 386 were found for the first time in rat plasma. The concentration of the potentially active metabolite was approximately equal to its parent compound concentration.
Keywords: m-Nisoldipine; Metabolites; Fragmentation pathways; Liquid chromatography–tandem mass spectrometry;

Metabolomic search for uremic toxins as indicators of the effect of an oral sorbent AST-120 by liquid chromatography/tandem mass spectrometry by Kaori Kikuchi; Yoshiharu Itoh; Ryoko Tateoka; Atsuko Ezawa; Kenjiro Murakami; Toshimitsu Niwa (2997-3002).
An oral sorbent AST-120 composed of spherical porous carbon particles has superior adsorption ability for certain small-molecular-weight organic compounds known to accumulate in patients with chronic renal failure (CRF). A metabolomic approach was applied to search for uremic toxins as possible indicators of the effect of AST-120. Serum metabolites in normal and CRF rats before and after administration of AST-120 for 3 days were analyzed by liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) and principal component analysis. Further, serum and urine levels of the indicators were quantified by selected reaction monitoring of LC/ESI-MS/MS. Indoxyl sulfate was the first principal serum metabolite, which could differentiate CRF from both normal and AST-120-administered CRF rats, followed by hippuric acid, phenyl sulfate and 4-ethylphenyl sulfate. CRF rats showed increased serum levels of indoxyl sulfate, hippuric acid, phenyl sulfate, 4-ethylphenyl sulfate and p-cresyl sulfate. Administration of AST-120 for 3 days to the CRF rats reduced the serum and urine levels of these metabolites. In conclusion, indoxyl sulfate is the best indicator of the effect of AST-120 in CRF rats. Hippuric acid, phenyl sulfate and 4-ethylphenyl sulfate are suggested as the additional indicators. 4-Ethylphenyl sulfate is a newly identified uremic substance.
Keywords: Oral sorbent AST-120; Metabolome; Indoxyl sulfate; Uremic toxin; Chronic renal failure; Mass spectrometry;

A simple and fast HPLC method based on an isocratic, reversed-phased ion-pair with amperometric end-point detection for simultaneous measurement of noradrenergic (MHPG/NA and A), dopaminergic (DOPAC, HVA/DA) and serotonergic (5-HIAA/5-HT) compounds in mouse brain tissue was developed. In order to improve the chromatographic resolution (Rs) with an acceptable total analysis time, experimental designs for multivariate optimization of the experimental conditions were applied. The optimal conditions for the separation of the eight neurotransmitters and metabolites, as well as two internal standards, i.e., DHBA and 5-HMT, were obtained using a mixture of methanol–phosphate–citric buffer (pH 3.2, 50 mM) (9:91, v/v) containing 2 mM OSA as mobile phase at 32 °C on a microbore ALF-115 column (150 mm × 1.0 mm, 3 μm particle size) filled with porous C18 silica stationary phase. In this study, a two-level fractional factorial experimental design (½ 2K) was employed to optimize the separation and capacity factor (k′) of each molecule, leading to a good separation of all biogenic amines and their metabolites in brain tissue. A simple method for the preparation of different bio-analytical samples in phosphate–citric buffer was also developed. Results show that all molecules of interest were stabilized for at least 24 h in the matrix conditions without any antioxidants. The method was fully validated according to the requirements of SFSTP (Société Française des Sciences et Techniques Pharmaceutiques). The acceptance limits were set at ±15% of the nominal concentration. The method was found accurate over a concentration range of 4–2000 ng/ml for MHPG, 1–450 ng/ml for NA, 1–700 ng/ml for A, 1–300 ng/ml for DOPAC, 1–300 ng/ml for 5-HIAA, 1–700 ng/ml for DA, 4–2800 ng/ml for HVA and 1–350 ng/ml for 5-HT. The assay limits of detection for MHPG, NA, A, DOPAC, 5-HIAA, DA, HVA and 5-HT were 2.6, 2.8, 4.1, 0.7, 0.6, 0.8, 4.2 and 1.4 pg, respectively. It was found that the mean inter- and intra-assay relative standard deviations (RSDs) over the range of standard curve were less than 3%, the absolute and the relative recoveries were around 100%, demonstrating the high precision and accuracy, and reliability of the analytical method described to apply in routine analysis of biogenic amines and their metabolites in brain tissue.
Keywords: Biogenic amines and metabolites; Mouse-brain tissue; Sample preparation; Experimental designs; Optimization of HPLC method; Validation;

A plasma global metabolic profiling approach applied to an exercise study monitoring the effects of glucose, galactose and fructose drinks during post-exercise recovery by Stephen J. Bruce; Isabelle Breton; Jacques Decombaz; Chris Boesch; Eva Scheurer; Ivan Montoliu; Serge Rezzi; Sunil Kochhar; Philippe A. Guy (3015-3023).
A global metabolic profiling methodology based on gas chromatography coupled to time-of-flight mass spectrometry (GC–TOFMS) for human plasma was applied to a human exercise study focused on the effects of beverages containing glucose, galactose, or fructose taken after exercise and throughout a recovery period of 6 h and 45 min. One group of 10 well trained male cyclists performed 3 experimental sessions on separate days (randomized, single center). After performing a standardized depletion protocol on a bicycle, subjects consumed one of three different beverages: maltodextrin (MD) + glucose (2:1 ratio), MD + galactose (2:1), and MD + fructose (2:1), consumed at an average of ∼1.25 g of carbohydrate (CHO) ingested per minute. Blood was taken straight after exercise and every 45 min within the recovery phase. With the resulting blood plasma, insulin, free fatty acid (FFA) profile, glucose, and GC–TOFMS global metabolic profiling measurements were performed. The resulting profiling data was able to match the results obtained from the other clinical measurements with the addition of being able to follow many different metabolites throughout the recovery period. The data quality was assessed, with all the labelled internal standards yielding values of <15% CV for all samples (n  = 335), apart from the labelled sucrose which gave a value of 15.19%. Differences between recovery treatments including the appearance of galactonic acid from the galactose based beverage were also highlighted.
Keywords: GC-TOF; Metabolomics; Post-exercise recovery; Galactonic acid; Glucose; Galactose; Fructose; Human nutrition; Sports drink;

Microwave irradiation for a fast gas chromatography–mass spectrometric analysis of polysaccharide-based plasma volume expanders in human urine by Monica Mazzarino; Francesca De Angelis; Teresa Di Cicco; Xavier de la Torre; Francesco Botrè (3024-3032).
In this contribution we tested the possibility to use microwave irradiation for the screening and confirmation pre-treatment steps of hydroxyethylstarch, with the aim to speed up gas chromatography–mass spectrometric procedures. Acid hydrolysis and derivatization processes were conducted in a temperature-controlled single beam microwave oven for organic synthesis. The kinetics of hydroxyethylstarch chemical hydrolysis and derivatization were investigated at different microwave power, incubation temperature and incubation time. The best hydrolysis conditions were found at a microwave power value of 1200 W (T 100 °C) with an incubation time of 2 min; whereas the best derivatization conditions were found at a microwave power value of 1020 W (T 100 °C) with an incubation time of 5 min. The effectiveness of this approach was evaluated by gas chromatography–mass spectrometry analyzing more than 20 different pools of blank urine samples spiked with hydroxyethylstarch at a concentration of 1 mg/mL. The results showed that the effect of microwave irradiation on the chemical hydrolysis process was very remarkable: the total sample preparation time can be shortened by 58 min compared to the reference method (2 min instead of 60 min). In addition to this, the time necessary for the derivatization process can also be drastically shortened with respect to the reference procedure (5 min instead of 30 min). The repeatability of the hydrolysis and derivatization recoveries, the limit of detection and the matrix interferences were comparable to the reference method accredited under the ISO 17025 guidelines and presently followed by the accredited sports anti-doping laboratory of Rome.
Keywords: Anti-doping analysis; Acid hydrolysis; Microwave irradiation; Plasma volume expander;

Quantitation of sorafenib and its active metabolite sorafenib N-oxide in human plasma by liquid chromatography–tandem mass spectrometry by Lie Li; Ming Zhao; Fariba Navid; Keith Pratz; B. Doug Smith; Michelle A. Rudek; Sharyn D. Baker (3033-3038).
A simple and rapid method with high performance liquid chromatography/tandem mass spectrometry is described for the quantitation of the kinase inhibitor sorafenib and its active metabolite sorafenib N-oxide in human plasma. A protein precipitation extraction procedure was applied to 50 μL of plasma. Chromatographic separation of the two analytes, and the internal standard [2H3 13C]-sorafenib, was achieved on a C18 analytical column and isocratic flow at 0.3 mL/min for 4 min. Mean within-run and between-run precision for all analytes were <6.9% and accuracy was <5.3%. Calibration curves were linear over the concentration range of 50–10,000 ng/mL for sorafenib and 10–2500 ng/mL for sorafenib N-oxide. This method allows a specific, sensitive, and reliable determination of the kinase inhibitor sorafenib and its active metabolite sorafenib N-oxide in human plasma in a single analytical run.
Keywords: Sorafenib N-oxide; HPLC; Tandem mass spectrometry;

Purification of optical imaging ligand-Cybesin by high-speed counter-current chromatography by Zhiyong Ma; Ying Ma; Xilin Sun; Yunpeng Ye; Baozhong Shen; Xiaoyuan Chen; Yoichiro Ito (3039-3043).
Fluorescent Cybesin (Cypate-Bombesin Peptide Analogue Conjugate) was synthesized from Indocyanine Green (ICG) and the bombesin receptor ligand as a contrast agent for detecting pancreas tumors. However, the LC–MS analysis indicated that the target compound was only a minor component in the reaction mixture. Since preparative HPLC can hardly separate such a small amount of the target compound directly from the original crude reaction mixture without a considerable adsorptive loss onto the solid support, high-speed counter-current chromatography (HSCCC) was used for purification since the method uses no solid support and promises high sample recovery. A suitable two-phase solvent system composed of hexane/ethyl acetate/methanol/methyl t-butyl ether/acetonitrile/water) at a volume ratio of 1:1:1:4:4:7 was selected based on the partition coefficient of Cybesin (K  ≈ 0.9) determined by LC–MS. The separation was performed in two steps using the same solvent system with lower aqueous mobile phase. From 400 mg of the crude reaction mixture the first separation yielded 7.7 mg of fractions containing the target compound at 12.8% purity, and in the second run 1 mg of Cybesin was obtained at purity of 94.0% with a sample recovery rate of over 95% based on the LC–MS analysis.
Keywords: High-speed counter-current chromatography; Spiral tube assembly; Isolation and purification; Cybesin; Cypate-Bombesin Peptide Analogue Conjugate; Pancreas tumor;