Journal of Chromatography B (v.879, #11-12)

Tyrosinemia is an inborn error of metabolism characterized by the accumulation of tyrosine as well as toxic by-products. NTBC or nitisinone is a drug currently used for the treatment of tyrosinemia that avoids the formation of these toxic substances. This paper presents the determination of NTBC in plasma and dry blood spots by high-performance liquid chromatography (HPLC) coupled to tandem mass spectrometry. The concentration of NTBC in matched plasma-dry blood spots was compared and the study of degradation of NTBC in plasma and dry spots at different temperatures is presented. Method: For sample preparation, plasma proteins were precipitated with acetonitrile and 3-mm discs were extracted with methanol. ESI(+) was used as inozation method and the analytes were detected by multiple reaction monitoring using the transitions 330 > 218 for NTBC and 340 > 228 for mesotrione, used as internal standard. Results: There is good correlation between concentrations obtained in dry blood spots and plasma (r 2  = 0.83), although values are 2.4 times higher in plasma samples. NTBC in plasma is stable at least for 45 days frozen at −30 °C and refrigerated at 4 °C. However, it shows slow decomposition at room temperature, approximately 30% after 45 days. The method shows good precision, accuracy and linearity and the detection limit is 50 nmol/L and paper samples are appropriate for the monitorization of NTBC.
Keywords: Tyrosinemia; Tandem mass spectrometry; Nitisinone; Plasma; Dry blood spots; Stability;

Determination of endocannabinoids in nematodes and human brain tissue by liquid chromatography electrospray ionization tandem mass spectrometry by Marko Lehtonen; Markus Storvik; Hanna Malinen; Petri Hyytiä; Merja Lakso; Seppo Auriola; Garry Wong; James C. Callaway (677-694).
A simple and highly sensitive liquid chromatography/tandem mass spectrometric (LC/MS/MS) method was developed to compare endogenous cannabinoid levels in nematodes and in brains of rats and humans, with and without prior exposure to ethanol. After liquid–liquid extraction of the lipid fraction from homogenized samples, a reversed-phase sub 2 μm column was used for separating analytes with an isocratic mobile phase. Deuterated internal standards were used in the analysis, and detection was made by triple quadrupole mass spectrometer with multiple reaction monitoring (MRM). Ionization was performed with positive electrospray ionization (ESI). The nematode Caenorhabditis elegans fat-3 mutant, that lacks the necessary enzyme to produce arachidonic acid, the biologic precursor to 2-arachidonoyl glycerol and anandamide, was used as an analyte-free surrogate material for selectivity and calibration studies. The matrix effect was further investigated by in-source multiple reaction monitoring (IS-MRM) and standard addition studies. Selectivity studies demonstrated that the method was free from matrix effects. Good accuracy and precision were obtained for concentrations within the calibration range of 0.4–70 nM and 40–11,000 nM for monitored N-acylethanolamides (NAEs) and acyl glycerols, respectively.
Keywords: Anandamide; 2-Arachidonoyl glycerol; Endocannabinoids; Brain; Tissue samples; LC–MS/MS;

A sensitive, precise and accurate quantitative liquid chromatography/tandem mass spectrometry (LC–MS/MS) method for the measurement of sunitinib (SU11248) and N-desethyl sunitinib (SU12662) in human plasma was developed and validated. All sample handling was done under strict light protection. The sample preparation method employed acetonitrile protein precipitation using d 5-SU11248 as an internal standard. The processed samples were chromatographed on a polymeric reversed-phase analytical column and analyzed by triple-quadrupole MS/MS in multiple reaction monitoring (MRM) mode using positive TurboIonSpray® (TISP). The LC–MS/MS method described in this paper presents high absolute recovery (86.2% SU11248, 84.8% SU12662), high sensitivity (lower limit of quantitation of 0.06 ng/mL for both analytes), high inter-day precision (1.6–6.1% SU11248, 1.1–5.3% SU12662) and high analytical recovery (99.8–109.1% SU11248, 99.9–106.2% SU12662), as well as excellent linearity over the concentration range 0.060–100 ng/mL (r 2  > 0.999) with a short runtime of only 4.0 min. Results on the stability of SU11248 and SU12662 in human plasma are presented. During validation plasma from intensive care patients receiving many drugs were tested for interference and incurred samples were analyzed. The method met all criteria of the EMA and FDA guidelines during validation and was successfully applied to a pharmacokinetic study in healthy human volunteers.
Keywords: Sunitinib; N-desethyl sunitinib; Tyrosine kinase inhibitors; LC–MS/MS analysis; Tandem mass spectrometry;

LC–MS/MS characterization of the urinary excretion profile of the mycotoxin deoxynivalenol in human and rat by Veronica M.T. Lattanzio; Michele Solfrizzo; Annalisa De Girolamo; Sofía N. Chulze; Adriana M. Torres; Angelo Visconti (707-715).
The understanding of mycotoxins transfer to biological fluids is challenged by the difficulties in performing and replicating in vivo experiments as well as the lack of suitable methods of analysis to detect simultaneously a range of chemically different metabolites at trace levels. LC–MS/MS has been used herein to study the urinary excretion profile of the mycotoxin deoxynivalenol in human and Wistar rat. Deoxynivalenol and deoxynivalenol glucuronide were found in both human and rat urines, whereas de-epoxydeoxynivalenol and its glucuronide conjugate were only detected in rat urine. The presence of two deoxynivalenol glucuronide isomers in Wistar rat urine has been shown for the first time. Structure confirmation of the detected metabolites was provided by the analysis of fragmentation patterns. A solid phase extraction clean up procedure allowing recoveries in the range 72–102% for deoxynivalenol, de-epoxydeoxynivalenol, and their glucuronide conjugates was optimized. A multiple reaction monitoring method for the simultaneous determination of all investigated metabolites was elaborated allowing the direct detection of deoxynivalenol metabolites without the hydrolysis step. Deoxynivalenol urinary levels in the range 0.003–0.008 μg/ml were detected in healthy human subjects, whereas deoxynivalenol and de-epoxynivalenol levels between 1.9–4.9 μg/ml and 1.6–5.9 μg/ml, respectively were found in administered rat urine. These findings emphasize the relevance of the highly selective and sensitive LC–MS/MS technique for the direct detection and characterization of deoxynivalenol metabolites in complex biological matrices.
Keywords: Deoxynivalenol; De-epoxydeoxynivalenol; Glucuronide; Human urine; Rat urine; LC–MS/MS;

Development of a high-performance liquid chromatography method for the simultaneous quantification of four organoarsenic compounds in the feeds of swine and chicken by Dongmei Chen; Huahai Zhang; Yanfei Tao; Yulian Wang; Lingli Huang; Zhenli Liu; Yuanhu Pan; Dapeng Peng; Xu Wang; Menghong Dai; Zonghui Yuan (716-720).
A high-performance liquid chromatography (HPLC) method with UV detection was developed for the simultaneous determination of arsanilic acid, roxarsone, nitarsone, and carbarsone in the feeds of swine and chicken. Feed samples were extracted with methanol/1% acetic acid (90:10, v/v) in an ultrasonic bath and the protein was precipitated with 2% Cu2SO4. The samples were further purified by solid phase extraction (SPE) on SAX cartridges. Separation was performed on a Zorbax SB-Aq C18 HPLC column using an isocratic procedure with methanol and 1% acetic acid (3:97, v/v) at a flow-rate of 0.7 mL min−1, and the UV detector was set at a wavelength of 260 nm. The recoveries of organoarsenic compounds spiked at levels of 2, 20 and 200 μg g−1 ranged from 81.2% to 91.3%; the inter-day relative standard deviation values were less than 7.0%. The limits of quantification for four organoarsenic compounds were 1.0–2.0 μg g−1. This simple and fast method could be applied to the determination of multi-residues of organic arsenic compounds in animal feeds.
Keywords: Arsanilic acid; Roxarsone; Nitarsone; Carbarsone; Animal feeds; Ultrasonic solvent extraction; HPLC; UV detection;

Partitioning of α-lactalbumin and β-lactoglobulin in aqueous two-phase systems of polyvinylpyrrolidone and potassium phosphate by Babak Mokhtarani; Hamid Reza Mortaheb; Morteza Mafi; Mohammad Hassan Amini (721-726).
In the present study, the partitioning of α-lactalbumin, β-lactoglobulin, and cheese whey proteins in aqueous two-phase system of polyvinylpyrrolidone–potassium phosphate is investigated. The partitioning of proteins in this system depends on the polymer and salt weight percents in feed, temperature, and pH. The orthogonal central composite design is used to study the effects of different parameters on partitioning of α-lactalbumin and β-lactoglobulin. A second order model is proposed to determine the impact of these parameters. The results of the model show that the weight percent of the salt in feed has a large effect on the protein partitioning. The weight percent of polyvinylpyrrolidone in the feed increases the partitioning coefficients. By increasing the temperature, the viscosity of polyvinylpyrrolidone is reduced and the protein can easily be transferred from one phase to the other phase. The pH of the aqueous two phase system can alter the protein partitioning coefficient through the variation of the protein net charge.
Keywords: Aqueous two phase system; Partitioning; Polyvinylpyrrolidone; Whey; α-Lactalbumin; β-Lactoglobulin;

A liquid-chromatography–tandem-mass-spectrometry method using pneumatically assisted electrospray ionisation (LC–ESI-MS/MS) was developed for the simultaneous determination of cathinone, methcathinone, ethcathinone, amfepramone, mephedrone, flephedrone, methedrone, methylone, butylone, cathine, norephedrine, ephedrine, pseudoephedrine, methylephedrine and methylpseudoephedrine in human live and post-mortem whole blood. The blood proteins were precipitated by the addition of methanol, and the extract was purified by ultrafiltration. The separation of diastereomeric ephedrines was achieved on an ethyl-linked phenyl column. Matrix-matched calibrants combined with the isotope dilution of selected substances were used for quantitative analysis. The relative intra-laboratory reproducibility standard deviations were generally better than 7% at concentrations of 20 μg/L, and the mean true recoveries were 87–106% in the concentration range of 10–250 μg/L. The detection limits were in the range of 0.5–3 μg/L. The cathinones were unstable in whole blood and sample extracts under neutral conditions, but the stability could be improved by the acidification of the sample matrix.
Keywords: Cathinones; Ephedrines; Khat; Whole blood; LC–MS/MS;

An effective way to determine the amount of different neurotransmitters is vital to the study of brain function. Here, a highly sensitive HPLC–MS/MS method was developed to simultaneously measure γ-aminobutyric acid, dopamine, epinephrine, norepinepherine, glutamate and serotonin in one sample. The quantification of the neurotransmitters was achieved by a tandem mass spectrometer using the selected reaction monitoring scan mode. The method validation included selectivity, linearity, accuracy, precision, stability, recovery and matrix effect. For the six neurotransmitters, the linear regression analysis was calibrated by deuterated internal standards with a R 2 of over 0.991, and the limit of detection (LOD) and the limit of quantification (LOQ) were from 2.5 to 500 pg/mg and 7.5 to 1000 pg/mg, respectively. This method was employed here to reveal different types and amounts of neurotransmitters simultaneously in adult and embryonic rat brains. Here, the change of dopamine concentration in embryonic and adult brain was from 0.071 to 0.760 ng/mg of brain tissue, GABA was from 207.643 to 445.148 ng/mg, glutamate was from 679.535 to 1408.920 ng/mg, serotonin was from 0.058 to 0.485 ng/mg and norepinepherine was from 0.054 to 0.290 ng/mg. For epinephrine, it was only detected in embryonic stage but not in adult, with the concentration at 0.241 ng/mg.
Keywords: Neurotransmitters analysis; LC–MS; Rat brain;

Quantification of dimethyl-ifosfamide and its N-deschloropropylated metabolites in mouse plasma by liquid chromatography–tandem mass spectrometry by Alain Deroussent; Stéphanie Rodriguez; Sonia Martelli; Atmane Seck; Estelle Dubus-Daudigeos; Didier Desmaële; Gilles Vassal; Angelo Paci (743-750).
Among antitumor oxazaphosphorine drugs, the prodrug ifosfamide (IFO) and its analogs require metabolic activation by specific liver cytochrome P450 (CYP) enzymes to become therapeutically active. New 7,9-dimethyl-ifosfamide analogs have shown greater cytotoxic activity than IFO, whereas side-chain oxidation still occurred leading to monochloroacetone after N-dechloropropylation. A sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay was developed and validated for the simultaneous quantitation of the prodrug 7S,9S-dimethyl-ifosfamide (diMeIFO) and its two inactive metabolites, N 2- and N 3-deschloropropyl-dimethylifosfamide (N 2-DCP-diMeIFO and N 3-DCP-diMeIFO) in mouse plasma. After protein precipitation with methanol, the analytes were separated by isocratic reversed-phase chromatography with (methanol/ammonium formate pH 5.5, 60:40, v/v) and detected by tandem mass spectrometry using multiple reaction monitoring of transitions ions m/z 289 → 168 for diMeIFO, m/z 213 → 168 for N 2-DCP-diMeIFO, m/z 213 → 92 for N 3-DCP-diMeIFO and m/z 261 → 154 for IFO (internal standard). The calibration curves were linear over the concentration range of 20–10,000 ng/mL for the three analytes. Mean extraction recoveries from mouse plasma were 99, 96, 99 and 100% for diMeIFO, N 2-DCP-diMeIFO, N 3-DCP-diMeIFO and IFO, respectively. The lower limit of quantitation for diMeIFO and its metabolites was 20 ng/mL in 50 μL plasma. The method was accurate with calculated bias from −5.8 to 4.0% for diMeIFO, from −1.1 to 10.6% for N 2-DCP-diMeIFO and from −6.9 to 9.8% for N 3-DCP-diMeIFO, and precise with coefficients of variation lower than 6.8%, 7.8% and 14.3%, respectively. The assay was successfully applied to a preliminary pharmacokinetic study of diMeIFO and of its metabolites in mice.
Keywords: Dimethyl-ifosfamide; Alkylating agents; Liquid chromatography; Tandem mass spectrometry; Mouse plasma; Pharmacokinetics;

A simple method has been developed for the simultaneous determination of lathosterol and cholesterol by high-performance liquid chromatography with electrochemical detection (HPLC–ECD). Lathosterol was found to be electrochemically oxidized and its current peak height was linearly related to the amount of lathosterol injected, ranging from 0.15 μmol/L to 300 μmol/L (r  = 0.995). Similar results were obtained with cholesterol from 15 μmol/L to 600 μmol/L (r  = 0.995). The separation was carried out with an ODS column, acetonitrile containing 30 mmol/L lithium perchlorate as a mobile phase, and an applied potential at +2.8 V vs. Ag/AgCl. The detection limit (S/N  = 3) of lathosterol as well as cholesterol was 0.03 μmol/L (0.15 pmol). Total lathosterol in control human and rat serum was determined by the present method with a recovery of more than 95.8% and an RSD (n  = 5) of less than 7.3%. The present method was applied to an experiment with rats to examine the effect of lathosterol feeding. There were no significant changes in serum lathosterol or cholesterol levels in rats fed with a high-lathosterol diet for six days. Therefore, we found this method to be both simple and useful for the simultaneous determination of lathosterol and cholesterol in serum.
Keywords: Lathosterol; Cholesterol; HPLC; Electrochemical detection;

Immobilization of matrix metalloproteinase 8 (MMP-8) for online drug screening by Francesco Mazzini; Elisa Nuti; Antonella Petri; Armando Rossello (756-762).
Matrix metalloproteinase 8 (MMP-8) has been reported to have a key role in several pathologic conditions, like heart diseases, osteoarthritis, multiple sclerosis, and various other inflammatory conditions. Therefore, there is a great interest regarding the development of MMP-8 selective inhibitors. In the recent years, immobilized enzyme reactors (IMERs) proved to be an efficient alternative to solution-based assays. Besides the recycling of the enzyme, IMER approach allows a simple way to determine affinity data and thus the ranking of inhibiting potency of the compounds under study, especially when coupled to MS. In this study, the immobilization of MMP-8 was investigated in terms of type of support, kinetic parameters, storage and pH stability. Epoxy activated silica resulted the best matrix for the preparation of an immobilized enzyme reactor (IMER) containing human MMP-8. The IMER was successfully used for the online screening of known MMP-8 inhibitors in zonal chromatography and inhibition experiments.
Keywords: Enzyme immobilization; Inhibitor; Metalloprotein; MMP-8; Online drug screening;

Higenamine is an active ingredient of Aconite root in Chinese herbal medicine and might be used as a new agent for a pharmaceutical stress test and was approved to undergo clinical pharmacokinetic study. Therefore, there exists a need to establish a sensitive and rapid method for the determination of higenamine in human plasma and urine. This paper described a sensitive and rapid method based on liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) for the determination of higenamine in human plasma and urine. Solid-phase extraction (SPE) was used to isolate the compounds from biological matrices followed by injection of the extracts onto an Atlantis dC18 column with isocratic elution. The mobile phase was 0.05% formic acid in water–methanol (40:60, v/v). The mass spectrometry was carried out using positive electrospray ionization (ESI) and data acquisition was carried out in the multiple reaction monitoring (MRM) mode. The method was fully validated over the concentration range of 0.100–50.0 ng/mL and 1.00–500 ng/mL in plasma and urine, respectively. The lower limits of quantification (LLOQs) were 0.100 and 1.00 ng/mL in plasma and urine, respectively. Inter- and intra-batch precision was less than 15% and the accuracy was within 85–115% for both plasma and urine. Extraction recovery was 82.1% and 56.6% in plasma and urine, respectively. Selectivity, matrix effects and stability were also validated in human plasma and urine. The method was applied to the pharmacokinetic study of higenamine hydrochloride in Chinese healthy subjects.
Keywords: Determination; Higenamine; LC–MS/MS; Human plasma and urine;

LC–ESI-MS/MS method for the quantification of entecavir in human plasma and its application to bioequivalence study by Balasekhara Reddy Challa; Bahlul Z. Awen; Babu Rao Chandu; Shaik Rihanaparveen (769-776).
Liquid chromatography–electrospray ionization-tandem mass spectrometry (LC–ESI-MS/MS) was used for a quantitative estimation of entecavir (EV) in human plasma using lamivudine (LM) as internal standard (IS). The method herein described is simple, sensitive, and specific. Chromatographic separation was performed on XBridge-C18, 4.6 mm × 50 mm, 5-μm column with an isocratic mobile phase composed of 10 mM ammonium hydrogen carbonate (pH 10.5):methanol (85:15 v/v), pumped at 0.3 ml/min. EV and LM were detected using proton adducts at m/z 278.1 → 152.1 and 230.2 → 112.0 in multiple reaction monitoring (MRM) positive mode. Solid phase extraction method was employed in the extraction of EV and LM from the biological matrix. This method was validated over a linear concentration range of 50.0–20000.0 pg/ml with a correlation coefficient (r) ≥0.9983. Intra and inter-day precision of EV was found within the range of 1.2–4.2 for EV and 4.4–4.5 for LM. EV was stable throughout three freeze/thaw cycles, bench top and postoperative studies. This method was successfully used in the analysis of plasma samples following oral administration of EV (0.5 mg) in 26 healthy human volunteers.
Keywords: Liquid chromatography; Mass spectrometry; Bioequivalence; Pharmacokinetics; Entecavir;

In this paper, a novel method is described for automated determination of dextromethorphan in biological fluids using molecularly imprinted solid-phase extraction (MISPE) as a sample clean-up technique combined with high performance liquid chromatography (HPLC). The water-compatible molecularly imprinted polymers (MIPs) were prepared using methacrylic acid as functional monomer, ethylene glycol dimethacrylate as cross-linker, chloroform as porogen and dextromethorphan as template molecule. These imprinted polymers were used as solid-phase extraction sorbent for the extraction of dextromethorphan from human plasma samples. Various parameters affecting the extraction efficiency of the MIP cartridges were evaluated. The high selectivity of the sorbent coupled to the high performance liquid chromatographic system permitted a simple and rapid analysis of this drug in plasma samples with limits of detection (LOD) and quantification (LOQ) of 0.12 ng/mL and 0.35 ng/mL, respectively. The MIP selectivity was evaluated by analyzing of the dextromethorphan in presence of several substances with similar molecular structures and properties. Results from the HPLC analyses showed that the recoveries of dextromethorphan using MIP cartridges from human plasma samples in the range of 1–50 ng/mL were higher than 87%.
Keywords: Molecularly imprinted polymer; Dextromethorphan; Human plasma; Automated; Sample clean-up;

In humans, concomitant dl-methylphenidate (dl-MPH) and ethanol results in the carboxylesterase 1 (hCES1) mediated biotransformation of MPH to the transesterification metabolite dl-ethylphenidate (dl-EPH). The separate enantiomers of MPH and EPH are found at low ng/ml to pg/ml plasma concentrations. Substantial pharmacological differences exist between d- and l-isomers of MPH and EPH, both in terms of pharmacological potencies and receptor selectivity, as well as in pharmacokinetic properties. Accordingly, a sensitive, accurate and precise enantiospecific analytical method is required in order to fully explore pharmacokinetic–pharmacodynamic correlations regarding the MPH–ethanol interaction. The present study describes a novel liquid chromatographic-tandem mass spectrometric method for simultaneous analysis of d- and l-MPH as well as d- and l-EPH concentrations from human plasma. This assay provides baseline resolution of the individual MPH and EPH isomers utilizing a vancomycin-based chiral column. The lower limit of quantification was 0.025 ng/ml for each isomer when extracting 0.5 ml plasma aliquots. Calibration curves were linear over the range from 0.025 ng/ml to 25 ng/ml for all analytes (r 2  > 0.995). Assay accuracy and precision were excellent and stability studies and assessment of potential matrix effects contributed to the validation of the method. Application of the method to human plasma samples collected after the administration of dl-MPH with or without ethanol is included, and the implications of this pharmacokinetic drug interaction discussed.
Keywords: Methylphenidate; Ethylphenidate; Ethanol; Enantiomer; LC–MS/MS; Drug interaction;

Quantitative analysis of a therapeutic protein through use of surrogate proteotypic peptides was evaluated for the measurement of Amevive (Alefacept) in human plasma using liquid chromatography tandem mass spectrometry. Signature peptides were obtained through in silico and iterative tuning processes to represent Alefacept for quantification. Horse heart myoglobin was chosen as a protein analogue internal standard to compensate for errors associated with matrix effects and to track recovery throughout the entire sample pretreatment process. Samples were prepared for analysis by selective precipitation of the target proteins with pH controlled at 5.1 and heat denaturation at 45 °C followed by enzymatic digestion, dilution, and filtration. On-line extraction of the signature peptides was carried out using a Phenomenex Gemini C18 security guard column (4.0 mm × 2.0 mm) as a loading column and a Gemini C18 (100 mm × 2.1 I.D., particle size 5 μm) as the analytical (eluting) column. Tandem mass spectrometric detection was performed on a hybrid triple quadrupole linear ion trap equipped with electrospray ionization to positively ionize signature peptides for Alefacept and myoglobin. The method was linear for Alefacept (protein) concentrations between 250 and 10,000 ng/mL. Precision and accuracy for inter- and intra-assay for the lower limit of quantification was less than 20% (16.2 and 10.3, respectively). The method was validated according to current FDA guidelines for bioanalytical method validation.
Keywords: Signature peptides; LC–MS/MS; Biological fluids; Matrix effects;

A simple and sensitive analytical method was developed for the simultaneous determination of clenbuterol, chloramphenicol and diethylstilbestrol in bovine milk by isotope dilution ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS). Samples were directly purified through HLB cartridge. The organic phase was dried under nitrogen and residues were redissolved in mobile phase. Samples were analyzed by UPLC–MS/MS on an Acquity UPLC® BEH C18 column with gradient elution. The samples were quantified using clenbuterol-D9, chloramphenicol-D5 and diethylstilbestrol-D8 as internal standards. The proposed method was validated according to the European Union regulation 2002/657/EC determining specificity, decision limit (CCα), detection capability (CCβ), trueness, precision, linearity and stability. The method is demonstrated to be suitable for the determination of clenbuterol, chloramphenicol and diethylstilbestrol in bovine milk. The total time required for the analysis of one sample was about 50 min.
Keywords: Clenbuterol; Chloramphenicol; Diethylstilbestrol; Milk; Ultraperformance liquid chromatography–tandem mass spectrometry; Isotope dilution;

An integrated platform consisting of monolithic immobilized pH gradient-based capillary isoelectric focusing (M-IPG CIEF) and capillary zone electrophoresis (CZE) coupled by a partially etched porous interface was established. Since carrier ampholytes (CAs) were immobilized on monolith in M-IPG CIEF to form a stable pH gradient, subsequent depletion of CAs at the interface to prevent the interference on CZE separation and detection were avoided. Moreover, a partially etched porous capillary column, which was facile for fabrication and durable for operation, was exploited as the interface to combine M-IPG CIEF and CZE. The RSD values in terms of the migration time for M-IPG CIEF separation, transfer protein from the first dimension to the second dimension, and CZE separation, were 2.4%, 3.9% and 2.3%, respectively. With a 6-protein mixture as the sample, two-dimensional capillary electrophoresis (2D-CE) separation was successfully completed within 116 min, yielding a peak capacity of ∼200 even with minute sample amount down to 5.0 μg/mL. The limit of detection was 0.2 μg/mL. In addition, proteins extracted from milk were used to test the performance of such a 2D-CE separation platform. We expect that such a novel 2D-CE system would provide a promising tool for protein separation with high throughput and high peak capacity.
Keywords: Two-dimensional capillary electrophoresis; Monolithic immobilized pH gradient-based capillary isoelectric focusing; Capillary zone electrophoresis; Partially etched porous interface; Protein;

Following an initial clean-up step on silica gel, high-speed counter-current chromatography (HSCCC) was used to separate cyclic peptides from an extract of the seeds of Vaccaria segetalis. The two-phase solvent system used for HSCCC separation was composed of petroleum ether–ethyl acetate–methanol–water at an optimized volume ratio of 0.5:3.5:1:5. From 190 mg of crude extract, 38.0 mg of segetalin B and 28.5 mg of segetalin A were obtained with purities of 98.1% and 95.6% as determined by HPLC, respectively. The chemical structures of the target compounds were confirmed by high resolution electrospray ionization time of flight MS (HRESI-TOF-MS) and 1H NMR analyses.
Keywords: Vaccaria segetalis; HSCCC; Preparative separation; Segetalin B; Segetalin A;

Remifentanil is a synthetic short-acting opioid with a short half-life that is being used during anaesthesia of small children. In this work an LC–MS/MS method for remifentanil quantification in 20 μL volume of human plasma was developed and validated in connection with a clinical study on neonatal children. Sample preparation was performed with micro extraction in packed syringe (MEPS), which is a miniaturization of solid phase extraction. For this method a mixed phase sorbent M1 (C8, cation exchange), and a protocol for basic compound extraction was followed. Remifentanil-13C6 was used as internal standard. For chromatographic separation, a C18 analytical column with gradient elution was used with mobile phase consisting of aqueous 0.1% formic acid and methanol. The total analysis time was 5.0 min and the measuring range was between 0.05 and 50 ng/mL. Precision and accuracy were with acceptance criteria of ±15%. Plasma samples were stable for 5 weeks at −20 °C and for 4 h at room temperature while 50% was lost after 24 h. This method was successfully applied for remifentanil determination in clinical samples and results agreed with a reference method. With this method using MEPS, a low limit of quantification and much reduced sample volume was obtained as compared with previous methods.
Keywords: Remifentanil; MEPS; LC–MS/MS; Low sample volume;

Quantification of steroid glycosides from Hoodia gordonii in porcine plasma using high performance liquid chromatography–mass spectrometry by Chris J. van Platerink; Hans-Gerd M. Janssen; Brigitte Graf; Leo Abrahamse; Johan Haverkamp (819-825).
An HPLC–ESI–MS/MS method using collision induced dissociation – multiple reaction monitoring was developed for the quantification of eight Hoodia gordonii steroid glycosides and their metabolites in porcine plasma samples. The method was validated for the three most important glycosides and was successfully applied also for the related glycosides and metabolites. The limits of quantification were 0.04 ng ml−1 for the two main steroid glycosides and 0.1 ng ml−1 for the detiglated metabolites. These limits are sufficiently low to allow monitoring the concentration–time profiles in plasma after feeding H. gordonii. The standard deviations of the intra-day measurements were better than 20% for concentrations below 5 ng ml−1 and better than 10% for concentrations above 5 ng ml−1. The method was successfully applied to plasma samples collected from a porcine pharmacokinetics study.
Keywords: Steroid glycosides; Hoodia gordonii; P57AS3; Quantification; Porcine plasma; Liquid–liquid extraction; HPLC; MS;