Journal of Chromatography B (v.877, #8-9)

Automated metal-free multiple-column nanoLC for improved phosphopeptide analysis sensitivity and throughput by Rui Zhao; Shi-Jian Ding; Yufeng Shen; David G. Camp II; Eric A. Livesay; Harold Udseth; Richard D. Smith (663-670).
We report on the development and characterization of automated metal-free multiple-column nanoLC instrumentation for sensitive and high-throughput analysis of phosphopeptides with mass spectrometry. The system employs a multiple-column capillary LC fluidic design developed for high-throughput analysis of peptides (Anal. Chem. 2001, 73, 3011–3021), incorporating modifications to achieve broad and sensitive analysis of phosphopeptides. The integrated nanoLC columns (50 μm i.d. × 30 cm containing 5 μm C18 particles) and the on-line solid phase extraction columns (150 μm i.d. × 4 cm containing 5 μm C18 particles) were connected to automatic switching valves with non-metal chromatographic accessories, and other modifications to avoid the exposure of the analyte to any metal surfaces during handling, separation, and electrospray ionization. The nanoLC developed provided a separation peak capacity of ∼250 for phosphopeptides (and ∼400 for normal peptides). A detection limit of 0.4 fmol was obtained when a linear ion trap tandem mass spectrometer (Finnegan LTQ) was coupled to a 50-μm i.d. column of the nanoLC. The separation power and sensitivity provided by the nanoLC–LTQ enabled identification of ∼4600 phosphopeptide candidates from ∼60 μg COS-7 cell tryptic digest followed by IMAC enrichment and ∼520 tyrosine phosphopeptides from ∼2 mg of human T cells digests followed by phosphotyrosine peptide immunoprecipitation.
Keywords: Metal free nano-LC; On line SPE column; Phosphopeptide; Automation; Mass spectrometer;

A rapid high-performance liquid chromatography–mass spectrometry (HPLC–MS) method was developed and validated for simultaneous quantification of 6-gingerol, 8-gingerol, 10-gingerol and 6-shogaol in rat plasma after oral administration of ginger oleoresin. Plasma samples extracted with a liquid–liquid extraction procedure were separated on an Agilent Zorbax StableBond-C18 column (4.6 mm × 50 mm, 1.8 μm) and detected by MS with electrospray ionization interface in positive selective ion monitoring (SIM) mode. Calibration curves (1/x 2 weighted) offered satisfactory linearity (r 2  > 0.995) in a wide linear range (0.0104–13.0 μg/mL for 6-gingerol, 0.00357–4.46 μg/mL for 8-gingerol, 0.00920–11.5 μg/mL for 10-gingerol and 0.00738–9.22 μg/mL for 6-shogaol). The lower limit of quantification (LLOQ) was in a range of 3.57–10.4 ng/mL. The analytes and internal standard can be baseline separated within 6 min. Inter- and intra-day assay variation was less than 15%. This developed method was successfully applied to pharmacokinetic studies of ginger oleoresin after oral administration to rats. Glucuronide of 6-gingerol was determined after β-glucuronidase hydrolysis for more information, and the intestinal glucuronidation was further confirmed by comparison of plasma samples of hepatic portal vein and femoral vein.
Keywords: Ginger; Pharmacokinetics; Rat plasma; HPLC–MS;

A simple, sensitive and high throughput liquid chromatography/positive-ion electrospray ionization mass spectrometry (LC–ESI-MS/MS) method has been developed for the simultaneous determination of valacyclovir and acyclovir in human plasma using fluconazole as internal standard (IS). The method involved solid phase extraction of the analytes and IS from 0.5 mL human plasma with no reconstitution and drying steps (direct injection of eluate). The chromatographic separation was achieved on a Gemini C18 analytical column using isocratic mobile phase, consisting of 0.1% formic acid and methanol (30:70 v/v), at a flow-rate of 0.8 mL/min. The precursor → product ion transition for valacyclovir (m/z 325.2 → 152.2), acyclovir (m/z 226.2 → 152.2) and IS (m/z 307.1 → 220.3) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) mode. The method was validated over the concentration range 5.0–1075 ng/mL and 47.6–10225 ng/mL for valacyclovir and acyclovir respectively. The mean recovery of valacyclovir (92.2%), acyclovir (84.2%) and IS (103.7%) from spiked plasma samples was consistent and reproducible. The bench top stability of valacyclovir and acyclovir was extensively evaluated in buffered and unbuffered plasma. It was successfully applied to a bioequivalence study in 41 healthy human subjects after oral administration of 1000 mg valacyclovir tablet formulation under fasting condition.
Keywords: Valacyclovir; Acyclovir; Solid phase extraction; LC–ESI-MS/MS; Bench top stability; High throughput;

To help ensure reliability of proinsulin measurements and define the optimal matrix for conducting routine bioanalysis of this prognostic biomarker, we undertook a systematic evaluation of its in vitro stability. For this study, we subjected mono-radioiodinated forms of hPI and its cleaved metabolites to size-exclusion chromatography (FPLC™-SEC employing a Superdex-75 10/30 HR column) to characterize their elution profiles following incubation in human serum and plasma. We determined that intact hPI is a substrate for serine-like protease(s) that are present in human serum. Furthermore, RIA analysis of the elution profile of unlabeled peptide demonstrated that the B–C junction is cleaved preferentially. Thus, in vitro degradation of hPI represents a potential pathway for the formation of cleaved metabolites. Our findings confirmed that EDTA plasma is the preferred matrix for quantitative determination of intact hPI and its cleaved metabolites. We concluded the SEC strategy employed in this study is broadly applicable to evaluating the in vitro stability of other peptides/proteins of diagnostic or therapeutic interest.
Keywords: Proinsulin; Insulin; Protease; Metabolism; Peptide; Size-exclusion chromatography; Immunoassay; Radioimmunoassay; Diabetes; Stability;

High-speed counter-current chromatography methods, combined with resin chromatography were applied to the separation and purification of flavonoid glycosides from the Chinese medicinal herb, Radix Astragali. Five flavonoid glycosides, namely calycosin-7-O-β-d-glucoside, ononin, (6aR, 11aR)-9,10-dimethoxypterocarpan-3-O-β-d-glucoside, (3R)-2′-hydroxy-3′,4′-dimethoxyisoflavan-7-O-β-d-glucoside and calycosin-7-O-β-d-glucoside-6′′-O-acetate, were obtained. Among them, calycosin-7-O-β-d-glucoside-6′′-O-acetate was preparatively separated from Radix Astragali for the first time. Their structures were identified by ESI–MS, 1H NMR, 13C NMR, and 2D NMR.
Keywords: High-speed counter-current chromatography; Radix Astragali; Isolation and purification; Calycosin-7-O-β-d-glucoside-6″-O-acetate;

Quantitative analysis of propofol in whole blood using capillary electrophoresis by Yu Hui; Koen Raedschelders; Hong Zhang; David M. Ansley; David D.Y. Chen (703-709).
We have developed a straightforward capillary electrophoresis method capable of quantifying clinically relevant propofol concentrations in whole blood from patients undergoing aortocoronary bypass grafting with cardiopulmonary bypass. The method utilizes 400 μL of whole blood and is capable of detecting propofol in the ng/mL range. Factors affecting reproducibility and reliability of analytical results for clinically relevant samples are discussed. The method was used to evaluate propofol concentrations in blood samples from 30 patients. The distribution in the whole blood concentration achieved in patients advocates the need for target-achieved monitoring techniques.
Keywords: Capillary electrophoresis; Propofol; Whole blood; Aortocoronary bypass grafting; Cardiopulmonary bypass; Method development;

Enumeration of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus is a priority due to their importance in yogurt production. Capillary electrophoresis (CE) of both bacteria could be achieved in 7.2 min with a resolution of 3.2 in the background electrolyte (BGE) containing 4.5 mM Tris(hydroxymethyl) amminomethane (TRIS)–4.5 mM boric acid–0.1 mM ethylenediamine tetraacetate (EDTA) (TBE) buffer (pH 8.4) and 0.05% (v/v) polyethylene oxide (PEO), using a capillary of 47.5 cm (effective length) × 100 μm i.d., injection of 50 mbar × 3 s followed by −5 kV × 120 s, a voltage and temperature of 20 kV and 25 °C, respectively. Appropriate amounts of PEO in the BGE, sample preparation (i.e. vortex) and introduction were key factors for their separation. A short hydrodynamic injection followed by applying reversed polarity voltage could compress the bacteria into narrow zones, which were detected as separated single peaks. Method linearity (r 2  > 0.99), precision (%RSDs < 9.3%), recovery (%R  = 91.7–106.7%) and limit of quantitation (1.0 × 106 colony forming unit per mL (CFU/mL)) were satisfactory. Results from the CE analysis of both bacteria in yogurt were not statistically different from those of the plate count method (P  > 0.05). The CE method can be used as an alternative for quantitation of L. delbrueckii subsp. bulgaricus and S. thermophilus in yogurt since it was reliable, simple, cost and labor effective and rapid, allowing the analysis of 3 samples/h (comparing to 2d/sample by plate count method).
Keywords: Lactobacillus delbrueckii subsp. bulgaricus; Streptococcus thermophilus; Capillary zone electrophoresis; Yogurt;

Application of liquid chromatography–mass spectrometry to measure short chain fatty acids in blood by Hans M.H. van Eijk; Johanne G. Bloemen; Cornelis H.C. Dejong (719-724).
A new liquid chromatography–mass spectrometry method is described to determine concentrations of the short chain fatty acids acetic acid, propionic acid and butyric acid (SCFAs) in human blood plasma. The method is based on reversed phase chromatography followed by post-column neutralization of the mobile phase with ammonia and a consecutive measurement of the SCFAs ammonia adducts using negative electro spray ionization. Sample preparation involved simple organic acid deproteinization, resulting in 100% recovery. SCFAs eluted baseline separated within a 25 min run cycle. A linear response was obtained in the range between 0 and 250 μmol/l (R 2 ranged from 0.997 to 0.9999). The limit of detection ranged from 0.05 μmol/l for propionic and butyric acid and 0.1 μmol/l for acetic acid. The method was tested by analyzing plasma of arterial blood, from portal vein and hepatic vein blood from patients undergoing a pylorus-preserving pancreaticoduodenectomy. As expected, the highest SCFA concentrations were found in portal plasma, hepatic vein levels were in between, while arterial concentrations were lowest. This newly developed method is suitable to determine SCFA concentrations in human plasma samples.
Keywords: Short chain fatty acids; Mass spectrometry; Liquid chromatography;

Metabolic profiling of transgenic rice with cryIAc and sck genes: An evaluation of unintended effects at metabolic level by using GC-FID and GC–MS by Jia Zhou; Chenfei Ma; Honglin Xu; Kailong Yuan; Xin Lu; Zhen Zhu; Yongning Wu; Guowang Xu (725-732).
The cryIAc and sck genes were introduced to the rice for the purpose of improving the insect resistance. Metabolic profiles of wild and transgenic rice were compared to assess the unintended effects related to gene modification. Wild samples with different sowing dates or sites were also examined to determine the environmental effects on metabolites. The polar compounds of grains were extracted, trimethylsilylated and analyzed by gas chromatography-flame ionization detection (GC-FID). Partial least squares-discriminant analysis (PLS-DA) and principal component analysis (PCA) were applied to differentiate transgenic and wild rice grains. The significantly distinguishable metabolites were picked out, and then identified by gas chromatography–mass spectrometry (GC–MS). It was found that both the environment and gene manipulation had remarkable impacts on the contents of glycerol-3-phosphate, citric acid, linoleic acid, oleic acid, hexadecanoic acid, 2,3-dihydroxypropyl ester, sucrose, 9-octadecenoic acid (Z)-, 2,3-dihydroxypropyl ester and so on. Sucrose, mannitol and glutamic acid had a significant increase in transgenic grains in contrast to those in non-genetically modified (GM) rice.
Keywords: cryiac and sck genes; Metabolic profiling; Unintended effect; GC-FID; GC–MS; Transgenic rice; Environmental effects;

Water-soluble salvianolic acid A (Sal A) and salvianolic acid B (Sal B) were successfully isolated and purified from the crude extract of Salvia miltiorrhiza by high-speed counter-current chromatography (HSCCC). The solvent system was n-hexane–ethyl acetate–methanol–water (3:6:6:10, v/v/v/v). 4.27 mg of Sal A and 32.09 mg of Sal B were obtained from 260 mg of the crude sample. The purities of Sal A and Sal B were 96.67% and 97.43%, respectively. Their structures were identified by 1H NMR and 13C NMR. Antioxidant activities of Sal A and Sal B were also evaluated and compared by the methods of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay and 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS• +) radical cation decolourisation assay. Both Sal A and Sal B showed high radical scavenging activities with their EC50 values being 1.43 ± 0.09 and 1.81 ± 0.01 μg/ml in DPPH radical method. The ABTS results showed that Sal A and Sal B exhibited high total antioxidant activities, their EC50 values were 1.35 ± 0.00 and 1.43 ± 0.01 μg/ml, respectively.
Keywords: HSCCC; Salvianolic acid A; Salvianolic acid B; Antioxidant activity; Salvia miltiorrhiza Bge.;

New ionic liquid-modified silica sorbents were developed by the surface chemical modification of the commercial silica using synthesized ionic liquids. The obtained ionic liquid-modified particles were successfully used as a special sorbent in solid-phase extraction process to isolation of cryptotanshinone, tanshinone I and tanshinone IIA from Salvia Miltiorrhiza Bunge. Different washing and elution solvents such as water, methanol and methanol–acetic acid (90/10, v/v) were evaluated. A comparison of ionic liquid-modified silica cartridges and traditional silica cartridge show that higher recovery was observed using ionic liquid-modified silica sorbents. A quantitative analysis was conducted by high-performance liquid chromatography using a C18 column (5 μm, 150 mm × 4.6 mm) with methanol–water (78:22, v/v, and containing 0.5% acetic acid) as a mobile phase. Good linearity was obtained from 0.5 × 10−4 to 0.5 mg/mL (r 2  > 0.999) with the relative standard deviations less than 4.8%.
Keywords: Ionic liquid-modified silica; Tanshinones; Solid-phase extraction; High-performance liquid chromatography; Salvia Miltiorrhiza Bunge;

Association of IGFBP-1, IGFBP-2 and IGFBP-3 with other proteins in human serum and placental cell membranes was investigated using affinity chromatography matrix with immobilized antibodies. Circulating IGFBP-1 was found to be predominantly bound to α2-macroglobulin and not in the binary complex with its ligand, IGFBP-2 complexes and/or polymers were detected, which was not acknowledged before, and IGFBP-3 molecular forms were differentiated into those that form binary/ternary complexes and those that form stable associations with other serum proteins. As for placental membranes, both IGFBP-1 dimers and high molecular mass IGFBP-1 associations, most probably with α2-macroglobulin, were recognized and resolved.
Keywords: Affinity chromatography; IGFBP-1; IGFBP-2; IGFBP-3; Molecular forms;

Novel liquid chromatographic determination of cystatin C in human urine by Othman Ibrahem Yousef Al-Musaimi; Manar Khalid Fayyad; Adel Khalil Mishal (747-750).
A rapid and sensitive method for quantitative determination of cystatin C (CC) protein in human urine via HPLC was developed and validated. Acetone has been used as a precipitating agent of CC protein from the urine biological matrix. Separation and detection were accomplished by ion pair liquid chromatography and UV detection. Gradient elution mode was utilized to elute CC with a UV detection of 224 nm. The analysis time was 14 min per sample using Ace C8 (150 × 4.6 mm i.d., 5 μm) as a chromatographic column with a flow rate of 1.0 mL/min. Calibration curve with good linearity (r 2  = 0.99) within the range from 0.390 to 0.001 mg/mL was obtained. Limits of detection and quantification were 0.001 and 0.002 mg/mL, respectively. Inter-assay and intra-assay variabilities were <15% for all levels and <20% at the limit of quantification level. Major advantages of the method: specific where no false positive results might be obtained and fast where sample pretreatment needs only 1 h.
Keywords: Cystatin C; Protein; HPLC; Chromatography;

Harpagoside, a major bioactive iridoid glucoside in genus Scrophularia, has been widely used in clinical practice for the treatment of pain in the joints and lower back for its neuroprotective and anti-inflammation activities. To investigate the pharmacokinetics and hepatobiliary excretion, an in vivo microdialysis method coupled with high performance liquid chromatography was developed to monitor the concentration of harpagoside in blood and bile. The harpagoside bile-to-blood distribution ratio (AUC bile/AUC blood) up to 986.28 ± 78.46 significantly decreased to 6.41 ± 0.56 or 221.20 ± 18.92 after co-administration of cyclosporin A or verapamil. The results indicated that harpagoside went through concentrative elimination from the bile which was probably regulated by P-glucoprotein, providing possible clinical trials of co-administration of transporter inhibitors to decrease drug efflux, thus to enhance the curative effects.
Keywords: Harpagoside; In vivo microdialysis; Pharmacokinetic; Hepatobiliary excretion; P-glucoprotein; Cyclosporin A; Verapamil;

Application of open tubular capillary columns coated with zirconium phosphonate for enrichment of phosphopeptides by Yanfeng Xue; Junying Wei; Huanhuan Han; Liyan Zhao; Dong Cao; Jinglan Wang; Xiaoming Yang; Yangjun Zhang; Xiaohong Qian (757-764).
A new approach utilizing open tubular capillary columns coated with zirconium phosphonate (ZrP-OTCC) for enrichment of phosphopeptides is described. The experimental conditions: interior diameter, length of capillary and flow rate was optimized using tryptic digest of α-casein (a phosphoprotein) as a model sample. The ZrP-OTCC was demonstrated to tolerate urea, sodium dodecyl sulphate (SDS), and NaCl. Further experimental results show that the ZrP-OTCC can trap the phosphopeptides even at the concentration of α-casein as low as 10−8  M. This column has also been successfully coupled online with nano-liquid chromatography for enrichment and then separation of phosphopeptides from a complex sample, and finally analyzed the phosphopeptides by mass spectrometry (MS).
Keywords: Open tubular capillary columns; Zirconium phosphonate; Phosphopeptides; Mass spectrometry;

A highly selective, sensitive and robust LC–MS/MS method was developed for the simultaneous quantification of cortisol, cortisone, prednisolone and prednisone in human plasma. Prednisolone, cortisol and cortisone have similar fragmentation pattern. These three compounds were chromatographically separated, thus eliminating the inherent interference that fragments derived from the M + 2 and M isotopes of prednisolone contribute in the MRM channels of cortisol and cortisone, respectively. Additionally, by using a small particle (1.8 μm) analytical column, interferences present in the plasma samples from post-transplant recipients were successfully resolved from cortisol after a simple extraction consisting of protein precipitation, evaporation and reconstitution. The chromatographic separation was achieved on a Zorbax-SB Phenyl column under isocratic conditions during a run time of 8 min. Intra-run and inter-run precision and accuracy within ±15% were achieved during a 3-run validation for quality control samples at five concentration levels in charcoal-stripped plasma as well as in normal plasma, over a 500-fold dynamic concentration range. The lower limit of quantitation was 0.500 ng/mL for cortisone and prednisone, 1.00 ng/mL for cortisol and 2.00 ng/mL for prednisolone. The performance of the small particle column was maintained during more than 1200 injections in terms of peak retention time, symmetry and backpressure.
Keywords: Assay; Concentration; Cortisol; LC–MS/MS; Pharmacokinetics; Prednisolone;

Single step determination of PCB 126 and 153 in rat tissues by using solid phase microextraction/gas chromatography–mass spectrometry: Comparison with solid phase extraction and liquid/liquid extraction by Diana Poli; Andrea Caglieri; Matteo Goldoni; Anna F. Castoldi; Teresa Coccini; Elisa Roda; Annabella Vitalone; Sandra Ceccatelli; Antonio Mutti (773-783).
A simple and reliable solid phase microextraction/gas chromatography–mass spectrometry (SPME/GC–MS) method was developed for the single-step determination of PCBs 126 and 153 in rat brain and serum, using liquid/liquid and solid phase extraction (SPE) as reference techniques. The multi-factor categorical experimental design used to study simultaneously the main parameters and their interactions affecting the efficiency of the method, showed that the use of an 85 μm PA exposed at 100 °C for 40 min was the optimum sampling condition for both PCBs. SPME was then validated by studying its linear dynamic (over two orders of magnitude), limits of detection (brain: 2 ng/g, serum: 0.2 ng/g) and analytical precision that was within 9% for SPME in both brain and serum. Finally, the method was used to determine the brain and blood target dose in mothers and pups after oral exposure of the mothers.
Keywords: Polychlorinated biphenyls; Solid phase microextraction; Brain and serum tissues; Gas chromatography–mass spectrometry;

3-Penten-2-one, a novel aldehyde adduct, is a biomarker for increased acetaldehyde in urine by Valerie Walker; Graham A. Mills; Elizabeth M. Stansbridge (784-790).
Diagnostic profiling of urine for volatile compounds of around 400 patients using headspace solid-phase microextraction (HS-SPME) in alkaline conditions identified 3-penten-2-one (approximately 1 to >6.3 μmol/L) in 26 patients. Five were in barbiturate coma. 3-Penten-2-one, previously of unknown origin, was shown to be formed by aldol condensation of acetaldehyde with acetone or acetoacetate during analysis. Semi-quantification of acetaldehyde using in-fibre derivatisation HS-SPME, showed high concentrations in five urine (33–348 μmol/L) and two plasma (17 and 43 μmol/L) samples. Hence, urinary 3-penten-2-one is a useful biomarker for increased accumulation of acetaldehyde during abnormal metabolic stress.
Keywords: 3-Penten-2-one; Acetaldehyde; Aldehydes; ALDH2; Biological fluids; Biomarker; Solid-phase microextraction;

A high throughput and specific method using ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) was developed for the simultaneous determination of diltiazem and its two metabolite (N-desmethyldiltiazem and O-desacetyldiltiazem) in human plasma. A one-step liquid–liquid extraction (LLE) with methyl-t-butyl ether (MTBE) involved for the extraction of diltiazem (DLTZ), metabolites (DMeD and DAcD) and internal standard. Analytes were chromatographed on a ACQUITY UPLC™ BEH C18 column (100 mm × 2.1 mm, i.d., 1.7 μm) with isocratic elution at a flow rate of 0.2 mL/min using 10 mM ammonium acetate buffer–acetonitrile (25:75, v/v). The Quattro Premier XE LC–MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. Using 300 μL plasma, the method was validated over the concentration range 0.48–639.9 ng/mL for DLTZ and 0.24–320.1 for DMeD and 0.24–320.7 ng/mL for DAcD, with a lower limit of quantification of 0.48 ng/mL for DLTZ and 0.24 ng/mL for metabolites. The intra- and inter-day precision and accuracy were within 10.0%. The recovery was 77.4%, 76.0%, 74.5% and 74.1% for DLTZ, DMeD, DAcD and Ziprasidone, respectively. Total run time was 2.0 min only.
Keywords: Diltiazem; O-desacetylediltiazem; N-desmethylediltiazem; LC–MS/MS; Human plasma; Acquity; UPLC;

Dried blood spots (DBSs) technology was evaluated in an assay for the quantitation of dextromethorphan (DM) and its metabolite, dextrorphan (DT), in human whole blood using high performance liquid chromatography with tandem mass spectrometry method (LC–MS/MS). Both the parent drug and metabolite were spiked in the blood matrix and subsequently allowed to dry on a specimen collection card. The dried blood spots were removed using a manual punch and then extracted into methyl tert-butyl ether (MTBE). The organic supernatant was transferred and evaporated and the residue was reconstituted in 20% acetonitrile. The overall method recovery of DM and DT was 87.8% and 95.4%, respectively. The assay was linear over the concentration range of 0.2–200 ng/mL for both analytes. Several factors that potentially affect DBS assay quantitation were investigated, such as punch size, DBS sample punch-out location, and the volume of the blood sample pipetted on the specimen collection cards. The study determined that punch size does not affect assay quantitation accuracy. Indeed, a larger punch size increases the sensitivity due to the larger sampled blood spots. Sampling from different location on the specimen collection cards shows no significant variation for both drugs. The study also shows that acceptable results can be achieved with some variation of the sample volume, which allows a simple blood sampling procedure at the test sites. To achieve the similar lower limit of quantitation (LLOQ) as the plasma assay, several blood spots at the same concentration level were stacked together and extracted. Bioanalytical assays using the DBS technique are promising given the advantages of the method over the plasma assay.
Keywords: Dried blood spot (DBS); Dextromethorphan; Dextrorphan; LC–MS/MS;

Solution isoelectric focusing (sIEF) is evaluated relative to ion exchange chromatography (IEC) as a preferred charge-based prefractionation tool for proteome mixtures. While IEC is extensively employed for proteome prefractionation prior to MS analysis, we demonstrate here that conventional salt gradient IEC has significant shortcomings compared to sIEF. Here, we critically evaluated a custom eight-channel sIEF device for intact protein separation, relative to strong cation exchange (SCX) and strong anion exchange (SAX) chromatography. The resolution, recovery, and uniformity of separation obtained with our sIEF device were comparable or superior to that of optimized IEC separations. Most importantly for intact proteins, sIEF separations strongly correlate with the proteins’ isoelectric point, which contrasts with IEC where no correlation was observed. To validate the sIEF platform for proteome analysis, prefractionation through sIEF resulted in the confident identification of a greater number of proteins from yeast (211) following LC–MS/MS, relative to those obtained through SAX (173) or SCX (148).
Keywords: Proteomics; Intact protein; Separation; Ion exchange; Solution isoelectric focusing; Mass spectrometry;

During our studies to establish a method for identifying tryptamine-related substances in human urine, we detected three large peaks of unknown origin in an HPLC chromatogram. Fluorometric HPLC and HPLC-TOF-MS/MS analyses led to the identification of these substances as 6-sulphatoxymelatonin, 5-sulphatoxydiacetyltryptamine, and reduced melatonin. This is the first report of the latter two compounds in human urine. Here, we report the results of two fluorometric HPLC assays of these three substances, as well as melatonin, 6-hydroxymelatonin, and 5-hydroxydiacetyltryptamine, using synthesized standards and discuss the possibility that 5-hydroxydiacetyltryptamine (the parent substance of 5-sulphatoxydiacetyltryptamine) and reduced melatonin have radical scavenging activity.
Keywords: Hydroxydiacetyltryptamine; Melatonin; Serotonin; Reduced melatonin; Hydroxytryptamine;

A shortcut from plasma to chromatographic analysis: Straightforward and fast sample preparation for analysis of green tea catechins in human plasma by Benno F. Zimmermann; Menelaos Papagiannopoulos; Sonja Brachmann; Mario Lorenz; Verena Stangl; Rudolf Galensa (823-826).
This paper describes a new and straightforward method for determination of the green tea catechins epicatechin, epicatechin gallate, epigallocatechin, and epigallocatechin gallate in human plasma. Sample preparation includes addition only of dimethylformamide and trichloroacetic acid. After centrifugation, the supernatant can be injected into the HPLC. If required, the glucuronides and sulphates of the catechins can be enzymatically hydrolysed before extraction. Recovery ranges from 92.9 to 98.2%; limits of detection, from 2.4 to 5.0 ng/mL; and relative standard deviations, from 3.1 to 8.6%. Twelve samples can be processed within 45 min, and are then ready to be injected into the HPLC. The method was successfully applied to human plasma. This method is suitable for studies on absorption, bioavailability, and kinetics of green tea catechins in plasma. Since manual work and time consumption are minimal, the procedure is especially useful for large numbers of samples.
Keywords: Green tea; Catechins; Epigallocatechin gallate; Human plasma; Sample preparation; EGCG;

An analytical procedure was developed for the detection and quantification of N-acetyl-S-(n-propyl)-l-cysteine (n-propylmercapturic acid, AcPrCys), a metabolite and biomarker for exposure to 1-bromopropane (1-BP). 1-BP is used as an industrial solvent and exposure is a health concern for industrial workers due to its toxicity. It has been associated with neurological disorders in both animals and humans. Urine sample preparation for the determination of AcPrCys consisted of solid phase extraction (SPE). Urine samples on preconditioned SPE (C18) columns were washed with 40% methanol/60% water solution prior to elution with acetone. Quantification was by means of a liquid chromatograph (LC) equipped with a mass spectrometer (MS) using an Aqua 3 μm C18 300A column and [d7]-AcPrCys was used as internal standard. Electrospray ionization (ESI) was used with the MS operated in the negative ion mode and selected ion monitoring (SIM) at m/z 204 for AcPrCys and m/z 211 for [d7]-AcPrCys. Demonstrated recovery of urine samples fortified at multiple levels (0.625–10 μg/ml) varied between 96 and 103% of theory with relative standard deviations (RSD) of 6.4% or less. The limit of detection (LOD) for the procedure was approximately 0.01 μg/ml AcPrCys in urine. These data will be discussed as well as other factors of the development of this test procedure.
Keywords: 1-Bromopropane; HPLC–MS;

The present study describes the simultaneous determination of four drugs, two local anaesthetics (lidocaine and bupivacaine) and two opium alkaloids (noscapine and papaverine) by capillary zone electrophoresis (CZE) with solid-phase extraction (SPE) procedure using Oasis HLB cartridges. Their recoveries ranged from 81 to 107% at the target concentrations of 2.0, 5.0 and 8.0 μg mL−1 in spiked urine samples. Coefficients of variation of the recoveries ranged from 2.1 to 11.3% at these concentrations. The quantitation limits of the method were approximately 300 ng mL−1 for the different compounds studied. The assay is very specific for these compounds and requires a short sample preparation procedure prior to the electrophoretic analysis.
Keywords: Capillary zone electrophoresis; Local anaesthetics; Opium alkaloids; Urine samples;

A sensitive high performance liquid chromatography method (HPLC) has been developed for the quantification of doxorubicin in mouse plasma and tissues. Samples of serum or tissue homogenates, 20 μl, were analyzed following a single step protein precipitation using perchloric acid (35%, v/v). Doxorubicin was separated from the internal standard, daunorubicin, on a Zorbax 300SB C18 column at 35 °C. Mobile phase was comprised of acetonitrile and water (25:75) containing 0.1% triethylamine, and was adjusted to pH 3 with phosphoric acid. Peaks eluting from the column were detected with a fluorescence detector with excitation and emission wavelengths of 480 and 560 nm, respectively. Standard curves were linear in the range 5–1000 ng/ml, and correlation coefficients were typically greater than 0.999. Intra-assay recoveries ranged from 94.7 to 99.9%, and inter-assay recoveries were in the range of 95.2–101%. The associated coefficient of variation (CV) was less than 10% in all cases. The method was successfully applied to investigate doxorubicin plasma pharmacokinetics and tissue distribution in athymic Foxnu mice.
Keywords: Doxorubicin; HPLC; Protein precipitation; Mouse plasma; Tissues;

A high-performance liquid chromatographic method with fluorescence detection for the determination of itopride in human plasma is reported. The sample preparation was based on liquid–liquid extraction of itopride from plasma with t-butylmethylether and dichloromethane (70:30, v/v) mixture followed by a back extraction of the analyte to the phosphate buffer (pH 3.2). Liquid chromatography was performed on an octadecylsilica column (55 mm × 4 mm, 3 μm particles), the mobile phase consisted of acetonitrile–triethylamine–15 mM dihydrogenpotassium phosphate (14.5:0.5:85, v/v/v), pH of the mobile phase was adjusted to 4.8. The run time was 3 min. The fluorimetric detector was operated at 250/342 nm (excitation/emission wavelength). Naratriptan was used as the internal standard. The limit of quantitation was 9.5 ng/ml using 0.5 ml of plasma. The method precision and inaccuracy were less than 8%. The assay was applied to the analysis of samples from a bioequivalence study.
Keywords: Itopride; Extraction; HPLC; Fluorescence;