Journal of Chromatography B (v.856, #1-2)

This study examines a novel sample preparation method for the determination of 11 hydroxy metabolites of polychlorinated biphenyls (PCBs) in plasma and organ tissues, followed by gas chromatography with mass spectrometric detection (GC/MS). The clean-up method was optimized to eliminate the interference matter by using a silica column and 10 mL of n-hexane/dichloromethane (4:6, v/v) as an eluent. Solid-phase and solvent extraction procedures were used for the plasma and tissues samples, respectively. Compared to C18 and C8 solid-phase, C2 showed higher extraction efficiency with n-hexane as the eluent for plasma. The hydroxy-PCB extraction recoveries achieved with this combined extraction and clean-up procedure from plasma ranged from 87 to 117%, while those from tissues ranged from 82 to 111%. The linear detector responses for propyl derivatives of hydroxy-PCBs were obtained with the coefficients of determination varying from 0.992 to 0.998 in the concentration range of 0.1–20 ng mL−1. The method detection limits ranged from 0.1 to 0.5 ng mL−1 in 1 mL of plasma and from 0.1 to 0.5 ng g−1 in 1 g of tissues. This procedure was successfully applied to the study of 3-OH-2,3′,4,4′,5-PeCB in rat plasma and liver samples after intraperitoneal injection (20 mg/kg) of 2,3′,4,4′,5-PeCB.
Keywords: Hydroxy-PCBs; GC/MS; SPE; Clean-up; Plasma; Tissue;

An evaporation-free solid-phase extraction (SPE) method was developed and validated for sumatriptan. High organic washing (50% methanol) and low organic elution (20% methanol) were used and the recovery was greater than 92%. The eluate was injected into a C18 column without evaporation and reconstitution. Sumatriptan was monitored in positive ion mode with mass transition of m/z 296.4–58.1 amu. The calibration curve was 1–100 ng/mL (r  ≥ 0.9923). The inter-day and intra-day precisions ranged from 4.53 to 9.12% and 1.72 to 6.93%, respectively. This method features reduced cost and pollution, clean extract, high speed, and most importantly overall method reliability.
Keywords: Evaporation-free; Sumatriptan; Solid-phase extraction; LC–MS/MS; Human plasma;

Novel LC–MS/MS method for assay of 7α-hydroxy-4-cholesten-3-one in human plasma by Anita Lövgren-Sandblom; Maura Heverin; Hanna Larsson; Eija Lundström; John Wahren; Ulf Diczfalusy; Ingemar Björkhem (15-19).
A new isotope dilution LC–MS/MS method for assay of 7α-hydroxy-4-cholesten-3-one without need for derivatization is described. This method was used in catheterization experiments on healthy fasting volunteers. The levels of this generally used marker for bile acid synthesis were slightly but significantly higher in the hepatic vein than in the brachial artery. In contrast, the levels of the precursor to 7α-hydroxy-4 cholesten-3-one, 7α-hydroxycholesterol, were the same in the two vessels. It is concluded that there is a net extrahepatic metabolism of 7α-hydroxy-4-cholesten-3-one. The similarity and very high correlation between the levels in the two vessels (r  = 0.97) are consistent with the contention that 7α-hydroxy-4-cholesten-3-one is a suitable marker for the activity of the hepatic cholesterol 7α-hydroxylase and thus bile acid synthesis.
Keywords: Liquid chromatography–mass spectrometry; Isotope dilution; CYP7A1; Bile acid synthesis;

A significant percentage of psychiatric patients who are treated with antipsychotics are treated with more than one antipsychotic drug in the clinic. Thus, it is advantageous to use a rapid and reliable assay that is suitable for determination of multiple antipsychotic drugs in plasma in a single run. A simple and sensitive HPLC-UV method was developed and validated for simultaneous quantification of olanzapine, haloperidol, chlorpromazine, ziprasidone, risperidone and its active metabolite 9-hydroxyrisperidone in rat plasma using imipramine as an internal standard (I.S.). The analytes were extracted from rat plasma using a single step liquid–liquid acid solution back extraction technique with wash procedure, which provided the very clear baseline for blank plasma extraction. The compounds were separated on an Agilent Eclipse XDB C8 (150 mm × 4.6 mm i.d., 5 μm) column using a mobile phase of acetonitrile/30 mM ammonium acetate including 0.05% triethylamine (pH 5.86 adjusted with acetic acid) with gradient elution. All of the analytes were monitored using UV detection. The method was validated and the linearity, lower limit of quantitation (LLOQ), precision, accuracy, recoveries, selectivity and stability were determined. The LLOQ was 2.0 ng/ml and correlation coefficient (R 2) values for the linear range of 2.0–500.0 ng/ml were 0.998 or greater for all the analytes. The precision and accuracy for intra-day and inter-day were better than 7.44%. The recovery was above 74.8% for all of the analytes. This validated method has been successfully used to quantify the plasma concentration of the analytes for pharmacological and toxicological studies following chronic treatment with antipsychotic drugs in the rat.
Keywords: Risperidone; 9-Hydroxyrisperidone; Olanzapine; Haloperidol; Chlorpromazine; Ziprasidone;

Pharmacokinetics of HZ08 in rats by liquid chromatography–tandem mass spectrometry by Jing-yan Weng; Min Song; Tai-jun Hang; Wen-long Huang; Yun Du (29-34).
A selective and sensitive liquid chromatographic method coupled with ion spray tandem mass spectrometry detection (LC–MS/MS) was developed for the determination and pharmacokinetic study of N-cyano-1-[(3,4-dimethoxyphenyl)methyl]-3,4-dihydro-6,7-dimethoxy-N′-octyl-2(1H)-isoquinoline-carboximidamide (HZ08, a candidate reversing agent for multidrug resistance of cancer) liposome injection in rat plasma. The analyte was extracted from plasma using liquid–liquid extraction by methyl tert-butyl ether with drotaverine as internal standard. The chromatographic separation was performed on a Kromasil-C18 column (150 mm × 4.6 mm, i.d., 5 μm) with gradient elution. The tandem mass detection was made with electrospray ionization in positive ion selected reaction monitoring mode with argon collision-induced dissociation. The ion transitions were m/z 523.1 to 342.1 for HZ08 at 27 eV and m/z 398.1 to 326.1 at 35 eV for the internal standard, respectively. The determination was validated to be accurate and precise for the analysis in the concentration range of 5–10,000 ng/ml for HZ08 with the lower limit of detection (LOD) being 1 ng/ml, when 0.1 ml of rat plasma sample was processed. The main pharmacokinetic parameters found for HZ08 after intravenous (i.v.) administration of its liposome injection at doses of 2, 4 and 8 mg/kg were as follows: C max (4511 ± 681), (5553 ± 1600) and (6444 ± 950) ng/ml, T max (0.033 ± 0), (0.056 ± 0.048) and (0.033 ± 0) h, t 1/2 (1.75 ± 0.19), (1.63 ± 0.12) and (1.56 ± 0.18) h, AUC0–6 (899 ± 112), (1238 ± 190) and (1707 ± 307) h ng/ml, AUC0-∞ (917 ± 110), (1256 ± 189) and (1723 ± 306) h ng/ml, MRT (1.14 ± 0.21), (1.01 ± 0.13) and (1.16 ± 0.17) h, CL (2.90 ± 0.15), (3.01 ± 0.74) and (4.11 ± 0.59) l/h/kg, respectively. The plasma concentration–time profiles of HZ08 were best fitted with two-compartment models. Linear pharmacokinetics was found for HZ08 in rats after intravenous administration of the liposome injection.
Keywords: HZ08; Rat plasma; Pharmacokinetics; LC–tandem MS;

A sensitive liquid chromatography/tandem mass spectrometric (LC–MS/MS) method was developed and validated for the determination of rosuvastatin in human plasma. The plasma samples were prepared using liquid–liquid extraction with ethyl ether. Chromatographic separation was accomplished on a Zorbax XDB-C18 (150 mm × 4.6 mm i.d., 5 μm) column. The mobile phase consisted of methanol–water (75:25, v/v, adjusted to pH 6 by aqueous ammonia). Detection of rosuvastatin and the internal standard (IS) hydrochlorothiazide was achieved by ESI MS/MS in the negative ion mode. The lower limit of quantification was 0.020 ng/ml by using 200 μl aliquots of plasma. The linear range of the method was from 0.020 to 60.0 ng/ml. The intra- and inter-day precisions were lower than 8.5% in terms of relative standard deviation (RSD), and the accuracy was within −0.3 to 1.9% in terms of relative error (RE). Compared with the existing methods, the validated method offered increased sensitivity. The method was successfully applied for the evaluation of pharmacokinetics of rosuvastatin after single oral doses of 5, 10 and 20 mg rosuvastatin to 10 healthy volunteers.
Keywords: Rosuvastatin; LC–MS/MS; Pharmacokinetics;

Ginger root powder is widely used as a dietary supplement as well as a spice and flavoring agent in foods and beverages. In this study, we developed a high-performance liquid chromatographic (HPLC) method that is suitable for the analysis of 6-gingerol, 6-shogaol, 8-gingerol, and 10-gingerol in a wide variety of ginger-containing dietary supplements, spices, teas, mints, and beverages. 6-Gingerol, 6-shogaol, 8-gingerol, and 10-gingerol were extracted from various ginger-containing products with ethyl acetate and analyzed by HPLC on a C-8 reversed phase column at 282 nm. The recoveries of 6-, 8-, and 10-gingerol, and 6-shogaol from the ginger dietary supplements and ginger-containing products were 94.7 ± 4.1, 93.6 ± 3.4, 94.9 ± 4.0, 97.1 ± 3.8%, respectively. The within-day coefficients of variation for 6-gingerol, 6-shogaol, 8-gingerol, and 10-gingerol standards at 50.0 μg/mL were 2.54, 2.38, 2.55, and 2.31%, respectively. The lower limit of quantitation was 25 ng injected. The standard curves for 6-, 8-, and 10-gingerol and 6-shogaol were linear from 10.0 to 1000 μg/mL. The variation (CV's) in the 6-gingerol, 6-shogaol, 8-gingerol, and 10-gingerol concentrations of nine different ginger root dietary supplements were 115.2, 45.7, 72.3, and 141.7%, respectively. The gingerol composition of various ginger-containing spices, teas, and beverages also were found to vary widely. The proposed method can be used for the analysis and standardization of 6-, 8-, and 10-gingerol in ginger-containing dietary supplements, spices, food products and beverages and as a method for determining the amounts of 6-shogaol as a marker for 6-gingerol stability.
Keywords: HPLC analysis; Ginger dietary supplements; 6-Gingerol; 6-Shogaol; 8-Gingerol; 10-Gingerol; Ginger spices; Ginger beverages;

Measurement of amino acid isotope enrichment by liquid chromatography mass spectroscopy after derivatization with 9-fluorenylmethylchloroformate by Hans M.H. van Eijk; Dennis P.L. Suylen; Cornelis H.C. Dejong; Yvette C. Luiking; Nicolaas E.P. Deutz (48-56).
An automated method is described to measure tracer-tracee-ratios (TTR) of plasma amino acids after their separation as 9-fluorenylmethylchloroformate derivatives. In a 45 min cycle, 5 μl plasma aliquots were derivatized, HPLC separated and subjected to electrospray ionization. By applying source collision induced dissociation, derivatives were dissociated at the peptide bond releasing the originating amino acids into the mass spectrometer. This approach enabled the determination of plasma amino acid TTRs with a standard deviation between 0.15 and 0.36%, which is sufficient to study the fate of infused tracers and their conversion products in an in vivo experiment in humans.
Keywords: Amino acid; Stable isotope; HPLC; Mass spectrometry;

Detection and quantification of aripiprazole and its metabolite, dehydroaripiprazole, by gas chromatography–mass spectrometry in blood samples of psychiatric patients by Hui-Ching Huang; Chin-Hung Liu; Tsuo-Hung Lan; Tsung-Ming Hu; Hsien-Jane Chiu; Yu-Chun Wu; Ying Lung Tseng (57-61).
Aripiprazole is a novel antipsychotic drug for the treatment of schizophrenia and schizoaffective disorders. In this study, a new method using gas chromatography–mass spectrometry (GC–MS) was developed and validated for the detection of aripiprazole and its main metabolite, dehydroaripiprazole, in plasma. Blood samples from seven psychiatric patients treated with aripiprazole (10–20 mg/day) underwent a solid-phase extraction (SPE) and N-methyl-N-trimethylsilytrifluoroacetamide (MSTFA) derivatization. The characteristic ions of mass spectra for aripiprazole and dehydroaripiprazole were m/z 306, 292, 218 and 304, 290, 218, respectively. Extraction recoveries from this method were 75.4% (n  = 5) for aripiprazole and 102.3% (n  = 5) for dehydroaripiprazole. The calibration curves of aripiprazole and dehydroaripiprazole were linear from 16 to 500 ng/ml (r 2  = 0.999) and 8 to 250 ng/ml (r 2  = 0.999), respectively. The respective limits of quantification (LOQs) for aripiprazole and dehydroaripiprazole evaluated in 0.5 ml of serum were 14.4 ng/ml and 6.9 ng/ml. Intra-assay and interassay precision and accuracy were within acceptable ranges. In this study, we also found that the mean trough concentrations in plasma at steady-state were 128.9 μg/l for aripiprazole and 30.1 μg/l for dehydroaripiprazole.
Keywords: Aripiprazole; Dehydroaripiprazole; Detection; GC–MS; Quantification;

Development of a capillary electrophoresis method for the screening of human urine for multiple drugs of abuse by Ahmed Alnajjar; Abubakr M. Idris; Marika Multzenberg; Bruce McCord (62-67).
A new capillary electrophoresis (CE) method was developed and validated for the screening of human urine for nineteen drugs of abuse. In order to achieve sufficient separation, the electrolyte composition was modified using β-cyclodextrin (β-CD) and organic solvents. To process each sample, a sequential injection-solid-phase extraction (SI-SPE) system was constructed. Using this device, matrix clean-up, extraction, and preconcentration of analytes were performed onto a C18 cartridge. Optimal separation and detection were obtained using a background electrolyte consisting of 100 mM phosphate adjusted to pH 6.0, with 20 mM β-CD, 5% acetonitrile and 20% isopropanol. Electrokinetic injection was performed at 5 kV for 10 s, separation voltage was 25 kV and column temperature was set to 25 °C. The separation was carried out in a 67.0 cm × 50 μm fused-silica capillary with UV detection at 214 nm. The combination of SI-SPE and sample stacking showed significant sensitivity enhancement with limits of detection in the range of 5–30 ng ml−1. A validation study showed good reproducibility of both migration time (RSD = 0.003–0.088%) and peak area (RSD = 0.54–4.8%). Overall, this automated and miniaturized SI-SPE system provides a rapid, sensitive, and robust procedure for analysis; as well as minimizes sample and solvent consumption.
Keywords: Capillary electrophoresis; Forensic analysis; Drugs of abuse; Sequential injection analysis; Solid-phase extraction;

A parallel chromatographic procedure for the purification of milligram amounts of plasmid DNA was developed. Initial studies showed that ion-exchange membrane capsules displayed high capacity for plasmid DNA. Interestingly, a weak anion exchanger (DEAE) proved to be superior to the strong quarternary ammonium group with respect to elution and regeneration properties and the 75 cm2 Sartobind D membrane capsule (MA75D, Sartorius) was selected for further studies. A method for reducing endotoxin levels by using CTAB as a precipitant was optimised. By introducing this step into the protocol, endotoxin levels could be reduced approximately 100-fold to ≤5 EU/mg plasmid. The parallel procedure was set up on a multi-channel peristaltic pump and evaluated with four different vectors (2.7–11.5 kbp). Starting with 5–10 g of E. coli cell paste (wet weight) generally saturated the membrane adsorber, resulting in plasmid DNA yields close to 10 mg.
Keywords: Purification; Plasmid DNA; Membranes; Multi-parallel;

Pseudomonas aeruginosa (PA) is the main cause of morbidity and mortality in cystic fibrosis (CF) patients. CF patients with chronic PA infections have a more rapid deterioration of their lung function and the bacteria become impossible to eradicate from the lungs. Antibiotic resistance among PA strains in CF patients is steadily increasing. Specific chicken (IgY) antibodies against PA have been shown to have potential to prevent PA infections in CF. Anti-Pseudomonas IgY reduces PA adhesion to epithelia, but the mechanism has not been fully elucidated. To gain further insight into the prophylactic effect of these antibodies, the immunoreactivity was investigated by 2D electrophoresis of PA strains, immunoblotting and MALDI-TOF-MS. To confirm the identity of the proteins, the tryptic peptides were analyzed by MALDI-TOF-MS to accurately measure their monoisotopic masses as well as determine their amino acid sequences. In order to facilitate fragmentation of the peptides they were N-terminally or C-terminally labeled. Several strains were investigated and anti-Pseudomonas IgY was immunoreactive against all of these strains, which strengthens its potential as a prophylactic treatment against PA. Flagellin was identified as the major antigen. Flagellin is the main protein of the flagella and is crucial for establishing infections in hosts as well as being involved in PA chemotaxis, motility, adhesion and inflammation. Furthermore, secreted flagellin elicits an inflammatory response. In conclusion, anti-Pseudomonas IgY binds flagellin, which may prevent PA infections in CF patients by hindering host invasion.
Keywords: 2D electrophoresis; Antigen; IgY; Mass spectrometry; Proteomics;

HPLC analysis of the novel antidepressant duloxetine in human plasma after an original solid-phase extraction procedure by Laura Mercolini; Roberto Mandrioli; Roberto Cazzolla; Mario Amore; Maria Augusta Raggi (81-87).
Duloxetine is the most recent serotonin and norepinephrine reuptake inhibitor (SNRI) drug introduced for the therapy of depression. Thus, it is evident that there is a need for having on hand new reliable analytical methods for the determination of duloxetine plasma levels in depressed patients. The present paper deals with the development of a rapid and sensitive high-performance liquid chromatographic method for duloxetine analysis in human plasma. The assays were carried out using a C8 reversed-phase column and a mobile phase composed of 60% aqueous phosphate buffer containing triethylamine at pH 3.0 and 40% acetonitrile. The UV detector was set at 230 nm and loxapine was used as the internal standard. An original pre-treatment of plasma samples was developed, based on solid-phase extraction (SPE) with mixed-mode reversed phase—strong cation exchange cartridges (30 mg, 1 mL). The extraction yields values were higher than 90%. Linearity was found in the 2–200 ng mL−1 duloxetine concentration range; the limit of quantitation was 2.0 ng mL−1 and the limit of detection was 0.7 ng mL−1. The method was applied to plasma samples from depressed patients undergoing therapy with duloxetine. Precision data and accuracy results were satisfactory and no interference from other drugs was found. Thus, the method seems to be suitable for the therapeutic drug monitoring of duloxetine in depressed patients’ plasma.
Keywords: Duloxetine; Antidepressant drug; High-performance liquid chromatography; Human plasma; Solid-phase extraction;

Analysis of the second generation antidepressant venlafaxine and its main active metabolite O-desmethylvenlafaxine in human plasma by HPLC with spectrofluorimetric detection by Roberto Mandrioli; Laura Mercolini; Roberta Cesta; Salvatore Fanali; Mario Amore; Maria Augusta Raggi (88-94).
A high-performance liquid chromatographic method has been developed for the determination of the recent serotonin and norepinephrine reuptake inhibitor (SNRI) venlafaxine and its main active metabolite, O-desmethylvenlafaxine, in human plasma. Separation was obtained by using a reversed-phase column (C8, 150 × 4.6 mm I.D., 5 μm) and a mobile phase composed of 75% aqueous phosphate buffer containing triethylamine at pH 6.8 and 25% acetonitrile. Fluorescence detection was used, exciting at λ  = 238 nm and monitoring the emission at λ  = 300 nm. Citalopram was used as the internal standard. A careful pre-treatment of plasma samples was developed, using solid-phase extraction with C1 cartridges (100 mg, 1 mL). The limit of quantification (LOQ) was 1.0 ng mL−1 and the limit of detection (LOD) was 0.3 ng mL−1 for both analytes. The method was applied with success to plasma samples taken from patients undergoing treatment with venlafaxine. Precision data, as well as accuracy results, were satisfactory and no interference from other drugs was found. Hence, the method is suitable for therapeutic drug monitoring of venlafaxine and its main metabolite in depressed patients’ plasma.
Keywords: Venlafaxine; High-performance liquid chromatography; Fluorescence detection; Human plasma; Solid-phase extraction; Therapeutic drug monitoring;

High performance liquid chromatographic measurement of iothalamate in human serum and urine for evaluation of glomerular filtration rate by Daoqin Bi; Kevin J. Leary; Julie A. Weitz; Svetlana A. Cherstniakova; Michael A. Reil; Michael J. Roy; Louis R. Cantilena (95-99).
A simple and sensitive HPLC-UV assay was developed for the measurement of iothalamate (IOT) in human serum and urine. Chromatographic separation was achieved using an embedded-carbamate-group bonded RP18 column and mobile phase consisting of 50 mM monobasic sodium phosphate and methanol (90:10, v/v) without the addition of ion-pair reagents. The assay demonstrated a high analytical reliability within the IOT concentration range of 1–150 μg/ml in serum and 25–1500 μg/ml in urine. The relative standard deviations (RSDs) for intra- and inter-day analysis were less than 5.1% in all cases. This method has been used for the evaluation of glomerular filtration rate (GFR) in subjects participating in a phase I clinical trial of a novel antimalarial medicine. The average baseline GFR was 100.41 ± 19.99 ml/min/1.73 m2 in 119 healthy volunteers. The assay may also allow the simultaneous measurements of p-aminohippuric acid (PAH), N-acetyl PAH (aPAH), and IOT with some modification. PAH, IOT, aPAH, and β-hydroxyethyl-theophylline internal standard peaks appeared approximately at 2.5, 3.7, 5.9, and 11.8 min, respectively, in an isocratic run.
Keywords: Iothalamate; BHET; GFR; HPLC-UV;

Liquid chromatography–tandem mass spectrometry analysis of protocatechuic aldehyde and its phase I and II metabolites in rat by Man Xu; Zichuan Zhang; Gang Fu; Shifeng Sun; Jianghao Sun; Min Yang; Aihua Liu; Jian Han; Dean Guo (100-107).
A method using liquid chromatography–electrospray ionization tandem mass spectrometry (LC–MS/MS) analysis was established for the identification of metabolites in rat after oral administration of protocatechuic aldehyde, a major bioactive phenolic acid in the roots of Salvia miltiorrhiza. Eleven metabolites in rat plasma and urine were firstly identified as protocatechuic aldehyde, protocatechuic acid and their methylated, glucuronized or glycine conjugates on the basis of their MS fragmentation behaviors, while nine of these metabolites (except protocatechuic aldehyde and protocatechuic acid) were detected in rat bile. In addition, the possible metabolic pathway was proposed for the first time. In the phase I metabolism, protocatechuic aldehyde could be oxidized to protocatechuic acid. The conjugates would be formed in rat intestine, liver and kidney and excreted from rat urine and bile. Enthrohepatic circulation played an important role in the metabolism of protocatechuic aldehyde. The results proved that the established method was simple, reliable and sensitive, revealing that it could be used to rapid screen and identify the structures of active components responsible for pharmacological effects of protocatechuic aldehyde and to better understand its in vivo metabolism.
Keywords: Protocatechuic aldehyde; Metabolites; HPLC–MS2; Salvia miltiorrhiza;

A novel affinity separation method in an aqueous two-phase system (ATPS) is suggested, using protein conjugated IgG as a ligand. For verification of the proposed approach, horseradish peroxidase (HRP) and human IgG was used as a ligand carrier and affinity ligand, respectively. The partition of the affinity ligand, human IgG, was controlled by the conjugation of HRP. Two ATPSs, one consisting of potassium phosphate (15%, w/w) and polyethylene glycol (PEG, M.W. 1450, 10%, w/w) and the other of dextran T500 (5%, w/w) and PEG (M.W. 8000, 5%, w/w), were used. The conjugated human IgG-HRP favored a PEG-rich top phase, whereas human IgG, rabbit anti-human IgG and goat anti-mouse IgG preferred a salt or dextran-rich bottom phase. Using the conjugated human IgG-HRP, rabbit anti-human IgG was successfully separated into a PEG-rich top phase from the mixture with goat anti-mouse IgG. The appropriate molar ratio between human IgG-HRP and rabbit anti-human IgG was around 3:1 and 1:1 for the salt and dextran-based ATPS, respectively. The dextran-based ATPS showed a better recovery yield and purity than the salt-based ATPS for the range of test conditions employed in this experiment. The yield and purity of the recovered rabbit anti-human IgG were 90.8 and 87.7%, respectively, in the dextran-based ATPS, while those in the salt-based ATPS were 78.2 and 73.2%.
Keywords: Affinity separation; Aqueous two-phase system; Protein conjugated IgG; Ligand carrier protein;

Immobilization of arginase and its application in an enzymatic chromatographic column: Thermodynamic studies of nor-NOHA/arginase binding and role of the reactive histidine residue by Teddy Bagnost; Yves-Claude Guillaume; Mireille Thomassin; Jean-François Robert; Alain Berthelot; Alain Xicluna; Claire André (113-120).
A biochromatographic approach is developed to measure for the first time changes in enthalpy, heat capacity change and protonation for the binding of nor-NOHA to arginase in a wide temperature range. For this, the arginase enzyme was immobilized on a chromatographic support. It was established that this novel arginase column was stable during an extended period of time. The affinity of nor-NOHA to arginase is high and changes slightly with the pH, because the number of protons linked to binding is low. The determination of the enthalpy change at different pH values suggested that the protonated group in the nor-NOHA–arginase complex exhibits a heat protonation of approximately −33 kJ/mol. This value agrees with the protonation of an imidazole group. Our result confirmed that active-site residue Hist 141 is protonated as imidazolium cation. Hist 141 can function as a general acid to protonate the leaving amino group of l-ornithine during catalysis. The thermodynamic data showed that nor-NOHA–arginase binding, for low temperature (<15 °C), is enthalpically unfavourable and being dominated by a positive entropy change. This result suggests that dehydration at the binding interface and charge–charge interactions contribute to the nor-NOHA–arginase complex formation. The temperature dependence of the free energy of binding is weak because of the enthalpy–entropy compensation caused by a large heat capacity change, ΔC p  = −2.43 kJ/mol/K, of arginase. Above 15 °C, the thermodynamic data ΔH and ΔS became negative due to van der Waals interactions and hydrogen bonding which are engaged at the complex interface confirming strong enzyme–inhibitor hydrogen bond networks. As well, by the use of these thermodynamic data and known correlations it was clearly demonstrated that the binding of nor-NOHA to arginase produces slight conformational changes in the vicinity of the active site. Our work indicated that our biochromatographic approach could soon become very attractive for studying other enzyme–ligand binding.
Keywords: Enzyme; HPLC; Ligand;

Oxidation of the guanosine moiety in DNA has become a hallmark biomarker in assessing oxidative stress. The oxidation of guanosine in the nucleotide triphosphate pool has been overlooked due to the lack of a reliable methodology. This method describes a sample processing and high performance liquid chromatography with electrochemical detection protocol for the analysis of the cellular pool of guanosine triphosphates and oxidized guanosine triphosphates. Validation of this method is demonstrated along with evaluation of these analytes in control and oxidizing conditions in vitro and in HEK 293T cells. Oxidation of this triphosphate pool occurred independently of oxidation to DNA.
Keywords: Nucleotide triphosphates; HPLC-EC; Oxidized nucleotides; GTP; Oxidative stress; Biomarker;

Development of a μ-turbulent flow chromatography focus mode method for drug quantitation in discovery bioanalysis by Paul Turnpenny; Daniela Fraier; Christophe Chassaing; Jonathan Duckworth (131-140).
An online turbulent flow chromatography method coupled to tandem mass spectrometry (TFC-MS/MS) has been developed within our bioanalytical group, suited to the analysis of mid to late stage discovery compounds. A dual column configuration utilising isocratic focusing of the analyte upon the analytical column maintained an excellent peak shape for a large proportion of compounds encountered and enabled consistent quantitation to sub-nanogram concentrations (<15 pg on column). Furthermore, the low sample injection volume coupled with rapid column washing using basic and acidic mobile phases, has proved advantageous in removing sample carryover and also the overall exposure to biological material; favourable for good system robustness. All the data discussed were generated with a method cycle time of 5 min providing accurate quantitation (acceptance criteria based upon FDA method validation guidelines) with multiple analytes and biological matrices.
Keywords: Focus mode; Tandem mass spectrometry; Turbulent flow chromatography (TFC); Online extraction; Low volume samples; Biological matrices; Analytical column;

A sensitive and specific method for the determination of 17α-hydroxyprogesterone caproate (17-OHPC) in human plasma using high-performance liquid chromatography and mass spectrometry has been developed and validated. Plasma samples were processed by a solid phase extraction (SPE) procedure using Oasis HLB extraction cartridge prior to chromatography. Medroxyprogesterone acetate (MPA) was used as the internal standard. Chromatography was performed using Waters C18 Symmetry analytical column, 3.5 μm, 2.1 mm × 10 mm, using a gradient elusion with a mobile phase consisting of acetonitrile [A] and 5% acetonitrile in water [B], with 0.1% formic acid being added to both [A] and [B], at a flow rate 0.2 ml/min. The retention times of 17-OHPC and MPA were 8.1 and 5.0 min, respectively, with a total run time of 15 min. Analysis was performed on Thermo Electron Finnigan TSQ Quantum Ultra mass spectrometer in a selected reaction-monitoring (SRM), positive mode using electron spray ionization (ESI) as an interface. Positive ions were measured using extracted ion chromatogram mode. The extracted ions following SRM transitions monitored were m/z 429.2 → 313.13 and 429.2 → 271.1, for 17-OHPC and m/z 385.1 → 276 for MPA. The extraction recoveries at concentrations of 5, 10 and 50 ng/ml were 97.1, 92.6 and 88.7%, respectively. The assay was linear over the range 0.5–50 ng/ml for 17-OHPC. The analysis of standard samples for 17-OHPC 0.5, 1, 2.5, 5, 10, 25 and 50 ng/ml demonstrated a relative standard deviation of 16.7, 12.4, 13.7, 1.4, 5.2, 3.7 and 5.3%, respectively (n  = 6). This method is simple, adaptable to routine application, and allows easy and accurate measurement of 17-OHPC in human plasma.
Keywords: 17-OHPC; MPA; Liquid chromatography–mass spectrometry; Pregnancy;

Lipophilicity and antiproliferative activity profiling of 2-benzylidencycloalkanones by Balázs Hallgas; Zsófia Dobos; Attila Agócs; Miklós Idei; György Kéri; Tamás Loránd; György Mészáros (148-155).
High performance liquid chromatographic (HPLC) method has been developed to separate the members of a library including 24 benzylidenecycloalkanone-type structures and to characterize their lipophilicity. The experimental lipophilicity data (k) of the compounds have been compared with their calculated lipophilicity parameters (CLOGP). In general, good correlations between the measured and calculated lipophilicities have been found and these results were in good accordance with our previously data obtained in case of structurally related molecular libraries. In addition, cytotoxicity screening has been performed to determine the antiproliferative activity of these compounds. Some of the investigated compounds possessed noticeable inhibitory potential. Based on the correlation between the antiproliferative activity and experimentally determined lipophilicity of the molecules investigated, limited structural demands to obtain more potent compounds can be exhibited to support the synthetic design.
Keywords: Cycloalkanone; Molecular library; Lipophilicity; Retention factor; CLOGP calculation; Drug-likeness; Antiproliferative screening; QSAR;

The development of a sensitive and solvent-free method for the measurement of estrone (E1) and 17β-estradiol (17β-E2) in human urine samples is described. The deconjugated estrogens were derivatized in situ with acetic acid anhydride and the derivatives were extracted directly from the aqueous samples using stir bar sorptive extraction (SBSE). The compounds containing a secondary alcohol function are further derivatized by headspace acylation prior to thermal desorption and gas chromatography/mass spectrometry (GC/MS). A number of experimental parameters, including salt addition, temperature and time, were optimized to increase the recovery of E1 and 17β-E2 by SBSE. The derivatization reactions were also optimized to obtain the highest yields of the acylated estrogens. Detection limits of 0.02 and 0.03 ng mL−1 were obtained for E1 and 17β-E2, respectively. The method was applied to determine the effect of conjugated equine estrogen intake on the excretion of E1 and 17β-E2 in human urine samples. Increased levels of the endogenous estrogens were detected after administering a standard dose of Premarin to a female volunteer. Routine monitoring of estrogen levels is recommended to avoid a high urinary excretion of E1 and 17β-E2, nowadays enlisted as endocrine disrupting chemicals (EDCs), during hormone replacement therapy.
Keywords: Stir bar sorptive extraction; Gas chromatography/mass spectrometry; Estrone; 17β-Estradiol; Conjugated equine estrogens; Urine samples;

Although it is accepted that trifluoroacetic acid (TFA) can cause suppression of an analyte during LC/MS analysis, this paper presents a relatively sensitive gradient method that uses a TFA mobile phase for the improved quantification of small, polar drug-like compounds. The described method was developed in a discovery drug metabolism and pharmacokinetics (DMPK) laboratory for the screening measurement of compound concentrations to calculate PK parameters and CNS exposure of compounds from a chemical series that had poor chromatography under generic methods using formic acid mobile phase. The samples were collected by a Culex automated sampling unit, and the plasma proteins were precipitated by a Tecan robot in 96-well plates. After centrifugation, the supernatant was removed, dried down using a SPE-Dry unit, and the samples were reconstituted in aqueous buffer on the robot. The samples were analyzed on an Agilent LC/MSD using a 5-min gradient on a 5 cm phenyl column. No additional steps, such as the “TFA-fix”, were necessary. Although sample batches were analyzed over 6 h, no drift or degradation of signal was observed. The improved chromatography resulted in a method that was selective, rugged, and had a dynamic range from 5 to 20,000 nM, which was sufficient to quantitate low volume, serial plasma samples collected out to 8 h postdose.
Keywords: Trifluoroacetic acid; Polar; Plasma; Mass selective detector; Automated; Pharmacokinetics;

A rapid and selective liquid chromatographic/tandem mass spectrometric method for determination of fosfomycin was developed and validated. Following protein-precipitation, the analyte and internal standard (fudosteine) were separated from human plasma using an isocratic mobile phase on an Ultimate™ XB-CN column. An API 4000 tandem mass spectrometer equipped with Turbo IonSpray ionization source was used as detector and was operated in the negative ion mode. Multiple reaction monitoring using the precursor to product ion combinations of m/z 137 → 79 and m/z 178 → 91 was performed to quantify fosfomycin and fudosteine, respectively. The method was linear in the concentration range of 0.10–12.0 μg/mL using 50 μL of plasma. The lower limit of quantification was 0.10 μg/mL. The intra- and inter-day relative standard deviation over the entire concentration range was less than 10.6%. Accuracy determined at three concentrations (0.25, 1.00 and 8.00 μg/mL for fosfomycin) ranged from −1.0% to −4.2% in terms of relative error. Each plasma sample was chromatographed within 5.0 min. The method was successfully used in a bioequivalence study of fosfomycin in human plasma after an oral administration of capsules containing 1.0 g fosfomycin (∼1.3 g calcium fosfomycin).
Keywords: Liquid chromatographic/tandem mass spectrometry; Fosfomycin; Plasma concentration;

An already well-described method for the determination of nitrofuran metabolites 3-amino-5-methyl-morpholino-2-oxazolidinone (AMOZ), 3-amino-2-oxazolidinone (AOZ), semicarbazide (SEM) and 1-aminohydantoin (AHD) was adapted to the needs of our laboratory and checked for its robustness regarding sample conditions and the processing step. Using the same data, the method was validated and the measurement uncertainty was estimated. All criteria and requirements of Commission Decision 2002/657/EC were fulfilled. The CCα determined lies between 0.1 and 0.7 μg/kg, the CCβ lies between 0.1 and 0.9 μg/kg, the measurement uncertainty was estimated as being between 7 and 17% taking into account matrix, time and sample preparation influences.
Keywords: Nitrofurans; Metabolite; Validation; Matrix-comprehensive in-house validation; Muscle; Shrimps; Poultry; LC–MS/MS; Residues; Measurement uncertainty; Robustness;

Quantitative determination of olmesartan in human plasma and urine by liquid chromatography coupled to tandem mass spectrometry by Dongyang Liu; Pei Hu; Nobuko Matsushima; Xiaoming Li; Li Li; Ji Jiang (190-197).
A specific, sensitive and fast method based on high performance liquid chromatography coupled to tandem mass spectrometry (HPLC–MS/MS) was developed for the determination of olmesartan in human plasma and urine. Solid-phase extraction (SPE) was used to isolate the compounds from biological matrix followed by injection of the extracts onto a C18 column with isocratic elution. The method was validated over the concentration range of 0.2–1000 and 5–10,000 ng/mL for olmesartan in human plasma and urine, respectively. The method was applied to the pharmacokinetic study of olmesartan medoxomil in healthy Chinese male and female subjects.
Keywords: Olmesartan; Urine; Plasma; Antihypertensive;

Validation and implementation of a liquid chromatography/tandem mass spectrometry assay to quantitate ON 01910.Na, a mitotic progression modulator, in human plasma by Jing Li; Ming Zhao; Antonio Jimeno; Ping He; M.V. Ramana Reddy; Manuel Hidalgo; Ross C. Donehower; Michelle A. Rudek (198-204).
A reverse-phase high performance liquid chromatographic method with tandem mass spectrometry (LC–MS/MS) was developed and validated for the quantitation of ON 01910.Na, a novel synthetic benzyl styryl sulfone, in human plasma. The assay involved a simple sample preparation with acetonitrile protein precipitation. ON 01910.Na and the internal standard temazepam were separated on a Waters X-Terra™ MS C18 column with mobile phase of acetonitrile containing 0.1% formic acid /10 mM ammonium acetate (55:45, v/v) using isocratic flow at 0.2 mL/min for 5 min. The analytes were monitored by tandem-mass spectrometry with electrospray positive ionization. Two calibration curves were generated over the range of 10–2000 ng/mL and 100–20000 ng/mL. The lower limit of quantitation (LLOQ) was 10 ng/mL for ON 01910.Na in human plasma. The accuracy and within- and between-day precisions were within the acceptance criteria for bioanalytical assays. ON 01910.Na was found stable in plasma at −70 °C for at least 1 year. The method was successfully applied to characterize the plasma concentration–time profiles of ON 01910.Na in the cancer patients in the Phase I study.
Keywords: ON 01910.Na; High performance liquid chromatography (HPLC); Mass spectrometry (MS); LC–MS/MS; Pharmacokinetics;

Urine profiling by SELDI-TOF/MS: Monitoring of the critical steps in sample collection, handling and analysis by Massimo Papale; Maria Carmela Pedicillo; Bradley J. Thatcher; Salvatore Di Paolo; Lorenzo Lo Muzio; Pantaleo Bufo; Maria Teresa Rocchetti; Marta Centra; Elena Ranieri; Loreto Gesualdo (205-213).
The topic of this study is the impact of several pre-analytical and analytical variables on proteomic profiling of human urine by surface enhanced laser desorption/ionization time of flight–mass spectrometry (SELDI-TOF–MS) in healthy subjects. Urine storage at room temperature caused a progressive degradation of proteins, which was prevented by the addition of protease inhibitors only up to 2 h from the collection. The timing of collection over the day had only a minor impact on protein profile, although influencing the intensity of peaks. Repeated freeze/thaw cycles (up to five) did not affect either the number or the intensity of the peaks. A comparison of the protein profile from eight different healthy individuals showed fairly consistent inter-subject similarities, along with between-subject differences, which were markedly dependent on the sex and the type of ProteinChip® array used. The addition of a variety of denaturing agents improved the quality of the spectra with all the chips tested (CM10, Q10 and H50), but not with the copper-coated IMAC-30 chip. Finally, SPA matrix allowed to achieve a better performance of SELDI-TOF/MS spectrum, as compared with CHCA, regardless of the ProteinChip® array used and even in the low m/z range (2500–10,000). In conclusion, we suggest that a careful choice of a number of pre-analytical and analytical conditions is required to accomplish and define a unifying protocol for the analysis of human urine by SELDI-TOF/MS, in physiological and in pathological states.
Keywords: Urine; Sample collection; SELDI-TOF/MS; Standardization;

Cytoplasmic expression is commonly used for production of recombinant human granulocyte macrophage-colony stimulating factor (rhGM-CSF) which most often comes with inclusion body formation. We expressed rhGM-CSF in periplasmic space of Escherichia coli and optimized its extraction by osmotic shock and purification by anion exchange chromatography. Our works show that MgCl2 at 2 mM in osmotic shock buffer improves extraction of the protein and reduces contamination with other proteins. To achieve a simplified purification procedure for rhGM-CSF, efforts were focused on the adjustment of pH of the buffers and application of proper concentration of salt. Following to measurement of the pI of 5.4 for rhGM-CSF by isoelectric focusing, the pH of dialysis buffer and buffers used in anion exchange chromatography were adjusted to 6.5 for optimal binding of the protein to the column and removal of proteins with higher pIs during washing of the column. In addition, it was found that appliance of NaCl at a concentration of 20 mM in dialysis and column washing buffers prior to elution with elution buffer containing 120 mM NaCl significantly improves purification of the protein. Starting with specific amount of total proteins obtained by osmotic shock, it was possible to recover 95% of which following to purification with a purification yield of 72% for rhGM-CSF along with appropriate biological activity.
Keywords: Ion exchange chromatography; Granulocyte macrophage colony-stimulating factor; Periplasmic; rhGM-CSF; Purification;

The method of high-performance liquid chromatography (HPLC) with UV–vis detection was used and validated for the simultaneous determination of six flavonoids (puerarin, rutin, morin, luteolin, quercetin, kaempferol) and troxerutin in rat urine and chicken plasma. Chromatographic separation was performed using a VP-ODS column (150 mm × 4.6 mm, 5.0 μm) maintained at 35.0 °C. The mobile phase was a mixture of water, methanol and acetic acid (57:43:1, v/v/v, pH 3.0) at the flow rate of 0.8 mL/min. Six flavonoids and troxerutin were analyzed simultaneously with good separation. On optimum conditions, calibration curves were found to be linear with the ranges of 0.10–70.00 μg/mL (puerarin, rutin, morin, luteolin, quercetin, kaempferol) and 0.50–350.00 μg/mL (troxerutin). The detection limits were 0.010–0.050 μg/mL. The method was validated for accuracy and precision, and it was successfully applied to determine drug concentrations in rat urine and chicken plasma samples from rat and chicken that had been orally administered with six flavonoids and troxerutin.
Keywords: Flavonoids; Troxerutin; Reversed-phase high-performance liquid chromatography; Rat urine; Chicken plasma;

Purification of recombinantly expressed and cytotoxic human amyloid-beta peptide 1–42 by Katja Wiesehan; Susanne Aileen Funke; Miriam Fries; Dieter Willbold (229-233).
The amyloid cascade hypothesis assigns the amyloid-beta peptide (Aβ) a central role in the pathogenesis of Alzheimer's disease (AD). Although there are strong efforts to biophysically characterize formation of Aβ aggregates and fibrils, as well as their prevention, progress is still severly hampered by the availability of tens of milligrams of recombinant Aβ(1–42). Here, we describe a reliable and easy procedure to recombinantly express and purify Aβ(1–42), which is fully cytotoxic and able to form fibrils without any further refolding steps. The yield of the procedure is 5–8 mg of tag-less peptide per liter culture volume.
Keywords: Purification; Amyloid-beta peptide; Cytotoxicity; Aggregation; Alzheimer's disease;

The presence of diadenosine oligophosphates (ApnA) in eukaryotic pathogens has been difficult technically to assess and thus is often overlooked. ApnA are a family of intercellular and intracellular signaling molecules and their biological activities differ relative to the number of phosphate moieties. The application of mass spectrometry to differentiate nucleotide phosphates has been limited by the high salt content in tissue extracts, enzymatic reactions or high performance liquid chromatography (HPLC) buffers, as well as the potential for sample loss when processing and desalting small biological samples. To address this problem a simple reverse phase HPLC (RP-HPLC) method using volatile organic buffers at low pH was developed to create elution profiles of adenosine and diadenosine phosphates. To test this method on a eukaryotic pathogen, small intravascular human filarial parasites (Brugia malayi) were extracted in phosphate buffered saline and a nucleotide phosphate profile was visualized by RP-HPLC. A major peak eluting at 10.4 min was analyzed directly by mass spectrometry and this confirmed the presence of significant quantities of diadenosine triphosphate, Ap3A. Application of this simplified RP-HPLC method will facilitate research on the normal and pathophysiological effects of ApnA particularly in situations when analysis of small biological samples is required.
Keywords: Diadenosine triphosphate; MALDI mass spectrometry; Filaria; Brugia malayi;

Poor availability of drug reference standards may severely complicate clinical and forensic toxicology investigations. To overcome this problem, a new approach is introduced for drug analysis without primary reference standards. Liquid chromatography–chemiluminescence nitrogen detection (LC–CLND) was employed as the analytical technique, based on the detector's equimolar response to nitrogen and using caffeine as single secondary standard. Liquid–liquid extraction recoveries for 33 basic lipophilic drugs were first established by LC–CLND in blood specimens spiked with the respective reference substances. The mean recovery by butyl chloride–isopropyl alcohol extraction for plasma and whole blood was 90 ± 18 and 84 ± 20%, respectively. The validity of the generic extraction recovery-corrected single-calibrant LC–CLND was then verified with proficiency test samples, including 20 different analyses. The mean accuracy was 24 and 17% for the plasma and the whole blood samples, respectively, and the maximum error was 31% for both specimens. All 20 analyses results by LC–CLND fell within the confidence range of the reference concentrations. LC–CLND proved to be an easy-to-use and robust technique, allowing analysis of 1000 injections of biological extracts without a need for major maintenance operations.
Keywords: LC–CLND; Whole blood; Plasma; Liquid–liquid extraction; Extraction recovery prediction; Single-calibrant quantification;

A simple, rapid and sensitive method was developed for routine analysis of riboflavin in beverage, green tea and urine by capillary electrophoresis with in-column optical fiber laser-induced fluorescence detection (LIF). The difference between the present detector in the study and others is that an optical fiber was adopted in the former, which can guide the excitation light into the capillary right at the detection window. The linearity of the method (r 2  = 0.998) was good over the concentration range from 0.05 to 20 μM for riboflavin. The limit of detection (LOD) was determined using linear regression analysis and was found to be 3.0 nM. The percent recoveries of riboflavin in beverage, green tea and urine samples were 95.3 ± 2.9, 105.5 ± 3.9 and 94.3 ± 1.7, respectively. These results of quantitative analysis of riboflavin in beverage and green tea samples is in agreement with that of obtained by the AOAC of fluorometric method. In the analysis of urine samples, all electropherograms of urine samples and corresponding concentrations of riboflavin in the period of 13 h after orally administrating the ingestion of vitamin B2 tablets were illustrated.
Keywords: Capillary electrophoresis; Laser-induced fluorescence detection; Optical fiber; Riboflavin; Urine; Beverage;

Quantitation of five nevirapine oxidative metabolites in human plasma using liquid chromatography–tandem mass spectrometry by Lois S. Rowland; Thomas R. MacGregor; Scot J. Campbell; Rand Jenkins; Amy B. Pearsall; Jennifer P. Morris (252-260).
A multiple-reaction-monitoring LC/MS/MS method for the analysis of nevirapine oxidative metabolites, 2-hydroxynevirapine, 3-hydroxynevirapine, 8-hydroxynevirapine, 12-hydroxynevirapine, and 4-carboxynevirapine, in human plasma was developed and validated. The metabolites were isolated from 50 μL heparinized plasma by enzymatic hydrolysis of the glucuronide conjugates to the free metabolite followed by protein precipitation with acetonitrile. Peaks were quantitated at 3.03 min for the 4-carboxynevirapine metabolite, at 3.72, 4.27, 5.27, and 5.73 min for the positional 2-hydroxynevirapine, 12-hydroxynevirapine, 3-hydroxynevirapine, and 8-hydroxynevirapine metabolites, respectively, and 2.30 min for the internal standard, pirenzepine. The assay was accurate and precise based on assay validation controls over the nominal range of 0.010–1.0 mg/L. The average accuracy at the lowest concentration quality control (QC) sample was 16% (difference from theoretical value) for 8-hydroxynevirapine, all others were closer to their known respective standards. Within- and between-day precisions were within 12% for quality control samples for all five metabolites. Repetitive thawing and freezing did not have an effect on any metabolite through a minimum of three cycles. Thawed samples, remaining in plasma for 4 h before extraction, were within 5% of theoretical value. Stability of the extracted samples on the autosampler at room temperature was evaluated for 48 h and was observed to be within 12% of a fresh analytical sample for 2-hydroxynevirapine and 3-hydroxynevirapine; other metabolites were within 6% of theoretical value. The utility of the analytical method was demonstrated using trough steady-state plasma samples collected from 48 patients in a hepatic impairment study.
Keywords: Nevirapine metabolites; 2-Hydroxynevirapine; 3-Hydroxynevirapine; 8-Hydroxynevirapine; 12-Hydroxynevirapine; 4-Carboxynevirapine;

A sensitive and rapid LC–MS/MS assay for the quantitative determination of 5-methylindirubine (5-MI) in murine plasma is described. A 50-μL-murine plasma aliquot was spiked with an internal standard, indirubine-3-monoxime (IMO), and extracted with 1.25 mL diethyl ether. Dried extracts were reconstituted in methanol–water (8:2, v/v) and 10 μL-volumes were injected onto the HPLC system. Separation was achieved on a Gemini C18 column (150 mm × 2.1 mm ID, particle size 5 μm) using an alkaline eluent (10 mM ammonium hydroxide–methanol (5:95, v/v)). Detection was performed by negative ion electrospray followed by tandem mass spectrometry. The assay quantifies 5-MI in a range from 1 to 500 ng/mL using 50 μL of murine EDTA plasma samples. Validation results demonstrate that 5-MI concentrations can be accurately and precisely quantified in murine plasma. This assay is used to support pre-clinical pharmacologic studies with 5-MI.
Keywords: 5-MI; Indirubin; Cyclin-dependent kinases; Liquid chromatography; Tandem mass spectrometry;

A validated, quantitative LC–APCI-MS/MS method for methadone, EDDP and EMDP in 200-μL plasma is presented. Specimen preparation was limited to protein precipitation and centrifugation. Chromatographic separation was achieved on a Synergi Hydro-RP 80A (50 mm × 2.0 mm, 4 μm) column with gradient elution. The assay was linear from 1 to 500 ng/mL, with intra- and inter-assay accuracy ≥87.5% and intra- and inter-assay precision <13.4% R.S.D. and recovery ≥87.5% for all analytes at 40 ng/mL. This analytical method is suitable for the accurate and precise determination of methadone and metabolites in human plasma specimens.
Keywords: Methadone; Plasma; LC–APCI-MS/MS; EDDP; EMDP;

Determination of arbidol in rat plasma by HPLC–UV using cloud-point extraction by Xiao Liu; Xiao-Hui Chen; Yuan-Yuan Zhang; Wen-Tao Liu; Kai-Shun Bi (273-277).
A method based on cloud-point extraction (CPE) was developed to determine arbidol in rat plasma by high performance liquid chromatography separation and ultraviolet detection (HPLC–UV). The non-ionic surfactant Triton X-114 was chosen as the extract solvent. Variable parameters affecting the CPE efficiency were evaluated and optimized. A Zorbax SB-C18 column (4.6 mm i.d. × 150 mm, 5 μm particle size) was used for isocratic elution separation at 40 °C with detection wavelength at 316 nm. Under the optimum conditions, the method was shown to be reproducible and reliable with intraday precision below 6.6%, interday precision below 8.8%, accuracy within ±5.0% and mean extraction recovery more than 89.7%, which were all calculated using a range of spiked samples at three concentrations of 0.2, 2 and 16 μg/ml for arbidol in plasma. The linear range was from 0.08 to 20 μg/ml. After strict validation, the method was successfully applied to the pharmacokinetic study of arbidol in rats after oral and intravenous administration, respectively.
Keywords: Cloud-point extraction; Arbidol; HPLC–UV; Triton X-114; Pharmacokinetics;

A method for determining triazine herbicides in infant nutrient cereal-based foods by pressurized microwave-assisted extraction (PMAE), coupled with high-performance liquid chromatography–electrospray ionization mass spectrometry (HPLC–ESI/MS), is described. The key parameters of PMAE, including extraction solvent, extraction time and temperature, were optimized. The isolation of the target compounds from the matrix was found to be efficient when 2 g of nutrient cereal samples was extracted with 20 mL of methanol for 10 min at 105 °C. Final determination was accomplished by HPLC–ESI/MS. The recoveries from 66.2 to 88.6% were obtained for three compounds at fortification levels (5–500 μg kg−1) with relative standard deviations (R.S.D.) ≤12.62%. Compared with atmospheric pressure microwave-assisted extraction (AMAE), ultrasonic extraction (UE) and soxhlet extraction (SE), the proposed method was more efficient, faster and more straightforward and required no additional cleanup steps. When the proposed method was applied to the aged spiked nutrient cereal samples, the results indicated that, although the recoveries of analytes were much lower than those obtained from fresh spiked samples, they were nevertheless satisfactory for the quantitative analysis of practical samples.
Keywords: Extraction methods; Infant nutrient cereal-based foods; Triazines;

Salmeterol is an inhaled bronchodilator drug used for treatment of asthma. Its concentrations in plasma are very low or undetectable by previously developed methods. The present paper describes a method for analysis of salmeterol in human plasma with 2.5 pg/mL lower limit of quantitation. Despite the basic character of the drug the method uses mixed mode anion-exchange solid phase extraction for sample preparation combined with a column switching approach to minimize matrix effects. Samples are separated and detected by LC/MS/MS. The method is easy to use, only requires 0.5 mL of plasma and was validated for use in bioanalytical applications. The method does not suffer from interference from co-administered fluticasone propionate.
Keywords: Salmeterol; Human plasma; Matrix effects; Liquid chromatography; Mass spectrometry; Solid-phase extraction;

A high-performance micellar electrokinetic capillary chromatography (MEKC) has been demonstrated for the determination of meropenem in human plasma and in cerebrospinal fluid (CSF) and application in meningitis patients after intravenous (IV) administration. Plasma sample was pretreated by means of solid-phase extraction (SPE) on C18 cartridge and CSF sample was by direct injection without sample pretreatment, with subsequent quantitation by MEKC. The separation of meropenem was carried out in an untreated fused-silica capillary (40.2 cm × 50 μm I.D., effective length 30 cm) and was performed at 25 °C using a background electrolyte consisting of Tris buffer (40 mM, pH 8.0) solution with sodium dodecyl sulfate (SDS) as the running buffer and on-column detection at 300 nm. Several parameters affecting the separation and sensitivity of the drug were studied, including pH, the concentrations of Tris buffer and surfactant. Using cefotaxime as an internal standard (IS), the linear ranges of the method for the determination of meropenem in plasma and in CSF were all over 0.5–50 μg/mL; the detection limits (signal-to-noise ratio = 3) of meropenem in plasma and in CSF were 0.2 μg/mL and 0.3 μg/mL, respectively.
Keywords: Meropenem; Plasma and cerebrospinal fluid; MEKC; Bacterial meningitis;

PR-104 is a dinitrobenzamide mustard pre-prodrug that is activated by reduction to a cytotoxic hydroxylamine metabolite in hypoxic tumour cells; it has recently commenced Phase I clinical trial. Here, we report two validated methods for the determination of PR-104 and its alcohol hydrolysis product, PR-104A in plasma and tissues across species. A high pH LC/MS/MS method was optimised for rapid and sensitive analysis of both analytes in rat, dog and human plasma. This assay was linear over the concentration range 0.005–2.5 μg/ml for PR-104 and 0.05–25 μg/ml for PR-104A (0.005–2.5 μg/ml for rat). A second method, using a low pH LC separation, was designed to provide higher chromatographic resolution, facilitating identification of metabolites. Both methods were successfully applied to the plasma pharmacokinetics of PR-104 and PR-104A in rats. In addition, cytotoxic reduced metabolites of PR-104A were identified in human tumour xenografts by the higher chromatographic resolution method.
Keywords: Nitrogen mustards; Prodrugs; PR-104; PR-104A; Metabolite identification;

Simultaneous determination of rivanol and mifepristone in human plasma by a HPLC-UV method with solid-phase extraction by Zhiyong Guo; Danyi Wei; Gengxin Yin; Sui Wang; Shasha Zhao; Yun Chu; Jinxia Zhai (312-317).
A HPLC method with UV detection was developed and validated for the simultaneous determination of rivanol and mifepristone in human plasma. Norethisterone was used as the internal standard. Separation was performed by a C18 reversed-phase column maintained at 20 °C. The mobile phase was a mixture of methanol–acetonitrile–0.05% sodium dodecylsulfonate in a 0.05 M phosphate buffer with the pH adjusted to 3.0 (30:30:40, v/v/v) at a flow rate of 0.8 ml/min. Dual wavelength mode was used, with mifepristone monitored at UV 302 nm, while rivanol and norethisterone at 272 nm. A reliable biological sample pre-treatment procedure by means of solid-phase extraction was used, which allowed to obtain good extraction efficiency (>93%) for both of the analytes and the internal standard. The calibration curves were both linear with the correlation coefficient r equal to 0.9999. For rivanol, the assay gave CV% values for precision always lower than 7.8% and mean accuracy values higher than 95.3%. As to mifepristone, precision was always lower than 10.1% and mean accuracy values were higher than 93.8%. The limit of detection for the assay of rivanol and mifepristone was 1.1 and 3 ng/ml, respectively. The method is simple, sensitive and accurate, and allow for simultaneous determination of nanogram levels of rivanol and mifepristone in human plasma. It could be applied to assess the plasma level of rivanol and mifepristone in women undergoing polypharmacy with the two drugs.
Keywords: Rivanol; Mifepristone; Human plasma; High performance liquid chromatography; Solid-phase extraction;

Type-2 diabetes is a disorder characterized by disrupted insulin production leading to high blood glucose levels. To control this disease, combination therapy is often used. Hypoglycemic agents such as metformin, glipizide, glyburide, repaglinide, rosiglitazone, nateglinide, and pioglitazone are widely prescribed to control blood sugar levels. These drugs provide the basis for the development of a quantitative multianalyte bioanalytical method. As an example, a highly sensitive and selective multi-drug method based on liquid chromatography tandem mass spectrometry (LC–MS/MS) was developed. This rapid, automated method consists of protein precipitation of 20 μL of plasma coupled with gradient HPLC elution of compounds using 10 mM ammonium formate buffer and 0.1% formic acid in acetonitrile as the mobile phases. MS/MS detection was performed using turbo ion spray in the positive ion multiple reaction monitoring (SRM) mode. A lower limit of quantitation (LLQ) in a range of 1.0–5.0 ng/mL was achieved for all analytes. The linearity of the method was observed over a 500-fold dynamic range. Drug recoveries ranged from 86.2 to 94.2% for all analytes of interest. Selectivity, sample dilution, intra-day and inter-day accuracy and precision, and stability assessment were evaluated for all compounds.
Keywords: Chromatography; Mass spectrometry; Hypoglycemic; Drugs; GLP; Analytical; Chemistry;

A sensitive and selective high-performance liquid chromatographic (HPLC) method has been developed for the simultaneous determination of bufuralol enantiomers in plasma and pharmaceutical formulations. Enantiomeric resolution was achieved on a vancomycin macrocyclic antibiotic chiral stationary phase (CSP) known as Chirobiotic V with UV detection set at 254 nm. The polar ionic mobile phase (PIM) consisting of methanol–glacial acetic acid–triethylamine (100:0.015:0.010, v/v/v) has been used at a flow rate of 0.5 ml/min. The method is highly specific where other coformulated compounds did not interfere. The stability of bufuralol enantiomers under different degrees of temperature was also studied. The results showed that the drug is stable for at least 7 days at 70 °C. The method was validated for its linearity, accuracy, precision and robustness. An experimental design was used during validation to evaluate method robustness. The calibration curves in plasma were linear over the range of 5–500 ng/ml for each enantiomer with detection limit of 2 ng/ml. The mean relative standard deviation (RSD) of the results of within-day precision and accuracy of the drug were ≤10%. There was no significant difference (p  > 0.05) between inter- and intra-day studies for each enantiomer which confirmed the reproducibility of the assay method. The mean extraction efficiency for S-(−)- and R-(+)-bufuralol from plasma was in the range 97–102% at 15–400 ng/ml level for each enantiomer. The overall recoveries of bufuralol enantiomers from pharmaceutical formulations was in the range 99.6–102.2% with %RSD ranging from 1.06 to 1.16%. The assay method proved to be suitable as chiral quality control for bufuralol formulations by HPLC and for therapeutic drug monitoring.
Keywords: Bufuralol enantiomers; Vancomycin chiral stationary phase; Plasma and pharmaceutical formulations;

A high-performance liquid chromatographic method for the determination of trimetazidine dihydrochloride (TMZ) in spiked human plasma is described. The method is based on the pre-column derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl) using the fluorimetric detection technique. Fluoxetine HCl (FLX) was used as internal standard. Both, TMZ and FLX were completely derivatized after heating at 50 °C for 20 min in borate buffer pH 8.0. Samples were analyzed by high performance liquid chromatography (HPLC) using Zorbax-TMS column (250 mm × 4.6 mm, i.d., 5 μm) and mobile phase consist of acetonitrile, methanol and 20 mM sodium acetate pH 4.7 (44:6:50; v/v/v). Fluorescence detector (FLD) was adjusted at excitation and emission wavelengths; 265 and 311 nm, respectively. The linearity of the method was in the range of 4.5–200 ng/ml. Limits of detection (LOD) and quantification (LOQ) were 1.5 and 4.5 ng/ml, respectively. Trimetazidine recovery was 96.5 ± 1.3% (n  = 6; RSD = 2.1%).
Keywords: Trimetazidine; Fluoxetine; FMOC; Plasma; Chromatography;

Endotoxin reduction in monoclonal antibody preparations using arginine by Ulrika Ritzén; Joke Rotticci-Mulder; Patrik Strömberg; Stefan R. Schmidt (343-347).
A monoclonal antibody preparation was found to be contaminated with endotoxin. Several commercial endotoxin removal steps were attempted but failed to produce a significant reduction due to the fact that the endotoxin was associated with the antibody. Here, several methods for endotoxin removal based on immobilizing monoclonal antibodies to chromatographic media have been evaluated. A crucial step in this process was to dissociate the endotoxin from the protein surface for subsequent removal. This was accomplished by introducing different buffer additives in the mobile phase. In agreement with previous reports, non-ionic detergents efficiently removed endotoxin, but it was also found that 0.5 M arginine performed equally well. Since arginine is a non-toxic common amino acid that can be readily removed, it was selected and successfully used in large-scale experiments. With this method, endotoxin could be reduced to <0.2 EU mg−1 with recovery of the target protein being >95%. Since this procedure is easily integrated into the existing processes of mAb purification, it offers advantages in speed, cost and effort.
Keywords: Endotoxin removal; Arginine; Affinity chromatography; Antibody purification;

Analysis of sofalcone in human plasma by high performance liquid chromatography by Aidong Wen; Zhipeng Wang; Taijun Hang; Yanyan Jia; Tingting Zhang; Yin Wu; Xuan Gao; Zhifu Yang (348-352).
A simple, rapid, sensitive and reliable high performance liquid chromatography (HPLC) method for the determination of the anti-ulcer drug sofalcone in human plasma was developed. Plasma was extracted with ethyl acetate under acidic conditions and sofalcone was determined by HPLC using a C18 column and (methanol–0.1% formic acid aqueous 80:20) mobile phase. The linear calibration curves of sofalcone in human plasma were obtained over the concentration range of 0.01–5.0 μg/ml. The lower limit of quantitation (LLOQ) was 10 ng/ml in human plasma. The precision measured for plasma was within 15%. Extraction recovery was over 85% in blood. The method was successfully applied to the identification and quantification of sofalcone in pharmacokinetic studies.
Keywords: Sofalcone; High performance liquid chromatography; Plasma;

Purification of bovine hemoglobin via fast performance liquid chromatography by Michael L. Dimino; Andre F. Palmer (353-357).
Bovine hemoglobin (bHb) was purified from bovine red blood cells (bRBCs) via anion exchange chromatography preceded by dialysis. This is a fast and effective way to obtain bHb from bRBCs using Q Sepharose XL, a strong anion exchange resin. This resin had double the binding capacity for bHb compared to three other anion exchange resins that were studied in this work. Methemoglobin levels remained below 2% with bHb concentrations between 0.7 and 1.7 mM. The high purity of bHb was confirmed via SDS-PAGE and size exclusion chromatography (SEC).
Keywords: Hemoglobin; Purification; Anion exchange chromatography; Oxygen carrier; Blood substitute;

Measurement of ribavirin and evaluation of its stability in human plasma by high-performance liquid chromatography with UV detection by Arianna Loregian; Maria Cristina Scarpa; Silvana Pagni; Saverio Giuseppe Parisi; Giorgio Palù (358-364).
A simple high-performance liquid chromatography method for the determination of the antiviral agent ribavirin in human plasma was developed and validated. The method involved solid-phase extraction on phenyl boronic acid cartridges, a reversed-phase liquid chromatography with a Waters Atlantis dC18 (150 mm × 3.9 mm, 5 μm) column and a mobile phase consisting of 10 mM potassium phosphate buffer (pH 4.0), and ultraviolet detection at 207 nm. This assay proved to be sensitive (lower limit of quantification of 0.05 μg/ml), linear (correlation coefficients ≥0.997), specific (no interference with various potentially co-administrated drugs), reproducible (both intra-day and inter-day coefficients of variation ≤4.3%), and accurate (deviations ranged from −5.6 to 2.2% and from −6.0 to 4.0% for intra-day and inter-day analysis, respectively). The method was applied to therapeutic monitoring of patients undergoing ribavirin treatment for hepatitis C and proved to be robust and reliable. Thus, this method provides a simple, sensitive, precise and reproducible assay for dosing ribavirin that can be readily adaptable to routine use by clinical laboratories with standard equipment. In addition, we evaluated the stability of ribavirin in plasma under various conditions, since no detailed study on thermal stability of ribavirin has been reported so far and discrepant data do exist on ribavirin stability upon conditions that clinical samples commonly experience. Ribavirin was stable in human plasma stored at room temperature for at least 24 h or at −20 °C for up to 1 month, after three freeze–thaw cycles, as well as in samples undergoing heat inactivation of infectious viruses for 60 min at 60 °C. The drug was also stable in processed samples stored at −20 °C for 3 days (as dried extracts) or at 20 °C for 4 days (as reconstituted samples).
Keywords: Ribavirin; Human plasma; Solid-phase extraction; High-performance liquid chromatography; UV method; Ribavirin stability; Therapeutic drug monitoring;

On-line coupling of solid-phase extraction and capillary electrophoresis for the determination of cefoperazone and ceftiofur in plasma by Patricia Puig; F.W. Alexander Tempels; Francesc Borrull; Marta Calull; Carme Aguilar; Govert W. Somsen; Gerhardus J. de Jong (365-370).
We present a method for determining two cephalosporins (cefoperazone and ceftiofur) in plasma by on-line solid-phase extraction (SPE)—capillary zone electrophoresis (CZE) with a T-split interface. Using this interface, a part of the SPE elution plug containing the cephalosporins is injected while the rest of the sample is flushed to waste. SPE was carried out using a C18 micro-precolumn and the cephalosporins presented good retention properties with breakthrough volumes above 1 ml. Using a desorption volume of 426 nl of acetonitrile, recoveries were 75 and 90%, for cefoperazone and ceftiofur, respectively. The resulting elution volume was about 1.8 μl. A deproteinization step was included prior to SPE for the analysis of plasma samples with recoveries of 90 and 57% for cefoperazone and ceftiofur, respectively. With UV detection at 254 nm, linear relationships between the injected concentration and peak area was measured between 10 and 500 ng ml−1 for standards, and 200 and 1500 ng ml−1 for plasma samples. Intra-day (n  = 5) and inter-day (n  = 5) peak area repeatability were lower than 12% RSD. The detection limits obtained for spiked plasma (100 ng ml−1 cefoperazone and ceftiofur) are sufficient for applying the method to pharmacokinetic studies.
Keywords: Solid-phase extraction; Capillary electrophoresis; On-line; Cephalosporins; Plasma samples;

High-performance liquid chromatography with ultraviolet detection for real-time therapeutic drug monitoring of meropenem in plasma by Kayo Ikeda; Kazuro Ikawa; Norifumi Morikawa; Mizuka Miki; Shin-Ichiro Nishimura; Masao Kobayashi (371-375).
A simple, rapid, and precise high-performance liquid chromatography (HPLC) method using ultrafiltration to remove plasma protein was developed to determine meropenem concentrations in human plasma in a clinical setting. Plasma was separated by centrifugation at 4 °C from blood collected in heparinized vacuum tubes, and meropenem was stabilized by immediately mixing the plasma with 1 M 3-morpholinopropanesulfonic acid buffer (pH 7.0) (1:1). Ultrafiltration was used for plasma deproteinization. Meropenem was detected by ultraviolet absorbance at 300 nm with no interfering plasma peak. The calibration curve of meropenem in human plasma was linear from 0.05 to 100 μg/mL. Intraday and interday precision was less than 7.17% (CV), and accuracy was between 97.7% and 106.3% over 0.05 μg/mL. The limit of detection was 0.01 μg/mL. The assay has been clinically applied to a real-time therapeutic drug monitoring in pediatric patients and pharmacokinetic studies.
Keywords: Meropenem; Ultrafiltration; HPLC; Therapeutic drug monitoring; Pediatrics;

Development and validation of a LC–MS/MS method for the determination of viaminate in human plasma by Jing Yin; Kun-Yi Ni; Yue Shen; Peng-Cheng Ma; Ling Cao; Wei-Peng Wang; Yu Wang (376-380).
A sensitive and specific method for determination of viaminate in human plasma by using high-performance liquid chromatography coupled with electrospray tandem mass spectrometry (LC–MS/MS) was developed in this study. The plasma samples were simply deproteinated, extracted, evaporated, and then reconstituted in 200 μl of methanol prior to analysis. Chromatographic separation was carried out on a Shimadzu VP-ODS column (250 mm × 2.0 mm, 5 μm) with a mobile phase of methanol–water (95:5, v/v) at a flow rate of 0.2 ml/min. Quantification was performed in the negative-ion electrospray ionization mode by selected ion monitoring of the product ions at m/z 164 for viaminate and m/z 109 for testosterone propionate which was used as the internal standard. The corresponding parent ions were m/z 446 and m/z 345. A linear calibration curve was observed within the concentration range of 0.10–200 ng/ml. The lowest limit of quantitation (LLOQ) was 0.1 ng/ml. The extraction-efficiency at three concentrations was 100.7, 93.6, and 99.7%. Practical utility of this new LC–MS/MS method was confirmed in pilot pharmacokinetic studies in humans following oral administration.
Keywords: LC–MS/MS; Viaminate; Human plasma;

A capillary zone electrophoresis method has been developed for the direct determination of norfloxacin in the physiological perfusate of isolated rat liver. Norfloxacin and the internal standard triamterene were detected using laser-induced fluorescence (LIF) detection with the excitation and emission wavelength of 325 and 435 nm, respectively. The background electrolyte (BGE) was 50 mM phosphate buffer (pH 4.6). The effect of pH and concentration of BGE on the electrophoretic migration and fluorescence response of analytes were examined. Calibration curves were linear over a wide range of 0.01–100 μg/mL. The limit of quantitation was 0.01 μg/mL. The intra- and inter-day relative standard deviation was 3.7%, or less, and the accuracy was 93.2% of the nominal concentration. No endogenous substances were found to interfere. The method was used to characterize the steady-state and transient pharmacokinetics of norfloxacin in the rat liver.
Keywords: Capillary electrophoresis; Hepatic disposition; Laser-induced fluorescence; Norfloxacin; Perfused rat liver;