Journal of Chromatography B (v.868, #1-2)

Highly active antiretroviral therapy (HAART) is the common treatment strategy for human immunodeficiency virus (HIV)-infected patients at present. Generally, HAART regimens apply multitherapy drugs that contain nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs), non-nucleoside/nucleotide reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs). Unlike NNRTIs and PIs, the active form of NRTIs is not the drug itself but its triphosphorylated (TP) metabolites in intracellular medium. Analysis of both the prodrugs or NRTIs and their intracellular metabolites is needed to provide overall information in pharmacokinetic and therapeutic effects to HIV-infected patients. Numerous publications have reported the assays for NRTIs and their phosphorylated metabolites in various biological matrices. The methods involved liquid chromatography (LC) with UV detection (LC-UV), LC with tandem mass spectrometry (LC–MS/MS), capillary electrophoresis/electrochromatography (CE/CEC) with UV detection (CE/CEC-UV) or/and MS/MS detection (CE-MS/MS). Due to the extremely low concentration of NRTIs and the phosphorylated metabolites as well as the complex biological matrices, sample pretreatment methods such as protein precipitation (PP), liquid–liquid extraction (LLE) and solid-phase extraction (SPE) have played important role in the successful analytical method development.
Keywords: Nucleoside reverse transcriptase inhibitors; Phosphorylated metabolites; High performance liquid chromatography; Tandem mass spectrometry; Electrophoresis/electrochromatography;

A sensitive and efficient liquid chromatography–mass spectrometry method was developed and validated for the simultaneous determination of two active chromones (prim-O-glucosylcimifugin and 4′-O-d-glucosyl-5-O-methylvisamminol) from Saposhnikovia root in rat plasma and urine. The plasma or urine samples were prepared by protein precipitation. Chromatographic separation of the two active chromones from matrix interferences was achieved on an Angilent TC-C18 column with a mobile phase consisted of methanol, water and 0.1% formic acid. Puerarin was added as the internal standard. The method was validated with the concentration range 1.0–100 ng/mL in rat plasma and 10–1000 ng/mL in urine for prim-O-glucosylcimifugin, 1.5–150 ng/mL in plasma and 15–1500 ng/mL in urine for 4′-O-d-glucosyl-5-O-methylvisamminol. The lower limit of quantitation (LLOQ) of prim-O-glucosylcimifugin and 4′-O-d-glucosyl-5-O-methylvisamminol was 1.0 and 1.5 ng/mL in plasma, 10 and 15 ng/mL in urine, respectively. The intra- and inter-day precision across three validation days over the entire concentration range was lower than 9.0% as terms of relative standard deviation (R.S.D.). Accuracy determined at three quality control concentrations (2.0, 25 and 75 ng/mL for prim-O-glucosylcimifugin; 3.0, 37.5 and 112.5 ng/mL for 4′-O-d-glucosyl-5-O-methylvisamminol) ranged from −1.9 to 3.9% as terms of relative error (R.E.). The LC–ESI–MS method was further applied to assess pharmacokinetics and urine excretion of the two chromones after oral administration of Fangfeng extract to rats. Practical utility of this new LC–MS method was confirmed in pilot pharmacokinetic studies in rats following oral administration.
Keywords: Saposhnikovia divaricata (Turcz.) Schischk.; Prim-O-glucosylcimifugin; 4′-O-d-glucosyl-5-O-methylvisamminol; LC–MS; Pharmacokinetics;

Many studies have demonstrated that the statin beneficial effects on cardiovascular diseases like coronary are linked to their hypocholesterolemic properties. These lipid-lowering drugs are the first-line pharmacologic therapy for hypercholesterolemia. In this paper, the interaction of a series of statin molecules STCOOH (pravastatin (prava), mevastatin (meva), simvastatin (simva) and fluvastatin (fluva)) with a phosphatidylcholine monolayer immobilized on to porous silica particles has been studied using a biochromatographic approach (molecular chromatography). The immobilized artificial membrane (IAM) provided a biophysical model system to study the binding of the statin molecules to a lipid membrane. For all the test statin molecules, linear retention plots were observed at all temperatures. An analysis of the thermodynamics (i.e., enthalpy (ΔH°), entropy (ΔS°*)) of the interaction of the statin molecules with the immobilized monolayer was also carried out. The ΔH° and ΔS°* values were negative due to van der Waals interactions and hydrogen bonding between the statin molecules with the polar head groups of the phospholipid monolayer (polar retention effect). The statin elution order was: Prava ⋘ Meva < Atorva ≪ Simva < Fluva. This result associated with IC50 data of each statin molecule confirmed that pravastatin, which exhibited the lowest association with the lipid monolayer, was taken up by a membrane transporter. In addition, the logarithm of the statin retention factor with the lipid membrane extrapolated to a total aqueous bulk solvent at pH 7.0 ( log k ′ w − IAM ) was measured and compared to the octanol–water partition coefficient (log  P). The observed significant correlation showed an affinity enhanced with the increase in the molecule lipophilicity and confirmed that the hydrophobic forces played an important role in the statin molecule–biomembrane association mechanism. As well, the affinity of STCOOH to IAM is high and changes slightly with the bulk solvent pH, because the number of protons linked to binding is low. This confirmed the importance to take into account the electrostatic interaction in this association mechanism. At pHs lower than ≈7.0, the binding process is accompanied by protons release and at higher pHs protons are taken up. A change in the phospholipid monolayer phosphate group pK a has been proposed to contribute to the positive number of protons exchanged at pHs higher than ≈7.0. This demonstrated that this phosphate residue could function as a general base/proton shuttle by facilitating the deprotonation of STCOOH, i.e., this hyper-reactive residue in the IAM surface could play a catalytic function.
Keywords: Statins; HPLC; IAM; Binding; Thermodynamic;

Urinary 5-HIAA measurement using automated on-line solid-phase extraction–high-performance liquid chromatography–tandem mass spectrometry by Wilhelmina H.A. de Jong; Kendon S. Graham; Elisabeth G.E. de Vries; Ido P. Kema (28-33).
Quantification of 5-hydroxyindole-3-acetic acid (5-HIAA) in urine is useful for diagnosis and follow-up of patients with carcinoid tumors and for monitoring serotonin (5-hydroxytryptamine) metabolism in various disorders. We describe an automated method (XLC–MS/MS) that incorporates on-line solid-phase extraction (SPE), high-performance liquid chromatography (HPLC) and tandem mass spectrometric (MS/MS) detection to measure urinary 5-HIAA. Automated pre-purification of urine was carried out with HySphere-Resin GP® SPE cartridges containing strong hydrophobic polystyrene resin. The analyte (5-HIAA) and internal standard (isotope-labelled 5-HIAA-d 2) were, after elution from the cartridge, separated by reversed-phase HPLC and detected with tandem MS. Total cycle time was 5 min. 5-HIAA and its deuterated internal standard (5-HIAA-d 2) were retained on and eluted from the SPE cartridges in high yields (81.5–98.0%). Absolute recovery was 96.5–99.6%. Intra-assay (n  = 20) and inter-assay (n  = 20) CVs for the measurement of 5-HIAA in urine in three concentration levels ranged from 0.8 to 1.4% and 1.7 to 4.2%, respectively. For urine samples from patients (n  = 78) with known or suspected metastatic carcinoid tumors, results obtained by XLC–MS/MS were highly correlated (R 2  = 0.99) with the routinely used fluorometric method. This XLC–MS/MS method demonstrated lower imprecision and time per analysis (high-throughput) than manual solvent extraction methods and higher sensitivity and specificity than non-mass spectrometric detection techniques.
Keywords: 5-HIAA; Automation; On-line; Mass spectrometry; Urine; Solid-phase extraction; Analysis;

A simple, sensitive and selective liquid chromatographic–tandem mass spectrometric (LC–MS/MS) method was developed for the determination of tacrolimus (FK506) in rabbit aqueous humor. After a simple protein-precipitation by methanol, the post-treatment samples were separated on a reversed-phase, Thermo-Hypersil-BDS-C18 column with a mobile phase of a mixture of 0.1% formic acid in water, methanol and acetonitrile (5:85:10, v/v/v). Tacrolimus and ritonavir (internal standard, IS) were all detected by the selected reaction-monitoring (SRM) mode. The method developed was validated in rabbit aqueous humor with a daily working range of 0.5–100 ng/ml with correlation coefficient, r  > 0.99 and a sensitivity of 0.5 ng/ml as lower limit of quantification, respectively. This method was fully validated for the accuracy, precision, possible matrix effect and stability. The method proved to be accurate and specific, and was applied to the pharmacokinetic study of tacrolimus in rabbit aqueous humor.
Keywords: Tacrolimus; LC–ESI-MS/MS; Aqueous humor;

Analysis of tetracycline residues in royal jelly by liquid chromatography–tandem mass spectrometry by Jin-Zhong Xu; Tao Ding; Bin Wu; Wen-Quan Yang; Xiao-Yan Zhang; Yan Liu; Chong-Yu Shen; Yuan Jiang (42-48).
A confirmatory method coupling liquid chromatography with tandem mass spectrometry (LC/MS/MS) was developed to determine the concentration of oxytetracycline (OTC), tetracycline (TC), chlortetracycline (CTC) and doxycycline (DC), which make up the tetracycline (TC) groups present in royal jelly. Sample preparation included deproteination, control of pH, extraction and clean-up on a solid-phase extraction (SPE) cartridge. The analyses were achieved by LC/MS/MS in selected reaction monitoring mode (SRM). The overall recovery of fortified royal jelly at the levels of 5.0, 10.0 and 40.0 μg/kg ranged from 62% to 115%, and the coefficients of variation ranged from 3.4% to 16.3% (n  = 6). The detection limits for TCs were under 1.0 μg/kg. The transformation between the TCs and its epimers (EpiTCs) was studied in standard solution and during the sample preparation process. This method can be used for the detection of tetracycline residues in royal jelly.
Keywords: Tetracycline; Residues; Royal jelly; Liquid chromatography–tandem mass spectrometry;

The recent commercial availability of small particle packed columns (<2 μm) and associated instrumentation capable of withstanding the high pressures of such columns, has lead to an increase in the application of so called ultra-performance liquid chromatography. The improved efficiency, resolution and peak capacity of these columns, when coupled to mass spectrometry, provides particular benefit for the identification of drug metabolites in complex biological samples. In this work, the ability of ViewLux™, a microplate imager, to act as a suitable radiodetection system for ultra-performance liquid chromatography methods is assessed. The system demonstrates robustness and sensitivity comparable to a microplate scintillation counter (TopCount®) more typically used for off-line metabolite radiodetection. The ViewLux is also used here to undertake successful metabolite profiling of actual samples, for two investigational drug candidates, using both 96- and 384-well yttrium silicate microplates.
Keywords: UPLC; Drug metabolism; ViewLux; LumaPlate-384; Metabolite profiling;

Improvement of the purification of Saint Louis encephalitis virus NS2B-NS3 recombinant protease expressed in Escherichia coli by Boris Pastorino; Dominique Rolland; Christophe N. Peyrefitte; Nathalie Wurtz; Lionel Almeras; Maël Bessaud; Hugues J. Tolou (58-63).
The NS2B-NS3 serine protease of Saint Louis Encephalitis virus (SLEV), a potential target for antiviral drug design, has been over-expressed as a recombinant His-tag protein in Escherichia coli for future structural determination. The production process resulted in a soluble protease with co-purification of DnaK, a bacterial molecular chaperone already described in E. coli protein expression. Two approaches were tested to remove this specific contaminant. The fusion protein bound to the purification resin was washed with MgATP plus soluble denatured E. coli proteins before elution, but this method proved to be poorly efficient due to a substantial loss of the targeted recombinant protease. After the immobilized metal affinity chromatography step, the use of gel permeation chromatography with addition of arginine in the mobile phase led to effective separation of the native viral protease from the DnaK aggregates. By this way, SLEV ΔNS2B-NS3pro protease was purified as a functional protein with a purity greater than 90% suitable for crystallization attempts.
Keywords: Arginine; Flavivirus; Flavivirin; NS2B-NS3 protease; DnaK contamination; Gel permeation chromatography;

Cloud point extraction-HPLC method for determination and pharmacokinetic study of flurbiprofen in rat plasma after oral and transdermal administration by Fei Han; Ran Yin; Xiao-lei Shi; Qiang Jia; Hong-zhuo Liu; Hui-min Yao; Lu Xu; San-ming Li (64-69).
A method based on cloud-point extraction (CPE) was developed for the determination of flurbiprofen (FP) in rat plasma after oral and transdermal administration by high-performance liquid chromatography coupled with UV detection (HPLC–UV). The non-ionic surfactant Genapol X-080 was chosen as the extract solvent. Variables parameter affecting the CPE efficiency were evaluated and optimized. Chromatography separation was performed on a Diamond C18 column (4.6 mm i.d. × 250 mm, 10 μm particle size) by isocratic elution with UV detection at 254 nm. The assay was linear over the range of 0.2–50 and 0.1–10 μg/ml for oral and transdermal administration, respectively, and the lower limit of quantification (LLOQ) was 0.1 μg/ml. The extraction recoveries were more than 84.5%, the accuracies were within ±3.8%, and the intra- and inter-day precisions were less than 10.1% in all cases. After strict validation, the method indicated good performance in terms of reproducibility, specificity, linearity, precision and accuracy, and it was successfully applied to the pharmacokinetic study of flurbiprofen in rats after oral and transdermal administration.
Keywords: Cloud-point extraction; Flurbiprofen; HPLC–UV; Genapol X-080; Pharmacokinetics;

A highly sensitive and specific LC–MS/MS method has been developed for simultaneous estimation of itraconazole (ITZ) and hydroxyitraconazole (OH-ITZ) with 500 μL of human plasma using fluconazole as an internal standard (IS). The API-4000 LC–MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. Solid phase extraction process was used to extract ITZ, OH-ITZ and IS from human plasma. The total run time was 3.0 min and the elution of ITZ, OH-ITZ and IS occurred at 2.08 min, 1.85 min and 1.29 min, respectively; this was achieved with a mobile phase consisting of 0.2% (v/v) ammonia solution:acetonitrile (20:80, v/v) at a flow rate of 0.50 mL/min on a HyPurity C18 (50 mm × 4.6 mm, 5 μm) column. The developed method was validated in human plasma with a lower limit of quantitation of 0.50 ng/mL for both ITZ and OH-ITZ. A linear response function was established for the range of concentrations 0.5–263 ng/mL (r  > 0.998) for both ITZ and OH-ITZ. The intra- and inter-day precision values for ITZ and OH-ITZ met the acceptance as per FDA guidelines. ITZ and OH-ITZ were stable in the battery of stability studies, viz., bench-top, auto-sampler, dry extract and freeze/thaw cycles. The developed assay method was applied to an oral bioequivalence study in humans.
Keywords: Itraconazole; Hydroxyitraconazole; Human plasma; Method validation; LC–MS/MS; Pharmacokinetics; Humans;

Bis(p-fluorobenzyl)trisulfide (BFTS) demonstrated a broad spectrum of anti-proliferative activity and in vivo anti-tumor efficacy in human xenograft mice models. BFTS is rapidly degraded to its major metabolite bis(p-fluorobenzyl)disulfide (BFDS) in blood. In this study, we developed a reliable procedure for stable storage and treatment of blood samples containing BFTS. An HPLC assay method was developed and validated for the quantitative determination of BFTS and BFDS in rat blood. The developed procedure and method have been successfully utilized to study the concentration–time curves of BFTS and BFDS in the blood of rats after vein injection of 12.5 mg kg−1 BFTS.
Keywords: Bis(p-fluorobenzyl)trisulfide; Disulfide; Determination; Blood; High-performance liquid chromatography;

A selective, rapid and sensitive ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) method was developed for the quantitative determination of mitiglinide in human plasma. With nateglinide as internal standard, sample pretreatment involved a one-step extraction with diethyl ether of 0.2 mL plasma. The separation was performed on an ACQUITY UPLC™ BEH C18 column (50 mm × 2.1 mm, i.d., 1.7 μm) with the mobile phase consisting of methanol and 10 mmol/L ammonium acetate (65:35, v/v) at a flow rate of 0.25 mL/min. The detection was carried out by means of electrospray ionization mass spectrometry in positive ion mode with multiple reaction monitoring (MRM). Linear calibration curves were obtained in the concentration range of 1.080–5400 ng/mL, with a lower limit of quantification of 1.080 ng/mL. The intra- and inter-day precision (RSD) values were below 15% and accuracy (RE) was from −3.5% to 7.3% at all QC levels. The method was fully validated and successfully applied to a clinical pharmacokinetic study of mitiglinide in 10 healthy volunteers following oral administration.
Keywords: Mitiglinide; UPLC/MS/MS; Human plasma; Pharmacokinetics;

Identification of volatile organic compounds secreted from cancer tissues and bacterial cultures by Bogusław Buszewski; Agnieszka Ulanowska; Tomasz Ligor; Marek Jackowski; Ewa Kłodzińska; Jacek Szeliga (88-94).
The early cancer diagnosis increases the possibility of total recovery. The infection of Helicobacter pylori is associated with gastric cancer, the second most common cancer in the world. The determination of volatile organic compounds (VOCs) excreted by stomach tissue and bacteria culture has been investigated. Solid-phase microextraction (SPME) was used for preconcentration and the determination was accomplished by gas chromatography coupled with mass spectrometry (GC/MS). The samples of tissue were taken from five patients (ten samples) with stomach cancer and normal (non-cancerous) segments from other parts of the stomach were used as a control. Eighteen compounds were identified in stomach tissue and seven of them were present both in healthy and cancer tissue. These compounds assumed to be endogenous and acetone ratio (AR) was calculated for ethanol, butane, carbon disulfide, 1-propanol, 2-butanone and 2-pentanone. The data shows that amount of 1-propanol and carbon disulfide in the gaseous composition is higher in cancer tissue than in normal tissue. Eight compounds were identified both in bacteria and tissue. These data suggest that bacteria present in the stomach might cause the increase in the concentration of 1-propanol and carbon disulfide in emission from cancer tissue.
Keywords: Gastric cancer; Helicobacter pylori; Volatile organic compounds; Gas chromatography; Mass spectrometry;

A sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and evaluated for the determination of pitavastatin in human plasma and urine. Samples were extracted using solid-phase extraction (SPE). The major benefit of the present method was the high sensitivity, with a lower limit of quantification (LLOQ) of 0.08 ng/mL. Pitavastatin and internal standard (IS, rosuvastatin) were separated on a C18 column with a mobile phase consisted of methanol/water (75:25, v/v) with 0.05% formic acid. Drug and IS were detected by LC/MS/MS with positive electrospray ionization (ESI). Accuracy and precision for the assay were determined by calculating the intra- and inter-batch variation of quality control (QC) samples at three concentration levels, with relative standard deviations (R.S.D.s) of less than 15%. The developed method was successfully applied to determine pitavastatin in human plasma and urine, and was proved to be suitable for use in Phase I clinical pharmacokinetic study after oral administration of pitavastatin (1, 2 and 4 mg) in healthy Chinese volunteers.
Keywords: Pitavastatin; Liquid chromatography/tandem mass spectrometry; Pharmacokinetics; Solid-phase extraction;

A sensitive assay for the quantitative analysis of vinorelbine in mouse and human EDTA plasma by high-performance liquid chromatography coupled with electrospray tandem mass spectrometry by Carola W.N. Damen; Hilde Rosing; Matthijs M. Tibben; Maria J. van Maanen; Jurjen S. Lagas; Alfred H. Schinkel; Jan H.M. Schellens; Jos H. Beijnen (102-109).
A sensitive, specific and fast high-performance liquid chromatography/tandem mass spectrometry (HPLC–MS/MS) assay for the determination of vinorelbine in mouse and human plasma is presented. A 200 μL aliquot was extracted with solid-phase extraction (SPE) using Bond-Elut C2 cartridges. Dried extracts were reconstituted in 100 μL 1 mM ammonium acetate pH 10.5–acetonitrile–methanol (21:9:70, v/v/v) containing the internal standard vintriptol (100 ng/mL) and 10 μL volumes were injected onto the HPLC system. Separation was achieved on a 50 mm × 2.0 mm i.d. Gemini C18 column using isocratic elution with 1 mM ammonium acetate pH 10.5–acetonitrile–methanol (21:9:70, v/v/v) at a flow rate of 0.4 mL/min. HPLC run time was only 5 min. Detection was performed using positive ion electrospray ionization followed by tandem mass spectrometry (ESI–MS/MS). The assay quantifies vinorelbine from 0.1 to 100 ng/mL using human plasma sample volumes of 200 μL. With this method vinorelbine can be measured in mouse plasma samples when these samples are diluted eight times in control human plasma. Calibration samples prepared in control human plasma can be used for the quantification of the drug. The lower limit of quantification in mouse plasma is 0.8 ng/mL. This assay is used to support preclinical and clinical pharmacologic studies with vinorelbine.
Keywords: Vinorelbine; Liquid chromatography; Mass spectrometry;

Development and validation of a sensitive liquid chromatography/mass spectrometry method for quantitation of flavopiridol in plasma enables accurate estimation of pharmacokinetic parameters with a clinically active dosing schedule by Mitch A. Phelps; Darlene M. Rozewski; Jeffrey S. Johnston; Katherine L. Farley; Katie A. Albanese; John C. Byrd; Thomas S. Lin; Michael R. Grever; James T. Dalton (110-115).
A high-performance liquid chromatographic assay with tandem mass spectrometric detection was developed and validated for quantitation of the broad spectrum kinase inhibitor, flavopiridol, in human plasma. Sample preparation conditions included liquid–liquid extraction in acetonitrile (ACN), drying, and reconstitution in 20/80 water/ACN. Flavopiridol and the internal standard (IS), genistein, were separated by reversed phase chromatography using a C-18 column and a gradient of water with 25 mM ammonium formate and ACN. Electrospray ionization and detection of flavopiridol and genistein were accomplished with single reaction monitoring of m/z 402.09 > 341.02 and 271.09 > 152.90, respectively in positive-ion mode [M+H]+ on a triple quadrupole mass spectrometer. Recovery was greater than 90% throughout the linear range of 3–1000 nM. Replicate sample analysis indicated within- and between-run accuracy and precision to be less than 13% throughout the linear range. This method has the lowest lower limit of quantitation (LLOQ) reported to date for flavopiridol, and it allows for more accurate determination of terminal phase concentrations and improved pharmacokinetic parameter estimation in patients receiving an active dosing schedule of flavopiridol.
Keywords: Flavopiridol; Quantitation; Pharmacokinetics; Plasma; LCMS; Genistein;

Development of an analytical method to confirm toxic trimethylated tin in human urine by Yoshihiro Suzuki; Yoko Endo; Masanori Ogawa; Yangho Kim; Nobuhiko Onda; Kenzo Yamanaka (116-119).
Dimethyltin dichloride (DMTC) is widely used as a heat stabilizer in manufacturing the polyvinyl chloride. We previously reported a case of acute DMTC poisoning with neurological manifestations very similar to trimethylated tin (TMT) encephalopathy, based on results of speciation analysis of methylated tins in the patient's urine with use of a combination of high performance liquid chromatography and inductively coupled plasma–mass spectrometry (HPLC–ICP–MS), which yielded peaks corresponding to DMT and TMT. In this study, we developed an analytical method to confirm TMT in urine using high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS), and found TMT molecular ion in the patient's urine.
Keywords: Dimethylated tin; Trimethylated tin; HPLC–MS/MS; HPLC–ICP–MS;