Journal of Chromatography B (v.863, #1)

Hydrophilic interaction chromatography (HILIC) is an effective technique for retaining and separating polar compounds. This approach offers several advantages for bioanalytical liquid chromatography/mass spectrometry, considering that a majority of active pharmaceutical ingredients are polar amines. HILIC employs high concentrations of relatively polar organic mobile phase components (i.e. acetonitrile), providing enhanced desolvation and electrospray ionization efficiency, as well as allowing direct injection of many protein precipitation, liquid/liquid, and solid phase extracts. A set of 30 probe compounds was evaluated to demonstrate a relationship between a compound's HILIC capacity factor (k′), and pH dependent distribution coefficient (D), using three sets of generic isocratic conditions. Plots of log  k′ versus log  D pH 3.0 produced correlation coefficients of 0.751, 0.696, and 0.689 at acetonitrile mobile phase concentrations of 85%, 90%, and 95% (v/v), respectively. For bioanalytical applications a k′ > 2 is typically targeted to ensure adequate retention of a given analyte relative to extracted matrix components. Using k′ ≥ 2 as a measure of HILIC applicability, the linear relationships for each of the three acetonitrile levels predicted whether or not HILIC was able to meet this criterion for at least 90% of the compounds tested. Overall, the relationship between k′ and log  D can serve as a valuable tool for identifying the applicability of HILIC and a starting point for the chromatographic conditions, prior to the initiation of any laboratory activities. Additionally, this relationship can assist with the selection of appropriate chemical analog internal standards.
Keywords: Capacity factor, k′; Hydrophilic interaction chromatography, HILIC; Log  D relationship;

Employing a silica gel column modified with carboxyl groups and an eluent consisting of 4.0 mM tartaric acid and 0.75 mM dipicolinic acid in 2% acetone, an otherwise difficult quantification of K+ at ppb level in presence of 6000 ppm NaCl was achieved by incorporating 0.75 mM 18-crown-6 ether in the mobile phase and subtracting the blank NaCl signal from each chromatogram. Optimized analytical conditions were established in terms of relative standard deviation (%) of retention time, peak area and calibration equations, and also by peak asymmetry factor. The net efflux of K+ into the gastric lumen under in vitro conditions of acid secretion was investigated in Ussing chamber model. The effects of the physiological secretagogue histamine and the antisecretory agents cimetidine, omeprazole and SCH28080 were studied. The decline of K+ efflux in presence of cimetidine, and the rise of the same in the presence of omeprazole and SCH28080 were conspicuously discernible, thereby validating the usefulness of ion chromatography based K+ quantification method under biological experimental conditions.
Keywords: Ion chromatography; Inorganic cations; Potassium efflux; 18-Crown-6 ether; Gastric antisecretory agents; Ussing chamber;

A reverse-phase high-performance liquid chromatography method with electrospray ionization and detection by tandem mass spectrometry is described for the simultaneous quantitative determination of decitabine (5-aza-2′-deoxycytidine) and vorinostat (Suberoylanalide hydroxamic acid, SAHA) in human plasma. The method involves a simple acetonitrile precipitation step and centrifugation followed by injection of the supernatant onto a C18 150 mm × 2.1 mm I.D., 3 μm HPLC column at 36 °C. Separation of decitabine, SAHA and their respective internal standards was achieved with a gradient elution and detection was via the mass spectrometer operated in selected reaction monitoring mode. The method was within the defined validation parameters for linearity, repeatability, reproducibility and stability. The limit of detection was determined as 1.0 and 0.125 ng ml−1 and lower limits of quantitation were 10 and 1 ng ml−1 for decitabine and SAHA, respectively. Effects of sample preparation on stability were also evaluated in human plasma. For clinical sample handling tetrahydrouridine, an inhibitor of cytidine deaminase was found to help prevent decitabine degradation. The method is currently being used in clinical pharmacokinetic studies for the evaluation of decitabine and SAHA combination therapies.
Keywords: Vorinostat; Decitabine; SAHA; Liquid chromatography; Mass spectrometry;

Determination of trichloroethylene in biological samples by headspace solid-phase microextraction gas chromatography/mass spectrometry by Yongzhen Liu; Srinivasa Muralidhara; James V. Bruckner; Michael G. Bartlett (26-35).
A simple, rapid and sensitive method for determination of trichloroethylene (TCE) in rat blood, liver, lung, kidney and brain, using headspace solid-phase microextraction (HS-SPME) and gas chromatography/mass spectrometry (GC/MS), is presented. A 100-μm polydimethylsiloxane (PDMS) fiber was selected for sampling. The major analytical parameters including extraction and desorption temperature, extraction and desorption time, salt addition, and sample preheating time were optimized for each of the biological matrices to enhance the extraction efficiency and sensitivity of the method. The lower limits of quantitation for TCE in blood and tissues were 0.25 ng/ml and 0.75 ng/g, respectively. The method showed good linearity over the range of 0.25–100 ng TCE/ml in blood and 0.75–300 ng TCE/g in tissues, with correlation coefficient (R 2) values higher than 0.994. The precision and accuracy for intra-day and inter-day measurements were less than 10%. The relative recoveries of TCE respect to deionized water from all matrices were greater than 55%. Stability tests including autosampler temperature and freeze and thaw of specimens were also investigated. This validated method was successfully applied to study the toxicokinetics of TCE following administration of a low oral dose.
Keywords: Trichloroethylene; GC–MS; EI; SPME;

A method for determination of a gamma-secretase inhibitor, cis-3-[4-[(4-chlorophenyl)sulfonyl]-4-(2,5-difluorophenyl)cyclohexyl]propanoic acid (A), in human plasma and cerebrospinal fluid (CSF) has been developed to support the clinical investigation of compound A for its potential treatment of Alzheimer's disease. The method is based on HPLC with atmospheric pressure chemical ionization tandem mass spectrometric detection (APCI-MS/MS) in the negative ionization mode using a heated nebulizer interface. The addition of phosphoric acid at the ratio of 10–30 μL per milliliter of human plasma or CSF was required during clinical sample collection to stabilize an acylglucuronide metabolite (C), which was potentially present in human plasma and CSF. Tween 20 (10% solution) was added at the ratio of 20 μL per milliliter of CSF during CSF sample collection to prevent the loss of compound A during the storage of clinical samples. The compound A and its analog internal standard (B) in treated plasma or CSF were isolated from human plasma or CSF using solid phase extraction (SPE) in the 96 well format. The isolated analyte and internal standard were chromatographed on a Phenomenex Synergi® Polar RP analytical column (50 mm × 3.0 mm, 4 μm), using a mobile phase consisting of 60/40 (v/v, %) acetonitrile/water at a flow-rate of 0.5 mL/min. Tandem mass spectrometric detection was performed using a Sciex API 3000 tandem mass spectrometer operated in the multiple reaction monitoring (MRM) mode using precursor to product ion transitions of 441 → 175 for A and 469 → 175 for B, respectively. The assays were validated over the concentration range of 0.5–200 ng/mL for human plasma and CSF. Replicate analyses (n  = 5) of spiked standards for both assays yielded a linear response with coefficients of variation less than 7% and accuracy within 5% of the nominal concentrations. In addition, the assays were automated to improve sample throughput by utilizing a Packard Multi PROBEII automated liquid handling system and a Tom-Tec Quadra 96 system. Numerous clinical studies have been supported using these assays.
Keywords: Gamma-secretase inhibitor; HPLC/MS/MS; CSF; Plasma; Acylglucuronide; Tween 20;

The comparison of two methods based on online solid phase extraction–liquid chromatography with UV (SPE–LC–UV) or mass spectrometry detection (SPE–LC–MS/MS) for the simultaneous quantification of sulfamethoxazole (SMZ) and trimethoprim (TMP) is presented. The methods were validated and proved to be accurate. The analysis of standard samples for SMZ at concentrations of 0.5, 1.5, 25 and 50 μg/mL demonstrated a relative standard deviation of less than 6% for both methods (n  = 18), while TMP samples at concentrations of 0.05, 0.15, 1.5 and 5.0 μg/mL were analyzed with R.S.D. of less than 4% (n  = 18). The method with mass spectrometric detection was approximately six times more sensitive than the method with ultraviolet detection. The total run time for the SPE–LC–MS/MS was 2.5 min per sample as opposed to 18.0 min for the SPE–LC–UV method. The method with MS detection in comparison with UV detection proved to be more rugged and was successfully applied to pharmacokinetics studies.
Keywords: Sulfamethoxazole; Trimethoprim; High-throughput; SPE–LC–MS/MS; SPE–LC–UV;

Quantification of puerarin in plasma by on-line solid-phase extraction column switching liquid chromatography–tandem mass spectrometry and its applications to a pharmacokinetic study by Qingqing Wang; Xiaoshuang Li; Shujia Dai; Lun Ou; Xiao Sun; Baozhen Zhu; Fang Chen; Mingmei Shang; Haifeng Song (55-63).
A highly precise, automatic and rapid method for quantification of puerarin in canine and human plasma using an on-line solid-phase extraction (SPE) column switching procedure combined with liquid chromatography/electrospray ionization tandem mass spectrometry (LC–ESI-MS) was developed. The eluent of SPE column consisted of acetonitrile/methanol/0.1% formic acid (25/25/50) at a flow rate of 0.2 mL min−1. Puerarin was analyzed by a linear ion trap mass spectrometer, LTQ-MS, operating in the negative ion and selective reaction monitoring (SRM) acquisition mode. Method validation results demonstrated that the linear calibration curve covered a wide range of 0.39–400.00 ng mL−1, the correlation coefficients (r 2) were above 0.999. The lower limit of detection (LLOD) with the signal-to-noise (S/N) ratio higher than 12 was 0.39 ng mL−1. The intra- and inter-batch precisions were less than 7.61% and 6.42%, respectively. The accuracy was well within the accept limit. The on-line SPE column switching HPLC–MS system was applied to pharmacokinetic (PK) study of puerarin after a single orally dose in beagles. And the optimum conditions were successfully utilized to quantify puerarin in human plasma, which indicated the feasibility and the reliability of this method for application in preclinical and clinical PK studies of isoflavone drugs.
Keywords: Puerarin; On-line solid-phase extraction column switching; HPLC–MS; Pharmacokinetic study;

On-line extraction assays using cohesive high-turbulence liquid chromatography (HTLC) coupled with tandem mass spectrometer (MS/MS) have been developed for the determination of MK-0974 in human plasma and urine. In this report, a four-step strategy for efficient method development of an on-line extraction assay was discussed. Several challenges – namely extraction recovery, carryover and analyte loss to urine container – were addressed. The assay procedures included sample preparation on a Packard MultiPROBE II liquid-handling system, direct injection on a Cohesive Flux 2300 system, on-line extraction with a Cohesive C18 column (0.5 mm × 50 mm, 50 μm), HPLC separation on a FluophasePFP column (50 mm × 3 mm, 5 μm) under cohesive quick-elution mode and MS detection on a Sciex API4000 in multiple-reaction monitoring (MRM) mode using positive ionization and turbo ion-spray. Since 37–80% analyte loss was observed in urine QCs, 1% BSA was added to bring urine QC accuracy back to ∼100% of nominal. Because of the nature of BSA, the urine assay was established by adapting the plasma method. Thus, the two assays were able to be validated side-by-side, which reduced the validation time by approximately two-fold. The linear dynamic ranges were 0.5–500 and 1–1000 nM for the plasma and urine assays, respectively. Obtained from five standard curves constructed with five lots of human plasma or urine, the intra-day precision (%CV) was <3.14 and <2.62%, and the accuracy was 98.3–101.0 and 99.13–100.64% of nominal for plasma and urine assays, respectively. Both plasma and urine QC samples were stable when kept at room temperature for 4 h, at −70 °C for 3 weeks, or after three freeze–thaw cycles. Both assays gave reasonable relative recovery (>88.8%) and acceptable matrix effect (<15%). The carryover from the upper limit of quantification (ULOQ) was able to be controlled at <20% of lower limit of quantification (LLOQ).
Keywords: On-line extraction; Cohesive technology; High-turbulence liquid chromatograph (HTLC); Parallel validation; CGRP receptor antagonist; MK-0974;

High-performance liquid chromatography method coupled with tandem mass spectrometry was developed for the quantitative determination of I3,II8-biapigenin. The procedure includes solid-phase extraction and separation on an XTerra MS C18. The assay was linear over a wide range; precision and accuracy were acceptable. Biapigenin was present in mouse and rat plasma after a standardized Hypericum perforatum extract. It was not detected in brain (<5 ng g−1), suggesting poor brain-to-blood permeability. Biapigenin concentrations were measurable in mice after intraperitoneal biapigenin (10 mg kg−1) but these amounted to about 2% of the equivalent systemic exposure, after correction for the contribution from residual blood.
Keywords: Biapigenin; Hypericum perforatum extract; LC/MS–MS; Rodents; Brain uptake and concentrations;

An advanced method for the determination of carboxyl methyl esterase activity using gas chromatography–chemical ionization–mass spectrometry by Yeon Jong Koo; Eunsil Yoon; Jong Tae Song; Hak Soo Seo; Jeong-Han Kim; Yin-Won Lee; Jong Seob Lee; Jong-Joo Cheong; Yang Do Choi (80-87).
We developed a quantitative method for the determination of methyl esterase activity, analyzing substrate specificity against three major signal molecules, jasmonic acid methyl ester (MeJA), salicylic acid methyl ester (MeSA), and indole-3-acetic acid methyl ester (MeIAA). We used a silylation reagent for chemical derivatization and used gas chromatography (GC)–mass spectroscopy in analyses, for high precision. To test this method, an Arabidopsis esterase gene, AtME8, was expressed in Escherichia coli, and then the kinetic parameters of the recombinant enzyme were determined for three substrates. Finally, this method was also applied to the direct quantification of phytohormones in petals from lilies and roses.
Keywords: Jasmonic acid methyl ester; Salicylic acid methyl ester; Indole 3-acetic acid methyl ester; Carboxyl methyl esterase; Gas chromatography–mass spectroscopy;

A new, simple, and reproducible isocratic high-performance liquid chromatography (HPLC) method has been developed for the determination of free and total tyrosine and tryptophan in a protein concentrate. To determine total amino acids, the method involves alkaline hydrolysis of the proteins with sodium hydroxide at 120 °C for 4 h in the absence of air. Best results were achieved with a SS Exil ODS column 5 μm (25 cm × 0.46 cm i.d.), with an eluent of methanol: 40 mM sodium acetate buffer (adjusted to pH 4.5 with acetic acid; 20:80, v/v), a flow rate of 0.80 mL/min at 26 °C, and with programmable fluorescence detection. Under optimum conditions excellent linearity was obtained, and the overall recovery was 90.5, and 95.9% for total tryptophan and tyrosine, respectively. The precision results showed that the relative standard deviation of the repeatability and reproducibility were ≤4.78 and ≤4.65, respectively. This method was used to quantify the cited analytes in the protein concentrate obtained during the lactic acid fermentation of shrimp waste.
Keywords: Tryptophan; Tyrosine; High-performance liquid chromatography (HPLC); Protein concentrate; Shrimp waste; Fluorescence detection;

Simultaneous quantification of 11 pivotal metabolites in neural tube defects by HPLC–electrospray tandem mass spectrometry by Yong Wang; Hong-Yang Zhang; Qiong-Lin Liang; Hui-Hua Yang; Yi-Ming Wang; Qing-Fei Liu; Ping Hu; Xiao-Ying Zheng; Xin-Ming Song; Gong Chen; Ting Zhang; Jian-Xin Wu; Guo-An Luo (94-100).
One-carbon metabolism that involves folate metabolism and homocysteine metabolism plays a powerful role in embryonic development. Any impairment to this metabolism during the neurulation process would trigger the occurrence of neural tube defects (NTDs). The great importance of one-carbon metabolism necessitates the establishment of methodology to determine the relative compounds involved in the metabolic cycles. We have developed a sensitive method for measurement of 11 pivotal compounds by using high-performance liquid chromatography coupled to mass spectrometry (HPLC–MS/MS) in sera of pregnant women. Use of an aqueous chromatography column increased retention time and separation of the polar compounds in the system, resulting in fewer co-elution and interference from the other compounds that can lead to ion suppression. Calibration curves suitable for the analysis of maternal serum were linear (r 2  > 0.997) with limits of detection from 0.05 to 1 ng/mL. Intra-day coefficients of variation (CVs) and inter-day CVs were both lower than 11%. With the developed method, 96 serum samples including 46 cases and 50 controls were analyzed. The established method provided a reliable method for quantifying most of the compounds involved in the one-carbon metabolism simultaneously, thus made it possible to elucidate NTDs with multiple factors instead of one single and provided a solid foundation for the diagnosis and prevention of NTDs as well as some other one-carbon metabolism related diseases.
Keywords: Neural tube defects; One-carbon metabolism; Tandem mass spectrometry; Folate; Homocysteine;

Capillary electrophoresis (CE) with amperometric detection (AD) has been developed for the separation and determination of disaccharide glycoside rutin, gentistic acid, quercetin, and gallic acid in the leaves of Ricinus communis Linn. for the first time. The effects of the acidity and the concentration of the running buffer, separation voltage, injection time, and detection potential were investigated to acquire the optimum conditions for the determination of the four analytes. The detection electrode was a 300 μm diameter carbon disc electrode at a detection potential of +0.90 V (versus saturated calomel electrode (SCE)). The four analytes could be well separated within 10 min in a 40 cm length fused silica capillary at a separation voltage of 15 kV in a 50 mM borate buffer (pH 9.0). The relation between peak current and analyte concentration was linear over about 3 orders of magnitude with detection limits (S/N = 3) ranging from 0.8 to 2.9 μM for all the analytes. The proposed method has been successfully applied to monitor flavones and phenolic acids in the real plant samples with satisfactory assay results.
Keywords: Ricinus communis Linn.; Disaccharide glycoside rutin; Gentistic acid; Quercetin; Gallic acid; Capillary electrophoresis; Amperometric detection;

There are no analytical methods that simultaneously quantify nicotine, cotinine, trans-3′-hydroxycotinine, nornicotine and norcotinine in human meconium. Such a method could improve identification of in utero tobacco exposure, determine if maternal dose–meconium concentration relationships exist, and whether nicotine meconium concentrations predict neonatal outcomes. The first liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry method for simultaneous quantification of these analytes in meconium was developed and validated. Specimen preparation included homogenization, enzyme hydrolysis and solid phase extraction. The linear range was 1.25 or 5–500 ng/g. Method applicability was evaluated with meconium collected from an in utero tobacco exposed infant.
Keywords: Meconium; Nicotine; Cotinine; trans-3-Hydroxycotinine; Tobacco;

A liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and fully validated, according to U.S. Food and Drug Administration guidance, for the simultaneous determination of phenylmercapturic acid, benzylmercapturic acid and o-methylbenzyl mercapturic acid in human urine as biomarkers of exposure to benzene, toluene and xylenes (BTX). After solid phase extraction and LC separation, samples were analyzed by a triple–quadrupole mass spectrometer operated in negative ion mode, using isotope-labeled analogs as internal standards (ISs). The method meets all the validation criteria required. The limits of detection of the three analytes, ranging from 0.30 to 0.40 μg l−1, and the high throughput make the method suitable for the routine biological monitoring of co-exposure to BTX both in the occupational and environmental settings. The validated method was applied to assess exposure to BTX in a group of 354 urban traffic wardens.
Keywords: Liquid chromatography–mass spectrometry; Mercapturic acids; Benzene; Toluene; Xylenes;

An analytical procedure for the determination of phencyclidine in oral fluid has been developed and validated using liquid chromatography with tandem mass spectral detection, following initial screening with enzyme linked immunosorbent assay. The oral fluid samples were collected using the Quantisal ™ device, and any drugs present were quantified using mixed mode solid-phase extraction followed by mass spectrometric detection in positive atmospheric pressure chemical ionization mode. For confirmation, two transitions were monitored and one ratio determined, which had to be within 20% of that of the known calibration standard. The monitoring of the qualifying transition and requirement for its presence within a specific ratio to the primary ion has the potential of limiting the sensitivity of the assay, however, the additional confidence in the final result as well as forensic defensibility were considered to be of greater importance. The limit of quantitation was 5 ng/mL; the intra-day precision of the assay (n  = 5) was 3.04%; inter-day precision 3.35% (n  = 5) at a concentration of 10 ng/mL. The accuracy was determined at four concentrations (5, 10, 20 and 40 ng/mL) within the linear range of the assay. The percentage recovery of phencyclidine from the oral fluid collection pad was 81.7% (n  = 6). The methods were applied to both proficiency specimens and to samples obtained during research studies in the USA.
Keywords: Oral fluid; Phencyclidine; LC/MS/MS;

The antimalarial drug amodiaquine is extensively metabolized to N-desethylamodiaquine (DEAQ) by cytochrome P450 2C8 (CYP2C8). DEAQ formation is an enzyme specific reaction that is used to quantify in vitro CYP2C8 activity. A rapid and sensitive method for the determination of DEAQ in human liver microsomes was developed using hydrophilic interaction liquid chromatography/tandem mass spectrometry (HILIC–MS/MS). Microsomal incubation samples were processed by protein precipitation with acetonitrile. The analytes were separated on a BETASIL Silica-100 (50 mm × 2.1 mm, 5 μm) column by isocratic elution at a flow rate of 220 μl/min with a mobile phase consisting of 85% acetonitrile containing 5 mM ammonium acetate and 0.1% formic acid. Detection was by positive electrospray ionization on a TSQ Quantum Discovery triple quadrupole mass spectrometer operated in the selective reaction monitoring mode. The precursor–product ion pair was m/z 328 → 283 for DEAQ and m/z 331 → 283 for DEAQ-d 3. The lower limit of quantification was 10 nM for DEAQ and linearity was observed over the concentration range of 10–1500 nM. Intra- and inter-day accuracy and precision were within 3.4 and 7.0%, respectively. The method was successfully applied to CYP2C8 drug metabolism studies in pooled human liver microsomes.
Keywords: Desethylamodiaquine; Cytochrome P450; CYP2C8; HILIC; Drug metabolism;

Interaction of pepsin with aromatic amino acids and their derivatives immobilized to Sepharose by Jana Frýdlová; Zdenka Kučerová; Marie Tichá (135-140).
The interaction of porcine pepsin A with immobilized derivatives of aromatic amino acids was investigated. Divinyl sulfone-activated Sepharose was used to immobilize N-acetyl-l-phenylalanine and 3,5-diiodo-l-tyrosine via their free carboxyl groups and l-tyrosine via its amino group. Immobilized l-tyrosine was iodinated after coupling. The optimum conditions for the separation of porcine pepsin A using the prepared affinity carriers were studied and the following parameters were established: enzyme recovery, reproducibility of analyses, capacity and dependence of the elution peak area on the concentration of the loaded enzyme. The ability of the prepared affinity carriers to retain various types of proteins was compared under optimum conditions for porcine pepsin A separation. While immobilized 3,5-diiodo-l-tyrosine and iodinated l-tyrosine-Sepharose adsorbed relatively high amounts of bovine serum albumin and ovalbumin, only negligible amounts of these proteins were adsorbed to immobilized N-acetyl-l-phenylalanine. The behavior of porcine pepsin A was the same as its complex with pepstatin A on the prepared affinity carriers, indicating that the enzyme active site is not involved in the studied interaction.
Keywords: Porcine pepsin A; Affinity chromatography; Immobilized derivatives of aromatic amino acids;

A sensitive and fast method was developed to quantitate the carcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) and eight of its oxidized metabolites by ultra-performance liquid chromatography (UPLC) coupling with mass spectrometry (MS). The UPLC method, using an acetonitrile:water gradient as a mobile phase, provided baseline separation of the BaP metabolites including three BaP diones. Linearity of detection was in the range of 0.2–5.0 ng/μL, and limits of detection (LOD) were lower than 0.01 ng/μL for BaP and all of the metabolites except BaP tetrol. In order to test this method in environmentally relevant samples, we exposed the small fish Fundulus heteroclitus to BaP and quantitated biliary BaP metabolites. Extraction recovery of all compounds varied from 65.4 ± 21.3% to 92.4 ± 3.0%. In exposed fish bile, the BaP diones, BaP-7,8-dihydrodiol, and 3-hydroxy BaP metabolites predominated, existing mainly as glucuronic acid conjugates. This UPLC–MS method will be useful for further defining the roles of cytochrome P450s with both in vivo and in vitro models in the understanding of the mechanisms of metabolic activation and detoxification of BaP.
Keywords: Benzo[a]pyrene; UPLC–MS; Metabolism;

An analytical method combining microwave assisted micellar extraction (MAME) and solid phase extraction (SPE) has been developed to extract and preconcentrate a selected group of eight pharmaceutical compounds in sediment samples prior to their determination using liquid chromatography with an UV–DAD detector. A non-ionic surfactant, Polyoxyethylene 10 lauryl ether (POLE) was used for the MAME extraction and the different parameters for the optimization process were studied. Then, SPE was used to clean-up and preconcentrate the target analytes in the extract, prior to their determination using HPLC–UV. The method was applied to the determination of the selected pharmaceuticals compounds in several kinds of sediment samples with different characteristics. Relative recoveries for spiked sediment samples were over 70% and relative standard deviations (RSDs) were under 11% for all recoveries tested. Detection limits between 4 and 167 ng g−1 were obtained. The method was validated using Soxhlet extraction procedure.
Keywords: Pharmaceuticals residues; Microwave assisted micellar extraction; Solid phase extraction; HPLC–UV–DAD;

Development and validation of a high performance liquid chromatography–tandem mass spectrometry for the determination of etodolac in human plasma by Hyun-Soo Lee; Il-Mo Kang; Heon-Woo Lee; Ji-Hyung Seo; Ju-Hee Ryu; Sang-Jun Choi; Myung-Jae Lee; Seo-Young Jeong; Young-Wuk Cho; Kyung-Tae Lee (158-162).
A simple and specific method using a one-step liquid–liquid extraction (LLE) with butyl acetate followed by high performance liquid chromatography (HPLC) coupled with positive ion electrospray ionization tandem mass spectrometry (ESI-MS/MS) detection was developed for the determination of etodolac in human plasma, using indomethacin as an internal standard (IS). Chromatographic separation was performed isocratically using a Capcellpak MGII C18 column with 65% acetonitrile and 35% water containing 10 mM ammonium formate (adjusted to pH 3.5 with formic acid). Acquisition was performed in multiple reaction monitoring (MRM) mode by monitoring the transitions: m/z 287.99 > 172.23 for etodolac and m/z 357.92 > 139.01 for IS. The method was validated to determine its selectivity, linearity, sensitivity, precision, accuracy, recovery and stability. The limit of quantitation (LLOQ) was 0.1 μg/mL with a relative standard deviation of less than 15%. The devised method provides an accurate, precise and sensitive tool for determining etodolac levels in plasma.
Keywords: Etodolac; Indomethacin; LC–MS/MS; Human plasma; Validation;

High-performance liquid chromatography spectrometric analysis of tripterin in rat plasma by Wenyan Wang; Ke Liu; Hongwei Dong; Wanhui Liu (163-166).
A quick, precise and reliable HPLC method has been developed to determine tripterin in rat plasma. After liquid–liquid extraction, the analytes was analyzed on a Discovery ODS C18 column (5 μm, 4.6 mm × 250 mm) with an isocratic elution consisting of methanol–water–phosphoric acid (87:13:0.2, v/v/v). Ultraviolet detection was at 425 nm. Using trioxymethylanthraquinone as an internal standard, the assay was linear over the concentration range of 0.025–1.60 μg/mL (r 2  = 0.9988). The extraction recovery of tripterin in rat plasma was more than 62%. The intra- and inter-day precision was less than 13% (CV). This validated method was successfully applied to the pharmacokinetics of tripterin in rats.
Keywords: Tripterin; HPLC; Rat; Plasma concentration;

Dissociation of neopterin and 7,8-dihydroneopterin from plasma components before HPLC analysis by Elizabeth A. Flavall; Elizabeth M. Crone; Grant A. Moore; Steven P. Gieseg (167-171).
Measurement of plasma neopterin by HPLC with fluorescence detection is used clinically as a marker of immune cell activation in the management of a number of disease pathologies. HPLC analysis of neopterin requires the acidic removal of plasma proteins but we have found that 7,8-dihydroneopterin is oxidised to neopterin with varying yield. Using acetonitrile as the precipitant, we have measured substantially higher quantities of both total neopterin (7,8-dihydroneopterin and neopterin) and neopterin from plasma of healthy and septicemia patient's. Total neopterin concentrations were on average 50% and 200% greater in healthy and septicemia subjects, respectively, when measured after acetonitrile precipitation compared to trichloroacetic acid. Our data suggests that some pterin co-precipitates with proteins during acid treatment.
Keywords: Neopterin; 7,8-Dihydroneopterin; Protein precipitation; HPLC; Acetonitrile; Trichloroacetic acid;

Determination of inositol hexanicotinate in rat plasma by high performance liquid chromatography with UV detection by Dong Liang; Jing Ma; Bo Wei; Ivy O. Poon; Edward C. Bell; Theodore R. Bates (172-176).
A HPLC method with UV detection at 262 nm was developed to analyze inositol hexanicotinate in rat plasma. Plasma samples were extracted with an equal volume of acetonitrile, followed by dilution with mobile phase buffer (5 mM phosphate buffer, pH 6.0) to eliminate any solvent effects. Inositol hexanicotinate and the internal standard (mebendazole) were separated isocratically using a mobile phase of acetonitrile/phosphate buffer (35:65, v/v, pH 6.0) at a flow rate of 1.0 mL/min and a reverse-phase XTerra® MS C18 column (4.6 mm × 150 mm, 3.5 μm). The standard curve was linear over a concentration range of 1.5–100.0 μg/mL of inositol hexanicotinate in rat plasma. The HPLC method was validated with intra- and inter-day precisions of 1.55–4.30% and 2.69–21.5%, respectively. The intra- and inter-day biases were −0.75 to 19.8% and 2.58–22.0%, respectively. At plasma concentrations of 1.5–100 μg/mL, the mean recovery of inositol hexanicotinate was 99.6%. The results of a stability study indicated that inositol hexanicotinate was unstable in rat plasma samples, but was stable in acetonitrile extracts of rat plasma for up to 24 h at 4 °C. The assay is simple, rapid, specific, sensitive, and reproducible and has been used successfully to analyze inositol hexanicotinate plasma concentrations in a pharmacokinetic study using the rat as an animal model.
Keywords: Inositol hexanicotinate; HPLC; Plasma;

Determination of lamotrigine in whole blood with on line solid phase extraction by Stefano Bompadre; Adriano Tagliabracci; Maurizio Battino; Raffaele Giorgetti (177-180).
A simple, sensitive and reproducible method was developed for the determination of lamotrigine in whole blood with on-line solid phase extraction followed by HPLC separation with UV detection. Whole blood samples were diluted 1:1 with water and then injected directly on a clean-up column dry-packed with 40 μm C8 silica and separated on a C18 reversed-phase column (150 × 4.6 mm) at room temperature. The extraction column was activated with methanol and conditioned with phosphate buffer of pH 4.5. Mobile phases consisted of phosphate buffer of pH 4.5 for the extraction column and of phosphate buffer of pH 4.5 – acetonitrile (60:40, v/v) for the analytical column. At a flow rate of 1.0 ml/min and a connection time of 1.0 min, the complete cycle time was 10.0 min. Detection was carried out at 260 nm. No internal standard was necessary. The method was linear over concentration range 0.2–20.0 μg/ml for lamotrigine. Recovery was 98%. Within-day and between-day coefficients of variation ranged from 1.8 to 6.7%.
Keywords: Lamotrigine; HPLC; Solid phase extraction;

This study involves the incorporation of a commercially available Phenomenex Onyx C18 monolith column into the separation and detection of oxidative DNA damage. It includes thorough investigation of monolith performance and a comparison of the performance of monolith columns with a commercially available packed Restek reverse phase Ultra C18 column for the separation of DNA bases and nucleosides. The performance of the monolith was examined using efficiency, resolution, plate height, asymmetry and retention times, and each case showed improved or at least comparable results in the separation of a mix of DNA bases and nucleosides. A 90% reduction, from just under 40 min to just under 4 min, was obtained in the elution time of this separation. To the best of our knowledge, this is the first report of a fast monolith column separation successfully coupled to both a UV–vis and EC detector, which is especially useful for the analysis of oxidative DNA damage. The determination of 8-oxoG and 8-OH-dG, oxidation products of guanine and 2′-deoxyguanosine, respectively, may be compromised by their ease of oxidation and therefore the fast separation, selective and sensitive detection, with no artifactual oxidation, detailed in this report, is ideal.
Keywords: HPLC-UV-EC; Oxidative DNA damage; Guanine; 8-Oxo-7,8-dihydroguanine; 2′-Deoxyguanosine; 8-Oxo-7,8-dihydro-2′-deoxyguanosine; Monolith;

In this study, a protein purified by fluorescein isothiocyanate (FITC)-affinity chromatography from human plasma was identified as albumin by MALDI-TOF-MS. Albumin was found to conjugate with FITC-labeled molecules through a copper-dependent reaction. The formation of this complex was confirmed by methods including a newly developed “charcoal-based fluorescence assay” (CFA), gel-filtration, affinity chromatography, and ultrafiltration. The binding was identified as disulfide bridge formation. This is the first to demonstrate that copper induces a covalent binding of FITC-labeled molecules with albumin. In addition, the developed CFA method facilitates the screening of small fluorescent dyes binding to macromolecules.
Keywords: Albumin; FITC; Copper; Affinity; Purification;