Journal of Chromatography B (v.877, #22)

CE offers a low running cost, short separation time, and a high-resolution technique that requires only a small amount of analyte. It has a wide variety of operation modes (CZE, CEC, CIEF, CITP, CAE, CGE and MEKC) that can be interfaced with MS for tissue and body fluid analysis, particularly urine and cerebrospinal fluid, to identify potential proteomic markers for the clinical diagnosis of many diseases (renal, genitourinary, vascular, diabetes mellitus, cancer, arthritis and neurological diseases) and for the monitoring of their therapeutic intervention. It has become evident that no one marker would be sufficient, but a combination of well-selected markers would be needed for that purpose. The potential of CE coupled to MS for studying the pathophysiology of these diseases and the development of biomarkers has been demonstrated. These biomarkers, when validated, will allow greater use of noninvasive methods for diagnosis of diseases, assessment of their progression, and monitoring individuals’ response to therapy.
Keywords: CSF; 2DE; Diagnoses; ESI; LC; MALDI; Plasma; SELDI; Urine; Validation;

Therapeutic Drug Monitoring of the new targeted anticancer agents imatinib, nilotinib, dasatinib, sunitinib, sorafenib and lapatinib by LC tandem mass spectrometry by A. Haouala; B. Zanolari; B. Rochat; M. Montemurro; K. Zaman; M.A. Duchosal; H.B. Ris; S. Leyvraz; N. Widmer; L.A. Decosterd (1982-1996).
The treatment of some cancer patients has shifted from traditional, non-specific cytotoxic chemotherapy to chronic treatment with molecular targeted therapies. Imatinib mesylate, a selective inhibitor of tyrosine kinases (TKIs) is the most prominent example of this new era and has opened the way to the development of several additional TKIs, including sunitinib, nilotinib, dasatinib, sorafenib and lapatinib, in the treatment of various hematological malignancies and solid tumors. All these agents are characterized by an important inter-individual pharmacokinetic variability, are at risk for drug interactions, and are not devoid of toxicity. Additionally, they are administered for prolonged periods, anticipating the careful monitoring of their plasma exposure via Therapeutic Drug Monitoring (TDM) to be an important component of patients’ follow-up. We have developed a liquid chromatography–tandem mass spectrometry method (LC–MS/MS) requiring 100 μL of plasma for the simultaneous determination of the six major TKIs currently in use. Plasma is purified by protein precipitation and the supernatant is diluted in ammonium formate 20 mM (pH 4.0) 1:2. Reverse-phase chromatographic separation of TKIs is obtained using a gradient elution of 20 mM ammonium formate pH 2.2 and acetonitrile containing 1% formic acid, followed by rinsing and re-equilibration to the initial solvent composition up to 20 min. Analyte quantification, using matrix-matched calibration samples, is performed by electro-spray ionization–triple quadrupole mass spectrometry by selected reaction monitoring detection using the positive mode. The method was validated according to FDA recommendations, including assessment of extraction yield, matrix effects variability (<9.6%), overall process efficiency (87.1–104.2%), as well as TKIs short- and long-term stability in plasma. The method is precise (inter-day CV%: 1.3–9.4%), accurate (−9.2 to +9.9%) and sensitive (lower limits of quantification comprised between 1 and 10 ng/mL). This is the first broad-range LC–MS/MS assay covering the major currently in-use TKIs. It is an improvement over previous methods in terms of convenience (a single extraction procedure for six major TKIs, reducing significantly the analytical time), sensitivity, selectivity and throughput. It may contribute to filling the current knowledge gaps in the pharmacokinetics/pharmacodynamics relationships of the latest TKIs developed after imatinib and better define their therapeutic ranges in different patient populations in order to evaluate whether a systematic TDM-guided dose adjustment of these anticancer drugs could contribute to minimize the risk of major adverse reactions and to increase the probability of efficient, long lasting, therapeutic response.
Keywords: LC–MS/MS; Anticancer targeted therapy; Tyrosine kinase inhibitors; Imatinib; Nilotinib; Dasatinib; Sunitinib; Sorafenib; Lapatinib; Plasma;

The preparation of a laboratory reference material (LRM) for the determination of naturally occurring heterocyclic amines (HAs) in processed foods is presented in this work. A LRM was prepared from raw chicken breast meat, which was fried under controlled cooking temperature and time. The cooked meat was ground, lyophilised, sieved, homogenised, bottled, and labelled. The HAs DMIP, PhIP, MeIQx, 4,8-DiMeIQx, Norharman and Harman were analysed in the LRM. Homogeneity and stability studies of the bulk LRM were carried out and no statistical differences were observed in the content of the studied HAs in between-bottle and within-bottle comparisons at different storage temperatures (−18, +4, +25 and +40 °C) and times (1, 3, 6 and 9 months) by means of HAs determination and analysis of the results. Consequently, the material can be considered homogeneous and stable and can be used in intercomparison exercises for the determination of HAs as well as for quality control purposes in the routine analysis of HAs in foodstuffs. This is the first LRM for the analysis of HAs where these analytes were naturally formed in the material.
Keywords: Fried chicken; Heterocyclic amines; Laboratory reference material; Liquid chromatography–mass spectrometry;

Significant electrospray ionization effects were observed for a hydrophobic analyte and its stable isotopically labeled internal standard (SIL IS) in human plasma extracts. Analyte and SIL IS were slightly offset in retention within the suppression region resulting in a differential suppression that biased calculated concentrations. Six abundant endogenous phospholipids were identified as potential contributors to ionization suppression. Chromatographic conditions were optimized using pH (relative to the log  D of the analytes), to better resolve nearby phospholipids from the analyte and SIL IS and minimize ionization suppression. The successful validation of this method demonstrates the value of investigating minor chromatographic changes to remediate detrimental ionization effects without further altering extraction procedures.
Keywords: Phospholipids; Matrix effects; Stable isotopically labeled (SIL) internal standard; Serum; log  D; LC–MS/MS;

Volatile compounds from human breath are a potential source of information for disease diagnosis. Breath may include volatile organic compounds (VOCs) originating in the nasal sinuses. If the sinuses are infected, disease-specific volatiles may enter exhaled air. Sinus infections are commonly caused by several known bacteria. We examined the volatiles characteristic of infectious bacteria in culture using solid-phase microextraction to collect and gas chromatography–mass spectrometry as well as gas chromatography with flame photometric detection to separate and analyze the resulting VOCs. Infected sinus mucus samples were also collected and their VOCs examined. Similar characteristic volatiles were seen from both cultures of individual “pure” bacteria and several mucus samples. However, the relative amounts of characteristic VOCs from individual bacteria differ greatly between cultures and sinus mucus. New compounds, not seen in culture were also seen in some mucus samples. Our results suggest an important role for growth substrate and environment. Our data further suggests that in some sinus mucus samples identification of bacteria-specific volatiles is possible and can suggest the identity of an infecting organism to physicians. Knowledge of these bacteria-related volatiles is necessary to create electronic nose-based, volatile-specific sensors for non-invasive examination for suspected sinus infection.
Keywords: Bacterial odors; Disease odors; GC/MS; Sinus disease; SPME–GC/MS;

A high performance liquid chromatography coupled with electrospray ionization/mass spectrometry method was developed for the determination of adenosine 5′-monophosphate (AMP), adenosine 5′-diphosphate (ADP), and adenosine 5′-triphosphate (ATP) in the extract of HepG-2 cells. The chromatographic conditions were optimized by using porous graphitic carbon as the stationary phase for the retention and separation of the AMP, ADP and ATP. Negative-ion mode ESI-MS in basic mobile phase was applied to improve the method sensitivity. An external calibration method with linear ranges from 0.22 to 57.80 μM for AMP, from 0.59 to 117.37 μM for ADP, and from 0.49 to 98.81 μM for ATP was used for quantitative analysis. The levels of ATP, ADP, and AMP in HepG-2 cells treated with benzo[a]pyrene with different time periods were determined. Total adenine nucleotides and the energy charge potential were calculated for the investigation of the effect of benzo[a]pyrene on cell energy metabolism.
Keywords: Adenosine nucleotides; HepG-2 cell; Benzo[a]pyrene; Porous graphitic carbon; LC–ESI-MS;

A liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed for the simultaneous determination of the 10 major components of Da-Cheng-Qi decoction (rhein, emodin, aloe-emodin, chrysophanol, rheochrysidin, naringin, naringenin, hesperidin, magnolol and honokiol) in dog plasma. Plasma samples were spiked with internal standard (ibuprofen), acidified with HCl and extracted twice by liquid–liquid extraction using ethyl acetate. Separation was performed on a YMC-Pack ODS-A C18 column (5 μm, 150 mm × 4.6 mm) and a C18 guard column (5 μm, 4.0 mm × 2.0 mm) with methanol–water (92:8, v/v) at a flow rate of 0.3 mL/min. The LC/MS system was operated under the multiple reaction monitoring mode using electrospray ionization in the negative ion mode. All analytes showed good linearity over a wide concentration range (r  > 0.99). The linear range of the calibration curves was 5000–19.53 ng/mL for rhein; 400–3.13 ng/mL for emodin; 800–3.13 ng/mL for aloe-emodin, chrysophanol, naringin, naringenin, hesperidin, magnolol and honokiol; 160–0.63 ng/mL for rheochrysidin. The lower limit of quantification was: 19.53 ng/mL for rhein; 3.13 ng/mL for emodin, aloe-emodin, chrysophanol, naringin, naringenin, hesperidin, magnolol and honokiol; 0.63 ng/mL for rheochrysidin. The overall mean accuracy for the 10 major components of Da-Cheng-Qi decoction was 90.40–108.60%. Intra-day and inter-day precision was ≤12.43% and ≤11.32%, respectively. We conclude that this method is appropriate for simultaneous determination of the 10 major components of Da-Cheng-Qi decoction in dog plasma and the investigation of the pharmacokinetics of Da-Cheng-Qi decoction in dog.
Keywords: Liquid chromatography tandem mass spectrometry; Da-Cheng-Qi decoction; Dog plasma;

A sensitive and specific liquid chromatography–tandem mass spectrometry method for the determination of intracellular and extracellular uric acid by Kyung Mee Kim; George N. Henderson; Xiaosen Ouyang; Reginald F. Frye; Yuri Y. Sautin; Daniel I. Feig; Richard J. Johnson (2032-2038).
Uric acid (UA) is known to be a major biological antioxidant in plasma. However, there is a strong correlation between UA levels and cardiovascular risk. Recent studies suggest that in the intracellular environment, UA can become a prooxidant that causes endothelial dysfunction. For conducting detailed studies of UA's role in human pathogenesis, there is a critical need for a sensitive and specific method for the determination of intracellular UA levels. We therefore developed a simple, sensitive method for determination of trace amounts of intracellular UA, as well as comparatively large amounts of UA in plasma and urine (for the determination of extracellular concentrations of UA), based on liquid chromatography and tandem mass spectrometry (LC–MS/MS). UA was separated from interferences by HPLC and quantified by mass spectrometry in the negative ESI mode using single reaction monitoring (SRM). For the identification and quantification of UA, the parent ions selected were m/z 167.0, which corresponds to the urate anion, and m/z 169.0, which corresponds to the 1,3-15N2-UA anion. 1,3-15N2-UA is used as an internal standard to ensure accuracy of the measurement. After precipitation of proteins with 10% TCA solution, UA was subjected to LC–MS/MS analysis. The correlation coefficient was 0.9998–1.0000 based on the calibration curve. The intra- and inter-day precision (C.V. %) ranged from 0.01 to 3.07 and 0.01 to 3.68 for in vivo and in vitro systems, respectively. Recovery tests of added standards have been successfully performed and the values ranged from 90.10 to 103.59% and 98.74 to 106.12% for in vivo and in vitro analyses, respectively. This study demonstrates that intracellular levels of UA can be measured using LC–MS/MS with isotope labeled UA as an internal standard.
Keywords: Uric acid; Urine; Plasma; Cell lysate; Cardiovascular disease; LC–MS/MS;

Immobilized analogues of sunflower trypsin inhibitor-1 constitute a versatile group of affinity sorbents for selective isolation of serine proteases by Hugo Juarez Vieira Pereira; Maria Cristina Oliveira Salgado; Eduardo Brandt Oliveira (2039-2044).
Sunflower trypsin inhibitor-1 (SFTI-1), a natural 14-residue cyclic peptide, and some of its synthetic acyclic variants are potent protease inhibitors displaying peculiar inhibitory profiles. Here we describe the synthesis and use of affinity sorbents prepared by coupling SFTI-1 analogues to agarose resin. Chymotrypsin- and trypsin-like proteases could then be selectively isolated from pancreatin; similarly, other proteases were obtained from distinct biological sources. The binding capacity of [Lys5]-SFTI-1-agarose for trypsin was estimated at over 10 mg/mL of packed gel. SFTI-1-based resins could find application either to improve the performance of current purification protocols or as novel protease-discovery tools in different areas of biological investigation.
Keywords: Proteolytic enzymes; Serine proteases; Affinity chromatography; SFTI-1; Bowman–Birk inhibitors;

Refolding of scFv mini-antibodies using size-exclusion chromatography via arginine solution layer by K.K. Fursova; A.G. Laman; B.S. Melnik; G.V. Semisotnov; P.Kh. Kopylov; N.V. Kiseleva; V.A. Nesmeyanov; F.A. Brovko (2045-2051).
The method for refolding of mini-antibodies using size-exclusion chromatography via arginine solution layer was developed. This method allows to refold scFv, to separate both aggregated protein and low molecular weight compounds and to isolate functionally active protein preparation in monomeric form. The comparison of various scFv preparations isolated either from inclusion bodies or from soluble fraction revealed that refolded mini-antibodies demonstrate higher antigen-binding activity. Mini-antibodies refolded in the presence of arginine also demonstrate higher electrophoretic mobility during native PAGE in comparison with soluble cytoplasmic antibodies. Both soluble as well as refolded antibodies had similar CD spectra. Refolded mini-antibodies are storage-stable.
Keywords: scFv; Refolding; Renaturation; Size-exclusion chromatography; Arginine;

Endogenous ethanolamide analysis in human plasma using HPLC tandem MS with electrospray ionization by Joe Palandra; Jeff Prusakiewicz; Josef S. Ozer; Yanhua Zhang; Timothy G. Heath (2052-2060).
A sensitive and selective liquid chromatography tandem mass spectrometry (LCMSMS) method has been developed for the simultaneous quantification in human plasma of the endocannabinoid anandamide (AEA) and three other related ethanolamides, linoleoyl ethanolamide (LEA), oleoyl ethanolamide (OEA), and palmitoyl ethanolamide (PEA). The analytical methodology requires 50 μL of human plasma which is processed via protein precipitation using a 96-well protein precipitation plate. Chromatographic separation of plasma extract was achieved with a Phenomenex Gemini C6-Phenyl HPLC column (2.1 mm × 50 mm, 5 μm) at a flow rate of 0.30 mL/min using gradient elution and a mobile phase consisting of acetonitrile and 5 mM ammonium formate. All four fatty acid ethanolamides were quantified by positive ion electrospray ionization tandem mass spectrometry, with the detection of ion current signal generated from the selected reaction monitoring (SRM) transition of [M + H]+  →  m/z 62. Deuterated anandamide (AEA-d8) was used as an internal standard for all four ethanolamides. The lower limit of quantitation was 0.05 ng/mL for AEA and LEA, 0.5 ng/mL for OEA and 1.0 ng/mL for PEA. Inter-assay precision and accuracy were typically within 12% for the four endogenous analytes and overall extraction recoveries ranged between 40% and 100%.
Keywords: Biomarker validation; Quantitative analysis; Electrospray mass spectrometry; Endogenous analysis; Multiplexing;

S-Adenosylmethionine (SAM) serves as a methyl donor in biological transmethylation reactions. S-Adenosylhomocysteine (SAH) is the product as well as the inhibitor of transmethylations and the ratio SAM/SAH is regarded as the measure of methylating capacity (“methylation index”). We present a rapid and sensitive LC–MS/MS method for SAM and SAH determination in mice tissues. The method is based on chromatographic separation on a Hypercarb column (30 mm × 2.1 mm, 3 μm particle size) filled with porous graphitic carbon stationary phase. Sufficient retention of SAM and SAH on the chromatographic packing allows simple sample preparation protocol avoiding solid phase extraction step. No significant matrix effects were observed by analysing the tissue extracts on LC–MS/MS. The intra-assay precision was less than 9%, the inter-assay precision was less than 13% and the accuracy was in the range 98–105% for both compounds. Stability of both metabolites during sample preparation and storage of tissue samples was studied: the SAM/SAH ratio in liver samples dropped by 34% and 48% after incubation of the tissues at 4 °C for 5 min and at 25 °C for 2 min, respectively. Storage of liver tissues at −80 °C for 2 months resulted in decrease of SAM/SAH ratio by 40%. These results demonstrate that preanalytical steps are critical for obtaining valid data of SAM and SAH in tissues.
Keywords: S-Adenosylmethionine; S-Adenosylhomocysteine; Mass spectrometry; Liquid chromatography; Mouse tissues;

Centrifugal partition chromatography method was applied to the separation and purification of a crude methanolic extract of a cyanobacterial lichen, Lichina pygmaea. A multiple dual-mode was used to separate two compounds of interest, namely mycosporine-serinol and a glutamic acid derivative. These compounds are described here for the first time in a lichen. Their structures were identified by UV, IR, ESI-MS, 1H NMR, 13C NMR, and 2D NMR.
Keywords: Centrifugal partition chromatography; Multiple-dual-mode; Lichina pygmaea; Purification; Isolation; Mycosporine;

The present manuscript describes development and validation of LC–MS/MS assay for the simultaneous quantitation of 97/78 and its active in-vivo metabolite 97/63 in monkey plasma using α-arteether as internal standard (IS). The method involves a single step protein precipitation using acetonitrile as extraction method. The analytes were separated on a Columbus C18 (50 mm × 2 mm i.d., 5 μm particle size) column by isocratic elution with acetonitrile:ammonium acetate buffer (pH 4, 10 mM) (80:20 v/v) at a flow rate of 0.45 mL/min, and analyzed by mass spectrometry in multiple reaction-monitoring (MRM) positive ion mode. The chromatographic run time was 4.0 min and the weighted (1/x 2) calibration curves were linear over a range of 1.56–200 ng/mL. The method was linear for both the analytes with correlation coefficients >0.995. The intra-day and inter-day accuracy (% bias) and precisions (% RSD) of the assay were less than 6.27%. Both analytes were stable after three freeze-thaw cycles (% deviation <8.2) and also for 30 days in plasma (% deviation <6.7). The absolute recoveries of 97/78, 97/63 and internal standard (IS), from spiked plasma samples were >90%. The validated assay method, described here, was successfully applied to the pharmacokinetic study of 97/78 and its active in-vivo metabolite 97/63 in Rhesus monkeys.
Keywords: LC–MS/MS; Validation; Monkey plasma; Antimalarial; Trioxane;

Combined gas chromatographic/mass spectrometric analysis of cholesterol precursors and plant sterols in cultured cells by Jure Acimovic; Anita Lövgren-Sandblom; Katalin Monostory; Damjana Rozman; Marko Golicnik; Dieter Lutjohann; Ingemar Björkhem (2081-2086).
We developed a powerful gas chromatographic/mass spectrometric method allowing quantitative analysis of 11 structurally similar cholesterol precursors and plant sterols (squalene, desmosterol, 7-dehydrocholesterol, lathosterol, zymosterol, dihydro-lanosterol, lanosterol, FF-MAS, T-MAS, campesterol, sitosterol) from cultured human hepatocytes in a single chromatographic run. Deuterium labelled cholesterol, sitosterol and lathosterol were used as internal standards. Care was taken to select ions for the detection that gave the most appropriate discrimination in the assay. Replicate analyses gave a coefficient of variation less than 6%. Recovery experiments were satisfactory for 7-dehydrocholesterol, campesterol, desmosterol, lathosterol, zymosterol and cholesterol with less than 7% difference between expected and found levels. For other sterols, the difference between expected and found levels varied between 10 and 16%. It is concluded that this method is suitable for studies on the effect of different inhibitors and stimulators of cholesterol synthesis in cultured cells. Additionally, the method is relevant also for clinical applications since abnormally increased late cholesterol intermediates in patients are representations of the inherited disorders linked to different enzyme defects in the post-squalene cholesterol biosynthesis.
Keywords: Gas chromatography/mass spectrometry; Human hepatocytes; Cholesterol biosynthesis intermediates;

Development and validation of an LC–MS/MS method for determination of methanesulfonamide in human urine by Roberto Anacardio; Frank G.P. Mullins; Sally Hannam; Muhammed S. Sheikh; Karen O'Shea; Andrea Aramini; Gaetano D’Anniballe; Loredana D’Anteo; Mauro P. Ferrari; Marcello Allegretti (2087-2092).
A sensitive and selective liquid chromatographic method coupled with tandem mass spectrometry (LC–MS/MS) was developed and validated for the quantification of methanesulfonamide (MSA) in human urine. MSA is a potential in vivo metabolite of reparixin, a specific inhibitor of the CXCL8 biological activity. In this study, a simple derivatization procedure with a new reagent, N-(4-methanesulfonyl-benzoyl)-imidazole, was set up to enable MSA and the internal standard (I.S.), ethanesulfonamide (ESA), to be analysed by LC–MS/MS. After derivatization, samples were evaporated and reconstituted in 30% acetonitrile, aq. MSA and I.S. derivatives were separated by reversed phased HPLC (high performance liquid chromatography) on a Luna 5 μ C18 column and quantitated by MS/MS using electrospray ionization (ESI) and multiple reaction monitoring (MR M) in the negative ion mode. The most intense [M−H] MRM transition of derivatized MSA at m/z 276.2 → 197.2 was used for quantitation and the transition at m/z 290.2 → 211.2 was used to monitor derivatized ESA. The method was linear over the concentration range from 1 to 100 μg/ml, with a lower limit of quantitation of 1 μg/ml. The intra- and inter-day precisions were less than 5.5% and 10.1%, respectively, and the accuracies were between −4.0% and +11.3%. The method was successfully applied to quantify levels of MSA in human urine after intravenous administration of reparixin to healthy volunteers.
Keywords: Methanesulfonamide (MSA); LC–MS/MS (liquid chromatography–mass/mass); Urine; Reparixin;

Ultra high performance liquid chromatography tandem mass spectrometric detection in clinical analysis of simvastatin and atorvastatin by Lucie Nováková; Hana Vlčková; Dalibor Šatínský; Petr Sadílek; Dagmar Solichová; Milan Bláha; Vladimír Bláha; Petr Solich (2093-2103).
Simvastatin and atorvastatin belong to the group of hypolipidemic drugs, more exactly to the second generation of inhibitors of microsomal 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. They induce a significant reduction in total cholesterol, low-density lipoprotein cholesterol and plasma triglycerides, therefore they are widely used in the treatment of hypercholesterolemia even of its severe form-familiar hypercholesterolemia. Simvastatin and atorvastatin as the most widely used statins in clinical treatment and their hydroxy-acid/lactone forms were determined by means of UPLC in connection with triple quadrupole mass spectrometer. Deuterium labeled reference standard compounds were used as internal standards for the quantitation. Separation was performed on Acquity BEH C18 (100 mm × 2.1 mm, 1.7 μm) using gradient elution by mobile phase containing acetonitrile and ammonium acetate pH 4.0, which is convenient in order to prevent interconversion of analytes. ESI in positive mode was used for the ionization of all compounds. Two SRM (selected reaction monitoring) transitions were carefully optimized for each analyte in order to get high sensitivity and selectivity. SPE on Discovery DSC-18 was used as a sample preparation step. Intra-day precision was generally within 10% RSD, while inter-day precision within 15% RSD. Method accuracy expressed as recovery ranged from 75 to 100%. The method was validated with the sensitivity reaching LOQ 0.08–5.46 nmol/l and LOD 0.01–1.80 nmol/l in biological samples. Atorvastatin, simvastatin, its metabolites and hydroxy-acid/lactone forms were monitored in human serum and in lipoprotein fractions (LDL, HDL and VLDL) at patients with end stage renal diseases.
Keywords: Atorvastatin; Simvastatin; Hemodialysis; UPLC; Tandem mass spectrometry; Bio-analytical method;

An ultra-performance liquid chromatography tandem mass spectrometry with multiple reaction monitoring method (UPLC-MRM/MS) is developed for fast and sensitive analysis of four genotoxic stereoisomers of anti-benzo[a]pyrene diol epoxide (BPDE)–N 2dG adducts (trans-(+), trans-(−), cis-(+) and cis-(−)), which result from environmental exposure to ubiquitous pollutant benzo[a]pyrene (B[a]P). The developed method displays a low limit of detection of <0.7 fmol (S/N = 3) for the four stereoisomers of anti-BPDE–N 2dG, a dynamic range of 2 orders of magnitude (2.3–630 fmol, R 2  ≥ 0.997), and one separation of 2–4 min. The developed method enables us to use the stereoisomers of anti-BPDE–N 2dG as a biomarker and to study the stereoselectivity of metabolic activation of B[a]P in human lung A549 cells. The UPLC-MRM/MS analysis of cellular DNA exposed to B[a]P show that activation of B[a]P in A549 cells predominantly induces trans-(+)-anti-BPDE–N 2dG with cis-(+)-anti-BPDE–N 2dG and one syn-BPDE–N 2dG as two minorities, while trans-(−)-anti-BPDE–N 2dG and cis-(−)-anti-BPDE–N 2dG are absent. The observed preferential formation of trans-(+)-anti-BPDE–N 2dG in B[a]P treated A549 cells may result from combined stereoselectivity of the metabolic activation of B[a]P and the reaction of anti-BPDE with dsDNA. The results also suggest that a number of key optical intermediates are formed during activation of B[a]P in A549 cells, including trans-(+)-B[a]P-7,8-dihydrodiol and trans-(−)-B[a]P-7,8-dihydrodiol and their corresponding downstream metabolites (+)-anti-BPDE and (+)-syn-BPDE.
Keywords: UPLC; ESI-MS/MS; Benzo[a]pyrene; DNA adducts;

An ultra-high pressure liquid chromatography-tandem mass spectrometry method has been developed and validated for identification and quantification of five major bioactive components in rat plasma after oral administration of Qihuotongqiao tablets. The analysis was performed on an Acquity UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 μm; Waters, USA) utilizing a gradient elution profile and a mobile phase consisting of (A) water containing 0.5 mM ammonium chloride and (B) acetonitrile. Electrospray ionization (ESI) tandem interface was employed prior to mass spectrometric detection. The calibration curve was linear over the range of 4.2–416.0 ng/mL for notoginsenoside R1, 38.4–3840.0 ng/mL for ginsenoside Rg1, 3.7–368.0 ng/mL for ginsenoside Re, 37.6–5640.0 ng/mL ginsenoside Rb1 and 4.5–448.0 ng/mL for icariin, respectively. The average accuracies ranged from 87.2 to 109.3% with RSD ≤ 13.7%. The results indicated that ultra-performance liquid chromatography-tandem mass spectrometry (UPLC–MS) provided improved chromatographic parameters resulting in significantly increased sample throughput including lower solvent consumption and lower limits of quantitation (LLOQ) for most of target analytes compared to previous method employing conventional high-performance liquid chromatography (HPLC) separation. So, the established method was validated, sensitive and reliable for the determination of five major bioactive components in rat plasma.
Keywords: Notoginsenoside R1; Ginsenoside Rg1, Re, Rb1; Icariin; Ultra-performance liquid chromatography-tandem mass spectrometry;

A sensitive, selective, and quantitative method for the simultaneous determination of gemcitabine and 2,2-difluoro-2-deoxyuridine (dFdU) has been developed and validated in human plasma in the presence of tetrahydrouridine, a cytidine deaminase inhibitor. The method employs derivatization of gemcitabine and dFdU with dansyl chloride to improve the chromatographic retention and separation. The derivatization was performed in plasma without prior sample clean-up, followed by extraction of the dansyl-derivatives using methyl tertiary-butyl ether (MTBE). Ultra performance liquid chromatography (UPLC) technology on a BEH C18 stationary phase column with 1.7 μm particle size was used for chromatographic separation coupled to tandem mass spectrometry. The method was validated over the concentration ranges of 20–5000 and 100–25,000 ng/mL for gemcitabine and dFdU, respectively. The results from assay validation show that the method is rugged, precise, accurate, and well-suited to support pharmacokinetic studies. In addition, the relatively small sample volume (50 μL) and a run time of 1.5 min facilitate automation and allow for high-throughput analysis.
Keywords: Gemcitabine; 2,2-Difluoro-2-deoxyuridine; Derivatization; Dansyl chloride; Mass spectrometry; UPLC;

Development and validation of a reversed-phase fluorescence HPLC method for determination of bucillamine in human plasma using pre-column derivatization with monobromobimane by Kang Choon Lee; Young Goo Chun; Insoo Kim; Beom Soo Shin; Eun-Seok Park; Sun Dong Yoo; Yu Seok Youn (2130-2134).
A simple, specific and sensitive derivatization with monobromobimane (mBrB) and the corresponding HPLC-fluorescence quantitation method for the analysis of bucillamine in human plasma was developed and validated. The analytical procedure involves a simple protein precipitation, pre-column fluorescence derivatization, and separation by reversed-phase high performance liquid chromatography (RP-HPLC). The calibration curve showed good linearity over a wide concentration range (50 ng/mL to 10 μg/mL) in human plasma (r 2  = 0.9998). The lower limit of quantitation (LLOQ) was 50 ng/mL. The average precision and accuracy at LLOQ were within 6.3% and 107.6%, respectively. This method was successfully applied to a pharmacokinetic study after oral administration of a dose (300 mg) of bucillamine to 20 healthy Korean volunteers.
Keywords: Pre-column fluorescence derivatization; RP-HPLC; Monobromobimane; Bucillamine; Pharmacokinetics;

Column chromatographic extraction and preparation of cordycepin from Cordyceps militaris waster medium by He Ni; Xiao-Hong Zhou; Hai-Hang Li; Wen-Fang Huang (2135-2141).
Large amounts of solid medium containing cordycepin, used in the industrial production of Cordyceps militaris through solid fermentation, are discarded as waste and contaminate the environment. We have developed a new column chromatographic extraction (CCE) method for the extraction of cordycepin from this waste and a preparation method for further separation and purification. Dried waste material was imbibed in four times its volume of water for 6 h, transferred to columns and eluted with water. Eluates were directly separated with macroporous resin DM130 columns followed by purification steps, including precipitation, crystallization, and polyamide column chromatography. Extraction rates of more than 97% were obtained with 12 volumes of water for a single column and 4 volumes of water for eluates circulated through 3 different columns designed to concentrate cordycepin. Cordycepin (98% pure) was obtained following the separation and purification processes, with an overall recovery rate of more than 90%. The CCE method has high extraction efficiency, uses a minimum volume of solvent and can be used for both quantitative analysis and large preparations of cordycepin from waste. The preparation method is simple, highly efficient, energy-saving, environmentally friendly, and has been demonstrated to be effective for large preparations of cordycepin from waste with low equipment and operating costs.
Keywords: Cordycepin production; Cordyceps militaris; Column chromatographic extraction; Macroporous resin; Solid fermentation; Medium waste;

As part of a study of the metabolism of aromatic compounds in marine gastropods, a sensitive and selective method was developed to detect, identify and quantify pyrene (PY) and four of its metabolites in tissues: 1-hydroxypyrene (PYOH), pyrene sulfate (PYOS), pyrene glucuronide (PYOG) and pyrenediol disulfate (PYDS). Liquid chromatography (LC) with fluorescence detection was first used to detect the PY derivatives in the visceral mass of whelks exposed to PYOH. The identification of metabolites was accomplished through a combination of retention time and spectral matching with standards, enzymatic hydrolysis, solid phase extraction and LC coupled with electrospray ionization mass spectrometry. In addition to four known PY derivatives, two novel metabolites were identified as pyrenediol glucuronide sulfate and a second isomer of PYDS. The methanol extraction of metabolites from tissue gave excellent mean recoveries, ranging from 67 to 97%, for the available standards PY, PYOH, PYOS and PYOG spiked in both the muscle and visceral mass of Buccinum spp. The mean recoveries of a surrogate standard, 2-hydroxyfluorene, spiked in all tissue samples were 100% and 95% for visceral and muscle tissue samples, respectively. The method limits of detection for these compounds were all below 0.2 ng/g of wet tissue, low enough to detect metabolites in reference animals. Results from the application of this method to the quantitative analysis of biotransformation products in the visceral mass of the whelk Neptunia lyrata exposed to PYOH contaminated food are also presented. This method will be useful to apply to the analysis of PY metabolites in soft tissues of other animals.
Keywords: Polycyclic aromatic hydrocarbons; Pyrene; Biotransformation; Mass spectrometry; Tissue; Metabolites;

On-line solid-phase extraction combined with liquid chromatography–tandem mass spectrometry for high throughput analysis of 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid in urine by María del Mar Ramírez Fernández; Sarah M.R. Wille; Nele Samyn; Michelle Wood; Manuel López-Rivadulla; Gert De Boeck (2153-2157).
A simple, rapid and highly sensitive method for the analysis of THC-COOH in urine, using automated on-line solid-phase extraction (SPE) combined with liquid chromatography (LC)–mass spectrometry (MS/MS), is developed and fully validated according to international guidelines. Chromatographic separation was achieved on an Atlantis dC18 column with an isocratical gradient, ensuring the elution of THC-COOH within 4.1 min. The total process time was 6 min and 500 μL of sample was required. SPE using C8 cartridges was highly effective, reproducible and led to significant decreases in the interferences present in the matrix. The method showed an excellent intra- and inter-assay precision (relative standard deviation (RSD) <7% and bias <13%) for four external quality control (QC) samples and three ‘in house’ QCs. Responses were linear over the investigated range (r 2  > 0.99, 5–200 μg/L). Limits of quantification (LOQ) and detection (LOD) were determined to be 5 μg/L and 0.25 μg/L, respectively. Furthermore, the analyte and the processed samples were demonstrated to be stable. Moreover, no carryover was observed after the analysis of high concentrated urine samples (5000 μg/L THC-COOH)). The method was subsequently applied to authentic samples previously screened by a routine immunoassay method.
Keywords: On-line SPE-LC–MS/MS; Urine; Cannabis; THC-COOH;

For the investigation of the metabolism and biosynthesis of carnitine, sensitive determination of carnitine and its metabolic precursors, trimethyllysine and γ-butyrobetaine, is required. We present here a new simplified method for the analysis of carnitine, its acetyl- and propyl esters, as well as trimethyllysine and γ-butyrobetaine without need for derivatization reactions by means of normal-phase LC and electrospray ionization tandem mass spectrometry. The limits of quantification were between 5 nM for acetyl carnitine and 70 nM for carnitine. Relative standard deviations in a fivefold determination of standard solutions were between <2% for carnitine and <10% for trimethyllysine. Quantifying the formation of deuterated carnitine from deuterated γ-butyrobetaine, this method is also suitable for the determination of the activity of γ-butyrobetaine dioxygenase in tissues.
Keywords: Carnitine; Acetyl carnitine; Propionyl carnitine; γ-Butyrobetaine; Trimethyllysine; γ-Butyrobetaine dioxygenase; Pig; Liquid chromatography; ESI; MS/MS; API 2000;

Measurement of serotonin in platelet depleted plasma by liquid chromatography tandem mass spectrometry by Phillip J. Monaghan; Heather A. Brown; Lesley A. Houghton; Brian G. Keevil (2163-2167).
5-Hydroxytryptamine (5-HT) in human platelet depleted plasma (PDP) is a biomarker in functional gastrointestinal disorders (FGID), with levels reflecting acute changes in circulating 5-HT concentration. PDP 5-HT is currently measured by reversed phase high performance liquid chromatography (HPLC) fluorimetric detection. We have developed a simple and rapid liquid chromatography tandem mass spectrometry (LC–MS/MS) method that is two times more rapid than the current HPLC methodology. Our method employs a simple protein precipitation requiring no further downstream sample preparation. 10 μL of extract was injected directly onto a SecurityGuard SCX cation exchange column followed by isocratic elution onto an Onyx Monolithic C18 analytical column and methanolic gradient elution. Eluant was connected directly to a Quattro Premier XE tandem mass spectrometer operating in ES+ mode. We detected multiple reaction monitoring transitions m/z 160 > 114.9 and m/z 164.1 > 118.9 for 5-HT and d4-5-HT, respectively. 5-HT and d4-5-HT co-eluted at 2.79 min and cycle time between injections was 6 min. Mean recovery was 98%, limit of detection 1.5 nmol/L, lower limit of quantification 5 nmol/L, linearity to 1000 nmol/L (r 2  = 0.999), imprecision <10% and bias <13.4%. 5-HT eluted with no ion suppression. No interference was found with l-tryptophan or 5-hydroxyindoleacetic acid (5-HIAA). This assay was compared to a previously published HPLC method. Passing-Bablok regression analysis showed LC–MS/MS = 0.91 (HPLC)–0.83, r 2  = 0.97, n  = 80. Bland Altman analysis showed general agreement, with a mean bias of 3.3 nmol/L. We have developed a simple and robust assay for PDP 5-HT that will increase throughput for clinical trials.
Keywords: Serotonin; LC–MS/MS; Protein precipitation; Functional gastrointestinal disorders;

We describe here a method for the determination of pantothenic acid, vitamin B5, in human urine by isocratic reversed-phase ion-pair HPLC with post-column derivatization. Pantothenic acid in urine was separated using a Tosoh ODS-80Ts (4.6 i.d. × 250 mm) column with phosphate–sodium hydroxide buffer (pH 7.0) containing dodecyltrimethylammonium chloride. Following the isolation of pantothenic acid it was decomposed to pantoic acid and β-alanine by alkali treatment. The product β-alanine was post-derivatized to the fluorescent 1-alkylthio-2-alkylisoindole with orthophthaldialdehyde in the presence of 3-mercaptopropionic acid. In the proposed method, a urine sample can be directly injected into a HPLC system without any pre-clean up treatment. The limit of detection was 3 pmol (ca. 650 pg) per 20 μL of urine at a signal-to-noise ratio of 5:1 and the limit of quantification was 5 pmol (ca. 1000 pg) per 20 μL of urine, which was sufficiently sensitive for the determination of pantothenic acid in human urine. The total time required for the analysis was ca. 25 min. The proposed method can be used to assess the pantothenic acid content of human urine as an alternative to the standard microbioassay for pantothenic acid.
Keywords: Pantothenic acid; Ion-pair HPLC; Vitamin; Human urine; Fluorometric; Post-column derivatization;

This paper describes the development of a simple and sensitive method for routine quantification of melatonin in low sample amounts by using standard equipment of HPLC with fluorescence detection. A double chloroform extraction with an intermediate cleaning step with 0.1N NaOH allowed to concentrate melatonin and to avoid interferences in extracts of the different tissues assayed. The analytical procedure was found to be precise and linear for a wide range of melatonin concentrations. The retention time of melatonin was about 9 min and the recoveries were in the range of 89–94%. The lower limit of quantification estimated on extracted samples was 8 pg/ml. This method was validated in daytime and nighttime samples of plasma, bile and intestinal tissues of trout.
Keywords: HPLC; Fluorescence detection; Melatonin; Plasma; Intestine; Bile;