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1.  Comparative Pharmacokinetics of Berberine, Palmatine and Jatrorrhizine in Rat Plasma after Oral Administration of Rhizoma coptidis and Zuojinwan Using Liquid Chromatography-Tandem Mass Spectrometry 
A selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/ MS) method was developed for the determination of berberine, palmatine and jatrorrhizine in rat plasma. Target compounds, together with the internal standard (metronidazole), were extracted from rat plasma samples by protein precipitation with acetonitrile-methanol (1:2, v/v). Chromatography was carried out using a C18 column (150 × 4.6mm, 5μm) under isocratic elution with water (containing 0.3% formic acid)-acetonitrile (30:70, v/v). The mass spectrometric detection was performed by selected reaction monitoring (SRM) mode via electrospray ionization (ESI) source operating in positive ionization mode. The method was linear over the concentration range of 0.2-100 ng/mL for all components. The intra- and inter-day precision values were less than 14.7% and the deviations were within ± 9.0%. The validated method was applied to the comparative pharmacokinetic studies of berberine, palmatine and jatrorrhizine after oral administration of Rhizoma coptidis and Zuojinwan. The results indicated that the pharmacokinetics of berberine, palmatine and jatrorrhizine were significantly different between different groups.
PMCID: PMC3813130  PMID: 24250523
Rhizoma coptidis; Zuojinwan preparation; Liquid chromatography tandem mass spectrometry; Pharmacokinetics
2.  Detection of 7-(2′-Carboxyethyl)guanine but not 7-Carboxymethylguanine in Human Liver DNA 
Chemical research in toxicology  2010;23(6):1089-1096.
7-Carboxymethylguanine (7-CMGua) and 7-(2′-carboxyethyl)guanine (7-CEGua) are DNA adducts that potentially could be formed upon metabolism of the carcinogenic nitrosamines N-nitrososarcosine (NSAR) and 3-(methylnitrosamino)propionic acid (MNPA), respectively, or from other sources such as nitrosation of glycine (7-CMGua) or reaction of DNA with acrylic acid (7-CEGua). Since both NSAR and MNPA have been detected in human urine, and there are plausible sources of exposure to other precursors to these adducts, we analyzed human liver DNA for 7-CMGua and 7-CEGua, using liquid chromatography-electrospray ionization-tandem mass spectrometry-selected reaction monitoring (LC-ESI-MS/MS-SRM). Human hepatic DNA was mixed with [15N5]7-CMGua and [15N5]7-CEGua as internal standards and enzymatically hydrolyzed. The hydrolysate was partially purified by solid-phase extraction and the resulting fraction was treated with acetyl chloride in methanol to convert 7-CMGua and 7-CEGua to their methyl esters. After a second solid-phase extraction, LC-ESI-MS/MS-SRM analysis was carried out using the transitions m/z 224 [M + H]+→ m/z 164 [(M + H) - HCOOCH3]+ and m/z 238 [M + H]+→ m/z 152 [BH]+ for the methyl esters of 7-CMGua and 7-CEGua, respectively. The method was sensitive, accurate, precise, and apparently free from artifact formation. 7-CEGua, as its methyl ester, was detected in all 24 human liver samples analyzed, mean ± S.D., 373 ± 320 fmol/μmol Gua (74.6 adducts per 109 nucleotides), range 17– 1189 fmol/μmol Gua, but the methyl ester of 7-CMGua was not detected in any sample. These results demonstrate the ubiquitous presence of 7-CEGua in human liver DNA. Acrylic acid may be a likely endogenous precursor to 7-CEGua.
doi:10.1021/tx100062v
PMCID: PMC3230219  PMID: 20438065
3.  Development of an online SPE–LC–MS-based assay using endogenous substrate for investigation of soluble epoxide hydrolase (sEH) inhibitors 
Analytical and Bioanalytical Chemistry  2011;400(5):1359-1366.
Soluble epoxide hydrolase (sEH) is a promising therapeutic target for the treatment of hypertension, pain, and inflammation-related diseases. In order to enable the development of sEH inhibitors (sEHIs), assays are needed for determination of their potency. Therefore, we developed a new method utilizing an epoxide of arachidonic acid (14(15)-EpETrE) as substrate. Incubation samples were directly injected without purification into an online solid phase extraction (SPE) liquid chromatography electrospray ionization tandem mass spectrometry (LC–ESI–MS–MS) setup allowing a total run time of only 108 s for a full gradient separation. Analytes were extracted from the matrix within 30 s by turbulent flow chromatography. Subsequently, a full gradient separation was carried out on a 50X2.1 mm RP-18 column filled with 1.7 μm core–shell particles. The analytes were detected with high sensitivity by ESI–MS–MS in SRM mode. The substrate 14(15)-EpETrE eluted at a stable retention time of 96 ± 1 s and its sEH hydrolysis product 14,15-DiHETrE at 63 ± 1 s with narrow peak width (full width at half maximum height: 1.5 ± 0.1 s). The analytical performance of the method was excellent, with a limit of detection of 2 fmol on column, a linear range of over three orders of magnitude, and a negligible carry-over of 0.1% for 14,15-DiHETrE. The enzyme assay was carried out in a 96-well plate format, and near perfect sigmoidal dose–response curves were obtained for 12 concentrations of each inhibitor in only 22 min, enabling precise determination of IC50 values. In contrast with other approaches, this method enables quantitative evaluation of potent sEHIs with picomolar potencies because only 33 pmol L−1 sEH were used in the reaction vessel. This was demonstrated by ranking ten compounds by their activity; in the fluorescence method all yielded IC50 ≤ 1 nmol L−1. Comparison of 13 inhibitors with IC50 values >1 nmol L−1 showed a good correlation with the fluorescence method (linear correlation coefficient 0.9, slope 0.95, Spearman’s rho 0.9). For individual compounds, however, up to eightfold differences in potencies between this and the fluorescence method were obtained. Therefore, enzyme assays using natural substrate, as described here, are indispensable for reliable determination of structure–activity relationships for sEH inhibition.
Electronic supplementary material
The online version of this article (doi:10.1007/s00216-011-4861-2) contains supplementary material, which is available to authorized users.
doi:10.1007/s00216-011-4861-2
PMCID: PMC3081056  PMID: 21479549
Soluble epoxide hydrolase (sEH); Natural substrate enzyme assay; Enzyme inhibitors turbulent-flow chromatography; Online-solid phase extraction; Liquid chromatography; Electrospray mass spectrometry tandem mass spectrometry
4.  Relative Determination Approach to the Metabolites of Protoberberine Alkaloids in Rat Urine by Liquid Chromatography Tandem Mass Spectrometry for the Comparative Studies on Rhizome coptidis and Zuojinwan Preparation  
The lack of authentic standards has limited the quantitative analysis of herbal drugs in biological samples. The present work demonstrated a practicable strategy for the assay of herbs and their metabolites independent of authentic standards. A liquid chromatography– electrospray ionization–mass spectrometry (LC–ESI–MS) method for the qualitative and quantitative determination of the metabolites after oral administration of Rhizome coptidis and Zuojinwan preparation in rat urine has been developed. Urine samples, extracted with a protein precipitation procedure were separated on a C18 column using a mixture of water (containing 0.1% formic acid) and acetonitrile (30:70, v/v) as mobile phase. The detection was performed via MS with electrospray ionization interface in positive selected reaction monitoring (SRM) mode. One urine sample after administration was selected as ‹standard›. The method validation was carried out according to a conventional method which was calibrated by authentic standards. The fully validated method was applied to the pharmacokinetic study of 2,9-demethyljateorhizine-3-sulfate, 13-methoxyjateorhizine-3- glucoronide and 6-methyljateorhizine-5-glucoronide in rat urine. The results could provide evidence to explain the combination of Rhizome coptidis and Evodiae fructus in terms of elimination.
PMCID: PMC3813152  PMID: 24250561
Metabolite; Determination; High performance liquid chromatography; Tandem mass spectrometry; Rhizome coptidis; Zuojinwan preparation
5.  LC–MS–MS Method for the Analysis of New Non-Imidazole Histamine H3 Receptor Antagonist 1-[3-(4-tert-Butylphenoxy)propyl]piperidine in Rat Serum—Application to Pharmacokinetic Studies 
Chromatographia  2011;73(9-10):913-919.
A sensitive and specific liquid chromatography electrospray ionisation–tandem mass spectrometry method for determination of new non-imidazole histamine H3 receptor antagonist 1-[3-(4-tert-butylphenoxy)propyl]piperidine (DL76) in rat serum has been developed and validated. Chromatography was performed on a XBridge™ C18 analytical column (2.1 × 30 mm, 3.5 µm, Waters, Ireland) with gradient elution using a mobile phase containing acetonitrile and water with an addition of 0.1% of formic acid. Detection was achieved by an Applied Biosystems MDS Sciex (Concord, Ontario, Canada) API 2000 triple quadrupole mass spectrometer. Electrospray ionization (ESI) was used for ion production. The limit of detection in the SRM mode was found to be 0.5 ng mL−1. The limit of quantification was 1 ng mL−1. The precision and accuracy for both intra- and inter-day determination of DL76 ranged from 1.65 to 15.09% and from 88.74 to 113.43%. The results of this analytical method validation allow to carry out pharmacokinetic studies in rats. The method was used for the pilot study of the pharmacokinetic behavior of DL76 in rats after intravenous administration.
doi:10.1007/s10337-011-1983-9
PMCID: PMC3073091  PMID: 21544189
Column liquid chromatography; Tandem MS detection; DL76 compound; Histamine H3 receptor antagonist; Rats serum
6.  Validation and application of a liquid chromatography-tandem mass spectrometric method for quantification of the drug transport probe fexofenadine in human plasma using 96-well filter plates 
A rapid method to determine fexofenadine concentrations in human plasma using protein precipitation in 96-well plates and liquid chromatography-tandem mass spectrometry was validated. Plasma proteins were precipitated with acetonitrile containing the internal standard fexofenadine-d6, mixed briefly, and then filtered into a collection plate. The resulting filtrate was diluted and injected onto a Phenomenex Gemini C18 (50 × 2.0 mm, 5 micron) analytical column. The mobile phase consisted of 0.1% formic acid, 5 mM ammonium acetate in deionized water and methanol (35:65, v/v). The flow rate was 0.2 ml/min and the total run time was 2 min. Detection of the analytes was achieved using positive ion electrospray ionization and high resolution multiple reaction monitoring mode (H-SRM). The linear standard curve ranged from 1 to 500 ng/ml and the precision and accuracy (intra- and inter-run) were within 4.3% and 8.0%, respectively. The method has been applied successfully to determine fexofenadine concentrations in human plasma samples obtained from subjects administered a single oral dose of fexofenadine. The method is rapid, sensitive, selective and directly applicable to human pharmacokinetic studies involving fexofenadine.
doi:10.1016/j.jchromb.2009.12.022
PMCID: PMC2818817  PMID: 20045385
Fexofenadine; fexofenadine-d6; protein precipitation; LC-MS; human plasma
7.  Detection and quantitation of N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine adducts in DNA using online column-switching liquid chromatography tandem mass spectrometry 
The heterocyclic aromatic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is formed by the grilled cooking of certain foods such as meats, poultry and fish. PhIP has been shown to induce tumours in the colon, prostate and mammary glands of rats and is regarded as a potential human dietary carcinogen. PhIP is metabolically activated via cytochrome P450 mediated oxidation to an N-hydroxylamino-PhIP intermediate that is subsequently converted to an ester by N-acetyltransferases or sulfotransferases and undergoes heterolytic cleavage to produce a PhIP-nitrenium ion, which reacts with DNA to form the N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP-C8-dG) adduct. Thus far, the detection and quantification of PhIP-DNA adducts has relied to a large extent on 32P-postlabelling methodologies. In order to expand the array of available techniques for the detection and improved quantification of PhIP-C8-dG adducts in DNA we have developed an online column-switching liquid chromatography (LC)-electrospray ionization (ESI)-tandem mass spectrometry (MS/MS) selected reaction monitoring (SRM) method incorporating an isotopically [13C10]-labelled PhIP-C8-dG internal standard for the analysis of DNA enzymatically hydrolysed to 2′-deoxynucleosides. A dose-dependent increase was observed for PhIP-C8-dG adducts when salmon testis DNA was reacted with N-acetoxy-PhIP. Analysis of DNA samples isolated from colon tissue of mice treated by oral gavage daily for 5 days with 50 mg/kg body weight of PhIP resulted in the detection of an average level of 14.8 ± 3.7 PhIP-C8-dG adducts per 106 2′-deoxynucleosides. The method required 50 μg of hydrolysed animal DNA on column and the limit of detection for PhIP-C8-dG was 2.5 fmol (1.5 PhIP-C8-dG adducts per 108 2′-deoxynucleosides). In summary, the LC-ESI-MS/MS SRM method provides for the rapid automation of the sample clean up and a reduction in matrix components that would otherwise interfere with the mass spectrometric analysis, with sufficient sensitivity and precision to analyse DNA adducts in animals exposed to PhIP.
doi:10.1016/j.jchromb.2010.06.008
PMCID: PMC2923026  PMID: 20598652
8.  Sensitive isotope dilution liquid chromatography/tandem mass spectrometry method for quantitative analysis of bumetanide in serum and brain tissue 
We have developed and validated a simple and sensitive stable isotope dilution liquid chromatography/tandem mass spectrometric (LC-MS/MS) method for the quantification of bumetanide in human serum. Samples were prepared with a simple acetonitrile based protein precipitation. The supernatant was then analyzed directly using LC-MS/MS. Chromatographic separation was achieved on a C18 reversed phase column using a methanol and water gradient. The detection was performed in selected reaction monitoring (SRM) mode via a positive electrospray ionization (ESI) interface. The method had a lower limit of quantification (LLOQ) of 1 ng/mL, linearity up to 1250 ng/mL, intra- and inter-day precision less than 10%, and accuracy within ±10%. This method was also demonstrated to be suitable for the analysis of bumetanide in rat serum and brain tissue. Bumetanide concentrations in rat serum and brain were determined for samples collected at several intervals following intraperitoneal (i.p.) injection of bumetanide, and were used to calculate bumetanide permeability through the blood brain barrier.
doi:10.1016/j.jchromb.2011.02.018
PMCID: PMC3727383  PMID: 21414852
bumetanide; liquid chromatography/tandem mass spectrometry (LC-MS/MS); stable isotope dilution
9.  Novel LC-ESI/MS/MSn Method for the Characterization and Quantification of 2′-Deoxyguanosine Adducts of the Dietary Carcinogen 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine by 2-D Linear Quadrupole Ion Trap Mass Spectrometry 
Chemical research in toxicology  2007;20(2):263-276.
An accurate and sensitive liquid chromatography-electrospray ionization/multi-stage mass spectrometry (LC-ESI/MS/MSn) technique has been developed for the characterization and quantification of 2′-deoxyguanosine (dG) adducts of the dietary mutagen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). PhIP is an animal and potential human carcinogen that occurs in grilled meats. Following enzymatic digestion and adduct enrichment by solid-phase extraction (SPE), PhIP—DNA adducts were analyzed by MS/MS and MSn scan modes on a 2-D linear quadrupole ion trap mass spectrometer (QIT/MS). The major DNA adduct, N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (dG-C8-PhIP), was detected in calf thymus (CT) DNA modified in vitro with a bioactivated form of PhIP and in the colon and liver of rats given PhIP as part of the diet. The lower limit of detection (LOD) was 1 adduct per 108 DNA bases, and the limit of quantification (LOQ) was 3 adducts per 108 DNA bases in both MS/MS and MS3 scan modes, using 27 μg of DNA for analysis. Measurements were based on isotope dilution with the internal standard, N-(deoxyguanosin-8-yl)-2-amino-1-(trideutero)methyl-6-phenylimidazo[4,5-b]pyridine (dG-C8-[2H3C]-PhIP). The selected reaction monitoring (SRM) scan mode in MS/MS was employed to monitor the loss of deoxyribose (dR) from the protonated molecules of the adducts ([M + H - 116]+). The consecutive reaction monitoring (CRM) scan modes in MS3 and MS4 were used to measure and further characterize product ions of the aglycone ion (BH2+) (Guanyl-PhIP). The MS3 scan mode was effective in eliminating isobaric interferences observed in the MS/MS scan mode and resulted in an improved signal-to-noise (S/N) ratio. Moreover, the product ion spectra obtained by the MSn scan modes provided rich structural information about the adduct and were used to corroborate the identity of dG-C8-PhIP. In addition, an isomeric dG-PhIP adduct was detected in vivo. This LCESI/MS/MSn method is the first reported application on the use of the MS3 scan mode for the analysis of DNA adducts in vivo.
doi:10.1021/tx0601713
PMCID: PMC2593646  PMID: 17305409
10.  Comparison of Gas Chromatography-Mass Spectrometry and Gas Chromatography-Tandem Mass Spectrometry with Electron Ionization and Negative-Ion Chemical Ionization for Analyses of Pesticides at Trace Levels in Atmospheric Samples 
A comparison of detection limits of gas chromatography-mass spectrometry (GC-MS) in selected ion monitoring (SIM) with gas chromatography-tandem mass spectrometry (GC-MS/MS) in selected reaction monitoring (SRM) mode with both electron ionization (EI) and negative-ion chemical ionization (NCI) are presented for over 50 pesticides ranging from organochlorines (OCs), organophosphorus pesticides (OPs) and pre-emergent herbicides used in the Canadian prairies (triallate, trifluralin, ethalfluralin). The developed GC-EI/SIM, GC-NCI/SIM, and GC-NCI/SRM are suitable for the determination of pesticides in air sample extracts at concentrations <100 pg μL−1 (<100 pg m−3 in air). No one method could be used to analyze the range of pre-emergent herbicides, OPs, and OCs investigated. In general GC-NCI/SIM provided the lowest method detection limits (MDLs commonly 2.5–10 pg μL−1) along with best confirmation (<25% RSD of ion ratio), while GC-NCI/SRM is recommended for use where added selectivity or confirmation is required (such as parathion-ethyl, tokuthion, carbofenothion). GC-EI/SRM at concentration <100 pg μL−1 was not suitable for most pesticides. GC-EI/SIM was more prone to interference issues than NCI methods, but gave good sensitivity (MDLs 1–10 pg μL−1) for pesticides with poor NCI response (OPs: sulfotep, phorate, aspon, ethion, and OCs: alachlor, aldrin, perthane, and DDE, DDD, DDT).
PMCID: PMC2701172  PMID: 19609395
gas chromatography; negative ion chemical ionization; mass spectrometry; pesticide analysis; atmospheric samples
11.  Pesticides in Surface Drinking-Water Supplies of the Northern Great Plains 
Environmental Health Perspectives  2007;115(8):1183-1191.
Background
Human health anomalies have been associated with pesticide exposure for people living in rural landscapes in the northern Great Plains of North America.
Objective
The objective of this study was to investigate the occurrence of 45 pesticides in drinking water from reservoirs in this area that received water primarily from snowmelt and rainfall runoff from agricultural crop lands.
Methods
Water from 15 reservoirs was sampled frequently during the spring pesticide application period (early May to mid-August) and less frequently for the remainder of the year. Drinking water was sampled in early July. Sample extracts were analyzed for pesticide content using mass spectrometric detection.
Results
We detected two insecticides and 27 herbicides in reservoir water. Consistent detection of a subset of 7 herbicides suggested that atmospheric deposition, either directly or in rain, was the principal pathway from fields to the reservoirs. However, the highest concentrations and number of herbicides in drinking water were associated with runoff from a localized 133-mm rainfall over 15 days toward the end of spring herbicide application. Water treatment removed from 14 to 86% of individual herbicides. Drinking water contained 3–15 herbicides (average, 6.4).
Conclusions
We estimated the mean annual calculated concentration of herbicides in drinking water to be 75 ng/L (2,4-dichlorophenoxy)acetic acid, 31 ng/L (2-chloro-4-methylphenoxy)acetic acid, 24 ng/L clopyralid, 11 ng/L dichlorprop, 4 ng/L dicamba, 3 ng/L mecoprop, and 1 ng/L bro-moxynil. The maximum total concentration of herbicides in drinking water was 2,423 ng/L. For the seven herbicides with established drinking water guidelines, all concentrations of the individual chemicals were well below their respective guideline. However, guidelines have not been established for the majority of the herbicides found in drinking water or for mixtures of pesticides.
doi:10.1289/ehp.9435
PMCID: PMC1940079  PMID: 17687445
drinking water; northern Great Plains; pesticides; reservoirs; water treatment
12.  Simplified method for determination of clarithromycin in human plasma using protein precipitation in a 96-well format and liquid chromatography-tandem mass spectrometry 
A simplified method to determine clarithromycin concentrations in human plasma using protein precipitation in a 96-well plate and liquid chromatography tandem mass spectrometry was developed and validated. Plasma proteins were precipitated with acetonitrile and roxithromycin was used as the internal standard. After vortex-mixing and centrifugation, the supernatants were directly injected onto a Phenomenex Luna Phenyl-Hexyl column (50 × 2.0 mm I.D., 3μm). The mobile phase consisted of water and methanol (30:70; v/v) containing 0.1% formic acid and 5 mM ammonium acetate. The flow rate was 0.22 mL/min and the total run time (injection to injection) was less than 3 minutes. Detection of the analytes was achieved using positive ion electrospray tandem mass spectrometry in selected reaction monitoring (SRM) mode. The linear standard curve ranged from 100 to 5,000 ng/mL and the precision and accuracy (inter- and intrarun) were within 8.3% and 6.3%, respectively. The method was successfully used to determine clarithromycin concentrations in human plasma samples obtained from healthy subjects who were given clarithromycin 500 mg for three days. The method is rapid, simple, precise and directly applicable to clarithromycin pharmacokinetic studies.
doi:10.1016/j.jchromb.2008.06.050
PMCID: PMC3832059  PMID: 18639501
clarithromycin; roxithromycin; protein precipitation; LC-MS; human plasma
13.  LC–MS/MS determination of carbamathione in microdialysis samples from rat brain and plasma 
A selective liquid chromatography–tandem mass spectrometric (LC–MS/MS) method was developed for the determination of S-(N, N-diethylcarbamoyl) glutathione (carbamathione) in microdialysis samples from rat brain and plasma. S-(N, N-Diethylcarbamoyl) glutathione (carbamathione) is a metabolite of disulfiram. This metabolite may be responsible for disulfiram’s effectiveness in the treatment of cocaine dependence. An analytical method using liquid chromatography–tandem mass spectrometric (LC–MS/MS) was developed to determine carbamathione in vivo using microdialysis sampling from rat brain and plasma. Chromatographic separations were carried out on an Alltech Altima C-18 (50 mm long × 2.1 mm i.d., 3 μm particles) analytical column at a flow rate of 0.3 ml/min. Solvent A consisted of 10 mM ammonium formate, methanol, and formic acid (99:1:0.06, v/v/v). Solvent B consisted of methanol, 10 mM ammonium formate and formic acid (99:1:0.06, v/v/v). A 20 min linear gradient from 95% aqueous to 95% organic was used. Tandem mass spectra were acquired on a Micromass Quattro Ultima “triple” quadrupole mass spectrometer equipped with an ESI interface. Quantitative mass spectrometric analysis was conducted in positive ion mode selected reaction monitoring (SRM) mode looking at the transition of m/z 407–100 and 175 for carbamathione and m/z 392–263 for the internal standard S-hexyl glutathione. The simultaneous collection of microdialysate from blood and brain was used to monitor carbamathione concentrations centrally and peripherally. Good linearity was obtained over a concentration range of 0.25–10,000 nM. The lowest limit of quantification (LLOQ) was determined to be 1 nM and the lowest limit of detection (LLOD) was calculated to be 0.25 nM. Intra- and inter-day accuracy and precision were determined and for all the samples evaluated, the variability was less that 10% (R.S.D.).
doi:10.1016/j.jpba.2009.07.026
PMCID: PMC2760994  PMID: 19709836
Disulfiram; Carbamathione; Microdialysis
14.  Quantitation of Melatonin and N-acetylserotonin in Human Plasma by Nanoflow LC-MS/MS and Electrospray LC-MS/MS 
Journal of Mass Spectrometry  2012;47(3):277-285.
Melatonin (MEL) and its chemical precursor N-acetylserotonin (NAS) are believed to be potential biomarkers for sleep-related disorders. Measurement of these compounds, however, has proven to be difficult due to their low circulating levels, especially that of NAS. Few methods offer the sensitivity, specificity and dynamic range needed to monitor MEL and its precursors and metabolites in small blood samples, such as those obtained from pediatric patients. In support of our ongoing study to determine the safety, tolerability, and PK dosing strategies for MEL in treating insomnia in children with autism spectrum disorder, two highly sensitive LC-MS/MS assays were developed for the quantitation of MEL and precursor NAS at pg/mL levels in small volumes of human plasma. A validated electrospray ionization (ESI) method was used to quantitate high levels of MEL in PK studies and a validated nanospray (nESI) method was developed for quantitation of MEL and NAS at endogenous levels. In both assays plasma samples were processed by centrifugal membrane dialysis after addition of stable isotopic internal standards, and the components were separated by either conventional LC using a Waters SymmetryShield RP18 column (2.1×100 mm, 3.5 μm) or on a polyimide-coated, fused-silica capillary self-packed with 17 cm AquaC18 (3 μm, 125 Å). Quantitation was done using the SRM transitions m/z 233→174 and m/z 219→160 for MEL and NAS, respectively. The analytical response ratio vs. concentration curves were linear for MEL (nanoflow LC: 11.7–1165 pg/mL, LC: 1165–116500 pg/mL) and for NAS (nanoflow LC: 11.0–1095 pg/mL).
doi:10.1002/jms.2051
PMCID: PMC3319463  PMID: 22431453
Melatonin; N-acetylserotonin; Autism Spectrum Disorder; Nanoflow LC-MS/MS
15.  Simultaneous Determination of Celecoxib, Erlotinib, and its Metabolite Desmethyl-Erlotinib (OSI-420) in Rat Plasma by Liquid chromatography/Tandem Mass Spectrometry with Positive/Negative Ion-Switching Electrospray Ionisation 
Scientia Pharmaceutica  2012;80(3):633-646.
A new method for the simultaneous determination of celecoxib, erlotinib, and its active metabolite desmethyl-erlotinib (OSI-420) in rat plasma, by liquid chromatography/tandem mass spectrometry with positive/negative ion-switching electrospray ionization mode, was developed and validated. Protein precipitation with methanol was selected as the method for preparing the samples. The analytes were separated on a reverse-phase C18 column (50mm×4.6mm i.d., 3μ) using methanol: 2 mM ammonium acetate buffer, and pH 4.0 as the mobile phase at a flow rate 0.8 mL/min. Sitagliptin and Efervirenz were used as the internal standards for quantification. The determination was carried out on a Theremo Finnigan Quantam ultra triple-quadrupole mass spectrometer, operated in selected reaction monitoring (SRM) mode using the following transitions monitored simultaneously: positive m/z 394.5→278.1 for erlotinib, m/z 380.3→278.1 for desmethyl erlotinib (OSI-420), and negative m/z −380.1→ −316.3 for celecoxib. The limits of quantification (LOQs) were 1.5 ng/mL for Celecoxib, erlotinib, and OSI-420. Within- and between-day accuracy and precision of the validated method were within the acceptable limits of < 15% at all concentrations. The quantitation method was successfully applied for the simultaneous estimation of celecoxib, erlotinib, and desmethyl erlotinib in a pharmacokinetic study in Wistar rats.
doi:10.3797/scipharm.1205-09
PMCID: PMC3447620  PMID: 23008811
Celecoxib; Erlotinib; Desmethyl erlotinib; OSI-420; LC-MS/MS; Bioanalytical
16.  Measuring long chain acyl-CoA concentrations and enrichment using LC/MS/MS with selected reaction monitoring 
Rapid communications in mass spectrometry : RCM  2011;25(15):10.1002/rcm.5110.
Long chain acyl-CoA's (LCACoA) are the activated form of long chain fatty acids and serve as key lipid metabolites. Excess accumulation of intracellular LCACoA, diacylglycerols (DAGs) and ceramides may create insulin resistance with respect to glucose metabolism. We present a new method to measure LCACoA concentrations and isotopic enrichment of palmitoyl-CoA ([U-13C]16-CoA) and oleoyl-CoA ([U-13C]18:1-CoA) using UPLC/MS/MS to quantitate 7 different LCACoA (C14-CoA, C16-CoA, C16:1-CoA, C18-CoA, C18:1-CoA, C18:2-CoA, C20-CoA). The molecules are separated on reverse-phase UPLC column using a binary gradient with ammonium hydroxide (NH4OH) in water and NH4OH in ACN. The LCACoA are quantified using selected reaction monitoring (SRM) on a triple quadrupole mass spectrometer in positive electrospray ionization (ESI) mode. All LCACoA ions except enriched palmitate and oleate were monitored as [M+2+H]+ and the [U13C]16-CoA and [U13C]18:1-CoA were monitored as [M+16+H]+ and [M+18+H]+ respectively. The method is simple, sensitive and efficient (run time as short as 5 min) and allowed us to measure the concentration and detect enrichment in intramyocellular [U13C]16-CoA and [U13C]18:1-CoA during a low dose intravenous infusion of [U13C]palmitate and [U13C]oleate in adults undergoing either a saline control experiment or an insulin/glucose infusion experiment. This technique should allow investigators to measure the trafficking of extracellular fatty acids to the intracellular LCACoA pool.
doi:10.1002/rcm.5110
PMCID: PMC3812934  PMID: 21735505
ultra-pressure liquid chromatography; muscle; free fatty acids; tandem mass spectrometry; stable isotopes
17.  Characterization of Three Berry Standard Reference Materials for Nutrients 
The National Institute of Standards and Technology (NIST) has been working with the National Institutes of Health Office of Dietary Supplements to produce Standard Reference Materials (SRMs) of interest to analysts of dietary supplements. Some of these SRMs are traditional foods including SRM 3281 Cranberry (Fruit), SRM 3282 Low-Calorie Cranberry Juice Cocktail, and SRM 3287 Blueberry (Fruit), which have been characterized for nine nutritional elements and sugars. The blueberries have also been characterized for proximates, two water-soluble vitamins, and amino acids. These new materials are intended for use in method development and validation as well as for quality assurance and traceability when assigning values to in-house control materials. Foods can be difficult to analyze because of matrix effects. With the addition of these three new SRMs, it is now possible to more closely match controls to matrices and analyte levels for fruit and vegetable test samples. Several nutritional elements in these three SRMs are present at lower levels than those in other food-matrix SRMs.
doi:10.1021/jf200883e
PMCID: PMC3153887  PMID: 21688777
18.  Development of an ultra fast online-solid phase extraction (SPE) liquid chromatography electrospray tandem mass spectrometry (LC-ESI-MS/MS) based approach for the determination of drugs in pharmacokinetic studies† 
High-throughput analyses of a large number of samples for pharmacokinetic (PK) studies are essential in drug development. Analysis of drug candidates from blood using LC-ESI-MS generally requires separation of the plasma fraction followed by various offline sample preparation procedures. This step is a bottleneck that impedes throughput. In order to overcome this difficulty and accelerate analysis in PK and other studies, we developed an approach allowing the direct analysis of low volumes of whole blood (10 μL) after dilution and centrifugation. Samples were injected in an online-SPE-LC-ESI-MS/MS setup allowing a total run time of only 126 s for a full gradient separation. Analytes were extracted from the matrix within 30 s by turbulent flow chromatography. Subsequently, a full gradient separation was carried out within 1.5 minutes on a 50 × 2.1 mm (1.7 μm) RP-18 column and the analytes were sensitively detected by ESI-MS/MS in SRM mode. The performance of this new ultra fast online SPE-LC-ESI-MS/MS approach was demonstrated by the analysis of diclofenac (DCF), a widely used anti-inflammatory drug. DCF eluted at stable retention times (±0.33%) with narrow peak width (FWHM 3.3 ± 0.15 s). The method displays excellent analytical performance, with a limit of detection of 6 fmol on column, a linear range of over four orders of magnitude and a negligible carry over of 0.12 ± 0.03% for DCF. The PK profile of DCF administered by topical and intraperitoneal routes in rats and by oral route in one human volunteer is investigated using this method. Finally, general applicability of the approach for drugs is demonstrated by analysis of rofecoxib and several inhibitors of the soluble epoxide hydrolase. This new method requires only readily available, off the shelf standard LC instrumentation, and is compliant with the requirements of green analytical chemistry.
doi:10.1039/C0AY00714E
PMCID: PMC3110015  PMID: 21660124
19.  Label-Free LC-MS Profiling of Skeletal Muscle Reveals Heart-Type Fatty Acid Binding Protein as a Candidate Biomarker of Aerobic Capacity 
Proteomes  2013;1(3):290-308.
Two-dimensional gel electrophoresis provides robust comparative analysis of skeletal muscle, but this technique is laborious and limited by its inability to resolve all proteins. In contrast, orthogonal separation by SDS-PAGE and reverse-phase liquid chromatography (RPLC) coupled to mass spectrometry (MS) affords deep mining of the muscle proteome, but differential analysis between samples is challenging due to the greater level of fractionation and the complexities of quantifying proteins based on the abundances of their tryptic peptides. Here we report simple, semi-automated and time efficient (i.e., 3 h per sample) proteome profiling of skeletal muscle by 1-dimensional RPLC electrospray ionisation tandem MS. Solei were analysed from rats (n = 5, in each group) bred as either high- or low-capacity runners (HCR and LCR, respectively) that exhibited a 6.4-fold difference (1,625 ± 112 m vs. 252 ± 43 m, p < 0.0001) in running capacity during a standardized treadmill test. Soluble muscle proteins were extracted, digested with trypsin and individual biological replicates (50 ng of tryptic peptides) subjected to LC-MS profiling. Proteins were identified by triplicate LC-MS/MS analysis of a pooled sample of each biological replicate. Differential expression profiling was performed on relative abundances (RA) of parent ions, which spanned three orders of magnitude. In total, 207 proteins were analysed, which encompassed almost all enzymes of the major metabolic pathways in skeletal muscle. The most abundant protein detected was type I myosin heavy chain (RA = 5,843 ± 897) and the least abundant protein detected was heat shock 70 kDa protein (RA = 2 ± 0.5). Sixteen proteins were significantly (p < 0.05) more abundant in HCR muscle and hierarchal clustering of the profiling data highlighted two protein subgroups, which encompassed proteins associated with either the respiratory chain or fatty acid oxidation. Heart-type fatty acid binding protein (FABPH) was 1.54-fold (p = 0.0064) more abundant in HCR than LCR soleus. This discovery was verified using selective reaction monitoring (SRM) of the y5 ion (551.21 m/z) of the doubly-charged peptide SLGVGFATR (454.19 m/z) of residues 23–31 of FABPH. SRM was conducted on technical replicates of each biological sample and exhibited a coefficient of variation of 20%. The abundance of FABPH measured by SRM was 2.84-fold greater (p = 0.0095) in HCR muscle. In addition, SRM of FABPH was performed in vastus lateralis samples of young and elderly humans with different habitual activity levels (collected during a previous study) finding FABPH abundance was 2.23-fold greater (p = 0.0396) in endurance-trained individuals regardless of differences in age. In summary, our findings in HCR/LCR rats provide protein-level confirmation for earlier transcriptome profiling work and show LC-MS is a viable means of profiling the abundance of almost all major metabolic enzymes of skeletal muscle in a highly parallel manner. Moreover, our approach is relatively more time efficient than techniques relying on orthogonal separations, and we demonstrate LC-MS profiling of the HCR/LCR selection model was able to highlight biomarkers that also exhibit differences in trained and untrained human muscle.
doi:10.3390/proteomes1030290
PMCID: PMC3997170  PMID: 24772389
aerobic capacity; animal selection model; exercise training; heart-type fatty acid binding protein; FABPH; Fabp3; human vastus lateralis; selective reaction monitoring
20.  Development and Comparison of Three Liquid Chromatography-Atmospheric Pressure Chemical Ionization/Mass Spectrometry Methods for Determining Vitamin D Metabolites in Human Serum 
Journal of Chromatography. a  2012;1240:132-139.
Liquid chromatographic methods with atmospheric pressure chemical ionization mass spectrometry were developed for the determination of the vitamin D metabolites 25-hydroxyvitamin D2 (25(OH)D2), 25-hydroxyvitamin D3 (25(OH)D3), and 3-epi-25-hydroxyvitamin-D3 (3-epi-25(OH)D3) in the four Levels of SRM 972, Vitamin D in Human Serum. One method utilized a C18 column, which separates 25(OH)D2 and 25(OH)D3, and one method utilized a CN column that also resolves the diastereomers 25(OH)D3 and 3-epi-25(OH)D3. Both methods utilized stable isotope labeled internal standards for quantitation of 25(OH)D2 and 25(OH)D3. These methods were subsequently used to evaluate SRM 909c Human Serum, and 25(OH)D3 was the only vitamin D metabolite detected in this material. However, SRM 909c samples contained matrix peaks that interfered with the determination of the [2H6]-25(OH)D3 peak area. The chromatographic conditions for the C18 column were modified to remove this interference, but conditions that separated the matrix peaks from [2H6]-25(OH)D3 on the CN column could not be identified. The alternate internal standard [2H3]-25(OH)D3 did not suffer from matrix interferences and was used for quantitation of 25(OH)D3 in SRM 909c. During the evaluation of SRM 909c samples, a third method was developed using a pentafluorophenylpropyl column that also separates the diastereomers 25(OH)D3 and 3-epi-25(OH)D3. The 25(OH)D3 was measured in SRM 909c using all three methods, and the results were compared.
doi:10.1016/j.chroma.2012.03.091
PMCID: PMC3348387  PMID: 22533908
LC-MS; 25-hydroxyvitamin D2; 25-hydroxyvitamin D3; 3-epi-25-hydroxyvitamin D3; Standard Reference Material; human serum
21.  Ultra high performance liquid chromatography tandem mass spectrometry for rapid analysis of trace organic contaminants in water 
Background
The widespread utilization of organic compounds in modern society and their dispersion through wastewater have resulted in extensive contamination of source and drinking waters. The vast majority of these compounds are not regulated in wastewater outfalls or in drinking water while trace amounts of certain compounds can impact aquatic wildlife. Hence it is prudent to monitor these contaminants in water sources until sufficient toxicological data relevant to humans becomes available. A method was developed for the analysis of 36 trace organic contaminants (TOrCs) including pharmaceuticals, pesticides, steroid hormones (androgens, progestins, and glucocorticoids), personal care products and polyfluorinated compounds (PFCs) using a single solid phase extraction (SPE) technique with ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). The method was applied to a variety of water matrices to demonstrate method performance and reliability.
Results
UHPLC-MS/MS in both positive and negative electrospray ionization (ESI) modes was employed to achieve optimum sensitivity while reducing sample analysis time (<20 min) compared with previously published methods. The detection limits for most compounds was lower than 1.0 picogram on the column while reporting limits in water ranged from 0.1 to 15 ng/L based on the extraction of a 1 L sample and concentration to 1 mL. Recoveries in ultrapure water for most compounds were between 90-110%, while recoveries in surface water and wastewater were in the range of 39-121% and 38-141% respectively. The analytical method was successfully applied to analyze samples across several different water matrices including wastewater, groundwater, surface water and drinking water at different stages of the treatment. Among several compounds detected in wastewater, sucralose and TCPP showed the highest concentrations.
Conclusion
The proposed method is sensitive, rapid and robust; hence it can be used to analyze a large variety of trace organic compounds in different water matrixes.
doi:10.1186/1752-153X-7-104
PMCID: PMC3707776  PMID: 23777604
Trace organic contaminant; Pharmaceutical; Personal-care product; Glucocorticoid; PFC; Solid-phase extraction; Ultra-high performance liquid chromatography; Tandem mass spectrometry; Water quality
22.  Validated method to measure yakuchinone A in plasma by LC-MS/MS and its application to a pharmacokinetic study in rats 
Background
Yakuchinone A has a plethora of beneficial biological effects. However, the pharmacokinetic (PK) data of yakuchinone A still remain unknown so far. Furthermore, the quantification of yakuchinone A in biological samples has not been reported in the literature. Therefore, in the present study we aimed to develop a new method for the fast, efficient and accurate assessment of yakuchinone A concentration in plasma, as a means for facilitating the PK evaluation of yakuchinone A.
Results
A liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method was developed and validated for the determination of yakuchinone A in rat plasma. Mass spectrometric and chromatographic conditions were optimized. Plasma samples were pretreated by protein precipitation with methanol. LC separation was performed on a Phenomenex Luna C18 column with gradient elution using a mobile phase consisting of methanol–water containing 0.5 mM formic acid (HCOOH) at a flow rate of 0.28 mL/min. ESI-MS spectra were acquired in positive ion multiple reaction monitoring mode (MRM). The precursor-to-product ion pairs used for MRM of yakuchinone A and yakuchinone B were m/z 313.1 → 137.0 and 311.2 → 117.1, respectively. Low concentration of HCOOH reduced the ion suppression caused by matrix components and clearly improved the analytical sensitivity. Yakuchinone A showed good linearity over a wide concentration range (r > 0.99). The accuracy, precision, stability and linearity were found to be within the acceptable criteria. This new method was successfully applied to analyze the rat plasma concentration of parent yakuchinone A after a single oral administration of SuoQuan capsules. Low systemic exposure to parent yakuchinone A was observed.
Conclusion
The proposed method is sensitive and reliable. It is hoped that this new method will prove useful for the future PK studies.
doi:10.1186/1752-153X-8-2
PMCID: PMC3896807  PMID: 24422995
Yakuchinone A; LC-MS/MS; Matrix effects; HCOOH; Pharmacokinetic study; SuoQuan capsules
23.  Analysis of Crotonaldehyde- and Acetaldehyde-Derived 1,N2-Propanodeoxyguanosine Adducts in DNA from Human Tissues using Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry 
Chemical research in toxicology  2006;19(10):1386-1392.
Crotonaldehyde, a mutagen and carcinogen, reacts with deoxyguanosine (dGuo) in DNA to generate a pair of diastereomeric 1,N2-propanodeoxyguanosine adducts (Cro-dGuo, 2), which occur in (6S, 8S) and (6R, 8R) configurations. They can also be formed through the consecutive reaction of two acetaldehyde molecules with dGuo. Cro-dGuo adducts inhibit DNA synthesis and induce miscoding in human cells. Considering their potential role in carcinogenesis, we have developed a sensitive and specific liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method to explore the presence of Cro-dGuo adducts in DNA from various human tissues, such as liver, lung, and blood. DNA was isolated from human tissues and enzymatically hydrolyzed to deoxyribonucleosides. [15N5]Cro-dGuo was synthesized and used as an internal standard. The Cro-dGuo adducts were enriched from the hydrolysate by solid phase extraction and analyzed by LC-ESI-MS/MS, using selected reaction monitoring (SRM). This method allows the quantitation of the Cro-dGuo adducts at a concentration of 4 fmol/μmol dGuo, corresponding to about 1 adduct per 109 normal nucleosides starting with 1 mg of DNA, with high accuracy and precision. DNA from human liver, lung and blood were analyzed. The Cro-dGuo adducts were detected more frequently in human lung DNA than in liver DNA, but were not detected in DNA from blood. The results of this study provide quantified data on Cro-dGuo adducts in human tissues. The higher frequency of Cro-dGuo in lung DNA than in the other tissues investigated is potentially important and deserves further study.
doi:10.1021/tx060154d
PMCID: PMC2596066  PMID: 17040109
24.  Analysis of r-7,t-8,9,c-10-Tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene in Human Urine: A Biomarker for Directly Assessing Carcinogenic Polycyclic Aromatic Hydrocarbon Exposure plus Metabolic Activation 
Polycyclic aromatic hydrocarbons (PAH) are believed to be causative agents for various types of cancers in humans. Benzo[a]pyrene (BaP) is a prototypic carcinogenic PAH, which requires metabolic activation to elicit its detrimental effects. The major end product of its diol epoxide metabolic activation pathway is r-7,t-8,9,c-10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (trans, anti-BaPT). Individual differences in exposure to, and metabolic activation of, carcinogenic PAH may influence cancer risk. Measurement of PAH metabolites in human urine could provide a direct way to assess individual differences in susceptibility to PAH-related cancer. In this paper, we describe a sensitive and reliable method for quantitation of trans, anti-BaPT in human urine using gas chromatography-negative ion chemical ionization-tandem mass spectrometry (GC-NICI-MS/MS). [13C6] trans, anti-BaPT was used as the internal standard. The urine was treated with β-glucuronidase and sulfatase, and then trans, anti-BaPT was enriched by solid-phase extraction with polymeric reversed phase and phenylboronic acid cartridges. The sample was silylated and analyzed by GC-NICI-MS/MS with selected reaction monitoring (SRM) for the trimethylsilyl (TMS) derivatives of trans, anti-BaPT (m/z 446→ m/z 255) and [13C6]trans, anti-BaPT (m/z 452→ m/z 261). The mean assay recovery was 44%. The instrumental on-column detection limit was about 20 amol of trans, anti-BaPT (as BaPT-TMS). trans, anti-BaPT was readily detected in all urine samples analyzed including 30 smokers (0.71 ± 0.64 fmol/mg creatinine) and 30 non-smokers (0.34 ± 0.2 fmol/mg creatinine) (P = 0.0018). The results of this study demonstrate a highly sensitive and selective method for quantitation of trans, anti-BaPT in human urine. This is to our knowledge the first study to show that smokers have significantly higher levels of trans, anti-BaPT in their urine than do non-smokers. This method may be useful as a direct phenotyping approach to assess individual differences in uptake and metabolic activation of carcinogenic PAH.
doi:10.1021/tx100287n
PMCID: PMC3064259  PMID: 21049951
benzo[a]pyrene metabolites; biomarker; metabolic activation; human urine
25.  Determination of cidofovir in human plasma after low dose drug administration using high performance liquid chromatography-tandem mass spectrometry 
Journal of pharmaceutical and biomedical analysis  2010;53(4):10.1016/j.jpba.2010.06.034.
A sensitive and specific method for the determination of cidofovir (CDV) in human plasma using high-performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) was developed and validated. Plasma samples were processed by a solid phase extraction (SPE) procedure using Varian® SAX extraction cartridges prior to chromatography. The internal standard was 13C5-Folic acid (13C5-FA). Chromatography was performed using a Luna C8(2) analytical column, 5 μm, 150 mm × 3.0 mm, using an isocratic elution with a mobile phase consisting of 43% methanol in water containing 12 mM ammonium acetate, at a flow rate of 0.3 mL/min. The retention times of CDV and 13C5-FA were 2.1 min and 1.9 min, respectively, with a total run time of 5 min. The analytes were detected by a Micromass Quattro Micro triple quadrupole mass spectrometer in positive electron spray ionization (ESI) mode using multiple reaction monitoring (MRM). The extracted ions monitored following MRM transitions were m/z 280.0 → 262.1 for CDV and m/z 447.0 → 294.8 for 13C5-FA (IS). The assay was linear over the range 20 - 1000 ng/mL. Accuracy (101.6 - 105.7%), intra-assay precision (4.1 - 5.4%), and inter-assay precision (5.6 - 6.8%) were within FDA limits. No significant variation in the concentration of CDV was observed with different sample storage conditions. This method is simple, adaptable to routine application, and allows easy and accurate measurement of CDV in human plasma.
doi:10.1016/j.jpba.2010.06.034
PMCID: PMC3654809  PMID: 20673618
cidofovir; BK virus; liquid chromatography–mass spectrometry

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