LC-MS/MS method for simultaneous analysis of uracil, 5,6-dihydrouracil, 5-fluorouracil and 5-fluoro-5,6-dihydrouracil in human plasma for therapeutic drug monitoring and toxicity prediction in cancer patients

The chemotherapeutic drug 5-fluorouracil (5-FU) is widely used for treating solid tumors. Response to 5-FU treatment is variable with 10-30% of patients experiencing serious toxicity partly explained by reduced activity of dihydropyrimidine dehydrogenase (DPD). DPD converts endogenous uracil (U) into 5,6-dihydrouracil (UH(2) ), and analogously, 5-FU into 5-fluoro-5,6-dihydrouracil (5-FUH(2) ). Combined quantification of U and UH(2) with 5-FU and 5-FUH(2) may provide a pre-therapeutic assessment of DPD activity and further guide drug dosing during therapy. Here, we report the development of a liquid chromatography-tandem mass spectrometry assay for simultaneous quantification of U, UH(2) , 5-FU and 5-FUH(2) in human plasma. Samples were prepared by liquid-liquid extraction with 10:1 ethyl acetate-2-propanol (v/v). The evaporated samples were reconstituted in 0.1% formic acid and 10 μL aliquots were injected into the HPLC system. Analyte separation was achieved on an Atlantis dC(18) column with a mobile phase consisting of 1.0 mm ammonium acetate, 0.5 mm formic acid and 3.3% methanol. Positively ionized analytes were detected by multiple reaction monitoring. The analytical response was linear in the range 0.01-10 μm for U, 0.1-10 μm for UH(2) , 0.1-75 μm for 5-FU and 0.75-75 μm for 5-FUH(2) , covering the expected concentration ranges in plasma. The method was validated following the FDA guidelines and applied to clinical samples obtained from ten 5-FU-treated colorectal cancer patients. The present method merges the analysis of 5-FU pharmacokinetics and DPD activity into a single assay representing a valuable tool to improve the efficacy and safety of 5-FU-based chemotherapy.

Enantioselective determination of ondansetron and 8-hydroxyondansetron in human plasma from recovered surgery patients by liquid chromatography-tandem mass spectrometry

A liquid chromatographic-mass spectrometric assay with atmospheric pressure chemical ionization for quantification of ondansetron and its main metabolite 8-hydroxyondansetron in human plasma was presented. The enantiomeric separation was achieved on a Chiralcel OD-R column containing cellulose tris-(3,5-dimethylphenylcarbamate). The validation data were within the required limits. The assay was successfully applied to authentic plasma samples. Quantitative results from postoperative patients receiving ondansetron demonstrated a great interindividual variability in postoperative plasma drug concentrations, the metabolites were not detected in their unconjugated form. A wide variation in the S-(+)-/R-(-)-ondansetron concentration ratio between 0.14 and 7.18 is indicative for a stereoselective disposition or metabolism. In further studies CYP2D6 and CYP3A4 genotype dependent metabolism of ondansetron enantiomers as well as of co-administered drugs and clinical efficacy of the medication should be tested.

Determination of free and total valproic acid in human plasma by capillary electrophoresis with contactless conductivity detection

A new approach for the determination of free and total valproic acid in small samples of 140 μL human plasma based on capillary electrophoresis with contactless conductivity detection is proposed. A dispersive liquid-liquid microextraction technique was employed in order to remove biological matrices prior to instrumental analysis. The free valproic acid was determined by isolating free valproic acid from protein-bound valproic acid by ultrafiltration under centrifugation of 100 μL sample. The filtrate was acidified to turn valproic acid into its protonated neutral form and then extracted. The determination of total valproic acid was carried out by acidifying 40 μL untreated plasma to release the protein-bound valproic acid prior to extraction. A solution consisting of 10 mM histidine, 10 mM 3-(N-morpholino)propanesulfonic acid and 10 μM hexadecyltrimethylammonium bromide of pH 6.5 was used as background electrolyte for the electrophoretic separation. The method showed good linearity in the range of 0.4-300 μg/mL with a correlation coefficient of 0.9996. The limit of detection was 0.08 μg/mL, and the reproducibility of the peak area was excellent (RSD=0.7-3.5%, n=3, for the concentration range from 1 to 150 μg/mL). The results for the free and total valproic acid concentration in human plasma were found to be comparable to those obtained with a standard immunoassay. The corresponding correlation coefficients were 0.9847 for free and 0.9521 for total valproic acid.

Proteome analysis of human plasma and amniotic fluid by Off-Gel isoelectric focusing followed by nano-LC-MS/MS

This paper presents a comparative proteomic analysis of human maternal plasma and amniotic fluid (AF) samples from the same patient at term of pregnancy in order to find specific AF proteins as markers of premature rupture of membranes, a complication frequently observed during pregnancy. Maternal plasma and the corresponding AF were immunodepleted in order to remove the six most abundant proteins before the systematic analysis of their protein composition. The protein samples were then fractionated by IEF Off-Gel electrophoresis (OGE), digested and analyzed with nano-LC-MS/MS separation, revealing a total of 73 and 69 proteins identified in maternal plasma and AF samples, respectively. The proteins identified in AF have been compared to those identified in the mother plasma as well as to the reference human plasma protein list reported by Anderson et al. (Mol. Cell. Proteomics 2004, 3, 311-326). This comparison showed that 26 proteins were exclusively present in AF and not in plasma among which 10 have already been described to be placenta or pregnancy specific. As a further validation of the method, plasma proteins fractionated by OGE and analysed by nano-LC-MS/MS have been compared to the Swiss 2-D PAGE reference map by reconstructing a map that matches 2-D gel and OGE experimental data. This representation shows that 36 of 49 reference proteins could be identified in both data sets, and that isoform shifts in pI are well conserved in the OGE data sets.

Determination of ajulemic acid and its glucuronide in human plasma by gas chromatography-mass spectrometry

A method using gas chromatography-mass spectrometry (GC-MS) and solid-phase extraction (SPE) was developed for the determination of ajulemic acid (AJA), a non-psychoactive synthetic cannabinoid with interesting therapeutic potential, in human plasma. When using two calibration graphs, the assay linearity ranged from 10 to 750 ng/ml, and 750 to 3000 ng/ml AJA. The intra- and inter-day precision (R.S.D., %), assessed across the linear ranges of the assay, was between 1.5 and 7.0, and 3.6 and 7.9, respectively. The limit of quantitation (LOQ) was 10 ng/ml. The amount of AJA glucuronide was determined by calculating the difference in the AJA concentration before ("free AJA") and after enzymatic hydrolysis ("total AJA"). The present method was used within a clinical study on 21 patients suffering from neuropathic pain with hyperalgesia and allodynia. For example, plasma levels of 599.4+/-37.2 ng/ml (mean+/-R.S.D., n=9) AJA were obtained for samples taken 2 h after the administration of an oral dose of 20 mg AJA. The mean AJA glucuronide concentration at 2h was 63.8+/-127.9 ng/ml.

Mass spectrometry-based analytical tools for the molecular protein characterization of human plasma lipoproteins

Lipoproteins are a heterogeneous population of blood plasma particles composed of apolipoproteins and lipids. Lipoproteins transport exogenous and endogenous triglycerides and cholesterol from sites of absorption and formation to sites of storage and usage. Three major classes of lipoproteins are distinguished according to their density: high-density (HDL), low-density (LDL) and very low-density lipoproteins (VLDL). While HDLs contain mainly apolipoproteins of lower molecular weight, the two other classes contain apolipoprotein B and apolipoprotein (a) together with triglycerides and cholesterol. HDL concentrations were found to be inversely related to coronary heart disease and LDL/VLDL concentrations directly related. Although many studies have been published in this area, few have concentrated on the exact protein composition of lipoprotein particles. Lipoproteins were separated by density gradient ultracentrifugation into different subclasses. Native gel electrophoresis revealed different gel migration behaviour of the particles, with less dense particles having higher apparent hydrodynamic radii than denser particles. Apolipoprotein composition profiles were measured by matrix-assisted laser desorption/ionization-mass spectrometry on a macromizer instrument, equipped with the recently introduced cryodetector technology, and revealed differences in apolipoprotein composition between HDL subclasses. By combining these profiles with protein identifications from native and denaturing polyacrylamide gels by liquid chromatography-tandem mass spectrometry, we characterized comprehensively the exact protein composition of different lipoprotein particles. We concluded that the differential display of protein weight information acquired by macromizer mass spectrometry is an excellent tool for revealing structural variations of different lipoprotein particles, and hence the foundation is laid for the screening of cardiovascular disease risk factors associated with lipoproteins.

Comparison of paired serum and lithium heparin plasma samples for the measurement of serum amyloid A in horses using an automated turbidimetric immunoassay

This study aimed to evaluate whether equine serum amyloid A (SAA) concentrations could be reliably measured in plasma with a turbidimetric immunoassay previously validated for equine SAA concentrations in serum. Paired serum and lithium-heparin samples obtained from 40 horses were evaluated. No difference was found in SAA concentrations between serum and plasma using a paired t test (P=0.48). The correlation between paired samples was 0.97 (Spearman's rank P<0.0001; 95% confidence interval 0.95-0.99). Passing-Bablok regression analyses revealed no differences between paired samples. Bland-Altman plots revealed a positive bias in plasma compared to serum but the difference was not considered clinically significant. The results indicate that lithium-heparin plasma samples are suitable for measurement of equine SAA using this method. Use of either serum or plasma allows for greater flexibility when it comes to sample collection although care should be taken when comparing data between measurements from different sample types.

Comparative methodology to investigate the presence of Escherichia coli K-12 strains in environmental and human stool samples

This study was undertaken to evaluate the specificity and efficiency of different methods to detect Escherichia coli K-12 strains. Another aim was to determine the frequency of E. coli K-12 strains among wild-type E. coli isolates from different sources. The detection of K-12 strains was performed both genotypically by K-12 specific polymerase chain reaction (PCR) and on the basis of phenotypical tests. In addition, the genome structures of E. coli strains were characterized by pulsed-field gel electrophoresis (PFGE). The most specific results could be obtained by the genotypical tests PCR and PFGE as well as by the K-12 specific phage assay. In total, 131 stool and 95 water isolates as well as 14 K-12 derivatives were examined by the different methods. No E. coli K-12 strains were detected among the wild-type isolates.

A quantitiative LC-MS/MS method for the measurement of arachidonic acid, prostanoids, endocannabinoids, N-acylethanolamines and steroids in human plasma.

Free arachidonic acid is functionally interlinked with different lipid signaling networks including those involving prostanoid pathways, the endocannabinoid system, N-acylethanolamines, as well as steroids. A sensitive and specific LC-MS/MS method for the quantification of arachidonic acid, prostaglandin E2, thromboxane B2, anandamide, 2-arachidonoylglycerol, noladin ether, lineoyl ethanolamide, oleoyl ethanolamide, palmitoyl ethanolamide, steroyl ethanolamide, aldosterone, cortisol, dehydroepiandrosterone, progesterone, and testosterone in human plasma was developed and validated. Analytes were extracted using acetonitrile precipitation followed by solid phase extraction. Separations were performed by UFLC using a C18 column and analyzed on a triple quadrupole MS with electron spray ionization. Analytes were run first in negative mode and, subsequently, in positive mode in two independent LC-MS/MS runs. For each analyte, two MRM transitions were collected in order to confirm identity. All analytes showed good linearity over the investigated concentration range (r>0.98). Validated LLOQs ranged from 0.1 to 190ng/mL and LODs ranged from 0.04 to 12.3ng/mL. Our data show that this LC-MS/MS method is suitable for the quantification of a diverse set of bioactive lipids in plasma from human donors (n=32). The determined plasma levels are in agreement with the literature, thus providing a versatile method to explore pathophysiological processes in which changes of these lipids are implicated.

Determination of voriconazole in human serum and plasma by micellar electrokinetic chromatography

The micellar electrokinetic capillary chromatography (MEKC) separation and analysis of voriconazole and UK 115794 (internal standard) were examined and an assay for determination of voriconazole in human plasma and serum was developed. The MEKC medium comprises a 2:15 (v/v) mixture of methanol and a pH 9.3 buffer composed of 5mM Na(2)B(4)O(7), 7 mM Na(2)HPO(4) and 54 mM SDS. Sample preparation is based upon liquid/liquid extraction with ethylacetate and dichloromethane (75%/25%) at physiological pH. Using this approach with 250 microl serum or plasma and reconstitution of the dried extract into 100 microl of a buffer composed of 0.5mM Na(2)B(4)O(7) and 0.7 mM Na(2)HPO(4) (pH 9.3), the detection and quantitation limits were determined to be 0.1 and 0.2 microg/ml, respectively, a sensitivity that is suitable for therapeutic drug monitoring of voriconazole (provisional therapeutic range: 1-6 microg/ml) in human plasma and serum samples. The method was validated and compared to an HPLC method, showing excellent agreement between the two for a set of 91 samples that stemmed from patients being treated with voriconazole. The MEKC assay is also demonstrated to be suitable to explore pharmacokinetic data of voriconazole.

Determination of Δ9-tetrahydrocannabinolic acid A (Δ9-THCA-A) in whole blood and plasma by LC–MS/MS and application in authentic samples from drivers suspected of driving under the influence of cannabis

Delta-9-tetrahydrocannabinolic acid A (THCA-A) is the biosynthetic precursor of delta-9-tetrahydrocannabinol (THC) in cannabis plants, and has no psychotropic effects. THCA-A can be detected in blood and urine, and several metabolites have been identified. THCA-A was also shown to be incorporated in hair by side stream smoke to a minor extent, but incorporation via blood stream or sweat seems unlikely. The detection of THCA-A in biological fluids may serve as a marker for differentiating between the intake of prescribed THC medication – containing only pure THC – and cannabis products containing THC besides THC-acid A and other cannabinoids. However, the knowledge about its usefulness in forensic cases is very limited. The aim of the present work was the development of a reliable method for THCA-A determination in human blood or plasma using LC–MS/MS and application to cases of driving under the influence of drugs. Fifty eight (58) authentic whole blood and the respective plasma samples were collected from drivers suspected of driving under the influence of cannabis from the region of Bern (Switzerland). Samples were first tested for THC, 11-OH-THC and THC-COOH, and then additionally for THCA-A. For this purpose, the existing LC–MS/MS method was modified and validated, and found to be selective and linear over a range of 1.0 to 200 ng/mL (the correlation coefficients were above 0.9980 in all validation runs). Limit of detection (LOD) and limit of quantification (LOQ) were 0.3 ng/mL and 1.0 ng/mL respectively. Intra- and inter-assay accuracy were equal or better than 90% and intra- and inter-assay precision were equal or better than 11.1%. The mean extraction efficiencies were satisfactory being equal or higher than 85.4%. THCA-A was stable in whole blood samples after 3 freeze/thaw cycles and storage at 4 °C for 7 days. Re-injection (autosampler) stability was also satisfactory. THC was present in all blood samples with levels ranging from 0.7 to 51 ng/mL. THCA-A concentrations ranged from 1.0 to 496 ng/mL in blood samples and from 1.4 to 824 ng/mL in plasma samples. The plasma:blood partition coefficient had a mean value of 1.7 (±0.21, SD). No correlation was found between the degree of intoxication or impairment stated in the police protocols or reports of medical examinations and the detected THCA-A-concentration in blood.

A plasma global metabolic profiling approach applied to an exercise study monitoring the effects of glucose, galactose and fructose drinks during post-exercise recovery

A global metabolic profiling methodology based on gas chromatography coupled to time-of-flight mass spectrometry (GC-TOFMS) for human plasma was applied to a human exercise study focused on the effects of beverages containing glucose, galactose, or fructose taken after exercise and throughout a recovery period of 6 h and 45 min. One group of 10 well trained male cyclists performed 3 experimental sessions on separate days (randomized, single center). After performing a standardized depletion protocol on a bicycle, subjects consumed one of three different beverages: maltodextrin (MD)+glucose (2:1 ratio), MD+galactose (2:1), and MD+fructose (2:1), consumed at an average of 1.25 g of carbohydrate (CHO) ingested per minute. Blood was taken straight after exercise and every 45 min within the recovery phase. With the resulting blood plasma, insulin, free fatty acid (FFA) profile, glucose, and GC-TOFMS global metabolic profiling measurements were performed. The resulting profiling data was able to match the results obtained from the other clinical measurements with the addition of being able to follow many different metabolites throughout the recovery period. The data quality was assessed, with all the labelled internal standards yielding values of <15% CV for all samples (n=335), apart from the labelled sucrose which gave a value of 15.19%. Differences between recovery treatments including the appearance of galactonic acid from the galactose based beverage were also highlighted.

Enamel matrix protein adsorption to root surfaces in the presence or absence of human blood

Background: The clinical use of an enamel matrix derivative (EMD) has been shown to promote formation of new cementum, periodontal ligament (PDL), and bone and to significantly enhance the clinical outcomes after regenerative periodontal surgery. It is currently unknown to what extent the bleeding during periodontal surgery may compete with EMD adsorption to root surfaces. The aim of this study is to evaluate the effect of blood interactions on EMD adsorption to root surfaces mimicking various clinical settings and to test their ability to influence human PDL cell attachment and proliferation. Methods: Teeth extracted for orthodontic reasons were subjected to ex vivo scaling and root planing and treated with 24% EDTA, EMD, and/or human blood in six clinically related settings to determine the ability of EMD to adsorb to root surfaces. Surfaces were analyzed for protein adsorption via scanning electron microscopy and immunohistochemical staining with an anti-EMD antibody. Primary human PDL cells were seeded on root surfaces and quantified for cell attachment and cell proliferation. Results: Plasma proteins from blood samples altered the ability of EMD to adsorb to root surfaces on human teeth. Samples coated with EMD lacking blood demonstrated a consistent even layer of EMD adsorption to the root surface. In vitro experiments with PDL cells demonstrated improved cell attachment and proliferation in all samples coated with EMD (irrespective of EDTA) when compared to samples containing human blood. Conclusion: Based on these findings, it is advised to minimize blood interactions during periodontal surgeries to allow better adsorption of EMD to root surfaces.

[(99m)Tc]Demomedin C, a radioligand based on human gastrin releasing peptide(18-27): synthesis and preclinical evaluation in gastrin releasing peptide receptor-expressing models

The synthesis and preclinical evaluation of [(99m)Tc]Demomedin C in GRPR-expressing models are reported. Demomedin C resulted by coupling a Boc-protected N(4)-chelator to neuromedin C (human GRP(18-27)), which, after (99m)Tc-labeling, afforded [(99m)Tc]Demomedin C. Demomedin C showed high affinity and selectivity for the GRPR during receptor autoradiography on human cancer samples (IC(50) in nM: GRPR, 1.4 ± 0.2; NMBR, 106 ± 18; and BB(3)R, >1000). It triggered GRPR internalization in HEK-GRPR cells and Ca(2+) release in PC-3 cells (EC(50) = 1.3 nM). [(99m)Tc]Demomedin C rapidly and specifically internalized at 37 °C in PC-3 cells and was stable in mouse plasma. [(99m)Tc]Demomedin C efficiently and specifically localized in human PC-3 implants in mice (9.84 ± 0.81%ID/g at 1 h pi; 6.36 ± 0.85%ID/g at 4 h pi, and 0.41 ± 0.07%ID/g at 4 h pi block). Thus, human GRP-based radioligands, such as [(99m)Tc]Demomedin C, can successfully target GRPR-expressing human tumors in vivo while displaying attractive biological features--e.g. higher GRPR-selectivity--vs their frog-homologues.