Large Number Of Plasma Samples Research Articles

Feasibility of an automated coagulation factor XIIIa test using its isopeptidase activity

Plasma transglutaminase FXIII provides mechanical and biochemical stability to blood clots. Congenital or acquired deficiency may be associated with bleeding diathesis and requires therefore careful monitoring. The precise automated measurement of a large number of plasma samples can provide new insights regarding the clinical relevance of certain FXIII levels. There is still the unmet diagnostic need for a reliable high-throughput method. Here we report the development and feasibility study of a promising prototype, adapting the precise FXIIIa isopeptidase assay principle on the optimized automated Ceveron s100 platform.

Abstract 3249: Detection of early-stage malignant lung cancer using methylation signatures in circulating tumor DNA

Abstract Low-dose computed tomography (LDCT) is recommended for early lung cancer screening in high-risk populations. Small lung nodules are identified by these CT scans in up to 50% of high-risk patients. While over 50% of lung nodules less than 1 cm are benign, discriminating benign nodules from malignant nodules remains extremely challenging without an invasive lung biopsy. Taking advantage of methylation signatures in circulating tumor DNA (ctDNA), we developed a non-invasive screening assay to identify patients with malignant lung nodules from those with benign lung nodules. Methods: We first adopted Singlera's MONOD+ assay as a marker screening technology to interrogate the methylation state of over 4,000,000 CpG sites across more than 200,000 methylation haplotype blocks in the human genome from as little as 10 ng of cell-free or tissue DNA. We first processed 129 fresh frozen and FFPE tissue samples from healthy lung, benign lung nodules, and lung cancer at various stages of malignancy. We identified a preliminary set of methylation markers capable of segregating lung nodules by malignancy and invasiveness. To examine the reproducibility of these markers in plasma, we applied the same screening assay to plasma cell-free DNA from 46 healthy patients and 175 lung nodule patients with either benign or malignant nodules (most at an early stage of Ia1/Ia2). We refined the marker set by removing methylation markers that were unstable in plasma samples. We next applied Singlera's MethylTitan assay to deeply interrogate approximately 1,000 methylation haplotype blocks enriched in malignant lung nodules in plasma samples, as MethylTitan's higher conversion rate allowed us to better handle the limited amount of DNA in plasma samples. We ran the MethylTitan assay on both healthy and lung nodule plasma samples; using this method, we confirmed that tissue-derived methylation signatures could be rapidly screened in plasma samples in a high-throughput and highly sensitive manner. Results: Using the MONOD+ marker screening assay on plasma samples, we were able to utilize cell-free DNA methylation to identify early-stage malignant lung nodule patients with a sensitivity of 80.25% and a specificity of 83.08%. We were able to further improve upon these results using the MethylTitan assay, as we could more efficiently and more accurately screen the methylation markers in a large number of plasma samples. Conclusion: In summary, we have applied Singlera's MONOD+ and MethylTitan methylation assays to non-invasively identify early stage lung cancer in patients with small lung nodules. We are currently expanding this study to a much larger cohort of lung nodule patients in order to clinically validate our early lung cancer screening assay. The approach of using MONOD+ to identify markers and MethylTitan to analyze plasma samples will allow us to noninvasively screen for additional cancer types in a high-throughput clinical setting. Citation Format: Ruijun Liu, Xiaojie Li, Athurva Gore, Zi Qin, Jeff Gole, Qiye He, Rui Liu, Shun Lu. Detection of early-stage malignant lung cancer using methylation signatures in circulating tumor DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3249.

Quantification of amlodipine and atorvastatin in human plasma by UPLC-MS/MS method and its application to a bioequivalence study.

A robust, rapid and sensitive UPLC-MS/MS method has been developed, optimized and validated for the determination of amlodipine (AML) and atorvastatin (ATO) in human plasma using eplerenone as an internal standard (IS). Multiple-reaction monitoring in positive electrospray ionization mode was utilized in Xevo TQD LC-MS/MS. Double extraction was used in sample preparation using diethyl ether and ethyl acetate. The prepared samples were analyzed using an Acquity UPLC BEH C18 (50 × 2.1 mm, 1.7 μm) column. Ammonium formate and acetonitrile, pumped isocraticaly at a flow rate of 0.25 mL/min, were used as a mobile phase. Method validation was done as per the US Food and Drug Administration guidelines. Linearity was achieved in the range of 0.1-10 ng/mL for AML and 0.05-50 ng/mL for ATO. Intra-day and inter-day accuracy and precision were calculated and found to be within the acceptable range. A short run time, of <1.5 min, permits analysis of a large number of plasma samples per batch. The developed and validated method was applied to estimate AML and ATO in a bioequivalence study in healthy human volunteers.

Multicentric study of the effect of pre-analytical variables in the quality of plasma samples stored in biobanks using different complementary proteomic methods

Analytical proteomics has experienced exponential progress in the last decade and can be expected to lead research studies on diagnostic and therapeutic biomarkers in the near future. Because the development of this type of analysis requires the use of a large number of human samples with a minimum of quality requirements, our objective was to identify appropriate indicators for quality control of plasma samples stored in biobanks for research in proteomics. To accomplish this, plasma samples from 100 healthy donors were obtained and processed according to the pre-analytical variables of: a) time delay for the first centrifugation of the original blood sample (4 or 24h) and b) number of freeze/thaw cycles (1, 2 or 3) of the processed plasma samples. The analyses of samples were performed by different and complementary methods such as SPE MALDI-TOF, DIGE, shotgun (iTRAQ, nLC MALDI TOF/TOF) and targeted nLC MS/MS proteomic techniques (SRM). In general, because the distribution of proteins in all samples was found to be very similar, the results shown that delayed processing of blood samples and the number of freeze/thaw cycles has little or no effect on the integrity of proteins in the plasma samples. SignificanceThe results of the present work indicate that blood proteins in plasma are broadly insensitive to such preanalytical variables as delayed processing or freeze/thaw cycles when analyzed at the peptide level. Although there are other studies related to protein stability of clinical samples with similar results, what is remarkable about our work is the large number of plasma samples examined and that our analyses assessed protein integrity by combining a wide set of complementary proteomic approaches performed at different proteomic platform participating laboratories that all yielded similar results. We believe our study is the most comprehensive performed to date to determine the changes in proteins induced by delayed sample processing and plasma freeze/thaw cycles.

MRM validation of targeted nonglycosylated peptides from N-glycoprotein biomarkers using direct trypsin digestion of undepleted human plasma

A rapid, simple, and reproducible MRM-based validation method for serological glycoprotein biomarkers in clinical use was developed by targeting the nonglycosylated tryptic peptides adjacent to N-glycosylation sites. Since changes in protein glycosylation are known to be associated with a variety of diseases, glycoproteins have been major targets in biomarker discovery. We previously found that nonglycosylated tryptic peptides adjacent to N-glycosylation sites differed in concentration between normal and hepatocellular carcinoma (HCC) plasma due to differences in steric hindrance of the glycan moiety in N-glycoproteins to tryptic digestion (Lee et al., 2011). To increase the feasibility and applicability of clinical validation of biomarker candidates (nonglycosylated tryptic peptides), we developed a method to effectively monitor nonglycosylated tryptic peptides from a large number of plasma samples and to reduce the total analysis time with maximizing the effect of steric hindrance by the glycans during digestion of glycoproteins. The AUC values of targeted nonglycosylated tryptic peptides were excellent (0.955 for GQYCYELDEK, 0.880 for FEDGVLDPDYPR and 0.907 for TEDTIFLR), indicating that these could be effective biomarkers for hepatocellular carcinoma. This method provides the necessary throughput required to validate glycoprotein biomarkers, as well as quantitative accuracy for human plasma analysis, and should be amenable to clinical use. Difficulties in verifying and validating putative protein biomarkers are often caused by complex sample preparation procedures required to determine their concentrations in a large number of plasma samples. To solve the difficulties, we developed MRM-based protein biomarker assays that greatly reduce complex, time-consuming, and less reproducible sample pretreatment steps in plasma for clinical implementation. First, we used undepleted human plasma samples without any enrichment procedures. Using nanoLC/MS/MS, we targeted nonglycosylated tryptic peptides adjacent to N-linked glycosylation sites in N-linked glycoprotein biomarkers, which could be detected in human plasma samples without depleting highly abundant proteins. Second, human plasma proteins were digested with trypsin without reduction and alkylation procedures to minimize sample preparation. Third, trypsin digestion times were shortened so as to obtain reproducible results with maximization of the steric hindrance effect of the glycans during enzyme digestion. Finally, this rapid and simple sample preparation method was applied to validate targeted nonglycosylated tryptic peptides as liver cancer biomarker candidates for diagnosis in 40 normal and 41 hepatocellular carcinoma (HCC) human plasma samples. This strategy provided the necessary throughput required to monitor protein biomarkers, as well as quantitative accuracy in human plasma analysis. From biomarker discovery to clinical implementation, our method will provide a biomarker study platform that is suitable for clinical deployment, and can be applied to high-throughput approaches.

Liquid chromatography–mass spectrometric determination of rufinamide in low volume plasma samples

Quantification of rufinamide in plasma was achieved using a selective and sensitive liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method. The chromatographic separation was achieved on a reversed phase column (Zorbax SB-C18 100mm×3mm, 3.5μm) under isocratic conditions. The mobile phase consisted of a mixture of water containing 0.1% formic acid and methanol (50:50, v/v). The mass spectrometric detection of the analyte was in multiple reaction monitoring mode (MRM) using an electrospray positive ionization (ESI positive). The monitored ions were 127m/z derived from 239m/z rufinamide and 108m/z derived from 251m/z the internal standard (lacosamide). Protein precipitation with methanol was applied for sample preparation using only 50μl aliquots. The concentration range was 40-2000ng/ml for rufinamide in plasma. The limit of detection was 1.25ng/ml and the lower limit of quantification was established at 5ng/ml rufinamide concentration. Selectivity and matrix effect was verified using individual human, rat and rabbit plasma samples. Short-term, post-preparative and freeze-thaw stability was also investigated. The proposed method provides accuracy, precision and high-throughput (short runtime 4.5min) for quantitative determination of rufinamide in plasma. This is the first reported liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for analysis of rufinamide from low volume plasma samples. The LC-MS/MS method was validated according to the current official guidelines and can be applied to accurately measure rufinamide level of large number of plasma samples from clinical studies or therapeutic drug monitoring.

Quantification of Acamprosate in human plasma by LC-ESI-MS/MS with solid phase extraction: Application to a bioequivalence study

BackgroundThe purpose of this investigation was to explore high selective, sensitive, rapid, stable, reproducible extraction method in long run with broader linear range. At the same time, it could be expected that, this method would be efficient in analyzing large numbers of plasma samples obtained for pharmacokinetic, bioavailability or bioequivalence studies. MethodsA simple, sensitive, selective and rapid high-performance liquid chromatography coupled with tandem mass spectrometry was developed and validated for quantification of Acamprosate in human plasma. The chromatography was performed by using waters atlantis HILIC, (2.1 mm × 50 mm, 3.0 μm) column connected with guard column waters atlantis HILIC, (2.1 mm × 10 mm, 3.0 μm). Acamprosate-d12 calcium trihydrate used as an IS. The extraction of drug and internal standard were obtained by solid phase extraction. The linearity was proved with concentration range 1.00–250.00 ng/ml for Acamprosate in human plasma. Results and discussionThe LOQ was demonstrated at 1.00 ng/ml. The within-batch, between-batch precision was found to be 2.21–4.07% and 2.00–3.20%. The within-batch, between-batch accuracy was found to be 96.26–102.00% and 98.27–102.00% for Acamprosate. Drug and IS were eluted within 3.0 min. ConclusionThe developed LC-MS/MS assay for Acamprosate is rapid, simple, sensitive, selective and suitable for routine measurement of sample analysis. The validated method was successfully applied in pharmacokinetic study of human plasma.

A simplified radiometabolite analysis procedure for PET radioligands using a solid phase extraction with micellar medium

A solid phase extraction method has been developed for simple and high-speed direct determination of PET radioligands in plasma. This methodology makes use of a micellar medium and a solid-phase extraction cartridge for displacement of plasma protein bound radioligand and separation of PET radioligands from their radiometabolites without significant preparation. The plasma samples taken from monkey or human during PET measurements were mixed with a micellar eluent containing an anionic surfactant sodium dodecyl sulphate and loaded onto SPE cartridges. The amount of radioactivity corresponding to parent radioligand (retained on the cartridge) and its radioactive metabolites (eluted with micellar eluent) was measured. Under the optimized conditions, excellent separation of target PET radioligands from their radiometabolites was achieved with a single elution and short run-time of 1 min. This method was successfully applied to study the metabolism for (11)C-labelled radioligands in human or monkey plasma. The amount of parent PET radioligands estimated by micellar solid phase extraction strongly corresponded with that determined by radio-LC. The improved throughput permitted the analysis of a large number of plasma samples (up to 13 samples per one PET study) for accurate estimation of metabolite-corrected input function during quantitative PET imaging studies. Solid phase extraction together with micellar medium is fast, sensitive and easy to use, and therefore it is an attractive alternative method to determine relative composition of PET radioligands in plasma.

Improved radiometabolite analysis procedure for positron emission tomography (PET) radioligands using a monolithic column coupled with direct injection micellar/high submicellar liquid chromatography

A high-speed and high-sensitive direct plasma radiometabolite analysis method for positron emission tomography (PET) radioligands has been developed. It was based on micellar/high submicellar liquid chromatography (LC) using a semi-preparative alkyl-bonded silica rod column in conjunction with steep gradient and high flow rate for rapid macromolecule removal from plasma without significant sample preparation and fast/efficient separation of PET radioligands from their radiometabolites. Excellent separation of target PET radioligand from its radiometabolites was achieved within 4min with a limit of detection of 1Bq (Bq) level. This method was successfully applied to study the radiometabolism for a wide variety of 11C and 18F labeled radioligands in human or monkey plasma. The improved sensitivity and throughput permitted the analysis of a large number of plasma samples for accurate determination PET radioligands during quantitative PET imaging studies. Micellar/high submicellar LC together with a monolithic column is an attractive alternative method to determine the radiometabolism of PET radioligands in plasma.

Early detection of colorectal cancer from patient blood plasma using microRNA-based q-rt-PCR

Abstract microRNAs (miRNAs) constitute a recently discovered class of small RNAs (typically 21-23 nt) that function as post-transcriptional regulators of gene expression. Current estimates indicate that more than one third of the cellular transcriptome is regulated by miRNAs, and that each miRNA potentially regulates hundreds of different mRNAs. As a consequence, miRNAs have been proposed to be master regulators of cellular state. This hypothesis has been borne out by a large number of studies demonstrating a causal link between miRNA dys-regulation and numerous disease states, including a diverse array of human cancers. Furthermore, the high stability of miRNA in common clinical source materials (e.g. FFPE blocks, plasma, serum, urine, saliva, etc.) and the ability of miRNA expression profiles to accurately classify discrete tissue types and disease states have positioned miRNA quantification as a promising new tool for a wide range of diagnostic applications. Colorectal cancer (CRC) ranks 4th in terms of prevalence and second in numbers of deaths among cancers of the western world. Although early detection of CRC leads to a favorable prognosis, and though CRC can easily be detected in the locally restricted state using a variety of diagnostic procedures, frequent late diagnosis means that CRC is still a leading cause of cancer mortality worldwide. Current procedures suffer from one or more disadvantages within areas such as cost, safety, inconvenience to the patient with the consequence of low compliance, lack of trained personnel, sensitivity etc, thereby precluding their adoption as a population screening tool. There is therefore an unmet need for a generally acceptable CRC screening assay. To facilitate discovery and clinical transfer of miRNA-based diagnostic markers, we developed a genome-wide LNA™-based miRNA q-rt-PCR platform with unparalleled sensitivity and robustness. The platform uses a single RT reaction to profile &amp;gt;700 human miRNAs from 2 predefined 384 well plates and thus allows high-throughput profiling of miRNAs from important clinical sources without the need for pre-amplification. Using this system, we have profiled a large number of plasma samples from localized and regional CRC patients, and from matched healthy controls. An extensive QC system has been implemented in order to secure technical excellence and reveal any unwanted bias in the dataset. We will present our approaches to data normalization and the results of signature development using linear classification methods. We show that we can detect the majority of cancer cases with good specificity, using a cross-validation approach. In summary, our results show that minimally invasive early detection of CRC using a clinically viable approach is feasible.

A rapid solid-phase extraction method for measurement of non-metabolised peripheral benzodiazepine receptor ligands, [18F]PBR102 and [18F]PBR111, in rat and primate plasma

To develop a rapid and reliable method for estimating non-metabolised PBR ligands fluoroethoxy ([(18)F]PBR102)- and fluoropropoxy ([(18)F]PBR111)-substituted 2-(6-chloro-2-phenyl)imidazo[1,2-a]pyridine-3-yl)-N,N-diethylacetamides in plasma. Rats and baboons were imaged with PET up to 2 h postinjection of [(18)F]PBR102 and [(18)F]PBR111 under baseline conditions, after pre-blocking or displacement with PK11195. Arterial plasma samples were directly analysed by reverse-phase solid-phase extraction (RP-SPE) and RP-HPLC and by normal-phase TLC. SPE cartridges were successively washed with acetonitrile/water mixtures. SPE eluant radioactivity was measured in a γ-counter to determine the parent compound fraction and then analysed by HPLC and TLC for validation. In SPE, hydrophilic and lipophilic radiolabelled metabolites were eluted in water and 20% acetonitrile/water. All non-metabolised [(18)F]PBR102 and [(18)F]PBR111 were in SPE acetonitrile fraction as confirmed by HPLC and TLC analysis. Unchanged (%) [(18)F]PBR102 and [(18)F]PBR111 from SPE analysis in rat and baboon plasma agreed with those from HPLC and TLC analysis. In rats and baboons, the fraction of unchanged tracer followed a bi-exponential decrease, with half-lives of 7 to 10 min for the fast component and >80 min for the slow component for both tracers. Direct plasma SPE analysis of [(18)F]PBR102 and [(18)F]PBR111 can reliably estimate parent compound fraction. SPE was superior to HPLC for samples with low activity; it allows rapid and accurate metabolite analysis of a large number of plasma samples for improved estimation of metabolite-corrected input function during quantitative PET imaging studies.

Estimating the Time of Last Cannabis Use from Plasma Δ9-Tetrahydrocannabinol and 11-nor-9-Carboxy-Δ9-Tetrahydrocannabinol Concentrations

Knowing the time cannabis was last used is important for determining impairment in accident investigations and clinical evaluations. Two models for predicting time of last cannabis use from single plasma cannabinoid concentrations-model I, using Delta(9)-tetrahydrocannabinol (THC), and model II, using the concentration ratio of 11-nor-9-carboxy-THC (THCCOOH) to THC-were developed and validated from controlled drug administration studies. Objectives of the current study were to extend the validation by use of a large number of plasma samples collected after administration of single and multiple doses of THC and to examine the effectiveness of the models at low plasma cannabinoid concentrations. Thirty-eight cannabis users each smoked a 2.64% THC cigarette in the morning, and 30 also smoked a second cigarette in the afternoon. Blood samples (n = 717) were collected at intervals after smoking, and plasma THC and THCCOOH concentrations measured by gas chromatography-mass spectrometry. Predicted times of cannabis smoking, based on each model, were compared with actual smoking times. The most accurate approach applied a combination of models I and II. For all 717 plasma samples, 99% of predicted times of last use were within the 95% confidence interval, 0.9% were overestimated, and none were underestimated. For 289 plasma samples collected after multiple doses, 97% were correct with no underestimates. All time estimates were correct for 76 plasma samples with THC concentrations between 0.5 and 2 mug/L, a concentration range not previously examined. This study extends the validation of the predictive models of time of last cannabis use to include multiple exposures and low THC concentrations. The models provide an objective and validated method for assessing the contribution of cannabis to accidents or clinical symptoms.

Simultaneous analysis of twenty‐one glucocorticoids in equine plasma by liquid chromatography/tandem mass spectrometry

A method for the simultaneous separation, identification, quantification and confirmation of the presence of 21 glucocorticoids (GCC) in equine plasma by liquid chromatography coupled with triple stage quadrupole tandem mass spectrometry (LC/TSQ-MS/MS) is described. Plasma sample augmented with the 21 GCC was extracted with methyl tert-butyl ether (MTBE) and analyzed by positive electrospray ionization. Desoxymetasone or dichlorisone acetate was used as the internal standard (IS). Quantification was performed by IS calibration. For each drug, one major product ion was chosen and used for screening for that drug. Analyte confirmation was performed by using the three most intense product ions formed from the precursor ion and the corresponding mass ratios. The recovery of the 21 GCC when spiked into blank plasma at 5 ng/mL was 45-200% with coefficient of variation (CV) from 0.3-18%. The limit of detection (LOD) and that of quantification (LOQ) for most of the analytes were 50-100 pg/mL and 1 ng/mL, respectively, whereas that of confirmation (LOC) was 100-300 pg/mL depending on the analyte. Intra- and inter-day precisions expressed as CV for quantification of 1 and 10 ng/mL was 1.0-17%, and 0.51-19%, respectively, and the accuracy was from 84-110%. The linear concentration range for quantification was 0.1-100 ng/mL (r(2) > 0.997). Estimated measurement uncertainty was from 11-37%. This study was undertaken to develop a method for simultaneous screening, identification, quantification and confirmation of these agents in post-race equine plasma samples. The method has been successfully applied to screening of a large number of plasma samples obtained from racehorses in competition and in pharmacokinetic studies of dexamethasone in the horse and concurrent changes in endogenous GCC, hydrocortisone and cortisone. The method is simple, sensitive, selective and reliably reproducible.

Application of liquid chromatography–mass spectrometry to monitoring plasma cyclophosphamide levels in phase I trial cancer patients*

A specific and efficient liquid chromatography-mass spectrometry (LC-MS) method was established for monitoring patient plasma cyclophosphamide levels in a phase I trial of an oral cyclophosphamide-based combination chemotherapy regimen. An Agilent 1100 Series LC-MSD system (Agilent Technologies, Avondale, PA, USA), with a single quadrupole mass detector using a positive atmospheric pressure chemical ionization (APCI) interface and single ion monitoring at m/z 261, was used. Chromatography was performed using a LUNA C8 5 microm 30 x 4.6 mm stainless steel column (Phenomenex, Torrance, CA, USA) and a mobile phase of aqueous acetonitrile pumped at a flow rate of 0.7 mL/min. High-throughput solid-phase sample extraction was performed using a Gilson ASPEC XL4 system (Gilson Medical, Middleton, WI, USA) controlled by prestored programs. The standard curve for cyclophosphamide was linear over the concentration range 0.026-1.08 microg/mL (r(2) > 0.994). Intra- and interassay accuracy and precision were 97-107 and 3-10%, respectively. The limit of detection was determined to be 0.01 microg/mL. Single ion monitoring at m/z 261 provided a high degree of specificity without interference from the matrix or other chemotherapy drugs. Automated sample processing allowed the analysis of a large number of plasma samples from a clinical trial of repeated daily oral dosing of cyclophosphamide. One hour after dosing, cyclophosphamide was detected in 98 of 106 plasma specimens at concentrations ranging between 0.03 and 4.88 microg/mL. Twenty-four hours after dosing, cyclophosphamide was detected in 72 of 77 plasma specimens at concentrations ranging between 0.06 and 3.13 microg/mL. There were no time-dependent changes in cyclophosphamide concentration during the 43 day period of repeated daily oral dosing. There was no correlation between cyclophosphamide dose and plasma concentration, despite the wide range of doses given in the clinical trial (50-125 mg/m(2)). We conclude that a solid-phase extraction LC-MS technique was validated for determining cyclophosphamide in human plasma. Interoccasion variability in the rate of oral absorption and in the clearance of systemically available drug may have contributed to the wide range of cyclophosphamide concentrations found at 1 and 24 h after tablet ingestion.

Evaluation of diabetes-related short-chain organic acids in rat plasma by capillary electrophoresis

A capillary zone electrophoresis method was optimised to analyse low-molecular-mass organic acids for the purpose of monitoring diabetes in rat plasma. The method included acetoacetic, 2-hydroxybutyric, lactic and uric acids. A variation in the background electrolyte allowed us to measure pyruvic acid in the same sample. Conditions have been optimised for measuring a large number of plasma samples corresponding to control and diabetic rats. Samples were mixed with acetonitrile (1:1, v/v) to precipitate proteins, centrifuged, diluted and injected. Tropic acid was chosen as an adequate internal standard. Separation was developed with reversed voltage by using a column cartridge pre-treated with polyacrylamide. Two electrophoretic buffers were employed: 0.150 M H 3PO 4 made up pH 6.20 with NaOH and 0.3 mM CaCl 2 for acetoacetic, hydroxybutyric, lactic and uric acids, and 200 mM phosphate–10 mM acetate pH 4.0 for pyruvic acid, both with direct detection at 200 nm. The method was validated for linearity, accuracy and precision and the limits of quantification were calculated. The method was successfully applied to analyse these organic acids in control and diabetic animals. Acetoacetic and hydroxybutyric acids were clearly increased in diabetic rats, meanwhile no statistically significant difference has been found with the other acids.

Automated isolation of high-purity plasma albumin for isotope ratio measurements

Measurement of the incorporation of labeled amino acids in plasma albumin, isolated from plasma sampled at different time points after infusion start is a well-known technique to study human albumin synthesis. Unfortunately, no chromatographic method has been described yet, enabling the automated isolation of high-purity albumin from large numbers of plasma samples as is required to study the kinetics of this process. Therefore, we developed a fast protein liquid chromatographic method, capable of processing 200 μl amounts of plasma in 74 min (injection to injection). The system can run unattended as the FPLC system is connected to a sample processor equipped with a polyether ether ketone (PEEK) sample loop and a cooled sample tray. Albumin isolation was divided into three steps. First, plasma samples were injected onto a 1-ml Blue Sepharose HiTrap affinity column, equilibrated with 50 mmol/l phosphate buffer (pH 7.0). After elution of non-binding protein, switching the solvent to phosphate buffer with 1.5 mol/l sodium chloride eluted albumin. The resulting albumin fraction was desalted on-line by directing it through two consecutive HiTrap 5-ml desalting columns, whereafter it was retained in the system within a 5-ml PTFE loop, connected to a motor valve. After switching this valve, thus bypassing the sample loop, the phosphate buffers were changed automatically to Tris buffers. Final purification involved elution of the captured fraction over a 1-ml ion-exchange Resource Q column, using a sodium chloride gradient, ranging from 0 to 0.5 mol/l in Tris buffer (20 mmol/l, pH 7.5). A more than 99% purity of the final albumin fraction was confirmed by capillary electrophoresis.

A Sensitive and specific ELISA for plasma pentosidine

Advanced glycation end products are formed by non-enzymatic glycation and oxidation reaction. Pentosidine is a well-known and characterized structure among them, and has been implicated in the pathogenesis of complications associated with chronic renal failure and long-term dialysis, such as dialysis-related amyloidosis and atherosclerosis. We established a highly sensitive and specific competitive enzyme-linked immunosorbent assay (ELISA) for plasma pentosidine and applied it to large numbers of plasma samples including haemodialysis (HD) and continuous ambulatory peritoneal dialysis (CAPD) patients. We compared their plasma pentosidine levels determined by the competitive ELISA with those determined by high-performance liquid chromatographic (HPLC) assay currently used. The plasma pentosidine levels determined by the ELISA were correlated well with those determined by sophisticated instrumental HPLC assay, both in non-diabetic and diabetic dialysis patients. Both analyses yielded comparable results, with over 8-fold higher plasma pentosidine levels in HD and CAPD patients, irrespective of the presence or absence of diabetes, as compared to normal subjects and non-uraemic diabetic patients. The competitive ELISA will provide a rapid and convenient determination of plasma pentosidine content and thus be useful to assess the carbonyl stress in uraemic patients.

Determination of phenethylamine, a phenethyl isothiocyanate marker, in dog plasma using solid-phase extraction and gas chromatography-mass spectrometry with chemical ionization

Phenethyl isothiocyanate is unstable in aqueous media and at low pH, and rapidly degrades to phenethylamine. Concentrations of phenethylamine, a phenethyl isothiocyanate marker, in dog plasma, were determined utilizing solid-phase extraction and gas chromatography–mass spectrometry with chemical ionization using acetone as the reagent gas. Deuterated d 5-amphetamine was used as an internal standard. After extraction, phenethylamine and d 5-amphetamine were derivatized using MBHFBA. Ions monitored for d 5-amphetamine were m/ z 337 and 338; and for phenethylamine were m/ z 318 and 319. Precision and accuracy were studied using control solutions prepared in naive dog plasma (80 and 300 ng/ml). Intra-day variability was determined using six replicates of each control solution analyzed on a single day. The relative standard deviation for the 80 ng/ml control was 12.9% and for the 300 ng/ml it was 12.1%. Relative accuracy was 10.9% for the low control and −4.1% for the high control. Inter-day variability was determined over a 6-day period. For the 80 and 300 ng/ml control solutions, the relative standard deviations were 15.8 and 9.1%, respectively, and relative accuracy values were 10.1 and −5.2%, respectively. Standard curves were prepared in naive dog plasma and were linear over the range of phenethylamine assayed (10–500 ng/ml). The results of this study indicate that the proposed method is simple, precise, accurate and sensitive enough for analysis of large numbers of plasma samples.

T- and B-cell responses of malaria immune individuals to synthetic peptides corresponding to non-repeat sequences in the N-terminal region of the Plasmodium falciparum antigen Pf155/RESA

While the C-terminal repeat region of Pf155/RESA, a Plasmodium falciparum vaccine candidate has been extensively studied for B- and T-cell reactivities, little is so far known about the non-repeat region in this respect. The present study aimed at investigating the non-repeat sequence 171-227 of Pf155/RESA for T- and B-cell epitopes. Eight overlapping peptides were synthesised and assayed for their ability to stimulate peripheral blood mononuclear cells obtained from P. falciparum-immune donors to proliferate and to induce secretion of interferon-gamma (IFN- γ) and/or interleukin 4 (IL-4) using the ELISPOT assay. The plasmas of the corresponding donors were tested for antibody reactivity with the same peptides in ELISA. The individual cellular responses to the different peptides varied and in general they were not correlated, emphasising the importance of including several parameters for T-cell activation. The most frequent T-cell responses (proliferation, IFN- γ and/or IL-4) were seen with two partially overlapping peptides corresponding to the sequences 171–185 and 181–195 that induced responses in 71 and 62% of the donors, respectively. Although, the frequency of responders was high, the magnitude of the responses was generally low. Two overlapping peptides corresponding to the sequence 186–206 bound antibodies from a large number of plasma samples. IL-4 producing cells were frequently found in donors whose sera contained antibodies to the corresponding peptide. However, there was no absolute correlation and many donors having anti-peptide antibodies could also be induced to produce IFN- γ. In conclusion, the non-repeat region of Pf155/RESA contains several epitopes inducing functionally distinct T-cell responses. The sequence 171–206 was found to contain both B- and T-cell epitopes recognised by almost all individuals naturally primed to malaria. Thus, this sequence should be a useful tool in future immuno-epidemiological studies and/or for inclusion into a subunit vaccine against the asexual blood stages of the P. falciparum parasite.

Determination of acyclovir in plasma by high performance liquid chromatography with UV detection. Method development and method validation

AbstractA HJPLC method for the determination of acyclovir in plasma is described. The method is simple and sensitive enough for bioequiva‐lence studies, where a large number of plasma samples with low acyclovir concertration are involved. The procedure is based on the deproteinization of plasma with perchloric acid and separation of acyclovir on a Hypersil ODS Column at pH 5.6 with UV detection. The calibration standards are linear up to at least 4000 ng/mL and the limit of quantification is 10 ng/mL.