Precipitation Of Plasma Proteins Research Articles

Unusual clotting dynamics of plasma supplemented with iron(III)

Iron salts are used in the treatment of iron deficiency anemia. Diabetic patients are frequently anemic and treatment includes administration of iron. Anemic patients on hemodialysis are at an increased risk of thromboembolic coronary events associated with the formation of dense fibrin clots resistant to fibrinolysis. Moreover, in chronic kidney disease patients, high labile plasma iron levels associated with iron supplementation are involved in complications found in dialyzed patients such as myocardial infarction. The aim of the present study was to investigate whether iron treatment is involved in the formation of the fibrin clots. Clotting of citrated plasma supplemented with Fe(3+) was investigated by thromboelastometry and electron microscopy. The results revealed that iron modifies coagulation in a complex manner. FeCl(3) stock solution underwent gradual chemical modification during storage and altered the coagulation profile over 29 days, suggesting that Fe(3+) interacts with both proteins of the coagulation cascade as well as the hydrolytic Fe(3+) species. Iron extends clotting of plasma by interacting with proteins of the coagulation cascade. Fe(3+) and/or its hydrolytic species interact with fibrinogen and/or fibrin changing their morphology and properties. In general FeCl(3) weakens the fibrin clot while at the same time precipitating plasma proteins immediately after application. Fe(3+) or its derivatives induced the formation of insoluble coagulums in non-enzymatic reactions including albumin and transferrin. Iron plays a role in coagulation and can precipitate plasma proteins. The formation of coagulums resistant to lysis in non‑enzymatic reactions can increase the risk of thrombosis, and extending clotting of plasma can prolong bleeding.

Identification of Niemann-Pick C1 disease biomarkers through sphingolipid profiling

Niemann-Pick type C (NPC)1 is a rare neurodegenerative disease for which treatment options are limited. A major barrier to development of effective treatments has been the lack of validated biomarkers to monitor disease progression or serve as outcome measures in clinical trials. Using targeted metabolomics to exploit the complex lipid storage phenotype that is the hallmark of NPC1 disease, we broadly surveyed Npc1(-/-) mouse tissues and identified elevated species across multiple sphingolipid classes that increased with disease progression. There was a striking accumulation of sphingoid bases, monohexosylceramides (MCs), and GM2 gangliosides in liver, and sphingoid bases and GM2 and GM3 gangliosides in brain. These lipids were modestly decreased following miglustat treatment, but markedly decreased in response to treatment with 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), two drugs that have shown efficacy in NPC1 animal models. Extending these studies to human subjects led to identification of sphingolipid classes that were significantly altered in the plasma of NPC1 patients. Plasma MCs and ceramides were elevated, whereas sphingoid bases were reduced in NPC1 subjects. Intervention with miglustat in NPC1 patients was accompanied by striking alterations in plasma (reductions in GM1 and GM3 gangliosides) and cerebrospinal fluid (CSF) (increased MCs) sphingolipids. Similar alterations were observed in the CSF from the NPC1 feline model following HP-β-CD treatment. Our findings suggest that these lipid biomarkers may prove useful as outcome measures for monitoring efficacy of therapy in clinical trials.

Determination of flomoxef in human plasma by liquid chromatography/electrospray ionization tandem mass spectrometry

A specific, sensitive, rapid and reproducible method for the determination of flomoxef in human plasma using high-performance liquid chromatography-tandem mass spectrometry was developed and validated. Flomoxef was detected using an electrospay ionization method operated in negative-ion mode. Chromatographic separation was performed in gradient elution mode on a Luna® C18(2) column (3 μM, 20 × 4.0 mm) at a flow rate of 1 mL/min and runtime 3.5 min. The mobile phase consisted of acetonitrile and water containing 0.1% formic acid as additive. Extraction of flomoxef from plasma and precipitation of plasma proteins was performed with acetonitrile with an absolute recovery of 86.4 ± 1.6%. The calibration curve was linear with a correlation coefficient of 0.999 over the concentration range 10-5000 ng/mL and the lower limit of quantification was 10 ng/mL. The intra- and inter-day precisions were <11.8%, while the accuracy ranged from 99.6 to 109.0%. A stability study of flomoxef revealed that it could be successfully analyzed at 4 ºС over 24 h, but it was unstable in solutions at room temperature during short-term storage (4 h) and several freeze-thaw cycles.

Determination of deltonin in rat plasma by using HPLC–MS/MS and the application of this method in pharmacokinetic studies

Deltonin is a naturally occurring spirostanol glycoside from Dioscorea zingiberensis C.H. Wright, which is used in traditional Chinese medicine. It exerts strong cytotoxic effect on C26 cells, inhibits C26 derived-tumor growth, and prolongs the survival of tumor-bearing mice after its oral administration, indicating its potential for use as an anti-tumor drug. To investigate the pharmacokinetic profiles of deltonin, a rapid, sensitive, and simplified high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay was developed and validated for the determination of deltonin in rat plasma. After acetonitrile-mediated plasma protein precipitation, chromatographic separation of deltonin was achieved using a reversed phase Hypersil Gold column (150mm×2.1mm, 5μm), with gradient elution using 0.1% formic acid and acetonitrile. Thereafter, deltonin was quantified using MS/MS with electrospray ionization (ESI) in positive multiple reaction monitoring (MRM) mode. The flow rate of the mobile phase was 200μL/min, and the retention time was 9.03min for deltonin and 6.31min for the internal standard (IS: 20(S)-ginsenoside Rb1). The linear range of the calibration curve was 2-5000ng/mL (r(2)>0.99), and the limit of detection (LOD) was 0.46ng/mL. The intra- and inter-day accuracies ranged from -2.8% to 11.1% and precisions (RSD) were within 13.1%. Deltonin was found to be stable under short-term temperature conditions, post-preparative temperature conditions, and after 3 freeze-thaw cycles conditions. The validated method was successfully applied to a pharmacokinetic study in rats after oral administration of deltonin (50 and 100mg/kg). The pharmacokinetics is characterized by high apparent clearance (CL/F) and apparent volume of distribution (Vd/F).

First liquid chromatography method for the simultaneous determination of levofloxacin, pazufloxacin, gatifloxacin, moxifloxacin and trovafloxacin in human plasma

For the first time a simple, selective and sensitive liquid chromatography method was developed and validated for the simultaneous determination of levofloxacin (LEV), pazufloxacin (PAZ), gatifloxacin (GAT), moxifloxacin (MOX) and trovafloxacin (TRO) in human plasma. Samples were pre-treated with acetonitrile for precipitation of plasma proteins followed by evaporation and reconstitution steps. Chromatographic separation of the analytes and norfloxacin, used as internal standard (IS), was performed under gradient elution on a LiChroCART(®) Purospher Star C18 column (55mm×4mm, 3μm). The mobile phase comprised a mixture of 0.1% aqueous formic acid adjusted to pH 3.0 with triethylamine, acetonitrile and methanol pumped at a flow rate of 1.0mL/min. The detector was set at excitation/emission wavelengths of 260/455nm. Calibration curves were linear (r(2)≥0.9923) in the ranges of 0.005-5μg/mL for GAT, 0.02-5μg/mL for LEV, PAZ and MOX and 0.04-5μg/mL for TRO. The intra and interday precision did not exceed 7.32% and the intra and interday accuracy ranged from -11.73 to 8.92%. The limits of quantification were established at 0.005μg/mL for GAT, 0.02μg/mL for LEV, PAZ and MOX and 0.04μg/mL for TRO. No endogenous or tested exogenous compounds were found to interfere at the retention times of the analytes and IS. Since the proposed method proved to be reliable for the quantitative determination of LEV, PAZ, GAT, MOX and TRO it may be a useful tool for routine analysis and to support clinical pharmacokinetic and toxicological studies involving these antibiotics.

Multiplex liquid chromatography-tandem mass spectrometry assay for simultaneous therapeutic drug monitoring of ribavirin, boceprevir, and telaprevir.

New directly acting antivirals (DAAs) that inhibit hepatitis C virus (HCV) replication are increasingly used for the treatment of chronic hepatitis C. A marked pharmacokinetic variability and a high potential for drug-drug interactions between DAAs and numerous drug classes have been identified. In addition, ribavirin (RBV), commonly associated with hemolytic anemia, often requires dose adjustment, advocating for therapeutic drug monitoring (TDM) in patients under combined antiviral therapy. However, an assay for the simultaneous analysis of RBV and DAAs constitutes an analytical challenge because of the large differences in polarity among these drugs, ranging from hydrophilic (RBV) to highly lipophilic (telaprevir [TVR]). Moreover, TVR is characterized by erratic behavior on standard octadecyl-based reversed-phase column chromatography and must be separated from VRT-127394, its inactive C-21 epimer metabolite. We have developed a convenient assay employing simple plasma protein precipitation, followed by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) for the simultaneous determination of levels of RBV, boceprevir, and TVR, as well as its metabolite VRT-127394, in plasma. This new, simple, rapid, and robust HPLC-MS/MS assay offers an efficient method of real-time TDM aimed at maximizing efficacy while minimizing the toxicity of antiviral therapy.

Fast liquid chromatography–quadrupole linear ion trap-mass spectrometry analysis of polyunsaturated fatty acids and eicosanoids in human plasma

Profiling of polyunsaturated fatty acids (PUFAs) and their oxidized metabolites, mainly eicosanoids, in human plasma by fast liquid chromatography-mass spectrometry is described. Sample preparation involved protein precipitation of 200μL plasma followed by on-line solid-phase extraction. 7 PUFAs and 94 oxidized metabolites were separated utilizing a C-18 column packed with 2.6μm core-shell particles in 7min. The analytes and deuterium-labeled standards were detected via scheduled multiple reaction monitoring transitions (123 sMRM). Simultaneously, linear ion trap fragment spectra were acquired for confirmation, if necessary. The lower limit of quantitation ranged between 200 and 1000ng/mL for the PUFAs and 10-1000pg/mL for the metabolites. The method was applied to a study on plasma samples from 50 healthy subjects.

Tracing and separating plasma components causing matrix effects in hydrophilic interaction chromatography–electrospray ionization mass spectrometry

Matrix effects on electrospray ionization were investigated for plasma samples analysed by hydrophilic interaction chromatography (HILIC) in gradient elution mode, and HILIC columns of different chemistries were tested for separation of plasma components and model analytes. By combining mass spectral data with post-column infusion traces, the following components of protein-precipitated plasma were identified and found to have significant effect on ionization: urea, creatinine, phosphocholine, lysophosphocholine, sphingomyelin, sodium ion, chloride ion, choline and proline betaine. The observed effect on ionization was both matrix-component and analyte dependent. The separation of identified plasma components and model analytes on eight columns was compared, using pair-wise linear correlation analysis and principal component analysis (PCA). Large changes in selectivity could be obtained by change of column, while smaller changes were seen when the mobile phase buffer was changed from ammonium formate pH 3.0 to ammonium acetate pH 4.5. While results from PCA and linear correlation analysis were largely in accord, linear correlation analysis was judged to be more straight-forward in terms of conduction and interpretation.

Fully validated method for rapid and simultaneous measurement of six antiepileptic drugs in serum and plasma using ultra-performance liquid chromatography–electrospray ionization tandem mass spectrometry

Therapeutic drug monitoring (TDM) may be very useful in the clinical management of antiepileptic drug therapy for multiple reasons, such as individual variability, metabolism, genetic factors or drug–drug or drug–food interactions. In addition, TDM is helpful to study the variation in pharmacokinetics that occurs between individuals. Here, we describe a rapid assay using ultra-performance liquid chromatography–electrospray ionization tandem mass spectrometry to measure the antiepileptic drugs lacosamide, lamotrigine, levetiracetam, primidone, topiramate, and zonisamide. After the addition of internal standards (ISs) and protein precipitation of serum or plasma, 1μl of sample was separated on a 2.1×50mm reverse phase column (Waters, Acquity UPLC BEH Phenyl, 1.7μm). Analytes were then ionized and detected by electrospray ionization mass spectrometry with multiple reaction monitoring. Runtime was 2.5min per injection. Matrix effects were investigated by systematical ion suppression and in-source fragmentation experiments. The calibration curves of the 6 antiepileptic drugs were linear over the working range between 0.05 and 50mg/L (r>0.99). The limit of detection (LOD) was <0.05mg/L, whereas the limit of quantification (LLOQ) was 0.10mg/L of all drugs measured in the assay. The intraassay and interassay coefficients of variation for all compounds were <15% for very low concentration (0.1mg/L) and <8% in the clinically relevant concentration range (>1.0mg/L). Mean recoveries were between 87.8 and 98.6% for all drugs. There were no significant ion suppressions detected at the elution times of the analytes. The mean differences between serum and heparinized plasma values were less than 6% for the 6 antiepileptic drugs. All drugs were stable in serum at −20°C, 4°C, and even at RT for at least 1 month. In summary, a specific and sensitive stable isotope dilution UPLC-MS/MS method was developed and validated for routine clinical monitoring of lacosamide, lamotrigine, levetiracetam, primidone, topiramate, and zonisamide.

A fast and reliable reversed phase high performance liquid chromatography method for simultaneous determination of selected anti-retroviral and lumefantrine in human plasma

A fast and reliable high performance liquid chromatography (HPLC) method with UV diode array detection for simultaneous quantitative analysis of the anti-retroviral drugs, nevirapine (NVP) and efavirenz (EFV) and the anti-malarial, lumefantrine (LUM) in human plasma has been developed and validated. The sample preparation consisted of a plasma protein precipitation with 0.5% acetic acid acetonitrile solution containing the internal standard halofantrine (HALO) prior the LC-analysis. Chromatographic separation was carried out on a Acclaim Polar Advantage C(16), column (150 mm × 4.6 mm, particle size, 3 μm) using a gradient of mobile phase made of 0.01% TFA in 0.1M ammonium acetate (solvent A) and 0.1% TFA in acetonitrile (solvent B). The separation of NVP, EFV, LUM and HALO was achieved within 17 min at a flow rate of 1.0 mL/min and detections were initially performed at three wavelengths, 275 nm (NVP), 255 nm (EFV), and 300 nm (LUM). The method selectivity was demonstrated in six different human plasma batches. In addition, several concomitant drugs were analyzed under our experimental conditions and none of them co-eluted with EFV, NVP and LUM. This demonstrated that our method is highly selective. Calibration graphs plotted with seven concentrations in duplicate for each compound were linear between the selected ranges with a regression coefficient (R(2)) greater than 0.998. Absolute extraction recovery for NVP, EFV and LUM were 99%, 98.6 and 102%, respectively. Inter- and intra-day coefficients of variation for LUM, EFV and NVP were ≤10%. The lower limits of quantification were 0.125 μg/mL for LUM and 0.250 μg/mL for both EFV and NVP. Intra- and inter-assay relative standard deviation values were found to be less than 15% at the concentrations examined (0.125-10.0 μg/mL for LUM and 0.250-15.0 μg/mL for both EFV and NVP). The present method was successfully implemented in Tanzania and only one wavelength (255 nm) was used to measure samples of patients receiving either NVP or EFV in combination with LUM. The concentration found in human plasma samples for all three compounds were within the calibration range. This makes our method particularly applicable and useful to resource-limited settings.

An HPLC-DAD method for the simultaneous quantification of opicapone (BIA 9-1067) and its active metabolite in human plasma

Opicapone (BIA 9-1067) is a novel catechol-O-methyltransferase inhibitor presently under clinical development as an adjuvant in the pharmacotherapy of Parkinson's disease. This report describes the development and validation of a bioanalytical assay for the simultaneous quantification of opicapone and its active metabolite (BIA 9-1079) in human plasma. The method herein reported is based on high-performance liquid chromatography coupled with diode-array detection (HPLC-DAD) and the sample preparation consists of a plasma protein precipitation step followed by liquid-liquid extraction. Chromatographic separation of the analytes (opicapone and BIA 9-1079) and the internal standard (tamoxifen) was achieved in less than 10 min on a reversed-phase C18 column at 25 °C by applying a gradient elution program using a mobile phase composed of 0.05 M monosodium phosphate solution adjusted to pH 2.45 (A) and acetonitrile (B) pumped at 0.8 mL min(-1). Opicapone and the internal standard were monitored at 271 nm while BIA 9-1079 was assessed at 257 nm. Calibration curves of both analytes were linear (r(2) ≥ 0.997) in the concentration range of 25-3000 ng mL(-1) and their limits of quantification were established to be 25 ng mL(-1). The overall precision did not exceed 13.2% and the accuracy was within ±11.1%. Several drugs potentially co-administered with opicapone were tested and they did not interfere at the retention times of the analytes (opicapone and BIA 9-1079) and internal standard. The method was then successfully applied for quantifying opicapone and its active metabolite (BIA 9-1079) in plasma samples obtained from a healthy subject enrolled in a clinical trial.

Determination of isotretinoin in human plasma: Application to pharmacokinetic study

A simple, rapid, sensitive and accurate HPLC–UV detection method was developed and validated for the determination of isotretinoin in human plasma. The method involves protein precipitation of plasma by acetonitrile using nifedipine as the internal standard (IS). Chromatographic separation was achieved on Agilent C18 column using an acetonitrile: aqueous 0.5% glacial acetic acid (70:30, v/v) mobile phase. Isotretinoin in the eluent was monitored at 350nm. Inter-assay of the coefficient of variations (CV%) over the range of plasma calibration curves (0.02–0.60μg/ml) ranged from 2.32% to 6.49% with good correlation coefficient (R2=0.999). The average recovery of the extraction procedure was 99.128%. The LLOD and LLOQ are 0.007 and 0.02μg/ml, respectively. This method was applied for pharmacokinetic study and the results indicated that this method is suitable for pharmacokinetic and bioavailability studies.

Survey of several methods deproteinizing human plasma before and within the chloroformate-mediated treatment of amino/carboxylic acids quantitated by gas chromatography

Trichloroacetic acid, perchloric acid, phosphotungstic acid, acetonitrile, methanol and some other organic solvents were evaluated for their ability to provide protein and lipid-free plasma supernatants. The residual proteins, total cholesterol and triacylglycerols were assayed in the supernatant on a Beckman Analyzer instrument. The free cholesterol and the neutral lipids were further analyzed by means of high-temperature GC analysis. The conditions for the deproteinizing step were optimized for minimal lipoprotein disruption. A substantial difference regarding contamination by the lipids was found if the plasma supernatant or the whole serum were treated with an alkyl chloroformate reagent. Three plasma sulfur amino acids and the aromatic ones were chosen as model compounds to evaluate compatibility of the precipitation methods with a subsequent methyl chloroformate-mediated derivatization and GC-MS analysis. The results of the total homocysteine assay matched well with that obtained using a commercial immunoassay. Precipitation with trichloroacetic acid has proven to be a method of choice for the analysis of the acido-basic analytes by GC-MS via chloroformates.

Comparison of Triple Quadrupole And High-Resolution Tof-Ms For Quantification of Peptides

With an increased interest in peptides and proteins as potential new drug candidates, new approaches for sensitive and selective quantitative analysis are required. LC-MS/MS analysis provides a good alternative to immunoassays with reduced method development times and increased specificity. We have evaluated two state-of-the-art triple quadrupole and high-resolution TOF mass spectrometers with respect to their performance for quantification of six peptides (glufibrinopeptide B, somatostatin, enfuvirtide, TRI1144, C34 and exenatide). The peptides were spiked into protein-precipitated plasma supernatant. Triple quadrupole quantification was performed in SRM mode, and in high-resolution, MS narrow-width extracted chromatograms were generated for quantification. Specificity, accuracy, reproducibility and robustness were found to be comparable between the two instruments. The triple quadrupole instrument is still the most sensitive instrument for quantification of peptides with a median factor of about four-times higher sensitivity (based on LLOQ evaluation). Based on sensitivity, the newest generation triple quadrupole MS systems are still the preferred technology for quantification of peptides. Since the sensitivity difference between triple quadrupole instruments and the new-generation high-resolution TOF-MS instruments is minor, the latter offer a useful alternative whenever additional selectivity is preferred or the use of a generic approach not requiring method optimization is advantageous.

Semi-automated in vivo solid-phase microextraction sampling and the diffusion-based interface calibration model to determine the pharmacokinetics of methoxyfenoterol and fenoterol in rats

In vivo solid-phase microextraction (SPME) can be used to sample the circulating blood of animals without the need to withdraw a representative blood sample. In this study, in vivo SPME in combination with liquid–chromatography tandem mass spectrometry (LC–MS/MS) was used to determine the pharmacokinetics of two drug analytes, R,R-fenoterol and R,R-methoxyfenoterol, administered as 5mgkg−1i.v. bolus doses to groups of 5 rats. This research illustrates, for the first time, the feasibility of the diffusion-based calibration interface model for in vivo SPME studies. To provide a constant sampling rate as required for the diffusion-based interface model, partial automation of the SPME sampling of the analytes from the circulating blood was accomplished using an automated blood sampling system. The use of the blood sampling system allowed automation of all SPME sampling steps in vivo, except for the insertion and removal of the SPME probe from the sampling interface. The results from in vivo SPME were compared to the conventional method based on blood withdrawal and sample clean up by plasma protein precipitation. Both whole blood and plasma concentrations were determined by the conventional method. The concentrations of methoxyfenoterol and fenoterol obtained by SPME generally concur with the whole blood concentrations determined by the conventional method indicating the utility of the proposed method. The proposed diffusion-based interface model has several advantages over other kinetic calibration models for in vivo SPME sampling including (i) it does not require the addition of a standard into the sample matrix during in vivo studies, (ii) it is simple and rapid and eliminates the need to pre-load appropriate standard onto the SPME extraction phase and (iii) the calibration constant for SPME can be calculated based on the diffusion coefficient, extraction time, fiber length and radius, and size of the boundary layer. In the current study, the experimental calibration constants of 338.9±30mm−3 and 298.5±25mm−3 are in excellent agreement with the theoretical calibration constants of 307.9mm−3 and 316.0mm−3 for fenoterol and methoxyfenoterol respectively.

Metabolic fingerprinting of high-fat plasma samples processed by centrifugation- and filtration-based protein precipitation delineates significant differences in metabolite information coverage

Metabolomics and metabolic fingerprinting are being extensively employed for improved understanding of biological changes induced by endogenous or exogenous factors. Blood serum or plasma samples are often employed for metabolomics studies. Plasma protein precipitation (PPP) is currently performed in most laboratories before LC–MS analysis. However, the impact of fat content in plasma samples on metabolite coverage has not previously been investigated. Here, we have studied whether PPP procedures influence coverage of plasma metabolites from high-fat plasma samples. An optimized UPLC-QTOF/MS metabolic fingerprinting approach and multivariate modeling (PCA and OPLS-DA) were utilized for finding characteristic metabolite changes induced by two PPP procedures; centrifugation and filtration. We used 12-h fasting samples and postprandial samples collected at 2h after a standardized high-fat protein-rich meal in obese non-diabetic subjects recruited in a dietary intervention. The two PPP procedures as well as external and internal standards (ISs) were used to track errors in response normalization and quantification. Remarkably and sometimes uniquely, the fPPP, but not the cPPP approach, recovered not only high molecular weight (HMW) lipophilic metabolites, but also small molecular weight (SMW) relatively polar metabolites. Characteristic SMW markers of postprandial samples were aromatic and branched-chain amino acids that were elevated (p<0.001) as a consequence of the protein challenge. In contrast, some HMW lipophilic species, e.g. acylcarnitines, were moderately lower (p<0.001) in postprandial samples. LysoPCs were largely unaffected. In conclusion, the fPPP procedure is recommended for processing high-fat plasma samples in metabolomics studies. While method improvements presented here were clear, use of several ISs revealed substantial challenges to untargeted metabolomics due to large and variable matrix effects.

HIGHLY SENSITIVE LC-MS/MS METHOD FOR THE DETERMINATION OF CLOZAPINE IN RAT PLASMA: APPLICATION TO A PRECLINICAL PHARMACOKINETIC STUDY

A highly sensitive, rapid assay method has been developed and validated for the quantification of clozapine in rat plasma using LC-MS/MS. The extraction process involved plasma protein precipitation with acetonitrile and elution with a mobile phase constituted by acetonitrile and acidic water (80/20), at 0.8 mL/min flow rate (split 1:3). Chromatographic separation was performed by C18 Symmetry column (4.6 × 75 mm, 3.5 µm). The lower limit of quantification was 1 ng/mL and the linearity was observed between 1–1000 ng/mL with a determination coefficient >0.9810. The intra- and inter-day precisions were less or equal to 8.38 and 4.36%, respectively. The method showed sensitivity, linearity, precision, accuracy, and specificity necessary for quantification of plasma clozapine in preclinical pharmacokinetics studies.

Plasma Preparation Method for Metabolomic Analysis Based on Rapid Resolution Liquid Chromatography-Mass Spectrometry

Abstract This study was undertaken to compare different methods for plasma preparation in metabolomic analysis. Plasma proteins were either precipitated with four different organic solvents or subjected to solid-phase extraction (SPE) on C 18 -boned phase, with consideration to the number of extracted standard compounds. The eluent was analyzed by RRLC-MS. The results indicated that acetonitrile could be used to effectively precipitate plasma proteins with the best extracting effect. Then, the optimal conditions for acetonitrile preparation were further investigated. A 3-fold volume of cold acetonitrile coupled with slow precipitation was employed. The results, according to the reproducibility, precision and freeze thawing stability of the sample, showed that the method is satisfactory and could be applied in metabolomic analysis of plasma preparation. Furthermore, the reproducibility of the method using principal component analysis (PCA) was sufficient to ensure data quality in metabolomic applications by carefully monitoring the quality control samples randomly inserted among the real sample queue, and the method would be applied in metabolomic studies.

Use of a novel technique, solid phase microextraction, to measure tranexamic acid in patients undergoing cardiac surgery

Solid phase microextraction (SPME) is a technique widely used and accepted in the field of food technology and in environmental and biological analyses. Despite its numerous advantages over older analytical methods, it has not been studied extensively in the medical sciences. Tranexamic acid (TXA) is currently the sole antifibrinolytic agent used during cardiac surgery involving the use of cardiopulmonary bypass (CPB). The current standard method of measuring TXA in plasma is based on plasma protein precipitation (PPP), but this analytical approach is time-consuming and not practical for routine use. The aim of the current study was to compare plasma TXA levels measured with the PPP method vs those acquired with the novel, highly efficient SPME technique. We also investigated the use of automated SPME with the aim of improving the technique so it could be used efficiently for measuring plasma TXA levels. With Research Ethics Board approval, we undertook a prospective, investigator-blinded study in ten patients undergoing cardiac surgery with CPB. An initial TXA bolus of 30mg·kg(-1) was infused over 15min followed by a 16mg·kg(-1)·hr(-1) infusion until chest closure with a 2mg·kg(-1) load in the pump prime. Each blood sample was divided into two portions and assigned a random number to blind the analyzing laboratory. The blood TXA concentration was measured using both PPP and SPME. Agreement between the two tests was analyzed using the Bland-Altman plot. Comparisons of plasma TXA concentrations measured with the two methods (PPP and SPME) showed good agreement. Absolute recovery of TXA for PPP was 64.9-78.2%; its precision, as a percentage of the relative standard deviation (RSD) was < 10% [with the exception of the lower limit of quantification (LLOQ), where the RSD was 18%]; and its accuracy, as the bias against the nominal concentration, was < 7% (for LLOQ it was 15%). Thus, extraction with SPME compared favourably with the PPP technique. Solid phase microextraction is a relatively simple, rapid extraction technique that can facilitate future pharmacokinetic studies analyzing TXA drug concentrations and drug dosing in various clinical settings.

Determinación de linezolid en plasma mediante cromatografía líquida de alta resolución para la monitorización terapéutica en pacientes

Introduction Linezolid is a synthetic antibiotic of the group of the oxazolidinones with Gram positive spectrum of activity. It is indicated in skin and soft tissue infections, community-acquired nosocomial infections and infections caused by methicillin-resistant and vancomycin-resistant Staphylococcus aureus and Enterococcus, respectively. The aim of this work is to develop and evaluate a high pressure (HPLC) method for the monitoring of plasma levels of linezolid in patients samples. Materials and methods The mobile phase consisted of a mixture of acetonitrile and water with a flow rate of 1 mL/min and a short C18 column. An ultraviolet/visible detector at 254 nm was used to detect the peaks. Sample treatment consisted of precipitation of plasma proteins with trichloroacetic acid and then injection of the supernatant. Results The method was linear and validated from 0,25 to 20 mg/L. The within-day and between-day coefficient of variation (CV) was less 1% and 1,6%, respectively. The accuracy varied between −4,3% and 0,4%. The average recovery was greater than 82%. No interferences were found with other drugs habitually used in the therapy combined with linezolid. Endogenous interferences of the biological matrix were not detected. Conclusions The method described to quantify linezolid in plasma samples is sensitive, reproducible, specific, rapid and needs very little sample, which makes it suitable for therapeutic drug monitoring.