Hemolytic Plasma Research Articles

A rapid and simple HPLC-MS/MS method for the therapeutic drug monitoring of six special-grade antimicrobials in pediatric patients

Meropenem, linezolid, fluconazole, voriconazole, posaconazole, and vancomycin are six important antimicrobials used for severe infections in critically ill patients listed in special-grade antimicrobials in China. The six antimicrobials' highly variable pharmacodynamics and pharmacokinetics in critically ill pediatric patients present significant challenges to clinicians in ensuring optimal therapeutic targets. Therefore, therapeutic drug monitoring of these antimicrobials in human plasma is necessary to obtain their plasma concentration. A rapid, simple, and sample-saving high-performance liquid chromatography-tandem mass spectrometry (HPLC–MS/MS) method was developed, which could simultaneously determine all six antimicrobials. It required only 10 μL of plasma and a one-step protein precipitation process. Chromatographic separation was achieved on a reversed-phase column (C18, 30 × 2.1 mm, 2.6 μm) via gradient elution using water and acetonitrile containing 0.1 % formic acid as mobile phase. The injection volume was 2 μL, and the total run time was only 2.5 min. Detection was done using a Triple Quad™ 4500MD tandem mass spectrometer coupled with an electrospray ionization (ESI) source in positive mode. The calibration curves ranged from 0.5 to 64 μg/mL for meropenem and fluconazole, 0.2–25.6 μg/mL for linezolid and voriconazole, 0.1–12.8 μg/mL for posaconazole and 1–128 μg/mL for vancomycin, with the coefficients of correlation all greater than 0.996. Furthermore, the method was validated rigorously according to the European Medicines Agency (EMA) guidelines, demonstrating excellent accuracy (from 93.0 % to 110.6 %) and precision (from 2.0 % to 12.8 %). Moreover, its applicability to various matrices (including serum, hemolytic plasma, and hyperlipidemic plasma) was evaluated. Thus, this method was successfully applied to routine therapeutic drug monitoring for critically ill pediatric patients and other patients in need.

Development and Validation of an LC-MS/MS Method for the Quantitative Determination of Contezolid in Human Plasma and Cerebrospinal Fluid

To develop and verify a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determining contezolid in plasma and cerebrospinal fluid (CSF). Protein precipitation was performed on samples using linezolid as the internal standard. We used an Agilent EclipsePlus C18 column operating at 0.4 mL/min in conjunction with acetonitrile and water mobile phases for the LC-MS/MS analysis. Using the precursor-product ion pairs 409.15→269.14 (contezolid) and 338.14→195.1 (linezolid), multiple reaction monitoring was used to quantify the compounds. Plasma linearity range was 50.0 to 5000 ng/mL, and CSF was 20.0 to 1000 ng/mL (r2 = 0.999). The inter-batch and intra-batch precisions were ≤2.57% and ≤5.79%, respectively. Plasma recovered 92.94%, and CSF recovered 97.83%. Plasma, CSF, hemolytic plasma, and hyperlipidemic plasma all showed a coefficient of variation ≤ 7.44%. The stability and dilution integrity of this method were also acceptable. The study also demonstrated that artificial CSF can be used as a matrix for the preparation of standard curve samples. A simple and accurate method was developed and validated for the determination of contezolid concentrations in human plasma and CSF, which may be useful for monitoring the therapeutic effect of central nervous system medications.

Method development and validation for simultaneous determination of six tyrosine kinase inhibitors and two active metabolites in human plasma/serum using UPLC–MS/MS for therapeutic drug monitoring

Over the past decades, therapeutic drug monitoring (TDM) of tyrosine kinase inhibitors (TKIs) and their main active metabolites has shown benefits in improving treatment efficacy and safety. Therefore, a sensitive, simple and economical ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the determination of six oral tyrosine kinase inhibitors (TKIs) and two active metabolites, including imatinib (IMA), N-desmethyl imatinib (NDIMA), sunitinib (SUNI), N-desethyl sunitinib (NDSUNI), regorafenib (REGO), nilotinib (NILO), dasatinib (DASA) and osimertinib (OSI) in human plasma/serum for therapeutic drug monitoring. The plasma/serum samples were deproteinated with acetonitrile after spiking with two deuterated internal standards (ISs, [2H8]-imatinib and [2H10]-sunitinib) and separated on a 40 °C ACQUITY UPLC® BEH C18 column (1.7 µm, 2.1 mm × 50 mm). The mobile phase was composed of acetonitrile (solution A) and water-formic acid-ammonium acetate (1 M) (994:1:5, v/v/v, solution B). Gradient elution was applied at a flow rate of 0.4 mL/min. Detection was carried out using a Triple Quad 5500 tandem mass spectrometer coupled with an electrospray ionization (ESI) source in positive mode. The method was validated over the calibration curve (CV) range of 2–400 ng/mL for NDSUNI and DASA, 2.5–500 ng/mL for SUNI, 10–2000 ng/mL NDIMA and OSI, 20–4000 ng/mL for NILO, 30–6000 ng/mL for REGO and 50–10000 ng/mL for IMA using linear regression and 1/x2 weighting. No difference was observed in the matrix effect (ME) among blank human plasma, hemolytic plasma, lipemic plasma, plasma spiked with ten commonly used drugs by cancer patients, or serum samples in general. The standard curve fitted by two standard curves of serum and plasma showed good linearity, and the precision and accuracy results of QC samples in both plasma and serum were acceptable and the concentration was comparable. To provide a clinical reference for the operation, the stability of the whole process from sample collection to drug detection was verified. SUNI and NDSUNI showed obvious photoisomerization under light exposure. Therefore, strict light protection was applied for all sample collection and handling steps of SUNI and NDSUNI. Compared with heparin anticoagulant tubes, the stability of the eight compounds in both whole blood and plasma was better in K3-EDTA and sodium citrate anticoagulant tubes. Given that all the analytes were stable in plasma at 4 °C for 48 h and in whole blood at room temperature for 48 h but OSI and REGO were unstable in whole blood and plasma at room temperature, samples should be centrifuged as soon as possible to be preserved as plasma at 4 °C when OSI or REGO is detected. In conclusion, this validated method can provide support for clinical practice, such as therapeutic drug monitoring (TDM) and pharmacokinetic studies of these six TKIs and two active metabolites.

Establishment and validation of an SIL‐IS LC–MS/MS method for the determination of ibuprofen in human plasma and its pharmacokinetic study

AbstractIn this work, we developed and validated a highly sensitive, rapid and stable LC–MS/MS method for the determination of ibuprofen in human plasma with ibuprofen‐d3 as a stable isotopically labeled internal standard (SIL‐IS). Human plasma samples were prepared by one‐step protein precipitation. The chromatographic separation was achieved on a Poroshell 120 EC‐C18 (2.1 × 50 mm, 2.7 μm). Aqueous solution (containing 0.05% acetic acid and 5 mm NH4Ac) and methanol were selected as the mobile phase with gradient elution. An electrospray ionization source was applied and operated in negative ion mode. Multiple reaction monitoring mode was used for quantification using target fragment ions m/z 205.0 → 161.1 for ibuprofen and m/z 208.0 → 164.0 for SIL‐IS, respectively. This method exhibited a linear range of 0.05–36 μg/ml for ibuprofen with correlation coefficient >0.99. Mean recoveries of ibuprofen in human plasma ranged from 78.4 to 80.9%. The RSD of intra‐ and inter‐day precision were both < 5%. The accuracy was between 88.2 and 103.67%. The matrix effect was negligible in human plasma, including lipidemia and hemolytic plasma. A simple, efficient and accurate LC–MS/MS method was successfully established and applied to a pharmacokinetic study in healthy Chinese volunteers after a single oral administration of ibuprofen granules.

Interpretation of Folate Results in Hemolytic Plasma Samples: A Practical Approach.

Folate analysis in plasma is affected by hemolysis, which can lead to biased results. However, the degree of hemolysis that is considered acceptable is unclear. We explored the relationship between folate concentration and degree of hemolysis. Heparin plasma samples (N=77, hemolysis index ≤10 μmol/L) were spiked with increasing amounts of corresponding patient-specific hemolysate. Subsequently, the folate concentration and hemolysis index were measured using two Roche Cobas platforms, and their incremental relationship was investigated. The folate concentration ranged from 2.9 to 30.9 nmol/L with a median (interquartile range) of 11.4 (8.6-19.1) nmol/L. The linear relationship between the increments in folate concentration and hemolysis index was approximated by the function y=1.86x+1.56 (R2=0.996), where x represents the laboratory-specific critical difference in folate concentration, which can be calculated from the analytical variation of the employed folate assay(s), and y represents the hemolysis threshold. The hemolysis threshold did not significantly differ between the tertiles of plasma folate concentration (P=0.10). In conclusion, we have provided an evidence-based approach that can be used to reliably interpret folate concentrations in hemolytic samples, independent of the patient’s folate status.

Method Development and Validation for Simultaneous Determination of Six Tyrosine Kinase Inhibitors and Two Active Metabolites in Human Plasma Using UPLC–MS/MS for Therapeutic Drug Monitoring

Over the past decades, therapeutic drug monitoring (TDM) of tyrosine kinase inhibitors (TKIs) and their main active metabolites has shown benefits in improving treatment efficacy and safety. Therefore, a sensitive, simple and economical ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the determination of six oral tyrosine kinase inhibitors (TKIs) and two active metabolites, including imatinib (IMA), N-desmethyl imatinib (NDIMA), sunitinib (SUNI), N-desethyl sunitinib (NDSUNI), regorafenib (REGO), nilotinib (NILO), dasatinib (DASA) and osimertinib (OSI) in human plasma for therapeutic drug monitoring. The plasma samples were deproteinated with acetonitrile after spiking with two deuterated internal standards (ISs, [ 2 H 8 ]-imatinib and [ 2 H 10 ]-sunitinib) and separated on a 40 °C ACQUITY UPLC® BEH C 18 column (1.7 μm, 2.1 mm×50 mm). The mobile phase was composed of acetonitrile (solution A) and water-formic acid-ammonium acetate (1M)(994:1:5, v/v/v, solution B). Gradient elution was applied at a flow rate of 0.4 mL/min. Detection was carried out using a Triple Quad 5500 tandem mass spectrometer coupled with an electrospray ionization (ESI) source in positive mode. The method was validated over the calibration curve (CV) range of 2-400 ng/mL for NDSUNI and DASA, 2.5-500 ng/mL for SUNI, 10-2000 ng/mL NDIMA and OSI, 20-4000 ng/mL for NILO, 30-6000 ng/mL for REGO and 50-10000 ng/mL for IMA using linear regression and 1/x 2 weighting. No difference was observed in the matrix effect (ME) among blank human plasma, hemolytic plasma and lipemic plasma samples in general. And the intra- and inter-batch precision and accuracy of all eight components were acceptable. To provide a clinical reference for the operation, the stability of the whole process from sample collection to drug detection was verified. SUNI and NDSUNI showed obvious photoisomerization under light exposure. Therefore, strict light protection was applied for all sample collection and handling steps of SUNI and NDSUNI. Compared with heparin anticoagulant tubes, the stability of the eight compounds in both whole blood and plasma was better in K3-EDTA and sodium citrate anticoagulant tubes. Given that all the analytes were stable in plasma at 4 °C for 48 h and in whole blood at room temperature for 48 h but OSI and REGO were unstable in whole blood and plasma at room temperature, samples should be centrifuged as soon as possible to be preserved as plasma at 4 °C when OSI or REGO is detected. In conclusion, this validated method can provide support for clinical practice, such as therapeutic drug monitoring (TDM) and pharmacokinetic studies of these six TKIs and two active metabolites.

Plasma chemistry in nesting leatherback sea turtles (Dermochelys coriacea) from Florida: Understanding the importance of sample hemolysis effects on blood analytes.

Plasma chemistry is widely used in diagnostic and research settings in sea turtles. However, plasma discolorations such as hemolysis are often not considered in data interpretation. The objectives of this study were to (1) evaluate the effects of moderate hemolysis on plasma electrolytes, minerals, and proteins using dry chemistry analysis (DCA) and protein electrophoresis from nesting leatherback sea turtles (Dermochelys coriacea) from Florida and to (2) establish blood analyte reference intervals. Twenty-six plasma samples with absence of hemolysis were selected and sub-divided into one non-hemolytic aliquot and an aliquot that was experimentally manipulated to mimic moderate hemolysis. Plasma samples were analyzed for hemoglobin using a handheld photometer; sodium, potassium, chloride, magnesium, calcium, phosphorus, and total protein using DCA; and protein electrophoresis. Packed cell volume and hemoglobin were measured in corresponding whole blood samples. Reference intervals were established. All analytes except calcium and pre-albumin were significantly higher and the calcium:phosphorus and albumin:globulin ratios were significantly lower in hemolytic plasma compared to non-hemolytic plasma. Alpha2-globulins and potassium were the analytes most impacted by hemolysis, averaging 3.3- and 2.0-fold higher in hemolyzed samples, respectively, indicating that (1) hemoglobin migrates into the alpha2-globulin region in this species and (2) notable intracellular potassium is released into plasma with hemolysis. Attempted conversion factors for compensation of hemolysis were considered inaccurate for 4 of 16 analytes due to non-significant regression lines. We also report that PCV provides an estimate of hemoglobin (g/L) using the formula: (2.59 × PCV) + 24.59. Given the spurious effects of hemolysis, the degree of this artifact should be reported with biochemistry data, and samples with moderate to severe hemolysis should be excluded from datasets when interpreting electrolyte, mineral, and protein results. This will ensure accurate data interpretation for individual turtles in rehabilitation or research settings and population-level data relevant to conservation-focused projects.

Effects of Quantification Methods, Isolation Kits, Plasma Biobanking, and Hemolysis on Cell-Free DNA Analysis in Plasma.

Cell-free DNA (cfDNA) has become a promising noninvasive clinical marker widely studied in early disease detection, monitoring, and therapy selection. However, there is lack of data on a number of cfDNA-associated procedural features such as blood plasma biobanking conditions, isolation, and quantification methods that should be taken into account as they can affect downstream applications. In this study cfDNA from 125 plasma samples from healthy individuals were isolated using three different commercial kits (bead and vacuum based). Yield of cfDNA, distribution of cfDNA fragments and absolute amount of miR-223 were estimated. Moreover, the impact of different plasma biobanking conditions and hemolytic plasma were evaluated. In conclusion, results showed that quantification method (fluorescence or microcapillary electrophoresis based) has a major impact in estimating cfDNA amount. Samples isolated by QIAamp showed a higher amount of larger (around 300 bp) DNA fragments and miRNA yield, suggesting possible applications for multiomics approach. On the other hand, the highest cfDNA yield was obtained in samples isolated by the MagMAX Isolation Kit. This kit also showed lowest coefficient of variation and low miRNA yield. Plasma storage conditions and hemolysis affected performance of isolation kits differently.

Use of basic mobile phase to improve chromatography and boost sensitivity for quantifying tetrahydrocurcumin in human plasma by LC-MS/MS.

Tetrahydrocurcumin (THC), a major metabolite of curcumin, is often quantified by LC-MS or LC-MS/MS using acidic mobile phases due to the concern of its instability in a basic medium. However, acidic mobile phases often lead to poor chromatography (e.g. split or double peaks) and reduced detection sensitivity in the commonly used negative ionization mode. To overcome these shortcomings, a basic mobile phase was used for the first time in the LC-MS/MS quantification of THC. In comparison with the acidic mobile phases, a single symmetrical chromatographic peak was obtained and the sensitivity increased by 7-fold or more under the equivalent conditions. The new LC-MS/MS method using the basic mobile phase has been successfully validated for the quantification of THC in human EDTA plasma over the concentration range of 5-2500ng/ml. The within-batch accuracy (% nominal concentration) was between 88.7 and 104.9 and the between-batch accuracy ranged from 96.7 to 108.6. The CVs for within- and between-batch precisions were equal to or less than 5.5% and 9.1%, respectively. No significant matrix interference or matrix effect was observed from normal or lipemic and hemolytic plasma matrices. In addition, the common stabilities with adequate durations were established, including up to 5days of post-preparative stability. Furthermore, when the validated method was applied to a clinical study, the passing rate of ISR samples was 83%, indicating the good reproducibility of the method. The success of the unconventional approach presented in this article demonstrates that a mobile phase could be selected based mainly on its merits to facilitate LC separation and/or MS detection. There is no need for excessive concern about the stability of the compound(s) of interest in the selected mobile phase because the run time of modern LC-MS or LC-MS/MS methods is typically only a few minutes.

Reference Intervals and Method Optimization for Variables Reflecting Hypocoagulatory and Hypercoagulatory States in Dogs using the STA Compact® Automated Analyzer

Reference intervals for coagulation parameters have been rarely determined in dogs for the STA Compact automated coagulation analyzer, so it is the aim of the current study to validate assays and establish reference ranges for its use in canine specimens. Coagulation parameters were assessed in 56 healthy dogs with a median age of 2 years and evenly distributed sex. The 95% reference intervals were as follows: 1-stage prothrombin time = 5.7-8.0 sec; activated partial thromboplastin time (APTT) = 10.0-14.3 sec; thrombin time (TT) = 11.9-18.3 sec; fibrinogen = 1.3-3.1 g/l; antithrombin (AT) = 107.9-128.0%; D-dimer = 0.023-0.65 microg/ml; anti-factor Xa = 0.04-0.26 IU/l; and activated protein C (APC) ratio = 2.0-3.0. Protein C and S activity was markedly below (<-20%) and factor VIII was 2- to 11-fold above the human calibration standard, so a standard curve had to be prepared from canine pooled plasma. Reference intervals for protein C, protein S, and factor VIII were 75.5-118.9%, 74.4-160.5%, and 70.9-136.4%, respectively, compared with a canine standard curve. Streptokinase-activated plasminogen assay was not suitable for dogs. There was no significant impact of sex on hemostasis test results. Factor VIII activity, AT, protein C, protein S, and APC ratio were overestimated in hemolytic plasma, whereas fibrinogen, TT, and APTT were underestimated. Lipemia resulted only in false-high D-dimers. This study provided useful reference intervals for dogs, but some human tests (i.e., protein C, protein S, factor VIII, and plasminogen) required modification.

Rapid, specific, and sensitive measurements of plasma sphingomyelin and phosphatidylcholine

Sphingomyelin (SM) and phosphatidylcholine (PC) are two major phospholipids on plasma lipoproteins. Their concentration is classically measured by lipid extraction, thin-layer chromatography, and phosphate determination on separated SM or PC spots. Here, we describe two rapid, specific, and sensitive enzymatic measurements for both phospholipids. Plasma was incubated with bacterial sphingomyelinase (for SM measurement) or bacterial PC-specific phospholipase D (for PC measurement), alkaline phosphatase, choline oxidase, peroxidase, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline, and 4-aminoantipyrine for 45 min. A blue dye, with an optimal absorption at 595 nm, was generated. PC levels did not influence SM measurement and vice versa. The linear range for the SM measurement was 0.5-5 microg, and that for PC was 2.5-20 microg. The mean percentage recovery was 98.0 +/- 5.2% for SM and 96.6 +/- 3.8% for PC. The interassay coefficient of variation of the assay was 1.7 +/- 0.05% for SM and 3.1 +/- 0.13% for PC. These two new methods are amenable to automation and can be adapted for large-scale, high-throughput assays.

Der Einfluß von Hämolyse auf Dichtemessungen an Kapillarblutplasma mittels der Biegeschwingermethode

Sampling of capillary blood often yields slightly hemolytic plasma. We investigated the influence of hemolysis on plasma density using the mechanical oscillator technique. Plasma density rose by approximately 0.1 milligram when the hemoglobin content increased by 0.5 milligram. These values correspond to 0.3% hemolysis and were not exceeded in more than 70% of the investigated capillary plasma samples. In general, hemolysis must be taken into account if plasma density measurements are to be made with an accuracy better than 0.5 milligram, which corresponds to 2-3% of the plasma protein content.

The interference of hemolysis on radioimmunoassay of insulin and its prevention by pre-incubation with anti-insulin serum.

Using the back-titration method it was studied the influence of hemolysis upon radioimmunoassay of insulin. The degradation of immunoassayable insulin was increased by incubation with the increasing degree of hemolysis of plasma and with incubation period. The degradation of insulin was prevented when immediately after preparation the hemolytic plasma was incubated with anti-insulin serum.

Experimental transfusion reactions and disseminated intravascular coagulation produced by incompatible plasma in monkeys.

Hemolytic plasma transfusion reactions were produced in six Macaca irus monkeys. The plasma for these transfusions was obtained from alloimmunized animals with agglutinins of high titer and, in some cases, hemolysins directed against the recipients' red blood cells. The transfusion reactions were characterized by varying degrees of intravascular hemolysis, hemoglobinemia and hemoglobinuria, and disseminated intravascular coagulation accompanied by marked falls in Factors VIII and V, and fibrinogen. Shortening of euglobulin clot lysis time was seen in three animals.Two animals died and had fibrin thrombi in renal glomeruli, myocardial veins, pulmonary arteries, and hepatic sinusoids.

Studies on the Anemia of Disseminated Malignant Neoplastic Disease. II. Study of the Life Span of the Erythrocyte

Abstract 1. A study of the life span of the erythrocyte in patients with advanced neoplastic disease was performed by transfusing blood from 23 hospitalized patients into 53 healthy volunteers. 2. The Ashby technic for red cell labeling was used alone in 16 instances, Cr51 technic in 52 instances, and a combination of the 2 technics in 8 instances. 3. Normal or nearly normal life spans were suggested by the slopes of curves in 15 instances where whole blood from patients was transfused into volunteers. 4. Some shortening of the life span of the patient’s red cells was noted when these cells, separated from the plasma, were transfused into volunteers or back into patients, possibly related to the handling during plasma removal. 5. Control studies using either Cr51 or Ashby labeling technic indicated a normal life span of approximately 120 days in the volunteers employed could be achieved by each method. 6. Values obtained with Cr51 were so similar to those using Ashby labeling that the easier Cr51 technic is recommended for future studies. 7. The normal erythrocyte transfused into patients with neoplastic disease may have a moderate to considerably shortened life span. 8. These studies seem to demonstrate an absence of an intrinsic defect in the erythrocytes of patients with neoplastic disease, and further favor the presence of a hemolytic plasma factor of considerable importance in the pathogenesis of the anemia of cancer.

TRANSFUSION REACTIONS TO A PLASMA CONSTITUENT OF WHOLE BLOOD: Their Pathogenesis and Treatment by Washed Red Blood Cell Transfusions

1. The clinical histories of 11 patients with a severe and hitherto undescribed type of reaction to in vitro compatible whole blood are presented. These patients all experienced beneficial responses to therapy with washed red blood cell transfusions. The technic of washing the blood is outlined.2. In 5 of the patients, sensitivity to a heat-labile constituent of fresh normal plasma was demonstrated. In the remaining 6 patients, although actual sensitivity to plasma was not proved, transfusions of washed red blood cells were well tolerated and were therapeutically effective.3. These uncommon reactions to the plasma in whole blood are differentiated from the usual pyrogenic, allergic or hemolytic plasma reactions by their ease of repetition in the absence of pyrogens, by lack of the usual allergic manifestations, and by the failure to demonstrate hemoglobinemia or hemoglobinuria.4. A simple diagnostic test (“plasma provocative test”) is described which appears to be of specific value in the differentiation of the reaction.5. It is suggested that in any instance of severe transfusion reaction to apparently compatible whole blood this type of sensitivity to the donated plasma should be borne in mind as a possible cause. The plasma provocative test should then be tried. If positive, the transfusion of well washed red blood cells will probably be effective and in some cases may be of life-saving importance.