Small Plasma Samples Research Articles

Development and Validation of a High-Performance Liquid Chromatography With Ultraviolet Detection Method to Facilitate Therapeutic Monitoring of Teicoplanin Using Dried Blood Spots.

In neonatal and pediatric intensive care units, Gram -positive infections are a significant cause of morbidity and mortality. The increase in infections caused by methicillin-resistant Staphylococcus aureus and methicillin-resistant coagulase-negative Staphylococci have led to the increased use of glycopeptides, which treat invasive infections caused by Gram -positive organisms, particularly those resistant to beta-lactam antibiotics. Teicoplanin has bacteriostatic activity against Gram -positive bacteria, but its pharmacokinetics in children is highly variable, with most children failing to reach target levels at the recommended dose. This study aimed to develop a cost-effective method for determining concentrations using dried blood spot (DBS). A method to determine the concentrations of teicoplanin in 20 µL blood or plasma using the Whatman 903 Protein Saver filter was evaluated. High-performance liquid chromatography with ultraviolet detection high-performance liquid chromatography with ultraviolet/vis was used, with internal standard ketoconazole. In addition, a method to quantify teicoplanin using 50 µL of liquid plasma was established to compare the results with the values obtained by DBS and dried plasma methods. The method was successfully developed and validated for 20 µL DBS. Furthermore, 50 µL of plasma was used to quantify teicoplanin with a lower limit of quantification of 10 mg/L. Precision and accuracy ranged from 2.3% to 10.7% and 95%-114.2%, respectively. A consistent factor (1.15) was used to calculate teicoplanin plasma concentrations from whole blood, indicating the reliability of the DBS method for therapeutic drug monitoring of teicoplanin. A simple, reliable, and cost-effective method using high-performance liquid chromatography with ultraviolet/vis was established to determine pediatric teicoplanin concentrations in both small plasma sample volumes and whole blood using DBS, and an accurate correlation factor for estimating teicoplanin plasma concentrations from DBS was identified. This method is suitable for the use in pediatrics.

Abstract 3624: A low-cost kit for gentle, effective and timely extracellular vesicle (GET EVs) isolation: Accelerating development of RNA-based liquid biopsies for neuroendocrine neoplasms

Abstract Extracellular vesicles (EVs) are cell fragments released by all cells, making them promising platforms for cancer biomarker development. Effective isolation of ultra-pure EVs for downstream analyses is key for EV-based liquid biopsy development. However, current EV isolation methods such as ultracentrifugation (UC) and other commercialized EV isolation kits are lackluster because of damage to EVs, length of time required, and low EV recovery rates. In response, we developed an “GET EV” kit which is based on the principles of lipid energy states, also known as the aqueous two-phase system (ATPS) to isolate EVs from any media. Nanoscale flow cytometry (nFC) analysis revealed that ATPS has greater EV enrichment capability (21.4 × vs. 10.9 × times fold enrichment) and higher EV recovery efficiency (97.6% vs. 69.3% recovery) than UC. Other EV isolation kits were tested and were inferior to ATPS in terms of EV recovery and EV-RNA recovery. EV subpopulations as determined by EV biomarkers (CD9, CD63, CD81, etc.) were confirmed by nFC. “Omics” studies (small RNA sequencing and proteomics) also confirmed the presence of expected EV markers. We developed an RNA-based liquid biopsy for Neuroendocrine Neoplasms (NENs) by quantitating the presence of EVs containing miR-375, an established biomarker for NENs. Using the GET EVs kit, we isolated EVs from healthy volunteer and patient plasma samples of NEN patients. RT-qPCR demonstrated enriched expression of miR-375 in EVs isolated from Lung and GI NEN patient plasma samples. Our study establishes the use of GET EV kits to efficiently, rapidly, and gently isolate EVs from small plasma samples (250 uL). This is a low-cost innovation that will finally enable the development of other RNA-based Liquid Biopsies for other disease sites with significant diagnostic and prognostic implications. Citation Format: Boyang Su, Morteza Jeyhani, Xiaojing Yang, Jina Nanayakkara, Reese Wunsche, Neil Renwich, Scott Tsai, Hon Leong. A low-cost kit for gentle, effective and timely extracellular vesicle (GET EVs) isolation: Accelerating development of RNA-based liquid biopsies for neuroendocrine neoplasms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3624.

Pesticides in small volume plasma samples: Method development and application to smallmouth bass (Micropterus dolomieu) from the Chesapeake Bay watershed, USA

Nontarget organisms are exposed to pesticides following applications in agricultural and urban settings, potentially resulting in deleterious effects. Direct measurements of pesticides in biological tissues may aid in characterizing exposure, accumulation, and potential toxicity versus analyses in environmental media alone (e.g., water, soil, and air). Plasma represents a nonlethal sampling medium that can be used to assess recent exposures to contaminants. Herein, a method was developed to test the extraction of 210 pesticides and their transformation products in small volume plasma samples (100 μL). Plasma samples were protein precipitated with 0.5 % formic acid in acetonitrile added to the sample (ratio of 3.5:1). Pass-through solid phase extraction was used for sample matrix and lipid removal and samples were analyzed by liquid chromatography and gas chromatography with tandem mass spectrometry. Recoveries of 70.0–129.8 % were achieved for 182 pesticides and degradates across the low (25 ng mL−1), medium (100 ng mL−1), and high (250 ng mL−1) spike levels. Method detection levels ranged 0.4–13.0 ng mL−1. Following development, the method was applied to smallmouth bass (Micropterus dolomieu) plasma samples (n = 10) collected from adults in the Chesapeake Bay watershed. Individual plasma samples resulted in four to seven analytes detected with summed concentrations ranging 16.4–95.0 ng mL−1. Biological multiresidue pesticide methods help elucidate recent exposures of bioactive compounds to nontarget organisms.

Development of a robust UPLC-MS/MS method for the quantification of riluzole in human plasma and its application in pharmacokinetics.

Introduction: The aim of the present study was to establish a simple method for the determination of riluzole in human plasma by ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and apply it for the determination of riluzole in amyotrophic lateral sclerosis (ALS) patients. Methods: Samples were prepared by protein precipitation and were then gradient-eluted on a column of ACQUITY UPLC® HSS T3 by using 0.1% formic acid acetonitrile and 0.1% formic acid water as the mobile phase. Detection was performed on a Xevo TQ-S tandem mass spectrometer in the multiple-reaction monitoring mode using positive electrospray ionization. Validation was performed in the range of 5-800ng/mL. Results and discussion: Three batches of precision accuracy, selectivity, matrix effects, extraction recovery, and stability were also verified and met the requirements. The results showed that the method was reliable and successfully applied to the pharmacokinetics study of riluzole in Chinese amyotrophic lateral sclerosis patients. Meanwhile, in comparison with other prior published methods, our method has the advantages of simple sample preparation, relatively short running time, and small plasma sample consumption, which represented a high-throughput sample determination potential.

ОПЫТ ПРИМЕНЕНИЯ ЭФМОРОКТОКОГА АЛЬФА У ДЕТЕЙ И ПОДРОСТКОВ: КЛИНИЧЕСКИЕ НАБЛЮДЕНИЯ

Despite availability of prophylactic therapy for hemophilia A with factor VIII concentrates with a standard half-life, patients continue to experience episodes of bleeding and joint damage. The reasons for this may be the relatively short half-life of the factor VIII drug and the low adherence of patients to treatment. The appearance of clotting factor concentrates with an extended half-life makes it possible to reduce the frequency of infusions and increase the residual activity of the deficient factor. The article presents clinical observations of the use of Efmoroctocog alfa (recombinant human coagulation factor VIII, Fc fusion protein (rFVIIIFc)) in a 16-year-old adolescent and a 7-year-old child with severe and moderate forms of hemophilia A. In order to select the most rational therapy regimen and evaluate the effectiveness of treatment, the authors have conducted individual assessments of patients’ pharmacokinetic (PK) parameters. The drug administration in both patients was twice per week. To assess individual PK, the WAPPS-Hemo was used in order to calculate individual PK parameters based on a small plasma samples collected during routine prophylactic treatment. During the PK study, 3 blood samples were taken to determine the level of factor VIII in a one-step method. In a 16-year-old patient, the half-life of Efmoroctocog alfa (t1/2) was 24.25 hours. The activity of factor VIII prior to the next injection was 7.4%. In addition, 81% of time the factor VIII activity was above 15%. In a 7-year-old patient, the t1/2 of the drug was 12.75 hours. The minimum residual activity of factor VIII was 2.1%, 86% of time the activity of factor VIII was above 3%. Conclusion: high efficacy and safety were demonstrated based on the results of the use of Efmoroctocog alfa in routine clinical practice in previously treated pediatric and adolescent patients with severe and moderate hemophilia A, as well as the reduction in the frequency of infusions per week from 3 to 2.

Quantification of N, N’ N’’-triethylenethiophosphoramide, N, N’ N’’-triethylenephosphoramide, cyclophosphamide, and 4-hydroxy-cyclophosphamide in microvolume human plasma to support neonatal and pediatric drug studies

N, N’ N’’-triethylenethiophosphoramide (thiotepa) and cyclophosphamide (CP) are alkylating agents used for a variety of malignant and non-malignant disorders. Both drugs are metabolized by cytochrome P450 enzymes to form active metabolites. To support pharmacokinetic studies of thiotepa and CP in children, we sought to develop assays to determine parent drug and metabolite concentration in small volume plasma samples. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used for assay development. CP metabolite 4-hydroxycyclophosphamide (4OHCP) was converted to the more stable semicarbazone derivative (4OHCP-SCZ) for quantitation. Samples (10 µL) were extracted by solid-phase extraction and injected onto the LC-MS/MS system equipped with a pentafluorophenyl reverse phase column (2.1 × 50 mm, 2.7 µm). Electrospray ionization in positive mode was used for detection. Multiple reaction monitoring of the precursor-to-product ion transitions m/z 190→147 for thiotepa, 174→131 for tepa, 261→233 for CP, and 334→221 for 4OHCP-SCZ was selected for quantification. The ion transitions m/z 202→155 for thiotepa-d12, 186→139 for tepa-d12, 267→237 for CP-d4, and 340→114 for 4OHCP-d4-SCZ were selected for the internal standard (IS) corresponding to each analyte. The less abundant IS ions from 37Cl were used for CP-d4 and 4OHCP-d4-SCZ to overcome the cross-talk interference from the analytes. Under optimized conditions, retention times were 0.67 min for tepa and its IS, 2.50 min for thiotepa and its IS, 2.52 min for 4OHCP-SCZ and its IS, and 2.86 min for CP and its IS. Total run time was 5 min per sample. The calibration ranges were 2.5-2,000ng/mL for thiotepa and tepa, 20-10,000ng/mL for CP and 20-5,000 ng/mL for 4OHCP; Dilution integrity for samples above the calibration range was validated with 10-fold dilution for thiotepa/tepa and 20-fold dilution for CP/4OHCP . Recoveries ranged from 86.3-93.4% for thiotepa, 86.3-89.0% for tepa, 90.2-107% for CP, and 99.3-115% for 4OHCP-SCZ. The IS normalized matrix effect was within (100±7) % for all 4 analytes. Plasma samples at room temperature were stable for at least 60 hours for thiotepa, 6 days for tepa, and 24 hours for CP and 4OHCP-SCZ. Plasma samples for thiotepa/tepa were stable after 4 freeze-thaw cycles, and for CP/4OHCP-SCZ were stable after 3 freeze-thaw cycles. The assays were validated and applied to clinical studies requiring small sample volumes.

Simultaneous detection of a panel of nine sedatives and metabolites in plasma from critically ill pediatric patients via UPLC-MS/MS

Sedative use can result in adverse drug reactions. Intensive care unit patients are especially at risk and pharmacokinetic modeling of drug concentrations is an approach to develop precision dosing strategies. However, limited blood sampling availability in critically ill children and need for multiple assays to quantify a variety of commonly used sedatives creates logistical challenges. The goal of this project was to develop a sensitive and selective assay for the simultaneous quantification of a panel of sedatives comprised of midazolam (MDZ), alpha hydroxymidazolam (1- OH MDZ), dexmedetomidine (DEX), morphine (MOR), morphine-3-glucuronide (M3G), morphine-6-glucuronide (M6G), fentanyl (FEN), norfentanyl (NF), and hydromorphone (HM) in small volume pediatric plasma samples. A sensitive and efficient ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method was developed following FDA guidance for bioanalytical validation. Minimal sample preparation consisting of simple protein precipitation extraction using acetonitrile with internal standards was utilized. Analyte separation was achieved using a gradient mixture of (A: 0.15% formic acid in water and B: Acetonitrile) and a Waters Acquity C18, 1.7 µm (2.1 × 100 mm) column. Assays were linear over the clinical concentration ranges: MDZ, MOR, HM: 0.5–125 ng/mL; 1-OH MDZ, M3G, M6G: 5–500 ng/mL; and DEX, FEN, NF: 0.05–7.5 ng/mL (R2 > 0.99 for all). Assay run time was 10 min and required only 100 μL of plasma. Initial testing of samples from pediatric patients demonstrates adequacy of assay to measure sedatives and metabolites at clinical concentrations confidently in low volumes of plasma. This novel highly-sensitive and specific method to measure a total of nine different analytes (five sedatives, four metabolites) simultaneously enables comprehensive analysis of a panel of sedatives in small volumes such as in pediatric ICU patients.

Enhancement of Acylcarnitine Levels in Small Intestine of Abdominal Irradiation Rats Might Relate to Fatty Acid β-Oxidation Pathway Disequilibration.

ObjectiveThis study aims to analyze the alteration of carnitine profile in the small intestine of abdominal irradiation-induced intestinal injury rats and explore the possible reason for the altered carnitine profile.MethodsThe abdomens of 15 male Sprague Dawley (SD) rats were irradiated with 0, 10, and 15 Gy of 60Co gamma rays. The carnitine profile in the small intestine and plasma samples of SD rats at 72 h after abdominal irradiated with 0 Gy or 10 Gy of 60Co gamma rays were measured by targeted metabolomics. The changes of fatty acid β-oxidation (FAO), including the expression of carnitine palmitoyltransferase 1 (CPT1) and acyl-CoA dehydrogenases, were analyzed in the small intestine samples of SD rats after exposed to 0, 10, and 15 Gy groups.ResultsThere were eleven acylcarnitines in the small intestine and fourteen acylcarnitines in the plasma of the rat model significantly enhanced, respectively (P < .05). The expression level and activity of CPT1 in the small intestine were remarkably increased (P < .05), and the activity of acyl-CoA dehydrogenase in the small intestine was noticeably reduced (P < .01) after abdominal irradiation.ConclusionThe enhanced acylcarnitine levels in the small intestine of abdominal irradiation rats might relate to the FAO pathway disequilibration.

Development of acoustically isolated extracellular plasma vesicles for biomarker discovery in allogeneic hematopoietic stem cell transplantation

BackgroundInfection and graft-versus-host disease (GvHD) are the major causes for mortality and morbidity of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Plasma-derived extracellular vesicles (EVs) contain disease-related proteins, DNAs and RNAs, and have recently been suggested as potential biomarker candidates for transplantation complications. However, EV isolation from small plasma volumes in clinical biomarker studies using conventional methods is challenging. We therefore investigated if EVs isolated by novel automated acoustic trapping could be developed as potential biomarkers for allo-HSCT complications by performing a clinical proof-of-principle study.ResultsPlasma samples were collected from twenty consecutive patients with high-risk/relapsed hematologic malignancies undergoing allo-HSCT before transplantation and post-transplant up to 12 weeks. EVs were isolated from small plasma sample volumes (150 μl) by an automated, acoustofluidic-based particle trapping device, which utilizes a local λ/2 ultrasonic standing wave in a borosilicate glass capillary to capture plasma EVs among pre-seeded polystyrene microbeads through sound scatter interactions. We found that EVs could be reliably isolated from all plasma samples (n = 173) and that EV numbers increased more than 2-fold in the majority of patients after transplantation. Also, sufficient quantities of RNA for downstream microRNA (miRNA) analysis were obtained from all samples and EV miRNA profiles were found to differ from whole plasma profiles. As a proof of principle, expression of platelet-specific miR-142-3p in EVs was shown to correlate with platelet count kinetics after transplantation as expected. Importantly, we identified plasma EV miRNAs that were consistently positively correlated with infection and GvHD, respectively, as well as miRNAs that were consistently negatively correlated with these complications.ConclusionsThis study demonstrates that acoustic enrichment of EVs in a clinical biomarker study setting is feasible and that downstream analysis of acoustically-enriched EVs presents a promising tool for biomarker development in allo-HSCT. Certainly, these findings warrant further exploration in larger studies, which will have significant implications not only for biomarker studies in transplantation but also for the broad field of EV-based biomarker discovery.

Characterizing imidacloprid and metabolites in songbird blood with applications for diagnosing field exposures

Neonicotinoids are the most widely used insecticides globally, but their rapid metabolism in vertebrates makes diagnosing wildlife exposure challenging. More detailed information on the pattern of imidacloprid metabolites over time could be used to better approximate the timing and level of exposure. Here, we applied recently developed sensitive analytical methods to measure imidacloprid (IMI) parent compound along with an expanded suite of metabolites (5-OH-IMI, IMI-olefin, desnitro-IMI, IMI-urea, 6-chloronicotinic acid, 5-AMCP, 6-OH nicotinic acid) and six other neonicotinoids in adult red-winged blackbirds (Agelaius phoeniceus) that were experimentally exposed to one of two field-realistic concentrations of imidacloprid (0.8 or 6.9 mg/kg bw). We measured concentrations in small (25 μL) plasma samples collected pre-exposure and at 1-, 6-, 24- and 48-h post-exposure. Imidacloprid was rapidly absorbed and metabolized within 48 h at both doses, with the largest decrease within 6 h post-exposure. The average proportion of parent IMI decreased from 68% of total detectable residues at 1-h to 34% at 6-h post-exposure. Two primary metabolites in blood were 5-OH-IMI and IMI-olefin, and 5-OH-IMI was the most persistent marker of exposure at 48-h. Desnitro-IMI was consistently detected following very recent (≤ 1-h) IMI exposure, and a higher ratio of parent IMI to metabolites also indicated recent exposure. Other metabolites were only detected in the higher dose group, and could be used as indicators of exposure to higher IMI concentrations. This sensitive analytical method and the observed metabolite patterns could be used to inform a growing body of field studies linking neonicotinoid exposure and effects in free-living birds.

Abstract 6252: Development and validation of an LC/MS/MS method for the determination of celastrol in rat plasma and its application to the pharmacokinetic evaluation of celastrol-encapsulated silk fibroin nanoparticles

Abstract Celastrol (CL), a compound isolated from Tripterygium wilfordii, possesses various bioactivities such as anti-tumor, anti-inflammatory and anti-obesity effects. Many new drug delivery systems of CL have been designed to overcome its poor water solubility and low bioavailability. However, few studies are available concerning its in vivo pharmacokinetics. In this study, a simple and sensitive LC-MS/MS method was developed and validated to determine CL in rat plasma using 18α-glycyrrhetinic acid as the internal standard. The separation of CL was performed on a Phenomenex Gemini C18 column (150 mm × 2 mm, 5 µm) following sample preparation using liquid-liquid extraction with the optimized extraction solvent of tert-butyl methyl ether and a small plasma sample volume of 50 μL. The analyte was quantitated under positive electrospray ionization and multiple reactions monitoring mode. The assay exhibited a good linearity in the concentration range of 0.5-500 ng/mL. The lower limit of quantification (LLOQ) was 0.5 ng/mL. The method was fully validated to meet the requirements for bioassay with accuracy of 91.1-110.0%, precision (RSD %) of less than 9.1 %, extraction recovery of 63.5-74.7%, and matrix effect of 87.3-101.2%. The developed method was successfully applied to the pharmacokinetic evaluation of CL-encapsulated silk fibroin nanoparticles (CL-SFNP). The results show, after intravenous (IV) and oral (PO) administration of equivalent CL (1 mg/kg and 3 mg/kg, respectively) in rats, the AUC values of CL were 8646.1±1998.9 ng/mL·h after IV dosing and 1065.5±494.6 ng/mL·h after PO dosing for CL-SFNP, which were significantly (P&amp;lt;0.05) higher than those for pure CL (4697.7±723.0 ng/mL·h and 441.9±82.6 ng/mL·h, respectively). The dose-normalized oral bioavailability of CL significantly (P&amp;lt;0.05) increased from 3.1% for pure CL to 7.6% for CL-SFNP. Citation Format: Shuyu Zhan, Amy Paik, Felicia Onyeabor, Baoyue Ding, Sunil Prabhu, Jeffrey Wang. Development and validation of an LC/MS/MS method for the determination of celastrol in rat plasma and its application to the pharmacokinetic evaluation of celastrol-encapsulated silk fibroin nanoparticles [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6252.

Bioanalytical method development and validation of corynantheidine, a kratom alkaloid, using UPLC-MS/MS, and its application to preclinical pharmacokinetic studies

Corynantheidine, a minor alkaloid found in Mitragyna speciosa (Korth.) Havil, has been shown to bind to opioid receptors and act as a functional opioid antagonist, but its unique contribution to the overall properties of kratom remains relatively unexplored. The first validated bioanalytical method for the quantification of corynantheidine in rat plasma is described. The method was linear in the dynamic range from 1–500 ng/mL, requires a small plasma sample volume (25 μL), and a simple protein precipitation method for extraction of the analyte. The separation was achieved with Waters BEH C18 2.1 × 50 mm column and the 3-minute gradient of 10 mM ammonium acetate buffer (pH = 3.5) and acetonitrile as mobile phase. The method was validated in terms of accuracy, precision, selectivity, sensitivity, recovery, stability, and dilution integrity. It was applied to the analysis of the male Sprague Dawley rat plasma samples obtained during pharmacokinetic studies of corynantheidine administered both intravenously (I.V.) and orally (P.O.) (2.5 mg/kg and 20 mg/kg, respectively). The non-compartmental analysis performed in Certara Phoenix® yielded the following parameters: clearance 884.1 ± 32.3 mL/h, apparent volume of distribution 8.0 ± 1.2 L, exposure up to the last measured time point 640.3 ± 24.0 h*ng/mL, and a mean residence time of 3.0 ± 0.2 h with I.V. dose. The maximum observed concentration after a P.O. dose of 213.4 ± 40.4 ng/mL was detected at 4.1 ± 1.3 h with a mean residence time of 8.8 ± 1.8 h. Absolute oral bioavailability was 49.9 ± 16.4 %. Corynantheidine demonstrated adequate oral bioavailability, prolonged absorption and exposure, and an extensive extravascular distribution. In addition, imaging mass spectrometry analysis of the brain tissue was performed to evaluate the distribution of the compound in the brain. Corynantheidine was detected in the corpus callosum and some regions of the hippocampus.

Detection and quantification of warfarin in pharmaceutical dosage form and in spiked human plasma using surface enhanced Raman scattering.

Analytical approaches for the quantitation of warfarin in plasma are high in demand. In this study, a novel surface enhanced Raman scattering (SERS) technique for the quantification of the widely used anticoagulant warfarin sodium in pharmaceutical dosage form and in spiked human plasma was developed. The colloidal-based SERS measurements were carefully optimized considering the laser wavelength, the type of metal nanoparticles, their surface functionalization and concentration as well as the time required for warfarin to associate with the metal surface. Poly(diallyldimethylammonium chloride) coated silver nanoparticles (PDDA-AgNPs) were established as a substrate which greatly enhanced the weak warfarin Raman signal with high reproducibility. The limit of detection was calculated in both water and human plasma to be 0.56 nM (0.17 ngmL-1) and 0.25 nM (0.08 ngmL-1) respectively, with a high degree of accuracy and reproducibility. The proposed method is simple, economical, and easily applied for routine application requiring only small plasma samples and also could be potentially useful for pharmacokinetic research on warfarin.

Establishment of reference values for the lysine acetylation marker Nɛ-acetyllysine in small volume human plasma samples by a multi-target LC-MS/MS method.

Cardiovascular disease (CVD) and chronic kidney disease (CKD) constitute substantial burdens for public health. The identification and validation of risk markers for CVD and CKD in epidemiological studies requires frequent adaption of existing analytical methods as well as development of new methods. In this study, an analytical procedure to simultaneously quantify ten endogenous biomarkers for CVD and CKD is described. An easy-to-handle sample preparation requiring only 20µL of human plasma is followed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The method was successfully validated according to established guidelines meeting required criteria for accuracy, precision, recovery, linearity, selectivity, and limits of quantification. The scalability of the method for application in larger cohorts was assessed using a set of plasma samples from healthy volunteers (n = 391) providing first reference values for the recently established biomarker Nɛ-acetyllysine (Nɛ-AcLys). Other biomarkers analyzed were creatinine, β-aminoisobutyric acid (β-AIB), carnitine, 1-methylnicotinamide (1-MNA), citrulline, symmetric dimethylarginine (SDMA), asymmetric dimethylarginine (ADMA), homoarginine (hArg), and ornithine. All obtained results are within reference values specified elsewhere. Overall, these results demonstrate the suitability of the method for simultaneous quantification of ten endogenous biomarkers for CVD and CKD in plasma samples from larger cohorts and allow validation of Nɛ-AcLys as a biomarker in large cohorts.

SAT-144 AN ULTRASENSITIVE SURFACE ENHANCED RAMAN SCATTERING (SERS)-BASED ASSAY FOR KIDNEY INJURY MOLECULE-1 (KIM-1)

Kidney injury molecule-1 (KIM-1) is a biomarker of acute and chronic kidney injury. However, dilution of urine samples to minimize the impact of pH and solute on results can push the concentration levels to below the lower limits detection of conventional ELISAs. Additionally, small samples from infants, children or from non-human renal injury models such as mice limits analyte detection. Here we develop an ultrasensitive (sub-pg/ml) assay utilizing surface enhanced Raman scattering (SERS) to measure KIM-1. Capture and detection antibodies of a commercially available KIM-1 ELISA kit (R&D Systems) were utilized. [email protected] nanocuboids ([email protected]) with 16 nm shell thickness, assembled on silicon wafers, were derivatized with cysteamine followed by glutaraldehyde to covalently attach the capture antibody. Separately, [email protected] were derivatized with the Raman reporter 4-aminothiophenol (pATP), glutaraldehyde followed by the detection antibody. After analyte capture on the silicon wafer, washing, incubating with the derivatized detection antibody and washing, the silicon wafers were subject to SERS. Among several prominent Raman bands, the intensity band at 1080 cm-1 of pATP was proportional to the concentration of human recombinant KIM-1 spiked into artificial urine. The urine of control individuals and patients with kidney disease was diluted prior to assay and the results compared to that using the conventional ELISA. The Raman intensity band at 1080 cm-1 of pATP was proportional to the KIM-1 concentration from to 400 pg/ml. As little as 1fg/ml above background could be reliably detected. The KIM-1 concentration, determined in patient urine using both the conventional ELISA and the SERS assay, was found to be synonymous with a correlation coefficient of 0.93 ( p<0.002). A similar strategy was used to develop a sub-pg/ml assay for the pro-inflammatory/myokine molecule IL-6. Using the power of SERS, capture and detection antibodies of a conventional ELISA kit were modified to measure sub-pg/ml amounts of KIM-1. This sensitivity allows dilution of samples to eliminate the influence of pH and/or solute concentration that would otherwise affect conventional ELISA results. Also the sensitivity of the assay allows the use of small samples of urine or plasma from infants, small children or from small animals such as mice with experimental renal diseases. It would be possible to detect preclinical changes in KIM-1 prior to disease manifestation. This assay strategy can be adapted to multiplexing to measure two or more analytes simultaneously in one sample by utilizing different Raman reporter molecules on different detection antibodies, an appropriate mix of capture antibodies on the silicon wafer, and monitoring non-overlapping Raman bands specific for each reporter.

A practical workflow for capillary microsampling in nonclinical studies.

The result of investigation on the procedure of sample handling and bioanalysis of small volume of plasma sample for nonclinical studies stored in 0.5-ml micronic tubes was reported. Results/methodology: Sample integrity of the small volume (25μl) during long-term storage and the feasibility and data reliability of performing multiple re-assays on the small volume sample using 5 μl aliquot per analysis was evaluated. Integrity was maintained in samples (25μl) stored for up to 1 month in 0.5-ml micronic tubes at -20°C. A 25 μl sample is sufficient for four-times of re-assays. This evaluation demonstrated the feasibility of this workflow of handling and bioanalysis on small volume plasma sample for GLP studies under the US FDA guidance.

Quantification of Cefepime, Meropenem, Piperacillin, and Tazobactam in Human Plasma Using a Sensitive and Robust Liquid Chromatography-Tandem Mass Spectrometry Method, Part 1: Assay Development and Validation.

The highly variable pharmacokinetics of β-lactam antibiotics and β-lactamase inhibitors poses a significant challenge to clinicians in ensuring appropriate antibiotic doses in critically ill patients. Therefore, routine monitoring of plasma concentrations is important for individualization of antimicrobial therapy. Accordingly, a simple and robust analytical method for the simultaneous measurement of multiple β-lactam antibiotics and β-lactamase inhibitors is highly desirable to ensure quick decisions on dose adjustments. In this study, a sensitive, simple, and robust method for the simultaneous quantification of cefepime, meropenem, piperacillin, and tazobactam in human plasma was developed and rigorously validated according to FDA guidance. Sample extraction was accomplished by simple protein precipitation. Chromatographic separation of analytes was achieved using stepwise gradient elution. Analytes were monitored using tandem mass spectrometry (MS/MS) with a turbo ion spray source in positive multiple-reaction-monitoring mode. The calibration curve ranged from 0.5 to 150 μg/ml for cefepime, 0.1 to 150 μg/ml for meropenem and piperacillin, and 0.25 to 150 μg/ml for tazobactam. Inter- and intraday precision and accuracy, sensitivity, selectivity, dilution integrity, matrix effect, extraction recovery, and hemolysis effect were investigated for all four analytes, and the results met the acceptance criteria. Compared to other reported methods, our method is more robust because of the combination of the following features: (i) a simple sample extraction procedure, (ii) a short sample run time, (iii) a wide dynamic range, and (iv) the small plasma sample volume needed. Since our method already covers β-lactams and a β-lactamase inhibitor with highly heterogeneous physicochemical properties, further antibiotic candidates may easily be incorporated into this multianalyte method.

English

Vitamin D (VD) deficiency and insufficiency have been linked to poor quality of life (QoL); general weakness, fatigue, and nonspecific pain. Despite abundance of sun light, there is a wide spread VD deficiency in the Arabian Gulf countries. However, the impact of VD deficiency on the quality of life has not been well documented in the Arabian Gulf countries, including the Kingdom of Saudi Arabia (KSA). This study aims to analyze the effect of crude vitamin D levels on quality of life in none pregnant Arab women in the Kingdom of Saudi Arabia (KSA). The study was a prospective cross sectional study, using a sample of 152 subjects of patients aged 13 years and above attending the outpatients’ clinic at King Faisal Hospital, Kingdom of Saudi Arabia (KSA). The study collected anthropometric and demographic variables, in addition to a small plasma sample from all subjects to measure VD levels, calcium (Ca), phosphorus (P), parathyroid hormone (PTH) and lipid profile by standard methods. Subjects were categorized according to VD levels as normal (>30 ng/ml), insufficiency (21 to 29 ng/ml), or deficient (≤ 20 ng/ml). The RAND Short Form -36 (SF-36), which consists of 36 questionnaire items was used to measure (QoL) across eight domains for both physically and emotional wellbeing: physical functioning; role limitations due to physical health; role limitations due to emotional problems; energy/fatigue; emotional well-being; social functioning; pain and general health. Logistic regression analysis was used to investigate the associations between VD levels and QoL. The mean scores in the total sample were 65.5 (SD ±14.6) for the physical component summary (PCS) score and 58.2 (SD ±15.2) for the mental component summary score (MCS). Vitamin D deficiency was overwhelmingly high among subjects across age and sex. The PCS and MCS QoL scores were also lower than the average. However, the study found no association between VD levels and QoL, presumably due to sample size and con-founders. Further studies are needed to identify the impact of VD levels on perceived QoL. Key words: Vitamin D, poor quality of life (QoL), kingdom of Saudi Arabia (KSA), physical component summary (PCS).

Establishing and Performing Targeted Multi-residue Analysis for Lipid Mediators and Fatty Acids in Small Clinical Plasma Samples.

LC-MS/MS- and GC-MS-based targeted metabolomics is typically conducted by analyzing and quantifying a cascade of metabolites with methods specifically developed for the metabolite class. Here we describe an approach for the development of multi-residue analytical profiles, calibration standards, and internal standard solutions in support of a fast, simple, and low-cost plasma sample preparation that captures and quantitates a range of metabolite cascades.

Determination of total and unbound docetaxel in plasma by ultrafiltration and UPLC-MS/MS: application to pharmacokinetic studies

A sensitive and specific liquid chromatographic/tandem mass spectrometric (LC-MS/MS) method was developed and validated for quantifying total and unbound docetaxel drug concentrations in plasma. Calibration curves for unbound and total docetaxel were linear over the respective ranges of 0.108~10.8 and 0.54~216 ng/mL. The intra- and interday assay accuracy and precision did not exceed 15%. The methods were validated to show the standard range linearity, sensitivity, selectivity, accuracy, precision, and stability of docetaxel in the matrices tested. In addition, this method is fast and simple with a short run time of 4.5 min and a small plasma sample volume (500 µL). The validated method was successfully applied to a pharmacokinetic study of a docetaxel micelle formulation in rat plasma after intravenous administration at a dose of 10 mg/kg. Docetaxel micelles slowly released their drug payload, and protein-bound, unbound, and micellar drug pools existed simultaneously. These various forms in plasma pools were also measured in the study. We confirmed that most of the docetaxel in plasma was micelle-associated (96.52% at 24 h and 83.14% at 72 h) after micellar docetaxel administration, as a result of sequestration of the drug in long-circulating micelles.