Analytes In Whole Blood Research Articles

Liquid-Liquid Extraction Solvent Selection for Comparing Illegal Drugs in Whole Blood and Dried Blood Spot with LC-MS/MS.

This study focused on the simultaneous detection of amphetamine, 3,4-methyl enedioxy methamphetamine, morphine, benzoylecgonine, and 11-nor-9-carboxy- tetrahydrocannabinol (Δ9-THC-COOH) in whole blood and DBS. It is aimed to select a solvent mixture for liquid-liquid extraction (LLE) technique employing LC-MS/MS. The obtained DBS results were compared with the whole blood samples results. A simple, rapid, and reliable LC-MS/MS method was developed and validated for all analytes in whole blood and DBS. LC was performed on a Hypersil Gold C18 column an initial step with a gradient of 0.01 % formic acid, 5 mM ammonium format buffer in water, and acetonitrile at 0.3 ml/min with 7.5-min runtime. A methanol:acetonitrile (40:60 v/v) mixture was selected for both matrices. LOQ values were 10-25 ng/mL; linear ranges were LOQ-500 ng/ml for all analytes; correlation coefficients were greater than 0.99, and all calibrator concentrations were within 20%. Analytical recovery in blood and DBS ranged from 84.9-113.2% of the expected concentration for both intra and-inter day. Analytes were stable for 1, 10, and 30 days after three freeze/thaw cycles. It was determined that the variances of the results obtained with the two matrices in the comparison study were equal for each analyte, and the results were highly correlated (r=0.9625). A sensitive, accurate, and reliable chromatographic method was developed to determine amphetamine, MDMA, morphine, benzoylecgonine, and cannabis, by performing the same preliminary steps with whole blood and dried blood spots. It was observed that the results obtained in these two matrices were compatible and interchangeable when statistically compared.

A Plasmonic Nanoledge Array Sensor for Selective Detection of Cardiovascular Disease Biomarkers in Human Whole Blood.

Optical sensors face challenges when detecting ultralow amounts of analytes in whole blood, including signal quenching due to optical absorption and false positives due to nonspecific binding. This study introduces gold nanoscale array features termed nanoledges (NLs), which interact with incident white light to produce a transmitted surface plasmon resonance (tSPR) signal. This extraordinary optical transmission (EOT) spectrum occurs in the near-infrared (NIR) region, thereby minimizing signal quenching caused by visible-light absorption from blood proteins and pigments. To develop a sensitive, selective, and label-free optical biosensor for detecting various levels of cardiac troponin I (cTnI) in very small volumes of whole blood samples, DNA aptamers are tethered to the NL surface, specifically binding to the cTnI biomarker. This biological binding activity alters the refractive index at the NL surface, causing a peak shift in the EOT spectrum and enabling quantification of cTnI levels. The NL array chip demonstrated high sensitivity for cTnI detection in buffer, human serum (HS), and human whole blood (HB), with detection limits of 0.079, 0.084, and 0.097 ng/mL, respectively. Control measurements using blank target mediums and those containing up to 125 ng/mL of other proteins, such as myoglobin, creatine kinase, and heparin, showed minimal interference and high specificity. The NL plasmonic array's performance in biosensing underscores its promise for clinical analysis and its potential development as a point-of-care platform for early cardiovascular disease (CVD) diagnostics.

The stability of 65 biochemistry analytes in plasma, serum, and whole blood.

The pre-analytical stability of various biochemical analytes requires careful consideration, as it can lead to the release of erroneous laboratory results. There is currently significant variability in the literature regarding the pre-analytical stability of various analytes. The aim of this study was to determine the pre-analytical stability of 65 analytes in whole blood, serum and plasma using a standardized approach. Blood samples were collected from 30 healthy volunteers (10 volunteers per analyte) into five vacutainers; either SST, Li-heparin, K2-EDTA, or Na-fluoride/K-oxalate. Several conditions were tested, including delayed centrifugation with storage of whole blood at room temperature (RT) for 8 h, delayed centrifugation with storage of whole blood at RT or 4 °C for 24 h, and immediate centrifugation with storage of plasma or serum at RT for 24 h. Percent deviation (% PD) from baseline was calculated for each analyte and compared to the maximum permissible instability (MPI) derived from intra- and inter-individual biological variation. The majority of the analytes evaluated remained stable across all vacutainer types, temperatures, and timepoints tested. Glucose, potassium, and aspartate aminotransferase, among others, were significantly impacted by delayed centrifugation, having been found to be unstable in whole blood specimens stored at room temperature for 8 h. The data presented provides insight into the pre-analytical variables that impact the stability of routine biochemical analytes. This study may help to reduce the frequency of erroneous laboratory results released due to exceeded stability and reduce unnecessary repeat phlebotomy for analytes that remain stable despite delayed processing.

Sample stability and heparin interference in ionized calcium and ionized magnesium measurements in horses using the Stat Profile Prime Plus co-oximetry electrolyte analyzer.

The determination of iCa and iMg is important in veterinary medicine, but their immediate determination in whole blood is not always possible. Their stability in other sample types and the existence of interferences must be evaluated before its use. We aimed to analyze the effects of storage time on the stability of iCa, iMg, and other analytes in whole blood, plasma, and serum samples in horses and assess the interference of heparin in these measurements. Whole blood, heparin-plasma, and serum samples from 10 horses were stored at 4°C and analyzed 1, 2, 3, 4, 5, 6, 7, 8, 24, 48, and 168 hours after sample collection using the Stat Profile Prime Plus Vet equipment (Nova Biomedical, Waltham, MA, USA). Results were analyzed by ANOVA or mixed-effect models. The concentration of iCa, iMg, total calcium (tCa), total magnesium (tMg), and the ratios iCa/tCa and iMg/tMg did not differ up to 168 hours when compared to the initial time. Total Ca, iMg, and tMg were not significantly different among sample types, but iCa concentrations were slightly but significantly lower in plasma. Freezing at -20°C did not affect iCa, iMg, tCa, and tMg. The pH increased in serum and plasma after 8 hours, and a mild negative correlation existed between plasma iCa concentration and pH. A negative correlation was observed also between the ratios iCa/tCa or iMg/tMg and pH in plasma and serum. A significant decrease in iCa and iMg was detected when comparing homemade syringes at high heparin concentration (~200-300 U heparin/mL) and commercial lithium-heparin tubes (20-30 U/mL). Samples stored at 4°C can be used to determine iCa and iMg concentrations up to 7 days after collection. Other metabolites are stable for up to 8 hours; heparin interference should be taken into account if using homemade heparin syringes.

Portable, Disposable, Biomimetic Electrochemical Sensors for Analyte Detection in a Single Drop of Whole Blood

Current diagnostics call for rapid, sensitive, and selective screening of physiologically important biomarkers. Point-of-care (POC) devices for the rapid, reliable, and easy acquisition of bioinformation at, or near the patient, offer opportunities for better healthcare management. Electrochemical biosensors with high sensitivity and ease of miniaturization are advantageous for such applications. We report a photolithographically micropatterned PEDOT:PSS and silk protein-based fully organic 3-electrode sensor (O3ES) for ultralow volume (single drop—10 µL) detection of analytes in whole blood. The O3ES produces reliable electrochemical signals in whole blood from a mouse model with minimal biofouling interference. The O3ES is demonstrated as a portable device for the simultaneous detection of dopamine, ascorbic acid and uric acid using voltammetry techniques. The O3ES displays excellent sensitivity towards each analyte in whole blood, and in the presence of each other. The water-based, ambient processing of the sensors allows the immobilization of enzymes in the organic working electrode. Amperometric detection of uric acid via uricase with high sensitivity in whole blood is demonstrated. Finally, the performance of the O3ES under enzymatic degradation is studied by monitoring sensitivity over an operating lifetime of ~14 days. This work demonstrates the realization of low-cost, disposable POC sensors capable of detecting blood metabolites using ultralow sample volumes.

Protective mechanism of dried blood spheroids: stabilization of labile analytes in whole blood, plasma, and serum.

Three-dimensional (3D) dried blood spheroids form when whole blood is deposited onto hydrophobic paper and allowed to dry in ambient air. The adsorbed 3D dried blood spheroid present at the surface of the hydrophobic paper is observed to offer enhanced stability for labile analytes that would otherwise degrade if stored in the traditional two-dimensional (2D) dried blood spot method. The protective mechanism for the dried blood spheroid microsampling platform was studied using scanning electron microscopy (SEM), which revealed the presence of a passivation thin film at the surface of the spheroid that serves to stabilize the interior of the spheroid against environmental stressors. Through time-course experiments based on sequential SEM analyses, we discovered that the surface protective thin film forms through the self-assembly of red blood cells following the evaporation of water from the blood sample. The bridging mechanism of red blood cell aggregation is evident in our experiments, which leads to the distinct rouleau conformation of stacked red blood cells in less than 60 min after creating the blood spheroid. The stack of self-assembled red blood cells at the exterior of the spheroid subsequently lyse to afford the surface protective layer detected to be approximately 30 μm in thickness after three weeks of storage in ambient air. We applied this mechanistic insight to plasma and serum to enhance stability when stored under ambient conditions. In addition to physical characterization of these thin biofilms, we also used paper spray (PS) mass spectrometry (MS) to examine chemical changes that occur in the stored biofluid. For example, we present stability data for cocaine spiked in whole blood, plasma, and serum when stored under ambient conditions on hydrophilic and hydrophobic paper substrates.

Development of “Quick-DB forensic”: A total workflow from QuEChERS-dSPE method to GC–MS/MS quantification of forensically relevant drugs and pesticides in whole blood

Trends in forensic toxicology show the advancement of rapid and sensitive analytical methods for qualitative and quantitative analysis of drugs of abuse. However, forensic toxicologists are continuously faced with the challenges of identifying and quantifying drug blood concentration while simultaneously struggling with manpower shortage. In view of developing a simple and productive toxicological analysis method encompassing total workflow from sample preparation to quantitative analysis, here we describe a simple, robust, and sensitive method for the simultaneous determination and quantification of 63 forensically relevant drugs and pesticides in human whole blood. The method is based on sample preparation by a modified QuEChERS extraction and dispersive solid-phase extraction (dSPE) clean-up followed by gas chromatography–tandem mass spectrometry (GC–MS/MS) analysis. Limits of detection of the target analytes in whole blood ranged in the few ng/mL-order levels. Intra- and inter-day validation result ranges were 0–24% for accuracy (% error) and 0.8–26% for precision (%RSD). Recovery rates ranged from 66% to 84% for barbiturates, 36% to 110% for benzodiazepines, 41% to 86% for tri/tetracyclic antidepressants, 15% to 81% for drugs of abuse, 28% to 44% for phenethylamines, and 25% to 118% for pesticides. The validated results were used to develop a user-friendly, systematic, and quantitative toxicological GC/MS/MS system and software “Quick-DB Forensic”.

Long-term stability of synthetic cathinones in dried blood spots and whole blood samples: a comparative study

The long-term stability of four synthetic cathinones in dried blood spots (DBS) were tested and compared with those of whole blood samples. A method to determine four cathinones (pentylone, butylone, mephedrone and benzedrone) in DBS was developed and validated. For comparison, the traditional liquid-liquid extraction for the same analytes in whole blood was also performed. The calibration curve was found to be linear over 25–1000 ng/mL for DBS samples, with a limit of quantification (LOQ) at 25 ng/mL. The interday imprecision and bias results [up to 7.1% relative standard deviation (RSD) and 5.8%, respectively] were much lower than the maximum data recommended by SWGTOX guidelines. The validation results were similar to those of whole blood samples, which showed interday imprecision and bias results of up to 8.1%RSD and 12.3%, respectively, with a linear calibration curve between 10 and 1000 ng/mL and LOQ of 10 ng/mL. A stability study to compare the degradation rate between both matrices at three storage temperatures [room temperature (25 °C), 4 and − 20 °C] and during 90 days proved the poor stability of cathinones in whole blood, where the methylenedioxy cathinones displayed better stability than those with ring substituents. DBS allowed detection of synthetic cathinones after much longer periods than in whole blood samples. DBS proved, therefore, to be an useful alternative technique to store blood samples. Extraction of DBS is easy and analytes remain stable for much longer periods.

Stability of routine biochemical analytes in whole blood and plasma/serum: focus on potassium stability from lithium heparin.

Blood specimens are transported from clinical departments to the biochemistry laboratory by hospital courier service, sometimes over long distances. The aim of this study was to assess the stability of common biochemical analytes in venous blood under our routine transport conditions and to evaluate analyte stability after prompt or delayed centrifugation. We investigated pre- and postanalytical contributions of 32 biochemical analytes in plasma and serum samples from 10 patients (healthy adults and patients from intensive care units). Differences in analyte concentrations between baseline (T0) and different time intervals (2, 4, 6, 8, 12 and 24 h) following storage after prompt and delayed centrifugation were reported. Evaluation was against the total change limit as described by Oddoze et al. (Oddoze C, Lombard E, Portugal H. Stability study of 81 analytes in human whole blood, in serum and in plasma. Clin Biochem 2012;45:464-9). The majority of analytes were stable with delayed separation up to 12 h, except for potassium, C-peptide, osteocalcin, parathyroid hormone (PTH), bicarbonate and LDH. After prompt centrifugation and storage at 4°C, stability was greatly increased up to 48 h for most analytes. LDH and bicarbonate had the lowest stability after centrifugation; therefore, no reanalysis of these analytes in a centrifuged tube can be allowed. Knowledge of analyte stability is crucial to interpret biological analysis with confidence. However, centrifugation prior to transport is time consuming, and the transfer of plasma or serum from a primary tube to a secondary tube increases the risk of preanalytical errors. For analytes that are stable in whole blood for 24 h or more, it seems that there is no benefit to centrifuge before transport.

Stability of Drugs, Drug Candidates, and Metabolites in Blood and Plasma.

Determination of drug or drug metabolite concentrations in biological samples, particularly in serum or plasma, is fundamental to describing the relationships between administered dose, route of administration, and time after dose for achieving the optimal clinical response. While a well-characterized, accurate analytical method is needed to define these parameters, it must also be established that the analyte concentration in the sample at the time of analysis is identical to the concentration at sample acquisition. This is necessitated by the fact that drugs and their metabolites are susceptible to degradation in samples due to metabolism or to physical and chemical processes, resulting in a lower measured concentration than was in the original sample. Careful examination of analyte stability during processing and storage and, if necessary, adjustment of procedures and conditions to maximize stability, are a critical component of method validation to ensure the accuracy of the data. The protocols provided in this unit address the stability of the analytes in whole blood and blood-derived samples prior to sample preparation for analysis. Issues addressed include sample acquisition, processing of whole blood, and storage of blood-derived samples. © 2016 by John Wiley & Sons, Inc.

Stability of Routine Biochemical Analytes in Whole Blood and Plasma From Lithium Heparin Gel Tubes During 6-hr Storage.

The stability of biochemical analytes has already been investigated, but results strongly differ depending on parameters, methodologies, and sample storage times. We investigated the stability for many biochemical parameters after different storage times of both whole blood and plasma, in order to define acceptable pre- and postcentrifugation delays in hospital laboratories. Twenty-four analytes were measured (Modular® Roche analyzer) in plasma obtained from blood collected into lithium heparin gel tubes, after 2-6 hr of storage at room temperature either before (n = 28: stability in whole blood) or after (n = 21: stability in plasma) centrifugation. Variations in concentrations were expressed as mean bias from baseline, using the analytical change limit (ACL%) or the reference change value (RCV%) as acceptance limit. In tubes stored before centrifugation, mean plasma concentrations significantly decreased after 3 hr for phosphorus (-6.1% [95% CI: -7.4 to -4.7%]; ACL 4.62%) and lactate dehydrogenase (LDH; -5.7% [95% CI: -7.4 to -4.1%]; ACL 5.17%), and slightly decreased after 6 hr for potassium (-2.9% [95% CI: -5.3 to -0.5%]; ACL 4.13%). In plasma stored after centrifugation, mean concentrations decreased after 6 hr for bicarbonates (-19.7% [95% CI: -22.9 to -16.5%]; ACL 15.4%), and moderately increased after 4 hr for LDH (+6.0% [95% CI: +4.3 to +7.6%]; ACL 5.17%). Based on RCV, all the analytes can be considered stable up to 6 hr, whether before or after centrifugation. This study proposes acceptable delays for most biochemical tests on lithium heparin gel tubes arriving at the laboratory or needing to be reanalyzed.

Pre-analytical stability before centrifugation of 7 biochemical analytes in whole blood

The pre-analytical stability of 7 biochemical parameters (parathyroid hormone -PTH-, vitamins A, C E and D, 1,25-dihydroxyvitamin D and insulin) at +4°C, was studied on whole blood samples before centrifugation. The impact of freezing at -20°C was also analyzed/performed for PTH and vitamin D. The differences in the results of assays for whole blood samples, being kept for different times between sampling time and analysis, from 9 healthy adults, were compaired by using a Student t test. The 7 analytes investigated remained stable up to 4 hours at +4°C in whole blood. This study showed that it is possible to accept uncentrifuged whole blood specimens kept at +4°C before analysis. PTH is affected by freezing whereas vitamin D is not.

Preanalytical standardization of sphingosine-1-phosphate, sphinganine-1-phosphate and sphingosine analysis in human plasma by liquid chromatography–tandem mass spectrometry

BackgroundPreanalytical standardization is required for a reliable quantification of the signaling molecules sphingosine-1-phosphate (S1P), sphinganine-1-phosphate (SA1P) and sphingosine (SPH). MethodsMethanolic protein precipitation of 15μL EDTA-plasma was applied prior to analysis. Sphingolipids were separated in 3min by hydrophilic interaction liquid chromatography (HILIC, SeQuant™ ZIC®-HILIC column) followed by tandem mass spectrometry. Stability of analytes in whole blood and plasma was investigated. Sphingolipid concentrations were determined in human plasma (n=50) and mice deficient in sphingosine kinase 1 (SK1) and 2 (SK2) (n=5). ResultsStoring EDTA whole blood >60min after blood withdrawal at room temperature resulted in an increase in S1P and SPH concentrations of ≥25%. Significant changes in SPH levels of +37% were observed after 60min of storage of EDTA plasma at room temperature. Repeated freeze–thaw cycles of EDTA plasma resulted in increased S1P and SPH levels. Concentrations in human EDTA plasma were between 55.5 and 145.2ng/mL for S1P and between 8.9 and 35.3ng/mL for SA1P. Concentrations of S1P were 36% lower and 96% higher in EDTA-plasma from SK1- and SK2-deficient mice, respectively, compared to the wild type. ConclusionsPreanalytical standardization is a precondition for the analysis of sphingolipids in human blood.

Screening for illicit and medicinal drugs in whole blood using fully automated SPE and ultra‐high‐performance liquid chromatography with TOF‐MS with data‐independent acquisition

A broad forensic screening method for 256 analytes in whole blood based on a fully automated SPE robotic extraction and ultra-high-performance liquid chromatography (UHPLC) with TOF-MS with data-independent acquisition has been developed. The limit of identification was evaluated for all 256 compounds and 95 of these compounds were validated with regard to matrix effects, extraction recovery, and process efficiency. The limit of identification ranged from 0.001 to 0.1 mg/kg, and the process efficiency exceeded 50% for 73 of the 95 analytes. As an example of application, 1335 forensic traffic cases were analyzed with the presented screening method. Of these, 992 cases (74%) were positive for one or more traffic-relevant drugs above the Danish legal limits. Commonly abused drugs such as amphetamine, cocaine, and frequent types of benzodiazepines were the major findings. Nineteen less frequently encountered drugs were detected e.g. buprenorphine, butylone, cathine, fentanyl, lysergic acid diethylamide, m-chlorophenylpiperazine, 3,4-methylenedioxypyrovalerone, mephedrone, 4-methylamphetamine, p-fluoroamphetamine, and p-methoxy-N-methylamphetamine. In conclusion, using UHPLC-TOF-MS screening with data-independent acquisition resulted in the detection of common drugs of abuse as well as new designer drugs and more rarely occurring drugs. Thus, TOF-MS screening of blood samples constitutes a practical way for screening traffic cases, with the exception of δ-9-tetrahydrocannabinol, which should be handled in a separate method.

Microextraction by Packed Sorbent for LC–MS/MS Determination of Drugs in Whole Blood Samples

Microextraction by packed sorbent (MEPS) is used as an online sample-preparation method. The determination of local anesthetics lidocaine, ropivacaine and bupivacaine directly in human blood was performed using MEPS online with LC-MS/MS. The range of the calibration curves in whole blood was 10-10000 nmol/l. The lower limit of quantification was set to 10.0 nmol/l. The accuracy of the quality control samples ranged from 85 to 97%. The interday precision of the studied analytes was within the range 1-5%. The regression correlation coefficient (r(2)) was over 0.995 for all runs. The present method is rapid, reliable and robust and may be used for therapeutic drug monitoring of studied analytes in whole blood. This assay allows the analysis of drugs in human blood directly. Sample preparation is simple and automated. The assay reduced the handling time and the cost, and could handle small volumes of whole blood samples (25 µl).

Determination of ciclosporin A and its six main metabolites in isolated T-lymphocytes and whole blood using liquid chromatography–tandem mass spectrometry

A specific and sensitive method for determination of intracellular ciclosporin A (CsA) and its six main metabolites AM1, AM9, AM1c, AM1c9, AM19 and AM4N, in isolated T-lymphocytes and whole blood is described. T-lymphocytes were separated from whole blood using Prepacyte ®. The analytes were extracted and purified from isolated lymphocytes and whole blood by protein precipitation followed by solid-phase extraction (SPE). The analytes and the internal standard, ciclosporin C (CsC), were separated on a reversed phase C8 column (30 mm × 2.1 mm, 3 μm) with a 10 mm × 2 mm, 5 μm Drop-In Guard Cartridge, using gradient elution chromatography and tandem ion trap mass spectrometry detection. The method has been validated in accordance with FDA guidelines and showed linear range from 0.25 to 500 ng/mL for CsA, 0.5 to 500 ng/mL for AM1, AM9 and AM19, 1 to 500 ng/mL for AM4N, AM1c and AM1c9 in intracellular matrix, and 2.5 to 3000 ng/mL for all analytes in whole blood. The applicability of the method is shown on patient samples.

Quantitative analysis of cocaine and its metabolites in whole blood and urine by high-performance liquid chromatography coupled with tandem mass spectrometry

Aim In forensic toxicology it is important to have specific and sensitive analysis for quantification of illicit drugs in biological matrices. This paper describes a quantitative method for determination of cocaine and its major metabolites (ecgonine methyl ester, benzoylecgonine, norcocaine and ethylene cocaine) in whole blood and urine by liquid chromatography coupled with tandem mass spectrometry LC/MS/MS. Method The sample pre-treatment (0.20 g) consisted of acid precipitation, followed by centrifugation and solid phase extraction of supernatant using mixed mode sorbent columns (SPEC ® MP1 Ansys Diag. Inc.). Chromatographic separation was performed at 30 °C on a reverse phase Zorbax C18 column with a gradient system consisting of formic acid, water and acetonitrile. The analysis was performed by positive electrospray ionisation with a triple quadropole mass spectrometer operating in multiple reaction monitoring (MRM) mode. Two MRM transitions of each analyte were established and identification criteria were set up based on the retention time and the ion ratio. The quantification was performed using deuterated internal analytes of cocaine, benzoylecgonine and ecgonine methyl ester. The calibration curves of extracted standards were linear over a working range of 0.001–2.00 mg/kg whole blood for all analytes. The limit of quantification was 0.008 mg/kg; the interday precision (measured by relative standard deviation—%RSD) was less than 10% and the accuracy (BIAS) less than 12% for all analytes in whole blood. Urine samples were estimated semi-quantitatively at a cut-off level of 0.15 mg/kg with an interday precision of 15%. Conclusion A liquid chromatography mass spectrometric (LC/MS/MS) method has been developed for confirmation and quantification of cocaine and its metabolites (ecgonine methyl ester, benzoylecgonine, norcocaine and ethylene cocaine) in whole blood and semi-quantitative in urine. The method is specific and sensitive and offers thereby an excellent alternative to other methods such as GC–MS that involves derivatisation.